A total of 234 patients (162 men and 72 women) with histologically or cytologically confirmed advanced gastric cancer registered from July 2005 to July 2008 at the Department of Oncology, Shandong Cancer Hospital and Institute were included in this study. The patients should have a histological diagnosis of gastric carcinoma with an unresectable primary tumor and/or metastases that were measurable or assessable by means of clinical examination, X-ray, computed tomography (CT) or ultrasound. The median age of the patients was 61.2 years (range, 27-79 years). There were 147 cases of antrum gastric cancer, 35 gastric cardia cancer, and 52 cases of other types, including 150 cases at stage IIIB and 84 cases at stage IV. Pathologically, 10 patients had well-differentiated adenocarcinoma, 45 had moderately differentiated adenocarcinoma, 161 poorly differentiated adenocarcinoma, and 18 signet ring cell carcinoma. Two hundred and six patients were treated with conventional and 5-fluorouracil (5-FU)-based chemotherapy. Among them, 96 cases were treated with capecitabine/fluorouracil + cisplatin/oxaliplatin regimen and 89 cases were treated with docetaxel + oxaliplatin + fluorouracil regimen. The median chemotherapy courses consisted of 5 (1-11) cycles. Follow-up time was calculated from the initiation of diagnosis to July 2009, with a median of 13.3 mo (range, 3.5-34.4 mo). Thirty-three patients were lost to follow-up and 188 patients died, including 3 patients who died from causes other than gastrointestinal carcinoma during the follow-up.

We also selected 243 control individuals (aged 26-79 years) who visited the Shandong Cancer Hospital and Institute between July 2005 and July 2008 for general physical exams. The control individuals were screened to ensure that none had ever been diagnosed with cancer or other serious diseases. The selected controls were age matched to the cases (± 5 years). All the subjects were unrelated ethnic Han Chinese, and written informed consent was obtained from each participant. The study was approved by the Review Board of Shandong Cancer Hospital and Institute. A 2-mL peripheral blood sample was collected from each study participant.

Genomic DNA was extracted from peripheral blood using a Genomic DNA Extraction Kit (Fastagen, Shanghai, China) according to the manufacturer’s protocol. IL-10 promoter polymorphisms were identified by PCR amplification and restriction analysis (PCR-RFLP), (Table 1). Each PCR reaction was performed in a GeneAmp PCR System 9600 thermocycler (Applied Biosystems, Foster, CA) at a final volume of 25 μL (containing 5 pmol for each primer, 50 ng genomic DNA, 1.5 mmol/L MgCl2, 5 mol/L dNTPs and 1 U of Taq DNA polymerase in PCR buffer containing 10 mmol/L Tris). PCR cycles used were as follows: 95°C for 5 min, 35 cycles of denaturing at 95°C for 40 s, annealing at the indicated temperature for 1 min, extension at 72°C for 40 s, and a single final extension at 72°C for 10 min. The amplified products were digested with corresponding restriction endonucleases (New England Biolabs, MA, USA), and separated by electrophoresis on a 10% polyacrylamide gel stained with sliver nitrate for visualization.

Statistical analysis was performed using SPSS 13.0 software (SPSS, Florida, USA). Demographic data between the study groups were compared using the Chi-square test and Student’s t test. Each polymorphism was tested for deviation from the Hardy-Weinberg equilibrium by comparing the observed and expected genotype frequencies using the χ² test. Genotype frequencies of IL-10 were compared between groups using the χ² test, and odds ratios (OR) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression with adjustment for age and sex. The haplotypes of IL-10 (-1082, -819 and -592) were analyzed using the SHEsis software (Bio-X Inc., Shanghai, China), which uses a full-precise-iteration (FPI) algorithm to reconstruct haplotypes. The Kaplan-Meier method with a log-rank test was used to evaluate the association between genotype and survival. Multivariate analysis was performed using Cox proportional hazards regression. Statistical significance was interpreted as P < 0.05.

The genotype and allele frequencies of the IL-10 SNP in 234 gastric cancer patients and 243 healthy controls are shown in Table 2. All genotype frequencies in both patient and control groups were in the Hardy-Weinberg equilibrium (P < 0.05). There were significant differences in the genotype and allele frequencies of the IL-10 promoter -1082 A/G polymorphism between gastric cancer and control groups. The -1082 AG genotypes were associated with a significantly increased risk of gastric cancer as compared with the -1082 AA genotypes (OR 1.9, 95% CI 1.1-3.4, P = 0.019). The -1082 G allele was associated with a significantly increased risk of gastric cancer as compared with the -1082 A allele (OR 1.2, 95% CI 0.6-3.2, P = 0.007). However, genotype and allele frequencies of the IL-10 -819 T/C and -592 A/C polymorphisms in gastric cancer patients were not significantly different compared with those in healthy controls (P > 0.05). The estimated haplotype frequencies of IL-10 polymorphisms in gastric cancer patients and controls are also shown in Table 2. Complete linkage disequilibrium was observed between locus -819T/C and locus -592A/C. Four possible haplotypes were demonstrated in our population. The most frequent haplotype in both patients (60.3%) and controls (64.6%) was the ATA (-1082A, -819T and -592A) haplotype. By haplotype analyses, we found that the GCC (-1082G, -819C and -592C) haplotype was associated with a significantly increased risk of gastric cancer as compared with the ATA haplotype (OR = 2.3, 95% CI 1.2-4.1, P = 0.005).

Of the 234 patients, 33 were lost to follow-up. We compared the IL-10 genotype with survival time in 201 patients with advanced gastric cancer. The median follow-up time was 13.3 mo (range, 3.0-30.3 mo), and the median survival time was 11.2 mo. The median survival time was 11.4 mo for patients with the IL-10 -1082AA genotype, 10.8 mo for patients with the GA genotype, and 11.0 mo for patients with the GG genotype. The IL-10 -1082 genotypes were not associated with the prognosis of gastric cancer (P =0.709, log-rank test). Similarly, the median survival time was 11.5 mo for patients with the IL-10 -819TT/-592AA genotype, 10.8 mo for patients with the -819TC/-592AC genotype, and 10.2 mo for patients with the -819CC/-592CC genotype (P = 0.090, log-rank test). When stratified by the clinical stage, the association between the IL-10 gene polymorphisms (-1082, -819 and -592) and the gastric cancer survival was not statistically significant either in stage IIIB or stage IV (Figure 1). Multivariate Cox regression was used to analyze the effect of different risk factors (IL-10-1082A/G, -819T/C and -592A/C genotype, chemotherapy regimen, age, gender, disease stage, and histology) on survival time. However, none of the genotypes of the three IL-10 sites was recognized as an independent prognostic indicator (Table 3).

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Figure 1 Kaplan-Meier survival plot for advanced gastric cancer patients by interleukin-10 genotype. A: Interleukin (IL)-10 -1082 genotype in stage IIIB; B: IL-10 -1082 genotype in stage IV; C: IL-10 -819/-592 genotype in stage IIIB; D: IL-10 -819/-592 genotype in stage IV.

The immune dysfunction may be linked to the incidence and clinical course of malignant cancers. Interleukin-10 (IL-10) is a pleiotropic immunoregulatory cytokine which is involved in inflammatory reaction and immune regulation and is considered to exert effects in malignant transformation. IL-10 promoter polymorphisms have been reported to determine inter-individual differences in IL-10 production and are associated with the risk and pathogenesis of several cancers.

Several studies have shown the possible involvement of IL-10 polymorphisms in the pathogenesis of gastric cancer, although its association with prognosis of gastric cancer was not well explored. The aim of this study was to explore the association between polymorphisms of IL-10 promoter and the prognosis of patients with advanced gastric cancer.

This study indicated that IL-10 promoter polymorphisms were associated with an increased risk of gastric cancer, but these polymorphisms did not influence the prognosis of the patients with advanced gastric cancer.

This is the first report of the association between IL-10 promoter polymorphism and prognosis of the Chinese patients with gastric cancer. The results of this study will help understand the genetic background of the immune-related gene and gastric cancer incidence and prognosis.

IL-10 is an important anti-inflammatory and immunosuppressive cytokine, which inhibits the Th1-type pathway activation, prevents antigen-presenting cells from obtaining access to tumor antigens, and down-regulates surface expression of costimulatory molecules CD80 or CD86 on tumor cells.