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

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.

Here, we discovered TGFBI as a new urinary biomarker for muscle invasive and high-grade urothelial carcinoma (UC). After biomarker identification using antibody arrays, results were verified in urine samples from a study population consisting of 303 patients with UC, and 128 urological and 58 population controls. The analyses of possible modifying factors (age, sex, smoking status, urinary leukocytes and erythrocytes, and history of UC) were calculated by multiple logistic regression. Additionally, we performed knockdown experiments with TGFBI siRNA in bladder cancer cells and investigated the effects on proliferation and migration by wound closure assays and BrdU cell cycle analysis. TGFBI concentrations in urine are generally increased in patients with UC when compared to urological and population controls (1321.0 versus 701.3 and 475.6 pg/mg creatinine, respectively). However, significantly increased TGFBI was predominantly found in muscle invasive (14,411.7 pg/mg creatinine), high-grade (8190.7 pg/mg) and de novo UC (1856.7 pg/mg; all p < 0.0001). Knockdown experiments in vitro led to a significant decline of cell proliferation and migration. In summary, our results suggest a critical role of TGFBI in UC tumorigenesis and particularly in high-risk UC patients with poor prognosis and an elevated risk of progression on the molecular level.

Limited data is available on the role, and extent of, postchemotherapy lymphadenectomy (PC-LND) in patients with clinical evidence of pelvic (cN1-3) or retroperitoneal (RP) lymph node spread from urothelial bladder carcinoma. To compare the outcomes of operated versus nonoperated patients after first-line chemotherapy. Data from 34 centers was collected, totaling 522 patients, treated between January 2000 and June 2015. Criteria for patient selection were the following: bladder primary tumor, lymph node metastases (pelvic±RP) only, first-line platinum-based chemotherapy given. LND (with cystectomy) versus observation after first-line chemotherapy for metastatic urothelial bladder carcinoma. Overall survival (OS) was the primary endpoint. Multiple propensity score techniques were adopted, including 1:1 propensity score matching and inverse probability of treatment weighting. Additionally, the inverse probability of treatment weighting analysis was performed with the inclusion of the covariates, that is, with doubly robust estimation. Overall, 242 (46.4%) patients received PC-LND and 280 (53.6%) observation after chemotherapy. There were 177 (33.9%) and 345 (66.1%) patients with either RP or pelvic LND only, respectively. Doubly robust estimation-adjusted comparison was not significant for improved OS for PC-LND (hazard ratio [HR]: 0.86, 95% confidence interval [CI]: 0.56-1.31, p=0.479), confirmed by matched analysis (HR: 0.91, 95% CI: 0.60-1.36, p=0.628). This was also observed in the RP subgroup (HR: 1.12, 95% CI: 0.68-1.84). The retrospective nature of the data and the heterogeneous patient population were the major limitations. Although there were substantial differences between the two groups, after accounting for major confounders we report a nonsignificant OS difference with PC-LND compared with observation only. These findings may be hypothesis-generating for future prospective trials. We found no differences in survival by adding postchemotherapy lymphadenectomy in patients with pelvic or retroperitoneal lymph node metastatic bladder cancer. The indication to perform postchemotherapy lymphadenectomy in the most suitable patients requires additional studies.