INFECTION WITH CagA1 HELICOBACTER PYLORISTRAINS AS A POSSIBLEPREDICTOR OF RISK IN THE DEVELOPMENT OF GASTRIC ADENOCARCINOMAIN MEXICOJavier TORRES1*, Guillermo I. PEREZ-PEREZ2, Yelda LEAL-HERRERA1 and Onofre MUNOZ1

1Unidad de Investigacio´n Medica en Enfermedades Infecciosas, Hospital de Pediatrı´a,Instituto Mexicano del Seguro Social, Mexico City, Mexico2Division of Infectious Diseases, Vanderbilt University School of Medicine, Nashville, TN, USA

Helicobacter pylori strains possessing the Cag pathogenicityisland have been associated with increased gastric inflamma-tion and with duodenal ulcer. In contrast, studies on theassociation of cagA1 H. pylori infections and gastric cancerhave shown conflicting results. The aim of our study was todetermine whether H. pylori and CagA status are associatedwith gastric cancer in Mexico. We selected serum samplesfrom 3 geographic areas with gastric cancer mortality ratesper 100,000 inhabitants of 2.5 (low risk), 4.5 (medium risk)and 6.4 (high risk). H. pylori infection was determined by thedetection of antibodies to H. pylori whole cell antigen by anenzyme-linked immunosorbent assay (ELISA). To study theprevalence of infection with cagA1 strains, serum IgG antibod-ies to CagA were determined by ELISA using a recombinantCagA antigen. Of the 2,775 individuals studied, 1,931 were H.pylori seropositive and 1,710 had antibodies against CagA.The risk for gastric cancer in the 3 populations studiedincreased proportionally as infection with cagA1 strains in-creased (p F 0.001 for trend). H. pylori infection also showedassociation with gastric cancer (p F 0.05). Individuals seroposi-tive for CagA, but seronegative for H. pylori whole cellantigen, were more frequent in areas with higher gastriccancer rates (p F 0.01). These results support the possiblerole of CagA(1) status as predictor of risk for gastric adeno-carcinoma in Mexico; this is in agreement with results inEuropean and American populations, but contrary to studiesin some Asian countries. Int. J. Cancer 78:298–300, 1998.r 1998 Wiley-Liss, Inc.

Helicobacter pylori infection is one of the most commonbacterial infections in humans, yet only a proportion of the infectedindividuals will present gastrointestinal disease. It is estimated that10–20% of the infected subjects will develop peptic ulcers and lessthan 1% will present gastric cancer after decades of infection(Kuipers et al., 1995). Disease outcome depends on factorsassociated with the host (e.g.,ethnicity, genetics, age of acquisitionof infection, diet), as well as on bacterial factors (e.g.,CagA, VacA,urease) (Correa, 1996).H. pylori strains possessing the Cagpathogenicity island (Censiniet al., 1996) have been associatedwith a stronger inflammatory reaction in the gastric mucosa and ahigher risk for duodenal ulcer (Crabtreeet al.,1993a; Peeket al.,1995). Seroepidemiological studies on the association of CagA(1)prevalence and gastric cancer rate have yielded conflicting results.Studies in Western countries have found a significant associationbetween CagA(1) seroprevalence and rates of gastric cancer(Crabtreeet al.,1993b; Webbet al.,1996). In contrast, Pe´rez-Perezet al.(1997a) failed to show any correlation between gastric cancerrates and prevalence of CagA(1) infection in 8 different popula-tions including Western and Eastern countries. Similar studies inChina have found no association between CagA(1) infection andgastric cancer rates (Mitchellet al.,1996). A possible explanationfor these discrepancies is the differences in the populations studied.However, a study in China demonstrated that in 2 villages withdifferent gastric cancer rates but similarH. pylori seroprevalence,CagA(1) prevalence was higher in the village with the highest rateof gastric cancer (Pe´rez-Perezet al.,1997b).

People from developing countries might have a higher risk ofdeveloping gastric cancer, as infection in these countries is more

common and is acquired at earlier ages than in developed countries(Neale and Logan, 1995; Sonnenberg, 1995). In Mexico,H. pyloriinfection is common in adults (80%) and is acquired early in life(Torreset al.,1996). These observations prompted us to investigatethe role of H. pylori and CagA infection as risk factors in thedevelopment of gastric cancer in 3 geographic areas of Mexicowith different gastric cancer mortality rates.

MATERIAL AND METHODS

Population studied

During 1987–1988, the Ministry of Health of Mexico performeda national serologic survey. The collection of the serum sampleswas done according to a master sampling frame based on theGeneral Population Census data. The survey included all 32 statesof Mexico and was designed to include individuals with age rangeof 1–90 years. All socioeconomic levels and all geographic zonesof the country were included (Tapia-Conyeret al.,1992). For thisstudy, we selected serum samples from 3 of the 32 states withcontrasting gastric cancer rates per 100,000 inhabitants. We studiedthe Estado de Mexico with a gastric cancer rate of 2.5 (low riskarea), Distrito Federal with a gastric cancer rate of 4.5 (mediumrisk area) and Chiapas with a gastric cancer rate of 6.4 (high riskarea). These populations were selected on the basis of the gastriccancer rates reported by the Ministry of Health for the year 1987.Cancer rates were calculated based on the registry of deaths withhistopathologic diagnosis of adenocarcinoma of the stomach per100,000 inhabitants.

A multistage stratified design was used to select serum samples.In stage I, the sampling unit was the state, stratifying by type ofpopulation (urban/rural). In stage II, the sample units were thehousehold characteristics. In stage III, sample units were character-istics of the individual (sex, age). In all stages, random selectionwas proportional to the size of the unit. The mean age and sex ratiosof these populations are shown in Table I.

Definition ofH. pylori infectionH. pylori infection was determined by the detection of IgG

antibodies to specificH. pylori antigens by an enzyme-linkedimmunosorbent assay (ELISA). This ELISA has been validated foruse in children and adults in Mexico (Camorlinga-Ponceet al.,1996) following procedures previously described (Blaseret al.,1991) and has a sensitivity of 85% and a specificity of 87%. In

Grant sponsor: CONACYT, Mexico; Grant number: 0524P-M; Grantsponsor: Iris and Homer Akers Fellowship in Infectious Diseases.

*Correspondence to: Unidad de Investigacio´n Medica en EnfermedadesInfecciosas, Hospital de Pediatrı´a, CMN Siglo XXI, IMSS, Av. Cuauhte-moc 330, Me´xico, D.F., C.P. 06725, Mexico. Fax: (5) 761-0952.E-mail: 105623.3140@compuserve.com

Received 12 March 1998; Revised 8 June 1998

Int. J. Cancer:78,298–300 (1998)

r 1998 Wiley-Liss, Inc.

Publication of the International Union Against CancerPublication de l’Union Internationale Contre le Cancer

brief, a sonicated whole cell antigen preparation was obtained froma pool of 3 strains ofH. pylori isolated from Mexican patients.ELISA plates were coated with 0.5 µg/well of the antigen. Serumsamples were diluted 1:1,000, and monoclonal antibodies (MAbs)conjugated to alkaline phosphatase, anti-human IgG (SouthernBiotech, Birmingham, AL) diluted 1:1,000 were used. For colordevelopment, p-nitrophenylphosphate (Southern Biotech) was usedas substrate and absorbance was read at 405 nm (iEMS analyzer,Labsystems, Helsinki, Finland). The mean6 3 standard deviation(SD) of the optical density (OD) values from 30H. pyloriuninfected Mexican patients was used to calculate the thresholdvalue. The result of each serum sample was expressed as the ratioof the OD value of the sample to the threshold value and expressedas ELISA units. Accordingly, serum samples with ELISA units$1.0 were considered seropositive. Each serum sample was testedin duplicate.

Definition of CagA prevalenceTo study the prevalence of infection withcagA1 strains, we

determined the presence of specific serum antibodies to CagAproduct. We used an ELISA that was previously validated in ourpopulation (Camorlinga-Ponceet al., 1996) with a sensitivity of83% and a specificity of 97%. We used a recombinant CagAantigen (kindly supplied by OraVax, Cambridge, MA) for theassay. Antigen concentration was 0.1 µg/well to coat microtiterplates, and serum at 1:200 dilution was added. Anti-human IgGMAbs conjugated to alkaline phosphatase (Southern Biotech) anddiluted 1:1,000 were used, and 1 mg/ml p-nitrophenylphosphate(Southern Biotech) was added as substrate. Absorbance was read at405 nm (iEMS analyzer, Labsystems). The cutoff value wasdefined as the mean OD6 3 SD of 25 serum samples fromH.pylori uninfected individuals.

Statistical analysisThe potential correlation betweenH. pylori and CagA seropreva-

lences and rate of mortality due to gastric cancer were analyzedusing the chi-square and Mantel-Haenszel trend tests;p , 0.05 wasconsidered significant.

RESULTS

Because of the design of the serosurvey, the populations of the 3areas studied were similar in age and gender distribution, asdescribed in Table I. Of the total of 2,775 individuals studied, 1,931(69.6%) wereH. pylori seropositive and 1,710 (61.6%) had apositive IgG antibody response against CagA. In 1,509 (88.3%) ofthe subjects studied, there was a concordance betweenH. pyloriand CagA serology. The possible correlation betweenH. pylori orCagA seroprevalences with gastric cancer is presented in Table II.Infection with cagA1 strains was significantly associated withhigher gastric cancer mortality rates. In contrast, infection withcagA2 strains was significantly more frequent in populations withlower gastric cancer rates. Interestingly, individuals seropositivefor CagA, but seronegative forH. pylori infection, were morecommonly detected in the populations with high gastric cancer

rates. CagA seropositivity, regardless ofH. pylori infection, had thestrongest association with cancer (p , 0.0001).H. pylori infectionregardless of CagA status was also associated with gastric cancer,although the association was weaker (p 5 0.002).

DISCUSSION

Infection with H. pylori cagA1 strains has been linked to anincreased risk of developing peptic ulcer disease and gastric cancer.In the United Kingdom, patients with gastric cancer had a higherIgG response to CagA antigen than patients with non-ulcerdyspepsia (Crabtreeet al., 1993b). In contrast, in a nestedcase-control study in Japanese Americans in Hawaii, infection withcagA1 H. pylori strains was found to only marginally increase therisk for developing gastric cancer (Blaseret al., 1995). Further-more, in a study performed in 17 centers in Europe, infection withcagA1 strains was significantly associated with a relative risk forgastric cancer (Webbet al.,1996). In the present study on Mexicanpopulations, infection withcagA1 H. pylori strains was signifi-cantly associated with an increased risk of mortality due to gastriccancer. The serum samples utilized in our study were collectedfrom asymptomatic subjects. Thus, the seroprevalence estimates ofH. pylori and CagA infection are not biased with respect todifferential inclusion of symptomatic patients. The observed asso-ciation describes the risk of gastric cancer in a healthy, asymptom-atic population.

This is not the first time than an association between CagA andgastric cancer has been reported. A similar association ofcagA1 H.pylori infection and gastric cancer was found in a study onasymptomatic individuals from 2 Chinese populations with verydifferent risks for gastric cancer (Pe´rez-Perez et al., 1997b).However, other studies did not find an association between gastriccancer rates and CagA seroprevalence. In a study performed withserum samples from 8 different countries, CagA seropositivity wasnot associated with the reported gastric cancer rates (Pe´rez-Perezetal., 1997a). An interesting observation made in that study was thatthe association between CagA seroprevalence and gastric cancerrates was quite strong when Asian populations were excluded fromthe analysis. In another study in China, infection withcagA1 strainswas similar in asymptomatic subjects and in patients with gastriccancer (Mitchellet al.,1996).

These conflicting results might reflect genetic differences in thepopulations studied, or perhaps they are caused by the differentgroups of patients selected for study. Studies with asymptomaticsubjects might better describe the role ofH. pylori infection as arisk factor for gastric cancer in the population, whereas studies onpatients with gastric cancer might not accurately describe this risk.In patients with gastric cancer, the gastric mucosa is no longersuitable forH. pylori infection and both infection and immuneresponse might be decreased or absent. Indeed, in patients withprecancerous lesions, the antibody response toH. pylori antigens,including CagA, has been found to be significantly higher than inpatients who already had gastric cancer (Klaamaset al., 1996;Zhanget al., 1996). In our study, infection withcagA2 H. pyloristrains was associated with lower rates of gastric cancer. Thiswould suggest that whereas infection withcagA1 strains predis-pose to more serious gastric diseases, infection withcagA2 strainsmay reduce the risk of gastric cancer in the host. A similarobservation has been reported for adenocarcinoma of the cardia inthe stomach and adenocarcinoma of the esophagus (Chowet al.,1997).

An important observation in our study is that there is asubstantial group of people who are seronegative forH. pylori, butseropositive for CagA. This type of response was significantly

TABLE I – CHARACTERISTICS OF THE STUDIED POPULATIONS

CharacteristicRisk of gastric cancer

Low Medium High

Number of persons 1,023 1,137 615Mean6 SD age (years) 246 18 276 19 256 19Male:female ratio 0.64 0.79 0.79Cancer rate mortality1 2.5 4.5 6.4

1Rates are per 100,000 inhabitants in each area.

299H. PYLORICagA AS RISK FOR GASTRIC CANCER

more frequent in populations from areas with high rates of gastriccancer than in populations from areas with low rates. The strongimmunogenicity of CagA has been documented; indeed, patientswith advanced gastric cancer lesions keep positive antibodyresponses to CagA even after the infection has cleared and theantibody responses to otherH. pylori antigens have declined(Klaamaset al.,1996). Thus, our observations suggest the possibil-ity that some of the individuals seropositive for CagA butseronegative for otherH. pylori antigens might have had an

infection and cleared it, probably due to changes in the gastricmucosa, such as atrophic gastritis that is commonly seen in gastriccancer.

In conclusion, our results show that IgG antibodies against CagAare a possible predictor of risk factor for gastric adenocarcinoma inMexico and support a role forcagA1 H. pylori infection in gastriccancer, as has been observed in other European and Americanpopulations.

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TABLE II – SEROPREVALENCE OFH. pylori AND CagA INFECTION IN POPULATIONS WITH DIFFERENT RISKS FORGASTRIC CANCER IN MEXICO

Seroprevalence status Gastric cancer risk population (% positive)p1

H. pylori CagA Low Medium High

(1) (1) 47.6 55.6 63.4 0.0006(1) (2) 18.6 15.2 9.6 0.0001(2) (1) 4.8 8.9 8.3 0.0043(2) (2) 29.0 20.3 18.7 0.0001(1) or (2)2 (1) 52.4 64.5 71.7 ,0.0001(1)3 (1) or (2) 66.2 70.8 73.0 0.002

1p-value for trend.–2CagA seropositive, regardless ofH. pylori infection.–3H. pylori infected, regardlessof serology to CagA.

300 TORRESET AL.