Development of new droplet digital PCR method for MSI assessment with automated analysis pipeline shows concordance in FFPE and plasma

Abstract Introduction: Microsatellite instability (MSI) arises when short tandem DNA repeat sequences undergo a length change due to impaired mismatch repair (MMR). MSI is present in many cancers and is an approved biomarker for immune checkpoint inhibitor therapy. In this study, we used 71 clinical specimens to evaluate a new droplet digital (ddPCR) method to assess MSI in tissue and in plasma. The ddPCR assay is designed to detect mutations in BAT25, BAT26, NR21, NR24 and Mono27, and is intended for use in FFPE. Our evaluation extended these analyses to cfDNA in plasma. Methods: DNA was extracted from ten (10) CRC FFPE samples, fifty-two (52) CRC liquid biopsies, and ten (10) Normal Healthy Donors (NHD) and tested using the new assay. DNA extracts were quantified by fluorescence, and duplicate ddPCR reactions were performed using 2.6 ng input DNA per well. MSI status was determined with a pre-commercial release auto-calling analysis software, which makes a positive call (MSI-High) when two or more of the five MSI markers (> 40%) are detected. Markers are called positive if the fractional abundance or copy number is greater than the previously established limit of blank (LOB) in FFPE and plasma, respectively. For FFPE, the LOBs (%) are 2.11 (BAT25), 0.34 (BAT26), 2.38 (NR21), 1.51 (NR24) and 0.56 (Mono27) based on analytic specimen evaluation. For plasma, a marker is positive if one (1) mutant copy is detected. Results: All 10 FFPE samples were deemed to be concordant with the reference Promega PCR assay at the microsatellite evaluation level. Additionally, the 8 cfDNA samples with reference MSI status yielded 87.5% (7/8) concordant results for microsatellite loci status. A survey of 44 plasma colorectal donor samples and 10 NHD samples with no reference results, yielded various microsatellite status as expected. The auto calling module for microsatellite loci status in these plasma samples accurately called 85.2 status (n = 54). At the marker level, 87% of BAT25, 98.1% of BAT26, 92.6% of NR21, 79.6% of NR24, and 61.1% of Mono27 calls were concordant between auto-calling and expert review. Conclusion: The new droplet digital MSI assay coupled with an automated calling module for MSI Status provides a convenient, cost-effective, and reproducible laboratory test method to determine MSI status in FFPE specimens. The utility of the test method and auto calling method for detection of MSI in cfDNA is less clear, with heterogenous clusters that require expert review appearing more often than in FFPE samples. Citation Format: Sarah Kreston, Mai Ho, Claire Gould, Ubaradka Sathyanarayana, Brittany D'Alessio, Janice Riley, Gary A. Pestano. Development of new droplet digital PCR method for MSI assessment with automated analysis pipeline shows concordance in FFPE and plasma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4339.