Detection of urothelial carcinoma in Lynch syndrome using microsatellite instability analysis of urine cell-free DNA.

BACKGROUND: Urothelial carcinoma is the third most common cancer in Lynch syndrome but there is no approved screening method. Lynch syndrome cancers are characterised by high levels of microsatellite instability (MSI/MSI-H). Here, we assess the feasibility of urine MSI analysis for non-invasive urothelial carcinoma screening. METHODS: We analysed urine cell-free DNA samples from two cohorts using an amplicon-sequencing MSI assay: (1) Sequential cases of upper tract urothelial carcinoma (UTUC) provided paired tumour/pre-operative urine samples for MSI analysis and mismatch repair protein immunohistochemistry (MMR IHC) and those with an MMR deficient (MMRd) tumour were offered constitutional Lynch syndrome testing (2) Eligible individuals with a diagnosis of MSH2-Lynch syndrome (aged 30-75 years, without recent cancer diagnosis) were offered urine MSI analysis via a clinic appointment or postal urine sample collection. All cases were followed up for at least 12 months. FINDINGS: Three of 50 cases of UTUC (6.00%) were MSI-H, MMRd by IHC and detectable by MSI-H or borderline-MSI-H urine signals. All occurred in individuals with new Lynch syndrome diagnoses. Urines and tumours from the remaining 47 patients were microsatellite stable (MSS) and tumours were MMR proficient by IHC. Urine MSI analysis achieved 100% sensitivity and specificity for symptomatic UTUC (95% CI 29.2%-100% and 92.4%-100% respectively). 81 of 142 eligible individuals with a diagnosis of MSH2-Lynch syndrome participated (57.0%) with one later excluded following urothelial carcinoma diagnosis between enrolment and sample collection. MSI-H urines were identified in 5/80 (6.25%), with urothelial carcinoma subsequently diagnosed in 4/5. None of the 75 participants with an MSS urine had a urothelial carcinoma diagnosis during follow up. Urine MSI analysis achieved 100% sensitivity, 98.7% specificity, 80% positive predictive value, and 100% negative predictive value for asymptomatic Lynch syndrome urothelial carcinoma (95% CIs: 39.8-100%, 92.9-99.9%, 28.4-99.5% and 95.2-100% respectively). INTERPRETATION: Urine MSI testing using a low-cost assay which is suitable for routine surveillance can detect asymptomatic Lynch syndrome urothelial carcinoma, facilitating early diagnosis. Further studies are needed to define accuracy and patient outcomes. FUNDING: Newcastle upon Tyne Hospitals NHS Charity/The Sir Bobby Robson Foundation, The Urology Foundation, The Barbour Foundation, Cancer Research UK.