Universal tumor screening for Lynch Syndrome (LS), which involves up to 6 sequential tests, is recommended by NCCN guidelines for all patients with colorectal cancer (CRC) at diagnosis. Microsatellite instability (MSI) testing is the first step in the genetic diagnosis for LS. Additionally, MSI status has been approved by FDA to select patients for immunotherapy treatments. Here we evaluate the performance of a single tumor sequencing test using Illumina TruSight™ Oncology 500 (TSO500) for MSI status determination and LS screening.
A total of 233 CRC subjects were screened through a commercial MSI-PCR assay run on tumor-normal DNA. Tumor DNA from 63 selected subjects was sequenced with TSO500. The MSI score was calculated using 130 homopolymer microsatellite loci targeted by the TSO500 panel. Subsequently, BRAF p.V600E status and potential mutations in MMR genes or EPCAM were analyzed from TSO500 results. For LS screening, a method with three filtering criteria was used: 1) MSI-high (MSI-H) status, 2) without BRAF p.V600E mutations, and 3) at least 1 MMR gene variant or EPCAM deletion inferred as germline small variant mutation by TSO500 germline classifier or copy number change. Finally, matched normal samples were sequenced with TSO500 to confirm any germline mutations linked to LS.
Using MSI-PCR, 45 of the 233 (19.3%) subjects were identified as MSI-H and 188 (80.7%) as microsatellite stable (MSS). TSO500 achieved an overall percent agreement (OPA) of 100.0% (95% CI: 94.3% - 100.0%) with MSI-PCR for the 63 subjects analyzed by both methods. Eight subjects were identified as LS positive through tumor sequencing by TSO500. Matched normal sequencing confirmed all 8 positive cases of identified potential LS mutations as germline. Overall, TSO500 tumor sequencing achieved an OPA of 100.0% (95% CI: 94.3% - 100.0%) with matched normal sequencing.
Collectively, our results demonstrated that MSI status can be accurately determined with tumor sequencing. Moreover, LS screening by TSO500 with secondary filtering algorithms can be used as a single upfront test to identify BRAF p.V600E status and potential pathogenic germline mutations linked to LS.
Illumina.
Illumina.
L. Liu: Shareholder / Stockholder / Stock options, Full / Part-time employment: Ilumina. C. Garbutt: Full / Part-time employment: Illumina. M. Golkaram: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. S. Kaplan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. L. Costa: Research grant / Funding (institution), PIC/IC/82821/2007: Faculdade de Medicina da Universidade de Lisboa. A. So: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. S. Zhang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. T. Pawlowski: Shareholder / Stockholder / Stock options, Full / Part-time employment: Illumina. All other authors have declared no conflicts of interest.