Purpose <p>The antibody–drug conjugates (ADCs) sacituzumab govitecan (SG) and datopotamab deruxtecan target trophoblast cell surface antigen 2 (TROP-2) and have shown significant efficacy in HER2-negative metastatic breast cancer (mBC). As TROP-2 may serve as a target across multiple treatment lines, detailed information on TROP-2 expression over time is of great interest.</p> Methods <p>TROP-2 expression was analyzed in breast cancer (BC) samples from patients treated at the University Hospital Würzburg between 2004 and 2025 at clinically indicated biopsy time points (TP), and, in a subset, before and after SG treatment. Expression was assessed immunohistochemically using the H-score (range 0–300).</p> Results <p>We evaluated 229 samples from 76 patients. Overall, patient-level TROP-2 expression was high (mean H-score 241.3 ± 73.2) and largely stable over time (TP1: 235.7 ± 60.8, n = 76; TP2: 244.6 ± 77.4, n = 72; TP3: 245.3 ± 84.4, n = 39; TP4: 244.9 ± 95.6, n = 8). However, 21 patients exhibited a decline in TROP-2 expression during disease progression, defined as a decrease of ≥ 50 H-score points between two consecutive TPs. Among 13 patients with paired biopsies obtained pre- and post-SG treatment, the mean TROP-2 H-score decreased from 227.0 ± 74.6 before SG to 202.9 ± 99.4 after SG (mean change − 24.1 ± 86.0; median 1.7, range − 170 to 110; Wilcoxon p = 0.542). TROP-2 H-score decreased in 6/13 patients and increased in 7/13 patients. Exploratory PFS analysis showed no statistically significant difference by post-SG TROP-2 change (log-rank p = 0.526).</p> Conclusion <p>TROP-2 expression was generally high and relatively stable; however, some patients showed declining levels over time.</p>