Proteomic profiling of circulating tumor cells (CTCs) in metastatic urothelial cancer (mUC) as associated with upregulation of EGFR, HER2, and B7-H3 in patients (pts) progressing on enfortumab vedotin (EV) plus pembrolizumab.

Abstract

4580 Background: Pts with mUC have poor prognosis, and while EV/pembro have improved outcomes, therapeutic targets arising after EV-pembro treatment are elusive. Proteomic profiling of circulating tumor cells (CTCs) offer a minimally invasive approach to capture tumor biology changes in real-time. We evaluated CTC burden and proteomic changes in relation to treatment response in mUC. Methods: Pts with mUC were prospectively enrolled at UT Southwestern Medical Center. Peripheral blood samples were collected at baseline, 4 weeks, and 12 weeks of treatment, as well as progression of disease. CTCs were isolated using a dendrimer-based microfluidic chip assay functionalized with antibodies against EpCAM, Trop-2, Nectin-4, and FGFR3, identifying nucleated CTCs (DAPI+/CK+, CD45-). After CTCs were selected within regions of interest, spatial proteomic profiling was performed via the Nanostring GeoMx Immuno-Oncology Proteome Atlas panel. CTC variation and surface proteomics after treatment were primary objectives. CTC changes were considered concordant if they decreased >10% in pts with complete or partial response (CR or PR), remained stable (within 10% of baseline) in pts with stable disease (SD), or increased >10% in pts with progressive disease (PD), as defined by RECIST 1.1. One pt underwent post-treatment biopsy with IHC analysis. Results: Thirty pts with mUC were enrolled, with a median age of 71.5 years, predominantly male (26/30, 87%) and Caucasian (24/30, 80%). Most received EV/pembro (26/30, 86.7%), while the rest were treated with chemotherapy (3/30, 10%) or SG (1/30, 3%). Median CTC counts (cells/ml) were assessed at baseline (30.3, IQR 18.5–62.9), at week 4 (22.3, IQR 6.0–37.0), and at week 12 (29.5, IQR 15.2–44.5). Twenty-two pts had sufficient radiographic and clinical follow up. CTC changes were concordant with treatment responses in 77% (17/22) of pts, including 12/22 pts with CR/PR, 3/22 with SD, and 7/22 with PD. Proteomic profiling of serial CTCs from 9 pts treated with EV/pembro revealed dynamic proteomic changes from baseline to week 12 and at disease progression. Together, these pts demonstrated upregulation of activated EGFR (phospho-Y1068), HER2 (phospho-Y877), STAT5, FAK, and vimentin. Pts with primary PD (n=3) showed B7-H3 upregulation on CTCs, whereas pts with secondary PD(n=2) showed higher expression of vimentin, c-MET, and MHC class I on CTCs. IHC of a post-EV/pembro progression bone metastasis confirmed increased HER2 expression (2+). Conclusions: CTCs with proteomic profiling can serve as a dynamic, noninvasive biomarker in mUC, identifying distinct therapeutic targets for pts progressing on EV/pembro. The emergence of actionable pathways, including EGFR, HER2, c-MET, and B7-H3, suggests opportunities for rational, biomarker-driven therapeutic strategies.