Noninvasive Detection of Bladder Cancer via Urinary DNA Mutation and Methylation Profiling Using Modified Real-Time...

Cystoscopy is the gold standard for bladder cancer (BC) diagnosis and surveillance but experiences invasiveness and patient discomfort. Existing urine-based assays with single-gene mutation or methylation biomarkers exhibit insufficient diagnostic efficacy, necessitating a reliable noninvasive detection approach. This study developed EpiMut-BC, a high-specificity urinary PCR assay integrating DNA mutation and methylation signatures based on pyrophosphorolysis-activated polymerization real-time quantitative PCR with engineered polymerases and blocked primers. A total of 502 participants from two Chinese tertiary hospitals, including 302 patients with histologically confirmed BC and 200 noncancer controls, were prospectively enrolled and divided into training (n = 93) and validation (n = 409) cohorts. All diagnostic outcomes were verified against cystoscopy and histopathology. Among 489 valid samples, EpiMut-BC achieved an overall sensitivity of 85.9%, a specificity of 95.8%, and an accuracy of 89.8%, with favorable predictive values and consistent specificity across diverse control subgroups. Notably, it yielded a sensitivity of 70.3% for early-stage tumors and 81.7% for superficial tumors. Validated in a large multicenter cohort, this noninvasive assay enables robust early detection of BC even with low-abundance tumor DNA, which can effectively reduce reliance on invasive cystoscopy and unnecessary repeated surgical resection.