Accurate Pan-Cancer Molecular Diagnosis of Microsatellite Instability by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing

Abstract

BACKGROUND: Microsatellite instability (MSI) is an emerging, actionable phenotype in oncology that informs tumor response to immune checkpoint pathway immunotherapy. However, there remains a need for MSI diagnostics that are low cost, highly accurate, and generalizable across cancer types. We developed a method for targeted high throughput sequencing of numerous microsatellite loci with pan-cancer informativity for MSI using single-molecule molecular inversion probes (smMIPs). METHODS: We designed a smMIP panel targeting 111 loci highly informative for MSI across cancers. We developed an analytical framework taking advantage of smMIP-mediated error correction to specifically and sensitively detect instability events without the need for typing matched normal material. RESULTS: Using synthetic DNA mixtures, smMIPs were sensitive to at least 1% MSI positive cells and were highly consistent across replicates. The fraction of identified unstable microsatellites discriminated tumors exhibiting MSI from those lacking MSI with high accuracy across colorectal (100% diagnostic sensitivity and specificity), prostate (100% diagnostic sensitivity and specificity), and endometrial cancers (95.8% diagnostic sensitivity and 100% specificity). MSI-PCR, the current standard-of-care molecular diagnostic for MSI, proved equally robust for colorectal tumors, but evidenced multiple false negative results in prostate (81.8% diagnostic sensitivity and 100% specificity) and endometrial (75.0% diagnostic sensitivity and 100% specificity) tumors. CONCLUSIONS: smMIP capture provides an accurate, diagnostically sensitive, and economical means to diagnose MSI across cancer types without reliance on patient-matched normal material. The assay is readily scalable to large numbers of clinical samples, enables automated and quantitative analysis of microsatellite instability, and is readily standardized across clinical laboratories.

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

“…Depending on the marker panel used, we estimate that 10 or 15 molecular barcodes per marker are required for correct classification of >95% of samples. We have, therefore, shown that it is possible to accurately determine MSI status using only six markers, a fraction of the number required by other NGS‐based MSI assays (Kautto et al, ; Waalkes et al, ; Zhu et al, ), and observed only a small reduction in assay robustness using this subset rather than the 24‐marker panel. The requirement of other NGS‐based MSI classifiers for larger marker panels may be explained by the classification method, as assessing the proportion of mutated microsatellites gives equal diagnostic weight to each marker and does not account for the variable influence of MMR deficiency on the mutation of individual microsatellites (Dietmaier et al, ).…”

“…However, the high cost of gene panel sequencing (Marino et al, ) may be a barrier to its widespread deployment for MSI testing, or for the detection of LS by MMR gene sequencing. Targeted NGS‐based MSI assays that use multiplex amplification of specific panels of microsatellites have been developed that, similar to gene panel‐based methods, classify samples by the proportion of microsatellites that are mutated (Gan et al, ; Hempelmann et al, ; Hempelmann, Scroggins, Pritchard, & Salipante, ; Waalkes et al, ). However, even when using the same method, different marker proportions can be used as a classification threshold with different marker sets (Hempelmann et al, ; Hempelmann et al, ; Kautto et al, ; Waalkes et al, ; Zhu et al, ), and thresholds can be uncertain when relatively few microsatellites (<20) are analyzed (Hempelmann et al, ).…”

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

“…Our smMIP-based MSI assay uses monomorphic and short (7-12bp) MNRs that have significantly lower PCR and sequencing error rates compared to longer markers 27 , including those used by the MSI Analysis System (Promega) and NGS-based assays 25,31 . Automated MSI classification from sequencing only tumour DNA achieved 100% sensitivity and 100% specificity relative to microsatellite FLA in 197 CRCs.…”

“…Here, we create a fully automatable, modular, and cheap MSI assay, suitable for high throughput MMR diagnostics and two-step screening for LS, by multiplex amplification of our markers and the BRAF V600E locus using single molecule molecular inversion probe (smMIP) technology 30 . smMIPs have previously been used to multiplex large panels of long (16-40bp) microsatellites 31 , and read-tagging with molecular barcodes provides a count of template molecules sequenced as a quality control 32 . We show that our smMIP-based MSI assay has 100% sensitivity and specificity for MMR deficiency relative to FLA, and detects BRAF V600E mutations missed by conventional techniques.…”

Clinical implications of the genetics of sporadic colorectal cancer

“…Next‐generation sequencing technology now allows rapid and cost‐effective sequencing of whole genomes . Early evidence suggests excellent specificity (100%) and sensitivity (98%); next‐generation sequencing could replace other methods of detecting dMMR.…”