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Relapsed medulloblastoma (rMB) is treatment-resistant and fatal in ~95% of cases. The epigenetic features of rMB, and any role as drivers of disease relapse/treatment-resistance have yet to be investigated. We therefore developed a pipeline to identify differentially methylated CpGs (DM-CpGs) and regions (DMRs) in a paired-rMB cohort. Our paired-rMB cohort (n=61, relapsed tumours matched with diagnosis counterparts) with available Illumina Methylation 450K/850K microarray data was processed in R-Studio. The packages Limma and DMRcate were used to perform a paired differential methylation analysis on a filtered selection of array probes (n=335,767), identifying DM-CpGs and DMRs with a 5% FDR. DMRs were further retained if they had a maximum-Δβ of >0.2 and correlated with locus-specific gene expression in a separate paired DNA-methylation array/RNA-seq cohort from medulloblastoma diagnosis samples (n=202). Finally, we created univariable Cox models to assess the prognostic potential of DM-CpGs/DMRs in an independent survival cohort of medulloblastoma diagnosis samples (n=498). Across the paired-rMB cohort, there were few significant differential methylation events initially identified at relapse (n=258 DM-CpGs, n=32 DMRs). Upon sub-analysis by molecular group, MBGroup4 (n=18 pairs) alone yielded significant findings (n=189 DM-CpGs, n=26 DMRs). Most changes involved hypermethylation events detected at relapse. Multiple DM-CpGs identified at relapse were prognostic for both overall and event-free survival when assessed in our independent cohort (n=22 whole cohort, n=13 Group 4, BH-adjusted p

Figure S2. Kaplan Meier Survival Analysis of Group 3 medulloblastomas from the Indian cohort comparing overall survival of ‘miR-204 high’ subset with that of ‘miR-204 low’ subset. (PPTX 69 kb)

Table S1. The nucleotide sequences of the primers used in the study. All sequences are given in 5â ˛ to 3â ˛ direction. (DOCX 15 kb)

Figure S1. Kaplan-Meier subgroup analysis. A, 10-year Kaplan-Meier subgroup analysis in the exploratory dataset. B, 10-year Kaplan-Meier subgroup analysis in the validation dataset. Figure S2. Association between the average relative expression of prognostic core genes and subtype and clinical characteristics. A, Boxplots of relative expression of 31 core genes from the top two PID pathways in SHH subgroup. B, Boxplots of relative expression of 20 core genes from the top two PID pathways in Group 3. C, Boxplots of relative expression of 31 core genes from the top two PID pathways in Group 4. A, large cell/anaplastic (LCA); C, Classic; D, Desmoplastic; M, Medulloblastoma with extensive nodularity (MBEN). *, Mann–Whitney U test p-value 0.4. Figure S6. Differential expressions of the prognostic genes identified in Group 4. A, Boxplots of gene expressions in four subgroups. B, Differential gene expressions with Mann–Whitney U test p-values according to the status of chromosome aberrations in Group 4. (PDF 1629 kb)

Figure S3. Methylation analysis of the CpG island from the promoter region of TRPM3/MIR204. A 203â bp region of the CpG island from the promoter region of the TRPM3 gene was PCR amplified from bisulfite converted genomic DNA of the medulloblastoma cell lines. Representative nucleotide sequence of this PCR product from the indicated medulloblastoma cell line is shown. Arrows indicate the CpG residues in the DNA sequence and their sequence in the bisulfite converted (BSP) DNA from the medulloblastoma cells. (PPTX 121 kb)

Figure S1. MiR-204 expression levels in the 8 subtypes of Group 3 / Group 4 medulloblastomas from Northcott et al. [34] data. (PPTX 39 kb)

Table S6. Univariate and multivariate analysis of ST and PF ependymomas in PFS and OS. (DOCX 44 kb)

Table S5. Validation results of PF-EPN subgroup prediction rule candidates. (DOCX 16 kb)

The Japan Pediatric Molecular Neuro-Oncology Group (JPMNG): participating centers and departments. (DOCX 17 kb)

PurposePosterior fossa ependymoma comprises two distinct molecular variants termed EPN_PFA and EPN_PFB that have a distinct biology and natural history. The therapeutic value of cytoreductive surgery and radiation therapy for posterior fossa ependymoma after accounting for molecular subgroup is not known.MethodsFour independent nonoverlapping retrospective cohorts of posterior fossa ependymomas (n = 820) were profiled using genome-wide methylation arrays. Risk stratification models were designed based on known clinical and newly described molecular biomarkers identified by multivariable Cox proportional hazards analyses.ResultsMolecular subgroup is a powerful independent predictor of outcome even when accounting for age or treatment regimen. Incompletely resected EPN_PFA ependymomas have a dismal prognosis, with a 5-year progression-free survival ranging from 26.1% to 56.8% across all four cohorts. Although first-line (adjuvant) radiation is clearly beneficial for completely resected EPN_PFA, a substantial proportion of patients with EPN_PFB can be cured with surgery alone, and patients with relapsed EPN_PFB can often be treated successfully with delayed external-beam irradiation.ConclusionThe most impactful biomarker for posterior fossa ependymoma is molecular subgroup affiliation, independent of other demographic or treatment variables. However, both EPN_PFA and EPN_PFB still benefit from increased extent of resection, with the survival rates being particularly poor for subtotally resected EPN_PFA, even with adjuvant radiation therapy. Patients with EPN_PFB who undergo gross total resection are at lower risk for relapse and should be considered for inclusion in a randomized clinical trial of observation alone with radiation reserved for those who experience recurrence.

The development of targeted anti-cancer therapies through the study of cancer genomes is intended to increase survival rates and decrease treatment-related toxicity. We treated a transposon-driven, functional genomic mouse model of medulloblastoma with 'humanized' in vivo therapy (microneurosurgical tumour resection followed by multi-fractionated, image-guided radiotherapy). Genetic events in recurrent murine medulloblastoma exhibit a very poor overlap with those in matched murine diagnostic samples (

PurposeMyxopapillary ependymoma (MPE) is a distinct histologic variant of ependymoma arising commonly in the spinal cord. Despite an overall favorable prognosis, distant metastases, subarachnoid dissemination, and late recurrences have been reported. Currently, the only effective treatment for MPE is gross-total resection. We characterized the genomic and transcriptional landscape of spinal ependymomas in an effort to delineate the genetic basis of this disease and identify new leads for therapy.Experimental designGene expression profiling was performed on 35 spinal ependymomas, and copy number profiling was done on an overlapping cohort of 46 spinal ependymomas. Functional validation experiments were performed on tumor lysates consisting of assays measuring pyruvate kinase M activity (PKM), hexokinase activity (HK), and lactate production.ResultsAt a gene expression level, we demonstrate that spinal grade II and MPE are molecularly and biologically distinct. These are supported by specific copy number alterations occurring in each histologic variant. Pathway analysis revealed that MPE are characterized by increased cellular metabolism, associated with upregulation of HIF1α. These findings were validated by Western blot analysis demonstrating increased protein expression of HIF1α, HK2, PDK1, and phosphorylation of PDHE1A. Functional assays were performed on MPE lysates, which demonstrated decreased PKM activity, increased HK activity, and elevated lactate production.ConclusionsOur findings suggest that MPE may be driven by a Warburg metabolic phenotype. The key enzymes promoting the Warburg phenotype: HK2, PKM2, and PDK are targetable by small-molecule inhibitors/activators, and should be considered for evaluation in future clinical trials for MPE.

Previously, we have identified nine distinct molecular groups of ependymoma across all age groups, three in each major anatomical compartment of the CNS: spinal, posterior fossa, and supratentorial. These nine molecular groups are genetically, epigenetically, transcriptionally, demographically, and clinically distinct. Pediatric intracranial ependymomas are either affiliated with the RELA and YAP1 supratentorial groups or with the posterior fossa PFA group. RELA and YAP1 ependymomas harbor recurrent and distinct fusion genes that drive the disease, but PFA ependymomas lack significant recurrent mutations, and the specific oncogenic events underlying these tumors remain undefined. Observing distinct outcomes among children with PFA ependymomas, we tested the hypothesis that further molecular diversity with clinical utility, including possible novel genetic alterations, may exist among these tumors. Genome-wide DNA methylation profiles of 675 pediatric PFA ependymomas were analyzed by unsupervised consensus hierarchical clustering and t-distributed stochastic neighbor embedding (tsne), which identified not only three major distinct molecular subgroups of PFA ependymoma: PFA-1, PFA-2, and PFA-3, but additional small subgroups that segregate within PFA-1 and PFA-2. Subgroups were characterized by distinct clinical and genetic characteristics; most PFA-3 tumors harbored 1q gain and occurred in children significantly older than those with PFA-1 and PFA-2 tumors. Some of the small subgroups demonstrated distinctive copy number alterations or gene mutations, e.g. H3 K27M. Outcome data revealed considerable heterogeneity among the different subgroups, overall survival at 5 years was >90% for one subgroup, but

Medulloblastoma (MB) is the most common malignant primary brain tumor and is currently classified into 5 distinct molecular subtypes based on genomic alterations, gene expression profile, response to treatment and cell of origin. This extensive heterogeneity has revealed a critical need for subtype-specific, functionally validated biomarkers and therapeutic strategies. Using an unbiased high throughput flow cytometry screen and gain/loss of function studies, we previously identified CD271/p75NTR as a candidate stem/progenitor cell marker, specifically in SHH MB. Here, we show that CD271+ and CD271- subpopulations do not exhibit a hierarchal organization; but are rather two co-existing cellular subsets. FACS sorting followed by re-culturing of CD271- and CD271+ cells from low passage primary cultures demonstrated that both cell populations can recapitulate the parental phenotype. However, global gene expression profiling by RNA sequencing revealed that genes/pathways associated with cell survival, proliferation and motility are downregulated in CD271- vs. CD271+ subpopulations. Cell cycle analysis supported these findings and demonstrated that CD271- cells exhibited a decrease in proliferation compared to CD271+ cells. Moreover, PI3K/AKT, JAK/STAT, and NFkB, are downregulated in CD271- vs. CD271+ cells, while pathways such as RAS/MYC are upregulated in CD271- vs. CD271+ cells. Immunohistochemical (IHC) analysis of CD271 levels across the MB subtypes demonstrated that nearly all SHH tumors express CD271, while Group 3 tumors exhibit moderate staining. In contrast, Group 4 MB and WNT do not express CD271. Importantly SHH MB exhibit a nodular CD271 staining pattern, further underscoring the heterogeneity within these tumors and supporting our findings that CD271- and CD271+ cells are distinct, yet clinically relevant, subpopulations. Our results suggest that CD271, in combination with other markers, could be effectively utilized as a diagnostic tool for SHH MB and that therapeutic strategies concomitantly targeting both CD271+ and CD271- subpopulations would be the most effective in SHH MB treatment.

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.

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 Parkinson's 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.

Despite decades of research, brain tumours remain among the deadliest of all forms of cancer. The ability of these tumours to resist almost all conventional and novel treatments relates, in part, to the unique cell-intrinsic and microenvironmental properties of neural tissues. In an attempt to encourage progress in our understanding and ability to successfully treat patients with brain tumours, Cancer Research UK convened an international panel of clinicians and laboratory-based scientists to identify challenges that must be overcome if we are to cure all patients with a brain tumour. The seven key challenges summarized in this Position Paper are intended to serve as foci for future research and investment.

BACKGROUND: Medulloblastoma 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. METHODS: In 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. FINDINGS: We 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. INTERPRETATION: Genetic 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. FUNDING: German 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.

Acknowledgements: The KiCS program is supported by the Garron Family Cancer Centre at The Hospital for Sick Children through funding from the SickKids Foundation. A.H. received funding from the Canadian Institutes for Health Research (grant no. 162267) and is the Tier 1 Canada Research Chair in Rare Childhood Brain Tumors. D.M. is supported by the CIBC Children’s Foundation Chair in Child Health Research. A.S. is partially supported by an Early Researcher Award from the Ontario Ministry of Research and Innovation; by the Canada Research Chair in Childhood Cancer Genomics; and by funding from the V Foundation and the Robert J. Arceci Innovation Award from the St. Baldrick’s Foundation. We would like to thank the Centre for Applied Genomics, The Hospital for Sick Children, for assistance with RNA sequencing and the Treehouse Childhood Cancer Initiative, University of California, Santa Cruz, for access to their public data repository., The causes of pediatric cancers' distinctiveness compared to adult-onset tumors of the same type are not completely clear and not fully explained by their genomes. In this study, we used an optimized multilevel RNA clustering approach to derive molecular definitions for most childhood cancers. Applying this method to 13,313 transcriptomes, we constructed a pediatric cancer atlas to explore age-associated changes. Tumor entities were sometimes unexpectedly grouped due to common lineages, drivers or stemness profiles. Some established entities were divided into subgroups that predicted outcome better than current diagnostic approaches. These definitions account for inter-tumoral and intra-tumoral heterogeneity and have the potential of enabling reproducible, quantifiable diagnostics. As a whole, childhood tumors had more transcriptional diversity than adult tumors, maintaining greater expression flexibility. To apply these insights, we designed an ensemble convolutional neural network classifier. We show that this tool was able to match or clarify the diagnosis for 85% of childhood tumors in a prospective cohort. If further validated, this framework could be extended to derive molecular definitions for all cancer types.

Despite decades of research, brain tumours remain among the deadliest of all forms of cancer. The ability of these tumours to resist almost all conventional and novel treatments relates, in part, to the unique cell-intrinsic and microenvironmental properties of neural tissues. In an attempt to encourage progress in our understanding and ability to successfully treat patients with brain tumours, Cancer Research UK convened an international panel of clinicians and laboratory-based scientists to identify challenges that must be overcome if we are to cure all patients with a brain tumour. The seven key challenges summarized in this Position Paper are intended to serve as foci for future research and investment.

Posterior fossa ependymoma comprise three distinct molecular variants, termed PF-EPN-A (PFA), PF-EPN-B (PFB), and PF-EPN-SE (subependymoma). Clinically, they are very disparate and PFB tumors are currently being considered for a trial of radiation avoidance. However, to move forward, unraveling the heterogeneity within PFB would be highly desirable. To discern the molecular heterogeneity within PFB, we performed an integrated analysis consisting of DNA methylation profiling, copy-number profiling, gene expression profiling, and clinical correlation across a cohort of 212 primary posterior fossa PFB tumors. Unsupervised spectral clustering and t-SNE analysis of genome-wide methylation data revealed five distinct subtypes of PFB tumors, termed PFB1-5, with distinct demographics, copy-number alterations, and gene expression profiles. All PFB subtypes were distinct from PFA and posterior fossa subependymomas. Of the five subtypes, PFB4 and PFB5 are more discrete, consisting of younger and older patients, respectively, with a strong female-gender enrichment in PFB5 (age: p = 0.011, gender: p = 0.04). Broad copy-number aberrations were common; however, many events such as chromosome 2 loss, 5 gain, and 17 loss were enriched in specific subtypes and 1q gain was enriched in PFB1. Late relapses were common across all five subtypes, but deaths were uncommon and present in only two subtypes (PFB1 and PFB3). Unlike the case in PFA ependymoma, 1q gain was not a robust marker of poor progression-free survival; however, chromosome 13q loss may represent a novel marker for risk stratification across the spectrum of PFB subtypes. Similar to PFA ependymoma, there exists a significant intertumoral heterogeneity within PFB, with distinct molecular subtypes identified. Even when accounting for this heterogeneity, extent of resection remains the strongest predictor of poor outcome. However, this biological heterogeneity must be accounted for in future preclinical modeling and personalized therapies.