Abstract 1286: Analytical validation of an integrated next-generation sequencing pan-cancer liquid biopsy approach for detection of microsatellite instability
Authors: Georgiadis Andrew , Wood Derrick , Murphy Derek , Parpart-Li Sonya , Riley David , Sengamalay Naomi , Novak Marian , Jones Sian , Angiuoli Samuel A. , Sausen Mark 
Published: August 2018

Abstract
Failure of the DNA mismatch repair (MMR) pathway during DNA replication in cancer leads to the increased accumulation of somatic mutations. The molecular hallmark of MMR deficiency is microsatellite instability (MSI), which presents as polymorphism of tandem nucleotide repeat lengths ubiquitously distributed throughout the genome. Furthermore, the presence of MMR-deficiency or MSI across solid tumors has been recently demonstrated to be a robust biomarker for immunotherapy response with checkpoint inhibition. We have previously developed technology to detect MSI in tumor tissue through hybrid-capture based targeted next-generation sequencing analyses of a 125 gene panel, with probes capturing mononucleotide repeat markers. Using a set of 32 MSI and 27 microsatellite stable (MSS) formalin-fixed paraffin-embedded tumor tissue specimens, we obtained 100% sensitivity and 100% specificity with a limit of detection of 20% tumor purity. The clinical importance of MSI status now requires a fast, objective, highly sensitive screening method, particularly in late-stage patients where tumor material may not be readily obtained. However, to extend this approach to a liquid biopsy panel requires technological advances to overcome the inherent challenges associated with low circulating tumor DNA (ctDNA) levels that are compounded by polymerase slippage in mononucleotide repeat regions during PCR amplification. To overcome these limitations, we applied a multifactorial error correction approach together with a novel peak finding algorithm to more accurately identify the specific mononucleotide sequences in cell-free DNA (cfDNA) analyses. By eliminating a significant majority of sequencing errors and polymerase slippage artifacts, we were able to reduce background error rates by &gt; 90%. Combined with implementation of a distribution modeling and a peak finding algorithm, we were able to accurately sequence the mononucleotide tracts to minimize false discovery rates for cfDNA analyses. To evaluate the performance of this approach in clinical samples, for a cohort of plasma samples obtained from 13 late-stage clinical cases with matched tumor tissue data available (9 MSI and 4 MSS) together with 66 healthy donor plasma samples, we obtained 89% sensitivity and 100% specificity, with a lowest reported mutant allele fraction of 0.4%. Taken together, this integrated next-generation sequencing liquid biopsy approach is valuable for the determination of MSI status to inform immunotherapy treatment.
Citation Format: Andrew Georgiadis, Derrick Wood, Derek Murphy, Sonya Parpart-Li, David Riley, Naomi Sengamalay, Marian Novak, Sian Jones, Samuel A. Angiuoli, Mark Sausen. Analytical validation of an integrated next-generation sequencing pan-cancer liquid biopsy approach for detection of microsatellite instability [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 1286.