Paired Nectin4 expression analysis pre and post enfortumab vedotin and pembrolizumab in urothelial carcinoma.

4586 Background: Enfortumab vedotin and pembrolizumab (EVP) transformed the treatment of urothelial carcinoma (UC), yet resistance remains a challenge. Mechanisms of resistance including changes in target expression are poorly defined. Methods: We analyzed an institutional retrospective cohort of patients (pts) who had paired tissue samples obtained pre- and post-EVP. Membranous (mNectin4) and cytoplasmic (cNectin4) expression were measured by immunohistochemistry (H-score). mNectin4 was categorized as negative (<15), weak (15-99), moderate (100-199) or strong (200-300). Changes in Nectin4 were assessed as absolute differences (post–pre) and as scaled log change (%) [100xlog((post + c)/(pre + c))] to capture relative expression changes. Nectin4 dynamics were compared between primary (resistant disease ≤6 months from EVP initiation) and acquired resistance (>6 months) using Wilcoxon rank-sum and fisher’s exact tests. Results: Of 46 pts with paired samples, 40 had evaluable Nectin4 IHC. Median age was 72, 70% were men, and 30% had upper tract primary. Median H-scores were similar pre- and post-EVP for both mNectin4 (195 [IQR 115, 260] vs 200 [IQR 85, 230]; p=0.5) and cNectin4 (0 [IQR 0, 40] vs 0 [IQR 0, 60]; p >0.9). Post-EVP mNectin4 was moderate/strong in 29 pts (73%). Complete loss of mNectin4 was uncommon: of five pts (13%) with negative mNectin4 post-EVP, two were negative at baseline and three had weak expression pre-EVP. Post-EVP samples obtained on EV (≤21d from last dose; n=21) vs off EV (>21d; n=19) showed similar changes from pre-EVP for mNectin4 (p=0.3) and cNectin4 (p=0.4). Nectin4 changes differed by resistance pattern, with primary resistance (n = 14) showing a greater relative decrease in mNectin4 expression (median log change −57 vs 3; p = 0.035) and a relative increase in cNectin4 expression (median log change 52 vs 0; p = 0.029) compared with acquired resistance (n = 21). Paired analyses of NECTIN4 copy number by FISH will be reported. Conclusions: In a paired analysis, most tumors retained substantial mNectin4 expression post-EVP and complete loss was rare, supporting the rationale for Nectin4-targeted strategies in the post-EVP setting. Distinct Nectin4 dynamics suggest greater relevance of target expression changes in primary vs acquired resistance; further studies are needed. All*N=40 Primary resistanceN=14 Acquired resistanceN=21 p mNectin4 TrendDecreaseIncreaseStable 21 (53%)17 (43%)2 (5.0%) 10 (71%)3 (21%)1 (7.1%) 10 (48%)11 (52%)0 (0%) 0.11 Absolute change, median [IQR] -13 [-60, 45] -48 [-100, 0] 10 [-50, 55] 0.2 Log change, median [IQR] -8 [-57, 23] -57 [-195, 0] 3 [-21, 21] 0.035 cNectin4 TrendDecreaseIncreaseStable 12 (30%)14 (35%)14 (35%) 3 (21%)8 (57%)3 (21%) 8 (38%)5 (24%)8 (38%) 0.2 Absolute change, median [IQR] 0 [-28, 25] 13 [0, 100] 0 [-30, 0] 0.055 Log change, median [IQR] 0 [-137, 67] 52 [0, 304] 0 [-256, 0] 0.029 *Including 5 pts not meeting resistance definitions.