High Sensitivity ctDNA Analysis Using a Novel Panel and NOIR-SS Technology for Monitoring Advanced Urothelial...

Advanced urothelial carcinoma (aUC) has a poor prognosis, and real-time monitoring of treatment response remains clinically challenging. Circulating tumor DNA (ctDNA) has emerged as a promising non-invasive biomarker that may reflect tumor burden and molecular response. In this study, we analyzed ctDNA dynamics using a urothelial carcinoma-specific mutation panel and the non-overlapping integrated read sequencing system (NOIR-SS), a high-fidelity deep sequencing platform incorporating molecular barcoding for enhanced analytical sensitivity. Tumor tissue and serial plasma samples were collected from 15 patients with aUC treated with dose-dense methotrexate, vinblastine, doxorubicin, and cisplatin (ddMVAC). The custom panel targeted hotspot mutations in TP53, FGFR3, KRAS/HRAS, and the TERT promoter. Based on tumor-derived mutations, a tumor-informed approach was used to track ctDNA in plasma. ctDNA was detectable in 10 of 15 patients prior to ddMVAC and showed a trend toward association with total tumor volume, especially in cases with liver metastases. In contrast, ctDNA detection was limited in cases with only pulmonary metastases. Longitudinal changes in variant allele frequency largely mirrored treatment response, although discrepancies were observed in two cases, likely reflecting subclonal resistance. While the NOIR-SS-based assay proved sensitive and informative, limitations include the cost and time required for sequencing, potential temporal discordance between tissue and plasma sampling, and the absence of correction for clonal hematopoiesis of indeterminate potential. Overall, ctDNA profiling using this targeted panel and NOIR-SS suggested the feasibility of sensitive, non-invasive molecular monitoring in aUC, and may have future clinical applicability if validated prospectively in larger cohorts.