LAUSR

Abstract Background Microsatellite instability (MSI) status has been approved by FDA to select for patients with metastatic tumors for cancer immunotherapy treatments. Additionally, MSI status is used in assessment of prognosis and treatment choices in certain cancer types, as well as the first step in the genetic diagnosis for Lynch syndrome. Circulating tumor DNA (ctDNA) is a noninvasive, real-time approach used for comprehensive genomic profiling of cancer. However, only a small fraction of cell-free DNA (cfDNA) fragments originate from tumor cells, requiring an ultra-sensitive method to detect MSI status from cfDNA. Here we evaluate the performance of Illumina TruSight™ Oncology 500 panel for MSI testing through cfDNA sequencing. Methods We developed a robust method to assess MSI status in cfDNA. For each MSI locus, we assessed the repeat length distribution of the test subject and a cohort of normal samples. By comparing allele distributions with information-theory based approach, we determined if each of the MSI locus was unstable. Final MSI score was calculated using the number of unstable sites divided by total tested sites. To assess the analytical performance of our method, we titrated MSI-high (MSI-H) cell lines into MSI stable (MSS) background at a series of concentrations ranging from 0.31% to 5.0%, representing low tumor fractions in cfDNA samples. Additionally, we processed 94 clinical samples with matched FFPE tumor and cfDNA to examine the concordance of MSI testing between FFPE and cfDNA. Results For titrated MSI-H samples with low tumor fraction, we achieved 100% sensitivity in samples at 0.625% MSI-H content titration into MSS background. Moreover, we achieved 98.9% overall percent agreement (93/94) for MSI status between matched FFPE and cfDNA samples with a wide range of tumor content. Conclusions Our evaluation indicates that we can accurately determine MSI status in cfDNA samples with a wide range of tumor content. Legal entity responsible for the study The authors. Funding Illumina. Disclosure S. Zhang: Full / Part-time employment: Illumina. J.S. LoCoco: Full / Part-time employment: Illumina. A. Mentzer: Full / Part-time employment: Illumina. B.M. Crain: Full / Part-time employment: Illumina. S. Katz: Full / Part-time employment: Illumina. G. Berry: Full / Part-time employment: Illumina. Y. Fu: Full / Part-time employment: Illumina. T. Jiang: Full / Part-time employment: Illumina. C. Zhao: Full / Part-time employment: Illumina. S. Bilke: Full / Part-time employment: Illumina. T. Pawlowski: Full / Part-time employment: Illumina. K. Kruglyak: Full / Part-time employment: Illumina.