1. MYELOPROLIFERATIVE DISORDERS EVOLVING CONCEPTS
ANTONIO PARREIRA
IPOFG - LISBOA

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

3. 1950

4. 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

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

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

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

8. 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

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

10. (acute leukemia – AML, ALL)
Chronic Myeloid Leukemia – clinical evolution
“Blast” crisis
(acute leukemia – AML, ALL)
Death
Accelerated
phase
Chronic phase
Clonal evolution
Diag
years

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

12. 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
Clinical examination and morphology
Cytogenetics
PCR/interphase cytogenetics
46,XY,t(9;22)(q34;q11)

14. 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

15. Historical perspective of Ph-negative Myeloproliferative Disorders
- First description of PV
1951 – PV, ET, IM linked as related disorders
Identification of EPO-independent erythroid colonies
1976 – Stem-cell origin of PV
– Dysregulated tyrosine kinases described in CML, CMML, Chronic eosinophilic leukemia and mastocytosis
2002 – Mitotic recombination of chromosome 9p as common lesion in PV
– 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

16. 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:

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

18. Polycythemia Rubra Vera - PV
Diag criteria
Absolute increase in RBCs, leukocytosis, trhombocytosis
2-3 /
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

19. Essential Thrombocythemia - ET
Clonal MPD
Platelets persistently > 600
Lack of positive diagn criteria
2.5 /
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

20. 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

21. 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).

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

23. 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:

24. 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:

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

26. 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:

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

28. 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