Mutations des Leucémies Aiguës Myéloïdes Intérêts cliniques

Christian Récher

DES hématologie

16 Janvier 2015

De la morphologie au séquençage

Classification

Morphologique

FAB

Cytogénétique Moléculaire

Mutations FLT3-ITD/KIT

NPM1 CEBPA

DNMT3A/IDH1/2 WT1/MLL-

PTDRAS/AML1 PHF6/TET2/ASXL1

1985-2000 1997-2010 1976

Séquençage du génome

Impact des mutations au sein des sous-groupes cytogénétiques

Les trois qualités d’un marqueur moléculaire

* Impact pronostique clair * Impact thérapeutique * Marqueur de maladie résiduelle

CBF

CN et Intermédiaire

CHU de Toulouse 2000-2009: 643 patients CTx intensive

Mutations de FLT3

o ITD: • 20-25% des LAM • LAM CN: 25-40 % • Taille variable: 3-400 bp • Domaine juxta membranaire • 30% intègre le domaine TKD1

• Feuillet β1 TKD1

o TKD: • 5-10% • D835Y le plus souvent • Résistance aux ITK

o Mutation ponctuelle du domaine JM*

• <1%

Membrane plasmique

TK1

TK2

ITD

D835Y

*

*

RTK de classe III FMS c-KIT

PDGFRA/B

FLT3

FLT3-ITD

• De novo

• Hyperleucocytose

• ↑ % de blastes sanguins et médullaires

• Caryotype normal (65-70%)

• Association aux mutations de NPM1

• Rechute:

– perte de la mutation ou émergence d’un autre mutant FLT3-ITD

– 88% des patients présentent la même mutation qu’au diagnostic

– Ratio ITD/wt ↑: perte de l’allèle sauvage, disomie uniparentale (UPD13q)

Pronostic

Kottaridis, Blood 2001

Pas d’impact sur la RC

Risque de rechute précoce ++ Time (years)

Ov

era

ll S

urv

iva

l

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0.0

0.2

0.4

0.6

0.8

1.0

FLT3 wtFLT3-ITD

Log-rank test: p-value = 0.0005

GOELAMS-LAM 2006 IR

FLT3-ITD et traitement de post-rémission Intérêt de l’allogreffe?

Gale, Blood 2005; Bornhauser, Blood 2007; Brunet, Current Op Oncol, 2013

≈30% de rechute post-allo

FLT3-ITD et post-rémission Intérêt de l’allogreffe?

Schlenk F, NEJM 2008

Hétérogénéité des mutations FLT3-ITD

Taille Ratio ITD/WT

Nombre de mutations/patients Localisation de l’insertion

Gale R, Blood 2008; Kayser S, Blood 2009

• 1 mutant (73%) • 2 mutants (21%) • 3 mutants (5%) • 4 mutants (1%)

Ratio ITD/wt

Rechute

Gale R, Blood, 2008

Influence du ratio ITD/wt sur la réponse à l’induction

Introduction rapide d’ITK à l’induction ?

* Taille et nombre d’ITD également associés à la RC

Schlenk, Blood 2014

Influence du ratio ITD/wt sur la survie Expérience du AMLSG

Patients non allogreffés

Schlenk, Blood 2014

Ratio ITD/wt et allogreffe AMLSG

ITD/wt >0,51 ITD/wt <0,51

Allogreffe à réserver pour les patients avec ratio >0.5?

Schlenk, Blood 2014

Ratio ITD/wt et allogreffe Expérience du MRC

Linch, Blood 2014

FLT3-ITD et allogreffe

• Plutôt oui ……mais

– Impact du ratio: seuil 0.5? Autre?

– 30 % de rechute (ITK post-greffe? Sorafenib, Metzelder, Leukemia 2012)

– Impact de NPM1

– Impact de la maladie résiduelle

• Choix du donneur

• Essai BIG-1: pas d’indication en RC1 si ratio <0.10 et mutation NPM1 et MRD2 <1%

Site d’insertion de l’ITD (TKD1)

Kayser S, Blood, 2009

Site d’insertion de l’ITD (TKD1)

Inhibiteurs de FLT3

Zarrinkar P, Blood 2010

Inhibiteurs de FLT3 en monothérapie

Kindler T, Blood 2010

PKC-412 (midostaurin) Phase III internationale CALGB 10603 (RATIFY)

Arm 1: Induction therapy *Midostaurin 50mg oral twice daily on days 8-21 *Cytarabine 200mg/m2 IV on days 1-7 *Daunorubicin 60mg/m2 IV on days 1-3. Consolidation therapy *Midostaurin 50mg oral, twice daily on days 8-21 *Cytarabine 3,000mg/m2 IV over 3 hours every 12 hours on days 1, 3, and 5. Both as part of a 28 day cycle for up to 4 cycles. Continuation therapy *Midostaurin 50mg oral, twice daily on days 1-14 as part of a 28 day cycle. Arm 2: Placebo

Inhibiteurs de FLT3: AC220: le plus prometteur?

Lestaurtinib (CEP-701)

Midostaurin (PKC-412)

Sorafenib

Quizartinib (AC220)

Pratz et al. Blood 2010;115(7):1425-32

ui artini IC50 cellular

assays IC50 nM

(Medium) IC50 nM

(Plasma)

Lestaurtinib 2 700

Midostaurin 12 1,000

Sorafinib 0.9 265

Quizartinib 0.6 18

AC220 (quizartinib)

Développement clinique

Phase Study Subject N (dose range)

Phase 1 CP0001: A first-in-human study in relapsed/refractory AML 76 (12 - 450 mg)

Phase 2 Study AC220-002: A monotherapy study in relapsed/refractory AML

333 (90, 135 & 200 mg)

Phase 2b

Study 2689-CL-2004: A randomized study in relapsed/refractory FLT3-ITD(+) AML

76 (30 & 60 mg)

Phase 1 2689-CL-0011: AC220 maintenance therapy in subjects post-allogeneic hematopoietic stem cell transplant (HSCT)

13 (40 & 60 mg)

Phase 1 Study 2689-CL-005: AC220 in combination with chemotherapy in newly diagnosed AML*

18 (40 & 60 mg)

Phase 2 (AC220-002)

FLT-ITD(+) (n=136)

FLT3-ITD(+)

(n=112)

FLT3-ITD(-)

(n=45)

Cohort 1 (n=157)

≥60 years, 1st relapse within 1 year, or refractory to 1st line treatment

Patients with AML or AML secondary to MDS (n=333)

(Included FLT3-ITD[+] and [-] patients with a 10% allelic ratio cutoff by a central laboratory)

Cohort 2 (n=176)

≥18 years, relapsed after, or refractory to 2nd line treatment or after HSCT

FLT3-ITD(-)

(n=40)

• A total of 333 patients enrolled in the Phase 2 • Initial dose 200 mg daily but reduced after 17 subjects enrolled due to QT prolongation; subsequently females received

90 mg and males 135 mg • Primary endpoint: Composite CR (CRc = CR + CRp + CRi)

Critères de réponse

• Complete Remission (CR) and Complete Remission with Incomplete Platelet Recovery (CRp): per IWG criteria (Cheson et al., Journal of Clinical Oncol. 2003 21: 4642 – 4649)

• Complete Remission with Incomplete Hematological Recovery (CRi): Same as CR except

for incomplete hematological recovery with residual neutropenia <1 × 109/L and/or platelet

recovery (<100 × 109/L). RBC and platelet transfusion independence is not required.

(Modified from IWG criteria)

• Partial Remission (PR): same criteria for CRi but only require a decrease of at least 50% in

bone marrow blasts with the total marrow blasts between 5% and 25% inclusive. (Modified

from IWG criteria)

Réponses FLT3-ITD+

Response Cohort 1 N = 112

%

Cohort 2 N = 136

%

CRc (CR+CRi+CRp) 56 46

CR 3 4

CRp 4 1

CRi 50 40

PR 21 28

CRc + PR 77 74

Median Duration of CRc (weeks) (95% CI)

12.1 (6.3, 15.7) 11.3 ( 8.1, 16.3)

Median Time to CRc (weeks) 4.3 4.3

Median OS (weeks) (95% CI) 25.4 (21.3, 29.7) 24.0 (21.1, 27.1)

Quizartinib et différenciation

Saxauer, Blood 2012

Taux de réponses par sous-groupes

CRc (%) NR (%) Best Response to Prior Therapy

Prior CR (N=172) 50 18

No prior CR (N=74) 50 13

Prior Allo-HSCT

No prior HSCT (N=207) 50 20

Prior HSCT (N=41) 51 7

Cytogenetic Risk Group*

Good (N=1) 100 (1/1) 0

Intermediate (N=108) 56 14

Poor (N=23) 39 26

Unknown (N=116) 47 20

ITD Ratio

>10 – 25% (N=54) 41 19

>25 – 50% (N=116) 47 21

>50% (N=78) 62 13

Réponses et ratio ITD

ITD “Positive” (ITD >10%)

ITD “Negative” (ITD ≤ 10%)

No Detectable ITD (=0)

Detectable ITD ≤10%

Number of Subjects 248 84 58 26

CRc % 50 33 28 46

PR % 25 12 10 15

Overall Survival (weeks) 24.6 22.7 19.6 25.1

Quizartinib et allogreffe

Facteurs affectant la survie

Hazard Ratio 95% CI (Wald) p-value

Age

< 60 years (n=102) 1

≥ 60 years (n=139) 1.180 (0.878, 1.586) 0.2712

Baseline Bone Marrow *

Blast Count (%) (n=241) 1.008* (1.002, 1.014) 0.0122

Prior CRc (Any)

Yes (n=165) 1

No (n=76) 1.175 (0.573, 2.409) 0.6605

Duration of CR1

≥ 26 weeks (n=64) 1

< 26 weeks (n=90) 1.236 (0.874, 1.750) 0.2309

No response (n=87) 0.960 (0.463, 1.991) 0.9122

FLT3-ITD Allele Burden

>10% to <25% (n=53) 1

≥ 25% to ≤ 50% (n=116) 1.333 (0.901, 1.971) 0.1500

> 50% (n=72) 1.934 (1.263, 2.962) 0.0024

Effets indésirables

Adverse Event Cohort 1 (N = 90) %

Cohort 2 (N = 100) %

Any event 100 100

Nausea 56 55

Diarrhea 41 41

Vomiting 41 41

Pyrexia 39 33

Fatigue 37 20

Anemia 34 18

Febrile Neutropenia 33 41

Thrombocytopenia 32 27

QT prolongation 30 25

Asthenia 27 18

Résistance au quizartinib Acquisition de nouvelles mutations dans le domaine TK (parfois polyclonales)

Smith, Nature 2012

Subject New FLT3

Mutation

Weeks on

Study

1 F691L 19

2 D835Y 6

3 D835V 23

4 D835Y 19

5 F691L 20

6 D835Y 12

7 D835Y 8

8 D835V,F691L 11

Nouveaux ITK pour les résistances au quizartinib

Shah, BJH 2013

NPM1

Falini NEJM 2005; Blood 2006

Caractéristiques des LAM NPM1c+

Mutations spécifiques des LAM, de novo le + svt

Mutations stables (rechute)

Mutations associées: FLT3-ITD (40%); IDH1/2; DNMT3A

Signature moléculaire unique (CD34 ↓ /gènes HOX ↑)

Profil microARN distinct

Profil de méthylation

Adultes (25-30% des cas) > enfant (6-8%)

Incidence plus élevée chez la femme

Association avec un caryotype normal (85% des cas)

Anomalies chromosomiques associées dans 15% des cas (+8; del9q; +4)

Tous types FAB mais plus souvent M4/M5 (M5b: 90%)

Hyperleucocytose

Dysplasie multilignée fréquente

Négativité du CD34 (90-95% des cas)

Taux de réponse complète élevé

Détection de NPM1c

Sarcomes myéloïdes

15% des sarcomes myéloïdes ont une mutation NPM1c

NPM1 et rechutes tardives (>5 ans)

Meloni, Haematologica 2009

CHU de Toulouse 2000-2013 1446 patients 924 CTx intensive 197 NPM1+ 70 rechutes 7 après 5 ans

Impact pronostique

Dohner Blood 2005 Schnittger Blood 2005 Verhaak Blood 2005 Schlenk NEJM 2008

Méta-analyse

Liu, Mol Clin Oncol 2014

Réponse

DFS

OS

NPM1 mitige le mauvais pronostic de FLT3-ITD …et vice versa

Gale R,Blood 2008

Implications pour la décision de greffe allogénique

L’allogreffe est réservée en RC1 aux patients qui n’ont pas le génotype FLT3wt/NPM1c

Schlenk F, NEJM 2008

Sujets âgés

Buchner, JCO 2009

Anomalies cytogénétiques et NPM1

Micol Blood 2009; Haferlach Blood 2009

Un rôle pour l’ATRA dans les LAM NPM1+/FLT3-ITD-? AMLSG

Schlenk et al, Haematologica, 2009

FLT3-ITD-/NPM1c avec ATRA *

NPM1mut n=129

NPM1mut + ATRA n=125

P<0.0001

NPM1WT + ATRA n=306 NPM1WT n=308

Time (years)

Even

t-fr

ee S

urv

ival

(%

)

0 1 2 3 4

0

20

40

60

80

100

n=868

Ida/AraC/VP16 + ATRA 45mg/m2 from day 6 to 8, and 15mg/m2 from day 9 to 21

Schlenk, ASH 2011

Sujets âgés

Sujets jeunes

Un rôle pour l’ATRA dans les LAM NPM1+/FLT3-ITD-? MRC

Burnett, Blood, 2010

ATRA 45 mg/m² D1-D60

Allogreffe et NPM1 SAL-AML 2003

NPM1+

CN NPM1+/FLT3-ITD-

Rollig, JCO 2014

Allogreffe et NPM1 SAL-AML 2003

Maladie résiduelle AMLSG

Krönke J, JCO 2010

245 LAM-NPM1c 18-60 ans Essais AMLSG 07-04 et AML HD98A Suivi en RT-PCR quantitative (moelle) MRD- (>3 log)

Post induction (2 cycles)

Fin de traitement

Maladie résiduelle MRC

ASH 2014: Abst#0070 Essai UK NCRI AML17: 341 patients avec mutation NPM1 MRD dans le sang après deux cures d’induction

MRD NPM1 indépendante de FLT3-ITD et DNMT3A

Mutations de CEBPA

Pabst ,Nature Genet 2001; Preudhomme, Blood, 2002; Green, JCO 2010

N-term. nonsense mutation C-term. missense mutation

* Monoallélique: 30-40% * Biallélique: 60-70% * 5-10% des LAM à caryotype normal

Impact pronostique

Preudhomme, Blood, 2002; Fröhling, JCO 2004, Schlenk, NEJM 2008. Wouters, Blood 2009

Mutations bi-alléliques: CEBPAdm

une entité clinico-biologique

• Hb +

• Plaquettes –

• FAB M1/M2

Dufour JCO 2009 ;Taskesen E, Blood 2011

Impact pronostique des CEBPAdm

Dufour JCO 2009; Wouters, Blood 2009, Taskesen E, Blood 2011

CEBPAdm: méta-analyse

Hong-Ying Li, Eur Journal of Haematol, Sept 2014

Le pronostic des CEBPAsm est lié aux mutations associées

Dufour JCO 2009; Renneville Blood, 2009 Taskesen E, Blood 2011

Pas d’impact du caryotype dans les CEBPAdm

Schlenk Blood 2013

Impact de l’auto et de l’allogreffe dans les CEBPAdm

(HOVON-AMLSG)

Schlenk Blood 2013

Taux élevé de RC2 et survie prolongée pour les patients en RC2

Voie Sox4: une cible pour les CEBPAdm

Mutations IDH1/IDH2

Isocitrate dehydrogenase (IDH) is a critical enzyme of the citric acid cycle

IDH mutations occur in a spectrum of solid and hematologic tumors1

IDH1 mutations: 6–10% of AML and 3% of MDS

IDH2 mutations: 9–13% of AML and 3–6% of MDS

IDH1/2 mutations confer a gain-of-function:

production of 2-hydroxyglutarate (2-HG)2

2-HG drives multiple oncogenic processes: increased histone and DNA methylation

impaired cellular differentiation

Clinical proof of concept established in hematologic cancers:

AG-221, IDH2m inhibitor (AACR 2014)

AG-120, IDH1m inhibitor (EORTC-NCI-AACR 2014)

Tumor cell

Mitochondrion

KG

IDH2

Isocitrate

Citrate

Citrate

Isocitrate

KG

IDH1

Epigenetic changes

Impaired cellular

differentiation

IDH2

mutant 2-HG

IDH1

mutant

NADPH

NADPH

Valeur pronostique dans les NPM1+/FLT3-ITD- ?

AMLSG Paschka, JCO 2010

CALGB Marcucci, JCO 2009

HOVON Abbas, Blood 2010

ECOG Patel, NEJM 2012

L’oncométabolite 2-HG prédit la présence d’une mutation IDH1/2 et la MRD

Ward, Cancer Cell 2010

Di Nardo, Blood 2013

L’oncométabolite 2-HG corrèle avec la réponse à la CTx

Janin, JCO 2013

67 67

Ongoing, first-in-human, dose escalation study [NCT01915498]:

AG-221: First-in-class, oral, potent, reversible, selective inhibitor of mutated IDH2

IDH2 mutation-positive relapsed or refractory AML, MDS, or untreated AML

AG-221 in continuous oral dosing QD or BID daily, 28-day cycles

Key outcome measures:

Assess safety and tolerability, including DLT

Determine MTD and recommended phase 2 dose

Explore PK and PD (2-HG)

Characterize differentiation effect and preliminary clinical activity

Response assessed by investigator using IWG AML and MDS criteria

Update since EHA presentation (data cut May 23, 2014):

Treated 38 additional patients, 73 total

Explored 4 additional dose cohorts (highest cumulative daily dose of 300 mg)

Initiated expansion cohorts at 100 mg PO QD in October 2014

In-parallel dose escalation continues in QD regimen

AG-221 phase 1 study design and status

MTD = maximum tolerated dose; DLT = dose limiting toxicities; PK = pharmacokinetics; PD = pharmacodynamics

68 68

Study Status: Dose Escalation

Activated in September 2013

As of 1 October 2014, 73 patients treated in 10 dose cohorts

Dose Escalation

Cohorts Treated

On Therapy

Discontinued

30 mg BID 7 1 6

50 mg BID 7 3 4

75 mg BID 7 3 4

100 mg BID 8 3 5

150 mg BID 5 4 1

50 mg QD 7 5 2

75 mg QD 5 3 2

100 mg QD 15 7 8

150 mg QD 6 4 2

200 mg QD 6 5 1

Total 73 38 35

69 69

Demographic characteristics

ITT population (n=73)

Age in years, median (range) 67 (33–90)

Diagnosis, n*

RR AML 55

MDS 6

Untreated AML 5

Other (5 CMML, 1 Myeloid sarcoma) 6

Men/women, n 39/34

ECOG performance status, n**

0 18 (25%)

1 39 (53%)

2 14 (19%)

Number of prior regimens, median (range)

RR AML 2 (1‒11)

Prior BMT, n 13 (18%)

IDH2 mutations, n***

R140 54 (74%)

R172 13 (18%)

Abnormal cytogenetics, n 21 (29%)

*Missing for 1 subject; **Missing for 2 subjects; ***Missing for 6 subjects

70

Excellent exposure to AG-221

High accumulation after multiple doses

Mean plasma half-life >40 hours

Sustained plasma 2-HG inhibition after

multiple doses

– Up to 98% in IDH2-R140Q subjects

PK/PD analysis

Excluded 1 patient from analysis:

very low pretreatment 2-HG level, no 2-HG inhibition

Plasma 2-HG inhibition in

IDH2-R140Q subjects

Plasma AG-221 exposure

THIS SLIDE NEEDS TO BE Q/C’ed

71 71

Best overall response by cumulative daily dose*

≤75 mg (n=9)

100 mg (n=14)

≥150 mg (n=22)

Total

(N=45 efficacy evaluable*)

CR Complete response

3 3 0 6

CRp CR, incomplete platelet recovery

1 1 2 4

mCR Marrow CR

0 0 1 1

CRi CR, incomplete hematologic recovery

0 2 2 4

PR Partial response

0 3 7 10

SD Stable disease

5 3 9 17

PD Progressive disease

0 1 1 2

NE Not evaluable

0 1 0 1

Overall Response Rate*** 4/9 (44%) 9/14

(64%)

12/22 (54%) 25/45 (56%) 95% CI (XX, XX)

* Includes patients with a Day 28 response assessment as of October 1, 2014. Excludes 12 on-going

patients with day 28 not yet available and 16 patients off study without evaluable day 28 assessment.

** ORR = CR + CRp + mCR + CRi + PRi

72 72

Duration of treatment & best overall response

All efficacy evaluable patients on study treatment as of 1 October 2014

18 Patients on study ≥ 4 months, 13 on-going

6 Patients on study ≥ 6.5 months, 4 on-going (1 CR, 3 PR, 2 SD)

5 Responders went on to transplant

N = 45 efficacy evaluable patients

73 73

Duration of treatment and best overall response

All responding patients on study treatment as of 1 October 2014 *censored at last response assessment

Duration of Response: 3-month 90%

N = 25 responders

74 74

AG-221, a potent, selective, oral inhibitor of mutated IDH2, is well tolerated in

patients with advanced hematologic malignancies

2-HG inhibition of >90% in patients with an IDH2-R140 or -R172 mutation

Consistent with preclinical models, AG-221 treatment leads to 2-HG lowering and

differentiation of leukemic blast cells, ultimately leading to objective responses

Overall response rate of 25/45 (56%) including 6 complete remissions

– Responses are durable, with a duration on study as long as 8+ months

Dose expansion at 100 mg PO QD in 4 cohorts of 25 patients in AML and advanced

hematologic malignancies each initiated in October 2014

These data provide continued validation of mutant IDH2 as a therapeutic cancer

target

Conclusions

DNMT3A

Impact pronostique mal défini

DNMT3A R882 vs autres (Marcucci, JCO 2012;

Gaidzik, Blood 2013)

Faux-sens vs autres (Gale, ASH 2014)

Impact des fortes doses d’anthracyclines

Patel, NEJM 2012

LaRochelle, Oncotarget 2011

Classification cytogénétique et moléculaire

Patel et al, NEJM 2012; Meyer, Lancet Oncol 2014

Classification moléculaire

393 pts avec caryotype normal et mutations NPM1

FLT3-ITD- DNMT3A non-R882- IDH1R132- 41%

FLT3-ITD+ 41%

FLT3-ITD- DNMT3A non-R882+ 8%

FLT3-ITD- IDH1R132+ 10%

Peterlin, Haematologica 2014

De la morphologie au séquençage en vie réelle

Classification

Morphologique

FAB 2 heures

Cytogénétique

3-5 jours ou >

Moléculaire

2j et >

1985-2000 1997-2010 1976