Abstract A09: A next generation sequencing based microsatellite instability assay suitable for routine risk stratification in colorectal cancer

Abstract

3-5% of CRCs show microsatellite instability (MSI) caused by germline defects in mismatch repair genes (MMR) predisposing to Lynch syndrome. In addition, 12% of sporadic CRCs show MSI. Currently, MSI is tested using a fragment analysis based assay not suitable for high throughput testing with suboptimal sensitivity and specificity. Knowledge of microsatellite instability affects prognosis (MSI is a positive prognostic factor in stage II CRC), risk stratification (for the affected and at risk relatives in Lynch syndrome), prediction of lymph node involvement (lymph node metastasis is unlikely in stage I MSI positive CRC) and treatment of CRCs (MMR deficient tumours showed observable benefit from PD-1 blocking agent pembrolizumab). For all these important benefits, MSI testing is now recommended for all newly diagnosed CRCs. As a result, development of a high throughput approach is desirable. We have developed and validated a high throughput sequence based MSI assay. In this study, we tested 17 short (7-12bp) mononucleotide markers (previously identified by our team via an in silico analyses of whole genome sequencing data). These 17 markers were able to discriminate between MSI-high (MSI-H) and microsatellite stable (MSS) cases. To define the optimal parameters to discriminate between MSI-H and MSS samples, we tested these 17 markers across a panel of 141 CRC samples. This allowed us to define a scoring scheme for the 17 markers using allelic imbalance based on a linked SNP (called weighted scoring scheme), which achieved 96% sensitivity and 100% specificity. This scoring scheme was then validated using an independent cohort of 70 CRCs without knowing their MSI status. The assay achieved a 100% sensitivity and specificity. We provide here a high throughput tool to detect microsatellite instability that is less costly, uses short mononucleotide markers (eliminating the need to test matched normal tissue) and is validated on formalin fixed paraffin embedded (FFPE) samples (similar to routine samples). The ability to test the microsatellite instability status in all the newly diagnosed CRC cases would have a great clinical impact on prognosis, risk stratification and treatment of CRCs. Citation Format: Ghanim Alhilal, Lisa Redford, Angel Alonso, Sira Moreno, Mark Arends, Anca Oniscu, Ottilia O9Brien, Stephanie Needham, John Burn, Michael Jackson, Mauro Santibanez-Koref. A next generation sequencing based microsatellite instability assay suitable for routine risk stratification in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Improving Cancer Risk Prediction for Prevention and Early Detection; Nov 16-19, 2016; Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(5 Suppl):Abstract nr A09.