Profiling Treatment-Associated Evolutionary Dynamics in Advanced Urothelial Cancer via Deep Whole Exome Sequencing of Cell-Free DNA

Circulating tumor DNA (ctDNA) has emerged as a promising biomarker for monitoring disease progression and treatment response in various cancers, including bladder cancer. In this study, we prospectively collected blood samples from a cohort of 70 patients with advanced bladder cancer to assess ctDNA dynamics during the course of treatment. Ultra-low pass whole genome sequencing (ULP-WGS; N = 213 samples) and deep 200X whole exome sequencing (D-WES; N = 47 samples with matched blood normals) were performed on cell-free DNA (cfDNA) specimens to quantify tumor fraction and identify somatic mutations. We observed that abundance of circulating tumor DNA correlated with clinical response, with increases in tumor fraction in the blood associated with disease progression and decreases in tumor fraction associated with responding disease. Longitudinal analysis of cfDNA D-WES during treatment revealed dynamic evolutionary competition between subclones – including higher nonsynonymous mutation burden in immunotherapy responsive subclones – and identified potential drivers of resistance and response to cytotoxic chemotherapy and PD-(L)1 therapy. Our findings suggest that cfDNA can serve as a valuable tool for monitoring treatment response in advanced bladder cancer, and that the addition of D-WES can potentially aid with anticipating tumor evolution on therapy as well as unraveling the biology underlying response and resistance.