Defining NECTIN4 Amplification and Protein Expression in Urothelial Carcinoma and Histologic Subtypes

Chromosome 1q23.3 is commonly amplified in metastatic urothelial carcinoma (UC). This region contains NECTIN4 that encodes for a membrane protein, and amplification may result in increased NECTIN4 expression. Enfortumab vedotin, an antibody-drug conjugate, targets NECTIN4. The objective of this study was to determine the frequency of NECTIN4 amplification by fluorescence in situ hybridization and protein expression by immunohistochemistry in primary UC and subtypes. Tissue microarrays (TMA) were constructed from 841 patients who underwent cystectomy between 2000 and 2020. In 218 patients, TMA were constructed from a concurrent pelvic lymph node metastasis. NECTIN4 amplification varied by subtype and was present in 18% of UCs, 13% of UCs with squamous differentiation, 25% of micropapillary carcinoma, 19% of plasmacytoid carcinoma, 17% of high-grade neuroendocrine carcinoma, 8% of pure squamous cell carcinoma, 6% of nested carcinoma, and 6% of sarcomatoid carcinoma. Tumors with NECTIN4 amplification had significantly higher H scores compared with nonamplified tumors (P < .0001), and H scores increased with increasing copy number in amplified tumors. NECTIN4 amplification was identified in 17% of lymph node metastases, and 70% of amplified primary tumors had an amplified metastasis, which increased to >90% when whole section fluorescence in situ hybridization was performed in discordant TMA cases. There was a moderate agreement between immunohistochemical staining of primary and lymph node metastases (kappa = 0.41). NECTIN4 amplifications vary in frequency among UC and subtypes, and amplifications result in higher protein expression. Bladder cancer with NECTIN4 amplification may have limited protein expression, whereas nonamplified tumors may show high expression. There was concordance of NECTIN4 amplification and protein expression between primary and lymph node metastases, but there were a small number of cases with amplified primary tumors and unamplified metastases, primary tumors with high protein expression, and metastasis with low expression.