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1.
Hum Mutat ; 41(1): 332-341, 2020 01.
Article in English | MEDLINE | ID: mdl-31471937

ABSTRACT

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.


Subject(s)
Genetic Markers , Genetic Predisposition to Disease , Genetic Testing , High-Throughput Screening Assays , Microsatellite Instability , Microsatellite Repeats , Alleles , Biomarkers, Tumor , Cell Line , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/genetics , DNA Mismatch Repair , Genetic Association Studies/methods , Genetic Testing/methods , Genetic Testing/standards , Genotype , High-Throughput Nucleotide Sequencing , High-Throughput Screening Assays/methods , High-Throughput Screening Assays/standards , Humans , Molecular Diagnostic Techniques , Phosphorylation , Reproducibility of Results
2.
PLoS One ; 13(8): e0203052, 2018.
Article in English | MEDLINE | ID: mdl-30157243

ABSTRACT

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.


Subject(s)
Colorectal Neoplasms/genetics , DNA Mismatch Repair , High-Throughput Nucleotide Sequencing , Microsatellite Instability , Microsatellite Repeats , Polymorphism, Genetic , Biomarkers, Tumor/genetics , Cohort Studies , Computer Simulation , Fixatives , Formaldehyde , Gene Frequency , Humans , Paraffin Embedding , Tissue Fixation
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