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

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. ) NECTIN4 amplification predicts response to EV in mUC and it occurs frequently across solid tumors (app. 25% mUC)


INTRODUCTION
The anti-NECTIN4 antibody-drug conjugate (ADC) enfortumab vedotin (EV) has been approved for previously treated patients with metastatic urothelial cancer (mUC). 1,2The combination of EV plus pembrolizumab (EV/P) was recently approved in metastatic, treatment-na ïve and cisplatin-ineligible patients with mUC.[18][19] The relationship between copy number variation (CNV), mRNA, and protein expression has been known for decades.As a prime example, anti-HER2-targeted therapy conquered modern oncologic therapy of certain breast cancer subtypes and subsequently other entities in an unprecedented success story.The HER2-targeted ADC trastuzumab deruxtecan (T-DXd) proved to be effective in various HER2-expressing solid cancers, also mUC, with a close correlation with expression status. 20,21owever, HER2-directed therapy is guided solely on the basis of biomarker testing that aims to identify HER2-overexpressing/ERBB2-amplified tumors.Unlike in this setting, anti-NECTIN4 EV, whose therapeutic efficacy has been shown to depend on the expression of its target, 7,10 is applied without previous tumor biomarker testing.Similar to HER2, whose expression is strongly linked to CNV of ERBB2, previous reports linked NECTIN4 gene expression to gains/amplifications of 1q23.3-where the NECTIN4 gene is located-occurring in approximately 15%-20% of mUC 22 with an enrichment of NECTIN4 amplifications in luminal molecular subtypes of mUC. 23Despite the frequency of NECTIN4 CNVs in mUC, to date, the link between NECTIN4 CNVs, membranous NECTIN4 protein expression, and especially the clinical potential of NECTIN4 CNVs to predict EV responses has not been assessed.Thus, we here assessed NECTIN4 CNVs and their association with membranous NECTIN4 protein expression in a multicenter cohort of n 5 108 EV-treated patients with mUC and correlated the results with EV responses and outcomes.Furthermore, we confirmed the correlation of NECTIN4 CNVs, mRNA, and protein expression in a The Cancer Genome Atlas (TCGA) pan-cancer analysis and explored the prevalence of NECTIN4 CNVs representing a potential tumoragnostic genomic biomarker to predict EV response in multiple cancer entities.

Multicenter EV-Treated mUC Cohort
We retrospectively reviewed medical records of n 5 108 EVtreated patients with mUC.All patients received EV as the standard of care.Treatment response was evaluated according to RECIST v.1.1 by site investigators. 26rogression-free survival (PFS) was defined as the time from EV initiation to radiologic or clinical progression or death from any cause.Representative formalin-fixed and paraffin-embedded (FFPE) tissue of the primary tumor (PRIM; transurethral resection of the bladder [TURB], cystectomy, or nephroureterectomy) and/or metastatic (MET) tissue was required for inclusion in our explorative biomarker study.When multiple tissue samples were available (matched PRIM 1 MET in n 5 27), we considered the one closest to EV start for our outcome analyses.The study was approved by the ethical review board of the Friedrich-Alexander-University Erlangen-N ürnberg (approval numbers: 329_16B and 97_18Bc) and the Medical Faculty of the University of Bonn (approval number: 372/ 21).Our biomarker study conforms to REMARK guidelines. 27

Non-EV-Treated mUC Cohort
Whole-genome sequencing (WGS) was previously conducted on fresh-frozen metastatic biopsy samples from 116 patients with mUC. 23These patients with mUC were enrolled in clinical trials (ClinicalTrials.govidentifiers: NCT01855477 and NCT02925234) for palliative systemic treatments, with none receiving EV (mUC-non-EV).This patient cohort was already described in detail by Nakauma-Gonz ález et al. 23 NECTIN4 CNVs were assessed using GISTIC 2.0. 28Sufficient clinical information on outcomes was available for n 5 103 patients.

NECTIN4 Fluorescence In Situ Hybridization
The NECTIN4 fluorescence in situ hybridization (FISH) probe was purchased from Empire Genomics (Catalog No. NECTIN4-20-GR, Empire Genomics, Buffalo, NY).The probe is designed to specifically target and bind to the NECTIN4 gene (NCBI Gene ID: 81607).The probe consisted of a fluorescently labeled DNA probe that specifically binds to the NECTIN4 gene.All hybridizations were performed in an accredited specialized laboratory for clinical molecular pathology (accredited according to DIN EN ISO/IEC 17020) using a standard protocol.
The slides were analyzed using a fluorescence microscope equipped with appropriate filter sets to detect the fluorescence signal from NECTIN4 and CEN1 probes.Representative tumor areas for formal analysis were chosen by an experienced board-certified pathologist (ME; blinded to patient outcomes), and at least 50 nonoverlapping nuclei per sample were assessed.Green (NECTIN4) and red (CEN1) signals were manually quantified.The NECTIN4/CEN1 ratio was calculated, and a ratio of ≥2.0 qualified tumors as NECTIN4amplified.Tumors with ratio values <2.0 were considered nonamplified.Furthermore, gene copy changes (≥4 NECTIN4 gene copies per nucleus) without qualifying for an amplification (NECTIN4/CEN1 ratio below <2.0) were considered as polysome tumors, and polysomy status was correlated with response to EV.
NECTIN4 CNV was correlated with NECTIN4 mRNA (log2transformed RSEM-normalized values) and membranous protein expression (H-score).Nonparametric Mann-Whitney test was used to compare two groups.For comparisons involving multiple groups, the nonparametric Kruskal-Wallis test was used.
The predictive value of NECTIN4 amplification for response to EV was assessed by comparing best overall response (BOR), progression-free survival (PFS), and overall survival (OS) between NECTIN4-amplified and nonamplified tumors.
To evaluate the survival after the start of EV treatment, univariable Kaplan-Meier regressions were performed, and significance was determined using the log-rank test.Multivariate Cox regression analyses were conducted to compare the prognostic value of NECTIN4 CNV with baseline patient characteristics (age, sex) and the Bellmunt risk factors (Eastern Cooperative Oncology Group >0, hemoglobin level <10 g/dL, and the presence of liver metastasis) 30 in relation to PFS and OS after EV initiation.
All P values were calculated as two-sided, and a significance level of P < .05 was used to determine statistical significance.

NECTIN4 Amplifications Predict Responses and Favorable Outcomes to EV in mUC
We first established a NECTIN4 FISH assay to examine NECTIN4 CNVs.FISH images and corresponding IHC stainings for a NECTIN4 nonamplified UC lacking membranous NECTIN4 expression and a NECTIN4-amplified UC that demonstrates pronounced membranous NECTIN4 expression, respectively, are illustrated in Figures 1A and 1B CEN1 ratio ≥2.0), consistent with amplification frequencies observed in the non-EV-treated metastatic biopsy mUC cohort (mUC-non-EV, 26%, 27 of 103).Regarding baseline characteristics, 25 of 28 patients with NECTIN4 amplifications were male (P 5 .043)and tended to be older (P 5 .20;Appendix Table A1).In the mUC-non-EV cohort (Clinical-Trials.govidentifiers: NCT01855477 and NCT02925234), 27 of 27 patients with NECTIN4 amplification were male (P 5 .001),and again, there was a nonsignificant trend toward a higher frequency of NECTIN4 amplification in older patients with mUC (P 5 .069;Appendix Table A2).In TCGA-BLCA, there was a significant correlation between NECTIN4 amplification and older age (P 5 .013),and there was a nonsignificant trend toward higher amplification frequency in males (P 5 .15;Appendix Table A3).Next, we evaluated whether NECTIN4 CNVs correlated with membranous NEC-TIN4 protein expression, the prerequisite for EV binding, known to be correlated with EV response. 10 1).In addition, NECTIN4 amplification was associated with prolonged PFS and OS compared with the patient subgroup of nonamplified tumors with strong membranous NECTIN4 expression (H-score ≥200; Appendix Fig A2).Furthermore, we explored whether polysome gene copy changes per nucleus (copy number ≥4.0) without qualifying for an amplification (NECTIN4/CEN1 ratio below <2.0) correlated with EV response and found that five of eight polysome tumors demonstrated an PR/CR or SD with disease control >6 months.
To rule out a prognostic bias of NECTIN4 CNVs, we assessed their prognostic impact in non-EV-treated UC patient cohorts.In the mUC-non-EV cohort, NECTIN4 amplifications were assessed via whole-genome DNA sequencing in 103 metastatic biopsy samples obtained before palliative systemic treatment.In this cohort, NECTIN4 amplifications were found in 26% of tumors and were not associated with OS (Fig 1I).In the TCGA-BLCA cohort of muscle-invasive bladder cancer, NECTIN4 amplifications were also not associated with disease-specific survival and OS (Appendix Fig A3A and A3B).

NECTIN4 Amplification Occurs Frequently Across Entities
In the TCGA Pan-Cancer cohort, NECTIN4 amplifications were observed in 25 of 32 cancer types including various solid entities with NECTIN4 amplification frequency > 5% (Fig 2A).

DISCUSSION
The identification of biomarkers to predict response to targeted therapies is crucial to improve the management of patients with cancer. 32Here, we provide data from a multicenter mUC patient cohort highlighting NECTIN4 amplifications as genomic biomarkers to predict EV responses and favorable outcomes.Importantly, in the non-EV-treated patients with mUC, NECTIN4 amplifications have no impact on OS, 33 suggesting that NECTIN4 amplifications are neither indicating aggressive nor favorable tumor biology, strengthening its potential value as a pure predictive biomarker. 34NECTIN4 amplification was strongly associated with EV sensitivity (BOR, 96%).However, the response rate of 32% in the nonamplified subgroup is comparable with the expected outcomes (BOR app.40%) observed in real-world settings and the pivotal phase III EV-301 study. 1,35,36With a median OS of 12 months (95% CI, 9.7 to NR) in our mUC-EV cohort, our data confirm the clinical activity of EV in previously treated patients with mUC (eg, EV-301, 12.9 months [95% CI, 10.6 to 15.2]).Therefore, EV again proves to be an effective drug in previously treated mUC also in the nonamplified context.
We recently showed that membranous NECTIN4 protein expression is volatile and often (>50%) decreases during metastatic progression of mUC. 10 By contrast, 88% of PRIM with NECTIN4 amplifications retains their NECTIN4 amplification and subsequently a stable high membranous NEC-TIN4 protein expression during metastatic progression.This is in line with previous results from the study by Faltas et al 37 demonstrating that early acquired genomic features including copy number alterations are rather stable during  metastatic progression in comparison with parental primary tumors.Thus, treatment decisions for the metastatic stage could be based on NECTIN4 amplification status in primary tumor material, facilitating implementation into clinical trials.It is worth noting that this consideration does not apply to the assessment of membranous NECTIN4 protein expression, which decreases substantially during metastasis in UC without NECTIN4 amplifications. 10This difference could be explained by the inability of NECTIN4amplified tumors to downregulate membranous expression of NECTIN4 at the transcriptional level.Because downregulation of the target is a known mechanism of resistance to ADCs, 38,39 this could explain, at least in part, the exceptional and durable clinical efficacy of EV in NECTIN4amplified tumors.Beside considerations of tissue choice for predictive biomarker testing, overcoming hurdles to implement biomarker tests into daily care is a major obstacle for biomarker-guided therapies.In the case of CNV assessment, a broad variety of cytogenetic and molecular techniques are available, including FISH/chromogenic in situ hybridization, SNP microarray, comparative genomic hybridization, multiplex ligation-dependent probe amplification, and sequencing methods like whole-exome or whole-genome sequencing. 40Among these options, FISH is the most frequently performed diagnostic assay to assess CNVs in clinical routine. 41Moreover, FISH as predictive biomarker assay has been proven to be a highly reproducible, easy-to-implement, fast, and cost-effective method in daily molecular pathology.Thus, we conclude that a NECTIN4 FISH assay could be quickly integrated into clinical trials and routine molecular pathology/daily patient care.
Other biomarkers were described to be associated with EV response and outcomes: Jindal et al 42 conducted a comprehensive biomarker analysis within the UNITE study cohort, which comprised 303 patients receiving EV monotherapy with available next-generation sequencing data across 16 US sites.Among these patients, 207 had their tumor mutational burden (TMB) assessed and 146 had their PD-L1 status evaluated.Multivariate analysis revealed that alterations in ERBB2, KDM6A, and PIK3CA were associated with favorable treatment outcomes on EV.Conversely, patients with low TMB (<10 Mut/Mb) and high PD-L1 (CPS ≥10) exhibited less favorable outcomes on EV. 42 It is known that alterations in ERBB2 and KDM6A are overrepresented in luminal differentiated UC, 43 which are known to be enriched for NECTIN4 amplification 23 and increased NECTIN4 mRNA and protein expression. 7,44herefore, the prognostic value of these genomic alterations may depend on luminal differentiation and concomitant higher NECTIN4 expression.Consistent with this, the absence of squamous differentiation has been shown to correlate with response to EV. 45 In addition, the occurrence of skin toxicity after initiation of EV treatment has been reported to be associated with favorable outcomes of EV treatment. 46In the context of ADC precision oncology, it is well established from several clinical trials that ADC response correlates with the respective target gene expression, for example, for HER2 14,20,21 and FOLR1-targeting ADC 47 ; we have demonstrated linear correlation also between membranous NECTIN-4 expression and EV response. 10Future biomarker analyses would therefore ideally need to integrate membranous NECTIN4 expression, NECTIN4 CNV, histomorphology, and further high throughput data to deepen our understanding of EVresponsive tumors.Rational biomarker-guided therapy selection is urgently required to establish the optimal therapy sequence for patients with (m)UC. 11,13,32,48Consideration of NECTIN4 amplifications as predictive biomarkers could potentially rationalize EV drug development-also at earlier disease stages-by defining the patient subgroup with the highest chance of durable benefit.In this context, a strategic focus on biomarker-guided trials could greatly enhance our understanding of the potential of EV or other anti-NECTIN4targeted therapies and open new avenues to optimize treatment and improve outcomes in patients with (m) UC. 48,49 A wide range of surface targets, such as HER2 or TROP2, are present in different types of cancers, and there has been a growing interest to expand the use of ADC beyond specific cancer types in a tumor-agnostic fashion. 16,17,50,51Of note, in our TCGA Pan-Cancer analysis, NECTIN4 amplifications can be found in 5%-10% of breast cancer and non-small cell lung cancer, both tumor types with a high impact on allcancer mortality, which are currently being evaluated for EV response in the multicohort phase II EV-202 trial (Clin-icalTrials.govidentifier: NCT04225117). 19Thus, NECTIN4 CNV may be a valuable predictive biomarker to streamline clinical development of NECTIN4-targeted therapies in tumor entities beyond UC. 52The frequent occurrence of NECTIN4 amplifications across solid cancer types could thus pave the way for basket trial designs studying the efficacy of EV on the basis of NECTIN4 CNV status in a tumor-agnostic study framework, 16,17 similar to the phase II DESTINY-PanTumor02 trial which assessed anti-HER2 ADC T-DXd in HER2-expressing solid tumors. 20though our study certainly has important strengths, its main limitation is the use of a retrospectively assembled patient cohort, which consists of both archived primary (TURB, cystectomy or nephroureterectomy) and metastatic tumor specimens with varying ranges between tumor sampling and start of EV treatment.Therefore, our data are hypothesis-generating and prospective confirmation in larger, biomarker-driven trials is mandatory.As the combination of EV/P is the new standard of care in the first-line treatment of mUC, the predictive value of NECTIN4 amplification in this new treatment setting should be further investigated.In addition, our study does not include correlative data on NECTIN4 CNVs and responses to EV in other cancer entities, as mUC is the only approved standard-ofcare setting for EV to date.
In conclusion, our study suggests that NECTIN4 amplification is a simple, valuable, and easy-to-implement predictive biomarker for EV in patients with mUC.The frequent occurrence of NECTIN4 amplifications in other cancer types suggests that this biomarker is a promising candidate with broader applicability for clinical development of NECTIN4-targeted ADCs in a tumor-agnostic context.Log R Ratio Log R Ratio

FIG 1 .
FIG 1. NECTIN4 amplification predicts EV response in mUC.(A and B) NECTIN4 FISH image (green signals 5 NECTIN4; red signals 5 centromere 1, 1,0003 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 5 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 .
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 NECTIN49 copy number variation and mRNA level in both (B) Pan-Cancer Study and (C) TCGA-BLCA.Standard TCGA study abbreviations were used. 31BLCA, Bladder Urothelial Carcinoma; TCGA, The Cancer Genome Atlas.

FIG A1 .
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.

TABLE 1 .
Multivariable Cox Regression Analyses in the Multicenter Enfortumab Vedotin Cohort NOTE.Significant P values are highlighted in bold.Abbreviations: CNV, copy number variation; ECOG, Eastern Cooperative Oncology Group; HR, hazard ratio; OS, overall survival; PFS, progression-free survival.

TABLE A1 .
Baseline Characteristics of the mUC-EV Cohort NOTE.Significant P values are highlighted in bold.Abbreviations: ECOG, Eastern Cooperative Oncology Group; mUC-EV, metastatic urothelial cancer-enfortumab vedotin.

TABLE A2 .
Baseline Characteristics of mUC-Non-EV a Pearson's chi-squared test; Wilcoxon rank-sum test.
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.