Bladder cancer (BC) is the ninth most common cancer worldwide, while being 4 times more common in men (1, 2). Around 75% of patients initially present with non-muscle invasive bladder cancer (NMIBC), which is treated with transurethral resection and adjuvant local intravesical therapy in higher risk stages. However, there is a risk that NMIBC can progress to a muscle-invasive stage later on (1). About 25% of patients present initially as muscle-invasive bladder cancer (MIBC) with significantly poorer prognosis compared to patients with NMIBC (3). For MIBC without any evidence of distant disease, curative radical cystectomy (RC), with neoadjuvant immunotherapy and platinum-based chemotherapy was long considered the gold standard (3, 4). Overall, 25-30% of patients experience disease recurrence after surgery, mostly within the first 2 years. This rate increases to above 50% in higher tumor stages (5-7). The search for additional markers to identify patients who have a higher risk of disease progression and therefore impaired survival, has been a steady focus of research for many years (8-11). So far, none have been established in clinical practice (12, 13).

A molecule that has been in the focus of oncological research, particularly in BC, is Nectin4 (14-17). Nectin4 is a cell surface adhesion molecule, related to the formation of junctions and adherence junctions (18, 19). It is the target molecule of the novel antibody-drug conjugate Enfortumab-Vedotin, which is now the first line treatment for metastatic BC in combination with Pembrolizumab (3, 20). A few recent studies have investigated a potential link between Nectin4 and its influence on disease progression in MIBC (21, 22). Due to the scarcity of data, no definitive conclusions were drawn so far, further investigations regarding this subject are therefore needed.

Another associated, less studied factor in BC is interferon regulatory factor 6 (IRF6). IRF6 is a transcription factor, which plays an essential role in keratinocyte proliferation, differentiation and migration (23-26) and regulates Nectin4 expression in mice (27). Since its discovery, several other functions have been uncovered including the regulation of inflammatory cytokines and protection against the development of liver steatosis (28-30). IRF6 has been evaluated in oncological research as a prognostic marker for several solid cancers (31-34). Regarding BC, no data exists on the prognostic value of IRF6 in MIBC before RC. In this study, we aimed to evaluate the potential of Nectin4 and IRF6 expression to predict oncological survival outcomes in patients with MIBC undergoing RC.

Bladder cancer tissue microarray and immunohistochemistry. Two tissue microarrays (TMAs) containing 326 tissue samples of formalin fixed cystectomy specimens from 163 BC patients who underwent RC, were analyzed. Per patient, two tissue cores were taken. After inspection of the whole cystectomy specimen, pathologists of the Institute of Pathology and Molecular Pathology of the University Hospital Zürich, Switzerland selected the core puncture site by choosing two central locations with histologically confirmed vital tumor. The samples were collected between 1990 and 2014. The TMAs were stored at room temperature at the Institute of Pathology and Molecular Pathology of the University Hospital Zürich, Switzerland. Both cohorts were approved by the Ethics Committee of the Canton of Zürich (StV-Nr. 25/2008 and 02/2009).

Immunohistochemical staining was performed on 2 µm - sections on the Leica BOND RX automated staining system (Biosystems Switzerland, Muttenz, Switzerland): Nectin4 (monoclonal EPR15613-68, 1:1’000, abcam, Cambridge, UK), IRF6 (monoclonal OTI2C10, 1:50, Novus Biologicals Inc., Abingdon, UK). The Nectin4 immunohistochemical staining was analyzed for membranous staining as previously described in literature and based on described protein location (15, 35, 36). IRF6 was analyzed for nuclear and cytoplasmic staining separately, as both locations of the protein have been described (36, 37). Staining intensity was rated in a semi-quantitative manner: negative (-), weak positive (+), medium positive (++) or strong positive (+++). Additionally, for each core it was documented what percentage of tumor cells expressed the examined staining intensity.

To quantify the staining intensity the H-score was used, a reliable, widely used and established method in histological oncological research (38, 39). The reported staining intensity (0-3) was multiplied by the percentage of positive cells in the tissue core (0-100%). The H-score average of the two tissue cores per patient was then calculated and used for further statistical analysis. Only patients, for whom 2 cores were available were used for further analysis.

Data acquisition. Patient data were collected by accessing the clinic's patient information system of the University Hospital of Zürich, the follow-up was conducted until December 2019. Follow-up care consisted of regular imaging examinations, every 6 months for 2 years after surgery and then yearly for a total of 5 years. Afterwards, a personalized approach depending on the patient’s age and general medical condition was chosen.

Progression-free survival (PFS) was defined as time in months from RC until detection of local or distant disease recurrence. Cancer Specific Survival (CSS) was defined as time in months from RC until death caused by BC.

Statistical analysis. To evaluate the prognostic performance of each marker, the H-score of Nectin4, IRF6c and IRF6n were analyzed as continuous variables to preserve quantitative information, as binary variables (positive vs. negative staining) to reflect potential clinical applicability, and as ranked variables [negative, weak (H-score=1-150), and strong staining (H-score=151-300)] to assess potential dose-response relationships.

Before association analysis, we tested for normal distribution of continuous variables using the Shapiro-Wilk test. Because H-scores were not normally distributed, non-parametric methods were used where appropriate. Binary pathological outcomes (lymph node status) were analyzed using binary logistic regression, whereas ordinal outcomes (T stage and AJCC stage) were evaluated using ordinal logistic regression. In both cases, significance was determined via the Wald Z-test. Potential associations between H-score and age were evaluated using Spearman’s rank correlation, while differences in H-score between female and male patients were assessed using the Mann-Whitney U-test.

We performed univariable Cox regression analysis to identify individual variables associated with PFS and CSS. Patients without disease progression or cancer-specific death at the end of follow-up were censored at the date of the last available follow-up. Multivariable Cox regression models were constructed to determine whether Nectin4 or IRF6 provided prognostic information beyond established clinicopathological predictors. AJCC stage was selected as the adjustment variable because it represents a validated and widely used composite prognostic classification incorporating pathological tumor and nodal stage (40-42).

Finally, Kaplan-Meier analysis was used to plot PFS curves for patients stratified into Nectin4 positive and Nectin4 negative cohorts. The log-rank test was then used to compare survival curves.

Only results with a p<0.05 were considered statistically significant. Data were analyzed with the statistic program R (R-Core-Team, Vienna, Austria, Version 4.4.1).

Immunohistochemistry. Figure 1 shows representative examples of scanned TMA sections showcasing the different staining intensities for Nectin4, IRF6c and IRF6n. The staining protocol resulted in overall good staining quality, which allowed accurate evaluation of the scanned TMA sections. For 151 out of the 163 patients within the TMA-set two tissue cores were available per patient and used for further statistical analysis. Nectin4 showed strong membranous staining. IRF6 staining was successful both in the cytoplasm and the nucleus.

Patient characteristics. One hundred and seventeen (77.5%) patients were male and the median age at cystectomy was 69 years [interquartile range (IQR)=60.9-75.0]. Almost all patients presented with high-grade disease (96.0%). Furthermore, 26 patients (17.2%) were diagnosed with an AJCC ≤I tumor, 33 (21.9%) with AJCC II, 57 (37.7%) with AJCC IIIA and 35 (23.3%) with AJCC IIIB. Final histopathology confirmed positive lymph nodes in 52 patients (34.4%).

Median follow-up time was 30.2 months (IQR=9.6-59.0). During the follow-up period, 74 patients (49%) experienced disease progression and 58 (38.4%) died of the BC. 40 (26.5%) patient samples stained positively for membranous Nectin4, 100 (66.2%) for IRF6c and 93 (61.6%) for IRF6n. Median H-score for Nectin4 was 0 (IQR=0-1.1), for IRF6c 60 (IQR=0-124.4) and for IRF6n 5 (IQR=0-45) (Table I).

Association analysis. Continuous variables were not normally distributed. Higher Nectin4 expression was significantly associated with lower pT-stages when analyzed as a continuous [odds ratio (OR)=0.99, 95% confidence interval (95%-CI)=0.98-0.99, p=0.017], binary (OR=0.19, 95%-CI=0.09-0.38, p<0.001) and ranked variable (OR=0.21, 95%-CI=0.11-0.41, p<0.001). A similar pattern was observed for AJCC-stages, with higher Nectin4 expression associated with lower stages when analyzed as a binary (OR=0.37, 95%-CI=0.18-0.74, p=0.006) and ranked variable (OR=0.38, 95%-CI=0.20-0.72, p=0.003). Likewise, higher IRF6c expression was inversely correlated with lower T-stages as a continuous (OR=0.99, 95%-CI=0.99-0.99, p=0.002) and ranked variable (OR=0.52, 95%-CI=0.36-0.96, p=0.001). IRF6c expression was also linked to lower AJCC stages in both continuous (OR=0.99, 95% CI=0.99-1.00, p=0.002) and ranked variable analyses (OR=0.55, 95% CI=0.36-0.83, p=0.005) (Table II).

IRF6c and Nectin4 expressions were not associated with patient age or sex (Table III). IRF6n expression did not correlate with any of the examined parameters (data not shown).

Uni- and multivariable Cox regression analysis. Uni- and multivariable Cox regression analyses, along with Kaplan-Meier and concordance index evaluations, were conducted to assess the prognostic significance of Nectin4 and IRF6 on PFS and CSS.

Univariable analysis revealed a significant association of Nectin4 expression and reduced risk of disease progression, both when analyzed as a binary [hazard ratio (HR)=0.50, 95%-CI=0.28-0.90, p=0.020] and ranked (HR=0.50, 95%-CI=0.28-0.88, p=0.018) variable. Similarly, IRF6c as a continuous (HR=1.00, 95%-CI=0.99-1.00, p=0.039) and ranked (HR=0.71, 95%-CI=0.52-0.96, p=0.028) variable was predictive for a longer PFS. Further, invasion of blood or lymph vessels, positive nodal status, higher pT-stages and AJCC-stages were significantly associated with a shorter PFS (all p<0.001) (Table IV).

In Kaplan-Meier analysis, patients with Nectin4 negative tumors had shorter PFS compared to Nectin4 positive tumors (p=0.018) (Figure 2).

Of the examined markers, only IRF6c was a marginally significant predictor for CSS when analyzed as a continuous variable (HR=0.99, 95%-CI=0.99-0.99, p=0.045). Again, invasion of blood and lymphatic vessels, positive nodal status, T-stages and AJCC-stages were significantly associated with a shorter CSS (all p<0.001) (Table V).

When correcting for AJCC-stages in a multivariable analysis, inclusion of Nectin4 and IRF6c to the model did not significantly improve prediction of PFS or CSS (Table VI and Table VII). Addition of these two markers also did not lead to a considerable increase of the concordance index of the AJCC model (Table VIII and Table IX).

IRF6n did not demonstrate predictive value for either PFS or CSS in univariable or multivariable (data not shown).

We showed that higher IRF6 and Nectin4 expressions are associated with a decreased risk of disease progression and a less aggressive tumor biology in patients undergoing RC for MIBC. However, both markers could not be confirmed as independent predictors when correcting for the AJCC tumor classification.

Several studies investigating the impact of Nectin4 on survival outcomes in BC have been published in recent years. Bahlinger et al. analyzed Nectin4 on a protein and RNA level in two cohorts of patients undergoing RC for MIBC and found Nectin4 expression not to be of value to predict CSS (22). Rodler et al. focused on a cohort of 110 RC patients with variant BC histology, namely squamous cell carcinoma, adenocarcinoma and sarcomatoid carcinoma. Again, Nectin4 expression was not predictive of either OS or PFS (21). Kobayashi et al. recently published a study analyzing TMAs of 93 patients undergoing RC and found higher Nectin4 expression to be marginally associated with CSS (p=0.046) (43). In metastatic BC, higher Nectin4 expression showed a non-significant trend towards better survival outcomes under platinum-based chemotherapy (44). Recently, Nectin4 expression was linked to longer PFS and CSS in upper tract urothelial carcinoma (UTUC) in univariable analysis (43). In other solid malignancies, the opposite effect has repeatedly been described. Nectin4 expression was linked to reduced overall survival in colorectal, pancreatic, breast and esophageal cancer patients undergoing surgery (16, 45-47).

Regarding CSS, our findings are in line with the findings reported by Bahlinger et al. (22), as we also found no significant association between CSS and Nectin4 expression. However, in contrast to Rodler et al. (21) we detected a significant association between higher Nectin4 expression and a longer PFS. They focused on BC with variant histology, whereas we analyzed an unselected cohort which could be a possible explanation.

In our cohort, higher Nectin4 expression was significantly associated with lower T- and AJCC-Stages. This mirrors several other recently published studies, where increased Nectin4 expression was found to be associated with lower T-stages and tumor grade in both BC and UTUC (43, 48, 49). In esophageal and breast cancer however, Nectin4 expression was higher in tumors with higher T-stages (46, 47). Contrary to pancreatic and ovarian cancer, in which no association between Nectin4 expression and tumor grade or stage was observed (16, 50).

Our findings and the available literature therefore suggests, that in BC and UTUC, unlike other solid cancers, Nectin4 expression is a marker for a less aggressive tumor biology and lower tumor stage. In our study, Nectin4 expression is further linked to a reduced risk of disease progression.

This study provides the first evidence that higher IRF6c H-scores are associated with reduced risk of disease progression in patients undergoing RC for MIBC. When analyzed as a continuous marker, IRF6c was also marginally associated with longer CSS. Studies in some other solid malignancies showed similar results, for example in gastric, colorectal and renal cell carcinoma (33, 51, 52). However, higher expression of IRF6 was associated with shorter OS and PFS or shorter OS in ovarian and pancreatic cancer respectively (34, 53).

In our cohort, higher IRF6 expression correlates to a lower rate of lymph node involvement, lower T- and AJCC-stages. This replicates findings made in a study by Jou et al. analyzing an RNA dataset; they found higher IRF6 expression in lower stage BC compared to higher stages (54). Similar results were observed in clear cell renal carcinoma, where higher IRF6 was associated with lower histological grade and tumor stage (51). In breast cancer, higher IRF6 expression was associated with a less aggressive tumor biology as well (55). In pancreatic cancer however, no association between IRF6 and tumor stages could be observed (53).

The different mechanisms of action of IRF6 have been investigated in different studies and cancer types. Most evidence exists for squamous cell carcinoma (SCC), where IRF6 exhibits tumor suppressing properties by modulating cell cycle genes, cell adhesion, proliferation and cell-to-cell contact (31, 32). Downregulation of IRF6 led to increased tumor formation via an increase in Rat Sarcoma (RAS) signaling (32). Regarding BC, in vitro experiments by Jou et al. showed that IRF6 overexpression resulted in reduced cell proliferation and increased apoptosis (54). In contrast, IRF6 knockdown increased proliferation in lung cancer and ovarian cancer cells (34, 56). These conflicting results of the in vitro studies in different cancer cell lines somewhat match the variable effects of IRF6 expression on patient survival in different solid malignancies.

We evaluated IRF6 expression in the nucleus and cytoplasm separately. IRF6c was associated with lower-risk pathological features and longer PFS, whereas IRF6n showed no such associations. To our knowledge, this is the first study to investigate the prognostic impact of IRF6 staining in cancer according to its subcellular localization. In ovarian cancer, Li et al. described both cytoplasmic and nuclear staining but assessed prognostic significance only at the RNA level (52). Botti et al. reported reduced nuclear staining in SCC compared to non-neoplastic keratinocytes. However, they did not examine whether nuclear and cytoplasmic staining differed regarding prognostic impact (31).

Previous studies have shown that transcription factors can have different functions depending on their location within the cell (57-60). Beyond regulating gene expression in the nucleus, they can influence cytoplasmic processes such as protein translation, mitochondrial function or signaling pathways (58, 59). This might offer an explanation for our observation that IRF6c, but not IRF6n, correlates with favorable features and longer PFS. As the impact of IRF6 on cancer development and patient survival appears to depend on its location within the cell as well as the type of cancer, further research seems to be needed to clarify its role.

The study cohort contains patients treated between 1990 and 2014, changes in staging practices, pathological assessment and perioperative systemic therapy during this period may have influenced clinical outcomes. Owing to the retrospective study design, detailed treatment information and other temporal variables were not consistently available for all patients and therefore could not be included in the analysis. Consequently, residual confounding related to treatment era cannot be excluded. However, we still believe that our results can be interpreted to provide insights into the impact of the evaluated markers on patient survival and pathological outcomes. The long follow-up time with a sufficient number of events allowed to conduct a reliable survival analysis. Still, statistical power to detect associations in subgroup analyses might be restricted due to sample size. An additional limitation of our study could be the relatively low proportion of Nectin4 positive tumors of 26.5% compared with the previously reported rates of 63-83% (43, 48, 49, 61). This discrepancy could be due to several factors, including a random predominance of tumor subtypes with inherently lower Nectin4 expression or cohort-specific differences (21, 48). Sampling and intratumoral heterogeneity could be another explanation, as a TMA-analysis of 314 patients used four cores per patient and found a Nectin4 positivity rate of 63% (48). Lastly, slide aging is known to lead to a loss of staining intensity and antigenicity, which given our older cohort, might contribute to the above-mentioned factors (62, 63). Even though the low positivity rate could limit the generalizability of our findings regarding Nectin4 and could reduce statistical power to detect associations with clinical outcomes, our results reveal promising associations with clinical outcomes justifying further exploration.

As no formal correction for multiple comparisons was applied, the observed associations, especially those with borderline significant p-values, should be interpreted cautiously and require validation in independent patient cohorts. Finally, as the prognostic significance of IRF6 subcellular localization was not previously explored, our findings lack external validation, which could represent a limitation and highlights the need for independent confirmation in future studies.

In summary, our study demonstrates that higher expression levels of IRF6 and Nectin4 are associated with lower-stage tumors and histopathological features linked to a less aggressive tumor biology. Additionally, while higher expression of both markers was associated with longer PFS in univariable analyses, when correcting for existing risk classification systems, this association did not remain significant in multivariable analysis. Our results provide important insights into the potential biological and prognostic relevance of IRF6 and Nectin4 in BC and suggest directions for future studies to explore their mechanistic roles and prognostic utility.

The Authors have no competing interests to declare.

Dominik Enderlin: Conceptualization, Methodology, Formal Analysis, Investigation, Data Curation, Writing – Original Draft, Visualization. Maria Gloor: Investigation, Writing – Original Draft, Writing – Review and Editing, Visualisation. Uwe Bieri: Investigation, Writing – Review and Editing. Alekhya Mazumdar: Writing – Review and Editing. Souzan Salemi: Writing – Review and Editing. Niels Rupp: Investigation, Writing – Review and Editing. Holger Moch: Writing – Review and Editing Daniel Eberli: Resources, Writing – Review and Editing. Cédric Poyet: Conceptualization, Methodology, Writing – Review and Editing, Supervision.

All Authors would like to thank Susanne Dettwiler, Fabiola Prutek and André Fitsche for their excellent technical support. Figure 1 was created in BioRender. Mazumdar, A. (2026) (https://BioRender.com/b10gnah).

This research has received no external funding.

During the preparation of this manuscript, a large language model (ChatGPT, OpenAI) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning–based image enhancement tools.