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 PRMT5-MAT2A 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 2,409 tumors profiled by whole-exome sequencing within the EXOMA2 study (NCT06550687) using the FACETS and TITAN algorithms. Tumors were categorized as wild-type, homozygous deletion, or probable heterozygous deleted. A subset of these cases with available tissue underwent orthogonal validation by immunohistochemistry (IHC) using three MTAP antibody clones (MTAP-RBT, 2G4, and 1813), with selected cases further analyzed by fluorescence in situ hybridization (FISH) and RNA sequencing. Our results show 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 by NGS; loss of protein expression in these cases correlated with homozygous genomic deletion confirmed by FISH. Moreover, heterozygous NGS calls were not associated with low tumor cellularity. These findings demonstrate that MTAP immunohistochemistry 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 improve the identification of MTAP-deficient tumors and facilitate more accurate, rapid, and cost-effective patient selection for emerging targeted therapies.