Abstract
Background: A possible anticancer role for somatostatin has been suggested. This study assessed the presence of somatostatin receptor subtype 2A (SSTR2A) in gastric cancer, correlating its expression with histological type, grade, human epidermal growth factor receptor 2 (HER2) expression, and disease outcome. Materials and Methods: Gastric cancer tissues of 51 consecutive patients were collected for immunohistochemical assessment of both SSTR2A and HER2 expression. Results: SSTR2A expression was observed in 38 (74.5%) cases. Prevalence of SSTR2A expression was significantly higher in well to moderately (G1–2) differentiated gastric cancer as compared to poorly (G3) differentiated tumors (96% vs. 52%; p<0.01), as well as in intestinal- than in diffuse-type cancer (97% vs. 20%; p<0.001). HER2 expression was positive in 18 (35%) patients, and it was associated with SSTR2A expression (r=0.50, p<0.001), being co-expressed in 17 (95%) out of 18 positive cases. RFS was significantly lower in patients with diffuse HER2 expression. Conclusion: This study demonstrated a co-localization between SSTR2A and HER2, potentially opening new therapeutic strategies for patients with gastric cancer.
Although advances in surgical and both adjuvant and neoadjuvant chemotherapy approaches have increased the survival of patients with locoregional gastric cancer, the prognosis of such a disease still remains poor, advocating therapeutic improvement (1, 2).
Several observations would suggest a possible antitumoral role for somatostatin (SST), experimental models showing its growth inhibition of different cancer cell lines (3). Of note, some in vitro studies demonstrated an antitumoral effect of SST analogs, such as octreotide, including towards gastric cancer cells (4-6). In addition, a randomized study found an increased survival following octreotide therapy as compared to supportive care in patients with advanced gastric cancer refractory to chemotherapy (7). The anticancer effect of SST may depend on suppression of both the synthesis and the release of cell growth factors and growth-promoting hormones (8). Moreover, an antiangiogenic effect – due to the inhibition of angiogenic factors such as vascular endothelial growth factor (VEGF) – has been also reported (9). Finally, SST exerts a direct effect on tumor cell growth through cell cycle arrest mediated by several signal transduction pathways, such as the inhibition of mitogen-activated protein kinase (MAPK) signaling transduction (6, 10). Biological effects of SST are mediated by a family of five G-protein coupled receptors (SSTR1 to 5). A wide distribution of SSTRs has been observed in normal tissues, including gastric foveolae. In detail, immunochemistry with chromogranin demonstrated that the SSTR-positive cells in gastric mucosa were not enterochromaffin-like cells, suggesting an action of SST even in non-endocrine cells (11). The presence of SSTRs has been reported in tumor cell lines and human primary tumors (6, 12). Although SSTR subtypes have been extensively studied in gastro-entero-pancreatic endocrine tumors, only a few studies have focused on their distribution in gastric cancer. A study reported SSTR3 protein expression in 62.5% of normal gastric mucosa and in 25% of gastric adenocarcinoma cases (13). Of note, both SSTR2 and SSTR3 genes were detected in gastric cancer cell lines, and 3H-thymidine incorporation in the same cells was significantly reduced by octreotide (6).
The overexpression of human epidermal growth factor receptor 2 (HER2; ERBB2), member of the type I receptor tyrosine kinase (RTK) family, has been reported in gastric adenocarcinoma, being an independent unfavorable prognostic factor in some retrospective studies (14, 15). Moreover a humanized monoclonal antibody against HER2 was found to be promising in therapy of patients with gastric cancer (16). Interestingly, a possible interaction between SSTRs and HER2 in regulating cell proliferation has been reported, potentially opening future therapeutic approaches (17). Therefore, we designed the present study aiming to assess the presence of SSTR2A in gastric cancer, and to correlate its expression with histological type, grade, HER2 expression, and disease outcome.
Materials and Methods
Patients. Consecutive patients with gastric cancer referred to a single institution between 2006 and 2010 were included in the study. There were no selection criteria for inclusion in the study. Tissue samples of gastric cancer were fixed in 10% phosphate-buffered formalin, embedded in paraffin, sectioned serially at a thickness of 4 μm and used for the routine histopathological diagnosis, and SSTR2A and HER2 detection. To better characterize cell types expressing SSTR2A, expression of vesicular monoamine transporter (VMAT2), a marker of the histamine-producing enterochromaffin-like (ECL) cells of the stomach, was also tested in SST2A positive cases (18). The study was conducted according to the informed consensus law in Italy.
Immunohistochemistry. Briefly, cut 4-μm sections were deparaffinized in xylene and then placed in a graded series of ethanol. Sections were reacted with biotinylated antirabbit antibody and streptavidin-biotin-peroxidase (Mack 4, Gramsh, Schwabhausen, Germany, for SSTR2A and AB1767 Chemicon International, Temecula, CA, for VMAT2). Non-specific binding was inhibited by incubation with normal goat serum for 60 min. To enhance antigen retrieval, slides were immersed in citrate buffer [0.01 M sodium citrate (pH 6.0)] and heated in a microwave oven at 600 W (three times for 5 min each). Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol for 30 min. Sections were then incubated with antibodies against: SSTR2A (SS800 P, 1:2000 dilution; Biotrend, Cologne, Germany), VMAT2 (AB1598 P, 1:1000 dilution; Millipore, Billerica, Massachusetts, USA) and HER2 (7269 M, 1:1000 dilution, Dako, Glostrup, Denmark) overnight at room temperature. Immunoreactivity was revealed with the chromogen diaminobenzidine and the sections were counterstained with Mayer's hematoxylin solution. Negative control sections were prepared by substituting the primary antibody with buffered saline. As previously described, a semi-quantitative approach was used for scoring both SSTR2A and HER2 (16, 19). All slides were reviewed by two observers who were unaware of clinical and pathological data.
Statistical analysis. The relationship between the different receptors and tumor and patient characteristics was performed using Fisher's exact test and multiple regression. In order to analyze the correlation between receptor expression and disease stage and depth of intestinal wall invasion, Spearman's test was used. Differences were considered statistically significant at p<0.05. As endpoint for the evaluation of the prognostic value of SSTR2A, HER2, and other clinicopathological characteristics, recurrence-free survival (RFS) was used. RFS was defined as the interval from the date of surgery to the date of disease recurrence or to the last follow-up. RFS was calculated by means of the Kaplan–Meier method, with the log-rank test for differences between groups.
Results
Overall, 51 patients with gastric cancer (male/female: 40/11; median age: 66 years, range: 42-85 years) were enrolled in the study. Surgically removed cancer tissue was available in 48 cases, whilst in the remaining three cases, specimens were obtained at upper endoscopy. Relevant data are shown in Table I. At a median follow-up time of 30 (range 6-68) months, 15 (29.4%) patients experienced disease recurrence, and 10 (19.6%) patients had died. Death was related to gastric cancer relapse in seven patients. The RFS curve according to stage of disease is provided in Figure 1A.
SSTR2A expression. SSTR2A expression was observed in 38 (74.5%) cases, including 34 cases of locoregional and 4 of metastatic disease. Positive cases exhibited mild SSTR2A immunostaining in 20 (53%) cases, moderate in 12 (31%), and marked in 6 (16%). All SSTR2A-positive cases were VMAT2 negative. As shown in Table I, prevalence of SSTR2A expression was significantly higher in well to moderately (G1-2) differentiated gastric cancer as compared to poorly differentiated tumors (96 versus 52%; p<0.01). Moreover, an inverse correlation between tumor grading and SSTR2A score was observed, a high grade being associated with a low SSTR2A staining score (r=−0.55, p<0.001). Overall, SSTR2A expression was detected in 97% of the intestinal-type gastric carcinomas as compared to only 20% of the diffuse-type (p<0.001) (Figure 2). No relationship between SSTR2A expression and RFS was observed.
HER2 expression. Immunostaining for HER2 was positive in 18 (35.3%) patients, with 7 (13.7%) cases which scored 2-3+ and 11 (21.6%) which scored 1+. According to the neoplasia grading, HER2 expression was detected in 60% and 12% of well- and poorly differentiated carcinomas, respectively, the difference being statistically significant (p<0.01). Moreover, an inverse correlation between neoplasia grade and HER2 score was observed, a lower grade being associated with a higher HER2 staining score (r=−0.42, p=0.002). HER2 expression was more frequently detected in intestinal- as compared to diffuse-type gastric cancer (45% vs. 13%, p=0.05), and it was associated with SSTR2A expression (r=0.50, p<0.001), being co-expressed in 17 out of 18 HER2-positive cases. No relationship between HER2 expression and other clinicopathological features was found. As far as survival is concerned, the RFS was significantly lower in patients with diffuse than in those with mild HER2 expression (Figure 1B).
Multivariate analysis. At multivariate analysis, only intestinal-type of cancer was significantly associated with presence of SSTR2A expression (p<0.001; odds ratio, OR=140, 95% confidence interval, CI=13-1477). By considering HER2 as a dependent variable, the model found that its expression was significantly associated with gastric cancer recurrence (p=0.018; OR=8.5, 95% CI=4-50). By considering gastric cancer recurrence as a dependent variable, both presence of HER2 (p=0.028; OR=14.5, 95% CI=1.3-160) and neoplasia staging >II (p=0.016; OR=20.5, 95% CI=1.7-245) were identified as independent predictive factors.
Discussion
SSTR2 has been widely documented not only in nervous and neuroendocrine tumors, but also in renal cell carcinoma, breast cancer, hepatocellular carcinoma, and lymphoma (20, 21). Of note, the presence of such an SSTR has also been detected in gastric cancer, both in vitro and in vivo (6, 13). In addition, the apoptotic rate of gastric cancer cells was significantly increased in a series of patients treated with octreotide and celecoxib (22). These observations could open the window to a new therapeutic strategy for gastric cancer, suggesting the potential use of drugs able to block SSTRs.
The present study demonstrated SSTR2A presence in as many as 74.5% of gastric carcinomas, a strong expression (moderate to marked) being detected in half of the positive cases. Moreover, we found that the SSTR2A expression was highly prevalent in well to moderately (G1-2) differentiated tumors, and in the intestinal-type gastric carcinomas. Our data is in agreement with that of a previous study showing a lower SSTR3 expression in poorly-differentiated gastric adenocarcinoma as compared to the well-differentiated type (5% vs. 20%) (13). In another study, SSTRs were identified in 67% of gastric carcinomas with a slightly higher proportion of positivity in well-differentiated tumors (23). Interestingly, a study reported that SSTR-positive breast tumors were less aggressive and patients with such tumors had a better RFS (24). To our knowledge, data about the outcome of gastric cancer patients in relation to SSTR2A are still lacking. Therefore, we are the first to report no relationship between SSTR2A expression and RFS in gastric adenocarcinoma.
A potential interaction between SSTRs and HER2 in regulating cell proliferation has been reported (17). Indeed, G-protein-coupled receptors, as well as RTKs constitute prominent families of cell-surface proteins implied in environmental signal transduction (25). It has been hypothesized that SSTRs would reverse the effects of ERBBs related to MAPK activation, and the consequent cell proliferation (26, 27). HER2 overexpression was reported in about 20% of gastric carcinomas (14-16), the positivity being more frequent in those located in the cardia and in the intestinal-type of gastric cancer (14, 15). Our data confirmed the higher prevalence of HER2 expression in the intestinal-type and in more differentiated tumors (28). Of note, we found a significant direct correlation between SSTR2A and HER2 expression, while HER2 co-localized with SSTR2A in almost all gastric cancer specimens. To our knowledge, a co-localization of SSTRs and ERBBs has been reported in breast cancer cell lines (29), but such a finding has not been previously reported in gastric cancer. Therefore, targeted therapies simultaneously blocking both SSTR (e.g. octreotide), and ERBB function (e.g. trastuzumab) could potentially improve therapeutic management of patients with gastric cancer.
SSTR2A expression according to the clinicopathological characteristics.
Data on the prognostic role of HER2 in gastric cancer are conflicting. Indeed, some studies demonstrated an association with worse survival (14, 15, 30) whilst another failed to confirm such an observation (31). In our study, a better RFS for HER2-negative cases (score 0 or 1+) as compared to the positive (2 or 3+) cases of gastric cancer was observed. Therefore, HER2 overexpression appeared to be associated with a worse prognosis, despite its significant association with more favorable histological categories, such as low grade and intestinal-type tumors.
In conclusion, this study demonstrated the presence of SST2A receptor in the majority of gastric carcinomas, particularly in well-differentiated and intestinal-type tumors. Co-localization of SSTR2A and HER2 expression was also documented. These observations, coupled with the potential interaction between the two factors in disturbing tumoral cell proliferation, constitute the rationale for considering a new therapeutic strategy for patients with gastric cancer prompting a clinical trial.
Kaplan-Meier curves depicting recurrence free survival (RFS) according to the stage of disease (A) (HR=1.96; 95% C.I.=1.23-3.12; p=0.003) and HER2 expression: score 0-1+ vs. 2-3+ (B) (HR=3.6; 95% C.I.=1.19-10.84; p=0.022).
SSTR2A expression in gastric cancer according to Lauren classification. A: A diffuse-type SSTR2A-positive case; B: an intestinal-type SSTR2A-positive case.
Acknowledgements
The Authors thank Dr. Federica Romei for reviewing the statistical analysis of the study.
- Received October 31, 2011.
- Revision received December 4, 2011.
- Accepted December 6, 2011.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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