Introduction: Nectin-4, a membrane protein involved in cell adhesion, has been recently introduced as a target of the novel antibody drug conjugate Enfortumab-Vedotin (EV). However, uniform Nectin-4 overexpression in BC was reported and no predictive capacity from Nectin-4 staining was reported. In the present trial we aimed to determine expression in two independent BC cohorts and further evaluate an alternative IHC interpretation approach for Nectin-4 in advanced BC. Methods: The study included two independent cohorts consisting of n=97 and 103 patients (70x male, median age 65.5 years, 31x T=2 – 43x T3 – 23x T4, 76x G>2, 11x M1 and 79x male, 69 years, 31x T=2 – 48x T3 – 24x T4, 78x G>2, 9x M1, median follow-up 44.5 months, respectively) who underwent radical cystectomy for invasive BC. Tissues from histologically proven BC and from benign urothelium (n=22 and 39) were processed to a tissue microarray and immunohistochemically stained by reported methods (polyclonal rabbit antibody, dilution 1:2000, incubation 16h at 4°, quantified by the histochemical scoring system / H-score 0-300). Results were transferred into four classes: Each cohort was divided into four 25%-classes of successive increasing staining and distribution was demonstrated for each the two BC cohorts. Results were compared to clinical and pathological features. Results: Mean expression scores in BC and parallel benign urothelium tissue were 133 and 81 / 157 and 114 (p < 0.005 / 0.002) with benign urothelium expression correlated with that of corresponding BC ( <0.02). The expressions within each patient cohort are lined up as follows: Expression in 25% of the cohort scored low (0-99), of 25% 100-119, of 25% 120-189 and of 25% strong (190-300) in the first cohort, the second distributed low (0-99) of 25%, 25% 100-165, 25% 166-209 and 25 % strong 210-300. There were no significant associations of Nectin-4 expression to demographic or clinical patients’ data. Moreover, there was no clinical association between Nectin-4 expression and survival. Conclusions: Nectin-4 overexpression in BC is accompanied by parallel strong expressing benign urothelium in the same individual. While the distributions of expression in independent BC cohorts are similar, the assumption of Nectin-4 overexpression in all BC may be overestimated. A considerable share of even advanced BCs exhibit Nectin-4 only to a low or a medium level. This observation may support efforts of adapted reintroduction of Nectin-4 as a predictive biomarker for EV. SOURCE OF Funding: Sources of Funding are coming from University Hospital Tuebingen
