Data was collected from patients admitted to the General Military Hospital of Beijing PLA (124 cases), the 281st Hospital of the PLA (107 cases), Zhejiang Cancer Hospital (99 cases) and Weifang People’s Hospital (96 cases) between 2011 and 2013. Written informed consent was obtained from all patients prior to their participation in the study. All patients were pathologically diagnosed with gastric cancer and Her-2 protein expression was detected using IHC. None of the patients received preoperative treatment with chemotherapy, radiotherapy or immunotherapy. Samples consisted of 424 cases of adenocarcinoma (including papillary adenocarcinoma, tubular adenocarcinoma, mucous adenocarcinoma and signet ring cell carcinoma), one case of gland scale cancer and one case of squamous carcinoma. The patients included 149 cases of intestinal type, 244 cases of diffuse type and 33 cases of mixed/unknown. The ages of the patients ranged between 27 and 84 years (median age, 59.2 years) and included 322 males and 104 females. According to the World Health Organization criteria (7), there were 192 poorly differentiated, 161 moderately differentiated and 73 highly differentiated cases. A total of 310 cases were observed with lymph node metastasis and 116 cases without lymph node metastasis. There were 93 cases in the cardia, 180 cases in the antrum and 153 cases in the stomach body. The cancer was classified as stage I in 40 cases, stage II in 108 cases, stage III in 248 cases and stage IV in 30 cases, respectively, according to the TNM Cancer Staging System of the American Joint Committee of Cancer (8).

The DNA extraction kit was purchased from Qiagen (Hilden, Germany) and a Her-2/neu FISH testing kit was obtained from Beijing ACCB Biotech Ltd.(Beijing, China). The Mx3000P qPCR system was obtained from Stratagene (La Jolla, CA, USA).

DNA extraction was performed using the QIAamp DNA FFPE Tissue kit (Qiagen). The tissue was sectioned into slices (10 μm). The concentration and purity of the DNA was then measured in accordance with the manufacturer’s instructions.

The PCR reaction volume (20 μl) included 0.3 μl Taq DNA polymerase, 0.4 μl substrate dNTP, 2.4 μl Mg²⁺, 2.0 μl buffer and 3.0 μl DNA. The primers were obtained from the real-time PCR kit used and their catalogue numbers were Q/HDYKB007. The cycling conditions were as follows: 95°C for 5 min, followed by 45 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 45 sec, for a total of 40 cycles.

Her-2 gene was amplified with dual-color FISH (Her-2 gene real-time PCR kit, Guangzhou LBP Medical Science Technology Co., Ltd., Guangzhou, China) in accordance with the manufacturer’s instructions. Briefly, hybridization buffer, a DNA probe and purified water were centrifuged and then heated to 65°C overnight in a water bath. Tissue sections (4 μm) were placed on slides and immersed in a denaturing bath (2× SSC) for 5 min at 73°C, followed by dehydration in increasing ethanol concentrations and then dried. The slides were incubated with the probe at 42°C for 30 min. The slides were then washed with 0.4× SSC/0.3% NP-40 for 2 min, air-dried in the dark, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and covered with a cover-slip. The slides were observed under an Olympus BX51 fluorescence microscope (Shanghai Pooher Photoelectric Technology Co., Ltd., Shanghai, China) equipped with a digital camera. A cell was considered to be amplified when a definite cluster of >10 signals for Her-2 was found. Known positive and negative cells were used as controls for each FISH assay. Gene amplification was scored when ≥20 cancer cell nuclei exhibited a Her-2/CEP17 ratio ≥2, or when a Her-2 signal cluster was observed (9).

The 4 μm thick tissue sections of malignant tumor cells on the slides were stained with brown staining (Zymed Corporation, Inc., San Francisco, CA, USA). Briefly, sections were deparaffinized in xylene and rehydrated in grade alcohols. The antigen retrieval was performed using the wet autoclaving method in the presence of citrate buffer pH 6.0. The sections were incubated overnight in primary antibody at a dilution of 1:100 in blocking buffer at 4°C. The sections were stained using a Polin-2 plus Polymer HRP Detection System(ZSBIO, Beijing, China). Strong brown staining in the cell membrane of malignant tumor cells indicates positivity in this staining method. The HercepTest™ Interpretation Guide (10) was used to grade the membrane staining. The staining was scored as negative (0) when no membrane staining was observed or when membranes were stained in ≤10% of tumor cells, weakly positive (+) if the focal membrane was stained in ≥10% of tumor cells, intermediately positive (++) if complete membranes were weakly-moderately stained in ≥10% of tumor cells and strongly positive (+++) if complete membranes were intensely stained in ≥10% of tumor cells.

Data were calculated using SPSS software, version 19.0 (SPSS, Inc., Chicago, IL, USA). χ² and Fisher’s exact tests were used to test for an association between Her-2 amplification or protein overexpression and clinicopathological parameters. The kappa test was used to measure the consistency. P<0.05 was considered to indicate a statistically significant difference.

Using IHC analysis, the rate of overexpression of Her-2 in cancerous tissues was found to be 13.38% (57/426). The overexpression of Her-2 was significantly correlated with the depth of invasion, lymph node metastasis and TNM stage (P<0.05), and no significant correlation was identified between the overexpression of Her-2 and age, gender, tumor location, differentiation degree and Lauren classification (P>0.05; Table I).

Using PCR analysis, the positive expression rate of Her-2/neu in cancerous tissue was found to be 11.17% (46/412). The expression of Her-2/neu was significantly correlated with the depth of invasion, lymphatic metastasis and TNM stage (P<0.05), and no significant correlation was identified between the positive expression of Her-2/neu and age, gender, tumor location, differentiation degree and Lauren classification (P>0.05; Fig. 1A–C; Table II).

There were 14 cases with a relative Her-2/neu gene copy number >2 and <4.5, the definite judgment for which was challenging. This range is used to determine whether the Her-2 gene is amplification-positive or negative. A Her-2 gene copy number >4.5 in the DNA sample that will be detected indicates an amplification-positive Her-2 gene and a value <2 an amplification-negative gene. Alternatively in situ hybridization can be used to detect the Her-2 gene without the use of the copy number. These comprised 9 females and 5 males. All cases were TNM stage II–III, including 3 cases of IIa, 5 cases of IIb, 4 cases of IIIa and 2 cases of IIIb. The expression of Her-2 was determined in 13 cases using IHC, and expression of Her-2/neu was determined in 8 cases using FISH (Table III; Fig 2A and B).

Her-2 overexpression was observed in 53 cases (12.86%) using IHC examination, Her-2/neu positive expression was observed in 46 cases via qPCR. In the kappa test, good consistency is indicated when 0.4<κ≤1 and poor consistency is indicated when 0≤κ≤0.4. For the studied population, there was a good consistency of diagnosis between IHC and qPCR (κ=0.828; P<0.001; Table IV).