stages type - BMJ

Gut 1996; 39: 434-438

Global DNA hypomethylation occurs in the earlystages of intestinal type gastric carcinoma

M Cravo, R Pinto, P Fidalgo, P Chaves, L Gloria, C Nobre-Leitao, F Costa Mira

AbstractBackground-Global DNA hypomethyl-ation has been found in the premalignantstages of some neoplasms and has beenimplicated as an important factor fortumour progression.Aims-The aim of this study was toevaluate whether DNA hypomethylationoccurs during the process of gastriccarcinogenesis.Methods-Gastric specimens were ob-tained from 49 patients and histologicallyclassified as: normal 10, superficialgastritis 14, chronic atrophic gastritis withintestinal metaplasia 15, and intestinaltype of gastric carcinoma 10. Global DNAmethylation was assessed by incubatingDNA with (3H)-S-adenosylmethionineand Sssl methylase. A higher incorpor-ation of (3H) methyl groups reflects alower degree ofintrinsic methylation.Results-A graduated increase in (3H)methyl group incorporation into DNA wasfound over the range extending fromnormal gastric mucosa, to superficialgastritis and to chronic atrophic gastritis(136 556 (24 085) v 235 725 (38 636) v 400 998(26 747 dpm/,ig/DNA respectively;p=00002). No further increase was foundin specimens from patients with carci-noma. No differences were found betweenextent of DNA methylation in neoplasticor non-neoplastic mucosa from patientswith gastric carcinoma. Hypomethylationof DNA increased substantially withsevere atrophy (p=0.01) or with type IIIintestinal metaplasia (p=0.15).Conclusions-Global DNA hypomethyl-ation occurs in the early stages of gastriccarcinogenesis, and it may be a novelbiomarker of gastric neoplasia, useful inmonitoring the response to chemo-preventive agents.(Gut 1996; 39: 434-438)

Keywords: DNA methylation, gastric carcinogenesis,intermediate biomarker.

According to previous epidemiological andmorphological studies, the intestinal type ofgastric cancer is preceded by a series ofprecursor lesions - namely, superficial gastritis,chronic gastritis, atrophy, intestinal meta-plasia, dysplasia, intramucosal carcinoma, andinvasion.'1 However, both chronic atrophicgastritis and intestinal metaplasia are signifi-cantly age related and extremely common inthe older age groups, both in benign andmalignant conditions, which may limit its

predictive value as an indicator of risk. This hasprompted a number of studies aimed atidentifying other phenotypic markers of riskthat may help to recognise subgroups ofpatients at increased risk who could then beincluded in surveillance and chemoprophy-lactic programmes. These studies found thatsevere atrophy and incomplete type IIIintestinal metaplasia are strongly associatedwith intestinal type gastric cancer.8 10

This model of gastric carcinogenesis inwhich the mucosa evolves through a series ofsequential changes, is extremely useful to studygenetic or epigenetic phenomena, which mayoccur at several stages of tumour developmentand which may also serve as intermediate bio-markers. Similar to colorectal carcinogenesis,several genetic events have been described inthis sequence of premalignant changes,"1 12although the exact order in which they occuris still debatable. Allelic deletions of APC/MCC and p53 tumour suppressor genes, aswell as mutations of K-ras proto-oncogene, oramplification ofc-erbB-2 oncogene, are amongthe most commonly described events. 3"15Alterations in DNA methylation patterns areknown to precede the development of a widevariety of human and animal cancers'624 andhave not been previously studied in gastriccarcinogenesis. Both global hypomethylationand regional hypermethylation may occursimultaneously during various stages oftumour

25-28progression.Potential mechanisms whereby dys-

regulation of DNA methylation patterns maypromote malignant transformation includealtered gene expression, increased rate ofmutations secondary to deamination of5-methylcytosine, or alterations in DNA con-formation and chromatin structure.29 3The aims of this study were to examine a

possible relation between global DNA methy-lation level and the histological changes thathave been identified as precursors of theintestinal type of gastric carcinoma.

Methods

PatientsThe study was approved by the ethics andscientific committee of our Institution.

Thirty nine patients referred to the gastro-enterology department of the InstitutoPortugues de Oncologia at Lisbon for an uppergastrointestinal endoscopy between June 1994and January 1995, were included in the study.Patients requiring emergency endoscopy wereexcluded, as well as patients with present orprevious neoplastic disease, previous gastric

Servi9o deGastrenterologiaM CravoR PintoP FidalgoL Gl6riaC Nobre-LeitaoF Costa Mira

Centro de PatobiologiaMolecularM Cravo

Servi9o de PatologiaMorfol6gicaP Chaves

Instituto Portugues deOncologia, FranciscoGentil, Lisbon,PortugalCorrespondence to:Dr M Cravo,Servico de Gastrenterologia,Instituto Portugues deOncologia Francisco Gentil,1093 Lisbon codex, Portugal.Accepted for publication16 April 1996

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Global DNA hypomethylation in early intestinal type gastric carcinoma

surgery, and gastric or duodenal ulcer. Patientswith normal endoscopy and those with antralabnormalities varying from mild erythema tofull blown endoscopic gastritis were included.Additionally, 10 other patients with a diagnosisof intestinal type gastric carcinoma of theantrum referred for surgery during the sameperiod, also entered the study. Thus a total of49 patients with either normal findings (n= 10)or with superficial gastritis (n= 14), chronicatrophic gastritis (n= 15), or intestinal typegastric carcinoma (n= 10) were included in thestudy. The Table shows the age and sexdistributions of the four groups.

In patients submitted to upper endoscopy,gastric biopsy specimens were taken as follows:a minimum of two from the antrum (mid-portion of antral lesser and greater curvatures),one from the transition zone, and at least onefrom the body, midanterior wall. Most of thetime additional specimens were taken and anymacroscopically identifiable lesions werebiopsied. Four to five biopsy specimens werealso taken from the antrum, frozen in liquidnitrogen, and kept at -70°C for later assess-ment of DNA methylation. In patients withgastric cancer submitted to surgery, twofragments of 5 mm each were sectioned fromthe surgical specimen at the time of operation(from neoplastic and non-neoplastic mucosa),immediately frozen in liquid nitrogen, andstored at -70°C for DNA extraction. All biopsyand surgical specimens were fixed in 10%formalin, embedded in paraffin wax, andstained with haematoxylin and eosin. Specialstainings for Helicobacter pylori detection werenot used. The non-neoplastic mucosa wasclassified according to Correa.34 The neo-plasms were histologically classified accordingto Lauren35 and staged according to UnionInternationale Contre Le Cancer criteria.36Biopsy specimens showing intestinal meta-plasia were stained with Alcian blue pH2.5/periodic acid Shiff' and high iron diamine/Alcian blue pH 2.52 to identify subtypes ofintestinal metaplasia.37 These were classified asdescribed by Filipe and Jass.'0

Global DNA methylation assayThe DNA from gastric biopsy specimens wasextracted with a previously described method.38

Clinical and pathological characteristics of the patients included in the study

Histology Normal SG CAG GC(n=10) (n=14) (n=15) (n=10)

Mean age (range) (y) 61 (39-82) 63 (20-81) 59 (38-73) 64 (44-74)Sex (M:F) 5:5 5:9 9:6 6:4Activity of gastritis:None 5 (36) 6 (40)Mild 4 (29) 5 (33)Moderate 2 (14) 1 (7)Severe 3 (21) 3 (20)

Degree of atrophy:Mild 7 (47)Moderate 3 (20)Severe 5 (33)

Helicobacter pylori:Positive 8 (57) 8 (53)Negative 6 (43) 7 (47)

SG=superficial gastritis; CAG=chronic atrophic gastritis; GC=gastric cancer.Values in parentheses are % unless otherwise stated.

Differences in the extent of in vivo DNAmethylation were determined by analysingDNA methyl accepting capacity in thepresence of (3H) S-adenosylmethionine andSssl methylase.39 Briefly 2 ,ug of DNA wereincubated with 5 puCi (3H)-methyl-S-adenosylmethionine((3H)-SAM, 3-10 mCimmol, Amersham, UK) and four units Ssslmethylase (New England Biolabs, Beverly,MA, USA) in 20:1 methylation buffer con-taining 5 mM sodium chloride, 10 mM TRIS,10 mM EDTA, and 1 mM dithiothreitol, pH8.0 for three hours at 37°C. The reaction wasstopped by incubating at 65°C for 20 minutes.The incubation mixtures were washed ontodisks of Whatman DE-8 1 paper (FisherScientific, Springfield, NJ, USA) and thensoaked in 50 ml of 5% dibasic sodium phos-phate. After drying, the radioactivity of theDNA retained in the discs was measured byscintillation counting. All measurements weredone in duplicate. The manner in which thisassay is performed produces a reciprocal re-lation between the endogenous DNA methy-lation status and the exogenous (3H)-methylincorporation. Therefore, a higher incorpor-ation of (3H) methyl groups into DNA reflectsa lower state of intrinsic methylation. Ssslmethylase is a bacterial DNA methyl-transferase that specifically catalyses thetransfer of methyl groups from S-adenosyl-methionine to cytosine residues in thecytosine-guanine doublet. In this regard, itshares the specificity of eukariotic DNAmethyltransferase.

Statistical analysisDifferences among groups were tested by oneway analysis of variance (ANOVA). Associ-ation between variables was assessed byregression analysis. Differences between pro-portions were determined using the x2 test.Results are presented as mean (SEM). All testsare bilateral and the significance level was setat 5%.

ResultsAccording to histological classification, 10patients were considered normal, 14 wereclassified as having superficial gastritis, and 15as having chronic atrophic gastritis. The Tableshows the degree of activity, atrophy, and theprevalence of H pylori infection in specimensfrom patients with chronic gastritis. As far aspatients with gastric cancer were concerned,one patient had stage I disease, four stage II,four stage III, and one stage IV disease. Thenon-neoplastic mucosa of these patients wasexamined as well. In nine of them, chronicatrophic gastritis was present, whereas super-ficial gastritis was found in only one.As shown in the Figure, there was a signifi-

cant and stepwise increase in (3H) methylgroup incorporation into DNA over the rangeextending from normal mucosa, to superficialgastritis and chronic atrophic gastritis(ANOVA, p=0 0002). However, no differenceswere found between the degree of DNA

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(H) methyl group incorporation into DNA from the fourstudy groups. A higher degree of (H) methyl groupincorporation reflects a lower degree of intrinsic DNAmethylation. Hypomethylation ofDNA increasedprogressively from controls, to superficial gastritis, to chronicatrophic gastritis (136 556 (24 085) v 235 725 (38 636) v400 998 (26 747) dpm/,ug/DNA respectively). Differenceswere significant byANOVA (p=0 0002). Nofurtherincrease wasfound in DNA from gastric carcinomas(424 482 (24 321) dpm/,g/DNA). Data are presented asmean (SEM).

methylation in samples from patients withchronic atrophic gastritis and patients withgastric cancer. Also, in samples from patientswith gastric carcinoma, the degree of tritiatedmethyl group incorporation was similar inDNA extracted from non-neoplastic mucosa

and in DNA from the neoplasm itself (408 605(32 322) v 424 482 (24 321) dpm/,g/DNArespectively).

Considering the group of patients withchronic gastritis (n=29), no correlation was

found between degree of activity of gastritisand extent of DNA hypomethylation(p=0.39).When searching for a correlation between

DNA methylation level and degree of atrophy,we found that DNA from patients withmoderate or severe atrophic gastritis (n=8)incorporated more (3H) methyl groups thanDNA from patients with mild atrophy (n=7)(440784 (37657) v 305573 (38584) dpm/,ug/DNA; p=0.01). Also, there was a trendtowards DNA from patients with type IIIintestinal metaplasia (n=5) to be more hypo-methylated than DNA from patients with typeI or II intestinal metaplasia (n=10), althoughit did not reach statistical significance (438 796(65 650) v 374 159 (21 607) dpm/4ig/DNA;p=0.15). H pylon infection did not seem toinfluence significantly the extent of DNAmethylation in gastric mucosa as DNA fromsamples of infected patients was less hypo-methylated than DNA from specimens ofpatients without histological evidence of Hpylori (323 674 (35 990) v 409 079 (29 915)dpm/nipg/DNA; p=0.09).

Considering cases with gastric carcinoma nocorrelation was found between DNA methyl-ation status and stage or grade of differ-

entiation. Hypomethylation ofDNA tended toincrease with aging (r=-025; p=0 14) and DNAfrom samples of female patients was morehypomethylated but not significantly so(p=0.07).

DiscussionColorectal and gastric carcinogenesis are twodistinct processes in the sense that in the firstthere is evidence that there is a field defect ofthe whole colonic mucosa that renders it moresusceptible to neoplastic transformation. In thestomach only the mucosa undergoing atrophyand metaplastic changes is at increased risk.None the less, in both models of tumori-genesis, the mucosa undergoes a series ofsequential changes before malignant transfor-mation takes place. Thus, in this respect andsimilarly to what has been found in colorectalcarcinogenesis, global DNA hypomethylationseems to occur early in the sequence of eventsthat have been postulated to precede thedevelopment of intestinal type of gastriccarcinoma. Our data show a graduated in-crease from normal mucosa, to superficial, andto atrophic gastritis, but there was no furtherincrease in DNA hypomethylation in samplestaken from patients with gastric carcinoma. Inthe colon, Feinberg et a12 also showed a signifi-cant reduction in 5-methylcytosine content incolonic adenomas and adenocarcinomas com-pared with normal, paired matched mucosa,but no differences existed between benign andmalignant neoplasms. By contrast, in cervicaldysplasia and carcinoma, Kim and colleagueshave shown that global DNA hypomethylationoccurs only in the later stages of carcinogenesis(in high grade squamous intraepithelial lesionsand invasive squamous cancer).24

Further supporting the hypothesis that DNAhypomethylation may be a significant event ingastric carcinogenesis, we found that the extentof DNA hypomethylation increased withdegree of atrophy and, more importantly,DNA from biopsy specimens showing type IIIintestinal metaplasia was substantially morehypomethylated than DNA from biopsy speci-mens with type I/II intestinal metaplasia. Dif-ferences did not reach statistical significanceprobably due to few patients with type IIIintestinal metaplasia. Previous retrospectiveand prospective studies'-3 have shown that,compared with type I/II intestinal metaplasia,this type of intestinal metaplasia with incom-plete cell differentiation and sulphomucinsecretion, carries with it a significant risk of theintestinal type of gastric cancer. Retrospectivestudies found that type I and II intestinalmetaplasia are common and prevalent in bothbenign and malignant conditions,40A2 whereasthe incidence of type III intestinal metaplasiain gastric carcinoma is significantly higher thanin chronic gastritis or gastric ulcer. In supportof these data, Rokkas et al,' in a prospectivestudy, found that endoscopic surveillance ofpatients with type III intestinal metaplasia ledto a significant increase in the rate of detectionof early gastric cancer. For these reasons,certain authors think that the presence of these

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Global DNA hypomethylation in early intestinal type gastric carcinoma 437

lesions should lead to prolonged endoscopicsurveillance for early detection of gastriccancer.Although global and gene specific DNA

hypomethylation have been proposed asessential steps in the development of severalhuman neoplasms, the exact part that hypo-methylation plays in carcinogenesis remainsunclear.'1'8 One possible mechanism might berelated to altered gene expression thataccompanies changes in DNA methylation asloss of DNA methyl groups has been shown,in some instances, to increase transcription ofcertain proto-oncogenes, thereby increasingthe risk of malignant transformation.29-33 Thusthe global DNA hypomethylation in the earlystages of gastric carcinogenesis found in thisstudy could be associated with hypomethyl-ation and increased transcription of certainoncogenes implicated in gastric cancer. Futurestudies examining the methylation status ofthese oncogenes are warranted.

Biopsy specimens from patients with chronicgastritis have a heavy inflammatory infiltrateand, therefore, we cannot exclude that thedifferences found between the several samplescould be ascribed to different proportions ofinflammatory cells present in the severalspecimens, especially in regard to the differ-ences found between normal subjects andthose with superficial gastritis. However, thefact that no correlation was found betweendegree of DNA methylation and activity ofgastritis is strongly against this hypothesis.Also, there was a trend towards specimens withH pylori, which are usually associated with adenser inflammatory infiltrate, to exhibit alower degree ofDNA hypomethylation. There-fore, it is highly unlikely that the increase inglobal DNA hypomethylation in specimenswith chronic gastritis is largely attributed todifferent proportions in inflammatory cells. Bycontrast, DNA from precursor lesions, whichhave been strongly associated with anincreased risk of malignant transformation,such as gastric atrophy and type III intestinalmetaplasia, were more heavily hypomethyl-ated. This suggests that the changes in DNAmethylation pattern may be a significantepigenetic phenomenon in the chain of eventsthat precede the development of gastriccancer.DNA hypomethylation has been experi-

mentally induced by severe folate deficiency.39Folate, in the form of methyltetrahydrofolate,is involved in the synthesis of methionine,which is a precursor of S-adenosylmethionine,the methyl group donor for several biologicaltransmethylation reactions, including that ofDNA.42 43 This is thought to be the mechanismby which folate deficiency would increase therisk ofmalignant transformation at several sitessuch as the colon, the cervix, and the bronchialepithelium.20 454 With regard tO gastriccarcinoma, epidemiological and case-cohortstudies have suggested that a low consumptionof fresh vegetables is associated tO an increasedrisk of developing gastric cancer.48~50 Althoughit iS usually accepted that this protective effectis related to the high content of vegetables in

ascorbic acid and carotenoids, it is possible thatthe high content in folate might also accountfor some of the protection found.Although the incidence of gastric

adenocarcinoma is declining worldwide,5' thistumour still remains a major health problem inmost countries. Portugal is one of theEuropean countries with the highest inci-dences. The poor prognosis of advancedgastric carcinoma has not changed sub-stantially over the years52 but this dismal out-come is in sharp contrast with the excellent fiveyear survival rate of surgically treated earlygastric cancer. Screening of large numbers ofasymptomatic patients to detect gastriccarcinoma in its early stages is not consideredfeasible apart from Japan, where about 25% ofall cases of gastric carcinoma are diagnosed inmass surveys.53 It is therefore necessary toidentify subgroups of patients with an in-creased risk of developing gastric carcinomasuch as those with chronic atrophic gastritisand type III intestinal metaplasia, in whom thedevelopment of surveillance programmescould be cost effective. Furthermore, in anattempt to reverse the process of malignanttransformation, these patients could beincluded in chemoprevention trials designed tochange the course of gastric carcinogenesis.Intermediate biomarkers such as global DNAmethylation level could be useful in monitoringthe response to these invervention studies.

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