Metastatic Bladder Cancer Expression and Subcellular Localization of Nectin-4 and Trop-2 in Variant Histology: A Rapid Autopsy Study

We sought to characterize the expression level and subcellular localization of Nectin-4 and Trop-2, targets of novel antibody-drug conjugates in metastatic urothelial cancer, within variant bladder cancer from matched primary and metastatic sites. We find significant levels of the targets in histologic variants. Additionally, high levels of cytoplasmic rather than membrane staining is seen, which may have important clinical implications. Background: Nectin-4 and Trop-2 are transmembrane targets of FDA-approved antibody-drug conjugates (ADC) Enfortumab-vedotin (EV) and Sacituzumab govitecan (SG), respectively, for the treatment of metastatic urothelial carcinoma (mUC). The expression and role of Nectin-4 and Trop-2 in mUC variant histology is poorly described. Materials and Methods: We evaluate membranous and cytoplasmic protein expression, and mRNA levels of Nectin-4 and Trop2 within matched primary and metastatic mUC samples to determine heterogeneity of ADC targets in mUC variants. Results: Patients with mUC were consented for rapid autopsy immediately after death. Tissues from matched primary and metastatic lesions were collected. A total of 67 specimens from 20 patients were analyzed: 27 were UC, 17 plasmacytoid (PUC), 18 UC with squamous differentiation (UCSD), and 5 neuroendocrine (NE); 10 from primary and 57 from metastatic sites. All histology except NE expressed moderate-high levels of Nectin-4 and Trop-2 by both immunohistochemistry and RNAseq. Nectin-4 demonstrated prominent cytoplasmic staining in metastatic PUC and UCSD. Trop-2 demonstrated strong cytoplasmic and membrane staining in primary and metastatic tumors. Interestingly, Nectin-4 and Trop-2 expression are positively correlated at both mRNA and protein levels. Conclusion: UC and non-NE variants express notable level of Nectin-4 and Trop-2 in both primary and metastatic lesions. Membrane staining of Nectin-4 and Trop-2 is present but cytoplasmic staining is a more common event in both mUC and mUC variant histology. These findings support evaluation of EV and SG in heavily treated variant histology BC and urge attention on the clinical relevance of cytoplasmic localization of ADC targets.