Abstract
Background: Fibroblast growth factors and their receptors regulate key cellular functions, such as proliferation, differentiation and survival. Herein, we studied the prevalence and prognostic role of fibroblast growth factor receptor 2 (FGFR2) amplification in patients with advanced gastric cancer (AGC) who received systemic chemotherapy. Patients and Methods: The gene copy number of FGFR2 was investigated in 80 patients with AGC who received systemic chemotherapy. FGFR2 gene status was assessed by dual-color fluorescence in-situ hybridization. Results: Among 80 patients, FGFR2 amplification was observed in seven cases (11.5%). Patients with FGFR2 amplification had significantly shorter overall survival (OS) than did those without FGFR2 amplification (9.1 vs. 16.5 months; p=0.037). In multivariate analysis, disease status and number of metastatic sites were associated with worse OS (p=0.015 and p=0.009, respectively). FGFR2 amplification tended to be correlated with a poorer outcome (p=0.080). Conclusion: FGFR2 amplification tended to result in a shorter survival period compared to cases without amplification.
Worldwide, gastric cancer is the fourth most common malignant disease in men and the fifth in women. Gastric cancer used to be the leading cause of cancer death in the world until the 1980s when it was overtaken by lung cancer (1). In Japan, gastric cancer is one of the three most frequent types of cancer. Although its incidence is declining rapidly, it still has the second highest mortality rate (2).
Systemic chemotherapy is of crucial importance in patients with advanced gastric cancer (AGC), in order to obtain palliation of symptoms and improved survival. Development of new-generation cytotoxic agents, including irinotecan, S-1, capecitabine, paclitaxel, docetaxel, and oxaliplatin, has been made intensively over the past decades; however, the prognosis for patients with AGC remains poor, with median survival times of 10-13 months (3, 4).
Fibroblast growth factor receptors (FGFR1 to -4) are transmembrane tyrosine kinase receptors (5). Fibroblast growth factors (FGFs) bind to FGFR, which subsequently undergoes phosphorylation at intracellular tyrosine residues, leading to the activation of downstream signaling. Signaling through this pathway plays crucial roles in regulating cellular proliferation, survival, migration and differentiation in several types of malignancies (6). There is a well-known association with dysregulation of the FGFR signaling pathway due to gene mutation, gene amplification, receptor overexpression, or aberrant transcriptional regulation. Activating mutations of the FGFR gene have been identified in many cancer types (7-9). Mutations of FGFR2 have been described in 12% of endometrial carcinoma (8), and that of FGFR4 in 8% of rhabdomyosarcoma (10). Among bladder cancer types, non-muscle-invasive bladder cancer has the best established link to FGFR3 mutations, but this abnormality has been identified in fewer than 20% of invasive high-grade bladder cancer (11). In contrast to activation by mutation, amplification of FGFR3 has been described only rarely in cancer. Conversely, amplifications of both FGFR1 and FGFR2 are more common. Approximately 5%-10% of gastric cancer cases show FGFR2 amplification, which is associated with poor-prognosis diffuse-type cancer (12, 13). FGFR2 protein overexpression was observed in about 30% of gastric carcinomas and was positively correlated with scirrhous cancer, a diffuse type, invasion depth, infiltration type and a poor prognosis (14).
Accordingly, the FGFR2 pathway has attracted considerable interest as an oncogenic pathway and as a target for the development of therapeutic agents (15). Although several studies have evaluated the status of FGFR2 amplification in resectable gastric cancer, no previous study has addressed the issue in patients with AGC who received chemotherapy. Therefore, we retrospectively evaluated the prevalence and prognostic role of FGFR2 amplification in patients with AGC who received systemic chemotherapy.
Patients and Methods
Patients. Patients with AGC who received systemic chemotherapy between January 2005 and December 2013 at the National Cancer Center Hospital were selected from our database.
The eligibility criteria for this study were as follows: received systemic chemotherapy; diagnosed with adenocarcinoma; Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0-2; age ≥18 years; adequate bone marrow, hepatic and renal functions; and provision of informed consent for tissue sample use for analysis. Patients who developed recurrence within 6 months after adjuvant S-1 chemotherapy was finished were excluded from the analysis.
The following clinical characteristics of patients were reviewed: Age, ECOG PS, endoscopic findings, primary site, tumor histology, history of gastrectomy, metastatic sites, presence of target lesion according to the response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (16), serum carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) levels, and chemotherapy regimen. This study was approved by the Institutional Review Board of the National Cancer Center, Tokyo, Japan (approval number 2012-221).
Fluorescence in situ hybridization analysis. Tumor specimens were taken by endoscopic biopsy or from resected primary lesions and the fluorescence in situ hybridization (FISH) method was as previously described (17). Tissue sections, 3- to 4-μm thick, and the FGFR2/CEP10 probes were subjected to pretreatment at 82°C for 5 minutes, and then hybridized at 45°C for 20 hours. Following this hybridization step, excess and unbound probes were removed with post-hybridization wash buffer, and nuclei were counterstained with 4’,6 diamidino-2-phenylindole (DAPI). Signals were enumerated in 50 tumor nuclei per each tissue section, using a fluorescence microscope with single-interference filter sets for green (FITC), red (Texas red), and blue (DAPI), as well as dual (red/green) band-pass filters. Amplification of FGFR2 was defined as a FGFR2: chromosome 10 ratio of >2, or tight FGFR2 gene clusters in >10% of the nuclei analyzed per tissue section.
Immunohistochemical staining. For human epidermal growth factor receptor type 2 (HER2) staining, a Hercep Test kit (Dako, Glostrup, Denmark) was used following the manufacturer's instructions. For staining analysis, the tumor specimens were scored according to modifications of criteria originally published by Hofmann and colleagues (18).
Statistical analysis. Differences between categorical variables were assessed using the Fisher's exact tests and the Mann–Whitney test. The tumor response was assessed according to RECIST (version 1.1) (16). The response rates were calculated without confirmation. Progression-free survival (PFS) was defined as the time from initiation of chemotherapy until detection of progression. Deaths of patients who died without evidence of a recurrence were treated as events. Patients who were lost to follow-up were treated as censored observations. The overall survival (OS) period was defined as the time from initiation of chemotherapy until the date of death or the most recent follow-up. Patients who were lost to follow-up were treated as censored cases. Median PFS and median survival time (MST) were calculated using the Kaplan-Meier method, and significance was determined using the log-rank test. For univariate and multivariate analyses, the Cox proportional regression model was used. All calculations were performed using SPSS version 17 (SPSS Inc., Chicago, IL, USA)
Results
Patients' characteristics. From January 2005 to December 2013, a total of 80 patients with AGC were enrolled in this study at our Institution. FISH was performed on the 80 patient tumor tissue samples. Among them, FGFR2 gene amplification status could not be identified in 19 patients because of insufficient quantity of tumor tissue. Most tumor specimens were taken by endoscopic biopsy. Thus, the other 61 patients were evaluated in detail.
The baseline characteristics of the 61 patients are summarized in Table I. Chemotherapy regimens were individually selected by the attending physician. The first-line treatment regimens consisted of the following: methotrexate plus 5-fluorouracil (5-FU) (n=4), cisplatin plus 5-FU (n=1), S-1 (n=1), S-1 plus cisplatin (SP) (n=37), SP plus trastuzumab (n=1), capecitabine plus cisplatin (XP) (n=2), XP plus trastuzumab (n=2), S-1 plus oxaliplatin (SOX) (n=3), docetaxel plus SP (DCS) (n=9), and irinotecan plus cisplatin (n=1). SOX and DCS regimens were frequently used in clinical trials at our Hospital. Among the 39 patients assessable for treatment response, there were 22 partial responses, providing an overall response rate of 56.4% [95% confidence interval (CI)=39.6-72.2%]. A total of 50 (82.0%) patients received second-line chemotherapy.
FGFR2 FISH results. To assess FGFR2 gene amplification status in gastric adenocarcinoma tissues, FISH was performed on the 80 patient tumor tissue samples. FGFR2 gene amplification was observed in seven cases (11.5%).
HER2 expression results. HER2 expression was assessed by immunohistochemical staining of 51 specimens of primary tumors. HER2 staining intensity was 3+ in seven cases (13.7%), 2+ in eight cases (15.7%), 1+ in five cases (9.8%), and negative in 31 cases (60.8%). All patients with HER2+ were characterized as HER2 non-amplification. From these results, the HER2-positivity rate was 13.7%.
Clinicopathological findings and treatment outcome in patients with FGFR2 amplification. The clinicopathological findings, including age, sex, location, histology, HER2 status, and treatment outcome, are summarized in Table II. Among the patients with FGFR2 amplification, three cases were intestinal-type gastric cancer, while the others were diffuse-type. None of the cases had both FGFR2 amplification and HER2 positivity. Five patients [SP (n=3), SOX (n=1), and DCS (n=1)] responded to the treatment (partial response), and another patient had stable disease.
FGFR2 amplification and PFS. The median PFS was 6.0 months and 7.1 months, respectively, in the patients with and without FGFR2 amplification (p=0.309; Figure 1).
The results of the univariate and multivariate analyses are summarized in Table III. Univariate analysis demonstrated a significantly shorter PFS in patients in two categories related to disease status, and number of metastatic sites. Multivariate analyses revealed that stage IV disease [hazard ratio (HR)=2.64; 95% CI=1.36-5.15 for stage IV vs. recurrent] was associated with worse PFS. There was no clear relationship between FGFR2 amplification and median duration of PFS.
Association between FGFR2 amplification and OS. The median OS among patients with FGFR2 amplification (9.1 months) was significantly shorter than that among those without amplification (16.5 months) (p=0.037; Figure 2). The results of the univariate and multivariate analyses are summarized in Table IV. The univariate analysis demonstrated a significantly shorter survival in patients in four categories related to disease status, number of metastatic sites, target lesion, and FGFR2 gene status. Multivariate analyses demonstrated that stage IV disease (HR=2.47; 95% CI=1.19-5.12 vs. recurrent), and two or more metastatic sites (HR=F2.82; 95% CI=1.30-6.15 vs. <2) were associated with a poor prognosis. FGFR2 amplification tended to be correlated with a poorer outcome (p=0.080).
Discussion
In this study, we analyzed the prevalence and prognostic role of FGFR2 amplification in patients with AGC who received systemic chemotherapy. FISH analysis showed that FGFR2 amplification in AGC was present in seven cases (11.5%), and that FGFR2 amplification tended to be correlated with shorter OS, indicating that this characteristic could serve as a prognostic factor for patients who receive systemic chemotherapy.
In gastric cancer, the reported incidence of FGFR2 overexpression varies widely. FGFR2 overexpression by immunohistochemical staining have been reported in 31-40% of cases (14, 20, 21). On the other hand, amplifications of FGFR2 have been described in approximately 5-10% of cases (22, 23). In this study, we found that the frequency of FGFR2 amplification in primary tumors was slightly higher than that found in the previous report. In our previous analysis, we reported that FGFR2 amplification was observed in patients with gastric cancer who had undergone surgery at a frequency of about 4.1%, using a real-time PCR-based copy number assay (12). One of the reasons for this discrepancy may be the different methods used for detecting FGFR2 gene amplification (a real-time PCR-based copy number assay vs. FISH).
Previous studies have analyzed the association between FGFR2 amplification in gastric cancer and histological sub-type. It has been reported that FGFR2 amplification occurs more frequently in diffuse-type gastric cancer (24). However, in our analysis, there was no association of FGFR2 amplification with histological subtype, which is in agreement with findings recently reported in two studies (25, 26). The reason for the lack of the association between FGFR2 amplification and histological type may be our insufficient number of cases with FGFR2 amplification. Additional large population studies are needed in order to define this association in gastric cancer.
Nagatsuma et al. reported that proteins related to tyrosine kinase receptors, such as HER2 and FGFR2, were overexpressed simultaneously in 22.7% of patients with gastric cancer when examined using immunohistochemical staining (20). On the other hand, Deng et al. found that gene amplifications of related tyrosine kinase receptors were usually mutually exclusive (13). A related retrospective study reported on the association between HER2 and FGFR2 amplification in 961 patients with resectable gastric cancer (26). That study revealed 5.6% of cases with FGFR2 amplification (54 out of 961 patients), but only three cases with amplification of both FGFR2 and HER2. These results agree with those of a previous study conducted on resectable gastric cancer tissue (25). Recently, Liu et al. analyzed the correlation between HER2 positivity and amplification of FGFR2 in 172 Chinese patients with resectable gastric cancer (27). Their HER2-positivity criteria were the same as those we used in the present study. Their results suggested that 13.4% of patients were HER2-positive (23 out of 172 patients) and that 5.2% had FGFR2 amplification (9 out of 172 patients). Among HER2-positive patients, there was no overlap between HER2 positivity and FGFR2 amplification. Our data also showed that there was no overlap between HER2 positivity and FGFR2 amplification, thus confirming previous results.
Several potential prognostic factors for short survival time in first-line chemotherapy of AGC have been proposed. These include PS >2, presence of liver, peritoneal or bone metastasis, and increased number of metastatic sites (28, 29). Recently, several studies reported that FGFR2 amplification is a molecular factor that is related to poor prognosis in patients with resectable gastric cancer (12, 25, 26). However, for the first-line chemotherapy of AGC, it remains unclear how FGFR2 gene status affects survival. In the present study, we demonstrated that FGFR2 amplification tended to be correlated with poorer outcomes. Furthermore, we examined the association between FGFR2 amplification and PFS, and found that there was no relation between FGFR2 amplification and median duration of PFS. To our knowledge, this report is the first to investigate the association between FGFR2 amplification and prognosis for PFS and OS in an AGC population receiving chemotherapy. Nevertheless, some limitations must be taken into account in interpreting our findings. These include the comparatively low frequency of FGFR2 amplification, the difference in first-line chemotherapy regimens, and insufficient sample size in our study. Moreover, there is a selection bias that the patients who had available tissue samples were enrolled into this retrospective study. Further research on a large population is needed in order to obtain more detailed information.
In the present analysis, no differences in median PFS time were observed between cases with FGFR2 amplification and those without. Furthermore, no statistically significant differences were seen between the two groups in the proportion of patients receiving second-line chemotherapy. Therefore, one of the reasons for the difference in OS between the two groups may be the presence of a malignant phenotype of FGFR2 amplification that has acquired ligand independency. In addition, the amplification may be associated with alterations in quality and quantity of downstream signaling.
In conclusion, the frequency of FGFR2 amplification in this study was 11.5%, which was slightly higher than in the previous report. For patients with AGC who received systemic chemotherapy, FGFR2 amplification tended to result in a shorter survival period compared to cases without amplification.
Acknowledgements
We thank Kumiko Hirayama for helping with collecting and organizing the clinical data.
Footnotes
Conflicts of Interest
The Authors declare that they have no competing interests with regard to this study.
- Received May 3, 2015.
- Revision received June 7, 2015.
- Accepted June 9, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved