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Serologic Response to Helicobacter pylori Proteins Associated With Risk of Colorectal Cancer Among Diverse Populations in the United States

Published:October 05, 2018DOI:https://doi.org/10.1053/j.gastro.2018.09.054

      Background & Aims

      Previous studies reported an association of the bacteria Helicobacter pylori, the primary cause of gastric cancer, and risk of colorectal cancer (CRC). However, these findings have been inconsistent, appear to vary with population characteristics, and may be specific for virulence factor VacA. To more thoroughly evaluate the potential association of H pylori antibodies with CRC risk, we assembled a large consortium of cohorts representing diverse populations in the United States.

      Methods

      We used H pylori multiplex serologic assays to analyze serum samples from 4063 incident cases of CRC, collected before diagnosis, and 4063 matched individuals without CRC (controls) from 10 prospective cohorts for antibody responses to 13 H pylori proteins, including virulence factors VacA and CagA. The association of seropositivity to H pylori proteins, as well as protein-specific antibody level, with odds of CRC was determined by conditional logistic regression.

      Results

      Overall, 40% of controls and 41% of cases were H pylori–seropositive (odds ratio [OR], 1.09; 95% CI, 0.99–1.20). H pylori VacA–specific seropositivity was associated with an 11% increased odds of CRC (OR, 1.11; 95% CI, 1.01–1.22), and this association was particularly strong among African Americans (OR, 1.45; 95% CI, 1.08–1.95). Additionally, odds of CRC increased with level of VacA antibody in the overall cohort (P = .008) and specifically among African Americans (P = .007).

      Conclusions

      In an analysis of a large consortium of cohorts representing diverse populations, we found serologic responses to H pylori VacA to associate with increased risk of CRC risk, particularly for African Americans. Future studies should seek to understand whether this marker is related to virulent H pylori strains carried in these populations.

      Graphical abstract

      Keywords

      Abbreviations used in this paper:

      BMI (body mass index), CI (confidence interval), CRC (colorectal cancer), MFI (median fluorescence intensity), OR (odds ratio), SCCS (Southern Community Cohort Study)
      See Covering the Cover synopsis on page 2.

       Background and Context

      Over 50% of the world’s population is infected with Helicobacter pylori, the main cause of gastric cancer. However, for specific populations and/or strains, H pylori infection may also be associated with increased risk of colorectal cancer.

       New Findings

      While H pylori sero-positivity was not associated with odds of colorectal cancer, H pylori VacA-specific sero-positivity was associated with 11% increased odds of colorectal cancer, particularly among African Americans.

       Limitations

      Serology indicates a past and/or acute infection and does not provide information on the current site-specific infection status of the individual.

       Impact

      Strong antibody responses to H pylori virulence factor VacA were significantly associated with odds of developing colorectal cancer, particularly for African Americans. Future studies are needed to assess if H pylori infection mechanistically increases CRC risk.
      It is currently estimated that at least 15% of all incident cancers are caused by infection, and the bacterium Helicobacter pylori is the leading single carcinogenic infectious agent, responsible for 770,000 cancer cases worldwide each year due to its established association with gastric cancer.
      • Plummer M.
      • de Martel C.
      • Vignat J.
      • et al.
      Global burden of cancers attributable to infections in 2012: a synthetic analysis.
      More than 50% of the world’s population is infected with this bacterium, and the prevalence of H pylori varies widely by geographic location, with the highest prevalence in East Asia, Africa, and parts of South America, and lowest prevalence in the United States, Oceania, and Western Europe.
      • Hooi J.K.Y.
      • Lai W.Y.
      • Ng W.K.
      • et al.
      Global prevalence of Helicobacter pylori infection: systematic review and meta-analysis.
      However, even within the United States, there remains great variation in both H pylori prevalence and gastric cancer incidence by race/ethnicity.
      • Grad Y.H.
      • Lipsitch M.
      • Aiello A.E.
      Secular trends in Helicobacter pylori seroprevalence in adults in the United States: evidence for sustained race/ethnic disparities.
      • Siegel R.L.
      • Miller K.D.
      • Jemal A.
      Cancer statistics, 2018.
      The mechanism by which infection with H pylori induces gastric cancer is understood to be due at least in part to chronic inflammation of the gastric mucosa. Several studies have investigated whether H pylori might also increase risk for developing colorectal cancer (CRC). The mechanism underlying this possible association has not yet been delineated.
      • de Korwin J.D.
      • Ianiro G.
      • Gibiino G.
      • et al.
      Helicobacter pylori infection and extragastric diseases in 2017.
      Nonetheless, the results of 2 meta-analyses found significant 30%–50% increased odds for CRC among those individuals with evidence of a current or past H pylori infection.
      • Zhao Y.
      • Wang X.
      • Wang Y.
      Helicobacter pylori infection and colorectal carcinoma risk: a meta-analysis.
      • Zumkeller N.
      • Brenner H.
      • Zwahlen M.
      • et al.
      Helicobacter pylori infection and colorectal cancer risk: a meta-analysis.
      H pylori strains are very diverse genetically,
      • Blaser M.J.
      The biology of cag in the Helicobacter pylori-human interaction.
      yet the majority of published studies examining the association between H pylori and CRC risk did not take into account this heterogeneity. H pylori strains exhibit variation in the presence or absence of virulence factors (such as the cag pathogenicity island, which encodes the oncogenic effector protein CagA), as well as allelic variation in the VacA.
      • Cover T.L.
      Helicobacter pylori diversity and gastric cancer risk.
      As the majority of the world’s population is infected with H pylori, but only a small proportion of those individuals develop gastric cancer, it is important that both bacterial virulence factors and host responses, such as the individual’s immune response to H pylori infection, are considered. Our group has utilized multiplex serology to assess immune response to 15 different H pylori proteins in a primarily low-income population in the southeast United States, and found an exceptionally high prevalence of antibodies to H pylori, with significantly higher prevalences among African Americans compared to whites for both CagA and VacA.
      • Epplein M.
      • Signorello L.B.
      • Zheng W.
      • et al.
      Race, African ancestry, and Helicobacter pylori infection in a low-income United States population.
      We also found that seropositivity to VacA was associated with a significant 84% increased CRC risk in this population, and there was also a strong dose–response association for CRC by increasing quartile of VacA antibody level.
      • Epplein M.
      • Pawlita M.
      • Michel A.
      • et al.
      Helicobacter pylori protein-specific antibodies and risk of colorectal cancer.
      In this study, we sought to thoroughly evaluate the novel association of Helicobacter pylori protein-specific antigen response and the odds of CRC through use of a consortium of nested case–control studies. We modeled the associations of serologic response of individual antigens with CRC incidence, with consideration of the heterogeneity of the outcome (by histologic site, stage, and age at onset of cancer), the time from antibody status assessment to cancer diagnosis, and the potential dose–response relationship between level of serologic response and risk of disease. Therefore, we created a consortium of 10 prospective cohort studies specifically chosen to highlight the diversity of the US population, including more than 4000 prospectively ascertained CRC cases and 1:1 matched controls. To date, this consortium provides the largest nested case–control study with prediagnostic serum to examine the association between H pylori and odds of CRC that addresses both H pylori protein expression diversity, as well as the racial/ethnic diversity in the United States.

      Materials and Methods

       Study Population

      This consortium was built using a nested case–control study design that includes prospectively ascertained colorectal cancer cases (and 1:1 matched controls) with available prediagnostic serum. These studies represent diverse populations within the United States, including primarily low-income African Americans and whites in the southeast recruited from community health clinics (Southern Community Cohort Study [SCCS], excluding CRC cases and controls participating in the hypothesis-generating earlier study
      • Epplein M.
      • Pawlita M.
      • Michel A.
      • et al.
      Helicobacter pylori protein-specific antibodies and risk of colorectal cancer.
      ); individuals of Native Hawaiian, Japanese, and European ancestry in Hawaii and individuals of African and Latino ancestry in Los Angeles, California (Multiethnic Cohort Study [MEC]
      • Kolonel L.N.
      • Henderson B.E.
      • Hankin J.H.
      • et al.
      A multiethnic cohort in Hawaii and Los Angeles: baseline characteristics.
      ); medical professionals from more than 14 states (Health Professionals Follow-up Study [HPFS],
      • Wei E.K.
      • Giovannucci E.
      • Fuchs C.S.
      • et al.
      Low plasma adiponectin levels and risk of colorectal cancer in men: a prospective study.
      Nurses’ Health Study [NHS],
      • Colditz G.A.
      • Hankinson S.E.
      The Nurses' Health Study: lifestyle and health among women.
      and Physicians’ Health Study [PHS]
      • Lee J.E.
      • Wei E.K.
      • Fuchs C.S.
      • et al.
      Plasma folate, methylenetetrahydrofolate reductase (MTHFR), and colorectal cancer risk in three large nested case-control studies.
      ); women visiting a breast screening center in New York City (New York University Women’s Health Study [NYU WHS]
      • Toniolo P.G.
      • Levitz M.
      • Zeleniuch-Jacquotte A.
      • et al.
      A prospective study of endogenous estrogens and breast cancer in postmenopausal women.
      ); postmenopausal women recruited at 40 clinical centers nationwide as part of a long-term national health study (Women’s Health Initiative [WHI]
      Design of the Women's Health Initiative clinical trial and observational study. The Women's Health Initiative Study Group.
      ); residents of suburban Washington County, Maryland, recruited using mobile trailers (Campaign Against Cancer and Stroke [CLUE II]
      • Huang H.Y.
      • Alberg A.J.
      • Norkus E.P.
      • et al.
      Prospective study of antioxidant micronutrients in the blood and the risk of developing prostate cancer.
      ); healthy individuals from 21 states recruited by the American Cancer Society (Cancer Prevention Study-II [CPSII]
      • Calle E.E.
      • Rodriguez C.
      • Jacobs E.J.
      • et al.
      The American Cancer Society Cancer Prevention Study II Nutrition Cohort: rationale, study design, and baseline characteristics.
      ); and participants in a large, nationwide cancer screening trial with recruitment sites in Washington, DC; Pittsburgh, PA; Birmingham, AL; St Louis, MO; Detroit, MI; Marshfield, WI; Minneapolis, MN; Denver, CO; Salt Lake City, UT; and Honolulu, HI (Prostate, Lung, Colorectal, and Ovarian Screening Study [PLCO]
      • Hayes R.B.
      • Reding D.
      • Kopp W.
      • et al.
      Etiologic and early marker studies in the prostate, lung, colorectal and ovarian (PLCO) cancer screening trial.
      ). The participants in these cohorts vary substantially by demographic and lifestyle factors (see Supplementary Table 1), and with this knowledge cases and controls in this consortium are matched not only by age, sex, and race, but also within each cohort.
      Cases included incident cancers of the colon and rectum as defined based on the International Classification of Diseases for Oncology (ICD-O-3, codes C180-189, C199, and C209). Controls were selected using incidence density sampling, with 1 control chosen at random for each CRC case from the appropriate risk sets consisting of all cohort members, who were alive and free of cancer (except non-melanoma skin cancer) at the time of diagnosis of the index case, and had provided blood specimens. Matching criteria included cohort, sex, race, date of birth, and date of blood collection.
      Covariates collected from participating cohorts included standard demographic and anthropometric variables, such as education as a measure of socioeconomic status, and height and weight. Data were harmonized for smoking variables (current/former/never, as well as pack-years); physical activity (metabolic equivalents/day); hormone therapy; diabetes history; CRC screening history; and family history of CRC. Where available, we collected data on use of antibiotics; personal history of inflammatory bowel disease and colorectal polyps; dietary intake of fruit, vegetables, and red meat; and average daily total energy intake. Clinical information ascertained for cases included tumor site, stage, histology, and date of diagnosis, as available.
      Of the 8420 samples that were assayed by multiplex serology (4210 cases and 4210 controls), 100 samples (and their 100 matched case or control counterparts) were excluded due to technical issues of insufficient volume (n = 27), pipetting errors (n = 52), and invalid measurements due to insufficient bead counts (n = 21). We also excluded from analyses 47 pairs that were mismatched by race and/or sex, resulting in a final study population of 4063 cases and 4063 controls. The median follow-up time for cases with valid serology results was 7 years (range, <1–40.2 years), the majority of cases were found in the colon (84%), and the median age at diagnosis was 73 years (range, 40–97 years).

       Multiplex Serology

      Serum samples were sent on dry ice to the German Cancer Research Center (Heidelberg, Germany) and analyzed in a 1:1000 final serum dilution. Multiplex serology was performed as described previously.
      • Michel A.
      • Waterboer T.
      • Kist M.
      • et al.
      Helicobacter pylori multiplex serology.
      Briefly, multiplex serology is a fluorescent bead-based suspension array. H pylori antigens were expressed as glutathione-S-transferase–tagged fusion proteins and affinity-purified on glutathione-casein coupled polystyrene beads (Luminex, Austin, TX) with distinct internal fluorescence. The antigen-loaded bead sets were mixed and incubated with serum. A Luminex flow cytometer distinguished between the bead set, and consequently the loaded antigen, as well as quantified the amount of bound serum antibody by a secondary antibody detecting human IgG, IgA, and IgM and a fluorescent reporter conjugate (Streptavidin-R-phycoerythrin). The output was the median fluorescence intensity (MFI) measured on at least 100 beads per set per sample. Net MFI were calculated by subtracting background values resulting from a serum-free reaction as well as from a bead-set loaded with glutathione-S-transferase tag only.
      All H pylori antigens, except for GroEL (strain G27), were produced as recombinant proteins derived from genes in strain 26695. This strain contains a type s1m1 vacA gene. Three proteins, HyuA, CagA, and VacA were expressed in 2 parts (N- and C-terminus) due to their large size; MFI values measured for each protein part were added to each other for analyses of the responses to full size proteins (Supplementary Table 2). Previous analyses with this assay applied in total 15 H pylori antigens; however, we excluded 2 previously noninformative antigens (CagD and CagM) from the present study, resulting in a total of 13 H pylori proteins assessed.
      • Epplein M.
      • Pawlita M.
      • Michel A.
      • et al.
      Helicobacter pylori protein-specific antibodies and risk of colorectal cancer.
      Antigen-specific cutoffs were applied as defined in the previous study and quality assured by visual inspection of percentile plots (Supplementary Table 2).
      • Epplein M.
      • Pawlita M.
      • Michel A.
      • et al.
      Helicobacter pylori protein-specific antibodies and risk of colorectal cancer.
      Overall, H pylori seropositivity (H pylori+) was defined as being positive to any 4 or more of the included 13 H pylori antigens.
      • Michel A.
      • Waterboer T.
      • Kist M.
      • et al.
      Helicobacter pylori multiplex serology.
      Individual H pylori antigens were only considered as seropositive when the sample was simultaneously considered overall H pylori–positive (H pylori+ antigen+). This prerequisite was applied to ensure that antigen-specific seropositivity did not result from cross-reactive antibody responses from infection with other pathogens expressing homologous proteins.
      Of 82 duplicates within the Women’s Health Initiative study set incorporated as blinded quality-control samples, correlations for antibody responses (MFI) to H pylori antigens ranged from 0.92 to 1.0 indicating a good reproducibility of the measured values.

       Statistical Analysis

      Conditional logistic regression was applied to estimate the odds ratios (OR) and 95% confidence intervals (CI) of CRC in relation to overall H pylori seropositivity, as well as to individual H pylori antigens. Potential confounders (apart from the matching variables age, sex, and race within each cohort) were defined a priori and included education; smoking; body mass index (BMI); family history of CRC; previous colonoscopy/sigmoidoscopy; hormone therapy; daily intake of fruit, vegetable, or red meat; aspirin use; and diabetes. While we were limited by values not missing at random for a number of these variables, overall, adjusting for any of them did not alter the main effect estimates by >10%, therefore main results are presented without further adjustment. The specific effects on the main results ORs from adjustment for the primary potential confounders of education, smoking, and BMI are shown in Supplementary Table 3.
      The matching variable race/ethnicity was strongly associated with H pylori seropositivity, and as we a priori believed that individuals of different race/ethnicity could be infected with different strains of H pylori, we therefore show results for the total cohort but also stratified by race/ethnicity. Additionally, stratified analyses by participating cohorts were performed to rule out that the difference in the association seen by race/ethnicity was study-specific.
      We further explored the dose–response relationship of antibody responses to VacA, which was a priori hypothesized to be specifically associated with CRC risk based on our previous study.
      • Epplein M.
      • Pawlita M.
      • Michel A.
      • et al.
      Helicobacter pylori protein-specific antibodies and risk of colorectal cancer.
      Antibody responses among H pylori VacA seropositive individuals were analyzed in race/ethnicity-specific quartiles defined based on the MFI distribution among controls. ORs and 95% CIs were calculated using conditional logistic regression models with individuals not H pylori+ VacA+ as the reference group, in the total cohort, as well as separately by race/ethnicity. A Cochrane–Armitage test was applied to test for a linear trend in increasing strength of the association.
      We also examined the association of H pylori+ VacA+ and the odds of CRC in separate models by certain case characteristics, including tumor site, stage, and time between blood draw and diagnosis. We did not identify substantial differences by case characteristics (data not shown).

      Results

      Baseline characteristics between CRC cases and controls in the consortium differed with respect to many known CRC risk factors. Specifically, CRC cases were more likely to be obese, diabetic, former smokers, to have a positive CRC family history, and to consume more red meat per day. History of endoscopic screening, regular use of aspirin or hormone therapy, as well as higher physical activity per day were less frequent among CRC cases than among their age, race, and sex-matched controls (Table 1).
      Table 1Baseline Characteristics of the Cohorts Participating in This Study
      VariableTotal (n = 8126)Controls (n = 4063)Cases (n = 4063)
      Study, n (%)
       CLUE982 (12)491 (12)491 (12)
       CPSII722 (9)361 (9)361 (9)
       HPFS302 (4)151 (4)151 (4)
       MEC1510 (19)755 (19)755 (19)
       NHS576 (7)288 (7)288 (7)
       NYUWHS572 (7)286 (7)286 (7)
       PHS360 (4)180 (4)180 (4)
       PLCO1240 (15)620 (15)620 (15)
       SCCS252 (3)126 (3)126 (3)
       WHI1610 (20)805 (20)805 (20)
      Age at blood draw, y, median (range)64 (18–89)64 (18–88)64 (18–89)
      Sex, n (%)
       Female5112 (63)2556 (63)2556 (63)
       Male3014 (37)1507 (37)1507 (37)
      Race/ethnicity, n (%)
       White6134 (75)3067 (75)3067 (75)
       African American798 (10)399 (10)399 (10)
       Asian American614 (8)307 (8)307 (8)
       Latino422 (5)211 (5)211 (5)
       Other/unknown/multiracial158 (2)79 (2)79 (2)
      Education, n (%)
       Less than HS972 (12)468 (12)504 (13)
       Completed HS or GED1668 (21)823 (20)845 (21)
       Post-HS training other than college362 (5)183 (5)179 (4)
       Some college1672 (21)845 (21)827 (21)
       College graduate1483 (19)756 (19)727 (18)
       Graduate school1861 (23)946 (24)915 (23)
       Missing1084266
      BMI,
      All studies except CLUE.
      n (%)
       <20 kg/m2194 (3)99 (3)95 (3)
       20–24.9 kg/m22314 (33)1226 (35)1088 (31)
       25–29.9 kg/m22852 (40)1456 (41)1396 (40)
       ≥30 kg/m21691 (24)748 (21)943 (27)
       Missing1075534541
      Smoking, n (%)
       Never3628 (45)1853 (46)1775 (44)
       Former3358 (42)1622 (40)1736 (43)
       Current1051 (13)546 (14)505 (13)
       Missing894247
      Family history of CRC,
      All studies except NYU WHS, CLUE, SCCS (<75% of data available).
      n (%)
       No5167 (85)2638 (87)2529 (84)
       Yes907 (15)408 (13)499 (16)
       Missing205210171035
      CRC screening, n (%)
       No3243 (53)1554 (51)1689 (55)
       Yes2853 (47)1494 (49)1359 (45)
       Missing203010151015
      Polyps, n (%)
       No4216 (86)2095 (86)2121 (87)
       Yes667 (14)349 (14)318 (13)
      Missing324316191624
      METs/d,
      All studies except CLUE, PLCO, PHS.
      n (%)
       Q11312 (24)612 (23)700 (26)
       Q21358 (25)667 (25)691 (26)
       Q31352 (25)692 (26)660 (24)
       Q41374 (25)723 (27)651 (24)
       Missing273013691361
      Fruit intake/d,
      All studies except WHI, CLUE.
      n (%)
       Q11307 (25)627 (24)680 (26)
       Q21321 (25)668 (25)653 (25)
       Q31328 (25)657 (25)671 (25)
       Q41350 (25)707 (27)643 (24)
       Missing282014041416
      Vegetable intake/d,
      All studies except WHI, CLUE.
      n (%)
       Q11316 (25)652 (25)664 (25)
       Q21312 (25)629 (24)683 (26)
       Q31345 (25)699 (26)646 (24)
       Q41333 (25)679 (26)654 (25)
      Missing282014041416
      Red meat intake/d,
      All studies except CLUE.
      n (%)
       Q11679 (24)880 (25)799 (23)
       Q21733 (25)897 (26)836 (24)
       Q31764 (26)835 (24)929 (27)
       Q41735 (25)849 (25)886 (27)
       Missing1215602613
      Ever regular aspirin use,
      All studies except WHI, CLUE, CPSII, NYU WHS.
      n (%)
       No1302 (44)614 (41)688 (46)
       Yes1690 (56)884 (59)806 (54)
       Missing513425652569
      Ever HT use,
      All studies except HPFS, PHS, CLUE (<75% of data available); women only.
      n (%)
       No2273 (51)1083 (48)1190 (54)
       Yes2164 (49)1150 (52)1014 (46)
       Missing368918301859
      Diabetes,
      All studies except CLUE.
      n (%)
       No6475 (91)3279 (92)3196 (91)
       Yes602 (9)271 (8)331 (9)
       Missing1049513536
      CLUE II, Campaign Against Cancer and Stroke; CPSII, Cancer Prevention Study-II; GED, General Educational Development Test; HPFS, Health Professionals Follow-up Study; HS, high school; HT, hormone therapy; MEC, Multiethnic Cohort Study; MET, metabolic equivalent of task (measure of physical activity); NHS, Nurses’ Health Study; NYU WHS, New York University Women’s Health Study; PHS, Physicians’ Health Study; PLCO, Prostate, Lung, Colorectal, and Ovarian Screening Study; Q, quartile; WHI, Women’s Health Initiative.
      a All studies except CLUE.
      b All studies except NYU WHS, CLUE, SCCS (<75% of data available).
      c All studies except CLUE, PLCO, PHS.
      d All studies except WHI, CLUE.
      e All studies except WHI, CLUE, CPSII, NYU WHS.
      f All studies except HPFS, PHS, CLUE (<75% of data available); women only.
      In the consortium overall, 40% of controls and 41% of CRC cases were assessed as H pylori seropositive, resulting in an OR of 1.09 (95% CI, 0.99–1.20). The prevalence of general H pylori seropositivity varied substantially by race/ethnicity with H pylori seroprevalence being lowest in whites (33% of controls and 35% of cases) and Asian Americans (39% of controls and 46% of cases) and higher in African Americans (65% of controls and 71% of cases) and Latinos (77% of controls and 74% of cases) (χ2 P value for difference in prevalence by race <.0001). The magnitude of the association of H pylori seropositivity with CRC risk also differed across race/ethnicity. We found no association of H pylori seropositivity with odds of CRC among whites (OR, 1.06; 95% CI, 0.95–1.18) or Latinos (OR, 0.84; 95% CI, 0.55–1.30), whereas suggestions of increased odds were seen with Asian Americans (OR, 1.30; 95% CI, 0.94–1.81) and African Americans (OR, 1.30; 95% CI, 0.97–1.76) (Table 2), although none reached statistical significance.
      Table 2Seropositivity to Helicobacter pylori Proteins and Colorectal Cancer Risk, Among All and by Race/Ethnicity
      VariableAllWhiteAfrican AmericanAsian AmericanLatinoOther/unknown/multiracial
      Cases/controls, n4063/40633067/3067399/399307/307211/21179/79
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      n (%)
      1682 (41)/1606 (40)1067 (35)/1027 (33)282 (71)/259 (65)140 (46)/121 (39)156 (74)/163 (77)37 (47)/36 (46)
      OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      1.09 (0.99–1.20)1.06 (0.95–1.18)1.30 (0.97–1.76)1.30 (0.94–1.81)0.84 (0.55–1.30)1.05 (0.57–1.94)
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      VacA+, n (%)
      1434 (35)/1350 (33)876 (29)/830 (27)267 (67)/233 (58)123 (40)/111 (36)134 (64)/145 (69)34 (43)/31 (39)
      OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      1.11 (1.011.22)1.08 (0.96–1.21)1.45 (1.081.95)1.20 (0.85–1.69)0.80 (0.53–1.19)1.18 (0.62–2.25)
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      GroEL+, n (%)
      1557 (38)/1467 (36)996 (32)/957 (31)268 (67)/239 (60)114 (37)/96 (31)146 (69)/143 (68)33 (42)/32 (41)
      OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      1.11 (1.011.22)1.06 (0.95–1.19)1.39 (1.031.87)1.32 (0.93–1.87)1.07 (0.70–1.64)1.06 (0.55–2.01)
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      Omp+, n (%)
      1400 (34)/1316 (32)870 (28)/812 (26)255 (64)/240 (60)116 (38)/100 (33)127 (60)/137 (65)32 (41)/27 (34)
      OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      1.11 (1.011.22)1.10 (0.98–1.24)1.18 (0.88–1.57)1.26 (0.90–1.76)0.83 (0.56–1.21)1.31 (0.69–2.52)
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      HcpC+, n (%)
      1407 (35)/1325 (33)880 (29)/847 (28)250 (63)/223 (56)114 (37)/98 (32)127 (60)/128 (61)36 (46)/29 (37)
      OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      1.10 (1.001.22)1.06 (0.94–1.19)1.33 (1.00–1.77)1.27 (0.90–1.79)0.98 (0.68–1.43)1.39 (0.76–2.55)
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      CagA+, n (%)
      1139 (28)/1074 (26)645 (21)/609 (20)244 (61)/221 (55)115 (37)/95 (31)104 (49)/120 (57)31 (39)/29 (37)
      OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      1.10 (0.99–1.22)1.08 (0.95–1.23)1.28 (0.96–1.70)1.39 (0.97–1.98)0.72 (0.49–1.08)1.12 (0.58–2.15)
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      HP0231+, n (%)
      782 (19)/729 (18)552 (18)/509 (17)126 (32)/105 (26)41 (13)/36 (12)51 (24)/62 (29)12 (15)/17 (22)
      OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      1.09 (0.98–1.22)1.10 (0.97–1.26)1.27 (0.94–1.70)1.17 (0.71–1.92)0.78 (0.51–1.19)0.67 (0.30–1.48)
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      HP0305+, n (%)
      886 (22)/870 (21)565 (18)/541 (18)163 (41)/161 (40)64 (21)/58 (19)73 (35)/91 (43)21 (27)/19 (24)
      OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      1.03 (0.92–1.15)1.06 (0.93–1.21),1.02 (0.77–1.36)1.14 (0.76–1.72)0.71 (0.48–1.04)1.17 (0.54–2.52)
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      urea+, n (%)
      883 (22)/869 (21)574 (19)/577 (19)130 (33)/133 (33)74 (24)/61 (20)90 (43)/78 (37)15 (19)/20 (25)
      OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      1.02 (0.92–1.14)0.99 (0.87–1.13)0.97 (0.72–1.29)1.28 (0.87–1.89)1.27 (0.86–1.89)0.74 (0.37–1.47)
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      HyuA+, n (%)
      802 (20)/789 (19)530 (17)/533 (17)112 (28)/108 (27)57 (19)/58 (19)85 (40)/74 (35)18 (23)/16 (20)
      OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      1.02 (0.91–1.14)0.99 (0.87–1.14)1.06 (0.77–1.46)0.98 (0.66–1.46)1.26 (0.84–1.88)1.15 (0.55–2.43)
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      Cad+, n (%)
      593 (15)/582 (14)386 (13)/380 (12)119 (30)/110 (28)23 (7)/29 (9)55 (26)/55 (26)10 (13)/8 (10)
      OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      1.02 (0.90–1.16)1.02 (0.88–1.19)1.13 (0.82–1.55)0.77 (0.43–1.38)1.00 (0.64–1.57)1.25 (0.49–3.17)
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      Catalase+, n (%)
      905 (22)/910 (22)604 (20)/603 (20)145 (36)/142 (36)52 (17)/59 (19)86 (41)/88 (42)18 (23)/18 (23)
      OR (95% CI)20.99 (0.89–1.10)1.00 (0.88–1.14)1.03 (0.77–1.39)0.85 (0.56–1.30)0.96 (0.65–1.42)1.00 (0.48–2.10)
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      HpaA+, n (%)
      628 (15)/638 (16)374 (12)/383 (12)140 (35)/136 (34)47 (15)/45 (15)55 (26)/61 (29)12 (15)/13 (16)
      OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      0.98 (0.87–1.11)0.97 (0.83–1.13)1.05 (0.78–1.40)1.05 (0.68–1.63)0.87 (0.57–1.33)0.91 (0.69–2.14)
      H pylori+,
      Overall H pylori–positive (≥4 antigens positive).
      NapA+, n (%)
      785 (19)/810 (20)501 (16)/552 (18)122 (31)/115 (30)55 (18)/41 (13)89 (42)/87 (41)18 (23)/15 (19)
      OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      0.96 (0.86–1.07)0.89 (0.78–1.02)1.09 (0.80–1.47)1.39 (0.91–2.13)1.04 (0.70–1.56)1.25 (0.59–2.67)
      NOTE. Bold type indicates significant associations at P < .05.
      a Overall H pylori–positive (≥4 antigens positive).
      b Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity.
      Seropositivity to 4 individual H pylori proteins included in the multiplex serology—VacA, GroEL, Omp, and HcpC—were significantly associated with a 10%–11% increased odds of CRC in the total consortium. Again, the strength of the association differed between races/ethnicities, with the strongest association of H pylori+ VacA+ with odds of CRC found among African Americans (OR, 1.45; 95% CI, 1.08–1.95). No association of the dichotomous measure of H pylori+ VacA+ and odds of CRC was found among Asian Americans (OR, 1.20; 95% CI, 0.85-1.69), whites (OR, 1.08; 95% CI, 0.96-1.21) or Latinos (OR, 0.80; 95% CI, 0.53-1.19) (Table 2). This race/ethnicity-specific association was generally consistent across different cohorts (Figure 1), although it only reached statistical significance for African Americans in the Multiethnic Cohort Study (OR, 1.91; 95% CI, 1.19–3.20).
      Figure thumbnail gr1
      Figure 1Forest plot of seropositivity to H pylori VacA and CRC risk by race/ethnicity and study. Conditional logistic regression models were applied to determine OR (diamonds) and 95% CI (horizontal lines), controls are matched to cases by study, age, sex, and race/ethnicity. No values are given when total case numbers are <20. Weight shows the contribution of each study to the number of participants per race/ethnicity in %. The vertical line at an OR of 1 serves as a reference for null association. Pos = antibody positive.
      In analyses of a dose-response relationship of VacA antibodies in quartiles with CRC risk, we found a significant trend of rising odds of CRC with increasing antibody response to VacA among all races/ethnicities combined (P = .008) and among African Americans alone (P = .007). Among all individuals, compared to individuals not H pylori+ VacA+, those in the fourth quartile of antibody response to VacA were at a 25% greater odds of developing CRC (OR, 1.25; 95% CI, 1.07–1.47). This association was particularly strong among Asian Americans (OR, 1.86; 95% CI, 1.06–3.25) and African Americans (OR, 1.70; 95% CI, 1.12–2.58) (Table 3).
      Table 3Colorectal Cancer Risk by Strength of Antibody Response to Helicobacter pylori VacA, Among All and by Race
      VariableAllWhiteAfrican AmericanAsian American
      Cases/controls, n (%)OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity;
      Cases/controls, n (%)OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity;
      Cases/controls, n (%)OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity;
      Cases/controls, n (%)OR (95% CI)
      Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity;
      H pylori
      Overall H pylori–positive (≥4 antigens positive).
      or VacA
      2629 (65)/2713 (67)1.00 (ref)2191 (71)/2237 (73)1.00 (ref)132 (33)/166 (42)1.00 (ref)184 (60)/196 (64)1.00 (ref)
      H pylori+
      Overall H pylori–positive (≥4 antigens positive).
      VacA+ MFI Q1
      Race-specific quartile borders based on MFI distribution in controls (All: 25th percentile: 162 MFI, 50th percentile: 404 MFI, 75th percentile: 1153 MFI; white: 25th percentile: 149 MFI, 50th percentile: 354 MFI, 75th percentile: 1009 MFI; African American: 25th percentile: 199 MFI, 50th percentile: 533 MFI, 75th percentile: 1313 MFI; Asian American: 25th percentile: 165 MFI, 50th percentile: 442 MFI, 75th percentile: 1027 MFI).
      330 (8)/338 (8)1.02 (0.87–1.20)196 (6)/208 (7)0.96 (0.78–1.18)58 (15)/59 (15)1.23 (0.81–1.88)29 (10)/28 (9)1.12 (0.63–2.00)
      H pylori+
      Overall H pylori–positive (≥4 antigens positive).
      VacA+ MFI Q2
      Race-specific quartile borders based on MFI distribution in controls (All: 25th percentile: 162 MFI, 50th percentile: 404 MFI, 75th percentile: 1153 MFI; white: 25th percentile: 149 MFI, 50th percentile: 354 MFI, 75th percentile: 1009 MFI; African American: 25th percentile: 199 MFI, 50th percentile: 533 MFI, 75th percentile: 1313 MFI; Asian American: 25th percentile: 165 MFI, 50th percentile: 442 MFI, 75th percentile: 1027 MFI).
      342 (8)/337 (8)1.06 (0.90–1.25)214 (7)/207 (7)1.06 (0.87–1.30)65 (16)/58 (15)1.44 (0.94–2.21)21 (7)/28 (9)0.80 (0.43–1.49)
      H pylori+
      Overall H pylori–positive (≥4 antigens positive).
      VacA+ MFI Q3
      Race-specific quartile borders based on MFI distribution in controls (All: 25th percentile: 162 MFI, 50th percentile: 404 MFI, 75th percentile: 1153 MFI; white: 25th percentile: 149 MFI, 50th percentile: 354 MFI, 75th percentile: 1009 MFI; African American: 25th percentile: 199 MFI, 50th percentile: 533 MFI, 75th percentile: 1313 MFI; Asian American: 25th percentile: 165 MFI, 50th percentile: 442 MFI, 75th percentile: 1027 MFI).
      359 (9)/338 (8)1.11 (0.95–1.30)226 (7)/208 (7)1.11 (0.91–1.35)67 (17)/58 (15)1.48 (0.97–2.26)29 (10)/28 (9)1.12 (0.64–1.96)
      H pylori+
      Overall H pylori–positive (≥4 antigens positive).
      VacA+ MFI Q4
      Race-specific quartile borders based on MFI distribution in controls (All: 25th percentile: 162 MFI, 50th percentile: 404 MFI, 75th percentile: 1153 MFI; white: 25th percentile: 149 MFI, 50th percentile: 354 MFI, 75th percentile: 1009 MFI; African American: 25th percentile: 199 MFI, 50th percentile: 533 MFI, 75th percentile: 1313 MFI; Asian American: 25th percentile: 165 MFI, 50th percentile: 442 MFI, 75th percentile: 1027 MFI).
      403 (10)/337 (8)1.25 (1.071.47)240 (8)/207 (7)1.18 (0.97–1.44)77 (19)/58 (15)1.70 (1.122.58)44 (14)/27 (9)1.86 (1.063.25)
      P trend
      Cochrane–Armitage trend test.
      0.0080.0610.0070.107
      NOTE. Bold type indicates significant associations at P < .05.
      a Conditional logistic regression model, controls are matched to cases by age, sex, and race/ethnicity;
      b Overall H pylori–positive (≥4 antigens positive).
      c Race-specific quartile borders based on MFI distribution in controls (All: 25th percentile: 162 MFI, 50th percentile: 404 MFI, 75th percentile: 1153 MFI; white: 25th percentile: 149 MFI, 50th percentile: 354 MFI, 75th percentile: 1009 MFI; African American: 25th percentile: 199 MFI, 50th percentile: 533 MFI, 75th percentile: 1313 MFI; Asian American: 25th percentile: 165 MFI, 50th percentile: 442 MFI, 75th percentile: 1027 MFI).
      d Cochrane–Armitage trend test.

      Discussion

      In this large prospective US cohort consortium, we found no overall association of H pylori seropositivity and odds of colorectal cancer. In line with our pilot data, we did find a significant 11% increased odds of developing CRC for individuals with antibody responses to the H pylori virulence factor VacA. When assessing this association separately by race/ethnicity, we found that the association was particularly strong for African Americans, among whom odds of CRC was significantly elevated by 45% and there was a strong dose–response relationship between VacA antibody level and the odds of developing CRC. We also found a significant VacA-associated increased odds among Asian Americans, notably those in the highest quartile of VacA antibody level. These findings suggest that serologic immune responses to H pylori proteins could serve as a marker for individuals at increased risk of developing CRC, possibly due to differences in strains carried by different ethnic groups, as we will discuss further.
      Previously published findings on the potential association between H pylori and CRC have varied widely, with results seemingly dependent on the racial/ethnic makeup of the study population. For example, 4 separate US studies reported different results according to the race/ethnicity examined. As mentioned, our pilot study, in an independent nested case–control population composed of 77% African Americans within the SCCS found no association overall between H pylori seropositivity and CRC, but did find significant associations with VacA (OR, 1.84; 95% CI 1.17–2.89), HP 0231 (OR, 1.74; 95% CI, 1.14–2.66), NapA (OR, 1.67; 95% CI, 1.10–2.53), HcpC (OR, 1.66; 95% CI, 1.13–2.43), and HP 0305 (OR, 1.63; 95% CI, 1.13–2.35).
      • Epplein M.
      • Pawlita M.
      • Michel A.
      • et al.
      Helicobacter pylori protein-specific antibodies and risk of colorectal cancer.
      In a retrospective clinic-based study of African-American patients undergoing bidrectional endoscopy on the same day, individuals with H pylori identified using immunohistochemistry staining on gastric biopsies were at greater odds of also having a colorectal adenoma (OR, 1.5; 95% CI, 1.1–2.0) or colorectal polyp (OR, 1.5; 95% CI, 1.1–2.0).
      • Brim H.
      • Zahaf M.
      • Laiyemo A.O.
      • et al.
      Gastric Helicobacter pylori infection associates with an increased risk of colorectal polyps in African Americans.
      In a nested case–control study of colorectal cancer risk among primarily elderly white Americans, no overall association was found with H pylori seropositivity, although 1 antigen, GroEL, did reach significance dichotomously (OR, 1.32; 95% CI, 1.03–1.70), but not in a dose–response manner by quartile.
      • Blase J.L.
      • Campbell P.T.
      • Gapstur S.M.
      • et al.
      Prediagnostic Helicobacter pylori antibodies and colorectal cancer risk in an elderly, Caucasian population.
      And, in another retrospective clinic-based study in the United States, this time among Hispanic patients undergoing bidirectional endoscopy, no association was found between H pylori found on biopsies and presence of colorectal adenoma.
      • Patel S.
      • Lipka S.
      • Shen H.
      • et al.
      The association of H. pylori and colorectal adenoma: does it exist in the US Hispanic population?.
      In addition, Asian studies have tended to report associations, whereas those in Western European populations have not.
      • Zhao Y.
      • Wang X.
      • Wang Y.
      Helicobacter pylori infection and colorectal carcinoma risk: a meta-analysis.
      • Zumkeller N.
      • Brenner H.
      • Zwahlen M.
      • et al.
      Helicobacter pylori infection and colorectal cancer risk: a meta-analysis.
      • Park H.
      • Park J.J.
      • Park Y.M.
      • et al.
      The association between Helicobacter pylori infection and the risk of advanced colorectal neoplasia may differ according to age and cigarette smoking.
      Our findings reinforce the notion that the association of H pylori with odds of CRC was most pronounced among African Americans, and that the strongest associations were specifically for antibody responses to the H pylori protein VacA, as we did not find strong dose–response associations for the other 3 antigens found to be associated with odds of CRC dichotomously.
      H pylori VacA is a secreted toxin that causes, among other effects, vacuolation in host cells.
      • McClain M.S.
      • Beckett A.C.
      • Cover T.L.
      Helicobacter pylori vacuolating toxin and gastric cancer.
      While every H pylori strain harbors a VacA gene, the encoded proteins vary in amino acid sequences. Subtypes of VacA proteins differ in avidity of host cell binding, ability to form membrane channels, and ability to induce cell vacuolation, and are associated with different levels of gastric cancer risk.
      • Ferreira R.M.
      • Machado J.C.
      • Figueiredo C.
      Clinical relevance of Helicobacter pylori vacA and cagA genotypes in gastric carcinoma.
      Heterogeneity in these vacA variants by race/ethnicity in the United States has been explored in one study, which found that African Americans, Latinos, and Asian Americans tend to harbor the most active forms of VacA. This could explain why the association of odds of CRC with antibody responses to VacA detected in African Americans and Asian Americans was stronger as compared to that in whites; however, it would not explain the lack of association in Latinos. Expression of other virulence factors, like CagA, could be an explanation for this observation. Previously, Parsonnet et al
      • Parsonnet J.
      • Replogle M.
      • Yang S.
      • et al.
      Seroprevalence of CagA-positive strains among Helicobacter pylori-infected, healthy young adults.
      found that African Americans more often harbored CagA-positive strains than whites or Latinos. Indeed, in our study, seroprevalence to CagA among CRC cases was 61% among African Americans compared to 49% in Latinos, although overall H pylori seroprevalence was similar (71% and 74%, respectively). Consequently, the observed race/ethnicity-dependent difference in association of antibody response to H pylori with odds of developing CRC might depend on the infecting strain. Typing of the infecting strain from gastroscopy samples could help to verify this hypothesis, although such samples/information are not available for the participating studies in this consortium. Additionally, genetics of the host, including host markers of inflammation such as polymorphisms in interleukin 1β, tumor necrosis factor–α, and interleukin 10, could be considered in this complex interplay of the bacterium and host’s immune response and would add another level of information on the observed race/ethnicity-dependent differences in the association between antibodies to H pylori and odds of CRC.
      It remains unclear how H pylori, or, as suggested here, potentially more virulent strains of the species could increase risk of developing CRC. It is hypothesized that, if H pylori is causally involved in colorectal carcinogenesis, it could be mediated via direct effects. This may be particularly true for VacA, a known gastric cancer virulence factor, which may also have the potential to exert its effects beyond the stomach, such as through cellular vacuolation, interference with cellular pathways, effects on epithelial permeability, as well as immunomodulatory and pro-inflammatory effects.
      • McClain M.S.
      • Beckett A.C.
      • Cover T.L.
      Helicobacter pylori vacuolating toxin and gastric cancer.
      Precedent for the ability of selected H pylori virulence factors to exert their effects beyond the stomach have already been shown in murine models of colitis.
      • Luther J.
      • Owyang S.Y.
      • Takeuchi T.
      • et al.
      Helicobacter pylori DNA decreases pro-inflammatory cytokine production by dendritic cells and attenuates dextran sodium sulphate-induced colitis.
      In contrast to other major H pylori virulence factors, such as CagA, which requires direct contact between the bacteria and host cells, VacA may disseminate more freely beyond the gastric niche. Antigens GroEl, OMP (HP 1564), and HcpC, which were significantly associated with odds of CRC as well, are reported to be released into the extracellular space, similar to VacA.
      • Dunn B.E.
      • Vakil N.B.
      • Schneider B.G.
      • et al.
      Localization of Helicobacter pylori urease and heat shock protein in human gastric biopsies.
      • Snider C.A.
      • Voss B.J.
      • McDonald W.H.
      • et al.
      Growth phase-dependent composition of the Helicobacter pylori exoproteome.
      Their potential biological function in carcinogenesis, and the reasons why antibody responses to this group of antigens specifically were associated with CRC risk, remain to be elucidated.
      Several indirect mechanisms could be considered as well, including H pylori infection and resulting changes in the gastric microbiome potentially leading to an altered intestinal microbiome that is associated with increased CRC risk.
      • Noto J.M.
      • Peek Jr., R.M.
      The gastric microbiome, its interaction with Helicobacter pylori, and its potential role in the progression to stomach cancer.
      Furthermore, chronic gastritis as induced by H pylori leads to increased gastrin production, a peptide hormone that functions as a mitogen.
      • Konturek S.J.
      • Konturek P.C.
      • Hartwich A.
      • et al.
      Helicobacter pylori infection and gastrin and cyclooxygenase expression in gastric and colorectal malignancies.
      • Sobhani I.
      • Lehy T.
      • Laurent-Puig P.
      • et al.
      Chronic endogenous hypergastrinemia in humans: evidence for a mitogenic effect on the colonic mucosa.
      Several human studies suggest an association between gastrin and CRC risk, although the findings remain inconclusive.
      • Strofilas A.
      • Lagoudianakis E.E.
      • Seretis C.
      • et al.
      Association of helicobacter pylori infection and colon cancer.
      • Thorburn C.M.
      • Friedman G.D.
      • Dickinson C.J.
      • et al.
      Gastrin and colorectal cancer: a prospective study.
      Another potential mechanism is that H pylori–induced gastric atrophy leads to increased levels of cyclooxygenase-2 and consequently urinary prostaglandin E2, inflammation-related biomarkers that have been identified as associated with colorectal adenoma and CRC risk.
      • Kountouras J.
      • Zavos C.
      • Chatzopoulos D.
      • et al.
      New aspects of Helicobacter pylori infection involvement in gastric oncogenesis.
      • Cai Q.
      • Gao Y.T.
      • Chow W.H.
      • et al.
      Prospective study of urinary prostaglandin E2 metabolite and colorectal cancer risk.
      • Bezawada N.
      • Song M.
      • Wu K.
      • et al.
      Urinary PGE-M levels are associated with risk of colorectal adenomas and chemopreventive response to anti-inflammatory drugs.
      • Davenport J.R.
      • Cai Q.
      • Ness R.M.
      • et al.
      Evaluation of pro-inflammatory markers plasma C-reactive protein and urinary prostaglandin-E2 metabolite in colorectal adenoma risk.
      The finding of a dose–response relationship of antibody response to VacA and odds of CRC supports a connection between severity of infection, inflammation, and resulting molecular consequences with increased CRC risk. It is noteworthy that the highest MFI values were found among African Americans. Furthermore, we have found that antibody responses to H pylori were higher in active infections among a subset of African Americans in the SCCS, as determined by urea breath test (unpublished data), and decrease after eradication of the bacterium,
      • Wang T.
      • Zhang Y.
      • Su H.
      • et al.
      Helicobacter pylori antibody responses in association with eradication outcome and recurrence: a population-based intervention trial with 7.3-year follow-up in China.
      and thus could be seen as a measure for an active infection. The dose–response relationship could therefore indicate that a more pronounced infection and inflammation induced by H pylori more strongly increases the risk of developing CRC. It is possible that VacA itself may not biologically be conferring risk, but rather, due to its high level of immunogenicity, may be a more sensitive biomarker to indicate a pronounced H pylori infection.
      A limitation of this study was that potentially informative variables like recent antibiotic use, and diagnosis of gastritis as well as inflammatory bowel disease were missing for the majority of study subjects. These variables could have added information on active vs past H pylori infections and/or eradication of H pylori infection at the time of blood draw, as well as on a potential link between H pylori infection and inflammatory diseases of the bowel. In this context, it is important to note that serologic analysis of H pylori infection as a systemic measure for past and/or acute infections does not provide information on the current site-specific infection status of the individual. And, while we were able to adjust for the main potential confounders of socioeconomic status in terms of education, BMI, and smoking status, finding that although these factors were associated with H pylori status, their adjustment had little impact on the associations between H pylori VacA and odds of CRC, there was still a significant amount of missing data on potential confounders, such as family history of CRC and CRC screening, which we were not able to effectively control for. We chose not to present a multiple testing correction with these analyses based on our a priori VacA-specific findings in the pilot data in the SCCS.
      • Epplein M.
      • Pawlita M.
      • Michel A.
      • et al.
      Helicobacter pylori protein-specific antibodies and risk of colorectal cancer.
      Thus, for the VacA analyses we are comfortable using uncorrected P values. Nonetheless, we appreciate that a Bonferroni-corrected P value would be .0038 (or .05/13), and considering this criteria none of the significant associations found when dichotomizing seropositivity for the other H pylori–specific antigens would be significant. This cohort consortium, however, also has several strengths; as demonstrated, participants in this study arose from diverse populations, with different underlying risk factors for CRC, varying prevalence of H pylori subtype–specific antibodies, and different baseline risks of disease. The heterogeneity of this population allowed for the potential of greater generalizability of the findings, as well as the power to explore subanalyses among more homogeneous populations and disease types.
      In conclusion, we assessed the prospective association of a serological immune response to H pylori proteins with the odds of developing CRC in a large US cohort consortium including more than 4000 CRC cases and their matched controls. We demonstrated that strong antibody responses to H pylori virulence factor VacA were significantly associated with odds of CRC and the association appeared to vary by race/ethnicity, with particularly strong associations observed for African Americans and Asian Americans. Future studies are needed to assess whether this association is causal and, if causally related, how H pylori infection, whose natural habitat is the gastric but not colonic mucosa, mechanistically increases CRC risk. In this context, it will also be important to investigate why this association exists only in certain racial/ethnic populations.

      Acknowledgments

      The Campaign Against Cancer and Stroke thanks the participants and staff for their contributions, as well as the Maryland Cancer Registry, Center for Cancer Surveillance and Control, Department of Health and Mental Hygiene. The authors would like to thank the participants and staff of the Nurses’ Health Study and the Health Professionals Follow-up Study for their valuable contributions, as well as the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, and WY. The authors assume full responsibility for analyses and interpretation of these data. The American Cancer Society funds the creation, maintenance, and updating of the Cancer Prevention Study-II cohort. The authors express sincere appreciation to all Cancer Prevention Study-II participants, and to each member of the study and biospecimen management group. The authors would like to acknowledge the contribution from central cancer registries supported through the Centers for Disease Control and Prevention's National Program of Cancer Registries and cancer registries supported by the National Cancer Institute's Surveillance Epidemiology and End Results Program.
      Authors contributions: JB contributed through literature search, data analysis, data interpretation, and manuscript writing. MGV contributed through literature search, data interpretation, and manuscript writing. WJB, KV, GYFH, RMP, and MP contributed through study design, data collection, data interpretation, and manuscript writing. LT, LLM, CH, YC, YB, HDS, SW-S, LET, TH, CU, FG, AZ-J, AH, and contributed through data collection, data interpretation, and manuscript writing. JDP, TLC, SB, MS, and TW contributed through data interpretation and manuscript writing. LHH and L-CH contributed through data analysis and data interpretation. ME was responsible for the entirety of the project, including obtaining funding, literature search, study design, data collection, data analysis, data interpretation, and manuscript writing.

      Supplementary Material

      Supplementary Table 1Baseline Characteristics of Participants in the Hp-CRC Consortium, by Cohort
      CharacteristicCLUE (n = 982)CPSII (n = 722)HPFS (n = 302)MEC (n = 1510)NHS (n = 576)NYU WHS (n = 572)PHS (n = 360)PLCO (n = 1240)SCCS (n = 252)WHI (n = 1610)Total (n = 8126)
      n9827223021510576572360124025216108126
      Age, y
       Mean (SD)52 (12.2)71 (5.5)65 (8.7)69 (8.2)58 (6.9)55 (7.7)69 (8.2)65 (5.1)55 (8.8)66 (6.9)63 (10.1)
       Median5371666959586964556664
       Q1, Q344, 6067, 7558, 7263, 7653, 6450, 6264, 7461, 6947, 6361, 7158, 71
       Range(18–82)(57–87)(47–81)(49–87)(43–70)(36–66)(51–89)(55–75)(40–76)(50–79)(18–89)
      Sex, n (%)
       Female554 (56)368 (51)0 (0)716 (47)576 (100)572 (100)0 (0)572 (46)144 (57)1610 (100)5112 (63)
       Male428 (44)354 (49)302 (100)794 (53)0 (0)0 (0)360 (100)668 (54)108 (43)0 (0)3014 (37)
      Race, n (%)
       White974 (99)712 (99)290 (96)226 (15)576 (100)446 (78)342 (95)1098 (89)74 (29)1396 (87)6134 (75)
       Black8 (1)6 (1)0 (0)286 (19)0 (0)62 (11)2 (1)98 (8)172 (68)164 (10)798 (10)
       Asian0 (0)2 (0)0 (0)570 (38)0 (0)6 (1)12 (3)24 (2)0 (0)0 (0)614 (8)
       Latino0 (0)0 (0)0 (0)332 (22)0 (0)20 (3)4 (1)16 (1)0 (0)50 (3)422 (5)
       Other/unknown/ multiracial0 (0)2 (0)12 (4)96 (6)0 (0)38 (7)0 (0)4 (0)6 (2)0 (0)158 (2)
      Education, n (%)
       Missing, n3 (0)2 (0)0 (0)9 (1)0 (0)73 (13)0 (0)1 (0)5 (2)15 (1)108 (1)
       Less than high school399 (41)22 (3)0 (0)215 (14)0 (0)35 (6)0 (0)119 (10)99 (39)83 (5)972 (12)
       Completed high school or GED356 (36)153 (21)0 (0)343 (23)0 (0)156 (27)0 (0)308 (25)76 (30)276 (17)1668 (21)
       Post high school training other than college0 (0)58 (8)0 (0)0 (0)0 (0)9 (2)0 (0)147 (12)6 (2)142 (9)362 (4)
       Some college122 (12)180 (25)0 (0)494 (33)0 (0)120 (21)0 (0)261 (21)50 (20)445 (28)1672 (21)
       College graduate53 (5)153 (21)0 (0)261 (17)531 (92)88 (15)0 (0)218 (18)10 (4)169 (10)1483 (18)
       Graduate school49 (5)154 (21)302 (100)188 (12)45 (8)91 (16)360 (100)186 (15)6 (2)480 (30)1861 (23)
      BMI
       Missing, n9825193460229251075
       <20 kg/m2, n (%)0 (0)260 (36)104 (37)443 (29)272 (48)268 (47)116 (32)330 (27)51 (21)470 (30)2314 (33)
       20 to <25 kg/m2, n (%)0 (0)19 (3)4 (1)65 (4)18 (3)24 (4)3 (1)18 (1)10 (4)33 (2)194 (3)
       25 to <30, n (%)0 (0)311 (43)144 (51)633 (42)182 (32)189 (33)193 (54)551 (45)70 (29)579 (37)2852 (40)
       ≥30 kg/m2, n (%)0 (0)127 (18)31 (11)366 (24)100 (17)85 (15)48 (13)319 (26)112 (46)503 (32)1691 (24)
      Smoking
       Missing, n011023411052689
       Never smoker, n (%)460 (47)325 (45)125 (43)594 (39)254 (44)255 (48)155 (43)572 (46)84 (34)804 (51)3628 (45)
       Former smoker, n (%)245 (25)375 (52)154 (53)675 (45)233 (41)183 (34)183 (51)555 (45)62 (25)693 (44)3358 (42)
       Current smoker, n (%)277 (28)21 (3)13 (4)239 (16)86 (15)93 (18)21 (6)113 (9)101 (41)87 (5)1051 (13)
      Family history of CRC
       Missing, n9820017257254442521292052
       No, n (%)0 (0)609 (84)249 (82)1292 (87)495 (86)0 (0)276 (90)1063 (89)0 (0)1183 (80)5167 (85)
       Yes, n (%)0 (0)113 (16)53 (18)201 (13)79 (14)0 (0)30 (10)133 (11)0 (0)298 (20)907 (15)
      CLUE II, Campaign Against Cancer and Stroke; CPSII, Cancer Prevention Study-II; GED, General Educational Development Test; HPFS, Health Professionals Follow-up Study; MEC, Multiethnic Cohort Study; NHS, Nurses’ Health Study; NYU WHS, New York University Women’s Health Study; PHS, Physicians’ Health Study; PLCO, Prostate, Lung, Colorectal, and Ovarian Screening Study; WHI, Women’s Health Initiative.
      Supplementary Table 2Helicobacter pylori Multiplex Serology Antigens
      Systematic nameNameFull nameAccession no.
      National Center for Biotechnology Information reference sequence.
      Selected amino acidsStrainCutoff (MFI)N+C
      HP0010GroELChaperonin GroELAM_9971631–547G2768
      HP0073UreAUrease alpha subunitNP_2068731–2382669595
      HP0231HP0231Hypothetical proteinNP_2070291–2652669595
      HP0243NapANeutrophil-activating proteinNP_2070411–1442669566
      HP0305HP0305Hypothetical proteinNP_2071031–1842669555
      HP0410HpaANeuraminyllactose-binding hemagglutinin homologNP_2072081–2492669573
      HP0547CagA N-TerminusCytotoxin-associated antigen ANP_2073431–65026695654
      CagA C-Terminus641v118626695
      HP0695HyuA N-TerminusHydantoin utilization protein ANP_2072081–22026695114
      HyuA C-Terminus210–713
      HP0875CatalaseCatalaseNP_2076691–50526695107
      HP0887VacA N-TerminusVacuolating cytotoxin ANP_20768034–3632669564
      VacA C-Terminus328–100826695
      HP1098HcpCConserved hypothetical secreted proteinNP_2078891–2902669530
      HP1104CadCinnamyl alcohol dehydrogenase ELI3-2NP_2078951–3482669588
      HP1564OmpOuter membrane proteinNP_2083551–27126695137
      a National Center for Biotechnology Information reference sequence.
      Supplementary Table 3Conditional Logistic Regression Models, With Adjustment For Potential Confounders
      VariableOdds of CRC
      Unadjusted model (1)Model + education (2)Model + BMI (3)Model + smoking (4)Fully adjusted (5)
      All
       n8,1267,9426,9607,9566,696
       HP+VacA+ OR1.11
      Significant at the 5% level.
      1.10
      Significant at the 10% level.
      1.11
      Significant at the 10% level.
      1.10
      Significant at the 10% level.
      1.09
       HP+VacA+ 95% CI(1.01–1.22)(0.99–1.21)(1.00–1.23)(1.00–1.21)(0.98–1.22)
      P value.038.069.062.059.117
      Black only
       n798762748776716
       HP+VacA+ OR1.45
      Significant at the 5% level.
      1.46
      Significant at the 5% level.
      1.48
      Significant at the 5% level.
      1.41
      Significant at the 5% level.
      1.48
      Significant at the 5% level.
       HP+VacA+ 95% CI(1.08–1.95)(1.08–1.98)(1.09–2.02)(1.05–1.90)(1.08–2.03)
      P value.013.014.011.023.015
      a Significant at the 5% level.
      b Significant at the 10% level.

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      Linked Article

      • Colon Cancer Risk and VacA Toxin of Helicobacter pylori
        GastroenterologyVol. 156Issue 8
        • Preview
          Butt et al1 observed an increased risk of developing colon rectal cancer in individuals possessing circulating antibodies to the vacuolating toxin (VacA) of Helicobacter pylori. Such a risk was particularly high for African Americans.1 Indeed, VacA forms chloride (Cl-) channels that become inserted into the cell and mitochondrial membranes,2 thereby reducing the membrane potential and mitochondrial energy production. The paradigm of altered Cl- handling is cystic fibrosis, and it is well-known that patients with this disease have a 5- to 10-fold increased risk of colon rectal cancer,3 as well of other gastrointestinal tract cancers,4 owing to the irregular activity of Cl- exchange by the cystic fibrosis transmembrane regulator.
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