Itzykson et al., Supplementary Material
Supplementary Figures 1-6
Supplementary Table 1
Supplementary Methods
Additional References
Study Group
Supplementary Figures.
Supplementary Figure 1. A-B. TaqMan® amplification plots of allele discrimination assay of
ASXL1 c.1934dupG -> p.G646WfsX12 for DNA from A. peripheral blood CD14+ cells and B.
skin fibroblasts from a representative patient (UPN #632). Green: wildtype allele; Blue:
mutant allele. C. Resulting allele discrimination plot: normalized vector signal for the mutant
probe plotted against that of the wildtype probe.
Supplementary Figure 1
A
C
Wildtype allele
CD14+
B
Fibroblasts
CD14+
Mutant allele
Fibroblasts
Supplementary Figure 2. A. Schematic representation of the experimental procedure
employed in Figures 1-3 to assess clonal architecture of recurrent mutations in CMML. B.
Simplified immunophenotypic steps of human granulomonocytic differentiation employed in
the study. The indicated functional properties are illustrated in Supplementary Figure 3E-F.
C. Flow cytometry analysis of bone marrow CD34+ cells selected by immunomagnetic
separation from representative CMML and age-matched control samples. Left panel:
CD34+/CD38- and CD34+/CD38+ gates in the overall CD34+ population. Middle panel: CMP
(CD45RA-/CD123+), GMP (CD45RA+/CD123+) and MEP (CD45RA+/CD123+) in the gated
CD34+/CD38+ population. Right panel: HSC (CD45RA-/CD90+), MPP (CD45RA-/CD90-), and
LMPP (Lymphoid-primed multipotent progenitor; CD45RA+/CD90-, according to Guardon et
al.46) in the gated CD34+/CD38- population.
PB
Sample
A
BM
Sample
±
Supplementary Figure 2
B
Immunomagnetic
Separation
18-genes mutations
screen
CD34+/CD38CD34+/CD38+
HSC, MPP,
CMP, GMP
Cell
sorting
CD34+/CD38-
CD34+
CD14+
HSC
CD90+
MPP
mutation-specific TaqMan®
discrimination assays
design
Serial
replating
CFU-GM
+
BFU-E
CD90-
Cloning
Liquid culture with broad cytokine panel
CMP
C
CMML
CMP
CD38
CD123
CD45RA
GMP
MPP HSC
CD45RA
CD90
CD123+
CD45RA-
GMP
Control
CD34
CD34+/CD38+
Collect clones, extract DNA & genotype
CFU-GM
+
BFU-E
CD123+
CD45RA+
CFU-GM
only
Supplementary Figure 3. A-B. Cloning efficacy according to sample’s mutational status.
Percentages represent the fraction of wells from 96-well plates (2-3 plates per sample)
containing ≥ 4 cells after 12 days of liquid culture in the presence of FBS 10% and cytokines
(SCF, FLT3-L, TPO, IL-3, IL-6, G-CSF, GM-CSF, EPO); 39 samples were cultured including
20 from bone marrow and 19 from peripheral blood CD34+/CD38- (A.) or CD34+/CD38+ (B.).
Mutations (not mutually exclusive) were present in methylation genes (n=21; all in TET2),
signalling genes (n=15; CBL n=4, NRAS n=3, KRAS n=6, JAK2 n=2), splicing genes (n=26,
SRSF2 n=21, ZRSR2 n=3, U2AF35 n=1, SF3B1 n=1), and ASXL1 (n=8); bar: median; ns:
not significant (Kruskal-Wallis test). C. Proportion of wildtype, single-mutated and double
mutated (white, gray and black bars respectively) clones grown in liquid culture for 12 days
with FBS 10% and cytokines (SCF, FLT3-L, TPO, IL-3, IL-6, G-CSF, GM-CSF, EPO) or
colonies grown in methylcellulose with FBS and cytokines (SCF, IL-3, G-CSF, EPO) for 14
days from sorted HSC, MPP, CMP and GMP from sample UPN#777 (first mutation: CBL
W408R, second mutation: TET2 S716X). Figures on top of the bars represent the number of
clones or colonies assessed. D. KRAS pyrosequencing of bulk CD34+/CD38- and
CD34+/CD38+ cells from UPN #752 harboring a KRAS G12A mutation. E. TET2 targeted
resequencing of bulk CMP cells of UPN#632 harboring a TET2 c.3467A>T (p.N1156I)
mutation (verified to be somatic by sequencing germline DNA from skin fibroblasts [data not
shown]; 353 reads at the mutated position). F. Comparison of the proportion of mutated cells
determined by single cell cloning (dark gray; 193, 88 and 38 clones screened for UPN#752
CD34+/CD38-, CD34+/CD38+ and UPN#632 CMP respectively) or bulk DNA sequencing (light
gray) for the KRAS and TET2 mutation in E-F. Proportion of mutated cells are the double of
proportion of alternative reads since both mutations are heterozygous G. Colonies from
purified CMP or GMP from representative control and CMML samples grown in
methylcellulose with 30% FBS and cytokines (SCF, IL-3, G-CSF and EPO), showing
erythroid and granulomonocytic colonies for CMP, and pure granulomonocytic colonies for
GMP (mean and standard deviation [SD] from triplicate). H. Serial replating in methycellulose
of individual clones from sorted HSC and MPP. Results are expressed as the percentage of
colonies relative to the initial number of clones seeded. Mean and SD from three
independent samples.
Supplementary Figure 2
A
B
ns
C
ns
105 75 117 95
56 66
28 31
80%
60%
40%
D
E
F
G
KRAS
#752
Clones
Colonies
Clones
Colonies
0%
Clones
Colonies
20%
Clones
Colonies
Percentage
100%
HSC
MPP
CMP
GMP
H
TET2
#632
250
100
60
40
20
150
100
50
control
GMP
CMP
GMP
CMP
CMP
CD34+/CD38+
80
HSC
60
40
20
MPP
0
0
CD34+/CD38-
CMP
CD34+/CD38+
CD34+/CD38-
0
Colonies (%)
80
Colonies (%)
Mutated cells (%)
100
200
CMML
0
1
2
3
Platings
4
5
Supplementary Figure 4. Architecture of 100 CD34+ clones in a sample (UPN#759)
harboring 16 mutations discovered by exome sequencing showing sequential acquisition of
mutations in 3 consecutive groups, along with loss of heterozygosity of TET2 variant.
Terminal branching of two subclones with wild-type or homozygous KRAS mutation through
possible mitotic recombination. Examples of Sanger sequences of the KRAS mutations in
representative heterozygous, homozygous and wild-type clones are shown. Top panel:
fraction (and standard deviation, by Wald’s method) of alternative (mutated) reads in the
mature CD14+ cells by exome sequencing; ns: not significant, chi-square test.
Supplementary Figure 4
ns
0.8
0.6
0.4
SH2B3
CTCF
0.0
KRAS
ASAP1
ADCY10
ATP2C2
CEP63
HECW2
SMOC2
SIPA1L2
0.2
TET2
U2AF1
BFSP2
TRAPPC6B
CTTNBP2
MYLK
Mutated clone size
in CD14+ cells
1.0
CTCF
SH2B3
SMOC2
SIPA1L2
HECW20
CEP63
ATP2C2
ADCY10
ASAP1
MYLK
CTTNBP2
TRAPPC6B
BFSP2
U2AF35
TET2 homozygous
Clone 4
N=7
Clone 1
N =10
Clone 2
N=1
Clone 3
N = 73
Clone 5
N=9
TET2 heterozygous
U2AF35
BFSP2
TRAPPC6B
CTTNBP2
MYLK
TET2 homozygous
U2AF35
BFSP2
TRAPPC6B
CTTNBP2
MYLK
KRAS heterozygous
ASAP1
ADCY10
ATP2C2
CEP63
HECW2
SIPA1L2
SMOC2
TET2 homozygous
U2AF35
BFSP2
TRAPPC6B
CTTNBP2
MYLK
KRAS heterozygous
ASAP1
ADCY10
ATP2C2
CEP63
HECW2
SIPA1L2
SMOC2
SH2B3
CTCF
TET2 homozygous
U2AF35
BFSP2
TRAPPC6B
CTTNBP2
MYLK
KRAS homozygous
ASAP1
ADCY10
ATP2C2
CEP63
HECW2
SIPA1L2
SMOC2
SH2B3
CTCF
Supplementary Figure 5. Unique somatic mosaicism in patient UPN#752 harboring two
independent subclones with NRAS G12D (dark grey) and KRAS G12A (light grey)
respectively. A. Unique mosaïcism in clonal architecture of CMML from UPN#752 with
respect to NRAS and KRAS genotypes (NRAS G12D: light grey, KRAS G12A: dark grey,
WT: wildtype, black). B. Proportion of the corresponding clones in the indicated bone marrow
CD34+ fractions of matched samples before and after investigational treatment with a MEK
inhibitor (MEKi). The total number of interrogated clones is indicated on top of the bars.
**P≤0.001 (Fisher exact test for the relative proportions of KRAS and NRAS mutated clones).
Supplementary Figure 5
B
60
MEKi
40
0
CD34+/CD38+
20
CD34+/CD38-
KRAS
G12A
**
80
CD34+/CD38+
WT
283 269
100
CD34+/CD38-
NRAS
G12D
193 88
Percentage of clones
A
Supplementary Figure 6. A. Cloning efficacy of CMP from 17 CMML and 5 control samples.
Percentages represent the fraction of wells from 96-well plates (2-3 plates per sample)
containing ≥ 4 cells after 12 days of liquid culture in the presence of FBS 10% and cytokines
(SCF, FLT3-L, TPO, IL-3, IL-6, G-CSF, GM-CSF, EPO); bar: median. B. Proportion of clones
with granulomonocytic (CD14+ and/or CD15+ and/or CD24+, grey), erythroid (GPA+, white)
and mixed (both GPA+ and + and/or CD15+ and/or CD24+ populations, black) phenotype after
12-day liquid culture of single CMP in the presence of 10% FBS and cytokines (SCF, FLT3L, TPO, IL-3, IL-6, G-CSF, GM-CSF, EPO); chi-square test for mean proportions from 7
CMML and 4 controls. C. 5.104 CMP from a representative sample were cultivated in bulk of
FBS 10% and cytokines (SCF, FLT3-L, TPO, IL-3, IL-6, G-CSF, GM-CSF, EPO). Cells with a
MEP, CMP, and GMP phenotype at day 3 of culture were sorted and seed in triplicate (250
cells/mL) in methylcellulose with FBS 30% and cytokines (SCF, IL-3, G-CSF and EPO).
Mean and SD of CFU-GM (grey) and BFU-E (white) colonies for each fraction. D. Gene
expression levels (relative to HPRT expression) of CEBPA (left panel) and CEBPB (right
panel) in sorted CMP fractions: mean and SEM from 4 control and 5 CMML samples each
analyzed in duplicate. Unpaired t tests.
Supplementary Figure 6
B
100
ns
CMP clones,%
100
80
60
40
80
60
40
20
20
0
Control
MEP
GMP
CMP
Number of colonies
gene expression (relative to HPRT)
D
C
CMML
Control
0
CMML
Cloning efficacy,%
A
CEBPA
CEBPB
ns
ns
Healthy CMML
Healthy CMML
Supplementary Tables:
Supplementary Table 1. Characteristics of the 5 patients with paired studies of clonal architecture with or without treatment.
INITIAL SAMPLE
UPN
507
AGE
70
GENDER
M
WHO
WBC
(G/L)
MONOCYTES
(G/L)
HB
(g/dL)
PLT
(G/L)
CMML-2
14.9
4.9
10.2
132
PAIRED SAMPLE
Tx
(IWG
response)
Interval
(months)
observation
WHO
WBC
(G/L)
MONOCYTES
(G/L)
HB
(g/dL)
PLT (G/L)
13.5
CMML-2
20.4
6.1
10.6
90
ESA (HI-E)
+5.8
CMML-2
29.2
8.2
12.7
83
516
76
M
CMML-1
46.2
7.4
13.7
54
HY (SD)
13.1
CMML-1
10.3
2.3
13.5
87
550
85
M
CMML-1
6.8
2.7
11.6
27
HMA x8 cycles
(SD)
14.2
CMML-1
6.4
2.1
13.2
21
ICx (PR)
1.4
CMML-1
17.1
12.0
9.4
116
HMA x3 cycles
(mCR)
+4.4
no blast
excess
2.2
0.3
8.1
74
ASCT
(relapse)
+6.6
RAEB-2
2.1
0.1
10.1
11
MEKi x2 (SD)
2.1
ND
7.4
(2%
blasts)
3.1
9.1
67
632
752
64
76
M
M
CMML-2
CMML-2
26.2
48.1
(7%
blasts)
10.0
17.3
11.7
9.2
34
69
HI-E: Hematological improvement of erythroid lineage; SD: stable disease; PR: partial remission; mCR: marrow complete response, all from
IWG 2006 criteria1; RAEB: Refractory Anemia with Excess of Blasts (WHO criteria3). ESA: Erythropoiesis Stimulating Agent; HMA:
hypomethylating agent, HY: hydroxyurea, ICx: Intensive chemotherapy, ASCT: Allogeneic Stem Cell Transplantation, MEKi: MEK inhibitor, HB:
haemoglobin, PLT: platelet count, WHO: World Health Organization, WBC: White Blood Cells count. Tx: Treatment. ND: not done.
Supplementary Table 2. Characteristic of patients with TET2-mutated myeloproliferative
neoplasm (MPD) or myelodysplastic syndrome (MDS).
UPN
MPN 001
MPN 004
MPN 005
MPN 020
MPN 035
MPN 096
MPN 099
MPN 120
MDS 028
MDS 003
MDS 009
MDS 664
MDS 541
Sex
M
M
F
M
M
F
M
F
M
M
M
M
M
WHO
PMF
PV
PV
PV
ET
ET
ET
PV
RCMD-RS
RAEB1
RARS-T
RAEB1
RA
Monocytes
(G/L)
1.44
1.35
0.24
0.38
3.93
0.8
1.81
0.28
0.27
0.17
1.0
0.31
0.97
TET2 mutation
TET2 defect
c.1669C>T
c.3707-3713del
del(4q24)
c.4619del
c.1061C>A + c.4075C>T
c.5541G>A
c.2030del
c.1378del
c.2131del
c.4870c>T
c.1973del
c.1680_1681ins
c.3764dupA
p.Q557X (4q24UPD)
p.P1237-S1239del (4q24UPD)
del(4q24)
p.P1540fsX31
p.[S354X]+[R1359Cys]
p.W1847X
p.L667fs
p.S460fs
p.E711fs
p.E1624X
p.H658fs
p.K561fs
p.Y1255X
PMF. Primary myelofibrosis ; PV : polycythemia vera ; ET : Essential thrombocytopenia ;
RCMD-RS : Refractory cytopenia with multilineage dysplasia and ring sideroblasts ; RAEB :
refractory anemia with excess of blasts. RA; refractory anemia.
Supplementary Methods
Patients
Between Feb 2007 and Jan 2011, thanks to the clinicians of the Groupe Francophone des
Myélodysplasies (GFM), we prospectively collected blood and bone marrow samples from
patients with CMML included in the PHRC MAD-06 or in the previously published decitabine
phase II trial (n=38; EudraCT #2008-000470-21, Braun et al. [9]). UPN #752 received the
MEK inhibitor GSK1120212 as a part of a phase I/II study (www.clinicaltrials.gov identifier
NCT00920140). All others patients received treatment according to European Medicines
Agency’s labeling (eg. Azacitidine, chemotherapy), or French Health Authorities’ (Agence
Française de Sécurité Sanitaire des Produits de Santé [AFSSAPS]) ‘temporary treatment
program’ for Erythropoiesis Stimulating Agents [ESAs]. CMML diagnosis was defined
according to WHO 2008 criteria (1). Response to therapy was classified according to
International Working Group revised criteria for myelodysplastic syndromes (2).
Control bone marrow samples were obtained from older (age > 50 years) patients
undergoing hip replacement surgery without a known diagnosis of malignancy or
inflammatory disease.
Flow cytometry and cell sorting or cloning
Enrichment of blood or bone marrow mononucleated cells for CD34+ cells was performed on
the AutoMacs system (CD34 MicroBead Kit, MiltenyiBiotec, Inc.) according to manufacturer’s
instructions. For blood samples, the CD34- fraction was then used to purify the CD3+
population, and the CD3- population was used to enrich the CD14+ population as previously
described (3). CD34+ from bone marrow samples were stained with FITC anti-CD45RA
(clone H100), PE anti-CD123 (clone 7G3), PerCP-Cy5.5 anti-CD38 (clone HIT2), APC antiCD90 (clone 5E10), all from BD Pharmingen, Inc., and PC-7 anti-CD34 (clone 581, Beckman
Coulter, Inc.) and sorted with a MoFlo cell sorter (Beckman Coulter) in the following fractions:
CD34+CD38-CD90+ (Hematopoietic Stem Cells, HSC), CD34+CD38-CD90- (Multipotent
Progenitors, MPP), CD34+CD38+CD45RA-CD123+ (Common Myeloid Progenitors, CMP),
CD34+CD38+
+
+
CD45RA+CD123+
-
-
(Granulocyte-Monocyte
Progenitors,
GMP)
and
+
CD34 CD38 CD45RA CD123 (Megakaryocyte-Erythrocyte Progenitors, MEP). CD34 from
peripheral blood samples were sorted as CD34+CD38- and CD34+CD38+ fractions. CD34+
sorted fractions were then cloned at one cell per well in 96-well plates. FACS analysis of
sorted CD34+ fractions after short-term culture or for cell-cycle analysis was performed with
the same panel on a LSR II analyzer (BD Biosciences). FACS analysis of clones at day 12 of
culture was performed with FITC anti-CD24, PE anti-CD14 (both from BD), PE-Cy5 anti-GPA
(clone CLB-ery-1, Invitrogen) and APC anti-CD15 (Myltenyi). All analyses were performed
with FlowJo 9.2 software (TreeStar Inc.,).
Liquid Cell culture
Short term culture of 5x104 cells from sorted CD34+ populations and single-cell culture of
CD34+ clones was performed for 3 and 12 days respectively, in MEM-alpha milieu (Life
Technologies) supplemented with 10% fetal bovine serum (FBS, StemCell Technologies,
Inc.) and recombinant human cytokines: Stem Cell Factor (SCF, 50 ng/mL, Biovitrum AB,
Inc.), FLT3-Ligand (50 ng/mL, Celldex Therapeutics, Inc.), pegylated thrombopoietin (TPO,
10 ng/mL, Kirin Laboratories, Inc.), interleukin-3 (IL-3, 10 ng/mL, Miltenyi Biotec), interleukin6 (IL-6, 10 ng/mL, gift from S. Burstein, Oklahoma City, OK, USA), granulocyte-macrophage
colony–stimulating factor (GM-CSF, 5 ng/mL, Peprotech, Inc.), erythropoietin (EPO, 1 IU/mL,
Amgen, Inc.) and granulocyte colony–stimulating factor (G-CSF,10 ng/mL, Amgen). Culture
of bone marrow CD34+CD38- MPN cells in B, NK lympho-myeloid differentiating conditions
was performed as described (4).
Gene mutation analysis
FLT3 internal tandem duplications (FLT3-ITD) and NPM1 exon 12 mutations were detected
by PCR and fragment analysis using a fluorescently labeled forward primer as previously
described (5, 6). PCR products were subjected to capillary electrophoresis on denaturing
polyacrylamide gel and analysed by the CEQTM 8000 Genetic Analysis System (Beckman
Coulter). Data were processed using Genetic Analysis System Software (Beckman Coulter).
FLT3 tyrosine kinase domain mutations (FLT3D835/I836) were screened by PCR and EcoRV
restriction enzyme digestion, as previously described (7). JAK2V617Fmutation analysis was
performed by TaqMan® single nucleotide polymorphism genotyping assay using the JAK2
MutaScreen® kit (Ipsogen, Inc.). Real-time PCR assays were performed on an ABI PRISM®
7900HT (Applied Biosystems).
The screening of NRAS, KRAS, and EZH2 mutations was performed by melting curve
analysis and suspected mutations were confirmed by direct sequencing as described in (8,
9). Screening for KIT exon 17 and DNMT3A exon 23 mutations was performed as previously
described (10, 11). Screening for mutations in: IDH1 at R132, IDH2 at R140 and R172, TET2
exon 3 to 11, c-CBL exon 8 and 9, RUNX1 exon 3 to 8, ASXL1 exon 12, SF3B1 exon 13 to
16, U2AF35 exon 6, ZRSR2 exon 1 to 11 and SRSF2 exon 2 was performed by bi-directional
direct sequencing, as described elsewhere (12-17). The complete list of primers is provided
at the end of this section. Seqscape (Applied Biosystems) was used to detect sequence
variations. Gene abnormalities were numbered according to EMBL nucleotide sequence
database. Patients with TET2 nonsense or frameshift or missense variations affecting
conserved positions were considered as mutations, according to (4). ASXL1 nonsense or
frameshift
(but
not
missense)
variations,
including
the
common
c.1934dupG;p.Gly646TrpfsX12 variant were considered as mutations. RUNX1 variations
outside of the RUNT and transactivation domains were considered non pathogenic.
Previously annotated single nucleotide polymorphisms (SNP) (http//www.hapmap.org) were
not considered pathogenic. Assessment of mutations in CD3+ cells was performed with the
same methods. The complete list of variants identified can be provided upon request.
Validation of the cloning strategy by resequencing
To validate the cloning strategy, bulk DNA from sorted CD34+ fractions was prepared as in
(18). KRAS pyrosequencing was performed as previously described (19). Ion Torrent
targeted resequencing of TET2 was performed using Ampliseq® from Life Technologies for
PCR multiplexing, One Touch from Life Technologies for emulsion PCR followed by PGM™
sequencing (Ion Torrent Systems). Detailed procedures are available upon request.
Statistical analyses
Prognostic information from patients was collected at the reference date of December 1st
2011. Overall and progression-free survivals were established by the Kaplan-Meier method
and defined as the time between sampling for genotyping and death from any cause (overall
survival), or death or AML transformation (progression-free survival). All variables with
P<0.05 in univariate analysis were included in a multivariate Cox model, after accounting for
interactions. The final model was retained after forward selection. The proportional hazard
hypothesis was verified by visual display of the Schönfeld residuals and a limited backward
selection was performed to retain significant parameters with P<0.05.
Exome sequencing and analysis
Primary fibroblasts were obtained from patient skin biopsy by mechanical dislocation
followed by culture in F-10 Glutamax® milieu (Gibco) supplemented with 20% FBS (Thermo
Scientific, Inc.), 1% penicilline/streptomycine and amphotericin B 750 ng/mL (Gibco). DNA
from primary fibroblasts and leukemic CD14+ cells was extracted using commercial kits
(Qiagen).
Genomic DNA was captured using Agilent in-solution enrichment methodology (SureSelect
Human All Exon Kits Version 2, Agilent Technologies, Inc.) with their biotinylated
oligonucleotides probes library (Human All Exon v2 – 46 Mb, Agilent Technologies), followed
by paired-end 75 bases massively parallel sequencing on a HiSEQ 2000 sequencer
(Illumina, Inc.) as described in details in (20).
Sequence capture, enrichment and elution were performed according to manufacturer’s
instruction and protocols (SureSelect, Agilent Technologies). Briefly, 3 µg of each genomic
DNA were fragmented by sonication and purified to yield fragments of 150-200 bp. Pairedend adaptor oligonucleotides from Illumina were ligated on repaired, A-tailed, DNA
fragments, then purified and enriched by 4 to 6 PCR cycles. 500ng of these purified Libraries
were hybridized to the SureSelect oligo probe capture library for 24 hr. After hybridization,
washing, and elution, the eluted fraction was PCR-amplified with 10 to 12 cycles, purified and
quantified by QPCR to obtain sufficient DNA template for downstream applications. Each
eluted-enriched DNA sample was then sequenced on an Illumina HiSEQ 2000 as paired-end
75b reads.
Image analysis and base calling is performed using Illumina Real Time Analysis (RTA)
Pipeline version 1.9 with default parameters.
The bioinfomatics analysis of sequencing data was based on the Illumina pipeline
(CASAVA1.7). CASAVA performs alignment of the reads to a reference genome (hg19) with
the alignment algorithm ELANDv2 (performs multiseed and gapped alignments), then calls
the SNPs based on the allele calls and read depth, and detects variants (SNPs&Indels). Only
the positions included in the bait coordinates ± 20bp were conserved. Genetic variation
annotation was performed using the IntegraGen in-house pipeline, which consists on gene
annotation (RefSeq), detection of known polymorphisms (dbSNP 131, 1000Genome)
followed by a mutation characterization (exonic, intronic, silent, nonsense….).
Clonogenic assays for TET2-mutated MDS and MPN samples
Sorted CD34+/CD38- cells were seeded at one cell per well for four to six weeks on a
confluent layer of MS-5 cells in 96-well plates in RPMI medium (Invitrogen, Cergy Pontoise,
France) supplemented with 10% human serum, 5% FCS, 10 ng/mL IL-3, 50 ng/mL SCF, 50
ng/mL fms-like tyrosine kinase 3 ligand (FLT3-L) (Immunex), 10 ng/mL thrombopoitin (Tpo)
(Kyrin laboratories, Tokyo, Japan), 20 ng/mL IL-7, 10 ng/mL IL-15 (Peprotech, London,
United Kingdom), and 5 ng/mL IL-2 (Chiron Laboratories, Suresnes, France), with weekly
media change, as previously described (18).
Sorted CD34+/CD38+ cells were seeded in methylcellulose with FCS and cytokines as
described in Methylcellulose colony-forming cell (CFC) assays. Individual colonies were
picked on day 14.
Sequencing Primers
SRSF2
SRSF2_ex1
ZRSR2
ZRSR2_ex1-2
ZRSR2_ex3
ZRSR2_ex4-5
ZRSR2_ex6
ZRSR2_ex7
ZRSR2_ex8
ZRSR2_ex9
ZRSR2_ex10
ZRSR2_ex11
U2AF35
U2AF35_ex2
U2AF35_ex6
SF3B1
SF3B1_ex13-14
SF3B1_ex15-16
KRAS
KRAS_ex2
KRAS_ex3
NRAS
NRAS_ex2
NRAS_ex3
CBL
CBL_ex8
CBL_ex9
RUNX1
RUNX1_ex3
RUNX1_ex4
RUNX1_ex5
RUNX1_ex6
RUNX1_ex7
RUNX1_ex8
EZH2
EZH2_ex2
EZH2_ex3
EZH2_ex4
EZH2_ex5
EZH2_ex6
EZH2_ex7
EZH2_ex8
EZH2_ex9
EZH2_ex10
EZH2_ex11
EZH2_ex12
EZH2_ex13
EZH2_ex14
EZH2_ex15
EZH2_ex16
EZH2_ex17
Forward primer
Reverse primer
GGCCGCCACTCAGAGCTA
ACCTCACAAAGGTCCGCG
TCTCGACTCTTAGGCCCGCCCTTT
GCTTGTGTTGTACCAAAGAAGG
TTTGCTCTCGTGTGTGTGTG
TGTTCCACTTGAGATTCTTAACCA
CTTGATTGCCTGTTCCAACT
ATGCCTGGTCTAAAGCAGTT
GCAAGAGTCAGCTAGTATCT
GAACTTGGTGGTCCTACAAT
AGTGCTGTTTCATCACTGTGC
CCTTCTGACACTGGGGCTTCAAG
ACAGAAGACTGGTACTGGTTAG
CCCAAACTCTGACATGCCTA
GATCTAACTAACCTACCACG
GTCTGATGACGGACATTTGA
TTATAGAGTGCTAGCGTGCC
TCCAGTGGAAAAATCCCAGA
TCCCCCAAAGAATATCCCTT
AACCCATCTGCGTTCATAGC
GCTGCTGACATATTCCATGTG
AAAGTCTTATTAAAGCGTGGATGG
TCTCAGACCTTCCACTGGAAGT
CGAACTGTGCTCAGTCACGTC
TGATGTGAAAGTGTAGCTTC
TGTTGGGGCATAGTTAAAACCT
GGCAACATAGTAAGACCCTGT
TGTTAGAACCATGAAACATATCCA
AAAGGTACTGGTGGAGTATTTGA
CAGACTGTGTTTCTCCCTTC
CATGAAAATGGTCAGAGAAACC
TAAACCCACCTATAATGGTG
GGCCGATATTAATCCGGTGT
CAAGTGGTTATAGATGGTGAAAC
TGGGTAAAGATGATCCGACA
CAAATGACTTGCTATTATTGATG
GGACCCAGACTAGATGCTTTCT
CTGGCTTTTGGGGTTAGGTT
GAAAATACATTTCCTAGAGATCAAAAA
TCGTTAAGTGTTTTACGGCTTT
AGCTGCTTGCTGAAGATCCG
CATCCCTGATGTCTGCATTTGTCC
TGTTCAGGCCACCAACCTCATTC
CCTGCTCCCCACAATAG
AATCCCACCCCACTTTACAT
TCCGTTCTCTTGCCCGC
GCCTGTCCTCCCACCACCCTCTC
GTGGGTTTGTTGCCATGAAACG
CCCAAGGAATCTGAGACATGGTCC
GGTGCAGGAGAGGCGGGCAG
CTCAGCTGCAAAGAATGTGT
GGCCTGGCGCCTCAGTA
GGTGATCATATTCAGGCTGG
TTTCTCCTTTCCTCTCCTTCA
GGCTACAGCTTAAGGTTGTCCT
AAATCTGGAGAACTGGGTAAAGAC
AGGCTATGCCTGTTTTGTCC
CTGACTGGCATTCCACAGAC
CATCAAAAGTAACACATGGAAACC
TCCATTAATTGACTTTTCCAGTG
TTCTCTTCCATCAAAATGAGTTTTAG
GAGTTGTCCTCATCTTTTCGC
AAGAATGGTTTGCCTAAATAAGAC
TCTTGGCTTTAACGCATTCC
TGATCGTTTCCATCTCCCTG
GAGAGTCAGTGAGATGCCCAG
TTTTTGATGATGTGATTGTGTTTT
TTCTGTCAGGCTTGATCACC
AAACTTATTGAACTTAGGAGGGG
TCCAATAGCATAAACCAAAAGATG
CTGTCTTGATTCACCTTGACAAT
TCATGCCCTATATGCTTCATAAAC
AAAAGAGAAAGAAGAAACTAAGCCC
AAGTGTAGTGGCTCATCCGC
TTGTAATAAATGATAGCACTCTCCAAG
ACCTCCACCAAAGTGCAAAG
TCCTCACAACACGAACTTTCAC
CCAAGAATTTTCTTTGTTTGGAC
CCTTGCCTGCAGTGTCTATC
CAAATTGGTTTAACATACAGAAGGC
AGGGAGTGCTCCCATGTTC
TTTGCCCCAGCTAAATCATC
TGGCAATTCATTTCCAATCA
CTCGTTTCTGAACACTCGGC
EZH2_ex18
EZH2_ex19
EZH2_ex20
ASXL1
ASXL1_ex12_1
ASXL1_ex12_2
ASXL1_ex12_3
ASXL1_ex12_4
ASXL1_ex12_5
ASXL1_ex12_6
DNMT3A
DNMT3A_ex23
KIT
KIT_ex17
TET2
TET2_fragment1
TET2_fragment2
TET2_fragment3
TET2_fragment4
TET2_fragment5
TET2_fragment6
TET2_fragment7
TET2_fragment8
TET2_fragment9
AGGCAAACCCTGAAGAACTG
CCGTCTTCATGCTCACTGAC
CTTCAGCAGGCTTTGTTGTG
TTCCAATTCTCACGTCAAAGGTA
AAAAACCCTCCTTTGTCCAGA
GGGGAGGAGGTAGCAGATG
AGGTCAGATCACCCAGTCAGTT
AGAGGACCTGCCTTCTCTGAGAAA
ACTTGAAAACCAAGGCTCTCGT
GGTGGACAAGGATGAGAAACCCAA
TGGATTCCAAAGAGCAGTTCTCTTC
ACAGGAAAGCTACTGGGCATAGTC
TAGCCCATCTGTGAGTCCAACTGT
TTCGATGGGATGGGTATCCAATGC
GCAACCATCCCATCTGTCCTTGTA
TGTCCTGTGACATAGCACGGACTT
CATGACAAAGGGCATCCCTTCCAA
CAAGAGTGCTCCTGCCTAAAGAGT
TCCTGCTGTGTGGTTAGACG
TTTTTCTCTTCTGGGTGCTGA
TGGTGTACTGAATACTTTAAAACAAAA
TGCAGGACTGTCAAGCAGAG
TGAACTTCCCACATTAGCTGGT
CAAAAGGCTAATGGAGAAAGACGTA
GCCAGTAAACTAGCTGCAATGCTAA
GACCAATGTCAGAACACCTCAA
TTGCAACATAAGCCTCATAAACAG
GCAACTTGCTCAGCAAAGGTACT
ATACTACATATAATACATTCTAATTCCCTCACTG
CATTTCTCAGGATGTGGTCATAGAAT
AGACTTTATGTATCTTTCATCTAGCTCTGG
TET2_fragment10
ATGCCACAGCTTAATACAGAGTTAGAT
TET2_fragment11
TET2_fragment12
GATGCTTTATTTAGTAATAAAGGCACCA
TGTCATTCCATTTTGTTTCTGGATA
TET2_fragment13
TET2_fragment14
TET2_fragment15
TET2_fragment16
TET2_fragment17
IDH1
IDH1-R132
IDH2
IDH2-R140
IDH2-R172
CTGGATCAACTAGGCCACCAAC
GCTCTTATCTTTGCTTAATGGGTGT
AATGGAAACCTATCAGTGGACAAC
CAGAGCTTTCTGGATCCTGACAT
TCTAAGCTCAGTCTACCACCCATCCATACA
GAAACTGTAGCACCATTAGGCATT
GCAGAAAAGGAATCCTTAGTGAACA
TGCCTCATTACGTTTTAGATGGG
TTGATTTTGAATACTGATTTTCACCA
ATTGGCCTGTGCATCTGACTAT
TGCTGCCAGACTCAAGATTTAAA
TGTTTACTGCTTTGTGTGTGAAGG
CCCAATTCTCAGGGTCAGATTTA
ACTCTCTTCCTTTCAACCAAAGATT
TGTCATATTGTTCACTTCATCTAAGCT
AAT
TTCAACAATTAAGAGGAAAAGTTAGAA
TAATATTT
AAATTACCCAGTCTTGCATATGTCTT
CCAAAATTAACAATGTTCATTTTACAA
TAAGAG
TGTACATTTGGTCTAATGGTACAACTG
TATATATCTGTTGTAAGGCCCTGTGA
GCCCACGTCCATGAGAACTATACTAC
TGCTCGCTGTCTGACCAGACCTCATCG
GTGGCACGGTCTTCAGAGA
TTCATACCTTGCTTAATGGGTGT
TGAAAGATGGCGGCTGCAGT
AGCCATCATCTGCAAAAC
GGGGTGAAGACCATTTTGAA
TGTGGCCTTGTACTGCAGAG
Mutation-specific Taqman® allele discrimination assays
GENE
ADCY10
AML1
ASAP1
ASXL1
ASXL1
ASXL1
ASXL1
ATP2C2
BFSP2
CBL
CBL
CBL
CBL
CEP63
CTCF
CTTNBP2
HECW2
JAK2
KRAS
KRAS
KRAS
KRAS
KRAS
KRAS
MYLK
NRAS
NRAS
RUNX1
RUNX1
SF3B1
SH2B3
SIPA1L2
SMOC2
SRSF2
VARIANT
c.3975G>A
c.930delC
c.1423A>G
c.1888_1910del
c.3194G>A
c.1924G>T
c.1900_1922del
c.248C->T
c.494G_>A
c.1219T>C
c.1222T>C
c.1259G>T
c.1211G>A
c.644G->A
c.1133C->T
c.4105G->A
c.2121A>T
c.1849G>T
c.34G>A
c.53C>A
c.34G>T
c.173C>T
c.35G>C
c.350A>G
c.4061G>C
c.35G>A
c.179C>G
c.171_174dupCCAC
c.1126dupT
c.1756G>C
c.10009insT
c.2800G>A
c.203G>A
c.284C>A
FORWARD PRIMER
ACTGATAATGTCGGTTGGGATTCTG
CCGACCTGACAGCGTTCA
CCATTTCCCTATGGATGCCAGAA
TCGCAGACATTAAAGCCCGT
GGACAGATGGGATGGTTGCT
AGGTCACCACTGCCATAGAGA
ACTGGGCTGTCGGAGTTCT
TGTCTCCTTTGGTGTGTGTGATC
CCCTGTGGACACCTCATGTG
GAGCCCTGTGGACACCTCAT
CCCTGTGGACACCTCATGTG
GCCCTGTGGACACCTCATG
GCCAATCAGAAATTCAACACTTAAGCA
ACGTCACATTCGCTCTCATACTG
TGTCTTTTCACAGAGGCTCTTGAC
CCCACTGGGCACCTGTAC
AAGCTTTCTCACAAGCATTTGGTTT
TGCTGAAAATGACTGAATATAAACTTGTG
TGCTGAAAATGACTGAATATAAACTTGTG
TGCTGAAAATGACTGAATATAAACTTGTG
GATGGAGAAACCTGTCTCTTGGAT
AGGCCTGCTGAAAATGACTGAATAT
GAGCCTGTTTTGTGTCTACTGTTCT
GATCTCGATGCTGTAGGACTGTAC
TGCTGAAAATGACTGAATATAAACTTGTG
GGTGAAACCTGTTTGTTGGACATAC
GACCGCAGCATGGTGGAG
GGGAGGCCCGTTCCAA
TTCAGATCCTCGTGGTCATTGAAC
TCAGCCCTAGAGCCTAGCA
GATGGCGGGAAAATAGAAAACATGT
GCATCTGACGGAAGGACCTT
GCTGCGGGTGCAAATGG
REVERSE PRIMER
MUTANT PROBE (FAM)
TGGAATTGTTTTCTTCCCAGGCT
CCCTTCAGATATGGGCAC
GGCGCCTGGATAGTGCAT
CAGTTCCCGCGCTGC
CACCTGGCTTTCAACCAACTTG
TTTGACCTGTGTAGAATG
CTCCACCCGGGCCAC
AGAGGTCACCACTGCC
GGCGGACCGCACATACT
CTCAGAGCTAGGTGTCT
CGCCGCCACCTCCA
CCCCTCAGATGGCA
direct sequencing
GTTGTCAGCAACAAACTCATTCCA
CGCTGCATCACCG
GCCGGGCATTTTCCAAGAC
CTCTAGGTGGATGAGGCA
CAGCCCTGACCTTCTGATTCC
CATCCTGTCTTACACCC
CAGCCCTGACCTTCTGATTCC
TGTCTTACATCCCGGC
CGATGGGTTCAGTACCTTTAATTTC
CTGTCCTTTCTGCCTAT
GTCGCTGTTTAGATCCGTACCT
TGCACATCCTATCTTAC
CAACTCATTGGCACAGATAGTGTCA
ACTGGAGCAGGCTAA
GCTGGCATAACTGCACAAACTG
ACTGAAACAGACGCTCT
GCTGTGGAATGGCGTCATC
AGTTCAAGAAACAATATT
CGAAGGGTCGCAGGAATCC
TGCTGGTTCTTTTCCTGTG
AGAAAGGCATTAGAAAGCCTGTAGTT
TCCACAGAAACATAC
AGCTGTATCGTCAAGGCACTCTT
TTGGAGCTAGTGGC
GGTCCTGCACCAGTAATATGCA
CAAGAGTGACTTGAC
AGCTGTATCGTCAAGGCACTCTT
TGGAGCTTGTGGCGT
GGTCCCTCATTGCACTGTACTC
TTGACCTGCTATGTCGAG
GCTGTATCGTCAAGGCACTCTT
TTGGAGCTGCTGGCGTA
GACTCTGAAGATGTACCTATGGTCCTA
AGGCAAATCACATCTATTT
ACATTCGGAGCTCCTCACTGA
CCTGGTATGCCTCCTC
AGCTGTATCGTCAAGGCACTCTT
TTGGAGCTGATGGC
TGGTCTCTCATGGCACTGTACT
CTTCTTGTCCACCTGTATC
GGGGCTGTCGGTGCG
TGGCCGACCCACCCG
CGAGGCGCCGTAGTACAG
CTCGCCCTCCTACCAC
CGGCCTTCCACTCTAGCAT
TTGATTGATGAACATTACT
CCTAGCTATTGGTTTACCCACCTT
CAGCACAGATTTCCCTTAA
TCGGTAGACAACTGTGCTGAAG
ACATCAGGAAAATTGT
GCAGTTTCCTCGATATGCAATCTCT
TGTGAATTTCAACATGCCAA
ACCGCCCCCGTACCT
CCGCCACCCGGACT
WT PROBE (VIC)
CCTTCAGATGTGGGCAC
CAGTTCCCCGCGCTGC
ATTTTGACCTGTATAGAATG
ACCACTGCCATAGAGAGG
CTCAGAGCTGGGTGTCT
CCCCCTCCGATGGCA
CGCTGCGTCACCG
CTCTAGGTGGGTGAGGCA
ATCCTGTCTTACATCC
CTGTCTTACATCCTGG
CTGTCCTTTCTGCCGAT
TGCACATCCTGTCTTAC
AACTGGAGCGGGCTAA
CTGAAACGGACGCTCT
AGTTCAAGAAGCAATATT
CTGCTGGTTCTTTACCTGTG
TCTCCACAGACACATAC
TAGTTGGAGCTGGTGGC
TAGGCAAGAGTGCCTTG
TTGGAGCTGGTGGC
TTGACCTGCTGTGTCGAG
TTGGAGCTGGTGGCGTA
AGGCAAATCACATTTATTT
CCTGGTATGGCTCCTC
TTGGAGCTGGTGGC
CTTCTTGTCCAGCTGTATC
TGGCCGACCACCCGGGCG
TCGCCCTCCTTACCAC
ATTGATTGATGAAGATTACT
CAGCACAGATTCCCTTAA
ACATCAGGGAAATTGT
TTGTGAATTTCAACGTGCCAA
CCGCCCCCCGGACT
DISEASE
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
PUTATIVE PROTEIN
M1325I
A311RfsX256
I475V
E635RfsX15
W1065X
G642X
E635RfsX15
T83M
G165D
S407P
W408R
R420L
C404Y
R215Q
P378L
A1369T
L707F
V617F
G12S
A18D
G12C
T58I
G12A
K117R
G1354A
G12D
A59G
G60PfsX52
Y376LfsX197
D586H
S337FfsX2
E934K
R68H
P95H
Mutation-specific Taqman® allele discrimination assays (continued)
GENE
VARIANT
FORWARD PRIMER
REVERSE PRIMER
MUTANT PROBE (FAM)
SRSF2
c.284C>T
GCTGCGGGTGCAAATGG
ACCGCCCCCGTACCT
CCGCCTCCCGGACT
SRSF2
c.284C>G
GCTGCGGGTGCAAATGG
ACCGCCCCCGTACCT
CGCCGCCCGGACT
TET2
c.2428 C>T
AAGCGAGTTCGAGACTCATAATGTC
TGCACTTGATTTCATGGTCTGACT
CGATTTATATTCTATACTTCC
TET2
c.2554G>T
CAAACAATACACACCTAGTTTCAGAGAA
TGGGTCTTGTTTCCTGCAAAA
CAGACTACACATCCTTAA
TET2
c.2268_2269insA
GCAACAGCAGCAAAAATTACAAAT
TGCTGATCATTGTTGCTTTGG
AAGAATAAAGAGGAAATAACTCCAG
TET2
c.2407C>T
CAAAATCAAGCGAGTTCGAGACT
GAAACCTGTATTTTGCATGCACTT
ATAATGTCCAAATGGG
TET2
c.4855delC
TCATCTCAAGCTGCAGGTTCA
TGATATGATGGATATTGGGTATTCTGA
GAACCCTTACCTGGGCT
TET2
c.2147C>G
GAATCAACAGGCTTCAGAGACTGA
GTGCTGCCTGTTTATGAGGCTTA
ATTTTCAAACTGACACCTT
TET2
c.1348A>T
CCCAACCAAAGTAACACAACACTT
ATGGATTAGGACTCTGGGAAGGT
TAAGGGAAGTGTAAATAG
TET2
c.4573_4574insA
GCGACTTTCAGGACCAGTCAT
TGTGGTGGCTGCTTCTGTAG
AGCAGTACCCAGCAGC
direct sequencing
TET2
c.5104C>T
TET2
c.4889C>A
ACCCTGGGCTTTTGAATCAGAATAC
GGGAGAATAGGAACCCAGATATGG
CATTGCATTGATATTATGGAT
TET2
c.3467A>T
GAGCAAATTATTGAAAAAGATGAAGGTCCTT
TTCTGTCCAAACCTTTCTTCCATGA
AGCAGGTCCTATTGTGGCA
TET2
c.4360delG
AGTGGTGCCATTCAGGTACTG
CAAGTCTTGACTGGCTCTGCTAA
CATCCTGATTTTCG
TET2
c.3723delT
CCTGGTGTGGGAAGGAATCC
CTCAGCGTCTCGGTAAGCT
TCTCTGGCGACAAAC
TET2
c.3139_40delAC
GTTTCACGCCAAGTCGTTATTTGA
TGGCCCTGACATTTCAACTTTTACT
CCATAAGGCTCTTTCTCAA
direct sequencing
TET2
c.2976_2977insGT
TET2
c.3569_3582del
GCAGCAGTGAAGAGAAGCTACT
ACAATCACTGCAGCCTCACA
TTTGGTGCAGGAGCGA
TET2
c.3641G>A
GCTATTAGGATTGAAAGAGTCATCTATACTGG AATGCCCAAGATTTAAGACCAAAG
GGCAAAAGTTATTGCTAAGTGGG
TET2
c.3764dupA
CTGACAAACTCTACTCGGAGCTTAC
GCACACCGGCGATTGG
CGTGCCGTATTTC
direct sequencing
TET2
c.1680_16781insTCAT
direct sequencing
TET2
c.1669C>T
direct sequencing
TET2
c.3707-3713del
direct sequencing
TET2
c.4619del
direct sequencing
TET2
c.1061C>A
direct sequencing
TET2
c.5541G>A
direct sequencing
TET2
c.2030del
direct sequencing
TET2
c.1378del
direct sequencing
TET2
c.2131del
direct sequencing
TET2
c.4870c>T
direct sequencing
TET2
c.1973del
TRAPPC6B
c.149G>A
CCTACCTGCAAGAAAATTTTCAAATCCA
GTTTCGAGTGGGACAAGGATTGA
CCTGCTCACTTTTCTA
U2AF35
c.470A->G
CCGTGACGGACTTCAGAGAA
ACTGGCCACTCCTCACTCA
TGCCGTCGGTATGAG
ZRSR2
498
GTCAGCTGCAATTTGGAACCT
AAATCAGGAAGACATCCACAAGCA
CAGTACCAGTCATAAGTAT
WT PROBE (VIC)
CCGCCCCCCGGACT
CCGCCCCCCGGACT
ACGATTTATATTCTGTACTTCC
CAGACTACACATCCTGAA
AAGAATAAAGAGGAAATACTCC
ATAATGTCTAAATGGGACTGG
CATGAACCCTTACCCTG
CAAACTCACACCTTTT
AGGGAAGTGAAAATA
GCAGTCCCAGCAGCC
CATTGCATTGATATGATGGAT
AGCAGGTCCTAATGTGGCA
CATCCTGACTTTTCG
TCTCTGGCTGACAAAC
CCATAAGGCTCTTACTCTCAA
TTTGGTGCGGGAGCGA
GTTCTCAGGGATGTCCTATTGC
CGTGCCGTTATTTC
CCTGCTCACCTTTCTA
CTGCCGTCAGTATGAG
CAGTACCAGTCGTAAGTAT
DISEASE
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
CMML
MDS
MDS
MDS
MDS
MPN
MPN
MPN
MPN
MPN
MPN
MPN
MPN
CMML
CMML
CMML
PUTATIVE PROTEIN
P95L
P95R
Q810X
E852X
L757TfsX12
Q803X
P1619LfsX4
S716X
K450X
S1525YfsX53
Q1702X
S1630X
N1156I
V1454SfsX4
D1242TfsX11
T1047SfsX9
M993VfsX15
S1190YfsX3
R1214Q
Y1255X
K561HfsX6
p.Q557X
p.P1237-S1239del
p.P1540fsX31
p.S354X
p.W1847X
p.L667fs
p.S460fs
p.E711fs
p.E1624X
p.H658fs
R50K
Q157R
splice n+1 exon 9
Primer list for variants identified in whole-exome sequencing
GENE
ADCY10
ASAP1
ATP2C2
BFSP2
CEP63
CTCF
CTTNBP2
HECW2
KRAS
MYLK
SH2B3
SIPA1L2
SMOC2
TET2
TRAPPC6B
U2AF35
FORWARD PRIMER
GAACCCAGAATACTCAATAATACGG
GAAATATGAACCCCCATTTCC
GGGAGTCTGTACCCTGTTGC
CTCCCAGTGACCTTGTCTCC
TAGGCTCAGCTTGTCAATCG
TTTTGTGCCTAACCTACTGTGC
TTCACAAGGGTGTGAATTTCC
CTCCTGGTCAGGTACAGTGG
AAGAAACCAAAGCCAAAAGC
CACGCACATTTGTTTCAAGC
ACCTGCCCAGATCCTTAACC
TGGTATAATCATGGTAGCAGTTCC
GCAGCCTTCTGTTTTCTGC
GCAACTTGCTCAGCAAAGGTACT
TTTTCAAATCCAACCTGCTC
AAAGTCTTATTAAAGCGTGGATGG
REVERSE PRIMER
TGGGACATGAGGATACAAGG
TGTTTTAGCTTCCTCTGTAGTTGG
GCAAAGAATGACACCTCTGG
GGCTCCAGAACAGCATTATCC
CAAGTCATTGACCCTCATGG
AATACAGTAAGGAGTGGAGAAGTCC
TGCTCCTAAGGGATGAATCG
TTCTCAGGAGGAGGAAGACG
TGACAAAAGTTGTGGACAGG
TGCAATTCTGGAAAGTGTGG
CCAGAAGACAATGGCCTAGC
GTGATTGGGTGGACATCTGG
GATGGATGTACCCATCTTTGG
TGCTGCCAGACTCAAGATTTAAA
ACAATTTCTGAATACTAACATCCTAGC
CGAACTGTGCTCAGTCACGTC
Additional References
1.
Vardiman JW, Thiele J, Arber DA, Brunning RD, Borowitz MJ, Porwit A, et al. The
2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms
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2.
Cheson BD, Greenberg PL, Bennett JM, Lowenberg B, Wijermans PW, Nimer SD, et
al. Clinical application and proposal for modification of the International Working Group
(IWG) response criteria in myelodysplasia. Blood. 2006 Jul 15;108(2):419-25.
3.
Droin N, Jacquel A, Hendra JB, Racoeur C, Truntzer C, Pecqueur D, et al. Alpha-
defensins secreted by dysplastic granulocytes inhibit the differentiation of monocytes in
chronic myelomonocytic leukemia. Blood. 2010 Jan 7;115(1):78-88.
4.
Delhommeau F, Dupont S, Della Valle V, James C, Trannoy S, Masse A, et al.
Mutation in TET2 in myeloid cancers. N Engl J Med. 2009 May 28;360(22):2289-301.
5.
Boissel N, Renneville A, Biggio V, Philippe N, Thomas X, Cayuela JM, et al.
Prevalence, clinical profile, and prognosis of NPM mutations in AML with normal karyotype.
Blood. 2005 Nov 15;106(10):3618-20.
6.
Kiyoi H, Naoe T, Yokota S, Nakao M, Minami S, Kuriyama K, et al. Internal tandem
duplication of FLT3 associated with leukocytosis in acute promyelocytic leukemia. Leukemia
Study Group of the Ministry of Health and Welfare (Kohseisho). Leukemia. 1997
Sep;11(9):1447-52.
7.
Abu-Duhier FM, Goodeve AC, Wilson GA, Care RS, Peake IR, Reilly JT. Identification
of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia. Br J Haematol. 2001
Jun;113(4):983-8.
8.
Braun T, Itzykson R, Renneville A, de Renzis B, Dreyfus F, Laribi K, et al. Molecular
predictors of response to decitabine in advanced chronic myelomonocytic leukemia: a phase
2 trial. Blood. 2011 Oct 6;118(14):3824-31.
9.
Ernst T, Chase AJ, Score J, Hidalgo-Curtis CE, Bryant C, Jones AV, et al. Inactivating
mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet.
2010 Aug;42(8):722-6.
10.
Boissel N, Leroy H, Brethon B, Philippe N, de Botton S, Auvrignon A, et al. Incidence
and prognostic impact of c-Kit, FLT3, and Ras gene mutations in core binding factor acute
myeloid leukemia (CBF-AML). Leukemia. 2006 Jun;20(6):965-70.
11.
Renneville A, Boissel N, Nibourel O, Berthon C, Helevaut N, Gardin C, et al.
Prognostic significance of DNA methyltransferase 3A mutations in cytogenetically normal
acute myeloid leukemia: a study by the Acute Leukemia French Association. Leukemia. 2012
Jan 13.
12.
Damm F, Kosmider O, Gelsi-Boyer V, Renneville A, Carbuccia N, Hidalgo-Curtis C, et
al. Mutations affecting mRNA splicing define distinct clinical phenotypes and correlate with
patient outcome in myelodysplastic syndromes. Blood. 2012 Feb 17.
13.
Gelsi-Boyer V, Trouplin V, Roquain J, Adelaide J, Carbuccia N, Esterni B, et al.
ASXL1 mutation is associated with poor prognosis and acute transformation in chronic
myelomonocytic leukaemia. Br J Haematol. 2010 Nov;151(4):365-75.
14.
Grossmann V, Kohlmann A, Eder C, Haferlach C, Kern W, Cross NC, et al. Molecular
profiling of chronic myelomonocytic leukemia reveals diverse mutations in >80% of patients
with TET2 and EZH2 being of high prognostic relevance. Leukemia. 2011 May;25(5):877-9.
15.
Kuo MC, Liang DC, Huang CF, Shih YS, Wu JH, Lin TL, et al. RUNX1 mutations are
frequent in chronic myelomonocytic leukemia and mutations at the C-terminal region might
predict acute myeloid leukemia transformation. Leukemia. 2009 Aug;23(8):1426-31.
16.
Boissel N, Nibourel O, Renneville A, Huchette P, Dombret H, Preudhomme C.
Differential prognosis impact of IDH2 mutations in cytogenetically normal acute myeloid
leukemia. Blood. 2011 Mar 31;117(13):3696-7.
17.
Boissel N, Nibourel O, Renneville A, Gardin C, Reman O, Contentin N, et al.
Prognostic impact of isocitrate dehydrogenase enzyme isoforms 1 and 2 mutations in acute
myeloid leukemia: a study by the Acute Leukemia French Association group. J Clin Oncol.
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Study group:
Daniel Re (Antibes), Mathilde Hunault-Berger (Angers), Borhane Slama (Avignon), Lionel
Adès, Thorsten Braun, Claude Gardin (Bobigny), Krimo Bouabdallah (Bordeaux), Benoit de
Renzis (Clermont-Ferrand), François Dreyfus, Didier Bouscary, Jerome Tamburini (Cochin,
Paris), Bertrand Joly (Corbeil-Essonnes), Andrea Toma (Créteil), Bernard Bonotte, JeanNoël Bastie (Dijon), Jean Gutnecht (Fréjus), Gérard Tertian (Le Kremlin-Bicêtre), Kamel
Laribi (Le Mans), Thomas Prébet, Norbert Vey (Marseille), Agnès Guerci (Nancy), Laurence
Legros (Nice), Eric Jourdan (Nimes), Laurence Sahnes (Perpignan), Aspasia Stamatoullas
(Rouen), Anne Vekhoff, Sandra Malak (Saint-Antoine, Paris), Bertrand Arnulf, Nicolas
Boissel, Emmanuel Raffoux (Saint-Louis, Paris), Odile Beyne-Rauzy, Christian Recher
(Toulouse), Stéphane de Botton (Villejuif), included patients.