Ventana benchmark system

This study used immunohistochemistry (IHC) to determine the status of ER, PR, and HER2. IHC was performed on formalin-fixed, paraffin-embedded tissue sections (thickness 4 μm) in the Central Pathology Laboratory at the hospital. ER and PR were determined using the Ventana Benchmark system (Ventana Medical Systems)^{32 (link)}. The percentage of positive-staining nuclei was recorded. In this study, we applied the National Comprehensive Cancer Network (NCCN) criteria to determine breast cancer's molecular phenotype. Both ER and PR status were determined for all invasive breast cancer and ductal carcinoma in situ (DCIS) using a cutoff value of ≥ 1% as a positive result³³ . HER2 status was reported as strong positive when the IHC score was 3 +^{34 (link)}. We defined the molecular subtype of breast cancer as (1) luminal A (ER-positive and/or PR-positive, and HER2-negative), (2) luminal B (ER-positive and/or PR-positive, and HER2-positive), (3) HER2/neu (ER-negative, PR-negative, and HER2-positive), and triple-negative (ER-negative, PR-negative, and HER2-negative).

Immunohistochemistry for cytokeratin (CK) 7, CK19 and H&E stainings were performed on 4 μm sections of formalin-fixed paraffin-embedded tumors and carried out with a standardized protocol on a Ventana Benchmark system (Ventana Medical Systems, Tucson, AZ, USA). Immunofluorescence was performed on 5 μm sections of formalin-fixed paraffin-embedded tumors. Briefly, tissue sections were deparaffinized and micro-wave heated for 10 min in 10 mM sodium citrate pH 6.0 for antigen unmasking. Sections were permeabilized for 5 min in phosphate-buffered saline (PBS)/0.3% Triton X-100 before blocking for 45 min in 0.3% milk/10% bovine serum albumin/0.3% Triton X-100 in PBS. Primary and secondary antibodies (listed in S1 Table) were diluted in blocking solution and incubated respectively at 4°C overnight and room temperature for 1 h. Pictures were taken with an Axiovert 200 fluorescent microscope using AxioVision system.

A neuropathologist identified histologically representative tumor regions that were stained by hematoxylin and eosin. Tissue sections were cut at 4 µm and the IHC was performed using the Ventana Benchmark system (Ventana Medical System, Tucson, AZ, USA). As a pre-treatment step, tissues were subjected to heat-induced epitope retrieval with the Cell Conditioning 2 solution (Ventana, Tucson, AZ, USA), 24 min for Ki-67 (30-9) (Ventana, Tucson, AZ, USA), 32 min for p53 (DO-7) (Ventana, Tucson, AZ, USA) and IDH1 (R132H) (Dianova, Hamburg, Germany). The antibody concentrations were 2 µg/ml for Ki-67, 184 µg/ml for p53, and 4 µg/ml for IDH1. Two independent observers evaluated the stained slides. Proliferation index was evaluated using Ki-67 antibody staining and calculated by determining the percentage of immunopositive nuclei. A total of 100-500 nuclei were counted. The tumors were divided into two groups, less aggressive (<15 %) and more aggressive ≥15 %). The consensus for p53 was scored in four different categories: no immunoreactivity (0 %), faint (≤50 %), moderate (50–75 %), and strong (≥75 %) immunoreactivity. IDH1 was scored in two categories: (0–10 %) for negative immunoreactivity, and (≥10 %) for positive immunoreactivity.

The ER and PR status, expression of human epidermal growth factor receptor 2 (HER2) and Ki67-proliferation index were measured in specimens of breast tumour tissue by immunohistochemistry methods. ER and PR status were quantitatively evaluated using the VENTANA BenchMark system (Ventana Medical Systems, Tucson, AZ, USA) with anti-ER clone SP1 and anti-PR clone 1E2 as the primary antibodies. Tumours were considered as positive for ER and PR if more than 1% of tumour nuclei were stained independently of staining intensity in accordance with the recommendations of the American Society of Clinical Oncology/College of American Pathologists. HER2 immunostaining was performed using VENTANA anti-HER2/neu (4B5) antibodies. The intensity of the HER2 expression was scored as HER2-negative = 0 or 1+ and HER2-positive = 3+. Tumours with scores of 2+ were taken as equivocal cases, which were further recommended for fluorescence in situ hybridisation (FISH) analysis. The Ki67 antigen staining was performed using the monoclonal mouse antibody (Auto-stainer Link 48, Agilent Technologies, Santa Clara, CA, USA). The Ki67 score was calculated as the percentage of immunostained cells. The optimal cut-off for a high versus low Ki67 score was defined as the 20% threshold.