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

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.

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

“…Nineteen and 73 CRC DNAs were provided by the Department of Molecular Pathology, University of Edinburgh, UK, and the Oncogenetics and Hereditary Cancer Group, Complejo Hospitalario de Navarra, Spain, respectively. These 92 samples were residual stocks from Redford et al (). An additional 128 CRC DNAs or CRC formalin‐fixed paraffin‐embedded (FFPE) tissue samples were provided by the Northern Genetics Service, Newcastle Hospitals NHS Foundation Trust, UK.…”

“…We have previously used amplicon sequencing of short (7–12 base pairs [bp]), monomorphic MNRs to classify the MSI status of CRCs, without needing matched normal tissue (Redford et al, ). Short MNRs were selected as longer (>15 bp) microsatellites are associated with increased PCR and sequencing error (Fazekas, Steeves, & Newmaster, ), and it has been reported that 9–15 bp microsatellites give the greatest differences in mutation frequencies between MSI‐H and microsatellite stable (MSS) samples using NGS (Maruvka et al, ).…”

“…Short MNRs were selected as longer (>15 bp) microsatellites are associated with increased PCR and sequencing error (Fazekas, Steeves, & Newmaster, ), and it has been reported that 9–15 bp microsatellites give the greatest differences in mutation frequencies between MSI‐H and microsatellite stable (MSS) samples using NGS (Maruvka et al, ). Our method for MSI detection accounts for the individual sensitivity and specificity of each marker, and achieved >97% accuracy in 209 CRCs with only 17 markers, using FLA as the reference method (Redford et al, ). However, the protocol required singleplex PCR amplification, followed by the second round of PCR to prepare the amplicons for sequencing.…”

A molecular inversion probe and sequencing-based microsatellite instability assay for high throughput cancer diagnostics and Lynch syndrome screening

“…A total of 24 MNRs with length 7-12bp and neighbouring SNP 29 were selected to test for MMR deficiency. BRAF V600E was included to screen for sporadic MMRd CRCs.…”

“…The MSI classifier used custom R scripts and the algorithm previously described by Redford et al 29 . In summary, to determine the relative probability that a sample is modelled by an MMRd versus an MMRp phenotype, both the proportion of reads containing microsatellite deletions and the allelic bias of deletions are used.…”