27 July 2021
AperTO - Archivio Istituzionale Open Access dell'Università di Torino
Original Citation:
Efficacy of two different doses of rabbit anti-T-lymphocyte globulin to prevent graft-versus-host disease in children with haematological malignancies transplanted from an unrelated donor: a multicentre, randomised, open-label, phase 3 trial
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DOI:10.1016/S1470-2045(17)30417-5 Terms of use:
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Long%term*results*of*an*open*label,*prospective,*randomized*clinical*trial*on*two* different*dosages*of*rabbit*anti%!human%T*lymphocyte*globulin*in*children*with* hematological*malignancies*given*allogeneic*hematopoietic*stem*cell* transplantation*from*an*unrelated*donor.* * *
Franco'Locatelli1,2,'Maria'Ester'Bernardo1,'Alice'Bertaina1,'Carla'Rognoni3,'Patrizia'Comoli4,' Attilio'Rovelli5,'Andrea'Pession6,'Franca'Fagioli7,'Claudio'Favre8,'Edoardo'Lanino9,'Giovanna' Giorgiani4,'Pietro'Merli1,'Arcangelo'Prete6,'Marco'Zecca4.'
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1.' Dipartimento' di' Oncoematologia' Pediatrica,' IRCCS' Ospedale' “Bambino' Gesù",' Roma,' Italy.'
2.' Università'degli'Studi'di'Pavia,'Italy.'
3.' Laboratorio'di'Bioinformatica'Medica'“Mario'Stefanelli”,'Università'of'Pavia,'Italy.' 4.' Oncoematologia'Pediatrica,'Fondazione'IRCCS'Policlinico'“San'Matteo”,'Pavia,'Italy.' 5.' Clinica'Pediatrica,'Fondazione'MBBM'/'A.O.'“San'Gerardo”,'Monza,'Italy.'
6.' Oncologia' ed' Ematologia' “Lalla' Seràgnoli”,' Clinica' Pediatrica,' Policlinico' Sant’Orsola' Malpighi,'Bologna,'Italy.' 7.' Oncoematologia'Pediatrica'e'Centro'Trapianti,'A.O.U.'Città'della'Salute'e'della'Scienza,' Torino,'Italy.' 8.' Dipartimento'di'Oncoematologia,'Tumori'pediatrici'e'Trapianto'di'cellule'staminali,'Azienda' OspedalieroZUniversitaria'Meyer,'Firenze,'Italy.' 9.' Dipartimento'di'Ematologia'e'Oncologia'Pediatrica,'Istituto'“G.'Gaslini”,'Genova,'Italy.' ' ' ' ' ' Corresponding'Author:'Prof.'Franco'Locatelli,'Dipartimento'di'Oncoematologia'Pediatrica,' IRCSS,'Ospedale'Pediatrico'Bambino'Gesù,'Piazza'Sant’Onofrio'4,'00165'Roma,'Italy.'Tel' +39'06'6859'2678.'Fax'+39'06'6859'2129.'EZmail:'franco.locatelli@opbg.net' ' Running'title:'Randomized'trial'on'ATG'in'children'given'UDZHSCT.' '
Abstract*
Background.' Although' rabbit' antiZhumanZTZlymphocyte' globulin' (ATLG)' is' largely' used' for' preventing' immuneZmediated' complications' in' patients' given' allogeneic' hematopoietic' stem' cell'transplantation'(HSCT)'from'an'unrelated'donor'(UD),'the'optimal'dosage'of'this'drug'in' children'is'still'undefined.'' Methods.*We'conducted'an'openZlabel,'randomized'trial'comparing'two'different'dosages'of' ATLG'(30'vs'15'mg/Kg,'over'3'days)'in'children'(aged'0Z18)'with'hematological'malignancies' transplanted'from'a'UD,'selected'using'highZresolution'typing'for'HLAZclass'I/II'loci.'All'patients' received'a'myeloablative'regimen'and'cyclosporineZA'plus'shortZterm'methotrexate'as'postZ transplantation' graftZversusZhost' disease' (GvHD)' prophylaxis.' The' study' (NCT00934557)' aimed'at'testing'whether'a'higher'dose'of'ATLG'was'superior'to'a'lower'dose'for'grade'IIZIV' acute'GvHD'prevention.''
Results.'From'01/2008'to'09/2012,'84'and'88'transplanted'children'belonging'to'the'30'and' the'15'mg/kg'group'were'included'in'this'analysis.'The'100Zday'cumulative'incidence'(CI)'of' grade' IIZIV' acute' GvHD' in' the' highZ' and' lowZATLG' group' was' 29%' and' 36%,' respectively' (P=NS).'The'CI'of'nonZrelapse'mortality'(NRM)'was'19%'and'9%'in'the'highZ'and'lowZATLG' group,'respectively'(P=0.09).'CI'of'disease'recurrence'was'17%'and'was'comparable'between' randomization'arms.'FiveZyear'eventZfree'survival'(EFS)'for'the'whole'cohort'was'69%j'it'was'
61%' and' 77%' for' children' given' highZ' and' lowZdose' ATLG,' respectively' (P=0.028).' In'
multivariate'analysis,'highZdose'ATLG'correlated'with'shorter'EFS'and'higher'NRM.''
Conclusions.' Children' with' hematological' malignancies' transplanted' from' a' UD' selected'
through'highZresolution'HLAZtyping'benefit'from'the'use'of'lowZdose'ATLG.'
INTRODUCTION* Acute'and'chronic'graftZversusZhost'disease'(GVHD)'remain'major'complications'of'allogeneic' hematopoietic'stem'cell'transplantation'(HSCT),'being'associated'with'an'increased'risk'of'nonZ relapse'mortality'(NRM)'and'reduction'in'quality'of'life'[1Z4].'AntiZTZlymphocyte'globulin'(ATLG)' has'been'widely'used'for'prevention'of'acute'and'chronic'GVHD'in'patients'given'allogeneic' HSCT.'In'particular,'in'the'last'years,'different'groups'have'shown'in'randomized'clinical'trials' that'the'addition'of'ATLG'to'GVHD'prophylaxis'is'able'to'decrease'the'risk'of'both'acute'and' chronic'GVHD'in'adults'receiving'either'HSCT'from'an'unrelated'donor'(UD)'[5Z8],'or'peripheral' blood' stem' cell' (PBSC)' transplantation' from' an' HLAZidentical' sibling' [9].' However,' recent' findings'have'suggested'that'use'of'ATLG'could'increase'the'risk'of'relapse'and'infections,' potentially'hampering'the'benefits'of'better'GVHD'prevention'[10].'In'fact,'ATLG'administration' has'been'demonstrated'to'be'associated'with'delayed'TZcell'reconstitution,'including'that'of' naïve'and'memory'T'lymphocytes,'and'with'an'increased'risk'of'infectious'complications'and' postZtransplant'lymphoproliferative'disease'[11Z13].'' Rabbit'ATLG'is'the'preferred'agent'for'approaches'of'GVHD'prevention'through'serotherapy' in' HSCT.' However,' two' diverse' ATLG' preparations' are' available' for' clinical' use' in' patients' given' allogeneic' HSCT' and' their' efficacy' can' be' markedly' different' and' have' an' impact' on' outcome,'since'they'differ'substantially'in'potency'and'dosing,'because'of'different'preparation' methods'[14,'15].''
In' children,' both' incidence' and' severity' of' acute' and' chronic' GVHD' are' lower' than' those' observed'in'adults'[4,'16]'and'none'of'the'previously'published'trials'has'focused'on'a'pediatric' population.' We' hypothesized' that' fine' tuning' of' GVHD' prophylaxis' and,' in' particular,' of' the' dosage'of'ATLG'administered'before'allogeneic'UDZHSCT,'could'play'an'important'role'on'the' outcome'of'in'children.'In'order'to'define'the'optimal'dose'of'rabbit'ATLG,'we'conducted'a' prospective,' multicenter,' clinical' trial' comparing' two' different' doses' of' the' drug' in' children' affected'by'hematological'malignancies'transplanted'with'either'bone'marrow'(BM)'or'PBSC' from'a'UD'selected'using'highZresolution'molecular'typing'for'class'I'and'II'HLA'loci.'
'
PATIENTS*and*METHODS* Study*design*
This' is' a' multicenter,' openZlabel,' phase' 3' study' enrolling' children' with' hematological' malignancies'given'allogeneic'HSCT'from'a'UD'and'randomly'assigned'to'receive'ATLG'at'a'
total'dose'of'either'30'mg/kg'or'15'mg/kg,'over'3'days,'namely'from'day'Z4'to'day'Z2.'The'study' was' sponsored' by' the' Fondazione' IRCCS' Policlinico' San' Matteo,' Pavia,' Italy' and' was' conducted'in'collaboration'with'the'HSCT'Working'Group'of'the'Italian'Association'for'Pediatric' Haematology'and'Oncology'(AIEOP).'The'institutional'review'board/ethical'committee'of'each' participating'center'approved'the'studyj'written'informed'consent'was'obtained'from'patient' parents'or'their'legal'guardians'before'enrollment.'The'trial'was'registered'at'ClinicalTrials.gov' (ClinicalTrials.gov'Identifier:'NCT00934557).' Inclusion'criteria'were:'i)'diagnosis'of'acute'leukemia'in'morphological'complete'remission'(CR)' or' PhiladelphiaZpositive' (Ph+)' chronic' myelogenous' leukemia' (CML)' in' chronic' phase,' nonZ Hodgkin'lymphoma'(NHL)'in'CR'or'myelodysplasiaj'ii)'age'at'transplantation'between'0'and'18' yearsj'iii)'a'life'expectancy'of'at'least'3'monthsj'iv)'the'availability'of'a'UD'selected'using'highZ resolution'molecular'typing'of'HLAZA,'B,'C'and'DRB1'loci,'with'no'more'than'2'allelic'disparities,' or'one'antigenic'differencej'v)'use'of'BMZderived'stem'cells'or'GZCSFZmobilized'PBSC.'
To' avoid' imbalance' between' the' 2' arms,' before' randomization,' patients' were' stratified' according'to'the'degree'of'compatibility'with'their'donor,'the'source'of'hematopoietic'stem'cells' employed'(BM'vs.'PBSC)'and'the'disease'risk'category'(standard'versus'high'risk,'SR'vs.'HR).' In'particular,'on'the'basis'of'compatibility'with'their'donor,'patients'were'divided'in'2'subgroups:' those'transplanted'from'a'UD'either'perfectly'matched'or'with'a'single'allelic'disparity'at'one'of' the'HLA'loci'(i.e.'A,'B,'C,'and'DRB1)'vs.'those'transplanted'from'a'UD'with'either'2'allelic' disparities'or'an'antigenic'disparity'at'the'HLA'loci.'SR'diseases'were'defined'as'follows:'ALL' in'1st'CRj'ALL'in'2nd'CR'belonging'to'the'BFM'S2'group'[17]j'AML'in'1st'CR'or'in'2nd'CR'if' relapsed'more'than'6'months'after'frontZline'treatment'discontinuationj'NHL'in'2nd'CRj'Ph+' CML'in'1st'chronic'phase'and'lowZgrade'myelodysplasia'(refractory'cytopenia).'HR'diseases' included:'ALL'in'2nd'CR'belonging'to'the'S3ZS4'group'[17]j'ALL'in'≥'3rd'CRj'AML'in'2nd'CR'if' relapsed'less'than'6'months'after'stop'therapyj'secondary'AMLj'NHL'in'3rd'CRj'Ph+'CML'in' 2nd' chronic' phase' and' highZgrade' myelodysplasia' (refractory' anemia' with' excess' of' blasts,' RAEB,'RAEB'in'transformation,'RAEBZt,'or'juvenile'myelomonocytic'leukemia,'JMML).'
Randomization*and*transplant*procedure*
Patients' were' randomly' assigned' to' either' of' the' 2' arms' with' a' 1:1' ratio,' according' the' stratification' criteria' reported' above.'The' randomization' code' was' generated' by' nQuery' Advisor,'with'the'use'of'a'block'randomization'plan'and'a'block'size'of'4.'
All'patients'were'given'a'myeloablative'regimen,'chosen'according'to'the'disease'and'the'policy' of'each'participating'center'(for'further'details'on'the'conditioning'regimens'used'see'also'Table' 1).'* GVHD*prophylaxis* GVHD'prophylaxis'consisted'of'the'combination'of'CyclosporineZA,'administered'intravenously' at'an'initial'dose'of'3'mg/kg/day'in'2'divided'doses,'starting'from'day'Z7'until'the'drug'could'be' administered'orally'at'a'dose'of'6'mg/kg/day,'and'shortZterm'methotrexate'(15'mg/m2'on'day' +1' and' 10' mg/m2' on' days' 3,' 6,' and' 11' after' transplantation,' respectively).' Rabbit' ATLG' (Grafalon®'kindly'provided'by'FRESENIUS/NEOVI)'was'given'on'day'Z4,'Z3'and'Z2'at'dose'of' either'10'or'5'mg/kg/day,'according'to'the'result'of'randomization.' Study*endpoints*and*definition*of*outcomes* The'primary'endpoint'of'the'study'was'100Zday'cumulative'incidence'of'grade'IIZIV'acute'GVHD' in'the'2'randomization'arms.'The'sample'size'was'calculated'based'on'expected'100Zday'rate' of'grade'IIZIV'acute'GVHD'of'20%'in'the'highZATLG'group'(30'mg/kg)'and'45%'in'the'lowZ ATLG'group'(15'mg/kg).'We'estimated'that'a'sample'size'of'170'patients'(85'patients'in'each' group)'would'give'the'study'at'least'90%'power'to'reject'the'null'hypothesis'in'a'logZrank'test' model.'Patients'surviving'for'more'than'7'and'100'days'after'HSCT'were'evaluated'for'acute' and'chronic'GVHD'occurrence,'respectively.'Severity'of'acute'GVHD'and'chronic'GVHD'was' assessed'according'to'the'Glucksberg'and'the'modified'Seattle'criteria'(for'categorization'of' chronic' GVHD' as' clinically' limited' or' extensive),' respectively' [18,' 19].' In' estimating' GVHD' occurrence,' both' rejection' and' NRM' were' considered' competing' events' [20].'Treatment' of' acute'and'chronic'GVHD'was'left'to'the'discretion'of'the'treating'physician.'
Secondary' endpoints' included' incidence' of' NRM' and' disease' recurrence' and' probability' of' overall'survival'(OS)'and'eventZfree'survival'(EFS)'in'the'2'arms.'We'also'evaluated'neutrophil' and'platelet'engraftment,'considering'both'rejection'and'death'as'competing'events.''
OS'was'defined'as'the'probability'of'survival,'regardless'of'disease'status,'from'the'time'of' HSCT'to'time'of'death'or'of'last'followZup'(surviving'patients'were'censored'at'last'followZup,' while'only'death'was'considered'an'event).'NRM'was'defined'as'the'probability'of'death'from' any'cause'other'than'disease'recurrence/progression.'EFS'was'defined'as'the'probability'of' survival'without'evidence'of'disease'at'any'time'after'transplantation.'In'estimating'EFS,'death,' relapse'and'rejection'with'or'without'autologous'hematopoietic'reconstitution'were'considered' events,'while'patients'who'were'alive,'with'sustained'donor'engraftment'and'diseaseZfree'were'
censored'at'last'followZup.'We'also'evaluated'the'composite'end'point'of'chronic'GVHD–free' and'relapseZfree'survival'[21].' Other'endpoints'included:'time'to'neutrophil'engraftment,'defined'as'time'from'HSCT'to'the' first'of'3'consecutive'days'with'an'absolute'neutrophil'count'greater'than'or'equal'to'0.5'x'109/L,' and'time'to'platelet'engraftment,'defined'as'time'from'HSCT'to'the'first'of'7'consecutive'days' with'an'unsupported'platelet'count'greater'than'or'equal'to'50'x'109/L.'Graft'failure'was'defined' as'either'lack'of'initial'engraftment'of'donor'cells'(primary'graft'failure)'or'loss'of'donor'cells' after'initial'engraftment'(secondary'graft'failure).'RegimenZrelated'toxicity'(RRT)'was'defined' as' nonZhematological' toxicities' due' to' the' preparative' regimen' documented' during' its' administration'and/or'during'the'first'30'days'after'transplantation,'and'was'graded'using'the' system'described'by'Bearman'et$al.$[22].'' Statistical*analysis* Quantitative'variables'were'reported'as'median'value'and'range,'while'categorical'variables' were'expressed'as'absolute'value'and'percentage.'Demographic'and'clinical'characteristics'of' patients'of'the'2'randomization'arms'were'compared'using'the'ChiZsquare'test'or'Fisher’s'exact' test' for' categorical' variables,' while' the' MannZWhitney' rank' sum' test' or' the' Student’s' TZtest' were'used'for'continuous'variables'as'appropriate'[23].'' Acute'and'chronic'GvHD,'rejection,'engraftment,'OS,'EFS,'NRM'and'relapse'incidence'were' estimated'from'the'date'of'transplantation'to'the'date'of'an'event'or'last'followZup.' Probabilities'of'OS'and'EFS'were'calculated'according'to'the'Kaplan'and'Meier'method'[24].' Acute'GvHD'and'chronic'GvHD,'NRM'and'relapse'were'calculated'as'cumulative'incidence' curves'in'order'to'adjust'the'estimates'for'competing'risks'[25].'All'results'were'expressed'as' probability'or'cumulative'incidence'(%)'and'95%'confidence'interval'(95%'CI)'[26,'27].''
The' significance' of' differences' between' OS' and' EFS' was' estimated' by' the' log–rank' test' (Mantel–Cox),' while' Gray’s' test' was' used' to' assess,' in' univariate' analyses,' differences' between' cumulative' incidences' [28].' Multivariate' analysis' was' performed' using' the' Cox' proportional'hazard'regression'model'[26,'27].''
P'values'<'0.05'were'considered'to'be'statistically'significant'and'reported'in'detailj'p'values' between'0.05'and'0.1'were'considered'not'statistically'significant'but'were'reported'in'detail'in' the'textj'P'values'≥'0.1'were'reported'as'nonZsignificant'(N.S.).'
Statistical'analysis'was'performed'using'NCSS'[NCSS'10'Statistical'Software'(2015).'NCSS,' LLC.' Kaysville,' UT,' ncss.com/software/ncss.]' and' R' 2.5.0' software' package' (http://www.RZ project.org)'[29,'30].'Data'were'analyzed'as'of'March'1st,'2016.' The'first,'second,'and'last'authors'designed'the'studyj'the'first'and'last'authors'wrote'the'first' draft,'and'all'the'coauthors'made'the'decision'to'submit'the'manuscript'for'publication,'while' no'one'who'is'not'an'author'contributed'to'the'writing'of'the'manuscript.'Neovii'Biotech,'which' provided'ATLG,'did'not'contribute'to'the'analysis'or'approve'the'manuscript'for'publication.' ' ' RESULTS* Patients* From'January'2008'to'August'2012,'a'total'of'187'patients'were'screened'for'inclusion'in'the' present'study,'at'7'centers'belonging'to'the'AIEOP'network.'Seven'patients'were'excluded' from'randomization,'because'they'did'not'fulfill'all'the'inclusion'criteria.'One'hundred'and'eighty' patients'were'enrolled'and'randomizedj'89'were'assigned'to'the'highZATLG'group'and'91'to' the'lowZATLG'group'(see'Figure'1).'Eight'patients,'5'belonging'to'the'highZATLG'group'and'3' to'the'lowZATLG'group,'did'not'proceed'to'transplantation'due'to'relapse'before'HSCT.'The' remaining'172'patients,'84'belonging'to'the'ATLG'30'mg/kg'group'and'88'belonging'to'the'15' mg/kg'group,'were'transplanted'and'included'in'this'analysis.'
Detailed' patient' and' transplant' characteristics' are' shown' in' table' 1.' The' two' randomization' groups'were'comparable'for'all'demographic'and'transplantZrelated'variables'evaluated'(Table' 1).' The'median'followZup'for'the'whole'study'population'was'3.4'years'(range,'1'day'–'7.9'years).' It'was'4.2'years'(range,'3.3'–'7.9)'for'patients'alive'and'0.7'years'(range,'1'day'–'4.2'years)'for' those'who'died.'Considering'only'surviving'patients,'the'median'followZup'was'4.2'years'(range,' 3.3'–'7.3)'for'the'30'mg/kg'arm'and'4.1'years'(range,'3.4'–'7.9)'for'the'15'mg/kg'arm'(P'='N.S.).' Engraftment*
Two' patients' (one' per' randomization' arm)' did' not' engraft.' The' median' time' to' neutrophil' engraftment'in'the'whole'study'population'was'20'days'(range,'9Z43),'with'no'difference'in'the' 2'randomization'arms'(data'not'shown).'The'median'time'to'platelet'engraftment'was'24'days' (range,'11Z96).'It'was'26'days'(range,'11Z96)'in'the'highZATLG'group'and'22'days'(range,'9Z 93)'in'the'lowZATLG'group'(MannZWhitney'P'='0.0107).'
Acute*and*chronic*GVHD* Figure'2'and'Table'2'summarize'the'cumulative'incidence'of'acute'and'chronic'GVHD.'In'detail,' 40'patients'(24%)'developed'grade'I'acute'GVHD,'44'(25%)'grade'II,'6'(3%)'grade'III'and'6' (3%)'grade'IV,'while'76'children'(44)'did'not'present'any'grade'of'acute'GVHD.'No'difference' was'observed'between'the'2'randomization'arms.'The'overall'100Zday'cumulative'incidence'of' grade'IIZIV'acute'GVHD'was'33%'(95%'CI,'26Z40),'while'that'of'grade'IIIZIV'acute'GVHD'was' 7%'(95%'CI,'4Z12).'The'cumulative'incidence'of'grade'IIZIV'and'of'grade'IIIZIV'acute'GvHD'was' comparable'between'the'2'randomization'arms'(see'also'Figure'2,'panel'A'and'B,'and'Table'2' for'further'details).''
One'hundred'and'fiftyZsix'patients'surviving'more'than'100'days'after'HSCT'were'evaluated' for' chronic' GVHD' occurrence.' Chronic' GVHD' vas' absent' in' 124' patients' (79%),' clinically' limited' in' 20' (13%)' and' clinically' extensive' in' 12' (8%).' The' overall'cumulative' incidence'of' chronic'GVHD'(limited'+'extensive)'was'21%'(95%'CI,'15Z28)'with'no'difference'was'observed'
between'the'2'randomization'arms'(see'also'Figure'2,'panel'C,'and'Table'2).'
The'overall'cumulative'incidence'of'extensive'chronic'GVHD'alone'was'8%'(95%'CI,'4Z14),'
being'6%'(95%'CI,'2Z14)'for'the'highZATLG'patients'and'10%'for'the'lowZATLG'group'(P=NS,' see'also'Figure'2,'panel'D).' NRM*and*infectious*complications* TwentyZtree'patients'(13%)'died'in'remission'due'to'transplantZrelated'causes,'15'in'the'highZ ATLG'group'and'8'in'the'lowZATLG'group.'The'overall'cumulative'incidence'of'NRM'was'14%' (95%'CI,'9Z21),'being'19%'for'patients'who'received'ATLG'at'a'total'dose'of'30'mg/kg'and'9%' for'those'given'15'mg/kg'(P'='0.09)'(Figure'3C).'Table'3'reports'in'detail'the'different'causes' of'death'in'the'2'arms.' Table'4'shows'the'occurrence'of'viral'infections'(HCMV,'EBV,'and'Adenovirus'both'reactivation' and' disease)' and' of' invasive' aspergillosis' in' the' whole' study' population' and' in' the' 2' randomization'arms.'Notably,'children'receiving'the'higher'ATLG'dose'had'a'greater'incidence' of'viral'reactivations'as'compared'to'those'who'received'the'lower'dosage.'The'difference'was' statistically' significant' for' EBV' reactivation' (37%' vs.' 23%j' P' =' 0.038)' and' for' Adenovirus' reactivation'(12%'vs.'1%j'P'='0.004).'Also'the'incidence'of'invasive'aspergillosis'was'slightly' higher'in'the'highZATLG'group'(7%'vs.'2%),'but'this'difference'was'not'statistically'significant.'
TwentyZnine'patients'(17%)'relapsed'at'a'median'of'6.6'months'after'HSCT'(range,'1.9Z21.8),' the'cumulative'incidence'of'relapse'being'15%'(95%'CI,'12Z24).'Seventeen'relapses'occurred' in' the' highZATLG' group' and' 12' in' the' lowZATLG' group.' Figure' 3D' shows' the' cumulative' incidence' of' relapse' of' the' study' population.' The' cumulative' incidence' of' relapse' was' 20%' (95%'CI,'13Z31)'for'the'highZATLG'group'and'14%'(95%'CI,'8Z23)'for'the'lowZATLG'group'(P=' N.S.).'
Survival*and*event%free*survival*
OneZhundred'and'twentyZthree'patients'(72%)'were'alive'at'time'of'the'last'followZup:'120'were' in' continuous' complete' remission' after' HSCT,' while' 3' were' alive' after' a' postZtransplant' leukemia'relapse.'
Figures' 3A' and' 3B' and' Table' 3' show' the' results' of' OS' and' EFS' analysis.' The' 5Zyear' OS' probability'was'70%'(95%'CI,'62Z77)'for'the'whole'study'population.'It'was'62%'(95%'CI,'50Z 73)'for'the'highZATLG'patients'and'78%'(95%'CI,'69Z87)'for'the'lowZATLG'patients'(P'='0.049).' Overall,'the'5Zyear'EFS'was'69%'(95%'CI,'62Z76)j'it'was'61%'(95%'CI,'50Z72)'and'77%'(95%' CI,'68Z85)'in'patients'who'received'ATLG'at'the'dose'of'30'mg/kg'or'of'15'mg/kg,'respectively' (P'='0.028).'
Table' 5' and' Figure' 4' summarize' the' results' of' survival' analysis' for' specific' subgroups' of' patients,' including' acute' leukemia' patients' and' subsets' of' patients' stratified' according' to' disease'risk'category,'degree'of'donor/recipient'HLA'compatibility,'and'source'of'stem'cells.' Children'with'SR'disease'had'a'better'EFS'probability'as'compared'with'HR'patients'(Figure' 4A),'as'well'as'those'transplanted'using'an'HLAZidentical'or'1'allelic'disparate'donor'had'a' better' outcome' that' those' transplanted' from' a' donor' showing' a' greater' disparity' with' the' recipient,'although'the'difference'in'univariate'analysis'is'not'statistically'significant'(Figure'4B).' Noteworthy,'in'all'subgroup'analyses,'we'observed'an'advantage'in'terms'of'EFS'for'patients' who'received'ATLG'at'the'dose'of'15'mg/kg'as'compared'to'those'who'received'the'30'mg/kg' dose.'This'advantage'was'statistically'significant'(P'<'0.05)'for'all'acute'leukemia'patients,'for' those' affected' by' ALL' and' for' those' receiving' a' transplant' from' a' 1Zantigen' or' 2Zallele' mismatched' donor.' The' advantage' was' not' statistically' significant' in' the' remaining' comparisons,'possibly'due'to'the'insufficient'number'of'patients'enrolled.'
Chronic' GVHDZfree' +' relapseZfree' survival' and' extended' chronic' GVHDZfree' +' relapseZfree' survival'were'also'evaluated'(see'Table'3'for'details).'In'both'analyses,'children'who'received'
lowZATLG' had' a' slightly' better' outcome,' even' though' in' both' cases' the' difference' was' not' statistically'significant.'
Multivariate*analysis*
Table' 6' summarizes' the' results' of' multivariate' analysis' of' EFS,' NRM' and' relapse.' In' multivariate' analysis' of' EFS,' the' following' variables' were' associated' with' worse' outcome:' ATLG'dose'of'30'mg/kg'(relative'risk'1.90j'P'='0.026),'HLA'disparity'>'1'allele'(relative'risk' 2.08j'P'='0.01)'and'HR'disease'(relative'risk'2.46j'P'='0.0015).'Only'HR'disease'group'resulted' significantly'associated'with'a'higher'risk'of'relapse'(relative'risk'='3.52j'P'='0.0012),'while'the' ATLG'dose'of'30'mg/kg'was'associated'with'a'higher'risk'of'NRM'(relative'risk'='1.80j'P'=' 0.047).' ' ' DISCUSSION*
Although' in$ vivo' serotherapy' has' been' largely' used' in' the' last' 2' decades' with' the' aim' of' reducing'the'incidence'and'severity'of'GVHD,'few'randomized'trials'have'been'conducted'[5Z 10]'and'none'of'these'in'pediatric'patients.'Moreover,'while'all'these'controlled'trials'compared' either'rabbitZantihuman'TZcell'line'(Jurkat)'globulin'(Grafalon®,''FRESENIUS/NEOVI)'versus' no'ATLG'[7,'9,'10]'or'rabbitZantihuman'thymocyte'globulin'(Thymoglobulin®,'Genzyme)'versus' no'ATLG'[5,'6,'8],'no'randomized'study'has'addressed'the'question'of'drug'dose.'Thus,'the' optimum' ATLG' therapeutic' window' and' the' timing' of' treatment' have' not' yet' been' defined.' Herein,' we' report' the' results' of' the' first' randomized' clinical' trial' designed' to' establish' the' optimal'dose'of'rabbit'ATLG'able'to'prevent'acute'GVHD,'while'maintaining'the'capacity'of' effectively' controlling/eradicating' infections' and' leukemia' reZgrowth' in' children' undergoing' transplantation'with'either'BMZ'or'PBSCZderived'grafts'from'a'UD.'
We'selected'to'compare'30'mg/kg'versus'15'mg/kg'of'ATLG'instead'of'the'dose'of'60'mg/kg' used'in'UDZHSCT'recipients'in'the'trial'published'by'Finke'et'al.'[7],'because'children'are'less' prone'to'develop'GVHD'than'adults.'Support'to'this'choice'is'also'given'by'a'retrospective' analysis'showing'that'30'mg/kg'of'ATLG'in'single'dose'were'equally'effective'for'preventing' GVHD' than' 60' mg/kg' administered' over' 3' days' in' adults' with' hematological' malignancies' transplanted'from'an'unrelated'volunteer'[31].'In'that'trial,'the'2Zyear'NRM'was'lower'for'the' ATGZ30'mg/kg'group'(12%'versus'33%,'P=0.02),'mainly'because'of'a'higher'incidence'of'fatal' infections'in'the'ATGZ60'mg/kg'group'[31].'
Our'longZterm'results'show'that,'in'an'era'in'which'highZresolution'molecular'typing'for'class'I' and'class'II'HLA'loci'has'significantly'improved'the'clinical'outcome'of'UDZHSCT'by'decreasing' the'risk'of'immunological'complications'[16,'32,'33],'lowering'the'ATLG'dose'to'15'mg/kg'did' not' affect' the' time' to' engraftment,' and' more' importantly,' the' incidence' of' acute' or' chronic' GVHD,'while'was'associated'with'an'improved'probability'of'EFS,'mainly'due'to'a'reduced'risk' of' NRM.' In' particular,' children' given' highZATLG' had' a' higher' incidence' of' infectionZrelated' deaths'(7'vs'3'in'the'lowZATLG'group)j'moreover,'they'also'experienced'an'increased'risk'of' adenovirus' and' EBV' infections.' Although' we' do' not' have' data' on' virusZspecific' immune'
reconstitution'in'the'2'randomization'arms,'several'studies'showed'that'ATLG'has'a'halfZlife'of' 7–14'days,'which'means'that'patients'are'exposed'to'ATLG'both'before'and'after'HSCT'[12,' 34,'35]'and'there'is'substantial'evidence'that'higher'doses'of'ATG'are'associated'with'slower' recovery'of'pathogenZspecific'immunity'and'higher'incidence'of'fatal'infections'[6,'10,'11,'13].' Adenovirus'infections'are'much'more'of'a'problem'in'pediatric'patients'(20Z26%)'undergoing' HSCT'than'in'adults'(9%),'possibly'because'of'the'reservoir'of'AdV'in'the'general'pediatric' population'[36,'37].'Higher'doses'of'ATLG'depleting'antiviral'T'cells'transferred'with'the'graft,' because' of' the' long' halfZlife' of' the' polyclonal' antibodies,' may' have' a' detrimental' effect' on' recovery' of' pathogenZspecific' TZcell' immunity,' which' was' demonstrated' to' be' crucial' for' clearing' the' viral' infection' and' preventing' dissemination' of' adenovirus' infection' [36,' 38].' Previous'reports'have'also'documented'that'both'TZcell'depletion'and'high'rabbit'ATLG'serum' levels' on' day' 7' after' HSCT' are' associated' with' increased' risk' of' EBV' infection' and' postZ transplantation'lymphoproliferative'disorder'[39Z41].' The'use'of'lowZdose'ATLG'was'associated'with'statistically'better'EFS'in'the'subgroups'of' children'with'acute'leukemia'or'with'ALL,'which'represents'the'more'frequent'indication'for'an' allograft'in'hematological'malignancies'of'childhood.'In'particular,'the'78%'probability'of'EFS' at'5'years'observed'in'the'50'children'with'ALL'given'lowZATLG'is'comparable'with'that'recently' reported'by'Peters'et'al.'[33]'in'306'children'transplanted'from'an'UD'using'a'standardized' transplantation'and'GVHD'prophylaxis'protocol'(71%'at'4'years).''
Remarkably,' the' advantage' of' using' lowZATLG' was' evident' also' in' patients' given' PBSC,' a' stem'cell'source'that,'in'comparison'to'BM,'was'shown'to'be'associated'with'more'chronic' GVHD' in' a' prospective,' randomized' trial' conducted' in' UDZHSCT' recipients' [42],' probably' reflecting'the'protective'effect'of'intensive'GVHD'prophylaxis'with'cyclosporine,'methotrexate,' and'rabbit'ATLG.''
In'our'cohort,'the'most'frequent'cause'of'treatment'failure'after'transplantation'was'disease' recurrence,'which'occurred'in'the'first'or'more'rarely'in'the'second'year'after'transplantation.' While'the'dose'of'ATLG'employed'did'not'influence'the'probability'of'relapse,'this'was'mainly' affected'by'the'disease'risk'category,'confirming'that'the'stratification'criterion'chosen'had'a' high'predictive'value'on'final'outcome.'''' In'this'study,'the'rate'of'the'composite'endZpoint'of'survival'free'from'chronic'GVHD'and'from' relapse'was'comparable'between'children'given'either'highZ'or'lowZATLG,'suggesting'that'a' lower'dose'of'serotherapy'does'not'affect'the'quality'of'life'of'surviving'patients.''
Different' types' of' ATLG' preparations' have' been' tested' as' part' of' conditioning' regimens' to' achieve'in$vivo'TZcell'depletion/modulation'and,'thus,'to'prevent'GVHD.'The'ATLG'preparation' used'in'our'study'is'a'polyclonal'antihuman'TZlymphocyte'immune'globulin'derived'from'rabbits' after'immunization'with'the'Jurkat'human'TZcell'line.'This'product'preferentially'targets'T'cellsj' however,'since'other'antigens'like'CD19'or'CD138'are'also'targeted'by'ATLG'[43,'44],'we' cannot' exclude' a' direct' antitumor' effect' of' the' drug' in' BZcell' ALL' and' to' a' lesser' extent' in' myeloid'leukemias.' Recent'data'on'pharmacokinetics'of'ATLG'in'the'pediatric'population'have'shown'that'older' children'have'a'disproportionately'higher'exposure'than'younger'children,'because'they'have' a'lower'clearance'of'the'drug'per'kg'[11,'12].'In'addition,'a'recipient'low'lymphocyte'count'at' time'of'ATLG'infusion'was'shown'to'translate'into'high'exposure'of'donor'T'cells'to'the'drug' [11,'12].'These'observations'provide'a'rationale'for'envisaging'for'the'future'more'sophisticated' strategies'on'ATLG'dose'and'timing'of'administration'based'on'pharmacokinetics'parameters.'' In'summary,'our'data'obtained'in'a'randomized'clinical'trial'with'a'long'followZup'indicate'that,' in'children'with'hematological'malignancies'transplanted'from'an'UD'selected'through'highZ resolution'HLA'typing,'the'use'of'low'dose'rabbit'ATLG'results'into'better'EFS.'Low'dose'ATLG' can'spare'lifeZthreatening'viral'infections,'without'significantly'increasing'the'incidence'of'acute' and'chronic'GvHD'and'without'adversely'affecting'other'outcomes,'like'engraftment'or'relapse.' ' '
!
Table&1.!Patient!and!transplant!characteristics.!
! ! ! ! ! ! ! ! ! ! !
! Randomization&arm& & ! ! & &
! 15&mg/kg& & 30&mg/kg& & Total& & P&
Number!of!patients! 88! (51%)! ! 84! (49%)! ! 172! (100%)! ! ! ! ! ! ! ! ! ! ! ! ! ! Gender:! ! ! ! ! ! ! ! ! ! ! !!!!!Male! 54! (61%)! ! 54! (64%)! ! 108! (63%)! ! 0.6919! !!!!!Female! 34! (39%)! ! 30! (36%)! ! 64! (37%)! ! ! ! ! ! ! ! ! ! ! ! ! ! Age!at!diagnosis!(years)! 7.5! (1J18)! ! 7.0! (1J18)! ! 7.0! (1J18)! ! 0.5961! ! ! ! ! ! ! ! ! ! ! ! Diagnosis:! ! ! ! ! ! ! ! ! ! ! !!!!!ALL! 50! (57%)! ! 44! (52%)! ! 94! (55%)! ! 0.9557! !!!!!AML! 21! (24%)! ! 21! (25%)! ! 42! (24%)! ! ! !!!!!CML! 1! (1%)! ! 2! (2%)! ! 3! (2%)! ! ! !!!!!MDS! 12! (14%)! ! 13! (16%)! ! 25! (14%)! ! ! !!!!!NHL! 4! (4%)! ! 4! (5%)! ! 8! (5%)! ! ! ! ! ! ! ! ! ! ! ! ! ! Age!at!HSCT!(years)! 9.0! (1J19)! ! 9.0! (1J19)! ! 9.0! (1J19)! ! 0.7656! ! ! ! ! ! ! ! ! ! ! ! Disease!risk!category:! ! ! ! ! ! ! ! ! ! ! !!!!!Standard!risk! 57! (65%)! ! 50! (60%)! ! 107! (62%)! ! 0.4779! !!!!!High!risk! 31! (35%)! ! 34! (40%)! ! 65! (38%)! ! ! ! ! ! ! ! ! ! ! ! ! ! Year&of&transplantation& ! ! ! ! ! ! ! ! ! ! !!!!!2008! 12! (14%)! ! 8! (9%)! ! 20! (12%)! ! 0.6042! !!!!!2009! 13! (15%)! ! 18! (21%)! ! 31! (18%)! ! ! !!!!!2010! 16! (18%)! ! 15! (18%)! ! 31! (18%)! ! ! !!!!!2011! 26! (30%)! ! 24! (29%)! ! 50! (29%)! ! ! !!!!!2012! 21! (23%)! ! 19! (23%)! ! 40! (23%)! ! !
! ! ! ! ! ! ! ! ! ! ! Donor&–&recipient&HLA&disparity& ! ! ! ! ! ! ! ! ! ! !!!!!Identical!or!1!allele!disparity! 64! (73%)! ! 61! (73%)! ! 125! (73%)! ! 0.8805! !!!!!1!Antigen!or!>!1!allele!disparity! 24! (27%)! ! 23! (27%)! ! 47! (27%)! ! ! ! ! ! ! ! ! ! ! ! ! ! Stem&cell&source& ! ! ! ! ! ! ! ! ! ! !!!!!Bone!marrow! 72! (82%)! ! 70! (83%)! ! 142! (79%)! ! 0.7935! !!!!!Peripheral!blood! 16! (18%)! ! 14! (17%)! ! 30! (21%)! ! ! ! ! ! ! ! ! ! ! ! ! ! Conditioning®imen& ! ! ! ! ! ! ! ! ! ! &&BusulfanHbased& 30! (34%)! ! 38! (45%)! ! 68! (40%)! ! 0.4709! !!!!!Busulfan!+!Cyclophosphamide!+!Melphalan! 18! ! ! 25! ! ! 43! ! ! ! !!!!!Busulfan!+!Thiotepa!+!Cyclophosphamide! 7! ! ! 8! ! ! 15! ! ! ! !!!!!Busulfan!+!Cyclophosphamide! 5! ! ! 5! ! ! 10! ! ! ! !!TreosulfanH&based& 12! (14%)! ! 10! (12%)! ! 22! (13%)! ! ! !!!!!Thiotepa!+!Treosulfan!+!Fludarabine! 7! ! ! 9! ! ! 16! ! ! ! !!!!!Treosulfan!+!Fludarabine! 6! ! ! 4! ! ! 10! ! ! ! !!TBIHbased& 45! (51%)! ! 33! (39%)! ! 78! (45%)! ! ! !!!!!TBI!+!Thiotepa!+!Cyclophosphamide! 29! ! ! 20! ! ! 49! ! ! ! !!!!!TBI!+!Thiotepa!+!Fludarabine! 11! ! ! 7! ! ! 18! ! ! ! !!!!!TBI!+!VP16! 5! ! ! 6! ! ! 11! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Cell&dose&infused& ! ! ! ! ! ! ! ! ! ! !!!!Bone!marrow!(TNC!x!108/kg)! 4! (2J7)! ! 5! (2J9)! ! 4! (2J9)! ! 0.3238! !!!!Peripheral!blood!(CD34+!cell!x!106/kg)! 7! (3J8)! ! 6! (3J8)! ! 6! (3J9)! ! 0.4256! ! !
! Table!2.!Overall!clinical!outcomes!and!outcomes!by!randomization!arm.! ! ! ! ! ! ! ! Outcome! Randomization! arm! N.!of! patients! Events! Probability! (%)! (95%!CI)! P! ! ! ! ! ! ! ! Grade!IIEIV!acute!GVHD! all!patients! 172! 56! 33%! (26>40)! ! ! 15!mg/kg! 88! 32! 36%! (28>48)! 0.2659! ! 30!mg/kg! 84! 24! 29%! (20>40)! ! ! ! ! ! ! ! ! Grade!IIIEIV!acute!GVHD! all!patients! 172! 12! 7%! (4>12)! ! ! 15!mg/kg! 88! 5! 6%! (2>13)! 0.4944! ! 30!mg/kg! 84! 7! 8%! (4>17)! ! ! ! ! ! ! ! ! Chronic!GVHD! all!patients! 156! 32! 21%! (15>28)! ! ! 15!mg/kg! 83! 18! 22%! (14>33)! 0.6923! ! 30!mg/kg! 73! 14! 19%! (12>31)! ! ! ! ! ! ! ! ! Extensive!chronic!GVHD! all!patients! 156! 12! 8%! (4>13)! ! ! 15!mg/kg! 83! 8! 10%! (5>19)! 0.3294! ! 30!mg/kg! 73! 4! 6%! (2>14)! ! ! ! ! ! ! ! ! Overall!survival! all!patients! 172! 49! 70%! (62>77)! ! ! 15!mg/kg! 88! 19! 78%! (69>87)! 0.0499! ! 30!mg/kg! 84! 30! 62%! (50>73)! ! ! ! ! ! ! ! ! EventEfree!survival!(EFS)! all!patients! 172! 52! 69%! (62>76)! ! ! 15!mg/kg! 88! 20! 77%! (68>85)! 0.0280! ! 30!mg/kg! 84! 32! 61%! (50>72)! ! ! ! ! ! ! ! ! NonErelapse!mortality! all!patients! 172! 23! 14%! (9>21)! ! ! 15!mg/kg! 88! 8! 9%! (5>18)! 0.0912! ! 30!mg/kg! 84! 15! 19%! (12>30)! ! ! ! ! ! ! ! ! Relapse! all!patients! 172! 29! 17%! (12>24)! ! ! 15!mg/kg! 88! 12! 14%! (8>23)! 0.2619! ! 30!mg/kg! 84! 17! 20%! (13>31)! ! ! ! ! ! ! ! ! CEGVHDEfree+relapseEfree! survival! all!patients! 172! 75! 56%! (49>64)! ! ! 15!mg/kg! 88! 34! 61%! (51>71)! 0.1851! ! 30!mg/kg! 84! 41! 51%! (40>62)! ! ! ! ! ! ! ! !
Extended!CEGVHDE free+relapseEfree!survival! all!patients! 172! 57! 67%! (60>74)! ! ! 15!mg/kg! 88! 25! 71%! (62>81)! 0.1688! ! 30!mg/kg! 84! 32! 62%! (51>72)! ! ! ! ! ! ! ! ! ! !
! Table!3.!Causes!of!death.! ! ! ! ! ! ! ! ! ! ! ! ! Randomization!arm! ! ! ! ! ! ! 15!mg/kg! ! 30!mg/kg! ! Total! ! P! Number!of!patients! 88! ! 84! ! 172! ! ! ! ! ! ! ! ! ! ! ! ! ! Number!of!deaths! 19! (22%)! ! 30! (36%)! ! 49! (28%)! ! 0.0402! ! ! ! ! ! ! ! ! ! ! ! Disease!progression! 11! (46%)! ! 15! (54%)! ! 26! (100%)! ! N.S.! Non?relapse!mortality! 8! (33%)! ! 15! (67%)! ! 23! (100%)! ! ! !!!!Acute!GVHD! 1! ! ! 2! ! ! 3! ! ! ! !!!!Chronic!GVHD! 3! ! ! 2! ! ! 5! ! ! ! !!!!Bacterial!infection/sepsis! 0! ! ! 2! ! ! 2! ! ! ! !!!!Fungal!infection! 2! ! ! 1! ! ! 2! ! ! ! !!!!Viral!infection! 1! ! ! 3! ! ! 4! ! ! ! !!!!Parasitic!infection! 0! ! ! 1! ! ! 1! ! ! ! !!!!Hemorrhage! 1! ! ! 3! ! ! 4! ! ! ! !!!!VenoNocclusive!disease! 0! ! ! 1! ! ! 1! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !
! Table&4.!Cumulative!incidence!of!infectious!complications!in!the!2!randomization!arms.! ! ! ! ! ! ! ! Type&of&infection& Randomization& arm& N.&of& patients& Events& Probability& (%)& (95%&CI)& P& ! ! ! ! ! ! ! HCMV&reactivation& all!patients! 172$ 60$ 35%$ (28,43)$ ! ! 15!mg/kg! 88$ 28$ 32%$ (23,43)$ N.S.! ! 30!mg/kg! 84$ 32$ 38%$ (29,50)$ ! ! ! ! ! ! ! ! HCMV&disease& all!patients! 172$ 0! 0%! ?!?! ! ! 15!mg/kg! 88$ 0! 0%! ?!?! N.S.! ! 30!mg/kg! 84$ 0! 0%! ?!?! ! ! ! ! ! ! ! ! EBV&reactivation& all!patients! 172$ 51$ 30%$ (24,37)$ $ ! 15!mg/kg! 88$ 20$ 23%$ (15,33)$ 0.0384& ! 30!mg/kg! 84$ 31$ 37%$ (28,49)$ $ ! ! ! ! ! ! ! EBV&PTLD& all!patients! 172$ 1! 1%! (0?4)! ! ! 15!mg/kg! 88$ 0! 0%! ?!?! N.S.! ! 30!mg/kg! 84$ 1! 2%! (0?8)! ! ! ! $ ! ! ! ! Adenovirus&reactivation& all!patients! 172$ 11$ 6%$ (4,11)$ $ ! 15!mg/kg! 88$ 1$ 1%$ (0.2,8)$ 0.0041& ! 30!mg/kg! 84$ 10$ 12%$ (7,21)$ $ ! ! ! ! ! ! ! Adenovirus&disease& all!patients! 172$ 5$ 3%$ (1,7)$ $ ! 15!mg/kg! 88$ 1$ 1%$ (0.2,8)$ N.S.$ ! 30!mg/kg! 84$ 4$ 5%$ (2,12)$ $ ! ! ! ! ! ! ! Invasive&aspergillosis& all!patients! 172$ 7$ 5%$ (2,10)$ $ ! 15!mg/kg! 88$ 2$ 2%$ (1,9)$ N.S.$ ! 30!mg/kg! 84$ 5$ 7%$ (3,18)$ $ ! ! ! ! ! ! ! ! ! ! !
! Table&5.!Specific!subgroup!analyses.! ! ! ! ! ! ! ! Outcome& Randomization& arm& N.&of& patients& Events& Probability& (%)& (95%&CI)& P& ! ! ! ! ! ! ! Acute&leukemia& ! ! ! ! ! ! !!!!Event?free!survival! all!patients! 136! 41! 69%! (61?77)! ! ! 15!mg/kg! 71! 16! 77%! (67?87)! 0.0499& ! 30!mg/kg! 65! 25! 60%! (48?73)! ! ! ! ! ! ! ! ! !!!!Non?relapse!mortality! all!patients! 136! 17! 13%! (8?21)! ! ! 15!mg/kg! 71! 6! 9%! (4?19)! N.S.! ! 30!mg/kg! 65! 11! 18%! (10?31)! ! ! ! ! ! ! ! ! !!!!Relapse! all!patients! 136! 24! 18%! (12?26)! ! ! 15!mg/kg! 71! 10! 14%! (8?25)! N.S.! ! 30!mg/kg! 65! 14! 22%! (14?35)! ! ! ! ! ! ! ! ! Acute&lymphoblastic&leukemia! ! ! ! ! ! !!!!Event?free!survival! all!patients! 94! 29! 68%! (58?78)! ! ! 15!mg/kg! 50! 11! 78%! (66?89)! 0.0496& ! 30!mg/kg! 44! 18! 58%! (43?73)! ! ! ! ! ! ! ! ! !!!!Non?relapse!mortality! all!patients! 94! 13! 15%! (9?25)! ! ! 15!mg/kg! 50! 4! 8%! (3?21)! 0.0825! ! 30!mg/kg! 44! 9! 22%! (12?39)! ! ! ! ! ! ! ! ! !!!!Relapse! all!patients! 94! 16! 17%! (11?27)! ! ! 15!mg/kg! 50! 7! 14%! (7?28)! N.S.! ! 30!mg/kg! 44! 9! 20%! (11?37)! ! ! ! ! ! ! ! ! Acute&myeloid&leukemia& ! ! ! ! ! ! !!!!Event?free!survival! all!patients! 42! 12! 70%! (56?85)! ! ! 15!mg/kg! 21! 5! 76%! (58?94)! N.S.! ! 30!mg/kg! 21! 7! 65%! (44?86)! ! ! ! ! ! ! ! ! !!!!Non?relapse!mortality! all!patients! 42! 4! 10%! (4?24)! ! ! 15!mg/kg! 21! 2! 10%! (3?36)! N.S.! ! 30!mg/kg! 21! 2! 10%! (3?36)! ! ! ! ! ! ! ! ! !!!!Relapse! all!patients! 42! 8! 20%! (11?38)! ! ! 15!mg/kg! 21! 3! 14%! (5?41)! N.S.!
! 30!mg/kg! 21! 5! 25%! (12?54)! ! ! ! ! ! ! ! ! SR&patients& ! ! ! ! ! ! !!!!Event?free!survival! all!patients! 107! 23! 78%! (69?86)! ! ! 15!mg/kg! 57! 9! 84%! (74?94)! N.S.! ! 30!mg/kg! 50! 14! 71%! (58?84)! ! ! ! ! ! ! ! ! !!!!Non?relapse!mortality! all!patients! 107! 12! 11%! (7?21)! ! ! 15!mg/kg! 57! 5! 9%! (4?22)! N.S.! ! 30!mg/kg! 50! 7! 15%! (8?31)! ! ! ! ! ! ! ! ! !!!!Relapse! all!patients! 107! 11! 10%! (6?18)! ! ! 15!mg/kg! 57! 4! 7%! (3?18)! N.S.! ! 30!mg/kg! 50! 7! 14%! (7?28)! ! ! ! ! ! ! ! ! HR&patients& ! ! ! ! ! ! !!!!Event?free!survival! all!patients! 65! 29! 55%! (43?67)! ! ! 15!mg/kg! 31! 11! 64%! (47?81)! N.S.! ! 30!mg/kg! 34! 18! 47%! (30?64)! ! ! ! ! ! ! ! ! !!!!Non?relapse!mortality! all!patients! 65! 11! 17%! (11?29)! ! ! 15!mg/kg! 31! 3! 10%! (3?28)! N.S.! ! 30!mg/kg! 34! 8! 24%! (13?43)! ! ! ! ! ! ! ! ! !!!!Relapse! all!patients! 65! 18! 28%! (19?41)! ! ! 15!mg/kg! 31! 8! 26%! (14?47)! N.S.! ! 30!mg/kg! 34! 19! 30%! (18?50)! ! ! ! ! ! ! ! ! HLAQidentical&or&1&allele&mismatched&donor& ! ! ! ! ! !!!!Event?free!survival! all!patients! 124! 31! 75%! (67?82)! ! ! 15!mg/kg! 63! 14! 77%! (67?88)! N.S.! ! 30!mg/kg! 61! 17! 72%! (61?83)! ! ! ! ! ! ! ! ! !!!!Non?relapse!mortality! all!patients! 124! 13! 11%! (6?18)! ! ! 15!mg/kg! 63! 5! 8%! (4?19)! N.S.! ! 30!mg/kg! 61! 8! 13%! (7?25)! ! ! ! ! ! ! ! ! !!!!Relapse! all!patients! 124! 18! 15%! (10?22)! ! ! 15!mg/kg! 63! 9! 14%! (8?26)! N.S.! ! 30!mg/kg! 61! 9! 15%! (8?27)! ! ! ! ! ! ! ! ! 1&Antigen&or&2QalleleQmismatched&donor& ! ! ! ! ! !!!!Event?free!survival! all!patients! 48! 21! 54%! (40?69)! ! ! 15!mg/kg! 25! 6! 76%! (59?93)! 0.0034&
! 30!mg/kg! 23! 15! 31%! (10?52)! ! ! ! ! ! ! ! ! !!!!Non?relapse!mortality! all!patients! 48! 10! 22%! (13?39)! ! ! 15!mg/kg! 25! 3! 12%! (4?35)! N.S.! ! 30!mg/kg! 23! 7! 34%! (18?64)! ! ! ! ! ! ! ! ! !!!!Relapse! all!patients! 48! 11! 23%! (14?39)! ! ! 15!mg/kg! 25! 3! 12%! (4?35)! 0.0549! ! 30!mg/kg! 23! 8! 35%! (20?62)! ! ! ! ! ! ! ! ! Bone&marrow&stem&cells& ! ! ! ! ! ! !!!!Event?free!survival! all!patients! 142! 45! 67%! (60?75)! ! ! 15!mg/kg! 72! 18! 74%! (64?85)! 0.0864! ! 30!mg/kg! 70! 27! 61%! (49?72)! ! ! ! ! ! ! ! ! !!!!Non?relapse!mortality! all!patients! 142! 23! 17%! (12?25)! ! ! 15!mg/kg! 72! 8! 11%! (6?22)! 0.0948! ! 30!mg/kg! 70! 15! 22%! (14?35)! ! ! ! ! ! ! ! ! !!!!Relapse! all!patients! 142! 22! 16%! (11?23)! ! ! 15!mg/kg! 72! 10! 14%! (8?25)! N.S.! ! 30!mg/kg! 70! 12! 17%! (10?29)! ! ! ! ! ! ! ! ! Peripheral&blood&stem&cells& ! ! ! ! ! ! !!!!Event?free!survival! all!patients! 30! 7! 76%! (61?92)! ! ! 15!mg/kg! 16! 2! 88%! (71?100)! N.S.! ! 30!mg/kg! 14! 5! 63%! (36?89)! ! ! ! ! ! ! ! ! !!!!Non?relapse!mortality! all!patients! 30! 0! 0%! ?!?! ! ! 15!mg/kg! 16! 0! 0%! ?!?! N.S.! ! 30!mg/kg! 14! 0! 0%! ?!?! ! ! ! ! ! ! ! ! !!!!Relapse! all!patients! 30! 7! 24%! (12?46)! ! ! 15!mg/kg! 16! 2! 13%! (3?46)! N.S.! ! 30!mg/kg! 14! 5! 38%! (19?76)! ! ! ! ! ! ! ! ! ! ! ! !
Table&6.!Multivariate!analysis!on!EFS,!TRM!and!relapse!risk!
! ! ! !
& Relative&risk& (95%&CI)& P&
! ! ! ! EVENTQFREE&SURVIVAL& ! ! ! ! ! ! ! &&Disease&risk&category& ! ! ! !!!!!!SR!vs.!HR! 2.46! (1.41?4.27)! 0.0015& ! ! ! ! &&HLA&compatibility& ! ! ! !!!!!>!1!allele!mismatch!vs.Identical!or!1!allele!mismatch! 2.08! (1.2?3.7)! 0.0111& ! ! ! ! &&Stem&cell&source& ! ! ! !!!!!Peripheral!blood!vs.!bone!marrow! 0.92! (0.41?2.07)! N.S.! ! ! ! ! &&Randomization&arm& ! ! ! !!!!!ATG!dose:!30!mg/kg!vs.!15!mg/kg! 1.90! (1.07?3.3)! 0.0256& ! ! ! ! ! ! ! ! NONQRELAPSE&MORTALITY& ! ! ! & ! ! ! &&Disease&risk&category& ! ! ! !!!!!!SR!vs.!HR! 1,56! (0.68?3,54)! N.S.! ! ! ! ! &&HLA&compatibility& ! ! ! !!!!!>!1!allele!mismatch!vs.Identical!or!1!allele!mismatch! 2.00! (0.87?4.59)! N.S.! ! ! ! ! &&Stem&cell&source& ! ! ! !!!!!Peripheral!blood!vs.!bone!marrow! 0.25! (0.03?1.88)! N.S.! ! ! ! ! &&Randomization&arm& ! ! ! !!!!!ATG!dose:!30!mg/kg!vs.!15!mg/kg! 1.80! (1.07?3.0)! 0.047& ! ! ! ! ! ! ! ! RELAPSE& ! ! ! ! ! ! ! &&Disease&risk&category& ! ! ! !!!!!!SR!vs.!HR! 3.52! (1.65?7.51)! 0.0012& !!!!!!! ! ! ! &&HLA&compatibility& ! ! ! !!!!!>!1!allele!mismatch!vs.Identical!or!1!allele!mismatch! 2.12! (0.94?4.58)! 0.0551! ! ! ! ! &&Stem&cell&source& ! ! ! !!!!!Peripheral!blood!vs.!bone!marrow! 1.95! (0.81?4.67)! N.S.! ! ! ! ! &&Randomization&arm& ! ! ! !!!!!ATG!dose:!30!mg/kg!vs.!15!mg/kg! 1.80! (0.85?3.81)! N.S.! ! ! ! ! ! & !
Figure&legends& ! Figure&1.!CONSORT!flow!diagram!of!the!whole!population!enrolled!into!the!randomized!trial.! ! Figure&2.!Cumulative!incidence!of!grade!II?IV!acute!GVHD!(A),!grade!III?IV!acute!GVHD!(B),! chronic!GVHD!(C)!and!extensive!chronic!GVHD!(D)!in!the!2!randomization!arms.! ! Figure&3.!Five?year!probability!of!overall!survival!(SURV)!(A),!event?free!survival!(EFS)!(B),! cumulative!incidence!of!non?relapse!mortality!(NRM)!(C)!and!relapse!(REL)!(D)!according!to! randomization!arm.! ! Figure&4.!Event?free!survival!(EFS)!according!to!disease!risk!category!(A)!and!according!to!the! degree!of!HLA!compatibility!in!the!donor/recipient!pair!(B).! ! ! !
187 patients screened 180 paziente0enrolled and0 randomized 91 patients allocated to the15 mg/kg0group 89 patients allocated to the 300mg/kg0group 880patients transplanted with ATG0150mg/kg 840patients transplanted with ATG0300mg/kg 680patients are0alive
and0disease free 520patients are0aliveand0disease free
8 patients died due0to0transplantE related causes 120patients relapsed 150patients died due0to0transplantE related causes 170patients relapsed 3 patients not transplanted due0 to0further relapse 5 patients not transplanted due0 to0further relapse 7 patients not elegible
!
Figure&1.&
Grade&II(IV´&GVHD&by&random 0 20 40 60 80 100 0 20 40 60 80 100 DAYS&AFTER&HSCT C U MU LA T IVE& IN C ID EN C E& (9 5% &C I)
15&mg/kg N&=&88O E&=&32
30&mg/kg N&=&84O E&=&24 15&mg/kg&=&36%&(28(48)
30&mg/kg&=&29%&(20(40)
Gray test&P&=&N.S.
Number of&patients at risk:
15&mg/kg 88 74 58 57 57 54 30&mg/kg 84 71 60 57 56 53
A
0 20 40 60 80 100 0 20 40 60 80 100 DAYS&AFTER&HSCT C U MU LA T IVE& IN C ID EN C E& (9 5% &C I)15&mg/kg N&=&88O E&=&5
30&mg/kg N&=&84O E&=&7
15&mg/kg&=&6%&(2(13) 30&mg/kg&=&8%&(4(17)
Gray test&P&=&N.S. Grade&III(IV´&GVHD&by&random
Number of&patients at risk:
15&mg/kg 88 85 83 82 81 79 30&mg/kg 84 78 74 72 71 68
B
0 20 40 60 80 100 0 1 2 3 4 5 YEARS&AFTER&HSCT C U MU LA T IVE& IN C ID EN C E& (9 5% &C I)15&mg/kg N&=&83O E&=&18
30&mg/kg N&=&73O E&=&14
15&mg/kg&=&22%&(14(33) 30&mg/kg&=&19%&(12(31)
Gray test&P&=&N.S. Chronic GVHD&by&random
Number of&patients at risk:
15&mg/kg 83 55 41 25 13 8 30&mg/kg 73 50 34 23 13 4
C
0 20 40 60 80 100 0 1 2 3 4 5 YEARS&AFTER&HSCT C U MU LA T IVE& IN C ID EN C E& (9 5% &C I)15&mg/kg N&=&83O E&=&8
30&mg/kg N&=&73O E&=&4
15&mg/kg&=&10%&(5(19) 30&mg/kg&=&6%&(2(14)
Gray test&P&=&N.S. Extensive chronic GVHD&by&random
Number of&patients at risk:
15&mg/kg 83 65 47 28 16 8
30&mg/kg 73 59 40 28 16 4
D
! Figure!2.!
! Figure!3.! 0 20 40 60 80 100 0 1 2 3 4 5 YEARS.AFTER.HSCT P RO B A B ILI T Y .( 95 %.CI) SURV.=.70%.(62@77) EFS.=.69%.(62@76) TRM.=.14%.(9@21) REL.=.17%.(12@24) SURV N.=.172C E.=.49 EFS N.=.172C E.=.52 REL N.=.172C E.=.29 TRM N.=.172C E.=.23
Number of.patients at risk:
SURV 172 136 98 61 33 12
EFS 172 130 92 59 32 12
REL 172 130 92 59 32 12
TRM 172 130 92 59 32 12
A
transplant@related mortality and.relapseOverall survival,.event@free.survival,0 20 40 60 80 100 0 1 2 3 4 5 YEARS.AFTER.HSCT P RO B A B ILI T Y .( 95 %.CI) 15.mg/kg N.=.88C E.=.20 30.mg/kg N.=.84C E.=.32 15.mg/kg.=.77%.(68@85) 30.mg/kg.=.61%.(50@72) Log@rank P.=.0.0280 Event@free.survival by.random
B
Number of.patients at risk:
15.mg/kg 88 72 52 30 17 8 30.mg/kg 84 59 41 30 16 4 15.mg/kg N.=.88C E.=.8 30.mg/kg N.=.84C E.=.15 30.mg/kg.=.19%.(12@30) 15.mg/kg.=.9%.(5@18) Gray test.P.=.0.0912 0 20 40 60 80 100 0 1 2 3 4 5 YEARS.AFTER.HSCT CUMULA T IV E .INCIDE NCE. (95 %.CI)
C
Number of.patients at risk:
15.mg/kg 88 72 52 30 17 8
30.mg/kg 84 59 41 30 16 4
Transplant@related mortality by.random
0 20 40 60 80 100 0 1 2 3 4 5 YEARS.AFTER.HSCT CUMULA T IV E .INCIDE NCE. (95 %.CI) 15.mg/kg N.=.88C E.=.12 30.mg/kg N.=.84C E.=.17 30.mg/kg.=.20%.(13@31) 15.mg/kg.=.14%.(8@23) Gray test.P.=.N.S.
D
Relapse.by.randomNumber of.patients at risk:
15.mg/kg 88 72 52 30 17 8
! Figure'4.' 0 20 40 60 80 100 0 1 2 3 4 5 YEARS.AFTER.HSCT P ROB A B IL IT Y .( 95 % .CI )
Good prognosis N.=.107H E.=.23 Poor prognosis N.=.65H E.=.29
Good prognosis patients =.78%.(69L86)
Poor prognosis patients =.55%.(43L67) LogLrank P.=.0.0011 EventLfree.survival by.prognosis group
A
Number of.patients at risk:
Good 107 88 52 63 25 8 Poor 65 43 30 17 8 4 0 20 40 60 80 100 0 1 2 3 4 5 YEARS.AFTER.HSCT P RO B A B IL IT Y .( 95 % .CI ) Id..or.1.allelic N.=.124H E.=.31 >.1.allelic N.=.48H E.=.21 Identical or.1.allelic =.75%.(67L82) >.1.allelic =.54%.(40L69) LogLrank P.=.0.212 EventLfree.survival by.HLA.compatibility
B
Number of.patients at risk:
Identical 124 99 72 45 24 10
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