Response to neoadjuvant treatment in rectal cancer surgery1049033/... · 2016-11-23 · Linköping...

LinköpingUniversityMedicalDissertationNo.1553

Responsetoneoadjuvanttreatmentinrectalcancersurgery

PerLoftås

Department for Clinical and Experimental Medicine, Linköping University, Linköping Division of Surgery

The Faculty of Medicine and Health Sciences, Linköping University SE-581 85 Linköping, Sweden

”Writing a book is an adventure. To begin with it is a toy and an amusement. Then it becomes a mistress, then it becomes a master, then it becomes a tyrant. The last phase is that just as you are about to be reconciled to your servitude, you kill the

monster and fling him to the public.” SirWinstonChurchill.

Tomyfamily

Contents1. Abstract……………………………………………………………. 9

2. Listofpublications…………………………………………... 11

3. Abbreviations………………………………………………… 13

4. SummaryinSwedish……………………………………….. 15

5. Introductionandbackground

Introduction……………………………………………………. 19

Molecularbackgroundtocolorectalcancer………. 21

Radiationtherapy……………………………………………. 25

Neoadjuvantchemotherapy…………………………….. 27Rectalcancerflowchart…………………………………... 28

Surgicaltreatment…………………………………………... 29

Magneticresonanceimaginginrectalcancer.…… 31

6. Aimsofthethesis……………………………………………. 33

7. Patientsandmethods

StudyI……………………………………………………………. 35StudyII…………………………………………………………… 37StudyIII………………………………………………………….. 39StudyIV………………………………………………………….. 41

8. Resultsanddiscussion

StudyI……………………………………………………………. 43StudyII…………………………………………………………… 45StudyIII………………………………………………………….. 47StudyIV………………………………………………………….. 51

9. Generaldiscussionandfutureperspectives……... 53

10. Acknowledgements…………………………………………. 57

11. References……………………………………………………… 61

12. StudiesI-IV…………………………………………………….. 67

AbstractRectalcancerisoneofthethreemostcommonmalignanciesinSwedenwithanannualincidenceofabout2000cases.Currenttreatmentconsistsofsurgicalresectionoftherectumincludingtheloco-regionallymphnodesinthemesorectum.Inadvancedcases,neoadjuvantchemo-radiotherapy(CRT)priortotheoperativetreatmentreduceslocalrecurrencesandenablessurgery.Theneoadjuvanttreatmentcanalsoeradicatethetumourcompletely,i.e.completeresponse.Thisresearchprojectwasdesignedtoinvestigatetheeffectsofpreoperativeradiotherapy/CRTandanalyzemethodstopredictresponsetoCRT.StudyIinvestigatedtheexpressionoftheFXYD-3proteinwithimmunohistochemistryinrectalcancer,withorwithoutpreoperativeradiotherapy.Theresultsfromthetotalcohortshowedthat,strongFXYD-3expressionwascorrelatedtoinfiltrativetumourgrowth(p=0.02).Intheradiotherapygroup,strongFXYD-3expressionwasrelatedtoanunfavourableprognosis(p=0.02).TumourswithstrongFXYD-3expressionhadlesstumournecrosis(p=0.02)afterradiotherapy.FXYD-3expressionintheprimarytumourwasincreasedcomparedtonormalmucosa(p=0.008).WeconcludedthatFXYD-3expressionwasaprognosticfactorinpatientsreceivingpreoperativeradiotherapyforrectalcancer.StudyIIinvestigatedFXYD-3expressionintumoursthatdevelopedlocalrecurrencesfollowingsurgeryandcomparedthiswithexpressionintumoursthatdidnotdeveloplocalrecurrences.TherewasnodifferenceintheexpressionofFXYD-3betweenthegroupthatdevelopedlocalrecurrencesandthegroupthatdidnotdeveloplocalrecurrences.TherewasnodifferenceinsurvivalbetweenthosewithstrongorweakFXYD-3expression.Weconcludedthatthisstudycouldnotconfirmthefindingsfromstudy1i.e.thatFXYD-3expressionhasprognosticsignificanceinrectalcancer.StudyIIIwasaregister-basedstudyontheincidenceandeffectsofcompleteresponsetoneoadjuvanttreatment.EightpercentofthepatientswithadequateCRTtoachievecompleteresponsealsohadacompletehistologicalresponseoftheluminaltumorintheresectedbowel.Sixteenpercentofthatgrouphadremaininglymphnodemetastasesintheoperativespecimen.Chemotherapytogetherwithradiotherapydoubledthechanceofcompleteresponseintheluminaltumour.Patientswithremaininglymphnodemetastaseshadalowersurvivalratecomparedtothosewithout.Weconcludedthatresidualnodalinvolvementafterneoadjuvanttreatmentwasanimportantfactorforreducedsurvivalaftercompleteresponseintheluminaltumour.StudyIVfolloweduptheresultsfromthepreviousstudybyre-evaluatingmagneticresonanceimaging(MRI)-imagesinpatientswithcompletetumourresponse.TwoexperiencedMRIradiologistsperformedblindedre-stagingofpostCRTMR-imagesfrompatientswithcompleteresponseintheluminaltumour.Onegroupwithlymphnodemetastasesandanotheronewithoutwerestudiedandtheresultscomparedwiththepathologyreports.Thesensitivity,specificity,andpositiveandnegativepredictedvaluesforcorrectstagingofpositivelymphnodeswas37%, 84%, 70% and 57%. Thesizeofthelargestlymphnode(4.5mm,p=0.04)seemedtoindicatepresenceofatumourpositivelymphnode.WeconcludedthatMRIcouldn’tcorrectlystagepatientsforlymphnodemetastasesinpatientswithcompleteresponsetoCRTintheluminaltumour.

Listofpublications 1. LoftåsP,ÖnnesjöS,WidegrenE,AdellG,KayedH,KleeffJ,ZentgrafH,SunX-F.ExpressionofFXYD-3isanIndependentPrognosticFactorinRectalCancerPatientsWithPreoperativeRadiotherapy.InternationalJournalofRadiationOncologyBiologyPhysics2009;75(1):137-142.2. LoftasP,ArbmanG,SunXF,EdlerD,SykE,HallbookO.FXYD-3expressioninrelationtolocalrecurrenceofrectalcancer.Radiationoncologyjournal2016;34(1):52-58.3. LoftasP,ArbmanG,FomichovV,HallbookO.Nodalinvolvementinluminalcompleteresponseafterneoadjuvanttreatmentforrectalcancer.Europeanjournalofsurgicaloncology:thejournaloftheEuropeanSocietyofSurgicalOncologyandtheBritishAssociationofSurgicalOncology2016;42(6):801-807.4.LoftåsP,Sturludóttir,HallböökO,AlmlövK,ArbmanG,BlomqvistL.AssessmentofremainingtumourpositivelymphnodeswithMRIinpatientswithcompleteluminalresponseafterneoadjuvanttreatmentofrectalcancerInmanuscript.

Abbreviations

• 3-D ThreeDimensional• 5-FU 5-Fluorouracil• AJCC AmericanJointCommitteeonCancer• AR AnteriorResection• APR AbdominoPerinealResection.• ATP AdenosinTriPhosphate• C.I ConfidenceInterval• cCR ClinicalCompleteResponse• CRM CircumferentialResectionMargin• CRT Chemo-RadioTherapy• DAB DiAmineBenzidinetetrahydrochloride• DCE DynamicContrastEnhanced• EMVI ExtraMuralVenousInvasion• FXYD-3 Probablyeponymfor“fixedID”• Gy Gray• IQR InterQuartileRange• kD/kDa kiloDalton• LCR LuminalCompleteResponse• LR LocalRecurrence• LRT LongtermRadiotherapyTreatment• MAT-8 MammaryTumourProtein• MCF-7 MichiganCancerFoundation• MRI MagneticResonanceImaging• mRNA MessengerRiboNukleinAcid• MDT MultiDisciplinaryTeam• MV MegaVolt• NCR NationalCancerRegistry• NPV NegativePredictiveValue• p21 Protein21• p53 Protein53• P73 Protein73• PBS PhosphateBufferedSaline• pCR pathologicCompleteResponse• PPV PositivePredictiveValue• PRL PhosphataseofRegeneratingLiver• R0 PathologicallyconfirmedRadicalResection• RT Radiotherapy• SCRCR SwedishColoRectalCancerRegister• SPSS StatisticalPackagefortheSocialScience• SRT Short-termRadiotherapyTreatment• T Tesla/Tumour• T1-T2-weigted Tissue-weighted• TME TotalMesorectalExcision• TNM Tumour,Node,Metastasis(classification)

Sammanfattningpåsvenska Ändtarms-ochtjocktarmscancerärtvåavdevanligastecancerformernaiSverigeochivästvärlden.Ändtarmscancerdrabbarvanligtvisindivideriåldern70-75år,mensjukdomenkanävendrabbabetydligtyngre.Denärnågotvanligarehosmänänhoskvinnor.Precissomfördeflestaandracancersjukdomarfinnsingenenskildorsaktillattmandrabbasavsjukdomen,menvästerländskkost,vissakroniskatarmsjukdomarsamtärftlighetkanökarisken.Somallaandracancersjukdomarkanändtarmscancerspridasigikroppenmeddottertumörer,vanligtvisileverochlungor.Iundantagsfallåterkommertumörenävenibäckenetefterbotandebehandling,såkallatlokalrecidiv.Behandlingenförändtarmscancerharlängevaritkirurgidåhelaellerdelaravändtarmenopererasbort.Iblandtasocksåändtarmsöppningenbortochpatientenfårenpermanenttarmöppningpåmagen,enstomi.Ompatientenstrålbehandlasföreoperationenminskarriskenattfååterfallibäckenet.Strålningenminskarocksåtumörensstorlekvilketgerbättreförutsättningarföroperation.Behandlingmedbådecellgifterochstrålningäreffektivareochkanävenpåverkatumörcellersomtagitsigvidareikroppen.Kombinationenavstrålningochcellgifteräriblandsålyckadatthelatumörenochäventumörsomspriditsigtillderegionalalymfkörtlarnaförsvinnerheltinnandenplaneradeoperationen.Hittillsharpatientenändåblivitopereradeftersomdetansesvaradensäkrastemetoden.Bådestrålningochcellgifterharsvåraochmångagångerlivslångabiverkningarochdessamåstevägasmotvinsternamedbehandlingen.Detinnebärattbarapatientermedmeravanceradsjukdomfårförbehandlingmedstrålningoch/ellercellgiftereftersomnyttanavbehandlingenidefallenvägertyngreänbiverkningarna.Denförstastudienfokuseradepåettprotein,FXYD-3.Allatumörerinnehållerintedettaprotein,medanendelharhögahalteravproteinet,andraavsevärtlägre.Syftetmedstudienvarattundersökaomtumörersomharhögahalteravdettaproteinharensämreprognosochdärmedettstörrebehovavstrålbehandling.StudienvisadeattFXYD-3varvanligareitumörvävnadäninormalvävnad.TumörersomhademycketFXYD-3växteocksåaggressivareäntumörersomintehadesåhögahalteravproteinet.HosdepatientersomstråladesinnanoperationenkundemanseattmycketFXYD-3itumörenmedfördesämreöverlevnadochsämresvarpåstrålbehandlingen.Dessaskillnaderkundemaninteseidenpatientgruppsominteficknågonstrålbehandling.

TolkningenavdessaresultatärattFXYD-3verkarkunnatydliggöravilkapatientersomharenmeraggressivsjukdomochsomdärförintesvararlikabrapåstrålbehandlingochdärmedocksåfårensämreprognos.DenandrastudienärenfortsättningpådenförstadärvivillebekräftaattdepatientersomharFXYD-3itumörenkanförutsägashasämreresultatavstrålbehandling.Dådettydligasteresultatetavstrålbehandlinginnanoperationvisatsigvaraenminskningavantaletåterfallibäckenet,lokalrecidiv,valdeviattanalyseratumörersomutvecklatlokalrecidiv.IdenhärstudiensamarbetadevimedKarolinskaInstitutetiStockholmsomtidigarestuderatsammapatientgrupp.VistuderademängdenFXYD-3hospatientermedlokalrecidiv,ochjämfördedemmedengrupppatientersominteutvecklatlokalrecidiv.VikundeintebekräftaatttumörermedmycketFXYD-3ihögregradriskeraråterfalltillbäckenet.TolkningenförsvårasdelsavattdetvarfåpatientermedmycketFXYD-3itumörensomfickstrålningochävenutveckladelokalrecidiv.Idenhärstudienkundeviintebekräftaresultatetidenförstastudien.Identredjestudienvaldeviattstuderadenpatientgruppsomfickettsåbraresultatavstrål-ochcellgiftsbehandlingeninnanoperationatthelatumörenförsvann,såkalladcompleteresponse.Vivillegenomregisterforskningtaredaförekomstochprognosfördessapatientersomopereradesutanattmanfannnågonkvarvarandetumörvidmikroskopiskundersökningavdenbortopereradetarmen,Knappt30procentfårensåpassavanceradbehandlingmedcellgifteroch/ellerstrålningattdetfinnsenteoretiskmöjlighetatttumörenkanförsvinna.Avdessahadeåttaprocentingenkvarvarandetumöridenbortopereradeändtarmenviddenefterföljandemikroskopiskaundersökningen.Avdepatienterdärtumörenförsvunnithelthade16procenttrotsdetenspridningavsjukdomentillregionalalymfkörtlar.Spridningtilldessalymfkörtlarinnebärenökadriskförspridningavsjukdomentillandraorgan.Detvisadesigocksåattdessapatienterhadesämreöverlevnadändepatientersomintehadenågonspridningtilllymfkörtlar,trotsattbådeändtarmmedlymfkörtlarvarbortopererade.Denfjärdestudienföljeruppresultatetavdentredje.Iochmedattantaletpatientermedcompleteresponseblirflersåharmaniandraländerbörjatavståfrånattopereradessapatienter.Dettaförattpatientenskaslippaenstoroperationmedstomisomföljdeftersomtumörenredantycksvaraborta.Detärrelativtenkeltattfastställaomtumörenitarmenförsvunnithelt,mendetärsvårareattmedmagnetröntgenskiljafriskalymfkörtlarfråndesomfåttspridningavtumörer.Dåvivetfråndenföregåendestudienattdetinteärheltovanligtmedspridningtilllymfkörtlarävenefteratttumörenförsvunnit,ochattdetdessutommedförsämreöverlevnad,ärdetviktigtattidentifieradepatientersomfåttensådanspridningtillkörtlarnainnandetfattasbeslutomattinteoperera.StudiengenomfördesisamarbetemedKarolinskaInstitutetiStockholm.Tvåerfarnaröntgenläkaremedstorkunskapiattbedömamagnetröntgenavdenhärtypenavpatienterfickbedömatidigaremagnetröntgenundersökningarfråntvågrupperavpatienter.Bådagruppernahadeändtarmscancerochsamtligahadefåttstrålbehandling,medellerutancellgiftsbehandlingsåatttumörenförsvunnitochdärefteropereratssåattoperationspreparatenkundeundersökas.Allapatienterhadeocksågenomgåttmagnetundersökningavbäckenetföreochefterstrål-/cellgiftsbehandlingen,meninnanoperationen.Hälftenavpatienternahadetumörkvarilymfkörtlarnamedandenandra

hälftenintehadedet.Röntgenläkarnasomgjordebedömningarnavissteintevilkapatientersomhadefriskalymfkörtlarochvilkasomintehadedet.Närvisammanställderesultatetvisadedetsigattmanintemedtillräckligstorsäkerhetvaresigkundebekräftaelleruteslutatumörilymfkörtlarnamedmagnetröntgenundersökning.BedömningavdenstörstalymfkörtelnsstorlekhosvarjepatientvisadesigkunnavaraettsättattidentifieradepatientersomhadeenspridningavtumörertilllymfkörtlarSlutsatsenblevattmagnetröntgenundersökninginteensamtkananvändasförattbedömaomkörtelnärfriskellersjukpådessapatienter,menattstorlekenpådenstörstakörtelnskullekunnaanvändasförattpåvisamisstankeomattpatientenharfåttenspridningavsjukdomentilllymfkörteln

Introduction.Colorectalcancerisanage-dependentmalignancythatisthesecondmostcommoncauseofcancer-relateddeathintheWesternworldandthesecondmostcommoncancerinwomenandmeninSweden.TheincidenceisalmostequalinmenandwomeninandisfairlystablethoughthereisaslightincreaseduetotheincreaseinaverageageinSweden.Rectalcanceraccountsforapproximately30%ofallcolorectalmalignancies(1,2).Theintroductionofpreoperativeradiotherapy(RT)inthetreatmentofrectalcancerhasreducedthefrequencyoflocalrecurrence,andRTisnowapartofthestandardtreatmentregimeforadvancedrectalcancer.Radiotherapyisoftencombinedwithchemotherapy,chemoradiotherapy(CRT).CRTinducesdownsizinganddownstagingofthetumourandregionallymphnodestherebyenablingsurgeryofanotherwiseinoperabletumour(1,3,4).Therearewell-knownshort-termandlong-termside-effectsofpelvicradiationandchemotherapy.FurthermorestudieshaveestablishedthattheresponsetoRTishighlyindividualandthatthismaypartlybeexplainedatthemolecularlevel(5-8).Inordertoavoidunnecessarysideeffectsitisimportanttodiscoverbiologicalfactorsthatcouldinfluencerecurrenceandsurvival,andtoidentifypatientsthatwillprobablynotbenefitfromRT/CRT.Anumberofclinicallyavailablebiomarkershavebeeninvestigatedastotheirabilitytopredictradiosensitivity,butsofarnonehascomeintoclinicaluse.Someareregardedaspromising.Examplesofsuchbiomarkersareepidermalgrowthfactor,(EGFR)thymidylatesynthase,p21andcarcinoembryonicantigen (CEA)(8-10).Adependablepredictorforneoadjuvanttreatmentresponsewouldspareprobablenon-respondersunnecessarysideeffectsandreduceunduetimewaitingforsurgery.Identificationofpotentialresponderswouldenablemoretailoredneoadjuvanttreatmentandindividualfollowupprogrammes.Inpatientswithanoutstandingresponsetoneoadjuvanttreatmenttheentireluminaltumourmayhavedisappearedpriortosurgery,aso-calledcompleteresponse,(ypT0N0M0).Extendedtimesbetweenchemoradiotherapyandsurgery,novelchemotherapeuticagentsandchangeinindicationsforneoadjuvanttreatmenthasledtoanincreaseinthefrequencyofcompleteresponse(11-15).Completeresponsecanoccurasaresultofstandardtreatmentfollowingnationalguidelinesforpreoperativedownstaging.Itcouldalsobeviewedasapreferredoutcomeinagoaldirectedtreatmentaimingtoavoidsurgery.Hencetheindicationsforneoadjuvanttreatmentcanbeextendedtoincludelessadvancedtumoursaswell(11).Therateofcompleteresponsevarieswiththeindication,asthereisabetterchanceofcompleteresponseinthelessadvancedtumours(11,16).Somesingleinstitutioncentershavepresentedhighcompleteresponseratesinsmalltumoursandinahighlyselectedpatientpopulation(12).Thereisaneedtoknowthefrequencyofcompleteresponseinalargeunselectedcohortfollowingnationalguidelineswherefactorsinfluencingincidenceandoutcomemaybeanalyzed.Whenconsideringaconservativewait-and-watchstrategyinpatientswithcompleteresponseboththesurgeonandthepatientneedtoknowtheindividualriskfactorsforrecurrentdiseaseandthelimitationsoftheimagingtechniquesusedforstagingandfollowup(17).Stagingandrestagingisofgreatimportancewhendecidingonneoadjuvanttreatmentandnon-surgicalmanagement.Asyettherearenoimplementedguidelinesforfollow-upafternon-surgicalmanagement.Highlyfrequentfollow-upsfor

manyyearsincludingphysicalexamination,endoscopy,CT-scansandMRIarenecessary(17,18)Figure1.MRIandendoscopicimagesofrectaltumoursbeforeandafterCRT,withluminalcompleteresponseinthesamepatient.

A. SagittalMRT2imageofrectalcancer(markedT).B. EndoscopicviewofthetumourinsamepatientC. PostCRTMRT2imagefromthesamepatientwithnovisibletumour,(arrow).D. PostCRTendoscopicviewshowingonlytelangiectasiaattheformertumoursite

(arrow).

Themolecularbackgroundtocolorectalcancer.Wedonotknowanysingleetiologyofcolorectalcanceroranyendogenousorexogenouscausativecarcinogensresponsibleforthedevelopmentofcolorectalcancer.Everyhumanisbuiltofapproximately60billioncellsthatareconstantlydividing.WitheverycelldivisionthereisariskforerrorintheDNAreplicationthatmaybeinheritedandmultiplied.Thelongerwelivethegreaterthenumberofmitosesthathaveoccurred,aswellasthegreatertheexposuretoenvironmentalcarcinogenssuchasradiation,chemicalsanddrugs.Veryeffectivecellularsystemexistsinthebodythatrepairorterminatemutationsinacellthatcouldotherwisebereplicatedandpassedontodaughtercells.Thissystemofrepairishowevernotfoolproof,andsomemutationsarenotcorrected.Thesemutationstogetherwithaccumulatedoncogeniceventseventuallyleadtothedevelopmentofcancer(19).In1990FearonandVogelsteinproposedtheadenomatocarcinomasequenceasamodelwithsuccessivegeneticmutationsleadingtocolorectalcancer.Thismodeldescribesthestepwisetransitionfromnormalepitheliumtocancerthroughmultiplemutationsinoncogenicandtumoursuppressorgenes(20)(Figure2and3).Figure2.Theadenoma-carcinomasequence.(ModifiedfromFearon-Vogelstein(20))

Normalepithelium

Earlyadenoma

Dysplasticadenoma

Carcinoma

Metastases

APC,COX2

Ki-RAS,DCC,SMAD4

ECMdegradation

Despitealmost25yearsofresearchintothedevelopmentofcancer,theVogelsteinmodel,withsomeimportantmodificationsstillstandsastheacceptedmodelforcolorectalcancerdevelopment,thoughthereareothermodels,suchasthetheoryofcellfusion(21,22).Weknowthatmanymutationstakeplaceinthevariousstepstowardsinvasivecancer,andthegreaterthenumberofmutationsoccurringthegreatertheriskforcancer.Furthermore,thecumulativenumberofmutationsismoreimportantthanthesequentialorder(20)(Figure2)Figure3.Commononcogenesincolorectalcancer.

Oncogenesincolorectalcancer

Protooncogenes

K-ras(Kirsten-ratsarcoma)

Supressorgenes

APC(AdenomatousPolyposisColi)

p53(Protein53kDaDCC(DeletedinColorectalCarcinoma)DNAmismatchrepairgenes.(Resposibleinhereditarynonpolyposiscoloncancer(HNPCC))hMSH2hMLH1hPMS1hPMS2

Thecolonandrectumareformedof107glandularcryptseachwiththousandsofdifferentiatedcellsofpolyclonaloriginandasmallnumberofstemcells(lessthan10percrypt).Thesestemcellsareprotectedfromthehostileintestinalenvironmentatthebottomofthecrypts.Thedifferentiatedcellsdividerapidlyandmigratetothetopofthecryptwhere1010cellsareshedeachday(19).Proliferationislimitedtothelowertwothirdsofthecryptsandthematurecellsareshedatthesurface.ThefirststepinthemonoclonalmalignanttransformationiscausedbyindividualmutationstobothallelesofthetumoursuppressorAdenomatousPolyposisColi(APC)gene.Thisinitialmutationshiftscellproliferationtotheupperportionofthecryptwherethedividingcellsaremoreexposedtothehazardouscontentsofthebowel.TheAPCgeneisarestrainerofproliferationandthelackofthisfunctionleadstouncontrolledcellgrowthandtheformationofapolyp(20).OverexpressionofCOX1andCOX2cyclooxygenases,catalyzesthearachidonicacidderivatemalondialdehydewhichitselfismutagenicandintensifiesthemalignantprocessbypromotingcellmigrationandproductionofangiogenicstimulatoryfactors(23).Thisisthoughttooccurafterthemutationofthesecondalleleofthegene.TheseinitialstepsofmalignanttransformationarereversibleasseenafteradministrationofNSAID,wherethereisareductioninbothsizeandnumberofpolypsthroughCOX2inhibition,thisledtotheconceptofCOX2inhibitorsinprophylaxisortreatmentofcolonadenomas(24).

FurthermutationsareneededtotransformtheadenomatoinvasivecancerandamutationoftheKi-rasproto-oncogeneincreasescelldivisionandleadstoincreaseddysplasiaintheadenoma.MutatedKi-rasisnotexpressedinearlybenignadenomasbutincreasesinexpressionindysplasticpolypsandcancersandisthusimportantformalignanttransformationinmany,butnotallcolorectalcancers(25).MutationsintheDeletedinColorectalCarcinoma(DCC)geneandSMAD4gene,bothofwhichplayaroleincelladhesionandstromalproliferation(20,26)arealsoimportantsteps.Oneofthefinalstepsonthepathtomalignancyisthemutationofbothalleles(doublehit)ofthetumoursuppressorgenep53.Thep53isknownasthe“guardianofthegenome”byrecognizingDNAdamageandinducingG1cellcyclearrestduringDNAreparationor,inthecaseofirreparabledamages,initiatingapoptosis(27).Mutationsofthep53genearefoundinin75%ofallcolorectalcancercellsbutarerareinadenomas(28).Mutationofp53resultsinreducedp21transcription,cumulativeDNAdamageanduncontrolledprematurereplicationofp53seemstohaveamoderateeffectinnormalcellsbuthasagreatereffectincellsstressedbyradiationorcytotoxicdrugs(29).

DNAMismatchRepairgenes(MMR)areresponsiblefortherepairofmismatchesinbasepairs.CellswithabnormallyfunctioningMMRareunabletocorrecterrorsthatoccurduringDNAreplicationandconsequentlyDNAerrorsaccumulate.Thisleadstothecreationofnovelmicrosatellitefragments.MicrosatellitesarecomposedofrepeatedDNAsequences.Thesesequencescanbemadeupofrepeatingunitsofonetosixbasepairsinlength.Mutationofthegenewillresultindefectiverepair,microsatelliteinstabilityandanincreaseindysplasia,bothofwhichareseenin13%ofsporadiccolorectalcarcinomasandinallHNPCC(30).

Theabilitytometastasizeistheleastunderstoodstageinthestepwiseprogressiontheory.Forcellstobeabletometastasizethecell-celladhesionsmustbedisrupted.ReductionincelladhesionsispartlyduereducedexpressionofEcadherin(31).VariousmetalloproteasestakepartinaproteolyticcascadethatdegradesECMcomponentsanddisruptsextracellularmatrix(ECM)cellinteraction(32).Intravasationofmalignantcellsintothebloodstreamandresistancetothemajorhistocompabilitycomplexsystem,whichremovescirculatingtumourcells,ispoorlyunderstood.Animalstudieshavedemonstratedthatonlyasmallportionofthemalignantcellsenteringthebloodstreamhavetheabilitytoestablishmetastasesbyavoidingbeingkilledbycirculatingimmunecellsorbyestablishingacolonyatadistantsite.Carcinoembryonicantigenthatisusedasatumourmarkerincolorectalcancerisbelievedtoplayaroleinthisprocessaswellasendothelialgrowthfactorthatisatargetfornovelcytotoxicdrugs(33,34).

Radiationtherapy.Theeffectsofradiationtherapymaybedividedintothreeseparatephases.ThephysicalphaseThephysicalphaseisshortandbasedontheinteractionsofchargedparticleswiththetissueDNA.Ittakesanelectron10-18secondstopassthroughtheDNAmoleculeandthejourneythroughtheentirecelltakesaround10-14s.Duringthisshorttimetheelectronorphotonwillinteractandejectelectronsfromatoms(ionization).Theseejectedelectronsmayionizenearbyatomsgivingrisetoanionizationcascade.ForeveryGrayofabsorbedradiation105ionizationstakeplaceforevery10µmoftissuepenetrated.ThechemicalphaseThechemicalphaseisthetimeperiodwhendamagedatomsreactwithcellularcomponents,breakingchemicalbondsandformingsplitmolecules,i.e.freeradicals.Duringthistimespanof1ms,protectivescavengersreactionsincludingsulphydrylcompounds,chemicallyinactivatethesefreeradicals.ThebiologicalphaseThisphaseincludesallotherprocessesthatfollow.MostdamagedDNAissuccessfullyrepaired.However,cancercellswithmutationsingenes,suchasp53arerapidlydividingandhaveadefectDNArepairsystemcausingincreasedcelldeathcomparedtonormaltissueafterradiation.Whenthemutationsleadtocelldeath,aprocessthattakestime,acellmayundergomanydivisionsbeforeitdies.Theearlyeffectsofradiationonskin,mucosaorbloodcomponentsareduetothekillingofstemcells.ThelateeffectsofradiationtherapysuchassecondarytumoursarealsoduetotheinitialDNAdamage(35).Radiationtherapyisusedasneoadjuvanttreatmentforrectalcancereitheraloneorincombinationwithchemotherapy.Twodifferentregimensexists,Short-termRadiotherapy(SRTe.g.5x5Gy)isdeliveredoverfivedaysfollowedbysurgerywithinfivedays.TheindicationisusuallyT3btumoursinthemiddleandlowerrectumaswellasmesorectalN+tumoursprovidedtheCRMisclear.SRThasnodownstagingeffectasthetimebetweenradiationandsurgeryistooshort.Itisusedtosterilizethepelviclymphnodesinordertoreducelocalrecurrencei.e.withinthepelvis(1).HoweversurgerymaybepostponedforseveralreasonstherebyincreasingthechancethatSRTalsohasadown-stagingeffect.Thisistermed“SRTwithdelay”whichisusuallydefinedasadelayofmorethanfourweeksbetweenradiotherapyandsurgery.LongTermRadiotherapy(LRT(e.g.28x1.8-2Gy),hasanintervalof6-8weeksbeforesurgeryandcanleadtosterilizationanddownstagingofboththetumourandadjacentlymphnodesandisindicatedforadvancedT3andT4tumours.Thereareseveralshort-andlong-termsideeffectsofpelvicradiationsuchasurinaryandfecalincontinence,sexualdysfunction,peripheralnervedamageandpathologicalfractures.Smallboweltoxicitywithanincreasedriskforbowelobstructionisprobablythemostfrequentsideeffect(5,36,37).Theincidenceofsevereradiotoxicitywithleukopenia,abdominalpainanddiarrheaisaround5%(38,39).ThelateeffectsofRTmayaddtothecomplicationsofpelvicsurgery.Bothirradiationandthesurgicaltraumainthevicinityofpelvicnervescanharmthenervescontrollingsexualfunctionandcontinenceofboththebowelandbladder.

TheincreasedriskfornervedamageafterRT,independentofsurgicaltrauma,hasbeendescribedinseveralstudies(5,36).Theriskforsecondarycancerisincreasedafterirradiation(36)SeveralstudieshavedemonstratedthatLRTandSRTwithdelayhavecomparabledownstagingeffectandreductionintherateoflocalrecurrence(38-40).Thereisacleardose-responsebetweentheRT-doseandthetumourdownstagingwhenthesamefractionationsarecompared(35,41).WithtailoredextendeddelaysbetweenCRTandsurgery,anincreaseinthefrequencyofcompletepathologicalresponse,(ypT0N0M0)isevident(11-15).

NeoadjuvantchemotherapyThefluoropyrimidines(5-fluorouracilandfluorodeoxyuridine),whengivenincombinationwithradiationincreasetheeffectivenessofneoadjuvanttreatment.IncreasedradiationsensitivityoccursincellsthatprogressinappropriatelyintotheSphaseduetoDNAdamageinflictedbyradiation.Thiscouldprovidethemolecularbasisfortheselectivekillingoftumourcellsratherthannormaltissuecells(42).FluoropyrimidinebasedchemotherapycanbeusedinconjunctionwithbothLRTandSRT,inthemostadvancedformsofrectalcancer(chemoradiotherapy,CRT),actingasaradio-sensitizertoincreasetheeffectofradiotherapy(43).CRTincreasestherateofcompleteresponsecomparedtoradiotherapyalone(40).Cisplatin,aplatinumbasedchemotherapy,hasmultipleeffectsoncells,oneofthembeingDNAdamagerepairinhibitionbuttheexactmechanismisnotknown(42).Theadditionofoxaliplatin,anotherplatinum-basedchemotherapyagent,to5-FUforadjuvanttreatment(44),reducestheriskforrecurrenceby25%inrectalcancer.Intheneoadjuvantsetting,oxaliplatinhasnotbeenshowntobeabetterradiosensitizer.Otherchemotherapeuticagentssuchasirinotecanormonoclonalantibodiessuchasbevacizumabhavenotbeenshowntohavethiseffecteither(45).Theside-effectsof5-FUbasedchemotherapyassingletherapyorincombinationwithothertherapiesintheneoadjuvantsettingareusuallywelltoleratedwithlessthan5%havingseveretoxicreactionsbecauseoftheshort-termexposure(46).Currenttreatmentschedulesincludenearly6weeksofneoadjuvantchemoradiation,6-8weeksofrecoverypriortosurgery,andanother4weeksofrecoverypriortoconsiderationforadjuvanttherapy.Inthiswaythereisaminimum4monthsdelaybeforeinitiationoffulldosesystemictherapy,(seeflowchartfollowingpage).Startingfulldosechemotherapyatanearlierstagehastheadvantageoftreatingmicro-metastaticdiseaseearlier,therebyreducingtheincidenceofdistantrecurrence.Furthermoreinmoststudiesradiotherapyhasnotimprovedsurvivalbutprimarilyreducedlocalrecurrence(1).Thusanalternativecouldbeneoadjuvantchemotherapyalone.Thereislimitedexperienceofchemotherapyassingletreatmentmodalitybutinitialreportsareencouragingintermsofbothdown-stagingandoutcomebutfurtherresearchwithlong-termfollow-upisneededandinitiated(43,46,47).

Figure4.ExampleofatheoreticalflowchartwithtreatmentoptionsforaSwedishpatientwithrectalcancer2016.

Surgicaltreatment.JacqueLisfrancperformedthefirstformalperinealresektionforrectalcancerin1826.GaussenbauerandHartmann1879addressedtheproblemwithlimitedexposurebyperformingresectionsviaanabdominalapproach(48).ErnestMilesstudiedthelymphaticspreadanddemonstratedanupwarddirectionoflymphflow.Between1899and1906heperformed57perinealresectionsofwhich95%developedearlyrecurrence.Inthepostmortemhefoundrecurrenceintheperitoneumandinlymphnodesaroundtheleftiliacartery.Hestated,“Ihavefailedinoneimportantaspect-namelythecompleteeradicationofthezoneofupwardspread”(48).In1907heperformedthefirstabdominoperinealresectionenablingresectionoftheupwardspreadtolymphnodes,theMiles´Operation(49).In1923hehadarecurrencerateof29.5%,whichwasoutstandingcomparedtopreviousresults(50).TheMiles´procedurebecamethegoldstandardandwasimprovedbyLloyd-Davisasaone-stageoperationincontrasttoMiles´two-stageprocedure.Moynihanadvocatedforthehightieoftheinferiormesentericarterysoastoincludelymphnodesthatcouldbeafuturelocusformetastaticgrowth,acontroversythatisnotyetsolved(51). In1948ClaudeDixonpresentedtheresultsof400patientsoperatedwithanteriorresectionformiddleandupperthirdrectalcancermaintainingbowelcontinuityinsteadofthepermanentstomausedintheMiles’operation(52).In1982HealddescribedthelymphaticspreadwithinthemesorectalsleeveandformulatedtheconceptofTotalMesorectalExcision(TME)withenblocresectionoftherectumandmesorectumandsharpdissectioninwelldefinedplanesasopposedtobluntmanualextractionusedpreviously.Inanarticlefrom1986hereportedon115patientswhereonly3localrecurrenceshadbeenseenafterafour-yearfollowup(53,54).TheTMEconceptrevolutionizedrectalcancersurgeryandisnowthegoldstandardworldwide.Anteriorresection.Anteriorresection(AR)isthemostcommonsurgicalprocedureforrectalcancerbeing40%ofthesurgicalproceduresforrectalcancerinSweden2015(2).Theprocedureisdecreasinginpopularityprobablyduetoagreaterawarenessamongstsurgeonsandpatientsofpoorpostoperativebowelfunctionwithaverylowanastomosisandthefearofcomplications.Theoperationincludesresectionoftherectum,mobilizationoftheleftcolon,anddivisionoftheinferiormesentericartery.Theleftcolicflexureisusuallytakendowntoallowatension-freeanastomosis.Therectumismobilizedcircumferentiallytothepelvicfloor,or5cmdistaltothetumouraccordingtotheTMEtechnique.Thedescendingcolonandthedistalendofrectumisdividedandacolorectalanastomosesiscreatedusingacircularstaplingdevice.Divertingileostomyisindicatedwithalowrectalanastomosis(2).Theprocedurecarriesa10-15%riskforanastomoticleakageleadingtopelvicabscessandsepticemia.Thereisariskfordamagingthenervescontrollingbladder,analsphincterandsexualfunction,ariskthatisalsoincreasedbypreoperativeradiation.Theoperationisasphincter–sparingprocedureifthedivertingstomaisreversed.RecentlytheLowAnteriorResectionSyndrome(LARS)withincontinence,urgency,andfrequentbowelmovementshasbeenhighlightedandhasbeenshowntocauseconsiderablefunctionallong-termbowelfunctionproblemsaftersurgery(55).

AbdominoperinealResectionAnabdominoperinealresection(APR)isindicatedforthemostdistaltumours.TheabdominalpartoftheoperationisperformedasinanAR.Thesecondpartoftheoperationisperformedwithaperinealapproach.Theanus,analcanalandpartsofthepelvicfloorareincludedintheresection.Thepatientisleftwithapermanentcolostomy.In2015,25%oftherectalcanceroperationsinSweden,wereAPR(2). Hartmann’sResection.InaHartmann’sresectionthesameabdominalprocedureasinARisperformed,butastheindicationsforthisprocedureareconcernsaboutanastomoticcomplicationsinoldage,poorhealthorexpectedpostoperativefunctionalproblems,noanastomosisisconstructed.Thepatientisleftwithacolostomyandtheclosedrectalstumpisleftinsitu.Hartmann’sresectionsisperformedinlessthan10%oftheabdominalresections(2).LocalExcisionandTransanalEndoscopicMicrosurgery(TEM).TheseoperationsareindicatedforT1cancersorasapalliativeprocedureinpatientsunfitforabdominalresection.TEMusesalargeendoscopeencompassinginstrumentsfordissectionandgasinsufflation.Full-thicknessresectionsmaybeobtainedandtheboweldefectisclosedorleftopenforsecondaryhealing.Themajordisadvantageofthistechniqueisthatevenifthetumourisradicallyresected,theregionallymphnodesareleftinsituandcannotbeexaminedpathologically.Minimallyinvasivetechniquessuchaslaparoscopicandroboticsurgeryhaveimprovedvisualizationandfurtherreducedtheperioperativetraumastillwithoutimprovedoncologicaloutcome(56,57).

Magneticresonanceimaginginrectalcancer.High-resolutionT2-weightedimagingisthekeysequenceinthemagneticresonance(MR)imagingevaluationofrectalcancerbothfortheprimarystagingandinrestagingafterchemoradiotherapy.Thetechniquecan,withhighaccuracydifferentiatebetweenrectaltumoursconfinedtotherectalwall(StageT2)fromthosethatextendbeyondthemuscularispropria(StageT3)(58,59).Severalstudieshaveshownthathigh-resolutionMRimagingisareliableandreproducibletechniquewithhighspecificityforassessingthedistanceofthetumourfromtheCRM(60).However,assessmentofnodalinvolvementremainsproblem.Priortostartingneoadjuvanttreatment,thesizeandmorphologyofnodesmaytoacertaindegreebeusedtodiscriminatebetweenbenignandmalignantlymphnodes.However,afterCRTtreatmentmorphologyisnolongerareliablecriterionandsizealonehassignificantlimitations.Assessmentofnodalstageseemstobehighlydependentupontheexperienceoftheradiologist(61-63).Diffusionweightedimaging(DWI)isanMRItechniquethatquantifiesthemovementofwatermoleculesatthecellularlevel.Sincethepropertiesofwatervaryinareasofnecrosis,inflammationandfibrosis,theDWItechniqueimprovesthestagingofcancerandfacilitateslymphnodedetection.ThereisnoconsensusontheuseofDWIinrectalcancerstaging,anditisnotreliablefordifferentiatingbetweenbenignandmalignantlymphnodes(64,65).

MRimagingofthepelviswithrectalcancer.A. SagittalT2viewofalargerectalcancer.B. TransverseT2viewofcircumferentialrectalcancer.C. TransverseT2viewwithalaterallymphnodeindicatedwitharrow.

Aimsofthethesis

Theaimsofthethesiswere;- toinvestigatetheexpressionofFXYD-3inrectalcancerandthe

relationshipofFXYD-3expressiontosurvivalinpatientswithorwithoutpreoperativeradiotherapy:

- toinvestigatethepotentialroleofFXYD-3asabiomarkerfordecreasedradio-sensitivity,usinglocalrecurrenceofrectalcancerasaproxy:

- toinvestigateinaprospectivenationalregister-basedstudytheimpactofneoadjuvanttherapyontheincidenceandoutcomeofpathologicallyconfirmedcompleteresponse:

- toassesstheaccuracyofMRIinpredictingthepresenceofremainingmesorectallymphnodemetastases(ypT0N+)inpatientswithcompletepathologicallyconfirmedluminalresponse(ypT0)afterneoadjuvantradiotherapyorchemo-radiotherapy.

PatientsandMethodsStudyIInstudyI,140specimensfrompatientsthatparticipatedinaSwedishrandomizedtrialonpreoperativeradiotherapy(RT)between1987and1990wereanalyzed.Allpatientshadprimaryrectalcarcinomaand65ofthe140received5x5GyRToverafive-dayperiod,followedbysurgerywithinaweek.Noneofthepatientshadadjuvantchemotherapyandthemeanfollow-uptimewas84months(66)(Table1).PatientswereanalyzedforexpressionofFXYD-3intheprimarytumour,normalmucosaandregionallymphnodemetastases.Immunohistochemicalanalysiswasperformedonparaffin-fixedsectionsusingamonoclonalanti-FXYD-3primaryantibody(Fig.1)Degreeofstainingwasgradedasnegative,weak,moderateorstrongbasedontheintensityofthestaining.Thestainedsectionsweremicroscopicallyexaminedandscoredindependentlybytwoinvestigatorsblindedtotheclinical,pathologicalandbiologicdata.Statistics.McNamara’smethodandChi-squarewereusedtotestthesignificanceofdifferencesinFXYD-3expressionbetweendistantnormalmucosa,adjacentnormalmucosa,theprimarytumourandlymphnodemetastases,andtheassociationofFXYD-3expressionwithclinical,pathologicandbiologicvariables.TherelationshipbetweenFXFD-3expressionandsurvivalwastestedusingCox’sproportionalhazardsmodel.SurvivalcurveswerecalculatedusingtheKaplan-Meiermethod.Alltestsweretwo-sidedandap-valueoflessthan0.05wasconsideredasstatisticallysignificant.Figure1.ImmunohistochemicalstainingforFXYD-3expressionfromthesamepatient.A:weakexpressionindistantnormalmucosa.B:Strongexpressioninprimarytumour,C:weakexpressioninlymphnodemetastases.

Table1.Characteristicsofpatientsandrectalcancers

Characteristics Non-Radiotherapy Radiotherapy No. % No. %Gender

Male 44 59 41 63Female 31 41 24 37

Age(years) ≤67 31 41 28 43>67 44 59 37 57

TNMstage I 20 27 21 32II 19 25 20 31III 54 41 17 26IV 5 7 7 11

Differentiation Well 5 7 5 8Moderately 53 71 40 62Poorly 17 23 20 31

Numbersoftumors Single 63 84 48 74Multiple* 10 13 16 25Unknown 2 3 1 2

Surgicaltype Rectalamputation 37 49 25 38Anteriorresection 38 51 40 62

Resectionmargin Tumourfree 73 97 60 92Tumourinvolvedmargin 2 3 5 8

Distancetoanalverge(cm) Mean 7.6 8.6

*Othercolorectalcancerorothertypeoftumourbesidesthepresentrectalcancer.

StudyIIDataonpatientswithrectalcancerfromthreehealthcareregionsduringthetimeperiod1985-2000wereextractedfromadatabase.Allpatientshadbeenoperatedforrectalcancer.ThemajorityofpatientsreceivingRTreceived5x5Gyfollowedbysurgerywithinaweek.Patientswithlocallyadvanceddiseasereceived25x1.8Gyandsurgeryafter6-8weeks.1180patientswereincludedandoperatedonwithradicalresection(R0)usingtheTMEtechnique(67,68).Forty-eightpatientswerediagnosedwithalocalrecurrence(LR)oftherectalcancerwithinthepelvis.Patientswithliverorlungmetastaseswerealsoincluded.Anestedcase-controlstudywasdesignedwithtwocontrolpatientsforeveryLRcase.ControlsandcaseswerematchedforgenderandpreoperativeRT(Table1).Themediantimeforfollow-upforthecasegroupwas79weeksandforthecontrolgroup316weeks.Wewereabletoretrieveformalin-fixedblocksfromtheprimarytumourof48casesand81controls.FXYD-3expressionwasanalyzedusingimmunohistochemistryontumortissuefromtheindexrectalcanceroperation.Theintensityofstainingwasgradedasnegative,weak,moderateorstrongbasedontheintensityofthestaining.Thecaseswithnegative,weakandmoderateintensityweregroupedtogetherastheweakFXYD-3expressiongroupandthecaseswithstrongintensityformedthestrongFXYD-3expressiongroup.ThestainingandexaminationprocesseswereperformedattheKarolinskaInstitute,Solnabytwohisto-pathologistsblindedforclinic-pathologicandbiologicdata.Statistics.TheChi-squaremethodwasusedtotestthesignificanceofthedifferencesinFXYD-3expressionbetweenthestrongFXYD-3expressiongroupandtheweakFXYD-3expressiongroup.ThefiguredepictingtimetolocalrecurrencewascalculatedusingtheKaplan-Meiermethod.Alltestsweretwo-sided,andavalueofp<0.05wasconsideredstatisticallysignificant.LogisticregressionandCoxregressionwereusedtoanalyzetheeffectofdifferencesbetweenthecasegroupandthecontrolgroup.Figure1ImmunohistochemistryshowingFXYD-3expressionintheprimarytumourfromdifferentpatients.AWeakFXYD-3expression.BStrongFXYD-3expression

Table1PatientcharacteristicsattheindexoperationandFXYD-3expressionCharacteristics Local

recurrenceGroup

ControlgroupP-valueP-value(Unadjusted)(Adjusted)*

Patientsn,(%) 48 81Timetorecurrence(mo)

median 18 ---Range 3-60

Age(y) Median 71 720.36-Range 34-85 34-95--

Gender(n) Male 24(50) 45(56)0.540.96Female 24(50) 36(44)--

Typeofsurgery(n) Anteriorresection 32(67) 65(80)0.080.88Abdominoperinealresection

16(33) 16(20)--

Distanceofprimarytumorfromanalverge(n)

11-15cm 15(31) 28(35)0.710.486-10cm 16(33) 31(38)--0-5cm 17(35) 22(27)--

Radiotherapy(n) Yes 18(38) 25(31)0.440.97No 30(62) 56(69)--

TNMstage(n) I 4(8) 26(32)0.010.02II 19(40) 36(44)--III 25(52) 19(23)--

Tumourdifferentiation

High 3(6) 6(8)-0.03Moderate 30(64) 61(81)--Poor 14(30) 8(11)--

FXYD-3Expression Weak(Grade0+1+2)

39(81%) 67(83%)

Strong(Grade3) 9(19%) 14(17%)p>0.05*Adjustedforallothervariablesinthetable

StudyIII.StudyIIIisaregister-basedstudyontheincidenceandoutcomeofcompletepathologicalresponseofrectalcancertoneoadjuvanttreatmentinSwedenbetween2007and2012.TheSwedishColo-RectalCancerRegister(SCRCR)isaprospectivepopulation-basedregistercollectingdatafromallcolorectalcancercasesinSweden(69).Thisisavalidatedregisterwith99.5%immediatecoverageandfive-yearfollow-updatafrom98%ofpatients.Thestudycohortconsistedof11226patientswithamedianageof71years59%weremales.Anabdominalprocedurewasperformedon7885ofthepatients.Twenty-sixpercent(2063patients)wereclassifiedashavingpotentialforpCRafterreceivinglong-termradiotherapytreatment(28x1.8Gy,LRT)orshort-termradiotherapytreatment(5x5Gy,SRT)withadelayofmorethanfourweeks,withorwithoutchemotherapybeforeabdominalsurgery.Thisgroupwaslabeled“potentialcompleteresponders”andformedthebasisofthisstudy(Table1).Amajorityofthe2063potentialresponders(84%)werecT3orcT4cancers,and56%hadreceivedneoadjuvantchemotherapy.PatientswithypT0wereidentifiedfromthatgroup.Thegroupof161patients(8%)withcompleteresponsewasfurtheranalyzedforlymphnodestatusandduringthatprocesserrorsandmisclassificationsintheregisterwerediscoveredandcorrected.

Statistics.Differencesbetweentheproportionsofgroupsweretestedwithatwo-sidedZ-testofproportion.Thenon-parametricMann-Whitneyu-testwasappliedfordifferencesbetweenmediantimes.OverallsurvivalwaspresentedasaKaplan-Meiercurve,anddifferencesinsurvivalweretestedwithGehan’sgeneralizedWilcoxontest.Five-yearsurvivalrateswerecalculatedusinglifetables.RelativerisksformortalitywerecomparedusinghazardratioscalculatedwithCOXregression.Binomiallogisticregressionwasimplementedtogetoddsratios.Table 1. Patient characteristics

Female M1*

Rectal cancer (2007-2012) n - % 11226 41% 20%

age (median)

Abdominally operated ** n - % 7885 40% 8%

age (median)

Anterior resection n - % 3940 42% 12%

age (median)

Abdominoperineal resection n - % 2837 38% 11%

age (median)

Hartmann's procedure n - % 1108 42% 8%

age (median)

Potential complete responders (Long-term RT or short-term RT with delay, +/- chemotherapy.)

n - % 2063 40% 13%

age (median)

Long-term radiation. n - % 1179 41% 10%

age (median)

Short-term radiation with delay. n - % 884 38% 16%

age (median)

*10oftheypTOpatientshaddistantmetastases(M1).*368patientswithrectalcancerhadalocalexcisionofthetumourandwerenotincludedinthiscohort

Study IV.

StudyIVwasafollow-upofthefindingsofstudyIIIonremainingloco-regionallymphnodemetastasesinluminalcompleteresponseandthefollowingreducedsurvival.TheSCRCRwasscrutinizedandfrom11226patientswithrectalcancerdiagnosedbetween2007and2012agroupof161patientswithluminalcompleteresponse(ypT0Nall)wereidentified.Fromthatgroup26patientswithypT0N+wereselectedinordertostudytheaccuracyofMRIindetectingremainingtumourpositivelymphnodes.WewereabletoretrievemedicalrecordsononcologicaltreatmentandcompleteMRIimagingfromthestagingandrestagingMRIinvestigationsfrom19patients.Another19patientswithypT0N0werematchedfortreatinghospitalandcomprisedthecontrolgroup(Table1).Allimageswereassessedbytworadiologistsblindedtosurgicalandpathologyfindingsfirstindependentlyandtheninconsensus.Imagesbeforeandafterneoadjuvanttreatmentwereassessedtogether. Extramuraldepthoftumourinvasionandinvolvementofthemesorectalfasciawerenotedaccordingtopreviouslyestablishedcriteria(60).Nodalnumber,sizeandpositionwereidentifiedineachpatientforbothbenignandtumourpositivelymphnodes.ThelymphnodesidentifiedonMRIwerejudgedtobemalignantorbenignusingmorphologiccriteria(62,70).Onthepre-treatmentMRIalymphnodewasconsideredtobemalignantifitshowedanirregularouterborderorinternalsignalheterogeneity.OnthepostCRTMRI,sizewasusedastheonlycriterion,wherelymphnodesexceeding5mmwereregardedasmalignant.Statistics

• Sensitivitymeasurestheproportionofpositivesthatarecorrectlyidentifiedassuch(e.g.thepercentageofsickpeoplewhoarecorrectlyidentifiedashavingthecondition).

• Specificitymeasurestheproportionofnegativesthatarecorrectlyidentifiedassuch(e.g.,thepercentageofhealthypeoplewhoarecorrectlyidentifiedasnothavingthecondition).

• Positivepredictivevalue=probabilityofdiseaseamongpatientswithapositivetest

• Negativepredictivevalue=probabilityofnodiseaseamongpatientswithanegativetest

TheStudent´sT-testwasusedtotestthesignificanceofthedifferencesbetweenthecasesandthecontrolgroup.Alltestsweretwo-sided,andavalueofp<0.05wasconsideredstatisticallysignificant.Logisticregressionwasusedtoanalyzetheeffectofdifferencesbetweenthecasegroupandthecontrolgroup.

Table1.Descriptionofthestudypopulation.

Gender All N+ N0 pFemale 17 11 6 0.10Male 21 8 13 Averageageatsurgery(range).years 57(37-74) 54(37-69) 61(37-74) 0.05 T-stage 0.18T1-T2 9 5 4 T3 14 8 6 T4 14 5 9 Tx 1 1 Radiation 0.7328x2Gy 27 13 14 5x5Gywithdelay 11 6 5 Chemotherapy 0.18None 7 3 4 5-FU/Capecitabine 18 7 11 5-FU/Capecitabine+Oxaliplatiine 13 9 4 Sum 38 19 19

ResultsanddiscussionStudyITheexpressionofFXYD-3wassignificantlyincreasedintheprimarytumourcomparedtonormalmucosa(p=0.008)(Fig1).Therewasnodifferenceinexpressionbetweentheprimarytumourandtumourpositivelymphnodes.TherewasahigherFXYD-3expressioninmenthaninwomen(p=0.04)andintumourswithinfiltrativegrowthcomparedtotumourswithexpansivegrowth(p=0.02).FXYD-3expressionwaspositivelyrelatedtobothphosphataseofregeneratingliver(PRL)(p=0.001)andp73(p=0.03).InpatientsreceivingRT,FXYD-3expressionwasstrongerintumourswithlessnecrosisthanintumourswithextensivenecrosis(p=0.02).Inbothuni-andmultivariateanalysispatientswithstrongFXYD-3expressionalone(P=0.02)orincombinationwithPRL(p=0.02)hadreducedsurvivalintheRTgroup,regardlessoftumourstageordifferentiation(Fig2).Fig. 1. Strong FXYD-3 expression in the radiotherapy (RT) group and non-RT group in distant normal mucosa, adjacent normal mucosa, primary tumour and tumour positive lymph nodes.

FXYD-3 expression

Distant normal mucosa Adjecent normal mucosa Primary tumor Metastasis

RTnon-RT

Figure2.Univariateanalysisofsurvivalvs.FXYD-3andPRLexpressionintheRTgroup

FXYD-3isatrans-membraneproteinnormallypresentinbodytissuesbutoverexpressedinseveralcancers.Increasedexpressioninearlytumourdifferentiationandtumourproliferationhasindicatedthatitcouldpossiblybeusedasatumourmarker(19,71-75).TherearealsoindicationsthatFXYD-3maybeinvolvedintumourresistanceto5-FUchemotherapy(76).AtthetimeofthestudynopreviousresearchhadanalyzedtheclinicalsignificanceofFXYD-3incolorectalcancer.OuraimwastodeterminewhethertherewasanincreasedexpressionofFXYD-3inrectalcancercomparedtonormalmucosa,andtoidentifydifferencesinoutcomebetweenpatientswithorwithoutRTandrelatethesetoFXYD-3expression.OurresultsshowedthattherewasincreasedFXYD-3expressionintheprimarytumourcomparedtonormalmucosa,andintumourswithinfiltrativegrowth.TheassociationofFXYD-3expressionwithp73andPRLisofinterestasinpreviousstudiesbothPRLandp73havebeenshowntoberelatedtoradio-sensitivity.Furthermore,overexpressionofp73isalsorelatedtoreducedsurvivalincolorectalcancer(6,77-79).LesstumournecrosisandpoorersurvivalwereseeninpatientswithincreasedFXYD-3expressionintheRTgroupbutnotinthegroupwithoutRT.Alowerdegreeoftumournecrosiscanbeseenasanindicatorofreducedradio-sensitivity.Therewasalsoanon-significantincreaseindistantmetastases(p=0.08)inthisgroup.TheseresultstakentogethersuggestedtousthatexpressionofFXYD-3couldbeaprognosticfactorandanindicatorofreducedradiosensitivity.SincethiswasthefirststudyonFXYD-3andcolorectaloutcome,ourfindingsrequiredconfirmation.Astudyonreducedradio-sensitivityusinglocalrecurrenceasaproxywasconsideredtobemoreaccuratesincefewstudieshavebeenabletoshowacorrelationbetweenRTandsurvival(1).WethereforewentontoattempttoconfirmthesignificanceofFXYD-3inrectalcancerbystudyingthefrequencyoflocalrecurrenceafterRT(studyII).

0 24 48 72 96 120 144 168 192

Follow-up time (months)

RT, n=57, P = .01

Both negative

FXYD3 and PRL expression

Either positive or both positive

RT, n=57, P = .01

StudyIITherewasnodifferenceinFXYD-3expressioninrelationtoRTneitherinthecontrolgroupnorinthelocalrecurrence(LR)group(p>0.05)(Table2).Therewasnosignificantdifferenceintimetodiagnosisofalocalrecurrence,betweentumourswithstrongFXYD-3expressionandthosewithweakFXYD-3expression(Figure1).Whenallpatientswereanalyzedtogether,i.e.boththosewithandwithoutLR,therewasnodifferenceinsurvivalintheradiatedgroupbetweenpatientswithstrongorweakFXYD-3expression(p=0.43).Therewasnodifferenceinsurvivalinthenon-radiatedgroupbetweenthepatientswithstrongorweakFXYD-3expression(p=0.30).TherewasatendencytowardsalowerincidenceofstrongFXYD-3expressioninpatientswithLRwhohadbeentreatedwithpreoperativeRT(Table3).Figure1.Timetolocalrecurrenceinall129patients.

p>0.05

Table1.FXYD-3Expressioninthecontrolgroupinrelationtopreoperativeradiotherapy(RT)(n=81)

p>0.05 Table2.FXYD-3expressioninthelocalrecurrencegroupinrelationtopreoperativeradiotherapy(RT)(n=48)

p>0.05

0 20 40 60

Time after surgery (months)

Weak FXYD-3 (n=106)

Strong FXYD-3 (n=23)

FXYD-3 expression No RT

Weak (0,1,2) 46 (84%) 21 (81%)

Strong (3) 9 (16%) 5 (19%)

FXYD-3 expression

No RT RT

Weak (0,1,2) 23 (77%) 16 (89%)

Strong (3) 7 (23%) 2 (11%)

TheresultsfromstudyIindicatedthatFXYD-3couldpossiblyserveasaprognosticindicatorforsurvivalandalsoanindicatorofreducedradio-sensitivity.InmanylargestudiesradiotherapyhasreducedtheincidenceofLRbutitseffectonsurvivalisnotsoclear(1,80-83).ItwasthusourambitiontoanalyseFXYD-3expressionintumoursfromrectalcancerpatientsthatdevelopedalocalrecurrenceandtoseeifthesetumourshaveastrongFXYD-3expression,orifstrongFXYD-3expressionisrelatedtotimetoLR.ResearchersattheKarolinskaInstituteinStockholmhadpreviouslyperformedastudyonacohortofpatientsregardingtherelationshipofLRtoRT.IncooperationwiththeKarolinskaInstitutewedesignedthisstudyusingtheirformerstudycohort,whichalsomadeitpossibletoconfirmourresultsfromstudyIatanotherlaboratory. WewerenotabletoconfirmourfindingsofFXYD-3asaprognosticfactorforreducedsurvivaloranindicatorofradio-resistanceinthisstudy.Severalfactorscouldhavecontributedtothis;FirstlytherewerefewofthepatientsinboththeLRgroup(36%)andthecontrolgroup(31%)thatreceivedRT(Figure1).Thisincombinationwiththefactthatonly19%intheLRgroupand17%inthecontrolgrouphadstrongFXYD-3expressionsmadeithardtoreachstatisticallysignificantresults.Furthermore,patientsandcontrolswereonlymatchedforgenderandRTwhilebothtumourstageanddifferentiationdifferedbetweenthegroups.Immunohistochemistryisasemi-quantitativemethodandanalysisofoldformalinfixedspecimencouldwellbeanissuetoo.Blindedexaminationsofimmunohistochimistryslideswereperformedatbothhospitalstoensureobjectivegrading.Eventhoughseveralfindings,togetherandindependentlyfromthestudyIindicatedthatFXYD-3couldbeaprognosticfactorandanindicatorofreducedradio-sensitivity,theresultsofstudyIIIcouldnotsupportthis.Thecollectionofalargercohortofpatientswithlocalrecurrenceinordertogaingreaterpowerwouldbealargelong-termproject.Still,alargeprospectivecohortwithsamplesfrompreoperativebiopsiesanalysedwithRNAtechniqueswouldbeofinterestinthesearchforpredictivemarkersofreducedresponsetoneoadjuvanttreatment.

StudyIII.Completeeradicationoftheluminaltumour,ypT0wasfoundin161patients(8%).TheypT0rateincreasedwithdecreasingcT-stage,thehighestratebeingincT1-2cancers(Tables2-3).Theadditionofchemotherapyresultedin10%ypT0comparedto5.1%inthegroupwithoutchemotherapy(p<0.00004)ThelengthofdelaybetweenCRTandsurgerydidnotaffecttheypT0rateregardlessofwhetherLRTorSRTwithdelaywasused.OftheypT0patients83%hadtumour-negativeregionallymphnodes(ypT0N0),12%had1-3tumourpositivelymphnodes(ypT0N1)and4%hadmorethanthreetumourpositivelymphnodes(ypT0N2).ThedistributionofpNstatusdependedsignificantlyonthecN-status(p=0,000).Therewasasignificantlyreducedriskfortumour-positivelymphnodesinyoungerpatientsandincT1-2tumourscomparedtocT3-4(p=0.01).TherewassignificantlygreatersurvivalinypT0comparedtoypT+patients(hazardratio0.38(C.I0.25-0.58))andsurvivalwassignificantlygreaterinpatientswithypT0N0comparedtoypT0N1-2(hazardratio0.36(C.I0.15-0.86)).(Figure1)

Table1.Oddsratiosofcompleteresponse(ypT0)inclinicaltumourstagescT-stage Completeresponse(ypT0)(n,%) Oddsratio(CI)ofhavingCR* 161 8% cT1-2 21 14% 1cT3 77 9% 0.401(0.230-0.698)cT4 58 6% 0.227(0.125-0.413)cTX 5 2% 0.179(0.058-0.555)*Adjustedfor:age(under/over66yearsp=0.019),gender(0.081),tumourlevel(under/over8cmp=0.020),preoperativechemotherapy(yes/nop=0.001)andpreoperativeradiotherapy(Long-termradiotherapyvs.short-termradiotherapywithdelay(p=0.666)

Table2Luminalcompleteresponse(ypT0)inrelationtodifferentneoadjuvanttreatments.Luminalcompleteresponse(ypT0)inrelationtodifferentneoadjuvanttreatments.

Preoperative treatment

Long radiotherapy

(25x1.8/2Gy) and

preop.

chemotherapy

radiotherapy

(25x1.8/2Gy)

radiotherapy

(5x5Gy) and wait

>4 weeks and

preop.

chemotherapy

Short radiotherapy

(5x5Gy) and delay

>4 weeks

Luminal complete

response

Luminal

complete

response

Luminal

complete

response

Luminal complete

response

Luminal

complete

response

No Yes No Yes No Yes No Yes No Yes

Clinical

stages.

14 6 8 0 3 3 108 12 133 21

70 % 30 % 100

0 % 50 % 50 % 90 % 10 % 86

cT3 304 45 79 6 70 8 368 18 821 77

87 % 13 % 93

7 % 90 % 10 % 95 % 5 % 91

cT4 533 43 74 4 69 7 174 4 850 58

92 % 8 % 95

5 % 91 % 9 % 98 % 2 % 94

cTX 41 2 14 1 2 0 32 1 89 4

95 % 5 % 93

7 % 100 % 0 % 97 % 3 % 96

Total 892 96 175 11 144 18 682 35 1893 160*

90 % 10 % 94

6 % 89 % 11 % 95 % 5 % 92

• InformationonclinicalT-stagesismissingforninepatients;oneofthemhadluminalcomplete

response.

Figure1cumulativesurvivalinrelationtoluminalcompleteresponseandnodalpathologystatus

Adjustedforadjuvantchemotherapyp=0000.HR(Noadjuvantchemotherapygiven):1.48(1.24-1.77)InthisregisterbasedstudywefoundalowypT0rate,(8%)comparedtootherstudiesthathaveshownratesof13-45%(84-86).OurresultsmayreflectthebroadinclusionofrectalcancerpatientsinSwedishnormalclinicalpracticewithahighproportionofT4tumoursandtheinclusionofpatientswithLRT,andSRTwithdelaybutwithoutchemotherapy.AsbothlocalandsystemicrecurrencesarereducedafterpCR(86-89)thesignificanceoftumourpositivenodesintheresectedspecimenisnotclear,buttheresultsofthisstudyindicateareductioninsurvivalinpatientswithypT0N1-2comparedtoypT0N0.Ourresultsshowthatthesurvivalbenefitfromcompleteresponseisdependentuponnodalinvolvement,witha survivalcurveforypT0N+similartothecurveforpatientswithypT+.WecouldnotconfirmpreviousfindingsofimprovedypT0rateswithincreaseddelaybetweenRTandsurgery(15).Therewas,howeveranarrowtimespanbetweenLRTandsurgeryforthegroupasawholeinthisstudyduetoadherencetoSwedishguidelines.Therationalebehindnon-surgicalmanagementisbasedontheclinicalabilitytoruleoutremainingtumourinthepelvis,includingtheregionallymphnodes.Aprerequisiteisthateventualregrowthcanbedetectedandtreatedwithresultsthatarenotinferiorto

HR :0.22(C .I.0.13-0.37)HR :0.54(C .I.0.47-0.63)HR :0.66(C .I.0.32-1.30)

Reference

Gehan's generaliz edWilcoxontes t,P -value= 0.0000

0year 1year 2year 4year 5year

pT0N0 133 132 119 85 91%(85%-97%) 63 38pT+N0 1052 1006 873 646 82%(80%-84%) 437 278pT0N+ 26 26 23 12 74%(56%-92%) 10 7

pT+N+ 817 763 596 391 67%(63%-71%) 237 131

3yearP atientsa trisk/ac tua l3-yearsurviva l(95%C .I.)

surgery.Foradvancedtumourstheriskforlymphnodemetastasesdespiteluminalcompleteresponseishighandthisstudyindicatesalessfavorableprognosisevenwithradicalsurgery.TheprognosisforpatientswithpT0N+issignificantlyworsethanforpT0N0evenwithradicalsurgery.Wealsofoundthatchemotherapyseemstohavelittleeffectonlymphnodemetastases.Whenconsideringa“watchfulwaiting”policythesefindingsshouldbetakenintoaccount.

StudyIV.Thirty-eightpatientswereevaluated,19withypT0N0and19withypT0N+.Themeantimebetweenpost-CRTMRIstagingandsurgerywas21days(6-68)withnostatisticaldifferencebetweentheN0andN+group(21and25daysp=0.27).Atthepre-CRTMRIstagingatotalnumberof314lymphnodesweredetected,and122(39%)oftheseweresuspectedtobemalignant.TheaveragenumberoflymphnodesintheypN0groupwas9andintheypN+group7(p=0.5).(Figure1).TheaveragesizeofthelymphnodesintheypN0groupwas4.0mmandintheypN+groupitwas3.8mm(p=0.65).Thetotalsumofsizeswas645and612mmrespectively(p=0.81).Atpost-CRTMRIstaging,107lymphnodesweredetectedand20(19%)oftheseweresuspectedtobemalignant.Twenty-nineoutof38patientshadnosuspectedmalignantnode.Onaveragetherewere2.7nodesdetectedintheypT0groupcomparedto2.9nodesintheypT+group(p=0.75)(Figure1).TheaveragelymphnodesizeintheypT0N0groupwas1.9mmand3.0mmintheypT0N+group(p=0.12).Thetotalsumofsizewas144and225mmrespectively(p=0.21).

WhencomparingtheaveragesizeofthelargestnodeforallpatientstherewasasignificantdifferencebetweentheypN0andypN+groups(2.6mmvs.4.5mm)(p=0.04)(Figure2).Nodeslargerthan8mmwereonlyfoundinN+patients(n=3).TwopatientswithnolymphnodedetectedonMRIstagingwerestagedypN+atthepathologyreport.Ofthe19patientswhowereypN+12wereMRIstagedasN0,and3ofthe19ypN0werestagedasN+.Sensitivity,specificity,PPVandNPVfortheexpertradiologiststocorrectlydetectlymphnodemetastasesinypT0patientswere37%,84%,70%and55%respectively.

Sizeandmorphologytogetherarethetwomostpromisinglymphnodefeaturesusedtodiscriminatebetweenmalignantandbenignnodes.Westillneedtofindathresholdvaluethatcanruleoutmalignantlymphnodesbysizealone.Thecut-offsizeisprobablyrelatedtopopulationfactorssuchasinitialtumourstage,CRTregimenandthetimebetweenCRTtreatmentandreevaluationMRI.TheresultsofthisstudyonypT0andlymphnodestagingshowsthatCRThasasubstantialeffectonthesizeandnumberoflymphnodesdetected.Thesmallnumbersofpatients,causingaType2errormadeithardtofindsignificantchangesbetweenthetwogroups.Withregardtosuspectedmalignantlymphnodes,wecouldseeatrendtowardstherebeingmoresuspectlymphnodesbothbeforeandafterCRTintheypT0N+group(Fig1).Furthermoretherewasnosignificantdifferenceinsizeofnodesbetweenthetwogroupsandwhichisincontrasttootherstudies(61,90).Weexcludedallbutcompleterespondersinourstudypopulationincontrasttootherstudies,whichcomprisedagreatervarietyinpostCRTT-stage.WhenweanalyzedtheaveragesizeofthelargestlymphnodewefoundthattheypT0N+grouphadasignificantlylargerparamountglandthantheypT0N0group.ThismayindicateN+stageinLCR.ThisisoneofthefirststudiesonluminalcompleteresponseandlymphnoderestagingbyMRI.Weshowedalargereductioninbothsuspectedmalignantandbenign-lookinglymphnodesafterCRT.Thesizeofthelargestlymphnodeseemedtopredictnodemalignancy.TheresultsofthisstudyindicatetheneedforabettermethodtoadequatelypredictlymphnodestatusinpatientswithluminalcompleteresponseafterCRTinrectalcancer.

Figure1.DetectedlymphnodesinrelationtoN-stage

Figure2.AveragesizeofthelargestlymphnodeinrelationtoN-stage.

51 5641

020406080100120140160180200

ypT0N0 ypT0N+

PreCRTMRItotalnumberoflymphnodes

PostCRTMRItotalnumberoflymphnodes

PreCRTMRIsuspectedlymphnodes

PostCRTMRIsuspectedlymphnodes

7,17,9

ypT0N0 ypT0N+

PreCRTMRIaveragelargestlymphnode.(p>0.05)

PostCRTMRIaveragelargestlymphnode.(p=0.04)

GeneraldiscussionandfutureperspectivesAfterClaudeDixon’sdevelopmentoftheanteriorresectionin1948(52)littlehappenedinrectalcancertreatmentforalmostfortyyearsuntilthemid1980’swithboththedevelopmentofTMEsurgeryandperioperativeradiotherapy(53,54).Duringtheyearsfollowingtherewasarapidevolutioninrectalcancertreatmentwithneoadjuvantandadjuvantchemotherapy,laparoscopy,enhancedrecoveryprogrammes,multidisciplinaryteamconferences,non-operativemanagementandroboticsurgery(43,45,57).Allthesemeasuresaimedatimprovingpatientoutcomeandminimizingmorbidity.Inoureffortstomaximizetheeffectsofantitumourtreatmentsthereisalwaystheriskthatthosepatientsnotresponding,gainnothingandenduppayingthepriceofside-effectsandalongertimewaitingforcurativetreatment.ItisthereforeimportanttofindindicatorsthatpredictorindicateresponsetoCRTtreatmentinperioperativeoncologicalmanagement.InstudyIweinvestigatedtheproteinFXYD-3thattheoreticallycouldserveasanindicatorofmalignantprogression.OurresultsshowedthattumourswithincreasedFXYD-3expressionshowedlessnecrosisafterRTandmoreinfiltrativegrowthcoupledwithreducedsurvival.ThissuggestedthatFXYD-3couldbeapredictorofreducedsurvivalasaresultoflowerradio-sensitivity.ToconfirmthesefindingswedesignedstudyIIonFXYD-3expressionrelatedtolocalrecurrencewherewecoulddirectlypinpointtheactualissueonradiosensitivity.Wealsoattemptedtoconfirmourfindingsbycomparisonofimmunohistochemistryresults,asemi-quantitativemethodatanotherrenownedcenter,(KarolinskaInstitue).InstudyIInoneofthefindingsfromstudyIonthespecificissuesofradio-sensitivityandlocalrecurrenceduetoover-expressionofFXYD-3couldbeconfirmed.ThismaybeduetothefactthatthereisnorelationshipbetweentheexpressionofFXYD-3andlocalrecurrence,itcouldalsobethatwedidnothavethestatisticalpowertoproveit.InconclusionthesignificanceofFXYD-3expressioninrectalcancerisstillnotclear.Theparadigmofnon-operativemanagementofrectalcancerwaspioneeredinBrazil(91).Culturaldifferencesmaytriggerandpropelsurgicaldevelopmentinonepartoftheworldthatwouldprobablynothaveevolvedinotherplaces.Oneexampleisthegeneralviewregardingstomas,wherebothsurgeonsandpatientsinnorthernEuropeseemtohaveamorepermissiveviewthanthosefromsouthernEuropeorSouthAmerica.Thisisprobablyreflectedintherateofcoloanalanastomosesandinverselyintherateofpermanentstomas(92).Culturaldifferenceslikethiscouldhavebeenacontributingfactorwhytheconceptofnon-operativemanagementwasfirstexploredinBrazil(12,13,91).Inthecaseofcompleteresponsewithabsenceofcancercellsintherectum,itisevenharderforapatienttoacceptapermanentstoma.Whennewtreatmentoptionsandtherapiesdevelopincentersoutsidethenationalwelfaresystem,conclusionsregardingtheeffectsofanewparadigmatthenationallevelaredifficulttodraw.Largenationalregisterscanbeusedtovalidatedatafromsmallcentersbyidentifyinglowfrequencyside-effects,aswellasculturaldifferencesinoutcomeandindications(69).InstudyIIIweaimedtoinvestigatetherateofcompletepathologicalresponseusingdatafromanationalregister.Wefoundalowrateofcompleteresponsecomparedtoothersingle-centerstudies(11,13,91),butourresultsreflectanolderpopulationwith

moreadvancedtumourstagesandtreatmentwithnointenttoachievecompleteresponseperse.Thefindingsofarelativelyhighrateofremainingtumourpositivelymphnodesinthesurgicalspecimenreflectedbyalowersurvivalrateisprobablyaconsequenceofthehighproportionofadvancedtumoursinourstudy.Thisstressestheimportanceofcarefulrestagingandpatientselectionwhenconsideringanon-operativemanagement.Inordertomakewell-foundeddecisionsonnon-operativemanagementthereisaneedtovalidatemethodsusedforrestagingafterneoadjuvanttreatment.InstudyIVwecametotheconclusionthatdespiteassessmentbyexpertradiologists,presentMRItechniqueseemnottobeadequateenoughtodiscriminatebetweenbenignandmalignantlymphnodes.Thisisafindingthatisinlinewithpreviousresearch(93).UnfortunatelyMRIisthebestwehaveinclinicalpracticeatthemoment.Thesearchfornewpredictorsofresponsetoneoadjuvantoradjuvanttreatmentwillgoon.Sofartherehasbeenmuchresearchbutwithdismalresults,whichleadsustobelievethatitwilltaketimebeforeapredictorofresponsetoneoadjuvanttreatmentcomesintoclinicaluse(9).Non-operativemanagementofrectalcancerwillprobablyhaveamajorimpactonfuturemanagement.Theconceptofneoadjuvanttreatmentusingonlychemotherapywillprobablycontinuetodevelop.Withearlyfulldosechemotherapywecanbeabletotreatmicro-metastaticdiseaseandatthesametimeomittingradiotherapywithitslong-termsideeffects.Althoughdatafromlargeprospectivestudiesisstilllacking. AstherateofbothlocalanddistantrecurrenceisreducedwithcompleteresponsetoCRT,outcomewillbeimproved(11,13,87).Ifthatholdstrueinnon-operativemanagement,rectalcancertreatmentwillundergomajorchangesinthenearfuture.Furthermorelong-termfollow-upanditsimpactonthehealthcaresystemanditsresources,andpatientconcernabouthavingtolivewithalife-longdiagnosisofcancerareissuesthatmustbediscussed.Withourpresentgoalofdevelopingdiagnostictoolsthatarenotdependentonionizingraytechniques,wewillcertainlyseerapidimprovementsinMRItechnologyinthenearfuture.Iaminterestedtocontinuetheresearchoncompleteresponseinrectalcancerinthefuture.Ibelieveitwouldbesafertoselectpatientsforanon-operativemanagementifwecouldbetteridentifypatientsatriskofhavingaregrowthoftheluminaltumourordevelopingmetastaticdisease.PresenceoftumourpositivelymphnodesafterCRTseemstobeariskfactorforreducedsurvival.ItisunfortunatelydifficulttoidentifythesetumourpositivelymphnodesafterCRTbyusingMRIonly.IwouldliketofurtheranalyzethefindingsfromstudyIVwherethesizeofthelargestlymphnode(4,5mm)seemedtoindicateN+stage.Ofinterestisthatthesizeof4.5mmalsohasbeensuggestedascut-offsizeforidentifyingmalignantnodesafterCRTinanotherstudy(61).Wehaveinitiatedaplanforastudywherewewouldidentifyandfollow-uppatientswhodevelopeddistantmetastasesafterCRTinoperatedrectalcancer.InitiallyweplantoretrospectivelyanalyzetherestagingMRIimagesandmeasurethesizeofthelargestlymphnode.Wewouldthenanalyzeifthereisacorrelationbetweenthesizeofthelargestlymphnodeandthedevelopmentofdistantmetastases.Acontrolgroupwiththesametreatmentbutnometastaseswouldbeusedforcomparison.Ifwecouldconfirmthatthesizeofthelargestlymphnodecorrelatestoreducedsurvivalitcouldindicateasentinelnodefunctioninrectalcancer.Identificationofasinglelargeindexlymphnode

asanindicatorforN+stagewouldbemorereproduciblethanmorphologiccriteriaofindividuallymphnodes.Wecouldusethispilot-studyforafuturelargerprospectivestudy.Inthatstudywecouldincludeotherfactorssuchasextra-muralvascularinvasion(EMVI)andtumourregression.Thesefactorscouldthenbeanalyzedandcomparedtooutcomeinordertosaferselectpatientsforanon-operativemanagementofrectalcancer.

AcknowledgementsTherearesomany thatdirectlyor indirectlyhavesupportedme through thisexcitingjourney. It isnotpossible to thankallofyou individuallybut Iammostgratefuland IthankyouallforallthehelpandsupportIhavereceived.Afewpersonsmustbementioned,aswithoutthemthisthesiswouldnothaveseenthelightofday.Olof Hallböök,mymain tutor, colleague, professor and friendwho has been a hugesource of knowledge and inspiration for me, both in research and daily surgical life.Longhoursofsurgerywithashortbreakforfoodandresearchdiscussion,thebetteroftwoworlds!Withgreatpatiencepushingmewhennecessarybutalwayssupportingmeand finding time for discussions despite tough schedules and grouchy section leader.YouhavealwaysbeentherefromthebasementofGrandCentralStationinNewYorktotheroofofIlDuomoDiMilano.Ihopewecanlookforwardtofutureprojectsandthatyou´llkeeppushingmetokeep“attentiontodetails”asIamnotreallythereyet…Xiao-FengSun,professorandmyfirsttutorwhotookmeonwhenIknewabsolutelynothingaboutresearchandevenlessoflabwork.Youtaughtmescientificwriting, labwork and all the other tricks of the trade in research. You were always extremelygenerous in sharingwithmeyour time andyourdeepknowledge.Without your kindand professional introduction to research I would probably have left the lab andreturnedbacktotheoperationtheatre,butIamverythankfulthatyoushowedmetheroadtoresearch.Gunnar Arbman, co-tutor and long-time colleague inNorrköping. Who alsowasmyfirsthead-of-thedepartment,andtheonewhoinspiredmetogoontoresearchusinghisuniqueregisterdata.MyintentionwastocombineyourclinicalexpertiseanddatawithXiao-Feng’smolecularknowledgeandlab.Asitturnedoutthatjobisstillnotdone!Your contribution to this thesis has been invaluable.With your laser-sharpmind youalwayscamewithaquickresponseandcrystalclearremarksregardingnewmaterial.Andnomatterhowmanytimes therestofusreadandrereadamanuscript, ithad topass through Gunnar’s hands before we could trust in it. I am very grateful foreverythingyouhavetaughtmeinsurgeryandscience.JohanDabrosinSöderholm,professor,forashortbutveryimportantperiodmymaintutor. You navigated through dire straits and put me in contact with people at theKarolinskaIntsituteandmadesurethattheshowwenton.IamverygratefulJohan.Victoria Fomichov, co-author and statistician. The process of boiling down 11226patients to the 26 that we were really interested in would not have been possiblewithoutadedicated,brightstatisticianwithvastexperience incancerregisters. Ihadone!Iamsurethatforawhileyouthought(orstilldo)thatthepersonwhosentyoue-mailquestionsonstatisticsandmethodswasatenyear-oldkidonhisfather’scomputer.Itwouldhavebeenfuntokeeponresearchingwithyou,butIthankyouforyoureffortsandwishyouthebestofluckwithyournewjob.

TheKarolinskateamI,DavidEdler,ErikSyk,PelleNilsson,andMarjaHallström.I amverygrateful forour cooperationon the2ndarticleand forwelcomingme to thefantasticteamsfromKarolinskaandErsta.Indifferentwaysyouhaveall inmadeveryimportantcontributions,butmostofallbyyourgenerositywithtimeandrecoursesandnot the least, patience. It ‘s always great to meet you at different surgical meetingswhereyoualwaystreatmeasifIwasa08!KarolinskaTeamII,ProfessorLennartBlomqvistandMargrétSturludottir,whentreadingonthiniceasasurgeoni.e.writinganarticleonMRIinterpretationyouneedsomeonetoholdonto.Lennartisaninternationalauthorityinthisfield.Iamextremelygrateful tohavebeenworkingwithyouandMargréton the4th article.Youhavebothmadeaverylargeandimportantcontributionandithasbeenaneye-openertositanddiscuss MRI set ups and interpretations with you. Without radiology we are blindwithoutradiologistswearelost.KarinAlmlöv,co-authorwhodidagreatjobwithfigures,numbersandpatientsinthe4tharticle,itisgreatfuntoworkwithyouandIhopewecancontinue.AnnaLindhoffLarssonandMalinOhlsson,researchnurseswhohavehelpedmewiththetediousworkofnaggingatotherhospitalstosenduspatientrecords,andMRIfiles.Nottomentionsortingthemoutintoperfectorder.Youhavebeenagreathelp.Peter Cox, not only an outstanding anesthesiologist but also a master of both theSwedishandEnglishlanguagewhonotonlyhelpedmedwithlanguageeditingbutwhoalso had many interesting questions and ideas about my research, which was veryrefreshing.Itisalwaysthesamepleasuretoworkwithyouontextsorinsurgery.NinaLönn,who tookmylongtimeratherblurryvisualideaoftheWaronCancerandturneditintoaworkofartonthecoverofthisbook.ThisisprobablytheonlythinginthisprocessthatendedupthewayIthoughtsixyearsagointhisprocess.Allmycolleagues,nursesandstaffattheDepartmentofSurgery,Linköpingwhohavereallymademefeelwelcome.Thecolorectalunit,OlofHallböök,DisaKalman,BärbelJung,AndersKald,PärMyrelid,HannaLjungbåge,EleonorAhlgrenandAnnaLevinandallthenurseswhoallmakeaperfectteamandwhohavesupportedmeallthewayinbothresearchandclinicalday-to-daylifesharingyourexperienceandfriendship.Iwillbeback!AllmycolleaguesandfriendsattheDepartmentofSurgeryinNorrköpingwhoformanyyearshavesupportedmeandincludedmeinawonderfulsurgicalteam.Afewindividualsmustbementionedthough,GunnarIhavementionedbefore,StaffanHaapaniemimylong-timepartnerinsurgerywhoinspiredmetodoresearchandcoveredformewhileIdelvedintobooks.HasseKrook,headofthedepartmentformanyyearswhoencouragedmetotaketheleapintoresearch.Thefantasticcolorectalnurses,whomadesurgicallifesomucheasier.Alltheresidents,severalofwhomhavemanagedtobecomeexcellentcolorectalsurgeonsduringthetimeittookmetowritethisbook,keeponpushing!

PerSandström,Professorforeditingandreadingmythesisandsharingyourexperienceonresearchwithme.

RuneSjödahl,professorwhohasbeenaninspirationinsurgery,leadershipandresearch.Ihavefeltyoursupportandinterestinmyresearchfromearlyon.Katarina,Kärling,TeresErwillandGunillaLinghammar.Greatgang,oneofyouwon’tmissme!ConnyWallon,headoftheDepartmentofSurgeryinLinköpingwhohastrustedandsupportedmeinclinicalwork,andhasmadethisthesispossiblebyallocatingtime,givingencouragementandbysteppingintohelpwithclinicalworkwheneveryouhadtheopportunitydespiteanoverbookedschedule.Allfriendswhohavepushedandsupportedandhelpedwheneverhelpwereneededinabusyfamilyschedule.TheCösterandPompermaier/WijkmanfamiliesaswellastheAldrinfamily,youhaveallbeenpartofthisjourneyandbeenimportantsupportforourfamily.JulieWilkandJonasMalmstedtforfriendshipandtheinspirationtoresearch.HannaandJohanBjörkdahl-Loftås.Foreverythingalltheway!MayLisandStigLoftås,wonderfulparentswhobroughtmeuptobelieveinmyselfnomatterwhat,andalwaysinspiredandsupportedmyfamilyandI.OnlyyoucouldhavebelievedIwouldenduphere,Iamsogratefultoyou,andmysistersSaraandAnnforbeingthebestfamilyonecouldaskfor.Aboveall-Lindamybelovedwife.Onlyyouknowwhatyouhavedonetohelpmethroughthis,andIloveyouforthat,andeverythingelse.Trustme,thereisalifeafterthisanditwillbegreattoliveitwithyou.Hanna,Ida,YlvaandEmil,wonderfulkidsthatgivemesomuchjoyandlaughtereveryday.Youhelpmegoon!

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