Fludarabine plus alemtuzumab (FA) front-line treatment in young patients with chronic lymphocytic leukemia (CLL) and an adverse biologic profile

ContentslistsavailableatScienceDirect

Leukemia

Research

j o u r n al hom ep a ge : w w w . e l s e v i e r . c o m / l o c a t e / l e u k r e s

Fludarabine

plus

alemtuzumab

(FA)

front-line

treatment

in

young

patients

with

chronic

lymphocytic

leukemia

(CLL)

and

an

adverse

biologic

profile

Francesca

R.

Mauro

_,

_Stefano

_Molica

_,

_Luca

_Laurenti

_,

_Agostino

_Cortelezzi

_,

Angelo

M.

Carella

_,

_Francesco

_Zaja

_,

_Annalisa

_Chiarenza

_,

_Francesco

_Angrilli

_,

Francesco

Nobile

_,

_Roberto

_Marasca

_,

_Caterina

_Musolino

_,

_Maura

_Brugiatelli

_,

Alfonso

Piciocchi

_,

_Marco

_Vignetti

_,

_Paola

_Fazi

_,

_Giuseppe

_Gentile

_,

Maria

S.

De

Propris

_,

_Irene

_Della

_Starza

_,

_Marilisa

_Marinelli

_,

_Sabina

_Chiaretti

_,

Ilaria

Del

Giudice

_,

_Mauro

_Nanni

_,

_Francesco

_Albano

_,

_Antonio

_Cuneo

_,

Anna

Guarini

_,

_Robin

_Foà

_,

_on

_behalf

_of

_the

_GIMEMA

_(Gruppo

_Italiano

_Malattie

EMatologiche

dell’Adulto)

Working

Party

for

chronic

lymphoproliferative

disorders

a_{Hematology,DepartmentofCellularBiotechnologiesandHematology,“Sapienza”University,Rome,Italy} b_{OncologiaMedica,AziendaOspedalieraPuglieseCiaccio,Catanzaro,Italy}

c_{DepartmentofHematology,Universita’CattolicadelSacroCuore,Rome,Italy}

d_{Ematology-BMTUnit,IRCCSCa’GrandaOspedaleMaggiorePoliclinicoFoundation,Milan,Italy} e_{U.O.C.Ematologia1,IRCCSSanMartino-IST,Genova,Italy}

f_{Hematology-BMTUnit,S.MariaMisericordiaHospital,UniversityofUdine,Italy} g_{DepartmentofHematology,HospitalFerrarotto,UniversityofCatania,Italy} h_{DepartmentofHematology,LocalHealthUnitofPescara,Italy}

i_{AziendaOspedaliera“Bianchi-Melacrino-Morelli”,ReggioCalabria,Italy}

j_{SectionofHematology,DepartmentofOncology,HematologyandRespiratoryDiseases,UniversityofModenaandReggioEmilia,Italy} k_{DivisionofHematology,MedicinalChemistrySection,UniversityofMessina,Italy}

l_{DepartmentofHematology,AziendaOspedalieraPapardo,Messina,Italy} m_{ItalianGroupforAdultHematologicDiseases(GIMEMA),Rome,Italy} n_{U.O.EmatologiaconTrapianto,UniversitàdegliStudiAldoMoro,Bari,Italy} o_{DepartmentofHematology,ArcispedaleSant’Anna,Ferrara,Italy}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received25August2013

Receivedinrevisedform9November2013 Accepted11November2013

Available online 19 November 2013 Keywords:

Chroniclymphocyticleukemia CLL Young Biology Fludarabine Alemtuzumab

a

b

s

t

r

a

c

t

In45,≤60yearsoldpatientswithCLLandanadversebiologicprofile,afront-linetreatmentwith Flu-darabineandCampath(Alemtuzumab®_{)wasgiven.Theoverallresponseratewas75.5%,thecomplete}

responserate(CR)24.4%withthelowestCRrates,16.7%and8.3%,in11qand17pdeletedcases.The 3-yearprogression-freesurvival(PFS)andoverallsurvivalwere42.5%and79.9%,respectively.PFSwas significantlyinfluencedbyCLLduration,beta2-microglobulin,andimprovedbypost-remissionalstem celltransplantation.Front-linefludarabineandalemtuzumabshowedamanageablesafetyprofileand evidenceofabenefitinasmallseriesofCLLpatientswithadversebiologicfeatures.

© 2013 Elsevier Ltd. All rights reserved.

∗ Correspondingauthorat:Hematology,DepartmentofCellularBiotechnologies andHematology,“Sapienza”University,ViaBenevento6,00161Rome,Italy. Tel.:+3906499741;fax:+390644241984.

E-mailaddress:mauro@bce.uniroma1.it(F.R.Mauro).

1. Introduction

Chroniclymphocyticleukemia(CLL)isanincurablediseasewith amedianageatthetimeofdiagnosisbetween65and70yearsand withabout22–30%ofpatientsyoungerthan60years[1,3].The vari-ableclinicalcourseofthediseaseisstronglyinfluencedbydifferent

0145-2126/$–seefrontmatter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2013.11.009

biologicmarkersthatdonotappeartodiffersubstantially accord-ingtoageatdiagnosis[4–8].However,Dohneretal.observedthat thepresenceofdeletion11qwasaprofoundstratifierofsurvival inpatientsaged<55years,butnotinpatientsaged>55years[9], whileinallagepatients17pdeletionand/orTP53mutationsare wellknownpoorprognosticmarkers[10–12].Ithasbeenestimated that60%ofpatients≤55yearsrequiretreatmentandthatthelife expectancyofyoungerCLLpatientsissignificantlyshorterthanthat oftheage-matchedgeneralpopulation[2,3].Asignificantclinical improvementintheoutcomeofCLLpatientshasbeenobtainedby thecombinationoftheanti-CD20monoclonalantibodyrituximab withfludarabine-basedregimens,inparticular,thefludarabineand cyclophosphamideregimen(FCR)[13,14].

Campath(Alemtuzumab®_{)a chimeric anti-CD52monoclonal}

antibodyiseffectivein themanagement ofCLLand hasshown activityregardlessofthegeneticriskgroups[15–18].Resultsfrom asingle-groupphase2studybyElteretal.suggestedthatthe flu-darabineandalemtuzumab(FA)combinationcouldimprovethe outcomeofpatientswithrelapsedorrefractoryCLL[19].Thesame authorsconfirmedtheefficacyoftheFAcombinationinastudy wherethisregimenwascomparedwithfludarabinemonotherapy inpatientswithrelapsedorrefractoryCLL[20].

Inordertoprolongtheresponseduration,apost-remissional treatmentwithalemtuzumab[21,22]and,inyoungerand phys-ically fit patients with poor prognosis, the role of a stem cell transplant(SCT)[23–26]havealsobeenexplored.

The GIMEMA (Gruppo Italiano Malattie EMatologiche dell’Adulto) promoted a prospective multicenter phase II trial toevaluateinCLLpatientsyoungerthan60yearstheefficacyand safetyofatreatmentapproachwiththeFAregimen.

2. Designandmethods

2.1. Studydesign

Aprospective,multicenter,phaseIItrialwasdesignedforyoung CLL,withadversebiologiccharacteristicsrequiringfront-line ther-apy.Theprimaryobjectivesofthestudyweretodefinetheoverall response (OR)and complete response (CR) rates afterFA regi-men.The secondary objectiveswere to assess thetoxicity, the progression-freesurvival(PFS),theoverallsurvival(OS),the rela-tionshipbetweenthebaselineclinicalandbiologicfeaturesandthe outcomeofthepatients.

2.2. Inclusionandexclusioncriteria

Inclusion criteria were age ≤60 years, no prior treatment, advancedBinetstage(C)orlessadvancedstage(BorA)[27]with clinicalsignsofactivediseaseaccordingtotheNCI-WGcriteria[28]

anda“highrisk”(HR)biologicprofile.Forthepurposeofthisstudy, aHRbiologicprofilewasdefinedbythepresenceof:(1),deletion 17p(_≥20%;HRasubset);or(2)deletion11qwith_≥1additional unfavorable factor (germline IGHV status, ZAP-70and/or CD38 expression;HRbsubset);or(3)germlineIGHVormutated VH3-21and≥2unfavorablefactors(ZAP-70and/orCD38expression, trisomy12;deletion6q;HRcsubset).

Institutional ethic committees approved the study. Patients signedawritteninformedconsent.TheEudraCTnumber registra-tionofthestudyis2005-002476-15.

2.3. Studytreatment

Patientsreceived4 monthlycoursesof theFAregimen (flu-darabine,30mg/m2iv;alemtuzumab,30mgiv,days1–3)[19,20]. Respondingpatientswithnoevidenceofresidualdiseaseatthe molecularlevel(mol-CR)underwentaperipheralbloodstemcell

(PBSC)mobilizationonly,whilepatientswithresidualdisease (par-tial response, PR; complete response, CR; cytometric complete response,cy-CR)underwentpost-remissionaltherapy.Inorderof priority,3 post-remissionaltreatmentoptionswereconsidered: areducedintensityallogeneicPBSCTor,intheabsenceofa sib-lingdonor,anautologousPBSCTor,intheabsenceofasufficient harvest,treatmentwithalemtuzumabsc,30mgweeklyfora max-imumof12weeks(SupplementaryTable1).

Supplementarymaterial relatedto this articlecanbe found, in the online version, at http://dx.doi.org/10.1016/j.leukres. 2013.11.009.

2.4. Supportivetreatment

Allpatientsreceivedbactrimandhighdosevalacyclovir pro-phylaxis (2g/8h) with weekly CMV antigenemia monitoring. Treatmentwasstoppedinthepresenceoflife-threateningadverse events,persistent(≥4weeks)grade≥2cytopenia,infectionorother toxicity.

2.5. Efficacyassessments

Responsewasdefinedbetweenthe8◦andthe12◦weekfromthe lastfludarabineandalemtuzumabadministration.Responsewas assessedaccordingtothe1996NCI-WGguidelines[28]andCRwas confirmedinallcasesbybonemarrowbiopsy.

Inaddition,inallCRpatients,computedtomography(CT)scans wereused.InpatientswithimagingconfirmedCR,acentralized evaluationoftheminimalresidualdisease(MRD)wasperformed bothonPBandbonemarrow(BM)byfourcolorflowcytometry.In patientswithnoevidenceofcytometricresidualdisease,molecular responsewasalsoassessedbypolymerasechainreaction(PCR).In asubsetofpatientsageneexpressionprofileanalysis(GEP)was performedaspreviouslydescribed[29]

StatisticalanalysisDifferencesinthedistributionsofprognostic factorswereanalyzedbythe␹2 _{orFisher’sexacttestandbythe}

Kruskal–Wallistest.Theprobabilityofsurvivalwasestimatedusing theKaplan–Meiermethod.Thelog-ranktestwasusedtocompare theeffectoftreatmentandriskfactorcategories,whileconfidence intervalswereestimated(95%CIs)usingtheSimonandLeemethod. LogisticregressionandCoxproportionalhazardregression mod-elswereperformed toexaminerisk factorsaffecting treatment responseandsurvivaloutcomes.Toascertainthepureimpactof theFAregimenandtheprognosticeffectofbaselineclinicaland biologicvariablespatientswhounderwentSCTwerecensoredat thetimeoftransplant.

3. Results

3.1. Studypopulation

BetweenDecember2005andFebruary2009,thebiologicprofile of86consecutiveyoungpatientswithCLLandaprogressivedisease wasassessed.Forty-fivepatients(52%)showedaHRbiologic pro-fileandweretreatedwiththeFAregimen.Theclinicalandbiologic characteristicsofthepatientsarereportedinTable1.The distribu-tionofthethreesubsetsofHRpatientswas:26.7%forthegroupof patientswithdeletion17p(HRasubset),40%forthegroup includ-ingpatientswithdeletion11qand1ormoreadditionaladverse biologicfactors(HRbsubset),and33.3%forthegrouprepresented bycaseswithunmutatedIGHVand2ormoreadditionaladverse biologicfactors(HRcsubset).

Table1

Baselineclinicalandbiologiccharacteristicsofpatients.

Patients(45)

n %

Gendermale/female 34/11 75.6/24.4

Medianage,years(range) 55.2(29.8–60.8)

ECOGperformancestatus0/1 42/3 95.3/6.7

Mediantimefromdiagnosis,months 9.0(0.6–639.8)

Medianlymphocytecount,×109_{/L 62.0(5.0–638)}

Binet’sstageA/B/C 6/30/9 13.3/66.7/20

Lymphnodesofatleastof5cmof diameter 16 35.6 Increased␤2M 22 48.9 IncreasedLDH 17 37.8 CD19/CD38≥30% 22 48.8 ZAP-70≥20% 27 60 IGVHunmutated/mutated 42/3 93.3/6.7

FISHabnormalities(hierarchcalmodel)

Noabnormalities 3 6.7 13q− 5 11.1 12+ 6 13.3 6q− 1 2.2 11q− 18 40.0 17p− 12 26.7

Abbreviations: ECOG, Eastern Cooperative Oncology Group; ␤2M, beta2-microglobulin; ZAP-70, zeta-chain-associated protein kinase 70; IGHV, immunoglobulinheavy-chain;FISH,fluorescenceinsituhybridization.

3.2. ResponsetoFAregimen

Themajorityofpatients(89%)completedtheplanned4courses

ofFAtreatment.

Two patients withdrew from treatment because of severe

reactionduringthefirstalemtuzumabinfusion.Onan

intention-to-treatbasis,34HRpatients(75.5%)respondedtoFAregimenwith

11(24.4%)CRsand23(51.1%)PRs(Table2).Outofthe11patients

whoachievedaCR,8(18%)showednoresidualdiseaseatthe cyto-metriclevel(cy-CR)and5bothatthecytometricandmolecular level(mol-CR).Atreatmentfailurewasrecordedin9cases(stable disease,5;progressivedisease,4).

Theeffectoftheclinicalandbiologicvariablesonresponseis detailedinTable2.Patientswithdeletion17por11qshowedthe lowestCRrates(HRavsHRbvsHRcsubset,8.3%vs16.7%vs46.7%; p=.07).TheproportionofpatientswhoobtainedaCRwas signifi-cantlylowerinthepresenceofashortintervalfromCLLdiagnosis, ≤12 months(p=.006),increased␤2M levels(p=.02). At multi-variateanalysis,theintervalbetweenCLLdiagnosisandtreatment (p=.02),increased␤2M(p<.027)anddeletion17p(p<.043) main-tainedasignificantandindependenteffectontheachievementof CR.

GEPanalysisrevealedadistinctivesignatureassociated with responsetotreatmentwith357differentiallyexpressedprobesets, themajoritybeingdownregulatedinpatientswhodidnotobtain aCR(Fig.1A).

Furthermore,DAVIDfunctionalannotationanalysisevidenced inpatientswhofailedtoreachaCRanoverrepresentation,ofgenes involvedintranslation,proteasomalcatabolicprocesses,RNA splic-ing,Rassignaling,cellularresponsetostress,DNArepairandBcell activation(Fig.1B).

Inaddition,inthecaseswhodidnotachieveaCRasignificant downmodulation(p=.014)ofthetranscriptencodingfortheCD52 antigenwasobserved.

Outofthe29patientswhoachievedaresponsewithFAand showedtheevidenceofresidualdisease,14underwentSCTandall arealive(mediantimefromSCT,40months).However,whilethe6 patientswhohadanallogeneicSCTareinpersistentCR(2in mol-CR),4outofthe8whohadanautologousSCThaverelapsedafter amediantimeof29months.

Table2

ResponsetoFluCambyclinicalandbiologiccharacteristics.

OR n(%) p-value CR n(%) p-value Allpatients 34(75.5) – 11(24.4) – Age >55 18/23(78.3) 0.7 4/23(17.4) 0.3 ≤55 16/22(72.7) 7/22(31.8) Gender Male 26/34(76.5) 1.0 9/34(75.6) 0.7 Female 8/11(72.7) 2/11(18.2) PBlymphocytes,×109_/L <60 17/22(77.3) 0.8 6/22(27.3) 0.7 ≥60 17/23(73.9) 5/23(21.7) Binet’sstage A 4/6(66.7) 0.5 2/6(33.3) 0.7 B 24/30(80.0) 8/30(26.7) C 6/9(66.7) 1/9(11.1) LDH Normal 22/28(78.6) 0.7 7/28(25.0) 1.0 Increased 12/17(37.8) 4/17(23.5) ˇ2M Normal 18/23(78.3) 0.7 9/23(39.1) 0.02 Increased 16/22(72.7) 2/22(9.1)

MonthsfromCLLdiagnosis

≤12 16/25(64.0)

0.08 2/25(8.0) 0.006

>12 18/20(90.0) 9/20(45.0)

Lymphnodesdiameter

<5cm 24/29(82.8) 0.2 10/29(34.5) 0.07 ≥5cm 10/16(62.5) 1/16(6.3) CD19/CD38 <30% 18/23(78.2) 1.0 6/23(26.0) 1.0 ≥30% 16/22(72.7) 5/22(22.7) ZAP-70 <20% 14/16(87.5) 0.2 4/16(25.0) 1.0 ≥20% 18/27(66.7) 6/27(22.2) IGHV Mutated 2/3(66.7) 1.0 1/3(33.3) 1.0 Unmutated 32/42(76.2) 10/42(23.8)

Highriskssubgroups

HRa 7/12(58.3) 0.3 1/12(8.3) 0.07 HRb 15/18(83.3) 3/18(16.7) HRc 12/15(80.0) 7/15(46.7)

Patientssubgroups:HRaincludes,patientswithdeletion17p(≥20%);HRb,patients withdeletion11qand1ormoreadditionaladversebiologicfactors;HRc,IGHV unmutatedpatientswith2ormoreadditionaladversebiologicfactors.

3.3. Survival

The3-yearPFSandOSare42.5%(95%CI:27.4–65.9)and79.9%

(95%CI: 66.6–95.9), respectively. Baseline clinical and biologic

parametersassociatedwithasignificantlyhigherPFSafterFluCam

werealongerintervalfromCLLdiagnosis(p=.0007),theabsence

ofbulkynodes(p=.02),orincreased␤2Mlevels(p=.05).Patients

withdeletion17por11qshowedashorter,thoughnotsignificantly

inferiorPFS(PFSat3years,17p-,51%vs11q-,28%,vsnodeletion

17por11q,60%;p=.85;Fig.2).Atmultivariateanalysis,the sig-nificantandindependentbaselinefactorsinfluencingPFSwerethe intervalfromCLLdiagnosis(p=.012;HR:3.2;95%CI:1.2–8.4)and thepresenceofbulkynodes(p=.016;HR:0.31;95%CI:0.12–0.77). AsignificantlylongerPFSwasobservedinpatientswhoachieveda CR(PFSat3years,CRvsPR,80.0%vs12.7%;p=.03)andinpatients whoafterresponsetoFAunderwentanallogeneicorautologous SCTascompared topatientswho didnotreceivefurther treat-ment(p<.02;HR:0.30;95%CI:0.11–0.84).Baselinevariableswith asignificanteffectonsurvivalprobabilityweretheageofpatients (p=.04),theintervalfromCLLdiagnosis(p=.01),andtheLDHvalue. Patientswithdeletion17pshowedthelowestOSprobability(OSat 3years,deletion17pvsdeletion11qvstrisomy12ordeletion13q, ornoabnormalities,69%vs85%vs83%).However,thedifference didnotreachstatisticalsignificance(p=.22),presumablybecause ofthelimitedsamplesize(Fig.2).

Fig.1. (A)Geneexpressionprofile.Relativelevelsofgeneexpression:redrepresentsthehighestlevelofexpressionandbluethelowestlevelofgeneexpression.(B)Functional annotationanalysisofdifferentiallyexpressedgenesfromthe2subsetsofpatients.

3.4. Safety

A grade III–IV granulocytopenia was recorded after 18% of coursesandin38%ofpatients.However,asevereinfectionwas recordedin a lowerproportion ofpatients, 13%.Threepatients (6.7%)experiencedaCMV reactivationthatrespondedto ganci-cloviriv,whilenocaseofsymptomaticCMVinfectionwasobserved. Alemtuzumab-relatedgrade3–4adverseinfusionreactionswere recordedin2patients(4.4%)whodiscontinuedtreatment. Dur-ingthefollow-up,a secondmalignancywasrecordedin2cases (prostatecarcinoma,Kaposisarcoma)and1patientdevelopeda Richter’ssyndrome.Sevenpatientshavedied(15.5%).Thecauseof deathwasaninfectionin1patientanddiseaseprogressionin6 (CLL,5cases,Richter’ssyndrome,1).

4. Discussion

Weknowtodayalargenumberofgeneticabnormalities asso-ciatedwith thepathogenesis and prognosisof CLL[30–33]. At the time this study was designed, in the pre-FCR era, there wasevidencethat bothdeletions17p and11qwere associated withapooroutcome aftertreatment, and a treatmentstrategy

including fludarabine combined with the monoclonalantibody alemtuzumabwasjudgedasanappropriate firstlinetreatment approachforyoungCLLpatientswithanoverallpoorprognostic likelihood.

Abouthalfof theyounger patientsweanalyzed at thetime offirst-linetreatmentshowedanadversebiologicprofile.Onan intention-to-treatbasis,aresponsewasobtainedafterFAby75.5% ofthesepatientswithaCRrateof24.4%,including18%of cytomet-ricCRsandasizablenumberofmolecularCRs.Ashorterinterval fromdiagnosisandbeta2-microglobulin,weresignificantly asso-ciatedwithanadverseoutcome.Thegeneticprofileidentifieda differentpatternofresponsetoFAregimen.Patientswith unmu-tatedIGHVandnodeletion11qor17pshowedthebestresponse intermsofOR,80%,andCR,47%,rateswhilepatientswithdeletion 11qshowedaverylowCRrate,17%,andpatientsharboringa dele-tion17ptheworseresponseintermsofboth,OR,58%andCR,8%, rates.

Interestingly,GEPanalysisrevealeda significant downmodu-lationoftranscriptsinvolvedintheDNArepairand/orapoptosis regulation and a significant downmodulation of the transcript encodingfortheCD52antigeninpatientswhoobtainedonlyaPR ornoresponse.

Fig.2.HRasubset,patientswithdeletion17p(≥20%).HRbsubset,deletion11q and≥1additionaladversebiologicfactor;HRcsubset,IGHVunmutatedand≥2 additionaladversebiologicfactors.(A)PFSat3years:HRa,51%;HRb,28%;HRc, 60%;p=.85.(B)OSat3years:HRa,69%;HRb,85%;HRc,83%;p=.22.

IfweconsidertheresultsoftheCLL8study[14],withtheFCR regimen,beingtodaythegoldstandardfront-linetherapyforfit patientswithCLL,patientswithunmutatedIGHV and no dele-tion11qor17pshowedasimilarCRrateafterFA.Unfortunately, patientswithdeletion 17pdidnot showabetterresponserate withFA,andpatientswithdeletion11qrevealedavery unsatisfac-toryCRrateandtheyappeartorespondbettertotheFCRregimen. Thisfindingfurthersuggeststhattreatmentcombinations includ-ingrituximabandalkylatingagentssuchascyclophosphamideor bendamustinearemoreeffectiveinpatientswiththe11qdeletion

[34,35].

Eventhoughtheanalysisofasmallcohortof45patientsshould beconsiderasexploratoryandnoconclusionscanbedrawn,itis worthnotingthattheintroductionofapost-remissionalallogeneic orautologousSCTshowedabenefitinprolongingresponseafter treatment.Thisobservationisinlinewiththatoftwostudiesthat reportedabenefitintheoutcomeofCLLpatientswhentheSCTwas performedinanearlyphaseofthedisease[25,26].

Thesafety profileofFAwasacceptable.Infectionswereless frequentthanexpectedconsideringtheimmunodepressiveeffect of both agents. The low cumulative doseof alemtuzumab, the extendedprophylaxisandtheabsenceofpriortreatmentsplayeda favorableeffectinrestraininginfections.Interestingly,onhighdose valacyclovir prophylaxis, no CMV symptomatic infections were observedandarelativelylowproportionofpatientsdevelopeda CMVreactivation.

In conclusion,inthis studyabouthalfofyoungCLLpatients requiringtherapyshowedadversebiologicfeatures.Inthis sub-setofpatientsFAregimengivenasfront-linetreatmentrevealed a manageablesafety profileandprovided evidenceof apatient benefit.FurtherconfirmatoryphaseIIIstudiesinlargerseriesof patientscomparingFAwiththegoldstandardregimenforCLLand anextendedfollow-uptoevaluatesurvivalendpointsand long-termtoxicityarerequired.

Acknowledgements

We thank the patients who agreed to participate in this study. We also thank Antonella Graziosiand LauraCollada Ali for the support in the study organization and data acquisi-tion, and all the study investigators who participated in this study:GiovannaMeloniandAnnaPaolaIori,Hematology, Depart-ment of Cellular Biotechnologies and Hematology, “Sapienza” University,Rome,Italy;FrancescoLauriaandFrancesco Forconi, Hematology, Department of Clinical Medicine and Immunolog-ical Sciences, University of Sienaand Department of Oncology, Azienda Ospedaliera Universitaria Senese, Siena, Italy; Davide Soligo, Hematology-BMT Unit, IRCCS Ca’Granda Ospedale Mag-giorePoliclinicoFoundation,Milan,Italy;AlbertoBosiandStefania Ciolli, Divisione di Ematologia, Università di Firenze, Firenze, Paolo Corradini, Division of Hematology, Istituto Nazionale dei Tumori, Milano, Italy; Rosanna Mirabelli, Oncologia Medica, AziendaOspedalieraPuglieseCiaccio, Catanzaro,Italy;Vincenzo Liso,GiorginaSpecchia,RitaRizzi,U.O.EmatologiaconTrapianto Università degli Studi Aldo Moro, Bari, Italy; Alessandro Levis, DivisionofHematology,Sant’AntonioeBiagioHospital, Alessan-dria, Italy; Massimo Massaia and Marta Coscia, Dipartimento di Medicina e Oncologia Sperimentale, Sezione di Ematologia, UniversityofTorino,Italy;FortunatoMorabitoandMassimo Gen-tile,HematologySection,CosenzaHospital,Cosenza,Italy;Sergio Amadori and Francesco Buccisano, Hematology, University Tor Vergata,Rome,Italy;GiovanniDelPoeta,Hematology,Ospedale S. Eugenio, Rome, Italy; Sergio Storti, Istituto di Ematologia-Campobasso,Italy;NicolaDiRenzo,DivisionediEmatologia,Lecce, Italy.

Fundingsources:ThisworkwassupportedbyGenzymewho pro-videdalemtuzumabandaresearchgrant,byAssociazioneItaliana perlaRicercasulCancro(AIRC),SpecialProgramMolecular Clin-icalOncology,5×1000,No.10007,Milan,Italy;and byRomAIL (AssociazioneItalianacontroleLeucemie,Romesection).

Author’s contributions: R.F. designed the research, wrote the manuscript and gave final approval of the manuscript. F.R.M. designed the research, provided clinical care to patients, col-lecteddata,analyzedandinterpreteddata,wrotethemanuscript and gave final approval of the manuscript. A.G. designed the research,performedresearch,collecteddata,analyzedand inter-preteddata,andgavefinalapprovalofthemanuscript.S.M.,A.C., L.L., A.M.C., F.Z.,A.C., F.A., F.N., R.M., C.M., M.V.,P.F., G.G., M.B., M.S.D., I.D.S.,M.M., I.D.G., S.C., M.N.,G.S., A.C.provided clinical caretopatients,collecteddata,andgavethefinalapprovalofthe manuscript.

Conflictofinterest:Theauthorsdeclarenocompetingfinancial interests.

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