A urine-based DNA methylation marker test to detect upper tract urothelial carcinoma: a prospective pilot study

Abstract Objectives: Upper tract urothelial carcinoma (UTUC) is an uncommon, yet lethal tumor of the urinary tract. Diagnosis and preoperative risk stratification of these patients present distinct challenges given the limitations of current available diagnostic tools, including endoscopic biopsy and imaging. Herein, we explore the feasibility and clinical performance of a urine-based epigenetic assay in the diagnosis of patients with UTUC. Methods: Under an institutional review board-approved protocol, voided urine samples were prospectively collected using the Bladder CARE Urine Collection Kit (Pangea Laboratory LLC, CA, USA) from primary UTUC patients before any genitourinary manipulation between November 2019 and March 2022. All patients were confirmed to have UTUC in the final specimen. A 1:1 age/sex-matched group of cancer-free healthy donors (control group) was also included in the study. “Healthy” status was based on self-reporting and defined as no history of any type of tumor. Urine samples were analyzed with Bladder CARE, a urine-based test that measures the methylation level of 3 urothelial cancer-specific biomarkers (TRNA-Cys, SIM2, and NKX1-1) and two internal control loci using methylation-sensitive restriction enzymes coupled with qPCR. Results were reported as Bladder CARE Index (BCI) score and categorized as “positive” (BCI > 5), “high risk” (2.5 < BCI ≤ 5) or “negative” (BCI ≤ 2.5). Association between BCI score and category, and clinical and pathological findings was assessed. Results: A total of 50 patients (40 males and 10 females) with a median age of 72 (64-79) years were included in the final analysis. Six patients received neoadjuvant chemotherapy. Surgical procedures included RNU (n=40), ureterectomy (n=7), and URS (n=3). All patients who underwent URS were confirmed to have UTUC through biopsy. Among UTUC patients, Bladder CARE test resulted positive in 47, high-risk in 1, and negative in 2 patients. Comparing Bladder CARE test results with tumor pathologic features demonstrated a statistically significant correlation between BCI values and tumor size (p = 0.009). UTUC patients had significantly higher BCI values compared to the controls (mean BCI 189.3 vs. 1.6, respectively; p < 0.0001). ROC curve showed an AUC of 0.9662 (95% CI = 0.9296-1). Sensitivity, specificity, PPV, and NPV of Bladder CARE to detect UTUC were 96%, 88%, 88.9%, and 95.7%, respectively. In comparison, the sensitivity of urine cytology in our cohort was 37%. Conclusions: In this prospective pilot study, the proposed urine-based epigenetic test (Bladder CARE) showed high sensitivity and negative predictive value in the diagnosis of patients with UTUC. In addition, the sensitivity of this test was exceedingly higher than the standard urine cytology. A larger sample size study to validate the accuracy of this test is the next step. Citation Format: Paolo Piatti, Alireza Ghoreifi, Sanam Seyedian, Yap Ching Chew, Benjamin Jara, Lucy Sanossian, Jeffrey Bhasin, Michael Basin, Taikun Yamada, Gerhard Fuchs, Sumeet Bhanvadia, Rene Sotelo, Andrew Hung, Monish Aron, Mihir Desai, Inderbir Gill, Siamak Daneshmand, Gangning Liang, Hooman Djaladat. A urine-based DNA methylation marker test to detect upper tract urothelial carcinoma: a prospective pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3296.