NECTIN4 Amplification Is Frequent in Solid Tumors and Predicts Enfortumab Vedotin Response in Metastatic Urothelial Cancer

Abstract

Purpose: The anti-NECTIN4 antibody-drug conjugate enfortumab vedotin (EV) is approved for patients with metastatic urothelial cancer (mUC). However, durable benefit is only achieved in a small, yet uncharacterized patient subset. NECTIN4 is located on chromosome 1q23.3, and 1q23.3 gains represent frequent copy number variations (CNVs) in urothelial cancer. Here, we aimed to evaluate NECTIN4 amplifications as a genomic biomarker to predict EV response in patients with mUC.

Materials and methods: We established a NECTIN4-specific fluorescence in situ hybridization (FISH) assay to assess the predictive value of NECTIN4 CNVs in a multicenter EV-treated mUC patient cohort (mUC-EV, n = 108). CNVs were correlated with membranous NECTIN4 protein expression, EV treatment responses, and outcomes. We also assessed the prognostic value of NECTIN4 CNVs measured in metastatic biopsies of non-EV-treated mUC (mUC-non-EV, n = 103). Furthermore, we queried The Cancer Genome Atlas (TCGA) data sets (10,712 patients across 32 cancer types) for NECTIN4 CNVs.

Results: NECTIN4 amplifications are frequent genomic events in muscle-invasive bladder cancer (TCGA bladder cancer data set: approximately 17%) and mUC (approximately 26% in our mUC cohorts). In mUC-EV, NECTIN4 amplification represents a stable genomic alteration during metastatic progression and associates with enhanced membranous NECTIN4 protein expression. Ninety-six percent (27 of 28) of patients with NECTIN4 amplifications demonstrated objective responses to EV compared with 32% (24 of 74) in the nonamplified subgroup (P < .001). In multivariable Cox analysis adjusted for age, sex, and Bellmunt risk factors, NECTIN4 amplifications led to a 92% risk reduction for death (hazard ratio, 0.08 [95% CI, 0.02 to 0.34]; P < .001). In the mUC-non-EV, NECTIN4 amplifications were not associated with outcomes. TCGA Pan-Cancer analysis demonstrated that NECTIN4 amplifications occur frequently in other cancers, for example, in 5%-10% of breast and lung cancers.

Conclusion: NECTIN4 amplifications are genomic predictors of EV responses and long-term survival in patients with mUC.

FIG 1.

NECTIN4 amplification predicts EV response in mUC. (A and B) NECTIN4 FISH image (green signals = NECTIN4; red signals = centromere 1, 1,000× oil immersion) and (A) corresponding immunohistochemical NECTIN4 staining on NECTIN4 nonamplified and (B) NECTIN4-amplified urothelial cancers. The gray dashed box demonstrates the two patient cases. (C) Membranous NECTIN4 expression is significantly associated with FISH-detected NECTIN4 amplification in our EV-treated UC cohort (mUC-EV). Statistical significance (***P < .001) was determined using the Mann-Whitney U test. (D) Sankey plot of NECTIN4 amplification status in the 27 matched PRIM and MET samples. (E) Evolution of membranous NECTIN4 expression during metastatic spread in the eight NECTIN4-amplified PRIMs. (F) BOR on the mUC-EV cohort on the basis of NECTIN4 copy number status; BOR was available for n = 65 patients. NECTIN4 amplification status is associated with both prolonged (G) PFS and (H) OS since EV therapy start compared with nonamplified tumors. (I) NECTIN4 amplification is not associated with OS in non–EV-treated mUC. The log-rank P value is shown. The dashed lines demonstrate median PFS and OS when reached. BOR, best overall response; EV, enfortumab vedotin; FISH, fluorescence in situ hybridization; OS, overall survival; MET, metastatic; PFS, progression-free survival.

FIG 2.

NECTIN4 amplifications occur frequently across solid tumors. (A) The frequency of NECTIN4 amplifications are depicted for 32 studies consisting of 10,712 samples/patients, with BLCA presenting the highest prevalence (17%). Positive correlation was observed between NECTIN4′ copy number variation and mRNA level in both (B) Pan-Cancer Study and (C) TCGA-BLCA. Standard TCGA study abbreviations were used. BLCA, Bladder Urothelial Carcinoma; TCGA, The Cancer Genome Atlas.

FIG A1.

Illustration of copy number variation profiles derived by Illumina SNP arrays. Upper panel: NECTIN4 nonamplified tumor profile; lower panel: NECTIN4 amplified tumor profile. The NECTIN4 gene location on Chr. 1 shows higher copy numbers in the amplified tumors. Chr.1, chromosome 1; SNP, single nucleotide polymorphisms.

FIG A2.

PFS (A) and OS (B) upon initiation of EV treatment stratified by presence of NECTIN4 gene amplification versus high membranous NECTIN4 protein expression without NECTIN4 gene amplification. EV, enfortumab vedotin; OS, overall survival; PFS, progression-free survival.

FIG A3.

(A) Disease-specific survival of n = 393 patients of the TCGA-BLCA cohort stratified by copy number alterations of NECTIN4 (data were missing for 14 patients). (B) Overall survival of n = 407 patients of the TCGA-BLCA cohort stratified by copy number alterations of NECTIN4. TCGA-BLCA, The Cancer Genome Atlas-bladder cancer.

FIG A4.

(A) NECTIN4 mRNA expression (log2 normalized RSEM values) in TCGA-BRCA cohort stratified by NECTIN4 copy number alterations. (B) NECTIN4 protein expression levels (Z-score scaled results from RPPA) in TCGA-BRCA cohort stratified by NECTIN4 copy number alterations. (C) NECTIN4 mRNA expression (log2 normalized RSEM values) in TCGA-LUAD cohort stratified by NECTIN4 copy number alterations. NS, not significant; RPPA, reverse-phase protein arrays; TCGA-BRCA, The Cancer Genome Atlas breast cancer; TCGA-LUAD; The Cancer Genome Atlas lung adenocarcinoma.