DNA methylation profiling reveals a novel subtype harboring IDH mutation in H3-altered gliomas

BACKGROUND: H3-altered gliomas, now recognized by WHO CNS5 as grade 4 neoplasms driven by histone H3 mutations (notably H3K27M and H3G34R/V), exhibit marked molecular heterogeneity, dismal survival, and resistance to standard therapies. Despite their classification as WHO grade 4 neoplasms, current stratification fails to capture their clinical and epigenetic diversity. METHODS: We performed genome-wide DNA methylation profiling on 49 representative cases from a clinical cohort of 375 H3-altered gliomas and validated subtype classifications using TCGA data. Single-cell RNA sequencing, ChIP-seq, and spatial transcriptomic analyses were employed to elucidate tumor-intrinsic programs and microenvironmental niches across subtypes. RESULTS: Unsupervised clustering identified four robust DNA methylation-defined subtypes: DMG_K27M, GBM_RTK, GBM_G34, and IDH/H3_comut, a novel subtype harboring co-occurring IDH and H3 mutations. The IDH/H3_comut subtype exhibited global DNA hypermethylation and significantly improved survival. In contrast, GBM_RTK tumors showed hypomethylation of cell-cycle enhancers and aggressive phenotypes, while GBM_G34 and DMG_K27M displayed subtype-specific epigenetic features. Single-cell transcriptomics revealed distinct lineage compositions and microenvironments, with IDH/H3_comut tumors enriched in NPC-like cells and GBM_RTK in astrocyte-like and vascular mimicry programs. Clonal and spatial analyses uncovered a compartmentalized coexistence of IDH- and H3-mutant cells, suggesting cooperative rather than exclusive evolution. CONCLUSIONS: Our study redefines H3-altered gliomas through methylation-based taxonomy, identifies a novel IDH/H3_comut subtype with favorable prognosis, and reveals subtype-specific therapeutic vulnerabilities, including potential responsiveness to hypomethylating agents or CDK4/6 inhibitors.