Spotlight Review

Leukemia (2008) 22, 1841–1848; doi:10.1038/leu.2008.233; published online 28 August 2008

Genetic complexity of myeloproliferative neoplasms

R Kralovics1,2

  1. 1Center for Molecular Medicine (CeMM), Austrian Academy of Sciences, Vienna, Austria
  2. 2Division of Hematology and Blood Coagulation, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria

Correspondence: Dr R Kralovics, Center for Molecular Medicine (CeMM), Austrian Academy of Sciences, Lazarettgasse 19/3, A-1090 Vienna, Austria. E-mail: robert.kralovics@cemm.oeaw.ac.at

Received 5 June 2008; Revised 21 July 2008; Accepted 30 July 2008; Published online 28 August 2008.

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Abstract

Oncogenic mutations in JAK2 and MPL genes have recently been identified in myeloproliferative neoplasms (MPNs). In addition to these mutations, cytogenetic aberrations are frequently present at diagnosis but their role in the pathogenesis remains unclear. Two models of MPN pathogenesis have recently emerged based on either a single-hit or a multi-hit concept. The first model proposes that the acquisition of JAK2 mutations is the disease-initiating event, causing both the onset of disease phenotype and establishment of clonal hematopoiesis. The second model postulates the existence of 'pre-JAK2' mutations that establish clonal hematopoiesis before acquisition of JAK2 mutations and onset of disease phenotype. In this review, the two models have been critically evaluated in the context of the latest findings. At present, neither of the two models can be universally applied to all MPN patients due to their genetic heterogeneity. It is likely that the disease pathogenesis in some patients follows the first, and in other patients, the second model. Thus, the somatic mutations in MPN do not seem to be acquired in a predetermined order as seen in other malignancies, but occur randomly. Furthermore, the role of uniparental disomy in MPN and certain aspects of MPN therapy are discussed.

Keywords:

uniparental disomy, clonality, myeloproliferative disorders, deletion, mitotic recombination, JAK2 V617F

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Introduction

Excessive production of terminally differentiated blood cells is the most prominent phenotypic feature of the three common myeloproliferative neoplasms (MPNs). Although increased erythrocyte mass in polycythemia vera (PV) is a phenotypic hallmark restricted to only a single MPN entity, the majority of clinical features of MPN is shared to a variable degree among the three MPN entities.1, 2 Thrombocythemia is a key feature of essential thrombocythemia (ET), and bone marrow fibrosis is the diagnostic prerequisite of primary myelofibrosis (PMF), but both of these phenotypes are often seen among all three MPNs. In addition, MPN has an inherent tendency toward thrombosis and bleeding,3, 4 and less frequently, toward leukemic transformation.5, 6, 7, 8

The increased production of blood cells in MPN was originally assigned to hypersensitivity of hematopoietic progenitors to cytokines. This hypothesis proved to be correct by demonstrating that PV erythroid progenitors exhibit growth in vitro in the absence of erythropoietin.9 Growth factor hypersensitivity of progenitors in MPN was later further elaborated in different culture systems and extended to ET and PMF.10 After abnormal cytokine responsiveness of hematopoietic progenitors in MPN had been established, a lot of attention was devoted to cytokine signaling. Initial studies targeted the cytokine receptors and their ligands, and later the individual signaling proteins and transcription factors were examined.11, 12, 13, 14, 15, 16, 17, 18, 19 The discovery of gain-of-function mutations in the Janus kinase 2 (JAK2) by different experimental approaches has been a foreseeable outcome of these efforts.20, 21, 22, 23, 24

The presence of the valine 617 to phenylalanine mutation of JAK2 (JAK2-V617F) has been detected across the MPN entities and at low frequency in other clonal myeloid disorders.1, 2 The highest frequency of JAK2-V617F is observed in PV followed by PMF and ET. The JAK2-V617F mutation was detected at low frequencies in acute myeloid leukemia (5%), myelodysplastic syndrome (3%), chronic myelomonocytic leukemia (6%), atypical myeloproliferative disorder (20%), hypereosinophilic syndrome (1%) and systemic mastocytosis (6%).25, 26, 27, 28, 29, 30 A thorough examination of the JAK2 gene in JAK2-V617F-negative PV patients led to the identification of various JAK2 mutations in exon 12 (JAK2-ex12).24 Interestingly, JAK2-ex12 has so far been found only in PV but the rarity of this mutation makes it difficult to predict whether other MPN entities could also harbor this type of JAK2 mutation at lower frequencies.24, 31 Sequence analysis of JAK/STAT pathway members in JAK2-V617F-negative PMF patients identified two gain-of-function mutations in the thrombopoietin receptor (MPL).32, 33 MPL mutations have not been found in PV, but PMF and ET patients carry at least five MPL variants (MPL-W515L, MPL-W515K, MPL-S505N, MPL-A506T and MPL-A519T) with a frequency of about 5% in PMF and between 1 and 9% in ET depending on the cohort of patients analyzed.33, 34, 35, 36

Functional studies of JAK2-V617F, JAK2-ex12 and MPL-W515L/K in cell lines and in animal models have established their oncogenic character and proven their disease causing potential. Both bone marrow transplant models20, 24, 32, 37, 38, 39 and transgenic mice40, 41, 42 expressing these mutations showed that JAK2-V617F, JAK2-ex12 and MPL-W515L are capable of inducing myeloproliferative phenotypes in mice resembling human MPN. Animals expressing JAK2-V617F in bone marrow transplant models induced erythrocytosis with mild leukocytosis in C57Bl/6.37 However, the mice developed erythrocytosis with dramatically increased leukocyte count and bone marrow fibrosis on a different genetic background (Balb/c).37 This observation provided evidence that the genetic background can modify the phenotype induced by JAK2-V617F. In a transgenic animal model, variable expression level of the JAK2-V617F transgene induced thrombocythemia in low expressing animals, whereas polycythemia was present in animals with high transgene expression.40 Although the animal models provided some clues as to how a single amino-acid substitution in JAK2 can result in three different phenotypes, this question has not yet been answered. It remains to be seen whether the genetic makeup of patients and/or the expression level of the mutant protein determine which MPN entity the patient will develop upon the acquisition of JAK2-V617F. A recent study reported that polymorphisms within the JAK/STAT signaling pathway could account for some phenotypic variability and provided evidence in patients that constitutive host factors contribute to MPN phenotype.43

Two possible models are currently postulated on the role of JAK2 mutations in MPN pathogenesis.1, 2, 23, 44, 45 The models address the primary versus secondary role of JAK2 mutations in the disease development (Figure 1). In model A, JAK2 mutations are thought to simultaneously induce clonal hematopoiesis and the onset of MPN phenotype. The development of a particular MPN entity is influenced by constitutive genetic factors of each patient. Model B advocates a 'multi-hit' pathogenesis model of MPN in which mutations acquired before JAK2-V617F occur and establish clonal hematopoiesis. These 'pre-JAK2' mutations provide a clonal background and/or facilitate the acquisition of JAK2-V617F. Since up to now, no evidence has been presented that clonal JAK2-V617F-negative MPN patients develop JAK2-V617F positivity after MPN diagnosis, the onset of MPN phenotype in model B had to be placed at the time of JAK2-V617F acquisition (Figure 1b). The animal models have also proven the clear association of JAK2-V617F with the onset of MPN phenotype. Model B implies that mutations occurring before the JAK2-V617F acquisition may determine which MPN entity will develop in the patient. Thus, acquisition of JAK2 mutations in some cases represents a rather late event in the clonal evolution of MPN stem/progenitor cells.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Outline of the two possible models of myeloproliferative neoplasm (MPN) pathogenesis. (a) In model A, the JAK2-V617F mutation (X) causes the onset of MPN phenotype and clonal hematopoiesis in a single hit. (b) In model B, the acquisition of an unknown somatic mutation results in clonal hematopoiesis (deletion on chromosome 20q; del20q, gray circle). The JAK2-V617F mutation is acquired later, on the background of clonal hematopoiesis, and at this point, the myeloproliferative disorder (MPD) phenotype appears. In some patients, uniparental disomy on chromosome 9p (9pUPD) results in transition of cells heterozygous for JAK2-V617F to homozygosity for the mutation (XX).

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The main purpose of this review is to draw attention to the genetic heterogeneity of MPN as it often provides an explanation to seemingly contradicting biological or clinical data. Initially, the two models of MPN pathogenesis are discussed in the context of new data emerging from analysis of clonality and studies of familial MPN. Next, the evidence of genetic heterogeneity in MPN is reviewed, including opinions on the potential roles of the common chromosomal aberrations and uniparental disomy (UPD) in the pathogenesis of MPN. Finally, the impact of genetic heterogeneity on MPN therapy is assessed and an outline of an updated model of MPN pathogenesis is presented.

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Clonality in MPN

Single cell origin of MPN hematopoiesis has been well established by studies of X chromosome inactivation.46, 47, 48, 49, 50 Clonal hematopoiesis in MPN provided one of the first experimental supports for Damashek's ideas on a common origin and pathogenetic relatedness of the chronic myeloid disorders.51 The early X inactivation studies relied on protein isoform analysis followed by analysis of differential methylation pattern of the human androgen receptor gene in women.52 Analysis of RNA transcription of the active X chromosome was the latest improvement of these methods allowing the analysis of clonality in cells without a nucleus.53 The major advantage of X chromosome-based clonality assays is that they provide evidence of clonal origin of cells in disorders where the somatic mutations causing clonality are unknown. The major disadvantage is the restriction to women, the lack of 100% informativity and a possible age-related distortion of X inactivation patterns.52

As the genetic cause of monoclonal hematopoiesis differentiates the current models of MPN pathogenesis (Figure 1), it became important to define the role of JAK2 mutations in clonality. When the abundance of the mutant JAK2 allele was compared with clonality in MPN patients in granulocytes and platelets, a number of ET, PMF and PV patients with monoclonal myeloid cells displayed significantly low abundance of the mutant JAK2 allele, suggesting that only a small proportion of clonal cells carried the JAK2-V617F mutation.44, 54 These studies provided evidence that somatic mutations precede the acquisition of JAK2-V617F and thus experimentally proved that the 'multi-hit' model of MPN pathogenesis is in place at least in a subset of MPN patients (Figure 1b). Moreover, deletions on chromosome 20q were proposed to be one type of 'pre-JAK2' somatic mutation.44 Similar clonality studies were not carried out in patients carrying MPL or JAK2-ex12 mutations. The presence of low mutational burden detected in some patients carrying MPL and JAK-ex12 mutations predicts that these mutations may also occur on a clonal background.33, 55

The presence of clonal hematopoiesis has been a fundamental part of MPN definition and played an important role in distinguishing MPN from reactive conditions such as secondary polycythemia or reactive thrombocythemia. The discovery of polyclonal ET56, 57 initially raised questions on the diagnostic criteria applied for ET and suggested the genetic heterogeneity of this MPN entity. Before the discovery of JAK2 mutations, polyclonal ET was suspected to represent a reactive condition mimicking ET. The presence of the JAK2-V617F mutation in polyclonal ET patients44, 54, 58 not only proved this concept wrong, but at the same time revealed an interesting fact about the ET progenitor pool. In ET, the cells acquiring somatic mutation(s) such as JAK2-V617F do not necessarily undergo selection to full clonality. A small population of JAK2-V617F-positive cells may contain progenitors with increased thrombopoietic capacity and/or an ability to induce normal megakaryocytic progenitors by stimulatory signals (most likely cytokines) resulting in excessive polyclonal thromobopoiesis. Thus, polyclonal ET patients may have certain elements of a reactive disorder. It remains to be seen whether polyclonal ET patients positive for the JAK2-V617F mutation develop to full clonality over extended periods of time. If this will be proven to be the case, polyclonal ET patients may merely represent an early disease phase that eventually changes to a clonal stage.

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Familial MPN

A second line of evidence supporting the multi-hit model of MPN pathogenesis comes from studies on the presence of JAK2 mutations in familial MPN. Familial MPN is characterized by an autosomal dominant inheritance with incomplete penetrance, variable presence of the three MPN entities in a single family, and with clinical and molecular features indistinguishable from sporadic MPN.10, 59 In all cases examined so far, JAK2-V617F and JAK2-ex12 mutations were acquired somatically in these families, never inherited through the germ line.60, 61 Thus, the unknown germline mutation inherited in these families predisposes carriers to somatically acquire JAK2 mutations and develop MPN. The penetrance of phenotype in familial MPN is incomplete because the germline mutation does not cause MPN on its own and the development of phenotype in familial MPN depends on the occurrence of somatic JAK2 mutation. Extrapolation of this finding led to the hypothesis that the only difference between sporadic and familial MPN is that the 'pre-JAK2' mutation is germ line inherited in familial MPN and somatically acquired in sporadic MPN. In this respect, familial MPN supports the multi-hit model of MPN pathogenesis. On the other hand, absence of germline JAK2 mutations in familial MPN may simply be due to incompatibility of JAK2-V617F mutation with embryogenesis as evidenced by the decreased viability of transgenic mice expressing JAK2-V617F in the germ line.40 In conclusion, absence of germline JAK2 mutations in familial MPN does not rule out the possibility that a proportion of sporadic MPN patients follow the single-hit model of MPN pathogenesis (Figure 1a). In support of this hypothesis are MPL mutations that seem to be somewhat different from JAK2. A gain-of-function mutation of MPL (MPL-S505N) has been described in a Japanese family with autosomal dominant thrombocytosis.62 The same MPL variant somatically acquired has been recently reported in two cases with sporadic MPN.34 Thus, activating mutations of MPL are likely to be tolerated through embryogenesis and MPN caused by MPL mutations may be a single-hit disease, following model A of MPN pathogenesis (Figure 1a).

The presence of incomplete penetrance in familial MPN often makes it difficult to determine the inheritance pattern of MPN. Although some families show a clear autosomal dominant inheritance pattern, in other families a recessive trait cannot be ruled out. What we learned from sporadic MPN suggests that familial MPN is likely to be heterogeneous also. Genetic heterogeneity of familial MPN might render classical linkage analysis of multiple families challenging. A recent study suggested a possible recessive mode of inheritance of familial MPN and offered an alternative, population-based approach to identify genetic factors playing a role in MPN predisposition.63 In case of recessive inheritance of familial MPN, the mutations/variants predisposing to MPN must be strong in effect and relatively common in the Western population.63 This issue will be resolved only after the predisposing mutations are identified.

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Cytogenetic aberrations of MPN are shared with other clonal myeloid disorders

Considerable attention has been devoted in the past to explaining the genetic basis of clonal hematopoiesis in MPN. The validity of this cytogenetic approach was demonstrated by the discovery of the Philadelphia chromosome and the subsequent identification of the BCR-ABL fusion gene in chronic myelogenous leukemia (CML)—a disease entity originally part of the MPN family. Although abnormal karyotypes are frequent at diagnosis in ET, PV and PMF, no specific cytogenetic aberrations were identified. Among the most frequent are 9pUPD, numerical aberrations of chromosomes 1, 8 and 9, deletions of chromosomes 5q, 13q and 20q and frequent gains of 9p.23, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 Except 9pUPD, most of these cytogenetic aberrations are often present in other clonal myeloid disorders (myelodysplastic syndrome, CML and acute myeloid leukemia) and therefore, their role in the pathogenesis of hematological malignancies may be more universal. Tumor growth is considered to be a result of clonal evolution driven by sequential acquisition of somatic mutations. Assuming that a 'multi-hit' concept also applies to clonal evolution in hematological malignancies, the role of the 'common' chromosomal aberrations may be linked with the early stages of clonal evolution or may even represent the first hits that establish a clone capable of further evolution. As many of these mutational events may not result in detectable hematological phenotypes, individuals with an established clonal hematopoiesis may escape detection. As clonal evolution driven by acquisition of additional mutations progresses, distinct hematological phenotypes may appear. It is likely that these later 'phenotype-initiating' mutational events define the individual phenotypes of hematological disorders.

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UPD and variability of mutational burden in MPN

Acquired UPD is a result of mitotic recombination caused by the exchange of chromosomal DNA between non-sister chromatids during mitosis. UPD is characterized by the presence of large regions of homozygosity extending from the recombination breakpoint to the chromosomal end and is not accompanied by changes in chromosomal copy number. Thus, classical cytogenetics, fluorescence in situ hybridization or comparative genomic hybridization do not detect UPD. Acquired UPD is detectable as loss of heterozygosity when genotypes of tumor- and non-tumor-derived DNA samples are compared by assessing polymorphic DNA markers such as single nucleotide polymorphisms (SNPs), microsatellite markers (simple repeat sequences), insertion–deletion polymorphisms or tandem repeats. UPD was first identified in MPN using a whole-genome microsatellite screening in which the short arm of chromosome 9 and the long arms of chromosomes 10 and 11 showed presence of loss of heterozygosity.64 Further mapping of the relevant chromosomal region on 9p represented one of the approaches that led to the identification of JAK2-V617F mutation in MPN.23

UPD is considered to be one of the genetic mechanisms involved in tumor suppressor inactivation as mutated or deleted alleles of tumor suppressors could become homozygous through mitotic recombination.75, 76, 77, 78 Interestingly, studies of UPD in MPN and other myeloid malignancies showed that UPD is often involved in oncogene amplification. Semi-dominant oncogenic mutations present in neoplastic tissues in a heterozygous state can become homozygous due to mitotic recombination. Semi-dominant mutations display lower phenotypic effects in a heterozygous state compared to a homozygous state. Fully dominant mutations do not exhibit a difference in phenotypic effect between heterozygous and homozygous states. Up to now, JAK2-V617F, JAK2-ex12 in MPN, and mutations in FLT3, KIT, CEBPA and RUNX1 genes in acute myeloid leukemia were often shown to acquire homozygosity due to mitotic recombination.79, 80, 81, 82, 83 MPL mutations in MPN can undergo mitotic recombination on chromosome 1p as patients either homozygous or with high MPL-W515L/K mutant allele burden are common.33 However, the association of homozygous MPL mutations with UPD on chromosome 1p has not formally been shown.

Differences in population size of mutant cells and the high frequency of 9pUPD in MPN (about 25%) result in great variability of JAK2-V617F mutational burden in patients. The mutational burden varies from a few percent to 100% (homozygosity). In patients, myeloid cell populations often represent a mixture of heterozygous and homozygous cells of variable ratios. Interestingly, patients with ET exhibit very low frequency of 9pUPD and as a result, the JAK2-V617F mutational burden in ET is low compared with PMF and PV.23, 84 This unique feature of ET is difficult to explain. One possibility is that the 'pre-JAK2' mutations in ET do not increase the frequency or suppress mitotic recombination. Another possibility is that JAK2-V617F behaves as a fully dominant mutation in ET, and therefore, 9pUPD does not provide a selective advantage to cells homozygous for JAK2-V617F. The latter possibility assumes that the hematopoietic microenvironment in ET is substantially different from that of PV and PMF where the selective pressure to acquire 9pUPD is present.

Mitotic recombination plays a role in DNA double-strand break repair mechanisms during the S phase of mitosis. Pathologic conditions such as Bloom and Werner syndromes caused by mutations in RecQ helicase family members BLM and WNR exhibit increased incidence of mitotic recombination.85 Primary cells of these patients display a marked hypersensitivity to DNA damage-inducing agents such as hydroxyurea.86, 87 Although hydroxyurea is successfully used in MPN therapy, we do not know whether MPN primary cells show increased susceptibility to this agent compared to normal cells. The increased mitotic recombination rate in PV and PMF suggests that a defect similar in effect to BLM and WNR mutations can be present in at least a proportion of MPN patients. Alternatively, the presence of heterozygous mutations in JAK2 and MPL may interfere with the function of RecQ helicases, perhaps at the gene expression level, as MPN exhibits marked deregulation of gene expression.88 Suppression of expression of RecQ helicases by JAK2 or MPL mutations may functionally mimic loss-of-function mutations, result in increased mitotic recombination rates and lead to UPD. In support of this hypothesis, a recent study showed that cells expressing JAK2-V617F have increased homologous recombination rates, increased Rad51 focus formation and increased genomic instability.89

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Secondary genetic effects of UPD on clinical phenotype

The gain of homozygosity along the short arm of chromosome 9 in patients with 9pUPD can influence the disease phenotype. Rare heterozygous SNPs that almost never occur in a homozygous state in the population become homozygous due to 9pUPD and as a result, the majority of myeloid cells can express many recessive alleles that exert influences on myeloid cell function. The chromosomal region between the 9p telomere and the JAK2 gene are always affected by homozygosity, whereas the region between JAK2 and the centromere are affected variably depending on the position of the mitotic recombination breakpoint (Figure 2). UPD as a genetic defect is best known in cases of various inherited syndromes such as Prader–Willi, Angelmann and Silver–Russell, associated with complex phenotypes ranging from aberrant growth to mental retardation. UPD in these congenital disorders leads to either loss or gain of expression of imprinted genes.91 Acquired 9pUPD in MPN can variably affect the maternal or the paternal chromosome.23 Thus, there is a 50% chance that imprinted genes can lose or gain expression in the 9pUPD-positive clone exerting an effect on myeloid hematopoietic cells. In addition, genes that exhibit random monoallelic expression can be affected the same way as imprinted genes.92 On chromosome 9p, CNTLN, KANK1, DMRT1, TOPORS and MLANA genes were reported to be either imprinted, differentially methylated or show monoallelic expression.92, 93, 94, 95 Taking these secondary influences of UPD into account, association of high allelic burden of JAK2-V617F mutation with clinical phenotypes may be partly influenced by homozygous SNPs and/or loss or gain of expression of monoallelically expressed genes. The region between the 9p telomere and JAK2 alone, which is always homozygous in all patients with 9pUPD, contains over 37 000 polymorphisms of which 129 are non-synonymous SNPs affecting the amino-acid sequence of proteins (data from http://www.ncbi.nlm.nih.gov/sites/entrez?db=snp). All these factors can potentially influence the clinical phenotype of MPN patients with 9pUPD.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Distribution of the mitotic recombination breakpoints in myeloproliferative neoplasm (MPN). The position of the JAK2 gene and the chromosome 9 centromere are indicated with an arrow. Chromosomal position is shown in mega base pairs (Mb). Data compiled from published studies.23, 90

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Clonal variability in MPN

The variability of somatic mutations and of chromosomal aberrations present in MPN clearly establishes the genetic heterogeneity of MPN. As a consequence, acquisition of several of these mutations/aberrations in the same patient contributes to clonal diversity among patients. A number of patients with a simultaneous presence of JAK2-V617F and MPL-W515L/K, and JAK2-V617F and JAK2-ex12 mutations have been reported.33, 55 However, only one study proved that two independent clones of progenitors co-existed, one positive for JAK2-V617F and the other for JAK2-ex12 mutations.96 Presence of del20q has been reported either as a mutation establishing clonal hematopoiesis before the acquisition of JAK2-V617F44 or, in a different recent study, del20q-positive progenitors have been observed as a minor clone arising from JAK2-V617F-positive cells.97 Figure 3 summarizes the clonal structures of MPN patients reported to date. Additional clonal and genetic variability of MPN will be described in the future as high-resolution karyotype analysis using SNP array technology becomes widely available. Whole-genome SNP array studies of few MPN patients were recently reported,90, 98 confirming 9pUPD to be the most frequent genomic aberration in MPN and demonstrating that 9pUPD can occur more than once in the same patient.

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Summary of clonal structures identified in myeloproliferative neoplasm (MPN) to date. Each black frame represents a clonal structure. The hierarchy of mutations (the acquisition order) in each clonal structure is shown by colored boxes. (a–c) The most common clonal structures in patients with a single mutation in either JAK2-V617F or JAK2-ex12. Patients homozygous for JAK2-V617F have a population of cells with uniparental disomy 9p (9pUPD).20, 21, 22, 23, 24 (d) Some patients with JAK2-ex12 acquire 9pUPD.55 (e) Deletions on chromosome 20q (del20q) were reported to occur on the background of JAK2-V617F-positive cells in some patients.97 (f) A patient with polycythemia vera with both JAK2-V617F and JAK2-ex12 mutations was reported.55 The two mutations were representing two clones of cells. (g, h) Simple clonal structure is likely in patients carrying a single MPL-W515L mutation. Homozygous patients for MPL-W515L are likely to have uniparental disomy on chromosome 1p (1pUPD).33 (I, j) Clonal structures of patients with a multi-hit pathogenesis, with JAK2-V617F occurring on the background of clonal cells established either by del20q or by unknown mutations.44, 54 (k) Two possible clonal structures in patients carrying both JAK2-V617F and MPL-W515L mutations.33

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Stochastic mutation acquisition model of MPN pathogenesis

The genetic data obtained from sporadic and familial MPN cases presented so far as well as data from animal models do not unequivocally confirm or exclude either of the two models of MPN pathogenesis (Figure 1). The genetic and clonal diversity observed in MPN patients suggests that the acquisition of mutations in MPN does not follow a particular order but it appears stochastic (Figure 4). If somatic mutations in MPN are acquired randomly, a population of patients acquires JAK2-V617F as the first mutation that establishes both the disease phenotype and clonal hematopoiesis. In another set of patients, the first mutations may be any of the chromosomal aberrations (del20q, del13q, and so on) that do not induce an apparent clinical phenotype but have the potential to establish clonal hematopoiesis. 'Phenotype-inducing mutations' such as JAK2-V617F are acquired later and the pathogenesis follows a multistep model. The only exception in this stochastic mutation acquisition model is the place of UPD that always occurs after the semi-dominant oncogenic mutation (9pUPD in JAK2 wild-type cells would not result in a selective advantage).

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Outline of the 'stochastic' mutation acquisition model of myeloproliferative neoplasm (MPN) pathogenesis. Two types of mutations are acquired in MPN. 'Phenotypic' mutations (squares) of JAK2 (V617F, exon 12 mutations—ex12) and MPL (such as W515L, W515K and S505N) cause both clonal hematopoiesis and onset of MPN phenotypes. 'Non-phenotypic' mutations such as deletions on chromosomes 20q and 13q (del20q and del13q) establish clonal hematopoiesis but do not cause any recognizable clinical phenotype. The stochastic mutation acquisition model of MPN pathogenesis proposes that the two types of mutations are acquired randomly in patients during the course of the disease (vertical arrows) without a predetermined order of acquisition. Acquisition of 'phenotypic' mutations is always associated with the appearance of disease phenotype (horizontal arrows). As mutations are acquired randomly, 'phenotypic' mutations are acquired first in a proportion of patients (lower panel left), whereas some patients acquire 'non-phenotypic' mutations first followed by acquisition of 'phenotypic' mutations (lower panel right).

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Genetic complexity of MPN and therapeutic strategies

The presence of a unique gain-of-function mutation of JAK2 kinase in more than half of MPN patients raised hopes that targeting JAK2-V617F in MPN with small molecule inhibitors may have similar success as imatinib in CML. This expectation is justified in many respects. JAK2-V617F is the mutation that beyond any doubt causes the clinical phenotype of MPN in patients. 'Addiction' of the MPN clone to survival mediated by the JAK/STAT signaling pathway can make it particularly sensitive to an anti-JAK2 therapy in patients carrying any of the JAK2 or MPL mutations. Thus, elimination of myeloid cells positive for these mutations should cause elimination of the MPN phenotype. It is questionable, however, whether the molecular remission will also restore polyclonal hematopoiesis in all the patients—the gold standard of 'complete cure'. The ability of JAK2 inhibitors to restore polyclonal hematopoiesis may vary depending on the genetic and clonal diversity of each patient. In patients with only a single mutation, such therapy should be successful in both remission of the MPN phenotype and restoration of polyclonal hematopoiesis. If patients carry multiple mutations and are clonally diverse, elimination of JAK2-V617F-positive cells may only reduce clonal diversity but polyclonal hematopoiesis is unlikely to be restored. Unfortunately, even in CML, it has not been formally shown that molecular remission induced by imatinib is accompanied by restoration of polyclonal hematopoiesis as clonality in CML is always assessed by the presence/absence of the BCR/ABL fusion.

Most cytostatic drugs (including hydroxyurea and interferon alpha) applied currently or in the future in MPN will impose a selective pressure on the MPN stem/progenitor pool resulting in changes in clonal structure and favoring subclones capable of survival and expansion in the presence of the drug. Kinase inhibitors targeting JAK2 often inhibit other kinases such as FLT3. This broad inhibitory effect of drugs may increase their cytotoxicity but it may also impose a selective pressure on clonal cells negative for JAK2-V617F and increase the chance of acquisition of leukemic mutations such as FLT3-ITD or other mutations facilitating survival in the presence of the drug. In this respect, post-MPN leukemic transformation in JAK2-V617F-positive MPN patients has been shown to frequently arise from the JAK2-V617F-negative cells,99, 100 demonstrating that JAK2-V617F-negative clonal cells have a potential to acquire various, often leukemogenic mutations.

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Conclusions

The data published so far on the genetic basis of MPN support the 'stochastic' model of MPN pathogenesis in which certain types of somatic mutations with different phenotypic effects are acquired in a random order. Five types of oncogenic mutations of JAK2 and MPL genes are clearly associated with the onset of MPN phenotype. Cytogenetic aberrations such as deletion of chromosome 20q or 13q are not likely to cause a specific hematopoietic phenotype. Patients with MPN not only display phenotypic variability but also genetic and clonal heterogeneity. Thus, individualized therapy taking genetic heterogeneity of patients into account is required to successfully treat MPN.

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