Highly sensitive duplex MSI test and BAT40 germline polymorphism.

Abstract

Tumors exhibiting DNA mismatch repair (MMR) deficiency and microsatellite instability (MSI) are responsive to immune checkpoint blockade. MSI is frequently diagnosed using five quasimonomorphic mononucleotide (pentaplex) markers; however, the assays have several technical limitations, including the lack of sensitivity of some of the markers. Although markers with increased sensitivity, such as CAT25 and BAT40, have been introduced, the majority of multiplex MSI tests have only been studied in Western populations and require further evaluation in an Asian cohort. This study tested the efficacy of BAT26, CAT25, and BAT40 mononucleotide MSI markers via triplex PCR on 300 samples from patients with advanced cancers from a Korean clinical population. The results were directly compared with those of a pentaplex MSI test and tumor mutation burden (TMB) status, and an additional 60 MSI-H cancers were used for further validation. Four (1.3%) out of 300 advanced tumors were MSI-high (MSI-H). In the pentaplex PCR assay, two colorectal cancers (0.7%) exhibited instability only with the BAT25 mononucleotide marker and were interpreted as MSI-low (MSI-L). In the triplex PCR assay, BAT40 was unstable in 64 cases (21%) and the results did not overlap with those of MSI-L from pentaplex. Given the high frequency of isolated BAT40 instability, we performed the same triplex PCR with DNA obtained from normal controls and found BAT40 polymorphisms in 37 cases (90%). Interestingly, the median TMB of the cases with BAT40 polymorphism was significantly higher (7.0 mt/Mb) than that of BAT40 wild-type cases (5.5 mt/Mb) (p=0.003). The triplex PCR results from 60 additional MSI-H cancers correlated perfectly (100%) with those of pentaplex PCR, and the results were consistent for two (BAT26 and CAT25) markers. BAT40 germline polymorphism is common in the Korean population and is associated with higher TMB values. The simple duplex (BAT26 and CAT25) MSI test provided the same sensitivity and specificity as pentaplex PCR tests.