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

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.

Reliable prediction of treatment responses is crucial for optimal patient care during treatment. However, in daily clinical practice, we almost exclusively rely on imaging-based staging with estimation of the tumor volume. Particularly in the era of immuno-oncology, imaging-only staging has several limitations, especially in the patient subset with stable disease (SD) while taking immune checkpoint inhibitors (ICIs), which encompasses a wide range of clinical outcomes across entities.¹ In this context, there is an unmet need for complementary markers that enable real-time assessment of therapy response and outcomes in conjunction with imaging.

Robust biomarkers to predict response to immune checkpoint blockade (ICB) in metastatic urothelial carcinoma (mUC) are still in demand. Recently, early C-reactive protein (CRP) kinetics and especially the novel CRP flare-response phenomenon has been associated with immunotherapy response. We conducted a multicentre observational study comprising 154 patients with mUC treated with ICB to evaluate the predictive value of a previously described on-treatment CRP kinetics: CRP flare responders (at least doubling of baseline CRP within the first month after initiation of ICB followed by a decline below baseline within three months), CRP responders (decline in baseline CRP by ≥ 30% within three months without a prior flare) and the remaining patients as CRP non-responders. CRP kinetics groups were correlated with baseline parameters, PD-L1 status, progression-free survival (PFS) and overall survival (OS). Objective response was observed in 57.1% of CRP responders, 45.8% of CRP flare responders and 17.9% of CRP non-responders (P < 0.001). CRP flare response was associated with prolonged PFS and OS (P < 0.001). In multivariable Cox regression analysis, CRP flare responders showed a risk reduction of ∼70% for tumour progression and death compared to CRP non-responders. Subgroup analysis of CRP flare responders revealed that patients with a long-flare response (completed flare-response kinetics ≥6 weeks on-treatment) showed even more favourable outcomes following ICB (HR = 0.18, 95%-CI: 0.07-0.48, P < 0.001). CRP (flare)response robustly predicts immunotherapy response and outcomes in mUC independent of PD-L1 status. Thus, early on-treatment CRP kinetics is a promising low-cost and easy-to-implement biomarker to optimise therapy monitoring in patients with mUC treated with ICB.

The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking.

Background: In metastatic clear cell renal cell carcinoma (ccRCC), different combination therapies, each including anti-PD-1 immune checkpoint blockade (ICB), are applied as first-line treatment. Robust predictive biomarkers for rational upfront therapy decisions are lacking, although they are urgently needed. Recently, we showed that CTLA4 promoter methylation predicts response to ICB in melanoma. Here, we aimed to investigate CTLA4 methylation in ccRCC and its utility to serve as a predictive biomarker for anti-PD-1 based ICB in metastatic ccRCC. Methods: CTLA4 methylation was analyzed with regard to transcriptional gene activity (mRNA expression), intratumoral immune cell composition, and clinical course in two ccRCC cohorts obtained from The Cancer Genome Atlas (TCGA cohort, n=533) and the University Hospital Bonn (UHB Non-ICB Cohort, n=116). In addition, CTLA4 methylation as well as CD8⁺ T cell infiltrates and PD-L1 expression were evaluated in pre-treatment samples from a multicenter cohort (RCC-ICB Cohort, n=71). Patients included in the RCC-ICB Cohort were treated with either first line anti-PD-1 based combination therapy (n=25) or monotherapy post–tyrosine kinase inhibition in second line or later. Analyses were performed with regard to treatment response according to RECIST, progression-free survival (PFS), event-free survival (EFS), and overall survival (OS) following treatment initiation. Results: CTLA4 promoter hypomethylation was significantly correlated with CTLA4 mRNA expression, lymphocyte infiltration, and poor OS in both primary ccRCC cohorts (TCGA: HR 0.30 (95% CI 0.18 to 0.49), p<0.001; UHB Non-ICB: HR 0.35 (95% CI 0.16 to 0.75), p=0.007). In contrast, CTLA4 promoter hypomethylation predicted response and, accordingly, favorable outcomes (PFS and OS) in patients with ICB-treated ccRCC, overcompensating the negative prognostic value of CTLA4 hypomethylation at initial diagnosis. Moreover, in multivariable Cox regression, CTLA4 promoter hypomethylation remained an independent predictor of improved outcome in ICB-treated ccRCC after co-adjustment of the International Metastatic Renal Cell Carcinoma Database Consortium score (HR 3.00 (95% CI 1.47 to 6.28), p=0.003). Conclusions: Our study suggests CTLA4 methylation as a powerful predictive biomarker for immunotherapy response in metastatic RCC.