Clinical utility of a urinary DNA methylation biomarker test in the diagnosis and surveillance of upper tract urothelial carcinoma: Results from a prospective trial.

Abstract

822 Background: DNA methylation, an epigenetic modification frequently altered during tumorigenesis, has emerged as a promising diagnostic and prognostic biomarker for various cancers. However, data on patients with upper tract urothelial carcinoma (UTUC), particularly in the postoperative setting, remain limited. This study aims to evaluate the accuracy of a urine-based epigenetic test for the diagnosis and surveillance of UTUC. Methods: In this prospective clinical trial, urine samples were collected from primary UTUC patients prior to surgery with curative intent (radical nephroureterectomy/distal ureterectomy) and during follow-up visits (every 3 months) at two academic centers, the University of Southern California and Duke University, between April 2024 and July 2025. Samples were analyzed using Bladder CARE, a urine-based test that measures the methylation levels of three urothelial cancer-specific DNA biomarkers (tRNA-Cys, SIM2, and NKX1-1) in a single polymerase chain reaction (qPCR). Results were reported as Bladder CARE Index (BCI) scores and categorized into three groups: positive ( > 10), low-positive (2.5–10), and negative ( < 2.5). Preoperative results were compared to those from 1:1 sex- and age-matched cancer-free healthy controls. In addition, all pre- and post-op urine sample findings were correlated with clinical and follow-up data. Results: A total of 38 patients were included, of whom 14 had at least one follow-up sample. Final pathology showed stage Ta (n = 11), T1 (n = 5), T2 (n = 4), T3 (n = 15), and T4 (n = 3) UTUC. Most tumors were high-grade (34/36; 89%). Pre-surgery BCI results were positive in 29, low-positive in 3, and negative in 6 patients. Urine cytology was available for 26 patients, with 14 (54%) false negatives. Pre-surgery BCI was significantly higher than in controls (median 85.3 vs. 1.7, p < 0.001). The Bladder CARE test showed 84% sensitivity, 92% specificity, 91% positive predictive value, and 85% negative predictive value for UTUC detection. Median BCI was higher in pT2–T4 vs. pTa/T1 (112.8 vs. 39.4) and in high-grade vs. low-grade UTUC (112.8 vs. 1.2); however, these differences were not statistically significant (p = 0.8 and 0.1, respectively). Among 22 follow-up samples, a BCI cutoff of 10 showed 91% concordance with urothelial recurrence status. Conclusions: Bladder CARE is an accurate, non-invasive, urine-based epigenetic test for the diagnosis of UTUC. It may also aid in monitoring disease recurrence following surgery; however, a larger sample size and longer follow-up are needed to validate these findings. Clinical trial information: NCT06805630 .