Immunohistochemistry Resolves Indeterminate MTAP Deletion Calls From Exome Sequencing: A Pan-Cancer Concordance Study

The deletion of the methylthioadenosine phosphorylase (MTAP) gene is a frequent genomic event across various malignancies, often associated with the loss of the adjacent CDKN2A locus. MTAP deficiency has recently emerged as a promising biomarker for novel targeted therapeutic strategies, including inhibitors of the protein arginine methyltransferase 5-methionine adenosyltransferase 2A axis. Although next-generation sequencing (NGS) allows detection of MTAP copy number alterations, interpretation may be challenging in samples with low tumor purity or borderline copy number scores. In this study, we analyzed MTAP copy number status in a cohort of 2409 tumors profiled using whole-exome sequencing within the EXOMA2 study (NCT06550687) using the Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing and Two-factor Inference of Tumor Architecture and Nucleotide-variant algorithms. Tumors were categorized as wild-type, homozygous deletion, or probable heterozygous deletion. A subset of these cases with available tissue underwent orthogonal validation using immunohistochemistry (IHC) with 3 MTAP antibody clones (MTAP-RBT, 2G4, and 1813), with selected cases further analyzed using fluorescence in situ hybridization (FISH) and RNA sequencing. Our results showed high concordance between IHC and genomic classification in tumors with either wild-type or homozygous deletion MTAP status. Importantly, IHC enabled reclassification of a substantial proportion of tumors initially classified as heterozygous using NGS; loss of protein expression in these cases correlated with homozygous genomic deletion confirmed using FISH. Moreover, heterozygous NGS calls were not associated with low tumor cellularity. These findings demonstrated that MTAP IHC represents a robust and accessible approach for assessing MTAP status and for resolving ambiguous NGS results, an issue not solely driven by low tumor cellularity. Incorporating protein-level assessment analyses with FISH confirmation improves the identification of MTAP-deficient tumors and facilitates a more accurate, rapid, and cost-effective patient selection for emerging targeted therapies.