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Abnormal expression of the cell cycle regulatory protein Bmi-1 has been studied in breast cancer, but the clinical relevance has not been fully elucidated. We studied the expression of Bmi-1 protein in breast cancer patients to define its clinical significance in breast cancer.Tissue microarrays were performed to evaluate the expression of Bmi-1 by immunohistochemistry in tumor tissues from 960 patients with stage I–III breast cancer. We assessed the relationship between the expression of Bmi-1 and pathologic prognostic indices as well as clinical long-term follow up outcome.Bmi-1 was expressed in 53.2% of breast cancer patients by immunohistochemistry, and the expression of Bmi-1 was significantly correlated with favorable prognostic indices at diagnosis. In univariate analysis, patients with Bmi-1 expression showed favorable relapse-free survival (88.6, ±, 2.7% vs. 72.3, ±, 4.3%, P, =, 0.041) and favorable overall survival (93.5, ±, 2.2% vs. 82.6, ±, 3.5%, P, <, 0.001) than patients without Bmi-1 expression. According to multivariate analyses, Bmi-1 expression was identified as independent prognostic factor for overall survival with a statistical significance (hazard ratio of Bmi-1 (−) patients compared to Bmi-1 (+) patients, 1.744; 95% CI, 1.013–3.003; P, =, 0.045). This correlation of Bmi-1 expression with favorable overall survival was maintained in patients underwent uniform chemotherapy, regardless of undergoing adjuvant chemotherapy. In a subset analysis according to ER status, Bmi-1 expression associated with favorable overall survival only in ER-positive patients.Bmi-1 expression assessed with Immunohistochemistry may be associated with favorable overall survival in breast cancer patients, especially in patients with ER-positive breast cancer.