Her2 clone 4b5

A formalin-fixed, paraffin-embedded tumor block was cut into 4-μm-thick sections for H and E and immunostaining. Immunohistochemistry was performed by using the rabbit monoclonal antibodies against HER 2 (clone 4B5, Ventana Medical System, Tucson, AZ, USA).

Nuclear staining values of 1% or higher were considered positive for ER (clone 6F11; dilution 1:200; Leica Biosystems, Wetzlar, Germany) and PR (clone 16; dilution 1:500; Leica Biosystems, Wetzlar, Germany) [26 (link)]. HER2 (clone 4B5; dilution 1:5; Ventana Medical System, Oro Valley, AZ, USA) staining was performed according to the 2018 American Society of Clinical Oncology/College of American Pathologists [27 (link)]. Only samples with strong and circumferential membranous HER2 immunoreactivity (3+) were considered positive, whereas those with 0 or 1+ HER2 staining were considered negative. Cases with equivocal HER2 expression (2+) were further evaluated for HER2 gene amplification via silver in situ hybridization (SISH). Positive nuclear Ki67 (clone MIB; dilution 1:1000; Abcam, Cambridge, UK) staining was assessed based on the percentage of positive tumor cells, defined as the Ki67 labelling index (LI). ER- and/or PR-positive and HER2-negative cases were selected for this study.

Expression of the basic biomarkers including ER, PR, HER2, p53, and Ki-67 was evaluated at the time of pathologic diagnosis in the surgical specimens. The antibodies stained were as follows; ER (clone SP1; 1:100 dilution; LabVision, Fremont, CA), PR (clone PgR 636; 1:70 dilution; Dako, Carpinteria, CA), HER2 (clone 4B5; ready to use; Ventana Medical Systems, Tuscon, AZ), p53 (clone D07; 1:600 dilution; Dako), and Ki-67 (clone MIB-1; 1:250 dilution; Dako). ER and PR staining in ≥ 1% of the tumor cells was determined as positive. Patients with ER- or PR-positive tumors were regarded as hormonal receptor (HR)-positive. HER2 positivity was defined as an immunohistochemical score of 3+ or the presence of HER2 gene amplification on fluorescence/silver in situ hybridization. For p53, staining in 10% or more of the tumor cells was considered positive. High Ki-67 proliferation index was defined as staining in ≥ 10% of tumor cells for DCIS and ≥ 20% for IBC.
Breast cancer subtype was determined with standard biomarker profiles according to 2011 St Gallen International Expert Consensus^{37 (link)}. Each subtype was defined as follows: luminal A (ER+ and/or PR+, HER2−, Ki-67 < 14%), luminal B (ER+ and/or PR+, HER2−, Ki-67 ≥ 14%; ER+ and/or PR+, HER2+), HER2+ (ER−, PR−, HER2+), and triple-negative (ER−, PR−, HER2−).

IHC primary antibodies used were: ERα (clone SP1, Ventana), PR (clone 1E2, Ventana), Ki67 (Clone 30–9, Ventana), Her2 (clone 4B5, Ventana). Tumors were considered positive for ER and PR when at least 1% of tumor cells showed unequivocal nuclear staining according to American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. PR expression was considered high in the presence of nuclear staining in 20% or more cells. We set a cut-off point to distinguish low versus high Ki67 expression at 20%. The original HER2/neu immunostained glass slides were concurrently reviewed by pathologists at a multiheaded microscope, and the consensus HER2/neu immunoreactivity was manually scored by conventional microscopy as 0, 1+, 2+, or 3+ according to the proposed HER/neu scoring system for breast cancer. According to the percentage of stained malignant cells, criteria for HER2/neu score assignment were: 0, no staining or staining in <10% of cells; 1, faint staining in ≥10% of cells; 2, moderate staining in ≥10% of cells; and 3, strong staining in ≥10% of cells. Tumors classified as 0, 1+, and 2+ were considered “negative” and those scored as 3+ were classified as “positive”.

Immunohistochemistry was performed using tissue arrays of type A and B3 thymomas and thymic carcinomas. 2 μm sections of the tissue arrays were stained on a Ventana Benchmark Ultra (Ventana, Tucson, AZ) with extended heat-induced epitope retrieval with CC1 buffer and the ultraView Universal DAB Detection Kit (Ventana). The following antibodies were employed: ALK (clone 1A4; Zytomed, Berlin, Germany), HER2 (clone 4B5; Ventana), HER3 (clone SP71; Abcam, Milton, UK), MET (clone SP44; Ventana), phospho-mTOR (clone 49F9; Cell Signalling, Danvers, MS), p16^INK4A (clone E6H4; Ventana), PDGFRA (rabbit polyclonal; Thermo Fisher Scientific), PDGFRB (clone 28E1, Cell Signalling), PD-L1 (clone E1L3N; Cell Signalling), PTEN (clone Y184; Abcam) and ROS1 (clone D4D6; Cell Signalling). An immunohistochemial score was determined by multiplying the percentage of positive cells by their respective staining intensity (0 = negative, 1 = weak, 2 = moderate, 3 = strong). Immunohistochemical score (maximum 300) = (% negative x 0) + (% weak x 1) + (% moderate x 2) + (% strong x 3).

A formalin-fixed, paraffin-embedded FFPE tumor block was cut into 4-μm-thick sections for hematoxylin and eosin (H&E) immunostaining. Immunohistochemistry was performed by using the antibodies for HER2 (clone 4B5, Ventana Medical System, Tucson, AZ, USA) and E-cadherin (clone NCH-38; Dako, Carpinteria, CA, USA). The E-CAD staining was scored on three scales: negative/weak staining (score 0) when less than 10% of tumor cells with strong IHC intensity (2–3+) or less than 30% of cells with weak intensity (1+) were present; reduced staining (score 1) when 10% to 90% of cells showed a strong IHC intensity or about 30% showed a weak intensity; normal staining (score 2) when more than 90% of cells showed a strong IHC intensity.