A practical targeted molecular workflow resolves the World Health Organization 2021 integrated diagnosis of diffuse gliomas

The 2021 World Health Organization (WHO) Classification of Tumors of the Central Nervous System requires molecular alterations for the integrated diagnosis of diffuse gliomas but the necessity of comprehensive genomic profiling in routine practice remains uncertain. We retrospectively evaluated a practical molecular diagnostic workflow using Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) in 307 consecutive diffuse gliomas diagnosed between March 2019 and January 2025. Histopathological diagnoses were established independently by 2 board-certified neuropathologists and compared with the final WHO 2021 integrated diagnoses. Targeted molecular testing identified diagnostically relevant mutations and copy-number alterations. Integrated diagnoses were established in 288 of 307 tumors (93.8%), leaving only 19 cases (6.2%) unresolved. Molecular findings changed the histopathological diagnosis in 60 tumors (19.5%), whereas 247 tumors (80.5%) showed concordant histopathological and integrated diagnoses. Reclassification most frequently involved molecular glioblastoma, H3K27-altered diffuse glioma, oligodendroglioma defined by whole-arm 1p/19q codeletion, astrocytoma upgraded to WHO grade 4 based on CDKN2A/B homozygous deletion, and diffuse hemispheric glioma, H3 G34-mutant. These results demonstrate that a workflow combining Sanger sequencing and MLPA provides a practical approach for establishing WHO 2021 integrated diagnoses in most diffuse gliomas, with only a small subset requiring additional molecular characterization.