Novel DNA methylation biomarkers in urine for non-invasive diagnosis of bladder urothelial carcinoma

Bladder urothelial carcinoma (BLCA) is a common malignant tumor with high invasiveness and recurrence rates, underscoring the need for early diagnosis and effective monitoring. Current diagnostic methods, such as invasive cystoscopy and low-sensitivity urine cytology, have limitations. Oncogene hypermethylation plays a key role in tumorigenesis and progression. However, DNA methylation in BLCA remain underexplored. Identifying and validating new DNA methylation markers in urine samples is crucial to enhance early detection accuracy. In this study, we identified three novel BLCA DNA methylation biomarkers (HIST1H3J, NKX2-4, and YBX3P1 genes), and compared with six known markers (ONECUT2, OTX1, POU4F2, SOX1, TWIST1, VIM). Real-time quantitative methylation-specific PCR (qMSP) was used to detect the methylation levels of biomarkers in 319 urine samples from patients with suspected BLCA. The individual biomarkers of HIST1H3J, NKX2-4, and YBX3P1 achieved Areas Under the Curve (AUCs) of 0.892, 0.914 and 0.871, with accuracies of 84.80%, 85.38% and 81.29%, respectively. In comparison, the six known markers exhibited AUCs ranging from 0.850 to 0.939 and the accuracies of 81.87%-88.30%. These methylation markers can not only identify high-grade BLCA but also low-grade BLCA, highlighting their potential clinical utility. Notably, a four-gene panel (ONECUT2, SOX1, TWIST1 and NKX2-4) significantly improved the detection performance, with an AUC of 0.971 and an accuracy of 92.39%. Our results provide three new DNA methylation markers for BLCA and propose a urine-based DNA methylation detection panel for non-invasive clinical diagnosis.