Prospective, blinded validation of a urinary cfDNA methylation assay in bladder cancer.

820 Background: Cystoscopy is the standard for local bladder cancer (BC) detection and follow-up, but is invasive and costly. Noninvasive, accurate molecular assays could transform screening and surveillance. To address this need, we evaluated the performance of a targeted 21-locus methylation assay for urinary cell-free DNA (cfDNA) as a noninvasive tool for disease assessment and monitoring in BC. Methods: Candidate loci were identified from TCGA Illumina HumanMethylation450K tumor–normal methylation array comparisons. Differentially methylated CpGs were expanded to CpG regions/blocks for primer design and down-selected to a 21-locus panel. The loci were incorporated into a targeted assay optimized for urinary cfDNA analysis of BC. Urine samples were prospectively collected from 53 individuals, including 37 referred for cystoscopy and 16 healthy volunteers. Investigators were blinded to the reason for referral, and samples were obtained immediately before any diagnostic procedures. cfDNA was extracted, PCR-amplified, and analyzed by deep bisulfite sequencing to quantify methylation across the 21 selected loci. Results: Data from 53 participants were analyzed. Diagnostic classification was based on cystoscopy and biopsy pathology obtained after urine collection: 21 had histologically confirmed active BC, 11 had a history of BC but no evidence of disease (NED), 5 had benign urologic findings, and 16 were healthy controls. The assay correctly classified 19 of 21 participants with active BC (sensitivity = 90.5%) and 28 of 32 non-cancer participants (specificity = 87.5%), yielding an overall diagnostic accuracy of AUC = 0.93. Cancer-derived urinary cfDNA levels were higher in participants with active BC than in all non-cancer groups combined. Participants with prior BC but NED showed intermediate methylation levels, consistent with residual epigenetic alterations despite clinical remission. The assay did not distinguish between non–muscle-invasive and muscle-invasive disease. Conclusions: This prospective, blinded study demonstrates the feasibility of a novel, low-cost urinary cfDNA methylation assay for disease assessment and monitoring in BC using a compact 21-locus panel. Deep bisulfite sequencing enabled accurate quantification of cancer-specific methylation patterns, supporting the potential of this minimally invasive approach for follow-up and surveillance of bladder cancer. Further studies are planned to assess clinical performance and longitudinal application in real-world settings.