Immunohistochemical reactions were performed automatically (Dako Autostainer immunostaining system; Dako) for CK5/6 (D5/16 B4; Dako; 1∶100), EGFR (2-18C9; Dako; 1∶100), AR (F.39.4.1; BioGenex; 1∶100), and p53 (mouse (Dako, M7001; 1∶100). P53 was considered in the analysis as a quality control marker of the immunohistochemical reactions because it is a driver gene in TNBCs [20] (link). Appropriate positive and negative control tissues were run concurrently.
The expression of CK5/6 was cytoplasmic, the expression of EGFR was both cytoplasmic and membranous, expression of AR and p53 was nuclear. Cytoplasmic expression in ≥10% of tumor cells for CK5/6, membranous staining in ≥10% of tumor cells for EGFR, and nuclear staining in ≥5% of tumor cells for AR and ≥50% for p53 were accepted as positive, as previously described [36] (link), [37] (link).
TNBCs were divided into subtypes of breast cancer as defined by their IHC profiles as basal-like triple negative (Core Basal; negative for ER, PR, and HER-2 and positive for CK5/6 and/or EGFR), and five negative (5NP; negative for ER, PR, HER-2, CK5/6, and EGFR). Slides were scored independently by three pathologists (SB, CI, MF) blinded to breast cancer subtype; one pathologist (MF) converted scores to numbers, selected cutoff values for each marker and entered data into Excel files.
The expression of CK5/6 was cytoplasmic, the expression of EGFR was both cytoplasmic and membranous, expression of AR and p53 was nuclear. Cytoplasmic expression in ≥10% of tumor cells for CK5/6, membranous staining in ≥10% of tumor cells for EGFR, and nuclear staining in ≥5% of tumor cells for AR and ≥50% for p53 were accepted as positive, as previously described [36] (link), [37] (link).
TNBCs were divided into subtypes of breast cancer as defined by their IHC profiles as basal-like triple negative (Core Basal; negative for ER, PR, and HER-2 and positive for CK5/6 and/or EGFR), and five negative (5NP; negative for ER, PR, HER-2, CK5/6, and EGFR). Slides were scored independently by three pathologists (SB, CI, MF) blinded to breast cancer subtype; one pathologist (MF) converted scores to numbers, selected cutoff values for each marker and entered data into Excel files.
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