The significance of urine extracellular vesicle DNA methylation detection in the diagnosis and classification of prostate cancer

This study confirmed the significance of EV DNA methylation targets in prostate cancer (PCa) diagnosis and classification. We developed and optimized a DNA methylation detection system for extracellular vesicles (EVs), performed an extensive genome-wide screening of differential methylation targets, and created a PCa-specific targeted sequencing panel. Notably, we constructed, for the first time, a differential diagnostic model for PCa based on urine EV DNA methylation. This model demonstrated unique advantages in distinguishing aggressive PCa cases, thereby providing new insights into tumor markers and offering a promising avenue for enhancing prostate cancer diagnosis. • EV DNA accurately reflects the methylation state of source cell genomic DNA, providing a reliable substitute for tissue detection in PCa. • Total EV without DNase I treatment is a preferable choice for detecting EV DNA methylation biomarkers. • A novel panel of six methylation sites (MACF1、LINC01359-1、LINC01359-2、ADCY4、GAPLINC、C19orf25) in urine EV DNA demonstrates unique advantages in distinguishing PCa patients, particularly those with aggressive disease. As one of the most common malignant tumors in men, prostate cancer (PCa) still lacks convenient, non-invasive and highly specific diagnostic markers. The advantages of Extracellular vesicle (EV) DNA in tumor diagnosis have gradually attracted the attention of researchers. However, methylation detection, which is more advantageous than mutation detection in tumor diagnosis, has not been widely practiced in EV DNA, and its value in PCa diagnosis also remains underexplored. This study aims to establish and optimize an EV DNA methylation detection system and evaluate its diagnostic and classification potential for PCa. We characterized EV DNA biological properties, optimized pretreatment strategies, validated its correlation with genomic DNA methylation, and explored urine EV DNA methylation targets in 86 benign prostatic hyperplasia (BPH) and 109 PCa patients across three cohorts (screening: 30 BPH/33 PCa; training: 27 BPH/30 PCa; validation: 29 BPH/46 PCa). Heterogeneous biological characteristics were observed among DNA from different subtypes of EV, but methylation profiles remained consistent across subtypes and post-DNase I treatment. EV DNA accurately reflected the methylation state of source cell genomic DNA. By combining our screening results with data from the TCGA database and previously reported, we developed a panel consisting of 667 PCa-specific methylation targets for detection. Among these, six methylation sites (MACF1、LINC01359-1、LINC01359-2、ADCY4、GAPLINC、C19orf25) demonstrated high diagnostic value for PCa, enabling construction of PCa and aggressive PCa differential diagnosis model with AUCs up to 0.74 and 0.91 respectively. The diagnostic value of these six markers was further confirmed using methylight PCR in the validation cohort which also displayed promising performance as a tool for diagnosing PCa. This study highlights the potential of urine EV DNA methylation as a novel diagnostic marker for PCa and lays a foundation for future EV DNA research.