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SNCAIP duplication may promote Group 4 medulloblastoma via induction of PRDM6, a poorly characterized member of the PRDF1 and RIZ1 homology domain-containing (PRDM) family of transcription factors. Here, we investigated the function of PRDM6 in human hindbrain neuroepithelial stem cells and tested PRDM6 as a driver of Group 4 medulloblastoma. We report that human PRDM6 localizes predominantly to the nucleus, where it causes widespread repression of chromatin accessibility and complex alterations of gene expression patterns. Genome-wide mapping of PRDM6 binding reveals that PRDM6 binds to chromatin regions marked by histone H3 lysine 27 trimethylation that are located within, or proximal to, genes. Moreover, we show that PRDM6 expression in neuroepithelial stem cells promotes medulloblastoma. Surprisingly, medulloblastomas derived from PRDM6-expressing neuroepithelial stem cells match human Group 3, but not Group 4, medulloblastoma. We conclude that PRDM6 expression has oncogenic potential but is insufficient to drive Group 4 medulloblastoma from neuroepithelial stem cells. We propose that both PRDM6 and additional factors, such as specific cell-of-origin features, are required for Group 4 medulloblastoma. Given the lack of PRDM6 expression in normal tissues and its oncogenic potential shown here, we suggest that PRDM6 inhibition may have therapeutic value in PRDM6-expressing medulloblastomas.

Methylation profiling has radically transformed our understanding of tumors previously called central nervous system primitive neuro-ectodermal tumors (CNS-PNET). While this marks a momentous step toward defining key differences, reclassification has thrown treatment into disarray. To shed light on response to therapy and guide clinical decision-making, we report outcomes and molecular features of children with CNS-PNETs from two multi-center risk-adapted studies (SJMB03 for patients ≥ 3 years; SJYC07 for patients

Medulloblastoma, a common pediatric malignant central nervous system tumour, represent a small proportion of brain tumours in adults. Previously it has been shown that in adults, Sonic Hedgehog (SHH)-activated tumours predominate, with Wingless-type (WNT) and Group 4 being less common, but molecular risk stratification remains a challenge. We performed an integrated analysis consisting of genome-wide methylation profiling, copy number profiling, somatic nucleotide variants and correlation of clinical variables across a cohort of 191 adult medulloblastoma cases identified through the Medulloblastoma Advanced Genomics International Consortium. We identified 30 WNT, 112 SHH, 6 Group 3, and 41 Group 4 tumours. Patients with SHH tumours were significantly older at diagnosis compared to other subgroups (p

Medulloblastoma (MB) is a malignant pediatric brain tumor arising in the cerebellum. Although abnormal GABAergic receptor activation has been described in MB, studies have not yet elucidated the contribution of receptor-independent GABA metabolism to MB pathogenesis. We find primary MB tumors globally display decreased expression of GABA transaminase (ABAT), the protein responsible for GABA metabolism, compared with normal cerebellum. However, less aggressive WNT and SHH subtypes express higher ABAT levels compared with metastatic G3 and G4 tumors. We show that elevated ABAT expression results in increased GABA catabolism, decreased tumor cell proliferation, and induction of metabolic and histone characteristics mirroring GABAergic neurons. Our studies suggest ABAT expression fluctuates depending on metabolite changes in the tumor microenvironment, with nutrient-poor conditions upregulating ABAT expression. We find metastatic MB cells require ABAT to maintain viability in the metabolite-scarce cerebrospinal fluid by using GABA as an energy source substitute, thereby facilitating leptomeningeal metastasis formation.

PURPOSE: We sought to investigate clinical outcomes of relapsed medulloblastoma and to compare molecular features between patient-matched diagnostic and relapsed tumors. METHODS: Children and infants enrolled on either SJMB03 (NCT00085202) or SJYC07 (NCT00602667) trials who experienced medulloblastoma relapse were analyzed for clinical outcomes, including anatomic and temporal patterns of relapse and postrelapse survival. A largely independent, paired molecular cohort was analyzed by DNA methylation array and next-generation sequencing. RESULTS: A total of 72 of 329 (22%) SJMB03 and 52 of 79 (66%) SJYC07 patients experienced relapse with significant representation of Group 3 and wingless tumors. Although most patients exhibited some distal disease (79%), 38% of patients with sonic hedgehog tumors experienced isolated local relapse. Time to relapse and postrelapse survival varied by molecular subgroup with longer latencies for patients with Group 4 tumors. Postrelapse radiation therapy among previously nonirradiated SJYC07 patients was associated with long-term survival. Reirradiation was only temporizing for SJMB03 patients. Among 127 patients with patient-matched tumor pairs, 9 (7%) experienced subsequent nonmedulloblastoma CNS malignancies. Subgroup (96%) and subtype (80%) stabilities were largely maintained among the remainder. Rare subgroup divergence was observed from Group 4 to Group 3 tumors, which is coincident with genetic alterations involving MYC, MYCN, and FBXW7. Subgroup-specific patterns of alteration were identified for driver genes and chromosome arms. CONCLUSION: Clinical behavior of relapsed medulloblastoma must be contextualized in terms of up-front therapies and molecular classifications. Group 4 tumors exhibit slower biological progression. Utility of radiation at relapse is dependent on patient age and prior treatments. Degree and patterns of molecular conservation at relapse vary by subgroup. Relapse tissue enables verification of molecular targets and identification of occult secondary malignancies.

Over the past decade, wingless-activated (WNT) medulloblastoma has been identified as a candidate for therapy de-escalation based on excellent survival; however, a paucity of relapses has precluded additional analyses of markers of relapse. To address this gap in knowledge, an international cohort of 93 molecularly confirmed WNT MB was assembled, where 5-year progression-free survival is 0.84 (95%, 0.763-0.925) with 15 relapsed individuals identified. Maintenance chemotherapy is identified as a strong predictor of relapse, with individuals receiving high doses of cyclophosphamide or ifosphamide having only one very late molecularly confirmed relapse (p = 0.032). The anatomical location of recurrence is metastatic in 12 of 15 relapses, with 8 of 12 metastatic relapses in the lateral ventricles. Maintenance chemotherapy, specifically cumulative cyclophosphamide doses, is a significant predictor of relapse across WNT MB. Future efforts to de-escalate therapy need to carefully consider not only the radiation dose but also the chemotherapy regimen and the propensity for metastatic relapses.

Cancer genomics has revealed many genes and core molecular processes that contribute to human malignancies, but the genetic and molecular bases of many rare cancers remains unclear. Genetic predisposition accounts for 5 to 10% of cancer diagnoses in children1,2, and genetic events that cooperate with known somatic driver events are poorly understood. Pathogenic germline variants in established cancer predisposition genes have been recently identified in 5% of patients with the malignant brain tumour medulloblastoma3. Here, by analysing all protein-coding genes, we identify and replicate rare germline loss-of-function variants across ELP1 in 14% of paediatric patients with the medulloblastoma subgroup Sonic Hedgehog (MBSHH). ELP1 was the most common medulloblastoma predisposition gene and increased the prevalence of genetic predisposition to 40% among paediatric patients with MBSHH. Parent-offspring and pedigree analyses identified two families with a history of paediatric medulloblastoma. ELP1-associated medulloblastomas were restricted to the molecular SHHα subtype4 and characterized by universal biallelic inactivation of ELP1 owing to somatic loss of chromosome arm 9q. Most ELP1-associated medulloblastomas also exhibited somatic alterations in PTCH1, which suggests that germline ELP1 loss-of-function variants predispose individuals to tumour development in combination with constitutive activation of SHH signalling. ELP1 is the largest subunit of the evolutionarily conserved Elongator complex, which catalyses translational elongation through tRNA modifications at the wobble (U34) position5,6. Tumours from patients with ELP1-associated MBSHH were characterized by a destabilized Elongator complex, loss of Elongator-dependent tRNA modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response, consistent with loss of protein homeostasis due to Elongator deficiency in model systems7-9. Thus, genetic predisposition to proteome instability may be a determinant in the pathogenesis of paediatric brain cancers. These results support investigation of the role of protein homeostasis in other cancer types and potential for therapeutic interference.

Pineoblastoma is a rare embryonal tumor of childhood that is conventionally treated with high-dose craniospinal irradiation (CSI). Multi-dimensional molecular evaluation of pineoblastoma and associated intertumoral heterogeneity is lacking. Herein, we report outcomes and molecular features of children with pineoblastoma from two multi-center, risk-adapted trials (SJMB03 for patients ≥ 3 years; SJYC07 for patients

Drugs that modify the epigenome are powerful tools for treating cancer, but these drugs often have pleiotropic effects, and identifying patients who will benefit from them remains a major clinical challenge. Here we show that medulloblastomas driven by the transcription factor Gfi1 are exquisitely dependent on the enzyme lysine demethylase 1 (Kdm1a/Lsd1). We demonstrate that Lsd1 physically associates with Gfi1, and that these proteins cooperate to inhibit genes involved in neuronal commitment and differentiation. We also show that Lsd1 is essential for Gfi1-mediated transformation: Gfi1 proteins that cannot recruit Lsd1 are unable to drive tumorigenesis, and genetic ablation of Lsd1 markedly impairs tumor growth in vivo. Finally, pharmacological inhibitors of Lsd1 potently inhibit growth of Gfi1-driven tumors. These studies provide important insight into the mechanisms by which Gfi1 contributes to tumorigenesis, and identify Lsd1 inhibitors as promising therapeutic agents for Gfi1-driven medulloblastoma.

There is a pressing need to identify therapeutic targets in tumors with low mutation rates such as the malignant pediatric brain tumor medulloblastoma. To address this challenge, we quantitatively profiled global proteomes and phospho-proteomes of 45 medulloblastoma samples. Integrated analyses revealed that tumors with similar RNA expression vary extensively at the post-transcriptional and post-translational levels. We identified distinct pathways associated with two subsets of SHH tumors, and found post-translational modifications of MYC that are associated with poor outcomes in group 3 tumors. We found kinases associated with subtypes and showed that inhibiting PRKDC sensitizes MYC-driven cells to radiation. Our study shows that proteomics enables a more comprehensive, functional readout, providing a foundation for future therapeutic strategies.

BACKGROUND: Young children with medulloblastoma have a poor overall survival compared with older children, due to use of radiation-sparing therapy in young children. Radiotherapy is omitted or reduced in these young patients to spare them from debilitating long-term side-effects. We aimed to estimate event-free survival and define the molecular characteristics associated with progression-free survival in young patients with medulloblastoma using a risk-stratified treatment strategy designed to defer, reduce, or delay radiation exposure. METHODS: In this multicentre, phase 2 trial, we enrolled children younger than 3 years with newly diagnosed medulloblastoma at six centres in the USA and Australia. Children aged 3-5 years with newly diagnosed, non-metastatic medulloblastoma without any high-risk features were also eligible. Eligible patients were required to start therapy within 31 days from definitive surgery, had a Lansky performance score of at least 30, and did not receive previous radiotherapy or chemotherapy. Patients were stratified postoperatively by clinical and histological criteria into low-risk, intermediate-risk, and high-risk treatment groups. All patients received identical induction chemotherapy (methotrexate, vincristine, cisplatin, and cyclophosphamide), with high-risk patients also receiving an additional five doses of vinblastine. Induction was followed by risk-adapted consolidation therapy: low-risk patients received cyclophosphamide (1500 mg/m2 on day 1), etoposide (100 mg/m2 on days 1 and 2), and carboplatin (area under the curve 5 mg/mL per min on day 2) for two 4-week cycles; intermediate-risk patients received focal radiation therapy (54 Gy with a clinical target volume of 5 mm over 6 weeks) to the tumour bed; and high-risk patients received chemotherapy with targeted intravenous topotecan (area under the curve 120-160 ng-h/mL intravenously on days 1-5) and cyclophosphamide (600 mg/m2 intravenously on days 1-5). After consolidation, all patients received maintenance chemotherapy with cyclophosphamide, topotecan, and erlotinib. The coprimary endpoints were event-free survival and patterns of methylation profiling associated with progression-free survival. Outcome and safety analyses were per protocol (all patients who received at least one dose of induction chemotherapy); biological analyses included all patients with tissue available for methylation profiling. This trial is registered with ClinicalTrials.gov, number NCT00602667, and was closed to accrual on April 19, 2017. FINDINGS: Between Nov 27, 2007, and April 19, 2017, we enrolled 81 patients with histologically confirmed medulloblastoma. Accrual to the low-risk group was suspended after an interim analysis on Dec 2, 2015, when the 1-year event-free survival was estimated to be below the stopping rule boundary. After a median follow-up of 5·5 years (IQR 2·7-7·3), 5-year event-free survival was 31·3% (95% CI 19·3-43·3) for the whole cohort, 55·3% (95% CI 33·3-77·3) in the low-risk cohort (n=23) versus 24·6% (3·6-45·6) in the intermediate-risk cohort (n=32; hazard ratio 2·50, 95% CI 1·19-5·27; p=0·016) and 16·7% (3·4-30·0) in the high-risk cohort (n=26; 3·55, 1·66-7·59; p=0·0011; overall p=0·0021). 5-year progression-free survival by methylation subgroup was 51·1% (95% CI 34·6-67·6) in the sonic hedgehog (SHH) subgroup (n=42), 8·3% (95% CI 0·0-24·0%) in the group 3 subgroup (n=24), and 13·3% (95% CI 0·0-37·6%) in the group 4 subgroup (n=10). Within the SHH subgroup, two distinct methylation subtypes were identified and named iSHH-I and iSHH-II. 5-year progression-free survival was 27·8% (95% CI 9·0-46·6; n=21) for iSHH-I and 75·4% (55·0-95·8; n=21) for iSHH-II. The most common adverse events were grade 3-4 febrile neutropenia (48 patients [59%]), neutropenia (21 [26%]), infection with neutropenia (20 [25%]), leucopenia (15 [19%]), vomiting (15 [19%]), and anorexia (13 [16%]). No treatment-related deaths occurred. INTERPRETATION: The risk-adapted approach did not improve event-free survival in young children with medulloblastoma. However, the methylation subgroup analyses showed that the SHH subgroup had improved progression-free survival compared with the group 3 subgroup. Moreover, within the SHH subgroup, the iSHH-II subtype had improved progression-free survival in the absence of radiation, intraventricular chemotherapy, or high-dose chemotherapy compared with the iSHH-I subtype. These findings support the development of a molecularly driven, risk-adapted, treatment approach in future trials in young children with medulloblastoma. FUNDING: American Lebanese Syrian Associated Charities, St Jude Childrens Research Hospital, NCI Cancer Center, Alexander and Margaret Stewart Trust, Sontag Foundation, and American Association for Cancer Research.

BackgroundMedulloblastoma is associated with rare hereditary cancer predisposition syndromes; however, consensus medulloblastoma predisposition genes have not been defined and screening guidelines for genetic counselling and testing for paediatric patients are not available. We aimed to assess and define these genes to provide evidence for future screening guidelines.MethodsIn this international, multicentre study, we analysed patients with medulloblastoma from retrospective cohorts (International Cancer Genome Consortium [ICGC] PedBrain, Medulloblastoma Advanced Genomics International Consortium [MAGIC], and the CEFALO series) and from prospective cohorts from four clinical studies (SJMB03, SJMB12, SJYC07, and I-HIT-MED). Whole-genome sequences and exome sequences from blood and tumour samples were analysed for rare damaging germline mutations in cancer predisposition genes. DNA methylation profiling was done to determine consensus molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGroup3), and group 4 (MBGroup4). Medulloblastoma predisposition genes were predicted on the basis of rare variant burden tests against controls without a cancer diagnosis from the Exome Aggregation Consortium (ExAC). Previously defined somatic mutational signatures were used to further classify medulloblastoma genomes into two groups, a clock-like group (signatures 1 and 5) and a homologous recombination repair deficiency-like group (signatures 3 and 8), and chromothripsis was investigated using previously established criteria. Progression-free survival and overall survival were modelled for patients with a genetic predisposition to medulloblastoma.FindingsWe included a total of 1022 patients with medulloblastoma from the retrospective cohorts (n=673) and the four prospective studies (n=349), from whom blood samples (n=1022) and tumour samples (n=800) were analysed for germline mutations in 110 cancer predisposition genes. In our rare variant burden analysis, we compared these against 53 105 sequenced controls from ExAC and identified APC, BRCA2, PALB2, PTCH1, SUFU, and TP53 as consensus medulloblastoma predisposition genes according to our rare variant burden analysis and estimated that germline mutations accounted for 6% of medulloblastoma diagnoses in the retrospective cohort. The prevalence of genetic predispositions differed between molecular subgroups in the retrospective cohort and was highest for patients in the MBSHH subgroup (20% in the retrospective cohort). These estimates were replicated in the prospective clinical cohort (germline mutations accounted for 5% of medulloblastoma diagnoses, with the highest prevalence [14%] in the MBSHH subgroup). Patients with germline APC mutations developed MBWNT and accounted for most (five [71%] of seven) cases of MBWNT that had no somatic CTNNB1 exon 3 mutations. Patients with germline mutations in SUFU and PTCH1 mostly developed infant MBSHH. Germline TP53 mutations presented only in childhood patients in the MBSHH subgroup and explained more than half (eight [57%] of 14) of all chromothripsis events in this subgroup. Germline mutations in PALB2 and BRCA2 were observed across the MBSHH, MBGroup3, and MBGroup4 molecular subgroups and were associated with mutational signatures typical of homologous recombination repair deficiency. In patients with a genetic predisposition to medulloblastoma, 5-year progression-free survival was 52% (95% CI 40-69) and 5-year overall survival was 65% (95% CI 52-81); these survival estimates differed significantly across patients with germline mutations in different medulloblastoma predisposition genes.InterpretationGenetic counselling and testing should be used as a standard-of-care procedure in patients with MBWNT and MBSHH because these patients have the highest prevalence of damaging germline mutations in known cancer predisposition genes. We propose criteria for routine genetic screening for patients with medulloblastoma based on clinical and molecular tumour characteristics.FundingGerman Cancer Aid; German Federal Ministry of Education and Research; German Childhood Cancer Foundation (Deutsche Kinderkrebsstiftung); European Research Council; National Institutes of Health; Canadian Institutes for Health Research; German Cancer Research Center; St Jude Comprehensive Cancer Center; American Lebanese Syrian Associated Charities; Swiss National Science Foundation; European Molecular Biology Organization; Cancer Research UK; Hertie Foundation; Alexander and Margaret Stewart Trust; V Foundation for Cancer Research; Sontag Foundation; Musicians Against Childhood Cancer; BC Cancer Foundation; Swedish Council for Health, Working Life and Welfare; Swedish Research Council; Swedish Cancer Society; the Swedish Radiation Protection Authority; Danish Strategic Research Council; Swiss Federal Office of Public Health; Swiss Research Foundation on Mobile Communication; Masaryk University; Ministry of Health of the Czech Republic; Research Council of Norway; Genome Canada; Genome BC; Terry Fox Research Institute; Ontario Institute for Cancer Research; Pediatric Oncology Group of Ontario; The Family of Kathleen Lorette and the Clark H Smith Brain Tumour Centre; Montreal Children's Hospital Foundation; The Hospital for Sick Children: Sonia and Arthur Labatt Brain Tumour Research Centre, Chief of Research Fund, Cancer Genetics Program, Garron Family Cancer Centre, MDT's Garron Family Endowment; BC Childhood Cancer Parents Association; Cure Search Foundation; Pediatric Brain Tumor Foundation; Brainchild; and the Government of Ontario.

Current therapies for medulloblastoma, a highly malignant childhood brain tumour, impose debilitating effects on the developing child, and highlight the need for molecularly targeted treatments with reduced toxicity. Previous studies have been unable to identify the full spectrum of driver genes and molecular processes that operate in medulloblastoma subgroups. Here we analyse the somatic landscape across 491 sequenced medulloblastoma samples and the molecular heterogeneity among 1,256 epigenetically analysed cases, and identify subgroup-specific driver alterations that include previously undiscovered actionable targets. Driver mutations were confidently assigned to most patients belonging to Group 3 and Group 4 medulloblastoma subgroups, greatly enhancing previous knowledge. New molecular subtypes were differentially enriched for specific driver events, including hotspot in-frame insertions that target KBTBD4 and 'enhancer hijacking' events that activate PRDM6. Thus, the application of integrative genomics to an extensive cohort of clinical samples derived from a single childhood cancer entity revealed a series of cancer genes and biologically relevant subtype diversity that represent attractive therapeutic targets for the treatment of patients with medulloblastoma.

DDX3X encodes a DEAD-box family RNA helicase (DDX3) commonly mutated in medulloblastoma, a highly aggressive cerebellar tumor affecting both children and adults. Despite being implicated in several facets of RNA metabolism, the nature and scope of DDX3's interactions with RNA remain unclear. Here, we show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5'UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex. Impairment of translation initiation is also evident in primary medulloblastomas harboring mutations in DDX3X, further highlighting DDX3's role in this process. Arsenite-induced stress shifts DDX3 binding from the 5'UTR into the coding region of mRNAs concomitant with a general reduction of translation, and both the shift of DDX3 on mRNA and decreased translation are blunted by expression of a catalytically-impaired, medulloblastoma-associated DDX3R534H variant. Furthermore, despite the global repression of translation induced by arsenite, translation is preserved on select genes involved in chromatin organization in DDX3R534H-expressing cells. Thus, DDX3 interacts extensively with RNA and ribosomal machinery to help remodel the translation landscape in response to stress, while cancer-related DDX3 variants adapt this response to selectively preserve translation.

BackgroundPatients with incomplete surgical resection of medulloblastoma are controversially regarded as having a marker of high-risk disease, which leads to patients undergoing aggressive surgical resections, so-called second-look surgeries, and intensified chemoradiotherapy. All previous studies assessing the clinical importance of extent of resection have not accounted for molecular subgroup. We analysed the prognostic value of extent of resection in a subgroup-specific manner.MethodsWe retrospectively identified patients who had a histological diagnosis of medulloblastoma and complete data about extent of resection and survival from centres participating in the Medulloblastoma Advanced Genomics International Consortium. We collected from resections done between April, 1997, and February, 2013, at 35 international institutions. We established medulloblastoma subgroup affiliation by gene expression profiling on frozen or formalin-fixed paraffin-embedded tissues. We classified extent of resection on the basis of postoperative imaging as gross total resection (no residual tumour), near-total resection (30 Gy vs no craniospinal irradiation). The primary analysis outcome was the effect of extent of resection by molecular subgroup and the effects of other clinical variables on overall and progression-free survival.FindingsWe included 787 patients with medulloblastoma (86 with WNT tumours, 242 with SHH tumours, 163 with group 3 tumours, and 296 with group 4 tumours) in our multivariable Cox models of progression-free and overall survival. We found that the prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. We identified a progression-free survival benefit for gross total resection over sub-total resection (hazard ratio [HR] 1·45, 95% CI 1·07-1·96, p=0·16) but no overall survival benefit (HR 1·23, 0·87-1·72, p=0·24). We saw no progression-free survival or overall survival benefit for gross total resection compared with near-total resection (HR 1·05, 0·71-1·53, p=0·8158 for progression-free survival and HR 1·14, 0·75-1·72, p=0·55 for overall survival). No significant survival benefit existed for greater extent of resection for patients with WNT, SHH, or group 3 tumours (HR 1·03, 0·67-1·58, p=0·89 for sub-total resection vs gross total resection). For patients with group 4 tumours, gross total resection conferred a benefit to progression-free survival compared with sub-total resection (HR 1·97, 1·22-3·17, p=0·0056), especially for those with metastatic disease (HR 2·22, 1·00-4·93, p=0·050). However, gross total resection had no effect on overall survival compared with sub-total resection in patients with group 4 tumours (HR 1·67, 0·93-2·99, p=0·084).InterpretationThe prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. Although maximum safe surgical resection should remain the standard of care, surgical removal of small residual portions of medulloblastoma is not recommended when the likelihood of neurological morbidity is high because there is no definitive benefit to gross total resection compared with near-total resection.FundingCanadian Cancer Society Research Institute, Terry Fox Research Institute, Canadian Institutes of Health Research, National Institutes of Health, Pediatric Brain Tumor Foundation, and the Garron Family Chair in Childhood Cancer Research.

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.

Three histological variants are known within the family of embryonal rosette-forming neuroepithelial brain tumors. These include embryonal tumor with abundant neuropil and true rosettes (ETANTR), ependymoblastoma (EBL), and medulloepithelioma (MEPL). In this study, we performed a comprehensive clinical, pathological, and molecular analysis of 97 cases of these rare brain neoplasms, including genome-wide DNA methylation and copy number profiling of 41 tumors. We identified uniform molecular signatures in all tumors irrespective of histological patterns, indicating that ETANTR, EBL, and MEPL comprise a single biological entity. As such, future WHO classification schemes should consider lumping these variants into a single diagnostic category, such as embryonal tumor with multilayered rosettes (ETMR). We recommend combined LIN28A immunohistochemistry and FISH analysis of the 19q13.42 locus for molecular diagnosis of this tumor category. Recognition of this distinct pediatric brain tumor entity based on the fact that the three histological variants are molecularly and clinically uniform will help to distinguish ETMR from other embryonal CNS tumors and to better understand the biology of these highly aggressive and therapy-resistant pediatric CNS malignancies, possibly leading to alternate treatment strategies.

BACKGROUND: LMD in children with recurrent medulloblastoma and other PNETs carries a poor prognosis and novel therapies are urgently needed to improve disease control. Somatostatin receptor-2 (SSR-2)is overexpressed in medulloblastoma and other central PNETs and can serve as a target for radionuclide tagged somatostatin analogues like 177Lu-DOTA-TATE that has shown considerable efficacy in adults with SSR-2 positive neuro-endocrine tumors. As a preliminary step prior to testing this agent in children with LMD, we performed an efficacy study of i.t. 177Lu-DOTA-TATE in athymic rats bearing LMD from MBL. METHODS: The subarachnoid space was accessed through the animal's cervical spine and a catheter was threaded along the dorsal aspect of spinal cord to the lumbar region and injected with 1 x 107 D341 human MBL cells and treatment initiated 3 days later. Groups of 10 animals received a single i.t. dose of 2, 3, or 5 mCi of 177Lu- DOTA-TATE or saline control. Animals were followed 300 days for survival. RESULTS: Treatment with 2 mCi resulted in an increase in median survival of 58.3% compared with saline control (p < 0.001). Treatment with 5.0 mCi of 177Lu-DOTA-TATE increased median survival by 75.0% compared with the saline control group while a single dose of 3.0 mCi 177Lu-DOTA-TATE increased median survival compared with saline controls by 519.4%. Long-term survivors were seen in 0 of 10 animals treated with saline, 4 of 11 treated with 3 mCi, and 3 of 12 treated with 5.0 mCi. CONCLUSION: Intrathecal 177Lu- DOTA TATE is efficacious in controlling LMD from medulloblastoma in athymic rats. A phase I trial of this agent is being planned in children with LMD from recurrent MBL and other CNS PNETs. INTRODUCTION: Medulloblastoma/PNET is the most common malignant brain tumor in children. For children older than 3 years, the treatment of high risk group includes surgery, craniospinal (CSI) radiation therapy (30-36 Gy) plus local boost radiotherapy (54-56 Gy) and adjuvant chemotherapy, such as cisplatinum, carboplatin, lomustine, cyclophosphamide, and vincristine. The results have demonstrated 5-year overall survival (OS) of 40-60%. This study aimed to evaluate the outcomes of high risk medulloblastoma/PNET patients who were treated with radiation and adjuvant chemotherapy. METHODS: Patients were diagnosed with high risk medulloblastoma/PNET according to the histopathology, medulloblastoma risk classification by an evidence of metastasis or the residual tumor more than 1.5 cm2 and evidence of residual tumor after surgery in PNET. Treatment protocol was CSI RT 36 Gy with local boost at tumor 54-56 Gy. Two to four weeks after RT, patients received 8 courses of chemotherapy consisting of cyclophosphamide 800 mg/m2, day 1-3 and vincristine 2 mg/m2, week 1-3, alternated with carboplatin 200 mg/m2, day 1-3 and etoposide 150 mg/m2, day 1-3. RESULTS: Total of 25 patients, male: female of 2.6:1 and mean ± SD for age of 9.7± 3.0 years, were enrolled. The 5-year progression free survival and OS were 41.6± 11.7% and 61.5± 12.9%,respectively. The age and sex did not determine the difference in outcomes. The hematotoxic side effect, according to the National Cancer Institute's Common Terminology Criteria, were grade 4 leucopenia 60%, grade 4 neutropenia 60%, grade 4 anemia 20%, grade 4 thrombocytopenia 16%, grade 3 leucopenia 20%, grade 3 neutropenia 20%, grade 3 anemia 40%, and grade 3 thrombocytopenia 36%. Febrile neutropenia was found in 11 patients (44%). CONCLUSION: The present study demonstrated the similar outcomes of high risk medulloblastoma/PNET with the previous studies. Although, the grade 3 and 4 hematologic toxicity was high, no treatment related death was found. OBJECTIVE: Recent investigations revealed an association between transcriptional subtypes and morphological features in medulloblastoma. Since both characteristics are of prognostic significance, a precise correlation between them should be well established. Therefore we re-examined paediatric nodular medulloblastoma tumours for correlation with molecular subtypes of disease. METHODS: Paediatric patients with previously diagnosed desmoplastic/nodular (D/N) or medulloblastoma with extensive nodularity (MBEN) histopathology were re-analysed by two neuropathologists. In addition to H&E-stained slides, reticulin preparations were simultaneously analysed from the same FFPE blocks. For identification of transcriptional subtypes of tumours immunohistopathological analyses were performed using a panel of representative antibodies. MYCC amplification was detected by FISH. RESULTS: Altogether 28 tumours with original MBEN or D/N diagnosis where molecular subtypes could be determined were identified. All tumours with MBEN histology belonged to SHH group and displayed distinctive reticulin-positive internodular reaction. However, only ∼60% of tumours with original D/N diagnosis were reticulin-positive. They belonged to SHH type, were mainly infantile and patients are still alive. Among reticulin-negative tumours only two were of the SHH type and were subsequently reclassified as classic and anaplastic tumours with pseudonodules. Importantly, all remaining reticulin-negative tumours with a presence of nodules in H&E staining belonged to the non-WNT,SHH type. Therefore the original diagnosis was again reclassified as classic or anaplastic tumours with pseudonodules. Patients from this group were only males, with median age 14 years old, one had MYCC amplification and two of them died because of disease. Therefore, non-WNT,SHH tumours did not display typical desmoplastic/nodular histology accompanied by reticulin positive reaction as opposite to truly D/N tumours being typical for SHH molecular group (p < 0.001). CONCLUSION: Reticulin staining is necessary to distinguish two different biologically and clinically group of nodular tumours which appear morphologically similar under H&E staining alone. BACKGROUND: In the PNET4 European randomised controlled trial, children with standard risk medulloblastoma were allocated to HFRT or to STRT. All received maintenance chemotherapy. Event-free survival was similar between the two treatment arms. HFRT was associated with worse growth and better questionnaire-based executive function 6.1 years post-diagnosis, especially in children aged 8 years at diagnosis. In children aged .10); WMI (5.20 [-2.07 to 12.47], p > .10), PSI (10.91 [-1.54 to 23.36], p = .08; FSIQ (5.28 [-4.23 to 14.79], p > .10). CONCLUSION: HFRT was associated with higher verbal IQ in children aged

PURPOSE: Medulloblastoma comprises four distinct molecular subgroups: WNT, SHH, Group 3, and Group 4. Current medulloblastoma protocols stratify patients based on clinical features: patient age, metastatic stage, extent of resection, and histologic variant. Stark prognostic and genetic differences among the four subgroups suggest that subgroup-specific molecular biomarkers could improve patient prognostication. PATIENTS AND METHODS: Molecular biomarkers were identified from a discovery set of 673 medulloblastomas from 43 cities around the world. Combined risk stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariable Cox proportional hazards analyses. Identified biomarkers were tested using fluorescent in situ hybridization (FISH) on a nonoverlapping medulloblastoma tissue microarray (n = 453), with subsequent validation of the risk stratification models. RESULTS: Subgroup information improves the predictive accuracy of a multivariable survival model compared with clinical biomarkers alone. Most previously published cytogenetic biomarkers are only prognostic within a single medulloblastoma subgroup. Profiling six FISH biomarkers (GLI2, MYC, chromosome 11 [chr11], chr14, 17p, and 17q) on formalin-fixed paraffin-embedded tissues, we can reliably and reproducibly identify very low-risk and very high-risk patients within SHH, Group 3, and Group 4 medulloblastomas. CONCLUSION: Combining subgroup and cytogenetic biomarkers with established clinical biomarkers substantially improves patient prognostication, even in the context of heterogeneous clinical therapies. The prognostic significance of most molecular biomarkers is restricted to a specific subgroup. We have identified a small panel of cytogenetic biomarkers that reliably identifies very high-risk and very low-risk groups of patients, making it an excellent tool for selecting patients for therapy intensification and therapy de-escalation in future clinical trials.

Smoothened (SMO) inhibitors recently entered clinical trials for sonic-hedgehog-driven medulloblastoma (SHH-MB). Clinical response is highly variable. To understand the mechanism(s) of primary resistance and identify pathways cooperating with aberrant SHH signaling, we sequenced and profiled a large cohort of SHH-MBs (n = 133). SHH pathway mutations involved PTCH1 (across all age groups), SUFU (infants, including germline), and SMO (adults). Children >3 years old harbored an excess of downstream MYCN and GLI2 amplifications and frequent TP53 mutations, often in the germline, all of which were rare in infants and adults. Functional assays in different SHH-MB xenograft models demonstrated that SHH-MBs harboring a PTCH1 mutation were responsive to SMO inhibition, whereas tumors harboring an SUFU mutation or MYCN amplification were primarily resistant.

Medulloblastoma is curable in approximately 70% of patients. Over the past decade, progress in improving survival using conventional therapies has stalled, resulting in reduced quality of life due to treatment-related side effects, which are a major concern in survivors. The vast amount of genomic and molecular data generated over the last 5-10 years encourages optimism that improved risk stratification and new molecular targets will improve outcomes. It is now clear that medulloblastoma is not a single-disease entity, but instead consists of at least four distinct molecular subgroups: WNT/Wingless, Sonic Hedgehog, Group 3, and Group 4. The Medulloblastoma Down Under 2013 meeting, which convened at Bunker Bay, Australia, brought together 50 leading clinicians and scientists. The 2-day agenda included focused sessions on pathology and molecular stratification, genomics and mouse models, high-throughput drug screening, and clinical trial design. The meeting established a global action plan to translate novel biologic insights and drug targeting into treatment regimens to improve outcomes. A consensus was reached in several key areas, with the most important being that a novel classification scheme for medulloblastoma based on the four molecular subgroups, as well as histopathologic features, should be presented for consideration in the upcoming fifth edition of the World Health Organizations classification of tumours of the central nervous system. Three other notable areas of agreement were as follows: (1) to establish a central repository of annotated mouse models that are readily accessible and freely available to the international research community; (2) to institute common eligibility criteria between the Childrens Oncology Group and the International Society of Paediatric Oncology Europe and initiate joint or parallel clinical trials; (3) to share preliminary high-throughput screening data across discovery labs to hasten the development of novel therapeutics. Medulloblastoma Down Under 2013 was an effective forum for meaningful discussion, which resulted in enhancing international collaborative clinical and translational research of this rare disease. This template could be applied to other fields to devise global action plans addressing all aspects of a disease, from improved disease classification, treatment stratification, and drug targeting to superior treatment regimens to be assessed in cooperative international clinical trials.

Telomerase reverse transcriptase (TERT) promoter mutations were recently shown to drive telomerase activity in various cancer types, including medulloblastoma. However, the clinical and biological implications of TERT mutations in medulloblastoma have not been described. Hence, we sought to describe these mutations and their impact in a subgroup-specific manner. We analyzed the TERT promoter by direct sequencing and genotyping in 466 medulloblastomas. The mutational distributions were determined according to subgroup affiliation, demographics, and clinical, prognostic, and molecular features. Integrated genomics approaches were used to identify specific somatic copy number alterations in TERT promoter-mutated and wild-type tumors. Overall, TERT promoter mutations were identified in 21 % of medulloblastomas. Strikingly, the highest frequencies of TERT mutations were observed in SHH (83 %; 55/66) and WNT (31 %; 4/13) medulloblastomas derived from adult patients. Group 3 and Group 4 harbored this alteration in

Medulloblastoma is the most common malignant brain tumor in children. Although aggressive surgery, radiation, and chemotherapy have improved outcomes, survivors suffer severe long-term side effects, and many patients still succumb to their disease. For patients whose tumors are driven by mutations in the sonic hedgehog (SHH) pathway, SHH antagonists offer some hope. However, many SHH-associated medulloblastomas do not respond to these drugs, and those that do may develop resistance. Therefore, more effective treatment strategies are needed for both SHH and non-SHH-associated medulloblastoma. One such strategy involves targeting the cells that are critical for maintaining tumor growth, known as tumor-propagating cells (TPC). We previously identified a population of TPCs in tumors from patched mutant mice, a model for SHH-dependent medulloblastoma. These cells express the surface antigen CD15/SSEA-1 and have elevated levels of genes associated with the G2-M phases of the cell cycle. Here, we show that CD15(+) cells progress more rapidly through the cell cycle than CD15(-) cells and contain an increased proportion of cells in G2-M, suggesting that they might be vulnerable to inhibitors of this phase. Indeed, exposure of tumor cells to inhibitors of Aurora kinase (Aurk) and Polo-like kinases (Plk), key regulators of G2-M, induces cell-cycle arrest, apoptosis, and enhanced sensitivity to conventional chemotherapy. Moreover, treatment of tumor-bearing mice with these agents significantly inhibits tumor progression. Importantly, cells from human patient-derived medulloblastoma xenografts are also sensitive to Aurk and Plk inhibitors. Our findings suggest that targeting G2-M regulators may represent a novel approach for treatment of human medulloblastoma.

PURPOSE: Reports detailing the prognostic impact of TP53 mutations in medulloblastoma offer conflicting conclusions. We resolve this issue through the inclusion of molecular subgroup profiles. PATIENTS AND METHODS: We determined subgroup affiliation, TP53 mutation status, and clinical outcome in a discovery cohort of 397 medulloblastomas. We subsequently validated our results on an independent cohort of 156 medulloblastomas. RESULTS: TP53 mutations are enriched in wingless (WNT; 16%) and sonic hedgehog (SHH; 21%) medulloblastomas and are virtually absent in subgroups 3 and 4 tumors (P < .001). Patients with SHH/TP53 mutant tumors are almost exclusively between ages 5 and 18 years, dramatically different from the general SHH distribution (P < .001). Children with SHH/TP53 mutant tumors harbor 56% germline TP53 mutations, which are not observed in children with WNT/TP53 mutant tumors. Five-year overall survival (OS; ± SE) was 41% ± 9% and 81% ± 5% for patients with SHH medulloblastomas with and without TP53 mutations, respectively (P < .001). Furthermore, TP53 mutations accounted for 72% of deaths in children older than 5 years with SHH medulloblastomas. In contrast, 5-year OS rates were 90% ± 9% and 97% ± 3% for patients with WNT tumors with and without TP53 mutations (P = .21). Multivariate analysis revealed that TP53 status was the most important risk factor for SHH medulloblastoma. Survival rates in the validation cohort mimicked the discovery results, revealing that poor survival of TP53 mutations is restricted to patients with SHH medulloblastomas (P = .012) and not WNT tumors. CONCLUSION: Subgroup-specific analysis reconciles prior conflicting publications and confirms that TP53 mutations are enriched among SHH medulloblastomas, in which they portend poor outcome and account for a large proportion of treatment failures in these patients.

Recent sequencing efforts have described the mutational landscape of the pediatric brain tumor medulloblastoma. Although MLL2 is among the most frequent somatic single nucleotide variants (SNV), the clinical and biological significance of these mutations remains uncharacterized. Through targeted re-sequencing, we identified mutations of MLL2 in 8 % (14/175) of MBs, the majority of which were loss of function. Notably, we also report mutations affecting the MLL2-binding partner KDM6A, in 4 % (7/175) of tumors. While MLL2 mutations were independent of age, gender, histological subtype, M-stage or molecular subgroup, KDM6A mutations were most commonly identified in Group 4 MBs, and were mutually exclusive with MLL2 mutations. Immunohistochemical staining for H3K4me3 and H3K27me3, the chromatin effectors of MLL2 and KDM6A activity, respectively, demonstrated alterations of the histone code in 24 % (53/220) of MBs across all subgroups. Correlating these MLL2- and KDM6A-driven histone marks with prognosis, we identified populations of MB with improved (K4+/K27-) and dismal (K4-/K27-) outcomes, observed primarily within Group 3 and 4 MBs. Group 3 and 4 MBs demonstrate somatic copy number aberrations, and transcriptional profiles that converge on modifiers of H3K27-methylation (EZH2, KDM6A, KDM6B), leading to silencing of PRC2-target genes. As PRC2-mediated aberrant methylation of H3K27 has recently been targeted for therapy in other diseases, it represents an actionable target for a substantial percentage of medulloblastoma patients with aggressive forms of the disease.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Very little is known about aggressive forms of this disease, such as metastatic or recurrent MBs. In order to identify pathways involved in aggressive MB pathophysiology, we performed unbiased, whole genome microarrays on MB tumors at both the human and murine levels. Primary human MBs were compared, transcriptomically, to their patient-matched recurrent or metastatic tumors. Expression profiling was also performed on murine tumors from two spontaneously developing MB mouse models (Ptch+/- and Smo/Smo) that present with differing clinical severities. At both the human and murine levels we identified transforming growth factor-beta (TGF-β) as a potential contributor to MB progression/metastasis. Smad3, a major downstream component of the TGF-β pathway, was also evaluated using immunohistochemistry in malignant human tissues and was shown to correlate with MB metastasis and survival. Similarly, Smad3 expression during development identified a subset of cerebellar neuronal precursors as putative cells of origin for the Smad3-positive MBs. To our knowledge, this is the first study that links TGF-β to MB pathogenesis. Our research suggests that canonical activation of this pathway leads to better prognosis for patients.

Embryonal tumor with multilayered rosettes (ETMR, previously known as ETANTR) is a highly aggressive embryonal CNS tumor, which almost exclusively affects infants and is associated with a dismal prognosis. Accurate diagnosis is of critical clinical importance because of its poor response to current treatment protocols and its distinct biology. Amplification of the miRNA cluster at 19q13.42 has been identified previously as a genetic hallmark for ETMR, but an immunohistochemistry-based assay for clinical routine diagnostics [such as INI-1 for atypical teratoid rhabdoid tumor (AT/RT)] is still lacking. In this study, we screened for an ETMR-specific marker using a gene-expression profiling dataset of more than 1,400 brain tumors and identified LIN28A as a highly specific marker for ETMR. The encoded protein binds small RNA and has been implicated in stem cell pluripotency, metabolism and tumorigenesis. Using an LIN28A specific antibody, we carried out immunohistochemical analysis of LIN28A in more than 800 childhood brain-tumor samples and confirmed its high specificity for ETMR. Strong LIN28A immunoexpression was found in all 37 ETMR samples tested, whereas focal reactivity was only present in a small (6/50) proportion of AT/RT samples. All other pediatric brain tumors were completely LIN28A-negative. In summary, we established LIN28A immunohistochemistry as a highly sensitive and specific, rapid, inexpensive diagnostic tool for routine pathological verification of ETMR.

Medulloblastoma (MB) is the most common pediatric CNS malignancy. We identify EAG2 as an overexpressed potassium channel in MBs across different molecular and histological subgroups. EAG2 knockdown not only impairs MB cell growth in vitro, but also reduces tumor burden in vivo and enhances survival in xenograft studies. Mechanistically, we demonstrate that EAG2 protein is confined intracellularly during interphase but is enriched in the plasma membrane during late G2 phase and mitosis. Disruption of EAG2 expression results in G2 arrest and mitotic catastrophe associated with failure of premitotic cytoplasmic condensation. While the tumor suppression function of EAG2 knockdown is independent of p53 activation, DNA damage checkpoint activation, or changes in the AKT pathway, this defective cell volume control is specifically associated with hyperactivation of the p38 MAPK pathway. Inhibition of the p38 pathway significantly rescues the growth defect and G2 arrest. Strikingly, ectopic membrane expression of EAG2 in cells at interphase results in cell volume reduction and mitotic-like morphology. Our study establishes the functional significance of EAG2 in promoting MB tumor progression via regulating cell volume dynamics, the perturbation of which activates the tumor suppressor p38 MAPK pathway, and provides clinical relevance for targeting this ion channel in human MBs.

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinsons disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-β signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.