Abstract
Background: Common single-nucleotide polymorphisms (SNPs) in pre-miRNAs (hsa-mir-196a2, hsa-mir-146a and hsa-mir-499) have been associated with various malignancies and their prognoses. We evaluated the associations of three selected SNPs (rs11614913, rs2910164, and rs3746444) in pre-miRNAs (hsa-mir-196a2, hsa-mir-146a and hsa-mir-499) with the prognosis of advanced gastric cancers (GCs) treated by chemotherapy. Materials and Methods: The rs11614913 (T>C), rs2910164 (C>T), and rs3746444 (A>G) SNPs were genotyped in 130 advanced GCs performing chemotherapy. Survival and response evaluation was based on overall survival (OS) and progression-free survival (PFS). Response rate (RR) was also evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST). Results: 63 patients performed gastrectomy after chemotherapy (neoadjuvant chemotherapy) and the remaining cases performed chemotherapy alone as treatment (chemotherapy alone). The majority of cases performed S-1-based chemotherapy as the first line treatment (n=119, 92%). The rs3746444 (A>G) SNP was significantly associated with OS by the log-rank test (p=0.018), while other SNPs were not associated with OS. The rs3746444 (A>G) SNP was also associated with OS and PFS among cases of neoadjuvant chemotherapy (p=0.038, 0.024, respectively). Multivariate survival analysis using the Cox's regression model revealed that non-responder by the RECIST (Hazard ratio (HR): 2.14 95%CI 1.06-4.19), upper third cancer (HR: 2.48 95%CI 1.12-5.49) and more advanced stage (HR: 4.12 95%CI 1.06-16.02) were predictive factors for worse OS, while the rs3746444 A allele carrier was predictive factor for better OS (HR: 0.33 95%CI 0.18-0.75). Conclusion: The rs3746444 A allele carrier in the hsa-mir-499 is associated with better prognosis in advanced GC performing chemotherapy.
Abbreviations: GC, gastric cancer; SNP, single-nucleotide polymorphism; OS, overall survival; PFS, progression-free survival; RR, Response rate; RECIST, Response Evaluation Criteria in Solid Tumors; HR, hazard ratio; CI, confidence interval.
Gastric cancer (GC) is one of the most common malignancies worldwide, accounting for approximately 70,000 new cases and 650,000 deaths per year (1, 2). Despite advance in strategy for early detection, many patients still have advanced disease at diagnosis and are not indicated for curative surgery. Chemotherapy is now recognized as the most effective treatment for patients with unresectable advanced or metastatic GC and many clinical trials have evaluated its efficacy and safety (3-5). Other than unresectable cases, chemotherapy can be also considered for potentially resectable cases to further improve their outcomes (6, 7).
The mechanisms of sensitivity or resistance to chemotherapy may involve multifactorial steps in which interactions of the biological behavior of the individual tumor, host immune systems and inter-individual differences in metabolic activity may all participate, and the process may be partly explained by genetics. Predictive factors for prognosis in advanced GC who receive chemotherapy, however, have not been truly understood.
MicroRNAs (miRNA) are 21- to 24-nucleotide-long small non-coding RNA gene products that regulate gene expression by base pairing with target mRNAs at the 3’-untranslated region, leading to mRNA cleavage or translational repression (8-10). Other than biological processes (11), several recent reports show that miRNAs participate in human tumorigenesis as tumor suppressors or oncogenes (12-14).
There are common single-nucleotide polymorphisms (SNPs) in miRNA sequences that identified four SNPs (rs11614913, rs2910164, and rs3746444) located at the pre-miRNA regions of miR-196a2, miR-146a, and miR-499, respectively (15). The association between common SNPs in miRNA and cancer predisposition and their prognosis has been extensively investigated in various cancers including GC (15-20). The current study evaluated the associations of three selected SNPs (rs11614913, rs2910164, and rs3746444) in pre-miRNAs (hsa-mir-196a2, hsa-mir-146a and hsa-mir-499) with the prognosis of advanced GC treated by chemotherapy. The result demonstrated that the rs3746444 SNP in the hsa-mir-499 is associated with OS and PFS, supporting its role in the mechanisms of response to chemotherapy in advanced GC patients.
Patients and Methods
Patients, survival and response evaluation. The studied population comprised of 130 Japanese patients with advanced GC performing chemotherapy from April 2003 to September 2012 in our Hospital. All GC were diagnosed histologically and were classified according to Lauren's classification (21). Detailed information about anatomic location, macroscopic types, depth, lymph node and other metastasis and peritoneal dissemination was obtained according to the Japanese classification of gastric carcinoma (22). Overall survival (OS) was defined as the time from start of initial administration of chemotherapy to the date of cancer-related death. If cancer-related death did not occurr, the OS was censored on the last date the patient has known to be alive. Tumor responses to chemotherapy were evaluated after 2 courses of the treatment using the guidelines of the Response Evaluation Criteria in Solid Tumors (RECIST) and classified as CR (complete response), PR (partial response), SD (stable disease) or PD (progressive disease). CR and PR were considered to be responders. All others were considered to be non-responders. Progression-free survival (PFS) was defined as the time of initial administration of chemotherapy to tumor progression or cancer-related death. Patients with no confirmation of progression or cancer-related death were censored at the date of the last objective tumor assessment. The Fujita Health University School of Medicine approved the protocol, and written informed consent was obtained from all participating subjects.
Genotyping. Genomic DNA was extracted from non-pathological gastric biopsies or peripheral blood using the standard protein precipitation method. The rs11614913 (C>T), rs2910164 (G>C), and rs3746444 (A>G) SNPs were determined by polymerase chain reaction (PCR)–based restricted fragment length polymorphism assays as described by Hu et al. (15). All genotypes were determined by reading the gel pictures by two independent investigators who were blinded to the names and phenotype of the patients.
Statistical analysis. Categorical variables among the two groups were assessed using the two-tailed Fisher's exact test. OS and PFS among the two groups were assessed using the Kaplan-Meier method and the log rank test. A p-value less than 0.05 was considered statistically significant. Multivariate survival analysis using the Cox's regression model was also performed for calculating the hazard ratio (HR) and 95% confidence interval (CI) with adjustment of clinicopatological factors.
Results
Clinicopathological characteristics of the patients. Clinicopathological characteristics of GC and information about treatment are shown in Tables I and Table II. Among all 130 patients, 63 patients were diagnosed as operable and performed gastrectomy with a D2 lymph node dissection after chemotherapy (neoadjuvant chemotherapy). For the remaining cases, chemotherapy alone was used for the treatment. For majority of the cases (n=119, 91.5%), S-1-based regimens were used for the first line chemotherapy. For 117 cases, tumor responses to chemotherapy were evaluated after 2 courses of the treatment using the RECIST. Thirty-four cases (26.2 %) and 83 (63.8%) were considered as responder and non-responder, respectively (Table II). OS, PFS, and RR could be assessed among 226, 216 and 204 cases, respectively. The median OS and median PFS in all cases were 17 months and 9.5 months during a median follow-up period of 17 months.
Association between SNPs in miRNAs and OS, PFS and RR of GC. Prevalence of rs11614913 (C>T), rs2910164 (G>C), and rs3746444 (A>G) SNPs are shown in Table III. Comparing the genotype frequency, the rs3746444 (A>G) SNP was significantly associated with OS by the log-rank test (p=0.018), while the same genotype was also weakly associated with PFS by the log-rank test (p=0.059). On the other hand, the rs11614913 (C>T) and rs2910164 (G>C) SNPs were not associated with OS and PFS (Figure 1). When subjects were divided into neoadjuvant chemotherapy and chemotherapy alone groups, the rs3746444 (A>G) SNP was also associated with OS and PFS among cases performing neoadjuvant chemotherapy (p=0.038, 0.024, respectively) (Figure 2). However, such association was not observed among chemotherapy-alone groups (p>0.10, data not shown). We also investigated whether SNPs in miRNAs would be associated with RR, however, we did not find any significant association between the three SNPs and RR (Table IV).
Multivariate survival analysis using the Cox's regression model. To evaluate independent prognostic factors that are associated with OS in GC patients who receive chemotherapy, multivariate survival analysis using the Cox's regression model was performed. For this analysis, all clinicopathological factors including gender, age, anatomic location, macroscopic and histological types, depth, information about metastasis and staging and response against treatment were included (Table V). This analysis demonstrated that non-responders by the RESIST (HR: 2.14 95%CI 1.06-4.19), upper third cancer (HR: 2.48 95%CI 1.12-5.49) and more advanced stage (HR: 4.12 95%CI 1.06-16.02) were predictive factors for worse OS, while the rs3746444 A allele carrier was predictive factor for better OS (HR: 0.33 95%CI 0.18-0.75).
Clinicopathological features of gastric cancers.
Discussion
Common single-nucleotide polymorphisms (SNPs) in pre-miRNAs (hsa-mir-196a2, hsa-mir-146a and hsa-mir-499) have been associated with various malignancies and their prognosis. Among these SNPs, rs3746444 (A>G) SNP was significantly associated with OS in advanced GC patients who receive chemotherapy. Multivariate survival analysis using the Cox's regression model revealed that the rs3746444 A allele carrier was an independent predictive factors for better OS. The study provides the first evidence that common SNPs in pre-miRNA, especially rs3746444 (A>G) in the hsa-mir-499 may be used as candidate biomarkers for prediction of prognosis in advanced GC performing chemotherapy.
miRNAs are regulators of gene expression that provide a regulation for a broad range of biological processes including cancer development, cellular differentiation, proliferation, apoptosis and metabolism (11). The hsa-mir-499, a gene with frequently increased expression in several cancers, may function to promote migration and invasion of malignant cells (23). A previous study demonstrated that the rs3746444 SNP in the miR-499 was associated with breast cancer susceptibility (19). hsa-mir-499 rs3746444 SNP is located in their corresponding 3p mature miRNA regions, and they may influence both the binding of target mRNAs to 3p mature miRNAs and pre-miRNA maturation of 5p and 3p miRNAs. The transcripts of genes participating in diverse cancer-related pathways could be targeted by hsa-mir-499 (19). It is also possible that hsa-mir-499 rs3746444 SNP influences the inter-individual biological behaviors in GC, which may correlate with prognosis in cases performing chemotherapy. Stratification analysis demonstrated that the rs3746444 SNP was associated with OS and PFS especially in cases of neoadjuvant chemotherapy, suggesting the strong influence of rs3746444 on the prognosis in GC performing preoperative chemotherapy. We have previously shown that the rs2910164 CC genotype in the miR-146a is associated with gastric cancer susceptibility (20). Moreover, rs11614913 CC genotype in the mir-196a2 was associated with polypoid or elevated-type morphology in early-stage cases, while the combined rs11614913 T allele carrier was weakly-associated with worse overall survival than in younger cases (24). How different miRNA SNPs are associated with different subtypes of gastric cancers need to be clarified.
Information about treatment of gastric cancers.
Prevalence of rs11614913, rs2910164, and rs3746444 polymorphisms in gastric cancers.
Association between rs11614913 (C>T), rs2910164 (G>C), and rs3746444 (A>G) SNPs with overall survival (OS: upper) and progression-free survival (PFS: lower). Statistical analysis was performed using the log-rank test.
Association between rs3746444 (A>G) SNP with overall survival (OS: left) and progression-free survival (PFS: right) in cases performing neoadjuvant chemotherapy. Statistical analysis was performed using the log-rank test.
We have shown that the rs3746444 A allele carrier was the independent predictive factors for better OS, while heterozygous AG genotype presented rather better OS and PFS (Figures 1 and 2). There are three potential speculations for the association we observed. First, it is suggested that the rs3746444 AG heterozygote genotype of the miR-499 gene, but not homozygotes (AA), may strongly alter the function or expression of miR-499 and modify the prognosis of GC altering the regulation of target mRNAs. The second, speculation for the associations we witnessed is that this polymorphism may be in linkage disequilibrium with other polymorphisms elsewhere in the miR-499 gene, demonstrating biologically-relevant variability. Finally, there exists the possibility that this polymorphism is in linkage disequilibrium with a genetic variation of another gene located near the miR-499 gene that is related to prognosis in GC patients. Since the detailed mechanisms by which miR-499 and their target mRNAs influence gastric cancinogenesis and confer different phenotypic differences are unknown, and the whole issue warrants further in vitro and in vivo studies to confirm our result.
Association between rs11614913, rs2910164, and rs3746444 polymorphisms and response to chemotherapy.
Multivariate survival analysis using Cox's regression model for adjustment of clinicopathological factors.
Footnotes
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Conflicts of Interest
Authors do not have any financial competing interests about this manuscript.
- Received April 21, 2014.
- Revision received June 21, 2014.
- Accepted June 23, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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