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A Study of Molecular Resection Margins for Esophageal Squamous Cell Carcinoma Using Large Pathologic Sections

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A Study of Molecular Resection Margins for Esophageal Squamous Cell Carcinoma Using Large Pathologic Sections

A Study of Molecular Resection Margins for

    Esophageal Squamous Cell Carcinoma

    Using Large Pathologic Sections

    AStudyofMolecularResectionMarginsfor EsophagealSquamousCellCarcinomaUsing LargePathologicSections

    GuoweiMa1,

    XuZhang~

    JunyeWang

    QiuliangWu'?0

    HaoLong'?

    PengLin1,

    JianhuaFu

    RichardMaIthaner4

    MeiqingZhao.

    LanjunZhang'?

    ZheshengWen'?

    ~ehuaRong',

    'StateKeyLaboratoryofOncologyin SouthernChina,Guangzhou,

    Guangdong,510060,China.

    DepartmentofThoracicSurgery.Can

    cerCenter,SunYat.SenUniversity, Guangzhou,Guangdong,510060,China. .PathologicalDepartment,CancerCen

    ter,SunYatSenUniversity,Guangzhou,

Guangdong,510060,China.

    DivisionofThoracicSurgeryandSurgi

    caIOncology.LondonHealthSciences

    Centre,UniversityofWesternOntario,

    Canada.

    COrresDOndenceto:TiehuaRong

    TeI:86-20.87343260

    Fax:86.20.87343298

    Email:rongth@sysucc.com

    ThisworkwassupportedbytheChina

    GuangdongProvinceMedicalScience

    ResearchGrant(No.B2003053).

    ReceivedSeptember12,2006;accepted

    October16.2006.

    CJCOhttp://www.qcocrlE-mail:cocr@!eyou.corn

    Tel(Fax):86-22-23522919

    315

    OBJECTlVEIthasbeenshownthatapp?cationofmolecularbiological

    techniquestosurgicaImarginsofsomecancerscouldpredictriskofIo

    caIrecurrenceHowever.theoptimalIengthofsurgicaIresectionwithtu

    morfreesurgicaImargimsforesophagealsquamouscelIcarcinomafES

    CC1isunknownThisstudywasconductedtoevaluatetheoptimalIength ofsurgicalresectionforESCCwithmolecularlytumorfreesurgicalmar

    aimsmarkedbyp53andKi67

    MHODSSurgicalspecimensfrOm70patientswithESCCwerecollect

    edforstudyThelengthsoftheuppermargin.tumOr.andlowermarginof everyspecimenweremeasuredduringtheoperationEachspecimen wasdividedintothreelargepathologicsections.stainedwithH&Eand immunohistochemicallYforp53andKi67,andexaminedmicrOscOpicaIIy. ThelengthsoftheupperandIowerresectionendsweremeasuredfor

p53andKi67positiveexpressionTheactualsurgicaIlengthswerecal

    culatedbytheprincipleofrationaIshrinkage

    RESUL1.sAllsurgicalmarginswerehistologicallytumOrfree,whilethe

    positiveratesofp53andKi67were66%and54%Thepositiveratesof p53andKi67intheupperresectionendwere17%and20%Themean lengthsoftheupperresectionendshowingp53andKi67positiveex

    pressionwere1.08?1.12cmand1.64?101cm.andthemaximum

    lengthswere373cmand326cmThepositive,[atesofp53andKi67in thelowerresectionendwere20%and23%ThemeanlengthsoftheIow

    erresectionendofp53andKi67withpositiveexpressionwere111?1.15

    cmand134~0.94cm.andthemaximumlengthswere373cmand361 Cm

    CONCLUSIONTheoptimallengthofsurgicalresectionwithmolecularly tumOrfreesurgicalmargimsofESCCisnotmorethan5cm

    KEYWORDS:esophagealcancer,surgery,polhology,molecularbiology. Esophagealcanceristhesixthmostcommoncauseofcancer--relat-.eddeathworldwide._1_

    Chinaisoneofthecountrieswithahigh

    incidenceofesophagealcancerandmorethan85percentof esophagealcancerinChinaissquamouscellcarcinoma.Theinci

    denceofesophagealadenocarcinomahasincreasedfromthe1970'sin theUnitedStatesandEurope.

    Traditionally,histopathologicalhematoxylinandeosin(H&E) stainingisusedtohistologicallyassessresectionmarginsinresections ofesophagealcarcinoma.The"molecularresectionmargin"concept wasintroducedintosurgicaloncologywiththedevelopmentof molecularbiology.Geneticandepigeneticalterationsarehallmarksof humancancers,includingprotooncogeneactivationandsuppressor

    geneinactivationviamutations,orloss,andpromoterhypermethyla

    316ChineseJournalofClinicalOncologr2006/Volume3/Number5

    tion.Sincegeneticalterationsprecedephenotypic changes,histologicassessmentalonemaybeinade' quatetoaccuratelydetectthepresenceoftransformed cellsinresectionmargins.[

    Brennanetalf8Jputforwardthe"molecularresec

    tionmargin''conceptforthefirsttimewhentheyused thepolymerasechainreaction(PCR)todetectp53 genemutationsintheresectionmargintissuesofthe headandneck.Theyfoundthateveniftheresection marginswerenegativeusingtraditionalhistopathologi

    calH&Estaining.therateof1ocalrecurrencewashigh inthosepatientswhoseresectionmarginswereposi

    tiveusingmolecularanalysis.

    Shaoeta1.[foundsimilarresultsinstudiesof

    esophagealsquamouscellcarcinoma.TheyusedPCR todetectp53genemutationsin23patientswith esophagealsquamouscellcarcinomawhoseresection marginswerenegativebyhistopathologicalexamina

    tion.Inl3casesofthesewithp53genemutationsin theresectionmargintissue,thesewerefollowedfor 3-16monthsafteroperation.6casesrecurred.Nocas

    esrecurredamongl0caseswithoutp53genemuta. tions.Masasyesvaetal,]10alsofoundthatmolecular

    marginanalysispredictedlocalrecurrenceaftersublo. borresectionoflungcancers.Otherresearchershave reportedsimilarresultssuggestingthattherateoflocal recurrencewashigherinthosepatientswhosemolecu. 1arresectionmarginwaspositive.[11,12] Theabovestudiesdemonstratedthatthepatients withpositivemolecularmarginshavemorelocalre.

    currencesevenwhentheirhistopathologicalresection marginswerenegative.Asignificantlimitationof thesemolecularresectionmarginstudiesistheyjust focusedontherelationshipbetweenlocalrecurrence andapositivemolecularmargin,Surgeonsalsoneed toknowhowtheycanachieveamarginthatisnega. tivebvbOthhistopathologyandmolecularanalysis. Doesthetraditional5.centimetremarginfor

    esophagealsquamouscellcarcinomastillachievea cleanmolecularresectionmargin?Whatistheopti. malresectionlengthtomakesurethatthemolecular resectionmarginiscleanforesophagealsquamouscell carcinoma?Thisstudywasdesignedtoanswerthese questions.

    Thep53gene,isawellknowntumorsuppressor

    gene,thatisbelievedtoserveasagatekeeperagainst carcinogenesis.It3Undernormalcircumstances.the

    functionofthep53proteinistopreventthepropaga

    tionofgeneticallydamagedcells.p53assistsinDNA repairbycausingG1arrestandinducingDNArepair genesordirectsapoptosisincells,whicharegenetical

    lydamagedbeyondrepair.B3]Cellswithalossofp53 functionarebelievedtoundergomalignanttransfor' mation.[t3Gaoeta1.[14demonstratedthatp53protein accumulationandgenemutationmayoccuratvery earlystagesofesophagealcarcinogenesis.Inprevious studies,thep53genehasbeenusedthemostoftenasa molecular.resectionmarginmarker.Wecontinuedto usethep53geneasamolecularmarginmarkerinour study,

    Ki67isanuclearnon.histoneproteinexpressed maximallyincellsintheG2andMphasesofthecell cycle.butabsentinrestingcells.[15Hence,Ki67can

    beemployedtomeasurethegrowthfractionofnormal tissuesandmalignanttumors.[16]Xueta1.]171showed thatexpressionofp53andKi67inthenormal,prema

    lignantandmalignantesophagealtissueshasobvious differences.WechoseKi67asanothermolecularre

    sectionmarkerinourresearch.

    Insummary,theaimofthisstudywastodetermine theoptimalmolecularresectionmarginlengthfor esophagealsquamouscellcarcinoma.Wechoosep53 andKi67asmolecularresectionmarginmarkersand largepathologicsectionsasamethodologyinthis study.

    Patientinformafion

    Specimensfrom70patientswithesophagealsquamous cellcarcinoma.whounderwentresectionsattheCan

    cerCenteroftheSunYat.senUniversitybetweenJuly, 1999toDecember.2000.werecollectedforstudy.All thehistopathologicalresectionmarginsinbothupper andlowerendswerenegative.Thereweretissuesfrom 53malesand17females.Thesitesoftumorswereup

    perthoracicinl2:middlethoracicin47andIower thoracicinll,ThreepatientswereinStageI,35inIIa, 4inIlban(128inIII.ThehistopathologicalGrades wereasfollows:GradeI.37;n.26;andIII,7. LencIthofspecimenmeasurement

    Duringtheoperation,thelengthoftheuppermargin, tumor.andlowermarginweremeasuredbeforethe

    esophagusvvrascut.Atiertheesophaguswiththetumor wasremoved,specimenswereopenedlongitudinally onthesideoppositethetumor.Thelengthoftheupper margin,tumorandlowermarginweremeasuredagain. Alargepathologicsectionwastakenandthelengthof uppermargin,tumorandlowermarginofthelarge pathologicsectionswasmeasuredforthethirdtime.

    Methodstotreatlargepathologicsections Thetechniqueoftreatingthelargepathologicspeci

    mensasbeenpreviouslyreported.[Inbrief:each specimenwaspinnedontoacorkboard.Afterformalin fixationforoneweek.thewholeesophaguswiththe tumormasswasslicedintoalongitudinalstripof about5mmthick.Theywerecomposedofthemaxi

    mallongitudinalpartofthetumor.apartofnormal esophagusproximalanddistaltothetumor.Thespeci

    menwasthendehydratedusing60%.70%,80%and 95%alcoholinturneachfor24handtwiceinl00% alcoholfor2h.Hyalinizationwasconductedbyplace

    ingthespecimenintocloveoilforlhandinto dimethylbenzenefor6h.Thespecimenswerethen placedinparaffinblocksandsliced.Thelargepatho

    logicsectionwasslicedtol0msections,andthe slicesputintowater,andthenplacedontoalargeglass slide.fFigs.12).

    GuoweiMaeta1.3l7

    H&EandimmunohistochemicaIIIHCJstaining Eachspecimenwasdividedintothreelargepathologic sections.Onelargepathologicsectionwasstainedwith

    H&E.theothertwosectionswerestainedforp53and Ki67.Themonoclonalantibodyusedinthisserieswas

    mousemonoclonalprimaryantip53antibodyfDAKO.

    dilutionl:l001andrabbitmonoclonalantiKi67anti.

    bodvfDAKO,dilutionl:80)fromtheDAKOCorp.

    fCarpinteriaCA).Theslideswereviewedandpho

    tographedunderalightmicroscope.Controlswerees

    tablishedbyreplacingtheprimaryantibodywithPBS

    andnormalmouseserum.Knownimmunostain

    ingpositiveslideswereusedaspositivecontrols.

    Scoringofthep53andKi67staining

    Theevaluationoftheimmunohistochemistrywasdone

    bytwopathologistsblindedtotheclinicalandpatho

    logiccharacteristicsofthepatients.Stainingforp53 ABCD

    F_g_1.Processofmakinganesophageallargepathologicsection A:Siteofthetumor.B:Resectionoftheesophagus,C:Specimenswereopenedlongitudinally,

    D:Alongitudinalstripabout5mmthickwassliced,E

    Alargepathologicparaffinblockwasmade.F:Sliceoflargepathologicsection. F.2.Photoofalargepathologicsectionofesophagealsquamouscel carcinoma.Thelargepathologicsectionincludedtheesophagea tumor,theuppermucosaandlowermucosaoftheesophagus. I.

    EsophagusduringoperationEsophagusonthelargesection Fig.3.Extrapolationactuallengthduringtheoperation. A:ProximaIsurgicalresectionlengthduringlheoperation,B:NotionaI safemarginlength.a:ProximaIsurgicalresectionlengthofthelarge pathologicsection.b:Lengthofpositiveexpressionofthelarge pathologicsectionAccordingtotheprincipleofrelativeshrinkage: B/A=b/a.Wecancalculatelhelengthofaloperation:B=Axb/a.

n???几二二]E

    ==f

    一一/

    ??,/一一一(一一(\一一,\

    318ChineseJournalofClinicalOncology2006/Volume3/Number5 Table1.p53ExpressionTable2.Ki67Expression Table3.Lengthdistributionofp53andKi67positiveexpressionbeyondtumor OrKi67wasscoredas+++ifmorethan50%Ofthe cellswereimmunostainedpositive;++if26%to50% ofcellswerepositive;+ifl1%to25%ofcellswere positive;and.iflessthan10%ofcellswerepositive. Thep53statusofthetumorandmucouslayerofthe upperandlowerends,Ki67statusoftumorandmu. COUSlayeroftheupperandlowerends,wereobserved andmeasured.

    Calculationsof1heactuallenglhduringopera- tions

    Duetotheshinkageoftheesophagusduringthepro- cessofmakingthelargeslice,thelengthoftheitems underthemicroscopewasnottheactuallengthprevi- ouslymeasuredduringtheoperation.[18,19]Wecalculat. edtheactuallengthduringtheoperationbytheprinci- pleofrationalshrinkage(Fig.31.

    Followupmethods

    Follow-upstartedaftersurgeryandcontinueduntilthe endofthestudyordeathofthepatient.Datafromall patientswhodiedfromothercausesorwerelostto follow-upwerecensored.

    RESUL1.S

Tumorpositiveexpression

    Ofthetotal70patientswithESCC,46(66%)showed positiveexpressionofp53(Table1)and38(54%)CX- pressedKi67(Table2).

    Lenglhofupperendpositiveexpression

    Inthemucouslayeroftheupperend,12patients showedpositiveexpressionofp53and14patients showedpositiveexpressionofKi67fTables1,21.Most p53positiveexpressionlengthswerelessthan2.0cm (Table3).MostKi67positiveCXpressionlengthswere lessthan3.0cm(Table3).Thelongestlengthsofpos- iriveexpressionofp53andKi67were3.33cmand 3.26cm(Table41.

    Table4.Lengthofp53andKi67positiveexpression

    beyondtumor(cm)

    Lengthsoflowerendpositiveexpression

    Inthemucouslayerofthelowerend,14patientsdis- playedpositiveexpressionofp53and16patientshad positiveexpressionofKi67(Tables1,2).Mostp53

    positiveexpressionlengthswere1essthan2.0cm fTable31andmostKi67positiveexpressionlengths werelessthan3.0cmfTable31.Thelongestlengthsof positiveexpressionofp53andKi67were3.73cmand theIongestIengthofpositiveexpressionofKi67inthe mucouslayerofthedistalendwas3.61cm(Table4). Resultsoffollow-up

    Thirtyfourpatientshaddiedwhenthestudyclosed. Onepatienthadananastomoticrecurrence.Three

    yearsurvivalwas48.4%.Themainmodeoftreatment

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