Liviu A. Sicinschi

Interleukin-1β and Interleukin-1 Receptor Antagonist Gene Polymorphisms and Gastric Cancer: A Meta-analysis

Background: Polymorphisms of interleukin-1B (IL1B) and its receptor antagonist (IL1RN) genes have been inconsistently associated with gastric cancer risk. We examined these associations by performing meta-analyses. Materials and Methods: Twenty-five studies testing the association between IL1B and/or IL1RN gene polymorphisms and gastric cancer were examined: 14 studies of IL1B-511, 14 studies of IL1B-31, 8 studies of IL1B+3954, and 23 studies of IL1RN. Overall and ethnicity-specific summary odds ratios and corresponding 95% confidence intervals for gastric cancer associated with these polymorphisms were estimated using fixed- and random-effects models. Heterogeneity and publication bias were evaluated. Results: IL1B-511T and IL1RN*2 were associated with gastric cancer risk in Caucasians, but not in Asians. For IL1B-511T, the association in Caucasians was stronger when intestinal-subtype and noncardia gastric cancer cases were examined. A nonsignificant trend was observed between IL1B-31C and gastric cancer in Caucasians. No significant association of IL1B+3954T and gastric cancer risk was detected. Studies with better methodologic characteristics reported stronger effects. There was no evidence of publication bias. Conclusion: IL1B-511T is associated with gastric cancer susceptibility in Caucasians. The meta-analyses suggest that the conflicting results among studies may be explained by variation in allele frequencies among the ethnic groups and variation in tumor types, as well as by the methodologic quality of the studies. (Cancer Epidemiol Biomarkers Prev 2006;15(9):1674–87)

Human and Helicobacter pylori coevolution shapes the risk of gastric disease

Significance Theory predicts that chronic pathogens with vertical or familial transmission should become less virulent over time because of coevolution. Although transmitted in this way, Helicobacter pylori is the major causative agent of gastric cancer. In two distinct Colombian populations with similar levels of H. pylori infection but different incidences of gastric cancer, we examined human and pathogen ancestry in matched samples to assess whether their genomic variation affects the severity of premalignant lesions. Interaction between human Amerindian ancestry and H. pylori African ancestry accounted for the geographic disparity in clinical presentation. We conclude that coevolutionary relationships are important determinants of gastric disease risk and that the historical colonization of the Americas continues to influence health in modern American populations. Helicobacter pylori is the principal cause of gastric cancer, the second leading cause of cancer mortality worldwide. However, H. pylori prevalence generally does not predict cancer incidence. To determine whether coevolution between host and pathogen influences disease risk, we examined the association between the severity of gastric lesions and patterns of genomic variation in matched human and H. pylori samples. Patients were recruited from two geographically distinct Colombian populations with significantly different incidences of gastric cancer, but virtually identical prevalence of H. pylori infection. All H. pylori isolates contained the genetic signatures of multiple ancestries, with an ancestral African cluster predominating in a low-risk, coastal population and a European cluster in a high-risk, mountain population. The human ancestry of the biopsied individuals also varied with geography, with mostly African ancestry in the coastal region (58%), and mostly Amerindian ancestry in the mountain region (67%). The interaction between the host and pathogen ancestries completely accounted for the difference in the severity of gastric lesions in the two regions of Colombia. In particular, African H. pylori ancestry was relatively benign in humans of African ancestry but was deleterious in individuals with substantial Amerindian ancestry. Thus, coevolution likely modulated disease risk, and the disruption of coevolved human and H. pylori genomes can explain the high incidence of gastric disease in the mountain population.

Phylogeographic origin of Helicobacter pylori is a determinant of gastric cancer risk

Background and aims Helicobacter pylori colonises the stomach in half of all humans, and is the principal cause of gastric cancer, the second leading cause of cancer death worldwide. While gastric cancer rates correlate with H pylori prevalence in some areas, there are regions where infection is nearly universal, but rates of gastric cancer are low. In the case of Colombia, there is a 25-fold increase in gastric cancer rate in the Andean mountain (high risk) region compared to the coastal (low risk) region, despite similarly high (∼90%) prevalence of H pylori in the two locations. Our aim was to investigate the ancestral origin of H pylori strains isolated from subjects in these high- and low-risk regions and to determine whether this is a predictive determinant of precancerous lesions. Methods Multi-locus sequence typing was used to investigate phylogeographic origins of infecting H pylori strains isolated from subjects in the Pacific coast and Andes Mountains in the state of Nariño, Colombia. We analysed 64 subjects infected with cagA+ vacA s1m1 strains. Gastric biopsy slides from each individual were scored for histological lesions and evaluated for DNA damage by immunohistochemistry. Results We show that strains from the high-risk region were all of European phylogeographic origin, whereas those from the low risk region were of either European (34%) or African origin (66%). European strain origin was strongly predictive of increased premalignant histological lesions and epithelial DNA damage, even in the low-risk region; African strain origin was associated with reduced severity of these parameters. Conclusion The phylogeographic origin of H pylori strains provides an explanation for geographic differences in cancer risk deriving from this infection.

Gastric cancer risk in a Mexican population: Role of Helicobacter pylori CagA positive infection and polymorphisms in interleukin‐1 and ‐10 genes

Several polymorphisms of the IL1B and IL10 gene promoters have been reported to be associated with gastric cancer risk in Caucasians. However, studies in other populations have shown differing results. We aimed to test for associations between polymorphisms in IL1B (−31 and +3954), IL10−592 and IL1RN variable number of tandem repeats (VNTR) and risk of gastric cancer in a Mexican population. DNA was extracted from sera of 183 gastric adenocarcinoma patients and 377 controls. The IL1B−31, IL1B+3954 and IL10−592 biallelic polymorphisms were discriminated using 5′ Nuclease (TaqMan) assays and Pyrosequencing. The IL1RN penta‐allelic VNTR polymorphism was genotyped using PCR followed by GeneScan analysis. A significant interaction was found between IL1B−31 and CagA status for the risk of intestinal‐type gastric cancer (p=0.023). Among CagA positive subjects, those with IL1B−31CC genotype had an increased risk of intestinal‐type gastric cancer (OR 3.19, 95%CI=1.05–9.68), compared to carriers of IL1B−31TT genotype. In contrast, among CagA negative subjects, no significant association of IL1B−31CC genotype with gastric cancer was observed. The IL10−592CC genotype was associated with more than doubling of the risk of the intestinal‐type gastric cancer (OR, 2.20, 95%CI=1.04–4.65). A nonsignificantly increased risk for intestinal‐type gastric cancer was found in IL1RN*2 carriers (OR 1.49, 95%CI=0.89–2.50). None of these polymorphisms was significantly related to the risk of diffuse‐type gastric cancer. No significant association was found between risk of gastric cancer and the IL1B+3954 polymorphism. Individuals carrying 2 or more of the risk‐associated alleles (IL1B−31C, IL1RN *2 and IL10−592C) were at increased risk for intestinal‐type gastric cancer, compared to those with 0 or 1 risk‐associated allele. The risk from multiple risk‐associated alleles was especially high in subjects infected with CagA positive H. pylori. Our results support the identification of the IL1B−31 promoter polymorphism as a useful marker for risk of intestinal type gastric cancer in persons with CagA positive H. pylori infections.

Promoter DNA Hypermethylation in Gastric Biopsies from Subjects at High and Low Risk for Gastric Cancer

Gene promoter CpG island hypermethylation is associated with Helicobacter pylori (H. pylori) infection and may be an important initiator of gastric carcinogenesis. To examine factors influencing methylation, we utilized bisulfite Pyrosequencing® technology for quantitative analysis of promoter DNA methylation in RPRM, APC, MGMT and TWIST1 genes using DNA from 86 gastric biopsies from Colombian residents of areas with high and low incidence of gastric cancer. H. pylori colonies were cultured from the same subjects, and gastric pathology was evaluated. Virulence factors cagA (including segments of the 3′ end, encoding EPIYA polymorphisms) and vacA s and m regions were characterized in the H. pylori strains. Using univariate analysis, we found significantly elevated levels of RPRM and TWIST1 promoter DNA methylation in biopsies from residents of the high‐risk region compared to those from residents of the low‐risk region. The presence of cagA and vacA s1m1 alleles were independently associated with elevated levels of promoter DNA methylation of RPRM and MGMT. Using multivariate analysis, DNA methylation of RPRM was associated with location of residence, cagA and vacA s1m1 status and methylation of TWIST1. We conclude that cagA and vacA virulence determinants are significantly associated with quantitative differences in promoter DNA methylation in these populations, but that other as yet undefined factors that differ between the populations may also contribute to variation in methylation status.

Association of interleukin-1beta gene polymorphisms with precancerous gastric lesions in African Americans and Caucasians.

OBJECTIVE: Interleukin-1β plays an important role in inflammation and gastric physiology. Polymorphisms of the IL1B gene have been associated with gastric atrophy and increased cancer risk, especially in Helicobacter pylori-infected subjects. The aim of this study was to evaluate the relationship between IL1B and IL1 receptor antagonist gene polymorphisms and the risk of multifocal atrophic gastritis in African Americans and Caucasians.METHODS: Genomic DNA was extracted from gastric biopsies of 269 adult outpatients (172 African Americans and 97 Caucasians) undergoing diagnostic upper gastrointestinal endoscopy. Histological diagnosis was evaluated according to the updated Sydney System and H. pylori status was assessed by Steiner silver stain. Polymorphisms of the IL1B gene (−511, −31, and +3954) and the IL1 receptor antagonist were investigated by PCR-RFLP. Logistic regression models were used to identify variables associated with multifocal atrophic gastritis in terms of odds ratios and 95% confidence intervals.RESULTS: Considering subjects with normal histology and nonatrophic gastritis as controls, a significant association was found between IL1B+3954T carrier and multiatrophic gastritis (OR 2.23, 95% CI 1.28, 3.88). Analyses stratified by ethnic group demonstrated similar associations in both African Americans (OR 2.23, 95% CI 1.14, 4.37) and Caucasians (OR 2.04, 95% CI 0.74, 5.65). A positive but not significant association was found between the allele 2 of the IL1RN and the presence of multifocal atrophic gastritis. The remaining proinflammatory polymorphisms were not associated with this precancerous lesion.CONCLUSIONS: Our results suggest that the presence of IL1B+3954T allele is a risk marker for multifocal atrophic gastritis in the population studied.

Detection and typing of Helicobacter pylori cagA/vacA genes by radioactive, one-step polymerase chain reaction in stool samples from children

The detection and molecular typing of Helicobacter pylori virulence genes in human stool specimens by polymerase chain reaction (PCR) require an adequate amount of bacterial DNA and an appropriately adjusted PCR protocol. DNA was isolated from stool samples of 39 H. pylori-infected and nine uninfected Colombian children using the QIAamp Kit following the manufacturers instructions but with modifications. DNA templates were amplified for the vacA s and m regions and for the cagA gene by PCR using radioactively labeled (32P) primers. The modifications in the standard Qiagen protocol of stool DNA extraction increased the final concentration of eluted total stool DNA 4.7 times (117 +/- 17 versus 22 +/- 3 ng/microl; P < 0.0001). Nevertheless, its amplification by regular PCR programs (30-40 cycles) did not generate visible signals because of the very low ratio of H. pylori DNA to other DNA. PCR for 80 cycles successfully amplified vacA in 36/39 samples (sensitivity, 92.3%) and cagA fragments in 21/39 (53.8%) fecal DNA samples. Both s and m vacA regions were amplified in 33/36 (91.7%) DNA samples. The s1m1 genotype was the most commonly isolated variant, accounting for 17/36 or 47.2% of positive samples. The s2m2 genotype was ascertained to be frequent also (14/36 or 38.9%). Almost all (94.1%) s1m1 genotypes were cagA positive. The majority of s2m2 genotypes (78.6%) were not associated with the cagA gene. Neither cagA nor vacA fragments were amplified from DNA isolates of H. pylori-uninfected children nor from DNA isolated from six gastrointestinal bacterial strains (specificity, 100%). The data suggest that the proposed modified technique of DNA extraction and PCR assay of stool samples may be an effective and reliable noninvasive tool for the detection and typing of H. pylori cagA/vacA virulence genes in infected individuals.

Eosinophils and mast cells in chronic gastritis: Possible implications in carcinogenesis

Eosinophils and mast cells participate in the immune response against Helicobacter pylori, but their involvement in the gastric precancerous process is unclear. This study aimed to estimate eosinophil and mast cell density in antral mucosa in subjects from 2 Colombian populations with contrasting gastric cancer risks. Gastric mucosa biopsies were collected from 117 adult males (72 from a high-risk area and 45 from a low-risk area). A histopathology score was used to quantify severity of the lesions. Quantitation of eosinophils in hematoxylin-eosin-stained sections and mast cells in immunostained sections for CD117/c-Kit was performed. Helicobacter pylori infection and genotyping were assessed in Steiner stain and polymerase chain reaction, respectively. Logistic regression models and semiparametric cubic smoothing splines were used for analysis of the results. Eosinophil density was significantly higher in subjects from the low-risk area as compared with subjects from the high-risk area. In both populations, eosinophil density increased with the histopathology score in the progression of lesions from normal morphology to multifocal atrophic gastritis. Intestinal metaplasia and dysplasia specimens showed further increase in eosinophil density in the high-risk area but an abrupt decrease in the low-risk area. Mast cell density increased in parallel to the histopathology score in both populations. Our results suggest that eosinophils play a dual role in chronic gastritis. In the low-risk area, elevated eosinophil density represents a T helper 2-biased response that may down-regulate the effects of proinflammatory cytokines preventing cancer development. In contrast, in the high-risk area, eosinophils might promote a T helper 1-type response leading to progression of precancerous lesions.

Non-invasive genotyping of Helicobacter pylori cagA, vacA, and hopQ from asymptomatic children.

Background:  Helicobacter pylori infection is usually acquired in childhood, but little is known about its natural history in asymptomatic children, primarily due to the paucity of non‐invasive diagnostic methods. H. pylori strains harboring cagA and specific alleles of hopQ and vacA are associated with increased risk for gastric cancer. Many studies of H. pylori virulence markers in children have the bias that symptomatic subjects are selected for endoscopy, and these children may harbor the most virulent strains. Our aim is to genotype cagA, hopQ, and vacA alleles in stool DNA samples of healthy Colombian children residing in an area with high incidence of gastric cancer, to avoid selection bias resulting from endoscopy.

Helicobacter pylori Genotyping and Sequencing Using Paraffin-Embedded Biopsies from Residents of Colombian Areas with Contrasting Gastric Cancer Risks

Background:   cagA‐positive and vacA s1 and m1 genotypes of Helicobacter pylori are associated with an elevated risk of gastric cancer (GC). We determined these genotypes using paraffin‐embedded gastric biopsy specimens harvested from infected individuals and compared genotype distributions in two Colombian populations residing in geographic regions with a high and low incidence of GC.