Abstract 942: Sensitive detection of microsatellite instability (MSI) in tumors and liquid biopsies using nuclease-based enrichment
Authors: Ladas Ioannis , Song Chen , Yu Fangyan , Leong Ka Wai , Troullinou Katerina , Kulke Matthew , Mamon Harvey , Makrigiorgos Mike G. 
Published: August 2018

The role of MSI in colorectal cancer (CRC) is well characterized, and colon tumors are classified as MSI-High or MSI-Stable by screening specific microsatellites. MSI-H compared to MSI-S is predictive for therapy outcome in chemotherapy and immunotherapy and has been associated with distinct characteristics and favorable results including better prognosis, a higher 5-year survival, and lesser metastasis. Several other tumors also present MSI changes reflecting mismatch repair deficiency. 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, thereby interrogating ‘systemic' MSI reflecting primary or secondary (occult) tumor status at the time of blood collection. However, presence of excess unaltered, wild-type DNA often masks alterations such as MSI. Using capillary 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 facilitating their detection. We recently developed nuclease-assisted minor-allele enrichment with probe-overlap, a single-step approach that removes WT-DNA and enriches mutation-containing alleles. Here we adapted NaME-PrO for detection of homopolymer indels, for MSI detection. The method employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating multiple micro-satellite targets. 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. The assay is applied at the genomic or circulating-DNA level prior to amplification, thereby avoiding polymerase-introduced ‘stutter peaks' arising from WT DNA. Inclusion of organic solvents allows homogeneous application of the method in closed tube reactions. We 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 enriched all altered targets and detected microsatellite alterations down to 0.01% altered allele frequency, thus improving detection sensitivity by &gt;100-fold relative to current approaches. We anticipate application of this highly- multiplex-able method either with standard 5-plex reactions in conjunction with capillary electrophoresis or with NGS-based detection of MSI on thousands of targets to enable sensitive detection in tumors and liquid biopsies.

Citation Format: Ioannis Ladas, Chen Song, Fangyan Yu, Ka Wai Leong, Katerina Troullinou, Matthew Kulke, Harvey Mamon, Mike G. Makrigiorgos. Sensitive detection of microsatellite instability (MSI) in tumors and liquid biopsies using nuclease-based enrichment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 942.