Prevalence
of
Helicobacter
pylori
genotypes
(
vac
A,
cag
A,
cag
E
and
vir
B11)
in
gastric
cancer
in
Brazilian’s
patients:
An
association
with
histopathological
parameters
Valeska
Portela
Lima
*
,
Isabelle
Joyce
de
Lima
Silva-Fernandes,
Markeˆnia
Ke´lia
Santos
Alves,
Silvia
Helena
Barem
Rabenhorst
Laborato´riodeGene´ticaMolecular–LABGEM,DepartamentodePatologiaeMedicinaLegal,UniversidadeFederaldoCeara´-UFC,Brazil
1. Introduction
Gastric cancer is the fourth most common cancer and the secondleadingcauseworldwideofcancerdeaths[1,2].About two-thirds of the cases occur in the developing world [3,4]. Adenocarcinoma,themostcommongastriccancertype,hasbeen classified by Lauren according to the clinical and histological features intotwo main types: diffuse and intestinal [5], which differintheirmolecularchangesandtheyareconsidereddistinct tumors,havingdistinctcarcinogeneticpathways[6,7].
Helicobacterpyloriisoneofthemostimportantenvironmental causativeagentsofgastriccarcinoma.Over50%ofthepopulationis reportedtobeinfectedbyH.pyloributonly1–2%willdevelopgastric cancer[8].TheassociationofH.pyloriwithgastriccancerisstrongest fornon-cardiacancer,anditholdsforbothintestinalanddiffuse histologicaltypes[9].Theseobservationsindicatethatbesidesthe hostparasiterelationshipingastriccancerdevelopment,thereisa needforstudiestoimproveunderstandingofH.pyloriitself.H.pylori isoneofthemostgeneticallydiversepathogenicbacteria[10].This featureresultsinpopulationsofbacteriawithregionaldifferences, withparticularattributesthatmakeitdifficulttoestablishmarkers
ofvirulence[10,11].However,thereisevidenceofdistinctgenetic lineagesthatmayhavearoleinitspathogenesis.
Some virulence factorsofH. pyloriarefrequentlyassociated withthemostseriousclinicaloutcomesandpathogenicbacteria. Theseinclude thecagpathogenicityisland (cag-PAI), whichhas about31genes,andthepresenceofallelicvariationofthevacA gene[11,12].ThevacAgeneproducesa95-kDahomonymsecreted proteinwhichinducesapoptosis,partlybyformingporesinthe mitochondrial and cytoplasm membrane, thereby allowing the efflux of cytochrome c, inducing apoptosis and increases the cellularpermeability[13].Italsoproducesimmunosuppressionby blockingantigenpresentationtoTcells[14,15]andthematuration ofmacrophagephagosomes[16].Thesignalsequence(s1a,s1b, s1c,s2) andthemiddleregion(m1,m2)ofvacAvaryamongH. pylori strains, with strains carrying the vacAs1m1 genotype consideredthemostpathogenicsubtype[17].
Amongcag-PAI genes,themoststudiedis cagA.Thisgeneis locatedontherighthalfofcag-PAI.Itencodesaprotein(CagA)that is injectedinto thecytoplasm ofthehost celland induces cell morphologic alterations, proliferation, adhesion and apoptosis
[18–20].Therighthalfofcag-PAIalsocontainsageneknownas cagE.Thisgeneencodesaproteinresponsiblefortheinductionof interleukin(IL)-8,producedbythegastricepithelialcells[21].Itis considered by same authors to be the best marker of cag-PAI
[22,23]. Additionally, another cag-PAI gene, virB11, can be an importantpotentialvirulentfactor,sinceitislocatedatacrucial point of the secretory apparatus system and exhibits ATPase activity,assistinginthetransportofCagAintothehostcell[24,25].
ARTICLE INFO
Articlehistory:
Accepted23February2011 Availableonline5April2011
Keywords:
Gastriccancer
Helicobacterpylori cagA
vacA
cagEandvirB11
ABSTRACT
Purpose:ToinvestigatethefrequencyandtheassociationofvacAalleles,cagA,cagEandvirB11genesof
Helicobacterpylorifrompatientswithgastriccancer,consideringtheclinichistopathologicalparameters.
Methods:OnehundredandonegastricadenocarcinomatissueswereassessedbyPCRtodetectH.pylori
andvacAalleles,cagA,cagEandvirB11.Results:Thedistributionofcasesaccordingtothepresenceofthe
genesstudiedshowedthatthegroupcontainingvacAs1m1,cagA,cagEandvirB11H.pylorigeneswas
significantlymorefrequent,followedbythegroupwithatleastonemarkerontherightsideandleftof
theisland. They werealso present in the earlystages and werethe most frequent in nearlyall
histopathologicalgrades.Conclusions:ThisstudyverifiedthatvacAs1m1andcag-PAIgenes,cagA,cagE
andvirB11areimportantH.pylorimarkersforgastriccancerdevelopment.Also,thisstudycorroborates
theimportanceofcagEandcagAtogetherascag-PAImarker.
ß 2011ElsevierLtd.Allrightsreserved.
*Corresponding authorat: Laborato´rio de Gene´tica Molecular – LABGEM, DepartamentodePatologiaeMedicinaLegal,UniversidadeFederaldoCeara´-UFC. RuaAlexandre Barau´na,949.CampusdePorangabussu.Fortaleza,Ceara´,CEP: 60183-630,Brazil.Tel.:+558588050432;fax:+558532673840.
E-mailaddress:[email protected](V.P.Lima).
ContentslistsavailableatScienceDirect
Cancer
Epidemiology
The
International
Journal
of
Cancer
Epidemiology,
Detection,
and
Prevention
j o urn a lhom e pa g e :ww w . ca nc e re pi d e mi ol o gy . ne t
Thisgeneislocatedonthelefthalfofcag-PAI.Therefore,itcanbe usedasamarkeroftheintegrityofthissideoftheisland.
Although several studies have described an association between H.pylorivacA s1m1and cagA withgastritis,duodenal ulcers,pepticulcersandgastriccancer,therelationbetweenthese geneswithmalignancyisstillcontroversial.In Western popula-tions,cagA-positivestrainsaremorecommonlyassociatedwith pepticulceration,atrophicgastritis andgastric adenocarcinoma thancagA-negativestrains[8,26].However,thisrelationshipisnot observedinmanyhighgastriccancerpopulations,includinginEast Asia,wherealmostallstrainsarecagA-positive.Also,inBrazil,the studiesanalyzingvacAs1m1andcagAassociationhavedescribed conflictingresults[27–32].
Arelevantpoint is thatsomestudieswhich haveassociated gastriccancerwithH.pyloriweredonewithoutconsideringtheH. pylorigenotypesinthecontextofthehistologicalsubtypes[31,33]. Also,reportsoftheassociationofH.pyloriwithhistologicaltypesof gastric cancers are controversial; somestudies have shown an associationbetween H.pyloriand intestinaltype [34,35],while others have observeda balanced distribution between thetwo histologicaltypes[36,37].Therefore,theaimofthisstudywasto investigatethefrequencyandtherelevanceoftheassociationof vacAalleles,cagA,cagEandvirB11genesofH.pyloriisolatedfrom patientswithgastriccancer,consideringtheclinical histopatho-logicalparameters.
2. Materialsandmethods
2.1. Clinicalspecimens
ThepresentstudywasapprovedbytheEthicsCommitteeofthe Hospital Complex of the Federal University of Ceara´ and all subjects signed the informed consent form before inclusion. Samples from one hundred and one patients with gastric adenocarcinomawhohadundergonegastrectomywerecollected fromtwohospitalsinCeara´ State,Brazil:WalterCantı´deoHospital attheFederalUniversityofCeara´ andSantaCasadeMiserico´rdia Hospital, both located in Fortaleza, the state capital. The histological classification was made according to the Lauren classificationandthetumorswerestagedaccordingtotheTNM criteriabytwopathologistsoftheteam.
2.2. DNAextraction
GenomicDNAwasextractedfromfrozentumortissueusingthe cetyltrimethyl ammonium bromide (CTAB) technique, adapted
fromthemethodofFosterandTwell[38].TheDNAextractionwas doneonlyinfragmentsthatshowedmorethan80%oftumorcells, and thequality wasanalyzedby 1%agarosegelelectrophoresis withethidiumbromide staining. Also, theamountofDNA was determinedusingtheNanoDropTM3300fluorospectrometer.
2.3. DetectionofH.pyloriandthepresenceofvacA,cagA,cagEand virB11genes
The H.pylori infectionwas detected by amplificationof the ureaseCgeneusingprimersforPCR,asdescribedbyLageetal.[39]. For theH.pylori-positive samples,thepresenceofthevacA and alleles, cagA, cagE and virB11 genes were identified using the primersetsfromthepublishedliterature.TheseareshowninTable 1.PCRmixtures,foramplificationofcagEandvirB11genes,were prepared in a volume of 20
m
L using MasterMix1 (Taq DNAPolymerase,dNTPs andMgCl2) accordingtothemanufacturer’s instructions(Promega1),withadditionof0.8%Tween20;0.3
m
M(virB11), 1
m
M (cagE) of each primer and 100ng of the DNAsample.
ThecagA,vacAs1/s2,vacAm1geneswereamplifiedina25
m
Lvolumecontaining2.5
m
Lof10PCRbuffer(Invitrogen1,Cergy Pontoise,France);1%Tween20,1.5mMofMgCl2(Invitrogen1),200
m
M (each) of dNTPs (Invitrogen1), 1U of Platinum Taqpolymerase(Invitrogen1);0.4
m
M(ureC, cagA, vacA s1/s2,vacAm1),0.3
m
M(vacAm2)foreachprimerand100ngofH.pyloriDNA.ThePCRproductswereanalyzedby1%agarosegel electropho-resiswithethidiumbromidestaining.Thesizeoftheamplification productwasusedtoconfirmtheidentityofthePCRproduct.The samplewasconsideredH.pyloripositivewhenanureCfragmentof 294bp was amplified. For confirmation of the specificity of reaction,PCRproductsfromureaseCgenewereclonedwithTOPO TACloningKit(Invitrogen1)andsequencedusingtheABIPRISM1
BigDye Terminator v.3.0 Cycle SequencingKit (Applied Biosys-tems)andABIPrism3100DNASequencer(AppliedBiosystems). vacA,cagA,cagEandvirB11geneswereconsideredpositivewhena specificfragmentweredetected(Table1).DNAse-freewaterwas used as a negative control. DNA preservation has also been confirmedbyamplificationofdifferentgenesinotherapproaches understudyinthelaboratory.Randomsampleswerereanalyzed forconfirmationoftheresults.
2.4. Statisticalanalyses
These werecarried outusing thestatisticalprogramsSPSS1
version15.0(Chicago,IL,USA).Statisticallysignificantdifferences
Table1
PCRprimersets,annealingtemperatureandsizeofthePCRproductsusedforgenotypingH.pylori.F–follow;R–reverse.
Gene Primersequence Annealing SizeofPCRproduct Reference
ureC F–50-AAGCTTTTAGGGGTGTTAGGGGTTT-30 558C 294bp [39]
R–50-AAGCTTACTTTCTAACACTAACGC-30
vacA [17]
s1/s2 F–50-ATGGAAATACAACAAACACAC-30 558C 259/286bp R–50-CTGCTTGAATGCGCCAAAC-30
m1 F–50GGTCAAAATGCGGTCATGG30 558C 290bp
R–50-CCATTGGTACCTGTAGAAAC-30
m2 F–50-GGAGCCCCAGGAAACATTG-30 528C 192bp
R–50-CATAACTAGCGCCTTGCAC-30
cagA F–50-ATAATGCTAAATTAGACAACTTGAGCGA-30 568C 297bp [40]
R–50-TTAGAATAATCAACAAACATAACGCCAT-30
cagE F–50-TTGAAAACTTCAAGGATAGGATAGAGC-30 508C 509bp [23]
R–50-GCCTAGCGTAATATCACCATTACCC-30
virB11 F–50-TTAAATCCTCTAAGGCATGCTAC-30 498C 491bp [23]
were evaluated by the chi-square test (
x
2). Correlations wereanalyzedbySpearman’srankcorrelationcoefficient.Theresults wereconsideredstatisticallysignificantwhenp-valueswereless than0.05.
3. Results
Amongthe101analyzedcases,68(67.3%)weremalesand33 (32.7%)females.Theaverageagewas62.7,rangingfrom23to90 yearsold.H.pyloriinfectionwasdetectedin94outof101(93%) gastricadenocarcinomas.Themalesweremorefrequent(68%)in theH.pylori(+)cases.TherateofH.pyloriinfectionintheantrum was statistically higher (p<0.001) than the cardia and body (53.2%, 26.6% and 17%, respectively). Among H. pylori positive cases,theintestinaland diffusehistologicalsubtypehadsimilar frequency,althoughtheintestinaltypewasslightlymorefrequent (58.5%and41.5%,respectively).
3.1. RelationshipbetweenH.pyloriinfectionandvacA,cagA,cagEand virB11geneswithgastricadenocarcinoma
The frequency of H. pylori genes is show on Table 2. The frequencyofvacAs1m1associatedwithcagAwas56.4%. Consider-ingtheintegrityofcag-PAIandtheassociationamongthecag-PAI genes,wefoundapositivecorrelationbetweencagAandvirB11 (r=0.229,p=0.027*),andbetweencagEandvirB11(r=0.728**; p<0.001).In6.4% (6/94)ofthecasesonlythecagAandvirB11 strainswerepresent,whileonlycagEandvirB11werepresentin 11.7%(11/94)ofthecases.
Most of the H. pylori strains cagA(+) were associated with cagE(+)(62.3%).ToinvestigatetherelationshipbetweenH.pylori infectionandthepresenceofvacAalleles,cagA,cagEandvirB11 genesingastrictumorigenesis,thecasesweredividedintothree groupsaccordingtovacAallelesandeachonewasthensubdivided into four subgroups according to the integrity of cag-PAI, considering the studied genes, the cases which no presented anycag-PAI geneswereclassifiedascag-PAI ( ).Thesegroups, theirfrequenciesandhistologicaltypesareshowninTable3.
No statistical difference was found between intestinal and diffuse histological types and the H. pylori groups. The most frequent subgroup in all adenocarcinomas cases was A1 (vacA s1m1,cagA,cagEandvirB11),whichrepresented36.2%(34/94)of H.pyloriinfected gastric cancer cases. There wasa statistically significantdifference in thefrequency ofcases foundin theA1 groupand the other groups: A2 (p=0.002), A3 (p=0.001), A4 (p=0.026),B3(p=0.038).Also, comparingtheA1 againsttheB
group(allBsub-groupstogether,sincethenumberofcaseswere insufficientforindividualstatisticalanalysis), theA1groupwas statisticallymorefrequent(p=0.001)thantheBgroup. Consider-ingtheH.pyloriAgroupswithatleastonemarkerintherightand leftsideofcag-PAI(A1andA2groups),asignificantnumberofthe caseswereincluded(51%; 48/94),withastatisticallysignificant differencewhencomparedtotheA3(p<0.001),A4(p=0.003),B3 (p=0.005)andB4(p=0.010)groups.
Inalmostallgroups,thepredominantanatomicalsitewasthe gastric antrum.In theH.pylori groups(other than groupA)in which it was possible to do statistical analysis, a significant differencewasfoundbetweengroupA3andthepresenceofthe tumorsinthecardiaregion(8/15;p=0.015).Interestingly,ofall gastriccancerslocatedinthecardia,70.8%(17/24)belongedtothe intestinalsubtype.
Fig.1showsthedistributionoftheH.pylorigenotypegroups accordingtothetumorstages.Duetoasmallnumberofcases,the subgroupsofgroupsBandCwerenotconsidered.Fromthisfigure, itispossibletoobservethattheA1groupwaspresentinalmostall stagesandalsowasthemostfrequentexcepttheIIItumorstage. AlthoughgroupsA4,BandCwerepresentinalmostallstages,they were less frequent in the majority of the grades. We didnot carefullyanalyzeotherriskfactors,likealcoholconsumptionand smoking,dueofthedifficultiesofmeasuringthelevelsofexposure. However, we observedthat thesefactors werefrequentin this group,since5/6caseshadatleastonesuchassociatedriskfactor.
Figs.2and 3showthedistributionoftheH.pylorigenotype groupsaccordingtotumorstageandhistologicalsubtypes.Asin
Fig.1,allcasesofBandCsubgroupsareshowntogether.Inboth histologicalsubtypesitispossibletoobservethattheA1groupwas presentfromearlierstageswithhighfrequency.Althoughinthe diffusetumorsthefrequenciesoftheA1andA2groupsincreased withtumorstage,intheintestinalsubtypetherewasadecreasein thefrequencyoftheA1subgroupingradesIIIandIV.Evenwhen
Table2
FrequencyofH.pylorigenes.
H.pylorigenes Frequency(n=94/100%)
vacA
s1m1 71/75,5%
s1m2 13/13.8%
s2m1 4/4.2%
s2m2 6/6.5%
cagA 61/64.9%
cagE 50/53.2%
virB11 57/60.6%
Table3
H.pyloriinfectioncasesingastriccancerdividedaccordingtoH.pylorigenotypesandhistologicaltypes.
Groups Histologicaltypes
n=94 Intestinal(n=55) Diffuse(n=39) Pvalue
GroupA:vacAs1m1 71/94(75.5%)
A1:cagA(+)andcagE(+)andvirB11(+) 34/94(36.2%) 21/55(38.2%) 13/39(33.3%) p=0.630 A2:cagA(+)andvirB11(+)orcagE(+)andvirB11(+) 14/94(14.9%) 8/55(14.5%) 6/39(15.4%) p=0.910 A3:cagA(+)orcagE(+)orvirB11(+)orcagA(+)andcagE(+) 15/94(16%) 10/55(18.2%) 5/39(12.8%) p=0.484
A4:cag-PAI( ) 08/94(8.5%) 3/55(5.5%) 5/39(12.8%) p=0.207
GroupB:vacAs1m2evacAs2m1 17/94(18.1%)
B1:cagA(+)andcagE(+)andvirB11(+) 02/94(2.1%) 1/55(1.8%) 1/39(2.6%) p=0.805 B2:cagA(+)andvirB11(+)orcagE(+)andvirB11(+) 02/94(2.1%) 1/55(1.8%) 1/39(2.6%) p=0.805 B3:cagA(+)orcagE(+)orvirB11(+)orcagA(+)andcagE(+) 07/94(7.4%) 5/55(9.1%) 2/39(5.1%) p=0.471
B4:cag-PAI( ) 06/94(6.4%) 4/55(7.3%) 2/39(5.1%) p=0.675
GroupC:vacAs2m2 06/94(6.4%)
C1:cagA(+)andcagE(+)andvirB11(+) 00/94(0%) 0/55(0%) 0/39(0%) -C2:cagA(+)andvirB11(+)orcagE(+)andvirB11(+) 01/94(1.1%) 0/55(0%) 1/39(2.6%) p=0.223 C3:cagA(+)orcagE(+)orvirB11(+)orcagA(+)andcagE(+) 02/94(2.1%) 1/55(1.8%) 1/39(2.6%) p=0.805
A1andA2wereconsideredtogether,thesametendencycanbe observed.
ThedistributionoftheH.pylorigroupsbygendershowsthat womenweremorefrequentthanmenintheA1(56.2%43.8%) andA2(26%16.7%)groups.However,thissexratiowasreversed
intheA3(25%13%)andA4(14.5%4.8%)groups,butwithout statisticalsignificance.
It is interesting to note that the C4 genotype group was significantlymorefrequent(p<0.001)intheyoungerpatients(up to44yearsold,sixcases)thantheoldestonesandfiveofthem were in the diffusetype, but no family history was described. Additionally,theA3groupwasassociatedwiththeolderpatients, thoseabove65yearsold(p=0.018).
4. Discussion
TheassociationofgastriccancerwithH.pylorihasbeenwell established[41].Someaspects,likehostgeneticprofile,lifestyle canexplainthegastriccarcinogenesis,butthebacterialgenotype seemstohavearelevantrole[13].H.pyloristrainshavesubstantial phenotypicandgenotypicdiversity,whichmayengender individ-ualhostinflammatoryresponsesthatinfluenceclinicaloutcome
[42–44].Thewellestablishedvirulencefactors,vacAalleles(s1m1) and cagA,althoughhavinganimpactonbacterialpathogenesis, cannot explain allgastric cancer development. Therefore,other potentialvirulencefactorsneedtobeinvestigated.Inthisrespect, cagEandvirB11genesareinterestingsincetheybelongstothecag -PAIandhaveanimportantrole[21–25]andalongwithcagAcould bemarkers ofthepathogenic strainforgastric cancer develop-ment.
Asexpected,acloseassociationofgastriccancerwithH.pylori infectionwasobservedinthisstudy.Similarfrequenciesobserved inthisstudy(93%)havebeenfoundinotherBrazilianstudiesfrom the southeast region, and in studies from Turkey and Hawaii
[28,36,45–48].Asinotherstudies,themostfrequentmosaicofthe vacAgenefoundinourstudywasvacAs1m1(75.5%).Thisfinding is consistentwiththepathogenicaspect of thisgene, sincethe productionofVacAtoxinisrelatedtotheallelicstructure,where the s1m1 and s1m2 genotype strains are high and moderate, respectively,producersofVacAprotein,whereass2m2genotype strainsdonotproduceVacA.Also,s1/m1istoxictoawiderrangeof epithelialcellsthans1/m2[49].
Arelativelyhighfrequency(64.9%)ofH.pylori-infectedgastric cancerswereinfectedbythecagA(+)variety.ThefrequencyofcagA isdiverseaccordingtoregion andassociateddisease.InEastern countries,almostallH.pyloristrainsarecagApositive(88.9%and 88.4%);andthereforethepresenceofthis geneisnotrelatedto gastrointestinaldiseases[50,51].On theotherhand,inWestern countriesthefrequencyofthecagAvarietyisbetween26.4%and Fig.1.DistributionofH.pylorigenotypegroupsA,BandCaccordingtothetumorstages.
Fig.3.DistributionofH.pylorigenotypegroupsA,BandCinthediffusecases accordingtothetumorstages.
90.4%,anditseemsthatthepresenceofthisgeneisassociatedwith thedevelopmentofmoreseveregastricdiseases,includinggastric cancer [47,52,53]. In Brazilian studies,a great variation is also reported (54.7–100%) of association with gastric cancer cases
[54,55].Therearesuggestionsthattheregionaldivergencecould bedueofpolymorphisminthecagAgene,butthissubjectisnot fullyelucidated.
AnassociationbetweenH.pyloristrainscarryingvacAs1m1and cagAgeneshasbeenrelatedwithclinicaloutcomeinsomestudies, butthepathogenicityofthesestrainsisstillcontroversial.Also, thereisalittleknowledgeabouttheinteractionbetweenthesetwo genesand thegastriccancer development. Recently,Yokoyama etal.[56]proposedamechanismbasedintheoppositeeffectsof thesegenesonthenuclearfactorofactivatedTcells(NFAT).They demonstratedthatCagAactivatesNFAT,whichinturnactivates manygenes,includingp21,acellcycleinhibitor.Conversely,they demonstratedthatVacAcounteractsthisactivation.Therefore,the nuclearactivationofNFATbyCagAiscounteractedbythepresence ofVacAs1m1, reducing thep21induction and thusstimulating deregulated cell growth. Since H. pylori-injected CagA also deregulates SHP-2 and other cellular target molecules that promotecellproliferation,thisprocesscouldleadto transforma-tioningastricepithelialcellsand,therefore,explaintheassociation ofthesegeneswiththepathogenicprocessandthehighfrequency oftheseassociationsfoundinthisstudy.
Despite the importance of cagA and vacA s1m1 genes for bacterialvirulence,theydonotappearsufficientforgastriccancer development.SootherH.pyloricag-PAIgenesmustbeinvolvedue oftheimportanceforthefunctionalityoftypeIVsystemsecretion. Additionally,thesegenescouldwork,alongwithcagA,asmarkers forcag-PAIintegrity.Studiesofcag-PAIintegrityassociatedwith diseasesarecontroversialbecausetherearenopatternsfor the selectedgenes [22,23,57,58]. In the present study, we did not analyzethecompositionofcag-PAIwasindepth.Wechose the genesbased for their location on the island as well as on the putativeroleoftheirproteins.
TheimportanceofcagEcanbeobservedbyitshighfrequencyin gastric cancer from India (100%), Turkey (81.8%) and Thailand (93.8%)[51,59,60]. Inthepresentstudy,53.2% thecaseshadH. pylorithat werecagE positive,which is lowerthan reported in theseother studies. Also, cagE has been suggested as a better markerfortheintegrityofcag-PAIthancagA[23,33].Interestingly, inourstudy,althoughcagAwasmorefrequentthancagE,mostof the time it was associated with cagE. Therefore, our data corroboratethesuggestionthatcagAisabettersinglemarkerof the pathogenicity island. However, the association with cagE improvedtheaccuracy,suggestingtheuseofbothasmarkersfor cag-PAIpresenceandalsoforthepathologicalsignificanceofthese genes.
In additiontocagE,virB11wasfoundinasignificant(60.6%) numberofthegastriccancercasesanalyzed.Sofar,thereareno studieslinkingthepresenceofgastriccancertovirB11. Neverthe-less Tomasini et al. [61] and Sozzi et al. [23] studied Italian dyspepticpatientsandfound90%and94.7%presence,respectively, ofthevirB11gene.
ToverifythemostfrequentH.pylorigenotypeingastriccancer andtheircontributiontothedevelopmentofgastriccancer,we dividedthecasesaccordingtovacAallelesandcag-PAIintegrity. Accordingthisdistribution,wefoundthatthevacAs1m1plusall cag-PAI genes studied (A1) was the most frequent genotype (36.2%),whichwasconsideredthemostpathogenicgenotypein this study.Additionally, consideringthecag-PAIintegrity using cagAand/orcagEasrightsidemarkers(A1+A2),51%ofthecases wereincluded.Sincegastriccancerisexpectedtoresultfrommore virulentstrains,thecombinationgenotypespresentedinthisstudy suggests that cagE and virB11 genes were involved in gastric
carcinogenesis.Also,virB11couldbeusedasleftmarkerforcag -PAI.Also,thesignificantfrequencyofmorevirulentstrainsinall tumorstages,includetheearlystage,indicatestheimportanceof theseH.pyloristrainstogastriccarcinogenesis.Thelowfrequency ofearlystagecasesfoundinthisstudyisexplainedbythefactthat thesamplesweretakenfrompatientstreatedbythepublichealth system,inwhichlow-incomeindividualswhoseekhospitalswhen thediseaseisalreadyinitsadvancedstagespredominate.
Inthisstudy,thefrequencyofH.pyloriA1andA2strainsdidnot differ between the intestinal and diffuse histological types, corroborating the suggestion of H.pylori’sinvolvement in both tumor subtypes [36,37,62,63]. Interestingly, we observed an increase in frequency of these groups with malignancyin the diffuse tumors, while in the intestinal tumors the highest frequencieswerein theearlystage,suggestingthat H.pyloriis involvedingastriccarcinogenesisindependentlyofthe histologi-calsubtype,althoughhavingdifferentcarcinogenicpathways.
Thegastriccancercaseslocatedintheantrumwere predomi-nantinpatientsinfectedwithmorepathogenicstrainsofgroupA, corroboratingwithsomestudiesthathavereportedthatH.pylori strainswereassociatedwiththegastricantrum[64].Noteworthy, samelessvirulentstrainwasrelatedtogastriccardia(A3andB4) andbody(C2andC4).Thisfactcanberelatedtoassociaterisks, suchasalcoholconsumption(atleast400mLaday)andsmoking (atleast2boxaday)inpatientsinfectedbylessvirulentstrains.In fact,53.8%ofpatientswithlessvirulentstrainsweresmokersand drinkers.
Inconclusion,thisstudyallowedverifyingthatcag-PAIgenesin association with vacAs1m1, are potentially important H. pylori strainmarkersforgastriccancerdevelopment,sincethisstrainwas themostfrequentandwaspresentfromtheinitialtumorigenic process. The H. pyloristrains seem to beimportant for gastric cancerdevelopmentinbothhistologicalsubtypes,butprobablyact through different carcinogenic pathways. The presence of less virulent strains in gastric cancer could be because of the association withhost genetic predisposition; other risk factors likesmokinganddrinkingorsomeotherbacterialfactorsthatwere not analyzed in this study. Also, this study corroborates the importanceofcagAas amarkerfor cag-PAI,buttheassociation withcagEseemsabettermarkerinthisrespectandalsosuggests virB11asaleftsidecag-PAImarker.
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