基于MTAP蛋白染色缺失检测纯合性甲硫腺苷磷酸化酶（MTAP）基因缺失的自动化免疫组织化学检测

Loss of both MTAP alleles on the short arm of chromosome 9 in tumor cells leading to accumulation of methylthioadenosine (MTA) creates an opportunity to disrupt cellular processes including protein methylation, RNA splicing, and DNA repair in the presence of an MTA-cooperative protein arginine methyltransferase 5 (PRMT5) inhibitor which selectively targets them for cell death. This study describes an automated immunohistochemical (IHC) assay based on the novel rabbit monoclonal antibody SP530. This MTAP (SP530) IHC assay is capable of detecting the MTAP enzyme with high sensitivity and specificity in formalin-fixed, paraffin-embedded (FFPE), tissue sections on the VENTANA BenchMark ULTRA and BenchMark ULTRA PLUS staining platforms in conjunction with the OptiView DAB IHC Detection Kit. In western blot analysis, the SP530 antibody recognized a single band of approximately 30kD whose intensity levels correlated with reported MTAP mRNA levels in cultured cancer cell lines. The MTAP (SP530) IHC assay produced staining in both nuclear and cytoplasmic compartments of tumor and normal cells consistent with prior literature and such staining was completely abrogated in the presence of a peptide encompassing the SP530 epitope. Specificity of the SP530 clone was comparable to other commercial monoclonal anti-MTAP antibodies in cancer cell lines with known MTAP genetic status or mRNA expression levels. The ability of the MTAP (SP530) IHC assay to detect biallelic MTAP loss in FFPE tumor sections was confirmed using a brightfield in situ hybridization (ISH) assay. The MTAP (SP530) IHC assay did not generate any staining in the nuclear or the cytoplasmic compartments of tumor cells in most tumor specimens with homozygous MTAP deletion, while non-neoplastic cells exhibited MTAP signal. In a small subset of specimens, the lack of MTAP ISH signals in tumor cells was associated with blush MTAP IHC staining of less than 1+ intensity in the cytoplasm or the nucleus. Such equivocal MTAP staining was not restricted to the SP530 clone, suggesting it is not off-target or background. In a cohort of 475 tumor specimens, pancreatic carcinoma, gastric carcinoma, non-small cell lung carcinoma, cholangiocarcinoma, and glioblastoma specimens exhibiting zero or lower than 1+ intensity MTAP staining in at least 75% of tumor cells were found at frequencies ranging from 9% to 56% in general agreement with previous literature. Vast majority of the specimens with 75% or more of tumor cells with zero or lower than 1+ staining exhibited a clonal pattern of deficient MTAP staining consistent with biallelic MTAP loss at an early stage in tumor development. Approximately 10% of the specimens displayed lack of nuclear MTAP staining in 20% to 75% of tumor cells and these exhibited subclonal or mosaic patterns, suggesting MTAP loss can occur at later stages of tumor development.