Association of Helicobacter pylori babA2 gene and gastric cancer risk: a meta-analysis

The association of Helicobacter pylori (H. pylori) babA2 gene with gastric cancer (GC) was reported by several studies, but results were inconsistent. This meta-analysis was performed to investigate the relationship between H. pylori babA2 gene and GC risk. Case-control studies involving the association between H. pylori babA2 gene and GC risk were systematically identified from PubMed databases. A meta-analysis was used to pool studies and to estimate odds ratios (ORs) with 95% confidence intervals (CIs) of H. pylori babA2 gene associated with GC risk. Twenty studies were identified with a total of 1289 GC cases and 1081 controls. H. pylori babA2 gene was associated with an increased risk of GC by 2.05 fold (95% CI, 1.30–3.24, P = 0.002). In subgroup analysis, we found that H. pylori babA2 gene was significantly associated with GC risk in Asian population (OR = 2.63, 95% CI: 1.36–5.09 P = 0.004) but not in South American population (OR = 1.35, 95% CI: 0.69–2.64, P = 0.379). This meta-analysis indicates that H. pylori babA2 gene may be associated with increased risk of GC, especially in Asian population.


Background
Gastric Cancer (GC) is the fifth most common cancer and the third leading cause of mortality worldwide [1][2][3], with approximately 42.5% of all cases diagnosed in China [4,5]. About 1 million incident cases of GC are annually projected, with the majority observed in Eastern Asia, Latin America and Eastern Europe [5]. In 2015, GC was the second most common cancer with about 6,791,000 new cases in China [4]. Genetic and environment factors are involved in GC development. H. pylori infection, cigarette smoking, low intake of fresh vegetables and fruits and salty foods are main risk factors of GC [6].
Helicobacter pylori (H. pylori) infection is the most common human infections inhabiting in the stomach. It is a gram-negative bacterium, which has epigenetic effects on gastric epithelial cells and indirect inflammatory response on the gastric mucosa [7]. Some studies showed that H. pylori alone or associated gene were strongly associated with gastric cancer risk [8][9][10]. According to the International Agency for Research on Cancer, H. pylori was defined as a class I carcinogen [11]. However, only a small fraction of infected patients develop severe diseases [12]. H. pylori is well distinguished to have a high level of genetic variations allowing it to be adapted to the host gastric epithelium [13].
The blood-group antigen-binding adhesin (babA) encoded by babA2 gene is a major adhesin on the outer membrane of H. pylori. BabA2 is characterized to be an active gene in the binding activity of Lewis-b blood group antigen on gastric epithelium and host cell and determine H. pylori colonization density [23,24]. The sequence of the three babA gene alleles have been identified (babA1, babA2 and babB), but only the babA2 is involve d in Lewis-b binding activity. To date, several studies have evaluated the effect of H. pylori babA2 gene on risk of GC [19,[25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42], but the results are conflicting possibly due to small sample size of single studies. In the present study, we conducted a meta-analysis to assess the association between H. pylori babA2 gene and GC risk based on published cases-control studies.

Search strategies
All relevant studies were identified from PubMed databases. The search strategy included the terms ("babA2" OR "antigen-binding adhesion gene") AND ("Helicobacter pylori" OR "H. pylori infection") AND ("genotype" OR "polymorphism") AND ("gastric cancer" OR "stomach cancer") in any text field of the database. In addition, we also collected additional studies from references of original and review articles.

Inclusion and exclusion criteria
Inclusion criteria to select studies for this meta-analysis were as follows: (1) study describing the relationship between H. pylori babA2 gene and GC, (2) studies that provided babA2 positive frequencies, (3) studies published in English with full text available. Exclusion criteria were as follow: (1) insufficient data to calculate

Data extraction
Data were extracted from each study independently by two investigators and contradictions between them were discussed to obtain agreement. The following information's were collected: first author's name, year of publication, country, ethnicity, sample size, type of study, source of sample, study quality assessment, babA2 positive frequencies, OR estimation and 95% CI for the association between H. pylori babA2 gene and GC.

Quality score assessment
Quality score of each included study was assessed by the same two authors independently using the Newcastle-Ottawa Quality Assessment Scale (NOS) for casecontrol studies [43]. The NOS is a validated quality assessment for case-control studies with three parameters for quality: selection, comparability and exposure.
The maximum score of each parameter is 4 for selection, 2 for comparability and 3 for exposure.

Statistical analysis
The pooled ORs with 95% CIs were used to indicate the effect of H. pylori babA2 gene effect on GC risk. χ 2 base on Q test and I 2 statistics were used to evaluate the statistical heterogeneity among included studies. The fixedeffects model was used when there was no significant heterogeneity (P ≥ 0.10 and I 2 ≤ 50%) [44,45] between studies, otherwise the random effect model was applied to provide more conservative estimates [46]. In addition, we performed subgroup analysis by ethnicity and quality score assessment. Ethnicities were divided into Asian, European, South American and North American. Moreover, sensitivity analyses were performed to estimate the effect of each included study on overall effect. We used Begg's test and Egger's test to estimate publication bias [47]. All the statistical analyses were performed using STATA 11.0.

Characteristics of selected studies
Literature research strategy is detailed in Fig. 1. There were 174 potentially relevant studies. After title and abstract evaluation, 24 articles with full-text assessment were included when duplicated studies were excluded. After full-text reviewed, a total of 19 eligible articles were included in this meta-analysis, and 5 articles were excluded because of the following reasons: two articles were reviews [48,49], and three articles had insufficient data [50][51][52]. One article included participants from two countries [19],  (Table 1).
In total, 1289 cases and 1081 controls were included in this meta-analysis (  (Fig. 3).

Publication bias and sensibility analysis
Publication bias was evaluated by Begg's and Egger's test.
The visual inspection of funnel plot revealed that there Fig. 2 Random-effects meta-analysis forest plot of the odds ratio of gastric cancer according to H.pylori infection with babA2 with respect to without babA2. The studies are sorted by publication year. The solid squares are centered on the odds ratio (OR) point estimate from each study, and the horizontal line through each square indicates the 95% confidence interval (CI) for the study. The area of each square represents the magnitude of association, and the horizontal tips of the diamond represent the 95% CI was no significant evidence of asymmetry distribution. And no significant publication bias was observed based on Begg's test (P = 0.284) or Egger's test (P = 0.288) (Figure S2). The impact of each study on the pooled OR was examined by repeating the meta-analysis while excluding individual study, which confirmed the stability of our results ( Figure S3).

Discussion
To date, numerous studies have assessed the association between H. pylori babA2 gene and GC risk, but results remained inconsistent. The controversial results of individual studies may be due to relatively small sample size. Meta-analysis is an important approach to pool multiple studies and therefore may result in more precise and robust conclusion. In this meta-analysis, we included 20 studies focusing on H. pylori babA2 gene and GC risk with a total of 1289 patients and 1081 controls. We found that H. pylori babA2 gene was significantly associated with risk of GC. There is evidence that H. pylori increase the risk of GC development through the sequence of atrophy and metaplasia originate from several studies. Chronic H. pylori induced inflammation which can probably lead to loss of normal gastric mucosal, with gastric gland destruction, and replacement by fibrosis [53]. The H. pylori strains virulence factors, host and environmental factors are main factors to contribute in clinical infection manifestations [54]. And it is well showed that gene encoding pathogenic H pylori factors are involved in GC development and colonization properties [29,49]. The increased GC risk was also associated with coexpression of H pylori vacAs1, cagA and babA2 genes [17,18,23,55]. Furthermore, interaction between host's immunological defenses and H pylori virulence factors may play an important role in the development of GC Fig. 3 Random-effects meta-analysis forest plot of the odds ratio of gastric cancer according to H.pylori infection with babA2 with respect to without babA2. The studies are divided into Asian and South American according to participants ethnicities and sorted by publication year. The solid squares are centered on the odds ratio (OR) point estimate from each study, and the horizontal line through each square indicates the 95% confidence interval (CI) for the study. The area of each square represents the magnitude of association, and the horizontal tips of the diamond represent the 95% CI [56,57]. It was showed that babA2 as a virulence marker could predict clinical outcome, which was dependent on the geographic origin of the H. pylori strains [27].
In our study, sub-group analysis according to geographical areas showed that H. pylori babA2 was not significantly associated with the risk of GC among South American. Our results are comparable to previous studies from South America [19,26,32,34,50]. The difference among populations may be due to the small sample size for each population, heterogeneity between studies and geographical factors. In other hand, H. pylori babA2 gene was closely involved in the risk of GC in Asian population, which was confirmed by original studies from Asian population [29,30,36,38].
Our meta-analysis showed some limitations. Firstly, we didn't obtain original data, which have limited further evaluation of potential gene-gene and gene-environmental interactions. Secondly, the sample sizes of most included studies are relative small. Thirdly, additional analysis based on other factors, such as age, gender, family history, other virulence factors, environment factors (e.g. alcohol intake, smoking, high BMI) and GC subtypes (e.g. intestinal, diffuse or mixed type), could not be analyzed because of the limited information obtained from included studies. Finally, high heterogeneity among studies indicates that the pooled estimation risk should be interpreted with caution.

Conclusions
Our results suggest that the presence of H. pylori with positive babA2 gene may contribute to increased risk of GC, especially in Asian population. Studies with large sample size are necessary to further elucidate the interaction among environmental factors, bacterial genotype and host factors on GC risk.
Additional file 1: Figure S1. Sub-group analysis of the association between H. pylori babA2 gene and gastric cancer risk according to study quality assessment. Figure S2. Funnel plot of case-control studies evaluating the association between H. pylori babA2 gene and gastric cancer risk. Each point represents a study to indicate an association. Figure S3. Influence of the summary OR coefficients on the association between H. pylori babA2 gene and gastric cancer risk.