Breast cancer samples were obtained from the Chinese PLA General Hospital, with the informed consent of patients and with institutional approval for experiments from the Chinese PLA General Hospital and Beijing Institute of Biotechnology. Immunohistochemical staining was performed as described previously (49) using rabbit anti-PES1 (A300-902A, Bethyl Laboratories) as primary antibody. Each specimen was assigned a score according to the intensity of the nuclear staining (no staining = 0, weak staining = 1, moderate staining = 2, strong staining = 3) and the extent of stained cells (0% = 0, 1 to 24% = 1, 25 to 49% = 2, 50 to 74% = 3, 75 to 100% = 4). The final immune reactive score was determined by multiplying the intensity score with the extent score, ranging from 0 (the minimum score) to 12 (the maximum score). The optimal cutoff value of the IHC scores was estimated using receiver operating characteristic curve analysis. We defined score 0 to 4 as low PES1 and score 5 to 12 as high PES1.

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