Mechanisms of Disease The Myeloproliferative Disorderssites.tufts.edu/imlib/wp-content/blogs.dir/1976/files/2014/05/The-Myeloproliferative...Ph-negative myeloproliferative disorders

review article

T h e n e w e ng l a nd j o u r na l o f m e dic i n e

n engl j med 355;23 www.nejm.org december 7, 20062452

Mechanisms of Disease

The Myeloproliferative DisordersPeter J. Campbell, F.R.A.C.P., F.R.C.P.A.,

and Anthony R. Green, F.R.C.Path., F.Med.Sci.

From the Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, Unit-ed Kingdom. Address reprint requests to Dr. Green at the Department of Haema-tology, Cambridge Institute for Medical Research, Hills Rd., Cambridge CB2 2XY, United Kingdom, or at [email protected].

N Engl J Med 2006;355:2452-66.Copyright © 2006 Massachusetts Medical Society.

T he myeloproliferative disorders comprise several clonal he-

matologic diseases that are thought to arise from a transformation in a he-matopoietic stem cell. The main clinical features of these diseases are the

overproduction of mature, functional blood cells and a long clinical course. Chron-ic myeloid leukemia (not discussed in detail here) is a myeloproliferative disorder that is defined by its causative molecular lesion, the BCR-ABL fusion gene, which most commonly results from the Philadelphia translocation (Ph).1 The three main Ph-negative myeloproliferative disorders — polycythemia vera, essential thrombocy-themia, and idiopathic myelofibrosis — are the focus of this article. Less common conditions that are also considered myeloproliferative disorders under the classifi-cation used by the World Health Organization include systemic mastocytosis, chron-ic eosinophilic leukemia, chronic myelomonocytic leukemia, and chronic neutro-philic leukemia.2

The cardinal features of the three main myeloproliferative disorders are an in-creased red-cell mass in polycythemia vera, a high platelet count in essential throm-bocythemia, and bone marrow fibrosis in idiopathic myelofibrosis (Fig. 1). These three disorders share many characteristics, including marrow hypercellularity, a propensity to thrombosis and hemorrhage, and a risk of leukemic transformation in the long term. The annual incidences of both polycythemia vera and essential thrombocythemia are 1 to 3 cases per 100,000 population; myelofibrosis is less common.3

Patho gene tic Fe at ur es

Clues to Molecular Mechanisms

Polycythemia vera, a condition of “persistent and excessive hypercellularity accom-panied by cyanosis,” was recognized by Vaquez in 1892,4 and idiopathic myelofibro-sis was described at about the same time (Table 1).23 Essential thrombocythemia was recognized in the 1930s.24 Dameshek, in 1951, was the first to appreciate the considerable overlap in the clinical and laboratory features of these conditions and proposed that they, together with chronic myeloid leukemia and other rarer disor-ders, constitute a spectrum of related diseases. He coined the term “myeloprolif-erative disorders” to embrace these related conditions.5 In 1974, a critical experi-ment showed that erythroid progenitors from the bone marrow of patients with polycythemia vera were capable of growing in vitro in the absence of erythropoie-tin.10 At about the same time, studies based on X-chromosome inactivation were performed in women with polycythemia vera or essential thrombocythemia who carried a polymorphic variant of the X-linked G6PD gene. The results suggested that each of these diseases originated from a clone of hematopoietic stem cells.11,25

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n engl j med 355;23 www.nejm.org december 7, 2006 2453

Other clues to the molecular mechanisms re-sponsible for the myeloproliferative disorders have been accumulating. These include the association of several chronic myeloid cancers with activated tyrosine kinases (Table 2)13-18,37 and the recog-nition of increased signaling through Janus ki-nases and signal transducers and activators of transcription (JAK–STAT) and phosphatidylino-sitol 3-kinase (PI3K) pathways in erythroid and

myeloid cells.21,22,54 An additional hint came with the discovery of recurrent mitotic recombination events affecting chromosome 9p, resulting in the loss of heterozygosity but a normal DNA copy number.20

The JAK2 V617F Mutation

In 2005, several groups reported a single, acquired point mutation in the Janus kinase 2 (JAK2) gene

PolycythemiaVera

Peripheral BloodBone Marrow

(hematoxylin and eosin)Bone Marrow

(reticulin stain)

EssentialThrombocythemia

IdiopathicMyelofibrosis

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 hypercel-lular 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 clus-ter 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.





in the majority of patients with Ph-negative myelo-proliferative disorders.6-9,19 JAK2, a cytoplasmic tyrosine kinase, is critical for instigating intracel-lular signaling by the receptors for erythropoi-etin, thrombopoietin, interleukin-3, granulocyte colony-stimulating factor (G-CSF), and granulo-cyte–macrophage colony-stimulating factor (GM-CSF).55,56 Mice that are deficient in Jak2 die at embryonic day 12.5, with a complete absence of definitive erythropoiesis,57,58 a finding that un-derscores the vital role of JAK2 as a transducer of signals evoked by the binding of erythropoietin to its receptor. JAK2 binds to the erythropoietin receptor in the endoplasmic reticulum and is required for its cell-surface expression.59 When erythropoietin binds to its receptor, it provokes a conformational change in the receptor 60-62 with consequent phosphorylation and activation of JAK2.63 The activated JAK2 then phosphorylates the receptor’s cytoplasmic domain, thereby pro-moting the docking of downstream effector pro-teins and the initiation of intracellular signaling cascades55,56 (Fig. 2A).

The JAK2 mutation in the myeloproliferative disorders is not in the germ line but, rather, is acquired.6-9,19 Sensitive methods demonstrate the mutation in more than 95% of patients with poly-cythemia vera6,26 and in 50 to 60% of patients with essential thrombocythemia6,26-28,31,64 or id-

iopathic myelofibrosis.6,26,29-31 A substantial pro-portion of patients with polycythemia vera or idiopathic myelofibrosis are homozygous for the JAK2 mutation as a result of mitotic recombina-tion affecting chromosome 9p,6-9 but this phe-nomenon is rarely detected in essential throm-bocythemia.65 The mutation is also found in a small minority of patients with the hypereosino-philic syndrome, chronic myelomonocytic leuke-mia, chronic neutrophilic leukemia, myelodyspla-sia, or acute myeloid leukemia,31-34,66-68 but not in patients with lymphoid or other cancers or in those without hematologic disorders.6-9,33,34,67,69 The presence of a mutant JAK2 in some patients with acute myeloid leukemia,33 especially older patients, raises the possibility that they may have had a preceding, undiagnosed myeloproliferative disorder. The JAK2 mutation is also found in more than 50% of patients with otherwise unexplained Budd–Chiari syndrome,70 which also suggests the presence of a masked myeloproliferative disorder in such cases.

The mutation in JAK2 substitutes a bulky phe-nylalanine for a conserved valine at position 617 of the JAK2 protein (V617F). This residue is lo-cated in the JH2, or pseudokinase, domain, which negatively regulates the kinase domain.71 Bio-chemical studies have shown that the JAK2 V617F mutation causes cytokine-independent activa-

Table 1. Milestones in the History of Philadelphia-Negative Myeloproliferative Disorders and Their Relationship with the V617F Mutation in JAK2.*

Year Historical MilestoneRelationship Later Established with V617F

Mutation in JAK2

1892 First description of polycythemia vera4

1951 Polycythemia vera, essential thrombocythemia, and idio-pathic myelofibrosis linked as related conditions5

Mutation found in all three disorders6-9

1974 Identification of erythropoietin-independent erythroid colonies10

Mutation associated with cytokine independence of primary erythroid progenitors and cell lines6-9

1976 Stem-cell origin of polycythemia vera11 Mutation found in multipotent progenitors and hematopoietic stem cells6,12

1983–2003 Dysregulated tyrosine kinases found in chronic myeloid leukemia,13,14 mastocytosis,15 chronic myelomonocy-tic leukemia,16,17 and chronic eosinophilic leukemia18

Tyrosine kinase function of JAK2 constitutively activated by mutation7-9,19

2002 Description of mitotic recombination involving chro-mosome 9p as the most common cytogenetic lesion in polycythemia vera20

Homozygosity of the mutation caused by mitotic recombination of chromosome 9p6-9

2001–2004 Erythropoietin-independent growth in poly cythemia vera dependent on JAK–STAT signaling21,22

STAT proteins constitutively activated by muta-tion7-9,19

2005 Description of the JAK2 V617F mutation6-9,19

* STAT denotes signal transducers and activators of transcription.





tion of JAK–STAT, PI3K, and AKT (also known as protein kinase B) pathways, and mitogen-acti-vated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK),7-9,19 all of which are implicated in erythropoietin-receptor signal-ing.22,63,72,73

The JAK2 mutation provides a unifying expla-nation for many features of the myeloprolifera-tive disorders (Table 1), but other mutations are probably associated with JAK2 V617F-negative myeloproliferative disorders. One example is the presence of an activating mutation in the throm-bopoietin receptor (MPL) gene in approximately 10% of patients with V617F-negative idiopathic myelofibrosis.35,36 This mutation is located in a

motif that is important for keeping the receptor inactive.74

Pathoph ysiol o gic a l Fe at ur es

Genetic Marker

The insights revealed by the foregoing molecular investigations are reshaping our understanding of the pathophysiology, classification, diagnosis, and treatment of these conditions. The JAK2 mu-tation is the first genetic marker that is directly associated with the pathogenesis of the myelopro-liferative disorders, and for this reason it is a pow-erful tool for analysis of the molecular and cel-lular basis of these disorders.

Table 2. Molecular Lesions Associated with the Myeloproliferative Disorders.*

Abnormality Association

Recurrent genetic lesions

BCR-ABL Chronic myeloid leukemia (100%)1

JAK2 V617F Polycythemia vera (95%),6,26 essential thrombocythemia (50–60%),27,28 idiopathic myelofibrosis (50–60%),29,30 and other myeloid disorders (1–5%)31-34

MPL W515K/L Idiopathic myelofibrosis (5%) and essential thrombocythemia (1%)35,36

KIT mutations Systemic mastocytosis15,37

FIP1L1–PDGFRA Chronic eosinophilic leukemia18,38

PDGFRB fusion genes Chronic myelomonocytic leukemia (rare)16

FGFR fusion genes Chronic myelomonocytic leukemia (rare)17

Other cytogenetic lesions

Trisomy 9 Amplifies JAK2 gene and associated with JAK2 V617F mutation39

Trisomy 8 Found in myeloproliferative disorders, myelodysplasia, and acute myeloid leukemias; target genes not identified

Trisomy 1q Caused by duplication, trisomy, or unbalanced translocations40

20q deletion Found in myeloproliferative disorders and myelodysplasia41; associated with JAK2 V617F muta-tion39 and precedes it in some cases42; target genes not identified41

5q and 7q deletions Thought to reflect changes secondary to cytotoxic therapy40; target genes not identified

13q deletion Associated with idiopathic myelofibrosis; overlap of commonly deleted region and the region for chronic lymphocytic leukemia40

Dysregulated genes and proteins

BCL-XL Overexpressed in polycythemia vera43 as a result of constitutive JAK–STAT signaling; antiapop-totic effects in erythroid cells44,45

NFE2 Up-regulated in JAK2-positive myeloproliferative diseases46,47; may affect erythroid differentiation48,49

PRV1 RNA levels increased in polycythemia vera50,51 but unlikely to play a direct role in disease patho-genesis since protein levels are not increased51

MPL Surface levels of protein decreased and aberrantly glycosylated in myeloproliferative disorders52,53; role in disease pathogenesis unclear

* FIP1L1 denotes FH interacting protein 1–like 1, PDGFRA platelet-derived growth-factor receptor alpha polypeptide, PDGFRB platelet-derived growth-factor receptor beta polypeptide, FGFR fibroblast growth-factor receptor, BCL-XL B-cell leukemia/lymphoma 2–like protein X long-transcript variant, NFE2 nuclear factor erythroid–derived 2, PRV1 polycythemia rubra vera 1, and MPL thrombopoietin receptor.





Mouse Models

Expression of the JAK2 V617F mutation in murine hematopoietic cells by means of a retroviral vec-tor recapitulates the features of polycythemia vera.7,75-78 The animals have erythrocytosis and leukocytosis, and there is evolution to postpoly-cythemic myelofibrosis.75-78 Thrombocytosis is not a reproducible feature in these mice, perhaps because high levels of mutant JAK2 generated by the retroviral vector inhibit the differentiation of megakaryocytes.76 Constitutive activation of signal transducers and activators of transcrip-tion 5 (STAT5), erythropoietin hypersensitivity, and cytokine independence are all present, as in the human disease. These results provide direct evi-dence of a causal link between the mutation and the disease.

Stem-Cell Biology

The myeloproliferative disorders arise from a mutant multipotent stem cell. Analyses of pat-terns of X-chromosome inactivation in female patients11,25,79 show a skewed pattern in myeloid cells but a balanced pattern in T cells and other cell types,79 suggesting that some myeloid cells in these women are clonally derived. However, skewed patterns of X-chromosome inactivation in granulocytes have been found in older women without a myeloproliferative disorder79 and are thought to reflect polymorphic X-linked genes that influence hematopoietic stem-cell behavior.80 Age-related skewing limits the use of the analysis of X-chromosome inactivation for diagnostic pur-poses, but such studies remain useful as research tools. For example, studies of X-chromosome inac-tivation indicate that in most patients with V617F-negative essential thrombocythemia or idiopathic myelofibrosis, there is a dominant clone of blood cells,26,28,81 implying that these disorders are clon-al hematologic diseases.

The presence of the JAK2 mutation in colo-nies of hematopoietic progenitor cells (grown in vitro)6,65 and in hematopoietic stem cells isolat-ed by fluorescence-activated cell sorting12,82 pro-vides direct evidence that polycythemia vera and related myeloproliferative disorders arise in a mul-tipotent progenitor or stem cell. In contrast to the normal-size myeloid stem-cell compartment in chronic myeloid leukemia,83 the stem-cell com-

partment in polycythemia vera is both expanded and characterized by preferential erythroid dif-ferentiation.12 Taken together, the data suggest that the JAK2 mutation affects the behavior of hematopoietic stem cells but probably also acts at later stages of differentiation, particularly in the erythroid, granulocytic, and megakaryocytic lineages (Fig. 2B).

Cooperating Mutations

The JAK2 mutation explains many of the cardinal features of the myeloproliferative disorders, as does the BCR-ABL fusion gene in chronic myeloid leukemia. It seems likely that, as in chronic myeloid leukemia, leukemic transformation of the myelo-proliferative disorders reflects the acquisition of additional mutations.84,85

Four lines of evidence suggest that cooperat-ing mutations occur at an early stage in V617F-positive myeloproliferative disorders and can even predate the JAK2 mutation. First, deletions of 20q and other cytogenetic abnormalities occur in 5 to

Figure 2 (facing page). Role of JAK2 in Pathway Signal-ing and Erythropoietin Binding, Stem-Cell Differentia-tion, 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 erythropoi-etin (Epo) to its receptor induces conformational chang-es 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 kinas-es and signal transducers and activators of transcrip-tion (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 im-portant for hematopoietic stem-cell biology and differ-entiation. Therefore, the JAK2 protein with the V617F mutation exerts its effects at various stages of differ-entiation and in various lineages. In Panel C, the devel-opment of homozygosity for the V617F mutation is a two-step process, with the initial point mutation fol-lowed by mitotic recombination of chromosome 9p be-tween the JAK2 locus and the centromere. This results in the loss of heterozygosity but a diploid DNA copy number.





A

B

C

Wild-type JAK2without erythropoietin

Wild-type JAK2with erythropoietin

JAK2 with V617F mutation without erythropoietin

Stem cell

Progenitor cells Terminally differentiated cells

Wild-type JAK2

Red cells

Megakaryocytes

Granulocytes

EPOR

MPL

G-CSFR

Heterozygous V167F

V617F

Homozygous V167F

Point mutation Mitotic recombination

No signal

JAK2

EPOR Epo

P

PI3KRAS–MAPKSTAT

JAK2V617F

P

PPP

P

P

P

Unknownreceptor

Chromosome 9

V617F

PI3KRAS–MAPKSTAT

V617F





10% of patients with a myeloproliferative disor-der.40 In one patient with polycythemia vera and in one with essential thrombocythemia, granulo-cytes with the 20q deletion outnumbered V617F-positive granulocytes,42 suggesting that the 20q deletion occurred before the JAK2 mutation. Pa-tients with molecularly defined 20q deletions are almost exclusively V617F-positive,39 which is con-sistent with the presence of a cooperating gene on 20q. Second, some women with polycythemia vera or essential thrombocythemia have more clonally derived granulocytes (estimated by pat-terns of X-chromosome inactivation) than JAK2-positive granulocytes.26,39,42 Although this find-ing has been interpreted as evidence of a clonal proliferation that preceded the JAK2 mutation, such a conclusion remains controversial.39 Third, in some patients with V617F-positive polycythe-mia vera or essential thrombocythemia that un-dergoes leukemic transformation, the leukemic cells lack the JAK2 mutation.39 This finding is consistent with the origin of the leukemia in a mutant clone that preceded the JAK2 mutation, although other interpretations are possible.39 Fourth, in familial clusters of the myeloprolifera-tive disorders, the JAK2 mutation is acquired,86 which suggests that the inherited predisposition is unrelated to the JAK2 mutation.

Homozygosity for V617F

Homozygosity for the V617F mutation plays a key role in the myeloproliferative disorders. Homozy-gosity results from mitotic recombination (Fig. 2C), with the breakpoints spread along chromo-some 9p between the JAK2 locus and the centro-mere,9 implying that there is no single fragile site that is prone to recombination. In polycythemia vera, the prevalence of blood cells that are homo-zygous for the JAK2 mutation increases with time,65 presumably owing to a proliferative or survival ad-vantage of mutant progenitor cells. Homozygos-ity for V617F is associated with more marked changes in the expression of downstream target genes than is heterozygosity for V617F.46 This re-flects increased signaling mediated by a double dose of V617F, possibly combined with a loss of inhibition by the wild-type allele.7

Homozygosity in granulocytes can be detect-ed in about 30% of patients with polycythemia vera.6-9 However, when colonies of hematopoi-etic progenitor cells derived from such patients

were studied, approximately 90% of them were homozygous for the V617F mutation. By contrast, homozygous progenitor colonies were not found in essential thrombocythemia.65 This difference suggests that V617F homozygosity promotes the development of polycythemia vera. Since there is substantial variation in the rate of mitotic recom-bination among persons without a myeloprolifera-tive disorder,87 genetic and environmental factors that determine susceptibility to mitotic recombi-nation may influence whether essential thrombo-cythemia or polycythemia vera develops.

Signaling Pathways

The mutant JAK2 protein activates multiple down-stream signaling pathways with effects on gene transcription,46 apoptosis, the cell cycle, and dif-ferentiation55,56 (Table 2). Effects on apoptosis include overexpression of the cell-survival protein BCL-X in erythroid precursor cells in polycythe-mia vera,43 probably as a result of enhanced JAK–STAT signaling.44,45,72 There is also a reduction in apoptosis induced by death receptors in JAK2 V617F-positive erythroid progenitors, an effect me-diated through PI3K–AKT and MAPK–ERK path-ways.88 With respect to the cell cycle, mutant JAK2 promotes G1/S phase transition in hematopoietic cell lines, accompanied by up-regulation of cyclin D2 and down-regulation of the inhibitor p27kip.89 Effects on erythroid differentiation may be medi-ated by nuclear factor erythroid-derived 2 (NF-E2), which is up-regulated in polycythemia vera46,47 and plays an important role in erythroid differentia-tion.48,49 Hematopoietic cells expressing JAK2 V617F become hypersensitive to insulin-like growth factor 1 (IGF-1), suggesting that IGF-1-receptor signaling influences cellular proliferation and dif-ferentiation in these diseases.90

Wild-type JAK2 is required for intracellular processing and cell-surface display of the eryth-ropoietin receptor59 and the thrombopoietin re-ceptor.91 Mutant JAK2 influences the display and stability of the erythropoietin receptor on the cell surface92 but reduces levels of cell-surface throm-bopoietin receptor (MPL) in cultured cells (Con-stantinescu S, Vainchenker W: personal communi-cation). The latter may explain the decreased levels and altered glycosylation of MPL in the my-eloproliferative disorders.50,52,53 It has been sug-gested that, unlike other kinases associated with hematologic cancers, the V617F protein requires





a type-1 cytokine receptor — such as the eryth-ropoietin receptor (EPOR), MPL, or G-CSF recep-tor — to act as a scaffold and docking site for downstream effector proteins. Such a mechanism may explain the involvement of the erythroid, megakaryocyte, and granulocyte lineages in the myeloproliferative disorders (Fig. 2B),93 but de-tailed structural and biochemical analyses are needed to explain why residue 617 is so critical for JAK2 activation.

Disease Evolution

The evolution of the myeloproliferative disor-ders into other forms is poorly understood. An increase in reticulin fibrosis may occur with time in patients with polycythemia vera94 or essential thrombocythemia,95,96 and in some of them a syndrome indistinguishable from idiopathic my-elofibrosis develops.97 The stromal cells and fi-broblasts responsible for the increased fibro-sis, angiogenesis, and formation of new bone do not derive from the myeloproliferative clone. They therefore represent a polyclonal reaction to cyto-kines — including transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) — produced by megakaryocytes, monocytes, or both from the myeloproliferative clone.98 Mouse models of myelofibrosis suggest that excessive MPL signaling,99 activation of nuclear factor κB (NF-κB),100 and low levels of the globin transcrip-tion factor 1 (GATA-1)101 also contribute to cyto-kine secretion and the development of reticulin fibrosis. In V617F-positive human disease, exces-sive MPL signaling may be particularly relevant, since the JAK2 protein is a key component of sig-nal transduction from the MPL receptor.102

Acute myeloid leukemia develops in a small minority of patients with a myeloproliferative dis-order, but transformation to acute lymphoblas-tic leukemia is extremely rare.94,103 Leukemic transformation can occur without exposure to cytoreductive therapy, but the incidence of this transformation increases after exposure to some cytoreductive agents103 and probably reflects ac-quisition of additional genetic lesions. The devel-opment of acute myeloid leukemia in the absence of the JAK2 mutation in patients with a V617F-positive myeloproliferative disorder demonstrates that mutant JAK2 is not necessarily involved in leukemic transformation.39

Molecul a r Cl a ssific ation

Prospective27,104 and retrospective28,64,105 studies of essential thrombocythemia have shown that the V617F mutation divides the disease into biologi-cally distinct subtypes. V617F-positive thrombo-cythemia resembles polycythemia vera. In contrast to V617F-negative essential thrombocythemia, V617F-positive thrombocythemia typically shows higher levels of hemoglobin and white cells, a more cellular bone marrow, an increased risk of venous thrombosis and transformation to poly-cythemia vera, and greater sensitivity to hy-droxyurea.27 These features suggest that V617F-positive thrombocythemia is a forme fruste of polycythemia vera, with the degree of erythro-cytosis inf luenced by factors such as low iron stores, low erythropoietin levels, sex, and homo-zygosity for V617F.27 V617F-negative patients, who have a more isolated thrombocytosis, can pres-ent with features of a bona fide myeloprolifera-tive disorder, such as splenomegaly, cytogenetic abnormalities, megakaryocyte dysplasia, a sus-ceptibility to leukemia or myelofibrosis,27 and clonal hematopoiesis.26,28,81 Fifty patients with V617F-negative essential thrombocythemia re-mained V617F-negative for more than 6 years, indicating that this disorder is distinct from, and not an early “pre-JAK2” phase of, V617F-positive thrombocythemia.39

V617F-positive patients with idiopathic myelo-fibrosis had a reduced transfusion requirement and greater white-cell counts than did patients without the mutation,29 analogous to the higher hemoglobin levels and white-cell counts in pa-tients with V617F-positive essential thrombo-cythemia. In this study, but not in a second,30 the V617F mutation was independently associated with poor survival.29

These results lay the foundation for a molecu-lar classification of the myeloproliferative disor-ders (Fig. 3). This classification is likely to evolve as additional genetic lesions are identified with-in the JAK2 V617F-negative category. The similari-ties between V617F-positive essential thrombo-cythemia and polycythemia suggest considerable overlap between these conditions. They may form a phenotypic continuum in which the degree of initial erythrocytosis or thrombocytosis depends on physiological or genetic modifiers.27

The accelerated phase of a myeloproliferative





disorder is analogous to the accelerated phase of chronic myeloid leukemia and is probably spurred by the acquisition of additional genetic changes. In the myeloproliferative disorders, the acceler-ated phase would encompass not only the trans-formation of polycythemia vera or essential throm-bocythemia into myelofibrosis, but also a rising white-cell count, increased blasts in the blood, and neutropenia or thrombocytopenia.94 It is im-portant to distinguish transformation to myelo-fibrosis from histologic detection of increased levels of reticulin. The former is a clinical syn-drome indicating a biologic change within the

myeloproliferative clone, whereas the latter may accompany chronic-phase polycythemia vera or essential thrombocythemia, with the degree of fibrosis reflecting an interplay between the dura-tion of the disease and physiological or genetic modifiers.

The disorder that is currently termed idiopathic myelofibrosis is clinically indistinguishable from the transformation of polycythemia vera or es-sential thrombocythemia to myelofibrosis,30,95 and the diagnostic criteria for the two conditions are similar.95,104,106 At least some patients who receive the diagnosis of idiopathic myelofibrosis

Myeloproliferative disorders

Chronic myeloidleukemia

BCR-ABL JAK2 V617F Unknown mutation

JAK2-positivethrombocythemia

JAK2-positivepolycythemia

JAK2-negativemyeloproliferative disorder

Accelerated phase

Blast crisis Leukemic transformation

myelofibrosiscytopenias

increasing blastsincreasing white cells

Chronic phase Chronic phase Chronic phase

Accelerated phase Accelerated phase

Leukemic transformation

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 pro-gression, from the chronic phase through the accelerated phase to blast crisis. Analogously, JAK2-positive thrombo-cythemia 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 myelofibro-sis or agnogenic myeloid metaplasia is clinically indistinguishable from the myelofibrotic transformation of polycy-themia 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.





probably are in the accelerated phase of previ-ously unrecognized polycythemia vera or essen-tial thrombocythemia.

Di agnosis

Until recently, robust tools for the diagnosis of the myeloproliferative disorders have been lack-ing. Criteria for the diagnosis of polycythemia vera are mostly modifications of 20-year-old stan-dards from the Polycythemia Vera Study Group.107 The available tests are expensive, not universally available, and lacking in sensitivity and specific-ity. They include a determination of red-cell mass to distinguish true erythrocytosis from relative polycythemia, identification of erythropoietin-independent erythroid colonies in vitro, cytoge-netic analysis of bone marrow cells, testing of erythropoietin levels, ultrasonography of the spleen, and testing for the overexpression of poly-cythemia rubra vera 1 (PRV1). There are no uni-versally accepted diagnostic tests for essential thrombocythemia.108,109

Testing for the JAK2 V617F mutation is now widely available and promises to simplify the diagnostic workup. Allele-specific polymerase-chain-reaction (PCR) assay,6,27,110 pyrosequenc-ing,31,66 restriction-enzyme digestion,6,110 and real-time PCR26 are all sufficiently sensitive to detect the presence of a heterozygous mutation in as few as 5 to 10% of cells. These assays have low rates of false positive results, making them useful diagnostic tools.

Proposed diagnostic criteria for V617F-posi-tive and V617-negative myeloproliferative disor-ders are outlined in Tables 3 and 4, respectively. The JAK2 mutation is not associated with causes of an absolute erythrocytosis other than polycy-themia vera.7,9,31,111,112 Therefore, in the absence of a coexisting secondary erythrocytosis, the pres-ence of the JAK2 mutation in a patient with an increased red-cell mass is sufficient for the di-agnosis of polycythemia vera. In this situation, the role of analysis of the red-cell mass is mainly to distinguish polycythemia vera from essential thrombocythemia. However, this distinction is becoming arbitrary, given the clinical and biologic similarities between the two conditions.27,28,64,105 Therefore, in patients with the JAK2 mutation, analysis of the red-cell mass is likely to become obsolete. Erythropoietin levels do not distinguish between polycythemia vera and V617F-positive

essential thrombocythemia27 and therefore have minimal use in patients with the JAK2 mutation. Cytogenetic studies in patients with the JAK2 mu-tation generally do not add useful diagnostic or prognostic information.

V617F-negative polycythemia vera represents less than 5% of all cases of polycythemia vera.6,26 In these rare cases, apparent erythrocytosis should be excluded by analysis of the red-cell mass and secondary erythrocytosis by measurement of eryth-ropoietin levels and oxygen saturation. Abnormal results on cytogenetic analysis or radiologic evi-dence of splenomegaly would support the diag-nosis of a myeloproliferative disorder.

The presence of a JAK2 mutation in a patient with thrombocytosis has a high positive predic-tive value for a myeloproliferative disorder; pa-tients with reactive or secondary thrombocytosis and persons without a hematologic disorder are V617F-negative6-9 (Table 3). Other causes of throm-

Table 3. Proposed Diagnostic Criteria for Myeloproliferative Diseases with JAK2 Mutation.

JAK2-positive polycythemia (diagnosis requires the presence of both cri-teria)*

A1. High hematocrit (>52% in men or >48% in women) or an increased red-cell mass (>25% above predicted value)

A2. Mutation in JAK2

JAK2-positive thrombocythemia (diagnosis requires the presence of all three criteria)

A1. Platelet count >450×109/liter


A3. No other myeloid cancer, especially JAK2-positive polycythemia, myelo-fibrosis, or myelodysplasia

JAK2-positive myelofibrosis (diagnosis requires the presence of A1 and A2 and any two B criteria)

A1. Reticulin grade 3 or higher (on a 0–4 scale)


B1. Palpable splenomegaly

B2. Otherwise unexplained anemia (hemoglobin <11.5 g/liter for men; <10 g/ liter for women)

B3. Teardrop red cells on peripheral blood film

B4. Leukoerythroblastic blood film (presence of at least 2 nucleated red cells or immature myeloid cells in peripheral blood film)

B5. Systemic symptoms (drenching night sweats, weight loss >10% over 6 mo, or diffuse bone pain)

B6. Histologic evidence of extramedullary hematopoiesis

* Dual pathology (secondary erythrocytosis or relative erythrocytosis) might rarely coexist with a JAK2-positive myeloproliferative disorder. In this situa-tion, it would be prudent to reduce the hematocrit to the same targets as those for polycythemia vera.





bocytosis must be excluded in patients without the mutation (Table 4). Cytogenetic analysis of bone marrow is sometimes helpful in JAK2-nega-tive thrombocythemia if it shows a clonal abnor-mality. Essential thrombocythemia has been divid-ed into histologically distinct subgroups (labeled “true” essential thrombocythemia, prefibrotic my-

elofibrosis, and early overt myelofibrosis) that are thought to have differing prognoses,2,96 but it is unclear whether this histologic classification is robust enough to be applied outside specialized centers.

Diagnostic criteria for idiopathic myelofibro-sis are presented in Tables 3 and 4, modified from the Italian Consensus Conference.113 The criteria proposed here define a clinical syndrome in which reticulin fibrosis of the marrow is accompanied by specific clinical or laboratory features and can be used for both idiopathic myelofibrosis and transformation from preceding essential throm-bocythemia or polycythemia vera.

Tr e atmen t

The JAK2 mutation is reshaping how we treat the myeloproliferative disorders. Moving to a new clas-sification could simplify therapy. In polycythe-mia vera, the hematocrit is the primary target of therapy, but the treatment of patients with throm-bocytosis is controversial.94,114 By contrast, in es-sential thrombocythemia, cytoreduction to nor-malize the platelet count reduces thrombosis in high-risk patients,115 but the hematocrit is not a therapeutic target. Given the many similarities be-tween JAK2-positive polycythemia and thrombo-cythemia, it seems logical to develop target levels for both the platelet count and the hematocrit in both conditions. The development of a unified treatment strategy is consistent with growing evi-dence that the increased risk of thrombosis is not exclusively caused by erythrocytosis or thrombo-cytosis but by interactions among white cells, red cells, platelets, and the endothelium. Abnormali-ties in the activation of leukocytes and platelets occur in both diseases.116,117

The JAK2 mutation also affects response to treatment. Among patients with essential throm-bocythemia, those with the V617F mutation are more sensitive to hydroxyurea (but not to ana-grelide) than are patients without the mutation.27 The response to therapy can now be directly moni-tored through quantification of JAK2-positive cells in peripheral blood.118,119

An understanding of the molecular pathogen-esis of the myeloproliferative disorders lays the foundation for the development of novel targeted therapies. Since JAK2 inhibitors reduce the growth of JAK2 V617F-positive cell lines and primary cells in vitro,8,12 there is considerable interest in de-

Table 4. Proposed Diagnostic Criteria for Myeloproliferative Diseases without JAK2 Mutation.

JAK2-negative polycythemia vera (diagnosis requires the presence of A1, A2, and A3, plus either another A criterion or two B criteria)

A1. Increased red-cell mass (>25% above predicted value) or a hematocrit ≥60% in men or >56% in women

A2. Absence of mutation in JAK2

A3. No causes of secondary erythrocytosis (normal arterial oxygen saturation and no elevation of serum erythropoietin)

A4. Palpable splenomegaly

A5. Presence of acquired genetic abnormality (excluding BCR-ABL) in hema-topoietic cells

B1. Thrombocytosis (platelets >450×109/liter)

B2. Neutrophilia (neutrophils >10×109/liter; >12.5×109/liter in smokers)

B3. Splenomegaly on radiography

B4. Endogenous erythroid colonies or low serum erythropoietin

JAK2-negative essential thrombocythemia (diagnosis requires the presence of all five criteria)*

A1. Platelet count >600×109/liter on two occasions at least 1 mo apart


A3. No reactive cause for thrombocytosis

A4. Normal ferritin (>20 μg/liter)

A5. No other myeloid disorder, especially chronic myeloid leukemia, myelofi-brosis, polycythemia vera, or myelodysplasia

JAK2-negative idiopathic myelofibrosis (diagnosis requires the presence of A1, A2, A3, and any two B criteria)

A1. Reticulin grade 3 or higher (on a 0–4 scale)


A3. Absence of BCR-ABL fusion gene

B1. Palpable splenomegaly

B2. Otherwise unexplained anemia (hemoglobin <11.5 g/liter for men or <10 g/liter for women)

B3. Teardrop red cells on peripheral blood film

B4. Leukoerythroblastic blood film (presence of at least 2 nucleated red cells or immature myeloid cells in peripheral blood film)

B5. Systemic symptoms (drenching night sweats, weight loss >10% over 6 mo, or diffuse bone pain)

B6. Histologic evidence of extramedullary hematopoiesis

* The platelet threshold is preferred in patients without the JAK2 mutation, given the difficulty in ruling out reactive thrombocytosis and the fact that 2.5% of persons without a myeloproliferative disorder have a platelet count above the normal range.





veloping compounds for clinical use. Such agents hold particular promise for the treatment of pa-tients whose disease is in the accelerated phase, including myelofibrosis, given the lack of satisfac-tory treatments currently available. These agents may also prove to be useful for reducing the risk of vascular events or long-term disease evolution in chronic-phase disease. With an eye to the suc-cess of imatinib in the treatment of chronic my-

eloid leukemia, we hope the next decade will see the development of well-tolerated, therapeutic JAK2 inhibitors.

No potential conflict of interest relevant to this article was reported.

We thank Gary Gilliland, William Vainchenker, Radek Skoda, Ruben Mesa, Tiziano Barbui, Wendy Erber, Claire Harrison, and Mary Frances McMullin for their constructive comments on the manuscript, and Wendy Erber for providing some of the light micrographs in Figure 1.

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