504 Background: Microsatellite instability (MSI) testing has become critically important in clinical cancer care of patients with cancer given the recent pan-tumor FDA approval of pembrolizumab for use in patients with MSI-High (MSI-H) tumors. We previously demonstrated the robustness of a novel proprietary algorithm for determination of MSI status via NGS from solid tumor biopsy specimens (J Clin Oncol 34, 2016 (suppl; abstr 1523)). Traditional MSI tests such as PCR or IHC are impractical for pan-tumor adoption, as MSI-H prevalence outside of gastrointestinal and endometrial cancers is usually < 1%. NGS-based ctDNA profiling provides an opportunity for both MSI and actionable alteration testing in patients in whom tissue-based biopsy is not available. Methods: Genomic DNA (gDNA) from five previously characterized MSI-H cell lines: (DLD1, 22Rv1, LNCap, RL952, CL188), and one MSS cell line (SCC9) was enzymatically-fragmented to simulate ctDNA and titrated to various dilution levels with DNA from a healthy hapmap subject (NA12878). Samples were screened with a 70-gene panel, FoundationOne Liquid, that includes 180 mononucleotide repeat sequences (8-26bp long in the human reference genome). Length variability in the 180 repeat loci was utilized to generate an overall MSI score via principal components analysis. The NGS based MSI algorithm was applied to all the samples. Results: Assessment of these six cell lines, targeting five dilution levels confirmed by SNP mixing ratios, show that our NGS based MSI test for liquid biopsies has 96% sensitivity at > 2% tumor fraction with 100% PPV. The regression intercept of the MSI-H dilution samples with the pre-established MSI-H calling threshold shows our method has a LOD of 1.03% tumor fraction. MSI-H prevalence data from liquid biopsies of gastrointestinal tumors obtained during clinical care will also be presented. Conclusions: These data demonstrate the feasibility of using NGS-based liquid biopsy assays for MSI testing. This ctDNA-based approach will allow for increased access to checkpoint inhibitors in a pan-tumor setting, which would be especially relevant for cancers where routine MSI testing is impractical or when tissue is not available.
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