Helicobacter pylori are gram-negative spiral microaerophilic

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1 GASTROENTEROLOGY 2005;128: Helicobacter pylori and MALT Lymphoma PEDRO FARINHA and RANDY D. GASCOYNE Department of Pathology and Laboratory Medicine, British Columbia Cancer Agency and the University of British Columbia, Vancouver, British Columbia, Canada Helicobacter pylori are gram-negative spiral microaerophilic organisms that belong to the Campylobacterales order, Helicobacteracea family. They are capable of colonizing the harsh environment of the human stomach. Over 50% of the world s population carries this infection. 1 H pylori have colonized the human stomach since time immemorial. 2 In virtually all infected individuals, H pylori causes inflammation in the form of chronic active gastritis, which progresses in 10% 20% of affected persons to peptic ulcer disease, gastric adenocarcinoma, and/or mucosa-associated lymphoid tissue (MALT) lymphoma. However, only a very small minority (1% 2%) of infected individuals will develop a malignant disease. 3 One of the most distinctive features of H pylori is the genetic diversity it displays between clinical isolates. 4 H pylori isolates are highly diverse, with evidence of a constantly changing genome, primarily caused by point mutations, substitutions, insertions, and/or deletions. 5,6 Moreover, mixed infections are frequent and may lead to exchange of DNA fragments between H pylori strains in a single host. This suggests co-evolution of H pylori with its human host and that disease may be caused both by strain-specific properties that increase virulence and by host susceptibility. Such strains possibly evolve through exercising significant flexibility in gene content and gene regulation. Among many of the host pathogen interactions that potentially could occur, some could prove beneficial in which the co-evolved bacteria and the host reach an almost symbiotic relationship. H pylori may not be just a bad bug in all instances. A large body of data have implicated H pylori in the pathogenesis of gastric MALT lymphoma, including epidemiologic, biological, and molecular genetic studies. Interestingly, both H pylori and MALT lymphoma were born almost simultaneously, when H pylori was described by Marshall and Warren 7 and MALT lymphoma was recognized as a distinct entity by Isaacson and Wright. 8 These latter investigators noted morphologic similarities between the histology of the condition known as immunoproliferative small intestinal disease, a subtype of primary intestinal B-cell lymphoma, and primary low-grade gastric B-cell lymphoma. Moreover, the histologic features differed from that of comparable nodal low-grade B-cell lymphomas in that the overall architecture and cytology of these lymphomas bore a resemblance to normal MALT tissue rather than recapitulating the structure of lymph nodes. Roughly one third of all non-hodgkin s lymphomas in adults are of extranodal origin. MALT lymphomas, by definition arising at mucosal/epithelial sites, collectively account for approximately 8% of all non-hodgkin s lymphomas, with gastric MALT lymphoma being the most common extranodal site. This review summarizes the published data that support a causative role for H pylori infection in the pathogenesis of gastric MALT lymphomas. The epidemiology of H pylori is discussed and its role in the pathogenesis, including factors affecting the infectious organism and the host. A major focus of this review addresses the relationship between the immune response, H pylori, and MALT lymphomas. This is followed by a description of the morphology, phenotype, and unifying concepts of the molecular genetics of MALT lymphomas. Treatment strategies of gastric MALT lymphoma are not discussed because these are beyond the scope of this review. Finally, we conclude with a brief discussion of H pylori negative MALT lymphomas, preventative measures for reducing infection, and the direction of future research into MALT lymphomas. Abbreviations used in this paper: CagA, cytotoxin-associated antigen A; CARD4, caspase recruitment domain; CIMP, CpG island methylator phenotype; DC-SIGN, dendritic cell specific intercellular adhesion molecule-3 grabbing nonintegrin; DLBCL, diffuse large B-cell lymphoma; DSB, double-strand DNA breaks; FOXP1, Forkhead box protein P1; GST, glutathione S-transferase; IAP, inhibitors of apoptosis; IFN, interferon; IGH, immunoglobulin heavy chain; IL, interleukin; LEL, lymphoepithelial lesion; MALT, mucosa-associated lymphoid tissue; MHC, major histocompatability complex; NF- B, nuclear factor B; ntx, neonatal thymectomized; PAI, pathogenicity island; PCR, polymerase chain reaction; ROS, reactive oxygen species; SS, Sjögren s syndrome; Th1, helper1 T cells; Th2, helper2 T cells; TRAF, tumor necrosis factor associated factor; Treg, regulatory T cells; TNF-, tumor necrosis factor by the American Gastroenterological Association /05/$30.00 doi: /j.gastro

2 1580 FARINHA AND GASCOYNE GASTROENTEROLOGY Vol. 128, No. 6 Epidemiology Infection with H pylori occurs worldwide and affects at least half the world s population, but the prevalence varies greatly among countries and within individuals in the same country. 3 The overall prevalence of H pylori infection is correlated strongly with socioeconomic status. 9 The prevalence among adults is approximately 80% in many developing countries and 50% in industrialized countries. Direct fecal-oral transmission predominates in industrialized countries whereas other transmission routes, such as contamination of water, may be more important in developing countries. 10 The bacteria is transmitted within families in early childhood. 11 Children often are infected by a strain with a genetic fingerprint identical to that of their parents, and they maintain this genotype even after moving to a different environment. Improved sanitation in industrialized countries reduced the rate of infection of H pylori over recent decades. In the United States, there has been a decrease in H pylori infection prevalence since the second half of the 19th century. It has been known for some time that even though the genome of H pylori is extraordinarily diverse, strains fall into distinct geographic groups. 2,12 By using distinct patterns of polymorphic sites based on the nucleotide sequences of 8 genes in 370 H pylori strains from 27 geographic and ethnic human populations, Falush et al 2 identified 7 H pylori populations and subpopulations. By comparing human migratory fluxes with these H pylori populations, some of the apparent inconsistencies could be explained by known human migration patterns (eg, the appearance of Helicobacter subpopulation West Africa (hspw Africa) strains in African-Americans in the United States as a result of the slave trade, and the isolation of H pylori East Asia strains from Native Americans introducing these strains into continental America some 12,000 years ago). In adults, H pylori infection usually is chronic and will not heal without specific therapy. In children, the elimination of the bacterium probably is relatively common, aided by the more frequent administration of antibiotics given for other reasons. 13 There currently is no evidence for zoonotic transmission, although H pylori is found in some nonhuman primates and occasionally in other animals. 14 Undoubtedly, management of colonized individuals will continue to evolve as new knowledge emerges and socioeconomic conditions change. Gastric MALT lymphoma develops in only a very small percentage of patients. The estimated incidence of this lymphoma in the United States is between 1 per 30,000 80,000. Similar numbers are reported in European countries, with recent reported incidence rates of.7.8 per 100,000 population, except in England where the incidence appears lower. 15 However, there are differences between populations within Europe, the United States, and other countries. A higher incidence (13 per 100,000) has been reported in Northeastern Italy, where the prevalence of H pylori is also very high, being detected in 80% of the adult population. 16 Developing countries, not surprisingly, show higher incidences, for example, in Tunisia, with incidence rates of 6.3 and 3.8 per 100,000, for men and women, respectively. 17 H pylori are not the only helicobacter species infecting humans. Humans also can become infected with Helicobacter heilmannii, a tightly coiled spiral bacterium larger than H pylori, found in dogs, cats, pigs, and nonhuman primates. The prevalence in humans is approximately.5%. H heilmannii causes only mild gastritis in most cases, but it also can induce acquired MALT in the stomach and has been found in association with gastric MALT lymphoma. 18 Evidence Linking H pylori Infection With Gastric MALT Lymphomas The first study linking H pylori with gastric MALT lymphoma occurred in These investigators showed that H pylori infection significantly increased the risk for gastric MALT lymphoma because the vast majority of gastric MALT lymphoma patients were infected with H pylori. The normal stomach is devoid of organized lymphoid tissue. It was shown that lymphoid follicles develop in H pylori infected persons and that the lymphoid tissue formed in response is identical morphologically to normal MALT. Moreover, case-control studies have shown an association between previous H pylori infection and the development of primary gastric lymphoma. 20 Direct evidence confirming the importance of H pylori in the pathogenesis of gastric MALT lymphoma was obtained from studies that detected the lymphoma B-cell clone in the biopsy specimens of chronic gastritis that preceded the development of lymphoma and from a series of in vitro studies showing that lymphoma growth could be stimulated in culture by H pylori strain-specific T cells when crude lymphoma cultures were exposed to the organism. 21 Finally, Wotherspoon et al 22 in 1993, and subsequently several other groups, showed that eradication of H pylori with antibiotics alone resulted in regression of gastric MALT lymphoma in 75% of cases. Most of these patients whose lymphomas responded to antibiotic eradication therapy have shown sustained clinical remissions. 23 These landmark studies showed that a ma-

3 May 2005 H PYLORI AND MALT LYMPHOMAS 1581 lignant tumor could be eradicated successfully by using antibiotic therapy alone. Moreover, very strong evidence based on seroepidemiologic case-control studies showed that infection with H pylori increased the risk for gastric carcinoma. Thus, in 1994, H pylori was classified as a type I (definitive) carcinogen by the International Agency for Research on Cancer. Pathogenesis In contrast to infection with other mucosal pathogens, only a small percentage of persons carrying H pylori ever develop clinical sequelae. H pylori induce gastric inflammation in virtually all hosts, and gastritis increases the risk for gastric and duodenal ulceration, distal gastric adenocarcinoma, and gastric mucosal lymphoproliferative disease of the MALT type. Recent investigations have emphasized that disease-specific mechanisms involve choreographed interactions between the pathogen and host, which, in turn, are dependent on strain-specific bacterial factors and induced host effectors. H pylori Bacteria indigenous to the human body are numerous and varied. They dwell in multiple niches on mucosal surfaces in various anatomic sites. Many of these sites are colonized persistently by several species. Such cohabitation implies an equilibrium in which the biologic cost of the colonization to the host is low. The different organisms compete for nutrients and the best locations, and participate in a number of shared activities such as waste management and genetic exchange. Colonization of the stomach by H pylori is an exception to these generalities because it can persist for decades and is not deterred by competing microbes. The H pylori genome (1.65 million bp) codes for about 1500 proteins. 5,6 H pylori research has benefited from genomic approaches more than any other bacterial pathogen. It was the first bacterial species to have the complete genome of 2 unrelated clinical isolates ( J99 and 26995) fully sequenced and compared. 5,6 Functional genomics has used the expression profiling of messenger RNA to provide a condition-dependent and time-specific genome scale portrait of the organism s transcriptome. 24 Comparison of the 2 completely sequenced H pylori organisms revealed a significant amount of genetic variation between genomes. The genome of strain J99 is smaller (1,643,831 bp) than that of strain (1,667,867 bp). 5,6 They contain 52 and 110 strain-specific genes, respectively, approximately half of which reside within a locus termed the plasticity zone. 25 These 2 H pylori genome sequencing projects showed that many genes could be switched on and off by base mispairing mediated mutagenesis. Proteins encoded by such genes include surface molecules responsible for control of entry of foreign DNA into the bacteria, and proteins affecting bacterial motility. Strains of H pylori have been shown to have a panmictic or freely recombining population structure over a period of years. The populations colonizing a given host experience genetic variation as a result of point mutations or recombination, which can involve exchanges across loci within a genome (intragenomic) or between differing organisms (intergenomic). 26 DNA microarray analysis has been used to study H pylori genes within the same host who had refused eradication therapy (9-year period). The genes of the later isolated strains were related closely but not identical to either the earlier isolates or even each other. Each of 13 new isolates tested had a unique complement of genes. 27 Considering that a single gastric biopsy specimen represents only a small percentage of gastric mucosal cells, the amount of variation within the total population of H pylori must be enormous. Thus, the genome of H pylori may change continuously during chronic colonization of an individual host by importing small pieces of foreign DNA from other H pylori strains during persistent or transient mixed infection. 28 These findings support microevolution of H pylori, that is, gradual small changes in its genome as an adaptive mechanism, with marked plasticity and a trend to adaptation to the host. A number of factors may impact this adaptation. These include ph levels within different gastric niches and their influence on the survival of H pylori and its ability to grow under acidic conditions, the distribution and viscosity of mucin glycoforms in the gastric mucosa, the adherence of H pylori to gastric mucins, and host genotype and physiology affecting niche characteristics. 29,30 After being ingested, the bacteria must evade the bactericidal activity of the gastric luminal contents and then enter the mucous layer. Urease production and motility are essential for this first step of infection. Urease hydrolyzes urea into carbon dioxide and ammonia, thereby allowing H pylori to survive in this acidic milieu. H pylori flagella have adapted similarly to the gastric microenvironment. Most strains of H pylori possess the cytotoxin-associated antigen (CagA), a kilodalton protein. 31 It is found in 60% 70% of H pylori strains in the industrialized world. 1 The caga gene is localized at one end of the cag pathogenicity island (cag-pai), a 37-kb genomic fragment containing 31 putative genes. Several of these genes encode components of a type IV secretion apparatus that translocates the 120- to 145-kilodalton CagA protein into the host cell. 32 After entering an epithelial

4 1582 FARINHA AND GASCOYNE GASTROENTEROLOGY Vol. 128, No. 6 cell, CagA is phosphorylated and binds to SHP-2 tyrosine phosphatase, leading to a growth factor like cellular response and cytokine production by the host cell. The deregulation of SHP-2 by CagA is an important mechanism by which CagA promotes gastric epithelial carcinogenesis. 33 In the lymphocytes, it has been shown that after entering the cells, CagA affects different pathways. Umehara et al 34 studied the effects of CagA protein on the growth and survival of B lymphocytes by using B-cell lines ectopically expressing CagA. The CagA protein was shown to suppress lymphoid cell proliferation through the suppression of the JAK-STAT pathway, which would be advantageous to the bacteria because it hampers the rapid expansion of H pylori specific immune cells. These investigators also provided evidence that CagA counteracts B-cell apoptosis induced by hydroxyurea by inhibiting P53 accumulation. This may contribute to the accumulation of genetic mutations in abnormal B cells by allowing escape from programmed cell death. Several other cag-pai genes such as cagg, cagh, cagi, cagl, and cagm, seem to be associated with particular epithelial cell responses relevant to H pylori pathogenesis. 35 Independent of cag-pai status, another type IV cluster, the comb gene cluster, has been linked to the natural transformation and competence of H pylori. 36 The comb gene cluster is essential for the bacterium to take up plasmid and chromosomal DNA via natural transformation. It appears that the comb transformation apparatus has evolved conservatively and typically is present in all strains. 37 A third cluster of type IV secretion genes has been described recently. 38 About half of all H pylori strains express the 95- kilodalton vacuolating cytotoxin, VacA, a secreted exotoxin. This toxin inserts itself into the epithelial cell membrane and forms anion-selective, voltage-dependent channels through which bicarbonate and organic anions can be released. 39 VacA also is targeted to the mitochondrial membrane, where it causes release of cytochrome c, resulting in apoptosis. 40 VacA inhibits the intracellular pathway of macrophage antigen presentation, resulting in a reduced amount of peptides able to bind major histocompatibility complex class II molecules, with consequent lower stimulation of an antigen-specific T-cell response. An alternative hypothesis is that VacA acts directly on T cells by inhibiting activation of intracellular signaling pathways. VacA expression is determined by genetic variation in the signal sequence (s1a, s1b, s1c, and s2) and midregion sequence (m1 and m2) of the vaca gene. Strains with the s1-type signaling sequence allele produce functional VacA toxin inducing epithelial vacuolization and cell death, whereas the s2-type signaling sequence has little cytotoxic activity. 41 H pylori can bind tightly to epithelial cells by multiple bacterial surface components. The best characterized adhesin, BabA, is a protein that binds to a Lewis B bloodgroup antigen and has 2 isoforms: baba1 and baba2. 42 The product of baba1, in contrast to baba2, cannot interact with Lewis B to enhance H pylori colonization to surface epithelium. 43 In European countries the presence of baba2 has been associated significantly with caga, the s1/m1 alleles of vaca, and oipa, which encodes an outermembrane protein that influences host cell release of interleukin-8 (IL-8) in vitro. Conversely, more than 90% of caga-negative strains are negative for baba2, had s2/m2 vaca alleles, and show an oipa off genotype, suggesting that the presence of genes located within cag-pai is linked to genes located outside the pathogenicity island, probably through the influence of a common selection pressure. 44 Strains bearing either caga, vaca s1m1, or baba2 genes show higher degrees of gastric antral and corpus colonization by H pylori and more severe inflammation. The clinical relevance of the baba2 genotype has been questioned after the results from Japanese individuals, in which no correlation was found between baba2 and caga genotypes and the baba2 genotype and clinical outcome. 45 These findings agree with previous results showing that genotype-phenotype correlations obtained in Western individuals are not applicable to Asian populations, suggesting that the clinical impact of H pylori genotypes with higher virulence is modulated by environmental and/or host factors. In gastric MALT lymphoma, several of these virulence factors have been investigated by several groups. The role of infection of caga strains of H pylori in the development of gastric MALT lymphoma is controversial. Eck et al 46 reported a very high correlation of caga H pylori with gastric MALT lymphoma. Other studies, however, did not support this correlation. 47,48 In other studies, caga was suggested to be associated preferentially with diffuse large B-cell lymphoma (DLBCL). 49,50 Recently, Ye et al 51 found a significant association of caga strains of H pylori with gastric MALT lymphoma, in particular the cases harboring the t(11;18)(q21;q21) (14 of 15; 93.3%). Importantly, H pylori strains harboring the caga island were associated with more apparent histologic inflammatory responses and induction of potent chemokines producing neutrophil activation. Activated neutrophils release reactive oxygen species (ROS), which can cause a wide range of DNA damage (see later). Lehours et al 52 studied some of the H pylori virulence factors described: caga, cage, vaca, IceA, baba, hopq,

5 May 2005 H PYLORI AND MALT LYMPHOMAS 1583 oipa, saba, and hopz were investigated in 43 H pylori gastric MALT strains in comparison with 39 strains associated only with gastritis. None of these genes considered individually was associated with MALT lymphoma. Nevertheless, a gene cluster comprised of the icea1 allele, saba on status, and hopz off status was identified in strains for which the likelihood of having a gastric MALT lymphoma was 10 times higher than that of the gastritis-associated strains. However, the low prevalence of such strains (25% of MALT strains) makes the association a low-sensitivity marker for MALT lymphoma H pylori strains. Genes linked to the development of gastric cancer have been assigned to the plasticity zone. 53 Lehours et al 54 attempted to identify the same correlation in gastric lymphoma. They studied genetic markers associated with H pylori strains isolated from patients with gastric MALT lymphoma in comparison with strains obtained from aged-matched patients with gastritis only, duodenal ulcer, or gastric adenocarcinoma. By using subtractive hybridization and multiple correspondence analyses, they failed to identify a gene cluster associated with the MALT lymphoma inducing strains when the analysis included all of the strains. However, when they performed dot-blot hybridization between MALT lymphoma strains and the gastritis strains only, they found one open reading frame located in the plasticity zone that was associated significantly with the MALT lymphoma, the open reading frame JHP950. These investigators concluded that open reading frame JHP950 is a specific marker for strains giving rise to an increased risk for MALT lymphoma. Flavodoxin-A protein and the 60-kilodalton heat shock protein of H pylori are both antigenic, resulting in an antibody response in some infected individuals. The presence of a serologic response has been reported to be associated with the development of gastric MALT lymphoma, but these results are controversial Despite the observation that H pylori genomic studies show trends for specific associations of open reading frame with the development of MALT lymphoma, to date no specific strains or factors of virulence have been identified that predict either disease or clinical outcome with epidemiologic consistency. Just as strains of H pylori with supposed virulence attributes may be responsible for disease, other strains may be protective where they have lost virulence through microevolutionary changes associated with life-long colonization of the gastric mucosa. A recent study has shown that H pylori can produce a cecropin-like peptide with high antimicrobial properties. 58 When released in the stomach it kills other microorganisms, preventing other organisms from colonizing the stomach. Children infected with H pylori were less likely to have diarrhea than children without the infection, again implying that some strains of H pylori may have beneficial properties for the human host. 59 Thus, the value of H pylori genotypes as predictors of disease outcome is limited because the pathogenic impact of bacterial virulence factor probably is most influenced by the host. Immune Response and H pylori H pylori cause continuous gastric inflammation in virtually all infected persons. 60 The host inflammatory response consists of both innate and adaptive immunity. It starts with recruitment of neutrophils, followed by T and B lymphocytes, plasma cells, and macrophages, all of which can damage the epithelium (Figure 1). 61 Most H pylori infected patients are unable to clear the pathogen, leading to the suspicion that H pylori somehow may hamper the host immune response. Because H pylori rarely, if ever, invade the gastric mucosa, the host response is triggered primarily by the interaction of the bacteria with the epithelial cells. As described, H pylori use the type IV secretion apparatus to mediate the translocation of protein effector molecules into the target host cells. 62 In vitro studies have shown that strains with functional cagpai induce cytoskeletal modifications and transcription factor nuclear factor B (NF- B) dependent proinflammatory responses in the epithelial cells. 63 Viala et al 64 identified the intracellular protein (nucleotide-binding oligomerization domain 1 [NOD1] or caspase recruitment domain 4 [CARD4]) as being a major pathogen-recognition molecule in gastric epithelial cells in mice. They showed that Nod1-deficient mice produced significantly lower amounts of Cxcl2, a NF- B dependent chemokine, which has biological activity in mice analogous to those of human IL-8. Although Toll-like receptors (a family of molecules that recognize conserved microbial components, including various products of gram-negative bacteria) 2 and 4 have been reported to mediate H pylori 60-kilodalton heat shock protein recognition by macrophages in vitro, H pylori recognition seems not to occur predominantly through this mechanism. 65 Instead, Nod1, an intracytoplasmic pathogen-recognition molecule with homology to plant resistance proteins, recognizes an internalized H pylori peptidoglycan and activates a proinflammatory signaling cascade. 66 Interestingly, Nod1-deficient mice cannot regulate the density of H pylori growing at their gastric epithelial surface, indicating that, after recognition of its cognate pathogen-associated molecular pattern, Nod1 activates the recruitment of inflammatory cells, particu-

6 1584 FARINHA AND GASCOYNE GASTROENTEROLOGY Vol. 128, No. 6 Figure 1. H pylori and MALT lymphoma. In the stomach, H pylori do not invade the epithelium, but bind to the host epithelial cells by multiple surface adhesion molecules. Binding of the organism translocates effector proteins into both epithelial and surrounding immune cells. These cells recognize H pylori by both membrane (MHC, DC-SIGN) and intracellular (CARD4) receptors that trigger cellular apoptosis, as well as an NF- B dependent inflammatory response. There is infiltration of the mucosa by neutrophils and macrophages (innate immunity) followed by lymphocytes and plasma cells (adaptive immunity). The inflammation is chronic in virtually all H pylori patients, which suggests H pylori somehow hamper the host immune response through a favorable balance between T-cells subsets (helper vs regulatory cells). The persistence of inflammation triggers the acquisition of reactive MALT and leads to the formation of genotoxic agents such as ROS. The combination of H pylori strain-specific virulence factors and host immune response features (eg, different cytokine polymorphisms and components of the immune microenvironment induced by Treg cells and alternatively activated macrophages) eventually will facilitate the emergence and proliferation of a malignant B-cell clone that resembles normal memory marginal zone B cells. In early phases the survival of the malignant cells is dependent on H pylori specific T cells and the lymphoma regresses with H pylori antibiotic therapy. Lymphoma cells can show different genomic abnormalities that proceed along 2 major pathways: (1) the H pylori dependent pathway that shows more often a methylator phenotype and is prone to accumulate further genomic abnormalities and eventually transform into a high-grade lymphoma (DLBCL); and (2) the H pylori independent pathway characterized by balanced translocations, the most common being t(11;18)(q32;q21), a nonmethylator phenotype, an advanced stage, and lack of response to antibiotic therapy. The t(11;18)(q32;q21) is often the only genomic abnormality found and these cases do not show clonal evolution or transformation into an aggressive lymphoma. larly neutrophils, which infiltrate the epithelium to eradicate the bacteria, thereby inducing chronic gastritis. In addition to the damage associated with CagPAImediated translocation of proteins, H pylori infection results in epithelial injury through several other mechanisms. The pathogen can bind directly to the surface of gastric epithelial cells through urease-targeting class II major histocompatibility complex (MHC) molecules, inducing their apoptosis. 67 Additionally, mucosal dendritic cells also recognize several H pylori components such as lipoproteins, lipopolysaccharide such as O-antigen Lewis blood group, and DNA. They are processed via intracellular signaling cascades leading to the production of regulatory cytokines and up-regulation of major histocompatibility complex class II molecules. Binding of these Lewis antigens of H pylori to the dendritic cell specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) was reported recently by Bergman et al. 68 DC-SIGN is involved in cell adhesion as well as

7 May 2005 H PYLORI AND MALT LYMPHOMAS 1585 antigen presentation to T cells. 69 H pylori targets DC-SIGN, and this interaction may contribute to the persistence of this pathogen by modeling the polarity of different T-cell subsets. Inflammation of the gastric mucosa follows and varies according to the host immune reaction against the pathogen. 70 The stimulation of isolated endothelial cells with H pylori contributes to the recruitment of neutrophils resulting from up-regulation of the adhesion molecules vascular cell adhesion molecule 1, intercellular adhesion molecule-1, E-selectin, and IL Neutrophils then increase the epithelial damage through the release of ROS. 72 The activation of macrophages results in the release of cytokines, including IL-1, IL-6, IL-8, IL-12, tumor necrosis factor- (TNF- ), and interferon- (IFN- ). Similarly, the cytotoxin VacA leads to disruption of the epithelial barrier, facilitating the translocation of bacterial antigens and leading to further activation of macrophages. 73 Cytokines produced by macrophages then can alter the secretion of mucus, contributing to H pylori mediated disruption of the mucous layer. H pylori infection typically induces a humoral response. 3 This antibody production does not lead to eradication of the infection but may contribute to damage of the surrounding tissues. Some H pylori infected patients have an autoantibody response directed against the H / K adenosine triphosphatase of gastric parietal cells that correlates with increased atrophy of the corpus, one of the typical morphologic features of H pylori induced chronic gastritis. 74 The chronic phase of H pylori gastritis associates an adaptive lymphocyte response with the initial innate response. Immature helper T cells can differentiate into 2 functional subtypes: helper1 T (Th1) cells that secrete IL-2 and IFN-, which elicit macrophage proliferation, and helper2 T (Th2) cells that secrete IL-4, IL-5, and IL-13, which act as growth/differentiation factors for B cells. 75 Along with these helper T cells there also are T regulatory (Treg) cells (mainly CD4 CD25 and IL- 10 secreting T cells) that control the former cells (Figure 1). 76 Because H pylori is noninvasive and induces a strong humoral response, a Th2-cell response would be expected. Paradoxically, the immune response resulting from H pylori infection is primarily a proinflammatory response (Th1), mediated by a cascade of cytokines including IL-1, IL-12, IL-18, TNF-, and INF-. 77 These cytokines regulate their own levels of expression by autocrine mechanisms, but they also influence downstream cytokine expression, thereby amplifying the inflammatory response. Studies in gene-targeted mice have shown that Th1 cytokines promote gastritis, whereas Th2 cytokines are protective against gastric inflammation. 78 This Th1 orientation may be caused by increased antral production of IL-18 in response to H pylori infection. 79 The resultant biased Th1 response, combined with Fas-mediated apoptosis of H pylori specific T-cell clones, may favor the persistence of H pylori. 80 However, this hypothesis is controversial. Panthel et al 81 assessed the ability of H pylori to colonize BALB/c (intrinsic Th2-cell responders) and C57BL/6J (an intrinsic Th1- cell responsive strain) wild-type mice, as compared with their IL-4 and IL-12 knockouts. Disruption of IL-12 in BALB/c mice resulted in enhanced colonization, and disruption of IL-4 in these mice had little effect. In contrast, colonization of C57BL/6J knockout mice deficient in either IL-4 or IL-12 was decreased. These findings support the hypothesis that H pylori benefits from a mechanism that modulates the host Th1-/Th2-cell balance during colonization, leading to suppression of Th1- cell responses. Bergman et al 68 also showed that H pylori targeted DC-SIGN could block a polarized Th1-cell response by phase-variable expression of the Lewis antigen by H pylori. CD4 CD25 Treg cells have been shown to reduce immunologic damage during H pylori infection in mice by reducing the activation of Th1 cells. Infection of athymic (nu/nu) mice with H pylori resulted in considerably lower colonization in mice previously reconstituted with lymph node cells depleted of Treg cells than in reconstituted controls. 82 Raghavan et al 82 suggested that IFN- producing CD4 T cells are reduced by the presence of Treg cells at the expense of a higher H pylori load. Establishing the link between a predominant Th1 response and the suppressive Treg cells, Kaser et al 83 reported that the Th1 cytokine IL-18 recruits a particular subset of dendritic cells, plasmacytoid DC, characterized by the expression of CD123 and blood DC antigen 2. These acute inflammatory cells are known to promote the development of Treg cells. 84 Their role in H pylori infection is unknown. Mechanisms used by H pylori to evade clearance may impair the macrophage response. Gobert et al 85 reported that H pylori arginase inhibits the antimicrobial effect of nitric oxide (essential component of innate immunity) produced by activated macrophages. Thus, bacterial arginase allows H pylori to evade the immune response. Furthermore, the same group showed that arginase II expression is up-regulated in both murine and human H pylori induced gastritis. 86 The arginase pathway leads to the metabolism of arginine with the generation of ornithine and polyamines. This pathway is used predominantly by the alternatively activated macrophage type of polarized macrophage, instead of the inducible NO syn-

8 1586 FARINHA AND GASCOYNE GASTROENTEROLOGY Vol. 128, No. 6 thase characteristic of the classically activated type macrophages. The classically activated macrophage is proinflammatory and produces IL-12 and TNF-, whereas alternatively activated macrophages typically produce IL The expression of arginase by macrophages seems to induce T-cell dysfunction/suppression. 88 These polarized alternatively activated macrophages have tissue repair/remodeling functions and could be responsible not only for the architectural features observed in chronic gastritis, but also might participate with Treg cells in immunosuppression toward H pylori. Finally, Zheng and Jones 89 reported that vaca H pylori strains also inhibit phagosome development after bacterial ingestion by macrophages, which is associated with the retention of coronin1 (or tryptophane aspartate containing coat protein, TACO), leading to a possible down-regulation of phagocytic macrophage function. 90 Immune Response and MALT Lymphoma Despite the ability of H pylori to infect chronically the stomach of all hosts, why do so few people develop lymphoma? A number of studies have indicated that genetic factors likely play a fundamental role in affecting different clinical outcomes after H pylori infection. For example, familial aggregation, interethnic variation of disease incidences, human leukocyte antigen typing, and animal models previously were shown to point to a genetic influence in gastric carcinogenesis susceptibility and/or resistance. 91 As described, the H pylori inflammatory immune response features are orchestrated by sequential elaboration of proinflammatory cytokines including IL-10, IFN-, TNF-, and IL It is probable that cytokine imbalance is responsible for disease progression and dictates the disease outcome after H pylori infection. Accordingly, factors involved in regulating cytokine responses may confer susceptibility to or protection against H pylori associated diseases. Specifically, single nucleotide polymorphisms in the coding or promoter regions of a cytokine or cytokine receptor may affect overall cytokine production directly, leading to either high or low production of a given cytokine. Therefore, variant cytokine alleles might contribute to interindividual differences in inflammatory responses and account for heterogeneous outcomes after an infectious event. Such a correlation has been reported in gastric carcinoma in which IL-1 polymorphisms confer a 2-fold increased risk for gastric adenocarcinoma after H pylori infection. 93 Recently, polymorphic alleles of several cytokines have been studied in MALT lymphoma. IL-1 mediates the Th1 immune response after H pylori infection, with IL-1 production typically up-regulated in the presence of H pylori. IL-1 is a powerful inhibitor of gastric acid secretion; thus, the hypochlorhydria associated with high-producing variants of IL-1 likely govern the extent of H pylori infection and the distribution of gastritis. The IL-1 gene cluster comprises 3 related genes: IL-1, IL-1, and IL-1 RN, which encode the proinflammatory cytokines IL-1 and IL-1 and their endogenous receptor antagonist IL-1ra, respectively. 94 Three bi-allelic polymorphisms in IL-1 have been described, all C to T base transitions found at positions 511, 31, and 3954 bp from the transcriptional start site. 95 The IL-1 RN(2) allele has been associated with chronic inflammatory conditions such as ulcerative colitis and autoimmune conditions such as Sjögren s syndrome (SS) and with enhanced IL-1 production in vitro. Studies reporting an increased associated risk for gastric carcinoma with inheritance of the IL-1-31 (CC)/IL-1 RN 2/2 genotype suggest that the IL-1-31 C allele in the context of H pylori infection is associated with a proinflammatory phenotype and therefore an increased risk for DNA damage and, ultimately, gastric carcinoma. 93,96 Rollinson et al 97 suggested a significant association between gastric MALT lymphoma and inheritance of the IL-1 RN 2/2 genotype, but no associated risk with the IL-1-31 genotype. The high IL-1 levels associated with the IL-1 RN(2) allele favor a proinflammatory response; at the same time, the concomitant inhibition of gastric acid facilitates widespread H pylori colonization of the gastric mucosa, promoting the development of MALT in response to infection. TNF- has been recognized as an important mediator in the pathogenesis of a wide variety of diseases. Multiple polymorphisms located in TNF- have been associated with increased malignancies, suggesting that genetic variants within TNF- may be relevant functionally in vivo. 98,99 TNF- and its receptors play an important role in the signaling system involved in the immune response to H pylori infection. In vitro studies have shown that TNF- production may promote the growth of lymphoid cells. 100 Clinical studies have shown high TNF- levels in patients with malignant lymphoma. 100 Previous studies also have documented that the TNF- 308G allele is a risk factor for duodenal ulcer whereas the 308A allele is associated with an increased risk for Epstein Barr virus associated gastric adenocarcinoma. 101,102 Wu et al 103 showed a strong correlation between this cytokine genotype and MALT lymphoma. Specifically, they found that individuals with the TNF- 857T allele were less likely to develop lymphoma, with a 3-fold decreased risk. However, it is noteworthy

9 May 2005 H PYLORI AND MALT LYMPHOMAS 1587 that published studies of the functional effect of the TNF- 857 C/T polymorphism have used different methodologies, and thus no clear consensus has emerged. 104 As a result, the functional relevance of the TNF- 857 C/T polymorphisms and their role in disease remain to be determined. Certain human leukocyte antigen class II antigens have been reported to predict the serologic response to H pylori infection in patients with gastrointestinal malignancies and have been associated with the development of atrophic gastritis, a premalignant condition, in H pylori positive patients. 105 On the other hand, many immune responses are controlled by the major histocompatibility complex, leading some investigators to postulate a role for autoimmune mechanisms in the development of lymphoma. 106 It is worth noting that patients with enteropathy-associated T-cell lymphoma often show the same human leukocyte antigen DQA and DQB antigens as patients with celiac disease, the underlying autoimmune disorder. 107 Reimer et al 108 investigated 46 patients with MALT lymphoma recruited in a prospective multicenter study with 13,000 voluntary stem cell donors from more than 40 German blood banks as the control group. They found that the frequency of human leukocyte antigen B35 was decreased significantly in patients with gastric MALT lymphoma compared with the control group, suggesting a protective role of this human leukocyte antigen against gastric MALT lymphoma. One of the major cellular defense mechanisms against DNA damage is enzymes with antioxidant activity, capable of neutralizing free radicals. These are glutathione S-transferase (GST) enzymes. There are 4 cytosolic GST families, including GST-T1 and GST M1. Independent gene deletions exist at both the GST-T1 and GST M1 loci, resulting in a lack of functional protein, and the null genotypes of both loci have been associated with several malignancies. 109 Associations between the GST-T1 null genotype and increased risks for astrocytoma, meningioma, myelodysplasia, and acute myeloid leukemia have been reported. 110 Rollinson et al 97 also described a significant association between the null genotype of GST-T1 and an increased risk for gastric MALT lymphoma. They postulate that the GST-T1 null genotype is associated with an increased risk because of a compromised antioxidant capacity and not because of the metabolism of a specific xenobiotic substrate. Thus, combinations of genetic variations for cytokines that promote the immune response to H pylori infection and the production of ROS and variants that decrease the levels of antioxidants are likely to play important roles in the development of MALT lymphoma (shown in Figure 1). MALT Lymphoma H pylori tumor cells show somatic hypermutation in the immunoglobulin genes that are characteristic of antigen selection. 111 MALT lymphoma cells resemble memory B cells still responsive to differentiation signals, such as CD40 costimulation and cytokines produced by antigen-stimulated helper T cells, and dependent for their growth on stimulation by H pylori specific T cells. 21 Thus, H pylori stimulation of lymphoma B cells is not direct, but occurs through tumor-infiltrating T cells, involving both CD40 and CD40L costimulatory molecules. 112,113 The surface immunoglobulin on gastric MALT lymphoma B cells does not recognize H pylori, but instead recognizes various autoantigens, suggesting that malignant cells are transformed from autoreactive B cells. 114 Initially, MALT lymphoma is sustained by H pylori induced T-cell help, remains localized, and regresses after H pylori eradication. 113 In chronic gastritis patients, the helper function toward B cells exerted by H pylori antigen stimulated gastric T-cell clones is regulated negatively by the concomitant cytolytic killing of B cells. 115 In MALT lymphoma, T cells are unable to express perforin-mediated cytotoxicity against autologous B cells, leading to B-cell proliferation. 116 The majority of T-helper clones from uncomplicated chronic gastritis induce Fas-Fas ligand-mediated apoptosis in target cells, whereas only a small fraction of H pylori specific gastric clones from MALT lymphoma are able to induce apoptosis in target cells, including autologous B cells. 116 Both defective perforin-mediated cytotoxicity and a decreased ability to induce Fas-Fas ligand mediated apoptosis are restricted to MALT lymphoma infiltrating T cells because H pylori specific helper T cells derived from the peripheral blood of the same patients expressed the same cytolytic potential and pro-apoptotic activity as that shown by helper T cells from chronic gastritis patients. 116 The reason why gastric T cells of MALT lymphoma, although delivering powerful help to B cells, apparently are deficient in mechanisms involved in the concomitant control of B-cell growth remains unclear. In a proportion of MALT lymphomas, resistance to Fas-mediated apoptosis appears to be caused by FAS mutations, suggesting that increased inhibition of apoptosis contributes to oncogenesis in MALT lymphoma. 106 Mutation of the FAS gene and its pathway-related components is associated with autoimmune disorders, including a rare disorder known as autoimmune lymphoproliferative syndrome. 117 The majority of MALT lymphomas with FAS mutations are restricted to patients with both MALT lymphoma and

10 1588 FARINHA AND GASCOYNE GASTROENTEROLOGY Vol. 128, No. 6 autoimmune disease, and rarely are found in typical gastric MALT lymphoma, which normally lacks prominent clinical autoimmune features. 118 Controversy remains about the predominant subset of infiltrating T cells in MALT lymphoma. In some studies, the dominant cytokine produced in cases of low-grade B-cell gastric lymphoma was IFN- regardless of stimulation with H pylori, indicating the dominance of an immature helper T-cell/1-type response similar to what is observed in H pylori gastritis. 116,119 However, on in vitro stimulation with H pylori, a strong IFN- response and production of Th2 cytokines was observed in tumorinfiltrating T cells from patients with gastric MALT lymphoma. 120 These studies indicate that both Th1- and Th2-type cytokines, both growth factors of B cells, are secreted by H pylori stimulated MALT lymphoma T cells. The polarization toward an IFN- dominated cytokine response in infiltrating T cells may contribute to ongoing tissue injury and the inhibition of a possibly beneficial Th2 cytokine response. This may lead to an inability of the immune system to eliminate H pylori and consequently may lead to a chronic inflammatory process. However, a Th2 response also seems to be present, so that a balanced Th1/Th2 may be beneficial in lymphomagenesis. The generation of abnormal B-cell clones and the progression toward B-cell lymphoma might occur within this background. These observations correlate with the findings recently reported in murine MALT lymphoma models (see later). Another hypothesis relates to the role of lymphomainfiltrating Treg cells in suppressing the T-cytotoxic functions that underlie immunosurveillance, and thus contribute to an immunosuppressive microenvironment favorable to the tumor (cancer immunoediting). 121 Increased levels of Treg cells in the duodenum that may suppress H pylori specific immune responses recently were shown in ulcer patients. 122 Although not reported in MALT lymphomas, this mechanism has been reported in other tumors as a mechanism of immune escape and tumoral progression. 123,124 Murine models recently have been shown to be useful mimics of both clinical and histopathologic characteristics of human MALT lymphomas, including the antibiotic-induced regression of disease. Thus, they may represent an important model to study the progression of MALT lymphoma events over time. 125 Fukui et al 126 used neonatal thymectomized (ntx) BALB/c mice, which after H pylori infection for 6 months showed reactive lymphoid follicles with prominent lymphoepithelial lesions (LELs) and evidence of B-cell monoclonality. These investigators used BALB/c mice, a Th2-dominant strain, and C57BL/6J mice, which are believed to be a Th1- dominant strain. In BALB/c mice, ntx depletes CD25 Treg cells, resulting in the development of Th1- dominant autoimmune atrophic gastritis with loss of parietal/chief cells. Gastric MALT lymphoma like lesions caused by the H pylori infection were seen only in the ntx BALB/c mice and not in the non-ntx animals. Moreover, lymphoid follicle formation by H pylori never was observed in C57BL/6 mice irrespective of ntx, although these mice developed severe active gastritis after H pylori infection (Th1-associated inflammation). These data show that in contrast to H pylori induced gastritis, both Th1 and Th2 responses are necessary for the development of MALT lymphoma like lesions induced by H pylori. In support of this hypothesis, Fukui et al 126 observed both IFN- (Th1-type cytokine) and IL-4 (Th2- type cytokine) in the gastric mucosa of only H pylori infected BALB/c mice with ntx that developed lymphoid tissue. Mueller et al 127 used a 38,000-element murine complementary DNA microarray and laser microdissection to identify molecular markers of the histopathologic stages of gastric MALT lymphoma and to define the cellular origin of the transcriptional responses. H. heilmanni infected BALB/c mice were followed-up for 12, 18, and 24 months after infection and compared with control animals. The majority of the infected mice developed gastric wall invasion with monoclonal B-cell populations showing numerous LELs, similar to human MALT lymphoma. A hierarchic unsupervised clustering analysis segregated, not surprisingly, uninfected from infected samples. Samples from animals classified with moderate and severe pathology cluster together in a group. This finding argues against the likelihood of a single definitive transforming event and in favor of a continuum of events for which malignant lymphoma represents the end point. Having determined the signature of infected vs uninfected stomachs, Mueller et al 127 next sought to identify genes that discriminated between mild and moderate to severe pathology. Mice that were uninfected and exhibited mild pathology were grouped together and compared with animals showing moderate and severe pathology. Additionally, they microdissected the tumors and studied separately the mucosal and lymphoid components. Interesting examples of genes that were up-regulated consistently in the severe pathology specimens have been associated with other human malignancies, including the laminin receptor-1, a protein implicated in basement membrane degradation and tumor dissemination; MDR-1, a multidrug resistance protein; and nibrin, a DNA double-strand repair protein. The defined gene signatures (mild/moderate vs severe) were useful in pre-