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Keywords:

  • Helicobacter pylori;
  • stomach cancer;
  • epidemiology;
  • pepsinogen;
  • chronic atrophic gastritis

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Helicobacter pylori (H. pylori), atrophic gastritis, dietary and life-style factors have been associated with gastric cancer (GC). These factors have been evaluated in a large case–control study nested in the European Prospective Investigation into Cancer and Nutrition carried out in 9 countries, including the Mediterranean area. Participants, enrolled in 1992–1998, provided life-style and dietary information and a blood sample (360,000; mean follow-up: 6.1 years). For 233 GC cases diagnosed after enrolment and their 910 controls individually-matched by center, gender, age and blood donation date H. pylori antibodies (antilysate and antiCagA) and plasma Pepsinogen A (PGA) were measured by ELISA methods. Severe chronic atrophic gastritis (SCAG) was defined as PGA circulating levels <22 μg/l. Overall, in a conditional logistic regression analysis adjusted for education, smoke, weight and consumption of total vegetables, fruit, red and preserved meat, H. pylori seropositivity was associated with GC risk. Subjects showing only antibodies anti-H. pylori lysate, however, were not at increased risk, while those with antiCagA antibodies had a 3.4-fold increased risk. Overall, the odds ratio associated with SCAG was 3.3 (95% CI 2.2–5.2). According to site, the risk of noncardia GC associated with CagA seropositivity showed a further increase (OR 6.5; 95% CI 3.3–12.6); on the other hand, a ten-fold increased risk of cardia GC was associated with SCAG (OR 11.0; 95% CI 3.0–40.9). These results support the causal relationship between H. pylori CagA+ strains infection, and GC in these European populations even after taking into account dietary habits. This association was limited to distal GC, while serologically defined SCAG was strongly associated with cardia GC, thus suggesting a divergent risk pattern for these 2 sites. © 2006 Wiley-Liss, Inc.

Despite a dramatic decline in incidence, gastric cancer (GC) remains the fourth more common cancer and the second lethal neoplasm worldwide.1 Nutritional, infectious, and genetic factors have been shown to play a role in gastric carcinogenesis.2, 3, 4 The relationship between Helicobacter pylori (H. pylori) infection and GC has been clarified in the first decade after its identification.5H. pylori has been classified by the International Agency for Research on Cancer (IARC) as a definite carcinogen to humans (Group 1).3H. pylori infection induces a chronic inflammation of the gastric mucosa that is intensified by the host inflammatory immune response with high levels of several cytokines. This chronic process leads, in a minority of infected individuals, to the development of GC through a series of intermediate progressive stages including mild and severe chronic gastritis, atrophic gastritis, gastric atrophy, characterized by hypochlorhydria, and intestinal metaplasia.6, 7, 8 Recent meta-analyses have estimated that the infection increases the GC risk by 2- to 3-fold9, 10, 11 with higher estimates for noncardia GC.

Up to 60% of the general population in most European areas is infected with H. pylori12 and 90–100% in less developed countries. It is clear that GC occurs only in a minority of infected individuals. Different outcomes of the infection have been related to variations of H. pylori pathogenicity, host susceptibility, environmental and life-style factors (particularly diet and age at infection) and interactions among all these factors.13 There is evidence that different strains of H. pylori may play a role in gastric carcinogenesis and subjects infected with cytotoxin CagA positive H. pylori strains (H. pylori CagA+) are at increased risk to develop GC in comparison with subjects infected with H. pylori CagA− strains.14, 15, 16 Also, only CagA positive strains were able to induce GC in experimental infected Mongolian gerbils.17 In a recent meta-analysis based on 16 case–control studies with age- and sex-matched controls, the infection with CagA positive strains was associated with an ∼2-fold increased risk of noncardia GC among H. pylori-infected subjects.18 This could be related to the active injection of CagA into the gastric epithelial cell through the Type IV secretion apparatus encoded by the cag pathogenicity island.19 The tyrosine phosphorylation of CagA and its interaction with membrane proteins would trigger a cascade of signal transductions leading to the induction of chronic inflammatory responses (e.g. through the production of IL-8) that would in turn favor the neoplastic transformation.20, 21, 22 Recently, it has also been reported that CagA is able to induce a transition from polarized epithelial cells (showing cell–cell adhesion) to a more invasive phenotype.23

Severe chronic atrophic gastritis (SCAG) is considered as a predisposing factor for GC, particularly of the intestinal type. Pepsinogen is secreted as 2 biochemical distinct groups of isoenzymes: Pepsinogen A (PGA) and Pepsinogen C. Pepsinogen C is secreted by corpus chief cells, antral glands and duodenal bulb, and PGA is secreted only by the chief cells of the gastric corpus and the fundus, and its serum level decreases with an increasing grade of corpus atrophy.24, 25 Traditionally, low levels of PGA have been used to identify SCAG in the frame of epidemiological projects or “screening” programs.26, 27, 28H. pylori infection is recognized as the main determinant of this precancerous lesion, although once that SCAG is established, the bacterium is less likely to survive and antibodies may be lost over time.

Our large case–control study nested in a cohort of 360,000 adults from several European countries (including the Mediterranean area), all with a blood sample donated at enrolment, offers the opportunity to evaluate the association between H. pylori infection and GC risk in a prospective design taking into account the effect of serologically defined SCAG and the role of dietary and other environmental factors in gastric carcinogenesis.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The EPIC cohort

EPIC is a multicenter prospective study coordinated by the IARC (International Agency on Research on Cancer, Lyon, France) aimed to study the association between diet and life-style habits and cancer, based on healthy adults who voluntarily agreed to participate in the study and to have their health status followed up. The enrolment started in 1992–1993 and was completed in 1998 in 23 collaborating centers in 10 European countries (Sweden, Denmark, Norway, Netherlands, UK, France, Germany, Spain, Italy and Greece). Overall ∼520,000 subjects (aged 35–74) of both genders have been recruited and blood samples at enrolment were obtained from over 380,000 subjects.

The study design has been reported in full elsewhere.29 Briefly, the volunteers were mostly enrolled from the general population residing in a specific area. Exceptions were the French cohort, based on female members of a national health insurance for school teachers and the Utrecht cohort (Netherlands), based on women attending the local breast cancer screening program. The 5 Spanish cohorts and 2 Italian cohorts (Ragusa and Turin) were mostly based on blood donors; most of the Oxford cohort in the UK was based on vegetarian and health conscious volunteers recruited across the whole country.

Detailed information on the consumption of a series of foods and drinks has been collected by country-specific food frequency questionnaires, validated in a pilot phase. For calibrating dietary measurements across countries and different questionnaires and to correct for systematic over- or under-estimation of dietary intakes,30 a 24-hr dietary recall has been obtained from a random sample (8%) of the cohort (36,994 participants).31 Detailed information has also been collected on education and other socioeconomic variables, smoking and alcohol drinking history, physical activity, reproductive history and medical history by a common life-style questionnaire developed in 2 separate versions for males and females. Anthropometric measurements (including weight and height) have been collected according to a standard protocol.

The EPIC cohort has been followed up since inception through population based cancer registries or other available sources as pathology hospital registries, health insurance records and active follow-up through participants and their next-of-kin. Mortality data were also obtained from mortality register at the regional or national level.

Design of the nested case–control study (EPIC-EURGAST)

GC cases

All newly diagnosed GC cases (ICD 10 C.16) identified in 9 national cohorts (except Norway, for a total of ∼225,000 women and 135,000 men) after the date of recruitment until the end of follow-up (December 1999 or September 2002 depending on the Centre), for which a blood sample was available, were included. All cases were histologically confirmed as adenocarcinomas. GC incidence rates in the different cohorts agreed with rates provided by local registries (Ferrari P, manuscript in preparation) and the male/female ratio was 2:1 as expected.

Matched controls

For each case, 4 controls, individually matched by centre, gender, age (±2.5 years) and blood donation date (±45 days), were randomly selected among those still at risk of GC (at the time of diagnosis of each case).

Blood samples

All individual available plasma samples were retrieved from the collaborative Biobank of the EPIC project (IARC, Lyon) or from local banks in Denmark and Sweden, aliquoted and shipped on dry ice to the study laboratories. Assays were performed blindly regarding the case–control status or any individual information.

Laboratory assays

Quantitation of anti-H. pylori and antiCagA IgG anti-bodies

For quantitation of anti-H. pylori IgG antibodies, ninety-six-well, flat-bottomed microtiter plates (Nunc, Roskilde, Denmark) were coated with H. pylori lysate (CCUG strain) prepared as already described in detail elsewhere.32 Hundred microliters of lysate at 1 μg/ml in PBS pH 7.4 were used and coating was allowed to proceed for 2 hr at room temperature. After 3 washes with PBS containing 0.05% (v/v) Tween-20, the plates were incubated with 250 μl/well of a solution of polyvinylpyrrolidone-15 (Serva, Heidelberg, Germany) for 1 hr at room temperature. After further washes, doubling dilutions of plasma samples (starting from 1:200), diluted in PBS-Tween-20 containing 2% (w/v) BSA (Sigma Chemical, St. Louis, MO) were added to the plates and incubation was allowed to proceed for 1 hr at room temperature. After extensive washing with PBS-Tween-20, bound IgG antibodies were quantified by the addition of an alkaline phosphatase-conjugated polyclonal affinity-purified goat antihuman IgG (Sigma Chemical), diluted 1:10,000 in PBS-Tween-20-BSA and incubated for 3 hr at 30°C. The plates were then washed, and 100 μl of p-nitrophenylphosphate (Sigma Chemical) at 1 mg/ml in diethanolamine buffer were added to each well. The chromogenic reaction was allowed to proceed for 1 hr in the dark at room temperature and was then stopped by the addition of 4 N NaOH. Optical densities were read at 405 and 650 nm. Antigen-specific IgG antibody titres were expressed as ELISA Units (EU), and were determined by interpolation relative to a standard curve constructed by a serial dilution of a standard positive control, which consisted of a pool of samples from subjects known to be positive for H. pylori infection and having antibodies as determined by Western blotting analyses. A cut-off value of 100 EU was defined using samples from individuals negative for H. pylori infection as determined by urea breath test, and by microbiological and other serological assays (Western blotting). Plasma samples giving EU values above 100 were considered as positive for anti-H. pylori IgG antibodies. In previous experiments, this assay exhibited specificity and sensitivity higher than 90% (Berti D and Del Giudice G, unpublished data).

For detection and quantitation of antiCagA IgG antibodies, ninety-six-well, flat-bottomed microtiter plates (Nunc) were coated with 100 μl of full-length recombinant CagA (lot 010101-CAG07/28) at the concentration of 0.9 μg/ml in PBS pH 7.4. Coating was allowed to proceed for 1.5 hr at 30°C. After 3 washes with PBS containing 0.05% (v/v) Tween-20, the plates were incubated with 250 μl/well of a solution of polyvinylpyrrolidone-15 (Serva) for 1.5 hr at room temperature. After further washes, doubling dilutions of plasma samples (starting from 1:400), diluted in PBS-Tween-20 containing 2% (w/v) BSA (Sigma Chemical) were added to the plates. Antibodies were allowed to react with the solid-phase antigens for 1 hr at 37°C. After extensive washing with PBS-Tween-20, bound IgG antibodies were quantified by the addition of an alkaline phosphatase-conjugated polyclonal affinity-purified goat antihuman IgG (Sigma Chemical), diluted 1:2,000 in PBS-Tween-20-BSA and incubated for 3 hr at room temperature. The plates were then washed, and 100 μl of p-nitrophenylphosphate (Sigma Chemical) at 1 mg/ml in diethanolamine buffer were added to each well. The chromogenic reaction was allowed to proceed for 40 min in the dark at room temperature and was then stopped by the addition of 4 N NaOH. Optical densities were read at 405 and 650 nm. Antigen-specific IgG antibody titres were expressed as ELISA Units (EU), and were determined by interpolation relative to a standard curve constructed by a serial dilution of a standard positive control. A cut-off value of 30 EU was defined using samples from individuals negative for H. pylori infection as determined by urea breath test, and by microbiological and by other serological assays (Western blotting). Plasma samples giving EU values above 30 were considered as positive for antiCagA IgG antibodies.

Determination of plasma pepsinogen A

Pepsinogen A (PGA) was assayed in the plasma samples by means of microplate-based quantitative enzyme immunoassay (ELISA) using a commercial kits (Biohit, Helsinki, Finland). The assay is based on a sandwich enzyme immunoassay technique with PGA-specific capture monoclonal antibodies adsorbed on a microwell plate and detection antibodies labeled with horseradish peroxidase. The concentrations of PGA in unknown samples were calculated on a calibration curve, made by using 3 calibrators (human serum samples with lot-specific PGA values) provided by the manufacturer. The coefficients of variations (intra- and inter-assay) did not exceed 5%. The reference range, calculated on a large series of control subjects, was 22–82 μg/l.24

Gastric cancer case definition

A panel of experienced pathologists met to re-evaluate GC diagnoses based on the material collected in each center for all GC identified. Original diagnostic reports were obtained from local pathology departments, translated and reviewed together with available H&E-stained slides. The Lauren classification was used for the histological type of GC. Briefly, among 233 GC cases, 91 (39.0%) were classified as intestinal adenocarcinoma, 89 (38.2%) as diffuse adenocarcinoma, 35 (15.0%) as adenocarcinoma NOS, 12 (5.2%) were unclassified adenocarcinomas, 2 (0.9%) were mixed adenocarcinomas and 4 (1.7%) gastric stumps.

According to the site within the stomach, 127 (54.5%) cases were classified as noncardia, 54 (23.2%) as cardia and 4 were considered mixed; for 44 cases the site was undetermined. An additional group of 16 cases were classified into a specific sub-group (gastro-oesophageal junction) but were not included in the main analyses.

Overall, the presence of antibodies to H. pylori lysate and full length recombinant CagA have been assessed for 233 cases and their 910 matched controls. Most serological assays were carried out for all the eligible 4 matched controls; for 12 cases, however, results were available only for 3 matched controls, 2 cases had only 2 matched controls and 2 cases had only 1 matched control. PGA measurements, on the other hand, were available for 231 cases and their 901 matched controls.

Statistical analysis

Overall H. pylori seropositivity was defined as the presence of at least 1 type of 2 specific antibodies (anti-H. pylori lysate and antiCagA IG antibodies); the latter results were used to create additional variables, as 1 term for each specific antibody or specific combinations in order to identify mutually exclusive subgroups among seropositive subjects (H. pylori seropositive but CagA negative vs. CagA positive). In addition, SCAG was serologically defined as a PGA circulating level lower than 22 μg/l.

Odds ratios (OR) for the association of H. pylori seropositivity or specific antiCagA antibodies with GC were calculated by multiple conditional logistic regression models. Confidence intervals (95% CI) were computed using the standard errors of the pertinent regression coefficients. The effects of additional potential confounders (other than the matching factors controlled for by design) like tobacco smoking, socio-economic level as defined by educational level, were examined by including in the logistic regression models terms for education (low/high), smoking history (current-/ex-/never-smoker) and weight. Adjustments for dietary variables were also performed adding to the models terms for selected food groups that were considered as risk or protective factors (red and preserved meat; total vegetables and fruit), using the predicted (calibrated) values for each individual on a continuous scale.30 The effects of H. pylori infection, CagA seropositivity and SCAG status were also evaluated adding the terms for H. pylori seropositivity (or CagA seropositivity) and for presence/absence of SCAG in the same model. Separate analyses were carried out according to gender, age classes (<60 years and ≥60 years), geographical area (countries in Central-Northern and Southern Europe), site of the tumor (cardia and non-cardia) and histotypes according to Lauren classification (intestinal and diffuse type). For the 2 latter sub-group analyses, the 2 main categories did not add to the total because of unclassified GC cases. The 2 broad geographical areas were defined a priori. We tested also a series of interactions between H. pylori seropositivity, CagA seropositivity and SCAG status and age and geographical area. All analyses were performed using the SAS statistical software (SAS/STAT version 9.1.3); a p-value <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

After an average follow-up of 6.1 years, 233 GC cases were identified (127 males) and with results available from laboratory assays for the analyses; among them, 54 (36 males) were classified as carcinomas of the gastric cardia. Eighty-two cases were identified in the 3 Mediterranean cohorts (Italy, Spain and Greece) and 151 in the 6 Central-Northern Europe cohorts (France, UK, The Netherlands, Germany, Sweden and Denmark) (Table I).

Table I. Distribution of 233 Gastric Cancer Cases and 910 Matched Controls, Prevalence OF H.pylori Seropositivity, Seroprevalence of Antibodies Anti-CagA and Prevalence of Severe Chronic Atrophic Gastritis (SCAG) (Serologically Defined, Pepsinogen A <22 μg/l) at Enrolment, According to Gender, Age, Country And Broad Geographical Area (EPIC-Eurgast 1993–2002)
 GC Cases (n)Controls (n)H. pylori1 seroprevalenceH. pylori CagA seroprevalenceSevere chronic atrophic gastritis
CasesControlsCasesControlsCasesControls
n%n%n%n%n%n%
  • 1

    Defined as presence of antibodies anti-H. pylori lysate and antiCagA.

Gender
 Males12749010481.933368.09070.923447.82318.3387.8
 Females1064209185.929269.57469.818644.32624.8358.4
Age
 <60 years1134259079.727163.88070.817841.91311.7317.4
 ≥60 years12048510587.535473.08470.024249.93630.0428.8
Mediterranean countries
 Italy431704093.010863.53172.16940.61125.61911.5
 Spain271072696.39689.72281.55955.1622.21110.3
 Greece12481191.74593.81083.33879.218.336.4
 Total823257793.924976.66376.816651.11822.03310.3
Central-Northern Europe
 France312266.7433.3266.718.3
 UK19751473.74560.01368.43242.7631.679.3
 The Netherlands19761684.25268.41157.93039.5421.122.7
 Germany311242787.17774.02271.04838.7723.3118.9
 Sweden552134785.515170.94581.812257.31324.1146.6
 Denmark24851250.04755.3833.32124.714.267.1
 Total15158511878.237664.310166.925443.43120.8406.9
Total23391019583.762568.716470.442046.24921.2738.1

H. pylori seropositivity

Overall, the prevalence of H. pylori seropositivity was 83.7% (195/233) among cases and 68.7 (625/910) among their matched controls, being higher among subjects older than 60 years and in Mediterranean countries (where 93.9% of cases and 76.6% of controls were seropositive).

The prevalence of specific antiCagA antibodies was 70.4% (164/233) among cases and 46.2% (420/910) among controls. The CagA seroprevalence tended to be higher in older controls and in those from Mediterranean cohorts (Table I). A small group of study subjects showed detectable levels of antiCagA antibodies (1.7 and 3.9% respectively, among cases and controls), while their specific antilysate antibodies were below the threshold value. Overall, the proportion of H. pylori seropositive subjects showing antiCagA antibodies was 84.1% (164/195) among cases and 67.2% (420/625) among controls with limited variation across the national cohorts.

The prevalence of H. pylori and CagA seropositivity among cardia GC cases was 64.8% (35/54) and 46.3% (25/54), and among noncardia GC cases 90.6% (115/127) and 78.7% (100/127), respectively.

Serologically defined SCAG

The prevalence of serologically defined SCAG was 21.2% among cases (49/231) and 8.1% (73/901) among controls (Table I). Again, some differences were evident among controls across different countries, with a higher prevalence in the Mediterranean cohorts. On the other hand, the prevalence of SCAG among GC cases was very similar in the 2 broad geographical areas. When SCAG prevalence was evaluated according to the presence of anti-H. pylori antibodies, we found that SCAG prevalence was 23.3% (45/193) and 10.5% (4/38) respectively, among H. pylori seropositive and seronegative GC cases (p = 0.08). SCAG prevalence was 9.1% (56/616) among H. pylori seropositive controls and 6.0% (17/285) among H. pylori negative controls (p = 0.11). According to antiCagA status, SCAG prevalence was 23.5% (38/162) and 15.9% (11/69) respectively among CagA seropositive and seronegative GC cases (p = 0.20), and 9.4% (39/414) and 7.0% (34/487) respectively among CagA seropositive and seronegative controls (p = 0.22).

H. pylori lysate and CagA seropositivity and GC risk

Overall, in a conditional multivariate model taking into account terms for education, smoking history, weight, and calibrated average daily consumption of total vegetables, total fruit, red and preserved meat, H. pylori seropositivity was associated with an approximate 2.5-fold risk of developing gastric cancer (OR 2.6; 95% CI 1.7–3.9) (Table II).

Table II. Association of H. pylorI Seropositivity, and Serologically Determined Severe Chronic Atrophic Gastritis (SCAG) with GC Risk Based on Separate Models: Overall, by Site (Noncardia and Cardia) and by Histotype (Diffuse and Intestinal). Nested Case–Control Study in the EPIC Cohorts (Each Matched Set with 1 GC Case and Age, Sex, Blood Donation Date and Center Matched Controls) (EPIC-Eurgast 1993–2002)
Variable(s) of interest in each separate modelGC(n = 233)Noncardia(n = 127)Cardia(n = 54)Diffuse type(n = 89)Intestinal type(n = 91)
OR195% CI1OR195% CI1OR195% CI1OR195% CI1OR195% CI1
  • 1

    Odds ratios (OR) and 95% confidence intervals (CI) estimated by a series of conditional logistic models including terms for education (low/high), smoking history (never-/ex-/current-smoker), weight (in continuous), total vegetables, fruit, red and preserved meat (calibrated values, in continuous) and the variable(s) of interest as indicated in each panel.

Antibodies anti-H.pylori (yes/no)2.61.7–3.94.72.5–9.00.80.4–1.84.21.9–9.22.61.4–4.9
Only antibodies anti-H.pylori lysate (yes/no)1.20.7–2.01.60.7–3.80.80.3–2.12.00.8–5.21.10.5–2.8
Antibodies antiCagA (yes/no)3.42.2–5.26.53.3–12.60.80.4–1.95.82.5–13.33.21.7–6.1
SCAG (yes/no)3.42.2–5.22.41.3–4.511.03.0–40.91.60.7–3.54.82.3–10.0
Antibodies anti-H.pylori (yes/no)2.61.7–3.95.12.6–9.80.70.3–1.64.11.8–9.12.51.3–4.7
SCAG (yes/no)3.32.1–5.22.71.5–5.111.93.1–45.61.70.7–3.84.52.2–9.4
Antibodies antiCagA (yes/no)3.12.2–4.45.43.2–9.10.90.5–2.04.02.2–7.42.81.6–4.7
SCAG (yes/no)3.32.1–5.22.61.3–4.911.02.9–40.71.70.7–3.94.22.0–9.1

When we included in the same model a specific term for those subjects showing only antilysate antibodies (that is H. pylori lysate seropositive but CagA seronegative subjects) and 1 for those showing antiCagA antibodies (independently from the antilysate antibodies status), results showed that the first group was not at increased risk, while CagA seropositive subjects showed an over 3-fold increased risk (OR 3.4; 95% CI 2.2–5.2).

When we restricted the analyses to noncardia GC (n = 127), the association with overall H. pylori seropositivity was more evident; on the other hand, a 6-fold increased risk emerged with CagA seropositivity (OR 6.5; 95% CI 3.3–12.6).

In the sub-group of cardia GC (n = 54) no association with H. pylori seropositivity was found overall and when H. pylori lysate and CagA were included separately.

In analyses carried out according to gastric cancer histotypes, H. pylori seropositivity was positively associated with both diffuse and intestinal type. When we included separately the terms for H. pylori lysate and CagA seropositivity in the model, the association with CagA seropositivity tended to be stronger for the diffuse than for the intestinal type.

No differences in the association between GC risk and H. pylori seropositivity emerged according to gender and age (Fig. 1). The effect of CagA tended to be stronger in younger subjects (Fig. 2).

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Figure 1. Estimates of gastric cancer risk associated with H. pylori seropositivity (antibodies anti-H. pylori lysate and antiCagA) overall and by sex, age group, area, site and histological type (EPIC-EURGAST 1993–2002). Odds ratios (OR) and 95% confidence intervals (CI) estimated by a series of conditional logistic models including terms for education (low/high), smoking history (never-/ex-/current-smoker) weight (in continuous), total vegetables, fruit, red and preserved meat (calibrated values, in continuous).

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thumbnail image

Figure 2. Estimates of gastric cancer risk associated with H. pylori CagA seropositivity (antibodies antiCagA) overall and by sex, area, age group, site and histological type (EPIC-EURGAST 1993–2002). Odds ratios (OR) and 95% confidence intervals (CI) estimated by a series of conditional logistic models including terms for education (low/high), smoking history (never-/ex-/current-smoker) weight (in continuous) total vegetables, fruit, red and preserved meat (calibrated values, in continuous) and the terms for H. pylori lysate seropositivity (yes/no) and CagA seropositivity (yes/no).

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In the Mediterranean cohorts, H. pylori seropositivity was overall associated with a 7.4-fold increase in GC risk; in the Central-Northern European cohorts the increase in GC risk among seropositive subjects was limited approximately to a 2-fold increase (Fig. 1).

When we considered the specific effects of H. pylori lysate and CagA seropositivity by geographical area, the association with GC risk appeared to be limited to antiCagA antibodies in the Central-Northern cohorts. In the Mediterranean cohorts, both H. pylori lysate and CagA seropositivity were associated with GC risk, respectively a 4-fold (OR 4.0; 95% CI 1.2–13.2, data not shown) and a 9-fold increase in comparison to seronegative subjects (Fig. 2).

Furthermore, specific models were developed to evaluate the effect of antiCagA antibodies among all H. pylori seropositive subjects; the results confirmed in the overall GC series a strongly increased risk for subjects with antiCagA antibodies in respect to H. pylori seropositive but CagA negative subjects (OR 3.0; 95% CI 1.9–4.6). This effect was particularly evident in younger subjects (OR 6.2; 95% CI 2.7–13.8).

Serologically defined SCAG and GC risk

A positive association between SCAG and gastric cancer risk was evident in the whole series (OR 3.4; 95% CI 2.2–5.2). This did not materially change when the term for H. pylori seropositivity was added to the model (Table II). Overall, the association with SCAG tended to be more evident in older subjects, while no differences emerged by gender (Fig. 3).

thumbnail image

Figure 3. Estimates of gastric cancer risk associated with serologically defined severe chronic atrophic gastritis (pepsinogen A <22 μg/l) overall and by sex, area, age group, site and histological type (EPIC-EURGAST 1993–2002). Odds ratios (OR) and 95% confidence intervals (CI) estimated by a series of conditional logistic models including terms for education (low/high), smoking history (never-/ex-/current-smoker) weight (in continuous) total vegetables, fruit, red and preserved meat (calibrated values, in continuous).

Download figure to PowerPoint

In the Central-Northern European cohorts, the strongest association with GC risk was actually shown by serologically defined SCAG (OR 4.2; 95% CI 2.3–7.7), that persisted substantially unchanged also in analyses taking into account CagA seropositivity. In the Mediterranean cohorts, the association with SCAG tended to be weaker (Fig. 3).

The risk of noncardia GC associated with SCAG was 2.4-fold increased (Table II). The results did not change when the other 48 GC cases of undetermined or mixed site were also considered in this category.

On the other hand, the presence of SCAG was associated with a ten-fold increased cardia GC risk (OR 11.0; 95% CI 3.0–40.9). This association persisted also when the separate terms for antilysate and antiCagA antibodies were added to the model (Table II).

According to histological subtypes, the association between GC risk and SCAG was statistically significant only for the intestinal type (Table II).

Interactions

Statistically significant interactions in modifying the risk to develop GC were found between 2 pairs of variables: H. pylori overall seropositivity and being enrolled in a Mediterranean cohort (p = 0.04) as well as the presence of a serologically defined SCAG and being older than 60 years old at enrolment (p = 0.015). When we examined the association of noncardia GC risk in the 2 different areas, these differences were much less evident, probably due to the lower proportion of cardia cases in the Mediterranean countries (12% in the Mediterranean area vs. 29% in Northern Central Europe). Also interactions between the 2 areas and specific antibodies (only antilysate vs. antiCagA) did not reach the level of statistical significance.

All analyses have also been repeated excluding GC cases diagnosed in the first year after the date of enrolment (n = 32), but results did not change.

We also repeated the analyses in 2 separate time periods (below and above the median latency between enrolment and diagnosis, ∼3 years), but the results were quite similar with no suggestion of further risk increase with increasing duration of follow-up (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this nested case–control study carried out in the frame of a specific project within the European Prospective Investigation into Cancer and Nutrition (EPIC-EURGAST), we have found that the presence of specific antibodies against H. pylori CagA was associated with an over 3-fold increased risk of developing gastric cancer. Overall, GC risk did not appear to be increased for subjects showing only antibodies against H. pylori lysate. These results were based on an average follow-up period of 6 years and were obtained taking into account the dietary habits reported at enrolment. These results were even more evident in the subgroup of noncardia GC cases, while, on the other hand, there was no evidence of any association between cardia GC risk and H. pylori or CagA seropositivity.

In addition, the presence of a serologically defined severe chronic atrophic gastritis (Pepsinogen A serum level lower than 22 μg/l) was overall associated with an increased GC risk of more than 3-fold, particularly among older individuals. This association persisted also after adjustment for H. pylori or CagA seropositivity. A specific and strong association of SCAG with GC of the cardia emerged, thus suggesting a possible divergent risk pattern for distal and cardia GC.

These results have been obtained taking into account a series of dietary determinants as consumption of total vegetables, fruit, red meat and preserved meat33, 34 in a large study population including also for the first time cohorts from Mediterranean countries.

The risk in H. pylori positive subjects we have found is quite similar to the recent estimate by a pooled analysis of 12 nested case–control studies within prospective cohorts9 and slightly higher than estimates from meta-analyses considering both case–control and cohort studies limited to studies in which H. pylori infection was determined by serological assays11 or more comprehensive.10

Several studies, both in animals and in humans, have suggested that the infection with H. pylori CagA positive strains increases the risk of GC over the risk associated with H. pylori infection alone and experimental studies have suggested the molecular basis for the pathological action of CagA.19, 22 Our result of an over 3-fold increased risk of GC associated with the presence of antiCagA antibodies in the present nested case–control study is consistent with the results of a recent meta-analysis addressing this issue.18 Our data showed very clearly that the risk of developing GC among H. pylori seropositive subjects was mostly related to an infection with H. pylori CagA+ strains.

A statistically significant association with the presence of anti-H. pylori lysate antibodies was evident only in a sub-group analysis for the Mediterranean cohorts, although a specific interaction did not emerge. Possible explanations for this finding (apart from chance) might be that in our cohorts enrolled in the Mediterranean countries the seroprevalence of H. pylori infection at baseline (1993–1998) appeared higher than in Northern-Central European cohorts, suggesting that the circulation of the bacterium might have been higher until recently. Our test to detect antibodies to H. pylori lysate was based on crude antigens from a specific strain (in contrast to the antiCagA test based on a full-length recombinant molecule) and this might also contribute to some variation in the ability to detect titres in different populations exposed to other strains.

As expected by the more frequently distal (noncardia) localization of the gastric pathology induced by the infection with H. pylori, when we considered separately cardia and noncardia GC cases, it was evident that the association between H. pylori CagA seropositivity and GC risk was confined to noncardia GC cases with a 6-fold increased risk.

On the other hand, the presence of serologically defined SCAG was overall associated with a 3-fold increased risk of GC. PGA is a marker of corpus atrophy and SCAG tended to be more clearly associated with GC in Northern Europe than in the Mediterranean countries. This may suggest that factors related to Pernicious anemia might play a role in noncardia GC associated with H. pylori infection.35

Our result of a strong association between serologically defined SCAG and adenocarcinoma of the gastric cardia is in agreement with the results of a recently published population-based case–control study carried out in Sweden.36 In the same study, no association was evident between cardia GC and H. pylori seropositivity. A number of studies reported a lack of association or a negative association between H. pylori and cardia GC37, 38, 39 with a few exceptions.40 Such association was not observed in a meta-analysis based on 42 studies (both case–control and cohort studies)10 and in a recent study with an extended follow-up period.41 In general, however, the anatomical site of cardia remains poorly defined and a number of studies may have been unable to differentiate between anatomical regions. In our study, all diagnoses have been revised by a panel of pathologists; in addition, we also performed an analysis taking into account 16 cases that had been defined as adenocarcinomas of the GEJ and results were weaker based on this less clear-cut definition of cardia GC. A complex interplay between gastro-oesophageal reflux disease and intestinal metaplasia has been recently postulated for cardia cancer42; our results would support a role for cardia metaplasia, a long term consequence of H. pylori infection possibly leading, over time and in presence of SCAG, to a reduction of antibody titres and sero-reversion in a proportion of subjects.

The different proportion of cardia and noncardia GC could contribute to explain the geographical differences in the estimates of the association between H. pylori seropositivity and GC that resulted higher in Mediterranean countries, where the proportion of cardia cases was lower than in Northern-Central Europe. A difference in the proportion of cardia and noncardia GC cases was also evident among subjects of different ages.

Diffuse gastric adenocarcinoma appeared to be more associated with H. pylori CagA seropositivity than the intestinal type, while the association with SCAG appeared more evident for the latter histological type. This stronger association between H. pylori seropositivity and diffuse GC was confirmed when we restricted the analysis to noncardia GC cases. On the other hand, the strong association between SCAG and intestinal adenocarcinoma persisted, even if less evident, in the sub-group of noncardia carcinomas, while in the cardia cases, no diffuse adenocarcinoma case occurred among subjects with serologically defined SCAG.

Our study design had several advantages. Its prospective nature implicates that blood samples used for H. pylori serology and Pepsinogen A determination were collected well before the development of the disease, thus reducing the misclassification due to the loss of anti-H. pylori antibodies once SCAG is established. A relevant point in assessing the magnitude of the association between H. pylori and GC is the possible attenuation of risk when H. pylori infection is assessed closer to the time of cancer diagnosis.9 We evaluated the association between GC and H. pylori seropositivity according to length of follow-up (below and above 3 years of follow-up) and results did not suggest differences in risk, although one has to consider that our follow-up is relatively short. An accurate ascertainment of disease through cancer registries and clinical records has been performed, and all cases were histologically confirmed. Dietary and life-style variables, used as adjusting variables, were collected through validated questionnaires and calibration of dietary variables allowed to reduce the effect of systematic over- or under-estimation of dietary intakes. The sample size of our study, however, was relatively modest and most subgroup analyses had limited power, showing overlapping results.

In conclusion, our results confirm a causal role of H. pylori infection in the pathogenesis of GC also after taking into account the dietary habits reported at baseline. Our results have also shown that CagA positive strains play a crucial role in noncardia gastric carcinogenesis, while SCAG results associated with a strongly increased risk of cardia cancer. Although a long term H. pylori infection is the main cause of SCAG, our findings might suggest 2 divergent risk patterns for noncardia (i.e. distal) and cardia gastric cancer.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank all study participants for their availability. We thank Dr. Daniele Casini (Research Center, Novartis Vaccines, Siena, Italy) and Mr. Adriano Tasinato (University Hospital, Padua, Italy) for technical support for H. pylori serology and plasma Pepsinogens determinations. The authors also thank the other members of the Panel of Pathologists for their valuable work: Dr. Roger Stenling, Umea, Sweden; Dr. U Mahlke, Postdam, Germany; Dr. Hendrik Bläker, Heildelberg; Germany; Dr. Vicki Save, Cambridge, United Kingdom; Dr. Claus Fenger, Copenhagen, Denmark; Dr. Julio Torrado, San Sebastian, Spain; Dr. Johan Offerhaus, Amsterdam, The Netherlands; Dr. Dimitrious Roukos, Ioannina, Greece. The authors thank Dr. Cátia Moutinho, Porto, Portugal, for her excellent technical support to the panel of pathologists.

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  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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