Immunohistochemical Analysis of Breast Tumor Markers

Immunohistochemistry was used to assess the expression of estrogen and progesterone receptors (ER and PR), HER2, and Ki-67 in breast tumors using the following antibodies: mouse anti-ER (Dako, Cat. # IR084, clone 1D5, RTU), mouse anti-PR (Dako, Cat. # IR068, clone PgR636, RTU), rabbit anti-HER2 (Dako, Cat. # A0485, 1:800), and mouse anti-Ki-67 (Dako, Cat. # IR626, clone MIB-1, RTU). Immunohistochemistry was performed as previously described (11 (link)). ER and PR immunostaining was scored using ASCO/CAP Recommendations (12 (link)). HER2 immunostaining was scored using St. Gallen recommendations (13 (link)). Ki-67 immunostaining was expressed as the percentage of positively stained cells. At least 10 fields of view and at least 1,000 cells at 400× magnification (field area = 0.196 mm²) were analyzed per sample. Molecular subtypes of the IC NST were categorized according to St. Gallen recommendations (13 (link)): luminal A (ER⁺ and/or PR⁺, HER2⁻, and Ki-67 < 20%), luminal B (ER⁺ and/or PR⁺, HER2^−/+, and Ki-67 ≥ 20%), HER2-positive (ER⁻, PR⁻, and HER2⁺), and triple-negative (ER⁻, PR⁻, and HER2⁻).