Workflow of molecular investigations in JAK2-negative MPNs - the Munich experience Susanne Schnittger
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Workflow of molecular investigations in JAK2-negative MPNs - the Munich experience
Susanne Schnittger
Cohort
• single centre experience to apply new markers in a daily diagnostic work flow
• total: 20,547 cases with suspected MPN investigated between 8/2005 und 9/2012
• individual patient specific combinations
JAK2V617F
• Most frequently tested marker
• Applied in 17,763 pts
• Melting curve analysis with a sensitivity of 1%
-(d/
dT) F
luor
esce
nce
(640
/530
) -100% WT
5% 1%
-100%Mut-
Schnittger, Leukemia 2006
• In 6,622/17,027 (34%) being JAK2V617F mutated a definite diagnosis of MPN could be made or confirmed
JAK2V617F
65% 58% 85% 24% 10%
42%
6%
0
2,000
4,000
6,000
8,000
10,000
12,000
V617Fpositive V617negative nu
mbers
*1: not futher specified
Unexplained* Leukocytosis Thrombocytosis Polyglobulia
Coincidence of BCR-ABL1 and JAK2V617F
n= %
BCR-ABL-/JAK2- 11.076 62.4%
BCR-ABL+/JAK2- 865 4.9%
BCR-ABL-/JAK2+ 5810 32.7%
BCR-ABL+/JAK2+ 12 0.07%
17.763 100,00
Scott et al., NEJM, 2007
H538DK539LI540S
H538QK539L
N542-E543del
WT
JAK2 Exon12 Mutations
• In 348 cases with JAK2V617F negative PV/Polyglobuly JAK2exon12 mutation was analyzed
• 64/348 (18.3%) were tested positive
• 64/348 (18.3%) were tested positive
• 50% with isolated erythrocytoses
• in median 11 years younger than JAK2V617F mutated PV (56 vs. 67 years, p=0.001)
• Females/Males: 2/1 (p=0.012)
JAK2 Exon12 Mutationen
JAK2 Exon12 Mutations
18.3%
0.6%
1.0%
0
400
800
1,200
1,600
2,000
2,400
2,800
3,200
3,600
positive
negative
n= 0/56 0/28 39/208 8/1452 17/1651 0/12
Cohort: n=2,524
Thrombopoietin Receptor
Amino acid exchange: MPLW515
Pardanani et al., Blood 2006 /Pikman et al., PloS Medicine 2006
MPLW515 Mutations
WT
MPLW515K MPLW515L
Analysis in JAK2V617F negative MPN (n=6660)
MPLW515 Mutations • 294/6660 (4.4%) • W515L , W515K, W515A, W515S und W515R
(Tryptophan> Leucine, Lysin, Alanin, Serin, Arginin)
positive
negative
0 300 600 900
1200 1500 1800 2100 2400 2700 3000
ET PV PMF CMML MPN-U MPN* unexplained thrombocytoses*
9.3%
8.5%
1.3%
number
CBL
L380G/P
I383M/T C384T
D388G D390V/Y
F418S
G397V H398A
C404T/W/Y
W408C/L C416R/S/Y
G415S
P417A/H R420L/Q I423N
I429F/N C419Y C403Y I375C Q379A Q367L V430M L370_C271del
C396Y
I393S
CBL Mutations
Patients with JAK2V617F negative leukocytosis
n=63/636
CBL Mutations in different Entities
13.0%
4.0%
9.2%
0
50
100
150
200
250
300
350
CBL mutated CBL wt
Total: 53/636 (9.9%) cases
- 26/199 CMML (13.0%) - 1/25 PMF (4.0%) - 27/293 MPN-U (9.2%)
Coincidence of other mutations with CBL
Results of TET2 Mutation Analysis
Exon 3 4 Exon11 5 6 7 8 9 10
AA
BOX1 BOX2
L18X L34F
Q108L A118LfsX10
C133WfsX11 L144X
P174H
C237X G355B Gln440X
S460F
P516QfsX23
R544X L615AfsX23
Q674X Q675X
S735R
K753RfsX14 N861TfsX11 N861TfsX12
Y867H E885X Glu885X
Q916X
Q939X
T940PfsX13
C973X P1012L
S1023HfsX3
K1038DfsX16 Q1084P Q1084PfsX19
L1101PfsX29 I1105RfsX23
M1164I R1176GfsX50
C1193S
L1199FfsX19 C1211Y L1212S R1216X
G1256R
N1266S C1271W
F1287LfsX76 F1287S
C1289W S1290X
N1296D N1299X
L1362P S1369X
D1376G A1341
A1434SfxX45
L1531HfsX40 Q1539SfsX32
Q1557CfsX21 F1585S
V1718L V1723S
R1739I
H1778LfsX42 L1819X
G1861R I1873T E1874K
H1893P
Trp1917GlyfsX33
TET2 Mutations according to Diagnosis
338/738 (45.8%)
21.4%
33.3%
32.3%
77.4% 19.8%
11.1%
33.3%
TET2 mutated
TET2 wt
19.8%
Association of other mutations with TET2
28.6%
1.8%
3.6%
8.9%
1.8% 1.8%
3.6%
0
2
4
6
8
10
12
14
16
18
n = 16 1 2 5 1 1 2
% mutated
D1 D2 CXC SET
Q10
9R
Y12
4C
G15
0E
A21
7V
Exon 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Q10
3G
M12
5T
D65
0N
N67
9S
N35
1X
R48
8Q
R67
6H
K14
7E
E36
1Tfs
X29
H68
0R
EZH2 Mutations
• a highly conserved histone H3 lysine 27 (H3K27) methyltransferase
• mutations abolish methyltransferase activity
• EZH2 has oncogenic activity Ernst et al., Nature Genetics, 2010
EZH2 Mutations
18.2%
6.3%
50.0%
4.8%
9.4%
7.9%
0
30
60
90
120
150
180
210
EZH2 mutated
EZH2 wt
• in total: 55/587 (9.4%) cases EZH2 mutated
FIP1L1-PDGFRA Fusion Gene in HES/CEL
• Analysis for FIP1L1-PDGFRA was performed in 2039 cases with suspected HES/CEL or unclear eosinophilia
• 49/2039 (2.4%) were tested positive
nested RT-PCR
• FIP1L1-PDGFRA: 49/2039 (2.4%) • ETV6-PDGFRB: 3/148 (2.0%) • PDGFRA: 2/365 (0.5%) • PDGFRB: 7/367 (1.9%)
• KITD816: 66/294 (22.4%)
Screening for PDGFR Rearrangements
Score et al., Leukemia 2006 Erben et al., Haematologica 2009
Konstitutional Mutations
untersucht mutiert VHL 146 2 EPOR 70 0 HIF2A 61 0 PHD2 35 1
Workflow MPN
Bench et al., BJH, 2012
PV ET PMF
JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
+ - 95% 5% 50% 50% 50%
JAK2exon12
- 10 %
+ MPLW515
BCR-ABL + CML -
- + 5-10 %
Workflow MPN (MLL)
PV ET PMF
JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
+ - 95% 5% 50% 50% 50%
JAK2exon12
- 10 %
+ MPLW515
BCR-ABL + CML -
- + 5-10 %
familiär VHL EPO HIF2A PHD2
Workflow MPN (MLL)
PV ET PMF MPN?
JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
- + 95% 5% 50% 50% 50% 50% 50%
JAK2exon12
- 10 %
+ MPLW515
BCR-ABL + CML -
- + 5-10 %
familiär VHL EPO HIF2A PHD2
familiär MPL THPO
Workflow MPN (MLL)
PV ET PMF MPN?
JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
- + 95% 5% 50% 50% 50% 50% 50%
JAK2exon12
- 10 %
+ MPLW515
Eosinophilie?
FIP1L1-PDGRFA PDGFRA PDGFRB
BCR-ABL + CML -
- + 5-10 %
familiär VHL EPO HIF2A PHD2
familiär MPL THPO
Workflow MPN (MLL)
PV ET PMF MPN?
JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
- + 95% 5% 50% 50% 50% 50% 50%
JAK2exon12
- 10 %
+ MPLW515
Eosinophilie?
FIP1L1-PDGRFA PDGFRA PDGFRB
BCR-ABL + CML -
- + 5-10 %
familiär VHL EPO HIF2A PHD2
+ Mastocytosis
familiär MPL THPO
Workflow MPN (MLL)
KIT
PV ET PMF MPN?
JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
- + 95% 5% 50% 50% 50% 50% 50%
JAK2exon12
- 10 %
+ MPLW515
Eosinophilie?
FIP1L1-PDGRFA PDGFRA PDGFRB
+NHL?
FGFR
BCR-ABL + CML -
- + 5-10 %
familiär VHL EPO HIF2A PHD2
familiär MPL THPO
Molecular Genetics in MPN
+ Mastocytosis
KIT
PV ET PMF MPN?
JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
- + 95% 5% 50% 50% 50% 50% 50%
JAK2exon12
- 10 %
+ MPLW515
Eosinophilie?
FIP1L1-PDGRFA PDGFRA PDGFRB
FGFR
BCR-ABL + CML -
- + 5-10 %
familiär VHL EPO HIF2A PHD2
CMML MPN/MDS
JAK2V617F
- + 10% 90%
+
familiär MPL THPO
+NHL?
Molecular Genetics in MPN
+ Mastocytosis
KIT
PV ET PMF MPN?
JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
- + 95% 5% 50% 50% 50% 50% 50%
JAK2exon12
- 10 %
+ MPLW515
Eosinophilie?
PDGFRA PDGFRB
FGFR
BCR-ABL + CML -
- + 5-10 %
familiär VHL EPO HIF2A PHD2
JAK2V617F
- + 10% 90%
CBL 20%
JAK2V617F
familiär MPL THPO
+NHL?
CMML MPN/MDS
Workflow MPN (MLL)
+ Mastocytosis
KIT
PV ET PMF MPN?
JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
- + 95% 5% 50% 50% 50% 50% 50%
JAK2exon12
- 10 %
+ MPLW515
Eosinophilie?
PDGFRA PDGFRB
FGFR
BCR-ABL + CML -
- + 5-10 %
familiär VHL EPO HIF2A PHD2
JAK2V617F
- + 10% 90%
CBL 20%
JAK2V617F
familiär MPL THPO
+NHL?
SRSF2 NRAS TET2 EZH2 ASXL1 RUNX1 (NPM1) (MLL-PTD)
CMML MPN/MDS
Workflow MPN (MLL)
+ Mastocytosis
KIT
PV ET PMF MPN?
JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
- + 95% 5% 50% 50% 50% 50% 50%
JAK2exon12
- 10 %
+ MPLW515
Eosinophilie?
PDGFRA PDGFRB
FGFR
BCR-ABL + CML -
- + 5-10 %
familiär VHL EPO HIF2A PHD2
JAK2V617F
- + 10% 90%
CBL 20%
TET2
TET2
TET2
TET2
TET2
JAK2V617F
familiär MPL THPO
+NHL?
SRSF2 NRAS TET2 EZH2 ASXL1 RUNX1 (NPM1) (MLL-PTD)
CMML MPN/MDS
Workflow MPN (MLL)
+ Mastocytosis
KIT
PV ET PMF MPN?
JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
- + 95% 5% 50% 50% 50% 50% 50%
JAK2exon12
- 10 %
+ MPLW515
Eosinophilie?
PDGFRA PDGFRB
FGFR
BCR-ABL + CML -
- + 5-10 %
familiär VHL EPO HIF2A PHD2
JAK2V617F
- + 10% 90%
CBL 20%
TET2
TET2
TET2
TET2
TET2
JAK2V617F
familiär MPL THPO
+NHL?
SRSF2 NRAS TET2 EZH2 ASXL1 RUNX1 (NPM1) (MLL-PTD)
CMML MPN/MDS
Workflow MPN (MLL)
+ Mastocytosis
KIT
PV ET PMF MPN?
JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
- + 95% 5% 50% 50% 50% 50% 50%
JAK2exon12
- 10 %
+ MPLW515
Eosinophilie?
PDGFRA PDGFRB
FGFR
BCR-ABL + CML -
- + 5-10 %
familiär VHL EPO HIF2A PHD2
JAK2V617F
- + 10% 90%
CBL 20%
TET2
TET2
TET2
TET2
TET2
JAK2V617F
familiär MPL THPO
+NHL?
SRSF2 NRAS TET2 EZH2 ASXL1 RUNX1 (NPM1) (MLL-PTD)
CMML MPN/MDS
Workflow MPN (MLL)
+ Mastocytosis
KIT
PV ET PMF MPN?
JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
+ - JAK2V617F
- + 95% 5% 50% 50% 50% 50% 50%
JAK2exon12
- 10 %
+ MPLW515
Eosinophilie?
PDGFRA PDGFRB
FGFR
BCR-ABL + CML -
- + 5-10 %
familiär VHL EPO HIF2A PHD2
JAK2V617F
- + 10% 90%
CBL 20%
TET2
TET2
TET2
TET2
TET2
JAK2V617F
familiär MPL THPO
+NHL?
SRSF2 NRAS TET2 EZH2 ASXL1 RUNX1 (NPM1) (MLL-PTD)
CMML MPN/MDS
Workflow MPN (MLL)
+ Mastocytosis
KIT
SUGGESTION WORKFLOW
Schnittger et al, Haematologica, 2012
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