Immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH)

IHC and FISH analysis were conducted on tissue sections (3–4 µm) and tissue microarray slides. IHC (Benchmark XT; Ventana Medical Systems, Inc., Tucson, AZ, USA) was performed on formalin-fixed and paraffin-embedded tissues of 757 patients using the avidin-biotin-immunoperoxidase technique (22). Tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. Antigen-retrieval was performed in citrate buffer (pH 6.0) at 120°C for 2.5 min. Subsequently, the slides were exposed to 3% hydrogen peroxide for 20 min and washed with PBS for 5 min 3 times. The further blocking of tissues was performed with normal goat serum (Abcam Inc., Cambridge, UK) for 30 min at room temperature. The sections were then incubated overnight at 4°C with the following primary antibodies: ER (cat no. NCL-L-PGR-312; dilution, 1:100), PR (cat no. NCL-L-ER-6F11; dilution, 1:80) (both Novocastra; Leica Biosystems GmbH, Wetzlar, Germany), HER2 (cat no. 800-2996; dilution 1:300; Ventana Medical Systems, Inc.). Following 5 washes with PBS, the slides were incubated with biotin-conjugated secondary antibody (cat no. ZB-2010; dilution, 1:200; OriGene Technologies, Inc., Beijing, China) for 30 min at room temperature. The sections with positive expression level was used as the positive control, the negative control was established with the primary antibody replaced by PBS. Detection was done by utilizing iView DAB Detection kit (Ventana Medical Systems, Inc.).

FISH analysis was conducted in line with the protocol of Abbott/Vysis PathVysion HER2 DNA Probe kit (cat no. 30161060/02J01-030; Abbott Molecular, Inc., Des Plaines, IL, USA), the Spectrum Orange fluorophore-labeled DNA probe and Spectrum Green fluorophore-labeled α-satellite DNA probes from this kit were used to assess the HER2 gene locus and chromosome 17, respectively. In total, 2 separate fields of ≥20 cells were counted and the average of the results of the selected tumor areas were used to calculate mean gene and chromosome 17 counts, which were used to calculate the ratio of HER2:CEP17 signal. Tumor cells from matching sites of IHC were scored for the number of red (HER2) and green (chromosome 17) signals. The slides were evaluated using an Olympus BX51 microscope (Olympus Corporation, Tokyo, Japan) with an oil-immersion objective lens and an appropriate filter set at a magnification of ×100. Immunoreactivity was assessed independently by ≥2 pathologists.