Large Cell/anaplastic Research Articles

Molecular Subgrouping Based on Immunohistochemistry in Medulloblastoma: A Single-Center Experience.

To investigate the efficacy of immunohistochemical methods to determine molecular subgroups and prognostic predictions of medulloblastomas (MBs). β-catenin, GAB1, YAP1, filamin A and p53 were immunohistochemically stained, and MYC and MYCN fluorescent in situ hybridization (FISH) procedures were applied to 218 cases in our series. Based on the histomorphological characteristics of the cases, 67.9% were deemed classic MB; 15.6% as desmoplastic/ nodular medulloblastoma (DNMB); 12.8% as large cell/anaplastic (LC/A) MB; 3.7% as medulloblastoma with extensive nodularity (MBEN). Molecular characteristics revealed that 50.5% had non-WNT/non-SHH; 33.9% had SHH-activated and TP53-wildtype; 8.7% had WNT-activated; 6.9% had SHH-activated and TP53-mutant. According to the survival curves, LC/A MBs or non-WNT/ non-SHH tumors showed the worst prognosis, whereas DNMBs and WNT-activated tumors showed the best prognosis. Classic MBs or SHH-activated tumors showed a moderate course. MYCN amplification was found to act as an independent poor prognostic factor in the study. The distribution of histological subtypes and molecular subgroups, amplification rates, and prognostic data obtained through immunohistochemical methods in our study were consistent with those reported in the literature. It was therefore hypothesized that the determination of molecular subgroups by immunohistochemical methods can be useful in daily diagnostic practice, especially in centers with limited access to molecular techniques.

MDB-25. LARGE CELL/ANAPLASTIC MEDULLOBLASTOMA REPRESENTS MORE THAN ONE DISEASE ENTITY

Abstract Medulloblastoma (MB) with predominant large cell or anaplastic cytological features have been identified to be associated to an aggressive clinical course and poor survival. Because cases can show mixed cytologies, these histologically defined MB types were merged in the recent WHO classification of tumors of the CNS to large cell/anaplastic (LC/A) MB. In this study, we collected FFPE samples of 137 LC/A MB of all age groups (0-39 years) diagnosed at the DGNN Brain Tumor Reference Center in Bonn, Germany, and performed a comprehensive histological, immunophenotypical, genetic and epigenetic analysis by next-generation panel sequencing (NGS), 450k/850k methylation bead-array hybridization and molecular inversion probe (MIP) technology. 73% of cases showed predominant anaplastic histology, 27% predominant large cell histology. Methylation-based subtyping and sequencing showed that the cohort of LC/A MB represented different biological entities: 6% WNT-MB, 7% SHH-MB-TP53-wildtype, 10% SHH-MB-TP53-mutant, and 77% non-WNT/non-SHH MB (with predominant epigenetic subtype II). High-resolution copy number profiling by MIP demonstrated MYC amplification in 40% of non-WNT/non-SHH MB. MYC amplification was highly associated to infant disease and predominant large cell histology. On the contrary, predominant anaplastic histology was highly associated to non-infant disease, MYCN amplification and SHH activation. By NGS we detected recurrent mutations in 37 MB-related genes; the frequency of mutations of individual MB-related genes differed significantly between the histologically and genetically defined MB types. Our data clearly demonstrate that large cell MB and anaplastic MB represent different biological entities. Therefore, and also in respect to the future development of targeted treatment approaches to these highly malignant MB types, they should be precisely diagnosed as separate MB entities.

MDB-45. SELECTIVE B7-H3 INHIBITORS NI1 AND NI4 REDUCE AGGRESSIVENESS OF GROUP 3 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is a primary childhood malignancy of the central nervous system (CNS). Clinically heterogenous MB are sub-grouped into four molecular subgroups, i.e. Wingless (WNT), Sonic Hedgehog (SHH), Group 3, and Group 4. Among all, Group 3 MB (G3MB) is known to be the most aggressive form, with less than 50% five-year survival. G3MB are associated with large-cell/anaplastic (LCA) histology, distinctive methylation profile, amplification of MYC, and isochromosome 17q (i17q). Targeting these high-risk molecular features unique to G3MB has been challenging, necessitating alternative approaches to therapy for G3-MB. One innovative approach is immunotherapy, which is largely impeded by inhibitory immune checkpoints. B7 homolog 3 (B7-H3/CD276), an immunosuppressive immune checkpoint protein and member of B7 superfamily, is significantly enriched in pediatric malignancies of CNS including G3MB. The enhanced expression of B7-H3/CD276 has been associated with aggressive and metastatic potential. B7-H3/CD276 is also a primary target of miR-1253, a novel tumor suppressor gene silenced in G3MB. Using a structure-based drug discovery pipeline, we screened the ChemBridge library of compounds to discover selective B7-H3 small-molecule inhibitors. Selective B7-H3 inhibitors Ni1 and Ni4 exhibited strong binding scores, CNS penetrance, and oral bioavailability. In vitro, both compounds had appreciably low IC50 values of 3µM (Ni1) and 0.75µM (Ni4). Further, both compounds inhibited wound healing and colony formation. Ni1 shifted the thermostability of B7-H3, suggesting in vitro biding. Ni1 has also undergone PK/PD and MTD studies in vivo, showing stability in serum, good blood-brain barrier permeability, and a half-life of 8 hours. We are now elucidating B7-H3-activated downstream signaling through JAK2/STAT3 signaling and testing the in vivo efficacy for Ni1 against G3MB tumors in a murine model.

Prognostic value of microRNA-125a expression status in molecular groups of pediatric medulloblastoma

PurposeMedulloblastoma (MB) is the most common malignant pediatric brain tumor. Current treatment allows decent survival rates but often with life-long morbidity. Molecular classification provides a base for novel therapeutic approaches. However, these groups are heterogeneous. MicroRNA-125a has a tumor suppressor function. It is downregulated in several tumors. The expression of microRNA-125a in MB patients remains unclear. Therefore, this study was designed to evaluate the expression of microRNA-125a in molecular groups of pediatric MB patients in Egyptian population and its clinical significance.MethodsFormalin-fixed, paraffin-embedded tissue blocks from 50 pediatric MB patients were retrospectively collected. Immunohistochemistry for β-catenin, GAB1, YAP1, and p53 was done for molecular classification. MicroRNA-125a expression analysis was done using qRT-PCR. Follow-up data were obtained from patients’ records.ResultsMicroRNA-125a expression was significantly lower in MB patients showing large cell/anaplastic (LC/A) histology and in the non-WNT/non-SHH group. Lower levels of microRNA-125a showed a tendency toward poor survival rates; however, difference was not significant. Infants and larger preoperative tumor size were significantly associated with lower survival rates. On a multivariate analysis, preoperative tumor size was an independent prognostic factor.ConclusionMicroRNA-125a expression was significantly lower in categories of pediatric MB patients with worse prognosis namely LC/A histology and the non-WNT/non-SHH group suggesting a pathogenetic role. MicroRNA-125a expression could represent a promising prognostic factor and a potential therapeutic target in the non-WNT/non-SHH group which represents the most common and the most heterogeneous group of pediatric MBs coupled with the highest rates of disseminated disease. Preoperative tumor size represents an independent prognostic factor.

Clinicomorphological and molecular analysis of medulloblastoma and association with survival: A single tertiary care center experience.

Medulloblastoma (MB) is a heterogeneous disease, displaying distinct genetic profiles, with specific molecular subgroups. Various clinical, pathological and molecular variables have been associated with disease outcome and therefore utilised in risk stratification of patients. To perform molecular classification of medulloblastoma using surrogate immunohistochemistry (IHC) and associate molecular subgroups, histopathological types, and available clinicopathological parameters with overall survival (OS) of MB patients. This study included 65 medulloblastoma patients. Immunohistochemical staining, using β-catenin YAP1 and GRB2-Associated Binding Protein 1 (GAB1) antibodies was used to classify MB cases into wingless signalling (WNT) activated, sonic hedgehog (SHH) activated, and non-WNT/non-SHH molecular subgroups. The relevant statistical analysis was done using GraphPad Prism version 9.3.0. Histological patterns included classic (40 cases, 62%), desmoplastic nodular (D/N) (14 cases, 22%), large cell/anaplastic (LC/A) (9 cases, 13%), medulloblastoma with extensive nodularity (MBEN) (1 case, 1.5%) and one special subtype, i.e., medulloblastoma with myogenic and melanotic differentiation. Molecular subgroups included WNT (4 cases, 6%), SHH (34 cases, 52%), and non-WNT/non-SHH (27 cases, 42%) subgroups. Histopathological types differed significantly according to tumor location, degree of anaplasia and molecular subgroups. Molecular subgroups differed significantly in age distribution and tumor location. The probability of survival was 78% and 68% after 1 and 2 years, respectively. Infants (<3 years of age), LC/A pattern, and TP53-mutant status among SHH subgroup conferred poor prognosis in our study. At the end of the study (at 65 months of maximum follow-up period) probability of survival was 51%. Immunohistochemical analysis helps in molecular classification of medulloblastoma in majority of the cases as well as helps in predicting prognosis and treatment response.

MODL-02. A novelCre-conditionalcMYC-driven MB Group 3 transgenic mouse model shows traceable leptomeningeal dissemination.

Medulloblastoma (MB), the most common embryonal tumour of the Central Nervous System, occurs in the cerebellum. Treatment regimens involve surgery, craniospinal radiotherapy, and chemotherapy. The greatest mortality is associated with disseminated disease, almost exclusively found in the leptomeningeal space. Unfortunately, knowledge about the aetiology of MB spread is limited and the need for kinder and efficacious therapy remains an unmet goal. Of the four molecular classified MB groups, Group3 (Gr3) MB presents with a high frequency of metastasis at diagnosis, with the worst overall survival. Gr3 MB tumours are dominated by primitive progenitor-like cells and cMYC deregulation; often, p53 deficiency is observed at relapse. To dissect the biology of primary and metastatic Gr3 MB, we have developed a new germline genetically engineered mouse model (GEMM), harbouring cMYC amplification in a Tamoxifen-inducible p53 functional background (Trp53ERTAM strain). A novel LSL-cMYC-CopGFP-Luciferase transgene was integrated into the Rosa-26 locus of the mouse genome. Transgenic mice were crossed with a strain expressing Cre recombinase under the Blbp promoter targeting embryonic neural progenitors, and subsequently bred to Trp53ERTAM mice. As result, the cMYC overexpression was sufficient to generate tumours. Tumour penetrance was observed in all the expected tumour bearing genotypes, with increased aggressiveness in a non-functional p53 background. Bioluminescence imaging demonstrated tumour onset in the brain and dissemination along the spinal cord. CopGFP positive tumour cells were isolated from primary and metastatic tumours. Pathological interrogation confirmed that tumours present large cell/anaplastic (LCA) histology. Analysis of preliminary transcriptional profiling data proved that tumours cluster with human Gr3 MB. Ongoing methylation profiling and multi-omics approaches will inform on the tumour cells of origin and clonal divergence of primary tumour versus metastasis. In conclusion, we have successfully developed a novel immunocompetent mouse model of metastatic Gr3 MB with which we can investigate therapeutic vulnerabilities of MB.

MTORC1 promotes malignant large cell/anaplastic histology and is a targetable vulnerability in SHH-TP53 mutant medulloblastoma.

Medulloblastoma (MB), one of the most malignant brain tumors of childhood, comprises distinct molecular subgroups, with p53 mutant sonic hedgehog–activated (SHH-activated) MB patients having a very severe outcome that is associated with unfavorable histological large cell/anaplastic (LC/A) features. To identify the molecular underpinnings of this phenotype, we analyzed a large cohort of MB developing in p53-deficient Ptch+/– SHH mice that, unexpectedly, showed LC/A traits that correlated with mTORC1 hyperactivation. Mechanistically, mTORC1 hyperactivation was mediated by a decrease in the p53-dependent expression of mTORC1 negative regulator Tsc2. Ectopic mTORC1 activation in mouse MB cancer stem cells (CSCs) promoted the in vivo acquisition of LC/A features and increased malignancy; accordingly, mTORC1 inhibition in p53-mutant Ptch+/– SHH MB and CSC-derived MB resulted in reduced tumor burden and aggressiveness. Most remarkably, mTORC1 hyperactivation was detected only in p53-mutant SHH MB patient samples, and treatment with rapamycin of a human preclinical model phenocopying this subgroup decreased tumor growth and malignancy. Thus, mTORC1 may act as a specific druggable target for this subset of SHH MB, resulting in the implementation of a stringent risk stratification and in the potentially rapid translation of this precision medicine approach into the clinical setting.

TMOD-03. A NOVEL MB GR3 TRANSGENIC MOUSE MODEL IS GENERATED BY MYCN AND P53 DEFECTS IN VENTRICULAR ZONE PROGENITORS.

Abstract Medulloblastoma (MB) represents the most common embryonal tumour of the Central Nervous System in childhood. MB occurs in the cerebellum and molecular features dictate the classification into four subgroups. Although Group3 (Gr3) MB tumours are dominated by primitive progenitor-like cells, the cells of origin remain unidentified. Gr3 MB is associated with relatively common MYC family member amplification and overexpression, often combined with p53 pathway defects at relapse. Molecularly stratified treatment is not yet available, causing Gr3 MB and its subsequent relapse to often represent an unstoppable progressive disease. The limited understanding of Gr3 tumorigenesis and targeted therapy development is also due to the lack of faithful in vivo models and consequently, their use in preclinical studies. We have now developed a new germline genetically engineered mouse model (GEMM), harbouring MYCN amplification in a p53 inactive background (tamoxifen-inducible p53 activation, Trp53ERTAM). The purpose of the GEMM is to investigate the developmental significance of MYC aberration in putative Gr3 MB cells of origin and exploit it in preclinical studies. A LSL-MYCN-Luciferase strain was crossed with mice expressing Cre recombinase under the Blbp promoter and subsequently to Trp53ERTAM inducible mice. As result, the MYCN overexpression alone did not generate tumours, conversely to the combination of MYCN with p53 deregulation. Tumours arise exclusively in the hindbrain of homozygote mice, with a penetrance of 100% and a latency of ~135 days. Pathology report suggests tumours are Gr3 MB with large cell/anaplastic (LCA) histology. Preliminary transcriptional profiling data analysis reveals that tumours share molecular features with human counterparts, clustering with Gr3 MB. Ongoing analysis will explore the tumour cells of origin, followed by tumour progression alteration restoring p53 activity and blood-brain barrier integrity status. In conclusion, we have developed a MYCN/Trp53ERTAM Gr3 MB GEMM arising from ventricular zone progenitor cells and resembling human cancer characteristics.

Molecular Subtyping of Paediatric Medulloblastoma by Immunohistochemistry

Four core molecular subgroups of medulloblastomas have recently been introduced disparate by their transcriptional profile, all of which have different prognostic value. We aimed to determine the histological variants and molecular subtypes of medulloblastomas by immunohistochemistry in our population, and to correlate that with clinicopathological parameters. Seventeen patients aged four-month to 14.3 years diagnosed with medulloblastoma were recruited from year 2002 to 2017. All medulloblastomas were assigned to various histological variants and molecular subgroups by immunohistochemistry surrogate markers (YAP-1 and beta catenin). They were then correlated with clinicopathological parameters and outcomes. Classic histology (76.5%) was the commonest, followed by large cell/anaplastic (LCA) (17.6%) and desmoplastic/nodular (DN) variants (59%). The most frequent molecular subgroup was non-SHH/WNT tumours (64.7%), seconded by SHH tumours (35.3%). Among the SHH tumours, 66.7% was classic histology and the remaining 33.3% was LCA variant. Interestingly, one DN histology demonstrated YAP-1 and beta catenin immunonegativity, denoting non-SHH/WNT molecular subgroup. Majority (88.2%) medulloblastomas were at midline 4th ventricle location, including DN variant. Estimated three-year diseasefree- survival (DFS) and overall-survival (OS) was 60% and 86.7%, respectively. Age &lt;3 years at diagnosis, tumour size &gt;5 cm, LCA histology and high risk group were inversely correlated with DFS, with early relapse. Infant &lt;3-year-old had worse OS. Other factors had no significant impact on DFS and OS. We had demonstrated the feasibility of simple immunohistochemistry-based surrogate markers (YAP-1 and beta catenin) to stratify medulloblastomas into distinct molecular subtypes. Prognostic and predictive values of YAP-1 immunomarker in medulloblastomas however await further investigations.

PATH-09. SJMB12 CLINICAL TRIAL: DISCREPANCY BETWEEN LOCAL AND CENTRAL PATHOLOGY IN ASSESSING ANAPLASTIC MEDULLOBLASTOMA – REPORT FROM A SINGLE SITE EXPERIENCE

INTRODUCTIONSJMB12 is a phase 2 clinical trial led by the St. Jude Children’s Research Hospital (St. Jude) that enrolls patients with medulloblastoma based on their biological subgroup. The large cell/anaplastic (LCA) histologic variant has been identified as an important independent risk factor associated with poor outcome. However, the histologic criteria for LCA is subjective, making the distinction between anaplastic and non-anaplastic medulloblastoma difficult in some cases.METHODSPathological central review was performed at St. Jude. For all patients enrolled in the study to date, concordance was assessed between the initial and central review diagnosis and histologic variant calls made at the Royal Children’s Hospital Melbourne (RCH) and at St. Jude, respectively.RESULTSSince the SJMB12 clinical trial opened locally in 2014, 34 patients were enrolled, and 31 were eligible for this retrospective study. A total of 12 (39%) cases with discordance were identified. The most frequent disagreement was between the designation of LCA (10 cases, 32%). In five cases the tumour was not designated as LCA variant locally. In five cases the initial designation of LCA was refuted centrally. Overall, this led to a change of treatment stratum for four patients (13%).CONCLUSIONA high discordance rate exists between neuropathologists in the designation of LCA variant. Differences in interpretation of the subjective histologic criteria and inconsistencies in the material submitted for central review contributed to the discordance. Incorporation of more objective histologic criteria and implementation of unbiased diagnostic tools may improve the generalisability of future risk stratification.

Medulloblastoma: Distinctive Histo-Molecular Correlation with Clinical Profile, Radiologic Characteristics, and Surgical Outcome

Objective: Medulloblastoma (MB) is a heterogenous tumor, and the prognosis is influenced by various clinical, histological, and molecular factors. The aim of the study is to determine the clinical profile and radiologic characteristics among the histo-molecular subgroups, the predictors of surgical outcome, and the pattern of relapse in pediatric and adult MB. Method: An analysis of 118 patients of MB who underwent surgical treatment at National Institute of Mental Health and Neurosciences, India, over a 7-year period (2005–2011) is presented. The clinical profile, radiologic characteristics, surgical nuances, and survival patterns are discussed. The relevant statistical analysis was done using SPSS software, version 22.0. Results: The mean age of the cohort was 12 years (12.3 ± 8.7). The primary manifestation was raised intracranial tension headache in 53 patients (44.9%), which was the predominant symptom in large cell/anaplastic (LCA)- and WNT-activated subgroups. The median preoperative Karnofsky performance score was 60 (60.6 ± 12.9). Vermian and hemispheric location of tumor was most commonly observed in non-WNT/non-SHH (groups 3 and 4; 91.7%) and SHH-activated (42.9%) subgroups, respectively. Ninety-two patients (78%) underwent preoperative ventriculoperitoneal shunts (VPS) for obstructive hydrocephalus (HCP) and 14 patients (11.8%) underwent VPS in the postoperative period. The median overall survival (OS) for the whole group was 82.1 ± 5.7 months and the median recurrence-free survival was 51.0 ± 4.8 months. While radiotherapy had a significant influence on OS, progression-free survival was influenced by radiotherapy as well as chemotherapy in both pediatric and adult cohort. Desmoplastic/nodular subtype and WNT-activated subgroup had the best prognosis; LCA and non-WNT/non-SHH had the worst prognosis. Conclusions: Majority of the patients were pediatric in the study. Age, hemispheric location of tumor, extent of resection, and adjuvant treatment status were the important clinical prognostic factors for survival. Surgery for MB is formidable, and VPS can be considered in persistent symptomatic and progressive HCP. Our study on pediatric and adult MB validates the prognostic significance of various clinical, radiologic, and histo-molecular parameters of MB.

C-Myc activation promotes cofilin-mediated F-actin cytoskeleton remodeling and telomere homeostasis as a response to oxidant-based DNA damage in medulloblastoma cells

Medulloblastoma (MB) is a common and highly aggressive pediatric brain tumor of a heterogeneous nature. According to transcriptome-based profiling, four molecular subgroups of MB have been revealed, namely WNT, SHH, Group 3 and Group 4. High MYC mRNA expression and MYC gene amplification in MB have been considered as indicators of poor prognosis. However, the role of c-Myc in MB biology is still not well established. In the present study, the effects of c-Myc activation in UW228-MycER MB cell line were investigated using 4-hydroxytamoxifen (4-OHT) induction system. Upon 4-OHT stimulation, an increase in metabolic activity, large-cell/anaplastic (LC/A) phenotype and oxidative stress-mediated DNA damage were observed. However, 53BP1 foci were not implicated in DNA damage response. Instead, cofilin nuclear translocation, changes in F-actin cytoskeleton and the levels of cytoskeletal proteins were shown. Moreover, the telomere length was found to be unaffected that may be associated with the upregulation of TRF proteins. Transcription of nascent RNA (synthesis of new rRNA) and the expression of RNA polymerase I-specific transcription initiation factor RRN3/TIF-IA were also elevated. Moreover, increased levels of DNMT2, a modulator of stress responses, were observed. A small fraction of cells responded differently as oncogene-induced senescence was also noticed. We postulate that c-Myc-mediated modulation of genetic stability of MB cells may trigger cellular heterogeneity and affect adaptive responses to changing environment.

Comparing children and adults with medulloblastoma: a SEER based analysis.

Medulloblastoma (MB) is a brain malignancy, which commonly occurs in children, but is rare in adults. The Surveillance, Epidemiology, and End Results (SEER) database was used to compare survival, clinical features, and prognostic factors of children and adults with MB from 1992 to 2013. Overall survival estimates were compared using the Kaplan–Meier method, and Cox Proportion Hazard Regression modeling was used to evaluate prognostic variables. We identified 616 children (63.8%) and 349 adults (36.2%) with diagnosis of MB. The estimated survival rates for children diagnosed with MB for 2, 5, and 10 years were 85.6%, 75.5%, and 67.9%, respectively; the corresponding estimates for adults were 84.9%, 74.2%, and 67.3%. Radiotherapy was the only identical prognostic factor observed in the two groups. Children MB patients were more likely to experience distal metastases that was associated with increased hazard of mortality, and be diagnosed after 2003. Among adult MB patients, gross total resection (GTR) was a favorable prognostic factor, while large cell/anaplastic (LC/A) histology was correlated with decreased survival. Our analysis highlighted that both groups had similar overall survival time, but the prognostic factors were not comparable, except radiotherapy which was associated with better survival.

A Retrospective Study of Progression-Free and Overall Survival in Pediatric Medulloblastoma Based on Molecular Subgroup Classification: A Single-Institution Experience.

Medulloblastoma (MB) has been classified into four core subgroups according to the transcriptional profile in recent years. However, some disagreement among researchers remains regarding the prognoses and most effective treatments of the different subgroups with different age distributions. The objective of this study was to analyze MB prognosis in children population based on the classification of four molecular subgroups. From January 2011 to January 2013, 84 consecutive MB patients aged underwent tumor removal at Beijing Tiantan Hospital. A total of 55 patients who ranged in age from 4 to 18 years underwent detailed follow-up. Molecular subgrouping was performed using RT-PCR. The 2-year progression-free survival (PFS) and overall survival (OS) rates for the entire cohort were 76.2 ± 5.8 and 81.8 ± 5.2%, respectively. Univariate analysis revealed that the Group 4 patients had a better survival (2-year OS, 90.6 ± 5.2%) than the SHH subgroup (P = 0.002) and Group 3 patients (P = 0.008). Only two of the 23 non-metastasized Group 4 patients relapsed, and chemotherapy did significantly affect these patients (PFS, P = 0.685). One out of five WNT patients had tumor relapse and died at last. Large cell/anaplastic (LC/A) histology and chemotherapy were independent risk factors in multivariate analysis. In our study, the non-metastasized Group 4 patients had an excellent prognosis. The SHH subgroup and Group 3 patients had worst prognoses. LC/A histology had a dismal prognosis in our cohorts, which warrants intensive treatment.

Large cell/anaplastic medulloblastoma is associated with poor prognosis—a retrospective analysis at a single institute

Medulloblastoma (MB) is the most commonly occurring malignant pediatric brain tumor worldwide. However, a recent study found that the treatment outcomes in those with high-risk disease receiving conventional treatment were suboptimal. This study aimed to assess outcomes and treatment strategies for specific histologic subtypes of pediatric MB. A total of 114 pediatric patients (age<20years) diagnosed with MB between March 1998 and August 2011 were retrospectively reviewed; 52 that were treated with surgery followed by adjuvant radiotherapy (RT) and chemotherapy (CHT) were included. The 5-year overall survival (OS) and relapse-free survival (RFS) rates were 73 and 69%, respectively. Median time to relapse was 17months with a median survival time of 6months after relapse. Patients of average risk had a better 5-year OS rate compared with high-risk patients (p=0.027). The 5-year RFS of high-risk patients was lower compared with average risk (p=0.038). A greater proportion of patients with large cell/anaplastic (LC/A) MB had recurrence than classic MB with 5-year RFS rate of 34 and 76%, respectively (p=0.001), and OS rate of 56 and 76%, respectively (p=0.04). High-risk group and histology of LC/A were the most significant factors associated with worse OS and RFS. Patients with LC/A-MB had higher relapse rates and worse survival than those with classic MB. LC/A-MB carries a high risk for recurrence and should be treated with the more aggressive strategies.

P11.02 Long-term Outcome of Infants and Young Children with Newly Diagnosed Non-Nodular/Desmoplastic Medulloblastoma Treated on “Head Start” III Protocol

Abstract Background: High-dose chemotherapy with autologous hematopoietic cell rescue (AuHCR) is a potentially curative approach used in the management young children with medulloblastoma), with the additional benefit of avoiding the late effects of craniospinal irradiation (CSI). We report the outcome of infants and young children with classic and large cell/anaplastic (LCA) medulloblastoma treated on “Head Start (HS)” III, a prospective, multi-national clinical trial. Methods: Between April 2003 and December 2009, 92 children with newly-diagnosed medulloblastoma were enrolled amongst 29 participating institutions. All children were to receive 5 induction cycles (vincristine, cisplatin, cyclophosphamide, etoposide, high dose methotrexate - cycles 1, 3, 5), and vincristine, cyclophosphamide, oral VP-16 and temozolomide (cycles 2, 4), followed by 1 consolidation cycle of myeloablative chemotherapy (thiotepa, carboplatin, etoposide) and AuHCR. Only children between 6-10 years old or with residual tumor pre-consolidation, were to receive irradiation after consolidation. Results: Of 92 children with medulloblastoma enrolled on HSIII, 52 patients had classic and 13 had LCA medulloblastoma. Eighteen patients with classic and 6 with LCA medulloblastoma had localized disease (M0) whereas 33 patients with classic and 7 patients with LCA medulloblastoma had evidence of dissemination (M+). The 2-year event-free (EFS) and overall survival (OS) rates (+/- SE) for patients with classic medulloblastoma (primary objective of the study) were 37 ± 7% and 62 ± 7%, and for patients with LCA medulloblastoma were 54 ± 14% and 62 ± 13%, respectively. The 5-year EFS and OS rates were 26 ± 6% and 53 ± 7% for classic medulloblastoma and 38 ± 13% and 46 ± 14% for LCA medulloblastoma patients, respectively. For patients with M0 medulloblastoma, the 5-year EFS and OS rates were 38 ± 12% and 66 ± 11% for patients with classic histology and 50 ± 20% and 67 ± 19% for patients with LCA histology. Similarly, for patients with M+ medulloblastoma, the 5-year EFS and OS rates were 21 ± 7% and 48 ± 9% for patients with classic histology and 29 ± 17% and 29 ± 17% for patients with LCA histology. In multivariate Cox regression analyses, no significant difference in EFS was found between classic and LCA tumors. 5-year irradiation free-EFS for non-nodular/desmoplastic patients was 29 +/- 6%. Conclusion: Encouraging survival figures are reported for patients with classic medulloblastoma, similar to other contemporary infant protocols, whereas patients with LCA medulloblastoma had equivalent survival when compared to patients with classic medulloblastoma when treated with high-dose chemotherapy with AuHCR. These are among the best survival data published so far on infants and young children with LCA medulloblastoma. Approximately third of the survivors were able to avoid irradiation.

Update on the integrated histopathological and genetic classification of medulloblastoma - a practical diagnostic guideline.

The revised WHO classification of tumors of the CNS 2016 has introduced the concept of the integrated diagnosis. The definition of medulloblastoma entities now requires a combination of the traditional histological information with additional molecular/genetic features. For definition of the histopathological component of the medulloblastoma diagnosis, the tumors should be assigned to one of the four entities classic, desmoplastic/nodular (DNMB), extensive nodular (MBEN), or large cell/anaplastic (LC/A) medulloblastoma. The genetically defined component comprises the four entities WNT-activated, SHH-activated and TP53 wildtype, SHH-activated and TP53 mutant, or non-WNT/non-SHH medulloblastoma. Robust and validated methods are available to allow a precise diagnosis of these medulloblastoma entities according to the updated WHO classification, and for differential diagnostic purposes. A combination of immunohistochemical markers including β-catenin, Yap1, p75-NGFR, Otx2, and p53, in combination with targeted sequencing and copy number assessment such as FISH analysis for MYC genes allows a precise assignment of patients for risk-adapted stratification. It also allows comparison to results of study cohorts in the past and provides a robust basis for further treatment refinement.

Somatic cell transfer of c-Myc and Bcl-2 induces large-cell anaplastic medulloblastomas in mice

A highly aggressive subgroup of the pediatric brain tumor medulloblastoma is characterized by overexpression of the proto-oncogene c-Myc, which encodes a transcription factor that normally maintains neural progenitor cells in an undifferentiated, proliferating state during embryonic development. Myc-driven medulloblastomas typically show a large-cell anaplastic (LCA) histological pattern, in which tumor cells display large, round nuclei with prominent nucleoli. This subgroup of medulloblastoma is therapeutically challenging because it is associated with a high rate of metastatic dissemination, which is a powerful predictor of short patient survival times. Genetically engineered mouse models have revealed important insights into the pathogenesis of medulloblastoma and served as preclinical testing platforms for new therapies. Here we report a new mouse model of Myc-driven medulloblastoma, in which tumors arise in situ after retroviral transfer and expression of Myc in Nestin-expressing neural progenitor cells in the cerebella of newborn mice. Tumor induction required concomitant loss of Tp53 or overexpression of the antiapoptotic protein Bcl-2. Like Myc-driven medulloblastomas in humans, the tumors induced in mice by Myc + Bcl-2 and Myc - Tp53 showed LCA cytoarchitecture and a high rate of metastatic dissemination to the spine. The fact that Myc - Tp53 tumors arose only in Tp53(-/-) mice, coupled with the inefficient germline transmission of the Tp53-null allele, made retroviral transfer of Myc + Bcl-2 a more practical method for generating LCA medulloblastomas. The high rate of spinal metastasis (87% of brain tumor-bearing mice) will be an asset for testing new therapies that target the most lethal aspect of medulloblastoma.

4. “Biphasic” histology is associated with the non-WNT/SHH molecular subtype of medulloblastoma

IntroductionIn 2007, Ellison et al coined the term “biphasic” medulloblastoma (B-MB) to characterize histology that mimicked the desmoplastic nodular (DN) variant on routine staining, but which lacked internodular reticulin deposition. Via interphase FISH, and utilizing markers for 9q22 and chromosome 17 alterations (ie, -17p and i17q), Ellison et al. suggested that B-MB and DN-MB were genetically different.MethodsWe performed a clinicopathologic review of MBs treated at BCCH from 1986-2011. Using nanoString’s n Counter Analysis System (nCAS), each tumor was molecularly subtyped (ie, WNT, SHH, group 3 or group 4). All original glass slides were reviewed to determine WHO histologic subtype [ie, classic, large cell anaplastic (LCA), DN, MB with extensive nodularity (MBEN)]. Tumors were also evaluated for nodularity (scattered vs. frequent) and advanced neuronal differentiation. Reticulin staining was assessed on all cases.Results20 B-MB were identified; by WHO definition, most of these resided within the classic category (N=19), while one was LCA. 13 of 20 B-MB displayed ‘scattered” nodules; by molecular subtype, these included eight group 4, four group 3 and one WNT tumors. Seven of the 20 B-MB exhibited “frequent” nodules; by molecular subtype, these included six group 4 and one group 3 tumors. Statistical analysis confirmed this non random distribution of B-MB across molecular subtypes.ConclusionOur data confirm the work of Ellison et al. that suggested B-MB is genetically different than DN-MB. In particular, B-MB resides in the non-WNT/SHH molecular category, but especially amongst group 4 when nodularity is “frequent”.

Abstract 2977: Myc and Miz1 in medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor, which arises in the cerebellum. Human MBs are classified into four subgroups: Wingless (WNT), Sonic Hedgehog (SHH), Group 3 (G3) and Group 4 (G4). In 2012, we developed a mouse model of the most aggressive and least curable G3 MB, by overexpressing MYC but not MYCN, in Trp53-null cerebellar granule neuron progenitors (GNPs), which were implanted into the cortices of recipient CD-1 nu/nu mice. Remarkably, enforced expression of MYCN in Trp53-null GNPs induced a SHH MB. This was unexpected since MYC and MYCN bind to the same E-box DNA binding sequences. We hypothesized that the difference between MYC- and MYCN-induced MBs might be due to their interaction with different partners in GNPs. Human G3 MBs are distinguished by MYC overexpression. The MYC proto-oncogene encodes a bHLH/leucine-zipper transcription factor that forms heterodimers with MAX to drive transcription. However, MYC can repress transcription when the MYC/MAX heterodimer is recruited to core promoter sequences by MIZ1, a member of the POZ-domain/zinc-finger transcription factor. MIZ1, ubiquitously expressed during embryonic development, can either be a transcription activator or repressor depending on its binding partners. To investigate whether the MYC/MIZ1 complex contributed to G3 MBs, we used a mutant of MYC which no longer binds to MIZ1 in which a Valine is substituted for an Aspartic acid amino acid at position 394 (MYCV394D). We enforced the expression of MYC or MYCV394D in GNPs purified from seven-day-old Trp53-null mouse pups using retroviral transduction, and implanted transduced GNPs into the cerebral cortices of recipient mice. MYCV394D-induced tumors developed later than MYC-induced G3 MBs. Histopathological analysis revealed that MYC-induced tumors exhibited a large cell/anaplastic (LCA) phenotype and were classified as G3 MBs; however, MYCV394D-induced tumors were described as unclassified primitive neuroectodermal tumors. In vitro, MYCV394D-induced tumors formed tumorspheres that were highly apoptotic and less proliferative compared to MYC tumorspheres. Global gene expression of MYCV394D tumors was distinct from murine G3 and SHH subgroup MBs. Particularly, genes needed for neuronal differentiation and which are strongly repressed by MYC in G3 MBs, were no longer repressed by the MYCV394D mutant, indicating that the MYC/MIZ1 complex was responsible for their repression. To analyze whether the MYC/MIZ1 complex was present on chromatin in G3 MBs, we performed ChIP-Seq for MYC and MIZ1. In contrast to Trp53-null GNPs, where MIZ1 only binds 140 promoters, 11.549 promoters were occupied by MIZ1 in G3 MBs, 9.353 of which were co-occupied by MYC (81%). Because as many of the joined binding sites neither contained a canonical E-box sequence nor a bona fide MIZ1 binding motif, we speculate that cooperative binding of oncogenic MYC with MIZ1 is necessary for binding. Our data suggest that the MYC-MIZ1 interaction is critical for the development of G3 MBs. Citation Format: BaoHan T. Vo, Elmar Wolf, Daisuke Kawauchi, Jerold Rehg, David Finkelstein, Brian Murphy, Martin Eilers, Martine F. Roussel. Myc and Miz1 in medulloblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2977. doi:10.1158/1538-7445.AM2014-2977