Expanding the spectrum of H3K27-altered gliomas: Hemispheric cases with midline epigenetic signatures.

Diffuse midline glioma, H3 K27-altered (DMG-H3 K27), is an aggressive CNS tumor defined by midline location, loss of H3 p.K28me3 (K27me3), and characteristic molecular alterations. Rare hemispheric gliomas with H3 K27M mutations challenge this definition and are designated "diffuse hemispheric glioma with H3 K27M mutation, NEC" (DHG-H3 K27). Their biological and clinicopathological features remain poorly defined. We retrospectively reviewed diffuse high-grade gliomas, NOS diagnosed between January 2020 and July 2025 at a single tertiary care center. Seventy-two cases underwent genome-wide DNA methylation profiling using the Illumina 935K array. Results: Three tumors radiologically interpreted as hemispheric clustered within the DMG-H3 K27 methylation class. Detailed histopathology, immunohistochemistry, radiological re-review, and whole-exome sequencing (WES; two cases) were performed. The three patients (ages 7, 15, and 33 years) presented with hemispheric, contrast-enhancing masses and underwent gross total resection. Histology showed high-grade astrocytic features in two tumors and low-grade morphology in one; all were IDH-wildtype. Methylation profiling assigned all cases to the DMG-H3 K27 class (calibrated scores ≥ 0.9) despite confirmed non-midline location. Immunohistochemistry demonstrated H3 K27M mutation with loss of H3 K27me3. WES identified H3-3A p.K28M mutations with additional alterations involving PIK3CA, FGFR1, BRAF, NF1, ATRX, and, in one case, PDGFRA, TERT, and TP53. Clinically, two patients died within 3-9 months, while one remains clinically stable despite radiological progression. DHG-H3 K27 represents a rare hemispheric glioma subgroup sharing molecular and epigenetic features of DMG-H3 K27 without midline involvement, underscoring biological heterogeneity and the importance of integrated molecular classification.