Skip to main content
NCBI home page
Neuro-Oncology logoLink to Neuro-Oncology
. 2012 Jun;14(Suppl 1):i7–i15. doi: 10.1093/neuonc/nos095

BIOLOGY

PMCID: PMC3483341
Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-01. ANTI-ANGIOGENIC ACTIVITY OF TISSUE FACTOR PATHWAY INHIBITOR TO TUMOR ANGIOGENESIS IN RETINOBLASTOMA VIA BLOCKADE OF ERK PATHWAY

Jin Hyoung Kim 1, Hyun Beom Song 1, Dong Hun Kim 2, Kyung Duk Park 3, Jeong Hun Kim 1

Abstract

PURPOSE: To investigate whether tissue factor (TF) regulates fibroblast growth factor (FGF)-2-induced angiogenesis in retinoblastoma. METHODS: In an orthotopic transplantation mouse model of retinoblastoma, immunofluorescence staining for TF and CD31 (an endothelial cell maker) was performed. With treatment of FGF-2 (10 ng/ml), TF expression in human umbilical vein endothelial cells (HUVECs) was measured by Western blotting. To confirm the role of TF in tumor angiogenesis in retinoblastoma, anti-angiogenic activity of TF pathway inhibitor (TFPI) was investigated by treating TFPI on FGF-2-induced proliferation, migration and in vitro tube formation of HUVECs. In addition, inhibition of ERK1/2 phosphorylation by TFPI was measured by Western blot analysis. RESULTS: TF was highly expressed on vascular endothelial cells of retinoblastoma, co-localized with CD31. With FGF-2-induced proliferation of HUVECs, TF expression was significantly up-regulated. Interestingly, TFPI effectively inhibited FGF-2-induced proliferation, migration and in vitro tube formation of HUVECs, which was accompanied by inhibition of ERK1/2 phosphorylation. CONCLUSIONS: TF is involved in tumor angiogenesis of retinoblastoma via extracellular signal-regulated kinase pathway.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-02. SHARING OF NUCLEAR EXCLUSION OF p53 IN TUMOR CELL AND TUMOR VASCULAR ENDOTHELIUM OF HUMAN RETINOBLASTOMA

Jeong Hun Kim 1, Byung Joo Lee 1, Dong Hun Kim 2, Jin Hyoung Kim 1

Abstract

PURPOSE: Nuclear exclusion of p53 has been suggested to be an important mechanism of tumor suppressor gene inactivation in the retinoblastoma. Here, we investigated whether the mature tumor vascular endothelium of human retinoblastoma also shows the cytogenetic abnormality of tumor cell by verifying the phenomenon of p53 nuclear exclusion in the tumor vascular endothelium of human retinoblastoma. METHODS: The formalin-fixed paraffin-embedded specimens of primarily enucleated eyes of retinoblastoma (7 eyes from 7 patients) were sectioned for immunofluorescent staining. Double immunofluorescence stain with anti-von Willebrand factor and anti-p53 antibodies and DAPI nuclear counterstain were performed to identify the presence of mature tumor vascular endothelium which shows nuclear exclusion of p53 protein. To determine the proportion of p53 nuclear exclusion among mature tumor vascular endothelium, two blinded investigators counted at least 10 randomly selected high power fields. RESULTS: Most of tumor cells from human retinoblastoma specimens showed nuclear exclusion of p53 protein. Overall, 45.1% of mature tumor vascular endothelium showed nuclear exclusion of p53, whereas the endothelium of pre-existing retinal vessels which supply the relatively preserved retina consistently showed negative stain for anti-p53 antibody. CONCLUSIONS: Some proportion of mature tumor vascular endothelium shares the same cytogenetic abnormality with retinoblastoma cells. These data suggest that an endothelial differentiation of retinoblastoma cell might partially contribute to the tumor vascularization in retinoblastoma.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-03. TARGET IDENTIFICATION AND PERSONALIZED THERAPY IN ETANTR

Soumen Khatua 1, Esra Kalkan 1, Robert Brown 1, Michael Pearlman 1, Tribhawan Vats 1

Abstract

Embryonal Tumors with Abundant Neuropil and True Rosettes (ETANTR) is a rare CNS tumor in children. Due to its rarity molecular pathways and treatment guidelines remain ill-defined. To our knowledge this is the second case report of this tumor presenting as an isolated spinal lesion and the first report of a personalized therapy approach in ETANTR. A four year male presented with headache, gluteal area discomfort and lower extremity weakness. MRI spine showed a lumbar mass. Surgical resection was performed and pathology was consistent with ETANTR. He was treated with chemoradiation followed by adjuvant chemotherapy with cisplatin, vincristine and cyclophosphamide. He is currently receiving maintenance therapy with cis-retinoic acid, melatonin, metformin and is clinically doing well. Our previous submission demonstrated an innovative approach of molecular tumor profiling using morphoproteomic (MP) analysis and quantifying immunohistochemistry (IHC) signaling intensity of major pathways identifying activation of ras/Raf kinase/ERK and Akt/mTOR pathway in this tumor. Use of melatonin, metformin and cisretinoic acid in maintenance chemotherapy is based on our MP data of ETANTR. Metformin inhibits components of the mTORC2 pathway and thereby down-regulating the expression of p-mTOR and p-Akt. We identified MTORC2 as a dominant complex in these tumors supported by nuclear compartmentalization of p-mTOR(Ser 2448) and correlative expression of its putative downstream effector phosphorylated Akt (Ser 473). Expression of melatonin 1a melatonin receptor known to have cystostatic and cytodifferentiating properties was negative in these tumors validating the supplement of melatonin. Melatonin is known to potentiate all-trans retinoic acid effect on retinoic acid receptor and their sequential use induces apoptosis in tumor cells. These preliminary data suggests the potential use of metformin, melatonin and cis-retinoic acid as a maintenance therapy in ETANTR. Larger studies are needed to evaluate and validate this innovative method of target identification and personalized therapy in these rare tumors.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-04. MicroRNA-9 IS A POTENTIAL TUMOR SUPPRESSOR TARGETING NOTCH PATHWAY IN MEDULLOBLASTOMA

Lucia Abela 1, Giulio Fiaschetti 1, Tarek Shalaby 1, Eveline Grunder 1, Min Ma 1, Jasmin Grählert 1, Martin Baumgartner 1, Ulrich Siler 2, Naosuke Nonoguchi 3, Hiroko Ohgaki 3, Michael Grotzer 1

Abstract

MicroRNA-9,(miR-9), which mediates neuronal differentiation is found to be dysexpressed in brain tumors, nevertheless the functional implications of altered miR-9 expression in medulloblastoma MB is yet unclear. In this study, we investigated the expression level and the functional role of miR-9 in MB. Compared to normal human cerebellum, miR-9 was found to be under-expressed in 22 out of 29 MB primary samples (2-90 fold) as well as in 6/6 MB cell lines tested (3-13 fold). Methylation-specific PCR analysis for miR-9-1 promoter showed methylation in 5/6 MB cell lines, but not in normal cerebellum, whilst ethylation-mediated silencing could be reverted through treatment with the demethylating agent 5-aza-2′-deoxycytidine. An in silico search revealed that the Hes1 oncogene, which is the principal effector of the Notch pathway and known to be elevated in MB with unfavourable prognosis, is a direct target of miR-9. Consistently, a Hes1 3′ UTR luciferase reporter assay experiment confirmed the inhibitory effect of miR-9 on Hes1 expression. Further analysis showed that increasing the expression of miR-9 in four MB cell lines down-regulates Hes1 protein and promotes mRNAs expression of its main responsive genes, P21 and Mash1. The resulting up-regulation of P21 increased the number of MB cells in the G1 phase of cell cycle and decreased cells in S phase, whereas the elevation of Mash1 increased protein expression of the neuronal differentiation markers Tuj1 and Nestin. In summary, our results suggest that miR-9 may act as a tumor suppressor and the failure of MB cells to express it may contribute to the disease through the over-expression of the Hes1. Moreover, the finding that miRNA-9 re-expression in MB cells results in the withdrawal from the cell cycle and in increased expression of neural-specific differentiation markers, qualifies miRNA-9 as a potential therapeutically efficacious remedy for the treatment of this disease.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-05. IDH1 MUTATION ANALYSIS IN PEDIATRIC GLIOMA PATIENTS

Jun-ichi Adachi 1, Tomonari Suzuki 1, Kohei Fukuoka 1, Takaaki Yanagisawa 1, Kazuhiko Mishima 1, Tomoyuki Koga 1, Masao Matsutani 1, Ryo Nishikawa 1

Abstract

PURPOSE: Isocitrate dehydrogenase 1 (IDH1) mutations are genetic alteration with a high prevalence (50-70% of cases) in WHO grade II or III gliomas and secondary glioblastoma. The acquisition of IDH1 mutations occurs early in the development of a glioma from a glial progenitor cell that can give rise to both astrocytes and oligodendrocytes. These results, however, are based on studies of adult gliomas, but not pediatric gliomas. In this study, we examined IDH1 mutations in pediatric glioma patients with the following goals: (1) to compare the prevalence of IDH1 mutations in pediatric gliomas with those of adult ones; (2) to evaluate the relationship between IDH1 mutations and loss of 1p/19q or O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation. MATERIALS & METHODS: We analyzed the mutational hot spot, codon 132 of IDH1 in 19 newly diagnosed pediatric gliomas treated in our hospital. Real-time PCR system followed by high-resolution melting (HRM) analysis were used to detect IDH1 mutaions. We estimated loss of 1p/19q and MGMT methylation status by fluorescence in situ hybridization and methylation specific HRM, respectively. RESULTS: Of the 19 cases, IDH1 was mutated in only one glioma (anaplastic astrocytoma). This case was accompanied with methylated MGMT. Four pediatric oligodendrogliomas with co-deletion of 1p/19q had no IDH1 mutations and no methylated MGMT. CONCLUSIONS: The frequency of IDH1 mutations in pediatric gliomas was significantly low irrespective of tumor grades or histology. All 1p/19q co-deleted adult oligodendrogliomas showed IDH1 mutations, whereas pediatric oligodendrogliomas did not. These results suggest that there is a difference between pediatric gliomas and adult ones in genetic significance of IDH1 mutataions in gliomagenesis.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-06. EXPRESSION OF β1- AND β2-ADRENERGIC RECEPTORS IN PEDIATRIC MALIGNANT BRAIN TUMORS

Iacopo Sardi 1, Laura Giunti 1, Cecilia Bresci 1, Stefania Cardellicchio 1, Martina Da Ros 1, Anna Maria Buccoliero 1, Silvia Farina 1, Maurizio Aricò 1, Lorenzo Genitori 1, Maura Massimino 2, Luca Filippi 1

Abstract

β-adrenergic receptors (β-ARs) are G-protein-coupled molecules that activate the protein kinase A pathway by accumulation of the second messenger cAMP. This signal transduction pathway seems to increase VEGF gene expression, resulting in enhanced tumor vascularization and more aggressive growth. Moreover, several epidemiologic studies documented a significantly lower risk of cancer development or recurrence in humans treated with β-blocking agents. In malignant brain tumors, such as medulloblastoma, glioblastoma and anaplastic ependymoma, hypervascularization might result also from an enhancement of the β-adrenergic signaling pathway. In this study we addressed the issue of the expression of β-ARs in a population of 12 children affected by malignant primary brain tumors. We found a significant expression of β1, β2 -ARs in all of 12 samples as well as the 3 cell lines tested, U87MG, T98G and DAOY. Median absolute β1-AR mRNA level standardized for GAPDH was 5.81 (range –7.91 to 11.20) for brain tumors and 8.50 (range 6.046 to 12.59) for cell lines (U87MG, DAOY and T98G) respectively; Median absolute β2-AR mRNA level was 4.74 (range –9.30 to 8.45) for tumor specimens and 7.62 (range 5.84 to 8.88) for cell lines. These qRT-PCR expression data were confirmed by immunohistochemistry analysis. Our study documented the presence of b1-R and b2-ARs both in malignant brain tumors of pediatric age and brain tumor cell lines. Even if further studies are needed to better define β-adrenoreceptor expression in pediatric CNS tumors, a possible effect of propranolol and other β-blockers on the natural history is conceivable. The demonstration of the presence of β-ARs on pediatric malignant brain tumors could be the basis for an experimental clinical use of propranolol.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-07. NOVEL EFFECT OF PID1 IN BRAIN TUMORS

Anat Erdreich-Epstein 1, Hong Zhou 1, Xiuhai Ren 1, Mathew Schur 1, Tom Belle Davidson 1, Lingyun Ji 1, Richard Sposto 1, Shahab Asgharzadeh 1

Abstract

PID1 (Phosphotyrosine Interaction Domain Containing 1) is a recently identified PTB domain-containing cytoplasmic protein that inhibits insulin-dependent signaling in adipocytes and myocytes. PID1 has never been reported in the context of cancer, and its function is poorly understood. We find that high tumor PID1 mRNA correlates with better progression-free survival in children with medulloblastoma and with better overall survival in glioma patients. Ectopic expression of PID1 inhibits colony formation and induces mitochondrial depolarization in cell lines of three types of brain tumors: medulloblastomas, glioblastomas, and ATRT. The growth inhibition caused by PID1 is due to increased cell death and/or decreased proliferation (depending on the cell line). We coimmunoprecipitated native PID1 with native LRP1 (Low Density Lipoprotein Receptor-Related Protein 1) in glioblastoma cell lines. Interestingly, level of expression of PID1 and LRP1 were correlated in glioblastoma cell lines. We are now investigating the mechanism(s) by which PID1 induces its inhibitory effect in the brain tumor cell lines.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-08. MAPPING THE CELLULAR AND MOLECULAR ORIGINS OF CHOROID PLEXUS CARCINOMA

Yiai Tong 1, Elsie White 1, Mohankumar Murugesan 1, Birgit Nimmervoll 1, Michael Wang 1, Diane Marino 2, David Ellison 1, David Finkelstein 1, Stanley Pounds 1, David Malkin 2, Richard Gilbertson 1

Abstract

Choroid plexus carcinoma (CPC) is a lethal, yet poorly understood pediatric brain tumor, for which treatment options are limited. There are no well characterized animal models that might advance knowledge of CPC. Here, we used in utero electroporation to develop a novel, highly flexible and predictable mouse model of CPC that accurately recapitulates the human disease. Pregnant mice carrying embryonic day (E) 12.5 embryos harboring homozygous floxed alleles of Rb, Tp53, Pten and the ROSA-YFP reporter (Rbflx/flx; Tp53flx/flx; Ptenflx/flx, YFP flx/flx) were anesthetized, the uterus externalized and 1.5 µg of DNA encoding Cre recombinase microinjected into the IV (or lateral) ventricle of each embryo. The choroid epithelium of each embryo was then electroporated to introduce Cre plasmid. The uterus was returned to maternal abdomen and the embryos developed and delivered normally. Serial analysis of developing mice revealed loss of polarity of choroid plexus epithelium cells and the formation of hyperplastic lesions that in 40% (n = 25/63) of mice developed into large intraventricular CPCs by 2 and 5 months of age. Cross species genomics allowed us to map a region of mouse chromosome 4 that was frequently gained in mouse CPCs, and that is syntenic with a portion human chromosome 1p that we observed to be frequently gained in a large cohort of human CPCs (>50). Expression profiling of human and mouse CPCs identified 21 genes within this region of inter-species common gain that were overexpressed in both mouse and human CPCs. Remarkably, introduction of several of these genes alone (e.g., RAD54L) into the embryonic mouse choroid plexus induced aberrant proliferation and early tumorigenesis. We identify embryonic choroid plexus epithelium as a source of CPC, provide a new model of this tumor, and map several genes on gained chromosome 1 that likely contribute to the development of the disease.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-09. MODELING BRAIN TUMORS IN ZEBRAFISH

Christopher Eden 1, Bensheng Ju 1, Mohankumar Murugesan 1, Timothy Phoenix 1, Helen Poppleton 1, Charles Lessman 2, Michael Taylor 1, Richard Gilbertson 2

Abstract

Brain tumors include some of the most deadly forms of childhood cancer. Few novel treatments of these diseases have been developed over the last 30 years. Recently, we have developed accurate mouse models of pediatric ependymoma (Johnson et al., Nature 2010), medulloblastoma (Gibson et al., Nature 2010; Kawauchi et al., Cancer Cell in press) and choroid plexus carcinoma (unpublished), and employed these in high throughput drug screens to identify new treatments (Atkinson et al., Cancer Cell, 2011). These in vitro screens have identified >200 potential new therapies. But testing the efficacy of all of these compounds in mouse models is both cost and time prohibitive. Therefore, we sought to develop an intermediate in vivo model that would accurately recapitulate the basic biology of brain tumors, while allowing for relatively large scale drug efficacy testing. We labeled stem cell cultures of mouse ependymomas, gliomas and choroid plexus carcinomas with red fluorescence protein and adapted these to grow at temperatures of 28 degrees Celsius. Adapted cells were then transplanted orthotopically into the brains of zebrafish. Live in vivo imaging of fluorescently labeled tumor cells in fish revealed accurate, quantifiable and reproducible brain tumor formation between 3 and 5 days. Histological review and gene expression profiling of these tumors confirmed that they retain the morphology and molecular characteristics of the parent tumors. Transplantation of tumor cells into Fli1-GFP fish that possess fluorescent endothelial cells, demonstrated the capacity of mouse xenotransplanted tumor cells to recruit fish vasculature by neoangiogenesis, and to metastasize through the neuroaxis. We show for the first time that large cohorts of tumor bearing fish can be produced rapidly with tumors that reproduce features of the parent tumor. Our results indicate that zebrafish may play a pivotal role in bridging the gap between in vitro and mouse drug screening.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-10. ONDANSETRON PRE-TREATMENT FACILITATES BLOOD-BRAIN BARRIER PENETRATION OF DOXORUBICIN IN RAT MODEL

Iacopo Sardi 1, Giancarlo la Marca 2, Stefania Cardellicchio 1, Martina Da Ros 1, Sabrina Malvagia 1, Laura Giunti 1, Valentina Fratoni 2, Silvia Farina 1, Maurizio Aricò 1, Lorenzo Genitori 1, Maura Massimino 3, Maria Grazia Giovannini 2

Abstract

Our group recently verified that morphine pre-treatment facilitates doxorubicin delivery beyond the blood brain barrier (BBB) to the brain in the absence of signs of increased acute systemic toxicity in a rat model. Thus, it was plausible that morphine and other drugs as ondansetron inhibiting P-gp (MDR-1) localized on BBB, neurons and glial cells could increase the access of doxorubicin to the brain competing with the same efflux transporter, that very efficiently removes these drugs from the CNS. Thus, we explored the feasibility of active modification of the BBB protection, by using ondansetron pretreatment, to allow doxorubicin accumulation into the brain in an animal model. Rats were pretreated with different doses of intraperitoneal ondansetron before injection of doxorubicin (12 mg/kg). Quantitative analysis of doxorubicin was performed by mass spectrometry. Acute hearth and kidney damage was analyzed by measuring doxorubicin accumulation, LDH activity and malondialdehyde plasma levels. The concentration of doxorubicin was significantly higher in all brain areas of rats pretreated with morphine than in control tissues (P <0.001). This was evident only at therapeutic ondansetron dose (2 mg/kg, three times over 24 hours), while lower doses were not associated with doxorubicin accumulation. Pretreatment with ondansetron did not induce an elevation of LDH activity or of lipid peroxidation compared to controls. Our data suggest that ondansetron pre-treatment is able to allow doxorubicin penetration inside the brain, by modulating the blood-brain barrier. This is not associated with acute cardiac or renal toxicity. This finding might provide the rationale for clinical applications in the treatment of refractory brain tumors and pave the way to novel applications of active but currently inapplicable drugs.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-11. KIAA1549-BRAF FUSION GENE OCCURENCE IN PEDIATRIC BRAIN TUMORS OF DIFFERENT HISTOGENESIS

Felice Giangaspero 1, Manuela Badiali 2, Vincent Gleize 3, Simona Paris 3, Loredana Moi 2, Selma Elhouadani 4, Antonietta Arcella 5, Roberta Morace 5, Manila Antonelli 1, Francesca Buttarelli 1, Karima Mokhtari 4, Marc Sanson 3

Abstract

BRAF-KIAA1549 fusion gene is considered a driver genetic events in pilocytic astrocytomas (PAs). However recent studies have shown that the occurrence of this fusion gene in diffuse gliomas of adults is an event which is independent from more specific alterations such as IDH mutations. The aim of our study was to screen the presence of this molecular alteration in a series of pediatric CNS tumors of different histogenesis. Sixty-nine CNS tumors in patients aged 0-18 years were studied. BRAF-KIAA1549 fusion gene was evaluated by RT-PCR and subsequent sequencing. We detected KIAA1549-BRAF fusion gene in 20 out 34 (58%) PAs and in 1 out 3 (33%) gangliogliomas. KIAA1549-BRAF fusion gene was not detected in two unclassified low grade gliomas and 1 low grade glioneuronal neoplasm. Among diffuse gliomas, BRAF-KIAA1549 fusion was found in 10 out 26 (38%). It was observed in 2 glioblastomas, 2 oligodendroglioma, 1 anaplastic astrocytoma, 1 oligoastrocytoma, 1 pleomorphic xantoastrocytoma and 1 ependymoma. Moreover we detected BRAF-KIAA1549 fusion in 1 out 5 (20%) supratentorial PNET. Our results indicate BRAF-KIAA1549 fusion is a molecular event which is highly frequent in PAs, but occurs also, although with a lower frequency, in pediatric neoplasms of different histogenesis and grading. Our results suggest that deregulation of Ras-RAF-ERK signaling pathway, in these tumors is related to KIAA1549-BRAF fusion gene as in PAs.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-12. VASCULOGENIC MIMICRY IN A THREE DIMENSIONAL PEDIATRIC BRAIN TUMOR CULTURE SYSTEM

Stuart Smith 1, Jennifer Ward 1, Martin Wilson 2, Cheryl Rahman 4, Felicity Rose 4, Andrew Peet 2, Donald Macarthur 3, Richard Grundy 1, Ruman Rahman 1

Abstract

INTRODUCTION: Angiogenesis is of critical importance in the rapid growth of high grade pediatric brain tumors, with studies suggesting tumor differentiation into functional vasculature is possible. Using a novel three dimensional (3D) culture system we demonstrate upregulation of angiogenic pathways and exhibition of vessel like qualities by tumour cells (so called vasculogenic mimicry) with gene expression and drug sensitivity closer to primary tumors than 2D cell culture. METHODS: The Rotary Cell Culture System (RCCS) was used to cultivate 0.2-0.8cm3 aggregates of high grade pediatric brain tumor cell lines, an endothelial cell line and primary pediatric tumor explants. Genome wide gene expression analysis comparing primary tumor with 2D and 3D culture was performed using the Affymetrix U133plus2 chip and Nanostring MicroRNA chip. Expression of 84 angiogenesis and 84 ECM related genes was assessed by RT-PCR. Immunohistochemistry was performed against vessel related antigens and components of angiogenic pathways. The hypoxyprobe system was utilized to assess hypoxia. Magic angle spinning magnetic resonance spectroscopy was performed to establish metabolic profiles. Drug testing was performed using the HDAC inhibitor vorinostat. RESULTS: Tumor aggregates possess a complex heterogeneous structure with distinct proliferating, hypoxic and necrotic regions, replicating primary tumor. Endothelial antigens such as CD105 and CD31 are expressed by tumor cells. Genome wide analysis and rt-PCR identified multiple pro-angiogenic genes upregulated in 3D culture with gene expression shifted towards that of primary tumour. Metabolic profiles demonstrated significant differences including elevated lipids in 3D compared to 2D cultures. Tumor drug sensitivity is significantly reduced in 3D culture. CONCLUSIONS: We demonstrate that 3D culture recapitulates many key characteristics of primary pediatric brain tumors. Our findings contribute to understanding the angiogenic process in pediatric brain tumors and mechanisms of resistance to current anti-angiogenic therapies. The 3D culture system permits evaluation of next-generation anti-angiogenic agents in a pathophysiologically relevant context.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-13. MicroRNA 218a ACTS AS A TUMOR SUPPRESSOR IN MEDULLOBLASTOMA BY TARGETING CDK6, RICTOR AND CATHESPIN B

Sujatha Venkatraman 1, Diane Birks 1, Ilango Balakrishnan 1, Irina Alimova 1, Peter Harris 1, Purvi Patel 1, Nicholas Foreman 1, Rajeev Vibhakar 1

Abstract

MicroRNAs (miRNAs) are a class of short non-coding RNAs that regulate cell homeostasis by inhibiting translation or degrading mRNA of target genes. They can act as tumor suppressor genes or oncogenes. The role of microRNAs in medulloblastoma has only recently been addressed. We hypothesized that microRNAs differentially expressed during normal CNS development might be abnormally regulated in medulloblastoma and are functionally important for medulloblastoma cell growth. We found that microRNA 218a is strongly down regulated in medulloblastoma compared to normal pediatric cerebellum. Re-expression of miR-218a in medulloblastoma cell lines decreased cancer cell growth, cell clonogenicity, tumorigenicity and increased cell differentiation. The mechanisms by which microRNAs exert their function are not well elucidated. Alterations of expression in a single microRNA can modify the expression of many critical genes involved in tumor cell proliferation and differentiation. Several databases based on bioinformatic algorithms are available for predicting the targets of selected microRNAs. However they fail to accurately identify which genes are targeted by a given microRNA in a particular cellular context. Therefore, we took an unbiased experimental approach to identify and validate the direct targets of microRNA 218a. We performed HITS-CLIP (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation), which is a genome-wide means of mapping protein–RNA binding sites. Using this technique we identified 692 unique genes that are bound by miR218. Interestingly bioinformatics analysis only predicted 11% of the experimentally identified 218 targets. Three targets, CDK6, Rictor and Cathespin B were successfully validated by biological assays in medulloblastoma cell lines. Identification of context specific microRNA targets will significantly impact future investigations to develop new therapeutic pathways.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-14. STUDY ON THE CHANGES OF OLIGODENDROCYTES IN DIFFERENT BRAIN REGIONS OF INTRAUTERINE ISCHEMIA WISTAR RAT

Hui Wu 1, Qi Zhou 1, Dongxuan Wang 1, Guohua Wang 1, Dan Dang 1

Abstract

OBJECTIVE: To observe the changes of oligodendrocytes of intrauterine ischemia in Wistar rats with different levels within 28 days after birth, at different time points and in different brain regions. METHODS: Full-term pregnant rats were subjected to bilateral uterine artery ligation for 15 or 25 minutes to induce mild or severe perinatal hypoxia-ischemia (HI), respectively. Changes in MBP-labeled oligodendrocytes in the striatum, cortex, and hippocampus of offspring were examined at the following postnatal time points: hours, 0.5, 3, 6, and 24; days, 3, 5, 7, 9, 14, 21, and 28. RESULTS: MBP-labeled cells started to appear at postnatal day 9 in the control group and began to appear at day 5 and gradually increased over time in the mild and severe HI groups. In the striatum, that was significantly higher in the mild HI group at all time points relative to the control group. That in the severe HI group was significantly lower than in the control and mild HI groups at days 21 and 28. In the cortex, that was significantly higher in the severe HI group than in the control and mild HI groups at days 9, 14, 21, and 28. In the hippocampus, that was significantly lower than that of the control and mild HI groups at days 21 and 28. CONCLUSIONS: HI can stimulate differentiation and maturation of oligodendrocytes in a time- and area-dependent manner. The changes of oligodendrocytes are different in different brain regions. In the same brain region, mild HI may stimulate hyperplasia of late oligodendrocytes, while severe HI may reduce the number of late oligodendrocytes.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-15. THE COMBINATION OF mTOR and HIF-1 INHIBITORS IS HIGHLY EFFICIENT IN PEDIATRIC MALIGNANT BRAIN TUMORS

Erwan Pencreach 1, Aurelia Nguyen 1, Eric Guerin 1, Christelle Lasthaus 1, Dominique Guenot 1, Natacha Entz-Werle 2

Abstract

Intratumoral hypoxia plays a fundamental role in tumor progression and resistance to therapies. Tumor cell adaptation to hypoxic environment is regulated partially by the Hypoxia Inducible Factor-1 (HIF-1), which is involved in gene expression of energy metabolism, angiogenesis or metastatic processes. HIF-1 accumulation in cells is driven in particular by oncogenic pathway activation, like mTOR pathway. Recently, studies demonstrated that irinotecan, inhibiting HIF-1, plus mTOR inhibitor are completely suppressing HIF-1 accumulation. Interestingly, mTOR/HIF-1 pathway activation has been implicated in pediatric brain tumors, such as medulloblastomas, ependymomas or high grade gliomas. Therefore, preclinical studies were launched to understand the efficacy of irinotecan-rapamycin combination in these brain tumors. MATERIALS: Two medulloblastoma, one ependymoma and three high grade glioma patient cell lines were developed in our Lab. These cell lines, molecularly well characterized, were cultured in normoxic and hypoxic (1%O2) conditions and, to go further, were also xenografted subcutaneously in nude mice. METHODS: To test the impact of irinotecan/rapamycin combination on cell lines, in vitro assays were performed, analyzing the drug effects, alone or in combination, with clonogenic and proliferation evaluations. After subcutaneous tumor development, irinotecan (10mg/kg), rapamycin (3mg/kg) or the combination were administered to mice every 5 days for 3 cycles. Histological, protein and glycolytic analyses were done to understand the sensitivity of each tumor type to treatments. RESULTS: The drug combination reduced cell proliferation in all cell lines, cultured in hypoxic conditions, and abrogated completely HIF-1 expression. In the in vivo experiment, the tumors disappeared with the drug combination. Histological analyses showed a major destruction of the tumors, which was correlated to anti-glycolytic and anti-angiogenic effects. CONCLUSION: These promising results encouraged the development of a phase I study in the pediatric setting including refractory tumors.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-16. EPIGENETIC DEREGULATION OF THE DOPAMINE RECEPTOR D4 IN PEDIATRIC BRAIN TUMORS

Rebekka Unland 1, Sabrina Schlosser 1, Nicole Farwick 1, Tanja Plagemann 1, Günther Richter 2, Heribert Juergens 1, Michael Fruehwald 3

Abstract

Epigeneitc alterations such as DNA methylation and histone modifications are common events in cancer. To discover novel methylation targets in pediatric brain tumors, genome wide methylation analysis was performed. AscI-RLGS of CNS tumors and non-malignant controls identified the dopamine receptor D4 (DRD4) as a novel methylation target. DRD4 is a G protein-coupled receptor and wildly expressed in the central nervous system. Tumor-specific methylation of DRD4 of > 35% was deteced in 18/24 medulloblastomas, 23/29 ependymomas, 6/6 high grade gliomas, 7/10 sPNET and 8/8 cell lines by calibrated COBRA (qCOBRA). Results were confirmed by bisulfite sequencing. Futhermore, real-time RT-PCR confirmed a significantly decreased expression of DRD4 in pediatric brain tumors and cell lines compared to non-malignant control tissues. To analyse the effects of epigenetically active compounds on DRD4 expression, medulloblastoma cell lines were treated with 5′-aza-2′-deoxycytidine and combinatorial treatment with 5′-aza-2′-deoxycytidine and trichostatin A. Quantitative real-time PCR and immunofluorecence staining confirmed a restoration of DRD4 in various medulloblastoma cell lines treated with 5′-aza-2′-deoxycytidine. Furthermore, the methylation status as well as histone modifications of DRD4 5′region were analyzed following treatment and epigenetic changes on DRD4 were observed. Methylation analysis by direct bisulfite sequencing and qCOBRA revealed no demethylation of the DRD4 5′region following treatment, whereas chromatin immunoprecipitation assays showed a significant decrease of trimethylated histone H3 at lysine 27 (H3K27me3) as well as of the histone methyltransferase enhancer of zeste homologue 2 (EZH2) at the DRD4 promoter. In addition, knockdown of EZH2 in vitro caused a restoration of DRD4, suggesting a functional role of EZH2 in the epigenetic deregulation of DRD4. Here, we present first functional analysis concerning the dopamine receptor D4 and it's epigenetic inhibition in pediatric brain tumors. Supported by DKH Project 109062

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-17. FUNCTIONAL GENOMICS ANALYSIS IDENTIFIES THE ONCOGENIC ROLE OF miR-221-222 IN PEDIATRIC EMBRYONAL BRAIN TUMORS

Chen-Li Chien 1, Yu-Hsiu Lee 2, Chen-I Lin 1, Jui-Yu Hsieh 2, Shih-Chieh Lin 3, Tai-Tong Wong 1, Donald Ming-Tak Ho 3, Hsei-Wei Wang 2

Abstract

Pediatric brain tumors are the most found solid tumors in children. Embryonal brain tumors, which include medulloblastoma (MB), atypical teratoid/rhabdoid tumor (AT/RT), and primitive neuroectodermal tumor (PNET), form the second most common pediatric brain tumors in both western countries and Taiwan. AT/RT is often misdiagnosed as MB. However, distinguishing AT/RT from MB is of clinical significance since AT/RT requires more extensive drug and irradiation treatment regimens with higher recurrence rate and lower survivals, yet the underlying pathogenesis mechanisms are largely unknown. To enhance current diagnosis, prognosis and therapy systems, tumor molecular properties of AT/RT and MB were examined. By using high throughput miRNA-Seq and gene expression microarray analyses, we studied the expression patterns of microRNAs (miRNAs) and messenger RNAs (mRNAs) in AT/RT and MB. We found miRNAs and mRNAs up- or down-regulated in AT/RTs when compared with MBs. Genes and miRNAs responsible for stemness (S100A4), tumorigenesis (miR-221/-222, miR-30a, IL7 and IL18) and angiogenesis (FGF2 and VEGFA) were abundant in AT/RT, while those associated with neuron differentiation in MB. Compared with MB, AT/RT recaptures ESC traits by forfeiting neurogenesis program. Since miRNA can function as oncomiR or tumor suppressor miRNA through blocking the expression of target mRNA which tumor suppressor gene or oncogene transcribed and also downstream protein translation, the roles of filterated miRNAs (such as miR-221/-222) and their downstream targets in AT/RT tumorigenesis and malignancy were evaluated by in vitro and in vivo functional assays. Overexpression of miR-221/-222 or knockdown of their downstream targets in embryonal brain tumor cells significantly enhanced cell proliferation and tumor formation in SCID mice. Thus, this study discloses the molecular compositions of embryonal brain tumors and suggests candidate drug targets, such as miR-221/-222, for AT/RT.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-18. RHPS4 G-QUADRUPLEX LIGAND EXERTS ANTI-PROLIFERATIVE EFFECTS IN CHILDHOOD BRAIN TUMOUR CELLS WITH ASSOCIATED TELOMERASE INHIBITION

Sunil Lagah 1, I-Li Tan 2, Stevens Malcolm 1, Richard Grundy 1, Ruman Rahman 1

Abstract

INTRODUCTION: The human telomeric 3' overhang can fold into a four-stranded DNA structure termed G-quadruplex (G4), a formation shown to inhibit telomerase. As telomerase activation is crucial for telomere length maintenance in most cancer cells, several classes of G4 ligands have been designed to directly disrupt telomeric structure. Here we investigate the anti-tumour effects of the G4 ligand RHPS4, on childhood brain tumour cells. METHODS: CNS PNET, medulloblastoma (MB), high grade glioma (HGG) and mixed glioneuronal cells were exposed to RHPS4 and anti-proliferative effects measured using the MTT assay. A modified TRAP assay was used to determine the effect of RHPS4 on telomerase activity and the TRF assay used to determine mean telomere length. Flow cytometry was conducted to establish cell cycle state upon RHPS4 exposure and caspase-3 protein expression used to confirm apoptosis. RESULTS: Proliferation was impaired in brain tumour cells upon acute RHPS4 exposure. Although all cell lines were sensitive to RHPS4, CNS PNET and MB cells exhibited 10-fold increased sensitivity compared to HGG and mixed glioneuronal cells (IC50 2.7 µM/2.2 µM vs. 26 µM/32 µM respectively). MB and HGG cells arrested in S-phase whereas CNS PNET cells showed an increased proportion of G1-phase cells. Caspase-3-dependent apoptosis was observed in a concentration-dependent manner. RHPS4-induced anti-cancer phenotypes were concomitant with telomerase inhibition, although mean telomere length did not correlate with RHPS4 sensitivity. CONCLUSIONS: RHPS4-mediated abrogation of tumour cell growth is associated with G1/S phase arrest, caspase-3-mediated apoptosis and telomerase inhibition. However, these effects are telomere length independent in this context. This suggests that RHPS4 induces an altered telomere state that impairs the access of telomerase to the telomere substrate, resulting in abrupt cessation of cell turnover. To our knowledge, this is the first report of G4 ligand-induced telomerase inhibition in childhood brain tumour cells and warrants in vivo validation as a therapeutic agent.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-19. CHEMOKINE EXPRESSION IN PAEDIATRIC HIGH GRADE GLIOMA IS INFLUENCED BY THE TUMOUR MICROENVIRONMENT, HYPOXIA AND DIFFERS FROM ADULT HIGH GRADE GLIOMA

Yvonne Majani 1, Stuart Smith 1, Richard Grundy 1, Ruman Rahman 1

Abstract

BACKGROUND: Chemokines are chemotactic cytokines involved in high grade glioma (HGG) angiogenesis, proliferation and invasion. Paediatric and adult HGG are similar histologically but differ genetically. We investigated whether chemokine biology differs between paediatric and adult HGG in 2D/ 3D culture and tested whether hypoxia influences chemokine expression. METHOD: Primary tumours, KNS42 (paediatric HGG) and U87 (adult HGG) cells cultured as 2D monolayers in normoxic or hypoxic (1% oxygen) conditions and tumour aggregates in a dynamic 3D culture system were used. RNA extraction was performed on cell pellets and tumour tissue followed by real-time array-PCR to analyze 84 chemokine-related genes. RESULTS: CXCL1, CXCL2, CXCL3. CXCL8, CCL1, CCL2, CCL3, CCL4, CCL8 and CCR6 were upregulated in KNS42 3D cultures compared to 2D cultures, whilst CXCL11 was downregulated (p < 0.02). There was no difference in chemokine expression between KNS42 2D cells in normoxia and hypoxia. CXCL8 was upregulated in U87 2D cells in normoxia compared to hypoxia, whilst CCL2 was downregulated (p < 0.05). In normoxia, CXCL6, CXCL11, CCL2 were upregulated in KNS42 2D cells compared to U87 2D cells, whilst CX3CL1, CXCL3, CXCL8 were downregulated. (p < 0.05). In hypoxia, CXCL6, CXCL10, CXCL11, CCL2, CCL7 and CXCR4, CCR10 were upregulated in KNS42 2D cells compared to U87 2D cells, whilst CXCL1, CXCL2, CXCL8, CXCL12 and CX3CL1 were downregulated (p < 0.01). Many chemokines were downregulated in all conditions when compared to a cohort of primary HGG. CONCLUSION: Chemokine expression in HGG is likely influenced by intrinsic cell-cell interactions and hypoxic gradients which partly affect the tumour microenvironment. 3D brain tumour cultures may reflect more physiologically relevant chemokine expression patterns. In addition we demonstrate that adult and paediatric HGG exhibit differential expression of chemokines and related receptors. Correlation between chemokine expression in primary tumours and 2D/3D cultures +/- hypoxia will be discussed.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-20. PRE-B LEUKEMIA HOMEOBOX INTERACTING PROTEIN (PBXIP1) AS A NOVEL ASTROCYTIC STEM CELL MARKER AND POTENTIAL NEW TREATMENT TARGET IN GLIAL AND EPENDYMAL TUMORS

Dannis G van Vuurden 1, Eleonora Aronica 5, Laurine E Wedekind 4, Esther Hulleman 1, Dennis Biesmans 4, Marianna Bugiani 3, W Peter Vandertop 7, Gertjan JL Kaspers 2, Thomas Würdinger 6, David P Noske 4, Petra M Van der Stoop 4

Abstract

BACKGROUND: Glial and ependymal brain tumors are malignancies that cause considerable mortality and morbidity in child- and adulthood. Innovative targets for therapy are needed to improve survival and reduce long term sequelae. AIM: To identify proteins that are abundantly expressed in brain tumor cells and not in normal brain tissues as possible candidates for targeted therapies. MATERIAL AND METHODS: We used the Human Protein Atlas for in silico protein analysis to identify proteins that are differentially overexpressed in high grade glioma (HGG). In silico genomics analysis and immunohistochemistry on glial tumors and tissues of pre- and postnatal human brain were used for validation and to study the role of the candidates during human brain development. RNA interference (RNAi) studies enabled to study their role in vitro. RESULTS: In silico proteomics and genomics analysis identified Pre-B-cell leukemia homeobox interacting protein 1 (PBXIP1) to be strongly overexpressed in adult and childhood HGG and in childhood ependymoma compared to non-malignant brain. Immunohistochemical studies confirmed specific overexpression of PBXIP1 in astrocytic and ependymal tumors, but not in oligodendroglioma and medulloblastoma, and in reactive astrogliosis. During human embryonal development, strong expression of PBXIP1 was observed in radial glia and in migrating astrocytic precursor cells and co-localisation with nestin and vimentin, suggesting PBXIP1 to be a stem cell / progenitor marker. PBXIP1 shRNA mediated knockdown in established and primary HGG cell lines strongly reduced proliferation and motility. Also disruption of lamellopodia, increase of filiopodial F-actin stress fibers and loss of polarity was observed in these cells. CONCLUSION: Our study shows that PBXIP1 is overexpressed in astrocytic and ependymal tumors and may be a marker for astrocytic stem-/progenitor cells. The strongly reduced viability upon loss of PBXIP1 expression warrants further studies to explore PBXIP1 as a novel therapeutic target in glial tumors.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-21. IN VITRO RADIOSENSITIZATION OF HIGH GRADE GLIOMA CELLS BY CI-1033 (CANERTINIB), A pan-ErbB FAMILY INHIBITOR

Dannis G van Vuurden 1, Shruti Shukla 1, Laurine E Wedekind 2, Gitta K Kuipers 3, Esther Hulleman 1, David P Noske 2, Thomas Würdinger 4, W Peter Vandertop 5, Ben J Slotman 3, Gertjan JL Kaspers 6, Jacqueline Cloos 7

Abstract

BACKGROUND: High grade gliomas (HGG) are highly invasive malignancies, resistant to treatment, causing considerable mortality in child- and adulthood, necessitating new therapies. Novel therapies directed against epidermal growth factor (EGF) family members (ErbB family) are potentially effective in HGG. AIM: To assess ErbB family expression in normal brain and pediatric and adult HGG in silico and to determine radiosensitizing property of the pan-ErbB inhibitor CI-1033, in HGG cells in vitro. MATERIAL AND METHODS: In silico mRNA array expression analysis was performed to assess EGFR, ErbB2, ErbB3, ErbB4 in normal brain, HGG and diffuse intrinsic pontine glioma (DIPG). ErbB protein expression was determined in HGG cell lines using Western blot. Sulforhodamine-B assay was used to assess IC50 values of CI-1033 before performing clonogenic assays to determine radiosensitization. The effect on cell cycle distribution and PI3K-Akt/Ras-MAPK signalling of CI-1033 as mono- and combination therapy with radiation was measured using flowcytometry. RESULTS: Compared to normal brain, EGFR and ErbB2 were overexpressed and ErbB4 downregulated in pediatric HGG/DIPG and adult HGG. ErbB3 was overexpressed in DIPG. Heterogeneous protein expression of EGFR, ErbB2 and ErbB4 was observed in HGG cell lines D384-MG, U251-MG and Gli-6 while ErbB3 expression was only observed in D384-MG cells. CI-1033 IC50 values of 1.0 µM, 3.1 µM and 6.1 µM were found in D384-MG, U251-MG and Gli-6 cells, respectively. In clonogenic assays, CI-1033 sensitized Gli-6 and D384-MG cell to radiation, with 24 hour and 48 hour pre-treatment respectively. CI-1033 reduced ERK and Akt phosphorylation, however its radiosensitization was not related to either Akt/ERK phosphorylation or cell cycle distribution. CONCLUSION: EGFR and ErbB2 are overexpressed in adult and childhood HGG/DIPG. CI-1033 radiosensitizes HGG cell lines in vitro, dependent on pre-incubation period of the drug. These results warrant further in vivo studies to assess the role of pan-ErbB family inhibition on radiosensitization.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-22. CELL-INTRINSIC SEX DIFFERENCES IN CYCLIC AMP SIGNALING IMPACT PEDIATRIC BRAIN TUMORIGENESIS

Tao Sun 1, Nicole Warrington 1, Jingqin Luo 2, Sara Ganzhorn 5, Uri Tabori 4, Todd Druley 1, David Gutmann 3, Joshua Rubin 1

Abstract

Childhood brain tumors occur more in boys than in girls. The incidence of pediatric brain tumor peaks in children younger than 4 years, when circulating sex hormones levels are equivalently low in males and females, suggesting that the sex difference in the incidence of pediatric brain tumors is unlikely to reflect the actions of sex hormones. In published and preliminary studies, we developed an induced mouse model of neurofibromatosis-1 (NF1) glioma that exhibits a sex-dependent pattern of tumorigenesis. In this model, we found that cyclic AMP (cAMP) suppression is sufficient to induce glioma, but only in male mice. The clinical relevance of this finding was strengthened by Affymetrix SNP array analysis on DNA samples from 89 NF1 patients with or without optic pathway glioma (OPG). Consistent with the results from our tumor induction model, multiple SNPs in several components of cAMP pathway were correlated with NF1 OPG in a sex-dependent manner. Based on these results, we hypothesize that cell-intrinsic sex differences in growth regulatory pathways, including cAMP pathway, confer male and female tumor progenitors with different tumorigenic susceptibility. In this regard, we found that male and female Nf1-/- astrocytes exhibited different levels of intracellular cAMP and the cAMP-regulated cell cycle inhibitor p21(Waf1/Cip1). Moreover, Nf1-/- astrocytes showed sex differences in cellular senescence following Nf1 loss, and in their susceptibility to oncogenic transformation in response to overexpression of a dominant-negative p53 (DN-p53) molecule. Finally, implantation of Nf1-/-; DN-p53 astrocytes into the flanks of immunodeficient mice resulted in a sexually dimorphic tumor growth pattern in which male Nf1-/-;DN-p53 astrocytes yielded significantly higher tumor volumes than female counterparts regardless of the sex of recipient mice. Collectively, these findings establish that cell-intrinsic differences between male and female astrocytes partly underlie the sexually-dimorphic patterns of gliomagenesis.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-23. A SPECIFIC REGION IN THE hTERT PROMOTER CAN PREDICT MALIGNANCY AND SURVIVAL IN PATIENTS WITH CHILDHOOD BRAIN TUMORS

Pedro Castelo-Branco 1, Sanaa Choufani 1, Stephen Mack 1, Denis Galagher 1, Cindy Zhang 1, Tatiana Lipman 1, Natalyia Zhukova 1, Dianna Martin 1, Diana Merino 1, Jonathan Wasserman 1, Cynthia Samuel 1, Noa Alon 1, Johann Hitzler 1, Jean CY Wang 3, David Malkin 1, Gordon Keller 3, Peter B Dirks 1, Stephan Pfister 2, Michael D Taylor 1, Rosanna Weksberg 1, Uri Tabori 1

Abstract

BACKGROUND: hTERT is expressed in 85% of all cancers and hTERT expression correlates with patient outcome in several childhood brain tumors. Analysis of hTERT promoter methylation in cancer has generated conflicting data and has never been done in CNS tumors. We searched for methylation differences in the hTERT promoter which distinguish normal tissue from cancer. METHODS: We performed methylation analysis of the entire hTERT promoter in multiple normal and malignant tissues including pluripotent, neural and cancer stem cells. Correlation between promoter methylation, hTERT expression and telomere status was performed. RESULTS: We identified a specific region upstream of the transcription start site (UPS) that is hypermethylated in 90% of malignant cancers that express high levels of hTERT. In contrast, UPS is hypomethylated in normal tissues and in low grade tumors lacking hTERT expression. Interestingly, the 10% of malignant tumors without UPS hypermethylation lacked hTERT expression and revealed an alternative lengthening of telomeres phenotype. We have also observed an increase in UPS methylation in paired samples from the same patients with tumors that transformed from low to high grade or from primary to metastatic. All choroid-plexus papillomas and atypical-papillomas with UPS hypomethylation did not recur, while atypical-papillomas with high UPS methylation progressed and transformed to carcinomas. Moreover, patients with ependymomas had 5-year overall survival of 45 + /-15% and 100% for methylated and hypomethylated tumors respectively (p = 0.0023). Finally, treatment with 5-Azacytidine resulted in UPS demethylation and a decrease in hTERT expression in glioma and medulloblastoma cells but not in embryonic stem cells. CONCLUSION: Hypermethylation of a specific region in the hTERT promoter is a cancer phenotype. It is a dynamic process which positively correlates with higher hTERT expression, tumor progression and poor prognosis. hTERT UPS methylation may also represent a diagnostic tool and a therapeutic target in nervous system tumors.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-24. CILENGITIDE TARGETS MORE EFFICIENTLY PEDIATRIC THAN ADULT GLIOMA CELLS IN VITRO THROUGH CELL DETACHMENT AND ANOÏKIS INDUCTION

Pierre Leblond 1, Samuel Meignan 2, Amélie Dewitte 2, Florence Le Tinier 1, Nicole Wattez 1, Eric Lartigau 1, Amélie Lansiaux 2

Abstract

Despite numerous clinical trials, the prognosis of children with high grade gliomas (HGG) remains poor, indicating a need for new treatments. Cilengitide (EMD 121974, Merck Kga, Darmstadt, Germany) is a selective antagonist of αvβ3 and αvβ5 integrins, known to be involved in tumor growth, angiogenesis and metastasis development. The aim was to investigate the efficacy of cilengitide in specific pediatric glioma cell lines. METHODS: We have a wide tumor cell panel including three high grade (SF188, KNS42, UW479) and two low grade (Res259, Res186) pediatric glioma cell lines compared with adult HGG cell line (U87MG). RESULTS: Flow cytometry analysis revealed various αvβ3 expression levels but no correlation with tumor grade. We noted a lack of αvβ3 expression in HGG UW479 cell line. Cell detachment assay showed that cilengitide have no effect on UW479 cells and on cells adhered on collagen I, a non specific matrix, demonstrating its specificity. Moreover, cilengitide presents a rapid and dose dependent action on cell detachment. Surprisingly, despite the strong detachment on U87MG cells, cilengitide presents a moderate toxicity for these cells compared to pediatric ones. Growth kinetics in non adherent conditions revealed that U87MG cells, forming floating cell clusters, are resistant to anoïkis and thus can grow in spite of cilengitide-induced detachment, contrary to pediatric cells. This result was confirmed by the inability to determine an IC50 value for U87MG and UW479 cells. Cilengitide action on glioma cells appears dependent of αvβ3 expression and sensibility to anoïkis, two properties found in the majority of pediatric cell lines. Thus cilengitide is able to target directly and efficiently the pediatric glioma cells. CONCLUSION: Considering the antiangiogenic effect of cilengitide published in vivo using U87MG cells, and its better toxicity observed on pediatric glioma cells, we can hope a strong effect on pediatric glioma in orthotopic model.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-25. IDENTIFICATION OF RADIATION-INDUCED GENE EXPRESSION PROFILES IN AN INTRACRANIAL XENOGRAFT MOUSE MODEL

Ryan Hanson 1, Ira Gordon 1, Shuping Zhao 1, Kevin Camphausen 1, Katherine Warren 1

Abstract

PURPOSE: Radiation therapy plays a central role in the treatment of brain tumors. To evade injury or death, cancer cells mount complex responses to radiation-induced damage. We hypothesized that the genetic response of tumor cells to radiation-induced damage is dose and schedule dependent. To evaluate differential gene expression after ionizing radiation (IR) in vivo, we generated radiation-induced gene expression profiles in a mouse model. METHODS: U87 cells grown in culture, collected by trypsinization and injected into the right caudate nucleus of 6-week-old female athymic nude mice. Radiation was delivered intracranially to anesthetized animals according to three different radiation schedules: 2 Gy, 10 Gy, and 2 Gy daily x 5. Tumors were harvested, homogenized and total cellular RNA was purified. Microarray was performed on DNA chips containing 7,680 human cDNA clones. Analysis of variance was performed using Partek Genomic Suite. Ingenuity Pathway Analysis was used to determine pertinent biological functions of upregulated genes. RESULTS: Comparison of profiles from irradiated to unirradiated tumors revealed changes in > 240 genes. Moreover, 21 genes involved in cell growth and proliferation increased in expression by ≥2-fold, regardless of the dose of IR (GAS1, LDOC1, MAF, NOV and SPP1). The transcriptional response of tumors to 2 Gy and 10 Gy yielded increased expression of genes relating to canonical pathways such as cell cycle, DNA repair, apoptosis, and free radical scavenging (CDC20, GADD45G, GLI1 and CYP1B1, respectively). Tumors treated with 2 Gy x 5 demonstrated elevated expression of genes associated with pathways not previously known to have a regulatory function in cellular radioresponse (DYRK3, FOXC1, RGS2 and SPRY). CONCLUSION: The transcriptional landscape of glioma is differentially affected by the dose of IR. Further characterization of the radiation-induced transcriptome may allow for the identification of targets that may be affected by ionizing radiation.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-26. DIFFERENCES IN CYCLIC AMP REGULATION AS A POTENTIAL MECHANISM FOR THE SEXUAL DISPARITY IN RATES OF PEDIATRIC BRAIN TUMORS

Nicole M Warrington 1, Tao Sun 1, David H Gutmann 2, Joshua B Rubin 1

Abstract

Gliomagenesis is well studied in models of the tumor predisposition syndrome, Neurofibromatosis 1 (NF1). We showed that cAMP suppression through forced expression of phosphodiesterase 4A1 is sufficient to drive gliomagenesis in a mouse model of NF1. We now report that the effects of PDE4A1 expression and cAMP suppression are sex-dependent. Expression of PDE4A1 resulted in glioma in 100% of male, but only 20% of female mice. As gliomas occur more commonly in males, we exploited this mouse model as a platform on which to investigate the molecular basis for the effects of sex on gliomagenesis. We found significant differences in cAMP regulation in male compared to female astrocytes, which appear to mediate an effect of sex on gliomagenesis. First, we measured expression of cAMP regulators in male and female Nf1-/- astrocytes. Male Nf1-/- astrocytes expressed greater levels of Gαi and adenylate cyclase (AC) 3, while female Nf1-/- astrocytes exhibited higher PDE4A1 expression. Consistent with the expression data, female astrocytes possessed higher basal PDE activity and lower levels of cAMP compared to male astrocytes, while male astrocytes exhibited greater basal AC activity. Next we determined that differences in cAMP regulation were biologically relevant as treatment with the AC activator forskolin inhibited male, but not female, Nf1-/- astrocyte growth, while treatment with the PDE4 inhibitor rolipram elevated cAMP levels in female, but not in male, cells. Finally, we explored the clinical relevance of the effects of sex and cAMP regulation through SNP array analysis of saliva DNA samples from NF1 patients with and without glioma. Polymorphisms in several cAMP regulator genes (GNAI, PDE4D, and AC8) were significantly associated with glioma risk in a sex-dependent manner. We conclude that sexual dimorphism in cAMP regulation impacts on gliomagenesis in NF1. Identification of polymorphisms in cAMP regulators may have prognostic value for patients with NF1.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-27. INVOLVEMENT OF DNA MISMATCH REPAIR (MMR), BASE EXCISION REPAIR (BER) SYSTEMS AND O6-METHYLGUANINE METHYLTRANSFERASE (MGMT) IN PEDIATRIC HIGH GRADE GLIOMA RESISTANCE TO TEMOZOLOMIDE (TMZ)

Aurelia Nguyen 1, Christelle Lasthaus 1, Madeleine Jaillet 1, Erwan Pencreach 1, Eric Guerin 1, Dominique Guenot 1, Natacha Entz-Werle 1

Abstract

Pediatric high-grade gliomas (pHGGs) remain tumors with a very dismal prognosis for which novel therapeutic strategies are needed. Currently, TMZ constitutes the standard of care for pHGGs even the tumor responses are rather disappointing comparatively to adult counterparts. In order to investigate the molecular characteristics explaining the TMZ resistance and highlighting new therapeutic targets, we studied 20 pHGGs for the expression of MGMT, for MMR dysfunction and for BER enzyme expressions. The cohort was also molecularly characterized for p53, PTEN, PDGFRA, MET and EGFR genes. MATERIAL AND METHODS: 20 fresh-frozen pHGGs (50% of grade III and 50% of grade IV gliomas) were collected at diagnosis in our centre during the 6 last years and provided good quality DNA and RNA extracts. Clinical database was established for all patients. The status of MGMT gene promoter methylation was determined by methylation-specific PCR, whereas the MMR phenotype presence was concluded on allelotyping analyses of the National Cancer Institute-recommended panel (BAT25, BAT26, D2S123, D5S346, and D17S250). The MGMT and BER enzyme expressions (PARP-1, MPG and APE-1) were studied by quantitative RT-PCR. The gene screening was performed by allelotyping analyses and/or quantitative PCR. RESULTS AND DISCUSSION: As already described in other pHGGs, a rearrangement of p53 gene locus and PTEN deletion were observed in almost 60%. PDGFRA was amplified in 10% and deleted in 10% pHGGs. EGFR and MET were rearranged in 40%pHGGs but amplified in fewer patients (10%). MGMT methylation and MSI phenotype were present in 15%, whereas concomitant over-expression of PARP-1 and MPG were observed in 40% of the cohort. Higher expression of MGMT and less frequent methylation of the MGMT gene promoter may partly account for TMZ resistance, but BER enzyme over-expressions seem to predominate and would explain more accurately the increase TMZ resistance in the pediatric setting.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-28. FOCUSED ULTRASOUND-MEDIATED DELIVERY OF DOXORUBICIN IN A MOUSE MODEL OF GLIOBLASTOMA

Zsofia Kovacs 1, Ernst Martin-Fiori 2, Tarek Shalaby 1, Michael Grotzer 1, Michele Bernasconi 1, Beat Werner 2

Abstract

BACKGROUND: Glioblastoma (GB) is the most malignant primary brain tumor. Here we demonstrate targeted delivery of Doxorubicin (DOX) into the brain of GB bearing mice by applying focused ultrasound (FUS) mediated transient disruption of the blood-brain barrier (BBB), and therapeutic benefit of DOX delivery in terms of tumor growth and survival time. METHODS: All FUS experiments were carried out in wild type C57BL/6J-Tyrc-J mice under MRI guidance. The FUS BBB disruption protocol consisted of a single element ultrasound transducer (Imasonic) that emitted ultrasound at peak negative pressure of 0.4 MPa and microbubbles (BR38®, Bracco) were injected into the tail vein (1 µL/s for 60 s). The feasibility of FUS mediated targeted trans-BBB delivery of DOX was demonstrated in 14 animals by injecting 8 mg/kg DOX immediately before FUS mediated BBB opening and subsequent measurement of DOX concentration in brain tissue homogenates by HPLC. Therapeutic effect of trans-BBB delivery of DOX was assessed in 7 animals by monitoring tumor growth and survival time in a syngenic mouse GB model. Tumors were induced by stereotactic injection of 105 GL261 mouse GB cells into the cortex. Treatment consisted of a single dose of 8 mg/kg DOX before application of FUS. RESULTS: HPLC quantification showed significantly elevated concentrations of DOX in sonicated brain tissue (173 ± 23.780 ng/ml) as compared to unsonicated tissue in the contralateral brain hemisphere (10.48 ± 5.097 ng/ml). Tumor bearing mice treated with DOX had a significantly slower tumor growth and survived significantly longer (35 ± 3 days) than the untreated littermates (21 ± 3 days). CONCLUSION: Microbubble enhanced FUS can be applied to focally open the BBB and efficiently elevate DOX concentration in the brain of tumor bearing mice and increase its therapeutic effect to slow down tumor growth and prolong survival time.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-29. Wnt/BETA-CATENIN SIGNALING REGULATES THE EXPRESSION OF O6-METHYLGUANINE DNA-METHYLTRANSFERASE IN MEDULLOBLASTOMA CELLS: A NEW STRATEGY TO INCREASE SENSITIVITY FOR DNA ALKYLATORS IN CANCER THERAPY

Cecilia Dyberg 1, Ninib Baryawno 1, Jelena Milosevic 1, Malin Wickström 1, Paul A Northcott 2, Michael D Taylor 2, Marcel Kool 3, Per Kogner 1, John Inge Johnsen 1

Abstract

BACKGROUND: A number of DNA-damaging agents attack the O6 position on guanine and thereby form the most potent cytotoxic DNA adducts known. The DNA-repair protein O6-alkylguanine (O6-AG) DNA alkyltransferase (AGT) encoded by the gene O6-Methylguanine (O6-MG)-DNA-methyltransferase (MGMT) repair DNA adducts caused by alkylating agents. MGMT has important implications in cancer treatment since its expression correlates inversely with sensitivity to agents that form O6-alkylguanine adducts, such as temozolamide. It is therefore of great interest to find agents that induce MGMT deficiency, increase the sensitivity and possible overcoming resistance to alkylating chemotherapeutic agents. METHODS: Cell lines from medulloblastomas and gliomas were examined for Wnt/beta-catenin activity and MGMT expression. We used western blot, Real-Time quantitative PCR (Q-PCR), siRNA knockdown, cDNA overexpression of beta-catenin and MGMT promotor reporter plasmids. Cell cytotoxicity and clonogenicity of chemotherapeutic drugs in combination with celecoxib were examined in cell lines using fluorometric microculture cytotoxicity assay and clonogenic assay, respectively. Compounds targeting Wnt signaling was investigated in combination with temolzolamide in vitro and in vivo. RESULTS: MGMT expression level was correlated to Wnt signaling activation both in primary tumors and cell lines of different origins. Transfection experiments revealed that beta-catenin directly regulates MGMT expression via Tcf/LEF binding. Wnt inhibiting drugs and compounds potentiates the cytotoxic effect of the DNA alkylating drug, temozolomide in cells with elevated MGMT expression in vitro and in vivo. CONCLUSIONS: Our data demonstrate that MGMT is a direct Wnt/beta-catenin target, and agents that inhibit Wnt signaling reduces the transcription of MGMT through a prostaglandin E2-Wnt/beta-catenin route and thus increases the sensitivity to temozolomide. This provides a rational approach for improved efficacy of chemotherapeutic drugs inducing DNA alkylation in cancer treatment. The data also suggest that Wnt/beta-catenin is an important target for therapeutic interventions.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-30. DETECTING REGIONS OF ACTIVE TUMOUR USING MAGNETIC RESONANCE SPECTROSCOPIC IMAGING

Martin Wilson 1, Greg Reynolds 2, Nigel Davies 1, Theo Arvanitis 1, Andrew Peet 1

Abstract

INTRODUCTION: Magnetic Resonance Spectroscopic Imaging (SI) provides information on the regional variation of a range of important metabolites. It is ideally suited for investigating large heterogeneous and diffuse infiltrating lesions either at diagnosis or follow-up. SI data is usually interpreted by viewing colour maps of single metabolites or their ratios. However, metabolite profiles have been shown to be much more specific than single metabolites for childhood brain tumours. We have used this property to develop a new SI analysis method for identifying regions of metabolically active tumour and applied it to scans collected at key clinical decision points where SI could provide important information. METHOD: 150 short echo-time (30ms) 2D SI datasets were acquired from 60 children with a brain tumour on a Siemens 1.5T Avanto with a voxel volume of either 1 or 3.4ml. SI data was analysed using the TARQUIN algorithm to extract metabolite quantities. Metabolite profiles from individual SI datasets were analysed using non-negative matrix factorisation to extract two components. The method was applied to selected datasets and the results matched to standard MRI and known clinical information. RESULTS: In each case analysed, metabolite profiles were identified which were characteristic of normal brain and tumour and correlated with the standard imaging characteristics. Regions containing active tumour tissue could easily be identified and used to aid non-invasive diagnosis, identify optimal biopsy sites or guide resection. It is particularly useful for identifying regions of brain infiltrated with tumour. Serial follow-up on and after treatment allowed evolution of the metabolite profile to be determined and regions of residual or new tumour to be detected. CONCLUSION: Tumour metabolite profiles can be automatically extracted from SI data and their distribution mapped within the brain. The data can provide important information on a range of clinical scenarios relevant to childhood brain tumours.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-31. IMPACT OF BRD4 INHIBITION ON MULTIPLE PEDIATRIC CANCERS FOCUSING ON EMBRYONIC TUMORS

Annabelle Zoghbi 1, Michael Meisterernst 1, Michael C Fruehwald 2, Kornelius Kerl 1

Abstract

INTRODUCTION: Epigenetic alterations are currently an area of great interest in cancer research. Recently, the focus on chromatin-modifying enzymes has shifted towards non-covalently binding proteins that alter histones and thus modify transcription. Particular interest is centered on bromodomains, specifically the BET family. One member of this family, Brd4, participates in “gene bookmarking”, a process whereby certain active genes are marked prior to mitosis, ensuring rapid induction of transcription after cell division. Specifically, Brd4 has been shown to bind to acetylated histones H3 and H4 to prevent their contraction during mitosis and subsequently enhance their accessibility to the transcription machinery. This guarantees that the transcriptional patterns are passed on to daughter cells. In this study we examined the growth inhibitory effect of the Brd4-targeting compound iBet on a range of pediatric tumor cell lines. METHODS: As a general screening method, cytotoxicity assays using XTT reagent were performed on multiple pediatric tumor cell lines including osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, neuroblastoma, medulloblastoma and rhabdoid tumors. Cells were treated with increasing concentrations of iBet in order to calculate the growth inhibitory concentration with only 50% viable cells remaining (GI50) for each cell line. FACS analysis was used to determine changes in cell-cycle and possible apoptosis induction following incubation with iBet. RESULTS: During proliferation analysis three tumor entities demonstrated sensitivity to Brd4 inhibition: neuroblastoma cell lines (IMR5, SY5Y and SHEP), medulloblastoma cell lines (UW228 and DAOY) and cell lines from rhabdoid tumors (A204, BT12, BT16 and MON), each revealing GI50 values below 1 µM iBet. Furthermore, FACS analysis demonstrated apoptosis induction and G1 arrest in each of these cell lines. CONCLUSION: Overall, a number of embryonal tumors, including medulloblastoma, rhabdoid tumors and neuroblastoma, demonstrated a significant response to treatment with the Brd4-inhibiting compound iBet.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-32. DECREASED 5-HYDROXYMETHYLCYTOSINE IS ASSOCIATED WITH NEURAL PROGENITOR PHENOTYPE IN NORMAL BRAIN AND SHORTER SURVIVAL IN MALIGNANT GLIOMA

Brent Orr 1, Michael Haffner 1, William Nelson 1, Srinivasan Yegnasubramanian 1, Charles Eberhart 1

Abstract

Epigenetic modification of DNA by cytosine methylation to produce 5-methylcytosine (5mC) has become well-recognized as an important epigenetic process in human health and disease. Recently, further modification of 5mC by the ten eleven translocated (TET) family of enzymes to produce 5-hydroxymethylcytosine (5hmC) has been described. In the present study, we used immunohistochemistry to evaluate the distribution of 5hmC in human brain during different periods of development and in a large series of gliomas (n = 225) including over 100 pediatric tumors. We found that during development, 5hmC levels are high in more differentiated compartments like the fetal cortex, but low in the periventricular progenitor cell regions. In fully patterned brains, we found 5hmC levels to be highest in the cortex, but present in all intrinsic cell types in the brain including stromal elements. In brain tumors, 5hmC levels were high in low grade tumors and reduced in malignant glioma, but did not exhibit any correlation with IDH1 mutation status. Additionally, we identified a significant relationship between low levels of 5hmC and reduced survival in malignant glioma. This observation was further supported by in silico analysis showing differential expression of genes involved in 5hmC homeostasis in aggressive subsets of glioblastoma. Finally, we show that several genes involved in regulating the levels of 5hmC are also prognostic in malignant glioma. These findings suggest that 5hmC regulation in malignant glioma may represent an important determinant of tumor differentiation and aggressive behavior, as well as a potential therapeutic target.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-33. YB-1 BRIDGES NEURAL STEM CELLS AND BRAIN TUMOR-INITIATING CELLS VIA ITS ROLES IN DIFFERENTIATION AND CELL GROWTH.

Abbas Fotovati 1, Samah Abu-Ali 1, Pei-Shan Wang 1, Loic Deleyrolle 1, Cathy Lee 1, Joanna Triscott 1, James Chen 1, Sonia Franciosi 1, Yasuhiro Nakamura 1, Yasuo Sugita 1, Takeshi Uchiumi 1, Michihiko Kuwano 1, Blair Leavitt 1, Sheila Singh 1, Alexa Jury 1, Chris Jones 1, Hiroaki Wakimoto 1, Brent Reynolds 1, Catherine Pallen 1, Sandra Dunn 1

Abstract

The Y-box binding protein 1 (YB-1) is upregulated in many human malignancies including glioblastoma (GBM). It is also essential for normal brain development, suggesting that YB-1 is part of a neural stem cell (NSC) network. Here, we show that YB-1 was highly expressed in the subventricular zone (SVZ) of mouse fetal brain tissues but not in terminally differentiated primary astrocytes. Conversely, YB-1 knockout mice had reduced Sox-2, nestin, and musashi-1 expression in the SVZ. Although primary murine neurospheres were rich in YB-1, its expression was lost during glial differentiation. Glial tumors often express NSC markers and tend to loose the cellular control that governs differentiation; therefore, we addressed whether YB-1 served a similar role in cancer cells. YB-1, Sox-2, musashi-1, Bmi-1, and nestin are coordinately expressed in SF188 cells and 9/9 GBM patient-derived primary brain tumor-initiating cells (BTIC). Silencing YB-1 with siRNA attenuated the expression of these NSC markers, reduced neurosphere growth, and triggered differentiation via coordinate loss of GSK3-β. Furthermore, differentiation of BTIC with 1% serum or bone morphogenetic protein-4 suppressed YB-1 protein expression. Likewise, YB-1 expression was lost during differentiation of normal human NSCs. Consistent with these observations, YB-1 expression increased with tumor grade (n = 49 cases). YB-1 was also coexpressed with Bmi-1 (Spearmans 0.80, P > 0.001) and Sox-2 (Spearmans 0.66, P > 0.001) based on the analysis of 282 cases of high-grade gliomas. These proteins were highly expressed in 10/15 (67%) of GBM patients that subsequently relapsed. In conclusion, YB-1 correlatively expresses with NSC markers where it functions to promote cell growth and inhibit differentiation.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-34. SPONTANEOUS BRAIN TUMORS IN DOGS ARE SIMILAR TO HUMAN BRAIN TUMORS: HISTOLOGY, MRI, AND ZINC TRANSPORT PROTEIN ANALYSIS

Stephen Fletcher 1, Jonathan Levine 2, Min Li 1

Abstract

The Texas Comparative Neuro-Oncology program over the past 7 years has advocated that spontaneous canine brain tumors provide an untapped resource for comparison to human brain tumors. Compelling evidence suggests that the incidence of spontaneous tumors in dogs may be up to 5 times that in humans. Even though the histological distribution is different, investigation of traditional characteristics of these tumors (imaging, histology, behavior and survival) suggests they share similarity. We have developed a comprehensive canine neurosurgical program that includes image guided biopsy and surgical removal of brain tumors for companion dogs. An extensive cadaveric validation study for accuracy and safety was performed prior to initiation of actual surgical management of these complex tumors. The goal in dogs presenting to our center was to establish a correct diagnois by stereotactic biopsy, or if appropriate, attempt to resect as much tumor as feasible. Subsequent therapy was dependant on the tumor type and desire of the owner to continue care. We present the imaging data, histological typing, survival, and in some the zinc transport protein data for a small group of canine brain tumors. Some of these characteristics parallel findings in human brain tumors. Further detailed parameters of comparison on tumor specimens are needed to validate the hypothesis that canine brain tumors are the best human brain tumor research model. If this proves a valid conclusion, the potential for initiating new therapies is enhanced.

Neuro Oncol. 2012 Jun;14(Suppl 1):i7–i15.

BI-35. WILMS' TUMOR 1(WT1) EXPRESSION IN PEDIATRIC BRAIN TUMORS

Naoki Kagawa 1, Ryuichi Hirayama 1, Yasuyoshi Chiba 1, Noriyuki Kijima 1, Hideyuki Arita 1, Manabu Kinoshita 1, Naoya Hashimoto 1, Shuichi Izumoto 2, Motohiko Maruno 3, Toshiki Yoshimine 1

Abstract

BACKGROUND: The WT1 is highly expressed in neoplasms of hematopoietic system and many solid tumors. Immunohistochemical studies have revealed adult malignant gliomas also express high levels of WT1 protein. We have reported relationship between response in phase II clinical trial on WT1-targeted immunotherapy and WT1 expression level and MIB1 labeling index in adult recurrent glioblastomas. But in pediatric brain tumors, correlation of WT1 expression with histopathological diagnosis or prognosis is not well known. MATERIALS AND METHODS: We retrospectively studied WT1 expression by immunohistochemistry in 44 formalin-fixed, paraffin-embedded tumor samples from pediatric cases (age, 0-20 years; 24 men, 20 women) and 6 normal brain tissues as controls. Histopathological diagnosis included 11 glioblastomas (2 diffuse intrinsic potine gliomas), 6 anaplastic astrocytomas, 5 diffuse astrocytomas, 5 central nervous system primitive neuroectodermal tumors (CNS PNET), 10 medulloblastoma, 6 anaplastic ependymomas (AE), one choroid plexus carcinoma (CPC) and one desmoplastic infantile astrocytoma (DIA). Correlation of WT1 with MIB1 labeling index, stem cell markers (nestin, musashi-1), some neural or glial differentiation markers was further studied. Whether WT1 expression in astrocytic tumors influenced overall survival (OS) and progression free survival (FPS) was analyzed statistically. RESULTS: Immunohistochemical analysis of WT1 in astrocytic tumors showed significant relationship between rate of WT1-positive cells and WHO grading. Furthermore, positive correlation of positive cell counts of WT1 with that of stem cell markers and prognosis (OS and PFS) in malignant gliomas was recognized. No significant correlation of it with sex, age, tumor location and MIB1 labeling index was shown. WT1 staining in AE and CNS PNETs was positive in 100% and 80% respectively, but that in 10 medulloblastomas, CPC and DIA was all negative. CONCLUSION: WT1 expression can be used as a prognositc marker in pediatric malignant astrocytic tumors. WT1 protein also is expressed in AEs and CNS PNETs.

Articles from Neuro-Oncology are provided here courtesy of Society for Neuro-Oncology and Oxford University Press

RESOURCES