Sequencing‐based microsatellite instability testing using as few as six markers for high‐throughput clinical diagnostics

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.

How Should We Test for Lynch Syndrome? A Review of Current Guidelines and Future Strategies

“…Alternatively, MSI assays that enrich and sequence specific microsatellites can be used, keeping costs equivalent to or less than MSI FLA or IHC, whilst leveraging the scalability and automatability of NGS and multiplexed analysis of BRAF c.1799T > A. Such assays can reduce LS screening to one, low cost, high throughput tumour test prior to germline MMR gene testing of select cases, and could facilitate widespread deployment of current guidance [ 127 , 128 , 129 ].…”

Constitutional mismatch repair deficiency is the diagnosis in 0.41% of pathogenic NF1/SPRED1 variant negative children suspected of sporadic neurofibromatosis type 1

“…These are facilitated by the assay’s low cost, at approximately $15–55 per sample for reagents, and scalability, being fully automatable and able to analyze 7–174 samples on a single MiSeq flow cell (Supplementary Information S 3 ). 25 , 29 In further support of its clinical utility, we have previously validated the MSI assay’s sensitivity and specificity using a blinded sample cohort, 25 and, here, we have demonstrated that it can be deployed in a separate laboratory from the laboratory in which it was developed (including perfect concordance of sample classification between laboratories), shown that control sample scoring is equivalent between laboratories, updated it with automatically flagged QCs based on sequencing metrics, and expanded the reference set of controls to make it more robust (Supplementary Information S 2 ). Therefore, while the MSI assay has not been formally accredited for clinical diagnostics, its performance in this study suggests it may not only be useful in research, but also in clinical settings as a prescreening assay on selected individuals or to resolve diagnostic uncertainties.…”

“…Therefore, while the MSI assay has not been formally accredited for clinical diagnostics, its performance in this study suggests it may not only be useful in research, but also in clinical settings as a prescreening assay on selected individuals or to resolve diagnostic uncertainties. 18 , 25 , 29 Having shown the feasibility of large-scale MSI analysis to screen for CMMRD, the prevalence of CMMRD in other patient groups could be defined using this screening pipeline. This is particularly important given the broad phenotypic spectrum of CMMRD, including the wide variety of associated cancer types, and the clinical implications of its differential diagnosis with respect to clinical surveillance, therapy, and genetic counseling.…”

“…The SNPs sequenced with each microsatellite were used to confirm the identity of any sample repeats as there is only a 3.6×10 −10 probability that any two individuals share the same genotype. 29 …”

“…The SNPs sequenced with each microsatellite were used to confirm the identity of any sample repeats as there is only a 3.6×10 −10 probability that any two individuals share the same genotype. 29 Germline MSI (gMSI) assay gMSI analysis was performed according to the protocols described by Ingham et al, 23 as adapted by Gallon et al 25…”