Dr H.-C. Chen, Department of Radiation Oncology, Chang Gung Memorial Hospital, Kaohsiung Hsien 833, Taiwan. E-mail: firstname.lastname@example.org
Background : The host genetic factors that determine the clinical outcomes of Helicobacter pylori-infected individuals remain unclear.
Aim : To elucidate the risks of host interleukin-1 (IL-1) genetic polymorphisms and H. pylori infection in the development of gastric cancer.
Methods : In a case–control study of 164 controls and 142 patients with gastric cancer, the IL-1B-511 biallelic polymorphisms and the IL-1RN penta-allelic variable number of tandem repeats were genotyped.
Results : The carriage of IL-1RN*2, male gender, old age and H. pylori infection independently increased the risk of gastric cancer, with odds ratios of 3.3 [95% confidence interval (CI), 1.4–7.7], 2.1 (95% CI, 1.2–3.8), 5.3 (95% CI, 3.1–9.0) and 2.2 (95% CI, 1.3–3.8), respectively. H. pylori-infected individuals who were carriers of IL-1RN*2 showed increased risks of both intestinal and diffuse types of gastric cancer, with odds ratios of 11.0 and 8.7, respectively. In addition, these individuals also had a higher score of intestinal metaplasia in the corpus than did uninfected non-carriers.
Conclusions : This study is the first to verify IL-1RN*2 as an independent factor governing the development of gastric cancer in Asian individuals. A combination of H. pylori testing and host genotyping may target the eradication of H. pylori to high-risk individuals.
Helicobacter pylori is a well-recognized pathogen that chronically infects more than 50% of the world's population. However, only approximately 20% of infected individuals develop clinically significant diseases, such as peptic ulcer, gastric adenocarcinoma or mucosa-associated lymphoid tissue lymphoma.1–4 It remains unclear which host, bacterial and environmental factors are the critical determinants that affect the outcomes of infected individuals.
It has been suggested that phenotypic or genotypic differences amongst bacterial strains may account for the development of severe diseases. Such associations with diseases have been proposed for the cag pathogenicity island, vacA and babA genotypes.5–9 Infection with cagA-positive strains has been reported to increase the risk of duodenal ulcer,5 atrophic gastritis10 and gastric adenocarcinoma.6 The vacAs1 and vacAm1 strains are also strongly associated with a higher degree of gastric inflammation and higher risks of peptic ulcer, atrophic gastritis and gastric adenocarcinoma.11,12 However, the predominant genotype of H. pylori that circulates in Asian countries is a cagA-positive, vacAs1 and babA2 genotype, independent of clinical outcomes.5,13,14 Therefore, bacterial virulent factors have so far failed to explain why the ulcer or gastric cancer phenotype develops in Asia.
Specific host characteristics that may determine the susceptibility to H. pylori-associated diseases include gender, human leucocyte antigen (HLA) genotype, blood group, gastric acid physiology and human genetic polymorphisms.15,16 Recently, polymorphisms of the interleukin-1β gene (IL-1B) and the IL-1 receptor antagonist gene (IL-1RN) have been associated with an increased risk of both hypochlorhydria and gastric adenocarcinoma.17,18H. pylori infection can elicit an impressive cytokine cascade of the gastric mucosa with IL-1β at its centre.19 IL-1β is also a powerful inhibitor of gastric acid secretion, being 100-fold more potent than proton pump inhibitors.20 The modulation of acid secretion and cytokine production by IL-1β may affect the outcomes of the hosts. Three diallelic polymorphisms with C–T base transitions in IL-1B have so far been reported.21 They are located − 511, − 31 and + 3954 base pairs (bp) from the transcription start site. A polymorphic allele with a T instead of a C at position − 511 of the regulatory region of the IL-1B gene is associated with increased IL-1β production.16,22 IL-1RN encodes the IL-1 receptor antagonist (IL-1ra), which is an anti-inflammatory cytokine and competes with IL-1β by binding to IL-1 receptors. The IL-1RN gene has a penta-allelic 86-bp tandem repeat in the second intron, resulting in a short allele (IL-1RN*2, two repeats) or a long allele (IL-1RN*L, three to six repeats).23 IL-1RN*2 is associated with increased production of IL-1ra and IL-1β.24,25 In a previous study, we demonstrated that IL-1 genetic polymorphisms influenced mucosal IL-1β levels of H. pylori-related gastritis and were related to gastric inflammation and gland atrophy.26
In this study, we assess the combined risk of IL-1 genetic polymorphisms and H. pylori infection for the development of gastric cancer, and investigate the relationship between IL-1 genetic polymorphisms and the histopathological features of gastritis in Taiwan.
Materials and methods
The study subjects included 142 unrelated patients with gastric adenocarcinoma and 164 healthy controls (Table 1, see later). Patients with gastric adenocarcinoma (mean age, 66.6 ± 14.7 years; range, 25–89years; male/female ratio, 111/31) were diagnosed and underwent resection of the cancer at the Kaohsiung Veterans General Hospital and Kaohsiung Chung Gung Memorial Hospital, Kaohsiung, Taiwan. The tumours were classified as intestinal (n = 83), diffuse (n = 42) or atypical (n = 17), according to Lauren's classification.27 One hundred and sixty-four consecutive, ethnically matched, asymptomatic healthy volunteers (mean age, 53.9 ± 13.4 years; range, 17–83 years; male/female ratio, 81/83), without a past history of peptic ulcer or gastrointestinal cancer, served as healthy controls; their endoscopic findings were normal or showed mild gastritis only. To minimize ethnic bias, all patients and control subjects were Han Chinese; aboriginal and alien populations were excluded. No subjects had received treatment for H. pylori infection. The presence of H. pylori infection was determined by rapid urease test.28,29 In addition, a serological test using a commercial immunoglobulin G enzyme-linked immunoabsorbent assay kit (Premier H. pylori; Meridian Diagnostics Inc., Cincinnati, OH, USA) was performed on all patients and controls. At least one test had to yield positive results for a subject to be designated as having the infection. To adjust for clinical characteristics, the following data were recorded for each age, sex, blood type, smoking history and alcohol consumption. The study was approved by the Medical Research Committee of the Kaohsiung Veterans General Hospital. All patients and controls gave informed consent.
Table 1. Demographic data of gastric cancer patients and healthy controls
Healthy controls (n = 164)
Gastric cancer patients (n = 142)
53.9 ± 13.4
66.6 ± 14.7
Helicobacter pylori infection
IL-1B and IL-1RN genotyping
Genomic DNA was extracted from 3 mL of whole blood using a QIAamp DNA Extraction Mini Kit (QIAGEN Inc., Valencia, CA, USA). DNA (30–100 ng) was used in a volume of 25 µL polymerase chain reaction (PCR) with 0.1 mm deoxynucleoside triphosphates (dNTPs), 0.05 µm of each primer, 2.5 U of AmpliTaq DNA polymerase (Perkin-Elmer Corp., Foster City, CA, USA) and 2.5 µL of 10 × PCR buffer including 1.0–3.0 mm MgCl2. The genotypes of IL-1B and IL-1RN were determined as described previously.30,31 The IL-1B-511(C/T) polymorphisms were characterized using polymerase chain reaction-restriction fragment length polymorphism.30 The PCR products of IL-1B-511 (304 bp) were digested overnight at 37 °C with 4 U Ava1, separated and checked by electrophoresis on 2.5% agarose gel: the 190- and 114-bp bands were genotype C/C, 304-, 190- and 114-bp bands genotype C/T and a single 304-bp band genotype T/T. IL-1RN polymorphisms were characterized by the penta-allelic variable number of tandem repeats in the second intron of IL-1RN.31 The PCR products of IL-1RN were separated and checked by electrophoresis on 2% agarose gel. For IL-1RN genotyping, the 240-bp product contained two 86-bp repeats (allele 2), the 326-bp product three 86-bp repeats (allele 4), the 412-bp product four 86-bp repeats (allele 1), the 498-bp product five 86-bp repeats (allele 3) and the 584-bp product six 86-bp repeats (allele 5).
A histological examination of the stomach was carried out during endoscopy in subjects who provided informed consent for a topographical histopathological study. Two specimens each were taken from the antrum (pyloric gland area) and corpus (fundic gland area) at standard topographical sites. The biopsy specimens were fixed in 10% buffered formalin, embedded in paraffin and sectioned. The sections were stained with a haematoxylin and eosin stain and a modified Giemsa stain, as described previously.32,33 Sections were examined blind to the patient's clinical diagnosis. The scores of acute inflammation (neutrophil infiltration), chronic inflammation (mononuclear cell infiltration), glandular atrophy and intestinal metaplasia were graded from 0 to 3 as described by the updated Sydney system.34
Statistical evaluations were performed using the SPSS/Windows computer software package (Chicago, IL, USA). Two-sample t-tests were used to compare the mean values of the variables considered to be continuous in the gastric cancer patients and healthy controls. The chi-squared test, with or without Yate's correction for continuity, and Fisher's exact test, when appropriate, were applied to analyse the categorized variables. Differences were considered to be significant at P < 0.05. Unconditional logistic regression analysis was carried out to quantify the effects of bacterial and host factors for gastric cancer as the dependent variable. Variables including age, sex, smoking history and H. pylori infection were adjusted in all the models. A stepwise forward conditional method was used to identify the independent risk factors for the development of gastric cancer. The studied variables included the following: age (< 60 or ≥ 60 years), sex, blood type (A type or non-A type), history of smoking (< 1 pack/week or ≥ 1 pack/week), alcohol consumption (< 80 g/day or ≥ 80 g/day), H. pylori status (presence or absence), carriers of IL-1B-511T (yes or no) and carriers of IL-1RN*2 (yes or no).
We estimated that a 20% difference in the susceptible factor could be present in gastric cancer patients. Based on this assumption, 95 subjects had to be studied in each group to yield a statistical power of 0.80 and an α value of 0.05.
Table 1 shows the demographic characteristics of gastric cancer patients and healthy controls. The patients with gastric cancer were significantly older than the healthy controls (P < 0.001). In addition, the rates of male gender, cigarette smoking and H. pylori infection of gastric cancer patients were significantly higher than those of healthy controls (P < 0.001, P < 0.01 and P < 0.001, respectively).
IL-1 loci polymorphisms and risk of gastric adenocarcinoma
Table 2 presents the IL-1B-511 and IL-1RN genetic polymorphisms in the gastric cancer patients and healthy controls. No significant differences were found between the groups in the genotype frequency of the IL-1B gene at position − 511. However, the carriage rate of IL-1RN*2 was markedly higher in gastric cancer patients than in healthy controls (15.5% vs. 5.5%, P = 0.003). The analysis of allele frequencies also revealed that the frequency of IL-1RN*2 in gastric cancer patients was significantly higher than that in healthy controls (Table 2; 8.2% vs. 2.7%, P = 0.001).
Table 2. Genotype and allele frequencies of IL-1B and IL-1RN in gastric cancer patients and healthy controls
Healthy controls (n = 164)
Gastric cancer patients (n = 142)
With reference to different histological types of gastric cancer, the carriage of IL-1RN*2 was associated with increased risks of intestinal, diffuse and atypical types, with adjusted odds ratios (ORs) of 3.3, 2.4 and 2.1 [95% confidence intervals (CIs) of 1.1–10.0, 0.7–8.2 and 0.3–13.5), respectively.
Combined risk of IL-1 genetic polymorphisms and H. pylori infection for gastric cancer
Table 3 shows the combined risk of H. pylori infection and IL-1B/IL-1RN genetic polymorphisms for the development of gastric cancer. H. pylori infection was associated with an increased risk of gastric cancer, with an adjusted OR of 2.1 (95% CI, 1.2–3.7) in non-carriers of IL-1RN*2. In subjects not infected with H. pylori, the carriage of IL-1RN*2 was also associated with a trend towards an increased risk of gastric cancer, with an adjusted OR of 2.2 (95% CI, 0.6–7.7). Interestingly, H. pylori-infected individuals who were carriers of IL-1RN*2 exhibited a very high risk of gastric cancer, with an OR of 8.1 (95% CI, 2.0–32.5). The data indicate a synergistic interaction between IL-1RN*2 and H. pylori infection in the development of gastric cancer.
Table 3. Combined risks of IL-1 genetic polymorphisms and Helicobacter pylori infection for gastric cancer in Taiwan
The relationship between H. pylori infection and IL-1B-511 genotypes was also examined to clarify the possible interactions of other IL-1 polymorphisms and bacterial factors in the development of gastric cancer, but no synergistic effects were found.
Risk of combined IL-1B-511 and IL-1RN genotypes for gastric cancer
Table 4 presents the risk of combined IL-1B-511 and IL-1RN genotypes for the development of gastric cancer. Compared with individuals with IL-1B-511C/C and IL-1RN*L/*L, the IL-1B-511T and IL-1RN*2 carriers had an increased risk of gastric cancer, with an OR of 2.9 (95% CI, 1.0–8.6). The risk was similar to that of individuals with IL-1B-511C/C and IL-1RN*2 (adjusted OR, 3.1). The data suggest that no synergistic effects between the two polymorphic genotypes exist in the development of gastric cancer.
Table 4. Risk of combined IL-1B-511 and IL-1RN genotypes for gastric cancer in Taiwan
Combined risk of IL-1 genetic polymorphisms and H. pylori infection for different subtypes of gastric cancer
Table 5 shows the combined risk of IL-1 genetic polymorphisms and H. pylori infection for different subtypes of gastric cancer. H. pylori infection was associated with an increased risk of intestinal-type gastric cancer, with an adjusted OR of 2.7 (95% CI, 1.2–5.7). The carriage of IL-1RN*2 was also associated with an increased risk of gastric cancer (adjusted OR, 2.7; 95% CI, 2.3–13.0). H. pylori-infected individuals who were carriers of IL-1RN*2 had a very high risk of intestinal-type gastric cancer, with an OR of 11.0 (95% CI, 2.2–56.1). A synergistic interaction between IL-1RN*2 and H. pylori infection was also observed in the development of diffuse-type gastric cancer. The cancer risk associated with H. pylori infection was substantially increased by the carriage of IL-1RN*2 (combined risk, 8.7; 95% CI, 1.5–48.7).
Table 5. Combined risks of IL-1RN polymorphism and Helicobacter pylori infection for intestinal and diffuse types of gastric cancer in Taiwan
In this study, the combined risk of IL-1 genetic polymorphisms and H. pylori infection for atypical-type gastric cancer was not analysed because the number of cases was too low.
Independent risk factors for the development of gastric cancer
A stepwise forward logistic regression analysis for genetic, clinical and bacterial variables was performed to search for independent risk factors of gastric cancer. Multivariate analysis indicated that the carriage of IL-1RN*2, male gender, old age and H. pylori infection were independent risk factors for the development of gastric cancer (Table 6). The adjusted ORs of the four parameters were 3.3, 2.1, 5.3 and 2.2, respectively (95% CIs of 1.4–7.7, 1.2–3.8, 3.1–9.0 and 1.3–3.8, respectively). The other factors (blood type, history of smoking, alcohol consumption and carriage of IL-1B-511T) did not increase the risk of gastric cancer.
Table 6. Independent risk factors for the development of gastric cancer in Taiwan
OR (95% CI)
CI, confidence interval; OR, odds ratio.
H. pylori infection
Effects of IL-1RN genotypes and H. pylori infection on histological gastritis
Figure 1 presents the relationship between H. pylori infection and the severity of gastritis. In the corpus, there were no differences in the scores of chronic inflammation, acute inflammation, glandular atrophy and intestinal metaplasia between H. pylori-positive and H. pylori-negative subjects. However, the chronic inflammation scores in the antrum were significantly higher in H. pylori-infected individuals than in non-infected individuals (2.5 ± 0.1 vs. 2.2 ± 0.1, P = 0.05).
Figure 2 shows the combined effects of IL-1RN genotypes and H. pylori infection on histological gastritis. Individuals who were H. pylori positive and IL-1RN*2 carriers had higher scores of intestinal metaplasia in the antrum and corpus than H. pylori-negative individuals who had IL-1RN*L/*L (antrum: 1.5 ± 0.4 vs. 0.7 ± 0.2, P = 0.075; corpus: 1.3 ± 0.5 vs. 0.4 ± 0.1, P = 0.022). In addition, these infected IL-1RN*2 carriers also showed a trend towards increased scores of glandular atrophy (1.6 ± 0.2 vs. 1.1 ± 0.1, P = 0.069) and chronic inflammation (2.6 ± 0.2 vs. 2.0 ± 0.1, P = 0.081) in the corpus.
On a global scale, gastric cancer remains the world's second most common malignancy, having been overtaken by lung cancer in the late 1980s.35 There is substantial international variation in gastric cancer incidence, with the highest rates reported from Japan and eastern Asia. The discovery of H. pylori in the early 1980s proved a turning point in the understanding of the pathogenesis of gastric cancer. It has been linked to gastric cancer by geographical correlation, case–control, nested cohort and animal studies.36,37 The eradication of H. pylori infection is also associated with a decreased recurrence of intestinal-type gastric cancer in patients who have undergone endoscopic resection of the primary tumour.38 However, it is still a matter of speculation why only a minority of individuals harbouring H. pylori develop gastric cancer.
This study demonstrated that H. pylori infection and the carriage of IL-1RN*2 independently increased the risk of gastric cancer in Taiwan. This report is the first to verify IL-1RN*2 as a risk factor of gastric cancer in Asian countries. In addition, an interaction was found between H. pylori infection and the IL-1RN genotype in the development of gastric cancer. Our data indicated that H. pylori infection increased the risk of gastric cancer 2.1-fold in non-carriers of IL-1RN*2. Meanwhile, IL-1RN*2 carriers without H. pylori infection showed an increased risk of gastric cancer, with an OR of 2.2. Notably, H. pylori-infected individuals who were carriers of IL-1RN*2 had a very high risk of gastric cancer, with an OR of 8.1 (95% CI, 2.0–32.5). These results imply a synergistic interaction between IL-1RN*2 and H. pylori infection in gastric carcinogenesis. These findings are important, especially in Asian countries where the incidence of gastric cancer is high. As proposed by Peek and Blaser, H. pylori-infected individuals with genetic polymorphisms that are associated with high levels of IL-1β expression are most likely to benefit from bacterial eradication, because such treatment can substantially reduce the risk of gastric cancer.39 According to the presented results, the eradication of H. pylori in infected IL-1RN*2 carriers has the potential to reduce the incidence of gastric cancer in Taiwan by 15% (22/142).
In this study, the carriage of IL-1RN*2 was associated with an increased risk of intestinal-type gastric cancer, with an adjusted OR of 3.3 (95% CI, 1.1–10.0), but the association was not statistically significant for diffuse-type gastric cancer (adjusted OR, 2.4; 95% CI, 0.7–8.2). The risks for both histological types of cancer in these carriers were markedly increased by H. pylori infection, with ORs of 11.0 and 8.7, respectively. Our work also demonstrated that H. pylori-infected IL-1RN*2 carriers had higher scores of intestinal metaplasia in the corpus than uninfected IL-1RN*L/*L carriers. In addition, these individuals also had higher scores of glandular atrophy and chronic inflammation. These findings agree with the pattern of gastritis typically associated with H. pylori-induced carcinogenesis, i.e. gastritis involving the acid-secreting corpus leading to hypochlorhydria, gastric atrophy and intestinal metaplasia.40 In a previous report, carriers of IL-1RN*2 exhibited higher mucosal IL-1β levels than non-carriers.26 IL-1β is the most powerful acid inhibitor. Beales and Calam have shown that IL-1β inhibits acid secretion in cultured rabbit parietal cells by multiple pathways.41 These findings, taken together, suggest that polymorphisms of the IL-1RN gene may contribute to the levels of IL-1β and histological changes in the gastric mucosa.
Recently, Figueiredo et al. have reported that H. pylori testing and IL-1B/IL-1RN genotyping may provide an opportunity to identify Caucasians at high risk for gastric cancer.42 In their study, IL-1B-511*T homozygous for IL-1RN*2 had an increased risk of gastric cancer. Infection with vacAs1-, vacAm1- and cagA-positive strains of H. pylori was also associated with an increased risk of gastric cancer. However, the IL-1B-511 genetic polymorphism was not associated with gastric cancer in the Japanese population43 or in this study. It is worth noting that the genotyping of H. pylori clinical isolates requires invasive procedures to obtain gastric specimens. In contrast, serological testing for H. pylori and blood sampling for IL-1RN genotyping may provide a non-invasive tool for assessing the risks of gastric cancer.
In conclusion, this study demonstrates that both H. pylori infection and the carriage of IL-1RN*2 independently increase the risk of gastric cancer in Taiwan. The two factors interact synergistically in gastric carcinogenesis. A combination of H. pylori testing and host genotyping may evaluate the gastric cancer risk and target the eradication of H. pylori to high-risk individuals.
This study was supported by grants from the Kaohsiung Veterans General Hospital (VGHSU91-2) and National Science Council (NSC89-2315-B-075B-001). We appreciate the invaluable comments of Professor David Y. Graham.