Genetic Complexity In MPN, MDS/MPN And MDS
Genetic complexity in MPN, MDS/MPNand MDSNick CrossWessex Regional Genetics Laboratory, SalisburyFaculty of Medicine, University of Southampton
Genetic complexity in chronic myeloid neoplasms Classes of mutations Complexity, subclonality and prognosis Specificity of mutations
Myeloproliferative disorders andmyelodysplastic tionEffective haemopoiesisDysplasiaIneffective haemopoiesis
Mutation discovery: cytogenetics, DNA arrays,exome/genome 333323424.124.224.3813der(13)der(8)normal 13der(8)normal 13normal 8normal 8der(8)der(13)der(13)52-M1516-L19Reiter et al., Blood 1998;92:1735-42; Gelsi-Boyer et al., Br J Haematol. 2009;145:788-800; Ernst et al., Nat Genet. 2010;42:722-6
Classes of somatically acquired driver mutation inMPN, MDS/MPN and MDSSIGNALLING EPIGENETICTK C1ASMC3RAD21CYTO5q-7/7q 8 19i(17q)del(11q)del(12p)del(20q)inv(3q)t(3;3)
No abnormality defines specific disease entitiesexcept BCR-ABL1SIGNALLING EPIGENETICTK ASMC3RAD21CYTO5q-7/7q 8 19i(17q)del(11q)del(12p)del(20q)inv(3q)t(3;3)
Drug targetable mutations?SIGNALLING EPIGENETICTK SMC3RAD21CYTO5q-7/7q 8 19i(17q)del(11q)del(12p)del(20q)inv(3q)t(3;3)
Classes of somatically acquired driver mutation inMPN, MDS/MPN and MDSMPNMDS/MPNMDSSignallingSplicing, cohesinsEpigenetic, transcription
Signalling abnormalities Activate growth factor signalling pathways Thought to be largely responsible for proliferative phenotypes Gain of function mutations in transducers of signalling– JAK2 V617F, KIT D816V, RAS mutations– Tyrosine kinase fusion genes e.g. BCR-ABL1, FIP1L1-PDGFRA Loss of function mutations of negative regulators of signalling,– eg CBL, SH2B3 (LNK)
Targeted therapy for myeloid disorders withactivated tyrosine kinasesmidostaurinMastocytosis withKIT D816VimatinibMPN withFGFR1Fusion genesMPN withPDGFRFusion genesimatinibJAK2 inhibitorsMPN withJAK2 V617FLess 3inhibitorsMPN withFLT3Fusion genesruxolitinibMPN withJAK2Fusion genesMore aggressive
Tyrosine kinase fusions in MLN-eo and related q12ETV612p13RET10q11N 7BCR22q11N 7q22N 19813q12CFS112q15September 2014: 60 tyrosine kinase fusion genes
Identification of BCR-ABL1 negative,imatinib responsive patients All known imatinib-responsive fusions are associated withcytogenetically visible abnormalities except for FIP1L1PDGFRA.– Screen patients with Eos-MPN or persistent unexplained eosinophiliafor FIP1L1-PDGFRA (and BCR-ABL1).– Only screen for other fusions if indicated by karyotype: abnormalities of4q11-12 (PDGFRA), 5q31-33 (PDGFRB), 9q34 (ABL)– If cytogenetics fails consider split apart FISH– Screen suspected mastocytosis for KIT D816V; only screen for FIP1L1PDGFRA if eosinophilia present.
Identification of BCR-ABL1 negative,imatinib responsive patients Most BCR-ABL1 negative imatinib responders are male ( 10:1ratio for PDGFR fusions) with eosinophilia. BCR-ABL1 negative imatinib responders are very rare.
JAK2 fusions: clinical responses to ruxolitinibSchwaab et al., Ann Hematol. 2015;94(2):233-8
STAT5 phosphoflow to identify potentialresponders to TKI therapy?Roberts et al., N Engl J Med. 2014;371:1005-15 Untreated vs imatinib, dasatinib,ruxolitinib, ponatinib (1 hour) Overnight fix, permeabilisationSample 1 and Sample 2 response with 080unt60dasatinib4020012
Signalling abnormalities in MDS/MPNGene Up to 50% of CMML cases haveabnormalities that activateTK/RAS signalling Higher frequency in proliferative(WBC 13.0 109/L) vs dysplasticCMML 10-60% of CNL/aCML cases haveactivating CSF3R mutationsMaxson et al.,NEJM 2013;368:11-20Mutated in T 1%Gelsi-Boyer et al. Br J Haematol. 2010;151:365-75Itzykson et al, J Clin Oncol, 2013;31:2428-36Haferlach et al. Leukemia. 2012;26:834-9Plon S. Genet Med. 2011;13:203-4
Epigenetic mutations Epigenetics: heritable (through cell generations) changes ingene expression without changes in DNA sequence Modification of:– DNA (methylation of CpG)DNMT3A, TET2, IDH1/2, WT1– histones (methylation, acetylation etc)EZH2, ASXL1Zhou et al. Nat Rev Genet. 2011;12:7-18
Epigenetic mutations in MPN and -10%CH3CH3DNMT3aCross, Am Soc Hematol Educ Program. 2011:208-14Jankowska et al., Blood. 2011;118:3932-41Grossmann et al. Leukemia. 2011;25:877-9Itzykson R et al, J Clin Oncol. 2013;31:2428-36
Epigenetic mutations in MPN and -10%5-10%40-60%CH3DNMT3aCross, Am Soc Hematol Educ Program. 2011:208-14Jankowska et al., Blood. 2011;118:3932-41Grossmann et al. Leukemia. 2011;25:877-9Itzykson R et al, J Clin Oncol. 2013;31:2428-36
Epigenetic mutations in MPN and VPMFCMMLCH31-5%5-10%5-10%40-60%CH3DNMT3aCross, Am Soc Hematol Educ Program. 2011:208-14Jankowska et al., Blood. 2011;118:3932-41Grossmann et al. Leukemia. 2011;25:877-9Itzykson R et al, J Clin Oncol. 2013;31:2428-36
Epigenetic mutations in MPN and /2:ETPVPMFCMML1-5%5-10%5-10%1-5%CH3CH3DNMT3aCross, Am Soc Hematol Educ Program. 2011:208-14Jankowska et al., Blood. 2011;118:3932-41Grossmann et al. Leukemia. 2011;25:877-9Itzykson R et al, J Clin Oncol. 2013;31:2428-36
Epigenetic mutations in MPN and NMT3aCross, Am Soc Hematol Educ Program. 2011:208-14Jankowska et al., Blood. 2011;118:3932-41Grossmann et al. Leukemia. 2011;25:877-9Itzykson R et al, J Clin Oncol. 2013;31:2428-36
Epigenetic mutations in MPN and 15%CH3DNMT3aCross, Am Soc Hematol Educ Program. 2011:208-14Jankowska et al., Blood. 2011;118:3932-41Grossmann et al. Leukemia. 2011;25:877-9Itzykson R et al, J Clin Oncol. 2013;31:2428-36
Epigenetic mutations in MPN and OHCH2OHCH3CH3DNMT3aCross, Am Soc Hematol Educ Program. 2011:208-14Jankowska et al., Blood. 2011;118:3932-41Grossmann et al. Leukemia. 2011;25:877-9Itzykson R et al, J Clin Oncol. 2013;31:2428-36
No clear association between mutations inepigenetic regulatorsEZH2TET2ASXL1Ernst et al., Nature Genetics. 2010; Grossmann et al., Leukemia 2011
TET2 mutations may be acquired before or afterJAK2 V617F in MPNDelhommeau et al. N Engl J Med. 2009;360(22):2289-301.Schaub et al. Blood. 2010;115(10):2003-7.
Several somatically mutated genes in myeloiddisorders are constitutionally mutated in raredevelopmental ver SyndromeSchinzel-GiedionBohring-Opitz SyndromeOvergrowth syndromeOFCD syndromeCornelia de LangeRubinstein-Taybi syndromeMPN/MDS/T-ALLaCMLMyeloid malignanciesMyeloid malignanciesAML, MDSMDS/MPN, MDSLymphoma, T-ALL, myeloid
Common consequences of EZH2, ASXL1 and othermutations?SETBP1Blood 2012;119:6099-108MLLSETDeregulation of HOXA gene expressionMay provide a stem cell advantage thatpromotes clonal expansion (landscapingmutations)EZH2EEDSUZ12ASXL1Cancer Cell 2012;22:180-93
Genetic complexity in chronic myeloid neoplasms Classes of mutations Complexity, subclonality and prognosis Specificity of mutations
MDS: prevalence of mutations13 genes30 genes 10% 5% 3%34% of totaloncogenic mutationsPapaemmanuil et al. Blood. 2013;122(22):3616-2
Prognostic significance of mutations in specific genes EZH2, RUNX1, TP53, ASXL1 mutations generally associated with poorprognosis in multiple studies (MPN, MDS/MPN, MDS) Is the level of mutation important, ie clonal vs subclonal?Papaemmanuil et al. Blood. 2013;122(22):3616-2
TP53 Mutation Status Divides MDS Patients withComplex Karyotypes into DistinctPrognostic Risk GroupsBejar et al., ASH 2014
Prognosis in MDS associated with somatic geneticcomplexity (111 candidate genes)Papaemmanuil et al. Blood. 2013;122(22):3616-2
Prognosis in CMML associated with somaticgenetic complexity (19 genes; 312 cases)Itzykson et al, J Clin Oncol, 2013;31:2428-36
Prognostic significance of somatic complexity inmyelofibrosis: ASXL1, EZH2, SRSF2, IDH1/2Lasho , Guglielmelli et al. Blood 2013;122:104 ASH 2013
Which genes should be screened for in diagnosticlabs. Who is going to pay? Is it worth paying for?
Is up front mutation profiling the best prognosticindicator? Collaboration between Jackie Boultwood, Andrea Pellagatti(Oxford) and Moritz Gerstung, Elli Papaemmanuil, PeterCampbell (Sanger Institute, Cambridge) 124 MDS cases: NGS mutation screen GEP microarray data(Affymetrix GeneChip Human Plus 2.0 arrays) on bone marrowCD34 cells from 124 MDS patients Built statistical models to disentangle the effect of 12 mutatedgenes and 4 cytogenetic alterations on gene expression,diagnostic clinical variables and outcome in patients with MDSGerstung et al., Nat Commun. 2015 Jan 9;6:5901
prognostic powerPrognostic power of gene expression, mutations andclinical parameters Genetics, gene expression, blood and bonemarrow counts all contain information forpredicting survival Prognostic power of expression data wasgreater than that of genetics, cytogenetics orIPSS score Prognostic information present in genetics andcytogenetics is mostly contained in expressionand blood/bone marrow count data and doesnot add independent prognostic information Gene expression data provide greatest prognostic informationGerstung et al., Nat Commun. 2015 Jan 9;6:5901
Molecular MRD status provides the most powerfulprognostic factor in many scenariose.g. NPM1 mutant AML
Genetic complexity in chronic myeloid neoplasms Classes of mutations Complexity, subclonality and prognosis Specificity of mutations
Somatic myeloid mutations in the generalpopulation n 17,182; 160 genes Mutations rare 40 years old butseen in 10% 70 years old DNMT3A, ASXL1, TET2 Increased risk of hematologicmalignancy (HR 11.1)Jaiswal et al., N Engl J Med. 2014 ;371(26):2488-98 n 12,380; WESMutations 1% 50 yrs; 10% 65 yrsDNMT3A, ASXL1, TET2Increased risk of hematologicmalignancy (HR 12.9) 42% of hematologic malignancies arosein persons who had clonalityGenovese et al., N Engl J Med. 2014;371(26):2477-87
Somatically acquired uniparental disomy (copynumber neutral LOH) in healthy individuals Study of 108 elderly men using the Illumina 1M-Duo beadchipanalysis plus 78 elderly monozygotic twins Somatic abnormalities seenin 3.4% of cases 60 years old Included abnormalitiesassociated with malignancy– del(5q), del(20q), 4q aUPD Blood counts normalForsberg et al., Am J Hum Genet. 2012 ;90:217-28
Is the aUPD at 4q in ULSAM-697 associated with anacquired TET2 mutation? Sanger sequencing entire TET2 coding sequence Found 21bp deletion that disrupts exon 4 Mutation found at ages 71, 82, 88 and 90 but absent infibroblasts
Why do some JAK2 V617F positive patientsdevelop PV and others develop ET? Strength of JAK2 signalling– Higher JAK2 V617F allele burden (%V617F) in PMF and PV compared to ET– Frequent homozygous JAK2 V617F clones in PMF and PV; rare in ET– JAK2 exon 12 mutations associated with erythroid phenotype and showstronger signalling in model systems Constitutional genetic differences?– MPN phenotype in retroviral transplant models dependent on mouse strainZaleskas et al., PLoS One. 2006;1:e18Godfrey et al., Blood. 2012 Sep 27;120(13):2704-7.Scott et al., N Engl J Med. 2007;356:459-68.
Constitutional genetic variation at HBSL1-MYBinfluences whether JAK2 V617F positive casesdevelop ET or PV n 1112 (556 ET, 556 PV) from the UK Top hit (excluding 9p) rs9399137 in HBSL1-MYB polymorphicintergenic regionTapper et al. Nat Commun 2015 in press
Summary Complex interplay between somatic and inherited variants inmyeloid disorders 40 recurrent somatically mutated genes identified that can begrouped into 5 principal functional classes Small subset of genes and overall somatic complexityprognostically significant [but GEP and MRD analysis may bemore powerful] Driver mutations may be present at high level in healthyelderly individuals
AcknowledgementsSalisburyAndy ChaseWill TapperAmy JonesJo ScoreCatherine BryantThomas ErnstWill LeungSanger/ICGCElli PapaemmanuilPeter CampbellJyoti NangaliaCarlo Gambacorti-PasseriniTony GreenMario CazzolaEva Hellstrom-LindbergDavid BowenJackie BoultwoodAndrea PellagattiMannheimAndreas ReiterJuliana SchwaabUppsala Jan DumanskiLars ForsbergGWASAlessandro VannucchiPaola GuglielmelliMario CazzolaGiovanni BarosiRobert KralovicsHeinz GisslingerKonny DöhnerFrank StegelmannAndreas HochhausKaterina ZoiHeike PahlSusanne SchnittgerDavid OscierAndrew DuncombeTony GreenAnna GodfreyClaire HarrisonRosemary GaleAdam MeadAnna SchuhAndrew JackPaul EvansJo EwingMike Griffiths
Itzykson R et al, J Clin Oncol. 2013;31:2428-36 . Epigenetic mutations in MPN and MDS/MPN
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MPN.37,38 Mutant IDH was documented in the presence or ab-sence of JAK2, MPL, and TET2 mutations. IDH mutations are MPN AML MDS MDS/MPN 2008 WHO classification of myeloid malignancies CML PV ET PMF BCR-ABL JAK2 V617F KIT D816V JAK2 Exon 12 MPL W515 Classic Non-classic MPN-eos FGFR1 rearranged PDGFR rearranged CNL CEL-NOS SM MPN-U CMML JMML aCML .
MPN: FLUKE 179/EDA2 7244193 60 C MPN: FLUKE-971 3317402 Magnetic Meter Hanger MPN: TPAK 1115341 CAT III 1000 V, CAT IV 600 V MPN: FLUKE-279FC/IFLEX 336328 CAT IV 600 V MPN: LVD2 7414358 CAT III 1000 V, CAT IV 600 V MPN: TL175 6161460 10A ac 600V ac 10A dc 600V dc
Bladder and Bowel Appliances: Intermittent Catheterization MDS Item H0100D 1 Inflammatory Bowel Disease MDS Item I8000 1 Aseptic Necrosis of Bone MDS Item I8000 1 Special Treatments/Programs: Suctioning Post-admit Code MDS Item O0100D2 1 Cardio-Respiratory Failure and Shock MDS Item I8000 1 Myelodysplastic Syndromes and Myelofibrosis MDS Item .
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