Spatial Expression of HER2, NECTIN4, and TROP-2 in Muscle-Invasive Bladder Cancer and Metastases: Implications for Pathological and Clinical Management

Muscle-invasive bladder cancer (MIBC) presents significant treatment challenges. Antibody-drug conjugates targeting human epidermal growth factor receptor 2 (HER2), trophoblast cell surface antigen 2 (TROP-2), and nectin cell adhesion molecule 4 (NECTIN4) offer promising therapeutic options. This study examined the spatial expression of HER2, TROP-2, and NECTIN4 in MIBC and metastases, their association with molecular subtypes, and clinical outcomes. Formalin-fixed, paraffin-embedded tissue samples from 251 patients with MIBC were analyzed using immunohistochemistry and tissue microarray analysis. Expression patterns between the tumor front and tumor center (TC) were compared, and statistical analyses assessed associations with molecular subtypes and clinical parameters. Additionally, 67 matched lymph node metastases and a secondary cohort comprising 16 distant metastases, including 7 matched primary tumors, were examined to explore the expression patterns in advanced tumor stages. In primary tumors, HER2 was predominantly negative (83%) but showed higher positivity in the TC. TROP-2 exhibited high overall expression (58% score 3+), whereas NECTIN4 displayed significant heterogeneity with stronger expression in the TC. Spatial overexpression of TROP-2 and NECTIN4 at the tumor front relative to the TC was associated with a better disease-free survival. Accurate assessment required 4 biopsies for HER2 and NECTIN4 and 3 for TROP-2. HER2 expression was associated with urothelial-like and genomically unstable molecular subtypes, whereas TROP-2 was widely expressed except in the mesenchymal-like subtype. NECTIN4 showed the absence of staining in basal, Mes-like, and Sc/NEC-like subtypes. Paired lymph node metastases showed higher expression scores for all 3 markers, whereas distant metastases showed reduced NECTIN4 expression. Additionally, lymph node metastases revealed a considerable heterogeneity for HER2 compared with their matched primary tumors. The spatial heterogeneity of HER2, TROP-2, and NECTIN4 expression necessitates multiple biopsies, particularly from the TC, for accurate evaluation. These findings underscore the need for personalized treatment strategies in MIBC, considering the increased risk of relapse associated with HER2 and NECTIN4 overexpression in the TC. Implementing a multibiopsy approach is critical to enhancing diagnostic accuracy.