molecular basis of chronic myelo-monocytic leukemia
DESCRIPTION
Molecular basis of Chronic Myelo-Monocytic Leukemia. Madrid, April 5 th , 2013. Overview. Disease definition & causes of death Genetic mutations Frequence Clonal architecture GM-CSF hypersensitivity Abnormal gene expression w/o mutation - PowerPoint PPT PresentationTRANSCRIPT
Molecular basis of Chronic Myelo-Monocytic
Leukemia
Madrid, April 5th, 2013
Overview
• Disease definition & causes of death
• Genetic mutations
• Frequence
• Clonal architecture
• GM-CSF hypersensitivity
• Abnormal gene expression w/o mutation
• Prognostic significance of gene mutation / deregulation
• Impact on current disease management
CMMLa disease defined by one positive
criteria
Clonal (oligoclonal?) disease of the HSC with monocytosis
- Monocytosis (> 1 G/L) that persists for at least 3 months
- NOT a CML Lack of Philadelphia chromosome Ph1 or BCR-ABL- NOT a MLN-Eo Lack of PDGFR/FGFR rearrangement and eosinophilia - NOT an AML Blood and bone marrow blast cells < 20%
- In most cases : cell dysplasia, at least one cell line
Tefferi A, Vardiman JW. Leukemia. 2008;22:14-22.
Persistant monocytosis > 1 G/L
Why do these patients die?
The best recognizedprognostic factor is blast cell count
Diagnostic feature
Peripheral blood blasts
Bone marrow blasts
CMML1
< 5%
< 10%
CMML2
5-19%
10-19%
Patients die in < 3 years from
Progression to AML (< 25%)Consequences of cytopenias
a-defensins HNP1-3
CD14-/CD24+Immature granulocytes
CD14+/CD24-Monocytes
Lymphocytes
Macrophages
CSF1
« Monocytes » include a fraction of dysplastic granulocytes
endowed with immunosuppressive properties
Droin N et al, Blood. 2010;115:78-88
Persistant monocytosis > 1 G/L
Why do these patients die?
Progression to acute leukemia
Consequences of cytopenias
Immune system deregulation
Molecular abnormalities in CMML cells
1 - Non specific cytogenetic abnormalities* 30-40 %
2 - Gene mutations (5-20 / patient) 100 % > 30 recurrently mutated genes
3 - Deregulated expression of genes (down, up) 100 %
* Such E et al, Haematologica. 2011;96:375-83
Gene tested (n) mutated (n,%)
ASXL1 312 125 (40%)TET2 262 151 (58%)SRSF2 220 101 (46%)RUNX1 263 39 (15%)
NRAS 263 29 (11%)
CBL 264 27 (10%)
JAK2 263 21 (8%)KRAS 263 20 (8%)ZRSF2 189 15 (8%)
IDH2 229 13 (6%)
SF3B1 220 13 (6%)
U2AF35 220 11 (5%)
EZH2 173 8 (5%)
FLT3 264 8 (3%)
DNMT3A 227 5 (2%)NPM1 260 3 (1%)IDH1 229 1 (<1%)
KIT 228 1 (<1%)
TP53 202 2 (1%)
Gene mutationsThe 3 most frequently
mutated genes are
TET2
SRSF2
ASXL1
Studied in sorted peripheral blood
monocytes Itzykson R et al, J Clin Oncol, 2013, in press
Damm F et al, Leukemia, 2013, in press
TET2
SRSF2
ASXL1
TET2 mutations associate with SRSF2 mutations
2 CMML groups ?
TET2 +/- SRSF2
ASXL1
Itzykson R et al, J Clin Oncol, 2013, in press
Gene mutations in CMML
Three frequently mutated genes
TET2 +/- SRSF2
&
ASXL1
120 3624 48 60
0%20
%40
%10
0%60
%80
%
5147
4236
3123
1914
84
1At risk Time (months)
Ove
rall
Surv
ival
(%)
0-1
2
3
≥4
Number of mutated genes
5124
3613
175
9 2 1
MonthsC
umul
ativ
e P
roba
bilit
y of
Sur
viva
l
0 6 12 18 24 30 36 42 48 54 60
0.0
0.2
0.4
0.6
0.8
1.0
120 3624 48 60
0%20
%40
%10
0%60
%80
%
5147
4236
3123
1914
84
1At risk Time (months)
Ove
rall
Surv
ival
(%)
0-1
2
3
≥4
Number of mutated genes
5124
3613
175
9 2 1
MonthsC
umul
ativ
e P
roba
bilit
y of
Sur
viva
l
0 6 12 18 24 30 36 42 48 54 60
0.0
0.2
0.4
0.6
0.8
1.0
1 2 3 4 5 6 7
0%
20%
40%
60%
Number of mutated genes
% o
f pat
ient
s
0 1 2 3 4 5 6
8
43 47
51
19
4 1
The number of mutations in a survey of 19 candidate genes affects the prognostic
~70% cases
Single cell analysis of the mutations
in 4 bone marrow cell compartments
19 genes sequenced on sorted CD14+ blood cells
and
HSC/MPP/CMP/GMP sorted
Seeded at 1 cell per well
Cultured for 12 days with cytokines
Colonies picked up
In each colony, mutation specific PCR
Early clonal dominance specifies CMML
among myeloid neoplasms
0
20
40
60
80
100
# 53
7#
500
# 42
8#
497
# 65
4#
536
# 53
1#
518
# 50
7#
501
# 46
2#
524
# 74
3#
514
# 73
1#
662
# 51
6#
550
# 63
2#
495
# 49
8#
586
# 64
4#
759
# 73
6#
658
63 61 9 102
22 46 16 160
20 67 46 48 67 37 119
95 51 49 56 72 10 49 32 93 70 28
HSC : CD34+/CD38-/CD90+
Black 0 mutationYellow 1 mutationOrange 2 mutationsRed 3 mutations
Clonal architecture in CMML (1/3)
TET2 SRSF2 KRAS
• Early clonal dominance in HSC• Linear acquisition of mutations, starting with epigenetic and splice genes• Growth advantage to the more mutated cells with differentiation
Itzykson R et al, Blood. 2013 Jan 14.
Clonal architecture in CMML (2/3)Growth advantage to the more mutated cells
with differentiation
KRAS homozygous
KRAS wildtype
Exome sequencing in blood leukemic cells (16 variants :14 SNP, 2 indels)Single cell analysis in CD34+ cells
Linear accumulation of mutations
with some branching
through HR
TET2 heterozygousU2AF35BFSP2TRAPPC6BCTTNBP2MYLK
TET2 homozygousU2AF35BFSP2TRAPPC6BCTTNBP2MYLKKRAS heterozygousASAP1ADCY10ATP2C2CEP63HECW2SIPA1L2SMOC2
TET2 homozygousU2AF35BFSP2TRAPPC6BCTTNBP2MYLKKRAS heterozygousASAP1ADCY10ATP2C2CEP63HECW2SIPA1L2SMOC2SH2B3CTCF
Clone 1N =10
Clone 2N = 1
Clone 3N = 73
Clone 4N = 7
Clone 5N = 9
TET2 homozygousU2AF35BFSP2TRAPPC6BCTTNBP2MYLKKRAS homozygousASAP1ADCY10ATP2C2CEP63HECW2SIPA1L2SMOC2SH2B3CTCF
Clonal architecture in CMML (3/3)
Clonal architecture in CMML
Early clonal dominance
Mostly linear acquisition of mutations
Some branching due to mitotic recombinations
Growth advantage to the most mutated cells
Limited effect of current therapeutic approaches
13 TET2mut chronic myeloid malignancies
(8 JAK2V617F MPN, 5 MDS)
CD34+/CD38- P=0.006 CD34+/CD38+ P=0.08
Early dominance of TET2 mutations may participate to the differentiation bias
Early dominance of TET2 mutations may participate to the differentiation bias
Sh:SCR
Sh:TET2
Sh:SCR
Sh:TET2
CD34+/CD38- CD34+/CD38+
ns*4.0
3.0
2.0
1.0
0.0(CD
33+)
GPA
- / G
PA+
Cord Blood CD34+
Proposed model to distinguish CMML from other chronic myeloid neoplasms
CD34+/CD38- CD34+/CD38+
TET2 genemutation
Other chronic myeloid neoplasms
CMML
CD34-
Clon
al E
xpan
sion
Myeloid Differentiation
Additional mutations?
Different HSC of origin?
Stochastic?
The mutated gene SF3B1 and ring sideroblasts
The altered pathway Signaling genes and proliferative forms of CMML
The allele burden JAK2 in ET versus PV
The early / late amplificationTET2 and monocytosis
Four parameters that affect the phenotype
in chronic myeloid diseases
JMML is a RAS diseaseHypersensitivity of progenenitorsto GM-CSF
Germline 10% Neurofibromatosis (NF1) Noonan syndrome (PTPN11, CBL)
Somatic 90% Inactivating homozygous mutations in PTPN11, NF1, CBLActivating mutations in NRAS, or KRAS
Usually only one mutation / patient by WGS
Loh ML. Br J Haematol. 2011;152:677-87.
In CMML, hypersensitivity of GM-CSF is inconstant
CONTROLCMML
0
20
40
60
80
Num
ber
of c
olon
ies
Serum-free + SCFGM-CSF 10 ng/mL
Mutation in a signaling geneNRAS, JAK2, CBL
6/7 cases
3/13 cases
Itzykson R et al, unpublished
GM-CSF hypersensitivity
Heterogeneous (contrary to JMML)
Mostly linked to mutations in signaling genes
Peripheral blood
0
1
2
3
4 CtrlD/D
1-13 14-26 27-39 ≥40
Mon
ocyt
es (k
/mm
3 )
Weeks
n=11n=23
n=12n=23
n=12n=20
n=9n=9
*
**
Gr1-FITC
Mac
1-A
lexa
647
Ctrl D/D
44% 1%3% 51%
Ctrl D/D
1 cm
Spleen
In the mouse, deletion of tif1g in myeloid cells (Transcription Intermediary Factor 1 g)
induces a CMML phenotype
Aucagne R et al, J Clin Invest. 2011;121:2361-70
TIF1g gene promoter is methylated in 35-40% of CMML
35%of patients
-139GGGAGGAYGT TYGTGYGTA YGTGYGYGTGT YGTAAT YGTTT TT TTTTAAA YGYGYGA YGYG-139GGGAGGACGTCCGTGCGTACGTGCGCGTGCCGCAACCGCCCTCCTTCAAACGCGCGACGCG unconverted
non methylatedmethylated
Control TIF1g low TIF1g norm
al expression (Subset # 1) (Part of subset # 2)
Aucagne R et al, J Clin Invest. 2011;121:2361-70
Cycles0
20
40
60
80
100
120
5 7 15
Tif1g
mR
NA
leve
l
0
CtrlDec.
170 TIF1g
HSC7072
Befo
re tr
eatm
ent
Afte
r 5 cy
cles
GTGTG70TTTTTG
-92AAATGTGTGATGTGAGGGTGGGGGCGCCGCGTGCGTGTGTG C C C C
T60TTGTTGGC
-92AAATGTGTGATGTGAGGGTGGGGGTGTTGTGTGTGTGTGTG
Bef
ore
deci
tabi
neA
fter
deci
tabi
ne
TIF1g expression increase in CD14+ cellsas a biomarker of HMA efficacy
Aucagne R et al, J Clin Invest. 2011;121:2361-70
Months
Cum
ulat
ive
Pro
babi
lity
of S
urvi
val
0 6 12 18 24 30
0.0
0.2
0.4
0.6
0.8
1.0
17 15 11 7 2 0
18 14 11 9 3 1
low TIF1ghigh TIF1g
Low TIF1g
Normal TIF1g
1.0
0.8
0.6
0.4
0.2
0.0 0 6 12 18 24 30
Months
Cum
ulati
ve p
roba
blili
ty o
f sur
viva
l
TIF1g expression level does not predict decitabine efficacy
0
50
100
150
200
250
300
mRN
A ex
pres
sion
(rel
ative
)
Cont
rol
Subs
et #
1
Subs
et #
2
Braun et al, Blood. 2011;118:3824-31.
Months
Cum
ulat
ive
Pro
babi
lity
of S
urvi
val
0 6 12 18 24 30
0.0
0.2
0.4
0.6
0.8
1.0
17 16 13 12 5 0
18 13 9 4 1
low MYBhigh MYB
Months
Cum
ulat
ive
Pro
babi
lity
of S
urvi
val
0 6 12 18 24 30
0.0
0.2
0.4
0.6
0.8
1.0
16 15 14 10 2 0
19 14 8 6 3 1
low CJUNhigh CJUN
P = 0.06
Cum
ulati
ve p
roba
blili
ty o
f sur
viva
l 1.0
0.8
0.6
0.4
0.2
0.0
Low
High
CJUN 1.0
0.8
0.6
0.4
0.2
0.0
CMYB
Low
HighP = 0.01
0 6 12 18 24 30 0 6 12 18 24 30 Months Months
Elevated CJUN and CMYB levels are negative prognostic factors
Braun et al, Blood. 2011;118:3824-31.
CMML: deregulated gene expression
Down
P15/INK4b – 50%
TIF1 g – 40%
miR-150
Up
cJUN
cMYB
Prognostic impactNo prognostic impact
wildtype
mutated
120 3624 48 60120 3624 48 60
Ove
rall
Surv
ival
(%)
100
80
60
40
20
0
187125
13468
8429
5010
196
51
187125
13468
8429
5010
196
51
187125
12656
7723
479
186
51
187125
12656
7723
479
186
51
120 3624 48 60120 3624 48 60
AM
L-fr
ee S
urvi
val (
%)
100
80
60
40
20
0
wildtype
mutated
Univariate analysisASXL1 : P < 0.0001
alsoSRSF2 : P = 0.03CBL : P = 0.003 IDH2 : P = 0.03
ASXL1 mutations have a negative prognostic impact
Itzykson R et al, J Clin Oncol, 2013, in press
120 3624 48 60
0%20
%40
%10
0%60
%80
%
5657
4630
2815
127
33
At risk Time (months)
Ove
rall
Surv
ival
(%)
Validation set (MLL)
52 24 8 2
low int high
Months
Cum
ulat
ive
Pro
babi
lity
of S
urvi
val
0 6 12 18 24 30 36 42 48 54 60
0.0
0.2
0.4
0.6
0.8
1.0
11 1
120 3624 48 60
0%20
%40
%10
0%60
%80
%
132106
10066
6736
4315
187
42
At risk Time (months)
Ove
rall
Surv
ival
(%)
low int high
Training set (GFM)
74 36 10 2
Months
Cum
ulat
ive
Pro
babi
lity
of S
urvi
val
0 6 12 18 24 30 36 42 48 54 60
0.0
0.2
0.4
0.6
0.8
1.0
A simplified prognostic score that includes ASXL1 mutations
Training (GFM) : 312 Validation (MLL) :165
Low
Int
High
Low
Int
High
Hyperleucocytosis (>15)Age (>65)
AnemiaThrombocytopenia (<100)
ASXL1 mutation
Low < 4Intermediate 4-8
High >8
Absence00000
Presence32222
Itzykson R et al, J Clin Oncol, 2013, in press
To refine the diagnosis
BCR-ABLPDGFR/FGFR
KIT
To capture prognostic informations
ASXL1
Molecular characterization of CMMLin clinical practice
Which molecular analyses ?
Monocytes > 1 G/L
Yes (MLN-Eo)Imatinib
Eliminate BCR-ABL
OthersASXL1, Complex
Karyotype
EosinophiliaPDGFR / FGFR
Mast cellsKIT
Yes (SM)TKI?
YesHSCT?
Which therapeutic proposal?
Leucocyte count
< 13 G/L > 13 G/L
0 ou 1 > 1
Poor prognostic
factors
HSCTHMA
HydroxyureaBest supportive
care
IPSS
Yes No
HSCTHMA
1 - CMML : a disease of the HSC in which gene mutations accumulate, mostly linearly, and the most mutated cells
accumulate with differentiation.
2 - High number of genetic abnormalities with mutations inTET2, SRSF2 and ASXL1 being the most prominent.
3 - Mutations in signaling genes and early clonal dominance of TET2 mutations favor GM amplification.
4 – Treatment of poor prognostic CMML: ABMT (rarely feasible) and hypomethylating agents
5 – Molecular informations may better guide the therapeutic choice in coming years, with ASXL1 mutations affecting
outcome
Gustave Roussy, Villejuif, France
Raphael Itzykson
Nathalie Droin
Hana Raslova
Margot MorabitoFrançois DelhommeauPhilippe RameauCédric OréarGuillaume Meurice
William VainchenkerOlivier Bernard
Marseille, France
Daniel BirnbaumVéronique Gelsi-Boyer
Groupe Francophone des Myélodysplasies
P. FenauxAnd all GFM clinical teams
Lille, FranceAline Renneville
Claude Preudhomme
Cochin, FranceOlivier Kosmider
Michaela Fontenay
Southampton, GBClaire Hidalgo-Curtis
Nicholas Cross
Dresden, Munich, Germany Uwe Platzbecker
Thorsten HaferlachSuzanne Schnittger
red blood cell
Haematopoietic stem cell
Lympho-myeloid progenitor cell
lymphoid progenitor cellMEP
BFU-E
CFU-E
CFU-MK
CFU-GM
CFU-MCFU-G
platelet granulocyteT cell B cell NK
erythro
MGK
CFU-BCFU-T
monocyte