Optimization of a urine based NGS assay for detecting minimal residual disease in bladder cancer

Background: Current clinical tools struggle to accurately distinguish minimal residual tumor from benign bladder tissue following neoadjuvant chemotherapy in radicl cystectomy patients. Identification of complete responders might permit avoidance of radical cystectomy. To address this need, we developed an NGS-based urine assay to support non-invasive detection of chemotherapy response. This study focuses on optimizing key pre-analytical factors, including preservative selection and DNA extraction methods. Methods: To overcome the logistical constraints of immediate -80 °C freezing, multiple urine preservatives were evaluated for their ability to maintain DNA integrity. Two DNA extraction strategies were compared: Sepharose-based extraction and a popular commercial kit. DNA from each condition was subjected quality control testing and to shallow pass whole-genome sequencing, followed by fragmentomics analysis, fragment end-motif profiling, and copy-number alteration (CNA) estimation. Results: Sepharose extraction combined with a specially designed salt buffer yielded the highest DNA recovery. Commercial products did not promote fragment stability. Fragmentomics analyses showed that cancer patients exhibited higher fraction of aberrant fragments (FAF) than healthy controls when processed with Sepharose extraction. Distinct fragment end-motif patterns were also observed between the two groups. CNA analysis demonstrated that buffer-preserved samples produced higher estimated tumor fractions than samples processed with immediate freezing, indicating improved tumor signal retention. Unexpectedly, Sepharose extraction yielded improved signal-to-noise ratio as well. Conclusions: The assay shows strong potential for detecting minimal residual bladder cancer and supporting non-invasive evaluation of complete response after chemotherapy. Salt buffer effectively enhances urinary DNA preservation and improves tumor-derived signal detection compared with immediate freezing. We are continuing to collect more urine samples to further refine and clinically validate the assay for clinical use.