Excessive reactive oxygen species (ROS) accumulation is a common phenomenon in carcinogenesis. However, the rationale behind ROS involvement in gastric cancer is unclear. In this study, the authors investigated the clinical significance of the single nucleotide polymorphisms (SNPs) of 2 ROS metabolic process-related genes: superoxide dismutase 2 (SOD2) and glutathione S-transferase π (GSTP1).
In total of 929 patients with gastric cancer who had definitive clinicopathologic and follow-up data were collected. SOD2 reference SNP 4880 (rs4880) and GSTP1 rs1695 genotyping were examined in DNA samples extracted from paraffin-embedded tumor tissue. Association of the 2 SNPs with each clinicopathologic feature was analyzed using the Pearson chi-square test and the independent Student t test. Gastric cancer-specific overall survival was analyzed using Kaplan-Meier curves and log-rank tests. Multivariate Cox regression analyses of these SNPs also were performed.
The SOD2 rs4880 CT + CC genotypes were significantly associated with a high level of lymph node metastasis (P = .023), whereas the GSTP1 rs1695 GA + GG genotypes were significantly associated with larger tumor size (>5 cm long; P = .048). Kaplan-Meier and Cox regression data indicated that the SOD2 rs4880 CT + CC genotypes alone (hazard ratio, 1.299; 95% confidence interval, 1.053-1.603; P = .015) and the GSTP1 rs1695 GA + GG combined genotypes (hazard ratio, 1.496; 95% CI, 1.078-2.074; P = .016) were independent predictors for overall survival.
Worldwide, gastric cancer is the third leading cause of cancer death in men and the fifth in women.1 The highest incidence and mortality rates are observed in East Asia, predominantly in China.1 Although its incidence rate has declined modestly in the recent decades, and remarkable progress has been achieved in comprehensive treatment strategies of combined surgery, chemotherapy, radiotherapy, and targeted therapy, the prognosis for patients still remains poor. The development of gastric cancer is a multistep, sequential process that initiates from chronic gastritis, atrophy, intestinal metaplasia, dysplasia, and finally malignant transformation to invasive gastric cancer.2 One mechanistic connection between chronic inflammation and gastric cancer may account for the generation of excessive reactive oxygen species (ROS)3 induced by Helicobacter pylori-infected gastric epithelial cells,4 activated inflammatory cells, and physical or chemical agents.3 Low levels of ROS are crucial in maintaining normal cellular physiologic functions, such as proliferation, apoptosis, cell cycle arrest, and senescence; whereas increased levels of ROS induce oxidative stress and cause an imbalanced hemostatic microenvironment, leading to DNA damage, tumorigenesis, and cancer progression.5 Hence, unchecked ROS generation and scavenging plays an important role in the development and progression of gastric cancer.
The products of the superoxide dismutase 2 (SOD2) and glutathione S-transferase π (GSTP1) genes are involved in the regulation of ROS metabolic processes. SOD2 is 1 of the major superoxide scavengers in mitochondria, and it catalyzes accumulated superoxide radicals into hydrogen peroxide (H2O2). GSTP1, a member of the glutathione S-transferase family of phase 2 detoxification isozymes, is a candidate enzyme protecting epithelial cells from ROS-induced oxidative stress and detoxifying free radicals by catalyzing the conjugation of hydrophobic and electrophilic compounds with reduced glutathione. Some single nucleotide polymorphisms (SNPs) within these genes have previously been reported, resulting in dysregulated protein translation and/or altered function. The 47cytosine-to-thymine (47C→T) transition at codon 16 in the SOD2 gene (reference SNP 4880 [rs4880]) creates a sense mutation of alanine (Ala)-to-valine (Val) in the SOD2 protein, which affects protein folding and localization.6 Miscoding of Ala to Val in SOD2 will result in SOD2 arresting in the inner mitochondrial membrane but not in the mitochondrial matrix, therefore reducing its functional activities.6 Carriage of 1 or 2 Ala-SOD2 allele(s) reportedly has been associated with greater risks of hepatocellular carcinoma,7 gastric cancer,8 prostate cancer9 and a poor prognosis in breast cancer.10 The SNP in the GSTP1 gene (rs1695) is a substitution of A (isoleucine [Iso]) to G (valine [Val]) at position 105 that results in miscoded GSTP1 protein with decreased enzymatic activity and less effective detoxification. A Val variant of GSTP1 was reported previously as a risk factor in esophageal cancer11 and in Asian-associated breast cancer,12, 13 but not in patients with ovarian cancer, in which it predicted a favorable clinical outcome after chemotherapy.14
Given evidence that the ROS metabolic process plays an obligatory role in gastric cancer, we surmised that the functional polymorphisms of the oxidative stress-related genes SOD2 (rs4880) and GSTP1 (rs1695) may have important clinical implications for prognosis. In the current study, we examined the clinical associations of these SOD2 (rs4880) and GSTP1 (rs1695) SNPs in a relatively large cohort of 929 postsurgical Chinese patients with stage I through IV gastric cancer.
MATERIALS AND METHODS
Patients and Clinical Samples
A retrospective cohort of 929 patients with gastric cancer who underwent surgery at the Yixing People's Hospital (Yixing, Jiangsu Province, China) between 1999 and 2006 who had a median follow-up of 35 months (range, 0-119 months) were recruited for this study.15 The demographic features and clinicopathologic data are summarized in Table 1. The median patient age was 61 years (range, 28-83 years). All patients were diagnosed with gastric carcinoma. None had received radiotherapy or chemotherapy before surgery, and not all of them had received adjuvant chemotherapy or radiotherapy. The surgical specimens were processed immediately after the operation, fixed in buffered paraformaldehyde, and then embedded in paraffin. The samples used for genotyping were reviewed and classified by 2 independent pathologists. The study protocol was approved by the Institutional Review Board of Nanjing Medical University (Nanjing, China), and all patients provided written informed consent on the use of clinical specimens for medical research.
Table 1. Clinical Correlations of Superoxide Dismutase 2 (SOD2) and Glutathione S-Transferase π 1 (GSTP1) Single Nucleotide Polymorphisms in Gastric Cancer
SOD2 rs4880, N = 908
GSTP1 rs1695, N = 900
No. of Patients (%)
Total, N = 929
Abbreviations: A, adenine; AJCC 7th, seventh edition of the American Joint Commission on Cancer (AJCC) Cancer Staging Manual (TNM classification); C, cytosine; G, guanine; rs1695, A→G substitution at codon 105 of the GSTP1 gene; rs4880, C→T transition at codon 16 of the SOD2 gene; SD, standard deviation; T, thymine.
Tumor size was measured by the length of the tumor.
Partial data were not available, and statistics were based on available data.
Invaded depth of tumor was defined according to the TNM classification (AJCC 7th) for gastric cancer as follows: T1, tumor invades lamina propria, muscularis mucosae, or submucosa; T2, tumor invades muscularis propria; T3, tumor penetrates subserosal connective tissue without invasion of visceral peritoneum or adjacent structures; T4, tumor invades serosa (visceral peritoneum) or adjacent structures.
Regional lymph nodes were defined according to the TNM classification (AJCC 7th) for gastric cancer as follows: N0, no regional lymph node metastasis; N1, metastasis in 1 or 2 regional lymph nodes; N2, metastasis in 3 to 6 regional lymph nodes; N3, metastasis in ≥7 regional lymph nodes.
Classification is based on the predominant pattern of tumor as tubular adenocarcinoma (well to moderately differentiated), poorly differentiated adenocarcinoma (poorly differentiated), mucinous carcinoma, and Signet-ring cell carcinoma.
Analysis of SOD2 and GSTP1 Single Nucleotide Polymorphisms
Genomic DNA was extracted from tumor specimens by proteinase K digestion, isopropanol extraction, and ethanol precipitation.15 The SOD2 (rs4880) and GSTP1 (rs1695) SNPs were examined by multiplex SNaPshot technology using an ABI fluorescence-based assay allelic discrimination method (Applied Biosystems, Foster City, Calif) as described previously.16 The primers were designed to anneal immediately adjacent to the nucleotide at the mutation site: SOD2 rs4880: forward, 5′-TCGGGGAGGCTGTGCTTCT-3′; reverse, 5′-CGGGCTGTGCTTTCTCGTCTT-3′; GSTP1 rs1695: forward, 5′-GTGAATGACGGCGTGGAGGAC-3′; reverse, 5′-CCCTGGTGCAGATGCTCWCAT-3′. The primers for extension were as follows: SOD2 rs4880S, 5′-TTTTTTTTTTTTTTTGGAGCCCAGATACCCCAAA-3′; and GSTP1 rs1695S, 5′-TTTTTTTTTTTTTTTTTTTTTTTTCATAGTTGGTGTAGATGAGGGAGA-3′. The SNPs were analyzed by using an ABI3130 genetic analyzer, and the genotypes were determined by using Genemapper4.0 software (Applied Biosystems). Genotyping was validated by sequencing a randomly selected 10% of samples, and the results were 100% concordant.
The SPSS statistical package for Window (version 16; SPSS, Inc., Chicago, Ill) was used for data analysis. The associations of each genotype or combinations of genotypes with clinicopathologic features were compared using the Pearson chi-square test for categorical variables and the Student t test for continuous data. Kaplan-Meier plots and log-rank tests were used for survival analysis. Overall survival was calculated from the date of surgery to the date of death or last follow-up (March 31, 2009). Cox regression was used in the univariate survival analysis to determine the association of individual clinicopathologic variables with overall survival. All variables with P < .1 in addition to age and sex subsequently were subjected to multivariate Cox regression analysis to determine the hazards ratios (HRs) and the independence of effects. Because of the exploratory nature of the study, the P values were not adjusted for multiple comparisons.
Patient Characteristics Related to SOD2 rs4880 and GSTP1 rs1695 Genotypes
Among 929 specimens from patients with gastric cancer, SOD2 rs4880 was successfully genotyped in 908 specimens, and GSTP1 rs1695 was successfully genotyped in 900 specimens. The frequency of each SOD2 rs4880 genotypes was 73.7% (669 specimens) for the TT variant, 24.1% (219 specimens) for the CT variant, and 2.2% (20 specimens) for the CC variant. Table 1 indicates that the risk for the CT and CC variants compared with that for the TT variant in SOD2 rs4880 was associated significantly with high levels of regional lymph node metastasis (P = .023) but not with age, sex, tumor size or location, cellular differentiation, depth of primary tumor invasion, the presence of distance metastasis, American Joint Committee on Cancer staging, or Lauren classification. Similar to the other ROS metabolic-related gene SNP, GSTP1 rs1695, the AA, GA, and GG genotypes were identified in 564 specimens (62.7%), 301 specimens (33.4%), and 35 specimens (3.9%), respectively. Compared with the AA genotype, the GA + GG genotypes were associated significantly with larger tumor size (>5 cm long; P = .048) but not with other clinicopathologic data.
Associations of SOD2 rs4880 and GSTP1 rs1695 With Overall Survival
All patients were closely monitored and followed after primary resection to ensure that sufficient and valid data were recorded. Cox regression analyses were used to assess associations of the SOD2 rs4880 and GSTP1 rs1695 genotypes with gastric cancer survival in different genetic models (Table 2). There were significant associations between the SOD2 rs4880 genotypes and gastric cancer postsurgical overall survival in codominant and dominant models. Figure 1 indicates that the SOD2 rs4880 CT + CC genotypes were correlated significantly with shorter overall survival (log-rank test, 6.769; P = .009). The median survival of patients who carried a SOD2 rs4880 TT genotype was 78 months, whereas that of patients who carried the CT + CC genotypes was only 48 months. For GSTP1 rs1695, patients who carried the AA genotype had a median survival of 77 months, and those who carried the GA + GG genotypes had a median survival of 60 month, which did not reach a statistically significant difference (log-rank test, 0.823; P = .364). In univariate Cox regression analysis, tumor size (P < .001), disease stage (stage III, P < .001; stage IV, P = .012) and Lauren classification (diffuse type, P < .001) were correlated significantly with overall survival (Table 3). Multivariate Cox regression revealed that, in addition to disease stage (P = .001), SOD2 rs4880 was an independent prognostic marker for postoperative overall survival in patients with gastric cancer (HR, 1.299; 95% CI, 1.053-1.603 P = .015) (Table 3).
Table 2. Single Nucleotide Polymorphisms and Gastric Cancer Postoperative Overall Survival in Patients With Gastric Cancer
Combined SOD2 rs4880 CT + CC and GSTP1 rs1695 GA + GG Single Nucleotide Polymorphisms Predict the Worst Clinical Outcome
Although GSTP1 rs1695 was not associated significantly with overall survival in univariate analysis, this functional SNP may result in a failure to protect cells in an excessive oxidative stress-producing environment because of decreased detoxification, and it was correlated significantly with tumor size. Therefore, we investigated whether combining the GSTP1 rs1695 GA + GG and the SOD2 rs4880 CT + CC genotypes would provide more prognostic information. Genotyping of both SOD2 rs4880 and GSTP1 rs1695 was performed successfully in 894 patients. Figure 2 demonstrates significant differences in overall survival (log-rank test, 7.968; P = .047) for the 4 genotypes in SOD2 rs4880 and GSTP1 rs1695 in assorted combinations. For patients who carried a SOD2 rs4880 TT genotype (n = 658), the GSTP1 rs1965 polymorphism was not an additional prognostic marker (subgroup analysis: log-rank test, 0.221; P = .638). However, for patients who carried SOD2 rs4880 CT + CC genotypes (n = 236) and had short overall survival, coexistence with GSTP1 rs1695 GA + GG genotypes revealed a trend toward an even poorer prognosis than that for patients who carried the GSTP1 rs1695 AA genotype (subgroup analysis: log-rank test, 1.252; P = .263). In general, patients who carried the SOD2 rs4880 TT and GSTP1 rs1695 AA (n = 411) genotypes had the longest median overall survival (98 months); conversely, patients who carried the SOD2 rs4880 CT + CC and GSTP1 rs1695 GA + GG (n = 85) genotypes had the shortest median overall survival (31 months). In addition, multivariate Cox regression analysis indicated that, along with disease stage (stage III, P < .001), SOD2 rs4880 and GSTP1 rs1695 combined SNPs independently predicted overall survival in patients with gastric cancer (SOD2 rs4880 CT + CC and GSTP1 rs1695 GA + GG; HR, 1.496; 95% CI, 1.078-2.074; P = .016) (Table 4).
Table 4. Cox Regression Analysis of Overall Survival: Superoxide Dismutase 2 (SOD2) and Glutathione S-Transferase π 1 (GSTP1)a
Abbreviations: A, adenine; AJCC, American Joint Commission on Cancer; C, cytosine; CI, confidence interval; G, guanine; HR, hazard ratio; rs1695, A→G substitution at codon 105 of the GSTP1 gene; rs4880, C→T transition at codon 16 of the SOD2 gene; T, thymine.
Partial data were not available, and statistics were based on available data.
Univariate analysis was performed using Cox regression.
In this study, we investigated the clinical significance of genetic polymorphisms of 2 ROS metabolic-related genes, SOD2 and GSTP1, in Chinese patients with gastric cancer. The homozygote Ala/Ala and the heterozygote Ala/Val in SOD2 were associated significantly with poor postsurgical overall survival.
SOD2 plays a complicated role in the ROS scavenging system. It acts in the front line to defend against ROS by catalyzing superoxide conversion to H2O2, which may be neutralized further to nontoxic H2O and oxygen by catalase (CAT) and glutathione peroxidase (GPX).17 H2O2 also can be converted to hypochlorous acid (HOCl) through a myeloperoxidase (MPO)-catalyzed reaction, causing secondary oxidative damage to the cells.18 Meanwhile, increased generation of H2O2 can abrogate tumor necrosis factor-alpha (TNFα)-mediated apoptosis to allow the survival of handicapped cells.19 Thus, SOD2 appears to play a pivotal role at this particular divergent point in the ROS metabolism pathway. Recently, it was reported that SOD2 rs4880 C alleles (Ala allele) were associated with less adjuvant chemotherapy-related adverse events but shorter disease-free survival than the TT genotype in patients with breast cancer.10, 20 In patients with gastric cancer, overexpression of SOD2 was observed in cancerous tissue and was associated with poor overall survival.21 In our study, patients who carried SOD2 rs4880 CT + CC genotypes had increased risks of regional lymph node metastasis and disease-related death. We surmise that patients who carry SOD2 rs4880 CT + CC genotypes may have active catalyzation, resulting in the accumulation of H2O2 and secondary ROS generation, which subsequently increase the chances of DNA damage and carcinogenesis in gastric mucosa. Nevertheless, whether intracellular ROS levels are changed in parallel with SOD2 rs4880 genotypes in the gastric cancer microenvironment needs further investigation.
Polymorphisms of GSTP1 have been associated with platinum-based chemotherapy efficacy in colon,22 lung23-25 and gastric cancer,26 revealing their clinical potential as a biomarker to predict platinum-related chemosensitivity. In our study, we observed an association of GSTP1 rs1695 GA + GG genotypes with tumor sizes >5 cm, but an association with overall survival was not observed. Although the risk of gastric cancer-specific death appeared to be driven primarily by the SOD2 rs4880 CC + CT genotypes, we observed an increased risk when GSTP1 rs1695 was added to the analysis. This suggests that the Val variant of GSTP1 with lower enzymatic activity may allow more prolonged exposure of cells to the toxic effects of excessive ROS. In our study, we observed a significant decrease in overall survival for patients who carried the SOD2 rs4880 CC + CT and GSTP1 rs1695 GA + GG genotypes (HR, 1.496; 95% CI, 1.078-2.074; P = .016) compared with patients who carried the SOD2 TT and GSTP1 AA genotypes. For patients with SOD2 rs4880 CC + CT genotypes, who had a poor prognosis, also carrying the GSTP1 rs1695 AA genotype would predict (although not with a statistically significant difference: log-rank test, 1.252; P = .263) a comparatively favorable outcome (median overall survival, 54 months) compared with those who carried the GA + GG genotypes (median overall survival, 31 months). Our findings suggest that an enhanced capability to eradicate excessive ROS at a certain extent may provide a protective effect against cancer development and, thus, may slow its pace toward malignant progression.
Consistent with previous research, our retrospective study demonstrated that large tumor size, advanced stage, and diffuse type of gastric cancer are correlated significantly with poor overall survival.27 Moreover, SOD2 rs4880 with or without GSTP1 rs1695 polymorphisms and American Joint Committee on Cancer stage were identified as independent prognostic markers of overall survival in the current Chinese cohort. Nevertheless, there are limitations in our study, because some potential confounding factors, such as chemotherapy regimens (which generally are given for postsurgical patients with stage II-IV disease and also are responsible for ROS generation) and H. pylori infection status, were not included for lack of definitive clinical and follow-up data. Further investigation is underway to clarify whether the associations between these polymorphisms and clinical outcomes are a consequence of drug metabolism.
In conclusion, our findings demonstrate the clinical significance of the SOD2 rs4880 and GSTP1 rs1695 SNPs in gastric cancer progression and their associations with regional lymph node metastasis and tumor size, respectively. These observations may be attributed to SOD2 CT + CC and GSTP1 GA + GG genotypes, representing impaired SOD2 and GSTP1 functions in catalyzing reactions to remove excessive ROS, which may provide a favorable microenvironment for gastric cancer development and progression. Analyses of their associations with specific chemotherapy reagent toxicity and response are underway and will be reported separately. Considering the connection of the SOD2 rs4880 polymorphism with overall survival in Chinese patients with gastric cancer, further investigations are needed to characterize whether the genotype can represent its activity in gastric tissue and whether reducing dysregulated ROS levels may be a new therapeutic direction.
We thank Dr. Da Ding and Ms. Yan Liu of Genesky Biotech (Shanghai, China) for excellent technical assistance. We also thank Dr. Stella Sun (Division of Neurosurgery, Department of Surgery, The University of Hong Kong, Hong Kong) for critical reading of the article.
The work was supported by grants from the National Natural Science Foundation of China to Dr. J. Chen (grant 81071641), Dr. Z. Xu (grant 81000880), Dr. H. Zhu (grant 81001274), and Dr. M. Wang (grant 81102089, BK2011773); by a grant from Jiangsu Provincial 12th Five-Year Program on Developing Health by Technology and Education Project to Dr. J. Chen; and by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine).