Distinct genomic landscape of Lynch syndrome–associated urothelial cancer.

4582 Background: Lynch syndrome (LS) is a hereditary cancer predisposition syndrome caused by DNA mismatch repair (MMR) deficiency and associated with a 10–25% lifetime risk of urothelial cancer (UC), particularly in the upper urinary tract. We aimed to investigate the somatic genomic landscape of LS-associated urothelial cancer (LS-UC) using targeted and whole-exome sequencing (WES). Methods: We analyzed 41 surgical tumor samples accrued to five Finnish biobanks between April 1987 - June 2022 and 3 urine DNA samples from 34 LS-UC patients, all enrolled in the Finnish Lynch Syndrome Registry. Tumors were profiled using the UroScout assay, targeting 25 UC-associated genes, to identify somatic mutations. Immunohistochemistry was performed to assess MMR protein loss, and WES was conducted on selected cases to investigate the broader mutation landscape. A comparative analysis of the genomic and mutational landscapes in LS-UC versus sporadic UC was performed. Results: We show that telomerase reverse transcriptase ( TERT ) promoter mutations found in 83% of sporadic UC are almost completely absent (5%) in LS-UC (p < 0.00001). Instead, all LS-UC exhibited a 5-methylcytosine deamination (CG > TG) and microsatellite instability driven mutation landscape, characterized by highly frequent ARID1A (82%), FGFR3 (80%), and KMT2D (78%) mutations, as well as preferential usage of CG > TG mutation hotspots. We propose that scarcity of TERT promoter mutations in LS-UC is due to inability to create the GABP binding motif 5’-GGAA through CG > TG mutation or microsatellite instability. Additionally, many mutation hotspots recurrently mutated in sporadic UC were not present in LS-UC. Conclusions: Our findings establish LS-UC as a distinct disease entity with a unique genomic signature driven by constrained hypermutation. Our proposed explanation that TERT mutations are absent in LS-UC due to constrained hypermutation is supported by our discovery that other UC driver genes also exhibit an altered mutation landscape in LS-UC. These insights advance the understanding of LS-UC tumorigenesis and support the development of tailored diagnostic and therapeutic approaches for this patient population.