Urinary cell TWIST1 methylation as a non-invasive dual biomarker: A Multicenter study for early detection and recurrence monitoring in bladder cancer.

OBJECTIVE

To develop and validate a non-invasive urine-based TWIST1 methylation-specific quantitative methylation‑specific polymerase chain reaction (qMSP) assay for bladder cancer (BC) diagnosis and surveillance to address the clinical need for a reliable complement invasive cystoscopy.

METHODS

This multicenter study included a three-phase design. The discovery phase identified hypermethylated CpG sites within the TWIST1 promoter in matched BC/normal tissue samples (n=6). A targeted qMSP assay was developed and subsequently validated in training (n=728) and independent multicenter validation cohorts (n=1,233).

RESULTS

The assay demonstrated 87.2% sensitivity and 90.8% specificity in the training cohort. Validation confirmed 88.2% sensitivity and 86.8% specificity for primary BC. Sensitivity remained high across tumor stages: 81.3% (Ta), 94.2% (T1), and 100.0% (T2-4). Muscle-invasive and high-grade tumors showed higher sensitivity (97.1% and 95.6%, respectively). Specificity was 91.7% in healthy controls and comparatively lower in upper-tract urothelial carcinomas. The assay exhibited high agreement with pyrosequencing (99.2% concordance), with a detection limit of 5% and low variability (coefficient of variation <10%).

CONCLUSION

The urinary TWIST1 methylation qPCR assay is a clinically robust, non-invasive tool for BC diagnosis and recurrence monitoring. The assay demonstrated high accuracy across disease stages and serves as a promising molecular adjunct to cystoscopy for bladder cancer surveillance. However, its performance in distinguishing BC from upper-tract tumors warrants further study.