Urine Methylation Test for Urothelial Cancer Diagnosis and Surveillance.

Importance: Urothelial carcinoma (UC) is characterized by frequent recurrence, and accurate noninvasive tests remain needed for early detection and postoperative surveillance. Objective: To develop and validate a urine-based assay targeting AL021918.2 and vimentin (VIM) methylation for UC detection and postoperative surveillance. Design, Setting, and Participants: This prospective, blinded, multicenter diagnostic study was performed at 3 tertiary medical centers in China. Samples were collected between December 1, 2021, and June 25, 2025. Consecutively enrolled adults with UC, benign urinary diseases, or nonurothelial malignant disease were included in a single-center training cohort, a 3-center validation cohort, and a prospective surveillance cohort. The surveillance cohort had a median follow-up of 6 months, completed December 13, 2023. Exposure: Urinary DNA methylation was assessed using quantitative methylation-specific polymerase chain reaction (PCR) analysis. Main Outcomes and Measures: The primary outcomes were the sensitivity and specificity of the methylation assay for UC detection. Secondary outcomes included diagnostic performance according to tumor stage and grade and comparisons with urine cytology and fluorescence in situ hybridization (FISH). Results: This analysis included a total of 2307 participants, consisting of 643 in the training cohort (median age, 64 [IQR, 56-71] years; 494 men [76.8%]), 1620 in the validation cohort (median age, 67 [IQR, 61-73] years; 1129 men [69.7%]), and 44 with UC in the surveillance cohort (median age, 62 [IQR, 59-72] years; 32 men [72.7%]). The assay demonstrated sensitivities of 91.2% (95% CI, 87.6%-93.9%) in the training cohort and 90.8% (95% CI, 88.4%-92.8%) in the validation cohort and specificities of 90.0% (95% CI, 85.9%-93.2%) in the training cohort and 93.2% (95% CI, 91.3%-94.7%) in the validation cohort. In the training and validation cohorts, sensitivity remained high for Ta UC (79.6% [95% CI, 64.3%-89.7%] and 84.3% [95% CI, 77.8%-89.2%], respectively) and low-grade UC (86.4% [95% CI, 77.0%-92.5%] and 82.5% [95% CI, 75.7%-87.8%], respectively). Compared with urine cytology and FISH, the methylation assay demonstrated higher sensitivity while maintaining comparable specificity. After surgery, urinary methylation results were negative in 30 of 32 patients (93.8%) with positive preoperative results. During surveillance, the assay detected all 9 recurrences with a median lead time of 4.5 (IQR, 1.5-5.5) months before clinical confirmation and remained negative in 29 of 35 patients (82.9%) without recurrence. Conclusions and Relevance: In this diagnostic study, a noninvasive PCR-based assay showed high diagnostic accuracy for UC detection and identified all observed recurrences during surveillance. The assay may serve as an adjunct for UC diagnosis and postoperative surveillance. Trial Registration: Chinese Clinical Trial Registry Identifier: ChiCTR2300078302.