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Role of host genes in sporadic gastric cancer
Best Practice & Research Clinical Gastroenterology Vol. 20, No. 4, pp. 675e686, 2006 doi:10.1016/j.bpg.2006.04.006 available online at http://www.sciencedirect.com
3 Role of host genes in sporadic gastric cancer Emad M. El-Omar*
BSc(Hons), MB ChB, MD, FRCP (Edin)
Professor of Gastroenterology/Honorary Consultant Physician Department of Medicine and Therapeutics, Aberdeen University, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK
Gastric cancer remains a major health problem particularly in the developing world. Helicobacter pylori (H. pylori) infection is the most recognised aetiological risk factor for this malignancy. The infection causes a chronic gastritis that is the precursor to all the pathophysiological abnormalities characteristic of gastric carcinogenesis. Genetic polymorphisms have emerged in recent years as important determinants of disease susceptibility and severity. In the case of H. pyloriinduced gastric cancer, host genetic polymorphisms play an important role both in the precancerous stages and in the transition to cancer. In particular, polymorphic genes of the adaptive and innate immune response are involved in all stages of the neoplastic process. This field is rapidly expanding and many other genetic determinants are currently being defined. The ultimate value of host genetics should be in understanding the pathogenesis of the disease, which would offer a true opportunity to defeat this global killer. Key words: gastric cancer; Helicobacter pylori; genetic polymorphisms; single nucleotide polymorphisms; cytokines; interleukins; toll-like receptors.
INTRODUCTION Gastric cancer remains a major health problem despite the recent decline in the incidence of this malignancy in the developed world. The global burden is shifting from the developed to the developing world and around 1.1 million new cases are expected in the year 2010.1 Helicobacter pylori (H. pylori) infection is the most recognised aetiological risk factor for this malignancy.2 As already outlined in earlier chapters, H. pylori infection causes a chronic gastritis that is the precursor to all the pathophysiological abnormalities characteristic of gastric carcinogenesis.3 The most important of these abnormalities are severe corpus-predominant gastritis, gastric atrophy and hypochlorhydria, which develop as a direct result of the chronic inflammation induced by the infection.4,5 It is important to appreciate however that this so-called ‘gastric cancer * Tel: þ44 1224 553021; Fax: þ44 1224 555766. E-mail address: [email protected]. 1521-6918/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved.
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phenotype’ is a relatively rare outcome whilst the overwhelming majority of infected subject worldwide do not develop any significant clinical disease. This second ‘benign’ phenotype is characterised by a mild mixed gastritis pattern that has little impact on gastric physiology, particularly on gastric acid secretion. Finally, there is a third phenotype, the so-called ‘duodenal ulcer phenotype’, which is characterised by an antrumpredominant gastritis, high gastric acid secretion and an increased risk of developing duodenal ulcers.6 The latter group account for a variable percentage of infected subjects and are relatively common in the West (up to 15%), and rare in the East. Furthermore, subjects who develop duodenal ulcers are actually protected from developing gastric cancer, suggesting that the two outcomes are mutually exclusive.7 It is clear therefore that H. pylori infection can lead to several divergent clinical outcomes. Explaining this apparent paradox is essential for understanding the pathogenesis of H. pylori-related disease in general and gastric cancer in particular. The last two decades have seen major advances in unravelling the contribution of bacterial virulence factors, environmental exposures and host genetic factors in the pathogenesis of H. pylori-induced diseases. The aim of this review is to focus on the role of host genetic factors in the pathogenesis of sporadic gastric adenocarcinoma. For excellent reviews of bacterial virulence factors the reader is directed to the following reports by Blaser & Atherton and Israel & Peek.8,9 GENETIC POLYMORPHISMS AND MULTIFACTORIAL HUMAN DISEASE Before discussing the role of host genetic factors in the pathogenesis of sporadic gastric cancer, it is essential to establish some basic principles of genetic epidemiology and the limitations of studying genetic polymorphisms in the context of complex multifactorial human diseases. The completion of the human genome project was a momentous occasion for humanity. It opened up the opportunity to dissect complex human traits and to understand basic pathways of health and disease. While linkage analysis studies are suitable for pursuing rare high-risk alleles in conditions that have a hereditary basis, population-based association studies are much more useful for examining genes with a role in commoner multifactorial diseases that have a strong environmental component.10 These association studies often estimate the risk of developing a certain disease in carriers and non-carriers of a particular genetic polymorphism. The overwhelming majority of polymorphisms studied are single nucleotide polymorphisms (SNPs) that occur with a frequency of >1% in the normal population (in contrast to ‘mutations’ that occur with a frequency of <1%). It is estimated that up to 10 million SNPs are probably present in the human genome though not all have thus far been identified. Naturally, most of these SNPs do not occur in coding sequences and even those that do, are not associated with any alteration in the amino acid sequence and are therefore of no functional consequence. A more useful definition of the variability of these SNPs involves haplotype analysis. Haplotypes are a combination of alleles at different markers along the same chromosome that are inherited as a unit. The HapMap project was set up to address this (www.hapmap.org) and is a valuable tool that will facilitate the study of genetic polymorphisms relevant to human health and disease. Other types of genetic variation include deletion and insertion polymorphisms and microsatellite repeat polymorphisms. There has been an exponential rise in the number of published genetic association studies. Quite often, a report of a single genetic marker is published with great
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promise only to be followed by several negative studies that fail to reproduce the original observation. There is no doubt that the strategy of genetic association studies could be a powerful tool for dissecting human diseases, provided certain principles are observed to minimise the chances of false positive, and negative, reports. The most important of these principles are (1) rigorous definition of disease phenotype, (2) choice of candidate genes that are plausibly linked to the pathophysiology of the disease under study, (3) selection of polymorphisms with known (or at least potentially) functional consequences, (4) choice of genetic markers that are reasonably frequent in the population under study (variant allele frequency of at least 5%), (5) appropriate selection of controls that are matched for ethnicity, age, gender, and environmental exposures, (6) design of studies that are adequately powered to produce a valid result. Even then, the statistical analyses of such studies have to take into account the real problem of false positive results by multiple testing. Appropriate corrections for multiple testing have to be applied, or alternatively, the positive findings should be regarded as preliminary and should be validated in an independent set of cases and controls. Finally, the genetic epidemiology has to involve basic science in order to unravel and validate the molecular mechanisms involved. Adherence to these basic principles will ensure that false positive trails are minimised and will offer a true opportunity to understand complex multifactorial human diseases. There has never been a more stressing time for adherence to these principles, as the advancement in genotyping technology has made possible the annotation of the entire human genome. Current technology allows us to genotype up to 500,000 SNPs in one run, but more awesome is the future prospect of replacing genotyping and haplotyping with direct low-cost sequencing of the entire genome, the so-called ‘$1000 genome’.11 Having set the background to the study of genetic polymorphisms, we can now examine the role of these in the pathogenesis of H. pylori-induced gastric cancer. H. pylori infection elicits a powerful humoural and cell-mediated immune response that causes inflammation in the gastric mucosa. The mediators of this inflammatory response belong to the two main arms of the immune system, namely adaptive and innate immunity. POLYMORPHISMS OF CYTOKINE GENES AND RISK OF GASTRIC CANCER Role of interleukin-1 (IL-1) genetic markers H. pylori causes its damage by initiating chronic inflammation in the gastric mucosa. This inflammation is mediated by an array of pro- and anti-inflammatory cytokines. Genetic polymorphisms directly influence inter-individual variation in the magnitude of cytokine response and this clearly contributes to an individual’s ultimate clinical outcome. In the case of H. pylori infection, we speculated that the most relevant candidate genes would be ones whose products were involved in handling the H. pylori attack (innate and adaptive immune responses) and ones that mediated the resulting inflammation. Because such a list of candidate genes would be prohibitively extensive, our initial search focussed on genes that were most relevant to gastric physiology, and in particular, gastric acid secretion. As mentioned above, H. pylori-induced gastritis is associated with three main phenotypes that correlate closely with clinical outcome: DU phenotype, benign phenotype and gastric cancer phenotype. Studies have shown that inhibition of gastric acid pharmacologically can lead to a shift from an antrum-predominant pattern (DU phenotype) to a corpus-predominant one with onset of gastric atrophy (gastric cancer
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phenotype).12,13 Thus it was clear that an endogenous agent that was upregulated in the presence of H. pylori, has a profound pro-inflammatory effect and was also an acid inhibitor, would be the most relevant host genetic factor to be studied. Interleukin-1 beta (IL-1b) fitted this profile perfectly, for not only is it one of the earliest and most important pro-inflammatory cytokines in the context of H. pylori infection, it is but also the most powerful acid inhibitor known.14 We have shown that pro-inflammatory IL-1 gene cluster polymorphisms (IL-1B encoding IL-1b and IL-1RN encoding its naturally occurring receptor antagonist) increase the risk of gastric cancer and its precursors in the presence of H. pylori.15 Individuals with the IL-1B-31*C or -511*T and IL-1RN*2/*2 genotypes are at increased risk of developing hypochlorhydria and gastric atrophy in response to H. pylori infection. This risk also extends to gastric cancer itself with a 2e3-fold increased risk of malignancy compared to subjects who have the less proinflammatory genotypes.15,16 Furthermore, the pro-inflammatory IL-1 genotypes increased the risk of both intestinal and diffuse types of gastric cancer but the risk was restricted to the non-cardia subsite. Indeed, the IL-1 markers had no effect on risk of cardia gastric adenocarcinoma, oesophageal adenocarcinoma or oesophageal squamous cell carcinoma.16 The latter findings are entirely in keeping with the proposed mechanism for the effect of these polymorphisms in gastric cancer, namely reduction of gastric acid secretion. Thus a high IL-1b genotype increases the risk of non-cardia gastric cancer, a disease characterised by hypochlorhydria, while it has no effect on cancers associated with high acid exposure such as oesophageal adenocarcinoma and some cardia cancers. Interestingly, the high IL-1b pro-inflammatory genotypes do protect against the development of erosive and non-erosive oesophagitis, suggesting again that they operate by reducing gastric acidity through induction of corpus atrophy.17,18 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.19e27 Machado et al were the first to confirm the association between IL-1 markers and gastric cancer in Caucasians and reported similar odds ratios to those reported by our group.19 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). They showed that for each combination of bacterial/host genotype, the odds of having gastric carcinoma were greatest in those with both bacterial and host high-risk genotypes.20 This highlights the important interaction between host and bacterium in the pathogenesis of gastric cancer. 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. However, even excluding the weaker studies, there is still the impression that not all Asian or Caucasian populations have demonstrated a predisposition for gastric cancer in association with ‘published’ pro-inflammatory IL-1 polymorphisms. In some instances, studies found that there was a positive association but with novel markers of the IL-1B gene.28 Other studies pointed 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 to high incidence areas.25 Finally some studies reported a positive association with IL-1 markers but this was confined to the opposite alleles from those reported by the majority of studies.29,30 In one study, the authors confirmed that the opposite alleles (previously found to associate with reduced IL-1b production) were in fact the high IL-1b alleles in their population.29 Taken at face value these findings still point to IL-1b being a crucial cytokine in the
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pathogenesis of H. pylori-induced gastric cancer and its precursors and variations in its gene act as host genetic factors that mediate this effect. A crucial piece of evidence that confirmed the unique role of IL-1b in H. pyloriinduced gastric carcinogenesis came from a transgenic mouse model in which IL-1b overproduction was targeted to the stomach by the Hþ/Kþ ATPase beta promoter. With overexpression of IL-1b confined to the stomach, these transgenic mice had a thickened gastric mucosa, produced lower amounts of gastric acid and developed severe gastritis followed by atrophy, intestinal metaplasia, dysplasia and adenocarcinoma. Crucially, these IL-1b 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.31 Role of other cytokine gene polymorphisms Soon after the IL-1 gene cluster polymorphisms were identified as risk factors for gastric cancer, the pro-inflammatory genotypes of tumour necrosis factor-a (TNF-A) and IL10 were reported as independent additional risk factors for non-cardia gastric cancer.16 TNF-a is another powerful pro-inflammatory cytokine that is produced in the gastric mucosa in response to H. pylori infection. Like IL-1b, it has an acid inhibitory effect, albeit much weaker.32 The TNF-A-308 G > A polymorphism is known to be involved in a number of inflammatory conditions. Carriage of the pro-inflammatory A allele increased the odds ratio for non-cardia gastric cancer to 2.2 (95% confidence intervals: 1.4e3.7). The role of the TNF-A-308 G > A polymorphism in gastric cancer was independently confirmed by a study from Machado et al.21 IL-10 is an anti-inflammatory cytokine that downregulates IL-1b, TNF-a, interferon-g and other pro-inflammatory cytokines. Relative deficiency of IL-10 may result in a T helper-1 (Th-1) -driven hyper-inflammatory response to H. pylori with greater damage to the gastric mucosa. We 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 noncardia gastric cancer with an odds ratio of 2.5 (95% CI: 1.1e5.7). We have studied the effect of having an increasing number of pro-inflammatory genotypes (IL-1B-511*T, IL-1RN*2*2, TNF-A-308*A, and IL-10 ATA/ATA) on the risk of non-gastric cancer. The risk increased progressively so that by the time three to 4 of these polymorphisms were present, the odds ratio for gastric cancer was increased to 27-fold (Table 1).16 The fact that H. pylori is a pre-requisite for the association of these polymorphisms with malignancy demonstrates that in this situation, inflammation is indeed driving carcinogenesis.
Table 1. Frequencies and age, sex and race-adjusted odds ratios (and Cornfield 95% confidence intervals) for the association of one to four pro-inflammatory polymorphisms in IL-1B, IL-1RN, IL-10 and TNF-A with non-cardia gastric cancer.16 Polymorphisms
Cases (N ¼ 188)
Controls (N ¼ 210)
Odds ratio (95% CI)
0 1 2 3 4
22 74 62 28 2
75 85 46 4 0
(Referent) 2.8 (1.6e5.1) 5.4 (2.7e10.6) 26.3 (7.1e97.1) N (undefined)
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Another important cytokine that is key in the pathogenesis of H. pylori-induced diseases is IL-8. This chemokines belongs to the CXC family and is a potent chemoattractant for neutrophils and lymphocytes. It also has effects on cell proliferation, migration and tumour 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 was also found 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 populations34e37 with no apparent effect in Caucasians.38 It is likely that other pro-inflammatory cytokine gene polymorphisms will be relevant to gastric cancer initiation and progression. This exciting field has expanded greatly over the past few years and the search is now fully on for the full complement of risk genotypes that dictate an individual’s likelihood of developing cancer. ROLE OF POLYMORPHISMS IN THE INNATE IMMUNE RESPONSE GENES Genetic polymorphisms of cytokines of the adaptive immune response clearly play an important role in the risk of H. pylori-induced gastric adenocarcinoma. However, H. pylori is initially handled by the innate immune response and it is conceivable that functionally relevant polymorphisms in genes of this arm of the immune system could affect the magnitude and subsequent direction of the host’s response against the infection. The majority of H. pylori cells do not invade the gastric mucosa but the inflammatory response against it is triggered through attachment of H. pylori to the gastric epithelia.39 Toll-like receptor 4 (TLR4), the lipopolysaccharide (LPS) receptor, was initially identified as the potential signalling receptor for H. pylori on gastric epithelial cells.40 TLR4 belongs to a family of pattern recognition receptors, of which there are currently 11 members, that activate pro-inflammatory signalling pathways in response to microbes or pathogen-associated molecular patterns (PAMPs).41 TLR4, in conjunction with CD14 and MD-2, transduces signals through MyD88, Toll/IL-1 receptor domain and TRAF6. This promotes transcription of genes, which are involved in immune activation including the transcription factor NF-kB and also MAP kinase pathways.42 Arbour et al described a functional polymorphism at position þ896 in exon 4 of the TLR4 gene (dbSNP ID: rs4986790).43 This A > G transition results in replacement of a conserved aspartic acid residue with glycine at amino acid 299 (Asp299Gly), and alteration in the extracellular domain of the TLR4 receptor. This renders carriers hyporesponsive to LPS challenge by either disrupting transport of TLR4 to the cell membrane or by impairing ligand binding or protein interactions.43 The mutation has been associated with a variety of inflammatory and infectious conditions including atherosclerosis, myocardial infarction, inflammatory bowel disease and septic shock.44e46 Recent work demonstrates that defective signalling through the TLR4 receptor ultimately leads to an exaggerated inflammatory response with severe tissue destruction, even though the initial immune response may be blunted. This is due to inadequate production of IL-10-secreting type 1 regulatory cells.47 We hypothesised that the TLR4 þ 896A > G polymorphism would be associated with an exaggerated and destructive chronic inflammatory phenotype in H. pylori-infected subjects. This phenotype would be characterised by gastric atrophy and hypochlorhydria, the hallmarks of subsequent increased risk of gastric cancer. We further
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hypothesised that the same polymorphism might increase the risk of gastric cancer itself. We proceeded to test the effect of this polymorphism on the H. pylori-induced gastric phenotype and the risk of developing pre-malignant and malignant outcomes. We assessed associations with pre-malignant gastric changes in relatives of gastric cancer patients, including those with hypochlorhydria and gastric atrophy. We also genotyped two independent Caucasian population-based caseecontrol studies of upper gastrointestinal tract cancer, initially in 312 non-cardia gastric cancer cases and 419 controls and then in 184 non-cardia gastric cancers, 123 cardia cancers, 159 oesophageal cancers, and 211 frequency-matched controls. TLR4 þ 896G carriers had a 7.7-fold (95% CI, 1.6e37.6) increased odds ratio for hypochlorhydria; the polymorphism was not associated with gastric acid output in the absence of H. pylori infection. Carriers also had significantly more severe gastric atrophy and inflammation.48 Sixteen percent of gastric cancer patients in the initial study and 15% of the non-cardia gastric cancer patients in the replication study had one or two TLR4 variant alleles vs. 8% of both control populations (combined OR ¼ 2.4; 95% CI ¼ 1.6e3.4).49 In contrast, prevalence of TLR4 þ 896G was not significantly increased in esophageal squamous cell (2%, OR ¼ 0.4) or adenocarcinoma (9%, OR ¼ 0.8) or gastric cardia cancer (11%, OR ¼ 1.2). The association of TLR4 þ 896A > G polymorphism with both gastric cancer and its precursor lesions implies that it is relevant to the entire multistage process of gastric carcinogenesis, which starts with H. pylori colonisation of the gastric mucosa. Subjects with this polymorphism have an increased risk of severe inflammation and subsequently, 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-colonisation 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. Evidence supporting this concept comes from the work of Sanduleanu et al who showed that pharmacological inhibition of acid secretion was associated with a higher prevalence of non-H. pylori bacteria.50 Furthermore, the simultaneous presence of H. pylori and non-H. pylori bacteria was associated with a markedly increased risk of atrophic gastritis, and with higher circulating levels of IL-1b and IL-8. Supportive evidence also comes from animal studies where hypochlorhydria was induced in mice either genetically (G/G gastrin-deficient mice) or pharmacologically (administration of omeprazole). Zavros et al found that genetic or chemical hypochlorhydria predisposes the stomach to bacterial overgrowth resulting in inflammation, which was not present in the wild type mice or those not treated with an acid inhibitor.51 We propose that subjects with a pro-inflammatory genetic makeup based on a combination of cytokine gene markers (e.g. IL-1b, TNF-a, IL-10, IL-8) and the innate immune response (e.g. TLR4), respond to H. pylori infection by creating an environment within the stomach that is chronically inflamed and with reduced acidity. This environment is conducive to the growth of other bacteria within the gastric milieu, leading to sustained inflammation and oxidative/genotoxic stress. Subjects with the same proinflammatory polymorphisms may respond in the same exaggerated manner to these non-H. pylori bacteria, thus maintaining the pro-neoplastic drive (Figure 1). This may explain why H. pylori is not required in the latter stages of gastric carcinogenesis and why it is often absent from gastric tumour tissue. The potential mechanism by which the TLR4 polymorphism increases the risk of gastric cancer and its precursors is intriguing and may lie in the nature of the host’s overall response to the H. pylori LPS attack. Failure to handle the invasion by appropriately recognising and activating the necessary pathways may lead to an imbalance of pro- and
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Pro-inflammatory cytokine gene polymorphisms
Innate immune response gene polymorphisms Gastric cancer phenotype
IL-1B-511*T IL-1-RN*2*2 IL-10 ATA haplotype TNF-A-308*A IL-8-251*A
Severe inflammation
atrophy
TLR4+896*G
Hypochlorhydria Esophagus Duodenum Lesser Curvature Pylorus
Cardiac End of Stomach
Greater Curvature
Bacterial overgrowth
Bacterial factors CagA vac A s1/m1
Environmental factors Smoking Diet
Figure 1. Role of host genetic factors in the pathogenesis of H. pylori-induced gastric cancer.
anti-inflammatory mediators. A very elegant demonstration of this phenomenon was recently reported by Higgins et al.47 The authors infected TLR4-defective C3H/HeJ mice and their wild type counterparts (C3H/HeN) with an aerosol of B. pertussis (a Gram-negative bacterium that causes whooping cough) and monitored the course of the infection and its consequences over several weeks. The course of the infection was more severe in the TLR4-defective than the wild type mice and this was associated with enhanced inflammatory cytokine production, cellular infiltration and severe pathological changes in the lungs. Most interestingly, Higgins et al showed that signalling through TLR4 in response to bacterial infection activated IL-10 production, which promoted IL-10-producing T cells and controlled inflammatory pathology during infection in normal, but not TLR4-defective mice. It is therefore likely that the severe tissue damage observed in the TLR4-defective mice was due to the deficiency of the anti-inflammatory IL-10, which in turn accentuated the pro-inflammatory destructive tissue response. ROLE OF HLA POLYMORPHISMS IN GASTRIC CANCER Several studies have examined the role of HLA class I and II alleles in gastric cancer, in Caucasian and non-Caucasian populations. Lee et al found that the HLA class II allele DQB1*0301 was more common in Caucasian patients with gastric adenocarcinoma than non-cancer controls.52 The mechanism linking HLA-DQB1*0301 with gastric adenocarcinoma was not clear but was thought to be independent of increased susceptibility to H. pylori infection. Azuma et al reported that the allele frequency of DQA1*0102 was significantly lower in the H. pylori-infected subjects with atrophic gastritis compared with infected subjects without atrophy and uninfected subjects.53 In addition, the allele frequency of DQA1*0102 was also significantly lower in the infected intestinal type gastric adenocarcinoma patients compared with all controls (infected and uninfected). They concluded that the HLA DQA1*0102 allele was
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protective against gastric atrophy and intestinal type gastric adenocarcinoma. Magnusson et al studied the effect of HLA class II alleles on risk of H. pylori infection and gastric cancer.54 They confirmed Azuma et al’s finding that the DQA1*0102 allele was associated with decreased risk of H. pylori infection but they found no protective effect on risk of gastric cancer. They also showed that the DRB1*1601 allele was significantly associated with an increased gastric cancer risk with an odds ratio of 8.7 (95% confidence interval 2.7e28.0) of 8.7. The effect of *1601 was more pronounced among Hp-negative subjects, and the association was stronger with the diffuse, rather than with the intestinal, histological type of gastric cancer.54 Interestingly, they failed to show any association between gastric cancer and the DQB1*0301 reported by Lee et al. These three studies highlight several problems pertinent to the study of HLA disease associations. The HLA system is highly polymorphic and the genetic variation is very much dependent on ethnicity and its background selective heritage. As such, HLA disease associations in certain ethnic groups are unlikely to be relevant to others, simply because certain alleles may be under/overrepresented in these groups independent of the disease under question. The other relevant point here is the power required for such association studies. If one is dealing with the most highly polymorphic genetic system, it follows that the power of the studies has to match the complexity of this system. The study by Lee et al was based on 52 gastric cancer cases and 260 non-cancer controls, while Azuma et al studied 82 cancer cases and 167 controls and Magnusson et al had 130 cancer cases and 263 population controls. It could be argued that all three studies were grossly underpowered to look at associations between gastric cancer and HLA markers. Indeed, the sample size required will likely be in the thousands to get closer to any true associations. WHAT IS THE VALUE OF IDENTIFYING HOST GENETIC FACTORS IN SPORADIC GASTRIC CANCER? Complex human diseases are often multifactorial and their pathogenesis combines the effects of host and environmental factors. In the past, our research knowledge and technical know-how were limited in their ability to probe disease pathogenesis. The genetic revolution over the past decade has enabled scientists for the first time to examine afresh a multitude of unanswered clinical problems. Defining host genetic factors that control basic physiological processes will explain many of the seemingly divergent phenotypic expressions of disease. Therefore, the most important benefit to the study of host genetics is the better understanding of disease pathogenesis. In the case of sporadic gastric cancer, host genetics has helped in confirming two very important facts: the first is the important role of the infection (in this case H. pylori), and second is the important role of chronic inflammation with its long-term deleterious effects on gastric physiology. As such, the most sensible and practical conclusions from this knowledge are to either avoid getting the infection in the first place, or to remove it or ameliorate its effects once it is established. The first is happening spontaneously, thanks to our improved hygiene and living conditions, but the second requires considerable thought and continued research. While most of us would recognise the obvious theoretical benefit to ridding the world of H. pylori in terms of preventing non-cardia gastric cancer and peptic ulcer disease, there remains considerable doubt about what might happen to those in whom the natural history of this chronic infection has been disrupted. Does eradication work at all stages of the disease
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process or is there a point of no return? Does the recovery of acid secretion lead to new problems? Is there an increased risk of acid-related cancer, e.g. oesophageal adenocarcinoma? These questions can only be answered through well-designed adequately powered and scientifically sound trials. Some of these trials are on going and the next few years promise to clear up some of the controversies. The other benefit to studying host genetic factors is in being able to predict clinical outcomes following certain exposures (microbial, chemical, dietary, pharmacological, etc). For example, if we could define who might develop an atrophic, hypochlorhydric response to H. pylori infection, this could form the basis for genetic screening so that these individuals could be offered eradication therapy. As things stand, this approach offers little benefit over simply checking for H. pylori infection itself. The reason is that the currently identified genetic risk markers are very common in the population and are not specific enough to act as predictors of gastric cancer risk. It may be that future advances in affordable high throughput genotyping could uncover a much more extensive genetic profile that satisfies the criteria for a screening test. If such a development was feasible, we must ensure that our governments enact laws that protect individuals from being discriminated against on the basis of their genetic heritage. These issues are not far off coming and the debate has to start now. SUMMARY 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 (Figure 1). 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 proinflammatory host genetic makeup is only relevant in the presence of infection, initially H. pylori but later could be 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. This genetic profile in combination with testing for the infection and its virulence factors may prove a useful tool in targeting the populations that require eradication therapy. Eradication studies aiming to prevent non-cardia gastric cancer should also focus on identifying who might develop an unfavourable outcome to this strategy. Host genetics will no doubt play its role in defining these patients as well.
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Role of host genes in sporadic gastric cancer 685 5. Macarthur M, Hold GL & El-Omar EM. Inflammation and Cancer II. Role of chronic inflammation and cytokine gene polymorphisms in the pathogenesis of gastrointestinal malignancy. Am J Physiol Gastrointest Liver Physiol 2004; 286(4): G515eG520. 6. El-Omar EM, Penman ID, Ardill JE et al. Helicobacter pylori infection and abnormalities of acid secretion in patients with duodenal ulcer disease. Gastroenterology 1995; 109(3): 681e691. 7. Hansson LE, Nyren O, Hsing AW et al. The risk of stomach cancer in patients with gastric or duodenal ulcer disease. N Engl J Med 1996; 335(4): 242e249. 8. Blaser MJ & Atherton JC. Helicobacter pylori persistence: biology and disease. J Clin Invest 2004; 113(3): 321e333. 9. Israel DA & Peek RM. Pathogenesis of Helicobacter pylori-induced gastric inflammation. Aliment Pharmacol Ther 2001; 15(9): 1271e1290. 10. Risch NJ. Searching for genetic determinants in the new millennium. Nature 2000; 405(6788): 847e856. 11. Collins FS, Green ED, Guttmacher AE et al. A vision for the future of genomics research. Nature 2003; 422(6934): 835e847. 12. Kuipers EJ, Lundell L, Klinkenberg-Knol EC et al. Atrophic gastritis and Helicobacter pylori infection in patients with reflux esophagitis treated with omeprazole or fundoplication. N Engl J Med 1996; 334(16): 1018e1022. 13. Kuipers EJ, Lee A, Klinkenberg-Knol EC et al. Review article: the development of atrophic gastritis e Helicobacter pylori and the effects of acid suppressive therapy. Aliment Pharmacol Ther 1995; 9(4): 331e340. 14. El-Omar EM. The importance of interleukin 1beta in Helicobacter pylori associated disease. Gut 2001; 48(6): 743e747. 15. El-Omar EM, Carrington M, Chow WH et al. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature 2000; 404(6776): 398e402. 16. El-Omar EM, Rabkin CS, Gammon MD et al. Increased risk of noncardia gastric cancer associated with proinflammatory cytokine gene polymorphisms. Gastroenterology 2003; 124(5): 1193e1201. 17. Ando T, El-Omar EM, Goto Y et al. Interleukin 1B proinflammatory genotypes protect against gastrooesophageal reflux disease through induction of corpus atrophy. Gut 2006; 55(2): 158e164. 18. Queiroz DM, Guerra JB, Rocha GA et al. IL1B and IL1RN polymorphic genes and Helicobacter pylori cagA strains decrease the risk of reflux esophagitis. Gastroenterology 2004; 127(1): 73e79. 19. Machado JC, Pharoah P, Sousa S et al. Interleukin 1B and interleukin 1RN polymorphisms are associated with increased risk of gastric carcinoma. Gastroenterology 2001; 121(4): 823e829. 20. Figueiredo C, Machado JC, Pharoah P et al. Helicobacter pylori and interleukin 1 genotyping: an opportunity to identify high-risk individuals for gastric carcinoma. J Natl Cancer Inst 2002; 94(22): 1680e1687. 21. Machado JC, Figueiredo C, Canedo P et al. A proinflammatory genetic profile increases the risk for chronic atrophic gastritis and gastric carcinoma. Gastroenterology 2003; 125(2): 364e371. 22. Rad R, Prinz C, Neu B et al. Synergistic effect of Helicobacter pylori virulence factors and interleukin-1 polymorphisms for the development of severe histological changes in the gastric mucosa. J Infect Dis 2003; 188(2): 272e281. 23. Palli D, Saieva C, Luzzi I et al. Interleukin-1 gene polymorphisms and gastric cancer risk in a high-risk Italian population. Am J Gastroenterol 2005; 100(9): 1941e1948. 24. Furuta T, El-Omar EM, Xiao F et al. Interleukin 1beta polymorphisms increase risk of hypochlorhydria and atrophic gastritis and reduce risk of duodenal ulcer recurrence in Japan. Gastroenterology 2002; 123(1): 92e105. 25. Zeng ZR, Hu PJ, Pang RP et al. The association of interleukin-1B gene polymorphism and gastric cancers in high and low prevalence regions in China. Gut 2003; 52(12): 1684e1689. 26. Garza-Gonzalez E, Bosques-Padilla FJ, El-Omar E et al. Role of the polymorphic IL-1B, IL-1RN and TNF-A genes in distal gastric cancer in Mexico. Int J Cancer 2005; 114(2): 237e241. 27. Warren BF. Cytokines in the cotton top tamarin model of human ulcerative colitis. Aliment Pharmacol Ther 1996; 10(supplement 2): 45e47. 28. Lee KA, Ki CS, Kim HJ et al. Novel interleukin 1beta polymorphism increased the risk of gastric cancer in a Korean population. J Gastroenterol 2004; 39(5): 429e433. 29. Chang YW, Jang JY, Kim NH et al. Interleukin-1B (IL-1B) polymorphisms and gastric mucosal levels of IL-1beta cytokine in Korean patients with gastric cancer. Int J Cancer 2005; 114(3): 465e471. 30. Yang J, Hu Z, Xu Y et al. Interleukin-1B gene promoter variants are associated with an increased risk of gastric cancer in a Chinese population. Cancer Lett 2004; 215(2): 191e198.
686 E. M. El-Omar 31. Tu S, Cui G, Takaishi S et al. Overexpression of human interleukin-1 beta in transgenic mice results in spontaneous gastric inflammation and carcinogenesis. Gastroenterology 2005; 128(4, supplement 2): A62 [ref type: abstract]. 32. Beales IL & Calam J. Interleukin 1 beta and tumour necrosis factor alpha inhibit acid secretion in cultured rabbit parietal cells by multiple pathways. Gut 1998; 42(2): 227e234. 33. Smith MG, Hold GL, Rabkin CS et al. The IL-8-251 promoter polymorphism is associated with high IL-8 production, severe inflammation and increased risk of pre-malignant changes in H-pylori positive subjects. Gastroenterology 2004; 124(4, supplement 2): A23 [ref type: abstract]. 34. Taguchi A, Ohmiya N, Shirai K et al. Interleukin-8 promoter polymorphism increases the risk of atrophic gastritis and gastric cancer in Japan. Cancer Epidemiol Biomarkers Prev 2005; 14(11 Pt 1): 2487e2493. 35. Lu W, Pan K, Zhang L et al. Genetic polymorphisms of interleukin (IL)-1B, IL-1RN, IL-8, IL-10 and tumor necrosis factor {alpha} and risk of gastric cancer in a Chinese population. Carcinogenesis 2005; 26(3): 631e636. 36. Lee WP, Tai DI, Lan KH et al. The -251T allele of the interleukin-8 promoter is associated with increased risk of gastric carcinoma featuring diffuse-type histopathology in Chinese population. Clin Cancer Res 2005; 11(18): 6431e6441. 37. Ohyauchi M, Imatani A, Yonechi M et al. The polymorphism interleukin 8-251 A/T influences the susceptibility of Helicobacter pylori related gastric diseases in the Japanese population. Gut 2005; 54(3): 330e335. 38. Smith MG, Hold GL, Rabkin CS et al. The interleukin-8-251 promoter polymorphism and risk of gastric cancer in Caucasian and Japanese populations. Gastroenterology 2006; 128(4, supplement 2): A-19 [ref type: abstract]. 39. Segal ED, Lange C, Covacci A et al. Induction of host signal transduction pathways by Helicobacter pylori. Proc Natl Acad Sci U S A 1997; 94(14): 7595e7599. 40. Su B, Ceponis PJ, Lebel S et al. Helicobacter pylori activates Toll-like receptor 4 expression in gastrointestinal epithelial cells. Infect Immun 2003; 71(6): 3496e3502. 41. Pasare C & Medzhitov R. Toll-like receptors: linking innate and adaptive immunity. Adv Exp Med Biol 2005; 560: 11e18. 42. Takeda K & Akira S. Toll-like receptors in innate immunity. Int Immunol 2005; 17(1): 1e14. 43. Arbour NC, Lorenz E, Schutte BC et al. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet 2000; 25(2): 187e191. 44. Franchimont D, Vermeire S, El Housni H et al. Deficient hostebacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn’s disease and ulcerative colitis. Gut 2004; 53(7): 987e992. 45. Lorenz E, Mira JP, Frees KL et al. Relevance of mutations in the TLR4 receptor in patients with gramnegative septic shock. Arch Intern Med 2002; 162(9): 1028e1032. 46. Kiechl S, Lorenz E, Reindl M et al. Toll-like receptor 4 polymorphisms and atherogenesis. N Engl J Med 2002; 347(3): 185e192. 47. Higgins SC, Lavelle EC, McCann C et al. Toll-like receptor 4-mediated innate IL-10 activates antigenspecific regulatory T cells and confers resistance to Bordetella pertussis by inhibiting inflammatory pathology. J Immunol 2003; 171(6): 3119e3127. 48. Hold GL, Smith MG, McColl KE et al. A functional Toll-like receptor 4 polymorphism increases risk of H. pylori-induced premalignant changes. Gastroenterology 2003; 124(4): A18 [ref type: abstract]. 49. Hold GL, Smith MG, Chow WH et al. A functional Toll-like receptor 4 polymorphism increases risk of gastric cancer. Gastroenterology 2004; 126(4, supplement 2): A245 [ref type: abstract]. 50. Sanduleanu S, Jonkers D, De Bruine A et al. Double gastric infection with Helicobacter pylori and nonHelicobacter pylori bacteria during acid-suppressive therapy: increase of pro-inflammatory cytokines and development of atrophic gastritis. Aliment Pharmacol Ther 2001; 15(8): 1163e1175. 51. Zavros Y, Rieder G, Ferguson A et al. Genetic or chemical hypochlorhydria is associated with inflammation that modulates parietal and G-cell populations in mice. Gastroenterology 2002; 122(1): 119e133. 52. Lee JE, Lowy AM, Thompson WA et al. Association of gastric adenocarcinoma with the HLA class II gene DQB10301. Gastroenterology 1996; 111(2): 426e432. 53. Azuma T, Ito S, Sato F et al. The role of the HLA-DQA1 gene in resistance to atrophic gastritis and gastric adenocarcinoma induced by Helicobacter pylori infection. Cancer 1998; 82(6): 1013e1018. 54. Magnusson PKE, Enroth H, Eriksson I et al. Gastric Cancer and Human Leukocyte Antigen: Distinct DQ and DR Alleles Are Associated with Development of Gastric Cancer and Infection by Helicobacter pylori. Cancer Res 2001; 61(6): 2684e2689.
3 Role of host genes in sporadic gastric cancer Emad M. El-Omar*
BSc(Hons), MB ChB, MD, FRCP (Edin)
Professor of Gastroenterology/Honorary Consultant Physician Department of Medicine and Therapeutics, Aberdeen University, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK
Gastric cancer remains a major health problem particularly in the developing world. Helicobacter pylori (H. pylori) infection is the most recognised aetiological risk factor for this malignancy. The infection causes a chronic gastritis that is the precursor to all the pathophysiological abnormalities characteristic of gastric carcinogenesis. Genetic polymorphisms have emerged in recent years as important determinants of disease susceptibility and severity. In the case of H. pyloriinduced gastric cancer, host genetic polymorphisms play an important role both in the precancerous stages and in the transition to cancer. In particular, polymorphic genes of the adaptive and innate immune response are involved in all stages of the neoplastic process. This field is rapidly expanding and many other genetic determinants are currently being defined. The ultimate value of host genetics should be in understanding the pathogenesis of the disease, which would offer a true opportunity to defeat this global killer. Key words: gastric cancer; Helicobacter pylori; genetic polymorphisms; single nucleotide polymorphisms; cytokines; interleukins; toll-like receptors.
INTRODUCTION Gastric cancer remains a major health problem despite the recent decline in the incidence of this malignancy in the developed world. The global burden is shifting from the developed to the developing world and around 1.1 million new cases are expected in the year 2010.1 Helicobacter pylori (H. pylori) infection is the most recognised aetiological risk factor for this malignancy.2 As already outlined in earlier chapters, H. pylori infection causes a chronic gastritis that is the precursor to all the pathophysiological abnormalities characteristic of gastric carcinogenesis.3 The most important of these abnormalities are severe corpus-predominant gastritis, gastric atrophy and hypochlorhydria, which develop as a direct result of the chronic inflammation induced by the infection.4,5 It is important to appreciate however that this so-called ‘gastric cancer * Tel: þ44 1224 553021; Fax: þ44 1224 555766. E-mail address: [email protected]. 1521-6918/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved.
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phenotype’ is a relatively rare outcome whilst the overwhelming majority of infected subject worldwide do not develop any significant clinical disease. This second ‘benign’ phenotype is characterised by a mild mixed gastritis pattern that has little impact on gastric physiology, particularly on gastric acid secretion. Finally, there is a third phenotype, the so-called ‘duodenal ulcer phenotype’, which is characterised by an antrumpredominant gastritis, high gastric acid secretion and an increased risk of developing duodenal ulcers.6 The latter group account for a variable percentage of infected subjects and are relatively common in the West (up to 15%), and rare in the East. Furthermore, subjects who develop duodenal ulcers are actually protected from developing gastric cancer, suggesting that the two outcomes are mutually exclusive.7 It is clear therefore that H. pylori infection can lead to several divergent clinical outcomes. Explaining this apparent paradox is essential for understanding the pathogenesis of H. pylori-related disease in general and gastric cancer in particular. The last two decades have seen major advances in unravelling the contribution of bacterial virulence factors, environmental exposures and host genetic factors in the pathogenesis of H. pylori-induced diseases. The aim of this review is to focus on the role of host genetic factors in the pathogenesis of sporadic gastric adenocarcinoma. For excellent reviews of bacterial virulence factors the reader is directed to the following reports by Blaser & Atherton and Israel & Peek.8,9 GENETIC POLYMORPHISMS AND MULTIFACTORIAL HUMAN DISEASE Before discussing the role of host genetic factors in the pathogenesis of sporadic gastric cancer, it is essential to establish some basic principles of genetic epidemiology and the limitations of studying genetic polymorphisms in the context of complex multifactorial human diseases. The completion of the human genome project was a momentous occasion for humanity. It opened up the opportunity to dissect complex human traits and to understand basic pathways of health and disease. While linkage analysis studies are suitable for pursuing rare high-risk alleles in conditions that have a hereditary basis, population-based association studies are much more useful for examining genes with a role in commoner multifactorial diseases that have a strong environmental component.10 These association studies often estimate the risk of developing a certain disease in carriers and non-carriers of a particular genetic polymorphism. The overwhelming majority of polymorphisms studied are single nucleotide polymorphisms (SNPs) that occur with a frequency of >1% in the normal population (in contrast to ‘mutations’ that occur with a frequency of <1%). It is estimated that up to 10 million SNPs are probably present in the human genome though not all have thus far been identified. Naturally, most of these SNPs do not occur in coding sequences and even those that do, are not associated with any alteration in the amino acid sequence and are therefore of no functional consequence. A more useful definition of the variability of these SNPs involves haplotype analysis. Haplotypes are a combination of alleles at different markers along the same chromosome that are inherited as a unit. The HapMap project was set up to address this (www.hapmap.org) and is a valuable tool that will facilitate the study of genetic polymorphisms relevant to human health and disease. Other types of genetic variation include deletion and insertion polymorphisms and microsatellite repeat polymorphisms. There has been an exponential rise in the number of published genetic association studies. Quite often, a report of a single genetic marker is published with great
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promise only to be followed by several negative studies that fail to reproduce the original observation. There is no doubt that the strategy of genetic association studies could be a powerful tool for dissecting human diseases, provided certain principles are observed to minimise the chances of false positive, and negative, reports. The most important of these principles are (1) rigorous definition of disease phenotype, (2) choice of candidate genes that are plausibly linked to the pathophysiology of the disease under study, (3) selection of polymorphisms with known (or at least potentially) functional consequences, (4) choice of genetic markers that are reasonably frequent in the population under study (variant allele frequency of at least 5%), (5) appropriate selection of controls that are matched for ethnicity, age, gender, and environmental exposures, (6) design of studies that are adequately powered to produce a valid result. Even then, the statistical analyses of such studies have to take into account the real problem of false positive results by multiple testing. Appropriate corrections for multiple testing have to be applied, or alternatively, the positive findings should be regarded as preliminary and should be validated in an independent set of cases and controls. Finally, the genetic epidemiology has to involve basic science in order to unravel and validate the molecular mechanisms involved. Adherence to these basic principles will ensure that false positive trails are minimised and will offer a true opportunity to understand complex multifactorial human diseases. There has never been a more stressing time for adherence to these principles, as the advancement in genotyping technology has made possible the annotation of the entire human genome. Current technology allows us to genotype up to 500,000 SNPs in one run, but more awesome is the future prospect of replacing genotyping and haplotyping with direct low-cost sequencing of the entire genome, the so-called ‘$1000 genome’.11 Having set the background to the study of genetic polymorphisms, we can now examine the role of these in the pathogenesis of H. pylori-induced gastric cancer. H. pylori infection elicits a powerful humoural and cell-mediated immune response that causes inflammation in the gastric mucosa. The mediators of this inflammatory response belong to the two main arms of the immune system, namely adaptive and innate immunity. POLYMORPHISMS OF CYTOKINE GENES AND RISK OF GASTRIC CANCER Role of interleukin-1 (IL-1) genetic markers H. pylori causes its damage by initiating chronic inflammation in the gastric mucosa. This inflammation is mediated by an array of pro- and anti-inflammatory cytokines. Genetic polymorphisms directly influence inter-individual variation in the magnitude of cytokine response and this clearly contributes to an individual’s ultimate clinical outcome. In the case of H. pylori infection, we speculated that the most relevant candidate genes would be ones whose products were involved in handling the H. pylori attack (innate and adaptive immune responses) and ones that mediated the resulting inflammation. Because such a list of candidate genes would be prohibitively extensive, our initial search focussed on genes that were most relevant to gastric physiology, and in particular, gastric acid secretion. As mentioned above, H. pylori-induced gastritis is associated with three main phenotypes that correlate closely with clinical outcome: DU phenotype, benign phenotype and gastric cancer phenotype. Studies have shown that inhibition of gastric acid pharmacologically can lead to a shift from an antrum-predominant pattern (DU phenotype) to a corpus-predominant one with onset of gastric atrophy (gastric cancer
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phenotype).12,13 Thus it was clear that an endogenous agent that was upregulated in the presence of H. pylori, has a profound pro-inflammatory effect and was also an acid inhibitor, would be the most relevant host genetic factor to be studied. Interleukin-1 beta (IL-1b) fitted this profile perfectly, for not only is it one of the earliest and most important pro-inflammatory cytokines in the context of H. pylori infection, it is but also the most powerful acid inhibitor known.14 We have shown that pro-inflammatory IL-1 gene cluster polymorphisms (IL-1B encoding IL-1b and IL-1RN encoding its naturally occurring receptor antagonist) increase the risk of gastric cancer and its precursors in the presence of H. pylori.15 Individuals with the IL-1B-31*C or -511*T and IL-1RN*2/*2 genotypes are at increased risk of developing hypochlorhydria and gastric atrophy in response to H. pylori infection. This risk also extends to gastric cancer itself with a 2e3-fold increased risk of malignancy compared to subjects who have the less proinflammatory genotypes.15,16 Furthermore, the pro-inflammatory IL-1 genotypes increased the risk of both intestinal and diffuse types of gastric cancer but the risk was restricted to the non-cardia subsite. Indeed, the IL-1 markers had no effect on risk of cardia gastric adenocarcinoma, oesophageal adenocarcinoma or oesophageal squamous cell carcinoma.16 The latter findings are entirely in keeping with the proposed mechanism for the effect of these polymorphisms in gastric cancer, namely reduction of gastric acid secretion. Thus a high IL-1b genotype increases the risk of non-cardia gastric cancer, a disease characterised by hypochlorhydria, while it has no effect on cancers associated with high acid exposure such as oesophageal adenocarcinoma and some cardia cancers. Interestingly, the high IL-1b pro-inflammatory genotypes do protect against the development of erosive and non-erosive oesophagitis, suggesting again that they operate by reducing gastric acidity through induction of corpus atrophy.17,18 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.19e27 Machado et al were the first to confirm the association between IL-1 markers and gastric cancer in Caucasians and reported similar odds ratios to those reported by our group.19 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). They showed that for each combination of bacterial/host genotype, the odds of having gastric carcinoma were greatest in those with both bacterial and host high-risk genotypes.20 This highlights the important interaction between host and bacterium in the pathogenesis of gastric cancer. 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. However, even excluding the weaker studies, there is still the impression that not all Asian or Caucasian populations have demonstrated a predisposition for gastric cancer in association with ‘published’ pro-inflammatory IL-1 polymorphisms. In some instances, studies found that there was a positive association but with novel markers of the IL-1B gene.28 Other studies pointed 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 to high incidence areas.25 Finally some studies reported a positive association with IL-1 markers but this was confined to the opposite alleles from those reported by the majority of studies.29,30 In one study, the authors confirmed that the opposite alleles (previously found to associate with reduced IL-1b production) were in fact the high IL-1b alleles in their population.29 Taken at face value these findings still point to IL-1b being a crucial cytokine in the
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pathogenesis of H. pylori-induced gastric cancer and its precursors and variations in its gene act as host genetic factors that mediate this effect. A crucial piece of evidence that confirmed the unique role of IL-1b in H. pyloriinduced gastric carcinogenesis came from a transgenic mouse model in which IL-1b overproduction was targeted to the stomach by the Hþ/Kþ ATPase beta promoter. With overexpression of IL-1b confined to the stomach, these transgenic mice had a thickened gastric mucosa, produced lower amounts of gastric acid and developed severe gastritis followed by atrophy, intestinal metaplasia, dysplasia and adenocarcinoma. Crucially, these IL-1b 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.31 Role of other cytokine gene polymorphisms Soon after the IL-1 gene cluster polymorphisms were identified as risk factors for gastric cancer, the pro-inflammatory genotypes of tumour necrosis factor-a (TNF-A) and IL10 were reported as independent additional risk factors for non-cardia gastric cancer.16 TNF-a is another powerful pro-inflammatory cytokine that is produced in the gastric mucosa in response to H. pylori infection. Like IL-1b, it has an acid inhibitory effect, albeit much weaker.32 The TNF-A-308 G > A polymorphism is known to be involved in a number of inflammatory conditions. Carriage of the pro-inflammatory A allele increased the odds ratio for non-cardia gastric cancer to 2.2 (95% confidence intervals: 1.4e3.7). The role of the TNF-A-308 G > A polymorphism in gastric cancer was independently confirmed by a study from Machado et al.21 IL-10 is an anti-inflammatory cytokine that downregulates IL-1b, TNF-a, interferon-g and other pro-inflammatory cytokines. Relative deficiency of IL-10 may result in a T helper-1 (Th-1) -driven hyper-inflammatory response to H. pylori with greater damage to the gastric mucosa. We 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 noncardia gastric cancer with an odds ratio of 2.5 (95% CI: 1.1e5.7). We have studied the effect of having an increasing number of pro-inflammatory genotypes (IL-1B-511*T, IL-1RN*2*2, TNF-A-308*A, and IL-10 ATA/ATA) on the risk of non-gastric cancer. The risk increased progressively so that by the time three to 4 of these polymorphisms were present, the odds ratio for gastric cancer was increased to 27-fold (Table 1).16 The fact that H. pylori is a pre-requisite for the association of these polymorphisms with malignancy demonstrates that in this situation, inflammation is indeed driving carcinogenesis.
Table 1. Frequencies and age, sex and race-adjusted odds ratios (and Cornfield 95% confidence intervals) for the association of one to four pro-inflammatory polymorphisms in IL-1B, IL-1RN, IL-10 and TNF-A with non-cardia gastric cancer.16 Polymorphisms
Cases (N ¼ 188)
Controls (N ¼ 210)
Odds ratio (95% CI)
0 1 2 3 4
22 74 62 28 2
75 85 46 4 0
(Referent) 2.8 (1.6e5.1) 5.4 (2.7e10.6) 26.3 (7.1e97.1) N (undefined)
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Another important cytokine that is key in the pathogenesis of H. pylori-induced diseases is IL-8. This chemokines belongs to the CXC family and is a potent chemoattractant for neutrophils and lymphocytes. It also has effects on cell proliferation, migration and tumour 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 was also found 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 populations34e37 with no apparent effect in Caucasians.38 It is likely that other pro-inflammatory cytokine gene polymorphisms will be relevant to gastric cancer initiation and progression. This exciting field has expanded greatly over the past few years and the search is now fully on for the full complement of risk genotypes that dictate an individual’s likelihood of developing cancer. ROLE OF POLYMORPHISMS IN THE INNATE IMMUNE RESPONSE GENES Genetic polymorphisms of cytokines of the adaptive immune response clearly play an important role in the risk of H. pylori-induced gastric adenocarcinoma. However, H. pylori is initially handled by the innate immune response and it is conceivable that functionally relevant polymorphisms in genes of this arm of the immune system could affect the magnitude and subsequent direction of the host’s response against the infection. The majority of H. pylori cells do not invade the gastric mucosa but the inflammatory response against it is triggered through attachment of H. pylori to the gastric epithelia.39 Toll-like receptor 4 (TLR4), the lipopolysaccharide (LPS) receptor, was initially identified as the potential signalling receptor for H. pylori on gastric epithelial cells.40 TLR4 belongs to a family of pattern recognition receptors, of which there are currently 11 members, that activate pro-inflammatory signalling pathways in response to microbes or pathogen-associated molecular patterns (PAMPs).41 TLR4, in conjunction with CD14 and MD-2, transduces signals through MyD88, Toll/IL-1 receptor domain and TRAF6. This promotes transcription of genes, which are involved in immune activation including the transcription factor NF-kB and also MAP kinase pathways.42 Arbour et al described a functional polymorphism at position þ896 in exon 4 of the TLR4 gene (dbSNP ID: rs4986790).43 This A > G transition results in replacement of a conserved aspartic acid residue with glycine at amino acid 299 (Asp299Gly), and alteration in the extracellular domain of the TLR4 receptor. This renders carriers hyporesponsive to LPS challenge by either disrupting transport of TLR4 to the cell membrane or by impairing ligand binding or protein interactions.43 The mutation has been associated with a variety of inflammatory and infectious conditions including atherosclerosis, myocardial infarction, inflammatory bowel disease and septic shock.44e46 Recent work demonstrates that defective signalling through the TLR4 receptor ultimately leads to an exaggerated inflammatory response with severe tissue destruction, even though the initial immune response may be blunted. This is due to inadequate production of IL-10-secreting type 1 regulatory cells.47 We hypothesised that the TLR4 þ 896A > G polymorphism would be associated with an exaggerated and destructive chronic inflammatory phenotype in H. pylori-infected subjects. This phenotype would be characterised by gastric atrophy and hypochlorhydria, the hallmarks of subsequent increased risk of gastric cancer. We further
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hypothesised that the same polymorphism might increase the risk of gastric cancer itself. We proceeded to test the effect of this polymorphism on the H. pylori-induced gastric phenotype and the risk of developing pre-malignant and malignant outcomes. We assessed associations with pre-malignant gastric changes in relatives of gastric cancer patients, including those with hypochlorhydria and gastric atrophy. We also genotyped two independent Caucasian population-based caseecontrol studies of upper gastrointestinal tract cancer, initially in 312 non-cardia gastric cancer cases and 419 controls and then in 184 non-cardia gastric cancers, 123 cardia cancers, 159 oesophageal cancers, and 211 frequency-matched controls. TLR4 þ 896G carriers had a 7.7-fold (95% CI, 1.6e37.6) increased odds ratio for hypochlorhydria; the polymorphism was not associated with gastric acid output in the absence of H. pylori infection. Carriers also had significantly more severe gastric atrophy and inflammation.48 Sixteen percent of gastric cancer patients in the initial study and 15% of the non-cardia gastric cancer patients in the replication study had one or two TLR4 variant alleles vs. 8% of both control populations (combined OR ¼ 2.4; 95% CI ¼ 1.6e3.4).49 In contrast, prevalence of TLR4 þ 896G was not significantly increased in esophageal squamous cell (2%, OR ¼ 0.4) or adenocarcinoma (9%, OR ¼ 0.8) or gastric cardia cancer (11%, OR ¼ 1.2). The association of TLR4 þ 896A > G polymorphism with both gastric cancer and its precursor lesions implies that it is relevant to the entire multistage process of gastric carcinogenesis, which starts with H. pylori colonisation of the gastric mucosa. Subjects with this polymorphism have an increased risk of severe inflammation and subsequently, 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-colonisation 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. Evidence supporting this concept comes from the work of Sanduleanu et al who showed that pharmacological inhibition of acid secretion was associated with a higher prevalence of non-H. pylori bacteria.50 Furthermore, the simultaneous presence of H. pylori and non-H. pylori bacteria was associated with a markedly increased risk of atrophic gastritis, and with higher circulating levels of IL-1b and IL-8. Supportive evidence also comes from animal studies where hypochlorhydria was induced in mice either genetically (G/G gastrin-deficient mice) or pharmacologically (administration of omeprazole). Zavros et al found that genetic or chemical hypochlorhydria predisposes the stomach to bacterial overgrowth resulting in inflammation, which was not present in the wild type mice or those not treated with an acid inhibitor.51 We propose that subjects with a pro-inflammatory genetic makeup based on a combination of cytokine gene markers (e.g. IL-1b, TNF-a, IL-10, IL-8) and the innate immune response (e.g. TLR4), respond to H. pylori infection by creating an environment within the stomach that is chronically inflamed and with reduced acidity. This environment is conducive to the growth of other bacteria within the gastric milieu, leading to sustained inflammation and oxidative/genotoxic stress. Subjects with the same proinflammatory polymorphisms may respond in the same exaggerated manner to these non-H. pylori bacteria, thus maintaining the pro-neoplastic drive (Figure 1). This may explain why H. pylori is not required in the latter stages of gastric carcinogenesis and why it is often absent from gastric tumour tissue. The potential mechanism by which the TLR4 polymorphism increases the risk of gastric cancer and its precursors is intriguing and may lie in the nature of the host’s overall response to the H. pylori LPS attack. Failure to handle the invasion by appropriately recognising and activating the necessary pathways may lead to an imbalance of pro- and
682 E. M. El-Omar
Pro-inflammatory cytokine gene polymorphisms
Innate immune response gene polymorphisms Gastric cancer phenotype
IL-1B-511*T IL-1-RN*2*2 IL-10 ATA haplotype TNF-A-308*A IL-8-251*A
Severe inflammation
atrophy
TLR4+896*G
Hypochlorhydria Esophagus Duodenum Lesser Curvature Pylorus
Cardiac End of Stomach
Greater Curvature
Bacterial overgrowth
Bacterial factors CagA vac A s1/m1
Environmental factors Smoking Diet
Figure 1. Role of host genetic factors in the pathogenesis of H. pylori-induced gastric cancer.
anti-inflammatory mediators. A very elegant demonstration of this phenomenon was recently reported by Higgins et al.47 The authors infected TLR4-defective C3H/HeJ mice and their wild type counterparts (C3H/HeN) with an aerosol of B. pertussis (a Gram-negative bacterium that causes whooping cough) and monitored the course of the infection and its consequences over several weeks. The course of the infection was more severe in the TLR4-defective than the wild type mice and this was associated with enhanced inflammatory cytokine production, cellular infiltration and severe pathological changes in the lungs. Most interestingly, Higgins et al showed that signalling through TLR4 in response to bacterial infection activated IL-10 production, which promoted IL-10-producing T cells and controlled inflammatory pathology during infection in normal, but not TLR4-defective mice. It is therefore likely that the severe tissue damage observed in the TLR4-defective mice was due to the deficiency of the anti-inflammatory IL-10, which in turn accentuated the pro-inflammatory destructive tissue response. ROLE OF HLA POLYMORPHISMS IN GASTRIC CANCER Several studies have examined the role of HLA class I and II alleles in gastric cancer, in Caucasian and non-Caucasian populations. Lee et al found that the HLA class II allele DQB1*0301 was more common in Caucasian patients with gastric adenocarcinoma than non-cancer controls.52 The mechanism linking HLA-DQB1*0301 with gastric adenocarcinoma was not clear but was thought to be independent of increased susceptibility to H. pylori infection. Azuma et al reported that the allele frequency of DQA1*0102 was significantly lower in the H. pylori-infected subjects with atrophic gastritis compared with infected subjects without atrophy and uninfected subjects.53 In addition, the allele frequency of DQA1*0102 was also significantly lower in the infected intestinal type gastric adenocarcinoma patients compared with all controls (infected and uninfected). They concluded that the HLA DQA1*0102 allele was
Role of host genes in sporadic gastric cancer 683
protective against gastric atrophy and intestinal type gastric adenocarcinoma. Magnusson et al studied the effect of HLA class II alleles on risk of H. pylori infection and gastric cancer.54 They confirmed Azuma et al’s finding that the DQA1*0102 allele was associated with decreased risk of H. pylori infection but they found no protective effect on risk of gastric cancer. They also showed that the DRB1*1601 allele was significantly associated with an increased gastric cancer risk with an odds ratio of 8.7 (95% confidence interval 2.7e28.0) of 8.7. The effect of *1601 was more pronounced among Hp-negative subjects, and the association was stronger with the diffuse, rather than with the intestinal, histological type of gastric cancer.54 Interestingly, they failed to show any association between gastric cancer and the DQB1*0301 reported by Lee et al. These three studies highlight several problems pertinent to the study of HLA disease associations. The HLA system is highly polymorphic and the genetic variation is very much dependent on ethnicity and its background selective heritage. As such, HLA disease associations in certain ethnic groups are unlikely to be relevant to others, simply because certain alleles may be under/overrepresented in these groups independent of the disease under question. The other relevant point here is the power required for such association studies. If one is dealing with the most highly polymorphic genetic system, it follows that the power of the studies has to match the complexity of this system. The study by Lee et al was based on 52 gastric cancer cases and 260 non-cancer controls, while Azuma et al studied 82 cancer cases and 167 controls and Magnusson et al had 130 cancer cases and 263 population controls. It could be argued that all three studies were grossly underpowered to look at associations between gastric cancer and HLA markers. Indeed, the sample size required will likely be in the thousands to get closer to any true associations. WHAT IS THE VALUE OF IDENTIFYING HOST GENETIC FACTORS IN SPORADIC GASTRIC CANCER? Complex human diseases are often multifactorial and their pathogenesis combines the effects of host and environmental factors. In the past, our research knowledge and technical know-how were limited in their ability to probe disease pathogenesis. The genetic revolution over the past decade has enabled scientists for the first time to examine afresh a multitude of unanswered clinical problems. Defining host genetic factors that control basic physiological processes will explain many of the seemingly divergent phenotypic expressions of disease. Therefore, the most important benefit to the study of host genetics is the better understanding of disease pathogenesis. In the case of sporadic gastric cancer, host genetics has helped in confirming two very important facts: the first is the important role of the infection (in this case H. pylori), and second is the important role of chronic inflammation with its long-term deleterious effects on gastric physiology. As such, the most sensible and practical conclusions from this knowledge are to either avoid getting the infection in the first place, or to remove it or ameliorate its effects once it is established. The first is happening spontaneously, thanks to our improved hygiene and living conditions, but the second requires considerable thought and continued research. While most of us would recognise the obvious theoretical benefit to ridding the world of H. pylori in terms of preventing non-cardia gastric cancer and peptic ulcer disease, there remains considerable doubt about what might happen to those in whom the natural history of this chronic infection has been disrupted. Does eradication work at all stages of the disease
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process or is there a point of no return? Does the recovery of acid secretion lead to new problems? Is there an increased risk of acid-related cancer, e.g. oesophageal adenocarcinoma? These questions can only be answered through well-designed adequately powered and scientifically sound trials. Some of these trials are on going and the next few years promise to clear up some of the controversies. The other benefit to studying host genetic factors is in being able to predict clinical outcomes following certain exposures (microbial, chemical, dietary, pharmacological, etc). For example, if we could define who might develop an atrophic, hypochlorhydric response to H. pylori infection, this could form the basis for genetic screening so that these individuals could be offered eradication therapy. As things stand, this approach offers little benefit over simply checking for H. pylori infection itself. The reason is that the currently identified genetic risk markers are very common in the population and are not specific enough to act as predictors of gastric cancer risk. It may be that future advances in affordable high throughput genotyping could uncover a much more extensive genetic profile that satisfies the criteria for a screening test. If such a development was feasible, we must ensure that our governments enact laws that protect individuals from being discriminated against on the basis of their genetic heritage. These issues are not far off coming and the debate has to start now. SUMMARY 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 (Figure 1). 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 proinflammatory host genetic makeup is only relevant in the presence of infection, initially H. pylori but later could be 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. This genetic profile in combination with testing for the infection and its virulence factors may prove a useful tool in targeting the populations that require eradication therapy. Eradication studies aiming to prevent non-cardia gastric cancer should also focus on identifying who might develop an unfavourable outcome to this strategy. Host genetics will no doubt play its role in defining these patients as well.
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