Juvenile and ChronicMyelo-MonocyticLeukemia
red blood cell
Haematopoietic stem cell
Lympho-myeloid progenitor cell
lymphoid progenitor cellMEP
BFU-E
CFU-E
CFU-MK
CFU-GM
CFU-M CFU-G
platelet granulocyte T cell B cell NK
erythro
MGK
CFU-BCFU-T
monocyte
MDS/MPN of the WHO classification
Clonal diseases of the HSC
Excluded in 2009 : PDGFBR rearrangement and eosinophilia
1. Chronic myelomonocytic leukemia
2. Juvenile myelomonocytic leukemia
3. Atypical chronic myeloid leukemia,
4. Refractory anemia with ring sideroblasts and thrombocytosis
5. MDS/MPN unclassifiable
No BCR-ABL or Ph1Bone marrow blast cells < 20%
JMML
An agressive myeloid malignancy of chilhood
MPN / MDS in the WHO classification
- Infant or young child (0-6), male > female, with fever and pallor, - Circulating WBC count > 10G/L; Peripheral monocytosis > 1 G/L- A few circulating myeloid precursor cells
- Sometimes : skin rash, cough, bloody stools
- Splenomegaly, hepatomegaly
- Lack of BCR-ABL- Less than 20% blast cells in the bone marrow
JMML
An agressive myeloid malignancy of chilhood
Cytogenetic abnormality (monosomy 7, 25%)
Hypersensitivity of myeloid progenitor cells to GM-CSF (not to IL-3 or G-CSF)
Increased HbF for age
Treatment : ABMT (EFS 52%)
AML4 / AML5 or spontaneous improvement
Genetic syndromes that predispose to JMML
Neurofibromatosis, type I (NF1) : prone to JMML in the first decade
- Autosomal dominant disorder (or spontaneous)- At least 6 café au lait macules, - At least 2 neurofibromas or 1 plexiform neurofibroma,- Lisch nodules (iris hamartomas), axillary or inguinal freckling, and/or optic gliomas
- NF1 encodes neurofibromin protein, a GTPase activating protein for Ras- Enhances the hydrolysis of the active, GTP-bound conformation of Ras
- In the bone marrow of children with JMML: loss of the wild type allele
- Mouse models of nf1 conditional deletion reproduce the disease
Genetic syndromes that predispose to JMML
Noonan syndrome (NS)
- Autosomal dominant (or spontaneous) disorder- Facial dysmorphism, short stature, webbed neck, cardiac anomalies,- Varying levels of impaired cognition,- Self-resolving myeloproliferative disorder in infancy that resembles JMML
- Germine mutations in PTPN11 (former SHP2) in 50% of cases- Encodes a non-receptor protein tyrosine phosphatase (also SHP2)- That connects tyrosine kinase receptors to Ras
- Other Noonan : germline mutations in SOS1, a RAS-GEF (Guanine nucleotide-exchange factor) KRAS
Gene mutations in JMML
SHP2Yp
Grb2
SOS1
Ras-GDP
Ras-GTP
NF1
CBL
Raf
Mutually exclusive mutations in
PTPN11 35%NF1 25%N/KRAS 20%CBL 10%
Other in SOS1, FLT3, ASXL1
No TET2 mutation
AKT STAT5
ERK
MEK
CMML
a disease of the lederly
defined by only one positive criteria
MPN / MDS in the WHO classification
Clonal disease of HSC with monocytosis
- Persistant monocytosis (> 1 G/L)
- Lack of Ph1 or Bcr-Abl
- Blood and bone marrow blast cells < 20%
- Cell dysplasia, at least one cell line
Molecular abnormalities identified
in human CMML
1 – Cytogenetic abnormalities : 15-40%
2 – Uniparental disomy : ~50 %
3 – Mutations in
Epigenetic genes : TET2 ; ASXL1 ; AML1/RUNX1; IDH1/2; EZH2; UTX
Signalling : N/KRAS; CBL; FLT3-ITD, JAK2; NOTCH2 /NCST / MALM
Splicing : SRSF2; ZRSR2; J2AF35; SF3B1
4 – Deregulated expression of
TIF1, miRNA, CJUN, CFOS
No specific mutationHigh frequency of TET2/IDH pathway mutations (> 70%)Combinations of mutations in signaling, epigenetic, splicingMore mutations to identify (NGS)
Molecular abnormalities identified
in human CMML
CML
BCR-ABL
TE
JAK2* hZ
PV
JAK2* HZ
MF
JAK2*other
CMML
TET2* - IDH1/2*other
TET2 rare JAK2 rare
Increasing complexityfrom myeloproliferative neoplasms
to myeloproliferative / myelodysplastic diseases
AML1 ASXL1 CBL JAK2 KRAS NRAS
29 MUTATIONS IN 17 PATIENTS
# CLONES
TET2
52 MUTATIONS IN 46 PATIENTS
Clonal expansion occurs in theCD34+/CD38- compartment
CMML : clonal amplification at the stem cell level
Stem cells
Progenitors
Maturecells
Normal MPN CMML
LMMP?
?
TIF1 involved in zebrafish erythropoiesis (severe anemia in « moonshine » mutant) human erythropoiesis (ex vivo differentiation of CD34+ cells)
TIF1 knock-out embryonic lethal in mice
TIF1 (Transcription Intermediary Factor 1 )
1127 AA1127 AARINGRING B1B1 B2B2 CoiledCoiled--coilcoil TSSTSS PHDPHD BromoBromo
RBCC ou TRIMRBCC ou TRIMTIF Signature TIF Signature SequenceSequence
FeaturesFeatures of of chromatinchromatinremodelingremodeling proteinprotein
MeMe33H3K4H3K4AcetylatedAcetylated
lysineslysines
TIF1- belongs to a group of four proteins (TIF1 ) - modulates the TGF- signaling pathway- is a transcriptional co-regulator (TAL1/PU1)
Ageing Tif1 / mice developa CMML-like disease
Peripheral blood
00
11
22
33
44 CtrlCtrl//
11--1313 1414--2626 2727--3939 ≥≥4040
Mon
ocyt
es (k
/mm
Mon
ocyt
es (k
/mm
33 ))
WeeksWeeks
n=11n=11n=23n=23
n=12n=12n=23n=23
n=12n=12n=20n=20
n=9n=9n=9n=9
**
****
Gr1Gr1--FITCFITC
Mac
1M
ac1 --
Ale
xa64
7A
lexa
647
CtrlCtrl //
44%44% 1%1%3%3% 51%51%
CtrlCtrl //
1 cm1 cm
Spleen
-139GGGAGGAYGT TYGTGYGTA YGTGYGYGTGT YGTAAT YGTTT TT TTTTAAA YGYGYGA YGYG
-139GGGAGGACGTCCGTGCGTACGTGCGCGTGCCGCAACCGCCCTCCTTCAAACGCGCGACGCG unconverted
non methylatedpartially methylatedtotally methylated
Control TIF1low
TIF1norm
alexpression (Subset# 1) (Part of subset# 2)
TIF1 gene promoter is methylatedin 35-50% of CMML
35%of patients
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 TIF1
Normal TIF1
1.0
0.8
0.6
0.4
0.2
0.0
0 6 12 18 24 30Months
Cum
ulat
ive
prob
ablil
ity
of s
urvi
val
TIF1 expression leveldoes not predict decitabine efficacy
0
50
100
150
200
250
300
mRN
Aex
pres
sion
(rel
ativ
e)
Cont
rol
Subs
et#
1
Subs
et#
2
Wild-type
Mutated
Months
19 15 11 7 219 17 14 12 5
0 6 12 18 24 300 6 12 18 24 30
Cum
ulat
ive
Prob
abili
tyof
Sur
viva
l
0.0
0.2
0.4
0.6
0.8
1.0
Mutated
Wild-type
TET2
13 10 9 7 325 22 16 12 4
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
27 24 18 15 6 0
10 7 6 3 1 1
AML1 WTAML1 MUT
Mutated
Wild-type
0 6 12 18 24 30
10 7 6 3 1 Mutated28 24 18 15 6 Wild-type
AML1ASXL1
Gene mutations in TET2, ASXL1, AML1 do not affect survival
Months
Cum
ulat
ive
Pro
babi
lity
of S
urvi
val
0 6 12 18 24 300.
00.
20.
40.
60.
81.
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
ulat
ive
prob
ablil
ity
of s
urvi
val 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 30Months Months
CJUN level correlates with responseCMYB level correlates with survival
The best recognizedprognostic factor is blast cell count
Diagnostic feature
Peripheral blood monocytes
Left shift and myelocytes
Peripheral blood blasts
Bone marrow blasts
Dysplasia
t(9;22) or BCR-ABL
PDGFRA/B
CMML1
> 1 G/L
< 10%
< 5%
< 10%
0, one, more
No
No
CMML2
> 1 G/L
> 10%
5-19%
10-19%
0, one, more
No
No
Forward scatter
Side
scat
ter
Healthy donor CMML patient
CD14
Cel
lnum
ber
CD14
Cells counted as « monocytes »in the peripheral bloodinclude 2 populations
TET2 mutation(s) in the two cell populations
NoneR1214W; L1420FSQ652XMutation splice donor site exon 6 + L1855FSR544X ; E1492FSNoneE320X ; R1359HK450X ; I1163FSH1219YF1104FS , D1384VNoneT1883FSNoneQ1501FS ; G1861RQ591XNoneL615FS ; K1422FSK1005FS ; S1324FSS354FS ; L615FS
UPN
4754717279818596
100136153107171174180194207211216
-défensinesHNP1-3
CD14-/CD24+
Granuleux immatures
CD14+/CD24-
Monocytes
Lymphocyte
ExosomesIL-13
STAT3STAT6
Macrophage
M-CSF
The immunosuppressive propertiesof the CMML immature granulocytes
Take home messages
1 – JMML / CMML are distinct diseases
2 – JMML is a RAS pathway disease with hypersensitivity to GM-CSF
3 – CMML is a TET2 disease with many different mutations
4 – Epigenetic changes in addition to mutations (TIF1, miRNA)
5 – Immunosuppressive dysplastic granulocytes
To further explore the disease pathogenesis : [email protected]