Objective: To develop and validate a non-invasive urine-based TWIST1 methylation-specific qPCR assay for bladder cancer (BC) diagnosis and surveillance, addressing the clinical need for a reliable alternative to 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 a training cohort (n=728) and an independent multicenter validation cohort (n=1,233).Results: The assay demonstrated 87.8% sensitivity and 90.7% 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 but 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 (CV<10%).Conclusion: The urinary TWIST1 methylation qPCR assay is a clinically robust, non-invasive tool for BC diagnosis and recurrence monitoring. It shows high accuracy across disease stages and may help reduce the need for repeated cystoscopy. However, its performance in distinguishing BC from upper tract tumors warrants further study.

Although single-cell analyses have advanced our understanding of pancreatic ductal adenocarcinoma (PDAC), most studies to date have focused on primary and metastatic tumors. Here, we map cell composition, phenotypic plasticity, and microenvironmental remodeling from human normal pancreas through preneoplastic lesions to PDAC, with the preneoplastic phase recognized as a critical window for carcinogenesis. We pinpoint genes that are persistently dysregulated throughout malignant transformation and are associated with a poor prognosis. Focusing on ductal and acinar cells as the principal origins of PDAC, we delineate malignant preneoplastic cell clusters that exhibit strong carcinogenic potential. Immune profiling reveals marked expansion and functional reprogramming of macrophages during disease progression. Integrative analysis with human PDAC bulk transcriptomic cohorts identifies candidate compounds, such as Brefeldin A, with potential for intervention in preneoplastic disease. Together, our study elucidates dynamic molecular and cellular mechanisms underlying PDAC carcinogenesis and provides actionable insights for early intervention and targeted therapy.