Clinical Application of Fluorescence

ReviewClinicalApplicationofFluorescenceImagingofLiverCancerUsingIndocyanineGreenNorihiroKokudoTakeakiIshizawaHepato-Biliary-PancreaticSurgeryDivision,DepartmentofSurgery,GraduateSchoolofMedicine,UniversityofTokyo,Tokyo,JapanKeyWordsFluorescenceimaging∙Hepatocellularcarcinoma∙Indocyaninegreen∙Metastaticlivercancer∙NavigationsurgeryAbstractRecently,fluorescenceimagingusingindocyaninegreen(ICG)hasbeenappliedtohepatobi-liarysurgery,notonlytovisualizethebileducts,butalsotoidentifylivercancerduringsur-gery.Inthistechnique,ICGisadministeredintravenouslyatadoseof0.5mg/kgbodyweightforroutineliverfunctiontestingbeforesurgery.Intraoperatively,livercancercanbereadilyidentifiedbyfluorescenceimagingontheliversurfacebeforeresectionandonthecutsur-faceoftheresectedspecimen.Thisisachievedbyvisualizingfluorescencefromtheareaofimpairedbileexcretioninhepatocellularcancertissueandintheliverparenchymasurround-ingmetastaticlivercancers.Livercancernavigationsurgery,firstdevelopedinJapan,isalsopossible,anditrepresentsoneofthefewfluorescenceimagingtechniquesforcancerthathavereachedthestageofclinicalapplication;withfurtherdevelopmentsinbasicresearch,fluorescenceimagingisexpectedtobecomeanindispensabletechniqueforthediagnosisandtreatmentoflivercancer.Copyright©2012S.KargerAG,BaselIntroductionAttemptshavelongbeenmadetolabelcancertissueswithfluorescentprobesandvisualizetheminrealtimeduringendoscopicexaminationsorsurgicaltreatment,and©2012S.KargerAG,Basel2235-1795/12/0011-0015$38.00/0www.karger.com/licLiverCancer2012;1:15–21DOI:10.1159/000339017Publishedonline:June8,2012NorihiroKokudo,MD,PhDHepato-Biliary-PancreaticSurgeryDivision,DepartmentofSurgery,GraduateSchoolofMedicine,UniversityofTokyo7-3-1Hongo,Bunkyo-ku,Tokyo113-8655(Japan)Tel.+81358008841,E-MailKOKUDO-2SU@h.u-tokyo.ac.jp15fluorescenceimagingremainsoneofthemostactivekeyareasofresearch.However,fewtechniqueshaveprogressedtoclinicaluse,despitetheenormousamountofbasicresearchbeingpublished.Applicationsotherthantheintraoperativeimagingoflivercancerwithindocyaninegreen(ICG)[1,2]arelimitedtothediagnosisofbraintumors(glioblastomas)[3],bladdercancer[4],andskintumors[5]andphotodynamictherapyusingtheporphyrinprecursor5-aminolevulinicacid.Herein,wereviewthehistoricaldevelopmentandmecha-nismoflivercancervisualizationusingICGfluorescenceimaging.OutlineofSurgicalNavigationwithICGPrincipleofICGFluorescenceImagingWhenprotein-boundICGisexposedtoexcitationlightat750–810nm,itemitsfluo-rescencethatpeaksatabout840nm[6].Becausethiswavelengthisbarelyabsorbedbyhemoglobinorwater,structuresthatcontainICGcanbevisualizedthrough5–10mmoftissuebycombineduseofacamerathatissensitivetoinfraredlightandanappropriatefil-ter(theICGfluorescenceimagingtechnique).Thesepropertiesareoptimalforperformingcontraststudiesofvascularstructures,andintheearly1990s,ICGfluorescenceimagingwasappliedclinicallytofundusangiographyinthefieldofophthalmology[7].However,probablybecauseICGwaswidelyadoptedasareagentformeasuringcardiacoutputormeasuringliverfunction,littleattentionwaspaidtoICG’sfluorescencepropertiesinthefieldsofthoracicsurgeryorgeneralsurgery,andtherehavebeenfewattemptstoapplyittosurgicalnavigation.ApplicationtoSurgicalNavigationAtthebeginningofthe21stcentury,severalsurgicalnavigationtechniquesutilizingthefluorescencepropertiesofICGweredevelopedinrapidsuccession.Coronaryarteriog-raphyduringcardiacbypasssurgeryusingICGfluorescenceimagingwasdevelopedfirst[8].Then,thefluorescencepropertiesofICGwereappliedtolymphangiography[9]andtheidentificationofsentinelnodesduringbreastcancerresections[10];atpresent,itsapplica-tionhasexpandedtocholangiographyduringhepatobiliarysurgery[11–13]andtoevalua-tionsofthebloodflowaftertheclippingofcerebralarterialaneurysms[14].Oneoftheseapplications,theidentificationofbreastcancersentinelnodes,wasthefirstapplicationofICGfluorescenceimagingtothesurgicaltreatmentofcancer.Howev-er,thismethodvisualizeslymphaticdrainageroutesfromthecancertissueandallowssearchesforlymphnodes,butdoesnotallowvisualizationoftheICGthathasbeentakenupspecificallybycancertissues.Thenatureoffluorescenceimagingofhepatocellularcar-cinoma(HCC)[1,2],whichwasfirstdescribedinJapanin2009,isverydifferentfromthatofothertechniques:itallowsvisualizationoftheHCCitselfbyfluorescenceimagingoftheICGthatistakenupandremainsinthecancertissuesasaresultofbiliaryexcretiondis-orders.Thisgiveshighlesion-to-backgroundcontrast[median(range),2.6(0.6–16.6)fold,unpublisheddata]forwell-ormoderatelydifferentiatedHCC,whichishigherthanthatforotherkindsofcancertissues(upto2fold)[15,16].LivercancerimagingbytheICGfluorescenceimagingtechniquewaspatentedbyagroupattheUniversityofRochester(US)in2008(WO2008/043101A2),althoughtherehavebeennodetailedreportsinrespectofsuchlivercancerimaging,exceptforarecentarticleontheapplicationofthistechniquetoroboticsurgeryforidentificationofrenalcancer[17].Basedonareadingofthepatentdetails,theirmethodprobablyutilizesthedifferenceinhemodynamicsbetweenlivercancertissueandthesurroundinglivertissue,16Kokudoetal.:ICGFluorescenceImagingofLiverCancerLiverCancer2012;1:15–21DOI:10.1159/000339017Publishedonline:June8,2012©2012S.KargerAG,Baselwww.karger.com/licwhichisdifferentfromtheJapanesetechniquethatisbasedonimpairedbiliaryexcretioninHCCtissues.DevelopmentofLiverCancerImagingUsingICGDiscoveryofFluorescenceImagingofLiverCancerTheauthorsoriginallydevelopedacholangiographicmethodbasedonICGfluores-cenceimaging[13,14].Duringtheprocessofthatdevelopment,wenoticedthattumorsontheliversurfaceemittedtheirownfluorescenceevenbeforeICGwasinjectedintothebileductforcholangiography.AllpatientsinourdepartmentareadministeredICGintrave-nouslytomeasuretheICGretentionrateat15minasaroutinepreoperativeliverfunctiontest[18,19].Thus,itwasassumedthattheintraoperativevisualizationoflivercancerbyICGfluorescenceimagingmusthavebeenbecausetheICGthathadbeeninjectedintrave-nouslypriortothesurgeryhadremainedinthelivercancerorsurroundinglivertissues.Aprospectiveclinicalstudywasimmediatelyinitiated,andthefollow-upresultsfor63HCCs(37cases)and28metastaticlivernodulesfromcolorectalcancer(12cases)werereportedin2009[1].Atthesametime,Gotohetal.reportedtheresultsofICGfluorescenceimagingof14HCCs(10cases)thatincluded4nodulesfirstdiscoveredbythistechnique[2].Yokoya-maetal.recentlyaddedareportonthepossibilityofapplyingICGfluorescenceimagingtotheintraoperativediagnosisofmicrometastasesofpancreaticcancer[20].MechanismofFluorescenceImagingofLiverCancerIntheauthors’study,well-differentiatedHCCtissueexhibitedintensefluorescenceonthecutsurfaceofsurgicallyresectedliverspecimens,whereasforpoorlydifferentiatedHCCandmetastasesofcolorectalcancer,thecancertissueitselfwasnotvisualized,butthesurroundingnoncanceroustissuesproducedaringoffluorescence(fig.1)[21].Moreover,observationsmadewithafluorescencemicroscopeconfirmedthepresenceofICGfluores-cenceinthecytoplasmandpseudoglandsinwell-differentiatedHCCs;fluorescencewasalsoseeninnontumorouslivertissuescompressedbythetumorincasesofpoorlydiffer-entiatedHCCsandcolorectalcancermetastases.Basedontheaboveresults,itissuggestedthatwell-differentiatedHCCtissuestakeupICG,butthebiliaryexcretionofICGisimpairedbecauseofmorphologicalorfunctionalabnormalities.Incontrast,poorlydifferentiatedHCCsandmetastaticlivercancersdonottakeupICG,buttheycanbevisualizedbyringsofflourescenceresultingfromimpairedbileexcretioninthesurroundingnontumoroustis-suescompressedbythetumors.Consequently,well-differentiatedHCCs,poorlydifferenti-atedHCCs,andmetastaticlivercancerscanallbevisualizedbyICGfluorescentimaging.TheexistenceofimpairedbileexcretioninHCCandinnoncanceroustissuessurround-ingthecancerhadbeensuggestedbytheresultsofmagneticresonanceimagingwithabile-excretedcontrastmedium[22].Moreover,Kitaoetal.demonstratedreducedexpres-sionoforganicaniontransportingpolypeptide8,whichisinvolvedinthecellularuptakeofcontrastmedia,inpoorlydifferentiatedHCCcomparedwiththatinwell-differentiatedHCC[23],andthedifferencesbetweentheenhancementpatternsofwell-andpoorlydiffer-entiatedHCCbyICGfluorescenceimagingmayalsobeattributabletoasimilarmechanism.Inaddition,throughapproachesutilizingimmunostainingandgeneexpressionanalyses,itishopedthatprogresswillbemadeinelucidatingthemechanismoflivercancerimag-ingbyICGfluorescenceimagingusingarecentlydevelopedfluorescenceendomicroscopysystem[24]andinmakingreal-timeobservationsofthepharmacokineticsofICGinlivercancersandthesurroundingtissues.Kokudoetal.:ICGFluorescenceImagingofLiverCancerLiverCancer2012;1:15–21DOI:10.1159/000339017Publishedonline:June8,2012©2012S.KargerAG,Baselwww.karger.com/lic17TechniquesandPrecautionsforICGFluorescenceImagingofLiverCancerThetechniqueofICGfluorescenceimagingoflivercancerisverysimple:onceICG(0.5mg/kgbodyweight)isadministeredwithin14dayspriortosurgery,wecanobtainfluo-rescenceimagesoflivercancersintraoperativelybysimplyplacingthecameraimagingheadofcommerciallyavailablefluorescenceimagingsystemsontheliversurfaceoronsurgicalspecimens(fig.2)[1,21].Inpatientswithbileductinfiltrationsoflivercancer,fluorescenceimagingisalsopossibleforbiliarycongestedregionsontheliversurface,whichmaybehelpfulforsurgeonsindeterminingthehepatictransectionline[25].ItshouldbenotedthatifICGisinjectedintravenouslyonthedaybeforesurgery,wash-outfromthenoncancerouslivertissueisinadequateandtheremaybemanyfalse-positivenodules;thepoorertheliverfunctionis,themoremarkedthistendencyis.FurtherstudiesareneededtodeterminetheoptimalintervalbetweenICGinjectionandsurgeryonthebasisofthepatient’sliverfunction.Moreover,thistechniquedoesnotuseanantigen–anti-bodyreactionthatisspecificforcancer,itjustallowsvisualizationoftheareasofimpairedbileexcretioninoraroundthetumortissues;consequently,benignlesionssuchasregen-eratingnodulesorbileductproliferationmayalsoexhibitfluorescence.Previousreportshaveindicatedthat40–50%ofthelesionsnewlydiscoveredbyfluorescenceimagingofresectedHCCspecimenswerenoncancerouslesions[1,2].Thus,theprincipalroleofthismethodliesinintraoperativelysearchingforsmalllivercancersneartheliversurfacethatweredetectedbypreoperativeimagingstudies;anyadditionalresectionoffluorescentar-easthatarenewlydiscoveredintraoperativelybytheICGfluorescenceimagingtechniqueshouldbeconsideredonlyaftertheyhavebeenadequatelyconfirmedbyinspectionandpalpationand/orbyanintraoperativeultrasoundexamination.18Kokudoetal.:ICGFluorescenceImagingofLiverCancerLiverCancer2012;1:15–21DOI:10.1159/000339017Publishedonline:June8,2012©2012S.KargerAG,Baselwww.karger.com/licFig.2. ICGfluorescenceimagingonliversurfaces(left)andtheirgrossappearances(right)(from[21]).aFluorescenceimagingpriortoliverresectionenabledvisualizationofthemetastasisofcolorectalcancerthatwaspalpablebutgrosslyunidentifiable(arrow),aswellastheothertwolesionsvisibleontheliversur-face.bFluorescenceimagingoftheresectedspecimen.Thear-rowindicatesagrosslyunidentifiabletumorthatwaslocated0.8cmbelowtheliversurface.Fig.1. Fluorescencepatternsoflivercancersoncutsur-faces(left)andtheirgrossap-pearance(right)(from[21]).aTotalfluorescencetype(well-differentiatedHCC,7mmindi-ameter).bPartialfluorescencetype(moderatelydifferenti-atedHCC,35mmindiameter).cRimfluorescencetype(poor-lydifferentiatedHCC,30mmindiameter).dRimfluorescencetype(metastasisofcolorectalcancer,25mmindiameter).ProblemswiththeICGFluorescenceImagingTechniqueandFutureProspectsProblemswithICGFluorescenceLiverCancerImagingICGfluorescenceimaginghastheadvantageofbeingaphenomenonthatiseasytoun-derstand–thereisfluorescencewherethereisbile–andofbeingsimpleandhighlysensi-tive;however,becauseitidentifieslivercancerbyvisualizingimpairedbiliaryexcretionofICG,itmaybelessspecificthanpre-clinicalfluorescenceimagingtechniquesusingacan-cer-specificantibody.Moreover,sincethetissuepenetrationofthefluorescenceemittedbyICGisonlyabout5–10mm,itisimpossibletovisualizetumorslocateddeepwithintheliver.AlthoughICGfluorescenceimagingisalreadyasufficientlypracticaltechniqueforhepaticsurgeons,itisimportanttounderstandthecharacteristicsofthetechniqueandtoutilizeitinacomplementarymannerwithinspectionandpalpationoranintraoperativeultrasoundexamination.NewTechniquesLikelyToBeDevelopedfortheClinicalSettingIfthedevelopmentprogressessatisfactorilyofanovelfluorescentprobe[26]thatisexcretedinthebilebythesamerouteasICGandwhosetissuepenetrationissuperiortothatofICG,thenthesensitivityofimagingforlivercancerwiththistechniquemayimprove.Moreover,ifICGcanbemodifiedwithacancer-specificantibodyorothertracer[27],itmightnotonlybepossibletouseitforsurgicalnavigation,butitmayevolveintoanewpre-operativeimagingtechniquethatsimultaneouslyallowstumordiagnosisandlocalization,evaluationofthesegmentalliverfunction.Inparticular,photodynamictherapyforcancerusingacompoundconsistingofatumor-specificantibodyandaphotosensitivesubstancehasrecentlyattractedmuchattention[28].SinceICGisalsoaphotosensitivesubstance,anovelphotodynamictherapythattakesadvantageofthispropertyofICGanditsaccumula-tioninlivercancermaybepossible.Itisalsoessentialtoimprovetheperformanceofimagingdevices.Itisalreadypossibletosimultaneouslyacquirecolorimagesandfluorescenceimages[29,30]andtoaddanin-fraredobservationsystemtolaparoscopicsurgerysystems[13,31,32]andtoaroboticsur-gerysystem[17].IfitbecomespossibletovisualizeICGfluorescencethatisemittedfromdeeperregions,thiswouldcontributetofurtherimprovementoftheaccuracyofresectionforlivercancer.ConclusionsICGfluorescenceimagingisquiteasimpletechniquethatenableshighlysensitiveiden-tificationoflivercancersinrealtimeduringsurgery.Thistechniqueisalsovaluablefromthestandpointofbeingoneofthefewfluorescenceimagingtechniquesforcancerthathavereachedclinicalapplication.Furtherbasicresearchshouldresultininnovativetechniquesinthenearfuture,andthistechniquemayevolvetoplayamajorclinicalroleinthediagno-sisandtreatmentoflivercancer.AcknowledgmentsThisworkwassupportedbygrantsfromtheTakedaScienceFoundation,theKanaeFoundationforthePromotionofMedicalScience,andtheMinistryofEducation,Culture,Sports,ScienceandTechnologyofJapan(No.23689060and23249067).Kokudoetal.:ICGFluorescenceImagingofLiverCancerLiverCancer2012;1:15–21DOI:10.1159/000339017Publishedonline:June8,2012©2012S.KargerAG,Baselwww.karger.com/lic19References1IshizawaT,FukushimaN,ShibaharaJ,etal:Real-timeidentificationoflivercancersbyusingindocyaninegreenfluorescentimaging.Cancer2009;115:2491–2504.2GotohK,YamadaT,IshikawaO,etal:Anovelimage-guidedsurgeryofhepatocellularcarcinomabyindo-cyaninegreenfluorescenceimagingnavigation.JSurgOncol2009;100:75–79.3StummerW,StockerS,WagnerS,etal:Intraoperativedetectionofmalignantgliomasby5-aminolevulinicacid-inducedporphyrinfluorescence.Neurosurgery1998;42:518–525.4KriegmairM,SteppH,SteinbachP,etal:Fluorescencecystoscopyfollowingintravesicalinstillationof5-aminolevulinicacid:anewprocedurewithhighsensitivityfordetectionofhardlyvisibleurothelialneoplasias.UrolInt1995;55:190–196.5MortonCA,BrownSB,CollinsS,etal:Guidelinesfortopicalphotodynamictherapy:reportofaworkshopoftheBritishPhotodermatologyGroup.BrJDermatol2002;146:552–567.6LandsmanML,KwantG,MookGA,etal:Light-absorbingproperties,stability,andspectralstabilizationofindocyaninegreen.JApplPhysiol1976;40:575–583.7GuyerDR,PuliafitoCA,MonésJM,etal:Digitalindocyanine-greenangiographyinchorioretinaldisor-ders.Ophthalmology1992;99:287–291.8RubensFD,RuelM,FremesSE:AnewandsimplifiedmethodforcoronaryandgraftimagingduringCABG.HeartSurgForum2002;5:141–144.9OgataF,AzumaR,KikuchiM,etal:Novellymphographyusingindocyaninegreendyefornear-infraredfluorescencelabeling.AnnPlastSurg2007;58:652–655.10KitaiT,InomotoT,MiwaM,etal:Fluorescencenavigationwithindocyaninegreenfordetectingsentinellymphnodesinbreastcancer.BreastCancer2005;12:211–215.11MitsuhashiN,KimuraF,ShimizuH,etal:Usefulnessofintraoperativefluorescenceimagingtoevaluatelocalanatomyinhepatobiliarysurgery.JHepatobiliaryPancreatSurg2008;15:508–514.12IshizawaT,TamuraS,MasudaK,etal:Intraoperativefluorescentcholangiographyusingindocyaninegreen:abiliaryroadmapforsafesurgery.JAmCollSurg2009;208:e1–e4.13IshizawaT,BandaiY,IjichiM,etal:Fluorescentcholangiographyilluminatingthebiliarytreeduringlapa-roscopiccholecystectomy.BrJSurg2010;97:1369–1377.14RaabeA,NakajiP,BeckJ,etal:Prospectiveevaluationofsurgicalmicroscope-integratedintraop-erativenear-infraredindocyaninegreenvideoangiographyduringaneurysmsurgery.JNeurosurg2005;103:982–989.15ThayerD,UnluMB,LinY,etal:Dual-contrastdynamicMRI-DOTforsmallanimalimaging.TechnolCancerResTreat2010;9:61–70.16KimTH,MountCW,DulkenBW,etal:Filamentous,mixedmicellesoftriblockcopolymersenhancetumorlocalizationofindocyaninegreeninamurinexenograftmodel.MolPharm2012;9:135–143.17TobisS,KnopfJ,SilversC,etal:Nearinfraredfluorescenceimagingwithroboticassistedlaparoscopicpartialnephrectomy:initialclinicalexperienceforrenalcorticaltumors.JUrol2011;186:47–52.18MakuuchiM,KosugeT,TakayamaT,etal:Surgeryforsmalllivercancers.SeminSurgOncol1993;9:298–304.19IshizawaT,HasegawaK,AokiT,etal:Neithermultipletumorsnorportalhypertensionaresurgicalcon-traindicationsforhepatocellularcarcinoma.Gastroenterology2008;134:1908–1916.20YokoyamaN,OtaniT,HashidateH,etal:Real-timedetectionofhepaticmicrometastasesfrompancreaticcancerbyintraoperativefluorescenceimaging:Preliminaryresultsofaprospectivestudy.Cancer2011;(inpress).21IshizawaT,HaradaN,MuraokaA,etal:ScientificbasisandclinicalapplicationofICGfluorescenceimag-ing:hepatobiliarycancer.OpenSurgOncolJ2010;2:31–36.22ChungJJ,KimMJ,KimKW:Mangafodipirtrisodium-enhancedMRIforthedetectionandcharacterizationoffocalhepaticlesions:isdelayedimaginguseful?JMagnResonImaging2006;23:706–711.23KitaoA,ZenY,MatsuiO,etal:Hepatocellularcarcinoma:signalintensityatgadoxeticacid-enhancedMRImaging–correlationwithmoleculartransportersandhistopathologicfeatures.Radiology2010;256:817–826.24GoetzM,DerisI,ViethM,etal:Near-infraredconfocalimagingduringmini-laparoscopy:anovelrigidendomicroscopewithincreasedimagingplanedepth.JHepatol2010;53:84–90.25HaradaN,IshizawaT,MuraokaA,etal:Fluorescencenavigationhepatectomybyvisualizationoflocal-izedcholestasisfrombileducttumorinfiltration.JAmCollSurg2010;210:e2–e6.26FigueiredoJL,SiegelC,NahrendorfM,etal:Intraoperativenear-infraredfluorescentcholangiography(NIRFC)inmousemodelsofbileductinjury.WorldJSurg2010;34:336–343.27OgawaM,KosakaN,ChoykePL,etal:Invivomolecularimagingofcancerwithaquenchingnear-in-fraredfluorescentprobeusingconjugatesofmonoclonalantibodiesandindocyaninegreen.CancerRes2009;69:1268–1272.28MitsunagaM,OgawaM,KosakaN,etal:Cancercell-selectiveinvivonearinfraredphotoimmunotherapytargetingspecificmembranemolecules.NatMed2011;17:1685–1691.29KawaguchiY,IshizawaT,MasudaK,etal:Hepatobiliarysurgeryguidedbyanovelfluorescentimagingtechniqueforvisualizinghepaticarteries,bileducts,andlivercancersoncolorimages.JAmCollSurg2011;212:e33–e39.30MatsuiA,WinerJH,LaurenceRG,etal:Predictingthesurvivalofexperimentalischaemicsmallbowelusingintraoperativenear-infraredfluorescenceangiography.BrJSurg2011;98:1725–1734.20Kokudoetal.:ICGFluorescenceImagingofLiverCancerLiverCancer2012;1:15–21DOI:10.1159/000339017Publishedonline:June8,2012©2012S.KargerAG,Baselwww.karger.com/lic31IshizawaT,BandaiY,HaradaN,etal:Indocyaninegreen-fluorescentimagingofhepatocellularcarcinomaduringlaparoscopichepatectomy:aninitialexperience.AsianJEndoscSurg2010;3:42–45.32IshizawaT,ZukerN,KokudoN,etal:Positiveandnegativestainingofhepaticsegmentsbyfluorescentimagingtechniqueduringlaparoscopichepatectomy.ArchSurg2012;(inpress).Kokudoetal.:ICGFluorescenceImagingofLiverCancerLiverCancer2012;1:15–21DOI:10.1159/000339017Publishedonline:June8,2012©2012S.KargerAG,Baselwww.karger.com/lic21