The Cancer Genome Atlas Research Network (NEJM, … Settembre/PDF/6... · The Cancer Genome Atlas...

200 The Cancer Genome Atlas Research Network

(NEJM, 368:2029-2074, 2013)

200 AML genome sequenced: 23

recurrently mutated genes in AML

Most common mutations in AML:

NPM1, FLT3, followed by DNMT3A

Other mutations occur at lower

frequency but may play a role

200 AML genomes

Frequency of NPM1, DNMT3A and FLT3-ITD mutations in

232 AML-NK (from Perugia and North Italian AML group)

NPM1- mutated 126/232 (54%)

DNMT3A-mutated 88/232 (38%)

FLT3-ITD 60/232 (26%)

IDH1/IDH2 (other studies) (15-20%)

Features NPM1* FLT3-ITD DNMT3A IDH1/2

Specificity AML AML, MDS AML, MDS, AML, MDS,

ETP-ALL MPN, PTCL MPN, gliomas

T-ALL

GEP Distinct No No No

Micro-RNA Distinct No No No**

Clonal hemopoiesis No No Yes Yes (IDH2)

Properties of most common mutations in AML

with normal cytogenetics

ETP: Early T-cell precursors ALL; * Falini B. et al (NEJM, 2005). Most of these features also applies

to double-CEBPA mut. AML; ** Only AML with R172 IDH2 mut.

Frequency 55-60% 25-30% 35-40% 10-15%

Unravelling the molecular pathogenesis of acute myeloid leukaemia with a normal

karyotype

George VassiliouWellcome Trust Sanger Institute

DisclosuresKymab: consultancy, Celgene: research grant

(Education Session, Sunday June 14)

Age-related clonal haemopoiesis (ARCH)

Busque et al, Nature Genetics 2012; Xie et al, Nature Medicine 2014; Genovese et al, NEJM 2014;Jaiswal et al, NEJM 2014; McKerrell et al, Cell Reports 2015

Polyclonal haemopoiesis

Clonal haemopoiesis

Cell with fitness mutation

1. Incidence rises with age:Under 30 years: <1% Over 90 years: >70%

2. Most commonly mutated genes:DNMT3A, JAK2, TET2, ASXL1, SF3B1, SRSF2 NPM1

1

2 2 22 2 2

2 2 22 2 22 2 2

2 2 22 2 22 2 2

2 2 22 2 22 2 2

2 2 22 2 22 2 2

2 2 2 2 2 32 2 2 2 2 2

2 2 22 2 22 2 3

2 2 22 2 22 2 3

1 1 11 1 11 1 1

1 1 11 1 11 1 1

1 1 11 1 11 1 1

1 1 11 1 11 1

1 1 1 1 1

2

21 1 1 1 1 1

3 3 33 3 33 3 3

3 3 33 3 33 3 3

3 3 33 3 33 3 3

3 3 33 3 33 3 3

3 3 33 3 33 3 3

3 3 3 3 3 33 3 3 3 3 3

3 3 3 3 3 33 3 33 3 33 3 3

3 3 33 3 33 3 3

3 3 33 3 33 3 3

3 3 33 3 33 3 3

3 3 33 3 33 3 3

3 3 33 3 33 3 3

3 3 3 3 3 33 3 3 3 3 3

3 3 3 3 3 33 3 33 3 33 3 3

3 3 33 3 33 3 3

3 3 3 3 3 3 3 3 3 3 3 3

333333

33

33334

3 3 33 3 33 3 33 3 33 3 33 3 3

3 3 33 3 3

3 3 33 3 33 3 33 3 33 3 3

333333

33

33333

3 3 33 3 3

3 3 33 3 3

3 3 3 3 3 33 3 3 3 3 3

4 4 44 4 4

4 44 4

4 4 4 4 44 4 4 4 43

3

33

33

33

3 3 3 3 3 3 3 3

Frank AMLPre-leukaemic haemopoiesis?subtle clinical features/ ? latency

Clonal haemopoiesis(subclinical)

Polyclonal haemopoiesis

Clinical relevance of clonal evolution model

Welch et al, Cell 2012; Ding et al, Nature 2012, Busque et al, Nature Genetics 2012; Xie et al, Nature Medicine 2014; Genovese et al, NEJM 2014; Jaiswal et al, NEJM 2014; McKerrell et al, Cell Reports 2015

DNMT3ANPM1cFLT3

DNMT3ANPM1c

DNMT3A

? intervenetargets of antileukaemic therapy

DNMT3A mutations in acute myeloid leukemia (AML): monitoring of minimal

residual disease (MRD). A study of the AML Study Group (AMLSG)

Gaidzig VI et al.Universitatklinikum Ulm, Germany

(Oral presentation (S451), Room Lehar 1+2, Saturday June 13)

Summary

• No prognostic impact of DNMT3Amut transcript levels at

diagnosis, after double induction, and end of treatment

• Only a small proportion (~10%) of the pts achieve MRD

negativity

• Detectable DNMT3Amut transcript levels in most patients >>

persistent clonal hematopoiesis

• In NPM1-mut/DNMT3A-mut AML, rare late relapse

characterized by loss of NPM1-mut but retention

of DNMT3A-mut are likely (secondary AML ?)

Therapeutic Targeting of AML with Aberrant Homeobox Gene Expression

Stefan FröhlingNational Center for Tumor Diseases (NCT)German Cancer Research Center (DKFZ)

Heidelberg University Hospital

________________________________________________

20th Congress of EHAVienna, June 13, 2015

• Common feature (50% of cases)

• Thought to reflect dysregulation of HOX pathways leading to abnormal self-renewal and leukemic transformation

• Most closely associated with MLL rearrangements

– High expression of HOXA genes

– HOXA9 as essential gene in MLL-rearranged AML

• Also observed in other AML subtypes

– High expression of HOXA genes in AML with MLL partial tandem duplication or PICALM-MLLT10 (CALM-AF10)

– High expression of HOXA and HOXB genes in NPM1mut AML

– Low or absent HOX gene expression in AML with PML-RARA, RUNX1-RUNX1T1 (AML1-ETO), or CEBPAmut

• Comprehensive pattern of HOX gene expression in normal versus malignant hematopoietic cells not fully established

“Aberrant” HOX Gene Expression in AML

t(9;11)

Targeting AML with HOX Gene Expression

Regulators of HOX Transcription – DOT1L

K79

DOT1L

H3K79 methyltransferase involved in transcriptional

activation and elongation

Associated with several MLL fusion partners (AF4,

AF9, AF10, AF17, ENL)

Aberrant H3K79me2/3 as shared mechanism of

oncogenic transcriptional activity in MLL leukemias

Dawson et al. NEJM 2012, Deshpande et al. Trends Immunol 2012


DOT1L Inhibition – Clinical Data

Phase 1 trial of EPZ-5676 in patients with advanced hematologic

malignancies (MLL-rearranged, 29 patients; MLL PTD, 5 patients)

Inhibition of H3K79 methylation in bone marrow (median, 52%) and

peripheral blood mononuclear cells (median, 43%)

Morphologic CR, 2/34 patients; PR, 1/34 patients; cytogenetic CR, 1/34

patients; resolution of leukemia cutis, 2/34 patients

Treatment-related increase in neutrophils and/or monocytes, 8/34 patients

(median onset, 15 days)

Identification of rearranged MLL in mature neutrophils

With permission from S. Armstrong

Stein, Tallman et al. ASH Annual Meeting 2014

t(11;19)+ neutrophil

ANC

x1000/µ

L

Targeting AML with HOX Gene ExpressionRole for DOT1L Inhibition in NPM1mut AML

Cell line Mutation IC50 [μM]

OCI-AML3

MOLM-13

HL-60

NPM1mut

MLL-AF9+

NRASmut

0.1

0.68

>10

0 5 10 150

50

100

EPZ004777 10µM in AML Cell Lines

Day of treatment

Cell c

ou

nt

(% v

eh

icle

)

OCI-AML3

OCI-AML2

MOLM13

HL60

0 5 10 150

50

100


Day of treatment

Cell c

ou

nt

(% v

eh

icle

)

OCI-AML3

OCI-AML2

MOLM13

HL600 5 10 15

0

50

100

10µM DFCI4777

Day of treatment

Cell c

ou

nt

(%

DM

SO

)

MOLM13

HL60

OCI-AML3

Cell

co

un

t

Day of treatment

0 5 10 150

50

100


Day of treatment

Cell c

ou

nt

(% v

eh

icle

)

OCI-AML3

OCI-AML2

MOLM13

HL60EPZ004777 [10 µM]

Kühn, Armstrong et al.

HOXA1

HOXA2

HOXA3

HOXA4

HOXA5

HOXA6

HOXA7

HOXA9

HOXA10

HOXA11

HOXA13

MEIS

10

3000

6000

9000

12000Normalized to ERCC spike-in controls

Genes

No

rmalized

Read

Co

un

t

OCI-AML3 DMSO

OCI-AML3 DFCI4777 [10uM]

HOXB1

HOXB2

HOXB3

HOXB4

HOXB5

HOXB6

HOXB7

HOXB8

HOXB9

HOXB130

500

1000

1500

2000

2500Normalized to ERCC spike-in controls

Genes

No

rmalized

Read

Co

un

t

OCI-AML3 DMSO


DMSO

EPZ004777 [10 µM]

HOXA1

HOXA2

HOXA3

HOXA4

HOXA5

HOXA6

HOXA7

HOXA9

HOXA10

HOXA11

HOXA13

MEIS

1

0

1000

2000

3000

4000

5000

Normalized to ERCC spike-in controls

Genes

No

rmalized

Read

Co

un

t

OCI-AML2 DMSO


H3K79me2

Histone 3

DMSO EPZ004777

No

rma

lize

d r

ea

d c

ou

nt

OCI-AML3 MOLM-13 HL-60


“Non-HOX” Targets – CDK6 in MLL-Rearranged AMLToxic

ity

RNAi screening

Validation Rescue

Kinase-enriched

library subset

10 AML cell lines

MLL-AF9, n=4 MLL WT, n=4

MLL-AF4, n=1

MLL-AF6, n=1

Placke et al. Blood 2014

Targeting AML with HOX Gene ExpressionPharmacologic CDK6 Inhibition in MLL-Rearranged AML

PD-0332991 (palbociclib): CDK4/6

inhibitor in clinical development (breast,

ovarian, lung cancer; HCC; GIST;

liposarcoma; GBM; MCL; myeloma)

AML cell lines

Primary human AML samples

Perspective: palbociclib treatment of

patients with MLL-rearranged AML

Placke et al. Blood 2014

What’s new in the WHO cassification ?

Clara Bloomfield Wthe Ohio State University, Columbus, Ohio, USA)

(Scientific Working Group, Saturday June 13)

Myeloid CAC Meeting Participants Chicago 3/31/14

AML WITH RECURRENT GENETIC ABNORMALITIES (WHO-2008)

a) AML with t(8;21)(q22;q22); RUNX1.RUNX1T1

b) AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22);CBFB-MYH11

c) Acute promyelocytic leukaemia (AML with t(15;17)(q22;q12);PML-RARA

d) AML with t(9;11)(p22;q23); MLLT3-MLL

e) AML with t(6;9) (p23;q34); DEK-NUP214 AML

f) AML with inv(3) (q21q26.2) or t(3;3) (q21;q62); RPN1-EVI1

g) AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MLK1

h) AML with mutated NPM1 (provisional entity)

i) AML with mutated CEBPA (provisional entity)

25%*

30%*

5%*

* Of all AML.

AML with MRC AML with mutated

NPM1

Not yet clear how it correlates with other WHO categories,

e.g. AML with MDS related changes (AML-MRC)

The same problems apply to CEBPA-mutated AML

NPM1-mutated AML : provisional entity (WHO-2008)

(Falini B et al., Blood 115:3776, 2010)

Multilineage dysplasia (MLD) in NPM1-mutated AML (n=318)

(Frequency of MLD: 23.3%)

Principal component analysis

(Falini B. et al. Blood 2010;115:3776-3786)©2010 by American Society of Hematology

No difference in GEP and survival in NPM1-mutated AML

with and without multilineage dysplasia

Survival curves from GIMEMA

AML with mutated NPM1 (WHO 2016)*

- Proposal: change from provisional to distinct entity

- Cases showing multilineage dysplasia as the only

AML-MRC defining criterion are classified as AML

with mutated NPM1

- The entity includes only de novo cases that lack

AML-MRC karyotype except del(9q) and previous

history of MDS or MDS/MPN, and are not therapy-

related **

** Almost all cases of NPM1-mutated AML, including the 15% with

abnormal karyotype (AK), will be classified as a distinct entity

(no previous history of MDS, AK different from that of AML-MRC)

CHROMOSOME ABNORMALITIES FOUND IN 15% of NPM1-MUTATED

AML ARE RARELY (<1%) THOSE DEFINING AML-MRC

Karyotype

AML

NPM1-mut* t(8;21) Inv16 t(15;17) 11q23/MLL

(N=632) (N=63) (N=37) (N=83) (N=83)

Additional

Abnormalities 93/632 44/63 13/37 39/83 28/83

(14.7%) (69.8%) (35.1%) (47%) (33.7%)

-X/-Y 11 32 1 3

+4 11 2 2

-7 3

+8 33 2 5 12 8

+13 2 2

+19 4

+21 5 4

+22 1 6 2

del(7q) 2

del(9q) 9 10 2

del(11q) 2

Ider(17)(q10)t(15;17) 7

Other 67 11 8 20 30

Total 142 59 22 44 52

(Blood 114:3024, 2009)

WHO 2016: proposed changes in the category of

“AML with recurrent genetic abnormalities”

- Switching from provisional to distinct entities:

AML with mutated NPM1 (distinct entity)

AML with double mutated CEBPA (distinct entity)*

* Same recommendations as for NPM1-mutated AML concerning

distinction from AML-MRC.

(Wouters BJ, et al. Blood. 2009;113:3088-91)

Ony double CEBPA mutations define a subgroup of AML with

a distinctive GEP and favorable outcome*

Subsequent studies (Bacher, Blood 119:4719; 2012; Schlenk RF Blood 122:

1576, 2013): no impact of MLD or accompanying chromosomal aberrations

Combining N- and C-terminal Cebpa mutations causes accelerated AML in the mouse

(Bereshchenko et al., Cancer Cell 2009)

E14.5

Fetal Liver cells

Lethally

irradiated recipients

+/+

K/L

L/L

K/K

+

K

L

Wild Type

Cebpa

N-terminal

mutations

C-terminal

mutations

p42

p30

Hematopoietic stem cells expansion

% s

urv

iva

l

+/+

L/L

K/L

K/K

Myeloid Leukemia Reduced K/L mice survival

% o

f G

MP

cells

Altered myeloid lineage commitment

p42

AML with mutated RUNX1

• Gene located at 21q22

• Encodes the alpha subunit of the core binding factor

• Mutation in 12.5-13.2% of AML

• More frequent in older male patients

• Frequent prior history of MDS, or prior exposure to radiation

• Immature morphology (60% M0) and phenotype

• Frequent associated MLL-PTD or ASXL1 mutations

• Rare CEBPA or NPM1 mutations

• Poor response to therapy with shortened survival

Tang et al. Blood 114:5352, 2009Mendler et al. JCO 30:3109, 2012

(by courtesy of Daniel Arber)

What about new translocations

in AML?

AML with BCR-ABL1

• Difficult to distinguish from myeloid blast

crisis of chronic myelogenous leukemia

• Criptic deletions of Ig and T cell receptors,

particularly IGH, recently shown to be

specific for de novo disease

• Important to recognize due to presence of

targeted (TKI) therapy

• Poor prognosis

Soupir CP, et al. Am J Clin Pathol 127:642, 2007Konoplev S, et al. Leuk Lymphoma 54:138, 2013Nacheva EP, et al. Br J Haematol 161:541, 2013

The Cancer Genome Atlas Research Network (NEJM, … Settembre/PDF/6... · The Cancer Genome Atlas...

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