Detection of Microsatellite Instability from Circulating Tumor DNA by Targeted Deep Sequencing

Currently, microsatellite instability (MSI) detection is limited to tissue samples with sufficient tumor content. Detection of MSI from blood has been explored but confounded by low sensitivity due to limited circulating tumor DNA (ctDNA). We developed a next-generation sequencing-based algorithm, blood MSI signature enrichment analysis, to detect MSI from blood. Blood MSI signature enrichment analysis development involved three major steps. First, marker sites that can effectively distinguish high MSI (MSI-H) from microsatellite stable tumors were extracted. Second, MSI signature enrichment analysis was performed based on hypergeometric probability, under the null hypothesis that plasma samples have similar MSI-H and microsatellite stable read coverage patterns for particular marker sites as the white blood cells from the training data set. Finally, enrichment scores of marker sites were normalized, and all markers were collectively considered to determine the MSI status of a plasma sample. In vitro dilution experiments with cell lines and in silico simulation experiments based on mixtures of MSI-H plasma and paired white blood cell DNA demonstrated 98% sensitivity and 100% specificity at a minimum of 1% ctDNA and 91.8% sensitivity and 100% specificity with 0.4% ctDNA. An independent validation cohort of 87 colorectal cancer patients with orthogonal confirmation of MSI status of tissues confirmed performance, achieving 94.1% sensitivity (16/17) and 100% specificity (27/27) for samples with ctDNA >0.4%.