Urine DNA methylation as a potential diagnostic and surveillance tool in non-muscle invasive bladder cancer in Thailand: A pilot study

Non-muscle invasive bladder cancer (NMIBC) exhibits high recurrence, necessitating frequent invasive surveillance. Urine DNA methylation profiling represents a promising non-invasive biomarker approach for early detection and monitoring. This pilot study aimed to investigate urine DNA methylation patterns in NMIBC among Thai patients. Five patients with high-grade NMIBC and three healthy volunteers were enrolled, and midstream urine and blood samples were collected prior to transurethral resection of bladder tumor (TUR-BT). Urine cell-free DNA (cfDNA) was extracted, mechanically sheared from an atypical peak of approximately 400 bp to an optimal target length of 150-200 bp, and analyzed using cell-free methylated DNA immunoprecipitation followed by high-throughput sequencing (cfMeDIP-seq). Differentially methylated regions (DMRs) were identified between NMIBC and controls, and principal component analysis (PCA) was performed. A total of 82 DMRs were identified (adjusted P < 0.001), including 76 hypermethylated and 6 hypomethylated regions, with IRX4, TBX5, and MIR124-3 representing the top hypermethylated genes. To account for cohort heterogeneity, a refined subset analysis excluding two outlier samples (UC2 and UC3) was performed, revealing 476 significant DMRs (449 hypermethylated). PCA demonstrated clear, robust segregation between NMIBC and controls, highlighting MIR124-3, IRX4, and LHX1-DT as the most significantly hypermethylated targets. Our findings demonstrate that urine cfDNA methylation profiling can robustly distinguish NMIBC from healthy controls, highlighting MIR124-3, IRX4, and LHX1-DT as highly promising, demographic-specific biomarkers. This non-invasive approach successfully bypasses the degradative limits of traditional bisulfite conversion and establishes a technically validated foundation for clinical surveillance and diagnostics