J Clin Pathol 1997;50:841-846

Altered microsatellites in incomplete-typeintestinal metaplasia adjacent to primary gastriccancers

T Hamamoto, H Yokozaki, S Semba,W Yasui, S Yunotani, K Miyazaki, E Tahara

First Department ofPathology, HiroshimaUniversity School ofMedicine, Hiroshima,JapanH YokozakiS SembaW YasuiE Tahara

Department ofSurgery, Saga MedicalSchool, Saga, JapanT HamamotoS YunotaniK Miyazaki

Correspondence to:Professor Tahara, FirstDepartment of Pathology,Hiroshima University Schoolof Medicine, 1-2-3 Kasumi,Minami-ku, Hiroshima 734,Japan.

Accepted for publication28 July 1997

AbstractAim-To investigate the presence of ge-netic instability in precancerous lesions ofthe stomach.Methods-Fifteen cases of sporadic gas-tric cancers with a background of intesti-nal metaplasia were studied bymicrosatellite assay at nine loci. Alteredmetaplastic mucosa was microdissected,reconstructed topographically, and exam-ined immunohistochemically with ananti-p53 antibody, comparing its positivearea with foci of microsatellite instabilityin each individual.Results-Alterations at one or more lociwere observed in seven of 15 cancers(46.7%) and four of 15 intestinal metapla-sias (26.7%). Two cases of replicationerror positive phenotype had no microsat-ellite alterations in their metaplastic mu-cosa. All the microsatellite alterations inthe metaplastic mucosa were restricted toincomplete-type intestinal metaplasiaaround the respective cancers. Moreover,in one case, an identical pattern ofmicro-satellite alteration was detected in thecancer tissue and in the adjacent meta-plastic mucosa, suggesting the sequentialdevelopment of gastric cancer from intes-tinal metaplasia. Frequent alteration wasfound at the locus D1S191 (lq), indicatingthat this locus might be altered early inthe development of intestinal-type gastriccancer. No significant association betweenmicrosatellite instability and p53 immuno-reactivity was observed in the cases exam-ined.Conclusion-These results indicate thatmicrosatellite instability may be an earlyevent in stomach carcinogenesis, espe-cially in intestinal-type cancers.(7 Clin Pathol 1997;50:841-846)

Keywords: microsatellite instability; replication error;intestinal metaplasia; gastric cancer

Gastric adenocarcinoma is classified histologi-cally into two subtypes: well differentiated orintestinal-type, and poorly differentiated ordiffuse-type.' 2 Frequently, the former is ac-companied by widespread intestinal metaplasiain the vicinity of the tumour, and is generallybelieved to arise via a multistage process,including chronic gastritis and intestinalmetaplasia.3 These morphological changesfrom normal gastric epithelium may occursequentially as a result of exogenous and

endogenous factors that cause genetic altera-tions. Several molecular genetic studies haverevealed that some well differentiated types ofgastric cancer may develop by a cumulativeseries of gene changes similar to those thatoccur in colorectal cancer.4 In fact, in additionto cancer tissue, some gastric intestinal meta-plasias show inactivation of the p535 and theAPC genes,6 and reactivation of telomerase,7indicating that metaplastic glands might be oneof the precancerous lesions of the stomach.Moreover, we have detected microsatelliteinstability in one in three intestinal metaplasiasof the stomach.8Mutations in mismatch repair genes, such as

hMSH2, hMLH1, hPMS 1, hPMS2, andGTBP, responsible for maintaining the fidelityof DNA replication increase spontaneousmutation rates greatly.91-2 Microsatellites aresimple oligonucleotide repeat units, distributedrandomly and widely throughout the genome.The appearances of additions or deletionswithin the microsatellites are known as replica-tion errors or microsatellite instability, and arebelieved to reflect derangement of the mis-match repair system.'3 14 Such genomic insta-bility at microsatellite loci has been observednot only in hereditary non-polyposis colorectalcancer (HNPCC) associated tumours but alsoin certain sporadic cancers.'5"'7 As for gastriccancer, microsatellite instability has beenreported with frequencies ranging from 18% to38%. 16 18-21However, little is known about genomic

instability in precancerous lesions of the stom-ach. To elucidate the role of genetic instabilityin the development of intestinal-type gastriccancer, we conducted a microsatellite assay onboth resected intestinal-type sporadic gastriccancer tissues and the surrounding intestinalmetaplastic mucosa. In addition, as microsatel-lite markers are useful as clonal markers, clon-ality of the somatic mutation within individualmetaplastic mucosas and cancer tissues wasalso analysed by microdissection and topo-graphical mapping.

MethodsPATIENTS AND TISSUE SAMPLESFifteen gastric cancers and correspondingsamples of intestinal metaplasia were studied.All samples were obtained from surgery atHiroshima University Hospital and HiroshimaMemorial Hospital. None of the patients metthe Amsterdam criteria for HNPCC22 accord-ing to their family history collected from areview of medical records. Either non-

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Table 1 Results of microsatellite assay in gastric cancers and intestinal metaplasia

Microsatellite lociIntestinal

Case metaplasia DISI9I D7S486 D11S29 TP53 Dl 7S855 BAT-RII BAT-25 BAT-26 BAT-40

538 Complete +T +T +T +T +T +T

805 Complete +T NI - NI ND - +T +T804 Complete +T - ND662 Complete ND - - ND ND753 Complete - NI- -336 Complete ND LOHM ND - NI834 Incomplete +TM ND - LOHTM545 Incomplete +TM - LOHT - - - - -

742 Incomplete +TM - LOHT - - - - -

969 Incomplete - - - +M ND - - - -

784 Incomplete - ND - ND337 Incomplete - ND - NI368 Incomplete - ND665 Incomplete -312 Incomplete - NI

+, microsatellite altered; -, microsatellite not altered; LOH, loss of heterozygosity; NI, not informative; ND, not determined; T,detected in tumour; M, detected in intestinal metaplasia; TM, detected in both tumour and intestinal metaplasia.

metaplastic normal mucosa or non-metastaticregional lymph node of the stomach was takenas a source of constitutive normal DNA.Tissues were fixed in 10% buffered formalinand embedded in paraffin. All tumours wereintestinal-type gastric cancers comprisingeight well differentiated and seven moderatelydifferentiated tubular adenocarcinomas.Among these 15 subjects, 13 had single earlycancers, one had single advanced cancer(extending into the muscularis propria), andone had double early cancers. Of the non-neoplastic mucosa, six revealed complete-typeintestinal metaplasia and nine had incomplete-type intestinal metaplasia. The definitions ofhistological classification, stage grouping, anddepth of tumour invasion were according tothose of the Japanese Research Society forGastric Cancer.23 Intestinal metaplasia wasclassified by the morphological definitionof Ming."4 Complete-type intestinal metapla-sia is characterised by the presence of gobletcells, absorptive-type enterocytes, and Panethcells, and incomplete-type intestinal metapla-sia is characterised by the loss of Paneth cells,with mucous columnar cells in place ofenterocytes.

DNA EXTRACTIONHaematoxylin and eosin stained serial 10 pimsections of cancer tissues and non-neoplasticmucosa were dissected finely under the micro-scope. After deparaffinisation, DNA was ex-tracted as described previously.8

MICROSATELLITE ANALYSISNine microsatellite loci containing (CA). dinu-cleotide or poly (A),, repeat sequences wereanalysed in this study. Markers for (CA).repeat were D1S191, D7S486, Dl 1S29, TP53and D17S855 (Research Genetics, Huntsville,

Table 2 Frequency of microsatellite instability in gastric cancer and intestinal metaplasia

Cases (n) MI at single locus MI at multiple loci MI at least one locus

Cancer 15 5 (33.3%) 2 (13.3%) 7 (46.7%)Intestinal

metaplasia 15 4 (26.7%) 0 4 (26.7%)Complete 9 0 0 0Incomplete 6 4 0 4

Alabama, USA) on chromosomes 1, 7, 11, and17; markers for poly (A), tract were BAT-RII,BAT-25, BAT-26, and BAT-4025 on chromo-somes 1, 2, and 4. The extracted DNA wasamplified by polymerase chain reaction (PCR)using the same conditions as describedpreviously.26 After 35-40 cycles of PCR with[r32P] ATP labelled primer, the aliquots werefractionated on 6% polyacrylamide/8 M urea/32% formamide gels. Following electrophore-sis, the gels were fixed briefly in 10%methanol/10% acetic acid, dried, and exposedat -80°C to x ray film. Tumours and metapla-sias were judged as microsatellite instabilitypositive if they demonstrated alterations in thesize of the microsatellite sequences, comparedwith the product from the matched normal tis-sue. All markers for unstable cases wererepeated for PCR and gel electrophoresis toconfirm the results.

IMMUNOHISTOCHEMISTRYA monoclonal antibody to p53 (DO7) waspurchased from Novocastra (Newcastle uponTyne, UK). A modification of the immuno-globulin enzyme bridge technique was used, asdescribed previously.27 Deparaffinised and re-hydrated sections were heated by microwaveoven for 10 minutes in citrate buffer to retrieveantigen and were treated with anti-p53 anti-body (diluted 1/1000) at 4°C for eight hours.The immunoreactivity in the tissues wasgraded from - to +++, according to thenumber of cells stained and the intensity of thereaction in individual cells. Grades weredefined as follows: -, almost no positive cells;+, 10-30% of tumour/metaplastic cells show-ing weak to moderate immunoreactivity; ++,30-60% of tumour/metaplastic cells showingmoderate immunoreactivity and/or 10-30% oftumour/metaplastic cells showing intense im-munoreactivity; +++, > 60% of tumour/metaplastic cells showing intense immunoreac-tivity.

ResultsMICROSATELLITE ALTERATIONS ININTESTINAL-TYPE GASTRIC CANCERSThe results of the microsatellite assay ingastric cancers and intestinal metaplasia are

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Microsatellite instability in intestinal metaplasia

834

T M N

545

T M N

742

T M N

969

T M N

i. Ei i.-

A

DlS191 D1S191 D1S191

TP53

Figure 1 Microsatellite alterations detected in intestinal metaplasia of the stomach at lociDISI91 and TP53. Cases are numbered according to table 1. T, tumour;M, intestinalmetaplasia; N, normal mucosa. Both the tumours and intestinal metaplasia from these fourcases (834, 545, 742, and 969) displayed altered alleles at respective loci.

3 10.4 11

5 X i. 12

6 \ 137 <, 14

B

1 2 3 4

summarised in tables 1 and 2. Seven of 15(46.7%) tumour specimens demonstratedaltered mobility of alleles at one or more loci.Out of the seven microsatellite instability posi-tive tumours, two tumour specimens (cases805 and 538) exhibited microsatellite instabil-ity at more than three loci. Among them, case

538 displayed one base pair insertion at theBAT-RII locus. They were interpreted as repli-cation error positive phenotypes, althoughobtained from non-HNPCC individuals.Their samples were microdissected and re-

examined by microsatellite assay at alteredloci. All the altered alleles in cancer tissuedemonstrated the same pattern of alteration ineach individual (data not shown). No signifi-cant difference in the incidence of micro-satellite instability was found between (CA)Lrepeat markers and poly (A). tract markers.Interestingly, frequent alteration (six of 15)was detected at the locus D l S 91 on the longarm of chromosome 1. Loss of hetero-zygosity was observed infrequently at severalloci, but was not regarded as microsatelliteinstability.

MICROSATELLITE ALTERATIONS IN INTESTINAL

METAPLASIA OF THE STOMACH

Four of 15 intestinal metaplasias (26.7%)revealed microsatellite instability at a singlelocus, as well as in corresponding cancer tissue(fig 1). All four were incomplete-typeintestinal metaplasia lacking Paneth cells(table 2). On the other hand, intestinalmetaplasias of two cases with sporadic replica-tion error positive phenotypes in the cancer

tissues did not harbour alteration at anymicrosatellite loci, which was confirmed usingmicrodissecting samples of case 538 (data notshown).

M T T T M M M T T

D1S191

Figure 2 (A) Topographical distribution of amicrosatellite instability positive lesions in case 834. Thetop panel is an illustration of a resected stomach and thebottom panel is a magnification of the lesions around thetumour. The square frame in the top panel indicates thearea examined by the microsatellite assay. A total of 80samples was analysed. Specimen numbers are indicated.Light grey, intestinal metaplasia; dark grey, tumour;cross-hatch, microsatellite instability altered lesion. (B) Arepresentative autoradiograph of a microsatellite assay ofcase 834. Each lane number corresponds to the specimennumber. T, tumour;M, intestinal metaplasia. A differentpattern of microsatellite alteration is seen in the field ofcancer (1 T and 4T) compared with that seen in theadjacent metaplastic mucosa (JM and 4M) at the locusD1S191.

TOPOGRAPHICAL DISTRIBUTION OFMICROSATELLITE ALTERATIONS IN INTESTINALMETAPLASIA OF THE STOMACHThe topographical distributions of microsatel-lite alteration in microsatellite instability posi-tive metaplasia was investigated. Distributionsof microsatellite instability were determinedusing microdissected samples. Representativecases of microsatellite instability positive meta-plasia are shown in figs 2 and 3. A mappingstudy demonstrated that all the microsatellitealterations in metaplastic mucosa were re-stricted to small areas, all of which wereadjacent to respective cancers (fig 2A). Analys-ing the pattern of altered alleles, three caseshad different alleles with altered microsatellites

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surrounding microsatellite instability negativemetaplastic glands (fig 3C).

Figure 3 (A) Topographical distribution of microsatellite instability and p53 positivelesions in case 545. A total of 47 samples was analysed. Specimen numbers are indicated.Medium grey, tumour; light grey, tubular adenoma; cross-hatch, microsatellite instabilityaltered lesion; dark grey, p53 positive lesion. (B) A representative autoradiograph of amicrosatellite assay of case 545. Each lane number corresponds to the specimen number.TX tumour; A, tubular adenoma; M, intestinal metaplasia. Identical and sequentialmicrosatellite alteration isfound in the tumour (7T) and adjacent intestinal metaplasia(8M) at the locus DISI91. (C) Microphotograph of microsatellite instability positivemetaplasia, specimen 8from case 545.

Table 3 Microsatellite instability and p53 status on the cases ofgastric cancers withintestinal metaplasia

Microsatellite instability p53 immunoreactivity

Case Tumour Intestinal metaplasia Tumour Intestinal metaplasia

538 + +805 +804 + ++662763336834 + +545 + + ++ +742 + + +++969 + +784 - -

337 - _ ++368 - -

665 - - ++312

in the field of cancer tissue and adjacent meta-plastic mucosa (fig 2B). Interestingly, in case

545, identical and sequential microsatellitealteration was observed in both the cancer tis-sue and adjacent intestinal metaplasia at thelocus DlS191 (figs 3A and B). Histologically,most of the altered epithelium demonstratedintestinal metaplasia with chronic inflamma-tion and mild atypia compared with the

MICROSATELLITE ALTERATIONS AND ABNORMAL

P53 EXPRESSIONWe performed p53 immunohistochemistry todetermine whether microsatellite instabilitycorrelated with p53 immunoreactivity in gas-tric intestinal metaplasia. Positive nuclearstaining for p53 was detected in six (40.0%) ofthe 15 cancers and one (6.7%) of the intestinalmetaplasias, respectively (table 3). Case 545showed positive immunostaining for p53 in thelower half of the incomplete-type metaplasticglands as well as in the cancer cells. Because thep53 positive area did not coincide with that ofthe microsatellite alterations (fig 3A), thereseemed to be no significant association be-tween microsatellite instability and abnormalaccumulation of p53 protein in intestinalmetaplasia of the stomach.

DiscussionIn this series of intestinal-type gastric cancers,we found microsatellite instability in seven of15 cancers (46.7%) in at least one locus afterexamining nine loci; this frequency is slightlyhigher than that reported previously.8 21 Twocases with microsatellite instability at morethan three loci were regarded as replicationerror positive cancers because widespread mic-rosatellite instability was comparable to thetypical microsatellite instability described inHNPCC associated tumours and representeddefects in the mismatch repair system.28 Inaddition, one of them had an alteration of 10base pair poly (A). tract of the transforminggrowth factor 3 type II receptor, which is acritical target of inactivation in mismatchrepair deficient tumours.25 29The main purpose of the present study was

to clarify the existence of microsatellite insta-bility in intestinal metaplasia, which is impli-cated in stomach carcinogenesis. We detectedmicrosatellite alterations in four of 15 foci ofmetaplasia (26.7%) at a single locus, indicatingthat irreversible genetic changes exist in meta-plastic glands of the stomach.

In addition, we investigated microsatelliteinstability in intestinal metaplasia of the stom-ach by analysing its topographical distributionand allelic pattern. First, microsatellite instabil-ity positive metaplasias were separated intosmall foci, but all of them were restricted toadjacent areas of the respective cancers. Also,the majority of altered alleles exhibited hetero-geneous patterns, suggesting that they mightnot have a common clonal origin. Interestingly,in case 545, an identical and sequential altera-tion pattern was observed in the cancer tissueas well as in the adjacent metaplastic mucosa,indicating single clonal origin and sequentialtumour development from intestinal metapla-sia. From the histological point of view, micro-satellite instability positive metaplasias of fourcases were incomplete-type with regenerativeatypia and chronic inflammation.Our observations suggest that microsatellite

instability may occur multiclonally withincertain fields of the gastric epithelium showing

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incomplete-type intestinal metaplasia, andsome of the gastric cancers may develop fromintestinal metaplasia. Therefore, microsatelliteinstability may represent an early geneticalteration in the development of intestinal-typegastric cancer. Moreover, p53 mutations havebeen detected in incomplete-type intestinalmetaplasia.30 Taken together, incomplete-typemetaplastic glands of the stomach can beviewed as genetically unstable.What is more interesting is the altered

number of microsatellite loci in intestinalmetaplasia. All microsatellite instabilities inintestinal metaplasia were detected at a singlelocus. On the other hand, we could not detectmicrosatellite instability in intestinal metapla-sia surrounding two replication error positivephenotype gastric cancers. Gleeson et aldescribed two types of genomic instability;widespread microsatellite instability and lowlevel microsatellite instability, based on themutated number of microsatellite markerstested and such low level instability may reflectthe inherent instability at microsatellitemarkers.28 Microsatellite instability in intesti-nal metaplasias may reflect the inherent insta-bility at each microsatellite locus. Seruca et alfound that most replication error positive gas-tric cancers displayed abundant T lymphocyteinfiltration.3" Gastric epithelium with continu-ous inflammation might have been exposed toa variety of carcinogens that could facilitateincreased mutagenesis, leading to the low levelmicrosatellite instability. In the present study,frequent instability in both intestinal-typecancer and intestinal metaplasia of the stom-ach was observed at D 1 S 191 on chromosomelq. It is likely that this locus is a specific frag-ile site that may be linked with stomachcarcinogenesis.However, it is still unclear whether micros-

atellite instability plays a pivotal role in stom-ach carcinogenesis, because most microsatel-lite instability occurs in non-coding DNA, thatis, within introns or intergenic regions of thegenome. On the other hand, alterations in thep53 gene are observed in over 60% of gastriccancer cases, regardless of histological type.Moreover, they take place even in the mucosalcancers and in intestinal metaplasia.32 33Among 15 intestinal metaplasias of thestomach, we observed positive nuclear stainingfor p53 in only one case (case 545), whichrevealed microsatellite instability at differentmetaplastic mucosa. However, it was difficultto distinguish morphologically between micro-satellite instability positive metaplastic glandsand p53 positive metaplastic glands. There-fore, there seems to be no apparent associationbetween microsatellite instability and p53alteration.

In conclusion, our results demonstrate thatmicrosatellite instability occurs frequently inthe incomplete type of intestinal metaplasia, acondition that can be viewed as a precancerouslesion of the stomach. Furthermore, it issuggested that microsatellite alterations couldbe useful as a biomarker for the clinical evalua-tion of the malignant potential of gastricnon-neoplastic mucosa.

This work was supported in part by a Grant-in-Aid for CancerResearch from the Ministry of Health and Welfare for the Com-prehensive 10-Year Strategy for Cancer Control and from theMinistry of Education, Science, Culture and Sports, Japan.

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