Basi biologiche e molecolari delle malattie mieloproliferative

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1 Basi biologiche e molecolari delle malattie mieloproliferative

2 Chronic Myeloproliferative Neoplasms BCR-ABL1 CML Classical CNL CEL-NOS Mastocytosis MPN-u JAK2 ex12 Polycythemia Vera >95% MPL W515L/K 3-8% Essential Thrombocythemia Primary Myelofibrosis 70% 60% JAK2 V617F Reviewed in Vannucchi et al. CA Cancer J Clin. 2009; 59(3):171-91

3 Sono malattie clonali derivate dalla trasformazione di una cellula progenitrice emopoietica pluripotente che, avendo conservato una pressochè normale capacità differenziativa, acquisce un vantaggio ed una indipendenza proliferativa, esprimendosi con un aumento preferenziale di una o più o tutte le linee emopoietiche mieloidi AA Lezioni di Ematologia prof A.M. Vannucchi 3

4 Predominanza clonale vs normale emopoiesi policlonale Cellula uovo X A X B Spermatozoo X A X B ZIGOTE X A X B X A X B X A X B EMBRIONE Lyonizzazione X A X B X A X B X A X B X A X A X B X B X A X A Emopoiesi policlonale Emopoiesi monoclonale AA Lezioni di Ematologia prof A.M. Vannucchi 4

5 Policitemia vera Trombocitemia essenziale Mielofibrosi [Primaria] LEUCEMIA ACUTA MIELOIDE AA Lezioni di Ematologia prof A.M. Vannucchi 5

6 La mutazione JAK2 V617F Una G-T mutazione a livello del nucleotide 1849 (nell esone 14) porta alla sostituzione di valina con fenilalanina in posizione 617 (V617F) Il codone mutato V617F si trova nel dominio pseudochinasico Ulteriori mutazioni/delezioni/inserzioni sono state dimostrate a livello dell esone 12 AA Lezioni di Ematologia prof A.M. Vannucchi 6

7 JAK2 functional domains James et al. Nature 2005; 434: 1144 ; Baxter et al. Lancet 2005; 365: 9464; Levine et al. Cancer Cell 2005; 7:387; Kralovics et al. NEJM. 2005: 352:1779; Scott et al. NEJM. 2007; 356:.459.

8 JAK2 signalling Vannucchi et al. CA Cancer J Clin. 2009; 59(3):171-91

9 FERM-domain SH2 Pseudokinase domain Kinase domain NH 2 COOH JH7 JH6 JH5 JH4 JH3 JH2 JH1 IL-3R IL-5R GM-CSFR IL-12R IL-23R IL-6R IL-11R OSMR LIFR d EpoR TpoR G-CSFR GHR PrlR IFNRs IL-10R IL-22R IL-28R IL-19R IL-20R IL-24R IL-29R IL-2R IL-4R IL-7R IL-9R IL-15R IL-21R ANGIOGENESIS INFLAMMATION IMMUNE RESPONSE PROLIFERATION APOPTOSIS JAK1 JAK2 JAK3 TYK2 Vannucchi AM, NEJM 2010;

10 Spectrum of MPNs with JAK2 V617F mutations Polycythemia Vera >95% Esential Thrombocythemia 50%-70% Primary Myelofibrosis 50%-60% MPN-MDS-U 12%-20% CNL 20% CMML 3%-13% JCMML 20% RARS-T 50%-70% MASTOCYTOSIS EOSINOPHILIC neoplasms Co-existing CML and MPN Very unfrequent Very unfrequent Rare, but well characterized

11 JAK2 V617F homozygosity and allele burden UPD at 9p24 PV WT Hetero JAK2 V617F allele burden (%) ET Homo BFU-E EEC CFU-G 0 PV ET PMF PPV/ET MF Adapted from Antonioli et al. Haematologica 2008; 93:41 Scott et al. Blood. 2006; 108:2435 The biallelic JAK2 V617F prevalence in ET is approximately 3-5% Dupont et al. Blood 2007; 110:1013 Kralovics et al. Exp Hematol 2002; 30:209 Kralovics et al. NEJM 2005; 352:1779

12 JAK2 V617F models: retroviral PV MF Zaleska, Plos One 2006

13 JAK2 V617F models: transgenic Retroviral transduction Transgenic mice VavCre; FF1 MxCre; FF1 wt V617F wt V617F wt V617F PV-like disease erythrocytosis NO thrombocytosis neutrophilia ET-like disease NO erythrocytosis thrombocytosis moderate neutrophilia PV-like disease erythrocytosis thrombocytosis neutrophilia Tiedt R, Blood 2008

14 JAK2 V617F models: conditional knock-in - ET 12 wks after transplantation Li, J. et al. Blood 2010;116:1528

15 JAK2V617F level determines disease severity KI mouse with JAK2V617F under endogenous JAK2 promoter control Heterozygous mice has features of PV homozygosity Significantly greater leukocytosis, reticulocytosis, thrombocytosis Marked expansion of erythroid progenitors and EPO-independent colonies Larger spleen size Bone marrow fibrosis Akada H, Blood 2010

16 MPL W515 mutations W515L and W515K promote G 1 /S transition after cytokine withdrawal Chalignè et al. Leukemia 2008; 22: 1557

17 MPL W515L murine model MPL WT MPL W515L

18 MPL Ser505Asn Hereditary Thrombocythemia: a phenotype close to ET First described in a japanese family with HT Relatively frequent in Italy (n=21 cases in 8 families) Median platelet count 795x10 9 /L (426-2,500) Splenomegaly in 45%, major thrombosis in 19%; BM fibrosis with age Overall survival and thrombosis-free survival significantly reduced vs nonthrombocytotic siblings Asn505 mutant produces homodimers and causes phosphorylation of intracellular signals Asn505 mutant causes TPO-independent cell growth Ding J. Blood 2004; 103:4198; Teofili L. Haematologica 2009; Ding J. Blood 2009

19 Genetic complexity of MPN 100% cells / alleles 100% cells / alleles Kralovics et al. Leukemia. 2008; 22(10):

20 Mutations in TET2 FRAMESHIFT, NONSENSE AND MISSENSE MUTATIONS TET2 mutations can be monoallelic or biallelic, less frequently, suggesting loss of function as the mechanism and supporting its role as a tumor suppressor gene TET2 mutations did not seem to affect survival, leukemic transformation, thrombosis risk or cytogenetic profile Delhommeau F et al. NEJM 2009; 22: 2289; Bacher U et al. Ann Hematol 2010; online

21 Mutations in TET2 MPN (n=791) 8%-13% SM (n=42) 29% CMML (n=279) 20%-51% MDS (n=279) 19%-26% RARS-T (n=19) 5 pts (25%) AML (n=128) 12%-20% Post-MPN AML (n=19) 32% Familial MPN (n=61) 20% rev in Bacher U et al. Ann Hematol 2010; online Kosmider O et al. Haematologica 2009;94:1676

22 Genome-wide SNP analysis of aupd discovered CBL mutations at 11q Cytosolic protein capable of dual fuction: -Negative regulation of kinase signaling mediate by E3 ubiquitin ligase activity -Adaptor protein function with a positive effect on downstream signaling Knockout mouse display : -Expanded hematopoietic stem cell pool -Splenomegaly -Growth factor sensitivity of hematopoietic progenitor cells Grand et al. Blood. 2009; 113: 6182 Makishima H, JCO. 2009; 27:6109

23 CBL mutations PV-ET (n=74-24) 0 PMF (n=53) 6% CMML (n=78) 13% MDS (n=116) 1% MDS/MPN (n=98) 10% acml (n=152) 8% RARS-T (n=19) 0 paml (n=97) 1% saml (n=110) 12% Makishima H, JCO. 2009; 27:6109 Grand et al. Blood. 2009; 113: 6182

24 Mutations of ASXL1 in MPNs A dditional S ex C ombs L ike 1 (20q11) Activator/suppressor activity of retinoic acid receptor transcription Not acquired during leukemic transformation PV (n=10) 0 ET (n=35) 1 PMF (n=10) 3 CMML (n=78) 13% PET-AML (n=1) 1 Overall (n=64) 5 (7.8%) Mostly heterozygous frameshit mutations in exon 12 leading to loss of PHD domain in 10% MDS and 40% CMML None of ASXL1 mut was JAK2 V617F pos, one was TET2 mut

25 LNK mutations in ET, PMF, PV Described in one ET and one PMF patient (Oh ST, 2010) Found in 2/8 patients with unexplained erythrocytosis, low sepo (Lasho T, 2010) Found in 8/61 pts (=13%) with MPN-blast phase (Pardanani A, 2010) 6 had diagnosis of PMF, 2 of PV Acquired in 5 at blast phase, 1 in both chronic and blast phase, in 2 lost at blastic tranformation 3 were JAK2V617F pos, 1 was IDH2R142Q mut Oh ST, Blood 2010; 116:988; Lasho T, NEJM 2010; 363:1189; Pardanani A, leukemia 2010; 24:1713.

26 EZH2 mutations L iperespressione della proteina EZH2 è stata associata all aggressività e alla prognosi infausta in diversi tumori solidi. Il gene EZH2 è localizzato in una regione cromosomica critica per i disordini mieloidi. Disomia uniparentale acquisita a livello del 7q in 12 pazienti con MPN/MDS Microdelezione al 7q36.1 in un paziente Microdelezione in eterozigosi (identificata mediante CGH) che coinvolge i geni C7orf33, CUL1, EZH2. EZH2 possibile gene coinvolto nella patogenesi delle neoplasie mieloidi Ernst et al., Nature Genetics 2010

27 Mappa sul braccio lungo del cromosoma 7 7q35-q36 È composto da 20 esoni Codifica per un membro delle Polycomb Group Protein (PcG), rappresenta la subunità catalitica del Polycomb Repressive Complex 2 (PRC2) È una metiltransferasi, metila la lisina 27 dell istone H3 (HEK27)

28 Ernst et al., Nature Genetics 2010 EZH2 mutations in myeloid disorders

29 Leukemic transformation in MPNs Leukemia occurs in 4% to 8% of PV or ET pts within 18 yrs from diagnosis, and in 8% to 23% of MF pts in the 10 yrs after diagnosis A variable proportion (from 9% to 53%) of saml from JAK2 V617F pos MPN are JAK2 WT However, JAK2 V617F pos saml was invariably preceded by PMF or myelofibrotic transformation of ET/PV Campbell PA, Blood 2006,108:3548; Theocarides A, Blood 2007,110:375; Beer PA, Blood 2010

30 Two routes to leukemic transformation in JAK2 mut MPNs (i) Normal HSC Pre-JAK2 mutation Founder clone JAK2 mutation MPN Other mutation(s) AML Normal HSC 1 JAK2 mutation MPN (ii) Normal HSC 2 Other mutation(s) AML

31 Green A, Beer P, NEJM 2010; 362: Somatic mutations of IDH1 and IDH2 in the leukemic transformation of myeloproliferative neoplasms

32 IDH1 and IDH2 mutations lead to biochemical gain of function IDH, Isocitrate dehydrogenase (IDH1 =cytoplasmic; IDH2 = mitocondrial) Mutations first discovered in gliomas and secondary glioblastomas Detected by sequencing an AML genome, preferentially clustering with cytogenetically normal AMLs Isocitrate NADP IDH1 IDH2 NADPH α-ketoglutarate IDH1 R132 IDH2 R172 2-hydroxyglutarate Yan H et al. NEJM 2009; 360:765-9 Mardis E et al, NEJM 2009; 361: Gross S et al, JEM, 2010; 207:339

33 Genetic analysis of leukemic transformation All four possible mutational combinations were observed JAK2 (36.8%) TET2 (26.3%) IDH1 (9.5%) ASXL1 (19.3%) In paired MPN saml samples: TET2 mut acquired at saml ASXL1 mut almost always in both Abdel-Wahab O, Cancer Research 2010; online

34 IKZF1 mutations in MPN Modulate expression of lineage-specific genes through chromatin remodeling > Lymphoid development and tumor suppression 19% Blast-phase MPN

35 Abnormal epigenetic gene regulation in MPN JAK2 goes nuclear microrna profile HEL UKE1 K562 CD34 + TET1 catalyzes conversion of 5-methyl-Cyt - 5-OH-methylCyt

36 Germline & somatic, and post-genetic, events concur to MPN pathogenesis Vannucchi et al. Haematologica. 2008; 93:972

37 Outline Somatic mutations JAK2 - TET2 MPL - ASXL1 CBL - IDH1/2 Germline genetic variations Clinical correlates of genotype

38 Increased risks of PV, ET and PMF among 24,577 first-degree relatives of 11,039 patients with myeloproliferative neoplasms in Sweden Any MPN PV ET PMF Proband Any PV N.A ET MF 2.7 NA NA NA Landgren O, Blood 2008, 112:2199

39 Germline genetics at the JAK2 locus Germline JAK2 haplotype 46/1 or GGCC is a very-low penetrance predisposition allele found in 50 of healthy subjects that accounts for 50% of risk in first-degree relatives based on a calculated RR of 5.7%, but does not account alone for familial MPNs nor does it explain the phenotypic diversity of JAK2 V617F mut MPNs Landgren et al. Blood 2008; 112:2199; Jones et al. Nat Genet 2009; 41:446; Kilpivara et al. Nat Genet. 2009; 41:455; Olcaydu et al. Nat Genet. 2009; 41:450; Olcaydu et al. Leukemia 2009; 23:1924

40 Germline genetics at the JAK2 locus Hypermutability hypothesis V617F in cis to both alleles of nearby heteroz SNP in 3% of MPN occasional cases with both V617F and exon 12 mutations 46/1 haplotype increased in JAK2 exon 12 mutations Fertile ground hypothesis 46/1 haplotype increased in MPL mut and JAK2 wt /MPL wt MPN Campbell et al. Nat Genet. 2009; 41: 385; Jones et al. Nat Genet. 2009; 41:446; Jones et al. EHA 2009; 135A

41 Proportion of JAK2 positive patients 46/1 (GGCC) haplotype and prognosis in PMF CT CC TT N= 130 TT CT CC N= 202 P=0.56 Overall Survival (months) Tefferi A et al, Leukemia 2009; 24:105 Guglielmelli P et al, Leukemia 2010,

42 Outline Somatic mutations JAK2 - TET2 MPL - ASXL1 CBL - IDH1/2 Germline genetic variations Clinical correlates of genotype

43 Diagnostic criteria for classic MPN according to 2008 WHO classification Diagnostic

44 Probability of thrombosis-free survival Thrombosis-free Survival in PV and ET PV n=397 CE=59 PV (14.9%) n=397 CE=59 (14.9%) ET ET n=637 n=637 CE=73 (11.5%) CE=73 (11.5%) Cause of death % Fatal thrombosis 41 Hemorrhages 4 AML/MF 13 Other cancers Other cause Time (months) Wasserman LR, J Mt Sinai Hsp 1959; Chievitz E, Ac Med Scand 1962; Vannucchi AM, Blood 2007; Marchioli R, JCO 2005

45 A continuum between JAK2V617F pos ET and PV JAK2V617F pos ET resembles PV: - increased erythropoiesis lower ferritin lower MCV lower sepo - higher leukocyte count - more venous thromboses - more frequent PV transformation Campbell P, Lancet 2006

46 JAK2 V617F Status and Thrombosis in ET Venous thrombosis 2.09 [1.44, 3.05] Arterial thrombosis 1.96 [1.43, 2.67] Thrombosis at presentation 1.88 [1.38, 2.56] Ziakas PD et al, Haematologica 2008

47 V617F Burden and Thrombosis in ET HR (95%CI) WT Hetero ( ) 3.97 Homo * ( ) P adjusted for age, sex, previous thrombosis and leukocytosis Vannucchi AM et al, Blood 2007

48 Relative Risk Relative Risk Relative Risk JAK2 V617F Burden and Thrombosis in PV Total thrombosis P= At diagnosis In follow-up P=0.003 V617F burden (%) Rate of total thrombosi s HR (95%CI) % % 1.2 ( ) % 1.7 ( ) % 3.56 ( ) P NS NS JAK2 V617F allele burden (%) Vannucchi AM et al, Leukemia 2007

49 Prognostic relevance of a low JAK2 V617F burden in PMF 1 st <25% 2 nd 26-50% 3 rd 51-75% 4 th % WT V617F + 2 nd 3 rd 4 th 1 st P=0.73 P= Guglielmelli P. Blood 2009;114:

50 The phenotype of MPL W515 mutant ET patients- I 776 pts PT-1 cohort 32 cases (4.1% of all ET), 8.5% of JAK2V617Fwt 1 pt coexpressed MPLW515 and JAK2V617F W515L was most common (80%), three S505N W515K allele burden greater than W515L (80% vs 0 had >50% mutant allele) 994 pts GIMEMA cohort 30 cases (3.0% of all ET), 5.2% of JAK2V617Fwt 8 pts coexpressed MPLW515 and JAK2V617F W515L was most common (80%) W515K allele burden greater than W515L (53±26% vs 37±23%) Beer P. Blood 2008; 112:141; Vannucchi AM. Blood 2008; 112:844

51 The phenotype of MPL W515 mutant ET patients- II PT-1 cohort Older than MPLwt/JAK2wt Lower Hb than MPLwt/JAK2V617F Higher platelet count than MPLwt/ JAK2V617F Endogenous Mk colonies but not EEC unlike JAK2V617F No impact on arterial & venous thrombosis, evolution to MF Reduced total, Gran & Ery cellularity GIMEMA cohort Older than MPLwt/JAK2wt Lower Hb than MPLwt/JAK2V617F Higher platelet count than MPLwt/ JAK2V617F More arterial events during FU More microvessel disturbances No difference in evolution to MF Reduced total and Ery cellularity Increased number of Mks, Mk clusters, small Mks, but not reticulin However, our findings did not result in the definition of a discrete clinical phenotype. Beer P. Blood 2008; 112:141; Vannucchi AM. Blood 2008; 112:844

52 LE IPER-EOSINOFILIE Eosinofilia: Lieve: x10 9 L Moderata: x10 9 L Grave: >5 x10 9 L AA Lezioni di Ematologia prof A.M. Vannucchi 52

53 EOSINOFILIA Eosinofilia reattiva o secondaria In associazione a cloni T-linfocitari Forme mieloproliferative (CEL, HES) Forme mieloproliferative molecolarmente caratterizzate AA Lezioni di Ematologia prof A.M. Vannucchi 53

54 INTERESSAMENTO D ORGANO / SISTEMA NELLE HES EMATOLOGICO 100% Leucocitosi con eosinofilia, anemia, piastrinopenia, mielofibrosi CARDIOVASCOLARE 60% Cardiomiopatia, pericardite costrittiva, endomiocardite, trombi murali, fibrosi endomiocardica, IMA CUTE 60% Orticaria, papule, noduli, orticaria, ulcere mucose, lesioni bollose orali, vasculite SISTEMA NERVOSO 55% Neuropatia periferica, encefalopatia, demenza, epilessia, encefalite a eosinofili POLMONE 50% Infiltrati polmonari, fibrosi, emboli, tromboembolie, ARDS MILZA 45% Ipersplenismo, infarti FEGATO 30% Epatomegalia, epatite cronica attiva, necrosi epatica, colangite sclerosante, S. Budd Chiari, colecistite, colestasi OCULARE 20% Trombi, arterite retinica, episclerite, cherotocongiuntivite sicca GASTROINTESTINALE 20% Ascite, diarrea, gastrite, colite, pancreatite MUSCOLOSCHELETRICO Artrite, sinovite, borsite, necrosi digitale, Raynaud RENALE S. nefrosica, glomerulonefrite, IRA con cristalluria di AA Lezioni di Ematologia prof A.M. Charcot Vannucchi Leyden 54

55 AA Lezioni di Ematologia prof A.M. Vannucchi 55

56 Terapia Steroidi Chemioterapici Interferone alpha Ciclosporina Imatinib Ab monoclonale anti-il5 (mepolizumab) AA Lezioni di Ematologia prof A.M. Vannucchi 56

57 Mastocitosi Mastocitosi cutanea Mastocitosi sistemica Indolente Aggressiva Associata a neoplasie ematologiche non-mastocitarie Leucemia a mastociti Sarcoma mastocitario Mastocitoma extracutaneo AA Lezioni di Ematologia prof A.M. Vannucchi 57

58 Mastocitosi Forme dovute alla proliferazione neoplastica clonale di mastociti che si accumulano in uno o più organi La classificazione dipende dalla sede e dalla aggressività (danno d organo) Associazione con mutazioni (D816V la più frequente) del gene KIT (che codifica per la citochina SCF) - resistenza ad imatinib AA Lezioni di Ematologia prof A.M. Vannucchi 58

59 CLASSIFICAZIONE WHO Cutaneous mastocytosis (skin-only disease) Both can manifest mast cell mediator release symptoms i Indolent SM ii Systemic mastocytosis (SM) Aggressive SM (cytopenia, bone disease, organomegaly, etc.) iii SM with associated hematologic disorder Travis et al. Medicine 1988;67:345 Valent et al. Leukemia Research 2001;25:603 iv Mast cell leukemia/sarcoma AA Lezioni di Ematologia prof A.M. Vannucchi 59

60 Mastocitosi cutanea AA Lezioni di Ematologia prof A.M. Vannucchi 60

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62 Mastocitosi sistemica : coinvolgimento midollare AA Lezioni di Ematologia prof A.M. Vannucchi 62

63 Cytofluorimetric features Expression of CD2 and/or CD25 in addition to normal mast cell markers is a minor diagnostic criterion Intensity of marker expression: mast cells: FCε, cyttriptasi activation: CD63, CD69, CD16, HLA-DR immaturity: HLA-DR, CD123, CD34 a) Distinction between normal and neoplastic MC b) 3 distinct immunophenotypic profiles correlated with prognosis: ISM/MMAS, WDSM, ASM/MCL

64 Mutazione c-kit D816V nel midollo osseo o in un altro organo extracutaneo Elettroforesi capillare dopo digestione enzimatica: WT unico amplificato di 173 bp MUT due amplificati, di 173 e 159 bp C-kit mutations Asp816Val (kinase domain) Val560Gly (JM domain) AA Lezioni di Ematologia prof A.M. Vannucchi 64