Er clone sp1

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.

The IHC analysis was performed utilizing specific antibodies against ER Clone SP1 (Ventana Medical Systems Inc., Tucson, AZ, USA); PR Clone 1E2 (Ventana Medical Systems); AR Clone SP107 (Cell-MarqueTM, Rocklin, CA, USA); HER2 PATHWAY Clone 4B5 (Ventana Medical Systems); Ki67 Clone 30-9 (Roche Diagnostics K.K., Tokyo, Japan). We used the Ventana Benchmark XT staining system with Optiview DAB detection kit for immunostaining on 3 µm-thick tissue sections of FFPE specimens. HER2 gene amplification by ultra-View SISH Detection Kit (Ventana Medical Systems) was performed in cases of HER2 with equivocal immunohistochemical score (IHC 2+). Evaluation of immunostaining and dual-probe in situ hybridization for HER2 was based on American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) recommendations^{1 (link)}. ER and PR expression were assessed positive if at least 1% immunostained tumor nuclei were detected in the sample, according to ASCO/CAP recommendations for immunohistochemical testing of hormone receptors in BC^{36 (link)}. The Ki67 cut-off was ≤ 20% and > 20%^{37 (link)}. AR expression was considered positive if at least 10% immunostained tumor nuclei were detected³⁸ . All IHC expressions were assessed using a semi-quantitative method.

Immunohistochemistry was performed on a Benchmark Ultra (Ventana, Tucson, AZ) automated stainer using the CC1 mild protocol for antigen retrieval and monoclonal antibodies against E-cadherin (clone ECH-6, Zytomed Systems, Berlin, Germany, 1:100), β-catenin (clone 14, Beckton Dickinson, Heidelberg, Germany, 1:100), CK7 (clone OV-TL12/30, DAKO, Glostrup, Denmark, 1:100), Epithelial membrane antibody (EMA, clone E29, DAKO, 1:600), ER (clone SP1, Ventana, undiluted ready-to-use), BCL2 (clone 124, DAKO, 1:100), GATA binding protein 3 (GATA3, clone L50-823, BioCare Medical, Concord, CA, 1:200), mammaglobin (MGBN, clone 304-1A5, Biologo, Kiel, Germany, 1:10) and v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2, clone 4B5, Ventana, undiluted ready-to-use). Detection of the immune reaction was achieved with the ultraView DAB kit (Ventana).