Utility of Urinary Extracellular Vesicles for Colorectal Cancer: Comparison of DNA Quality and MRD Detection

This study evaluated the feasibility of urinary extracellular vesicles (EVs) as a liquid biopsy source for colorectal cancer (CRC) using two predefined cohorts: 97 patients for analytical validation and 63 patients for postoperative survival analysis. We compared urinary EVs, urinary cell-free tumor DNA (ctDNA), plasma EVs, and plasma ctDNA across multiple molecular dimensions, and assessed the utility of urinary EV-DNA for minimal residual disease (MRD) detection. Urinary EVs exhibited larger particle size (median 175.6 nm) and markedly higher purity, demonstrated by a substantially broader proteomic profile (4674 vs. 476 proteins) and significantly higher expression of EV-specific markers (CD63, CD81, CD9). DNA extracted from urinary EVs showed the highest concentration and best preservation of nucleic acid integrity, with the greatest Long/Short fragment ratio, outperforming all plasma-derived samples. Mutation detection sensitivity was comparable across sample types but was highest in plasma ctDNA (39.2%) and urinary EV-DNA (34.0%), exceeding urinary ctDNA (21.6%). In the survival cohort, MRD positivity-defined as detectable RAS/BRAF mutations in urinary EV-DNA 1 month after curative surgery-was strongly associated with inferior outcomes (HR for recurrence 5.25, 95% CI 1.74-15.80), and overall survival was significantly worse in MRD-positive patients. These findings indicate that urinary EVs provide highly pure vesicles with superior DNA quality, making them a robust and completely non-invasive source for molecular profiling. Urinary EV-DNA-based MRD assessment shows significant prognostic value and may serve as a practical tool for postoperative monitoring and risk stratification in colorectal cancer.