Clone pgr636

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⁻).

We collected factors responsible for progressive BCs including conventional tumor attributes (size, lymph node involvement, and histological grade), three immunohistochemical markers (ER, PR, HER2), triple negative (defined by these three IHC markers), surgical treatment and adjuvant therapy. Conventional tumor attributes have been collected since the dawn of the service screening. Surgical treatment (breast-conserving surgery, mastectomy, or others), and adjuvant therapy (radiotherapy, chemotherapy, or hormone therapy) had been collected since 1996.
Data on tumor phenotypes related to IHC markers including ER, PR and HER2 status were collected retrospectively for the period of 1996 to 1998 by standard antibody staining in the largest invasive tumor component for each patient and was described in full in previous studies [22 (link)]. The antibodies (supplier, type, dilution) used for staining are delineated as follows: ER (clone SP1; Ventana Medical Systems, Tucson, AZ, USA; 1:200 dilution), PR (clone PgR 636; Dako, Glostrup, Denmark; 1:50 dilution), and HER2 (code A 0485; Dako; 1:250 dilution). The cut-off point for ER and PR positivity is nuclear staining >10% of tumor cells. The criteria of HER2 positivity was offered by manufacturer. Triple negative BC is defined as a breast tumor with all ER, PR, and HER2 being negative.

TNBC status was evaluated by immunohistochemistry using specific antibodies against monoclonal rabbit ER antibody, Clone SP1 (Neomarker) and monoclonal mouse anti-human PR antibody, Clone PgR 636 (Dako). ER and PgR expression was interpreted as positive if at least 1% immunostained tumor nuclei were detected in the sample, according with ASCO/CAP recommendations for immunohistochemical testing of hormone receptors in breast cancer [17 (link)]. HER2 protein expression was determined using FDA approved HercepTest™ (K5206 DAKO) and evaluated according to the manufacturer’s instructions. HER2 gene amplification was determined by ultra-View SISH Detection Kit (Ventana Medical Systems, Tucson, USA). Tumors were classified according to the 2013 ASCO/CAP recommendations [18 (link)]. Given that the study included patients diagnosed over almost 20 years in different hospital centers, all surgical specimens of TNBC patients were reviewed independently by three experienced pathologists to achieve a consensus on morphologic criteria and to standardize the results at the current guidelines recommendations for ER, PgR and HER2 immunohistochemistry [17 (link)].