Susanne Schnittger Workflow of molecular investigations in JAK2negative MPNs the Munich experience
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
Most frequently tested marker Applied in 17,763 pts Melting curve analysis with a sensitivity of 1% (d/dt) Fluorescence (640/530) 5% 1% 100%Mut 100% WT
In 6,622/17,027 (34%) being mutated a definite diagnosis of MPN could be made or confirmed
Coincidence of BCRABL1 and n= % BCRABL/JAK2 11.076 62.4% BCRABL/JAK2 865 4.9% BCRABL/JAK2 5810 32.7% BCRABL/JAK2 12 0.07% 17.763 100,00
JAK2 Exon12 Mutations H538QK539L WT H538DK539LI540S N542E543del Scott et al., NEJM, 2007 In 348 cases with negative PV/Polyglobuly JAK2exon12 mutation was analyzed 64/348 (18.3%) were tested positive
JAK2 Exon12 Mutationen 64/348 (18.3%) were tested positive 50% with isolated erythrocytoses in median 11 years younger than mutated PV (56 vs. 67 years, p=0.001) Females/Males: 2/1 (p=0.012)
JAK2 Exon12 Mutations Cohort: n=2,524 3,600 1.0% 3,200 2,800 2,400 2,000 1,600 1,200 800 0.6% positive negative 400 18.3% 0 n= 0/56 0/28 39/208 8/1452 17/1651 0/12
MPLW515 Mutations WT MPLW515K MPLW515L Thrombopoietin Receptor Amino acid exchange: MPLW515 Pardanani et al., Blood 2006 /Pikman et al., PloS Medicine 2006
MPLW515 Mutations 294/6660 (4.4%) W515L, W515K, W515A, W515S und W515R (Tryptophan> Leucine, Lysin, Alanin, Serin, Arginin) 3000 2700 2400 2100 1800 1500 positive negative 1200 900 600 300 0 ET PV PMF CMML MPNU MPN* unexplained thrombocytoses*
CBL Mutations CBL C404T/W/Y F418S I383M/T I393S H398A P417A/H R420L/Q L370_C271del C384T D390V/Y G397V C416R/S/Y I423N V430M Q367L I375C Q379A L380G/P D388G C396Y C403Y W408C/L G415S C419Y I429F/N
CBL Mutations in different Entities Total: 53/636 (9.9%) cases 26/199 CMML (13.0%) 1/25 PMF (4.0%) 27/293 MPNU (9.2%) 350 9.2% 300 250 13.0% 200 150 CBL mutated CBL wt 100 50 4.0% 0
Coincidence of other mutations with CBL
Results of Mutation Analysis Exon 3 4 5 6 7 8 9 10 Exon11 AA BOX1 BOX2 L18X Q108L C237X L34F A118LfsX10 C133WfsX11 L144X P174H G355B P516QfsX23 Q674X K753RfsX14 Q916X P1012L M1164I G1256R L1362P L1531HfsX40 V1718L H1778LfsX42 Trp1917GlyfsX33 Gln440X R544X Q675X N861TfsX11 C973X R1176GfsX50 S1369X Q1539SfsX32 V1723S L1819X N861TfsX12 N1296D A1434SfxX45 R1739I G1861R S460F L615AfsX23 S735R S1023HfsX3 C1193S N1299X Q1557CfsX21 I1873T Y867H Q939X K1038DfsX16 L1199FfsX19 F1585S E1874K E885X Q1084P C1211Y A1341 H1893P Glu885X Q1084PfsX19 L1212S D1376G R1216X T940PfsX13 L1101PfsX29 N1266S I1105RfsX23 C1271W F1287LfsX76 F1287S C1289W S1290X
Mutations according to Diagnosis 338/738 (45.8%) 77.4% 19.8% mutated 21.4% 32.3% wt 33.3% 11.1% 33.3%
Association of other mutations with 18 28.6% 16 14 12 10 8 6 8.9% 4 2 1.8% 3.6% 1.8% 1.8% 3.6% 0 n = 16 1 2 5 1 1 2
EZH2 Mutations Ernst et al., Nature Genetics, 2010 Exon 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 D1 D2 CXC SET Q103G Q109R Y124C M125T K147E G150E A217V N351X R488Q E361TfsX29 D650N R676H N679S H680R
EZH2 Mutations in total: 55/587 (9.4%) cases EZH2 mutated 210 9.4% 180 150 7.9% 120 EZH2 mutated 90 4.8% EZH2 wt 60 30 18.2% 6.3% 50.0% 0
FIP1L1PDGFRA Fusion Gene in HES/CEL Analysis for FIP1L1PDGFRA was performed in 2039 cases with suspected HES/CEL or unclear eosinophilia nested RTPCR 49/2039 (2.4%) were tested positive
Screening for PDGFR Rearrangements Score et al., Leukemia 2006 Erben et al., Haematologica 2009 FIP1L1PDGFRA: 49/2039 (2.4%) ETV6PDGFRB: 3/148 (2.0%) PDGFRA: 2/365 (0.5%) PDGFRB: 7/367 (1.9%) KITD816: 66/294 (22.4%)
Konstitutional Mutations untersucht mutiert VHL 146 2 EPOR 70 0 HIF2A 61 0 PHD2 35 1
Workflow MPN Bench et al., BJH, 2012
Workflow MPN (MLL) CML BCRABL PV ET PMF 95% 5% 50% 50% 50% 10 % JAK2exon12 510 % MPLW515
Workflow MPN (MLL) CML BCRABL PV ET PMF 95% 5% 50% 50% 50% 10 % JAK2exon12 510 % MPLW515 VHL EPO HIF2A PHD2
Workflow MPN (MLL) CML BCRABL PV ET PMF MPN? 95% 5% 50% 50% 50% 50% 50% 10 % JAK2exon12 510 % MPLW515 VHL EPO HIF2A PHD2 MPL THPO
Workflow MPN (MLL) CML BCRABL PV ET PMF MPN? 95% 5% 50% 50% 50% 50% 50% 10 % JAK2exon12 510 % MPLW515 Eosinophilie? VHL EPO HIF2A PHD2 MPL THPO FIP1L1PDGRFA PDGFRA PDGFRB
Workflow MPN (MLL) CML BCRABL PV ET PMF MPN? 95% 5% 50% 50% 50% 50% 50% 10 % JAK2exon12 510 % MPLW515 Eosinophilie? VHL EPO HIF2A PHD2 MPL THPO FIP1L1PDGRFA PDGFRA PDGFRB KIT Mastocytosis
Molecular Genetics in MPN CML BCRABL PV ET PMF MPN? 95% 5% 50% 50% 50% 50% 50% 10 % JAK2exon12 VHL EPO HIF2A PHD2 510 % MPLW515 MPL THPO Eosinophilie? FIP1L1PDGRFA PDGFRA PDGFRB NHL? FGFR KIT Mastocytosis
Molecular Genetics in MPN CML BCRABL PV ET PMF MPN? CMML MPN/MDS 95% 5% 50% 50% 50% 50% 50% 90% 10% 10 % JAK2exon12 VHL EPO HIF2A PHD2 510 % MPLW515 MPL THPO Eosinophilie? FIP1L1PDGRFA PDGFRA PDGFRB NHL? FGFR KIT Mastocytosis
Workflow MPN (MLL) CML BCRABL PV ET PMF MPN? CMML MPN/MDS 95% 5% 50% 50% 50% 50% 50% 90% 10% 10 % JAK2exon12 VHL EPO HIF2A PHD2 510 % MPLW515 MPL THPO Eosinophilie? PDGFRA PDGFRB NHL? FGFR KIT Mastocytosis CBL 20%
Workflow MPN (MLL) CML BCRABL PV ET PMF MPN? CMML MPN/MDS 95% 5% 50% 50% 50% 50% 50% 90% 10% 10 % JAK2exon12 VHL EPO HIF2A PHD2 510 % MPLW515 MPL THPO Eosinophilie? PDGFRA PDGFRB NHL? FGFR KIT Mastocytosis CBL 20% SRSF2 NRAS EZH2 ASXL1 RUNX1 (NPM1) (MLLPTD)
Workflow MPN (MLL) CML BCRABL PV ET PMF MPN? CMML MPN/MDS 95% 5% 50% 50% 50% 50% 50% 90% 10% 10 % JAK2exon12 VHL EPO HIF2A PHD2 510 % MPLW515 MPL THPO Eosinophilie? PDGFRA PDGFRB NHL? FGFR KIT Mastocytosis CBL 20% SRSF2 NRAS EZH2 ASXL1 RUNX1 (NPM1) (MLLPTD)
Workflow MPN (MLL) CML BCRABL PV ET PMF MPN? CMML MPN/MDS 95% 5% 50% 50% 50% 50% 50% 90% 10% 10 % JAK2exon12 VHL EPO HIF2A PHD2 510 % MPLW515 MPL THPO Eosinophilie? PDGFRA PDGFRB NHL? FGFR KIT Mastocytosis CBL 20% SRSF2 NRAS EZH2 ASXL1 RUNX1 (NPM1) (MLLPTD)
Workflow MPN (MLL) CML BCRABL PV ET PMF MPN? CMML MPN/MDS 95% 5% 50% 50% 50% 50% 50% 90% 10% 10 % JAK2exon12 VHL EPO HIF2A PHD2 510 % MPLW515 MPL THPO Eosinophilie? PDGFRA PDGFRB NHL? FGFR KIT Mastocytosis CBL 20% SRSF2 NRAS EZH2 ASXL1 RUNX1 (NPM1) (MLLPTD)
Workflow MPN (MLL) CML BCRABL PV ET PMF MPN? CMML MPN/MDS 95% 5% 50% 50% 50% 50% 50% 90% 10% 10 % JAK2exon12 VHL EPO HIF2A PHD2 510 % MPLW515 MPL THPO Eosinophilie? PDGFRA PDGFRB NHL? FGFR KIT Mastocytosis CBL 20% SRSF2 NRAS EZH2 ASXL1 RUNX1 (NPM1) (MLLPTD)
SUGGESTION WORKFLOW Schnittger et al, Haematologica, 2012