LA MALATTIA MINIMA RESIDUA NELLA

LEUCEMIA ACUTA MIELOIDE

Francesco Lo-Coco Università Tor Vergata, Roma

Progetto di Formazione Nazionale SIE Napoli, 25 Ottobre 2018

Disclosures

Norelevantconflictsofinteresttodeclare ResearchSupport/P.I.

Employee Norelevantconflictsofinteresttodeclare

Norelevantconflictsofinteresttodeclare Stockholder

Consultant Teva,Orsenix

AdvisoryBoard Novar5s,Teva,Orsenix

SpeakersBureau Novar5s,Teva

Other(Specify) Norelevantconflictsofinteresttodeclare

•  Advancedmedianage(70yrs)•  Currenttherapypoorlytoleratedinelderly•  Dismalprognosis(50-80%relapserate)

•  Targetedtherapystronglyneeded

AcuteMyeloidLeukemia

Age CR% ED% Cure%

<15 90 <5 >60

<60 75 <10 45

>60 50 ~20 <15

AMLTreatmentOutcome

Gene=cheterogeneityofAML

Dohner et al, European LeukemiaNet, 2017

HeterogeneityofNKAML

Schlenk et al. N Engl J Med 2008

Muta=onalpaDerninAMLbyWGS

The Cancer Genome Atlas Research Network. N Engl J Med 2013

MolecularComplexityofAMLasdefinedbyNGS:Implica=onsforTreatment

•  Inhibitorsagainstonetargetwillnotsuppressallleukemogenicclones

•  ClearingallmutaUonsincreasesoverallsurvival

Patel, et. al. NEJM 2012

ComprehensiveprognosisassessmentinAML

• Pre-treatment prognostic factors •  Karyotype •  Molecular genetics •  Clinical parameters

• Post-treatment prognostic factors •  MRD detection

•  Flow-cytometry •  Molecular biology

Prognos=cfactorsinAMLandtypeoffailure

Characteristic Unfavorable

category Early death

Death during

remission Resistance/

Relapse Age Older × × Performance status >2 × × Comorbidities increasing × × Previous HD/t-AML Yes × Cytogenetics Unfavorable × Mutational status* FLT3-ITD; KMT2A × WBC count Hyperleucocytosis × × Quality of response MRD+ × *Other mutations have been recently added : RUNX1, ASXL1, TP53

CanMRDimproveoutcomedetermina=oninAML?

Thismodalitymaynotonlycapturedifferencesintreatmentresponsethatreflecttheunderlyingmolecularheterogeneity,butalsointer-

pa=entvariabilityindrugavailabilityandmetabolism,whichmayalsosignificantlyinfluenceoutcome

10 12

10 10

10 8

10 6

10 4

10 2

10 0

Time

No.

of l

euke

mic

cel

ls

Relapse

Cure

CR

MRD

Grimwade, Best Pract Hematol 2012

Hourigan, et al, Nature Reviews Clinical Oncology 2013

Measurable Residual Disease

Differences according to !  disease type !  drug availability !  individual metabolism

NGS: !  Clonal heterogeneity and evolution

dD-PCR

QRT-PCR

Ding L, et al. Nature 2012

ClonalHeterogeneityandClonalEvolu=on

-> one subclone within the founding clone evolved to become the dominant clone at relapse by acquiring additional mutations

Technical Platforms for MRD detection in AML

!  PCR, RT-PCR

!  RQ-PCR

!  Digital PCR

!  NGS

!  Flow cytometry

MRDdetec=oninAML:PCRvsMPFC

PRO CONTRA SENSITIVITY APPLICABILITY

Flow(MPFC)

1.  Fast,lessexpensive

2.  Singlecellanalysis3.  Cellviability

1.  Lessleukemiaspecific2.  Subclonesexpansion3.  Phenotypicshi\4.  Complexanalysis

104-105 Wide(>90%)

PCR/RQ-PCR

1.  HighDNAstability2.  Specific3.  Verylow

backgroundinnormalcells

4.  QuanUtaUon

1.  Timeconsuming,expensive

2.  FalseposiUve3.  RNAinstability(sample

quality)4.  Cellviabilitynot

determinable

104-106 Specificsubgroups:(~60%)

CBF-AML,NPM1mutWT1

Modified from Buccisano, CCO 2009

E2A/PBX1

MLL/AF4

BCR/ABL

TEL/AML1

PML/RARα

SIL/TAL

CBFb/MYH11

RUNX1/ RUNX1T1

Standardiza=onofQ-PCRforfusiontranscripts

Gabert et al., Leukemia 2003

•  NPM1

•  RUNX1

•  ASXL1

•  MLL-PTD

•  CEBPA ?

•  FLT3 ?

•  DNMT3a ?

MoleculartargetsforMRDinAML

•  PML-RARA

•  CBFB-MYH11

•  RUNX1-RUNX1T1

•  MLL-fusion partner

•  DEK-NUP214

•  BCR-ABL

•  Others

•  WT1

•  BAALC

•  ERG

•  MN1

Fusion genes Mutations Overexpression

MRDdetermina=oninCBF-AMLpredictsrelapse

•  Upfront genetics and MRD determination in CBF acute myeloid leukemia. •  198 patients •  RQ-PCR transcripts determination after first consolidation •  a less than 3-log MRD reduction or a level <0.1% was associated with

a higher specific hazard of relapse

Jourdan et al, Blood 2013

RecurrentTransloca=ons:t(8;21)(RUNX1-RUNX1T1)CBF2006trial,n=94pts

Willekens et al, Haematologica 2016

!  MRD>0.001%consideredposiUve!  BMpersistently+at2yrsin8%ofpt!  PBattheendofconsolidaUonpredicUve,anUcipaUngrelapse

by4mos!  Duringfollow-up:100%relapserateinBM>500copies,PB>100copies!  RisingMRDlevelsaccuratelypredict

relapse(AML-MRC15,LiuYin,Blood2012)

CIR OS

BM

PB

Kronke et al, JCO 2009

RQ-PCRforNPM1mut:impactofMRDonsurvival

NPM1muta=onsMRDbyRQ-PCRinPBa\er2CyclesofCHT

ThepresenceofMRDbyquan=ta=onofNPM1-mutatedtranscriptsprovidedpowerfulprognos=cinforma=onindependentofotherriskfactors.

Ivey A et al., NEJM 2016

Overall Survival Cumulative incidence of relapse

MRDasaPredictorofOutcomeinDevelopmentandValida=onCohorts

Ivey A et al, NEJM 2016

NPM1mutisastablemarker,DNMT3AmutandFLT3ITDarenot

Ottone et al, Am J Hematol 2018

1

10

100

1000

10000

100000

1 2 3 4

NPM1

DNMT3A

• Texto1

! Texto21

10

100

1000

10000

100000

Esordio Induz ione P ostc ons

NPM1

DNMT3A

1

10

100

1000

10000

100000

E sor dio Induzione Postcons

NPM1

DNMT 3A

UPN_243

UPN_277

UPN_82

0,1

1

10

100

1000

10000

100000

Esordio Induzione Post cons Relapse

NPM1

DNMT3A

UPN_43

Relapsed Relapsed

ParalelMRDassessmentofDNMT3AandNPM1

ClonalHematopoiesisandBlood-CancerRiskInferredfromBloodDNASequence

Giulio Genovese, Ph.D., Anna K. Kähler, Ph.D., Robert E. Handsaker, B.S., Johan Lindberg, Ph.D., Samuel A. Rose, B.S., Samuel F. Bakhoum, M.D., Ph.D., Kimberly

Chambert, M.S., Eran Mick, B.S., Benjamin M. Neale, Ph.D., Menachem Fromer, Ph.D., Shaun M. Purcell, Ph.D., Oscar Svantesson, M.S., Mikael

Landén, Ph.D., Martin Höglund, M.D., Ph.D., Sören Lehmann, M.D., Ph.D., Stacey B. Gabriel, Ph.D., Jennifer L. Moran, Ph.D., Eric S. Lander, Ph.D., Patrick F.

Sullivan, M.D., Pamela Sklar, M.D., Ph.D., Henrik Grönberg, M.D., Ph.D., Christina M. Hultman, Ph.D., and Steven A. McCarroll, Ph.D.

N Engl J Med Volume 371(26):2477-2487

December 25, 2014

Weanalyzeddatafromwhole-exomesequencingofDNAinperipheral-bloodcellsfrom12,380persons,unselectedforcancerorhematologicphenotypes.

Clonalhematopoiesiswithsoma=cmuta=onsin:

•  DNMT3A,ASXL1,andTET2,genesimplicatedinhematologiccancers.

•  Riskofhematologiccancerincreasedintheseindividuals

Genovese G, Jaiswal G, et al. N Engl J Med 2014

NGSandClonalhematopoiesis(CHIP)

UPN Karyotype FLT3-ITD NPM1 Karyotype FLT3-ITD NPM1 routine RT-PCR

routine RT-PCR

104 46,XY negative positive 46,XY positive positive 213 46,XX negative positive 46,XX positive positive 241 46,XX negative positive 46,XX positive positive

DIAGNOSIS RELAPSE

FLT3-ITD patient-specific RQ-PCR

UPN FLT3-ITD FLT3-ITD/ABLx104 routine RT-PCR RQ-PCR

104 negative 82.00 213 negative 37.98 241 negative 1719.21

Exon 14 Exon 15 ITD

Primer forward Patients specific

Primer reverse

Probe

Clonal evolution of FLT3-ITD mutations in AML

Iden=fica=onofan=gensassociatedwithFLT3-ITD(MPFCanalysisoftotalBMcellsfromFLT3-ITDmutpts)

*Pearson's chi-squared test

Antigen FLT3 mutated FLT3 wt p value* (% of pts/tot) (% of pts/tot)

CD99 73% (27/37) 48% (44/91) 0.02

CD123 100% (38/38) 83% (75/90) 0.02

CD11b 92% (35/38) 66% (59/90) 0.004

CD7 68% (26/38) 46% (41/89) 0.003

CD25 87% (33/38) 40% (36/90) 0.000003

Battistini, Ottone et al, Clin Cancer Res 2015

Athresholdof11.7%cellsstainingposi=veforthisan=genicprofilepredictsFLT3

mutwithspecificityandsensi=vity>90%

ReceiverOpera=ngCharacteris=c(ROC)curve

analysis

Battistini, Ottone et al, Clin Cancer Res 2015

ITD-mutations Exons 14/15

Suitability of alterations for MRD detection

Preleukemic Leukemic Post-Onset Drivers

FLT3-ITD FLT3-TKD RAS PTPN11 KIT

DNMT3A TET2 IDH1/2 SF3B1

NPM1 Translocations RUNX1 CEBPA

Sensitive Potentially unspecific

Specific Lost in 10-90%

Sensitive specific

by courtesy of C. Thiede

Are MRD studies still valuable and cost effective in APL ?

J Clin Oncol 2003

MRDtodirectpre-emp=vetreatmentinAPL

Lo-Coco et al. Blood, 1999 Esteve et al. Leukemia, 2006

0 1 2 3 4 5 6 7 8

Years

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Pro

babi

lity

Hematological relapse (n=33; 24 events)

Molecular failure (n=16; 5 events)

p=0.008

0 1 2 3 4 5 6 7 8

Years

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Pro

babi

lity

Hematological relapse (n=33; 24 events)

Molecular failure (n=16; 5 events)

p=0.008

Grimwade et al, JCO 2009

Pre-emptive ATO therapy

MRCAML-15TrialforNewlyDiagnosedAPL

Cicconietal,Leukemia2016

Diagnosis

Post-induc=on

PostIIIconsolida=on

Kine=csofPML/RARaclearanceinAPL0406

PML/RARA +ve patients by RQ-PCR

PrimaryEndpoint:Rateofmolecularcompleteremission

SecondaryEndpoints: 2yearEFS,Safety,QoL

ORH-2014 in APL: Pivotal Registration Study A Randomized, Multi-center, Phase III Study to Compare

ORH-2014 and i.v. ATO in Low-Intermediate Risk APL

Agreement with FDA on study design, population, and endpoints

ATRA-CHTATRA-ATOWT1FLT3 -ITDDNMT3AETV6FLT3-TKDTET2ASXL1JAK2RUNX1SRSF2TP53U2AF1PMLRARA

single mutation

wild-type

multiple mutations

not analysed

Muta=onalpaDerninrelapsedAPL

PMLandRARAmuta=onsatrelapse

B2 LBD PML RARA

A216V (2)

A216T

(1)

L224I

(1)

T291A (1)

T285I

(1)

C235F (1)

F286del (1)

R272Q (1)

I222_D223insE (1)

W225C (1)

S287L

(1)

B2 PML

A216V (2)

A216T

(1)

E224G (1)

L220P (1)

L290V (1)

Madan et al, Leukemia 2016

Iaccarino et al, BJH 2016 Zhu et al, NEJM 2014

Lehmann-Che et al, NEJM 2014 Goto et al, Blood 2011

Earlyandsensi=vedetec=onofPMLA216VbyddPCR

posiUvebyddPCR

posiUvebySanger

negaUvebyddPCR

negaUvebySanger

41

• WT1overexpressedin>90%AMLs

• Suitable“universal”MRDmarkerforAML• ComparisonofsensiUvity&specifiUyof9differentRQ-PCRassaysby11Europeanlabs(LeukemiaNet)

Op=mizedRQ-PCRofWT1(Cillonietal,JCO2009)

1

10

100

1000

10000

100000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Normal range (<20 copies/104 copies of ABL)

WT1levelinPBofAMLpa=entsattheendoftheinduc=ontherapypredictsforrelapse

Cilloni et al, Haematologica 2008

GlobalimmunophenotypicapproachtoAML

Multiple staining at diagnosis

Identification of “leukemia-associated” phenotypes

Definition of a patient’s “immunologic fingerprint”

Immunologic fingerprint used during follow-up

Venditti, Blood 2000; Venditti, Leukemia 2003; Buccisano, Leukemia 2006; Maurillo, JCO 2008; Buccisano, Blood 2010

Leukemia-associatedImmunophenotypes

LAIPtype Examples

Asynchronousan=genexpression

CD34+CD14+CD34+CD15+CD33+

Lackofan=genexpressionCD34+CD13-CD33+Hla-DR-CD34+CD13+

Cross-lineagean=genexpression

CD34+CD13+CD19+

CD34+CD13+CD2+

An=genoverexpression

HLA-DR++CD34+++CD13++

CD64++CD4++CD45++

Average3LAIPperpa=ent

• 85%ofpediatricAML

• 80-95%ofadultAML

Pitfalls: immunophenotypic shifts, complex analysis

Laboratory diagnostics of hematological malignancies has three major applications:

•  Diagnosis •  Prognostic (sub)classification •  Evaluation of treatment effectiveness via detection of MRD

MPFC

MRDAssessmentbyFlowCytometryinAML

Terwijn M et al, JCO 2013

AML02:Aprospec=ve,mul=centerstudyofrisk/MRD-directedtherapy

•  LevelsofMRDwereusedtoallocateGOandtodeterminetheUmingofinducUon2

•  BothMRDandgeneUcabnormaliUesatdiagnosiswereusedtodeterminethefinalriskclassificaUon

SR (with donor)

HR

Enrollment, Randomization, Initial Risk Assignment

H-ADE ADE ± GO

Final Risk Assignment

SCT

CI CII CIII

ADE

MRD MRD

LR SR (w/o donor)

SR (with donor)

HR

Rubnitz et al, Lancet Oncology 2010

AML02:Mainconclusions

71% ± 4%OS

63% ± 4% EFS

00.10.20.30.40.50.60.70.80.9

1

0 1 2 3 4 5 6 7

19% ± 3%

Years on Study

9% ± 2%

Relapse

Death

•  Risk- and MRD-adapted therapy resulted in 71% OS •  Day 22 MRD >1% significantly associated with worse OS, EFS, CIR

N=230 CR rate 94% MDR+ 37% (Ind1) MDR+ 20% (Ind2) St. Jude AML Trials

(Rubnitz et al, Lancet Oncology 2010

IntegratedRisk-Score(TorVergata)

Low-Risk High-Risk

Favorable K / MRD-

Int K / MRD-

Adverse K FLT3+ Good K / MRD+

Int K / MRD+

Buccisano et. al. Blood 2010

GIMEMAAML1310:astudyofrisk-adaptedandMRD-directedtherapyforadultAML

Low-risk: CBF/Kitwt; NPM1+/FLT3- Int-risk: all others High-risk: Adverse K; FLT3+

Diagnosis

Low-risk

Int-risk

High-risk

MRD-

MRD+

MRD marker LAIP Risk stratif

CG, molecular MRD assess

LAIP

FLA-Ida salvage No CR CR

CR In

duct

ion

(1 o

r 2 c

ours

es)

Con

solid

atio

n 1

autoSCT

alloSCT

alloSCT: MRD, MUD, UCB, HRD

GIMEMA AML1310

342 post consolidation

177 candidates to AutoSCT

110 (62%) received AutoSCT

165 candidates to AlloSCT

110 (67%) received AlloSCT

81 not in CR post induction

23 (CR post salvage) candidates to

AlloSCT

16 (70%) received AlloSCT

AML1310: results

GIMEMA AML1310

52

AML1310: intermediate-risk OS and DFS by MRD status

Venditti A et al, submitted

Responseto

therapy

CRMRD-asanewresponsecriterion

MolecularMRD:ELN2017recommenda=onsforAML

Transcripts

-NPM1-RUNX1-RUNX1T1-CBFB-MYH11-PML-RARA

When

-aoer2cyclesofstandardinduc=on/consolida=on-aoertheendoftreatment-every3monthsfor24monthsaoerendoftreatment

ModifiedfromSchuurhuisGJ,etalELN-MRDWP,Blood2018

Source

-inPBandBMorPBevery4-6weeks

!  PBinsteadofBM?Inpar=cularatprolongedfollow-up?!  Absolutecopynumbersshouldalsobereported

Defini=onoffailure

-FailuretoachieveMRD-nega=veCR(molecularresistance)-RisingMRDlevels(>1Log)duringoraoertherapy(molecularprogression)-Molecularrelapse

Clinical Impact of MRD in AML is Increasingly Recognized

Anetworkoflaboratorieslicencedforthe

moleculardiagnosisofAML

Aimingatdefiningandspreadingdiagnos=c

standardsforAML,includingmoleculargene=cs,

cytogene=cs,andimmunophenotype

Researchprojects

LabnetAML

Diagnos=caLAM:PannelloBase

Cario=po •  G-banding

QRT-PCRQ-LAMP

•  PML/RARA(bcr1-2ebcr3)

•  BCR/ABL(p190ep210)

•  RUNX1/RUNX1T1•  CBFb/MYH11

PCR-ECPCR-RLFP

•  FLT3-ITD•  FLT3-D835eI836•  NPM1

Tempi

15gg

24ore

72-96ore

72-96ore

ProssimiStep:PannelloAvanzato

! 

Risulta=StandardizzazioneMRD

BCR-ABL p190

BCR-ABL p210 b3a2

PML-RARA bcr1

PML-RARA bcr3

CBFb/MYH11 type A

RUNX1/RUNX1T1

NPM1

Undiluited

10-2

10-3

10-1

Conclusive remarks (I)

•  NGS may provide in the future more comprehensive data on clonal heterogeneity / evolution and overall effects of treatments

•  MRD assessment by MPFC / RQ-PCR allows better strategies for delivering treatment in AML

•  RQ-PCR provides valuable prognostic information in CBF and NPM1 +ve AML

Conclusive remarks (II)

•  MRD –ve remission as a new treatment objective in AML to accelerate drug approval

•  Importance of reference laboratories

TorVergataTeam

LauraCicconi MariadomenicaDivonaTizianaOpone SerenaLavorgnaEmilianoFabiani GiuliaFalconiNélidaI.Noguera ValenUnaAlfonsoSerenaTravaglini LiciaIaccarino

AdrianoVendir FrancescoBuccisano

MariaTeresaVoso