Detection of Nectin-4 in serum from patients with MBC. Serum Nectin-4 was detected by ELISA using a pair of anti-Nectin-4 mAbs. A: Percentage of Nectin-4 positive sera in healthy donors (n = 45), and in patients with non-metastatic breast tumors compared with CEA and CA15.3 levels (n = 53). B: Percentage of sera positive for Nectin-4, CEA and CA15.3 and combinations of markers. The association of Nectin-4 with CEA and CA15.3 increases the percentage of patients detected. White bars; patients with metachronous MBC. Grey bars; patients with synchronous MBC. Black bars; percentage including both groups. C: ROC curves were calculated to estimate the accuracy of the association of these markers in breast cancer diagnosis.