Helicobacter Pylori in Gastric Cancer

Emad M El-Omar, Professor of Gastroenterology/Honorary Consultant Physician
Department of Medicine and Therapeutics, Aberdeen University and Aberdeen Royal Infirmary, UK
Gastrointestinal Cancer Abstracts 10 Spring 2003
(With thanks to Euromed Communications Ltd and Aventis)

Introduction

The role of infectious agents in carcinogenesis has commanded significant scientific interest culminating in 5 Nobel prizes in the 20th century 1 . Infections can cause cancer by a variety of mechanisms including direct transformation of cells, induction of immunosuppression with consequent reduced cancer immunosurveillance, or by causing chronic inflammation. The latter is becoming increasingly recognised as an essential component of many epithelial cancers by virtue of its combined effects of generating genotoxic by-products and increased cellular proliferation, thus maximising the potential for DNA damage.

In this article, I hope to demonstrate how interactions between an infectious agent, host genetic makeup, and environmental factors could influence the pathogenesis of a cancer. The infectious agent in question is Helicobacter pylori (H. pylori), the world's commonest chronic bacterial infection and the malignancy is gastric cancer, second only to lung cancer in its global incidence and impact. We demonstrate how this gastric infection could be utilised as a paradigm for gene-environment interactions in human disease, one that could help unravel a multitude of other microbial-induced malignancies.

Gastric cancer is a major global health problem that claimed 800,000 lives in 1998. It remains the world's second commonest malignancy, having only been overtaken by lung cancer in the late 1980s. In 1994, the International Agency for Research on Cancer (IARC) declared H. pylori a Group l (definite) carcinogen 2 . This bold statement was met with considerable scepticism but in the ensuing 8 years evidence from epidemiological and interventional studies in humans as well as experiments in rodents, has convinced many that this bacterial infection is indeed the key factor in the initiation of the neoplastic process in the stomach.

H. pylori is a gram negative, spiral shaped, microaerophilic, urease-positive bacillus, which is known to chronically infect the stomachs of over half the world's population. The infection is acquired during childhood, most probably via the faecal/oral or gastric/oral routes, and if not treated with antibiotics, will persist throughout life. 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 (Figure 1). This immune response is largely ineffective however as most infections become chronically established with little evidence that spontaneous clearance occurs.

H. pylori has to survive in one of the harshest and least hospitable niches in the human body. Gastric acidity acts as a formidable first-line of defence against food-borne pathogens and the constant outpouring of gastric secretions, coupled with regular peristalsis, ensure that gastric contents, including microbial agents, are constantly flushed away. Despite this, H. pylori seems well equipped and adapted for habitation within this harsh environment. Recent studies show that H. pylori maintains its periplasmic pH within viable limits through possession of an acid-induced urea channel that regulates intra-bacterial urease activity 3 . Essential nourishment for H. pylori is drawn from host gastric tissue through the inflammatory exudate it induces. In order to understand how this bacterium can predispose to gastric cancer, it is necessary to understand the basic pathophysiologic consequences of its presence within the human stomach.

The key pathophysiologic event in H. pylori infection is the initiation of an inflammatory response 4 . This response is most probably triggered by the bacterium's lipopolysaccharide, urease, and/or cytotoxins and is mediated by cytokines. The cytokine repertoire comprises a multitude of pro- and anti-inflammatory mediators whose function is to co-ordinate an effective immune/inflammatory response against invading pathogens without causing undue damage to the host.

Figure 1 H. pylori organisms overlying the gastric epithelial cells. Both cellular and humoral immune responses are activated but the organisms still manage to persist lifelong unless eradicated with antibiotics.

In addition to their pro- or anti-inflammatory properties, some H. pylori-induced cytokines have direct effects on gastric epithelial cells that have a profound effect on gastric physiology. For example, the pro-inflammatory cytokine interleukin-1 beta (IL-1beta ) is the most potent of known agents that are gastric cytoprotective, antiulcer, antisecretory, and inhibitors of gastric emptying 5 . Wolfe and Nompleggi 6 estimated that on a molar basis, IL-beta is 100 times more potent than both prostaglandins and the PPI omeprazole and 6000 times more potent than cimetidine in inhibiting acid secretion. Another important pro-inflammatory cytokine that is up-regulated by H. pylori infection is tumor necrosis factor alpha (TNF-alpha ), which also inhibits gastric acid secretion, but to a lesser extent than IL-1beta 7.

In physiologic terms, the stomach could 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. H. pylori infection is first established in parts of the stomach that have a higher pH such as the antrum. This is most likely due to the bacterium's attempt at energy preservation, for although H. pylori is well equipped for survival at low pH, this is achieved at a high cost of energy expenditure. Thus, high acid production by the parietal cells probably protects the corpus mucosa from initial colonisation. Both animal and human ingestion studies suggest that successful colonisation of the gastric mucosa is best achieved with the aid of acid suppression 8 . Furthermore, pharmacologic inhibition of acid secretion in infected subjects leads to redistribution of the infection and its associated gastritis from an antral to a corpus-predominant pattern 9 . Thus lack of gastric acid extends the area of colonisation and also maximises the tissue damage resulting from this colonisation.

There are three main gastric phenotypes that result from chronic H. pylori infection:

1. the commonest by far is a mild pangastritis that does not affect gastric physiology and is not associated with significant human disease,

2. a corpus-predominant gastritis associated with gastric atrophy, hypochlorhydria and increased risk of gastric cancer (the gastric cancer phenotype) 10 , and

3. an antral-predominant gastritis associated with high gastric acid secretion and increased risk of duodenal ulcer disease (the DU phenotype) 11 .

There is accumulating evidence that acid secretory capacity is crucial in determining the distribution and natural history of H. pylori infection 12 . In hosts with low secretory capacity (genetically determined or secondary to pharmacologic inhibition) the organism is capable of colonising a wider niche than would be possible in the presence of high volumes of acid. Colonisation of a wider niche including the corpus mucosca, leads to corpus gastritis with resultant functional inhibition of acid secretion. This inhibition is mediated by H. pylori-induced inflammatory cytokines (such as IL-1beta and TNF-alpha ) and the net effect is the establishment of a more aggressive gastritis that accelerates the development of gastric atrophy. Once atrophy develops, acid secretion is not only attenuated by the functional inhibition caused by inflammatory mediators but by a more permanent morphological change that is harder to reverse. This situation is very relevant to the subgroup of humans who develop the gastric cancer phenotype in the presence of chronic H. pylori infection.

In contrast to subjects who have an increased risk of gastric cancer, subjects who develop duodenal ulcer disease are known to have a large parietal cell mass that is relatively free of H. pylori-induced inflammatory activity. This pattern of antral-predominant gastritis with high acid output characterises the duodenal ulcer diathesis.

The effect of acid secretion on changing the distribution of H. pylori colonisation and gastritis is most markedly exposed in subjects in whom acid secretion is manipulated by pharmacological means. Thus H. pylori infected subjects on long-term proton pump inhibitors undergo a shift in the pattern of gastritis from antral to corpus-predominant, and they have a higher risk of developing gastric atrophy, a precursor lesion for gastric neoplasia 9 . This observation provided a clue as to the role of potential endogenous substances that could also inhibit acid secretion, such as IL-1beta and TNF-alpha . These two cytokines were therefore prime candidates as host genetic factors that may increase risk of gastric cancer.

The key question in H. pylori research is how this infection could be associated with mutually exclusive clinical outcomes such as gastric cancer and duodenal ulcer disease. A large volume of research has focussed on the role of bacterial virulence factors (e.g. cagA, vacA, iceA, etc) in the pathogenesis of these diseases and although these factors undoubtedly contribute to the degree of tissue damage, they do not distinguish between the two key outcomes 13 . This prompted us to concentrate on the host genetic factors that may be relevant to this process. The search for the appropriate candidate genes had to stem from a profound understanding of gastric physiology and how it is disrupted by H. pylori infection. Since H. pylori achieves most of its damage through induction of chronic inflammation, it was 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-1alpha and IL-beta as well as their endogenous receptor antagonist IL-1ra, respectively 14 . IL-1beta is up-regulated in the presence of H. pylori and plays a central role in initiating and amplifying the inflammatory response to this infection 15 . As mentioned above, IL-beta 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 16 . 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 16 . The presence of such highly prevalent and functional genetic polymorphisms provided for an ideal opportunity to design the appropriate epidemiologic studies to test for the role of these candidate loci.

We first studied the correlation of these high IL-1beta genotypes (two polymorphisms in the IL-1B and IL-1RN genes) with hypochlorhydria and gastric atrophy in a Caucasian population of gastric cancer relatives from Scotland. These relatives are known to be at increased risk of developing the same cancer and have a higher prevalence of the precancerous abnormalities but only in the presence of H. pylori infection. We found that the high IL-lbeta genetic markers significantly increase the risk of these precancerousconditions. In a logistic regression model including both genotypes, the estimated age-adjusted odds ratios for IL-1B-511/-31*2+ and IL-1RN*21*2 were 7.5 (95% C.I: 1.8-31) and 2.1% (95% C.I: 0.7-6.3 respectively) 17 . We proceeded to examine the association between the same IL-1beta genetic polymorphisms and gastric cancer itself utilising another Caucasian case-control study comprising 366 gastric cancer patients and 429 population controls from Poland. We confirmed the same positive association between these genotypes and gastric cancer. In a logistic regression model including both genotypes, the estimated odds ratios for IL-1B-511/-31T+ and IL-1RN*21*2 were 1.6 (95% C.I. 1.2-2.2) and 2.9 (95% C.I 1.9-4.4) respectively 17 .We have since confirmed our initial observations utilising another Caucasian case-control study from the USA 18 . In this study the pro-inflammatory IL-1 genotypes (IL-1B-511 and IL-1RN) conferred similar odds ratios for non-cardia gastric adenocarcinoma as in the Polish study. In yet another Caucasian population from Portugal, Machado et al. have independently confirmed our findings in relation to the IL-1 gene cluster markers reporting similar odds ratios 19 . Such independent confirmation strengthens the confidence in the role played by IL-beta in this human disease.

Although IL-beta was the perfect candidate gene, other genes involved in the H. pylori-induced gastritis cascade are also legitimate targets. Our most recent search has confirmed a positive but weaker role for polymorphisms in the TNF-A gene that correlate with high TNF-alpha levels 18 . The TNF-alpha polymorphism increases the risk of gastric cancer and its precursors in a similar fashion to the IL-lb polymorphisms. There is no doubt that other genetic markers will be uncovered that have a direct effect on the host's response to H. pylori infection.

But how do these IL-lbeta /TNF-alpha polymorphisms explain the divergent outcome to H. pylori infection? We speculate that the effect of these polymorphisms operates early in the disease process and requires the presence of H. pylori infection. When H. pylori infection challenges the gastric mucosa, a vigorous inflammatory response with a high IL-1beta /TNF-alpha component may appear to be beneficial, but it has the unfortunate effect of switching acid secretion off, thus allowing the infection to extend its colonisation and damaging inflammation to the corpus mucosa, an area that is usually well protected by secretion of acid. A decreased flow of acid will also undermine attempts to flush out these toxic substances causing further damage to the mucosa. More inflammation in the corpus leads to more inhibition of acid secretion and a continuing cycle that accelerates glandular loss and onset of gastric atrophy. It is apparent that this vicious cycle ultimately succeeds in driving the infection out but at a very high price for the host. This is amply demonstrated by the finding that H. pylori density becomes progressively lower with the progression from mild gastritis through severe gastritis, atrophy and intestinal metaplasia. Indeed, by the time gastric cancer develops, it is extremely hard to demonstrate any evidence of the infection 20 .

Why do only a few H. pylori infected subjects with these polymorphisms develop gastric cancer? The answer lies in the polygenic and multifactorial nature of most complex human diseases. These genetic factors operate only in the presence of an infectious agent and lead to the development of an atrophic phenotype. Progression of atrophy towards cancer depends on other components of the host genetic constitution acting epistatically, as well as by dietary and other factors in the environment. For example, it is known that men are twice as likely to develop distal gastric adenocarcinoma compared to women. The gender difference in risk raises the interesting possibility that either hormonal factors such as oestrogens or perhaps the lower body content of iron in females (carcinogenic in other tissues such as liver) may explain the difference. Furthermore, while H. pylori infection and host genetics interact to initiate a hypochlorhydric and atrophic phenotype, environmental co-factors may mediate subsequent neoplastic transformation, even after disappearance of the infection. Diet may be particularly relevant, with greater consumption of fresh fruits and vegetables shown to protect against risk of gastric as well as several other cancers. Dietary vitamin C reduces the formation of N-nitroso- compounds and scavenges mutagenic reactive oxygen metabolites generated by gastric inflammation 21 and supplemental vitamin C is associated with significantly lower risk of non-cardia gastric cancer 22 . Furthermore, vitamin C concentrations and bioavailability are reduced in the presence of H. pylori infection 23 . Another important co-factor is cigarette smoking, which was found to nearly double the risk of transition from atrophic gastritis to dysplasia in a high-risk population 24 . Thus, cytokine gene polymorphisms represent only one component of a complex interplay among host, pathogen, and environmental factors involved in gastric carcinogenesis (Figure 2).

	Helicobacter pylori infection
* Low inflammatory genetic makeup * High parietal cell mass + Environmental factors (eg smoking)	* Low inflammatory genetic makeup * Healthy diet	* Pro-inflammatory genetic makeup    (IL-1beta/TNF-A) + Environmental factors (eg high salt diet,    smoking, lack of vit C)
Antral-predominant Gastritis      No atrophy, high acid secretion	Mild pangastritis Gastritis      Normal gastric physiology	Corpus-predominant Gastritis           Atrophy and Hypochlorhydria
Duodenal ulcer disease	No serious GI disease	Gastric Cancer
Figure 2 Contribution of cytokine gene polymorphisms to distribution of gastritis and clinical outcome of H.pylori infection

Figure 2 Contribution of cytokine gene polymorphisms to distribution of gastritis and clinical outcome of H.pylori infection

Gastric cancer has a complex multifactorial aetiology but recent work shows that the key pathophysiological events are largely initiated by H. pylori infection. In genetically predisposed hosts, this infection induces a hypochlorhydric and atrophic phenotype that facilitates genotoxic damage and neoplastic transformation. The study of this relatively simple model will open the way to understanding more complex malignancies where interactions between the host, microbes and the environment are thought to play a role.

1. Kuper H, Adami HO, Trichopoulos D. Infections as a major preventable cause of human cancer. J Intern Med2000; 248(3): 171-183
2. IARC monographs on the evaluation of carcinogenic risks to humans. Vol. 61. Schistosomes, liver flukes and Helicobacter pylori. Lyon France IARC, 1994.177-240.
3. Scott DR, Weeks D, Hong C et al. The role of internal urease in acid resistance of Helicobacter pylori. Gastroenterology 1998; 114(1): 58-70.
4. Israel DA, Peek RM. pathogenesis of Helicobacter pylori-induced gastric inflammation. Aliment Pharmacol Ther 2001; 15(9): 1271-1290.
5. Robert A, Olafsson AS, Lancaster C, Zhang WR. lnterleukin-1 is cytoprotective, antisecretory, stimulates PGE2 synthesis by the stomach, and retards gastric emptying. Life Sd 1991; 48(2): 123-134.
6. Wolfe MM, Nompleggi DJ. Cytokine inhibition of gastric acid secretion - a little goes a long way. Gastroenterology 1992; 102(6): 2177-2178.
7. 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): 227-234.
8. Danon SJ, O'Rourke JL, Moss ND, Lee A. The importance of local acid production in the distribution of Helicobacter felis in the mouse stomach Gastroenterology 1995; 108(5): 1386-1395.
9. 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): 1018-1022.
10. El-Omar EM, Oien K, El Nujumi A et al. Helicobacter pylori infection and chronic gastric acid hyposecretion. Gastroenterology 1997- 113(1)-15-24.
11. 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): 681-691.
12. McColl KE, El-Omar E, Gillen D. Helicobacter pylori gastritis and gastric physiology. Gastroenterol Clin North Am 2000; 29(3): 687-703.
13. Graham DY, Yamaoka Y. Disease-specific Helicobacter pylori virulence factors: the unfulfilled promise. Helicobacter 2000; 5 Suppl 1: S3-S9.
14. Dinarello CA. Biologic basis for interleukin-1 in disease Blood 1996-87(6): 2095-2147.
15. El-Omar EM. The importance of interleukin 1beta in Helicobacter pylori associated disease. Gut 2001; 48(6): 743-747.
16. Bidwell J, Keen L, Gallagher G et al. Cytokine gene polymorphism in human disease: on-line databases, supplement 1. Genes Immun 2001;2(2): 61-70.
17. El-Omar EM, Carrington M, Chow WH et al. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature 2000; 404(6776):398-402.
18. El-Omar EM, Chow WH, Gammon MD, Vaughan TL, Risch HA, Fraumeni JF, Jr. Pro-inflammatory genotypes of IL-1beta, TNF-alpha and IL-10 increase risk of distal gastric cancer but not of cardia or oesophageal adenocarcinoma. Gastroenterology 120[5, suppl.1], A86. 2001.
19. Machado JC, Pharoah P, Sousa Sefa/. Interleukin 1Band interleukin 1RNpolymorphisms are associated with increased risk of gastric carcinoma. Gastroenterology 2001; 121(4):823-829.
20. Kuipers EJ. Review article: exploring the link between Helicobacter pylori and gastric cancer. Aliment Pharmacol Ther 1999; 13 Suppl 1:3-11.
21. Correa P. Human gastric carcinogenesis: a multistep and multifactorial process - First American Cancer Society Award Lecture on Cancer Epidemiology and Prevention. Cancer Res 1992; 52(24): 6735-6740
22. Mayne ST, Risch HA, Dubrow R et al. Nutrient intake and risk of subtypes of esophageal and gastric cancer. Cancer Epidemiol Biomarkers 2001; 10(10): 1055-1062.
23. Banerjee S, Hawksby C, Miller S et al. Effect of Helicobacter pylori and its eradication on gastric juice ascorbic acid. Gut 1994; 35(3); 317-322
24. Kneller RW, You WC, Chang YS et al. Cigarette smoking and other risk factors for progression of precancerous stomach lesions. J Natl Cancer Inst 1992:84(16): 1261-1266.