Duplex-Seq derived urinary cell-free DNA of Lynch syndrome patients with upper tract urothelial carcinoma.

Abstract

838 Background: There is a critical need to develop urinary biomarkers for upper tract urothelial carcinoma (UTUC), especially for patients with Lynch Syndrome (LS), who have a 22-fold higher risk of developing UTUC. Urinary cell-free DNA (uDNA) represents a promising biomarker, but its utility has been limited thus far in predicting UTUC. However, LS-associated UTUC has three unique genomic features that may be targeted to improve biomarker capabilities; (1) somatic inactivation of the functional allele, (2) hypermutator phenotype, and (3) microsatellite instability (MSI). Herein, we apply Duplex-Seq, which is an ultra-accurate next-generation sequencing technology, to assess genomic alterations in uDNA with the goal of developing a more reliable biomarker for LS-associated UTUC. Methods: We performed Duplex-Seq on uDNA from 17 participants enrolled in a prospectively maintained biorepository at the University of Washington. The cohort included MSH2- mutated LS patients with active UTUC (LS+UTUC) (n = 3), MSH2 -mutated LS patients without UTUC (LS-UTUC) (n = 8), and non-LS patients with active UTUC (NonLS+UTUC) (n = 6). Sequencing targeted the 10 most frequently mutated genes in UTUC, MSH2 , and 17 poly-G microsatellites. Results: All LS+UTUC patients (3/3) exhibited second-site somatic inactivating mutations consistent with biallelic loss (VAF range: 0.00031-0.00075), and demonstrated markedly elevated mutation burdens (6.18±1.97x10 -8 ) indicative of a strong hypermutator phenotype. In contrast, none of the LS−UTUC patients (0/8) had second-site mutations; seven also had substantially lower mutation burdens (2.13 ± 1.60×10⁻⁸). NonLS+UTUC patients (6/6) had no MSH2 inactivating mutations and minimal mutation loads (1.26 ± 0.35×10⁻⁸). MSI status was inconclusive between LS+UTUC and LS-UTUC but suggested a slight increase relative to NonLS+UTUC patients. Conclusions: Duplex-Seq identified distinct mutational profiles in uDNA from LS-associated UTUC, distinguishing them from LS patients without UTUC and non-LS UTUC cases. These findings support the potential of uDNA as a non-invasive biomarker for early detection and surveillance of UTUC in LS patients, potentially guiding screening and clinical management in this high-risk population.