A0485

Tumors were histopathologically characterized on HE-stained sections following the routine method for histological description of neoplasms [5 (link)]. Immunohistochemical characterization of estrogen and progesterone receptors (ER, Ref. M7047, Dako; PR, Ref. 790-2223, Ventana, Oro Valley, AZ, USA) and human epidermal receptor-2 (HER-2, Ref. A0485, Dako, Santa Clara, CA, USA) was performed. Paraffin sections were placed in a PT module, heated for 20 min at 95 °C, and cooled down to 60 °C. Then, slides were rinsed in warm tap water and placed in an automatic immunostainer device (Lab Vision Corp., Fremont, CA, USA) for immunohistochemistry using a peroxidase detection system. After immunostaining, the slides were counterstained with hematoxylin and permanently mounted with Depex. Corresponding negative control slides were prepared by replacing the primary antibody with nonreactive antibody. Slides from human and canine mammary tumors with previously demonstrated reactivity to the primary antibody and tissue internal controls were used as positive controls [5 (link)].
For estrogen receptor, progesterone receptor, and HER-2 evaluation, 3+ positive scoring was considered, following the recommended guidelines of the American Society of Cancer Oncology (ASCO).

Immunoreactions were revealed by Bond Polymer Refine Detection on an automated autostainer (Bond^™ Max, Leica Biosystem, Milan, Italy). ER and PgR expression was evaluated using mAb 6F11 (Leica, Novocastra) and mAb 1A6 (Leica, Novocastra) respectively. Cell proliferation and HER2 overexpression were tested using the Ki-67 mAb (MIB1, Dako, Milan, Italy) and the polyclonal antibody A0485 (Dako), respectively. Tumors were also characterized for cytokeratin 19 expression (Dako). HER2 IHC positivity was defined according to ASCO/CAP guidelines [20 (link)]. ER and PgR were considered positive when >1% and ≥20% of the neoplastic cells showed distinct nuclear immunoreactivity, respectively. Ki-67, based on the median value of our series, was regarded as high if more than 15% of the cell nuclei were immunostained. Evaluation of the IHC results, blinded to all patient data, was performed independently and in blinded manner by two investigators (SB and MM).

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⁻).