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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
Targeting AML with HOX Gene Expression
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
EPZ004777 10µM in AML Cell Lines
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
EPZ004777 10µM in AML Cell Lines
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
OCI-AML3 DFCI4777 [10uM]
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
OCI-AML2 DFCI4777 [10uM]
H3K79me2
Histone 3
DMSO EPZ004777
No
rma
lize
d r
ea
d c
ou
nt
OCI-AML3 MOLM-13 HL-60
Targeting AML with HOX Gene Expression
“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