International guidelines for the diagnosis of Lynch syndrome (LS) recommend molecular screening of colorectal cancers (CRCs) to identify patients for germline mismatch repair (MMR) gene testing. As our understanding of the LS phenotype and diagnostic technologies have advanced, there is a need to review these guidelines and new screening opportunities. We discuss the barriers to implementation of current guidelines, as well as guideline limitations, and highlight new technologies and knowledge that may address these. We also discuss alternative screening strategies to increase the rate of LS diagnoses. In particular, the focus of current guidance on CRCs means that approximately half of Lynch-spectrum tumours occurring in unknown male LS carriers, and only one-third in female LS carriers, will trigger testing for LS. There is increasing pressure to expand guidelines to include molecular screening of endometrial cancers, the most frequent cancer in female LS carriers. Furthermore, we collate the evidence to support MMR deficiency testing of other Lynch-spectrum tumours to screen for LS. However, a reliance on tumour tissue limits preoperative testing and, therefore, diagnosis prior to malignancy. The recent successes of functional assays to detect microsatellite instability or MMR deficiency in non-neoplastic tissues suggest that future diagnostic pipelines could become independent of tumour tissue.

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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.

Lynch syndrome (LS) is a hereditary cancer syndrome caused by pathogenic germline variants in genes that encode mismatch repair (MMR) proteins. LS is characterized by an increased risk of multiple cancers, including colorectal and endometrial cancer. Muir–Torre syndrome (MTS) is an allelic, phenotypic variant of LS characterized by the presence of skin tumours, including keratoacanthomas and sebaceous tumours. Recently, an association has been proposed between MTS/LS and cutaneous squamous cell carcinoma (cSCC). MTS/LS tumours demonstrate a mutation signature termed microsatellite instability (MSI) that can be used to screen sporadic tumours for underlying MTS/LS. We have previously shown that amplicon-sequencing of microsatellites can detect increased MSI in cSCC in patients with LS. In this study, we assayed a pilot cohort sporadic cSCC in the general population to further explore this potential association. DNA was extracted from cSCC samples. We used an established molecular inversion probe-based protocol to amplify informative microsatellites and sequenced these using the Illumina MiSeq platform. An MSI score was calculated for each tumour sample using a naïve Bayesian approach based on microsatellite deletion frequencies. We found that 5% (n = 1/19) of cSCCs were MSI-high (95% confidence interval 0.1–26.0), which is equivalent to estimated frequencies among unselected sporadic cSCCs in recent literature. We plan to expand our pilot cohort to include a larger series of cSCC and to also carry out somatic sequencing of MSH2, MSH6, MLH1 and PMS2 in tumours to determine if MSI status correlates with MMR gene variant status. Ultimately, this work aims to improve identification of new patients with LS presenting with cSCC and allow them to benefit equitably from preventative cancer surveillance and aspirin chemoprevention.