MYELOPROLIFERATIVE DISORDERS EVOLVING CONCEPTS ANTONIO PARREIRA IPOFG - LISBOA

Neoplastic Myeloid disorders MPD - PV - ET - MF AML CML CMML MDS - RA - RARS - RAEB – I - RAEB - II

1950

Myeloproliferative disorders - MPD Neoplastic (clonal) disorders of hematopoietic stem cells Dysregulated proliferation of one / several cell lineages Thrombotic and hemorrhagic complications Fibrosis is frequent Progression to AML may occur

Myeloproliferative disorders - MPD Red cells - Polycythemia Rubra Vera – PV White cells - Chronic Myeloid Leukemia – CML Platelets - Essential Thrombocytemia – ET Stroma - Idiopathic Myelofibrosis - IM

Chronic Myeloid Leukemia 15-20% of adult leukemia (1.6/100000) Increased WBC

Nowell & Ungeford. J Nat Cancer Inst, 1960 David Hungeford

1973. Rowley J. Nature. 1973 Jun 1;243(5405):290-3. CML was the first human malignancy shown to be ‘due’ to acquisition of a fusion protein with activated tyrosine kinase activity

MITOGENIC ACTIVATION - PROLIFERATION APTOPTOSIS INHIBITION ALTERED ADHESION MITOGENIC ACTIVATION - PROLIFERATION APTOPTOSIS INHIBITION

(acute leukemia – AML, ALL) Chronic Myeloid Leukemia – clinical evolution “Blast” crisis (acute leukemia – AML, ALL) Death Accelerated phase Chronic phase Clonal evolution Diag 1 2 3 4 5 years

Ren R. NATURE REVIEWS | CANCER VOLUME 5 | MARCH 2005

Clinical examination and morphology 50 years Morphology Molecular lesion Pathophysiology Rational treatment Bcr-Abl # Ph t(9;22) Imatinib in vitro Imatinib therapy New TK inhibitors 1960 1973 1986 1996 2001 Clinical examination and morphology Cytogenetics PCR/interphase cytogenetics 46,XY,t(9;22)(q34;q11)

57 y ♀ pt, Ph+ CML in chronic phase BCR-ABL Real-time quantitative PCR follow-up GLIVEC 400 mg E255V DASATINIB 140 mg/day GLIVEC 600 mg IPOFG 2006

Historical perspective of Ph-negative Myeloproliferative Disorders - First description of PV 1951 – PV, ET, IM linked as related disorders 1974 - Identification of EPO-independent erythroid colonies 1976 – Stem-cell origin of PV 1983-2003 – Dysregulated tyrosine kinases described in CML, CMML, Chronic eosinophilic leukemia and mastocytosis 2002 – Mitotic recombination of chromosome 9p as common lesion in PV 2001-2004 – EPO independent growth in PV dependent on JAK-STAT signaling 2005 – Description of JAK2 V617F mutation. Adapted from Campbell and Green, NEJM, 2006;355,2452

Laboratory Features of Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis Figure 1. Laboratory Features of Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis. Polycythemia vera is characterized by an increased hematocrit in the peripheral blood (test tube on left); a hypercellular marrow with increased numbers of erythroid, megakaryocytic, and granulocytic precursor cells; and a variable increase in the number of reticulin fibers. Essential thrombocythemia is characterized by an increase in the number of platelets in the peripheral blood and an increased number of megakaryocytes in the marrow, which tend to cluster together and have hyperlobated nuclei. Idiopathic myelofibrosis is characterized by the presence of immature red and white cells (a so-called leukoerythroblastic blood film) and "teardrop" red cells, disordered cellular architecture, dysplastic megakaryocytes, new bone formation in the marrow, and the formation of collagen fibers. Campbell P and Green A. N Engl J Med 2006;355:2452-2466

Polycythemia True / Absolute Primary Polycythemia Secondary polycythemia Epo dependent Hypoxia dependent Hypoxia independent Epo independent Apparent / Relative Reduction in plasma volume

Polycythemia Rubra Vera - PV Diag criteria Absolute increase in RBCs, leukocytosis, trhombocytosis 2-3 / 100.000 Median age of presentation – 55-70; M/F: 1.2 Plethora, generalized pruritus, unusual thrombosis, gout Splenomegaly A1 – raised red cell mass A2 - Normal O2 sat and low EPO A3 – palpable spleen A4 – No BCR-ABL fusion B1 – Thrombocytosis > 400 B2 – Neutrophilia > 10 B3 – Radiological splenomegaly B4 – Endogenous erythroid colonies A1+A2+ either one A or two B

Essential Thrombocythemia - ET Clonal MPD Platelets persistently > 600 Lack of positive diagn criteria 2.5 / 100.000 M/F: 2:1 Median age diagn: 60 Transformation Headache, lightheadness Syncope Erythromelalgia (burning hands and feet wih erythema) Transient visual disturbancies Thrombosis and haemorrhage

Idiopathic myelofibrosis - IM Normocytic anemia, poikilocytosis Low WBC / Platelets Hepatosplenomegaly Extramedulllary hematopoesis BM fibrosis Rare, M/F: 2:1 Median age diagn: 60 Transformation agnogenic myeloid metaplasia or myelofibrosis with myeloid metaplasia

JAK2 V617F MUTATION JAK2 protein is a cytoplasmic tyrosine kinase A single point mutation (G T) in the JH2 pseudokinase domain of the JAK2 gene results in a valine to phenylalanine substitution at amino acid 617 (V617F). The mutation leads to the constitutive activation of the JAK2 tyrosine kinase AAT TAT GGA GTA TGT GTC TGT GGA GAC GAG wild type mutant 617 codon valine phenylalanine AAT TAT GGA GTA TGT TTC TGT GGA GAC GAG Two independent studies in 2005 demonstrated that a significant proportion of patients with PV, ET and IM have acquired this somatic mutation (Baxter et al, Lancet 2005, Levine et al, Cancer Cell 2005).

Campbell P and Green A. N Engl J Med 2006;355:2452-2466

Role of JAK2 in Pathway Signaling and Erythropoietin Binding, Stem-Cell Differentiation, and Development of Homozygosity for the V617F Mutation Figure 2. Role of JAK2 in Pathway Signaling and Erythropoietin Binding, Stem-Cell Differentiation, and Development of Homozygosity for the V617F Mutation. In Panel A, in the absence of ligand, the erythropoietin receptor (EPOR) binds JAK2 as an inactive dimer. In cells with wild-type JAK2 protein, the binding of erythropoietin (Epo) to its receptor induces conformational changes in the receptor, resulting in phosphorylation (P) of JAK2 and the cytoplasmic tail of the receptor. This leads to signaling through pathways made up of Janus kinases and signal transducers and activators of transcription (JAK-STAT), phosphatidylinositol 3 kinase (PI3K), and RAS and mitogen-activated protein kinase (RAS-MAPK). In cells with the V617F mutation, the signaling is constitutively increased, even in the absence of erythropoietin. In Panel B, the JAK2 protein binds to multiple cytokine receptors -- EPOR, thrombopoietin receptor (MPL), granulocyte colony-stimulating factor receptor (G-CSFR), and probably others -- that are important for hematopoietic stem-cell biology and differentiation. Therefore, the JAK2 protein with the V617F mutation exerts its effects at various stages of differentiation and in various lineages. In Panel C, the development of homozygosity for the V617F mutation is a two-step process, with the initial point mutation followed by mitotic recombination of chromosome 9p between the JAK2 locus and the centromere. This results in the loss of heterozygosity but a diploid DNA copy number. Campbell P and Green A. N Engl J Med 2006;355:2452-2466

Possible Roles of the JAK2 V617F Mutation in Myeloproliferative Diseases Figure 5. Possible Roles of the JAK2 V617F Mutation in Myeloproliferative Diseases. In model A, a mutation (V617F) of one allele of JAK2 on chromosome 9p (red dot), alone or in combination with a hypothetical preexisting mutation in an unknown gene ("X"), initiates the onset of the myeloproliferative disease (dashed arrow). In model B, the heterozygous V617F mutation on 9p occurs after the initiation of the myeloproliferative disease (dashed arrow), which was provoked by one or more mutations in an unknown gene or genes. Cells that are heterozygous for the V617F mutation have a proliferative advantage over cells bearing only the wild-type allele. Mitotic recombination between homologous regions of the two chromosomes 9 in a cell heterozygous for V617F results in loss of heterozygosity of 9p (9pLOH). One of the daughter cells is homozygous for V617F and gains an additional proliferative advantage. This cell establishes a subclone that outcompetes both cells that are heterozygous for V617F and cells that are homozygous for wild-type JAK2. Kralovics R et al. N Engl J Med 2005;352:1779-1790

Incidence of JAK2 V617F mutation in MPD Ross and Wernig, Hematology 2006 JAK2 mutation JAK2 homozygosity Polycythemia Vera 90 - 95% 25 - 30% Essential Thrombocytemia 35 – 50% 1 – 3 % Idiopathic Myelofibrosis 50 – 60% 10 – 29%

Classification of the Myeloproliferative Disorders on the Basis of Molecular Pathogenetic Characteristics JAK2 exon 12 mutations Scott et al, 2007 Figure 3. Classification of the Myeloproliferative Disorders on the Basis of Molecular Pathogenetic Characteristics. Chronic myeloid leukemia is defined by the BCR-ABL fusion gene and characterized by three stages of disease progression, from the chronic phase through the accelerated phase to blast crisis. Analogously, JAK2-positive thrombocythemia and polycythemia have overlapping phenotypes in the chronic phase and can progress to an accelerated phase, which is manifested as myelofibrosis or other complications. Leukemic transformation can also occur. JAK2-negative disease follows similar patterns of progression. The disorder that is currently called idiopathic myelofibrosis or agnogenic myeloid metaplasia is clinically indistinguishable from the myelofibrotic transformation of polycythemia vera or essential thrombocythemia. Therefore, this disorder may be an accelerated phase of polycythemia vera or thrombocythemia. Many patients with polycythemia vera who do not have the V617F mutation have other JAK2 mutations, and therefore truly JAK2-negative polycythemia vera is probably extremely rare. Campbell P and Green A. N Engl J Med 2006;355:2452-2466

Diagnostic algorithm for suspected PV in routin clinical practice – Tefferi, ASH 2006

Diagnostic criteria of MPD JAK2 V617F + (proposed by Campbell and Green, 2006) JAK2+ Polycythemia (Diagn if both criteria) A1 High hematocrit (>52% M; >48% F) or increased red cell mass (>25% predicted value) A2 Mutation in JAK2 JAK2+ Thrombocytemia (Diagn if both criteria) A1 Platelet count > 450x10e9/L A3 No other myeloid cancer, especially JAK2 PV, IM or MDS JAK2+ Myelofibrosis Diagn if A1 and A2 and any two of B criteria) A1 Reticulin grade 3 or higher B1 Palpable spenomegaly B2 Otherwise unexplained anemia B3 Teardrop cells on peripheral blood smear B4 Leucoerythroblastic blood film B5 Systemic symptoms B6 Histological evidence of extramedullary hematopoiesis