<div>Abstract<p><b>Purpose:</b> Currently, there are no molecular biomarkers for the early detection of breast cancer. This study focused on identifying surface proteins found on circulating extracellular vesicles (EVs) for detecting early-stage breast cancer.</p><p><b>Experimental Design:</b> Circulating EVs, isolated from the plasma of 10 patients with breast cancer (stages I and II) and 5 healthy controls, were analyzed using LC-MS/MS. Developmental endothelial locus-1 protein (Del-1) was selected as a candidate biomarker. Two different ELISAs were used to measure Del-1 in plasma samples from healthy controls (<i>n</i> = 81), patients with breast cancer (<i>n</i> = 269), breast cancer patients after surgical resection (<i>n</i> = 50), patients with benign breast tumors (<i>n</i> = 64), and patients with noncancerous diseases (<i>n</i> = 98) in two cohorts.</p><p><b>Results:</b> Plasma Del-1 levels were significantly higher (<i>P</i> < 0.0001) in patients with breast cancer than in all controls and returned to almost normal after tumor removal. The diagnostic accuracy of Del-1 was AUC, 0.961 [95% confidence interval (CI), 0.924–0.983], sensitivity of 94.70%, and specificity of 86.36% in test cohort and 0.968 (0.933–0.988), 92.31%, and 86.62% in validation cohort for early-stage breast cancer by one type of ELISA. Furthermore, Del-1 maintained diagnostic accuracy for patients with early-stage breast cancer using the other type of ELISA [0.946 (0.905–0.972), 90.90%, and 77.14% in the test cohort; 0.943 (0.900–0.971), 89.23%, and 80.99% in the validation cohort].</p><p><b>Conclusions:</b> Del-1 on circulating EVs is a promising marker to improve identification of patients with early-stage breast cancer and distinguish breast cancer from benign breast tumors and noncancerous diseases. <i>Clin Cancer Res; 22(7); 1757–66. ©2015 AACR</i>.</p></div>