Development of a urine-based DNA methylation assay for early endometrial cancer detection: a multicenter diagnostic study

Abstract

Endometrial cancer (EC) incidence is rising globally, yet diagnosis still depends on invasive procedures, which limit accessibility and delay early detection. A noninvasive, accurate, and accessible diagnostic assay is urgently needed to enable early EC detection. In this multicenter diagnostic study across six hospitals in China (May 2021 - May 2026), we integrated whole-epigenome methylation profiling of 1,211 datasets followed by targeted bisulfite sequencing of 367 samples to identify EC-specific urine DNA methylation biomarkers. A qPCR-based urinary methylation assay (Urine-qEC) targeting three genes, VWC2 , ZNF132 , and ZSCAN12 , was subsequently developed and validated in a cross-sectional multicenter cohort ( n = 587). Urine hypermethylation signatures exhibited concordance with EC tumors and significantly diminished after tumor resection. The Urine-qEC assay achieved an AUC of 0.976 (89.4% sensitivity, 97.0% specificity) in the training cohort and 0.938 (85.5% sensitivity, 91.1% specificity) in the multicenter validation, with ZSCAN12 being the main contributor ( ZSCAN12 alone led to an AUC of 0.961 and 0.928, respectively). Sensitivity remained high across clinical stages—84.2% sensitivity for stage I ( n = 165, 68.8% of cases), 87.9% for stage II, and 94.4% for advanced-stage (III/IV) (92.2% specificity). Specificity was consistent across control subgroups, reaching 93.0% against symptomatic controls with abnormal uterine bleeding and 91.0% against benign controls. The Urine-qEC assay demonstrates the technical feasibility of a urine-based methylation approach for noninvasive endometrial cancer detection. These findings support further prospective validation in larger, diverse populations to establish clinical utility.