Comparison of immunohistochemistry, next generation sequencing and fluorescence in situ hybridization for detection of MTAP loss in pleural mesothelioma

9p21 deletions involving MTAP/CDKN2A genes are detected in diffuse pleural mesotheliomas (DPM) but absent in benign mesothelial proliferations. Loss of MTAP expression by immunohistochemistry (IHC) is well accepted as a surrogate for 9p21 deletion to support a diagnosis of DPM. Accurate interpretation can be critical in the diagnosis of DPM, but variations in antibody performance may impact interpretation. The objectives of this study are to compare the performance of MTAP monoclonal antibodies (mAbs) EPR6893 and 1813 and to compare MTAP expression by IHC with 9p21 copy number status in DPM. Cytoplasmic expression of MTAP IHC with mAbs EPR6893 (ab126770; Abcam, Cambridge, MA, USA) and 1813 (NBP2-75730, Novus Biologicals, Centennial, CO, USA) was evaluated in 56 DPM (47 epithelioid, 7 biphasic, and 2 sarcomatoid) profiled by targeted NGS. 9p21 copy number status was assessed by FACETS analysis and also by CDKN2A FISH in discrepant cases when material was available. MTAP mAb 1813 showed stronger immunoreactivity, more specific staining and no equivocal interpretations compared to mAb EPR6893 which showed equivocal staining in 19 (34%) of cases due to weak or heterogenous immunoreactivity, lack of definitive internal positive control, and/or non-specific background staining. MTAP expression with mAb 1813 showed near perfect agreement with 9p21 copy number by combined FACETS/FISH calls (Kappa=0.85; 95% IC: 0.71-0.99, P<0.001). MTAP IHC with mAb 1813 was 96% sensitive, 86% specific and 93% accurate for 9p21 homozygous deletion. The findings of this study suggest that interpretation of MTAP IHC is improved with mAb 1813 since mAb EPR6893 was often limited by equivocal interpretations. We show that MTAP IHC and molecular assays are complementary in detecting 9p21 homozygous deletion. MTAP IHC may be particularly useful for low tumor purity samples and in low-resource settings.