Detection of Microsatellite Instability in Colonoscopic Biopsies and Postal Urine Samples from Lynch Syndrome Cancer Patients Using a Multiplex PCR Assay

Abstract

Identification of mismatch repair (MMR)-deficient colorectal cancers (CRCs) is recommended for Lynch syndrome (LS) screening, and supports targeting of immune checkpoint inhibitors. Microsatellite instability (MSI) analysis is commonly used to test for MMR deficiency. Testing biopsies prior to tumour resection can inform surgical and therapeutic decisions, but can be limited by DNA quantity. MSI analysis of voided urine could also provide much needed surveillance for genitourinary tract cancers in LS. Here, we reconfigure an existing molecular inversion probe-based MSI and BRAF c.1799T > A assay to a multiplex PCR (mPCR) format, and demonstrate that it can sample >140 unique molecules per marker from <1 ng of DNA and classify CRCs with 96–100% sensitivity and specificity. We also show that it can detect increased MSI within individual and composite CRC biopsies from LS patients, and within preoperative urine cell free DNA (cfDNA) from two LS patients, one with an upper tract urothelial cancer, the other an undiagnosed endometrial cancer. Approximately 60–70% of the urine cfDNAs were tumour-derived. Our results suggest that mPCR sequence-based analysis of MSI and mutation hotspots in CRC biopsies could facilitate presurgery decision making, and could enable postal-based screening for urinary tract and endometrial tumours in LS patients.

A Focused Clinical Review of Lynch Syndrome

“…In addition, novel microsatellite analysis of urine has to been used to detect cancers of the urogenital tract and endometrium in those with LS with some success. 106 Larger studies are needed however, to confirm if this could be a viable non-invasive means of cancer surveillance. Finally, the development of technologies that utilise cell-free DNA could mean in the near future a blood test could be used to screen for all LS-associated cancers through the detection of molecular markers.…”

Transforming the care of people with Lynch syndrome: a system-wide approach

“…The National Bowel Cancer Screening programme has accepted responsibility for all LS carriers which should ensure regular high-quality colonoscopies for all and initial MMR plus BRAF screening can now be done more rapidly on biopsy blocks. 7 Growing numbers of coloproctology nurse practitioners have embraced the mainstreaming, with others soon to follow, ensuring eligible patients are offered germline testing while still under their care. The next traffic jam is getting the germline gene sequencing done in a timely fashion, but this will improve as technology advances alongside international efforts to curate the many thousands of gene variants in these genes.…”