Microsatellite instability (MSI) testing of colorectal cancers (CRCs) is used to screen for Lynch syndrome (LS), a hereditary cancer‐predisposition, and can be used to predict response to immunotherapy. Here, we present a single‐molecule molecular inversion probe and sequencing‐based MSI assay and demonstrate its clinical validity according to existing guidelines. We amplified 24 microsatellites in multiplex and trained a classifier using 98 CRCs, which accommodates marker specific sensitivities to MSI. Sample classification achieved 100% concordance with the MSI Analysis System v1.2 (Promega) in three independent cohorts, totaling 220 CRCs. Backward–forward stepwise selection was used to identify a 6‐marker subset of equal accuracy to the 24‐marker panel. Assessment of assay detection limits showed that the 24‐marker panel is marginally more robust to sample variables than the 6‐marker subset, detecting as little as 3% high levels of MSI DNA in sample mixtures, and requiring a minimum of 10 template molecules to be sequenced per marker for >95% accuracy. BRAF c.1799 mutation analysis was also included to streamline LS testing, with all c.1799T>A variants being correctly identified. The assay, therefore, provides a cheap, robust, automatable, and scalable MSI test with internal quality controls, suitable for clinical cancer diagnostics.

Somatic mutations in mononucleotide repeats are commonly used to assess the mismatch repair status of tumours. Current tests focus on repeats with a length above 15bp, which tend to be somatically more unstable than shorter ones. These longer repeats also have a substantially higher PCR error rate and tests that use capillary electrophoresis for fragment size analysis often require expert interpretation. In this communication, we present a panel of 17 short repeats (length 7–12bp) for sequence-based microsatellite instability (MSI) testing. Using a simple scoring procedure that incorporates the allelic distribution of the mutant repeats, and analysis of two cohort of tumours totalling 209 samples, we show that this panel is able to discriminate between MMR proficient and deficient tumours, even when constitutional DNA is not available. In the training cohort, the method achieved 100% concordance with fragment analysis, while in the testing cohort, 4 discordant samples were observed (corresponding to 97% concordance). Of these, 2 showed discrepancies between fragment analysis and immunohistochemistry and one was reclassified after re-testing using fragment analysis. These results indicate that our approach offers the option of a reliable, scalable routine test for MSI.

Somatic mutations in mononucleotide repeats are commonly used to assess the mismatch repair status of tumours. Current tests focus on repeats with a length above 15bp, which tend to be somatically more unstable than shorter ones. These longer repeats also have a substantially higher PCR error rate, and tests that use capillary electrophoresis for fragment size analysis often require expert interpretation. In this communication, we present a panel of 17 short repeats (length 7–12bp) for sequence-based microsatellite instability (MSI) testing. Using a simple scoring procedure that incorporates the allelic distribution of the mutant repeats, and analysis of two cohort of tumours totalling 209 samples, we show that this panel is able to discriminate between MMR proficient and deficient tumours, even when constitutional DNA is not available. In the training cohort, the method achieved 100% concordance with fragment analysis, while in the testing cohort, 4 discordant samples were observed (corresponding to 97% concordance). Of these, 2 showed discrepancies between fragment analysis and immunohistochemistry and one was reclassified after re-testing using fragment analysis. These results indicate that our approach offers the option of a reliable, scalable routine test for MSI.

Background

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 O'Brien, 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.