Circulating Tumour DNA (ctDNA) in Bladder Cancer: ctDNA for Risk Stratification, Disease Monitoring, and Precision Oncology in Urothelial Bladder Cancer

Abstract

Bladder cancer management relies heavily on invasive diagnostic and surveillance procedures, particularly cystoscopy, despite substantial biological heterogeneity in disease behaviour. Circulating tumour DNA (ctDNA) has emerged as a potential biomarker for risk stratification, minimal residual disease detection, and treatment monitoring; however, its clinical utility in early-stage bladder cancer and in real-world sample types remains incompletely defined. This thesis investigated the feasibility, analytical performance, and clinical relevance of tumour-informed ctDNA detection across muscle-invasive and high-risk non–muscle-invasive bladder cancer (NMIBC), with a particular focus on serum-based analysis and international applicability. An initial methodological phase optimised cfDNA extraction from human serum, addressing key pre-analytical challenges associated with low DNA yield and genomic DNA contamination. Mock cfDNA and genomic DNA models were developed to evaluate fragmentation, size selection, and recovery efficiency, resulting in an optimised workflow capable of preserving low-frequency tumour-derived variants. This protocol enabled reliable downstream sequencing from archival serum samples, expanding the potential use of legacy biobank material for translational ctDNA research. Using the GALEAS™ Bladder targeted sequencing panel, tumour-informed ctDNA detection was subsequently evaluated in multiple clinical cohorts. In muscle-invasive bladder cancer, tumour-informed ctDNA was detectable in archival serum samples and was associated with significantly shorter disease-specific and overall survival. In a high-risk pT1 NMIBC cohort, baseline serum ctDNA was detected in a minority of patients; however, ctDNA positivity identified a subgroup with significantly worse progression-free, disease-specific, and overall survival, independent of European Association of Urology risk classification. A harmonised genomic analysis of Saudi Arabian and UK tumour cohorts using the same targeted panel demonstrated broadly similar distributions of recurrent bladder cancer driver mutations, with modest differences in gene-specific mutation frequencies. Overall, this thesis demonstrates that tumour-informed ctDNA analysis can be applied to real-world serum samples and that ctDNA positivity identifies patients with adverse clinical outcomes across bladder cancer stages. The work defines the strengths and limitations of fixed-panel ctDNA assays relative to ultra-sensitive minimal residual disease technologies and provides a practical framework for the future integration of ctDNA into bladder cancer prognostication, surveillance, and clinical trial design.