Radiation therapy combined with intracerebral administration of carboplatin for the treatment of brain tumors

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Title:
Radiation therapy combined with intracerebral administration of carboplatin for the treatment of brain tumors
Series Title:
Radiation Oncology
Physical Description:
Mixed Material
Creator:
Weilian Yang
Rolf F Barth
Tianyao Huo
Robin J Nakkula
Michael Weldon
Nilendu Gupta
Lawrence Agius
John C Grecula
Publisher:
Radiation Oncology
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Subjects / Keywords:
F98 glioma
Carboplatin
Convection enhanced delivery
Radiotherapy
Brain tumors

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Abstract:
Background: In this study we determined if treatment combining radiation therapy (RT) with intracerebral (i.c.) administration of carboplatin to F98 glioma bearing rats could improve survival over that previously reported by us with a 15 Gy dose (5 Gy × 3) of 6 MV photons. Methods: First, in order to reduce tumor interstitial pressure, a biodistribution study was carried out to determine if pretreatment with dexamethasone alone or in combination with mannitol and furosemide (DMF) would increase carboplatin uptake following convection enhanced delivery (CED). Next, therapy studies were carried out in rats that had received carboplatin either by CED over 30 min (20 μg) or by Alzet pumps over 7 d (84 μg), followed by RT using a LINAC to deliver either 20 Gy (5 Gy × 4) or 15 Gy (7.5 Gy × 2) dose at 6 or 24 hrs after drug administration. Finally, a study was carried out to determine if efficacy could be improved by decreasing the time interval between drug administration and RT. Results: Tumor carboplatin values for D and DMF-treated rats were 9.4 ±4.4 and 12.4 ±3.2 μg/g, respectively, which were not significantly different (P = 0.14). The best survival data were obtained by combining pump delivery with 5 Gy × 4 of X-irradiation with a mean survival time (MST) of 107.7 d and a 43% cure rate vs. 83.6 d with CED vs. 30-35 d for RT alone and 24.6 d for untreated controls. Treatment-related mortality was observed when RT was initiated 6 h after CED of carboplatin and RT was started 7 d after tumor implantation. Dividing carboplatin into two 10 μg doses and RT into two 7.5 Gy fractions, administered 24 hrs later, yielded survival data (MST 82.1 d with a 25% cure rate) equivalent to that previously reported with 5 Gy × 3 and 20 μg of carboplatin. Conclusions: Although the best survival data were obtained by pump delivery, CED was highly effective in combination with 20 Gy, or as previously reported, 15 Gy, and the latter would be preferable since it would produce less late tissue effects.

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University of Florida
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University of Florida
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AA00020076:00001

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RESEARCHOpenAccessRadiationtherapycombinedwithintracerebral administrationofcarboplatinforthetreatmentof braintumorsWeilianYang1,RolfFBarth1*,TianyaoHuo1,4,RobinJNakkula1,MichaelWeldon2,NilenduGupta2,LawrenceAgius3andJohnCGrecula2AbstractBackground: Inthisstudywedeterminediftreatmentcombiningradiationtherapy(RT)withintracerebral(i.c.) administrationofcarboplatintoF98gliomabearingratscouldimprovesurvivaloverthatpreviouslyreportedby uswitha15Gydose(5Gy3)of6MVphotons. Methods: First, inordertoreducetumorinterstitialpressure,abiodistributionstudywascarriedouttodetermine ifpretreatmentwithdexamethasonealoneorincombinationwithmannitolandfurosemide(DMF)wouldincrease carboplatinuptakefollowingconvectionenhanceddelivery(CED). Next, therapystudieswerecarriedoutinratsthat hadreceivedcarboplatineitherbyCEDover30min(20 g)orbyAlzetpumpsover7d(84 g),followedbyRT usingaLINACtodelivereither20Gy(5Gy4)or15Gy(7.5Gy2)doseat6or24hrsafterdrugadministration. Finally, astudywascarriedouttodetermineifefficacycouldbeimprovedbydecreasingthetimeintervalbetween drugadministrationandRT. Results: TumorcarboplatinvaluesforDandDMF-treatedratswere9.44.4and12.43.2 g/g,respectively,which werenotsignificantlydifferent(P=0.14).Thebestsurvivaldatawereobtainedbycombiningpumpdeliverywith5 Gy4ofX-irradiationwithameansurvivaltime(MST)of107.7danda43%curerate vs. 83.6dwithCED vs. 30-35 dforRTaloneand24.6dforuntreatedcontrols.Treatment-relatedmortalitywasobservedwhenRTwasinitiated 6hafterCEDofcarboplatinandRTwasstarted7daftertumorimplantation.Dividingcarboplatinintotwo10 g dosesandRTintotwo7.5Gyfractions,administered24hrslater,yieldedsurvivaldata(MST82.1dwitha25% curerate)equivalenttothatpreviouslyreportedwith5Gy3and20 gofcarboplatin. Conclusions: Althoughthebestsurvivaldatawereobtainedbypumpdelivery,CEDwashighlyeffectivein combinationwith20Gy,oraspreviouslyreported,15Gy,andthelatterwouldbepreferablesinceitwould producelesslatetissueeffects. Keywords: F98glioma,Carboplatin,Convectionenhanceddelivery,Radiotherapy,BraintumorsBackgroundAlthoughcisplatinandcarboplatinarehighlyeffective anti-cancerdrugs,whichhavebeenusedclinicallyto treatavarietyofmalignancies,theyhavenotbeeneffectiveinpatientswithhighgradegliomas[1].Despitethe factthattheirtumoricidalactivitycanbedemonstrated invitro [2],theirsystemictoxicity,highwatersolubility, andinabilitytoeffectivelypenetratetheblood-brain barrier(BBB)havelimitedtheirusefulnessfortreating patientswithbraintumors.AEuropeanOrganization forResearchandTreatmentofCancer(EORTC)clinical trialinpatientswithsupratentorialmalignantgliomas, whohadreceivedacombinationofcisplatinandRT, failedtodemonstrateanyimprovementineitherprogressionfreeoroverallsurvival[3].Thisbroughttoanend anyfurtherclinicalstudiestoinvestigatethecombination ofplatinum(Pt)compoundsandphotonradiationtotreat highgradegliomas. *Correspondence: rolf.barth@osumc.edu1DepartmentofPathology,TheOhioStateUniversity,Columbus,OH43210, USA Fulllistofauthorinformationisavailableattheendofthearticle 2014Yangetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Yang etal.RadiationOncology 2014, 9 :25 http://www.ro-journal.com/content/9/1/25

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Extensiveexperimentalstudieshavebeencarriedout byElleaumeandherresearchteaminFrance[4-8]and byusintheUnitedStates[9]toevaluateintracerebral(i.c.)deliveryofeithercisplatinorcarboplatinin combinationwithRT.Thiscombinationhasproduced thebestsurvivaldatathateverhavebeenreported withtheF98ratgliomamodel[10].Thepurposeof thepresentstudywastodetermineiftherapeuticefficacycouldbeimprovedoverthatpreviouslyreported by[9]usingtwodifferentRTregimensincombinationwithi.c.administrationofcarboplatinbyeither convectionenhanceddelivery(CED)orAlzetpump delivery.Bothoftheseapproachescandeliveratherapeuticagentdirectlytothesiteofthebraintumor, completelybypassingtheBBBandallowingoneto obtainadrugconcentrationthatcanbeupto1,000 greaterthanthatachievedbysystemicadministration ofcarboplatin[9].InthepresentstudythebestsurvivaldataandcurerateswereobtainedbyadministeringcarboplatinviaAlzetosmoticpumpsorCEDin combinationwitha5Gy4fractionateddoseof6 MVphotonswithnotreatment-relatedmortality.However,equivalentsurvivaldatawerereportedpreviouslyby us[9]usingafractionated15Gydose(5Gy3),andthe latterappearstobetheoptimumregimensinceitwould reducelongtermradiationrelatedeffectsonthebrain.MethodsF98ratgliomamodelTheF98ratgliomahasbeenpropagated invitro and invivo since1971and,asdescribedinarecentreview [10],ithasbeenusedinawidevarietyofstudiesin experimentalneuro-oncology .Itisaradioresistanttumor thatisinvariablyfatalwithaninoculumofasfewas100 cells.F98cellsweregrowninDulbecco ’ smodifiedEagle ’ s medium(DMEM)containing5%fetalbovineserum,as previouslydescribed[9].Allanimalstudieswerecarried outinaccordancewiththe GuidefortheCareandUseof LaboratoryAnimals (NationalAcademyPress,Washington, DC,1996)andtheprotocolwasapprovedbytheInstitutionalLaboratoryAnimalCareandUseCommittee.Male Fischerrats(AnimalProductionBranch,NationalCancer Institute,Frederick,MD)weighing~200 – 220gwereused inthepresentstudy.Astereotacticimplantationprocedurewasemployed[11].F98gliomacellsweresuspended inDMEMcontaininglowgellingtemperatureagaroseata concentrationofeither103cells/10 lfortherapystudies or105cells/10 lforbiodistributionstudies.Thesewere injectedintotherightcaudatenucleusover10-15sec.BiodistributionofcarboplatininF98gliomabearingratsAgreaternumberofcellswereusedforbiodistribution studiesinordertohavealargertumormassfor determinationofcarboplatinconcentrations.Inorderto reducetumorinterstitialpressure[12],abiodistribution studywascarriedouttodetermineifpretreatmentwith dexamethasone(D),aloneorincombinationwithmannitolandfurosemide(DMF)wouldincreasethetumor uptakeofcarboplatininF98gliomabearingratsfollowingi.c.convectionenhanceddelivery(CED)compared tountreatedrats.Elevento13daftertumorimplantation, biodistributionstudieswereinitiatedineitheruntreated ratsorthosethathadreceivedeitherintraperitoneal(i.p.) D(3mg/kgb.w.)daily3aloneorincombinationwith intravenous(i.v.)M(2.5g/kgb.w.)at0.25ml/minover10 minandi.v.F(2mg/kgb.w.)over15min.Asreportedby Boucheretal.,thisregimenproducedamarkedreduction intheintratumoralinterstitialfluidpressureinF98glioma bearingrats[12].Carboplatin(HospiraInc.LakeForrest, IL)wasadministeredbyCED(0.33 l/minover30min) [9].ImmediatelyfollowingterminationofCED,samplesof bloodweretakenandtheanimalswereeuthanized,their brainswereremoved,tumorswerecarefullydissectedout, weighed,frozen,andstoredat 20Candeventually processedforPtdeterminationsbymeansofinductively coupledplasma – opticalemissionspectroscopy(ICP-OES) [9].BasedontheatomicweightofPt(195.1),theconcentrationsofcarboplatin(M.W.371Da)werecalculatedby multiplyingthePtvaluesby1.90.TherapystudiesThefirstsetofexperimentswascarriedoutusinga 20 gdoseofcarboplatin,administeredi.c.byCED (0.33 l/min)over30minateither7dor13dafter tumorcellimplantationoralternativelybyinfusion usingAlzetosmoticpumps(model#2001,DurectCorp., Cupertino,CA)over7d(84 gin168 lat1 l/hr).In ordertodetermineifthetimeintervalbetweendrug administrationandRTwouldimprovesurvivaltimes,rats wereirradiatedateither6or24hrlaterwith6MV photonsusingaSiemensMevatronlinearaccelerator. Theratswereirradiatedundercontinuousisoflurane anesthesiainaspeciallyfabricatedcircularplexiglas chamberdividedinto4compartmentsradiatingoutfrom thecenter.Theanimals ’ bodieswereshieldedusinga pinwheel-shapedcerrobendblocksothatonlytheright cerebralhemisphereoftheirheads,whichwerepointed inwardstowardsthecenter,wasirradiated.Thechamber hadaninletandoutletforadministrationofisoflurane andoxygenviaananesthesiamachine(modelAestiva/5 7900,GEDatex-Ohmeda).Thetumorbearingrightcerebralhemispherewasirradiatedineitherfour5Gyortwo 7.5Gyfractions.Theradiationdosewascalculatedto mid-pointofthebrainalongtheanterior-posterioraxis.In thesecondstudy,ratsreceivedtwodivideddosesof carboplatin(10 gin20 l)byCEDondays13and15 followingtumorimplantationandtheywereirradiated with7.5Gyondays14and16.Yang etal.RadiationOncology 2014, 9 :25 Page2of9 http://www.ro-journal.com/content/9/1/25

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EvaluationoftherapeuticresponseAllexperimentalanimalswereweighed3timesperweek andtheirclinicalstatuswasevaluatedatthesametime. Oncetheanimalshadprogressivelygrowingtumors,as evidencedbysustainedweightloss(20%oftheirbody weightfollowingtreatment)theywereeuthanizedby exposuretoCO2.Survivaltimesweredeterminedby adding1-2daystothetimebetweentumorimplantationandeuthanization.Ratssurviving>180dwere designatedas “ cured ” andwereeuthanized.Thebrainsof allanimalsinthetherapystudieswereremovedafter death,andprocessedforneuropathologicexamination, stainedwithhematoxylinandeosin(H&E),andexamined microscopically.Neuropathologicevaluationofgliomabearingrats followingCEDofcarboplatinandX-irradiationFourgroups,consistingof6 – 7tumorbearingratseach, receivedCEDofcarboplatin(20 gin10 l)ond13or 14followingimplantationof104F98gliomacellsand wereirradiatedwithfour5Gyfractionsbeginning24hr followingCED.Cohortsof6 – 7ratswereeuthanizedat 1,2,3,or4weeksfollowingterminationofX-irradiation (25,32,39,and46dfollowingimplantation).Their brainswereremoved,fixedin10%bufferedformalin, sectionedcoronally,andthenprocessedforneuropathologicexamination.StatisticalevaluationofdataThemeancarboplatinconcentrationsstandarddeviationswerecomputedfortumorandtherightandleft cerebralhemispheresforbothuntreatedandDMFtreatedanimals.Atwo-tailed,twosamplettestwasused todeterminestatisticalsignificance(P-value 0.05).To studytheeffectsoftreatmentonsurvival,theMST, standarderror(SE),andmediansurvivaltime(MeST) werecalculatedforeachgroupandKaplan-Meier survivalcurveswereplotted.AnoverallLogRanktest wasperformedtotestforequalityofsurvivalcurvesover thesixgroups,andbetweenindividualgroups[13].The overall levelwas0.05,andmultiplecomparisonswere adjustedusingtheBonferronicorrection[14].Thepercentincreasedlifespanwasdetermined,aspreviously described[15].Inordertodeterminethesamplesize thatwouldhavebeenrequiredtodemonstratestatistical significancefollowingCEDofcarboplatintountreated andDMF-treatedrats,samplesizecalculationswerecarriedoutusingSAS9.3(SASInstitute,Cary,NC).Proc Powerprocedurewithaone-wayANOVA.Samplesizes werecalculatedwithpower=0.8,basedonthegroup meansandpooledstandarddeviationofthePtconcentrationintumorofuntreatedratsvsDMF-treated ratswithan level=0.05.ResultsBiodistributionstudiesThebiodistributiondatafortwogroupsofeightF98gliomabearingratsthathadreceived20 gofcarboplatin eitheraloneorfollowingpre-treatmentwitheitherD aloneorDMFaresummarizedinTable1.Sincethe carboplatinvaluesfollowingtheadministrationofdexamethasoneDaloneorDMFwereequivalent,thesevalues werecombined.Themeantumorvaluesandrangesforrats thatreceivedcarboplatinalonewere9.414.40 g/gand 3.02 – 17.10 g/g,respectively.Thecorrespondingvaluesfor ratspre-treatedwithDMFwere12.433.17 g/gand 8.55 – 18.51 g/g.Althoughthesedifferencesincarboplatinvalueswerenotsignificant(P=0.14)therewasa suggestionthatDMFtreatmentincreasedthetumor carboplatinconcentrations.Incontrasttothebroad rangesthatwereseeninthetumorcarboplatinvalues, thenormalbrainvaluesfortheright(tumorbearing) andleftcerebralhemisphereswereallinaverynarrow range.Thecarboplatinvaluesinnormalbrainofuntreated vs. DMFtreatedratswerenotsignificantlydifferent(P=0.22and0.52forrightandleftcerebral hemispheres,respectively).TherapeuticefficacyofX-irradiationincombinationwith i.c.carboplatinInthefirstseriesofexperiments,aradiationdoseof 20Gyinfour5Gyfractionswasadministeredtothe right,tumorbearingcerebralhemisphereincombinationwith20 gofcarboplatin,administeredbyCED or84 gbyAlzetpumpinfusion.Thesurvivaldata aresummarizedinTable2andKaplan-Meiersurvival plotsareshowninFigure1.Thebestsurvivaldata withnotreatment-relatedtoxicitywereseeninrats thatreceivedcarboplatinviaAlzetpumpsbetween days7and13followingtumorimplantationwithaMSTof 107.720.7dandacurerate(i.e.,survival>180d)of43%. RatsthatreceivedcarboplatinbyCEDonday13,followed 24hrslaterbytheinitiationofRT,hadaMSTof83.6 21.5d,witha25%curerate.IfRTwasinitiated6hrs followingCED,theMSTwas80.618.5d.However,there were2of8treatment-relatedearlydeaths(d27and29) andifthesewereexcluded,thecureratewas29%.If administrationofcarboplatinbyCEDwascarriedoutond 7followingimplantationandRTwasinitiatedond8,the MSTwas72.117.3dandacurerateof25%with2or8 earlytreatment-relateddeaths(d9and10),whichwere attributedtocerebraledema.Incontrast,untreatedcontrolshadaMSTof24.61.1dandX-irradiatedratshad anMSTof35.31.8d,bothwithinaverynarrowrange comparedtoratsthatreceivedRTincombinationwith carboplatin,whichhadverybroadranges.UsingtheLog Ranktest,theoveralldifferencebetweenthevarioustreatmentgroupsanduntreatedcontrolswashighlysignificantYang etal.RadiationOncology 2014, 9 :25 Page3of9 http://www.ro-journal.com/content/9/1/25

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(P<0.0001),aswerethedifferencesbetweenanimalsthat wereX-irradiatedvs.thosethatreceivedcombination therapy(P 0.003). Inthesecondseriesofexperiments,a15Gydose (7.5Gy2fraction)incombinationwithcarboplatin (10 g/d2d)wascarriedouttodetermineifthe survivaldatacouldbeimprovedifthecarboplatin dosewasdividedsothatahigherdrugconcentration wouldbeavailableatthetimethatRTwasinitiated. ThesedataaresummarizedinTable3andFigure1. TheMSToftheseratswas82.115.5dwitha25% curerateandnotreatment-relatedearlydeaths.Rats thatreceivedadivideddoseofcarboplatinaloneby CEDhadaMSTof46.87.5d,andthosethatonlyhad receivedRThadaMSTof30.01.9d(P=0.0006). NeuropathologicexaminationofthebrainsofthenonsurvivingratsthatreceivedcarboplatinbyeitherCEDor Alzetpumpdelivery,followedbyX-irradiation,revealed bothmacroscopicandmicroscopicdepositsoftumorat thetimesoftheirdeaths.Incontrast,thebrainsofthe long-termsurvivors(>180d)showednomicroscopic evidenceoftumor. Asummaryoftheradiationdosingschedulesandthe equivalentdosescomparedtoconventional2GyfractionsareshowninTable4.Basedonthesecalculations, a15Gydoseinthree5Gyfractions,aspreviously Table2SurvivaldataofF98ratsfollowingCEDofcarboplatinincombinationwithX-irradiationSurvivaltimes(days)b%Increasedlifespan TreatmentgroupaNMeanSEMedianRangeMeanMedian Untreatedcontrols524.61.12521 – 2800 X-irradiation535.31.835.531 – 394342 CEDofcarboplatin(onday13)+X-irradiation(6hlater)8c80.618.5c4827->180(2)22792 CEDofcarboplatin(onday13)+X-irradiation883.621.555.539->180(2)240122 CEDofcarboplatin(onday7)+X-irradiation8d72.117.3d499->180(2)19396 Alzetpumpdeliverye(d7-13)+X-irradiation8107.7216242->180(3)337148aStereotacticintracerebralimplantationof103F98gliomacellswascarriedoutond0.Carboplatin(20 gin10 L)wasadministeredbyconvectionenhanced deliveryataflowrateof0.33 L/min.for30min.ond13followingimplantation.Alternatively,theyreceived84 gofthedrugbyAlzetpumpsover7d (168 lat1 l/h).Animalsreceived20Gyof6MVLINACX-rays,deliveredin5Gyfractionsbeginning24hrsafterCEDunlessotherwiseindicated.bNindicatesthenumberofanimalspergroupandSEdesignatesthestandarderror.Survivalof>180dindicatesthattheseanimalshavebeencuredofthei r tumors.Thenumberinparenthesisindicatesthenumberoflong-termsurvivors.cThreetreatment-relatedearlydeathsond27and29wereincludedinthesecalculations.dTwotreatment-relatedearlydeathsond9and10wereincludedinthesecalculations.eCarboplatinwasadministeredbyAlzetpump(84 gover168hr)followed24hrlaterbyX-irradiation. Table1BiodistributionofcarboplatininF98gliomabearingratsfollowingCEDineitheruntreatedordexamethasone, mannitol,andfurosemidetreatedratsaCarboplatinconcentration( g/gtissue)bAnimalno.UntreatedDMFtreated TumorRBrainLBrainTumorRBrainLBrain 13.021.411.358.551.631.01 26.340.820.4510.161.220.78 37.921.551.0010.631.831.12 48.151.130.9210.641.790.83 58.841.620.8812.853.101.34 69.840.920.3013.320.950.99 714.102.050.8414.811.300.82 817.101.741.3418.512.280.96 MeanSDc9.414.401.410.420.890.3712.433.171.760.680.980.18 Median8.501.480.9011.751.710.98 Tumor:brainratios6.7:110.6:17.1:112.7:1aRatsreceivedeitheri.p.dexamethasone(D)(3mg/kgb.v.dailyfor3days)ori.p.dexamethasone(D)followedbyi.v.mannitol(M)(2.5g/kgb.w.)at0. 25ml/min over10min,followed15minlaterbyi.v.furosemide(F)(2mg/kgb.w.).Sincethecarboplatinvalueswereequivalent,thesewerecombined.bRatswereeuthanizedat~20minfollowingterminationofCED,theirbrainsremoved,tumorsdissectedout,aswellastheremainderofthetumorbearing right andnon-tumorbearingleftcerebralhemispheres.PlatinumconcentrationsweredeterminedbymeansofICP-OESandthenconvertedtocarboplatinby multiplyingby1.9.cSDdesignatesthestandarddeviation.Yang etal.RadiationOncology 2014, 9 :25 Page4of9 http://www.ro-journal.com/content/9/1/25

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reported[9],wasthebestintermsofnormaltissue sparingandproducedidenticalMSTsinF98glioma bearingratscomparedto20Gyinfour5Gyfractions (83.4dvs.83.6d,respectively).Neuropathologicevaluationfollowingcombination therapyAtoneweekfollowingadministrationofcarboplatin(20 g) byCEDandterminationofX-irradiation(5Gy4),the brainsofonly2of7ratsweremicroscopicallypositivefor tumor.However,itshouldbepointedoutthatsinceas fewas100tumorcellscanbefatal,itismorethanlikely thannotthatahighpercentageoftheseanimalswould havediedoftheirbraintumorsiftheyhadbeenfollowed foralongerperiodoftime.Thebrainsoftheotherrats showedfocalchangesconsistingofgliosis,scattered infiltratesoflymphocytesandmacrophages(Figure2B) comparedtothoseofuntreatedrats(Figure2A),which hadhighlyinfiltrativetumors.At2weeks,thebrainsof5 of6ratsweremicroscopicallypositivefortumor.However,therewasconsiderablevariabilityintheanatomic locationsofthesemicroscopicfocioftumorcells,includingsubcortical,leptomeningeal,andthewalloftheright lateralventriclesuggestingthatthewholecerebralhemispherewasinvolved.At3weeksfollowingtreatment,the brainsof5of7ratsweremicroscopicallypositivefor tumor,butonlyoneoftheseanimalshadmacroscopically visible(1 – 2mm)tumor,extendingfromthewallofthe lateralventricletothecorticalsurface.At4weeks,2of8 ratspresumptivelyhaddiedoftheirtumors,basedona Days after implantation 020406080180 Percent survival 0 20 40 60 80 100 Figure1 Kaplan-MeiersurvivalplotsofF98gliomabearingratsfollowingadministrationofcarboplatinbyCEDorAlzetpumpsin combinationwithX-irradiation. Survivaltimesindaysafterimplantationhavebeenplottedforuntreatedanimals( ),X-irradiationonly (5Gy4)( ),CEDofcarboplatin(20 g)+X-irradiation6hlater( ),CEDofcarboplatin(20 g)at7d+X-irradiation( ),CEDofcarboplatin (20 g)+X-irradiation24hlater( ),carboplatin(84 g)administeredbyAlzetpumps+X-irradiation( ),andCEDofcarboplatin(10 g2)on days13and15+X-irradiation(7.5Gy2)ondays14and16( ). Table3SurvivaltimeofratsbearingF98gliomasfollowingCEDofcarboplatin(10 g2)and15GyX-irradiation (7.5Gy2)Survivaltimes(days)c%Increasedlifespan TreatmentgroupaNMeanSEMedianRangeMeanMedian Untreated524.61.12521 – 2800 X-irradiationb430.01.93028 – 322220 CEDofcarboplatin546.87.54333 – 679072 CEDofcarboplatin+X-ray1282.115.554.535>180(3)234118aStereotacticintracerebralimplantationof103F98gliomacellswascarriedoutond.0.Carboplatin(10 gin10 L)wasadministeredbyconvectionenhanced deliveryataflowrateof0.33 L/min.for30minonday13thand15th.bAnimalsreceived15Gyof6MVLINACX-rays,deliveredintwo7.5Gyfractionsonday14thand16th.cSurvivalof>180d.indicatesthattheseanimalshavebeencuredoftheirtumors.Thenumberinparenthesisindicatesthenumberoflong-termsurvivor s.Yang etal.RadiationOncology 2014, 9 :25 Page5of9 http://www.ro-journal.com/content/9/1/25

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20%lossofbodyweight.Theremaining6ratsallshowed microscopicevidenceoftumor,andin3animalsthe tumorsweremacroscopicallyevident.Itisnoteworthy thatthemicroscopictumordepositsweretoosmalltobe detectedbymagneticresonanceimaging(MRI),wherethe limitofresolutionis>1mm,whichwouldhavebeen equivalentto106tumorcells.Therefore,neuropathologic examinationwasamorepowerfulapproachtodetecta muchsmallertumorcellburden.Incontrasttotherats thatreceivedbothcarboplatinandX-irradiation,the brainsofanimalsthatreceivedafractionated20Gydose ofX-irradiationaloneallhadmacroscopictumorsatthe timeofdeath(3,3,5,and7mmingreatestdimensionon days31,34,37,and39,respectively).DiscussionAspreviouslyreportedbyus[9],a20 gdoseofcarboplatinadministeredbyCEDover30min,oran84 g dosegivenover7dbyAlzetpumps,incombination withX-irradiation,wereshowntobenon-neurotoxic. Doublingthedoseofcarboplatinto40 gbyCEDor 168 gbyAlzetpumpifusionwasassociatedwithsignificantneurotoxicity[9].Therefore,thelowerdoses wereselectedforthestudiesdescribedinthepresent Table4ComparisonofradiationdosingparadignsEquivalentdoseaPhysicaldoseandfractionationregimenaTotaldose15Gy(5Gy3)b15Gy(7.5Gy2)20Gy(5Gy4) Acuterespondingtissues( / =10)18.75Gy21.88Gy25Gy Laterespondingtissues( / =2)26.25Gy35.63Gy35GyaUsingan / ratioof10foracute(tumor)effectsand2forlate(normalbrain)effects,thedosesindicatedwereatconventional2Gyfractions.bDoseregimenutilizedinourpreviouslypublisheddata[ 9 ]. A B C D Figure2 Neuropathologiccorrelates.(A) BrainofanuntreatedF98gliomabearingratthatdiedond.26followingtumorimplantation.The tumormeasured4mmindiameter,hadacentralzoneofnecrosis,andwashighlyinfiltrativeofnormalbrain. (B) BrainofanF98gliomabearing ratthatreceived20 gofcarboplatinbyCED,followed24hrslaterbythefirstoffour5Gyfractionsof6MVphotons,andwaseuthanizedone weeklater(25daftertumorimplantation).Notumorcellswereidentifiedinthisandtwoadditionalsections.Thereisafocalareaofgliosis.The superficialwhitemattershowsdebris,whichpossiblycouldhaverepresentedtumorcellsthathadbeenkilledasaresultoftreatment,scattered lymphocytesandmacrophages,andprominentwhitemattervacuolation.Otherwise,theremainderofthebrainwashistologicallyunremarkable. (C) BrainofanF98gliomabearingratthatreceivedfour5Gyfractions,administeredondays14,15,16and17followingtumorimplantationand diedond34.Thetumormeasured5mmwithaprominent,necroticcentralcoreandwasinfiltrativeofsurroundingwhitematter.Thereare scattereddilated(ectatic)vesselsandprominentareasofhemorrhage,bothofwhichmayberadiationrelated. (D) BrainofanF98glioma bearingratthatreceived20 gofcarboplatinond7afterimplantationfollowedbyfour5Gyfractionsof6MVphotonsanddiedond.44.The tumormeasured3mmwithacentralnecroticcore.Therearefocalcollagendepositswithhyalinizationandmultiplemicroscopicfociof hemorrhage,whichmostlikelyareradiationrelatedchanges.AllsectionswerestainedwithH&Eandphotographedatamagnificationof200x. Yang etal.RadiationOncology 2014, 9 :25 Page6of9 http://www.ro-journal.com/content/9/1/25

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study.Similarly,inapreviousstudyusingtheF98glioma model[16]weobservedthatanX-raydoseof22.5Gy, deliveredtothewholebrain,resultedina30%rateof radiationrelateddeaths.Therefore,wechoseamaximumdoseof20Gy,deliveredtothetumorbearing cerebralhemisphere,infourconsecutive5Gyfractions. Asshowninthepresentstudy,thiswaswelltolerated. Basedonthepreviousfindings[9],inthepresentstudy thedoseofcarboplatinwassetateither20 gbyCED or84 gbyAlzetpumpdeliveryincombinationwitha maximumradiationdoseof20Gytothetumorbearing cerebralhemisphere.However,inordertodetermineif thesurvivaldatacouldbefurtherimprovedoverthatpreviouslyreportedbyus[9],wealsoinitiatedX-irradiation ateither6or24hrsfollowingadministrationofcarboplatinordivideditintotwo10 gdosesondays1and3with a24hrintervalbetweendrugadministrationbyCEDand X-irradiationwithtwo7.5Gyfractions. Turningnexttothebiodistributiondata,thepurpose ofDMFtreatmentwastoreduceinterstitialpressure withinthetumor[12]andtherebyenhancetheuptake andpossiblythemicrodistributionofcarboplatin.Althoughthedifferencesinmeantumorcarboplatinvalues forDaloneandDMF-treatedratswerenotstatistically significant,therewasasuggestionthatthoseanimals thathadreceivedDMFhadhighercarboplatinvalues withanarrowerrangecomparedtothosethatdidnot. However,giventhebroadrange,itwouldhaverequired twogroupsof102animalseachtoattainalevelofstatisticalsignificance(power=0.8, =0.05)anditwould havebeenhighlyunlikelyinsmallcohortsofanimalsto demonstrateanysignificantdifferencesinMSTs.Therefore,suchstudieswerenotcarriedout.Thisisnot dissimilarfromwhatwasobservedintheEORTCtrial evaluatingthecombinationofRTandtemozolomide [17,18]where573patientswererequiredtodemonstrate asmalldifference(2.5months)inoverallmediansurvivaltime.Sincethereductionininterstitialpressureis transient[12],asingleDMFtreatmentwouldnothave beenusefulinincreasingcarboplatinconcentrationin animalsthathadreceivedthedrugbyAlzetpumpinfusions.Becauseofthetransiencyoftheeffect,weelected todeterminedrugconcentrationsatashorttimeintervalfollowingterminationofCED.However,itispossible thatthedifferencesindrugconcentrationsmighthave increasedoverlongertimeintervalsifthereweredifferencesinthedruguptakeandefflux. Asindicatedearlier,thepurposeofthepresentstudy wastodetermineifmodificationoftheRTregimenand thedosingparadigmofcarboplatinwouldresultin improvedsurvivaldatacomparedtothosepreviously reported[5-9].Theveryclear-cutanswertothisquestion isthatsustaineddeliveryofcarboplatin(84 gin168 l) byAlzetpumpsincombinationwitheitheratotaldoseof 20Gyor,aspreviouslyreported[9],15Gyinthree5Gy fractions,producedthegreatestincreasesinMSTs(107.7d vs. 111.8d,respectively),withnotreatment-relateddeaths. However,thismighthavebeenduetothehighertotaldose ofthedruganditsimprovedtumormicrodistribution.Increasingtheradiationdoseto20GyandadministeringcarboplatinbyCEDresultedinanidenticalMSTasthat previouslyreportedbyus(83.6d vs. 83.4d,respectively) [9].However,asshowninTable4,the20Gydosewould haveresultedin35Gytolaterespondingtissuessuchas vascularendotheliumcomparedto26.25Gywiththe15 Gydose.Itisnoteworthythatanadditionaldoseof6.25 Gyof6MVphotonstothetumor,atconventional2Gy fractions(Table4),didnothaveanyeffectontheMST, suggestingthatmajordeterminantofefficacywasdrug ratherthanradiation-related.Aspreviouslyreported[16], ratsthatreceivedanX-raydoseof22.5Gyinthree7.5Gy fractionshadaMSTof53.24.6dwithsignificantradiation-relatedmorbidity.Modifyingthecarboplatindosing paradigmtotwo10 gdosesandtheRTregimentotwo 7.5GyfractionsresultedinanalmostidenticalMST (82.1d vs. 83.4d)asthatpreviouslyreported[9]. Twopossiblewaysthatthiscombinationtherapymightbe improvedwouldbetoincreasetumoruptakeandthe microdistributionofcarboplatinortoadministeraneutralizingagenttodecreasesystemictoxicityassociated withahigherdoseofchemotherapy,aspreviouslyreportedwith “ RADPLAT ” inthetreatmentofadvanced squamouscarcinomasoftheheadandneck[19].AlthoughDMFtreatmentmarginallyincreasedthetumor drugconcentration,itisunlikelythatthiswouldhave resultedinasignificantincreaseinMSTcomparedto animalsthatdidnotreceiveDMFtreatment. Themajorproblem,whichhaslimitedthestrong synergyofRTincombinationwithi.c.administrationof carboplatin[9],isthetremendousvariabilityfollowing CEDofthedrug(Table1).Wehypothesizethatthose animalsthatwerecuredoftheirtumorshadhigher tumorconcentrationsandbettermicrodistributionsof carboplatin.Althoughtheremayhavebeensomedifferencesinthesizesofthetumorsatthetimethatthedrug wasadministered,thisdoesnotseemtobeanadequate explanationforthebroadrangesintumorcarboplatin concentrationsthatwereobserved.Ifthisisthecasefor aratbraintumormodelwhereeachtumorhasavolume of~25 – 30mm3inacerebralhemispherethatweighs600 mg[9],theproblemisordersofmagnitudegreaterinthe caseofapatientwithahighlyinfiltrativegliomainacerebralhemispherethatweighs~600g.AlthoughCEDis superiortodirectintratumoralinjection[20-24],itisstill aworkinprogress[20],andmorelikelythannotitcanbe significantlyimproved.Ashasbeenreportedpreviouslyby us[9],a20 gdoseofcarboplatinadministeredi.c.by CEDresultedinatumordrugconcentrationof10.4 g/g,Yang etal.RadiationOncology 2014, 9 :25 Page7of9 http://www.ro-journal.com/content/9/1/25

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whichwasequaltothatattainedbyadoseof20mg (20,000 g)administeredintravenously.Themajorquestionthatmustbeaddressed,therefore,is, “ Howcan administrationofatherapeuticagenttoabraintumorbe improvedtoachieveahigherandmorehomogeneous distributionofvarioustherapeuticagents? ” Basicallythis isaproblemthatwillrequirediverseexpertisetosolve butitisnotunreasonabletoexpectthatsuchimprovementswillbemadeandthatCEDultimatelywillproveto behighlyeffectiveclinically[25,26].ConclusionsOurresultsprovidestrong proof-of-principle thati.c.CED ofcarboplatinaloneorincombinationwithX-irradiation canresultinsignificantprolongationsinMSTsandcures ofabraintumorthathasbeenincurablebyallother therapeuticstrategiesexceptforone[15].Asafirststepin clinicallytranslatingthesefindingsaPhaseItrialcurrently isinprogressatTheOhioStateUniversitytoevaluatei.c. CEDofcarboplatin(72mlover3days)inpatientswith recurrenthighgradegliomas.Abbreviations RT: Radiationtherapy;DMF:Dexamethasone,mannitol,andfurosemide; CED:Convectionenhanceddelivery;LINAC:Linearaccelerator;MST:Mean survivaltime;EORTC:EuropeanOrganizationforResearchandTreatmentof Cancer;BBB:Blood-brainbarrier;i.c.:Intracerebral;i.v.:Intravenenous; i.p.:Intraperitoneal;ICP-OES:Inductivelycoupledplasma – Opticalemission spectroscopy;SE:Standarderror;MST:Meansurvivaltime;MeST:Median survivaltime;SD:Standarddeviation;ANOVA:Analysisofvariance; MRI:Magneticresonanceimaging. Competinginterests Theauthorsdeclarethattherearenocompetinginterests. Authors ’ contributions WY:Carriedoutalloftheanimalstudiesandcontributedtothedesignof experiments,andtheevaluationofdata.RFB:Hadoverallresponsibilityfor theproject,evaluateddataandwrotealmosttheentiremanuscript. TH:Assistedincarryingoutanimalstudies,performedcarboplatin determinations,analyzedthedataandwrotethesectiononstatistical analysis;RJN:Assistedincarryingouttheanimalstudies.MW:Carriedoutthe animalradiationswiththeassistanceofNG,WY,THandRJN.LAandRFB reviewedbrainslidesandwrotethesectiononneuropathologicevaluation. JCGcontributedtothedesignofexperimentsandwritingthemanuscript. Allauthorsreadandapprovedthefinalmanuscript. Acknowledgments WethankMr.JimSommerfeldfordesigningandfabricatingaratradiation chamber,Dr.HamdyAwadforassistanceinhelpingussetupa procedureforcontinuousinhalationalanesthesia,Mr.AndyPultzfor providinguswithcarboplatin,Ms.SaraLimforhelpingtopreparesurvival plots,Mr.ShawnScullyandDr.AdrianSuarezforassistancewith photomicroscopyandMrs.HeidiBosworthforexpertsecretarialassistance. SupportforthisprojectwasprovidedbytheMusellaFoundation,Voices AgainstBrainCancer,TheOhioStateUniversityDepartmentofPathology, andtheKevinJ.MullinMemorialFundforBrainTumorResearch. Authordetails1DepartmentofPathology,TheOhioStateUniversity,Columbus,OH43210, USA.2DepartmentofRadiationOncology,TheOhioStateUniversity, Columbus,OH43210,USA.3DepartmentofPathology,MaterDeiHospital, UniversityofMaltaMedicalSchool,Msida,Malta.4Currentaddress: DepartmentofHealthOutcomesandPolicy,CollegeofMedicine,University ofFlorida,Gainesville,FL,USA. Received:16September2013Accepted:31December2013 Published:14January2014 References1.KellandL: Theresurgenceofplatinum-basedcancerchemotherapy. NatRevCancer 2007, 7: 573 – 584. 2.StewartDJ,MolepoJM,EapenL,MontpetitVA,GoelR,WongPT,PopovicP, TaylorKD,RaaphorstGP: Cisplatinandradiationinthetreatmentof tumorsofthecentralnervoussystem:pharmacologicalconsiderations andresultsofearlystudies. IntJRadiatOncolBiolPhys 1994, 28: 531 – 542. 3.Anonymous: Cisplatindoesnotenhancetheeffectofradiationtherapy inmalignantgliomas.EORTCBrainTumorGroup. 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NEnglJMed 2005, 352: 987 – 996. 18.StuppR,HegiME,MasonWP,vandenBentMJ,TaphoornMJ,JanzerRC, LudwinSK,AllgeierA,FisherB,BelangerK, etal : Effectsofradiotherapy withconcomitantandadjuvanttemozolomideversusradiotherapy aloneonsurvivalinglioblastomainarandomisedphaseIIIstudy:5-year analysisoftheEORTC-NCICtrial. LancetOncol 2009, 10: 459 – 466.Yang etal.RadiationOncology 2014, 9 :25 Page8of9 http://www.ro-journal.com/content/9/1/25

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19.RabbaniA,HinermanRW,SchmalfussIM,AmdurRJ,MorrisCG,PetersKR, RobbinsKT,MendenhallWM: Radiotherapyandconcomitantintraarterial cisplatin(RADPLAT)foradvancedsquamouscellcarcinomasofthehead andneck. AmJClinOncol 2007, 30: 283 – 286. 20.KunwarS,ChangS,WestphalM,VogelbaumM,SampsonJ,BarnettG, ShaffreyM,RamZ,PiepmeierJ,PradosM, etal : PhaseIIIrandomizedtrial ofCEDofIL13-PE38QQRvsGliadelwafersforrecurrentglioblastoma. NeuroOncol 2010, 12: 871 – 881. 21.FergusonS,LesniakMS: Convectionenhanceddrugdeliveryofnovel therapeuticagentstomalignantbraintumors. CurrDrugDeliv 2007, 4: 169 – 180. 22.LopezKA,WaziriAE,CanollPD,BruceJN: Convection-enhanceddeliveryin thetreatmentofmalignantglioma. NeurolRes 2006, 28: 542 – 548. 23.RaizerJ: Issuesindevelopingdrugsforprimarybraintumors:barriers andtoxicities. ToxicolPathol 2011, 39: 152 – 157. 24.MehtaAI,ChoiBD,AjayD,RaghavanR,BradyM,FriedmanAH,PastanI, BignerDD,SampsonJH: Convectionenhanceddeliveryof macromoleculesforbraintumors. CurrDrugDiscovTechnol 2012, 9: 305 – 310. 25.SampsonJH,RaghavanR,BradyM,FriedmanAH,BignerD: Convectionenhanceddelivery. JNeurosurg 2011, 115: 463 – 464.discussion465-466. 26.AsthagiriA,WalbridgeS,HeissJ,LonserR: Responsetoeditorial, convection-enhanceddelivery. JNeurosurg 2011, 115: 467 – 473.doi:10.1186/1748-717X-9-25 Citethisarticleas: Yang etal. : Radiationtherapycombinedwith intracerebraladministrationofcarboplatinforthetreatmentofbrain tumors. RadiationOncology 2014 9 :25. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Yang etal.RadiationOncology 2014, 9 :25 Page9of9 http://www.ro-journal.com/content/9/1/25