Helicobacter pylori infection, host genetics and gastric cancer
Correspondence to: Emad M. EL-OMAR, Division of Applied Medicine, Institute of Medical Sciences, School of Medicine and Dentistry, Aberdeen University, Foresterhill, Aberdeen AB25 2ZD UK. Email: email@example.com
Helicobacter pylori infects half the world's population and is responsible for a considerable global health burden, including peptic ulcer disease and gastric cancer. The infection causes a chronic gastritis, the severity and distribution of which determine the clinical outcome. Bacterial, environmental and host genetic factors combine to define the degree of gastric damage. Most patients have a limited mild pan-gastritis with no significant clinical consequences. Antral-predominant gastritis is associated with high gastric acid output and an increased risk of duodenal ulcers. Corpus-predominant gastritis is associated with a reduction in gastric acid, multifocal gastric atrophy and an increased risk of gastric cancer. Host genetic factors are particularly important in defining the severity and extent of Helicobacter-induced gastritis. The most relevant and consistent genetic factors uncovered thus far are in the interleukin-1 and tumor necrosis factor-A gene clusters. These cytokines appear to play a key role in the pathophysiology of gastric cancer and their roles have been confirmed in animal models that mimic human gastric neoplasia. More genetic factors have also been uncovered and, with advancing technology, there is every prospect of defining a full genetic risk profile in the next decade. This will aid in targeting the testing and treatment of Helicobacter pylori, which offers a true opportunity to prevent and defeat this global killer.
Infection with Helicobacter pylori (H. pylori) is the commonest single chronic bacterial infection in the world, affecting at least half of all humans. Recognizing it as the prime etiological agent in peptic ulcer disease (PUD) rapidly led to the cure and virtual disappearance of this important human disease. In 1994 the International Agency for Research on Cancer declared H. pylori a Group I (definite) human carcinogen.1 The past 15 years have seen major advances in understanding the key factors involved in H. pylori-induced gastric carcinogenesis. Thus H. pylori infection is associated with two major human diseases, gastric cancer and PUD, and exerts a significant global burden on mortality, morbidity and health-care economics.
Importantly, most infected individuals develop no significant disease. Perhaps the most challenging scientific conundrum is to explain why certain individuals develop serious sequelae while the majority does not. There has been considerable momentum towards understanding the bacterial and host genetic factors that may account for this individual predisposition.
In this review, we concentrate on the role of host genetic factors in influencing the clinical outcome of H. pylori infection. In order to facilitate this, it is essential to give a brief account of the nature of this infection and its pathophysiological effects in the stomach.
PATHOPHYSIOLOGY OF H. PYLORI INFECTION
H. pylori is a gram negative, spiral-shaped, microaerophilic urease-positive bacillus that is acquired during childhood, probably via the fecal/oral or gastric/oral routes. Once acquired, the infection persists throughout life unless treated with antibiotics. Although the bacteria mainly reside on the surface mucus gel layer, with little invasion of the gastric glands, the host responds with an impressive humoral and cell-mediated immune response. In spite of this sophisticated immune response most infections become chronically established, with little evidence that spontaneous clearance occurs.2
In physiological terms the stomach may be divided into two main compartments: an acidic proximal corpus that contains the acid-producing parietal cells and a less acidic distal antrum that does not have parietal cells but contains the endocrine cells that control acid secretion. Both animal and human ingestion studies suggest that successful colonization of the gastric mucosa is best achieved with the aid of acid suppression.3,4 Furthermore, the pharmacological inhibition of acid secretion in infected patients leads to the redistribution of the infection and its associated gastritis from an antral to a corpus-predominant pattern.5 Thus, lack of gastric acid extends the area of colonization and also maximizes the tissue damage resulting from this colonization.
The key patho-physiological event in H. pylori infection is the initiation of an inflammatory response.6 This response is probably triggered by the bacterium's lipopolysaccharide (LPS), urease and/or cytotoxins with the ensuing inflammatory response mediated by cytokines. The cytokine repertoire comprises a multitude of pro- and anti-inflammatory mediators whose function is to co-ordinate an effective immune or inflammatory response against invading pathogens without causing undue damage to the host.
In addition to their pro- or anti-inflammatory properties, some H. pylori-induced cytokines have direct effects on the gastric epithelial cells that regulate the physiological processes of the stomach. For example, the pro-inflammatory cytokine interleukin-1 beta (IL-1β) is the most potent of known agents that are gastric cyto-protective, antiulcer, antisecretory and inhibitors of gastric emptying.7 Another important pro-inflammatory cytokine that is upregulated by H. pylori infection is tumor necrosis factor alpha (TNF-α), which also inhibits gastric acid secretion but to a lesser extent than IL-1β.8
There are three main gastric phenotypes that result from chronic H. pylori infection: (i) the commonest by far is a mild pan-gastritis that does not affect gastric physiology and is not associated with significant human disease; (ii) a corpus-predominant gastritis associated with progressive gastric atrophy and multifocal intestinal metaplasia, hypochlorhydria and increased risk of gastric cancer (the gastric cancer phenotype);9 and (iii) an antral-predominant gastritis associated with high gastric acid secretion and increased risk of duodenal ulcer disease (the DU phenotype).10 It is fascinating to note that the determinants of these outcomes are the severity and extent of the gastritis, which in turn are determined by host, bacterial and environmental factors.
ROLE OF GENETIC POLYMORPHISMS IN THE PATHOGENESIS OF GASTRIC CANCER AND ITS PRECURSORS
IL-1 gene cluster polymorphisms increase the risk of gastric cancer and its precursors
One of the paradoxes of H. pylori infection is its association with mutually exclusive clinical outcomes such as gastric cancer and duodenal ulcer disease. A large volume of research has focused on the role of bacterial virulence factors (e.g., cagA, vacA, BabA and OipA) in the pathogenesis of these diseases, and although these factors undoubtedly contribute to the degree of tissue damage they do not readily distinguish between the two key outcomes of gastric cancer and PUD.11 This prompted some researchers to consider the host genetic factors that may be relevant to this process. Crucially, the search for the appropriate candidate genes had to stem from a profound understanding of gastric physiology and how this is disrupted by H. pylori infection. Since H. pylori achieve most damage through induction of chronic inflammation, it is reasonable to consider genes that control this process as appropriate candidates.
The IL-1 gene cluster on chromosome 2q contains three related genes within a 430-kb region, IL-1A, IL-1B and IL-1RN, which encode for the pro-inflammatory cytokines IL-1α and IL-1β, as well as their endogenous receptor antagonist IL-1ra, respectively.12 IL-1β is upregulated in the presence of H. pylori and plays a central role in initiating and amplifying the inflammatory response to this infection.13 As mentioned earlier, IL-1β is also an extremely potent inhibitor of gastric acid secretion. Three diallelic polymorphisms in IL-1B have been reported, all representing C–T or T–C transitions, at positions −511, −31, and +3954 bp from the transcriptional start site.14 The IL-1RN gene has a penta-allelic 86 bp tandem repeat (VNTR) in intron 2, of which the less common allele 2 (IL-1RN*2) is associated with a wide range of chronic inflammatory and autoimmune conditions.14 The presence of such highly prevalent and functional genetic polymorphisms provides an ideal opportunity to design epidemiologic studies to test the role of these candidate loci.
El-Omar et al. first studied the correlation of these high IL-1β genotypes (two polymorphisms in the IL-1B and IL-1RN genes) with hypochlorhydria and gastric atrophy in a Caucasian population of gastric cancer first-degree relatives from Scotland. These relatives were known to be at increased risk of developing the same cancer and had a high prevalence of precancerous abnormalities (hypochlorhydria and gastric atrophy) but only in the presence of H. pylori infection. In these individuals, high IL-1β genetic markers were associated with an increased risk of precancerous conditions.15 The association between the same IL-1β genetic polymorphisms and gastric cancer itself was subsequently examined using two independent Caucasian case-control studies from Poland and the USA.15,16
In the above studies the pro-inflammatory IL-1 genotypes were associated with an increased risk of both intestinal and diffuse types of gastric cancer; however, the risk was restricted to the non-cardia sub-site. Indeed, the IL-1 markers had no effect on risk of cardia gastric adenocarcinomas, esophageal adenocarcinomas or esophageal squamous cell carcinomas.16 The latter findings are entirely in keeping with the proposed mechanism for the effect of these polymorphisms in gastric cancer, namely the reduction of gastric acid secretion. Thus, a high IL-1β genotype appears to increase the risk of non-cardia gastric cancer, a disease characterized by hypochlorhydria, while it has no effect on cancers associated with high acid exposure such as esophageal adenocarcinomas and some cardia cancers.
The association between IL-1 gene cluster polymorphisms and gastric cancer and its precursors has been confirmed independently by other groups covering Caucasian, Asian and Hispanic populations.17–23 Machado et al. were the first to confirm the association between IL-1 markers and gastric cancer in Caucasians and reported similar modest odds ratios to those reported by El-Omar et al.17 Furthermore, the same group subsequently reported on the combined effects of pro-inflammatory IL-1 genotypes and H. pylori bacterial virulence factors (cagA positive, VacA s1 and VacA m1), reporting that for each combination of bacterial and host genotype the odds of having a gastric carcinoma were greatest in those with both bacterial and host high-risk genotypes.18 This highlights a potentially important interaction between host and bacterium in the pathogenesis of gastric cancer.
Discrepancy between different populations relating to IL-1 polymorphisms and gastric cancer risk
Unlike the studies mentioned above, some reports, particularly from Asian countries, failed to find an association between IL-1 markers and gastric cancer risk. A number of these studies were underpowered, while others used inappropriate controls and mixed phenotypes; however, even excluding the weaker studies, there remains the impression that not all Asian or Caucasian populations demonstrate a predisposition for gastric cancer in association with published pro-inflammatory IL-1 polymorphisms. In some instances, studies found a positive association, but with novel markers of the IL-1β gene.24 Other studies point to the importance of the background prevalence of gastric cancer in the population, with the positive associations being easier to demonstrate in low-incidence compared with high-incidence areas.23 Finally, some studies reported a positive association with IL-1 markers, but this was confined to the opposite alleles from those reported by most of the studies.25,26 In one study the authors confirmed that the opposite alleles (previously found to associate with reduced IL-1β production) were, in fact, the high IL-1β alleles in their population.25
Other potential explanations for the lack of association with the IL-1 markers in some populations should also be considered. One very interesting concept that has emerged recently is that of the haplotype context. Chen et al. studied the significance of the haplotype structure in gene regulation by testing whether individual single nucleotide polymorphisms (SNP) within a gene promoter region (IL-1β in this case) might affect promoter function and, if so, whether function was dependent on the haplotype context.27 They very elegantly showed that significant interactions between SNP according to haplotype had a profound influence on the functionality of the gene promoter. As haplotype context is influenced by ethnic background and past selective pressures that are unique to different populations, this may explain why some studies may demonstrate a positive association with a particular marker in some but not all populations.
Lee et al. recently reported on another potential explanation for variation in association with IL-1β and gastric cancer.28 They studied the interaction between SNP in IL-1β and general transcription factor (GTF2A1) genes reporting an association between carriage of the IL-1β-31C allele and gastric cancer among Koreans, which was observed only in subjects with GTF2A1 GG genotype; this suggested potential synergy between the two genetic markers. Taken at face value these findings still point to IL-1β being a crucial cytokine in the pathogenesis of H. pylori-induced gastric cancer and its precursors and variations in its gene act as host genetic factors that mediate this effect.
Finally, and potentially the most interesting explanation for the discrepant results, the role of diet as a modifier of genetic risk has been examined. In a fascinating paper, Shen et al. studied the effect of IL-1β genetic variants on the risk of metabolic syndrome.29 They reported that IL-1β genetic variants were associated with measures of both chronic inflammation and risk of developing metabolic syndrome, and that a genetic influence was more evident among subjects with low (n-3) polyunsaturated fatty acids (PUFA) intake. We may therefore hypothesize that a varied diet, excessive or deficient in certain nutrients, may potentially modulate genetic risk; since no two populations consume exactly the same diet, it is plausible that at least some of the discrepancy in genetic association studies may be explained by this hitherto ignored factor.
Confirmation of the role of IL-1β in gastric cancer pathogenesis
A crucial piece of evidence that confirmed the apparent role of IL-1β in H. pylori-induced gastric carcinogenesis came from a transgenic mouse model in which IL-1β overproduction was targeted to the stomach by the H+/K+ ATPase beta promoter.30 With the overexpression of IL-1β confined to the stomach, these transgenic mice had a thickened gastric mucosa, produced lower amounts of gastric acid and developed severe gastritis followed by gastric atrophy, intestinal metaplasia, dysplasia and adenocarcinomas. Crucially, these IL-1β transgenic mice proceeded through a multistage process that mimicked human gastric neoplasia. These changes occurred even in the absence of H. pylori infection, which, when introduced, led to an acceleration of these abnormalities. Most interestingly, the pathological changes, including the progression to gastric cancer were prevented by infusion of IL-1 receptor antagonist, proving beyond doubt that IL-1β is responsible for the pathological effects.30
Another crucial piece of evidence came from recent elegant work by Stoicov et al.31 T-bet is a central regulator of the cytokine environment during Helicobacter infection and T-bet knockout (KO) mice maintain infection for 15 months at levels similar to wild type mice and develop significant inflammation with a blunted T-helper 1 (Th1). Furthermore, this blunted response is associated with the preservation of parietal and chief cells and protection from the development of gastric cancer. Crucially however, T-bet KO mice respond to Helicobacter infection with a markedly blunted IL-1β and TNF-α and elevated IL-10 levels. This mirrors the situation in humans who are protected against gastric cancer, and merits further research.
ROLE OF OTHER INFLAMMATORY CYTOKINE GENE POLYMORPHISMS IN GASTRIC CANCER RISK
TNF-α, IL-10 and IL-8
Soon after the IL-1 gene cluster polymorphisms were identified as risk factors for gastric cancer, the pro-inflammatory genotypes of tumor necrosis factor-α (TNF-α) and IL-10 were reported as independent additional risk factors for non-cardia gastric cancer.16 TNF-α is another powerful pro-inflammatory cytokine that is produced in the gastric mucosa in response to H. pylori infection. Like IL-1β, it has an inhibitory, albeit weaker, effect on acid production.8 The TNF-α-308 G > A polymorphism is known to be involved in a number of inflammatory conditions. Carrying the pro-inflammatory A allele increased the risk of non-cardia gastric cancer. This association was independently confirmed by a study from Machado et al.19 The same group also investigated TNF-α extended haplotypes and showed that the association between the TNF-α-308G > A polymorphism and increased risk of gastric carcinoma is dependent on a linkage disequilibrium with an as yet unidentified locus.32
Another interleukin, IL-10, is an anti-inflammatory cytokine that downregulates IL-1β, TNF-α, interferon-γ and other pro-inflammatory cytokines. A relative deficiency of IL-10 may result in a T helper-1-driven hyper-inflammatory response to H. pylori with greater damage to the gastric mucosa. El-Omar et al. reported that homozygosity for the low-IL-10 ATA haplotype (based on three promoter polymorphisms at positions −592, −819 and −1082) increased the risk of non-cardia gastric cancer with an odds ratio of 2.5 (95%CI: 1.1–5.7).16 The results have not been widely replicated.
Interestingly, there seems to be a cumulative effect in carrying more than one pro-inflammatory cytokine gene polymorphism. El-Omar et al. studied the effect of having multiple pro-inflammatory genotypes (IL-1β-511*T, IL-1RN*2*2, TNF-α-308*A and IL-10 ATA/ATA) on the risk of non-gastric cancer. With the addition of each relevant polymorphism the risk of non-gastric cancer increased progressively such that when three to four of these polymorphisms were present the risk for gastric cancer was increased 27-fold.16 The fact that H. pylori is a prerequisite for the association of these polymorphisms with malignancy demonstrates that in this situation, infection-induced inflammation may indeed be contributing to carcinogenesis.
Another cytokine that has an important role in the pathogenesis of H. pylori-induced diseases is IL-8. This chemokine belongs to the CXC family and is a potent chemoattractant for neutrophils and lymphocytes. It also has effects on cell proliferation, migration and tumor angiogenesis. The gene has a well-established promoter polymorphism at position −251 (IL-8–251 T > A). The A allele is associated with increased production of IL-8 in H. pylori-infected gastric mucosa.33 It has been reported to increase the risk of severe inflammation and precancerous gastric abnormalities in Caucasian33 and Asian populations.34 However, the same polymorphism was only found to increase risk of gastric cancer in some Asian populations34–37 and had no apparent effect in Caucasians.38
Other immune/inflammatory gene polymorphisms
Since the original publications on IL-1β and TNF-α polymorphisms many other cytokine gene polymorphisms have been associated with the increased risk of gastric cancer. The list is far too extensive to describe but the following section details a few notable ones.
Hou et al. studied the association between gastric cancer and several variants in genes responsible for Th1 cell-mediated response.39 They reported that carrying the C allele of the interferon gamma receptor 2 (IFNGR2) (Ex7–128) rs4986958 polymorphism was associated with an increased risk of gastric cancer compared with the TT genotype. Furthermore, there was an additive effect when the IFNGR2 polymorphism was combined with the TNF-α-308 TT genotype (OR 5.5, 95%CI 1.5–19.4).
The interferon gamma receptor 1 (IFNGR1) –56C/T gene polymorphism was studied by Canedo et al. In patients with early onset gastric cancer (less than 40 years of age at the time of diagnosis) there was a significant over-representation of the IFNGR1–56*T/*T homozygous genotype with an OR of 4.1 (95%CI 1.6–10.6).40 This result was replicated in a second independent case-control study.
Mahajan et al. studied polymorphisms in the Th1 IL7R gene and one polymorphism in the Th2 IL5 gene.41 The OR for IL7R rs1494555 were 1.4 (95%CI 1.0–1.9) for A/G and 1.5 (95%CI 1.0–2.4) for G/G carriers compared with A/A carriers (P = 0.04). The OR for IL5 rs2069812 were 0.9 (95%CI 0.7–1.3) for C/T and 0.6 (95%CI 0.3–1.0) T/T carriers compared with C/C carriers (P = 0.03). These results suggest that IL5 rs2069812 and IL7R rs1389832, rs1494556 and rs1494555 polymorphisms may contribute to the risk of gastric cancer.
ROLE OF POLYMORPHISMS IN THE INNATE IMMUNE RESPONSE GENES
Genetic polymorphisms of the cytokines and chemokines discussed above clearly play an important role in the risk of H. pylori-induced gastric adenocarcinomas. However, H. pylori is initially handled by the innate immune response and it is conceivable that functionally relevant polymorphisms in the genes of this arm of the immune system may affect the magnitude and subsequent direction of the host's response against the infection. Most H. pylori cells do not invade the gastric mucosa but the inflammatory response against it is triggered through the attachment of H. pylori to the gastric epithelia.42 Toll-like receptor 4 (TLR4), the LPS receptor, was initially identified as the potential signaling receptor for H. pylori on gastric epithelial cells.43
Recently, Hold et al. have shown that the TLR4+896A > G polymorphism was associated with an exaggerated and destructive chronic inflammatory phenotype in H. pylori-infected patients. This phenotype was characterized by gastric atrophy and hypochlorhydria, the hallmarks of subsequent increased risk of gastric cancer. They further showed that the same polymorphism increased the risk of non-cardia gastric cancer.44 In contrast, prevalence of TLR4+896G was not significantly increased in esophageal squamous cell or adenocarcinoma or gastric cardia cancer.44 The association of TLR4+896A > G polymorphism with both gastric cancer and its precursor lesions implies that it is relevant to the entire multistage process of gastric carcinogenesis that starts with the H. pylori colonization of the gastric mucosa. Patients with this polymorphism have an increased risk of severe inflammation and subsequent development of hypochlorhydria and gastric atrophy, which are regarded as the most important precancerous abnormalities. This severe inflammation is initiated by H. pylori infection but it is entirely feasible that subsequent co-colonization of an achlorhydric stomach by a variety of other bacteria may sustain and enhance the microbial inflammatory stimulus and continue to drive the carcinogenic process.
Santini et al. showed that another TLR4 polymorphism increases the risk of intestinal type gastric cancer. Thus, the TLR4 Thr399Ile was associated with an increased hazard ratio of 5.38, 95%CI 1.652–8.145, P = 0.006.45 More work is required on elucidating the full impact of polymorphisms in the toll-like receptors and their associated pathways.
There are other reports of innate immune response gene polymorphisms being associated with increased risk of gastric cancer. Mannose binding lectin is an antigen-recognition molecule involved in systemic and mucosal innate immunity. It is able to bind to a range of microbes and subsequently kill them by activating the complement system and promoting complement-independent opsonophagocytosis. Baccarelli et al. showed that polymorphisms in the mannose binding lectin-2 gene (MBL2) were associated with increased risk of gastric cancer.46 In haplotype analysis, the HYD haplotype was associated with an increased risk of stomach cancer when compared with HYA, the most common haplotype (OR = 1.9, 95%CI 1.1–3.2; P = 0.02). Further analyses to examine the joint effect of MBL2 and IL-1β polymorphisms indicated that the combination of at-risk IL-1β genotypes (CT or TT at location –511) and HYD MBL2 haplotype was associated with a 3.5-fold risk (OR = 3.5, 95%CI 1.6–7.6; P = 0.001). The findings suggest that the codon 52 D MBL2 variant, causing a cysteine to arginine replacement, is specifically associated with gastric cancer risk.
Genome-wide association studies and gastric cancer
Genotyping technology has advanced dramatically in the past 5 years and it is now possible to study hundreds of thousands of SNP simultaneously. This approach, termed genome-wide association studies, was recently used by Sakamoto et al. to study gastric cancer in the Japanese population.47 Employing a two-stage genome-wide association study (stage 1: 85 576 SNP on 188 cases and 752 references; stage 2: 2753 SNP on 749 cases and 750 controls) identified a significant association between an intronic SNP (rs2976392) in the prostate stem cell antigen (PSCA) and diffuse-type gastric cancer (allele-specific OR = 1.62, 95%CI = 1.38–1.89, P = 1.11 × 10−9). The association was far less significant in intestinal types of gastric cancer. The PSCA gene is possibly involved in regulating gastric epithelial cell proliferation and it will be very interesting to find out how it influences susceptibility to diffuse types of gastric cancer.
Overall contribution of a host pro-inflammatory genetic makeup to pathogenesis of gastric cancer
It appears that patients with a pro-inflammatory genetic make-up based on a combination of markers from cytokine or chemokine genes (e.g., IL-1β, TNF-α, IL-10 and IL-8) and the innate immune response (e.g., TLR4 and MBL2), respond to H. pylori infection by creating a gastric environment that is chronically inflamed and with reduced acidity. The damage is exacerbated if the infecting organisms are particularly virulent and particularly if CagA positive. This bacterial protein has recently been shown to act as an oncoprotein and there is no doubt that its presence further heightens the inflammatory process.48 This environment is conducive to the growth of other non-H. pylori bacteria in the gastric milieu, leading to sustained inflammation and oxidative, genotoxic or oncogenic stress. Patients with the same pro-inflammatory polymorphisms may respond in the same exaggerated manner to these non-H. pylori bacteria, thus maintaining the pro-neoplastic drive. This may explain why H. pylori is not required in the latter stages of gastric carcinogenesis and why it is often absent from gastric tumor tissue.
H. pylori infection is an important organism and, while it may not cause any clinical problems in most infected patients, it has the potential to leave the host with devastating consequences. Sporadic gastric cancer is a common cancer with a grave prognosis, particularly in the West. A major advance in the fight against this global killer came with the recognition of the role of H. pylori infection in its pathogenesis. The cancer represents a classic example of an inflammation-induced malignancy. Host genetic factors interacting with bacterial virulence and environmental factors play an important role in the pathogenesis of this cancer. In particular, genetic polymorphisms in the adaptive and innate immune response genes seem to increase the risk of cancer, largely through induction of severe gastritis, which progresses to atrophy and hypochlorhydria. The pro-inflammatory host genetic make-up is only relevant in the presence of infection, initially with H. pylori but potentially a later, similar pro-inflammatory pathogenic role is assumed by other bacteria that thrive in an achlorhydric environment. Future research must focus on defining a more comprehensive genetic profile that better predicts the clinical outcome of H. pylori infection, including gastric cancer.