TROP2 Expression Across Molecular Subtypes of Urothelial Carcinoma and Enfortumab Vedotin-resistant Cells

Abstract

Sacituzumab govitecan (SG) is an antibody-drug conjugate (ADC) targeting TROP2, which has recently been approved for treatment-refractory metastatic urothelial cancer (UC). However, the variability of TROP2 expression across different bladder cancer (BC) subtypes, as well as after enfortumab vedotin (EV) exposure, remains unknown. Using gene expression data from four clinical cohorts with >1400 patient samples of muscle-invasive BC and a BC tissue microarray, we found that TROP2 mRNA and protein are highly expressed across basal, luminal, and stroma-rich subtypes, but depleted in the neuroendocrine subtype. In addition, TROP2 mRNA levels are correlated with NECTIN4 mRNA but are more highly expressed than NECTIN4 mRNA in patient cohorts and BC cell lines. Moreover, CRISPR/Cas9-mediated knockdown of TROP2 demonstrates that its expression is one factor governing SG sensitivity. After prolonged EV exposure, cells can downregulate NECTIN4, leading to EV resistance, but retain TROP2 expression and remain sensitive to SG, suggesting nonoverlapping resistance mechanisms to these ADCs. While our findings warrant further validation, they have significant implications for biomarker development, patient selection, and treatment sequencing in the clinic as well as clinical trial design and stratification for metastatic BC patients. PATIENT SUMMARY: In this report, we investigated the expression levels of the drug target TROP2 across different molecular subtypes of bladder cancer in multiple patient cohorts and cell lines. We found high levels of TROP2 in most subtypes except in the neuroendocrine subtype. Overall, TROP2 gene expression is higher than NECTIN4 gene expression, and cells resistant to enfortumab vedotin (EV), a NECTIN4-targeting antibody-drug conjugate, remain sensitive to sacituzumab govitecan (SG). Our findings suggest that SG may be effective across most bladder cancer subtypes, including the bladder cancers previously treated with EV.

Keywords: Antibody-drug conjugate; Bladder cancer; Enfortumab vedotin; Molecular subtypes; Sacituzumab govitecan; Urothelial cancer.

Fig. 1 –

TROP2 mRNA and TROP2 protein expression across the molecular subtypes of muscle-invasive bladder cancer (MIBC). Violin plots showing TROP2 mRNA expression levels by consensus molecular subtypes in the (A) Seiler, (B) TCGA, (C) Sjödahl, and (D) Decipher cohorts. (E) Immunohistochemistry for TROP2 was performed using a bladder cancer TMA (n = 80 samples, in duplicate). H scores for TROP2 were assigned in a blinded manner, and subtypes were determined previously. The average TROP2 H score ± SEM is shown for each subtype. The p value from Kruskal-Wallis testing is shown for each cohort in panels A–E. (F) Scatter plot showing the correlation between TROP2 protein (H score) and TROP2 mRNA expression levels. The Spearman’s rho coefficient is shown (p < 0.0001). Ba/Sq = basal/Squamous; LumNS = luminal nonspecified; LumP = luminal papillary; LumU = luminal unstable; NE = neuroendocrine; SEM = standard error of the mean; TCGA = The Cancer Genome Atlas; TMA = tissue microarray.

Fig. 2 –

TROP2 and NECTIN4 mRNA expression in MIBC and correlation to antibody-drug conjugate response. Violin plots of NECTIN4 and TROP2 mRNA levels in the (A) TCGA and (B) Sjödahlcohorts. The p value from Wilcoxon rank-sum testing is shown for each cohort. (C) Box and whisker plot of NECTIN4 and TROP2 mRNA expression in 35 urothelial carcinoma cell lines. (D) NECTIN4 and (E) TROP2 surface protein expression in seven bladder cancer cell lines. (F) Dose-response curves to the antibody drug conjugates (ADCs) enfortumab vedotin (EV) and sacituzumab govitecan (SG) in the UMUC-1 cell line. (G) NECTIN4 and (H) TROP2 surface protein expression in 647V control (black) and two 647V EV-resistant lines (purple and magenta) cell lines. Dose-response curves to (I) EV and (K) SG in 647V control (black) and two 647V EV-resistant lines (purple and magenta). BC = bladder cancer; MIBC = muscle-invasive bladder cancer; TCGA = The Cancer Genome Atlas. **** p < 0.0001 by Wilcoxon rank-sum test.