Single-cell transcriptomic characterization of NECTIN-4 expression in penile squamous cell carcinoma.

Abstract

9 Background: NECTIN-4, a cell adhesion molecule and target of the antibody–drug conjugate enfortumab vedotin, has emerged as a promising therapeutic target within genitourinary malignancies. Given the limited treatment options and high morbidity associated with penile cancer, there is a critical need to identify novel therapeutic opportunities. We investigated NECTIN-4 expression and its associations with HPV status, disease stage, lymphovascular invasion (LVI), and copy number variation (CNV) using single-cell RNA sequencing (scRNA-seq) of human penile squamous cell carcinoma (PSCC) tissues. Methods: scRNA-seq was performed on ten PSCC tissue samples spanning stages pT1–pT3, pN0–pN3, and M0–M1, including five penile primaries, four lymph node metastases, and one distant metastasis. HPV status was determined by high-risk HPV in situ hybridization and p16 immunohistochemistry. Single-cell suspensions were processed using Cell Ranger (v7.1.0, 10x Genomics) and analyzed with Seurat V4. Cells with < 200 detected genes, log10(genes/UMI) < 0.8, > 15% mitochondrial reads, or doublets were excluded. Malignant cells were identified using inferCNV, and group comparisons were performed using the Wilcoxon rank-sum test. Results: NECTIN-4 transcripts were detected exclusively in epithelial cells and were absent in endothelial and immune populations. Median expression was higher in HPV-positive versus HPV-negative tumors (1.19 [IQR 2.07] vs 0.53 [1.59]; p < 2.2×10⁻ 16 ; n = 9227 vs 1101) and in LVI-positive versus LVI-negative tumors (1.33 [2.22] vs 0.71 [1.41]; p < 2.2×10⁻ 16 ; n = 7744 vs 2584). Advanced disease demonstrated elevated NECTIN-4 expression compared to localized tumors (1.19 [2.06] vs 0.00 [1.54]; p < 2.2×10⁻ 16 ; n = 9398 vs 930). NECTIN-4 levels correlated inversely with CNV burden, decreasing from low (1.47 [2.41]) to median (1.13 [2.00]; p < 2.2×10⁻ 16 ) to high (1.03 [1.58]; p < 3.4×10⁻ 10 ) CNV categories. Conclusions: NECTIN-4 shows variable expression across PSCC with increased expression in HPV-positive, LVI-positive, and advanced-stage tumors, and an inverse correlation with CNV burden. These findings highlight NECTIN-4 as a potential biomarker of tumor aggressiveness and a promising therapeutic target in PSCC. Ongoing and future studies evaluating enfortumab vedotin and other NECTIN-4–directed therapies may provide new treatment avenues for patients with this rare malignancy.