Development and validation of a next generation sequencing based microsatellite instability assay for routine clinical use

PhD ThesisColorectal cancer (CRC) is the second most common cancer in both men and women. Approximately 3-5% of CRCs show microsatellite instability (MSI) caused by germline defects in mismatch repair genes. In addition, 12% of sporadic CRCs show MSI. Currently, MSI is tested using a fragment analysis based assay not suitable for high throughput testing. Knowledge of microsatellite instability affects prognosis, surveillance and treatment of CRCs and MSI testing is now recommended for all newly diagnosed CRCs. As a result, development of high throughput approaches is desirable. The focus of my work was to develop and validate a high throughput sequence based MSI assay. Initially, I tested 25 (7-9bp) mononucleotide markers, previously identified from in silico analyses, using a cohort of 55 CRCs, and selected 8 markers which collectively could discriminate between MSI-high (MSI-H) and microsatellite stable (MSS) cases. To define the optimal parameters to discriminate between MSI-H and MSS samples, I tested these 8 markers and 9 long (8-12bp) mononucleotide markers identified in a parallel study, across a panel of 141 CRC samples. This allowed development of a scoring scheme for the 17 markers, which achieved 96% sensitivity and 100% specificity. I validated this scheme using an independent cohort of 70 CRCs without knowing their MSI status. The assay achieved a 100% sensitivity and specificity. Finally, I assessed the ability of short repeats to allow inference of the clonal variation within both FFPE (7) and fresh (4) MSI-H CRCs by analysing multiple samples from each cancer. I was able to infer the lineage relationship between primary tumour and lymph node metastasis in three cases and to construct phylogenetic trees for all cancers for which multiple samples were available illustrating the utility of these markers for understanding of CRC clonal variation.Higher Committee for Education Development in Iraq (HCED Iraq

A novel panel of short mononucleotide repeats linked to informative polymorphisms enabling effective high volume low cost discrimination between mismatch repair deficient and proficient tumours

<div><p>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.</p></div