Tissue microarrays were constructed by extracting 2-mm diameter cores of histologically confirmed invasive breast carcinoma areas, as previously described.30 (link) Five-micrometer tissue sections were cut and stained using the purified goat polyclonal antibody raised against the recombinant human Nectin-4 extracellular domain (1:60 dilution, 60 min, AF2659, R&D Systems Inc., Minneapolis, MN, USA). Whole sections of non-neoplastic breast tissues from 30 patients were also stained. As positive controls of Nectin-4 expression, skin and nipple sections were used.18 (link) Antigen retrieval was performed by microwave treatment at 750 W for 10 min in 1 M urea buffer (pH 8.0). The LSAB kit (K0679, Dako, Glostrup, Denmark) was used for signal amplification. In control sections, the specific primary antibody was replaced with isotype-matched immunoglobulins. The following antibodies were used for the identification of tumor subtypes, as previously detailed:31 (link) the anti-ER-α MoAb 6F11 (Novocastra, Menarini, Florence, Italy), the anti-PR MoAb 1A6 (Menarini), the anti-Ki67 MoAb MIB-1 (Dako) and the anti-HER-2 (Herceptest, Dako). Immunohistochemical analysis was done by two pathologists (MP, RL) by consensus without knowledge of the clinicopathological information.