Blood- and urine-based ctDNA profiling and monitoring in bladder carcinoma in situ.

846 Background: Carcinoma in situ (CIS) of the bladder is a high-grade, flat urothelial carcinoma with significant risk of recurrence and progression, requiring intensive and invasive surveillance. Liquid biopsy offers a non-invasive alternative for disease detection and monitoring. This study utilizes a next-generation sequencing (NGS)-based ctDNA assay to profile somatic mutations and copy number alterations in bladder CIS patients, using both blood and urine samples for longitudinal monitoring. Methods: In this ongoing prospective study, 48 patients with CIS bladder cancer were enrolled, and paired blood and urine samples were collected prior to initiation of first-line treatment. Additionally, patients with suspected disease recurrence were encouraged to provide blood and urine samples during follow-up visits at the cancer center. The study utilizes PredicineCARE, a targeted NGS liquid biopsy assay, to detect somatic alterations in ctDNA and to enable longitudinal monitoring of molecular recurrence. Results: Of the 48 patients enrolled, baseline blood samples were collected from 46 patients, and matched urine samples were obtained from 12 out of these 46 patients. In the blood samples, the assay detected 42 somatic mutations and 37 copy number variations (CNVs). The five most frequently altered genes in blood were TP53 (17%), FGFR1 (11%), PTEN (9%), FGFR3 (7%), and POLE (7%). In the urine samples, 58 somatic mutations and 13 CNVs were identified, with the most commonly altered genes being ATM (33%), TP53 (33%), ARID1A (25%), BRCA2 (25%), and ERBB2 (25%). Tumor fraction was estimated based on the identified somatic mutations to assess molecular tumor burden. Urine samples demonstrated a higher ctDNA signal than blood, with a median tumor fraction of 4.1% versus 3.1% in positive samples (p < 0.001). In addition, follow-up blood samples were collected from five patients with suspected recurrent disease, with one of these patients also providing a urine sample at the second time point. Tumor fraction analysis revealed molecular progression in four patients, indicated by increased tumor fraction, while one patient showed a decreased tumor fraction (down to 0), suggesting a complete molecular response. The urine result from one patient also indicated molecular progression, supporting the findings observed in blood. These molecular findings were concordant with subsequent clinical diagnoses. Conclusions: This study demonstrates the feasibility of using a targeted NGS-based liquid biopsy assay to detect and monitor ctDNA in both blood and urine of patients with bladder CIS. Urine samples showed higher sensitivity than blood, and longitudinal ctDNA dynamics correlated with clinical outcomes, supporting the potential of liquid biopsy for non-invasive disease surveillance in bladder cancer.