NECTIN4 Heterogeneity and Molecular Diversity in Bladder Cancers: Deconstructing the Activity of An Antibody–Drug Conjugate

In this issue of Clinical Cancer Research, Chu and colleagues demonstrated heterogeneity in NECTIN4 mRNA expression across different molecular subtypes of urothelial cancers of the bladder (1). NECTIN4, also known as poliovirus receptor-related protein 4 (PRR4), encodes for Nectin-4, a protein which contains two immunoglobulin-like (Ig-like) C2-type domains and one Ig-like V-type domain. It is a single-pass type I membrane protein which is involved in cell adhesion through transhomophilic and transheterophilic interactions, which was first discovered via bioinformatics analysis. Nectin-4 is overexpressed in various tumors, with highest frequencies for overall expression observed in bladder, breast, and pancreatic tumors by IHC (2), and is a target of a novel antibody–drug conjugate, enfortumab vedotin (EV; ref. 3). With nectin-4 as a cell surface target, a high concentration of monomethyl auristatin (MMAE)—a highly toxic antimitotic chemotherapeutic agent when administered systemically—could be safely delivered and internalized selectively to the cancer cells, leading to subsequent cell kill. Indeed, the strategy has been effective: EV improved overall survival in a phase III trial for metastatic urothelial cancer (EV301) compared with conventional chemotherapy. Nearly all patients enrolled in various EV trials expressed nectin-4 per IHC on their tumors, with a moderate to strong H-score observed in 2 of 3 of cases. Nevertheless, rate of clinically observed responses was 41%–52%, lower than would be expected on the basis of the IHC expression.

In this important work, Chu and colleagues strove to solve a piece of the puzzle underlying this discrepancy in NECTIN4 gene expression and observed clinical benefits. Using seven clinical datasets with comprehensive transcriptomic data, majority of which were from pretreatment transurethral resection or radical cystectomy specimens, as well as a post-cisplatin-based neoadjuvant chemotherapy cohort, the authors demonstrated differential levels of NECTIN4 mRNA expression across different molecular subtypes (Fig. 1). Six molecular subtypes have been described in urothelial cancers based on transcriptomic expression: luminal (papillary, non-specified, and unstable), basal/squamous, stroma rich, and neuroendocrine like. Each are individually characterized by different oncogenic mechanisms, extent of immune and stromal cell infiltration, associated histomorphology, and clinical characteristics including natural history and response to systemic therapies (4). In this analysis of over 1,500 tumors, luminal subtype tumors were associated with higher NECTIN4 gene expression compared with basal/squamous and stromal-rich subtypes, while the neuroendocrine-like tumors have the lowest level of nectin4 gene expression. These differential expression levels were witnessed irrespective of prior cisplatin exposure. This observation was replicated immunohistochemically in 80 tumors in a tissue microarray.

The role of nectin-4 protein in urothelial carcinogenesis and disease progression remains undefined. Of the seven datasets which served as the basis of the authors' analysis, survival data were available for some of these datasets. NECTIN4 mRNA transcript levels were not associated with survival among these cases. Furthermore, nectin-4 did not appear to be essential for cell survival based on RNAi knockdown and CRISPR knockouts across hundreds of cell lines. More importantly, this study represented one of the first to show the crucial role of nectin-4 in EV-related cytotoxicity. The investigators showed that sensitivity to EV correlated strongly with NECTIN4 mRNA and protein levels, using luminal cell lines with high endogenous nectin-4 (HT-1376 and HT-1197) and basal cell lines with minimal endogenous NECTIN4 expression (UMUC-3 and TCCSUP). Furthermore, knockdown of NECTIN4 in luminal cell lines led to abrogation of nectin-4 protein expression and resistance to EV. Importantly, upregulating NECTIN4 expression resulted in markedly increased sensitivity to EV as evidenced by proliferation and clonogenic assays. These findings strongly suggest the importance of nectin-4 in EV-induced cell death.

Across different cell lines, NECTIN4 gene expression generally correlated strongly with protein levels. However, in clinical specimens, this relationship may prove to be more complicated. In the phase II EV201, all evaluable patients had detectable nectin-4 on archival or fresh tumor samples by IHC as determined by H-score, with a median H-score of 290 (range, 14–300). In a recent IHC survey of nectin-4 expression across a wide array of variant morphology of urothelial cancers and non-urothelial histologies, over 80% of urothelial carcinoma demonstrated moderate to strong nectin4 expression and six of 10 squamous cell differentiation was associated with moderate to strong nectin4 staining (5). Nectin-4 staining by IHC was minimal among small cell cancers of the bladder, consistent with Chu and colleagues' observation that NECTIN4 gene expression was the lowest among the neuroendocrine-like subtype. What remains unknown is the minimal amount of transcript needed for significant protein expression, and what, if any, post-translational mechanisms may modulate this expression. This poses a space for further exploration.

The high prevalence of nectin-4 IHC expression in tumor biopsies renders dichotomization and correlation with antitumor effect impossible, and it is unclear whether the difference in its mRNA level may demonstrate any meaningful association with clinical activity of EV. While the authors have demonstrated IHC heterogeneity per molecular subtypes in a smaller tissue microarray series, the correlation remains to be evaluated at an individual case level. As these gene expression studies were performed on primary tumors, it is also unclear whether metastatic tumors and intrapatient heterogeneity could explain clinical results.

Despite the clinical activity seen with EV in advanced treatment-refractory urothelial cancers, most patients do not experience durable responses. The biological and mechanistic gap between high rate of nectin-4 overexpression in most urothelial cancers and the lower observed response rate of 30%–50% still requires explanation. Chu and colleagues have taken the first step to investigate heterogeneity in NECTIN4 transcriptomic levels as potential mechanism for primary and acquired resistance. Other resistance mechanisms are also likely to exist. As a target of EV, nectin-4 may be downregulated or receptor internalization may be decreased; resistance may also involve upregulation of drug efflux pumps or inherent resistance to MMAE. Furthermore, as an antibody-bound therapy, the interaction of its molecular size and the physical or mechanical environment of the tumor may also interfere with effective drug delivery.

Finally, the different levels of NECTIN4 gene expression across different molecular subtypes further reaffirm the biological diversity among bladder and urinary tract cancers. More than mere scientific curiosity, different survival outcomes have been observed across molecular subgroups. Emerging data, including retrospective analyses of large clinical trials, have shown differential response to different therapies, such as platinum-based chemotherapy and immune checkpoint inhibitors, leading to proposals of molecularly guided therapeutic paradigms. Chu and colleagues' findings may lay the groundwork to further understand the interactions between the molecular underpinnings of these tumors and our systemic therapeutic armamentarium.

Chu and colleagues have embarked on a laudable effort as the first step in elucidating the heterogenous clinical response to EV. However, much work remains to be done to improve patients' response to EV in the clinic. In addition to serving as a launching pad for various future lines of investigation, this work further highlights the heterogeneity of cancers and the need for prospective molecular subtyping as potential biomarker in clinical trials.

M.Y. Teo reports other support from Seattle Genetics, Astellas, Clovis Oncology, and Pharmacyclics and grants from Bristol Myers Squibb outside the submitted work. J.E. Rosenberg reports personal fees and other support from Seattle Genetics, Astellas, Roche/Genentech, Bayer, QED Therapeutics, and AstraZeneca, and personal fees from Gilead/Immunomedics, BMS, GSK, Merck, Mirati, Pharmacyclics, Janssen, Tyra Biosciences, Bioclin, Boehringer Ingelheim, and Eli Lilly outside the submitted work.