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Keywords:

  • gastric carcinoma;
  • cytokine genotype;
  • Helicobacter pylori;
  • cigarette smoking;
  • single nucleotide polymorphism

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The association of cytokine genotypes with gastric carcinoma (GC) may be influenced by environmental factors and varies among different populations. Few studies have addressed the impact of different cytokine genotypes on the development and progression of GC. We analyzed 11 functional polymorphisms in tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-4 and IL-10 genes in 220 Taiwanese Chinese with GC and in 230 healthy controls. The risk of genotypes was adjusted with confounding environmental risks. Our results revealed that the frequency of Helicobacter pylori infection [odds ratio (OR) 1.7, 95% confidence interval (CI) 1.19–2.56], cigarette smoking (OR 2.02, 95% CI 1.38–2.95) and high IL-10 producer genotype (OR 2.67, 95% CI 1.29–5.50) was significantly increased in the entire GC patients. Among different subtypes of GC, a higher risk of developing diffuse type (OR 1.64, 95% CI 1.01–2.67) or cardia cancer (OR 2.44, 95% CI 1.13–2.67) was observed for the CT/CC genotype of IL-4 at the position −590, whereas the high IL-10 producer genotype was significantly linked with the risk of cardia cancer (OR 3.21, 95% CI 1.06–9.73) or advanced stage (OR 2.29, 95% CI 1.12–4.64). No association was noted between GC and controls in the distribution of IL-1 and TNF-α genotypes. Logistic regression analyses revealed that H. pylori infection (OR 1.7, 95% CI 1.14–2.52), cigarette smoking (OR 1.87, 95% CI 1.27–-2.96) and IL-10 genotype (OR 2.54, 95% CI 1.24–5.61) are independent risks for GC. Independent effects of IL-10 genotype, H. pylori infection and cigarette smoking indicate that carcinogenesis of GC is influenced by a variety of host and environmental factors. © 2003 Wiley-Liss, Inc.

Gastric carcinoma (GC) is a common and complex multifactorial disease in which environmental and host-related factors interact.1Helicobacter pylori infection and cigarette smoking are among important environmental risks for GC. However, only a small portion of H. pylori-infected patients or cigarette smokers develop GC, indicating that additional factors must be involved in determining the fate after exposure to environmental factors.2, 3 Recently, studies of twins, familial clustering, ethnic differences and human leukocyte antigen (HLA) genes have provided evidence that host factors play a significant role in the pathogenesis of GC.4, 5 Specifically, the host response to environmental triggers, rather than bacteria or environmental factors per se, may be responsible for the outcome of disease.6

In the multistage model of gastric carcinogenesis, gastric inflammation is a prerequisite for the development of GC.7 Accordingly, factors involved in initiation and regulation of the inflammatory response may confer susceptibility to or protection against GC. In this respect, cytokines that play a crucial role in regulating inflammation are potential candidates for correlating with such variation. The in vitro maximal capacity to produce different cytokines varies among different individuals. Family studies indicate that much of this variability is genetically determined.8 Such interindividual differences can be attributed to several molecular mechanisms, including single nucleotide polymorphisms (SNPs) in the coding or promoter regions of cytokine or cytokine receptor genes. These polymorphisms may affect the overall expression and secretion of cytokines. The observed genetically determined differences in cytokine production in inflammatory response might account for some of the heterogeneity of infectious diseases.8, 9 For H. pylori infection, tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β are 2 prototypic proinflammatory cytokines that have been implicated in the pathogenesis of GC and H. pylori-associated diseases.10, 11, 12, 13

Multiple polymorphisms have been found in both TNF-α and IL-1 gene clusters. Two polymorphisms located in the TNF-α promoter (−238 and −308 positions) have been studied.10, 11 The expression of TNF-α-308 A allele is a risk factor for duodenal ulcer,10 whereas that of TNF-α−238 A allele is associated with a decreased risk of GC.11 The IL-1 gene family includes IL-α, IL-1β and the IL-1 receptor antagonist (IL-1Ra). The IL-1β gene has polymorphisms at positions −511, and −31, and there is a variable number of tandem repeats (VNTRs) in IL-1RN.12 A recent breakthrough in the investigation of H. pylori-associated diseases is the discovery that polymorphisms of IL-1β and IL-1RN are strongly linked to the occurrence of hypochlorhydria and GC for Caucasians.12, 13 This implies that genetic host factors, such as cytokine polymorphisms, may render an individual susceptible to GC through modulation of gastric inflammation in a permissive cytokine environment. However, ethnicity might greatly affect the genotype status,14, 15 and a recent study from Japan revealed no difference in IL-1 genotypes between GC and controls.14 Therefore, the importance of cytokine polymorphisms needs to be replicated in other ethnically diverse populations.

An array of cytokines, which can be either a T-helper cell type 1 (Th1) response promoting cell-mediated immunity or a T-helper cell type 2 (Th2) response promoting humoral immunity, may determine the fate of inflammation.9 In contrast to proinflammatory cytokines such as TNF-α and IL-1, which are considered Th1 cytokines, relatively little is known about the role of antiinflammatory cytokine genotypes in the pathogenesis of GC. In the Helicobacter felis murine model of infection, gastric inflammation is more severe in IL-10 knockout animals.16 Likewise, a more severe gastric inflammation was observed in IL-4 gene-deficient mice infected with H. pylori.17 Therefore, the balance between Th1 and Th2 cytokines is critical as to the influence on the outcome of H. pylori infection.6 IL-10 and IL-4 also have many polymorphisms. Three SNPs, located at positions −1082 (G to A), −819 (C to T) and −592 (C to A), have been identified for IL-10. These polymorphisms were found to be in strong linkage disequilibrium, and 3 of 8 possible haplotypes (GCC, ACC and ATA) segregate in the Caucasian population.18 The GCC and ATA haplotypes are associated with high and low IL-10 production in peripheral blood cell cultures, respectively.19, 20 There are 3 SNPs that may affect IL-4 function. A nucleotide substitution (C [RIGHTWARDS ARROW] T) at position −590 of the IL-4 gene promoter is associated with increased IL-4 gene expression.21 Amino acid substitutions in the IL-4 receptor gene at position 50 (valine [RIGHTWARDS ARROW] isoleucine) and position 576 (glutamine [RIGHTWARDS ARROW] arginine) produce receptors that exhibit enhanced signal transduction in vitro.22 Whether these polymorphisms of IL-4 and IL-10 genes were related to the risk of GC remains unknown.

Furthermore, recent studies have revealed that environmental factors, such as cigarette smoking, may induce cytokine expression and are associated with certain cytokine genotypes.23H. pylori infection and cigarette smoking may act independently or in synergy with cytokine genotypes to confer individual susceptibility to developing GC. Nevertheless, studies concerning interaction of cytokine genes and environmental factors in gastric carcinogenesis are lacking.

In the present case-control association study, we investigated a total of 11 polymorphisms, including 2 in the TNF-α gene, 2 in the IL-1 gene, 1 in the IL-1RN gene, 1 in the IL-4 gene, 2 in the IL-4 receptor gene, and 3 in the IL-10 gene, and compared their allele frequencies in affecting disease susceptibility and severity of GC. We further investigated the interaction between these cytokine genotypes and other environmental risks such as H. pylori and cigarette smoking in gastric carcinogenesis.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Study subjects

Since 1998, blood samples (serum, erythrocytes and buffy coat) have been collected from individuals participating in a national project for the investigation of risk factors of GC in Taiwan. Our study protocol was approved by the Department of Health, Executive Yuan, Taiwan. A full verbal explanation of the study was given to all participants. They gave their consent to participate in our study on a voluntary basis. Patients with newly diagnosed gastric cancer were enrolled from the inpatient units and outpatient cancer clinics of 3 major tertiary care hospitals in Taipei, Tao-Yuan and Lo-Tung. In Taiwan, more than 90% of the residents are Fukienese or Hakka, whose ancestors immigrated to Taiwan from Southern China. To minimize ethnic biases within the population studied, all patients and controls were from Han Chinese, and the aboriginal and alien populations were excluded. The eligible patients had been histopathologically confirmed to have gastric adenocarcinoma by endoscopic biopsy or surgical specimens.

On an average, fewer than 5% of the hospital patients meeting the diagnostic criteria refused to participate the study when they were approached. The major reason for nonparticipation was terminal stage of the disease. At recruitment, a well-trained research assistant interviewed each study subject to obtain information on ethnic status, socioeconomic characteristics, relevant medical history and lifetime habit of cigarette smoking. Blood specimens, including serum and white blood cells from study subjects, were collected, separated and stored at −70°C until subsequent analysis. From January 1998 to December 2000, a total of 292 cases were enrolled.

Individuals who were not inflicted with GC and were noted to have minimal gastritis or normal appearance of the gastric mucosa after the gastroscopic examination, were randomly selected from health examination clinics in the same hospital as controls. The controls were matched by gender, ethnicity, age (± 3 years), and the date of blood collection and interview by questionnaire (± 3 months).

At the time of the present study, lymphocyte DNA was available for 254 cases and 264 controls. The DNA that was of poor quality in 15 and 17 of them was excluded from the study. After genotyping, 220 cases and 230 controls with complete data of histopathologic characteristics and cytokine genotypes were subjected to final analysis. Among 220 GC cases, the subtype distribution was 112 intestinal and 108 diffuse GC, 30 cardia and 190 noncardia GC and 29 early and 191 advanced GC.

Laboratory analysis

Serum samples were tested for the status of H. pylori using a standard enzyme-linked immunosorbent assay.24 Genomic DNA was isolated from cryopreserved white blood cells by a standard proteinase K digestion and phenol-chloroform method. Polymorphism analyses for IL-4 (−590 C [RIGHTWARDS ARROW] T), IL-4 receptor (Ile50val), IL-4 receptor (Q576R), and IL-1RN (intron 2 VNTR) were performed in duplicate according to modified protocols based on previously reported assays.25, 26, 27, 28 Briefly, PCR amplification of the promoter or coding regions of the genes was performed using specifically designed pairs of oligonucleotide primers. Polymorphisms were then identified by restriction enzyme length polymorphism (IL-4 and IL-4 receptor) or by direct size fractionation by VNTR (IL-1RN). Assays for TNF-α (−238, −308), IL-1 (−31, −511), and IL-10 (−592, −819, −1082) polymorphisms were performed by PCR followed by direct sequencing (ABI Prism 377 DNA sequencer, PE Biosystems, Foster City, CA). The primer sequences, annealing temperatures for PCR and detection methods used in assays are listed in Table I. All laboratory assays were conducted and interpreted blindly without the knowledge of the case or control status.

Table I. Primer Sequences and Methods Used for Detection of Cytokine Gene Polymorphisms
Gene1PrimersPCR conditionDetection method
  • 1

    TNF-α, tumor necrosis factor-α; IL, interleukin.

TNF-α(−238, −308)5′-CAAACACAGGCCTCAGGACTC-3′95°C 40 secDirect sequencing with primer 5′-TCTGGAAGTTAGAAGGAAAC-3′
 5′-AGGGAGCGTCTGCTGGGCTG-3′65°C 1 min 
  72°C 30 sec 
  35 cycles 
IL-1β (−31, −511)5′-CTCAGAGGCTCCTGCAATTG-3′95°C 45 secDirect sequencing with primer 5′-TCGTTCTGCAGTTGATGTCCA-3′
 5′-AGATAAGCAGTATCCATTCCC-3′55°C 1 min 
  72°C 1 min 
  35 cycles 
IL-IRN (intron 2 VNTR)5′-CTCAGCAACACTCCTAT-3′95°C 1 minSize fractionation in 2.5% agarose gel electrophoresis
 5′-TCCTGGTCTGCAGGTAA-3′58°C 1 min 
  72°C 1 min 
  35 cycles 
IL-4 (−590)5′-ACTAGGCCTCACCTGATACG-3′95°C 30 secBsmF1 digestion, 6%
  55°C 30 sec 
 5′-GTTGTAATGCAGTCCTCCTG3′72°C 30 secPolyacrylamide gel electrophoresis
  35 cycles 
IL-4R (Ile50 Val)5′-GGCAGGTGTGAGGAGCATCC-3′95°C 30 secRsaI digestion, 3% agarose & 1% NuSieve agarose gel electrophoresis
  55°C 30 sec 
 5′-GCCTCCGTTGTTCTCAGGTA-3′72°C 1 min1% NuSieve agarose gel electrophoresis
  35 cycles 
IL-4R (Q576R)5′-GCCCCCACCAGTGGCTACC-3′94°C 30 secMspI digestion, 6%
  55°C 30 sec 
 5′-GCCTTGTAACCAGCCTCTCCT-3′72°C 1 minPolyacrylamide gel electrophoresis
  40 cycles 
IL-10 (−592, −819, −1082)5′-ATCCAAGACAACACTACTAA-3′94°C 40 secDirect sequencing with primer 5′-TAAATATCCTCAAAGTTCC-3′ and 5′-TTGGCCTTAGAGTTTCTTTT-3′
 5′-TAAATATCCTCAAAGTTCC-3′54°C 45 sec 
  72°C 30 s 
  35 cycles 

Statistical analysis

The demographic characteristics and environmental factors as well as the gene frequencies of TNF-α, IL-1, IL-4 and IL-10 in patients and controls were compared and tested using χ2 statistics. In a matched case-control study, assuming the proportion exposed in the control group is more than 0.1 and the control-to-case ratio is 1, the statistical power for detecting an OR of 2.0 will be more than 80% on the basis of 200 cases and 200 controls using a 2-sided test at the α = 0.05 level of significance. Since environmental variables, including cigarette smoking and H. pylori infection, were the main risk factor for GC in our study, we also dichotomized these and investigated their effects on the risk of GC. Logistic regression analyses were used to evaluate the effect of genotypes, H. pylori infection and habit of cigarette smoking.

The data were analyzed using the SAS statistical package (SAS Institute, Cary, NC). ORs and 95% confidence intervals (CIs) are presented.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Demographic comparison of the studied population and controls is summarized in Table II. There were no differences in the distribution of age, gender, ethnicity or blood types between cases and controls. The risk of GC was significantly higher for those who had H. pylori infection (OR, 1.74; 95% CI 1.19–2.56; p = 0.005) or who had the habit of cigarette smoking (OR, 2.02, 95% CI 1.38–2.95; p = 0.0003).

Table II. Distribution of Cases and Controls According to Selected Sociodemographic Characteristics
CharacteristicsNo. of casesNo. of controlsOdds ratio (95% CI:)p-value
  • 1

    Subjects who had smoked cigarettes more than 4 days a week for more than 6 months were classified as cigarette smokers and others as nonsmokers.

Age (y)60.9 ± 12.660.7 ± 13.4
Gender    
 Female8488  
 Male136142
Ethnicity    
 Fukienese143149  
 Hakka2223
 Mainland China5558  
Blood type    
 A6959Referent 
 B56520.92 (0.55–1.54)0.75
 O81980.71 (0.45–1.11)0.14
 AB14210.57 (0.27–1.22)0.15
H. pylori    
 Seronegative69102Referent 
 Seropositive1511281.74 (1.19–2.56)0.005
Smoking1    
 No107151Referent 
 Yes113792.02 (1.38–2.95)0.0003

The distribution of cytokine gene polymorphisms among subjects with GC and controls is summarized in Tables III and IV. Because there were no differences in the frequencies of analyzed cytokine genotypes among patients from Fukien, Hakka and Mainland China (data not shown), we combined and analyzed them as a single group. No statistical significance was noted in the overall risk of developing GC for TNF-α, IL-1 and IL-4. A positive association between the IL-10 genotype and the overall risk of developing GC was noted. The IL-10 genotype containing the G allele at −1082, C allele at −819 or C allele at −592 positions was more frequent in cases than in controls. Because previous reports have documented that the IL-10 haplotype containing the G allele is associated with increased production of IL-10,19, 20 we further analyzed the OR of the IL-10 haplotype based on the IL-10 producing capability. The haplotype containing G allele had a higher OR (2.67; 95% CI 1.29–5.50) compared with ATA haplotype.

Table III. Association Between TNF-α and IL-1 Genotypes and Development of Gastric Carcinoma
Genotype1No. of casesNo. of controlsOdds ratio2 (95%CI)p-value
  • 1

    TNF-α, tumor necrosis factor-α; IL, interleukin.–Unadjusted.

TNF    
 −238    
  AA34Referent 
  AG422.67 (0.27–25.63)0.40
  GG2132241.27 (0.28–5.73)0.76
 −308    
  AA1316Referent 
  AG31291.32 (0.54–3.20)0.55
  GG1761851.17 (0.55–2.50)0.68
IL-1    
 −31    
  CC4742Referent 
  CT1021250.73 (0.45–1.19)0.21
  TT71631.01 (0.56–1.72)0.98
 −511    
  CC6961Referent 
  CT1061240.76 (0.49–1.16)0.20
  TT45450.88 (0.52–1.51)0.65
IL-1 RN    
 11185195Referent 
 1231301.09 (0.63–1.87)0.76
 13221.05 (0.15–7.56)0.96
 15111.05 (0.06–16.98)0.97
 22120.53 (0.05–5.86)0.60
Table IV. Association Between Interleukin (IL)-4 and IL-10 Genotypes and Development of Gastric Carcinoma
GenotypeNo. of casesNo. of controlsOdds ratio1 (95%CI)p-value
  • 1

    Unadjusted.

IL-4    
 −590    
  TT146163Referent 
  CT69551.40 (0.92–2.13)0.11
  CC5120.47 (0.16–1.35)0.16
IL-4 receptor    
 Ile50Val    
  II5152Referent 
  IV1201191.03 (0.65–1.63)0.91
  VV49590.85 (0.49–1.45)0.55
 Q576R    
  QQ160164Referent 
  QR57610.96 (0.63–1.46)0.84
  RR350.62 (0.14–2.62)0.51
IL-10    
 −592    
  AA88127Referent 
  AC105831.83 (1.23–2.71)0.003
  CC27201.95 (1.03–3.69)0.04
 −819    
  TT88127Referent 
  TC105831.83 (1.23–2.71)0.003
  CC27201.95 (1.03–3.69)0.04
 −1082    
  AA195217Referent 
  AG23112.14 (1.07–4.30)0.04
  GG22  
 −592/−819/−1082    
  AA/TT/AA88127Referent 
  AC/TC/AA90741.50 (1.00–2.31)0.048
  CC/CC/AA1716  
  AC/TC/GA1472.67 (1.29–5.50)0.008
  CC/CC/GA83  
  CC/CC/GG21  
  AC/TC/GG01  
  AC/TC/GA11  

In an effort to isolate the contribution of the cytokine genotype to different subtypes of GC, stratification analysis was also performed according to histology (intestinal vs. diffuse), stage (early vs. advanced) and location (cardia vs. non-cardia) of tumors. A higher risk of developing diffuse type (OR, 1.64; 95% CI 1.01–2.67) or cardia type cancer (OR, 2.44; 95% CI 1.13–5.27) was observed for the CT/CC genotype of IL-4 at the position −590. The effect of high IL-10 producing genotype was more prominent for the advanced stage (OR, 2.29, 95% CI 1.12–4.64) and the cardia location (OR, 3.21, 95% CI 1.06–9.73) compared with the early stage (OR, 1.19; 95% CI 0.26–5.56) and the non-cardia location (OR, 1.96, 95% CI 0.95–4.06).

The data in Table V show the effect of combined contributions of habitual smoking, H. pylori infection and high IL-10 producer genotype to the risk of developing GC.Compared with individuals carrying a low IL-10 producer genotype, a significantly higher risk of developing GC was observed in those with a high IL-10 producer genotype (OR 2.14; 95%CI 1.07–4.30, p = 0.03), and who were smokers (OR 3.05; 95% CI 1.08–8.62, p = 0.035) or H. pylori-seropositive (OR 2.32; 95% CI 0.98–5.50, p = 0.055). A much higher risk was observed for those with a high IL-10 producer genotype and who were both smoker and H. pylori-seropositive (OR 6.00, 95% CI 1.31–27.37, p = 0.02).

Table V. Combined Effects of High Interleukin (IL)-10 Producer Genotype and Environmental Risk Factors on the Risk of Gastric Carcinoma
H. pyloriSmokingHigh IL-10 producer genotype1Odds ration (95%CI)p-value
  • 1

    Any one of two alleles containing GCC was defined as a high IL-10 producer genotype.

+/−+/−1 
+2.14 (1.07–4.30)0.03
++2.32 (0.98–5.50)0.055
++3.05 (1.08–8.62)0.035
+++6.00 (1.31–27.37)0.02

Table VI shows results of the logistic regression analysis for significant risks for GC.Subjects who were infected with H. pylori and smoked heavily had 1.7- and 1.8- fold increased risks (95% CI 1.14–2.52 and 1.27–2.76), respectively. After adjusting for H. pylori infection and cigarette smoking, we found that subjects with a high IL-10 producer genotype had 2.54-fold increased GC risk (95% CI 1.24–5.61) compared with the AA/TT/AA genotype of IL-10.

Table VI. Logistic Regression Analysis of Significant Risk Factors for Gastric Carcinoma1
VariablesOR (95%CI)p-value
  • 1

    Two hundred and twenty gastric carcinoma patients and 230 controls with similar distribution of age, gender, and ethnicity.

  • 2

    Using the AA/TT/AA genotype as the referent.

Smoking1.87 (1.27–2.76)0.002
H. pylori infection1.70 (1.14–2.52)0.009
High interleukin-10 (IL-10) producer genotype22.54 (1.24–5.61)0.008

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The pivotal roles of different cytokines in regulating antimicrobial immunity and inflammation make them attractive candidates for being genetic host markers in evaluating individual susceptibility to GC development. They may influence the risk of developing GC by altering the quality and vigor of inflammatory responses produced by the host after exposure to various environmental or infectious triggers. The numbers of potential cytokine genes that need to be investigated are extensive. Here we chose to study 11 polymorphisms of the TNF-α, IL-1β, IL-1RN, IL-4, IL-4 receptor and IL-10 genes that are important in the H. pylori-associated inflammation.10, 11, 12, 13, 16, 17 Our results indicate that GC is significantly associated with the polymorphisms in the promoter region of the IL-10 gene. Such an association still existed even after being corrected for potential confounding variables such as H. pylori infection and cigarette smoking. The latter 2 environmental factors were also found to be high risks for developing GC. Specifically, individuals genetically predisposed to produce more IL-10 are at a higher risk of developing GC, in particular advanced and cardia subtypes.

The association of IL-10 genotypes with GC appears to be biologically and clinically important. IL-10 is a key immunosuppressive cytokine that gears the immune response toward a Th2 cell response.9 The haplotype alleles formed in the promoter region of the IL-10 gene at positions −1082, −819 and −592 are related to high IL-10 producing capability.19, 20 Compared with the ATA haplotype, the GCC haplotype is associated with a higher production of IL-10 in culture of stimulated peripheral blood mononuclear cells.19, 20 These high IL-10 producing haplotypes have been implicated in determining the severity of several autoimmune inflammatory diseases and chronic hepatitis C.19, 20 Our study was the first to show that such IL-10 haplotypes are linked to susceptibility and severity of GC. The finding that there was an increased risk of GC in high IL-10 producer haplotype was in agreement with the concept that Th2 cytokines including IL-10 are highly expressed in patients with GC.29, 30, 31 It is also intriguing to note that a more advanced stage and cardia location of GC tended to be associated with a high IL-10 producer genotype. This notion could partly be explained by reported findings that increased expression of mRNA and elevated serum levels of IL-10 are correlated with advanced stage and progression of GC.30, 31

Although numerous studies have addressed the potential impact of different risk factors on the development of GC,1, 2, 3 few have focused on the relationship between genotypes and environmental factors. In the present study, we noted that H. pylori infection, cigarette smoking and IL-10 genotypes are independent risk factors of GC. Both habitual smoking and H. pylori infection are known to affect local chemokine or cytokine expression in the stomach.23, 32 Shimoyama et al.23 have shown that smoking may enhance 2 C-X-X chemokines (growth-related oncogen-α and epithelial neutrophil activating protein 78) expression. Infection of H. pylori could lead to upregulation of TNF-α, IL-1 and IL-8 in gastric epithelia.32 Therefore, smoking and H. pylori infection may cooperate with cytokine genotype to exert their effects on gastric inflammation. Specifically, they modify the GC risk among those with high IL-10 producer haplotypes. Such an additive effect was more prominent in smoking than H. pylori infection, based on a greater increase of ORs for smoker. The combined effects highlight the notion that GC is a multifactorial disease that involves both environmental and genetic factors.1, 2 If our interpretation is correct, we also surmise that a certain portion of patients with a genetic predisposition favoring a Th2-type inflammatory response may be adversely affected by H. pylori infection and habitual smoking in the development and progression of GC.

An unexplained finding was that the IL-4 genotype was not found to be related to an overall risk of GC, and instead the low CT/CC genotype at position −590 was associated with cardia and diffuse subtypes of GC. This finding substantiates the likelihood of intrinsic heterogeneity in GC tumorigenesis and phenotype,2, 7 subclassified according to the anatomic location and the histologic changes. It also implies that different host susceptibility plays a role in such heterogeneity and that stratification of GC into different subtypes is mandatory for meaningful genetic analyses. Although IL-4 has been reported to be associated with growth and progression of GC in culture,33 interpretation should be exercised with caution in our study because of limited case numbers of cardia cancer. It needs to be replicated in other population and further studies.

Our findings did not reveal any significant association between IL-1 or TNF-α polymorphisms and GC. Previously, Jang et al.11 suggested that the −238A allele of TNF-α has a protective function against GC development, whereas El-Omar et al.12 and Machado et al.13 demonstrated that IL-1 polymorphisms are associated with increased risks of GC. Both the control populations in these latter studies consisted of Caucasians. However, 1 recent study from Japan did not show such an increased risk.14 One possible explanation is the ethnic variability in allele frequencies of cytokine genotype. In the control populations studied by El-Omar et al.,12 the frequencies at the −31 position of IL-1β were C/C 10.7%, C/T 38.2% and T/T 51%. However, our control population exhibited a remarkable difference, at 18.2%, 54.3% and 27.4%, respectively, which was similar to rates reported in a Japanese study and a previous study from Taiwan.14, 15 Besides the aforementioned ethnic variability in allele frequencies of IL-1, there might be a similar situation in other cytokine genes such as TNF-α and IL-10. The distribution of TNF-α and IL-10 genotypes in the control population of our study was consistent with what has been previously reported with a very high frequency (>90%) of G allele for TNF-α (−238, −308) and the A allele for IL-10 (−1082) in Chinese.28, 34 In contrast, the reported frequencies of the G allele for TNF-α (∼80%) and the A allele for IL-10 (∼50%) in Caucasians were relatively lower than in Chinese.28, 34 The reported frequency of the −238A in TNF-α varies significantly between ethnic groups, and it is rare (<3%)in the Chinese and Japanese.18, 28 Intriguingly, a new haplotype (GTA) of the IL-10 gene, which was not present in reports from Caucasians,18 was noted in our results. This haplotype was first reported by Mok et al.,35 who have demonstrated that 1 in 83 Southern Chinese had such a haplotype.

These results further indicate that a significant difference exists in the distribution of various cytokine genotypes in different ethnic groups. Another explanation is that the lack of associations may reflect genetic heterogeneity in the pathogenesis of GC. Taken together, ethnic differences as well as genetic heterogeneity may account for the variability in different studies of host susceptibility and should be taken into consideration in future studies of any candidate gene.

There are several limitations to our study. First, although we enrolled cases from 3 different hospitals located in different regions of Taiwan, inherent selection bias cannot be completely excluded in hospital-based case-control studies, and more cases are needed to confirm the preliminary findings, especially in circumstances of stratification analysis. Second, it is important to note that in vitro expression of selected cytokines is stimulus-dependent, and different experimental systems may yield different results for each different polymorphism. Determination of the exact functional consequence of each cytokine polymorphism was not performed in our study. More detailed in vitro and in vivo studies are needed to explain how genetic heterogeneity in the IL-10 or IL-4 promoter actually modifies GC development and severity. Third, although the effects of IL-10 and IL-4 alleles identified in our study are important in determining GC outcomes, they only account for a small percentage of GC patients. Other genes not investigated in our study may act either alone or in concert with those studied here and environmental factors in pathogenesis of GC. Fourth, cross-sectional determination of H. pylori status by serology may underestimate the rate of H. pylori infection in patients with GC.36 Finally, some other variables, such as dietary habits, were not investigated in our study. Therefore, studies using a larger number of subjects in different populations are needed to elucidate further gene-gene and gene-environment interactions in GC susceptibility.

In conclusion, our study provides evidence that genetically determined differences in cytokine production via promoter polymorphisms, notably IL-10 and somewhat IL-4, could contribute to individual susceptibility to GC development and progression. The independent effect of IL-10 genotype, H. pylori infection and cigarette smoking indicates that gastric carcinogenesis is induced in multiple steps involving both genetic and environmental factors. Remarkable differences in the distribution of cytokine genotypes in the control population between Taiwanese Chinese and Caucasians underscore the importance of genetic variation in different ethnic groups. Further studies in a larger number but ethnically diverse cohort are mandatory to elucidate gene-gene and gene-environment interactions in the pathogenesis of GC.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES