Herceptest 2

HER2 IHC was performed as described previously[18 (link)]. In brief, tumor specimens were fixed in 10% neutral-buffered formalin overnight and embedded in paraffin blocks. Sections (4 μm) were cut from the tissue paraffin blocks, and IHC was performed using HercepTest II™ (Dako, Denmark) according to the manufacturer’s instructions. This process was conducted at the Department of Pathology, Chinese People’s Liberation Army General Hospital. IHC sections were reviewed by experienced pathologists and were scored for HER2 expression according to the criteria used in the ToGA trial[1 (link)]. If tissue HER2 expression was scored as 2+, a further FISH assay was performed with Abbott-Vysis PathVysion™ (Abbott Laboratories, United States) according to the manufacturer’s protocol. Tumor specimens with a HER2:CEP17 signal ratio ≥ 2.0 or 3+ IHC staining intensity were judged as HER2-positive.

To determine MET expression status by immunohistochemistry (IHC), monoclonal antibody (mAb) for MET, MET4 mAb,26 was used. Epitope sequence of MET4 revealed that DVLPEFR on amino acid residue 236–242 was the specific recognition site of MET. To retrieve MET antigen, tissue slide sections were treated at 98°C for 20 min in Target Retrieval Solution, pH9 (DAKO, Santa Clara, CA). ENVISION+ System (DAKO) was used for the secondary antibody reaction and DAB+ was used for color development. In the final step, the nuclei were lightly counterstained with Meyer hematoxylin.
Regarding the tissue staining of HGF/SF, anti‐HGF/SF mAb (clone 7‐2) was purchased from Enzo Life Sciences (Ann Arbor, MI, USA) and the samples were stained according to the company's instruction. Antigen retrieval was performed by boiling the sample slides in the 10 mM citrate buffer (pH 6) in a microwave for 7–10 min.
For the evaluation of Her2/neu IHC status HercepTest II (DAKO) was used. For the evaluation of tumor cell proliferation, we performed Ki‐67 staining using anti‐human Ki‐67 antibody (M7240, DAKO). E‐cadherin expression in the proliferative margin of tumor cells was also investigated using E‐cadherin antibody (H‐108, Santa Cruz Biotechnology, CA, USA).