Expression of HER2/c-erbB-2, EGFR protein in gastric carcinoma and its clinical significance

2.1 Patients

A total of 67 patients who suffered from gastric cancer and underwent general surgery from February 2012 to March 2016 in the People’s Hospital of Lishui, Zhejiang, China, were retrospectively analyzed. All patients had confirmed pathology diagnosis of gastric cancer and completed survival data. Gastric benign carcinoma and gastric cancer without enough survival data were excluded from the study. The immunohistochemical assay was performed to examine HER2/c-erbB-2 and EGFR protein expression in surgically resected cancerous tissues and paired normal gastric tissues in 67 patients with gastric cancer. The average age of the included 67 patients with gastric cancer was 62.3 ± 15.4 years (rang 43~79) with 41 males and 26 females. 32 patients were in I/II clinical stages, and other 35 were in the III/IV clinical stages. 17 cases were well/moderate differentiated adenocarcinomas, 50 cases were poor differentiated adenocarcinomas, 48 cases had regional lymph node metastases, and 19 cases without regional lymph node metastases.

Informed consent: Informed consent has been obtained from all individuals included in this study.

Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by Medical Ethics Committee of Lishui Peoples’ Hospital of Zhejiang Province.

2.2 Immunohistochemical assay

Immunohistochemical assay was performed by the EnVision two-step method. The operation was carried out in accordance with laboratory procedures and instruction of the kit. Dewaxing and hydration were routinely performed, followed by 3% hydrogen peroxide application for 8 min to remove endogenous enzyme activity, citrate high-pressure antigen retrieval, and reagent A application at room temperature for 15 min. Primary antibody incubation was conducted overnight at 4 °C, and secondary antibody incubation was performed at 37 °C for 20 min. Reagent C incubation was implemented at 37 °C for 10 min. DAB was used for coloring and hematoxylin counterstaining.

2.3 Positive standards

The HER2/c-erbB-2, EGFR positive cells had mainly brownish yellow particles in the membrane and cytoplasm. The positive expression score of HER2/c-erbB-2 was follows: positive cells <5% was (−), 5%–25% was (+), 25%–50% was (++), and positive cells >50% was (+++).

For EGFR positive expression, the cells were mainly divided into 0 to 3 levels as follows: (i) Level 0, no brown-yellow particle-like precipitation observed in the cell membrane and the cytoplasm; (ii) level 1, the cell membrane and the cytoplasm showing a brown-yellow particle-like precipitation phenomenon with the percentage of positive cells was 10% or less recorded as “+”; (ii) level 2, cell membrane and cytoplasm exhibiting a brown-yellow particle-like precipitation phenomenon with the percentage of positive cells 10%–50%, recorded as “++;” (iv) level 3, the cell membrane and the cytoplasm displaying a brown-yellow particle-like precipitation phenomenon with the percentage of positive cells above 50% recorded as “+++.”

2.4 Statistical analysis

The statistical analysis was performed by STATA11.0 software. The measurement data were expressed with x̄ ± s and the comparison between groups was made by student-t test of the sample mean. The enumeration data were expressed with number and compared by chi-square test. The survival data was expressed with the median and compared by log-rank test. P<0.05 meant a statistical difference.

3.1 HER2/c-erbB-2 expression

HER2/c-erbB-2 protein mainly expressed in the cell membrane of gastric cancer cells with brownish yellow particles, (Figure 1). EGFR was mainly expressed in the membrane and cytoplasm of gastric cancer cells with uniform distribution of brown and yellow granules, (Figure 2). The positive rates of HER2/c-erbB-2 in the cancer and paired normal gastric tissues were 32.8% (22/67) and 4.5% (3/67), respectively with statistical difference (p<0.05). And the positive rates of EGFR in cancer and paired normal gastric tissues were 41.8% (28/67) and 5.9 (4/67), respectively with statistical difference (p<0.05). The HER2/c-erbB-2 and EGFR positive expression rate in the cancer tissues were significantly higher than that of normal gastric tissues.

Figure 1

HER2/c-erbB-2 expression in gastric cancer and normal gastric tissue (A: HER2/c-erbB-2 positive expression in gastric cancer tissue; B: HER2/c-erbB-2 negative expression in gastric cancer tissue; C: HER2/c-erbB-2 positive expression in normal gastric tissue; D: HER2/c-erbB-2 negative expression in normal gastric tissue).

Figure 2

EGFR expression in gastric cancer and normal gastric tissue (A: EGFR positive expression in gastric cancer tissue; B: EGFR positive expression in normal gastric tissue).

3.2 Correlation between HER2/c-erbB-2expression and patients’ clinical features

HER2/c-erbB-2 positive expression in cancer tissue was significant correlated with the pathology grading (p<0.05), tumor invasion depth (p<0.05) and local regional lymph node metastasis (p<0.05); However, HER2/c-erbB-2 expression was not correlated with the patients age, gender, tumor location and tumor diameter (p>0.05), Table 1.

3.3 Correlation between EGFR expression and patients’ clinical features

EGFR positive expression in cancer tissue was significant correlated with the tumor invasion depth (p<0.05) and local regional lymph node metastasis (p<0.05), but was not correlated with the patients age, gender, tumor location, tumor diameter and pathology grading, (p>0.05), Table 2.

3.4 HER2/c-erbB-2 expression and prognosis

The median survival time of were 13.14 and 23.6 months respectively for HER2/c-erbB-2 positive and negative expression groups respectively. The median survival time in positive group was significant lower than that of negative group with statistical difference (HR=2.26, 9%CI:1.06-4.80, p<0.05), Figure 3.

Figure 3

Survival curve of patients in HER2/c-erbB-2 positive and negative groups

3.5 EGFR expression and prognosis

The median survival time of were 15.47 and 22.87 months respectively for EGFR positive and negative expression groups respectively without statistical difference (HR=1.78, 9%CI:0.96-3.29, p>0.05), Figure 2.