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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.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

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.

Ependymal tumors across age groups are currently classified and graded solely by histopathology. It is, however, commonly accepted that this classification scheme has limited clinical utility based on its lack of reproducibility in predicting patients' outcome. We aimed at establishing a uniform molecular classification using DNA methylation profiling. Nine molecular subgroups were identified in a large cohort of 500 tumors, 3 in each anatomical compartment of the CNS, spine, posterior fossa, supratentorial. Two supratentorial subgroups are characterized by prototypic fusion genes involving RELA and YAP1, respectively. Regarding clinical associations, the molecular classification proposed herein outperforms the current histopathological classification and thus might serve as a basis for the next World Health Organization classification of CNS tumors.

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.

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.