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Two recurrent mutations, K27M and G34R/V, in H3F3A, encoding non-canonical histone H3.3, are reported in pediatric and young adult gliomas, whereas G34W mutation is prevalent in bone tumors. In contrast to K27M mutation, it remains elusive how G34 mutations affect the epigenome. Here we performed whole-genome bisulfite sequencing of four G34R-mutated gliomas and the G34V-mutated glioma cell line KNS-42 for comparison with gliomas harboring K27M and no mutations in H3F3A and with G34W-mutated bone tumors. G34R-mutated gliomas exhibited lower global methylation levels, similar CpG island (CGI) methylation levels, and compromised hypermethylation of telomere-proximal CGIs, compared to the other two glioma subgroups. Hypermethylated regions specific to G34R-mutated gliomas were enriched for CGIs, including those of OLIG1, OLIG2, and canonical histone genes in the HIST1 cluster. They were notably hypermethylated in osteosarcomas with, but not without, G34W mutation. Independent component analysis revealed that G34 mutation-specific components shared a significant similarity between glioma and osteosarcoma, suggesting that G34 mutations exert characteristic methylomic effects regardless of the tumor tissue-of-origin. CRISPR/Cas9-mediated disruption of G34V-allele in KNS-42 cells led to demethylation of a subset of CGIs hypermethylated in G34R-mutated gliomas. These findings will provide a basis for elucidating epigenomic roles of G34 oncohistone in tumorigenesis.

A recurrent glycine-to-arginine/valine alteration at codon 34 (G34R/V) within H3F3A, a gene that encodes the replication-independent histone variant H3.3, reportedly occurs exclusively in pediatric glioblastomas. However, the clinicopathological and biological significances of this mutation have not been completely elucidated; especially, no such data exist for tumor samples from Japanese patients. We analyzed 411 consecutive glioma cases representing patients of all ages. Our results demonstrated that 14 patients (3.4%) harbored H3F3A mutations, of which four had G34R mutations and 10 had K27M mutations. G34R-mutant tumors were located in the parietal region in two patients and the basal ganglia in one patient. One patient showed multi-lobular extension similar to the pattern observed in gliomatosis cerebri. Regarding neuroradiological features, intratumoral calcification was evident in two cases and all cases showed no or scarce contrast enhancement on MRI. Histopathologically, the four G34R-mutant cases included three glioblastomas and one astroblastoma. We have also investigated alterations in histone methylation including H3K27me3, H3K9me3, and H3K4me3 in G34R-mutant samples by immunohistochemistry. These results indicate that G34R-mutant tumors are likely to show extensive infiltration and alterations in global histone trimethylation might also play an important role in G34R mutant tumors.

Brain tumors harbor various BRAF alterations, the vast majority of which are the BRAF kinase-activating V600E mutation. BRAF mutations are most frequently detected in certain subtypes of low-grade glioma, such as pilocytic astrocytoma (PA), pleomorphic xanthoastrocytoma (PXA), ganglioglioma (GG) and dysembryoplastic neuroepithelial tumor (DNT). However, it is unclear whether gliomas harboring BRAF mutations can be invariably regarded as these glioma subtypes or their derivatives. To address this question, we analyzed 274 gliomas in our institutional case series. We performed high-resolution melting analyses and subsequent direct Sanger sequencing on DNA isolated from snap-frozen tumor tissues. As expected, BRAF mutations were detected in the aforementioned low-grade gliomas: in 4/27 PAs, 2/3 PXAs, 4/8 GGs, and 1/6 DNTs. In addition to these gliomas, 1/2 astroblastomas (ABs) and 2/122 glioblastomas (GBs) harbored BRAF mutations. Pathological investigation of the two GBs revealed that one was a GB displaying epithelial features that presumably arose from a precedent GG, whereas the other GB, which harbored a rare G596 A mutation, showed marked epithelial features, including astroblastic rosettes. Our results indicate that in addition to being present in established BRAF-associated gliomas, BRAF mutations might be associated with epithelial features in high-grade gliomas, including sheet-like arrangement of polygonal tumor cells with a plump cytoplasm and astroblastic rosettes, and thus could potentially serve as a genetic marker for these features., (© 2016 Japanese Society of Neuropathology.)

Isocitrate dehydrogenase (IDH) mutation is a good prognostic marker for glioblastoma (GBM). Although it is infrequent in primary tumors, it is found in most lower-grade gliomas. Thus, it is unclear whether IDH mutation is a marker for a specific phenotype of apparently primary de novo GBMs (pGBMs), or a marker for secondary tumors (sGBMs). We addressed this issue by analyzing clinical, radiographic and molecular findings in our institutional case series. Our cases included 92 pGBMs, with five cases of IDH1 mutations at R132 and no IDH2 mutations. The median overall survival of these five patients was 29 months (range: 4 to >40 months), which is considered good prognoses. Clinical and radiographic characteristics were distinct from IDH-wildtype (IDH-wt) pGBMs. IDH-mutant (IDH-mut) tumors consistently involved insular lesions and were subdivided into: (i) the two cases of elderly patients with long clinical histories and features implying multistep tumor development; and (ii) the three cases of younger patients with diffusely swelling insular tumors, slight contrast enhancement and no necrosis. Genetic and expression analyses of IDH-mut pGBMs were similar to those of sGBMs, suggesting that they are indeed distinct from their IDH-wt counterparts. TERT promoter mutation, a genetic marker of oligodendroglial derivation, was detected in one long-surviving case, but genetic alterations in the astrocyte-sGBM pathway were generally prevalent in IDH-mut pGBMs. Our results present a unique phenotype of IDH-mut pGBMs arising from insular cortex region, the molecular backgrounds of which are similar to sGBMs., (© 2017 Japanese Society of Neuropathology.)