Membranous NECTIN-4 Expression Frequently Decreases during Metastatic Spread of Urothelial Carcinoma and Is Associated with Enfortumab Vedotin Resistance

Abstract

Purpose: The antibody-drug conjugate enfortumab vedotin (EV) releases a cytotoxic agent into tumor cells via binding to the membrane receptor NECTIN-4. EV was recently approved for patients with metastatic urothelial carcinoma (mUC) without prior assessment of the tumor receptor status as ubiquitous NECTIN-4 expression is assumed. Our objective was to determine the prevalence of membranous NECTIN-4 protein expression in primary tumors (PRIM) and patient-matched distant metastases (MET).

Experimental design: Membranous NECTIN-4 protein expression was measured (H-score) by IHC in PRIM and corresponding MET (N = 137) and in a multicenter EV-treated cohort (N = 47). Progression-free survival (PFS) after initiation of EV treatment was assessed for the NECTIN-4-negative/weak (H-score 0-99) versus moderate/strong (H-score 100-300) subgroup. The specificity of the NECTIN-4 IHC staining protocol was validated by establishing CRISPR-Cas9-induced polyclonal NECTIN-4 knockouts.

Results: In our cohort, membranous NECTIN-4 expression significantly decreased during metastatic spread (Wilcoxon matched pairs P < 0.001; median H-score = 40; interquartile range, 0-140), with 39.4% of MET lacking membranous NECTIN-4 expression. In our multicenter EV cohort, absence or weak membranous NECTIN-4 expression (34.0% of the cohort) was associated with a significantly shortened PFS on EV (log-rank P < 0.001).

Conclusions: Membranous NECTIN-4 expression is frequently decreased or absent in mUC tissue. Of note, the clinical benefit of EV strongly depends on membranous NECTIN-4 expression. Thus, our results are of highest clinical relevance and argue for a critical reconsideration of the current practice and suggest that the NECTIN-4 receptor status should be determined (ideally in a metastatic/progressive lesion) before initiation of EV. See related commentary by Aggen et al., p. 1377.

Figure 1.

Membranous NECTIN-4 protein expression patterns assessed by IHC. A and B, Membranous NECTIN-4 expression decreases significantly during metastatic spread of urothelial carcinoma, with 39.4% of MET lacking NECTIN-4 expression. P values were calculated by a nonparametric Mann–Whitney test. C–E, Representative IHC stainings for NECTIN-4. Each panel resembles pairs of matched PRIM and distant MET. C, Concordantly negative PRIM and MET (membranous H-score 0). D, Strongly and homogenously positive PRIM (membranous H-score 300) with strong reduction of membranous expression in corresponding MET (membranous H-score 20; positive tumor cells marked with arrow). E, Strongly but inhomogenously positive PRIM (membranous H-score 270) with negative corresponding MET (membranous H-score 0). All pictures were taken on an Axio Imager A1 microscope (Carl Zeiss AG, Jena, Germany) under 200× magnification using a Gryphax Arktur camera (Jenoptik, Jena, Germany). F, The differential expression of membranous NECTIN-4 is not dependent on the time interval between PRIM and MET biopsy. Intergroup comparison was calculated by nonparametric Kruskal–Wallis test.

Figure 2.

Comparison of NECTIN-4 expression with previously published UC cohorts and validation of NECTIN-4 IHC staining protocol. A, Comparison of expression data obtained from our cohort with the UC cohort reported by Challita-Eid and colleagues including a total of 524 patients (including 25 metastases). B, NECTIN-4 expression in our PRIM and MET subcohort is significantly lower than in the phase I EV-101 trial (Cutoff NECTIN-4 H-score 150), where only 5/152 cases had a NECTIN-4 expression < 150. C, Western blot comparing NECTIN-4 protein expression in polyclonal HT1376 NECTIN-4 KO cultures (KO-1 and KO-2) compared with control (CTRL). Detection of β-actin served as loading control. D, Normalized histogram illustrating membranous NECTIN-4 expression detected by flow cytometry in HT1376 CTRL, KO-1, and KO-2 cultures (plus unstained control as reference). E, Pie charts summarizing out-of-frame (KO), in-frame indel, and no-indel (WT allele) frequencies of the polyclonal HT1376 KO-1 and KO-2 determined by targeted amplicon NGS. F, Membranous NECTIN-4 protein expression patterns of our HT1376 cell culture models (CTRL vs. KO-1 vs. KO-2) assessed by IHC. The red arrow depicts single isolated NECTIN-4–positive clones of HT1376 KO-1 and a strong NECTIN-4–positive cell cluster of HT1376 KO-2.

Figure 3.

Membranous NECTIN-4 predicts response and outcomes for patients with mUC treated with EV. A, Membranous NECTIN-4 expression predicts EV response (mixed, mixed response; SD, stable disease; PR, partial response; CR, complete response); radiologic response data were available for N = 41. P values were calculated by a nonparametric Mann–Whitney test. B, Absence or weak membranous NECTIN-4 expression (H-score 0–99) is associated with shortened PFS on EV.