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. 2012 Jun;14(Suppl 1):i82–i105. doi: 10.1093/neuonc/nos093

MEDULLOBLASTOMA

PMCID: PMC3483339
Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-01. INVOLVEMENTS OF hsa-miR-383 AND ITS TARGET PEROXIREDOXIN 3 (PRDX3) IN CONTROLS OF MEDULLOBLASTOMA CELL GROWTH

Kay Ka-Wai Li 1, Jesse Chung-Sean Pang 1, Ho-Keung Ng 1

Abstract

Medulloblastoma (MB) is the most common children brain cancer. Recent advances in cancer biology strMBongly suggest that impaired microRNAs expression is one of the critical events driving cancer development. Previous studies of microRNA expression profiling suggested hsa-miR-383 as one of the down-regulated microRNAs in MB. However, the functions of this microRNA in MB remain unclear. In this study, we demonstrated frequent down-regulation of hsa-miR-383 expression in MB by quantitative stem-loop-RT-PCR analysis. Twenty-three out of 29 (79%) MB samples expressed >2-fold of the lower level of hsa-miR-383 compared with normal cerebellar samples and also limited/non-detectable levels were found in 4 MB cell lines (DAOY, ONS-76, D283, and D458). Ectopic expression of hsa-miR-383 by microRNA mimic significantly inhibited MB cell growth along with increase of PARP cleavage, suggesting induction of apoptosis in the hsa-miR-383-mimic-treated MB cells and tumor suppressive roles of hsa-miR-383. By transcriptome analysis of hsa-miR-383-mimic-treated MB cells and computational prediction of hsa-miR-383 targets, we identified Peroxiredoxin 3 (PRDX3) as one of the targets with significant down-regulation of expression in the mimic-treated MB cells. Down-regulation was verified at both RNA and protein levels. In addition, the mimic significantly reduced luciferase activity of the reporter that was constructed with the 3'UTR of PRDX3 in MB cells. Site-directed mutation of the predicted recognition site abrogated the reduction, and this demonstrated the specificity of hsa-miR-383-mediated repression on PRDX3 expression in MB cells. Furthermore, siRNA knockdown of PRDX3 resulted in cell growth inhibition and induction of PARP cleavage, mimicking the effects of the hsa-miR-383 restoration by microRNA mimic in MB cells. In conclusion, hsa-miR-383 may function as tumor suppressive microRNA in MB and this is mediated through its target PRDX3 to control MB cell growth.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-02. QUESTIONABLE ROLE OF CRANIOSPINAL IRRADIATION (CSI) IN NON CEREBELLAR PNET(NCPNET) WHEN USING A HIGH-DOSE CHEMOTHERAPY (HDCT) STRATEGY

Maura Massimino 1, Lorenza Gandola 1, Veronica Biassoni 1, Filippo Spreafico 1, Elisabetta Schiavello 1, Geraldina Poggi 2, Michela Casanova 1, Emilia Pecori 1, Marco Vajna De Pava 1, Andrea Ferrari 1, Cristina Meazza 1, Monica Terenziani 1, Daniela Polastri 1, Roberto Luksch 1, Marta Podda 1, Piergiorgio Modena 5, Manila Antonelli 3, Felice Giangaspero 4

Abstract

BACKGROUND: A prospective intensive chemo-radiotherapy trial was launched for ncPNET. After introducing HDCT, we amended CSI to focal RT for selected cases. METHODS: From 1997 to 2010 we enrolled 25 consecutive patients in a pre-radiation schedule cointaining HDMTX, HDVP16, HDCTX and HDCBDCA, followed by HART-CSI at total doses 31-39 Gy, implemented with 2 hd thiotepa courses following CSI after the first 5 patients. Six children received the same chemotherapy but only focal RT at 54 Gy. RESULTS: 16 were males, median age was 7 years. Median follow-up was 42 mos (11-152). 5year PFS and OS were 56% and 47%, respectively, being PFS 80% for pineal tumors (n 10) vs 43% for other sites (p 0.05). Residual disease (n 22), metastases (4), response to pre-RT CT were not prognostically significant. Patients receiving HDCT had a PFS of 64% vs 37.5% (p 0.09). Response to RT in children with residual tumor was significant (3 year PFS 57% vs 42%, p 0.03) while it was not total CSI dose when used. Relapses were local in 6, local and disseminated in 2 and disseminated only in one case, not submitted to HDCT. Hence our decision to give only focal radiation to subsequent children providing they had no progression during pre-RT CT. Only 1/6 relapsed so far, locally (ETANTR diagnosis). CONCLUSIONS: This intensive schedule obtained a very satisfying outcome in pineoblastoma and allowed a further treatment stratification in radiation delivery, thus potentially saving a subgroup of children from full dose and CSI. Local relapse is in fact the main cause of treatment failure in ncPNET.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-03. MEDULLOBLASTOMA BELOW THE AGE OF 3 YEARS: TREATMENT AND PROGNOSTIC FACTORS

Soha Ahmed 1, Mohamed S Zaghloul 1, Amr G Mousa 1, Eman Eldebawy 1, Mohamed Elbeltagy 1, Madeha Awaad 1

Abstract

BACKGROUND: Medulloblastoma patients below 3 years had inferior survival rates due to several reasons. AIM: To investigate the treatment end-results of medulloblastoma under 3 years of age and determine the factors that affects its prognosis. PATIENTS AND METHODS: Eighteen children below the age of 3 years were treated at Children's Cancer Hospital, Egypt during the period from July 2007 and December 2010. Safe maximum resections were attempted in all patients. Gross total resection was performed in 10 children (56%), subtotal excision in 7 children (39%) and biopsy in one patient. Fourteen children (78%) proved to be non-metastatic, while 4 belonged to M3 category (spinal seedling). Eight out of the 18 (44%) children received infantile medulloblastoma chemotherapy protocol, while the other 10 received other chemotherapy protocols. All the 4 metastatic children received craniospinal irradiation (CSI) with boost to the seedling site. Six out of the M0 patients received posterior fossa (PF) irradiation (5580 cgy), while the other 8 received CSI, as they reached the age of 3 years, with booster dose up to 5580 cGy to PF. RESULTS: The 3-year overall survival (OS) for all children was 55 ± 16%. The OS for non-metastatic was 61 ± 15% and 50 ± 29% for M children. The infantile chemotherapy protocol led to 3-year OS of 71 ± 17% compared to 24 ± 18% for other protocols. The OS for CSI was 71 ± 17% compared to 49 ± 25% for conformal PF irradiation. None of the CSI group developed CNS relapse, while only one (17%) who received PF irradiation had spinal relapse. It is worthnoting that non of the these detected differences were statistically significant. All children tolerated treatment with minimal immediate toxicity and acceptable, so far, late effects. CONCLUSIONS: The 3-year OS of children below 3 years were modest with improved OS in non-metastatic patients who received infantile protocol and CSI.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-04. MEDULLOBLASTOMA HISTOLOGICAL VARIANTS AS THE MOST POWERFUL CLINICAL PROGNOSTIC INDICATOR: A 10-YEAR MONO-INSTITUTIONAL EXPERIENCE

Maura Massimino 1, Lorenza Gandola 1, Veronica Biassoni 1, Manila Antonelli 2, Elisabetta Schiavello 1, Francesca Buttarelli 2, Filippo Spreafico 1, Paola Collini 1, Bianca Pollo 1, Carlo Patriarca 3, Felice Giangaspero 2

Abstract

BACKGROUND: Histological classification in medulloblastoma has aroused in importance and newer treatment protocols will include histology as risk factor. We centrally revised all our medulloblastoma cases of the last ten years and re-assessed their histology to identify its prognostic importance. METHODS: Patients' samples were reviewed according to the two subsequent WHO classifications 2000 and 2007. Consecutive patients were 125. RESULTS: Male were 99, non-metastatic were 82, primary tumor was completely resected in 97, 10 patients were under 3-years of age. Median follow-up was 96 months. Treatment applied was institutional driven, hyperfractionated accelerated radiotherapy (HART) based, for 39 non metastatic cases before 2003, thereafter European PNET IV protocol was applied in 31, a HART based strategy was applied to 39 metastatic medulloblastoma children, a infants-tailored in 10 (high-dose-chemotherapy based), finally it was patient conditions tailored in 7. Five years PFS/EFS/OS were 76%, 73% and 81%, respectively. Histology was classic in 93 cases, desmoplastic in 20, anaplastic/large cell in 7/2 and with extensive nodularity (MBEN) in the last three. Prognostic stratification according to both presence of residual disease and metastases was not prognostic either, as were not age and the different protocols adopted. Histology gave 82% 5-year PFS for desmoplastic + MBEN variant, 78% for classic, and 44% for anaplastic/large cell one (P = 0.009). CONCLUSIONS: "Tailored" treatments to recognized risk factors have flattened prognostic differences, while histologic revised anaplasia, that was not considered as such when designing previous trials, remained the most powerful prognostic factor and deserve appropriate treatment intensification.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-05. THE APPLICATION OF NANOPARTICLE LIPOSOME-IMPRAMINE BLUE IN THE TREATMENT OF MEDULLOBLASTOMA IN THE SmoA1 TRANSGENIC MICE

Tobey MacDonald 1, Jingbo Liu 1, Jenny Munson 2, Jaekeun Park 3, Kenty Wang 4, Baowei Fei 4, Ravi Bellamkonda 2, Jack Arbiser 5

Abstract

Medulloblastoma is one of the most common malignant tumors in children. Its lethality is associated with tumor metastasis and the side effects of available treatment severely affect survivor's quality of life. Impramine blue (IB), a new anti-tumor drug, is encapsulated with liposome to form a liposomal nanoparticle (Liposome-IB), which has the advantage of reaching tumor via the enhanced permeability and retention effect. In vitro studies demonstrated that Liposome-IB inhibit the growth and migration of several medulloblastoma cells including Daoy (human) and Ps125 (mouse) in a dose-dependent manner. To determine the responsiveness of medulloblastom to liposome-IB in vivo, we carried out a preclinical survival study using the SmoA transgenic mouse model of medulloblastoma. Magnetic resonance imaging (MRI) was utilized for screening and detecting tumor in mice aged 12-15 week old. The tumor bearing mice were divided into treatment and control groups randomly. The mice in the treatment group received two doses of Liposome-IB by tail vein injection with a 5 day interval. The dosage of Liposome-IB is 4.5mg/kg (liposome-IB /body weight). The mice in the control group received the same amount of Liposome-only. The tumor progression was monitored by MRI at the different time points after the Liposome-IB treatment. The results showed that the tumor volume increased dramatically in the control group compared with Liposome-IB treatment group. The Liposome-IB treated mice survived significantly longer (median survivals of 82 ± 21.9 days) than the control mice (median survivals of 25 ± 15.5 days; P = 0.024). In conclusion, the nanoparticle Liposome-IB is effective in the treatment of mouse medulloblastoma in vivo. It can significantly delay the tumor progression and prolong the tumor bearing mice's survival time. The results provide valuable data in supporting the translation from the preclinical animal model trial to the development of clinical trial and protocol to the cancer patients.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-06. HOW TO REDUCE CHEMOTHERAPY FOR LOWER-RISK MEDULLOBLASTOMAS - IMPORTANCE OF INTERACTIONS BETWEEN SURGERY AND ADJUVANT THERAPY

Akira Gomi 1, Takashi Yamaguchi 1, Toshihiro Mashiko 1, Keiji Oguro 1

Abstract

PURPOSE: ICE chemotherapy, comprising ifosfamide, cisplatin, and etoposide, is one of the most common regimens for medulloblastoma in Japan. However, hematological and hearing toxicities are frequently encountered. We report herein a single-institute experience of reduced chemotherapy for lower-risk medulloblastomas. METHODS: Thirteen patients with medulloblastoma treated at our institute from 2000 to 2011 were retrospectively reviewed. Adjuvant treatment was selected based on our new clinical classification. Tumors that have been completely resected, with no residual tumor apparent on MRI, in patients >3 years old with no leptomeningeal dissemination (M0) are classified as “lower risk”. Patients with residual tumors <1.5 cm2 who are >3 years old and show no or only focal dissemination (M0-2) are classified as “average risk”. Patients with residual tumors ≥1.5 cm2, age <3 years or M3 are classified as “high risk”. Seven patients were categorized to the lower-risk group, with 4 in the average-risk group and 2 in the high-risk group. Adjuvant treatment after surgery consists of radiotherapy, 24 Gy to the whole brain and spine plus 30 Gy to the posterior fossa, followed by chemotherapy. Chemotherapy for lower-risk patients consists of cisplatin and etoposide (CE chemotherapy), while ICE is used for average-risk ICE. RESULTS: Five-year survival and 5-year progression-free survival rates for the 11 patients of lower- and average-risk groups were 100% and 75%, respectively. Performance status scores for 10 of these 11 patients (91.0%) were 90-100%. Surgical complications, including post-operative intracranial hematoma and meningitis, were seen in recurrent cases and the low performance status cases. Introduction of radiation was delayed in these cases, with intervals between surgery and radiation of >40 days, compared to <28 days in the other 8 cases. CONCLUSIONS: For the lower-risk group, reduced chemotherapy was acceptable. Surgery with no complications that might delay adjuvant therapy is essential to reduce chemotherapy required.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-07. GORLIN'S SYNDROME (GS) AND DESMOPLASTIC MEDULLOBLASTOMA (DMB): REPORT OF 3 CASES WITH UNIQUE TUMOR LOCATION, CLINICAL COURSE, AND NOVEL MUTATION

Aravind Somasundaram 1, Ronnie Neuberg 2, Gerald Grant 1, Herbert Fuchs 1, Tim Driscoll 1, Oren Becher 1, Roger McLendon 1, Thomas Cummings 1, Sridharan Gururangan 1

Abstract

GS is a genetic condition associated with DMB in 5% of cases. DMB in GS usually occurs in infants and has a good prognosis. Radiotherapy (RT) is usually avoided in these children due to the higher risk of secondary cancers following RT exposure. We present three cases of GS with DMB with unique disease features. Patient #1 with frontal bossing, facial dysmorphism, bifid right 3rd rib, and a spontaneous germline PTCH mutation (C > T, exon 18) underwent gross total resection (GTR) of cerebellar DMB at age 2 years but focally relapsed in the right lateral ventricle following induction chemotherapy (IC). She underwent re-resection, IC, and high dose chemotherapy (HDC) + autologous stem cell rescue (ASCR), and no RT. She is 84+ months from HDC with no evidence of disease (NED). Patient #2 with mild frontal bossing and large hands and an inherited germline PTCH mutation (C > A, exon 12), underwent partial resection of a pineal tumor at age 20 months and found to be a DMB. He underwent IC followed by HDC + ASCR but had disseminated recurrence 6 months following treatment and subsequently died following palliative RT. Patient # 3 with frontal bossing, macrocephaly, synorphis, bifid right 3rd rib, and spontaneous germline mutation, c.1670 C > G, p. Thr557Arg), presented at age 2.5 years with a cerebellar DMB with excessive nodularity. She underwent GTR of tumor and IC followed by HDC + ASCR and no RT. She is now 10 + months following ASCR with NED. Although pts with GS and DMB have a good prognosis, disease progression can occur following initial therapy but can still be controlled using HDC + ASCR without RT. The association of GS with DMB in the pineal region or due to exon 12 1670 C > G, p. Thr557Arg mutation have not been previously reported.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-08. MYC AMPLIFICATION CAN BE SAFELY ASSESSED BY ARRAY-CGH IN MEDULLOBLASTOMAS

Franck Bourdeaut 1, Camille Grison 1, Francois Doz 1, Gaelle Pierron 1, Olivier Delattre 1, Jerome Couturier 1

Abstract

As risk factors for poor outcome, MYC and MYCN amplifications are routinely assessed in medulloblastomas. FISH is considered as the technique of reference. Recently, array-CGH has been developed as an alternative technique to evaluate genomic abnormalities in many other tumour types. However, this technique has not been validated in medulloblastomas. MATERIAL AND METHODS: we systematically compared FISH and array-CGH on a retrospective series of medulloblastomas referred to our institution from 2007 to 2011. FISH were performed on frozen section, using MYC and MYCN probes (Vysis, Abbott Molecular, Des Plaines, IL). Array-CGH were done on home-made BAC-arrays until 2009, and then on 4*72K arrays NimbelGen (Madison, WI). Before DNA extraction, the tumour cellularity was evaluated on a matched frozen section; all tumours showed >80% tumour cells. RESULTS: we screened 72 tumours by FISH; MYC and MYCN amplifications were evidenced in 4 and 7 cases, respectively. In all cases showing amplification by FISH, array-CGH unambiguously revealed the abnormality. Array-CGH was also done on 25 tumours showing no amplification by FISH; consistently, no amplification was detected. Interestingly, one tumour showed a focal MYC amplification by FISH, observed in approximately 20% of tumour cells; this subclonal amplification was clearly evidenced on array-CGH. CONCLUSION: our analysis confirms that array-CGH is as safe as FISH for the detection of MYC genes amplification. Even in case of focal amplification, a theoretical event which is very rare in routine practice, array-CGH is sensitive enough to detect the subclonal abnormality. Since the percentage of tumour cells reaches at least 80% in virtually all medulloblastomas, the issue of contamination by normal cells is marginal. Given their cost effective price in comparison to two FISH tests and the wide genomic information provided by array-CGHs, this reproducible technique can be safely retained as an alternative to FISH for daily routine medulloblastomas investigation.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-09. MEDULLOBLASTOMA EXOME SEQUENCING UNCOVERS SUBTYPE-SPECIFIC SOMATIC MUTATIONS WITHIN A BROAD LANDSCAPE OF GENETIC HETEROGENEITY

Yoon-Jae Cho 1, Trevor Pugh 3, Shyamal Dilhan Weeraratne 2, Tenley Archer 2, Daniel Pomeranz Krummel 4, Daniel Auclair 3, Kristian Cibulkis 3, Michael Lawrence 3, Heidi Greulich 3, Aaron McKenna 3, Alex Ramos 3, Erica Shefler 3, Andrey Sivachenko 3, Vladimir Amani 2, Jessica Pierre-Francois 2, Natalia Teider 2, Peter Northcott 5, Michael Taylor 5, Matthew Meyerson 3, Scott Pomeroy 2

Abstract

Medulloblastomas are the most common malignant brain tumors in children. Identifying and understanding the genetic events that drive these tumors is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma based on transcriptional and copy number profiles. Here, we utilize whole exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas exhibit low mutation rates consistent with other pediatric tumors, with a median of 0.35 non-silent mutations per megabase; yet, medulloblastomas display a significant breadth of mutations with 78% of mutated genes altered in single tumors. Nonetheless, twelve genes are mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Novel findings include recurrent somatic mutations in nuclear co-repressor (N-CoR) complex genes GPS2, BCOR, and LDB1, and in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations. We further reveal mutant DDX3X co-localizes with and potentiates mutant but not wild type beta-catenin. Together, our study reveals the broad heterogeneity of somatic mutations across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic beta-catenin signaling in medulloblastoma.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-10. HIF1α DOWNSTREAM OF SONIC HEDGEHOG MITOGENIC AND ONCOGENIC SIGNALING AND BRAIN DEVELOPMENT AND MEDULLOBLASTOMA

Chad Potts 1, Holly Cline 1, Rachel Rotenberry 1, Cemile Guldal 2, Bobby Bhatia 2, Zaher Nahle 1, Anna Kenney 1

Abstract

Medulloblastoma originates during post-natal development in the cerebellum. Cerebellar granule neuron precursors (CGNPs) are proposed cells-of-origin for medulloblastomas of the Sonic hedgehog (SHH) subclass. Shh signaling promotes CGNP proliferation and inhibits their differentiation. To carry out these functions, the Shh pathway interacts with cooperating intracellular signalling pathways. We have previously shown that interactions between Shh and insulin-like growth factor (IGF) are required for the full mitogenic effect of Shh in CGNPs. IGF activity is prominent in human SHH medulloblastomas. IGF and Shh drive mTOR activity, which is associated in certain contexts with increased translation of hypoxia inducible factor 1α (HIF1α). We asked whether Shh regulates HIF1α in CGNPs during cerebellar development and in mouse models of SHH medulloblastoma. We observed increased HIF1α and its transcriptional targets PHD2 and PHD3 in Shh-treated CGNPs. In vivo, HIF1α localized to the nuclei of cells in the EGL. There was no significant effect of Shh treatment on HIF1α expression, but exposure of CGNPs to the mTOR inhibitor rapamycin resulted in reduced HIF1α protein levels. We also observed high levels of HIF1α in mouse medulloblastomas, where it co-localized with its targets pyruvate kinase M2 and Oct4 in the perivascular niche (PVN) and tumor bulk. IGF2, an established HIF1α target, was highly expressed in the PVN. When tumor-bearing mice were treated with an mTOR inhibitor HIF1α levels were reduced. These results are consistent with HIF1α regulating VEGF, which we have previously shown to be elevated in mouse medulloblastomas in an mTOR-dependent manner. Moreover, the expression of HIF1α in both the PVN and tumor bulk is consistent with mTOR activity being widely distributed throughout mouse medulloblastomas. These results suggest a role for HIF1α in Shh mitogenic/oncogenic signaling in the developing cerebellum and medulloblastoma.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-11. IMPAIRED p53 TRANSCRIPTIONAL ACTIVITY DURING LONG TERM HYPOXIA INCREASES CHEMORESISTANCE IN MEDULLOBLASTOMA

Yuen Ngan Fan 1, Barry Pizer 1, Violaine See 1

Abstract

INTRODUCTION: Neuronal proliferation, differentiation and migration are coordinated during cerebellar development. Disruption of these processes can lead to Medulloblastoma (MB), the most common malignant paediatric brain tumour. Etoposide induces DNA damages thus activating the p53 apoptotic pathway. However, some MB cells are resistant to etoposide, and we have previously shown a correlation between p53 activity in MB and glioblastoma cells and the cell sensitivity to chemotherapeutic intervention (Meley et al, Cell Death and Disease 2010). Hypoxia is a well-characterized parameter of the tumour microenvironment that influences cancer progression and response to treatment. The aim of this study was to elucidate the molecular mechanisms underlying the resistance in chronic hypoxia. We have explored how hypoxia affects p53 activity and cell sensitivity to chemotherapeutic treatment. METHODS AND RESULTS: We used etoposide, a classical chemotherapeutic agent for these tumors and demonstrated an impairment of cell cycle arrest in chronic hypoxia compared to normoxic cells. This was correlated with a loss of p53 activation and p53-target gene expression. We have further shown that the reduced p53 activation was correlated with a decrease in histone H2AX phosphorylation, likely due to a reduced activation of the upstream DNA damage sensors: ATM and ATR. Interestingly, the cells remained resistant to etoposide even after a 24h re-oxygenation, suggesting that the cells retain a memory of their environment. CONCLUSION: We have demonstrated for the first time a molecular mechanism that explains the drug resistance observed in medulloblastoma cells submitted to a long-term hypoxic environment. Hypoxia triggers a decrease of ATM/ATR levels at late time-points (> 3 days), which is correlated with a reduction of etoposide-induced H2AX foci formation, p53 activation and cell cycle arrest. These phenomenons are lasting up to 24h after re-oxygenation, demonstrating that the cells retain a memory of the hypoxic tumour environment.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-12. OUTCOME OF SUPRATENTORIAL PRIMITIVE NEUROECTODERMAL TUMORS IN KUMAMOTO UNIVERSITY HOSPITAL, KUMAMOTO, JAPAN

Keishi Makino 1, Hideo Nakamura 1, Jun-ichi Kuratsu 1

Abstract

BACKGROUNDS AND PURPOSE: Supratentorial primitive neuroectodermal tumors (SPNET) are rare and represent less than 2.5% of childhood brain tumors. However, the outcome is very poor despite more intensive treatments, including radiotherapy and chemotherapy after surgery. The purpose of this study was to evaluate outcome of SPNET and to identify prognostic factors for overall survival in our hospital. METHODS: We reviewed the outcome of 6 consecutive patients under 15 years (mean age, 2; range, 1 – 7 years) with SPNET who were treated in our hospital between 1989 and 2010. Tumor locations were 4 cerebral hemispheres, 1 thalamus and 1 ventricle. All patients underwent surgery. For adjuvant treatments, chemotherapy with irradiation was performed for over 3years, but only chemotherapy was done for less than 3 years. RESULTS: Two patients had near-total resection (NTR); three subtotal resection (STR) and one underwent biopsy. Of the 6 patients, 5 had developed disease progression and 4 had died at the time of last follow-up. Median progression free survival was 4.6 (range, 3.4 – 28.3) months and median overall survival was 28.9 (range, 9.7 – 49.5) months. Only 2 patients with NTR are still alive (49.5 and 28.3 months). One (1 year) had disease progression after 8 cycles chemotherapies near the surgical cavity. The region was completely removed by second surgery. Another one (7 years) is still disease free after initial treatment with irradiation and 8 cycles chemotherapies. CONCLUSIONS: This study was small populations. Although outcome of SPNET was poor, there was a trend toward better survival of those patients with NTR compared with those with STR or biopsy.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-13. KINASE REGULATION OF MEDULLOBLASTOMA CELL MOTILITY

Jasmin Grählert 1, Min Ma 1, Giulio Fiaschetti 1, Tarek Shalaby 1, Michael Grotzer 2, Martin Baumgartner 1

Abstract

Metastatic dissemination of tumor cells is a hallmark of Medulloblastoma (MB), the most common malignant brain tumor in children, and results in high mortality and severe treatment-associated long-term side effects. Therefore, the identification of novel treatment options effectively targeting MB metastasis with reduced side effects is of highest priority. Mechanisms that control motility and metastasis of MB are poorly understood and this lack of fundamental knowledge hampers the development of specific treatments targeting MB metastasis. Metastatic dissemination depends on acquired cell motility of tumor cells and the capability to detach from the primary tumor and seed at distant locations. Thus, molecular determinants such as protein kinases that control MB cell motility are potential novel drugable targets. To determine protein kinases involved in MB cell motility regulation, we combine in vitro cell culture systems suitable for advanced microscopy analysis that mimic MB cell dissemination in vivo with targeted knock-down or pharmaceutical inhibition of protein kinases. We particularly focus on kinases involved in cytoskeleton dynamics because increased motile properties of MB cells depend on dynamic remodeling of the cytoskeleton. In an alternative approach, we found that the expression of the c-Met receptor tyrosine kinase is increased, that c-Met is permanently activated in several MB tumor cell lines and that stimulation by its ligand HGF promotes MB cell migration and scattering. Importantly, we also detected increased expression of CD44 and of its tumor-specific CD44v6 isoform. Furthermore, we found that MB cell adhesion promotes c-Met activation independent of HGF and that invasive MB cell behavior requires functional cell-matrix interaction. Based on these observations, we will discuss the impact of environmental cues triggering a motile phenotype in MB and the role of protein kinases relaying these signals to cellular effectors that mediate the dynamic changes in the actin cytoskeleton required to drive cell motility.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-14. BIOMARKER-DRIVEN STRATIFICATION OF DISEASE-RISK IN NON-METASTATIC MEDULLOBLASTOMA: THE SIOP-EUROPE PNET4 CLINICAL TRIAL

Steven Clifford 1, Goran Gustafsson 2, David Ellison 3, Dominique Figarella-Branger 4, Francois Doz 5, Stefan Rutkowski 6, Birgitta Lannering 2, Torsten Pietsch 7

Abstract

The accurate stratification of disease-risk, and delivery of risk-adapted therapies, are major goals in medulloblastoma. Patients aged <3 years and/or with metastatic disease at diagnosis have been considered ‘high-risk’, and their exclusion defined the ‘standard-risk’ group treated on the recent SIOP-PNET4 trial. A series of validated molecular and pathological prognostic markers have now been identified for medulloblastoma. However, whilst favourable-risk WNT subgroup patients will receive reduced therapy in the forthcoming PNET5 clinical trial, risk-biomarkers within the remaining ‘standard-risk’ group are less well defined. We undertook a prospective study of biomarkers of reported biological or prognostic significance, alongside clinical and pathological variables, within the PNET4 cohort. Formalin-fixed paraffin-embedded (FFPE) tumour tissue was collected for centralised pathology review (n = 338) and analysis (up to n = 236) of WNT subgroup status (β-catenin immunohistochemistry, CTNNB1 mutation)) and copy-number alterations (chromosome 17, MYC/MYCN, PTCH1 and DNA ploidy status). Five-year event-free survivals with standard (77 ± 4%) and hyperfractionated (78 ± 4%) radiotherapy were equivalent (p = 0.82). β-catenin nuclear positivity predicted a favourable outcome (p = 0.048), while chromosome 17p loss or 17q gain (p = 0.01), and residual tumour following surgery (p = 0.01) were associated with a poor prognosis. Chromosome 17p/q defects were heterogenous, and predicted a poor prognosis when they occurred against a diploid, but not polyploid, genetic background. All three factors were supported as independent markers in multivariate testing. Notably, previously identified poor prognosis markers (MYC/MYCN amplification, LCA disease) were not associated with adverse outcome in this ‘standard-risk’ cohort. In survival models of non-metastatic, completely resected disease, chromosome 17p/q defects predicted a poor outcome (56 ± 12%), while cases without 17p/q defects did not diverge significantly from the WNT subgroup. These findings demonstrate assessment of disease-risk in ‘standard-risk’ medulloblastoma requires distinct biomarkers and stratification schemes. The routine testing of chromosome 17 and WNT subgroup status provides a strong basis for improved patient stratification in future clinical trials.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-15. INHIBITION OF NOTCH SIGNALING PATHWAY VIA DEPLETION OF ITS LIGANDS AS NOVEL THERAPEUTIC APPROACH FOR CHILDHOOD MEDULLOBLASTOMA

Giulio Fiaschetti 1, Tarek Shalaby 1, Martin Baumgartner 1, Michael Grotzer 1

Abstract

The Notch pathway is required for proper cerebellum development and it is often aberrantly activated in medulloblastoma (MB), the most common malignant childhood brain tumor. Recent approaches attempting to inhibit this pathway employed small molecule inhibitors of the gamma-secretase, a protease complex required for Notch signal transmission. Although very effective, these molecules lack substrate specificity. As novel strategy to inhibit the activation of Notch signaling, we propose the depletion of Notch ligands, which initiate Notch signaling. Indeed, siRNA-mediated depletion of the ligand JAG1, but not JAG2, triggers increased MB cell death, whereas double silencing of both ligands reverses this effect. Therefore, the two ligands trigger distinct actions and JAG2 appears to counter JAG1 functions. Expression profiles in two MB patient datasets confirmed the opposite correlation between these two ligands and revealed that JAG2 is highly expressed in the most aggressive MB histological subtype (anaplastic). This subset of tumors is commonly characterized by increased expression of the oncogene MYC. Therefore, we explored the correlation between increased Notch ligands expression and MYC and found that in primary MB samples the expression of MYC correlates positively with that of JAG2. We confirmed that JAG2 is a MYC target gene by proving that MYC transcription factor binds its promoter. We are currently characterizing the functional significance of the different ligands for the activation of the Notch pathway in MB to determine the tumorigenic potential of Notch ligands and potential ways to interfere with it. Specifically, we are evaluating whether the Notch ligands mediate some of the oncogenic properties of MYC in MB, and exploring their depletion as a novel therapeutic approach. Therefore, we will test whether interference with the function of Notch by blocking its ligands would be a suitable strategy towards the development of tailored therapies to treat the most aggressive anaplastic MB.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-16. OUTCOME OF PATIENTS WITH RECURRENT DESMOPLASTIC MEDULLOBLASTOMAS TREATED WITH A MULTIMODAL THERAPY: A REPORT OF THE HIT-REZ-97 AND HIT-REZ-2005 STUDIES

Gudrun Fleischhack 1, Nele Siegler 1, Martina Zimmermann 2, Stefan Rutkowski 3, Monika Warmuth-Metz 4, Rolf-Dieter Kortmann 5, Torsten Pietsch 6, Andreas Faldum 7, Udo Bode 2

Abstract

BACKGROUND: Desmoplastic medulloblastoma (DMB) is most frequently diagnosed in young children and adult patients and was associated with an improved prognosis in newly diagnosed infants. The clinical behaviour of recurrent DMB is unclear. This analysis was set up to investigate the treatment and outcome in patients with recurrent DMB treated in the studies HIT-REZ-97/HIT-REZ-2005. METHODS: Thirteen patients treated in Germany and Austria between 1999 and 2011 were included in this analysis. All patients received an oral (temozolomide or trofosfamide/etoposide) or intravenous (carboplatin/etoposide) chemotherapy. Local therapy such as surgical resection, radiotherapy and intraventricular chemotherapy were administered where applicable. RESULTS: At study entry patients were in median 11 years old (range, 3.9 - 24.6) and showed following Chang stages: 4 M2, 7 M3, and 2 M4. Progression-free survival after primary diagnosis was in median 1.9 years (range, 0.7 to 7.4). At first relapse all patients received chemotherapy (iv 2, oral 8, intraventricular 4, high dose 3), 2 patients underwent subtotal resection of focal tumor lesions, 3 patients received focal (2) and craniospinal (1) radiotherapy. Treatment after further progress/multiple relapses consisted of systemic chemotherapy in 4 patients, tumor resection and focal radiotherapy in 1 patient each. The median follow-up was 21 months. Ten patients died from progressive disease, one patient is lost to follow up, one patient each is alive in SD and in PD. Overall survival was 26% and 17% at 3 and 5 years, respectively, and did not correlate with the age at primary diagnosis. CONCLUSIONS: Multimodal therapy led to long-term survival in a few patients with recurrent desmoplastic MB. Younger age is not of prognostic significance in the relapse situation.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-17. CLINICAL CHARACTERISTICS AND TREATMENT OUTCOME OF THE AVERAGE-RISK MEDULLOBLASTOMA IN CHILDREN: 10 YEARS' EXPERIENCE AT A SINGLE CENTER

Jong Hyung Yoon 1, Hyoung Jin Kang 2, Kyung Duk Park 2, Sung-Hye Park 3, Ji Hoon Phi 4, Seung-Ki Kim 4, Kyu-Chang Wang 4, Il Han Kim 5, Hee Young Shin 2, Hyo Seop Ahn 2

Abstract

Medulloblastoma (MBL) is the most common malignant brain tumor of childhood, and especially the average-risk (AVR) MBL (patient with not younger than 3-year-old of age at diagnosis, size of the residual tumor <1.5 cm2 on postoperative imaging, and M0 staging at diagnosis), is widely studied in Western countries, although there is no previous in-depth report of the characteristics and outcome, and prognostic factors of AVR MBL in Korean children. Therefore, retrospective study is performed for the patients diagnosed as AVR MBL in Seoul National University Children's Hospital for 10 years and their clinical manifestations, treatments, outcomes and prognostic factors were analyzed. A total 47 patients with male:female ratio of 33:14 were evaluated, and their median age was 8.9 years old. Histologic subtypes were classic type in 30 patients, desmoplastic in 5, large cell/anaplastic (LCA) in 8, and others in 4. All patients underwent operation and radiotherapy, and 46 patients also had chemotherapy as first-line treatment. With median follow-up of 41 months, 13 patients experienced relapse or progression of the tumor, and 3-year and 5-year event-free survival rates (EFS) were 74.5 ± 7.1% and 65.3 ± 8.7%, respectively, and overall survival rates (OS) were 82.8 ± 6.0% and 72.5 ± 7.7%, respectively. The sex, age at diagnosis, time intervals between operation and radiotherapy, up-front or deferred radiotherapy, chemotherapeutic regimens had no relationship with EFS or OS. However, histological subtype was related to EFS with borderline significance (P = 0.056), and especially LCA subtype is associated with poorer EFS, compared to other subtypes (P = 0.013). In conclusion, the treatment outcome of the AVR MBL is not so inferior to that of the Western countries, but still not satisfactory, warranting further investigation for efficient treatment. Further investigations of the optimal treatments and biologic research are essential for the better treatment outcome, and large-scaled study about more intensive treatment in LCA subtype would be needed.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-18. FORETINIB, A MULTI-KINASE INHIBITOR OF cMET AND PDGFRβ, CROSSES THE BLOOD BRAIN BARRIER AND REDUCES DISSEMINATION IN MEDULLOBLASTOMA XENOGRAFTS

Claudia Faria 1, Brian Golbourn 1, Christian Smith 1, James Rutka 1

Abstract

Medulloblastoma (MB) is the most common malignant brain tumor in childhood accounting for around 10% of all pediatric cancer deaths. Dissemination occurs in 30% of the patients at the time of diagnosis and essentially defines children with incurable disease given that metastatic disease is refractory to current treatments. Therefore, there is an urgent need for new therapies to treat metastatic MB. The hepatocyte growth factor (HGF)/cMET signaling pathway has been recently implicated in the pathogenesis of MB. Overexpression of cMET and HGF is associated with group C tumors which have high incidence of metastasis and poor outcome. Moreover, overexpression of the platelet-derived growth factor receptor (PDGFR) was also identified in metastatic MB. Using foretinib, a multikinase inhibitor of cMET and PDGFRβ, we aimed to target two important pathways involved in MB dissemination. Using three MB cell lines (Daoy, ONS-76 and D425) we performed dose-response experiments with foretinib, measuring downstream targets of cMET and PDGFRβ activation. We showed that foretinib inhibits proliferation, migration, invasion and colony forming of MB cell lines in a dose-dependent manner. By high-performance liquid chromatography and mass spectrometry we quantified the amount of foretinib that crosses the blood brain barrier and determined the best concentration of drug to use in vivo. We have created a disseminated mouse model of MB injecting MB cells in the fourth ventricle of nude mice, mimicking the process of dissemination in children. The cells were previously transfected with a luciferase expressing vector allowing weekly monitoring of tumor growth and dissemination by bioluminescence imaging. Foretinib was able to reduce tumor growth and metastasis in xenografts, increasing survival when compared to controls. Altogether these results suggest that small molecule inhibitors of cMET and PDGFR may represent a new therapeutic strategy in the treatment of disseminated MB.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-19. PEDIATRIC AND ADULT MEDULLOBLASTOMA: WHAT MAKES THE DIFFERENCE IN OUTCOME?

Brittany Dalia Greene 1, Anthony Whitton 2, Sheila Singh 3, Katrin Scheinemann 1

Abstract

BACKGROUND: Medulloblastoma accounts for 20-25% of primary central nervous system tumours in children, but less than 1% in adults. Pediatric treatment protocols have been established over a long time and progression free survival has improved significantly over the last decades. In contrary outcomes for adult medulloblastoma are reported as poor despite using often adapted pediatric treatment protocols. METHODS: In a retrospective, population-based (catchment area 2.4 million) study between 1988 and 2011 we compared diagnosis, clinical course, survival, and health and social outcomes of pediatric and adult patients treated at McMaster Children's Hospital and Juravinski Cancer Center in Hamilton, Ontario. RESULTS: Sixty-one patients were diagnosed with medulloblastoma: 50 children, ranging from 6 months to 17.92 years of age at diagnosis (mean 7.99 years) and 11 adults (ages 18.67 to 50.5 years at the age of diagnosis, mean 34.70 years). The pediatric patients have been followed for an average of 5.49 years (0.08–23.25 years), the adult patient for an average of 4.73 years (0.17–15.75). Currently 30 pediatric and 8 adult patients are alive. Chemotherapy dose reductions were more frequent in the adult population. Standard protocol was discontinued in 50% of adults treated with chemotherapy due to unacceptable toxicity or progression. While nutritional interventions, such as nasogastric and gastric feeding tubes, were ubiquitous in the pediatric population, only 18% of the adult population received nutritional support. Among survivors, 71.4% of adult population was able to return to school or work, compared to 95% of the pediatric population. CONCLUSIONS: Our data suggests that adult medulloblastoma patients experience greater treatment toxicity leading to significant treatment changes. Reduced daily functioning in the adult survivor population suggests that treatment toxicity has long-term effects on the patients' quality of life. The role of nutritional support in mitigating chemotherapy toxicity for medulloblastoma patients should be explored further.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-20. EVALUATION OF MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (MLPA) AS A METHOD FOR DETECTION OF MYC/MYCN ONCOGENE AMPLIFICATION IN MEDULLOBLASTOMA

Rebecca Hill 1, Janet Lindsey 1, Christopher Howell 1, Sarra Ryan 1, Keir Shiels 1, Emily Shrimpton 1, Simon Bailey 1, Steven Clifford 1

Abstract

Molecular and histopathological biomarkers will, for the first time, be used to guide medulloblastoma treatment stratification in the upcoming pan-European SIOP-PNET5 clinical trial. Amongst these, MYC/MYCN oncogene amplification will define high-risk patients, and tumours with ≥5% of cells displaying amplification by fluorescence in-situ hybridisation (FISH) will be classed as ‘amplified'. MLPA is a quantitative PCR-based technique for the detection of genomic copy number variation, which is becoming increasingly adopted into routine molecular diagnostic laboratories. Here, we report the development and validation of MLPA methods for the assessment of MYC and MYCN amplification status in medulloblastoma. MLPA methods were developed and 290 medulloblastomas, including tumours previously identified as MYC (n = 7) or MYCN (n = 12) amplified by FISH, and 7 diploid control samples, were selected for analysis. Tumour sample results were compared against the control cohort, to identify those with an elevated MYC or MYCN copy number. All copy number-elevated samples with available material (MYC n = 28, MYCN n = 17) were validated by FISH, alongside a series of samples with no evidence of copy number elevation (MYC n = 23, MYCN n = 26). MLPA reliably detected high copy number elevations when MYC amplification was present in >30% cells and MYCN in >10% cells by FISH. MLPA also robustly detected tumours with no MYC/MYCN amplification by FISH. In tumours with lower-level amplifications, MLPA delivered intermediate copy number scores and amplification status could not be assigned confidently. Based on our findings, MLPA has clear potential application to support FISH methods for the detection of MYC/MYCN amplification in clinical studies, principally (i) as a screening method to prioritise intermediate or highly elevated cases for FISH analysis, and (ii) for the identification of high-level amplification in cases where FISH analysis was unsuccessful. Its low DNA requirement (20-100ng) and application in limited clinical material further support its assimilation into routine molecular diagnostics services.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-21. EPIGENETIC SUB-CLASSIFICATION AND RISK-STRATIFICATION OF MEDULLOBLASTOMA USING DNA METHYLOMICS

Ed Schwalbe 1, Janet Lindsey 1, Daniel Williamson 1, Dolores Hamilton 1, Paul Northcott 2, Kieran O'Toole 1, Sarah Leigh Nicholson 1, Meryl Lusher 1, Richard Gilbertson 3, Peter Hauser 5, Michael Taylor 2, Roger Taylor 4, David Ellison 3, Simon Bailey 1, Steven Clifford 1

Abstract

We investigated the potential of epigenetics to advance our understanding of medulloblastoma biology and its clinical application. Genome-wide DNA methylation array profiling was undertaken in 230 medulloblastomas derived predominantly from the SIOP-CCLG PNET3 clinical trial. Application of consensus clustering approaches identified four methylomic subgroups in a training sub-cohort of snap-frozen tumours (n = 100), which validated in a test sub-cohort of FFPE tumours (n = 130). Subgroups were characterised by significant relationships to specific clinico-pathological and molecular disease features. Two subgroups were characterised by WNT and SHH pathway activation, while two further subgroups showed significant relationships to the Group 3 and Group 4 transcriptomic subgroups, assessed by Nanostring assay. Subgroups showed equivalent clinico-pathological and molecular characteristics to these previously defined transcriptomic subgroups. In this large, clinical-trials based cohort (n = 191; age 3.0-16.0yrs at diagnosis), WNT subgroup patients showed expected favourable outcomes, however survival was equivalent in the remaining subgroups. We therefore investigated whether DNA methylation biomarkers offered potential for improved disease prognostication, with particular focus on non-WNT patients (n = 163). Using the Cox-Boost algorithm, which adds high-dimensional molecular data to mandatory clinical covariates to form cross-validated survival models, MXI1 and IL8 methylation were identified as independently prognostic biomarkers, and validated using alternative, non-array methods. These were used to develop a novel risk-stratification scheme, based on the cumulative assessment of disease-risk, using clinico-pathological (metastatic disease, LCA pathology) and methylomic variables (WNT subgroup, MXI1, IL8 methylation). Importantly, this scheme assigned 46% of cases to a low-risk group (>90% survival) which could potentially be treated less intensively, with the aim of reducing therapy-associated effects. DNA methylomics demonstrate significant potential for use in the improved management of medulloblastoma. The molecular sub-classification and risk-stratification models developed here may be assessed in routinely collected clinical material using minimal signature-based assays. Together, these provide a strong basis for their application in future clinical studies.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-22. ADULT SHH-MEDULLOBLASTOMAS SHOW A DISTINCT MUTATION SPECTRUM, TRANSCRIPTOME, AND METHYLOME

Marcel Kool 1, David TW Jones 1, Nathalie Jäger 1, Volker Hovestadt 1, Ulrich Schüller 3, Nada Jabado 4, Arie Perry 5, Cynthia Cowdrey 5, Sydney Croul 6, V Peter Collins 7, Yoon-Jae Cho 8, Scott Pomeroy 9, Roland Eils 1, Andrey Korshunov 1, Peter Lichter 1, Stefan Pfister 1

Abstract

Medulloblastoma is a rare primary brain tumor in adults, whereas it constitutes the most common malignant brain tumor in children. The current consensus is that there are four major subgroups named WNT, SHH, Group 3 and Group 4, each of them showing an age-specific distribution. Interestingly, SHH medulloblastomas show a bimodal age distribution. They frequently occur in infants, much less in older children, but are by far the most frequently occurring subgroup in adults, suggesting that distinct subsets may exist within the SHH subgroup. Expression profiling revealed considerable transcriptional differences between infant and adult SHH medulloblastomas (Northcott et al., 2011). Moreover, recent whole genome sequencing data from our group demonstrated that SHH medulloblastomas in older children frequently have TP53 mutations and are characterized by chromotripsis (Rausch et al., 2012). To investigate these molecular differences between pediatric and adult medulloblastomas further, we performed whole genome sequencing (WGS) for 28 tumor-normal pairs of adult patients. In addition, we examined the tumors by expression profiling using Affymetrix 133 plus 2.0 arrays and DNA methylation analyses using Illumina 450K arrays. Results of this study showed that adult SHH medulloblastomas indeed form a distinct molecular subgroup. They do not only show transcriptional differences, but also strongly differ in DNA methylation as compared to pediatric SHH medulloblastomas. Most strikingly, WGS data revealed three interesting aspects: 1. adult medulloblastomas have on average more mutations than pediatric medulloblastomas; 2. the observed correlation between mutation rate and patient age in pediatric medulloblastomas holds true in the adult cohort; 3. beside similar gene mutations affecting the SHH pathway, adult SHH medulloblastomas also harbor several recurrent mutations that were not detected in pediatric SHH medulloblastomas. These data will help to better understand the biology of adult medulloblastoma and may help predicting responsiveness to targeted SHH inhibition in a clinical setting.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-23. INTEGRATIVE GENOMICS IDENTIFIES ACTIONABLE TARGETS FOR THERAPY IN MEDULLOBLASTOMA SUBGROUPS

Paul Northcott 1, David Shih 1, Michael Taylor 1; MAGIC Consortium1

Abstract

The application of genomics to the study of medulloblastoma has recently led to a significant enhancement in our understanding of its pathogenesis, implicating previously uncharacterized molecular processes and the existence of distinct biological subgroups. Despite these advances, few genomic studies have profiled sufficient cases to identify recurrent genetic events, including those restricted to a particular molecular subgroup. To specifically address these issues we have performed a comprehensive copy number analysis of 1,250 medulloblastomas and summarized their genomes by molecular subgroup. The most prevalent oncogenic events observed in medulloblastoma included those targeting known oncogenes and tumor suppressors such as members of the MYC family (MYCN, MYC, and MYCL1), cell cycle regulators (CCND2 and CDK6), and genes involved in RTK/PI3K/mTOR signaling (IRS2, PTEN, and TSC1). Analysis of the cohort according to subgroup identified multiple novel candidates that appear to be targeted in a subgroup-restricted manner. PPM1D, MDM4, and LMO4 were revealed as novel targets in SHH medulloblastoma, providing insight into the pathways that cooperate with SHH signaling in this subgroup. Similarly, we identified genetic events restricted to the poor prognosis Group 3 and Group 4 medulloblastomas, including recurrent amplification of members of the TGFβ pathway (ACVR2A and ACVR2B) in Group 3 and apparent deregulation of the RB pathway (CDK6 and RB1) in Group 4. Definitive elucidation of the genetic events contributing to the initiation, maintenance, and progression of medulloblastoma will be essential for the future development of rationally designed targeted therapies. Our current study of >1,200 medulloblastoma genomes has shown that medulloblastoma subgroups exhibit distinct genomics and implicated novel actionable targets for therapy within the subgroups. Prospective functional validation of our findings and the development of appropriate preclinical models recapitulating the genetics we have observed in subgroups of the human disease will be necessary for advances in the treatment of medulloblastoma.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-24. ESTABLISHMENT OF MEDULLOBLASTOMA AND EPENDYMOMA CELL LINES IN SERUM FREE CELL CULTURE MEDIUM

Anna Darabi 1, Emma Sandén 1, Edward Visse 1, Peter Siesjö 1

Abstract

INTRODUCTION: Primary brain tumors, including medulloblastomas (MB) and ependymomas (EP), are the most common solid tumors in children. Less than 60% of children survive 5 years after diagnosis and the survivors display a substantial morbidity from long-term cognitive and neurological sequels after radio- and chemotherapy. There is subsequently an urge for improved therapies. In vitro cultured tumor cells are indispensable tools for the development of new therapies. Culturing of tumors in serum free culture medium has been shown to preserve rare tumor-initiating cells better than serum containing medium. Since there is a lack of MB and EP cell lines established in serum free medium, we wanted to define such a protocol. METHODS: MB and EP tumor tissues from pediatric brain tumor patients were collected. Tumors were single-cell dissociated in TrypLE™-Express. Ultra Culture™ serum-free culture medium containing L-glutamine, penicillin-streptomycin, bFGF and EGF was used. Cells were seeded in Corning CellBind™ (CB-adherent) or Corning Ultra-Low™ (UL-non-adherent) flasks. RESULTS: MB initially formed spheres in both UL and CB but attached after some time in CB. EP also formed spheres in UL but proliferated best when cultured adherently in CB. Common concentrations for bFGF and EGF are 20ng/mL, but to optimize proliferation we have increased the concentration to 50ng/mL. It was possible to achieve >10 milj MB cells within 2 month and >20 milj EP cells within 1 month of culture in CB (passage 4-5). MB and EP cells have been passaged >10 times. MB and EP cells formed spheres when transferred from adherent to non-adherent conditions. CONCLUSIONS: We have been able to establish MB and EP cell lines by culturing the cells adherently in serum-free culture medium supplemented with high concentrations of bFGF and EGF. Sphere forming capacity was maintained when transferred from adherent to non-adherent conditions.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-25. INTEGRATED GENOMIC ANALYSIS IDENTIFIES THE MITOTIC CHECKPOINT AS A NOVEL THERAPEUTIC TARGET IN MEDULLOBLASTOMA

Peter Harris 1, Sujatha Venkataraman 1, Irina Alimova 1, Diane Birks 1, Brian Cristiano 1, Andrew Donson 1, Nicholas Foreman 1, Rajeev Vibhakar 1

Abstract

Medulloblastoma is the most common type of malignant brain tumor that afflicts children. Although recent advances in chemotherapy and radiation have improved outcomes, high-risk patients do poorly with significant morbidity. To identify new molecular targets, we performed an integrated genomic analysis using structural and functional methods to identify new signaling pathways in medulloblastoma. We profiled gene expression in 24 patient specimens. Pathway analysis and gene set enrichment analysis (GSEA) indicated that cell cycle related genes and in particular cell cycle kinases were associated with disease development. We then performed a kinome wide siRNA screen to identify kinases that when inhibited could prevent cell proliferation. Integrating the two analyses revealed that kinases involved in the regulation of the G2-M cell cycle checkpoint were critical determinants of medulloblastoma cell viability. One of these was the Wee1 kinase. Wee 1 participates in the G2-M checkpoint to prevent mitosis in the presence of DNA damage. RNAi-mediated knockdown of WEE1 kinase was sufficient to suppress medulloblastoma cell proliferation. These data prompted us to examine the effects of a small-molecule inhibitor of WEE1, MK1775 in medulloblastoma cell lines. MK1775 inhibited the growth of medulloblastoma cell lines and induced apoptosis at nanomolar concentrations. Further MK1775 was synergistic with cisplatin in killing medulloblastoma cells. Taken together, these findings highlight mitotic kinases and in particular WEE1 as a rational therapeutic target for medulloblastoma.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-26. TREATMENT AND OUTCOME OF MEDULLOBLASTOMA RECURRENCE IN PATIENTS PREVIOUSLY TREATED WITH STANDARD-RISK PROTOCOLS: A SINGLE-CENTER EXPERIENCE

Daniele Bertin 1, Stefano Vallero 1, Maria Eleonora Basso 1, Erica Romano 1, Paola Peretta 2, Isabella Morra 3, Anna Mussano 4, Franca Fagioli 1

Abstract

Between 1999 and 2006, 20 patients with non-metastatic medulloblastoma were treated at our Institution according to standard-risk protocols (AIEOP SNC99 from 1999 to 2003 and SIOP PNET4 from 2004 to 2006). Among these patients 6 patients relapsed with at a median time of 16.5 months (minimum 7, maximum 54 months). Two recurrences were diffuse leptomeningeal, 3 localized supratentorial (2 frontal and one ventricular) and 1 in posterior fossa. These patients were treated with a high-intensity chemotherapy schedule encompassing a mobilizing course with cyclophosphamide and etoposide with peripheral blood stem cells (PBSC) harvesting, followed by 2 to 4 CECaT courses (cyclophosphamide, etoposide, carbopatin and thiotepa) and high-dose chemotherapy with autologous PBSC rescue. All patients had gone through neurosurgery, chemo- and craniospinal radiotherapy as part of the first-line therapy, but only 2 of them received surgery and 3 received local radiotherapy to the relapse site during the second-line treatment. Out of 6 relapsed patients, the 2 patients with diffuse relapse died due to disease progression (one before HDCT and one 8 month after the second stop-therapy), the one with posterior fossa relapse is alive with persistent disease (one year after second stop-therapy), and the 3 with supratentorial relapse are alive and disease-free, with a follow-up respectively of 11, 11 and 8 years. The treatment of relapsed medulloblastoma is often challenging, since first-line protocols encompass neurosurgery, chemotherapy and craniospinal irradiation, leaving few additional weapons to second-line therapy. Nonetheless, our data seem to support the hypothesis that relapsed patients with standard-risk medulloblastoma deserve a second-line treatment with chemotherapic intensification, especially in case of focal relapses. In some cases surgery and irradiation of the relapse site should also be considered as part of the secondary treatment. However, wider studies are needed in order to identify the best therapeutic management for patients with relapsed medulloblastoma.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-27. PHARMACOLOGICAL ACTIVATION OF THE p53 PATHWAY BY NUTLIN-3 EXERTS ANTI-TUMORAL EFFECTS IN MEDULLOBLASTOMAS

Annette Künkele 1, Katleen De Preter 2, Lukas Heukamp 3, Theresa Thor 1, Kristian Pajtler 1, Wolfgang Hartmann 3, Michel Mittelbronn 4, Michael Grotzer 5, Hedwig Deubzer 6, Frank Speleman 2, Alexander Schramm 1, Angelika Eggert 1, Johannes Schulte 1

Abstract

Medulloblastomas account for 20% of pediatric brain tumors. With an overall survival of 40-70% their treatment is still a challenge. The majority of medulloblastomas lack p53 mutations, but even in cancers retaining wild-type p53, the tumor surveillance function of p53 is inhibited by the oncoprotein, MDM2. Deregulation of the MDM2/p53 balance leads to malignant transformation. We here analyzed MDM2 mRNA and protein expression in primary medulloblastomas and normal cerebellum, and assessed the mutational status of p53 and MDM2 expression in six medulloblastoma cell lines. MDM2 expression was elevated in medulloblastomas and associated with adverse patient outcome. Four of six medulloblastoma cell lines expressed wild-type p53 and high levels of MDM2. The tumor-promoting p53-MDM2 interaction can be inhibited by the small molecule, nutlin-3, restoring p53 function. Targeting the p53-MDM2 axis using nutlin-3 significantly reduced cell viability, and induced either cell cycle arrest or apoptosis and expression of the p53 target gene, p21, in these four cell lines. In contrast, DAOY and UW-228 cells harboring TP53 mutations were almost unaffected by nutlin-3 treatment. MDM2 knockdown in medulloblastoma cells by siRNA mimicked nutlin-3 treatment, whereas expression of dominant negative p53 abrogated nutlin-3 effects. Oral nutlin-3 treatment of mice with established medulloblastoma xenografts inhibited tumor growth and significantly increased survival. Thus, nutlin-3 reduced medulloblastoma cell viability in vitro and in vivo by re-activating p53 function. We suggest that inhibition of the MDM2-p53 interaction with nutlin-3 is a promising therapeutic option for medulloblastomas with functional p53 that should be further evaluated in clinical trials.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-28. Wnt POSITIVE MEDULLOBLASTOMA: A PILOT STUDY ASSESSING THE EFFICACY OF A SURGERY AND CHEMOTHERAPY ONLY APPROACH IN THE UPFRONT THERAPY OF CHILDREN WITH LOW RISK, Wnt POSITIVE MEDULLOBLASTOMA

Pratiti Bandopadhayay 1, Mark Kieran 1, Peter Manley 1, Nathan Robison 1, Susan Chi 1

Abstract

BACKGROUND: Medulloblastoma includes a heterogeneous group of tumors that carry distinct genomic signatures and prognostic features. The current therapeutic approach to standard risk (SR) medulloblastoma involves maximal safe surgical resection, adjuvant radiation therapy (craniospinal irradiation of 23.40Gy with focal boost of 30.6Gy and concurrent Vincristine) followed by maintenance chemotherapy. The 5-year overall survival rates for standard risk medulloblastoma range between 70-80%, however long term morbidity from therapy, in particular radiation therapy, remains of concern. Wnt positive medulloblastomas account for approximately 10% of all pediatric medulloblastomas. Retrospective analysis has demonstrated long-term progression-free survival rates of up to 90% in children with non-metastatic Wnt positive medulloblastoma. The Wnt positive subgroup of tumors is identified by immunohistochemistry staining for nuclear b catenin accumulation and FISH for monosomy 6. METHODS: We hypothesize that children with Wnt positive medulloblastoma can be successfully treated without the use of radiation therapy. We have proposed a multi-institutional pilot study investigating a surgery and chemotherapy only approach to the treatment of Wnt positive medulloblastoma. Children with non- metastatic, completely resected, nuclear b catenin expression and monosomy 6 medulloblastoma will be treated with chemotherapy only. Primary outcome measure will be progression-free survival. DISCUSSION: If successful, this trial will provide a foundation on which to develop risk-adapted, lower morbidity approaches in sub-groups of medulloblastoma. If proven unsuccessful, this pilot study will demonstrate the need for radiation therapy for children with wnt positive medulloblastoma while allowing salvage of this radiation naive cohort with craniospinal radiation therapy. It will also evaluate real time at-risk stratification of medulloblastoma in a multi-institutional, international pilot study. The results of this pilot study will therefore provide efficacy data to enable the design of larger cooperative studies of risk-adapted therapy for pediatric medulloblastoma.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-29. miR-34a IS DISPENSABLE FOR NORMAL DEVELOPMENT BUT ITS LOSS ACCELERATES MEDULLOBLASTOMA FORMATION

Theresa Thor 1, Pieter Mestdagh 2, Jo Vandesomple 2, Helmut Fuchs 3, Valerie Gailus Durner 3, Martin Hrabe de Angelis 3, Lukas Heukamp 4, Annette Künkele 1, Kristian Pajtler 1, Angelika Eggert 1, Alexander Schramm 1, Johannes H Schulte 1

Abstract

MicroRNAs (miRNAs) are small, noncoding RNAs that inhibit translation or initiate degradation of target mRNA by sequence-specific interaction and are involved in regulating important functions in development and differentiation, including cell cycle control and apoptosis. Previous studies have evaluated the role of miRNAs in the initiation and progression of cancer. MiR-34a was found to be downregulated in several tumor entities, including medulloblastomas. Our analysis of miR-34a expression in a cohort of human medulloblastomas and medulloblastoma cell lines indeed revealed significant downregulation compared to normal human cerebellar tissue. Re-expression of mir-34a in human medulloblastoma cells in vitro reduced cell viability and proliferation and induced apoptosis. Among the targets we found downregulated by miR-34a in human medulloblastoma cells were MYCN, SIRT1 and E2F3. The well-established medulloblastoma mouse model ND2:SmoA1 is based on activation of the SHH pathway by transgenical expression of a constitutively active form of Smoothened in mouse cerebellar granule neuron precursors. Analysis of miR-34a in ND2:SmoA1-derived medulloblastomas revealed significant suppression of miR-34a compared to samples of murine cerebellum. We next aimed to analyze miR-34a function in vivo by targeted transgenesis in mice. As expected, tissues from mice with constitutive homozygous deletion of the miR-34a gene did not express miR-34a transcripts as determined by stem-loop PCR. Mice were viable and fertile, and a comprehensive standardized phenotype analysis including > 300 single parameters performed by the German Mouse Clinic (GMC) revealed no apparent phenotype. However, crossbreeding ND2:SmoA1 mice with miR-34a knockout mice significantly accelerated medulloblastoma growth in mice deficient for miR-34a. Interestingly, MYCN and SIRT1 were much more strongly expressed in medulloblastomas derived from these mice. Taken together, we demonstrate that miR-34a is dispensable for normal development, but that its loss accelerates medulloblastoma formation in mice. Strategies aiming to re-express miR-34a in tumors could be effective therapeutic strategies in the future.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-30. TREATMENT FOR MEDULLOBLASTOMA: A SINGLE INSTITUTION EXPERIENCE OF 19 PATIENTS

Naoyuki Ohe 1, Hirohito Yano 1, Noriyuki Nakayama 1, Toru Iwama 1

Abstract

OBJECTIVE: The prognosis of medulloblastoma patients is improving by the multidisciplinary therapy. We evaluated the curative effects for the medullobalastoma patients with three different protocols in Gifu University Hospital. MATERIALS: Between January 1976 and December 2011, 19 medulloblastoma patients (11 boys and 8 girls) with median age of 7.0 years ( range 4-16 years ) received initial treatment with surgery followed by chemotherapy and craniospinal radiotherapy in our institution. The early 7 cases (group A) were underwent chemotherapy with nimustine (ACNU) and vincristine (VCR) and the total 39 Gy of fractionated external beam radiation therapy (EBRT) to the posterior fossa with craniospinal radiation. The middle 7 cases (group B) were with cisplatin (CDDP) and etoposide (VP-16) and total 52-56Gy of EBRT to the posterior fossa with craniospinal radiation. The recent 5 cases (group C) were not high risk group, treated with cyclophosphamide (CPM), VP-16, VCR and carboplatin (CBDCA) and total 50Gy of EBRT to the posterior fossa with craniospinal radiation. RESULTS: Median progression-free survival (PFS) was 47 months and 5- and 10-year overall survival (OS) rate was 71 and 50%. The significant difference was not recognized between group A, group B and group C in PFS (p = 0.696 ). All patients, who passed from a first operation in 81 months, were alive. There were 3 patients with the long-term survival for more than 25 years. CONCLUSION: The treatment results for the medulloblastoma are improved, and some patients were considered to be cured during a long-term follow-up. In addition, it was considered that the appropriate therapies for the histologically and molecularly different cases might contribute to a good functional prognosis.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-31. TRANSCRIPTIONAL PROFILES OF MEDULLOBLASTOMA TUMOURS: CORRELATION WITH CLINICAL AND HISTOPATHOLOGICAL FEATURES. AN EXPERIENCE FROM A SINGLE INSTITUTION

Maria Lastowska 1, Marta Perek-Polnik 1, Wieslawa Grajkowska 1, Katarzyna Malczyk 1, Bozena Cukrowska 1, Bozenna Dembowska-Baginska 1, Danuta Perek 1

Abstract

The integrated use of technologies for whole genome and transcriptome analyses in medulloblastoma revealed the existence of at least four sub-types of tumours. Since the biological heterogeneity of the disease is clearly of clinical relevance, molecular sub-types of tumours should be identified and further tested in the cohort of uniformly treated patients. METHODS: Fifty-one patients with known transcriptional profiles of tumours detected by expression microarrays were investigated for clinical and histopathological correlations. Subsequently, using a panel of representative antibodies for identification of transcriptional sub-types, extended analyses was performed in the groups of uniformly treated patients. Further, an association between the biological type of tumour and the site on diagnosis was assessed using MRI analysis. RESULTS: Our analysis revealed two groups of patients with an excellent survival rate who belonged to either the WNT group or to the group of infants with desmoplastic/nodular or MBEN histopathology. All the latter patients were spared intracranial irradiation and 30% of them relapsed; however, after the introduction of second line therapy, all are disease free. This result is in striking contrast with the outcome for infants with classic or anaplastic histopathology, who performed badly on the same treatment (p < 0.0001). Among older patients, those with transcriptional group C tumours had a worse survival rate and on average relapsed one year earlier than patients from group D. Almost half of the group C tumours displayed anaplastic histopathological features. MRI images indicate that all three WNT tumours analysed so far were attached to the dorsal surface of the brain stem pointing to this region as a potential site of origin for tumours of the WNT type. CONCLUSION: Our preliminary analysis confirmed that the biological characteristic of the medulloblastoma tumour is related to other clinical and histopathological parameters and should be taken into account in treatment stratification schemes.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-32. ABCB1 AND DRUG RESISTANCE IN MEDULLOBLASTOMA

Ramadhan T Othman 1, Lisa Storer 1, Richard Grundy 1, Ian Kerr 1, Beth Coyle 1

Abstract

BACKGROUND: Medulloblastoma is the most common paediatric central nervous system tumour. Children with medulloblastoma are treated with neurosurgery, craniospinal radiotherapy (except for children under three years of age) and chemotherapy. Although the initial response to treatment with chemotherapy is good, subsequent relapse and progression of disease is a common problem. Over expression of the multidrug transporter ABCB1 is a possible cause of drug resistance in this tumour. The aims of our study were to evaluate the expression and function of ABCB1 in medulloblastoma derived cell lines. MATERIALS AND METHODS: ABCB1 expression was assessed by quantitative reverse transcription polymerase chain reaction (QRT-PCR) and Western blotting in each cell line. ABCB1 function was assessed by flow cytometry. The clonogenic assay was used to assess cell survival in the response to chemotherapeutics (etoposide and temozolomide) in the presence and absence of ABCB1 inhibitor (Verapamil, Vardenafil and elacridar). RESULTS: ABCB1 is expressed at a low level in medulloblastoma derived cell lines with a higher level observed in those from recurrent tumours. A significant reduction in cell survival was observed when cells were treated with a combination of ABCB1 inhibitors plus etoposide compared to etoposide alone. CONCLUSIONS: ABCB1 is expressed in primary medulloblastoma cell lines; the use of ABCB1 inhibitors increases the cytotoxicity of drugs in vitro. ABCB1 may therefore represent a promising clinical target and warrants further preclinical evaluation in addition to evaluating ABCB1 expression in medulloblastoma tissue samples to assess the role of ABCB1 as a prognostic marker.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-33. A CHEMICAL SMALL MOLECULE SCREEN IDENTIFIES QUERCETIN AS A PUTATIVE RADIOSENSITIZER FOR THE TREATMENT OF MEDULLOBLASTOMA

Esther Hulleman 1, Tonny Lagerweij 1, Dennis Biesmans 1, Matheus HW Crommentuijn 2, Jacqueline Cloos 3, Bakhos A Tannous 4, W Peter Vandertop 5, David P Noske 5, Gertjan JL Kaspers 6, Tom Wurdinger 7

Abstract

Although treatment of medulloblastoma in children has greatly improved in the past decades, current therapy - consisting of surgery, chemotherapy and radiotherapy - still fails in approximately 20% of patients. In addition, medulloblastoma patients who survive often suffer from severe sequelae, largely resulting from the adverse effects of radiation on developing normal brain tissue. In order to identify more effective therapeutic drugs that diminish the long-term side effects, we set out to identify novel radiosensitizers for medulloblastoma. Therefore, a chemical small molecule library (TimTec-960K) was screened for compounds that reduce cell survival in irradiated medulloblastoma cells, but do not affect cell growth in the absence of ionizing radiation. This screen identified the flavonoid quercetin as a radiosensitizer for medulloblastoma. Treatment of DAOY and D283-med cells with micromolar concentrations of quercetin 30 minutes prior to irradiation (3 Gray) inhibited cell proliferation more effectively than monotherapy of each modality (21% cell survival). The sensitizing effect was not found on normal human fibroblasts, suggesting that quercetin acts specifically on medulloblastoma cells. Importantly, clonogenic assays showed that the radiosensitizing effect of quercetin also occurred at radiobiologically relevant irradiation doses used in fractionated radiation schemes or lower (1-2 Gray). Finally, in vivo experiments confirmed the radiosensitizing properties of quercetin, as administration of this flavonoid at the time of irradiation significantly prolonged survival in orthotopically xenografted mice. All animals treated with the combination of quercetin and irradiation survived for more than 30 days after treatment, with a median survival time of 40 days. Animals that did not receive irradiation (either vehicle- or quercetin treated) had a median survival of 15 days, while irradiated animals survived slightly longer (median of 18 days). Together, these findings indicate that the use of quercetin as radiosensitizer may be a valid strategy in the treatment of medulloblastoma patients.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-34. HIGH-DOSE BUSULFAN-THIOTEPA WITH AUTOLOGOUS STEM CELL TRANSPLANTATION FOLLOWED BY POSTERIOR FOSSA IRRADIATION IN YOUNG CHILDREN WITH CLASSICAL OR INCOMPLETELY RESECTED MEDULLOSTOMA

Guillaume Bergthold 1, Maria El Kababri 2, Pascale Varlet 3, Fréderic Dhermain 1, Christian Sainte-Rose 4, Marie-Anne Raquin 1, Dominique Valteau-Couanet 1, Jacques Grill 1, Christelle Dufour 1

Abstract

PURPOSE: To evaluate the outcome of young children with newly diagnosed classical or incompletely resected medulloblastoma (MB) treated by combination of high-dose busulfan-thiotepa with autologous stem cell rescue followed by posterior fossa irradiation. PATIENTS AND METHODS: Between September 1994 and January 2010, 19 children younger than 5 years of age with newly diagnosed localised classical or incompletely resected MB were treated at the Institute Gustave Roussy. Treatment strategy consisted in busulfan at a dose of 600 mg/m2 and thiotepa at a dose of 900 mg/m2 followed by autologous stem cell transplantation (ASCT). Posterior fossa radiotherapy was delivered at doses from 50 grays (Gy) to 55 Gy 70 days after ASCT. RESULTS: The median follow-up was 40,5 months. The 3-year event-free survival and overall survival were 64,9% (95% CI 40,7-83,2 %) and 89,2 % (95% CI 67,8-97%), respectively. Acute toxicity consisted mainly in hepatic veno-occlusive disease (31,6% of patients) and bone marrow aplasia. No toxic death occurred. At the most recent evaluation, among the 15 children who had at least one Intellectual Quotient (IQ) examination after treatment at a median follow-up of 4.5 years (range, 1 months - 13 years) after diagnosis, the mean estimated full scale IQ was 78 (range, 44-109). CONCLUSION: This treatment strategy may improve the survival of young children with incompletely resected or classical medulloblastoma. The acute toxicity was manageable.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-35. INVESTIGATING THE EFFICACY OF THE IRREVERSIBLE pan-ErbB INHIBITOR PF-00299804 USING PAEDIATRIC EMBRYONAL TUMOR MODELS

Chantel Burchill 1, Hilary Hii 1, Peter Dallas 1, Catherine Cole 2, Raelene Endersby 1, Nicholas Gottardo 3

Abstract

Medulloblastoma and pineoblastoma are malignant embryonal brain tumors with a propensity to disseminate throughout the CNS. Despite significant improvements in therapy, many children remain incurable and survivors are often left with devastating late-effects, highlighting the need for innovative therapeutic strategies that target tumorigenic signaling pathways. To evaluate the efficacy of potential new therapies, as single agents and in combination with conventional chemotherapeutic drugs, we have developed in vitro models and orthotopic xenograft systems with a panel of cell lines representing various medulloblastoma subtypes and pineoblastoma. In addition, this study utilizes a transgenic spontaneous mouse model of medulloblastoma, the ND2-SmoA1 mouse, which represents the Sonic Hedgehog subgroup of human medulloblastomas. Previous studies have revealed that over-expression of the transmembrane receptors ErbB2 and ErbB4 is associated with poor prognosis in children with medulloblastoma, suggesting that inhibition of this pathway may be of therapeutic benefit. To determine if the ErbB pathway is deregulated in our models, we profiled expression of ErbB1-4. Immunoblot and immunohistochemical analysis revealed variable expression of the ErbB receptors in both in vitro and in vivo models, as observed in children with medulloblastoma, indicating that these are ideal systems to examine inhibitors of this pathway. We therefore tested a novel irreversible pan-ErbB inhibitor, PF-00299804 (Pfizer Inc.), which has shown anti-tumor activity in lung cancers harboring deregulated ErbB family receptors. Standard in vitro cell proliferation assays (Alamar blue) revealed significant variability among brain tumor cell lines in response to PF-00299804 (ED50 values ranged between 10-55 µM) with nodular desmoplastic medulloblastoma cells being the most sensitive. Drug synergism was observed upon combined treatment of cells with PF-00299804 and cyclophosphamide in vitro, providing the basis for combinatorial pre-clinical in vivo testing to determine if this is a potential candidate drug to propose for future clinical trials in a subset of patients with this disease.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-36. RESULTS OF HIGH-DOSE CHEMOTHERAPY WITH AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION IN THE TREATMENT OF PEDIATRIC BRAIN TUMORS

Asmik Gevorgian 1, Elena Morozova 1, Ilya Kazantsev 1, Tatjana Youhta 2, Svetlana Safonova 2, Andrey Kozlov 1, Yury Punanov 2, Boris Afanasyev 1, Olga Zheludkova 3

Abstract

AIM: Central nervous system (CNS) tumors are the second most common pediatric malignancies with an about 30% 5-year overall survival rate in high-risk group. The aim of this study was to assess the effectiveness of single or tandem high-dose chemotherapy (HDCT) with autologous hematopoietic stem-cell transplantation (auto-HSCT) in this patient group. METHODS: From October 2006 to September 2011, 12 pediatric patients with high-risk medulloblastoma (N = 7), supratentorial PNET (N = 3), and germinoma (N = 2) received HDCT with auto-HSCT after induction chemotherapy and surgical treatment. At the moment of HDCT 4 patients were in complete remission (CR), 4 patients were in partial remission (PR) and 4 patients had stable disease (SD). Patients with germinoma received single auto-HSCT, patients with medulloblastoma or supratentorial PNET received tandem auto-HSCT. The conditioning regimen for single auto-HDCT consisted of cisplatin, etoposide, and ifosfamide. In tandem HDCT, the first preparation regimen was carboplatin and etoposide, the second was thiotepa and cyclophosphamide. Bone marrow (n = 5), peripheral blood stem cells (n = 4) or both (n = 3) were used for stem cell sources. The mean transplanted CD34+ cell dose was 5.27 x 10^6/kg (range, 1.0–8.9 x 10^6/kg). RESULTS: The median follow-up was 22 months (range, 3–72). All patients with SD at the moment of transplantation died due to progression. Three of 7 patients with CR or PR relapsed during 1-9 months after HDCT, the other 5 patients are currently in CR. There was no unacceptable toxicity during conditioning regimens. The median time to engraftment was 16 (range 12–30) and 17 (range 13–86) days after the first and second auto-HSCT respectively. CONCLUSIONS: HDCT with auto-SCT in pediatric patients with high-risk CNS tumors may be a feasible option for patients in CR or PR after induction chemotherapy. It is ineffective as a salvage therapy in refractory patients.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-37. DIRECTIONS OF THE COG FOR MEDULLOBLASTOMA

Roger Packer 1, Amar Gajjar 2, Jeffrey Michalski 4, Regina Jakacki 5, Nick Gottardo 7, Nancy Tarbell 6, Gilbert Vezina 1, James Olson 3

Abstract

Over the past decade, advances have been rapidly made in the biologic understanding of medulloblastoma. Concurrently, improved outcomes have been reported in sequential COG studies, including a greater than 75% 10-year overall survival for children with non-disseminated disease treated with 2400 cGy of craniospinal radiotherapy (CSRT) and post-RT cis-platin based chemotherapy and over 70% 5-year PFS for “high-risk” disease treated in a pilot study using 3600cGy CSRT and carboplatin radiosensitization. A study is near completion prospectively comparing 1800cGy to 2340 cGy CSRT for children between 3 and 8 years of age with nondisseminated disease. For children with high-risk disease, a second study is underway evaluating the benefits of carboplatin radiosensitization and the use of retinoic acid. Despite encouraging survival data, survivors are at risk for posterior-fossa mutism syndrome, neurocognitive sequelae, and an alarming occurrence of secondary malignant tumors. The next generation of studies must: incorporate biologic tissue assessment in real-time; determine what platform is optimal for biologic analysis; use such analysis to guide therapy; and develop means for centralized real-time neuroradiographic review. Pressing therapeutic issues include: 1) how far can therapy be reduced for WNT-driven tumors?; 2) what is the significance of extent of resection with current therapies?; 3) what is the significance of histologic anaplasia?; 4) for the biologically-worse (? CMYC amplified) tumors, what approaches can be used?; 5) how can biologic therapy be implemented and with what agents?; 6) how can intellectual sequelae be safely decreased?; and 7) are secondary malignant tumors increasing in incidence and what can be done to prevent their occurrence?

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-38. TREATMENT OF YOUNG CHILDREN WITH DISSEMINATED MEDULLOBLASTOMA WITHIN THE PROSPECTIVE MULTICENTER TRIAL HIT 2000

Carsten Friedrich 1, André O von Bueren 1, Katja von Hoff 1, Nicolas U Gerber 2, Martin Benesch 3, Andreas Faldum 4, Torsten Pietsch 5, Monika Warmuth-Metz 6, Joachim Kuehl 7, Rolf D Kortmann 8, Stefan Rutkowski 1

Abstract

PURPOSE: To improve the prognosis of young children with disseminated medulloblastoma, patients were treated by a shorter and more intense induction chemotherapy compared to the previous trial HIT-SKK’92, followed by tandem high-dose chemotherapy (HDCT) with/without craniospinal radiotherapy (CSI). PROCEDURES: From 2001-2005, 20 eligible children aged < 4 years (median age: 2.8 years, range 0.2-3.6) with metastatic medulloblastoma (isolated M1, 2; M2-3, 18) confirmed by central pathological and neuroradiological review were prospectively treated according to HIT 2000. Patients received 3-4 postoperative courses of carboplatin and etoposide. In case of complete or partial neuroradiologic response patients were treated with tandem-HDCT (1. course: carboplatin, etoposide; 2. course: thiotepa, cyclophosphamide) and ASCT. CSI was applied to patients with poor response to induction or residual disease after HDCT, and optional to patients with residual disease before HDCT. RESULTS: Neuroradiologic responses to induction were complete in 2 and partial in 7 patients; 3 had stable and 5 progressive diseases (3, not assessable). One patient with desmoplastic medulloblastoma died of sepsis after one course of induction. Thirteen patients received tandem-HDCT (including 2 patients with stable disease after induction) followed by CSI in 4 patients. One patient with poor response to induction received CSI only. Five-year event-free survival, overall survival, CSI-free survival rates were 29% ± 10%, 39% ± 11% and 15 ± 8, respectively (median follow-up: 6.6 years). Eight of 20 patients survived (classic medulloblastoma, n = 6/15; desmoplastic medulloblastoma, n = 2/3; large-cell medulloblastoma, n = 0/2); four of them in first remission. CSI was omitted in two surviving patients (classic medulloblastoma, n = 1; desmoplastic medulloblastoma, n = 1). CONCLUSIONS: Survival rates of young patients with disseminated medulloblastoma remain low. To improve initial response rates, the induction chemotherapy has been further intensified since 2005. Whether CSI can be omitted in a subgroup of patients (e.g. desmoplastic medulloblastoma) remains to be investigated. Supported by Deutsche Kinderkrebsstiftung

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-39. TOLERABILITY OF TEMOZOLOMIDE IN CONJUNCTION WITH CRANIOSPINAL IRRADIATION FOR THE TREATMENT OF PEDIATRIC CNS EMBRYONAL TUMORS

Fatema Malbari 1, Mark Atlas 1

Abstract

OBJECTIVE: To evaluate tolerability of temozolomide in conjunction with craniospinal irradiation for treatment of pediatric CNS embryonal tumors. METHODS: Retrospective pilot study, ages 3-22, with an embryonal tumor from 1/1/02-1/1/12 who received craniospinal irradiation (CSI) with temozolomide (90mg/m2/day x 42 days). Eight charts reviewed; five received 23.4 Gy, three received 36 Gy for high risk disease. All completed local boost to 54Gy. Information obtained included: tumor type, treatment, side effects, neurotoxicity, symptoms and exam: at diagnosis, after surgery, radiation with temozolomide, and chemotherapy. Primary outcome variables are delays/changes in treatment and side effects: bone marrow suppression, neurologic, GI. RESULTS: Of eight patients, seven completed CSI with minimal side effects: nausea, vomiting, that responded well to antiemetics. One patient required PRBC transfusion. No patients required platelet transfusions. One patient developed a rash which self resolved in five days. Two patients experienced delay in treatment secondary to side effects. One patient suffered from severe esophagitis, after 13 treatments of 23.4 Gy prior to posterior fossa boost, requiring admission and delay of radiation for two weeks. Severe esophagitis and thrombocytopenia (grade 2) resulted in discontinuation of temozolomide after 22 days. A second patient suffered from thrombocytopenia (grade 2) which recovered after temozolomide was held for five days. This patient also missed one dose of CSI secondary to viral gastroenteritis requiring admission for 24 hours. No patients suffered neurotoxicity. CONCLUSIONS: Patients receiving CSI with temozolomide was well tolerated in seven of eights patients without significant side effects. One patient suffered from severe esophagitis which may be from CSI, but is unclear if temozolomide contributed. No patients suffered neurotoxicity. A prospective study of temozolomide in conjunction with CSI + boost in embryonal tumors is warranted.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-40. CLINICALLY-READY ONCOLYTIC ENGINEERED HERPES SIMPLEX VIRUSES KILL MEDULLOBLASTOMA NEUROSPHERES FROM PATIENT-DERIVED XENOLINES

Gregory Friedman 1, Virginia Kelly 1, Alexander Bray 1, Kevin Cassady 1, James Markert 1, Yancey Gillespie 1

Abstract

Despite advances in surgery, radiation and chemotherapy, both treatment-related toxicities in the developing brain and death rates remain high in children with medulloblastoma, the most frequent malignant childhood brain tumor. Therapeutic resistance of some aggressive pediatric medulloblastomas has recently been attributed to a radiation- and chemotherapy-resistant cell subpopulation termed ‘brain tumor stem cells' (BTSC). A novel, targeted therapy such as oncolytic engineered herpes simplex virus (oHSV) that kills both tumor cells and BTSC while sparing normal cells may decrease toxicity and improve survival for children with resistant medulloblastoma. Medulloblastoma xenolines (BT-45, BT-28), established in athymic nude mice directly from patient tumors and maintained by in vivo serial passage, were disaggregated, and grown as neurospheres in serumless, growth factor-defined Neurobasal medium to sustain the BTSC phenotype. FACS analysis was performed to monitor expression of HSV-entry molecules CD111 (nectin-1), CD112 (nectin-2), CD270 (HVEM) and SDC2 (heparin sulfate proteoglycan). Neurosphere cytotoxicity by clinically-ready oHSV M002, which produces IL-12 thereby eliciting an immune response through T-cell and NK cell activation, and C134, which expresses the human cytomegalovirus IRS-1 gene that greatly improves virus replication without restoring wild-type neurovirulence, was measured (AlamarBlue assay) 72 hours after infection. Dissociated neurospheres maintained in parallel highly expressed CD111 (BT-45, 92.4 ± 8.1%; BT-28, 87.6 ± 9.4%), the adhesion molecule that most efficiently mediates HSV-entry, CD112 (93.7 ± 2.6%; 98.9 ± 0.9%) and CD270 (66.0 ± 11.6%; 90.8 ± 9.8%). SDC2 was expressed at lower levels (20.6 ± 7.1%; 21.5 ± 3.7%). Neurospheres from both xenolines were highly sensitive to killing by M002 (BT-45, LD50 = 0.58 PFU/cell; BT-28, 8.3 PFU/cell); and C134 (2.2 and 2.7 PFU/cell respectively). These findings suggest that pediatric medulloblastoma cells including the BTSC subpopulation may be ideal targets for clinically-ready oHSV M002 or C134. IND-directed in vivo safety and efficacy trials of both viruses are underway.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-41. HYPERFRACTIONATED ACCELERATED RADIOTHERAPY (HART) WITH CHEMOTHERAPY FOR M 1-3 MEDULLOBLASTOMA (MB) - A CHILDREN'S CANCER AND LEUKAEMIA GROUP (CCLG)/NATIONAL CANCER RESEARCH NETWORK (NCRN) STUDY

Roger Taylor 1, Andrew Howman 2, Elena Brogden 2, Kathryn Robinson 3, Deborah Jones 2, Michael Gibson 2, Sylwia Bujkiewicz 3, Dipayan Mitra 4, Frank Saran 5, Antony Michalski 6, Barry Pizer 7

Abstract

The purpose was to evaluate the feasibility and toxicity of HART (1.24 Gy b.i.d.) followed by chemotherapy for M1-3 MB. The aim of HART was to improve the therapeutic ratio for RT by introducing acceleration in addition to hyperfractionation to minimise tumour cell repopulation during RT. Between February 2002 and May 2008 36 patients were entered. 2 were ineligible for RT, leaving 34 patients (22 male,12 female) aged 3-15 (median:7) with metastatic MB (M1:9; M2:3, M3:22). Following maximum resection of primary tumour, patients were treated by HART followed by 8 cycles of chemotherapy. Craniospinal radiotherapy (CSRT) dose was 39.68 Gy in 32 fractions, followed by a whole posterior fossa boost of 22.32 Gy in 18 fractions and where appropriate, boosts to metastases of 9.92 Gy in 8 fractions. Chemotherapy comprised vincristine (VCR) 1.5 mg/m2 x3, CCNU 75 mg/m2 and cisplatin 70 mg/m2. The first 7 patients did not receive VCR concurrent with RT. The remainder received VCR 1.5 mg/m2 weekly x 8 doses starting during the first week of RT. HART was delivered with a median duration of 34 days, range 31-38 days. Common grade 3-4 toxicities during HART included mucositis (8 patients), nausea (10), anaemia (5), thrombocytopaenia (2), leukopaenia (24). 22 patients received all 8 courses of adjuvant chemotherapy. 28 patients had carboplatin substituted for cisplatin for at least 1 course. Exploratory analysis showed that with a median follow-up of 4 years EFS and OS were 69% and 81% at 2 years and 59% and 71% at 3 years. Of 10 relapses, 1 was outside the CNS, 1 posterior fossa alone and 8 leptomeningeal with 3 of these also associated with posterior fossa relapse. The HART regimen was well tolerated and may have a place in the multi-modality management of patients with high risk MB/PNET.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-42. ICGC PEDBRAIN TUMOR - DISSECTING THE GENOMIC COMPLEXITY UNDERLYING MEDULLOBLASTOMA

David T W Jones 1, Natalie Jäger 1, Marcel Kool 1, Thomas Zichner 2, Barbara Hutter 1, Marc Sultan 3, Yoon-Jae Cho 4, Trevor J Pugh 5, Hans-Jörg Warnatz 3, Guido Reifenberger 6, Paul A Northcott 7, Michael D Taylor 7, Matthew Meyerson 8, Scott L Pomeroy 9, Marie-Laure Yaspo 3, Jan O Korbel 2, Andrey Korshunov 10, Roland Eils 1, Stefan M Pfister 1, Peter Lichter 1

Abstract

Despite advances in treatment for medulloblastoma over the past few decades, approximately 40% of children who develop this aggressively-growing malignancy will experience tumor recurrence, and 30% will die from their disease. Those children who do survive often suffer from severe tumor- or treatment-related morbidity, including neurocognitive dysfunction. Four biologically distinct subgroups are currently discriminated (WNT, SHH, Group 3 and Group 4), but the genetic events driving this distinction remain unclear. In this study, part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project (www.pedbraintumor.org), we have utilised an integrated next-generation sequencing based approach to identify alterations at the DNA and RNA level. We have sequenced samples from a total of 125 medulloblastoma patients, including whole genome sequencing (WGS) of 39 tumors and matched germline material. This was supplemented with large-insert mate-pair sequencing for structural variant detection, and strand-specific RNA-sequencing (RNASeq) data, in 36 and 28 cases, respectively. A target-capture approach was used for whole exome sequencing (WES) or for the sequencing of 2,734 target genes in an additional 86 tumor-normal pairs. These cases were further supplemented with low-coverage whole-genome sequencing for DNA copy-number profiling. Strikingly, tetraploidy was found to be a common early event in clinically challenging Group 3 & 4 tumors. For non-tetraploid tumors, a clear correlation of patients' age and mutation number was observed. Beside alterations affecting known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4), several novel recurrent mutations were identified (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA-sequencing data confirmed these alterations, and additionally revealed the expression of several novel fusion genes. Across all subgroups, genes encoding chromatin modifiers were altered in one-third of tumors. These findings provide a detailed insight into medulloblastoma tumorigenesis, and disclose novel targets for therapeutic approaches, especially for Group 3 & 4 patients.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-43. LYSIN-SPECIFIC HISTONE DEMETHYLASE 1 (LSD1) IS A PROMISING NEW EPIGENETIC THERAPY TARGET CRUCIALLY INVOLVED IN THE MIGRATION OF MEDULLOBLASTOMA CELLS

Kristian W Pajtler 1, Christina Weingarten 1, Theresa Thor 1, Annette Kuenkele 1, Gudrun Fleischhack 1, Lukas C Heukamp 3, Reinhard Buettner 3, Jutta Kirfel 2, Angelika Eggert 1, Alexander Schramm 1, Johannes H Schulte 1

Abstract

Epigenetic modifications including DNA methylation and histone acetylation are thought to play important roles in the onset and progression of cancer including brain tumors. More recently, also histone methylation has been identified as a major determinant of oncogenesis as well as embryonal development and differentiation. The prototype of the new class of histone demethylating enzymes is LSD1, which controls broad gene expression programs and is involved in malignant progression of several cancers. As enzymes controlling epigenetic alterations are of considerable interest as targets for cancer therapy, we here examined the role of LSD1 in medulloblastoma. Re-analysis of microarray data and immunohistochemical analysis of 87 medulloblastomas in a tissue microarray revealed upregulation of LSD1 mRNA and protein in medulloblastomas compared to normal cerebellar tissue. High LSD1 transcript levels in primary medulloblastomas positively correlated with reduced overall patient survival. LSD1 was also strongly expressed in human medulloblastoma cell lines and medulloblastomas derived from PTCH + /-/SmoA1 mice. Interestingly, downregulation of LSD1 in human medulloblastoma cells using siRNA significantly reduced cell migration. Furthermore, LSD1 expression increased in the distinct phase of granule precursor cell migration during cerebellar development in mice. BMP2 was identified as a major LSD1 target gene, as it was most prominently up-regulated upon LSD1 knockdown. Consequently, the Smad signaling pathway was reactivated in LSD1-knockdown cells. We conclude that LSD1 plays a crucial role for the migration of both MB and potential MB precursor cells, suggesting a deregulation of a physiological function of LSD1 during MB development. This work lays the foundation for further preclinical and clinical evaluations of LSD1 as a promising new epigenetic therapy target for MB.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-44. DIFFERENT INDUCTION CHEMOTHERAPY REGIMENS IN THE TREATMENT OF YOUNG CHILDREN WITH INTRACRANIAL CENTRAL NERVOUS SYSTEM PRIMITIVE NEUROECTODERMAL TUMORS/PINEOBLASTOMAS: RESULTS OF THE PROSPECTIVE MULTICENTER TRIAL HIT 2000

Carsten Friedrich 1, André O von Bueren 1, Katja von Hoff 1, Nicolas U Gerber 2, Martin Benesch 3, Robert Kwiecien 4, Torsten Pietsch 5, Monika Warmuth-Metz 6, Andreas Faldum 4, Joachim Kuehl 7, Rolf D Kortmann 8, Stefan Rutkowski 1

Abstract

BACKGROUND: Primitive neuroectodermal tumors of the central nervous system (CNS-PNET) and pineoblastomas are associated with an unfavorable outcome when compared to patients with medulloblastoma, especially in young children in whom craniospinal radiotherapy (CSI) is often omitted or delayed. Only a few prospective trials have been reported so far. PROCEDURES: Between January 2001 and January 2005, 17 eligible children aged <4 years with CNS-PNET not other specified (n = 8), ependymoblastoma (n = 1) or pineoblastoma (n = 8) confirmed by central pathologic review were prospectively treated in the trial HIT 2000. In nonmetastatic disease (n = 11), up to 5 postoperative cycles of HIT-SKK systemic multiagent chemotherapy (vincristine, cyclophosphamide, methotrexate, carboplatin, etoposide, 10 months duration) followed by CSI were given. In metastatic disease (M1-M3, n = 6), treatment consisted of a shorter induction chemotherapy (2-3 months) with carboplatin and etoposide, followed by tandem high-dose chemotherapy (HDCT) in patients who showed good response to induction chemotherapy. During induction and HDCT patients received intraventricular methotrexate. CSI was: 1) mandatory after poor response to induction chemotherapy or if no remission could be achieved following tandem-HDCT; 2) optional in case of residual disease before tandem-HDCT. RESULTS: Five-year event-free survival and overall survival rates ± standard error for all eligible patients were 24% ± 10% and 40% ± 12%, respectively (median follow-up of survivors: 8.3 years). Only one patient with nonmetastatic disease remained free of relapse/progressive disease during induction chemotherapy. Three of 6 patients with metastatic disease responded to induction chemotherapy and received tandem-HDCT followed by preventive CSI, and remain in continuous complete remission after 2.1, 6.5, 9.3 years. CONCLUSIONS: Short intensive induction chemotherapy followed by tandem-HDCT in young children with CNS-PNET/pineoblastomas seems to be superior to the prolonged and less intensive induction regimen. Neuropsychological outcomes and the impact of additional CSI should be considered. Supported by Deutsche Kinderkrebsstiftung

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-45. A NOVEL CASE-ONLY ANALYSIS OF GENE-GENE INTERACTIONS AND PEDIATRIC MEDULLOBLASTOMA RISK

Philip Lupo 1, Michael Scheurer 2

Abstract

BACKGROUND: The discovery of risk factors for pediatric medulloblastoma is complicated by several issues. For example, as medulloblastoma is a complex trait, the identification of risk factors may require the simultaneous assessment of multiple factors (e.g., gene-gene interactions). However, the ability to evaluate the role of gene-gene interactions is often limited by small sample sizes. The case-only study design is an efficient and powerful design for identifying interactions. We conducted a case-only study to evaluate candidate gene-gene interactions and pediatric medulloblastoma risk. METHODS: Cases (n = 98) were obtained from Texas Children's Hospital and MD Anderson Cancer Center. Key single nucleotide polymorphisms (SNPs) were selected from the Illumina Human 1M Quad SNP Chip in 11 detoxification genes and four DNA repair genes. We assumed a log-additive model of inheritance where genotypes for each SNP were classified based on the number of minor alleles (i.e., 0, 1, or 2). Ordinal logistic regression was used to evaluate the role of SNP-SNP interactions on disease risk, where one SNP was included as the independent variable and the other was the dependent variable. RESULTS: Among the 348 SNPs identified, those with a minor allele frequency <10% (n = 226) were eliminated from the analysis. From the remaining 122 SNPs, tagSNPs with high linkage disequilibrium (r 2 >0.8) were identified for each gene, leaving 22 SNPs for pairwise comparisons. Ten interactions were significantly associated with pediatric medulloblastoma risk at q <0.05 after adjusting for the false discovery rate. The interaction between OGG1 rs159150 and rs293795 approached genomewide significance (p = 2.82 x 10 − 8). CONCLUSIONS: To our knowledge, this is the first assessment of SNP-SNP interactions in detoxification and DNA repair genes and the risk of pediatric medulloblastoma risk. Although our results suggest genetic variation in these pathways is associated with disease risk, they are exploratory and must be validated in additional studies.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-46. NOVEL IMMUNE CHARACTERISTICS OF MEDULLOBLASTOMA CORRELATE WITH MYC AMPLIFICATION STATUS

Allison Martin 1, Christopher Nirschl 1, Magda Polanczyk 1, Kenneth J Cohen 1, Drew M Pardoll 1, Charles G Drake 1, Michael Lim 1

Abstract

The need to improve risk stratification for medulloblastoma, the most common malignant central nervous system in children, has long been recognized. Recently this tumor was categorized into four molecularly distinct groups with the goal of tailoring therapy to risk. Patients with the worst clinical prognosis were categorized as having Group C tumors whose molecular signature is dominated by MYC amplification. Despite the exciting progress made with regard to its molecular characteristics; little remains known about the immunology of medulloblastoma. B7H1 is an important immune regulatory molecule expressed on the cell surface of some solid tumors. Data from our group and others has shown that B7H1 expression correlates strongly with outcome in several solid tumors including melanoma and renal cell carcinoma. To date expression of this molecule has not been well studied in CNS tumors either at baseline or under the influence of cytokines. In this study we seek to characterize the ability of human medulloblastomas to express the immune regulatory ligands of PD-1, B7H1 and B7DC by flow cytometric analysis. We demonstrate for the first time that certain human medulloblastoma cell lines have constitutive expression of these ligands while others have inducible expression mediated by the pro-inflammatory cytokine, interferon gamma. Strikingly, the pattern of these immune phenotypes correlates with the MYC amplification status of the tumor. Those tumors with constitutive ligand expression are generally negative for MYC amplification while those that have inducible ligand expression are positive for MYC amplification. This is the first demonstration that medulloblastoma may have a unique immune phenotype corresponding with MYC expression patterns. These findings suggest that not only do medulloblastomas interact with the immune system in a previously unidentified way but that this immune phenotype may play a role in the prognostic significance of MYC amplification.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-47. Bmi1 AND FoxG1 REGULATE SELF-RENEWAL IN CD15+ non-Shh/Wnt SUBTYPE MEDULLOBLASTOMA STEM CELLS THROUGH RECIPROCAL PROMOTER OCCUPANCY

Branavan Manoranjan 1, Robin Hallett 2, Xin Wang 1, Chitra Venugopal 1, Nicole McFarlane 1, Katrin Sheinemann 3, John Hassell 2, Sheila Singh 1

Abstract

Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant tumor. By merging cancer genomics and developmental biology, recent studies have demonstrated the presence of several MB molecular subtypes, each distinct in terms of prognosis and predicted therapeutic response. Subtypes A and B are characterized by a relatively good clinical outcome and activation of the Wnt and Shh signaling pathways, respectively. In contrast, subtypes C and D (also known as “non-Shh/Wnt subtypes”) have yet to be characterized by a specific signaling pathway phenotype and are distinguished by metastatic disease and poor patient outcome. Current gene expression platforms have not detected brain tumor-initiating cell (BTIC) self-renewal genes in subtype C and D MBs, as BTICs typically comprise a minority of tumor cells and may therefore not be detected on bulk tumor analyses. Since previous studies have shown increasing BTIC frequency to be associated with increasing tumor aggressiveness and poor patient outcome, we recombined all poor-outcome subtypes into one group (non-Shh/Wnt) and investigated the subtype-specific gene expression profile of candidate genes previously implicated in normal and malignant stem cell populations within 251 primary human MBs from four non-overlapping MB transcriptional databases (Amsterdam, Memphis, Toronto, Boston). We then assessed the functional relevance of two genes, Bmi1 and FoxG1, which were preferentially expressed in non-Shh/Wnt MBs, and showed these genes to function as mediators of MB stem cell self-renewal and proliferation, while identifying their transcriptional regulation through reciprocal promoter occupancy in CD15+ MB stem cells in contrast to CD15- cells. Our work demonstrates the application of stem cell data gathered from genomic platforms to guide functional in vitro studies, which may then be used to identify novel subtype-specific BTIC self-renewal pathways amenable to therapeutic targeting.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-48. DEVELOPMENT OF IN VITRO AND IN VIVO CHEMO- AND RADIOTHERAPY MODELS FOR DETECTION OF TREATMENT-REFRACTORY MEDULLOBLASTOMA BRAIN TUMOR-INITIATING CELLS

Chitra Venugopal 1, Branavan Manoranjan 1, Nicole McFarlane 1, Anthony Whitton 2, Kathleen Delaney 3, Katrin Scheinemann 2, Sheila Singh 1

Abstract

The most clinically compelling component of the Cancer Stem Cell (CSC) hypothesis holds that CSCs evade current therapy, and are responsible for disease recurrence. Although there is strong evidence that tumor-initiating cell (TIC) populations are chemo- and radio-resistant, no studies prospectively demonstrate these treatment-resistant TICs to be exclusively causative in solid tumor relapse and recurrence. In the present study, we aim to identify the treatment-refractory brain tumor-initiating cell (BTIC) in medulloblastoma (MB), the most frequent malignant childhood brain tumor. We have validated Daoy cells, a MB cell line cultured in neural stem cell conditions, to act as a suitable surrogate for primary human MB BTICs. We first irradiated Daoy cells with 10 Gy at a rate of 0.7 Gy/min and found an enrichment of CD15+ and CD133+ BTICs by flow analysis post-radiotherapy. The irradiated cells also displayed an enhance self-renewal and proliferative capacity. Our initial in vitro chemotherapy studies were performed using cisplatin, vincristine, and cyclophosphamide, the current chemotherapeutic drugs for MB, and we have determined their respective IC50 levels. Similarly, we also found the drug-treated cells showed an increase in self-renewal and proliferation post-treatment. These in vitro studies confirm the presence of a treatment-refractory population with stem cell properties. Our future studies include generation of in vivo xenografts with MB BTICs in mice and treatment using clinically utilized doses of chemotherapy and radiation. Comparative BTIC profiles of surviving and relapsed mice will identify BTICs that respond to or evade specific therapy. Profiling genomic changes in “treatment-responsive” tumors against those that fail therapy will generate a differential profile of the refractory BTIC, which may guide future therapeutic approaches targeting this cell, and will serve as a model for targeting such CSCs in other TIC-driven solid tumors.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-49. Bmi1 REGULATION OF SONIC HEDGEHOG SIGNALING THROUGH THE E3 UBIQUITIN LIGASE CULLIN3 IN MEDULLOBLASTOMA STEM CELLS

Branavan Manoranjan 1, Robin Hallett 2, Chitra Venugopal 1, Nicole McFarlane 1, John Hassell 2, Katrin Scheinemann 3, Sandra Dunn 4, Sheila Singh 1

Abstract

Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant tumor. The recent identification of brain tumor-initiating cells (BTICs) has provided hope for targeted therapy in MB. Several developmental signaling pathways have been implicated in MB pathogenesis, of which Sonic hedgehog (Shh) signaling has proven to be the most susceptible to therapeutic interventions. We have recently identified the Shh effector proteins Gli1/2 to activate the master stem cell regulatory gene, Bmi1, by binding to its promoter and regulate the in vitro and in vivo self-renewal of MB BTICs. Additionally, our data alludes to the presence of an uncharacterized positive feedback loop through which Bmi1 regulates the Shh pathway. Recent data in the literature suggests the E3 ubiquitin ligase, Cullin3, to ubiquitinate and degrade histone deacetylase 1 (HDAC1). This mechanism has been shown to inhibit the deacetylation-mediated transcriptional activation of Gli1/2 in Shh-driven MB. As Cullin3 levels appear to be elevated in CD15- sort-enriched cells when compared to their CD15+ fraction, and Bmi1 appears to preferentially bind to the Cullin3 promoter in CD15+ cells when compared to the CD15- population, we hypothesize that Bmi1 regulates the Shh pathway through the transcriptional silencing of Cullin3 in CD15+ MB BTICs. Current Bmi1 knockdown and overexpression studies will be used to assess the resulting acetylation status of Gli1/2 in order to establish a functional role for Bmi1 in regulating the deacetylation-mediated transcriptional activation of Gli1/2. Future work will investigate the therapeutic potential of small molecule HDAC1 inhibitors, which may also function as Bmi1 signaling inhibitors in reducing the in vitro self-renewal capacity of MB BTICs and in vivo tumorigenic potential in our MB BTIC human-mouse xenograft model.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-50. BAX-RESISTANCE PLAYS AN ESSENTIAL ROLE IN MEDULLOBLASTOMA FORMATION

Idoia Garcia 1, Andrew J Crowther 1, Vivian Gama 1, C Ryan Miller 1, Mohanish Deshmukh 1, Timothy R Gershon 1

Abstract

Dysregulation of neural progenitors is essential to medulloblastoma pathogenesis. Failure to switch-off developmental proliferation of cerebellar granule neuron progenitors (CGNPs) promotes tumor formation. Previous work has defined molecular pathways that converge on p27/KIP to down-regulate CGNP proliferation through differentiation. Here we show that Bax-dependent apoptosis operates in parallel to regulate CGNPs, and that loss of programmed cell death (PCD) through Bax deletion accelerates medulloblastoma tumorigenesis. Genetic deletion of Bax prolonged cerebellar neurogenesis. Moreover, when bred into medulloblastoma-prone ND2:SmoA1 transgenic mice, Bax deletion significantly increased the frequency of medulloblastoma while shortening animal survival. Thus Bax functioned as a tumor suppressor, exerting a potent brake on medulloblastoma formation. Importantly, Bax deficiency greatly altered tumor pathology, causing increased markedly differentiation despite shorter survival time. The altered histology of Bax-deficient medulloblastomas revealed the continued influence of Bax on tumor behavior. We identified Bcl-2 and p27 as genes differentially expressed in response to Bax in medulloblastoma: tumors with intact Bax consistently up-regulated Bcl-2 and translocated p27 to the cytoplasm, while Bax-deficient tumors never expressed detectable Bcl-2 and maintained nuclear localization of p27. These findings suggest that Bcl-2 and cytoplasmic p27 act to block Bax-mediated tumor suppression. Immunohistochemical analysis of human medulloblastoma samples, confirmed the interrelation of Bax, p27 and Bcl-2 in human disease. Bax was expressed in all patient-derived tumors, accompanied by cytoplasmic p27 expression. Moreover, we found an inverse correlation between Bcl-2 and apoptosis, demonstrating the importance of Bcl-2 to mitigate PCD in the presence of Bax. Our results demonstrate the importance of Bax-mediated apoptosis in both preventing medulloblastoma and defining tumor phenotype. We have further identified specific mechanisms through which tumors adapt to the selective pressure imposed by Bax and develop Bax resistance, allowing malignant growth to proceed. Targeting mechanisms that reverse Bax-resistance may be a potent new avenue for medulloblastoma therapy.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-51. ASPM, A TARGET OF SONIC HEDGEHOG, IS A KEY ELEMENT IN CEREBELLAR DEVELOPMENT AND MEDULLOBLASTOMA PATHOGENESIS

Idoia Garcia 1, Andrew J Crowther 1, Timothy R Gershon 1

Abstract

Medulloblastoma derives from cerebellar granule neuron progenitors (CGNPs), undifferentiated cells that proliferate in the postnatal cerebellum in response to Sonic Hedgehog (Shh). While hyperstimulation of the Shh signaling promotes medulloblastoma in both mice and humans, Shh-pathway activation does not account for the majority of human disease. Identifying molecular mechanisms that are universally required for medulloblastoma is critical to the development of targeted therapies. Aspm, Abnormal spindle-like microcephaly-associated, is a gene mutated in familial microcephaly that maintains the undifferentiated state of neural progenitors. Interestingly, our expression microarray analysis identified Aspm as a developmentally-regulated gene induced by Shh in CGNPs. Here, we examined whether Aspm supports growth in medulloblastoma. We developed transgenic Aspm-GFP reporter mice, and confirmed that proliferating CGNPs express Aspm in response to Shh. Importantly, when bred into medulloblastoma-prone ND2:SmoA1 transgenic mice, the Aspm-GFP transgene drove robust, homogeneous expression of GFP in the resulting medulloblastomas. Thus expression of Aspm paralleled the progenitor state, driven by Shh and up-regulated in medulloblastoma. To determine if ASPM was essential to CGNP growth, we generated mice with genetic deletion of Aspm. Proliferative capacity and self-renewal of CGNPs were markedly reduced by loss of Aspm. While Aspm deficient CGNPs were able to proliferate when exposed to Shh in culture, the period of CGNP proliferation in vivo was shortened, as CGNPs terminally differentiated precociously. Thus ASPM was required to sustain progenitor phenotype over time. Our results demonstrate for the first time that Aspm, a previous unappreciated target of Shh, is integral to the proliferation of CGNPs and medulloblastoma cells. Self-renewal and sustained progenitor phenotype are essential to tumor growth. The reduced self-renewal and early loss of progenitor state seen in the ASPM knock-out CGNPs suggest that Aspm may be an ideal, entirely novel target for medulloblastoma therapy, relevant across tumor subtypes.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-52. HYPERFRACTIONATED CRANIOSPINAL RADIOCHEMOTHERAPY FOLLOWED BY MAINTENANCE CHEMOTHERAPY IN CHILDREN OLDER THAN 4 YEARS WITH SUPRATENTORIAL CENTRAL NERVOUS SYSTEM PRIMITIVE NEUROECTODERMAL TUMOR (STPNET) AND PINEOBLASTOMA

Nicolas U Gerber 1, Katja von Hoff 2, Carsten Friedrich 2, André O von Bueren 2, Wiebke Treulieb 2, Martin Benesch 3, Andreas Faldum 4, Torsten Pietsch 5, Monika Warmuth-Metz 6, Stefan Rutkowski 2, Rolf D Kortmann 7

Abstract

BACKGROUND: The prognosis for children aged ≥4 years with stPNET or pineoblastoma is poor with 5-year progression-free (PFS) and overall survival rates (OS) below 50% in most published series. Here we report the results of patients between 4 and 21 years with non-metastatic stPNET or pineoblastoma diagnosed from January 2001 to December 2005 and treated within the prospective GPOH-trial HIT 2000. METHODS: After surgery, children received hyperfractionated radiation therapy (craniospinal axis, 36 Gy; tumor region, 68 Gy; any residual tumor, 72 Gy, fractionated at 2 x 1 Gy per day on 5 days per week) accompanied by weekly intra-venous vincristine. Thereafter, 8 cycles of maintenance chemotherapy (CCNU, cisplatin, and vincristine) were given. RESULTS: 33 patients (17, stPNET; 16, pineoblastoma) were enrolled. Age at diagnosis was 12.2 years (range, 4.0 - 20.7). Gross-total tumor resection was achieved in 15, partial resection in 6 patients (indeterminate/not assessed, 12 patients). Median follow-up time of the 19 surviving patients was 7.0 (range, 3.2 - 10.0) years. Among the evaluable patients with residual tumor, the combined response rate to therapy was 85% (7/15, complete response; 4/15, partial response). Fifteen out of 33 patients (45%; 9/17 [53%], stPNET; 6/16 [38%], pineoblastoma) showed a tumor progression/relapse (7/15, local; 6/15, distant; 2/15, combined), median time to progression was 1.3 years (range, 0.3 - 6.7). 5-year PFS (± standard error) was 57.6% (±8.6%) for the entire cohort; stPNET, 47.1% (±12.1); pineoblastoma, 68.2% (±11.8%; p = 0.260). 5-year OS was 56.7% (±8.8%); stPNET, 47.1% (±12.1); pineoblastoma, 68.2% (±11.8%; p = 0.259). Of the 19 survivors, 15 (79.0%, 45.4% of all patients) were in complete remission at the last follow-up visit. CONCLUSION: Post-operative hyperfractionated radiotherapy with local dose escalation followed by maintenance chemotherapy was feasible without major toxicity. Survival rates compare favorably to most other series, including the previous trial HIT'91. Supported by Deutsche Kinderkrebsstiftung.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-53. NUTLIN-3 INCREASES CHEMOTHERAPY AND RADIOTHERAPY INDUCED APOPTOSIS OF HUMAN MEDULLOBLASTOMA CELLS THROUGH p53 SIGNALING PATHWAYS

Angelica Zin 1, Massimiliano De Bortoli 1, Paolo Bonvini 1, Elisabetta Viscardi 1, Giorgio Perilongo 1, Angelo Rosolen 1

Abstract

INTRODUCTION: Prognosis of disseminated or relapsed medulloblastoma is dismal and survivors often suffer from long-term sequelae of chemo- and radiotherapy. Thus, research to identify new treatment strategies is warranted. Recently, small-molecule antagonists of MDM2 including Nutlin-3, have been developed to inhibit p53-MDM2 interaction and to increase non-ubiquitinated (active) p53, which in turn increases apoptosis in cancer cells. MATERIALS AND METHODS: We studied the effects of Nutlin-3, alone and in combination with other anticancer agents (Etoposide, Cisplatin, Vincristine) or with radiotherapy on medulloblastoma cell lines (MB1603 and Daoy), and assessed whether the co-treatments induced increased anti-tumor response. Citotoxicity was measured by MTT assay whereas Western blotting, terminal deoxynucleotidyl transferase nick-end-labeling (TUNEL) and nuclear morphology were used to analyze the induction of apoptosis. RESULTS: Nutlin-3 induced low level of apoptosis in medulloblastoma cells carrying wild type p53. However, combination treatments with Nutlin-3 and chemo- or radio-therapy increased remarkably the efficacy of each chemotherapy agent and the effects of irradiation. We demonstrated that those effects were p53-dependent by studying expression levels of p53 molecular targets and apoptosis related proteins. Analysis of drug response-curves in a p53 wild type medulloblastoma cell line showed clear anti-tumor effects of Nutlin-3 combined with chemotherapy, after only 16 hours, and high levels of apoptosis after 72 hours. Nutlin-3 did not show any effects when used in medulloblastoma cells with mutated p53. CONCLUSION: This study suggests that novel tools in the treatment of patients with medulloblastoma, such as MDM2 antagonists, may increase response to chemo- and radiotherapy.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-54. INITIAL RELAPSE OF STANDARD RISK MEDULLOBLASTOMA IN FRONTAL HORN OF LATERAL VENTRICLE: SITE-SPECIFIC OR RANDOM PATTERN?

Eileen Connolly 1, Chi Zhang 1, Richard Anderson 1, Neil Feldstein 1, Eileen Stark 1, James Garvin 1

Abstract

In the past decade, with the addition of maintenance chemotherapy and despite the reduction in prophylactic radiation dose to the neuraxis, the risk of recurrence of childhood standard-risk medulloblastoma has decreased from about 35% to about 20%. The most frequent type of relapse is isolated neuraxis not involving the posterior fossa, but most reports do not indicate specific site(s) of relapse. In a retrospective review of 18 patients age 3-19 years diagnosed between 2006 and 2011 with standard-risk medulloblastoma, a total of three relapses occurred. We noted that all 3 relapses occurred initially isolated to the frontal horn of one of the lateral ventricles. All three patients received conventional craniospinal irradiation 23.4Gy followed by a 30.6Gy posterior fossa boost with weekly vincristine followed by maintenance vincristine, cisplatin, lomustine, and cyclophosphamide. Ages at diagnosis were 5-12 years; all were males. Primary sites at diagnosis were vermis (1) and 4th ventricle (2). Histologies were classic (2) and nodular (1). No patients had a ventricular-peritoneal shunt. Radiotherapy technique was 3D-CRT using photon (1) or proton (2). Interval from diagnosis to relapse was 19-23 months. Subsequent relapses occurred in 2 of 3 patients (frontal horn/4th ventricle in both). While the finding of initial frontal horn relapse could be coincidental, and the suggested pattern needs to be confirmed in a larger series, the frontal horn has certain properties which could predispose to site-specifictumor recurrence (lack of choroid plexus in contrast to the remainder of the ventricular system; CSF flow artifact with reported incidence of 14%), The possibility of site-specific recurrence could have implications for treatment, and also for refinement of clinical/molecular predictive models for relapse vs. no relapse in standard-risk medulloblastoma.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-55. INTENSIVE CHEMOTHERAPY (HEADSTART I AND II) AND TANDEM TRANSPLANTS FOR YOUNG CHILDREN WITH MEDULLBOASTLOMA AND PINEABLASTOMA

Matthew M K Shing 1, Vincent Lee 1, Frankie W T Cheng 1, Alex W K Leung 1, X L Zhu 1, H T Wong 1, Michael Kam 1, C K Li 1

Abstract

PATIENTS AND METHOD: Five children younger (< 3 years of age) presented between November 2009 and May 2011. Headstart I(non-metastatic) chemotherapy was given in 2 patients, consisting of five cycles of vincristine 0.05 mg/kg on day 1, 8 and 15(cycles 1 to 3), cisplatin 3.5 mg/kg on day 1, etoposide 4 mg/kg on day 2 and 3, cyclophosphamide 65 mg/kg on day 2 & 3. Three patients (metastatic diseases) were given five cycles of Headstart II (methotrexate 400 mg/kg was added on day 4). Afterwards, they were treated with three tandem transplants i.e. carboplatin 17 mg/kg/dose for 2 days and thiotepa 10 mg/kg for 1 day and autologous peripheral stem cell rescue. RESULTS: The mean age was 23 months of age (17 to 28 months). Four patients had medulloblastoma (classical: 2, desmoplastic: 1 anaplastic: 1; disease stages (Chang's) T3M3: 3, T3M0: 1). Gross total resection of the primary tumour site was performed in three patients. One patient had biopsy of the temporal lobe metastasis. The fifth patient had pineablastoma and had gross total resection. After chemotherapy, four patients had complete response. One patient had stable disease. The patient with T3M0 disease had local relapse soon after the third tandem transplant. He was treated with second operation and conformal radiotherapy to the tumour bed. He died after widespread metastatic disease. Another patient with T3M3 disease showed good response to Headstart II chemotherapy. He had leptomeningeal relapse after the third tandem transplant. Craniospinal radiotherapy (3600 cGY) and posterior fossa boost (1800 cCY) were given. There was no response and he died. The remaining three patients were alive for 10 months, 10 months and 12 months (complete remission: 2 and progression free: 1). They did not receive radiotherapy. CONCLUSIONS: Medulloblastoma and pinealoblastoma in young children appear to respond to intensive chemotherapy and tandem transplants.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-56. COMBINED Shh AND CXCR4 ANTAGONISM EXHIBITS SUPERIOR ANTI-TUMOR EFFECT FOR Shh-DRIVEN MEDULLOBLASTOMA

Stacey Ward 1, Rajarshi Sengupta 1, Kristen Kroll 1, Joshua Rubin 1

Abstract

Recent advances in molecular subtyping of cancers have identified specific patterns of growth-promoting pathway activation that may refine the development of more targeted approaches to treatment. We identified a subset of Sonic hedgehog (Shh)-driven medulloblastomas that also expressed high levels of CXCR4. These cells exhibited a requirement for CXCR4 activation for maximal growth in vitro. We report here that CXCR4 antagonism can be used to inhibit medulloblastoma growth and the combination of Shh and CXCR4 inhibitors together are superior to either alone. Clinical evaluation suggests that the utility of Shh-antagonists for treating medulloblastomas may be limited by the development of resistance through mutation of the target protein, Smoothened (Smo). We have published that CXCR4 is highly expressed in 80% of Shh-driven medulloblastoma. We hypothesized that targeting CXCR4 might augment the efficacy of Shh antagonism and circumvent the mechanism of resistance. To evaluate the anti-tumor efficacy of CXCR4 antagonism for Shh-driven medulloblastoma we created nude mice which were xenotransplanted with medulloblastoma cells derived from spontaneously-occuring tumors in SmoA1 mice. These mice were treated with vehicle, the CXCR4 antagonist AMD3100, or the Shh antagonist cyclopamine. While the vehicle-treated tumors grew rapidly, the cyclopamine and the AMD singly-treated tumors demonstrated transient growth arrest. In contrast, tumor-bearing animals treated with both cyclopamine and AMD3100 exhibited a significantly different response, with little to no growth over a four-week treatment period. In fact, one doubly-treated mouse tumor almost completely regressed in size, with few remnant cells remaining at the end of treatment. We are currently investigating the molecular mechanism of this inhibition. These data expand our understanding of the complex nature of growth regulation in tumors and indicate that combined CXCR4 and Shh antagonism should be clinically evaluated for the treatment of those Shh-driven medulloblastomas that co-expresses CXCR4.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-57. NOVEL PEPTIDE BASED DRUGS FOR THE TREATMENT OF SONIC HEDGEHOG DEPENDENT MEDULLOBLASTOMA

Peter Dallas 1, Nadia Milech 1, Brooke Longville 1, Richard Hopkins 1, Julius Varano della Vergiliana 1, Raelene Endersby 1, Nicholas Gottardo 2

Abstract

Medulloblastoma (MB), the most common malignant brain tumour in children, can be cured in up to 85% of patients using a combination of surgery, radiotherapy and chemotherapy. However, a significant number of children succumb to the disease, and survivors often face permanent treatment related disabilities, highlighting the need for more effective and less toxic therapies. The sonic hedgehog (SHH) pathway is a critical cell fate regulator in multiple mammalian organ systems, and hyperactive SHH signalling has been linked to SHH MB, one of the four main molecular subgroups of MB that have been characterised recently. Clinical trial data demonstrated that targeted inhibition of Smoothened (SMO), an integral component of the SHH pathway, can be effective for the treatment of patients with basal cell carcinoma and MB. This success suggests that inhibitors of the SHH pathway may be useful for the treatment of other solid tumours including lung, pancreas, prostate, and breast which have also been linked to deregulated SHH signalling. To identify novel SHH pathway blockers, we assessed a unique panel of peptide-based biopharmaceuticals generated from many different species of bacteria, and referred to as Phylomers, which target intracellular components of the pathway. An initial yeast two hybrid screen of a Phylomer library yielded ∼50 putative “interactors” with a C-terminal fragment of human SMO. Preliminary functional analyses of these interactors using SHH responsive cell lines revealed that a subset of these hits exhibit SHH pathway blocking activity. Ultimately, the unique properties of Phylomers may provide more clinically effective treatment for MB and other cancers driven by aberrant SHH pathway activity.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-58. DNA COPY NUMBER ABERRATIONS IN CENTRAL PRIMITIVE NEUROECTODERMAL TUMORS AND TUMORS OF THE PINEAL REGION: AN INDIVIDUAL PATIENT DATA META-ANALYSIS

André O von Bueren 1, Joachim Gerss 2, Christian Hagel 3, Haoyang Cai 4, Marc Remke 5, Martin Hasselblatt 6, Burt G Feuerstein 7, Sarah Pernet 8, Olivier Delattre 8, Andrey Korshunov 9, Stefan Rutkowski 1, Stefan M Pfister 5, Michael Baudis 4

Abstract

PURPOSE: To assess frequency, association with clinical characteristics, and prognostic impact of DNA copy number alterations (CNA) in central primitive neuroectodermal tumors (CNS-PNET) and tumors of the pineal region. EXPERIMENTAL DESIGN: Searches of PubMed, Medline, and Embase - after the original description of comparative genomic hybridization (CGH) in 1992 and July 2010 - identified 15 studies including patients with CNS-PNET and/or tumors of the pineal region whose tumors were investigated for CNA by chromosomal CGH, array based CGH, or single-color oligonucleotide array technologies (e.g. genomic single nucleotide polymorphism (SNP) arrays). One additional case study was identified from contact with scientists. Individual patient data were extracted from publications or were obtained via direct communication with investigators. Genome-wide aberrations were converted to a digitized format suitable for global data mining and subgroup identification. Summary profiles for genomic imbalances were generated from case-specific data. Overall survival (OS) was analyzed using Kaplan-Meier survival curves, by univariable, and multivariable Cox regression models. RESULTS: In their overall CNA profiles, low grade tumors of the pineal region clearly diverged from CNS-PNET and pineoblastoma. At a median follow-up of 89 months, 7-year OS rates of CNS-PNET, pineoblastoma, and low grade tumors of the pineal region were 22.9% ± 6%, 0% ± 0%, and 87.5% ± 12%, respectively. Multivariable analysis revealed that histology (CNS-PNET), age (≤ 2.5 years), and recurrent CNAs (gains of 3p1, 13q1, and 15q2) were associated with unfavorable OS. CONCLUSIONS: DNA copy number profiling suggests a close relation between CNS-PNET and pineoblastoma. Low grade tumors of the pineal region differed from CNS-PNET and pineoblastoma. Certain circumscribed CNAs may have an impact on the prognosis. Due to their high biological and clinical variability, a coordinated prospective validation in future studies is necessary to establish genomic risk factors.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-59. MOLECULAR TARGETING OF POLO-LIKE KINASE 1 (PLK1) ELIMINATES MEDULLOBLASTOMA BRAIN TUMOUR CELLS

Cathy Lee 1, Abbas Fotovati 1, Joanna Triscott 1, Sandra Dunn 1

Abstract

Medulloblastoma (MDB) is the most common pediatric brain malignancy. Although the cure rate of MDB has improved over the years, children surviving the disease often experience adverse long-term sequelae from surgery or radiation. In this study, we sought to determine whether polo-like kinase 1 (PLK1) would be a potential therapeutic target for the treatment of pediatric MDB, providing we have substansive pre-clinical evidence suggesting that inhibition of this disease-relevant kinase effectively suppresses the growth of rapidly growing brain tumour cells. Herein, we showed that biological and pharmacological inhibition of PLK1 significanly hindered the proliferation of Daoy cells and induced cell cycle arrest accompanied by apoptosis. Furthermore, PLK1 inhibitor BI2536 repressed the expansion of putative cancer stem and progenitor cell population in Daoys. To investigate the relevance of targeting PLK1 in clinical setting, we turned to primary MDB cells and first assessed its level in post-surgical tumour specimens. PLK1 level was found to be elevated in the primary medulloblastoma cells specimens examined and inhibition of this kinase by BI2536 reduced the proliferation/self-renewal of these cells in vitro. Together, these results indicate the pivotal role of PLK1 in sustaining growth and survival of MDB cells and the molecular targeting of this kinase may be a promising avenue for treating this disease.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-60. TARGETING KCNJ2 IN HIGH-RISK MEDULLOBLASTOMAS

Francesca Valdora 1, Florian Freier 1, Claudia Seyler 2, Nathan Brady 3, Sebastian Bender 1, Paul Northcott 5, Marcel Kool 1, David Jones 1, Simona Coco 7, Gian Paolo Tonini 7, Wolfram Scheurlen 9, Michael Boutros 4, Michael Taylor 5, Hugo Katus 2, Andreas Kulozik 10, Edgar Zitron 2, Andrey Korshunov 8, Peter Lichter 11, Stefan Pfister 1, Marc Remke 5

Abstract

Medulloblastoma constitutes the most common malignant brain tumor in childhood. Integrated genomic approaches revealed four major biological disease variants: WNT (wingless), SHH (sonic hedgehog), group 3, and group 4. Treatment failure mainly occurs in children harboring metastatic tumors, which typically carry an isochromosome 17 or gain of 17q. Thus, novel therapeutic options for these patients are urgently warranted. We identified potassium inwardly-rectifying channel J2 (KCNJ2) as one of the most upregulated genes on chromosome 17q in tumors with 17q gain by using gene expression profiling of 64 cases. We validated our findings on KCNJ2 transcript levels using three independent studies showing a similar gene expression pattern. High KCNJ2 expression was significantly associated with non-WNT/non-SHH grouping, anaplastic histology, metastatic dissemination, and poor clinical outcome. KCNJ2 protein expression was analyzed by immunohistochemistry in a large independent cohort of patients (n = 199). Underlining our previous findings, high protein expression was significantly correlated with 17q gain, metastatic dissemination, and inferior overall and progression-free survival (p < 0.0001). To functionally validate the potential role of KCNJ2 in medulloblastoma biology, we performed knockdown experiments by small interfering RNA-mediated silencing in two well characterized medulloblastoma cell lines. Knockdown of KCNJ2 resulted in a reduced proliferation rate and induction of apoptosis. Treatment of the medulloblastoma cell lines with Amiodarone and SR 59230A, two inhibitors of this class of Kir channels, phenocopied these promising anti-proliferative and pro-apoptotic effects in a time- and dose-dependent manner. Notably, SR 59230A demonstrated reduced toxicity in neuronal stem cell cultures compared to the medulloblastoma cell lines. Whole cell patch clamp results demonstrated a remarkable current reduction upon inhibitor treatment with SR 59230A. In summary, we could delineate KCNJ2 expression as a characteristic feature of medulloblastomas with dismal prognosis. Thus, pharmacological inhibition of this candidate gene may constitute a new therapeutic option for these patients with high-risk medulloblastomas.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-61. DELINEATING THE GENOMIC DIFFERENCES BETWEEN PRIMARY AND RECURRENT MEDULLOBLASTOMA

David J H Shih 1, Paul A Northcott 1, Timothy Van Meter 2, Ian F Pollack 3, Erwin Van Meir 4, Charles G Eberhart 5, Xing Fan 6, Olivier Dellatre 7, V Peter Collins 8, David T W Jones 9, Steven C Clifford 10, Stefan M Pfister 9, Michael D Taylor 1

Abstract

Medulloblastoma is the most common solid childhood malignancy. While survival rates for medulloblastoma patients have improved over the years, recurrent medulloblastomas currently have no effective treatment option. Further, it is unknown whether recurrent medulloblastomas are biologically different from their primary counterparts. Clinicians largely assume recurrent medulloblastomas are identical to their primary tumours, and enroll patients in clinical trials based on diagnostic tests of the primary tumours. To delineate the genomic differences between primary and recurrent medulloblastoma, we performed a genome-wide copy-number profiling analysis on 21 pairs/sets of primary and recurrent samples, and compared the somatic copy-number aberrations of recurrent tumours to their matched primary. In >80% of cases, recurrent tumours have acquired additional alterations, have arisen from a cancer clone distinct from the dominant clone in the primary tumour, or appear to be de novo tumours. Importantly, we found that recurrent tumours can ‘lose' critical amplicons, such as MYCN and GLI2, which characterize the molecular subgroup of the primary tumours. Such genomic differences would likely lead to differential response to targeted therapy and impact the outcome of clinical trials. Current clinical trials of targeted therapy in medulloblastoma involve target identification in the untreated primary tumour, followed by administration of the targeted therapy at the time of recurrence when the target may no longer be present. This schema is a recipe for certain failure. Future clinical trials of targeted agents for recurrent medulloblastoma should mandate re-biopsy to ascertain the continued presence of the target, or the targeted agent should be used upfront on the untreated primary tumour.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-62. FEASIBILITY AND TOXICITY OF INTRAVENTRICULAR METHOTREXATE FOR PRIMARY THERAPY OF PATIENTS WITH CHILDHOOD MEDULLOBLASTOMA TREATED WITHIN THE HIT 2000 TRIAL

Raisa Pompe 1, André O von Bueren 1, Katja von Hoff 1, Carsten Friedrich 1, Wiebke Treulieb 1, Christine Lindow 1, Frank Deinlein 2, Joachim Kuehl 2, Stefan Rutkowski 1

Abstract

PURPOSE: To assess the feasibility and toxicity of intraventricular methotrexate treatment in the primary therapy of medulloblastoma patients. METHODS: We enrolled 240 patients < 21 years from January 2001 - December 2007 (203 patients with complete and 37 with incomplete central review of histology, imaging, and/or CSF). Patients were treated according to age and metastasis status with 2-3 cycles of systemic chemotherapy and intraventricular methotrexate in three different treatment arms (150 children > 4 years with metastatic; 59 <4 years with non-metastatic; 31 <4 years with metastatic medulloblastoma) in 61 centers. RESULTS: Of 526 treatment cycles including intraventricular methotrexate treatment, 233 (44%) were reduced or omitted. Dose reduction was most frequently observed during the first cycle of chemotherapy. Of 220 evaluable patients with information on intraventricular access device, 9 never received one (reasons: VP-shunt = 7; EVD = 1; individual decision = 1) and it was implanted with delay in 39/211 (18%). Reservoir associated complications were documented for 55/211 (26%) patients (infection (25/55), intraventricular access device malfunction (14/55), CSF-leakage (7/55), combined complications (7/55), other (3/55)) and was followed by reservoir explantation in 39/211 (18%). Toxicity potentially associated to intraventricular methotrexate treatment was documented for 47 (11%) of 440 applied treatment cycles (15 without toxicity data) and included most commonly infection (32), neurotoxicity (seizure, 9; brain edema with following death, 1), combined toxicities (infection and neurotoxicity, 1), other (4). Methotrexate CSF levels were determined in 313 of 440 cycles and were elevated in 120 (38%) of 313, resulting in a documented dose reduction in 33/313 and leucovorine rescue in 4/313 cycles. Symptoms related to increased methotrexate CSF levels were reported for one patient (transient headache, nausea and nystagmus). CONCLUSIONS: Intraventricular methotrexate therapy was feasible and well tolerated by most patients. Evaluation of efficacy and possible late effects on neuropsychological outcome is needed. Supported by Deutsche Kinderkrebsstiftung

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-63. OUTCOME ANALYSIS BASED ON CLINICO-PATHOLOGIC FACTORS AND MOLECULAR SUB-GROUPING IN INDIAN PATIENTS WITH MEDULLOBLASTOMA TREATED ON PROSPECTIVE CLINICAL TRIALS

Tejpal Gupta 1, Rahul Krishnatry 2, Neelam Shirsat 1, Sridhar Epari 1, Ratika Kunder 1, Purna Kurkure 2, Tushar Vora 1, Aliasgar Moiyadi 1, Rakesh Jalali 2

Abstract

AIMS: To identify and validate clinico-pathologic factors including molecular sub-grouping that correlate with outcome in Indian patients with medulloblastoma. METHODS: Patients with medulloblastoma accrued and treated post-operatively on prospective clinical trials with available tumor tissues (either fresh-frozen or paraffin blocks) were identified from a prospective neuro-oncology database. Molecular sub-classification was done by mRNA expression levels of select protein-coding marker genes by real-time reverse transcriptase polymerase chain reaction. Univariate and multivariate analysis was done to identify factors associated with poor outcomes. RESULTS: Electronic search identified 40 patients with complete clinical details and analyzable tissues. The median age of the study cohort was 10 years (inter-quartile range 8-14 years) with a predominant male distribution (n = 29). Post-operative adjuvant therapy was based on conventional clinico-pathologic risk-stratification. Twenty six (65%) patients were classified as high-risk, including 10 (25%) with metastatic disease at presentation. Molecular analysis identified four distinct sub-groups viz. WNT (n = 10), SHH (n = 6), C (n = 11), and D (n = 13) as per consensus classification. There was substantially high incidence (55%) of metastases at presentation in molecular sub-group C. With a median follow-up of 26 months (inter-quartile range 9-40 months), the 5-year (+standard error) event-free survival and overall survival for the entire cohort was 53.2% (+9.7%) and 62.1% (+9.3%) respectively. On univariate analysis, risk-strata (average-risk vs high-risk metastatic, p = 0.017); histology (classical vs anaplastic, p = 0.016); and molecular sub-groups (WNT vs C, p = 0.003, and WNT vs SHH, p = 0.005) were significant prognostic factors. Cox regression analysis identified molecular sub-group C as the worst prognostic factor independently associated with significantly poor survival (p = 0.015). CONCLUSION: Molecular sub-group C, high-risk metastatic disease, and anaplastic histology are associated with significantly poorer outcomes in Indian patients with medulloblastoma consistent with published data from the western world.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-64. PEDIATRIC NEURO-ONCOLOGY IN THE AGE OF MOLECULAR CHARACTERIZATION: WHAT'S THE CLINICIAN TO DO?

Kenneth Cohen 1

Abstract

The recent characterization of molecularly diverse subtypes of medulloblastoma has been appropriately heralded as a major breakthrough in our understanding of this family of tumors. Phenotypically distinct pathologic variants have now been linked to these molecular signatures, and retrospective analyses have suggested favorable outcomes for some subtypes (e.g. Wnt positive tumors) and dismal outcomes for other subtypes (e.g. c-Myc expressing tumors). These findings, while offering the promise of more tailored therapy for each subtype, pose substantial challenges for the clinician. For example, the retrospective finding that current standard-risk therapy for medulloblastoma leads to very high (≥ 90%) EFS for patients with Wnt positive tumors might lead the practitioner to conclude that a reduction in the current intensity of combination chemotradiotherapy and adjuvant chemotherapy is warranted. Indeed, various strategies are being tested including radiotherapy reduction, chemotherapy reduction, and radiotherapy elimination. While each approach has some justification, none is evidently more rationale than any other, and thoughtful clinician-scientists can persuasively argue the merits of any of these approaches. Given the absence of a definitive "right" strategy, how does the clinician help a patient/family reflect on the risks and benefits of each approach? Moreover, if any of these strategies fail, do we risk being unable to salvage, or being forced to aggressively treat an otherwise readily curable patient had current standard risk therapies been given? The author will reflect on the ethics, regulatory considerations, and challenges of evolving molecular science in the treatment of children with brain tumors.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-65. TREATMENT RESULTS OF METASTATIC MEDULLOBLASTOMA (M2 AND M3 ACCORDING TO CHANG CLASSIFICATION) IN CHILDREN OVER 3 YEARS OF AGE. AN EXPERIENCE FROM A SINGLE INSTITUTION

Danuta Perek 1, Marta Perek-Polnik 1, Bozenna Dembowska-Baginska 1, Monika Drogosiewicz 1, Wieslawa Grajkowska 1, Maria Lastowska 1, Marzanna Chojnacka 2, Iwona Filipek 1, Magdalena Tarasinska 1, Marcin Roszkowski 1

Abstract

AIM: Analysis of treatment results of patients with M2 and M3 Medulloblastoma treated according to Polish Pediatric Neurooncology Group protocol. MATERIAL AND METHOD: 153 Medulloblastoma patients older than 3 years were treated in the Children's Memorial Health Institute between 1999 and 2009. 59 pts presented with metastases visible on MRI were treated according to high risk protocol (4 courses of pre-irradiation chemotherapy, craniospinal irradiation of 35 Gy, tumor bed boost up to 55Gy and individual boost to the remains of metastases followed by 8 courses of chemotherapy). Analysis included: age, gender, stage of disease, location of metastases, pathological subtypes, molecular subtypes in the M2&3 group, survival according to pathological and molecular subtype and location of metastases. The results were evaluated and compared in 2 groups M0&1 and M2&3. RESULTS: 5years OS was 87% in M0&1 group and 62% in M2&3 (p = 0,005), 5years PFS was 80% and 59% respectively (p = 0,003). There was no correlation between age and gender and disease stage (mean age 9,4 in both groups, 105 boys, 65 M0&1, 40 M2&3; 48 girls 29 M0&1, 19 M2&3), metastases were localized in brain in 7 pts, spine 35 and in 17 pts in both. There was a higher proportion of LCA subtype in the metastatic group (3,8% v/s 16,9, p = 0,007). 5 years OS was 64,3% in classic Medulloblastoma and 29,1% in anaplastic, PFS was 62% and 0 respectively. 5 years OS was 71,4% in pts with brain, 67,7% in pts with spine and 47,5% in pts with metastases in both sites. The preliminary results of molecular analysis will be presented. CONCLUSIONS: The strategy of conventional chemotherapy administered both before and after individualized irradiation is an option of treatment that offers long time remission to approximately 60% of M2&3 patients, but is ineffective in patients with LCA Medulloblastoma.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-66. OUTCOME OF PEDIATRIC CNS PNET IN HUNGARY WITH THE HUNGARIAN MBL2004/2008 BRAIN TUMOR TREATMENT SCHEDULE

Peter Hauser 1, Zsuzsanna Jakab 1, László Bognár 2, Balázs Markia 3, Zsuzsanna Gyorsok 3, Gábor Ottóffy 4, Kálmán Nagy 5, Judit Cservenyák 5, Péter Masát 6, Eszter Turányi 7, Júlia Vízkeleti 8, Gergely Kriván 9, Krisztián Kállay 9, Dezso Schuler 1, Miklós Garami 1

Abstract

BACKGROUND: Survival of newly diagnosed CNS PNET is poor despite multimodal intensive therapy. In 2004 intensified PNET therapy, containing high-dose chemotherapy with autologous stem cell rescue (AuBMT) was introduced in Hungary (MBL2004/2008). We retrospectively analyzed data of Hungarian patients treated with MBL2004/2008 in terms of outcome. PATIENTS AND METHOD: Between 2004 and 2010, 15 patients were diagnosed with CNS PNET in Hungary. Two patients had early refusal of therapy and one was lost in the postoperative period. All other patients were treated with the Hungarian MBL2004 or MBL2008 schedule (MTX or vincristin(VCR) + cyclophosphamide(CP)+ etoposide (VP16) followed by in both schedule by irradiation (booster + CSI) with concomitant VCR (older than 3 years) and 9 times VCR + VP16+ CP/cisplatin/carboplatin containing cycles, then AuBMT (busulphan + thiotepa + etoposid). Event free (EFS) and overall survival (OS) were assessed by the Kaplan-Meier method. For statistical analysis log rank test was applied. RESULT: Mean age of patients was 6.78 years (0.02 to 19.09 years). 3 of 12 patients had metastasis at diagnosis and 6 had incomplete tumor removal, 3 patients did not received irradiation due to young age. Two patients refused AuBMT (1 in CR, 1 has died), and 4 patients did not have AuBMT due to early progression. 6 of 12 patients accomplished treatment schedule (2 pts had recurrence, 4 in CR). There was no early termination of therapy due to side effects. With a mean follow-up time of 2.77 years Estimated 5-year EFS and OS of all patients are 36 % and 40%. Estimated EFS and OS after AuBMT are 63% and 78%. Patients younger than 3 years or after complete tumor removal have better survival. CONCLUSION: Based on this retrospective national overview, we conclude that the Hungarian MBL2004/2008 schedules are feasible. Patients accomplished full treatment schedule have a remarkable survival rate.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-67. EXPRESSION PROFILING REVEALS REGULATOR GENES OF EARLY NEURAL PATTERNING AS PUTATIVE CELLULAR TARGETS MEDIATING CYTOPATHIC EFFECTS OF PARVOVIRUS H-1PV ON PEDIATRIC MEDULLOBLASTOMA CELLS

Jeannine Lacroix 1, Franziska Schlund 1, Kathrin Adolph 1, Barbara Leuchs 1, Sebastian Bender 3, Thomas Hielscher 4, Stefan Pfister 3, Olaf Witt 5, Jörg R Schlehofer 1, Jean Rommelaere 1, Hendrik Witt 2

Abstract

PURPOSE: Cure rates below 30% in high-risk medulloblastoma patients indicate the need of new therapeutic approaches such as oncolytic virotherapy. Here, we assess the therapeutic efficacy of the oncolytic parvovirus H-1PV to high-risk medulloblastoma in vitro and characterize cellular target genes mediating its cytotoxicity. EXPERIMENTAL DESIGN: Seven medulloblastoma cell lines were analyzed by array cGH and by whole genome oligonucleotide microarrays and the results were matched to molecular and cytogenetic risk factors previously described. Subsequently, the efficacy of H-1PV entry, replication, and the cytotoxicity of H-1PV infection were investigated in these cell lines. Changes in gene expression profiles of H-1PV infected MB cells in course of repeated infectious cycles were determined and correlated to treatment response. RESULTS: In contrast to non-transformed infant astrocytes and neurons, six out seven medulloblastoma cell lines sustained lytic H-1PV infection and efficient viral replication. The cytotoxic effects induced by H-1PV were observed at LD50s below 0.05 p. f. u. per cell. Gene expression patterns in the responsive MB cell lines were analyzed in order to identify candidate target genes mediating the cytotoxic effects of H-1PV. H-1PV induced down-regulation of key regulators of early neurogenesis known to confer poor prognosis in medulloblastoma such as ZIC1, FOXG1B, MYC, NF1A and NF1B. In MB cell lines with genomic MYC amplification, MYC was the gene most significantly repressed after infection with H-1PV. CONCLUSION: By targeting genes of functional relevance and inducing cell death at very low titers, H1-PV may represent a promising treatment option for medulloblastoma patients.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-68. GABRA5 IS REGULATED BY C-MYC IN MEDULLOBLASTOMA

Konstantin Leskov 1, Nina Ma 1, Charles Eberhart 2, Duncan Stearns 1

Abstract

Overexpression of the MYC oncogene has been associated with dismal prognosis in medulloblastoma (MB), yet the mechanisms by which this occurs are not understood. Identifying genes that are transcriptionally regulated by MYC in MB may improve therapeutic interventions for the highest risk patients. Overexpression of MYC in the MB cell line DAOY results in a dramatic increase in the mRNA levels of the GABRA5 gene. This phenomenon was reproduced using an inducible MycER construct. Silencing of MYC in the MB cell line D425 resulted in a dramatic decrease in GABRA5 expression at both the mRNA and protein levels. Examination of the published sequence of the GABRA5 promoter revealed several an E-boxes (CACGTG) located at -1918? that could act as MYC binding/regulatory sites. Chromatin immunoprecipitation and DNA binding pull-down assays using MycER-DAOY and D425 cell lines documented an inducible binding to of the GABRA5 promoter at this location. Preliminary experiments using the cloned GABRA5 promoter also indicate an inducible increase in transcription in cell lines containing MycER. Expression analysis of a series of primary MB tumors shows a strong correlation between higher levels of GABRA5 expression and overexpression of MYC. GABRA5 has previously been identified as a marker for several molecularly classified subsets of MB (Cho et al JCO 2011), including the so-called c1 group which is characterized by MYC amplification. Our data support the concept that GABRA5 is a direct transcriptional target of c-Myc and may therefore be useful in identifying high risk tumors. GABRA5 is not normally found in the mature cerebellum. We speculate that the aberrant expression in medulloblastoma may represent a reprogramming of normal neurotransmitter receptor to a more progenitor cell phenotype. The functional consequences of GABRA5 expression in medulloblastoma are being investigated.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-69. FEASIBILITY PILOT OF ATTENUATED MAINTENANCE CHEMOTHERAPY FOR ADOLESCENTS AND ADULTS WITH NEWLY DIAGNOSED LOCALIZED MEDULLOBLASTOMA AND OTHER PRIMITIVE NEUROECTODERMAL TUMORS

Jennifer N Dagri 1, Joseph Torkildson 2, Anna Evans 1, Lynn S Ashby 3, Branko Zakotnik 4, Robert J Brown 1, Girish Dhall 1, Jana Portnow 5, Jonathan L Finlay 1

Abstract

BACKGROUND: Newly-diagnosed adolescents and adults with medulloblastoma experience substantial toxicities with “standard” maintenance chemotherapy regimens including CCNU, cisplatin and vincristine. We evaluated an attenuated maintenance chemotherapy regimen targeting adolescents and adults. METHODS: Eleven patients with newly-diagnosed medulloblastoma and 1 suprasellar atypical teratoid/rhabdoid tumor (AT/RT) were treated between 2004 and 2010. Eight patients were between 17 and 50 years old. Eleven received 2340cGy craniospinal irradiation, 1 AT/RT 3600cGy, all with posterior fossa or tumor bed boosts. Ten patients received weekly vincristine x 6 (1.5mg/M2, max. 2mg) during irradiation. Maintenance chemotherapy commenced 4-6 weeks following irradiation: Cycle A: vincristine (days 1, 8), oral temozolomide (150mg/m2 days 1-5) and oral etoposide (50mg/M2 days 1-21), Cycle B: vincristine (days 1, 8), carboplatin day 1 (560mg/M2). Cycle C: vincristine (days 1, 8), cyclophosphamide (600mg/M2 on days 1 and 2). Cycles A, B and C were repeated for 9 total cycles. RESULTS: A total of 87 cycles were administered. Grades III/IV neutropenia occurred in 60% cycles, anemia in 33% cycles and thrombocytopenia in 29% cycles. 8 and 8 patients received a total of 28 packed red blood cell (PRBC) and platelet transfusions respectively. 59% of platelet and 48% of PRBC transfusions were in Cycle B. Grades III/IV transaminitis occurred in 8% cycles. There was 1 grade III sensory and 4 grade III motor neuropathies and one grade III hypocalcemia. No grades I-IV ototoxicities or nephrotoxicities were observed. No grades III/IV weight loss were observed. Two patients had single hospitalizations for chemotherapy-related toxicities (1 and 13 days’ duration). After 4 years median follow-up, no patient with medulloblastoma relapsed; the ATRT patient recurred 24 months from diagnosis. CONCLUSIONS: This attenuated maintenance chemotherapy regimen was well tolerated by adolescents and adults. A prospective North American clinical trial is planned for adult medulloblastoma/PNET to determine the efficacy of this regimen.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-70. TREATMENT OF EXTRANEURAL MEDULLOBLASTOMA - AN EVIDENCE REVIEW AND UK SURVEY

Martin McCabe 1, Barry Pizer 2

Abstract

BACKGROUND: Extraneural metastases from medulloblastoma are associated with a particularly dismal outlook and there is no consensus on how best to treat them. METHODS: PubMed was searched using the terms: metastatic, metastas*, extraneural, extracranial, medulloblastoma, medulloblastomas, PNET, PNETs. Clinical trial reports over the last decade were examined for cases of extraneural medulloblastomas. Where explicit treatment details and survival data were available we recorded demographic data, initial treatment data, relapse data and survival. In addition, UK paediatric and clinical oncologists responsible for treating medulloblastoma were surveyed for their views on optimal management of extraneural disease. RESULTS: There were 102 individuals with extraneural disease from 46 reports with adequate data. 90% were diagnosed at relapse. Only 6/844 metastatic patients registered on prospective clinical trials had extraneural disease. 77% of extraneural metastases occurred within 4 years of diagnosis. Bone, bone marrow and lymph node disease accounted for 85% of cases. Most were treated with chemotherapy +/- irradiation. 22% were given several sequential chemotherapy regimens. A minority (n = 13) had high dose therapy, using 9 conditioning regimes. Median survival following extraneural disease was 1.01 years. 13/13 disease-free survivors (median 1.83 years after diagnosis of extraneural disease) were given one or more of cyclophosphamide, doxorubicin, vincristine and etoposide at standard dose +/- irradiation +/- high dose therapy, compared to 29/68 non-survivors (chi-square p = 0.025). 3 of 4 disease-free 5-year survivors had standard dose chemotherapy and irradiation. UK SURVEY: Experience of extraneural disease is limited. Of 18 respondents to an online survey, most would treat extraneural disease at initial presentation with intensive induction therapy and myeloablative therapy. Most would treat extraneural disease at relapse with either palliative chemotherapy or symptom control only.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-71. ANTINEOPLASTIC ACTION OF 5-AZA-2'DEOXYCYTIDINE AND SODIUM 4-PHENYLBUTYRATE ON MEDULLOBLASTOMA CELL MODELS

Ana-Maria Marino 1, Ninib Baryawno 1, Tomas P Ekstrom 1, Arne Ostman 1, John-Inge Johnsen 1

Abstract

Aberrant epigenetic alterations are known to be involved in cancer development. Epigenetic drugs have been shown to enhance drug sensitivity. Promising pre-clinical and clinical studies have demonstrated that combination therapy using DNA methyltransferase inhibitors and histone deacetylase inhibitors with existing chemotherapeutic agents have the potential to overcome drug resistance, induce apoptosis and inhibit proliferation. Therefore we hypothesized that combination treatment using sodium 4-phenylbutyrate (a HDAC inhibitor), and dacogen (5-aza-2'deoxycytidine a DNA methylation inhibitor)) could sensitize medulloblastoma tumor cells to conventional chemotherapy and radiotherapy. We have already seen additive effects after combination treatments with sodium 4-phenylbutyrate and dacogen in 2 medulloblastoma cell lines. Additive effects were also seen after combining 4-phenylbutyrate with Imatinib and dacogen with Imatinib. However we observed very good results when we combined the 3 drugs at very low concentrations. Clonogenic assays also indicate a reduction of cell proliferation and cell cycle analysis has shown arrest at G1 and G2 (is currently been repeated). The results makes us hypothesize that sodium 4-phenylbutyrate might be inhibiting HDAC1 and HDAC2, this is further been explored. The combination will hopefully lead to lower the dosage of chemotherapy and radiation therapy, enhance tumor cell death and lower side effects of current medulloblastoma therapy.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-72. WHOLE GENOME SEQUENCING IDENTIFIES NOVEL MUTATIONS IN DISTINCT SUBGROUPS OF MEDULLOBLASTOMA

Giles Robinson 1, Matthew Parker 1, Tanya Kranenburg 1, Charles Lu 2, Timothy Pheonix 1, Robert Huether 1, John Easton 1, Arzu Onar 1, Ching Lau 3, Eric Bouffet 4, Sridharan Gururangan 5, Tim Hassall 6, Richard Cohn 7, Amar Gajjar 1, David Ellison 1, Elaine Mardis 2, Richard Wilson 2, James Downing 1, Jinghui Zhang 1, Richard Gilbertson 1

Abstract

BACKGROUND: Medulloblastoma is a malignant childhood brain tumor comprising four discrete subgroups (SHH-subgroup, WNT-subgroup, subgroup-3 and subgroup-4). The genetic alterations that drive these subgroups and that might serve as treatment targets are largely unknown. METHODS: We sequenced the entire genomes of 37 tumors and matched normal blood. 136 somatically mutated genes identified in this discovery cohort were sequenced in an additional 56 medulloblastomas. All tumors were classified into the 4 subgroups by expression profiling and immunohistochemistry. All mutations were validated by custom capture, 454, or Sanger sequencing. RESULTS: Recurrent mutations were detected in 49 genes: 41 are not yet implicated in medulloblastoma. Several target distinct components of the epigenetic machinery in different disease subgroups, e.g., regulators of H3K27 and H3K4 trimethylation in subgroup-3 and 4 (e.g., KDM6A and ZMYM3), and CTNNB1-associated chromatin remodellers in WNT-subgroup tumors (e.g., SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumors, identified genes that maintain this cell lineage (DDX3X) as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis. CONCLUSIONS: We have identified several new recurrent somatic mutations that are enriched in specific subgroups of medulloblastoma. Alterations affecting subgroup-3 and 4 tumors appear to disrupt chromatin marking, most notably H3K27me3, potentially preserving a stem cell-like state in tumor cells. Mutations in WNT subgroup tumors affect binding partners of CTNNB1 that regulate WNT-response gene transcription. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-73. LOSS OF 10Q IS AN INDICATOR OF POOR OUTCOME IN SHH SUBGROUP MEDULLOBLASTOMA

Giles Robinson 1, James Dalton 1, Terri O'Neill 1, William Yong 2, Murali Chingtagumpala 3, Eric Bouffet 4, Daniel Bowers 5, Stewart Kellie 6, Sridharan Gururangan 7, Paul Fisher 8, Anne Bendel 9, Michael Fisher 10, Tim Hassall 11, Cynthia Wetmore 1, Alberto Broniscer 1, Steven Clifford 12, Richard Gilbertson 1, Amar Gajjar 1, David Ellison 1

Abstract

BACKGROUND: One of the best characterized molecular subgroups of medulloblastoma, the SHH subgroup is also the most heterogeneous. Age at diagnosis varies widely, as demonstrated by its bimodal distribution in infants (<3yrs old) and adults (>16yrs old), and all three major histologic subtypes are represented. We have studied cytogenetic abnormalities common to medulloblastoma across a large series of SHH tumors, focusing particularly on chromosome 10q and outcome indicators. METHODS: Using an immunohistochemical assay on archived medulloblastomas, we identified 123 SHH tumors. Interphase FISH was undertaken using probes to chromosomes 17, 9q (PTCH1), 10q (PTEN, SUFU, DMBT1), NMYC, and MYC. RESULTS: 28 (23%) tumors had 10q loss (12 - isolated 10q loss; 16 - combined 9q/10q loss), 51 (41%) had 9q loss (35- isolated; 16 - combined 9q/10q), and 59 (49%) had neither. 10q loss was associated with significantly worse outcome (p-value < 0.05) than any other cytogenetic variable; 50% (9/18) of patients 10q loss (isolated or combined) had progressive disease or died of disease, as compared with 18% (12/69) of those without 10q loss. The following high-risk variables were more frequent in tumors with 10q loss; large-cell/anaplastic (LC/A) morphology 32% (9/28) versus 7% (7/95); and NMYC/MYC amplification 30% (8/27) versus 5% (5/79). No association was seen with metastatic disease. iFISH to three loci on 10q identified the PTEN locus as the putative target of the deletion; two tumors exhibited an isolated PTEN/10q23 loss and three displayed biallelic PTEN/10q23 loss in the setting of a broader mono-allelic 10q loss. CONCLUSIONS: 10q loss is an indicator of poor outcome in SHH medulloblastoma and is associated with recognized high-risk disease features, LC/A and NMYC/MYC amplification, but not metastatic disease. These data provide important new insights into the SHH subgroup and highlight the PI3K pathway as a target for developmental therapeutics.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-74. WNT ACTIVATION BY LITHIUM ABROGATES MUTANT TP53 RADIATION RESISTANCE IN MEDULLOBLASTOMA

Nataliya Zhukova 1, Dianna Martin 6, Tatiana Lipman 6, Pedro Castelo-Branco 6, Cindy Zhang 6, Mihael Fraser 2, Berivan Baskin 7, Peter Ray 7, Eric Bouffet 1, Benjamin Alman 8, Vijay Ramaswamy 9, Peter Dirks 1, Steve Clifford 3, Stefan Rutkowski 4, Stefan Pfister 5, Robert Bristow 2, Michael Taylor 9, David Malkin 1, Cynthia Hawkins 10, Uri Tabori 1

Abstract

Although survival has improved dramatically for children with medulloblastoma (MB), 30% of patients will die of their tumour indicating a need for novel therapies. While patients whose tumors exhibit WNT activation demonstrate improved survival, results are conflicting for patients with TP53 mutations conferring radioresistance. We hypothesized that the adverse survival associated with TP53 mutations is modulated by other genetic alterations: activation of WNT or SHH signalling. This is especially important since WNT activation can be achieved with administration of Lithium. METHODS: In a collaborative effort, we compiled data from four published studies to determine the role of TP53 mutations according to molecular subgroups. Human MB cell lines harbouring either wild-type or mutant TP53 and wild-type or mutant CTNNB1 were used to investigate differences in radiation resistance and effects of Lithium as a radiosensitizer. Normal neuronal stem cells (NNSC) were used to assess effects of Lithium/radiation on ‘normal' brain tissue. Clonogenic assays were used to determine radiosensitivity. RESULTS: Survival for children with mutant TP53 MB was dependent on molecular subgroups with 5-year OS of 87% (+/-11%) and 12% (+/-11%) for WNT and SHH subgroups, respectively (p = 0.0003). Confirming our clinical observations, TP53 mutant MB cells were more resistant to radiation than TP53 wild-type lines (p < 0.01), while insertion of a CTNNB1 mutation increased radiosenstivity especially in TP53 mutant cells (p < 0.01). Treatment with Lithium sensitized MB cells to radiation (p < 0.01), accompanied by increased DNA damage and delayed repair as shown by γH2AX immunofluorescence. In contrast, Lithium treatment of NNSC did not sensitize them to radiation. CONCLUSIONS: Detrimental effects of TP53 mutation may be modulated by other genetic alterations. Constitutive activation of WNT pathway by Lithium sensitizes TP53 mut MB cells to radiation. Knowing the safety of Lithium, its neuroprotective capacity in normal neuronal cells, oral administration of Lithium may represent an attractive novel therapy for MB patients.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-75. OUTCOME OF INFANTS AND YOUNG CHILDREN WITH NEWLY DIAGNOSED MEDULLOBLASTOMA TREATED ON “HEAD START” III PROTOCOL: FINAL UPDATE FROM THE “HEAD START” CONSORTIUM

Girish Dhall 1, Lingyun Ji 1, Kelley Haley 1, Sharon Gardner 2, Richard Sposto 1, Jonathan Finlay 1

Abstract

BACKGROUND: High-dose chemotherapy with autologous hematopoietic cell rescue (AuHCR) is a potentially curative approach for young children with medulloblastoma (MB), while avoiding late effects of irradiation. We report the outcome of newly-diagnosed young children with MB treated on "Head Start" III, a prospective, multi-national clinical trial. METHODS: Between April 2003 and December 2009, 92 children were enrolled at 29 institutions. All children received 5 induction cycles (vincristine, cisplatin, cyclophosphamide, etoposide, high-dose methotrexate - [cycles 1, 3, 5], and vincristine, cyclophosphamide, oral VP-16, temozolomide [cycles 2, 4]), followed by 1 myeloablative chemotherapy cycle (thiotepa, carboplatin, etoposide) with AuHCR. Children between 6 -10 years old or with residual tumor pre-consolidation, received irradiation after consolidation. RESULTS: 3-year event-free (EFS) and overall survival (OS) rates (+/- SE) for all patients, patients with localized (M0) disease (n = 39), and patients with disseminated (M1+) disease (n = 51) were 49 +/- 5% and 66 +/- 5%, 64 +/- 8% and 79 +/- 7%, and 37 +/- 7% and 56 +/- 7%, respectively. The 3-year EFS and OS (+/-SE) for all patients with desmoplastic, classical and anaplastic MB were 89 +/- 6% and 88 +/- 7%, 29 +/- 7% and 58 +/- 7%, 45 +/- 14% and 51 +/- 15%, respectively and for M0 patients were 93 +/- 6% and 93 +/- 6%, 44 +/- 12% and 71 +/- 11%, 50 +/- 20% and 67 +/- 19%, respectively. 3-year irradiation free EFS was 50 +/- 6%. In multivariate Cox regression analyses, histology was the only significant independent predictor of EFS with Desmoplastic MB patients having significantly better EFS than classic MB (HR = 0.11, 95%CI = 0.033, 0.35). No significant difference was found between anaplastic and classic tumors. CONCLUSION: We report the best survival data published so far on young children with desmoplastic and anaplastic MB, while avoiding irradiation in half of the patients.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-76. COMBINATION THERAPY DEVELOPMENT SPECIFICALLY FOR PEDIATRIC SUPRATENTORIAL PRIMITIVE NEUROECTODERMAL TUMOR USING A PATIENT-DERIVED XENOGRAFT MODEL

Sarah Leary 1, Andrew Strand 2, Sally Ditzler 2, Greg Heinicke 2, Lindsey Conrad 2, Andrew Richards 2, Kyle Pedro 2, Sue Knoblaugh 2, Bonnie Cole 1, James Olson 2

Abstract

Supratentorial primitive neuroectodermal tumors (sPNET) are currently treated with therapy designed for patients with medulloblastoma, but are biologically distinct tumors with an inferior survival rate. The study of sPNET has been limited by a lack of preclinical models. Patient-derived xenografts (PDX) and stem cell cultures of sPNET were established at the time of therapeutic tumor resection of a recurrent pediatric tumor. Human tumor at diagnosis and recurrence as well as PDX tumor were evaluated by standard histology and immunohistochemistry. Cultured cells were screened with a siRNA panel against protein kinases as well as a panel of FDA-approved cancer drugs. PDX mice were treated with conventional chemotherapeutic agents and small molecule inhibitors as single agents and in combination. PDX tumors recapitulated human tumor by standard histological evaluation and protein expression by immunohistochemistry demonstrating EGFR and c-kit expression. Tissue and cell based screens predicted inefficacy of standard platinum chemotherapy and efficacy of several alternative therapeutics tested in the PDX model. Combination therapy with erlotinib and dasatinib resulted in rapid tumor response in PDX tumors. Mean tumor volume was 11% of initial volume after 28 days of therapy. Combination therapy was superior to vehicle control, cytotoxic therapy, and therapy with either agent alone (all p values < 0.01). We report the first mouse model of sPNET, which was used to efficiently identify therapeutics warranting further study in this disease. PDX models offer an opportunity to study biology and prioritize therapeutics for rare tumors for which no genetically engineered models exist.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-77. A COMBINATION OF BEVACIZUMAB AND ISOTRETINOIN IN CHILDREN WITH RECURRENT MEDULLOBLASTOMA

Maxim Yankelevich 1, Michail Budarin 3, Andre Konski 2, George Mentkevich 3

Abstract

BACKGROUND: Attempts to salvage patients with recurrent medulloblastoma using chemotherapy fail in majority of the cases. As an alternative approach, we evaluated a combination of two potentially effective biological agents, bevacizumab and isotretinoin. METHODS: Four pediatric patients were enrolled from 2006 to 2009. Two patients were treated at first recurrence, two at third recurrence; all had primary M+ stage. All 4 patients had craniospinal irradiation (CSI) and chemotherapy for their primary disease. Three out of 4 patients received repeat irradiation in a form of gamma-knife (2) or CSI (1) prior to initiation of biotherapy. Biotherapy was started 4-6 weeks after irradiation with bevacizumab given at 10 mg/kg every 2 weeks, and isotretinoin 160 mg/m2/day x 14 days with 2 week intervals. After 8 weeks of therapy the disease was evaluated by MRI. In case of radiological response or stabilization patients received second 8-week course. RESULTS: All patients tolerated therapy well and completed 16 weeks of treatment. All had dry skin and cheilitis, three had occasional episodes of epistaxis, and two had headaches. MRI after 8 weeks of biotherapy demonstrated partial response in a patient who did not receive irradiation upfront (a 9 year-old girl whose metastatic tumors had progressed in spite of several lines of chemotherapy and second CSI). The other 3 patients already had partial responses after irradiation by the time biotherapy was started. Although they all had further improvement of their tumors, continuous effect of RT could not be ruled out. Three out of 4 patients developed disease progression at +7, +6, +4 months from the start of biotherapy. One patient is alive in CR at +42 months. CONCLUSION: The combination of repeat irradiation followed by bevacizumab and isotretinoin was safe and resulted in objective responses and sustained remissions in this group of refractory patients.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-78. MEDULLOBLASTOMA WITH UNUSUAL MULTILINEAL DIFFERENTIATION: CLINICAL, HISTOPATHOLOGICAL AND MOLECULAR GENETIC FINDINGS.

Harald Stefanits 1, Georg Ebetsberger-Dachs 2, Serge Weis 3, Christine Haberler 1

Abstract

We present an unusual tumor in a 3.9-year-old boy who presented with a two-week history of nausea and vertigo. MRI revealed a 5x5.5x5cm sized tumor located in the fourth ventricle and spinal leptomeningeal dissemination. Histopathologically, the tumor showed undifferentiated, primitive neuroectodermal-like tumor cells with focal anaplastic features, and admixed rhabdomyoblastic and pigmented elements arranged in tubular structures, as well as cartilage. Immunohistochemically, widespread expression of synaptophysin was present in the undifferentiated PNET-like cells. The pigmented cells were immunoreactive for HMB45 and cytokeratin. The rhabdomyoblastic and also (a considerable percentage of) the small PNET-like cells expressed desmin and skeletal muscle actin. Immunohistochemical nuclear expression of SMARCB1 was retained, thus excluding an AT/RT. The patient was treated according to the MET-HIT 2000-BIS4 protocol, but showed tumor progression after 6 months and died 8 months postoperatively. Sequencing and FISH analyses did neither detect a CTNNB1 mutation nor MYCC/MYCN amplification. A pangenomic SNP array showed partial gain of chromosome 1q and 17q, monosomy of chromosome 8 and focal loss of chromosome 21q. Differential diagnostic considerations include 1) medulloblastoma with an unusual multilineal differentiation, 2) unusual distinct primitive neuroepithelial tumor,3) immature teratoma. Although, the histopathological features suggested a teratoid tumor, the third (endo/mesodermal) cell lineage required for this diagnosis was not present. The combination of rhabdomyoblastic and melanotic differentiation in medulloblastoma is known but exceptional and additional cartilage has only been reported in two cases. Regarding the molecular profile this tumor could be compatible with a type C or D molecular subtype, initial metastatic disease like in our tumor is characteristic for type C tumors. In conclusion, we consider this tumor rather as a medulloblastoma, but it remains to be clarified whether tumors with multilineal differentiation and aggressive biological behavior fall into one of the known molecular subgroups or represent a distinct variant.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-79. THE POTENTIAL ONCOGENIC SIGNIFICANCE OF WIP1 IN MEDULLOBLASTOMA

Jelena Milosevic 1, Ninib Baryawno 1, Baldur Sveinbjörnsson 2, Tommy Martinsson 3, Michael Grotzer 4, John Inge Johnsen 1, Per Kogner 1

Abstract

BACKGROUND: The most common cytogenetic lesions affecting medulloblastoma (MB) are the gain of 17q and i17q, consisting of 17p deletion with duplication of 17q, in approximately one-third of cases. In MB, gain and amplification of wild-type p53-induced phosphatase 1 (Wip1) is prevalent in the tumors with 17q gain. Wip1 is a serine/threonine phosphatase linked to the regulation of cellular stress responses. Wip1 was described as a gatekeeper in the Mdm2-p53 regulatory loop promoting Mdm2-mediated proteolysis of p53. METHODS: Comparative genomic hybridization (CGH) was used to analyze 31 MB tumors and 11 MB cell lines. MB cells were examined for Wip1 expression using immunoblotting, immunohistochemistry and qPCR. Stable Wip1 knock-down cell lines were generated by shRNA transfections. Histone H2AX phosphorylation of Wip1 knock-down was examined using flow cytometry. RESULTS: CGH analysis revealed that Wip1 is present in at least one extra genomic copy in the majority of primary MB, and in all 19 samples containing 17q gain. Results from our MB cell lines demonstrate 17q aberrations. These findings have been confirmed by analyzing the expression pattern of Wip1. Moreover, mRNA levels of Wip1 determined by qPCR correlate to Wip1 protein expression. Transfection experiments with shRNA against Wip1 showed substantial decrease in cell viability and cell growth in MB cell lines. D283-MED and D324-MED showed a substantial increase of gamma-H2AX expression after Wip1 knock-down compared to the non silencing transfection. CONCLUSIONS: Our study indicates an important oncogenic role of Wip1 in MB development and a potential therapeutic target. Our results showed that Wip1 is present in at least one extra genomic copy in the majority of MB tumors and cell lines. Wip1 knocked cell lines exhibited decreased cell growth compared to non-silenced cells. Moreover, knock-down of Wip1 induced phosphorylation of histone H2AX indicating a role of Wip1 in the DNA damage response.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-80. FUNCTIONAL GENOMICS OF METASTATIC RECURRENT MEDULLOBLASTOMA

Livia Garzia 1, Sorana Morrisy 1, Salomeh Jelveh 3, Patricia Lindsay 2, Richard Hill 3, Michael Taylor 1

Abstract

Medulloblastoma (MB) is the most common paediatric malignant brain tumor. By the way of optimal surgery, radiation, and chemotherapy, medulloblastoma can be treated in a good fraction of children but despite the best therapy the disease recurs in 40% of the cases. Our laboratory developed a novel murine model of MB with leptomeningeal spread which is highly penetrant, has a short latency, and involves random secondary genetic events. The model is based on mobilizing the Sleeping Beauty transposon in the cerebella of Ptch + /- mice. We used this well characterized model as a translational functional genomics tool to identify the genes and pathways important for radiation resistance and recurrence of MB in vitro and in vivo. We established proliferative cultures of murine MB primary cells and treated them with different doses of ionizing radiation (IR). The resistant cells were deep sequenced on the Ilumina platform to determine the sites of transposon insertion. Preliminary results indicate that the clonal profiles of IR treated cells differ from the sham treated counterpart. Many factors composing the physiological context around tumor cells can also influence radiosensitivity. For this reason we also developed a protocol to study the genetic differences between primary and recurrent (radioresistant) tumors in vivo, using our SB mouse model. We perform sub-total surgical removal of the tumor, and then treated the mice by multi-fractionated CT-guided craniospinal irradiation. About 70% of the mice treated with surgery and CSI recur, we have sequenced the first batch of recurrent mice, identifying several genes known to play a role in cell-cycle, apoptosis and radiation resistance, such as Trp53, as potential drivers of Medulloblatoma resistance. Our screen has identified novel targets for radiation sensitization that could be useful to prevent recurrence of medulloblastoma after radiation, and which should be tested in the context of a clinical trial.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-81. CXCR7 AND PEDIATRIC MEDULLOBLASTOMA

Asher Marks 1, Huizhen Zhang 1, Brian Rood 1

Abstract

BACKGROUND: Recently, CXCR4, a receptor of ligand CXCL12, has been shown to be a putative therapeutic target for the treatment of medulloblastoma. In addition, chemokine receptor 7 (CXCR7), also a receptor of CXCL12, has been of increasing interest due to its reported contribution to cell proliferation, survival, and migration. CXCR7 and CXCR4 have been shown to interact through CXCL12 in various physiologic and pathologic pathways. In an effort to better define the relationship between CXCR7, CXCR4, and CXCL12 in medulloblastoma, we set out to further elucidate the role CXCR7 plays in the pathogenesis of pediatric medulloblastoma. METHODS: Gene and protein expression of CXCR7/CXCR7 and CXCR4/CXCR4 was quantified in three medulloblastoma cell lines through the use of qRT-PCR and western blot. Twenty-six clinical samples of medulloblastoma were studied via immunohistochemistry. RESULTS: qRT-PCR demonstrated that mRNA expression of CXCR4 and CXCR7 in medulloblastoma cell lines is inversely related, although this correlation is not maintained in their protein expression. Western blot studies found a notable absence of CXCR7 protein in medulloblastoma cell lines in contrast to normal cerebellum. Immunohistochemistry revealed similar results with appreciable levels of CXCR4 expression and minimal expression of CXCR7 in clinical FFPE samples. CONCLUSION: These studies identify a significant decrease of CXCR7 protein in the context of an increase of CXCR4 protein in medulloblastoma. Previous studies of the CXCR7/CXCR4 system have suggested that CXCR7 acts as a modifier of CXCR4 activity through the binding of CXCL12. In the presence of CXCR4 over-expression, this competitive balance may be lost, contributing to the malignant phenotype of CXCR4-rich tumors. To better describe the regulatory relationship between CXCR4 and CXCR7, our future work will focus on knocking down CXCR4 at the expression level and inhibiting it at the protein level to test whether its absence affects the transcription/translation of CXCR7.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-82. A SIGNIFICANT PROPORTION OF MEDULLOBLASTOMA DO NOT SUB-GROUP ROBUSTLY: IDENTIFICATION OF A MOLECULAR NOS (NOT OTHERWISE SPECIFIED) SUBGROUP

Daniel Williamson 1, Steve Clifford 1

Abstract

There is a broad consensus that medulloblastoma can be grouped into molecular subgroups including WNT, SHH and at least two further subgroups. Future clinical and biological studies will need to sub-classify medulloblastoma accurately and uniformly in order to contextualise new findings. We have re-examined the biological subgroups within medulloblastoma using all available published molecular profiling data in order to reach a consensus about how many expression sub-groups exist and which genes most robustly define them. We designed a novel, unbiased method which emphasises the robustness of classification and selects only features for which there is maximal evidence across five independent profiling studies. Briefly, metagenes, reflecting key expression features are extracted from one study. Following thousands of resamplings these metagenes are validated by projecting the results upon every other study in turn. Thus we can measure the inter-study robustness of sub-group classification and select only those genes with consistent evidence of sub-group specificity across all published studies. We further integrated expression, genomic and clinicopathological data to reveal four robust patient sub-groups defined by five discrete biological effects with unique clinical features. Our study highlights the dangers of overfitting and choosing a limited set of genes or antibodies to sub-classify medulloblastoma; several previously proposed sub-type specific genes lack cross-study robustness. Most importantly, we demonstrate that there exists a disparate but significant proportion (∼5-10%) of medulloblastoma which cannot be confidently classified into any one subgroup on the basis of current methods. There are no obvious underlying pathological or sample quality anomalies in these unclassifiable tumours. Our results suggest that any antibody or gene classifier scheme proposed for uniform sub-grouping should carefully consider and validate the robustness of gene selection across independent studies. Furthermore, our findings indicate that any scheme for sub-grouping medulloblastoma should incorporate molecular NOS as a necessary and legitimate sub-group.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-83. STUDY OF BIOLOGICAL FACTORS OF SPANISH CHILDHOOD MEDULLOBLASTOMAS, FOR FUTURE THERAPEUTIC STRATIFICATION

Olaia Aurtenetxe 1, Ayman Gaffar 1, Jose Ignacio Lopez 1, Alejandro Urberuaga 1, Aurora Navajas 1

Abstract

INTRODUCTION: The Spanish participation in SIOP collaborative protocols, and consequently, the centralization of biological studies designed for Standard Risk childhood medulloblastoma (MB), in future protocols, made necessary the development of techniques in the reference hospital (Cruces University Hospital). We have studied retrospectively a non-uniformly treated cohort using combined clinical, pathologic and molecular variables. METHODS: Paraffin-embedded (FFPE) tissue samples from 49 pediatric patients submitted from 13 Spanish hospitals were analyzed using fluorescent in situ hybridization (FISH) to study the expression of MYCC and MYCN using Vysis specific probes and immunohistochemistry with a SIOP-E BTG operative protocol, standarized for β-catenin. Detailed clinical data were available for all these patients. RESULTS: Mean age at diagnosis was six years (1-16). Classic MB dominated the study, contributing 58% of all tumors, followed by Nodular/Desmoplastic (ND) MB which contributed 32% of the cohort and finally, Large cell/Anaplastic (LCA) MB (10%). A combination of metastatic disease and LCA phenotype were the clinicopathologic variables associated with poorer overall survival. All MYCC and MYCN amplified tumors, within classic and LCA histological subtypes were metastatic tumors and were associated to poor outcome, except one patient with localized classic MB and MYCN amplification that remains alive. MYCN and MYCC amplification was not found in ND tumors. Survival for this group was 75%, 67% for classic type and less than 20% for LCA tumors. β-catenin nuclear expression only was found in two localized tumors. COMMENTS: High Risk disease has been defined by clinical findings (M+ disease), pathological subtypes (LCA variant) and in recent years molecular markers (MYCC and MYCN amplification). Our results in a cohort of medulloblastomas support thiscriteria, although we were not able to validate the prognostic role of β-catenin nuclear expression in this study that could be different with the increased number of samples that are entering the study.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-84. OUTCOMES OF CHILDREN TREATED WITH A CHEMOTHERAPY-ONLY INFANT PROTOCOL AT BRITISH COLUMBIA'S CHILDREN'S HOSPITAL

Katrina O'Halloran 3, Juliette Hukin 1, Ash Singhal 1, Chris Dunham 1, Karen Goddard 2, Shahrad R Rassekh 1

Abstract

BACKGROUND: Conventional therapy in children with malignant brain tumors includes surgery, chemotherapy and radiation therapy and is 70-80% curative. However, radiation severely damages a child's developing brain resulting in catastrophic cognitive outcomes. Infant protocols deliver surgery and intensive chemotherapy with an aim to avoid or delay brain irradiation in children. The published literature suggests that this approach is 55-65% curative. BC Children's Hospital was an early adopter of using this approach and also included children over the age of 3 on these protocols. Recent studies have shown that medulloblastoma can be classified into four sub-groups which are prognostic when using chemotherapy with adjuvant radiation. However, the prognosis of these four groups in those treated with chemotherapy alone is unknown. METHODS: A retrospective chart review of 38 patients with malignant brain tumors treated according to a chemotherapy only protocol was performed investigating survival and late sequelae of therapy, including hearing loss. Neurocognitive data was also obtained where available. Archived tumor specimens are currently being analyzed in order to determine the molecular subgroups of each tumor in the cohort. RESULTS: 38 patients were identified of which 32% had metastatic disease at time of presentation. Event free survival was 29%, with an overall survival of 55%. The best results were seen in non-metastatic medulloblastoma with EFS of 54% and OS of 69%. The main toxicity is hearing loss. Complete audiograms were done in 32 patients with 31 showing evidence of at least Grade 1 toxicity. CONCLUSIONS: Our results are comparable with the published literature with regards to survival using a chemotherapy only approach. Further work will report on neurocognitive outcomes and will also investigate possible molecular markers to predict successful outcomes using chemotherapy alone. Some degree of hearing loss is seen in almost all subjects.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-85. META-ANALYSIS OF HEAD START (HS) I, II AND III: OUTCOME OF CHILDREN WITH NEWLY DIAGNOSED CENTRAL NERVOUS SYSTEM (CNS) PRIMITIVE NEUROECTODERMAL TUMORS (PNET)

Tom B Davidson 1, Jason R Fangusaro 4, Lingyun Ji 2, Richard Sposto 2, Sharon L Gardner 3, Jeffrey C Allen 3, Ira J Dunkel 5, Girish Dhall 2, Jonathan L Finlay 2

Abstract

Outcomes for children with CNS-PNET remain poor. In order to improve overall survival (OS), event-free survival (EFS) and to decrease irradiation (RT) exposure in children with newly diagnosed CNS-PNET, the HS consortium investigated the use of intensified induction chemotherapy followed by consolidation with myeloablative chemotherapy and autologous hematopoietic cell rescue (AuHCR). Between 1991 and 2009, 95 children with CNS-PNET were enrolled among 22 institutions on 3 serial studies (HS I, II, III). Patients on HS I and patients with localized disease on HS II were to receive 5 induction cycles (vincristine, cisplatin, cyclophosphamide, etoposide). Patients on HSII with disseminated disease were to receive high-dose methotrexate during induction. Patient on HSIII were to receive vincristine, cisplatin, cyclophosphamide, etoposide, high-dose methotrexate in cycles 1, 3, and 5; vincristine, cyclophosphamide, oral etoposide, temozolomide in cycles 2 and 4. In HS I-III, induction was followed by consolidation (thiotepa, carboplatin, etoposide) with AuHCR. Patients with residual tumor after induction and patients >6 years were to receive RT. The 5-year EFS and OS rates (±SE) for patients in the three protocols were 34 ± 5% and 40% ± 5%, respectively. The 5-year EFS and OS (±SE) for the 3 subtypes, non-pineal supratentorial PNET, pineal PNET and other PNET were 48 ± 10% and 52 ± 10%, 9 ± 6% and 13 ± 7%, 39 ± 7% and 48 ± 7% (p = 0.017, p = 0.005). The 5-year EFS and OS (±SE) for patients with localized and metastatic disease were 39 ± 6% and 45 ± 7% versus 25 ± 8% and 28 ± 8% (p = 0.042, p = 0.042). Using multivariate Cox regression models, disease subtype was the only significant predictor of EFS and OS. Extent of resection, metastatic status, age and sex were not significant predictors of outcome. 33 ± 6% of children survived 5-year event-free without irradiation. The present meta-analysis represents the largest series of young children with CNS-PNET. Innovative strategies need to be incorporated to further improve the outcome of these children.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-86. TEN YEARS OF PAEDIATRIC POSTERIOR FOSSA TUMOURS, THE LEEDS EXPERIENCE

M Trivedi 1, A Tyagi 1, J Goodden 1, P Chumas 1, R O'kane 1, D Crimmins 1, M Elliott 1, S Picton 1

Abstract

INTRODUCTION: Posterior fossa tumours in paediatric age group remains an important topic in terms of surgical resection, morbidity and debate over reasons for residual tumour. METHOD: Patients aged 0-18 years were identified from the neuro-oncology database, from January 2001 to December 2010. Retrospective data was collected for age, sex, tumour location, histopathology, surgical resection, residual tumour, and post-operative morbidity and mortality. RESULTS: Eighty patients were identified - 36 pilocytic astrocytoma (45%), 30 medulloblastoma (37.5%) and 4 ependymoma (5%), 3 diffuse astrocytoma (3.8%), 2 teratoma (2.5%), and single cases (1.25% each) of anaplastic astrocytoma, pilomyxoid astrocytoma, ganglioglioma, gangliocytoma, and glioblastoma multiforme. Mean age 7.9 years (range 0.2-17.8 years). Male: Female ratio 1.7:1. Seventy were cerebellar & fourth ventricular (87.5%), 4 were brainstem (5%), 4 were cervicomedullary tumours (5%), and 2 were CP-angle tumours (2.5%). There was one biopsy of a brainstem tumour (1.25%). At first surgery, gross total resection was achieved in 62 (77.5%) patients, while 15 (18.8%) had incomplete resection. Six patients (7.5%) had second-look surgery, with complete resection in four cases. Two patients had insufficient notes. Fourteen cases had incomplete resection due to either cardiovascular instability during surgery or because tumour was adherent to or invading the brainstem / cranial nerves / vessels. Treatment for disease recurrence is examined and correlated to tumour location and type. 30-day mortality was Zero in this series. However, 12 patients (15%) have succumbed to their tumour. Cerebellar mutism occurred in six patients (7.5%). CONCLUSION: Complete surgical resection is the main aim for posterior fossa tumours. This can sometimes be challenging to achieve due to tumour location with tumour being left in-situ due to cardiovascular instability or brainstem invasion. Where unexpected tumour remnant is identified on post-operative MRI scan, intra-operative imaging might have been able to improve resection rates.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-87. STUDY OF MSI PHENOTYPE AND NOVEL TARGET GENES ASSOCIATED WITH MSI PHENOTYPE IN MEDULLOBLASTOMAS

Dina Sofia Silva 1, Marta Viana-Pereira 1, João N Stavale 2, Susana Malheiro 2, Gisele Caravina Almeida 3, Carlos Clara 4, Chris Jones 5, Rui M Reis 1,6

Abstract

Brain tumours stand as the second most frequent malignancy in paediatric patients, and medulloblastomas account for the most frequent malignant type of brain tumours in children. Recently, we showed that microsatellite instability (MSI) - a particular type of genetic instability that is characterized by frameshift mutations in microsatellite regions along the genome - occurs in a subset of medulloblastomas (11%, in a set of 36 samples) patients. Yet, further validation studies are required to confirm our previous findings. Therefore, in this work our aim is to extend the study of MSI in a larger set of medulloblastomas and to study the potential target genes in MSI positive cases. We have analysed a series of 62 medulloblastomas regarding their MSI status and found 14,5% (9/62) of our cases exhibiting a MSI-Low phenotype. In those MSI positive cases, we screened for alterations in several MSI target genes, including, DNAPkcs, BRCA1, BRCA2, WISP3, BLM, PTENex7, PTENex8, ATM, RAD50, MSH6, BAX, ATR, TCF4, XRCC2, MBD4, MRE11, MSH3, TGFBR2, EGFR, HSP110, GLYR1, ABCC5, ROCK1, XPO5 and TARBP2. With the exception of a MRE11 polymorphism, no other mutation was found. In conclusion, these data confirms that MSI is present in a subset of medulloblastomas. The absence of mutations in potential target genes suggests that further studies are needed to explore the true target genes of MSI phenotype in brain tumours and, in particular, to address the biological meaning of MSI in medulloblastomas.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-88. FUNCTIONAL SYNERGY OF THE ONCOGENIC C19MC miRNAs

Tara Spence 1,2, Patrick Sin-Chan 1,2, Daniel Picard 1,2, King Ching Ho 1, Mei Lu 1, Annie Huang 1,2

Abstract

CNS-PNETs are highly aggressive tumours with poorly defined biology. In prior work, we discovered a novel oncogenic miRNA cluster that spans 54 miRNAs, on chr19q13.41 (C19MC), in a distinct sub-group of CNS-PNET. Substantial studies have now documented the universal fatality of C19MC amplified tumours. In order to advance therapeutics for these devastating tumours, it will be important to define the oncogenic mechanisms of the C19MC oncomiRs. MicroRNAs have been shown to be important mediators of cellular transformation; however, studies to date indicate that most miRNAs individually mediate modest cellular and molecular changes in the transformed cell. We previously reported that 5 of the C19MC miRNAs (miR-520g, 517c, 517a, 519a2 and 512-3p) were specifically upregulated in C19MC amplified tumours. Informatic analyses suggest that the 5 oncomiRs may target common critical cell cycle regulatory and neural differentiation pathways. We hypothesize that C19MC oncogenic effects may be primarily mediated via synergistic effects of these 5 C19MC oncomiRs on critical cellular transformation pathways. In our prior studies we established that miR-520g and 517c have oncogenic activity in vitro and in vivo, and inhibit differentiation of hNSCs. We also observed that co-expression of miR-520g and 517c synergistically enhances cell transformation in vitro. We have now examined the cellular effects of miR-517a, 519a2 and 512-3p and show they individually also enhance cellular transformation. We further demonstrate using a 5 miRNA expression construct that co-expression of the 5 C19MC oncomiRs have additive effects on cell transformation in vitro. Using reporter assays and immuno-blot analyses, we demonstrate that the 5 oncomiRs synergistically regulate key cell cycle regulatory genes, and alter neural differentiation. Collectively, our findings underscore C19MC as a potent oncogenic locus with important roles in CNS-PNET pathogenesis.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-89. PRE-CLINICAL STUDY OF HISTONE METHYL TRANSFERASE INHIBITOR IN PEDIATRIC BRAIN TUMORS

Sunil Bochare 1, Soumen Khatua 1, Vidya Gopalakrishnan 1

Abstract

Medulloblastoma (MB) and atypical teratoid rhabdoid tumor (ATRT) are malignant pediatric brain tumors. Survival in children with standard-risk MB and older children with ATRT is around 70%. However, survivors face long-term neurocognitive sequelae, underscoring the need for novel targeted therapies. Studies have implicated enzymes controlling histone methylation in the pathogenesis of MBs. ATRTs often exhibit loss of the chromatin remodeler INI1. Its loss in other cells promotes increased recruitment of the histone methyl transferase G9a to chromatin and an elevation in global histone methylation. Since G9a-mediated histone methylation represses the expression of genes involved in cell proliferation and apoptosis, we hypothesized that its pharmacological inhibition will block MB and ATRT growth. OBJECTIVE: To test the effect of G9a inhibition on MB and ATRT growth in-vitro and in-vivo. DESIGN/METHOD: Established MB and ATRT cell-lines and primary tumor-derived cultures were queried for G9a expression by Q-RT-PCR. We performed a dose-response study over time with the G9a inhibitor BIX-01294. Cell growth was assessed by MTT assays and IC50 values established. Cell cycle progression was examined by flow-cytometry and loss of G9a activity measured by immunofluorescence assays (IFA) and Western blotting. Effect of drug treatment on tumorigenic potential was assessed by soft agar assays and in mouse orthotopic models. RESULTS: G9a expression was elevated in tumors compared to normal cerebellum. BIX-01294 treatment decreased MB and ATRT growth in-vitro. The IC50 was found to be 3-6mM and 2mM for MB and ATRT cells respectively at 48-hrs. Flow cytometry revealed an increase in cells with sub-G1 DNA content. Loss of histone H3-Lysine-(K)-9 dimethylation was confirmed by Western blotting and IFA. Results of anchorage independent growth and in-vivo assays will be presented. CONCLUSION: In pre-clinical studies, inhibition of G9a blocked growth of MB and ATRT cells. Gene expression profiling will provide information on the underlying molecular mechanisms.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-90. THROMBOSPONDIN 1 - A CANDIDATE THERAPEUTIC TARGET IN MEDULLOBLASTOMA

Tiffany SY Chan 1, Daniel Picard 1, Stefan Pfister 3, Cynthia Hawkins 1, Annie Huang 1

Abstract

MYCC amplification/activation is one of the strongest predictor of poor survival in Medulloblastoma (MB), the most common pediatric malignant brain tumor. Yet how MYCC confers aggressive and metastatic MB phenotypes remains poorly understood. In prior ChIP-chip analysis, we demonstrated that Myc profoundly altered cell differentiation and migration programs in MB. Furthermore, we discovered thrombospondin-1 (TSP-1), a potent anti-angiogenic tumour suppressor, is robustly repressed by Myc via direct and oncoMiR-17 ∼ 92-mediated post-transcriptional mechanisms. In recent studies we sought to define the therapeutic roles of TSP-1 in Myc-induced MB. We observed, using orthotopic xenograft assay, that TSP-1 expression prolongs survival (p < 0.02, n = 11) and correlates with significantly diminished metastases (p < 0.005). Using micro-vessel density assays, we showed that TSP expression does not correlate with tumour angiogenesis in both primary MB (n = 80, p > 0.3) and MB xenografts (n = 10, p > 0.5), thus anti-tumour effect of TSP-1 appears to be independent of its potent anti-angiogenic properties. Interestingly, we observed that TSP-1 reconstitution sensitizes MB cells to Etoposide (∼5-fold decrease in IC25; p < 0.05) and radiation (∼2-fold decrease in IC25; p < 0.05). Importantly, we demonstrate that ABT-898, a pharmological peptide mimetic of TSP-1, can recapitulate the effects of TSP-1 expression. In MB cells, ABT-898 treatment resulted potent inhibition of MB cell migration (p < 0.05), and increased sensitivity of MB cells to Etoposide and radiation (p < 0.05). Our collective findings further underscore TSP-1 as a critical Myc target in MB and highlight synthetic TSP-1 peptide mimetics as potential novel therapeutics in MB.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-91. Myc INTERACTOR- JPO2 PROMOTES MEDULLOBLASTOMA CELL MIGRATION AND INVASION VIA AKT SIGNALING

Tiffany SY Chan 1, Daniel Picard 1, King Ching Ho 1, Annie Huang 1

Abstract

MYCC amplification/activation is one of the strongest predictor of poor survival in medulloblastoma (MB), however mechanism by which MYCC confers aggressive and metastatic MB phenotypes remains poorly understood. In prior studies we identified JPO2 as a novel Myc-interacting and co-transforming protein that is frequently up-regulated in metastatic MB. Recently, we demonstrated that JPO2 enhances Myc transformation in part by acting as a transcriptional co-repressor of thrombospondin-1 (TSP-1), a critical Myc-silenced anti-migratory/metastatic tumour suppressor in MB cells. Furthemore, studies in our lab demonstrated that JPO2 has Myc-independent oncogenic activity in vitro and in vivo. In this study we sought to further characterize the mechanism underlying JPO2 transforming activity on MB cells. We demonstrate using JPO2 over-expression and knockdown, that JPO2 have limited effects on cell proliferation, and cell response to various death triggers including serum starvation and chemotherapeutic agents (VP-16, 5'FU, Docetaxel). Significantly we observed that JPO2 robustly enhances cell migration/invasion of several MB cell lines (UW228, UW426 and DAOY cells; p < 0.01) while JPO2 knockdown significantly diminished cell migration /invasion in D283, D341 and ONS76 MB cells (p < 0.05). We demonstrate that JPO2 induced MB cell migration correlates with increased AKT activation and can be reversed by phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002. Interestingly, anti-migratory effect of LY294002 was accompanied by decreased JPO2 protein expression thus indicating JPO2 acts via a PI3K associated feed-forward signaling loop to promote MB migratory phenotypes. As PI3K/AKT signaling is also known to have a critical role in Myc tumourigenesis, our findings suggest that JPO2 may promote MB tumourigenesis by enhancing the Myc:AKT signaling axis in MB cells.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-92. MARKERS OF CELL LINEAGE CORRELATE WITH SURVIVAL AND METASTATIC POTENTIAL IN CNS-PNET

Daniel Picard 1, Suzanne Millar 2, Cynthia Hawkins 1, Hazel Rogers 2, Seoung Ki Kim 3, Young Shin Ra 4, Jason Fangusaro 5, Helen Toledano 6, Hideo Nakamura 7, Timothy Van Meter 17, Scott Pomeroy 11, H K Ng 12, Chris Jones 13, Amar Gajjar 14, Steve Clifford 8, Stefan Pfister 15, Charles Eberhart 16, Eric Bouffet 1, Richard Grundy 2, Annie Huang 1

Abstract

CNS-PNETs are lethal brain tumours for which molecular features remain poorly defined. We comprehensively analysed a large cohort of 125 primary CNS-PNETs to identify molecular markers for clinically relevant tumour sub-groups. Tumour transcriptional and copy number profiles were examined using clustering, gene and pathway enrichment analyses to discover sub-groups and define group-specific molecular features in relation to clinical phenotypes. Unsupervised cluster analyses of expression profiles indicated CNS-PNETs comprised 3 molecular sub-groups with differential enrichment of lineage and developmental signaling genes. Group 1 tumours displayed primitive neural lineage and activated WNT signature, group 2 tumour profiles were enriched for oligo-neural lineage genes while group 3 tumours were enriched for mesenchymal lineage genes and activated TGF-b signaling. Tumour sub-groups differed in: gender (p = 0.03), age at diagnosis (p = 0.05), length of survival (p = 0.02) and incidence of metastases (p = 0.03). Group 1 comprised predominantly females (62%) with bimodal age peaks, however a majority (77%) were < 48 months at diagnosis. Group 2 tumours correlated with older age of presentation with 72% presenting at >48months without gender pre-dilection, while group 3 tumours primarily presented in children between 48-96 months of age and occurred more frequently in males (63%). Survival analysis across all ages indicated group 1 tumours with primitive neural lineage expression signatures fared the worst with a median survival of 11months. Group 3 tumours had the highest incidence of metastases - 53% vs 26% and 15% in group1 and 2 respectively. Interestingly, despite frequent metastases, survival analysis showed group 3 tumours had the best overall survival (35-40% OS) when analyses was restricted to patients >48months of age to correct for potential treatment biases in younger children. Collectively, our findings provide one of the most comprehensive insights into the molecular make-up of CNS-PNET, and identify promising diagnostic and prognostic markers for CNS-PNET.

Neuro Oncol. 2012 Jun;14(Suppl 1):i82–i105.

MB-93. LEVERAGING EXPRESSION OF A5/GABA-A RECEPTOR IN MEDULLOBLASTOMA AS A NOVEL THERAPEUTIC TARGET

Soma Sengupta 1, Shyamal Dilhan Weeraratne 1, Jillian Phallen 2, Hongyu Sun 1, Sundari Rallapalli 3, Vladimir Amani 1, Jessica Pierre-Francois 1, Natalia Teider 1, James Cook 3, Frances Jensen 1, Michael Lim 2, Scott Pomeroy 1, Yoon-Jae Cho 4

Abstract

Neural tumors often display molecular markers suggestive of their developmental lineage, including the expression of neurotransmitter receptors. Although neurotransmitter receptors have been well-characterized in normal neurophysiological, developmental and pharmacological settings, their importance in the maintenance and progression of brain tumors, and importantly, the effect of their ‘targeting’ in brain cancers remains obscure. We previously demonstrated high levels of GABRA5 expression in a particularly aggressive molecular subtype of medulloblastoma. GABRA5 encodes for the A5-subunit of the GABA-A receptor complex and we hypothesized that modulation of its activity would alter medulloblastoma cell survival. We treated GABRA5 expressing medulloblastoma cells with a series of GABA-A receptor agonists and antagonists and then assayed for proliferation, cell cycle distribution, apoptosis, and changes in electrophysiological properties, in vitro and in/ex vivo. While GABA-A receptor antagonists, such as picrotoxin, had limited effects on cell survival, QHii066, a highly specific A5/GABA-A receptor agonist, significantly decreased medulloblastoma cell survival through the induction of apoptosis. Furthermore, QHii066 markedly sensitized GABRA5 positive medulloblastoma cells to radiation and chemotherapy, in vitro and in vivo. Thus, our results provide a novel strategy for the treatment and management of a highly lethal subtype of medulloblastoma, and provide a paradigm to approach the treatment of other tumors of neural origin.

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

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