A comprehensive multi-omics analysis exploring the correlation between NECTIN4 and tumor microenvironment profiles in bladder cancer.

74 Background: NECTIN4 is a transmembrane protein that is found in the adherent junctions and highly expressed in urothelial carcinoma (UC). NECTIN4 is a TIGIT ligand and the TIGIT-NECTIN4 interaction inhibits natural killer cell activity. Therefore, Enfortumab vedotin (EV), an antibody-drug conjugate targeting NECTIN4, potentially induces immunogenic cell death. In fact, an encouraging result has been recently reported from a clinical trial of combined EV and pembrolizumab as the first line treatment for platinum-ineligible UC patients. However, there has been no study exploring the correlation between NECTIN4 expression and tumor microenvironment (TME) so far. Methods: To explore the potential association between NECTIN4 and TME, we analyzed a comprehensive dataset of whole-exome and RNA-sequencing, immunohistochemistry, and clinicopathological record from 389 bladder cancers (BC) and 35 adjacent normal tissues, which enables us to evaluate the potential relationship between genetic and transcriptomic profile and TME. Results: NECTIN4 mRNA expression was positively correlated with NECTIN4 H-score, gender, T-stage, tumor grade, lymph vascular invasion and tumor mutation burden, and was negatively correlated with CD274 mRNA expression levels in BC. Our cohort included 44 patients (44/389 = 11.3%) who were diagnosed as having variant histology. NECTIN4 expression levels of samples with variant histology (especially small cell carcinoma, sarcomatoid and adenocarcinoma) were significantly lower compared to conventional BC. When we classified 378 patients into three groups according to H-score, 93 patients were included in low group (H-score:0), 202 patients in medium group (H-score:10-190) and 83 patients in high group (H-score:200-300). Overall survival was significantly worse in the low group compared to medium/high groups (p<0.0001). CIBERSORTx analysis suggested increased proportions of M0 macrophages and neutrophils in low group. Gene set enrichment analysis identified that EMT-related programs and immune-related cytokines were activated in the low group. 45 platinum-refractory BC patients were treated with pembrolizumab and objective response rate was 57.1% in H-score high group, which was significantly higher compared to low/medium groups (p=0.0498). We also compared 73 immune check point related gene expression in H-score low and high groups and found that putative immune checkpoint gene expression was significantly different between the two groups, which collectively suggests that differential immune therapy strategies according to NECTIN4 expression levels would be beneficial for patients with BC. Conclusions: NECTIN4 expression levels are closely associated with tumor pathology and microenvironment. NECTIN4 expression levels may be a predictive marker that potentially contributes to the precision immune therapy in BC.