Dynamics of nectin-4 (N4) expression in urothelial carcinoma (UC): Across disease states.

3131 Background: Enfortumab vedotin (EV), which targets N4, has revolutionized advanced UC management. Despite high prevalence of N4 in UC, the temporal stability of N4 is poorly characterized. Conflicting reports describe both decreased and increased expression in metastatic (met) UC compared to primary tumors raising uncertainty as to whether changes reflect antigen loss or quantitative modulation—an issue central to resistance to EV. Methods: This single-center retrospective study identified pts with met UC who received treatment (trt) with EV. Demographic, clinicopathologic, trt and survival data (from time of met diagnosis) were recorded. N4 expression was evaluated via immunohistochemistry (IHC) (N4 Clone EPR15163-68) H-Score in available primary (TURBT, cystectomy/nephroureterectomy) and met tissue. H-Score was assessed as N4 positive (+) vs negative (neg,-) (>0 vs 0) and ordinally (neg, low <100, medium 100-200, high >200). Concordance between specimen pairs was assessed using percent agreement, McNemar’s test, and weighted Cohen’s kappa. Overall survival (OS) was evaluated via Kaplan–Meier methods and exploratory Cox regression. Results: We identified 141 pts (median age 70; 71% male; primary: bladder (83%), upper tract (15%), synchronous (2.1%), unknown (0.7%). 93% of primary tumors were N4+ (n=44). In matched TURBT-Cystectomy pairs (n=21), binary status was stable (95% agreement), but ordinal concordance was poor (27% agreement, κ= −0.05) and significant antigenic dampening was observed: High N4 expression decreased from 90% at TURBT to 29% at cystectomy, with a reciprocal increase in medium-level expression from 10% to 48% (p<0.001). 89% of met sites (n=27) were N4+. There were no differences in receipt of prior neoadjuvant chemotherapy (NAC) or 1st line trt between met N4+ and N4- groups. In matched cystectomy and metastases pairs (n=11), binary N4 concordance remained high (82% agreement) with poor ordinal concordance (27% agreement, p=0.12). Neither binary nor ordinal metastatic expression, nor changes in H-score over time, were associated with OS. There were no differences in OS in binary met N4+ vs N4- (p=0.82) nor ordinally classified groups (p=0.43). Conclusions: N4 is a stable binary target in UC, maintaining positivity across primary and metastatic sites despite significant quantitative dampening in expression intensity. These findings, although exploratory suggest that tumor evolution and therapeutic resistance may be characterized by dynamic antigen density modulation rather than categorical loss, an interpretation that may help reconcile prior discordant reports of expression changes in metastatic disease. Future studies should evaluate whether the degree of H-score shift (rather than absolute value) correlates with the durability of response to EV as well as the impact of neoadjuvant and subsequent sequential treatments on N4 expression intensity.