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. 2009 Oct;11(5):564–699. doi: 10.1215/15228517-2009-034

Abstracts from the 2009 Joint Meeting of the Society for Neuro-Oncology (SNO) and the American Association of Neurological Surgeons/Congress of Neurological Surgeons (AANS/CNS) Section on Tumors: October 22–24, 2009: New Orleans, LA

PMCID: PMC2765346
Neuro-Oncol. 2009 Oct;11(5):564.

1. JNK2ALPHA2 Regulates GBM Cell Survival by Phosphorylating BCL-2

Ryan Nitta, Albert J Wong

INTRODUCTION

Activation of the JNK pathway has been implicated in glioblastoma multiforme (GBM). Expression and activity of a specific JNK isoform, JNK2α2, are increased in 86% of primary GBMs. Since we have shown JNK2α2 is constitutively active and induces glial tumor formation, we studied the connection between JNK2α2 and a proto-oncogene, BCL-2. BCL-2 was the first gene associated with regulating apoptosis and is important for GBM cell survival. The JNK family was shown to directly phosphorylate BCL-2 at Ser70, thereby regulating its antiapoptotic ability. Enhanced phosphorylation of Ser70 increased resistance to apoptosis and increased tumor formation. We hypothesized that JNK2α2-induced tumorigenesis may result from altered levels of BCL-2 Ser70 phosphorylation.

METHODS

We used retrovirus to introduce shRNAs targeted to the JNK2α gene to reduce expression in two GBM cell lines, U87-MG and U251. Western blot and quantitative polymerase chain reaction analyses were conducted to verify a 4-fold isoform-specific knockdown. Apoptosis was induced using nocodazole and okadaic acid, and cell survival was monitored using the CellTiter Blue assay.

RESULTS

We initially determined that decreased expression of endogenous JNK2α2 by shRNA significantly reduced tumor volume by 3-fold, while the incidence of tumors was reduced by 50%. In addition, when stimulated with okadaic acid, JNK2α2-deficient cells had 4-fold less BCL-2 Ser70 phosphorylation, while cells overexpressing JNK2α2 had a 10-fold increase. Subsequent analysis showed that cells lacking JNK2α2 were more sensitive to nocodazole-induced apoptotic cell death. Interestingly, the control cells had a 10-fold increase in Ser70 phosphorylation when grown in nocodazole, while the JNK2α2-deficient cells had no significant change.

CONCLUSION

Overall, we determined that JNK2α2 was directly involved in glial tumorigenesis and that JNK2α2 was important for GBM cell survival by regulating BCL-2 activity. These results show a novel connection between JNK2α2 and the proto-oncogene BCL-2.

Neuro-Oncol. 2009 Oct;11(5):564.

2. CD44 AND CD155: Is their Coexpression Indicative of a Combined Role in Modulating Glioma Invasion?

Geoffrey J Pilkington, Zaynah Maherally

INTRODUCTION

Increased expression levels of the cell surface molecules CD44 (hyaluronic acid/lymphocyte homing receptor) and CD155 (poliovirus receptor) have been reported on human glioma cells. While CD44 has long been associated with tumor invasion, CD155 has more recently attracted interest for its similar biological role. Here we report the colocalization and proposed associated roles of CD44 and CD155 in glioma invasion.

METHODS

Immunocytochemical (qualitative) and flow cytometric (quantitative) expression of CD44 and CD155 was established for both established cell lines and early-passage cultures of glioma biopsy-derived tissue. Total internal reflected fluorescence (TIRF) microscopy served to give high-signal/low-noise imaging of double-labeled cells. The effects of monoclonal antibody-blocking and siRNA-silencing approaches to both CD44 and CD155, individually and together, were assessed using Transwell assay and live cell imaging for invasion.

RESULTS

CD44 was generally expressed evenly across the cell surface, while CD155 accumulated in “zones” over the cell surface, frequently at the leading edge of invadopodia. TIRF microscopy revealed close proximity between the two epitopes, albeit at distinct sites on the cell surface. CD44 blocking and silencing resulted in a higher level of inhibition of invasion than CD155; such interference with combined CD44/CD155 resulted in 100% inhibition of invasion within the time frame of the studies.

CONCLUSION

Joint CD44/CD155 approaches may merit further study in targeting infiltrating glioma cells in therapeutic protocols.

Neuro-Oncol. 2009 Oct;11(5):564.

3. The Role of ID-1 in Modulating Brain Tumor Invasion and Dispersal

Liliana Soroceanu, Max Ferretti, Darryl Lau, Pierre-Yves Desprez, Sean D McAllister

INTRODUCTION

Id-1, the inhibitor of basic helix-loop-helix transcription factors, regulates various aspects of cell proliferation, differentiation, migration, and invasion in several types of tissues. We set out to investigate whether Id-1 modulates the dispersal and invasiveness of high-grade gliomas (GBM).

METHODS

Western blot analysis, immunofluorescence, and invasion assays were used.

RESULTS

In SF210 and U87 glioma cells, Id-1 protein was not detected, whereas significant levels of Id-1 were expressed in SF126 and U251 glioma cell lines. We next determined whether there was a correlation between expression levels of Id-1 and the magnitude of GBM cell invasion. Cell lines expressing Id-1 exhibited a 5- to 7-fold increase in cell invasion compared to GBM cells in which Id-1 could not be detected, demonstrating a strong positive correlation between Id-1 expression levels and GBM cell invasiveness. We measured Id-1 levels in several primary GBM-derived cultures and found that more than 75% of the cultures expressed Id-1 protein. Immunohistochemical analyses of tissue microarrays containing different grades of glioma documented that Id-1 expression levels correlated positively with tumor grade. Using antisense approaches, we also determined that Id-1 knockdown sig-nificantly inhibited GBM cell invasion. Overall, these data suggested that Id-1 promotes an invasive phenotype in GBM cells. Moreover, we recently found that cannabidiol (CBD), a nontoxic cannabinoid, was an effective inhibitor of GBM cell aggressiveness. Prolonged CBD treatment induced a concentration-dependent downregulation of Id-1 gene expression and a corresponding inhibition of cell invasion.

CONCLUSION

Taken together, these data suggest that Id-1 expression is a hallmark of the most malignant and invasive human gliomas. Furthermore, our preliminary data suggest that the nonpsychoactive compound CBD can reduce Id-1 expression and corresponding tumor cell dispersal. The use of CBD may therefore represent a novel therapeutic approach for GBM patients.

Neuro-Oncol. 2009 Oct;11(5):565.

4. Targeting Myosin II Activity Blocks Glioma Invasion Even in the Presence of Multiple Motogenic Signals

Steven Rosenfeld, Sanja Ivkovic, Christopher Beadle, Peter D Canoll

INTRODUCTION

There remains a pressing need to identify new strategies to block glioma invasion of brain, particularly ones that are likely to work within a microenvironment in which promigratory ligands and their receptors stimulate brain invasion. We had previously shown that brain invasion by glioma cells has an absolute requirement for the molecular motor myosin II. In this study, we examined how necessary myosin II is under the more realistic setting of epidermal growth factor receptor (EGFR) overexpression, a promigratory molecular alteration that is found in approximately 50% of malignant gliomas.

METHODS

We infected glial progenitor cells with two retroviruses, encoding for 1) PDGF-GFP and 2) an EGFR-red fusion protein, which signals in a ligand-dependent manner. This results in formation of tumors that have all the histologic features of human glioblastoma and consist of green, PDGF-secreting tumor cells and red, EGFR-expressing tumor cells. By contrast, injecting rats with the EGFR-expressing retrovirus alone drives the infected glial progenitors to diffusely invade white matter in a pattern reminiscent of gliomatosis cerebri. We examined the migratory patterns of both types of tumor cell in brain slices from tumor-bearing rats using time-lapse microscopy.

RESULTS

Addition of EGF to the brain slices markedly enhanced the migration of the EGFR-expressing tumor cells. Addition of the EGFR tyrosine kinase inhibitor Iressa had only a minimal inhibitory effect on the invasive behavior of these cells, although it completely blocked their migration in the absence of PDGF secretion. By contrast, blebbistatin, a highly specific allosteric inhibitor of myosin II, completely stopped brain invasion of both the EGFR-expressing and PDGF-secreting tumor cells, even in the presence of added EGF. Our results in brain slices complement our Transwell invasion studies, which demonstrate that while the promigratory cytokines PDGF and lysophosphatidic acid completely overcome the antiinvasive effects of upstream signal transduction inhibitors, they cannot overcome the antiinvasive effects of blebbistatin.

CONCLUSION

Our results indicate that while glioma invasion is highly resistant to inhibitors of signal transduction pathways that are frequently abnormal in these tumors, it remains highly sensitive to an antiinvasion strategy that directly targets the molecular motor myosin II.

Neuro-Oncol. 2009 Oct;11(5):565.

5. Different Changes in Protein and Phosphoprotein Levels Result from Serum Starvation of High-Grade Glioma and Adenocarcinoma Cell Lines

Victor A Levin, Sonali Panchabhai, Li Shen, Steven M Kornblau, Yihua Qiu, Keith A Baggerly

INTRODUCTION

Changes in protein and phosphoprotein levels after serum starvation may reflect the adapted phenotype of the metabolically stressed cancer cell trying to adjust to a changed environment in vivo by inducing signal transduction to support tumor cell growth and existence.

METHODS

We used reverse-phase protein microarrays to interrogate five high-grade glioma cell lines and seven adenocarcinoma cell lines for differences in the expression of 81 proteins and 25 phosphoproteins. Cell lines were studied in the well-fed condition of growth with 10% FBS and in the starved condition with 0.5% FBS. Protein expression levels were normalized to β-actin and trichotomized as increased (+1, upper 75th quartile), decreased (−1, lowest 25th quartile), or unchanged (0, others) to focus on the patterns of the biggest changes in protein and phosphoprotein levels to understand starved-fed differences among the cell lines and gain insight into the effects of serum starvation on cellular responses.

RESULTS

Expression of proteins and phosphoproteins 24 h after FBS starvation increased more often in glioma lines than in adenocarcinoma lines, which appeared to have fewer increased protein scores and more decreased scores. Many proteins increased in gliomas were downstream targets of the PTEN/PI-3 kinase/AKT, EGFR/MAPK/Stat, and transcription activator/polyamine signaling pathways. In adenocarcinomas, expression of proteins and phosphoproteins generally increased in apoptosis pathways, while there were minor fluctuations in the other pathways listed above. Contrawise, gliomas became resistant to apoptosis after 24 h of serum starvation and upregulated transcription activators and polyamines more than adenocarciomas.

CONCLUSION

High-grade gliomas respond differently to stress induced by serum starvation than adenocarcinomas, based on changes in the expression of proteins and phosphoproteins involved in signaling pathways. Information obtained from these studies also includes differences in vulnerability to apoptosis-differential regulation of transcription activators and polyamines. These data, together with ongoing hypoxia studies, may guide future targeted chemotherapy for gliomas.

Neuro-Oncol. 2009 Oct;11(5):565.

6. CA IX Mediates Invasion of Glycolytic Glioblastoma Cells by Modulation of Cathepsin B Secretion

Martin A Proescholdt, Marsha J Merrill, Munzir Al-Zabin, Eva-Maria Störr, Annette Lohmeier, Alexander T Brawanski

INTRODUCTION

Malignant gliomas show high rates of glycolysis caused by hypoxia and specific gene alterations leading to high lactic acid levels. We hypothesized that the hypoxia-inducible carbonic anhydrase (CA) IX is involved in the extrusion of hydrogen ions into the extracellular space. This may cause the activation of cathepsin B owing to the extracellular acidification, which ultimately leads to increased invasion of glioblastoma cells under glycolytic conditions. We therefore induced glycolysis in glioblastoma cells and observed their invasive potential in the presence of a specific CA inhibitor. To detect cathepsin B activation, we analyzed the expression and subcellular distribution of cathepsin B and the secretion of cathepsin B into the supernatant.

METHODS

U251 glioblastoma cells were cultured in a BioCoat Matrigel invasion chamber. The chambers were incubated in a 5% CO2 modular with either 21% oxygen and 25 mM glucose in the culture medium (control) or 0% oxygen plus 125 mM glucose (glycolysis). CA activity was inhibited by 40 mM acetazolamide. Invasion was quantified by counting the cells that invaded into the lower culture compartment. Cathepsin B’s expression was investigated by Western blot, its subcellular distribution by immunofluorescent staining, and its secretion into the supernatant using a cathepsin B activity assay.

RESULTS

Glycolysis caused a significant increase of cathepsin B secretion combined with a massive invasion of glioblastoma cells. Acetazolamide reduced both the cathepsin B secretion and the invasive potential. In addition, the subcellular redistribution of cathepsin B caused by glycolysis was attenuated by acetazolamide.

CONCLUSION

Our data demonstrate that glycolysis leads to enhanced invasion of glioblastoma cells, which is mediated by increased cathepsin B secretion. Inhibition of CA reduces the invasive potential by reducing the cathepsin B secretion. This indicates a link between glycolytic metabolism, pericellular acidification moderated by CAs, cathepsin B activity, and the invasive potential of glioblastoma cells.

Neuro-Oncol. 2009 Oct;11(5):565.

7. PDGFR-α Stimulates Glioma Cell Invasion Through Tyrosine Phosphorylation of a Bipartite Guanine Nucleotide Exchange Factor, Dock180

Haizhong Feng, Bo Hu, Kunwei Liu, Andrius Kazauskas, Kodi Ravichandran, Kristiina Vuori, Ryo Nishikawa, Motoo Nagane, Shi Yuan Cheng

INTRODUCTION

Platelet-derived growth factor receptor (PDGFR)-α ranks third among the top amplified genes in glioblastomas and is overex-pressed in low-grade gliomas. Dock180 is a guanine nucleotide exchange factor (GEF) for Rac1 that mediates growth factor-induced cell migration in drosophila and is critical in glioma cell invasion. However, the mechanisms by which PDGFR-α promotes glioma invasion and whether Dock180 is involved in PDGFR-α signaling are largely unknown.

METHODS

PDGFR-α, PDGF-A, and Dock180 expression was analyzed by immunohistochemical staining. The effect of activation of PDGFR-α on glioma cell invasion was assessed by Boyden chamber assays in vitro and in an intracranial glioma mouse model. The effect of tyrosine phosphorylation of Dock180 on PDGFR-α signaling was examined by expression of various deletion or point mutants of Dock180 with PDGFR-α, CrkII, and p130cas in 293T cells and glioma cells, followed by Western blot analysis.

RESULTS

PDGFR-α, PDGF-A, and Dock180 were coexpressed in invasive areas but not in the central regions of primary glioma tissue specimens. Expression of PDGF-A by glioma LN444 and LN443 cells significantly promoted glioma growth and invasion in the brain. Depletion of shRNA of Dock180 inhibited PDGF-A-promoted cell migration in vitro and glioma invasion in the brain. PDGF-A stimulated cell motility and tyrosine phosphorylation of Dock180, and AG1296, an inhibitor of PDGFR-α, inhibited PDGF-A stimulation. Tyrosine 1811 (Y1811) of Dock180, located within the CrkII-binding domain at its C-terminus, was identified as a major phosphorylation site. Mutation of Y1811 to F1811 (phenylalanine) significantly attenuated PDGFR-α-induced phosphorylation and decreased Rac1 activity. Coexpression of Dock180, PDGFR-α, and CrkII promoted PDGF-A-induced association of Dock180, CrkII, and p130Cas, whereas the Y1811F mutant disrupted their association.

CONCLUSION

Our results suggest an unanticipated mechanism by which PDGFR-α stimulates glioma cell invasion through phosphorylation at Y1811 of the Rac1 GEF Dock180, enhancing the activation of the CrkII/p130cas/Rac1 pathway.

Neuro-Oncol. 2009 Oct;11(5):566.

8. Metabolic Targeting of Malignant Glioma: Downregulation of Glioma-Expressed Erythropoietic Factors via siRNA in Vitro and in Vivo

Todd Brendon Francis, Murali Guthikonda, Setti S Rengachary, Saroj Mathupala

INTRODUCTION

Malignant astrocytoma (glioblastoma in particular) metabolizes glucose predominantly via “aerobic glycolysis” (i.e., lactic acid fermentation), regardless of the oxygen tension in the tumor tissue. To facilitate this aberrant phenotype, glioblastoma upregulates several fetal-type glycolytic enzyme isoforms that show much greater affinity for glucose and less sensitivity to metabolic regulation. This altered metabolic profile results in a large glucose-to-lactate flux in glioblastoma, with the lactate being rapidly shunted out of the tumor cells via specific transmembrane transporters. Targeting these transporters via siRNA or small-molecule drugs has been shown to cause cell death in glioblastoma via the inhibition of glycolysis. Analysis of the promoter of the lactate transporter gene that is predominantly expressed in glioblastoma indicates multiple response elements for GATA-1, a transcription factor that previously had been thought to be involved only in erythropoiesis and that is expressed by glioblastoma. Thus, we investigated whether regulation of GATA-1 expression can adversely affect glioblastoma.

METHODS

U87-MG glioblastoma tumor cell lines were engineered with regulated expression systems to both silence (via siRNA, utilizing a tetracycline-regulated expression system) and over-express GATA-1. We have tested the effect of downregulation of GATA-1 on U87-MG glioblastoma cells in vitro on cell proliferation and cell cycling as a preliminary evaluation of the therapeutic utility of GATA-1 targeting as an adjuvant metabolic therapy against glioblastoma.

RESULTS

Western blot analysis was used to demonstrate regulation of GATA-1 expression in the engineered glioblastoma cells at the protein level. While GATA-1 over-expression enhanced the proliferation of the cells, siRNA-mediated silencing indicated the opposite effect.

CONCLUSION

Our studies indicate that GATA-1 is important for the modulation of glioblastoma cell proliferation, with its silencing demonstrating a negative effect on tumor growth and its overexpression enhancing proliferation. Thus, targeting GATA-1 may be a novel adjuvant clinical therapeutic avenue against glioblastoma.

Neuro-Oncol. 2009 Oct;11(5):566.

9. Interleukin-17A Promotes Autoimmunity by Synergizing with Interleukin-6 via a Positive-Feedback Loop of Interleukin-6 Induction in Primary Astrocytes

Xiangyu Ma, Hongwei Qin, Etty Benveniste

INTRODUCTION

Interleukin-17A (IL-17A) and IL-6 are important in many disorders characterized by immune self-recognition, and IL-6 is known to induce the differentiation of T helper 17 (Th17) cells.

METHODS

Primary astrocyte cell culture was performed, and mRNA extraction, RT-PCR, and Western blot were used to analyze the samples.

RESULTS

A positive-feedback loop of IL-6 signaling enhanced by IL-17A in astrocytes, which was a target of TH17 and IL-17A in CNS and also IL-17, strongly synergized with IL-6 for induction of CCL20. This positive feedback involved the activation of nuclear factor (NF)-κB, signal transducer and activator of transcription 3, and extracellular signal-regulated kinases (ERK) pathways in primary astrocytes. The NF-κB and ERK pathway inhibitors BAY11 and U0126 reversed the synergistic effect of IL-6 and IL-17A on IL-6 and CCL20 mRNA abundance.

CONCLUSION

The synergistic effect of IL-6 and IL-17A on IL-6 and CCL20 may play a key role in neuroautoimmunity.

Neuro-Oncol. 2009 Oct;11(5):566.

10. Role of AQP1 in Invasion of GBM

Gay M Samuelson

INTRODUCTION

There has been little improvement in glioblastoma multiforme (GBM) patient prognosis over the past 20 years, despite advances in imaging, neurosurgery, postoperative care, radiation delivery, and chemotherapy. One reason for treatment failure is the invasive nature of glioma cells. Thus, it is essential to understand the mechanisms underlying invasion so that therapeutic targets can be developed and their impact assessed, noninvasively, during treatment.

METHODS

Aquaporin 1 (AQP1) overexpression correlates inversely with GBM survival and drives migration of invading glioma cells. Our goal is to determine relationships between AQP1 expression and brain tumor invasion in tissue culture by wound assay, in orthotopic murine brain slices by confocal microscopy, and in rodent xenografts by diffusion-weighted magnetic resonance imaging (DW-MRI). Modulating aquaporin channels alters water diffusion through cells, and these changes have been detected with DW-MRI.

RESULTS

Our data on 168 GBM patients show AQP1 over-expression correlates inversely with survival. Wound assay data indicate AQP1 overexpression drives migration and tetraethylammonium chloride, an inhibitor of AQP1 water permeation, inhibits migration. Histologic data in rodent xenografts demonstrate aggressive invasive behavior of AQP1 overexpressors compared to empty vector controls. We will also present preliminary MRI results using an orthotopic rodent model in which we modulate levels of AQP1 expression.

CONCLUSION

Our data support our hypotheses that the water permeation function of AQP1 drives migration and that DW-MRI can be used to measure the water permeation and, hence, invasion.

Neuro-Oncol. 2009 Oct;11(5):566.

11. Tumor Heterogeneity in Glioblastoma is Actively Maintained by a Paracrine Circuit Driven by Mutant EGFR

Frank Furnari, Maria del Mar Inda, Rudy Bonavia, Dinah Sah, Akitake Mukasa, Yoshitake Narita, Terrance G Johns, Robert Bachoo, Philipp Hadwiger, Pamela Tan, Ron A DePinho, Webster Cavenee

INTRODUCTION

A powerful oncogenic event in glioblastoma (GBM) is the amplification and rearrangement of the epidermal growth factor receptor (EGFR) (EGFRvIII, de2-7EGFR [deltaEGFR]). Yet notably, only a minority of primary GBM cells possess deltaEGFR, while the remainder maintain expression of wild-type EGFR (wtEGFR). The disconnect between tumorigenic potential and the frequencies and proportions of these receptors might result simply from a stochastic process wherein independent genetic events arise in GBM, rapidly fatal tumors that never have the time to become homogeneous. Another possibility would be that deltaEGFR occurs later in tumor progression, and the minority of cells that express it not only enhance their own intrinsic tumorigenic abilities but also potentiate the proliferation of neighboring majority cells expressing amplified wtEGFR. If the latter is true, then elucidation of such a potentiation loop would provide a potential novel therapeutic point of attack for high-grade disease.

METHODS

We engrafted wtEGFR-expressing glioma cells into mice and examined the ability of small numbers of coinjected deltaEGFR-expressing cells to influence tumorigenic growth. We also examined conditioned media produced from deltaEGFR-expressing cells (deltaEGFR-CM) for the ability to activate signaling pathways in wtEGFR-expressing cells and for the presence of potential paracrine factors.

RESULTS

Heterogeneous tumors produced from mixtures of delta-EGFR- and wtEGFR-expressing cells had enhanced tumor growth owing to increased growth of the wtEGFR-expressing cells. In vitro exposure of the wtEGFR-expressing cells to deltaEGFR-CM caused the activation of wtEGFR and the gliomagenic signature molecules Akt, MAPK, and STAT3, elicited in major part by abundant interleukin-6 (IL-6) secreted by deltaEGFR-expressing cells. Conversely, siRNA knock-down of IL-6 expression in deltaEGFR-expressing cells ablated their ability to enhance the growth of wtEGFR-expressing cells in vivo.

CONCLUSION

Tumor heterogeneity may not simply represent a stochastically occurring feature but rather may be the consequence of mechanisms actively maintaining the heterogeneous state. These observations also provide novel strategies for more effective therapeutic combinations.

Neuro-Oncol. 2009 Oct;11(5):566–567.

12. IGFBP2 is a Candidate Biomarker for Ink4a-Arf Status and a Therapeutic Target for High-Grade Gliomas

Lynette Moore, Kristen M Holmes, Sarah M Smith, Ying Wu, Elena Tchougounova, Lene Uhrbom, Raymond Sawaya, Janet M Bruner, Gregory N Fuller, Wei Zhang

INTRODUCTION

Loss of Ink4a-Arf and overexpression of insulin-like growth factor binding protein 2 (IGFBP2) are two of the most frequent events in glioma; however, little is known about their relationship. Here we propose that loss of Ink4a-Arf is a key event leading to IGFBP2 overexpression in gliomas.

METHODS

These studies utilized transgenic mice expressing the avian viral receptor tv-a driven by the glial progenitor-specific nestin promoter (Ntv-a mice), which were injected with PDGFB in the avian viral vector RCAS (replication competent avian SLV long terminal repeat with splice acceptor) to induce gliomas.

RESULTS

We identified an inverse relationship between IGFBP2 and p16INK4a protein levels in human primary glioma samples and 90 cancer cell lines. We extended these studies to the Ntv-a mouse glioma model by crossing Ntv-a mice with Ink4a-Arf-null mice to generate Ntv-a; Ink4a-Arf−/− mice. Previous studies revealed that overexpression of PDGFB in wild-type mice promoted the development of low-grade oligodendroglioma. Combining IGFBP2 with PDGFB in this system promoted progression to anaplastic oligodendroglioma (AO). We hypothesized that loss of Ink4a-Arf bypasses the requirement of exogenously introduced IGFBP2 for glioma progression. Of the Ntv-a; Ink4a-Arf−/− mice injected with RCAS-PDGFB, 97% developed tumors, and in contrast to Ntv-a wild-type mice, all tumors were classified as AO. Tumors from these mice demonstrated upregulation of endogenous IGFBP2, especially at the leading edges of the tumors. We utilized an IGFBP2 antisense RCAS construct to decrease IGFBP2 levels. Delivery of PDGFB and the IGFBP2 antisense to the Ntv-a; Ink4a-Arf−/− mice conferred a survival benefit to these animals.

CONCLUSION

These observations suggest IGFBP2 may serve as a marker and therapeutic target for Ink4a-Arf-deleted gliomas. This study provides a more in-depth understanding of the intricate interplay between tumor suppressor loss and oncogene overexpression that leads to glioma initiation and maintenance.

Neuro-Oncol. 2009 Oct;11(5):567.

13. ADAR3 Expression is Decreased in Glioblastomas and it Regulates Glioma Cell Migration and Response to Radio- and Chemotherapy

Susan Finniss, Simona Cazacu, Ninette Amariglio, Gideon Rechavi, Tom Mikkelsen, Chaya Brodie

INTRODUCTION

Adenosine to inosine (A-to-I) RNA editing is a site-specific modification in stem loop structures within precursor mRNAs, catalyzed by members of the double-stranded RNA (dsRNA)-specific ADAR family, and it is essential for the normal development of both invertebrates and vertebrates. ADAR family members ADAR1 and ADAR2 are ubiquitously expressed and catalytically active; the expression of ADAR3 is limited to the brain and is not directly involved in RNA editing.

METHODS

We recently reported that brain tumors exhibit significant global hypo-editing of Alu repetitive elements. In addition, we found reduced RNA levels of all three ADAR proteins in these tumors.

RESULTS

In this study, we further found that ADAR3 expression was significantly lower in glioma cell lines than in normal human astrocytes. In addition, ADAR3 expression correlated with patient survival. Using ADAR3-GFP, we found that this protein was localized to the nucleoli of both astrocytes and glioma cells. We demonstrated that overexpression of ADAR1 and ADAR2 decreased the proliferation of glioma cells. In contrast, overexpression of ADAR3 did not significantly affect glioma cell proliferation or the expression of ADAR1 and ADAR2. However, ADAR3 decreased the migration of glioma cells, whereas it did not alter the migration of normal human astrocytes. In addition, overexpression of ADAR3 increased the sensitivity of glioma cells to γ-radiation and temozolomide.

CONCLUSION

In summary, we found that the expression of ADAR3, which localizes to chromosome region 10p15, frequently deleted in glioblastoma, was significantly decreased in astrocytic tumors. We also found that overexpression of ADAR3 in glioma cells selectively decreased the migration of these cells and increased their radiosensitivity and chemosensitivity. We propose that the effect of ADAR3 may be mediated by mechanisms that are associated with its ssRNA and dsDNA binding modules and that ADAR3 may serve as a therapeutic target and a diagnostic marker in astrocytic tumors.

Neuro-Oncol. 2009 Oct;11(5):567.

14. PH-Sensitive Liposomes as a Potent Novel Treatment for Primary Brain Tumors

Marie-Belle Poirier, Marc-Andre Bellavance, Fernand J Gobeil, David Fortin

INTRODUCTION

The treatment of many CNS diseases, such as primary brain tumors, is considerably impaired due to the limited delivery of therapeutics engendered by the blood-brain barrier (BBB). Liposomes represent a drug delivery system that could enhance the distribution of therapeutic agents across the BBB to primary brain tumors. Here we have generated different liposomal formulations and assessed their lipofection efficiency and intracellular fate in vitro.

METHODS

Briefly, different mixtures of lipids were dissolved in chloroform and dried by rotary evaporation. The resulting lipid-film was suspended in HEPES buffer containing propidium iodide (PI). Liposomes were then extruded through 200/100 nm pores and purified by exclusion chromatography. Liposome concentration was adjusted to 0.1 mM following enzymatic quantification, and morphological analyses were carried by transmission electron microscopy. Time course analyses (at 0, 1, 2, 4, 6, 12, and 24 h) of liposomes or free-PI uptake were performed on F98 and U118 cells using flow cytometry (FAC-Scan). Intracellular fate was studied by confocal microscopy using DHPE-OregonGreen and PI.

RESULTS

Among the liposomal formulations tested, a mixture of DPPC:DC-Chol:DOPE:DHPE-OregonGreen with a mean diameter of 100 nm depicted the best lipofection efficiency. FAC-Scan established that nearly 100% of exposed cells had uptaken liposomes after 2 h and that intracellular delivery of PI occurred in 95% after 6 h in both cell lines. Addition of DPPE-PEG2000 to the formulation restrained liposome uptake in U118 cells and severely hampered intracellular cellular delivery of PI in both cell lines, presumably by preventing its endosomal escape. Free PI did not stain F98 or U118 significantly.

CONCLUSION

These promising results reveal high lipofection levels of F98 and U118 cells and effective intracellular delivery only by PEG-free formulations. The underlying mechanisms of internalization are currently being addressed. Next we will establish the effectiveness of liposomes to circumvent the BBB and deliver their contents to primary brain tumors using our F98/implanted rat model.

Neuro-Oncol. 2009 Oct;11(5):567.

15. In Vitro Selection for Invasion Alters Cell Phenotype and Mirna Expression in Glioblastoma Cell Lines

Gail S Martin, Kevin J Shultz, Matthew K Ronck, L Gerard Toussaint

INTRODUCTION

Glioblastoma (GBM) patients suffer from a disease that is currently incurable. Local therapies—surgery and targeted radiation—cannot treat cells invading far from the original tumor nidus. In fact, invasive GBM cells are relatively resistant to conventional adjuvant therapies. In these assays, we aim to identify critical miRNA species that support the invasive phenotype.

METHODS

GBM cell lines were selected in vitro for an invasive phenotype using serial passaging through Matrigel. The miRNA expression profile of the invasive lines was compared to that of the parental cell population. We selected miRNA species with expression profiles correlating with invasion. Using antagomirs and expression vectors, we investigated the effects of knock-down and overexpression on the in vitro phenotype. In vivo assays are pending.

RESULTS

Serial selection of GBM cell lines produces subpopulations with the expected phenotype: enhanced invasion. These invasive cells differentially up- and downregulate a repertoire of miRNA species compared to their nonselected parental lines. Enhanced expression or repression of certain miRNAs identified in this screen reproduced the invasive phenotype.

CONCLUSION

A simple in vivo selection algorithm easily produces subpopulations of cells capable of enhanced invasion. From these subpopulations, meaningful information can be gleaned regarding the miRNA control of tumor dispersal. In vivo confirmation of these data is ongoing and could lead to new therapeutic targets in GBM.

Neuro-Oncol. 2009 Oct;11(5):567.

16. Low-Level Expression of Collapsin Response Mediator Protein1 in Human Gliomas with Mutant EGFRVIII Promotes Invasion

Joydeep Mukherjee, Leroi DeSouza, Johann Micaleff, Zia Karim, Sidney E Croul, KW Michael Siu, Abhijit Guha

INTRODUCTION

Glioblastoma multiforme (GBM) is the most common and lethal primary human brain tumor. GBMs are characterized by a variety of genetic alterations, among which oncogenic mutations of epidermal growth factor receptor (EGFRvIII) are most common. GBMs harboring EGFRvIII have increased proliferation and invasive characteristics compared to those expressing wild-type EGFR (wtEGFR).

METHODS

To identify the molecular basis of this increased tumorgenic phenotype, we utilized iTRAQ-labeling differential proteomic analysis. Among several differentially expressed proteins, we selected for further study collapsin response mediator protein1 (CRMP1), a protein implicated in cellular invasion that was markedly decreased in GBMs expressing EGFRvIII.

RESULTS

The differential expression of CRMP1 was confirmed in a panel of human GBM cell lines and operative specimens expressing wtEGFR or mutant EGFRvIII. In human GBM samples, decreased expression of CRMP1 correlated with EGFRvIII positivity. Knockdown of CRMP1 resulted in increased invasion of wtEGFR-expressing human GBM cells (U87 and U373) compared to those found in isogenic GBM cells. Exogenous expression of EGFRvIII in these wtEGFR-expressing GBM cells promoted their ability to invade and was accompanied by decreased expression of CRMP1. Rescuing CRMP1 expression decreased invasion of the EGFRvIII-expressing GBM cells by tilting the balance between Rac and Rho.

CONCLUSION

Collectively, these results demonstrate that the loss of CRMP1 contributed to the increased invasive phenotype of human GBM expressing mutant EGFRvIII. Further investigation is needed to elucidate the complete mechanism by which CRMP1 modulates glioma invasion and migration.

Neuro-Oncol. 2009 Oct;11(5):568.

17. Activation of the PI3K/MTOR Pathway Occurs in the Majority of Adult Low-Grade Gliomas and Predicts Patient Survival

Sean M McBride, Daniel A Perez, Mei-Yin C Polley, Scott R VandenBerg, Justin S Smith, Shichun Zheng, Kathleen L Lamborn, John Wiencke, Susan Chang, Michael Prados, Mitchel S Berger, David Stokoe, Daphne Haas-Kogan

INTRODUCTION

Recent evidence suggests the Akt-mTOR pathway plays a role in the development of low-grade gliomas (LGG). This pathway amplifies protein transcription, intensifies cell growth, and halts apoptosis. PTEN inhibits the pathway; PRAS40 is a downstream inhibitor that, when phosphorylated, is itself inhibited; and S6, when phosphorylated, increases ribosomal biogenesis. We sought to evaluate whether the activation of this pathway correlates with survival in LGG by examining expression patterns of the above-mentioned proteins. Such a relationship would provide rationale for selectively targeting this pathway with the mTOR inhibitor rapamycin.

METHODS

Forty-five LGG tumor specimens from newly diagnosed patients were analyzed for methylation of the putative 5′-promoter region of PTEN using methylation-specific PCR as well as phosphorylation of S6 and PRAS40 and expression of PTEN protein using immunohistochemistry. Relationships between molecular markers and overall survival were assessed using Kaplan-Meier methods and the exact log-rank test. Correlation between molecular markers was determined using the Mann-Whitney U and Spearman rank correlation tests.

RESULTS

Eight of 26 patients with methylated PTEN died, compared to 1 of 19 without methylation. There was a trend toward statistical significance, with PTEN-methylated patients having decreased survival (p = 0.128). Eight of 29 patients that expressed phospho-S6 died, whereas all nine patients lacking p-S6 expression were alive at last follow-up. There was an inverse relationship between expression of phospho-S6 and survival (p = 0.029). There was a trend toward decreased survival in patients expressing phospho-PRAS40 (p = 0.077). Analyses of relationships between molecular markers demonstrated a statistically significant positive correlation between expression of p-S6(235) and p-PRAS40 (p = 0.04); expression of p-S6(240) correlated positively with PTEN methylation (p = 0.04) and negatively with PTEN expression (p = 0.03).

CONCLUSION

Survival of LGG patients correlates with phosphorylation of S6 protein. This relationship supports the use of selective mTOR inhibitors in the treatment of LGG.

Neuro-Oncol. 2009 Oct;11(5):568.

18. Suppression of Flips Protein Synthesis and Stability Synergistically Sensitize GBM Cells to Trail-Induced Apoptosis

Amith Panner, Courtney Crane, Andrew T Parsa, Russ Pieper

INTRODUCTION

We previously showed that the antiapoptotic protein FLIPs is a key controller of TRAIL-induced apoptosis in glioblastoma (GBM) cells and that levels of FLIPs are controlled by two distinct PTEN-regulated pathways: a PTEN-Akt-mTOR pathway that controls the translation of FLIPs mRNA and a PTEN-USP8-AIP4 pathway that controls FLIPs protein ubiquitination and stability. The relative contribution and therapeutic potential of manipulation of each pathway, however, remains unknown.

METHODS

To address the relative contributions of FLIPs mRNA translation and FLIPs stability to GBM apoptotic sensitivity, we pharmacologically and/or genetically manipulated both pathways in an independent manner in xenograft cells derived from primary human GBM and monitored the effects on TRAIL sensitivity.

RESULTS

Exposure of PTEN-mutant, TRAIL-resistant GBM cells to a concentration of rapamy-cin that maximally suppressed pS6K levels did not effect FLIPs protein stability but did suppress levels of FLIPs and TRAIL resistance. Similar overexpression of USP8 and subsequent destabilization of FLIPs protein did not alter pS6K levels but did sensitize cells to TRAIL-induced apoptosis to a similar extent as did rapamycin. Overexpression of USP8 in combination with rapamycin additively or synergistically increased TRAIL sensitivity.

CONCLUSION

These results suggest that suppression of FLIPs production and stability synergistically sensitizes GBM cells to TRAIL-induced apoptosis. Maximal sensitization of GBM cells to TRAIL-induced apoptosis may therefore be best achieved by combining inhibitors of protein translation, such as mTOR inhibitors, with agents that can selectively target the FLIPs ubiquitination pathway.

Neuro-Oncol. 2009 Oct;11(5):568.

19. Fibulin-3, An ECM Protein Specifically Upregulated in Malignant Gliomas, Promotes Tumor Invasion and Resistance to Apoptosis

Bin Hu, Joshua Saldivar, Hosung Sim, Keerthi Thirtamara-Rajamani, Mariano S Viapiano

INTRODUCTION

A fundamental challenge in treating malignant gliomas is their distinctive ability to infiltrate normal neural tissue, which makes them virtually impossible to eliminate by conventional therapies. Glioma cells are thought to produce unique molecular changes in their surrounding extracellular matrix (ECM) that form a microenvironment permissive for cell motility. We have identified a novel ECM protein, fibulin-3, that has a unique expression and protumoral role in gliomas compared to other solid tumors.

METHODS

Fibulin-3 expression in solid tumors was compared by meta-analysis of microarray data. Expression in gliomas and glioma cells was validated by qRT-PCR and Western blotting. Glioma cell lines expressing fibulin-3 or specific siRNAs were tested for changes in proliferation, adhesion, and migration. Rat glioma cells were implanted intracranially, and tumors were evaluated for total volume, invasive profile, metalloprotease expression, TUNEL, and Ki67 staining.

RESULTS

Fibulin-3 is downregulated in peripheral tumors and is thought to inhibit tumor growth. However, we found fibulin-3 was highly upregulated in gliomas, although the protein was undetectable in normal brain or cultured astrocytes. Gain-of-function experiments revealed that fibulin-3 did not affect cell morphology or proliferation but enhanced cell adhesion and promoted cell motility and invasion. These effects were reversed by fibulin-3 knockdown, which in addition increased the proportion of apoptotic cells and reduced cell growth in vitro. Orthotopic implantation of fibulin-3– overexpressing glioma cells resulted in diffuse tumors that were quantitatively more invasive and had reduced proportion of TUNEL-positive cells compared to controls. Tumors and cultured cells overexpressing fibulin-3 also showed elevated expression of matrix metalloproteases (MMP-2/9 and ADAMTS-5).

CONCLUSION

Taken together, our results suggest that fibulin-3 has a unique expression and protumoral role in gliomas and could be a potential target against tumor progression. Strategies against this glioma-specific matrix component could disrupt invasive mechanisms and restrict dissemination of these tumors.

Neuro-Oncol. 2009 Oct;11(5):568.

20. The Death Domain Kinase RIP1 Links Inflammatory and Oncogenic Signaling Pathways in Glioblastoma

Seongmi Park, Kimmo Hatanpaa, Yang Xie, Deepti Ramnarain, Bruce E Mickey, Amyn Habib

INTRODUCTION

Inflammation appears to play a causal role in certain human cancers, but its role in glioma is unknown. The mechanisms used by inflammatory networks to influence cancers are under intense investigation. In addition to their role in generating the inflammatory response, individual components of inflammatory signaling pathways may directly engage in a cross-talk with oncogenic and tumor suppressor signaling pathways. Recent work has shown that the death domain kinase receptor interacting protein (RIP, RIP1), known for its role in NF-κB activation, is a key node in this cross-talk and impacts several signaling pathways of central importance in gliomagenesis and resistance to treatment.

METHODS

We tested the effect of increasing or silencing RIP1 levels in glioblastoma (GBM) cell lines. RIP1 levels in gliomas were determined by Western blot and immunohistochemistry.

RESULTS

RIP1 negatively regulates p53. Loss of RIP1 from cells results in augmented induction of p53 in response to DNA damage, while increased RIP1 levels lead to the inhibition of DNA-damage-induced p53 induction by enhancing levels of cellular mdm2. The key signal generated by RIP1 to upregulate mdm2 and inhibit p53 is the activation of NF-κB. Secondly, RIP1 activates the PI3K-Akt pathway via a dual mechanism by removing the two major brakes on PI3K-Akt activity. RIP1 downregulates PTEN and also inhibits a negative feedback loop that extends from mTOR via S6K-IRS1 to negatively regulate PI3K-Akt activity. RIP1 is overexpressed in about 30% of GBMs but not in grade II–III gliomas and confers a worse prognosis in this disease. A high RIP1 level is associated with decreased overall and progression-free survival (n = 70). In addition, progression from grade II–III glioma to secondary GBM is associated with an increase in RIP1 levels.

CONCLUSION

RIP1 may play an important role in gliomagenesis and resistance to treatment.

Neuro-Oncol. 2009 Oct;11(5):569.

21. Insulin-Like Growth Factor 2 Binding Protein 3 (IGF2BP3/IMP3)—A Novel RNA Binding Protein with a Role in Glioma Development and Progression

Suvasini Ramaswamy, Kumar Somasundaram, Vani Santosh, Balaram Thota, A Arivazhagan

INTRODUCTION

Glioblastoma (GBM, World Health Organization grade IV astrocytoma) is one of the most aggressive forms of infiltrating astrocytic neoplasm and is associated with a median survival duration of less than a year, making it one of the most daunting therapeutic challenges. This underscores the need for the development of novel treatment protocols for astrocytoma, particularly GBM, which necessitates a greater understanding of the principal mechanisms of astrocytoma development and progression. The insulin-like growth factor (IGF) pathway is known to be hyperactivated in a variety of cancers by several means, making this system an attractive target for therapeutic intervention.

METHODS

We carried out real-time transcript profiling of members of the IGF pathway across different astrocytoma grades. Differentially regulated genes were subsequently validated on an independent set of 110 tumor samples by RT-qPCR and immunohistochemistry. To understand the physiological function of the candidate gene, knock-down- and overexpression-based approaches were adopted.

RESULTS

We found IGF2BP3 (IGF2 mRNA binding protein 3/IMP3) to be upregulated in GBM as compared to normal brain and grade II/III astrocytoma samples (p < 0.0001). IGF2BP3 is an RNA-binding protein that activates the translation of IGF2 mRNA. Concordantly, increased amounts of IGF2 protein were found in grade IV astrocytomas without a corresponding increase in transcript levels. Signaling downstream of the IGFIR was also found to be hyperactivated in the GBM samples, as validated by immunohistochemistry. Overexpression and knock-down studies in an ex vivo system established a proprolifera-tive role for IGF2BP3, aiding in migration, invasion, chemoresistance, and anchorage-independent growth. We also explored a diagnostic and prognostic utility for IGF2BP3.

CONCLUSION

We identify and characterize IGF2BP3 as a GBM-specific diagnostic and potentially prognostic marker. IGF2BP3 activates the translation of IGF2 mRNA in the higher grades of astrocytoma, thereby activating the proproliferative IGF pathway, which ultimately leads to the development of more recalcitrant tumors.

Neuro-Oncol. 2009 Oct;11(5):569.

22. Lipidomics in a Systems Biological Approach to Cancer Research: Response, Targets, and Biomarkers

Mark R Emmett, Huan He, Alan G Marshall, Carol L Nilsson, Roger Kroes, Joe Moskal, Morris Groves, Charles A Conrad

INTRODUCTION

Glioblastoma multiforme (GBM), malignant primary brain tumors, are characterized by a low survival rate, high invasivity, and resistance to apoptosis, necessitating a better understanding of the disease on a molecular level. Galectin-1 (Gal-1) levels correlate with decreased invasivity and a greater tendency toward apoptosis and have been defined as a therapeutic target for GBM. Gal-1 is known to bind terminal β-galactose on both glycoproteins and glycolipids. A method for lipid profiling has been developed and implemented in the lipidomic studies of cell culture and biological fluids.

METHODS

2 × 106 cells were mixed with methanol:chloroform and incubated overnight. The supernatant was collected and partitioned with chloroform and H2O. The upper layer was collected and separated by nano-LC in a self-packed phenylhexyl column with a gradient system of H2O, methanol, and 10 mM NH4OAc. Effluent was analyzed online by negative-ion microelectrospray with a hybrid LTQ 14.5 tesla FT-ICR MS. Structural assignment was based on accurate mass and tandem mass spectrometry.

RESULTS

This novel lipidomics methodology was used to further access potential Gal-1 ligands in cultured GBM cell lines in response to gene therapy and/or chemotherapy treatments. This method permits the identification of not only the targeted gangliosides but also phospholipids, sphingomyelin, and sulfatides of various ceramide lengths and with varying degrees of modification (hydroxylation and double-bond saturation). GBM cultures induced to apoptosis by gene therapy and/or chemotherapy showed high expression of short-chain sulfatides and increased hydroxylation of the phospholipid fatty acyl tails.

CONCLUSION

Lipidomic analysis has been extended to cancer stem cells, patient blood samples, and patient cerebrospinal fluid samples. These lipidomic analyses are crucial to our interinstitutional, multidisciplinary systems biological approach to the study of GBM, which correlates phenotypic, genomic, transcriptomic, proteomic, and lipidomic data sets. The systems biological approach is essential to discovering new biomarkers and identifying novel therapeutic targets for the treatment of glioblastoma.

Neuro-Oncol. 2009 Oct;11(5):569.

23. WT-1 Expression in Glioblastoma Cells Fosters Cell Survival: Identifying Underlying Mechanisms

Archana Chidambaram, Nitya Moothathu, Paul Dent, Adley Yacoub, Helen L Fillmore, William C Broaddus

INTRODUCTION

Wilm’s tumor-1 (WT-1), a zinc-finger transcription factor, is expressed by cancer cells arising from several different tissue types. Previous studies in our laboratory have established the aberrant expression of this protein in approximately 80% of glioma cells from primary tumor specimens and glioma cell lines and have also proven its necessity for maintaining cellular proliferation in vitro and in vivo. This study was undertaken to determine the presence of WT-1 in two hitherto unexamined glioblastoma cell lines, GBM-6 and GBM-12; to ascertain the importance of WT-1 in these cells; and to study the mechanism(s) by which WT-1 generally mediates its effects in gliomagenesis.

METHODS

We used phase contrast microscopy, ATP viability assay, real-time RT-PCR, and Western blotting.

RESULTS

a) WT-1 was observed to be expressed in both the GBM-6 and GBM-12 xenograft cell lines. Furthermore, its importance in cell survival was underscored by the finding that WT-1 silencing led to a striking decrease in cell viability qualitatively (determined by phase contrast microscopy) and quantitatively (determined by ATP viability assay). b) We assessed the effects of WT-1 silencing on the expression levels of two of WT-1’s known target genes, insulin-like growth factor-1 receptor (IGF-1R) and platelet-derived growth factor-A (PDGF-A). We found an increase and decrease, respectively, of both mRNA and protein (using real-time RT-PCR and Western blotting, respectively). Our current efforts are focused on further characterizing the roles of IGF-1R and PDGF-A in mediating the effects of WT-1 in gliomas and identifying other gene targets for this complex transcription factor using the gene-array technique.

CONCLUSION

WT-1 overexpression in the majority of gliomas suggests an important role for this protein in promoting the glial tumor phenotype. Identifying the downstream factors that help this protein promote oncogenesis, which constitutes the scope of this study, will provide important insights for the development of a multimolecular targeting strategy against these aggressive tumors.

Neuro-Oncol. 2009 Oct;11(5):569.

24. Nitric Oxide (NO) Mediates Glioma Cell Motility through Matrix Metalloproteinase (MMP)-1

Nicholas A Pullen, Helen L Fillmore

INTRODUCTION

Work by multiple investigators has shown that constitutive nitric oxide synthase (cNOS) isoforms are associated with high-grade primary CNS tumors. In particular, endothelial (e)NOS is observed with tumor-associated endothelial cell foci in glioblastoma multiforme (GBM). We hypothesized that NO, especially produced from cNOS, fulfills important roles in maintaining the insidious nature of high-grade gliomas. Previous work from our laboratories as well as those of other investigators has confirmed that matrix metalloproteinase-1 (MMP-1) is expressed in human GBM but not in normal brain parenchyma. Herein we present our in vitro NO investigations with regard to MMP-1 in human GBM cell lines, considering the overlap of NO and MMP-1 production.

METHODS

We used real-time polymerase chain reaction, MMP-1 immunoblot, ATP viability assay, transient siRNA transfection, membrane motility assay, stable shRNA expression though lentiviral transduction, and intracranial implantation of human cell lines into nude mice with end point immunohistochemistry.

RESULTS

We observed significant increases of MMP-1 at the message (p < 0.001) and mature protein (U-87MG, p < 0.001; T98G, p < 0.05) levels when GBM cell lines were exposed to the NO donor sodium nitroprusside (SNP) at a concentration consistent with constitutive NO production. We recorded no remarkable changes in cell line viability with constitutive-like concentrations. However, as expected, inducible-like NO concentration decreased culture viability at least 80% (p < 0.01), yet a small population of cells remained viable throughout 5- to 6-day exposure time courses. NO donation resulted in increased cell motility, markedly in T98G (p < 0.001), while the application of the NOS inhibitor L-NAME produced an opposite effect (p < 0.001). Furthermore, transient inhibition of MMP-1 production through siRNA resulted in abrogation of the NO-induced motile response (p < 0.05). We are currently investigating the effects of altered MMP-1 expression in an orthotopic tumor model.

CONCLUSION

Production of MMP-1 is associated with enhanced glioma cell motility. This axis is mediated in part by NO.

Neuro-Oncol. 2009 Oct;11(5):570.

25. Transcriptional Profiling Reveals the Phosphatase SHP2 as an Important Marker for the Classical Subgroup of Glioblastoma

Lisa-Marie Sturla, Pascal Zinn, David Kozono, Masayuki Nitta, Kimberly Ng, Ruprecht Wiedemeyer, Yonghong Xiao, Ekkehard Matthias Kasper, Lynda Chin, Clark C Chen

INTRODUCTION

Transcriptional profiling has transformed the nature of oncologic investigation, revealing tumor classifications based on underlying molecular pathways rather than histology. The Tumor Cancer Genome Atlas (TCGA) project profiled glioblastomas by transcriptome analysis, identifying four distinct subgroups. Since phosphatases play a critical role in pathway modulation, we wished to determine whether the expression of phosphatases differs between the four subgroups.

METHODS

We examined the expression of the 191 phosphatases in the TCGA1 database using comparative marker analysis, principal component analysis, and integrated pathway analysis.

RESULTS

We found that the four subgroups differed significantly with regard to expression level of specific phosphatases by comparative marker analysis. Surprisingly, the four subgroups can be defined solely on the basis of phosphatase expression by principal component analysis. This result suggests that critical phosphatases are responsible for the modulation of specific molecular pathways within each subgroup. To further validate this hypothesis, we performed integrated pathway analysis using the phosphatases overexpressed in each subgroup. Overall, this analysis validated our hypothesis. For example, the classical subgroup was defined by receptor tyrosine kinase (RTK) signaling pathway overexpression. It is known that SHP2 (PTPN11) modulates signaling from several RTKs, and our analysis revealed SHP2 to be highly overexpressed in the classical subgroup. It also constitutes one of the 12 phosphatases that define this group. Integrated pathway analysis also revealed an over-representation of SHP2 in the pathways activated in the classical group. We confirmed biological significance by siRNA knockdown of SHP2. In glioblastoma cell lines with overactive RTKs, SHP2 knockdown significantly reduced cell viability and enhanced cellular senescence.

CONCLUSION

Our data support the notion that the subgroups defined by TCGA reflect distinct biology that is, in part, mediated by phosphatases. The data support targeting of select phosphatases in TCGA subgroups as a strategy for glioblastoma therapy.

Neuro-Oncol. 2009 Oct;11(5):570.

26. RTVP-1 Regulates Glioma Cell Spreading and Migration via Interaction with N-Wasp

Amotz Ziv-Av, Hae-Kyung Lee, Cunli Xiang, Susan Finniss, Mira Barda-Saad, Maor Hazani, Chaya Brodie

INTRODUCTION

Glioma, the most frequent primary brain tumor, is characterized by increased proliferation and invasion into the surrounding normal brain tissue. In a recent study, we reported that RTVP-1 is highly expressed in gliomas, that it is not expressed in the normal brain, and that its expression correlates with the degree of malignancy of astrocytic tumors. In addition, RTVP-1 is involved in the regulation of the growth, survival, and migration of glioma cells.

METHODS

To further delineate the molecular mechanisms underlying the effect of RTVP-1 in glioma cells, we performed a pull-down assay using His-tagged RTVP-1 followed by mass spectroscopy and found that RTVP-1 was associated with the actin polymerization regulator N-WASP. Using confocal microscopy and various spreading and migration assays, we further characterized the RTVP-1 and N-WASP interaction.

RESULTS

The association of RTVP-1 and N-WASP was further validated by coimmunoprecipitation and FRET analysis. Since N-WASP regulates cell spreading and migration, we examined the role of RTVP-1 in these processes. We found that overexpression of RTVP-1 increased and its silencing decreased cell spreading and migration of glioma cells. The effect of RTVP-1 was mediated by N-WASP, since silencing of N-WASP abolished RTVP-1 effects. Moreover, the association of RTVP-1 and N-WASP was increased during cell spreading and appeared to be localized to areas of contact between the spreading cells and the extra-cellular matrix. Another protein, hnRNPK, was found by the pull-down assay to interact with RTVP-1. This protein has recently been reported to associate with and inhibit the effect of N-WASP on cell spreading. Using coimmunoprecipitation and FRET analysis, we validated the interactions of hnRNPK with N-WASP and RTVP-1 in glioma cells. In addition, using coimmunoprecipitation studies, we found that overexpression of RTVP-1 decreased the association of N-WASP and hnRNPK, therefore suggesting that RTVP-1 may activate N-WASP by decreasing its association with hnRNPK.

CONCLUSION

In summary, we report that RTVP-1 regulates glioma cell spreading and migration and that these effects are mediated via interacting with N-WASP and interfering with the inhibitory effect of hnRNPK on the migratory effect of N-WASP.

Neuro-Oncol. 2009 Oct;11(5):570.

27. Molecular Targets and Promoter Occupancy of a Transcriptional Regulator, AEBP1, that is Differentially Expressed in Primary and Secondary Glioblastoma

Jayashree S Ladha, D Sainitin, Satyanarayan Rao

INTRODUCTION

Earlier microarray expression profiling of graded patient glioma samples from our group led to the identification of differential gene signatures associated with primary and secondary glioblastoma (GBM). One such gene, AEBP1, was found to be upregulated more than 4-fold in the majority of primary GBMs as compared with secondary GBMs, thus qualifying AEBP1 to be a primary GBM-specific marker. AEBP1 is a transcriptional repressor with a carboxypeptidase domain and is known to bind to a regulatory sequence called AE-1 element, first shown to bind a proximal promoter of the aP2 gene that codes for a-FABP.

METHODS

To elucidate the role of AEBP1 overexpression in GBM, we employed a complementary gene-silencing approach to identify genes affected by AEBP1 by microarray analysis. To define targets of this transcription factor, we used a combination of gene expression data and network analysis using the AEBP1 interactome, followed by prediction of the consensus transcription factor binding site and promoter occupancy analysis of the differentially expressed genes. A colony suppression assay using shRNA and puromycin selection was performed to elucidate its functional role.

RESULTS

Silencing identified about 689 genes that were differentially regulated by expression (1.5-fold, p = 0.05) and belong to several Gene Ontology (GO) categories, such as transcription regulation, cell growth and proliferation, differentiation, signaling, apoptosis, and ubiquitin related. A combinatorial approach enabled us to delineate primary transcriptional targets from the secondary ones, further narrowing down to 36 genes in distributed GO categories that could potentially be regulated by AEBP1. We also confined the transcription factor binding site for AEBP1 to be “XGAAATX” by using a position-weighted matrix. Chromatin immunoprecipitation verified identified targets as being bound by AEBP1, further validating the approach used in this study. Our study and evidence in literature show overexpression of AEBP1 correlated with epidermal growth factor receptor (EGFR) amplification, thus defining a subset of primary gliomas. A colony-suppression assay showed maximal suppression of growth when AEBP1 was silenced.

CONCLUSION

AEBP1 is a growth-promoting protein. Involvement of the identified genes regulated by AEBP1 forms the basis of understanding their role in a subset of primary GBMs with EGFR amplification.

Neuro-Oncol. 2009 Oct;11(5):570.

28. Inhibition of P38A/B Promotes Self-Renewal of Human Glioma Stem Cells by Enhancing Expression of EGFR and CD133

Akio Soeda, Dae-Hee Lee, Arlan H Mintz, Johnathan A Engh, Ian F Pollack, Amin B Kassam, Deric M Park

INTRODUCTION

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases activated in a variety of cancers. EGFR signaling also appears to be important for self-renewal of human glioma stem cells, in part by its promoting CD133 expression. A stress-activated protein kinase class of MAPK, p38, has a suppressive effect on tumorigenesis and promotes internalization and ubiquitin ligase-mediated degradation of EGFR. Furthermore, self-renewal capacity of hematopoietic stem cells is restricted by reactive oxygen species stimulation of p38. In this study, we investigated the role of p38 signaling on the self-renewal of human glioma stem cells with the hypothesis that inhibition may lead to enhanced self-renewal capacity by retention of EGFR.

METHODS

Three tumorigenic human glioma-derived stem cell lines were established from acutely resected surgical specimens. The p38 pathway was manipulated with anisomycin, an inducer of p38 MAPK phosphorylation, and SB 203580, a selective inhibitor of the p38-α (MAPK14) and -β (MAPK11) isoforms. The self-renewal capacity was assessed by neurosphere-forming assay.

RESULTS

Inhibition of p38 led to enhanced growth and neurosphere formation by the glioma stem cells. Immunocytochemistry and flow cytometry revealed increased expression of EGFR and CD133 upon treatment with SB 203580. Detailed analyses showed p38 inhibition in the glioma stem cells resulted in a decrease in EGFR-Y1045 phosphorylation, the docking site for ubiquitin ligase Cbl, and prolonged the presence of detectable recombinant EGF in culture. Treatment with the p38 inhibitor, SB 203580, also resulted in decreased phosphorylation of Akt (T308 and S473 sites) and Erk1/2. Activation of the p38 pathway by anisomycin resulted in poor self-renewal and cell death.

CONCLUSION

Inhibition of the p38 pathway promotes glioma stem cell self-renewal, in spite of decreased Akt and Erk1/2 activations. Activation of the p38 pathway may represent a potential therapeutic target.

Neuro-Oncol. 2009 Oct;11(5):571.

29. Knockdown of Krüppel-Like Factor 8 (Klf-8) Reduces Proliferation in Glioma Cells

Oliver Schnell, Alexander Romagna, Valerie Albrecht, Niklas Thon, Nikolaus Plesnila, Roland Goldbrunner, Jörg-Christian Tonn, Christian Schichor

INTRODUCTION

Krüppel-like factor (KLF-8) has been identified as an integrin-dependent downstream molecule stimulating cyclin D1 and subsequently G1/S-progression in mouse fibroblast NIH3T3 tumor cells. For glial tumors, the mechanisms used by integrins, especially αvβ3 integrin, to promote tumor cell proliferation are still unclear. Here we investigate the expression and functional impact of suspected downstream signaling molecules of αvβ3 integrin.

METHODS

Sections from de novo glioblastomas (WHO grade IV) and low-grade gliomas (WHO grade II) (n = 5 each) as well as glioma cell line U87 were subjected to immunohistochemical/cytochemical staining for integrin αvβ3, the phosphorylated focal adhesion kinase (pFAK), KLF-8, and cyclin D1. To determine whether these molecules were expressed in a WHO grade-dependent manner, we also performed Western blot analysis in the same entities. For analysis of the functional impact of KLF-8 on cell cycle progression in gliomas, U87 cells were transduced with shRNA for KLF-8 knockdown.

RESULTS

Integrin αvβ3, pFAK, KLF-8, and cyclin D1 could be detected in low-grade gliomas and glioblastomas but were much more prominent in glioblasto-mas, as confirmed by Western blot and immunhistochemistry. In U87 cells, αvβ3-integrin and pFAK were found to be colocalized on cell membranes, whereas cyclin D1 and KLF-8 were detected preferentially in the nucleus. Transduction of U87 cells with shRNA-expressing adenoviral vectors at a low multiplicity of infection led to a more than 80% knockdown of KLF-8 mRNA, as quantified by qRT-PCR. Moreover, preliminary data showed a reduction of the proliferation rate of KLF-8-knockdown—U87 cells of approximately 25% compared to cells transduced with a scramble shRNA.

CONCLUSION

KLF-8 is expressed grade-dependently in gliomas. If the strong proliferation-enhancing function of KLF-8 can be confirmed, inhibiting downstream integrin signaling by blocking KLF-8 may represent a novel antiproliferative treatment strategy for malignant gliomas.

Neuro-Oncol. 2009 Oct;11(5):571.

30. CD99 Modulates the Migration and Invasiveness of Human Malignant Glioma Cells

Ho Jun Seol, Christian Smith, Young Cheol Seo, James T Rutka

INTRODUCTION

Malignant glioma is the most common primary human brain tumor, and its migration and invasiveness away from the primary tumor mass is considered a leading cause of tumor recurrence and treatment failure. Recently, gene expression profiling revealed that CD99 is highly expressed in malignant glioma compared to normal brain. CD99 has been implicated in cell adhesion and migration in various cells, although the function of this gene is not understood clearly. There are two isoforms of wild-type CD99 and a splicing variant. Several investigators suggest that wild-type CD99 may be an oncosuppressor in some tumors, as opposed to the splicing variant.

METHODS

This report validates CD99 expression in glioma and investigates the role of CD99 in glioma migration and invasion using in vitro migration and invasion assays and an in vivo orthotopic xenograft model system.

RESULTS

Real-time polymerase chain reaction and Western blot revealed that only wild-type CD99 is expressed in human glioma cells and tissues. In addition, we found that glioma cells with wild-type CD99 are resistant to anoikis and are capable of anchorage-independent growth, in contrast to previous reports in other tumors. Using immunohistochemistry, we validated that CD99 demonstrates higher expression in gliomas than in nonneoplastic brain. To assess the role of CD99 in glioma migration and invasion, we inhibited CD99 expression by siRNA and demonstrated decreased glioma cell migration and invasion. In contrast, when CD99 was overexpressed in glioma cells, we observed enhancement of cell migration and invasiveness. An orthotopic brain tumor model harboring CD99-overexpressing glioma cells resulted in a significantly decreased survival rate. Moreover, higher invasiveness was observed in overexpressing mice by pathological review.

CONCLUSION

Taken together, our findings suggest that CD99 plays an important role in the migration and invasion of human gliomas. CD99 could be a target to inhibit migration and invasion, especially in CD99-expressing gliomas.

Neuro-Oncol. 2009 Oct;11(5):571.

31. Molecular Features of Olfactory Sensory Neurogenesis are Partially Recapitulated in Human Esthesioneuroblastoma Tumors

Leila Khorasani, Maria Elias, Supata Ray, Mei Deng, Tom Reh, Neal Futran, Andrei Mihkeev, Philip J Horner, Robert C Rostomily

INTRODUCTION

Uncoupling of differentiation and cell cycle withdrawal are presumed to contribute to the malignant phenotype of many cancers. In a prior study of the pediatric brain tumor medulloblastoma, ubiquitous expression of high levels of NeuroD1 suggested that failure of NeuroD1 to activate its normal terminal differentiation and cell cycle withdrawal programs might contribute to the malignant phenotype. Like medulloblastoma, esthesioneuroblastoma (ENB), a rare neuroblastic cancer arising in the nasal vault, is presumed to originate from neuronal precursor cells whose terminal differentiation is under the control of NeuroD1.

METHODS

To extend our observations in medulloblastoma to esthesioneuroblastoma, we profiled the expression of a panel of genes known to govern olfactory sensory neuron (OSN) maturation in six human ENB tumor samples and NeuroD1 expression in a total of 10 samples.

RESULTS

Ubiquitous expression of the OSN-specific marker, OMP, in all samples supported the likely origin of ENB from OSN lineage cells, while variable expression patterns of maturational, stage-specific bHLH genes indicated partial conservation of the OSN neurogenic cascade. Most notably, NeuroD1, detected at the mRNA or protein level in 9 of 10 samples, was highly coexpressed in mitotic Mib-1-positive cells, with a mean NeuroD1/Mib-1 labeling index of 77% (range, 51–90%).

CONCLUSION

These data demonstrated the partial recapitulation of molecular regulation of OSN neurogenesis in ENB and supported the possibility that functional impairment of NeuroD1 may contribute to their malignant phenotype. Taken together with our previous observations in medulloblastoma, these data warrant further investigation of the potential role that NeuroD1 function plays in the deregulation of differentiation and cell cycle withdrawal in neuroblastic malignancies.

Neuro-Oncol. 2009 Oct;11(5):571.

32. Mutant Epidermal Growth Factor Receptor (EGFR) Signaling in Glioblastoma Maintenance and the Emergence of EGFR-Independent Tumor Growth-Promoting Mechanisms

Jill Wykosky, Akitake Mukasa, Lynda Chin, Webster Cavenee, Frank Furnari

INTRODUCTION

A major setback in the use of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) for the treatment of glioblastoma (GBM) is the occurrence of resistance. Although it is known that EGFR and mutant EGFRvIII (EGFR*) confer enhanced tumorigenicity, the question remains whether EGFR*-induced signaling is required for glioma maintenance. Thus, we set out to define the importance of EGFR* for GBM maintenance, with the ultimate goal of uncovering molecular mechanisms of glioma resistance to TKIs.

METHODS

U373 cells were engineered to express doxycycline-repressible EGFR*. Following the establishment of subcutaneous tumors in nude mice, EGFR* was silenced and the effects on tumor growth were observed. Tumors were analyzed by Western blotting. Gene expression was analyzed by microarrays, and in vitro effects of TKIs were determined by soft agar colony formation assays.

RESULTS

Suppression of EGFR* attenuated GBM tumor growth. After a period of stasis, however, tumors regained the ability to grow rapidly, which is strikingly similar to the clinical scenario of resistance. These aggressive tumors, which remained silenced for EGFR*, employed distinctly different signaling pathways, and we identified genes specifically expressed at higher levels compared to the initial EGFR*-dependent tumors. One of these genes, referred to as SE*E (substitute for EGFR* expression)-1, was highly expressed in the tumors that had escaped the requirement for EGFR* expression, and knockdown of the gene suppressed GBM tumor growth. Moreover, cells derived from EGFR*-independent tumors were resistant to the in vitro growth-inhibitory effects of the TKI gefitinib, even despite reexpression of the receptor.

CONCLUSION

EGFR* is required for maintenance of glioma growth, and tumors that lose the signaling imparted by EGFR* undergo selective pressure to employ alternative pathways to regain the ability to grow aggressively. The genes and pathways identified point to potential targets for the development of agents that could be used in combination with TKIs to circumvent resistance while improving clinical response.

Neuro-Oncol. 2009 Oct;11(5):572.

33. The Role of the Guanine-Nucleotide-Exchange Factor Swap-70 in the Migration and Invasiveness of Human Malignant Glioma Cells

Ho Jun Seol, Christian Smith, Bodour Salhia, James T Rutka

INTRODUCTION

Malignant glioma is the most common primary human brain tumor. Its invasiveness and tendency to migrate away from the primary tumor mass is considered a leading cause of tumor recurrence and treatment failure. The molecular pathogenesis of malignant glioma invasion is currently under investigation. Previously, we reported gene expression array data for several Rac guanine nucleotide exchange factors in human gliomas and compared these data to nonneoplastic human brain tissue. Here, we report that the guanine nucleotide exchange factor SWAP-70 is highly expressed in malignant gliomas. SWAP-70 is a multifunctional signaling protein involved in membrane ruffling that works cooperatively with activated Rac.

METHODS

This report validates SWAP-70 expression in glioma and investigates the role of SWAP-70 in glioma migration and invasion using in vitro migration and invasion assays and an in vivo orthotopic xenograft model system.

RESULTS

Using a glioma tissue microarray, we validated that SWAP-70 demonstrates higher expression in malignant gliomas than it does in low-grade gliomas and nonneoplastic brain tissue. Through immunoflorescence and Western blot analysis, we showed that SWAP-70 redistributes within membrane ruffles in response to specific growth factors, such as epidermal growth factor (EGF). To assess the role of SWAP-70 in glioma migration and invasion, we inhibited its expression by siRNA. The results showed decreased glioma cell migration and invasion, even when stimulated with the Rac-activating growth factor EGF. In contrast, when SWAP-70 was overexpressed in glioma cells and treated with EGF, we observed membrane ruffle formation followed by enhanced cell migration and invasiveness. In addition, SWAP-70 over-expression led to increased levels of active Rac, even in low-serum conditions.

CONCLUSION

Taken together, our findings suggest that the guanine nucleotide exchange factor SWAP-70 plays an important role in the migration and invasion of human gliomas into surrounding tissue.

Neuro-Oncol. 2009 Oct;11(5):572.

34. Cadherin-Dependent Adhesion of Human Glioblastoma Cells Promotes Neurite Outgrowth and Increased Migratory Capacity

Christopher Paul Cifarelli

INTRODUCTION

The current management of primary CNS tumors involves a multimodal approach, incorporating cytoreductive techniques including surgical resection, radiotherapy, and antiproliferative chemotherapeutic agents. Despite these attempts, the majority of patients with a diagnosis of a high-grade glioma have a dismal prognosis, with the leading cause of treatment failure and tumor recurrence being attributed to local invasion of adjacent brain parenchyma. The current study examined the capacity of glioma tumor cells to undergo neurite outgrowth and local migration, specifically focusing on the role of the cadherin cell-adhesion system.

METHODS

Using recombinant cadherin ectodomain protein, U373MG human glioblastoma cells were assessed for their ability to adhere and migrate in a cadherin-dependent manner in culture. Adhesion was evaluated via growth assessment and neurite length at 72 h of growth on an immobilized cadherin substrate and compared to other matrix adhesion proteins, such as collagen and vitronectin. Migratory capacity was measured via modified Transwell assays, using recombinant cadherin ectodomain in comparison with collagen and vitronectin.

RESULTS

Cadherin-adherent cells adopted a fasciculated morphology, with a significant increase in neurite extension, measuring 104 ± 13.3 μm in length, compared to background adhesion on BSA and nonfunctional cadherin ectodomain controls measuring 55 ± 4.4 μm and 47 ± 3.84 μm, respectively. Significant increases in neurite length over controls also were seen in the vitronectin (81 ± 4.69 μm) and collagen (91 ± 7.7 μm) groups. With respect to migration, U373 cells demonstrated increased invasion in response to cadherin ectodomain exposure, whereas vitronectin and collagen were not potent initiators of migration through the Transwell barrier. Both adhesion and migration outcomes were seen in the absence of any relative changes in cell proliferation, indicating a primary role for the cadherin-based adhesion system in tumor invasion.

CONCLUSION

Cadherin-based adhesion promotes increased adhesion, neurite outgrowth, and migration in U373MG glioblastoma cells, providing a novel therapeutic target for addressing local tumor invasion.

Neuro-Oncol. 2009 Oct;11(5):572.

35. HSP27 siRNA Suppression of Glioma Survival and Sensitization to Subsequent Temozolomide Treatment Occurs via Suppression of Sparc and Pakt and is Blocked with Forced Sparc Expression

Chad R Schultz, William A Golembieski, Daniel King, Sandra A Rempel

INTRODUCTION

Secreted protein acidic and rich in cysteine (SPARC) regulates cell adhesion, migration, and survival. We have previously demonstrated that SPARC is highly expressed in human gliomas and that it promotes brain tumor invasion in vitro and in vivo. We also demonstrated that HSP27 mediates the SPARC-induced changes in migration and invasion via p38 MAPK and MAPKAPK2 activation. HSP27 inhibition was sufficient to suppress the SPARC-induced changes. SPARC also mediates cell survival via ILK and AKT activation. Because HSP27 also regulates AKT activation, we hypothesized that the HSP27 inhibition would suppress SPARC-induced survival and that this would sensitize glioma cells to subsequent temozolomide (TMZ) treatment.

METHODS

U87 cells (low in endogenous SPARC) expressing control and SPARC vectors and LN443 cells (high in endogenous SPARC) were untreated or treated with control or HSP27 siRNAs for 72 h. Cells were harvested and plated either in 6-well plates for Western blot analyses or in triplicate in 60-mm tissue culture dishes for the clonogenic assay. Cells were plated ± fibronectin (100 μg/ml) and ± TMZ (0, 20, 40, 60, 80, or 100 μM).

RESULTS

SPARC-expressing cells displayed a plating advantage (2- to 10-fold, depending on the ECM) compared to control cells. Furthermore, SPARC protected glioma cells from temozolomide treatment. HSP27 siRNA treatment removed the SPARC-induced plating and survival advantages via suppressing SPARC and pAKT expression. The inhibition of HSP27, and subsequent suppression of SPARC and pAKT, sensitized the cells to subsequent TMZ treatment. In contrast, U87 cells forced to overexpress SPARC maintained high SPARC and pAKT levels (despite the inhibition of HSP27 expression), which protected cells in the face of increasing TMZ treatment. In summary, the data indicate that HSP27 inhibition suppresses SPARC-induced survival.

CONCLUSION

We conclude that HSP27 inhibition provides a novel therapeutic strategy to suppress SPARC-induced signaling pathways governing migration and invasion, as well as survival, in gliomas.

Neuro-Oncol. 2009 Oct;11(5):572.

36. Perfusion-Weighted Imaging Identifies Mr Surrogates of Malignant Glioma Molecular Subtypes

Gurpreet S Kapoor, Elana S Tykocinski, Ryan Grant, Jaroslaw Krejza, Leif-Erik Bohman, Timothy A Gocke, Sanjeev Chawla, Casey H Halpern, Robert Gray Whitmore, Gregory G Heuer, Joanna Lopinto, Justin Plaum, Eileen Maloney-Wilensky, Kevin D Judy, Elias R Melhem, Donald M O’Rourke

INTRODUCTION

Coexpression of EGFRvIII mutation and PTEN by glioblastomas (GBMs) is associated with therapeutic response to EGFR kinase inhibitors. Similarly, chemosensitivity of oligodendrogliomas (OLs) and prolonged patient survival are predicted by 1p and 19q loss of heterozygosity (LOH). We have utilized MR perfusion-weighted imaging (MR-PWI) to predict and identify MR surrogates of EGFRvIII-expressing GBMs and 1p/19q-deleted OLs.

METHODS

Forty-four patients with OL tumors and 35 of 97 patients with primary GBMs were retrospectively reviewed for preoperative MR-PWI. OLs were stratified as group 1 (1p or 1p/19q LOH), and group 2 (19q LOH or intact alleles). Primary GBM tumors were grouped as EGFRvIII(−) (n = 72) and EGFRvIII (+) (n = 25) tumors using an EGFRvIII-specific RT-PCR assay. The relative cerebral blood volumes (rCBV) were calculated in relation to contralateral white matter. Tumor tissues were analyzed for VEGF expression using real-time PCR to correlate with changes in rCBV.

RESULTS

The group 1 grade II OLs showed significantly greater rCBV than group 2 tumors (p = 0.013). In grade III neoplasms, the differences between group 1 and group 2 were not significant. Grade III neoplasms showed a significantly higher rCBV than grade II neoplasms. Multivariate logistic regression analysis showed significant association of rCBV with 1p19q LOH and the expression of EGFR and VEGF in grade II OLs. EGFRvIII(+) GBMs showed statistically significant higher maximum rCBV when compared to EGFRvIII(−) tumors (p = 0.0017). Expression of VEGF was higher in EGFRvIII(+) GBMs compared to EGFRvIII(−) tumors. Accuracy of rCBV in detecting EGFRvIII mutation was higher (Az = 0.82) than that of VEGF (Az = 0.59). Multivariate logistic regression analysis revealed that rCBV assessment was a significant independent predictor of EGFRvIII mutation (p = 0.014).

CONCLUSION

EGFRvIII mutation in GBMs and 1p/19q deletions in OLs are associated with elevated rCBV. Collectively, our data suggest that MR-PWI may provide noninvasive surrogate markers for malignant glioma molecular subtypes.

Neuro-Oncol. 2009 Oct;11(5):573.

37. Microrna Alterations in Glioblastoma: Effects on Proliferation, Migration, Metabolism, and Self-Renewal

Jakub Godlewski, Oskar Nowicki, Shante Williams, Agnieszka Bronisz, Kaveh Asadi-Moghaddam, Michael DeLay, E Antonio Chiocca, Sean Lawler

INTRODUCTION

Many microRNA alterations in glioblastoma have been documented. The functional consequences of these alterations are only just beginning to be examined. We have carried out phenotypic studies on several microRNAs in glioblastoma and examined their effects on muliple parameters. We have also identified relevant targets that explain the phenotypic observations. These targets and pathways are giving us new insights into the fundamental biology underlying glioblastoma.

METHODS

We carried out profiling of microRNA expression in glioblastoma, surrounding normal tissue, and migrating glioma cells in the spheroid assay. Alterations were validated by polymerase chain reaction, and functional studies were carried out using transfection studies and assays for proliferation, invasion, survival, and self-renewal.

RESULTS

Many microRNA alterations were identified. These include downregulation of miR-128 in glioblastoma compared with normal brain and miR-451 in glioma migration. When overexpressed in glioma cells, these microRNAs have distinct effects. MiR-128 blocks proliferation, and miR-451 blocks migration but increases growth. We have carried out analysis of targets that potentially mediate these effects and identified Bmi1 is a direct target and major mediator of the effects of miR-128. Interestingly, Bmi1 was the first gene associated with neural stem cell self-renewal. Detailed analysis of miR-128 effects shows that it affects chromatin structure, blocks glioma stem cell self-renewal, and downregulates CD133 levels, consistent with its effects on Bmi1. We have identified several targets of miR-451 whose cumulative effects can explain the phenotypes we have observed and highlight some important pathways in glioma regulation that so far have been overlooked.

CONCLUSION

MicroRNAs affect glioma biology on many levels. This reflects their importance as regulators of gene expression and reveals novel therapeutic targets and potential strategies for treating this devastating disease.

Neuro-Oncol. 2009 Oct;11(5):573.

38. Identification of Novel Targets in GBM Using High-Throughput siRNA Kinome Array Screening

Deepti Ramnarain, Tiffany Lafortune, Jun Yao, Geoffrey Bartholomeusz, WK Alfred Yung, Dimpy Koul

INTRODUCTION

Constitutive activation of the PI3K/Akt pathway seems to be a prerequisite for a wide spectrum of cancers, including glioblastomas. Studies have demonstrated that beyond the well-characterized mutations and genetic alterations of the epidermal growth factor receptor and PI3K signaling pathways, a large number of aberrations exist, presumably targeting yet-to-be discovered glioma-relevant oncogenes and tumor suppressors. Therefore, developing a systematic and rational platform for identifying novel targets that have a synergistic effect on tumors treated with PI3K inhibitors or gliomas in which PI3K has been silenced by employing functional RNAi screening has been used in this study.

METHODS

Inhibitors of PI3 kinases are highly promising to several cancers. This has led to PI3K inhibitors entering clinical trials as potential therapy for cancer in carriers of PI3K pathway activation/mutations. To discover new determinants of sensitivity to these drugs, we performed a PI3K-inhibitor (PX866) synthetic lethal short interfering RNA (siRNA) kinome screen using ON-TARGETplus SMARTpools (Dharmacon) targeting 714 genes.

RESULTS

We identified 10 genes in the primary screen whose silencing strongly sensitized to PI3K inhibition. First, a secondary screen of positive hits from the original, triplicate library screen was done using four individual ON-TARGETplus siRNAs that made up the SMARTpool. Hits that showed effects with each individual ON-TARGETplus siRNA were evaluated further. With this strategy, the probability that any siRNA showed consistent synthetic lethality through all levels of screening because of off-target effects was significantly reduced. We identified a number of genes whose silencing strongly sensitized to PI3K inhibition, including cell division cycle 2L5 gene, riboflavin kinase, Fas activated Ser/Thr kinase and homeo domain interacting protein domain-1. The biological validation of these genes is of potential interest, and further validations are in progress to identify a novel target gene.

CONCLUSION

These results highlight the potential of synthetic lethal siRNA screens with signaling inhibitors to define new determinants of sensitivity and potential therapeutic targets.

Neuro-Oncol. 2009 Oct;11(5):573.

39. The Mechanism by which HFE Gene Variants Confer Drug Resistance in Cancer

Sang Y Lee, Becky Slagle-Webb, Elana Farace, Jonas Sheehan, James R Connor

INTRODUCTION

HFE is a protein involved in cellular iron uptake and mediating inflammatory responses; it may also be part of the innate immune system. HFE allelic variants have been associated with a variety of cancers. Therefore, we hypothesized that there are differences in sensitivity to treatment based on HFE genotype.

METHODS

We determined the association between drug resistance and HFE polymorphisms using a human neuroblastoma cell line and glioma cell lines. The neuroblastoma cells were stably transfected with the different HFE alleles, whereas the glioma cells were purchased from ATCC and genotyped. The drug resistance mechanism of the C282Y HFE-variant cells was determined by functional gene arrays, qPCR, and siRNA techniques. The clinical relevance of these observations was examined by performing a survival analysis on brain tumor patients.

RESULTS

The expression of p16INK4A (cyclin dependent kinase inhibitor 2A) gene and protein was significantly increased in the neuroblastoma C282Y cells compared to the wild-type cell lines. C282Y-expressing astrocytoma cells also expressed high levels of p16INK4A protein. Therefore, we further determined the role of p16INK4A in temozolomide-resistant and C282Y-expressing cells using p16INK4A siRNA. Decreasing the expression of p16INK4A protein following transfection with the siRNA was associated with increased sensitivity of the C282Y-expressing cells to temozolomide. At present, we are expanding other possible drug resistance mechanisms from the gene array data in tetracycline-inducible cells. The survival analysis of brain tumor patients indicated that C282Y polymorphism showed a trend (p < 0.07) toward being an independent predictor of shorter length of survival, even after age, grade, and baseline functional status were accounted for. The clinical sample size continues to be expanded.

CONCLUSION

These data suggest the sensitivity to available treatment strategies can be affected by the HFE genotype, and the mechanism of resistance appears to be via expression of levels of p16INK4A.

Neuro-Oncol. 2009 Oct;11(5):573.

40. Adenovirus-Induced Autophagy Requires Expression of Stress-Related Kinases but No Downregulation of AKT

Erin White, Hong Jiang, Candelaria Gomez-Manzano, Juan Fueyo

INTRODUCTION

Our group has reported previously that the Delta-24-RGD adenovirus, which is currently being tested in a phase I clinical trial, induces autophagy in brain tumor stem cells and glioma xenografts. Because induction of autophagy correlates with high viral production and a high antiglioma effect, understanding the mechanism underlying Delta-24-RGD-induced autophagy can provide novel avenues to increase the oncolytic potency.

METHODS

We observed that after Delta-24-RGD infection, glioma cells expressed high levels of phosphorylated eIF2α as assessed by Western blot. The eIF2α protein is the substrate of four stress-related kinases: GCN2, PKR, PERK, and HIR. Using acrydine orange staining of the acidic vesicle organelles, electron microscopy, detection of autophagosomes, and immunoblot analysis of LC3-I conversion to LC3-II, we examined the role of the four kinases in the adenovirus-induced autophagy.

RESULTS

We found that GCN2-knockout mouse embryonic fibroblasts were resistant to the induction of autophagy by Delta-24-RGD and significantly more resistant to adenovirus-induced cell death. It is important to note, that we found that during Delta-24-RGD infection the AKT pathway was activated before autophagic biomarkers became positive. Using an antibody array, we found that AKT was phosphorylated along with downstream targets mTOR and P70S6K, which progressively acquired higher levels of phosphorylation that remained high until 48 h after infection, the latest point examined.

CONCLUSION

This paradoxal increase of AKT activity in cells undergoing autophagy clearly indicates that Delta-24-RGD triggers different pathways other than inactivation of AKT to induce autophagy and immediately suggests a strong rationale for the therapeutic combination of AKT inhibitors and Delta-24-RGD for the treatment of malignant gliomas.

Neuro-Oncol. 2009 Oct;11(5):573–574.

41. ATP Citrate Lyase is Coexpressed with Markers of Hypoxia, HIF1A, and VEGFA in Brain Tumors

Marie E Beckner

INTRODUCTION

ATP citrate lyase (ACLY) breaks down cytoplasmic citric acid, the first Krebs cycle intermediate, that enters cytoplasm via transporters in mitochondrial and plasma membranes. Citric acid inhibits glycolysis via phosphofructokinase, thus providing ACLY with indirect control on glycolysis.

METHODS

The NIH’s Respository of Molecular Brain Neoplasia Data (REMBRANDT) of expression data (Affymetrix) was queried for ACLY’s relationships with hypoxia inducible factor (HIF1A) and its effector, vascular endothelial growth factor (VEGFA). Upregulated gene expressions based on median reporter values were analyzed with the chi-square test to detect potential relationships. As a second method, previously obtained real-time quantitative polymerase chain reaction (RQ-PCR) data were reanalyzed with correlation.

RESULTS

Of 202 brain tumor cases (all gliomas) included in REMBRANDT, 52 had at least 2× elevations of ACLY and none had 2× downregulated levels compared to normal. HIF1A was elevated 2× and 3× or more in 104 and 20 cases, respectively. VEGFA was elevated 2× and 10× or more in 125 and 59 cases, respectively. Coexpression of 2× increased ACLY with 2× and 3× increased HIF1A and 2× and 10× increased VEGFA occurred in 37, 11, 51, and 34 cases, respectively. Significant associations with increased ACLY were indicated with p-values of 0.000988 and 0.00162 for 2× and 3× increased HIF1A, respectively, and 4.49E-10 and 2.88E-11 for 2× and 10× increased VEGFA, respectively. Although initial correlative analysis of RQ-PCR data from 22 additional glioblastomas revealed only a weak relationship for ACLY and HIF1A, reanalysis revealed that eliminating cases with the five highest values for ACLY and one outlier in the remaining cases led to a robust correlation coefficient, r = 0.900.

CONCLUSION

Coexpression of ACLY with mediators of adaptations to hypoxia support its role in the control of glycolysis in brain tumors. Additional RQ-PCR studies are in progress.

Neuro-Oncol. 2009 Oct;11(5):574.

42. PTEN Deficiency and SHP-2 Activation are Coupled to Enhance EGFRvIII Transformation of Glioblastoma (GBM) Cells

Yi Zhan, Donald M O’Rourke

INTRODUCTION

The protein tyrosine phosphatase (PTPase) SHP-2 is required for cell transformation in different cancers, including human glioblastomas (GBMs). Phosphorylation of SHP-2 at Tyr542 regulates and increases its PTPase activity. Our previous work demonstrated that EGFRvIII expression increased SHP-2 Tyr542 in PTEN-deficient U87MG.EGFRvIII but not PTEN-intact LN229.EGFRvIII cells. Consistent with this, complete inhibition of U87MG.EGFRvIII transformation required higher concentration of the SHP-2 inhibitor NSC-87877 (200–400 μM) compared to that of LN229.EGFRvIII (50–100 μM). More interestingly, PTEN deficiency resulted in resistance to Tarceva treatment, and our results showed that tarceva increased SHP-2 Tyr542 in U87MG.EGFRvIII cells. Moreover, only EGFRvIII, but not wild-type EGFR, constitutive expression resulted in SHP-2 Tyr542 and translocation to the perinuclear region in U87MG cells. Interestingly, EGF treatment resulted in transient SHP-2 Tyr542 phosphorylation and translocation to the perinuclear region. These results suggest that EGFRvIII initiates distinct signals from wild-type EGFR to transform GBM cells.

METHODS

Western blot analysis, immunofluorescence, and soft agar Assay were used.

RESULTS

To further study how PTEN is coupled to SHP-2 to regulate EGFRvIII-induced GBM transformation, we introduced PTEN into U87MG and U87MG.EGFRvIII cells. Stable PTEN-expressing U87MG clones grew very slowly and showed a completely untransformed phenotype. On the other hand, PTEN-expressing U87MG.EGFRvIII clones grew more slowly and showed a partially untransformed phenotype. In these PTEN-expressing U87MG.EGFRvIII clones, PTEN expression also partially reversed SHP-2 Tyr542 localization from the perinuclear region to the cell membrane, which was observed in parental U87MG cells and correlated to a more untransformed phenotype. More interestingly, the SHP-2 inhibitor NSC-87877 completely inhibited U87MG.EGFRvIII expressing PTEN clones grown in soft agar at much lower concentrations (50–100 μM) compared to parental U87MG.EGFRvIII cells (200–400 μM).

CONCLUSION

These results suggest that SHP-2 is a downstream effector of PTEN and that PTEN deficiency leads to enhanced EGFRvIII transformation through SHP-2 activation. Therefore, targeting SHP-2 may provide a new and effective therapeutic approach for the treatment of glioblastoma.

Neuro-Oncol. 2009 Oct;11(5):574.

43. The Role of ILK and NfκB in the IGFBP2 Pathway

Kristen M Holmes, Sarah M Smith, Lynette Moore, David Cogdell, Ronaldo Hashimoto, Ilya Shmulevich, Kenneth Hess, Gregory N Fuller, Wei Zhang

INTRODUCTION

The identification and functional characterization of key glioma pathways remains a critical step toward the development of efficacious therapy. Insulin-like growth factor binding protein 2 (IGFBP2) is an oncogene that is associated with poor survival and advanced grade in gliomas. Our laboratory discovered that IGFBP2 drives glioma progression in the RCAS/tv-a mouse model.

METHODS

Probabilistic Boolean network modeling of transcriptome data from 25 glioma patient samples of different grades revealed a relationship between IGFBP2, ILK, and NFκB. Utilizing the glial-specific transgenic mouse model, a mutant form of IGFBP2 (RGE) was injected in combination with PDGFB.

RESULTS

The IGFBP2 (RGE) was not able to promote glioma progression, indicating that integrin binding is essential in the IGFBP2 pathway. Subsequent mouse experiments validated the IGFBP2-ILK link by demonstrating that ILK initiated progression to a similar extent as IGFBP2, and a kinase dead form of ILK was sufficient to block IGFBP2-mediated progression. Immu-nostaining of NFκB subunit p65 of IGFBP2-overexpressing SNB19 glioma cells demonstrated that p65 was localized to the nucleus; in contrast, p65 was retained in the cytoplasm in nonintegrin-binding IGFBP2 (RGE)- overexpressing SNB19 cells. Further evidence of an NFκB role in the IGFBP2 pathway comes from EMSA experiments, in which a stronger NFκB binding band was observed in IGFBP2-overexpressing SNB19 nuclear extracts.

CONCLUSION

The IGFBP2 pathway is a critical pathway in mediating glioma progression. Further studies of the IGFBP2-ILK-NFκB pathway will reveal its significance in glioma progression and potentially provide new treatment strategies.

Neuro-Oncol. 2009 Oct;11(5):574.

44. Targeted Inhibition of the Hedgehog Pathway in Established Malignant Glioma Xenografts Enhances Survival

Michael K Cooper, Anuraag Sarangi, Juan G Valadez, Sarah Zieber, Ty Abel, Reid C Thompson

INTRODUCTION

Hedgehog pathway activity has been demonstrated in malignant glioma. However, the biological role of Hedgehog signaling in gliomas is poorly understood.

METHODS

To study the role of Hedgehog signaling in glioma growth, we transplanted CD133-enriched cells from freshly resected patient specimens into the striatum of NOD/SCID mice. Direct orthotopic xenografts generated from an anaplastic oligoastrocytoma, an analplastic astrocytoma, and a primary glioblastoma multiforme (GBM) were assayed for the operational status of the Hedgehog pathway and survival following pharmacological inhibition of the pathway.

RESULTS

An operational Hedgehog pathway was assayed only in the anaplastic oligoastrocytoma and astrocytoma xenografts. Furthermore, pharmacological inhibition of the Hedgehog pathway in established orthotopic xenografts conferred a significant survival advantage. Correspondingly, a survival benefit was observed only in tumors with an operational Hedgehog pathway. Pathway inhibition was measured in transplanted human tumor cells and not in host mouse brain. We also found that Hedgehog pathway component and gene target expression segregate to CD133+ tumor-initiating cells.

CONCLUSION

These data indicate that Hedgehog signaling regulates the growth of select malignant gliomas. Treated mice eventually succumb to disease; thus, targeting the Hedgehog pathway in CD133+ cells produces significant but incomplete tumor regression. Therefore, our studies suggest that more complete tumor regression may require the inclusion of other therapeutic targets, including CD133– cells.

Neuro-Oncol. 2009 Oct;11(5):574–575.

45. Modeling the Effects of Progenitor Cell Recruitment on Glioma Growth and Progression

Susan Massey, Peter D Canoll, Kristin R Swanson

INTRODUCTION

We have recently shown that infecting glial progenitors in the subcortical white matter of adult rats with retroviruses that express platelet-derived growth factor (PDGF-IRES-GFP) induces the formation of tumors with the histological features of glioblastoma. Surprisingly, the retrovirus infected cells account for only a minority of cells within these tumors (~15%), while the majority are uninfected glial progenitors that have been driven to proliferate massively via paracrine growth factor stimulation.

METHODS

In the current study, we have created and parameterized a mathematical model of glioma growth and progression that incorporates the effects of paracrine PDGF signaling and progenitor cell recruitment. Using this model, we have generated simulations of brain tumor growth with different levels of paracrine signaling, ranging from no recruitment to levels of recruitment equal to those seen in the PDGF-retrovirus-driven rat model.

RESULTS

Simulations generated by our model exhibit a similar range of radial expansion rates as observed in measurements of human gliomas. We saw faster rates of expansion of the MRI-detectable tumors for simulations with higher levels of PDGF-driven recruitment, paired with a lower number of PDGF-IRES-GFP+ cells lying outside the MRI-detectable tumor, indicating that high levels of recruitment will lead to higher-grade tumors with a less infiltrative growth pattern.

CONCLUSION

This model demonstrates the feasibility of a recruitment mechanism acting in human glioma. Moreover, model results indicate that the effects of paracrine signals on the glial progenitors could explain the differing rates of progression observed in human GBM patients. Given that current antivascular agents may also have effects on paracrine PDGF signaling and recruitment in gliomas, these experimental and mathematical modeling approaches provide unique tools for understanding the overall effects of such agents on glioma growth dynamics.

Neuro-Oncol. 2009 Oct;11(5):575.

46. IKBA is An EGFR-Regulating Tumor Suppressor in Glioblastomas

Markus Bredel, Ajay Yadav, Jaclyn Renfrow, Angel A Alvarez, Denise Scholtens, David Lin, Xiaolin He, James P Chandler, Claudia Bredel, Heidi S Phillips, Hannes Vogel, Pierre Robe, Bret Mobley, Adrienne C Scheck, Branimir I Sikic, Kenneth D Aldape, Arnab Chakravarti, Griffith R Harsh

INTRODUCTION

Constitutive activation of either epidermal growth factor receptor (EGFR) or NF-κB has been implicated in the oncogenesis of several human cancers, including glioblastoma multiforme.

METHODS

We conducted multidimensional analysis involving gene and transcript data for the NF-κB-negative modulator NFKBIA and clinical patient profiles of 458 glioblastomas/high-grade gliomas from multiple institutions in the United States and the Cancer Genome Atlas Pilot Project. We also conducted functional analyses using LN229, U87, and U118 glioblastoma cells and human embryonic kidney 293T cells with transgene phenotypes for IκBa. IκBa promoter and coding sequence and promoter methylation analyses in a resistance model of 15 glioblastomas cell lines with in vitro and/or in vivo resistance to O6-alkylating agents were conducted.

RESULTS

We describe an interaction between EGFR and NF-κB signaling in glioblastomas, in which NFKBIA binds to EGFR and attenuates EGFR transforming signaling through receptor ubiquitination and internalization. We show that NFKBIA constrains the malignant phenotype of glioblastoma cells. We found the NFKBIA gene at chromosome 14q13.2 was deleted in 25% of glioblastomas; most of this subset lacked EGFR amplification at chromosome 7p12, which occurred in about 40% of cases (p = 0.002, chi-square test). Loss of NFKBIA associates independently with patient survival (Cox model, p = 0.015; HR, 0.43; 95% confidence interval, 0.22–0.85) in 195 glioblastoma patients. This association with survival is validated using gene expression data in three independent malignant glioma studies, comprising 49 (log-rank, p = 0.0002), 47 (p = 0.015), and 191 glioblastomas (p = 0.001), and 76 (p = 0.024) and 70 (p = 0.010) high-grade gliomas.

CONCLUSION

Loss of NFKBIA inhibition of EGFR following deletion of NFKBIA is an alternative mechanism for tumorigenic increase in EGFR signaling and thus an alternative route to malignancy for tumors with wild-type EGFR status, obviating the genetic pressure for EGFR amplification. These data suggest a tumor suppressor role of NFKBIA in glioblastomas.

Neuro-Oncol. 2009 Oct;11(5):575.

48. Bone Morphogenetic Protein and Glial Tumors

Maryam Rahman, Aamy Amundson Smith, Shanshan Wang, Dennis Steindler, Brent A Reynolds

INTRODUCTION

Glial tumors of the CNS are infiltrative by nature and complete surgical resection is rare. Similar to other cancers, such as leukemia and those of the breast and colon, tumor initiation and growth have been attributed to a select population of tumor-initiating cells (TICs). Medical treatment for these tumors has traditionally included toxic therapy that results in cellular apoptosis. However, tumor control may be achieved with differentiation therapy (bone morphogenetic protein [BMP]) that results in differentiation of TICs and decrease in tumor growth.

METHODS

Human glial tumors (glioblastoma multiforme, ependymoma, anaplastic ependymoma, anaplastic pleomorphic xanthoastrocytoma, and juvenile pilocytic astrocytoma) were obtained as fresh surgical specimens. The tissue was placed in culture as a single cell suspension. After establishing tumor cell lines, the cells were exposed to BMP and subsequently analyzed for the number of cells in culture. After cellular fixation, immunohistochemistry was performed and analyzed with flow cytometry for cell cycle and proliferative markers.

RESULTS

The glial tumors had variable responses to brief BMP exposure in terms of cell growth and expression of markers. Of note, the ependymoma cell line demonstrated an increase in number of cells after BMP exposure compared to control, indicating a proliferative response to BMP (n = 3).

CONCLUSION

BMP results in variable growth responses in glial tumors. Expression of BMP receptors may predict how a tumor will respond to BMP.

Neuro-Oncol. 2009 Oct;11(5):575.

49. Abrogation of MAPK and AKT Signaling by Vandetanib (Zactima) Synergistically Potentiates Histone Deacetylase Inhibitor-Induced Apoptosis in Human Glioma Cells

Esther P Jane, Daniel R Premkumar, Steven O Addo-Yobo, Ian F Pollack

INTRODUCTION

Vandetanib is a multitargeted tyrosine kinase inhibitor. Our initial studies demonstrated that this agent blocks vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor (PDGFR) phosphorylation and MAPK-mediated signaling in glioma cell lines in a dose-dependent manner. Despite these effects, we observed that vandetanib had little effect on apoptosis induction at clinically achievable concentrations. Because histone deacetylase inhibitors (HDACI) have been suggested to regulate signaling protein transcription and downstream interactions via modulation of protein chaperone function through hsp90, we investigated whether combining vandetanib with an HDACI could synergistically potentiate signaling pathway inhibition and apoptosis induction in a panel of malignant human glioma cell lines.

METHODS

We conducted cell proliferation and cytotoxicity assays, clonogenic growth assay, immu-noprecipitation and Western blotting analysis, adenovirus infection, and analysis of combinatorial effects.

RESULTS

Proliferation assays, apoptosis induction studies, and Western immunoblot analysis were conducted in cells treated with vandetanib and HDACIs as single agents or in combination. Vandetanib and SAHA reduced proliferation in all cell lines when used as single agents, and the combination produced marked potentiation of growth inhibition as assessed by combinatorial methods. These effects were paralleled by potentiation of AKT signaling inhibition and apoptosis induction.

CONCLUSION

Our results indicate that inhibition of histone deacetylation enhances the antiproliferative effect of vandetanib in malignant human glioma cell lines by enhancing the inhibition of MAPK, AKT, and other downstream effectors, which may have application in combinatorial therapeutics for these tumors.

Neuro-Oncol. 2009 Oct;11(5):575.

50. Deacetylation of GD3A Leads to Mitochondrially Mediated Apoptosis: Therapeutic Potential in Glioma

Geoffrey J Pilkington, Suzanne Birks

INTRODUCTION

GD3 is upregulated in neoplastic glial cells and plays a pivotal role in the regulation of glioma growth and invasion. While in nonneoplastic cells the buildup of GD3 induces mitochondrially mediated apoptosis, this does not occur in tumor cells owing to the acetylation of the terminal sialic acid residue to form GD3A. Influenza C virus hemaglutinin esterase (HE) deacetylates GD3A and restores proapoptotic GD3. Baculovirus, an insect-specific virus, is rapidly gaining credibility as a vector for gene therapy, and we will attempt to use it as a delivery system for the esterase gene in human glioma.

METHODS

Glioblastoma multiforme biopsy-derived early passage cells were incubated with 10 mU recombinant HE. GD3 expression studies were carried out by flow cytometry and immunocytochemistry. Apoptosis was assayed using a) annexin V, b) JC-1 mitochondrial membrane probe, c) Western blotting for cytochrome C release and caspase activation, and d) time-lapse microscopy. The modified Transwell-Boyden chamber invasion assay and time-lapse microscopy were used to determine the invasive potential of cells incubated with or without esterase. Transduction efficiency of baculovirus was determined and HE cDNA was used to produce pBacPAK-CHE-CMV and recombinant baculovirus (AcCHE-CMV).

RESULTS

Exogenous addition of esterase resulted in decreased expression of GD3A with an increase in apoptotic and dead cells. Baculovirus transduced brain tumor cells, and the expression of DsRed under the CMV promoter indicated that the same promoter can drive the HE gene.

CONCLUSION

GD3/GD3A may provide a target for modulating mitochondrially mediated apoptosis in central nervous system neoplasms, and the baculovirus may facilitate efficient, nonpathogenic delivery of acteylesterase cDNA in future therapeutic strategies.

Neuro-Oncol. 2009 Oct;11(5):575–576.

51. Ciliopathy and Medulloblastoma: Evidence from Human Cases and a Mouse Model of Ciliary Loss

David Gate, Moise Danielpour, Jennifer Lang, K Pattie Figueroa, Serguei Bannykh, Joshua Breunig, Terrence Town

INTRODUCTION

Cilia are evolutionarily conserved organelles present on virtually all mammalian cells. A growing number of clinical disorders, collectively referred to as ciliopathies, are linked to dysfunctional cilia. Mutations in the patched gene, a key mediator of Sonic hedgehog (Shh) signaling, which localizes to the primary (nonmotile) cilium, account for up to 14% of medulloblastomas. Work characterizing additional Shh pathway-associated genes including smoothened (Smo), suppressor of fused, and Gli1 suggests that mutations in this gene family will likely account for approximately 25% of medulloblastomas.

METHODS

We utilized epifluorescence, confocal microscopy, and Western blot to evaluate abundance of primary cilia, Shh pathway activation, and cellular hyper-plasia in human medulloblastoma tissue, mouse medulloblastoma models, and cilia-deficient stumpy mutant mice.

RESULTS

We found a remarkable difference between pediatric and adult forms of the disease. In pediatric patients (n = 6), primary cilia were nearly absent, while in adult cases (n = 3), we noted that 12%–22% of tumor cells were ciliated (average length = 1.4 μm) versus 32% of normal cerebellar cells in controls (average length = 1.9 μm). Further, we observed primary cilia on CD133+ cancer stem-like cells in adult medulloblastomas and on CD15+ stem-like cells in medulloblastomas in mutant SmoA1 mice. Additionally, activated Smo protein was overrepresented in medulloblastoma tissue samples. To evaluate the putative role of cilia in cerebellar development and medulloblastoma formation, we characterized a novel mouse deficient in a gene termed stumpy. These animals lack cilia in all brain regions and have marked developmental defects in the cerebellum, including heterotopia and “islands” of hyperproliferation reminiscent of tumor-like growth in cerebellar germinal areas. Stumpy deficient animals also have abnormal Shh pathway signaling.

CONCLUSION

These results suggest that dysregulation of primary cilia and associated Shh signaling play a role in medulloblasoma formation. Adult and pediatric medulloblastomas may have different etiologies with respect to primary cilia and Shh signaling.

Neuro-Oncol. 2009 Oct;11(5):576.

52. P16INK4A Expression is Related to the Resistance to HSP90-Targeted Therapy

Siying Liu, Sang Y Lee, Jonas Sheehan, James R Connor

INTRODUCTION

p16INK4A encodes cyclin-dependent kinase inhibitor 2A (CDKN2A), which causes cell cycle arrest and is involved in the chemoresistance mechanism to alkylating agents. We determined whether p16INK4A is associated with resistance to the emerging tumor therapy that targets heat shock protein 90 (HSP90), a molecular chaperone that ensures proper folding, localization, and activity of various oncogenic client proteins.

METHODS

The sensitivity of HSP90 inhibitors (geldanamycin [GA] and its derivatives 17-allylaminogeldanamycin [17-AAG] and 17-dimethylaminoethylamino-17-demethoxygeldanamycin [17-DMAG]) was determined using SRB cytotoxicity assay in human glioma cell lines U251, T98G, and CCF-STTG1. Western blot was used to assess protein expression with or without 17-DMAG treatment. In CCF-STTG1 cells, p16INK4A gene knock-down was performed using commercial siRNA for 4 days before 17-DMAG treatment to assess 17-DMAG sensitivity. The cervical cancer cell line MS751 was also used to extend our findings beyond gliomas.

RESULTS

CCF-STTG1 cells were relatively resistant to the HSP90 inhibitors (IC50 > 1 μM) compared with other glioma cells (IC50 in the order of 1 to 102 nM range). The expression level of HSP70 and HSP90 was induced by the 17-DMAG treatment; however, a difference was not observed between the glioma cells. Interestingly, p16INK4A was only expressed in CCF-STTG1 cells, and the expression level did not change with 17-DMAG treatment. p16INK4A siRNA enhanced the cytotoxicity of 17-DMAG in CCF-STTG1 cells. Similar to CCF-STTG1 cells, the cervical cancer cell line MS751, which highly expresses p16INK4A, is also resistant to HSP90 inhibitors (IC50 > 1 μM) and is sensitized by administration of siRNA for p16INK4A.

CONCLUSION

These data suggest that p16INK4A expression is related to the resistance to HSP90 inhibitors in some cancer cells.

Neuro-Oncol. 2009 Oct;11(5):576.

53. The Role of a Novel Neurofibromin 1 (NF1) Interaction with Leucine-Rich Pentatricopeptide Repeat Cassette (LRPPRC) in Subcellular Transport of RNA Granules

Vedant Arun, Abhijit Guha

INTRODUCTION

Neurofibromatosis 1 (NF1) is the most common tumor-predisposing syndrome in humans, with an incidence of 1 in 3,500 live births. Neurofibromin, encoded by Nf1, acts as a p21-Ras-GAP to directly interact with and inactivate p21-Ras through its GAP-related domain (GRD). Evidence suggests that non-Ras-GAP functions mediated through interactions with domains outside of the GRD are of importance.

METHODS

The large size and thereby lack of full-length Nf1 cDNA led us to use GST-fusion proteins of neurofibromin domains, coupled with gel-based mass-specrometric (MS) protemic analysis. We identified leucine-rich pentatricopeptide repeat cassette (LRPPRC) and dynein as previously unreported NF1-TBD-interacting proteins. LRPPRC was of interest as it is mutated in the Leigh syndrome French Canadian (LSFC) variant, a cytochrome-oxidase-deficiency syndrome characterized by neurodegen-eration and developmental, mental, and growth retardation that thereby has some similarities with nontumor manifestations of NF1.

RESULTS

We have established that LRPPRC and neurofibromin interact in vitro by immunoprecipitation and reverse bait immunoprecipitation. A novel proximity ligation assay was used to further confirm the interaction in situ in cell lines and clinical tissue cryosections. Toward elucidating the functional relevance of the interaction, we established that the two proteins complex with microtubules and the kinesin motor protein, which indicates a role in microtubule-dependant intracellular transport.

CONCLUSION

Since LRPPRC binds with mRNA and is involved in its nucleocytosolic shuttling, we hypothesize that the cargo transported by this complex could be RNA granules. MS analysis of NF1 immunoprecipitates revealed a number of proteins that are a part of the RNA granule complex, including RNA binding proteins and proteins involved in mRNA translation. Current studies are focused on continued investigation of the above hypothesis and understanding the biological relevance of this novel interaction toward some of the overlap of neurological and developmental manifestations between the LSFC and NF1.

Neuro-Oncol. 2009 Oct;11(5):576.

54. In Situ Evaluation of Mutant EGFR and ERBB Family Member Dimerization and Activation in Human Gbms

Aaron Gajadhar, Abhijit Guha

INTRODUCTION

Aberrations in epidermal growth factor receptor (EGFR/ErbB1) signaling are the most common oncogenic stimuli in human glioblastomas (GBMs). Interactions between mutant and wild-type ErbB family members in GBMs are of biological and potential therapeutic importance. Utilizing our novel in situ platform for EGFR dimerization analysis, we seek to directly ascertain the dimerization capacity and activation status among EGFR mutants and ErbB members in GBM operative specimens and whether this feature has important prognostic or diagnostic value in GBMs harboring them.

METHODS

We have recently described our work developing and optimizing a novel in situ proximity ligation assay (PLA) for dimerization analysis of EGFR mutants and ErbB members prevalent in GBMs. The ability to directly analyze cell lines in situ and tissue sections from clinical specimens allows detection and resolution of individual EGFR dimers in intact cells and tissue samples.

RESULTS

Preliminary in vitro results demonstrate the homodimeric and heterodimeric abilities of EGFRvIII and EGFRc958 mutants, the two most common mutants associated with EGFR amplification in GBMs. We propose to use this innovative PLA platform to directly assay GBM operative specimens in a tissue microarray format (TMA). We will initially assess the EGFR wild-type and EGFRvIII heterodimer by PLA-screening the GBM TMA from Dr. Paul Mischel (UCLA) composed of 90 GBMs (primary, n = 74; secondary, n = 16) to investigate the presence of this heterodimer. We will further measure the phosho-activation status of detected heterodimers using a complementary PLA approach. Interaction and activation status of this heterodimer will be clinically correlated with regard to molecular alterations (e.g., ErbB2, p53, PTEN, Ink4A/Arf), treatment regimen, and overall survival data.

CONCLUSION

This in situ PLA EGFR dimerization platform will provide unique insight into the clinical relevance of mutant EGFR and ErbB member dimerization and hopefully guide the development of rational targeted therapy.

Neuro-Oncol. 2009 Oct;11(5):576–577.

55. Hexokinase 2 Promotes Tumor Cell Survival Through Metabolic Remodeling in Glioblastoma Multiforme

Amparo Wolf, Cynthia Hawkins, Abhijit Guha

INTRODUCTION

Tumor cells commonly undergo metabolic remodeling, including a switch to aerobic glycolysis referred to as the Warburg effect. The underlying mechanism of this switch and its necessity for tumor growth has yet to be deciphered. Herein we show that the rate-limiting glycolytic enzyme hexokinase 2 (HK2) plays a crucial role in the Warburg effect in glioblastoma multiforme (GBM).

METHODS

Human GBM cell lines and laser-capture microdissected GBM specimens were used in vitro and in vivo.

RESULTS

Human GBMs preferentially overexpressed HK2 but not the normal brain hexokinase 1 (HK1) isoform, particularly in perinecrotic regions resistant to apoptosis, and HK2’s expression predicted poor overall survival of GBM patients. HK2 expression in vitro was found to provide a survival advantage via the intrinsic apoptotic pathway as well as an invasive advantage. Furthermore, stable loss of HK2 in GBM cells, unlike glycolytic enzymes HK1 or PKM2, resulted in a more “normalized” mitochondrial metabolism associated with reduced tumorigenesis in an in vivo subcutaneous xenograft model. However, injection of GBM cells with stable loss of HK2 within the well-vascularized intracranial environment resulted in heightened perivascular invasion deep within both hemispheres, analogous to recent reports on the effect of antiangiogenic therapies in GBMs.

CONCLUSION

Targeting key enzymes employed by the tumor to modulate its energy metabolism, such as HK2 in GBMs, may favorably alter their therapeutic sensitivity with the potential caveat of increasing the tumor’s invasive nature.

Neuro-Oncol. 2009 Oct;11(5):577.

56. HUR Controls RNA Stability and Growth in Gliomas, Providing a Potential New Therapeutic Target

David S Baskin, Federico Bolognani

INTRODUCTION

Gene expression is controlled, in part, by regulation of mRNA decay, which is altered in brain tumors. The control of mRNA stability depends on sequences in the transcript and on RNA-binding proteins that bind to these sequences. HuR is an RNA-binding protein found in gliomas that increases the half-life and steady-state levels of a number of glioma RNAs. We examined whether changes in HuR levels were associated with changes in glioma proliferation.

METHODS

We examined HuR levels and cellular localization in postsurgical glioma specimens and cells in culture using immunohistochemistry and Western blot. The half-lives of HuR target mRNAs COX-2, VEGF, and c-myc were determined using actinomycin D transcriptional arrest and real-time RT-PCR in U87 and U118 glioma cell lines and in normal human astrocytes.

RESULTS

Immunohistochemistry on tissue microarrays revealed that HuR was overexpressed in human gliomas and was abnormally translocated from the nucleus to the cytosol at high levels. Moreover, HuR levels and cytosolic localization correlated with tumor grade. Furthermore, the tumor-associated HuR target mRNAs COX-2, VEGF, and c-myc decayed more slowly in glioma cell lines than in astrocytes. Finally, cells transfected with HuR grew faster than control cells, whereas cells transfected with a HuR RNA decoy grew more slowly.

CONCLUSION

Our data demonstrate that abnormalities in posttranscriptional control of mRNA stability, mediated by HuR, are necessary to sustain the rapid growth of glioblastoma multiforme cells. This important regulatory protein is a potential new therapeutic target, and RNA-binding proteins in general may prove to be important for the treatment of tumors.

Neuro-Oncol. 2009 Oct;11(5):577.

57. Alpha-Actinin 1 and Alpha-Actinin 4: Contrasting and Crucial Roles in the Survival, Motility, and RHOA Signaling of Astrocytoma Cells

Quincy Quick, Omar Skalli

INTRODUCTION

α-Actinin is a prominent actin filament-associated protein for which different isoforms exist. Here, we have examined the contribution of the two highly homologous nonmuscle α-actinin isoforms 1 and 4 to the properties of astrocytoma cells.

METHODS

α-Actinin 1 and 4 expression levels were examined in normal and neoplastic brain tissues. Functional studies of α-actinin 1 and 4 were performed in U-373 MG astrocytoma cells by using shRNAs.

RESULTS

α-Actinin 1 levels were higher in astrocytomas than in normal brains, whereas α-actinin 4 levels were elevated in high-grade astrocytomas compared to normal brains and low-grade astrocytomas, suggesting that α-actinin 1 and 4 play roles in astrocytoma development and progression, respectively. RNAi also demonstrated important and contrasted contributions of α-actinin 1 and 4 to the malignant behavior of U-373 MG astrocytoma cells. Decreased clonogenic survival and increased apoptosis were induced by antagonizing α-actinin 1 but to a lesser extent by antagonizing α-actinin 4. On the other hand, downregulation of α-actinin 4 but not α-actinin 1 reduced cell motility, cortical actin, and RhoA levels. These divergent effects on motility may be because, compared to α-actinin 1, a larger fraction of α-actinin 4 is associated with actin filaments.

CONCLUSION

Collectively, these findings suggest that α-actinin 1 and 4 are differentially regulated during the development and progression of astrocytomas because each of these isoforms uniquely contributes to distinct malignant properties of astrocytoma cells.

Neuro-Oncol. 2009 Oct;11(5):577.

58. Cytoskeletal Protein Synemin Affects the Proliferation and Sensitivity to Cisplatin of Astrocytoma Cells Through the AKT Pathways

Aaron Pitre, Omar Skalli

INTRODUCTION

We have shown previously that the intermediate filament protein synemin is expressed in astrocytomas but not in normal astrocytes and that it is involved in determining the motility and proliferation of astrocytoma cells.

METHODS

The mechanisms by which synemin influences the proliferation of astrocytoma cells was examined in A172 astrocytoma by using an shRNA-based RNAi approach.

RESULTS

We demonstrate now that the inhibition of proliferation after synemin down-regulation is due to G1 arrest rather than to increased apoptosis. Synemin-silenced cells, however, are more sensitive to cisplatin-induced apoptosis. This suggests that synemin participates in signal transduction pathways important for the proliferation and survival of astrocytoma cells. Support for this hypothesis is provided by the findings that synemin downregulation is accompanied by changes in the expression levels or phosphorylation states of several key players in cell cycle and survival pathways. Synemin-silenced astrocytoma cells showed an increase in the protein levels of the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1, along with hypophosphorylation of the tumor suppressor Rb. Immunofluorescence studies also showed that synemin downregulation induced the translocation of p21Cip1 from the cytoplasm to the nucleus, an event associated with cell cycle arrest. Finally, in synemin-silenced cells, there was a sharp decrease in Akt phosphorylation (that is, a decrease in Akt activation) at both the Ser473 and Thr308 sites.

CONCLUSION

Since Akt is an upstream regulator of the CDK inhibitors (CDKIs), these results suggest that synemin influences the proliferation of astrocytoma cells through the Akt pathways. It remains to be determined whether it is also through this pathway that synemin downregulation sensitizes synemin-silenced cells to cisplatin.

Neuro-Oncol. 2009 Oct;11(5):577.

59. C6 Glioma Conditioned Media Regulates Matrix Molecules MMP-9 and SPARC Expression in Normoxic and Hypoxic Brain Endothelial Cells in Culture

Sandra J Hill-Felberg, Virginia Mazack, Steven A Toms

INTRODUCTION

Tumor cell survival and growth is dependent on the stimulation of proliferation, blood vessel formation, and metabolic adaption by brain endothelial cells to the hypoxic state created during tumorigenesis. In this study, we investigated the effects of C6 astroglioma conditioned media on hypoxic-induced changes in brain endothelial cells’ expression of extracellular matrix molecules in culture.

METHODS

Rat brain endothelial RBE4 cells were grown in culture for 3 days (n = 8). On day 3, the media from RBE4 cells (n = 4) was removed and replaced with 1-day C6 condition RBE4 media. RBE4 cells (n = 4; two control, two C6 media) were then placed in anaerobic bags for 18 h to produce hypoxic conditions. Total cell lysate from both hypoxic and normoxic conditions were harvested and processed for SDS-PAGE for matrix metalloproteinase-9 (MMP-9) and secreted protein acidic and rich in cysteine (SPARC).

RESULTS

We found no change in MMP-9 expression on normoxic endothelial cells grown in 1-day C6 condition media, whereas we found a 1.6-fold increase in MMP-9 with hypoxia alone and a 1.4-fold (~29%) additional increase with C6 condition media added. C6 condition media, on the other hand, had a 2.6-fold decrease in the expression of SPARC in normoxic cells. Hypoxia alone caused a dramatic 5-fold (~80%) decrease in SPARC expression, while C6 condition media had no additional reduction in SPARC expression in hypoxic RBE4 cells.

CONCLUSION

We demonstrated that C6 condition media appears to drive the protein level in the same direction as hypoxia. This occurs by further increasing the expression with MMP-9 or by decreasing the expression with SPARC. Experiments with hypoxic glioma cells are under way to determine whether it enhances or alters this effect.

Neuro-Oncol. 2009 Oct;11(5):577–578.

60. EGF Receptor Family Activation and Lapatinib in Vestibular Schwannoma

Sylwia Ammoun, Clare Cunliffe, Jeffrey C Allen, David Zagzag, Oliver Hanemann, Matthias Karajannis

INTRODUCTION

Vestibular schwannomas (VS) arising sporadically or in patients with neurofibromatosis-2 (NF2) lack expression of merlin, a putative tumor suppressor gene. Surgery is the mainstay of therapy, and no effective medical treatment is known. Recent evidence suggests that merlin deficiency may result in abnormal activation of receptor tyrosine kinases and downstream signaling, promoting tumor growth. Although small-molecule RTK inhibitors are widely available for clinical use, no such therapy has been validated in patients with VS.

METHODS

To screen for RTK activation, surgical VS specimens from four NF2 patients and four non-NF2 patients were analyzed by phospho-RTK arrays. Downstream signaling pathway activation was analyzed by phospho-MAPK arrays. Activated RTKs and downstream kinases were validated immunohistochemically in five NF2 and four non-NF2 VS samples. Based on the findings, the small-molecule EGFR/ErbB2 inhibitor lapatinib was selected for evaluation in a preclinical VS model.

RESULTS

Phospho-RTK arrays and immunohistochemistry showed EGFR family receptors and Erk1/2 downstream signaling in all samples analyzed, as well as expression of the inhibitor of apoptosis (IAP) family protein survivin. In the preclinical VS model, EGFR/ErbB2-targeted therapy with lapatinib inhibited ErbB2 phosphorylation and downstream Erk1/2 activation, resulting in decreased proliferation. In addition, survivin was downregulated by lapatinib in vitro.

CONCLUSION

EGFR family receptor activation is a consistent feature of both sporadic and NF2-related VS. Molecular targeted therapy with lapatinib, a small-molecule EGFR/ErB2 inhibitor, has antiproliferative activity in a preclinical VS model. Based on these findings, a clinical trial with lapatinib for the treatment of VS is currently under way. Survivin expression has been linked to ErbB2 pathway activation in other tumors and may represent a novel target for the treatment of VS.

Neuro-Oncol. 2009 Oct;11(5):578.

61. CD44 Loss of Function in Spontaneous Murine Glioma Implicates CD44 in Tumor Maintenance

Stacy A Decker, Jim B McCarthy, Eva A Turley, Colleen Forster, Karen S SantaCruz, John R Ohlfest

INTRODUCTION

As gliomas develop and progress, tumor cells interact with the immune system and the extracellular matrix (ECM). Gliomas evolve in a hyaluronan (HA)-rich ECM and overexpress the primary HA receptor, CD44. Glioma models that utilize tumor cell implantation into adult mouse brains damage tissue and cause the release of chemokines and mitogens. In contrast, spontaneous models of glioma have the advantage of recapitulating the development of human tumors. For this reason, a spontaneous model is essential for understanding interactions between the ECM and gliomas. We recently developed a novel oncogene-induced glioma model that can be immediately applied to any transgenic mouse. In this model, tumor burden is quantified by bioluminescent imaging.

METHODS

Spontaneous malignant glioma was induced by plasmid DNA transfection into the lateral cerebral ventricle of neonatal CD44+/+ and CD44−/− mice with SV40LgT and NRASG12V. Tumor growth was monitored weekly by bioimaging and correlated with animal survival. Immunohistochemistry was employed to detect CD44 expression in tumor and normal brain.

RESULTS

Wild-type (CD44+/+) animals developed rapidly progressing tumor, as assessed by bioimaging (median survival of 37 days). In contrast, CD44−/− animals exhibited two distinct tumor phenotypes: the majority of mice developed tumors that progressed slower than in CD44+/+ mice (median survival of 59 days), whereas a fraction developed tumors that spontaneously regressed, as measured by bioimaging. Immunohistochemistry revealed that CD44 was markedly overexpressed in wild-type tumors, and individual CD44+ tumor cells were documented infiltrating the normal brain far away from the main tumor mass.

CONCLUSION

These data demonstrate that CD44 accelerates glioma development and growth kinetics. Moreover, the spontaneous regression of CD44−/− tumors suggests CD44 may play a crucial role in the self-renewal of tumor- propagating cells. Together our data suggest CD44 is an attractive therapeutic target for glioma therapy.

Neuro-Oncol. 2009 Oct;11(5):578.

62. Chemokines Control Cell Migration of Glioblastoma Multiforme Xenografts

Brian D Milligan, John R Henley

INTRODUCTION

Glioblastoma multiforme (GBM) is a CNS neoplasm that kills primarily through invasion of normal brain. Several chemokine receptors are reportedly expressed in GBM, but functional data demonstrating a role in invasion are limited to cell lines. Unlike cell lines, heterotopic GBM xenografts maintain the characteristic mutations in pathways that interact with chemokine signaling. This study tested the hypothesis that invasive GBM xenograft cells express functional chemokine receptors that control cell migration.

METHODS

Chemokine receptor expression was defined by RT-PCR using gene-specific primers for CXCR1, CXCR2, CXCR4, CCR3, and CCR5. For functional experiments, we used two well-characterized xenograft tumors: GBM6 (EGFR viii, PTEN wild-type (wt), PDGFR amplified) and GBM8 (EGFR wt, PTEN−/−, PDGFR wt). Tumors were harvested and enzymatically dissociated before culturing under stem-cell conditions. In the functional gap migration assay, we created gaps in a cell monolayer and added chemokine or inhibitor. Process protrusion and cell migration into the gap were quantified by analysis of actin and nuclear immunostaining.

RESULTS

Five GBM xenograft tumor lines and five fresh surgical GBM specimens expressed a varied array of chemokine receptors. CXCL12 (activating CXCR4) increased process protrusion in GBM6 (140%) and GBM8 (125%) compared to controls. In GBM8, CCL5 (activating CCR3/CCR5) was sufficient to increase process protrusion by 40%, and CXCL8 (activating CXCR1/CXCR2) decreased nuclear migration by 25%. Pharmacologic inhibition of protein kinase B/Akt but not phosphoinositide-3-kinase reduced process protrusion and nuclear migration by 30%–60% from baseline in both tumors.

CONCLUSION

GBM xenograft tumor cells express multiple, functional chemokine receptors that regulate process protrusion. The perivascular CNS localization of CXCL12 may account for the perivascular satellitosis and perivas-cular invasion observed in GBM. Antiinvasive therapy for GBM may target CXCR4 and CCR3/5 expression, receptor activation, and downstream signaling, as well as the CNS expression or distribution of cognate ligands.

Neuro-Oncol. 2009 Oct;11(5):578.

63. The Role of Cytoplasmic ECT2 in Glioma Invasion and Migration

Adrienne Weeks, Stacey Ivanchuk, Ho Jun Seol, Christian Smith, Soma Mondal, James T Rutka

INTRODUCTION

Glioblastoma multiforme is the most malignant and aggressive brain tumor. Treatment failures are, in part, due to the tumor’s highly invasive nature and tendency for local recurrence. Our lab has previously shown that the cytokinetic protein ECT2 is a negative prognostic indicator and that loss of ECT2 function results in a less invasive cell phenotype.

METHODS

We employed shRNA, immunofluorescence, and orthotopic mouse xenograft techniques to elucidate ECT2’s role in invasion and migration.

RESULTS

We now show that ECT2 exerts its effect on migration in association with Rac and the actin cytoskeleton. ECT2 expression is increased at the leading edge of glioma cells. Loss of ECT2 using shRNA results in depletion of activated Rac. Previous literature has shown ECT2 to be predominantly nuclear during interphase and that loss of the nuclear localization signal results in a cytoplasmic form of ECT2 that can transform cells into an oncogenic phenotype. We have shown that glioma cells have an increased cytoplasmic ECT2 compared to normal human astrocytes.

CONCLUSION

Further research into this cytoplasmic component of ECT2 is currently under way to better understand ECT’s role in glioma biology.

Neuro-Oncol. 2009 Oct;11(5):578.

64. Nanoparticle Transport into the Central Nervous System

Arnold B Etame, Stephen Perrault, Christian Smith, Warren C W Chan, James T Rutka

INTRODUCTION

There has been considerable interest in nanoparticles and their potential diagnostic and therapeutic applications within the CNS. Traditionally, the blood-brain barrier (BBB) has been the greatest impediment to the delivery of therapeutic agents into the CNS. Nano-particles, however, represent a potential avenue for drug delivery into the brain.

METHODS

The mechanisms of nanoparticle transport across the BBB as well as factors that influence this process have not been fully elucidated. We therefore sought to examine nanoparticle transport across an in vitro BBB. Rat astrocytes as well as rat brain endothelial cells were cocultured in a characterized system. Gold nanoparticles of varying sizes, surface areas, shapes, and functionalized groups were tested.

RESULTS

Size and functionalized groups appeared to be the key determinants in the transport of nanoparticles across the BBB. Furthermore, internalization of nanoparticles by the cellular components of the BBB was also noted.

CONCLUSION

Ultimately, such studies will identify the ideal nanoparticle characteristics for delivery of specific agents into the central nervous sytem.

Neuro-Oncol. 2009 Oct;11(5):578–579.

65. Epigenetic Regulation of Glial Fibrillary Acidic Protein by Methylation in Malignant Gliomas

Andres Restrepo, Sameer Agnihotri, Christian Smith, Paul N Kongkham, James T Rutka

INTRODUCTION

Glial fibrillary acidic protein (GFAP) is an intermediate filament expressed predominantly in glial cells that helps to stabilize and maintain the glial cytoskeleton. The expression of GFAP is progressively lost in increasing grades of glioma, suggesting the gene is important for maintaining normal glial cell morphology or in regulating astrocytoma cell growth. Similarly, many of the cell lines derived from astrocytomas are also GFAP negative. Analysis of the human GFAP gene revealed no genomic aberrations, suggesting that epigenetic mechanisms, such as promoter methylation, result in the silencing of GFAP expression.

METHODS

In this study, we utilized known GFAP-negative glioma cell lines treated with 5-aza-2′-deoxycytidine to examine GFAP promoter hypermethylation.

RESULTS

Bisulfite sequencing was performed on primary glioblastoma samples and cell lines with a strong correlation between methylation status and GFAP expression. A gene reporter assay with the 2 kb GFAP promoter cloned upstream of a luciferase gene showed that methylation of the GFAP promoter downregulated the expression of the luciferase gene.

CONCLUSION

Taken together, these results indicate that the GFAP gene is regulated by methylation in malignant glioma, and further investigation into the functional consequences of GFAP inactivation will provide a greater understanding of glioma pathogenesis.

Neuro-Oncol. 2009 Oct;11(5):579.

66. Studies on the Release and Function of Monomeric Ephrina1 in Glioblastoma Multiforme

Amanda Beauchamp, Denise M Gibo, Jill Wykosky, Waldemar Debinski

INTRODUCTION

We have recently discovered that a monomeric functional form of ephrinA1 is released by proteolytic cleavage from glioblastoma multiforme (GBM) cells. Activation of the EphA2 receptor by ephrinA1 leads to a decrease in the oncogenic properties of GBM cells. However, the exact mechanism of the release of ephrinA1 from the cell membrane is unknown, as are the exact downstream effects of EphA2 receptor activation by soluble ephrinA1.

METHODS

To further understand the mechanism of release of ephrinA1 from cancer cells, we used broad-spectrum inhibitors of serine proteases and matrix metalloprotein-ases (MMP) or generated ephrinA1 mutants interfering with the potential mechanism of ephrinA1 release. To determine functional effects of ephrinA1 on EphA2-expressing cells, U-251 MG GBM cells were treated with homodimeric or monomeric recombinant ephrinA1. We utilized a PCR microarray (RT2 Profiler PCR Array: Human Signal Transduction PathwayFinder, SABiosciences) and phosphorylation antibody array (Human RTK Phosphorylation Antibody Array I, RayBiotech Inc.).

RESULTS

GM-6001, an MMP inhibitor, and AEBSF, a serine protease inhibitor, decreased significantly the release of ephrinA1 from GBM cells, whereas hirudin, a specific thrombin inhibitor, was ineffective, as expected. EphrinA1 (2 h) caused the upregulation of genes involved in the TGFβ pathway (cdkn1a, cdkn1b), p53 pathway (bax, cdkn1a), and stress pathway (hspb1, hsp90aa2, tp53). EphrinA1 (10 min) also led to a decrease in phosphorylation of FAK, JAK2, and VEGFR2, and to an increase in phosphorylation of JAK1 and FGRFs.

CONCLUSION

The proteolytic cleavage releasing ephrinA1 takes place in response not only to an MMP, but also to serine protease(s), and the exact nature of the proteases involved is under further investigation. Moreover, ephrinA1 is able to induce significant genomic and protein changes downstream of EphA2 activation within the GBM cell that could be important to the overall tumor-suppressing properties of ephrinA1.

Neuro-Oncol. 2009 Oct;11(5):579.

67. Functional and Structural Characterization of the High-Affinity EPHA2 Binding Site of EphrinA1

Carla Maria Lema Tome, Enzo Palma, Jill Wykosky, Waldemar Debinski

INTRODUCTION

We have demonstrated that the EphA2 receptor, a receptor tyrosine kinase, but not its preferred ligand, ephrinA1, is over-expressed in glioblastoma multiforme (GBM). EphA2 expression is linked to malignancy grade and patient survival. Binding of ephrinA1 to EphA2 results in receptor activation, internalization, and downregulation, which mediate a reversal of malignant properties in GBM cells. Moreover, ephrinA1 in GBM cells is released into the extracellular environment as a functional, soluble, monomeric protein and thus can mediate its effects in a paracrine manner. Here, the structure-function relationship of ephrinA1 was studied, with the goal of developing an optimal soluble ligand for EphA2.

METHODS

The region selected for study is the GH loop of ephrinA1, a highly conserved region known to mediate high-affinity binding of ephrinA5 to EphA3. Alanine substitution mutants of the ephrinA1 G-H loop (H-108FQRFTPFTLGKEFKE123G-OH) were generated by site-directed mutagenesis. Sequence-verified ephrinA1-mutant plasmids were then transfected into U-251 MG cells. Mutant ephrinA1 expression in the media was verified by Western blotting. Conditioned media were assayed for ephrinA1 content using a sandwich enzyme-linked immunosorbent assay. Functional activity of each mutant was assessed by EphA2 downregulation in U-251 MG cells.

RESULTS

Alanine substitutions at F111, T112, F114, L116, K118, E119, and F120 had a deleterious effect on EphA2 downregulation when compared to that induced by wild-type ephrinA1. This supports the hypothesis of the G-H loop being the principal binding site of ephrinA1 to the EphA2 receptor. On the other hand, alanine substitutions at positions P113 and T115 enhanced the EphA2 receptor downregulation.

CONCLUSION

The G-H loop in ephrinA1 is mainly responsible for the binding to the EphA2 receptor. Our data suggest that it is possible to identify monomeric ephrinA1 variants with either superagonist or antagonist activities. These engineered mutants of ephrinA1 should prove useful in the design of novel anti-GBM treatment strategies.

Neuro-Oncol. 2009 Oct;11(5):579.

68. Glioblastoma Multiforme Xenograft Cell Polarity Correlates with Migratory Behavior

Brian D Milligan, John R Henley

INTRODUCTION

The glioblastoma multiforme (GBM) heterotopic xenograft system may be a more biologically relevant model of human disease when compared to cell lines or extended primary cell culture and, as such, is increasingly used to study glioma pathobiology and invasion. However, the morphology and motile phenotype of GBM xenograft cells remain undefined. The aim of this study was to test the hypothesis that invasive GBM xenograft cells are highly polarized, and that this polarity correlates with migratory behavior.

METHODS

GBM xenograft tumors were passaged in nude mouse flank, harvested, and subjected to enzymatic dissociation before culturing under stem-cell conditions. Cell process polarity and morphology during migration were characterized using live-cell phase-contrast imaging. For the gap migration assay, cells were immunostained for actin and nuclei to highlight process protrusion and cell migration into gaps created in a tumor-cell monolayer. Immunohistochemistry (IHC) further highlighted process polarity (phalloidin), as well as Golgi apparatus (anti-GM130) and microtubule organizing center (MTOC, anticentrin) position.

RESULTS

IHC demonstrated that GBM xenograft tumor cells are elongate and highly polarized. The leading pseudopodial edge is characterized by actin-rich lamellipodia. Numerous filopodia can be found around the entire cell perimeter. Time-lapse microscopy revealed that both migrating xenograft GBM cells and acutely dissociated primary tumor cells have a long, dominant leading process and a slim trailing process. Migrating cells can extend processes and translocate nuclei in a temporally distinct or synchronized manner. Xenograft GBM cells frequently reorient the Golgi apparatus and MTOC toward the leading process and direction of migration.

CONCLUSION

Like other migrating cell types, invasive GBM xenograft cells polarize the actin cytoskeleton and intracellular organelles relative to the direction of migration. Characterization of the morphology and intracellular polarity of migrating GBM cells is an early step toward understanding cell migration in this invasive, deadly tumor.

Neuro-Oncol. 2009 Oct;11(5):579.

69. Galectins Mediating Resistance to Radiotherapy

Tim Lautenschlaeger, Disha Patel, Kamalakannan Palanichamy, Arnab Chakravarti

INTRODUCTION

We found galectins to upregulated in glioma by microarray analysis. Here we investigate the role of galectins in mediating resistance to radiotherapy in vitro.

METHODS

Previously we found using microarrays that the galectin family (LGALS1–14) was consistently upregulated in glioma. In this study, we investigated the role of galectins in mediating resistance to radiotherapy by RNAi-mediated silencing of galectins in glioma cell lines. We performed the clonogenic survival assay, viability assays, DNA damage assays, and apoptosis assays. To verify the silencing of galectin genes, we used real-time polymerase chain reaction and Western blotting. To evaluate the activation of signaling cascades, we used Western blotting and enzyme-linked immunosorbent assay. We focused in our study on controlling intracellular protein and mRNA levels.

RESULTS

RNAi-mediated silencing of galectins (1, 3, 8, and 9) was efficient. Silencing of galectins resulted in decreased resistance to radiotherapy. Clonogenic survival was decreased after the silencing of LGALS1 and LGALS3. Both galectin 1 and 3 affected the ras-pathway. Whereas both galectins facilitated the activation of different ras proteins, galectin 1’s effect on radiosensitivity seems to have resulted from its control of PI3K activity. Galectin 3, on the other hand, controlled ERK1/2 MAPK. Combined silencing of both genes was particularly effective in sensitizing glioma cells to radiation. DNA damage after 6 Gy radiation was increased after silencing of all tested galectin family genes.

CONCLUSION

Intracellular levels of galectins are important for the radioresistance of gliomas. All galectins decreased at least one parameter that was measured to determine radiosensitivity. Galectins seem to mediate or facilitate the activation of different prosurvival and proliferation pathways. Galectins are an attractive target to sensitize cells to irradiation.

Neuro-Oncol. 2009 Oct;11(5):579–580.

70. Inhibition of the Hedgehog Pathway Can Target Glioblastoma Multiforme Stem Cells Independently of GLI

David Hsieh, Katie Miller, Ron Ellsworth, Daruka Mahadevan, Michael A Badruddoja, Abhay Sanan, Baldassarre Stea

INTRODUCTION

Small-molecule inhibitors of the Hedgehog pathway have demonstrated efficacy in targeting a stem cell population in glioblastoma multiforme, suggesting that Hedgehog antagonists may have extensive therapeutic utility. The majority of hedgehog pathway inhibitors target Smoothened protein, which is responsible for downstream signal transduction and activation of the Gli family proteins. The purpose of our study was to elucidate the downstream biochemical mechanism in which Smoothened inhibitors may affect self-renewal and proliferation of glioblastoma (GBM) stem cells.

METHODS

GBM stem cells were extracted from human specimens, cultured in serum-free media conditioned with vitamin B27, bFGF, and EGF, and identified by positive nestin, CD133, Sox, and Oct staining. Cell viability and proliferation were analyzed by Trypan blue and MTS. RT-PCR and qPCR were used to analyze Gli-mediated transcripts.

RESULTS

RT-PCR revealed that multiple modulators of the pathway are overexpressed in glioblastoma stem cells. Potentiation and inhibition of Gli proteins have been considered to be the primary effectors of Hedgehog signaling; however, it was determined that Gli1 activity could not be completely inhibited by cyclopamine, a Smoothened inhibitor, and suppression of Gli1 activity did not correlate to dose-dependent repression of glioblastoma stem cell proliferation by cyclopamine. Comparisons between IC50 doses of a Gli1 inhibitor, Gant61, and cyclopamine also revealed differing degrees of repression of Gli-regulated transcripts. Lastly, cyclopamine was capable of selectively targeting the cancer stem cell population that expressed nestin (up to 80% loss of nestin-expressing cells) while Gant61 was much less discriminate (less than 14% loss of nes-tin-expression cells).

CONCLUSION

Our results suggest that Gli is not the only effector of Hedgehog signaling in glioblastoma stem cells and that Smoothened inhibitors may be, in part, acting independently of Gli-mediated transcription through an uncharacterized signal activity. Our work warrants investigation of Gli-independent Hedgehog signaling, which may yield additional therapeutic targets for the treatment of GBM and eradication of the stem cell population.

Neuro-Oncol. 2009 Oct;11(5):580.

71. Sonic Hedgehog: Hippo Pathway Cross-Talk in Neural Precursor Proliferation and Pediatric Brain Tumorigenesis

Africa Fernandez-Lopez, Anna Kenney

INTRODUCTION

Medulloblastoma, the most common solid pediatric tumor, originates in the cerebellum during postnatal development. It is postulated that cerebellar granule neuron precursors (CGNPs) are cells-of-origin for certain classes of medulloblastomas. These cells undergo a rapid postnatal expansion phase in the external granule layer (EGL) of the cerebellum. This expansion requires Sonic hedgehog (Shh) and insulin-like growth factor (IGF) signaling. Previous work from our lab identified insulin receptor substrate 1 (IRS1) as a novel node of cross talk between Shh and IGF pathways in proliferating CGNPs. IRS1 is upregulated by Shh in CGNPs in vitro, and IRS1 overexpression is sufficient to maintain CGNP proliferation in the absence of Shh.

METHODS

Using mass spectrometry, we found that IRS1 interacts in CGNPs with Yes-associated protein 1 (YAP1), a transcriptional cofactor recently described as having a potent oncogenic potential. YAP1 is inhibited by the Hippo pathway, a signaling cascade involved in organ size regulation and cell contact inhibition but poorly understood in mammalian cells.

RESULTS

YAP1 expression is induced in the presence of Shh, and it is localized mainly in the nucleus of proliferating CGNPs. Overexpression of Yap1 is sufficient to drive CGNP proliferation in the absence of Shh and can synergize with exogenous Shh to further increase proliferation. We have found that YAP1 is highly expressed in human and mouse medulloblastomas, specifically in those of the Shh and Wnt-driven subclasses. While it is expressed diffusely throughout the tumors, YAP1 is most highly expressed in the cells surrounding the blood vessels, and these YAP1-positive cells survive radiation treatment, suggesting that YAP1 expression is part of the program used by tumor-repopulating cells.

CONCLUSION

Characterizing this new cross talk between Shh and Hippo pathways will increase our understanding of how the combined output of signaling pathways results in neural precursor proliferation during cerebellum development or, in the case of medulloblastoma, aberrant proliferation and tumor growth.

Neuro-Oncol. 2009 Oct;11(5):580.

72. ATOH1 Collaborates with GLI1 to Induce Medulloblastoma by Regulating Hes Family Members

Haotian Zhao

INTRODUCTION

Medulloblastomas (MBs), the most common malignant pediatric brain cancer, are embryonal tumors of the cerebellum. The cerebellar granule neuron progenitors (GNPs) undergo a short period of postnatal proliferation stimulated by the Sonic hedgehog/Patched (Shh/Ptch) signaling pathway before their differentiation into postmitotic neurons to form the mature cerebellar cortex. Despite the pleiotropic effects of Shh during nervous system development, tumor formation caused by aberrant Shh signaling is restricted to the GNP lineage. However, the molecular nature of this GNP-specific oncogenic addition of Shh remains unclear. Atonal homolog 1 (Atoh1/Math1) is a basic-helix-loop-helix proneural transcription factor essential for GNP histogenesis. In this study, we examined the role of Atoh1 in MB development.

METHODS

GNPs from neonatal cerebella of Ptch1± tumor-prone mice or wild-type mice were infected with retroviruses encoding Atoh1 or Gli1, a direct effector of Shh/Ptch signaling. Infected GNPs were evaluated for their proliferation and differentiation in vitro or in vivo by orthotopic transplantation into the cerebra of immunodeficient recipients. Atoh1 downstream targets were characterized by Affymetrix Gene Chip analysis using a tamoxifen-regulated Atoh1 construct expressed in primary GNPs.

RESULTS

Retro-virus-mediated expression of Atoh1 in GNPs from Ptch1± tumor-prone mice greatly accelerated MB development. Remarkably, although unable to induce transformation by itself, enforced expression of Atoh1 in wild-type primary GNPs resulted in a more than 800-fold increase in Gli1-dependent tumorigenicity. Atoh1 overexpression did not stimulate the proliferation of primary GNP in the absence of Shh. However, Atoh1 significantly enhanced the Shh-induced mitogenic response of GNPs and inhibited their cell cycle exit and differentiation in response to Shh withdrawal. Micro-array analysis revealed reciprocal regulation between Atoh1 and members of the Hairy/Enhancer of split (Hes) family and that suppression of Hes1 and Hes5 functions are required for MB development.

CONCLUSION

Atoh1 determines the GNP lineage’s specificity for Shh pathway-mediated tumorigenesis by regulating members of the Hes family.

Neuro-Oncol. 2009 Oct;11(5):580.

73. Diffuse Pontine Glioma: New Insights From Primary Cell Culture of Postmortem Tissue

Michelle Monje, Siddhartha S Mitra, Michael SB Edwards, Paul Fisher, Albert J Wong, Philip Beachy

INTRODUCTION

Gliomas of the brainstem comprise 15%–20% of brain tumors in children, and the majority of these are diffusely infiltrative gliomas of the ventral pons, a devastating cancer that occurs in school-age children (peak incidence at age 7 years) and almost invariably results in death. Advances in the understanding of and treatment for diffuse intrinsic pontine gliomas are desperately needed, but both have stalled for more than three decades. Contributing to this dismal progress is a dearth of available tumor tissue for study (biopsies are rare in this location, as diagnosis can be made radiologically) and lack of a valid experimental model.

METHODS

A 5-year-old male with a diffuse ventral pontine glioma who received minimal treatment donated tissue for postmortem analysis. The tissue was obtained under sterile conditions 14 h after death; the body was kept in a cool environment from the time of death until autopsy. The tumor was apparent on gross examination and was isolated from surrounding normal tissue, minced and processed for cell culture under stem cell conditions in serum-free media. Additional tumor samples were fixed in 4% paraformaldehyde and prepared for immunohistochemistry and confocal microscopy.

RESULTS

Here, we report primary cell culture of early postmortem ventral pontine glioma under stem cell conditions and the generation of pontine glioma tumor spheres. The tumor sphere cells coexpressed GFAP and nestin. Immunocytochemical markers of endothelial cells were absent. Tumorigenicity of the cultured cells was tested by transplantation to the brains of NOD/SCID/IL2 gamma chain (null) mice. Ongoing work is directed toward characterizing the activity of classical signaling pathways in these tumor cells.

CONCLUSION

Postmortem tumor cell harvest and culture is feasible and circumvents a major obstacle in the study of diffuse ventral pontine glioma. Basic questions regarding diffuse pontine glioma cell biology can now be addressed experimentally.

Neuro-Oncol. 2009 Oct;11(5):580–581.

74. Rac1-Pak1 Signaling is Critical for PDGFR-Mediated Medulloblastoma Cell Migration

Tobey MacDonald

INTRODUCTION

We previously demonstrated that the platelet-derived growth factor receptor (PDGFR) is overexpressed in metastatic medulloblastoma (MB) and that PDGF treatment promotes MB cell migration. The mechanism by which PDGFR mediates migration in MB cells is unclear, and whether inhibition of PDGFR signaling blocks MB migration is unknown. Understanding the signaling mechanisms may uncover novel therapeutic targets.

METHODS

We thus investigated the signaling activity of the major chemotaxis effectors Rac1 and Pak1 in Daoy and D556 medulloblastoma cells to determine whether Rac1-Pak1 activation is critically involved in PDGFR-mediated MB cell migration and whether Rac1 and phosphorylated Pak1 is detectable in MB tumors.

RESULTS

We demonstrated that treatment of Daoy and D556 MB cells with PDGF-BB induces concomitant activation of PDGFRβ, Ras, and Rac1 but inhibits Rho activity, which in turn significantly enhances cell migration. Conversely, the Rac1 inhibitors, NSC23766 and Tris-dipalladium, abolish PDGF-mediated cell migration. Rac1 activation by PDGF is further enhanced in the presence of the Rho kinase inhibitor, Y27632, confirming that Rho-Rock feedback signaling inhibits PDGF-induced Rac1 activity in MB cells. Furthermore, we demonstrated that PDGF-BB significantly increased the level of phosphorylated Pak1 and that siRNA knock-down of PDGFRβ, Rac1, or Pak1 abrogated PDGF-mediated activation of each of these three targets, respectively, and significantly inhibited Pak1 phosphorylation, resulting in abolished cell migration. We also showed that Erk inhibition suppressed Pak1 phosphorylation, indicating that Pak1 activity in medulloblastoma cells was critically regulated by both Erk and Rac activity. Combined treatment with PDGFR and Rac inhibitors effectively blocked cell migration, proliferation, and soft agar colony formation. Finally, using tissue microarrays, we showed that increased phosphorylated Pak1 immunopositivity correlated with worse clinical outcome.

CONCLUSION

These results indicate that specific inhibition of Rac1-Pak1 signaling via Rac1 and PDGFR/Erk inhibitors may be a novel therapeutic strategy for MB. Validation of these responses in vivo is currently under way using combined PDGFR and Rac inhibitors in a mouse model of medulloblastoma.

Neuro-Oncol. 2009 Oct;11(5):581.

75. Combination of Retinoic Acid and Rapamycin Inhibits Medulloblastoma Growth Through Upregulation of Specific Tumor Suppressors and Downregulation of Specific Risk Markers

Timothy R Gershon, Arash Shirazi, Bobby Bhatia, Anna Kenney

INTRODUCTION

Cerebellar granule-cell precursors (CGCPs) are neuroblasts that proliferate in response to Sonic Hedgehog (Shh) and can serve as cells of origin for medulloblastoma with aberrant Shh signaling. The intracellular kinase mTOR, activated by IGF, cooperates with Shh in CGCP proliferation. We have previously found that constitutive activation of the mTOR pathway caused by dominant TSC2 mutation causes CGCP hyperproliferation and synergizes with hedgehog signaling to promote medulloblastoma. Retinoic acid (RA), an endogenous molecule with established antineoplastic properties, may downregulate CGCP proliferation. We investigated the antiproliferative potential of combined mTOR inhibition and RA stimulation on CGCPs and medulloblastoma cells.

METHODS

We measured proliferation in CGCPs and CGCP-derived murine medulloblastoma cells (PZp53med). We compared effects of all-trans-RA (ATRA) and the mTOR inhibitor rapamycin separately and in combination on CGCPs from wild-type animals and from mice with constitutive mTOR activation and on PZp53med cells. Lastly, we performed an expression microarray comparison of PZPmed cells grown in ATRA, rapamycin, or both in combination.

RESULTS

ATRA and rapamycin inhibited CGCPs and PZPmed cells. CGCPs from transgenic mice with mTOR hyperactivation were resistant to ATRA, while treatment of CGCPs and PZPmed cells with rapamycin increased ATRA sensitivity. ATRA plus rapamycin yielded a vastly stronger antiproliferative effect. Microarray analysis revealed that ATRA and rapamycin synergized to increase expression of tumor suppressors, including p15-ink4b and Dkk3, and to decrease expression of genes associated with proliferation and poor prognosis, including CyclinD2 and EEF1D.

CONCLUSION

CGCPs and CGCP-derived medulloblastoma cells integrate signals through mTOR and RA pathways. These signaling pathways can be manipulated by pharmacologic agents in combinations that confer a dramatically enhanced antineoplastic effect. The synergistic regulation of proliferation is accompanied by synergistic changes in specific genes that regulate cell proliferation. These changes in gene expression may mediate the observed antineoplastic effect.

Neuro-Oncol. 2009 Oct;11(5):581.

76. Activation of the Hedgehog Pathway in Pilocytic Astrocytomas Correlates with Earlier Age at Onset and Aggressive Histologic Features

Sarah Zieber Rush, Juan G Valadez, Ty Abel, Matthew M Pearson, Michael K Cooper

INTRODUCTION

Pilocytic astrocytomas are the most commonly diagnosed primary brain tumor in children, yet little is known about molecular mechanisms regulating their behavior. Recent studies have demonstrated that the Hedgehog signaling pathway is activated in adult astrocytomas. Whether Hedgehog signaling is activated in lower-grade pediatric gliomas is unknown.

METHODS

To evaluate the Hedgehog pathway in pilocytic astrocytomas, 16 samples were analyzed by qRT- PCR measuring expression levels of the Hedgehog gene target PTCH. Compared to control temporal lobe specimens, PTCH RNA expression was elevated and correlated inversely with age. To corroborate these findings, a tissue microarray was constructed with 13 pediatric pilocytic astrocytoma specimens.

RESULTS

Histological review identified aggressive features such as infiltration, increased proliferation, and pilomyxoid features in eight of the samples. The tissue array was stained for Gli-1, PTCH, and Ki67 and scored in a blinded fashion. Gli-1 and PTCH staining produced concordant results, and specimens were scored as strongly positive, positive, or negative. An inverse relationship between Gli-1/PTCH staining and age was observed. The average age at diagnosis was 4.7 years for the four specimens scored as strongly positive for Gli-1 and PTCH. These four tumor specimens also demonstrated the most aggressive features, including increased proliferation, infiltration, and an average MIB-1 index of 11. The five tumors scored as positive for Gli-1 and PTCH staining had an average patient age of 5.5 years at diagnosis, demonstrated less aggressive features, and had a lower average MIB-1 index of 2.8. Specimens with negative staining came from patients with an average age of 9.7 years at diagnosis, had classic histological features, and had an average MIB-1 index of 1.3.

CONCLUSION

These findings suggest the Hedgehog pathway is activated in pediatric pilocytic astrocytomas. Furthermore, activation of the Hedgehog pathway may be associated with a younger age of diagnosis and more aggressive features.

Neuro-Oncol. 2009 Oct;11(5):581.

77. Subcellular Localization of Y-Box Protein 1 Regulates Proliferation, Invasion, and Increased Mesenchymal Phenotype in Astrocytomas

Caroline Sollier, Damien Faury, Noha Gerges, Xiao-Yang Liu, Brian Meehan, Janusz Rak, Steffen Albrecht, Nada Jabado

INTRODUCTION

Y-box protein 1(YB1) is a DNA/RNA-binding protein implicated in cancer progression. It is mandated for embryonic development and acts as a transcriptional/translational regulator in the cytoplasm. Akt-dependent phosphorylation of S102YB1 induces nuclear-translocation and potentially oncogenicity. We previously established elevated YB1 levels in glioblastoma (GBM), possibly driving oncogenesis. We hypothesized that dysregulated YB1 expression drives oncogenesis in GBM. The dual YB1 subcellular localization may be associated with dual function, with active Akt potentially engaging YB1 to promote the tumorigenic potential of astrocytomas.

METHODS

We stably overexpressed HA-tagged wild-type (WT) and S102A YB1 (preventing nuclear shuttling) in pGBM(SF188/clone5) and aGBM(U251-U87) cell lines and Htert-immortalized astrocytes. In parallel, we stably knocked down YB1 expression (shRNA) in these cell lines. We investigated the effects of overexpression/silencing of YB1 on cell signaling (Ras/Akt pathways), proliferation (monolayer/soft agar assays, xenograft models), invasion, and EGFR/Met levels in transfectant cell lines.

RESULTS

Complete stable silencing of YB1 was never achieved, indicative of the role of this protein in cell survival. In all shYB1-stable clones, residual YB1 was nuclear, and cells showed increased proliferation (monolayer/soft agar/xenografts), increased EGFR levels, and sustained activation of Ras compared to empty-vector transfectant cells. Ectopic HA-tagged YB1 was predominantly cytoplasmic in three GBM cell lines, similar to endogenous YB1, and moderately increased cell proliferation in only one GBM cell line. Both WT and S102A YB1 increased cell migration in all cell lines. In HTert-immortalized astrocytes, silencing YB1 increased proliferation and decreased cell migration, while ectopic expression provoked the reverse, in addition to the induction of marked mesenchymal features in these cells. In one GBM cell line, overexpressed HA-tagged YB1 was predominantly nuclear, similar to endogenous YB1. This led to increased cell proliferation and EGFR levels and had little effect on cell migration.

CONCLUSION

Our results suggest that YB1 modulates cellular proliferation and increased mesenchymal phenotype properties, based on its subcellular localization. While ascertaining the role of nuclear YB1 in driving cell growth, our data argue for caution in targeting YB1 for therapeutic intervention.

Neuro-Oncol. 2009 Oct;11(5):581–582.

78. Effects of CMET Oncogenic Activation on Medulloblastoma Pathogenesis

Sara Onvani, Paul N Kongkham, Christian Smith, James T Rutka

INTRODUCTION

HGF/cMET signaling plays a role in cerebral development, and the overactivation of this pathway has been implicated in several human malignancies. We have identified SPINT2 as an inhibitor of the HGF/cMET pathway, which is silenced by methylation in 33% of primary human medulloblastoma (MB) tumors. In addition, aberrant expression of the HGF receptor, cMET tyrosine kinase, has been associated with these tumors.

METHODS

We have made an expression construct containing a mutant, constitutively active form of cMET. We will examine the effects of overactive cMET expression on MB cells in vitro to study the role of aberrant HGF/cMET signaling on MB pathogenesis.

RESULTS

To confirm pathway activation, we will characterize cMET phosphorylation and downstream targets. HGF/cMET signaling mediates several normal cellular activities, including growth, proliferation, motility, and survival, and therefore we will examine the effects of constitutive overactivation of this pathway on MB proliferation and apoptosis. In addition, effects on cell motility and invasion will be analyzed using in vitro migration and invasion assays.

CONCLUSION

Greater understanding of the role of HGF/cMET signaling with respect to MB pathogenesis may reveal novel therapeutic targets.

Neuro-Oncol. 2009 Oct;11(5):582.

79. Patterns of Microrna Dysregulation among Pediatric Brain Tumors

Diane Birks, Rajeev Vibhakar, Andrew M Donson, Valerie Nicole Barton, Michael H Handler, Nicholas K Foreman

INTRODUCTION

Pediatric brain tumors continue to cause significant morbidity as a result of treatment, and many aggressive tumors have a dismal prognosis. A better understanding of the biology of these malignancies is needed to assist in the development of less-toxic therapies and to provide better markers for disease stratification. MicroRNAs are short, nonprotein coding RNAs that posttranscriptionally regulate gene expression. They play important roles in many processes, including cell proliferation, differentiation, apoptosis, and development. Much research also points to their involvement in cancer. To date, only research on microRNA expression in medulloblastomas has been published in the area of pediatric CNS tumors.

METHODS

To address the role of microRNAs in a wider set of pediatric brain tumors, this study used microarray technology to profile the expression of 470 human microRNAs in five common childhood brain tumors: atypical teratoid/rhabdoid tumors (AT/RTs), pediatric glioblastomas (GBMs), medulloblastomas (MEDs), ependymomas (EPNs), and pilocytic astrocytomas (PAs).

RESULTS

Three microRNAs, miR-129, miR-25, and miR-142-5p, showed significant dysregulation in all of these tumor types compared to normal pediatric brain. MicroRNAs that down-regulate p21 and p57 were strongly overexpressed among all grade IV tumor types but not in EPNs or PAs. Dysregulated microRNAs in pediatric GBMs showed both similarities and differences to those found in adult GBMs. Finally, microRNA expression patterns of AT/RTs showed little similarity to MEDs but instead demonstrated a strong correspondence to those of pediatric GBMs. This finding is of particular interest, since the cell of origin for AT/RTs is currently unknown.

CONCLUSION

MicroRNAs show significant dysregulation in all pediatric CNS tumor types examined, and common expression patterns exist among these types. These findings contribute to further insights into the etiology and biology of pediatric brain tumors.

Neuro-Oncol. 2009 Oct;11(5):582.

80. PI3 Kinase-Mediated Deregulation of Actin Turnover by Slingshot 1 Phosphatase Impacts Cell Cycle Progression in Pediatric Ependymomas

Timothy Van Meter, Nilesh Mepani, Morgan McCrocklin, Renu Kadian, Catherine I Dumur, Gary W Tye

INTRODUCTION

To better understand the specific impact of PI3 kinase in pediatric brain tumors, genomic profiling studies were performed on clinical samples previously identified as having elevated PI3 kinase activity. Pathway-specific analysis led to the discovery of elevated Slingshot 1 (SSH1) in pediatric ependymomas and prompted the investigation of the biological impact on cytoskeletal signaling in these tumors.

METHODS

Protein complexes regulating actin dynamics during cell motility and cell cycle progression were studied in established and primary ependymoma-derived cell lines and in tissue lysates prepared from snap-frozen biopsies, as well as in a series of rat brain tissues during successive developmental stages from E15 to adult.

RESULTS

Epidermal growth factor receptor activation and serum stimulation models demonstrated that tyrosine kinase receptor-mediated dephosphorylation of Cofilin was dependent on PI3 kinase signaling, revealing a novel function for this pathway in pediatric ependymomas. Rapid dephosphorylation of Cofilin and LIM kinases 1 and 2 preceded cell rounding and cell cycle progression as well as cytokinesis, shown using kinase inhibitors and by overexpression of full-length and dominant-negative phosphatase-dead mutant forms of SSH1 or LIM kinases or SSH1 shRNA. Growth of tumor cells was significantly augmented by SSH1 overexpression.

CONCLUSION

Together these studies characterize the impact of Slingshot 1 on cell cycle in vitro and in vivo in novel models of pediatric ependymoma. Targeting SSH1 could provide a novel avenue for suppressing tumor growth in these common pediatric cancers.

Neuro-Oncol. 2009 Oct;11(5):582.

81. Treatment Disparities Following Diagnosis of a Glioblastoma Multiforme

Paula R Sherwood, Bassam Dahman, Heidi Donovan, Arlan H Mintz, Charles Given, Cathy Bradley

INTRODUCTION

Radiation is clearly associated with survival following diagnosis of a glioblastoma multiforme (GBM), becoming the standard of care. However, there is a dearth of research identifying disparities in receiving radiation. The purpose of this analysis was to identify differences in receiving radiation and in survival based on age, gender, income, residence, and insurance status (receiving Medicare and Medicaid versus Medicare only).

METHODS

Statewide Medicaid and Medicare data were merged with tumor registry data to extract a sample with a first GBM diagnosis between 1996 and 2000. Patients were matched to the Medicare Denominator File using Social Security numbers. All claims for inpatient, outpatient, and physician services were collected. Adjusted logistic regression was used to evaluate predictors of reporting at least one claim for radiation. Survival curves were constructed with the Kaplan-Meier estimation method and compared with the log rank test.

RESULTS

African Americans were less likely to receive radiation (OR = 0.13; CI = 0.04–0.44) than Caucasians. Radiation was less likely in persons receiving both Medicare and Medicaid versus Medicare only (OR = 0.47; CI = 0.24–0.92); persons >75 years (OR = 0.43; 95% CI = 0.24–0.75) and 70–74 years (OR = 0.60; CI = 0.37–0.96) versus those 65–69 years. Those undergoing surgical intervention were more likely to report radiation (OR = 4.85; CI = 2.51–9.25). Longer survival was seen in persons 70–74 years (HR = 1.29; CI = 1.01–1.64) and those >75 years (HR = 1.38; CI = 1.09–1.75) versus those 65–69 years; persons in metropolitan areas (HR = 1.47; CI = 1.16–1.88); receiving radiation (HR = 0.55; CI = 0.45–0.75) or chemotherapy (HR = 0.59; CI = 0.46–0.75); and persons with an income of $25,000–$35,000/year (HR = 0.77; CI = 0.60–0.99) or >$35,000/year (HR = 0.71; CI = 0.55–0.93) versus those with incomes <$25,000/year.

CONCLUSION

Data suggest that ethnic, economic, and insurance disparities may exist in receiving radiation after a GBM diagnosis. A national database is needed to explore the nature of these potential disparities. Once results are explored on a larger scale, research is needed to understand the source of disparity so interventions can be implemented to ensure all persons receive quality care.

Neuro-Oncol. 2009 Oct;11(5):582.

82. Survival Among Patients with Primary Central Nervous System Lymphoma From 1973 to 2005

Andrew Norden, Jan Drappatz, Patrick Wen, Elizabeth Claus

INTRODUCTION

Primary CNS lymphoma (PCNSL) is a rare, extra-nodal non-Hodgkin’s lymphoma that represents 3% of all malignant CNS tumors. Despite advances in chemotherapy and radiation for this disease, survival trends over time are uncertain.

METHODS

We used 1973–2005 data from the Surveillance, Epidemiology, and End Results (SEER) cancer registry, which covers 26% of the United States population, to identify patients with PCNSL as the first primary cancer. Patients reported to have died of the human immunodeficiency virus and cases identified posthumously were excluded. Survival was evaluated using the Kaplan-Meier method, and univariate survival differences were examined with two-sided log-rank tests. A Cox proportional hazards regression model was constructed to evaluate multivariate predictors of survival.

RESULTS

This study included 1,967 PCNSL patients with a median age at diagnosis of 63 years. Of these patients, 55% were men, 86% were Caucasian, and 67% received radiation therapy. Of the tumors, 74% occurred in the brain, 5% in meninges, 9% in the spine, and the remainder in uncertain locations. Median survival was 12 months (95% confidence interval [CI] 11, 14). For patients diagnosed in the intervals 1973–1979, 1980–1989, 1990–1999, and 2000–2005, median survival was 7.5 (95% CI 6, 14), 12 (95% CI 9, 16), 10 (95% CI 8, 13), and 18 months (95% CI 14, 25), respectively (log-rank test p = 0.002). Univariate predictors of reduced survival included nonblack race, previous marriage (i.e., separated, divorced, or widowed), advanced age, no radiation, and earlier year of diagnosis. Multivariate predictors of reduced survival included male sex, previous marriage, advanced age, no radiation, and earlier year of diagnosis. The hazard ratio for the year of diagnosis was 0.979 (95% CI 0.973, 0.986; p < 0.0001).

CONCLUSION

Survival among patients with PCNSL is improving with time. The reason for this improvement cannot be determined with certainty from these data, but increasing use of methotrexate-based chemotherapy is a potential explanation. Survival differences based on gender, race, and marital status are intriguing and warrant further study.

Neuro-Oncol. 2009 Oct;11(5):583.

83. Evaluation of Concomitant Temozolomide and Radiotherapy Treatment in Patients with Glioblastoma Multiforme in Two Canadian Tertiary Care Centers

Ibrahim Mohammed Alnaami, Vivek Mehta, Sita Gourishnkar, Ambikaipakan Senthilselvan

INTRODUCTION

The glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults and is the most aggressive brain tumor leading to morbidity and mortality, with 9–12 months survival after combined surgical and radiotherapy (RT) management. In 2004, a landmark paper on the history of GBM was published by Stupp et al., who found that concomitant and adjuvant temozolomide (TMZ) and RT for newly diagnosed GBM resulted in a clinically meaningful and statistically significant survival benefit with minimal additional toxicity, where the median survival was 14.6 months with RT plus TMZ and 12.1 months with RT alone.

METHODS

In a retrospective cohort study, we analyzed 346 GBM patients from two Canadian tertiary care centers from 2000 to 2006 and compared survival pre- and postintroduction of concomitant TMZ management.

RESULTS

The patient group included 216 men and 130 women. The mean survival was 12 months in the nonconcomitant TMZ group and 18.6 months in the concomitant TMZ group. From multivariate analysis, we found age, type of surgery, concomitant TMZ, and time to RT [(hazard ratio [HR] 1.02, 95% confidence interval [CI] 1.01–1.03), (HR 0.50, 95% CI 0.37–0.66), (HR 1.87, 95% CI 1.33–2.63), and (HR 0.99, 95% CI 0.98–0.99), respectively], are all significant predictors of survival.

CONCLUSION

Concomitanant TMZ is showing an excellent outcome outside the RCT trial. We encourge the use of TMZ in those two Canadian centers and we hope that would encourge other institutions to report their results in an effort to generalize the results.

Neuro-Oncol. 2009 Oct;11(5):583.

84. A Comparison of CNS Cancer Identification Using Mortality and Incidence Data

Jeanine M Buchanich, Ada O Youk, Gary M Marsh, Steven E Lacey, Kathleen J Kennedy, Nurtan A Esmen, Zb Bornemann, Frank Lieberman, Mary Lou Fleissner

INTRODUCTION

As part of an exploratory investigation of an unusual occurrence of glioblastoma at one jet engine manufacturing facility located in North Haven, Connecticut, we conducted cancer incidence and mortality tracing to identify CNS tumors. We compare the results of the two types of tracing here.

METHODS

To identify CNS tumor deaths, we used our standard two-stage vital status tracing protocol to identify deaths among cohort members with unconfirmed vital status (not known from company-held records to be alive as of 12/31/2004). To identify incident CNS tumor cases, we matched our cohort to 19 state cancer registries, requesting all CNS tumor matches and their diagnostic data from 1/1/1976, or the registry start date through 2004.

RESULTS

We identified 597 CNS neoplasm deaths with underlying (UCOD) or contributory (CCOD) CNS causes from mortality tracing and 722 incident CNS neoplasm cases through cancer incidence tracing. Of the incident CNS cases, 178 were alive as of 12/31/2004 and not included in these comparisons. Of the 746 total cases or death identified by either source, 57% were identified in both the mortality and cancer incidence tracing, 23% were identified only through mortality, and 20% were identified only through cancer incidence tracing. We identified 86% of the malignant incident CNS cases, 20% of the benign incident CNS cases, and 67% of the uncertain CNS cases as CNS neoplasm deaths using UCOD or CCOD. “Other types of cancer” were the UCOD most commonly reported for the cases not identified as dying from CNS cancer.

CONCLUSION

Researchers should consider tracing for cancer incidence in retrospective cohort studies. Death certificates may not be the most accurate way of identifying cancer cases, particularly if studying CNS cancer.

Neuro-Oncol. 2009 Oct;11(5):583.

85. Long-Term Health Experience of Jet Engine Manufacturing Workers: Incidence of Nonmalignant Central Nervous System Neoplasms

Jeanine M Buchanich, Ada O Youk, Gary M Marsh, Steven E Lacey, Kathleen J Kennedy, Nurtan A Esmen, Frank Lieberman, Mary Lou Fleissner

INTRODUCTION

As part of an exploratory investigation of an unusual occurrence of glioblastoma at one jet engine manufacturing facility located in North Haven (NH), Connecticut (CT), we examined incidence rates for nonmalignant CNS neoplasms at eight of the company’s CT facilities.

METHODS

Subjects were 212,513 workers ever employed in one or more of the manufacturing facilities from 1952 to 2001 and at risk during the 1976–2004 observation period. By tracing through 19 state cancer registries or by using medical records to confirm the histologic diagnosis of CNS neoplasm deaths, we identified 199 nonmalignant CNS neoplasm cases of three histologies: nerve sheath, meningioma, and pituitary. We computed standardized incidence ratios (SIRs) based on CT state and national (SEER 9) rates and modeled internal relative risks. Reporting for nonmalignant CNS cases was only mandated in 2004. To determine the extent to which nonmalignant cases may have been underascertained, we also performed sensitivity analyses.

RESULTS

Preliminary results indicate no elevations at the total cohort level for the nonmalignant CNS neoplasm histologies examined relative to the general populations of the U.S. or CT. SIRs are higher for all three histologies among long-term (5+ years of employment) compared to short-term employees. Based on preliminary results, rates of meningioma appear elevated among workers in the only NH plant group compared to the other plant groups.

CONCLUSION

At the total cohort level, preliminary results indicate that incidence rates for the nonmalignant CNS neoplasm histologies examined were not elevated relative to the general populations of the United States and Connecticut. The excesses in certain subgroups of workers may reflect external occupational factors, nonoccupational factors or workplace factors unique to the facility not measured in the current study. We will further explore reasons for the excesses using the companion exposure assessment component.

Neuro-Oncol. 2009 Oct;11(5):583.

86. Long-Term Health Experience of Jet Engine Manufacturing Workers: III. Incidence of Malignant Central Nervous System Neoplasms

Gary M Marsh, Jeanine M Buchanich, Ada O Youk, Michael Cunningham, Frank Lieberman, Kathleen J Kennedy, Steven E Lacey, Roger P Hancock, Nurtan A Esmen, Mary Lou Fleissner

INTRODUCTION

As part of an exploratory investigation of an unusual occurrence of glioblastoma at one jet engine manufacturing facility located in North Haven (NH), Connecticut (CT), we examined incidence rates for malignant CNS neoplasms at eight of the company’s CT facilities.

METHODS

Subjects were 212,513 workers ever employed in one or more of the manufacturing facilities from 1952 to 2001 and at risk during the 1976–2004 observation period. We identified 722 cases of CNS neoplasms by tracing through 19 state cancer registries or by using medical records to confirm the histologic diagnosis of CNS neoplasm deaths. We computed standardized incidence ratios (SIRs) based on CT state and national (SEER 9) rates and modeled internal relative risks.

RESULTS

Using CT state rates, we found overall deficits in cases in most histology categories, including glioma (427 cases, SIR = 0.76, CI = 0.69–0.84) and glioblastoma (275 cases, SIR = 0.77, CI = 0.68–0.87). A not statistically significant 8% excess in glioblastoma cases was found in the only NH plant group (43 cases, SIR = 1.08, CI = 0.78–1.46) compared with deficits in the partial and non-NH groups. For the combined NH plant group, we found higher risks of glioblastoma for salaried workers, older workers, older and more recent hires and the most recent time period, but no clear association with duration of employment or time since first employment. Findings for the other malignant histology categories were essentially unremarkable.

CONCLUSION

At the total cohort level, incidence rates for the malignant CNS neoplasm histologies examined were not elevated relative to the general populations of the United States and CT. The glioblastoma excesses in certain subgroups of workers from NH may reflect external occupational factors, nonoccupational factors or workplace factors unique to NH that were not measured in the current study. We will explore reasons for the NH excesses in our case-control and exposure assessment studies.

Neuro-Oncol. 2009 Oct;11(5):583–584.

87. Incidence, Survival, and Initial Treatment in Adults Diagnosed with Glioblastoma 1981–2006 in the United States

Wei Dong, Laura Chu, Asha Das, Meghna Samant, Michael D Taylor

INTRODUCTION

Glioblastoma (GBM), the most common primary brain tumor in adults, is very aggressive with limited treatment options. Although a significant unmet medical need, GBM is not well studied potentially because of its lower incidence compared to other malignancies and lack of effective therapies. The objective of this study was to examine GBM incidence, survival and initial treatment (i.e., within 4 months (m) after diagnosis) to help further understand its natural history and define target patient populations for potentially effective therapies.

METHODS

The study population included all newly diagnosed adult (⩾18 years) GBM patients in the Surveillance, Epidemiology and End Results (SEER) program diagnosed between 1981 and 2006. Incidence was calculated as the number of new cases of GBM in SEER divided by the SEER population and age-adjusted to the 2000 U.S. standard population. Median survival was estimated using the Kaplan-Meier method.

RESULTS

Between 1981 and 2006, a total of 28,913 GBM cases were identified (median age at diagnosis, 64 years) with 2,202 incident cases occurring in 2006. Age-adjusted incidence rate for 2006 was 3.9 per 100,000 adults. The 1-year survival rate was ~27.1%. About 85% of patients received radiotherapy and/or surgery as initial therapy. Chemotherapy data were not available from SEER. Median survival for all patients was 6 months during 1981–1985, 1991–1995, 1996–2000, and 7 months in 1986–1990 and 2001–2006. Median survival was longer for patients who received initial radiation and surgical resection (12 months in 1996–2000 and 14 months in 2001–2006). Additional analysis confirmed that GBM has the worst survival compared to all other brain tumor histologies.

CONCLUSION

GBM affects ~10,000 adults annually, accounting for 50% of all primary brain tumor cases and 62% of all deaths due to primary brain tumor. Survival remains low despite some advances in radiotherapy and chemotherapy. GBM remains a significant unmet medical need where effective therapies are urgently needed.

Neuro-Oncol. 2009 Oct;11(5):584.

88. Exposure Reconstruction for Epidemiologic Studies of Brain Cancer Mortality and Incidence Among a Cohort of Jet Engine Manufacturing Workers: An Update

Kathleen J Kennedy, Steven E Lacey, Roger P Hancock, Nurtan A Esmen, Frank Lieberman, Jeanine M Buchanich, Ada O Youk, Michael Cunningham, Gary M Marsh

INTRODUCTION

An exposure reconstruction is under way to inform brain cancer mortality and incidence studies. The study was initiated because of concern over a perceived excess of glioblastoma cases at a jet engine manufacturing plant in the United States. The study cohort includes more than 220,000 individuals ever employed 1 day or more at any of eight of the company’s plants.

METHODS

Quantitative and semiquantitative methods are being utilized to reconstruct exposures to jet engine parts, processes, and several specific agents of interest for the period 1952–2001 for eight of the company’s plants involved in original jet engine manufacturing, overhaul and repair, and assembly. The exposure data developed will ultimately be merged with the mortality and incidence data to allow further investigation into whether there is an association between occupational exposures specific to any job category and brain cancer.

RESULTS

A job dictionary that includes more than 250,000 unique work history entries from company employment records was developed. The dictionary enables the cohort to be properly stratified into job categories so that exposure estimates may be assigned. Company industrial hygiene data have been utilized to inform exposure estimates for specific agents of interest.

CONCLUSION

Exposure reconstruction for these large retrospective brain cancer epidemiologic studies comprises several vital components. Methods and issues surrounding these components, including job dictionary construction, exposure assignment, and combination of the exposure and mortality and incidence data sets, will be presented.

Neuro-Oncol. 2009 Oct;11(5):584.

89. Detection of Circulating Tissue Factor Activity in the Plasma of a Patient with Glioblastoma Multiforme: A Potential Cause of Cancer-Associated Hypercoagulability?

Nicholas George Avgeropoulos, Enriqueta Coll, Ali Amirkhosravi, Hina Desai, Liza Robles-Carrillo, Mildred Amaya, Alane Drexler, Casey Kulscar, George C Bobustuc, Sergey Bushnev, John Francis

INTRODUCTION

Patients with glioblastoma multiforme (GBM) are at high risk of venous thromboembolism (VTE). Although clinical factors such as immobility, paresis, and hypovolemia contribute to this risk, they do not correlate with occurrence of VTE. Indeed, biological factors may be more significant than clinical factors in predicting postoperative thrombosis. Tissue factor (TF), the initiator of coagulation in vivo, is expressed in all gliomas, and correlates with tumor grade. TF is normally expressed extravascularly but has recently been shown to be present in blood. Whether blood-borne TF is functionally active remains unclear. We report the presence of active TF in cell-free plasma of a 38-year-old male patient with GBM.

METHODS

We collected pre- and postoperative (day 3) blood samples and performed whole blood coagulation analysis by thromboelastography (TEG). We determined platelet activation by measuring platelet surface P-selectin and monocyte-platelet conjugates. Plasma microparticles (MP) were isolated by high-speed centrifugation of cell-free plasma. Procoagulant activities of MP and particle-free supernatant (SN) were measured by thrombin generation assay.

RESULTS

Preoperative TEG revealed a hypercoagulable state as evidenced by a shortened clot time (6.8 min; normal, 9–27 min) and elevated clot rate and strength values. Postoperatively, the clot time normalized (10 min), indicating the reduction of a plasmatic procoagulant trigger. Other TEG parameters remained elevated. Platelet counts were normal in both samples, although platelet activation decreased by >50% postoperatively. TF-dependent procoagulant activity was detected in MP (but not in SN). Consistent with the TEG clot times, this TF activity was reduced postoperatively, but remained higher than that of control plasma.

CONCLUSION

This is the first report of TF activity in the plasma of a patient with GBM. The postoperative reduction of this circulating MP-associated TF activity suggests that the observed hyperco-agulability could be at least partially due to TF shed from the tumor.

Neuro-Oncol. 2009 Oct;11(5):584.

90. Factors Influencing the Care of Trauma Patients with Incidentally Identified Central Nervous System Tumors

Maya Babu, William T Curry, Brian V Nahed

INTRODUCTION

While fears have risen over the shortage of neurosurgeons across the country, few studies have actually explored the impact of the number of neurosurgeons in a facility on patient care. In this study, we evaluated the influence of the number of neurosurgeons, number of trauma surgeons, and trauma center level on hospital admission for patients presenting with trauma and incidentally identified primary or secondary brain and spinal cord tumors.

METHODS

Subjects included patients with primary or secondary brain and spinal cord tumors, which were identified when presenting with acute trauma. We gathered data from the 2002–2006 American College of Surgeons National Trauma Databank and used Stata statistical software analysis; regression analyses explored the relationship between treatment facility, number of surgeons, admissions, injury severity score, and socioeconomic factors (race, gender, age, and insurance status).

RESULTS

(1) The number of neurosurgeons practicing in a treatment facility was strongly related to the number of days between presentation in the emergency department and admission to the hospital (coefficient = 1.211, p-value = 0.004) when controlling for trauma center level, number of trauma surgeons, injury severity score, and patient demographics. When controlling for presenting Glasgow Coma Score, this relationship became marginally significant (coefficient = 0.789, p-value = 0.08). The number of neurosurgeons in a facility did not affect the time between presentation and first procedure (p-value = 0.391) or the length of hospital stay for the admitted patient (p-value = 0.087). (2) The level of the admitting trauma center was strongly related to the time between presentation and the first procedure performed for these patients (coefficient = 0.332, p-value = 0.016).

CONCLUSION

The number of neurosurgeons available to treat patients with complex neurological disease is an important determinant of admission time to the hospital.

Neuro-Oncol. 2009 Oct;11(5):584.

91. High Mobility Group-A2 (HMGA2) Gene Polymorphism is Associated with Long-Term Survival in Glioblastoma

Yanhong Liu, George Alexiou, Carol Etzel, Sanjay Shete, Melissa L Bondy

INTRODUCTION

Glioblastoma (GBM) is the most common and lethal type of brain cancer in adults. Median survival time for patients with GBM is approximately 1 year, and 90% die within 3 years after diagnosis. The purpose of this study was to determine whether genetic polymorphisms in a top-ranking glioma genomewide association (GWA) study predict GBM survival.

METHODS

The study included 323 primary GBM patients who were enrolled in epidemiologic studies at M.D. Anderson Cancer Center from 1992 to 2008, with follow-up until April 2009. The patients were divided into two subgroups: short-term survivors with a survival time of <1 year (n = 216) and long-term survivors with a survival time of ⩾3 years (n = 107). For SNP selection, we focused on the top-ranking 100 SNPs from our glioma GWA study. We compared genotype frequencies in short-term and long-term survivors using the chi-square test. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) by unconditional logistic regression analysis with adjustment for diagnosis age, gender, extent of surgical resection, radiation therapy, and chemotherapy.

RESULTS

We found four variants, HMGA2 rs1563834 (adjusted OR, 17.39; 95% CI, 1.80–167.72), RTEL1 rs2297440 (adjusted OR, 5.71; 95% CI, 1.00–33.78), rs10464870 (adjusted OR, 4.70; 95% CI, 1.44–15.51), and FAM rs7325927 (adjusted OR, 0.34; 95% CI, 0.12–0.95), were significantly different between the short-term and long-term survivors after correcting for multiple testing (p = 0.009, 0.034, 0.005, and 0.045, respectively). Further analysis indicated the effect of HMGA2 rs1563834 and RTEL1 rs2297440 were more evident in patients who survived longer than 5 years (n = 59; adjusted OR, 19.08; 95% CI, 1.30–279.12; and OR 7.83; 95% CI, 1.03–59.80, respectively). Moreover, we found a significant gene-dosage effect for increased risk with increasing numbers of the above mentioned three risk variants (ptrend < 0.0000).

CONCLUSION

Polymorphism in HMGA2 gene may be the important genetic predictor for GBM survival but requires replication.

Neuro-Oncol. 2009 Oct;11(5):585.

92. A Novel Approach to Exploring Potential Pairwise Interactions Among Single-Nucleotide Polymorphisms of Inflammation-Related Genes in Gliomagenesis: An Exploratory Case-Only Study

Michael E Scheurer, E Amirian, Anthony M D’Amelio Jr, Carol Etzel, Yanhong Liu, Richard Houlston, Melissa L Bondy

INTRODUCTION

Despite extensive research on the topic, glioma etiology remains largely unknown. Exploration of potential interactions between single-nucleotide polymorphisms (SNPs) of immune-related genes is a promising new area of glioma research. The case-only study design is a powerful and efficient design for exploring possible multiplicative interactions between factors that are independent of one another. The purpose of our study was to use this exploratory design to identify potential pairwise SNP-SNP interactions from genes involved in several different immune-related pathways for investigation in future studies.

METHODS

We examined potential interaction between only SNPs located on different chromosomes. The study population consisted of 1,949 histologically con-firmed, non-Hispanic white glioma cases originating from both the United States (1,308 participants) and the United Kingdom (641 participants). We utilized unconditional logistic regression to calculate the p-values for each interaction in the U.S. data set. We then validated the interactions detected in the U.S. data with a p-value less than that corrected with the Benjamini and Hockberg method using the U.K. data.

RESULTS

Using this data-mining strategy, we found 12 significant SNP-SNP interactions among polymorphisms of immune-related genes in both data sets. Genes implicated in the significant interactions included CSNK2A1, IL16, TXN, CUX1, THSD4, and PTPN11. Six of the 12 significant interactions contained SNPs from the CSNK2A1 gene on chromosome 20 and the IL16 gene on chromosome 15.

CONCLUSION

We conducted this exploratory study for the purpose of hypothesis generation, and the interactions implicated by this study provided promising new hypotheses that can now be tested utilizing more traditional study designs.

Neuro-Oncol. 2009 Oct;11(5):585.

93. Trends in Survival of Patients with Glioblastoma Multiforme: Surveillance, Epidemiology, and End Results (SEER) Program 1973–2005

Ciara Harraher, Andrew Norden, Elizabeth Claus

INTRODUCTION

Previous studies have suggested that treatment of primary malignant gliomas differs by race and marital status. The objective of this study was to examine differences in survival by race and sex in patients diagnosed with glioblastoma multiforme (GBM) using recent population-based data.

METHODS

We used the Surveillance, Epidemiology, and End Results (SEER) Registry, 1973–2005 and analyzed 17,663 patients diagnosed with GBM (ICD-O 9440). We used chi-square tests to determine the statistical significance of prognostic factors by race and sex. We used the Kaplan-Meier method to estimate crude survival by race and sex. We calculated differences in survival by prognostic variables using Cox proportional hazards models.

RESULTS

The crude survival of patients with GBM was 13.80 months, with 50% of patients alive at 7 months. Survival in male patients and black patients increased (log rank p = 0.0006 and log rank p = 0.0351, respectively). Univariate models showed that female patients had a 5% increased hazard of death (HR, 1.05) compared to men, and patients who were single had a 9% increased hazard of death compared to those who were married (HR:1.09). A diagnosis of GBM in 1974 (HR, 1.25) had an increased hazard of death compared to a diagnosis in 2004 (HR, 0.94). The multivariate model demonstrated that every 1-year increase in the age of diagnosis increased the hazard of death by 3% (HR, 1.03). Radiation treatment (HR, 0.48), any surgical treatment (HR, 0.74), marital status (HR, 0.95), and total surgical resection (HR, 0.79) significantly decreased the hazard of death from GBM. Race, tumor location, partial surgical resection, and year of diagnosis were not significant predictors of survival.

CONCLUSION

Race does not appear to affect survival in patients diagnosed with GBM, even after adjusting for the type of treatment and other prognostic variables. Married patients who undergo total surgical resection and radiation have significantly increased survival.

Neuro-Oncol. 2009 Oct;11(5):585.

94. Analysis of Survival and Prognostic Factors in Ependymoma—A 30-Year Population-Based Study

Bradly Biagioni, Paula de Robles, Qiuli Duan, Mark G Hamilton, Jacob Easaw

INTRODUCTION

Ependymoma is a rare tumor of the CNS. Its rarity makes determining prognostic factors difficult, with most studies limited by their small size. We, therefore, undertook an analysis of all cases of ependymoma in Alberta, Canada, diagnosed in the past 30 years.

METHODS

We performed a population-based review using the Alberta Cancer Registry. The major outcomes of interest were median overall survival (mOS) and median progression-free survival (mPFS).

RESULTS

We analyzed 176 cases; 62 (35%) were children (age = 18), and 114 were adults (65%). The median age at diagnosis was 32. Of the pediatric cases, 78% had brain ependymoma and 22% had spinal tumors, whereas in adults, 37% had brain ependymomas and 63% had spinal tumors. In children, the mPFS was 1.88 years; in adults younger than 50 it was 13.23 years; and in adults older than 50, it was 5.03 years. Patients older than 18 years (p = 0.003), patients who had a gross total resection of their tumors (p < 0.001), and patients who had a WHO grade I tumor (p = 0.07) were found to have a significantly longer mPFS on multivariate analysis. The 5-year and 10-year overall survival rates were 61.7% and 37.7%, respectively. In children, the mOS was 5.32 years (range, 0–30) which compared unfavorably with the adult population. Adults 19–50 years old had a mOS of 10.6 years (0.6–32), and adults >50 had an mOS of 5.7 years (0.1–24). Patients older than 18 years (p = 0.003), patients who had a gross total resection of their tumor (p = 0.002), and patients who had a WHO grade I tumor (p = 0.092) were found to have a significantly increased mOS on multivariate analysis.

CONCLUSION

In our population-based study of childhood and adult ependymoma, age, extent of resection, and pathological grade independently influenced overall and progression-free survival. Adjuvant radiotherapy did not show any benefit for survival.

Neuro-Oncol. 2009 Oct;11(5):585.

95. Descriptive Epidemiology of Primary Central Nervous System Non-Hodgkin’s Lymphomas

Bridget J McCarthy, Therese A Dolecek, John Lee Villano

INTRODUCTION

Primary CNS lymphomas (PCNSL) are a rare subtype of brain and CNS tumors, and few population-based studies have described their epidemiology.

METHODS

We used the Surveillance, Epidemiology, and End Results Program limited-use 9-registries database to identify PCNSLs from 1973–2006. PCNSLs were defined by ICD-O-3 histology codes 9590–9599 and 9670–9729 in brain/CNS primary sites (C70.0–72.9 and 75.1–75.3). Estimates of incidence rates, trends, and survival analyses were determined using SEER*Stat version 6.5.1. Rates were age-adjusted to the 2000 U.S. standard, trends were expressed as annual percentage change, and survival was calculated using the Kaplan Meier method.

RESULTS

We identified a total of 3,525 malignant PCNSLs, accounting for 0.002% of all reported non-Hodgkin’s lymphomas (NHLs) and 6.5% of all reported malignant brain tumors. The most frequently reported primary sites were the brain lobes (27.2%), brain-NOS (25.6%), nervous system-NOS (11.7%), and overlapping brain/CNS (11.5%). The average annual age-adjusted incidence rate of PCNSLs was 0.47/100,000 person-years (95% CI, 0.45–0.48) and has been significantly increasing over time by approximately 3% per year (p < 0.02). We observed PCNSL incidence to be 2.26 times greater in males than females (p < 0.001) and 1.14 times higher in blacks than whites (p < 0.02). PCNSL incidence increased with age, with average annual rates of 0.02/100,000 person-years for ages 0–19, peaking in those 65–74 years (1.32/100,000) and slightly declining for those aged 75+ (1.28/100,000). Over time, trends in incidence rates increased significantly in those aged 45+ years (p < 0.002). Five-year survival was 18.6%, with females having significantly better survival at 5 years than males (23.7% vs. 16.1%, respectively).

CONCLUSION

Incidence rate patterns by age and gender for PCNSL are similar to all NHLs. Increasing trends in incidence, especially among older ages, have been observed. Descriptive characteristics of PCNSL may provide clues to etiology.

Neuro-Oncol. 2009 Oct;11(5):585–586.

96. Mode of Delivery and Prior Pregnancy Losses as Risk Factors for Pediatric Central Nervous System Tumors: A California Cancer Registry Study

Sonia Partap, Julie Von Behren, Jane MacLean, Paul Fisher, Peggy Reynolds

INTRODUCTION

Birth characteristics may serve as surrogate markers for the etiology of childhood CNS tumors. Fifteen percent of human cancers are attributed to infections, and vaginal delivery has been considered a potential conduit for perinatal infection. Prior pregnancy loss has been used as a proxy for fetal genetic abnormalities, since 60% of pregnancy wastage has cytogenic aberrations. Past epidemiologic studies with limited power have examined both delivery and fetal loss as risk factors for childhood CNS tumors and yielded conflicting results.

METHODS

We obtained data on 4,560 cases of CNS cancers from the population-based California Cancer Registry. Cases included children ages 0–14 years diagnosed from 1988 through 2006 and matched via probabilistic record linkage to a California birth certificate. Four controls, matched on gender and birth date, were selected from state birth files for each case. We assessed cases and controls for mode of delivery and fetal miscarriages as recorded on birth certificates.

RESULTS

We found no statistically significant association between delivery method and CNS tumors overall (OR = 1.05; 95% CI, 0.95–1.15) or within tumor subtype, nor did we find any relationship between CNS tumors and one prior pregnancy loss or multiple miscarriages before 20 weeks gestation. Offspring in mothers who had two or more fetal losses after 20 weeks of gestation had an increased risk for CNS tumors (OR = 3.13; 95% CI, 1.32–7.41), and a 10-fold risk for high-grade glioma (OR = 10.10; 95% CI, 1.02–99.60).

CONCLUSION

In this very large study of pediatric CNS tumors, we found no relationship between the mode of delivery and CNS tumors. There was no association with a maternal history of one pregnancy loss; however, having two or more pregnancy losses at greater than 20 weeks gestation increased the risk of childhood CNS tumors, especially high-grade glioma.

Neuro-Oncol. 2009 Oct;11(5):586.

97. Association of Pediatric Brain Tumors with Horseshoe Kidneys

Jeffrey Carlton Murray, Carlos A Galliani, Amy Rowell, Daniel Oshman, John H Honeycutt, David J Donahue, Richards Roberts

INTRODUCTION

Brain tumors are the most common childhood solid tumor. The exact oncogenic mechanisms for most tumor types remain elusive, despite known gene mutations for some types and the occasional association with recognized genetic syndromes. Horseshoe (HS) congenital fusion malformations occur with the lung, adrenal glands, and kidneys. The HS kidney is the most common HS configuration. We have observed several children with brain tumors having HS kidneys, discovered incidentally via spine MRI performed as part of neuraxis metastatic evaluations.

METHODS

We performed an institutional review board-approved review of our brain tumor registry from 1998 to the present and queried our diagnostic imaging department’s database, cross-referencing for all coincident HS kidney and brain tumor diagnoses. We abstracted records for age at diagnosis, gender, tumor histology, and other anomalies.

RESULTS

Six patients with a coexisting brain tumor and HS kidney were identified: 1–19 years old; four males, two females; four medulloblastoma, one midline cerebellar hemangioma, and one pineal germ cell tumor. Other identified features included prematurity, club foot, atypical cystic lung and HS kidney changes, macrocephaly, and hypotonia.

CONCLUSION

The coexistence of brain tumors and HS kidneys in children has not been reported from large pediatric cancer registries. While there is no unifying explanation for midline visceral fusion anomalies such as HS kidney, defects in HOX or other homeobox genes, key to caudal-rostral visceral development and anatomic organ positioning, may be explanatory. HOX genes have also been implicated in malignant transformation. Also, there may be “genetic overlap” between malformation phenomena and pediatric tumor development. Having two rare midline conditions simultaneously in our reported cohort, we postulate that there may be a causal relationship (HOX defect?). Our observation warrants further cancer registry analysis so the association between brain tumors and visceral malformations can be better understood.

Neuro-Oncol. 2009 Oct;11(5):586.

98. Embryonic Stem Cell (ESC)-Mediated Gene Delivery Induces Growth Suppression, Apoptosis, and Radiosensitization and Overcomes Temozolomide Resistance in Malignant Gliomas

Luni Emdad, Mahmud Uzzaman, Zulekha Qadeer, Isabelle M Germano

INTRODUCTION

Malignant gliomas are aggressive tumors currently lacking effective treatment modalities. Adenovirus-mediated gene transfer shows a promising approach to proapoptotic gene delivery. However, lack of tissue penetration is currently a limiting factor. We recently published a new method of gene delivery using embryonic stem cell (ESC)-derived astrocytes. The aim of this study is to assess the proapoptotic effects of ESC-mediated transgene mda-7/IL-24 delivery on malignant gliomas in vitro as a single therapy and in combination with ionizing radiation (IR) and temozolomide (TMZ).

METHODS

ESC-derived astrocytes were engineered to express tet-inducible mda-7/IL-24. RT-PCR and ELISA were used to quantify mda-7/IL-24 expression. Malignant glioma cells U87 and T98G were used for trans-well and coculture experiments. Doxycycline (Dox) was used for gene induction. In another set of experiments, glioma cells were also exposed to IR (6 Gy) and TMZ (100 μM). Cell viability and apoptosis were detected by colony formation, MTT, TUNEL, and FACS analysis. Western blot analysis was performed to detect changes in signaling pathways following treatment with various agents.

RESULTS

ESC-mediated transgene mda-7/IL-24 delivery resulted in a significant increase in apoptotic rate in both glioma cell lines 24 h after Dox induction (p < 0.05). This increase was not observed when Dox was omitted. Cotreatment with IR and TMZ increased the apoptotic rate in both cell lines. Triple therapy resulted in a significant decrease in colony survival compared with each treatment alone or in combinations of two. Signaling analysis of triple therapy showed increased expression of GADD 153 and JNK activation with concomitant decrease of the antiapoptotic gene Bcl-xl.

CONCLUSION

Our data suggest that ESC-mediated mda-7/IL-24 transgene delivery can be used to augment the apoptotic effects of conventional treatment for malignant brain tumors. These results suggest that ESC-derived astrocytes could be a promising tool for adjuvant treatment of malignant gliomas.

Acknowledgements

The present research was supported by a grant from the National Institutes of Health and the National Cancer Institute (R01 CA129489-01A1).

Neuro-Oncol. 2009 Oct;11(5):586.

99. Mesenchymal Stem Cells Display Tumor-Specific Tropism in a Spontaneous Brain Tumor Model

Tiffany A Doucette, Yuhui Yang, Joy Gumin, Naoki Shinojima, Seok-Gu Kang, Wei Zhang, Ganesh Rao, Frederick F Lang

INTRODUCTION

Bone marrow-derived mesenchymal stem cells (MSCs) are effective vehicles for systemic delivery of therapy because they exhibit tumor-specific tropism. However, the tropism of MSCs for gliomas has been demonstrated in orthotopic xenografts in immunodeficient mice or syngeneic rat models, neither recapitulating human gliomas well. In contrast, the RCAS/tv-a system results in spontaneous gliomas in immu-nocompetent mice better representing human disease. We hypothesized that MSCs are capable of localizing to tumors using the RCAS/tv-a mouse model of anaplastic oligodendroglioma (AO).

METHODS

Newborn Ntv-a mice were injected in the right frontal lobe with RCAS-PDGFB and RCAS-IGFBP2 to induce AOs. MSCs were harvested from the long bones of Ntv-a mice. After verification as MSCs, the cells were transduced with adenovirus carrying the luciferase gene and injected intraarterially into mice (105 to 106 cells/100 μl) at 6–10 weeks of age. Bioluminescent imaging (BLI) was used to track MSCs by treating the animals (n = 9) with luciferin (150 mg/ml i.p.) and imaging after MSC injection. Following imaging, the brains were removed and the location of the MSCs was correlated with the location of the tumor.

RESULTS

After intraarterial injection, BLI revealed MSCs in the right frontal lobe in five mice by day 1 of imaging. No MSCs were localized to the left frontal lobe or posterior fossa. On histological examination, tumors were detected within the right frontal lobe in all five mice and were correlated with the location of the MSCs. In contrast, MSCs were not detected by BLI in the remaining mice. Histological analysis revealed that none of these animals had developed tumors. Therefore, MSCs were only detected in tumor-bearing mice.

CONCLUSION

Using the RCAS/tv-a system, our results show that MSCs are capable of localizing to spontaneously forming gliomas in immunocompetent mice. These results provide strong support for the use of engineered MSCs for targeted delivery of therapeutic agents to brain tumors.

Neuro-Oncol. 2009 Oct;11(5):586–587.

100. The Green Tea Extract EGCG (Epigallocatechin Gallate) Enhances Temozolomide Cytotoxicity in Malignant Gliomas

Weijun Wang, Encouse B Golden, Walavan Sivakumar, Florence M Hofman, Axel Schonthal, Stan G Louie, Thomas C Chen

INTRODUCTION

Temozolomide (TMZ) is currently the chemotherapeutic standard of care for malignant glioma patients. We have recently demonstrated that TMZ induces upregulation of glucose-regulated protein-78 (GRP78) expression in malignant gliomas, leading to chemoresistance. Epigallocatechin gallate (EGCG), the main constituent of green tea, has been demonstrated to functionally inactivate GRP78. We hypothesize that EGCG, by inhibiting GRP78 activity, would enhance TMZ cytotoxicity in vitro and in vivo.

METHODS

Human glioma cells (U87, U251, LN229) were treated with EGCG in the presence or absence of TMZ. Cytotoxicity studies were performed using colony formation assays (CFA). Western blots were used to correlate GRP78 levels and the proapoptotic protein CHOP/GADD-153 with cytotoxicity in the presence or absence of TMZ. Survival studies were performed using U-87 or U-251 glioma cells implanted in the right frontal lobe of nude mice. Kaplan-Meier method was used to construct a survival curve for the mice treated with TMZ alone, EGCG alone, and TMZ with EGCG.

RESULTS

EGCG by itself did not cause any glioma cell death; however, EGCG additively/synergistically enhanced the cytotoxic effects of TMZ in CFA. The mechanism of this additive/synergistic effect was determined to be secondary to down-regulation of GRP78 in glioma cells, with induction of CHOP. Inhibition of CHOP expression with siRNA reduced the cytotoxic effects of TMZ in glioma cells in vitro. In vivo studies demonstrated that EGCG in combination with TMZ significantly prolonged the survival of mice bearing intracranial U-87 or U-251 glioma xenografts compared with mice treated with TMZ or EGCG alone.

CONCLUSION

EGCG can be used to block the cytoprotective effects of GRP78, thereby enhancing the cytotoxic effects of TMZ in the treatment of glioblastomas.

Neuro-Oncol. 2009 Oct;11(5):587.

101. Intratumoral Administration of Bevacizumab (Avastin): A Novel Approach to Glioblastoma Therapy

Walavan Sivakumar, Weijun Wang, Encouse B Golden, Axel Schonthal, Stan G Louie, Florence M Hofman, Thomas C Chen

INTRODUCTION

Glioblastoma multiforme (GBM) patients have a poor prognosis despite advances in surgery, radiation therapy, and chemotherapy. In recurrent GBM, a common second-line therapy is bevacizumab (Avastin, an inhibitor of vascular endothelial growth factor [VEGF]) with irinotecan (Camptosar, a topoisomerase I inhibitor). Despite a promising initial response, cessation or resistance to bevacizumab treatment results in an explosive recurrence of GBM that is even less responsive to treatment. Long-term intravenous bevacizumab use is limited due to systemic side effects, including poor wound healing, renal complications, and potential intracranial hemorrhage. We sought to determine whether bevacizumab could be administered safely and efficaciously via continuous metronomic intratumoral delivery, with and without irinotecan, in xenograft intracranial glioma models.

METHODS

Right frontal lobes of nude mice were injected with bevacizumab to determine whether intraparenchymal hemorrhage occurred. Forty-eight hours after injection, mice were euthanized, and their brains were harvested to determine whether intratumoral hemorrhage occurred. Intracranial tumors were formed by stereotactic injection of U-87 glioma cells into the right frontal lobe of nude mice. Alzet pumps containing bevacizumab (two doses of 10 mg/kg) were implanted, where the needle was inserted directly into the tumor and the entire dose was administered over 4 weeks. Mice were divided into four treatment groups: PBS, intratumoral bevacizumab, intraperitoneal irinotecan, and a combination of intratumoral bevacizumab and intraperitoneal irino-tecan.

RESULTS

Intratumoral delivery of bevacizumab did not cause intratumoral or intraparenchymal hemorrhage. CD-31 (endothelial cell marker) and VEGF staining were significantly decreased in intratumoral bevacizumab-treated tumors compared with controls, demonstrating anti-angiogenesis. Mice treated with intratumoral bevacizumab in combination with irinotecan survived significantly longer (p < 0.05) than mice treated with intratumoral bevacizumab, i.v. bevacizumab or irinotecan alone, or i.v. bevacizumab in combination with irinotecan.

CONCLUSION

Intra-tumoral bevacizumab delivery is well tolerated, does not induce hemorrhage, inhibits angiogenesis, and prolongs survival when given alone; when combined with irinotecan, intratumoral bevacizumab delivery has an even greater effect on survival in an intracranial glioma xenograft model.

Neuro-Oncol. 2009 Oct;11(5):587.

102. Vicrostatin, a Recombinant Monomeric Disintegrin, Inhibits Glioma Cell Function In Vitro

Walavan Sivakumar, Don J Phillips, Stephen Swenson, Radu Minea, Francis S Markland, Thomas C Chen

INTRODUCTION

The prognosis associated with glioblastoma multiforme (GBM) is dismal and has failed to improve significantly in the past 50 years. Much of this prognosis is due to GBM’s invasive nature, since the tumor typically has infiltrated into normal brain by the time of diagnosis. Our group has worked on the antiinvasion molecule, contortrostatin (CN), a novel disintegrin purified from the copperhead snake; CN specifically inhibits GBM invasion by binding to the vimentin receptors avb3 and avb5, which are overexpressed on the invading glioma cell surface. Inhibition of these receptors prevents binding to the extracellular matrix, preventing glioma cell migration. Recently, we synthesized vicrostatin (VN), a recombinant monomeric disintegrin similar to CN. In this study, we examined the functional activity of VN for inhibiting glioma cell invasion and compared its activity with native CN.

METHODS

We studied human glioma cell lines: U87, U251, A172, and LN229. We used the MTT colorimetric assay, migration scratch assay, and in vitro invasion assay to evaluate VN’s direct cytotoxicity and its ability to inhibit glioma migration and invasion compared with CN.

RESULTS

MTT studies demonstrated that VN does not have any direct cytotoxicity after 48 h on glioma cells at concentrations ranging from 0 to 1 μM. Migration scratch assays showed a comparable inhibition of migration at 24 h between VN and CN in U87 and A172 cells. The antiinvasive effect of VN on glioma cells was investigated using Boyden chambers with Matrigel-coated filters. VN-treated cells were less invasive than untreated cells at all concentrations tested, and inhibition was dose dependent. VN-treated cells showed patterns of inhibition similar to CN-treated cells.

CONCLUSION

VN was capable of inhibiting glioma cell function in all of the completed in vitro assays. Activity of VN was similar to CN throughout all studies, validating VN as a potential novel antiinvasion therapy for GBMs; in vivo studies are currently under way.

Neuro-Oncol. 2009 Oct;11(5):587.

103. Chloroquine Enhances the Cytotoxic Effects of Temozolomide in Glioblastoma Cells by Blocking Autophagy

Encouse B Golden, Hee-Yeon Cho, Stan G Louie, Florence M Hofman, Axel Schonthal, Thomas C Chen

INTRODUCTION

Recently, chloroquine (CQ) in combination with conventional glioblastoma therapy was reported to be beneficial to patients with newly diagnosed disease by prolonging their median time of survival from 11.4 to 25 months. However, the mechanism by which CQ enhances the therapeutic efficacy of temozolomide (TMZ) is poorly understood.

METHODS

Cytotoxicity was determined by colony formation assay (CFA). Relative amounts of protein were determined via Western blot analysis with specific antibodies to glucose-regulated protein 78 (GRP78, a marker of endoplasmic reticulum [ER] stress), beclin (a marker for autophagosome formation), LC3B-II (a marker for autophagosome levels), CHOP/GADD-153 (an ER stress proapoptotic protein), and cleaved PARP (a marker for apoptosis). A targeted knock-down of protein expression was implemented with siRNA. CHOP/GADD-153 induction in tumor tissue from drug-treated animals was determined by immunohistochemical analysis.

RESULTS

Inhibition of autophagy with CQ enhanced TMZ cytotoxicity in vitro and in vivo. CQ blocked autophagosome degradation and induced the expression of CHOP/GADD-153 in glioma cells, thereby promoting apoptosis. A knock-down of GRP78 or treatment with 3-MA blocked autophagy and enhanced TMZ cytotoxicity in CFAs. However, a knock-down of beclin did not enhance TMZ cytotoxicity, suggesting that other mechanisms could be involved also. When CQ was combined with TMZ to treat glioma cells, LC3B-II, CHOP/GADD-153, and cleaved PARP increased compared with untreated cells or cells treated with individual drugs. Furthermore, these molecular mechanisms appeared to take place in vivo as well. Subcutaneously implanted U87MG tumors from mice treated with CQ combined with TMZ displayed higher levels of CHOP/GADD-153 than did untreated tumors or tumors treated with individual drugs.

CONCLUSION

Our results demonstrate that CQ enhances TMZ therapy via its ability to block autophagy and trigger ER stress, which results in increased chemosensitization of glioma cells.

Neuro-Oncol. 2009 Oct;11(5):587.

104. Improved Antitumor Effects with Chloroquine Analogs via Inhibition of Autophagy

Encouse B Golden, Hee-Yeon Cho, Stan G Louie, Florence M Hofman, Axel Schonthal, Thomas C Chen

INTRODUCTION

Recently, chloroquine (CQ), an inhibitor of autophagy, has shown clinical benefit in the treatment of glioblastoma patients. However, CQ has limited CNS penetration. Therefore, we evaluated whether other quinoline-based antimalarial compounds, such as quinine (QN), hydroxychloroquine (HCQ), quinacrine (QNX), and meflo-quine (MFQ), with improved CNS penetration compared with CQ, would be able to induce similar apoptotic activity in CQ using in vitro assays.

METHODS

Cytotoxicity of the various compounds was determined by short-term MTT and long-term colony forming assays. Protein markers of autophagy and cell death were determined via Western blot analysis.

RESULTS

We found that all of the quinoline-based antimalarials tested displayed similar antitumor activity compared with CQ in several glioblastoma cell lines. QNX was the most potent compound, followed by MFQ > HCQ > CQ > QN. All of the compounds tested were able to block autophagy, indicated by an increase in the conversion of LC3B-I to LC3B-II (a marker for autophagosome accumulation) and an increase in the levels of polyubiquitinated proteins. In addition, an increase in CHOP/GADD-153 (an ER stress proapoptotic protein) and PARP cleavage (a marker for apoptosis) revealed that apoptotic cell death ensued. Importantly, these cytotoxic effects were also discovered in TMZ-resistant glioma cell lines, indicating that the compounds tested were able to overcome drug resistance. Finally, the antitumoral activity of the most potent compound, QNX, correlated with its accumulation in lysosomes and the resultant inhibition of autophagy.

CONCLUSION

The quinoline-based antima-larial agents presented here represent a novel class of autophagy inhibitors that demonstrate the ability to accumulate in lysosomes and block autophagosome degradation, thereby inducing apoptosis in glioma cells. This finding suggests that we have a novel class of autophagy inhibitors that may have clinical utility.

Neuro-Oncol. 2009 Oct;11(5):588.

105. Bevacizumab Decreases Tumor Delivery of [3H]Methotrexate in a Malignant Glioma Model

Eric M Thompson, Leslie L Muldoon, Michael Pagel, Edward A Neuwelt

INTRODUCTION

In patients with recurrent malignant gliomas, bevacizumab plus several chemotherapeutics has demonstrated improved progression-free survival, overall survival, and imaging responses compared with chemotherapy alone. Bevacizumab induces tumor blood vessel “normalization,” which is thought to improve chemotherapy delivery to tumors. However, bevacizumab also may stabilize the blood-brain barrier, reducing chemotherapy delivery in the CNS. The primary objective of this preclinical study was to determine whether chemotherapy delivery into malignant gliomas is increased or decreased by pretreatment with bevacizumab.

METHODS

UW28 human malignant glioma cells were implanted stereotactically into the right caudate of male nude rats. Two weeks later, postcontrast T1-weighted and T2-weighted magnetic resonance images were obtained to verify tumor presence. Twelve animals were then randomized into control and treatment (24-h pretreatment with 10 mg/kg bevacizumab i.v.) groups. All rats received [3H]methotrexate (25 μCi i.v.) plus 140 mg/kg methotrexate i.v. Ten minutes after radiotracer injection, the animals were perfused and sacrificed. The brains were grossly dissected into four areas: tumor, brain adjacent to tumor, brain distal to tumor, and left hemisphere. Radioactivity from each area was measured by scintillation counter.

RESULTS

The difference of [3H]methotrexate delivery among the four brain locations was significant (p = 0.0244). The total amount of [3H]methotrexate delivered in the bevacizumab group was lower than the control group but did not achieve significance (p = 0.154). In the tumor area, there was a suggestive but nonconclusive trend for decreased [3H]methotrexate delivery in the bevacizumab group compared with the control group (p = 0.0901).

CONCLUSION

The administration of bevacizumab 24 h prior to chemotherapy appears to decrease the delivery of [3H]methotrexate into the tumor compared with controls. However, a greater number of animals are needed to achieve statistical significance. We hypothesize that bevacizumab stabilizes the blood-brain barrier, causing decreased chemotherapy tumor delivery, and that patient response is secondary to other mechanisms.

Neuro-Oncol. 2009 Oct;11(5):588.

106. Chronic Dosing with MLN0518 (Tandutinib), A Small Molecule PDGFR Alpha/Beta Inhibitor, Reduces Tumor Hypoxia and Perfusion in C6 Glioma Xenografts

Jessica KR Boult, Simon P Robinson, Simon Walker-Samuel, Daniel P Bradley

INTRODUCTION

Glioblastoma multiforme (GBM) relies heavily on angiogenesis for growth and represents one of the most vascularized human tumors. MLN0518 (Tandutinib) is a type III receptor tyrosine kinase inhibitor (RTKi) with activity against PDGFR α/β, FLT3, and c-KIT in the submicromolar range. MLN0518 is currently in phase I/II trials as a monotherapy and in combination with bevacizumab for recurrent GBM. A phase I study of tandutinib use in patients with newly diagnosed GBM is planned.

METHODS

C6 xenografts were grown subcutaneously in nude mice (n = 14); when these tumors reached 200 × 100 mm3, they were stratified to receive either vehicle or 20 mg/kg MLN0518 subcutaneously twice a day for 10 days. At the study’s end, tumors were excised and the total endothelial cell concentration (CD31), degree of perfused vasculature (Hoechst 33342), and hypoxia (pimonidazole) were quantified by immunohistochemistry and fluorescence microscopy.

RESULTS

Tumors in mice treated with MLN0518 grew slower than vehicle-treated tumors (volume-doubling time 6.3 ± 0.6 days vs. 4.7 ± 0.3 days, p < 0.05). CD31 staining revealed an endothelial cell area in the treated tumors that was 21% lower than the vehicle. Analysis of the distribution of staining demonstrated that the percentage of the total vessels perfused at the time of Hoechst 33342 injection was 24.2% lower in MLN0518 than in vehicle-treated tumors (p < 0.05), which together equated to a 41% reduction in an overall perfused vessel area in treated versus control tumors (p < 0.05). Finally and most strikingly, MLN0518-treated tumors were 23.4% less hypoxic than were control tumors as assessed by pimonidazole adduct formation (p < 0.05).

CONCLUSION

MLN0518 chronically limits the growth of C6 glioma xenografts and reduces both the mean perfused vessel fraction and hypoxic area. Tumor hypoxia is well known for promoting a more metastatic, malignant, angiogenic, and chemo-/radio-resistant lesion. Thus, MLN0518 is a promising agent for the treatment of well-vascularized tumors such as GBM as a monotherapy and in combination with other antitumor agents.

Neuro-Oncol. 2009 Oct;11(5):588.

107. New Anticancer Etoposide and Doxorubicin Derivatives with Increased Brain Penetration for the Treatment of Brain Cancers

Reinhard Gabathuler, Michel Demeule, Christian Che, Gaogiang Yang, Anthony Regina, Richard Beliveau, Jean-Paul Castaigne

INTRODUCTION

The blood-brain barrier (BBB) is mainly formed by brain capillary endothelial cells, which are sealed closely by tight junctions and are expressing high levels of active efflux transport proteins. As a result, the overwhelming majority of small molecules, proteins, and peptides do not cross the BBB. In the present study, we provide experimental evidence that a new engineered peptide compound (EPiC) platform provides a noninvasive and flexible manner to create new drugs that have access to the CNS using the low-density lipoprotein receptor-related protein (LRP).

METHODS

The lead peptide was evaluated in vivo by in situ brain perfusion in mice. Angiopep-2 peptide (An2) was detected very rapidly in the brain parenchyma. Using An2 properties, we created a portfolio of new drug entities composed of chemotherapeutics, siRNA, peptides, and mAbs. These new compounds were evaluated for in vitro cytotoxic activity and for brain uptake in vivo using in situ mice brain perfusion and mice xenograft tumor models.

RESULTS

Our data reflect the brain uptake of new chemical entities synthesized from etoposide and doxorubicin. In vitro, both Etop-An2 and Doxo-An2 derivatives inhibited cancer cell proliferation. Furthermore, we showed by mice in situ brain perfusion that these new etoposide (Etop-An2) and doxorubicin (Doxo-An2) derivatives enter the brain at a rate 20- to 100-fold higher than unmodified drugs. Interestingly, these derivatives also bypass the drug efflux pump P-gp at the BBB. Finally, in vivo studies using xenograft tumor models showed that both new drugs inhibit tumor growth and i.v. administration of the Etop-An2 results in 20- to 50-times higher distribution in the brain and brain tumor than with etoposide.

CONCLUSION

In conclusion, these data indicate that the modification of drugs using our EPiC platform enhances their entry into the brain and validates the use of Angiochem’s technology for the treatment of brain diseases.

Neuro-Oncol. 2009 Oct;11(5):588.

108. Irreversible Electroporation for Intracranial Surgery: A Pilot Study in a Canine Model

Amritraj Ganesh Loganathan, Thomas L Ellis, John H Rossmeisl, Paulo A Garcia, John Robertson, Rafael V Davalos

INTRODUCTION

Irreversible electroporation (IRE) is a new technique used to ablate tissue using short, intense electric pulses. This technique is unique because the pulses can be adapted to destroy cells without thermally damaging the extracellular matrix, neurons, and major blood vessels. The volume of ablated tissue can be predicted with numerical modeling and can be confirmed with imaging such as ultrasound and MRI. IRE has been shown to successfully ablate cancer cells in vivo but has not been shown previously to treat brain cancer. We present preliminary data on the safety of using IRE for canine brain surgery.

METHODS

Three healthy canines underwent general anesthesia and craniectomy to expose their temporal lobes. An IRE lesion was created using a blunt-tip electrode. Postoperative MRI was obtained immediately after and 48 h after the surgery. The animals were killed and the brains harvested for histological analysis.

RESULTS

The zone of ablation was visible on intraoperative ultrasound and postoperative MRI. Using 3-D modeling software, we correlated the volume computed from the reconstructed lesion with the volume of brain exposed to a given electric-field strength. In doing so, we were able to calculate the electrical parameters necessary for focal ablation of brain tissue and to provide guidelines for successful treatment planning and execution. Histopathological analysis confirmed a sharp demarcation between the zone of ablation and normal brain.

CONCLUSION

This pilot study demonstrates the first use of IRE for intracranial surgery. IRE offers advantages over traditional surgical techniques, including its minimal invasiveness, coupled with its adaptability. Additionally, IRE generates little heat and can spare major blood vessels. These initial results are the first step towards using IRE as a treatment for brain cancer. Future efforts using IRE may focus on delivery of targeted chemotherapeutic or antineoplastic agents within electroporated areas of brain.

Neuro-Oncol. 2009 Oct;11(5):588–589.

109. Human Bone Marrow-Derived Mesenchymal Stromal Cells Expressing S-Trail: A Cellular Delivery Vehicle for Human Glioma Therapy

Lata G Menon, Kathleen M Kelly, Hongwei Yang, Seung-Ki Kim, Peter Mclaren Black, Rona S Carroll

INTRODUCTION

Glioblastoma (GBM) is among the most aggressive and treatment-resistant of all human cancers. Conventional therapeutic approaches are unsuccessful because of the diffuse infiltrative invasion of glioma tumor cells into the normal brain parenchyma. Stem cell-based therapies provide a promising approach for the treatment of malignant gliomas because of their migratory ability to invade tumor cells.

METHODS

We have used human bone marrow-derived mesenchymal stromal cells (hMSCs) as cellular vehicles for the targeted delivery and local production of the biologic agent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) at the glioma tumor site. hMSCs were transduced with a lentivirus expressing secretable TRAIL (hMSC S-TRAIL) and mCherry (red fluorescent protein). For the in vivo assessment of therapeutic efficacy, hMSCs were injected ipsilaterally into an established intracranial glioma tumor in a mouse xenograft model.

RESULTS

Our results demonstrated clearly the retention of tumor tropic ability, biologic activity, and migratory ability of hMSC S-TRAIL cells in vitro and in vivo. Genetically engineered hMSC S-TRAIL cells were effective in inhibiting intracranial U87 glioma tumor growth (81.6%) in vivo, which resulted in a significant increase in animal survival. Immunohistochemical studies demonstrated a significant, 8-fold increase in tumor cell apoptosis in the hMSC S-TRAIL-treated group compared with controls.

CONCLUSION

In summary, our studies confirm that hMSCs expressing S-TRAIL serve as an excellent cellular local drug delivery system for the tumor site-specific targeted delivery and therapeutic efficacy in an orthotopic glioma tumor model. Based on our studies, we hypothesize that hMSCs expressing S-TRAIL represent an effective and novel approach for local treatment of human malignant gliomas.

Neuro-Oncol. 2009 Oct;11(5):589.

110. A Local Cell-Based Delivery System for Human Gliomas using Human Bone Marrow-Derived Mesenchymal Stromal Cells

Lata G Menon, Myriam Armant, Christian Badr, Tannous A Bakhos, Hongwei Yang, John Pratt, A Gregory Sorensen, Peter Mclaren Black, Rona S Carroll

INTRODUCTION

Malignant gliomas are the most common type of primary brain tumor, of which glioblastoma multiforme (GBM) is the most aggressive. Potential therapies fail due to the infiltrative manner in which GBM cells invade the normal brain parenchyma. Attempts to use human mesenchymal stromal cells (MSC) as a cellular delivery vehicle have combined the tumor tropic ability and the ease of genetic modification. We have modified MSC to express yeast cytosine deaminase (CD); CD is a microbial enzyme that converts the nontoxic prodrug (5-FC) to the highly cytotoxic 5-fluorouracil (5-FU).

METHODS

Sensitivity to 5-FU and 5-FC, in vitro and in vivo migration assays, bioactivity and coculture assays for CD activity, and in vivo therapeutic efficacy of MSC transduced with a lentivirus expressing CD (MSC-CD) were studied. For in vivo tracking of migration, Feridex-labeled hMSC were injected contralateral to the tumor and migration followed by MRI. For therapeutic efficacy studiesC-CD cells were injected ipsilaterally into the established intracranial glioma in an orthotopic mouse xenograft model or intratumorally into a subcutaneous model. Five days later, mice were treated with 5-FC (500 mg/kg by weight) for 10 days.

RESULTS

Our results demonstrate the retention of the tumor tropic ability of MSC-CD cells by in vitro and in vivo migration assays. In vitro assays confirmed the expression and retention of the biological activity of CD released. A dose-response increase in U87 cell death was observed after coculture with increasing numbers of MSC-CD cells and 5-FC (100 μg/ml). In the orthotopic model, 80% of untreated mice developed a tumor, while only 20% developed a tumor in the treated groups.

CONCLUSION

Our study demonstrates the therapeutic efficacy of MSC-CD cells and confirms that MSC can serve as a powerful cell-based delivery vehicle for the site-specific release of prodrug enzyme.

Neuro-Oncol. 2009 Oct;11(5):589.

111. CXCR7 Regulates Glioma Cell Proliferation, Migration, Invasion, and Tumor Development

Yang Liu, Eleanor B Carson-Walter, Melissa Whiteman, Kevin A Walter

INTRODUCTION

Chemokines and their receptors play critical roles in many physiological and pathological processes, including cancer, and exert their effects through multiple mechanisms. CXCL12/SDF-1 and its receptor, CXCR4, contribute to the growth of primary tumors as well as progression to metastatic disease. Recently, it has been reported that CXCR7 serves as a second receptor for CXCL12, exerting an important role in tumor vascular formation and promoting the growth of breast, lung, and prostate cancers. However, the functional contribution of CXCR7 to malignant brain tumors has not been explored.

METHODS

We used serial analysis of gene expression (SAGE) to identify CXCR7 as a glioma endothelial marker gene. We examined cellular localization of CXCR7 in GBM specimens using in situ hybridization and immunohistochemistry. We suppressed expression of CXCR7 in U251MG GBM cells using RNAi and measured the results using XTT proliferation assays, migration assays, and transwell invasion assays. We examined the effects of CXCR7 inhibition on in vivo growth using U251MG intracranial xenografts.

RESULTS

CXCR7 mRNA was expressed at levels nine times higher in brain tumors than normal brain samples by SAGE analysis and was localized to vascular regions within glioma samples. Inhibition of CXCR7 expression significantly impeded glioma cell proliferation, migration, and invasion in vitro. In vivo, siRNA-mediated knockdown of CXCR7 expression in U251MG cells significantly inhibited intracranial xenograft growth and improved mouse survival. Preliminary results with a small molecule inhibitor of CXCR7 supported these results.

CONCLUSION

CXCR7 may play an important role in promoting GBM growth, migration, and invasion in vitro and in vivo, implying that it is a potential therapeutic target for the treatment of malignant glioma.

Neuro-Oncol. 2009 Oct;11(5):589.

112. Glioma Therapy using Second-Generation HIF-Activated Adenoviruses with Improved Replication, Oncolytic, and Antitumor Efficacy

Tiffani Cherry, Sharon L Longo, David Padalino, Dawn E Post

INTRODUCTION

HYPR-Ad#1 and HYPR-Ad-IL4 are targeted oncolytic adenoviruses that specifically kill hypoxic/HIF-active tumor cells. The interleukin-4 (IL4) adjuvant therapeutic gene was chosen because of its ability to inhibit tumor angiogenesis and induce a host antitumor immune response. Published studies with HYPR-Ad#1 and HYPR-Ad-IL4 have demonstrated that treatment with a HIF-activated oncolytic Ad can lead to a measurable therapeutic response. However, these viruses exhibited attenuated viral replication, oncolytic, and cellular egress activity compared with a wild-type adenovirus in vitro.

METHODS

To improve the therapeutic efficacy of the HYPR-Ads, we created HIF-Ad and HIF-Ad-IL4 using an alternative Ad5 genome backbone plasmid (pAd329). The use of pAd329 introduced two important changes: (i) a modified HIF-responsive promoter to regulate the Ad E1A replication gene and (ii) insertion of the Ad E3 gene region.

RESULTS

In vitro studies demonstrated the conditional activation of HIF-Ad and HIF-Ad-IL4 by hypoxia. These viruses conditionally expressed E1A and IL4 under hypoxia. HIF-Ad and HIF-Ad-IL4 exhibited a 6- to 17-fold increase in viral replication under hypoxia compared with the HYPR-Ads. The rate and magnitude of hypoxic tumor cell death following HIF-Ad and HIF-Ad-IL4 infection was greater than that observed with HYPR-Ad#1 and HYPR-Ad-IL4. Finally, HIF-Ad and HIF-Ad-IL4 exhibited a 97- to 870-fold increase in cellular egress under hypoxia compared with the HYPR-Ads. Pilot antitumor efficacy studies demonstrated that HIF-Ad treatment led to a 2-fold reduction in tumor growth, which was equivalent to reduction seen with a wild-type adenovirus (Ad5WT). HIF-Ad-IL4 treatment led to tumor regressions in all mice, and its antitumor efficacy was 6-fold greater than Ad5WT treatment. Finally, the antitumor activity of HIF-Ad-IL4 was superior to that obtained with HYPR-Ad-IL4.

CONCLUSION

These preliminary results demonstrate that HIF-Ad and HIF-Ad-IL4 have improved replication, oncolytic, and antitumor activity. These second-generation HIF-activated oncolytic Ads have the potential to be clinically efficacious and improve the survival of cancer patients.

Neuro-Oncol. 2009 Oct;11(5):589–590.

113. HSP90 Inhibitor NXD30001 Increased Survival of GL261 Glioma-Bearing Mice

Eugene Lukyanov, Ruihong Chen, Zahara M Jaffer, Nicolas Winssinger, Allan E Rubenstein, David Zagzag, Elizabeth W Newcomb

INTRODUCTION

Heat shock protein 90 (Hsp90) functions to maintain the conformation and activity of many oncoproteins and has thus become an attractive target for cancer therapy. Inhibitors of Hsp90 have demonstrated inhibition of cancer cell growth in various tumor models. Several inhibitors have been tested in clinical trials. We have shown that 17-AAG has preclinical efficacy in GL261 cells and GL261 gliomas. NexGenix Pharmaceuticals has developed a novel radicicol-based series of Hsp90 inhibitors, for which the prototype is NXD30001. NXD30001 efficiently depletes Hsp90 client oncoproteins, inhibits tumor cell growth in vitro and in vivo, readily crosses the blood-brain barrier, and accumulates in the brain. Taken together, these characteristics make NXD30001 an attractive therapeutic candidate for treatment of gliomas. Its use in treating brain tumors has not been explored; therefore, we have performed a pilot study to test its antitumor activity in the GL261 orthotopic glioma model.

METHODS

Female C57BL/6 mice were implanted intracerebrally with GL261 glioma cells (1 × 105). On day 14, animals were randomized into treatment groups (n = 5) as follows: 1) vehicle only and 2) 50 mg/kg of NXD30001. Drug or vehicle was administered i.p. three times a week. The mice were closely monitored for changes in body weight, behavior, and neurological status. Animals were killed when they became moribund, and their brains were harvested for histopathological analysis.

RESULTS

Treatment of established intracranial gliomas with NXD30001 significantly improved mice survival (p < 0.02). Median survival in controls was 21.5 days and in animals receiving 50 mg/kg of NXD30001 was 29 days. No drug-related or vehicle-related toxicities were observed.

CONCLUSION

Taken together, these pilot results suggest that NXD30001 warrants further study for its potential as a novel therapeutic agent for treatment of malignant gliomas.

Neuro-Oncol. 2009 Oct;11(5):590.

114. CDK Inhibitor Enhances Action of Temozolomide in Glioma Cells

Yuichi Hirose, Hirotoshi Sano

INTRODUCTION

Recent progress in chemotherapy for malignant gliomas was led by DNA-methylating agent temozolomide (TMZ); however, drug resistance is still a major problem. Previous studies revealed that TMZ induces prolonged G2/M arrest of human glioma cells followed by senescence-like phenomenon (in p53-wild type cells) or mitotic catastrophe (in p53-nonfunctional cells) and suggested that G2 checkpoint system might be linked to a DNA repair mechanism.

METHODS

We tested the effect of a cdk inhibitor flavopiridol (FP), which has been known to inhibit the action of cdc2 (cdk1), a key protein in G2 checkpoint pathway, on TMZ-treated U87MG human glioma cells. The drug toxicity and activation of cell-signaling proteins were evaluated by colony formation efficiency and immunoblotting, respectively. Furthermore, to analyze the action of cdk inhibitor in TMZ-resistant cells, we treated U87MG cells repeatedly with dose escalation to establish TMZ-resistant clones and used U87MG cells transfected with fusion protein of myristoylated Akt and murine estrogen receptor (U87MG-AktER cells), of which Akt activity can be enhanced by exogeneous treatment with estrogen analogue.

RESULTS

FP potentiated the cytotoxicity of TMZ at a lower concentration (<10 nM). This effect was clearly associated with suppression of key proteins at G2-M transition (Plk1, Aurora, and Pin1), with accumulation of the cells exclusively at the G2 phase, and with increased expression of γ-H2AX, a DNA double-strand break marker. FP also enhanced cytotoxicity of TMZ on U87MG-AktER cells with overactivated Akt. We established TMZ-resistant clones, which showed activation of G2 checkpoint in response to TMZ, and found that FP treatment resensitized these clones to TMZ.

CONCLUSION

Our results suggest that TMZ resistance could be promoted by enhanced DNA repair activity in G2-M transition and that cdk inhibitor could suppress this activity leading to potentiation of TMZ action on glioma cells.

Neuro-Oncol. 2009 Oct;11(5):590.

115. Enhanced Survival and Cure of F98 Glioma-Bearing Rats Following Convection-Enhanced Delivery of Carboplatin in Combination with X-Irradiation

Rolf Barth, Weilian Yang, Tianyao Huo, Nilendu Gupta, Michael Weldon, John Grecula, Julia Rousseau, Helene Elleaume

INTRODUCTION

The purpose of this study was to further evaluate the therapeutic efficacy of convection-enhanced delivery (CED) of carboplatin combined with 6 MV photon irradiation for treatment of the F98 glioma-bearing rat.

METHODS

One hundred three tumor cells were implanted into the brains of Fischer rats, and 13 or 17 days later, carboplatin (20 μg/10 μl) was administered by either CED over 30 min (0.33 μl/min) or by prolonged infusion using Alzet osmotic pumps (1 μg/μl/h for 7 days) beginning 6 days following implantation. As determined by MRI prior to treatment, the tumor volume was ~20–25 mm3 on day 13 and ~60–80 mm3 on day 17. Rats received a 15 Gy fractionated dose (5 Gy × 3) of 6 MV photons beginning on the day after drug administration. Other groups of rats received either carboplatin or X-irradiation alone.

RESULTS

Rats bearing small tumors, treated with carboplatin and X-irradiation, had a MST of 83.4 days following CED and 111.8 days following pump administration; 40% of the latter survived >180 days compared with 55.2 days for CED and 77.2 days for pump delivery of carboplatin alone. X-irradiated or untreated control rats had MSTs of 31.8 days and 24.2 days, respectively. The tumor carboplatin concentration following CED (20 μg in 10 μl) was 10.4 μg/g compared with 1.2 μg/g in normal brain and 0.3 μg/g in blood.

CONCLUSION

In the present study, we have demonstrated the superiority of combined chemotherapy and radiotherapy compared with monotherapy. Treatment of animals bearing small tumors yielded greater MSTs than animals with large tumors, and sustained delivery using Alzet pumps was more effective than short-term CED. Only a modest increase in MST was observed in animals that received temozolomide plus X-irradiation. These survival data, as well as those previously reported by us, are the best ever obtained with the F98 glioma model, and they should provide a platform for a phase I clinical trial. (Supported by the Grey Ribbon Crusade, the Musella Foundation, and the O.S.U. Department of Pathology.)

Neuro-Oncol. 2009 Oct;11(5):590.

116. IL-13 Receptor Targeted Quantum Dots for GBM Imaging

AB Madhankumar, Becky Slagle-Webb, Jonas Sheehan, James R Connor

INTRODUCTION

Glioblastoma multiforme (GBM) is an aggressive high-grade brain tumor that is highly infiltrative in nature. Because of GBM’s diffusive nature, it can be difficult to demark tumor tissue from normal tissue during surgery. Although MRI images can differentiate between the normal tissue and tumor tissue, the boundaries of the tumor are difficult to identify. GBM tumors are known to overexpress IL13Rα2, a receptor for IL-13. The purpose of the current work is to utilize quantum dots conjugated with IL-13 to target tumor cells and to visualize high-grade astrocytomas in a much more reliable manner, particularly to distinctly differentiate the tumor border from normal healthy tissue.

METHODS

Quantum dots, a semiconductor material composed of CdSe modified with ZnS and a polymer coating, were used. We conjugated recombinant human IL-13 protein to the quantum dots and characterized them for physico-chemical properties and cytotoxicity in the in vitro cell culture models. The binding affinity of the IL-13 conjugated quantum dots (IL-13QD) towards well-established glioma cells and tumor sections were investigated using fluorescent microscopy and flow cytometry techniques. Experiments are in progress to determine the in vivo tumor-targeting ability of IL-13QD in intracranial glioma tumor-bearing mice.

RESULTS

IL-13QD demonstrated significant binding affinity towards U251 and U87 human glioma cell lines. Binding experiments with several high-grade tumor sections revealed the selective uptake of IL-13QD compared with normal human cortex. IL-13QD had negligible cytotoxicity on U251 and U87 glioma cells up to a concentration of 10 nM.

CONCLUSION

The data suggest the possibility of using the targeted quantum dots as a diagnostic agent for identifying high-grade glioma tumors. Quantum dots that target glioma tumor receptors may serve as a marker for tumor boundaries when administered in vivo.

Neuro-Oncol. 2009 Oct;11(5):590.

117. Human Glioblastoma Stem Cells Targeted Through Treatment with Temozolomide and Gamma-Secretase Inhibitors

Alonzo H Ross, Candace Gilbert, Alicia Mihaliak, Li Li, Marie-Claire Daou, Richard Moser

INTRODUCTION

Gliomas possess a population of cancer stem cells (CSCs) resistant to therapy, which allow neurosphere formation, multi-lineage differentiation, and tumor initiation. Our laboratory found that although chemotherapy drastically reduces neurosphere formation, some cells enter a reversible quiescent state and form neurospheres after a short recovery period.

METHODS

Cell lines and primary cultures were grown as neurospheres for this study.

RESULTS

We investigated targeting the chemoresistant CSCs by combining temozolomide (TMZ) chemotherapy and γ-secretase inhibitors (GSIs) to suppress the Notch signaling pathway, which is activated in brain CSCs. Neurosphere cell lines and primary glioma cultures were treated with dimethyl sulfoxide (DMSO), GSI only, TMZ only, or TMZ + GSI. Treatment with GSI only had insignificant effects on neurosphere formation compared with control cultures. TMZ-only resulted in a significant decrease in neurosphere formation, but an additional week allowed for a 2- to 3-fold recovery in the number and size of neurospheres. Treatment with TMZ + GSI resulted in a similar decrease in initial neurosphere formation. However, after their second week in culture, TMZ + GSI cells showed no change in sphere number or size, demonstrating an inhibition of neurosphere recovery. To determine if the treated cultures retained self-renewal properties, the remaining spheres were analyzed for secondary sphere formation. TMZ-only cultures had a high secondary sphere formation, 2- to 10-fold greater than the number of recovery spheres. In contrast, TMZ + DAPT cultures had limited self-renewal capabilities, with up to a 6-fold decrease in sphere formation. TMZ + GSI cells had a 2-fold increase in senescence, demonstrated by β-galactosidase expression, compared with TMZ-only cultures. When treated cells were subcutaneously engrafted into immunodeficient mice, TMZ-only treatment increased tumor latency, but the tumor incidence was similar to DMSO and DAPT-only controls. Notably, TMZ + DAPT cells showed greatly reduced tumor formation.

CONCLUSION

These studies suggest that inhibiting the Notch signaling pathway in combination with chemotherapy can cause quiescent cells to transition into a senescence, thereby enhancing treatment.

Neuro-Oncol. 2009 Oct;11(5):591.

118. Effects of Kava Kava Compounds on Glioblastoma Multiforme (GBM)

Mariana Rossette, Courtney Dawson, Dongqin Zhu, Hannah Caldas

INTRODUCTION

In some kava-drinking countries in the South Pacific, an unbalanced proportion of brain cancer incidence exists between males and females. The lower incidence rates for males in these areas are thought to be associated with kava consumption, since kava drinking is traditionally only performed by males.

METHODS

We have utilized individual compounds of interest from kava extract (chalcones: flavokawain A and B; kavalactone: kawain) to determine the efficacy of these natural compounds at containing brain cancer cell growth.

RESULTS

We identified flavokawain B as a potent inhibitor of glioma cell growth through a mechanism different from that previously described for flavokawain A. Furthermore, we determined that kava chalcones typically associated with liver toxicity in kava preparations do not possess the same effect as kava chalcones in brain cancer cells.

CONCLUSION

Our data suggests that kava chalcones are an attractive compound to be applied in the treatment of GBM.

Neuro-Oncol. 2009 Oct;11(5):591.

119. Combined Anti-VEGF and Radiotherapy for Metastatic Brain Melanoma

Ilya G Lim, Iman Osman, David Zagzag, Eugene Lukyanov, Erik C Parker, Mary Louise Devitt, Daniel Santovasi, Elizabeth W Newcomb

INTRODUCTION

Patients with melanoma metastases to the brain have a dismal prognosis, as available treatment modalities are of limited clinical efficacy. Vascular endothelial growth factor (VEGF), a potent angiogenic factor, is reported to play an important role in the growth and metastatic progression of melanoma. In several tumor models, the addition of antiangiogenic treatment to radiotherapy has been shown to improve tumor response to treatment. The aim of our study was to assess the anti-tumor efficacy of anti-VEGF antibody (B20–4.1.1, Genentech), an antian-giogenic drug, in combination with fractionated whole brain radiotherapy (WBRT) in a B16-F10 melanoma brain metastasis model, with the end point being overall survival.

METHODS

Metastatic tumors to the brain were established by infusion of EGFP-B16-F10 mouse melanoma cells (5 × 104) into the left internal carotid artery of 55 female C57BL/6 mice. On day 4, the mice were randomized into treatment groups as follows: 1) no treatment (n = 27); 2) WBRT alone (n = 8) with a total dose of 30 Gy (10 × 3 Gy); 3) anti-VEGF antibody alone (n = 10) (5 mg/kg i.p. 2×/week); and 4) combination of WBRT and anti-VEGF antibody treatment (n = 10). Drug injection was started 3 days prior to the radiation treatment. Mice were killed when moribund.

RESULTS

Treatment with WBRT or anti-VEGF antibody alone significantly increased survival time. The median survival was 18 days for the untreated group, 26 days for WBRT alone, and 27 days for mice treated with anti-VEGF antibody (p < 0.0001). In the combination group, 70% of mice remained alive on day 42, after 6 weeks of anti-VEGF antibody treatment. The analysis for tumor size, location, invasiveness, vascular density, and extent of necrosis is in progress.

CONCLUSION

Our results indicate that the combination of anti-VEGF antibody with fractionated WBRT significantly improves survival in the B16-F10 melanoma brain metastases model. These promising results warrant further studies to explore this therapeutic strategy.

Neuro-Oncol. 2009 Oct;11(5):591.

120. Efficient Suicide Gene Therapy of Invasive, Cancer Stem-Like Glioblastoma Xenografts using Lentiviral Vectors

Hrvoje Miletic, Peter Huszthy, Tsanan Giroglou, Oleg Tsinkalovsky, Philipp Euskirchen, Kai Ove Skaftnesmo, Rolf Bjerkvig, Dorothee von Laer

INTRODUCTION

Glioblastoma is the most frequent and most malignant primary brain tumor with a poor prognosis. The translation of therapeutic strategies for glioblastoma from the experimental phase into the clinic has been limited by insufficient animal models, which lack important features of human tumors. Lentiviral gene therapy is an attractive therapeutic option for human glioblastoma, which we validated in a clinically relevant animal model.

METHODS

We used a rodent xenograft model that recapitulates the invasive and angiogenic features of human glioblastoma to analyze the transduction pattern and therapeutic efficacy of lentiviral pseudotyped vectors.

RESULTS

Both lymphocytic chorio-meningitis virus glycoprotein (LCMV-GP) and vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors very efficiently transduced human glioblastoma cells in vitro and in vivo. In contrast, pseudo-typed γ-retroviral vectors, similar to those evaluated for clinical therapy of glioblastoma, showed inefficient gene transfer in vitro and in vivo. Both pseudotyped lentiviral vectors transduced cancer stem-like cells characterized by their CD133-, nestin-, and SOX2-expression and the ability to form spheroids in neural stem cell medium and to express astrocytic and neuronal differentiation markers under serum conditions. In a therapeutic approach using the suicide gene herpes simplex virus thymidine kinase (HSV-tk) fused to eGFP, both lentiviral vectors mediated a complete remission of solid tumors as seen on MRI resulting in a highly significant survival benefit (p < 0.001) compared with control groups. In all recurrent tumors, surviving eGFP-positive tumor cells were found, advocating a prodrug application for several cycles to even enhance and prolong the therapeutic effect.

CONCLUSION

In conclusion, lentiviral pseudotyped vectors are promising candidates for gene therapy of gliomas in patients. The inefficient gene delivery by γ-retroviral vectors is in line with the results obtained in clinical therapy for GBM and thus confirms the high relevance of the invasive glioma animal model for translational research.

Neuro-Oncol. 2009 Oct;11(5):591.

121. Can Small-Molecule Tyrosine Kinase Inhibitors alter Signaling in Gliomas? Evaluation of the ability of Erlotinib to achieve clinically relevant tissue concentrations in an orthotopic rat xenograft model

Michael A Vogelbaum, Juan Manuel Palomo, Sagar Agarwal, Gazanfar Ramathula, Jennifer Cutter, John R Ohlfest, William Elmquist

INTRODUCTION

We evaluated the ability of erlotinib to penetrate the blood-brain-barrier (BBB) and produce significant inhibition of EGFR tyrosine kinase activity in glioma xenografts with and without inhibition of active drug efflux.

METHODS

Athymic nude rats bearing orthotopic human gliomas (U87MG) with or without transgenic overexpression of wild-type EGFR were treated with erlotinib at doses ranging from 20 to 40 mg/kg/day for 5 days. Thirty minutes after the last treatment, multiple samples, including blood, normal brain, and tumor, were harvested for analysis of erlotinib concentration by LC/MS/MS, with or without prior saline perfusion. Evaluation of EGFR and Akt tyrosine phosphorylation was performed by Western blot. A subset of rats was also treated with the Pgp/Bcrp dual inhibitor GF120918 on the final day of erlotinib treatment.

RESULTS

Erlotinib failed to produce dephosphorylation of EGFR until animals were exposed to lethal doses. No change in Akt phosphorylation was observed at any dose. Drug concentration analysis indicated that erlotinib penetrated the tumor core but not the brain tissue immediately surrounding the tumor mass or normal brain tissue (approximately 10% of plasma concentration). Intravascular saline perfusion prior to harvesting significantly influenced the erlotinib tissue distribution values in the brain surrounding the tumor and contralateral brain, but not the tumor core. Treatment with GF120918 significantly enhanced erlotinib distribution into the tumor core (2.5 fold), brain tissue surrounding the tumor (12-fold), and normal brain tissue (10-fold).

CONCLUSION

Erlotinib is excluded from the brain by active efflux at the BBB, especially at the tumor edge and normal brain, but this may be overcome by an active efflux inhibitor. Concentrations that reach glioma tissue in this model fail to induce EGFR signaling changes at nontoxic doses. Evaluations of the ability of a drug to penetrate the brain, which are limited to assessment of drug concentrations only, may provide misleading results because of the detection of circulating drug within microvessels.

Neuro-Oncol. 2009 Oct;11(5):591–592.

122. Targeted Liposomal Doxorubicin as Potential Therapy for Malignant Peripheral Nerve Sheath Tumors

AB Madhankumar, Kimberly Harbaugh, Becky Slagle-Webb, Min Pang, James R Connor

INTRODUCTION

We have previously demonstrated the therapeutic effect of IL13-conjugated liposomal doxorubicin (IL13LDXR) on glioma tumors. These tumors overexpress IL13 receptor a2, a receptor for IL13. Recently, we found that malignant peripheral nerve sheath tumors (MPNST) also express the receptors for IL13. This observation prompted us to investigate the therapeutic value of IL13LDXR in MPNST models.

METHODS

IL13LDXR was formulated and characterized in our laboratory using our previously established procedure. The cytotoxic potential of IL13LDXR was studied in well-established MPNST cell lines: sNF96.2, ST88-14, and #215 cell line, a cell line that was cultured in our laboratory from a MPNST obtained from a patient biopsy. Immunohistochemistry was performed on malignant and benign peripheral nerve sheath tumor (PNST) sections to determine the expression of IL13a2 receptor. In addition, we investigated the correlation of IL13Rα2 receptor expression with P-glycoprotein a drug resistance-associated protein by immunocytochemistry. IL13LDXR was exposed to MPNST cells at 37°C, and the doxorubicin (DXR) accumulation in the cells was observed under fluorescent microscopy. The results were quantified by flow cytometry.

RESULTS

We confirmed the expression of IL13Rα2 in PNST tumors and in established MPNST cell lines. We also demonstrated the expression of P-glycoprotein, a multidrug-resistant protein in MPNST cell lines by immunocytochemistry. The flow cytometry analysis revealed the ability of IL13LDXR to overcome the MDR and showed significant accumulation of doxorubicin in the nucleus of MPNST cell lines resulting in their cytotoxicity.

CONCLUSION

Consistent expression of IL13Rα2 receptors in PNST and MPNST cells and tissues indicate a viable approach to use this receptor as a target for MPNST therapy. The in vitro doxorubicin accumulation property and its cytotoxic potential on MPNST cells when delivered though the targeted liposomes envisage IL13LDXR as a potential drug for MPNST.

Neuro-Oncol. 2009 Oct;11(5):592.

123. IL13-Conjugated Liposomal Doxorubicin as a Therapeutic Agent for High-Grade Astrocytomas

AB Madhankumar, Becky Slagle-Webb, Jonas Sheehan, Qing X Yang, Patti Miller, James R Connor

INTRODUCTION

The uniform overexpression of IL13Rα2 receptors on high-grade GBM tumors was previously established. We have demonstrated that by conjugating interleukin-13 to liposomes carrying doxorubicin, it can be selectively targeted towards high-grade astrocytoma in subcutaneous glioma tumor models in mice. In this study, we determined the tumor regression property and the in vivo toxicological studies in intracranial glioma tumor-bearing mice after administration of IL13-conjugated liposomal doxorubicin.

METHODS

IL13-conjugated and -unconjugated liposomal doxorubicin were administered to intracranial glioma mice for 7 weeks at a concentration of 15 mg/kg body weight. The mice were imaged weekly with T1 contrast MRI imaging to visualize the tumor regression. After 7 weeks of injections, serum analysis was performed on the mice treated with IL13LDXR to determine the enzyme levels corresponding with liver and kidney functions like SGOT, SGPT, and creatine. We are in the process of determining the pharmacokinetics and biodistribution studies on treated mice in collaboration with the nanocharacterization laboratory of the National Cancer Institute.

RESULTS

In vivo experiment confirmed that IL13-conjugated liposomal doxorubicin (IL13LDXR) is effective in treating intracranial tumor-bearing mice with minimum toxicity. After 7 weeks of injections of IL13LDXR at a concentration of 15 mg/kg body weight, the serum SGOT, SGPT, creatine, and BUN levels were found to be comparable with control mice treated with PBS, indicating minimum toxicity. The same experiment was performed in male athymic nude mice for 7 weeks. No significant toxicity was seen in the male mice treated with 15 mg/kg body weight of IL13LDXR. The survival study indicated that the mice treated with untargeted liposomal doxorubicin survived only 25 days (mean survival), whereas the mean survival of the mice treated with IL13-conjugated liposomal doxorubicin was 142 days.

CONCLUSION

The tumor regression property and lack of extratumoral toxicity, coupled with the survival data, provide compelling evidence for the efficacy of liposomal encapsulation of chemotoxin and IL13-targeted delivery.

Neuro-Oncol. 2009 Oct;11(5):592.

124. Novel Magnetic/Ultrasound-Focusing System Enhances Nanoparticle Drug Delivery for Glioma Treatment

Pin-Yuan Chen, Hao-Li Liu, Mu-Yi Hua, Kuo-Chen Wei, Chiungyin Huang, Hung-Wei Yang

INTRODUCTION

Malignant glioma is a common and severe primary brain tumor with a high recurrence rate and a poor prognosis. Intravenously administered chemotherapeutic drugs have limited use because of their adverse systemic effects and poor blood-brain barrier penetration.

METHODS

We developed a novel magnetic nanoparticle composited by poly[aniline-co-N-(1-one-butyric acid)] aniline (SPAnH) and Fe3O4 that can carry 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). We used focused ultrasound to transiently permeate the blood-brain barrier, and magnetic targeting enhanced localization of a chemotherapeutic agent immobilized on the nanoparticle into the brain tumor of a rodent.

RESULTS

The size of the particles was approximately 5–20 nm. Typical superparamagnetic behavior of the nanoparticles permitted the distribution of the particles in the brain to be monitored by MRI. By using the magnetic/ultrasound-enhanced system, successful tumor treatment and prolongation of the survival of the rats could be achieved.

CONCLUSION

In conclusion, our novel magnetic/ultrasound system provides a more effective means of brain tumor treatment, with lower therapeutic doses and potentially fewer side effects.

Neuro-Oncol. 2009 Oct;11(5):592.

125. Combination Treatment of Glioblastoma Multiforme (GBM) In Vitro with Disulfiram and Copper Increases Sensitivity to Cytotoxic Drugs by Blocking NF8 B Activity and Increasing Levels of Intracellular Reactive Oxygen Species (ROS)

Sarah Brown, Weiguang Wang, John L Darling

INTRODUCTION

Although the influence of NFκB in modulating drug sensitivity of glioblastoma multiforme (GBM) cells is unknown, DNA damage induced by chemotherapy triggers nuclear NFκB activation and plays an antiapoptotic role in these cells. Disulfiram (DS) is an antialco-holism drug that enhances cytotoxic drug efficacy by inhibiting the NFκB pathway, and copper (Cu) is a potent oxidant leading to the generation of intracellular ROS.

METHODS

To determine if a combination of DS and Cu can simultaneously induce ROS and block NFκB activation leading to enhanced cytotoxicity, NFκB activity was determined in GBM cell lines using dual-reporter luciferase assays, Western blotting, and immunofluorescence and chemosensitivity determined by MTT assays.

RESULTS

NFκB activity was 10- to 55-fold higher in GBM cell lines than in controls. Treatment with BCNU, CCNU, and VCR produced a further 2- to 2.5-fold increase. GBM cell lines were sensitive to DS (ID50s 15–40 μM) and were significantly sensitized by Cu used in combination (ID50s, 0.3–1 μM). DS and Cu also increased the sensitivity of the cell lines to chemotherapeutic drugs. To achieve this, Cu was of equal or higher concentration than DS. This effect was reversed when DS concentration was higher than Cu.

CONCLUSION

Further studies will elucidate the molecular mechanisms involved in NFκB-induced drug resistance and the role of Cu and ROS levels in the chemosensitivity of GBM. This study was supported by a grant from the Samantha Dickson Brain Tumour Trust.

Neuro-Oncol. 2009 Oct;11(5):592.

126. Does Cytomegalovirus Play a Role in Malignant Glioma Pathogenesis?

Charles Cobbs

INTRODUCTION

We first demonstrated that cytomegalovirus (CMV) infection occurred specifically in >90% of malignant gliomas in 2002. Since that time, two reports have refuted these findings and four other groups have confirmed them. Two clinical trials for malignant gliomas based on CMV antiviral strategies have occurred. New evidence has demonstrated potential roles for CMV in glioma pathogenesis.

METHODS

We will review evidence for the presence of CMV in malignant gliomas. Evidence that CMV viral expression can contribute to tumor etiology and/ or pathogenesis will be examined. We will review preliminary results from two clinical trials (at Duke University and Karolinska Institute) currently under way to treat malignant gliomas based on CMV-specific antiviral strategies.

RESULTS

Sufficient data existed to confirm a strong association between CMV infection and malignant glioma. Current evidence suggested that viral load could predict patient survival. Preliminary data from clinical trials are discussed.

CONCLUSION

CMV infection is strongly associated with malignant glioma, and sufficient evidence exists to suggest that CMV may play an active role in malignant glioma pathogenesis. Overall, these findings indicate that CMV infection may represent a novel paradigm in our understanding of malignant glioma biology and an infectious agent in cancer. Furthermore, these data suggest that future therapeutic efforts aimed at CMV infection might influence survival in patients with malignant gliomas.

Neuro-Oncol. 2009 Oct;11(5):592–593.

127. MPC-6827: Antitumor Activity in an Orthotopic Brain Model and in Combination with Bevacizumab

Margaret K Yu, Vijay Baichwal, Joshua Jones, Gary Mather, Robert Carlson

INTRODUCTION

MPC-6827 as monotherapy has shown efficacy in a broad spectrum of xenograft models and synergy with carboplatin in a human ovarian xenograft model. Based on findings in mice and dogs that MPC-6827 can achieve concentrations in the brain that are 14- to 50-fold higher than plasma, determination of activity against brain tumors is of particular interest. MPC-6827 is currently being tested in a phase 1b/2 trial in combination with carboplatin for recurrent/relapsed GBM. We report here the effects of MPC-6827 monotherapy in an orthotopic glioma model and the effects of MPC-6827 in combination with bevacizumab in a subcutaneous glioblastoma xenograft model.

METHODS

For the orthotopic model, nu/nu mice were intracranially implanted with D54 human glioma cells overexpressing luciferase (D54-luc), and tumor growth was monitored using the IVIS luminescence imaging system. Survival was monitored, and mice were euthanized when original body weight loss was greater than 20%. For the subcutaneous glioblastoma xenograft model, tumor-bearing mice were randomized to receive vehicle, MPC-6827 (5 mg/kg, i.v., every week × 3 weeks), bevacizumab (10 mg/kg i.p., BIW × 3 weeks), or MPC-6827 plus bevacizumab (identical dosing as monotherapy).

RESULTS

In the orthotopic model, 5 mg/kg of MPC-6827 displayed a reduction in tumor burden on day 48 (p = 0.0097) and an increase in median survival time from 56 days to 68 days (p = 0.039) compared with vehicle. For the subcutaneous glioblastoma xenografts, median tumor volumes on day 18 in the MPC-6827 + bevacizumab combination group increased by 63% compared with 264% (p = 0.006) for the bevacizumab monotherapy group.

CONCLUSION

MPC-6827 showed significant activity in an orthotopic human glioma model as monotherapy and enhanced the effect of bevacizumab in a subcutaneous glioblastoma xenograft model. These results contribute to the rationale for studying MPC-6827 in human brain tumor clinical trials.

Neuro-Oncol. 2009 Oct;11(5):593.

128. Conjugation of a Functionalized Gadolinium Endohedral Fulleride with an IL-13 Peptide for the Targeting and Imaging of Glioma Tumor Cells Expressing the IL-13RA2 Receptor

Helen L Fillmore, Michael D Shultz, Scott Henderson, Patricia Cooper, William C Broaddus, ZhiJian Chen, Shu Chun-Ying, Harry C Dorn, Frank Corwin, Jerry I Hirsch, John D Wilson, Fatouros P Panos

INTRODUCTION

Because of the ability of glioma cells to aggressively infiltrate normal brain tissue and to survive and escape exposure to current adjuvant therapies, a great need exists for specific targeted platforms to deliver nano-therapeutics and imaging agents. These molecules target tumor cells specifically, while leaving normal brain cells alone. To seek out and destroy the highly invasive glioma cells, we chose to target a receptor that is selectively expressed on these tumor cells. One such receptor is the IL-13Rα2 receptor, which has been shown to be overexpressed in GBM. We report the successful conjugation of a functionalized metallofullerene to IL-13 peptides and evaluate its ability to specifically bind glioma cells.

METHODS

Conjugation was confirmed using polyacrylamide electrophoresis (PAGE). Cellular uptake of Gd3+ was measured by inductively coupled plasma mass spectrometry (ICP-MS). A xenograft orthotopic glioma model was used to study the in vivo effects of direct infusion of product using magnetic resonance imaging and immunohistochemistry.

RESULTS

Cellular uptake of the conjugated IL-13 metallofullerene in glioma cells significantly increased, as determined by Gd3+ uptake measured by (ICP-MS) when compared with metallofullerene alone, suggesting that the conjugation of the IL-13 peptide enhances cellular uptake. In a xenograft orthotopic glioma model, infusion of the conjugated product demonstrated similar water proton relaxivity to the metallofullerene alone, as seen on T1-weighted magnetic resonance images. The targeting conjugated product was able to diffuse into the brain parenchyma and bind to glioma cells and not host cells, as observed with confocal microscopy.

CONCLUSION

These studies suggest that the IL-13 conjugated metallofullerenes have a higher affinity to glioma cells than to host cells, and this potential platform could deliver imaging and therapeutic agents directly to glioma cells.

Neuro-Oncol. 2009 Oct;11(5):593.

129. Inhibition of PI3K/mTOR Pathways in Glioblastoma and Implications for Combination Therapy with Erlotinib, Temozolomide, and Radiation

Gautam Prasad, Theo Sottero, Xiaodong Yang, Sabine Mueller, C David James, Mei-Yin C Polley, Mitchel S Berger, Dana Aftab, Michael Prados, Daphne Haas-Kogan

INTRODUCTION

Because of their molecular heterogeneity and infiltrative nature, glioblastomas (GBM) have been notoriously difficult to treat. To overcome these hurdles, combining targeted agents with conventional therapy is being explored. Using a novel, orally bioavailable PI3K/ mTOR inhibitor (XL765), we evaluated its preclinical potential in vitro and in vivo.

METHODS

We used a series of in vivo serially passaged human GBM xenografts that are more genetically stable than GBM cell lines. In vitro, XL765 was evaluated by Western blot and viability assays. We combined XL765 with erlotinib (ERL), temozolomide (TMZ), and radiation (XRT) to test for combinatorial effects. Finally, we injected a xenograft (EGFR VIII, PTEN wt; altered to express luciferase) intracranially into nude mice and tested various combinations of the above agents. The Kaplan-Meier method was used to estimate survival, and exact log-rank test was used to compare survival distributions between the two treatment groups. Differences in tumor volumes based on bioluminescence were compared using Wilcoxon rank sum test (at the last time points, all mice were alive).

RESULTS

In all xenografts tested in vitro, XL765 inhibited the PI3K pathway in a concentration- and time-dependent manner, and these molecular changes correlated with increasing cytotoxicity. In vivo, XL765 administered by oral gavage had significantly lower tumor bioluminescence than did control (p = 0.0011). The median survival was 67.5 and 55 days for XL765 and control, respectively (p = 0.062). Compared with TMZ alone, XL765 + TMZ had a trend towards lower tumor bioluminescence and improved survival, even though statistical significance was not reached (p = 0.06 and 0.19, respectively). XL765 + ERL did not demonstrate a statistically significant difference in tumor bioluminescence or survival compared with ERL alone (p = 0.97 and 0.45).

CONCLUSION

XL765 shows activity as a monotherapy and in combination with conventional therapeutics in preclinical models. Phase Ib/II trials combining XL765 with ERL or TMZ are ongoing.

Neuro-Oncol. 2009 Oct;11(5):593.

130. Neural Stem Cell-Based Gene Therapy for Malignant Brain Tumors by Double-Stranded Adeno-Associated Virus Vectors

Chung-Che Wu, Kai-Yun Chen, Yung-Hsiao Chiang

INTRODUCTION

Despite intensive efforts exploring gene therapeutic approaches for human cancers, the clinical results in the treatment of malignant brain tumors are still poor. We showed previously that a combined therapeutic strategy using an adenoviral vector carrying the thymidine kinase (suicidal) gene and an adeno-associated virus (AAV) vector carrying the angiostatin gene achieved better therapeutic results. However, the malignant brain tumor could not be completely eradicated. Here we have developed a safe and effective neural stem cell (NSC)-based gene therapy protocol to treat malignant glioma.

METHODS

The double-stranded AAV (dsAAV) vector carrying the eGFP gene was used to determine the transduction efficiency of different serotypes (types 1, 2, 5, 7, 8, 9, rh2, rh8, rh10, rh39, and rh43) AAV in NSCs. The angiostatin and decorin genes were used as therapeutic genes. Green fluorescence was observed by the direct inverted fluorescence microscopy. The expressions of dsAAV-angiostatin and dsAAV-decorin were detected by Western blot analysis. To investigate the therapeutic effects of the engineered NSCs in the U87MG/nude mice model, ipsilateral or contralateral injections of genetically modified NSCs were applied. The tumor size and survival of animals with brain tumors were analyzed 4 weeks after the injection.

RESULTS

The NSCs were successfully gene-modified by dsAAV-eGFP, and its expression persisted longer than 28 days. By immunostaining and flow cytometry analysis, we found that dsAAV2 and dsAAV7 had higher transduction efficiency in NSCs. Overexpression of decorin by dsAAV7 may arrest the cell cycle of U87MG cell in subG1 phase. The survival rate of mice with brain tumors treated with dsAAV7-decorin was higher than that of mice treated with dsAAV2-eGFP. In addition, immunohistochemistry data showed that genetically modified NSCs had the capacity to migrate into brain tumors and inhibit tumor growth. The therapeutic effect of NSCs-dsAAV7-decorin in mice with brain tumors was better than was the effect of NSCs-dsAAV2-angiostatin.

CONCLUSION

This study indicates that effective glioma suppression might be achieved by using genetically modified NSCs.

Neuro-Oncol. 2009 Oct;11(5):593–594.

131. Chromatin Remodeling-Mediated Overexpression of MGMT As a Novel Mechanism of Temozolomide Resistance in Glioblastoma Xenografts

Gaspar J Kitange, Ann C Mladek, Brett Carlson, Mark Schroeder, Jann Sarkaria

INTRODUCTION

Temozolomide (TMZ) is considered the most effective drug for treatment of glioblastoma (GBM), but tumor resistance limits its efficacy and presents a significant hurdle in GBM therapy.

METHODS

To study the mechanisms of TMZ resistance, we developed a panel of primary GBM xenograft lines that are established from patient tumor samples and maintained by serial transplantation in nude mice. Ten of these primary lines were subjected to in vivo selection with escalating doses of TMZ to establish corresponding TMZ-resistant lines.

RESULTS

Characterization of parental and TMZ-resistant lines using real-time RT-PCR and Western blotting revealed increased expression of O6-methyl-guanine methyltransferase (MGMT) in 6 of the 10 (60%) TMZ-resistant xenografts compared with the parental lines. This increase in expression was independent of MGMT promoter methylation status, as determined by MS-PCR. Moreover, bisulfite sequencing revealed no significant differences in methylation status across the MGMT promoter region between the parental and derivative TMZ-resistant xenografts, which suggested that additional epigenetic mechanisms might modulate the upregulation of MGMT expression. Consistent with this hypothesis, chromatin immunoprecipitation (ChIP) demonstrated increased acetylation of lysine 9 of histone H3 (H3K9) and decreased methylation of H3K9 within the MGMT promoter region in the MGMT-upregulated TMZ-resistant line GBM12TMZ compared with the parental line GBM12. Recruitment of transcription repressor MeCP2 protein to MGMT promoter was lost in the TMZ-resistant GBM12TMZ line relative to parental GBM12. In contrast, in GBM22 TMZ-resistant xenografts, in which MGMT was not upregulated, neither acetylation and methylation of H3K9 nor the binding of MeCP2 to the MGMT promoter were noticeably altered compared with the parental line.

CONCLUSION

Our findings suggest that chromatin remodeling without a corresponding change in promoter methylation can lead to increased MGMT expression in association with TMZ resistance in a subset of GBM lines.

Neuro-Oncol. 2009 Oct;11(5):594.

132. Silencing H-Ferritin Sensitizes Gliomas to Treatment Agents

Xiaoli Liu, AB Madhankumar, Becky Slagle-Webb, Jonas Sheehan, James R Connor

INTRODUCTION

Tumor cells have a robust iron requirement that is consistent with their rapid growth and high metabolic rates. The high levels of iron uptake require a mechanism to limit iron-induced oxidative damage. This function is the responsibility of ferritin, an iron-storage protein. We hypothesize that limiting the expression of ferritin using siRNA would increase the vulnerability of gliomas to chemotoxins and radiation.

METHODS

We used U251 human astrocytoma cells and athymic nude mice for in vitro and in vivo studies. The cells and animals were exposed to carmustine (BCNU) or radiation in the presence of H-ferritin and nonspecific siRNA. The siRNAs were delivered via nanovesicles. A superhelical plasmid DNA, pUC19, was chosen to determine the effect of H-ferritin on DNA. The form DNA was examined after treatment of BCNU or radiation with or without H-ferritin or the other subunit, L-ferritin.

RESULTS

Our studies demonstrated that silencing H-ferritin reduced the LD50 for BCNU dosage from >100 μM to 38 μM in U251 cells and led to a 50% increase in cell death at 20 Gy. Intratumor injections of liposomes containing H-ferritin siRNA reduced the effective in vivo dose of BCNU for tumor suppression by more than 50%. The superhelical plasmid DNA was converted to a supercoiled form by BCNU. The BCNU effect was inhibited by H-ferritin but not L-ferritin. H-ferritin increased the conversion of relaxation and linear forms of DNA, even in the presence of BCNU or radiation. The H-ferritin effect could be inhibited by the presence of EDTA and EGTA.

CONCLUSION

The protective mechanism of H-ferritin for cancer cells appears to be a direct action on the DNA that involves maintaining DNA in a relaxed form, with promotes transcription even in the presence of radiation and chemotoxins. Silencing H-ferritin renders tumor cells more vulnerable to chemotherapy.

Neuro-Oncol. 2009 Oct;11(5):594.

133. CDK4/6 Small-Molecule Inhibitor PD-0332991 Acts in Combination with Radiation Therapy to Significantly Extend the Survival of Mice with Intracranial Glioblastoma Xenografts

Karine Michaud, Tomoko Ozawa, Michael Prados, C David James

INTRODUCTION

The purpose of this study was to characterize the response of glioblastoma (GBM) to CDK4/6 inhibitor monotherapy and to CDK4/6 inhibitor + radiation combination therapy, using an orthotopic glioblastoma xenograft therapy-response model.

METHODS

Luciferase-modified U87 GBM cells, which lack p16 function, were injected into the brains of athymic nu/nu mice. Mice were monitored 1–2× weekly by bioluminescence imaging (BLI) until logarithmic tumor growth was evident, at which time treatment was initiated. Monitoring by BLI was continued until mice presented neurological symptoms indicative of excessive tumor burden, at which point they were euthanized. In the initial experiment, mice were randomized to PD 0332991 and vehicle-only treatment groups. In a second experiment, we assessed the combination of PD 0332991 and radiation therapy versus either treatment as a monotherapy. In this second experiment, mice were randomized to four different groups: control, PD 0332991 (150 mg/kg daily for 14 days) alone, PD 0332991 and concurrent radiation therapy (2 G/day × 5 days), and PD 0332991 treatment followed by radiation therapy.

RESULTS

BLI monitoring revealed sustained anti-tumor activity of PD 0332991 as a single agent for the entire period of therapy administration, which was followed by rapid tumor growth after completion of the treatment regimen. CDK4 inhibitor extended median survival by 18 days (60%) relative to vehicle treatment. Comparison of MIB-1 staining in tumors from the brains of mice that were killed while on therapy versus the MIB-1 staining in tumors from the control (untreated) group revealed a greater than 8-fold reduction in tumor MIB-1 positivity resulting from CDK4/6-inhibitor treatment. The combination of PD 0332991 and radiation therapy resulted in significantly increased survival benefit compared with either therapy alone.

CONCLUSION

Our results indicate that oral administration of CDK4/6 inhibitor PD-0332991 results in substantial antiproliferative activity against orthotopic GBM xenografts lacking p16 function. In addition, combined antitumor activity and survival benefit are seen when PD-0332991 is used in combination with radiation therapy. Our results support investigation of this therapy for treating GBM patients in the context of a clinical trial.

Neuro-Oncol. 2009 Oct;11(5):594.

134. Cyr61 Mediates Hepatocyte Growth Factor-Induced Glioma Cell Growth, Migration, and Akt Activation

C Rory Goodwin, Bachchu Lal, Linda Zhou, Sandra Ho, Taeger Alexandra, John Laterra

INTRODUCTION

Tumor cell responses to the growth factor-inducible early response gene product CCN1/Cyr61 overlap with those induced by the hepatocyte growth factor (HGF):c-Met signaling pathway. In this study, we investigated whether Cyr61 is a downstream effector of HGF/c-Met pathway activation in human glioma cells.

METHODS

Cyr61 and Met expressions in 112 human primary gliomas were determined via semiquantitative immunohistochemical analysis. U373wt or U87MG glioma cells were treated with control or Cyr61 siRNAs and assayed for cell migration via modified wound-scratch assay, cell growth via MTT assay, or Akt activation via immunoblot analysis. Preestablished subcutaneous U87 glioma xenografts were treated with Cyr61 siRNA or control siRNA by direct intratumoral delivery.

RESULTS

Semiquantitative immunohistochemical analysis of human glioma and normal brain specimens showed that levels of tumor-associated Cyr61 protein correlated with tumor grade (p < 0.001) and with c-Met protein expression (R2 = 0.4791, p < 0.001). Purified HGF rapidly upregulated Cyr61 mRNA (peak at 30 min) and protein expression (peak at 2 h) in HGF-/c-Met+ human glioma cell lines via a transcription- and translation-dependent mechanism. Conversely, HGF/c-Met pathway inhibitors reduced Cyr61 expression in HGF+/c-Met+ human glioma cell lines in vitro and in HGF+/c-Met+ glioma xenografts. Targeting Cyr61 expression with siRNA inhibited HGF-induced cell migration (p < 0.01) and cell growth (p < 0.001) in vitro. Cyr61 siRNA had no effect on the early phase of HGF-induced Akt activation (phospho-Ser-473) 30 min poststimulation with HGF. Cyr61 siRNA inhibited a second phase of Akt phosphorylation measured 12 h after cell stimulation with HGF and also inhibited HGF-induced phosphorylation of the Akt target GSK3. Cyr61 siRNA inhibited xenograft growth by up to 35% in a dose-dependent manner (p < 0.05).

CONCLUSION

These results identify a Cyr61-dependent pathway by which c-Met activation mediates cell growth, cell migration, and long-lasting signaling events in glioma cell lines and possibly astroglial malignancies.

Neuro-Oncol. 2009 Oct;11(5):594.

135. LCMV-Pseudotyped VSV-Vectors for Treatment of Malignant Glioma

Isabel Vogler, Inna Kneiske, Alexander Muik, Dorothee von Laer

INTRODUCTION

Malignant glioma is the most frequent primary brain tumor and still has a dismal prognosis despite advances in neurosurgical resection and adjuvant radiotherapy and chemotherapy. In a novel approach, we use vesicular stomatitis virus (VSV) armed with a suicide gene for oncolytic virotherapy. We developed a new vector with improved glioma cell specificity by incorporating LCMV glycoprotein (GP) into VSV particles. To diminish toxicity of native VSV, several point mutations have been introduced into the matrix protein, resulting in a noncytopathic (ncp) VSV variant. Treatment of gliomas with LCMV-pseudotyped (ncp) VSV vectors are expected to lead to preferential infection of malignant glioma cells, while sparing healthy neurons.

METHODS

To generate LCMV-pseudotyped VSV vectors, BHK-21 cells were stably transduced with a lentiviral vector coding for optimized LCMV-GP and puromycin. Following antibiotic selection, the cells were infected with VSV-deltaG(G) non- and cytopathic variants, followed by harvest the next day of pseudotyped vector containing supernatants. The VSVcp(LCMV) and VSVncp(LCMV) vector supernatants were titrated on seven glioblastoma, four neuroblastoma, and two medulloblastoma cell lines, as well as on primary blood lymphocytes, monocytes, and macrophages. As a reference, a corresponding VSV-deltaG(G) vector was titrated in parallel.

RESULTS

LCMV-pseudo-typed VSV vectors were able to infect diverse tumor cell lines, including glioblastoma and medulloblastoma lines. Glioma cell lines were found to be especially susceptible to LCMV GP pseudotypes. Primary blood cells allowed only low levels of infection with VSV.

CONCLUSION

Provided that a reduced neurotoxicity can be shown in vivo, a clinical implementation of LCMV-pseudotyped VSV vectors appears warranted.

Neuro-Oncol. 2009 Oct;11(5):595.

136. Development of Antitumor Compounds for Temozolomide-Resistant Tumors

Sang Y Lee, Becky Slagle-Webb, Elias Rizk, Patti Miller, Xuesheng Zhang, Padma Ponnuru, Jonas Sheehan, James R Connor

INTRODUCTION

Gliomas represent 42% of all primary CNS tumors, and more than 75% of gliomas are malignant. Temozolomide is the standard alkylation agent used for brain tumor chemotherapy. However, about 50% of brain tumors are reportedly temozolomide-resistant. We found that the C282Y HFE variants expressing neuroblastoma and glioma cells were resistant to the temozolomide and γ-radiation. The resistant cell lines offered an opportunity to screen a drug library for new cytotoxic compounds.

METHODS

We used the temozolomide-resistant tumor cell lines to screen 15,000 compounds in our Drug Discovery Core. The toxic effect of the chemotype compound was determined both in vitro and in vivo tumor models. The toxic mechanism was probed using a functional gene array.

RESULTS

Chemotype compounds designated as E and E3 were cytotoxic to the temozolomide-resistant glioma cell lines at an approximate LD50 of 3.0–4.3 μM, but temozolomide-sensitive SW-1,088 astrocytoma cells were even more sensitive (LD50, 1.2–1.7 μM). At concentrations that were toxic to the temozolomide-resistant glioma cells, the chemotype compound E was not toxic to human astrocytes cultured from amyotrophic lateral sclerosis postmortem samples and bovine retinal endothelial cells. Compound E also completely inhibited tumor growth in vivo in the athymic nude mouse subcutaneous model as well as in an intracranial brain tumor model. The tumor growth did not recur over a period of 13 weeks when the compound E was discontinued. The animals receiving compound E had no loss of body weight and no detectable liver or kidney toxicity. The cytotoxicity of the compound E was mediated by apoptosis, mainly through the tumor necrosis factor (TNF) ligand and receptor family pathway.

CONCLUSION

We have identified a new chemotype compound that is effective against temozolomide-resistant tumors in both cell culture and in vivo.

Neuro-Oncol. 2009 Oct;11(5):595.

137. Clinically Achievable Concentrations of Digitoxin Inhibit HIF-1a and Induce Apoptosis of Human Glioma Stem Cells

Dae-Hee Lee, Akio Soeda, Arlan H Mintz, Johnathan A Engh, Merrill J Egorin, Ian F Pollack, Amin B Kassam, Deric M Park

INTRODUCTION

Solid tumors are characterized by regions of poor oxygenation that result from rapid growth of tumor cells and functionally impaired vasculature. Tumor tissue hypoxia further contributes to tumor growth by 1) selecting for more aggressive cancer cells; 2) activating adaptive programs that promote tumor cell survival and invasion; 3) promoting angiogenesis; and 4) maintaining the stem-like state. A hypoxic environment also serves as a barrier to radiation therapy. Therefore, a therapeutic strategy capable of addressing hypoxia may lead to more effective and durable outcomes. Cardiac glycosides have been identified recently as hypoxia-inducible factor 1α (HIF-1a) inhibitors with direct antitumor properties. Because our previous observation found that hypoxia selectively expands the human malignant glioma-derived tumorigenic CD133+ stem cells (GCSC), we investigated the antitumor properties of the cardiac glycosides digoxin and digitoxin on GCSC.

METHODS

Three human GCSC lines capable of forming tumors in immunodeficient animals were established from acutely resected surgical specimens. The effect of cardiac glycosides on in vitro growth in 1% and 20% oxygen environments, expression of HIF-1a and vascular endothelial growth factor (VEGF), and the mechanism of growth inhibition were investigated.

RESULTS

Both digoxin and digitoxin showed antitumor activity and inhibition of HIF-1a. However, digitoxin was capable of exerting an antitumor effect at clinically achievable concentrations of 10–25 nM. Antitumor activity with poly(ADP-ribose) polymerase (PARP) cleavage was observed at 1% and 20% oxygen tensions. Digitoxin treatment of GCSC propagated at 1% oxygen tension inhibited VEGF production.

CONCLUSION

Clinically achievable concentrations of the cardiac glycoside digitoxin, which is capable of penetrating the blood-brain barrier, show in vitro antitumor activity against GCSC cultured at 1% oxygen tension. In addition, digitoxin induces apoptosis and downregulates HIF-1a and expression of VEGF.

Neuro-Oncol. 2009 Oct;11(5):595.

138. Response and Escape of Orthotopic GBM Models to Treatment with Anti-VEGF Antibodies

C David James, Merry Nishimura, Tomoko Ozawa, Li Peng, Mary Ludlam, Dara Kallop, Joan M Greve, Samir Kharbanda, Zora Modrusan, Michelle Niannini, Frank Peale, Ian Kasman, Heidi S Phillips

INTRODUCTION

Radiographic responses of glioblastomas (GBMs) to bevacizumab treatment may reflect alterations in tumor growth, a vascular leak, or a combination of effects. Our current study was designed to examine responses of orthotopic GBM models to treatment with anti-vascular endothelial growth factor (VEGF) antibody.

METHODS

U87 and G55 human GBM cell lines, modified to express firefly luciferase, were implanted into the striatum of CD-1 nude mice. Beginning 7–10 days after implantation, host mice were dosed twice weekly with 5 mg/kg of anti-VEGF monoclonal antibody or control. Dosing continued until mice became symptomatic, at which point they were euthanized.

RESULTS

Serial optical imaging of luciferase signal revealed that growth of both U87 and G55 grafts was suppressed in response to treatment with anti-VEGF. In both models, growth suppression was accompanied by a significant prolongation of survival. Progressive tumor growth was evident, however, in all treatment groups, and all mice eventually died of disease. For U87 grafts, volumetric assessments with T2-weighted MRI and bioluminescent signal showed comparable changes in response to anti-VEGF treatment. Tissue harvested at the end of the study showed only subtle signs of increased invasion for the U87 line, whereas grafts of the G55 line showed profound exophytic growth at time points after control mice had died. Increased immunohistochemical signal for caspase-3 was observed in G55 grafts, suggesting increased apoptotic activity resulting from anti-VEGF treatment. Surprisingly, gene expression profiling revealed minimal changes in grafts of either xenograft line following growth escape from anti-VEGF treatment. Profiling of mouse tumor stroma in U87 xenografts revealed that anti-VEGF treatment led to relative increases and decreases in genes associated with host brain and angiogenesis, respectively.

CONCLUSION

Our findings demonstrate that anti-VEGF treatment exerts growth suppression and survival benefit in two orthotopic GBM models, and suggest that tumors can escape treatment without major changes in transcriptional activity.

Neuro-Oncol. 2009 Oct;11(5):595.

139. Glioblastoma Multiforme Sensitivity to Temozolomide in An Ex Vivo Invasion Assay: Correlation with Patient Survival

Joseph F Megyesi, Penny Costello, Erin Dyer, Warren McDonald, Robert Hammond, David Macdonald

INTRODUCTION

Glioblastoma multiforme (GBM) tumor progression is dependent on the tumor’s ability to invade and grow into surrounding tissue. GBM surgical samples were assessed while exposed to a panel of clinically relevant chemotherapies, including temozolomide (TMZ), using an ex vivo invasion and growth model. The invasion and growth of representative tumor tissue fragments are hypothesized to be reflective of clinical response to therapy. GBM patients in this study went on to receive TMZ postoperatively.

METHODS

Operative tissue specimens were placed into a collagen matrix and were monitored for invasion in the presence of chemotherapies, including TMZ, for 5 days postsurgery. All samples were preserved for further examination of tumor growth, invasion, and viability. Patient outcomes were measured from the operation/assay date onward.

RESULTS

One hundred twenty-eight CNS patient tumors were assessed, of which 58 were GBMs. Each patient’s tumor displayed a unique invasion and response profile. We found a significant correlation between a TMZ invasion inhibition of 20% or greater to GBM patient outcome. Mean survival time for GBM patients whose tumors were not significantly sensitive to TMZ in the invasion assay was 181.7 ± 43 days. GBM patients’ mean survival for invasion assay responders to TMZ was 290 ± 33 days. Results will continue to be compared to patient response, time to recurrence, and survival for up to 2 years.

CONCLUSION

Individual response to chemotherapy in patients with a brain tumor is highly variable, both clinically and in our ex vivo assessment. Prescreening responsiveness to chemotherapies could lead to more individualized and effective treatment of brain tumors.

Neuro-Oncol. 2009 Oct;11(5):596.

140. Induction of Tumor-Specific T Cell Responses to IGF-1R Antisense-Treated Glioma Cells

Jinliang Li, Kirsten Hooper, Larry Harshyne, Craig D Hooper, Phyllis Flomenberg, David W Andrews

INTRODUCTION

We have identified an insulin-like growth factor type 1 receptor (IGF-1R) antisense oligodeoxynucleotide (AS ODN) that causes selective apoptosis of tumor cells and induces dendritic cell maturation. In a previous phase I trial, clinical responses were detected in 8 of 12 glioma patients treated with autologous tumor cells incubated ex vivo with the IGF-1R AS ODN. The goal of this study was to determine if this treatment induced a tumor-specific T cell response in a murine model.

METHODS

The C57BL/6 (B6) glioma cell line GL261 was treated overnight with the IGF-1R AS ODN and injected subcutaneously into the flank of B6 mice. Mice were challenged with GL261 in the opposite flank after 2 weeks. Naive mice were injected with tumor cells as a control. Splenocytes from surviving mice were harvested, stimulated in vitro for 1 week, and assayed for tumor-specific T cell responses by IFN-γ ELISPOT assay.

RESULTS

Most of mice pretreated with AS ODN-treated glioma GL261 cells were resistant to tumor challenge. T cell lines derived from treated mice exhibited IFN-γ responses to a Kb-restricted epitope from the tumor antigen TRP-2. T cell lines also recognized GL261 targets.

CONCLUSION

Immunization of mice with IGF-1R antisense-treated syngeneic glioma cells was protective against tumor challenge and induced a tumor-specific T cell response. This strategy will be further evaluated in a phase II clinical trial in patients with malignant glioma.

Neuro-Oncol. 2009 Oct;11(5):596.

141. The Therapeutic Effect of 5-FC/CD::UPRT Combinative Gene on Rat Encephalic Glioma

Weixing Hu

INTRODUCTION

Glioblastoma multiforme is the most common tumor of the CNS. Treatment generally consists of both surgery and radiotherapy, but the prognosis is very poor. Nonetheless, gene therapy remains a promising treatment. In this study, we evaluated the suicide gene system 5-FU/CD::UPRT therapy strategy on encephalic glioma xenografts in Sprague Dawley rats. That shows an increased antitumor activity and improved survival in rats.

METHODS

Reconstructed malignant glioma cells C6-CD::UPRT, C6-pLXSN, and C6 were engrafted to the brain by a third-dimension direction finder. When tumors had grown for 3 weeks, rats were treated with 5-FC. The therapeutic effects were determined by measuring tumor size and survival period, and by examining the tumor using an electron microscope and FCM assays.

RESULTS

Compared with the control group, the growth of tumors in the test group was significantly suppressed. The average survival period of the control group was about 32 days and in the test group extended to 85 days. The percentage of apoptotic cells was 19.36% according to the results of the flow cytometry assays.

CONCLUSION

5-FC/CD::UPRT combinative gene has a significant therapeutic effect on encephalic gliomas in mice.

Neuro-Oncol. 2009 Oct;11(5):596.

142. Sunitinib Inhibits Glioma Growth by Blocking Progenitor Cell Recruitment

Aaron M Tannenbaum, Kim A Lopez, Marcela Assanah, James G Christensen, Jeffrey N Bruce, Peter D Canoll

INTRODUCTION

Several recent studies using animal models of glioblastoma have shown that glioma cells have the ability to recruit nonneoplastic glial progenitor cells from the surrounding brain tissue by paracrine growth factor signaling. This massive cellular recruitment contributes significantly to tumor size and rate of growth and introduces the inhibition of cellular recruitment as a potential therapeutic target. We have shown that infecting glial progenitors with a platelet-derived growth factor (PDGF)-expressing retrovirus drives the formation of brain tumors that recapitulate the histologic features of human glioblastoma. These tumors contain a mixture of retrovirus-infected and -uninfected cells. In this study, we administered sunitinib (Sutent), an orally active multitarget tyrosine kinase inhibitor, to tumor-bearing animals and analyzed the effects on recruitment, tumor growth, and animal survival.

METHODS

A PDGF-expressing retrovirus was injected into the subcortical white matter of adult rats. Animals were treated with either 20 mg/kg of sunitinib or vehicle orally starting at 7 dpi and continued until animals showed signs of tumor-induced morbidity. Animals were killed and histologic and immunofluorescence analyses were performed to evaluate tumor size, grade, proliferation index, and abundance of recruited progenitors. Effects on survival were evaluated using the Kaplan-Meier method.

RESULTS

Sunitinib-treated animals showed a significant survival advantage (mean survival: sunitinib, 55.6 ± 1.6 days vs. control, 17.4 ± 1.0 days; p < 0.0001). The sunitinib-treated tumors contained significantly fewer recruited progenitors, and a marked reduction was seen in proliferation index. Furthermore, the treated tumors had less vascular proliferation and showed a complete absence of pseudo-palisading necrosis.

CONCLUSION

Paracrine PDGF signaling by glioma cells causes the massive recruitment of glial progenitors, which contributes to tumor growth and progression. Sunitinib effectively blocks the recruitment of these cells, leading to more slowly proliferating, lower-grade tumors and increased survival.

Neuro-Oncol. 2009 Oct;11(5):596.

143. Chromatography-Based Rapid Assessment of MGMT (CRAM): A Rapid and Quantitative Assay of MGMT Activity in Tumor Tissue

Cliff Robinson, Juan Manuel Palomo, Michael A Vogelbaum

INTRODUCTION

Expression of the DNA repair enzyme O6-methyl-guanine methyltransferase (MGMT) in gliomas correlates with resistance to alkylating agents. Analysis of MGMT promoter methylation detects only epigenetic modification of MGMT production but not enzymatic activity. The gold standard MGMT activity assay is time-consuming and employs radioactivity. We developed a method for rapidly assessing MGMT activity through a thin-layer chromatography (TLC)-based visualization of the reaction between cell lysate with fluorescently labeled O6BG (F-O6BG). A single fluorescently labeled benzyl group is irreversibly transferred from F-O6BG to the active site of a single molecule of functional MGMT. Unre-acted F-O6BG is rapidly separated from the reaction product by TLC, and fluorescently labeled MGMT retained at the origin is visualized on the same plate.

METHODS

Snap-frozen GBM cell-line pellets and tumor samples from the Cleveland Clinic Brain Tumor Tissue Bank were lysed via sonication in reaction buffer, and total protein was measured. Protein was contacted with F-06BG (BG-vista Green, Covalys, Switzerland) in reaction buffer. Samples were spotted onto a silica gel TLC plate and run for 10 min. The plate origin was scanned at 450 nm/520 nm on a fluorescence imager, and sample intensity was measured by densitometry. MGMT activity of each sample was also measured using a validated high-performance liquid chromatography-based variation on the methyl group transfer method as described by Gerson et al.

RESULTS

The amount of fluorescently labeled benzyl group transferred to MGMT as measured by the chromatography-based rapid assessment of MGMT (CRAM) assay was significantly correlated with activity as measured by the HPLC method (R2 = 0.91, p = 0.003). Multiple runs were performed, and the CRAM assay performed reliably in both cellular and tissue lysates.

CONCLUSION

MGMT activity can be rapidly and quantitatively assayed by the CRAM assay. This assay can be performed in under 60 min, which may also allow for intraoperative therapeutic decision making.

Neuro-Oncol. 2009 Oct;11(5):596–597.

144. Effects of the RTOG 0525 Dose-Intense Temozolomide Schedule on GBM MGMT Activity and Overall Survival in An Orthotopic Xenograft Model

Cliff Robinson, Juan Manuel Palomo, Gazanfar Rahmathulla, Mary V McGraw, Lili Liu, Jon Donze, Michael A Vogelbaum

INTRODUCTION

Expression of O6-methylguanine methyltransferase (MGMT) in glioblastoma multiforme (GBM) correlates with temozolomide (TMZ) resistance. Dose-intense schedules of TMZ, which deplete MGMT in peripheral blood mononuclear cells, may also enhance tumor cytotoxicity. Radiation Therapy Oncology Group (RTOG) 0525 randomized patients with GBM to adjuvant TMZ given on a standard (STD-TMZ; 200 mg/m2 × 5 days every 28 days) or dose-intense (DI-TMZ; 100 mg/m2 × 21 days every 28 days) schedule. No published data exist evaluating the effect of DI-TMZ schedules on intracranial tumor MGMT activity. Using an orthotopic xenograft model, we evaluated the impact of DI-TMZ on tumor MGMT activity and overall survival.

METHODS

GBM43 cells (courtesy of Jann Sarkaria at the Mayo Clinic, unmethylated MGMT promoter) were implanted intracranially in the striatum of immunodeficient Nu/Nu mice and RNu rats. Three weeks after inoculation, animals received TMZ 200 mg/m2 × 5 days i.p. (group 1) or 100 mg/m2 × 21 days i.p. (group 2). At serial time points, brain tumors were extracted, and MGMT activity was assayed by HPLC. For survival evaluation, mice were inoculated with GBM43 and randomized to group 1, group 2, or no drug (group 3). Survival was evaluated using the Kaplan-Meier method.

RESULTS

Tumor MGMT activity was depleted by day 6 in both groups. Activity returned to baseline by day 22 in group 1 but remained suppressed in group 2. By day 29, tumor MGMT activity had returned to baseline in both groups. Median survival (MS) was 64, 42, and 28 days for groups 1, 2, and 3, respectively. MS of groups 1 and 2 was significantly greater than was group 3 (1 vs. 3, HR 4.60, p < 0.0001; 2 vs. 3, HR 3.69, p = 0.0001). MS of group 1 versus 2 was not different (HR 1.52, p = 0.124).

CONCLUSION

DI-TMZ prolongs MGMT activity depletion in orthotopic GBM compared with STD-TMZ; however, DI-TMZ did not improve survival in this model.

Neuro-Oncol. 2009 Oct;11(5):597.

145. Electrochemotherapy for Brain Tumors Investigated In Vivo

Birgit Agerholm Larsen, Helle Iversen, Per Ibsen, Jakob Moeller, Lasse Staal, Leif G Salford, Julie Gehl

INTRODUCTION

Delivery of molecules, such as chemotherapeutic drugs, is markedly increased by use of electric pulses (electrotransfer). For example, the cytotoxic effect of bleomycin can be increased 300-fold. Several tissues like skin, liver, and muscles have been targeted successfully by drug and gene electrotransfer. Few attempts have been made to target brain tissue. The aim of this study is to perform preclinical animal experiments using electrochemotherapy to treat brain tumors.

METHODS

Electrochemotherapy uses electric pulses to create an electric field for transient permeability of cell membranes, improving uptake of chemotherapeutic drugs. Fischer rat glial cell derived tumor cells (N32) were inoculated through a burr hole in the skull of Fischer rats 9 days prior to electrochemotherapy. Intratumor bleomycin (Baxter) was given, and newly developed electrodes specific for brain electrotransfer (Pulse, Denmark) were inserted through the burr hole. High-voltage pulses from a pulse generator (Cliniporator, Igea, Italy) were given in the area of interest. MRI was performed before and after electrochemotherapy. Seven days after electrochemotherapy, the rats were killed and underwent paraformaldehyde perfusion for later pathological analyses.

RESULTS

Electrochemotherapy given to Fischer rats inoculated with brain tumor cells showed necrotic tissues in the area of treatment, the area where tumor cells were deposited the week before. Six out of nine rats treated with a four-pin electrode and nine out of ten rats treated with a eight-pin electrode had severely affected and necrotic brain tissue. Posttreatment rats showed no obvious behavioral adverse effects. MRI confirmed presence of tumors >2 mm in diameter before electro-chemotherapy was performed, and pathological staining (HE, PAS, NF, and GFAP) of paraformaldehyde-perfused rat brain was used to define and characterize the treated area. Glial cells were substantially more affected than neurons.

CONCLUSION

We have been able to eliminate rat brain tumors in areas targeted with electrochemotherapy.

Neuro-Oncol. 2009 Oct;11(5):597.

146. Dasatinib Enhances NVP-AEW541-Induced Cell Proliferation Inhibition in Malignant Human Glioma Cell Lines via Bcl-2 Downregulation and Bax Activation

Daniel R Premkumar, Esther P Jane, Ian F Pollack

INTRODUCTION

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-IR) have been implicated in the tumorigenesis of a variety of human cancers. The combination of the highly selective IGF-1R inhibitor, NVP-AEW541, and dasatinib, which multitarget kinase inhibitors, caused inhibition of cell proliferation and induction of apoptosis in glioma cell lines. Our study demonstrated a significant synergistic antiproliferative inhibition (combination index) according to the Chou and Talalay model for drug-drug interaction.

METHODS

We performed cell proliferation and cytotoxicity assays, a clonogenic growth assay, immunocytochemistry, immunoprecipitation, and Western blotting. We analyzed the combined effects.

RESULTS

We evaluated the effects of the combination of a small-molecule receptor tyrosine kinase inhibitor, NVP-AEW541 and dasatinib, which inhibits IGF-1R, EGFR, and PDGFR tyrosine kinases in a panel of malignant human glioma cell lines. Proliferation assays and Western immunoblot analysis were conducted. The cytotoxicity of this combination for glioma cells was associated with translocation of cytochrome c and apoptosis inducing factor (AIF) from the mitochondria to the nucleus after treating the cells with NVP-AEW541 and dasatinib. The synergistic effects on inhibition of proliferation corresponded with a reduction of Akt and mitogen-activated protein kinases (MAPK) and a decrease in cell cycle regulatory protein levels. Conversely, the increase in apoptosis was associated with decreased levels of the antiapoptotic protein Bcl-2, as well as activation of the proapoptotic proteins such as Bax, caspases, and poly (ADP-ribose) polymerase (PARP).

CONCLUSION

The present data show that inhibition of the IGF-IR transduction cascade enhances the antiproliferative and proapoptotic effects of EGFR inhibition in malignant human glioma cell lines. A clinical application of combination therapy targeting both EGFR and IGF-IR could be a promising therapeutic strategy.

Neuro-Oncol. 2009 Oct;11(5):597.

147. Targeted Delivery of Chemotherapy by Localized Opening of the Blood-Brain Barrier using Photochemical Internalization

Henry Hirschberg, Francisco Uzal, Michael Gach, Steen Madsen

INTRODUCTION

The therapeutic goal following surgical resection is the elimination of infiltrating tumor cells remaining in the margins of the resection cavity, while minimizing damage to normal brain. Presently, this goal has proven difficult to reach, since infiltrating tumor cells in normal brain are protected by the blood-brain barrier (BBB). Photochemical internalization (PCI) is a novel laser light-mediated photodynamic therapy technology that can enhance the delivery of macromolecules in a site-specific manner and therefore can be used to a target BBB opening in limited regions of the brain.

METHODS

PCI-mediated delivery of Clostridium perfringens prototoxin (Clp), an agent know to open the BBB, was performed on normal Fisher rats. Surface-light irradiation was given approximately 60 min after Clp administration at increasing light levels. Postcontrast T1 MRI scans were used to monitor the degree of localized BBB disruption. F98 tumor cells were implanted in the brain to mimic infiltrating tumor cells. The animals were treated 24 h later with PCI-mediated BBB opening followed by the i.v. administration of bleomycin.

RESULTS

PCI mediated BBB opening at low-light levels demonstrated significant MRI enhancement localized to the illuminated region of the brain. The BBB disruption was reversible, and no tissue damage was observed on histological sections taken 3 weeks posttreatment. No effect was observed if photosensitizer and light was given in the absence Clp. The survival of animals implanted with F98 tumor cells was significantly extended following PCI-BBB opening and bleomycin therapy compared with bleomycin-only controls; 30% of implanted animals never developed tumors.

CONCLUSION

PCI was highly effective in opening the BBB in a limited and targeted region of the brain in a transitory manner. PCI-mediated BBB opening clearly increased the efficacy of bleomycin therapy in tumor-bearing rats.

Neuro-Oncol. 2009 Oct;11(5):597.

148. Selection of a Linear Heptapeptide that Binds Interleukin 13 Receptor Alpha 2 at a Site Distinct from the Native Ligand

Hetal Pandya, Shivank Garg, Denise M Gibo, Steven Kridel, Waldemar Debinski

INTRODUCTION

One of our goals is to identify specific delivery vectors to glioblastoma multiforme (GBM). Interleukin 13 receptor-α2 (IL-13Rα2), an IL-13 plasma membrane receptor, is overexpressed in >75% of GBM patients. Here, we explored whether smaller peptides could be identified that bind IL-13Rα2 specifically. Smaller compounds may offer better uptake by tumors from circulation or better distribution when used with convection-enhanced delivery.

METHODS

To isolate the peptides binding to IL-13Rα2, we screened a cyclic heptapeptide phage display library, Ph.D-C7C (New England Biolabs) with a diversity of 1.2 × 109 different peptides. The phage library was repetitively bio-panned using GBM cell lines, which overexpress or do not express IL-13Rα2.

RESULTS

We identified three different peptide phage clones binding IL-13Rα2 using various ELISA experiments. Peptide#1 phage demonstrated high affin-ity and specificity to the IL-13Rα2 protein. Peptide#1 was synthesized in disulfide-constrained and linear forms, with or without biotin attached. The experiments with the synthetic peptides reproduced those obtained with peptide phages. The linear Peptide#1 exhibited higher binding to the receptor than the disulphide-constrained peptide, as expected from reducing/nonreducing condition assays performed previously, but unexpectedly considering the nature of the phage library. Furthermore, an excess of IL-13, unlike antibody to the receptor, did not block the peptide’s binding to IL-13Rα2. The affinity of Peptide#1 was found to be in the high nanomolar, low micromolar range. Also, fluorescent binding experiments confirmed that the linear Peptide#1 binds to GBM cells with greater affinity than the disulphide-constrained peptide.

CONCLUSION

We were able to select a specific linear heptapeptide from a cyclic phage-display library that binds to IL-13Rα2. This peptide binds to the receptor at a site other than that of the native ligand-binding site. The Peptide#1 has potential to be further developed for diagnostic, imaging, and therapeutic interventions for GBM.

Neuro-Oncol. 2009 Oct;11(5):597–598.

149. Trans Sodium Crocetinate Increases Brain Tumor Oxygenation

Jason P Sheehan, Jonathan H Sherman, Christopher Paul Cifarelli, Kasandra Dassoulas, Claire Olson, Jessica Rainey, Shaojie Han

INTRODUCTION

Glioblastoma multiforme (GBM) tumors typically exhibit regions of hypoxia. Hypoxic regions within the tumor make cells less sensitive to radiosurgery and radiation therapy. Trans sodium crocetinate (TSC) has been shown to be a radiosensitizer. The goal of this research was to elucidate the underlying mechanism of TSC’s radiosensi-tizing effect.

METHODS

A rat C6 glioma model was utilized. C6 glioma cells were stereotactically injected into the rat brain to create a tumor. Two weeks later, MRI was used to confirm the presence of a glioma. Following demonstration on MRI of a brain tumor, animals were randomized into one of two groups: 1) TSC alone (100 mcg/kg) or 2) saline control. Licox probes were inserted into the brain tumor and contralateral cerebral hemisphere. Tissue oxygenation measurements were recorded pre- and postintravenous infusion of either TSC or saline.

RESULTS

Not surprisingly, tissue oxygenation measurements revealed that the brain tumor was hypoxic relative to the contralateral cerebral hemisphere brain tissue. Two to eight minutes after TSC was infused, tissue oxygenation measurements in the brain tumor increased above baseline by as much as 60%. After this temporary elevation following TSC infusion, tumor oxygenation measurements returned to baseline. No significant elevations in tissue oxygenation were seen on the contralateral side. Similarly, the saline vehicle was not observed to increase tissue oxygenation in either the brain tumor or the contralateral brain tissue.

CONCLUSION

TSC transiently improves tissue oxygenation in hypoxic gliomas. Such an effect is one potential mechanism for the radiosensitization previously observed by TSC.

Neuro-Oncol. 2009 Oct;11(5):598.

150. Potentiation of Etoposide and Temozolomide Cytotoxicity by Curcumin and Turmeric Force in Brain Tumor Cell Lines

Cheppail Ramachandran, Anna P Resek, Anat Aviram, Enrique A Escalon, Ziad Khatib, Steven J Melnick

INTRODUCTION

Chemotherapeutic options for the treatment of brain tumors, especially glioblastomas, are very much limited because of blood-brain barrier and the resistance of tumor cells to most of the chemotherapeutic drugs.

METHODS

Cytotoxicity studies were performed with etoposide (ETP), temozolomide (TMZ), curcumin, Turmeric Force, and their combinations (72 h) in U-87MG glioblastoma and D-283 medullo-blastoma cell lines using the MTT assay. The surviving cells (percentage of untreated control) at various drug concentrations were analyzed by CalcuSyn software to ascertain the synergism, additivity, or antagonism between the combination treatments.

RESULTS

U-87 MG glioblastoma cell line is generally resistant to several cancer drugs including ETP and TMZ. Curcumin and Turmeric Force have IC50 values of 37.33 and 30.75 μg/ml, respectively, against this cell line. When curcumin or Turmeric Force were combined with ETP and TMZ, increased chemotherapeutic efficiency was noted. Turmeric Force as a single agent is highly potent against D283 cell line with IC50, IC75, and IC90 values of 1.55, 5.10, and 7.00 μg/ml, respectively. Although both curcumin and Turmeric Force potentiate ETP and TMZ cytotoxicity, Turmeric Force appears to be a better adjuvant than pure curcumin based on combination index values. CalcuSyn analysis of cytotoxicity data showed that Turmeric Force acts synergistically with ETP and TMZ with combination index (CI) values of 0.383 and 0.569, respectively, at IC50 level in U87 glioblastoma cell line. The CI values for ETP + Turmeric Force and TMZ + Turmeric Force combinations are 0.041 and 0.004, respectively, indicating very strong synergism in D283 medulloblastoma cell line. The synergistic effect of curcumin with ETP and TMZ was inferior to Turmeric Force.

CONCLUSION

Turmeric Force may be useful as an adjuvant agent with ETP and TMZ for brain tumor chemotherapy, since the combination regimen appears to be more effective in brain tumor cell lines than either agent alone.

Neuro-Oncol. 2009 Oct;11(5):598.

151. The Expression Level of Hypoxia-Activated Proteins in A Rodent Glioma Model in Response to Two Drug Delivery Methods

Akihide Kondo, Stewart Goldman, Rishi R Lulla, Veena Rajaram, Marcelo B Soares, Tadanori Tomita

INTRODUCTION

Recent evidence has shown that tumor cells in hypoxic conditions may survive and proliferate by activation of diverse signaling pathways, otherwise termed “death resistance” or “malignant mutation.” These changes may result in poor response to treatment, development of metastases, and recurrence of cancer. Though some studies have investigated chemotherapy’s effect on hypoxia-induced pathways, no previous work has considered the effect of different drug delivery methods for chemotherapy. In our study, we evaluated tumor control, morphology, and expression level of hypoxia activated proteins in a rodent glioma model in response to two drug delivery methods.

METHODS

Rat 9L gliosarcoma cells were transduced with the luciferase gene, permitting cells to be monitored using bioluminescence imaging (BLI). Fifty thousand transduced tumor cells were inoculated into the right striatum of 15 Fisher 344 rats. Seven days after inoculation, animals were randomly divided into three groups. Group 1 received no treatment. Group 2 received 60 mg/kg of carboplatin via single i.p. bolus. Group 3 received 10 mg/kg of carboplatin i.p. five times per week (metronomic administration). Tumor growth was measured every 2–3 days with BLI. At 28 days after treatment, tumor tissues were harvested and evaluated. We examined tumor morphology, vascularity, and proliferation using immunohistochemical stains and expression levels of hypoxia-activated proteins HIF-1α, cMet, and ERK1/2 with Western blot analysis.

RESULTS

Both bolus and metronomic administration of carboplatin controlled tumor growth in our model. However, expression levels of hypoxia-activated protein HIF-1α, cMet, and ERK1/2 were lower in tumors treated with metronomic chemotherapy.

CONCLUSION

Our data suggests that bolus administration of carboplatin may cause unfavorable oxygen conditions in the tumor bed and result in activation of hypoxic pathways leading to death resistance and malignant mutation. Metronomic chemotherapy may provide a therapeutic benefit in this regard.

Neuro-Oncol. 2009 Oct;11(5):598.

152. Human Schwannomas Express Activated Platelet-Derived Growth Factor Receptors and CKIT and are Growth Inhibited by Gleevec (Imatinib Mesylate)

Joydeep Mukherjee, Deepak Kamnasaran, Anandh Balasubramaniam, Ivan Radovanovic, Gelareh Zadeh, Tim-Rasmus Kiehl, Abhijit Guha

INTRODUCTION

Schwannomas, although benign, can be fatal or give rise to significant morbidity due to an unpredictable growth rate. They can reoccur after surgery or radiation-current treatments, each with inherent significant risks. These risks are further amplified in neurofibromatosis type2 (NF2), a germline predisposition syndrome characterized by multiple schwannomas, underlying the need of biological targeted therapies.

METHODS

Gleevec (STI571, imatinib mesylate), in addition to the bcr-abl oncogene in chronic myelogenous leukemia, inhibits c-kit and platelet-derived growth factor receptor (PDGFR) signaling, thereby expanding its use to several malignant and benign human diseases.

RESULTS

In the present study, human sporadic and NF2-associated schwannomas had increased expression along with activation of PDGFR-α, PDGFR-β, and c-kit receptors, compared with normal or traumatic nerve. Using the human NF2-null HEI-193 schwannoma cell line, Gleevec inhibited schwannoma viability, proliferation, and anchorage-independent growth, as well as induced apoptosis in a dose-dependent manner (IC50, 5–10 μmol/L). These antitumorigenic effects were correlated with inhibition of PDGFR-α, PDGFR-β, and c-kit activation/phosphorylation and major downstream signaling pathways.

CONCLUSION

Lack of robust xeno-graft or transgenic models of schwannomas prevents extension of these studies in vivo. However, the established and long track record and tolerable toxicity of Gleevec already in clinical use plus our preclinical data indicate that Gleevec should be evaluated in human schwannomas with proven progressive growth.

Neuro-Oncol. 2009 Oct;11(5):598–599.

153. Chimeric HSV, C134, Produces Superior Preclinical Results for Oncolytic HSV Therapy of Malignant Gliomas

Kevin A Cassady, David F Bauer, Gregory K Friedman, James M Markert, G Yancey Gillespie

INTRODUCTION

The chimeric herpes simplex virus (HSV), C134, is a novel Deltagamma134.5-based oncolytic HSV that is capable of improved late viral protein synthesis and viral replication in tumors, while maintaining the avirulence of a Deltagamma134.5 vector. Previous studies have shown that C134-treated animals show improved survival and tumor reduction over nonchimeric Deltagamma134.5 recombinants in both xenogeneic human gliomas and syngeneic murine gliomas implanted orthotopically. A principal advantage of the C134 chimeric HSV over Delta-gamma134.5 in brain tumor therapy is its improved replication and direct oncolytic activity in the tumor. C134 has improved survival or reduced tumor volumes over other Deltagamma134.5 HSVs in both syngeneic and xenogeneic tumor models (U87, U251, and N2A).

METHODS

We performed a survival study and murine and nonhuman primate safety studies (lethal dose, viral recovery, and immunohistochemistry). Explant xenograft infection was evaluated using immunohistochemistry and flow cytometry.

RESULTS

Chimeric HSV C134 was more effective than Delta-gamma134.5 in additional tumor models. The virus infected, replicated, and spread in CD133+ human glioma xenografts better than did several Deltagamma134.5 viruses. The virus also improved survival of nude mice implanted with a glioma cell line (D54MG), in which C134 did not possess a significant in vitro replication advantage (<1 log) over Deltagamma134.5 viruses. The virus was safe in additional neurotoxicity studies (immunohistochemistry, virus recovery) in mice and in the highly sensitive nonhuman primate Aotus nancymae.

CONCLUSION

These studies confirm that the C134-recombinant overcomes the major limitation of inadequate late gene expression characteristic of Deltagamma134.5 HSVs, resulting in a markedly improved oncolytic HSV. These studies provided enough proof of efficacy and neurosafety in two animal models that the National Institutes of Heath Rapid Access to Interventional Development (NIH-RAID) program has approved production and certification of cGMP C134 necessary for FDA and investigational new drug approval. When RAID-produced and -qualified C134 becomes available, we will proceed with phase I clinical testing in patients with recurrent anaplastic gliomas.

Neuro-Oncol. 2009 Oct;11(5):599.

154. Susceptibility of Malignant Peripheral Nerve Sheath Tumors to Gamma-1-34.5 Herpes Simplex Virus can be Enhanced by CMV IRS-1 Gene Expression

Gregory K Friedman, Kevin A Cassady, Jacqueline N Parker, Steven L Carroll, James M Markert, G Yancey Gillespie

INTRODUCTION

HSV oncolytic therapy has shown promise in various neurological tumors. We investigated its role in malignant peripheral nerve sheath tumors (MPNST), the most common malignancy occurring in neurofibromatosis type 1.

METHODS

Relative infectivity of wild-type or green fluorescent protein (GFP)-expressing γ-1-34.5-deleted herpes simplex virus (HSV) (C101 or C154) 30 h postinfection was quantified by fluorescence-activated cell sorting (FACS) in two human and murine cell lines. Cytotoxicity was measured by the MTT assay 72 h postinfection.

RESULTS

Infection rates with C101 were 55.7% of ST88-14 cells vs. 6.7% for STS-26T (human) and 48.3% for A18 vs. 0.8% for A202 (murine). Modification of γ-1-34.5-deleted HSV-GFP to incorporate the cytomegalo-virus (CMV) gene IRS1 (C154), which enhances virus replication in tumor cells by encoding a HCMV PKR-evasion gene, improved infectivity: 87.0% of ST88-14 cells vs. 17.0% of STS-26T cells. Notably, absolute infectivity produced by HSV C154 was similar to that seen after infection with wild-type virus in both the human and murine tumor cells. Unexpectedly, when infecting cells with the original γ-1-34.5-deleted HSV, R3616, viral recovery was higher in ST88-14 than in STS-26T, consistent with infectivity using C101; however, cytotoxicity with R3616 was similar among the human cell lines. The basis for this imperfect correlation between infectivity and cytotoxicity is being defined, but may be partly related to tumor genotype, for example, activation status of PKR, MAPK, or p38. Activated p38 detected in uninfected cells correlated with infectability in ST88-14 and A18 cell lines (present in both) and STS-26T (absent), suggesting that early infectability may be enhanced by preinfection p38 activation.

CONCLUSION

These studies demonstrate that infectivity of MPNST cells to engineered HSV is improved with the chimeric virus C154. Differential sensitivities to γ-1-34.5-deleted HSV by both human and murine MPNST cell lines can be exploited for future studies to define molecular bases for sensitivity and resistance to engineered HSV therapy.

Neuro-Oncol. 2009 Oct;11(5):599.

155. A Potential New Therapeutic Agent: CCN3 Can Suppress Glioma Growth Through Inhibition of Notch Signal Transduction

Kartik Krishnan, Holly Dobrenen, Stacey Rittmueller

INTRODUCTION

In an effort to identify targets for directed treatment of brain tumors, we identified the secreted protein CCN3 as turned off during oncogenic transformation. The work described here seeks to elucidate and validate CCN3 as a potential novel antitumor target in brain tumors.

METHODS

In these studies, we used the established glioma cell lines, U87, U118, and 1321N1. The CCN3 cDNA was purchased, cloned into an inducible expression vector, and transfected using FuGene 6 (Roche, Indianapolis, IN). Growth analysis was accomplished using the ViCell cell counting apparatus (Beckman-Coulter, Fullerton, CA). Molecular studies were performed using manufacturers’ and previously reported techniques, including RT-PCR (NEB, Ipswich, MA), Western immunoblot (Invitrogen, Carlsbad, CA), and qPCR (ABI, Foster City, CA).

RESULTS

First, overex-pression of CCN3 in glioma cell lines resulted in their growth suppression. Further, treating these cells with media containing CCN3 also resulted in slower growth. Moreover, we found that CCN3 treatment of glioma cells resulted in inhibition of the Notch signal transduction pathway. The inhibition of Notch signaling by CCN3 is equivalent to the inhibition achieved by γ-secretase inhibitors. In addition, we found that the IGF domain of the CCN3 molecule may be able to slow cancer cell growth by itself. Finally, we have begun to investigate the ability of CCN3 to slow tumor growth in mouse models. Preliminary results of these experiments are promising.

CONCLUSION

The studies reported here demonstrate that CCN3 can slow the growth of glioma cell lines in vitro through inhibition of Notch signal transduction. While Notch signaling has been shown to play a role in some brain tumors, especially medulloblastoma, its role in glial neoplasms is not well explored. Work presented here demonstrates that Notch signaling may be a potent new target in the brain tumor treatment.

Neuro-Oncol. 2009 Oct;11(5):599.

156. Chemotherapeutic Ablation of Recruited Endogenous Glial Progenitors and its Implications on Tumor Recurrence and Survival in a PDGF-Driven Rodent Glioma Model

Kim A Lopez, Aaron M Tannenbaum, Marcela Assanah, Katy Linskey, Peter D Canoll, Jeffrey N Bruce

INTRODUCTION

Platelet-derived growth factor (PDGF)-driven animal glioma models have shown the significant contribution of endogenous glial progenitors, recruited through paracrine growth factor signaling, to the cellular composition of gliomas. In this study, by administering topo-tecan via convection-enhanced delivery (using Alzet osmotic mini-pumps), we sought to determine the effects of chemotherapy on both the tumor-initiated as well as the recruited populations in a rodent glioma model.

METHODS

The PDGF-IRES-GFP retrovirus was used to infect white matter glial progenitors, inducing them to overexpress PDGF (as well as label them with GFP). We then used PDGFR-a as the putative marker for recruited cells (i.e., GFP[−]/PDGFR-a[+]). Pumps were implanted at 7 days postinjection of retrovirus.

RESULTS

In phosphate buffered saline-treated animals, GFP(+) cells/HPF increased ~4-fold from day 1 to day 7 post-implantation. This increase coincided with an ~4-fold increase in recruited progenitors. In topotecan-treated animals, GFP(+) cells/HPF decreased ~10-fold from day 1 to day 7, and recruited progenitors decreased ~80-fold. Topotecan provided a significant survival advantage. Longer survival was associated with longer treatment duration (i.e., 7 days > 4 days > 1 day). Locoregional ablation of glial progenitors related anatomically to the distribution of gadodiamide as seen on MRI. Progenitors were depleted ipsilateral to the site of topotecan infusion up to the corpus callosum. Viable progenitors were identified distal to the corpus callosum at the region of the contralateral ventricle. Despite significant decreases in tumor-initiating GFP(+) cells upon treatment, tumors recurred in all animals. Recurrent tumors had an increased proportion of GFP(+) cells concentrated in central regions of the tumors, while more recruited cells were observed at the infiltrative tumor edge.

CONCLUSION

Convection-enhanced delivery (CED) of topotecan is an effective treatment in our tumor model, and survival is linked to the ability of topotecan to ablate both tumor-initiating and recruited cells. Regions of progenitor ablation corresponded with the predicted anatomical distribution of topotecan. While topotecan CED is effective, all treated tumors recurred. Histopathologic changes in recurrent tumors point to a decrease in recruitment, most likely due to chemothera-peutic ablation of the recruitable progenitor pool.

Neuro-Oncol. 2009 Oct;11(5):599.

157. Aurka is Associated with Survival Benefit and Cell Growth Suppression in Glioblastoma

Valerie Nicole Barton, Andrew M Donson, Nicholas K Foreman, Diane Birks, Rajeev Vibhakar

INTRODUCTION

Despite advances in therapy, the outcome of glioblastoma multiforme (GBM) tumors remains poor. The focus of neu-rooncology research has thus turned towards identifying novel therapeutic targets. Aurora kinase A (AURKA) regulates chromosome segregation and cytokinesis. Because defects in these processes can lead to genetic abnormalities associated with cancer, AURKA is a suspected oncogene that is implicated in many types of cancer. However, the role of AURKA in glioblastoma cell growth and survival benefit is undefined.

METHODS

AURKA gene expression in GBM patients and cell lines was measured using microarray technology and quantitative real time PCR (qRTPCR), respectively. The relationship between AURKA gene expression and patient outcome was analyzed using a Kaplan-Meier estimate of survival. Cell proliferation was measured using a MTT assay after adding an AURKA inhibitor at different concentrations. Cell cycle analysis was analyzed by flow cytometry using propidium iodide.

RESULTS

AURKA mRNA was overexpressed in GBM patients compared with normal controls (13-fold, p = 4.3E-9) as measured by microarray. Glioblastoma patients with low AURKA mRNA expression had a more favorable prognosis than those patients with high AURKA expression (p = 0.05). AURKA expression in GBM cell lines was confirmed by qRTPCR and Western blot analysis. Inhibition of AURKA induced cell death in all cell lines. Cell arrest at G2/M phase was significantly increased in cell lines treated with the AURKA inhibitor.

CONCLUSION

Our data suggest that AURKA expression may serve as a prognostic marker in glioblastoma. Suppression of glioblastoma cell growth by C1368 indicates that AURKA inhibitors warrant further investigation as a novel therapeutic target.

Neuro-Oncol. 2009 Oct;11(5):600.

158. Mapping Invasion at Tumor Boundaries using Diffusion-Weighted MRI

Peter S LaViolette, Benjamin M Ellingson, Scott D Rand, Jennifer M Connelly, Mark Malkin, Kathleen M Schmainda

INTRODUCTION

The degree of tumor invasion is difficult to assess in many types of brain cancer and is thought to be the primary reason for failure of surgical excision and focal radiation therapy. Diffusion weighted imaging (DWI) measures the movement of water molecules and has been described as an indirect proxy of cellular density. Based on a recent report, we hypothesized that diffusion characteristics at the edge of a contrast-enhanced tumor may be useful for quantifying the degree of tumor invasiveness, and these characteristics may potentially guide surgical resection and radiation planning and predict locations of radiographic recurrence.

METHODS

One patient with clinical MRI, including DWI, was analyzed for this study. DWI was used to compute apparent diffusion coefficient (ADC) maps. A region of interest was drawn within the surgical boundary and then isotropically inflated in 1-mm steps. At each increment, ADC values were sampled. Spatial differences in ADC were calculated by comparing sampled ADC values with those in the previous spatial increment. The resulting spatial diffusion differences were then compared with follow-up functional diffusion maps (FDMs) (shown to be predictive of tumor cell invasion) at the time of disease progression, 4 months later, using an ADC threshold 0.4 × 10−3 mm2/s.

RESULTS

This technique clearly demonstrated regions of spatially decreasing ADC values in regions that later showed a lower ADC, consistent with an increase in cellularity at time of recurrence. These results were consistent with a less-defined tumor boundary and increased invasiveness.

CONCLUSION

This study introduces a new technique for quantifying degree of tumor invasion. Unlike FDMs, spatially changing ADC values are calculable using only one time point. Though more cases need to be assessed, this technique shows promise as a noninvasive biomarker for detecting sites of tumor burden that are not visible with standard anatomic MRI.

Neuro-Oncol. 2009 Oct;11(5):600.

159. Liposomal Platinum Compounds to Improve the Concomitant Treatment of F98 Glioma Cells with Ionizing Radiation

Gabriel Charest, Léon Sanche, Benoit Paquette, David Mathieu, David Fortin

INTRODUCTION

Concomitant radiotherapy with platinum compounds to treat patients with glioblastoma has been hampered by severe adverse effects. To limit them, a potential approach consists in incorporating the platinum agents in a liposome.

METHODS

In this study, Lipoplatin and Lipoxal (the liposomal formulations of cisplatin and oxaliplatin) as well as carboplatin, oxaliplatin, and cisplatin were tested on the F98 glioma cells for their ability to improve the cell uptake and increase the synergic effect when combined with ionizing radiation. These platinum agents were also assessed in Fisher rats implanted with a F98 malignant glial tumor.

RESULTS

Our in vitro results demonstrated that Lipoplatin, the liposomal formulation of cisplatin, improved the cell uptake by 3-fold, and its radiosensitizing potential was enhanced by 14-fold, while treatment with cisplatin did not result in a synergic effect. With Lipoxal, a synergic effect was measured only when incubated at a concentration higher than its IC50. The concomitant treatment of F98 cells with carboplatin and radiation produced the highest radiosensitizing effect (30-fold increase). Our preliminary data also showed that Lipoplatin and Lipoxal considerably reduced the toxicity observed with their liposome-free analogs and improved the median survival of Fisher rats implanted with a F98 tumor.

CONCLUSION

Among the five platinum compounds tested, Lipoplatin and carboplatin showed the best radiosensitizing effects in vitro. Lipoplatin seems the most promising, since it led to the best cellular incorporation and also reduced the toxicity in the Fischer rat implanted with a F98 tumor and significantly increased the median survival.

Neuro-Oncol. 2009 Oct;11(5):600.

160. Sorafenib as a Novel Therapeutic Option For Low-Grade Glioma

Marc Remke, Till Milde, Dominik Sturm, Aurélie Ernst, Hendrik Witt, Anna Schöttler, Christel Herold-Mende, Andreas E Kulozik, Andrey Korshunov, Olaf Witt, Peter Lichter, Stefan Pfister

INTRODUCTION

Low-grade gliomas (LGG) comprise the most common primary brain tumors in children. Despite excellent overall survival rates reported for LGG patients, recurrent disease remains a great clinical challenge, mostly due to low efficacy of current treatment strategies encompassing adjuvant radiation and/or chemotherapy. Tandem duplications leading to B-raf oncogene (BRAF) fusion transcripts with constitutive kinase activity and activating mutations of BRAF have recently been identified by us and others as a hallmark of juvenile pilocytic astrocytoma (JPA) tumorigenesis, and additional mechanisms leading to activating of the MAPK pathway are suspected in LGG. We therefore investigated the therapeutic potential of sorafenib (BAY 43-9006), a multikinase inhibitor with selectivity against BRAF among other targets, in pediatric and adult LGG.

METHODS

Antiproliferative effects of sorafenib alone and in combination with the HDAC inhibitor valproic acid and/or temozolamide were evaluated in a panel of LGG cell lines after 12- to 140-h exposure using MTS assays. Sorafenib in vitro testing included concentrations from 0.1 to 20 μM. To measure cell cycle distribution and apoptosis induction upon inhibitor administration, we performed flow cytometry analyses. Activation of MAPK and PI3K pathways was assessed by Western blotting.

RESULTS

Proliferation of all LGG cell lines was effectively blocked by sorafenib administration. Intriguingly, antiproliferative effects were most prominent in the JPA-derived cell line NCH492 with an IC50 of 4 μM, a concentration well within the range achievable in patients. In addition, flow cytometry analysis revealed a G2/M arrest without induction of apoptosis. Notably, additional therapeutic effects were observed upon coadministration of sorafenib with valproic acid and/or temozolomide. As a likely underlying mechanism of sorafenib-induced cell cycle arrest, we identified effective abrogation of ERK1/2 phosphorylation, the final effector of BRAF-mediated MAPK activation.

CONCLUSION

We have demonstrated direct antiproliferative effects of sorafenib against LGG cell lines at concentrations that are readily achieved in patients. As a next step, testing of sorafenib using in vivo xenograft models currently in development may thus provide a further rationale for the use of this targeted therapy in patients with LGG.

Neuro-Oncol. 2009 Oct;11(5):600.

161. Novel Modifications in Interleukin 13 Molecule for Specific Targeting of GBM

Waldemar Debinski, Tiefu Liu, Denise M Gibo

INTRODUCTION

huIL13-PE38QQR, a wild type interleukin 13-based and Pseudomonas exotoxin A-containing cytotoxin demonstrated efficacy in clinical trials in patients with recurrent glioblastoma multiforme (GBM). IL-13 is a compact core-bundle of four antiparallel α-helices (A–D), and we have previously engineered the cytokine to bind specifically to its GBM-associated receptor by substitution mutagenesis in the helices A, C, and D. In the current study, we circularly permutated (cp) IL-13 at the helix B, C, and D in an attempt to position the fused to the cytokine toxin moieties in varying spacial configurations. We also used diphtheria toxin (DT390) that can be fused to IL-13 at its C-terminal end.

METHODS

We have cloned DT.IL-13 cytotoxins in which the new N-termini start at the residues preceding the B, C, and D helices. We have produced these recombinant cytotoxins in a prokaryotic protein expression system and purified to homogeneity using FPLC. The cytotoxins were tested in cell viability assays on cells expressing various levels of the IL-13 receptor-α2 (IL-13Rα2), the GBM-associated receptor for the cytokine.

RESULTS

DT.cpBIL-13 and DT.cpDIL-13 cytotoxins effectively targeted and killed IL-13Rα2 expressing GBM cells, as did the wild type IL-13-based cytotoxin. Their actions were neutralized in the presence of an excess of IL-13 alone. However, we could not generate an active DT.cpCIL-13 cytotoxin that included the preservation or nonpreservation of the connecting loop of α-helix C. This is likely related to the presence of a disulphide bond involving a connecting loop between helices B and C.

CONCLUSION

We have successfully obtained DT.IL-13 cytotoxins in cpB and cpD helix forms. The cpDIL-13 form represents the first successful attempt at making any cytokine of this topology have the N-terminal end preceding the D helix. The potency and specificity of the new cpIL-13 cytotoxins warrant further preclinical evaluation.

Neuro-Oncol. 2009 Oct;11(5):600–601.

162. Using the Natural Remedies Zeng Sheng Ping and Curcumin to Treat Medulloblastoma and Glioblastoma

Kah Jing Lim, Anirban Maitra, Savita Bisht, Eli E Bar, Charles G Eberhart

INTRODUCTION

We attempted to treat malignant brain tumor cells with zeng sheng ping (ZSP), a traditional Chinese medicine composed of six herbs, and curcumin, a compound derived from the Indian spice turmeric.

METHODS/RESULTS

ZSP extracts were found to cause a 73% reduction in growth of the glioblastoma cell line U87 and an 81% reduction in the medulloblastoma cell line DAOY. However, ZSP was also found to reduce viability in human cortical neuronal neurospheres as well as NIH 3T3 cells, raising the possibility that the effects lack specificity. We are therefore currently investigating whether ZSP can prevent engraftment of DAOY cells in nude mice. Curcumin was delivered in a nanoparticle- encapsulated formulation (nanocurcumin) to increase solubility and bioavailability. Nanocurcumin caused a dose-dependent decrease in cell growth measured by MTT in multiple brain cancer cell lines. Growth reductions using 10 μM nanocurcumin concentrations were 49% for DAOY, 87% for PFSK, and 31% for U87. A GBM neurosphere line (HSR-GBM1) had its growth inhibited 44%, reduced by 20 μM nanocurcumin. Importantly, the same doses of nanocurcumin did not cause a comparable inhibition in the growth of NIH 3T3 cells or nonneoplastic human fetal cortical neurospheres. The reductions in viable cell mass that we observed were associated with a combination of G2/M arrest and induction of apoptosis. The proportion of G2/M cells increased between 25% and 106% in the various lines, while the percentage of apoptotic cells increased at least 2-fold. Curcumin has been suggested to act by regulating Notch signaling activity and expression of Bcl2 and p21. We did not find significant changes in the expression of Notch signaling pathway targets or of Bcl2 and phospho-Akt following nanocurcumin treatment of brain tumor cell lines, but P21 protein levels were somewhat increased.

CONCLUSION

In summary, our data suggest that both ZSP and curcumin can inhibit the growth of malignant brain tumor cells.

Neuro-Oncol. 2009 Oct;11(5):601.

163. Convection-Enhanced Delivery of Etoposide Prolongs Survival in a Retrovirally Induced Rat Glioma Model

Aaron M Tannenbaum, Katy Linskey, Kim A Lopez, Marcela Assanah, Peter D Canoll, Jeffrey N Bruce

INTRODUCTION

Topoisomerase inhibitors are potentially effective drugs for the treatment of malignant gliomas because of their specificity for rapidly dividing cells. Etoposide inhibits the action of topoisomerase IIa, causing single- and double-stranded DNA breaks, which eventually lead to apoptosis. Systemic administration of etoposide has dose-limiting toxicity due to its inability to cross the blood-brain barrier (BBB). Convection-enhanced delivery (CED) provides a means to bypass the BBB and administer chemotherapeutics directly into the tumor bed. Previous work from our laboratory has demonstrated the in vivo safety and effectiveness of CED of topotecan, a topoisomerase I inhibitor. In this study, we set out to determine whether targeting topoisomerase IIa by CED of etopo-side will incur a similarly safe and robust therapeutic effect.

METHODS

As previously described, PDGF-IRES-GFP retrovirus was injected into the subcortical white matter of adult rats. At 7 days postinjection (dpi), animals were treated by continuous CED of either etoposide (272 μM) or vehicle (<0.5% dimethyl sulfoxide in phosphate-buffered saline) for 7 days (2 ml, 10 μl/h). Animals were killed at first sign of tumor burden.

RESULTS

CED of etoposide was well tolerated, as all animals survived treatment. Etoposide-treated animals showed a significant survival advantage versus control (median = 61 dpi vs. 16 dpi; p < 0.0001). Four of 15 animals were regarded as long-term survivors (>100 dpi).

CONCLUSION

Targeting topoisomerase IIa by CED of etoposide is safe and effective in a preclinical rodent glioma model. Animals given etoposide had a significant survival advantage over vehicle-treated controls, and in some cases, etoposide treatment led to long-term survival. Future studies should explore whether the combined inhibition of topoisomerases I and IIa would provide a more robust therapeutic benefit than monotherapy. Furthermore, reproducibility of tumor recurrence in our model may lend itself to studying recurrence mechanisms associated with topoisomerase inhibition.

Neuro-Oncol. 2009 Oct;11(5):601.

164. The Ketogenic Diet as an Adjuvant Therapy for Malignant Gliomas

Adrienne C Scheck, Mohammed G Abdelwahab, Phillip Stafford, Do-Young Kim, Katie Fenton, Deepti Boddupalli, Sergey V Kushchayev, Mark C Preul, Jong M Rho

INTRODUCTION

The high-fat, low-carbohydrate ketogenic diet (KD) is an established nonpharmacologic treatment for refractory epilepsy and has more recently been suggested as a treatment for malignant brain tumors. The goal of our studies is to demonstrate the mechanism(s) of tumor inhibition and determine if the KD can potentiate the action of radiotherapy and chemotherapy in vivo.

METHODS

An immunocompetent, syngeneic intracranial tumor model was used to study the effect of the KD as an adjuvant to standard therapy in vivo. GL261 cells were stably transfected with luciferase (GL261-L1 cells) and stereotactically implanted into the right hemisphere of albino C57Bl/6 mice. Animals were fed a standard diet (SD) for 3 days, after which half of the animals were randomized to the ketogenic Bio-Serv F3666 diet (Bio-serv, Frenchtown, NJ). Tumors were imaged in vivo every 3 days.

RESULTS

Median survival of animals fed KD was 25 days compared with 19 days for animals on SD. Median survival for animals given combination therapies were: KD plus radiation, 37 days; KD plus temozolomide, 38 days; SD plus radiation, 31 days; SD plus temozolomide, 32 days (p < 0.0001). In vivo imaging of reactive oxygen species (ROS) suggested a reduction in ROS in the tumors of animals fed KD. Gene expression profiling analysis of tumor and nontumor tissue from animals fed SD versus KD suggest the KD is driving gene expression back toward nontumor conditions. Individual genes upregulated in tumor tissue from SD versus KD include Rex2, RIK, HTF4, EIF4, LIN7, and RAD54. Genes upregulated in tumor tissue from KD versus SD include H2-Aa, EGR3, SYNDAPIN, NF66, and HPA1. Genes downregulated in tissue from animals fed KD relative to animals fed SD included COX2, HSP70, FOSB, and insulin-like growth factor 1 (IGF1).

CONCLUSION

These data suggest the utility of combining the KD with standard therapies for the treatment of brain tumors.

Neuro-Oncol. 2009 Oct;11(5):601.

165. Deep Sequencing of the DNA Methylome of Glioblastomas

Raman P Nagarajan, Ting Wang, Brett E Johnson, Chibo Hong, Shaun Fouse, David Haussler, Martin Hirst, Marco A Marra, Joseph Costello

INTRODUCTION

Alterations in the methylation status of CpG sites in DNA play an important role in the incidence, growth rate, and therapeutic response of glioblastoma multiforme (GBM). There are 29 million CpG sites in the human genome, yet current assays being applied to GBM assess less than 30,000 sites, primarily in canonical promoters. To move methylation mapping beyond these small-scale and solely promoter-based sampling methods, we designed two new complementary, genomewide methods (MeDIP-seq and MRE-seq) to characterize GBM methylomes. Together these methods allow the discovery of epigenetic alterations in GBM at an unprecedented depth.

METHODS

For methyl-DNA immu-noprecipitation-high throughput sequencing (MeDIP-seq), sonicated DNA was enriched for methylated fragments by immunoprecipitation with a monoclonal antibody against methylcytidine, followed by sequencing with an Illumina Genome Analyzer II (IGAII). In methyl-sensitive restriction enzyme-seq (MRE-seq), unmethylated CpG sites were identified at single CpG resolution by digesting DNA with HpaII, Hin6I, and AciI, size selecting fragments, generating a library, and IGAII sequencing.

RESULTS

MeDIP-seq and MRE-seq were first performed on a control human brain to test their performance and provide a baseline for comparison with GBMs. MeDIP-seq assayed approximately 24 million CpGs at 100–300 base pair resolution, while MRE-seq assayed approximately 1.6 million unmethylated sites at single CpG resolution. The methylation status of 22 of 23 randomly selected loci was well validated with bisulfite sequencing. At the genomewide level, the two assays were significantly inversely correlated, as predicted. MRE-seq and MeDIP-seq data will be presented for primary GBMs, including two samples in The Cancer Genome Atlas (TCGA) consortium for which there is expression, mutation, copy number, and limited DNA methylation data.

CONCLUSION

Two complementary methods, MeDIP-seq and MRE-seq, were developed, optimized, and employed to analyze DNA methylation in normal cells and GBMs. Together these methods will provide unprecedented insights into methylation alterations in the GBM methylome and their effect on gene expression and potential interaction with genetic alterations.

Neuro-Oncol. 2009 Oct;11(5):601–602.

166. YKL-40 Expression in High-Grade Gliomas Differs According to Molecular Subtype

Craig Horbinski, Guoji Wang, Colleen Lovell, Mark Stauffer, Clayton A Wiley

INTRODUCTION

YKL-40 is a secreted chitinase-like molecule that is produced in both inflammatory and neoplastic conditions that feature extra-cellular matrix (ECM) remodeling. Its regulation is unclear, but its expression correlates with activation of the MAPK and Akt pathways and may contribute to adjuvant therapy resistance (1, 2). Whether YKL-40 expression correlates with any hallmark molecular abnormalities in high-grade gliomas is unknown.

METHODS

A rank-order list of YKL-40 expression was determined by immunohistochemical analysis in 81 high-grade adult gliomas, including 28 anaplastic oligodendrogliomas (AOs) and 53 glio-blastoma multiformes (GBMs). Loss of heterozygosity (LOH) on 1p/19q, 9p, 10q, and 17p was performed using PCR-based microsatellite analysis. EGFR amplification, CDKN2A deletion, and codeletion of 1p36 and 19q13 were assessed with fluorescent in situ hybridization (FISH). P53 protein accumulation, Ki67 proliferation index (PI), and EGFR expression were quantified by immunohistochemical analysis. YKL-40 expression was compared with glioma class, key molecular alterations, and immunohistochemistry using a series of Spearman rank correlations.

RESULTS

YKL-40 expression was higher in GBMs than AOs (p < 0.0001). The strongest staining in GBM cells was frequently seen around areas of neovascularization. Among GBMs, YKL-40 expression was lower in tumors with EGFR amplification (p = 0.003), elevated EGFR expression (p = 0.001), or 9p LOH (p = 0.04). Among AOs, YKL-40 expression was lower in tumors with 1p19q codeletion (p = 0.02) but was higher with 10q LOH (p = 0.03).

CONCLUSION

Our data indicate that YKL-40 expression is stronger in GBMs than AOs, but that YKL-40 expression may be negatively regulated by EGFR in GBMs. Moreover, YKL-40 may play a role in the ECM remodeling that occurs during neovascularization. YKL-40 appears to be reduced in AOs with 1p19q codeletion, perhaps accounting for, in part, the increased sensitivity to adjuvant therapy seen in this subset.

Neuro-Oncol. 2009 Oct;11(5):602.

167. A Microrna Expression Signature Accurately Discriminates Glioblastoma from Anaplastic Astrocytoma

AM Soumya, Vani Santosh, Kumar Somasundaram

INTRODUCTION

While glioblastoma (GBM; grade IV) and ana-plastic astrocytoma (AA; grade III) represent malignant forms of glioma, there exists a substantial difference between them in their histological features, clinical behavior, prognosis, and ascribed treatment protocol. It is important to distinguish these two tumors accurately since the treatment protocols and survival times are distinctly different in these patients. Since histological grading can be subjective and does not permit accurate prediction of clinical behavior, there is a need for developing more accurate histology-independent molecular classifiers.

METHODS

We carried out large-scale genomewide miRNA (n = 756) expression profiling of 26 GBMs, 13 AAs, and 7 normal brain samples. Further, we also carried out real-time RT-qPCR validation of selected miRNAs from an independent set of tumor samples and functional characterization of selected miRNAs.

RESULTS

We identified several differentially regulated miRNAs between the groups tested, which were found to be sufficient for differentiating glioma grades and normal brain as recognized by principal component analysis (PCA). More important, we identified the most discriminatory 24-miRNA expression signature by using prediction analysis of microarrays (PAM), which precisely distinguished GBM from AA with an accuracy of 95%. The differential expression pattern of six miRNAs was validated further by real-time RT-qPCR on an independent set of GBM tumors and normal samples. Inhibition of two GBM-upregulated miRNAs (miR-21 and miR-23a) and exogenous overexpression of three GBM-downregulated miRNAs (miR-129, miR-218, and miR-219) resulted in reduced soft agar colony formation and showed varying effects on cell proliferation and chemosensitivity.

CONCLUSION

Taken together, we have identified a miRNA expression signature that accurately discriminates GBM from AA. We also demonstrate the involvement of miRNAs and their importance during glioma development.

Neuro-Oncol. 2009 Oct;11(5):602.

168. Intrinsic Gene Expression Profiles of Gliomas are a Better Predictor of Survival than Histology

Lonneke Gravendeel, Mathilde Kouwenhoven, Olivier Gevaert, Johan de Rooi, Andrew Stubbs, J Duijm, Anneleen Daemen, Fonnet Bleeker, Linda BC Bralten, Nanne Kloosterhof, Bart De Moor, Peter van der Spek, Johan Kros, Peter Sillevis Smitt, Martin van den Bent, Pim French

INTRODUCTION

Gliomas are the most common primary brain tumors with heterogeneous morphologies and largely variable prognoses. Treatment decisions in glioma patients rely mainly on histological classification and clinical parameters. However, differences between histological subclasses and grades are subtle, and classifying gliomas is subject to a large interobserver variability.

METHODS

To improve current classification standards, we have performed gene expression profiling on a large cohort of glioma samples of all histological subtypes and grades.

RESULTS

Expression profiling identified seven distinct molecular subgroups that correlate with survival. These subgroups include two favorable prognostic subgroups (median survival > 4.7 years), two subgroups with an intermediate prognosis (median survival = 1–4 years), two subgroups with a poor prognosis (median survival < 1 year), and one control group. The intrinsic molecular subtypes of glioma are different from the histological subgroups and correlate better with patient survival. The prognostic value of the molecular subgroups was validated on three large, independent sample cohorts (TCGA, REMBRANDT, and GSE12907). The power of intrinsic subtyping is demonstrated by its ability to identify a subset of prognostically favorable tumors within an external data set that contains only histologically confirmed glioblastomas. Specific genetic changes (EGFR amplification, IDH1 mutation, and 1p/19q LOH) segregate into distinct molecular subgroups. We identified a subgroup with molecular features associated with secondary glioblastoma, suggesting that different genetic changes drive the gene expression profiles.

CONCLUSION

Our data provides compelling evidence that expression profiling is a more accurate and objective method for classification of gliomas than histological classification. Molecular classification, therefore, may aid diagnosis and can guide clinical decision making.

Neuro-Oncol. 2009 Oct;11(5):602.

169. 500k Single-Nucleotide Polymorphism (SNP) Array Analysis Identifies Multiple Unique Genomic Changes in Aggressive Meninigiomas

Hongwei Yang, Stephane Goutagny, Jonathan Dreyfuss, Jennifer Chan, Peter Park, Peter Mclaren Black, Michel Kalamarides, Rona S Carroll, Mark D Johnson

INTRODUCTION

Meningiomas are the most common primary brain tumor. Although most meningiomas are slow growing lesions, about 10% are atypical meningiomas (WHO grade II) that display increased growth and recurrence rates, and 3% are anaplastic meningiomas (WHO grade III). Importantly, 10–30% of WHO grade I meningiomas behave aggressively, leading to recurrence or progression to higher grades (WHO grade II or grade III). Molecular markers to predict the clinical behavior of meningiomas are needed.

METHODS

Eighteen meningiomas that progressed to a higher grade at the time of recurrence and 10 clinically benign meningiomas were selected and reviewed by pathologists. DNA was extracted from the tumors and applied to the 500k SNP hybridization array. The data were analyzed with the dCHIP program.

RESULTS

In addition to monosomy 22, we observed frequent loss of chromosomes 9p, 3q, 10q, and 16q and gain of chromosomes 1q, 9q, and 17q in atypical and malignant tumors. Meningiomas that progress histologically and clinically to a higher grade display a unique genomic signature at the time of initial diagnosis.

CONCLUSION

Grade I meningiomas that progress to higher grade may represent a distinct molecular subclass. Several candidate genes identified in the present study may potentially serve as predictive markers for the progressive behavior of meningiomas.

Neuro-Oncol. 2009 Oct;11(5):602.

170. Identification and Functional Analysis of Novel GBM Tumor Suppressor Genes

David Solomon, Jung-Sik Kim, Taeyeon Kim, Todd Waldman

INTRODUCTION

Glioblastoma multiforme (GBM) is the most common and most consistently devastating primary tumor of the CNS. In an effort to more completely understand the pathogenesis of GBM and to identify new molecular targets, we have been working to identify the genes that drive the pathogenesis of GBM.

METHODS

We performed high-resolution copy number analysis of human GBM cell lines, primary cultures, primary xenografts, and primary tumors to identify novel regions of amplification and deletion. The genes in these intervals have been identified and sequenced in an effort to identify somatic mutations. Once the target of the deletions/amplification were identified, wild-type and mutant forms of the gene were expressed by lentiviral infection in the appropriate GBM cells, and the cancer-relevant phenotypes were assessed.

RESULTS

Using this approach, we have identified several novel GBM tumor-suppressor genes that are commonly inactivated by homozygous deletion and/or point mutations. These include p18INK4c (a cdk inhibitor) and PTPRD (a receptor tyrosine phosphatase). We have also performed a comparison of copy number data obtained from the four different GBM sample types and concluded that, while primary tumors are ideal for the identification of novel amplifications, primary xenografts and cell lines are better for the identification of homozygous deletions.

CONCLUSION

These data point to pathways controlled by PTPRD and p18INK4c as novel molecular targets for GBM drug development. They also suggest that ongoing cancer genomics projects should consider employing a wide range of sample types for the identification of novel oncogenes and tumor suppressor genes. In addition to these results, our newest results identifying novel GBM oncogenes and tumor suppressors will be presented.

Neuro-Oncol. 2009 Oct;11(5):602–603.

171. Micro-RNA-128 (MiRNA-128; CHR 2q21.3) Downregulation Targets BMI-1-Mediated Polycomb Silencing and Transcription Factor E2F3a, Key Regulators of Cell Cycle Progression

Jian Guo Cui, Yuan Yuan Li, Ali Mahta, Prerna Sethi, Yuhai Zhao, Frank Culicchia, Walter J Lukiw

INTRODUCTION

Emerging evidence suggests that micro RNA (miRNA) complexity and the abundance of specific brain-enriched miRNA species are significantly altered in glioma and glioblastoma multiforme (GBM).

METHODS

We used total RNA isolation, DNA miRNA panels, high-density fluorescent miRNA assays, Northern blot analysis, qRT-PCR, human brain biopsy samples, glioma/GBM cell cultures, human brain primary cultures, Western blot analysis, bioinformatics, transfection vectors, and statistical analysis.

RESULTS

Brain-enriched miRNA-128 was found to be significantly downregulated in glioma and GBM as compared with miRNA-128 levels in age-matched control brain samples from the same anatomical area. MiRNA-128 levels are also reduced in GBM tumor cell lines CRL-2020 (DBTRG-05MG), CRL-1690 (T98G), CRL-2365 (M059K), and CRL-2366 (M059J) (American Tissue Culture Collection, Bethesda) was compared with human primary neural cell controls. MiRNA-128 downregulation was found to correlate with WHO tumor grade. The Bmi-1 gene, encoding a transcription repressor and a confirmed miRNA-128 target, promotes stem cell renewal by acting as a part of the polycomb-silencing complex that alters chromatin structure and inhibits the expression of known tumor-suppressor genes involved in senescence and differentiation. Another related miRNA-128 target includes the gene encoding transcription factor E2F3a, which is critical for the control of cell cycle progression and neural cell development.

CONCLUSION

Our data suggest that downregulation of miRNA-128 may contribute to several key proliferative aspects of glioma and GBM by allowing increased expression of Bmi-1 and E2F3a, resulting in the maintenance of the undifferentiated self-renewing state of brain tumor cells.

Neuro-Oncol. 2009 Oct;11(5):603.

172. CASPR2 Functions as a Tumor-Suppressor Gene in Glioma

Linda BC Bralten, Lonneke Gravendeel, Nanne Kloosterhof, Andrea Sacchetti, Martin van den Bent, Johan Kros, Casper Hoogenraad, Peter Sillevis Smitt, Pim French

INTRODUCTION

We have developed a strategy that uses exon-level expression profiling of glial brain tumors to screen for genetic changes that result in fusion genes. One of the genes identified encodes CASPR2, a neural cell-adhesion molecule.

METHODS

See Results.

RESULTS

CASPR2 was identified in GBM142 based on high expression of 3′ exons and the absence of expression of 5′ exons. 5′-RACE PCR identified a fusion gene between CASPR2 and an expressed sequence tag (EST) close to the NCOA1 gene. In a set of 25 glioblastoma multiforme (GBM) samples, this translocation was unique. Western blotting did not detect expression of a fusion protein. Indeed, the predicted transcript does not harbor a consensus translation initiation site. This translocation, therefore, appears to disrupt the protein expression, despite high mRNA expression. Because causative genetic changes often have a significant mutation frequency, we screened for additional mutations. Direct sequencing of 24 GBMs and 19 oligodendrogliomas (ODs) identified one nonsomatic missense mutation within the conserved signal-peptide. 250K Nsp arrays (21 GBMs and 19 ODs) identified two aberrations in the genomic region of this gene, one intragenic deletion (OD53) and one larger deletion (GBM139). In a set of 272 glioma samples, high CASPR2 expression was associated with longer survival (p < 0.0001). Multivariate analysis indicated CASPR2 mRNA expression as an independent prognostic factor. Finally, we performed functional analysis on U87 cells stably expressing CASPR2-GFP and found that CASPR2-expressing cells had a reduced proliferation rate compared to control (Ds-Red) transfected cells (p = 0.017). FACS analysis using mixed populations of CASPR2 and Ds-Red-expressing cells confirmed the reduced proliferation rate.

CONCLUSION

The reduced proliferation rate of CASPR2-expressing cells, the absence of protein expression resulting from the translocation in GBM142, the intragenic deletion in OD53, and the positive correlation between CASPR2 expression and survival indicates that CASPR2 functions as a tumor-suppressor gene in gliomas.

Neuro-Oncol. 2009 Oct;11(5):603.

173. Genomic and Transcriptomic Profiling as Tools to Improve Diagnosis and to Individualize Treatment of Malignant Gliomas

Mahfoud Assem, Zita Sibenaller, Timothy C Ryken, Supreet Agarwal, Joyce Marner

INTRODUCTION

Malignant gliomas are the most frequent type of primitive brain tumors. The outcome of patients with these tumors did not improve despite new therapeutics, underscoring the need for a better understanding and fresh approach to their treatment. Compounding the problem is the inability to predict sensitivity or resistance to chemotherapy regimens, which may result in early and inappropriate chemotherapy treatment. The lack of reproducibility in the classification of many grade II and III gliomas presents an opportunity where genomics may be paramount for accurate diagnosis, and therefore, therapeutic decisions. The aim of our work is to identify polymorphic molecular markers that correlate with classification and therapeutic outcome.

METHODS

DNA and RNA from 60 glioma tumor specimens were harvested at The University of Iowa and subjected to copy number variation (CNV), loss of heterozygosity (LOH), and gene expression analyses using the GeneChipR Human SNP Mapping 6.0 and Gene 1.0 arrays. The data generated were analyzed with the PartekR genomics software suite. Histology, diagnosis, pathology, and predictive clinical and outcome data were recorded and the associations between clinical and biological elements were studied.

RESULTS

Our studies revealed more than 4,000 CNVs and 350 differentially expressed genes. Some of these genetic alterations were concordant with previously published studies; chromosomes 1p and/or 19q deletions and chromosome 7 gain. Losses of chromosomes 10, 13, 14, and 22 and gains of chromosomes 19 and 20 were additionally found. Several alterations correlated significantly with histology and grade. Moreover, LOH was detected at numerous chromosomes. Interestingly, several of these regions harbor genes with potential tumor suppressor properties. Furthermore, gene expression analysis revealed many genetic signatures representing key pathways that can be used for targeted and individualized therapies.

CONCLUSION

These novel genetic signatures may lead to critical insights into diagnosis, classification, prognosis, and prediction in designing individualized therapies based on the genetic makeup of the tumor and the patient.

Neuro-Oncol. 2009 Oct;11(5):603.

174. Sequential Stratification of Glioblastoma Patients using Survival-Related Genes and its Therapeutic Implications

Jun Yao, Yong-Wan Kim, Dimpy Koul, Jonas Silva Almeida, John N Weinstein, WK Alfred Yung

INTRODUCTION

We developed a novel algorithm to identify survival-related genes by analyzing microarray data sets.

METHODS

TCGA glioblastoma multiforme (GBM) patient samples with microarray U133A gene expression data were analyzed using this approach.

RESULTS

The first tier analysis using all GBM patients (n = 252) revealed a strong enrichment of chromosomal 10 genes within the top survival genes, suggestive of loss of PTEN in relation to patient survival. This is consistent with the previous finding of chromosome 10 loss of heterozygosity (LOH) as a poor prognostic marker. In fact, expression levels from whole chromosome 10 genes could be used to effectively stratify GBM patients into good and poor prognostic groups (n = 53 and 199; median survival, 607 and 345 days, respectively). The same approach was then applied to the poor prognosis group only to further stratify GBM patients into three groups (groups A, B, C; n = 85, 51, 63, respectively). Among these, the group A tumors had elevated expression of collagens, reminiscent of tumor stem cells with activation of the TGF-β pathway. Group B tumors were enriched in EGFR amplification and p16 deletion. Interestingly, these tumors had high overall NF1 expression and were devoid of p53 mutations, suggesting that p53/NF1 inactivation is functionally redundant with the simultaneous EGFR amplification and p16 deletion. Group C was enriched in tumors with higher expression of PDGFRA as well as an activated cell cycle gene expression signature and was associated with the worst clinical outcome (median survival 290 days).

CONCLUSION

Our results demonstrate the power of our novel approach, allowing us to gain insights into the underlying mechanisms governing GBM patient survival, and provide a solid rationale for patient selection for combined targeted therapy for the PI3K, EGFR, TGF-β, and PDGFRA pathways in GBM patients.

Neuro-Oncol. 2009 Oct;11(5):603–604.

175. Germline Polymorphisms in 9p21 are Associated with Somatic Deletion in High-Grade Gliomas

Robert Jenkins, Paul Decker, Thomas Kollmeyer, Jeanette Keefe, Karla V Ballman, Daniel H Lachance, Ping Yang

INTRODUCTION

Somatic deletions of 9p21 are very common in gliomas. A recent genomewide association study (GWAS) has shown that single-nucleotide polymorphisms (SNPs) within 9p21 are associated with the development of high-grade gliomas (Wrensch et al., in press). These observations generate two alternative hypotheses: 1) patients inherit an inactivating alteration within 9p21 and somatic deletion eliminates the wild-type allele or 2) the associated SNP(s) are in linkage disequilibrium with another alteration, which facilitates somatic 9p21 deletion. These hypotheses can be distinguished by comparing the germline allele/genotype prevalence stratified by glioma 9p deletion status.

METHODS

Germline genomewide SNP genotyping was performed on 301 Mayo Clinic patients with high-grade gliomas. From this data set, four 9p21 SNPs were evaluated. We performed 9p21 fluorescence in situ hybridization (FISH) analysis on all available gliomas. We also compared the TCGA 9p21 glioblastoma CGHa and germline SNP data.

RESULTS

Tumor material was available for 167 high-grade glioma patients from the Mayo Clinic. Evaluable CGHa and SNP data were available for 80 TCGA patients. The prevalence of hemi- or homozygous 9p21 deletion was 50% (84/167) by FISH and 52% (42/80) by CGHa in the Mayo Clinic and TCGA gliomas, respectively. For the four 9p21 SNPs, the prevalence of the homozygous at-risk genotype (e.g., AA) ranged from 33% to 35% in Mayo Clinic patients whose tumor contained 9p21 deletion. The prevalence of the at-risk genotype ranged from 18% to 20% in patients whose tumor lacked 9p21 deletion. The differences in prevalence were statistically different for each SNP tested (p-values, 0.024–0.042). The results were similar when the TCGA data were examined.

CONCLUSION

Our results support the hypothesis that germline alteration(s) within 9p21 facilitate the acquisition of somatic 9p21 deletion in high-grade gliomas. These observations have implications for our understanding of high-grade glioma genetics and for the management of patients with these tumors.

Neuro-Oncol. 2009 Oct;11(5):604.

176. Novel Genes Associated with Meningioma Progression Identified with Microarray-Based Gene Expression Profiling

Elisa Perez-Magan, Yolanda Ruano, Bárbara Meléndez, Rocio Juarez, Gerardo Perez-Bautista, Teresa Ribalta, Concepción Fiaño, Ángel Rodríguez de Lope

INTRODUCTION

The majority of meningiomas are probably benign, but a number of tumors are histologically and/or clinically more aggressive. Recurrence rates after radical removal are sometimes unexpectedly high. Understanding the potential behavior of these tumors in individual patients is critical for rational therapeutic decision making. The aim of this study was to identify gene expression profiles and candidate markers associated with primary and recurrent meningiomas.

METHODS

We analyzed 44 meningiomas (21 grade I, 12 grade II, 3 grade III, and 8 relapses) using oligo microarrays. We compared the expression profiles of meningiomas to those of normal arachnoidal tissues to identify genes and pathways potentially associated with meningioma. To identify specific genes potentially associated with recurrence, we searched for genes that were differentially expressed between primary and recurrent tumors. A TMA with 110 meningiomas was used for validation.

RESULTS

An unsupervised hierarchical clustering of the samples revealed two main groups: one composed of all the WHO grade III and most of the relapsed tumors and the other containing most of the WHO grade I and II tumors. The molecular signature was composed of a total of 166 altered genes, and most of them had reduced expression. Differential gene expression between the primary and relapsed tumors was seen for a total of 309 genes (FDR < 0.20). The altered genes were related to previously described signaling pathways, such as Notch, TGF-β, and WNT, and with other novel pathways, such as the PPAR and PDGF signaling pathways, regulation of the actin cytoskeleton, and oxidative phosphorylation. Of interest, the genes TMEM30B (14q23.1), SFRP1 (8p12-p11.1), and connective tissue growth factor (CTGF [6q23.1]) show the highest differences in expression levels between the primary and recurrent tumors.

CONCLUSION

We identified the novel candidate genes TMEM30B, SFRP1, and CTGF, which could act as tumor-suppressor genes and contribute to meningioma progression and may be useful for therapeutic decision making in individual patients.

Neuro-Oncol. 2009 Oct;11(5):604.

177. Identification of Exosomal RNA Levels of EGFRvIII From the Sera of Patients with Glioblastoma

Bob S Carter, Fred Hochberg, Johan Skog, Ayguen Sahin, Lin Dan Zhu, Ryan Lessard, Xandra Breakefield

INTRODUCTION

In this report, we describe the development of a novel biomaker for human glioblastoma. It is recognized that different patterns of genetic mutations in gliomas can confer markedly different prognoses and sensitivity to different treatment programs. We hypothesized that serum exosomal RNA could be used as a biomarker for the genetic changes that occur in human glioblastoma and other cancers. Exosomes are small vesicular bodies released by mammalian cells, which encapsulate mRNA, microRNA, and protein. Tumor RNA within exosomes is protected from circulating serum RNases. We describe the development, validation against tumor tissue, and potential use of this novel biomarker assay in glioblastoma.

METHODS

Sera were collected from 20 patients with glioblastoma prior to resection, and serum exosomes were isolated and RNA harvested. We compared techniques to optimize the yield and integrity of the exosomal RNA. Levels of epidermal growth factor (EGFR) and EGFRvIII mRNAs in the blood were measured by qRT-PCR and compared with the RNA profile of matched tumor tissue. Correlation between serum exosomal RNA and tumor tissue RNA was made.

RESULTS

Exosomal RNA was reflective of the tumor transcriptome, and we specifically identified tumor-specific EGFRvIII mutations with a high correlation. Normal individuals did not express EGFRvIII in serum exosomes. The level of tumor-specific RNA was correlated with disease volume, and we were not able to detect tumor-specific RNA in patients who had gross total resection of their tumor.

CONCLUSION

The ability to sample the tumor transcriptome in serum exosomes opens new possibilities for diagnosing and monitoring glioblastoma as treatment progresses. We will present our recent findings on optimizing this technique for clinical use.

Neuro-Oncol. 2009 Oct;11(5):604.

178. Identification of Prognostic Gene Signatures of Glioblastoma Based on the Cancer Genome Atlas Analysis

Yong-Wan Kim, Dimpy Koul, Jun Yao, Jonas Silva Almeida, Oliver Bogler, Howard Colman, Kenneth D Aldape, WK Alfred Yung

INTRODUCTION

Glioblastomas have few prognostic markers and predictors of therapeutic response. One way to identify potential markers is to search The Cancer Genome Atlas (TCGA) for novel molecular aberrations in GBM tissues.

METHODS

Here, we describe our comparative analysis of gene expression levels and copy number variations (CNVs) using the 173 TCGA GBM patients. Our goal was to identify novel gene sets that could classify GBMs into prognostic groups.

RESULTS

Using a hierarchical clustering analysis, we identified 42 probe gene sets that could be used to divide the tumor samples into three groups and showed a significantly (p = 0.0006) improved 2-year overall survival. Kaplan-Meier plots showed that the median survival of group 3 was markedly longer (127 weeks) than that of groups 1 and 2 (47 and 52 weeks, respectively). On biologic pathway analysis, we found that the IL-17 pathway was significantly associated with the probe sets (p < 0.0084) and the IL-17 pathway network displayed high-level functions in cell death and inflammatory response. We then used the 42 probe sets to stratify the patients according to survival in other public GBM gene expression data sets. A parallel CNV analysis of the Agilent 244K oligonucleotide array data on 170 matched GBM tumors showed a predominant copy number signature defined by chromosome 7 gain/chromosome 10 loss, which is a genetic signature for poor prognosis. For patients who did not have chromosome 7gain/chromosome 10 loss or homozygous deletion of PTEN and CDKN2A/2B, the median survival was longer (70 weeks) than for those who showed both of these alterations (49 weeks). The 42-gene signature and CNV data were the dominant characteristics that permitted the stratification of patients into different survival groups.

CONCLUSION

Our results identified specific patterns of gene expression and DNA copy number events in a new prognostic group of GBM tumors based on 42 probe gene sets.

Neuro-Oncol. 2009 Oct;11(5):604.

179. Neurospheres from Glioblastoma Multiforme Express High Levels of EZH2 and Arise More Frequently from Tumors with Low/Absent Mgmt Methylation

Francesca Orzan, Serena Pellegatta, Sara Guzzetti, Giulia Villa, Francesca Menghi, Luigi Poliani, Federica Pisati, Valentina Caldera, Elena Stucchi, Rossana Bettolini, Paola Porrati, Marica Eoli, Mariangela Farinotti, Carlo E Marras, Patrizia Tunici, Mariagrazia Bruzzone, Davide Schiffer, Gaetano Finocchiaro

INTRODUCTION

Glioblastoma multiforme (GBM) may contain a fraction of cells expressing stem cell programs that, in vitro, are contained in neurospheres (NSs). Here we have looked for clinical implications of NS formation and for NS expression of genes important for glioma and stem cell biology.

METHODS

We studied correlations between in vitro GBM-NS formation and: i) overall survival of patients, ii) MGMT methylation, and iii) other genetic features related to glioma subtyping. We also investigated the expression of stem cell genes, including radial glia and polycomb genes, in GBM-NSs and primary GBMs using DNA microarray, quantitative PCR, and immunohistochemical analysis.

RESULTS

NSs could be obtained from 40 of 76 GBMs studied; this feature correlated with lower rates of overall survival (p = 0.0084) and lower rates of MGMT methylation (p = 0.0040), as determined in a subgroup of 36 cases. In vivo, GBM-NSs were tumorigenic in all mice tested. Using DNA microarray and real-time PCR we found that the radial glia gene FABP7 is upregulated in human GBM-NSs and in primary GBM specimens. We also found that enhancer of zeste 2 (EZH2), a critical component of the polycomb repressive complex 2, is highly expressed in GBM-NSs and is expressed significantly more in GBMs than in lower-grade gliomas (p = 0.0013).

CONCLUSION

The results show that NSs are an important in vitro model of a fraction of highly aggressive GBMs. Genes that are active in radial glia and epigenetic control, like FABP7 and EZH2, are highly expressed in NSs and their role in GBM biology deserves further investigation.

Neuro-Oncol. 2009 Oct;11(5):605.

180. A Distinct Pattern of Aberrant DNA Methylation Distinguishes Tumorigenic CD1331 GBM Cells from Patient-Matched Nontumorigenic GBM Cells

Shaun Fouse, Ruihuan Chen, Samir Kharbanda, Nir Oksenberg, Ivan Smirnov, Malek Fahem, Michael Prados, Heidi S Phillips, Joseph Costello

INTRODUCTION

Human glioblastoma multiformes (GBMs) contain a subset of cells termed cancer stem cells (CSC). These CSC express the cell surface marker CD133, whereas CD133-negative (CD133−) cells derived from the same tumor do not exhibit the same tumorigenic potential. Interestingly, the CD133+ cells can give rise to both CD133+ and CD133− cells, suggesting the CSC phenotype may be reversible. DNA methylation, a reversible epigenetic modification that regulates gene expression, is heterogeneous within GBMs. We asked whether the heterogeneity of GBM DNA methylation might be derived from differences in methylation in tumorigenic CD133+ cells relative to nontumorigenic CD133− cells from the same tumor.

METHODS

Primary GBMs were grown under neurosphere conditions and sorted by FACS for CD133+ and CD133− populations. Methylation analysis of 150,000 CpG sites was performed on clones of CD133+ and CD133− cells using the Affymetrix mTACL array system. To determine if aberrant hypo- or hypermethylation events were associated with altered gene expression, RNA from the sorted populations was hybridized to Affymetrix gene expression microarrays. We also tested for correlations between methylation and gene expression.

RESULTS

Intracerebral injection of CD133+ cells resulted in tumors in mice while CD133− cells did not during the same time period. Hierarchical clustering and principle component analyses showed that CD133+ and CD133− subpopulations have distinct methylation patterns. The altered methylation correlated with altered expression for a subset of genes.

CONCLUSION

Tumorigenic CD133+ tumor cells appear to have distinct DNA methylation profiles relative to nontumorigenic CD133− cells from the same GBM. These populations also have different gene expression profiles that correlate with differences in methylation. These studies suggest that intratumoral heterogeneity of DNA methylation may reflect phenotypically distinct cell subpopulations. Further studies will determine whether the tumorigenic phenotype of CD133+ cells is regulated by epigenetic mechanisms.

Neuro-Oncol. 2009 Oct;11(5):605.

181. Characterization of Gene Expression-Derived Glioblastoma Multiforme Subgroups

Jason Reeves, Charles William Kanaly, Micheal Gatza, Joseph Lucas, Joseph Nevins, John H Sampson

INTRODUCTION

Previous studies have shown that glioblastoma multiforme (GBM) is comprised of multiple molecular subgroups within the same histological classification. In order to better understand the molecular mechanisms by which GBMs arise, we have used gene expression data to define subgroups based on a priori analysis of gene activity and then explored the molecular characteristics of each subgroup.

METHODS

Gene expression data from more than 300 GBM samples from The Cancer Genome Atlas (TCGA) were analyzed by modeling activity of key tumor suppressors and oncogenes. Once activity of these genes was predicted, subgroups were generated based on predicted gene activities using agglomerative hierarchical k-means and affinity propagation clustering methods. Results were validated by analysis of more than 450 previously published samples from six data sets and internal cross-validation of TCGA data. Once the subgroups were established, patterns of clinical characteristics, promotor methylation, copy number, and gene sequence mutations were analyzed to determine subgroup-specific characteristics.

RESULTS

This analysis has shown evidence for at least six subgroups within the clinical diagnosis of GBM. Samples identified as progressive GBM primarily cluster together within one subgroup, with de novo GBM accounting for the bulk of the additional groups. Within each subgroup distinct patterns of clinical characteristics and molecular events were detected.

CONCLUSION

These results confirm that within the same histopathological diagnosis several distinct groups of GBM exist. Furthermore, we characterized, at a genome-wide level, the patterns of molecular alterations that are present within these distinct subgroups of GBM. These characteristics robustly define these groups and provide insight into how they arise and progress within the same histopathology along with the potential therapeutic opportunities that are unique to each subgroup.

Neuro-Oncol. 2009 Oct;11(5):605.

182. Microrna-10a is Highly Expressed in Glioblastoma Multiforme and May Contribute to its Malignant Behavior by Regulating HOXD10

Arti B Gaur, Mark A Israel

INTRODUCTION

MicroRNAs are small noncoding RNAs that play a critical role in developmental and physiological processes and are implicated in the pathogenesis of several human diseases, including cancer. They function by regulating target gene expression posttranscriptionally. We sought to elucidate the role of a potentially oncogenic microRNA, microRNA-10b (mir-10b), in mediating the pathology of glioblastoma multiforme (GBM).

METHODS

We have previously assessed the expression pattern of 241 mature human microRNAs in the NCI60 panel of cell lines and in a set of corresponding normal tissues. Our results provided evidence that microRNA expression patterns mark specific biological characteristics of tumors and/or mediate biological activities important for their pathobiology. In CNS tumor-derived cell lines, we identified mir-10b as a candidate oncogene, as it is expressed at higher levels in human GBM lines and primary GBM tissue when compared with normal brain tissue.

RESULTS

Using the MiRanda target prediction algorithm we identified a member of the homeobox family, Homeobox D10 (HOXD10), as a potential target of mir-10b. Our in vitro studies demonstrate that downregulation of mir-10b in GBM-derived cell lines results in increased expression levels of HOXD10. Additionally, our data indicate that downregulation of mir-10b in human GBM lines leads to reduced proliferation and reduced colony formation in soft agar. Importantly, using a xenograft model in immune-deficient nude mice, we have observed that human GBM lines with down-regulated mir-10b levels result in reduced tumor growth in vivo. Further experiments are ongoing to identify the specific role of HOXD10 in GBM pathology.

CONCLUSION

We interpret these findings as indicating that expression levels of mir-10b are associated with pathologic characteristics of GBM. Further experiments are ongoing to identify the specific role of HOXD10 in GBM pathology. This work will help elucidate the various aspects of cell growth and differentiation that may play a role in GBM oncogenesis and could potentially lead to new therapeutic strategies.

Neuro-Oncol. 2009 Oct;11(5):605.

183. Functional Genomic Network Analysis of the Core Genes and Pathways Essential for Glioblastoma Cell Survival

Nikhil Thaker, Peter McDonald, Carolyn Kitchens, Tong Ying Shun, John Lazo, Ian F Pollack

INTRODUCTION

Identification of the genes and pathways essential for glioblastoma multiforme (GBM) cell survival may facilitate the drug discovery effort. We implemented a genomic screening approach and a protein-protein interaction network database to investigate the gene nodes and networks that are essential for GBM and cancer cell survival.

METHODS

We employed a high-throughput short interfering RNA screen targeting 5,520 unique human genes in the T98G glioma cell line. Pathway and network analyses were performed using Ingenuity Pathways Analysis. The gene list was uploaded to this Web-based application and used to generate biological networks. Fisher’s exact test was used with α = 0.05 to calculate the probability that each function assigned to the set of survival genes was due to chance.

RESULTS

We identified 55 survival genes and classified these genes according to their biological functions, which included proteases, kinases, transferases, signaling molecules, and transcription regulators. Thirty-three survival genes were reportedly overexpressed in GBM in the Oncomine database. The most significant network analysis identified a network of genes connected through the proteasome complex, NF-κB, and histone H3. We identified a gene network associated with cell-to-cell signaling, cancer, and the cell cycle that centered on β-estradiol and HNF4A. We organized the survival genes into the most highly represented cellular pathways, including protein ubiquitination, purine metabolism, nucleotide excision repair, pyrimidine metabolism, and NF-κB. We also uncovered similar core pathways within a panel of cancer tissues, including colorectal and pancreatic cancers.

CONCLUSION

We have applied a functional network analysis to investigate the molecular connectivity of genes central to cell survival. The proteasome complex, NF-κB, and histone H3 appear to be most significantly involved in the regulation of glioma cell survival. Future research will scrutinize the mechanisms of cytotoxicity associated with these gene pathways and will help elucidate the role of these genes in the treatment of glioma.

Neuro-Oncol. 2009 Oct;11(5):606.

184. Quantitative Proteomic Analysis of Oligodendrogliomas with and without 1p/19q Deletion

Leila Khorasani, Robert C Rostomily, Donald Born, Richard Beyer, Min Shin, Jinghua Jin, Ellsworth C Alvord Jr, Catherine Pan, Josh Sonnen, Thomas Montine, Jing Zhang

INTRODUCTION

Approximately 50%–80% of oligodendrogliomas demonstrate combined loss of chromosome 1p and 19q. Chromosome 1p/19q deletion, appearing early in tumorigenesis, is associated with improved clinical outcomes, including response to chemotherapy and radiation. Although many hypotheses have been proposed, the molecular mechanisms underlying improved clinical outcomes for oligodendrogliomas with 1p/19q deletion have not been fully characterized.

METHODS

To investigate the molecular differences between oligodendrogliomas with and without 1p/19q deletion, we employed an unbiased proteomic approach using microcapillary liquid chromatography mass spectrometry (LC-MS) with isotope-coded affinity tags (ICAT) on patient samples of grade II oligodendrogliomas. Following conventional biochemical separation of pooled tumor tissue from five samples each of 1p/19q-deleted and -undeleted grade II oligodendrogliomas into nuclei-, mitochondria-, and cytosol-enriched fractions, relative changes in protein abundance were quantified.

RESULTS

Our results demonstrated that among the >200 proteins identified in the cytosolic fraction, about 30 proteins changed significantly in their relative abundance (>100%) in morphological oligodendrogliomas with 1p/19q deletion as compared to those without 1p/19q deletion. Some of these proteins appear to be involved in signal transduction whereas others have been implicated in tumorigenesis.

CONCLUSION

Our data suggest that among the many changes noted in protein expression between tumors with and without 1p19q codeletion, metabolic reprogramming and differential invasiveness are potential consequences of 1p19q deletion that contribute to the well-documented differences in prognosis and treatment response for these glioma subtypes. It is anticipated that this type of analysis will allow us to generate a roster of proteins that may be developed into specific biomarker panels for predicting oligodendrogliomas with better prognoses and/or identify new therapeutic targets for this glioma.

Neuro-Oncol. 2009 Oct;11(5):606.

185. Deubiquitinating Enzyme-Based Analysis of the Tumor Cancer Genome Atlas (TCGA) Transcriptome Profiles of Glioblastoma

Pascal O Zinn, Lisa-Marie Sturla, David Kozono, Masayuki Nitta, Kimberly Ng, Peter C Warnke, Ruprecht Wiedemeyer, Yonghong Xiao, Lynda Chin, Clark C Chen, Ekkehard Matthias Kasper

INTRODUCTION

The Cancer Genome Atlas (TCGA) project classified glioblastomas into four subgroups based on transcriptome analysis. These subgroups include proneural, neural, mesenchymal, and classical. The subgroups carry prognostic value and likely reflect differing underlying biology. Deubiquitinating enzymes (DUBs) play a critical role in several biochemical pathways relevant to cancer, such as DNA damage repair, apoptosis and cell-cycle progression. We therefore determined whether the expression profile of DUBs differed significantly among the four subgroups.

METHODS

We examined the expression profile of the 89 DUBs available in the TCGA database. Relevant DUBs over- and underexpressed in the fourglioblastoma subgroups were identified and analyzed by comparative marker selection.

RESULTS

Our data show that the four subgroups differ significantly with regard to the expression levels of particular DUBs. This result suggests that specific DUBs are responsible for modulation of molecular pathways critical to each subgroup. We identified the following DUBs as significantly group-defining using comparative marker selection. The proneural subgroup was defined by overexpression of USP43, USP49, USP22, USP11, USP51, SENP1, SENP7, USP13, USP7, USP46, USP34, USP20, USP37, USP21, USP10, USP31, USP54, ZRANB1, UCHL1, USP24, STAMBP, USP47, SENP6, USP3, USP19, and SENP2. The neural subgroup was defined by USP33, PSMD14, COPS5, OTUB1, USP45, JOSD2, and DUB3. The mesenchymal subgroup was defined by TNFAIP3, VCPIP1, USP25, ATG4A, JOSD1, OTUD5, USP9X, JOSD2, and USP12. Finally, the classical subgroup was defined by USP4 and USP42.

CONCLUSION

Our data support the concept that TCGA-defined subgroups reflect distinct biologies, which are, in part, mediated by DUBs. The data support glioblastoma subtype-specific targeting of relevant DUBs as a therapeutic strategy.

Neuro-Oncol. 2009 Oct;11(5):606.

186. Biomarker Development using Absolute Copy Number of Methylated Tumor Suppressor Genes in the Plasma of Patients with Malignant Glioma

Kyle Weaver, Larry Pierce

INTRODUCTION

Therapy for malignant glioma continues to improve. However, biomarker development to guide the utilization of these therapies lags behind. Recent work demonstrates that malignant gliomas release their DNA into the blood, and the methylated tumor suppressor gene promoters from intracranial tumors can be isolated from plasma. We demonstrate that the presence of this tumor DNA in blood has the potential to be developed as a biomarker to noninvasively diagnose and guide many of these emerging therapies.

METHODS

DNA from preoperative blood samples and tumors obtained at resection from 39 patients undergoing craniotomy for high-grade glioma was isolated, purified, and subjected to bisulfate treatment. Normalization of reaction efficiency was achieved using a novel S. cerevisiae β-actin spike-in method. Absolute copy number of methylated MGMT promoter per unit volume was calculated for all matched sample pairs using quantitative PCR. Patients undergoing craniotomy for nonneoplastic diagnoses were used as controls.

RESULTS

The total copy number of methylated MGMT promoter DNA in plasma (ng/ml) and tumor (mg) correlated poorly when compared directly (R2 = − 0.13). However, when corrected for patient plasma volume and preoperative T1 postcontrast tumor volume, the R 2 increased to 0.70. There was complete concordance between absolute copy number in tumor and plasma when a cutoff of 200 copies was used, with no tumor demonstrating a smaller copy number in tissue than in plasma. No control patient demonstrated a copy number in plasma above the accepted sensitivity of the assay.

CONCLUSION

Gliomas release their abnormal DNA into the blood. This may reflect the epigenetic aberrations in the lesion and correlates reliably with tissue genetics and accepted clinical surveillance parameters. Ongoing work with MGMT and other genes of interest addresses further development with respect to survival, tumor burden, and response to therapy.

Neuro-Oncol. 2009 Oct;11(5):606.

187. Model of a Cooperative Genetic Landscape in Gliomas

Markus Bredel, Denise Scholtens, Griffith R Harsh, Claudia Bredel, James P Chandler, Jaclyn Renfrow, Ajay Yadav, Hannes Vogel, Adrienne C Scheck, Robert Tibshirani, Branimir I Sikic

INTRODUCTION

Gliomas, in particular glioblastomas, are among the deadliest of human tumors. Gliomas emerge through the accumulation of recurrent chromosomal alterations, some of which target yet-to-be-discovered cancer genes. A persistent question concerns the biological basis for the coselection of these alterations during gliomagenesis. We here describe a network model of a cooperative genetic landscape in gliomas and evaluate its clinical relevance.

METHODS

Multidimensional genomic profiles and clinical profiles of 501 human gliomas (45 tumors in an initial discovery set collected between 2001 and 2004 and 456 tumors in validation sets made public between 2006 and 2008) from multiple academic centers in the United States and The Cancer Genome Atlas (TCGA) pilot project.

RESULTS

Gliomas select for a nonrandom genetic landscape, a consistent pattern of chromosomal alterations that involves altered regions (“territories”) on chromosomes 1p, 7, 8q, 9p, 10, 12q, 13q, 19q, 20, and 22q (false discovery rate-corrected p-value < 0.05). A network model shows that these territories harbor genes with putative synergistic, tumor-promoting relationships. The coalteration of the most interactive of these genes in glioblastoma associates with unfavorable patient survival. A multigene risk-scoring model based on seven landscape genes (POLD2, CYCS, MYC, AKR1C3, YME1L1, ANXA7, and PDCD4) is associated with the duration of overall survival in 189 glioblastoma samples from the TCGA (log-rank p = 0.023, comparing three survival curves for patients with 0–2, 3–4, and 5–7 dosage-altered genes). These associations with survival rates were validated using gene expression data from three independent glioma studies, comprising 76 (p = 0.003), 70 (p = 0.008), and 191 glioblastomas (p = 0.002).

CONCLUSION

The alteration of multiple networking genes by recurrent chromosomal aberrations in gliomas deregulates critical signaling pathways through multiple, cooperative mechanisms. These mutations, which are likely due to nonrandom selection of a distinct genetic landscape during gliomagenesis, are associated with patient prognosis.

Neuro-Oncol. 2009 Oct;11(5):607.

188. Monosomy 10 Dysregulates EGFR Signaling in Glioblastomas

Ajay Yadav, Jaclyn Renfrow, Denise Scholtens, Hehuang Xie, George E Duran, Claudia Bredel, Hannes Vogel, James P Chandler, Arnab Chakravarti, Pierre Robe, Sunit Das, Adrienne C Scheck, John A Kessler, Bento Soares, Branimir I Sikic, Griffith R Harsh, Markus Bredel

INTRODUCTION

Glioblastomas frequently possess both monosomy of chromosome 10 and gains of the epidermal growth factor receptor (EGFR) gene locus on chromosome 7, a coincidence whose mechanism is poorly understood. We assessed whether the coselection of EGFR gains on 7p12 and monosomy of chromosome 10 in glioblastomas promotes EGFR signaling through loss of annexin 7 (ANXA7), a candidate tumor suppressor gene on 10q21.1-q21.2.

METHODS

We performed multidimensional analysis of gene, coding sequence, promoter methylation, transcript, and protein data for ANXA7 (and EGFR) and clinical patient data involving publicly available (made public between 2006 and 2008) and unpublished (tumors collected between 2001 and 2008) profiles of 543 high-grade gliomas from medical centers in the United States and the Cancer Genome Atlas (TCGA) pilot project. We also performed functional analyses using LN229 and U87 glioblastoma cells and human embryonic kidney 293T (clone tsA54) cells with knockdown and transgene phenotypes for ANXA7 and EGFR.

RESULTS

Heterozygous ANXA7 gene deletion is associated with a significant loss of ANXA7 expression (p < 0.00001) and a significant reduction of ANXA7 protein expression (p = 0.004). ANXA7 loss-of-function stabilizes the EGFR protein and augments EGFR-transforming signaling in glioblastoma cells. Combined ANXA7 knockdown and EGFR overexpression promotes tumorigenicity in a synergistic fashion. The loss of ANXA7 in ~75% of glioblastomas in the TCGA coupled with the infrequency of ANXA7 mutation (~6% of tumors) indicates its role as a haploinsufficiency gene. ANXA7 gene expression, dichotomized at the median, associates with patient survival in 191 glioblastomas (log-rank p = 0.008) and in a separate group of 180 high-grade gliomas (p = 0.00003). Deletion of the ANXA7 gene associates with poor patient survival in 189 glioblastomas (log-rank p = 0.042).

CONCLUSION

Haploinsufficiency of the tumor suppressor ANXA7 caused by monosomy of chromosome 10 provides a clinically relevant mechanism to augment EGFR signaling in glioblastomas.

Neuro-Oncol. 2009 Oct;11(5):607.

189. A Comprehensive View of the Structure and Expression of the Ependymoma Genome

Karen D Wright, Vikki Rand, Sarah E Leary, Robert A Johnson, Helen Poppleton, Mohankumar Murugesan, Stephen Mack, Beth Coyle, G Yancey Gillespie, Jeffrey Allen, Amar Gajjar, Frederick A Boop, Robert A Sanford, Michael D Taylor, Richard Grundy, Richard J Gilbertson

INTRODUCTION

To identify genetic alterations that underlie ependymoma tumorigenesis, we conducted an extensive and comprehensive analysis of the structure and expression of 204 ependymoma genomes.

METHODS

Fifty-six supratentorial (ST), 110 posterior fossa (PF) and 38 spinal (SP) ependymomas were profiled using 500K single-nucleotide polymorphism (SNP), U133Plus2 Affymetrix gene, and microRNA (miRNA) expression microarrays. Real-time polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization were used to validate selected genetic alterations.

RESULTS

Gene expression profiles segregated tumors by site and identified disease subgroups within each anatomical region (four ST, three PF, one SP). Gene expression subgroups also displayed significantly distinct miRNA expression profiles indicating that these are likely to be biologically discrete types of ependymoma. Manual and algorithmic genomewide review of the SNP arrays identified large chromosomal changes as well as 256 nonrandom regions of focal amplification or deletion containing 1,194 genes. Large chromosomal changes occurred more frequently in SP tumors (p < 0.0001); ST tumors averaged more focal changes (n = 13.2) than either PF (n = 6.2) or SP tumors (n = 3.0) (p < 0.0001). Integration of gene expression and SNP profiles identified regions of copy number-driven gene expression among large and focal genetic changes. Large deletions or gains of chromosome 6, 9, 10, and 16 contributed significantly to the gene expression subgroups. Sixty-eight of 256 focal changes (32 deletions and 36 amplifications) were selected for further study, which included copy number-driven gene expression changes. Sixty-three (93%, n = 63/68) were validated by RT-PCR. Validated alterations targeted specific cellular functions (e.g., cell adhesion, cell cycle, and neuronal development) and pathways (e.g., NOTCH, EPHRIN, and TP53). These genes have been further analyzed functionally in our mouse model systems (see submitted abstract Johnson, Gilbertson et al.).

CONCLUSION

We present a highly comprehensive view of the ependymoma genome, including 63 previously unrecognized candidate tumor suppressor genes and oncogenes that may afford diagnostic and therapeutic targets.

Neuro-Oncol. 2009 Oct;11(5):607.

190. Novel Characterization of the Medulloblastoma Genome using Transposon-Mediated Insertional Mutagenesis

Jon D Larson, Fausto J Rodriguez, Addison M Demer, Aaron M Bender, Robert Jenkins, Adam Dupuy, Neal Copeland, Nancy Jenkins, Michael D Taylor, David A Largaespada

INTRODUCTION

Medulloblastoma is a primitive neuroectodermal tumor of the cerebellum that accounts for ~20% of pediatric primary brain tumors in the United States. Although current medulloblastoma treatments are effective, the overall 5-year survival rate is only sixty percent. Current understanding of the heterogeneous genetic basis for medulloblastoma has provided insight into its histogenesis and pathology; however, this knowledge is incomplete as distinct molecules and signaling pathways provide variable levels of disease risk and severity.

METHODS

To address this, we are using Sleeping Beauty (SB) transposon-mediated insertional mutagenesis in mice as a forward genetics method to direct the identification and characterization of novel genetic mutations required for tumor formation. The T2/Onc transposon is provided as a multicopy chromosomal concatomer capable of activating oncogenes or silencing tumor suppressor genes upon transposase-mediated mobilization and reintegration within or near such genes. We are conditionally activating this system in glial and neural cells throughout the mouse brain where transposition is tracked by transposon-specific sequence. Furthermore, we combined this strategy with conditional activation of a dominant-negative form of TRP53 or conditional PTEN loss. These experiments are significant because of accumulating evidence that the TRP53 and PTEN pathways are directly affected in human medulloblastoma, and will provide a novel model to identify additional mutations that cooperate with these altered pathways during tumor formation.

RESULTS

These experiments generated genetically heterogeneous medulloblastoma in mice that suffer brain-specific transposition. Histological and immunohistochemical analysis revealed these tumors are similar to human medulloblastoma. Common integration sites throughout the genome of multiple tumors identified genes that potentially play a previously unreported causal role in medulloblastoma development.

CONCLUSION

We are testing how expression levels of these genes influence cellular transformation in vitro and in vivo. This report illustrates the utility of the SB system to further understand the genetic causes of medulloblastoma.

Neuro-Oncol. 2009 Oct;11(5):607.

191. A Murine Insertional Mutagenesis Screen to Identify Oncogenes and Tumor Suppressor Genes Involved in Glial and Schwann Cell Tumor Initiation and Progression

Eric Rahrmann, Vincent W Keng, Fausto J Rodriguez, Adam Dupuy, Lino Tessarollo, Nancy Jenkins, Neal Copeland, Nancy Ratner, Robert Jenkins, David A Largaespada

INTRODUCTION

Glial cell malignancies are serious clinical problems. Despite some information on the genetic basis of these cancers, the diversity of mutations that cause glial cell tumors remains incompletely understood. We are using the conditional Sleeping Beauty (SB) transposon-based somatic mutagenesis system to model glial tumors in mice to identify new genes and pathways that cause these tumors. We are focusing on tumors derived from oligodendritic and Schwann cell lineages by targeting the SB system to 3′-cyclic nucleotide 3′-phosphodiesterase (CNP)-positive cells. Since loss of p53 function and/or overexpression of epidermal growth factor receptor (EGFR) are associated with malignant peripheral nerve sheath tumors (MPNSTs) and oligodendroglioma, CNP-EGFR and conditional p53R270H alleles are also included.

METHODS

To create mutations in glial cells, three transgenes were introduced into mice. The first transgene conditionally expresses the Sleeping Beauty (SB11) transposase enzyme when Cre recombinase is also present within the cell. The second transgene is an oligodendritic and Schwann cell specific Cre-recombinase controlled by the CNP promoter. The third transgene is a concatomer of oncogenic transposons capable of activating and/or disrupting endogenous gene expression. Mice possessing all three transgenes experience transposition and insertional mutations in oligodendritic and Schwann cell lineage cells.

RESULTS

SB is expressed and active in the oligodendrocyte lineage with variegated expression in other tissue types. Furthermore, mice with active transposition develop a variety of tumors including bony nodules found in the spinal column leading to paralysis and gliomas with oligoden-droglioma features. Mice that also harbor both a conditional dominant-negative p53 allele and CNP-EGFR transgene are developing MPNSTs.

CONCLUSION

SB insertional mutagenesis can cause nervous system tumors. The next step will be to identify candidate brain cancer genes among a large number of tumors generated, which will be studied for their role in tumor initiation and progression.

Neuro-Oncol. 2009 Oct;11(5):608.

192. IDH1 and IDH2 Mutations Play A Fundamental Role in Glioma Development

Hai Yan, Donald W Parsons, Genglin Jin, Zachary J Reitman, Roger McLendon, David A Reardon, Allan H Friedman, Henry Friedman, James Herndon, Gregory J Riggins, Sion Jones, Ken Kinzler, Bert Vogelstein, Victor Velculescu, Ahmed B Rasheed, Ivan Kos, Ines Batinic-Haberle, Darell Bigner

INTRODUCTION

Gliomas are the most common type of primary brain tumors. A comprehensive elucidation of genetic alterations in gliomas would provide novel targets for diagnostic, prognostic, or therapeutic purposes, as well as allow subclassification of patients who may preferentially respond to particular targeted therapies.

METHODS

We determined the sequence of the isocitrate dehydrogenase 1 (IDH1) gene and the related IDH2 gene in 445 CNS tumors and 494 non-CNS tumors. The enzymatic activity of normal and mutant IDH1 and IDH2 was determined. Kaplan-Meier survival curves were plotted according to IDH mutation status.

RESULTS

The genomewide mutational analysis of glioblastomas (WHO grade IV glioma) revealed somatic mutations of IDH1 in a fraction of such tumors. We then identified mutations that affected the same amino acid of IDH1, R132, in more than 70% of WHO grades II and III astrocytomas and oligodendrogliomas and in glioblastomas that developed from these lower-grade lesions. Tumors without mutations in IDH1 often had mutations affecting R172, the analogous amino acid in IDH2, but IDH mutations were not found in any other type of cancer. Tumors with these mutations had distinctive genetic and clinical characteristics, and patients with such tumors had better outcomes than those with wild-type IDH genes. The IDH mutations changed the enzymatic activity of the encoded protein. Glioma-derived IDH1 mutants have recently been shown to heterodimerize with and dominantly inhibit the activity of wild-type IDH1, and we show that increasing amounts of exogenous IDH2R172K dominantly inhibit the activity of exogenous wild-type IDH2, suggesting that glioma-derived IDH2 mutants also exhibit dominant-negative activity.

CONCLUSION

Mutations of the NADP+-dependent isocitrate dehydrogenases encoded by IDH1 and IDH2 occur as the earliest genetic event in a majority of malignant gliomas and have important implications for the pathogenesis, diagnosis, and treatment of these tumors.

Neuro-Oncol. 2009 Oct;11(5):608.

193. Identification of CDK6 Oncogene Amplification, 10q Deletion, and 17q Gain as Novel Molecular Markers for Adverse Prognosis in Adult Medulloblastoma

Stefan Pfister, Marc Remke, Axel Benner, Wiebke Werft, Marina Ryzhova, Hendrik Witt, Dominik Sturm, Anna Schöttler, Andrea Wittmann, Wolfram Scheurlen, Andreas E Kulozik, Peter Lichter, Andrey Korshunov

INTRODUCTION

While in children medulloblastoma (MB) comprises the most common malignant brain tumor, it only accounts for 1% of intracranial malignancies in adults. The infrequent occurrence of MB in adults poses the question of whether these tumors are the same in adults and children in terms of biological and clinical peculiarities. This study is aimed at the identification of molecular pathomechanims and novel molecular biomarkers in adult medulloblastoma.

METHODS

Array comparative genomic hybridization (CGH) was performed for a total 34 adult medulloblastoma samples and the results were compared with data from 101 pediatric patients. Selected genomic regions were further investigated by FISH analysis in an independent cohort of 415 samples (112 adult and 303 pediatric). All 146 adult patients received a standard treatment regimen (Philadelphia protocol). To identify novel prognostic markers, DNA copy-number information was correlated with survival data using log rank and chi-square tests.

RESULTS

Deletion of chromosome 10q, gain of chromosome 17q, and oncogene amplification of CDK6 were identified as novel adverse prognostic markers in adult medulloblastoma. Except in one case, CDK6 amplification was only observed in adult patients (9% in adults vs. 0.2% in children), whereas amplifications of MYC or MYCN were significantly overrepresented in the pediatric cohort. 10q deletion showed a similar frequency in adults and children (20%–25%), however, were only associated with dismal prognosis in adults. Monosomy of chromosome 6, in contrast to the pediatric cohort, was not significantly associated with good prognosis, although nuclear β-catenin accumulation was detected in most cases (r = 0.68). Based on these results, we propose a molecular staging system for adult medulloblastoma: i) cases with oncogene amplification (11% of cases, 5-year OS = 0%), ii) cases with chromosome 17q gain and/or 10q deletion without oncogene amplification (30% of cases, 5-year OS = 41%), and iii) cases without oncogene amplification or 17q gain/10q deletion (59% of cases, 5-year OS = 96%).

CONCLUSION

We report on the largest cohort of adult medulloblastoma investigated for genomic imbalances to date. Based on our results, we propose a model for the molecular risk stratification of adult medulloblastoma comprising three distinct genomic risk groups with significantly different survival and tumor biology.

Neuro-Oncol. 2009 Oct;11(5):608.

194. Genome-Wide Screen Reveals Core Genes and Pathways Essential For Glioblastoma Cell Survival

Nikhil Thaker, Peter McDonald, Tong Ying Shun, John Lazo, Ian F Pollack

INTRODUCTION

Target identification and validation remain difficult steps in the drug discovery process, and identifying the genes and pathways that are most essential for cancer cell survival may greatly facilitate this process. Glioblastoma multiforme (GBM) is an excellent model for identifying these essential genes because of its well-characterized radio-resistance and chemoresistance profiles. Thus, annotation of these essential genes may provide insight into the core regulators of cell survival and may help uncover nodes of chemosensitivity.

METHODS

We implemented an siRNA library targeting 5,520 unique human genes in the T98G GBM cell line. Cells were reverse transfected, and siRNA complexes were prepared by combining siRNA in pools of three unique duplexes with DharmaFECT2 transfection reagent. Cells were incubated for 96 h to allow for gene silencing, and cell viability was measured with a resazurin fluorescent dye assay. We developed two orthogonal statistical analysis methods to capture high-confidence hits. This screen was performed in eight replicates.

RESULTS

We identified 55 genes that were essential for glioma cell survival. Interestingly, 22% of these “survival” genes were constituents of the proteasome complex, and we chose the proteasome component PSMB4 for further validation. We confirmed the on-target effect by observing protein knockdown with two of three unpooled siRNA sequences, which was consistent with cell viability data. Protein knockdown was observed by 48 h and appeared to occur before induction of cell death. The generality of this cytotoxic effect was also confirmed in a series of glioma and nonglioma cell lines with the PSMB4 siRNA and the proteasome inhibitor MG-132.

CONCLUSION

We identified the proteasome complex as an essential regulator of glioma cell survival. Proteasome inhibition, through either small molecule inhibition or therapeutic RNAi, may be an effective treatment for GBM and may be enhanced with the development of second-generation proteasome inhibitors that can penetrate the blood-brain barrier.

Neuro-Oncol. 2009 Oct;11(5):608.

195. Genomic DNA Analysis of Diffuse Intrinsic Pontine Glioma (DIPG) Tissue Obtained from Autopsy

Katherine E Warren, Keith Killian, Miia Suuriniemi, Yonghong Wang, Martha Quezado, Paul Meltzer

INTRODUCTION

The utility of biopsy in children with DIPG has been debated. The prognosis remains dismal and progress has been hindered by the limited tissue available for study. We performed comparative genomic hybridization (CGH) studies on autopsy specimens to examine the feasibility of determining DNA genomic copy-number aberrations on formalin-fixed, paraffin-embedded (FFPE) blocks.

METHODS

FFPE blocks obtained from autopsies of 14 pediatric DIPG patients were reviewed by a pathologist. Regions of the block were marked for processing and punched with a 0.6-mm tissue microarray needle. DNA was extracted from the core and labeled by chemical coupling with Cy5 (Agilent) and hybridized to 105K oligonucleotide CGH arrays (Agilent). After hybridization and washing, arrays were scanned and data segmented and processed with Nexus software.

RESULTS

Histological examination showed intrapatient and interpatient variability. All samples demonstrated abnormal DNA. Frequent DNA structural aberrations included gain of 1q, gain of 7p, and loss of 10q. High copy-number amplification of known oncogenes was observed, including PDGFRA, EGFR, CCND1, and MYCN. Homozygous deletions of known tumor suppressor genes included PTEN and CDKN2A. Additional regions of high copy number amplification and homozygous deletion were observed. Geographical variation in the CGH pattern, corresponding to distinct areas of low-grade and high-grade tumor was observed in one patient, with the high-grade region containing many more aberrations.

CONCLUSION

Satisfactory CGH results can be obtained from DIPG autopsy material. The sample set was highly informative, with the majority of specimens showing at least one abnormality related to a known cancer gene. Aberrations in candidate drug targets (KIT and EGFR) were observed. This study establishes the feasibility of genomic DNA analysis from DIPG autopsy material and suggests significant heterogeneity among DIPG patients, supporting the need for biopsy and a larger scale study to define the spectrum of molecular abnormalities in DIPG.

Neuro-Oncol. 2009 Oct;11(5):609.

196. Deregulated Expression of Minichromosome Maintenance Proteins MCM2, MCM3, and MCM7 and their Biological and Clinical Significance in Medulloblastoma

Kin Mang-Lau, Queeny Kwan Yi Chan, Ho Keung-Ng

INTRODUCTION

Minichromosome maintenance proteins MCM2–7 play important roles in DNA replication licensing to ensure precise chromosomal duplication during normal cell division. Deregulation of expression of these MCMs results in genetic instability, which frequently occurs in cancers. In this study, we investigated the abnormalities of these MCM proteins in medulloblastoma.

METHODS

Statistical analysis of publicly available DNA microarray data for 60 medulloblastomas and two normal cerebellum samples indicated significant upregulation of MCM2, MCM3, MCM5, and MCM7 expression in medulloblastoma. Using quantitative reverse transcription-polymerase chain reaction, we demonstrated that human medulloblastoma cell lines (n = 7) expressed abnormally high levels of MCM2 and MCM3 transcripts as compared with five normal cerebellum samples. These upregulated genes were also observed in our 31 medulloblastoma samples.

RESULTS

In addition, MCM7 expression was found to be upregulated in our clinical samples, as two of the medulloblastoma cell lines (DAOY and ONS76) showed this upregulation. With immunohistochemical analysis on tissue microarray, we found 37 (51%), 42 (58%), and 54 (74%) of 73 medulloblastoma samples with strong nuclear immunostaining for MCM2, MCM3, and MCM7 respectively. Kaplan-Meier survival analysis using the log-rank test showed that patients with high expression of MCM3 showed poor survival. To determine the biological significance of deregulation of these MCMs, the effects of specific knockdown of MCM7 by siRNA on the growth, migration, and invasion of DAOY medulloblastoma cells were investigated. Treatment of siRNA targeting these MCMs significantly induced cell growth inhibition, suggesting that deregulation of these MCMs might be favorable for increased proliferation or survival of medulloblastoma cells. In addition, we demonstrated that these MCMs might be involved in the regulation of cell migration and invasion as the knockdowns significantly inhibited cell migration and invasion using wound healing and transwell migration/invasion assays. Furthermore, the knockdowns reduced the filopodial cells with blocking the cdc42 activation in the cells.

CONCLUSION

Taken together, deregulation of MCM2, MCM3, and MCM7 expression may play a role in the development of medulloblastoma, and this is the first study to demonstrate MCMs’ regulation of medulloblastoma cell migration and invasion.

Neuro-Oncol. 2009 Oct;11(5):609.

197. An Epigenetic Genome-Wide Screen Identifies Members of the SFRP Family of WNT Signaling Inhibitors as Novel Tumor Suppressor Genes in Medulloblastoma

Paul N Kongkham, Paul A Northcott, Michael D Taylor, James T Rutka

INTRODUCTION

Medulloblastomas (MB) are the most common pediatric nervous system malignancy. Despite treatment, mortality rates remain at 30%. Known mutations account for only a subset of MB cases. We hypothesize that screening for tumor suppressor genes (TSGs) silenced by promoter CpG island methylation may uncover TSGs not previously described in MB.

METHODS

We performed an epigenomewide screen of nine MB cell lines treated with 5-aza 2′deoxycytidine (5-aza-dC) using Affymetrix HG U133 plus 2.0 expression arrays, identifying genes with increased expression following treatment.

RESULTS

We identified three members of the SFRP family of Wnt signaling inhibitors (SFRP1, SFRP2, and SFRP3) as putative TSGs silenced by promoter methylation in MB. We confirmed methylation of the SFRP1, SFRP2, and SFRP3 promoter regions using bisulfite sequencing and found aberrant methylation in 12/51, 2/51, and 8/51 primary MB tumors by methylation-specific PCR, respectively. TSG function was assessed through stable reexpression of SFRP1, SFRP2, and SFRP3 in the D283 and ONS76 MB cell lines. Reexpression of each gene reduced MB cell proliferation as assessed by MTS assay. SFRP1, SFRP2, and SFRP3 reexpression in D283 cells reduced their ability to form colonies in soft agar. Reexpression of these Wnt signaling inhibitors also limited Wnt signaling axis activity, as evidenced by reduced phospho-DVL2 levels by Western blotting. In vivo, SFRP1 reexpression limited tumor growth of ONS76 cells in a flank xenograft model. Using an orthotopic intracerebellar xenograft model, mice engrafted with D283 cells reexpressing SFRP1 demonstrated reduced tumor burden and increased overall survival compared to controls.

CONCLUSION

Aberrant Wnt signaling is known to play a role in the pathogenesis of a subset of MB cases. We have identified, for the first time, a novel mechanism for the loss of normal pathway inhibition that contributes to upregulated Wnt signaling in this disease.

Neuro-Oncol. 2009 Oct;11(5):609.

198. Paraffin-Friendly Assays for Identification of BRAF Mutations in Pediatric Low-Grade Astrocytomas

Mark W Kieran, Laura E MacConaill, Jennifer Chan, Sandro Santagata, Deborah J Goff, Natalie Vena, Azra Ligon, Charles D Stiles, Levi Garraway, Keith L Ligon

INTRODUCTION

In pediatric gliomas, two distinct classes of activating mutations in the protein kinase BRAF have been identified, suggesting that inhibitors of BRAF, already in adult clinical trials, may be promising therapeutics for these tumors. As such, there is an emerging need for clinically robust paraffin-based technologies to identify such events in children who might benefit from these targeted agents. In this study, we sought to develop a set of robust paraffin-based technologies for BRAF mutation detection and to better characterize the BRAF mutation spectrum in pediatric glioma subtypes.

METHODS

BRAF activation events in pediatric gliomas are reported to occur as tandem duplications of the BRAF locus or the point mutation BRAFV600E. To detect BRAF duplications, we developed a robust BRAF fluorescence in situ hybridization (FISH) assay and compared the results to those obtained using FFPE-based whole genome Agilent array comparative genomic hybridization (CGH; 1 million-feature resolution). To detect the BRAFV600E mutations, we developed a paraffin protocol for the OncoMap platform, a mass spectroscopy-based genotyping method that simultaneously queries mutations in ~1,000 known oncogenes and tumor suppressors.

RESULTS

BRAF duplication was detected in 100% of “classic” piloyctic astrocytomas by paraffin FISH (n = 23) and whole genome array CGH (n = 3) with comparable efficiency. Duplication events were rarely detected in nonpilocytic gliomas using either platform. Application of OncoMap to a series of 127 pediatric gliomas demonstrated BRAFV600E mutations in 30% of nonpilocytic gliomas (21/66), including 57% of gangliogliomas (8/14), a tumor not previously reported to have the BRAFV600E mutation. Cross-validation studies with Sanger and Solexa sequencing demonstrated higher sensitivity using OncoMap than conventional Sanger sequencing.

CONCLUSION

We have established and validated protocols for detecting the two most common types of BRAF mutations in clinical archival FFPE pediatric glioma samples. Such assays appear sufficiently robust for migration into clinical lab environments to enact future personalized medicine approaches for pediatric glioma patients and other cancers.

Neuro-Oncol. 2009 Oct;11(5):609.

199. Unique Molecular Characteristics of Pediatric Myxopapillary Ependymoma

Valerie Nicole Barton, Andrew M Donson, Betty K Kleinschmidt-DeMasters, Diane Birks, Nicholas K Foreman

INTRODUCTION

Myxopapillary ependymomas (MEPNs) are slow-growing spinal tumors. Surgical resection generally results in a favorable prognosis, but recurrence is common, and the role of adjuvant chemotherapy or radiation therapy is unclear in the clinical management of recurrent tumors. This is particularly relevant to pediatric MEPNs, which have a higher rate of local recurrence, are more likely to develop metastases, and display a more aggressive course of disease. Clinical trial design for MEPN requires a better understanding of tumor biology, which is currently limited.

METHODS

To better characterize these tumors, we compared the gene expression profiles of five pediatric MEPNs and 16 pediatric intracranial EPNs. Genes of interest were further examined by immunohistochemical analysis. Normal adult and fetal ependyma was used as a control.

RESULTS

In a clustering analysis of EPN gene expression profiles, pediatric MEPN formed a distinct subgroup within pediatric intracranial EPNs suggesting that gene expression of MEPN is distinct from other EPNs. Unique molecular features of MEPN were identified including overexpression of multiple genes of the homeobox family (HOX), neurofilament light (NEFL), and platelet-derived growth factor receptor-α (PDGFRA).

CONCLUSION

To our knowledge, this is the largest study to examine the molecular signature of pediatric MEPN as compared with intracranial EPN by microarray. In addition to defining up- and downregulated genes in MEPN, our analysis revealed several potential therapeutic targets. The overexpression of HOX genes in MEPN, combined with their potential oncogenic function, implies that the HOX family may be involved in the tumorigenesis of MEPN. High expression of HOX family genes and PDGFRA in MEPN suggests that therapeutics targeting these would be appropriate candidates for future clinical trails.

Neuro-Oncol. 2009 Oct;11(5):610.

200. Molecular Characterization of Pediatric Glioblastoma using An Integrative Genomics Approach

Dominik Sturm, Marc Remke, Hendrik Witt, Sebastian Barbus, Bernhard Radlwimmer, Till Milde, Michael Frühwald, Christoph Kramm, Guido Reifenberger, Jörg Felsberg, Andreas von Deimling, Christian Hartmann, Andrey Korshunov, Andreas E Kulozik, Olaf Witt, Marina Rhyzova, Peter Lichter, Stefan Pfister

INTRODUCTION

In contrast to their adult counterparts, glioblastoma multiformes (GBMs) rarely occur in children and comprise a largely different entity in terms of tumor biology and patient outcome. Whereas genetic and epigenetic properties have been extensively studied in adult GBM, little is known about the molecular pathogenesis of pediatric GBM.

METHODS

To identify DNA copy-number aberrations, 24 pediatric GBM samples were analyzed by array-based comparative genomic hybridization (array-CGH). In addition, the mutational status of TP53, IDH1, and IDH2 was determined by direct genomic sequencing. To account for epigenetic regulatory events, we are conducting a genomewide methylation study and investigating MGMT promoter methylation by sodium bisulfite sequencing. Results of genomewide mRNA expression profiling will be correlated to DNA copy-number alterations and methylation status. Molecular findings will be correlated with clinical parameters including patient survival. Clinically and biologically relevant findings will be validated in an independent series of 50 paraffin-embedded tumor samples from pediatric GBM patients.

RESULTS

Array-CGH revealed well-known high-level amplifications targeting RB signaling (CDK4, 17%; CCND2, 4%), homozygous deletion of CDKN2A (18%), receptor tyrosine kinase signaling (EGFR, 9%; PDGFRA, 8%), and p53 signaling (MYC/MYCN, 8%; MDM2, 4%; MDM4, 4%). Novel focal amplifications, including NTRK1, EPHA2, EPS8, and FGFR3 among others, were identified. Loss-of-function mutations of TP53 were observed in 47% of the cases. Remarkably, we detected alternative mechanisms to overcome p53-mediated cell cycle arrest (in the presence of wild-type p53), such as amplification of EP400 and amplification of a miRNA cluster at 19q13.4 (including miRNA-372 and miRNA-373). Mutations of IDH1 occurred in 5% of the samples, whereas no IDH2 mutations were identified.

CONCLUSION

We report on one of the largest cohorts of pediatric GBM investigated for genetic alterations to date, which together with integrative analyses of mRNA expression and DNA methylation patterns will identify frequently altered signaling pathways. We will next correlate these alterations with clinical outcome and may, thus, identify tumor-derived biomarkers to tailor treatment intensity to molecular risk. Furthermore, these observations may open the avenue to developing novel therapeutic strategies.

Neuro-Oncol. 2009 Oct;11(5):610.

201. Massively Parallel Paired-End Sequencing in High-Risk Medulloblastoma

Stefan Pfister, Andreas Schattl, Andrey Korshunov, Marc Remke, Hendrik Witt, Adrian Stuetz, Wolfram Scheurlen, Andreas E Kulozik, Peter Lichter, Jan Korbel

INTRODUCTION

Molecular pathways such as WNT and SHH could recently be attributed to standard-risk medulloblastoma, whereas the molecular pathomechanisms underlying intermediate and high-risk medulloblastoma are still poorly understood. Isochromosome 17 formation comprises a hallmark cytogenetic lesion within these latter two clinical subgroups. This project aims to identify somatically acquired structural genomic rearrangements (SRs) in high-risk medulloblastoma by high-resolution paired-end mapping (PEM). PEM is capable of identifying ~100 somatically acquired SRs and hundreds of germline structural variants in a single tumor genome.

METHODS

To comprehensively fine-map SRs in medulloblastoma, we investigated a classic medulloblastoma harboring an isochromosome 17q by using PEM. PEM involves end-sequencing of randomly sampled size-selected genomic DNA fragments of an individual tumor and comparing these with the human reference genome. SRs are identified computationally by using Paired-End-Mapper. To validate the impact of identified candidate SRs in medulloblastoma, more than 250 primary medulloblastoma samples will be analyzed for the presence of these SRs by PCR-based assays and genomic sequencing.

RESULTS

We have generated PEM libraries with insert sizes of 500 bp and 3 kb for the tumor, and a 3-kb library for the corresponding germline DNA. So far, 50.8 M and 33.6 M Illumina paired-end reads were sequenced from the tumor-derived 3-kb and 500-bp libraries, reaching 7-fold and 1-fold coverage, respectively. The 3-kb protocol has yielded 3,203 SRs so far, including 1,376 insertions, 1,341 deletions, 269 inversions, and 217 complex rearrangements, out of which approximately half were previously reported as germline aberrations. Thus, a large fraction of events may either represent somatic SRs specific to the tumor, novel germline variants, or false positives. Currently, more than 50 SRs targeting at least one exon of a known protein-coding gene are validated by PCR-based methods applied to both the tumor and the germline sample. Tumor-specific events will be investigated in a large cohort of primary medulloblastoma samples and presented at the meeting.

CONCLUSION

Applying PEM in a prototype primary medulloblastoma will likely identify interesting somatically acquired SRs. The relevance of these findings will be validated in a large collection of primary medulloblastoma samples.

Neuro-Oncol. 2009 Oct;11(5):610.

202. Abnormalities of the MAPK Pathway in Pediatric Low-Grade Gliomas

David Ellison, Tim Forshew, Ruth Tatevossian, Andrew Lawson, Jing Ma, Geoff Neale, James Dalton, Alberto Broniscer, Amar Gajjar, Simon Bailey, Denise Sheer

INTRODUCTION

Low-grade gliomas (LGGs) have the highest frequency among pediatric brain tumors. Although 16% of patients with LGGs have NF-1, underlying genetic defects remain poorly defined in these tumors.

METHODS

Detailed histopathologic review of 50 pediatric LGGs preceded analysis of DNA from frozen tumor samples on Affymetrix SNP 6.0 arrays. Sequence analysis was subsequently performed on genes involved in the MAPK pathway and Western blotting was used to confirm pathway activation.

RESULTS

Focal CN gains at 7q34 were associated with five fusion-gene variants between BRAF and KIAA1549 in 30/32 posterior fossa pilocytic astrocytomas (PAs), while in 1/2 remaining PAs a fusion gene between SRGAP3 and RAF1 was associated with focal gain at 3p25. The resulting fusion genes lack the autoinhibitory domains of BRAF and RAF1, respectively, conferring constitutive kinase activity. An activating mutation of KRAS was identified in the remaining PA without a BRAF or RAF1 fusion. Thus, we identified activating mutations in constituents of the MAPK pathway in all 32 posterior fossa PAs. In contrast, BRAF and KIAA1549 fusion genes were found in only 2 of 18 non-PA LGGs, one hypothalamic pilomyxoid astrocytoma and one diffuse astrocytoma. BRAF V600E mutations were identified in a single example of a pleomorphic xanthoastrocytoma and 1 of 11 diffuse cerebral astrocytomas. Protein lysates from BRAF-KIAA1549 fusion-gene tumors were probed with antibodies specific to MEK1/2, phospho-MEK1/2, ERK1/2 and phospho-ERK1/2. Western blot analysis showed no differences in levels of MEK and ERK between tumor samples and normal brain controls, but higher levels of phospho-MEK and phospho-ERK were detected in tumor samples when compared to controls.

CONCLUSION

Our results suggest that activating mutations of the MAPK pathway constitute a relatively specific marker of posterior fossa PAs. In addition, identifying MAPK pathway activation in LGGs will facilitate the use of targeted drug therapies for disease that is not amenable to surgical resection.

Neuro-Oncol. 2009 Oct;11(5):610.

203. Age Drives Genomic Subgrouping of Medulloblastoma

Paul A Northcott, Stefan Pfister, Andrey Korshunov, Pim French, Michael D Taylor

INTRODUCTION

We reanalyzed our data set of 103 human medulloblastomas with paired copy-number and expression data.

METHODS

Affymetrix SNP and Exon array platforms were used.

RESULTS

Unsupervised hierarchical clustering of expression data demonstrated four distinct, nonoverlapping subgroups. The known Wnt/Monosomy-6 subgroup comprised 8%, and the SHH subgroup comprised 32% of tumors. We identify two additional, nonoverlapping molecular subgroups. Group C (26%) was comprised of childhood, classic subgroup tumors (age range, 2–10 years) with high expression of MYCC. Conversely, group D (34%) includes patients of all ages with classic histology, but low levels of MYCC expression. Group C tumors were only found between the ages of 2 and 10 years. The absence of group C tumors in teenagers may account for the different prognosis known to occur in that age group. SHH-driven tumors are found in infants and adults, but only rarely in children or teenagers. Our data demonstrate that a number of hitherto neglected regions of genomic gain/loss are highly restricted to biological subgroups of medulloblastomas and that they have a relatively high frequency within these subgroups. It is well known that monosomy 6 is found only in Wnt-driven medulloblastomas. We now demonstrate 2p, 3q, and 20q gain and 9q loss in SHH-group tumors, 10q loss and 1q,13 gain in group C tumors, and i17q, which is largely restricted to group D tumors. Many of these copy-number aberrations seem rare and unimportant when analyzing all medulloblastomas, but become of obvious importance when analyzed in a subgroup-specific manner. Analysis of SHH-driven tumors demonstrates two clinical peaks, one in infants and another in adulthood. Unsupervised hierarchical clustering of SHH-driven medulloblastomas based on their expression profiles demonstrates that infant SHH medulloblastomas are biologically distinct from adult SHH medulloblastomas.

CONCLUSION

Our data demonstrate that the four subgroups of medulloblastoma (Wnt/SHH/C/D) have specific clinical, expression, and genetic features suggesting that they represent separate and distinct diseases.

Neuro-Oncol. 2009 Oct;11(5):611.

204. Epigenetic Silencing of Gene Expression in Pediatric Intracranial Ependymoma

Tracy J Warr, Katherine Karakoula, Kim Phipps, William Harkness, Richard Hayward, Dominic Thompson, Thomas Jacques, David GT Thomas

INTRODUCTION

Transcriptional silencing of genes through promotor hypermethylation is widespread in the pathogenesis of many tumor types, including adult brain tumors. Previously, we have demonstrated that aberrant DNA methylation is an important mechanism in the downregulation of a putative tumor suppressor gene, Chibby, on chromosome 22 in pediatric intracranial ependymoma. We have now investigated whether aberrant methylation is the predominant mechanism driving downregulation of multiple genes by determining global gene expression changes in pediatric ependymoma cells following pharmacological unmasking of methylated genes with the demethylating agent 5-aza-2′-deoxycytidine (5Aza-dC).

METHODS

Three short-term cell cultures derived from pediatric ependymomas were treated with 5Aza-dC or mocked treated with DMSO for 96 h. Global expression microarray analysis was carried out using U133A plus 2.0 Arrays (Affymetrix). The methylation and expression status of target candidate genes was validated by bisulfite sequencing, methylation-specific PCR, and real-time Q-PCR in a larger panel of pediatric ependymomas.

RESULTS

Five hundred and ninety genes were upregulated 3-fold compared to the mock-treated control cells. Genes that had been silenced by hypermethylation included some with tumor suppressor functions and genes involved in cell adhesion, angiogenesis, transcriptional regulation, apoptosis, and signal transduction pathways. One gene, SFN, is involved in the p53-mediated response of cells to DNA damage and is epigenetically silenced in a number of other types of cancers. Bisulphite sequencing and methylation-specific PCR revealed hypermethylation of the promotor region of SFN in 60% of ependymomas.

CONCLUSION

Pharmacological unmasking of methylated genes followed by global gene expression analysis has identified a number of genes in which gene silencing through DNA methylation appears to be the predominant mechanism for downregulation of expression in pediatric ependymomas.

Neuro-Oncol. 2009 Oct;11(5):611.

205. Comparative Genomic Hybridization (CGH) of Tubers and Subependymal Giant Cell Tumors (SGCTS) in Patients with Tuberous Sclerosis Complex

Robert John Bollo, Ingeborg Fischer, Adelou Olasunkanmi, Veronique Haegeli, Martha Eugenia Ruiz-Tachiquin, Christopher Leo Campbell, Alexander Pearlman, Harold Ostrer, Howard L Weiner, Robert J Schneider

INTRODUCTION

Tuberous sclerosis complex (TSC) is a multisystem genetic disorder associated with two pathognomonic cerebral lesions: neuroglial hamartomas (tubers) and SGCTs. Our experience suggests divergent clinical phenotypes; patients often present with tuber-associated epilepsy or hydrocephalus caused by an enlarging SGCT, but rarely both. We hypothesize that these distinct phenotypes reflect unique genetic changes in tubers versus SGCTs.

METHODS

CGH was performed on eight tubers (mean age, 4.8 years), six SGCTs (mean age, 8.9 years), and one control sample of normal cortex. One SGCT and one tuber were resected from monozygotic twins harboring the same TSC2 mutation. DNA was extracted from paraffin-embedded tissue. Samples were analyzed on the Affymetrix GeneChip human mapping 500K Nsp assay kit. Copy-number regions of amplifications and deletions were reported when event loci overlapped in multiple test samples but not control. Array probes were mapped to genes within 500 kb, and a z-score was calculated for each gene and assigned a Bonferoni adjusted p-value used to rank significant genes (p < 0.05).

RESULTS

Of the genes with significant copy-number variation, 435 were observed in the entire set, 382 in tubers, and 300 in SGCTs. Two hundred and sixty-nine genes were significantly altered in tubers alone, 740 in SGCTs, and 39 were unique to the combined set of tubers and SGCTs. Genes with significant variation exclusively in tubers included IMPA2, a know risk factor for febrile seizures and schizophrenia, as well as CDC16, PTK2, PPP2R2D, THADA, and CASP3, highlighting cellular proliferation, neural development, and apoptosis pathways. Genes exclusively varied in SGCTs included SDC4, MATN4, DCC, PPFIA1, and NPHP4, reflecting genes involved in cell-matrix interactions and axonal guidance, as well as nuclear transcription factors.

CONCLUSION

Tubers and SGCTs from patients with TSC display distinct profiles of genetic variation and suggest a distinct pathogenesis, possibly underlying observed differences in clinical phenotype, even among monozygotic twins.

Neuro-Oncol. 2009 Oct;11(5):611.

206. Elevated p-STAT-3 Expression in Peripheral Blood Mononuclear Cells in Glioma Patients

William E Humphries, Yongtao Wang, Wei Qiao, Chantal Reina-Ortiz, Lamonne Crutcher, Jun Wei, Ling-Yuan Kong, Raymond Sawaya, Ganesh Rao, Jeffrey S Weinberg, Sujit S Prabhu, Gregory N Fuller, Amy B Heimberger

INTRODUCTION

Phosphorylation of the signal transducer and activator of transcription 3 (p-STAT-3) is frequent in malignant gliomas, propagates tumorigenesis, and is a key regulator of immunosuppression in cancer patients. Tumor expression of p-STAT-3 can induce p-STAT-3 expression in tumor-associated immune cells that can return to the circulatory system. We hypothesized that the percentage of peripheral blood mononuclear cells (PBMCs) expressing p-STAT-3 would be higher in glioma patients, would correlate with the extent of tumor-expressed p-STAT-3, and would correlate with the level of immune suppressive regulatory T cells (Tregs).

METHODS

The percentages of PBMCs expressing p-STAT-3 in normal donors (n = 23) and in patients with primary CNS gliomas (n = 45) were compared using a mixed linear model. The amount of p-STAT-3 expression in the tumor tissue was determined by immunohistochemistry and the fraction of FoxP3+ Tregs within the systemic CD4 compartment was determined by flow cytometry.

RESULTS

The average percentage of PBMCs expressing p-STAT-3 was higher in patients with anaplastic gliomas (14.3 + 9.4%, p = 0.01) and glioblastoma multiforme (GBM) (11.8 + 13.4%, p = 0.007) than in healthy volunteers (4.9 + 3.4%). Patients with recurrent GBM had the highest percentage of PBMCs expressing p-STAT-3 (18.8 + 17.1%, p = 0.0130), while the percentage of PBMCs expressing p-STAT 3 in patients with low-grade astrocytoma (5.4 + 7.4%, p = 0.95) and patients with GBM in remission (3.9 + 3.5%, p = 0.55) did not significantly differ from the levels in healthy volunteers. There was no correlation between p-STAT-3 expression in the tumor and the percentage of PBMCs expressing p-STAT-3. Furthermore, the percentage of PBMCs expressing p-STAT-3 did not directly correlate with the number of systemic FoxP3+ Tregs.

CONCLUSION

The percentage of PBMCs expressing p-STAT3 in malignant glioma patients has the potential to serve as an indicator of tumor progression and recurrence.

Neuro-Oncol. 2009 Oct;11(5):611.

207. Type-1 Dendritic Cell Vaccines in Combination with Poly-ICLC—Association Between Positive Tetramer Response and 6-Month Progression-Free Survival

Hideho Okada, Frank Lieberman, Ryo Ueda, Aki Hoji, Pawel Kalinski, Arlan H Mintz, Amin B Kassam, Johnathan A Engh, Deric M Park, David L Bartlett, Herbert Zeh, Theresa L Whiteside, Lisa H Butterfield, Ronald L Hamilton, Andres M Salazar, Ian F Pollack

INTRODUCTION

Our previous preclinical studies have demonstrated that intramuscular administration of Toll-like receptor 3-ligand poly-ICLC remarkably enhances the induction of type-1 cytotoxic T-lymphocytes (CTLs) and improves the therapeutic efficacy of vaccinations against glioma-associated antigen (GAA)-derived CD8+ T cell epitopes.

METHODS

We developed a phase I/II trial. Human leukocyte antigen (HLA)-A2+ participants with recurrent malignant glioma received intra-lymphnode injections of type-1 dendritic cells (DCs) loaded with HLA-A2-binding GAA-peptides EphA2 (883–891), IL-13Rα2 (345–353:1A9V), YKL-40 (202–211), and GP100 (209–217: 2M) at 2-week intervals. Participants also received twice weekly intramuscular injections of 20 μg/kg of poly-ICLC. Participants who demonstrated a positive radiological response or stable disease without major adverse events were allowed to receive booster vaccines. Primary end points were assessments of safety and immunological responses. Clinical and radiological responses were also evaluated.

RESULTS

To date, 15 participants (eight with glioblastoma multiforme [GBM], five with anaplastic astrocytoma [AA] and two with anaplastic oligodendroglioma [AO]) have received vaccinations with no major adverse events. Increased CD8+ cells reactive to EphA2- or IL-13Rα2-tetramers were detected in postvaccine peripheral blood mononuclear cells (PBMC) in 9 of 11 participants evaluated. These patients also demonstrated upregulation of a chemokine receptor CXCR3 on CD8+ PBMC following vaccines, indicating that the vaccine regimen induced type-1 CTL responses. Five patients achieved progression free at 6 months (two GBM, two AA, and one AO). Among these, four patients were currently progression free at 17 (AA), 15 (GBM), 14 (AA), or 9 (AO) months after the first vaccine and are receiving booster vaccines every 3 months. Although the trial enrolled mixed tumor types, Fisher’s exact test indicated an association between positive tetramer response and 6-month progression-free survival, suggesting a possible correlation between antigen-specific responses and clinical response.

CONCLUSION

These interim data demonstrate the safety and immunological and clinical activity of poly-ICLC-assisted type-1 DC-based vaccines.

Neuro-Oncol. 2009 Oct;11(5):612.

208. HLA Class I Restricted Killing of HCMV-Infected Human Glioma Cells Mediated by pp65 Specific CD81 T Lymphocytes

Gerald P Linette, Michelle Becker-Hapak, Zhikang Qian, Michael R Chicoine, Dong Yu, Beatriz Carreno

INTRODUCTION

Emerging evidence implicates human cytomegalovirus (HCMV) as a possible cofactor involved in glioma pathogenesis. HCMV encodes a series of immune evasion (US2-US11) proteins that downregulate HLA class I antigen presentation. pp65 (UL83, major tegument) and pp72 (UL123, immediate-early 1) are the major target viral antigens recognized by T cells from HCMV-seropositive healthy individuals. The goal of this study was to determine the sensitivity of HCMV-infected glioma cells to antigen-specific killing by virus-specific CD8+ T lymphocytes.

METHODS

Highly enriched oligoclonal HCMV-specific CD8+ T cell lines were established from HLA-A*0201+ seropositive donors. Standard 5-h 51Cr release assays using the T98 glioma line infected with the HCMV AD169 strain as target cells were used as the primary model system to assess HLA-restricted killing. Downregulation of HLA class I was determined by flow cytometry of infected T98 stained with the W6/32 mAb. AD169-GFP was used to measure viral infectivity of human glioma cells at various time points after infection.

RESULTS

AD169 infection of human T98 glioma confirmed 40–50% GFP+ cells at 72 h after infection. HLA class I downregulation was observed after AD169 infection of T98. HCMV pp65-specific T cells recognized ~10 pM viral peptide-pulsed T2 (TAP-deficient, A*0201+) target cells at a 5:1 effector-to-target ratio. HCMV pp65-specific T cells recognized AD169-infected T98 target cells in a dose-dependent manner. In contrast, HCMV pp72 (immediate-early 1)-specific T cells did not recognize AD169-infected T98 cells.

CONCLUSION

HCMV-infected human glioma is recognized by pp65-specific (but not IE-1 specific) CD8+ T lymphocytes despite US2-11-induced HLA class I downregulation. This observation provides support for immunotherapy approaches targeted to HCMV, particularly for adoptive T-cell therapy with autologous pp65-specific T cells in patients with HCMV-associated glioblastoma.

Neuro-Oncol. 2009 Oct;11(5):612.

210. Integration of DC Vaccination in the Primary Treatment of Adults with Glioblastoma: Final Results of the HGG-2006 Phase I/II Trial

Hilko Ardon, Stefaan W Van Gool, Wim Maes, Marc Van Haaren, Frank Van Calenbergh, Johannes JL van Loon, Jan Goffin, Steven De Vleeschouwer

INTRODUCTION

Despite neurosurgery, radiochemotherapy (RCT), and maintenance temozolomide chemotherapy (TZMm), the prognosis of patients with GBM remains poor. Because immunotherapy opened new perspectives for patients at the time of relapse, we aimed to integrate immunotherapy in the primary postoperative multimodal treatment in 60 adults with newly diagnosed WHO grade IV glioblastoma (GB) on reference pathology as a phase I/II protocol (HGG-2006).

METHODS

After total or subtotal resection, leukapheresis was performed prior to RCT, and 4 weekly vaccinations with autologous GB lysate-loaded dendritic cells were given after RCT. Boosts with GB lysates were given during TMZm. Immunomonitoring, feasibility, toxicity, progression-free survival (PFS), and overall survival (OS) were assessed. National Cancer Institute Common Toxicity Criteria were used to grade any adverse events.

RESULTS

With a median FU of 16 months, the preliminary median PFS and OS of 66 per-protocol treated patients were 11 and 18 months, respectively. At the time of the joint meeting, final data on PFS at 6 months and updated PFS and OS data will be available for the full study group. Serious adverse events during FU included an ischemic cerebral event, epilepsia with humerus fracture after a fall due to an epileptic fit, grade IV thrombopenia, a short bowel perforation, and cerebral abscess. ELISPOT for tumor antigen-reacting IFN-γ-producing and/or IL-10-producing T cells was performed on stored blood samples at different time points, and the evolutionary patterns of regulatory T cells, effector T cells, and NK cells during the vaccination were recorded.

CONCLUSION

Tumor vaccination integrated within the standard primary postoperative treatment for patients with newly diagnosed malignant glioma is feasible, well tolerated, and possibly beneficial for patients with minimal residual tumor burden. These early experiences with postoperative radio-chemo-immunotherapy provide hope that integrating immunotherapy as a fourth treatment modality in patients with a primary diagnosis of high-grade glioma might result in better disease control.

Neuro-Oncol. 2009 Oct;11(5):612.

211. Validation of RPA Classification as a Prognostic Tool in Adult Patients with Relapsed High-Grade Glioma Vaccinated at Time of Recurrence

Steven De Vleeschouwer, Hilko Ardon, Frank Van Calenbergh, Johannes JL van Loon, Jan Goffin, Stefaan W Van Gool

INTRODUCTION

Recursive partitioning analysis (RPA) in newly diagnosed high-grade glioma (HGG) patients was originally validated by the RTOG as a prognostic tool. It classifies patients into six prognostic classes according to age, pathology, performance status, mental status, extent of surgical treatment, and intensity of radiotherapy. We aimed to validate this classification as a prognostic tool in more than 120 adult patients with a relapsed HGG who were reoperated on and vaccinated at recurrence.

METHODS

More than 120 adult patients with a relapsed HGG were reoperated on and vaccinated using autologous dendritic cell-based vaccines. After reoperation but before vaccination, each patient was classified as RPA class I–VI. The prognostic value of the RPA classification was assessed after log rank analysis was used to create Kaplan Meier overall survival (OS) estimates.

RESULTS

The median OS after the prevaccine reoperation for RPA class I–VI patients was not yet reached (>36 months in class I patients [n = 4]; 14.2 months in class II patients [n = 10]; 14 months in class III patients [n = 28]; 10.6 months in class IV patients [n = 51]; 6 months in class V patients [n = 15]; and 4 months in class VI patients [n = 9]). At last follow-up, 75.0%, 50.0%, 35.7%, 11.7%, 0%, and 0% of RPA class I–VI patients, respectively, were still alive. These differences in OS were highly significant (p < 0.0001). Subgroup analysis revealed a significantly longer progression-free survival for the patients receiving a Toll-like receptor 7 agonist during vaccination.

CONCLUSION

We validated the original RTOG RPA classification as a good prognostic tool in a large group of patients with relapsed HGG who were reoperated on and vaccinated at the time of recurrence. The use of RPA classification to compare new treatments in relapsed HGG is strongly recommended to estimate the real value of the tested therapy.

Neuro-Oncol. 2009 Oct;11(5):612.

212. Immunotherapy with CpG-ODN in Neoplastic Meningitis: Phase I Trial

Renata Ursu, Sophie Taillibert, Olivier Bailon, Irene Coman, Johan Le Guilloux, Catherine Belin, Khé Hoang-Xuan, Antoine Carpentier

INTRODUCTION

Phosphorothioate oligodeoxynucleotides containing CpG motifs (CpG-ODNs) are immunostimulating agents that have antitumor effects in animal models. Neoplastic meningitis is a devastating disease without efficient therapy. A phase I trial was conducted in patients with neoplastic meningitis to define the safety profile of subcutaneous injections, with or without intrathecal administration of a CpG ODN.

METHODS

Cohorts of 3–6 patients were treated for 5 weeks with escalating doses of CpG-28 (level 1, 0.1 mg/kg/week subcutaneously; level 2, 0.3 mg/kg/week subcutaneously; level 3, 0.3 mg/kg subcutaneously associated with 3 mg intrathecally every other week). The diagnosis of neoplastic meningitis was based on CSF cytology or clinical symptoms with meningeal enhancement on MRI. The primary end point was tolerance. Secondary end points were time until neurological progression and survival.

RESULTS

Fifteen patients were treated between March 2007 and May 2009. Primary cancers included malignant glioma, breast cancer, melanocytoma, ependymoma, and lung cancer. The median age was 56 years, and the median KPS was 70%. Adverse effects possibly or probably related to the study drug were moderate and consisted in grade 2 lymphopenia, anemia, and neutropenia; local erythema at injections sites; and fever, seizure, and back pain. The median time until neurological progression was 6 weeks. The median survival was 9 weeks.

CONCLUSION

CpG-28 was well tolerated at doses of up to 0.3 mg/kg subcutaneously and 3 mg intrathecally. Main side effects were limited to local erythema, lymphopenia, and fever. Escalating doses of CpG-28 for intrathecal administration is ongoing. Updated results will be presented.

Neuro-Oncol. 2009 Oct;11(5):612–613.

213. Myeloid-Derived Suppressor Cells: Another Hurdle for Glioblastoma Immunotherapy?

Wim Maes, Hilko Ardon, Marc Van Haaren, Steven De Vleeschouwer, Stefaan W Van Gool

INTRODUCTION

The interaction between glioblastoma (GB) tumor cells and the immune system is complex. We studied the role of effector T cells, memory T cells, and regulatory T cells (Tregs) in the adaptive immune response induced by vaccination with dendritic cells (DC) loaded with GB tumor antigens. Myeloid cells have potential immunosuppressive and tumor-promoting effects. We therefore analyzed the presence and phenotype of myeloid cells both in an experimental glioma model and in GB patients.

METHODS

In an orthotopic GL261 mouse model, brain-infiltrating immune cells were isolated out of tumor-bearing animals, and circulating cells in blood were analyzed with flow cytometry. In patients with suspected GB, immune cells isolated out of the tumor and circulating cells in the blood were analyzed with flow cytometry.

RESULTS

Within the brain-infiltrating immune cells in GL261-bearing mice, a well defined subpopulation of CD11b+/CD45hi cells that expressed Gr-1 as a marker of myeloid-derived suppressor cells (MDSCs) could be detected. Fewer of these cells were present after prophylactic DC vaccination, and the cells were almost completely absent when Tregs were depleted prior to tumor inoculation with or without prophylactic vaccination. In GB patients’ tumor samples, an enrichment of CD11b+/CD33+ myeloid cells was detected as compared to the peripheral blood circulation. Current studies are focused on the functional role of these cells.

CONCLUSION

Myeloid cells with an MDSC phenotype are clearly detected in the GL261 glioma mouse model. Likewise, their human counterparts are also enriched in GB tumor samples. Their functional role in tumor biology and immunotherapy has yet to be defined.

Neuro-Oncol. 2009 Oct;11(5):613.

214. Influence of a Synthetic Peptide Mimetic of Macrophage-Activating Factor in Response to Radiotherapy in a Murine Glioma Model

Mohammed G Abdelwahab, Sergey V Kushchayev, Yevgeniya Kushchayeva, Tejas Sankar, Ruolan Liu, Fung-Do Shi, Adrienne Scheck, Laura Eggink, Kenneth Hoober, Mark C Preul

INTRODUCTION

Microglial cells are a predominant immune subset in gliomas. Yet little is known about the immune performance and interactions of CNS microglia/macrophages in glioma. Microglial activation is a promising immunotherapeutic approach to glioma treatment as an adjuvant therapy to existing treatment modalities. A novel peptide mimetic of human macrophage activating factor (L4MAF) that directly stimulates blood macrophages and microglial cells to attack the tumor, activate leukocytes, and increase the release of cytokines IL-15, IL-21, eotaxin, and TIM2 was developed. The efficacy of this peptide when combined with radiotherapy in a murine glioma model was investigated.

METHODS

C57BL/6 mice underwent stereotactic intracranial implantation of 105 GL261 glioma cells. Animals were MRI randomized 7 days after implantation and were divided into a cranial radiation group (4 Gy on days 7 and 9) and a group that received radiation +L4MAF administered subcutaneously on alternate days. Animals were followed with serial MRI and euthanized when they became moribund. Blood, spleen, and brain tissue were analyzed for FACS analysis.

RESULTS

FACS analysis revealed a decrease in CD11b-high/CD45-high (macrophages) and an increase in CD11b-high/CD45-high (macrophages) in the tumor hemisphere in L4MAF-treated animals and a decrease in CD11b-high/CD45-low (microglia) in tumor-free hemisphere in L4MAF-treated animals. Mean survival was higher in the peptide plus radiation group (29 days) than in the radiation group (21 days; p < 0.05). MR image analysis showed that the increase in tumor size was also reduced in the peptide plus radiation group (p < 0.05).

CONCLUSION

Microglial activation, achieved by administering a novel peptide mimetic of MAF in this model, improves the response to radiation in glioma. A redistribution of mononuclear cells that correspond to microglia and macrophages was also found. Further investigations are under way to clarify the effect of L4MAF on the tumor microenvironment.

Neuro-Oncol. 2009 Oct;11(5):613.

215. Novel Synthetic Peptide Mimetic Activates Macrophage/Microglia in a Murine Glioblastoma Model

Sergey V Kushchayev, Mohammed G Abdelwahab, Yevgeniya Kushchayeva, Tejas Sankar, Ruolan Liu, Fu-Dong Shi, Adrienne Scheck, Laura Eggink, Kenneth Hoober, Mark C Preul

INTRODUCTION

A novel peptide mimetic of human macrophage-activating factor (L4MAF) that directly activate macrophages, microglia, and leukocytes was synthesized. The use of L4MAF as immunotherapy in murine glioblastoma (GBM) was investigated.

METHODS

Naive C57BL/6 mice were treated with L4MAF to identify systematic immunological changes. Mice also underwent intracranial implantation of 105 GL261 glioma cells. Animals were MRI randomized 7 days after implantation and were divided into a group that received subcutaneous L4MAF treatment on alternate days and a control group with sham injections. Animals were followed with serial MRI. Animals were killed; blood, spleen, and brain tissue were analyzed for FACS analysis.

RESULTS

Preliminary FACS analysis of tissues from naive mice revealed that L4MAF-treated mice had fewer CD11-low/CD45-high cells (CD11-low/CD45-high cells [microglia], CD11-high/CD45-high [macrophages]) in the brain. In spleen, L4MAF decreased the number of CD8+ and CD4+ cells. L4MAF also decreases the number of CD8+/CD4+, CD8+, and CD4+ cells in the blood of both treatment groups relative to control animals. Furthermore, FACS analysis of GBM mice treated with L4MAF revealed a decrease in the number of CD11b-high/CD45-high cells (macrophages) and slight increase in the number of CD11b-high/CD45-low cells (microglia) in tumor tissue in L4MAF-treated animals; an increase in the number of macrophages and microglia in peritumoral brain tissue in L4MAF-treated animals; and a slight increase in the number of macrophages and microglia in peritumoral brain tissue in L4MAF-treated animals. MR image analysis showed that the increase in tumor size was also reduced in the peptide group (p < 0.05). The mean survival was higher in the peptide group (22 days) than in the control group (20 days).

CONCLUSION

L4MAF provides significant macrophage/microglial activation with subsequent changes in the immune system of naive and GBM mice. Further investigations are under way to clarify the effect of L4MAF on the tumor microenvironment.

Neuro-Oncol. 2009 Oct;11(5):613.

216. Recognition and Killing of Brain Tumor Stem Cells by CD81 Cytolytic T Cells

Christine Brown, Renate Starr, Catalina Martinez, Massimo D’Apuzzo, Behnam Badie, Michael Hudecek, Riddell Stanley, Michael Jensen

INTRODUCTION

Our laboratory constructed a prototype IL13 (E13Y)-zetakine chimeric antigen receptor that endows cytotoxic T lymphocytes (CTLs) with potent HLA-independent IL13a2-specific antiglioma effector functioning. Our studies have culminated in the initiation of an FDA/OBA-authorized pilot feasibility/safety clinical trial (BB IND#9149) of intracranial adoptive therapy with autologous IL13-zetakine+ CD8+ CTL clones targeting recurrent or progressive malignant glioma. Compelling evidence now suggests that brain tumors contain a subset of neoplastic cells with stem cell-like properties that have the preferential ability to drive tumor formation and resist conventional therapies such as radiotherapy and chemotherapy. This subpopulation of stem/progenitor cells thus represents a critical cellular target for curative brain tumor therapies.

METHODS

We isolated and expanded CD133+ brain tumor stem cells (BTSCs) from human high-grade gliomas and evaluated their immunologic recognition and elimination by CD8+ CTLs.

RESULTS

Compared to serum-differentiated CD133low tumor cells and established glioma cell lines, BTSCs are equivalent with respect to expression levels of HLA class I and ICAM-1, similar in their ability to trigger degranulation and cytokine synthesis by antigen-specific CD8+ CTLs, and equally susceptible to perforin- dependent CTL-mediated cytolysis. BTSCs are also competent in processing and presenting endogenously expressed antigens. Importantly, CTLs can eliminate all BTSC engraftment potential in an antigen-specific manner. Moreover, we found that chimeric immunoreceptor-redirected IL13Ra2-specific CTL, which is currently being used in FDA-authorized clinical trials, can target and kill BTSCs in vitro and reduce the engrafted potential of the glioma stem/progenitor population in an orthotopic murine tumor model.

CONCLUSION

Current models now predict that curative therapies for many cancers will require the elimination of the stem/initiating tumor population, and these studies lay the foundation for an immunotherapeutic approach to achieve this goal for high-grade gliomas.

Neuro-Oncol. 2009 Oct;11(5):613–614.

217. Tumor Infiltrating IFN-a-Producing Plasmacytoid Dendritic Cells Mediate GBM Regression Induced by Adenoviral Vectors Expressing Flt3L in Combination with HSV1-TK (1GCV)

Marianela Candolfi, Kader Yagiz, Gwendalyn King, David Foulad, Ali Azadmehr, Patricia Lin, AKM Ghulam Muhammad, James Curtin, Chunyan Liu, Kurt Kroeger, Pedro R Lowenstein, Maria Castro

INTRODUCTION

The immunosuppressive tumor microenvironment poses a challenge when inducing antitumor immune responses. We therefore aimed to modify the tumor microenvironment of intracranial glioblastomas (GBM) using adenoviral vectors (Ads) expressing HSV1-TK (Ad-TK) and Flt3L (Ad-Flt3L). This approach combined the cytotoxicity of TK+gan-cyclovir, which leads to the release of inflammatory molecules and tumor antigens, with the immunostimulatory effects of Flt3L, which recruits antigen- presenting cells (APCs) into the tumor microenvironment, leading to long-term survival in 70% of GBM-bearing rats. Therapeutic efficacy depends on CD4+ and CD8+ T cells.

METHODS

We characterized the APCs infiltrating the GBM microenvironment in response to Ad-TK/GCV+Ad-Flt3L delivered into the tumor mass. Also, to mimic the effects of Ad-Flt3L, we injected bone marrow-derived plasmacytoid dendritic cells (pDCs) or an Ad expressing IFN-α (Ad-IFN-a), into the tumor mass. Bone marrow-derived pDCs were loaded with GBM cell debris, activated with CpG 2216, and injected into GBMs treated with Ad-TK alone. Alternatively, tumors were treated with Ad-IFN-a instead of pDCs. To determine whether the therapeutic efficacy of Ad-Flt3L was dependent on IFN-α, we used an Ad vector expressing B16R, which blocks IFN-α.

RESULTS

We found that pDCs, B cells, conventional DCs, and macrophages infiltrated the tumor. Tumor pDCs were capable of in vitro phagocytosis of PKH67-labeled GBM cell debris and 1-μm FluoSpheres. Incubation of tumor pDCs with the TLR-9 agonist ODN Cpg2216 (50 μg/ml) stimulated the release of IFN-α, TNF-α, and IL-6, and upregulated MHCII. Tumor-bearing rats that received single treatments of Ad-TK, pDCs, or Ad-IFN-a succumbed to the tumor. However, 35% of the rats treated with the combination of Ad-TK+pDCs, and 50% of the rats treated with Ad-TK+Ad-IFN-a survived more than 90 days. Administering Ad-B16R with Ad-TK/GCV+Ad-Flt3L completely abrogated therapeutic efficacy.

CONCLUSION

Tumor- infiltrating pDCs are capable of antigen presentation and IFN-α release and mediate a specific anti-GBM immune response and long-term memory.

Neuro-Oncol. 2009 Oct;11(5):614.

218. Generation of IL13.E11K.R107K Designer T Cells Specific for Malignant Glioma

Seogkyoung Kong, Richard Junghans, Prakash Sampath

INTRODUCTION

Malignant glioma (MG) is the most common primary adult brain tumor. The prognosis of patients with MG has remained dismal because of the failure to eradicate local tumor growth as indicated by the development of 90% of MG recurrences at or adjacent to the site of origin. Therefore, adjuvant therapies designed to increase local control are critical. Genetically modified T cells are one promising immunological approach for the treatment of recurrent MG. T cells encoding a chimeric T-cell receptor comprising an extracellular domain specifically targeting MG, the cytoplasmic CD28 costimulation domain, and the CD3 zeta chain can target and selectively lyse MG. With the CD28 costimulation molecule, second generation designer T cells show sustained proliferation, enhanced survival, and antitumor efficacy in vivo. IL13 receptor a2 is overexpressed in 50–75% of MGs but is minimally or not expressed in normal brain cells.

METHODS

To achieve specific targeting and selectively enhance the cytotoxicity of IL13 receptor a2, we mutagenized human IL13. The mutations were made to enhance cytotoxicity against IL13 receptor a2, the MG-associated receptor, but not against IL13 receptor a1, which is expressed in normal brain cells and other tissues. We generated IL13.E11K.R107K (both glutamic acid at position 11 and arginine at 107 changed to lysine).

RESULTS

The IL13.E11K.R107K designer T cells were specifically proliferated on contact with IL13 receptor a2+ glioma cells. The IL13.E11K. R107K designer T cells showed specific targeting and selectively enhanced cytotoxicity of IL13 receptor a2+ glioma cells compared to wild-type IL13 designer T cells in preclinical in vitro testing. We also demonstrated the in vivo antiglioma efficacy of the IL13.E11K.R107K designer T cells employing a glioma xenograft model established with human glioma cells in immunodeficient rodents.

CONCLUSION

IL13.E11K.R107K designer T cells have significant potential for the treatment of recurrent MG.

Neuro-Oncol. 2009 Oct;11(5):614.

219. B Cells’ Presentation of Tumor Antigen is Required for Therapeutic Efficacy of an Immunotherapeutic approach that Combines Immune-Stimulatory and Conditional Cytotoxic Adenoviral Vectors for Glioblastoma

Marianela Candolfi, James Curtin, Kader Yagiz, Hikmat Assi, Kurt Kroeger, Matthew Tesarfreud, Andrew Alden, Chunyan Liu, R Dunn, Pedro Lowenstein, Maria Castro

INTRODUCTION

We developed adenoviral vectors (Ads) expressing HSV1-TK (Ad-TK; a conditional cytotoxic molecule) and Flt3L (Ad-Flt3L; an immune-stimulatory cytokine), which increase the survival of GBM rodent models. CD4+ and CD8+ T cells are necessary for Ad-TK+Ad-Flt3L-mediated tumor regression and immunological memory. We investigated whether B cells play a role in GBM regression following Ad-TK+Ad-Flt3L.

METHODS

We studied the efficacy of Ad-TK+Ad-Flt3L in tumor-bearing Bcell-deficient Igh6−/− mice and in wild-type mice depleted of B cells. B cells were collected and characterized from brain tumor, lymph nodes, and spleen.

RESULTS

While Ad-TK+Ad-Flt3L induced tumor regression and long-term survival in 60% of the wild-type mice, the treatment failed in B-cell-deficient Igh6−/− mice. Similarly, Ad-TK+Ad-Flt3L treatment failed in wild-type tumor-bearing mice depleted of total or marginal zone B cells. Ad-TK+Ad-Flt3L increased the number of MZB cells in the spleen but did not change the B-cell content in the tumor mass or draining lymph nodes. We could not detect circulating antibodies against tumor cells. We also studied whether B cells could present tumor antigen during the onset of the antitumoral immune response induced by Ad-TK+Ad-Flt3L. We found that B cells purified from the draining lymph nodes of Ad-TK+Ad-Flt3L-treated mice, but not from control tumor-bearing mice, increased allogeneic T-cell proliferation. T-cell clonal expansion was also studied by ELISPOT using splenocytes from tumor-bearing wild-type or Igh6−/− mice treated with saline or Ad-TK+Ad-Flt3L and stimulated with Trp2180-188, an MHCI-associated GL26 tumor antigen. Trp2-specific T-cell precursors were detected only in Ad-TK+Ad-Flt3L-treated wild-type mice, but we did not observe any increase in the frequency of tumor antigen-specific T cells in Igh6−/− mice.

CONCLUSION

Our results indicate that B cells play an important role in facilitating brain tumor regression, most likely by acting as antigen-presenting cells rather than antibody-producing cells.

Neuro-Oncol. 2009 Oct;11(5):614.

220. Survvax-M, A Survivin-Specific Vaccine for Glioma

Michael J Ciesielski, Molly Orton, Tara Barone, Stephan Munich, Robert A Fenstermaker

INTRODUCTION

SurvVax-M is a KLH-linked survivin-specific modified peptide vaccine developed in our laboratory. The SurvVax-M peptide (SVN53-67/M57) contains an amino acid alteration that improves MHC I binding and is more effective than wild-type peptides at eliciting CTL responses against survivin-expressing tumor cells.

METHODS

We have shown that immunization with SurvVax-M leads to increased in vivo antitumor efficacy in murine glioma models. Our recent data show that SurvVax-M is also effective against murine prostate, ovarian, and breast carcinomas. We present results utilizing PBMC-derived CTL from patients whose DC are primed to SVN53-67/M57 ex vivo. CTL were tested against autologous and allogeneic HLA-matched glioma, CNS lymphoma, and CLL.

RESULTS

Our results indicate that patients displaying HLA-A*0201, A*0301, A*01, A*24, A*2901, and A*3002 are capable of mounting an immune response to SVN53-67/M57 consisting of CTL. The SVN53-67/M57-stimulated CTL produce a stronger lytic response than wild-type survivin-stimulated CTL. While the SVN53-67/M57 peptide can be processed into multiple MHC class I epitopes, it also represents an MHC class II ligand. In ex vivo assays, we found that SVN53-67/M57 could stimulate strong cytokine support (CD4+) that was characteristically different from that stimulated by wild-type survivin, predominantly in the form of enhanced IFN-γ secretion. CTL were tested for target specificity via HLA-A*0201 pentamer analysis, which showed that cells stimulated with SVN53-67/M57 produced CD8+ T cells that were cross-reactive to both SVN53-67/M57-derived epitopes as well as wild-type survivin epitopes. We have also begun to characterize antisurvivin antibodies found in patient sera in the absence of vaccination, leading to the possibility that the SVN53-67/M57 vaccine might be acting to redirect preexisting humoral immunity.

CONCLUSION

SurvVax-M is a promiscuous peptide immunogen capable of producing multiple tumor-reactive CTL and cytokine support within a broad range of HLA haplotypes. We are currently developing SurvVax-M for clinical trials.

Neuro-Oncol. 2009 Oct;11(5):614.

221. Systemic Effect of Malignant Gliomas: NKG2D Ligand Expression on Circulating Monocytes

Courtney Crane, Seunggu J Han, Brian Ahn, Lewis L Lanier, Andrew T Parsa

INTRODUCTION

We previously demonstrated that in patients with malignant glioma, expression of the activating receptor NKG2D is decreased on the surface of natural killer and CD8+ T cells in a TGFβ-mediated fashion. In such patients, tumor cells express NKG2D ligands, suggesting that decreased NKG2D expression may be relevant to tumor immune escape. While examining the expression of NKG2D ligands in patient tumor samples, we observed that circulating monocytes in these patients, but not in meningioma patients or healthy controls, also expressed NKG2D ligands.

METHODS

We examined circulating and tumor-infiltrating myeloid cells from patients with malignant glioma to determine NKG2D ligand expression. Healthy controls were used to identify a mechanism for this expression.

RESULTS

When IL-2 activated, NK cells produced proinflammatory cytokines and lysed NKG2D ligand-positive monocytes, though these cells were not deleted in vivo. Furthermore, we found that the mechanism of NKG2D ligand induction on circulating monocytes is a glioma-derived soluble factor, macrophage colony-stimulating factor (M-CSF, CSF-1). Glioma patient monocytes had significantly higher expression of the MCSF receptor (CD115), indicating that monocytes in these patients can be directly affected by the presence of M-CSF. We found that M-CSF is sufficient to upregulate NKG2D ligands on monocytes, expressed by primary glioma cell lines at the transcript and protein level, and present in the sera of glioma patients, indicating that M-CSF secretion by tumor cells may not only affect local microglia and macrophages but also circulating monocytes.

CONCLUSION

This work provides evidence that a tumor can have a systemic impact on immune cells that could otherwise contribute to tumor recognition and elimination by the immune system. As immune cells that may function to present antigen and activate T cells, the effects of glioma on monocytes may play an important role in tumor persistence and progression.

Neuro-Oncol. 2009 Oct;11(5):615.

222. Cellular Immunotherapy Trials Targeting Human Cytomegalovirus Antigens in Glioblastoma

Duane A Mitchell, Gary E Archer, Darell Bigner, Henry Friedman, Denise Lally-Goss, Beth Perry, James Herndon, Sharon McGehee-Norman, Roger McLendon, David A Reardon, Janet Ottinger, Kent Weinhold, John H Sampson

INTRODUCTION

Conventional therapies for GBM fail to target tumor cells exclusively. Immunologic targeting of tumor-specific proteins may allow more precise eradication of neoplastic cells. The discovery and recent confirmation that GBM, but not surrounding normal brain tissue, serves as a refuge for cytomegalovirus (CMV) reactivation provides an unparalleled opportunity to subvert, as tumor-specific antigens, the highly immunogenic viral proteins expressed by CMV.

METHODS

A randomized, prospective phase I/II clinical trial was undertaken to assess the immunogenicity and efficacy of targeting the immunodominant CMV integument protein, pp65, in patients with newly diagnosed GBM using pp65 RNA-transfected dendritic cells (DCs). After resection and radiation with concurrent TMZ (75 mg/m2/day), patients received subsequent monthly cycles of TMZ (200 mg/m2) simultaneous with intradermal DC vaccinations. Patients received vaccinations until evidence of tumor progression or death.

RESULTS

Thirteen patients were treated with pp65 RNA-pulsed DC vaccines. There were no vaccine-related adverse events. One nearly complete response was observed. The median progression-free survival (PFS) was 15.4 months (CI95: 10.0, 8), and the median overall survival (OS) was 20.6 months. Increases in CMV-specific delayed-type hypersensitivity (DTH) reactions, humoral responses, and circulating CMV-specific T cells were observed after vaccination; however, polyfunctional immune responses remained impaired. Ex vivo expansion of CMV-specific T cells, however, restored polyfunctional immune responses in these patients. Therefore, a phase I clinical trial of adoptive cellular therapy targeting CMV antigens in patients with newly diagnosed GBM has been initiated at our center (FDA IND 13,240, Duke IRB 0580; PI: Duane A. Mitchell).

CONCLUSION

Immunotherapy targeting CMV pp65 is safe in patients with GBM and produces promising immunologic, radiographic, and clinical responses. Adoptive T-cell therapy, which may improve the induction of polyfunctional immune responses in these patients, is under current evaluation.

Neuro-Oncol. 2009 Oct;11(5):615.

223. STAT-3 Blockade Enhances IFN-α Immunotherapy for Intracerebral Malignancy

Ling-Yuan Kong, Jun Wei, Alexander Gelbard, Tony Wang, Qiao Wei, Izabela Fokt, Willem W Overwijk, Waldemar Priebe, Amy B Heimberger

INTRODUCTION

Activation by phosphorylation of the signal transducer and activator of transcription 3 (STAT-3) has been identified as a key mediator that drives the fundamental components of malignancy, including immunosuppression. We hypothesized that WP1193, an orally administered inhibitor of p-STAT-3 signaling, would enhance the antitumor activity of IFN-α against established intracerebral tumors by inhibiting regulatory T cells (Tregs) and enhancing T-cell cytotoxicity.

METHODS

Combination therapy of WP1193 with IFN-α was investigated in an established syngeneic intracerebral murine tumor model. The immunological in vivo mechanisms of efficacy were investigated using T-cell cytotoxic assays, ELISA, and the presence of Tregs in vivo.

RESULTS

IFN-α immunotherapy was synergistic, with WP1193 demonstrating marked in vivo efficacy against established intracerebral tumors. The median overall survival for tumor-bearing mice without treatment was 17 days. Overall survival in these mice was enhanced by either subtherapeutic WP1193 (30 mg/kg; overall survival, 18.5 days) or IFN-α alone (overall survival, 27.5 days). Moreover, the median overall survival (40 days; p < 0.001) was significantly longer for the combination therapy (p < 0.02). Twenty-five percent of the mice treated with the combination therapy had long-term survival (>84 days; p < 0.001 compared with the control group), and there was at least a 235% increase in the median survival time. Both IFN-α and WP1193 inhibited Tregs (CD4+Foxp3+); however, the combination was not additive or synergistic. The in vivo administration of WP1193 markedly enhanced cytotoxic antitumor T-cell activities and increased IFN-α-induced IL-1 and IFN-γ production.

CONCLUSION

The immune modulatory effects of STAT-3 blockade can enhance the therapeutic efficacy of IFN-α immunotherapy by enhancing cytotoxic T-cell activities. Combination therapy is a potential treatment of malignancy in the CNS, a site typically refractory to this type of immunotherapeutic approach.

Neuro-Oncol. 2009 Oct;11(5):615.

224. Gamma Interferon-Mediated Superinduction of B7-H1 in PTEN-Deficient Glioma Patients: An Immunoresistant Phenotype that Can Confound Response to Cancer Vaccine Therapy

Seunggu J Han, Isaac Yang, Brian Ahn, James S Waldron, Courtney Crane, Shanna Fang, Russell O Pieper, Andrew T Parsa

INTRODUCTION

B7-H1 is a recently discovered protein that confers immunoresistance in PTEN-negative glioma cells. Gamma interferon induces B7-H1 expression in glioma cells through transcriptional and posttranscriptional regulation. We propose that the combination of PTEN deficiency and gamma interferon exposure induces an exceptionally immunoresistant phenotype in glioma cells. This paradoxical induction of immunoresistance is relevant in the context of T-cell-based immunotherapies involving the local secretion of gamma interferon by activated T cells.

METHODS

To test this hypothesis of paradoxical immunoresistance induced by gamma interferon, the expression profile of B7H1 in human astrocytes was determined. The implications of this B7-H1 superinduction were tested using autologous tumor cells from patients with GBM. The GBM patients were characterized for PTEN status, and their T cells were selected for apoptosis assays after 1:1 coculture with autologous glioma cells.

RESULTS

Glioma cells with PTEN dysfunction had a significantly higher level of B7-H1 expression at baseline than tumors with wild-type PTEN. In PTEN-deficient samples, exposure to gamma interferon also resulted in a significantly greater magnitude of increase in transcript and protein expression in B7-H1. Glioma cells lacking PTEN (n = 9) had a significantly greater degree of immunoresistance at baseline than glioma cells with wild-type PTEN (n = 13) as measured by T-cell apoptosis (45 ± 6% vs. 12 ± 4%, p < 0.001). Treating glioma cells with gamma interferon resulted in a substantial increase in T-cell apoptosis in PTEN-negative glioma (72% ± 5%) compared to PTEN-positive glioma (20% ± 5%;p < 0.001). Inhibiting the PI3K pathway abrogated these effects of gamma interferon on the superinduction of B7-H1 and increased T-cell apoptosis.

CONCLUSION

Exposure to gamma interferon results in a superinduction of B7-H1 that correlates with T-cell apoptosis in PTEN-deficient gliomas. This combination of loss of PTEN function and exposure to gamma interferon may identify a subset of patients who are particularly resistant to immunotherapy.

Neuro-Oncol. 2009 Oct;11(5):615.

225. The Degree of CD11b1 Microglia and Macrophages Associated with High-Grade Glioma Correlates with Tumor Treatment Effect

Seunggu J Han, Courtney Crane, James S Waldron, Brian Ahn, Isaac Yang, Andrew T Parsa

INTRODUCTION

Recent evidence suggests that microglia and infiltrating macrophages are the predominant immune cell types locally associated with malignant gliomas. However, their role in glioma growth, progression, and response to treatment remains largely unclear. We present an association between a tumor treatment effect and an increased presence of microglia and infiltrating macrophages.

METHODS

Lymphocytes isolated from operative samples or samples of stereotactic biopsy of contrast-enhancing lesions in patients with glioblastoma were analyzed by flow cytometry for CD11b and CD45. Levels of tumor-associated microglia or macrophages were analyzed in patients who had multiple resections and/or biopsies at points of true recurrence and compared to times when biopsy showed treatment effect. A percentage of the CD11b-positive population, representing microglia and infiltrating macrophages, of the total CD45 population was measured.

RESULTS

Tissue samples acquired at two different times for three patients were analyzed. In all three patients, tissue acquired at one point represented a treatment effect with little to no viable tumor. In all three patients, the percentage of CD11b+ subset of the total CD45+ population at points of true tumor progression was lower than at other points when biopsy of the enhancing lesion showed treatment effect with little viable tumor. The degrees of increase in the CD11b+ subpopulation were 9%, 14%, and 15%.

CONCLUSION

There is a predominant CD11b+/CD45+ population associated with malignant glioma tissue. The presence of tumor necrosis from treatment effect and the relative absence of viable tumor is associated with an increased degree of tumor-associated microglia and macrophages, suggesting a role of these subsets of immune cells in glioma growth, progression, and necrosis.

Neuro-Oncol. 2009 Oct;11(5):616.

226. In Vivo Formation of Antigen-Specific Immunological Synapses between Antitumor T Cells and Target Glioma Cells: A Detailed Quantitative and Qualitative Characterization of the Kinetics, Distribution, and Microanatomy of the Antitumor Immune Response

Jieping Yang, Nicholas Sanderson, Mariana Puntel, Robert J Barrett, Kurt M Kroeger, Maria Castro, Pedro R Lowenstein

INTRODUCTION

We have shown that Ad-Flt3L and Ad-TK treatment of brain gliomas induces an antigen specific T-cell-dependent antitu-mor immune response in several syngeneic rodent glioma models. Although we also demonstrated that antiviral CD8+ T cells establish “classical” immunological synapses in vivo with Ad-infected brain astrocytes, the existence of immunological synapses between T cells and brain glioma cells remains unknown.

METHODS

To study antitumor antigen-specific immune responses, we established a syngeneic mouse glioma model using GL26 glioma cells expressing ovalbumin (OVA) as a surrogate tumor antigen. Antitumor-specific immune responses were characterized by flow cytometry, ELISPOT assays, immunohistochemistry, and specific SIIN-FEKL H2-Kb tetramers to determine the distribution and morphology of antigen-specific immunological synapses. To characterize immunological synapses, we quantified the immunocytochemical distribution of CD3-epsilon and LFA-1, or SIINFEKL H2-Kb tetramers and LFA-1- and OVA-positive cells. Antigen-specific immunological synapses were also characterized in Rag1−/− mice implanted with GL26-cOVA tumors treated with Flt3L/TK and adoptively transferred with splenocytes from OT-I mice.

RESULTS

Using flow cytometry, we determined that the vast majority of tumor antigen-specific T cells were localized to the brain tumors. Restimulation with tumor antigen indicated that 18% of the brain-infiltrating T cells expressed IFN-γ. Intriguingly, we were unable to detect quantitative or qualitative differences between immunological synapses from the brains of saline- or Flt3L/TK-treated mice. Importantly, tetramer-positive immunological synapses differed in structure from typical “classical” immunological synapses and were mainly found in immunized animals.

CONCLUSION

Our data suggest that “classical” immunological synapses may not represent the immunologically active synapses that mediate brain tumor regression. Nevertheless, it is likely that the immunologically active synapses are those identified by tetramer immunolabeling. Continued evaluation of complex cellular and molecular basis of T cell-tumor cell interactions is needed to further improve the design and clinical efficacy of novel glioma immunotherapy approaches.

Neuro-Oncol. 2009 Oct;11(5):616.

227. Adjunctive use of the HDAC Inhibitor, LBH589, Together with Immunotherapy is Associated with an Increased Effector T Cell: Regulatory T-Cell Ratio and Enhanced Antitumor Immunity for Intracranial Tumors

Horacio Soto, Dominique Lisiero, Linda M Liau, Robert M Prins

INTRODUCTION

Epigenetic modifying agents are known to increase expression of proapoptotic molecules in tumors, a process that may also enhance the immune recognition of tumor cells. We examined whether a histone deacetylase inhibitor, LBH589 (Novartis), could synergize with adoptive transfer immunotherapy. We hypothesized that adjunctive use of LBH589 may result in immunosensitization and facilitate a more effective and durable antitumor immune response.

METHODS

The effects of LBH589 were examined in an in vitro cell culture system and used in combination with immunotherapy for both in vivo subcutaneous and intracranial tumor models.

RESULTS

In vitro data suggested that LBH589 had no direct effect on the modulation of tumor cell expression of proapoptotic factors, MHC class I, or tumor-associated antigens. However, in vivo data suggested that LBH589 effectively synergized with adoptive immunotherapy by decreasing tumor burden and increasing survival to subcutaneous and intracranial tumors (p < 0.011). Mice treated with a combination of immunotherapy and LBH589 demonstrated a significant expansion of effector T cells while showing a decrease in the overall numbers of CD4+FoxP3+ T-regulatory cells (p < 0.002). Furthermore, groups treated with LBH589 and immunotherapy not only increased their overall numbers of effector T cells but also increased secretion of interferon-γ and TNF-α in response to ex vivo peptide restimulation. It appears that the synergism of LBH589 is derived from an increase in the number and functionality of tumor-specific CD8+ T cells coupled with a decrease in T-regulatory cells.

CONCLUSION

These results suggest the use of epigenetic modifying agents such as LBH589 can facilitate the expansion of effector cells and decrease the number of regulatory T cells. Furthermore, this combination provides a novel combination treatment that may increase the functional effectiveness of an immunotherapy treatment.

Neuro-Oncol. 2009 Oct;11(5):616.

228. Impact of Gliomas on Regulatory T-Cell Survival and Function

Brian Ahn, Courtney Crane, Seunggu J Han, Andrew T Parsa

INTRODUCTION

In patients with glioma, the presence of the tumor can significantly impact immune cell population and function within the tumor microenvironment. The mechanisms by which this occurs, however, have yet to be expounded. While surveying the T-cell populations of patients with malignant glioma, we observed that immunosuppressive regulatory T cells (Tregs) were overrepresented in the tumor microenvironment when compared to peripheral blood. We hypothesized that the presence of a tumor may have an effect on Treg survival, migration, expansion, and/or induction of a regulatory phenotype from non-Treg T cells.

METHODS

T-cell populations in GBM patients were analyzed for frequencies of Treg. We assayed the impact of soluble factors produced by tumor cell lines on Tregs from non-GBM patients in vitro to determine mechanisms by which gliomas influence T-cell populations.

RESULTS

We found that tumor-derived soluble factors allowed for up to a 1.8-fold preferential proliferation (45.63 ± 2.03 vs. 25.43 ± 1.02; p < 0.001) and a 2.5-fold increased chemotaxis (5.19 ± 0.37 vs. 2.01 ± 0.22; p < 0.001) of Tregs when compared to non-Treg T cells, indicating that these mechanisms may contribute to the observed increase in Treg numbers. Non-Treg CD4 T cells also exhibited a significantly increased expression of proapoptotic transcripts in the presence of tumor-derived factors, while Treg did not, suggesting that survival of this T-cell subset in the tumor site may be preferred. The induction of a functionally suppressive Treg phenotype in CD25-, FoxP3- CD4 non-Treg T cells occurred only transiently following stimulation, and may indicate that in an ongoing immune response, proinflammatory T cells may help suppress T-cell responses.

CONCLUSION

Together, these data suggest that tumor burden may induce increased Treg infiltration, proliferation, and survival, which could potentially negate antitumor immune responses. Consideration of Treg functions in clinical trial design may therefore improve upon efficacy of current immunotherapies.

Neuro-Oncol. 2009 Oct;11(5):616.

229. Third-Generation Chimeric Immune Receptor-Expressing T Cells Extend Survival in an Intracranial Glioblastoma Model

Ayguen Sahin, Carlos E Sanchez, Steven Maxfield, Szofia S Bullain, Peter Waterman, Ralph Weissleder, Bob S Carter

INTRODUCTION

Using chimeric immune receptor (CIR) technology, we previously engineered a “first-generation” CIR targeting EGFRvIII mutant protein (G1). We recently demonstrated the ability to retarget T cells to kill EGFRvIII-expressing human glioblastoma cells in vitro and prolong the survival of mice bearing such tumors upon adoptive transfer in vivo. However, to overcome the problem of reduced activation and survival of the first generation T cells, we created a “third-generation” (G3) tripartite CIR that contained costimulatory factors CD28 and OX40.

METHODS

To provide antigen-specific stimulation and investigate the optimal growth condition in vitro, we generated two new K562-based artificial antigen-presenting cell lines (aAPC) expressing EGFRvIII lacking its intracellular domain (KEAS and KJEAS). KEAS cells only express EGFRvIII on their surface, whereas KJEAS cells were engineered using a K562 cell line already stably expressing additional costimulatory molecules CD32, CD80, 41bbl. We implanted 25,000 U87vIIIffluc cells mixed with 25,000 G1 or G3 or non-CIR (GFP) T cells intracranially into six adult NOD/SCID mice per group. Bioluminescence imaging (BLI) was used at biweekly time points to follow tumor size.

RESULTS

Our in vitro results showed that the highest cell growth was achieved in the G3-T cell/KJEAS group compared to the nonspecific T cells (GFP) and G1-T cells in only 5 weeks of culturing. In vivo results demonstrated a prolonged survival in G3 mice compared to G1 and GFP control groups. Whereas mean survival of G1 and GFP groups was about 30 days, two mice in the G3 group are currently alive (day 58).

CONCLUSION

We conclude that the survival benefit of G3 was clearly superior to that of G1 and GFP control groups and therefore shows a most effective antitumor strategy specific for glioblastoma.

Neuro-Oncol. 2009 Oct;11(5):616–617.

230. Cancer Stem-Like Cells from Glioblastoma Patients Mediate Immunosuppression that is Reversed with STAT-3 Blockade

Jun Wei, Jason Barr, Ling-Yuan Kong, David S Yang, Wei Sun, Amit Sharma, Joy Gumin, Verlene Henry, Howard Colman, Waldemar Priebe, Raymond Sawaya, Frederick F Lang, Amy B Heimberger

INTRODUCTION

Glioblastoma multiforme (GBM) is a lethal cancer that responds poorly to therapy. GBM cancer stem-like cells (gCSCs) have been shown to mediate resistance to both chemotherapy and radiation; however, it is unknown whether gCSCs contribute to profound immunosuppression in GBM patients. Recent studies have found that the activated form of signal transducer and activator of transcription 3 (STAT-3) is a key mediator responsible for immunosuppression in GBM. This study was designed to investigate the immunosuppressive properties of gCSCs and to determine whether inhibition of p-STAT3 could reverse this immunosuppression.

METHODS

gCSCs were isolated from newly diagnosed GBM patients, and single colonies of gCSCs were generated from sorted CD133+ gCSCs and phenotypically characterized. Immunosuppressive cytokines and chemokines elaborated by the gCSCs were determined by enzyme-linked immunosorbent assay. After coculture with gCSCs or conditioned media derived from gCSCs, T-cell proliferation and elaboration of proinflammatory cytokines were determined by flow cytometry. To clarify the mechanisms of inhibition on T-cell effector responses, T-cell apoptosis and the induction of FoxP3+ regulatory T cells were assessed. Furthermore, the gCSCs were differentiated or treated with STAT-3 inhibitors (siRNA and WP1066), and the immunosuppressive phenotype and functional assays were reevaluated.

RESULTS

We found that the gCSCs expressed CTLA-4 and B7-H1-inducers of T-cell apoptosis and inhibitors of T-cell proliferation. Furthermore, the gCSCs, by both direct cell-to-cell contact and secreted products, markedly inhibited T-cell proliferation and activation. The gCSCs also induce T-regulatory cells and trigger T-cell apoptosis. These immunosuppressive properties were diminished when the gCSCs were induced to differentiate or were treated with STAT-3 inhibition.

CONCLUSION

gCSCs contribute to GBMs’ evasion of the immune system, and blockade of the STAT-3 pathway has therapeutic potential.

Neuro-Oncol. 2009 Oct;11(5):617.

231. A Pivotal Role of Type-1 Interferon Signaling in Immunosurveillance Against Glioma: Mouse Studies Leading to Discovery of Novel Prognostic Markers in Humans

Mitsugu Fujita, Michael E Scheurer, Stacy A Decker, Flavia Popescu, Heather A McDonald, Gary Kohanbash, Hisashi Kato, Melissa L Bondy, John R Ohlfest, Hideho Okada

INTRODUCTION

Type I interferons (IFNs) have emerged as central coordinators between host immune systems and cancers. We hypothesized that type I IFNs might play a pivotal role in immunosurveillance protecting against glioma through promotion of type-1 adaptive immunity and suppression of immunoregulatory cells.

METHODS

We employed a novel Sleeping Beauty (SB) transposon-mediated de novo mouse glioma model. Malignant gliomas were induced by SB-mediated transfection of NRas and a short hairpin RNA against P53 into endogenous periventricular cells of Ifnar1−/− (deficient for α-subunit for type I IFN receptor) or wild-type (WT) mice. To evaluate the impact of IFN single nucleotide polymorphisms (SNPs) on prognosis in humans, we performed a survival analysis in a population of 291 patients with low-grade and anaplastic gliomas.

RESULTS

Ifnar1−/− mice exhibited accelerated tumor growth and death. Analyses of tumor-infiltrating lymphocytes in Ifnar1−/− mice revealed an increase of cells positive for CD11b+Gr-1+ and CD4+FoxP3+, markers representing myeloid-derived suppressor cells and regulatory T cells, respectively, but a decrease in cytotoxic T cells (CTL) compared to WT mice. Ifnar1−/− mouse-derived glioma tissues exhibited a decrease in a CTL-attracting chemokine, CXCL10, but an increase in the expression levels of CCL2 and CCL22, both of which are known to attract regulatory immune cell populations. These data suggested a dynamic role of type-1 IFN signaling in the immunological surveillance against glioma. In humans, we also found significant effects on survival for type-1 IFN-related SNPs in FNAR1 and IFNA8. Cox regression analysis showed highly elevated hazard ratios (HRs) for both genes (IFNAR1 HR = 3.6 [95% CI = 1.5–8.3]); IFNA8 HR = 2.5 [95% CI = 1.1–5.9]).

CONCLUSION

The novel SB-induced mouse glioma model allowed us to discover a pivotal role for the type-1 IFN pathway in the immunological surveillance against glioma and led us to discover relevant human SNPs that may represent novel prognostic markers.

Neuro-Oncol. 2009 Oct;11(5):617.

232. Systemic Inhibition of Transforming Growth Factor-2 in Glioma-Bearing Mice Improves the Therapeutic Efficacy of Glioma-Associated Antigen Peptide Vaccines

Ryo Ueda, Mitsugu Fujita, Xinmei Zhu, Kotaro Sasaki, Edward R Kastenhuber, Gary Kohanbash, Heather A McDonald, Jay Harper, Scott Lonning, Hideho Okada

INTRODUCTION

A variety of cancers, including malignant gliomas, overexpress transforming growth factor β (TGF-β), which helps tumors evade effective immune surveillance through a variety of mechanisms, including inhibiting CD8+ cytotoxic T lymphocytes (CTL) and enhancing the generation of regulatory T cells (Tregs). We hypothesized that inhibition of TGF-β would improve the efficacy of vaccines targeting glioma-associated antigen (GAA)-derived CTL epitopes by reversal of immunosuppression.

METHODS

In mice bearing intracranial (i.c.) GL261 gliomas, intraperitoneal (i.p.) administration of the TGF-β neutralizing monoclonal antibody (mAb), 1D11, was combined with subcutaneous vaccinations of synthetic peptides for GAA-derived CTL epitopes GARC-1 (77–85) and EphA2 (671–679) emulsified in incomplete Freund’s adjuvant (IFA).

RESULTS

Mice receiving the combination regimen exhibited significantly prolonged survival compared with mice receiving either 1D11 alone, GAA-vaccines alone, or mock-treatments alone. TGF-β neutralization enhanced the systemic induction of antigen-specific CTLs in GL261-bearing mice. Flow cytometric analyses of brain-infiltrating lymphocytes (BILs) revealed that i.p. 1D11 treatment suppressed phosphorylation of Smad2, increased GAA-reactive/interferon (IFN)-γ-producing CD8+ T cells, and reduced CD4+/FoxP3+ Treg cells in the glioma microenvironment. Systemic 1D11 administration also upregulated plasma levels of interleukin (IL)-12, macrophage inflammatory protein (MIP)-1a and IFN-inducible protein (IP)-10, suggesting a systemic promotion of type-1 cytokine/chemokine production. Furthermore, 1D11 treatment upregulated plasma IL-15 levels and promoted the persistence of GAA-reactive CD8+ T cells in the GL261-bearing mice.

CONCLUSION

These data suggest that systemic inhibition of TGF-β by 1D11 can reverse the suppressive immunological environment of i.c. tumor-bearing mice both systemically and locally, thereby promoting the therapeutic efficacy of GAA-vaccines.

Neuro-Oncol. 2009 Oct;11(5):617.

233. Cmv-Specific HER2-Redirected T Cells for the Adoptive Immunotherapy of Glioblastoma

Nabil Ahmed, Vita Salsman, Yvonne Kew, Donald Shaffer, Suzanne Powell, Robert G Grossman, Catherine M Bollard, Helen E Heslop, Stephen Gottschalk

INTRODUCTION

We demonstrated that T cells from GBM patients expressing a HER2-specific chimeric-antigen-receptor (CAR) kill autologous HER2-positive GBM cells ex vivo. The adoptive transfer of HER2-specific T cells resulted in the regression of established GBM xenografts, yet tumors recurred in a number of treated animals. This limitation in T-cell efficacy is likely due to limited T-cell persistence and/or antigen-loss variants in treated tumors. Since the majority of GBMs are CMVpp65-positive, we expressed HER2.CARs in CMV-specific cytotoxic-T-lymphocytes (CTLs) to a) enhance their persistence in vivo following native α-β-TCR engagement with CMV-antigens presented by APCs and b) improve their direct antitumor activity by simultaneously targeting CMV and HER2.

METHODS

We determined the precursor frequency of CMV-specific T cells in 12 GBM patients using IFN-γ-ELISPOT. To generate CMV-CTL ex vivo, peripheral blood mononuclear cells were transduced with an adenovirus vector encoding the immunodominant CMV-pp65 antigen (Ad5f35pp65) then stimulated with autologous EBV-transformed Ad5f35pp65-transduced irradiated lymphoblastic cell lines. CTLs were then transduced with a retroviral vector encoding a second-generation HER2.CAR (FRP5.CD28. zeta). To identify HER2.CAR.CMV.CTL, we used pentamer analysis for CMVpp65 in flow cytometry. We performed 51Cr-release assays to characterize the functionality of HER2.CAR CMV.CTLs ex vivo.

RESULTS

CMV-specific T cells were detectable in the blood of GBM patients by IFN-γ-ELIspot after CMVpp65-PepMixes stimulation. We were able to generate CTLs that are CMV-specific via α-β-TCR-recognition and specific for HER2 through CAR recognition. Between 6% and 40% of CTLs generated were CMV-specific as detected by Pentamer analysis. Approximately 50% of CMVpp65-specific CTLs expressed HER2.CARs on the cell surface. CMV-specific CTLs expressing the HER2-specific CAR killed the HER2-positive GBM-cells as well as OKT3-blasted T cells pulsed with CMVpp65 PepMixes in 51Cr-release assays. Neither HER2 cell lines nor autologous OKT3-blasted T cells were killed.

CONCLUSION

Our data demonstrate the feasibility of generating CAR-expressing CMVpp65-specific CTLs that are functional against HER2 and CMV.

Neuro-Oncol. 2009 Oct;11(5):617–618.

234. Induction of Antiglioma Memory-NK Cell Response Following Multiple Low-Dose Intracerebral CpG Therapies

Darya Alizadeh, Leying Zhang, Behnam Badie

INTRODUCTION

Although immunotherapy is a promising treatment option for malignant brain tumors, the immunosuppressive glioma microenvironment has hindered the efficacy of this approach. Stimulation of toll-like receptor-9 (TLR-9) by CpG oligodeoxynucleotides (CpG) has been shown to counteract this immunosuppression and inhibit tumor growth in glioma models. These studies, however, have used high doses of CpG (10-ug range) that can be associated with CNS toxicity. We hypothesized that repeated low-dose CpG could also lead to immune evasion and tumor eradication.

METHODS

Mice bearing 4-day-old intracranial GL261 gliomas received a single intratumoral injection or multiple (two or four) intratumoral injections of CpG (3 μg) every 4 days. Tumor growth was measured by bioluminescent imaging, histology, and animal survival. Flow cytometry and cytotoxicity assays were used to assess antiglioma immune response.

RESULTS

Two or four injections of CpG, but not a single injection of CpG, resulted in cure rates of 50% and 75%, respectively. Flow cytometry demonstrated an increase in brain infiltration of CD8+ NK cells and CD11b+ cells in all groups. Interestingly, blood Gr1+ myeloid-suppressive cells, which increased with a single CpG injection, did not change in multiple-injected hosts. Furthermore, “cured” animals that received multiple CpG injections did not develop secondary tumors after intracranial rechallenge with GL261 cells 3 months after the initial treatment. Finally, splenocytes from these mice demonstrated in vitro cytotoxicity against GL261 cells and contained higher frequencies of NK+CD107a, but not CD8+CD107a, suggesting induction of NK-mediated immunity against gliomas.

CONCLUSION

These findings suggest that multiple low-dose intratumoral injections of CpG can generate long-term antitumor immunity through the induction of memory-like NK cells. Future studies will investigate the role of Gr1+ myeloid-suppressive cells on CpG-mediated NK cell activation in glioma models.

Neuro-Oncol. 2009 Oct;11(5):618.

235. Depletion of Gr11 Myeloid Suppressive Cells Enhances Glioma CpG Therapy

Darya Alizadeh, Leying Zhang, Behnam Badie

INTRODUCTION

Activation of the innate immune system could be a promising treatment option for patients with malignant brain tumors. Through activation of toll-like receptor-9, CpG oligodeoxynucleotides have been shown to stimulate this system and inhibit tumor growth in glioma models. These studies, however, have used high doses of CpG (10-ug range), which can be associated with CNS toxicity. Because CpG has been shown to enhance Gr1+ myeloid-suppressive cell trafficking, we hypothesized that depletion of this cell population may potentiate CpG antiglioma response.

METHODS

Mice bearing 4-day-old intracranial GL261 gliomas received a single intratumoral injection of CpG (3 μg) with or without Gr1+ depletion using monoclonal Ab. Tumor growth was measured by bioluminescent imaging, histology, and animal survival. Flow cytometry and cytotoxicity assays were used to assess antiglioma immune response.

RESULTS

A 6-fold increase in tumor-associated Gr1+ cells was seen after a single intratumoral CpG injection. Most of the Gr1+ cells were Ly6G+CD11b+ cells consistent with granulocyte phenotype. Interestingly, concurrent Gr1+ depletion with CpG therapy significantly decreased tumor growth (p < 0.05) and improved animal survival over vehicle, CpG, or Gr1+-depleted groups (p = 0.02).

CONCLUSION

These findings suggest that depletion of myeloid-suppressive cells can enhance antiglioma CpG treatment response. The role of Gr1+ myeloid-suppressive cells on tumor immune evasion will be investigated in future studies.

Neuro-Oncol. 2009 Oct;11(5):618.

236. Immunotherapy of Malignant Brain Tumors Requires Antibody Response

Katherine A Murphy, Charlie Seiler, Mike Olin, Patrick Grogan, Colleen Forster, Karen A Santa Cruz, Wei Chen, John R Ohlfest

INTRODUCTION

Immunotherapy using vaccination shows promise as an adjuvant therapy for glioma patients. The importance of antibody response in immunotherapy has been controversial because antibodies do not penetrate the intact blood brain barrier (BBB) in “normal brain” structures in which infiltrating glioma cells remain after surgery. We hypothesized that antibody-secreting plasma cells infiltrate the brain parenchyma following vaccination and release antibody at the tumor site, thereby circumventing the BBB.

METHODS

Wild-type or B/plasma cell-deficient mice bearing GL261 gliomas were vaccinated with GL261 tumor lysate mixed with an adjuvant, CpG oligodeoxynucleotide (ODN). Antibody response was detected in the serum and in brain homogenates by Western blot. Immunohistochemistry was used to detect plasma cells in the brains of tumor-bearing mice. Glioma patient serum and autologous tumor cells were used to assay for spontaneously occurring antibody-dependent cell-mediated cytotoxicity (ADCC).

RESULTS

Lysate/CpG ODN vaccinations significantly increased the survival of glioma-bearing mice (p < 0.05), whereas the same therapy failed in B/plasma cell-deficient mice. Vaccination induced tumor-reactive antibody responses against multiple tumor antigens. Moreover, IgG was detected in the tumor-bearing hemisphere and contralateral hemisphere to a lesser extent, but not in normal brain controls. Cells consistent with plasma cell morphology and immunophenotype were detected with increased frequency in the tumor but not normal brain of vaccinated mice. In human correlative studies, spontaneous glioma-reactive IgG was identified in two of two patients assayed, and ADCC that was dependent on natural killer cells was documented.

CONCLUSION

Plasma cells that secrete tumor-reactive antibody are induced by vaccination and appear to infiltrate the brain to release antibody at the tumor site in mice. Our data implicate antibody response as crucial for the efficacy of vaccination through this mechanism. The detection of glioma-reactive IgG and ADCC in patients suggests this mechanism could be clinically relevant and exploited for therapeutic intent.

Neuro-Oncol. 2009 Oct;11(5):618.

237. CTLA-4 Blockade Augments Vaccine-Induced Antitumor Immunity Against Intracranial Glioma

William T Curry, Zachary Rollins Barnard, Pankaj K Agarwalla, Peter Fecci, Glenn Dranoff

INTRODUCTION

Previous studies have demonstrated antitumor immunity following vaccination with irradiated tumor cells engineered to secrete GM-CSF. Antibody blockade of the costimulatory molecule CTLA-4 (CD152) may enhance T-lymphocyte responses. We hypothesized that following cell-based immunotherapy with CTLA-4 blockade engenders more effective antitumor immunity and results in better tumor control and survival in a syngeneic glioma model.

METHODS

Animal experiments: 75,000 GL261 glioma cells were implanted into the right frontal lobes of C57/Bl6 mice. On days 3, 6, and 9, mice were treated with subcutaneous injections of GM-CSF-expressing irradiated GL261 cells and then with intraperitoneal delivery of anti-CTLA-4 antibody on days 12, 15, and 18. Control groups were treated appropriately. All implanted tumor cells expressed luciferase via lentiviral transfection. Tumor growth was followed by bioluminescence, and survival was documented via Kaplan-Meier analysis. ELISPOT: Splenocytes were harvested from treated mice (n = 3) on day 22 and restimulated in vitro with GL261 cells. Cytokine production was measured after 24 h on plates coated with IFN-γ antibody. Flow Cytometry: Brains, spleens, ipsilateral cervical lymph nodes, blood, and bone marrow were harvested on day 22 for tissue processing and stained with CD3, CD4, CD8, and Foxp3 antibodies prior to flow cytometry.

RESULTS

Survival analysis demonstrated a significant increase in survival for animals treated with combination immunotherapy versus either anti-CTLA-4 (p = 0.004) or cell-based vaccination alone (p = 0.03), which corresponded with rates of tumor growth as measured by bioluminescence. By ELISPOT, splenocytes harvested from animals in the combination therapy group produced significantly higher levels of IFN-γ in response to tumor-specific stimulation as compared to splenocytes from controls. Flow cytometry analysis showed a significant increase in the effector T lymphocyte to regulatory T lymphocyte ratio (p = 0.02) in the brains of combination-treated animals.

CONCLUSION

Combination immunotherapy utilizing irradiated GM-CSF-expressing glioma cells and CTLA-4 blockade significantly improves the survival of glioma-bearing mice.

Neuro-Oncol. 2009 Oct;11(5):618.

238. Antitumor Responses in Dogs with Spontaneous Astrocytoma and Papillary Meningioma Treated by Immunotherapy

G Elizabeth Pluhar, Wei Chen, Patrick Grogan, Charlie Seiler, Mike Olin, Cathy Carlson, Karen A Santa Cruz, Maria Castro, Pedro Lowenstein, Stephen J Haines, Matthew Allan Hunt, John R Ohlfest

INTRODUCTION

WHO grade II–III astrocytoma and meningioma are often incurable with standard therapies. One major hurdle in developing adjuvant therapy for these tumor types is the paucity of biologically relevant animal models. Pet dogs spontaneously develop these tumors, which are amenable to surgical resection plus adjuvant therapy similar to human treatments. Although canine and human tumors exhibit many histological similarities, the disease progression is much more rapid in dogs. Therefore, these dogs represent an attractive model to test experimental therapies. We initiated a canine brain tumor clinical trials program and have treated several dogs including one with gemistocytic astrocytoma and two with papillary meningioma.

METHODS

The dog with astrocytoma was treated with surgical debulking, intracavitary interferon gene transfer, and vaccination with glioma cell lysate mixed with an adjuvant, CpG oligode-oxynucleotide (ODN). Dogs with papillary meningioma were treated with surgical debulking and tumor lysate/CpG ODN vaccination.

RESULTS

In the dog with astrocytoma, vaccination induced a tumor-reactive IgG response that persisted for more than 250 days, consistent with no evidence of tumor as documented by MRI. Repeated vaccinations induced temporary blindness and left-sided hemiparesis suggestive of inflammation in the tumor-infiltrated right hemisphere. Residual tumor volume decreased by more than 50% in both dogs with papillary meningioma. Tumor regression correlated with the complete resolution of neurological symptoms and improved overall health. An apparent delayed type hypersensitivity reaction that increased in severity following each vaccination at the injection sites occurred in one dog. Lymphopenia and mild elevation in liver enzymes occurred after lysate/CpG vaccinations in all cases. In several dogs, vaccination induced an IgG response against multiple tumor antigens, yet only a fraction of these were sustained as a memory response.

CONCLUSION

Tumor lysate/CpG vaccination induces clinically relevant antitumor immunity in dogs with spontaneous tumors that are often incurable and warrants testing in patients with similar histology.

Neuro-Oncol. 2009 Oct;11(5):619.

239. Novel Bicistronic High-Capacity Helper-Dependent Adenoviral Vector Coexpressing Flt3L and TK (1GCV) Induces Intracranial GBM Regression and Anti-GBM Immunological Memory

AKM Ghulam Muhammad, Kurt Kroeger, Mariana Puntel, Marianela Candolfi, Kader Yagiz, Chunyan Liu, Alireza Salem, Maricel Gozo, Greg Baker, David Foulad, Wei Xiong, Dona Palmer, Philip Ng, Pedro Lowenstein, Maria Castro

INTRODUCTION

The combined immunotherapy/conditional cytotoxic approach involving intratumoral delivery of first-generation adenoviral vectors (Ad) encoding the conditional cytotoxic gene, herpes simplex virus type 1 thymidine kinase (TK) and the immune stimulatory, Fms-like tyrosine kinase 3 ligand (Flt3L) generates an anti-GBM immune response and memory resulting in significantly improved survival in several intracranial syngeneic rodent GBM models. We engineered a novel, bicistronic high-capacity (HC-Ad) vector capable of inducible expression of Flt3L using the TetOn regulatory system and constitutive expression of TK.

METHODS

Lewis rats bearing large syngeneic intracranial GBMs were treated with the HC-Ad-TetON-Flt3L/TK. Efficacy, neuropathology, biodistribution, and systemic side effects were assessed at short-term (5 days), medium-term (1 month), and long-term (6 month) time points.

RESULTS

more than 80% of the treated rats survived long-term. Real-time quantitative PCR (qPCR) revealed that HC-Ad genomes were restricted to the brain injection site. Using ELISA, the levels of Flt3L in the serum could be detected at 5 days only. At the 6-month time point, Nissl staining showed an absence of residual tumor. Immunocytochemistry with antibodies against myelin basic protein or tyrosine hydroxylase showed absence of demyelination or striatal damage, respectively; CD68+ macrophages/activated microglia, CD8+ T cells, and MHC II immunopositive cells were observed at the site of tumor regression. Long-term survivors survived a second tumor implanted in the contralateral hemisphere, indicating that this therapeutic modality induces long-term anti-GBM immunological memory.

CONCLUSION

Our results indicate that tumor regression in response to HC-Ad-mediated gene therapy does not lead to overt adverse neuropathological side effects to the normal surrounding brain parenchyma and no “off-target” leakage of the therapeutic HC-Ad vector. Thus, the high therapeutic efficacy and safety profile of the bicistronic HC-Ad vector makes it an excellent candidate to be tested as an adjuvant therapy for human GBM in phase I clinical trials.

Neuro-Oncol. 2009 Oct;11(5):619.

240. Systemic Ad-Flt3L Delivery Coupled with Intratumoral Ad-HSV1-TK Eradicates Intracranial GBM and Generates Long-Term Immunological Memory in a Syngeneic Rat Model

AKM Ghulam Muhammad, Marianela Candolfi, Kader Yagiz, Mariana Puntel, Nicholas Sanderson, Chunyan Liu, Kurt Kroeger, WS Nichols, Pedro Lowenstein, Maria Castro

INTRODUCTION

Aiming to make the combined gene therapy for GBM less invasive, we delivered the adenoviral vector (Ad) expressing fms-like tyrosine kinase 3 ligand (Flt3L) systemically into GBM-bearing Lewis rats.

METHODS

Six days after GBM implantation into the striatum, intravenous predosing with empty Ad (Ad-0; 4 × 1010 vp) was performed to saturate the liver reticulo-endothelium system/Kuffer cells. Within 4–6 h, Ad-Flt3L (5 × 109 pfu) was injected via the tail vein followed by intratumoral delivery of Ad-TK (1 × 108 pfu). The controls received either Ad-Flt3L or Ad-0 in the tail vein followed by intratumoral saline or Ad-TK. After 24 h, ganciclovir was injected daily for 10 days.

RESULTS

GBM-bearing control rats succumbed by day 22, whereas ~80% of the rats treated with Ad-Flt3L (systemic)+Ad-TK (intratumoral) survived >60 days (p < 0.01; log-rank test). The long-term survivors were rechallenged with 4,500 GBM cells in the brain, and 70% of them survived long-term (>120 days). Serum biochemistry and H&E-stained liver specimens did not reveal hepatotoxicity. Nissl staining on long-term survivors treated with Ad-Flt3L+Ad-TK displayed no residual tumor. Immunohistochemistry against myelin basic protein or tyrosine hydroxylase showed an absence of demyelination or overt striatal damage, respectively; CD68+ macrophages, few CD8+ T cells, and MHC II-immunopositive cells were detected. Positive delayed-type hypersensitivity reactions in long-term survivors indicated the presence of systemic anti-GBM cellular immune responses.

CONCLUSION

These findings highlight the potential of systemic delivery of Ad-Flt3L in the current clinical setting in which GBM antigens would become available from dead or dying GBM tumor cells because of surgery, radiotherapy, and/or chemotherapy in human GBM patients. Hence, the novel treatment strategy should render our combined gene therapy strategy even safer and less invasive. In conclusion, gene therapy aimed at mounting immune stimulation using systemic Ad-Flt3L coupled with conditional cytotoxic treatment (Ad-TK/GCV) elicits tumor regression and immunological memory inhibiting tumor recurrence, highlighting its prospect as a novel adjuvant treatment strategy for human GBM.

Neuro-Oncol. 2009 Oct;11(5):619.

241. PKH-26 In Vitro Cytotoxicity Assay Demonstrates Increasing Tumor Cell Kill using Increasing Numbers of Splenocytes from a Mouse Treated with a Vaccine Incorporating a Selectively Replicating HSV-1 that Expresses IL-12

David F Bauer, Jacqueline N Parker, James M Markert

INTRODUCTION

High-grade intracranial neoplasms carry poor prognosis with current therapy. Novel strategies, including oncolytic viral therapy using conditionally replicating tumor-selective herpes simplex virus type 1 vectors (HSV-1), have been investigated and have had encouraging results in phase I clinical trials in both the U.S. and the U.K. M002 is a γ-1-34.5-deleted HSV-1 vector that expresses IL-12 and selectively replicates in tumor cells. We previously reported that M002 significantly improves the survival of tumor-bearing mice in multiple experimental murine brain tumor models. The current study aimed to ascertain whether tumor cells previously infected with M002 could be used as a vaccine against developing tumors in the brain.

METHODS

For vaccination, Neuro-2a cells were infected using a high multiplicity of infection (m.o.i. = 5) of M002, treated with 10 Gy gamma radiation, then injected into the gastrocnemius muscle of A/J mice. A boost vaccination was administered 7 days later. Kaplan-Meier analysis was performed on survival experiments. For the in vitro cytotoxicity assay, splenocytes were extracted from the vaccinated mouse 10 days after the boost vaccination. Cells from the murine neuroblastoma cell line, Neuro-2a, were grown in vitro. The Neuro-2a cells were labeled with PKH-26, a fluorescent cell membrane-labeling dye. In small capped plastic tubes, 10,000 PKH-26-labeled Neuro-2a cells (target cells) underwent a 4-h incubation at 37ºC with increasing numbers of spleno-cytes (effector cells). All samples were done in triplicate. After incubation, samples were placed on ice and taken for flow cytometry analysis. Just prior to flow cytometry, To-Pro-3 Iodide was added to label dead cells.

RESULTS

This vaccine increased the survival of mice given intracranial Neuro-2a tumors. In the in vitro cytotoxicity assay, increasing numbers of splenocytes caused increasing numbers of Neuro-2a cells to die. Appropriate negative and positive controls for this assay were confirmed.

CONCLUSION

A tumor vaccine incorporating a selectively replicating HSV-1 that expresses IL-12 has been shown to be effective at killing tumor cells in vivo and in vitro. Future studies include flow cytometry analysis of immune infiltrate to confirm the mechanism of this vaccine.

Neuro-Oncol. 2009 Oct;11(5):619.

242. Immunotherapeutic Targeting of Brain Tumor Stem Cells

Joseph L Lasky, Rachana Patel, Linda M Liau

INTRODUCTION

Immunotherapy provides a theoretically appealing approach to developing a targeted, nontoxic treatment for malignant brain tumors. Efforts to develop these approaches have been hampered in part because of the lack of knowledge of the proper antigenic targets in these malignancies. This project focused on identifying and generating immune responses against epitopes derived from brain tumor stem cell (BTSC)-related genes to test the hypothesis that targeting putative BTSC-like cells using immunotherapy results in objective responses in vitro and in animal models.

METHODS

Using computer-based prediction algorithms and previously published analyses, we picked two candidate epitopes from three putative BTSC-related genes: Bmi1, SOX2, and MELK. Peripheral blood mononuclear cells (PBMCs) of type HLA-A*0201 were obtained from normal donors and used to generate mature dendritic cells (DCs) and autologous T lymphocytes. The DCs were pulsed with one of the peptide-epitopes and cocultured with lymphocytes to generate cytotoxic T cells capable of responding to these antigens. The function of these T cells were evaluated using assays for interferon (IFN)-gamma production in response to pulsed T2 cells as well as granzyme B production in response to serum- or neurosphere (BTSC)-cultured brain tumor cell lines.

RESULTS

T cells were successfully expanded using the coculture methodology. The two epitopes from MELK were able to generate means of 7.5 pg/ml and 10.5 pg/ml, respectively, of IFN-γ after four restimulations. The epitopes from Sox2 generated means of 26.9 pg/ml and 51.3 pg/ml IFN-γ. These were all significantly different from the control nonspecific T cells (mean, 3.9 pg/ml IFN-γ). Bmi-1 (2), SOX2 (2), and MELK (1) epitopes were used to generate T cells for the granzyme B ELISPOT assay. All five epitopes generated a granzyme B response above control.

CONCLUSION

Human immune responses can be generated in vitro to BTSC-related antigens. Further studies are needed to determine whether these responses can be generated in vivo.

Neuro-Oncol. 2009 Oct;11(5):620.

243. Immune Gene and Cell Enrichment is Associated with a Good Prognosis in Ependymoma

Andrew M Donson, Diane Birks, Valerie Nicole Barton, Betty K Kleinschmidt-DeMasters, Michael H Handler, Allen E Waziri, Nicholas K Foreman

INTRODUCTION

Approximately 50% of children with ependymoma will suffer from tumor recurrences that will ultimately lead to death. Development of more effective therapies and patient stratification in ependymoma mandates better prognostication. To address this need, tumor gene expression microarray profiles were used to identify prognostic markers in 19 pediatric ependymoma patients who received uniform therapy with known clinical outcomes.

METHODS

Gene expression microarray data were combined with computer-based gene ontology analyses Gene Set Enrichment Analysis (GSEA) and Database for Annotation, Visualization and Integrated Discovery (DAVID) to identify sets of functionally related genes that were associated with a good clinical outcome. Results of ontological analyses were further evaluated using immunohistochemistry and quantitative PCR.

RESULTS

Ontology analyses revealed that genes associated with nonrecurrent EPN were predominantly immune-related. In addition, increased expression of immune-related genes was correlated with longer time to progression in recurrent ependymoma. Ninety-five percent (19/20) of genes that correlated both with nonrecurrence and a longer time to progression were associated with immune function. Of these, a number of genes that are critically involved in both innate and adaptive immune responses were identified. Histological analysis of immune function proteins whose expression correlated with a good outcome revealed that they were restricted to a subpopulation of tumor-infiltrating cells. Analysis of tumor-infiltrating immune cells showed increased infiltration of CD4+ T cells in the nonrecurrent ependymoma group. No genomic sequences for SV40, BK, JC, or Merkel polyomaviruses were found in nonrecurrent ependymoma.

CONCLUSION

The findings of this study reveal that upregulation of immune-related genes is a significant factor that distinguishes good prognosis ependymoma and provides preliminary evidence of a beneficial host proinflammatory and/or antigen-specific response.

Neuro-Oncol. 2009 Oct;11(5):620.

244. First-Line Therapy with Bevacizumab and Irinotecan versus Bevacizumab and Temozolomide and Delayed Concomitant Radiotherapy in Newly Diagnosed Primary Glioblastoma Multiforme: Early Results from a Randomized Phase II Study

Kenneth F Hofland, Hans Skovgaard Poulsen, Morten P Sørensen, Anders Ask, Anne K Bertelsen, Silke Spahn-Horn, Steinbjørn Hansen, Henrik P Schulz, Ulrik Lassen

INTRODUCTION

Concomitant temozolomide and radiotherapy is the standard of care in patients with newly diagnosed glioblastoma multiforme (GBM) and good performance status. Bevacizumab and irinote-can have proven efficacious in patients with recurrent disease, achieving complete response in a significant number of patients. We tested the use of bevacizumab in combination with temozolomide or irinotecan with concomitant radiotherapy as first-line therapy in patients with newly diagnosed GBM.

METHODS

Patients with newly diagnosed GBM (previously untreated except for primary surgery/biopsy) were randomized to bevacizumab (B) and irinotecan (I) on days 1 and 14 (BI regimen) or to bevacizumab on days 1 and 14 and temozolomide (T) on days 1–5 in a 28-day cycle (BT regimen) for 8 weeks, followed by radiotherapy (60 Gy/30 fractions) and concomitant BI or BT. Postradiotherapy, chemotherapy was continued for 8 weeks. Toxicity was assessed every 2 weeks. Response was assessed every 8 weeks. Study end points were safety, response, and 6- and 12-month progression-free survival.

RESULTS

As of May 5, 2009, seven patients have been included—four patients in the BT arm and three patients in the BI arm. Two patients in the BT arm and one patient in the BI arm have completed concomitant chemoradiation. The other three patients have started neoadjuvant chemotherapy and presently await the concomitant chemoradiation. To date, toxicity has been mild (CTC I–II) during the neo-adjuvant chemotherapy as well as during the concomitant chemoradiation. There has been no indication of any unexpected interaction with chemotherapy and radiotherapy. However, one patient in the BT arm experienced gastric bleeding 6 weeks after completing radiotherapy. This patient continues treatment without bevacizumab.

CONCLUSION

This neoadjuvant and concomitant approach as first-line therapy for patients with newly diagnosed GBM seems safe and feasible. Accrual is ongoing, and updated results with safety data will be presented at the meeting.

Neuro-Oncol. 2009 Oct;11(5):620.

245. Methotrexate-Induced Isolated CNS Hodgkin-Like Lymphoproliferative Disorder: A Case Report

Jennifer M Connelly, Alexandra Harrington, Steven Kroft, Khang-Cheng Ho, Timothy Fenske, Mark Malkin

INTRODUCTION

Methotrexate (MTX) is one of the most common disease-modifying agents used in the treatment of rheumatoid arthritis (RA). Recent studies have shown that patients treated with MTX can develop immunosuppression-associated lymphoproliferative disorders. We describe the first case of MTX-induced isolated CNS Hodgkin-like lymphoproliferative disorder (HLD).

METHODS

Case report.

RESULTS

A 53-year-old female with a 3-year history of RA treated with MTX presented with blurred vision, ataxia, and fatigue. Magnetic resonance imaging (MRI) demonstrated enhancing lesions of the left thalamus and right occipital lobe. CSF analysis and bone marrow biopsy were negative. The patient was started on prednisone and improved clinically and radiographically. Prednisone was slowly tapered, and she remained asymptomatic for 1 year, at which time she presented with similar symptoms. An MRI demonstrated several new enhancing areas. MTX was discontinued, and a brain biopsy was scheduled. However, a repeat MRI showed resolution of the lesions, and her clinical symptoms resolved over 6 weeks. MTX was resumed to control her RA. Six months later, however, she presented with ataxia and increased fatigue. An MRI showed recurrence of enhancing lesions, now involving bifrontal and left parietal lobes. She subsequently underwent a right frontal brain biopsy, which showed focally dense lymphohistiocytic infiltrates in a background of gliosis. The infiltrates were predominantly composed of small lymphocytes and histiocytes mixed with a few large atypical cells, resembling Reed-Sternberg cells, having enlarged irregular nuclei, vesicular chromatin, and prominent nucleoli. The large atypical cells stained CD20(+), CD30(+), CD15(−), and CD45(−). In situ hybridization staining for EBV was negative. CSF analysis and bone marrow biopsy were negative again. MTX was again discontinued, and dexamethasone and rituximab were initiated. Six weeks later, an MRI showed complete resolution of the enhancing lesions, and the patient improved clinically.

CONCLUSION

To our knowledge, this is the first reported case of an MTX-induced isolated CNS HLD. Although rare, it is important to recognize this CNS pathology within the spectrum of complications arising from the use of MTX among patients with RA.

Neuro-Oncol. 2009 Oct;11(5):620.

246. Phase II Trial of Single-Agent Bevacizumab Followed by Bevacizumab Plus Irinotecan at Tumor Progression in Recurrent Anaplastic Glioma

Teri Nguyen Kreisl, John A Butman, Dima Hammoud, Fabio M Iwamoto, Lyndon Kim, Paul Duic, Paul Albert, Howard A Fine

INTRODUCTION

Bevacizumab is evolving as the standard second-line chemotherapy for temozolomide-resistant glioblastoma. We present data for recurrent anaplastic glioma (AG) patients treated with bevacizumab alone and in combination with irinotecan.

METHODS

Patients with recurrent AG after radiation were eligible for this National Cancer Institute institutional review board-approved study of single-agent bevacizumab (10 mg/kg) administered biweekly. After tumor progression, eligible patients were enrolled in a companion study where they were treated with bevacizumab plus irinotecan (340 mg/m2 or 125 mg/m2) every 2 weeks, depending on use of EIAEDs. Evaluations for response and toxicity were obtained every 4 weeks, including cycle one fluorodeoxyglucose positron emission tomography (FDG-PET) brain imaging and perfusion magnetic resonance imaging (MRI) within 96 h of the start of treatment.

RESULTS

Thirty-one patients (21 men; 10 women) were enrolled (median Karnofsky performance score = 90; median prior chemotherapies = 2; 12 (39%) AO or AOA histology). grade 3 was most common, and treatment-associated toxicities were hypophosphatemia (26%), hypertension (19%), and thromboembolic events (16%). Median progression-free survival (PFS) was 3.7 months; median overall survival was 12.4 months (PFS6, 24.1%; 95% CI, 10.1–41.3). Radiographic response rate was 58% using standard bidi-mensional criteria and 81% using qualitative scoring criteria. Patients on steroids at study entry had an average 40% reduction in steroid dose, and 48% of patients had clinical improvement. Patient outcomes correlated to early MRI response, and PET imaging data will be presented. Ten patients received irinotecan after single-agent bevacizumab failure. One patient achieved a partial response, and two others achieved stable disease with combination therapy. Only one patient, who had mostly necrotic disease at study entry, had PFS greater than 6 months.

CONCLUSION

Bevacizumab is an effective drug in treating recurrent AG patients. A significant number of patients achieved prolonged PFS and clinical benefit in terms of reduced steroid requirements and improved neurological symptoms. However, numbers were too small to assess the efficacy of adding irinotecan to bevacizumab.

Neuro-Oncol. 2009 Oct;11(5):621.

247. High-Dose Methotrexate and Rituximab with Deferred Radiotherapy for Newly Diagnosed Primary CNS Lymphoma

Marc C Chamberlain, Sandra K Johnston

INTRODUCTION

We conducted a prospective phase II study of high-dose methotrexate (HD-MTX) and rituximab in adult patients with newly diagnosed primary CNS lymphoma (PCNSL); our primary objective was to evaluate median progression-free survival (PFS).

METHODS

Forty patients (25 men; 15 women) with newly diagnosed PCNSL, ranging in age from 18 to 93 years (median, 61.5 years), were treated. Surgery, CSF-flow cytometry, or vitrectomy was used to diagnose aggressive B-cell lymphoma. All patients were treated with biweekly HD-MTX/rituximab (8 gm/m2/dose; 37 mg/m2/dose) for four to six cycles (induction), following the best radiographic response every 4 weeks with HD-MTX (8 gm/m2/dose) for four cycles (maintenance). Neurological and neuroradiographic evaluations were performed every 4 weeks during HD-MTX/rituximab (induction therapy) and every 8 weeks during HD-MTX (maintenance therapy).

RESULTS

All patients were evaluable. A total of 303 cycles of HD-MTX (median, 8 cycles; range, 4–10 cycles) were administered. HD-MTX/rituximab-related toxicity included 16 grade 3 adverse events (AEs) in 13 patients (32.5%) and 2 grade 4 AEs, including renal (nine patients; 22.5%), neutropenia (five patients; 12.5%), anemia (three patients; 7.5%), and thrombocytopenia (two patients; 5%). Following four to six cycles of HD-MTX/rituximab, eight patients (20%) demonstrated progressive disease and discontinued therapy. Thirty-two patients (80%) demonstrated a partial (8/40; 20%) or complete (24/40; 60%) radiographic response to HD-MTX/rituximab. At the conclusion of induction and maintenance HD-MTX (6–10 months of total therapy), 28 patients (70%) demonstrated either a partial (1/28) or complete (27/28) response. Overall survival of these 28 patients ranged from 11 to 72 months (median, 31 months). Survival in the entire cohort ranged from 6 to 72 months, with an estimated median survival of 29.0 months (95% CI, 20.73–37.27).

CONCLUSION

HD-MTX/rituximab and deferred WBI demonstrated efficacy similar to other HD-MTX regimens that include up-front radiotherapy; toxicity levels were acceptable in this cohort of adult patients with newly diagnosed PCNSL.

Neuro-Oncol. 2009 Oct;11(5):621.

248. Phase II and Pharmacogenomics (PGx) Study of Enzastaurin (Enz) Plus Temozolomide (TMZ) and Radiation Therapy (RT) in Patients with Glioblastoma Multiforme (GBM) Or Gliosarcoma (GS)

Nicholas A Butowski, Kathleen L Lamborn, Mei-Yin C Polley, Emily Hsieh, Valerie Kivett, Margaretta S Page, Rupa Parvataneni, Steve Nicol, Don Thornton, Michael Prados

INTRODUCTION

Enzastaurin (ENZ) suppresses signaling through PKCβ and PI3K/AKT pathways. The primary end point of this single-arm trial was overall survival (OS). A secondary objective was progression-free survival (PFS). Magnetic resonance spectroscopy (MRS), perfusion imaging (rCBV), and PGx were evaluated for outcome-predictive value.

METHODS

Patients with newly diagnosed glioblastoma multiforme (GBM) or gliosarcoma (GS) and a Karnofsky performance score (KPS) >60 were enrolled in the trial. Treatment started less than 5 weeks after diagnosis, with radiation therapy (RT; 60 Gy) given over 6 weeks and temozolomide (TMZ; 75 mg/m2) given daily during RT and then as adjuvant therapy at 200 mg/m2 on days 1–5 of a 28-day cycle. ENZ (250 mg/day) was given daily during RT and as adjuvant therapy. Twelve adjuvant cycles were planned. PGx parameters included MIB, MGMT, MMR, PKC isoforms, MAPK, pCREB, EGFR, PTEN, GSK3β, VEGF, and pS6. MRS/rCBV were performed at baseline and at scheduled intervals. PFS and OS were estimated using the Kaplan-Meier method. Correlation of biomarker and imaging data with clinical outcomes was assessed using Cox proportional hazard models.

RESULTS

For this analysis, 66 patients were included—60 phase II patients (enrolled from September 2007 to November 2008) and 6 patients from a prior phase I trial. Forty-four patients progressed, and 20 patients died. At the time of this analysis, median follow-up for survivors and nonprogressors was 47 weeks (range, 22–130 weeks) and 43 weeks (range, 18–130 weeks), respectively. Median PFS was 36 weeks (95% CI, 30–50 weeks), and the 6-month PFS rate was 65% (95% CI, 54–78%). The 52-week OS rate was 73% (95% CI, 61–86%). Hazard ratios for PFS and OS correlated to biomarkers included MGMT-unmethylated versus methylated patients (2.18 [95% CI, 0.92–5.17] and 2.06 [95% CI, 0.64–6.6], respectively) and patients with a higher MIB index (1.02 [95% CI, 0.99–1.04] and 1.01 [95% CI, 0.98–1.05], respectively). Statistical significance for these biomarkers was not reached.

CONCLUSION

ENZ plus TMZ during and following RT shows promising activity in patients with GBM. An end point and biomarker update will be provided.

Neuro-Oncol. 2009 Oct;11(5):621.

249. Six-Month Progression-Free Survival as an Alternative Primary Efficacy End Point to Overall Survival in Newly Diagnosed Glioblastoma Patients Receiving Temozolomide

Mei-Yin C Polley, Kathleen L Lamborn, Susan Chang, Nicholas A Butowski, Jennifer L Clarke, Michael Prados

INTRODUCTION

Our objective was to assess 6-month progression-free survival (PFS) as an alternative primary efficacy end point to overall survival in newly diagnosed glioblastoma multiforme (GBM) patients receiving temozolomide (TMZ).

METHODS

A total of 183 patients with newly diagnosed GBM enrolled in three phase II protocols at UCSF were included. Patients were treated with interventions based on the Stupp regimen, each with the added component of a second oral agent given concurrently with radiotherapy and TMZ, followed by its coadministration with adjuvant TMZ. We examined whether progression status at 2, 4, and 6 months predicted subsequent survival using the landmark analysis. The hazard ratios of death as a function of progression status were estimated based on the Cox proportional hazards model after adjustment for putative prognostic factors.

RESULTS

Progression status at 2, 4, and 6 months were all consistently found to be strong predictors of subsequent survival in all studies. The study-specific hazard ratios associated with progression status at 6 months ranged from 2.03 to 3.39. The hazard ratios associated with the earlier time points (2-month and 4-month progression) all exceeded 2 in magnitude, ranging from 2.29 to 4.73. p-values were statistically significant for all time points.

CONCLUSION

In this work, we demonstrated a strong association between the end points of PFS at 2, 4, and 6 months and survival. Patients who showed signs of early progression were at significantly higher risk of earlier death. Our analysis suggests that 6-month PFS may be an appropriate primary end point in the context of phase II up-front GBM trials in the TMZ era.

Neuro-Oncol. 2009 Oct;11(5):621.

250. Intratumoral Application of Trabedersen (Ap 12009) in Recurrent Or Refractory High-Grade Glioma Patients

Ulrich Bogdahn, Ashok Kumar Mahapatra, Ashish Suri, Chandra Mouli, Kolluri VR Sastry, Neelam Venkataramana, Victor Emeljanovich Oliushine, VA Parfenov, Irina Poverennova, Guenther Stockhammer, Thomas Schneider, Peter Hau, Hubert Heinrichs, Karl-Hermann Schlingensiepen

INTRODUCTION

TGF-β2 regulates key mechanisms of cancerogenesis, namely, immunosuppression, metastasis, angiogenesis, and proliferation. Trabedersen is a TGF-β2-specific inhibitor and was developed for the treatment of patients with recurrent or refractory high-grade glioma (HGG).

METHODS

To date, clinical studies in HGG have included three phase I/II studies and one randomized, active-controlled, open-label, multinational dose-finding phase IIb study. These studies were performed in adult patients with recurrent or refractory HGG (anaplastic astrocytoma [AA] WHO grade III and glioblastoma multiforme [GBM] WHO grade IV). Trabedersen was administered intratumorally by convection-enhanced delivery.

RESULTS

In the phase IIb study, a total of 145 patients were randomized to either one of the two doses of trabedersen (10 μM or 80 μM) or to standard chemotherapy (TMZ or PCV). As in the previous phase I/II studies, treatment with trabedersen led to long-lasting remission in patients with recurrent or refractory AA or GBM. Overall, the highest efficacy was observed in AA patients treated with the lower dose of trabedersen. At 24 months, the 10 μM trabedersen group had the highest survival rate (83%) compared to the 80 μM trabedersen (53%) and standard chemotherapy (42%) groups. In GBM patients, trabedersen was as efficacious as standard chemotherapy, and in a subgroup with prespecified prognostic factors (age = 55 years; Karnofsky performance score > 80%), trabedersen patients had a markedly higher 2-year survival rate than patients on standard chemotherapy (44.4% vs. 13.3%).

CONCLUSION

Trabedersen treatment is safe and has a clear clinical benefit in HGG. A randomized, multinational, active-controlled phase III study (SAPPHIRE) with trabedersen as mono-therapy in patients with recurrent or refractory AA (WHO grade III) was initiated, and patient enrollment is ongoing. In this pivotal study, patients are randomized either to treatment with 10 μM trabedersen or to standard chemotherapy (TMZ or BCNU). A phase III study of patients with GBM is in preparation.

Neuro-Oncol. 2009 Oct;11(5):622.

251. Phase II Trials of Erlotinib or Gefitinib in Patients with Recurrent Meningiomas

Andrew Norden, Jeffrey J Raizer, Kathleen L Lamborn, Lauren E Abrey, Susan Chang, Mark R Gilbert, Timothy F Cloughesy, Michael Prados, Frank Lieberman, Patrick Wen

INTRODUCTION

No effective treatment is available for recurrent meningiomas when surgical and radiation options are exhausted. The epidermal growth factor receptor (EGFR) is often overexpressed in meningiomas and may promote tumor growth. In open-label, single-arm phase II studies of the EGFR inhibitors gefitinib (NABTC 00-01) and erlotinib (NABTC 01-03) in recurrent malignant gliomas, we included exploratory subsets of recurrent meningioma patients. We have pooled the data and report the results here.

METHODS

Patients with recurrent, histologically confirmed meningiomas and no more than two previous chemotherapy regimens were treated with gefitinib (500 mg/day) or erlotinib (150 mg/day) until tumor progression or unacceptable toxicity was reached.

RESULTS

Twenty-five eligible patients were enrolled, with a median age of 57 years (range, 29–81 years) and median Karnofsky performance status (KPS) score of 90 (range, 60–100). Sixteen patients (64%) received gefitinib, and nine received (36%) erlotinib. Eight patients (32%) had benign tumors, nine (36%) had atypical tumors, and eight (32%) had malignant tumors. For benign tumors, 6-month progression-free survival (PFS6) was 29%, 12-month PFS (PFS12) was 0%, 6-month overall survival (OS6) was 63%, and 12-month OS (OS12) was 50%. For atypical/malignant tumors, PFS6 was 25%, PFS12 was 19%, OS6 was 81%, and OS12 was 68%. There were no significant PFS or OS differences by histology. Of 21 evaluable patients, there were no responses; 8 patients (38%) had stable disease, and 13 (62%) had progressive disease. Treatment was well tolerated. Rash was not a significant predictor of PFS or OS.

CONCLUSION

Neither gefitinib nor erlotinib appear to have significant activity against recurrent meningioma. The role of EGFR inhibitors in meningiomas is unclear, but evaluation of EGFR inhibitors in combination with other targeted molecular agents may be warranted.

Neuro-Oncol. 2009 Oct;11(5):622.

252. Characterization of Antineoplastic Agents’ Delivery to the CNS Enhanced by Osmotic BBBD

Marie Blanchette, Luc Tremblay, Martin Lepage, David Fortin

INTRODUCTION

Treatment of malignant astrocytomas is hampered by the blood-brain barrier (BBB), which limits the CNS delivery of antineo-plastic agents. Osmotic BBB disruption (BBBD) increases the delivery of compounds to the CNS. However, its dynamic process remains unclear. The objective of this study was to determine the dynamics of the accumulation, elimination, spatial distribution, and therapeutic window for small and large molecules after BBBD using dynamic magnetic resonance imaging (MRI).

METHODS

Healthy and glioma-bearing rats were imaged before, during, and after BBBD with a 7-tesla MRI animal scanner. Images were produced every ~50 s, allowing a dynamic study. Two different contrast agents (Magnevist [0.5 kDa] or Gadomer [17 kDa]) were administered at different times post-BBBD (1, 2, 3, 5, 7, 10, 15, 20, 30, 45, 60 min). The concentration of the contrast agents in the CNS was determined as a function of time postinjection.

RESULTS

The mean maximal concentration in the treated hemisphere was reached when the contrast agent was administered 3 min post-BBBD and was about 0.1 mM for Magnevist and 0.037 mM for Gadomer. Magnevist distribution into the CNS was regulated by two mechanisms: 1) the direct delivery via the BBBD process, and 2) diffusion of the molecule in the interstitial fluid. The diffusion process was not observed for Gadomer. The BBB was functional within 10 min for Gadomer but was still permeable to Magnevist 30 min post-BBBD. Preliminary data suggest that the distribution of Gadomer is different in glioma-bearing animals than in healthy animals; this was not observed with Magnevist.

CONCLUSION

Following the BBBD, molecules that do not normally reach the CNS were observed over a large area of the CNS. Parameters that govern the dynamic process of the BBBD were unveiled and may have major clinical implications for the treatment of malignant astrocytomas.

Neuro-Oncol. 2009 Oct;11(5):622.

253. New Therapeutic Approach for Brain Tumors: Intranasal Administration of Ras Inhibitor Monoterpene Perillyl Alcohol

Clovis O Fonseca

INTRODUCTION

Malignant gliomas commonly overexpress the oncogenes EGFR and PDGFR and contain mutations and deletions of the tumor suppressor genes PTEN and TP53. Some of these alterations lead to activation of the P13K/Akt and Ras/MAPK pathways, which provide targets for therapy. Perillyl alcohol (POH), the isoprenoid of greatest clinical interest, was initially considered to inhibit farnesyl protein transferase. Follow-up studies revealed that POH suppresses the synthesis of small G proteins, including Ras. Intranasal delivery allows drugs that do not cross the blood-brain barrier to enter the CNS. Moreover, it eliminates the need for systemic delivery, thereby reducing unwanted systemic side effects.

METHODS

A cohort of 89 patients with progressive disease after prior surgery, radiotherapy, and at least temozolomide-based chemotherapy were enrolled; 69 of the patients had GBM, 13 had AA, and 7 had AO. The following parameters were assessed: safety for POH, initial symptoms, PFS at 6 months, overall survival, topography and tumor size, presence of midline shift, and extent of peritumoral edema. Statistical analysis was carried out with log-rank tests, and overall survival time was assessed with the Kaplan-Meier method, including 95% confidence intervals.

RESULTS

There were no toxicity events. Progression-free survival (partial response and stable disease) at 6 months was 48.2% for patients with GBM, 60% for patients with AA, and 66.6% for patients with AO. Patients with recurrent gliomas with localization in the basal ganglia survived significantly longer (0.0003) than patients with tumors at lobar localization. Presence of peritumoral tumor brain edema contributed to symptoms likely to be implicated in the morbidity and invading potential of malignant gliomas. The median survival rate of patients with secondary GBM was 11.2 months, significantly longer (p = 0.0002) than those with primary GBM (5.9 months). Among primary GBM patients, those with tumors in a deep location survived longer than those with tumors in a lobar location (p = 0.0083).

CONCLUSION

Here, we are investigating a novel therapeutic strategy using intranasal delivery of POH, an inhibitor of Ras. The treatment was well tolerated and suggests improvement in tumor response rates.

Neuro-Oncol. 2009 Oct;11(5):622.

254. Alternating Weekly Temozolomide as Salvage therapy in Malignant Gliomas

Alison B Allen, Denise M Damek

INTRODUCTION

The current standard of care for patients with histologically proven malignant glioma is concomitant and adjuvant chemoradiotherapy with temozolomide. Adjuvant dosing of temozolomide is typically on a 5 days on, 28 days off dosing schedule. However, when patients have radiographic progression of disease, many oncologists turn to an alternate dosing schedule as salvage therapy. There is limited data on the efficacy of this treatment approach.

METHODS

The institutional review board approved a retrospective case series reporting consecutive patients with radiographically progressive and standard-dose temozolomide-refractory malignant glioma who were treated with an alternating weekly temozolomide dosing schedule as salvage therapy.

RESULTS

Eighteen patients (10 men; 8 women), ages 26–53 years (median, 44.5 years), with radiographically progressive malignant glioma (13 anaplastic astrocytoma [AA]; five glioblastoma [GBM]) were treated with alternating weekly temozolomide (150 mg/m2/day for 7 days every other week) as salvage therapy. All patients had previously been treated with surgery, involved field external beam irradiation (deferred in one patient because of lesion size), and adjuvant temozolomide chemotherapy utilizing the standard 5 days every 28 days dosing schedule. Thirteen of these patients additionally received temozolomide concurrent with radiation therapy. A total of 163 cycles of salvage temozolomide (median, 6 cycles) were administered. NCI-CTC grade 3 toxicities were seen in four patients (lymphopenia in two patients, neutropenia in one patient, and autoimmune hemolytic anemia in one patient). The progression-free survival at 12 months was 77% in patients with AA and 20% in patients with GBM. Eleven patients (10 AA; 1 GBM) had no tumor progression 15–86 months after initiating salvage therapy. In the seven patients with progressive disease (four AA; three GBM), time to tumor progression ranged from 1 to 5 months (median, 2 months).

CONCLUSION

Temozolomide on an alternating weekly schedule is a well-tolerated, effective salvage strategy for patients with AA.

Neuro-Oncol. 2009 Oct;11(5):622–623.

255. Proposal for Updated Response Criteria in Malignant Gliomas

Patrick Y Wen, David Macdonald, Martin van den Bent, David A Reardon, Evanthia Galanis, Timothy F Cloughesy, Kathleen L Lamborn, John Degroot, Mark R Gilbert, Wolfgang Wick, Christina Tsien, Tom Mikkelsen, Marc C Chamberlain, Eric T Wong, A Gregory Sorensen, Michael A Vogelbaum, Susan Chang

INTRODUCTION

Currently, response assessment in malignant glioma trials is based primarily on measurement of cross-sectional areas of contrast-enhancing tumors (Macdonald et al. J Clin Oncol 1990;8:1277–80). Limitations of the “Macdonald criteria” include interobserver variability, difficulty in measuring tumors with complex shapes and components (i.e., cystic or postoperative cavities), nontumor factors producing imaging changes (e.g., postoperative enhancement, infarction, seizures), reaction to local therapies (convection-enhanced delivery, chemotherapy wafers, radiosurgery, etc.), posttreatment changes that may mimic tumors (i.e., pseudoprogression, radiation necrosis), and lack of applicability to nonenhancing tumors. Recently, the introduction of antiangiogenic therapies directly affecting the permeability of tumor vasculature and degree of contrast enhancement has made the limitations of the Macdonald criteria more apparent. Anti-VEGF therapies may reduce contrast enhancement within days, producing a pseudoresponse, while some patients develop progressive disease characterized by increased nonenhancing tumors, which is currently not accounted for by the Macdonald criteria.

METHODS

The Response Assessment in Neuro-Oncology (RANO) working group is an ongoing international multidisciplinary consensus-building effort to develop new response criteria to address these issues. Three committees (high-grade glioma, low-grade glioma, and surgical therapies) are evaluating current response criteria, new imaging modalities, and clinical, neurocognitive, and quality of life end points, with the aim of proposing new response guidelines.

RESULTS

Details of the proposed criteria for malignant gliomas will be presented. In addition to updating specific definitions of measurable tumor, response, progression, and clinical deterioration, increasing nonenhancing tumors will be incorporated into the definition of progressive disease. The new criteria also excludes most patients who are within 3 months of completing radiation therapy from recurrent glioma clinical trials to avoid patients with pseudoprogression.

CONCLUSION

These updated criteria represent work in progress and will be revised as more advanced imaging techniques and other end points are validated; hopefully, the updated criteria will improve the assessment of novel therapies in malignant gliomas.

Neuro-Oncol. 2009 Oct;11(5):623.

256. Ang1005: Development of A New Engineered Peptide Compound (EPiC) for the Treatment of Advanced Solid Tumors and Brain Metastases

Razelle Kurzrock, Nashat Gabrail, Stacy Moulder, Andrew Brenner, Zhong Guo, Kelly M Elian, Wendy Churchill, Paula Bento, Dimitri Fitsialos, Bruno Fraitag, Betty Lawrence, Jean-Paul Castaigne, John Sarantopoulos

INTRODUCTION

In the United States, approximately 170,000 patients with systemic tumors develop brain metastases annually, and this incidence is expected to rise. Challenges to treatment stem from the inability of most drugs to cross the blood-brain barrier (BBB). Angiochem is developing a broad pipeline of new drugs uniquely capable of crossing the BBB to treat brain diseases. ANG1005 is the first drug from the engineered peptide compounds (EPiC) platform to be developed clinically. Studies show that ANG1005 enters the brain by targeting low-density lipoprotein receptor-related protein (LRP), one of the most highly expressed receptors on the BBB, and ANG1005 also enters tumor cells via the LRP, which is upregulated in various cancer cells, including metastatic cancer cells.

METHODS

As of May 19, 2009, 48 patients with advanced solid tumors/brain metastases have received ANG1005 by intravenous infusion at doses of 30–700 mg/m2 q21d without premedication. Study objectives include characterizing the safety/tolerability of ANG1005, identifying the maximum tolerated dose (MTD), and obtaining preliminary PK and antitumor information. Severity of adverse events (AEs) is assessed using CTCAE, v.3.

RESULTS

Currently, 650 mg/m2 is being explored as the MTD. Although some patients have developed neutropenia, leucopenia, thrombocytopenia, and anemia, these cases have been milder than expected based on clinical observation with paclitaxel and have been manageable with standard treatments. Preliminary neurocognitive data showed no evidence of CNS toxicity; in fact, data from one patient with a minor tumor response showed significant improvement in memory, processing speed, and executive function after 6, 12, and 24 weeks of therapy. Biological data show that ANG1005 does not elicit an immune response, even in patients who have received six treatment cycles. Pharmacokinetic data show a linear relationship between dose and bioavailability. Radiology data indicate potential efficacy in tumor regression/slowing tumor progression, mostly at doses >300 mg/m2.

CONCLUSION

Taken together, the data presented offer encouragement to patients suffering from brain metastases.

Neuro-Oncol. 2009 Oct;11(5):623.

257. Patterns of Recurrence of Primary Glioblastoma Multiforme Treated with Chemoradiation in the Temozolomide Era

Jiwon Oh, Arjun Sahgal, May N Tsao, Paul Sanghera, Phil Davey, Kelvin Lam, Richard Aviv, Sean Symons, James R Perry

INTRODUCTION

Temozolomide (TMZ) is now widely accepted as the standard of care when used concurrently with radiation in the treatment of glioblastoma multiforme (GBM). There is controversy as to whether the recurrence pattern of GBM has shifted from predominantly local recurrence, as observed historically in 80% to 90% of patients treated with radiation alone, to greater distant recurrence with the use of TMZ. The purpose of this study was to evaluate patterns of recurrence of GBM in patients treated with radiation therapy (RT) and concurrent and adjuvant TMZ chemotherapy.

METHODS

A retrospective chart review was performed of patients with newly diagnosed GBM treated with RT/TMZ between 2003 and 2007. Of 111 patients meeting the inclusion criteria, 67 had tumor recurrence and adequate baseline and recurrence scans for analysis during the study period. Statistical analyses included the Kaplan-Meier method for survival, log-rank test for differences in prognostic factors, and multivariate Cox proportional hazards model for describing the effect of the recurrence pattern on survival and time to progression (TTP).

RESULTS

Fifty-eight patients (86.6%) had local recurrence, while nine patients (13.4%) had recurrence that included a distant noncontiguous focus of disease. Overall median survival was 17 months, and median TTP was 6.8 months. The local and distant recurrence groups were comparable with respect to potential prognostic variables, including age (p = 0.14), performance status (p = 0.83), and extent of surgery (p = 0.26). There was no statistically significant difference in median survival (p = 0.35) or TTP (p = 0.95) with respect to location of recurrence.

CONCLUSION

In this study, the pattern of recurrence of GBM treated with RT/TMZ chemoradiation was predominantly local, which is similar to what was seen prior to the use of TMZ. Understanding the effects of TMZ treatment on recurrence patterns has implications for the development of treatment protocols and tumor surveillance strategies. Large prospective studies are needed to definitively characterize patterns of recurrence in GBM patients treated with TMZ.

Neuro-Oncol. 2009 Oct;11(5):623.

258. Ang1005: A Promising New Engineered Peptide Compound (EPiC) for Patients with Malignant Glioma

Jan Drappatz, Andrew Brenner, Steven Rosenfeld, David Schiff, Patrick Y Wen, Tom Mikkelsen, Morris Groves, Eric T Wong, April Eichler, Danielle Bouchard, Ann Neale, Jean-Paul Castaigne

INTRODUCTION

Malignant glioma (MG) is an aggressive and fatal cancer whose treatment is limited by the inability of drugs to cross the blood-brain barrier (BBB). ANG1005 is a novel taxane derivative that was created from the engineered peptide compound (EPiC) platform and that is uniquely capable of crossing the BBB. Studies show that ANG1005 enters the brain by targeting low-density lipoprotein receptor-related protein (LRP), one of the most highly expressed receptors on the BBB, and ANG1005 also enters tumor cells via the LRP, which is upregulated in various cancer cells, including MG cells.

METHODS

As of May 19, 2009, 37 patients with recurrent/progressive MG (WHO grades III–IV) have received ANG1005 by intravenous infusion at doses of 30–550 mg/m2 once q21d without premedication. Study objectives include characterizing the safety/tolerability of ANG1005, identifying the maximum tolerated dose (MTD), and obtaining preliminary PK and antitumor information in patients with MG. Drug penetration into extracted MG tumors was measured in a subgroup of patients undergoing debulking surgery following a single dose of ANG1005.

RESULTS

Dose escalation is ongoing. Few, if any, patients have experienced neutropenia, leucopenia, thrombocytopenia, anemia, infusion reactions, mucositis, or peripheral neuropathy at a severity of grade 2 (CTCAE, v. 3). Neurocognitive data show that ANG1005 does not cause CNS toxicity at these doses. Biological data show that ANG1005 does not elicit an immune response even in patients who have received multiple treatment cycles. Magnetic resonance imaging data indicate potential efficacy in tumor regression/slowing tumor progression. Pharmacokinetic data show a linear relationship between dose and bioavailability. Data from extracted tumors (n = 3) showed that ANG1005 uptake was 20× to 40× greater than reported following an equivalent dose of paclitaxel, and ANG1005 concentration in tumor relative to blood exceeded 25%.

CONCLUSION

Data to date demonstrate that ANG1005 has an excellent safety/tolerability profile, penetrates tumors, and shows promise as a potential treatment option for recurrent/progressive MG.

Neuro-Oncol. 2009 Oct;11(5):623–624.

259. Evaluation of Objective Response as a Predictor of Survival in Bevacizumab-Treated Patients with Glioblastoma at First or Second Relapse in the Brain Study

Michael Prados, Timothy F Cloughesy, Meghna Samant, Liang Fang, Asha Das, Henry Friedman; for the BRAIN Investigators

INTRODUCTION

Bevacizumab (BEV), a humanized monoclonal antibody, inhibits the activity of human vascular endothelial growth factor (VEGF). In the BRAIN study (ClinicalTrials.gov; NCT00345163), treatment with BEV alone or in combination with irinotecan demonstrated a clinically meaningful improvement in objective response (OR) rates (28.2% and 37.8%, respectively) in patients with relapsed glioblastoma (GBM) compared to prior studies conducted with other agents in this disease setting. This exploratory analysis by the BRAIN study was performed to determine if response status was associated with greater residual survival.

METHODS

BRAIN was a phase II, open-label, multicenter, randomized, noncomparative trial of 167 patients that evaluated the efficacy and safety of BEV alone and in combination with irinotecan in patients with relapsed GBM (Cloughesy T et al. JCO. 2008;26:2010b). OR rate and 6-month progression-free survival, as assessed by an independent review facility, were coprimary end points of the study. Pooled data from both treatment arms were used to explore the association of OR with survival. Landmark analyses were conducted at 9, 18, and 26 weeks using Cox regression models adjusted for important prognostic factors, including age, baseline Karnofsky performance score, first versus second relapse, and treatment arm. Analysis at each time point required that patients survive until the respective landmark. Hazard ratios for survival between the responder and nonresponder groups were calculated. The goodness of fit, robustness, predictive accuracy, and validation of these models were tested.

RESULTS

There was a statistically significant association between OR and survival. Nonresponders were approximately twice as likely to die as responders in a given time period.

CONCLUSION

In BRAIN, OR rate was clinically compelling when compared to historical controls. This exploratory landmark analysis with pooled treatment arms suggests that patients with an OR had longer residual survival than those who did not have an OR. While these conclusions are limited in the absence of a control arm, OR could be considered a potential predictor of survival in this study of BEV-treated patients with relapsed GBM.

Neuro-Oncol. 2009 Oct;11(5):624.

260. Phase II Trial of Talampanel, A Glutamate Receptor Inhibitor, for Adults with Recurrent Malignant Gliomas

Fabio M Iwamoto

INTRODUCTION

Glioma cells secrete glutamate and also express AMPA glutamate receptors, which contribute to proliferation, migration, and neurotoxicity of malignant gliomas. Talampanel is an oral AMPA-receptor inhibitor with excellent CNS penetration and good tolerability in clinical trials for epilepsy and other neurological disorders.

METHODS

We conducted a phase II trial to evaluate the efficacy of talampanel in patients with recurrent malignant glioma as measured by 6-month progression-free survival (PFS6).

RESULTS

Thirty patients (22 glioblastomas [GBM]; 8 anaplastic gliomas [AG]; 63% men), with a median age of 51 years (range, 20–67 years) and median Karnofsky performance score of 80, were included. Patients tolerated treatment well and most adverse events were mild and reversible; the most common toxicities were fatigue (27%), dizziness (23%), and ataxia (17%). There was only one partial response (5%) in the GBM stratum and none among AG patients. With a median follow-up of 13 months, 28 patients (93%) had died. The PFS6 was 4.6% for the initial 22 GBM patients, and the study was terminated early because of treatment futility; PFS6 was 0% for the eight AG patients. Median PFS was 5.9 weeks for GBM patients and 8.9 weeks for AG patients. Median overall survival was 13 weeks for GBM patients and 14 months for AG patients.

CONCLUSION

Talampanel was well tolerated but had no significant activity as a single agent in unselected recurrent malignant gliomas.

Neuro-Oncol. 2009 Oct;11(5):624.

261. Reversible Blindness from Optic Nerve Infiltration by Recurrent Anaplastic Astrocytoma Responsive to Chemotherapy Treatment

Mimi Sohn, Pamela S Chavis, Pierre Giglio

INTRODUCTION

Anaplastic astrocytomas are malignant gliomas with poor prognoses. Despite therapy, progression is universal. Progression usually occurs within the original tumor bed, but distant and multifocal progression is occasionally noted. To our knowledge, progression with optic nerve involvement has never been described. Herein, we describe a case of anaplastic astrocytoma progression causing bilateral optic nerve infiltration and blindness. Additionally, we describe improvement in vision and neuroimaging findings after treatment.

METHODS

Review of the patient’s medical record, and review of the medical literature for published cases of glioma infiltration of the optic nerve(s) causing blindness.

RESULTS

A 26-year-old female was diagnosed with a left temporal mass in February 2007. Pathology showed anaplastic astrocytoma. The patient received radiation therapy and temozolomide (TMZ) chemotherapy. In December 2007, she experienced progression, and TMZ was increased. In April 2008, she presented with visual complaints and was diagnosed with bilateral papilledema. Magnetic resonance imaging (MRI) showed bilateral optic nerve enhancement, indicating involvement of the optic nerves. There was no light perception (NLP) in the right eye, and 20/25 −2 in the left eye. The patient was treated with high-dose steroids but showed no improvement. Late in April 2008, her vision deteriorated to light perception (LP) in the left eye and NLP in the right eye. She was started on bevacizumab/irinotecan in early May. After one cycle, there was no improvement in vision in the right eye, but vision improved to 20/400 in the left eye. An MRI showed response with resolution of optic nerve enhancement. Chemotherapy continued, and bevacizumab was increased in June 2008. After an additional cycle, vision improved to 20/100 −1 in the left eye, but showed no improvement in the right eye. Tumor progression occurred in September 2008, with vision deterioration and recurrent optic nerve enhancement. The treatment was changed to VP-16/bevacizumab, but further progression was noted. The patient was admitted to hospice care and died.

CONCLUSION

Anaplastic astrocytomas rarely progress with optic nerve involvement. We describe such a case showing improvement in vision from response to treatment. We speculate improvement was unilateral due to earlier and more severe right optic nerve involvement causing irreversible ischemic damage.

Neuro-Oncol. 2009 Oct;11(5):624.

262. Bevacizumab for Recurrent Ependymoma

Richard M Green, Timothy F Cloughesy, Roger Stupp, Lisa M DeAngelis, Emily A Woyshner, Douglas E Ney, Andrew B Lassman

INTRODUCTION

Ependymoma is a rare type of glioma, representing 5% of all CNS malignancies. Radiotherapy (RT) is commonly administered, but there is no standard chemotherapy. At recurrence, ependymoma is notoriously refractory to therapy, and the prognosis is poor. In recurrent glioblastoma, encouraging responses with bevacizumab have been observed.

METHODS

In this institutional review board-approved study, we retrospectively analyzed the records of eight adult patients treated for recurrent ependymoma and anaplastic ependymoma with chemotherapy regimens containing bevacizumab. We determined radiographic response (Macdonald criteria), median time to progression (TTP), and median overall survival (OS; Kaplan-Meier method).

RESULTS

There were four men and four women in the study, with a median age of 40 years (range, 20–65 years). Prior treatment included surgery (n = 8), RT (n = 8), temozolomide (n = 5), and carboplatin (n = 4). Bevacizumab (5–15 mg/kg every 2–3 weeks) was administered alone (n = 2) or concurrently with cytotoxic chemotherapy, including irinotecan (n = 3), carboplatin (n = 2), or temozolomide (n = 1). Six patients (75%) achieved a partial response of enhancing disease, typically with concurrently improved edema, and one remained stable for more than 8 months. Leptomeningeal tumor also improved. Median TTP and OS were 6.4 months (95% CI, 1.4–7.4) and 9.4 months (95% CI, from 7.0 to not reached), respectively, with a median follow-up of 5.2 months for the five (63%) surviving patients.

CONCLUSION

Our findings indicate bevacizumab has efficacy in the treatment of recurrent ependymoma. A prospective study is warranted.

Neuro-Oncol. 2009 Oct;11(5):624.

263. Temozolomide and Bevacizumab for Recurrent Adult Suprasellar Pilocytic Astrocytoma

Richard M Green, Emily A Woyshner, Timothy F Cloughesy

INTRODUCTION

Pilocytic astrocytoma (PA) is a WHO grade I tumor that occurs most frequently in children and young adults. Maximal surgical resection is the standard initial therapy. Although relatively well circumscribed, these tumors are often not completely resectable given their frequent location in critical midline structures, such as the optic pathways, hypothalamus, and brainstem. Radiation therapy (RT) is often used after incomplete surgical resection and to treat tumor growth following resection. No accepted chemotherapy regimen for recurrent tumors is available.

METHODS

We retrospectively reviewed cases of suprasellar PA treated with temozolomide (TMZ) and bevacizumab (Bev). We identified four patients (two men, two women); the average age was 39 years. All patients had undergone biopsy followed by up-front radiation therapy for a residual tumor. Three patients were treated for their first disease recurrence; the fourth was treated for her second recurrence, having been treated with carboplatin for her first recurrence. Two patients exhibited visual field deficits from optic nerve and/or chiasmatic compression; two exhibited extraocular movement paresis. Imaging showed cystic contrast-enhancing tumors in all patients. All patients were treated with TMZ 150–200 mg/m2/day on days 1–5 and Bev 5 mg/kg on days 1 and 14 of a 28-day cycle. Magnetic resonance scans were performed every 8 weeks and graded according to the Macdonald criteria.

RESULTS

All patients (100%) achieved a partial response on follow-up imaging. Neurological deficits improved significantly in all patients. One patient experienced symptomatic intratumoral hemorrhage, leading to discontinuation of Bev. After an average follow-up period of 486 days, one patient is not on active therapy, two patients are still being treated, and the fourth is receiving Bev 5 mg/kg every 21 days. No patients have yet developed recurrent disease.

CONCLUSION

In adult suprasellar PA recurrent after RT, the combination of TMZ and Bev appears to be effective and deserves further investigation.

Neuro-Oncol. 2009 Oct;11(5):625.

264. Phase II Trial of Sunitinib (Su011248) in Patients with Recurrent or Inoperable Meningioma

Thomas J Kaley, Patrick Y Wen, David Schiff, Sasan Karimi, Frank Lieberman, Andrew B Lassman, Craig P Nolan, A Omuro, Igor Gavrilovic, Andrew Norden, Jan Drappatz, Benjamin Purow, Vineet Punia, Subramanian Hariharan, Lauren E Abrey

INTRODUCTION

Medical therapies for meningiomas are limited, as antihormone agents, immunomodulators, and traditional chemotherapeutics have all proven ineffective. Sunitinib malate (SU011248) is a small molecule multiple tyrosine kinase inhibitor that targets VEGF and PDGF receptors, which are abundant in meningiomas.

METHODS

This phase II trial for recurrent meningioma has a planned sample size of 50 patients: 20 with benign tumors, 20 with atypical/malignant meningioma, and 10 with hemangiopericytoma/hemangioblastoma. Sunitinib is administered orally at 50 mg/day for 4 weeks followed by a 2-week rest (one cycle). Magnetic resonance imaging (MRI) is performed every cycle for the first two cycles and then every two cycles thereafter. The primary end point is 6-month progression-free survival, with secondary end points of response rate, safety, progression-free survival (PFS), and overall survival (OS). Exploratory objectives include analysis of tumoral molecular characteristics and MRI-perfusion.

RESULTS

To date, 26 patients (15 male; 11 female), with a median age of 60 years (range, 32–85 years) and median Karnofsky performance score of 80, have been enrolled, including 20 patients with malignant/atypical meningioma, 3 with hemangiopericytoma, and 3 with hemangioblastoma. For meningioma patients, mPFS is 7 months, and mOS has not been reached. Eighteen patients are alive, and eight remain on treatment. Initial radiographic response in the meningioma patients include 1 PR, 13 SD (some with a minor reduction in tumor size), and 3 PD; it is too early to assess two patients. The patient with PR had an intratumoral hemorrhage (grade 2). One patient with hemangiopericytoma had a hemorrhage at progression. Ten patients required dose reduction for toxicity. To date, grade 3 and 4 toxicities include myelosuppression (n = 9), headache (n = 4), fatigue (n = 5), nausea/vomiting (n = 5), gastrointestinal perforation (n = 1), hypertension (n = 3), prolonged QTc (n = 2), dehydration (n = 2), elevated ALT/AST/amylase/lipase (n = 1 each), and gait difficulty (n = 2). MRI-perfusion demonstrated decreased perfusion after treatment in most patients.

CONCLUSION

Sunitinib may be active in recurrent atypical/malignant meningioma patients who are not eligible for further surgery or radiotherapy.

Neuro-Oncol. 2009 Oct;11(5):625.

265. Prolonged Pancytopenia from Combined Myelotoxic Effect of Temozolomide and Levetiracetam in a Patient with Glioblastoma Multiforme

Alexandra Flowers

INTRODUCTION

Radiation therapy (RT) and concomitant chemotherapy with temozolomide (TMZ) is now standard of care for patients with glioblastoma multiforme (GBM). Pancytopenia from TMZ is rare and usually transient. Levetiracetam (LEV) is a nonenzyme inducing antiepileptic drug (AED) with low side effects and is preferred to other AEDs. There are rare reports of pancytopenia in patients taking LEV. We report a patient with GBM who had prolonged pancytopenia from combined myelotoxic effects of TMZ and LEV.

METHODS

Our patient was a 69-year-old woman with left parietal GBM diagnosed in September 2008. She underwent resection followed by RT plus TMZ (100 mg/day), which was completed in December 2008. She was also on LEV (500 mg/bid). She had complete blood cell (CBC) counts taken weekly. Baseline CBC was normal with WBC = 16.4 (on Dex), Hct = 38.4, Plt = 209, and creatinine = 0.6. At the end of RT plus TMZ, on December 1, 2008, she had Plt = 43K, WBC = 5.9, and Hct = 36.2. On December 10, 2008, she had pancytopenia, with WBC = 1.9, ALC = 0.1, ANC = 1.7, Plt = 6K, and Hct = 23; on December 19, her WBC was 0.8. She required multiple transfusions and had two hospitalizations for sepsis, including multiple brain abscesses. Neutropenia resolved with Neupogen support over 1 month; she continued to require blood and platelet transfusions. In March 2009, the patient was switched from LEV to Lyrica; the anemia resolved, and the platelet count stabilized, with a slow trend toward normalization. The patient no longer needs transfusions. Bone marrow biopsy cytogenetics was not consistent with myelodysplastic syndrome.

RESULTS

Discontinuation of LEV led to rapid improvement and stabilization of Hct and platelet counts, indicating a myelosuppressive effect of LEV.

CONCLUSION

While pancytopenia was reported in patients taking TMZ, it is usually transient. There are rare instances of pancytopenia from LEV. To our knowledge, this is the first report of prolonged pancytopenia from the combined myelotoxic effects of TMZ and LEV. Further studies to identify patients at risk are warranted.

Neuro-Oncol. 2009 Oct;11(5):625.

266. Late Relapse in Primary Central Nervous System Lymphoma (PCNSL)

Lakshmi Nayak, Marc Ladanyi, Cyrus Hedvat, Lauren E Abrey, Lisa M DeAngelis

INTRODUCTION

Progression of primary CNS lymphoma (PCNSL) after initial therapy occurs within 2 years in 80% of patients, and some studies suggest relapse is rare after 5 years. Our objective was to evaluate relapse >5 years after initial diagnosis of PCNSL.

METHODS

We identified eight patients from our database of 441 whose PCNSL relapsed 5 years after initial diagnosis. Immunosuppressed patients and those with a prior history of systemic lymphoma were excluded.

RESULTS

At initial diagnosis, median age was 48 years (range, 35–72 years), four of the patients had a single lesion, two had leptomeningeal, and none had ocular involvement. All patients achieved a complete response to initial therapy (seven received high-dose methotrexate) within 3–9 months. Median time to relapse was 7.3 years (range, 5.2–13.8 years). Six patients had relapse in the brain, one had ocular relapse, and one had systemic relapse. The histological specimens at initial diagnosis and relapse were examined for clonal rearrangement in three patients; one was uninformative. One patient had the identical clone at diagnosis and at relapse 13.8 years later. One patient proved to have a T-cell lymphoma in the original specimen but clonality could not be established on his recurrence obtained at autopsy. All patients received salvage therapy; seven patients received systemic chemotherapy, and one received intraocular chemotherapy. Seven patients had complete response to salvage therapy, and one had partial response. Two patients relapsed again, one of whom died. The median progression-free survival after relapse was 41.6 months. Four patients are alive, two were lost to follow-up, and two died.

CONCLUSION

Late relapse from PCNSL is uncommon but possible. Long-term persistence of the PCNSL clone, despite initial treatment with systemic chemotherapy, raises questions regarding the source of these tumor cells. Patients with late relapse have a particularly good response to salvage therapy.

Neuro-Oncol. 2009 Oct;11(5):625.

267. Prognostic Factors Influencing the Clinical Outcomes of Glioblastoma Multiforme

Li Shouwei

INTRODUCTION

Glioblastoma multiforme (GBM) is the most malignant kind of astrocytic tumor and is associated with a poor prognosis. In this retrospective study, we assessed the clinical, radiological, genetic molecular, and treatment factors that influence clinical outcomes of patients with GBM.

METHODS

A total of 116 patients with GBM who received surgery and radiation between 2006 and 2007 were included in this study. Kaplan-Meier survival analysis and Cox regression analysis were used to find the factors independently influencing patients’ progression-free survival (PFS) times and overall survival (OS) times.

RESULTS

Age, preoperative Karnofsky performance scale (KPS) score, KPS score change at 2 weeks postoperation, neurological deficit symptoms, tumor resection extent, maximal tumor diameter, involvement of eloquent cortex or deep structure, involvement of brain lobe, Ki-67 expression level, and adjuvant chemotherapy were statistically significant factors (p < 0.05) for both PFS and OS in the univariate analysis. Cox proportional hazards modeling revealed that age = 50 years, preoperative KPS score = 80, KPS score change after operation = 0, involvement of single frontal lobe, non-eloquent area or deep structure involvement, low Ki-67 expression, and adjuvant chemotherapy were independent favorable factors (p < 0.05) for patients’ clinical outcomes.

CONCLUSION

Age at diagnosis, preoperative KPS score, KPS score change at 2 weeks postoperation, involvement of the brain lobe, involvement of eloquent cortex or deep structure, Ki-67 expression level, and adjuvant chemotherapy correlate significantly with the prognosis of patients with GBM.

Neuro-Oncol. 2009 Oct;11(5):625–626.

268. Bevacizumab Plus Carboplatin Increases Survival in Patients with Recurrent Malignant Glioma

Eric M Thompson, Edit Dosa, Dale F Kraemer, Edward A Neuwelt

INTRODUCTION

Bevacizumab plus irinotecan improves imaging responses and increases progression-free survival (PFS) and overall survival (OS) in patients with recurrent malignant glioma (WHO grade 3 or 4). However, the efficacy of bevacizumab plus carboplatin is not well characterized in this patient population. The primary objective of this study was to estimate OS, PFS, imaging responses, and toxicities of bevacizumab plus carboplatin for treatment of malignant glioma at first recurrence. The secondary objective was to estimate the agreement between postcontrast T1-weighted magnetic resonance images (MRI) and T2-weighted images.

METHODS

A retrospective analysis of nine patients who received bevacizumab (10 mg/kg IV) and carboplatin (AUC 5 IV) for recurrent malignant glioma (WHO grade 3 or 4) is presented. All patients received this regimen at first recurrence. Macdonald criteria employing both postcontrast T1- and T2-weighted images were used to determine progression and imaging responses.

RESULTS

The median age and Karnofsky performance score were 51 years and 70, respectively. Median PFS was not attained, but three patients were progression-free for 336 days or longer (95% CI, 94 to not attained). Six-month PFS was 44.4%. Median OS was not attained; however, five patients survived 301 days or longer, (95% CI, 136 to not attained). Six-month OS was 66.7%. On contrast-enhanced T1-weighted MRI, 55.6% had partial response (PR), and 44.4% had stable disease (SD). On T2-weighted MRI, 33.3% had PR, and 66.7% had SD. The agreement between contrast-enhanced T1-weighted and T2-weighted images was moderate (κ = 0.5714). There were three grade 3 and 4 toxicities, including hyponatremia and thrombocytopenia.

CONCLUSION

We conclude that the combination of bevacizumab plus carboplatin increases PFS and OS compared to historical data and has similar PFS, OS, and imaging responses compared to other bevacizumab plus chemotherapy regimens for recurrent malignant glioma. Agreement between postcontrast T1- and T2-weighted images is only moderate in the context of bevacizumab therapy.

Neuro-Oncol. 2009 Oct;11(5):626.

269. Levetiracetam Enhances P53-Mediated MGMT Inhibition and Sensitizes Glioblastoma Cells to Temozolomide

George C Bobustuc, Cheryl H Baker, Arati Limaye, Wayne D Jenkins, Nicholas George Avgeropoulos, Gary Pearl, Santhi D Konduri

INTRODUCTION

Antiepileptic drugs (AEDs) are frequently used to treat seizures in glioma patients. AEDs may have an underrecognized impact in modulating O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that has an important role in tumor cell resistance to alkylating agents. We report that levetiracetam (LEV) is the most potent MGMT inhibitor among several AEDs with diverse MGMT regulatory actions.

METHODS

Human glioblastoma cells were treated with levetiracetam, either alone or in combination with temozolomide or BCNU. Tumor cell proliferation was monitored. Protein expression patterns and mRNA message of MGMT and p53 were determined by Western blot and real-time RT-PCR analysis. Chromatin immunoprecipitation assays were performed to investigate the binding of p53 on the MGMT promoter and the modulatory role of the mSin3A/HDAC1 corepressor system. Immunohistochemical studies were performed to investigate the effect of levetiracetam on MGMT expression in glioblastoma patients.

RESULTS

Levetiracetam is the most potent MGMT inhibitor among several AEDs (phenytoin, valproic acid, and phenobarbital) and has diverse MGMT regulatory actions. In vitro, at concentrations within the human therapeutic range for seizure prophylaxis, levetiracetam significantly decreases MGMT protein and mRNA expression levels. Chromatin immunoprecipitation (ChIP) analysis reveals that levetiracetam enhances p53 binding on the MGMT promoter by recruiting the mSin3A/HDAC1 corepressor complex. However, levetiracetam does not exert any MGMT inhibitory activity when the expression of p53, mSin3A, or HDAC1 is abrogated. Levetiracetam inhibits malignant glioma cell proliferation and increases glioma cell sensitivity to the monofunctional alkylating agent temozolomide (TMZ). In four newly diagnosed patients who had two craniotomies 7–14 days apart, prior to initiation of any tumor-specific treatment, samples obtained before and after levetiracetam treatment showed inhibition of MGMT expression.

CONCLUSION

Our results suggest that the choice of AED in patients with malignant gliomas may have an underrecognized impact in clinical practice and research trial design.

Neuro-Oncol. 2009 Oct;11(5):626.

270. Patterns of Progression in Patients with Glioblastoma at First or Second Relapse Treated with Bevacizumab Alone or in Combination with Irinotecan in the Brain Study

Whitney B Pope, Qi Xia, Asha Das, Julie Hambleton, Hyun Kim, Matthew Brown, Jonathan Goldin, Timothy F Cloughesy

INTRODUCTION

In the phase II BRAIN study (ClinicalTrials.gov; NCT00345163) of patients with glioblastoma (GBM) at first or second relapse, bevacizumab (BEV) significantly improved objective response rate (ORR) and 6-month progression-free survival (PFS) compared to historical controls. Early observations raised a concern that recurrence following antiangiogenic therapies may be more invasive and diffuse than typically seen. Therefore, in the BRAIN study we analyzed patterns of GBM progression before and after BEV treatment.

METHODS

BRAIN, a phase II, open-label, multicenter, randomized, noncomparative trial of 167 patients, evaluated efficacy and safety of BEV alone (n = 85) and in combination with irinotecan (IRI) (n = 82). Primary efficacy end points were ORR and 6-month PFS. An independent neuroradiologist (WP) reviewed magnetic resonance imaging scans at baseline and time of progression, characterizing disease as local, distant, diffuse, or multifocal. Median PFS and overall survival (OS) were estimated by the Kaplan-Meier method.

RESULTS

In the BEV arm, among patients with postbaseline scans (n = 79), baseline tumor patterns prior to BEV treatment were local (n = 57; 72%), distant (n = 2; 3%), diffuse (n = 15; 19%), and multifocal (n = 5; 6%). Among patients in the BEV arm with disease progression on BEV treatment (n = 67), 55 (82%) had no shift in disease pattern, 11 (16%) shifted from local to diffuse, and 1 (2%) shifted from multifocal to diffuse. Of patients with local disease at baseline who later progressed (n = 48), 37 (77%) progressed locally, and 11 (23%) progressed diffusely. There was no significant difference in median PFS for patients with local disease at baseline who progressed locally (3.1 months) versus diffusely (3.7 months). Median OS was 9.3 months for both groups. Additionally, analysis of the BEV-IRI arm will be presented.

CONCLUSION

The majority of BEV-treated patients did not experience a change in tumor pattern at the time of progression. Patients with a progression-pattern shift from local to diffuse had similar efficacy outcomes compared with patients with a pattern of local progression.

Neuro-Oncol. 2009 Oct;11(5):626.

271. Corticosteroid use in Patients with Glioblastoma at First or Second Relapse Treated with Bevacizumab in the Brain Study

James Vredenburgh, Timothy F Cloughesy, Meghna Samant, Liang Fang, Asha Das, Henry Friedman; for the BRAIN Investigators

INTRODUCTION

Patients with glioblastoma suffer from symptomatic peritumoral edema requiring corticosteroids, which contributes to treatment morbidity. Corticosteroid discontinuation or reduction to physiologic levels is clinically beneficial and seldom occurs with ineffective therapies. We conducted an exploratory analysis of patterns of corticosteroid use in the BRAIN study (ClinicalTrials.gov; NCT00345163).

METHODS

BRAIN, a phase II, open-label, multicenter, randomized, noncomparative trial of glioblastoma at first or second relapse evaluated efficacy and safety of bevacizumab (BEV) alone (n = 85) and with irinotecan (IRI) (n = 82). Coprimary end points of objective response (OR) rate and 6-month (6m) progression free survival (PFS) were determined by an independent review facility. According to modified Macdonald criteria, patients with OR had stable or reduced corticosteroid doses. Longitudinal steroid dosing was recorded. For patients with baseline corticosteroid use, sustained reductions were defined as ⩾50% corticosteroid dose reduction for ⩾50% of the time during the course of treatment. Sustained reduction in corticosteroid dose by response and PFS status for patients with corticosteroid use at baseline was summarized.

RESULTS

OR rate (BEV 28.2% and BEV-IRI 37.8%) and 6-month PFS (BEV 42.6% and BEV-IRI 50.3%) were compelling. At baseline, 50.6% of patients receiving BEV and 52.4% receiving BEV-IRI took systemic corticosteroid for nonchemoprophylaxis. More than 75% of patients receiving BEV and >65% of patients receiving BEV-IRI without corticosteroid at baseline did not use corticosteroid postbaseline. When compared to baseline, responders (complete or partial response; BEV 57% and BEV-IRI 64%) and nonresponders (stable or progressive disease; BEV 17% and BEV-IRI 38%) had sustained corticosteroid reduction. Similarly, patients with PFS >6m (BEV 58% and BEV-IRI 86%) and PFS ⩽ 6 months (BEV 19% and BEV-IRI 28%) had sustained corticosteroid reduction.

CONCLUSION

In BRAIN, OR rate and 6-month PFS with BEV treatment were clinically compelling. A majority of patients had decreased corticosteroid use. Patients with OR or PFS > 6 months had the associated benefit of corticosteroid reduction.

Neuro-Oncol. 2009 Oct;11(5):626–627.

272. Bevacizumab for Active Brain Metastases from Non-Small-Cell Lung Cancer

Kevin C DeBraganca, Douglas E Ney, Mark G Kris, Craig P Nolan, Andrew B Lassman

INTRODUCTION

Bevacizumab is effective for the treatment of non-small-cell lung cancer (NSCLC) but has been contraindicated in patients with brain metastases because of potential intracranial hemorrhage (ICH). Although several trials are currently exploring the safety of bevacizumab in patients with inactive, previously treated brain metastases, the safety and efficacy in the treatment of active brain metastases is unknown. The Food and Drug Administration recently approved the use of bevacizumab in patients with progressive glioblastoma given the high response rate and low incidence of ICH.

METHODS

We retrospectively identified patients treated with bevacizumab for active brain metastases refractory to other therapies. Magnetic resonance imaging (MRI) scans performed at least 8 weeks after initiating bevacizumab were assessed for response.

RESULTS

Two patients were identified for analysis. Patient A (age 60) and patient B (age 77) each had two or more brain metastases in multiple lobes. In both patients, prior treatment for brain metastases included surgical resection, whole-brain radiotherapy, and temozolomide. Patient A previously responded to bevacizumab for extracranial metastatic disease but presented with brain metastases 11 months after discontinuing bevacizumab. In both patients, MRIs demonstrated decreased enhancing disease and improved peritumoral edema during bevacizumab, and corticosteroid dependence was reduced. Patient A improved clinically, and patient B stabilized. Currently, both patients are alive and without CNS progression 7+ and 5+ months after initiating bevacizumab. Neither experienced ICH. A pulmonary embolus developed in patient A after three doses of bevacizumab, and she continues treatment concurrently with low-molecular-weight heparin anticoagulation. One additional patient (not included in this analysis) has initiated bevacizumab.

CONCLUSION

Bevacizumab may be effective in the treatment of active brain metastases from NSCLC and deserves further study.

Neuro-Oncol. 2009 Oct;11(5):627.

273. Correlation Between P53 Expression Level and Progression-Free Survival in Low O6-Methylguanine-DNA Methyltransferase Expressing Glioblastoma Treated with Adjuvant Temozolomide Chemotherapy

Li Shouwei

INTRODUCTION

This study evaluated the role of p53 to clinical outcomes in low O6-methylguanine-DNA methyltransferase (MGMT) expressing glioblastomas (GBM) treated with temozolomide (TMZ) chemotherapy.

METHODS

The authors reviewed the clinical outcomes of 46 low MGMT expressing GBM patients who had undergone surgery, conventional local radiotherapy, and TMZ chemotherapy. Correlation between p53 expression levels and clinical outcomes were analyzed, using the univariate and multivariate Cox model.

RESULTS

Median progression-free survival (PFS) time was 315 days from the initiation of surgery. No correlation existed between p53 expression level and patient age, gender, Karnofsky performance status (KPS), or tumor resection extent. Patients with low p53 expression had a significantly improved PFS (360 days) compared to those with high expression (246 days) (p = 0.015). On both univariate and multivariate analyses, low p53 expression persisted as a significant, independent, favorable prognostic factor for PFS (p = 0.017).

CONCLUSION

Low MGMT expressing GBM patients with different p53 expression levels had statistically significant different PFS time when treated with TMZ chemotherapy. P53 may be an important stratification variable in future therapeutic trials of GBM.

Neuro-Oncol. 2009 Oct;11(5):627.

274. Antiangiogenic Therapy with Bevacizumab in Recurrent Malignant Gliomas: Analysis of the Response and Core Pathway Aberrations

Li Shouwei

INTRODUCTION

Bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor, has shown promising activity in recurrent malignant gliomas. We reported the treatment response for the combination of bevacizumab and chemotherapy in a series of six patients with recurrent malignant glioma and investigated the molecular alterations in cancer pathways using the surgical biopsies from these patients.

METHODS

Standard therapy with primary resection followed by adjuvant chemoradiotherapy had failed in all patients. Bevacizumab was administered at a dose of 10 mg/kg every 2 weeks. Concomitantly, four patients received temozolomide (50 mg/m2/day), one patient irinotecan (125 mg/m2 every 2 weeks), and one patient topotecan (1.2 mg/m2/day). Response to therapy was mainly determined by magnetic resonance imaging. The expression of Ras, phosphorylated mitogen activated protein kinase (p-MAPK), phosphorylated AKT (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) were semiquantitatively assessed by immunohistochemistry using surgical biopsies before the initial treatment.

RESULTS

Five of the six patients had a radiographic response. Three had complete response, and two had partial response. Only one patient had progressive disease. The 6-month progression-free survival (PFS) was 33%, and the median PFS was 15 weeks, with a range from 6 to more than 60 weeks. Of the three core pathways analyzed in this study, the Ras/MAPK and phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR pathways were more likely to be associated with the treatment response to bevacizumab. In two younger patients (age < 50 years) with complete response, simultaneous overexpression of p-MAPK, p-AKT, and p-mTOR might be the crucial feature.

CONCLUSION

Bevacizumab in combination with chemotherapeutic agents may be an effective strategy for patients with recurrent malignant glioma. Activated MAPK and AKT might be possible biomarkers for selecting suitable patients for this targeted therapy.

Neuro-Oncol. 2009 Oct;11(5):627.

275. High-Dose Methotrexate, Rituximab, and Temozolomide (M/R/T) for Patients with Primary CNS Lymphoma (PCNSL)

Elizabeth Gerstner, Fred Hochberg, Scott Plotkin, April Eichler, Tracy Batchelor

INTRODUCTION

Primary CNS lymphoma (PCNSL) is a rare form of non-Hodgkin lymphoma. The backbone of treatment is high-dose methotrexate (HD-MTX), but there remains debate about what additional chemotherapies should be added to HD-MTX.

METHODS

We evaluated patients with PCNSL treated at our hospital with combination methotrexate, rituximab, and temozolomide (M/R/T) at initial diagnosis or at relapse. Patients were treated in 28-day cycles as follows: HD-MTX (8 g/m2; dose adjusted based on creatinine clearance) on days 1 and 15; rituximab (375 mg/m2) on days 3, 10, 17, and 24; and temozolomide (150–200 mg/m2) on days 7–11. HD-MTX was given every 2 weeks until complete response (CR), and for two additional treatments followed by monthly treatments for 11 months. Rituximab was given weekly for 8 weeks, and temozolomide was given during induction and then for 6 months after CR. Magnetic resonance imaging of the brain was done every month to assess response.

RESULTS

Fifteen patients were treated from February 2006 to May 2009. Nine patients received M/R/T as first-line therapy at the time of initial diagnosis, and six received it at relapse. The median age of patients in the first-line group was 58 (range, 47–73 years) and 60 (range, 46–76 years) in the relapsed group. CSF cytology was atypical in 4 of 14 patients (three first-line; one relapse). For first-line therapy, there were eight CRs (median cycles to CR = 2.5) and one partial response (PR) (patient still in induction). For second-line therapy, there were three CRs (median cycles to CR = 4) and three PRs (one still in induction; one died of an unrelated heart attack mid-treatment). With a median follow-up of 8.9 months in the first-line group and 5.6 months in the relapse group, only two patients have progressed, one of whom had clinical progression after a PR. Treatment was well tolerated, with grade 3 transaminitis in one patient and grade 3 hematological toxicities in eight patients.

CONCLUSION

Combination M/R/T is well tolerated and resulted in a promising response rate, thus warranting further evaluation in large scale, prospective studies.

Neuro-Oncol. 2009 Oct;11(5):627.

276. Up-Front Chemotherapy For 1p/19q Codeleted Gliomas Followed by Second-Look Removal and/or Radiotherapy at Maximum Response

Hikaru Sasaki, Yuichi Hirose, Kazunari Yoshida, Takeshi Kawase

INTRODUCTION

In grade 2–3 gliomas, it has been reported that the presence of 1p/19q codeletion correlates with increased response to chemotherapy and survival regardless of histology.

METHODS

According to Keio protocol (February 2008), gliomas (grade 2–3) with 1p and 19q codeletion were treated with up-front chemotherapy regardless of histology. At maximum response, patients were reevaluated to see if effective removal was possible, and second-look removal was considered if near total removal seemed possible. Grade 3 tumors were irradiated after maximum response or after second-look removal, if performed.

RESULTS

Nineteen gliomas with codeletion have been treated (nine PR/MR; eight SD; two NE). There was no PD at 9 months after initiation of chemotherapy, and three tumors that did not undergo either second-look removal or radiotherapy showed local recurrence—two after PR (AO at 12 months; OD at 17 months) and one after SD (OA at 23 months). The recurrences occurred within the area that was to be resected if second-look removal was performed. Second-look removal was performed in two tumors at maximum response (DA; OA). In an AO, radiotherapy was given at maximum response. The three cases that underwent removal or radiotherapy at maximum response have been progression-free for 1 year 8 months, 3 years 2 months, and 2 years 3 months.

CONCLUSION

Up-front chemotherapy for 1p/19q codeleted gliomas followed by second-look removal and/or radiotherapy is among the therapeutic approaches making the most of predictive response to chemotherapy; it is worthy of being tested in a clinical trial.

Neuro-Oncol. 2009 Oct;11(5):627–628.

277. Lymphomatosis Cerebri (LC) Presenting with Orthostatic Hypotension (OH), Anorexia, and Paraparesis

Anjeni Keswani, Eileen Biggio, Sean Grimm

INTRODUCTION

Primary CNS lymphoma (PCNSL) usually appears as enhancing mass lesions on neuroimaging. In rare instances, PCNSL occurs as lymphomatosis cerebri (LC), a diffuse infiltrative variant without mass lesion or blood-brain barrier compromise. In all published cases, LC presented as a rapidly progressive dementia.

METHODS

Case report.

RESULTS

A 58-year-old woman presented with subacute onset paraparesis, orthostatic hypotension (OH), and an 80-lb weight loss. Her symptoms progressed over 6 months, and she developed headaches. Magnetic resonance imaging (MRI) of the brain revealed venous sinus thrombosis and nonenhancing foci of FLAIR hyperintensity in periventricular white matter (WM), subcortical WM, and pons. An MRI of the spine revealed enhancement of lumbar nerve roots. Cerebrospinal fluid suggested a malignancy. Cytology was negative despite multiple samples. Bone marrow biopsy, body CT, and body PET were unremarkable. She had subtle radiologic response but no clinical improvement following steroids. Two months later, she developed encephalopathy, quadriparesis, and progression of the WM lesions. She developed fluctuating hypotension and died. Systemic autopsy was unremarkable. Gross appearance of the brain and spinal cord was normal, without discrete mass. Microscopic inspection revealed diffuse infiltration of the brain by large B-cell lymphoma.

CONCLUSION

Instead of dementia (as in all prior published cases), this patient with LC presented with anorexia and OH, symptoms usually associated with systemic disease. Diencephalic infiltration is a CNS cause of anorexia. Entities such as lymphoma, leukemia, or histiocytosis should be considered in patients with weight loss and WM lesions. Rarely, OH has a CNS cause. Recognition that the brainstem WM lesions were tumor infiltration rather than chronic vascular disease may have prompted earlier diagnosis. LC has a variable presentation. A high index of suspicion is necessary to make the diagnosis. Early recognition is important since treatment can lead to prolonged survival or cure.

Neuro-Oncol. 2009 Oct;11(5):628.

278. Preclinical and Clinical Rationale for a Phase III Trial of Bevacizumab Combined with the Current Standard of Care in Patients with Newly Diagnosed Glioblastoma Multiforme (ND-GBM)

Olivier Chinot, Thibault de La Motte Rouge, Ali Zeaiter, Nicola Moore, Asha Das, Raji Swamy, Timothy F Cloughesy

INTRODUCTION

Glioblastoma multiforme (GBM) is characterized by microvascular proliferation and high vascular endothelial growth factor (VEGF) expression, which correlate with outcome. In preclinical models, monoclonal antibodies that target VEGF enhance tumor responses to radiation, with supraadditive effects observed, particularly in the U87 GBM model, under both normoxic and hypoxic conditions. In orthotopic glioma models, the addition of bevacizumab (Avastin) to temozolomide increases survival compared with either drug alone. In clinical trials involving patients with relapsed GBM, 6-month progression-free survival (PFS) and response rates with bevacizumab ± irinotecan compare favorably with historical controls. Toxicity is consistent with bevacizumab use in other indications, and no new safety signals were identified. These data provide a strong rationale for a prospective, randomized trial evaluating bevacizumab combined with the current standard of care in newly diagnosed GBM (nd-GBM).

METHODS

BO21990 (AVAGLIO) is a randomized, double-blind, placebo-controlled, global phase III trial that aims to recruit 920 patients. Patients ⩾18 years with histologically confirmed nd-GBM and WHO performance status ⩽2 are eligible. Initial treatment comprises radiation with concurrent oral temozolomide plus intravenous bevacizumab or placebo (10 mg/kg q2w) for 6 weeks. After a 28-day treatment break, patients receive temozolomide plus bevacizumab or placebo (10 mg/kg q2w) for six cycles. Patients continue on bevacizumab or placebo (15 mg/kg q3w) until disease progression or unacceptable toxicity. Coprimary end points are overall survival and PFS. Secondary end points are 1- and 2-year survival, health-related quality of life, and safety.

RESULTS

A summary of the preclinical and preliminary clinical data supporting the evaluation of bevacizumab in nd-GBM and BO21990 study design details will be presented.

CONCLUSION

A strong rationale underpins this phase III study that will evaluate the efficacy and safety of bevacizumab combined with the current standard of care in nd-GBM. This trial is sponsored by F. Hoffmann-La Roche Ltd.

Neuro-Oncol. 2009 Oct;11(5):628.

279. Initial Experience with Elective Discontinuation of Bevacizumab For Malignant Gliomas

Roy G Torcuator, Ravneet Thind, Joseph Anderson, Thomas Doyle, Randa Loutfi, Tom Mikkelsen

INTRODUCTION

Bevacizumab is a humanized monoclonal antibody targeting VEGF receptors. Although the results with bevacizumab therapy are promising, there are many unanswered questions, one of which is duration of antiangiogenic therapy.

METHODS

We retrospectively reviewed six patients with malignant gliomas who were treated with bevacizumab and irinotecan at Henry Ford Hospital from 2006 to 2009 and who electively stopped bevacizumab. Clinical data was gathered from the Hermelin Brain Tumor Database.

RESULTS

Six patients (three male, three female) were identified as having received bevacizumab and irinotecan and who electively discontinued treatment. Patients were diagnosed as having AO = 3, AA = 1, GBM = 1, and diffuse brainstem glioma = 1. The median duration of bevacizumab plus irinotecan treatment was 11.5 months (range, 5–12 months). The radiographic response rate on therapy was 100%, with five partial responses and one complete response. Three of the six patients discontinued treatment because of fatigue, and the remaining three patients electively requested to stop treatment as the tumor had shown sustained radiographic response. The patients are currently being followed clinically and with interval MRIs. The median time of follow-up off-treatment is 12 months (range, 4–16 months). Only one patient progressed, with new contrast enhancement noted at 4 months off bevacizumab. The patient was restarted on bevacizumab and irinotecan with reresponse noted in the follow-up MRI. The remaining five patients continue to be followed off-treatment with no new clinical or radiographic changes.

CONCLUSION

The two main reasons for discontinuing treatment are disease progression and unacceptable toxicities. However, it is difficult to address the issue of duration of treatment for patients who are clinically stable with ongoing sustained radiologic response. Furthermore, there is no published clinical experience with patients on bevacizumab who decided to electively discontinue treatment. Based on this small number of six patients, it may be safe to electively discontinue bevacizumab for patients with sustained radiographic response.

Neuro-Oncol. 2009 Oct;11(5):628.

280. Feasibility of Combined Treatment with Bevacizumab, Radiotherapy, and Temozolomide in Resectable and Unresectable Newly Diagnosed Glioblastoma Multiforme

Myra E van Linde, Joost JC Verhoeff, Lukas JA Stalpers, Jaap C Reijneveld, Dirk J Richel, Wouter Ralph Van Furth

INTRODUCTION

The current standard of care in first-line treatment for glioblastoma multiforme (GBM) is maximal resection when feasible, followed by irradiation plus concomitant and adjuvant temozolomide. Angiogenesis inhibition is a rational treatment strategy for GBM, given the characteristic high degree of endothelial proliferation and vascular permeability and the expression of proangiogenic growth factors, such as the vascular endothelial growth factor (VEGF). Remarkably high response rates have been shown in phase II studies using bevacizumab, a monoclonal antibody against VEGF, in combination with irinotecan in patients with recurrent GBM. In this study, we investigated the feasibility and safety of adding bevacizumab to the standard chemoradiotherapy in newly diagnosed GBM patients who had optimal resection or biopsy only.

METHODS

Patients received 30 fractions of 2 Gy irradiation concomitant with daily oral temozolomide (75 mg/m2) and intravenous bevacizumab (10 mg/kg) at days 1, 14, and 28, followed by 6 monthly cycles of temozolomide (150–200 mg/m2) on days 1–5.

RESULTS

Twenty primary GBM patients, 14 with an optimal resection, and 6 with a biopsy only, were included between November 2007 and May 2009. As of May 2009, seven patients completed the whole treatment and are in follow-up, seven patients are still receiving treatment, and treatment was prematurely stopped in six patients because of progression during the temozolomide cycles. In the group of six patients who underwent biopsy only, two completed the whole treatment. One of them still has ongoing stable disease and has been in follow-up since June 2008; the other patient developed progression during follow-up. No grade III/IV hematologic or nonhematologic toxicities were seen, and we found no differences in toxicities between the patients who underwent biopsy and the patients who underwent resection. Radiographic responses as well as the 6-month progression-free survival will be presented.

CONCLUSION

The combination of bevacizumab, radiotherapy, and temozolomide is safe and feasible in resectable and unresectable newly diagnosed GBM.

Neuro-Oncol. 2009 Oct;11(5):628–629.

281. Common Variable Immunodeficiency Disorders (CVID) May Predispose Patients to Low-Grade CNS Lymphoproliferative Disorders (LPD)

Pedro Ciarlini, Tamila L Kindwall-Keller, Robert J Maciunas, Mark L Cohen, Lisa R Rogers

INTRODUCTION

Patients with common variable immunodeficiency disorders (CVID) have poorly functioning B cells and low levels of IgG, IgA, or IgM. Not only are these patients more likely to have significant infections, but they may be predisposed to lymphoproliferative disorders (LPD). CNS involvement by low-grade LPD is rare. The majority of parenchymal CNS LPD are high-grade B-cell lymphomas.

METHODS

We retrospectively reviewed the clinical course of three patients—two with inflammatory myofibroblastic tumors (IMF), an inflammatory pseudotumor characterized by proliferation of polyclonal plasma cells, and one with an atypical lymphoplasmacytic infiltrate (ALPI). All three patients had CNS involvement during their clinical course.

RESULTS

Age at diagnosis ranged from 5 to 32 years. Initial tumor presentation was systemic in two patients, both with lung involvement and splenomegaly, and each developed CNS disease (1-year and 13-years later). The other patient presented with a right parietooccipital lesion and new lung lesion. Initial treatment was surgery with or without prednisone for the two patients with IMF. The patient with ALPI was treated with six cycles of rituximab-CHOP. This patient had a CNS relapse and failed to respond to one cycle of high-dose methotrexate (8 gm/m2) and two cycles of methotrexate, vincristine, procarbazine, and rituximab (one cycle was without procarbazine). The patient subsequently received whole-brain irradiation with a response. The two patients with IMF had an indolent course with no evidence of disease at 17 and 21 years. The patient (ALPI), with 2-year follow-up, is currently being evaluated for CNS recurrence. All patients had suspected immunodeficiencies; two with IgA deficiencies and one with a possible IgG4 deficiency.

CONCLUSION

CVID predisposes patients to LPD and other tumors. Patients with low-grade LPD or an atypical inflammatory proliferation of plasma cells in their CNS should be evaluated for immunodeficiencies.

Neuro-Oncol. 2009 Oct;11(5):629.

282. Progression of Neurofibromatosis Type 2-Related Meningiomas on Bevacizumab Therapy

Deva Sanjeeva Jeyaretna, Anat Stemmer-Rachamimov, Gordon J Harris, Wenli Cai, Xiaozhou Ma, Frederick George Barker, Emmanuelle di Tomaso, Scott Plotkin

INTRODUCTION

Meningiomas occur in >50% of patients with neurofibromatosis 2 (NF2) and comprise 30% of all brain tumors. Treatment results with surgery and radiation are good for sporadic meningiomas, but new options are needed for patients with multiple tumors or tumors that recur after standard therapy.

METHODS

We determined the expression pattern of vascular endothelial growth factor (VEGF) and its receptors VEGFR2, neuropilin 1 (NRP1), and neuropilin 2 (NRP2), and measured morphometric parameters of tumor vessels in paraffin-embedded tissues of 13 NF2-related and 13 sporadic meningiomas. “Positive” immunohistochemical staining was defined as 2+ or 3+ expression using a semiquantitative scale. We retrospectively reviewed the response of five intracranial meningiomas in five patients treated for NF2-related vestibular schwannomas using bevacizumab. Patients underwent magnetic resonance imaging (MRI) scans at months 1 and 3 and every 3 months thereafter. Radiographic response was defined as 20% or greater shrinkage and progressive disease as 20% or greater growth in volume, both compared to baseline.

RESULTS

VEGF was expressed in 24 of 26 (92%) meningiomas; VEGFR2 was expressed in 20 of 26 (77%) tumor vessels. Microvascular density, but not average perimeter or diameter, was significantly greater in sporadic meningiomas than in NF2-related meningiomas (52 vessels/mm2 vs. 32 vessels/mm2; t-test, p = 0.015). Before treatment, the median annual volumetric growth rate was 25% (range, 2% to 101%). The median best response to treatment was −11% volumetric reduction (range, −26% to 71%). All five tumors progressed during bevacizumab treatment after a median of 8.2 months (range, 4.6–13.5 months). One patient developed distant metastases to bone and liver from an atypical meningioma and subsequently died of progressive brain disease. The single tumor with objective radiographic responses progressed on the subsequent MRI scan.

CONCLUSION

Sporadic and NF2-related meningiomas express VEGF and VEGFR2, but durable responses to bevacizumab treatment were not seen in this small series of NF2 patients.

Neuro-Oncol. 2009 Oct;11(5):629.

283. International Retrospective Study of 1,0001 Adults with Anaplastic Oligodendroglial Tumors

Andrew B Lassman, Katherine S Panageas, Fabio M Iwamoto, Lisa M DeAngelis, Andreana L Rivera, Kenneth D Aldape, Timothy F Cloughesy, April Eichler, David N Louis, Nina Paleologos, Barbara Fisher, Lynn Ashby, Gloria B Roldan, Gregory Cairncross, Patrick Y Wen, David Schiff, Brandon G Rocque, H Ian Robins, Marc C Chamberlain, Warren Mason, Susan Weaver, Richard M Green, Francois G Kamar, Suresh Jhanwar, Marc K Rosenblum, Lauren E Abrey

INTRODUCTION

Treatment for anaplastic oligodendroglioma is controversial. Early results of randomized trials suggest procarbazine-lomustine-vincristine (PCV) chemotherapy (CT) before/after radiotherapy (RT) improves progression-free but not overall survival (OS) compared with RT alone. We sought to compare CT alone with CT and RT and temozolomide with PCV.

METHODS

We identified adults diagnosed with anaplastic oligodendroglioma/oligoastrocytoma from 1981 to 2007, capturing initial treatment strategy. Median time to progression (TTP) and overall survival (OS) were estimated by the Kaplan-Meier method and compared with the log-rank test.

RESULTS

There were 590 (56%) men and 456 (44%) women, with a median age of 42 years (range, 18–88 years), treated with RT alone (n = 201; 20%), CT and RT (sequential or concurrent; n = 538; 51%), CT alone (n = 205; 20%), or other strategies (n = 102; 10%). Median TTP and OS were 3 and 6 years, respectively, with 5-year median follow-up among 523 (50%) surviving patients. We found that 1p/19q codeletion (n = 311; 48% of 647 tested) correlated with longer OS (8.6 vs. 3.6 years; p < 0.0001) than no deletion (n = 246; 38%), the strongest predictor of OS in a multivariate model accounting for treatment, age, extent of resection, Karnofsky performance score (KPS), and histology. Median TTP was longer (p = 0.0002) following CT and RT than CT alone (4.0 vs. 2.8 years), but without improved OS (7.1 vs. 7.0 years), regardless of deletion status. In codeleted cases, median TTP was longer (p = 0.017) following PCV alone (7.6 years; n = 21) than temozolomide alone (3.2 years; n = 69), remaining significant when controlling for confounders; median OS was longer (10.4 vs. 7.1 years) but did not reach significance (p = 0.18) on early analysis (77% surviving).

CONCLUSION

Independent predictors of longer OS were 1p/19q codeletion, KPS, extent of resection, and AO histology. CT and RT lengthened TTP but not OS compared with CT alone. Among codeleted cases, median TTP was longer by >4 years following PCV alone than temozolomide alone, but had more toxicity and an unclear OS benefit on early analysis.

Neuro-Oncol. 2009 Oct;11(5):629.

284. Chemotherapy Treatment for Radiation-Induced Fibrosarcoma: Case Report and Review of the Literature

Daniela A Bota, Daniela Alexandru

INTRODUCTION

Secondary fibrosarcoma of the brain is an exceedingly rare tumor, occurring almost exclusively as a complication of therapeutic radiation to a preexisting intracranial tumor. These tumors tend to spread rapidly with extensive involvement of the optic nerves, hypothalamus, and brainstem, which makes total surgical resection impossible. Current treatment options encompass subtotal resection attempts, Glia-Site brachytherapy system, and chemotherapy with classical chemotherapy agents such as cisplatin, etoposide, and adriamycin. The results of the current therapy are disappointing, with very limited survival.

METHODS

Case report and review of the literature.

RESULTS

We report the case of a 48-year-old man who presented with a brainstem lesion 20 years after he received radiation therapy for a pituitary tumor. A subtotal resection of the new tumor was performed. Microscopical analysis of the specimen revealed spindle cell proliferation, with areas positive for vimentin. We chose to treat the patient with cyclophosphamide and imatinib chemotherapy, which the patient tolerated with no side effects. He was followed clinically and with serial imaging over a period of 6 months. During this time his disease was stable. The review of the literature shows that radiation-induced fibrosarcoma of the brain appears after a latency period of 2.5–20 years. Surgical treatment is very limited, especially when the tumor is in highly eloquent areas. We found only four case reports of using chemotherapy for the treatment of secondary fibrosarcomas of the brain. The progression-free survival of the patients described in these case reports were similar to the one described by us. However, the drugs previously used had potentially more side effects and impact on the patient’s quality of life.

CONCLUSION

Postradiation treatment of fibrosarcoma is not well defined, and this severely impacts patient prognosis. New research on the molecular pathology of radiation-induced brain fibrosarcoma and more targeted therapies are currently needed.

Neuro-Oncol. 2009 Oct;11(5):629–630.

285. Outcome in Adults with Medulloblastoma Treated with Modified Protocol POG 9031

Pamela Z New, Farhat Shaheen, Ching Lau

INTRODUCTION

Adult medulloblastoma is a rare tumor (1% of brain tumors.) It has been observed that this tumor in adults, though histologically the same, behaves differently than the pediatric counterpart. Survival may also be longer (65–84% at 5 years). Adjuvant chemotherapy seems to prolong survival compared to radiation and surgery alone. No preferred treatment regimen exists for this disease in adults as its rarity has precluded adequate trials.

METHODS

From 1991 to 2008, eight patients (two female, six male), ranging in age from 19 to 36, underwent treatment on the RT1 arm of POG 9032 with minor changes. All patients underwent gross total/near total tumor resection followed by craniospinal radiotherapy totaling 5,000 cGy to the tumor bed and 3,800 cGy to the spinal axis. At the conclusion of radiation, patients received adjuvant chemotherapy of cisplatin and etoposide at 100 mg/m2 for four cycles followed by cyclophosphamide (1,000 mg/m2) and vincristine (2 mg) monthly for eight cycles.

RESULTS

One patient stopped therapy after the second course of treatment and died 9 months postdiagnosis. One patient continues on treatment. Seven of eight patients experienced grade 3 or 4 neutropenia or thrombocytopenia, requiring delay in treatment or dose reduction. Thus far, the 5-year survival rate on this regimen is 75%. Five patients remain progression free.

CONCLUSION

Adults diagnosed with medulloblastoma have been treated with various pediatric protocols over the years. Hematologic toxicity has always been problematic when following craniospinal radiation with chemotherapy. Modified POG 9031 appears to be effective in this patient population, although the cohort is small. Significant toxicity impacted quality of life. A look at newer forms of treatment, targeted therapy, and DNA repair mechanisms is warranted and should involve a dedicated consortium for adequate patient accrual.

Neuro-Oncol. 2009 Oct;11(5):630.

286. Ventriculolumbar Perfusion Therapy for the Treatment of Leptomeningeal Carcinomatosis: A Pilot Study with Pharmacokinetic Data

Ho-Shin Gwak, Sang Hoon Shin, Heon Yoo, Ji Yeon Han, Heung Tae Kim, Jin Soo Lee, Seung Hoon Lee

INTRODUCTION

Conventional intraventricular methotrexate (MTX) via Ommaya reservoirs prolonged patient survival but failed to improve morbidity from leptomeningeal seeding (LMS). We adopted ventriculolumbar perfusion (VLP) therapy for the purpose of improving LMS symptoms and obtaining pharmacokinetic data to set up a sophisticated protocol.

METHODS

Basic techniques of VLP therapy are the same as previously reported. Briefly, prewarmed artificial CSF is infused via a preinstalled chemoport at Kocher’s point. A steady CSF flow throughout the lumbar drainage was confirmed overnight before intraventricular MTX injection. MTX (20 mg) was injected into the ventricle via the chemoport per 12 h for 3 consecutive days. The MTX level was checked at CSF from the lumbar drainage. The cytology, tumor marker, ventricular pressure, and LMS-related symptoms were monitored throughout and 2 weeks after treatment. All patients agreed to be enrolled into the experimental protocol. The side effects were assessed in terms of Common Terminology Criteria for Adverse Events (CTCAE, version 3.0).

RESULTS

Between October 2008 and March 2009, 12 cytologically proven LMS patient who had LMS-related symptoms were enrolled in the study. Among nine patients who had increased ventricular pressure (> 15 cm H2O), seven patients showed normalized ventricular pressure and improved headache (78%) after VLP. Five patients had cauda equina involvement (voiding difficulty, etc.) and were immobilized. Three patients did not experience voiding difficulty, and two patients were able to walk after VLP. Cytologic negative conversion was achieved in three patients (33%). The peak MTX concentration in CSF was 134.4 (±60.6) μM at 90 min. The peak serum concentration at 90 min was < 0.5 μM. All patients suffered from disturbance of the normal sleep cycle, but grade 3 insomnia occurred in only one patient. Two cases of delirium/confusion were noticed in a patient whose peak MTX concentration > 300 μM.

CONCLUSION

The improved VLP techniques enhanced both intraventricular accessibility and stability for perfusion. The apparent VLP-related complications appeared to be related to the peak MTX concentration, which is affected by not only the amount of MTX injected but also by the steady CSF flow. The improvements of pre-VLP LMS-related symptoms encourage us to launch a sophisticated phase I clinical trial.

Neuro-Oncol. 2009 Oct;11(5):630.

287. The Role of Demographic and Genetic Factors in Temozolomide-Related Severe Myelotoxicity

Ravneet Thind, Roy G Torcuator, Thomas Doyle, Joseph Anderson, Randa Loutfi, Ali Haythem, Sandy Remer, Lonni Schultz, Tom Mikkelsen

INTRODUCTION

Glioblastoma has a median survival of 12–14 months despite treatment with maximal surgical resection, radiation, and chemotherapy. Hegi et al. reported a survival benefit in patients treated with radiation and temozolomide (TMZ) with methylated MGMT. TMZ-related grade 3 or 4 myelosuppression is relatively uncommon, with an overall incidence of 4% to −8%. The relationship between MGMT status and TMZ-induced myelosuppression has not been defined.

METHODS

We conducted a retrospective review to define the incidence, duration, demographic, and genetic factors related to TMZ-induced pancytopenia. Of 500 adult glioma patients at our institution who received TMZ, 25 (5%) developed severe (grade 3 or 4) myelosuppression.

RESULTS

Of the 25 patients who developed severe myelosuppression, 14 (56%) were female, and 17 (68%) were older than 50 years of age. The rate of myelosuppression was higher in females than in males (8% vs. 3.4%; p = 0.032, Fisher’s exact test). The median body surface area was 1.96 m2 (range, 1.47–2.48 m2). MGMT status was known for 13 patients, five were methylated and eight were not. For patients with MGMT methylation, the median recovery time was 12 days (n = 5; range, 7–122 days) compared to 35 days (n = 8; range, 25–98 days) for patients with unmethylated MGMT. This difference was not statistically significant (p = 0.42, Wilcoxon two-sample test).

CONCLUSION

We have described demographic factors associated with severe myelosuppression at our institution. The majority of the patients were female and older than 50 years of age. This data does not show an association between MGMT status, cumulative dose, or duration of myelosuppression. Thus, the effect of MGMT status on incidence of myelosuppression remains undetermined. Updated results will be presented. While relatively uncommon, severe myelotoxicity is a concern in select patients as it may cause significant treatment delays and should be considered when contemplating empirical dose escalations or duration of TMZ. MGMT status may not discriminate those patients at risk.

Neuro-Oncol. 2009 Oct;11(5):630.

288. Prognosis and Response to Treatment of Pleomorphic Xanthoastrocytoma with Anaplastic Features

Denise M Damek, Kevin O Lillehei, Bette K Kleinschmidt-DeMasters

INTRODUCTION

Pleomorphic xanthoastrocytomas (PXA) are rare, WHO grade II tumors that demonstrate a relatively favorable clinical course. PXA with anaplastic features (PXA-A) designates a subset with more aggressive histopathologic features of increased mitotic activity, necrosis, microvascular proliferation, and marked cellular anaplasia that has yet to be assigned a formal WHO grade. These tumors are thought to manifest poorer prognosis, although few studies have documented response to treatment and clinical outcome.

METHODS

An institutional review board-approved retrospective case series studied patients with PXA-A accrued at the University of Colorado hospital over the past 10 years.

RESULTS

Ten patients (four female; six male), ages 18–68 (median, 34.5 years), were identified. All were treated as high-grade gliomas with surgery, external beam irradiation, and chemotherapy, except for one patient who refused brain irradiation and chemotherapy. PXA-A occurred de novo in all but one patient. Median survival exceeded 39 months (range, 14–108+ months). Four patients succumbed to progressive PXA-A at 19, 20, 20, and 23 months after diagnosis; one of these patients had refused treatment beyond surgical resection. Another patient withdrew care after an unrelated medical illness. Two patients died without evidence of disease 14 and 150 months after diagnosis (the latter patient from radiation-induced CNS sarcoma). Four patients are alive without evidence of active disease 56–108 months after diagnosis (median, 95.5 months). We identified no specific histological feature, imaging characteristic (location or cyst formation), or treatment factor (extent of resection or chemotherapy regimen) predictive of outcome. In this small patient cohort, age over 50 was associated with poorer prognosis.

CONCLUSION

PXA-A does not portend a universally poor prognosis. Malignant glioma treatment regimens provided durable disease control in the majority of our patients.

Neuro-Oncol. 2009 Oct;11(5):630.

289. Reresponse to Bevacizumab Treatment for Recurrent Malignant Gliomas Previously Treated with Bevacizumab-Based Regimen

Ravneet Thind, Roy G Torcuator, Thomas Doyle, Joseph Anderson, Tom Mikkelsen

INTRODUCTION

Glioblastoma multiforme (GBM) carries a dismal prognosis despite the current standard of multimodality treatment. Recent studies have shown promising results with bevacizumab and irinotecan for recurrent GBM. However, patients ultimately progress and rarely respond to a second bevacizumab-containing regimen. Here we report three patients with recurrent malignant glioma who showed reresponse to bevacizumab.

METHODS

We reviewed the records of three patients at Hermelin Brain Tumour Centre who showed reresponse to bevacizumab.

RESULTS

Case 1 was a 69-year-old male with left occipital GBM, treated with radiation and temozolomide and then started on bevacizumab and irinotecan for progression after 2 years with initial response. Upon further progression, chemotherapy was changed to temozolomide initially and then to BCNU with maintenance of bevacizumab with note of major partial response. Case 2 was a 22-year-old female with right thalamic diffuse fibrillary astrocytoma treated with EBRT and temozolomide. She had early progression and was started on bevacizumab and irinotecan with major response; hence, treatment was discontinued after 1 year. In 6 months, she progressed again, and bevacizumab and irinotecan were reinitiated with note of partial response. Case 3 was a 50-year-old male with left parietooccipital anaplastic oligodendroglioma treated with EBRT and temozolomide initially. Progression was noted after 3 years; hence, bevacizumab and irinotecan were started with initial response. Upon further progression, he received fractionated stereotactic radiotherapy and ANG1005. Therapy was again changed to procarbazine and CCNU, and later bevacizumab was reinitiated with temozolomide for progressive disease and partial response was noted.

CONCLUSION

The optimal chemotherapeutic agent to combine with bevacizumab is unknown. The treatment options for patients who progress on the initial bevacizumab regimen are limited. Hence, the common practice is to maintain bevacizumab and change the chemotherapy. However, the response rate is dismal with very short time to progression. We therefore present our experience with reresponse to bevacizumab with chemotherapy.

Neuro-Oncol. 2009 Oct;11(5):631.

290. Bevacizumab Plus Temozolomide for Recurrent Malignant Glioma: Update

Michael A Badruddoja

INTRODUCTION

Bevacizumab (BEV) is a monoclonal antibody targeting vascular endothelial growth factor receptor (VEGF-R) that has recently been FDA approved as monotherapy for the treatment of recurrent glioblastoma multiforme. Bevacizumab acts synergistically with chemotherapy in the treatment of other solid tumors, such as colorectal, breast, lung, and renal cell carcinoma. Temozolomide (TMZ) is an oral alkylating agent with activity in the treatment of malignant glioma.

METHODS

This study evaluates the efficacy, toxicity, and radiographic changes associated with the use of BEV plus TMZ in 13 patients with malignant glioma.

RESULTS

Data to be reported will represent 30-month follow-up.

CONCLUSION

Bevacizumab plus Temodar is a well-tolerated regimen for patients with recurrent malignant gliomas. Radiographic response rates are very promising. Survival data for this group of patients will be reported as 30-month follow-up. Alternate dosing regimens of temozolomide may improve survival when added to bevacizumab.

Neuro-Oncol. 2009 Oct;11(5):631.

291. Rapid Response of Bevacizumab in Adult Progressive Pilocytic Astrocytomas of Brainstem

Rebecca Naor, Jeremy Rudnick, Joanna Wilson, Surasak Phuphanich

INTRODUCTION

Pilocytic astrocytomas are categorized as WHO grade I tumors; they represent 5% of gliomas and are usually found in children and young adults. As a variant of low-grade astrocytomas, they rarely undergo malignant transformation. The definitive treatment for pilocytic astrocytoma is surgical resection. There is evidence for treatment with radiation for nonsurgical candidates, postoperative residual tumors, and recurrent pilocytic astrocytomas. The role of chemotherapy is controversial.

METHODS

We describe a case of a 55-year-old right-handed male who presented with left-sided ptosis, facial numbness, balance disturbance, and facial asymmetry. Magnetic resonance imaging (MRI) demonstrated an enhancing lesion in the left brainstem extending into the pons and cerebral peduncle. He underwent a left retrosigmoid biopsy. Pathology demonstrated pilocytic astrocytoma grade I with atypical features and an elevated Ki-67. Since he was not a surgical candidate, his pathology demonstrated atypical features, and he was clinically symptomatic, we treated the patient with radiation and temozolomide (TMZ) for 6 weeks and four cycles of adjuvant TMZ. His 6-month follow-up MRI demonstrated increasing enhancement, with T2/FLAIR changes in the brainstem consistent with tumor progression. Clinically he developed dysphagia, third nerve palsy, and jaw deviation.

RESULTS

The patient was started on off-label bevacizumab (10 mg/kg) every 2 weeks and TMZ (200 mg/m2/day) for 5 days every 28 days. Two weeks after the first dose, MRI demonstrated approximately 70% decrease in enhancement, FLAIR signal, partial tumor response, and improved dysphagia and balance.

CONCLUSION

Bevacizumab has not been used to treat pilocytic astrocytomas, but these highly vascular tumors may benefit from antiangiogenic therapy, as seen is this case. This is the first case demonstrating significant benefit from bevacizumab treatment in correlation with clinical improvement. We will continue this treatment regimen.

Neuro-Oncol. 2009 Oct;11(5):631.

292. Phase I Study of Metronomic Temozolomide and Arterial Spin Labeling Perfusion Measurements for Recurrent Glioblastoma Multiforme

Eric T Wong, Loretta Barron, Julianne Bloom, David Hackney, David Alsop

INTRODUCTION

Metronomic temozolomide (TMZ) has an antiangiogenic effect in animal models. However, its toxicity and efficacy in patients with malignant gliomas are unknown. Therefore, we conducted a phase I study of metronomic TMZ for malignant gliomas and measured tumor perfusion by arterial spin labeling (ASL) magnetic resonance imaging (MRI).

METHODS

Using a 3+3 dose escalation schema, subjects with recurrent glioblastoma multiforme (GBM) and anaplastic glioma (AG) were treated with daily TMZ at 25 mg/m2/day or 50 mg/m2/day. Entry criteria included normal laboratory profiles, Karnofsky performance status of 60 or greater, and nonenzyme-inducing anticonvulsants. Each cycle consisted of 4 weeks of daily TMZ, and ASL studies were performed every 8 weeks. Response was evaluated according to Macdonald’s criteria.

RESULTS

Three subjects with recurrent GBM were enrolled in the 25 mg/m2/day cohort while another three were enrolled in the 50 mg/m2/day cohort. The recurrent AG arm is still enrolling patients. No clinically significant treatment-related toxicity, including opportunistic infection, was encountered. Two subjects had a grade 1 leukopenia, but their TMZ treatment was not interrupted. One subject had CD4/CD8 < 1.0 at baseline, but the others had CD4/CD8 ranges from 1.1 to 2.7 at baseline and throughout treatment. The six subjects completed 1–6 cycles of metronomic TMZ. Four had progressive disease after one cycle, while one had stable disease after 4+ cycles and another had a partial response after six cycles. One had markedly decreased tumor perfusion on ASL despite partial response on MRI. All had increased ASL perfusion at the time of disease progression.

CONCLUSION

Metronomic TMZ is well tolerated in our GBM cohort. ASL perfusion measurement may be a more sensitive marker of treatment response and disease progression than gadolinium-enhanced T1 images. Further studies are needed to determine the efficacy of metronomic TMZ for GBM.

Neuro-Oncol. 2009 Oct;11(5):631.

293. Bevacizumab and Chemotherapy for Recurrent Heavily Pretreated Low-Grade Gliomas

Phioanh Nghiemphu, Mimie Tran, Albert Lai, Timothy F Cloughesy, Richard M Green

INTRODUCTION

Bevacizumab is a monoclonal antibody to vascular endothelial growth factor that has been shown to be effective in treating recurrent malignant gliomas and is now approved for the treatment of recurrent glioblastoma. Its effectiveness has not been studied for treatment of low-grade gliomas, although some activities in low-grade as well as high-grade oligodendrogliomas have been demonstrated.

METHODS

We conducted a retrospective analysis of 24 consecutive patients with low-grade gliomas treated with bevacizumab and chemotherapy after failed treatment with radiotherapy and temozolomide chemotherapy. We measured progression-free survival (PFS) and response on magnetic resonance imaging (MRI) in both contrast-enhancing (CE) and noncontrast-enhancing (NCE) tumors.

RESULTS

There were 14 low-grade oligodendrogliomas (LO), 3 low-grade mixed gliomas (LMG), and 7 low-grade astrocytomas (LA). These patients were treated with bevacizumab at 5–10 mg/kg every 2 weeks in combination with chemotherapy, including irinotecan (14), carboplatin (7), and lomustine (3). The median age was 47 (range, 26–68 years). All patients had CE tumors on progression, and 19 patients also had NCE tumor growth. The response rate was 63%, but response was mainly for CE tumors and edema. Only two patients had a clear NCE tumor response. The median PFS was 158 days, and 6-month PFS was 38%. Only one patient discontinued therapy because of an intracranial hemorrhage, and this patient had an LO.

CONCLUSION

Overall, bevacizumab has some activity in treating heavily pretreated, recurrent low-grade gliomas, similar to previous data seen with bevacizumab treatment for recurrent malignant gliomas.

Neuro-Oncol. 2009 Oct;11(5):631–632.

294. Reversible Nodular Enhancement Following Resection with Biodegradable Carmustine (BCNU) Wafer Placement Followed by Vaccination with Dendritic Cells Pulsed with Tumor Lysate for Patients with Newly Diagnosed Glioma

Jeremy D Rudnick, Surasak Phuphanich, Rebecca Naor, Anne Luptrawan, Miriam Nuno, Franklin G Moser, Ray M Chu, Keith L Black, John S Yu

INTRODUCTION

Our prior immunotherapy trials demonstrated efficacy in generating a tumor-specific immune response in malignant glioma and potential for high tumor-specific toxicity and sustained tumoricidal activity. Immunotherapy may synergize with chemotherapy, and biodegradable carmustine (BCNU) wafers extend overall survival from 11.6 to 13.9 months. This is a subset analysis of imaging findings from our vaccine trial. Postsurgical nodular enhancement is well defined after BCNU wafer placement, but the natural history of this enhancement is yet to be elucidated. This relationship has not been correlated with O6-meth-ylguanine methyltransferase (MGMT), a predictor of pseudoprogression, or studied in the absence of cytotoxic chemotherapy.

METHODS

Patients with high-grade glioma histology were studied after resection (no residual enhancement) and biodegradable BCNU wafer placement. Screening leu-kapheresis was used to isolate mononuclear cells, which were differentiated into dendritic cells, pulsed with tumor lysate, and then three intradermal vaccines were administered at 2-week intervals. Magnetic resonance imaging (MRI) was obtained preoperatively, < 24 h postoperatively, following concurrent chemoirradiation, and postvaccination, per standard protocol. Immunohistochemistry was performed with MGMT analysis.

RESULTS

Six patients underwent surgery between May 2007 and September 2008; mean age was 58 (range, 26–74 years). Notably, 66% (4/6) developed nodular enhancement within 1 month after completion of chemoirradiation. The mean duration for the resolution of enhancement was 11 weeks (range, 4–18 weeks). MGMT expression was elevated (>20%) in one of four of the nodular enhancing patients and one of two patients without enhancement.

CONCLUSION

Although not powered for statistical significance, nodular enhancement is common after BCNU wafer placement and chemoirradiation. This may be a distinct phenomenon from pseudoprogression with specific imaging characteristics and may be mistaken for tumor progression. There is no correlate with MGMT status. Resolution of enhancement occurs within 3 months and prior to the initiation of chemotherapy. The natural history and radiographic features will be presented.

Neuro-Oncol. 2009 Oct;11(5):632.

295. Phase I Trial of Arsenic Trioxide (ATO) and Temozolomide (TMZ) in Combination with Radiation Therapy (RT)

Sean Grimm, Maryanne Marymont, James P Chandler, Kenji Muro, Steve Newman, Robert M Levy, Jeff Raizer

INTRODUCTION

Arsenic trioxide (ATO) causes G2/M arrest and cell death in glioma cell lines and is synergistic with radiation therapy (RT) in animal models. The study objective was to determine the safety and maximum tolerated dose (MTD) of ATO in combination with temozolomide (TMZ) and RT.

METHODS

A standard 3+3 dose escalation study was performed to determine MTD and dose-limiting toxicity (DLT). Newly diagnosed glioblastoma multiforme (GBM), anaplastic astrocytoma (AA), or anaplastic oligoastrocytoma (AOA) patients were enrolled. All received daily ATO, 1–2 h prior to RT for 5 days during the first week, then twice weekly until completion of RT. All received TMZ daily for 42 days. The dose levels (DL) were (1) TMZ 60 mg/m2/ATO 0.2 mg/kg; (2) TMZ 75 mg/m2/ATO 0.2 mg/kg; and (3) TMZ 75 mg/m2/ATO 0.25 mg/kg. All patients received RT 5,940–6,120 cGy in 28–33 fractions. DLT was determined by toxicity measurements, using the National Cancer Institute’s CTCAE v3, from enrollment until 3 weeks after completing RT.

RESULTS

Seventeen patients (13 GBM, 2 AA, 2 AOA) were accrued. Of these, 65% were men, and the median age was 52 (range, 25–80 years). Median Karnofsky performance score (KPS) was 90% (range, 70–100%). DL-1 included two additional patients because two received TMZ incorrectly (5 days per week instead of 7). One DLT occurred at DL-2 (grade 4 transaminase elevation), and two DLTs occurred at DL-3 (grade 4 neutropenia and grade 3 QTc prolongation). Grade 3 toxicities included leukopenia (2), thrombocytopenia (2), anemia (1), and QTc prolongation (1). Grade 4 toxicities included leukopenia (1) and liver transaminase elevation (1). MTD was ATO 0.2 mg/kg and TMZ 75 mg/m2.

CONCLUSION

ATO in combination with TMZ and RT is safe and well tolerated. The MTD is ATO 0.2 mg/kg and TMZ 75 mg/m2.

Neuro-Oncol. 2009 Oct;11(5):632.

296. Long-Term Follow-Up of Pituitary Carcinoma Treated with Combination Chemotherapy using Temozolomide and Carboplatin

Erik C Parker, Roberto Steven Zaninovich, David L Kleinberg, Robert E Elliott, Stephen Rush, David Zagzag, Michael L Gruber

INTRODUCTION

Pituitary carcinoma is an exceedingly rare disease with historically poor response to treatment. Recent reports have demonstrated response of aggressive pituitary adenomas to treatment with temozolomide chemotherapy. We present a case with a nearly complete response and long-term control of pituitary carcinoma treated with temozolomide in combination with carboplatin.

METHODS

The patient is a 68-year-old female who has a recurrent prolactin-producing pituitary carcinoma that was found to be refractory to medical management. She had undergone three prior resections of the primary sellar lesion as well as external beam radiation. CNS metastasis was documented by biopsy of a posterior fossa nodule. Magnetic resonance imaging (MRI) demonstrated progression of the sellar tumor as well as two additional metastatic lesions involving the brainstem and the right L1 nerve root. She experienced significant visual loss as well as right lower extremity radiculopathy. She underwent treatment with six cycles of chemotherapy using temozolomide in combination with carboplatin. These 28-day cycles consisted of treatment with carboplatin (dose of AUC = 6) on day 1 followed by temozolomide (150 mg/m2/day) on days 2–6, with the patient off treatment for the remainder of the cycle.

RESULTS

The patient tolerated the chemotherapy regimen well and suffered no morbidity related to treatment. She experienced resolution of her radicular pain. Vision has remained stable. MRI reveals nearly complete response of all three tumor sites. Follow-up over 27 months has demonstrated no tumor progression.

CONCLUSION

This patient with pituitary carcinoma has had a dramatic response to this chemotherapy regimen and has experienced relatively long-term tumor control. This report adds to the experience of similar cases treated with temozolomide alone that have recently been reported. The use of temozolomide in combination with carboplatin should be considered in the treatment of pituitary carcinoma.

Neuro-Oncol. 2009 Oct;11(5):632.

297. Updated Results of Phase II Trial of Bevacizumab in Combination with Temozolomide and Regional Radiation Therapy For Upfront Treatment of Patients with Newly Diagnosed Glioblastoma Multiforme

Albert Lai, Phioanh Nghiemphu, Richard M Green, Liz Spier, Scott Peak, Surasak Phuphanich, Lou Fehrenbacher, Tatjana Kolevska, Jonathan A Polikoff, Timothy F Cloughesy

INTRODUCTION

Bevacizumab (BV) is a humanized monoclonal antibody directed against the vascular endothelial growth factor (VEGF) that has recently gained FDA approval for treatment of recurrent glioblastoma (GBM). We are conducting a 70-patient phase II trial to determine the safety and efficacy of BV combined with standard of care radiation (RT) and temozolomide (TMZ) for newly diagnosed GBM.

METHODS

Newly diagnosed GBM patients with no prior treatments are eligible. All patients are required to have resections to ensure that frozen tissue (>200 mg) was collected. Therapy begins between 3 and 6 weeks of surgery with BV (10 mg/kg every 2 weeks), TMZ (75 mg/m2 daily), and external beam RT (30 × 200 Gy) administered on the same day. After completion of RT, patients are placed on a maintenance phase of BV (10 mg/kg every 2 weeks) and TMZ (150–200 mg/m2 for 5 days out of every 28 days) until progression or 24 months; patients are then maintained on BV only. Primary outcome measure is overall survival; the secondary outcome measure is progression-free survival.

RESULTS

Seventy patients were enrolled between August 2006 and November 2008 at UCLA and Kaiser Permanente (KP) (Northern and Southern California). The median age was 57 years (range, 31–76 years). MGMT promoter methylation was detected in 30 of the 70 (43%) patients. Fifty of the 70 patients are off study. To date, severe adverse events have included ischemic stroke, pulmonary embolus, wound breakdown, gastrointestinal bleeding/perforation, and renal dysfunction. Patients with MGMT promoter methylation have improved outcome with this regimen. Preliminary TTP by Kaplan-Meier analysis compared to that of a UCLA/KP control group receiving a conventional RT/TMZ regimen indicated that BV provides significant benefits in progression-free survival, particularly in RPA class V versus class III/IV patients.

CONCLUSION

Addition of BV to the standard regimen of TMZ and RT for newly diagnosed GBM is tolerable and shows promising efficacy as measured by PFS. More detailed safety, survival, and correlative tissue analysis will be presented.

Neuro-Oncol. 2009 Oct;11(5):632.

298. Neurolymphomatosis as Initial Presentation of Primary CNS Lymphoma (PCNSL)

Eugenio Guevara

INTRODUCTION

Primary CNS lymphoma (PCNSL) in immunocompetent hosts represents 4–7% of newly diagnosed primary CNS tumors, and its incidence is increasing. Neurolymphomatosis (NL), the infiltration of nerve roots or peripheral nerves by lymphoma cells, is a very rare and distinct neurological complication of lymphoma. PCNSL and NL coexist in about 10% of cases. The most common pattern of NL is painful nerve/root involvement, but cervical radiculopathy or brachial plexopathy are very rare.

METHODS

A case report.

RESULTS

A 57-year-old fisherman had acute neck pain irradiated to shoulders and arms with occasional cramps. In less than 24 h, he developed bilateral and asymmetric weakness of the shoulder girdle, progressing to complete paralysis of the left arm and shoulder. The patient had a history of previously treated bladder cancer and was an active smoker. He experienced weakness in his right leg 2 days before admission. His general physical exam was normal, including normal mental status and cranial nerves. He had motor deficits referable to cervical roots C4–C7, with bilateral atrophy of deltoid muscles, hyporeflexia, an absent left pectoralis reflex, and weakness 3/5 in flexion/extension of right hip and knee. A sensory exam showed deficits referable to the left cervical roots above. All laboratory data were normal or negative; CSF results were protein 321 and negative cytology. Magnetic resonance imaging (MRI) of the brain demonstrated multiple contrast enhancing lesions around the fourth ventricle and lateral ventricles, and leptomeningeal enhancement on MRI cervical spine. MRI of brachial plexus was normal. Stereotactic brain biopsy confirmed PCNSL. Treatment with intravenous methotrexate (3.5 g/m2) was followed within 15 days by improvement in neurologic function.

CONCLUSION

We present here an immunocompetent patient whose initial symptoms and peripheral deficits led to a diagnosis of PCNSL. This pathway to diagnosis of PCNSL is exceptional.

Neuro-Oncol. 2009 Oct;11(5):632–633.

299. Primary Amyloidoma of the Brain

Manmeet Ahluwalia, Atul Sofat, P Kanneganti, L Mechtler

INTRODUCTION

Cerebral amyloidoma (CB) is a term used for the tumor-like deposition of fibrillar protein with cross-beta sheet conformation in the brain parenchyma. CB is extremely rare, and only 27 cases have been reported in the literature so far. We report an additional two cases.

METHODS

We retrospectively reviewed medical records at our institutions for diagnosis of CB.

RESULTS

In our first case study, a 65-year-old female presented with left-side hemiparesis. Her medical history was significant for complex partial seizures since she was 25-years-old. Seizures were of 15–20 s duration and not preceded by aura. She had left facial droop, and motor-strength was 4/5 (left-side). Magnetic resonance imaging (MRI) showed a large enhancing right frontoparietal mass extending across midline into left frontal white matter, which is suggestive of an infiltrative glioma. The patient was initiated on steroids and underwent stereotactic biopsy that showed brain parenchyma with extensive deposition of eosinophilic, hyaline extracellular material. Congo red stain was strongly positive, with green birefringence consistent with CB. She was managed conservatively with anticonvulsants. Despite treatment, she had complex partial seizures at least twice a month. In our second case study, a 42-year-old male patient presented with complaints of seizures of 1–2 min duration. There was no preceding aura or postictal state. On examination, there were no lateralizing or localizing signs. MRI was suggestive of a benign brain tumor, and the patient underwent a brain biopsy that was consistent with cerebral amyloidoma. He was managed conservatively with anticonvulsants for the last 17 years with minimal progression.

CONCLUSION

CB has been described as a chameleon-like lesion because it can mimic different brain pathologies, such as gliomas. CB has shown to follow a benign course in all of the 27 cases described so far. There is no standard treatment guideline to manage CB. Some advocate complete resection for the fear of progression, while others favor a “wait-and-see” approach with supportive treatment, as adopted by us in these two cases.

Neuro-Oncol. 2009 Oct;11(5):633.

300. Pemetrexed as Salvage Therapy for CNS Lymphoma

Jan Drappatz, Andrew Norden, David C Fisher, Ann Lacasce, Philippe Armand, Abigail Ciampa, Lisa Doherty, Patrick Y Wen

INTRODUCTION

High-dose methotrexate-based regimens are effective for CNS lymphoma, but a significant number of patients relapse. There is currently no standard therapy for relapsed disease. Pemetrexed is a multitargeted antifolate agent approved for first-line treatment of malignant pleural mesothelioma and second-line treatment for locally advanced or metastatic non-small-cell lung cancer. Preliminary data from a phase I study suggests that pemetrexed may be effective in recurrent CNS lymphoma.

METHODS

We performed a retrospective chart review of patients with relapsed CNS lymphoma to identify patients who received pemetrexed as salvage chemotherapy and recorded the demographics, adverse events, and responses.

RESULTS

To date, seven patients (five female; two male) with recurrent CNS lymphoma have been treated with pemetrexed. Median age was 60 (range, 53–84 years). Two patients had relapsed systemic lymphoma (one with diffuse large B-cell lymphoma and one with transformed marginal zone lymphoma); all others had primary diffuse large B-cell CNS lymphoma. All patients were heavily pretreated, including two patients who were treated following relapse after autologous stem cell transplant. With the exception of one elderly female, all patients received pemetrexed at 900 mg/m2 every 3 weeks. Patients received dexamethasone at 4 mg twice daily the day before, the day of, and the day after each infusion and supplemental folic acid and cyanocobalamin. Five patients achieved a complete radiographic response. One patient had stable disease after two doses and continues on treatment. With a median follow-up of 5 months, median progression-free survival is 5 months (range, 2–12 months). One patient died from progressive disease. No significant toxicities have been observed.

CONCLUSION

Pemetrexed appears active in relapsed CNS lymphoma. The treatment has been well tolerated. The optimal dose and schedule need to be determined, as well as activity in a larger number of patients.

Neuro-Oncol. 2009 Oct;11(5):633.

301. Does Survival of Newly Diagnosed GBM Patients Vary as a Function of the Reason for Withdrawal from Active Treatment? An NCCTG Database Study

Kurt A Jaeckle, Wenting Wu, Paul D Brown, Jan Buckner

INTRODUCTION

Protocol treatment is usually stopped because of tumor progression, but there are other reasons for withdrawal, including refusal, adverse events, or completion of therapy. It is unclear whether survival varies as a function of reason for withdrawal.

METHODS

We correlated the reasons for stopping protocol therapy with posttreatment overall survival (PTOS), measured from time of withdrawal, using a database from 16 prospective NCCTG trials of newly diagnosed GBM The analysis excluded 72 patients (4.5%) with missing data and 132 (8.21%) who died on study. The first analysis involved the entire group (n = 1,404), and the second analysis involved a subgroup (n = 365) treated in trials specifying the duration of prescribed therapy. Progression was defined by standard NCCTG criteria. PTOS was estimated by Kaplan-Meier method and compared by log-rank test.

RESULTS

The median PTOS, as a function of reason for protocol treatment withdrawal, is presented in Table 1. There were differences in median PTOS (months; 95% CI) between those completing protocol therapy (14.4; 12.2, 27.5) versus all other reasons (4.7; 4.4, 5.0) (p = 0.001), those refusing therapy (6.2; 4.6, 7.5) versus others (4.7; 4.4, 5.0) (p = 0.01), and those with tumor progression (4.5; 4.3, 4.9) versus others (6.2; 5.0, 7.0) (p < 0.0001). In the second analysis, patients completing all prescribed protocol therapy lived longer posttreatment than those with progressive disease (median PTOS 14.4 vs. 6.3 months; p = 0.0006). If patients completing therapy were excluded, there were no differences in PTOS between those with progression and those refusing therapy (p = 0.23) or those who went off therapy because of adverse events (p = 0.68).

CONCLUSION

GBM patients completing protocol therapy live longer posttreatment than those withdrawing from treatment for other reasons. Patients stopping therapy because of adverse events or refusal do not live substantially longer posttreatment than patients with progressive disease. Completion of prescribed protocol therapy may identify a group with favorable prognosis or possibly indicate efficacy of treatment.

Neuro-Oncol. 2009 Oct;11(5):633.

302. A Phase II Study of Bevacizumab Plus Etoposide among Recurrent Malignant Glioma Patients

Annick Desjardins, James J Vredenburgh, Sridharan Gururangan, Katherine B Peters, Allan H Friedman, Henry Friedman, David A Reardon

INTRODUCTION

Bevacizumab (BV), a neutralizing monoclonal antibody to vascular endothelial growth factor (VEGF), has demonstrated a significant therapeutic benefit for recurrent malignant glioma (MG) patients, leading to its expedited approval by the U.S. Food and Drug Administration. Daily protracted (metronomic) chemotherapy may exert an antiangiogenic effect by inducing apoptosis in tumor vascular endothelial cells. Combining antiangiogenic therapeutics, including VEGF-targeting agents and metronomic chemotherapy, may increase antitumor efficacy. In this study, we evaluated the efficacy of BV plus etoposide (E), a topoisomerase inhibitor, among recurrent MG patients.

METHODS

Recurrent MG adult patients with no more than three prior episodes of recurrence were eligible, while those with prior BV treatment or prior intracranial hemorrhage were excluded. The primary outcome measure was 6-month progression-free survival (6-PFS). BV was dosed at 10 mg/kg intravenously every other week. Etoposide was orally administered daily (50 mg/m2) for days 1–21 of each 28-day cycle.

RESULTS

Fifty-nine patients (grade 4 MG, n = 27; grade 3 MG, n = 32) with a median of two prior progressions were enrolled on study. A median overall survival (OS) for grade 4 MG of 46.4 weeks was observed and has not been reached yet for grade 3 MG. The 6-PFS was 44.4% for grade 4 MG and 40.6% for grade 3 MG. The most common toxicities were neutropenia (48%), fatigue (33%), and infection (31%); toxicities were grade 2 in most cases. One patient developed grade 1 intracranial hemorrhage, and one patient had a grade 4 gastrointestinal perforation. Radiographic response was achieved in 23% and 22% of grade 4 MG and grade 3 MG patients, respectively, while 70% and 66% of each cohort achieved stable disease.

CONCLUSION

The combination of bevacizumab and etoposide is well tolerated in recurrent MG patients and is associated with an encouraging rate of PFS, OS, and radiographic response.

Neuro-Oncol. 2009 Oct;11(5):633–634.

303. Sunitinib Plus Irinotecan for Patients with Recurrent Malignant Glioma: A Phase I Trial

Annick Desjardins, James J Vredenburgh, Katherine B Peters, Allan H Friedman, Sridharan Gururangan, Henry Friedman, David A Reardon

INTRODUCTION

Recent studies have shown the significant antitumor benefit associated with antiangiogenic therapies in recurrent malignant gliomas (MG). Sunitinib is a tyrosine kinase inhibitor targeting VEGFR2 and PDGFR, two key mediators of angiogenesis. As the first published studies of VEGF-directed therapies were in combination with irinotecan, a topoisomerase-1 inhibitor, we decided to evaluate the maximum tolerated dose (MTD) and dose limiting toxicity (DLT) of sunitinib with the same agent.

METHODS

Adult recurrent MG patients with a Karnofsky performance score (KPS) of at least 70%; adequate hematologic, renal, and hepatic function; and at least 2 weeks from prior surgery, 12 weeks from prior radiation therapy, and 4 weeks from prior chemotherapy or bevacizumab were eligible. Patients were excluded if they had prior treatment with sunitinib, prior progression or greater than grade 3 toxicity to irinotecan, concurrent use of enzyme inducing antiepileptic drugs or warfarin, > grade 1 intratumoral hemorrhage, or were homozygous for the TA7 (*28) allele of UGT1A1. Sunitinib was administered once daily for the first 28 days of each 42-day cycle, and irinotecan was administered every 2 weeks. The initial sunitinib and irinotecan doses were 25 mg/day and 75 mg/m2, respectively.

RESULTS

Thirteen patients (seven grade 4 MG and six grade 3 MG) have been enrolled thus far. One DLT has occurred on dose level 1 (grade 3 pancreatitis), and one patient on dose level 2 has experienced grade 3 neutropenia and grade 4 thrombocytopenia. Enrollment is ongoing on dose level 3. Evidence of therapeutic benefit to date includes six patients with completion of at least three cycles, including two patients still on study after five and six cycles.

CONCLUSION

The MTD is yet to be determined, but no unexpected toxicity has been observed thus far. PK analyses will be completed. Early evidence of antitumor benefit has been observed.

Neuro-Oncol. 2009 Oct;11(5):634.

304. Vandetanib, Imatinib Mesylate, and Hydroxyurea for Recurrent Malignant Glioma: A Phase I Study

Annick Desjardins, James J Vredenburgh, Sridharan Gururangan, Katherine B Peters, Allan H Friedman, Henry Friedman, David A Reardon

INTRODUCTION

Malignant glioma (MG) is characterized by frequent aberrant activation of EGFR, VEGFR, and PDGFR. We determined the maximum tolerated dose (MTD) and dose level toxicity (DLT) of vandetanib (V), a once-daily, oral selective inhibitor of VEGFR and EGFR when combined with imatinib mesylate (IM), a multikinase inhibitor including PDGFR, and hydroxyurea (H).

METHODS

Adult patients with recurrent MG with less than three prior recurrences, KPS of at least 60%, and adequate organ function were stratified based on concurrent enzyme-inducing anticonvulsant use (EIAC). Both arms were independently escalated using a 3+3 design. V was increased by 100 mg in successive cohorts beginning at 100 mg and 200 mg for patients not on and on EIAC, respectively, with established dose levels of IM and H. Evaluations were after every other 28-day cycle.

RESULTS

Twenty-seven patients (20 grade 4 MG, 7 grade 3 MG) have been enrolled thus far. Only one DLT (reversible grade 4 transaminase elevation; dose level 1) occurred among 18 non-EIAC patients. Accrual is ongoing for this arm. Of the nine patients accrued on the EIAC arm, one patient had a DLT at dose level 1 (grade 3 electrolyte disturbance) and two of three patients experienced a DLT at dose level 2 (first patient: grade 3 hypophosphatemia and grade 3 thrombocytopenia; second patient: grade 3 thrombocytopenia). Durable responses and stable disease have been observed.

CONCLUSION

Accrual is ongoing for the non-EIAC arm. The MTD was defined at first dose level for the EIAC arm. The combination of V, IM, and H is well tolerated in recurrent MG patients, and toxicities have been within what was expected for these agents; however, we have seen a higher rate of electrolyte disturbance. An update of outcome, toxicity, and pharmacokinetic analyses will be presented.

Neuro-Oncol. 2009 Oct;11(5):634.

305. Bevacizumab (BV) in Combination to Temozolomide (TMZ) and Radiation Therapy (RT) Followed by BV, TMZ, and Irinotecan (CPT-11) for Newly Diagnosed Glioblastoma Multiforme (GBM): A Phase 2 Trial

Annick Desjardins, David A Reardon, Katherine B Peters, James Herndon, John Kirkpatrick, Sridharan Gururangan, John H Sampson, Allan H Friedman, Henry Friedman, James J Vredenburgh

INTRODUCTION

Newly diagnosed glioblastoma multiforme (GBM) patients receiving temozolomide (TMZ) and radiation therapy (RT) followed by 6 monthly cycles of TMZ have a median progression-free survival and median survival of 6.9 and 15.8 months, respectively. Bevacizumab (BV), a humanized antibody to VEGF, has demonstrated a significant therapeutic benefit for recurrent GBM. This study aims to evaluate the benefit of incorporating BV with RT and TMZ, and irinotecan (CPT-11) and BV to TMZ post-RT therapy for newly diagnosed GBM patients.

METHODS

Patients received standard RT and TMZ at 75 mg/m2/day, with BV at 10 mg/kg every 14 days beginning at least 28 days postoperatively. Afterward, patients received six cycles of BV, TMZ, and CPT-11 (28-day cycle). BV was given at a dose of 10 mg/kg on days 1 and 15, TMZ at 200 mg/m2 on days 1–5, and CPT-11 on days 1 and 15 at 125 mg/m2 for patients not on an enzyme inducing antiepileptic drug (EIAED) and at 340 mg/m2 for patients on an EIAED.

RESULTS

For the first 75 patients enrolled, three patients did not complete RT; one each because of pancytopenia, grade 2 CNS hemorrhage, and pulmonary embolism (PE). Five patients developed thrombocytopenia for which TMZ was held (grade 3, n = 1; grade 4, n = 4). There were no other grade 3 or greater toxicities, including no wound dehiscence during RT. Forty-three patients have completed six cycles of BV, TMZ, and CPT-11. Seventeen patients discontinued treatment because of toxicity—seven with fatigue; two with PEs; four with grade 4 thrombocytopenia; two with diarrhea; and one each with a rectal abscess and sepsis. There have been 16 deaths—14 from tumor progression and one each from sepsis and PEs. At a median follow-up of 15 months, 79% remain alive, and 60% are progression-free.

CONCLUSION

Adding BV to TMZ and RT followed by BV and TMZ with CPT-11 is tolerable and efficacious.

Neuro-Oncol. 2009 Oct;11(5):634.

306. Phase I Study of Vandetanib with Radiation Therapy and Temozolomide for Newly Diagnosed Glioblastoma

Jan Drappatz, Andrew Norden, Eric T Wong, Lisa Doherty, Debra LaFrankie, Abigail Ciampa, Santosh Kesari, Christine Sceppa, Mary Gerard, Phuong Phan, Andrew B Lassman, David Schiff, Tracy Batchelor, Keith L Ligon, Geoffrey Young, Alona Muzikansky, Stephanie Weiss, Patrick Y Wen

INTRODUCTION

Glioblastoma (GBM) is the most common malignant primary brain tumor. Despite standard therapy consisting of surgical resection followed by radiotherapy (RT) with concurrent and adjuvant temozolomide (TMZ), median survival is only 15 months. There is a need for more effective therapies. Increasing evidence suggests that angiogenesis inhibition may potentiate the effects of RT and chemotherapy in patients with GBM. In addition, inhibition of the epidermal growth factor receptor (EGFR) may be of therapeutic benefit, as EGFR is often upregulated in GBM and contributes to radiation resistance. We conducted a phase I study of vandetanib (ZD6474), an inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and EGFR, in patients with newly diagnosed GBM in combination with RT and TMZ.

METHODS

Thirteen newly diagnosed GBM patients were treated with vandetanib, radiotherapy, and concurrent and adjuvant TMZ, using a standard 3+3 dose escalation design. The maximum tolerated dose (MTD) was defined as the dose with <1/6 dose limiting toxicities (DLT) during the first 12 weeks of therapy. Eligible patients were adults with newly diagnosed GBM, Karnofsky performance status = 60, normal organ function, and not taking enzyme-inducing antiepileptic drugs or QTc prolonging drugs.

RESULTS

Six patients were treated with vandetanib at a dose of 200 mg daily. Three of the six patients developed DLTs within the first 12 weeks—gastrointestinal hemorrhage and thrombocytopenia in one patient, neutropenia in a second patient, and diverticulitis with gastrointestinal perforation in a third patient. Seven patients were treated at 100 mg daily with no DLTs observed, establishing this dose as the MTD in combination with TMZ and RT.

CONCLUSION

Vandetanib may be safely combined with RT and TMZ in GBM patients. A phase II study in which patients are randomized to vandetanib 100 mg daily with RT and TMZ, or RT and TMZ alone, is under way.

Neuro-Oncol. 2009 Oct;11(5):634.

307. Does Bevacizumab Increase the Risk of Leptomeningeal Gliomatosis?

Maciej M Mrugala, Jeremy D Rudnick, Jason K Rockhill, Lawrence D Recht

INTRODUCTION

Bevacizumab, a humanized monoclonal antibody against VEGF, is frequently used in therapy of malignant glioma. It is predominantly utilized in the recurrent disease setting; however, applications in newly diagnosed patients are being investigated. Therapy with bevacizumab can be associated with dramatic radiographic responses and clinical improvement. In select patients receiving bevacizumab, diffuse nonenhancing tumor growth has been described. Leptomeningeal gliomatosis (LG) is a rare complication in patients with primary glial brain tumors. The risk factors for developing LG are poorly defined. It has been suggested that younger age might increase the risk of dissemination in glioma patients. Development of LG does not correlate with the length of the illness, and early cases have been described.

METHODS

This is a retrospective case series review.

RESULTS

We present seven cases of LG in patients with primary brain tumors who received bevacizumab as part of therapy at three different institutions. All patients were young, with median age of 28. There were five men and two women. All patients were diagnosed with high-grade glioma (primary glioblastoma in five cases and secondary glioblastoma in 2) and received temozolomide as their primary therapy. All patients received bevacizumab at the time of recurrence. Imaging was consistent with LG in all cases. In one patient, LG was discovered before administration of bevacizumab, but progression was rapid after the drug was introduced. Postmortem confirmation of LG was available in one case.

CONCLUSION

The incidence of LG in patients treated with bevacizumab appears increased, especially among young patients with recurrent high-grade glioma. Further study is needed to assess what underlies this propensity and whether it can be prevented.

Neuro-Oncol. 2009 Oct;11(5):634–635.

308. Primary Central Nervous System Lymphoma (PCNSL) Treated Only with Chemotherapy

Tatsuya Abe, Kenji Sugita, Masaki Morishige, Yasutomo Momii, Mitsuhiro Anan, Kumi Murata, Tohru Kamida, Takamitsu Hikawa, Minoru Fujiki, Hidenori Kobayashi

INTRODUCTION

Primary CNS lymphoma (PCNSL) is sensitive to radiochemotherapy. However, many elderly patients who had whole brain radiotherapy developed dementia. To reduce the incidence of neurologic toxicity of radiotherapy, patients were managed initially only with chemotherapy in our hospital.

METHODS

After the pathological findings were reviewed, the patient had an operation, putting Ommaya’s reservoir via lateral ventricle. The patients were treated with a combination of intrathecal chemotherapy and systemic R-CHOP chemotherapy for two or three cycles. Intrathecal infusions of arabinoside (20 mg), methotrexate (2.5 mg), and nimustine (5 mg) were first given via Ommaya’s reservoir on day 1, cyclophosphamide, Adriamycin and vincristine infusions were given systematically on day 2, and prednisolone was given orally on day 2 for 14 days. In addition, immunotherapy with rituximab, anti-CD20 monoclonal antibodies, every month (a total of eight times) was performed. In cases with no response to the CHOP chemotherapy, patients were treated with etoposide (VP-16) and carboplatin (CBDCA) for two or three cycles.

RESULTS

Of patients treated only with chemotherapy, 5% showed complete remission. In addition, 90% of these patients treated with rituximab showed no sign of recurrence for more than 2 years.

CONCLUSION

Systemic and intrathecal chemotherapy and repeated immunotherapy with rituximab were effective. However, if the tumor was to relapse, then radiotherapy would be absolutely required. Therefore, close monitoring of the patient with surveillance imaging and clinical examinations will continue to be carried out in order to detect any possible recurrence.

Neuro-Oncol. 2009 Oct;11(5):635.

309. Salvage Therapy with Single-Agent Bevacizumab for Recurrent Glioblastoma

Marc C Chamberlain, Sandra K Johnston

INTRODUCTION

We conducted a retrospective evaluation of single-agent bevacizumab in adults with recurrent glioblastoma (GBM) with an objective of determining progression-free survival (PFS). Background: There is no standard therapy for recurrent GBM after failure of alkylator-based chemotherapy.

METHODS

Fifty adults, ages 36–70 years (median, 64), with recurrent GBM were treated. All patients had previously been treated with surgery, concurrent radiotherapy and temozolomide, and postradiotherapy temozolomide, and in 34 patients, one salvage regimen (PCV, 21; cyclophosphamide, 13). Thirteen patients underwent repeat surgery. Patients were treated at first or second recurrence with bevacizumab, once every 2 weeks, defined as a single cycle. Neurological evaluation was performed every 2 weeks, and neuroradiographic assessment was performed following the initial two cycles of bevacizumab and subsequently after every four cycles of bevacizumab.

RESULTS

A total of 468 cycles of bevacizumab (median, 2 cycles; range, 1–30) was administered. Bevacizumab-related toxicity included fatigue (16 patients; 4 grade 3), leukopenia (9 patients; 1 grade 3), anemia (5 patients; 0 grade 3), hypertension (7 patients; 1 grade 3), deep vein thrombosis (4 patients; 1 grade 3), and wound dehiscence (two patients; one grade 3). Twenty-one patients (42%) demonstrated a partial radiographic response, and 29 (58%) demonstrated progressive disease following one to two cycles of bevacizumab. Time to tumor progression ranged from 0.5 to 15 months (median, 1.0 months). Survival ranged from 2 to 17 months (median, 8.5 months). Six-month and 12-month PFS were 42% and 22%, respectively.

CONCLUSION

Single-agent bevacizumab demonstrated efficacy and acceptable toxicity in this cohort of adults with recurrent alkylator-refractory GBM.

Neuro-Oncol. 2009 Oct;11(5):635.

310. Unusual Case of a Sella Region and Cavernous Sinus Atypical Teratoid/Rhabdoid Tumor Presenting with Acute Cavernous Sinus Syndrome in an Adult

Lara J Kunschner, Stevens Tracey, Pu Cunfeng

INTRODUCTION

Atypical teratoid/rhabdoid tumor (ATRT) is an aggressive primary CNS malignancy found predominantly in children and usually seen in the infratentorial region. Less than 30 cases of ATRT in adults are reported in the English literature, and only one prior case was described as presenting with a mass in the sella. Diagnosis can be difficult because of the histological and molecular genotypic heterogeneity seen in these tumors. Diffuse loss of nuclear INI-1 protein has been characterized as the gold standard immunohistochemical finding for this tumor when seen in a rhabdoid tumor with diffuse positivity for EMA and vimentin.

METHODS

We present a case report, including detailed radiographic and pathological characterization of a sellar region ATRT in a 51-year-old woman. Multimodality treatment is reviewed and discussed with reference to previously described adult cases.

RESULTS

A 51-year-old woman presented with an acute cavernous sinus syndrome (painful ophthalmoplegia, ptosis, and chemosis). Magnetic resonance imaging (MRI) of the brain revealed a large, enhancing, sella region mass with extension into the right cavernous sinus. Partial surgical resection was achieved; however, residual tumor remained in the cavernous sinus. Pathological diagnosis was difficult, leading to multiple neuropathological opinions and a consensus diagnosis of ATRT based on the characteristics of a highly mitotically active tumor with prominent rhabdoid morphology, focal staining for keratin, S100, and lack of staining for INI-1. EMA staining was negative. We will discuss the pathological features that make this a unique case. The clinical management of the case will be presented with updated follow-up after multimodality treatment involving radiotherapy and combination alkylating chemotherapy.

CONCLUSION

This case exemplifies an unusual etiology for a sella mass. While no specific guidelines are available with regard to treatment of this entity in the adult, and only general guidelines for children, we will provide updated treatment and outcome information at the time of presentation. Brief review of the very few adult ATRT cases in the literature will also be noted.

Neuro-Oncol. 2009 Oct;11(5):635.

311. Multiply Recurrent Disseminated Medulloblastoma Treated with a Bevacizumab-Based Regimen

Rimas V Lukas, Martin Kelly Nicholas

INTRODUCTION

There is no standard of care for recurrent medulloblastoma. We report a case of response to a bevacizumab-based regimen.

METHODS

A case report.

RESULTS

The patient was diagnosed with medulloblastoma at 19 years old. He underwent a gross-radiographic resection, craniospinal radiation, and eight cycles of cisplatin, cyclophosphamide, and vincristine. He remained disease-free for 5 years. Upon focal recurrence, he received stereotactic radiosurgery followed by ifosfamide, carboplatin, and etoposide with autologous stem cell transplant. Tumor dissemination occurred 1 year later, evidenced by diffuse CSF involvement and focal tumor nodules. The patient began treatment with temozolomide (150–200 mg/m2) for 5 days every 28 days and intravenous bevacizumab (10 mg/kg) every 2 weeks. Clinical improvement and partial radiographic response lasted approximately 7 months. At progression, his regimen was changed to cis-retinoic acid (cRA) (100 mg/m2) for 20 days every 28 days with biweekly intrathecal (IT) thiotepa. Disease progressed after 2 months. cRA was continued, and bevacizumab was resumed at the previous dose and schedule. Biweekly IT liposomal cytarabine was substituted for thiotepa. Bevacizumab was discontinued 2.5 months later for financial reasons. Clinical and radiographic progression were evident after 4 months. The patient continued on a short course of cRA and oral celecoxib (400 mg twice daily) before choosing hospice care. He remains clinically stable more than 17 months after initiating treatment with bevacizumab and temozolomide for his second relapse.

CONCLUSION

To our knowledge, this is the first report of the antitumor activity of bevacizumab used in combination with chemotherapy for medulloblastoma. It is also the first report of the combined use of bevacizumab with intrathecal chemotherapy in any malignancy. The patient developed significant hearing loss, but there were no serious unexpected toxicities. The use of bevacizumab in combination with systemic and IT chemotherapy regimens warrants further investigation in medulloblastoma.

Neuro-Oncol. 2009 Oct;11(5):635–636.

312. Outcome of Children and Adults with Intracranial Ependymoma: A Multiinstitutional Experience with 43 Patients

Kuniaki Saito, Yoshitaka Narita, Soichiro Shibui, Yoshihumi Okada, Keisuke Ueki, Motoo Nagane, Kazuhiko Mishima, Ryo Nishikawa, Akitake Mukasa, Tomoyuki Koga, Tomoki Todo, Nobusada Shinoura, Nobuhito Saito, Masao Matsutani

INTRODUCTION

Intracranial ependymomas are relatively rare central nervous tumors, accounting for 2% and 6–12% of all intracranial tumors in adults and children, respectively. The rarity of these tumors makes prognosis and treatment decisions difficult. We reviewed our histologically confirmed cases to better characterize the natural history and treatment outcomes of these rare tumors.

METHODS

We performed a retrospective analysis of 43 patients with intracranial ependymoma between 1979 and 2008. The medical record was reviewed to obtain demographic, clinical, radiographic, pathologic, treatment, and outcome data.

RESULTS

Histological findings were ependymoma (WHO grade 2) in 21 patients and anaplastic ependymoma (WHO grade 3) in 22 patients. The tumor was located in supratentorial in 23 patients and infratentorial in 20 patients. About 50% of the patients underwent gross total or subtotal removal. Local irradiation and whole-brain plus whole-spine irradiation were performed in 77% and 9% of the patients, respectively. More than 80% of the patients with anaplastic ependymoma underwent chemotherapy. Common chemotherapies included ICE, ACNU+VCR, TMZ, PAV, ACNU+VP-16, and so on. The median overall survival and progression-free survival for the entire group was 86 months and 34 months, respectively. The patients with grade 2 ependymoma had better survival rates compared to those with anaplastic ependymoma (p = 0.01). In the anaplastic ependymoma group, the extent of resection was a predictive factor of survival (p = 0.026). We also found that in anaplastic ependymoma, infratentorial tumor location tended to be associated with better outcome (borderline significance).

CONCLUSION

This report provides further information on the natural history and response to therapies of intracranial ependymomas and the influence of tumor grade. Detailed information will be included in the poster presentation.

Neuro-Oncol. 2009 Oct;11(5):636.

313. Electrolyte Imbalances in Patients Admitted with Chemotherapy Induced Febrile Neutropenia: Patterns and Impact on Outcomes. A Single Center Study from Pakistan

Samira Ahmad Bawany

INTRODUCTION

Febrile neutropenia (FN) and myelosuppression remain challenging oncologic medical emergencies and dose-limiting toxicities of chemotherapy. Various factors are associated with and affect the outcome of patients with FN. Electrolyte abnormalities are commonly observed, but the real incidence and impact has been only scarcely studied. At the molecular level, too, electrolyte imbalances can occur in these patients because of the fluid shift mechanism from intracellular to extra-cellular compartments as a result of catabolic response sepsis. The aim of the study was to observe the trends of electrolyte abnormalities in patients admitted with chemotherapy-induced FN.

METHODS

This was a retrospective study of 215 patients admitted with FN between January 2007 and August 2008. Data was collected using Aga Khan University Hospital patients’ records, both hard copy and electronic. Data was entered and analyzed using SPSS 16.0.

RESULTS

FN was equally distributed among patients with solid or hematological malignancies. A total of 83.5% patients demonstrated electrolyte abnormalities. Hypokalemia was seen in 48% and hyponatremia was seen in 67.9% of patients. About 12% patients with electrolyte deficit also had coexisting vomiting or diarrhea or both. Overall, 65% of patients received intravenous electrolyte replacement therapy, and disturbances in potassium levels were more critical as compared to disturbances of sodium, requiring significantly greater replacement of the former as compared to the latter (p = 0.022). Average length of hospital stay was 5.1 days in patients having electrolyte abnormalities compared to 4.7 days in those who had none, but the difference was not statistically significant (p = 0.69). However, in patients who were successfully discharged, the average length of stay was 5.4 days, whereas it was 11.4 days in patients who died (p < 0.0001).

CONCLUSION

This analysis, perhaps the first of its kind, suggests that electrolyte abnormalities are frequently observed in patients presenting with FN. These abnormalities can negatively affect outcomes. Therefore, the incidence and impact of electrolyte abnormalities on FN outcomes should be studied further, and the molecular mechanisms underlying this response should be elaborated.

Neuro-Oncol. 2009 Oct;11(5):636.

314. Epstein-Barr Virus-Associated Intracranial Leiomyosarcoma in an HIV-Positive Adolescent

Sweta Gupta, Sachin Jogal, Peter Havens, James Southern, Selim Firat

INTRODUCTION

Leiomyosarcoma is an extremely rare tumor in children. The incidence is less than two cases per 10 million children per year. However, in children with human immunodeficiency virus (HIV) infection, leiomyomas and leiomyosarcomas are the second most common malignancy after non-Hodgkin lymphoma (NHL), constituting 3% to 17% of all cancers. In this population, leiomyosarcomas are commonly found in visceral organs. Primary intracranial leiomyosarcomas are extremely rare (about 3 in 25,000 brain tumor biopsies). In adults with acquired immunodeficiency syndrome (AIDS) or HIV, there have been several reports of primary CNS leiomyosarcomas. However, in children with AIDS or HIV, there is only one documented report of an intracranial leiomyoma. Most of the tumors in immune deficient patients are associated with the Epstein-Barr virus (EVB). We describe the course and outcome of a pediatric patient with HIV who developed an EBV-positive primary intracranial leiomyosarcoma.

METHODS

Case report.

RESULTS

A 17-year-old African-American female with HIV infection presented with an unresectable intracranial neoplasm mass in the right paracentral prepontine with mass-effect upon the brainstem. Stereotactic biopsy revealed this to be an EBV-associated leiomyosarcoma. Radiation therapy and gemcitabine were used to shrink the mass, with the aim of making it surgically resectable. Prolonged neutropenia and recurrent skin infections led to the discontinuation of gemcitabine. The mass stabilized after radiation therapy and has not shown progression in 8 months of follow-up. EBV has been demonstrated in most smooth muscle tumors associated with AIDS and other immunocompromised states. This is the first documented case to describe an EBV-positive intracranial leiomyosarcoma in a pediatric HIV patient.

CONCLUSION

Our patient illustrated the possibility of an unusual presentation and site of a soft tissue sarcoma in the pediatric HIV population. These types of tumors should be in the differential diagnosis of intracranial lesions in HIV patients.

Neuro-Oncol. 2009 Oct;11(5):636.

315. Metastatic Pilocytic Astrocytomas in Children: A Case Series

Hillary N Bess, Jeffrey Bennett, David W Pincus, Stacie Stapleton, Jeffrey Schwartz, Amy Amundson Smith

INTRODUCTION

Dissemination of juvenile pilocytic astrocytomas (JPA) is reported to occur in approximately 3%–5% of cases. However, the clinical behavior of these tumors can be variable, and this entity is generally poorly understood.

METHODS

We performed a retrospective chart review of all the patients seen at the University of Florida Pediatric Neuro-Oncology Clinic between 2001 and 2008.

RESULTS

There were six children, with ages at presentation ranging from 23 months to 14 years (median, 7 years), with the diagnosis of metastatic JPA. Primary tumor locations were cerebellar in four patients and diencephalon in two. Five of the six had evidence of metastatic disease at initial presentation. One child had a cerebellar primary that was subtotally resected and, after three operations for locally progressive disease over a 2-year time period, developed drop metastasis to the spine. Three children presented with diffuse leptomeningeal disease, while two had isolated metastatic lesions to the spine. Four children were treated with chemotherapy as up-front treatment and three of the four had PR/SD for 6–8 months. The initial chemotherapy regimens used were a) carboplatin and vincristine; b) 6TG, procarbazine, vincristine, and CCNU; and c) carboplatin, vincristine, and temozolomide. Four children have received craniospinal radiation therapy doses ranging from 36 Gy to 51 Gy; three of the four have progressed at 6, 9, and 31 months postradiation. One child has stable disease 12 months post-RT. All children are alive with disease. Time from diagnosis to present ranges from 10 months to 11 years 6 months. Two children are undergoing active treatment for a recurrence.

CONCLUSION

Metastatic JPA is a poorly understood entity with variable clinical behavior. We clearly need a better understanding of the biology of these tumors so that we may find improved treatment approaches.

Neuro-Oncol. 2009 Oct;11(5):636.

316. Pilot Study of Systemic and Intrathecal Chemotherapy Followed by Conformal Radiation for Infants with Embryonal Intracranial Central Nervous System Tumors: A Pediatric Brain Tumor Consortium Trial

Susan M Blaney, Mehmet Kocak, Richard Heideman, Amar Gajjar, Mark W Kieran, Peter Phillips, Roger McLendon, James Boyett, Larry Kun

INTRODUCTION

A pilot study was performed to investigate the feasibility of adding intrathecal mafosfamide to an intensive regimen of concomitant multiagent systemic chemotherapy followed by conformal radiation therapy for children < 3 years of age with newly diagnosed embryonal tumors. Progression-free survival (PFS) was estimated in M0 patients with medulloblastoma, supratentorial PNET, and ATRT.

METHODS

Twenty weeks of systemic multiagent chemotherapy (cyclophosphamide, cisplatin, vincristine, and oral etoposide) commenced within 35 days of definitive surgery. Intrathecal mafosfamide was administered to those children without evidence of CSF flow obstruction as assessed by radionuclide imaging. Seventy-one (76%) of the patients who received intrathecal mafosfamide at the maximum tolerated dose (MTD) of 14 mg were evaluable for feasibility assessment; M0 patients subsequently received local conformal XRT and 20 additional weeks of systemic chemotherapy (vincristine, cyclophosphamide, and oral etoposide).

RESULTS

Ninety-four infants and children (median age at diagnosis, 1.6 years; range, 0.1–3.0; 52 male) were enrolled. Forty-six patients completed the duration of prescribed protocol therapy. The 1-year PFS for patients with medulloblastoma, supratentorial PNET, and ATRT was 68 ± 9%, 67 ± 15%, and 27 ± 11%, respectively, and the 5-year PFS was 57 ± 17%, 28 ± 24%, and 9 ± 9%, respectively. Desmoplastic histology was not associated with PFS. M0 medulloblastoma patients who received XRT had 5-year PFS of 85 ± 16.5% and overall survival of 94.7 ± 8.9%.

CONCLUSION

The addition of intrathecal mafosfamide to a regimen of systemic chemotherapy was feasible. This treatment regimen appears to be associated with PFS that is at least equivalent to prior infant brain tumor trials. The survival for children with medulloblastoma patients who received local conformal XRT at week 20 was excellent.

Neuro-Oncol. 2009 Oct;11(5):636–637.

317. Retrospective Analysis of Neurofibromatosis Type 1 Associated Optic Glioma Visual Outcomes

Michael J Fisher, Laura Balcer, Robert Listernick, David H Gutmann, Roger J Packer, Rosalie Ferner, Robert Hoffman, Uri Tabori, Eric Bouffet, Darren Hargrave, Joel Charrow, Janice Lasky Zeid, Anne Albers, Annie Kuo, Michael Loguidice, Angie Miller, Grant Liu

INTRODUCTION

Optic gliomas (OPGs) arise in 15%–20% of children with NF1; nearly one-half become symptomatic. Disagreement persists regarding indications to initiate treatment. Additionally, there is little information regarding ophthalmologic outcomes after chemotherapy. Much of the literature on chemotherapy has focused on radiographic outcomes with a 5-year EFS of 68.7% in a large Children’s Oncology Group study using carboplatin with vincristine.

METHODS

A retrospective multicenter (n = 9) study was undertaken to define reasons for treatment and visual outcomes following chemotherapy for NF1-associated OPG and to identify risks for visual loss. Subjects included children undergoing initial treatment for OPGs with chemotherapy between January 1997 and December 2007.

RESULTS

Preliminary data on 79 subjects from six sites is available. Approximately one-third had tumors of the anterior visual pathway (nerves and/or chiasm). Common primary reasons to initiate treatment were: visual acuity (VA) loss (44), tumor growth (42), size/extent (11), tumor location (9), tumor enhancement (8), progressive proptosis (7), visual field loss (6), and inability to obtain reliable VA (5). For most subjects, more than one factor spurred treatment (mean, 1.85; range, 1–7). Twenty-three subjects were treated for a single reason: VA decline (14), tumor growth (8), and progressive proptosis (1). Sixty-four subjects were evaluable for VA outcomes. At completion of chemotherapy, VA improved (33%), remained stable (42%), or declined (25%). Notably, VA improved in 42% of anterior-OPGs, but only 28% of posterior pathway (hypothalamus and/or optic tracts/radiations) tumors. In contrast, VA worsened in 30% of posterior- and 17% of anterior-OPGs.

CONCLUSION

Visual acuity decline and tumor progression were the primary reasons to initiate treatment. Of concern, one-fourth of subjects showed decline in VA by the end of treatment; outcomes for subjects with posterior pathway tumors were particularly poor. Data from remaining sites and additional ophthalmologic outcomes (visual fields, proptosis, optic disc swelling/pallor, etc.) is forthcoming.

Neuro-Oncol. 2009 Oct;11(5):637.

318. Carboplatin and Vincristine Chemotherapy for NF1 Children with Progressive Optic Pathway Gliomas

Jeffrey C Allen, Jeena Mathew, Erin Hartnett, Matthias Karajannis, Sharon Gardner, Joan Ater

INTRODUCTION

The management of optic pathway gliomas (OPG) in children with NF1 requires special consideration related to their unpredictable and often less aggressive natural history and the reluctance of neurooncologists to administer radiotherapy and mutagenic chemotherapy. In the recently completed low-grade astrocytoma phase III Children’s Oncology Group protocol 9952, 129 of 401 children had NF1 and were assigned to the carboplatin/vincristine treatment arm. The NF1 cohort with OPG experienced a longer 5-year EFS than those without NF1, but little information is available about the types of objective responses they experienced and their durability.

METHODS

We reviewed our institutional experience over the past 10 years in children with NF1 with radiographically and/or clinically progressive OPG whose initial treatment involved the weekly administration of carboplatin and vincristine for 48 weeks.

RESULTS

There were 10 patients whose mean age at diagnosis was 5.1 years (range, 2–11 years). The primary tumor location was chiasm/optic tract (n = 9) and optic nerve (n = 1). Five patients had histologically confirmed JPA. Chemotherapy was terminated prematurely in two patients because of an allergic reaction to carboplatin. All 10 patients were evaluable for response, and two are still undergoing therapy. The maximum responses were: partial (n = 4), minor (n = 2), and stable disease (n = 4). Six patients have remained in continuous first remission for 3+, 6+, 19+, 28+, 62+, and 78+ months, and four have progressed off therapy at 10, 14, 44, and 48 months.

CONCLUSION

Carboplatin/vincristine has a high likelihood (100%) of stabilizing or remitting OPG in children with NF1, but disease progression off therapy is common (40%). There is a need for new nonmutagenic treatment alternatives.

Neuro-Oncol. 2009 Oct;11(5):637.

319. Prolonged Progression-Free Survival in Two Pediatric Patients with Recurrent/Refractory Medulloblastoma Treated with a Combination of Bevacizumab and Irinotecan

Jason Fangusaro, Stewart Goldman, Dolly G Aguilera

INTRODUCTION

Patients with recurrent/refractory medulloblastoma have a very poor prognosis. Treatment strategies for these patients have included repeat surgical resection, repeat irradiation when feasible, early-phase trials, and high-dose chemotherapy with autologous hematopoietic cell rescue. Although there has been some survival reported utilizing these strategies, there is no current standard of care because no single treatment modality has proven superior. In fact, the far majority of patients will succumb to their disease regardless of the treatment employed. We report on two patients who show prolonged progression-free survival (PFS) utilizing a combination of bevacizumab and irinotecan.

METHODS

Patient 1 is a 5-year-old female diagnosed with M3 anaplastic medulloblastoma and initially treated as per CCG 99701 with chemotherapy and irradiation. At the end of therapy, she had persistent lesions in her spine that were biopsied and revealed refractory medulloblastoma. She was treated with a combination of bevacizumab and irinotecan. Patient 2 is a 5-year-old male treated for M3 anaplastic medulloblastoma also treated as per CCG 99701. Six months posttreatment, he developed new brain lesions consistent with recurrent disease. He was also treated with a combination of bevacizumab and irinotecan.

RESULTS

Patient 1 has remained on this regimen for approximately 1 year with stable disease. Patient 2 had a partial response after 3 months of therapy, and after 6 months, he had a complete response. He continues on therapy currently with a progression-free survival of 9 months postrecurrence. Both patients have maintained excellent quality of life.

CONCLUSION

This combination may be an alternative that offers sustained PFS in a subset of patients. Current prospective trials utilizing this combination in recurrent medulloblastoma will better answer whether this combination has utility in a broader group of patients.

Neuro-Oncol. 2009 Oct;11(5):637.

320. Urine c-Met Levels Correlate with Tumor Size in a Mouse U87 Glioma Model

Leor Zach, Pamela Tulleners, Tamalee Scott, Malissa Kirk, Donald Bottaro, Kevin Camphausen

INTRODUCTION

c-Met is a transmembrane receptor protein tyrosine kinase that signals proliferation, motility, cell adhesion, invasion, and anti-apoptotic responses in several human tumors, including glioblastoma multiforme (GBM). The U87 GBM cell line expresses high levels of c-MET and is widely used as an experimental intracranial (IC) glioma model. We sought to investigate urine c-MET levels as a screening tool for early selection of successful IC implants in mice.

METHODS

The human glioma U87 cell line was grown in RPMI 1640 containing glutamate (5 mmol/L) and 5% fetal bovine serum at 37°C in 5% CO2 and 95% room air. Tumor cells (5 × 106 cells) were injected intracranially into mice. The mice were euthanized at three time points (20, 29, and 45 days post-IC implant) and their brains were processed and stained with standard hematoxylin and eosin (H&E) to allow tumor volume measurements. Urine was collected 24 h prior to euthanasia and c-MET levels were measured using a standard human c-MET ELISA kit.

RESULTS

The mean tumor volume was highly correlated to the time of harvest (R2 = 0.9). Urinary human c-MET levels were correlated with both the time of euthanasia (R2 = 0.57) and tumor volume (R2 = 0.48). Furthermore, no human c-MET was found in the urine of mice without a tumor.

CONCLUSION

Human urinary c-MET levels’ correlation with the presence and volume of experimental IC-implanted U87 glioma tumors is a simple, noninvasive, inexpensive, and reliable tool, which allows early distinction (at least 10 days before actual symptoms are expected) between successful and futile IC implants and can be an adjunct in choosing the correct time points for interventions in a mouse glioma experimental model. Preliminary data indicate that c-MET is also detectable in the urine of human subjects with high-grade glial tumors.

Neuro-Oncol. 2009 Oct;11(5):637–638.

321. Imaging Tumor Permeability and Vascular Properties in a Rat Model of Breast Cancer Brain Metastases

Leslie L Muldoon, Seymur Gahramanov, Y Jeffrey Wu, Edward A Neuwelt

INTRODUCTION

We assessed tumor cell localization in the cerebral vasculature and the permeability of metastases in rat brain after intraarterial infusion of MDA-MB-231BR human breast cancer cells expressing HER2.

METHODS

MDA-MB231BR-HER2 cells were infused into the right internal carotid artery in female nude rats, with or without pretreatment with cyclophosphamide (CTX 100 mg/kg IP). To assess tumor cell localization, cells were labeled with quantum dots (n = 4) or ferumoxides iron oxide nanoparticles (n = 4) for histology or magnetic resonance imaging (MRI), respectively. For long-term assessment of tumor growth, eight rats received a second dose of CTX 2 weeks after tumor cell infusion. Development of brain metastases was followed with 3T MRI using T2- and T1-weighted sequences ± gadolinium contrast. Selected metastases were assessed by MRI at 12 T using dynamic contrast-enhanced (DCE) MRI with gadolinium, dynamic susceptibility contrast (DSC) MRI, and perfusion with the blood pool iron oxide nanoparticle ferumoxytol.

RESULTS

In the absence of CTX pretreatment, breast cancer cells were found within the vasculature but not bound to the capillary endothelium nor infiltrated into the brain. Breast cancer cells administered 24 h after CTX were found lining blood vessel walls at 1 and 3 days after inoculation, with parenchymal localization at 1 week. At 8 weeks after tumor cell infusion in rats treated with double-dose CTX, all animals showed multiple millimeter-sized brain tumors located throughout the brain. Individual metastases with similar gross morphology showed markedly variable vascular properties. Tumor relative cerebral blood volume (rCBV) and permeability markers such as rate constant for passive contrast agent leakage across the blood-brain barrier (Ktrans) and the interstitial space volume fraction (ve) showed differences between tumors that did not depend on size or location.

CONCLUSION

Tumor cell binding to brain vasculature and development of breast cancer brain metastases after intraarterial infusion in the nude rat was markedly enhanced by treatment with CTX. Brain metastases have consistently inconsistent rCBV, permeability, and morphology. Marked variability in the permeability of individual metastases may impact drug delivery and efficacy.

Neuro-Oncol. 2009 Oct;11(5):638.

322. Effects of Receptor Tyrosine Kinase Inhibitors on Meningiomas

Li Li, Marie-Claire Daou, Alicia Mihaliak, Richard Moser, Thomas Smith, Alonzo H Ross

INTRODUCTION

Meningiomas are common tumors of the CNS and are thought to arise from arachnoid cap cells of the meninges. Most meningiomas are benign tumors that are efficiently treated by surgical resection. However, about 15–20% of these tumors are very aggressive and have a high chance of recurrence after surgical removal and hence, may require adjuvant therapy.

METHODS

We used commercial protein arrays to assess activation of receptor tyrosine kinases.

RESULTS

Our lab generated 18 primary meningioma cell lines from patient specimens. These cells can grow in DMEM with 10% FBS and be passaged at least eight times. Using these cell lines, we assessed which cell surface receptor tyrosine kinase receptors are activated. Phospho-EGF receptor protein was highly expressed in all cell lines. Activated PDGF receptor, insulin receptor, Axl, and Eph were moderately expressed. These receptor tyrosine kinases are potential targets for directed therapies. Antireceptor tyrosine kinase inhibitors, SU11274, AG1478, imatinib, and PD-089828 inhibited meningioma cell proliferation and induced apoptosis.

CONCLUSION

Our data suggest that receptor tyrosine kinase inhibitors alone or in combination with other inhibitors of cell growth could provide a tool for treatment of meningiomas.

Neuro-Oncol. 2009 Oct;11(5):638.

323. Treating a PDGF-Driven Model of Glioblastoma with Sunitinib Slows Tumor Growth and Prolongs Survival But Induces a Marked Increase in Tumor Cell Invasion

Julia Sisti, Liang Lei, Marie Roguski, Jeffrey N Bruce, Peter D Canoll

INTRODUCTION

Recent studies have suggested that paracrine growth factor signaling plays an important role in the growth and progression of glioblastoma multiforme (GBM) through the recruitment of highly proliferative glial progenitors. Infecting glial progenitors with a PDGF-expressing retrovirus has been shown to cause massive recruitment of uninfected cells, consistently leading to the formation of malignant tumors resembling GBM. The current study evaluates the effects of blocking cellular recruitment in a PDGF-driven murine model of GBM by administration of the multitarget tyrosine kinase inhibitor sunitinib (Sutent).

METHODS

Glioma cells harboring genetic deletion of p53 and overexpression of PDGF were infected with a firefly luciferase-expressing lentivirus. Cells were then stereotactically injected into the subcortical white matter of adult mice. Tumor growth was monitored by luciferase imaging twice weekly. Beginning one-week postinjection, mice received either vehicle or sunitinib (60 mg/kg) by oral gavage and were sacrificed at the first signs of tumor-induced morbidity. Brains were examined to evaluate tumor size, histological grade, degree of invasion, and proliferation index of tumor cells and blood vessels. Kaplan-Meier analysis was performed to evaluate effects on survival.

RESULTS

Sunitinib-treated mice survived significantly longer than their vehicle-treated counterparts (sunitinib, 37.8 ± 1.6 days vs. vehicle, 24.6 ± 3.6 days; p = 0.017). Serial luciferase imaging revealed a reduced rate of tumor growth in the sunitinib-treated brains as compared with vehicle-treated brains. Histologically, the sunitinib-treated tumors showed less vascular proliferation and a lack of palisading necrosis but had a far more diffusely infiltrative growth pattern than their vehicle-treated counterparts.

CONCLUSION

Treatment with sunitinib results in increased survival and a vastly altered pattern of tumor progression, possibly by inhibiting cellular recruitment of glial progenitor cells by blocking paracrine PDGF signaling.

Neuro-Oncol. 2009 Oct;11(5):638.

324. Concurrent Temozolomide-External-Beam Radiation Therapy is Effective for Experimental Glioblastomas in An Orthotopic, Genetically Engineered Syngeneic Mouse Allograft Model System

Ryan Bash, Natalie O Karpinich, Mark Vitucci, Olguitza Y Guzman, Kristen K White, Jared L Snider, Terry Van Dyke, Ryan Miller

INTRODUCTION

Neither diagnosis of glioblastoma (GBM) by microscopic morphology nor empiric therapy with concurrent fractionated radiation (XRT) and temozolomide (TMZ) is based upon the molecular mechanisms of gliomagenesis. Genetically engineered mice (GEM) are well suited for mechanistic studies and are logical model systems for preclinical cancer drug development.

METHODS

We developed an orthotopic allograft model of GBM using AdCre-infected primary astrocytes from a conditional GEM (TRP−/−) with inactivated RB and PTEN and constitutively activated KRAS (KRASG12D). Proliferation in vitro was determined by the MTS assay. Orthotopic growth in syngeneic mice was determined by serial magnetic resonance imaging, survival, and histopathology. Expression profiles of untreated TRP−/− allografts and human GBMs from TCGA were compared by unsupervised hierarchical clustering. Finally, the efficacy of TMZ (100 mg/kg, every day, days 6–10), XRT (5 Gy fractions, every other day, days 6–10), and combined TMZ-XRT on established TRP−/− allografts was defined.

RESULTS

TRP−/− cells are sensitive to TMZ (IC50 = 190 μM) and XRT. Allografts grew rapidly (2 day doubling time) as diffusely infiltrative masses with secondary structures of Scherer and yielded reproducible mortality (five experiments, n = 42; median survival, 21 days; 95% CI 20–22). Comparative genomics showed that untreated TRP−/− allografts were most similar to proneural (PN) human GBM. TMZ alone was ineffective (22 day median survival (MS), log-rank p = 0.16). Fractionated XRT was effective (MS 27 days, p = 0.0004), but the TMZ-XRT combination was superior (MS 32 days, p = 0.0001 vs. control, p = 0.01 versus XRT alone).

CONCLUSION

We established a genetically tractable model system uniquely suited for investigation of the molecular basis of gliomagenesis and for preclinical drug development. The qualitatively similar response of TRP−/−, PN subtype-like GEM, and human GBM to standard-of-care TMZ-XRT suggests that this model system will serve as a facile platform for biomarker development to further stratify human PN GBM for TMZ-XRT versus alternative therapies.

Neuro-Oncol. 2009 Oct;11(5):638.

325. Analysis of Intracranial Fluorescence-Labeled Liposome Biodistribution and Retention in the Brains of Naive Versus Tumor-Bearing Athymic Mice

Laura Serwer, Rintaro Hashizume, Kevin Weng, John Park, Daryl C Drummond, Charles O Noble, Mitchel S Berger, C David James

INTRODUCTION

This study was done to compare the biodistribution and sites of retention for fluorescence-labeled liposomes (FLLs) in athymic mice, both with and without intracranial glioblastoma (GBM) xenograft following intravenous versus convection-enhanced delivery (CED) of FLLs.

METHODS

Quantum dot 800 (QD-800) FLLs, or car-bocyanine (DiI) liposomes, either unmodified or modified with anti-EGFR targeting Fab' fragments, were administered by CED or by tail-vein injection in mice with or without established supratentorial, EGFR-amplified GBM, with subsequent monitoring of mice for liposome localization by optical imaging.

RESULTS

In our initial experiment involving CED administration of QD-800 FLLs into mice with EGFR-amplified xeno-grafts, optical imaging revealed a detectable intracranial signal during a 4-day period of observation. During this period of observation, the rate of signal decrease was more rapid for mice injected with unmodified FLLs, suggesting that EGFR targeting against EGFR-amplified tumors promotes increased retention of liposome-associated fluorescence. In contrast to the results from CED, tail-vein injection of both nontargeted and EGFR-targeted FLLs did not result in detectable signal in the brains of mice with intracranial tumors. This experiment was followed by the injection of DiI liposomes in the brains of naive mice, with monitoring by fluorescence optical imaging, which has revealed sustained detectable signal for >35 days following injection.

CONCLUSION

1) Liposome CED is feasible in mice, with detectable intracranial fluorescence of FLLs, indicative of successful localized delivery. 2) CED-administered FLL signal can be followed longitudinally in living mice, either with or without established brain tumor, for extended periods of time. 3) Tumor-cell epitope targeting (EGFR in the current study) appears to promote more durable retention of QD-800 FLL signal following CED of FLLs. 4) In comparison with intravenous administration, CED administration of FLLs promotes superior local accumulation and retention of fluorescence signal in mice with established intracranial tumors.

Neuro-Oncol. 2009 Oct;11(5):639.

326. Deletions of P53 and PTEN Cooperate to Facilitate Tumorigenesis in a PDGF-Driven Murine Model of Glioma

Marie Roguski, Julia Sisti, Jason A Ellis, Liang Lei, Roland Zemla, Jeffrey N Bruce, Peter D Canoll

INTRODUCTION

Glioblastomas frequently harbor genetic mutations and deletions in the tumor suppressor genes PTEN and p53, and recent studies have shown that these lesions often occur in the same tumor. We have previously shown that genetic deletion of PTEN in glial progenitors in the subcortical white matter or adult mice, while insufficient to induce tumor formation by itself, greatly facilitates PDGF-driven gliomagenesis. In this study, we tested the effects of genetically deleting p53 in adult glial progenitors, either alone, or in combination with deletion of PTEN in this model.

METHODS

PDGF- and Cre-expressing retroviruses were stereotactically injected into the subcortical white matter of 6-week-old PTENlox/lox, p53lox/lox, and dual PTENlox/lox p53lox/lox mice. Mice were sacrificed at first signs of tumor morbidity. Survival was analyzed by Kaplan Meier analysis. Histological and immunohistochemical analysis was performed on brains to evaluate tumor grade and location.

RESULTS

The mean survival of p53-only deleted mice was 36.9 days postinjection (95% CI, 33.74–40.01), and the mean survival of PTEN-deleted mice was 78.0 days postinjection (95% CI, 63.2–92.8). Deletion of both p53 and PTEN further facilitated tumorigenesis and reduced the mean survival of p53- and PTEN-deleted mice to 27.5 days postinjection (95% CI, 23.73–31.33). The survival curves of these three groups were significantly different (p < 0.0001). The tumors that formed in all genetic backgrounds showed the histological features of glioblastoma, including marked vascular proliferation and pseudopalisading necrosis. Interestingly, compared to the PTEN-deleted tumors, the p53-deleted tumors displayed a more invasive phenotype, with tumor cells consistently extending into the leptomeningeal space.

CONCLUSION

Genetic deletion of either PTEN or p53 facilitated PDGF-driven gliomagenesis, with deletion of p53 showing a greater effect on survival. Furthermore, deleting both tumor suppressors resulted in the shortest survival, demonstrating that genetic deletions in PTEN and p53 cooperate to facilitate PDGF-driven gliomagenesis.

Neuro-Oncol. 2009 Oct;11(5):639.

327. The Effect of the CXCR4 Antagonist AMD3100 on Glioma Migration and Tumor Invasion in the GL261 Murine Model

Daniel Santovasi, Eugene Lukyanov, Mine Esencay, David Zagzag, Ilya G Lim, Elizabeth W Newcomb

INTRODUCTION

The natural history of glioblastoma involves diffuse, invasive local spread. Presently, there is no therapy that specifically targets invading glioma cells. Using an in vitro assay and our previously described murine model, we tested AMD3100, the small molecule CXCR4 antagonist, to observe its effects on glioma tumor cell migration and invasion.

METHODS

To evaluate the effect of AMD3100 on GL261 migration, GL261 cells (5 × 104) were plated in Boyden chambers in the presence or absence of 100 nM AMD3100 and cultured in normoxic or hypoxic conditions for 16 h. They were then fixed, stained, and counted. For in vivo studies, female C57BL/6 mice were implanted intracerebrally with GL261 glioma cells (1 × 105). On day 14, animals were randomized into treatment groups as follows: 1) vehicle only (n = 10) and 2) AMD3100 1.25 mg/kg in PBS (n = 7), and both were administered subcutaneously twice daily (9 am and 5 pm) for 2 weeks. Animals were sacrificed on day 28 and the brains were harvested for histopathological analysis. Gross tumor volume was measured and extent of invasion was quantified as the mean distance of the invading tumor edge from the tumor core using Adobe Photoshop CS3.

RESULTS

AMD3100 treatment significantly reduced the migration of GL261 cells in the Boyden chamber by 17% compared with untreated controls cultured in hypoxic conditions (p < 0.0001). Analysis of the brains of AMD3100-treated mice compared with controls demonstrated a significant reduction in the mean depth of invasion from 178 μm to 79 μm (p < 0.0001).

CONCLUSION

The CXCR4 antagonist AMD3100 significantly decreases GL261 migration and invasion. These results suggest CXCR4 antagonists warrant further testing in combination with existing therapeutic modalities.

Neuro-Oncol. 2009 Oct;11(5):639.

328. Novel Monoclonal Antibody 13R2.C3 Against Human and Canine Interleukin 13 Receptor Alpha-2

Waldemar Debinski, Peter Dickinson, Denise M Gibo

INTRODUCTION

Interleukin 13 receptor-α2 (IL-13Ra2), an IL-13 plasma membrane receptor, is overexpressed in a vast majority of patients with glioblastoma multiforme (GBM), mainly because of PI3-K and ERK activation. IL-13Ra2 is also overexpressed in dogs with spontaneous malignant gliomas. We have thus decided to generate a monoclonal antibody against the human receptor that would cross-react with the canine receptor as a potentially useful tool to study a highly clinically relevant model of human disease.

METHODS

A 15-amino acid peptide spanning the region of complete homology between the human and canine extracellular portions of IL-13Ra2 was synthesized for immunization. A standard protocol was used for the generation and purification of monoclonal antibody. The selected clone, 13R2.C3, was used for Western blot analysis of human and canine tumor lysates.

RESULTS

13R2.C3 antibody revealed a single protein band of expected molecular size for IL-13Ra2 in Western blots of cell and tissue lysates. IL-13Ra2 protein was readily detected in human GBM with 9/14 showing high and 4/14 showing moderate overexpression. This overexpression is higher than seen in studies using a polyclonal antibody. All (6/6) canine GBM tumors were positive for IL-13Ra2 with 50% exhibiting high overexpression. Human and canine astrocytomas and oligodendrogliomas were all positive for IL-13Ra2 to various degrees. Interestingly, human meningiomas exhibited strong positivity (5/6). In addition, IL-13Ra2 was highly expressed in canine choroid plexus papillomas. Normal human and canine brain samples were negative for IL-13Ra2 with the13R2.C3 antibody.

CONCLUSION

We have obtained a monoclonal antibody against IL-13Ra2 that cross-reacts with human and canine receptors. The 13R2.C3 antibody detects immunoreactive IL-13Ra2 in up to 90% of human GBMs. In addition, other brain tumors, such as high-grade oligodendrogliomas, meningiomas, and canine choroid plexus papillomas, appear to express the receptor at high levels, and thus, may be appropriate candidates for IL-13Ra2-targeted therapies.

Neuro-Oncol. 2009 Oct;11(5):639.

329. Elucidating the Genetic Complexity of NF1 Syndrome using a Conditional Sleeping Beauty Transposon Somatic Insertional Mutagenesis Screen in Mice

Vincent W Keng, Eric Rahrmann, Barbara Ryan, Kevin Silverstein, Aaron Sarver, Walter Jessen, Adam Dupuy, Nancy Jenkins, Neal Copeland, Nancy Ratner, David A Largaespada

INTRODUCTION

Neurofibromatosis type 1 (NF1) syndrome is an autosomal dominant inherited disease in which a majority of patients develop plexiform and/or dermal neurofibromas. Of great concern is that ~10% of NF1 patients develop malignant peripheral nerve sheath tumors (MPNST), which often develop from plexiform neurofibromas and have a poor prognosis. In order to further understand the genetic changes that drive plexiform neurofibroma and MPNST, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen for mutations that can promote progressive neurofibroma and/or MPNST formation in the context of somatic inactivation of the Nf1 gene in Schwann cells and their precursors, using a conditional NF1-flox/flox allele.

METHODS

We used a conditional SB expression system where the SB transposase (SB11) is knocked into the Rosa26 locus but its expression is initially blocked by a floxed-stop cassette (lsl) in the Rosa26-lsl-SB11 transgene. The desert hedgehog regulatory sequence driving Cre recombinase was used to elicit recombination of the NF1-flox/flox allele and Rosa26-lsl-SB11 transgene, allowing for inactivation of the NF1 gene and the initiation of SB transposase expression in Schwann cells. When combined with a transposon mutagenic vector (T2/Onc) capable of either disrupting tumor suppressor genes or inducing oncogenes, we have a powerful conditional SB somatic mutagenesis forward genetics screen for cancer-associated genes.

RESULTS

From neurofibromas isolated from experimental mice, we identified candidate genes involved in tumorigenesis using a high-throughput pyrosequencing technique. After searching for the human homologs of candidate genes in expression microarray analyses of human cell lines, neurofibroma, and MPNST samples, initial results indicate distinct changes in the expression profiles of certain genes during disease progression from neurofibromas to MPNST.

CONCLUSION

These genes may be important mutational drivers for disease progression from benign neurofibroma to MPNST. Therefore, genes identified in this screen will give a better understanding of the underlying genetic mechanisms of neurofibromatosis.

Neuro-Oncol. 2009 Oct;11(5):639–640.

330. Three End Points of Tumor Radiation Therapy in Vivo and their Statistical Estimation

Eugene Demidenko

INTRODUCTION

The existing practice of tumor growth delay data analysis from animal experiments suffers from imprecision and large standard errors that yield low power and statistically insignificant treatment effect. The goal of the present paper is to review the existing end points of tumor radiation therapy in experimental radiobiology with an emphasis on their efficient estimation for statistically significant identification of the treatment effect. Doubling time, tumor growth delay, and cancer-cell surviving fraction in vivo are mathematically defined using exponential growth and regrowth models with tumor volume measurements obtained from animal experiments.

METHODS

A statistical-model-based approach is used to define and efficiently estimate the three end points of tumor therapy in experimental cancer research.

RESULTS

The relationship between cancer cell surviving fraction, tumor growth delay, and rate of growth is rigorously established. The log scale is advocated for plotting the tumor volume data and the respective analysis. The geometric mean should be used to display the mean tumor volume data, and the t-test for the group comparison should be done for the log volume to comply with the Gaussian distribution assumption.

CONCLUSION

The existing practice of tumor growth delay data analysis should be changed to a more sophisticated analysis based on the modeling approach on the log scale. The author suggests a complete set of instructions and the code based on the public-domain statistical package R on his Web site, www.dartmouth.edu/~eugened.

Neuro-Oncol. 2009 Oct;11(5):640.

331. Morphologic, Immunohistochemical, and Therapeutic Response Characterization of Tumors Arising from the Spontaneous Transformation of P53-Null Subventricular Zone Cells

C David James, Rintaro Hashizume, Yasuyuki Aoki, Claudia Petritsch, Jeanette Hyer, J Graeme Hodgson, Joanna Phillips

INTRODUCTION

The subventricular zone (SVZ) is an extensive germinal layer that concentrates neural and glial progenitor cells and has been considered a source of astrocytoma precursor cells. Inactivation of p53 is a relevant tumor suppressor gene alteration for the development of astrocytoma.

METHODS

Athymic mice were injected with luciferase-modified p53-null SVZ cells, or the same cells modified with human telomerase reverse transcriptase (hTERT) and human papilloma virus E6/E7. Cells were monitored for proliferation by bioluminescence imaging (BLI), and resultant tumors were harvested for use in orthotopic allograft therapy response experiments and for histopathologic as well as immunohistochemical characterization.

RESULTS

BLI revealed that subcutaneous (SC) injection of p53-null + hTERT + E6/E7 cells resulted in immediate tumor establishment with progressive growth. In contrast, SC injection of p53-null cells showed no indication of growth for 100 days but thereafter revealed establishment of a rapidly growing tumor that was used as a source of cells for intracranial injection in a series of 20 mice, 10 of which were treated with temozolomide. Temozolomide treatment significantly extended the survival of mice with an IC tumor (p < 0.0001). p53-null SVZ tumors show high cellularity and immunohistochemical evidence of neuronal and glial differentiation.

CONCLUSION

This study makes the following points regarding the use of mouse p53-null SVZ cells: 1) p53-null SVZ cells have the ability to spontaneously transform in vivo and the transformed p53-null SVZ cells can be used in orthotopic animal model therapeutic response studies. The intracranial tumors generated by spontaneously transformed p53-null SVZ cells have both neuronal and glial immunohistochemical characteristics. We are currently conducting analyses to determine gene alterations and gene expression changes that occur during the spontaneous transformation of p53 null cells and will report the results of these analyses at the meeting.

Neuro-Oncol. 2009 Oct;11(5):640.

332. Berubicin: A Novel Topoisomerase II Inhibitor with Activity in Ependymoma

Dimpy Koul, Izabela Fokt, Su Guan, Charles A Conrad, Timothy Madden, WK Alfred Yung, Waldemar Priebe

INTRODUCTION

Ependymomas are rare CNS tumors, occuring predominantly in children. Tumor size can make gross total resection exceptionally difficult, and postoperative radiation may be of benefit, but CNS toxicity prevents its widespread application in patients <2 years old. Development of new therapies for this tumor is limited by the absence of optimal in vivo and in vitro model systems. Given these limitations, the development of new agents with novel antineoplastic actions is urgently needed. Previous studies of berubicin in patients with glioblastoma demonstrated that berubicin is active and crosses the blood-brain barrier to reach therapeutic concentrations in the CNS.

METHODS

In vitro studies to assess the cytotoxicity and growth inhibitory effects of berubicin were performed in ependymoma cell lines. These studies demonstrated the potent cytotoxic activity of berubicin against this tumor type.

RESULTS

Berubicin, in vitro, possesses growth inhibitory activities in ependymoma cell lines with IC50s ranging from 50 to 100 nM. Treatment with berubicin induced a strong apoptotic response in all ependymoma cell lines tested. Cancer stem cells isolated from ependymoma tumor specimens demonstrated extreme sensitivity (10-fold more than glioma cell lines) to berubicin. Berubicin treatment did not result in any change in ABCG2 (BCRP) and MDR-1 expression levels in the ependymal cells. Previous phase I clinical studies of berubicin showed the dose-limiting toxicity was myelosuppression, with an MTD of 7.5 mg/m2/day given for 3 consecutive days and every 21 days. Pharmacokinetic studies showed dose-independent clearance, with a mean half-life of 36 h and a large volume of distribution.

CONCLUSION

Berubicin is a new anthracycline derivative that demonstrates excellent in vivo activity in human brain tumors (primarily gliomas), in vitro activity against a variety of ependymoma cell lines, and favorable drug-like properties. These data suggest that a clinical trial of berubicin in patients with recurrent ependymal tumors is warranted.

Neuro-Oncol. 2009 Oct;11(5):640.

333. IκB Kinase Inhibitor, BMS-345541, for the Treatment of Methylator-Resistant Tumors

Bakhtiar Yamini, Nassir H Monim Mansour, Charles Kulwin, Xiaohong Yu, Elliot Arsoniadis, Ralph R Weichselbaum, Adam Schmitt

INTRODUCTION

Resistance of malignant gliomas to chemotherapeutics is an important cause of poor therapeutic response and early tumor recurrence. An acquired mismatch repair (MMR) deficiency is often seen in tumors that are treated with ionizing radiation and temozolomide (TMZ). As glioblastoma multiforme (GBM) recurrence is virtually universal, and as TMZ is an important antiglioma agent, resistance to TMZ is a significant obstacle to the overall management of malignant glioma. We recently reported a link between TMZ and nuclear factor-κB (NF-κB) showing that, in MMR-proficient cells, TMZ blocks NF-κB activity. This finding suggests that TMZ may induce cytotoxicity by inhibition of NF-κB. We hypothesize that inhibition of NF-κB may have a significant antiglioma effect in combination with TMZ in tumors that are both sensitive and resistant to TMZ caused by MMR deficiency. The therapeutic efficacy of combining TMZ with the selective NF-κB inhibitor, BMS-345541, was examined both in cells and animals.

METHODS

The antiglioma effect of the combination of BMS-345541 and TMZ was examined using both an Annexin V binding assay and colony forming assay. Subsequently, animal xenograft models, using U87 glioma cells and MMR-deficient HCT116 cells, were assessed for antitumor effects. Pathological and immunohistochemical examination of tumors was used to assess apoptosis.

RESULTS

Combination treatment with BMS-345541 and TMZ results in a significant increase in cytotoxicity and tumor growth delay compared with that seen with either agent alone (p < 0.05). Examination of activated caspase 3 demonstrates that induction of apoptosis, and not necrosis or vascular thrombosis, contributes to the delay in tumor growth seen following combination treatment. The combination of BMS-345541 and TMZ also demonstrated significant activity against an MMR-deficient tumor as compared with either agent alone (p < 0.05).

CONCLUSION

This study demonstrates that the inhibition of NF-κB in combination with TMZ can be a promising strategy in the treatment of sensitive and resistant malignant gliomas.

Neuro-Oncol. 2009 Oct;11(5):640.

334. Hyperpolarized 13c MR Metabolic Imaging of Brain Tumors

Ilwoo Park, Peder EZ Larson, Matthew L Zierhut, Simon Hu, Robert Bok, Tomoko Ozawa, John Kurhanewicz, Daniel B Vigneron, Scott R VandenBerg, C David James, Sarah J Nelson

INTRODUCTION

The Dynamic Nuclear Polarization (DNP) method for dissolution of hyperpolarized compounds has allowed for in vivo metabolic imaging with a more than 50,000-fold signal increase. The purpose of this study was to explore the feasibility of using 13C magnetic resonance spectroscopic imaging (MRSI) with hyperpolarized [1-13C]-pyruvate as a substrate for evaluation of in vivo brain tumors by comparing hyperpolarized 13C-MRSI data from rats with and without intracranial human xeno-graft tumors. In conducting this study, we characterized and compared 13C-imaging parameters with results from histology/immunochemistry for two different tumor types.

METHODS

Nine athymic rats with intracranial implantation of human glioblastoma cells (four U-251 MG and five U-87 MG xenograft model) and six normal rats were included in the study. 13C-2D-MRSI data were acquired using a 3T with a custom 1H/13C rat coil. Proliferation (MIB-1), hypoxia (CA-9), and percent necrosis were evaluated from the resected brains of rats with tumors.

RESULTS

The 13C lactate and pyruvate data exhibited significantly higher SNR in the tumors than normal brain tissue. Statistical analysis showed significant differences in all 13C imaging parameters (p < 0.0004) between tumor and normal brain. The SNR of lactate, pyruvate, and total carbon were significantly different between the brains of rats with U-251 MG and U-87 MG tumors. The histology/immunochemistry also confirmed distinct patterns between the two tumor types. There was a significant difference in the percent necrosis (p < 0.02) and a strong trend toward differences (p = 0.06) in CA-9 index between the U-251 MG and U-87 MG tumors. The MIB-1 index (%) and the SNR of lactate appeared to be correlated (r = 0.8) for each type of tumor.

CONCLUSION

The results from this study suggest that this technique may be useful in assessing prognosis and monitoring response to therapy in preclinical models, and ultimately, in patients with brain tumors.

Neuro-Oncol. 2009 Oct;11(5):641.

335. Selective Induction of Optic Nerve Tumors by Notch3

Charles G Eberhart, Tarran Pierfelice, Karisa Schreck, Louis Dang, Nicholas Gaiano

INTRODUCTION

It is not clear in what cells Notch activation is sufficient to induce tumorigenesis in the brain. We have previously shown that expression of the Notch3 intracellular domain (NICD3) can drive formation of choroid plexus papillomas when fetal (embryonic day 9.5) mice are injected in the CNS with a retrovirus encoding this constitutively active receptor fragment. Herein, we report an expanded spectrum of tumors, including optic nerve gliomas.

METHODS

Retroviral constructs were injected into the ventricles of fetal mice on embryonic day 9.5 under ultrasound guidance. Tumors were analyzed by an experienced neuropathologist (CGE) using standard techniques. PLAP histochemical stains were used to confirm viral infection.

RESULTS

NICD3 expression was sufficient to induce invasive GFAP-immunopositive glial tumors of the optic nerve and retina in 35 of 48 animals examined (73%). The morphology was relatively bland and, in some ways, resembled human optic nerve gliomas. However, the tumors contained more mitotic figures than pilocytic tumors arising at this site and commonly invaded out of the eye into the orbital fat, a feature not seen in humans. Interestingly, invasive gliomas were not identified intracranially, suggesting that optic nerve astrocytic/glial precursors are especially sensitive to transformation by NICD3. Indeed, introduction of active forms of the Notch1 and Notch2 receptors (NICD1 and NICD2) resulted in an increased number of infected cells with astrocytic morphology in the optic nerve as compared with control vector, but no invasive tumors formed.

CONCLUSION

Our findings highlight the fact that glial cells in different anatomical locations of the CNS can be dramatically different in their susceptibility to transformation by activated Notch receptors and suggest that optic nerve gliomas may be caused in some cases by activation of the Notch pathway.

Neuro-Oncol. 2009 Oct;11(5):641.

336. Bioluminescent Medulloblastomas Induced by Specific Somatic Cell Introduction of FoxG1, Mycn, and Sonic Hedgehog in a New Transgenic Mouse Model System

Gregory M Shackleford, Shahab Asgarzadeh, Anat Erdreich-Epstein, Ignacio Gonzalez-Gomez, Rex Moats, Robert C Seeger, Xiang-He Shi, Kimberly Mittelholtz

INTRODUCTION

We have generated a new transgenic mouse model system for medulloblastoma that enables multiple genes-of-choice to be introduced into neonatal cerebellar external granular layer (EGL) cells using retroviral vectors and, moreover, allows tumor growth and regression to be monitored in vivo with bioluminescence imaging. The transgenic mice express genes for the avian retrovirus receptor, Tva, and an eGFP-luciferase fusion protein specifically in the EGL cells of the developing cerebellum via a Barhl1 promoter. Since Barhl1 and, thus, the transgene are expressed in EGL cells for only a few weeks after birth, tumors produced in these Barhl1-positive cells were expected to continue to express luciferase whereas the developing normal cerebellar cells would lose background expression as they mature and differentiate.

METHODS

As proof of principle, neonatal transgenic cerebella were injected with a mixture of cells producing avian viral vectors containing sonic hedgehog (Shh) and a stabilized mutant of Mycn (T50A,S54A). Viruses containing Shh and the forkhead box G1 protein (FoxG1), a winged-helix transcription factor associated with high-risk medulloblastomas, were also injected experimentally.

RESULTS

The Shh plus Mycn (T50A,S54A) viruses caused medulloblastomas in approximately 50% of mice within 3 months. Time course studies showed that tumors continued to express the luciferase-containing transgene but normal cells did not, thus, allowing imaging of bioluminescent tumors on a bioluminescence-negative background. Tumors could be detected by imaging as early as 4 weeks of age. In ongoing experiments, Shh plus FoxG1 viruses are also causing medulloblastomas more frequently than either virus alone.

CONCLUSION

FoxG1 cooperates oncogenically with Shh to cause medulloblastomas in this model. This single new transgenic mouse model enables multiple genetic models for medulloblastoma to be produced by retroviral infection of specific somatic cells and promises to allow facile bioluminescent analysis of tumor growth and therapy-induced regression in biological and preclinical studies.

Neuro-Oncol. 2009 Oct;11(5):641.

337. Derailed Receptor Tyrosine Kinase Endocytosis in Glioblastoma: Sorting Nexin 3 Disrupts EGFR and MET Trafficking Promoting Cell Proliferation and Tumorigenicity

Dongh-Anh Khuong-Kahn, Takrima Haque, Damien Faury, Brian Meehan, Janusz Rak, Steffen Albrecht, Nada Jabado

INTRODUCTION

Amplification/mutation or rearrangement of receptor tyrosine kinases (RTKs) occurs in a large proportion of adult glioblastoma multiformes (GBMs) (~60%) and plays a major role in gliomagenesis. Unlike adult GBMs, pediatric GBMs show no genetic amplification/mutation of RTKs. Using microarray data we generated, we identified and validated overexpression of sorting nexin 3 (SNX3), a protein involved in the regulation of intracellular trafficking and signaling of EGF-EGFR complexes from within the endosomes, as potentially involved in oncogeneis of pediatric GBM. Based on our preliminary data, we hypothesize that the paradigm established for adult GBMs, whereby oncogenic RTK signaling drives tumorigenesis and Ras activation applies in a subset of pediatric astrocytomas. However, oncogenic upregulation and activation of RTKs in children is driven by unique mechanisms, which involve dysregulated SNX3 expression/activation.

METHODS

We stably overexpressed C-myc-tagged SNX3 in pGBM (SF188, SJ-G2) and adult GBM (U87) cell lines. In parallel, we knocked-down SNX3 expression in these cell lines. We investigated the effects of overexpression/silencing of SNX3 on EGFR and cMet levels and activation, cell signaling (Ras and Akt pathways), cell proliferation (monolayer/soft agar assays, xenograft mouse models), and cell invasion (matrigel assays).

RESULTS

Myc-tagged SNX3 overexpression increased RTK levels and delayed their degradation following EGF/HGF stimulation as compared with empty-vector transfectants. This led to increased and sustained activation of the Ras pathway in SNX3 transfectants, a drastic increase in cell proliferation in monolayer and soft agar assays, and in invasion in matrigel assays. Importantly, it increased tumor formation and tumor size in a xenograft nude mouse model. Silencing SNX3 in SF188 led to the reverse in knocked-down cell lines.

CONCLUSION

RTKs are major oncogenes in GBM. Key findings in this study are that multiple biological events can lead to the same phenotype. In this instance, perturbing endo-somal recycling in pediatric GBM may lead to increased RTK signaling and cell proliferation similar to oncogenic mutations/genetic amplification affecting these molecules in adult GBM. Importantly, we also show that gliomagenesis in children is driven by unique mechanisms.

Neuro-Oncol. 2009 Oct;11(5):641.

338. Neurocognitive Function in Patients with Glioblastoma at First or Second Relapse Treated with Bevacizumab Alone or in Combination with Irinotecan in the Brain Study

Jeffrey S Wefel, Timothy F Cloughesy, Jim Zazzali, Maoxia Zheng, Meghna Samant, Asha Das, Henry Friedman; for the BRAIN Investigators

INTRODUCTION

Patients with glioblastoma (GBM) suffer from neurocognitive decline due to the disease and its treatment. We analyzed neurocognitive function of patients with recurrent GBM who participated in BRAIN, a noncomparative clinical trial of bevacizumab (BEV) alone or in combination with irinotecan (BEV + CPT-11) (ClinicalTrials.gov no. NCT00345163).

METHODS

Patients receiving BEV (n = 85) or BEV+ CPT-11 (n = 82) were assessed with the Hopkins Verbal Learning Test-Revised, Trail Making Test parts A and B, and the Controlled Oral Word Association Test. Brain imaging and neurocognitive assessments were conducted at baseline and then every 6 weeks while patients remained on the study drug, up to 52 weeks. Neurocognitive function was categorized as improved, stable, or declined, using the reliable change index (RCI). Changes from baseline neurocognitive function were confirmed at the next assessment, when available.

RESULTS

Neurocognitive testing was completed by 85 (98%) patients at baseline and 80 (94%) patients at week 24. Of the BEV and BEV+CPT-11 patients who had an independent review facility (IRF)-determined objective response (OR), 75.0% and 60.7%, respectively, demonstrated stable or improved performance on all tests at the time of response relative to baseline. Of the BEV and BEV+CPT-11 patients who were progression free for 6 months, 70.4% and 70.0%, respectively, demonstrated stable or improved performance on all tests at week 24 relative to baseline.

CONCLUSION

The majority of patients with an IRF-determined OR had improved or stable neurocognitive function at the time of response, compared to baseline. Similar improvements and stability in neurocognitive function were observed for patients who were progression free for 6 months. Given the exploratory nature of these analyses, absence of historical controls, and noncomparative treatment arms, the results should be interpreted cautiously.

Neuro-Oncol. 2009 Oct;11(5):642.

339. Pilot Study of Sensory Information Processing and Motor Processing: Relationship to Executive Control in Adults with Primary Brain Tumors

Deborah H Allen, Barbara W Carlson, Matthew G Ewend, Sharon Cush, Renee D Raynor, Annick Desjardins, William B Nix, Virginia J Neelon

INTRODUCTION

Increasing emphasis is being placed on maintaining independent function following treatment in adult survivors of primary brain tumors (PBT). In particular, disturbances in executive control function (ECF) are thought to be important to independent function, yet the level of ECF disturbance to independent function remains unclear. One reason for this uncertainty may be that ECF includes both cognitive and noncognitive domains, like psychomotor control and sensory perceptual function, are not often captured using standardized test batteries. Thus, the aim of this pilot study is to describe differences in ECF as determined by a test battery that measures sensory and motor processing in addition to cognitive components of ECF in adults with PBT and healthy controls.

METHODS

The sample consists of 11 adults with PBT treatment completed and 11 age-matched, gender-matched, healthy controls, ages 30–55 years, with Mini Mental State Examination scores greater than 24. All subjects underwent a test battery that includes the EXIT-25, Quantitative Sensory Test (QST), Digitized version of the Trails B (DTB), OARS Activities of Daily Living Scale (OARS), and Center for Epidemiological Studies-Depression Scale (CESD).

RESULTS

Enrollment is ongoing with full results expected in September 2009. Analysis for the Trails B relates to the standard measure of completion time between groups and additional measures from the digitized version on individual segment strokes from one number/letter to the next (number of pauses, peak velocities, time between segments, depression depth). The relationship between performance on the DTB and the EXIT-25, QST, CESD, and OARS will be examined.

CONCLUSION

Adding measures of psychomotor control and sensory perceptual function to the standard measures of ECF may provide a more clinically relevant evaluation of ECF in adult survivors with PBT. In addition, this pilot will serve as the framework for a larger study to evaluate changes in ECF over time in adults with PBT.

Neuro-Oncol. 2009 Oct;11(5):642.

340. Neurocognitive Outcomes Associated with Combined Levothyroxine/Liothyronine Supplementation in Hypothyroid Patients with Primary Brain Tumors

Jeffrey S Wefel, Surabhi Patwardhan, Victor A Levin

INTRODUCTION

Impaired neurocognitive function (NCF) is common in primary brain tumor (PBT) patients and reflects tumor burden and treatment effects. PBTs and their treatment can impair the hypothalamic-pituitary axis, increasing the possibility of central hypothyroidism and associated impaired NCF. To investigate whether the addition of synthetic triiodothyronine improved NCF, we conducted a single-arm, open-label pilot study of liothyronine added to levothyroxine for hypothyroid PBT patients previously treated with external beam irradiation.

METHODS

PBT patients on stable doses of levothyroxine were placed on liothyronine with the objective of maintaining a treatment ratio between levothyroxine and liothyronine of 4:1 or 5:1. Assessment of NCF was performed before starting liothyronine and after 8 weeks of therapy. NCF domains assessed included attention, processing speed, memory, executive function, and motor dexterity. Symptom and quality of life questionnaires were administered.

RESULTS

Ten PBT patients participated; the average age was 47 years and 70% were women. The majority (60%) of patients had anaplastic astrocytoma or anaplastic oligoastrocytoma. Frontal lobe tumor location was seen in 60%. Paired t-tests and effect sizes were calculated. Paired t-test revealed a statistically significant decline in memory function (t = 2.497, p < 0.05, Cohen’s d = 0.83). Graphomotor speed demonstrated statistically significant improvement (t = −2.648, p < 0.05, Cohen’s d = 1.05). A trend toward improvement in executive function was also seen (t = 2.239, p = 0.052, Cohen’s d = 0.71).

CONCLUSION

In this small pilot study, liothyronine improved measures requiring psychomotor processing speed, but did not improve memory function. Benefit in executive function was also indicated. Given the mixed pattern of NCF outcomes, there is mild enthusiasm to pursue a larger placebo-controlled trial to evaluate the potential efficacy of combined levothyroxine and higher dose liothyronine to palliate cognitive dysfunction associated with hypothyroidism in irradiated PBT patients.

Neuro-Oncol. 2009 Oct;11(5):642.

341. Sexuality and Intimacy Perceived by Brain Tumor Patients and their Partners

Laurie Lyn Rice, Jeffrey Raizer, Czarina Serra

INTRODUCTION

This pilot study seeks to systematically explore the issues of sexuality and intimacy in patients diagnosed with primary brain tumors and their partners. Brain tumors may lead to physical and psychosocial concerns for patients and their partners. Intimacy and sexuality are significant concerns that are rarely addressed. Brain tumor treatments may be focused on survival and considered more important than addressing sexual function and intimacy. A previous study revealed that palliative care patients’ intimacy and sexuality were affected more than those of patients without cancer. An Australian study found mismatched expectations between patients and health care professionals and unmet patient needs in the areas of intimacy and sexuality. The professionals felt that the patients shared their professional focus on fighting the disease.

METHODS

Patients were identified from the neurooncology or neurosurgery clinic. Sixteen patient/caregiver dyads were administered questionnaires: Female Sexuality Function Index (FSFI), Sexual Health Inventory for Men (SHIM), Emotional Index Scale (EIS), BN20 and European Organisation for Research and Treatment of Cancer Quality of Life (EORTC QOL). These were completed at one setting without discussion between the dyads.

RESULTS

Demographics: patients: men 11, women 5; caregiver group: men 6; women 8. Working status: patients: yes 8, no 7, retired 1; caregivers: yes 10, retired 2. Age (mean): patients: 43, caregivers: 40. Length of diagnosis: mean 48 months. FSFI: sexual dysfunction in both patients and caregivers; SHIM issues: patients more than caregivers; EIS: misunderstandings between patient and caregiver; BN20 issues: future uncertainty, communication deficits, headaches and drowsiness; EORTC issues: areas of cognitive and social functioning, nausea/vomiting and constipation.

CONCLUSION

Patients experience mild erectile dysfunction and female sexual dysfunction. Health care providers need to be aware that sexual and intimacy issues should be addressed at some point during ongoing care. Miscommunications exist between caregivers and patients, in which counseling and intervention may help prevent caregiver burnout and improve relationships.

Neuro-Oncol. 2009 Oct;11(5):642.

342. Understanding the Molecular Bases of Chemotherapy-Induced Cognitive Impairment: Normal Neural Precursors Have Differential Sensitivity to Commonly used Chemotherapy Drugs

Kelsey Albert, Philip Schwartz, Daniela A Bota

INTRODUCTION

The role of neural stem/precursor cells (NPCs) in memory and attention is increasingly recognized. However, there is a need to identify the role NPCs play in the development of chemotherapy-induced cognitive decline. In this study, we first report that nonspecific DNA-damaging chemotherapy agents are toxic to NPCs, and that more targeted drugs do not affect the normal cells while killing the malignant glioma stem/precursor cells (GPCs).

METHODS

We used three human NPC cultures, and multiple GPC cultures. All these cells form spheres and differentiate in appropiate conditions.

RESULTS

Treatment of GPCs with erlotinib caused cell death at low concentrations, with 4 μM causing a 50% decrease in viability. In contrast, treatment of NPCs with erlotinib as high as 50 μM did not affect cell viability, which correlates with low epidermal growth factor receptor (EGFR) expression. GPCs were very sensitive to bortezomib, with doses as low as 0.5 nM causing more than 80% cell death, proportional with the base-line proteosome levels. At similar doses, NPCs were less affected, with viabilities of more than 70%. The GPCs were resistant to temozolomide, with doses as high as 750 mM producing only a 25% decrease in viability. NPCs, on the other hand, experienced a 50% decrease in viability at a dose of 100 mM. Finally, cisplatin treatment at 5 mM led to viability of less than 20% of NPCs but 70% of GPCs. The NPCs’ sensitivity to temozolomide and cisplatin correlates with low expression of the drug resistence gene ABCG2.

CONCLUSION

We have identified that EGFR-TK inhibition as well as proteosome inhibition are effective against GPCs, while producing minimal effects on NPCs. In contrast, temozolomide and cisplatin are more toxic for NPCs than for GPCs. As the ultimate goal of chemotherapy is the eradication of GPCs while maintaining patient cognition, that is, preserving normal NPCs, the development and use of more selective drugs is warranted.

Neuro-Oncol. 2009 Oct;11(5):643.

343. Symptoms of Inattention in Survivors of Pediatric Brain Tumors: Do Survivors Meet Symptom Criteria for ADHD?

Kristina K Hardy, Victoria W Willard, Taryn M Allen, Sridharan Gururangan, Melanie J Bonner

INTRODUCTION

Survivors of pediatric brain tumors, especially those treated with cranial radiation, often go on to experience declines in working memory and inattention. Symptoms are associated with academic problems and difficulty with social relations. This pattern of symptoms may be similar to those of children diagnosed with attention deficit hyper-activity disorder predominantly inattentive subtype (ADHD-IN). Indeed, many of the interventions aimed at improving functioning in survivors (e.g., school accommodations and stimulant medication) have been adapted from those used for ADHD. Moreover, in many school systems, having such a diagnostic label may be the only mechanism through which survivors gain access to supportive services. However, no studies have directly compared survivors with healthy children meeting criteria for ADHD.

METHODS

Thirty-four survivors were age-, sex-, and race-matched with a comparison group of children diagnosed with ADHD-IN. Both groups completed the Conners’ Parent Rating Scale, a widely used questionnaire assessing ADHD symptoms.

RESULTS

ADHD-IN diagnosis is considered when children exhibit at least six of nine possible symptoms of inattention. Although children with ADHD experienced significantly greater ADHD inattentive symptoms than did survivors (mean = 6.8 vs. 3.1, respectively; t = 6.19, p < 0.001), 22% of survivors exhibited at least six symptoms. Symptoms endorsed most frequently in survivors included “makes careless mistakes,” (50.0%) “doesn’t follow through on tasks,” (39.9%) “is forgetful” (38.2%), and “trouble organizing tasks” (44.1%). Moreover, children who were younger at diagnosis experienced significantly greater symptoms of inattention.

CONCLUSION

Survivors exhibit fewer symptoms of inattention than do youth with ADHD, though several symptoms are experienced by a majority of survivors. It is reasonable to model interventions for survivors on the ADHD population, though diagnostically, only about one-fifth of survivors actually meet symptom-criteria for the disorder. Future research should examine whether ADHD treatments are effectively targeting the scope and severity of problems survivors experience.

Neuro-Oncol. 2009 Oct;11(5):643.

344. Neurocognitive Outcome 12 Months Following Cerebellar Mutism Syndrome (CMS) in Pediatric Patients with Embryonal Tumors: Results of a Prospective Study with Matched Controls

Shawna L Palmer, Tim Hassall, Murali Chintagumpala, Sridharan Gururangan, Eric Bouffet, David Ashley, Richard Cohn, Amar Gajjar

INTRODUCTION

Neurocognitive deficits are well documented among children treated for embryonal tumors. In comparison, few studies exist specifically examining the cognitive outcomes for those children who are diagnosed with cerebellar mutism syndrome (CMS), a distinct postoperative syndrome that typically arises 24–48 h following resection and is characterized by mutism, emotional lability, hypotonia, and ataxia.

METHODS

Children enrolled in an ongoing IRB-approved treatment protocol for treatment of embryonal tumors, diagnosed with postoperative CMS, who were at least 12 months postdiagnosis and had completed prospectively planned neuropsychological evaluation using the Woodcock-Johnson Tests of Cognitive Abilities-Third Edition, were considered eligible (n = 24). The cognitive outcomes of these patients were compared to 24 patients from the same protocol matched with regard to primary diagnosis, age at diagnosis (3.25–16.88 years), and risk/corresponding treatment (16 average-risk pairs, 8 high-risk pairs). Sixteen were also matched according to gender, and 17 were also matched according to race. High-risk patients received 36–39.6 Gy craniospinal irradiation (CSI) and 3-D conformal boost to the primary site to 55.8–59.4 Gy. Average-risk patients received 23.4 Gy CSI and 3-D conformal boost to the primary site to 55.8 Gy.

RESULTS

Using paired sample t-tests, significant group differences were found in cognitive outcomes. While the matched control patients exhibited performance in the average range (M = 100, SD = 15) for all outcomes, patients who developed CMS postsurgery were found to have significantly lower performance in decision speed (p < 0.001), visual matching (p < 0.001), processing speed (p < 0.001), attention (p < 0.001), working memory (p = 0.028), and cognitive efficiency (p < 0.001).

CONCLUSION

Patients treated for embryonal tumors who experience postoperative CMS show an increased risk for neurocognitive impairment. These impairments are evident as early as 12 months following diagnosis. This study highlights the need for careful neuropsychological evaluation and, based on the findings, critical support for these patients and their families.

Neuro-Oncol. 2009 Oct;11(5):643.

345. Sluggish Cognitive Tempo in Survivors of Pediatric Brain Tumors

Victoria W Willard, Taryn M Allen, Kristina K Hardy, Kendra A Sawyer, Sridharan Gururangan, Melanie J Bonner

INTRODUCTION

Neurocognitive deficits, including deficits in attention, working memory, and processing speed, experienced by survivors of pediatric brain tumor (BT) have been well-documented in the literature over the past three decades. Sluggish Cognitive Tempo (SCT) has been identified as a cognitive style exhibited by a subset of children with attention deficit hyperactivity disorder (ADHD). SCT is characterized by symptoms of lethargy, the appearance of confusion, frequent daydreaming or staring into space, and fatigue. These symptoms are associated with cognitive and academic deficits in children with ADHD. Moreover, symptoms of SCT have been documented in survivors of acute lymphoblastic leukemia (ALL) but have never been assessed in a BT population. Thus, our objective was to evaluate the presence of SCT symptoms in a sample of pediatric BT survivors compared to healthy controls.

METHODS

Participants were 50 survivors of pediatric BT (at least 1 year off-therapy; mean age = 12.8, SD = 3.56) who had completed a routine assessment of neurocognitive functioning. Parents completed the Child Behavior Checklist (CBCL), a measure of emotional and behavioral functioning which includes five items assessing SCT. Parents of 50 healthy controls (mean age = 10.2, SD = 3.32) also completed the CBCL.

RESULTS

Survivors of pediatric BT exhibited significantly greater levels of SCT compared to controls (mean = 3.1 vs. 1.3, respectively; t = 4.01, p < 0.001). In contrast to previous work, IQ scores were unrelated to levels of SCT symptoms. Higher levels of SCT symptoms correlated with more math achievement problems, participation in fewer extracurricular activities, greater attention problems, and increased internalizing difficulties.

CONCLUSION

Survivors of pediatric BT exhibit high levels of SCT symptoms compared to healthy controls. SCT may be a useful way to characterize the broad cognitive style exhibited by many survivors and may also allow clinicians to make use of existing strategies to improve these symptoms.

Neuro-Oncol. 2009 Oct;11(5):643.

346. The Imaging and Neuropathological Effects of Bevacizumab (Avastin) in Patients with Leptomeningeal Carcinomatosis

Denise M Damek, Bette K Kleinschmidt-DeMasters

INTRODUCTION

Bevacizumab is an antiangiogenic drug known to prolong progression-free survival and reduce edema and mass effect in patients harboring malignant gliomas. A dramatic reduction of tumor enhancement after gadolinium administration is often seen on serial imaging studies in treated patients. However, it remains unclear whether this represents a true antitumor effect or an artifact of the drug’s effect on vascular permeability around the tumor. In addition, multifocal tumor spread within the brain is increasingly recognized as a late sequela in patients receiving bevacizumab. Minimal information exists about the effect of the drug on patients with metastatic brain disease.

METHODS

We report two patients with known non-small-cell lung carcinomas who had received bevacizumab for their systemic disease and then developed cognitive deficits with unusual neuroimaging abnormalities, ultimately proven to be secondary to leptomeningeal carcinomatosis (LC).

RESULTS

The most prominent clinical and neurological examination findings in both of these patients were symptoms and signs of white matter dementia. Neuroimaging features included minimal to absent leptomeningeal enhancement and unusual perivascular and punctate hemorrhagic lesions. Neuropathological assessment confirmed LC, and in an autopsy case, further disclosed extraordinary perivascular spread of individual tumor cells along blood vessels to the depth of capillaries. Small infarctions were associated with perivascular tumor deposits and vasculopathy.

CONCLUSION

Bevacizumab alters neuroimaging characteristics in metastatic leptomeningeal disease. It may also alter the pattern of tumor spread of LC, promoting growth of tumor cells along blood vessels similar to vascular “cooption” by tumors seen in preclinical animal models of bevacizumab. More cases will need to be studied to confirm this latter finding. Our findings alert other clinicians and pathologists to look for similar unusual neuroimaging findings and tissue alterations in the brain of LC patients treated with bevacizumab.

Neuro-Oncol. 2009 Oct;11(5):644.

347. Upregulated Glioma Endothelial Cell Marker Genes as Tumor Biomarkers in Patient Peripheral Blood

Melissa Whiteman, Eleanor B Carson-Walter, Kevin A Walter

INTRODUCTION

Recent evidence suggests that bone marrow-derived circulating endothelial progenitor cells (cEPCs) can be recruited to the neovasculature of various tumors, including glioblastoma multiforme (GBM), and be incorporated into tumor microvessels. cEPCs may, therefore, contribute to tumor vasculogenesis and serve as tumor biomarkers for diagnosis or monitoring of response. There are no definitive markers for cEPC and the identification of new, specific markers for these cells is required. Previously, we identified 21 glioma endothelial marker genes (GEMs) specific to the tumor vasculature. We now evaluate expression of these GEMs and known cEPC markers in the blood and brain tumor vessels in GBM and metastatic (MET) patient samples.

METHODS

Peripheral blood mononuclear cells (PBMCs), which contain cEPCs, were obtained from tumor patients and healthy donors. qRT-PCR was utilized to analyze expression of cEPC-specific markers in the blood of tumor patients and controls. Patient matched blood and tissue samples were used to correlate the presence and expression profile of cEPC and GEM markers compared to nonneoplastic controls. To evaluate tracking of cEPCs to the tumors, CFSE-labeled PBMC were administered via the carotid artery to mice bearing intracranial U251MG xenografts. Incorporation of cEPCs in the tumor vasculature and confirmation of cEPC and GEM expression were assessed by coimmunofluorescence.

RESULTS

Known cEPC markers and GEMs were upregulated in the blood of tumor patients but not in control blood specimens. GBM and MET patient tumor sections showed robust staining for the cEPC markers CD133 and VEGFR2, and GEMs, TEM1 and CXCR7. Labeled PBMC administered via the carotid artery tracked specifically to intracranial GBM xenografts and also exhibited GEM and cEPC marker coexpression.

CONCLUSION

Circulating peripheral blood cells track to GBM xenografts and coexpress cEPC and glioma endothelial markers. These cellular markers can be measured in patient peripheral blood and hold potential as tumor biomarkers.

Neuro-Oncol. 2009 Oct;11(5):644.

348. Epstein-Barr Virus-Related Smooth Muscle Tumor Masquerading as an Acoustic Neuroma

David D Gonda, Charles Benjamin Newman, Homeyra Masoumi, Min S Park

INTRODUCTION

The authors present the case of a primary smooth muscle tumor of the cerebellopontine angle masquerading in its presentation as an acoustic neuroma.

METHODS

A 48-year-old HIV-positive man with a CD4 count of 47 presented with a 2-year history of progressive hearing loss on his left side and associated left-sided facial pain. Imaging revealed a cerebellopontine angle mass centered at the internal auditory canal and extending into Meckel’s cave.

RESULTS

The patient underwent a two-stage procedure for resection of the tumor. A left retrosigmoid cran-iotomy for resection of the cerebellopontine angle tumor was performed initially. Two weeks later a left frontotemporal craniotomy was performed for excision of the supratentorial component. The histologic specimens resembled a schwannoma with interwoven fascicles of fusiform spindle cells with elongate bipolar nuclei and rudimentary nuclear stacking suggesting the appearance of Verocay bodies. In consideration of the patient’s HIV status, they performed additional immunohistochemical staining procedures, revealing strong cytoplasmic immunolabeling of the fascicular spindle cell neoplasm for smooth muscle actin and negative results with the S-100. Latent membrane protein 1, normally discovered in Epstein-Barr virus (EBV)-associated lymphomas, was negative. Chromogenic in situ hybridization for EBV-encoded RNAs was positive. Thus, the tumor was accurately identified as an EBV-related smooth muscle tumor.

CONCLUSION

On a clinical, radiological, and even histologic examination EBV-related smooth muscle tumors can appear extremely similar to a schwannoma unless more detailed staining and examination is performed. The differential diagnosis of cerebellopontine angle tumors in immunocompromised patients must include primary smooth muscle tumors, as the incidence is becoming increasingly recognized in this population.

Neuro-Oncol. 2009 Oct;11(5):644.

349. Adult Ependymal Tumors: Prognosis and the University of Texas M.D. Anderson Cancer Center Experience

Terri S Armstrong, Elizabeth Vera-Bolanos, Nebiyou Bekele, Kenneth D Aldape, Mark R Gilbert

INTRODUCTION

Ependymomas in adults are rare and often misdiagnosed. There are limited reports with centralized confirmation of diagnosis and evaluation of prognosis. This study reports on a series of adult patients with confirmed ependymoma treated at M.D. Anderson Cancer Center.

METHODS

Patients who were older than 17 years and had a diagnosis of ependymoma were identified through the M.D. Anderson Neuro-Oncology Database, with clinical data collected retrospectively. Descriptive statistics were used to describe the clinical data, Kaplan-Meier methods were used to generate survival curves, and Cox proportional hazards models were used to evaluate the association of clinical characteristics with survival.

RESULTS

This series included 123 adult patients (51% men; median age, 39 years; range, 18–72). Forty patients had tumors in the brain, 80 in the spine (three had both). The majority were grade I/II lesions (108), and 15 were grade III lesions (anaplastic). Seventeen patients had previously been misdiagnosed and 18 patients had a second cancer diagnosis. The most common presenting symptom was pain, with patients reporting an average of four symptoms prior to diagnosis. Sixty-three percent of patients had a gross total resection, 49% received radiation therapy, and 13% received chemotherapy. Patients were followed for an average of 5.5 years, and 13% had died. Median time to recurrence was 21 months for grade II brain tumors and 18 months for grade III tumors. Worse outcomes, as measured by overall survival and progression-free survival, were associated with brain location (p = 0.01, p = 0.04) and tumor anaplasia (p = 0.0025, p = 0.001). An analysis of tumor tissue correlative markers is under way.

CONCLUSION

In this study of patients with central pathologic review, tumor grade and brain location are associated with a worse prognosis. Nearly one-fourth of patients were previously misdiagnosed. Results of this study have lead to a multicenter study to further define important diagnostic and prognostic variables for adults with ependymoma.

Neuro-Oncol. 2009 Oct;11(5):644.

350. An Association Between Notch1 and Prognosis in Glioblastoma

Adam Wu, Ian E McCutcheon, Gregory N Fuller, Hannah Cheung, Gilbert J Cote

INTRODUCTION

The role of the notch pathway in astrocytoma has attracted recent interest. Notch activity supports survival and proliferation of glioma cancer stem cells, but also mediates terminal differentiation of astrocytic precursors. We recently demonstrated an association between increasing notch1IC positivity and grade of astrocytoma. However, among glioblastomas, there was a significant notch1IC-negative subpopulation. We, therefore, investigated the association between notch1IC positivity in glioblastoma and prognosis.

METHODS

Notch1IC levels were determined by immunohistochemical analysis on biopsies from patients undergoing surgery at our institution. Clinical data was then obtained from the institutional database and MRI images were used to determine degree of necrosis and volumetric extent of resection. Progression-free survival (PFS) and overall survival (OS) times of notch1IC-positive tumors were compared with notch1IC-negative tumors using Kaplan-Meier and Cox-regression analysis.

RESULTS

The notch1IC-positive and notch1IC-negative groups did not differ significantly in age, KPS, degree of necrosis, use of adjuvant chemotherapy and radiation, or extent of resection. Notch1IC positivity was associated with improved prognosis (PFS 9.6 vs. 4.7 months, HR 1.75, p = 0.046; OS 22.0 vs. 18.7 months, HR 1.67, p = 0.083), which was most significant in newly diagnosed glioblastomas (PFS 9.6 vs. 2.6 months, p = 0.026; OS 22.0 vs. 8.3 months, p = 0.026) but not in recurrent glioblastomas (PFS 10.5 vs. 4.7 months, p = 0.278; OS 26.0 vs. 19.0 months, p = 0.228).

CONCLUSION

Notch1IC positivity is associated with improved prognosis in glioblastoma. The progression of astrocytoma may involve aberrant notch pathway regulation resulting in an increasing imbalance between gliomagenesis restraint and promotion. Notch1IC-negative tumors may represent a more advanced stage of malignancy where elevated distal notch activity is dissociated from proximal regulation, and Notch1IC may be suppressed by aberrant feedback inhibition or a more malignant pathway of gliomagenesis independent of notch activity.

Neuro-Oncol. 2009 Oct;11(5):644–645.

351. Expression Profile of Micrornas in Malignant Glioma

Pin-Yuan Chen, Hua-Chien Chen, Su-Jen Chen, Kuo-Chen Wei, Chiungyin Huang, Hong-Chieh Tsai, Shu-Mei Chen

INTRODUCTION

MicroRNAs (miRNAs) are short noncoding RNAs encoded by the human genome. To date, more than 900 miRNAs have been identified in the human genome. miRNAs negatively regulate protein-coding genes by inducing mRNA degradation or repressing mRNA translation. These regulators have been shown to be involved in a diverse set of biological functions, and dysregulated miRNA expression has been linked to numerous human disorders, including cancer. Recent studies have demonstrated that miRNAs exhibit distinct expression patterns in various tumors, and tumor-specific miRNA expression signatures may be used as prognostic markers. Malignant glioma is a common primary brain tumor with a poor prognosis. The expression pattern of miRNAs in malignant glioma has not been fully studied.

METHODS

Using a stem-loop RT-qPCR analysis, we quantified the expression levels of 270 human miRNAs in clinical tissues from 41 patients with different grades of glioma. We identified 47 miRNAs differentially expressed in GBM samples (p < 0.01, ⩾3-fold change).

RESULTS

Hierarchical clustering analysis showed that these 47 miRNAs can distinguish malignant gliomas from normal brain tissue. Several GBM upregulated miRNAs, including miR-21 and miR-10b, were known oncogenic miRNAs in other tumor types. Several downregulated miRNAs, including miR-124, were brain-enriched miRNAs and have been shown to inhibit proliferation of GBM cells in culture. Some of these GBM-specific differentially expressed miRNAs showed grade-dependent alterations. Alterations of a few miRNAs were also found to be correlated with patients’ overall survival.

CONCLUSION

In conclusion, malignant glioma has a distinct miRNA expression signature that may have both diagnostic and prognostic value. Further studies with a larger sample size are needed to validate our data.

Neuro-Oncol. 2009 Oct;11(5):645.

352. Melanotic Glioblastoma Multiforme

Sushila Jaiswal, Vinita Agrawal, Rabi Sahu, Awadhesh K Jaiswal, Sanjay Behari

INTRODUCTION

Pigmented neoplasm of neuroepithelial tumors include astrocytoma, ganglioglioma, ependymoma, subependymoma, choroids plexus tumors, primitive pineal tumor, medulloblastoma, medulloepithelioma, and central neurocytoma. Only five cases of pigmented astrocytic tumors have been reported to date and all were histologically proven to be low-grade tumors. The authors present here the first case in the literature where a tumor exhibiting melanin pigment was histologically proven to be WHO grade IV.

METHODS

The data and information presented were derived from a review of patient medical records and radiographic and histological studies, as well as a review of the relevant medical literature.

RESULTS

A 54-year-old man presented to us with the features of raised intracranial pressure for 2 months. Neurological examination revealed bilateral papilloedema. MRI of the head showed a mass in the right posterior temporal region which was iso- to hyperintense on T1 and hyperintense on T2 weighted images. The periphery of the mass was enhancing on contrast. He underwent right temporal craniotomy and tumor decompression. Intraoperatively the tumor was intraaxial, soft, and vascular with necrotic areas in the center. Histopathology showed tumor cells displaying round to oval nuclei with moderate nuclear pleomorphism. There were mitotic figures, areas of endovascular proliferation and necrosis. Focal areas show clusters of cells showing intracytoplasmic melanin pigmentation highlighted on Masson Fontana staining. The glial cells were positive for GFAP, foam cells were positive CD68, and melanin-containing cells were positive for HMB45. These findings were suggestive of glioblastoma multiforme with melanotic areas. The patient received radiotherapy and chemotherapy following surgery. At follow-up after 4 months, he was symptom free and doing well.

CONCLUSION

Only five cases of pigmented astrocytic tumors have been reported to date and all were histologically low grade. This unusual case provides, for the first time histological evidence of melaninization in glioblastoma multiforme.

Neuro-Oncol. 2009 Oct;11(5):645.

353. Long-Term Survivors with Cystic Glioblastoma with Oligodendroglial Components

Yoshitaka Narita, Yasuji Miyakita, Makoto Ohno, Aya Shinomiya, Soichiro Shibui

INTRODUCTION

Cystic glioblastoma (GBM) patients were reported to have better prognoses. We examined whether cystic GBM patients survive longer than others and elucidated its characteristics.

METHODS

Ninety-six newly diagnosed GBM patients were treated in the National Cancer Center Hospital in Japan from 1996 to 2005. Cystic GBM was defined as a glioblastoma with a large serous cyst more than 3 cm in diameter. Clinical history, MRI findings, MIB-1 staining index, oligodendroglial component, 1p/19q LOH, and MGMT promoter methylation status were examined to elucidate the characteristics of cystic GBM.

RESULTS

Twelve patients (12.5%) were diagnosed with large cystic GBM. The average age of cystic GBM and noncystic (classical) GBM were 52.8 and 58.5 years old, respectively. The mean duration of diagnosis from initial clinical symptoms was 1 month in cystic GBM patients. Overall survival of cystic and classical GBM patients was 24.1 and 13.4 months, respectively (p < 0.05). Three patients among the 12 cystic GBM patients survived more than 5 years. Two of those three patients were diagnosed with GBM with oligodendroglial components.

CONCLUSION

Cystic GBM with oligodendroglial components may be one of characteristics of long-term survivors of glioblastoma.

Neuro-Oncol. 2009 Oct;11(5):645.

354. Survival Prediction by Multiple O6-Methylguanine-DNA Methyltransferase Testings for Glioblastoma Treated with Temozolomide

Motoo Nagane, Keiichi Kobayashi, Saki Shimizu, Andreas Waha, Yoshiaki Shiokawa

INTRODUCTION

O6-methylguanine-DNA methyltransferase (MGMT) is implicated in resistance to temozolomide (TMZ). MGMT status has been shown to correlate with survival of patients with glioblastoma (GBM). We evaluated the relationship between promoter methylation status and protein expression of the MGMT gene in patients with GBM treated with TMZ, and we also analyzed their prognostic value regarding response to TMZ and survival.

METHODS

We analyzed 47 GBM tissues from 43 patients treated with TMZ from September 2003 to January 2009. Among them, 22 patients with recurrent GBM treated with standard TMZ single therapy were subjected to survival analyses. TMZ was given daily orally at 75 mg/m2 during radiotherapy, or as a standard 5/28-day regimen. MGMT protein expression was quantified by Western blotting, and promoter methylation status was determined by both methylation-specific PCR (MSP) and quantitative pyrosequencing.

RESULTS

MGMT promoter methylation was detected in 19/47 cases (40.4%) by MSP and 16/47 cases (34%) by pyrosequencing, and the results were significantly correlated with each other (p < 0.001). The methylation status correlated significantly with MGMT protein expression as well. Upon TMZ treatment for recurrent GBM, patients with both methylated MGMT promoter as determined by MSP and low MGMT protein expression had significantly improved progression-free survival (PFS) and overall survival (OS), whereas methylation status by pyrosequencing data did not. MGMT status was the only significant prognostic factor for PFS and OS.

CONCLUSION

The three MGMT assays revealed similar methylation/expression patterns and were suggested to be important prognostic factors for patients treated with TMZ. Whether precise quantitation of the methylation status provides any clinically relevant information on the treatment strategy for GBM needs to be further investigated.

Neuro-Oncol. 2009 Oct;11(5):645.

355. A Novel Brain Neoplasm with Combined Gliosarcoma and Primitive Neuroectodermal Tumor (PNET) Components

Kamal RM Woods, Arie Perry, Frank PK Hsu, Austin RT Colohan, Ravi Raghavan

INTRODUCTION

Malignant gliomas (WHO grade IV) are often grouped together as a single clinicopathologic entity with poor overall response to treatment. Improved subclassification is needed to facilitate an understanding of the biology and management of this group of tumors. Rare brain neoplasms with both glioblastoma and PNET features (MG-PNET) were recently described by a consortium of institutions. We present an even rarer case of MG-PNET containing gliosarcomatous elements.

METHODS

A 65-year-old male with headaches for 2 weeks had an MRI showing a left parietal ring-enhancing tumor. He underwent near-total surgical resection of this tumor. Starting 2 weeks postoperatively, he received daily temozolomide concurrent with proton beam radiation over 6 weeks, followed by temozolomide only on days 1–5 monthly for 6 months. MRI at 5 months postoperatively showed increased residual tumor with extension to the corpus callosum and contralateral hemisphere. At this time, another craniotomy was performed with subtotal resection of tumor and brachytherapy with Gliadel wafers.

RESULTS

Pathology studies performed on a specimen from the first resection revealed a MG-PNET containing both glial fibrillary acid protein (GFAP) and desmin positive gliosarcoma elements, as well as a PNET component strongly expressing synaptophysin. Fluorescent in situ hybridization (FISH) identified glioma-like 10q deletions, and PNET-like c-myc amplifications. A specimen obtained from the second resection showed foci of residual glial elements with radiation-associated changes.

CONCLUSION

The MG-PNET subtype of malignant gliomas is a heterogeneous group of neoplasms, which rarely can have gliosarcomatous elements. An understanding of the unique genetics, biology, and clinical behavior of such tumor subtypes remains crucial as we aim to fine-tune the treatment of malignant gliomas.

Neuro-Oncol. 2009 Oct;11(5):646.

356. IDH1132 Mutation is Strongly Associated with the Proneural Gene Expression Signature in High-Grade Astrocytomas

Samir Kharbanda, Kanan Pujara, Somasekar Seshagiri, Robert Soriano, Zora Modrusan, Thomas Wu, William Forrest, Kenneth D Aldape, Albert Lai, Timothy F Cloughesy, Heidi S Phillips

INTRODUCTION

Mutation of amino acid residue 132 of isocitrate dehydrogenase 1 (IDH1132mut) occurs in a majority of grade II and III astrocytic and oligodendroglial tumors and secondary glioblastomas (GBMs) but is relatively infrequent in primary GBMs.

METHODS

We investigate here the relationship between IDH1 mutation status and gene expression signature in a series of grade III and IV gliomas. Similar to previously published observations, IDH1132mut was associated with a younger GBM patient population and found to be a robust positive prognostic indicator within both grade III and IV newly diagnosed astrocytomas. To determine whether IDH1132mut is associated with prognostic molecular signatures of high-grade glioma, IDH1132 mutation status was determined in a series of tumors that had been previously profiled and classified as belonging to the proneural, proliferative or mesenchymal molecular subtype.

RESULTS

Analysis of 43 grade III gliomas and 99 GBMs revealed highly significant differences between IDH1132mut and IDH1132WT samples in their correlation with proneural and mesenchymal signatures, within both grade III and IV tumors. Among GBMs, while most tumors with the wild-type IDH1 sequence were classified as mesenchymal (62%), the majority of GBMs with IDH1132mut were proneural (78%), and no instances of mesenchymal-subtype tumors with IDH1132mut were observed. Analysis of 22 matched sample pairs from primary and recurrent high-grade astrocytoma revealed that, while nearly all IDH1132WT tumors (13/14) displayed the mesenchymal phenotype, either on initial presentation or upon recurrence, none of the IDH1132mut cases (0/8) showed the mesenchymal signature at either timepoint in the disease process. Consistent with the predominance of the proneural expression signature in IDH1132mut GBMs, these tumors express lower levels of VEGF and other angiogenic genes than GBMs with wild-type IDH1.

CONCLUSION

These findings suggest that the better prognosis of IDH132mut tumors may be attributed, in part, to lower levels of angiogenesis relative to IDH1132WT tumors.

Neuro-Oncol. 2009 Oct;11(5):646.

357. GBM Histologic Changes Following Radiation and Chemotherapy

Carolyn Quinsey, Scott D Rand, Benjamin M Ellingson, Kang-Cheng Ho, Dick Krouwer, Kathleen M Schmainda

INTRODUCTION

Histologic changes following radiation and chemotherapy are predictable, yet they have not been quantified in glioblastoma multiforme (GBM) treatment. Tissue cellularity is predictably decreased and blood vessels predictably thicken; however, the degree is not well characterized. Quantifying this information may lead to a better understanding of the relationship between histologic changes and clinical outcomes to continue to improve treatment efficacy.

METHODS

Thirty-six patients with GBM underwent resection. The untreated group of 19 patients underwent resection without prior chemotherapy or radiation therapy. The treated group, 16 patients and 20 resections, received variable amounts and dosing schedules of chemotherapy and radiation. For each biopsy sample analyzed, two photographed and calibrated areas were measured manually for cellular area (cells counted/area), blood vessel area, blood vessel diameter, blood vessel thickness, and endothelial thickness. These histologic measurements were then compared with the Mann-Whitney U-test (significance = 0.05).

RESULTS

There is a statistically significant reduction in cell count per area in the treated as compared with the untreated group by a factor of 0.45 in the median (p = 0.001). Median blood vessel luminal area increased in the untreated group and blood vessel and endothelial thickness increased in the treated group; however, these findings were not statistically significant.

CONCLUSION

Tissue cellularity following chemotherapy and radiation treatment of GBM is expected to be 55% of that of untreated GBM in the median sense across multiple patients. It remains to be seen whether longitudinal percentage differences in cellularity between untreated and treated tissue samples in the same patient are correlated with 1) differences in ADC measurements obtained from serial MRI exams at initial giloma diagnosis and GBM progression or recurrence and 2) duration of glioma progression-free survival. Exact cellularity quantification could be correlated with ADC measurements to increase sensitivity of imaging for GBM recurrence.

Neuro-Oncol. 2009 Oct;11(5):646.

358. Evaluation of CD44 as a Biomarker for Brain Tumor Prognosis

Chiungyin Huang, Pin-Yuan Chen, Li-ying Feng, Kuo-Chen Wei

INTRODUCTION

Brain tumors are among the leading cause of death of young children and adults. Glioblastomas and astrocytomas, which account for more than 40% of all CNS tumors, are the most common brain tumors that affect adults between 45 and 60 years of age. The poor prognosis of patients with glioblastomas (~1 year) is due to recurrence of tumor cells after surgery and radiation treatments. Recurrence of gliomas occurs in the majority of patients. Surgery followed by radiation and chemotherapy is the common treatment currently available. Although these procedures are beneficial for the initial treatment, they often do not completely remove all the tumor cells; therefore, the recurrence rate is very high and a reliable prognostic marker is critically needed. Thus, there is an urgent need to develop therapeutic strategies, as well as some diagnostic/prognostic markers.

METHODS

We used a microarray/bioinformatics approach to examine clinical specimens from glioblastoma multiforme patients, followed by validation of the potential markers with different set of samples. Clincal data were also analyzed to evaluate the prognostic value of these markers.

RESULTS

We identified five potential prognostic markers of malignant glioma (CD44, GADD45A, FN1, CD63, and SPP1) that show statistically significant correlations. The expression of these potential markers was analyzed by real-time RT-PCR. Further statistical analysis of the first marker, CD44, in various tumor collections, confirmed that it was a poor prognostic marker for patients’ survival rates. By immunostaining and Western blot analysis, we also demonstrated that the expression of CD44 is gradually increased from the peripheral region to the tumor core at the translational level.

CONCLUSION

Our results suggest CD44 might be a good candidate for further development of prognosis and treatment targets.

Neuro-Oncol. 2009 Oct;11(5):646.

359. Prognostic Significance of Immunohistochemical Expression of MGMT in Newly Diagnosed Glioblastoma Patients Treated with Temazolamide

Anandh Balasubramaniam, Vani Santosh, A Arivazhagan, T Balaram, BA Chandramouli, AS Hegde, Kumaravel Somasundaram, P Kondaiah, MRS Rao

INTRODUCTION

The value of the expression of O6-methylguanine-DNA methyltransferase (MGMT) protein in tumor tissues of glioblastoma to assess the response to temozolamide (TMZ) therapy has been debated in view of confounding features that include high levels of MGMT protein in infiltrating lymphocytes, microglia, and endothelial cells. This is in contrast to the strong correlation of the methylation status of the MGMT gene promoter with TMZ therapy and patient outcome. The objective of this study was to analyze the prognostic value of MGMT protein expression in the tumor tissue with response to TMZ and patient survival.

METHODS

In this prospective study, 96 patients with newly diagnosed glioblastoma were studied. This group included adult patients with cerebral lobar tumors, with a KPS score of 70. All patients underwent maximal safe resection, as assessed by postoperative MRI scans. They received uniform adjuvant treatment comprised of radiotherapy with concomitant temozolamide, followed by cyclical temozolamide. The patients were followed-up clinically and by radiological imaging. The median survival time in this cohort was 19 months. Histological sections of tumors were immunostained using a mouse monoclonal anti-MGMT antibody and the extent of nuclear immunoreactivity was assessed by a visual semiquantitative method.

RESULTS

The extent of MGMT nuclear immunoreactivity varied substantially, ranging from nil to 65%, with a mean value of 31.5%. For analysis, 30% cell positivity of MGMT expression was taken as the cutoff. On univariate analysis, it was noted that the median survival of patients with MGMT expression > 30% was 14 months versus the median survival of patients with < 30% MGMT expression being 21 months (p = 0.025).

CONCLUSION

Despite the established fallacy of correlating MGMT expression by immunohistochemical analysis with TMZ therapy response, we have noted that MGMT protein expression in glioblastoma tissue correlates with patient outcome in a cohort of uniformly treated glioblastoma patients.

Neuro-Oncol. 2009 Oct;11(5):646–647.

360. BEHAB/Brevican Mediates Glioma Malignancy

Wenya Linda Bi, Russell Matthews

INTRODUCTION

A fundamental challenge to the treatment of malignant gliomas is their ability to insidiously invade neural parenchyma, preventing a surgical cure. One major barrier to movement is posed by the molecules that comprise the neural extracellular matrix (ECM). Gliomas are able to overcome this barrier through a variety of mechanisms, from proteolytic cleavage of and modified interactions with the normal ECM to the production of unique matrix elements. The ECM molecule, brain-enriched hyaluronan-binding protein (BEHAB)/brevican, is highly upregulated and uniquely processed in gliomas and also increases tumor invasion when overexpressed. We further investigated the contribution of BEHAB/brevican and its isoforms to tumor progression to gain insight into its overall impact on gliomas and its potential as a therapeutic target.

METHODS

Expression of BEHAB/brevican in a rat intracranial model of glioma was knocked down using RNA interference. Animals with low versus high expression of BEHAB/brevican were compared for survival, tumor volume, tumor invasion, and histology. Furthermore, human surgical samples of low-grade and high-grade gliomas were analyzed by Western blotting for BEHAB/brevican expression. Levels of each protein isoform were quantified and compared to patient age, tumor grade, proliferation index, chromosome 1p and 19q expression profiles, and association with epilepsy by statistical analyses.

RESULTS

In rats harboring intracranial gliomas, knockdown of BEHAB/brevican significantly increased survival while decreasing tumor volume and spread. In human tissues, an underglycosylated form of BEHAB/brevican was demonstrated to be a specific and sensitive marker of glioma. Furthermore, the expression levels of BEHAB/brevican correlated directly with tumor grade and inversely with clinical markers of favorable prognoses.

CONCLUSION

Taken together, these findings strongly support the role of BEHAB/brevican, or its processing, in promoting a malignant course in gliomas. This work also points to BEHAB/brevican as a promising candidate for diagnostic or therapeutic development.

Neuro-Oncol. 2009 Oct;11(5):647.

361. Ki-67 Proliferative Index Changes Following Resection and/or Radiation Therapy in Intracranial Tumors

Matthew Justin Tormenti, Amir Faraji, John Flickinger, Johnathan A Engh

INTRODUCTION

The Ki-67 proliferation index is a widely accepted assay for cycling cells within tumor specimens of multiple histological subtypes. While it is not a substitute for WHO (World Health Organization) grading, the Ki-67 index is thought to predict the biological activity of selected tumors. In the case of intracranial tumors, many lesions may be resected multiple times, with radiation and/or chemotherapy juxtaposed between surgical procedures.

METHODS

A retrospective review of 3,776 consecutive patients undergoing intracranial surgical resection at the University of Pittsburgh Medical Center from 2003 to 2008 was undertaken. Of these patients, 604 had multiple resections. Multiple Ki-67 values were available in the pathology reports of 98 patients with varying pathologies who suffered a recurrence or progression after their initial resection. Evidence of radiation therapy and chemotherapy in the interval between resections was also recorded. Chi-squared and logistic regression analyses were performed.

RESULTS

Median Ki-67 values for both initial and subsequent surgeries were 10%. The median time interval between surgical procedures was 9 months. For tumors with an initial Ki-67 that was less than 10%, there was a significant number of tumors with a higher Ki-67 found during subsequent resection (p = 0.005). There was also a trend for Ki-67 to increase when the interval between surgery was greater than the median of 9 months; however, this was not significant (p = 0.059). Neither the pathological diagnosis nor adjuvant therapy had any effect on subsequent Ki-67 values.

CONCLUSION

Neither surgical manipulation, irradiation, nor chemotherapy had any significant effect on the proliferation index. An initial Ki-67 of <10% was predictive of an increased index value at recurrence. There was a nonsignificant trend for the Ki-67 value to increase if the interval between initial resection and reoperation was greater than 9 months.

Neuro-Oncol. 2009 Oct;11(5):647.

362. CD133 Survival Analysis in Glioma Patients: Relationship with CDK4, EGFR, P53, MDM2, and PTEN Molecules

Yolanda Ruano, Ángel Rodríguez de Lope, Elisa Perez-Magan, Manuela Mollejo, Yolanda Campos-Martín, Juan Fernando García, David Fuentes, José Luis Hernández-Moneo, Bárbara Meléndez

INTRODUCTION

Little is still understood regarding the molecular characteristics and regulatory mechanisms that control glioma-initiating cell (GIC) biology. Several markers, most of them previously described for neuroprogenitor cells, have been reported to identify GICs. Specifically, a subpopulation of cells in human glioma tumors expressing the cell surface protein CD133 (prominin-1) have been described to be enriched for GICs. In this study, we aimed to analyze CD133 expression in glioma tumors to investigate the relationship of CD133 with the clinical outcome of the patients and other molecules such as CDK4, EGFR, p53, MDM2, and PTEN.

METHODS

To evaluate expression of CD133, we performed immunohistochemical analysis in tissue microarrays (TMAs) containing 90 gliomas of different subtypes and grades: 26 oligodendrogliomas (OG), 20 mixed oligoastrocytomas (OA), and 44 astrocytomas (As). Additionally, immunophenotypic and molecular analysis by fluorescence in situ hybridization (FISH) of CDK4, EGFR, p53, MDM2, and PTEN were performed to correlate them with CD133.

RESULTS

CD133 immunopositivity was detected in 11 of 26 OGs (42.3%), 11 of 20 OAs (55%), and 9 of 44 As (20.4%). Among the As group, we detected CD133 protein expression in 6 of 35 glioblastomas (GBMs), which was significantly associated with a worse clinical outcome (log rank p < 0.05). In addition, CD133 protein expression was positively correlated with p53 in oligodendroglial tumors (OGs and OAs) (? = 0.479; p < 0.05).

CONCLUSION

The analysis of GIC markers such as CD133 may predict the survival of GBM patients. Moreover, a higher proportion of CD133-positive cases was detected in OGs and OAs, which were significantly correlated with p53 immunopositivity. CD133 should be balanced with other molecular markers to predict patient outcome.

Neuro-Oncol. 2009 Oct;11(5):647.

363. The Combination of Genomics and Proteomics: A New Approach to Identify Crucial Proteins to Glioma Angiogenesis

Johan M Kros

INTRODUCTION

The process of angiogenesis is essential for organ growth and repair. Growth of a vascular system is one of the earliest events in organogenesis. Neoangiogenesis in adulthood occurs in the female reproductive organs, in the placenta during pregnancy, and under reactive conditions like wound healing. Neoangiogenesis also plays a crucial role in tumorigenesis and is notoriously present in gliomas. Gliomas are among the most vascularized tumors in humans. Nevertheless, antiangiogenic therapeutic strategies have remained relatively unsuccessful so far. The differences in protein expression patterns between the blood vessels in gliomas and in normal tissues with high-rate physiological angiogenesis may offer specific targets for more successful antiangiogenic therapeutic strategies.

METHODS

In order to identify proteins specifically expressed in glioma angiogenesis, we microdissected blood vessels from glioblastoma and endometrial samples to extract both RNA and proteins. We measured the enzymatically generated peptide profiles using the Orbitrap. In addition, we performed RNA microarrays using the same specimens.

RESULTS

This combination approach allowed us to identify several proteins essential to angiogenesis in glioma. The RNA expression profiles served as a database to direct the identification of more differentially expressed proteins.

CONCLUSION

We conclude that the integration of the proteomics and genomics approach through bioinformatics yields a comprehensive database of gene functions that serves as a powerful tool for the identification of specific gene expression profiles and proteins.

Neuro-Oncol. 2009 Oct;11(5):647.

364. Prognosis Analysis of 203 Patients with Low-Grade Gliomas

Yi Lin, Tao Jiang, Yuling Yang, Na Liu, Guilin Li

INTRODUCTION

To assess the prognostic predictors of low-grade gliomas in a Chinese population, we compared the clinicopathological factors of the patients with early recurrence of disease with those who did not have recurrence.

METHODS

We analyzed the clinical and pathological factors of 203 low-grade glioma patients who had a follow-up for a period of more than 3 years. In these patients, 53 had a disease recurrence.

RESULTS

Patients with astrocytomas are more likely to have disease recurrence (37/115) than those with oligodendrocytomas (11/62) and the mixed-type tumors (5/26). Tumors with nuclear atypia are more likely to recur (8/16) than those without atypia (45/187). The appearance of gemistocytes does not affect the timing of recurrence. Age does not differ between the early-recurrence group and the rest of patients. The mitotic index (Ki67) and KPS score are the strongest predictors of early recurrence.

CONCLUSION

Age and gemistocytic component are not indicator of early tumor recurrence. Mitotic index, KPS, and nuclear atypia are still the strongest prognostic predictors of low-grade gliomas.

Neuro-Oncol. 2009 Oct;11(5):647–648.

365. Preoperational Plasma IgE Level does not Predict Survival in GBM Patients

Yi Lin, Tao Jiang, Yuling Yang, Baoshi Chen, Xiaoguang Qiu

INTRODUCTION

Adults with glioma are 1.5- to 4-fold less likely than controls to have allergies. IgE is the antibody characteristic of allergic diseases. It was reported that serum IgE levels are lower in glioma patients than controls, and glioblastoma patients with elevated IgE live longer than those with normal IgE levels. To determine the plasma IgE levels in patients with different grades of gliomas and their relationship with patient outcome, we assessed the plasma IgE levels in 155 glioma patients.

METHODS

Using the ELISA method, we determined the plasma IgE levels of 155 glioma patients and 25 normal controls. The relationship between the clinicopathological characteristics of the patients and their IgE levels are analyzed.

RESULTS

Plasma IgE concentrations in glioma patients were significantly lower than normal subjects (t = −2.598, p = 0.010). Moreover, plasma IgE concentrations in low-grade gliomas were significantly lower than those in high-grade gliomas (t = −2.469, p = 0.015). In low-grade gliomas, the plasma IgE levels in patients with astrocytomas were significantly lower than those with an oligodendritic component (oligodendrogliomas and oligoastrocytomas; p < 0.001). Plasma IgE levels increased after successful removal of glioblastoma (t = −5.749, p < 0.001), but did not decrease after tumor recurrence. We further analyzed the prognosis in 40 glioblastoma muliforme (GBM) patients. Preoperational plasma IgE levels did not correlate with GBM patients’ survival. However, the magnitude of pre-post IgE elevation seemed to correlate with GBM patients’ survival (n = 7, r = 0.900, p = 0.006).

CONCLUSION

Our research provides new insight for our understanding of the function of the immune system and its action during the initiation and development of gliomas. Plasma IgE levels have the potential to become surrogate biomarkers for gliomas. More cases and longer follow-up periods are needed to further confirm these initial findings.

Neuro-Oncol. 2009 Oct;11(5):648.

366. Molecular Classification of Glioma and Development of a Prognostic Metagene Expression Signature

Erik P Sulman, Vinicius Bonato, Maria M De La Garza, Marisol Guerrero, Bradely M Broom, Kim-Anh Do, Veera Baladandayuthapani, V Peter Collins, Lawrence P Petalidis, Mitchel S Berger, Stanley F Nelson, Timothy F Cloughesy, Heidi S Phillips, Kenneth D Aldape

INTRODUCTION

Reproducible and accurate multigene survival prediction models in glioma are both rare and necessary for the personalization of treatment. A major limitation of the microarray analyses used for predictive modeling is a small sample size relative to the large number of variables. Pooling of data sets into one large analysis may minimize this problem.

METHODS

Affymetrix U133-based expression data (.CEL files, n = 783) from infiltrating gliomas (WHO grade II–IV) were obtained from available external and internal sources, and annotated with diagnosis/grade, patient age, and survival time.

RESULTS

Initial analysis indicated a degree of batch effect, the major determinant of which was the specific U133 GeneChip type (HG-U133Av2 vs. HG-U133Plus2 vs. HT-HG-U133A). Following normalization, unsupervised hierarchical clustering identified two major subclasses, corresponding to our previously described mesenchymal and proneural classes, with the mesenchymal class showing a significantly worse outcome. Further subclassification indicated that samples were best viewed as a continuum according to the degree to which they expressed mesenchymal genes, and survival was highly associated with the degree of mesenchymal gene expression. To identify a survival prediction model, samples were randomized to training (60%) and testing (40%) groups. Metagenes (groups of coexpressed genes) were created using unsupervised methods and subjected to a random forest partition tree model with cross-validation in the training set. Application of this model to the test set showed an area under the receiver operator curve of >80% for survival prediction accuracy.

CONCLUSION

This study represents the largest analysis of microarray data from diffuse glioma to date. Unsupervised clustering of these data indicates two major groups. The use of metagenes may shed light on biologically relevant pathways in these tumors. A survival prediction model developed from one subset of these data performed well on the remaining subset, indicating its potential utility as a clinical test across all diffuse glioma grades.

Neuro-Oncol. 2009 Oct;11(5):648.

367. Prognostic and Predictive Value of MGMT Evaluated by Immunohistochemistry in a Series of Glioblastoma Multiforme

Søren Møller, Helle Broholm, Hans Skovgaard Poulsen

INTRODUCTION

The presence of the repair enzyme MGMT (methylguanine methyltransferase) in glioblastoma cells has been correlated to prognosis and response to chemotherapy. Controversy exists as to the best method of assessment. In works based on the EORTC/NCIC trial 26981/22981 population, the value of immunohistochemical tissue microarray (IHC-TMA) has been questioned and methylation-specific polymerase chain reaction (PCR) has shown greater accuracy and consistency. However, PCR is complicated and has practical disadvantages. TMA evaluates only small areas of a tumor and, therefore, has the potential to overlook focal areas of MGMT positivity. We assessed the potential of IHC analysis of histological tumor slides as a prognostic and predictive factor in glioblastoma.

METHODS

A retrospective analysis of 93 consecutive glioblastoma patients treated at Copenhagen University Hospital between 2005 and 2008 was performed. Patients received standard chemoradiation and adjuvant treatment (Stupp/EORTC regimen). Histological tumor slides were analyzed by IHC (Zymed Clone MT23.2 antibody). The presence of MGMT staining was evaluated semiquantitatively (0%, 0–10%, 10–50%, >50% positive cells) and was also dichotomized (negative or positive). In heterogeneous tumors, focal areas of positivity translated into overall positivity.

RESULTS

Clinical outcomes resembled the EORTC/NCIC trial 26981/22981 results with regards to time-to-progression and overall survival. MGMT staining was, as of writing, available for 63 of the 93 patients. MGMT staining was positive in 60.3% of tumors and negative in 39.7% of tumors. No correlation between MGMT positivity and clinical outcomes were found, even when using a variety of cutoffs.

CONCLUSION

At this moment, the value of MGMT IHC analysis of histological tumor slides as a positive or predictive factor in glioblastoma seems limited. Updated material, including staining of the remaining specimens, will be presented.

Neuro-Oncol. 2009 Oct;11(5):648.

368. Clinical Pathological Correlation to Biomarker Profile in a Consecutive Series of 250 Glioblastomas

Bianca Pollo, Emanuela Maderna, Sara Guzzetti, Ilaria Grecchi, Rita Farina, Gaetano Finocchiaro, Andrea Salmaggi

INTRODUCTION

The application of molecular biology technologies to cerebral tumors identified genetic and molecular alterations specific for some oncotypes, allowing suggestions for tumor biological behavior and therapeutic strategies. This issue has changed the clinical management of patients and has required the implementation of some biomarkers to routine laboratory tests.

METHODS

In order to give early information about bio-markers, the aim of this study was to verify the transferability of molecular biology data to routine diagnosis by performing an immunohistochemical study on surgical specimens from 250 glioblastoma patients who had surgery at our institute. The results were related to the clinical data obtained from the Tumor Register. The patients’ characteristics were as follows: 96 women and 154 men with median age of 58 years (range, 18–84 years), median time to tumor progression was 8.8 months (range, 1–23 months), median survival time was 12 months (range, 1–33 months). The specimens were fixed in Carnoy’s alcohol-based fixative and processed for histology and immunhistochemical analysis for EGFR, p53, PTEN, YKL-40, and MGMT.

RESULTS

The immunohistochemical results showed that 58% of glioblastoma tumor cells were positive for PTEN, 32% were positive for EGFR, 37% were positive for p53, and 60% were positive for YKL-40. A correlation between EGFR and YKL-40 expression and shorter time to tumor progression was found. MGMT was positive (more than 20% of neoplastic cells immunostained) in 65% of glioblastomas. Correlation of these results to molecular data by methylation-specific PCR was assessed for 100 cases with 90% correspondence.

CONCLUSION

The immunohistochemical profile of diagnostic, prognostic, and predictive biomarkers in glioblastoma patients could be, with adequate technical handling and knowledge, a useful, easy, and fast tool for the pathologist in routine clinical practice, supporting oncologists in making treatment decisions.

Neuro-Oncol. 2009 Oct;11(5):648–649.

369. Opportunities for Personalized Treatment and Management of Malignant Gliomas: The Clinical Significance of Mathematical Models

Addie E Boone, Russ Rockne, Maciej M Mrugala, Jason K Rockhill, Ellsworth C Alvord Jr, Kristin R Swanson

INTRODUCTION

Due to the invasive and infiltrative nature of malignant gliomas, and in spite of the advances in imaging technologies, general medical treatment and management of newly diagnosed malignant gliomas varies little from one patient to the next and, often times, results in tumor recurrence, neurological and/or cognitive deficit, and poor quality of life with modest improvement in survival.

METHODS

Recently, notable progress has been made in the application of mathematical models to clinical data in understanding the in vivo dynamics of malignant glioma growth and invasion patterns (Harpold et al. J Neuropathol Exp Neurol. 2007;66(1):1–9). These mathematical models use patient-specific clinical data to assess net rates of glioma cell migration and proliferation in vivo, in combination with known variations in migration rates between grey and white matter (Swanson et al. Cell Prolif. 2000;33(5):317–29).

RESULTS

In this respect, applications of patient-specific modeling offer a significant opportunity to individualize malignant glioma treatment and management by assessing patient-specific growth kinetics and therapy response noninvasively, using only clinically available data (Szeto et al. Cancer Res. 2009;69:4502–9).

CONCLUSION

Thus, we see that mathematical modeling has the ability to offer clinicians the opportunity to individualize the treatment and management of malignant gliomas. These patient-specific models would complement existing diagnostic tools to provide vital data not currently available by quantifying more fully the extent of glioma cell infiltration, offering opportunities to achieve maximal surgical resection and radiotherapy coverage. Furthermore, these patient-specific models would make it possible to assess tumor response to various treatment options through virtual control simulations, obtaining response metrics, before any treatment has been implemented, leading to improved decision making and outcomes as measured by tumor recurrence, extent of surgical resection, response to radiotherapy and chemotherapy, and overall quality of life for the patient.

Neuro-Oncol. 2009 Oct;11(5):649.

370. Treatment of High-Grade Glioma with Bevacizumab and Irinotecan with or without Cetuximab Showed No Correlation Between EGFR- and Angiogenesis-Related Biomarkers and Response to Treatment

Benedikte Hasselbalch, Jesper Eriksen, Helle Broholm, Ulrik Lassen, Morten P Sørensen, Mats Holmberg, Steinbjørn Hansen, Hans Skovgaard Poulsen

INTRODUCTION

Recent data has shown that bevacizumab (B) and irinotecan (I), as well as cetuximab (C) combined with B and I, induce significant responses in recurrent high-grade gliomas (HGGs). HGG tumors are known to be hypoxic, which, among others, stimulates transcription of vascular endothelial factor (VEGF). This eventually induces angiogenesis and HGGs are known to be highly vascularized, with pronounced tumor vascularity significantly correlated with poor survival. HGGs and especially primary glioblastoma multiformes (GBMs) are characterized by frequent amplification/overexpression of the epidermal growth factor receptor (EGFR) and expression of the mutated EGFR version III (EGFRvIII), leading to dysregulated downstream signaling through the PI3K/AKT pathway. Furthermore, GBMs have inactivating mutations of the tumor suppressor PTEN, leading to elevated activity of AKT (pAKT). Signaling through the PI3K/AKT pathway has been shown to induce the expression of VEGF either by stimulating its transcription directly or by upregulation of HIF-1a. Moreover, the hypoxia-inducible carbonic anhydrase 9 (CA9) has been shown to be associated with poor survival outcome.

METHODS

At recurrence, 39 patients with primary GBM were treated with CBI and 39 patients with HGG were treated with BI. Immunohistochemical (IHC) analysis was performed on tumor tissue obtained prior to treatment. Staining was performed with the following antibodies: EGFR, EGFRvIII, CA9, and CD34. Semiquantitative scoring was used for evaluation of the slides. The Pearson chi-square test was used for correlation between the level of the different markers investigated and response. The Kaplan-Meier method and log-rank test were performed to compare survival in responders and nonresponders versus the markers investigated.

RESULTS

No significant correlations were found between treatment response and expression of the biomarkers tested. Further IHC analyses regarding pAKT, PTEN, VEGF, and HIF-1a will be presented at the meeting.

CONCLUSION

Expression of the EGFR- and angiogenesis-related biomarkers tested so far fails to correlate with response to treatment when tested by IHC analysis.

Neuro-Oncol. 2009 Oct;11(5):649.

371. Using 3-D 1H-Mr Spectroscopy for Meningioma Surgery: Integrated Biochemical Image Guidance and Determination of Biochemical Markers of Clinically Aggressive Tumors in Vivo that Provide a Resection Advantage

Nina Z Moore, Robert WJ Ryan, Tejas Sankar, Katie Fenton, Berkay Kanberoglu, Adrienne C Scheck, Kris A Smith, Peter Nakaji, Josef Debbins, Robert F Spetzler, Mark C Preul

INTRODUCTION

Our ex vivo MRS imaging of resected meningioma tissue demonstrated a heterogeneous metabolite profile throughout that correlated with genetic alterations and tumor recurrence. We propose that utilizing 3-D 1H-MRS coregistered with an image-guided surgery system (IGS) may permit in vivo grading of intracranial meningiomas by allowing identification and sampling of tumor-specific voxels and regions.

METHODS

Multivoxel 3-D 1H-MRS (3T) was performed preoperatively on patients with recurrent and newly diagnosed meningiomas. Voxels of biochemical interest were selected, and masks of the regions were generated for overlay within the IGS-STEALTH for resection guidance. On the same tissue, fluorescent in situ hybridization was used to detect genetic alterations of chromosomes 1p, 14q, and 22q and ex vivo NMR biochemical profiles were compared with the in vivo spectra.

RESULTS

3-D 1H-MRS showed spectral heterogeneity intratumorally, correlating with genetic alterations in resected tissue and with ex vivo NMR. Alanine, choline, creatine, and lactate concentrations varied within the tumors. Alanine correlates with chromosomal aberrations, is higher in low-grade meningiomas, and is reduced in recurrent and higher-grade tissue. Recurrent meningiomas show alanine borders with significantly reduced alanine centrally. Newly diagnosed tumors have a greater combination of metabolite values.

CONCLUSION

This novel integration of 3-D 1H-MRS within a standard IGS allows the surgeon a significant advantage to intraoperatively and preferentially examine tumor metabolic regions of interest and selectively resect tumoral regions. Metabolite levels preoperatively predict more aggressive meningiomas and more aggressive intratumoral regions. This information should help guide operative planning to target regions for the most extensive resection and use of adjuvant therapy.

Neuro-Oncol. 2009 Oct;11(5):649.

372. Determination of the Methylation Status within Different Regions of Glioblastoma Multiforme Tumors

Mark G Hamilton, Gloria B Roldan, Tony Magliocco, Ian Parney, Jacob Easaw

INTRODUCTION

Epigenetic silencing of the MGMT gene through promoter methylation is correlated with improved survival in glioblastoma multiforme (GBM) patients receiving concurrent radiotherapy. When evaluating the MGMT promoter methylation status, the possibility of intratumoral heterogeneity is concerning. This study was designed to determine whether the methylation status of the MGMT promoter is homogeneous in different areas of GBM tumors and compare methylation status from paraffin-embedded versus fresh-frozen tissue blocks.

METHODS

Using intraoperative navigation, intraoperative biopsies were obtained from the nonenhancing outer region, enhancing rim, and hypoxic core of the tumor. Patients with a confirmed pathological diagnosis of GBM were included for evaluation and analysis. Methylation-specific PCR (MS-PCR) was used for the qualitative analysis of the methylation status of DNA obtained from fresh-frozen tissue and from paraffin-embedded samples from each region of the same GBM.

RESULTS

Ten patients diagnosed with GBM were included in this study. All patients were male; the median age was 62 years (range, 30–74), and the median Karnofsky score was 80 (range, 70–90). In all patients, samples were taken from the enhancing rim and the necrotic center. It was not considered safe or feasible to obtain samples from the area immediately adjacent to the tumor in four cases. All patients had homogeneous MGMT promoter methylation status in every sample, when considering frozen or paraffin-embedded tissue samples. However, discrepancies in the MGMT promoter status between frozen and paraffin-embedded blocks were noted in four patients.

CONCLUSION

MGMT promoter methylation status is homogeneous within GBM tumors. However, the observation that the evaluation of the methylation status of the MGMT promoter varied depending on whether the DNA was extracted from paraffin-embedded or frozen tissue samples is concerning. Although the reason for this is unclear, we postulate that the timing between resection and fixation and the process of fixation itself may potentially alter the methylation status in paraffin-embedded tumors.

Neuro-Oncol. 2009 Oct;11(5):649–650.

373. Immunohistochemical Evaluation of Multiple Biological Markers in Glioblastoma: Correlation with Histology and Patient Survival

Vani Santosh, T Balram, M Vrinda, BM Shilpa, A Arivazhagan, B Anandh, BA Chandramouli, AS Hegde, Kumaravel Somasundaram, P Kondaiah, MRS Rao

INTRODUCTION

Use of biomarkers for screening patients with glioblastoma may be used for prognostication, modification of treatments, and to improve outcomes. In our earlier gene expression profiling study, we have identified several glioblastoma-specific candidate genes.

METHODS

To evaluate biomarkers for diagnostic and prognostic assessment in glioblastoma, we have assessed the expression of short listed genes at the protein level by immunohistochemical analysis of samples of diffusely infiltrating astrocytoma of all grades (II, III, and IV) and glioblastoma, and correlated their expression with histological parameters. Expression of some markers and histology were further correlated with survival in 83 adult patients with newly diagnosed glioblastoma. All patients received a uniform treatment protocol, including surgery, radiotherapy with concomitant temozolamide, and followed by cyclical temozolamide.

RESULTS

We found PBEF-1 (n = 163), YKL-40 (n = 62), IGFBP-2 (n = 197), FSTL-1 (n = 110), β2M (n = 88), FABP-7 (n = 97), and AEBP-1 (n = 30) to be glioblastoma-specific biomarkers, while GADD 45à (n = 84), MMP9 (n = 78), IGFBP-3-5–7 (n = 197), IGFBP-4 (n = 75), COL6A1 (n = 100), Col IV (n = 100), and SOD2 (n = 30) were expressed in all grades of astrocytoma, with the highest expression in glioblastoma. Histological correlation with biomarker expression showed intense cytoplasmic or membrane staining in the tumor cells with most markers. IGFBP-4 was labeled in the nucleus, and for AEBP-1 and FABP7, nuclear and cytoplasmic staining was evident. Overexpression of MMP9 and COL6A1 was additionally noted in the invasive front of the tumor. High expression of IGFBP-2 was evident in the perinecrotic pseudopalisading cells. IGFBP-7 and Col IV staining was prominent in the endothelial cells and basement membrane of tumor vessels, respectively. Survival analysis showed that patients = 45 years had better prognosis (p = 0.014). Among histological parameters, focal mesenchymal differentiation in the tumor portended poor prognosis (p = 0.016). Overexpression of IGFBP-2 (p = 0.030), −3 (p = 0.037), −5 (p = 0.002), and MMP-9 (p = 0.008) correlated inversely with survival by univariate analysis compared to PBEF-1 (p = 0.120).

CONCLUSION

Our study underscores the biological significance and clinical utility of glioblastoma biomarkers, which could add prognostic power to solely histologic observation.

Neuro-Oncol. 2009 Oct;11(5):650.

374. Identification and Validation of a Blood-Based Proteomic Signature for Glioblastoma

Baogang J Xu, Qi An, Kyle D Weaver, Supriya Srinivasa Gowda, Larry Pierce, Ty Abel, Donald Hucks, Reid C Thompson

INTRODUCTION

The diagnosis of glioblastoma multiforme (GBM) requires an invasive procedure. A reliable, noninvasive, and convenient method for diagnosing GBM and monitoring for recurrence is needed. We investigated the use of a molecular proteomic biomarker-based detection method for glioblastoma using patients’ blood samples.

METHODS

Using a multiplex immunoassay (Luminex, Austin, TX), 79 proteins associated with glioma were examined in the plasma from 28 high-grade glioma patients and 367 healthy controls. Differentially expressed proteins were selected using the Wilcoxon rank-sum test. Receiver operating characteristic (ROC) curves were constructed for these proteins to determine their diagnostic accuracy. A second cohort of serum samples from 20 different GBM patients and 1,278 healthy controls was similarly analyzed. Immunohistochemical analysis was performed to examine the expression of the protein biomarkers in GBM tissues.

RESULTS

A total of 40 and 25 proteins were found to be significantly differentially expressed between the glioma and control groups (p < 0.0005) using the plasma or serum samples, respectively. With ROC analyses, 19 and 9 proteins were found with an area under the curve greater than 85% using the plasma or serum samples, respectively. Five protein biomarkers with an average of 87.2% sensitivity and 87.4% specificity were identified in these two different cohorts of patients. Immunohistochemical analysis showed that glioblastoma tissue positively expressed the identified protein biomarkers. The differential expression of the blood-based protein biomarkers, both overexpressed and underexpressed, were found to be consistent with previously published studies performed at the GBM-tissue level, suggesting the blood-based bio-markers may originate from the tumor.

CONCLUSION

A blood-based proteomic signature for glioblastoma was identified, which may offer an unprecedented noninvasive approach for diagnosing GBM and monitoring for recurrence.

Neuro-Oncol. 2009 Oct;11(5):650.

375. Pilomyxoid Astrocytoma in Two Different Age Groups

Abdullah Omar A Alobaid, Boleslaw Lach, Sheila K Singh, Kesava Reddy

INTRODUCTION

Pilomyxoid astrocytoma (PMA), a newly identified tumor entity, was previously considered to be a variant of pilocytic astrocytoma. It is mainly a pediatric disease, but some adult cases have been reported. This article presents two cases of PMA, believed to be the only cases seen in our center. A review of the literature about PMA, regarding its presentation, diagnosis, management, and prognosis is also included in this article.

METHODS

We performed a chart review from our electronic system.

RESULTS

First case: A 19-year-old girl presented with a 3-month history of progressive, bifrontal headache, decrease vision in her left eye, right homonymous hemianopsia, and decreased visual acuity in her left eye. MRI with contrast revealed a massive tumor arising from the sella, with a multicystic appearance. She underwent a two-stage procedure: transnasal-transsphenoidal bilateral optic nerve decompression and left pterional craniotomy subtotal resection of the tumor. After 9 months she developed a recurrence and was treated by radiation and Mitrex tubular retraction resection. Second case: A 15-month-old boy presented with failure to thrive, abnormal head posturing, frequent falls, horizontal and vertical nystagmus, and dilated nonreactive bilateral pupils. MRI revealed a large suprasellar mass involving the chiasma and the hypothalamus. A companied right subfrontal and pterional approach had been done to debulk the tumor. Postoperatively, he developed left-sided weakness. Two months later, he had evidence of recurrence with massive tumor growth, hydrocephalus, and drop metastasis to the spine. He underwent a second debulking procedure, but he suffered then altos of complications including septicemia, and he end up in a semivegetative state, and he passed away.

CONCLUSION

PMA is a rare, pediatric disease, but it should be considered in adults especially those in their late teens and early twenties. Further studies are required regarding other locations, prognosis, and optimal treatment.

Neuro-Oncol. 2009 Oct;11(5):650.

376. Clinicopathological Characteristics of Adamantinomatous and Papillary Craniopharyngiomas: UCSF Experience 1985–2005

Jonathan Louie, Michele M Bloomer, Tarik Tihan, Nalin Gupta

INTRODUCTION

Craniopharyngiomas are considered WHO grade I neoplasms, which implies low growth potential and possible cure by surgery alone. We present our experience with craniopharyngiomas in an attempt to reconfirm the current WHO grading.

METHODS

We performed a retrospective review of all patients with craniopharyngioma diagnosed and treated at UCSF between 1985 and 2005.

RESULTS

We identified 81 patients based on the inclusion criteria. There were 43 males and 38 females with a median age of 33 years. Twenty-eight patients were younger than 20 years. Seventy tumors were classified as adamantinoma-tous. The mean tumor size was 3.11 ± 1.5 cm and the mean volume was 25 ± 4.9 cm3. Fifty-four patients (67%) suffered some form of visual-field defect, and 41 (51%) suffered endocrine deficiency. Gross total resection was achieved in 10 patients, subtotal surgery in 61, and biopsy in four. One case was diagnosed at autopsy, and the extent of surgery was not recorded in five patients. During a median follow up of 82 months, clinical and radiological progression occurred in 37 patients (46%). Thirty-five patients required additional surgery and 20 required additional radiotherapy. At the end of the follow-up period, nine patients were dead, 16 were alive with disease, and 56 were lost to follow-up.

CONCLUSION

Our group includes a greater percentage of subtotal resections, reflecting the conservative surgical approach used at UCSF. Even with this approach, the long-term outcome is similar to the earlier series. Despite the large percentage of patients lost to follow-up, many tumors progressed and needed subsequent surgical/medical interventions. The recurrence patterns, visual, and endocrinologic morbidities suggest that craniopharyngiomas behave more aggressively than a typical WHO grade I neoplasm. Consideration should be given to assign a WHO grade II for typical adamantinomatous cranio-pharyngioma. Our data are not sufficient to suggest the same designation for the papillary variant.

Neuro-Oncol. 2009 Oct;11(5):650.

377. Microrna Signature Discriminates Molecular Risk Groups in Medulloblastoma

Marc Remke, Alfa HC Bai, Paul A Northcott, Grischa Toedt, Mirco Castoldi, Armin Pscherer, Martina U Muckenthaler, Andreas E Kulozik, Peter Lichter, Michael D Taylor, Stefan Pfister

INTRODUCTION

Medulloblastoma comprises the most common malignant brain tumor in children. Current treatment approaches are based on clinical and histopathological parameters. Identification of novel tumor-derived biomarkers may improve the risk assessment of medulloblastoma patients and serve as a basis for more refined risk-adapted treatment strategies.

METHODS

Array-comparative genomic hybridization (CGH) or fluorescence in situ hybridization (FISH) was performed to identify tumor-specific DNA copy-number alterations of prognostic value in 340 samples from medulloblastoma patients. To further elucidate the peculiarities in tumor biology of these distinct prognostic subgroups, we next aimed to discriminate patients of favorable and unfavorable molecular risk groups by a miRNA expression signature. Based on genomewide miRNA expression profiles performed in a screening set of 33 medulloblastoma samples, a risk-associated signature was determined by PAM. The predictive power of this classifier was tested in an independent validation set of 90 medulloblastomas on a different microarray platform.

RESULTS

Tumors harboring MYC or MYCN amplification, 6 gain, and/or 17q gain were grouped into the high-molecular risk group. Low molecular risk was noted for medulloblastomas with monosomy 6 and balanced copy-number status for 6q, 17q, MYC, and MYCN. This set of genomic imbalances was significantly associated with overall and progression-free survival (p < 0.001). PAM revealed a specific miRNA signature for discrimination between these molecular risk groups consisting of 18 miRNAs, with an overall error rate of 0.09 in the screening set. Remarkably, this classifier identified a group of patients with favorable prognosis in the independent validation set. This miRNA signature contains both positive and negative regulators of sonic hedgehog signaling, such as miR-199b and miR-326, respectively. Interestingly, we identified miR-155, a regulator of apoptosis, to be among the most strongly downregulated miRNAs in high-molecular risk medulloblastomas.

CONCLUSION

Genomewide profiling of miRNAs allows the discrimination of molecular risk groups in medulloblastoma. Future challenges are to elucidate the functional role of single, deregulated miRNAs, leading to a better understanding of medulloblastoma biology.

Neuro-Oncol. 2009 Oct;11(5):651.

378. Claudin 6 Immunostaining Distinguishes Atypical Teratoid Rhabdoid Tumors from Other CNS Rhabdoid/Epithelioid Neoplasms

BK DeMasters, Diane Birks, Kathy Newell, Kevin Lillehei

INTRODUCTION

Claudin 6 (CLDN6) is a key component of tight junctions. We have previously shown that positive cytoplasmic immunohistochemical (IHC) reactivity characterizes AT/RTs and complements the loss of the nuclear INI-1 protein identified by BAF47 IHC analysis in making the diagnosis. To date, broader application of the CLDN6 antibody on histologically similar tumors in the CNS has not been performed.

METHODS

IHC analysis for BAF47 and CLDN6 was compared in seven AT/RTs, two rhabdoid glioblastomas, seven epithelioid glioblastomas, and one pleomorphic xanthoastrocytoma with a striking epithelioid glioneuronal component (PXA-E).

RESULTS

None of the epithelioid glioblastomas or the PXA-E showed immunoreactivity with either BAF47 or INI-1. INI-1 nuclear protein was focally lost in the two true rhabdoid glioblastomas (both of which also had monosomy 22). In contrast, only the seven AT/RTs showed positive CLDN6 cytoplasmic expression.

CONCLUSION

INI-1 protein can occasionally be lost in CNS tumors with the rhabdoid phenotype, but these tumors, as well as other CNS epithelioid phenocopies, retain CLDN6. Thus, in the CNS, CLDN6 expression appears to have greater fidelity for true rhabdoid neoplasms than does INI-1 nuclear protein staining and serves as a better discriminator of AT/RTs from other CNS rhabdoid/epithelioid neoplasms.

Neuro-Oncol. 2009 Oct;11(5):651.

379. Transformation of Juvenile Pilocytic Astrocytoma (JPA) to Anaplastic Pilocytic Astrocytoma (APA) in Two Patients with Neurofibromatosis Type I (NF-1)

Katherine B Peters, Thomas J Cummings, Sridharan Gururangan

INTRODUCTION

JPAs that occur in patients with NF-1 tend to have a more indolent course than sporadic tumors. Furthermore, transformation of JPA to APA is extremely rare and is usually seen in the context of prior radiotherapy exposure.

METHODS

We identified two older patients with NF-1 who initially were diagnosed with JPA that transformed to APA and evaluated risk factors for such transformation.

RESULTS

Both patients, ages 21 and 18 years, initially had histologically confirmed JPA in the left thalamus and cerebellar vermis, respectively. Both patients had received chemotherapy only for the treatment of their tumors (carboplatin and vin-cristine alternating with temozolomide for a mean number of 5 courses; range, 4–6 cycles) and were found to have converted to APA at progression. The time interval from initial diagnosis to conversion was 10 years and 2 years, respectively. One patient died despite surgical resection and focal radiotherapy. The other patient had progression of disease despite focal radiotherapy but responded well to bevacizumab and irinotecan. On this current regimen, he now has stable disease. MGMT expression as assessed by immunohistochemical analysis was 25% in both patients on transformation. Prior to transformation, one patient’s MGMT was 50% based on JPA pathology.

CONCLUSION

Although JPAs can be indolent in patients with NF-1, transformation to APA can occur rarely in the context of alkylator chemotherapy.

Neuro-Oncol. 2009 Oct;11(5):651.

380. Innovative Molecular Risk Stratification of Pediatric Intracranial Ependymoma Based on DNA Copy Number Alterations and mRNA Expression of Candidate Genes

Hendrik Witt, Sebastian Bender, Till Milde, Axel Benner, Marc Remke, Andreas von Deimling, Christian Hartmann, Andreas E Kulozik, Olaf Witt, Peter Lichter, Stefan Pfister, Andrey Korshunov

INTRODUCTION

Ependymoma remains a significant clinical challenge in pediatric oncology, as a subgroup of patients experience multiple relapses and ultimately succumb to their disease. The main problem in classifying ependymoma is that these high-risk tumors cannot be reliably detected at the time of primary diagnosis. Neither clinical variables nor histopathological grading or molecular markers have so far been successful in defining a well-circumscribed group of “high-risk” patients. Thus, an innovative stratification model of ependymoma is desperately needed.

METHODS

We herein combine genomewide mRNA expression profiling (Agilent 44k platform) with array-comparative genomic hybridization (CGH) (10k BAC arrays) in primary tumor samples from 78 patients with a median follow-up of more than 8 years. RNA expression and DNA copy-number status were correlated with clinical characteristics and survival data using significance analysis of microarrays (SAM) and Kaplan-Meier analysis, respectively. For validation of selected candidate genes, protein expression was investigated by immunohistochemical analysis on tissue microarrays (n = 202 primary ependymoma samples) and QRT-PCR.

RESULTS

On the level of DNA copy-number aberrations, we were able to define a stratification model for three subgroups of ependymoma patients: the high-risk group (29% of patients), including tumors with chromosome 1q gain and/or 9p loss and homozygous CDKN2A deletions, have a 5-year overall survival (OS) of 45%; the low-risk group (40% of patients), including tumors with gain of either of chromosomes 9, 15q, 18 or a combination thereof, have a 5-year OS of 100%; the intermediate risk group (31% of patients), including tumors without any aberration mentioned previously, have a 5-year OS of 75%. By integrating these data with genomewide mRNA expression profiling, we identified several interesting candidate genes that are potentially targeted by 1q gain. The most robust cytogenetic markers for poor outcome in our cohort are LEMD, SHC1, RFWD2, and ASPM, which are all located at 1q and showed a strong correlation with 1q copy-number status and consequently, patient outcome.

CONCLUSION

In summary, by combining array-CGH and mRNA expression profiling in a large cohort of patients, we were able to identify novel biomarkers that can be used for outcome prediction in ependymoma patients and have a high potential to be useful markers for stratifying patients in risk-adapted treatment protocols.

Neuro-Oncol. 2009 Oct;11(5):651.

381. Adhesion Molecule Expression in Primary, Recurrent, and Metastatic Medulloblastomas

Sidney E Croul, Darren Partridge

INTRODUCTION

Medulloblastoma spreads by leptomeningeal dissemination rather than the infiltration that characterizes other CNS tumors. Adhesion of tumor cells to the meninges is necessary for this spread and may contribute to the survival and proliferation of the tumor implants. The mechanisms accounting for this are not known.

METHODS

This study represents an immunohistochemical study of molecules that may govern medulloblastoma adhesion to leptomeninges. Search of the UHN surgical archives uncovered 57 adult medulloblastomas from 42 patients. In addition to primary resections for all patients, there were 13 recurrences in the posterior fossa and two instances of leptomeningeal dissemination. Immunohistochemical analysis was performed with antibodies to transmembrane adhesion molecules β1 integrin, NCAM, L1CAM, and N-cadherin and to extracellular matrix molecules, collagens I and IV, tenascin, fibronectin, vitronectin, thrombospondin and laminin.

RESULTS

Evaluation of the stained sections showed increased reactivity of β1 integrin in recurrent and leptomeningeal tumors as compared with their matched primary resections. There was little difference in the reactivity of NCAM, L1CAM, and N-cadherin between primary and recurrent tumors. Of the extracellular matrix molecules, the greatest difference was seen in tenascin, the staining of which was increased in recurrent tumors.

CONCLUSION

The current study supports previous in vitro studies from our group demonstrating that β1 integrin and tenascin play critical roles in medulloblastoma adherence to the leptomeningeal extracellular matrix. Further studies in our laboratory will be directed toward elucidating the mechanism by which the expressions of β1 integrin and tenascin is increased in these tumors and the importance of that expression for medulloblastoma metastasis.

Neuro-Oncol. 2009 Oct;11(5):651–652.

382. Identification of SGK1 as a Novel Prognostic Marker for Disease Outcome in Medulloblastoma

Dominik Sturm, Marc Remke, Sabrina Pleier, Hendrik Witt, Guido Reifenberger, Jörg Felsberg, Andreas E Kulozik, Andrey Korshunov, Peter Lichter, Stefan Pfister

INTRODUCTION

We have recently demonstrated that DNA copy-number aberrations affecting chromosome 6q clearly distinguish different clinical and biological subgroups of medulloblastoma. However, the underlying molecular mechanisms accounting for the prognostic impact of these aberrations have not yet been identified.

METHODS

To identify potential candidate genes on chromosome 6q targeted by these genomic aberrations, we conducted a genomewide mRNA expression profiling study (Agilent, 48k) for 47 well-characterized medulloblastoma samples and correlated gene expression to chromosome 6q DNA copy-number status as determined by array-based comparative genomic hybridization (10k). Real-time quantitative PCR (RTQ-PCR) was used to validate expression profiling results. Using a tissue-microarray representing a large independent cohort of uniformly treated medulloblastoma patients (n = 260), we examined SGK1 protein expression by immunohistochemical analysis. Finally, our findings on DNA, RNA, and protein levels were correlated with clinical follow-up data.

RESULTS

Gene expression of the AKT homolog SGK1 showed a strikingly close correlation with 6q DNA copy-number status as indicated by expression profiling and validated by RTQ-PCR. SGK1 mRNA levels were significantly upregulated in tumors with gain of chromosome 6q (median log2 ratio when normalized against normal cerebellum = 3.88) and, in contrast, downregulated in cases carrying deletions of this region (median log2 ratio = −1.97). Assessment of SGK1 protein expression by immunohistochemical analysis and correlation with patient survival data revealed high SGK1 protein levels to be closely associated with poor overall and progression-free survival (Kaplan-Meier method; p < 0.001).

CONCLUSION

Our search for an interesting novel candidate gene on chromosome 6q led to the identification of SGK1 as a novel prognostic marker in medulloblastoma, reflecting the astonishing differences in overall and progression-free survival of cases with chromosome 6q gain or loss, who are known to have either a very poor or particularly good prognosis, respectively. The fact that SGK1 encodes for a serum- and glucocorticoid-inducible kinase, which is both structurally and functionally homologous to the antiapoptotic kinase AKT, further underlines the potential biological role of this gene in the pathogenesis of medulloblastoma.

Neuro-Oncol. 2009 Oct;11(5):652.

383. Rest Expression and its Prognostic Relevance in Ependymomas

Dolly G Aguilera, Veena Rajaram, Jason Fangusaro, Tadanori Tomita, Vidya Gopalakrishnan, Sijin Wen, Stewart Goldman

INTRODUCTION

Repressor element silencing transcription factor (REST) is a transcriptional factor and a repressor of neurogenesis. REST is overexpressed in medulloblastoma and plays a role in its tumorigenesis. REST expression in other pediatric brain tumors is not known. In this pilot study, we have examined REST expression in ependymomas and studied its relevance as a prognostic factor.

METHODS

With IRB approval, we conducted this study with 24 patients diagnosed with ependymoma between 1995 and 2004 for whom tumor tissue was available for immunohistochemical staining. REST grading was defined on a scale from 0 to 4. Positive controls included placenta and normal ependymal lining. Survival functions were estimated using the Kaplan-Meier method and compared using the log-rank test.

RESULTS

Twenty-four patients with median age 5 years, 16 anaplastic ependymomas, 7 ependymomas, and 1 mixopapillary ependymoma were analyzed. Of these ependymomas, 75% were supratentorial and 20% were infratentorial. On average, patients had three surgical resections and 2.5 chemotherapy regimens. REST staining was positive in 75% and negative in 25%. The degree of staining was as follows: +/++ in 12.5% and +++/++++ in 58%. Median follow-up was 124 months. Overall survival (OS) was not affected by gender (p = 0.76), age (<5 vs. >5 years; p = 0.31), or location (supratentorial vs. infratentorial; p = 0.46). Patients with REST-positive staining had a trend toward better OS (p = 0.09). OS for patients who underwent chemotherapy was no different than for those who did not have chemotherapy (p = 0.40). The median survival was 114 months for patients with radiation therapy versus 9 months for patients without radiation therapy (p = 0.006).

CONCLUSION

This pilot study demonstrated overexpression of REST in ependymomas. There is a trend towards improved OS for patients with REST positivity by immunohistochemical analysis. These results require further confirmation in a larger sample size. The importance of REST expression in ependymomas is currently under investigation.

Neuro-Oncol. 2009 Oct;11(5):652.

384. The Matricellular Protein Spondin-1 is a Novel Angiogenic Mediator in Pediatric Brain Cancers

Timothy Van Meter, Catherine I Dumur, Nilesh Mepani, Morgan McCrocklin, Renu Kadian, Annie Huang, Michael D Taylor, William C Broaddus, Gary W Tye

INTRODUCTION

We have recently shown that the extracellular matrix protein spondin-1 is dramatically elevated in a subset of pediatric ependymomas. The role of spondin-1 in neuroblast migration during development is well documented in the cortex and spinal cord, but expression is downregulated postnatally. Spondin-1 expression has also been recently described as a poor prognostic marker in certain carcinomas and is likely reactivated by epigenetic changes during tumorigenesis. Aberrant reexpression is pronounced in certain pediatric ependymomas and also in a subset of supratentorial PNETs with a poor overall survival signature. Targeted genomic profiling studies in our laboratories have demonstrated that spondin-1 overexpression correlates with expression of glial progenitor cell markers in primary pediatric ependymomas but is negatively correlated with angiogenic mediators prominent in adult gliomas, such as VEGF-A, thrombospondin-1, or angiopoietin-1.

METHODS

Functional studies were performed with shRNA and cDNA overexpression in primary and established ependymoma lines.

RESULTS

Neutralizing antibodies directed at integrin binding sites in spondin-1 suppressed neurite extension of primary embryonic rat dorsal root ganglion cells, which were used as a control, and was used to diminish spondin-1-mediated endothelial cell sprouting in three-dimensional fibrin matrices in coculture experiments with ependymoma cells. Furthermore, perturbing spondin-1 expression impacts angiogenesis in ependymoma xenografts, as shown by quantitative imaging studies of microvessel density. Genomic profiling, promoter analysis, and ChIP assays were used to identify Sox-5 as a novel coex-pressed transcriptional regulator of spondin-1 in subgroups of pediatric brain tumors.

CONCLUSION

These studies suggest that significant differences may exist in angiogenic programs in ependymomas, with important implications for successfully generating clinically appropriate therapeutics for pediatric brain tumors.

Neuro-Oncol. 2009 Oct;11(5):652.

385. Universal Poor Survival in Children with Medulloblastoma Harboring Somatic TP53 Mutations

Uri Tabori, Nataliya Zhukova, Berivan Baskin, Michael D Taylor, Eric Bouffet, Peter Ray, Noa Alon, David Malkin, Cynthia Hawkins

INTRODUCTION

Medulloblastoma is the prototype of treatment success in modern pediatric neurooncology. Unfortunately, 20–30% of tumors recur despite maximal resection and multimodal therapy. Multiple biologic prognostic markers have been investigated to predict recurrences, but there is still controversy regarding their clinical utility. Since p53 immunopositivity is an adverse prognostic marker in pediatric medulloblastoma and TP53 mutations are associated with chemo/radiation therapy resistance we aimed to determine the extent and role of TP53 mutations in pediatric medulloblastoma treatment failure.

METHODS

One hundred eight of 111 consecutive patients diagnosed with medulloblastoma in our institution between 1995 and 2007 were included. Median follow-up was 5.3 years in survivors. All samples were graded histologically, immunostained, and scored by two blinded reviewers for p53 and other prognostic markers. For 49 patients, frozen material was available for TP53 sequencing. The main outcome measures were overall and progression-free survival. We also performed radiosensitivity assays on TP53-WT and -mutated medulloblastoma and patient-derived lymphoblastoid cell lines.

RESULTS

Sixteen percent of sequenced medulloblastomas harbored a TP53 mutation. As a screening test, p53 immunohistochemistry was 100% sensitive and 83% specific for a TP53 mutation. Strikingly, all mutated tumors recurred early, resulting in a 5-year survival of 0% for patients with TP53-mutated versus 74 ± 8% for patients with wild-type medulloblastomas (p < 0.0001). Furthermore, 75% of recurrences in average-risk patients were associated with TP53 mutations. On multivariate analysis TP53 mutation status was the most significant adverse prognostic factor (hazard ratio 10.4, p = 0.003). Medulloblastoma and lymphoblastoid cell lines harboring TP53 mutations had significantly higher radioresistance than WT cell lines as determined by cell survival and clonogenic assays. These cells also exhibited less gH2AX and 53BP1 immunofluorescence, indicating less DNA damage and lower levels of apoptosis by an Annexin PE assay.

CONCLUSION

Lack of long-term survivors and radioresistance in TP53-mutated medulloblastomas and patient-derived cell lines highlight the role of TP53 mutations in medulloblastoma resistance to conventional therapies and the need for alternative treatments for such children.

Neuro-Oncol. 2009 Oct;11(5):652–653.

386. Patient and Caregiver Congruence in Symptom Report and Association with Patient Neurocognitive Status

Terri S Armstrong, Jeffrey Wefel, Ibrahima Gning, Elizabeth Vera-Bolanos, Alvina Acquaye, Tito Mendoza, Mark R Gilbert, Anne Kayl, Charles Cleeland

INTRODUCTION

Evaluating the severity of symptoms in primary brain tumor (PBT) patients is important in clinical care and research. These patients often develop neurocognitive (NC) impairment, making self reporting difficult. This study was conducted to evaluate the congruence of symptom reporting in patient and caregiver dyads, examining the potential impact of NC impairment.

METHODS

Patients with PBT referred for NC testing and their caregivers were included in this study. These dyads completed the M.D. Anderson Symptom Inventory-Brain Tumor Module (MDASI-BT) prior to testing, and impairment was categorized based on NC test scores.

RESULTS

Fifty-two dyads (patient-caregiver pairs) participated in the study; median patient age was 49, and 52% were male. Multiple tumor types were included; glioblastoma multiforme was the most common. NC testing was performed primarily for postoperative follow-up (35%) and suspected NC deficits (27%). Caregivers were mostly female spouses (62%) who lived with the patient (82%). The most severe symptoms reported by patients and caregivers were fatigue, difficulty remembering, distress, and difficulty sleeping. Independent sample t-tests using mean overall symptom severity showed no significant differences between caregivers and patients (1.87 vs. 1.82), and the mean differences for individual symptoms were less than one point. Subscale correlations between the dyads were highly significant for symptom severity (0.525, p < 0.0001) and interference (0.644, p < 0.000001). No significant differences were seen between patients and caregivers as a function of NC status (p < 0.01), reason for referral, treatment, or functional status. Further tests based on the 90% confidence interval (CI) showed equivalency between patients and caregivers for overall severity, severity of Core items, Brain Tumor items, and Interference subscales. All computed 90% CIs were contained within the interval [−1, 1].

CONCLUSION

Caregivers and PBT patients have highly congruent assessments of symptom severity, regardless of NC function or disease status. These findings suggest that caregivers may serve as proxy report of symptoms for patients during the course of the disease.

Neuro-Oncol. 2009 Oct;11(5):653.

387. A New Paradigm of Care for Primary Brain Cancer

Cynthia D Harrison, Alixis VanHorn, Concetta Sapienza, Jon Levenson, Steven Rosenfeld, Kenneth Prager

INTRODUCTION

Individuals diagnosed with aggressive brain tumors have a tremendous need for information, guidance, and support. They are challenged by a bewildering amount of information regarding their diagnosis and treatment. At the same time, patients with aggressive brain tumors are hampered by cognitive deficits at every stage of the disease. Health care providers are equally challenged about how to provide necessary information and support in the limited time periods alloted for patient visits. Ethical and legal dilemmas abound because of issues of patient competency and conflicting family interests. Mood, for both patients and families, can become unstable and influence treatment decisions and response, and it is not surprising that patient’s needs are sometimes inadequately met. This deficit creates the need for a new paradigm of care for patients with primary brain cancer.

METHODS

We have designed a novel program to meet these needs and have created a format that is reproducible in other neurooncology centers. It consists of four interventions: 1) structured meetings with patients and their families at sentinel times in their disease course: at diagnosis, when treatment regimes change, when tumor progression occurs, and when requested by patients or families; 2) a patient resource manual that contains information supplemental to the topics discussed during sentinel visits as well as other educational material; 3) a dedicated interdisciplinary committee composed of social work, nursing, psychiatry, ethics, and law that meets regularly to review cases and make recommendations; and 4) support groups for families and patients.

RESULTS

The schedule and content of our sentinel visits, the format of the patient resource manual, and the composition and function of the interdisciplinary committee and support groups were standardized and easily transferable.

CONCLUSION

The program is an affordable, flexible, and reproducible model of services that constitutes a new paradigm of care for patients with aggressive brain tumors.

Neuro-Oncol. 2009 Oct;11(5):653.

388. Palliative Home Care for Brain Tumor Patients: Results and Cost/Utility Analysis of 6 Years of a Pilot Project

Andrea Pace, Cherubino Di Lorenzo, Lara Guariglia, Carmine Carapella Maria, Alfredo Pompili

INTRODUCTION

The complex needs of patients with advanced malignant brain tumors are not well documented, and these patients require management of late clinical complications, rehabilitation, and psychosocial problems. In October 2000, in the Regina Elena National Cancer Institute of Rome, we started (with the financial support of the Regional Health System) a palliative home-care pilot project for patients affected by malignant brain tumors who had been discharged from the hospital.

METHODS

The aims of this model of assistance were to meet patients’ care needs during the evolution of the disease, to provide rehabilitation at home, to improve patients’ quality of life with palliative care, and to facilitate death at home. Neurooncologic home staff include one neurologist, five nurses, two rehabilitation therapists, one psychologist, and one social worker.

RESULTS

In the first 6 years of our program, 450 patients were assisted at home, and of those 450 patients, 271 died. The most frequent symptoms observed in the last 4 weeks of life were: epilepsy, 30%; headache, 36%; drowsiness, 85%; dysphagia, 85%; death rattle, 12%; and agitation and delirium, 15%. Among the 182 patients who died at home (182/271, 69%), the most frequent symptoms of the terminal phase were lethargy (85.5%), dysphagia (81%), headache (39%), and seizures (19%). A cost/utility analysis showed that in the group of patients that received continued home care, the hospital readmission rate and the median time spent in hospital in the last 2 months of life were significantly lower than were in the control group (p < 0.05).

CONCLUSION

Future clinical research strategies in neurooncology should include a new model of care for brain tumor patients, with special attention paid to palliative home-care models.

Neuro-Oncol. 2009 Oct;11(5):653.

389. A Private Caregiver Listserve: Maximum Benefit for Minimum $$$$$

Michele R Lucas

INTRODUCTION

The diagnosis of a brain tumor impacts not only the patient but also the entire family. As many patients are cognitively and/or physically impaired, the family has to assimilate the patient’s responsibilities and assume any financial consequences. Incorporating all that is entailed in assuring quality care for the patient is overwhelming for the majority of families. As caregiver stress and anxiety directly influences the care they can provide, it is in the best interest of the patient to acknowledge the caregiver burden and to provide outlets for their fears, frustrations, and concerns. The dilemma becomes how and when to do this within a healthcare environment already short on resources.

METHODS

In August 2006, a private caregiver listserve was offered to the primary caregivers of the patients in a large metropolitan brain tumor clinic.

RESULTS

For the first few years, the postings were sporadic and superficial. The caregivers mostly exchanged lighthearted encouragement to one another and did not utilize the forum in the manner in which it had been envisioned. The hope had been that they would be able to express the depth of their despair, their fears, burden, and anger. From the group’s inception, it had been decided that there would be no intervention to encourage or direct the use of the listserve in any way. Then a year and a half ago, everything changed. The exchanges were serious, expressive, heartbreaking, and honest. They listened to and supported each other. They felt this group was often the only place that their experience was understood. One recent quote was as follows, “I am so grateful for this group. It’s an outlet for all the horrible scary feelings that come up. I feel so much less alone knowing that I can communicate with all of you.”

CONCLUSION

For $15 per month and a few hours per week of monitoring for accuracy of posts, many caregivers can be provided the support they desperately need while caring for their loved ones.

Neuro-Oncol. 2009 Oct;11(5):653.

390. Assessing Information Needs, Support Needs, and Likeliness to use Services among Brain Tumor Patients and Caregivers

Jenette A Spezeski, Harriet Patterson

INTRODUCTION

Cognitive, physical, and psychosocial repercussions of brain tumors greatly affect patients’ and caregivers’ quality of life. We assessed information and support needs of brain tumor patients and family caregivers and explored their likeliness to use supportive services.

METHODS

We pilot-tested and conducted a national online survey containing closed- and open-ended questions. Frequencies were produced for quantitative items; chi-square tests were performed to examine relationships between variables. Qualitative data were analyzed for common emerging themes.

RESULTS

Seven hundred nine brain tumor patients and 702 family caregivers participated in the survey. The most common diagnosis represented among patients was meningioma (29.9%), whereas most caregivers represented glioblastoma (49.3%). Needs: Half of respondents reported difficulty in finding information about cognitive changes (58.7%), clinical trials (52.3%), and fatigue (48.5%). One in three caregivers felt unprepared to cope with the patient’s personality changes (33.3%), cognitive changes (32.6%), physical changes (32.4%), and changes in family roles (29.5%). Respondents identified challenges including: managing care and treatment decisions, adjusting to physical and cognitive changes, and coping with the psychosocial aftermath. Most respondents reported never seeing a neuropsychologist (75.9%), counselor/therapist (67.0%), or social worker (52.6%) within 6 months of diagnosis. Significantly more patients with low-grade tumors (90.2%) rarely or never saw a social worker than did patients with high-grade tumors (72.4%) (p = 0.000). Services: The preferred ways to receive information were: meeting with healthcare providers (78.5%), Internet (73.9%), and brochures/booklets (70.1%). Most respondents indicated they would likely use in-person counseling (73.3%), in-person support groups (71.0%), or online support groups (66.3%), if available for free in their community. Caregivers were significantly more likely to say they would use in-person counseling than would patients (82.2% vs. 71.4%) (p = 0.000).

CONCLUSION

People affected by brain tumors experience substantial unmet needs. Oncology professionals can provide information, support, and referrals to help families anticipate and cope with the tumor and its effects.

Neuro-Oncol. 2009 Oct;11(5):654.

391. Congruence Between Patient and Caregiver Reports of Patient Symptoms in Persons with a Primary Malignant Brain Tumor

Mary Beth Baer, Chien-Wen Kuo, Sherry Fox, Frank Lieberman, Arlan H Mintz, Amin B Kassam, Heidi Donovan, Paula R Sherwood

INTRODUCTION

Multiple studies in oncology have reported discrepancies between patient and family caregiver reports of patients’ symptom severity. However, few studies have examined symptom congruence in neurooncology or have identified variables that predict discrepancies. The purpose of this study was to evaluate congruence between patient and care-giver reports of patients’ physical and emotional symptoms, and to identify variables associated with incongruence.

METHODS

Data were obtained from 35 patients with a primary malignant brain tumor and their family caregivers recruited for an ongoing National Cancer Institute-funded study (R01-118711). Symptom severity was obtained via interview using the M.D. Anderson Symptom Inventory-Brain Tumor Module (MDASI-BT) for physical symptoms and a composite scale of 18 psychological symptoms. During separate interviews, both patients and caregivers were asked to rate patients’ symptom severity. Wilcoxon matched-pairs signed-rank tests were used to compare discrepancies between patient and caregiver reports.

RESULTS

Summing severity scores for the MDASI-BT and emotional symptom scale for both caregiver and patient revealed that symptom reports significantly differed (p < 0.01 and p = 0.05 respectively). When severity scores for individual symptoms were examined, caregivers consistently rated patients’ fear (p < 0.01), anxiety (p = 0.04), and loneliness (p = 0.01) to be more severe than patient reports. Caregivers also reported higher symptom severity for patients regarding difficulty concentrating (p = 0.03), fatigue (p = 0.02), and pain (p = 0.01). Regression analyses revealed that caregivers who were older were significantly (p = 0.05) more likely to report patients feeling less calm and peaceful compared with patient reports.

CONCLUSION

Data suggest patient and caregiver reports of patients’ symptom severity in neurooncology may not be equivalent, particularly in emotional symptoms, fatigue, and pain. Because symptom management interventions are often based on patient or caregiver reports, symptoms that are likely to be incongruent should be assessed in more depth by treating clinicians. Future research with larger samples should investigate the role of sociodemographic and treatment characteristics in predicting incongruence.

Neuro-Oncol. 2009 Oct;11(5):654.

392. Internet usage by Neurooncology Patients and their Families During the First 4 Months Following Diagnosis

Kelley Wachter, Chien-Wen Kuo, Allison Hricik, Sherry Fox, Arlan H Mintz, Frank Lieberman, Amin B Kassam, Paula R Sherwood

INTRODUCTION

Health information is accessed from the Internet by more than 10 million people every day and can be a frequently used source of information by people with a brain tumor and their families. However, neurooncology practitioners know little about how and when patients and families access the Internet for information. The purpose of this study was to identify how frequently patients and their families access the Internet after the diagnosis of a brain tumor and what information they are seeking at diagnosis and at 4 months after diagnosis.

METHODS

As part of a descriptive, longitudinal, ongoing National Cancer Institute-funded study (R01-118711), 60 patients with a primary malignant brain tumor and their family caregivers (dyads) were recruited within a month of diagnosis. Data regarding type, frequency, and purpose of Internet access were gathered from telephone interviews at diagnosis and 4 months after diagnosis. Descriptive analyses were used to characterize the data, and two-sample proportion tests were used to evaluate differences over time.

RESULTS

Almost all dyads reported having home Internet connections at diagnosis (93%) and at 4 months after diagnosis (98%); the most common type of connection was cable or DSL (91%). The proportion of dyads that reported using the Internet to seek disease information was significantly greater at baseline (88%) than it was 4 months after diagnosis (65%) (p = 0.01). The main reasons for accessing the Internet (to obtain information on disease processes and treatment options) did not change over time.

CONCLUSION

Data suggest that neurooncology dyads are using the Internet consistently and frequently to obtain information on disease and treatment options. Practitioners need to be cognizant that the Internet is a large source of information and to implement ways to ensure the information available to dyads is accurate and current.

Neuro-Oncol. 2009 Oct;11(5):654.

393. Symptom Clusters in Patients with Brain Metastases

Terri S Armstrong, Ibrahima Gning, Elizabeth Vera-Bolanos

INTRODUCTION

Brain metastases are common and are associated with significant morbidity. Patients experience concomitant symptoms related to their systemic disease, brain metastases, and treatment. The purpose of this study was to identify symptom clusters in patients with brain metastases in relation to treatment status.

METHODS

One hundred twenty-four patients with brain metastases participated in this study. Patients completed the M.D. Anderson Symptom Inventory-Brain Tumor Module (MDASI-BT). Descriptive statistics were used to report the characteristics of the sample, overall symptom severity, and association between symptoms and functional status. Hierarchical cluster analysis was used to identify clusters of symptoms, which occurred in relation to treatment status (within 1 month of surgery and within 1 month of radiation therapy).

RESULTS

Sixty-six females and 58 males participated in the study. Of the 124 participants, 80% were Caucasian, and 28% were at least 65 years old with a median age of 57. The majority had metastases from either breast (24%) or lung (31%) cancer. Patients with a poor performance status had significantly higher overall mean symptom severity (p = 0.001), and interference with daily function (p < 0.00001). Six symptom clusters were identified: a cognitive cluster (difficulty understanding, speaking, concentrating, and remembering); a focal neurologic cluster (numbness, weakness, and seizures); a fatigue cluster (fatigue and difficulty sleeping); a constitutional symptom cluster (dry mouth, irritability, drowsiness, and diarrhea/constipation); a pain cluster (pain, shortness of breath, distress, and sadness); and an interference cluster (walking, enjoying life, activity, alteration in mood, and working). Patients within 1 month of surgery had higher symptom severity for interference items (mean difference 1.68, p < 0.05). Patients undergoing radiation therapy experienced a significantly higher burden for the gastrointestinal cluster (mean difference 1.16, p < 0.05).

CONCLUSION

Patients with brain metastases experience six symptom clusters with relationships demonstrated for treatment status. By identifying these clusters, we can then explore underlying biologic mechanisms and targeted preventative treatments to improve symptomatology.

Neuro-Oncol. 2009 Oct;11(5):654.

394. The use of Video to Facilitate End-of-Life Discussions with Cancer Patients: A Randomized Controlled Trial

Areej El-Jawahri, Lisa Podgurski, April Eichler, Scott Plotkin, Jennifer Temel, Susan L Mitchell, Yuchiao Chang, Michael J Barry, Angelo E Volandes

INTRODUCTION

Cancer patients have a strong desire to be involved in their end-of-life decision-making. Despite the importance of advance care planning (ACP) for patients with malignant glioma, ACP discussions are often avoided and when initiated are often ineffective. We therefore examined the use of a video for patients with malignant glioma to facilitate a more comprehensive understanding of goals-of-care options.

METHODS

In a randomized controlled trial, 50 subjects with malignant glioma were randomized to a verbal narrative of goals-of-care options at the end of life or a video after the same verbal narrative. The video depicted three levels of medical care: life-prolonging care (cardiopulmonary resuscitation [CPR], intubation), basic care (hospitalization, no CPR), and comfort care (symptom relief) (the video is available online: http://gallery.mac.com/avolandes#100044). Study end points were subjects’ end-of-life care preferences, change in knowledge scores, and comfort level for patients randomized to the video.

RESULTS

Fifty subjects were randomized to either the verbal narrative (n = 27) or the video (n = 23). After the verbal description, 7 (26%) participants preferred life-prolonging care, 14 (52%) preferred basic care, and 6 (22%) preferred comfort care. After watching the video, no participants preferred life-prolonging care, 1 (4%) preferred basic care, 21 (92%) preferred comfort care, and 1 (4%) was uncertain (p < 0.0001). The mean increase in knowledge score in the video-intervention group was 1.9 compared with 0.85 in the verbal group (p = 0.004). Most subjects (83%) reported being “very comfortable” watching the video, and 19 (83%) subjects stated that they would “definitely” recommend it to other cancer patients.

CONCLUSION

Compared with patients with malignant glioma who only heard a verbal description, patients who viewed a goals-of-care video were more likely to prefer comfort care, were more informed regarding their goals-of-care options, and reported feeling comfortable watching the video. These results indicate that using an ACP video may improve patients’ understanding and confidence regarding their end-of-life decisions.

Neuro-Oncol. 2009 Oct;11(5):655.

395. A Prospective Evaluation and Literature Review of Levetiracetam use in Patients with Brain Tumors and Seizures

Allen K Sills, L Madison Michael, Justin B Usery, Christopher K Finch

INTRODUCTION

Treatment of seizures related to brain tumors is often challenging due to tumor growth, anticonvulsant resistance, and pharmacokinetic or pharmacodynamic drug interactions. Levetiracetam has become a popular agent in tumor patients due to avoidance of serum drug concentration monitoring, limited side-effect profile, lack of known drug interactions, and 1:1 intravenous to oral conversion. The objective of this study was to determine safety and tolerability of i.v. and oral levetiracetam as monotherapy for seizures in brain tumor patients undergoing surgical resection.

METHODS

Patients undergoing craniotomy with >1 seizure within 1 month of surgery were enrolled to receive i.v. levetiracetam for 48 h at the time of craniotomy. Patients made the transition to equivalent oral levetiracetam and were followed for 1 month after discharge. Patients were assessed daily in the hospital and then provided a seizure diary and a 1-month supply of levetiracetam upon discharge. Patients were contacted weekly to assess cognitive status and seizure frequency.

RESULTS

Seventeen patients were enrolled, and the types of seizures experienced were tonic clonic (8), partial (8), and complex partial with secondary generalization (1). The most common tumor was glioblastoma multiforme (7). Levetiracetam was well tolerated with no medication discontinuation during the study period. Adverse effects included: somnolence (3), nausea/vomiting (1), insomnia (1), and aphasia (1). Eleven patients were evaluable for telephone interview on cognitive status (TICS) scores (64.7%). Average cognitive status score was 33.3. Two patients were deemed to have cognitive impairment upon study entry (18.2%). Eleven of 12 patients (91.7%) completing the 4-week study period achieved >50% reduction in number of seizures. Total drug interactions avoided was 92 (p = 0.0016), with dexamethasone (31), acetaminophen (25), and fentanyl (12) being the most common.

CONCLUSION

Levetiracetam monotherapy was found to be safe and well tolerated in this patient population. Nearly all patients achieved >50% reduction in seizure frequency after surgery with levetiracetam monotherapy. Levetiracetam also has the potential to produce fewer drug interactions in brain tumor patients when compared with older anticonvulsants.

Neuro-Oncol. 2009 Oct;11(5):655.

396. Endocrine Recovery after Surgery in Pituitary Adenomas Presenting with Hormone Deficiency

Brandon C Gabel, Gregory G Heuer, M Sean Grady, Kevin D Judy

INTRODUCTION

Patients with pituitary adenomas often develop symptoms and signs of hormone deficiency. Compression or destruction of normal pituitary tissue by the adenoma is believed to cause the endocrine dysfunction. Hypopituitarism of any type can cause life changing, and sometimes life threatening problems. In regards to return of endocrine function, surgical resection of adenomas has yielded conflicting results in the literature. This study was designed to determine the rate of hormone recovery after surgical resection.

METHODS

Retrospective data were collected for all patients who underwent resection of pituitary adenomas between 1996 and 2002 within the University of Pennsylvania Health System and had a drop in at least one or more pituitary hormones preoperatively. The patients were evaluated and treated by an interdisciplinary team that consisted of neurosurgeons, otorhinolaryngologists, and endocrinologists.

RESULTS

Twenty-four patients were identified who underwent resection of their pituitary adenoma, had a drop in at least one pituitary hormone preoperatively, and had follow-up data available at the time of this study. Of these 24 patients, 5 had total correction of their hormone deficiency, 8 had at least partial correction, and 11 had no correction of their endocrine abnormality. Average follow-up time for all patients was 1.65 years.

CONCLUSION

In the patients with available data, surgical resection of adenomas, with a preoperative drop in at least one hormone, resulted in total correction of the endocrine abnormality in only 21% (5/24) of patients. Forty-six percent (11/24) of patients had no correction of their initial hormone dysfunction. Given the morbidities of transsphenoidal pituitary surgery, further study is needed to determine whether resection is appropriate in the absence of other nonhormone related signs and symptoms.

Neuro-Oncol. 2009 Oct;11(5):655.

397. The Danish Cancer Pathway Program Speeding up the Diagnostic Process: 1-Month Status after Implementation in a Regional Neurology Department

Emilie Lund Laursen

INTRODUCTION

The Danish Integrated Cancer Pathway integrates the organizational, clinical, and monitoring aspects of 34 types of cancer and aims to standardize, optimize, and speed up diagnostics and treatment for cancer patients according to national evidence-based clinical guidelines. We implemented the brain cancer pathway in April 2009. The initial neurological core of the pathway consists of neurological examination by a senior neurologist and MRI-C within 72 h of referral for suspected brain malignancy.

METHODS

We included all patients with suspected brain malignancy admitted to our department in April 2009. The inclusion and exclusion criteria to the pathway are described. Age, gender, debut symptoms, previous malignant illness, referral indications and manner, radiological diagnosis, and time perspective of the different parts of the pathway were noted.

RESULTS

Fourteen patients (seven men and seven women; mean age, 50 years) were enrolled: 35.7% upon referral and the rest after admission. All met as least one criterion for enrollment. The majority of patients were referred from general practice. Fifty-seven percent had diagnostic imaging performed on the day of admission, and 35.7% had an MRI performed within 48 h. Mean time from referral to primary MRI was 3 days and to primary computed axial tomography (CAT) scan was 0.3 days. Seventy-one percent had intracranial space-occupying lesions (six primary tumors, three metastases, one arachnoid cyst). Enrollment in the pathway was noted in 78.5% of patient files and exit from the pathway was noted in 50%. A senior neurologist examined all enrolled patients, and physical and occupational therapists saw 64.3% within 24 h. Mean time from radiological diagnosis to neurosurgical consult was 12.6 days.

CONCLUSION

The enrollment criteria result in high diagnostic precision. For a more expedient diagnostic process, the cooperation between specialist departments must be refined. As a whole, the pathway has resulted in increased diagnostic speed and has facilitated the way to appropriate treatment.

Neuro-Oncol. 2009 Oct;11(5):655.

398. Longitudinal Study of Quality of Life in Newly Diagnosed Malignant Glioma Patients

Rupa Parvataneni, Margaretta S Page, Kathleen L Lamborn, Mei-Yin C Polley, Michael Prados, Jennifer L Clarke, Emily Hsieh, Valerie Kivett, Teresa Urquhart, Jane Rabbitt, Anne Fedoroff, Emelia Clow, Nicholas A Butowski, Raymond Liu, Susan Chang

INTRODUCTION

Developing interventions that improve the quality of life (QOL) in patients with malignant glioma requires an improved understanding of factors that influence QOL over time. The primary objectives of this study are to prospectively describe changes in QOL and symptom burden over time in newly diagnosed patients.

METHODS

Questionnaires will be administered to 100 newly diagnosed malignant glioma patients ⩾age 18 and Karnofsky performance status (KPS) ⩾50%. These questionnaires are completed before and after radiation, then every 2 months until patient withdrawal. Pittsburgh Quality Sleep Index (PSQI), Epworth Sleepiness Scale (ESS), Lee Fatigue Scale (LFS), Hospital Anxiety and Depression Scale (HADS), Medical Outcomes Study- Cognitive Scale (MOS-Cog), and Functional Assessment of Cancer Therapy- Brain (FACT-Br) are all used. Descriptive statistics will summarize baseline and postradiation QOL and the symptom-burden status of the patients. A paired t-test will identify areas with significant changes between baseline and postradiation status.

RESULTS

Forty-eight eligible patients were enrolled from March 2008 to April 2009. Baseline characteristics for these patients included: median (range) age 55 (24–71), KPS 90 (60–100), 62.5% male, and 91.6% grade 4 tumor. Twenty-five patients completed both the pre- and postradiation questionnaires. For those 25 patients, the median (range) time to complete the questionnaires preradiation was 5 days (0–18) and postradiation was 13 days (1–33). To date, analyses have been completed on the following instruments: HADS, PSQI hours of sleep, LFS, ESS total score, and MOS-Cog. Mean difference between pre- and postradiation was statistically significant for an increased hours of sleep/night (1.06, p = 0.02), increased fatigue (9.58, p = 0.007), and reduced anxiety (−1.84, p = 0.01).

CONCLUSION

Insight gained about QOL and symptom changes in this population may generate future interventions that improve QOL. We plan to present data on additional patients enrolled and analysis of all tools administered at baseline and after radiation.

Neuro-Oncol. 2009 Oct;11(5):655–656.

399. Measuring Health Status in Recurrent Glioblastoma: Validation of the Euroqol EQ-5D

Martin van den Bent, Astra M Liepa, Vinay K Puduvalli, Antoine Carpentier, Lawrence Cher, Warren Mason, Shengyan Hong, Wolfgang Wick

INTRODUCTION

The EuroQoL EQ-5D is a patient-reported measure of health status used to indirectly obtain utility values, which are incorporated into economic evaluations to derive quality-adjusted life years (QALYs). Limited data on health status or utility values for CNS tumors have been reported. Here we explore properties of the EQ-5D in patients with recurrent glioblastoma.

METHODS

Patients participating in an open-label phase III study comparing enzastaurin and lomustine completed the EQ-5D prior to randomization, during the study, and at discontinuation. Data from treatment arms were pooled. To assess criterion validity, patients were grouped by baseline Karnofsky performance status (KPS) and neurological functional status (NFS); baseline EQ-5D scores were analyzed using analysis of variance. To assess responsiveness to change, EQ-5D scores at discontinuation (progression) were compared with baseline values for patients with data at both time points using a paired t-test.

RESULTS

Baseline EQ-5D data were available from 248 of 266 randomized patients. Mean EQ-5D index scores ranged from 0.55 for patients with KPS = 70–0.88 for KPS = 100 (p < 0.001). Based on NFS, mean index scores ranged from 0.50 for partially disabled patients with moderate symptoms to 0.81 for fully active patients with no symptoms (p < 0.001). Mean EQ-5D visual analogue scale scores were similarly different when grouped by KPS and NFS (both p < 0.001). Based on 64 patients, the mean baseline index score was 0.77 and the discontinuation score was 0.70 (p = 0.033). This decrease in score was associated with increased impairment in all five dimensions, typically changing from “no problems” to “some problems.”

CONCLUSION

The EQ-5D demonstrated properties of validity to measure health status in patients with recurrent glioblastoma, was significantly associated with clinical ratings of functional status, and was responsive to change in clinical status. These results can be used to inform clinical trial designs and economic evaluations for recurrent glioblastoma.

Neuro-Oncol. 2009 Oct;11(5):656.

400. A Phase II Single-Arm Trial of Palonosetron (PALO) For the Prevention of Acute and Delayed Chemotherapy-Induced Nausea and Vomiting (CINV) in Malignant Glioma (MG) Patients Receiving Irinotecan in Combination with Bevacizumab

Mary Lou Affronti, Alise Brickhouse, Jennifer Marcello, James Herndon, David A Reardon, Annick Desjardins, James J Vredenburgh

INTRODUCTION

CINV, while significantly reduced since the advent of first-generation serotonin subtype 3 (5HT3) receptor antagonists, continues to rank as the most distressing side effect of chemotherapy with a reported incidence of 50–76%. Irinotecan in combination with bevacizumab is a highly effective regimen in malignant glioma (MG) patients but has a low CINV complete response (CR) of 55% with ondansetron. Thus, we conducted a phase II trial to determine the efficacy of a second-generation 5HT3 receptor antagonist intravenous palonosetron (PALO) (0.25 mg) and dexamethasone (10 mg) in preventing acute CINV in bimonthly doses of irinotecan/bevacizumab.

METHODS

Primary end point was the proportion of patients achieving an acute CR (no emetic episode or antiemetic medication 24 h after chemotherapy). Secondary end points included: delayed CR (day 2–5), overall CR (days 1–5), quality of life (QOL), fatigue, and toxicity. A two-stage O’Brien and Fleming design was used to determine if the true PALO CINV CR rate is >65%; HO: p < 0.55 versus H1: p > 65%, where p is the proportion who did not experience CINV. Validated tools to assess QOL and fatigue included Osoba survey, FLIE, and FACIT-Fatigue.

RESULTS

Of the 44 patients required for the first stage, 34 were enrolled. Seventy-four percent were diagnosed with a glioblastoma. Mean age was 51 (standard deviation, 13.3; range, 29–75); 74% were male and 91% were Caucasian. Fifty-nine percent had a KPS of >90; AC use was 24% enzyme-inducing antiepileptic drugs (EIAED), 32% no EIAED, and 41% none (missing 3%). Forty-one percent of patients were on oral steroids, and 50% had prior chemotherapy. Acute CR was 91% (95% CI, 72–99), delayed CR was 88% (95% CI, 68–97), and overall CR was 83% (95% CI, 63–95). Updated results for CR rates, fatigue, and QOL during the first stage will be presented.

CONCLUSION

Preliminary data suggests that PALO is a highly effective and tolerable antiemetic when used with irinotecan and bevacizumab compared with historical pilot data using ondansetron.

***

Neuro-Oncol. 2009 Oct;11(5):656.

401. Gabapentin Ameliorates Bortezomib-Induced Peripheral Neuropathy, Leading to Better Tolerance and Dose Intensity and Less Interruption of Therapy

Philip A Haddad

INTRODUCTION

Bortezomib (B), a proteasome inhibitor, is one of the major active agents in the treatment of multiple myeloma. Bortezomib peripheral neuropathy (PN), primarily a small fiber and painful, axonal, sensory distal neuropathy, is a frequent adverse event that often causes dose reduction and interruption of therapy. Such PN occurs at a median of 53 days from the initiation of therapy and fortunately improves and/or resolves in the majority of cases at a median of 3 months after B discontinuation. Gabapentin (G) has been reported to ameliorate the symptoms, although its impact on treatment has not been documented and theoretical concerns about masking symptoms of potentially ongoing neurological damage have not been addressed.

METHODS

We retrospectively reviewed our experience over the past 3 years with patients who received G as an only intervention for B-related NCI-CTC grade 1 PN in patients with multiple myeloma (MM) and its impact on symptom tolerance, dose reduction, and interruption of B therapy.

RESULTS

We identified eight (seven MM, one POEMS) patients that met our criteria. The majority of patients had subjective symptomatic improvement at G dose of 300 mg tid (two at 200 mg three times a day, two at 600 mg three times a day). None needed B interruption due to PN, although five patients required only one level of dose reduction per manufacturer’s recommendation (one at third cycle, two at fifth, one at sixth, one at seventh). All patients reported improvement of symptoms after B therapy ended, and none progressed to have a neurological deficit.

CONCLUSION

Symptomatic G intervention at NCI-CTC grade 1 PN seems to improve symptom tolerability without masking neurologic damage, delays dose reduction, and limits patients to level 1 reductions and lessens interruption of B therapy. Further clinical exploration of such an intervention is warranted in B related PN.

Neuro-Oncol. 2009 Oct;11(5):656.

402. Pilot Study of Paroxetine to Prevent Development of Depression in Primary Brain Tumors

Elana Farace, Jonas Sheehan, Vernon M Chinchilli, James Hegarty

INTRODUCTION

Whether from disruption of neurotransmitters, frustration from loss of function, or a normal response to coping, brain tumor patients have a high incidence of depression. Our previous research has shown that >50% of patients meet the DSM-IV criteria for major depression and that depression significantly lowers quality of life (QOL) and may even impact length of survival; despite these findings, limited research had been done on the prevention of depression in this population.

METHODS

We performed a pilot-randomized, double-blind, placebo-controlled trial of paroxetine with primary brain tumor patients enrolled at surgery before chemotherapy or radiation had begun. Subjects were excluded for current antidepressant use, history of mania or suicide attempts, or current clinical depression. Subjects were randomized 1:1 to paroxetine (10 mg daily) or placebo; after 2 weeks, the dose was increased to 20 mg and continued until outcomes were assessed at 3 months. Outcome measures included depression symptoms (BDI-2 and PANAS) and health-related QOL: EORTCQOL and Fox QOL.

RESULTS

Seven men and six women were enrolled: seven to active treatment and six to placebo. Data were analyzed using a repeated-measures analysis of variance with the treatment group as the dependent factor. The primary outcome was the BDI 2 score. Results showed that patients on active paroxetine had significantly fewer depressive symptoms than did the placebo group (p = 0.018). On average, the active paroxetine group score decreased by three points, while the placebo group increased on average by one point. Patients on the active paroxetine also improved in their positive affect, although the improvement was not significant.

CONCLUSION

The results of this study have the potential to improve the care of brain tumor survivors by potentially identifying a mechanism to prevent the development of depression. In this group of patients with poor prognosis, prevention of depression is of utmost importance, as they do not have sufficient time to be diagnosed and treated during their relatively short remaining life span.

Neuro-Oncol. 2009 Oct;11(5):656.

403. Steroid Requirements during Radiotherapy for Malignant Gliomas

Athina Marantidou, Christine Levy, Olivier Bailon, Renata Ursu, Johan Le Guilloux, Irene Coman, Catherine Belin, Antoine Carpentier

INTRODUCTION

Radiotherapy (RT) is the mainstay of treatment for high-grade gliomas. However, even with optimal RT regimens, toxicities may develop such as brain edema and worsening of neurological symptoms. Steroids are currently used to manage these complications. Steroid requirements and the factors predicting such requirements have been mainly described in patients with brain metastasis but have not been specifically described in adult patients with malignant gliomas.

METHODS

We evaluated prospectively all patients with malignant gliomas scheduled for RT in a single center from July 2006 to September 2008. For all patients, we recorded the following: age, sex, initial Karnofsky performance status (KPS), tumor localization and histology, type of surgical resection, clinical target volume, total dose and duration of RT, concomitant treatment with temozolomide and steroid dosage during RT and at 1 and 3 months after RT.

RESULTS

Out of 51 patients, 27 (53%) required installation or increase of steroids during RT. The median time to steroid increase was 5 days. Only 14% of patients were free of steroids by the end of RT and 33% at 3 months after RT. The mean maximum dosage of prednisone was 57 ± 51 mg during RT and 29 ± 49 mg 3 months after RT. Surprisingly, the initial KPS of 80% was the only variable in the multivariate analysis associated with higher steroid requirements, both during RT and 3 months after. A tendency for a higher steroid dosage was also seen during RT when RT was associated with temozolomide.

CONCLUSION

In our series, almost all patients required steroids during RT. Poor initial KPS and combination use of temozolomide were associated with higher steroid requirements.

Neuro-Oncol. 2009 Oct;11(5):657.

404. Perceived Quality of Life and Emotional Impact of Treatment in Brain Tumor Patients

Allen K Sills, Michael DeCuypere, Allen Redmond

INTRODUCTION

A patient’s perception of treatment and quality of life (QOL) significantly affect clinical outcome and treatment satisfaction. Unfortunately, these clinical features are rarely considered in brain tumor therapy. This study surveys satisfaction and emotional impact of treatments reported by patients with primary and secondary brain tumors within 1 year of their most recent treatment.

METHODS

A total of 105 patients were surveyed and stratified by brain tumor pathology and treatment. Therapeutic modalities included surgery, conformal external-beam radiotherapy, whole-brain radiation therapy, stereotactic radiosurgery (SRS), and chemotherapy. In addition, a brief questionnaire was provided to the patient’s caregiver to draw observational data regarding treatment and emotional well-being of the patient.

RESULTS

Of the 105 patients surveyed, 20% reported depression symptoms directly related to diagnosis. Similarly, 18% of caregivers felt the patient suffered from depressive symptoms at some point during the course of treatment. Overall treatment satisfaction was highest for surgery (n = 82) and SRS (n = 30), with 80% of both groups reporting favorable impact on QOL. Treatment satisfaction was rated lowest for radiation therapy, with only 50% of patients receiving this treatment (n = 41 total encounters) reporting favorable impact on QOL. Nine percent of surgical patients and 4% of patients receiving chemotherapy (n = 25) stated that these interventions had a negative impact on QOL, while 20% of those receiving radiation therapy perceived a negative effect. However, 27% of surgical patients, 20% of SRS patients, and 42% of chemotherapy patients reported unpleasant side effects associated with treatment. Furthermore, 24% of surgical patients, 25% of chemotherapy patients, and 20% of radiation therapy patients reported considerable treatment discomfort. While associated with high treatment satisfaction, 33% of patients receiving SRS reported significant pain and distress. Finally, 90% of patients receiving surgery and 83% of patients receiving SRS felt well educated by their physician, while only 56% of patients receiving radiation therapy reported similar results.

CONCLUSION

These findings underscore the importance of physician counseling in the treatment of brain tumors and demonstrate that physician-directed patient education may increase treatment satisfaction and perceived QOL in the face of unpleasant side effects associated with therapy.

Neuro-Oncol. 2009 Oct;11(5):657.

405. Toxicity Profile for use of Dronabinol in Adult Patients with Primary Malignant Gliomas to Manage CINV

Deborah H Allen, James J Vredenburgh, Bonnie Dryman, Karen Carter, Maria W Freeman

INTRODUCTION

Up to 50% of neurooncology patients report experiencing chemotherapy-induced nausea and vomiting (CINV), despite 5HT3 or steroid therapy. Adjuvant benzodiazepines frequently alter mental status. Dronabinol has been suggested to provide a safe alternative with little neurologic toxicity (TOX). This study purposed to describe the TOX profile associated with dronabinol administration in neurooncology patients.

METHODS

An exploratory study was conducted of adult patients with WHO grade III/IV primary gliomas being treated with adjuvant chemotherapy; a power analysis was performed for 0.80 effect size, determining a sample size of 34 subjects. Ongoing enrollment is expected to be completed in September, and our current subject accrual is 31 of 34. Subjects take dronabinol 5 mg twice a day 24 h prior to, during, and 48 h after completion of oral/i.v. chemotherapy. Subjects continue their established antiemetic regimen. Between chemotherapy doses, dronabinol is reduced to 2.5 mg daily. Intolerance of dronabinol is defined as two or more grade 3 or greater nonhematologic toxicities according to the CTC v.3 handbook. Modified Functional Living Index-Emesis, Functional Assessment of Cancer Therapy-Brain Tumor, Mini Mental Status Exam, and CINV visual analog scales are collected at specific points for two cycles for TOX and QOL data.

RESULTS

Subjects are predominately male (n = 18), with a mean age of 46 years. Eleven subjects withdrew due to >grade 2 neurologic TOX of somnolence or fatigue (n = 4), and nine subjects had >grade 2 persistent CINV. Increased dosages were required in eight subjects to better manage CINV. Twenty subjects have not reported TOX or worsening baseline data profiles.

CONCLUSION

While persistent CINV was reported, dronabinol doses initiated in this study are low to develop the TOX profile. As there were few CNS TOXs reported, dose escalations for CINV management are warranted. Dronabinol can be offered as a safe adjunct to standard antiemetic therapy during treatment for primary glioma.

Neuro-Oncol. 2009 Oct;11(5):657.

406. Seizures in Patients with Glioma Treated with Phenytoin and Levetiracetam

Ryan Thomas Merrell, Daniel H Lachance, S Keith Anderson

INTRODUCTION

With the demonstrated efficacy of second-generation antiepileptic medications (AEDs), which are relatively free of enzyme-inducing properties, there is interest in comparing these medications with traditionally used first-generation AEDs in patients with glioma. We compared seizure outcomes and side effects in patients with gliomas being treated with phenytoin and levetiracetam monotherapy.

METHODS

We retrospectively reviewed 500 consecutive patients with glioma who were treated from 2001 to 2008 at three Mayo Clinic campuses. Subjects had at least one clinical seizure and had to be followed for at least 6 months. Seizure outcomes and AED side effects were compared between cohorts treated with phenytoin or levetiracetam and were defined by time to second seizure and seizure frequency.

RESULTS

Seventy-six patients (34 females were identified; 25 treated with phenytoin and 51 with levetiracetam). Sixty-four percent had grade 4 astrocytoma. No difference was seen in seizure outcome between the phenytoin and levetiracetam groups when comparing time to second seizure (p = 0.584), second seizure rates (p = 0.561), and average seizures per month (p = 0.776). When adjusted for age, gender, type of seizure, type of glioma, and dosage using univariate and multivariate models, no differences were seen between the treatment groups and none of these covariates were statistically significant in explaining the second seizure rates between treatment groups (all p-values > 0.05). The incidence of side effects in the levetiracetam group was 5.9% vs, 20% in the phenytoin group (p = 0.106). Additionally, 36% of the patients in the phenytoin group had dose adjustments not related to breakthrough seizures compared with only 9.8% in the levetiracetam group (p = 0.010).

CONCLUSION

In this study, glioma patients treated with levetiracetam and phenytoin had similar seizure control. Patients treated with levetiracetam experienced fewer side effects and required fewer non-seizure-related dose adjustments than patients treated with phenytoin. Levetiracetam is a safe, effective, and preferred alternative for seizure management in patients with glioma.

Neuro-Oncol. 2009 Oct;11(5):657.

407. Phase I Trial of Topotecan by Convection-Enhanced Delivery for Recurrent Malignant Gliomas: Radiographic and Quality of Life Analysis

Jeffrey N Bruce, Robert Fine, Peter D Canoll, Michael B Sisti, Guy M McKhann, Casilda Balmaceda, Aaron M Tannenbaum, Benjamin C Kennedy, Steven Rosenfeld, Steven Sands, Rose Lai, Candy Yanes, Venkat Seshan, Robert DeLaPaz

INTRODUCTION

The failure of most chemotherapy drugs in the treatment of gliomas is caused by inadequate delivery rather than lack of antitumor activity. Convection-enhanced delivery (CED) is a strategy designed to overcome the limitations of conventional systemic drug delivery. This strategy is ideal for the topoisomerase inhibitor Topotecan, as it is cytotoxic to glioma cells, which express high levels of topoisomerase, while remaining nontoxic to normal brain where topoisomerase expression is low. We performed a phase I dose-escalation trial of Topotecan by convection-enhanced delivery in patients with recurrent malignant gliomas.

METHODS

Two catheters were stereotactically implanted intratumorally or peritumorally in 16 patients with radiographically localized recurrent malignant gliomas (10 patients with glioblastoma multiforme [GBM] and 6 patients with anaplastic glioma). Over 100 h, 40 ml Topotecan was infused in a dose-escalating strategy (dose range = 0.02–0.133 mg/ml) until a maximum tolerated dose was established. Patients were assessed clinically, radiographically, and with neuropsychological testing for toxicity and antitumor response.

RESULTS

Toxicity was only seen at the highest dose concentration (0.133 mg/ml), including one patient with parietal lobe syndrome and one with left upper-extremity weakness. Quality of life measurements verified the treatment’s tolerability. Tumor regression was seen in 25% of patients. Median overall survival was 59 weeks (range = 8–250 weeks) and median progression-free survival was 20 weeks (range, 4–112 weeks). The 6-month progression-free survival was 44% and 6-month overall survival was 75%.

CONCLUSION

CED of Topotecan can cause tumor regression without excessive toxicity in selected patients with recurrent malignant gliomas refractory to conventional therapy. Quality of life measurements demonstrate advantages of CED over systemic delivery of chemotherapy. The established maximum tolerated dose of 0.1 mg/ml will be used in a multicenter phase II trial.

Neuro-Oncol. 2009 Oct;11(5):658.

408. Measuring the Impact of age-at-Diagnosis on the Quality of Life of Neurooncology Patients

Nadia Jackie Veilleux, David Fortin, Marie Boudrias, David Mathieu, Philippe Goffaux

INTRODUCTION

Quality of life (QOL) has recently emerged as a central theme in neurooncology. Unfortunately, QOL studies in neurooncology have largely ignored the moderating effects of age, preferring instead to focus on tumor- or treatment-related factors. This focus is regrettable, since age-at-diagnosis is often recognized as an important determinant of the way patients interpret and deal with life-altering events. In fact, life-stage studies conducted in other fields of oncology reveal that older and younger adults (typically using 40 to 50 years of age as the cut off) are differentially affected by their malignancy. Given the absence of life-stage studies in neuro-oncology, we measured and compared the QOL of young (⩽40) and old (>40) patients to determine how age impacts specific dimensions of QOL following the diagnosis of a brain tumor. We also isolated specific items that were predictive of overall QOL in younger and older patients, respectively.

METHODS

One hundred thirty patients (42 young, 88 old) diagnosed with supratentorial gliomas participated in this study. QOL was measured using the Sherbrooke Neurooncology Assessment Scale—a questionnaire recently developed to assess both core and disease-specific concerns.

RESULTS

Multivariate analyses revealed that, independently from the effects attributable to histotype, older adults reported poorer functional well-being and suffered from greater neurocognitive decline than did younger adults. Analyses also showed that for younger adults, better QOL was associated with continued support from friends, preserved physical strength, and absence of pain; whereas, for older adults, a preserved appreciation for life and continued ability to concentrate were important QOL predictors.

CONCLUSION

These results indicate that younger and older adults are differentially affected by their condition and that the factors predictive of QOL are radically different depending on age. Paying attention to age, therefore, is critical when considering a patient’s perspective, and when establishing tailored therapies and support programs.

Neuro-Oncol. 2009 Oct;11(5):658.

409. Temozolomide-Induced Semen Analysis: Changes in Men with Newly Diagnosed or Recurrent Malignant Glioma

Glenn J Lesser, Robert Blackwood, Michele Harmon

INTRODUCTION

Cumulative exposure to alkylating agents may produce azoospermia and impaired reproductive function. We initiated a pilot study to examine the effects of temozolomide (TMZ) on sperm, semen integrity, and sex steroid levels in men with newly diagnosed or recurrent malignant glioma (MG) scheduled to receive TMZ.

METHODS

Eligible patients had no known fertility problems or impotence and could not have received prior chemotherapy. A semen analysis and blood sample for measurement of serum testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were obtained at baseline, then following 3 months and at least 6 months of TMZ.

RESULTS

Thirteen men were recruited, with a mean age of 42 years (28–58); 3 had recurrent and 10 had newly diagnosed MG. Four were unable to ejaculate or had azoospermia at baseline. Only four patients provided samples at baseline and following 6 months of TMZ. Reasons for noncompliance included clinical deterioration, modification of chemotherapy regimen, and inability to ejaculate. Of the nine viable baseline samples, 100% had normal sperm density and pH, while 78% had normal sperm forms (>30%) and motility (>50%). Two of the four patients with paired baseline and 6-month samples were previously treated with brain irradiation and received 6 months of standard monthly TMZ (150–200 mg/m2/day × 5 days), while two patients with newly diagnosed disease received standard concurrent chemoradiotherapy and adjuvant TMZ. Three of these four patients demonstrated low sperm motility at 6 months (two low at baseline); three patients had an abnormally low percentage of normal forms at 6 months (one abnormal at baseline), and two developed an abnormally low sperm density at the 6-month time point. Testosterone, LH, and FSH levels at baseline, 3 months, and 6 months were within the normal range in all four patients.

CONCLUSION

A significant number of MG patients are unable to ejaculate or have azoospermia prior to therapy. Although some changes were seen in semen analysis parameters following 6 months of TMZ, the small sample size precludes any firm conclusions regarding the importance and duration of these findings and their relation to TMZ exposure.

Neuro-Oncol. 2009 Oct;11(5):658.

410. Brain Metastases from Melanoma: Seizure Incidence and Associated Morbidity and Mortality

Tulika Ranjan, Edward Avila, Lindsay Jacks

INTRODUCTION

Melanoma, a highly malignant tumor and the third leading cause of brain metastases, is more likely to invade the brain cortex and the lesions are mostly hemorrhagic with a higher risk for seizure incidence.

METHODS

We retrospectively reviewed 91 charts of patients with histological diagnosis of melanoma and brain metastases confirmed by MRI and treated at MSKCC during 2006–2008. We sought information regarding age, sex, Karnofsky Performance Status (KPS), number and location of brain metastases, extent of systemic disease, interval from initial diagnosis to brain metastases, incidence of hemorrhage and seizure, and anticonvulsant use. T1, T2, and GRE sequences of the MRI were used to identify hemorrhage.

RESULTS

Data from 91 patients were analyzed. Of those 91 patients, 63 were men and 28 were women (median age was 57 years). The median interval between the initial diagnosis of melanoma and the diagnosis of brain metastases was 3 years. Ninety-one percent of patients had active systemic disease. Median KPS at the time of melanoma diagnosis was 100 and at the time of brain metastases was 80. The median KPS of patients with and without seizures was 80. Of the 91 evaluable patients, 20 (22%) had single brain metastases and 67 (74%) had multiple. Seventy-three patients (80%) had cerebral hemorrhage. In total 23 patients (25%) had seizures. Three patients had a seizure at the time of diagnosis of brain metastases. Seven patients were on prophylactic anticonvulsants with no seizures. Of the 81 patients not receiving prophylactic anticonvulsants, 20 patients developed seizures. In patients with seizures, 3 (13%) had single metastases and 18 (78%) had multiple metastases. Forty-three percent had cortical lesions, and 57% had both cortical and subcortical lesions. No patients with only subcortical lesions (7%) had seizures. Of the 23 patients with seizures, 22 (96%) had hemorrhage.

CONCLUSION

In this cohort of patients with melanoma and brain metastases, the incidence of seizure was 25%. Patients with multiple brain metastases, cortical lesions, and hemorrhagic lesions were at higher risk for seizures. Seizure incidence did not affect the KPS.

Neuro-Oncol. 2009 Oct;11(5):658.

411. Early Surgical Intervention in 1,089 Adult Ganglioglioma Patients is Associated with Improved Clinical Seizure Outcomes

Isaac Yang, Shanna Fang, Seunggu J Han, Edward F Chang, Tarik Tihan, Andrew T Parsa

INTRODUCTION

Gangliogliomas are CNS tumors most commonly affecting children and young adults. Here we analyze the effects of early surgical intervention on the clinical outcome of patients with gangliogliomas. Surgical resection is the most curative form of therapy, but the clinical benefit of differential timing for surgical resection of these slow-growing tumors remains unclear.

METHODS

Inclusion criteria for articles were 1) outcomes were reported specifically for gangliogliomas, 2) data were reported for adult patients older than the age of 18, 3) treatment data was included for the management of gangliogliomas, and 4) ganglioglioma was the only pathology diagnosis for the evaluation of the tumor. Cases without treatment information, pediatric patients at time of diagnosis or surgery, and other glial tumor variants were excluded in this comprehensive retrospective analysis. Data were systematically analyzed as a whole, then stratified into groups of early and late treatment intervention.

RESULTS

The query identified a total of 1,089 cases of ganglioglioma that met our inclusion and exclusion criteria. The mean reported age of the patients at time of diagnosis was 40.4 years, and the average reported time between onset of symptoms or seizure and surgical intervention was 4.7 years. Mean length of reported follow-up time was 40.7 months (±34.0 months). There was a 55% prevalence in males, representing a statistically significant predilection (51–59%, 95% confidence interval). Our analysis revealed that an improved seizure control rate (78% vs. 48%) was significantly associated with the early intervention group (p = 0.0001).

CONCLUSION

Our findings suggest that an early intervention of less than 3 years from the onset of symptoms to surgical intervention is significantly associated with improved seizure control. This critical finding may significantly improve patient intervention, as the mean time to intervention was more than 4 years in this analysis.

Neuro-Oncol. 2009 Oct;11(5):658–659.

413. Mobility in Brain Tumor Patients: Assessment and Relationship to Quality of Life: A Pilot Study

N Scott Litofsky, Jeffrey Krug

INTRODUCTION

Impaired ambulation and standing balance negatively impact quality of life (QOL) in patients who undergo surgical resection of brain tumors. A means of quantifying mobility is a key step towards routinely assessing mobility function and direct therapy to positively impact QOL. We hypothesized that the Timed-up-and-go (TUG) and the Tinetti Performance-Oriented Mobility Assessment tests can measure mobility in brain tumor patients undergoing surgical resection. Furthermore, we hypothesized that mobility function measured by these tests will correlate with QOL.

METHODS

Nine adult patients with primary brain tumors were evaluated by a physical therapist prior to surgery, after surgery just prior to hospital discharge, and 3 months postoperatively with TUG and Tinetti tests. Patients concurrently completed a SF-36 survey to assess QOL. Differences between time points were analyzed by analysis of variance; Spearman correlations determined associations between tests.

RESULTS

Three patients with glioblastoma multiforme and six patients with meningioma (mean age, 49.2 years) were evaluated. TUG (p = 0.03), Tinetti (p = 0.05), and SF-36 (p = 0.04) deteriorated from presurgery to immediate postsurgery. TUG (p = 0.003), Tinetti (p < 0.001), and SF-36 (p < 0.006) improved 3 months postoperatively. TUG and Tinetti correlated with each other at all time points (r = −0.992, p = 0.00; r = −0.833, p = 0.01; r = −0.966, p = 0.0). SF-36 correlated with TUG and Tinetti immediately after surgery (r = −0.762, p = 0.028; r = 0.883, p = 0.002) and 3 months postoperatively (r = −0.845, p = 0.004; r = 0.849, p = 0.004).

CONCLUSION

The TUG and Tinetti tests measure mobility deficits and improvements in patients who undergo surgery for primary brain tumors. These mobility assessments correlate with QOL. Outcomes of interventions to improve mobility can therefore be quantified. These interventions will likely lead to improvement in patients’ QOL.

Neuro-Oncol. 2009 Oct;11(5):659.

414. Endocrine Function after Craniospinal Irradiation for Medulloblastoma in Children and Adults

Sonia Beltran, Mohamad El Rawas, Pascale Berlier, Marie Pierre Sunyach, Francoise Borson-Chazot, Didier Frappaz

INTRODUCTION

Craniospinal radiation (CSI) is the cornerstone of the treatment of medulloblastoma but may damage hypothalamic pituitary function. Many publications report toxicity of CSI in children, but there is little reported on adults. Objective: To study the prevalence of pituitary deficits, following CSI in children (<15 years at radiotherapy) and adults.

METHODS

A series of 30 patients (16 children, 14 adults) treated by surgery, radiotherapy, and chemotherapy for a medulloblastoma was retrospectively studied. The children’s group was composed of five pubescent girls and 11 boys (three pubescent). Age at radiotherapy ranged between 5 and 12 years. Delay between CSI and evaluation was 1–9 years. Adult patients were four women (three menopausal) and ten men. Age at radiotherapy ranged between 15 and 40 years. Delay between CSI and evaluation was 1–24 years. Growth hormone (GH) and adrenal axes were assessed using stimulation tests (insulin tolerance, glucagon-propranolol, and Synacthene test). Gonadotropin and thyrotropin functions were tested using baseline blood measurements.

RESULTS

Hypopituitarism was present in 57.2% of adult patients and 87.5% of children. The most frequent deficit was GH deficiency, observed in 35.7% of adult patients and 62.5% of children; GH deficiency was significantly associated with the young age at radiotherapy (p < 0.05). ACTH deficiency was rare and was observed in 7.1% of adults and 12.5% of children. Hypothyroidism from central or peripheral origins was recorded in 21.4% of adults and 31.2% of children. Finally, gonadotropin deficiency was observed in 35.7% of adults and 6.2% of children. Two children developed peripheral gonadal insufficiency.

CONCLUSION

Both adult and children treated with CSI for a medulloblastoma are at high risk of hypopituitarism (especially GH deficiency), which depends on age at time of radiotherapy. Long-term surveillance and periodic evaluation are needed in children and adults treated with CSI.

Neuro-Oncol. 2009 Oct;11(5):659.

415. Cavernous Hemangiomas after Cranial Radiation in Childhood Cancer Survivors

Jill Lunsford Lee, Dominique Pichard, David Nascene, Alexander McKinney, Christopher Moertel, Daniel Mulrooney

INTRODUCTION

Radiation therapy, an effective treatment modality for a variety of childhood malignancies, may result in significant late adverse outcomes, particularly when directed towards the CNS. We report an increased number of cavernous hemangiomas following cranial radiation. An unclear understanding of their long-term course and a gap in the pediatric oncology literature prompted us to report our experience among adult survivors of childhood cancer evaluated in our Long-Term Follow-Up Clinic.

METHODS

A retrospective review of medical records and MRI images was completed on six clinic patients incidentally found to have cavernous hemangiomas on surveillance MRI, completed due to increased risk for subsequent neoplasms following radiation exposure for a pediatric malignancy. All images were examined by two neuroradiologists, and diagnosis was based on the presence of hypointense “popcorn”-like lesions on T2 star gradient echo imaging. T2 star images are more sensitive in detecting hemorrhagic lesions than are spin echo T2-weighed imaging.

RESULTS

Diagnoses included brain tumor (n = 1), solid tumors (n = 2), and leukemias (n = 3). Mean age at cancer diagnosis was 7.3 years, mean age at cavernoma diagnosis was 27.8 years, and mean time from radiation exposure was 20.2 years. Radiation doses ranged from 1,050 to 5,371 cGy, and foci varied from wide to narrow. Two survivors had single lesions, and four survivors had multiple lesions. Most were asymptomatic, but two patients (one ALL and one AML) had seizures preceding the diagnosis of cavernous hemangioma.

CONCLUSION

Surveillance MRI imaging for new primary CNS neoplasms has become commonplace following CNS-directed radiation therapy for childhood malignancies. The risk of vascular injury is also increased, and its progression and standard for surveillance have not yet been determined. MRI surveillance should include T2 star-weighted images to detect the presence of cavernous hemangiomas following cranial radiation. More research is needed to determine appropriate long-term management of these lesions.

Neuro-Oncol. 2009 Oct;11(5):659.

416. Life after Brain Cancer: Self- and Proxy-Evaluated Quality of Life Among Pediatric Survivors of Low-Grade Glioma

Lindy Musial-Bright, Louisa Panteli, Guenter Henze, Pablo Hernaiz Driever

INTRODUCTION

Despite improved management of pediatric low-grade glioma (LGG), many survivors experience some morbidity including motor, sensory, audio, visual and cognitive deficits, endocrine deficiencies, and epilepsy. Assessing the subjective impact that the effects of LGG and its treatment have on a patient can be tricky and has become the goal of pediatric quality of life (QoL) research. This single institution study looks at LGG survivors’ self- and parent-evaluated quality of life.

METHODS

Forty-nine (59.8%) of 82 LGG survivors and their families seen for follow-up care between November 2003 and November 2004 completed the KINDL quality of life survey. The KINDL is a psychometrically tested and kid-friendly 24-item survey covering six domains of QoL (physical and emotional well-being, self-esteem, family, friends, and school). Parent- and self-evaluated QoL data were compared with published norms. Differences between the self- and proxy-evaluation and between boys’ and girls’ self-evaluations were compared by Wilcoxon and Mann-Whitney U tests, respectively. Correlations between age, tumor localization, and modified Rankin disability scores with QoL were investigated with Spearman’s rho.

RESULTS

LGG survivors, ages 4–17 years old, rated total QoL significantly higher than their parents. Eleven- to 17-year-old LGG survivors rated total QoL, physical well-being, and self-esteem significantly higher than their peers. The parents’ evaluation of their children’s total QoL did not differ significantly from published norms. Girls rated total QoL significantly lower than boys, as did their parents. No significant correlations were found between self-evaluated total QoL and age, tumor localization, or disability, although there was a weak yet significant correlation between the friends subscale and disability score.

CONCLUSION

Despite tumor and treatment-associated morbidity, LGG patients but not their parents rate their total QoL better than their peers. These results may reflect successful management of LGG, patients’ successful coping mechanisms, or the difficulties pediatric oncologists face in assessing QoL in pediatric patient populations.

Neuro-Oncol. 2009 Oct;11(5):659–660.

417. Bevacizumab In Recurrent High-Grade Pediatric Gliomas: Do the Clinical Results Hold up?

Ashwatha Narayana, Jeena Mathew, Jennifer Zeng, Shahzad Raza, Sharon Gardner, Matthias Karajannis, David Zagzag, Howard L Weiner, Jeffrey H Wisoff, Jeffrey Allen

INTRODUCTION

Bevacizumab, a monoclonal antibody against vascular endothelial growth factor, has recently been approved for the treatment of recurrent adult high-grade glioma (HGG). However, very little data exist on pediatric gliomas treated with bevacizumab therapy. We report the preliminary results of bevacizumab therapy in recurrent pediatric HGG.

METHODS

Ten patients with recurrent nonbrainstem HGG received 10 mg/kg of bevacizumab every 2 weeks, along with 125 mg/m2 of CPT-11. The median age was 14 (range, 4–22). The pathology was WHO grade III in seven patients and grade IV in three patients. The median number of bevacizumab infusions was three (range, 2–18). Radiological response, progression-free survival (PFS), overall survival (OS), and toxicities were analyzed.

RESULTS

The median duration of follow-up was 5.3 months (range, 1–12). The median number of recurrences before starting bevacizumab was two (range, 1–3). Nine patients tolerated therapy. Therapy was discontinued in one patient due to anaphylactic reaction. Two grade-III nonhematological toxicities were seen that included delayed wound healing and severe fatigue. One patient experienced grade-IV intratumoral hemorrhage. Partial radiographic responses were noted in four of the seven evaluable patients at the time of first evaluation. The median PFS and OS were 2.2 and 5.6 months, respectively. The 6-month PFS and OS were 29.6% and 43%, respectively. In our published data of 61 recurrent adult HGG patients treated with bevacizumab, the median PFS and OS were 5 and 9 months, respectively, and the 6-month PFS and OS were 45% and 61.7%, respectively.

CONCLUSION

Treatment tolerance, toxicity, and radiological responses were comparable with adults treated with bevacizumab therapy. However, the duration of response and survival appeared inferior compared with the adults. Differences in the genetic make-up of pediatric gliomas might account for this difference and need to be further defined in future prospective trials.

Neuro-Oncol. 2009 Oct;11(5):660.

418. Carboplatin and Ototoxicity: Hearing Loss Rates among Survivors of Childhood Medulloblastoma

Lindy Musial-Bright, Guenter Henze, Ruediger Fengler, Pablo Hernaiz Driever

INTRODUCTION

Health care providers shift their focus from merely improving patients’ overall survival to safeguarding their quality of life after antineoplastic treatment. Because of its localization and management, patients with medulloblastoma are exposed to many potentially ototoxic treatments including neurosurgery, radiation therapy, and platinum chemotherapy. The favorable toxicity profile of carboplatin led us to substitute this chemotherapeutic agent for cisplatin in the chemotherapy protocols of HIT’91, HIT-MED’99, and HIT-2000 randomized prospective trials. This is the first retrospective analysis of this concept and its consequences in terms of overall survival and ototoxicity rates.

METHODS

Twenty-four medulloblastoma patients were treated between April 1999 and June 2006 according to HIT protocols with carboplatin substitution. Nineteen (79%) of those 24 patients had adequate baseline and posttreatment audiological data to be included in this analysis. The mean age at diagnosis was 9.3 (range, 3.5–18.9) years, and the mean time to follow-up was 30.8 (range, 8.1–111.3) months. The mean carboplatin cumulative dose was 2,131 (range, 830–4,312) mg/m2. The most common cause of dose reduction was hemotoxicity (66.7%).

RESULTS

Hearing loss greater than 20dB was observed in two (10.5%) of 19 patients. Both patients had grade-2 ototoxicity, according to Brock’s scale. No significant differences existed between the patients’ baseline and posttreatment audiograms at any frequency. The observed hearing loss was weakly but significantly correlated to younger age at diagnosis and cumulative carboplatin dose (p < 0.05). The 24 patients were alive at the time of assessment.

CONCLUSION

The encouraging survival and low hearing-loss rates (100% and 10.5%, respectively) in this patient cohort suggest that protocols containing carboplatin may offer a viable alternative to standard cisplatin protocols and warrant further investigation.

Neuro-Oncol. 2009 Oct;11(5):660.

419. Benefit Finding in Caregivers of Pediatric Cancer Survivors: a Mixed Methods Approach

Sarah A Hostetter, Katherine C Hutchinson, Kristina K Hardy, Victoria W Willard, Sridharan Gururangan, Melanie J Bonner

INTRODUCTION

There has been recent recognition that both positive and negative results accompany serious illness. “Benefit finding,” or the positive effects that result from stressors, has been linked to decreased depression, increased well-being, and increased life satisfaction and has been implicated as a component in the coping process. While benefit finding has been studied in adult cancer survivors, this construct has rarely been applied to pediatric cancer survivors and has not been extended to their caregivers. This mixed methods study describes qualitative themes related to benefit finding reported by caregivers of pediatric cancer survivors, while also examining the relationship between qualitative themes and quantitative measures of psychosocial adjustment and coping.

METHODS

Caregivers of pediatric cancer survivors (n = 36; 50% brain tumor and 50% other cancer) were recruited during regularly scheduled clinic visits to complete quantitative measures including a demographic questionnaire and a qualitative questionnaire about their experience caring for a pediatric cancer survivor. Qualitative questionnaires were transcribed and coded for salient themes, including social support and personal growth.

RESULTS

Correlation matrices were created to evaluate associations between qualitative themes and quantitative measures of stress and coping. Total benefits reported and personal growth benefits specifically were correlated with availability of emotional resources (r = 0.439, p = 0.008; r = 0.408, p = 0.015). A t-test was conducted to determine whether there were any significant differences in benefit finding between the brain tumor group and the other cancer diagnosis group. No significant differences were found between the two.

CONCLUSION

Despite the stress of their child’s illness, many caregivers of pediatric cancer survivors report finding benefits associated with their experience. Benefit finding in this population is associated with better adjustment. The role of benefit finding in the coping process appears to be consistent across both brain tumor and other cancer diagnoses.

Neuro-Oncol. 2009 Oct;11(5):660.

420. Radiosurgery Achieves Long-Term Tumor Control of Petroclival Meningiomas

Thomas J Flannery, Douglas Kondziolka, Hideyuki Kano, L Dade Lunsford, Ajay Niranjan, Sait Sirin, Matthew J Tormenti, John Flickinger

INTRODUCTION

Because of their critical location adjacent to brain, cranial nerve, and vascular structures, petroclival meningiomas remain a clinical challenge. We evaluated clinical and imaging outcomes in petroclival meningioma patients who underwent Gamma Knife radiosurgery (GKRS) over a 21-year period.

METHODS

Radiosurgery was used either as primary (n = 105, 58%) or adjuvant management (n = 77, 42%) for petroclival meningiomas involving the region between the petrous apex and the upper two-thirds of the clivus. In patients with prior resection, the WHO grade was grade I (n = 72), grade II (n = 3), and grade III (n = 2). The most common symptoms patients presented with were trigeminal nerve dysfunction, balance problems, diplopia, and hearing loss. The median tumor volume was 6.0 cm3 (range, 0.3–32.5 cm3), and the median margin dose was 13 Gy (range, 9–18 Gy).

RESULTS

Clinical follow-up was available in 172 patients (94.5%; median follow-up, 78 months). Neurological status improved in 44 patients (26%), remained stable in 102 patients (59%), and worsened in 26 patients (15%). Imaging follow-up was available in 168 patients (median follow-up, 64 months). Tumor volume decreased in 77 patients (46%), remained stable in 74 patients (44%), and increased in 16 patients (10%) who underwent additional management. Significant risk factors for symptom and tumor progression were a tumor volume equal to 8 cm3, (p = 0.007 and 0.001, respectively) and male gender (p = 0.02 and 0.02, respectively). Eight patients underwent repeat radiosurgery for tumor progression, four patients underwent resection for progressive disease, and four patients underwent fractionated radiotherapy for progressive disease. Cerebrospinal fluid diversion was performed in seven patients (4%).

CONCLUSION

In this 21-year experience, Gamma Knife radiosurgery for petroclival meningiomas achieved freedom from the need for surgical resection in 98% of patients and was associated with a low risk of adverse effects from radiation. We suggest that radiosurgery be the preferred initial option for patients with smaller-volume tumors.

Neuro-Oncol. 2009 Oct;11(5):660.

421. Adjuvant Radiosurgery after Resection of a Brain Metastasis: Patterns of Failure Analysis

Courtney A Jensen, Michael Chan, Thomas McCoy, Edward G Shaw, Thomas L Ellis, Kevin P McMullen, Michael Munley, J Daniel Bourland, Ken Ekstrand, Allan F deGuzman, Stephen B Tatter

INTRODUCTION

The current standard of care for the postoperative management of resected brain metastasis is whole brain radiotherapy (WBRT), which can be associated with significant neurocognitive toxicity. At our institution, we have used cavity-directed radiosurgery (RS) to delay or prevent the need for WBRT in this case.

METHODS

Between April 2001 and March 2009, 106 resection cavities in 101 patients without prior WBRT were treated with cavity-directed RS and RS treatment of any synchronous brain metastases. A median dose of 17.5 Gy to the 50% isodose line was prescribed to the rim of enhancement around the resection cavity. Patients were followed radiographically, and new brain metastases were generally treated with additional RS with salvage WBRT typically reserved for local failure at a resection cavity or numerous brain failures. Local and distant brain failures were determined radiographically. Kaplan-Meier curves were generated to estimate local and distant brain failure rates, overall survival, and time to salvage WBRT. Univariate and multivariable analyses were performed using Cox proportional hazards regression.

RESULTS

The median overall survival was 10.9 months. The 1-year local brain failure rate was 20.3%. Distant brain failure occurred in 50% of patients at a median of 6.2 months. Fifty percent of patients were treated with salvage WBRT at a median of 9.9 months. Univariate analysis revealed that the only factor significantly predictive of local failure was a tumor diameter greater than 3 cm on preoperative magnetic resonance imaging (HR = 4.61; p = 0.047) and that the only factor significantly predictive of the need for salvage WBRT was subtotal resection (HR = 3.53; p = 0.039).

CONCLUSION

While cavity-directed RS delayed the need for WBRT in most patients, patients with tumors greater than 3 cm on preoperative magnetic resonance imaging or patients with subtotal resection of tumor should be considered for WBRT as part of their postoperative management.

Neuro-Oncol. 2009 Oct;11(5):661.

422. The Effects of Glycemic Control on Volumetric White Matter Changes after Whole Brain Radiation

Nicholas J Szerlip, Charles Rutter, Nilam Ram, Susannah Yovino, Esther Kim, William Warren Maggio, William Regine

INTRODUCTION

Whole brain radiotherapy (WBRT) is one of the most effective modalities for the treatment of brain metastases. With increasing cancer control, there is growing concern regarding the long-term effects of treatment. These effects are seen as white matter changes (WMCs) on brain MRI. Given that the severity of WMCs are implicated in patients’ cognitive and functional decline, our objective was to identify clinical factors associated with worsening WMCs.

METHODS

In a retrospective review, we obtained the serial magnetic resonance images of 22 patients who were treated at a single institution between 2002 and 2007 and who survived more than 1 year after WBRT. Segmentation software was used to calculate volumetric WMCs over time. Changes related to a tumor, its secondary effects, surgery or stereotactic radiosurgery were excluded. Multivariate analysis was used to identify factors that influenced the rate at which WMCs accumulated.

RESULTS

The majority of the patients (63%, n = 14) were women; the median age was 51 years. The predominant histology was non-small-cell, and the mean total radiation dose was 36 Gy. Patients accumulated, on average, WMCs at a rate of 0.057% of total brain volume per month. Greater rates of accumulation were independently associated with age, (β = 0.003, p < 0.0001), hyperglycemia (β = 0.047, p < 0.0001), and hypertension diagnosis (β = 0.046, p < 0.05). In multivariate analyses all effects remained independent and significant (p < 0.05).

CONCLUSION

Long-term survivors of cancer allow us to observe late effects of treatment modalities. WBRT appears to be related to a continued and steady rate of white matter damage over time. Our results suggest that WMCs may be enhanced by hyperglycemia and hypertension. This study has a potentially significant clinical impact by clearly identifying hyperglycemia as a target for intervention to decrease WMCs in patients receiving WBRT.

Neuro-Oncol. 2009 Oct;11(5):661.

423. Outcomes of Stereotactic Radiosurgery for Prolactin-Secreting Pituitary Adenoma: Tumor Control and Biochemical Remission

Shota Tanaka, Michael J Link, Bruce E Pollock

INTRODUCTION

To evaluate the outcomes of stereotactic radiosurgery (SRS) for patients with prolactin (PRL)-secreting pituitary adenomas.

METHODS

The clinical and dosimetric data of 26 patients with prolactinomas who underwent SRS between April 1994 and March 2006 were retrieved. One patient with no follow-up information was excluded. The median age was 42 years (range, 17–85 years). Ten patients (40%) had undergone transsphenoidal surgery prior to SRS; one patient (4%) had undergone fractionated radiotherapy. The median preoperative PRL level was 103 ng/ml (range, 25–1,690 ng/ml). The median prescribed isodose volume was 2.4 cm3 (range, 0.4–29 cm3), and the median margin dose was 25 Gy (range, 16–30 Gy). The median follow-up was 48 months (range, 3–129 months).

RESULTS

Twenty-four patients (96%) had tumor control with stable or decreased tumor volumes. The median PRL level at last follow-up was lower than the median PRL level before SRS (103 ng/ml vs. 29 ng/ml; p = 0.02). Three patients (12%) achieved biochemical remission (a normal PRL level without dopamine agonists) at a median of 24 months (range, 6–44 months) after SRS; the biochemical remission rate was 14% at 2 years and 20% at 4 years. Ten patients (40%) experienced clinical remission (no symptoms or signs without dopamine agonists) at a median of 15 months (range, 3–97 months); the clinical remission rate was 20% at 2 years and 37% at 4 years. None of the factors tested, including the presence of dopamine agonists at the time of SRS, was significantly associated with biomechanical or clinical remission. Eight of 23 patients (35%) with normal or partial preoperative anterior pituitary function developed new anterior pituitary deficits at a median of 19 months (range, 4–40 months). The incidence of new anterior pituitary deficits was 25% at 2 years and 45% at 4 years.

CONCLUSION

Although the number of patients with prolactinomas reached biochemical remission after SRS was low, approximately half of the patients had clinical remission after SRS. Radiosurgery should be considered for patients with PRL-secreting pituitary adenomas who have failed medical therapy and are considered poor candidates for surgical resection.

Neuro-Oncol. 2009 Oct;11(5):661.

424. Prognostic Factors and Outcomes of Patients Treated with Reirradiation for Recurrent Glioblastoma

Beth Beadle, Anita Mahajan, Eric L Chang, Shiao Y Woo, Erik P Sulman, Mary F McAleer, Moshe Maor, Dima Suki, Mark R Gilbert, Christopher E Pelloski

INTRODUCTION

Despite aggressive primary treatment of glioblastoma (GBM), recurrence develops in nearly all patients. Optimal management of recurrence is controversial. In this study, we reviewed the outcomes of patients with recurrent GBM treated with reirradiation (reXRT) to clarify its role in the salvage setting.

METHODS

The medical records of 82 patients treated with reXRT for recurrent GBM from 1991 to 2007 at The University of Texas M.D. Anderson Cancer Center were studied. The end points analyzed were median survival time after recurrence (MSr) and the change in clinical status after reXRT.

RESULTS

The median time to progression after the first radiation course was 31 weeks; the MSr was 53 weeks. Patients who had a longer interval between the first radiation course and reXRT had improved outcomes (MSr of 101 weeks if the interval was longer than the median [21 weeks] vs. an MSr of 46 weeks if the interval was shorter than the median; p < 0.0001). After recurrence, patients who received temozolomide (MSr of 65 weeks vs. 37 weeks; p < 0.0001) and patients who received surgery (MSr of 56 weeks vs. 46 weeks; p = 0.037) had improved outcome. On multivariate analysis, a longer delay between the first and second courses of radiation (HR = 0.92; 95% CI, 0.88–0.96; p < 0.001) and the use of salvage temozolomide (HR = 0.50; 95% CI, 0.31–0.81; p = 0.005) were correlated with improved MSr. ReXRT improved the clinical status of patients with recurrent GBM, as judged by Karnofsky performance status, neurologic examination, steroid use, and symptom reporting; approximately 10% of patients demonstrated improvement and 86–89% of patients demonstrated stabilization in these categories. The rates of radiographic evidence of disease regression and stabilization were 29% and 52%, respectively.

CONCLUSION

In selected patients with recurrent GBM, reXRT appears to be safe and provides a clinical benefit. While the optimum salvage treatment for GBM remains unknown, the inclusion of reXRT, along with surgery and chemotherapy, may be warranted.

Neuro-Oncol. 2009 Oct;11(5):661.

425. Semicontinuous Low-Dose-Rate Teletherapy (SLDR) for Previously Irradiated Recurrent Glioma: 40 versus 100 cGy/h

Malika L Siker, Selim Firat, Wade M Mueller, Christopher J Schultz

INTRODUCTION

Semicontinuous low-dose-rate teletherapy (SLDR) is a unique irradiation strategy that potentially exploits the inverse dose-rate effect observed in glial cell lines (defined at approximately 40 cGy/hr) that may occur because of the synchronization of tumor cells into sensitive regions of the cell cycle. We compared the clinical outcomes of previously irradiated patients with recurrent gliomas who were treated with SLDR using two different dose rates.

METHODS

SLDR was delivered using a modified cobalt treatment unit at an approximate dose-rate of 40 cGy/h or 100 cGy/h. SLDR was delivered 6–8 h/day over 12 days for 40 cGy/h patients and delivered 4–6 h/day over 19 days for 100 cGy/h patients, both receiving a total dose of 25–30 Gy. Overall survival from the time of SLDR initiation and toxicity outcomes were compared for all patients, patients with glioblastoma (GBM), and patients who underwent reresection prior to SLDR.

RESULTS

Twenty patients received SLDR at 40 cGy/h (12 GBM, 7 grade II, and 1 ependymoma), and 32 patients received SLDR at 100 cGy/h (21 GBM, 5 grade III, and 6 grade II). Treatment was well tolerated with no acute grade 3 or higher CNS toxicities in the absence of radiologic evidence of disease progression. There was no difference in the median overall survival for all patients (6.72 vs. 6.48 months, p = 0.34) or patients with GBM (6.54 vs. 5.98 months, p = 0.29) treated at 40 and 100 cGy/h, respectively. For patients who underwent reresection, there was a significant improvement in survival for patients treated at 40 cGy/h compared to patients treated at 100 cGy/h (7.82 vs. 6.35 months, p = 0.05).

CONCLUSION

Patients who underwent reresection prior to SLDR treated at 40 cGy/h demonstrated significantly superior survival, potentially giving in vivo support of the inverse dose-rate phenomenon previously shown in vitro with SLDR. Additional studies are needed to better define this treatment strategy.

Neuro-Oncol. 2009 Oct;11(5):662.

426. A Phase I Trial of Dose Per Fraction Escalation of Hypofractionated IMRT with Temozolomide Chemotherapy for Patients with Newly Diagnosed Glioblastoma Multiforme

Changhu Chen, Denise M Damek, Allen E Waziri, Laurie Elizabeth Gaspar, Kevin O Lillehei, Madeleine Kane, Monica Robischon, Brian Kavanagh

INTRODUCTION

This was a phase I safety and toxicity study.

METHODS

Patients with a newly diagnosed glioblastoma were prospectively treated with postoperative intensity-modulated radiotherapy (IMRT) and concurrent temozolomide chemotherapy followed by adjuvant temozolomide chemotherapy. All patients received a total dose of 60 Gy IMRT to the surgical cavity plus residual tumor with a 5 mm margin, but the IMRT fraction size was escalated from 3 Gy per fraction in cohort 1, to 4 Gy per fraction in cohort 2, to 5 Gy per fraction in cohort 3, and to 6 Gy per fraction in cohort 4. Concurrent temozolomide was given at 75 mg/m2/day for 28 consecutive days starting on day 1 of IMRT. Adjuvant temozolomide chemotherapy was given at 150–200 mg/m2/day for 5 days every 28 days. Dose-limiting toxicity was defined as any grade 3 and 4 nonhematological toxicity as defined in CTCAE v3.

RESULTS

Seventeen patients (12 men and 5 women) were enrolled in the study. The median age was 69 years (range, 34–84 years), and the median Karnofsky performance status was 80 (range, 60–90). Three patients each were treated on cohorts 1 and 2, four patients were treated on cohort 3, and seven patients were treated on cohort 4. All patients received IMRT and concurrent temozolomide chemotherapy. The median number of adjuvant temozolomide chemotherapy cycles was 8 (range, 0–11 cycles). With a median follow-up of 11.6 months (range, 0.9–28.5 months) and a minimum follow-up of 6.5 months of live patients, dose-limiting toxicity was observed in one patient treated on cohort 1 who lost vision in the left eye 7 months after radiotherapy. Other nonhematological toxicity included grade 1 headache in one patient, grade 2 insomnia in one patient and grade 2 simple seizures in two patients. Hematological toxicity included grade 2, 3, or 4 neutropenia in six patients; and grade 1, 2, or 4 thrombocytopenia in four patients.

CONCLUSION

We found that 60 Gy IMRT delivered over 2 weeks with temozolomide chemotherapy was well tolerated.

Neuro-Oncol. 2009 Oct;11(5):662.

428. A Phase I Study of Sorafenib and Radiation Therapy for the Treatment of Patients with Brain Metastases and Sorafenib, Temozolomide and Radiation Therapy for the Treatment of Primary Brain Tumors

Robert Den, Maria Werner-Wasik, Mitchell Machtay, David W Andrews, Hanna Li, Rita Axelrod, Jon Glass, Adam P Dicker

INTRODUCTION

Preclinical studies have shown that combining sorafenib with ionizing radiation may enhance the therapeutic ratio. This phase I open label dose-escalation trial evaluated sorafenib combined with radiation therapy (RT) alone for the treatment of patients with brain metastases (BM) or combined with RT and temozolomide for the treatment of patients with high-grade gliomas (HGG).

METHODS

Eligible patients required a minimum 2-week course of RT to the brain, had a WHO performance status of 0–2, and had a life expectancy >3 months. Previous RT was allowed. During RT, sorafenib was administered daily for the duration of RT and could be continued for 1 month following completion of RT at the discretion of the treating clinician. The primary end point was acute toxicity documented according to the Common Toxicity Criteria version 3.

RESULTS

A total of 15 patients (9 HGG and 6 BM) were enrolled to the first dose cohort (200 mg BID). In the BM cohort, two patients had melanoma, three patients had non-small-cell lung cancer, and one patient had renal cell carcinoma. Common acute toxicities included rash, hyperglycemia, and pain. One patient had transient grade 3 lymphopenia, and one patient had a grade 4 seizure. In the HGG cohort, six patients had primary tumors, and three patients had recurrent disease. Common acute toxicities were rash, hyperglycemia, nausea, seizure, and fatigue. One patient had grade 4 lymphopenia, and one patient had grade 4 thrombocytopenia. One patient died of intercurrent disease while receiving RT. At the 1-month follow-up, magnetic resonance imaging revealed that four of five (80%) patients in the BM cohort and three of eight (38%) patients in the HGG cohort had a complete or partial response.

CONCLUSION

Sorafenib at a 200-mg twice-daily dose schedule with RT alone or with temozolomide yields acceptable toxicities and adverse events.

Neuro-Oncol. 2009 Oct;11(5):662.

431. Tumor Control by Whole Brain Irradiation of Anti-VEGF-Treated Mice Bearing Intracerebral Glioma

Joost JC Verhoeff, Lukas JA Stalpers, An Claes, Koos E Hovinga, William P Vandertop, Dirk J Richel, William PJ Leenders, Wouter Ralph Van Furth

INTRODUCTION

Tumor angiogenesis and invasion are key features of glioblastoma multiforme (GBM). Angiogenesis inhibitors increase the progression-free survival (PFS) of recurrent GBM patients. VEGF inhibition controls the bulk tumor growth by inhibiting angiogenesis but does not inhibit the invasive tumor component. We investigated whether invasive GBM growth can be controlled by combining anti-VEGF treatment with irradiation of the tumor and the surrounding brain in an orthotopic murine model.

METHODS

The GBM cell line U251-NG2 was inoculated through a guide screw into the right frontal lobe of 53 athymic nude mice. Pegaptanib (a slow-releasing aptamer against VEGF) was injected in the tumor bed with or without subsequent irradiation treatment with implanted I-125 seeds. Pegaptanib and/or irradiation were compared with sham-treated controls, resulting in four groups comprising 10–15 mice each. After 6 weeks of treatment, histological analysis was performed on all brains.

RESULTS

VEGF inhibition by locally deposited pegaptanib decreased tumor blood vessel density and increased tumor hypoxia. Pegaptanib treatment still allowed the formation of tumor satellites. Irradiation decreased tumor size and suppressed the formation of satellites. Combined pegaptanib plus irradiation further increased PFS. Tumor size directly correlated with PFS.

CONCLUSION

The antitumor effects of local VEGF inhibition are partially circumvented by the formation of invasive tumor satellites. Additional irradiation is effective in slowing the proliferation of these invasive tumor components.

Neuro-Oncol. 2009 Oct;11(5):662.

432. A Meta-Analysis of Stereotactic Radiosurgery for Intracranial Metastatic Disease

Justin F Fraser, Susan C Pannullo, William Stewart Cobb, Jennifer A Moliterno, Philip E Stieg

INTRODUCTION

Stereotactic radiosurgery (SRS) is an important tool in the neurosurgical management of intracranial metastases. We aimed to provide a quantitative meta-analysis of SRS applications in neurooncology, and benchmarks for expected outcomes.

METHODS

The meta-analysis was conducted in accordance with established standards for observational data. Search terms were recorded, and data were extracted to summarize demographics and outcomes. The meta-analysis was statistically appropriate, and clinical outcomes such as survival, cause of death, local control, and complications were summarized.

RESULTS

Of the 57 studies we screened, 27 studies were included in the meta-analysis. The most common reasons for exclusion were publication from a group with a study already included in the meta-analysis and application of radiosurgery as an adjunct rather than primary treatment modality. The patients (n = 2,679) had mean/median ages ranging from 49.1 to 63.1 years. The mean/median tumors volumes ranged from 0.3 to 9.7 cm3. Overall median survival ranged from 5 to 14 months. One-year survival rates ranged from 15% to 54.9%. The percentages of patients who died from nonneurologic causes ranged from 42.9% to 95%. Local tumor control rates ranged from 59.6% to 96.8%. The overall complication rate was 10.0% without significant publication bias.

CONCLUSION

While local control rates are relatively high in lesions treated with SRS, the median survival and 1-year survival rates are relatively low. As most recorded deaths were due to non-neurologic disease, control of systemic disease most likely accounts for the discordance between local control rates and survival rates.

Neuro-Oncol. 2009 Oct;11(5):662–663.

433. Adjuvant Whole Brain Radiotherapy Versus Observation after Radiosurgery or Surgical Resection of 1–3 Cerebral Metastases—Results of the EORTC 22952-26001 Study

Riccardo Soffietti, Rolf-Peter Mueller, MU Abacioglu, S Villa, Francois Fauchon, Brigitta Baumert, Laura Fariselli, T Tzuk-Shina, Laurence Collette, Martin Kocher

INTRODUCTION

The role of prophylactic whole brain radiotherapy (WBRT) after either surgery or radiosurgery of brain metastases is still debated. The EORTC conducted a phase III trial to define the role of adjuvant WBRT after local treatment (surgery or radiosurgery) of a limited number of brain metastases in solid tumors with stable systemic disease.

METHODS

Patients eligible for radiosurgery had one to three metastases of solid tumors (SCLC excluded) <3.5 cm in diameter (<2.5 cm for 2–3 lesions) located outside the brain stem. For surgery, a complete resection of the metastatic lesions was mandatory. Only patients with absent or stable systemic disease or with asymptomatic primary tumors and in good condition (WHO PS 0–2) were included. Patients were randomized to receive either WBRT or observation (OBS). The primary end point was survival. Secondary end points were the time to intracranial progression, neurologic death, and overall survival.

RESULTS

Between 1996 and 2007, 359 patients were recruited. The median survival time was 10.0 months in the OBS arm and 9.5 months in the WBRT arm. The overall survival time was 10.9 months in the OBS and 10.7 months in the WBRT arm. The cumulative incidence of intracranial progression among OBS patients at 6 and 24 months was 39.7% and 54.2%, respectively; among WBRT patients, the incidence at 6 and 24 months was only 15.2% and 31.2%, respectively. Relapses at sites treated initially with surgery or radiosurgery (incidence at 24 months = 31.3% and 16.4%, respectively) and relapses at new intracranial sites (incidence at 24 months = 32.4% vs. 17.6%, respectively) were significantly reduced. The median progression-free survival was 3.4 months in the OBS arm and 4.6 months in the WBRT arm.

CONCLUSION

After radiosurgery or surgery for a limited number of brain metastases, adjuvant WBRT does not prolong the period of functional independence and overall survival. Adjuvant WBRT significantly reduces the risk of neurologic death and prolongs progression-free survival.

Neuro-Oncol. 2009 Oct;11(5):663.

434. Single-Versus Multiple-Dose Radiopharmaceutical Injections: Dosimetry Analysis of Intracavitary 131I-TM601 in High-Grade Glioma Patients

Sui Shen, LB Nabors, Jeff Raizer, John Fiveash, Stewart Spies, Ruthanna Costello, Alison M O’Neill

INTRODUCTION

TM601, or synthetic chlorotoxin, is a peptide that specifically targets tumor cells. Radiolabeled TM601 can deliver localized radiation to tumor with the potential to reduce the incidence of normal brain radionecrosis. We report the difference in biological equivalent dose (BED) between single and multiple injections based on a dosimetry substudy of a phase II trial in which intracavitary 131I-TM601 was given weekly for either 3 or 6 weeks.

METHODS

As part of a dosimetry substudy, 5 of 76 patients treated in this phase II trial underwent additional imaging after receiving 1, 3, or 6 doses of 40 mCi/0.8 mg 131I-TM601 intracavitarily. For each imaging study, five sequential SPECT images were registered with MRI to determine the radiation dose to the 2-cm tumor cavity margin. BED was calculated from the measured dose rate and clearance half-life for 131I-TM601 in the 2-cm cavity margin, assuming an α/β of 3 and repair time of 1.5 h for normal tissue.

RESULTS

In 11 imaging studies of five subjects, the median effective clearance half-life for 131I-TM601 in the 2-cm margin was 48 h (range, 35–74 h). The median radiation dose to the cavity margin was 1.2 Gy/mCi (range, 0.5–3.4 Gy/mCi), and its median intrapatient variation was 14.7% (range, 7.1–18.9%). The median total dose to the cavity margin was 278 Gy (range, 72–396 Gy). The median BED to the cavity margin was 414 Gy (range, 96–709 Gy). If the same dose were administered as a single injection, the BED to the cavity margin would increase 1.5–2.0 times for three injections and 2.6 times for six injections.

CONCLUSION

For multiple dose delivery, the intrapatient variation in 131I-TM601 dosimetry demonstrated less variability than that of interpatient variation. Fractionation in future studies will minimize the risk of normal tissue toxicity.

Neuro-Oncol. 2009 Oct;11(5):663.

435. A Phase I Trial Using Patupilone (Epothilone B) and Concurrent Radiotherapy for CNS Malignancies

Shannon E Fogh, Mitchell Machtay, Maria Werner-Wasik, Walter Curran, Roseann Bonanni, Rita Axelrod, David W Andrews, Adam P Dicker

INTRODUCTION

On the basis of preclinical data indicating the radiosensitizing potential of Epothilone B, this study was designed to evaluate the toxicity and response rate of Patupilone (EPO906; Novartis), an anti-microtubule stabilizing agent, with concurrent radiotherapy in the treatment of patients with CNS malignancies.

METHODS

This phase I study evaluated the toxicities associated with Patupilone in combination with radiotherapy for the treatment of CNS malignancies to establish maximum tolerated dose (MTD). Eligible patients had recurrent gliomas (10 patients), primary brain tumors (5 patients), or metastatic brain tumors (17 patients). Dose escalation occurred if no dose-limiting toxicities (DLTs), defined as any grade 4 or 5 toxicity or grade 3 toxicity requiring hospitalization, occurred. Radiation treatment was determined according to the patient’s diagnosis.

RESULTS

Thirty-two patients were enrolled. Five patients were treated with weekly Patupilone at 1.5 mg/m2, four patients at 2.0 mg/m2, four patients at 2.5 mg/m2 and one patient at 4 mg/m2. Seven patients were treated in the 6 mg/m2 q3wk group, six patients were treated in the 8 mg/m2 q3wk group, and five patients were treated in the 10 mg/m2 q3wk group. No DLTs were observed in patients who received weekly Patupilone or in patients in the 6 mg/m2 cohort. One DLT (pneumonia/infection) was observed in the 8 mg/m2 q3wk group. In the subsequent dose level (10 mg/m2), two grade 4 DLTs (renal failure and pulmonary hemorrhage) occurred. Thus, we recommend 8 mg/m2 q3wk as the MTD and recommended phase II dose. The median overall survival was 12 months for recurrent gliomas, 15 months for primary brain tumors, and 4 months for CNS metastasis.

CONCLUSION

Combined with a variety of radiation doses and fractionation schedules, concurrent Patupilone is well tolerated and safe in patients with CNS malignancies at an MTD of 8 mg/m2 q3wk. A phase II study to characterize efficacy is warranted.

Neuro-Oncol. 2009 Oct;11(5):663.

436. Early Prediction of Radiotherapy Response of Glioma by Diffusion Magnetic Resonance Imaging

Li Shouwei

INTRODUCTION

To evaluate the role of diffusion MRI in the early detection of treatment response to radiation for glioma.

METHODS

Conventional and diffusion MRI were acquired before and at 3 weeks after initiating radiotherapy for newly diagnosed glioma patients who had partial tumor resection. ANOVA was used to compare diffusion MRI parameters among different treatment response groups. A receiver operating characteristic (ROC) curve was used to analyze the sensitivity and specificity of the parameter.

RESULTS

Pretreatment apparent diffusion coefficient (ADC), intertreatment ADC, ADC subtraction value, pretreatment accumulated ADC value, intertreatment accumulated ADC value were not significantly different among patients in the partial response (PR) group, stable disease (SD) group, and progressive disease (PD) group (p > 0.05). The difference in the accumulated ADC change percentages among the groups was significant (p < 0.05). The predictive value and overall accuracy for early detection of treatment response by the accumulated ADC change percentage was 92.7%. The sensitivity and specificity for predicting PD patients were 81.9% and 96.2%, respectively.

CONCLUSION

Diffusion MRI could predict glioma patients’ response to radiotherapy. The accumulated ADC change percentage was a good parameter for detecting PD early in newly diagnosed glioma patients undergoing radiotherapy.

Neuro-Oncol. 2009 Oct;11(5):663.

437. Leading-Edge Gamma Knife Stereotactic Radiosurgery for Patients with Recurrent Glioblastoma Multiforme

Christopher M Duma, Brian Kim, Peter Chen, Marianne Plunkett, Ralph McIntosh, Robert O Dillman, Ramin Khadem

INTRODUCTION

Glioblastoma multiforme (GBM) spreads along predictable white matter pathways, has a predominantly local pattern of recurrence, and responds uniquely to high-dose radiation. Astrocytes migrate down white matter pathways by projecting invadopodia and expressing proteins capable of breaking down surrounding extracellular matrix. Laboratory data show that large, single-fraction doses of radiation are more effective than low doses at interfering with the migratory ability of dedifferentiated astrocytes. RTOG 93–05 addressed the radiosurgical treatment of the enhancing nidus of GBM on magnetic resonance imaging. We hypothesized that targeting the local white matter pathways of spread (the “leading edge”) may prove more effective in the treatment of GBM.

METHODS

Between March 14, 1998 and June 16, 2008, 41 patients with recurrent GBM were treated to the FLAIR- or MR SPECT-defined leading edge with Gamma Knife radiosurgery (LEGRS). The median age was 56 years, and 58% of the patients were men. LEGRS took place a median of 4.3 months from original diagnosis (range, 2.8–24 months). The median Karnofsky performance status (KPS) was 80; all patients had a KPS > 70. An average volume of 39 cm3 of leading-edge tissue was targeted, using a median dose of 10 Gy (range, 8–12 Gy) at the 50% isodose line.

RESULTS

The median survival from the time of recurrence and LEGRS was 12.6 months. With 24 patients (77%) deceased, and a median follow-up of 27.8 months for the seven survivors, the median overall survival from diagnosis was 19.3 months, and the 2- and 3-year survival rates were 36% and 19.5%, respectively. The rate of clinically significant radionecrosis was 7%.

CONCLUSION

These results compare favorably to those reported from phase II trials in patients with recurrent malignant gliomas. LEGRS may be an effective salvage therapy for patients with recurrent GBM. A prospective multicenter study should follow. Up-front leading-edge radiosurgery should also be considered for this disease.

Neuro-Oncol. 2009 Oct;11(5):664.

438. Radiosurgery with or without Whole-Brain Radiotherapy for Cerebral Metastatic Melanoma: the Thomas Jefferson University Experience

Kimberly Ann Tefft, Shannon E Fogh, Maria Werner-Wasik, Beverly Downes-Phillips, David W Andrews, Mitchell Machtay

INTRODUCTION

It is unclear if whole-brain radiotherapy (WBRT) added to radiosurgery (SRS) for cerebral metastatic melanoma is beneficial. We sought to determine the outcomes of SRS alone vs. SRS plus WBRT.

METHODS

We retrospectively reviewed 35 patients with 64 intracerebral melanoma metastases who underwent Linac- or Gamma Knife- based radiosurgery from 1996 to 2006 at TJUH. All patients had <5 metastases. The median prescribed SRS dose was 21 Gy (range, 15–25 Gy) to the 50–90% isodose line. WBRT, when given, was delivered in 2.5- to 3-Gy fractions to 30–37.5 Gy. Patients were selected for SRS alone vs. SRS plus WBRT based on patient and/or physician preference; in general, patients who selected SRS alone were concerned about toxicity from WBRT. Survival was calculated from the date of diagnosis of brain metastases, and the 2-sided log rank test was used for comparison between the groups.

RESULTS

Seventeen patients received SRS alone, and 18 patients received SRS plus WBRT. Patients who received SRS alone were older (median age, 67 years) than patients who received SRS plus WBRT (median age, 48 years). The total tumor volume was similar between the two groups, with an SRS median volume of 3.4 cm3 (range, 0.2–38.5 cm3) vs. SRS plus WBRT median volume of 4.8 cm3 (range, 0.4–24.2 cm3). The median survival time was 6 months in the SRS group (range, 1–29 months), compared with 12.5 months in the SRS plus WBRT group (range, 1–128 months; p = 0.09). The 1- and 2-year survival rates for the SRS group were 18% and 6%, respectively. The 1- and 2-year survival rates for the SRS plus WBRT group were 55% and 16%, respectively. Although all patients ultimately died from metastatic melanoma, three patients in the SRS plus WBRT group had survival times that exceeded 5 years.

CONCLUSION

Within the limits of the small sample size and potential selection bias, our study suggests SRS plus WBRT is associated with better survival than SRS alone.

Neuro-Oncol. 2009 Oct;11(5):664.

439. Phase II Trial of Conventional Radiotherapy with Stereotactic Radiosurgery to High-Risk Tumor Regions as Determined by MR Spectroscopy in Patients with Glioblastoma Multiforme

Douglas B Einstein, Barry Wessels, Barbara Bangert, Pingfu Fu, A Dennis Nelson, Mark L Cohen, Stephen Sagar, Jonathan Lewin, Jordonna Williams, Robert Vinkler, Yiran Zheng, Robert J Maciunas

INTRODUCTION

The objective of this phase II trial was to determine the efficacy of a radiosurgical boost directed only to high-risk regions determined by magnetic resonance (MR) spectroscopy in addition to standard radiotherapy for glioblastoma patients.

METHODS

The 34 patients enrolled underwent surgical resection for glioblastoma followed by Gamma Knife radiosurgery directed toward areas of high biological activity determined by MR spectroscopy within 2 cm of the postoperative enhancing surgical bed. The radiosurgery dose used was based on the maximum diameter of the combined high biological voxels with doses of 15 Gy, 18 Gy, and 24 Gy utilized for total treated voxel diameter between 3 and 4 cm, 2 and 3 cm, and 1 and 2 cm, respectively. All patients received conformal fractionated radiotherapy after radiosurgery to a total dose of 60 Gy. The primary end point was overall survival, which was measured from the date of Gamma Knife treatment to the date of death, and censored at the date of last follow-up for survivors.

RESULTS

Using Kaplan-Meier methods, the median overall survival time for the entire cohort was 15.8 months (95% CI, 11–19.9 months). The median survival for RPA class 3 patients has not yet been reached; 75% of patients are still alive 18 months after treatment. The median survivals for RPA class 4, 5, and 6 patients were 18.7 months (95% CI, 14.5–29.1 months), 12.5 months (95% CI, 9.2–18.5 months), and 3.9 months (95% CI, 2.7–5.1 months), respectively. RTOG historical control median survivals for patients with RPA class 3, 4, 5, and 6 patients treated with conformal radiotherapy alone were 17.9, 11.1, 8.9, and 4.6 months, respectively.

CONCLUSION

MR spectroscopy-targeted radiosurgery directed only to areas of high biologic activity combined with conformal brain radiotherapy for patients with glioblastoma multiforme is feasible, with overall patient survivals that compare favorably to historical controls of patients treated with conformal radiotherapy alone.

Neuro-Oncol. 2009 Oct;11(5):664.

440. Proton Therapy for High-Grade Meningiomas: the UPFTI Experience

Robert S Malyapa, Zuofeng Li, Daniel Yeung, William M Mendenhall, Nancy P Mendenhall

INTRODUCTION

High-grade meningiomas are aggressive tumors requiring radiation doses above 60 Gy to reduce local recurrence. These tumors often occur where delivering high-dose radiation without compromising the tolerance of adjacent critical structures is impossible. Proton therapy, with its ability to stop treatment beams completely within the treated volume, allows the delivery of the high curative dose while respecting tolerance doses of nearby critical organs. Advantages of proton therapy for high-grade meningiomas are demonstrated by three case studies at UFPTI.

METHODS

Immobilization for patients with high-grade meningiomas employs a base-of-skull frame with a facemask and bite plate. Computed tomography and magnetic resonance simulation images of patients are imported into the Eclipse proton therapy treatment planning system for treatment planning. Proton therapy treatments were delivered using double- scattering proton beams. Orthogonal X-ray images were acquired daily to enable accurate and reproducible patient positioning using the Digital Imaging Positioning System. Patient 1 was a 48-year-old man with an atypical meningioma with rhabdoid features arising in the posterior fossa, with extension to the neck through the jugular foramen, s/p biopsy. Patient 2 was a 56-year-old man with atypical meningioma, s/p four surgeries, presenting with multifocal recurrence in the parasagittal and right temporal regions. Patient 3 was a 30-year-old woman, s/p subtotal resection, with recurrent atypical meningioma involving the right orbit and sphenoid wing, extending to involve the cavernous sinus and middle cranial and infratemporal fossa. Proton treatment plans were optimized to deliver the prescription doses of 61.2 CGE at 1.8 CGE/fraction for patients 1 and 2, and 60.0 CGE at 1.2 CGE/fraction for patient 3.

RESULTS

Dosimetry analysis of patient treatment plans demonstrated that proton therapy is able to deliver the prescribed tumor doses while respecting all critical organ tolerances.

CONCLUSION

Proton therapy shows clear dosimetric advantages over traditional radiotherapy techniques.

Neuro-Oncol. 2009 Oct;11(5):664.

441. The Role of Fractionated Stereotactic Radiosurgery for Malignant Gliomas that had Progressed on Bevacizumab

Roy G Torcuator, Ravneet Thind, Joseph Anderson, Thomas Doyle, Ali Haythem, Sam Ryu, Rajan Jain, YS Mohan, Lonni Schultz, Mark L Rosenblum, Tom Mikkelsen

INTRODUCTION

Bevacizumab and irinotecan are effective against malignant gliomas. However, Quant reported poor response to a second bevacizumab regimen with a median progression-free survival (PFS) of 37.5 days and a 6-month PFS rate of 2%. Patel reported radiographic response and improved survival using salvage reirradiation for recurrent gliomas in the prebevacizumab era. The efficacy of reradiation in patients in whom bevacizumab therapy fails is currently not described.

METHODS

We retrospectively reviewed the records of patients treated with fractionated stereotactic radiosurgery/stereotactic radiosurgery (FSRT/SRS) after progression on an initial bevacizumab regimen followed by bevacizumab with another chemotherapy and compared those patients to patients whose disease progressed but was maintained with bevacizumab with alternative chemotherapy without reradiation.

RESULTS

Group 1 (FSRT/SRS) consisted of 23 patients, 16 of whom were treated with FSRT with a median dose of 36 Gy. Of these 16 patients, 12 patients were maintained on bevacizumab combined with a different chemotherapy. Seven of the 23 were treated with SRS with median dose of 18 Gy, and six of these seven patients were maintained on bevacizumab with a different chemotherapy. The median time to progression (TTP) for group 1 was 2.6 months. Group 2 (no FSRT/SRS) consisted of 23 patients. All patients were maintained on bevacizumab, but chemotherapy was changed. Twenty of these 23 patients demonstrated progressive disease on the first follow-up magnetic resonance imaging study. The median TTP for group 2 was 1.7 months.

CONCLUSION

This study confirms the limited response patients have to a second bevacizumab-containing chemotherapy after progression on an initial bevacizumab regimen. After observing early progression on the second bevacizumab regimen when simply changing chemotherapy, we offered FSRT or SRS followed by bevacizumab combined with a different chemotherapy. While patients numbers are small, these results suggest an improved median TTP for the FSRT/SRS group (2.6 months vs. 1.7 months; p = 0.009) with a radiographic response rate of 22% (p = 0.049).

Neuro-Oncol. 2009 Oct;11(5):665.

442. Cranial and Thoracic Stevens-Johnson Syndrome after Concurrent Phenytoin and Radiation Treatment

Jay-Jiguang Zhu, Abdullah Kandil, Tomas Dvorak, Carl B Heilman, Gary Strauss, Julian K Wu, John Mignano

INTRODUCTION

Stevens Johnson syndrome (SJS) is a rare, cutaneous, cell-mediated hypersensitivity reaction that is usually drug- or virus-induced. Historically, SJS was considered to be on a spectrum of erythema multiforme (EM), but now it is considered clinically distinct from EM and is part of the SJS-toxic epidermal necrolysis (TEN) spectrum. SJS is characterized by heterogenous, cutaneous, bullous eruptions and can result in sloughing of 10% of the epidermis. Several antiepileptic drugs (AEDs) have been associated with SJS, including phenobarbital, phenytoin, carbamazepine, and lamotrigine. Certain HLA types are sometimes associated with an increased risk of SJS, including HLA B1502. Combining radiation with antiseizure drugs increases the risk of SJS. Investigators from the Mayo Clinic suggested an acronym “EMPACT” (EM associated with phenytoin and cranial radiotherapy) to describe this reaction.

METHODS

This was a single case report.

RESULTS

A 46-year-old man with progressive metastatic prostate cancer involving the cranium and thoracic spine developed SJS at the radiation sites after undergoing whole brain and thoracic radiation while receiving phenytoin for seizure prophylaxis. The patient’s HLA typing was negative for HLA B1502. The patient recovered without sequelae after receiving supportive care and antibiotics.

CONCLUSION

Prophylactic use of AED should be discouraged during radiotherapy. The EMPACT acryname should be modified to include other body sites irradiated in addition to the cranium.

Neuro-Oncol. 2009 Oct;11(5):665.

443. Delayed Cerebral Radiation Necrosis after Concurrent Hyperfractionated Chemoradiation Therapy: A Case Report

Evan J Wuthrick, Shibu Anamalayil, Michael Wagner, Leslie Tupchong, James J Evans, Adam P Dicker, Yaacov R Lawrence

INTRODUCTION

Cerebral necrosis is a potentially devastating complication of radiotherapy. Risk factors include total radiation dose, fraction size, treatment duration, irradiated volume, and possibly concurrent chemotherapy. The median interval between the conclusion of fractionated radiotherapy and the development of necrosis is reported to be 1 year.

METHODS

This was a case report.

RESULTS

A 79-year-old man presented with acute lethargy, fever, and seizures. Contrast-enhanced MRI magnetic resonance imaging revealed a left frontal lobe lesion measuring 3.8 × 2.9 cm with faint rim enhancement and no diffusion restriction that had not been present 1 year previously. The lesion was hypointense on T1-weighted images and hyperintense on T2-weighted images and caused a 5-mm mid-line shift. T2 and FLAIR sequences revealed hyperintensity throughout the bilateral frontal and into the right posterior temporal white matter compatible with radiation changes. Nineteen years previously the patient presented with epistaxis and diplopia and was diagnosed with an extensive, poorly differentiated, ethmoid sinus tumor that had invaded through the cribriform plate into both frontal lobes. The patient was enrolled in a phase 1 trial investigating hyperfractionated radiotherapy with concomitant chemotherapy. He was treated from November 29, 1989, to January 10, 1990, with 160-cGy fractions twice daily over 41 days using anterior and right and left lateral fields, together with two cycles of 5-fluorouracil and carboplatin. In 2009, a biopsy of the patient’s left frontal cortex revealed increased cellularity, reactive glial cells, macrophage infiltration, microglial cells, hemosiderin deposition, focal necrosis, and vascular changes suggestive of radiation necrosis.

CONCLUSION

Cerebral necrosis can develop two decades after the administration of radiotherapy. Many clinical trials are currently combining targeted agents with radiotherapy to augment the antitumor effect. We do not know to what degree or over what time course normal tissue toxicity will develop. Our case emphasizes the need for long-term follow-up of patients enrolled to such trials.

Neuro-Oncol. 2009 Oct;11(5):665.

444. The Effect of Delaying Radiotherapy (RT) in Patients with Newly Diagnosed Glioblastoma Multiforme (GBM) Receiving Temozolomide (TMZ): A Secondary Analysis Based on the UCSF Experience

Nicholas A Butowski, Mei-Yin C Polley, Kathleen L Lamborn, Susan Chang, Michael Prados, Jennifer L Clarke, Kim Huang, Patricia Sneed

INTRODUCTION

The effect of delayed initiation of radiotherapy (RT) after surgery on the outcome of patients with glioblastoma multiforme (GBM) remains controversial. Most neurooncologists believe that timely initiation of RT may result in clinical benefits. However, a recent report suggested that delayed RT may be positively associated with survival for GBM patients. To further explore this issue, we analyzed three clinical trials for newly diagnosed GBM patients receiving temozolomide (TMZ) conducted at UCSF.

METHODS

From 2000 through 2007, 193 adult patients with supratentorial GBM were enrolled in three clinical trials consisting of RT plus TMZ and an experimental agent. Timing of RT was defined as the interval between definitive surgery and the commencement of RT, which was intended to be within 5 weeks per protocol eligibility. Overall survival (OS) and progression-free survival (PFS), measured from study registration, were estimated using the Kaplan-Meier method. The Cox proportional hazards model was used to assess the effect of RT timing (in days) on clinical outcomes, adjusting for treatment protocol, age, Karnofsky performance status, and the extent of resection.

RESULTS

The median wait time between surgery and RT was 30 days (range, 11–43 days). There was no significant difference in timing of RT with respect to baseline variables, except for the extent of resection: patients who had undergone biopsy were given RT earlier (median, 25 days; range, 14–34 days) than patients who underwent more extensive surgery. Unexpectedly, delayed RT was predictive of prolonged survival after adjusting for protocol and baseline variables even though the statistical significance was marginal (HR = 0.97; p = 0.047). Delayed RT was not predictive of PFS (HR = 0.98; p = 0.16).

CONCLUSION

Delayed RT may be associated with prolonged survival but not PFS. Caution should be exercised in interpreting these results since it is well known that secondary analyses may be influenced by unknown confounding factors. Future studies are needed to validate these results.

Neuro-Oncol. 2009 Oct;11(5):665.

445. The Implications of Identifying Additional Cerebral Metastases during Stereotactic Radiosurgery

Toral Ramanlal Patel, Maxwell SH Laurans, Ali Kemal Ozturk, Jonathan PS Knisely, Veronica Chiang

INTRODUCTION

Radiosurgery with or without whole brain radiation remains the standard treatment for patients with multiple brain metastases. Given the median survival of less than 12 months in the majority of these patients, strategies to predict patients with improved survival may rest on accurately identifying prognostic factors.

METHODS

We reviewed 180 radiosurgical procedures performed at a single institution in 133 consecutive patients with brain metastases diagnosed by MRI. SPGR sequence MRIs were then performed, using single- or double-dose gadolinium, for treatment. Multivariate analysis was used to identify variables significantly affecting outcome, including the difference in the number of metastases detected between the initial diagnostic MRI and the subsequent treatment MRI. Kaplan-Meier analysis was used to examine the effect of a change in the number of metastases on overall survival.

RESULTS

The median number of preoperatively identified lesions was one (range, 1–11 lesions). The median number of lesions on the treatment scan was 2 (range, 1–21 lesions). The overall median survival was 9.85 months. Additional lesions were identified in 42% of the treatment MRIs. Multivariate stepwise linear regression demonstrated that the greater the number of initial lesions, the greater the likelihood that additional lesions would be found on the treatment MRI (p = 0.003). This relationship was independent of other factors analyzed, including age, sex, time between scans, dose of gadolinium, whole brain radiotherapy, and primary pathology. Kaplan-Meier analysis demonstrated a significant decrease in survival for patients in whom additional metastases were identified (median, 6.84 months; hazard ratio 1.51; p = 0.033) when compared to patients in whom no additional metastases were identified (median, 11.61 months).

CONCLUSION

These data highlight the dynamic nature of metastatic intracranial disease. Identifying additional cerebral metastasis at the time of radiosurgical intervention yields important prognostic information that may be useful in directing patient care.

Neuro-Oncol. 2009 Oct;11(5):665–666.

446. Efficacy and Safety of Fractionated Stereotactic Radiotherapy in the Treatment of Craniocervical Meningiomas

Mina Kim, Shannon E Fogh, Maria Werner-Wasik, David W Andrews

INTRODUCTION

Craniocervical meningiomas, because of their proximity to critical neurovascular structures, represent a treatment challenge. We analyzed the safety, tolerability, and efficacy of high-precision fractionated stereotactic radiotherapy for treating patients with craniocervical meningiomas.

METHODS

Thirty-five patients with benign craniocervical meningiomas were treated with stereotactic five conformal dynamic arc fractionated radiotherapy between 1998 and 2007. Patients received a course of radiotherapy as primary treatment, as adjuvant therapy following subtotal resection, or as treatment for recurrent disease. The median target volume was 9.98 ml (range, 0.66 ml–62.2 ml). The mean radiation dose was 52.7 Gy ± 1.7 Gy. Follow-up examinations, including magnetic resonance imaging and physical exams, were performed first at 3 months posttreatment then annually.

RESULTS

With a median follow-up of 52 months (range, 11–143 months) overall survival was 91% at 5 years and 86% at 10 years. Local tumor failure was observed in 4 of the 35 patients. A volume reduction was observed in five patients (14%). Preexisting cranial nerve symptoms resolved completely in 8 patients (23%) and partially in 20 patients (54%). Posttreatment edema that resolved with a short course of steroids was observed in one patient. No other clinically significant treatment-induced toxicity was observed. No treatment-related deaths occurred.

CONCLUSION

Fractionated stereotactic radiotherapy is safe and effective with minimal toxicity in the treatment of craniocervical meningiomas, including subtotally resected or unresectable meningiomas. In addition, this therapy demonstrated excellent symptom improvement. Overall morbidity and the incidence of side effects using this approach are low. This is the largest study to examine efficacy and tolerability in patients with craniocervical meningiomas.

Neuro-Oncol. 2009 Oct;11(5):666.

447. Gamma Knife Radiosurgery for Cerebral Metastases: the Chus Experience

David Mathieu, Nicolas Dea, Martin Borduas, Brendan Kenny, David Fortin

INTRODUCTION

Brain metastases are the most frequent cerebral tumors in adults. Microsurgery and standard fractionated radiotherapy have been the mainstay of treatment for years, but Gamma Knife radiosurgery (GKRS) has emerged as a minimally invasive treatment option. Brain metastases are now the most commonly treated lesions in most radiosurgery centers.

METHODS

The charts of all brain metastases patients treated by GKRS at the Centre Hospitalier Universitaire de Sherbrooke from August 2004 to April 2008 were retrospectively reviewed. An analysis of survival, tumor response, and complications was performed.

RESULTS

295 patients were included in the analysis, for a total of 650 metastases treated. The median age was 59 years, and women constituted 60% of the study population. The median Karnofsky performance status was 80% and most patients (71.2%) were in RPA class 2. In most patients (60.3%), the primary cancer was non-small-cell lung cancer. Radiosurgery was the primary treatment in 67.2% of patients, in combination with whole brain radiotherapy in 24.2% of patients and for the postoperative irradiation of the tumor bed in 7.2% of patients. The median marginal radiation dose was 18 Gy at the 50% isodose. The median survival after GKRS was 8 months. Variables that significantly influenced survival in multivariate analysis were the extent of systemic disease and the RPA class. Postradiosurgery imaging was available for 222 patients (476 metastases). Immediate tumor control was achieved in 91% of metastases, and 75% of metastases were still controlled at the last follow-up. The median time to progression was 25 months. Actuarial tumor control at 1 year was 72%. During follow-up, 42% of patients developed new metastases, which did not impact survival. After radiosurgery, seizures occurred in 5% of patients, and new focal neurologic deficit occurred in 3.6% of patients. Radiation-induced T2 changes on MRI magnetic resonance imaging occurred in 5.9% of patients.

CONCLUSION

GKRS is a safe and effective treatment for brain metastases.

Neuro-Oncol. 2009 Oct;11(5):666.

448. Analysis of Factors Associated with Necrosis after Radiotherapy in Patients with Oligodendrogliomas

Anita Mahajan, John de Groot, Jeffrey S Weinberg, Radhe Mohan, Shiao Y Woo

INTRODUCTION

Progressive MRI changes were noted in four of seven patients treated with proton radiotherapy (PRT) for anaplastic oligodendroglioma (AO) or oligodendroglioma (OG). The PRT dosimetry has been verified. In this analysis, we compared this incidence to that of patients treated with photon radiotherapy (XRT) at our institution.

METHODS

A retrospective analysis of all patients with AO or OG treated with XRT and PRT from 1999 through 2008 was performed. The patient treatment history and MRI outcomes were reviewed, and factors associated with progressive MRI changes were evaluated.

RESULTS

Sixteen patients with AO (3 who underwent PRT and 13 who underwent XRT) were treated with 57 Gy/CGE in 30 fractions. Progressive MRI changes were noted in two of three patients who underwent PRT and 4 of 13 patients who underwent XRT (OR, 0.22; p = 0.3). The two patients who underwent PRT in whom progressive MRI changes were noted had biopsies that revealed one necrosis and one small amount of viable tumor. Only one of the four patients who underwent XRT had a biopsy, which revealed active tumor. The mean time to MRI changes was 215 days and 323 days for the PRT and XRT groups, respectively, with a range of 58d-657 days for the whole group. The mean GTV of the XRT patients with MRI changes was 114 cm3 versus 36 cm3 for patients with no change (p < 0.01). The mean CTV for the XRT patients with changes was 312 cm3 versus 186 cm3 (p = 0.02). This relationship was not observed in the PRT group. For the 12 patients with OG (four patients who underwent PRT and eight patients who underwent XRT; median dose, 54 Gy/CGE; range, 50.4–57 Gy/CGE), one PRT patient treated with 50.4CGE developed progressive, pathologically confirmed necrosis 223 days after PRT with a GTV and CTV of 33 cm3 and 105 cm3, respectively. No OG patients who underwent XRT have had progressive MRI changes at a median follow-up of 7.1 years.

CONCLUSION

The overall incidence of progressive MRI changes is comparable among patients with OA or OG treated with PRT or XRT. Patients with larger tumors or treatment volumes appear to be at a greater risk.

Neuro-Oncol. 2009 Oct;11(5):666.

449. A Phase I Study of at-101 Plus Radiotherapy and Temozolomide and at-101 Plus Adjuvant Temozolomide for Patients with Newly Diagnosed Glioblastoma Multiforme

John Fiveash, Louis B Nabors, Tom Mikkelsen, David M Peereboom, Glenn J Lesser, Sajeel A Chowdhary, Stuart Grossman, Xiaubu Ye, Lance Leopold, Myrna Rosenfeld

INTRODUCTION

AT-101 is an oral Bcl-2 family protein inhibitor (Bcl-2, Bcl-XL, Mcl-1, Bcl-W) and potent inducer of proapoptotic proteins. The objectives of this trial were to determine the maximum tolerated dose (MTD) of AT-101 when given concurrently with temozolomide (TMZ) and radiotherapy (RT) (arm 1) and to determine the MTD of AT-101 when given with adjuvant TMZ after completion of standard chemoradiation (arm 2).

METHODS

After maximal safe resection, patients with newly diagnosed glioblastoma multiforme (GBM) received concurrent RT (60 Gy) and TMZ (75 mg/m2/day) followed by TMZ (150–200 mg/m2) on days 1–5 every 28 days for six cycles. In arm 1, AT-101 (starting dose, 20 mg) was administered Monday through Friday during RT. In arm 2, AT-101 (starting dose, 20 mg) was administered concurrently with each adjuvant cycle of TMZ on days 1–21 every 28 days for a maximum of six cycles.

RESULTS

Sixteen patients were enrolled in the two study arms. In arm 1, AT-101 was escalated from 20 mg to 30 mg when one of the six patients experienced a dose-limiting toxicity (DLT; grade 3 GI ulcer). In arm 2, one patient treated with 20 mg AT-101 experienced a DLT (grade 3 ileus, nausea and diarrhea). The cohort was expanded without observation of DLT. Pharmacokinetics results were consistent with drug levels from non-CNS studies. To date, six patients are still alive. The median survival times for arm 1 and arm 2 are 15.2 months and 18.2 months, respectively.

CONCLUSION

AT-101 can be administered safely with RT and TMZ in patients with newly diagnosed GBM without toxicity unique to patients with CNS tumors. Because of toxicity observed in non-CNS AT-101 clinical trials, further dose escalation was not attempted. The recommended AT-101 dose for future studies is 20 mg on days Monday through Friday given concurrently with RT and TMZ and 20 mg on days 1–21 for each 28-day cycle of TMZ.

Neuro-Oncol. 2009 Oct;11(5):666–667.

450. Balloon Brachytherapy versus Chemotherapy for Recurrent Malignant Gliomas

Samuel Lewis Cooper, Sunil J Patel, Pierre Giglio, Joseph Jenrette, Amy Wahlquist, Elizabeth Garrett-Mayer, John Fortney

INTRODUCTION

The purpose of this study was to report on a retrospective analysis of a single institution’s experience using balloon brachytherapy vs. chemotherapy in the treatment of patients with recurrent malignant gliomas.

METHODS

Between 2001 and 2009, 30 patients with recurrent malignant gliomas were treated with balloon brachytherapy, and 31 patients were treated with chemotherapy. Twenty-four brachytherapy patients and 23 chemotherapy patients had glioblastoma multiforme (GBM). Six brachytherapy patients and eight chemotherapy patients had recurrent malignant gliomas. The median age at diagnosis was 54 years for brachytherapy patients and 49 years for chemotherapy patients. The median Karnofsky performance status (KPS) was 80 for both groups. All brachytherapy patients underwent surgery for reresection with placement of balloon catheter. The chemotherapy group received various chemotherapy regimens; the majority were bevacizumab-based (21 patients).

RESULTS

At the time of analysis, 29 of the brachytherapy patients and 24 of the chemotherapy patients had died. One brachytherapy patient was lost to follow-up, and seven chemotherapy patients were alive with disease. The median progression free survival (PFS) and survival following salvage for patients with GBM were 3.4 and 7.5 months, respectively, for the brachytherapy group compared to 5.0 and 8.2 months, respectively, for the chemotherapy group (p = 0.74). For all recurrent gliomas, the median PFS and survival following salvage were 3.5 and 7.4 months, respectively, for the brachytherapy group and 4.9 and 8.7 months, respectively, for the chemotherapy group (p = 0.35). Six-month survival was 65.5% for the brachytherapy group and 77.4% for the chemotherapy group (p = 0.39). In the brachytherapy group, four patients had seven grade 3 toxicities and one grade 4 toxicity. In the chemotherapy group, seven patients had grade 3 toxicity and two patients had grade 4 toxicities.

CONCLUSION

Balloon brachytherapy after reresection for recurrent malignant glioma seems to lead to a benefit similar to that of current chemotherapy, but this could be confounded by brachytherapy patients that subsequently received chemotherapy.

Neuro-Oncol. 2009 Oct;11(5):667.

451. Reirradiation of Large-Volume Recurrent High-Grade Glioma with Pulsed Reduced-Dose-Rate Radiation

Jarrod Atkison, Wolfgang Tomé, Songwon Seo, H Ian Robins, Karl Rassmussen, James S Welsh, Peter A Mahler, Steven P Howard

INTRODUCTION

The use of reirradiation in the management of recurrent high-grade glioma remains limited because of concerns of exceeding tissue tolerance. Pulsed reduced-dose-rate radiotherapy (PRDR), a reirradiation technique, reduces the effective dose-rate and increases treatment time with the objective of improving therapeutic index.

METHODS

This technology was applied to a series of 94 patients with previously irradiated recurrent high-grade glioma: 68 patients had glioblastoma multiforme (GBM); 8 patients had anaplastic astrocytoma (AA); 6 patients had anaplastic oligodendroglioma (AO)/mixed AA; and 12 patients had presumed transformed GBM by history and MRI. Using PRDR, a median dose of 50 Gy, (range, 20–60 Gy) was delivered in 1.8- to 2.0-Gy fractions. PRDR was delivered via a series of 0.2-Gy pulses separated by 3-min intervals, creating an apparent dose rate of 0.0667 Gy/min. The mean irradiated volume was 396.2 ml. Median survival was calculated from the time of PRDR initiation. Outcome variables included age, Karnofsky performance status (KPS), the number of prior relapses, prior temozolomide therapy, and the time from initial radiotherapy to PRDR.

RESULTS

Therapy was well tolerated. Multivariate analysis revealed the time from initial-radiotherapy to PRDR, KPS, and age to be significant predictors of survival. In the GBM cohort (n = 86), the median time between first XRT and PRDR was 14 months. Forty-three patients receiving PRDR within 14 months of first XRT had a median survival of 19 weeks; 43 patients treated for >14 months after XRT had a median survival of 29 weeks (Mann-Whitney p = 0.021). These data compare favorably to historical data sets evaluating chemotherapy in patients with recurrent high-grade glioma, as only 16% of patients were treated at first relapse (with 46% at second relapse, 32% at third or fourth relapse, and 4% at fourth or fifth relapse).

CONCLUSION

PRDR is a promising reirradiation strategy that allows safe retreatment of larger target volumes to high doses with palliative benefit.

Neuro-Oncol. 2009 Oct;11(5):667.

452. Impact of Her2/Neu Status on Overall Survival and Distant Brain Failure in Patients with Breast Cancer Brain Metastases

Aleksandar F Dragovic, Stephen M Sawrie, Ruby F Meredith, Sharon A Spencer, James M Markert, Barton L Guthrie, John Fiveash

INTRODUCTION

Her2/neu positivity has been shown in recent studies to predict the development of brain metastases in patients with breast cancer, but it is unknown whether this translates into poorer outcomes.

METHODS

Seventy-six patients with primary breast cancer who subsequently developed brain metastases were reviewed. Factors including age, ER/PR status, Her2/neu status, Karnofsky performance status (KPS), presence of extracranial metastatic disease, number of brain metastases, and use of whole-brain radiation therapy (WBRT) were analyzed for predictive value by Kaplan-Meier method. All patients were assessed for overall survival (OS), and the subset of patients who did not receive WBRT was assessed for distant brain failure (DBF) after focal therapy only for brain metastases.

RESULTS

The median age was 50 years (range, 27–78 years). Fifty-seven percent of patients were Her2/neu positive, while 25% of patients had “triple-negative” disease (ER-, PR-, and Her2/neu-negative). KPS was ⩾ 80 in 66% of patients. Sixty-seven percent of patients had ⩽3 initial brain metastases. Eighty-five percent had extracranial metastases at the time of brain metastasis diagnosis. The median follow-up was 41 weeks, and the median survival (MS) for all patients was 80 weeks. The only statistically significant predictor of OS was Her2/neu positivity, with a median survival of 86 weeks compared to 51 weeks in Her2/neu-negative patients (p = 0.03, log-rank). Of 33 evaluable patients who did not receive WBRT as part of their initial therapy for brain metastases, the 1-year freedom from DBF was 52%. DBF was not predicted by any of the factors assessed above, including Her2/neu status.

CONCLUSION

Her2/neu-positive patients with metastatic breast cancer in the brain have higher survival compared to Her2/neu-negative patients with metastatic breast cancer in the brain. Future versions of disease-specific prognostic indices (e.g., GPA) should incorporate molecular markers such as Her2/neu.

Neuro-Oncol. 2009 Oct;11(5):667.

453. Involved-Field Radiation Therapy with Temozolomide for Multicentric High-Grade Gliomas

Donato J Perretta, Shahzad Raza, Michael L Gruber, Deborah Gruber, Ashwatha Narayana

INTRODUCTION

Multicentric high-grade gliomas (HGG) at the time of initial diagnosis are usually associated with poor prognosis and treated with whole brain radiotherapy (WBRT) alone for palliation. The purpose of this study was to evaluate the outcomes of patients who received involved field radiotherapy (IFRT) with concomitant temozolomide and compare them to those of patients who were treated with hypofractionated WBRT.

METHODS

Fourteen patients with multicentric HGG were treated from January 2006 through May 2009. Surgery was limited to biopsy or partial resection of the tumor in 13 patients. Seven patients received wide-field IFRT to a dose of 5,940 cGy over 33 fractions. Seven patients received hypofractionated WBRT to 40.05 Gy over 15 fractions. Time to recurrence, survival, and quality of life were monitored for these patients.

RESULTS

The mean age for the IFRT treatment group was 30 years, with a median Karnofsky performance status (KPS) of 90. The pathology was WHO grade III in six patients. The mean age of the WBRT group was 64 years, with a median KPS of 70. Pathology was grade IV in six patients. With a median follow-up of 7 months, (range, 2–30 months) 11 patients developed recurrent disease. The median progression-free survival (PFS) was 5 months, and the median overall survival (OS) for the entire group was 13 months. The median PFS for the subgroups of patients who received IFRT and patients who received WBRT were 11 and 3 months, respectively. The 6-month PFS in these two groups was 50.0% and 14.3%, respectively. However, the 1-year OS for the two groups was similar (58% vs. 57%).

CONCLUSION

Age, KPS, and tumor grade have prognostic significance in multicentric HGG. The subgroups of patients with young age, high performance status, and grade III tumors have a longer time to progression and may require more aggressive therapy.

Neuro-Oncol. 2009 Oct;11(5):667.

454. Multiparameter Functional MR Imaging as an Early Biomarker of Tumor Response to Whole Brain Radiation in Brain Metastases

Christina Tsien, Felix Feng, Diana Gomez Hassan, James Hayman, Theodore Lawrence, Yue Cao

INTRODUCTION

The response of metastatic lesions to WBRT is markedly heterogeneous. Therefore, we conducted a prospective study to evaluate multiparameters of functional MRI as early biomarkers of response. We hypothesized that changes in vascular volume and diffusion in metastatic lesions are better predictors of response than conventional MRI.

METHODS

Twenty-two patients treated with WBRT for brain metastases were enrolled in a prospective MRI study. Histology included melanoma in 15 patients, non-small-cell lung cancer in five patients, renal cell carcinoma in one patient, and squamous cell carcinoma of the head and neck in one patient. Dynamic contrast enhanced (DCE) and diffusion-weighted (DW) MRI were performed prior to, at the end of, and 1 month after completing WBRT. Changes in neovascular volume and apparent diffusion coefficient (ADC) immediately following WBRT were evaluated for association with the subsequent volumetric changes 1 month after WBRT.

RESULTS

Of 35 analyzed lesions, 17 lesions (47%) progressed 1 month after WBRT. The remaining lesions were either stable or decreased. Volumetric changes at the completion of WBRT did not correlate with subsequent volumetric changes 1 month following WBRT (p > 0.5). In contrast, decreases in neovascular volume at the completion of RT was significantly correlated with response 1 month following WBRT (p < 0.005), and this association was greatest for metastases greater than 1 cm3 (p < 0.002). Increases in mean ADC at the completion of RT correlated with tumor response 1 month following WBRT for lesions 1 cm3 or less in volume (p < 0.05).

CONCLUSION

In this study, we demonstrate that multiparameters of functional MRI analogous to prior work in high-grade gliomas can provide early and improved assessment of treatment response to WBRT in brain metastases. This requires further validation but may provide the means for individualizing therapy in patients with brain metastases by identifying patients who require treatment intensification with stereotactic RT.

Neuro-Oncol. 2009 Oct;11(5):667–668.

455. Efficacy of Gamma Knife Radiosurgery for Recurrent Malignant Gliomas

Robert E Elliott, Erik C Parker, Stephen Rush, Stephen Kalhorn, Yaron A Moshel, Ashwatha Narayana, Bernadine Donahue, John G Golfinos

INTRODUCTION

The role of Gamma Knife radiosurgery (GKRS) in the treatment of high-grade gliomas (HGGs) and their recurrence is unclear. We reviewed our experience with GKRS for recurrent HGGs in a group of high-functioning patients following radical resection.

METHODS

We retrospectively analyzed 26 consecutive adults (8 women and 18 men; mean age, 55.3 years) who underwent GKRS for recurrent HGGs between April 2004 and March 2009. Eight patients (31%) were RTOG class III, and 18 patients (69%) were RTOG class IV. Twenty-four patients initially underwent gross total resection, and two patients underwent near-total resection. The most common tumor locations were in the temporal (42%) and frontal (38%) regions. Most lesions (69%) were less than 1 cm3 (range, 0.02–22.3 cm3). The mean treatment dose was 15 Gy (range, 10–18 Gy). Pathology included GBM in 16 patients, anaplastic astrocytoma in five patients, and anaplastic mixed in five patients. All patients underwent external beam RT and chemotherapy postoperatively. Twenty-three patients underwent GKRS for recurrence in the tumor bed and three patients underwent GKRS for recurrences outside the tumor bed. The mean time to recurrence was 22.2 months.

RESULTS

Fifteen patients (58%) were alive at a mean follow-up of 24.4 months. Fifteen tumors (62%) decreased, and nine tumors (38%) were unchanged for the observation period. Eight patients (33%) experienced local failure, and 18 patients (75%) experienced distant progression. Kaplan-Meier-estimated times to local failure and distant progression were 38.8 and 18.6 months, respectively. Complications included radiation necrosis requiring steroids and transient worsening of hemiparesis in 7.7% of patients. There was no difference in OS among tumor types. Estimated OS was 69.4 months from the time of surgical diagnosis and 39.1 months from time of GKRS.

CONCLUSION

GKRS provided excellent local tumor control in this group of high-functioning patients with small recurrent HGGs after radical resection. Meaningful survival times after GKRS were seen. Prospective data are needed to further characterize the subpopulation of patients with recurrent HGGs who may benefit from radiosurgery.

Neuro-Oncol. 2009 Oct;11(5):668.

456. Prospective Evaluation of Radiosurgery for Hemangioblastomas in Von Hippel-Lindau Disease

Ashok Rajappa Asthagiri, Gautam Unmeel Mehta, Leor Zach, Xiaobai Li, John A Butman, Kevin Camphausen, Russell R Lonser

INTRODUCTION

To determine the effectiveness of stereotactic radiosurgery (SRS) in treating hemangioblastomas of CNS in patients with von Hippel-Lindau disease (VHL), we analyzed the long-term results in VHL patients treated with SRS.

METHODS

Patients enrolled in a prospective VHL natural history study who underwent SRS of CNS heman-gioblastomas were included. Treatment regimens, serial clinical evaluations and longitudinal imaging data were analyzed.

RESULTS

Twenty VHL patients (10 men and 10 women) underwent SRS treatment for 44 CNS hemangioblastomas (39 cerebellar, 5 brainstem). The mean age at treatment was 37.5 ± 12.0 years (range, 13–67 years). The mean follow-up was 8.5 ± 3.2 years (range, 3.0–17.6 years). All patients were alive at last follow-up. The mean treated tumor volume was 0.5 ± 0.7 cm3 (range, 0.01–3.6 cm3). The mean prescription dose was 18.9 Gy (range, 12–24 Gy) at the tumor margin. The local control rates at 2, 5, 10, and 15 years after SRS were 91%, 83%, 61%, and 51%, respectively. Univariate analysis did not identify variables associated with worse tumor control at last follow-up (p > 0.05). Thirty-three percent of SRS-treated small (less than 1.0 cm diameter), asymptomatic tumors progressed over long-term follow-up. There were no long-term adverse radiation effects.

CONCLUSION

While SRS for hemangioblastomas in VHL patients has a low risk for adverse radiation effects, it is associated with diminishing control over the long term. These results indicate that SRS should not be used to prophylactically treat asymptomatic patients and should instead be reserved for treating patients with tumors that are not surgically resectable.

Neuro-Oncol. 2009 Oct;11(5):668.

457. Fractionated Stereotactic Radiosurgery for Meningiomas

Mario Ammirati, H Zarzour, J Grecula

INTRODUCTION

Meningiomas are the most common benign primary tumor of the brain. When radiotherapy is considered, adequate control of benign meningiomas has been shown to be the same with single-dose radiosurgery (15 Gy) or fractionated doses of radiotherapy (54 Gy). Large size and proximity to the optic apparatus and brain stem are dose limiting when considering a single dose. New regimens with fractionated stereotactic radiosurgery (FRSR) have been proposed to treat benign meningiomas.

METHODS

Hypothesizing that 15 Gy in one fraction is biologically effective as 54 Gy in 30 fractions, we calculated an α/β for meningioma. BED1 = BED2, α/β is approximately = 3.276. Estimation of brain stem/optic nerve/chiasm tolerance was calculated for our protocol using Emami’s work and established clinical tolerance of the optic nerves and chiasm to multiple and single-dose (15–25 Gy) treatments. We treated five patients (four women and one man) with FSRS, all of whom had shown neuroradiological evidence of tumor growth. The mean patient age was 58.4 years. Meningiomas were orbital-cavernous sinus, falx, petroclival, and tentorial. The median volume was 3.88 cm3 (range, 0.48–24.9 cm3). All meningiomas were treated without surgery with five fractions of 6 Gy per day to a total dose 30 Gy or five fractions of 5 Gy per day to a total dose 25 Gy. FSRS was delivered using intensity-modulated radiotherapy (IMRT). We used a minimally invasive fixation device (Talon system).

RESULTS

The median follow-up was 8 months (range, 3–22 months). All patients had a complete radiological and neurological evaluation every 6 months. The tumor control rate was 100%; all patients noticed symptom regression. No visual or any other side effect was reported.

CONCLUSION

Our results suggest that FSRS be considered as a primary treatment for meningiomas deemed to be unsuitable for surgical removal, especially when they are large and close to organ at risk. These are the clinical situations in which single-fraction radiosurgery may be not as effective as it is for smaller tumors.

Neuro-Oncol. 2009 Oct;11(5):668.

458. Reducing the use of Whole Brain Radiotherapy for Patients with One to Three Brain Metastases <2 cm in Size

Robert E Elliott, Stephen Rush, Amr Morsi, Nisha Mehta, Jeri Spriet, Ashwatha Narayana, Bernadine Donahue, John G Golfinos

INTRODUCTION

To determine our ability in limiting the use of whole brain radiotherapy (WBRT) in selected cancer patients with brain metastases (BM), we retrospectively reviewed the efficacy of 20-Gy Gamma Knife radiosurgery (GKRS) as the sole treatment for patients with 1–3 brain metastases ⩽2 cm who required subsequent WBRT.

METHODS

We retrospectively reviewed 99 consecutive adults (69 women and 30 men; mean age, 60.8 years) who received GKRS for 1–3 BMs ⩽ 2 cm between February 2001 and January 2009. All patients received 20 Gy at the 50% isodose line and had a Karnofsky performance status equal to 60. Five patients lacked follow-up and were excluded. RPA class was I in 15 patients (17%), II in 73 patients (81%), III in two patients (2%) and unavailable for four patients. The most common cancer types were non-small-cell lung cancer (47%), breast cancer (20%), and melanoma (19%). Sixty-three patients (67%) had one metastasis, 19 patients (20%) had two metastases, and 12 patients (13%) had three metastases. Posttreatment, patients underwent MRI at 6 weeks and every 3 months thereafter unless they were unable to do so or refused.

RESULTS

Follow-up ranged from 4 to 91 months. Nineteen patients (20%) are alive, and the mean survival for the group is 18.8 months (median, 14 months). Thirty-nine patients (41%) underwent a total of 46 salvage treatments (26 patients underwent GKRS, 9 patients underwent surgical resection, and 11 patients underwent WBRT). Salvage therapy was focal (GKRS, surgery) when possible. Indications for WBRT included multiple metastases (seven patients), carcinomatous meningitis (two patients), and recurrence at a surgical margin (two patients). Seven patients (7.4%) suffered neurologic deaths. Risk factors for poor OS included low KPS, high RPA class, and decreased interval between cancer diagnosis and brain disease (p = 0.002). Complications occurred in 10% of patients and included symptomatic edema (four patients), seizures (four patients), and hemorrhage of a lesion requiring evacuation (one patient).

CONCLUSION

The use of 20-Gy GKRS for selected patients with BMs, close follow-up, and individualized interventions allowed for a low subsequent need (11%) for WBRT with a low rate of neurologic deaths from uncontrolled BMs. Using focal treatments for recurrences when possible may significantly reduce the use of WBRT in these patients.

Neuro-Oncol. 2009 Oct;11(5):668–669.

459. Postoperative Stereotactic Radiosurgery (SRS) without Whole-Brain Radiation Therapy in the Treatment of Metastases to the Brain

Anthony Paravarti, Alan C Hartford, Nathan E Simmons, Zhongze Li, William J Spire, Amita Kulkarni, Camilo Fadul, Harker Rhodes, Marc Bellerive, Kadir Erkmen, Jonathan Friedman, David Gladstone, Eugen Hug, David W Roberts

INTRODUCTION

Following a surgical resection of metastases, postoperative radiotherapy to the whole brain lowers the risk of tumor recurrence at the site of the original metastasis and other sites in the brain. One recent series has studied resection followed by stereotactic radiosurgery (SRS) to the surgical bed, demonstrating 74% local control at 12 months. For several years, our institution has offered patients the option of SRS of the surgical bed as an alternative to whole brain radiotherapy.

METHODS

We performed a retrospective, IRB-approved chart review of patients undergoing surgical resection for brain metastases followed by focal stereotactic radiosurgery to the surgical resection cavity plus margin without whole brain radiotherapy. All patients were treated with LINAC-based stereotactic isocentric static beams and dynamic arc techniques while immobilized within an externally fixed, BRW-stereotactic headframe.

RESULTS

Forty-one patients with a total of 43 lesions underwent SRS between February 2002 and September 2008. The mean dose to the resection cavity was 12.1 Gy (range, 8.1–20 Gy). With a mean follow-up of 13.1 months (range, 1.2–58.4 months), actuarial local control at the resection cavity site was 85% at 1 year and 68% at 2 years. No local recurrence was seen for lesions with a preoperative diameter of up to 2.0 cm. Actuarial overall survival rates at 1 year and 2 years were 51% and 32%, respectively. A log-rank test showed a significantly higher likelihood of intracranial disease control for tumors 2.0 cm or less in diameter (p = 0.02). Further analyses are ongoing.

CONCLUSION

In this series, patients who underwent postoperative SRS achieved actuarial local control rates comparable to other studies of postoperative WBRT and SRS. We found that the size of the resected tumor had a negative impact on the rate of intracranial disease control. We conclude that SRS to the surgical bed without WBRT is an effective treatment for resected brain metastases, especially in patients with smaller tumors. Close follow-up is advisable, especially for patients with large tumors at time of initial treatment.

Neuro-Oncol. 2009 Oct;11(5):669.

460. The Effect of Surgery and SRS on Single Intracranial Metastasis and SRS on Synchronous Intracranial Lesions

Timothy R Smith, Kenji Muro, Sunit Das, Martin Pham

INTRODUCTION

Patients with systemic cancer and a single brain metastasis who receive treatment with surgical resection plus radiotherapy live longer and have a higher quality of life than patients treated with radio-therapy alone. This study looked at the effect of surgery and stereotactic radiosurgery (SRS) on a single intracranial metastasis as well as SRS treatment of synchronous intracranial lesions.

METHODS

To assess the effect of SRS on the natural history of metastatic brain tumors, a retrospective cohort was analyzed for time to local progression, time to nonresected synchronous intracranial lesion progression, time to new intracranial lesion development, time to systemic progression, and overall survival after surgical resection and subsequent SRS. The medical records of 151 patients with brain metastases who were treated with complete surgical resections as verified by postoperative magnetic resonance imaging between July 1998 and July 2006 were examined.

RESULTS

The patients had an mean age of 46.2 years (range, 16–82 years). The majority of patients (62.7%) were female. The mean number of intracranial lesions treated with SRS per patient was 2.5 (SD, 2.3 lesions). Of the patients, 17.3% were diagnosed with a metastatic brain tumor less than 3 months before surgery; 55.3% greater than 3 months, and 26.7% concurrently. Primary cancers included lung (43.3% of patients), breast (21.3% of patients), melanoma (10.0% of patients), renal cell (6.7% of patients), and colon (6.7% of patients). The mean time between surgery and SRS was 3.2 weeks. At 1 year, local progression of dually treated lesions occurred in 9.3% of patients, synchronous progression occurred in 13.9% of patients, and new intracranial lesions developed in 13.9% of patients. Systemic progression occurred in 33.8% of patients. Overall 1-year survival was 98%. Of the patients who died, 22.6% of the deaths were attributable to neurologic complications.

CONCLUSION

These findings confirm that the 1-year outcomes for patients with multiple intracranial metastases treated with surgical resection and SRS are similar to established outcomes for patients with single metastases treated with surgery and SRS.

Neuro-Oncol. 2009 Oct;11(5):669.

461. An Imaging Assessment of Malignant Glioma Patients Treated by Boron Neutron Capture Therapy using Functional Diffusion Map

Ryo Hiramatsu, Shinji Kawabata, Shin-Ichi Miyatake, Yoshitaka Yamada, Toshihiko Kuroiwa

INTRODUCTION

Boron neutron capture therapy (BNCT) produced a good improvement in patients with malignant gliomas (MGs), as evidenced by neuroimages and survival time. Assessing radiotherapy and chemotherapy efficacy in MG patients is traditionally accomplished by measuring changes in tumor size on gadolinium-enhanced T1-weighted MR images 10 weeks after treatment initiation. Hamstra et al. reported that diffusion MRI using a functional diffusion map (fDM) provided an earlier assessment of high-grade glioma in patients treated with conventional radiotherapy. The fDM analysis revealed that the volume of tumors with increased diffusion by fDM at 3 weeks was the strongest predictor of patient survival.

METHODS

Serial MR images, including diffusion-weighted images, of MG patients were retrospectively reviewed. fDM analysis was applied to test the efficacy of this approach to assess BNCT response. Nineteen MG patients, including 10 newly diagnosed glioblastoma patients treated by BNCT, were analyzed. The fDM provides the ability to objectively segment the tumor into three different patterns of apparent diffusion coefficient (ADC) change based on the change in ADC values. The total volume (decrease = %Vd, increase = %Vi, stable = %Vs) of the voxels within each of the three patterns was calculated for each patient at several days and/or weeks.

RESULTS

Among newly diagnosed patients, there was good correlation between the %Vd at 2 days after BNCT and survival time from BNCT (r2 = 0.7656). However, there was no correlation between survival time from BNCT and the %Vi at 2 days, 7 days, or 28 days after BNCT.

CONCLUSION

BNCT differs from conventional radiotherapy by the point of localized irradiation with high-dose X-rays. So, MG cells treated with BNCT may swell after BNCT. The fDM analysis provided an early biomarker for predicting BNCT response in patients with malignant brain tumor.

Neuro-Oncol. 2009 Oct;11(5):669.

462. Cone-Beam CT-Guided Stereotactic Radiosurgery for Spinal Metastases

Trang K Nguyen, Jean-Paul Wolinsky, Daniel M Sciubba, Yi Le, Eric Ford, Eric Tryggestad, Ali Bydon, Timothy F Witham, Daniele Rigamonti, John H Wong, Ziya L Gokaslan, Lawrence Kleinberg, Michael Lim

INTRODUCTION

Spinal tumors are a painful and debilitating condition for many cancer patients. We report our experience using the Elekta Synergy S system for treating metastatic spinal lesions.

METHODS

Between July 2008 and March 2009, 10 lesions (two cervical, three thoracic, four lumbar, one sacral) in nine patients were treated using cone-beam CT image-guided stereotactic radiotherapy with Synergy S (Elekta, Stockholm, Sweden). Three patients had previously undergone conventional radiation and surgical resection, and one patient had undergone prior radiation and vertebroplasty. The mean tumor volume was 106.5 cm3 (range, 9.7–259.7 cm3). The dose ranged from 1,400 to 2,100 cGy prescribed to the 78–90% isodose line delivered in 1–4 fractions. The mean maximum dose to the spinal cord was 1,092 cGy (range, 0–1,978 cGy). A retrospective analysis of patients’ pre- and postradiation pain scores (0–10), neurological examinations, and treatment effects was performed.

RESULTS

Eight patients had decreased or stable pain scores following treatment. One patient had a higher pain score after treatment that was adequately managed with medications. The average change in pain scores was a decrease of 3. There were no cases of radiation-induced myelopathy. One patient had a pathological fracture 3 months after receiving radiation and underwent vertebroplasty. Eight of the tumors remained stable in size. One patient was lost to follow-up, and one patient had tumor progression.

CONCLUSION

Our preliminary experience demonstrates that metastatic spinal lesions can be safely treated with Synergy S radiosurgery while alleviating pain and achieving effective local tumor control.

Neuro-Oncol. 2009 Oct;11(5):669.

463. The use of a Virtual Frame System for Gamma Knife Radiosurgery Planning

Trang K Nguyen, Eric Ford, Eric Tryggestad, Daniele Rigamonti, John H Wong, Lawrence Kleinberg, Michael Lim

INTRODUCTION

Gamma Knife (Elekta Instrument AB, Stockholm, Sweden) radiosurgery is highly dependent on the appropriate placement of the stereotactic frame to reach targeted lesions. In some cases, radiosurgery is aborted the day of treatment because of suboptimal frame placement. We report our experience using a computerized virtual frame as a pretreatment tool to determine the accessibility of the tumors and as a guide for optimal frame placement.

METHODS

IRB approval was first obtained. The 16 patients who were recruited for the study were simulated using the virtual frame program on the Pinnacle Radiation Treatment Planning System (RTPS; Philips Radiation Oncology Systems, Madison, WI) prior to their Gamma Knife treatment. The program contains a triangular mesh representation of the frame hardware that can input DICOM-formatted MR scans. The software was used to perform a determination of lesion accessibility and optimal frame placement. The simulation then suggested a frame position based on the relationship to anatomical landmarks. On the day of treatment, the frame was placed at the suggested position. We then assessed our ability to replicate the frame position as well as tumor accessibility.

RESULTS

All 16 presimulated patients had successful treatment with Gamma Knife radiosurgery. A total of 45 lesions were intended for treatment. Although all 45 lesions were reachable on the day of treatment, not all lesions were treated. One lesion was not treated because of its increased size, which was not amenable to Gamma Knife radiation. Another lesion was reachable, but not treated because of excess toxicity to the optic nerve. A total of 14 new metastatic lesions were found on the day of treatment. We also found that a 7.9-mm average difference between the suggested frame placement and what was actually achieved.

CONCLUSION

The use of a virtual frame system allowed for successful treatment planning and frame placement for patients undergoing Gamma Knife radiation.

Neuro-Oncol. 2009 Oct;11(5):670.

464. A New in vitro Assay using Gamma Knife Radiosurgery on Cells from Primary and Recurrent GBMs to Detect the Development of Radiation Resistance in Recurrent Tumors

Kris A Smith, Adrienne C Scheck, Ryan S Smith

INTRODUCTION

Glioblastomas (GBMs) typically recur within a year of initial treatment with high-dose radiation. The mechanisms of radiation resistance in GBMs are not well understood.

METHODS

Cells from six paired primary and recurrent tumors were grown to a confluent monolayer in Opticell cassettes. The center of the cassette was treated in an adapted Gamma Knife phantom with an 18-mm collimator with a maximum dose of 24 Gy. Thus, the 50% isodose line (12 Gy) was delivered to an 18-mm diameter circle. Selected isodose lines (90%, 50%, 30%, 20%, and 10%) were then plotted outward toward the edges of the cassette. Cell lines were treated, observed for 18 days, fixed with methanol, and stained. Cell death and resistant colony formation were observed over time and correlated to the radiation dose received. The diameter of the clearing zone and the number of surviving colonies within the isodose lines were quantified as markers of radiation response. Radiation colony-forming assays (CFAs) were performed on these same cell lines in six groups of 3, with conventional radiation at doses of 0, 1, 2, 4, 8, or 16 Gy. CFA dose responses were correlated to the diameter of the mean clearing zone dose for each cell line. Radioresistant colonies were recultured for analysis and retreatment.

RESULTS

Correlation between the radiation CFAs and clearing zone diameters was very strong, thus validating measures of radioresponse in this novel assay. A two-way ANOVA was used to determine the statistical significance between the survival curves (p-value < 0.0001). Recurrent tumors were shown to be significantly more radioresistant than primary tumors.

CONCLUSION

A new radiosurgical bioassay has been shown to differentiate radiation response between individual cell lines and to document the development of radiation resistance of tumor cells from individual patients. Mechanisms of radioresistance can be assessed from cells isolated in this assay, hopefully resulting in improved counterresistance strategies.

Neuro-Oncol. 2009 Oct;11(5):670.

465. Selective Sensitization of Epidermal Growth Factor Receptor-Overexpressing Glioblastoma Cells to Ionizing Radiation by Inhibition of DNA Repair

Kimberly Ng, Masayuki Nitta, David Kozono, Lisa-Marie Sturla, Pascal Zinn, Clark C Chen

INTRODUCTION

While oncogenic activation of epidermal growth factor receptor (EGFR) is often observed during glioblastoma pathogenesis, therapeutic strategies involving direct EGFR inhibition have been met with limited success. We propose an alternate approach to glioblastoma therapy based on the inhibition of DNA repair.

METHODS

Tissue culture and intracranial mouse xenograft models were used. DNA damage accumulation was measured by γ-H2AX, point mutation, and chromosome instability assays. A library of siRNAs directed against DNA repair genes was purchased from Qiagen.

RESULTS

U87-harboring EGFRvIII, a constitutively active and oncogenic form of EGFR, exhibited increased accumulation of reactive oxygen species and γ-H2AX, suggesting an accumulation of double-stranded DNA breaks (DSBs). This accumulation was accompanied by increased genomic instability as evidenced by increased point mutation formation as well as chromosomal instability. We hypothesized that this genomic instability arose as a result of aberrant DNA repair utilization in response to excessive DSB accumulation. We reasoned that the silencing of such processes should cause selective toxicity to the U87-EGFRvIII cells. We further reasoned that this selective toxicity would be magnified in the presence of exogenously administered DNA damage, such as ionizing radiation. Therefore, we conducted an siRNA screen using a library directed against DNA repair genes to identify gene silencing that selectively sensitized the U87-EGFRvIII cells to ionizing radiation. We found that silencing of poly-ADP ribose polymerase 1 (PARP1) selectively radiosensitized U87 cells that overexpressed EGFRvIII. To exclude “off-target” effects, we recapitulated these results with three pharmacologic inhibitors of PARP1 (3-aminobenzamide, NU1025, and 4-amino-1,8-naphthylamide). We further confirmed our results in an independent GBM cell line (U373MG) with tetracycline-repressible EGFRvIII expression.

CONCLUSION

Our result suggests that EGFR hyperactivation in glioblastoma is associated with increased reliance on specific DNA repair processes. Inhibition of these processes can be exploited as strategy for tumor-specific radiation sensitization.

Neuro-Oncol. 2009 Oct;11(5):670.

466. Outcomes of Stereotactic Radiosurgery for Skull Base Meningiomas

Tomoyuki Koga, Masahiro Shin, Nobuhito Saito

INTRODUCTION

Meningiomas involving the skull base still remain a therapeutic challenge despite advances in microsurgical techniques. To safely achieve tumor control, the combination of microsurgery and stereotactic radiosurgery (SRS) is often utilized to treat meningiomas. We evaluated the long-term outcomes of SRS to address how SRS can be used to effectively and safely treat skull base meningiomas.

METHODS

During the 19-year period since the Gamma Knife unit was installed at our institution, 153 patients with skull base meningiomas were treated by SRS using Gamma Knife. Sixty-four (42%) patients had cavernous sinus lesions, and 35 patients (23%) had petroclival tumors. The median tumor volume was 4.1 cm3 (range, 0.33–45 cm3). Ninety-eight patients (64%) underwent microsurgery prior to SRS. These patients were followed for 2–204 months (mean, 55 months), and 22 of these patients were followed for more than 10 years.

RESULTS

Local tumor control rates at 3, 5, and 10 years after SRS were 93%, 91%, and 82%, respectively. The control rates were lower in patients with lesions larger than 8 cm3 (p = 0.048), and in patients with tumors pathologically diagnosed as WHO grade II or III (p = 0.006). The 10-year local control rate was 91% for tumors gross-totally or subtotally removed prior to SRS and 81% for lesions partially resected or biopsied, although there was no statistical significance. Neurological deterioration including facial hypesthesia, sensory disturbance, visual deficit, and motor paresis, which were observed in 24 patients (16%).

CONCLUSION

Sufficient volume reduction by microsurgery enabled SRS to be more effectively utilized. For those patients in whom tumors were radiologically diagnosed as higher-grade lesions, surgery was necessary for pathological diagnosis and optimal tumor reduction. The proper combination of microsurgery and SRS for skull base meningiomas could provide effective growth control and low morbidity.

Neuro-Oncol. 2009 Oct;11(5):670.

467. Evolving Relapse Patterns in Diffuse Intrinsic Pontine Gliomas

Rajni A Sethi, Bernadine Donahue, Shahzad Raza, Jeena Chacko-Mathew, Matthias Karajannis, Sharon Gardner, Jeffrey H Wisoff, Jeffrey C Allen, Ashwatha Narayana

INTRODUCTION

Diffuse intrinsic pontine gliomas (DIPGs) remain difficult to control. Failure has been predominantly local, with leptomeningeal dissemination (LD) occurring in 4–33% of patients in series from the pre-MRI era. Treatment has historically involved focal irradiation with or without adjuvant chemotherapy. Routine MRI surveys of the entire CNS following initial treatment may reveal other patterns of relapse.

METHODS

Fifteen consecutive pediatric patients with DIPG treated from 2006 through 2009 were retrospectively reviewed. Treatment regimens, imaging surveillance, recurrence patterns, and survival were recorded. Results were compared with those from an earlier prospective series of 18 patients treated at our institution between 1990 and 1995 on a phase I–II trial of hyperfractionated radiotherapy with concurrent carboplatin, which utilized routine MRI surveillance.

RESULTS

Thirteen patients received involved-field radiotherapy to 54 Gy, and two patients received craniospinal irradiation for LD at presentation. Twelve patients received adjuvant chemotherapy. All patients underwent brain and spine MRI at presentation and periodically during follow-up. Two patients did not progress after a follow-up of 10 months. Five patients (33%) progressed locally without LD. One patient developed intracranial metastasis. Seven patients (47%) developed LD (two patients at initial presentation, one patient at the only site of recurrence, two patients concurrently with local treatment failure, and two patients preceding local treatment failure), and five of these patients were symptomatic. The median time to develop LD was 5 months, and the median survival after LD was 4 months.

CONCLUSION

All DIPG patients should undergo baseline spine MRI. The high incidence of LD at relapse in these contemporary series likely reflects more consistent CNS imaging surveillance. CNS prophylaxis should be considered in future clinical trials.

Neuro-Oncol. 2009 Oct;11(5):671.

468. Predicting MGMT Gene Promoter Methylation Status of Glioblastomas using MRI Texture

Ilya Levner, Paula de Robles, Sylvia Drabycz, Gloria B Roldan, Ross Mitchell, Gregory Cairncross

INTRODUCTION

In glioblastoma (GBM), promoter methylation of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) is associated with benefit from chemotherapy. Because MGMT promoter methylation status cannot be determined in all cases, a surrogate for its detection would be a useful clinical tool. Correlations between methylation status and magnetic resonance (MR) imaging features in GBMs have recently been reported, suggesting that noninvasive MGMT methylation status detection might be possible. The aim of our study was to assess whether MGMT methylation status in GBM could be predicted by a novel space/frequency texture analysis of standard MR imaging followed by neural network classification of texture features.

METHODS

We conducted a retrospective analysis of presurgical MR images (FLAIR, T2, and T1 postcontrast) from 59 patients with newly diagnosed GBM. Tumor boundaries were manually outlined on the T1 postcontrast images and then utilized for creating the region of interest (ROI) for the analysis of texture. Quantitative texture features for each ROI were obtained using a space-frequency technique based on the two-dimensional Discrete Ortho-normal Stockwell Transform (DOST). These features were then presented to a L1-regularized artificial neural network (ANN) to predict the MGMT promoter methylation status of the GBMs.

RESULTS

Blinded classification of MGMT gene promoter methylation based on DOST/ANN texture analysis of standard MR imaging reached an average accuracy of 87.7%, with a sensitivity of 85.4% and specificity of 90%. The positive predictive value with this method was 89.3%, and the negative predicted value was 86.2%.

CONCLUSION

Our new method’s accuracy for noninvasive prediction of MGMT gene promoter methylation status in 59 patients with newly diagnosed GBM is encouraging and warrants further validation in a new cohort of patients.

Neuro-Oncol. 2009 Oct;11(5):671.

469. Detecting Recurrence in Malignant Glioma Patients on Bevacizumab: MRI, SPECT CT, or PET—which is better?

Govardhanan Nagaiah, Alejandro Torres-Trejo, Mohammed Almubarak, Warren W Boling, Gary Marano, Ramin Altaha

INTRODUCTION

The reliability of MRI changes in bevacizumab-treated malignant glioma patients is not established. Therefore, we wanted to compare the utility of SPECT CT, MRI, and PET in evaluating tumor recurrence in this patient group.

METHODS

After IRB approval, we conducted a retrospective analysis of MRI, SPECT CT, and PET scan results from all patients treated with bevacizumab between January 2005 to February 2009 at the Mary Babb Randolph Cancer Center, Morgantown, WV.

RESULTS

Thirteen patients were identified, with a mean age of 60 years (range, 43–79). The male:female ratio was 7:6. Radiologist-reported progression by MRI, SPECT CT, or PET scans after first exposure to bevacizumab was counted as date of progression. One patient who did not show progression by MRI but progressed by PET within 1 week of starting bevacizumab and died was excluded from our analysis. Four patients have not progressed on bevacizumab and irinotecan as of reporting and hence were not included in the statistical analysis. Six patients had MRI that showed progression, and seven patients had SPECT CT that showed progression. The mean time to progression by MRI and SPECT was 48 weeks and 42 weeks, respectively. The mean time to progression by PET was 34 weeks, but only four patients in this cohort had PET scans. Time to progression by MRI significantly correlated with time to progression by SPECT CT (p < 0.01). The two patients who had all three tests done (SPECT CT, MRI, and PET) showed progression in all of the tests at the same time. Not all patients received MRI, SPECT CT, and PET, and these data are not a comparison of MRI/SPECT CT/PET on the same patients.

CONCLUSION

From our small case series, it can be concluded that both MRI and SPECT CT have similar utility in detecting the progression of malignant gliomas in patients being treated with bevacizumab.

Neuro-Oncol. 2009 Oct;11(5):671.

470. A Knowledge-based Framework for Quantifying Enhancing Brain Tissue

Eric J Leach, Pingkun Yan, David J Rippe, Nicholas George Avgeropoulos, Shandong Wu, Mubarak Shah

INTRODUCTION

Dynamic contrast-enhanced magnetic resonance (MR) imaging is commonly used to visualize enhancing tissue for diagnosis. Currently, enhancing tissue is measured using bidirectional measurements taken separately in two-dimensional slices. It is not only time-intensive to manually calculate the volume, but it is also common to find large variance among intra- and interoperator interpretation of tumor delineation, yielding results not being reproducible. This can greatly affect the evaluation of treatment plans. Thus, we propose a new knowledge-based framework to automatically quantify the volume of enhancing tissue, which would allow physicians to better assess the efficacy of treatment.

METHODS

Our method follows from the fact that radiologists determine the enhancing tissue by analyzing both T1 and T1E images and by applying their expert medical knowledge at the same time. First, initial detection of enhancing regions is obtained by comparing the difference between the aligned set of T1 and T1E MR images. Then, a new probabilistic framework is developed to automatically determine the true enhancing tissue by considering the anatomical knowledge from an aligned probabilistic brain atlas and the prior distribution of tumor in the brain. The false enhancing tissues can be successfully removed and the true enhancing regions then correctly measured.

RESULTS

Our method was tested on MR images, and the enhancing tissue labeling results were validated by comparing to the ground truth, which was manually labeled by radiologists. Experiments show the system has a high recognition rate (>90%) and a close estimation of tumor size.

CONCLUSION

Our approach provides an objective and consistent way to evaluate tumor progression and to assess the treatment plan, with three main advantages: i) the measurement is user independent, ii) the results are invariant to the image contrast change, and iii) using the segmented regions instead of independent pixels is much less sensitive to small registration errors and image noise.

Neuro-Oncol. 2009 Oct;11(5):671.

471. Relationship between Early Perfusion Parameters Derived from DSC-MRI and Time to Progression in Patients with Glioblastoma Multiforme following Antiangiogenic Therapy

Emma Essock-Burns, Janine M Lupo, Soonmee Cha, Susan Chang, Nick A Butowski, Sarah J Nelson

INTRODUCTION

Glioblastoma multiforme (GBM) is the most malignant subtype of glioma, characterized by extensive neovasculature and angiogenesis. Antiangiogenic therapy aims to impede the tumor’s efficiency in recruiting new vasculature and, when used as an adjuvant therapy, can transiently normalize the tumor’s tortuous vasculature, assisting the delivery of chemotherapeutics.

METHODS

Twenty-six patients with newly diagnosed GBM were imaged prior to receiving therapy and scanned serially at 1, 2, 4, and 6 months on a 3T scanner. Perfusion imaging utilized a dynamic gradient-echo, echo-planar sequence (TE/TR = 54/1,500 ms, flip angle = 35°, 26 × 26 cm2 FOV, 128 × 128 matrix) to track an injected bolus of gadolinium contrast agent over time. Peak height (PH) and percent recovery (%REC) maps were calculated from the dynamic curves to assess blood volume and leakiness, respectively. T2-weighted images, postcontrast T1-weighted images, and Choline-to-NAA index maps derived from lactate-edited 3-D MRSI were used to define a putative tumor region. The 90th percentile PH and 25th percentile %REC within this region were tracked serially for each patient. Patients were divided based on time to radiological progression (TTP). “Long-TTP” patients (n = 6) and “short-TTP” patients (n = 5) were >1 or <1 standard deviation of the mean TTP, respectively, and the rest were deemed “median-TTP” (n = 15).

RESULTS

The 25th percentile of %REC within the tumor at 2 months posttherapy correlated with patients’ TTP (p = 0.05), with more leaky tumors having shorter TTP. Long-TTP patients showed a greater drop in 90th percentile PH between the baseline and 1 month posttherapy scans relative to short-TTP patients (p < 0.05). The reduction in CE volume between 1 and 2 months correlated with TTP (p < 0.05), although no correlation was found prior to 2 months.

CONCLUSION

Perfusion parameters are useful in assessing early response to antiangiogenic therapy (prior to CE) and may be predictive of TTP. An initially well-vascularized tumor may indicate better response and be advantageous in selecting patients for anti-angiogenic therapy.

Neuro-Oncol. 2009 Oct;11(5):672.

472. Retrospective Study to Identify If 99m Technetium-MIBI SPECT has the Same Correlation as F-18-Fluoro-Deoxy-D-Glucose PET with the Final Histological Diagnosis According to World Health Organization (WHO) Criteria in Patients with Brain Tumors

Alejandro Torres-Trejo, Mark Haut, Terrence D Julien, Charles L Rosen, Warren W Boling, Ramin Altaha, Kenneth Fallon, Kymberly Gyure, Gary Marano

INTRODUCTION

F-18-fluoro-deoxy-D-glucose PET (F-18-FDG-PET) is known to correlate with the histology and proliferative index of brain tumors (BT) and can differentiate between tumor and radiation necrosis (RN). 99m technetium-MIBI SPECT (99mTc-MIBI-SPECT) has also been reported to correlate with the histology of BT.

METHODS

In order to corroborate the above reports, and with IRB approval, we identified nine patients with BT who underwent 99mTc-MIBI-SPECT and F-18-FDG-PET perioperatively. We compared the results of 99mTc-MIBI-SPECT with F-18-FDG-PET and correlated the results with the histology. In patients 1–7, imaging was performed preoperatively, and in patients 8 and 9, imaging was performed postoperatively. Age range, 20–60 years. Gender: eight females and one male. Histology: patient 1, low-grade astrocytoma (LGA-WHO-II); patient 2, transformed to anaplastic oligoastrocytoma (AOA-WHO-III); patient 3, recurrent anaplastic astrocytoma (AA-WHO-III) with RN; patient 4, recurrent GBM (WHO-IV) with RN; patient 5, recurrent GBM with RN; patient 6, B-cell primary CNS lymphoma (B-cell PCNSL); patient 7, choroid plexus papilloma (CPP-WHO-I); patient 8, hypothalamic low-grade glioma (HLGG-WHO-II); and patient 9, recurrent anaplastic astrocytoma (AA-WHO-III) with RN.

RESULTS

Six of nine tumors had the expected correlation with the F-18-FDG-PET, and 5/9 tumors had expected correlation with the 9mTc-MIBI-SPECT. Only 2/9 patients had discrepancy between both images in relationship to the histology.

CONCLUSION

Based on these studies, we can conclude that both images can be used to make noninvasive analysis of brain tumors. However, caution should be exercised in the interpretation of these images in patients with recurrent anaplastic gliomas (AG) and in patients with low-grade tumors. In two-thirds of patients with recurrent AG, both image results were those of possible RN when the final pathology showed viable tumor. The 9mTc-MIBI-SPECT gave results of a high-grade tumor in two-thirds of cases when the final pathology was that of a low-grade tumor (WHO I and II). Larger studies are needed to corroborate these findings.

Neuro-Oncol. 2009 Oct;11(5):672.

473. Novel MRI Changes in Patients on Bevacizumab

Stephen Futterer, Sean Grimm, James P Chandler, Kenji Muro, Achilles Karagianis, Maryanne Marymont, Jeff Raizer

INTRODUCTION

Bevacizumab was recently approved by the Food and Drug Administration for the treatment of recurrent glioblastoma (GBM), which will result in its widespread use. Bevacizumab alters the magnetic resonance (MR) imaging appearance by a reduction of lesion enhancement; some patients on treatment have progression of infiltrative, nonenhancing tumor. We have observed additional MR imaging findings not previously described in patients receiving bevacizumab.

METHODS

We retrospectively reviewed serial brain MR exams on patients with malignant CNS tumors who received bevacizumab.

RESULTS

We analyzed serial brain MR exams in six patients with malignant gliomas and one with recurrent malignant meningioma. All patients had a history of RT and later bevacizumab-based treatment, except one who had concurrent RT with bevacizumab (meningioma). Two patients had small areas of deep white matter hemorrhage, which did not involve the primary tumor bed. All patients demonstrated areas of diffusion restriction, of which only two were confined to the surgical tumor bed. The other five had DWI restriction away from the resection bed. Difffusion restriction was seen in the corpus callosum (n = 2), parasagittal aspect of the contralateral hemisphere (n = 2), and cortical/gyral distribution (n = 3). These areas showed variable precontrast T1 shortening (laminar necrosis pattern) and enhancement (presumed subacute ischemia). Two patients had similar changes in the deep gray nuclei. These areas were not hyperperfusing.

CONCLUSION

We describe several novel imaging findings on MR exams in patients with malignant CNS tumors treated with bevacizumab and prior radiation. We surmise that these complex signal changes represent subclinical drug toxicity, likely potentiated by a background radiation vasculitis, as they appear to occur within or near the RT ports. We found that these findings were often mistaken for recurrent tumor. Such confusion might lead to an underestimation of drug efficacy and premature termination of treatment as it did in one of our patients; hence, clinicians need to be aware of these changes.

Neuro-Oncol. 2009 Oct;11(5):672.

474. Long-Term Clinical Monitoring of Gliomas using Functional Diffusion Maps (fDMs) in Regions of Flair Abnormality

Benjamin M Ellingson, Mark Malkin, Scott D Rand, Jennifer M Connelly, Kathleen M Schmainda

INTRODUCTION

Diffusion-weighted magnetic resonance imaging (DWI) is a sensitive imaging biomarker for tumor cellularity. Functional diffusion maps (fDMs) exploit this sensitivity by calculating voxel-wise changes in diffusivity over time in the same patient. Currently, fDMs have only been applied to contrast-enhanced regions on T1-weighted images, which represent regions of blood-brain barrier breakdown and not necessarily the boundary of infiltrating gliomas. Since up to 45% of gliomas do not enhance, the utility of fDMs as biomarkers for early tumor invasion and in monitoring tumor progression in glioma patients exhibiting little or no contrast enhancement is of significant clinical interest. In the current study, we demonstrate long-term clinical monitoring of glioma patients using fDM analysis in regions with abnormal FLAIR signal.

METHODS

A consecutive series of 33 patients with recurrent malignant gliomas were enrolled in the study. The fDMs were calculated by first coregistering sequential apparent diffusion coefficient (ADC) maps to the same coordinate system, subtracting current ADC maps from baseline ADC maps, and stratifying voxels into three types: no change in ADC (green), increasing ADC (red, e.g., hypocellularity, red), or decreasing ADC (blue, e.g., hypercellularity, blue). The total volume of voxels with decreasing ADC in FLAIR abnormal regions was used to track the tumor status.

RESULTS

The utility of fDMs in spatially localizing and quantitatively tracking presumed brain tumor progression is demonstrated graphically in two patients. In general, we found the total volume of voxels with decreasing ADC appeared to reflect histological grade of the tumor, consistent with expected hypercelluarity, whereas the rate of change in volume with decreasing ADC, which may be indicative of tumor growth, was sensitive to tumor progression.

CONCLUSION

Functional diffusion maps applied to FLAIR-abnormal areas are valuable for the long-term clinical monitoring of gliomas.

Neuro-Oncol. 2009 Oct;11(5):672.

475. Evaluation of Cytotoxic and Antiangiogenic Treatments using Functional Diffusion Maps (fDMs) in Flair-Abnormal Regions

Benjamin M Ellingson, Mark Malkin, Scott D Rand, Jennifer M Connelly, Devyani Bedekar, Kathleen M Schmainda

INTRODUCTION

Functional diffusion maps (fDMs) exploit the sensitivity of diffusion MRI to tissue cellularity by calculating voxel-wise changes in diffusivity within contrast-enhancing regions over time in the same patient. Although fDMs have been examined for use with cytotoxic treatments, their utility in the evaluation of antiangiogenic treatments has not been studied. To address the challenges associated with the lack of contrast enhancement, which often follows antiangiogenic treatments, we applied the fDM technique to regions of fluid-attenuated inversion recovery (FLAIR) image abnormality.

METHODS

We examined fDMs in eight patients treated with bevacizumab+irinotecan and 12 treated with temozolomide concurrently with standard radiotherapy. Each scan was classified as responsive disease (RD), stable disease (SD), or progressive disease (PD) based on neurological and radiographic status. The fDMs were calculated by coregistering sequential apparent diffusion coefficient (ADC) maps, subtracting current ADC maps from baseline ADC maps, and stratifying voxels into three categories: no change in ADC (green), increasing ADC (red), or decreasing ADC (blue). The volume of voxels with decreasing ADC, which may be indicative of hypercellularity in FLAIR-abnormal regions, was used to track presumed tumor.

RESULTS

The rate of change in decreasing ADC (“hypercellular”) volume was higher in PD compared to both SD and RD (ANOVA, p < 0.001; Tukey, p < 0.001), suggesting this may be a predictor of progression. The rate of change of decreasing ADC was significantly higher in patients with PD after treatment with antiangiogenic drugs compared to standard treatment (Tukey, p < 0.025).

CONCLUSION

Given that decreasing ADC volumes may be a marker of increasing cellularity, the results of the current study support the hypothesis that recurrence after treatment with bevacizumab results in growth of an infiltrative nonenhancing tumor phenotype, and further support the use of fDMs applied to FLAIR regions as a valuable imaging biomarker for assessing brain tumor cellularity.

Neuro-Oncol. 2009 Oct;11(5):672–673.

476. Hybrid Functional Diffusion and Perfusion Maps for the Evaluation of Gliomas

Benjamin M Ellingson, Mark Malkin, Scott D Rand, Jennifer M Connelly, Devyani Bedekar, Kathleen M Schmainda

INTRODUCTION

In vivo identification of brain regions regions containing high cell density and vascularity consistent with tumor are critical for the assessment and treatment of human gliomas. Diffusion and perfusion magnetic resonance imaging (MRI) are noninvasive, in vivo techniques that can be used to elicit insight into the cellular and vascular microenvironments. Functional diffusion maps (fDMs) and functional perfusion maps (fPMs) were developed to examine voxel-wise changes in apparent diffusion coefficient (ADC) and relative cerebral blood volume (rCBV) during serial MRI comparisons. In the current study, we have merged these two techniques into a hybrid functional diffusion and perfusion map (fDM+fPM) to localize and quantify regions with both increasing cellularity and increasing vascularity in gliomas.

METHODS

Ten patients with recurrent glioblastomas were enrolled in the study. Regions of “hyprecellularity” were isolated by coregistering sequential ADC maps, subtracting current ADC maps from baseline ADC maps, and labeling voxels with decreasing ADC based on a 0.55 μm2/s change in ADC relative to baseline images. Similarly, “hypervascular” regions were isolated by registering sequential rCBV maps and identifying voxels with increasing rCBV based on a 100% change in standardized rCBV from baseline. To generate hybrid maps, the “hypercellular” voxels (blue) and the “hypervascular” voxels (red) were combined in the same image space. If a voxel demonstrated both hypercellularity (blue) and hypervascularity (red), it was labeled yellow for visualization.

RESULTS

Hybrid fDM+fPMs were useful for spatially localizing suspected hypercellular and hypervascular regions in gliomas. In some tumors, and sometimes in different regions of the same tumor, areas exhibiting an increase in hypercellularity preceded an increase in hypervascularity. In other tumors, increasing vascularity preceded increasing cellularity.

CONCLUSION

Hybrid fDM+fPMs may be useful for spatially localizing high-risk regions of growing tumors in vivo and studying temporal changes in the cellular and vascular phenotypic growth patterns in human gliomas.

Neuro-Oncol. 2009 Oct;11(5):673.

477. Evaluation of Long-Term Effects of Radiation Therapy in Patients with Gliomas using Susceptibility-Weighted Imaging at 7 Tesla

Janine M Lupo, Susan Chang, Bert Jimenez, Sarah J Nelson

INTRODUCTION

Susceptibility-weighted imaging (SWI) is a powerful tool for imaging microhemorrhages. With this technique, the postprocessing of the phase information is used to amplify contrast between tissues that have different susceptibilities in the magnitude image. At 7T, heightened susceptibility effects provide enhanced sensitivity to these hemorrhages, which could potentially be useful for examining changes in normal brain tissue after exposure to radiotherapy.

METHODS

Five patients with long-term stable gliomas (one juvenile pilocytic astrocytoma, two grade II, and two grade III), treated with fractionated external beam radiation therapy (x-RT) for at least 5 years (median, 9 years; range, 5–22 years) prior to the time of imaging, were recruited for this study. High-resolution SWI was performed in a 7T scanner with volume excitation and 8-channel phased-array reception and employed a 3-D SPGR sequence with TE/TR = 16/50 ms, flip angle = 15 degrees, BW = 62.5 kHz, 24 × 24 cm3 FOV, and 2 mm slice thickness. To keep the scan time under 6 min, a GRAPPA-based parallel imaging acquisition was utilized with a 3-fold reduction, 512 × 144 acquired matrix, and 16 autocalibrating lines. Standard SWI postprocessing was performed on the reconstructed k-space data for each coil, and then combined, intensity corrected, and projected through 8-mm-thick slabs.

RESULTS

7T SWI revealed hemorrhagic lesions in four of five patients, with three patients’ lesions extending beyond the T2 lesion and two patients having >20 microbleeds. These lesions were located further from the original tumor in patients who received x-RT longer ago, and the patient without hemorrhages underwent x-RT most recently. All patients were asymptomatic except for one, who also possessed the highest lesion count and years since x-RT.

CONCLUSION

High-field SWI has potential for visualizing hemorrhages associated with long-term effects of radiotherapy on normal brain tissue. This may be important in further assessing the utility of this treatment in patients with lower-grade tumors with longer survival rates.

Neuro-Oncol. 2009 Oct;11(5):673.

478. Standardization Reduces Variability in rCBV Measurements

Devyani Bedekar, Todd Jensen, Benjamin M Ellingson, Scott D Rand, Kathleen M Schmainda

INTRODUCTION

Relative cerebral blood volume (rCBV) measurement using DSC (dynamic susceptibility contrast) magenetic resonance imaging (MRI) suffers from interpatient and interstudy variability for the same tissue type. Traditionally, rCBV values are normalized (NRMrCBV) to normal-appearing white matter (NAWM), but this technique of normalization is very subjective and introduces user-dependent variability. In this study, we demonstrate that a method called standardization, applied to rCBV (STDrCBV) maps, is an objective means of translating all rCBV values to a consistent scale. This approach reduces interpatient and interstudy variability for the same tissue type, thus enabling easy and accurate visual comparison across studies.

METHODS

All MRI studies were performed on either a 1.5-T GE Signa or a CV scanner. A 0.10-mmol/kg dose of gadodiamide (Omniscan; Nycomed Amersham, Princeton, NJ) was administered to diminish T1 effects that might result from agent extravasation. Next, GRE EPI images were acquired for 1 min before and 2 min after a 0.1-mmol/kg bolus injection (TE/TR = 30/1,100 ms). Finally, postcontrast T1 images were acquired (SE, TE/TR = 4.668/34 ms). Multiple scans were acquired for three subjects. Upon coregistration, STDrCBV and NRM-rCBV values for NAWM region were tracked for all subjects. Coefficient of variance (CV) for STDrCBV and NRMrCBV for NAWM region and percentage difference with respect to the first date were calculated.

RESULTS

Data collected demonstrate that standardization of rCBV maps reduced interstudy and interpatient variability better than the normalized rCBV maps, thus enabling objective visual comparison between patients and robust longitudinal tracking of tumor progression. The CV for STDrCBV and NRMrCBV for all 13 dates was 16.43% and 22.25%, respectively.

CONCLUSION

From a clinical standpoint, standardization should prove very helpful to radiologists and researchers alike as it makes it unnecessary to subjectively change windowing and determine reference ROIs.

Neuro-Oncol. 2009 Oct;11(5):673.

479. Identifying Pseudoprogression in Glioblastoma: Utility of Conventional MR Findings

Akash Shah, Ajay Gupta, Robert Young, Kathryn Beal, Sasan Karimi, Weiji Shi, Zhigang Zhang, Andre Holodny

INTRODUCTION

Patients with glioblastoma multiforme (GBM) treated with combination radiation therapy (RT) and chemotherapy may demonstrate pseudoprogression (PsP), the transient worsening of magenetic resonance imaging (MRI) abnormalities owing to potentiated radiation-induced cell killing without a worse prognosis. We examine the utility of conventional MRI in differentiating PsP from true progression.

METHODS

We retrospectively reviewed 200 patients with GBM. We identified 39 patients (31 males, 8 females; mean age 54 years) who 1) completed RT with concurrent/adjuvant temozolomide; 2) had no change in treatment for at least 3 months after RT; and 3) had new or increased enhancing abnormalities on their first post-RT MRI. At the 3 month post-RT MRI, patients were classified as true progression if the abnormalities continued to worsen or PsP if the abnormalities improved or stabilized. Diagnosis was confirmed by pathology from repeat surgical resection in eight patients. The following MRI findings were examined by a CAQ neuroradiologist: 1) corpus callosum involvement, 2) midline involvement, 3) subependymal spread, 4) ill-defined enhancing margins, 5) diffusion restriction, 6) increased enhancement, 7) new enhancement, 8) marginal enhancement around surgical cavity, 9) new discrete enhancing nodule, and 10) lesion quotient (T2 nodule/enhancing lesion). Univariate analysis based on Fisher exact (categorical variable) and Wilcoxon rank sum (continuous variable) tests were performed to study the correlation between the MRI findings and PsP.

RESULTS

At 3 months, 23/39 patients (59%) were determined to have PsP. No significant correlation was found between any MRI finding and PsP (p-values > 0.19).

CONCLUSION

About half of all GBM patients with worsening abnormalities on their initial post-RT MRI were diagnosed with PsP after 3 months of follow-up. No conventional MRI finding was found as a significant predictor of PsP. Future studies using advanced MRI techniques to examine the biophysiologic properties of the worsening abnormalities may lead to the earlier and more accurate diagnosis of PsP.

Neuro-Oncol. 2009 Oct;11(5):673–674.

480. MRI Measures of rCBV Predict Response to Bevacizumab Treatment More Reliably than Standard MRI in Patients with Recurrent High-Grade Gliomas

Kathleen M Schmainda, Devyani Bedekar, Scott D Rand, Jennifer M Connelly, Hendrikus G Krouwer, Mark Malkin

INTRODUCTION

The anti-VEGF antibody bevacizumab (Avastin) is in clinical testing for the treatment of gliomas, with promising results. In these studies, standard measures of tumor volume derived from contrast- enhancing T1-weighted images or abnormal volumes on T2-weighted (FLAIR) images, have been used to evaluate response. However, since antiangiogenic therapies also decrease blood brain barrier permeability, these measures are often unreliable. In this study, we evaluate dynamic susceptibility contrast magnetic resonance imaging (DSC-MRI) measures of relative cerebral blood volume (rCBV) as a preferred alternative.

METHODS

Seven patients with recurrent high-grade glioma receiving a regimen of bevacizumab plus irinotecan were enrolled in this study. All patients had previous resection(s) and adjuvant therapy including radiation and chemotherapy. Both standard MRI and DSC-MRI were performed before and periodically throughout treatment. The rCBV were calculated as previously described. All image series were standardized and registered across studies. T1+C and FLAIR ROIs were determined, along with mean rCBV in these areas. The number of days from the start of treatment (day 0) to maximum response (nadir) was determined, as well as the number of days prior to neurologic deterioration (as indicated by KPS) when the parameter of interest increased by at least 25%.

RESULTS

Six of seven patients showed a response to treatment, with survival ranging from 151 to 601 days and one patient still alive 1,440 days after diagnosis. For all patients, the nadir was earlier or similar to that of T1+C or FLAIR volumes. Increases in rCBV denoting progression preceded neurologic deterioration by 63–204 days in all but one patient and occurred earlier than increases in T1+C volumes (1–105 days).

CONCLUSION

This study demonstrates that rCBV is a reliable indicator of response to treatment as well as being an earlier and more reliable predictor of disease progression compared to standard MRI measures of enhancing tumor volume.

Neuro-Oncol. 2009 Oct;11(5):674.

481. Early Prognostic Value of 3-D 1H-MRSI and Diffusion Tensor Imaging for Newly Diagnosed GBM Patients Receiving RT, Temozolamide, and PKC Inhibitor

Adam Elkhaled, Inas Khayal, Nick A Butowski, Susan Chang, Sarah J Nelson

INTRODUCTION

Glioblastoma multiforme (GBM) demonstrates marked vascularization and proliferative capacity, in contradistinction with low-grade gliomas. The dynamic nature of this disease, which is characterized by relatively short time to progression and survival, emphasizes the need for prognostic tools that can detect significant changes early in treatment and evaluate the likelihood of therapeutic response.

METHODS

Fifteen patients with newly diagnosed GBM were imaged on a 3-T scanner prior to treatment and then at 1- and 2-month follow-up. The spectroscopic imaging consisted of a lactate-edited echo-planar pulse sequence (TE/TR = 144 ms/1,140 ms, FOV = 16 × 16 × 16, voxel = 1 cc) and the diffusion tensor imaging was acquired using six gradient directions in the axial plane (TR/TE = 7,000/76 ms, voxel = 0.9 × 0.9 × 3 mm, b = 1,000 s/mm3). The MRSI and DTI data were registered to the pre-RT baseline images. Automated algorithms determined maps of the choline-to-NAA index (CNI) and apparent diffusion coefficient (ADC). Patients were segregated into two groups based on time to radiological progression (TTP): early progression (1–6 months, n = 7) and late progression (7–12 months, n = 8). Nonparametric comparisons were made between groups at each scan interval and across time points for the same group (Wilcoxon Ranked-Sum/Signed-Rank tests).

RESULTS

CNI values decreased globally for all patients over the duration of observed scans. The opposite trend was seen from the net increase of ADC by month 2 within the contrast-enhancing lesion, irrespective of the TTP group. The maximum CNI value at the baseline scan provided the best classification of early progression (p < 0.05) (sensitivity/specificity/accuracy = 100/80/88.2% for cutoff = 6.00). At the 1- and 2-month scan intervals, respectively, lower median contrast-enhancing ADC (p = 0.05) and larger contrast-enhancing volume (p < 0.01) were most predictive of advancing disease (sensitivity/specificity/accuracy = 87.5/80/83.3% for cutoffs = 1.63, 4.90 cc). Group assignment according to the prescribed cutoff values consistently defined the boundary separating early from late progression.

CONCLUSION

Parameters derived from MRSI and DTI provided information at baseline and early follow-up MR examinations that may be valuable in predicting time to radiological progression for patients with GBM.

Neuro-Oncol. 2009 Oct;11(5):674.

482. Diffusion-Weighted Magnetic Resonance Imaging for the Evaluation of Tumor Progression in Patients Treated with Bevacizumab

Kevin Becker, Tina Mayer, Jill Lacy, Joachim M Baehring

INTRODUCTION

Bevacizumab, a humanized monoclonal antibody directed against vascular endothelial growth factor, has shown effectiveness in relapsed glioblastoma multiforme. Conventional outcome measures (Macdonald criteria) have proven problematic in patients treated with antiangiogenic agents. In this study, we sought to evaluate the role of diffusion-weighted magnetic resonance imaging (DW-MRI) in patients treated with bevacizumab.

METHODS

We performed a retrospective analysis of patients with relapsed malignant glioma treated with bevacizumab between 12/05 and 5/09. Comprehensive MRI studies were obtained at the time of disease progression, during treatment with bevacizumab, and at time of treatment failure. These images were assessed for evidence of disease progression or treatment-related changes.

RESULTS

73 patients were identified, of whom 26 have been analyzed so far. Two individuals were excluded because of insufficient imaging data. The following DW-MRI abnormalities were identified: 1) nodular growth with restricted water diffusion (ADC+), negative by T1-weighted sequences with gadolinium (T1W−); 2) nodular growth ADC+, T1W+; 3) nodular growth ADC, T1W+; 4) gliomatosis ADC+; 5) gliomatosis ADC; 6) hemorrhage; and 7) calcification. In 21% of cases (5/24), nodular growth occurred in the absence of contrast enhancement. 63% of patients showed tumor progression as demonstrated by ADC+ and T1W+, albeit with reduced contrast enhancement compared to prior to initiation of bevacizumab. Gliomatosis was identified in 11 cases (four ADC+, seven ADC−). ADC+ secondary to intratumoral hemorrhage was seen in five cases, and calcification was seen in one. Two individuals had unexplained diffusion abnormalities.

CONCLUSION

We found diffusion-weighted MRI to be a useful addition to conventional sequences in the evaluation of patients with relapsed malignant glioma treated with bevacizumab. Whether it may serve as a more accurate outcome measure than postgadolinium T1-weighted sequences in this patient population will require prospective evaluation correlating DWI and ADC findings with performance status and survival.

Neuro-Oncol. 2009 Oct;11(5):674.

483. The use of Preoperative Magnetic Resonance Flair and Ependymal Proximity as Surrogate Markers of Brain Tumor Origin

Timothy R Smith, Kenji Muro, Sunit Das, Martin Pham

INTRODUCTION

Adult neural stem cells (NSCs) are known to reside in the subventricular zone (SVZ). NSCs proliferate in the SVZ and give rise to progeny that differentiate and migrate throughout the brain. Some investigators have hypothesized that glioma behavior and grade might correlate with the identity of the tumor cell of origin and that these differences might account for variations in tumor cell proliferation and migration. To test this possibility, we evaluated three preoperative radiographic features (magnetic resonance FLAIR characteristics, tumor proximity to ventricular ependyma, and subependymal representation) as surrogate markers of tumor origin using a retrospective cohort design.

METHODS

The medical records of 228 patients who underwent surgical resection of a glioma from January 2004 to August 2008 were reviewed. Thirty-one patients were excluded because of a lack of access to preoperative magnetic resonance (MR) imaging.

RESULTS

Average patient age was 54.5 years (SD 15.3 years) with a male (62.9%) predominance. 71.6% of gliomas were grade IV, 21.3% were grade III, and 7.1% were grade II. Mean survival was 11.2 months (SD 10.5 months), with a mean follow-up of 12.8 months (SD 11.3 months). Glioma tumor grade was significantly correlated to FLAIR signal proximity to the ependymal surface (p < 0.01) and inversely with proximity of tumor mass to the ependyma (p < 0.01). The mean distance of tumor-associated FLAIR signal from the ependymal surface for glioblastoma (GBM) was 1.2 mm (SD 3.3 mm), compared to 4.8 mm (SD 6.5 mm) for anaplastic astrocytomas and 6.6 mm (SD 6.7 mm, p < 0.01) for low-grade gliomas. Conversely, the mean distance of the enhancing tumor mass from the ependyma for GBM was 7.3 mm (SD 9.4), for grade III glioma was 2.3 mm (SD 4.9 mm), and for grade II glioma was 3.8 mm (SD 6.8 mm, p < 0.05). These findings suggest that higher-grade gliomas might arise from less-differentiated neuroepithelial cells in the SVZ that possess greater migratory potential.

CONCLUSION

Glioma grade correlates directly with FLAIR signal proximity to the ependymal surface and inversely with proximity of the tumor mass to the ependyma.

Neuro-Oncol. 2009 Oct;11(5):674–675.

484. Characterization of Recurrent Low-Grade Gliomas using Diffusion-Weighted MR Imaging

Colleen Cloyd, Susan Chang, Soonmee Cha, Mitchel S Berger, Michael William McDermott, Andrew T Parsa, Manish Kumar Aghi, Sarah J Nelson

INTRODUCTION

The diagnosis of low-grade gliomas typically relies on the presence of new contrast enhancement, increased T2 hyperintensity, and presentation of clinical symptoms. However, these measures are often not sufficient for determining tumor recurrence. Incorporating functional imaging parameters derived from diffusion-weighted imaging (DWI), perfusion-weighted imaging (PWI), and magnetic resonance (MR) spectroscopy may aid in distinguishing tumor recurrence from treatment effect, characterizing progression to a higher classification, and determining the appropriate treatment strategy.

METHODS

Presurgical 3T magnetic resonance imaging (MRI) scans were obtained from 32 patients with recurrent low-grade glioma. Anatomical T2-weighted FLAIR images and pre/postcontrast T1-weighted SPGR images were acquired and used to define volumetric regions of interest of normal-appearing white matter (NAWM), nonenhancing T2-hyperintensity (NEL), gadolinium contrast-enhanced lesion (CEL), and necrosis (NEC). An apparent diffusion coefficient (ADC) volume map was derived from the DWI, aligned to the anatomical images, and normalized to the NAWM. Patients were stratified based on whether surgical pathology displayed an upgrade in World Health Organization classification compared to a previous pathological sample. The results from this recurrent cohort were also compared to previously published data from patients with newly diagnosed glioma.

RESULTS

New contrast enhancement was found in 10 of 16 patients with tumor reclassification and in 2 of 16 stable patients. Reclassified patients exhibited significantly larger T2 lesions and lower volumes with abnormal nADC (<1.5) volumes than those who remained the same grade (p < 0.05). The nADC values in the NEL of the reclassified cohort were lower than in the patients with stable classification (p < 0.05) and were consistent with values previously observed in newly diagnosed glioma of the corresponding higher grade.

CONCLUSION

Recurrent low-grade gliomas exhibit different imaging characteristics than newly diagnosed gliomas. The characterization of ADC parameters in recurrent low-grade gliomas may be useful in predicting whether the lesion has progressed to a higher grade.

Neuro-Oncol. 2009 Oct;11(5):675.

485. Correlation between MRI Sequences is Variable in Patients with Progressive Glioblastoma Multiforme Treated with Bevacizumab plus Chemotherapy

Eric M Thompson, Edit Dosa, Edward A Neuwelt

INTRODUCTION

Macdonald criteria using postcontrast T1-weighted (T1+Gd) magnetic resonance images (MRI) to evaluate tumor response and progression in the context of antiangiogenic therapy has not been validated. The primary objective of this study was to determine whether a reliable and predictable relationship over time exists between T1+Gd, T2-weighted, and fluid-attenuated inversion-recovery (FLAIR) MRI in patients with progressive glioblastoma multiforme (GBM) receiving bevacizumab.

METHODS

A retrospective analysis of 10 patients who received bevacizumab plus carboplatin, irinotecan, or temozolomide for progressive GBM is presented. A single radiologist evaluated all images from each patient’s last prebevacizumab MRI to January 1, 2009. Lesion volumes were manually acquired using ImageJ software. Correlation between T1+Gd, T2, and FLAIR MRI using pairwise Pearson product moment correlation (r) was performed for each patient.

RESULTS

Following bevacizumab, two patients had no decrease in size of tumor-related T1 enhancement, while three patients had no decrease in size of T2 and FLAIR hyperintensity. Of the patients who radiographically responded to bevacizumab, the mean amount of decrease in T1+Gd enhancement, T2 hyperintensity, and FLAIR hyperintensity was 61.0 ± 24.3%, 63.1 ± 24.5%, and 62.9 ± 24.8%, respectively. Correlation between T1+Gd and T2 ranged from 0.18 to 0.99, between T1+Gd and FLAIR ranged from 0.20 to 0.98, and between T2 and FLAIR ranged from 0.66 to 0.99. Four patients had a significant correlation (p < 0.05) between T1+Gd and T2, four patients had a significant correlation between T1+Gd and FLAIR, and six patients had a significant correlation between T2 and FLAIR.

CONCLUSION

In patients with progressive GBM receiving bevacizumab plus chemotherapy, we conclude that correlation between T1+Gd and both T2 and FLAIR was variable, but correlation between T2 and FLAIR was generally > 0.87. More sophisticated imaging techniques are needed to accurately evaluate imaging response to therapy and disease progression in patients receiving antiangiogenic agents.

Neuro-Oncol. 2009 Oct;11(5):675.

486. Molecular Image-Guided Brain Tumor Resection with Miniature Dual-Axis Confocal Microscopy

Jonathan TC Liu, Henry Haeberle, Michael J Mandella, Gordon S Kino, Christopher H Contag

INTRODUCTION

Numerous studies have indicated a correlation between the outcome of brain tumor patients and the degree of surgical resection. Real-time image guidance is necessary to allow for complete resections in a larger proportion of patients and to reduce the debilitating effects of overaggressive resections. If high detection sensitivity is desired (sensitive enough to detect a few tumor cells), a high-resolution imaging device is essential. This is because each pixel in a low-resolution image averages over many cells and hinders one’s ability to detect tumors with single-cell sensitivity. Furthermore, if accurate detection of cell-surface biomarkers is desired, then subsurface imaging is advantageous. This is because surface cells are often disturbed by prior surgical procedures or by the imaging device itself and will not be labeled as well as the undisturbed cells below the tissue surface. Confocal microscopy, if modified for deep-tissue imaging, allows one to image subsurface cells that are in their natural undisturbed tissue microenvironment, where cell-surface proteins may be accurately labeled with exogenous contrast agents.

METHODS

We have been developing a miniature confocal microscope with a 2-mm-diameter tip, along with fluorescent peptide contrast agents, to provide in vivo histopathology for guiding brain tumor resection.

RESULTS

The microscope we have developed utilizes a novel dual-axis architecture to efficiently reject out-of-focus light for high-contrast optical sectioning within tissues. Imaging studies have been performed with tissues from a transgenic mouse (Ptc ± p53−/− Math1-GFP) that spontaneously develops medulloblastoma. We have also developed a ratiometric imaging method to quantify the level of specific versus nonspecific binding of molecularly targeted contrast agents.

CONCLUSION

This technique will allow surgeons to unambiguously distinguish between normal and cancerous tissues for chemically specific and spatially precise tumor debulking.

Neuro-Oncol. 2009 Oct;11(5):675.

487. Kinetics of α-[C-11]Methyl-L-tryptophan in Low-Grade Brain Tumors: A Dynamic PET Study

Csaba Juhasz, Sandeep Mittal, Sandeep Sood, Diane C Chugani, Harry T Chugani, Pulak Chakraborty, Geoffrey R Barger

INTRODUCTION

α-[C-11]Methyl-L-tryptophan (AMT) is a positron emission tomography (PET) tracer for tryptophan catabolism via the immunomodulatory kynurenine pathway. Previous studies demonstrated increased AMT uptake in brain tumors of various grades. In the present study, we analyzed tracer kinetics of AMT in low-grade brain tumors to better understand mechanisms of increased tryptophan accumulation and to determine whether tumor types can be differentiated by AMT kinetics.

METHODS

Twenty-five patients (mean age, 29 years) underwent dynamic AMT PET scanning before tumor resection. Grade I–II tumors included 15 gliomas (five astrocytomas, seven oligodendrogliomas, three mixed gliomas) and 10 glioneuronal tumors (seven DNETs, three gangliogliomas); 13 tumors showed gadolinium enhancement on magnetic resonance imaging (MRI). FDG PET done in 20 patients showed decreased tumor metabolism in 18 and increased metabolism in two. Kinetic AMT parameters were determined by the Patlak graphical approach and included the unidirectional uptake rate (K), the transport rate (estimated by the volume of distribution, VD), and the calculated metabolic rate (k3 = K/VD). Increased AMT uptake was defined by a tumor/cortex ratio > 1.10.

RESULTS

AMT PET showed increased uptake in 23/25 cases (92%), including 10/12 tumors with no gadolinium enhancement. No increases were seen in an oligodendroglioma and a DNET. In the 23 AMT-positive cases, increased AMT uptake was due to increased transport (VD; n = 17) and/or increased unidirectional uptake (K; n = 18). High metabolic rates (k3) were found in 14 cases. AMT kinetic parameters were similar in contrast-enhancing versus nonenhancing tumors. Pure astrocytomas showed higher tumor/cortex k3 ratios (mean, 1.66) than other gliomas (mean, 0.99; p = 0.008) and glioneuronal tumors (mean, 0.80; p = 0.026).

CONCLUSION

AMT PET identifies most low-grade gliomas and glioneuronal tumors by increased uptake, regardless of contrast enhancement and FDG uptake. Increased AMT uptake can occur because of increased tryptophan transport and/or high metabolic rate; the latter may differentiate pure astrocytomas from other low-grade gliomas and glioneuronal tumors.

Neuro-Oncol. 2009 Oct;11(5):675.

488. Imaging and Biologic Correlates in a Phase II Study of Bevacizumab (BVZ) 1 Irinotecan (CPT-11) in Children with Recurrent Malignant Gliomas and Diffuse Brainstem Gliomas (PBTC-022)

Tina Young Poussaint, Sridharan Gururangan, Richard J Gilbertson, Arzu Onar, Sridhar Vajapeyam, James Boyett, Larry Kun

INTRODUCTION

We recently completed a phase II study of bevacizumab (BVZ) + (CPT-11) in children with recurrent malignant glioma (Stratum A) and diffuse brainstem glioma (Stratum B). We report imaging and biologic correlates.

METHODS

BVZ was given on day 1 and day 15 followed by BVZ+CPT-11 every 2 weeks until disease progression, unacceptable toxicity, or 2-year duration. MR T1 permeability, T2* perfusion and diffusion were documented at baseline and day 15 and every 2–3 months during the first year. Estimates of VEGF-R2 phosphorylation (pVEGFR2) in peripheral blood mononuclear cells (PBMC) before and after two doses of BVZ were correlated with MR parameters.

RESULTS

The 31 evaluable patients (age range, 2.9–20.1 years) had no sustained objective responses. Baseline CBVmax was higher in Stratum A (n = 8) than in Stratum B (n = 7) (p = 0.016). Changes in enhancement, CBVmax, and Kps-max between baseline and after two doses of BVZ were not significant (Stratum A, median change CBVmax = − 0.45 [p = 1.0], Kps-max = − 0.79 [p = 0.73]; Stratum B, median change CBVmax = − 1.28 [p = 1.0], Kps-max = − 3.1 [p = 0.45]). There was no correlation among MR parameters and PFS. Median diffusion values (DV) decreased between baseline and day 15 for Stratum A (n = 17; p = 0.001) but not Stratum B (n = 14; p = 0.083). DV decreased during the first four courses for Stratum A (p = 0.004) but not Stratum B (p = 0.6). No significant increase in nonenhancing infiltrating FLAIR signal at progression was observed. PBMC pVEGFR2 levels decreased in eight patients after BVZ and increased in two (overall slope, − 0.2004; p = 0.3701). Small numbers of biology and permeability samples limited further analyses.

CONCLUSION

There were no significant changes in permeability parameters in either stratum following BVZ ± CPT-11. A decrease in diffusion in Stratum A between the pretreatment and day 15 MR scans and over four courses of therapy reflects increased intratumoral cellularity and decreased edema.

Neuro-Oncol. 2009 Oct;11(5):676.

489. Optimization of Glioblastoma Multiforme Stem Cell Isolation and Transfection

Robel T Beyene, Demirkan Gursel, Brian Howard, Margarita Arango, Michael Gordon Kaplitt, John A Boockvar

INTRODUCTION

Brain tumor stem cells (TSCs) are thought to be the tumor initiating population responsible for glioblastoma multiforme (GBM), and evidence increasingly suggests that they are the ideal cell population for in vitro study of GBMs. High-yield generation of these stem cells, which form tumor spheres when grown in culture, from gross tumor patient tissue can be difficult. Once tumor spheres are isolated, successful transfection and infection, especially in the center of the spheres, has not been previously described. The purpose of this study was to develop an optimized protocol for the generation of TSCs and their subsequent transfection or infection.

METHODS

GBM patient tissue was collected and enzymatically and mechanically dissociated; red blood cells (RBCs) and dead cells were removed before remaining live cells were moved to stem cell media to generate tumor spheres. These cells were then analyzed to ensure they met the stem cell criteria. Some of these cells were then either transfected with plasmids carrying genes that encode fluorescent proteins or infected with adeno-associated virus (AAV) carrying PTEN. Live spheres and cryosections of embedded spheres were analyzed to establish successful transfection or infection.

RESULTS

Meticulous mechanical and enzymatic dissociation of gross patient GBM tissue, followed by the removal of RBCs and dead cells, was able to produce a high yield of tumor stem cells. A large portion of these cells were found to meet the criteria to designate them stem cells, such as multipotentiality and self-renewal. Plasmid transfection and AAV infection of tumor spheres was successful using techniques previously used on isolated cells, and cryosectioning showed that these procedures were effective even at the center of the neurospheres.

CONCLUSION

This study can be used as a methodological guide to optimize the generation of tumor stem cells from patient gross GBM tissue and to show that GBM tumor spheres can be transfected with plasmids or infected with viruses. This may be important from a therapeutic perspective if gene therapy is successful in replacing genes lost in GBM progression or knockdown genes that are overexpressed.

Neuro-Oncol. 2009 Oct;11(5):676.

490. A Self-Assembling IKVAV Nanogel for the Treatment of GBM

Sunit Das, Maya Srikanth, Samuel I Stupp, John A Kessler

INTRODUCTION

Brain tumor stem cells (BTSCs) represent a small proportion of all glioma cells but are likely responsible for tumor recurrence and progression. In addition, the treatment of primary brain tumors is complicated by factors intrinsic to the location of tumor cells within the brain parenchyma. One approach to developing new therapies against BTSCs is to activate biological pathways that drive these stem-like cells to differentiate and presumably exit the cell cycle (i.e., differentiation therapy). Activation of the β-integrin signaling pathway appears to exert a particularly profound role in driving NSCs to undergo neuronal differentiation. Toward that end, we have designed a self-assembling, peptide amphiphile (PA) nanogel that can be safely delivered into the brain parenchyma to deliver a β-integrin signal to BTSCs.

METHODS

In collaboration with the lab of Dr. Sam Stupp (Northwestern University), we developed a self-assembling nanofiber gel conjugated to an IKVAV epitope. BTSCs isolated from patients undergoing craniotomy for resection of GBM at Northwestern Memorial Hospital were treated in vitro with the IKVAV-PA nanogel. A nanogel conjugated to a scrambled, biologically inactive epitope was used as a control.

RESULTS

Treatment of BTSCs with the IKVAV-PA caused these cells to extend processes that are immunopositive for Tuj1 and express the nuclear protein NeuN. In addition, IKVAV PA-treated BTSCs no longer expressed nestin and Sox2.

CONCLUSION

Treatment of BTSCs with the IKVAV-PA nanogel results in expression of neuronal markers and loss of markers of stem cell-like phenotype. We are currently investigating whether these cells truly undergo terminal differentiation and whether differentiation renders these cells more sensitive to DNA damage secondary to chemotherapy and radiation. We propose that the nanofiber gel may be an effective adjuvant therapy in patients with GBM.

Neuro-Oncol. 2009 Oct;11(5):676.

491. Notch Inhibition Reduces Glioblastoma Radioresistance by Targeting Cancer Stem Cells

Koos E Hovinga, Rong Wang, Hamideh Moayedpardazi, Fumiko Shimizu, Jayanthi Menon, Tamara Major, Viviane S Tabar

INTRODUCTION

Notch signaling is crucial for stem cell self-renewal and cell fate determination in the CNS. Stem-like cells have been described in glioblastoma (GBM). These “cancer stem cells” (CSC) share properties with normal stem cells, such as self-replication through asymmetric cell division, very slow proliferation rates, and radioresistance. We asked whether Notch inhibition can reduce GBM radioresistance by modulating the “stemness” of CSCs and coaxing them into a progenitor-like state, thus sensitizing them to radiation.

METHODS

To maintain the CSCs in their original niche, we placed GBM obtained from surgical specimens in a 3-D organotypic culture (explants). Explants were treated with the Notch inhibitor DAPT, radiation, or a combination thereof. Proliferation, neurosphere forming capacity, and the percentage of CD133+ cells were assessed.

RESULTS

The explant model maintained the cytoarchitecture of GBM and its high proliferation rate. The radioresistant phenotype was preserved in the explants, as shown by a decrease in BrdU uptake in the immediate posttreatment phase but a recovery to near original proliferation rates after 5 days. Neurosphere formation, a test used as a surrogate for CSC function, decreased dramatically after Notch inhibition (82.3% decrease; p < 0.0018) and was associated with a decrease in CD133+ cells as demonstrated by FACS analysis (50% decrease). The combination of radiation and DAPT resulted in a profound decrease in proliferation (Ki67 = 3.4% vs. control [14.2%; p = 0.0004] or vs. separate treatments [7.2% and 7.3% DAPT and radiation resp.]) and in neurosphere formation (71.6% decrease; p < 0.014).

CONCLUSION

Our data demonstrates that the combination of Notch inhibition and radiation exerts a significant antiproliferative effect on GBM. This is likely due to a selective decrease in the stem cell population within the tumor, as demonstrated by a decrease in neurosphere formation and CD133. Preclinical studies are ongoing.

Neuro-Oncol. 2009 Oct;11(5):676.

492. Hypoxia-Induced Upregulation of Erythropoietin is Related to Glioma Driven Recruitment of Mesenchymal Stem Cells

Florian Faber, Kim Garnier, Valerie Albrecht, Ketai Guo, Jörg-Christian Tonn, Christian Schichor

INTRODUCTION

Necrosis and hypoxia are critical processes during the growth of high-grade gliomas. Moreover, emerging data suggest that mesenchymal stem cells (MSC) may contribute to tumorigenesis in glioblastoma. The aim of the study was to characterize the hypoxia-inducible hormone erythropoietin (EPO) and erythropoietin receptor (EPOR) in glioma isolated mesenchymal stem cells and glioma isolated endothelial cells (EC) under hypoxic conditions.

METHODS

We performed consecutive immunohistochemical studies of HIF1-α and EPOR in 10 glioblastoma samples. We analyzed the expression of erythropoietin receptor and its ligand erythropoietin in mesenchymal stem cells and endothelial cells isolated from glioblastoma, as well as in bone marrow isolated mesenchymal stem cells and glioma cell lines under normoxic and hypoxic conditions by RT-PCR. Furthermore, we characterized their EPO-induced migratory behavior by a modified Boyden chamber assay.

RESULTS

HIF1-α as a marker for hypoxia and EPOR were observed frequently in isolated glioblastoma samples. EPO and EPOR expression was detected heterogeneously and increased under hypoxic conditions in glioma cell lines, but particularly in tumor- and bone marrow-isolated MSCs and also in tumor-isolated ECs. Erythropoietin showed a promigratory effect on tumor- isolated MSCs as well as on bone marrow-isolated MSCs. In addition, the hypoxia-induced upregulation of EPOR was conducive to the migration toward the EPO-gradient.

CONCLUSION

Hypoxia-inducible expression of EPO and EPOR is not only limited to glioma cells but mainly upregulated in MSCs isolated from glioblastomas and bone marrow. Furthermore, our results suggest that EPO is an essential hypoxia related recruitment factor in gliomas. These data support the hypothesis that gliomas use physiological pathways related to hypoxic conditions in order to recruit mesenchymal progenitor cells.

Neuro-Oncol. 2009 Oct;11(5):676–677.

493. The Effect of Bone Morphogenetic Protein 4 on Glioblastoma Stem Cells and its Implication for Therapy

Hiroaki Wakimoto, Sasa Jereb, Toshihiko Kuroda, Santosh Kesari, Robert L Martuza, Samuel Rabkin

INTRODUCTION

Glioblastoma stem cells (GSC) or glioblastoma-initiating cells represent a critical target for successful glioma therapy. The ability to differentiate is one of the characteristics of GSC and provides an opportunity for therapeutic intervention. Bone morphogenetic protein 4 (BMP4), a member of TGF-β superfamily, has been shown to induce differentiation in GSC.

METHODS

Recombinant BMP4, ciliary neurotrophic factor (CNTF), and brain-derived neurotrophic factor (BDNF), cytokines that play a role in neural development, were tested for their ability to affect proliferation, self-renewal, and cell fate in two human glioblastoma-derived GSC lines, GBM8 and BT74, in vitro. The effect of BMP4 on the tumorigenicity of GSC was examined using an orthotopic xenograft assay in mice.

RESULTS

BMP4 suppressed proliferation of GBM8 and BT74, and CNTF inhibited growth of BT74 but not GBM8, while BDNF had no effect. Self-renewal, assessed by neurosphere formation, was disrupted by the exposure of GSC to BMP4 and/or CNTF. Treatment of GSC with BMP4 significantly reduced the fraction of CD133-positive cells. Immunocytochemistry and quantitative RT-PCR revealed that both BMP4 and CNTF upregulate the expression of glial fibrillary acidic protein (GFAP), indicating that the cytokines mediate induction of differentiation towards an astrocytic lineage. Intracerebral implantation of highly tumorigenic GBM8 pretreated with BMP4 resulted in impaired aggressive behavior in vivo, with the tumors derived from BMP4-treated cells exhibiting a marked reduction in the Ki-67 proliferation index compared to untreated tumor cells.

CONCLUSION

This study shows that BMP4 can abrogate the stem-like phenotype of GSC by either repressing their self-renewal or forcing astrocytic differentiation, thus providing an insight into the biology of GSC. Furthermore, this study suggests that local administration of BMP4 or its in situ production by gene therapy vectors may represent a promising strategy to treat glioblastoma.

Neuro-Oncol. 2009 Oct;11(5):677.

494. Therapeutic Application of CD1331 Glioma Tumor Stem Cells

Yung-Hsiao Chiang, Kai-Yun Chen

INTRODUCTION

Malignant gliomas are common primary tumors seen in the brain. There are very effective treatments available to control the disease. Even though temozolomide showed its effectiveness to prolong a patient’s life after diagnosis of malignant gliomas, the time of disease control is still limited. Tumors will recur and take the patient’s life. Recently, studies support cancer stem cells in gliomas that are functionally defined by their capacity for self-renewal, and tumor formation might be responsible for tumor maintenance and progression. Here we hypothesized that depletion of glioma stem cells (GSC) may control the growth of malignant glioma, and we offer a potential way to treat glioma.

METHODS

CD133 was recently identified as an important cancer stem cell marker, with increased expression in various human malignancies, including glioma. We isolated CD133 positive glioma stem cells (GSC-CD133+) from brain tumor specimens by the magnetic sorting method. By real-time PCR, we examined the expression of embryonic stem cell markers (Oct-4, Nanog, Sox-2, Klf-4, and c-Myc). To investigate the changes of GSC cell biology by different CD133 expression in vitro and in vivo, we used a lentiviral vector expressing shRNA to knockdown CD133 expression (shCD133). We further injected CD133 knockdown GSC-CD133+ into the brains of nude mice to verify their tumorigenicity.

RESULTS

We successfully isolated GSC-CD133+ and demonstrated that GSC-CD133+ are capable of self-renewal and express high levels of embryonic stem cell markers. Silencing of CD133 in GSC-CD133+ significantly reduced proliferation about 40–50% within 3 days, with decreasing expression of XIAP, Bcl-2, and c-IAP and increasing expression of ATG5, Beclin, and LC3. Importantly, GSCs with decreased CD133 levels have lower tumorigenicity when xenotransplanted into the brains of nude mice.

CONCLUSION

These results support an important role of CD133 in regulating proliferation and survival of GSCs. Targeting CD133 may offer improved therapeutic approaches for gliomas.

Neuro-Oncol. 2009 Oct;11(5):677.

495. A New Protocol for Isolation Emanated from the Integrative Analysis of Publicly Available Gene Expression Data: Mesenchymal Stem Cells Contribute to Glioblastoma

Valerie Albrecht, Ketai Guo, Kathrin Juerchott, Roland Goldbrunner, Jörg-Christian Tonn, Christian Schichor

INTRODUCTION

Malignant gliomas have been described as heterogeneous tumors that recruit a broad variety of host progenitor cells. Analyzing selected publicly available array data for glioblastoma (GBM) and brain and mesenchymal stem cells, we identified high positive correlation in gene expression for glioblastoma and bone marrow-derived mesenchymal stromal cells (bmMSCs). It was emphasized that hMSCs are incorporated in glioma tissue. Purification, cultivation, and characterization of MSCs from glioblastoma tissue (gbMSCs) are prerequisites for further analysis. Therefore, we established a new protocol for isolation and purification of gbMSCs from glioblastoma tissue.

METHODS

Publicly available expression data generated in individual experiments with normal brain, glioblastoma, and bmMSC material on Affymetrix hg U133 Plus 2.0 arrays were integrated in a combined expression matrix. The correlations of the rank-transformed expression profiles of the three groups were analyzed. A new protocol for isolation of gbMSCs was designed, using a gradient-based isolation technique combined with magnetic bead negative sorting. The isolated cells (gbMSCs) were characterized, using extensive FACS analysis. Multipotency of the cells was confirmed via differentiation assays.

RESULTS

The correlation of the glioblastoma samples to the bmMSC samples was significantly higher than the correlation of the normal brain group to the bmMSC samples. Sixty-six probe sets with high expression in bmMSC and low expression in brain were identified. The majority of probes (67%) showed significantly higher expression in glioblastoma tissue compared to normal brain. FACS analysis of gbMSCs displayed close similarities to bmMSCs. Differentiation assays showed the osteogenic and adipogenic differentiation potential of gbMSCs, thereby displaying multi-potency.

CONCLUSION

Our new approach of comparative analysis of publicly available expression data sets showed close similarities between mesenchymal stem cells and glioblastoma tissue. Therefore, we established a new protocol for isolation and expansion of mesenchymal stem cells from tumor tissue, demonstrating the presence of gbMSCs in malignant gliomas.

Neuro-Oncol. 2009 Oct;11(5):677.

496. Trim3, the Human Homolog of Drosophila Brat, Regulates Neural Differentiation and Myc Expression and May Control Glioma Stem Cells Properties

Gang Chen, Xiang Fan, Fahmia Rahman, Yuan Rong, Carol Tucker-Burden, Scott Dougan, Costas George Hadjipanayis, Erwin Van Meir, Daniel Brat

INTRODUCTION

Glioblastoma (GBM) is the most common and malignant primary brain tumor. A CD133+ stem cell compartment has been described, yet mechanisms underlying its specialized properties are unknown. Drosophila brain tumor (brat) directs asymmetric neuroblast division and guides neural differentiation at least partially through its translational suppression of Myc. Mutations in brat result in a massively enlarged fly brain, consisting of a neuroblast cell population with neoplastic growth properties. We investigated the role of brat homologs in directing zebra fish neural differentiation and in human GBMs for a potential role in modulating c-Myc and stem cell properties.

METHODS

We used morpholino oligo in Zebra fish, in situ hybridization, RT-PCR, Western blot, and bioinformatics.

RESULTS

In zebra fish, we used a morpholino oligo directed at the brat homolog Trim3b and found a CNS phenotype characterized by an enlarged head, ventricular expansion, slightly fewer overall cells, and an enrichment of neuroblastic cells lacking differentiation. In situ hybridization for Notch 1a and nestin showed modestly reduced expression compared to age-matched controls, consistent with a lower cell number. However, GFAP and Hu were far more reduced, suggesting impaired differentiation. We found that the mRNA expression of the human homolog of brat, Trim3, was markedly reduced in human GBM cell lines and specimens compared to astrocytes and normal brain, respectively, consistent with mining of gene expression data sets (oncomine). Since Drosophila brat guides neural differentiation through its repression of Myc, we explored whether human Trim3 might regulate glioma c-Myc. We found that the forced expression of Trim3 in human GBM cell lines caused reduced c-Myc protein expression. This inverse relationship between Trim3 and c-Myc was also noted in GBM samples.

CONCLUSION

We suggest that Trim3 expression is reduced in human GBMs and results in upregulated c-Myc, which could potentially enrich the stem cell compartment of these neoplasms.

Neuro-Oncol. 2009 Oct;11(5):677–678.

497. Glioma Tropism by Human Umbilical Cord Blood Stem Cells Toward Glioma Cancer Cells is Dependent on Nuclear Localization of uPA

Christopher S Gondi, Bharathi Gorantla, Dzung H Dinh, Jeffrey Klopfenstein, Meena Gujrati, Jasti S Rao

INTRODUCTION

Brain metastasis is known to be dependent on multiple cell-cell and cell-matrix interactions, which allow tumor cells to form secondary tumors. Generally, adhesion molecules have been identified as crucial chemo attraction factors. These chemo attractants are also thought to be involved in mediating stem cell tropism toward gliomas. Stem cells of various lineages have been recognized as attractive vehicles to improve the delivery of therapeutic genes to glioma cells.

METHODS

CD133 and CD44 enriched U87 human glioma cells and 5,310 human glioma xenograft cells, both showing cancer stem cell properties, were used. Simultaneous expression of c-Myc and CD44 was used to identify cancer stem cells. Involvement of uPA in hUCB tropism toward glioma cells was determined by migration assay after uPA downregulation or TSA treatment. Involvement of nucleolin and nuclear localization of uPA was also determined.

RESULTS

Migration of hUCBs toward U87 normal cells was completed in 36 h and toward U87S was completed in > 24 h. Similarly, migration of hUCB-spheroids toward 5310 glioma cells was complete in 40 h and toward 5310S cells in > 30 h. hUCB-spheroids downregulated for uPA showed decreased migration toward U87 cells by 68 ± 3% and toward U87S by 56 ± 3%. In case, 5310 migration was reduced by 62 ± 5% and 5310S by 58 ± 3%. Brain slice culture studies showed similar reduction of migration by hUCBs after uPA-downregulation (U87 = 59 ± 2%; U87S = 68 ± 6%; 5310 = 68 ± 8%; 5310S = 73 ± 6%). TSA treatment caused increased migration in both in vitro (U87 = 123 ± 2%; U87S = 135 ± 3%; 5310 = 130 ± 4%; 5310S = 126 ± 6%) and ex vivo (U87 = 113 ± 6%; U87S = 145 ± 3%; 5310 = 122 ± 4%; 5310S = 136 ± 6%) conditions. Nucleolin downregulation inhibited uPA nuclear localization and completely inhibited hUCB migration in both in vitro and ex vivo conditions. Interaction of uPA with CREB1 transcription-factor was also observed.

CONCLUSION

Nuclear localization of uPA is necessary for hUCB glioma tropism.

Neuro-Oncol. 2009 Oct;11(5):678.

498. Adipose-Derived Stem Cells as Therapeutic Delivery Vehicles of Myxoma Virus for Glioblastoma Multiforme (GBM)

Fernanda Dreher, Dongqin Zhu, Darnell Josiah, Michael Callahan, Grant McFadden, Hannah Caldas

INTRODUCTION

The overall prognosis for GBM has changed little in the past two decades, despite major improvements in neuroimaging, neurosurgery, and radiation treatment techniques. Treatment difficulty is due to GBM’s exceptional migratory nature and penchant to integrate extensively into normal brain tissue. Long-term survivors are very rare, and median survival for patients with GBM is only about 1 year. Adult stem cells, such as those derived from mesenchymal tissues, have the ability to home to cancer cells and tumors. Myxoma virus, a poxvirus that is highly rabbit specific, displays potent oncolytic activity towards human malignant gliomas; however, it suffers from limited biodistribution, greatly limiting its clinical application. GBM cells are not constrained only to the radiographically visible tumor mass, and tumor cells can be some distance away from the main mass, even to the opposite cerebral hemisphere. Collectively, mesenchymal stem cell’s ability to home to cancer cells while transporting myxoma virus may represent a novel strategic therapeutic solution for GBM.

METHODS

Adipose-derived stem cells were infected with myxoma virus and injected into tumor-bearing animals via internal carotid injection. The animals were followed until an experimental end point at which time brains were removed and analyzed histologically.

RESULTS

Adipose-derived stem cells successfully delivered myxoma virus to brain tumors after systemic delivery, resulting in a decrease in tumor size and extending the life span of treated animals.

CONCLUSION

Adipose-derived stem cells are promising therapeutic vectors to deliver oncolytic myxoma virus to GBM and has the potential to be translated into a novel brain tumor-targeted therapy.

Neuro-Oncol. 2009 Oct;11(5):678.

499. CD133 Cells in Glioblastomas are Multipotent and Generate Tumor Endothelium

Rong Wang, Koos E Hovinga, Jayanthi Menon, Viviane S Tabar

INTRODUCTION

The origin of endothelial cells within glioblastomas remains unclear. Studies have suggested the bone marrow or circulating endothelial progenitors as a potential source of recruitment to tumor neoangiogenesis. However, the extent, if any, of contribution from these sources is probably small. Here we demonstrate that endothelial cells within glioblastoma tissue harbor the same genetic aberrations as the tumor cell population, raising the possibility of a shared lineage.

METHODS

Freshly obtained surgical specimens of GBM were used.

RESULTS

Analysis of the putative cancer stem cell (CD133+) fraction demonstrated the presence of endothelial progenitor markers. In vitro and in vivo data demonstrated that CD133+ cells exhibit remarkable plasticity by differentiating into glioma cells, endothelial cells, and pericytes. The transition from CD133+ to a mature endothelial cell occurs via a tumor endothelial progenitor characterized by the concomitant expression of vascular E-cadherin and CD133. CGH analysis shows identical genetic aberrations in CD133+, CD133+/VE-cadherin+, and the more mature endothelial (CD105+) cell fractions. This is further confirmed by FISH and RT-qPCR for several exons within the EGFR amplicon, which confirms the presence of identical genomic changes in cancer stem cells, endothelial progenitors, and the tumor cell population. Karyotype analysis ruled out cell fusion as a potential explanation of endothelial marker expression by tumor cells. Our data also suggest that the transition from cancer stem cell to endothelial progenitor is VEGF-independent and not responsive to bevacizumab.

CONCLUSION

This data demonstrates that cancer stem cells within a solid tumor harbor progenitors capable of generating tumor vasculature and pericytes. The findings have significant implications to the biology of solid tumors, angiogenesis, and therapeutic targeting. This data highlights a novel aspect of cancer biology that ties tumor stem cells directly to angiogenesis. It also addresses a previously unresolved question as to the origin of endothelial cells within a glioblastoma.

Neuro-Oncol. 2009 Oct;11(5):678.

500. Glioma Cells and Adult Human Mesenchymal Stem Cells form a Structural as well as a Functional Syncytium in vitro

Valerie Albrecht, Benjamin Korte, Alexander Buchner, Rainer Riesenberg, Josef Mysliewitz, Igor Paron, Jörg-Christian Tonn, Christian Schichor

INTRODUCTION

Human mesenchymal stem cells (hMSCs) have come into focus as a source for cell-based treatment strategies in glioma patients. The capacity of hMSCs as a treatment vector is based on their property of being recruited to malignant gliomas. The recruitment mechanism is driven by glioma-secreted factors that lead to accumulation of both administered as well as bone marrow derived hMSCs within the tumor.

METHODS

The expression of connexin 43 was detected with Alexa488 labeled anti-Cx43 antibody. Proteins were separated by SDS-PAGE and visualized by immunoblotting. hMSCs and U87 glioma cells were stained with different fluorescent dyes (Vybrant DiI or DiO, rhodamine, calcein) and cocultured from 30 min to 48 h. Inhibition of gap junction coupling was performed by treating cells with increasing concentrations of AGA and measuring calcine-positive glioma cells per hMSC.

RESULTS

Based on the finding that glioma cells and hMSCs express connexons, we found an intense interaction via gap-junction coupling. Besides this functional syncytium formation, we detected signs of cell fusion events, also referred to as structural syncytium. These cell-cellular interactions resulted in markedly enhanced migration in vitro. Taken together, our data show that glioma cells display signs of functional as well as structural syncytium formation with MSCs in vitro.

CONCLUSION

Our results show that hMSCs form a gap junction-mediated functional syncytium as well as a fusion-mediated structural syncytium with malignant glioma cells. Syncytium formation led to increased migration on extracellular matrix-components. Characterization of fused cells on a genetic level and in vivo investigations should be subject to further studies. Nevertheless, this phenomenon should be taken into account if data on glioma-stem cell interaction have to be interpreted.

Neuro-Oncol. 2009 Oct;11(5):678.

501. Tumor Initiation is Independent of Differentiation in Glioblastoma Stem Cells

Veerakumar Balasubramaniyan, Brian Vaillant, Shuzhen Wang, Krishna Bhat, Joy Gumin, Ke Sai, Jun Yao, Frederick F Lang, Kenneth D Aldape, Howard Colman

INTRODUCTION

Prior data indicates that glioblastoma tumors with a “mesenchymal/angiogenic” gene expression phenotype have a worse prognosis than those with a more “proneural” profile. Recent findings have highlighted the importance of tumor stem cells in the initiation and treatment resistance of glioblastoma. One of the hallmarks of tumor stem cells is plasticity in differentiation. However, the relationship between glioblastoma stem cell (GSC) differentiation capacity and tumor molecular phenotype remains unclear. The current study investigated the effects of differentiation on the GSC molecular phenotype and tumor initiation capacity.

METHODS

Four GSCs isolated from human GBM tumors and maintained as neurosphere cultures were analyzed for self-renewal and tumorigenicity after intracranial injection. Differentiation of GSCs was induced by removal of growth factors, addition of retinoic acid, or addition of FBS (2% or 10%). Global gene expression profiling was performed using the Affymetrix U133 Plus 2.0.

RESULTS

All control GSCs demonstrated the key properties of self-renewal, multilineage differentiation, and tumor initiation. After differentiation for 10–14 days, GSCs expressed a combination of stem cell and multiple neural lineage markers, indicating an aberrant differentiation capacity compared with normal neural stem cells. Surprisingly, both neurosphere and tumor initiation capacity were maintained after both short-term and long-term differentiation. Furthermore, long-term differentiation (>40 days) of GSCs resulted in intracranial xenograft tumors with a significantly shorter median survival time than control (undifferentiated) GSCs (p < 0.005; log-rank). Long-term differentiation was also associated with an increase in expression (>2-fold) of multiple mesenchymal genes and functional groups (p < 0.01; DAVID) that were also increased in human GBM tumors with poor survival.

CONCLUSION

These data demonstrate that differentiation in GSCs is aberrant and does not block tumorigenicity. In addition, long-term differentiation of GSCs results in more aggressive tumors and increased mesenchymal gene expression in a pattern similar to that observed in resistant human tumors.

Neuro-Oncol. 2009 Oct;11(5):679.

502. Hypoxia Increases the Expression of the Stem Cell Markers Side Population and CD133 and Promotes Clonogenicity in Tumor Neurospheres

Charles G Eberhart, Alex Y Lin, William Matsui, Sara GM Piccirillo, Angelo L Vescovi, Eli E Bar

INTRODUCTION

Lack of oxygen promotes the expansion of non-neoplastic stem/precursor cell populations in the brain; therefore, we examined the potential role of hypoxia in supporting cancer stem cells (CSC) in gliomas. A full understanding of how hypoxia regulates CSC biology may be critical if we are to therapeutically target stem-like tumor cells and their niche/microenvironment.

METHODS

See results.

RESULTS

We found that when GBM-derived neurosphere cultures were grown in 1% oxygen for 3 days, HIF1-α protein levels increased dramatically, and mRNA encoding of other hypoxic response genes, such as HIG2, LOX, and VEGF, was induced more than 10-fold. Interestingly, hypoxia exposure increased the percentage of side population-positive cells from 0.8% in normoxia to 5.8% under hypoxia. In addition, mRNA levels of the stem cell marker CD133 increased more than 5-fold, and the percentage of CD133-positive cells measured by flow cytometry was induced 2-fold. This rise in CD133-positive fraction was paralleled by an approximately 2-fold increase in clonogenicity when single cells previously grown in hypoxic conditions were seeded in methylcellulose. We believe these CSC changes were HIF1-dependant, as when stabilized HIF1-α is expressed in normoxic glioma cells CD133 protein levels are induced. It has recently been shown that the drug digoxin can lower HIF1 protein levels in vitro and in vivo. In order to determine if digoxin might be useful in suppressing the progrowth hypoxic response in brain tumors, we used it to treat a number of malignant brain tumor cell lines. We found 100 nM of digoxin was able to suppress HIF1-α protein levels and also significantly slowed cell growth in vitro. Finally, intraperitoneal injections of digoxin (2 mg/kg/day) was able to significantly slow the growth of subcutaneous glioblastoma xenografts in nude mice.

CONCLUSION

These data suggest that agents targeting HIF factors may represent effective novel glioblastoma therapies and may regulate the fraction of stem-like cancer cells.

Neuro-Oncol. 2009 Oct;11(5):679.

503. Resistance of Glioblastoma Cancer Stem Cells to TGF-Beta Signaling

Praveen Kumar, Christoph P Beier, Ines Aschenbrenner, Dagmar Beier

INTRODUCTION

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor and has dismal prognosis despite multimodal therapy. GBMs are maintained by a rare subpopulation of cells referred to as cancer stem cells (CSCs), which can be propagated in vitro with a remarkable genetic and transcriptional stability. CSC lines therefore serve as a powerful tool to investigate the tumor characteristics in vitro. Transforming growth factor (TGF)-β plays a major and rather controversial role in tumor development and progression. While it acts as a tumor suppressor factor in the initial stages of tumor development, there is complete role reversal in the later stages, wherein TGF-β provides oncogenic cues by modulating immunosuppression, invasion, metastasis, and angiogenesis. The role of TGF-β has been investigated in normal stem cells, but its role in cancer stem cells is still elusive. We have, therefore, investigated the effects of TGF-β in nine different CD133+ and CD133- CSC lines.

METHODS

Microarray, Western blot, RT-PCR, immunocytochemistry, FACS, clonogenicity assay, and ELISA.

RESULTS

The CSC lines show a heterogeneous response pattern with resistant CSC lines (six of nine) and CSC lines in which TFG-β induced proliferation (one out of 9) or growth arrest of progenitor cells (two out of nine). Notably, TGF-β did not change the differentiation capacity, the self-renewal, or the expression of stem cell markers in all cell lines. In addition, we compared the transcriptional profiles of responding and nonresponding CSC and identified the loss of TGF-β receptor 2 as a crucial factor for the development of TGF-β resistance.

CONCLUSION

Although a heterogeneous effect of TGF-β towards CSC lines was expected, the resistance of CSC against TGF-β suggests that TGF-β acts mainly on the tumor stroma rather than on GBM CSC itself.

Neuro-Oncol. 2009 Oct;11(5):679.

504. Role of Micrornas in Regulating Aggressive Brain Tumor-Initiating Cancer Stem Cells and Tumor Progression

Kamalakannan Palanichamy, Deva Sanjeeva Jeyaretna, Shin-Ei Noda, Michael Siedow, Tim Lautenschlaeger, Sean Lawler, Arnab Chakravarti

INTRODUCTION

Glioblastoma multiforme is a neoplasm of the CNS with a median survival of about 15 months with the standard care of surgery (depending on feasibility), followed by radiation and chemotherapy. Resistance to treatment represents a major obstacle to recovery. It is becoming better understood now that local relapse, distant metastasis, and recurrence are due to a small subpopulation of cells within the tumor, referred to as brain tumor initiating cancer stem cells (CSCs). Our continuing investigation on identifying the molecular and cellular mechanisms underlying the radiation and chemotherapy resistance in gliomas has led to the current work.

METHODS

The microRNAs were isolated using triazol and mirvana extraction kits and were analyzed using the SA-Bioscience cancer MiRNA array. The miRNA expressions were further validated using TaqMan probes. We are currently comparing this data with applied biosystems TILDA cards Sanger miRBase v. 10.

RESULTS

We isolated the CSC subcompartment from the tumor mass. Growth factor responsive neurosphere (CSCs) and differentiated cells obtained under serum conditions were characterized for their radiation and chemotherapy sensitivities and analyzed for their transcriptomic and proteomic profiles. MicroRNA analysis of the CSC and mature cells showed differential regulation of tumor suppressor and oncogenic microRNAs. Our studies showed marked downregulation of tumor suppressors and upregulation of oncogenes, leading to an enhanced oncogenic activity in CSCs. Surprisingly, when the differentiated cells obtained from the same tumor were analyzed, the results were opposite, confirming that these CSCs possess enhanced tumorigenicity compared to the differentiated phenotype. Further transcriptomic and proteomic profiling showed a higher expression of chemokine receptors, leading to increased metastatic potential and prosurvival proteins, such as inhibition of apoptosis, Akt, etc., in the CSCs when compared to differentiated cells. It is noteworthy to mention that CSCs are consistently capable of inducing tumors upon xenotransplantation that resemble the original malignancies with as few as 50 cells.

CONCLUSION

The results show that the occurrence of miRNA in glioblastoma CSCs opens new avenues for understanding of oncogenesis and, likely, for management of glioblastomas. Further studies are under way to exploit these targets for therapeutic benefit.

Neuro-Oncol. 2009 Oct;11(5):679.

505. Sox2 Plays a Pivotal Role in the Self-Renewal Property of Brain Tumor Stem Cells

Marta M Alonso, Dan Liu, Jing Xu, Sudheer K Pabbissety, Howard Colman, WK Alfred Yung, Juan Fueyo, Candelaria Gomez-Manzano

INTRODUCTION

Brain tumor stem cells (BTSCs), recently described as a key population in brain tumors, possess traits reminiscent of those ascribed to normal stem cells, such as the property of self-renewal. This intrinsic property of normal neural stem cells is regulated by a plethora of factors. Among these factors, several lines of evidence suggest that Sox2 maintains the developmental potential and proliferation of neural stem cells. Interestingly, Sox2 has been found overexpressed in poorly differentiated gliomas. However, the role that this molecule plays in brain tumors remains under investigation.

METHODS

In this study, we sought to ascertain whether Sox2 is expressed in gliomas and the characterization of its potential role in the control of self-renewal in BTSCs.

RESULTS

Our results showed Sox2 expression in human surgical malignant gliomas. Importantly, Sox 2 expression was significantly higher in the BTSC lines than in established glioma cell lines. Downmodulation of Sox2 in BTSCs by transfection-specific siRNA resulted in the loss of self-renewal properties, as shown by the limited dilution assay. Moreover, we observed a modification in the pattern of expression of markers related to stem cells properties (e.g., Nanog, OCT4) and differentiation (e.g., BMP4, ID2). In addition, transfer of siRNA against Sox2 in BTSCs resulted in loss of viability and reduced proliferation. Intriguingly, in established malignant glioma cell lines the modulation of Sox2 did not result in the acquisition of self-renewal capacity, even though we observed an increased proliferation rate of these cell lines.

CONCLUSION

We conclude that Sox2 is expressed in BTSCs in culture and is present in human surgical malignant gliomas. Of interest, Sox2 plays an essential role in regulating self-renewal, perhaps the most critical characteristic of stemness. Therefore, Sox2 may become a key target for the development of molecular therapies aiming to eradicate BTSCs.

Neuro-Oncol. 2009 Oct;11(5):679–680.

506. Inhibition of SRC-Family Kinases Modulates EGF Receptor Family Signaling in Glioblastoma Cancer Stem Cells

Paul A Clark, Mari Iida, Deric L Wheeler, John S Kuo

INTRODUCTION

Aberrant epidermal growth factor receptor (EGFR) signaling enhances proliferation of glioblastoma multiforme (GBM) cells. However, anti-EGFR therapies do not show clinical efficacy for GBM, implying mechanisms exist that compensate for EGFR signal inhibition. We isolated GBM cancer stem cells that grow without EGF signaling as a model for anti-EGFR resistance. Overcoming such therapeutic resistance is critical to improve effectiveness of anti-EGFR targeted therapies.

METHODS

GBM CSC lines (BT12.1, BT22) were isolated in stem media containing 50 ng/ml EGFR and basic fibroblast growth factor (bFGF) and validated for self-renewal, multipotent differentiation, and tumor initiation properties. Matched long-term GBM CSC lines were established in no growth factor (NGF) media (absent EGF and bFGF) and characterized. Control human fetal neural stem cells perish in NGF media. EGFR signaling components were analyzed from GBM CSC in standard, NGF, and ± dasatinib (SRC inhibitor) media.

RESULTS

Upregulation of activated HER2 and HER3 (EGFR family receptors), activated SRC, and downstream effectors (AKT, MAPK, PI3K) was found in NGF GBM CSC lines compared to controls on Western analysis; this may represent mechanisms to compensate for absent EGF signaling, thereby simulating resistance to anti-EGFR therapy. Since SRC family kinases are known to modulate EGFR family receptor dimerization and activate signaling, its role in NGF GBM CSC was examined via dasatinib inhibition. Dasatinib-mediated SRC inhibition prevented HER2, HER3, and downstream effector (PI3K, AKT) phosphorylation. Addition of 100 nM dasatinib decreased GBM CSC viability in NGF media by 30%–40% (p < 0.05).

CONCLUSION

GBM CSC grown without exogenous EGF models GBM that survives anti-EGFR therapies. We find compensatory activation of HER2, HER3, SRC, AKT, and MAPK in GBM CSC that likely contributes to therapeutic escape, and dasatinib-mediated SRC inhibition can overcome this mechanism. Therefore, combining SRC-inhibition and anti-EGFR therapies may be a viable GBM treatment.

Neuro-Oncol. 2009 Oct;11(5):680.

507. Neural Stem Cell-Mediated Tumor Selective Therapy for High-Grade Glioma Clinical Trials

Karen S Aboody, Jana Portnow, Joseph Najbauer, Marianne Metz, Elizabeth Garcia, Seung U Kim

INTRODUCTION

Despite aggressive chemotherapy treatments and advances in imaging, surgical, and radiation techniques, gliomas remain virtually incurable. Neural stem cells (NSCs) provide an unprecedented means to achieve tumor-selective treatment for glioma therapy because of their inherent tumor-tropic properties and potential use as vehicles for delivering therapeutic agents directly to invasive brain tumor cells. NSCs can cross the BBB, selectively target brain tumor cells, mediate tumor-localized production of antitumor agents, and limit toxicity to normal tissues. We now propose the clinical use of a well-characterized, v-myc immortalized, clonal human NSC line that has been modified to stably express the 5-fluorocytosine (5-FC) prodrug activating enzyme, cytosine deaminase, (HB1.F3.CD NSCs), which we postulate will localize to tumor cells and convert oral 5-FC to the active chemotherapeutic 5-fluorouracil, killing surrounding tumor cells.

METHODS

We have completed preclinical biodistribution and safety studies of this HB1.F3.CD cell line (Master Cell Bank established at COH) in immunocompromised and immunocompetent, tumor and nontumor bearing mouse models. Chromosomal and afunctional stability were assessed by karyotype, LAM-PCR, and Boyden chamber assays; CD expression by flow cytometry, immunochemistry, and LC-MS/MS.

RESULTS

Results indicate the HB1.F3.CD cell line is non-toxic, nonimmunogenic, and nontumorigenic. It was chromosomally and functionally stable. Identification of a single copy and insertion site for both CD and v-myc genes was determined. No statistically significant differences between NSC treated and control animals were found in regard to clinical observations, blood counts, liver and kidney function, or organ pathology. An IND for a first in human phase I study of NSC-mediated CD-5/FC therapy in recurrent high-grade glioma patients has been submitted to the FDA.

CONCLUSION

Clinical use of this stable and expandable NSC line will circumvent the problems associated with characterization, senescence, and replenishment sources of primary stem cell pools. We believe NSC-mediated therapy will improve clinical outcome for glioma patients.

Neuro-Oncol. 2009 Oct;11(5):680.

508. Identification of Glioblastoma Cancer Stem Cell-Specific Biomarkers using Differential Gene Expression and Microrna Array Analysis

Michael Zorniak, Raman Kutty, Clive N Svendsen, John S Kuo

INTRODUCTION

Glioblastoma (GBM) cancer stem cells (CSCs) exhibit stem-like properties, are highly efficient at forming tumor xenografts, and survive many current therapies. Identifying GBM CSC-specific molecular markers could yield clinically useful biomarkers and uncover novel therapeutic targets for treating GBM.

METHODS

Six independent human GBM CSC lines (derived from three GBMs) were isolated and validated for self-renewal, multipotent differentiation, and tumor initiation properties. These properties were maintained in either standard stem cell media (+EGF, +bFGF) or minimal stem cell media (− EGF, − bFGF), with the minimal media GBM CSCs modeling cells surviving anti-EGF therapy. Differentially upregulated, GBM CSC-specific candidate markers were identified via Affymetrix U133 Plus 2.0 gene expression and LC Sciences microRNA arrays by comparing GBM CSC ± GF, human fetal neural stem cells (hNSCs), normal brain tissue, and primary/recurrent GBM cells. Results were validated by qPCR and Western blot analysis by comparison to hNSCs, normal human astrocytes, U87 glioma cells, and matched serum-cultured GBM cells.

RESULTS

A candidate GBM-CSC-specific signature of 19 upregulated known and novel plasma membrane-associated genes has been identified. Preferential upregulation of seven plasma-membrane-linked genes was validated by qPCR. Cadherin-19 is one of seven candidate membrane proteins that showed enhanced GBM CSC-specific expression and was significantly elevated in minimal media GBM CSC lines, suggesting linkage with anti-EGF therapy resistance. In parallel, a novel microRNA signature for GBM CSCs ± GFs was also generated and is being validated for linkage to GBM CSC properties. Both strategies are yielding candidate GBM CSC molecular markers and potential oncogenic mechanisms for further investigation.

CONCLUSION

We aim to study the candidate GBM CSC plasma membrane and microRNA markers to better understand GBM CSC biology and possibly uncover novel therapeutic targets.

Neuro-Oncol. 2009 Oct;11(5):680.

509. Oncogenic EGF Receptor Variant Expressed in CNS Tumor Stem Cells can be Utilized for Targeted Cancer Stem Cell Therapy

Siddhartha S Mitra, Shuangyin Han, Albert J Wong

INTRODUCTION

Tumor stem cells (TSCs) are hypothesized to be the source of tumor initiation/relapse in malignant brain tumors. However, a tumor-specific marker has not been identified in TSCs. Previously we presented evidence that EGFRvIII, a common oncogenic alteration of the EGF receptor, is expressed in a significant fraction of the CD133+ TSC population.

METHODS

Freshly resected human brain tumors were analyzed for CD133 and EGFRvIII coexpression by FACS. Cells expressing CD133 and EGFRvIII were sorted and analyzed for self-renewal and tumorigenicity. To target EGFRvIII+CD133+ cells, we engineered a bispecific recombinant antibody (BsAb) that recognizes both CD133 and EGFRvIII and analyzed its targeting and cytotoxic specificity.

RESULTS

A limiting dilution assay showed that frequency of self-renewing cells was 1/25 for EGFRvIII+CD133+ cells, 1/40 for EGFRvIII+CD133− cells, and 1/60 for EGFRvIII-CD133+ cells. Furthermore, while typical cell culture conditions result in the loss of EGFRvIII expression, this expression was maintained under conditions that lead to tumor sphere formation. Orthotopic transplantation into NOD-SCID mice of sorted EGFRvIII+CD133+ cells was able to initiate high-grade tumor formation of 1,000 cells in 30 days whereas the EGFRvIII− CD133+ population was not. Additionally, limiting dilution analysis of two medulloblastomas and three GBM that express only EGFRvIII but not CD133 showed a high frequency of self-renewing cells in the EGFRvIII+ (1/30) compared to EGFRvIII− (1/250) cells. This strongly suggests that EGFRvIII may mark glioma stem cells even in the absence of CD133 expression. Using ELISA and live cell pull-down assays, we showed that EGFRvIII+/CD133+ cells can be specifically targeted by using the BsAb, whereas normal neural stem cells can not. Antibody mediated cytotoxic lysis of EGFRvIII+/CD133+ cells severely reduced the self-renewal potential of neurospheres.

CONCLUSION

We conclude that EGFRvIII is preferentially expressed in brain tumor stem cells, and these cells can be targeted using a recombinant BsAb approach. This BsAb is specific for cancer stem cells only, does not target normal human neural stem cells, and severely inhibits neurosphere formation in primary human tumors.

Neuro-Oncol. 2009 Oct;11(5):680–681.

510. Endogenous Sulfated Glycosphingolipids: A Potential New “Stemness” Biomarker Detected in Glioma Cancer Stem Cells

Huan He, Mark R Emmett, Carol L Nilsson, Alan G Marshall, Howard Colman, Charles A Conrad

INTRODUCTION

Relapse of glioblastoma multiforme (GBM) after initial surgical removal may be because of the presence of cancer stem cells (CSCs) that possess the ability of unlimited self-renewal and multilineage differentiation. CSCs are enriched from clinical tumor samples based on CD133 expression. The establishment of supplemental markers of “stemness” would provide better classification of CSCs. Identification of unique proteins or glycolipids expressed by CSCs of “stemness” would be valuable. Cellular polar lipid extraction followed by nano-liquid chromatography (nano-LC) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and tandem MS can help to identify glycosphingolipids (GSLs) that may play a role in cell adhesion and migration of CSCs.

METHODS

In our study, 2 × 106 cells were mixed with methanol chloroform and incubated overnight. The supernatant was collected and partitioned with chloroform and H2O. The upper layer was collected and separated by nano-LC in a self-packed phenylhexyl column with a gradient system of H2O, methanol, and 10 mM NH4OAc. Effluent was analyzed on-line by negative-ion microelectrospray, with a hybrid LTQ 14.5 tesla FT-ICR MS and 9.4 tesla FT-ICR MS. Structural assignment was based on accurate mass and tandem mass spectrometry.

RESULTS

In CSCs, we identified a class of endogenous acidic GSLs that share the same or similar epitopes with several nervous system glycoproteins. This class of GSL has been found in adult peripheral nervous system (PNS) and embryonic CNS but not in adult CNS. The presence of this class of GSL in the CSCs may account for the unusual regeneration and adhesion phenotype of CSCs. High mass accuracy and resolving power of 14.5 T FT-ICR MS, combined with different tandem mass techniques, helped us assign possible GSL structures.

CONCLUSION

In several CSC cell lines, we identified a class of endogenous sulfated GSL, which may serve as a new small molecule based biomarker for CSCs.

Neuro-Oncol. 2009 Oct;11(5):681.

511. Development of a Three-Dimensional Extracellular Matrix as a Delivery System for the Transplantation of Glioma-Targeting Neural Stem Cells

Nils Ole Schmidt, Katharina Hansen, Franz-Josef Mueller, Markus Messing, Frank Zeigler, Jeanne Loring, Katrin Lamszus, Manfred Westphal

INTRODUCTION

Neural stem cells (NSCs) display inherent pathotropic properties that can be exploited for targeted delivery of therapeutic genes to invasive malignancies in the CNS. Optimizing transplantation efficiency will be essential for developing relevant NSC-based brain tumor therapies. To date, the real-world issue of handling and affixing NSC in the context of the neurosurgical resection cavity has not been addressed. Stem cell transplantation using biocompatible devices is a promising approach to counteract poor NSC graft survival and integration in various types of neurological disorders.

METHODS

Here we report the development of a three-dimensional substrate which is based on extracellular matrix purified from tissue-engineered skin cultures (3DECM).

RESULTS

3DECM enables the expansion of embedded NSC in vitro while retaining their uncommitted differentiation status. When implanted in intracerebral glioma models, NSCs were able to migrate out of the 3DECM to targeted glioma growing in the contralateral hemisphere. Direct application of a 3DECM implant into a tumor resection cavity led to a marked NSC infiltration of recurrent glioma. In the absence of tumor, the 3DECM-embedded NSC did not display any distant migration. The semisolid consistency of the 3DECM implants allowed simple handling during the surgical procedure of intracerebral and intracavitary application and ensured continuous contact with the surrounding brain parenchyma.

CONCLUSION

Here we demonstrate proof-of-concept of a matrix-supported transplantation of tumor-targeting NSC. The semisolid 3DECM as a delivery system for NSC has the potential to increase transplantation efficiency by reducing metabolic stress and providing mechanical support, especially when administered to the surgical resection cavity after brain tumor removal.

Neuro-Oncol. 2009 Oct;11(5):681.

512. Kruppel-Like Factor 9 Modulates the Growth and Differentiation of Glioblastoma-Derived Neoplastic Stem Cells

Mingyao Ying, Shuli Xia, John Laterra

INTRODUCTION

Glioblastoma (GBM) is the most prevalent brain tumor, with high frequency of recurrence. During the past decade, the identification of neoplastic stem-like cells in diverse human cancers, including GBM, represents an important conceptual advance in cancer biology and suggests new therapeutic strategies.

METHODS

We used lentivirus-based gene overexpression and knockdown methods to modulate Kruppel-like factor 9 (KLF9) expression in GBM-SCs.

RESULTS

We found that retinoic acid (RA) induces growth arrest and cell differentiation of GBM-derived stem-like cells (GBM-SCs). RA treatment also inhibited the growth of GBM-SC xenografts in mice. We studied the gene expression changes during RA-induced GBM-SC differentiation and found that KLF9 expression increased 8-fold after RA treatment. KLF proteins are evolutionarily conserved zinc finger-containing transcription factors that bind to CACCC elements and GC-rich DNA regions. KLF9 functions as a progesterone receptor cofactor to regulate progesterone-responsive genes, but its function in cancer and stem cell biology is essentially unknown. We found that KLF9 overexpression induced GBM-SC differentiation and inhibited GBM-derived neurosphere formation in clonogenic and neurosphere cell growth assays. Knockdown of KLF9 suppressed RA-induced differentiation of GBM-SCs.

CONCLUSION

These data indicate that KLF9 and possibly other KLF family members modulate transcriptional mechanisms that regulate GBM-SC growth, differentiation, and possibly tumor initiation. These findings provide new insights pertaining to the molecular regulation of the GBM-SC phenotype and could lead to innovative therapeutic strategies for targeting the stem-like cell pool in malignant brain tumors.

Neuro-Oncol. 2009 Oct;11(5):681.

513. EGFR Signaling Controls the Chromatin Binding Status of BMI1 Through A CK2/PP2A Phosphorylation/Dephosphorylation Pathway and Affects Astrocytoma Proliferation and Neural Stem Cell Differentiation

Jessica Hawes, Robert Tuskan, Keiko Akagi, Karlyne M Reilly

INTRODUCTION

Studies of brain tumors demonstrate the importance of a rare tumor cell population with the capability of regenerating tumors upon transplantation. These cancer stem cells (CSC) are believed to be more resistant to therapy than the bulk of brain tumor cells. Studies in mouse brain tumor models demonstrate that neural stem cells (NSC) can be an important initiating cell type for brain tumors, but the relationship between NSC and CSC is unclear. EGF is a critical growth factor for NSC maintenance in culture, and EGFR signaling is upregulated in glioblastoma. Similarly, the chromatin modulator BMI1 is necessary for NSC maintenance and has been implicated in glioblastoma. We present studies linking cell surface EGF signaling and nuclear BMI1 chromatin binding.

METHODS

Human and mouse astrocytoma/glioblastoma cells were examined for BMI1 status upon EGF stimulation. Mutant BMI1 was used to examine the effects EGF-responsiveness on tumor growth and NSC maintenance. Inhibitors were used to identify the signal transduction pathways that link EGFR signaling to BMI1.

RESULTS

BMI1 is specifically phosphorylated by EGFR signaling, but not by other receptors tested. We have identified the sites that are phosphorylated in response to EGFR signaling and show that CK2 and PP2A are necessary for control of BMI1 phosphorylation. This phosphorylation alters the chromatin binding state of BMI1 and transcriptional regulation in astrocytoma cells. Furthermore, EGF-induced phosphorylation of BMI1 is important for astrocytoma growth in vitro and in vivo, as well as maintenance of NSC.

CONCLUSION

Our data link signals outside the cell to the nucleus, which is important for NSC maintenance and astrocytoma proliferation. These data represent a common pathway in NSC and CSC that may explain the importance of EGF signaling for NSC maintenance and glioma genesis and suggest a novel downstream pathway (CK2/PP2A) involved in these processes.

Neuro-Oncol. 2009 Oct;11(5):681.

514. Identification of Target Compounds for Brain Tumor Stem Cells

Ichiro Nakano, Paul Mischel, Harley I Kornblum

INTRODUCTION

Emerging evidence has suggested that neural stem cell-like tumor cells, called brain tumor stem cells (BTSCs) represent a minority of tumor cells, which are responsible for cancer initiation, propagation, and maintenance. Although BTSCs are likely the therapeutic target, these BTSCs are relatively resistant to the current standard-of-care therapy, including radiation and chemotherapy with temozolomide. Novel therapeutic strategies that can effectively and selectively eradicate BTSCs are, therefore, crucially required.

METHODS

We designed a large-scale screening of chemotherapeutic compounds aimed at identifying target compounds for BTSC survival.

RESULTS

Here, we demonstrate that thiazole antibiotic, siomycin A, potently inhibits self-renewal of BTSCs and induces apoptosis of BTSCs in culture but not their nonstem counterparts or normal neural progenitors. More importantly, siomycin A treatment reduced the ability of stem-like GBM cells to recapitulate tumors in the orthotropic model of brain tumors using immunodeficient mice.

CONCLUSION

Together, these findings indicate that treatment with siomycin A may afford us a novel avenue toward the mechanism-based targeted therapy for GBM without affecting the normal cells in the brain.

Neuro-Oncol. 2009 Oct;11(5):681–682.

517. Radial Glia are Susceptible to Transformation into Ependymoma by Candidate Human Ependymoma Tumor Suppressor Genes (TSG) and Oncogenes

Robert A Johnson, Karen D Wright, Helen Poppleton, Sarah E Leary, Mohankumar Murugesan, Christopher J Eden, Richard J Gilbertson

INTRODUCTION

Ependymomas are tumors that arise throughout the neural axis of children and adults. We previously identified radial glia (RG) as candidate cells of origin of this disease. Here, we tested if RG are susceptible to transformation into ependymoma by candidate ependymoma oncogenes and TSGs identified in a genomic analysis of more than 200 ependymomas (Wright et al., cosubmitted abstract).

METHODS

Green fluorescent protein-positive RG were isolated from the forebrain, hind-brain, and spine of embryonic (E14.5) and adult wild-type and Ink4a/ARF(−/−) Blbp-eGFP transgenic mice. The immunophenotype, gene expression profile and potency of >200 cultures of RG from >1,000 mice were characterized. Isolated RG were then transduced with retroviruses carrying candidate oncogenes of human ependymoma and implanted into the cortex of immunocompromised mice to test for tumor formation.

RESULTS

GFP+ cells from all three regions of E14.5 and adult mice were bona fide RG (Nestin, RC2, Blbp, and GFAP), molecularly distinct, and multipotent. Microarray expression analysis identified 12 distinct types of RG that display unique gene expression profiles according to region, age, and genetic background. Expression profiles were stable through multiple passages. Comparative genomic analysis revealed remarkable similarities in gene expression between mouse RG and tumors arising in humans from the corresponding part of the CNS. The NOTCH- and Ephrin-signal pathways are activated in human cerebral tumors; therefore, we used retroviruses to activate these same pathways in cerebral RG. Remarkably, these cells (>70/100 implants), but not controls, transduced RG (0/100) generated ependymomas in mice that were practically indistinguishable at the histologic and transcriptome level from the human disease.

CONCLUSION

RG are cells of origin of ependymoma. We have developed the first mouse model for ependymoma that histologically and molecularly recapitulates subgroups of the human disease. Our system provides a flexible platform to generate diverse subgroups of human ependymoma.

Neuro-Oncol. 2009 Oct;11(5):682.

518. Human Neural Progenitor Cell Binding and Encirclement of Tumor Cells in vitro are Cell Autonomous and Resembles Developmental Migration

Michael Barish, Nousha Khosh, Karen S Aboody

INTRODUCTION

Neuroprogenitor cells (NPCs), endogenous or introduced, home to tumor sites in rodent models of human glioma. Near tumor sites, individual NPCs are in close contact with single tumor cells, raising the possibility of contact-activated tumor cell-directed therapies. An unresolved question is the extent to which NPC-tumor cell binding is an intrinsic property of NPCs or a consequence of unique tumor cell properties.

METHODS

Individual immortalized neuroprogenitor cells grown in matrices devoid of growth factors or other cues will bind to and encircle human glioma cells, indicating that this process can occur independently of the tumor microenvironment. In the present experiments, we have compared the properties of NPC binding to a variety of potential targets, including glioma, breast cancer, fibroblast cells, and astrocytes. F3 NPCs were derived from retroviral v-myc transfection of 15 week-old human telencephalon. We cocultured GFP-expressing F3 cells (F3-GFP) and targeted cells in peptide hydrogel (Puramatrix; 3DM Inc.). Potential NPC targets were (human) glioma (U251, U87), breast cancer (MCF-7, SK-BR-3A-MB-231), fibroblast (3T3) cells, and primary mouse cortical astrocytes. Target cells were marked by Cell Tracker Orange (CMRA; Molecular Probes) or by dsRed/mRFP expression. Cell-cell interactions were imaged by confocal microscopy of fixed cultures or wide-field time-lapse imaging of living cultures.

RESULTS

We observed that F3-GFP cells will avidly contact cocultured U251-ds red cells and in time-lapse sequences will either proceed to encircle the glioma cells or move off them. Analyses of fixed cultures indicate that at the steady state, F3-GFP cells will bind to and encircle all of the potential targets listed above, although small consistent differences are seen between lines.

CONCLUSION

Taken together, our observations suggest that F3-GFP binding to target cells, including tumor cells, is largely driven autonomously by the NPCs.

Neuro-Oncol. 2009 Oct;11(5):682.

519. Activated EGFR Signaling Increases Proliferation, Survival, and Migration and Blocks Neuronal Differentiation in Neural Stem Cells

Angel Ayuso-Sacido, Jennifer A Moliterno, John A Boockvar

INTRODUCTION

Recent evidence supports that transformation of undifferentiated neural stem cell (NSC) precursors may contribute to the development of glioblastoma multiforme (GBM). The overexpression and mutation of the epidermal growth factor receptor (EGFR), along with other cellular pathway mutations, plays a significant role in GBM progression. Though EGFR signaling is important in determining neural cell fate and conferring astrocyte differentiation, there is limited understanding of its role in NSC and tumor stem cell (TSC) biology. We hypothesized that EGFR expression and mutation leads to enhanced cellular proliferation, survival, and migration in NSC.

METHODS

Stable subclones of C17.2 murine NSC lines were immortalized from a neural progenitor cell with a retrovirus carrying v-myc. They were transfected to overexpress either the wtEGFR or the most common variant, EGFRvIII.

RESULTS

Activated wtEGFR signaling in these cells induced behaviors characteristic of GBM TSCs, including enhanced proliferation, survival, and migration, even in the absence of EGF ligand. wtEGFR activation was also found to block neuronal differentiation and was associated with a dramatic increase in chemotaxis in the presence of EGF. EGFRvIII expression led to an increase in NSC proliferation and survival while simultaneously blocking neuronal differentiation and promoting glial fate.

CONCLUSION

Our findings suggest that activated EGFR signaling enhances the survival of NSCs. wtEGFR overexpression in GBM may increase TSC proliferation in the presence of EGF, thereby increasing TSC chemotaxis and migration through the parenchyma. The mere expression of the mutated EGFRvIII in GBM TSCs, irrespective of exposure to EGF ligand, may increase the cells’ proliferation and survival. Evaluation of patients’ TSC subpopulations for activated EGFR may offer further insight into its role in TSC biology. Molecular therapies targeted to inhibit EGFR-mediated TSC proliferation, survival, and migration may ultimately improve anticancer strategies in patients with GBM.

Neuro-Oncol. 2009 Oct;11(5):682.

520. Modeling High-Grade Brain Tumors using Human Neural Stem Cells: Does Location Matter?

Eric Raabe, Guido Nikkhah, Jarek Maciaczyk, Charles G Eberhart

INTRODUCTION

Brain tumors have heterogeneous histological appearances and clinical outcomes. It is not clear if these differences are due to distinct oncogenic alterations, to intrinsic properties of the cells from which the tumor arose, or a combination of these factors. To address these questions, we are using fetal stem/progenitor cells derived from human cortex, thalamus, and cerebellum to generate malignant brain tumor models.

METHODS

Neurospheres were derived from human fetal autopsy tissues and grown in serum-free media supplemented with defined growth factors. Oncogenes and other genetic elements were introduced using lentiviral vectors.

RESULTS

To prove that human fetal neurosphere cells can be transformed, we first introduced SV40 large T antigen. SV40 T-Ag expressing stem/progenitor cells showed increased proliferation and decreased differentiation capacity relative to GFP-infected controls. SV40 T-Ag infected cells derived from cortex and thalamus formed many large colonies in soft agarose, while control cells and SV-40 T-Ag infected cerebellar cells did not. We also introduced dominant-negative (DN) TP53, constitutively active c-myc, and hTERT into stem/progenitor cells, singly and in combination. Cortical DN TP53/c-myc/hTERT infected cells proliferated at an increased rate and increased levels of apoptosis compared to control GFP-infected cells. These characteristics are similar to those exhibited by high-grade gliomas, and we are currently investigating the potential of the various lines to form tumors in vivo.

CONCLUSION

Human neural stem/progenitor cells derived from cortex and thalamus can be transformed by SV40 T-Ag in terms of their ability to grow in soft agarose, suggesting that such neurospheres can be used to generate model systems for the study of brain tumors. Cerebellar progenitors cannot form colonies after SV40 T-Ag infection, suggesting that the anatomical origin of neural stem/progenitor cells may affect response to a given oncogenic stimulus.

Neuro-Oncol. 2009 Oct;11(5):682.

521. Integrin Alpha3 Prevents Neurosphere Formation and Proliferation of Glioma Stem Cells

Mitsutoshi Nakada, Emi Nambu, Yuya Yoshida, Natsuki Furuyama, Daisuke Kita, Yutaka Hayashi, Yasuhiko Hayashi, Naoyuki Uchiyama, Junichiro Hamada

INTRODUCTION

CD133+ cells have been identified as exhibiting cancer stem cell properties in glioblastoma. One of the definitions of a glioma stem cell is neurosphere formation in stem cell medium. To identify the genes which contribute the character of a glioma stem cell, we investigated the genes that are associated with sphere formation of glioma stem cells.

METHODS

CD133+ cells were isolated from glioblastoma surgical specimens and glioma cell lines (U87, U251, T98G, SNB19) with magnetic affinity cell sorting (MACS). Cells have been characterized by differentiation assays and immunocytochemistry. Kept in stem cell medium or 0.1% FBS DMEM, sphere formation, cell morphology, adhesion activity, and proliferation rates were analyzed. The expression level of adhesion molecules identified so far was screened in CD133+ and CD133- cells. The function of candidate adhesion molecule in glioma stem cells was analyzed by manipulating gene expression.

RESULTS

The ratio of CD133+ cells were 39% (n = 11, 0.2–87%) in glioblastoma tissues and 0.66% (0.35–0.9%) in glioma cell lines. CD133+ cells showed neurosphere formation in stem cell medium but not in DMEM containing FBS. CD133+ cells demonstrated round cell shape and were easily detachable on fibronectin-coated dish in 0.1% FBS DMEM, whereas CD133- cells showed spread and spindle-shape. QRT-PCR showed a significant low level of integrin α3 in glioma stem cells, both from surgical specimens and from cell lines, whereas it has high expression in CD133- cells. CD133+ cells sorted by MACS from the cells transfected with integrin α3 prevent neurosphere formation in stem cell medium. The depletion of integrin α3 in CD133- cells showed round cell shape and a marked increase in proliferation rate.

CONCLUSION

These findings showed for the first time that integrin α3 is downregulated in glioma stem cells and is related to neurosphere formation and proliferation rate.

Neuro-Oncol. 2009 Oct;11(5):683.

522. Light Microscope Imaging of Novel Patient-Derived Tumor Cell Models of Glioma Toward Describing the in vivo Niche Environment

Christine Brown, Michael Barish

INTRODUCTION

Brain tumors can be thought of as heterogeneous tissues developing within a complex brain environment. We are working to achieve understanding of tumor initiation, progression, and dissemination using appropriate biological models together with powerful imaging techniques.

METHODS

We have derived pools of patient-derived glioma cells, lentivirus-transduced them to express fluorescent tracking proteins, and implanted them in immunodeficient mice. Optionally, cell-based therapeutics (IL13R2a-directed zetakine-cytotoxic T cells or tumor-homing F3 immortalized neural progenitor cells) expressing complementary fluorescent marker proteins were administered by intracranial or intravascular routes. Tumor morphology with cell resolution was assessed by multiphoton confocal microscopy of thick (up to 1 mm) brain sections.

RESULTS

Patient-derived glioma line PBT003 cells formed self-renewing multipotent tumor spheres in culture. They were also highly tumorigenic, forming large masses detectable with MRI and bioluminescence. Conventional postmortem histology revealed a disseminating tumor with the main mass showing prominent microvasculature and pseudopalisading necrosis, hallmarks of high-grade glioma. When imaged by two-photon confocal microscopy in thick sections, PBT003 appeared as a “spaghetti-like” mass with emerging tendrils. This appearance could correlate with the structure of the tumor vasculature.

CONCLUSION

Multiphoton imaging of experimental glioma modeling human disease can reveal tumor macrostructure at cellular resolution. More sophisticated extraction of quantitative information from these images, together with immunochemical localization of the vasculature and other structures, will enhance development and optimization of cell-based therapies.

Neuro-Oncol. 2009 Oct;11(5):683.

523. Nestin1 Progenitors as Targets of Transformation in Medulloblastoma

Zengjie Yang, Robert Wechsler-Reya

INTRODUCTION

Medulloblastoma is the most common malignant brain tumor in children. Effective approaches to prevention and treatment of medulloblastoma require advances in our understanding of the cellular and molecular basis of the disease. Our previous studies have demonstrated that deletion of patched (an antagonist of the Sonic hedgehog signaling pathway) in granule neuron precursors (GNPs) leads to medulloblastoma, suggesting that GNPs can represent cells of origin for the disease. However, after patched deletion, only a small percentage of GNPs become transformed and give rise to tumors; the remaining cells proliferate transiently and then differentiate. These results suggest that only a subset of GNPs in cerebellum may be susceptible to transformation. Our preliminary studies indicate that GNPs expressing Nestin (a marker often associated with stem cells) may represent such a subset.

METHODS

Microdissection and FAC sorting are applied to isolate the Nestin expressing cells, and gene expression pattern of these nestin+ cells will be analyzed by microarray analysis. The distinct functional characteristics of these Nestin+ cells are being investigated by intracranial transplantation, in vitro differentiation assay, and confocal live cell imaging.

RESULTS

Genetic lineage tracing studies suggest that Nestin+ GNPs give rise to a distinct subset of granule neurons. Although Nestin+ cells represent only 5% of GNPs, deletion of patched in these cells results in rapid tumor formation comparable to that seen when patched is deleted in all GNPs.

CONCLUSION

Understanding the unique properties of Nestin+ cells will provide insight into the mechanisms that control normal cerebellar development and the manner in which these mechanisms are dysregulated in medulloblastoma. Moreover, identifying the properties that make Nestin+ cells susceptible to transformation may allow us to develop more effective methods for treating this highly malignant tumor.

Neuro-Oncol. 2009 Oct;11(5):683.

524. Modeling Large Cell-Anaplastic Medulloblastoma using Cerebellar Stem Cells

Yanxin Pei, Robert Wechsler-Reya

INTRODUCTION

Large cell-anaplastic (LCA) medulloblastoma is among the most aggressive pediatric brain tumors and is associated with a much poorer prognosis than other subtypes of the disease. Although LCA tumors frequently contain amplification of the myc oncogene, the role of myc in tumor formation is poorly understood, and the cell type from which these tumors originate remains unknown.

METHODS

We have recently identified a population of multipotent neural stem cells in the neonatal cerebellum.

RESULTS

Our preliminary studies indicate that these cells are exquisitely sensitive to the effects of c-myc. Infection of cerebellar stem cells with c-myc-encoding retroviruses leads to a marked increase in proliferation and self-renewal in vitro and (in combination with a dominant negative form of the tumor suppressor p53) allows these cells to form tumors following transplantation into the cerebellum.

CONCLUSION

These tumors consist of large, poorly differentiated cells with a high proliferative index, characteristics commonly associated with human large cell-anaplastic medulloblastoma. We are currently characterizing these tumors to determine whether they resemble human LCA tumors at a molecular level. In addition, we are developing transgenic mice that overexpress c-myc and inactivate p53 in cerebellar stem cells and testing whether these animals develop medulloblastoma. Using these models, we will investigate the molecular mechanisms of tumor progression and evaluate the efficacy of targeted therapies for treating LCA medulloblastoma.

Neuro-Oncol. 2009 Oct;11(5):683.

525. Biologic and Therapeutic Role of Telomere Maintenance in Tumor-Initiating Cells

Pedro Castello-Branco, Cindy Zhang, Loen Hansford, Tatiana Lipman, Ian D Clarke, Calvin Harley, Robert Tressler, Ning Go, Katia Bassett, David Kaplan, Peter B Dirks, Uri Tabori

INTRODUCTION

Tumor-initiating cells (TIC) are a small subpopulation of tumor cells which share similar characteristics with normal tissue stem cells. They are resistant to conventional chemoradiotherapies and can explain tumor recurrence even after maximal therapy and minimal disease. We still lack understanding regarding their endless replicative potential and resistance to therapy. Pediatric neuronal tumors share unique tendency to spontaneously growth arrest and relentless growth in same tumor types.

METHODS

We previously showed that telomerase predicts recurrence in pediatric neural tumors. We also recently established unique normal and malignant stem cell lines from these tumors and corresponding tissues. We therefore used these tumors to examine the role of telomere maintenance in tumor subpopulations and corresponding tissue stem cells.

RESULTS

First, we revealed that TIC isolated from fresh glioblastomas have telomerase activity whereas mature glioblastoma cells do not. Strikingly, TIC lines derived from glioblastomas and neuroblastomas were also telomerase-positive while normal neural and neural crest stem cell lines lack detectable telomerase activity in these experiments. Next, we used GRN163L, a novel telomerase inhibitor, to treat glioblastoma and neuroblastoma TIC. GRN163L treatment resulted in growth arrest of both TIC lines accompanied by induction of DNA/telomere damage, senescence, and loss of stemness. Treatment of normal neuronal stem cells with GRN163L had no effect on proliferation and was not associated with telomere shortening or dysfunction. In vivo telomerase inhibition of mice injected with neuroblastoma TIC revealed significant inhibition of tumor growth. Furthermore, pretreatment before implantation resulted in lack of tumor development (p < 0.001).

CONCLUSION

We show that telomerase is only active in the TIC subpopulation. Telomerase inhibition of TIC results in loss of their proliferative capacity and stemness in vitro and in vivo. Since normal tissue stem cells have relatively long telomeres and lack telomerase activity, telomerase inhibition can open new therapeutic avenues for these devastating tumors.

Neuro-Oncol. 2009 Oct;11(5):683–684.

526. Outcome of an Aggressive Treatment Paradigm of Patients with Rapid Early Recurrent GBM

Erez Nossek, Razi Sitt, Carmit Ben Harosh, Felix Bokstein, Deborah T Blumenthal, Ben Korn, Zvi Ram

INTRODUCTION

Glioblastoma multiforme (GBM) is associated with a short median survival. In view of the grim prognosis of patients with GBM, early and rapid recurrence of tumors may indicate an aggressive tumor biology and presents a therapeutic dilemma. It is unclear whether aggressive treatment in these patients may result in any clinical benefit. We have retrospectively evaluated the outcome of aggressive treatment of patients with clinically significant early (within 3 months from diagnosis) rapid tumor recurrence after an initial extensive tumor resection.

METHODS

Between 2003–2008, 376 patients with newly diagnosed, de Novo GBM had been treated in the Dpartment of Neurosurgery at the Tel Aviv Medical Center. Of these patients, 270 underwent aggressive (> 90%) tumor resection. Of those, 22 patients (mean age, 58 years; range, 34–77 years) had experienced a clinically significant rapid and early recurrence of their tumors (mean time to recurrence, 6 weeks; range, 1–14 weeks). Two patients had large (>4 cm) tumor recurrence with minimal symptoms. Eighteen of the 22 patients had received a second aggressive resection at the time of recurrence. Four patients were not reoperated because of tumor location or per the patient/family decision. Following the second operation, the patients had completed radiation therapy and were treated with a variety of chemotherapeutic protocols. Four of the patients had subsequently received additional surgical resection.

RESULTS

In this selective group of patients, an aggressive therapeutic approach resulted in median survival of 39 weeks (95% CI, 18–60 weeks). In comparison, the median survival of patients with newly diagnosed GBM who did not exhibit early rapid recurrence was 52 weeks (95% CI, 42–62 weeks).

CONCLUSION

Our data indicate that early and rapid recurrence of GBM is associated with a worse prognosis. However, aggressive treatment does extend survival and should be considered a valid therapeutic option.

Neuro-Oncol. 2009 Oct;11(5):684.

527. Trial to Survive: the Impact of Enrollment in Clinical Trials on Survival of Patients with GBM

Tal Shahar, Erez Nossek, Deborah T Blumenthal, Felix Bokstein, Sigal Freedman, Carmit Ben Harosh, Razi Sitt, Benjamin W Corn, Andrew A Kanner, Zvi Ram

INTRODUCTION

Multiple clinical trials for gliobastoma multiforme (GBM) therapy are being explored. The actual impact on survival of being enrolled in a clinical study has not been fully explored.

METHODS

We conducted a retrospective analysis of 583 patients with de novo, newly diagnosed GBM treated between 1995 and 2008, in whom complete medical records and follow up data were available. Of these patients, 106 (62 treatment, 44 control) had been enrolled in at least 1 of 10 prospective randomized clinical studies involving tumor resection. Additionally, 166 nontrial patients were matched to the study group (age, KPS, number of operations) and served as a comparative arm. We performed survival analysis with statistical adjustments to overcome possible selection biases. We used the multivariable COX proportional hazards model and recursive partitioning analysis used to identify various predictors of survival.

RESULTS

The overall median survival for nontrial, control and treatment groups was 11.63 months (95% CI, 10.47–12.80), 17.00 months (95% CI, 15.18–18.82) and 15.57 months (95% CI, 13.06–18.08) respectively, (means 14.29 months (95% CI, 12.41–16.16), 16.73 months (95% CI, 14.13–19.33) and 19.72 months (95% CI, 15.62–23.82), respectively (p = 0.014, log-rank scale). Multivariate analysis showed that participation in clinical trial, regardless of being in either the treatment or control arms, was a significant predictor of survival (p = 0.01). Younger age, higher KPS at diagnosis, and number of surgical resections were also significant predictors of better outcome.

CONCLUSION

Our data indicate that being enrolled in a clinical study, regardless whether in the treatment or control arms, carries a significant survival advantage for patients with GBM. This observation remains valid even when possible selection bias and coexisting confounding factors in nontrial patients are taken into account. This observation raises some practical and ethical issues related to the quality of care given to patients with GBM who receive standard therapy and underscores the limited value of using historical data as controls in clinical trials.

Neuro-Oncol. 2009 Oct;11(5):684.

528. Electrophysiological Mapping and Tumor Resection with High-Field Intraoperative MRI: A Recent Mayo Clinic Experience

Jason M Hoover, Ian Parney, Fredric B Meyer

INTRODUCTION

Anatomic guidance obtained with high field intra-operative magnetic resonance imaging (iMRI) can facilitate glioma resection. However, this result can come at the expense of functional information, as adapting speech and motor electrophysiological mapping (EPM) to the high field iMRI environment has been challenging. To overcome this challenge, we developed techniques enabling EPM in high field iMRI. In this study, we describe indications, techniques, and outcomes for glioma resection performed with mapping in high field iMRI.

METHODS

Adult patients with newly diagnosed, resectable brain lesions compatible with glioma that encroach on motor/speech cortex were eligible for this study. A total of 20 patients were enrolled. All patients underwent craniotomy and resection in 1.5 T iMRI and were imaged before, during, and at surgery completion. Preoperative and interdissection images were registered to a standard image guidance system. Patients requiring motor mapping received a general anesthetic. We performed speech mapping under a local anesthetic. We used somatosensory evoked potentials (SSEP) to identify phase reversal between motor/sensory cortex, and we performed mapping using an Ojemann Stimulator. We obtained interdissection images using a minimal draping and patient transfer technique.

RESULTS

Most patients had tumors involving left frontal (50%) or temporal lobes (50%). Grade II gliomas equaled 67%. iMRI images were obtained in all cases. All patients had motor mapping. Two-thirds had speech mapping while awake. We identified functionally critical areas intratumorally in 16%. Postoperative images confirmed extensive resection in all cases. Mean volume was 19.6 ml preoperatively and 2.2 ml postoperatively. Patients had either no permanent neurological deficits or mild transient deficits (33%). The median length of stay was 2 days.

CONCLUSION

Our techniques allow the combination of high-field iMRI and EPM. This combination facilitated extensive glioma resection without significant morbidity for tumors involving eloquent cortex.

Neuro-Oncol. 2009 Oct;11(5):684.

529. Management after Nondiagnostic Frameless Stereotactic Needle Biopsy: A Retrospective Review of 28 Patients

Ellen L Air, Ronald E Warnick, Christopher M McPherson

INTRODUCTION

Frameless stereotactic needle biopsy is an accepted procedure for the diagnosis of intracranial lesions; however, 2%–15% of cases are nondiagnostic. No recommendations exist to guide subsequent care. Through analysis of our experience in such cases, we aimed to develop a paradigm for the management of patients after nondiagnostic biopsy.

METHODS

We reviewed all frameless stereotactic needle biopsies performed from January 2000 through December 2006 to identify the subset for which no definitive diagnosis could be made based on permanent pathologic samples. We further examined the postoperative treatment plans and clinical courses of these patients.

RESULTS

Of 284 total stereotactic biopsies performed, 10.2% (29 biopsies in 28 patients) did not yield a definitive pathologic diagnosis. Follow-up information was not available for seven patients. In the remaining patients, lesion location and characteristics guided the decision to recommend further surgery or initiate empiric treatment. Five patients had further surgery, either repeat biopsy or resection, soon after the initial biopsy. In all five cases, the second procedure provided diagnostic pathologic tissue. Sixteen patients received empiric treatment based on their clinical situations. Of the 16 patients empirically treated, seven (43.7%) subsequently required a change in treatment plan due to lack of improvement, and five underwent a second biopsy, of which four were diagnostic. Evolving clinical information precipitated a change in treatment plans for two patients. In all, 10 patients had a second surgery prompted by the need for better diagnostic information, for which the diagnostic yield was 90%.

CONCLUSION

Based on this review, we recommend repeat surgery in the majority of cases of nondiagnostic biopsies, especially for lesions considered potentially neoplastic or infectious. Empiric management is an option, especially for lesions considered likely to be neurodegenerative, but requires close follow-up.

Neuro-Oncol. 2009 Oct;11(5):684.

530. Emergence of Neurometastatic Merkel Cell Carcinoma: A Surgical Case Series with use of Intracavitary BCNU

Thomasina L Bailey, Christine Boudreau, William G Ellis, Rudolph J Schrot

INTRODUCTION

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine neoplasm of likely viral origin and is known for its aggressive behavior. Its incidence has increased in the last 15 years. Although it has the potential to metastasize, CNS involvement is rare.

METHODS

We describe three consecutive surgical cases of MCC presenting at a single institution within 1 year and review the literature.

RESULTS

Case 1: A 75-year-old woman with MCC of the nasal ala developed a right parietal intraparenchymal mass. After gross total resection and implantation of Gliadel wafers, her survival was 5 months after CNS involvement. Case 2: A 76-year-old woman with a painful scalp mass developed a subgaleal and epidural mass by direct extension. She underwent craniectomy, resection, and reconstructive cranioplasty but was lost to follow-up. Case 3: A 51-year-old woman with MCC of the left thigh developed a right parietal intraparenchymal mass. After gross total resection and implantation of Gliadel wafers and adjuvant radiation therapy, she is alive and working 15 months after CNS involvement. Pathological features, including CD10 positivity, were consistent with MCC in all cases. Overall, we identified 25 cases of MCC with CNS involvement: 20% had direct extension from a lesion, 72% had distant intraparenchymal involvement, and 16% had leptomeningeal disease.

CONCLUSION

Neurometastatic MCC may be emerging as a more common entity than previously thought. CNS involvement is usually intraparenchymal, but it may also exist through direct extension from a lesion or through CSF dissemination. A literature review revealed 22 neurometastatic MCC cases over 23 years. To this number, we add three cases that presented within 1 year at a single institution. Intracavitary BCNU may help prevent local recurrence. The succession of cases we encountered in a short time highlights the increasing incidence of MCC and the increasing need to address neurometastatic MCC.

***

Neuro-Oncol. 2009 Oct;11(5):685.

531. Meningiomas in Patients with Uterine Sarcoma Treated with Long-Term Megestrol Acetate

Thomas J Gruber, Robert A Fenstermaker, Andrew J Fabiano, Shashikant B Lele, George Deeb

INTRODUCTION

The authors report a series of four patients with uterine sarcoma, including one with mullerian adenosarcoma and three with low-grade endometrial stromal sarcoma (LGESS), who developed intracranial meningiomas while receiving the progesterone agonist megestrol acetate (Megace).

METHODS

We reviewed the hospital records, imaging studies and pathology of four patients who were treated for uterine sarcomas and who subsequently developed intracranial meningiomas.

RESULTS

All patients underwent surgery for their gynecologic cancers and were maintained on chronic hormonal suppression therapy with megestrol acetate. Each of the four patients later developed neurologic symptoms due to intracranial meningiomas; three had multiple tumors. Immunohistopathology of all excised tumors demonstrated strong positivity for progesterone receptors.

CONCLUSION

Patients with the uterine sarcoma subtypes LGESS and müllerian adenosarcoma may be predisposed to develop meningiomas, particularly in the setting of long-term treatment with megestrol acetate. LGESS or müllerian adenosarcoma may be predisposed to meningioma formation or the development of meningiomas in these patients may be causally related to prolonged therapy with megestrol acetate.

Neuro-Oncol. 2009 Oct;11(5):685.

532. Single Center, Retrospective Review of Ommaya Reservoir Placement: Indications and Complications

David E Connor, Anil Nanda

INTRODUCTION

Ommaya reservoir placement is often required to provide optimal treatment of cancer patients with leptomeningeal disease. In this report, we review our 18-year, single-center experience regarding the indications for and complications associated with placement of Ommaya reservoirs.

METHODS

We conducted a retrospective analysis of 62 patients treated at the Louisiana State University Health Sciences Center-Shreveport (LSU-HSC-Shreveport) from November 1991 to December 2007. We obtained and analyzed data regarding indications for placement, need for catheter revision and immediate and long-term postoperative complications. All procedures were followed by postoperative CT scan of the head to assess for optimal placement of the catheter.

RESULTS

Of 62 patients selected for Ommaya placement, 35 (56.4%) were women and 28 (44.6%) were men. The mean age at presentation was 43 years. Indications for placement included lymphoma (41.9%), metastatic cancer with evidence of leptomeningeal disease (24.2%), leukemia (22.6%), recurrent dilatation of cystic lesions (8%) and temporary treatment of obstructive hydrocephalus (3.2%). Repositioning of one catheter (1.6%) was required for poor placement. Historical data has reported serious complications requiring surgical intervention in the range of 6%–9%. In our series, postoperative complications included one subdural hematoma (1.6%) and one superficial wound dehiscence (1.6%). Delayed complications related to the usage of reservoirs included ventriculitis or frank abscess in three patients (4.8%); these were seen an average of 205 days after Ommaya placement. We also observed an isolated complication related to inadvertent intrathecal administration of vincristine, which resulted in death.

CONCLUSION

Ommaya reservoir placement has been a safe and effective adjunct to the treatment of oncologic patients at LSU-HSC in Shreveport. In our series, complication rates and the need for revision compare favorably with established norms in the literature.

Neuro-Oncol. 2009 Oct;11(5):685.

533. Rare Nerve Lesions of Nonnerve Sheath Origin: A 17-Year Retrospective Series

Tobin Strom, Bette K Kleinschmidt-DeMasters, Andrew M Donson, Nicholas K Foreman, Kevin Lillehei

INTRODUCTION

Context: Peripheral nerve masses are frequently encountered in surgical pathology practice. However, once a peripheral nerve mass is determined not to be a nerve sheath neoplasm, differential diagnostic considerations drop off sharply. Objective: Review our experience with surgically resected nerve masses.

METHODS

Data sources: Retrospective search of pathology database. We studied rare neoplasms by cytogenetic analysis or gene microarray.

RESULTS

We identified 458 cases. After elimination of common lesions (mostly nerve sheath tumors), 37 cases (8%) remained, almost all of which were of nonnerve sheath origin, for example, hemangioma, metastatic neuroendocrine pancreatic carcinoma, meningiomas invading nerve fascicles, and primary extrarenal rhabdoid tumor and Ewing’s sarcoma of the nerve. The latter showed rearrangement of the EWSR1 locus (22q12). The gene expression pattern of an undifferentiated sarcoma, presenting as rope-like nerve enlargement, clustered with malignant peripheral nerve sheath neoplasms, but not other sarcomas or neuroepithelial tumors.

CONCLUSION

Diverse benign and malignant conditions can affect peripheral nerve.

Neuro-Oncol. 2009 Oct;11(5):685.

534. Pleomorphic Xanthoastrocytoma of Cervical Spinal Cord with Leptomeningeal Dissemination Presenting as Recurrent Aseptic Meningitis

Farhan Siddiq, Kyle M Uittenbogaard, Koijan Kainth, H Brent Clark, Christopher Moertel, Matthew Allan Hunt

INTRODUCTION

Plemorphic xanthoastrocytoma (PXA) has been reported in the spinal cord only twice before. Here, we present a case of PXA with atypical presentation and features.

METHODS

Case report.

RESULTS

A 23-year-old woman presented with a history of recurrent episode of aseptic meningitis with neutrophilic pleocytosis, elevated protein, and decreased glucose in cerebrospinal fluid on lumbar puncture. Upon further investigation, magnetic resonance imaging (MRI) revealed an enhancing, 4 × 7 cm bulky lesion on the anterolateral aspect of the cervical spinal cord extending from C4 to C7 with additional leptomeningeal enhancement. At surgery, the tumor had a good surgical plane laterally; however, it was adherent to the leptomeninges, neuronal tissue, and C7 ventral nerve root anteriorly, preventing it from being completely resected. The histopathology was consistent with PXA, with astrocytes both spindled and larger pleomorphic astrocytes intermingled with eosinophilic granular bodies and frequent Rosenthal fibers. Lymphocytic infiltrates and areas of fibrosis also were present. Immunohistochemistry for S100 protein and GFAP was positive, while some larger cells labeled for neuron-specific enolase. Synaptophysin staining was seen in only rare cells and chromogranin staining was absent. Mitoses were rare but there was a mildly increased Ki-67 labeling index. There was minimal vascular proliferation, and necrosis was absent. Spinal cord location of this rare tumor has only been described in literature twice previously. Rosenthal fibers have not been commonly described with this tumor. Leptomeningeal spread has been previously described with intracranial tumors but not for spinal PXA. Surgical resection has been the traditional treatment of choice for these tumors when possible. This case represents a previously undescribed presentation of spinal PXA, and correlates with the findings of two previously reported cases of spinal PXA.

CONCLUSION

Spinal cord PXA are rare tumors that may present with leptomeningeal spread.

Neuro-Oncol. 2009 Oct;11(5):685–686.

535. Results of Phase I Study of Multimodality Treatment for Patients with Newly Diagnosed Glioblastoma Multiforme using Concurrent BCNU Wafers and Permanent I-125 Seeds

Christopher M McPherson, Margie Gerena-Lewis, John C Breneman, Ronald E Warnick

INTRODUCTION

We previously demonstrated the efficacy of combination therapy with permanent, low activity I-125 seeds and BCNU wafers in patients who underwent treatment of recurrent glioblastoma multiforme (GBM) with median survival of 69 weeks. Based on these findings, we then designed and implemented a prospective phase I trial to determine whether this treatment could be effective in patients with newly diagnosed GBM.

METHODS

Patients with newly diagnosed GBM whose tumors were deemed amenable to gross total resection were included. The study was designed as a cohort dose escalation of I-125 seeds with three cohorts of six patients; doses would be administered at 3,000 cGy, 6,000 cGy, and 9,000 cGy. At the time of surgery, treatment plans specified that each cohort of six patients would receive BCNU wafers, with a maximum of eight wafers placed. Postoperatively, patients received standard fractionated radiation to 5,940 cGy followed by temozolomide chemotherapy.

RESULTS

Of six patients enrolled in this first cohort, only five patients were able to complete postoperative radiation. Of the five patients included, median age was 55 years (range, 46–64 years) and median KPS was 90 (range, 70–90). All patients were in the lowest dose cohort and received I-125 seeds at the 3,000 cGy dose prescription, and all patients received maximal BCNU wafer placement (eight wafers). All patients have reached end point (median follow-up, 26 weeks). Two patients developed local disease progression (median, 34.4 weeks). Three patients developed radiation toxicity, which was documented on follow-up magnetic resonance imaging (MRI) and confirmed by MRI spectroscopy (median, 20 weeks); these patients then underwent treatment for radiation toxicity with steroids and Avastin.

CONCLUSION

Enrollment for this phase I study was closed early after completing only the first cohort of patients because of the high incidence of early radiation toxicity, specifically three (60%) of five patients. Therefore, we do not recommend combination seeds and wafer therapy for newly diagnosed GBM patients. Rather, we continue to use this multimodality treatment as a salvage therapy for patients with recurrent GBM.

Neuro-Oncol. 2009 Oct;11(5):686.

536. Maintain the Plane: Technique for Maximizing Resection of Contrast-Enhancing High-Grade Glial Neoplasms

Yaron A Moshel, Robert E Elliott, Amr Morsi, Donato J Perretta, Erik C Parker, Patrick J Kelly, John G Golfinos

INTRODUCTION

The benefits of gross total resection (GTR) of the magnetic resonance imaging (MRI)-defined, contrast-enhancing portion of high-grade gliomas remains controversial. Several recent studies have suggested survival benefit with such resection. Our objective is to describe the risks and benefits of an “extratumoral” dissection technique along the tumor-white matter pseudo-plane that allows more frequent GTR.

METHODS

A total of 200 patients who underwent 240 microsurgical “extra-tumoral” resections of intraxial high-grade contrast enhancing gliomas between 2004 and 2008 were available for review. We retrospectively reviewed patient demographics, KPS score, pathology and neurological outcomes. We evaluate pre- and postoperative MRIs independently to determine the anatomical extensions of the lesion and the degree of resection.

RESULTS

Patients included 122 (61%) men and 78 (39%) women (mean age, 54 years). Forty-seven patients (24%) had more than one surgery and 32 (24%) underwent prior chemotherapy/radiation. There were 106 (53%) patients with L-sided tumors and 94 (47%) with R-sided tumors. Ninety-four 94 (47%) patients had tumors in eloquent locations and 106 (53%) had noneloquent tumor locations. Of the 200 patients, tumors were superficially located in 114 (57%), deep-seated in 25 (13%) and superficial and deep in 60 (30%) patients. Tumors were frontal (64, 32%), temporal (68, 34%), parietal (36, 18%), occipital (11, 5.3%), corpus callosal (3, 1.3%), central (7, 3.3%), insular (8, 4%) or thalamic/basal ganglionic (4, 2%) in location. GTR of the imaging defined lesion volume was achieved in 161 (80%) patients, near total resection in 27 (14%) and subtotal in 12 (6%).

CONCLUSION

The “extratumoral” white matter dissection technique resulted in higher rates of GTR of the MRI-defined, contrast-enhancing tumor compared to rates reported in the literature. Lesions located in or extending into the basal ganglia and insula were most predictive of an ultimate incomplete surgical resection. The impact of such “extratumoral” resection on survival will be analyzed.

Neuro-Oncol. 2009 Oct;11(5):686.

537. Renal Cell Cancer Metastasis to the Calvarium Presenting as an Isolated Large Expansile Lesion

Todd Brendon Francis, Sandeep Mittal, Aashit K Shah, Murali Guthikonda

The incidence of renal cell metastasis to the calvarium is very low. Furthermore, very few case reports exist in the literature describing the presentation of renal cell cancer as a large exophytic calvarial mass. We present a 69-year-old woman who presented with a rapidly growing, firm, nontender mass in the left parietal region. The mass was presumed to be an aggressive meningioma. Her neurological exam was normal. A gross total resection of the mass was performed and revealed metastatic renal cell carcinoma, clear cell type. The patient also had a left radical nephrectomy and bilateral adrenalectomy, followed by cranioplasty for correction of her skull defect. She has since made a full recovery.

Neuro-Oncol. 2009 Oct;11(5):686.

538. A Rare Case of Adrenal Ganglioneuroma

Todd Brendon Francis, Sandeep Mittal, Madhu Prasad, Murali Guthikonda

Ganglioneuroma is a very rare sympathetic tumor of the adrenal gland, occuring more frequently in children and young adults. The largest series in the literature described eight patients with ganglioneuroma, 42% of whom were under 20 years of age. We present a 48-year-old female with a history of cervical cancer managed with radiation. She subsequently developed hydronephrosis and underwent left ureteral stent placement. A CT scan of the abdomen revealed a right adrenal mass that was then biopsied, demonstrating the ganglioneuroma. The patient then underwent laparoscopic right adrenalectomy. A complete metabolic workup was performed on the patient and was essentially negative. The patient made a full recovery.

Neuro-Oncol. 2009 Oct;11(5):686.

539. The Outcome of Initial Radiological and Clinical Surveillance in Vestibular Schwannoma: A Prospective Cohort Study in a Single Center

Sarah R Eljamel, Musheer Hussain, M Sam Eljamel

INTRODUCTION

Early diagnosis of vestibular schwannoma (VS) had increased recently because aging population and increased magnetic resonance imaging (MRI). Some authors have recommended initial observation in all small VS and intervention if they grew. Others recommended intervention for symptomatic deterioration, and some recommended intervention in all tumors = 1.5cm. We prospectively evaluated 54 VS that had initial MRI surveillance to find out when this policy should be abandoned.

METHODS

Fifty-four VS managed in Dundee University (1998–2008) had initial yearly MRI. MRI data were collected prospectively and analyzed by Kodak CareStream. The widest diameter in X, Y, and Z were obtained in each scan. One had one MRI only and was excluded.

RESULTS

Median age was 59 years (26–86); 25 patients were men; 18 of the VS were extracanalicular, 18 intracanalicular and 17 both; 21 were = 1 cm, 22 were 1.1–2 cm and 10 were >2 cm. Year 1 (53 patients): 24.5% grew by = 2 mm, 69.8% were unchanged and 5.7% shrank. Year 2 (42 patients): 9.5% grew, 35.7% were unchanged, 16.7% shrank and 14.3% continued to grow (CTG). Year 3 (29 patients): 10.3% grew, 44.8% were unchanged, 19% shrank and 11.9% CTG. Year 4 (22 patients): none grew, 36.4% were unchanged, 22.7% shrank and 40.9% CTG. Year 5 (11 patients): 9% grew, 45.5% were unchanged, 18.2% shrunk and 27.3% CTG. No further growth or shrinkage was noted after year 5. Age, gender, and VS side or size at presentation did not predict future growth (p>0.05). However, growth in the first year was a strong predictor of future growth (p < 0.001).

CONCLUSION

Initial radiological surveillance is justified in patients with small VS. Yearly MRI needs to be continued for at least 5 years. The main predictor of VS future growth is growth in the first year of follow-up. The published results of SRS-tumor control rate and shrinkage rates need to take into consideration that up to 40% of VS do not grow and up to 20% may shrink spontaneously in the first 5 years after diagnosis without any intervention.

Neuro-Oncol. 2009 Oct;11(5):686.

540. Comparison of Awake Craniotomy Versus Craniotomies Under General Anesthesia for Supratentorial Glioma Resection

Pier Paolo Peruzzi, Adolfo Viloria, Sergio Bergese, E Antonio Chiocca

INTRODUCTION

Awake surgery for glioma resection has long been used to address lesions in the so-called “eloquent areas” of the brain, that is, those directly involved in movement or speech and whose compromise by surgical injury would confer unacceptable patient morbidity. On the contrary, in the U.S., the present standard for all other surgically accessible gliomas is general endotracheal anesthesia. The aim of this study is to compare the two modalities with the final end point of assessing whether either one is superior to the other in terms of perioperative morbidity, patient functional status, length of hospital stay and overall hospital costs.

METHODS

We retrospectively evaluated 24 patients who underwent surgery under conscious sedation (CS) and 24 patients undergoing surgery for cerebral gliomas under general anesthesia (GA) at Ohio State University in the period between January 2006 and January 2009 for operative morbidity, length of postoperative hospital stay, need for rehabilitation and postoperative functional status. Selection criteria for both groups were 1) gross total resection; 2) WHO grade II–IV; 3) supratentorial tumor; 4) KPS 70–100; and 5) operations performed by the same surgeon.

RESULTS

Average hospital stay, including the day of surgery was 3.5 and 4.5 days for patients undergoing CS vs. GA craniotomies, respectively. Five of 24 patients in the CS group (21%) had worsening of their preoperative neurological status, versus 7 of 24 (29%) in the GA group. Also, the need for inpatient rehabilitation was 1 in 24 (4%) and 5 in 24 (20%), respectively. Finally, the average hospital expense for CS was $12,000 per person, versus $15,000 for the patients receiving surgery with GA, with an overall 20% cost reduction in the former group.

CONCLUSION

Under similar circumstances, namely preoperative KPS, extent of resection, hystologic grade, age, and same operating surgeon, patients undergoing surgery with CS for glioma resection have a shorter hospital stay, lower operative morbidity, faster resumption of preoperative activities and lower hospital costs compared to patients receiving craniotomies under GA.

Neuro-Oncol. 2009 Oct;11(5):686–687.

541. Disconnection Syndrome after Tumor Resection

Peter Campbell, David W Andrews

INTRODUCTION

Interhemispheric disconnection syndromes were initially reported in patients after corpus callosotomy for epilepsy control. Few cases of disconnection syndromes have been described in patients with tumors involving the corpus callosum.

METHODS

We reviewed our cases from June 2005 to October 2007 in patients with tumors involving the corpus callosum.

RESULTS

We evaluated four patients. Patient 1: A 49-year-old woman had a headache evaluation which ultimately revealed a 3.8-cm oligodendroglioma. Intraoperatively, SSEPs were lost in the left arm and leg during resection near the splenium. Postoperative neurologic examination demonstrated brief left leg greater than arm weakness, temporary left-sided sensory loss and a left upper quadrant visual field defect. At 6-month follow-u,p the patient had continued visual field loss. Patient 2: A 45-year-old woman with a 3.6 cm falcine meningioma presented with headache and diplopia. Postoperative neurologic exam demonstrated left leg greater than arm weakness (1/5) and bilateral 6th nerve palsies. Six-month follow-up revealed a normal neurologic exam. Patient 3: A 53-year-old woman presented with a 4.3 cm pineal region teratoma and obstructive hydrocephalus. Postoperative exam demonstrated left leg greater than arm weakness. Six-month follow-up confirmed resolution of weakness but continued left numbness and difficulty with left-sided coordination. Patient 4: A 47-year-old female presented with headache and right-sided weakness. A 7.6-cm oligodendroglioma involved of the spenium of the corpus callosum. Postoperative exam demonstrated continued right-sided weakness with nonfluent speech, difficulty with comprehension, naming and left hand ideomotor apraxia. She was unable to read or write, but she could copy a triangle. At 6-month follow-up, the patient continued to have right-sided weakness, but the left hand apraxia and reading difficulties had resolved. At 18 months, the weakness had resolved.

CONCLUSION

We reviewed the findings of four patients with various tumors involving the corpus callosum and their postoperative neurologic examinations. Postoperative findings may indicate motor and/or sensory manifestations or strong signs of hemispheric disconnection. These patients experienced a near complete resolution at 6-month follow-up.

Neuro-Oncol. 2009 Oct;11(5):687.

542. Prognostic Impact of MGMT Silencing in Patients Harboring Recurrent Glioblastoma Multiforme treated with Surgery and BCNU Wafers Implantation: A Prospective Patient Cohort

Philippe Metellus, Bema Coulibaly, Isabelle Nanni, Frederic Fina, Nathalie Eudes, Nathalie Baeza, L’houcine Ouafik, Olivier Chinot, Dominique Figarella-Branger

INTRODUCTION

The O6-methylguanine DNA methyltransferase (MGMT) is a key enzyme in the DNA repair process after alkylating agent action. Epigenetic silencing of the MGMT gene by promoter methylation has been associated with longer survival in patients with newly diagnosed glioblastoma multiforme (GBM) who receive alkylating agents. Here, we evaluated the prognostic value of different biomarkers in recurrent GBM and analyzed the changes in MGMT status between primary and recurrent tumors.

METHODS

Twenty-two patients harboring recurrent GBM treated by surgery with BCNU wafers implantation were enrolled prospectively between 2005 and 2007. We tested the relationship between MGMT silencing in the tumor at recurrence and survival, taking into account other clinical recognized prognostic factors. MGMT status was determined by methylation-specific polymerase-chain-reaction, a high-throughput quantitative methylation assay, and immunohistochemistry. Besides, expression analysis of hMLH1, hMSH2 and TNFAIP3 at recurrence was conducted with regard to prognosis impact.

RESULTS

Median progression-free survival (PFS) and OS after recurrence were 3.6 and 9.9 months, respectively. The 6-month PFS rate after recurrence was 27.2%. On multivariate analysis, only age (p = 0.04) and MGMT promoter hypermethylation at recurrence as determined by MethyLight (p = 0.0012) and MSP (p = 0.004) were correlated with a better PFS. On multivariate analysis, only MGMT promoter hypermethylation at recurrence as determined by MethyLight (p = 0.019) and MSP (p = 0.046) were associated with a better OS.

CONCLUSION

MGMT methylation status is an important prognostic factor in recurrent GBM treated with surgery plus BCNU wafers implantation and therefore is useful in predicting the outcome of GBM therapy at recurrence.

Neuro-Oncol. 2009 Oct;11(5):687.

543. Adult Intracranial Ependymomas: Long-Term Outcome and Prognostic Factors Analysis in a Retrospective Series of 114 WHO Grade II Patients

Philippe Metellus, Jacques Guyotat, Marylin Barrie, Anne Durand, Roch Giorgi, Anne Jouvet, Olivier Chinot, Dominique Figarella-Branger

INTRODUCTION

Ependymomas account for 2% of all intracranial tumors in adults. Low-grade (WHO grade II) ependymomas represent the most frequent (80%) histological subtype in adults. The predictive value of surgery, location and patient-related prognostic factors for these tumors is still unresolved. We report here a retrospective series of 114 adult patients with low-grade ependymomas to address the role of these different prognostic factors in tumor control.

METHODS

We report a retrospective study of a homogenous population of 114 adult patients harboring WHO grade II ependymomas from 24 French neurosurgical centers between 1990 and 2004. All clinicoradiological and follow-up data were analyzed and a central pathologic review was performed by two confirmed neuropathologists.

RESULTS

The 5- and 10-year OS rates for the entire population were 86.1% ± 3.5 and 81.0% ± 4.4, respectively. The 5- and 10-year progression-free survival rates for the entire cohort were 74.6% ± 4.5 and 58.9% ± 6.0, respectively. On multivariate analysis, gross total removal and infratentorial location were found to be associated with a longer overall and progression-free survival. Preoperative KPS scores >80 were also found to be associated with a longer overall survival on multivariate analysis. In patients with incompletely resected tumors, postoperative radiotherapy provided a better PFS but not OS (p < 0.05).

CONCLUSION

Extent of surgery as well as tumor location is a major prognostic factor in adult WHO grade II ependymomas. KPS preoperative scores also represent an independent prognostic factor on overall survival. The impact of adjuvant radiotherapy for patients with incompletely resected low-grade ependymomas seems to be beneficial but needs to be investigated further.

Neuro-Oncol. 2009 Oct;11(5):687.

544. The Transpalpebral Approach to Anterior Cranial Fossa Meningiomas

Khaled M Aziz, Sanjay Bhatia, Erik Happ

INTRODUCTION

We describe our experience with a transpalpebral incision which utilizes normal skin fold in the upper eyelid to obtain access to subfrontal-supraorbital corridor. This approach minimizes the cosmetic problems with the traditional supraciliary approach.

METHODS

The incision for this approach is placed in the superior palpebral sulcus above the tarsal plate. The incision is made by sharply cutting the skin and identifying the orbicularis oculi muscle below. The orbicularis oculi muscle is split and the plane between the orbicularis oculi and the orbital septum is developed superiorly and laterally until the periosteum of the orbital rim is identified. The periosteum is divided sharply and elevated to identify the supraorbital foramen, supraorbital rim, frontal bone and fronto-zygomatic suture from medial to lateral. The anterior fibers of the temporal muscle are elevated from the bone behind the frontal process of the zygoma, to provide room for placing a burr hole. The burr hole is drilled on the fronto-sphenoid suture. The dura is separated and a craniotome is used to create a bone flap about 25 mm in height, extending to the supraorbital notch medially and the temporal burr hole laterally. The anterior and posterior ethmoidal arteries can be cauterized on the medial orbital wall during the approach. We review our experience with four anterior cranial fossa meningiomas.

RESULTS

Four anterior cranial fossa meningiomas (two palnum, one tuburculum, and one olfactory groove) were resected with this approach. The maximum dimension of the meningiomas on MRI ranged from 1.5 to 5.8 cm. The volumes ranged from 2.7 cm3 to 200 cm3. Total resection was achieved in three meningiomas. In the largest meningioma, the tumor was deliberately left on the optic nerve and ACOM complex. The average age of the patients was 45 years. One patient had a postoperative infection requiring further surgeries.

CONCLUSION

The transpalpebral approach provides an excellent cosmetic result, without any restrictions to the subfrontal-supraorbital corridor

Neuro-Oncol. 2009 Oct;11(5):687–688.

545. Treatment of Primary High-Grade Glioma with Sitimagene Ceradenovec (Cerepro) and Ganciclovir Gene Medicine: Update of Phase III Results

Zvi Ram, Manfred Westphal, Peter C Warnke, Philippe Menei, Seppo Ylä-Herttuala

INTRODUCTION

High-grade glioma (HGG) continues to have a dismal prognosis with limited survival, despite multimodal therapy. This study assesses the impact of the delivery of the HSV-tk gene into the healthy tumor bed of resected HGG using a serotype 5 adenoviral vector (Cerepro, sitimagene ceradenovec). This gene induces the expression of thymidine kinase, which phosphorylates the systemically administered ganciclovir, making it locally effective against malignant cells.

METHODS

We recruited 250 patients with operable HGG from 38 centers in Europe and Israel between November 2005 and April 2007. Patients were randomized to receive standard therapy, with or without sitimagene ceradenovec injections. On days 5–19, postoperatively, the active group received ganciclovir (5 mg/kg intravenously twice daily). Adjuvant treatment with temozolomide was permitted, but not obligatory, depending on local treatment practice. We are currently following up with patients for reintervention-free survival, overall survival (OS) and magnetic resonance imaging (MRI) progression, as well as standard safety monitoring. The primary outcome measure is time from surgery until reintervention or death. The principal secondary outcome is time to all-cause mortality OS.

RESULTS

We completed the study in May 2008. A statistically significant improvement existed in the Cerepro primary end point between the active versus control arms (HR = 1.33 p < 0.05). The safety profile was as expected for local therapy in the patient population. Updated results from the OS outcome will be available in Q3 2009 and will be presented at the meeting, along with demographics of the study population, the efficacy of therapy (as Kaplan-Meier survival plots and statistical analysis) and overall safety profile. Of all patient tumor samples, 75% were available for an MGMT assay. The results incorporating the MGMT analysis will be presented.

CONCLUSION

Conclusions will be derived from the results to provide an overall risk and benefit assessment of this form of gene medicine in operable HGG.

Neuro-Oncol. 2009 Oct;11(5):688.

546. Hematological Malignancies Presenting as Neurosurgical Emergency

Pari Kodaiarasan

INTRODUCTION

Hematological malignancies have several clinical presentations, but primary presentation as neurosurgical emergency is not common. Here, we present a few hematological malignancies that required emergency neurosurgical intervention with excellent neurosurgical outcomes.

METHODS

This is a retrospective analysis of seven patients who were treated in our hospital between January 2007 and March 2009 for hematological malignancies who required neurosurgical intervention. We analyzed the clinical features, radiological findings, management and outcomes. The follow-up period ranged from 4 weeks to 24 months.

RESULTS

Patients’ ages ranged from 23 to 65 years with a M:F ratio of 3:4. The disease spectrum included non-Hodgkin’s lymphoma (NHL) (4), leukemia (2), and Hodgkin’s lymphoma (1). All patients with intracranial involvement (subdural hematoma-2, cerebellar lesion with hydrocephalus) had features of raised ICP with acute neurological deterioration. One young male presented with acute onset severe headache and bilateral ophthalmoplegia and ptosis diagnosed as sellar, infrasellar, and parasellar NHL. Patients with spine involvement presented with paraparesis with bladder dysfunction. Lymphoma was the predominant primary hematological malignancy causing neurological complications. All of the patients underwent emergency surgical procedures. One case of leukemia (52/f, GCS 3/15) with subacute subdural hematoma deteriorated and died. All other patients improved clinically and were referred for further treatment. One female with cerebellar NHL developed spinal metastasis after cranial radiotherapy. Another young male with D11 vertebral body collapse had relapse (mediastinal) after chemotherapy during follow-up.

CONCLUSION

Prompt and optimal neurosurgical intervention definitely improves the quality of life and decreases morbidity despite the natural course of the primary hematological disorder.

Neuro-Oncol. 2009 Oct;11(5):688.

547. Managing the Difficult Meningiomas in the First Year of Complex Cranial Practice

A Samy K Youssef

INTRODUCTION

Meningiomas are intuitively benign lesions that attract most neurosurgeons to manage. The great majority are of benign pathological nature and in accessible anatomical locations. Over the years, meningiomas have become popular among most neurosurgeons, which tempted practitioners with diverse experiences and institutional levels to deal with these lesions. However, tertiary referral centers remain the final destination for some categories of complex meningiomas that require a multidisciplinary approach of management that is not available in most hospitals. For example, parasagittal, falcotentorial and skull base meningiomas are complex by location and require different management strategies. Starting an academic practice in complex cranial surgery faces the challenge of managing such difficult lesions.

METHODS

In this presentation, we review the different categories of complex meningiomas, identify the management challenges and discuss the multidiscipinary aproach of management in light of recent advances in microsurgery and adjuvant therapy techniques. We present a diverse clinical series of complex meningiomas and share the unique experience of the first year in an academic practice of complex cranial surgery.

RESULTS

The challenge posed by difficult meningiomas at the beginning of an academic practice in complex cranial surgery can be overcome by applying a multidisciplinary team approach towards management. Surgical resection ranged between Simpson grades I and III. Complications were limited to postoperative hydrocephalus and reversible cranial nerve deficits.

CONCLUSION

Meningiomas are generally benign but can pose challenges to neurosurgeons. Management of difficult meningiomas requires the resources available in academic tertiary referral centers. The presence of residents, fellows and senior-level experience was invaluable in achieving optimum outcomes.

Neuro-Oncol. 2009 Oct;11(5):688.

548. Cervical Radiculopathy and Fever due to Foramen Magnum Meningioma

Alexa L Reeves, Marc A Vanefsky

INTRODUCTION

Etiologies of cervical radiculopathies and fevers include foramen magnum tumors extending to the cervical spine and compressing brainstem autoregulatory centers.

METHODS

A 64-year-old right-handed woman with a C6 radiculopathy and X-ray showing degeneration at the C6–C7 disc space underwent conservative management for 3 years. She developed dizziness and numbness spread to the third digit of her right hand, left hand and throat. Cervical spine magnetic resonance imaging (MRI) revealed a homogenously enhancing mass, 3.9 cm anterior-posteriorly, 3.0 cm transversely, and 3.7 cm in the rostrocaudal direction compressing her brainstem. On neurosurgical evaluation, she had difficulty with writing and proprioception. Hoffmann’s and Babinski’s reflexes were present on the right. Past medical history was significant for chronic idiopathic fevers.

RESULTS

The patient underwent a suboccipital craniotomy and C1 laminectomy with somatosensory evoked potential (SSEP) and EMG monitoring of CN IX, X, and XI. She was placed prone on the Wilson frame with a Mayfield head holder in the Concord position. The head was elevated over the heart. A midline incision was created and the paraspinous muscles were stripped from C2 to the inion; four burr holes were placed to create a square cranial flap. The dura was opened in a Y-shaped fashion and after frozen biopsy confirmed meningioma, the tumor was internally decompressed and gross total resection attained. Intraoperative stimulation of a C2 rootlet and CN XI confirmed their location and avoided their inadvertent ligation.

CONCLUSION

The tumor extended caudally to C2, rostrally displaced the cerebellum, and ventrally to the clivus. Rarely is the clivus exposed from the suboccipital approach. Fever etiology included direct compression of the raphe pallidus and magnus nuclei, which are centers for excitatory sympathetic premotor neurons involved in thermoregulation. Expression of vesicular glutamate transporter 3 may allow these neurons to mediate fever.

Neuro-Oncol. 2009 Oct;11(5):688.

549. Rare Case of Intradural and Extradural Meningioma of the Thoracic Spine with Dural Ossification

Azedine Medhkour, Sunil A Mutgi

INTRODUCTION

Spinal meningiomas are rare. Approximately 2.7%–15% of spinal meningiomas are both intradural and extradural. Dural ossification associated with extradural and intradural meningioma is extremely rare. We recently treated a 61-year-old female with thoracic spinal meningioma with intradural and extradural components and dural ossification.

METHODS

We report a 61-year-old female who presented with numbness and tingling from her torso to her feet bilaterally. She was originally diagnosed with peripheral neuropathy, later evaluated with magnetic resonance imaging (MRI) and found to have a mass lesion at the level of T4–T5 with cord compression. Intraoperatively, the tumor was visible epidurally at T4–T5 in an en-plaque shape with a small anterior-lateral component. The superior aspect of the tumor tracked towards the T4 root. Intraoperative ultrasound confirmed an intradural component. Micro-surgical technique allowed for total resection. Removal of the partially ossified dura was extremely challenging. A thin layer of dura, free of any visible tumor, obviated the need for a dural graft.

RESULTS

The pathology reported the tumor as a benign meningioma, psammomatous type. The report confirmed the presence of dural ossification and many areas of intratumoral calcifications. The postoperative course was uneventful with a rapid regression of many of the preoperative neurologic deficits.

CONCLUSION

Thoracic spinal meningiomas can be challenging. Their resection can lead to disastrous outcomes. Resection of the extradural or intradural component, particularly the ossified portion, should be done without any manipulation of the spinal cord. Resection of the calcified dura should be extremely meticulous. When feasible, dural graft should be applied.

Neuro-Oncol. 2009 Oct;11(5):688–689.

550. Subarachnoid Metastasis of Atypical Meningioma

Alexa L Reeves, Devin K Binder, Ronald Kim

INTRODUCTION

Meningiomas originate from arachnoid tissue and most commonly attach to the inner surface of the dura mater as well-circumscribed, distinct tumors. Atypical meningiomas (WHO grade II) account for 4.7%–7.2% of meningiomas. They typically spread directly or seed the cerebrospinal fluid. Subarachnoid spread is rare, and no report exists of a subarachnoid metastasis of an atypical intraaxial meningioma.

METHODS

A 28-year-old female experienced intense neck pain, bilateral auditory deficits and visual changes postpartum. A right posterior fossa mass was discovered on magnetic resonance imaging (MRI), but the patient refused surgical resection. Her vision worsened and on exam a few months later, had severe papilledema with flame-shaped hemorrhages in both eyes. The patient then opted for treatment. EVD was placed and suboccipital craniotomy was performed for resection of tumor.

RESULTS

A neuropathologist diagnosed an atypical meningioma from the primary tumor cavity and distant site. The patient recovered from surgery without complication or recurrence 6 months later.

CONCLUSION

A diagnosis of atypical meningioma increases the odds of recurrence by more than eight times and makes them more likely to metastasize. Brain invasion is possible in any grade and positively correlates with recurrence. Spread via the subarachnoid space could be explained by reconsidering the clonality of multiple meningiomas. Common chromosomal deletions associated with atypical meningiomas include 1p, 14q, and 18q, as well as the loss of progesterone receptor expression in addition to the classic deletion of the long arm of chromosome 22 and mutations of NF2. Although some studies have suggested that some meningiomas could be polyclonal, most indicate they are monoclonal as shown by experiments done by Jacoby et al. with Southern blot analysis and X-chromosome inactivation. They and others have shown that most meningiomas have only a single mutation in the NF2 gene. However, the possibility that these meningiomas could be polyclonal and represent a genetic mosaic with separate NF2 mutations exists.

Neuro-Oncol. 2009 Oct;11(5):689.

551. Angioleiomyomas in the Cavernous Sinus Mimicking Cavernous Hemangiomas: Two Case Reports and Literature Review

Zhengyan Liu, Liangfu Zhou, Ying Mao

INTRODUCTION

Angioleiomyomas are benign neoplasms with smooth muscle and vascular components. The fact that they are extremely rare in intracranial locations often causes misdiagnosis before treatment. In recent years, we surgically treated two cases of angioleiomyomas that were both diagnosed as cavernous hemangiomas before surgery. Here, we present the patients’ clinical, radiological and operative data, emphasizing the pathological and differential diagnosis and management of this rare lesion.

METHODS

The first case was a 41-year-old male patient, complaining of bilateral eyesight decrease for 2 years and temporal visual field deficit in the left eye. The second case was a 46-year-old female patient, suffering from horizontal diplopia and facial numbness in the left side. Preoperative magnetic resonance imaging (MRI) revealed an occupying lesion in the cavernous sinus and intrasellar region (case 1) with hyposignal on T1WI and hypersignal on T2WI, and homogeneous enhancement after Gd-DTPA injection. These two patients were both preoperatively diagnosed as cavernous hemangiomas based on radiological findings.

RESULTS

For case 1, intranasal transsphenoidal sinus approach was performed to remove the tumor in the intrasellar and suprasellar region and the optic nerves were decompressed completely. A small piece of tumor originating from the cavernous sinus remained intact. For the second case, the tumor was removed “en bloc” by modified pterional craniotomy with an extradural approach to the cavernous sinus. Cranial nerves were identified and protected very well. Both cases were pathologically diagnosed as angioleiomyomas. Immunohistochemical assessment revealed SMA, Vimentin and CD34 were strongly expressed. Postoperative short-term follow-up found the two patients improved in neurological deficits.

CONCLUSION

Angioleiomyomas are rare tumors, causing difficulties in making correct diagnosis before surgery, especially in differentiating angioleiomyomas from cavernous hemangiomas. Angioleiomyomas are also benign tumors with good prognosis if removed with mini-invasive technology.

Neuro-Oncol. 2009 Oct;11(5):689.

552. Mini-Invasive Unilateral Approach for the Removal of Intradural Extramedullary Tumors

Alfredo Pompili, Fabrizio Caroli, Fabio Cattani, Laura Raus, Stefano Telera, Antonello Vidiri

INTRODUCTION

Spinal intradural tumors are generally removed by uni- or multilevel laminectomy with midline dural incision. Delayed postoperative pain, kyphosis, and spinal instability that occur in 6% of the patients may be reduced by a more conservative unilateral microsurgery, sparing bilateral damage.

METHODS

Sixty patients, 38 with neurinoma, 9 meningioma, 5 caudal ependymoma, 2 conus medullaris dermoid tumor, and 6 miscellaneous (intramedullary metastases, radionecrosis, neuro-cytoma, non-Hodgkin’s lymphoma) were operated upon between June 2000 and December 2008. There were 64 tumors (double meningioma, double ependymoma, triple neurinoma). Surgery was performed in the prone position with a unilateral approach. The extension of the laminectomy was generally kept to one level. When necessary, it was extended craniocaudally for 1.5–2 cm. The dura was opened paramedially and the tumor dissected and removed either “en bloc” or piecemeal. Dural closure was done with 5–0 or 6–0 stitches. We suggest nonresorbable material. Tumor dimensions ranged from 1.2 to 2.8 cm in maximum diameter. Duration of surgery ranged from 120 to 300 min (average, 160).

RESULTS

All the patients were mobilized on day 2–3 and discharged on day 4–5. No important complications due to the technique were observed, and postoperative pain was minimal. Three patients had persistent ortostatic headache. One patient had a pseudomeningocele under the muscular plane that required reoperation. Overall, neurological results were good: those with preoperative pain improved dramatically, and those with neurological focal deficit recovered almost completely or completely. One patient with a melanocytic malignant schwannoma died of intradural dissemination after 1 year.

CONCLUSION

Hospital stay and postoperative pain may be reduced and stability preserved with an appropriate microsurgical mininvasive technique. In select patients, it can be used also for small intramedullary lesions, although in our cases it was done because of preoperative misdiagnosis. In case of operative difficulty, it can be converted rapidly into a traditional approach.

Neuro-Oncol. 2009 Oct;11(5):689.

553. Stereotactic Biopsy of Intracranial Tumors with Intraoperative MRI

Michael Schulder, David Spiro, John C Quinn

INTRODUCTION

Stereotactic brain biopsy (SBBx) remains an important tool in the management of patients with brain tumors. Despite advances in technology, biopsy techniques based on preoperative imaging have about an 8% likelihood of being nondiagnostic. Intraoperative magnetic resonance imaging (iMRI) offers a way to improve these results.

METHODS

Biopsies were performed using the PoleStar N20 iMRI (Medtronic Navigation), a compact, mobile system based on a 0.15 tesla magnet. Images acquired in the operating room were used to create a surgical plan. A skull-mounted guide (Navigus, Medtronic Navigation) was used to define a trajectory and measure the distance to the target. An MRI-compatible biopsy cannula was inserted and a repeat image obtained. If the cannula position was acceptable, then a biopsy was taken. If necessary, repeat targeting and imaging were done. A frozen section was examined to ensure that lesional tissue was obtained.

RESULTS

iMRI-guided SBBx was performed on 35 patients. Of these patients, 22 proved to have gliomas (8 low-grade and 13 high-grade), with other tumors diagnosed in two patients. Inflammatory or infectious lesions were confirmed in 11, five of whom were presumed to have tumors preoperatively. iMRI setup and imaging added a mean of 1.1 h under general anesthesia to the time needed to do a frameless SBBx. Of note is that over the last 12 months, four “conventional” SBBx (two framed, two frameless) at the authors’ institution failed to yield a diagnosis. All underwent successful repeat biopsies (two with iMRI).

CONCLUSION

SBBx can be done in a low field iMRI environment with a technique that provides improved diagnostic yield compared to “conventional” procedures using framed or frameless approaches. An added benefit is ruling out hemorrhage before closing the incision. This justifies the relatively short additional time incurred in the OR for iMRI setup and imaging.

Neuro-Oncol. 2009 Oct;11(5):689.

554. Intraoperative MRI for Surgery of Intracranial Meningiomas

Michael Schulder, David Spiro

INTRODUCTION

Intraoperative MRI (iMRI) ideally can be applied to the surgery of any patient with an intracranial tumor. Meningiomas are extraaxial tumors whose borders can almost always be defined by visual inspection. We analyzed our experience using iMRI on patients with meningiomas.

METHODS

Surgery was done using the compact, mobile Pole-Star N20 iMRI (Medtronic Navigation), based on a 0.15 tesla magnet. The system has an integrated infrared-based navigation tool. iMRI was used if the specific OR was available and if there was no patient-related contraindication to its use (cardiac pacemaker, very large body habitus).

RESULTS

Sixty-three operations for meningiomas were done on 60 patients. Of these, 33 operations were done on 32 patients for tumors that were above the skull base (group A: 13 parasagittal, 12 convexity, 5 anterior falcine, and two intraventricular), and 30 on 28 patients with skull base tumors (group B: 12 midline anterior, 12 sphenoid wing, and 8 lateral posterior fossa). In four group A surgeries, intraoperative imaging led to additional tumor resection; in one patient with a deep falcine tumor, unnecessary dissection was avoided after imaging confirmed complete resection. In one patient with a parasagittal tumor, further resection after imaging may have led to a surgical cure. In group B operations, additional tumor was removed after imaging in four patients, with additional dissection avoided in 1. No surgical cures were achieved (or intended) in these patients. Additional time incurred from setup and imaging with iMRI averaged 1.3 h (1.8 h for patients with skull base meningiomas).

CONCLUSION

iMRI is not likely to have a significant impact on surgery in patients with intracranial meningiomas. In all but a few of these patients, frameless stereotaxy with preoperative imaging can be used without incurring the additional time and other potential limitations of the iMRI environment.

Neuro-Oncol. 2009 Oct;11(5):690.

555. Neurochemical Profile of Human Brain Tumors: Differentiation of Low-Grade from High-Grade Neoplasms with High-Resolution Magic Angle Spinning 1H-MRS

Caitlin C Shapiro, Sandeep Mittal, Farhad Ghoddoussi, Matthew Galloway

INTRODUCTION

At present, clinical scanners cannot resolve the constituents of the choline peak and typically do not asses levels of neuro-chemicals such as alanine or taurine. Therefore, we used high-field, high-resolution 1H-MRS to determine the neurochemical profile in brain tissue freshly excised from patients with various brain neoplasms. Specifically, we hypothesized that neurochemical profiles would distinguish aggressive tumors based on energy status as well as membrane turnover.

METHODS

Tumor specimens were obtained during surgery from patients harboring glioblastomas, low-grade gliomas, meningiomas, and brain metastases. Tissues were resected without prior arterial occlusion to minimize hypoxia. Samples were promptly frozen on solid CO2 and neurochemical profiles in intact tissue specimens were obtained with high-resolution magic angle spinning 1H-MRS at 11.7 tesla. LCModel was used for postprocessing and quantitation. Noncancerous cortical gray matter from four patients was also analyzed.

RESULTS

Compared to low-grade gliomas, choline and phosphorylcholine, glycine, alanine, and lactate (as well as the alanine [ALA]/lactate [LAC] ratio) were significantly increased in glioblastomas; levels of myo-inositol were substantially reduced. Compared to noncancerous cortex, meningioma had markedly reduced creatine, N-acetylaspartate, LAC, and INS, whereas ALA, glutathione, taurine, choline, and phosphorylethanolamine (PEA) increased. ALA increased 5-fold in meningioma and the ALA/LAC ratio distinguished this nonneuronal tumor; taurine increased 8-fold in the meningioma.

CONCLUSION

Increased levels of energy-related neurochemicals (ALA, LAC) and CHO-containing compounds in glioblastomas are consistent with increased cellular proliferation and increased membrane turnover, respectively, in these aggressive tumors. Increased levels of total cholines in meningioma (compared to noncancerous cortex) are consistent with established clinical MRS observations and increased ALA/LAC is consistent with shifted energy homeostasis. Similarly, increases in taurine may reflect apoptosis-induced cell shrinkage in the meningioma. Overall, the results demonstrate the utility of HR-MAS 1H-MRS to provide insight into the diagnostic and neurobiological characterization of brain tumors related to levels of anaplasia.

Neuro-Oncol. 2009 Oct;11(5):690.

556. Three-Stage Resection of a Multilevel Cervical Spine Chorodoma in a 74-Year-Old Male

Terrence Pencek, Gordon KT Chu

INTRODUCTION

Chordoma is a malignant tumor most common in the clivus and sacral region. Cervical spine chordomas are considered rare tumors and found in patients age 6–60 years of age. En bloc resection has been considered for tumors with surgical resection time up to 40 h.

METHODS

We report a 74-year-old man who underwent a three-stage retrocarotid, retrojugular resection with C3–C4 spondylectomy with left vertebral artery dissection from C2 to C4. We will describe the otolaryngoligical and neurosurgical exposure as well as the medial carotid instrumentation approach. This patient underwent a 3-h and a 9-h resection with a 4-h posterior instrumentation and tolerated it with a residual left Horner’s syndrome and mild left deltoid weakness. This was carried out with concomitant anterior instrumented fusion from C2 to C5. The tumor had extended into the soft tissues of the neck with epidural and cervical cord compression. We also carried out lymph node dissection, with 18 nodes negative. The posterior instrumented fusion was carried out from C2 to C6.

RESULTS

The patient underwent a staged operation for resection of a cervical spine chordoma with minimal neurologic changes.

CONCLUSION

We believe that this is the oldest individual described and do not feel that age should be a factor in determining the extent of resection in this minimally radiosensitive tumor.

Neuro-Oncol. 2009 Oct;11(5):690.

557. Hot-Spot Areas in FET-PET Delineate Malignant Tumor Parts within Suspected WHO Grade II Glioma

Mathias Kunz, Rupert Egensperger, Gabriele Poepperl, Julia Geisler, Markus Holtmannspoetter, Hans Kretzschmar, Jörg-Christian Tonn, Friedrich-Wilhelm Kreth

INTRODUCTION

Metabolic imaging such as [F-18]fluoroethyl-tyrosine (FET) positron emission tomography (PET) often reveals heterogeneous findings in suspected WHO grade II gliomas. The extent to which metabolic active areas (hot-spots) correlate with a malignant phenotype remains unclear; conversely, it is unclear whether nonhotspot areas indicate more benign tumor parts. Thus, a prospective correlative topographical analysis of FET-PET imaging and neuropathological findings was conducted.

METHODS

Adult patients with a magnetic resonance imaging (MRI)-based suspicion of a supratentorial WHO grade II glioma were included after prior informed consent. Maps of FET uptake kinetics were generated for each tumor. Areas with suspected malignant biology (hot-spots) were outlined three-dimensionally. For histological evaluation and correlative analysis, a PET-guided stereotactic biopsy technique was used. Tissue samples were taken in 1-mm steps throughout each tumor and evaluated independently by two neuropathologists unaware of the PET findings. Grading was done according to WHO criteria.

RESULTS

Fifty-two patients were included. Nineteen samples per tumor (median) were evaluated. Nine patients exhibited a heterogeneous uptake kinetic within the tumor: grade II characteristics were seen outside and grade III characteristics within the hot-spot in these tumors. Thirty-two tumors showed a kinetic indicative of low-grade glioma throughout the whole tumor: Stepwise histological evaluation confirmed grade II characteristics in 31 patients and displayed grade III characteristics in one patient. In 11 tumors, hot-spot areas covered the entire tumor volume: Histological evaluation revealed malignant glioma characteristics throughout the tumor in 10 patients and WHO grade II characteristics in 1patient. Hot-spot areas were associated with a significant higher Ki-67 labeling index (15% vs. 4%, p < 0.01).

CONCLUSION

Homogeneously or heterogeneously distributed histological characteristics within suspected low-grade gliomas can be successfully delineated by dynamic FET-PET evaluation; areas with a malignant phenotype can be reliably identified. This finding has implications for biopsy and treatment planning.

Neuro-Oncol. 2009 Oct;11(5):690.

558. Assessment of the Peritumoral Microenvironment following Craniotomy using Intracerebral Microdialysis

Behnam Badie, Mike Yue Chen, Timothy W Synold, Jana Portnow

INTRODUCTION

Local inflammatory response following surgery for malignant brain tumors can affect the efficacy of targeted agents injected into the peritumoral tissue. To better understand the peritumoral microenvironment responses to surgery, we used intracerebral microdialysis to examine extracellular levels of cytokines, chemokines and growth factors that are secreted shortly after tumor resection.

METHODS

Five patients undergoing craniotomy for resection of primary or metastatic brain tumors were enrolled in this IRB-approved study. All patients received perioperative steroids. A CMA 71 microdialysis catheter (Solna, Sweden) with a 100-kDa molecular weight cutoff semipermeable membrane was inserted within 5 mm of the resection edge after tumor removal. Following postoperative CT placement confirmation, catheters were perfused with 2% Dextran 40. Dialysate samples were collected at 6-h intervals continuously for 96 h and analyzed for 30 cytokines and chemokines using the Protein Multiplex Immunoassay Kit.

RESULTS

There were no complications from catheter insertion. Dialysate samples could not be collected from two patients due to catheter failure. Among the proinflammatory cytokines measured, IL-6 levels were the highest (400–1,500 pg/ml) in all three patients up to 48 h after surgery, with a gradual drop over the next 2 days. In one patient, transient increases in proinflammatory IL-4 and IL-12 levels (50 pg/ml) were also observed within the first 24 h of surgery Levels of IL-1a, IL-2, IL-5, IL-7, IL-10, IL-13, IL-15, IL-17, TNFα, IFN-γ, EGF, and VEGF remained low (<10 pg/ml) or undetectable in all three patients. Among the chemokines evaluated, MCP-1 (3,000–30,000 pg/ml) and IL-8 (200–1,500 pg/ ml), levels were the highest and remained elevated (100–500 pg/ml) during the monitoring period. Interestingly, levels of IL-1ra (a secreted IL-1 antagonist) also remained elevated in two patients during this postoperative period.

CONCLUSION

To our knowledge, this is the first report of intracerebral microdialysis assessment of peritumoral microenvironmental changes after tumor resection. Our preliminary findings indicate a transient proinflammatory response followed by sustained release of cytokines and/or chemokines involved in monocytes chemotaxis and activation in peritumoral brain tissue. This data will serve as a baseline to compare cytokine and chemokine changes in response to targeted therapies in future studies.

Neuro-Oncol. 2009 Oct;11(5):690–691.

559. Spinal Cord Radionecrosis after Spine Irradiation for Metastatic Breast Cancer

Alfredo Pompili, Fabrizio Caroli, Fabio Cattani, Maddalena Giovannetti, Laura Raus, Stefano Telera, Antonello Vidiri

INTRODUCTION

Cerebral radionecrosis is a well-known complication after whole brain radiotherapy and/or radiosurgery. Quite rare is spinal cord radionecrosis after irradiation of the spine for metastatic disease.

METHODS

In 2008, we observed and operated upon two females with irradiated spine for metastatic breast cancer that developed severe neurological impairment due to spinal cord lesion.

RESULTS

The first patient, 61 years old, had a cervical enhancing lesion on the right portion of the C5-C6 cord. Irradiation had been done in 2007 with a total multifractioned dose of 21 Gy. At operation, only necrotic tissue was found in the cord, just under the pia mater. The severe neurological conditions were unchanged. A therapeutic assay with a high dose of corticosteroid and hyperbaric oxygen had no results. The second patient, 59 years old, had been irradiated on the dorso-lumbar spine in 2007 (total dose 30 Gy in 10 fractions). In 2008, she developed a severe paraparesis. MRI showed cord alterations at D10–D11 on T2 sequences. At operation, we found plastic arachnoiditis and intramedullary subpial necrosis. Despite aggressive medical therapies, this patient did not improve. Neither hystology showed any malignant cells, but only necrotic material.

CONCLUSION

Spinal radiotherapy is frequently used in metastatic cancer disease, either for therapeutic purposes or as palliation for intractable pain. An increase of spinal radionecrosis may be expected in the next years as spinal radiotherapy is more and more widely used. A severe neurological deterioration in these patients should raise the suspicion of this complication. Differential diagnosis should be discussed versus intramedullary metastases (first patient) and/or spinal cord ischemia (second patient). In our cases, the doses and techniques used for irradiation were “standard”; thus, there should be some individual sensibility. Nevertheless, it is a severe event in the clinical history of these patients because at present there is no demonstrated effective treatment.

Neuro-Oncol. 2009 Oct;11(5):691.

560. Solution of Brain Shift by Bipolar Needle Electrode in Subcortical Tumor Resection near the Motor Tract

Fumio Yamaguchi, Toyoyuki Kojima, Hiroshi Takahashi, Akira Teramoto

INTRODUCTION

Brain shift is a significant problem when neuro-navigator is used for brain tumor resection near the motor tract. Moreover, tractography integrated into neuronavigator is not reliable by the influence of brain shift. Electrical stimulation of white matter is necessary to prevent postoperative neurological deterioration in these cases. However, it is sometimes too late to prevent destruction of the pyramidal tract after resection of the adjacent tissue that is important for blood circulation of this tract. To overcome this problem, bipolar needle electrode has been used and evaluated.

METHODS

Glioma patients underwent removal of tumor located adjacent to pyramidal tract. We used a NY Tract Finder bipolar needle electrode and a new electrode NY Tract Finder II, which was modified for navigation-assisted detection of motor tract in cerebral white matter Motor-evoked potentials were induced by electrical stimulation with these electrodes when they were inserted toward the estimated motor pathway from the tumor resection cavity with guidance by a neuronavigator. The scale on the needle indicated the approximate distance from the tumor cavity wall to the pyramidal tract.

RESULTS

We detected pyramidal tracts in all cases. Postoperative MRI revealed that the tumor was resected close to the primary motor area and pyramidal tract even with brain shifts. None of the patients showed postoperative neurological deterioration using this method. The Karnofsky performance scale of those patients did not change or increase.

CONCLUSION

This technique may be a feasible method for detecting and sparing the motor pathways. Especially when brain shift is not ignored near motor tract, electrophysiological monitoring is a reliable technique. Even if brain shift exists; neuronavigator is still useful to estimate the approximate direction of the pyramidal tract from the tumor resection cavity wall. Also, this may be an effective method to avoid damage to the pyramidal tract and adjacent tissue, important for regional blood circulation.

Neuro-Oncol. 2009 Oct;11(5):691.

561. Primary Intraosseous Paraganglioma of the Sacrum

Thomas P Lo, Ravi Raghavan, Walter Johnson

INTRODUCTION

Primary intraosseous sacral paragangliomas are among the rarest of lesions to affect the sacrum, as there are only three other cases reported to date and these masses are typically found in the adrenal medulla, carotid body and glomus jugulare. Its origin is controversial, as CNS tissue lacks neuroendocrine cells, and some have proposed faulty cellular migration, residual neuroblasts and ependymal cells as potential etiologies. Reported outcomes have been favorable, however, with authors describing either one or a combination of preoperative embolization, surgical resection, radioactive injections and/or stereotactic radiosurgery in the current treatment regimen.

METHODS

A 63-year-old female dentist, neurologically intact, with a history of hypercholesterolemia, presented to the clinic with sacral pain associated with tenderness to palpation. She was recently traveling in Vietnam and urged to get an MRI, which showed an erosive 3.3 × 2.2 × 2.2 cm midline sacral mass at the level of S3 with some brightly enhancing features but was not hyperintense on T2 imaging. Her management included open complete resection without preoperative embolization, followed by oncologic evaluation for chemotherapy and radiation therapy.

RESULTS

A highly vascular lesion was found intraoperatively that both frozen and final immunomorphological evaluation confirmed as a paraganglioma. She was referred to the Comprehensive Brain Tumor Program treatment center at UC Irvine for further chemotherapy and radiation therapy, and postoperative clinic followup approximately 2 months later showed complete resolution of her presenting back pain without new deficit.

CONCLUSION

Intraosseous sacral paragangliomas are among the rarest of lesions at this spinal location and should be included in the differential diagnosis of sacral lesions. Three other cases have been reported thus far, and these, taken in unison with this study, show that satisfactory postoperative outcomes are achievable despite management variations employing anything from a single to a combination of the aforementioned modalities.

Neuro-Oncol. 2009 Oct;11(5):691.

562. Chemoport as a Durable and Easily Accessable Replacement for the Ommaya Reservoir in the Treatment of Leptomeningeal Carcinomatosis

Ho-Shin Gwak, Heon Yoo, Seung Hoon Lee, Sang Hoon Shin, Hee Seok Yang, Ji Yeon Han, Heung Tae Kim, Jin Soo Lee

INTRODUCTION

Ommaya reservoirs are frequently used to deliver intraventricular chemotherapy in cancer patients with leptomeningeal metastases. But the Ommaya reservoir has a few limitations and complications. We introduced the technique of chemoport insertion in the replacement of conventional Ommaya reservoir to enhance intraventricular accessibility and reduce Ommaya-related complications.

METHODS

Basic techniques of intraventricular chemoport placement are the same with Ommaya reservoir. A hockey stick incision is centered at Kocher’s point (10 cm posterior from the nasion and 3 cm lateral to the midline), generally on the right (nondominant) side). A burrhole is drilled at Kocher’s point and the outer layer of the posterior part of the skull is engraved in the shape of chemoport so that the chemoport can be set down inside it, thereby reducing the height of whole device over the scalp closure. A ventricular catheter is inserted thorough single burrhole following a planned trajectory and depth, assisted by a stereotactic system. After confirmation of ventricular access by CSF flow, the catheter is then connected to the chemoport reservoir and the chemoport is secured with a 4-0 silk suture to the periosteal apron.

RESULTS

Between February 2007 and December 2008, we placed chemoport reservoirs in 80 patients for the treatment of leptomeningeal metastases after confirmation of CSF cytology at the National Cancer Center. Intraventricular methotrexate injection was then followed 5 days after the insertion repeatedly (twice a week at the most). Only one intraventricular hemorrhage occurred as installation-related complication. We observed no CSF or chemotherapeutic agent leakage in a total of 726 chemoport punctures and methotrexate injections (average, 9.1 injections per patient). We received no formal report of failure to puncture the chemoport from the physician, while four cases of catheter malfuction were reported and the catheters reinstalled. The causes were two cases of malposition and two cases of catheter obstruction from the cellular debris. The median survival of patients treated for leptomeningeal metastases was 9 months. One case of CSF infection occurred to the patient who had prolonged extraventricular drainage due to hydrocephalus.

CONCLUSION

We recommend this new technique as a more durable and easily accessible tool for intraventricular chemotherapy for the treatment of leptomeningeal metastases.

Neuro-Oncol. 2009 Oct;11(5):691–692.

563. Intracerebral Hemorrhage Secondary to Thrombocytopenia in a Patient Treated with Temozolomide

Durga R Sure, Ian F Dunn, William S Anderson

INTRODUCTION

Thrombocytopenia-related intracerebral hemorrhage secondary to chemotherapy-induced myelosuppression can be very difficult to manage and may result in the death of the patient. Temozolomide is an orally available cytotoxic drug used in high-grade gliomas. We are presenting this complication in a patient with high-grade glioma treated with temozolomide.

METHODS

We present a chart review of the patient with intracerebral hemorrhage as a complication after a fall in the setting of temozolomide induced thrombocytopenia. A literature search shows possible complications of temozolomide therapy and patients’ management.

RESULTS

A 26-year-old male with no significant past medical history presented with gradually worsening headaches and nausea. He was found to have a right parietooccipital lesion on MRI. After surgical resection, pathology onfirmed glioblastoma multiforme. He was treated with radiation and chemotherapy, including initial temozolomide, which was subsequently discontinued. Approximately 4 years after the surgery, the patient was restarted on temozolomide therapy in the context of further tumor growth. He subsequently presented with a large right flank hematoma and a right cerebellar bleed with platelet counts of approximately 10,000/μl. He had rapid replacements of platelets and evacuation of the hematoma twice with eventual loss of neurologic examination. Hematoma recurrence was immediate after the last evacuation. Care was eventually withdrawn.

CONCLUSION

A dose-dependent severe and possibly irreversible myelotoxicity can occur with temozolomide treatment resulting in thrombocy-topenic intracerebral hemorrhage. Close monitoring of hematological labs with prophylactic transfusions as needed can help in avoiding fatal outcomes. Identifying and treating underlying associated conditions that affect platelet function and monitoring for immunologic platelet refractoriness well in advance in patients receiving prophylactic transfusions helps in the management of acute conditions. A multidisciplinary approach in management is strongly advised.

Neuro-Oncol. 2009 Oct;11(5):692.

564. Gamma Knife Radiosurgery to the Resection Cavity following Surgical Excision of Brain Metastases Results in Excellent Local Control

Abdullah Kandil, Abraham Boskovitz, Tomas Dvorak, Kevin C Yao, John Mignano, Julian K Wu

INTRODUCTION

Historically, management of resected brain metastases included adjuvant whole brain radiation therapy (WBRT). Concerns about neurocognitive deterioration after WBRT recently led to the use of focal radiotherapeutic techniques. The purpose of this study was to analyze the outcomes of patients with brain metastases treated with surgery and adjuvant Gamma Knife radiosurgery (GKRS) to the surgical resection cavity at our institution.

METHODS

We retrospectively reviewed medical records of patients who underwent surgical resection of a brain metastasis followed by GKRS between 2000 and 2005. GKRS had to be administered within 3 months postoperatively. We reviewed clinical, radiodosimetric, and outcome data.

RESULTS

Follow-up data was available for 17 patients. Mean age was 59.6 years (37–74). Primary tumor was lung (10), melanoma (4), breast (2) and rectum (1) carcinoma. A mean dose of 19.8 Gy (15–24) was administered to a mean targeted postoperative cavity volume (TV) of 7.9 cm3 (0.3–51.4 cm3). A median of 98% (88%–100%) of TV was included within 50% prescribed isodose volume (PIV). On average, PIV exceeded TV by 1.94-fold. Mean Paddick conformality index was 0.55 (0.18–0.79). At a median follow-up of 22.1 months, eight patients were alive. Local control was achieved in 94% (one failure); regional progression was found in 53% of the cases.

CONCLUSION

Although limited by its small size, this study suggests that adjuvant GKRS to the surgical resection cavity of a brain metastasis provides excellent local control. Local failure was seen in one case, which at a median follow-up of 22.1 months is comparable to or better than data in the literature for adjuvant WBRT or stereotactic radiosurgery. These results may be due to the PIV being significantly larger than the TV as a margin surrounding the resection cavity was included within the field, which is reflected by a relatively low conformality index. No neurotoxicity was observed.

Neuro-Oncol. 2009 Oct;11(5):692.

565. Olfactory Changes following Transnasal Endoscopic Resection of Pituitary Tumors

Philip V Theodosopoulos, Lee Zimmer, Catherine Hart

INTRODUCTION

Given that the transnasal endoscopic approach to the sella involves resection of anatomical structures known to contain olfactory nerve fibers, we evaluated the effect of this approach on olfaction.

METHODS

Study design: prospective case series. From October 2007 to December 2008, 50 adults diagnosed with a pituitary mass and scheduled to undergo transnasal endoscopic resection at a single tertiary care institution underwent preoperative olfactory evaluation using the University of Pennsylvania Smell Identification Test. We performed repeat testing at 1 and 3 months postoperatively. Paired Student’s t-tests were used to compare pre- and postoperative scores. All procedures were performed by the senior author utilizing the same operative technique. A clinically significant change was assumed to be 25% based on previous olfactory studies.

RESULTS

Fifty consecutive patients were enrolled. Thirty-five patients (21 men, 14 women) have completed olfactory evaluation at 1 month postoperatively and 27 (17 men, 10 women) have completed a 3-month evaluation to date. The average score (out of 40) was 31.2 preoperatively, 30.7 at 1 month and 32.6 at 3 months postoperatively. No significant difference was found between preoperative, 1-month postoperative, and 3-month postoperative scores (p = 0.21, p = 0.14), respectively. Two of 27 patients experienced improvement of greater than 25% following surgery. The remaining 25 patients did not experience significant change.

CONCLUSION

No clinically significant difference was found between preoperative and postoperative olfactory testing scores. Although the transnasal, endoscopic approach to the sella removes structures known to have olfactory nerve fibers, it has no effect on olfaction.

Neuro-Oncol. 2009 Oct;11(5):692.

566. Phase II Prospective Trial of Stereotactic Radiosurgery following Surgical Resection for Brain Metastases: Predictors of Local Control

Cameron Brennan, Kelvin Chan, Ilya Laufer, Timothy Chan, Yoshiya Yamada, Ashwatha Narayana, Stella Lymberis, Viviane S Tabar, Philip H Gutin, Kathryn Beal

INTRODUCTION

Postoperative whole brain irradiation has been shown to improve local control after resection of brain metastases. We investigated whether focal stereotactic radiosurgery (SRS) might provide comparable efficacy in the first phase II prospective study of postoperative radiosurgery for brain metastases.

METHODS

Fifty patients were enrolled (median age = 59 years). Eligibility criteria included confirmed malignancy with one or two intraparenchymal brain metastases, age >18, and KPS >70. Following surgical resection (median 31 days), 40 patients were treated with SRS to the surgical bed: median dose = 1,800 cGy (1,500–2,200 based on cavity size). Pathology included NSCLCA (29), breast cancer (9), melanoma (4), gastrointestinal (4) and other cancer (4).

RESULTS

Median follow-up was 69 weeks. Forty patients received SRS; 10 did not due to early progression (4), medical decline (3), further surgery (1), large cavity size (1) and dropout (1). Of 40 treated patients, 8 experienced local recurrence and 21 had CNS progression elsewhere. Thirteen (32%) received salvage WBRT median 43 weeks later; 7 patients received SRS to other sites, and 11 had further neurosurgery. Overall survival was 51.4 weeks. For the 40 SRS-treated patients, median survival was 70 weeks and median local control was 90 weeks (range, 3–176). Surprisingly, neither postoperative cavity volume nor treatment dose predicted local control. Failure after radiation was best predicted by preresection tumor volume (p = 0.004). Tumors >3 cm (n = 8) had regrowth after a median 45 weeks, whereas 91% of smaller tumors never recurred (n = 32). Superficial location, scored by dural and/or pial contact on preoperative MRI, also predicted early failure, independent of tumor size in multivariate analysis. Local control was 100% for tumors <3 cm in size, deeply located (without gross dural/ pial contact) (n = 15).

CONCLUSION

Postoperative SRS is feasible and offers good local control rates for selected patients who may want to avoid or delay WBRT. Preoperative tumor size and superficial location appear to influence local control.

Neuro-Oncol. 2009 Oct;11(5):692.

567. Impact of Stereotactic Radiosurgery Treatment on Survival Time of Breast Cancer Patients with Neurologically Asymptomatic Parenchymal Brain Metastases

Zhiyuan Xu, Kapila Navaratne, Mary V McGraw, Robert John Weil

INTRODUCTION

The purpose of this retrospective study was to explore the impact of stereotactic radiosurgery (SRS) treatment modality on survival of breast cancer patients harboring neurologically asymptomatic parenchymal brain metastases.

METHODS

A total of 147 consecutive patients with breast cancer and parenchymal brain metastases who underwent SRS in the Brain Tumor and Neuro-Oncology Center at Cleve-land Clinic between 1999 and 2009 were studied. We identified estrogen receptor (ER), progesterone receptor (PR), and HER2 expression status. We calculated overall survival (OS), neurological survival (NS), and survival after stereotactic radiosurgery (SRSS) by the Kaplan-Meier method. We utilized Cox proportional hazards regression models to determine the relationship between ER, PR, and distant metastasis at sites such as lung, liver, and bone.

RESULTS

Patient characteristics, including median age at the time of diagnosis of initial breast cancer and median interval time from breast cancer diagnosis to CNS parenchymal metastasis, are shown in Tables 1 and 2. Survival times are showed in Table 3. Cox proportional hazards regression study demonstrated no significant correlation between ER, PR, and distant metastases.

CONCLUSION

SRS prolonged survival time, including NS and SRSS, but not OS, in neurologically asymptomatic patients with breast cancer metastatic to the brain as compared with those who were symptomatic. Our results suggest that MRI screening for asymptomatic brain parenchymal metastases may be of value in elongating survival in a subset of patients with metastatic breast cancer and are amenable to SRS. To the best of our knowledge, this is the first study addressing the importance of symptomatology evaluation in relation to survival benefit in patients with brain metastases from breast cancer.

Neuro-Oncol. 2009 Oct;11(5):692–693.

568. Stereotactic Radiosurgical Treatment Might Prolong Survival of HER2-Overexpressing Breast Cancer Patients with Parenchymal Brain Metastases

Zhiyuan Xu, Kapila Navaratne, Mary V McGraw, Robert John Weil

INTRODUCTION

We studied the effect of stereotactic radiosurgery (SRS) treatment on survival of patients harboring HER2 overexpressing breast cancer with brain metastases.

METHODS

We reviewed a total of 259 consecutive patients diagnosed with breast cancer and brain metastases referred to the Brain Tumor and Neuro-Oncology Center at Cleveland Clinic between 1999 and 2009. We identified estrogen receptors (ER), progesterone receptors (PR), and HER2 expression status. We calculated overall survival (OS), neurological survival (NS), and survival after stereotactic radiosurgery (SRSS) by the Kaplan-Meier method. We utilized Cox proportional hazards regression models to determine the relationship between ER, PR, and distant metastasis at sites such as lung, liver, and bone.

RESULTS

Median age at the time of diagnosis of initial breast cancer was 46 years (range, 21–76, mean ± SD, 47.06 ± 10.64). Median interval time from breast cancer diagnosis to CNS parenchymal metastasis was 37 (−2 to 407) months. Among them, 82 patients were identified as HER2-expression positive, 89 HER2 negative, and the remainder status unknown. The present study demonstrates that SRS confers OS benefit in HER2 over-expressing patients with brain metastatic breast cancer compared to those who have negative HER2 expression (p = 0.0057, survival 82 months vs. 72 months, Hazard ratio, 1.822; 95% CI, 1.202–2.954). The same holds true for survival benefit in NS (survival time, 25 months vs. 14 months; hazard ratio, 1.929; 95% CI of ratio: 1.418–2.906; p = 0.0001) and SRSS (survival time, 22 months vs. 12 months; hazard ratio, 1.972; 95% CI of ratio, 1.321–3.428; p = 0.0019). Cox proportional hazards regression study demonstrated no significant correlation between ER, PR, and distant metastases.

CONCLUSION

SRS confers a survival benefit, including OS, NS, and SRSS, in patients with HER2 overexpressing breast cancer meta-static to brain as compared with patients bearing no HER2 expression. Our results suggest that SRS may prolong survival in patients with HER2 overexpression and CNS metastases. Whether HER2 overexpression could potentiate the SRS’s effect warrants further investigation.

Neuro-Oncol. 2009 Oct;11(5):693.

569. The Usefulness of DTI Merged Neuronavigation in Brain Tumor Surgery

Jin Mo Cho, Won Seok Chang, Jong Hee Chang, Sun Ho Kim, Kyu Sung Lee

INTRODUCTION

Functional neuronavigation (FNN) is a new modality that merges functional imaging such as diffusion tensor imaging (DTI) with conventional neuronavigation imaging. The authors report the efficacy of DTI-integrated FNN for brain tumor surgery.

METHODS

We performed 62 craniotomies and 6 stereotactic biopsies using FNN for the patients with a brain tumor adjacent to the eloquent area such as motor, vision, or language from November 2008 to January 2009. In each case, we got preoperative and postoperative DTI tractography and checked neurologic status.

RESULTS

This study included 40 men and 22 women and the mean age was 41.5 years. The location of the tumor was close to the corticospinal tract in 42 patients, the optic pathway in 12 cases, and language associated area in 8 cases. The pathologic diagnosis was glioma or metastatic tumor. All patients underwent grossly total resection or successful biopsy and the adjacent important white matter tract was preserved, which was confirmed by postoperative MRI and DTI. There was no permanent neurological deterioration postoperatively.

CONCLUSION

DTI could be reliably and easily integrated into navigational data sets and FNN could be more helpful than conventional neuronavigation for maximal safe resection or biopsy of tumor and preserving brain function. However, there are still limitations, including white matter tract shift during surgery and DTI itself.

Neuro-Oncol. 2009 Oct;11(5):693.

570. Phase I/II Results of AdV-tk Gene-Mediated Cytotoxic Immunotherapy Adjuvant to Standard Upfront Surgery and Chemoradiation for Malignant Gliomas

E Antonio Chiocca, Balveen Kaur, Jayson Hardcastle, Susan Bell, Robert Cavaliere, Simon Lo, Behnam Badie, Jana Portnow, Carissa Monterroso, Laura Aguilar, Estuardo Aguilar-Cordova, David S Baskin, Todd Wilson Trask, Pamela Z New

INTRODUCTION

Despite aggressive combination therapies, most patients with malignant gliomas still survive less than 2 years. Patients with unmethylated MGMT, resistant to temozolomide, fare worse. AdV-tk, an adenoviral vector containing the Herpes virus thymidine-kinase gene plus prodrug (GliAtak, Advantagene), kills tumor cells and elicits antitumor immune responses. Preclinical studies demonstrated synergy with surgery and chemoradiation.

METHODS

A phase-1b/2a trial of AdV-tk plus valacyclovir (Valtrex, GSK) in combination with standard care was conducted in newly diagnosed, resectable disease. AdV-tk was delivered to tumor cells via tumor bed injection followed by valacyclovir for 14 days. Synergy with radiation was maximized by starting radiation early to overlap with HSV-tk activity. The phase-1b evaluated three dose levels of AdV-tk. The phase-2a expanded dose level 3.

RESULTS

Median survival is 13.6 months and progression-free survival is 7.7 months with 23 evaluable subjects to date. No dose limiting toxicity has been observed. Radiation started on average by day 7 (range, 4–12). Temozolomide was administered after valacyclovir without increased toxicity. In the phase-1b, 2-year survival is 33% with 4 of 12 patients still alive (range, 29–38 months). Two have more than double the expected survival based on RPA classification and Gorlia nomogram. Seven of 11 phase-2a patients are alive (range, 5–21 months). Analysis of MGMT methylation status is under way. Health-related quality of life was stable or improved in 14 of 16 patients evaluated. Significant CD3+ T cell infiltrate was found in four reresection cases (3–9 months) including a new tumor focus outside the primary treated site.

CONCLUSION

AdV-tk/prodrug can be safely delivered with standard therapy in patients with newly diagnosed malignant gliomas. Intratumoral immune responses are generated despite ongoing temozolomide therapy. A randomized trial with the same chemoradiation schedule in control and treatment groups, to avoid confounding variables between arms, is planned.

Neuro-Oncol. 2009 Oct;11(5):693.

571. Multiple Dysembryoplastic Neuroepithelial Tumors of the Cerebellum and Brainstem

Frederic Pierre Collignon

INTRODUCTION

Dysembryoplastic neuroepithelial tumors (DNETs) are clinicopathologically unique tumors with a mixed glial-neuronal component, characterized by their intracortical location, multinodular architecture and heterogeneous cellular composition. They are mostly located in the temporal lobe and associated with intractable complex partial seizure in young patients. However, due to the putative origin of DNETs in secondary germinal layers, they can occur in unusual areas of the CNS, such as the caudate nucleus, lateral ventricles and posterior fossa.

METHODS

A 27-year-old male developed 6 weeks before admission an acute vertical diplopia due to a right fourth-nerve palsy. He had a 5-year history of vertigo while in neck extension. Magnetic resonance imaging (MRI) of the brain revealed multiple foci of abnormal signals in the right cerebellar hemisphere, vermis, and brainstem. Via a right paramedian supracer-ebellar and infratentorial approach, the vermian and the quadrigeminal lesions were completely resected.

RESULTS

Microscopically, the specimen disclosed fragments of cerebellar cortex and white matter distorted by alignments of small oligodendrocyte-like cells running along bundles of axons and capillaries. They were separated by abundant myxoid material and mature neurons, resuming the characteristic features of the specific glio-neuronal element distinctive of DNETs. MRI performed 1 year after surgery shows the stability of the remaining lesions.

CONCLUSION

Dys-embryoplastic neuroepithelial tumors, although extremely rare, must be discussed in the differential diagnosis of multiple cystic lesions located in the posterior fossa. They occur in young patients and behave as benign and stable lesions. The recognition of such rare tumors is needed in order to avoid unnecessary neoadjuvant therapy.

Neuro-Oncol. 2009 Oct;11(5):693–694.

572. Changes in Perilesional Edema following Thermal Coagulation from Laser Interstitial Therapy

Shakeel A Chowdhary, Jeffrey L Sunshine, Gene H Barnett, Sunil Manjila, Dawn Diorio, Barbara Dabb, Steve E Jones, Michael Phillips, Mark Griswald, Andrew E Sloan

INTRODUCTION

Prognosis for patients suffering from recurrent glioblastoma multiforme (GBM) is poor. With radiation therapy, median survival is less than 5 months, and a recent meta-analysis suggests that repeat resection increases survival by only 8 weeks. Laser interstitial therapy (LITT) has been previously described as an alternative for reducing tumor burden. Initial results of the phase I AutoLITT (Monteris Medical) thermal dose escalation trial demonstrate reduction in target tissue enhancement, and we report that during the early stages, a small but significant increase in perilesional edema can be appreciated.

METHODS

Two consecutive patients at Case Medical Center, following burrhole craniotomies, were transferred to a Siemen 1.5T magnetic resonance imaging (MRI) scanner and secured to a three-pin head fixation device. A rigid, gas-cooled, side-firing probe was coupled with a diode laser of 1,064 nm wavelength operated at 12-W pulse mode to deliver treatment under real-time MR thermometry guided by stereotaxis with 3 image-plane coverage orthogonal to probe, with integrated software and preclinical results, providing predictive thermal damage feedback. The neurological exam and imaging results were analyzed at fixed intervals after treatment.

RESULTS

Treatment necrosis was evident at 24 h. This was followed by a period of increased perilesional edema averaging 10% in width in the axial plane. During this time, the treated lesion size remained stable in one patient and slightly increased in the other. Decreasing edema was noted prior to identification of reduced contrast enhancement.

CONCLUSION

Treatment with AutoLITT is associated with increased perilesional edema that resolves within 1 week. This process may be a direct reaction of nearby tissue to the products of coagulation necrosis or secondary to transient increased mass effect from slight enlargement of the treatment bed seen immediately following thermal ablation. This phenomenon should be taken to into account when planning for larger lesions situated near regions of eloquence.

Neuro-Oncol. 2009 Oct;11(5):694.

573. The Percutaneous Approach to the Foramen Ovale: The Pyramid Concept and its Anatomical Nuances

Jorge E Alvernia, Nguyen Dang, David Z Cai, Marc Sindou

INTRODUCTION

Meckel’s cave can be reached by percutaneous access through the foramen ovale (FO). This approach described by Hartel and Taptas has been used for treating trigeminal neuralgia. Biopsies of lesions located in the parasellar region are of prime importance and were initially described by the senior author. Appropriate knowledge of the anatomical surroundings improves the target accuracy and prevents complications. We performed a cadaveric study of the area traversed by needle.

METHODS

We fixed three cadaveric heads (six sides) in formaldehyde and injected them with latex. We then described in detail the anatomical trajectories.

RESULTS

An “inverted pyramid” subdivided into three segments is deduced according to the anatomical landmarks and needle’s trajectories described by Hartel. The inferior third begins at cutaneous penetration and ends when the parotid duct (PD) is reached. The middle third begins at the PD and ends at the external pterygoid muscle (PM). The superior third starts from the PM and ends at the FO. In regard to the needle’s axis, the sphenopalatin artery runs posteromedially, the middle meningeal artery posterolaterally, and the internal maxillary artery (MA) postero-medial. In regard to the needle’s axis at the base of this pyramid, the middle meningeal artery was anterior and the prestyloid portion of the IJV and the ICA posterior. The MA was found to have two main anatomical courses; in half of the cases it traveled though the middle and in the other half through the upper third.

CONCLUSION

This approach to the FO is widely used but has risks that need to be considered. A CT-angiogram of the subtemporal fossa is recommended preoperatively when biopsies through this route are attempted. When the MA traveled through the upper third, a close proximity was observed between the artery and needle, so particular attention needs to be paid when performing this approach.

Neuro-Oncol. 2009 Oct;11(5):694.

574. Intraventricular Inflammatory Pseudotumor: Report of Two Cases and Review of the Literature

Ravi Hemant Gandhi, Yu-Hung Kuo

INTRODUCTION

An inflammatory pseudotumor (IP) is a nonneo-plastic lesion characterized by collagenous stroma and polyclonal mononu-clear infiltrates. It is best characterized in the lung but can occur in the CNS, mimicking a neoplastic process.

METHODS

We carried out a prospective case series with review of the literature.

RESULTS

The first patient is a 70-year-old woman who presented with headache and vision changes from temporal horn entrapment due to an atrial choroid plexus mass. Resection of an encapsulated, firm mass restored CSF flow and resolved her symptoms. The second patient is a 52-year-old woman who presented with lightheadedness. Imaging showed a heterogeneously enhancing right choroid plexus mass with mild temporal entrapment, which was hyperactive upon positron emission tomography (PET) scanning. Resection of an encapsulated, firm mass resolved her symptoms. Histopathologically, both specimens revealed a plasma cell granuloma variant of inflammatory pseudotumor. Medline search identified 42 cases of CNS IP, only six of which were located within the ventricle. Most patients present due to CSF obstruction or mass effect with headache and seizures most common. Radiographically, the lesions have a variable appearance though most enhance with gadolinium. Complete resection was achieved in 67% with a 12% rate of recurrence. With incomplete resection or biopsy alone, progression is seen despite steroid or radiation administration. Malignant transformation is only reported once.

CONCLUSION

CNS IP is a rare entity that can not be diagnosed through clinical presentation or radiographic characteristics. The available literature suggests that complete resection with close follow-up is necessary due to the high likelihood of recurrence.

Neuro-Oncol. 2009 Oct;11(5):694.

575. Management of Atypical Meningiomas following Gross Total Resection

Loyola V Gressot, Eric G St Clair, Franco DeMonte, Raymond Sawaya

INTRODUCTION

The indication for radiation therapy following gross total resection of atypical intracranial meningiomas remains controversial.

METHODS

We performed a retrospective analysis of the postoperative management of 40 WHO grade II (atypical) meningiomas that underwent initial gross total resection (GTR) in 36 consecutive patients between 1991 and 2008. Data was collected regarding the administration or not of postoperative radiation therapy (XRT), interval for tumor recurrence and subsequent treatments.

RESULTS

A total of five lesions underwent radiation therapy (mean dose, 58.2 Gy) after GTR (group 1). Recurrence was observed in three cases (60%) in a median time of 19.0 months (range, 14–48 months) after XRT. Reresection was performed in one lesion and stereotactic radiosurgery (SRS) followed by chemotherapy in another. Both lesions have good local control after 10 months of follow-up. The third patient died of systemic aspergillosis shortly after recurrence diagnosis and treatment with imitinab. Thirty-five lesions underwent GTR not followed by XRT (group 2). Recurrence was observed in five cases (14%) in a median time of 25.0 months (range, 14–32 months) after surgery. Two patients underwent reresection, two patients underwent XRT and one patient underwent SRS. The three recurrent lesions treated with radiation only had good local control at median follow-up of nine months (range, 4–10 months). Thirty lesions (86%) did not recur after the initial GTR with median follow-up of 26.5 months. Paired t-test of time to recurrence for the three patients with immediate XRT (group 1) and the five patients without immediate XRT (group 2) did not show significant difference in time to recurrence (group 1 mean, 27.0 months; group 2 mean, 24.6 months; p = 0.79).

CONCLUSION

Our experience suggests that it is reasonable to withhold radiation following GTR of atypical meningiomas and follow with surveillance imaging.

Neuro-Oncol. 2009 Oct;11(5):694.

576. Utilization of Intraoperative Cortical Mapping in Glioma Surgery with High-Field Intraoperative Magnetic Resonance Imaging

Mustafa A Hatiboglu, Sujit S Prabhu, Dima Suki, Ganesh Rao, Sudhakar Tummala, Raymond Sawaya, Jeffrey S Weinberg

INTRODUCTION

Maximum tumor resection while avoiding neurological deficit is a tenet of brain tumor surgery. While intraoperative magnetic resonance imaging (iMRI) helps to maximize tumor resection, intraoperative cortical mapping provides a dynamic means of evaluating brain function. The purpose of this retrospective study was to assess the feasibility and safety of cortical mapping in iMRI suite.

METHODS

We performed cortical mapping with somatosensory evoked potentials (SSEPs), continuous motor evoked potentials (CMEPs) and subcortical direct electrical stimulation in patients undergoing glioma surgery with iMRI-guidance (1.5 T) at M.D. Anderson Cancer Center from September 2006 through December 2008. We documented intraoperative surgical and electrophysiological findings prospectively. We retrospectively reviewed all data including pre- and postoperative neurological evaluations with Institutional Review Board approval.

RESULTS

Ten patients with no postoperative follow-up were excluded. Forty patients with gliomas were identified (three WHO grade I, five grade II, 10 grade III, and 22 grade IV). SSEPs were successfully recorded in 36 patients. CMEPs and subcortical stimulation were performed in 19 and four patients, respectively. All patients tolerated the monitoring well without technical difficulty. Thirty-six (90%) had gross-total resection, three (7.5%) subtotal and one (2.5%) a partial resection of the tumor. A new or worsening of existing neurological deficit occurred in 14 (35%) patients. Follow-up at 6 months showed a persistent deficit in three patients (7.5%). Three out of four patients who lost CMEPs and one patient who lost subcortical stimulation response during surgery had immediate postoperative neurological deterioration.

CONCLUSION

Our results suggest that intraoperative cortical mapping can be used safely and without any technical difficulty within a high-field iMRI. Maximal tumor resection with an acceptable postoperative complication rate can be achieved in tumors near or in eloquent brain using this technique.

Neuro-Oncol. 2009 Oct;11(5):694–695.

577. Dysplastic Gangliocytoma of the Cerebellum (Lhermitte-Duclos Disease): A Case Report

Jennifer Pick, Stephanie Baker, Anuradha Gupta, David Masel, Angel F Campos-Gines, Ellen Dudrey, Glen Friedman, Juan Herrada

INTRODUCTION

Dysplastic gangliocytoma (Lhermitte-Duclos disease) is a rare disorder of unknown pathogenesis, characterized by a slowly progressive unilateral tumoral growth of the cerebellar cortex. Lhermitte- Duclos disease has been linked to the multiple hamartoma-neoplasia complex (Cowden disease), a rare autosomal dominant familial cancer syndrome with several manifestations, including multiple hamartomatous lesions (in skin, oral mucosa, breast and intestine) and an increased predisposition to early breast and thyroid cancer. Both conditions are thought to be caused by mutations in the PTEN gene, which controls cell apoptosis, migration and differentiation.

METHODS

Case report.

RESULTS

In February 2009, a 35-year-old white man was evaluated for acute onset of dizziness and headache. Computed tomography and magnetic resonance imaging of the brain showed a 4.5 × 3.4 × 3.3 cm cerebellar mass, causing compression of the fourth ventricle and tonsillar herniation into the foramen magnum. The lesion was surgically removed. Histological examinations revealed dysplastic ganglion cells arranged in bands that replaced the normal granular cell layer. Immunohistochemical stains on paraffin-embeded tissue showed that the atypical ganglion cells stained positive for synaptophysin, neurofilament and Neu-N. MIB-1 labeling index was low. At that time, the diagnosis of Lhermitte-Duclos disease was made. No evidence of hamartomas or malignancy was found.

CONCLUSION

Lhermitte-Duclos disease should be considered in young adults presenting with clinical and radiological evidence of cerebellar tumors. Because of its association with Cowden disease, clinicians should exclude concomitant malignancies.

Neuro-Oncol. 2009 Oct;11(5):695.

578. Cervical Cancer Brain Metastasis: Clinical Characteristics, Response to Treatment, and Outcome of Patients Treated at a Large U.S. Cancer Center

David S Rosen, Jeffrey S Weinberg

INTRODUCTION

Cervical cancer is diagnosed in approximately 500,000 women per year. However, it is rarely associated with brain metastasis, and consequently, a paucity of data exists on the clinical characteristics, response to treatment, and outcome of patients with brain metastasis from cervical cancer. We reviewed a series of patients treated at a large U.S. cancer center for brain metastasis from cervical cancer.

METHODS

We retrospectively reviewed a prospectively maintained database to identify patients treated for brain metastasis from cervical cancer at our institution between 1993 and 2008. We reviewed patient records to collect clinical characteristics and outcome data and performed statistical analysis to identify significant characteristics of the cohort.

RESULTS

Sixteen patients were identified with a median age at diagnosis of brain metastasis of 38 years (range, 26–77 years) and median duration from diagnosis of cervical cancer of 31 months (range, 0–216 months). Eight patients (50%) had a histologic diagnosis of squamous cell carcinoma, three (19%) had adeno-carcinoma, and three (19%) had neuroendocrine carcinoma. Ten patients (60%) presented with a single metastasis, and six patients (38%) had multiple metastases. Two patients (13%) developed leptomeningeal disease. Nine patients (56%) were treated with microsurgical resection, five patients (31%) had stereotactic radiosurgery, and two patients (13%) had intrathecal chemotherapy through an Ommaya reservoir. The overall median survival was 99 days (range, 12–523) after treatment. Patients undergoing microsurgical resection had a significantly longer survival than stereotactic radiosurgery patients (99 vs. 43 days, p = 0.01). In addition, patients with adenocarcinoma had a significantly longer survival than patients with squamous cell or neuroendocrine carcinoma (284 vs. 98, 99 days, p = 0.05).

CONCLUSION

Patients with cervical cancer brain metastasis have a poor prognosis; however, adenocarcinoma histology and treatment by microsurgical resection may be associated with longer survival.

Neuro-Oncol. 2009 Oct;11(5):695.

579. Ommaya Reservoir Placement using Stereotactic Navigation: the New Standard of Care?

Raghuram Sampath, Rishi Kumar Wadhwa, Tamir Tawfik, Bharat Guthikonda

INTRODUCTION

Ommaya reservoirs play an important role in treating leptomeningeal disease and hematological malignancies. Ideal placement of Ommaya reservoir catheters is essential to ensure safe intra-ventricular drug delivery without intraparenchymal leakage. This may be difficult using a freehand technique, as patients requiring intrathecal chemotherapy often have small ventricles. We present our experience with Ommaya reservoir placement using frameless stereotactic guidance.

METHODS

We performed a retrospective analysis of prospectively collected data of all patients undergoing stereotactic guided placement of Ommaya reservoirs by a single surgeon between November 2007 and January 2009. Navigation was used to preset a surgical plan consisting of a traditional frontal entry point and target point at the ipsilateral foramen of Munro. The navigation probe was initially passed along this trajectory. The precut ventricular catheter attached to the Ommaya reservoir was then passed without a stylet along the created path. Postoperative CT scans and clinical follow-up were assessed.

RESULTS

Thirteen patients (mean age, 45 years) underwent 14 Ommaya reservoir placements. Diagnoses included ALL (4), Burkitt’s lymphoma (2), non-Hodgkin’s lymphoma (2), T-cell lymphoma (2), leptomeningeal carcinomatosis (2), and CML (1). All catheters were placed with single pass. One patient had a poorly placed Ommaya reservoir requiring repositioning (malposition rate, 7%). Mean surgical time was 42 min and mean total OR time was 82 min. No surgical or chemotherapy complications were seen. Mean follow-up time was only 4.5 months, as several patients died from their primary malignancy.

CONCLUSION

Placement of Ommaya reservoirs using stereotactic guidance allowed excellent positioning with the ability to safely administer intrathecal chemotherapy without intraparenchymal toxicity. The malposition rate was low and no other complications were noted. As many patients needing an Ommaya resesrvoir will have small ventricles, we recommend routine use of stereotactic guidance for increasing the accuracy of Ommaya reservoir placement.

Neuro-Oncol. 2009 Oct;11(5):695.

581. The Effective Use of the Hemostatic Agent Vitagel in Sealing Ependymal Breaches in Tumoral Resections that involve the Ventricular System

Edward M Marchan, David W Andrews

INTRODUCTION

Vitagel is a composite liquid hemostatic agent combining the biomaterials thrombin and collagen with the patient’s autologous plasma for soft-tissue healing. Major setbacks in tumoral resections encompassing the ventricular system are porencephalic cyst formation, ventriculitis, hydrocephalus, and cerebrospinal fluid (CSF) leakage from the wound.

METHODS

We have reviewed a series of 31 patients where we utilized Vitagel applied over successive layers of GelFoam and Surgicel and studied its ability to reconstruct the ventricular surface and prevent these complications. We studied 18 men and 13 women. Mean age was 54.3, and 33% were redo craniotomies for tumor. All patients had resections that included an ependymal breach as an intended part of the resection since all tumors involved or were near the ependyma.

RESULTS

Of all patients, 61% had evidence of tumor periependymally or had penetrated into the ventricle. The pathological specimens included 66% glioblastoma, 16% anaplastic oligodendroglioma, 6% metastasis, 3% gliosarcoma, 3% recurrent gemistocytic, 3% ependymoma, and 3% anaplastic astrocytoma. In 87% of patients, magnetic resonance imaging revealed sealing of the ventricular wall with no evidence of CSF extravasation into the resection cavity. The four patients who had documented evidence of persistent communication had radiographic evidence of residual tumor penetrating the ependymal surface into the ventricle. We observed no evidence of poren-cephalic cyst formation, hydrocephalus, trapped horn, connection to the subdural or epidural space, or CSF wound leak after placement of Vita-gel, nor was there evidence of infection attributed to Vitagel. One patient developed a bone plate infection, and reexploration of the Vitagel cavity revealed no evidence of infection in the cavity.

CONCLUSION

Based on our experience, we believe tumors involving or near the ependyma should be aggressively resected, and Vitagel should be used routinely as an effective means of repairing an ependymal breach.

Neuro-Oncol. 2009 Oct;11(5):695.

582. A New Strategy for Intracranial Giant Vasogenic Tumor Surgery: Radiofrequency Thermocoagulation-Assisted Surgery

Weixing Hu

INTRODUCTION

Intracranial giant vasogenic tumors require surgery because of their enormous occupancy; however, sometimes the surgery must be abandoned because of uncontrolled bleeding on the operative surface. We present an innovative new strategy for intracranial skull base/ deep-seated giant vasogenic tumor surgery. Radiofrequency thermoco-agulation (RFT) was used to clot the interior of the tumor before resection, facilitating the surgery with no bleeding.

METHODS

MRI, CTA revealed all these huge tumors with abundant blood supply in 24 patients: 13 tumors in middle fossa, 8 tumors based on cerebellar tentoria and three cerebellar substantial hemangioblastoma. Diameters ranged from 5.6 to 9.2 cm (mean, 6.5 cm). Surgical strategy: Tumor was well exposed and tumor-brain interface was achieved by blunt dissection. The surface of n tumor was devascularized. RFT was used to coagulate the interior of tumor to destroy/block blood circulation within tumors. Surrounding tissues were irrigated with 4°C NS preventing thermal damage. Tumor was then resected in a piecemeal fashion from thermocoagulated regions to complete removal.

RESULTS

With RFT assistance, 16 of the 24 tumors were removed completely and 8 subtotally with no bleeding. Surrounding tissues were kept intact. Patient recovery was uneventful; two experienced oculomotor damages, but no other complications or no tumor recurrences were reported following a 1–4 year follow-up. Pathology: 4 extracerebral angiocavernoma, 7 hemangiopericytoma, 10 vasogenic meningioma, and 3 prenchymatous hemangioblastomas.

CONCLUSION

RFT is extremely effective in blocking the blood supply and controlling bleeding during surgical excision of intracranial giant vasogenic tumors. The thermocoagulated tissues were curettaged with no blood loss, which supplied effective operatiing space and improved the ease and safety of surgery.

Neuro-Oncol. 2009 Oct;11(5):696.

583. Gliomatosis Cerebri and Central Neurogenic Hyperventilation: A Case Study, Discussion of Pathophysiology, and Review of the Literature

Nnenna Mbabuike, Daniela A Bota, Mark E Linskey

INTRODUCTION

Central neurogenic hyperventilation (CNH) is a rare syndrome first described in 1959 among nine patients with brain neoplasms exhibiting high arterial pH, low PaCO2, high PaO2, persistence through sleep and sedation, and no peripheral cause for hyperventilation. Since then, 24 cases have been reported, including this one involving aggressive infiltrating brain neoplasms, none of which have been able to propose a clear mechanism for CNH.

METHODS

We present the first case of gliomatosis cerebri associated with this CNH, a discussion of pathophysiology, and review of the literature.

RESULTS

Unlike other reports, we were able to follow the path of the tumor from initial presentation through the time the syndrome appeared and disappeared. Through the use of sequential magnetic resonance images, we were able to follow the spread of the tumor to areas involved in respiratory modulation to postulate a cause for CNH in our patient involving the interruption of projection input fibers to the medulla.

CONCLUSION

We concluded that CNH in our case may have resulted from a disruption in respiratory modulation as a result of infiltration of the tumor. We also reviewed the current literature and noted possible protective factors of age, sex, and the pathology of astrocytoma linked to survival of CNH.

Neuro-Oncol. 2009 Oct;11(5):696.

584. Improvement of Methods of Neurosurgical Removal of Brain Metastatic Tumors

Anna L Kizokyan

INTRODUCTION

Cerebral metastatic cancer is a difficult and unsolved problem of contemporary neurooncology. Urgency of this issue increases parallel to the increase of oncological morbidity throughout the world. Metastatic tumors are encountered in 35–50% of all brain tumors. The frequency of metastatic involvement of the brain is 5–10 times more often than primary ones. There is need for further development of approaches to diagnostics and surgical treatment of metastatic brain tumors. The absence of strict indications and criteria to surgical treatment is the explanation for unsatisfactory results of surgical treatment of metastatic brain tumors. According to the literature, the average length of life of nonoperated patients ranges from 4 to 8 weeks.

METHODS

We performed a retrospective analysis of 107 patients (68 men and 39 women; average age, 53.3 ± 11.7 years; range, 28–69 years) with brain metastatic tumors, treated from 1986 to 2007 at the neurosurgical department of Fanardjian National Center of Oncology in Yerevan, Armenia.

RESULTS

Definitive indications and requirements to selection of patients for surgical treatment from patients with cerebral solitary metastatic lesions are worked out.

CONCLUSION

Thanks to selection of the patients to the surgical treatment for brain metastatic tumor, the survival of the operated patients was increased, treatment of primary tumors was possible, and significant improvement of quality of life was achieved.

Neuro-Oncol. 2009 Oct;11(5):696.

585. Transclinoidal Pretemporal Approach for More Radical and Safer Resection of Retrochiasmatic Craniopharyngiomas

Ali F Krisht, Kerem Bikmaz, Sanford PC Hsu, Brian Liao

INTRODUCTION

Retrochiasmatic craniopharyngiomas are a treatment challenge because of their hidden location and their significant attachments to the surrounding neurovascular structures. As a result, they are more difficult to remove and have a high rate of recurrence. We present our experience with a transclinoidal pretemporal approach to retrochiasmatic craniopharyngiomas in 15 patients and detail the surgical steps and their advantages.

METHODS

We utilized the transclinoidal pretemporal approach in 15 patients with retrochiasmatic craniopharyngiomas. The approach involves the removal of the anterior clinoid process and utilizing the pretemporal route. The approach provided a shorter working distance to the sella and parasellar region and significantly widened the opticocarotid window especially after cutting the falciform ligament and the internal carotid dural ring to mobilize the optic nerve away from the internal carotid artery. The approach provided the advantage of a direct lateral-to-medial and inferior-to-superior angle of approach to the under surface of the optic chiasm and into the hypothalamic and third ventricular region.

RESULTS

Gross total resection was achieved in 13 patients. In two patients, residuals were left attached to the basilar apex perforators, necessitating surgery and Gamma Knife in one. The pituitary stalk was anatomically preserved in 13 patients and it was functional in 50% of the patients. Eleven patients presented with visual deficits, nine of whom had postoperative improvement. None of the patients developed worsening of vision. One patient with a history of fibromuscular dysplasia and carotid occlusion developed transient hemiparasis due to hypovolemia from diabetes insipidus. One patient with morbid obesity due to the hypothalamic syndrome preoperatively was very immobile and died of pulmonary embolism at home 1 month postoperation. The mean follow-up was 74.6 months; we observed was no evidence of recurrence or regrowth in any of the surviving patients at the time of the most recent follow-up.

CONCLUSION

The pretemporal transclinoidal approach to the suprasellar and retrochiasmatic region has the potential to help achieve a more radical and safer resection of retrochiasmiatic craniopharyngiomas.

Neuro-Oncol. 2009 Oct;11(5):696.

586. Surgical, Radiological, and Endocrinological Cure in Either Hormonally Active or Nonfunctional Invasive Pituitary Adenomas with Radical Surgical Removal

Ali F Krisht, Brian Liao

INTRODUCTION

Giant and invasive pituitary adenomas comprise up to 5% of all pituitary adenomas. Most of these are agressive in clinical course and with cavernous sinus invasion. Traditionally, these invasive adenomas are resected with transsphenoidal approaches followed by irradiation for the residuals, but high recurrence and complication rates were addressed in the literature, especially for younger patients. Based on this, we utilized epidural transcavernous approaches to cure locally infiltrative cavernous sinus and to avoid the risks of irradiation therapy.

METHODS

We adopt a pretemporal, predominantly epidural, transcavernous approach for the giant invasive adenomas with cavernous sinus invasions, either hormonally active or inactive ones. Tumor resections were completed after full visualization of the intracavernous cranial nerves and the carotid artery. Pituitary glands and stalks were preserved carefully in all cases. Both endocrinological and radiological images were followed for at least 2 years.

RESULTS

Between 1997 and 2004, a total of 12 cases received such surgical resections. Among them, eight patients were hormonally active and four cases were nonfunctional ones. Of the eight hormone-active adenomas, two were prolactinomas, three ACTH adenomas, two GH adenomas and one TSH adenoma. All patients were hormonally cured and became independent from medicine. During a mean follow-up of 27 months, none of the 12 cases had clinical or radiological recurrence. There was no surgical related mortality. Two patients developed transient ocular neuropathy, one in the occulomotor nerve and the other in abducence nerves neuropathies. None of the patients developed new pituitary hormonal dysfunction.

CONCLUSION

Locally infiltrative, cavernous sinus pituitary adenomas can now be cured by utilizing epidural pretemporal transcavernous approaches. With good neuroanatomy knowledge and microneurosurgery techniques, these treatment modalities are safe. Besides, these can not only prevent patients from the risks of irradiation therapy but also obtain a high quality of life to the patients in long-term follow-up.

Neuro-Oncol. 2009 Oct;11(5):696–697.

587. Toward Zero Mortality in Neuro-Oncology Practice: A Single Neurosurgeon’s Experience in a Peripheral Center in India

Pari Kodaiarasan

INTRODUCTION

Mortality and morbidity is very high in neurosurgical practice. This is a major cause of concern and fear even in the mind of medical fraternity. With the advancement of neuroimaging, neuroanesthesia and neurosurgical techniques, mortality and morbidity has reduced many-fold. Here, I present my personal experience in neurooncology from a peripheral center in India.

METHODS

This is a retrospective analysis of 110 tumors (92 cranial and 18 spinal) operated by me from January 2006 to April 2009 in VMH, Salem, Tamilnadu. Follow-up ranges from 40 months to 2 weeks.

RESULTS

Age ranges from 3 years to 84 years with a male:female ratio of 3:2. Cranial disease spectrum includes gliomas (37), meningiomas (17), pituitary adenomas (10), metastasis (6), medullo-blastomas (3), vestibular schwannomas (3) and miscellaneous tumors (16). Spinal tumors were neurofibromas (7), lymphomas (3), ependymomas (2) and miscellaneous (epidermoid, metastasis, angioma, Schwannian tumor, sacral chordoma and hemangioma). There were 23 tumors in the “giant” category and others were in medium and small size. Most of the surgeries were performed as elective procedures and a few cases were as emergency. Gross total microsurgical excision was achieved in most cases except pituitary macroadenomas and gliomas of eloquent areas. There is no neurosurgical procedure related to intra/perioperative mortality. The only mortality in this series (84/M) is because of acute myocardial infarction. One case of giant pituitary adenoma and petrous apex meningioma required staged procedures. Another case of oligodendroglioma deep seated, underwent a second surgery after negative biopsy. Other complications (transient motor weakness, 3; bone flap osteomyelitis, 2; wound infection, 2; transient lower cranial nerve palsies, 2; mutism, 1; pseudomeningocele, 1; diabetes insipidus, 1) managed without significant morbidity.

CONCLUSION

The aim in tumor surgery is maximum possible excision without causing further deficit irrespective of the site or size of the tumor. It is important to know when not to operate and when to stop; only then can we achieve results competitive with other disciplines of surgery.

Neuro-Oncol. 2009 Oct;11(5):697.

588. Tumor Paint: A Scorpion Toxin Peptide that Targets Fluorescent Dye to Brain Tumor Cells for Real-Time Intraoperative Imaging

Muharrem Akcan, Mark R Stroud, Stacey A Hansen, Norelle Daly, David Craik, Jim Olson

INTRODUCTION

We previously reported the development of a chlorotoxin:Cy5.5 bioconjugate that, upon intravenous administration, crosses the blood brain barrier and illuminates gliomas or medulloblastomas with near infrared fluorescence that can be detected in real time. Prior to advancing this molecule to the FDA for regulatory approval to begin human trials, we wished to optimize the targeting molecule and, if possible, identify the molecular target of chlorotoxin. The molecular target of chlorotoxin was previously thought to be matrix metalloproteinase-2 (MMP2).

METHODS

1) To definitively establish whether MMP2 is the target, we crossed Smo/Smo medulloblastoma mice onto a background that genetically lacks both copies of the MMP2 gene. 2) Toward the goal of developing an improved imaging agent, we synthesized a cyclic peptide that is more resistant to proteolysis than chlorotoxin. 3) We also engineered the peptide in a manner that prevents poly-labeling with near infrared fluorescent molecules because mono-labeled products are more suitable for molecular imaging from a regulatory perspective.

RESULTS

1) Tumor paint illuminated medulloblastomas in mice lacking MMP2 to the same extent as in MMP2-expressing mice, showing that MMP2 is not the target of chlorotoxin. 2) We have generated a peptide that has a prolonged serum half-life compared to chlorotoxin. 3) A strategy for successfully limiting dye conjugation to a single site has been established. 4) Four other near infrared dyes illuminate tumors when conjugated to chlorotoxin; some are superior to others.

CONCLUSION

We have learned that MMP2 is not the target of chlorotoxin. We hope to identify the target prior to the meeting. We have generated new targeting molecules that are superior to chlorotoxin and have identified alternatives to Cy5.5 for near infrared imaging.

Neuro-Oncol. 2009 Oct;11(5):697.

589. Development and Progress on an Implantable Brain Tumor Pump for Metronomic Convection-Enhanced Delivery of Biological Agents

Thomas C Chen, Josh Shachar, Winston Wu, Leslie Farkas, Brett Jordan, Kyle Zimmerman

INTRODUCTION

Convection-enhanced delivery (CED) has been used with limited success in the treatment of malignant gliomas. One reason for this lack of success is in the limitation of CED techniques, as multiple externalized catheters are inserted into the tumor for limited time periods (i.e., 5–7 days) and removed; repeat infusions require another surgery for catheter placement.

METHODS

An implantable brain tumor pump has been constructed in benchtop format. It consists of five components: 1) an intratumoral delivery catheter (for unresectable tumors) or an intracranial pouch (for tumor cavity in resected tumors), 2) a reservoir for tapping the catheter or intracranial pouch, 3) a connecting catheter to link the reservoir, 4) an internalized pump with canisters containing biological agents of choice, capable of delivering drugs at microliters/hour, and 5) multiple sensors for measuring intratumoral pH, pO2, temperature, pressure, and spectral analysis of vascular endothelial growth factor (VEGF) levels of tumor fluid.

RESULTS

The magnetic breather pump will be implanted much like a shunt, with the delivery pump secured at the chest wall. An accelerometer has been added to ensure that constant delivery of fluids occurs in all positions. The canisters in the delivery pump have been constructed to function like bellows to ensure constant fluid delivery. The canisters in the delivery pump may be accessed via subcutaneous ports to refill the drugs and can be wirelessly reprogrammed to deliver at specific time intervals and different cycles using a Medical Implant Communication Service (MICS) interface.

CONCLUSION

The benchtop pathfinder has been completed with all five components of the brain tumor pump connected to demonstrate proof of principle in vitro. The next step is miniaturization, and in vivo testing in large animal models. Successful completion of animal studies will result in human phase I studies.

Neuro-Oncol. 2009 Oct;11(5):697.

590. Role of Tumor Markers in Diagnosis and Management of Pineal Region Tumors

Srinivasa Rao Ravuri

INTRODUCTION

Germ cell tumors are highly sensitive to radio-therapy and chemotherapy and account for more than 50% of pineal region tumors. The aim of this presentation is to evaluate the role of tumor markers in the diagnosis and management of pineal region tumors.

METHODS

Twenty-five patients with pineal region tumors admitted over the past 4 years were analyzed. Tumor marker (AFP, β-HCG, and CEA) analysis of cerebrospinal fluid was done for all. They were divided into two groups depending on tumor marker levels. Group I (elevated tumor marker levels) were treated with direct radiotherapy or chemotherapy. Group II (insignificant tumor marker levels) were treated with definitive surgery/biopsy followed by radiotherapy or chemotherapy as necessary.

RESULTS

Ten patients had elevated tumor marker levels, of which eight patients were referred for direct radiotherapy and one for direct chemotherapy. Definitive surgery was performed for one patient, for which HPE revealed pinealoblastoma. Fifteen patients had insignificant tumor marker levels. Surgery was done in ten patients and one of them was an immature teratoma. One patient was put on ATT based on imageological characteristics and another patient under observation as there was benign pineal cyst.

CONCLUSION

Tumor marker study is useful for differentiating germ cell tumors from nongerm cell tumors of the pineal region.

Neuro-Oncol. 2009 Oct;11(5):697.

591. Results of Transphenoidal Microsurgery of Centrally Located Pituitary Macroadenoma in the Modern Neurosurgical Era

Carlos S Rizo, Ali F Krisht

INTRODUCTION

The preoperative and postoperative improved visualization of pituitary macroadenomas is expected to impact the long-term outcome of the treatment of these patients. This paper looks at the potential of this impact in 187 patients with predominantly centrally located pituitary macroadenomas.

METHODS

The preoperative and postoperative improved visualization of pituitary macroadenomas is expected to impact the long-term outcome of the treatment of these patients. This paper looks at the potential of this impact in 187 patients with predominantly centrally located pituitary macroadenomas.

RESULTS

The female/male ratio was 106/81. Median age was 56.12 years. There were 111 nonfunctional tumors (59%), 14.43% prolactinomas, 10.65% ACTH-producing adenomas, 8.02% growth hormone-producing adenomas, and 3.74% growth hormone + prolactin-secreting adenomas, and 3.74% LH- and FSH-producing adenomas. Gross total resection confirmed by postoperative MRI was achieved in 93.58% of patients. Preservation of no change or preservation of pituitary functions was achieved in 90% of patients. Overall surgical morbidity was 2.13% with two patients having cerebrospinal fluid rhinorrhea, two transient cranial nerve palsies. There was 0 mortality. The average follow-up was 4.95 years. Recurrence occurred in 2.13% of patients. Radio-therapy was utilized in 4.8% of patients, all of which used Gamma Knife radiosurgery.

CONCLUSION

Advances in preoperative and postoperative visualization led to a significant positive impact on centrally located pituitary macroadenomas. The transsphenoidal approach continues to be the gold standard approach leading to a high rate of recurrence-free survival with a very low surgical and hormonal complication rate. Conventional radiotherapy should be ruled out for the overall majority of these patients with occasional use of radiosurgery.

Neuro-Oncol. 2009 Oct;11(5):697–698.

592. Time-Resolved Fluorescence Spectroscopy for Intraoperative Delineation of Human Gliomas

Pramod Butte, Adam N Mamelak, Serguei Bannykh, Keith L Black, Laura Marcu

INTRODUCTION

Time-resolved laser-induced fluorescence spectroscopy (tr-LIFS) is a technique where biological tissue is excited with laser pulse and intrinsic fluorescence emission decay is measured in wavelength. Tr-LIFS promises intraoperative delineation of the tumor from normal brain tissue, thus facilitating complete resection. The study goal was to determine the Tr-LIFS characteristics that accurately discriminate normal brain tissue vs. tumor intraoperatively.

METHODS

Sixty patients undergoing surgery for glioma (WHO grade I–IV) were enrolled. During tumor resection a Tr-LIFS prototype apparatus (gated detection, fast digitizer) recorded fluorescence. Time-resolved fluorescence was induced using a pulsed N2 laser (337 nm excitation wavelength, 0.7 ns pulse width) and recorded in 360–550 nm wavelength range (10-nm interval). Biopsies from sites where Tr-LIFS measurement were subject to pathological analysis, performed without knowledge of the Tr-LIFS data, and conversely Tr-LIFS data collected without knowledge of pathology. Using parameters derived from spectroscopic data from each specimen, a classification algorithm was developed.

RESULTS

We measured and biopsied 102 regions of tumor and normal brain. Normal cortex (n = 33) and normal white matter (n = 12) exhibited fluorescence emission at, 390 nm (t, 2 ± 0.09 ns) and 460 nm (t, 0.8 ± 0.07 ns). High grade glioma (III and IV) (n = 25) demonstrated emission peaks at 460 nm, with large variation at 390 nm. Low grade glioma (I and II) samples (n = 10) demonstrated a peak fluorescence emission at 460 nm. Using these parameters for classification, normal brain tissues were identified with more than 80% sensitivity and 80–98% specificity. Low-grade glioma could be classified with 100% sensitivity and 98% specificity.

CONCLUSION

Tr-LIFS-derived parameters allow for discrimination of glioma from the normal brain tissue providing real-time discrimination during surgery, thereby facilitating maximal cytoreductive surgery while minimizing injury to normal tissues.

Neuro-Oncol. 2009 Oct;11(5):698.

593. Patient-Specific Virtual Reality Systems for Brain Tumor Surgery

Robert DiRaddo, Boguslaw Tomanek, Denis Laroche, Sebastien Delorme, Rolando Del Maestro

INTRODUCTION

Rapidly evolving surgical techniques, decreasing resident workloads and patient safety concerns are strongly driving the need for innovative training approaches. The potential for error and the need for training are highest during the surgeon’s learning curve. The objective of this research is to develop an interactive virtual reality (VR)-based simulator to aid neurosurgery residents and neurosurgeons in preparation for brain tumor resection.

METHODS

The system creates a brain model from medical images, providing planning and rehearsal prior to the actual surgery. The immersion experience is maximized using highly realistic visual feedback of tool-specific tissue behavior, including dissection and bleeding, and high-fidelity touch feedback with tissues using haptic devices connected to a real-time computational engine. Enhanced magnetic resonance imaging is being developed to provide input information on critical anatomical structures, functional areas and tissue biomechanics. In collaboration with clinicians, surgical skills metrics and simulation scenarios for brain tumor resection are being defined and implemented.

RESULTS

Two simulation scenarios have been implemented in the system. In a ventriculostomy scenario, the trainee must drive a tube through a whole in the skull until its tip is felt to be inside a ventricle. In a tumor debulking scenario, the trainee must debulk a tumor using an aspirator without removing healthy brain tissue.

CONCLUSION

The system will allow learning of new surgical techniques, training on new devices such as medical robots, performing a surgical skills warm-up or rehearsing a procedure for a patient-specific anatomy in a safe environment outside the operating room. In addition, it will provide an objective measure of surgical performance without requiring the presence of an external observer. Clinical adoption of VR simulation will result in accelerated training, rapid adoption of new surgical techniques and better surgeries with minimal risk.

Neuro-Oncol. 2009 Oct;11(5):698.

594. Impact of Movable Ceiling-Mounted Intraoperative Magnetic Resonance Imaging (iMRI) on the Extent of Resection of Malignant Gliomas: Initial One-Year Experience at a Single Center

Eriks Lusis, John Evans, Michael C Zahner, Ralph G Dacey, Keith M Rich, Joshua Dowling, Gregory J Zipfel, Jeffrey R Leonard, Matthew D Smyth, David D Limbrick, Eric C Leuthardt, Justin Brown, Todd J Stewart, Michael R Chicoine

INTRODUCTION

Here, we assess malignant glioma (WHO grade III or IV) resections during the first year after implementation of a movable ceiling-mounted intraoperative magnetic resonance imaging (iMRI).

METHODS

We reviewed the malignant glioma database of surgeries with or without iMRI since installation of the iMRI device (iMRI use according to surgeon preference). Assessments included additional resections prompted by iMRI, histopathology of specimens removed after iMRI, and the number of cases in which postoperative MRI indicated gross or near total resection for both iMRI and non-iMRI cases.

RESULTS

Sixty patients underwent resection for malignant gliomas, including 24 surgeries using iMRI (10 grade III and 14 grade IV) and 36 surgeries without iMRI (4 grade III and 32 grade IV). Intraoperative MRI prompted additional resection in 17 of 24 (71%) of cases (8 of 10 grade III tumors and 9 of 14 grade IV tumors). Separate post-iMRI histopathology demonstrated tumor in 10 specimens (five grade III and five grade IV) after iMRI (59%). Postoperative MRI showed gross or near total resection in 22 of 24 (92%) iMRI cases (9 of 10 grade III tumors [90%] and 13 of 14 grade IV tumors [93%]), and in 25 of 36 (69%) non-iMRI cases (three of 4 grade III tumors [75%] and 22 of 32 of grade IV tumors [68%]).

CONCLUSION

A movable high field strength iMRI integrated into management of high-grade gliomas improves the extent of malignant glioma resection as confirmed both histopathologically and radiographically.

Neuro-Oncol. 2009 Oct;11(5):698.

595. Combined Systemic and Intra-CSF Chemotherapy in the Treatment of Primary Brain Tumor Leptomeningeal Metastases

Ibrahim Jalloh, Ian Graham, Juliet Clayton, Nicole Whitehead, Timothy Jaspan, Richard Grundy, David A Walker, Michael Vloeburghs

INTRODUCTION

Combination chemotherapy with systemic and intra-cerebrospinal fluid (CSF) agents enables cytotoxic concentrations to be achieved within both the CSF spaces and brain parenchyma. This allows not only the control of bulk disease but also treatment of leptomeningeal deposits and prevention of CSF dissemination. Concern regarding the surgical risk of CSF access devices, disturbed CSF dynamics and potential myelotoxicity from intra-CSF therapy, has led to reluctance to pursue intra-CSF treatments.

METHODS

Eight patients with leptomeningeal metastases (three male; age range, 2–21 years) were treated over a 5-year period with combination intra-CSF and systemic chemotherapy regimes. All the patients had recurrent disease except one who, at presentation, had established leptomeningeal spread. Five patients had primitive neuroectodermal tumors (PNETs), two patients had sarcomas, and one patient had an anaplastic ependymoma. Patients received intra-CSF agents either via a lumbar puncture (one patient), an intraventricular Ommaya reservoir (four patients) or a lumbar intrathecal catheter with abdominal access port (three patients). Radionucleotide tracer or gadolinium contrast studies were used to confirm adequate CSF distribution throughout the brain and spine. All patients received either etoposide or topotecan administered systemically and into the CSF.

RESULTS

We observed good radiological and/or cytological responses in six patients. One patient demonstrated a complete remission. Three patients achieved a significant leptomeningeal response while bulk disease progressed. We observed drug-related adverse events in two patients (acute renal failure, hearing loss) and three device-related adverse events (CSF leak, infection, ).

CONCLUSION

This patient series clearly illustrates the safe and effective use of combined systemic and intra-CSF chemotherapy. The differential response observed between bulk and leptomeningeal disease highlights the preferential pharmacokinetic effect of direct CSF treatment over systemic regimes. With this pharmacokinetic advantage and with the development of new intra-CSF agents, direct CSF treatment of leptomeningeal metastasis may well improve prognosis in these often challenging cases.

Neuro-Oncol. 2009 Oct;11(5):698.

596. Risk Factors for Decreased Extent of Resection and Survival Following Radical Resection of Craniopharyngiomas in Children

Robert E Elliott, Kevin Hsieh, Jessica Wisoff, Jeffrey H Wisoff

INTRODUCTION

We report on the risk factors (RF) for decreased extent of resection (EOR) and worse survival in children treated with attempted radical resection.

METHODS

We performed a retrospective analysis of 86 children with craniopharyngiomas who underwent 103 operations over 22 years. Mean age at surgery and follow-up were 9.6 and 9.0 years, respectively. We used uni- and multivariate logistic regression analysis to determine RF for decreased EOR (odds ratios, OR) and multivariate Cox proportional analysis to determine predictors of decreased survival (hazard ratios, HR).

RESULTS

All 57 children with primary tumors underwent gross-total resection (GTR). GTR was achieved in significantly fewer children with recurrent tumors (18/29, 62%). Both overall survival (OS) and recurrence-free survival (RFS) were significantly better in patients with primary tumors at the time of NYU presentation (log-rank test = 14.2/p = 0.0002 and log-rank test = 10.46/p = 0.0012). We observed no significant differences in the neurological, endocrinological, visual or functional outcomes between patients with primary and recurrent tumors. Factors negatively affecting OS and RFS include subtotal resection (HR = 5.15/p = 0.004), tumor size = 5 cm (HR = 3.38/p = 0.03), presence of hydrocephalus (HR = 4.00/p = 0.02) or ventriculoperitoneal shunt (VPS; HR = 5.26/p = 0.003). Age = 5 did not impact survival. Among patients who had GTR, only the presence of VPS predicted worse OS (HR = 4.34/p = 0.04). Multivariate analysis found only prior radiation therapy (OR = 8.93/p = 0.031) and increasing tumor size (OR = 2.05/p = 0.045) were RF for incomplete resection at reoperation. Previous radical resection did not negatively impact EOR.

CONCLUSION

In the hands of surgeons with experience with craniopharyngiomas, resection at presentation offers an excellent chance for disease control potentially avoiding cranial irradiation and the need for VPS. While GTR does not preclude recurrence and is more difficult to achieve in recurrent tumors, especially large and previously irradiated tumors, radical resection is still possible in patients with recurrent craniopharyngiomas with morbidity similar to that of primary tumors.

Neuro-Oncol. 2009 Oct;11(5):699.

598. Endonasal Endoscopic-Assisted Microsurgical Management of Pediatric Sellar Region Tumors

James L Frazier, Kaisorn L Chaichana, George I Jallo, Alfredo Quinones-Hinojosa

INTRODUCTION

Sellar/parasellar lesions have been approached through either a transsphenoidal approach and/or craniotomy. Transsphenoidal microsurgery is an established technique, and the introduction and evolution of endonasal endoscopic techniques has provided minimally invasive methods alone or in combination with the microscope for removal of these lesions. The authors have implemented the use of endonasal endoscopic-assisted microsurgery in the pediatric population.

METHODS

Ten consecutive patients (3–17 years) who had either a craniopharyngioma (5), rathke cleft cyst (1), germinoma (1), fibrosis (1), or pituitary adenoma (2) underwent endonasal endoscopic-assisted microsurgery. The patient with fibrosis had a diagnostic biopsy. Patients presented with headaches (6), visual problems (6), and/or emesis (1). Preoperatively, median size of the lesions was 2.5 + 1.6 cm. We observed suprasellar extension and optic chiasm compression in seven cases and cavernous sinus invasion in 1. We observed endocrinopathy in five patients preoperatively.

RESULTS

Postoperatively, gross total resection was observed in 6 of the 10 cases, and a second operation was required in one case with an initial subtotal resection. One patient had near-total resection of the lesion. The mean postoperative hospital stay was 5 ± 2 days. Postoperative cerebrospinal fluid leak was observed in one patient. Vision was stable or improved in six patients with preoperative problems, and there was no worsening of vision in any cases. Two patients had diabetes insipidus (one preoperatively) while three others had panhypopituitarism (two preoperatively).

CONCLUSION

Transnasal endoscopic-assisted microsurgery is safe and effective for extirpation of lesions in the sellar/suprasellar region and will be enhanced with the evolution of endoscopic techniques.

Articles from Neuro-Oncology are provided here courtesy of Society for Neuro-Oncology and Oxford University Press

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