Esophageal cancers are the sixth most commonly occurring

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1 GASTROENTEROLOGY 2010;139:73 83 CLINICAL Association of Helicobacter pylori Infection With Reduced Risk for Esophageal Cancer Is Independent of Environmental and Genetic Modifiers DAVID C. WHITEMAN, PRIYA PARMAR, PAUL FAHEY, SUZANNE P. MOORE, MITCHELL STARK, ZHEN ZHEN ZHAO, GRANT W. MONTGOMERY, ADÈLE C. GREEN, NICHOLAS K. HAYWARD, and PENELOPE M. WEBB for the Australian Cancer Study Queensland Institute of Medical Research, Brisbane, Australia This article has an accompanying continuing medical education activity on page e11. Learning Objective: Upon completion of reading this article, successful learners will be able to identify the strengths and limitations of case-control studies in examining risk factors for esophageal cancer and to interpret findings of a case-control study. See editorial on page 17. BACKGROUND & AIMS: Infection with Helicobacter pylori is associated with reduced risk of esophageal adenocarcinoma (EAC), but it is not clear whether this reduction is modified by genotype, other host characteristics, or environmental factors. Furthermore, little is known about the association between H pylori and adenocarcinomas of the esophagogastric junction (EGJAC) or squamous cell carcinomas (ESCC). We sought to measure the association between H pylori infection and esophageal cancer and identify potential modifiers. METHODS: In an Australian, population-based, case-control study, we compared the prevalence of H pylori seropositivity and single nucleotide polymorphisms in interleukin (IL)-1B ( 31, 511) and tumor necrosis factor (TNF)- ( 308, 238) among 260 EAC, 298 EGJAC, and 208 ESCC patients and 1346 controls. To estimate relative risks, we calculated odds ratios (OR) and 95% confidence intervals (CI) using multivariable logistic regression in the entire sample and within strata of phenotypic and genotypic risk factors. RESULTS: H pylori infection was associated with significantly reduced risks of EAC (OR, 0.45; 95% CI: ) and EGJAC (OR, 0.41; 95% CI: ) but not ESCC (OR, 1.04; 95% CI: ). For each cancer subtype, risks were of similar magnitude across strata of reflux frequency and smoking status. We found no evidence that polymorphisms in IL-1B or TNF- modified the association between H pylori and EAC or EGJAC. CON- CLUSIONS: H pylori infection is inversely associated with risks of EAC and EGJAC (but not ESCC); the reduction in risk is similar across subgroups of potential modifiers. Keywords: Esophageal Neoplasms; Case-Control Studies; Genetic and Environmental Modifiers; Acid Reflux. Esophageal cancers are the sixth most commonly occurring cancers worldwide, 1 and their very high mortality rate accentuates their public health importance. Recent rapid rises in the incidence of adenocarcinomas of the esophagus (EAC) and esophago-gastric junction (EGJAC) and contrasting declines in the incidence of esophageal squamous cell carcinomas (ESCC) suggest population-wide changes in exposure to causal factors. 2,3 Chronic, frequent reflux of gastric acid into the distal esophagus is the primary factor underlying most cases of EAC. 4 Other factors, including obesity and smoking, have also been shown to significantly increase a person s risk of these cancers 5 10 and may act synergistically through inflammatory pathways to enhance the carcinogenic effects of gastroesophageal reflux On the other hand, ESCC has been most strongly associated with smoking and alcohol intake in Western populations 10,14 17 and with other environmental factors such as diet, 18,19 infections, 20 and thermal injury 21 in high-incidence populations. Recently, attention has focused on a possible role of Helicobacter pylori in the changing epidemiology of EAC. 22 Infection with H pylori is causally associated with ulceration, atrophy, and carcinoma of the stomach and with ulceration of the duodenum. A developing body of observational data suggests that people with evidence of H Abbreviations used in this paper: BMI, body mass index; CI, confidence interval; EAC, esophageal adenocarcinoma; EGJAC, esophagogastric junction adenocarcinoma; ESCC, esophageal squamous cell carcinoma; IL, interleukin; NSAIDs, nonsteroidal anti-inflammatory drugs; OR, odds ratio; PPI, proton pump inhibitors; SNP, single nucleotide polymorphism; TNF, tumor necrosis factor by the AGA Institute /$36.00 doi: /j.gastro

2 74 WHITEMAN ET AL GASTROENTEROLOGY Vol. 139, No. 1 pylori infection have lower than average risks of EAC; 23 hypoacidity induced by atrophic gastritis has been proffered as one explanation for this inverse association. 22 Other physiologic sequelae of H pylori infection that also might explain the inverse association include reduced ghrelin synthesis among infected persons 24 (speculated to induce early satiety and thereby prevent obesity) and rapid gastric emptying 25 (thereby reducing the likelihood of gastroesophageal reflux). The relationship between H pylori and upper gastrointestinal cancer may be more complicated than first appreciated however because it is now apparent that, for a subset of people, infection with H pylori leads to hyperacidity and antral predominant, nonatrophic gastritis. 26 There is evidence to suggest that infections among this group of patients may actually increase the risk of EAC. 27,28 Host cytokine responses appear to underpin these divergent clinical pathways following H pylori colonization, and the evidence to date most strongly implicates polymorphisms in the genes encoding interleukin (IL)-1B and tumor necrosis factor (TNA). 29 IL-1B is among the most potent inhibitors of gastric acid yet identified, and the T allele at the 31 locus of IL1B confers enhanced activity. The common TNF- 308A/G (rs ) polymorphism is associated with an increased production of TNF-, 30 and a recent meta-analysis concluded that the TNF- 308AA genotype is associated with a moderately increased risk of gastric cancer. 31 There are grounds for predicting that another polymorphism at TNF- 238 A/G (rs361525) also has functional significance, 30 although epidemiologic evidence for an association with cancers of the gastrointestinal tract is so far lacking. It has been hypothesized that the association between H pylori infection and the risk of adenocarcinomas of the esophagus and esophago-gastric junction is modified by polymorphisms in these proinflammatory genes that regulate gastric acid secretion. No studies to date have tested this hypothesis. Here, we report the findings of an investigation into the association between H pylori infection and cancers of the esophagus conducted within a population having a low prevalence of infection ( 25%). In particular, we sought to assess whether the effects of H pylori infection were modified by genes and other factors known or strongly suspected to be associated with risks of cancers of the esophagus. Materials and Methods Study Design and Participants We used data from a nationwide case-control study of esophageal cancer conducted in Australia, the details of which have been described in full elsewhere. 13 In summary, eligible case patients were people aged years with a histologically confirmed primary in vasive cancer of the esophagus or esophago-gastric junction diagnosed between July 1, 2002 (July 1, 2001, in Queensland), and June 30, 2005, in the mainland states of Australia. Patients were recruited either through major treatment centers or state-based cancer registries. A total of 1577 patients with esophageal cancer received an invitation to participate in the study, of whom 1102 patients (858 through clinics and 244 through cancer registries) returned a completed questionnaire (70% of all invited; 35% of all living and deceased persons in mainland Australia who had been diagnosed with incident esophageal cancer). Details of the histologic type and anatomic site of each patient s tumor were abstracted from diagnostic pathology reports. All of the squamous cell carcinomas were considered esophageal in origin; anatomic sites of adenocarcinomas were categorized according to the World Health Organization classification into esophageal and esophago-gastric junction tumors. 32 Eight case patients were deemed ineligible on review and were excluded from the analysis. Full questionnaire data were available for 365 EAC, 426 EGJAC, and 303 ESCC patients. Potential controls were randomly selected from the Australian Electoral Roll within strata of age (in 5-year age groups) and state of residence to match the distribution of the case series. We aimed for similar numbers of male cases and controls in each stratum of age and state; female controls were intentionally over-sampled at all ages to accommodate their simultaneous enrolment in a parallel case-control study of ovarian cancer. 33 Of 3258 potentially eligible control participants who were contacted and invited to participate, 175 were excluded (16 deceased, 61 were too ill, 98 were unable to read or write in English), and 41 were lost to follow-up shortly after initial contact. Of 3042 remaining controls, 1680 (55%) accepted the initial invitation, and 1580 returned the completed questionnaires (51% of all potentially eligible controls contacted). Data Collection Participants self-completed a health and lifestyle questionnaire asking about their social background and general health. We assessed the frequency of symptoms of gastroesophageal reflux 10 years before diagnosis, defined as the presence of heartburn ( a burning pain behind the breastbone after eating ) or acid reflux ( a sour taste from acid or bile rising up into the mouth or throat ). For analysis, we used the highest reported frequency for either symptom and defined frequent symptoms as those occurring at least weekly during the 10 years before diagnosis, consistent with previous reports. 34,35 Participants were asked Has a doctor ever told you that you have a Helicobacter pylori infection? (an infection of the lining of your stomach that can cause ulcers). (Note: this can ONLY be diagnosed by putting a

3 July 2010 HELICOBACTER PYLORI AND ESOPHAGEAL CARCINOMAS 75 tube down your throat, or by a blood or breath test.) ; those who responded affirmatively were also asked whether they had received specific treatment for this infection. Height and weight 1 year ago (1 year before diagnosis for cases) were elicited and used to calculate the body mass index (BMI; in kg/m 2 ). Participants were asked whether, over their whole life, they had ever smoked more than 100 cigarettes, cigars, or pipes; positive responses elicited further questions regarding consumption and duration of smoking. We derived the number of packyears of tobacco exposure by dividing the number of cigarettes smoked on a typical day by 20 and multiplying by the total number of years smoked. We asked participants to report the frequency with which they consumed different classes of alcohol (light beer, regular beer, white wine, red wine, port/sherry, and spirits/liqueurs) at ages 20 29, 30 49, and 50 years, as applicable. For these analyses, total alcohol consumption was summed across all age groups from which we calculated the average number of standard drinks (10 g ethanol) consumed per week between age 20 years and current age. We also asked participants whether they had ever used aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs) during the past 5 years and, if so, the frequency of use on a 7-point scale ranging from less than once a month up to 2 or more times/per day. Finally, participants were asked whether they had every used acid-suppressant medications and were given lists of generic and trade names for H2-receptor antagonists and proton pump inhibitors (PPIs). Blood Collection Facilities for the systematic collection of blood became available 6 months after the study commenced, and no attempt was made to re-contact those participants who had already completed data collection. Blood samples for H pylori analysis and genotyping were available for 1400 controls and 269 EAC, 307 EGJAC, and 218 ESCC cases. Serologic Methods Following collection from study participants, serum samples were stored at 80 C and thawed immediately prior to testing. We used a commercially available, rapid enzyme-linked immunosorbent assay kit (Genesis Diagnostics Ltd, Littleport, Cambridge, UK) to detect immunoglobulin G antibodies to H pylori according to the manufacturer s instructions. Briefly, diluted serum samples were incubated with partially purified H pylori antigens immobilized on microtiter wells. After washing away unbound serum components, rabbit anti-human immunoglobulin G conjugated to horseradish peroxidase was added to the wells. Unbound conjugate was removed by washing, and a solution containing 3,3=,5,5=tetramethylbenzidine and enzyme substrate was added to trace specific antibody binding. The optical densities of the standards, controls, and samples were measured using a microplate reader at 450 nm. An index of 0.9 was considered negative, an index of 1.1 was considered positive, and values between 0.9 and 1.1 were equivocal. Appropriate positive and negative controls were included in each rack. Genotyping Assays We tested for all 3 genotypes at each of 4 single nucleotide polymorphisms (SNPs); 2 in the IL-1B gene (IL-1B 31, rs ; IL-1B 511, rs16944), and 2 in the TNF- gene (TNF- 308, rs ; TNF- 238, rs361525) using the Sequenom iplex protocol (Sequenom, San Diego, CA). The polymerase chain reaction reactions and primer extension reactions were conducted on Sequenom platform as described previously. 36 iplex products were diluted and desalted with 15 L sterile water and 3 L of resin prior to spotting onto a SpectroChip (Sequenom) for analysis in the Compact Mass Spectrometer, using the MassARRAY Workstation software version 3.3 (Sequenom). Genotype accuracy was calculated for all SNPs tested at 99.95%. Statistical Analysis We estimated the relative risks of esophageal cancer associated with H pylori infection by calculating the odds ratio (OR) and 95% confidence interval (95% CI) using multivariable logistic regression analysis in SAS version 9.1 (SAS Institute, Inc, Cary, NC). Our approach was, first, to fit simple age- and sex-adjusted models for each exposure. We then additionally adjusted for those variables that were significantly associated with risk of esophageal cancers in our data set, namely education, smoking, alcohol consumption, frequency of aspirin/ NSAID use, and BMI. Fully adjusted models included the preceding variables as well as a term for frequency of gastroesophageal reflux symptoms. To explore whether the associations between H pylori infection and cancer risk were modified by exposure to known or suspected causal factors, we repeated the above analyses for genotypes at each locus (3 genotypes each for IL-1B 31 and IL-1B 511; combined genotypes for TNF- 308; TNF- 238) and within strata of the frequency of symptoms of gastroesophageal reflux ( never, less than weekly, at least weekly ), smoking status ( never, ever ), and BMI ( 25.0, , 30 [kg/ m 2 ]). To assess the statistical significance of differences in associations across the strata of genotype or host characteristics, we assessed the P value for the type III analysis of effects for the interaction terms. For all analyses, statistical significance was determined at.05, and all tests for statistical significance were 2 sided. The study was approved by the Human Research Ethics Committee of the Queensland Institute of Medical Research and

4 76 WHITEMAN ET AL GASTROENTEROLOGY Vol. 139, No. 1 Table 1. Demographic Characteristics of Controls and Cases of Esophageal Adenocarcinoma, Adenocarcinoma of Esophago- Gastric Junction, and Esophageal Squamous Cell Carcinoma Distribution of controls and cases Exposure Category Control EAC EGJAC ESCC n 1355 n 269 n 307 n 218 Count (%) Count (%) Count (%) Count (%) Age, y (15) 21 (8) 28 (8) 14 (6) (26) 75 (28) 88 (29) 57 (26) (35) 103 (39) 100 (33) 78 (36) (23) 70 (26) 91 (30) 69 (32) Sex Female 459 (34) 22 (8) 38 (12) 92 (42) Male 896 (66) 247 (92) 269 (88) 126 (58) Education School only 547 (40) 123 (46) 125 (41) 121 (56) Tech/diploma 604 (45) 129 (48) 148 (48) 80 (38) University 204 (15) 17 (6) 34 (11) 17 (8) P value a BMI (kg/m 2 ) (35) 44 (17) 76 (26) 113 (55) (44) 114 (44) 126 (42) 61 (30) (21) 101 (39) 96 (32) 33 (16) P value Smoking history Never 610 (45) 65 (24) 75 (24) 53 (24) 1 29 pack-years 516 (38) 109 (41) 128 (42) 82 (38) 30 pack-years 229 (17) 95 (35) 104 (34) 82 (38) P value Mean alcohol consumption (standard drinks/week) Frequency of symptoms of heartburn or reflux Frequency of use of aspirin/nsaids past 5 years None 143 (11) 20 (8) 27 (9) 28 (13) 1 86 (6) 6 (2) 12 (4) 13 (6) (31) 60 (23) 77 (25) 50 (23) (32) 99 (37) 106 (35) 42 (19) (19) 82 (31) 83 (27) 85 (39) P value Never 578 (43) 59 (22) 83 (27) 101 (47) 1/week 597 (44) 96 (36) 116 (38) 54 (25) 1/week 174 (13) 112 (42) 107 (35) 59 (28) P value Never 276 (20) 63 (23) 78 (25) 43 (20) 1/week 711 (52) 126 (49) 153 (50) 121 (57) 1/week 368 (27) 80 (30) 76 (25) 50 (23) P value Self-reported prior H pylori infection No 1237 (93) 248 (93) 278 (91) 194 (90) Yes 93 (7) 20 (7) 27 (9) 22 (10) P value EAC, esophageal adenocarcinoma; EGJAC, adenocarcinoma of esophago-gastric junction; ESCC, esophageal squamous cell carcinoma. a P value for 2 test comparing each group of cases to the controls for the distribution of each categorical variable. participating hospitals, and all participants gave their informed consent to take part. Results Demographic characteristics of cases and controls are shown in Table 1. As anticipated, higher proportions of cases were ever smokers, and reported having had reflux symptoms. The prevalence of overweight and obesity was higher among EAC and EGJAC cases than controls; patients with ESCC were least likely to be overweight or obese. Predictors of H pylori Infection Among Controls The overall prevalence of H pylori antibodies among controls was 23%. The prevalence of H pylori seropositivity increased markedly with age ( 40 years, 5%; years, 12%; years, 19%; years, 25%; years, 31%; P trend.001) and was significantly associated with low levels of education (school only, 28%; technical college, 22%; university, 14%; P.001) and high levels of smoking (never smokers, 21%; 1 29 pack-years, 23%; 30 pack-years, 29%; P trend.03). We found similar prevalences of seropositivity by sex and across categories of BMI, alcohol intake, and gastroesophageal reflux symptom frequency (not shown). H pylori Infection and Risk of Esophageal Cancers Patients with EAC and EGJAC were significantly less likely than controls to have antibodies to H pylori

5 July 2010 HELICOBACTER PYLORI AND ESOPHAGEAL CARCINOMAS 77 Table 2. Relative risks of Esophageal Adenocarcinoma, Adenocarcinoma of Esophago-Gastric Junction, and Esophageal Squamous Cell Carcinoma Associated With H pylori Seropositivity Controls Cases Minimally adjusted Partially adjusted Fully adjusted H pylori serostatus N % N % OR a 95% CI OR b 95% CI OR c 95% CI EAC Negative Reference 1.00 Reference 1.00 Reference Positive Equivocal EGJAC Negative Reference 1.00 Reference 1.00 Reference Positive Equivocal ESCC Negative Reference 1.00 Reference 1.00 Reference Positive Equivocal a Odds ratio for H pylori ve vs H pylori ve, adjusted for sex and age. b Odds ratio for H pylori ve vs H pylori ve, adjusted for sex, age, educational level, smoking history, BMI category, mean lifetime alcohol intake, frequency of aspirin/nsaid use in past 5 years. c Odds ratio for H pylori ve vs H pylori ve, adjusted for sex, age, educational level, smoking history, BMI category, mean lifetime alcohol intake, frequency of aspirin/nsaid use in past 5 years, and frequency of reflux/heartburn symptoms in 10 years before study. (Table 2), whereas the prevalence of seropositivity among patients with ESCC was very similar to controls. Adjustment for potentially confounding factors made little difference to the risk estimates for any of the cancers. Analyses Stratified by Patient Characteristics We repeated the multivariable analyses for H pylori within strata of known causal factors for esophageal cancer (Table 3). Whereas there was some variability in the magnitude of risk estimates across strata Table 3. Relative risks of Esophageal Adenocarcinoma, Adenocarcinoma of Esophago-Gastric Junction, and Esophageal Squamous Cell Carcinoma Associated With H pylori Seropositivity, Stratified by Other Causal Factors EAC EGJAC ESCC Odds ratios a 95% CI Odds ratios a 95% CI Odds ratios a 95% CI GER never GER weekly GER weekly P b.51 P.16 P.20 Never smoker pack-years pack-years P.23 P.58 P.82 BMI BMI BMI P.08 P.71 P.86 Never used H2 blockers Ever used H2 blockers P.05 P.89 P.13 Never used PPI Ever used PPI P.29 P.35 P.98 Never diagnosed with H pylori Ever diagnosed with H pylori P.25 P.13 P.05 GER, gastroesophageal reflux; EAC, esophageal adenocarcinoma; EGJAC, adenocarcinoma of esophago-gastric junction; ESCC, esophageal squamous cell carcinoma. a Odds ratio for H pylori ve vs H pylori ve, adjusted for age, sex, educational level, smoking status, mean lifetime alcohol intake, BMI category, frequency of reflux/heartburn symptoms in 10 years before study, and frequency of aspirin/nsaid use in the past 5 years. b P value for the type III analysis of effects for the addition of the interaction term to the saturated model.

6 78 WHITEMAN ET AL GASTROENTEROLOGY Vol. 139, No. 1 of gastroesophageal reflux symptom frequency and cumulative smoking history, these were within the bounds of random variation, and the interaction terms were uniformly nonsignificant. Overall, 7% of population controls and 7% 10% of patients with esophageal cancer reported that they been diagnosed previously with H pylori. There was no evidence that the magnitude of the associations between H pylori serology and cancer risk differed according to prior diagnosis H pylori (not shown). For BMI however, the interaction term included in EAC model approached statistical significance (P.08), although inspection of the stratum-specific risk estimates does not suggest a consistent pattern of effect modification. There was some evidence that the inverse association between H pylori and EAC was stronger among those who reported never using H2-antagonists (OR, 0.35) than those who ever reported using them (OR, 0.77) (test for interaction, P.05). There was no statistical evidence for there being a difference in the magnitude of effects for EGJAC or ESCC nor for any of the cancers when stratified by PPI use (Table 3). Cytokine Genotypes and Risk of Esophageal Cancers All SNPs were in Hardy Weinberg equilibrium. Genotype and minor allele frequencies for genes hypothesized to determine host responses to H pylori infection are presented in Table 4. Distributions of IL-1B 31, IL-1B 511, TNF- 308, and TNF- 238 among patients with EAC, EGJAC, or ESCC were not statistically significantly different from population controls. Analyses Stratified by Genotype In stratified analyses, we found no evidence that the inverse associations between H pylori infection and EAC/EGJAC were modified by the presence of polymorphisms at IL-1B 31, IL-1B 511, TNF- 308, and TNF- 238 (Table 5). Similarly, the null associations for ESCC were observed within all genotypic strata. Discussion We found that patients with EAC or EGJAC were significantly less likely than population controls to have serologic evidence of H pylori infection, whereas patients with ESCC were no different from controls in this respect. For the first time, we assessed whether polymorphisms in the interleukin-1 gene cluster and TNF- modify the association between H pylori infection and risk of cancer. Previous research suggested that these genes greatly influence gastric acid production and thereby modify the carcinogenic potential of H pylori infection. We found little evidence to support this hypothesis with respect to cancers of the Table 4. Genotype frequencies for IL-1B 31, IL-1B 511, TNF- 308 and TNF- 238 Among Controls and Cases of Esophageal Adenocarcinoma, Adenocarcinoma of Esophago-Gastric Junction, and Esophageal Squamous Cell Carcinoma Control EAC EGJAC ESCC Genotype n 1316 % n 260 % n 298 % n 208 % IL-1B 31 CC rs TC TT Missing MAF IL-1B 511 CC rs16944 CT TT Missing MAF TNF- 308 AA rs GA GG Missing MAF TNF- 238 AA rs GA GG Missing MAF Total MAF, Minor allele frequency; EAC, esophageal adenocarcinoma; EGJAC, adenocarcinoma of esophago-gastric junction; ESCC, esophageal squamous cell carcinoma.

7 July 2010 HELICOBACTER PYLORI AND ESOPHAGEAL CARCINOMAS 79 Table 5. Relative Risks of Esophageal Adenocarcinoma, Adenocarcinoma of Esophago-Gastric Junction, and Esophageal Squamous Cell Carcinoma Associated With H pylori Seropositivity, Stratified by Genotype EAC EGJAC ESCC Genotype Odds ratios a 95% CI Odds ratios a 95% CI Odds ratios a 95% CI IL-1B 31 CC rs CT TT P b.53 P.44 P.76 IL-1B 511 CC rs16944 CT TT P.38 P.62 P.84 TNF- 308 AA/GA rs GG P.82 P.12 P.94 TNF- 238 AA/GA rs GG P.88 P.95 P.89 EAC, esophageal adenocarcinoma; EGJAC, adenocarcinoma of esophago-gastric junction; ESCC, esophageal squamous cell carcinoma. a Odds ratio for H pylori ve vs H pylori ve, adjusted for age, sex, educational level, smoking status, mean lifetime alcohol intake, BMI category, frequency of reflux/heartburn symptoms in 10 years before study, and frequency of aspirin/nsaid use in the past 5 years. b P value for the type III analysis of effects for the addition of the interaction term to the saturated model. esophagus or esophago-gastric junction; the inverse associations observed between H pylori and EAC and EGJAC were not significantly different across groups with polymorphisms in IL-1B 31, IL-1B 511, TNF- 308, or TNF Other factors known to be strongly associated with risk of esophageal adenocarcinomas, including frequency of symptoms of gastroesophageal reflux, smoking, and body mass index, did not substantially modify these associations. A notable feature of the present study was the overall low prevalence of H pylori antibodies. At 23% among controls, the prevalence was less than that observed among comparable population-based studies in Sweden 37 and the United States. 38 The prevalence of H pylori infection appears to be declining with successive birth cohorts in Western populations, 39 commonly ascribed to smaller families and improved sanitation and hygiene resulting in lower levels of bacterial colonization during childhood. 40 Seroprevalence data for the Australian population with which to compare our sample are scarce because earlier reports have tended to focus on particular subgroups within the community. The most comprehensive data come from a 2002 survey of 2413 sera retrieved from 37 major diagnostic laboratories around the nation, which reported the prevalence of H pylori antibodies at 15%. 41 That sample was restricted to people less than age 60 years however, whereas our sample was predominantly older than 60 years. The age-specific prevalences among people age years, the largest overlapping age category between the 2 studies, were similar (this study, 23% vs laboratory survey, 19%). Although the 2 studies differed in design and purpose, their concordant data suggest that the prevalence of H pylori infection in the Australian population is markedly lower than in the United States, 42 United Kingdom, and other Western populations. 43 Notwithstanding the current low prevalence of infection in the Australian population, the 50% 60% risk reductions we observed for EAC were remarkably similar to those reported in other studies. 23,44 Relatively few studies have examined separately those adenocarcinomas occurring at the junction of the esophagus and stomach. The difficulties of determining the precise site of origin of such tumors are well-known and have been outlined previously 45 and no doubt contribute to the differing findings across studies; however, our finding of substantially lower risks of EGJAC cancers associated with H pylori is similar to that of Kamangar et al. 46 The definition of cardia cancer used in that study was essentially the same as the definition we used for EGJAC, and it is possible that in both studies this category comprises a mixture of distal EACs, genuine junctional cancers, and some gastric cancers. We explored whether these inverse associations were confounded or modified by other factors also known to be strongly associated with risk of adenocarcinomas but found no evidence of such effects. Whereas there was a marginally significant difference in the magnitude of the inverse association for EAC between those who self-reported use of H2 antagonists and those who did not, no such effect was seen for PPIs or for EGJAC and ESCC. Of note, we observed lower risks of EAC and EGJAC associated with H pylori infection regard-

8 80 WHITEMAN ET AL GASTROENTEROLOGY Vol. 139, No. 1 less of frequency of gastroesophageal reflux, a finding consistent with the notion that H pylori may influence cancer risks through pathways other than gastric acid. Host responses to H pylori infections are determined, at least in part, by families of cytokines. With respect to gastric cancer, there is strong evidence that SNPs in IL-1B, and to a lesser extent TNF-, confer substantially increased risks of the disease, most likely through acid suppression and gastric atrophy. 29 We therefore hypothesized that the same proinflammatory genotypes that inhibit gastric acid in the presence of H pylori infection would be especially protective against EAC and EGJAC, but our data provide no support for this hypothesis. One previous study has genotyped patients with EAC and ESCC and controls for these SNPs, and, whereas the tabulated risk estimates for IL-1B 511 suggested significant positive associations between the TT genotype and both ESCC and EAC, the authors concluded that there was in fact no evidence of an association. 47 We found no association between evidence of H pylori infection with squamous cell cancers of the esophagus. Whereas a number of studies have reported positive associations with infection, particularly with the caga strain, 37 a recent meta-analysis concluded that there was no association (caga ve vs H pylori-ve: summary OR, 1.01; 95% CI: ). 23 However, it was noted that there was considerable heterogeneity between studies in the magnitude of associations, although the source was not determined. Adding our data to future meta-analyses would likely strengthen the case for no association. Is the inverse association between H pylori infection and EAC and EGJAC evidence of a biologically protective effect? Several potential mechanisms have been proposed to explain the association, including hypoacidity subsequent to prolonged infection, 48 dysregulation of host cytokine or immune responses, disturbances to microbial flora, 22 and changes in the expression of locally acting hormones relating to obesity pathways (notably leptin and ghrelin). 24,49,50 Each of these hypotheses is plausible, and all may indeed play a role. Assays for other markers of gastric function, notably the ratio of pepsinogens I and II, could be informative in determining whether the protective effect is mediated through gastric atrophy for example. The blood samples collected from participants in this study precluded their use for pepsinogen assays on 2 grounds. First, the interval between collection and processing was greater than 10 hours for most participants, and, second, participants were not fasted prior to blood draw. Two previous studies have reported strongly protective effects of H pylori among those without serologic evidence of gastric atrophy however, 37,51 suggesting that this is not the only pathway through which H pylori mediates its apparent protective effects. Perhaps the definitive test for a protective role for this organism in EAC/EGJAC would be to examine gastric biopsy samples from study participants for histologic evidence of H pylori-induced corpus atrophy. Such biopsy samples were not available for cases or controls in this study but could be considered in future studies. Our stratified analyses failed to identify particular subgroups of the population for whom infection with H pylori was more or less protective, and so we have no strong evidence to refute any of the hypotheses above. Indeed, such mechanistic questions involving the interplay of numerous pathways, all of which may interact, may be beyond the resolution of epidemiologic studies. Whatever the precise mechanism, it seems likely that H pylori acts early in the causal pathway to EAC, given the findings of recent studies reporting that patients with Barrett s esophagus, the precancerous precursor to EAC, also have lower rates of H pylori infection than the general population. 52 Arguably the most compelling evidence to date comes from a community-based case-control study of H pylori and Barrett s esophagus conducted in northern California, a population with similarly low prevalences of H pylori infection as we found in Australia. 53 In that study, the overall association with Barrett s esophagus was OR, 0.42; (95% CI: ), similar in magnitude to the association reported here. A limitation of the study was the low rates of participation, raising concerns about possibly biased selection of cases and controls. The cases who participated were very similar in age and sex distribution to those notified to the Australian Institute of Health and Welfare; however, further details about the characteristics of non-participating cases were not available because of Australian privacy laws. It is likely that cases with long survival were over-represented in our study sample, but this would apply to all patients with esophageal cancer recruited into the study and could not explain the inverse associations between H pylori infection status and EAC and EGJAC. The participation fraction among controls was also less than ideal, and a spurious positive association might be explained if the prevalences of H pylori infection or other factors among participating controls were unrepresentative. We have shown that the age-specific prevalence of H pylori infection among our controls was similar to the only other recent population study in Australia, and the distribution of BMI was also similar to the Australian National Health Survey 2004, a representative survey of the Australian adult population. Frequent symptoms of reflux were reported by 12% of controls, remarkably similar to other population surveys. 34,54,55 Whereas we have shown previously that the proportion

9 July 2010 HELICOBACTER PYLORI AND ESOPHAGEAL CARCINOMAS 81 of current smokers among our control series was somewhat lower than the population average, 56 this would most likely bias our study towards the null because H pylori infection is positively associated with smoking. We therefore consider the likelihood of biased selection on the basis of these factors to be no greater than for previous studies and unlikely to account for the inverse associations we observed. We cannot exclude misclassification of H pylori exposure, particularly false negative results, as an explanation, although the kit we used has a sensitivity of 91% and specificity of 100%. Moreover, for misclassification of H pylori infection status to wholly explain these findings would require that patients with EAC and EGJAC, but not ESCC, were incorrectly categorized, a highly improbable scenario. 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Prevalence of symptoms and use of medication for gastroesophageal reflux in an Australian community. World J Surg 2009;33: Nilsson M, Johnsen R, Ye W, et al. Prevalence of gastro-oesophageal reflux symptoms and the influence of age and sex. Scand J Gastroenterol 2004;39: Pandeya N, Williams GM, Green AC, et al. Do low control response rates always affect the findings? Assessments of smoking and obesity in two Australian case-control studies of cancer. Aust N Z J Public Health 2009;33: Received August 13, Accepted April 1, Reprint requests Address requests for reprints to: David Whiteman, MBBS, PhD, head, Cancer Control Laboratory Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Queensland 4029, Australia. david.whiteman@qimr.edu.au; fax (61) Acknowledgments The Australian Cancer Study: Oesophageal Cancer. Investigators: David C. Whiteman, MBBS, PhD; Penelope M. Webb, MA, DPhil; Adele C. Green, MBBS, PhD; Nicholas K. Hayward, PhD; Peter G. Parsons, PhD; David M. Purdie, PhD. Clinical collaborators: B. Mark Smithers, FRACS; David Gotley, FRACS, PhD; Andrew Clouston, FRACP, PhD; Ian Brown, FRACP. Project manager: Suzanne Moore RN, MPH. Database: Karen Harrap, BIT, Troy Sadkowski BIT. Research nurses: Suzanne O Brien, RN, MPH; Ellen Minehan, RN; Deborah Roffe, RN; Sue O Keefe, RN; Suzanne Lipshut, RN; Gabby Connor, RN; Hayley Berry, RN; Frances Walker, RN; Teresa Barnes, RN; Janine Thomas, RN; Linda Terry, RN, MPH; Michael Connard, BSc; Leanne Bowes, BSc; MaryRose Malt, RN; Jo White, RN. Laboratory assistants: Lauren Aoude, BEng; Peter Schultz, BSc; Loralie Parsonson, BSc; Stephen Walsh, BSc. Clinical contributors: Australian Capital Territory: Charles Mosse, FRACS; Noel Tait, FRACS; New South Wales: Chris Bambach, FRACS; Andrew Biankan, FRACS; Roy Brancatisano, FRACS, Max Coleman FRACS, Michael Cox FRACS, Stephen Deane FRACS; Gregory L. Falk, FRACS; James Gallagher, FRACS; Mike Hollands, FRACS; Tom Hugh, FRACS; David Hunt, FRACS; John Jorgensen, FRACS; Christopher Martin, FRACS; Mark Richardson, FRACS; Garrett Smith, FRACS; Ross Smith, FRACS; David Storey, FRACS. Queensland: John Avramovic, FRACS; John Croese, FRACP; Justin D Arcy, FRACS; Stephen Fairley, FRACP; John Hansen, FRACS; John Masson, FRACP; Les Nathanson, FRACS; Barry O Loughlin, FRACS; Leigh Rutherford, FRACS; Richard Turner, FRACS; Morgan Windsor, FRACS. South Australia: Justin Bessell, FRACS; Peter Devitt, FRACS; Glyn Jamieson, FRACS; David Watson, FRACS. Victoria: Stephen Blamey, FRACS; Alex Boussioutas, FRACP; Richard Cade, FRACS; Gary Crosthwaite, FRACS; Ian Faragher, FRACS; John Gribbin, FRACS; Geoff Hebbard, FRACP; George Kiroff, FRACS; Bruce Mann, FRACS; Bob Millar, FRACS; Paul O Brien, FRACS; Robert Thomas, FRACS; Simon Wood, FRACS. Western Australia: Steve Archer, FRACS; Kingsley Faulkner, FRACS; Jeff Hamdorf, FRACS. The authors thank Shahram Sadeghi, MD, PhD, and Harish Babu, MD, for their assistance with pathology abstractions and Nirmala Pandeya, M Med Sc, provided assistance with data cleaning and statistical programming.