e15117 Background: The role of MSI in colorectal cancer (CRC) is well characterized, and tumors are classified as MSI-High or MSI-Stable by screening specific microsatellites. MSI-H reflects mismatch repair deficiency, is predictive for CRC therapy outcome in chemotherapy and immunotherapy and has a higher 5-year survival. While tumor testing is the gold standard, a convenient approach to screen for MSI before and during cancer treatment is screening circulating DNA (liquid biopsy) using a blood draw. However, using electrophoresis or next generation sequencing for MSI detection presents challenges at low levels of MSI due to polymerase slippage (‘stutter’) that generate high false positive rates at positions of homo-polymers. We present a new approach for enrichment of altered micro-satellites prior to DNA-amplification thereby reducing stutter from wild-type alleles and facilitating detection of MSI. The method employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating multiple micro-satellite targets (‘NaMe-PrO’, nuclease-assisted minor-allele enrichment with probe-overlap). Following DNA denaturation, the probes form double-stranded regions with their targets, thereby guiding nuclease digestion to selected sites. Microsatellite indels create ‘bulges’ that inhibit digestion, thus subsequent amplification yields DNA with microsatellite alterations enhanced at multiple targets. Methods: We first validated the method by evaluating 5 MSI targets simultaneously, NR27, NR21, NR24, BAT25 and BAT26 using DNA from tumor biopsies and circulating-DNA from colorectal cancer patients. The technique detected microsatellite alterations down to 0.01% altered allele frequency, thus improving detection sensitivity by > 100-fold relative to current approaches. Next, a clinical study was performed. We screened microsatellites in circulating DNA from groups of early stage colon cancer patients with known MSI status in the tumor. Results: Combination of NAME-PRO with capillary electrophoresis demonstrated 100% sensitivity (10/10) and 90% specificity (9/10) in detecting MSI status in the blood, thereby opening the possibility of early cancer detection for MSI positive tumors, or for application in minimal residual disease detection. Conclusions: We anticipate application of this highly- multiplex-able method either with standard 5-plex reactions or with NGS-based detection of MSI on thousands of targets to enable sensitive detection in tumors and liquid biopsies.
Sensitive Detection of Microsatellite Instability (MSI) in Liquid Biopsies from Early Stage Colon Cancer Patients using Nuclease-based Enrichment and Standard-Marker or NGS based approaches
