Prognostic importance of PTEN, EGFR, HER-2, and IGF-1R in gastric cancer patients treated with postoperative chemoradiation

Background/aim This study aimed to describe the prognostic importance of epidermal growth factor (EGFR), phosphatase and tensin homolog (PTEN), human EGF receptor-2 (HER-2), and insulin-like growth factor 1 receptor (IGF-1R) in gastric cancer patients treated with postoperative chemoradiation therapy. Materials and methods Sixty-nine patients treated with adjuvant chemoradiation therapy were retrospectively evaluated. Tumor samples were stained immunohistochemically. Results All patients were treated with 3D conformal radiation therapy with concomitant and adjuvant chemotherapy. Perineural invasion (PNI) (P = 0.042), prechemoradiation therapy albumin levels below 3.5 mg/dL (P = 0.011), and EGFR positivity (P = 0.008) had negative effects on overall survival (OS). The median OS was 26 months for patients with PNI (+), 34.9 months for those with PNI (–), 19.5 months for those with albumin levels below 3.5 mg/dL, and 33.2 months for those with albumin levels above 3.5 mg/dL. IGF-1R (+) (P = 0.035) and history of cigarette smoking (P = 0.033) were observed to have a statistically significantly negative effect on disease-free survival (DFS). The median DFS was 29.2 months for IGF-1R (+) patients, 37.9 months for those with IGF-1R (-), and 26.3 and 40.59 months for smokers and nonsmokers, respectively. Conclusion IGF-1R and EGFR may be used for patient selection in future prospective studies that evaluate the prognostic importance of these receptors.

clinical role of EGFR in gastric cancer is not clear. HER-2 is a transmembrane TK receptor [8,9]. Intestinal-type and well-differentiated gastric cancers have a higher rate of HER-2 positivity than poorly differentiated and diffusedtype gastric cancers [7]. The overexpression of HER-2 and EGFR is associated with poor prognosis and has been reported in approximately 30%-50% of gastric cancers [10,11]. PTEN is a tumor-suppressor gene localized on chromosome 10 [12]. Studies have reported that inactivation of the PTEN gene is closely associated with the progression and incidence of gastric cancer [13]. The PTEN protein affects apoptosis, migration, metabolism, transcription, and translation and also inhabits cellular invasion and metastasis [14]. Due to genetic modification, PTEN gene inactivation might be observed in endometrial carcinoma, glioblastoma multiforme, prostate, skin, and breast cancers [3]. IGF-1R is a membrane cell receptor that is frequently overexpressed in human cancers [11]. IGF-1R has a role in cellular proliferation, malignant transformation, differentiation, and prevention of apoptosis [15]. There is a relationship between IGF-1R and survival in breast cancer and solid tumors, but IGF-1R expression in gastric cancer is poorly understood. IGF-1R expression is a predictor for poor results [11]. The overexpression of IGF-1 was distinct in large tumors with high mitotic activity, and it was correlated with high risk of malignancy. The overexpression of IGF-1R was correlated with the overexpression of IGF, high mitotic activity, and high risk of malignancy [16].
The present study was designed to define the prognostic importance of HER-2, IGF-1R, PTEN, and EGFR in gastric cancer patients treated with postoperative chemoradiation therapy.

Materials and methods
The hospital records of 69 patients with nonmetastatic gastric cancer were retrospectively evaluated between January 2010 and October 2013. The study was approved by the Ethics Committee of Necmettin Erbakan University. Paraffin-embedded tumor samples were immunohistochemically stained for PTEN, EGFR, HER-2, and IGF-1R. All patients were treated with 3D conformal radiation therapy with concomitant and adjuvant chemotherapy. Formalin-fixed paraffin-embedded (FFPE) tumor samples were immunohistochemically stained for PTEN, EGFR, HER-2, and IGF-1R. Sections of tumor blocks of 4 µm were stained with hematoxylin and eosin, and the samples were evaluated by a pathologist. All reagents and equipment for immunohistochemistry (IHC) were manufactured by Ventana Medical Systems (Tucson, AZ, USA). FFPE sections were stained automatically with Ventana Benchmark XD by using primary antibodies directed against EGFR, PTEN, HER-2, and IGF-1R.
A scoring system based on the variability of staining intensity was used. Brown membranous staining was considered positive for HER-2. The results were evaluated as follows: 0, no staining or membranous reactivity in <10% of cells; (+), faintly to barely perceptible membranous reactivity in >10% of cells; (++), basolateral membranous staining of weak to moderate intensity in >10% of cells; and (+++), basolateral membranous staining of strong intensity in >10% of cells [9]. EGFR and IGF-1R tumor cell membrane immunostaining was scored as 0, 1+ , 2+, or 3+. The results were evaluated as follows: 0, no membranous reactivity within the tumor; 1+, 2+, or 3+, depending on the intensity above the background level [1,15]. For EGFR, IGF-1R, and HER-2, an IHC score of 3+ was defined as positive. The intensity of staining for PTEN was scored using a 0 to 3 grading system: 0, negative; 1, weak; 2, moderate; and 3, strong. The level of PTEN staining was evaluated by calculating the immunoreactive score (IRS) from the staining intensity and the percentage of PTEN-positive cells [17]. IRS = 0 and IRS = 2-3 were considered as negative and positive, respectively. The total PTEN expression was calculated as cytoplasmic PTEN expression + nuclear PTEN expression.

Statistical analysis
Statistical analysis was performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Primary and secondary outcomes were OS and disease-free survival (DFS). The time from diagnosis to the date of the patient's death or last followup was defined as OS. The time from diagnosis to the date of the documented progression, distant metastases, or recurrence was defined as DFS. Survival was calculated with the Kaplan-Meier method and a log-rank test. Fisher's exact test was used to analyze HER-2, IGF-1R, PTEN, and EGFR (positive or negative) and univariate correlations between clinicopathological variations. Multivariate analysis of the data was performed using the Cox proportional hazards model. P < 0.05 was accepted as statistically significant.
Perineural invasion (PNI) (log-rank; P = 0.042), prechemoradiation therapy albumin level below 3.5 mg/ dL, and EGFR positivity were found to have negative effects on OS. The median OS was 26 months for patients with PNI (+), 34.9 months for those with PNI (-), 19.5 months for those with albumin levels of <3.5 mg/dL, and 33.2 months for those with albumin levels of >3.5 mg/dL. OS was 16.2 and 32.1 months in EGFR (+) and EGFR (-) patients, respectively (P = 0.008) (Figure 3).

Discussion
In our study, 24 of 69 cases (34.8%) of gastric cancer were positive for IGF-1R expression. We demonstrated that IGF-1R expression was correlated with prognosis and OS in gastric cancer. IGF-1R positivity had a prognostic effect on DFS in both univariate and multivariate analysis. The positive EGFR expression rate was 14.5% in gastric cancer tissue. EGFR positivity had a negative impact on OS. The   median OS rates were 16.2 and 32.1 months in EGFR (+) and EGFR (-) patients, respectively (P = 0.008). In multivariate analysis, EGFR positivity had a negative effect on DFS (P = 0.038). In our study, HER-2 and PTEN were not prognostic factors in gastric cancer patients. The correlation between IGF-1R expression and outcome was shown for breast and other solid tumors. However, the role of IGF-1R expression is not clearly defined in gastric cancers. Matsubara et al. showed that IGF-1R expression was a significant independent predictor of poor outcomes. However, EGFR and HER-2 were not related to OS [11,18].
EGFR is the most important member of the growth factor receptor family. EGFR expression was found to be related to survival in gastric cancer patients [6,11,18,19]. Atmaca et al. reported similar median OS in patients with positive EGFR compared to those with negative tumors [6]. Zhang et al. detected a positive correlation between high EGFR expression and cetuximab response in gastric cancer [20]. Cetuximab, lapatinib, and panitumumab are anti-EGFR inhibitors. The EXPAND study, a randomized phase 3 trial, compared capecitabine plus cisplatin alone versus cisplatin + capecitabine + cetuximab therapy. The median progression-free survival (PFS) and OS were 5.6 and 10.7 months, respectively, in the capecitabine plus cisplatin group compared to 4.4 and 9.4 months in patients receiving capecitabine plus cisplatin and cetuximab (P = 0.315 and P = 0.954). The addition of cetuximab to capecitabine + cisplatin provided no additional benefit to the first-line treatment of advanced gastric cancer [21].
The REAL-3 study, a randomized phase 3 trial, compared panitumumab plus chemotherapy with chemotherapy in 553 patients with advanced esophageal and gastric cancer. However, the survival rate in the  combination arm was lower than that in the chemotherapyalone arm (PFS, 6.0 months vs. 7.4 months, P = 0.068; OS, 8.8 months vs. 11.3 months, P = 0.013) [22].
The overexpression of HER-2 occurs in 10%-30% of gastric cancers [23]. While HER-2 overexpression is associated with aggressive tumor biology in breast cancer [24], its prognostic importance in gastric cancer is less clear. It was suggested in early studies of gastric cancer that HER-2 had a negative effect on prognosis [9]. However, later studies did not find any prognostic effect of HER-2 [1]. Trastuzumab (a monoclonal antibody targeted against HER-2) causes antibody-dependent cellular cytotoxicity and inhibits HER-2 receptor signalization [9]. Trastuzumab is a standard therapy for early-stage and metastatic HER-2-positive breast cancers [9]. In the ToGA study, the OS for HER-2-positive gastric cancers was significantly improved when trastuzumab was used in combination with chemotherapy rather than chemotherapy alone. The overall HER-2 positivity rate was 22.1%, and the median OS was significantly improved in the trastuzumab group compared to the chemotherapy control group (13.8 vs. 11.1 months, P = 0.0046) [10]. Trastuzumab plus chemotherapy was indicated as a novel standard in HER-2-positive gastric and gastroesophageal junction tumors in the ToGA study. Masorini et al. showed positivity of EGFR and HER-2 in 75 (9%) and 113 (13.6%) patients, respectively. The overall and relapse-free survival rates were significantly lower in EGFRpositive patients compared to EGFR-negative patients, regardless of HER-2 status. EGFR positivity was associated with poor outcomes in multivariate analysis [1].
PTEN gene mutation is rarely seen in gastric cancer. However, loss of heterozygosity (LOH) is rather common [13]. The LOH rate is significantly higher in advanced gastric cancer than early-stage gastric cancer (63% and 18%, respectively) and is also significantly higher in poorly differentiated tumors than well-differentiated gastric tumors (69% and 29%, respectively) [13]. Zhu et al. stated in their study that the loss of cytoplasmic PTEN expression in gastric carcinoma was more common in comparison to the adjacent nonneoplastic tissues. The loss of cytoplasmic PTEN was correlated with histological stage. Total loss of PTEN expression or nuclear loss of PTEN was correlated with tumor grade. PTEN expression was not significantly related to the 3-year survival and OS rates [25]. Li et al. showed that the loss of cytoplasmic PTEN expression was associated with distant metastasis and advanced clinical stage in gastric cancer patients [14].
Cigarette smoking and alcohol consumption are considered potential risk factors for gastric cancer. The mechanism of how tobacco smoking increases the risk of gastric cancer is not well understood. The risk of gastric cancer development is 1.45 times higher in smokers than in nonsmokers [26]. Han et al. found an association between cigarette smoking after surgery and poor survival and cancer recurrence. However, they did not indicate a relationship between alcohol consumption and survival [27]. In certain studies, alcohol consumption was found to be significantly associated with poor outcomes in gastric cancer patients [28]. Cigarette smoking was an independent risk factor for DFS, but alcohol consumption was not associated with survival in our study.
PNI is a poor prognostic factor in certain cancers, such as head/neck and prostate cancers [29]. It is also correlated with an aggressive tumor genotype [17]. The prognostic significance of PNI in gastric cancer has been investigated in several studies. Duroker et al. stated that PNI did not add any additional information to classic prognostic factors [30]. Bilici et al. showed that PNI (-) patients had longer survival than PNI (+) patients, and PNI was found to be a prognostic factor for the curative treatment of patients with gastric cancer [31]. In the study of Deng et al., PNI positivity was shown to be an independent prognostic factor affecting OS and DFS of patients with gastric cancer who underwent curative resection [17]. In our study, there was a significant difference between PNI (+) and PNI (-) patients in terms of OS. The median OS was 26.07 and 34.9 months in PNI (+) and PNI (-) patients, respectively (P = 0.042, log-rank).
The statistical power of our study is weak because distant metastasis developed in only 12 patients. The power of HER-2 as a prognostic factor was also weak, as HER-2 positivity was only seen in nine of 69 patients. In conclusion, this study suggests that IGF-1R, EGFR, and cigarette smoking can be used as additional prognostic factors to the previously known prognostic factors in gastric cancer patients treated with chemoradiation therapy. IGF-1R and EGFR may be used for patient selection in future prospective studies that evaluate the prognostic importance of these receptors.