Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

Glioblastoma (GBM) is a brain tumor that carries a dismal prognosis and displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical amino acids (K27 and G34) of histone H3.3 in one-third of pediatric GBM. Here, we show that each H3F3A mutation defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and that they are mutually exclusive with IDH1 mutations, which characterize a third mutation-defined subgroup. Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM and/or established transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of transcription factors OLIG1, OLIG2, and FOXG1, possibly reflecting different cellular origins.

Summary Medulloblastoma, the most common malignant pediatric brain tumour, is currently treated with non-specific cytotoxic therapies including surgery, whole brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, prior attempts to identify targets for therapy have been underpowered due to small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1087 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 the Parkinson’s disease gene SNCAIP, 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 TGFβ signaling in Group 3, and NF-κB signaling in Group 4 suggest future avenues for rational, targeted therapy.

Midline pediatric high-grade astrocytomas (pHGAs) are incurable with few treatment targets identified. Most tumors harbor K27M mutations on histone 3 variants. In 40 treatment-naïve midline pHGAs, 39 analyzed by whole-exome sequencing, we find additional somatic mutations specific to tumor location. Gain-of-function mutations in ACVR1 occur in tumors of the pons in conjunction with H3.1 K27M, while FGFR1 mutations/fusions occur in thalamic tumors associated with H3.3 K27M. Hyper-activation of the bone morphogenetic protein (BMP)/ACVR1 developmental pathway in pHGAs harbouring ACVR1 mutations led to increased phospho-SMAD1/5/8 expression and up-regulation of BMP downstream early response genes in tumour cells. Global DNA methylation profiles were significantly associated with the K27M mutation regardless of the mutant H3 variant and irrespective of tumor location, supporting its role in driving the epigenetic phenotype. This significantly expands the potential treatment targets and further justifies pre-treatment biopsy in pHGA as a means to orient therapeutic efforts in this disease.

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