Assessment of microsatellite instability (MSI) in cell free DNA (cfDNA) of colorectal cancers (CRC) patients (pts).

Abstract

672 Background: While there is accumulating evidence that tumor specific mutations can be accurately detected in cfDNA, the determination of MSI status in cfDNA has not been investigated, perhaps because the complexity of detecting tumor instability embedded within stutter amplification patterns generated from normal cfDNA was considered too challenging to allow the detection of altered microsatellite profiles. We explored the possibility of detecting MSI in cfDNA. Methods: Circulating cfDNA was isolated from Streck tubes from 13 patients with CRC; 6 pts had serial collections. Isolation and testing of cfDNA for MSI was performed using kits purchased from Promega. Microsatellite alterations were scored by comparing the amplicon stutter profiles of 5 mononucleotide markers generated from cfDNA to those present in genomic DNA extracted from buffy coats from the same pts. MSI was diagnosed based on a shift in the size of a predominant stutter peak together with a commensurate shift in the stutter profile resulting in the appearance of at least one new stutter in tumor-derived DNA compared to matched germline DNA. MSI was scored high when 30% or more of the markers showed MSI or low if present in less than 30% of the biomarkers examined. Results: No MSI was detected in 3 pts with microsatellite stable tumors and 2 Lynch syndrome pts with no evidence of disease. Five of six pts with MSI high tumors (sporadic and Lynch syndrome) had a detectable MSI high biomarker profile in cfDNA at baseline. Four pts with positive MSI in cfDNA started immunotherapy and had a decline in the relative amounts of tumor-derived microsatellite alleles in their subsequent specimens. The pt with MSI high in the primary tumor and no MSI in cfDNA had rapid progression on immunotherapy. Two other pts with MSI high who responded deeply to check point inhibitors had no evidence of MSI in cfDNA at the time of their specimen collection. In each case with MSI in cfDNA, the electrophoretic profile matched that in the respective primary tumor. Conclusions: Detection of MSI in cfDNA is feasible and may be reflective of burden of the disease. A prospective study to establish the validity of cfDNA in the diagnosis and follow up care in pts with metastatic CRC is planned.