A Practical Guide to Molecular Testing in Gliomas: Implementing the WHO 2021 Classification in Resource-Variable Settings

Abstract The 2021 World Health Organization (WHO) classification of central nervous system tumors has fundamentally transformed glioma diagnostics by placing molecular alterations at the center of tumor taxonomy. Accurate classification now requires demonstration of key genetic markers, including isocitrate dehydrogenase (IDH) mutation status, 1p/19q codeletion, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) expression, H3 K27 alterations, and CDKN2A/B homozygous deletion in IDH-mutant astrocytomas, rather than relying on histomorphology alone. Access to comprehensive genomic profiling, however, remains limited across many South Asian and other low- and middle-income country settings. This narrative review proposes a practical, three-tier molecular testing strategy applicable across diverse resource environments. Tier one consists of a core immunohistochemistry (IHC) panel comprising IDH1 R132H, ATRX, p53, H3 K27M, and H3 K27me3 (mandatory for midline tumors), combined with mandatory 1p/19q codeletion testing and IDH sequencing when IHC is negative; this tier is feasible at all centers or via referral to regional molecular hubs. Tier two introduces additional selective molecular assays at tertiary centers, including telomerase reverse transcriptase (TERT) promoter mutation testing, BRAF V600E assessment, and CDKN2A/B homozygous deletion (with p16 and methylthioadenosine phosphorylase as IHC surrogate markers). Tier three reserves next-generation sequencing for diagnostically ambiguous cases, pediatric gliomas with atypical features, and recurrent disease. A stepwise laboratory flowchart, recognition of common interpretative pitfalls, and adoption of an integrated diagnostic approach support accurate WHO 2021-aligned classification while optimizing cost and resource utilization. This pragmatic framework aims to improve equity, accuracy, and outcomes in glioma care across India and other resource-variable healthcare settings.