Abstract 1966: Evaluation of circulating tumor cells as a real-time biomarker in metastatic urothelial carcinoma

Abstract Background: Patients with metastatic urothelial carcinoma (mUC) have poor prognosis and need biomarkers to guide treatment and monitor treatment responses. Circulating tumor cells (CTCs) offer a minimally invasive approach to capture tumor dynamics in real-time. This study aim to correlate mUC CTCs with treatment response and evaluate therapy-induced molecular alterations. Methods: Patients with mUC were prospectively enrolled at the University of Texas Southwestern Medical Center. Peripheral blood samples were collected at baseline, 4 weeks, and 12 weeks of treatment. CTCs were isolated using a dendrimer-based assay functionalized with antibodies against EpCAM, Trop-2, Nectin-4, and FGFR3, identifying nucleated CTCs (DAPI+/CK+, CD45-). Single cell proteomic profiling of CTCs was performed via the Nanostring GeoMx Immuno-Oncology (IO) Proteome Atlas panel. The primary objective was to assess CTC variation as a biomarker for treatment response. CTC changes were considered concordant if they decreased in patients with complete or partial response (CR or PR), remained stable in those with stable disease (SD), or increased in patients with progressive disease (PD), as defined by RECIST 1.1. Results: Eighteen patients with mUC were enrolled with median age of 76 years-old, 89% male and 61% Caucasian. Among the cohort, 78% (n=14) received enfortumab vedotin (EV) and pembrolizumab (P), 17% (n=3) received chemotherapy, and 5% (n=1) received sacituzumab govitecan. The median CTC counts (cells/ml) were assessed at baseline (39.3, IQR 26.5-64.4), week 4 (31.05, IQR 7.6-43.6), and week 12 (32.5, IQR 23.25-44.98). 13 patients had sufficient radiographic and clinical follow up. CTC changes were concordant with treatment responses in 77% (10/13) of patients, including 6/7 patients with PR, 1/2 with SD, and 3/4 with PD. Preliminary proteomic profiling of three patients who responded to 1L EV-P showed 58 upregulated and 70 downregulated proteins from baseline to week 4. We found that post-therapy, proteins related to antigen presentation (HLA-A, HLA-C, HLA-E), metabolism (beta glucuronidase), GLB1/Beta-galactosidase (Hexokinase 1), cell death (pro-apoptotic protein Bax, Smac/Diablo), and epithelial-mesenchymal transition (c-MET, N-cadherin, and vimentin) were upregulated, while proliferation signals (Ki67, Her2/ERBB2), metastasis (E-Cadherin, Phospho S431 Cytokeratin 8, c-Jun), and DNA damage repair (BRCA1, γH2A, ChK1, cleaved PARP1) were downregulated. Conclusion and Future Directions: Novel capture of CTCs in patients with mUC can be potential biomarkers for monitoring treatment response and detecting therapy-induced molecular alterations. Some on treatment molecular expression changes may be further targeted with targeted treatments. We continue to characterize CTCs to identify key molecular pathways that contribute to treatment sensitivity and resistance. Citation Format: Wadih Issa, Navneet Kaur, Qinhan Zhou, Andrew DeVilbiss, Ze Yu, Damla Gunenc, Michael Poellmann, Changchuan Jiang, Qian Qin, Jue Wang, Suzanne Cole, Seungpyo Hong, Chao Xing, Andrew Z. Wang, Tian Zhang. Evaluation of circulating tumor cells as a real-time biomarker in metastatic urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1966.