A Gain-of-Function Mutation of JAK2 in Myeloproliferative Disorders
Robert Kralovics, Ph.D., Francesco Passamonti, M.D., Andreas S. Buser, M.D., Soon-Siong Teo, B.S., Ralph Tiedt, Ph.D., Jakob R. Passweg, M.D., Andre Tichelli, M.D., Mario Cazzola, M.D., and Radek C. Skoda, M.D.
Background Polycythemia vera, essential thrombocythemia, andidiopathic myelofibrosis are clonal myeloproliferative disordersarising from a multipotent progenitor. The loss of heterozygosity(LOH) on the short arm of chromosome 9 (9pLOH) in myeloproliferativedisorders suggests that 9p harbors a mutation that contributesto the cause of clonal expansion of hematopoietic cells in thesediseases.
Methods We performed microsatellite mapping of the 9pLOH regionand DNA sequencing in 244 patients with myeloproliferative disorders(128 with polycythemia vera, 93 with essential thrombocythemia,and 23 with idiopathic myelofibrosis).
Results Microsatellite mapping identified a 9pLOH region thatincluded the Janus kinase 2 (JAK2) gene. In patients with 9pLOH,JAK2 had a homozygous GT transversion, causing phenylalanineto be substituted for valine at position 617 of JAK2 (V617F).All 51 patients with 9pLOH had the V617F mutation. Of 193 patientswithout 9pLOH, 66 were heterozygous for V617F and 127 did nothave the mutation. The frequency of V617F was 65 percent amongpatients with polycythemia vera (83 of 128), 57 percent amongpatients with idiopathic myelofibrosis (13 of 23), and 23 percentamong patients with essential thrombocythemia (21 of 93). V617Fis a somatic mutation present in hematopoietic cells. Mitoticrecombination probably causes both 9pLOH and the transitionfrom heterozygosity to homozygosity for V617F. Genetic evidenceand in vitro functional studies indicate that V617F gives hematopoieticprecursors proliferative and survival advantages. Patients withthe V617F mutation had a significantly longer duration of diseaseand a higher rate of complications (fibrosis, hemorrhage, andthrombosis) and treatment with cytoreductive therapy than patientswith wild-type JAK2.
Conclusions A high proportion of patients with myeloproliferativedisorders carry a dominant gain-of-function mutation of JAK2.
The myeloproliferative disorders are a heterogeneous group ofdiseases characterized by excessive production of blood cellsby hematopoietic precursors. In addition to thrombotic and hemorrhagiccomplications, leukemic transformation can occur.1 Typically,the myeloproliferative disorders encompass four related entities2:chronic myelogenous leukemia (CML), polycythemia vera, essentialthrombocythemia, and idiopathic myelofibrosis. Clonal hematopoiesisis a key feature of these disorders.3,4,5 The lesion is believedto involve the hematopoietic stem cell, since all myeloid lineagesand, frequently, the B-cell lineage are monoclonal. T cells,however, are polyclonal.4,6
Progenitor cells in polycythemia vera form erythroid coloniesin the absence of exogenous erythropoietin. These endogenouserythroid colonies7 have been used in an auxiliary diagnosticassay to distinguish polycythemia vera from secondary erythrocytosis,8but they also occur in some cases of essential thrombocythemiaand idiopathic myelofibrosis. More important, the presence ofendogenous erythroid colonies is a hallmark of abnormal in vitrogrowth of hematopoietic progenitors, and this finding has beenthe basis of many studies of signaling by cytokine receptorsof hematopoietic cells. These receptors transduce signals byactivating members of the Janus kinase (JAK) family of proteins,which phosphorylate cytoplasmic targets, including the signaltransducers and activators of transcription (STATs).9 Constitutiveactivation of the STAT3 protein has been found in 30 percentof patients with polycythemia vera,10 and a decrease in thelevel of the thrombopoietin receptor protein in platelets isa feature of both polycythemia vera and essential thrombocythemia.11,12Only the late steps of differentiation of endogenous erythroidcolonies in polycythemia vera are erythropoietin-independent,and they can be blocked by inhibitors of JAK2, phosphatidylinositol3' kinase, or kinases of the Src family.13 Recently, the tyrosinekinase inhibitor imatinib mesylate has been reported to produceclinical responses in patients with polycythemia vera.14,15These data suggest the involvement of a kinase in the pathogenesisof myeloproliferative disorders.
Cytogenetic abnormalities occur in only 10 to 15 percent ofpatients with myeloproliferative disorders.16,17,18 The mostcommon abnormalities are deletions in chromosome 20q.19,20 Fluorescencein situ hybridization and comparative genomic hybridizationsuggested a role for chromosome 9p.20,21,22,23 Using genome-widemicrosatellite screening, we identified loss of heterozygosity(LOH) on the short arm of chromosome 9 (9pLOH) in six patientswith polycythemia vera24; using microsatellite markers withinthe LOH region, we found 9pLOH in 13 of 43 patients with polycythemiavera and 1 of 15 patients with essential thrombocythemia.24,25Markers from the 9pLOH region did not cosegregate with the phenotypein four families with polycythemia vera, suggesting that a somaticevent causes 9pLOH.26 In the present study, we increased thenumber of microsatellite markers in order to map a minimal genomicregion shared by all patients with 9pLOH and myeloproliferativedisorders to identify potential candidate genes.
Methods
Subjects
We evaluated 244 patients with myeloproliferative disordersfrom Switzerland and Italy: 128 patients with polycythemia vera,93 with essential thrombocythemia, and 23 with idiopathic myelofibrosis.25,27,28We studied 41 healthy persons, 9 patients with chronic myelogenousleukemia, and 11 patients with secondary erythrocytosis as controls.The study was approved by the local ethics committees, and allsamples were obtained after subjects provided written informedconsent. In addition, we used DNA from 30 archival samples.The diagnostic criteria of the World Health Organization (WHO)were followed for all Swiss patients, whereas the PolycythemiaVera Study Group (PVSG) criteria were used for all Italian patients.29,30,31The main difference between the two classifications is the useof bone marrow histologic findings as a diagnostic criterionin the WHO classification to distinguish early stages of idiopathicmyelofibrosis from essential thrombocythemia or polycythemiavera. The presence of endogenous erythroid colonies is a majorWHO criterion and a minor PVSG criterion.
Isolation of Cells and DNA
Granulocytes were isolated,24 and analysis of cytospin preparationsverified that the purity exceeded 90 percent. Peripheral-bloodCD4+ T cells were isolated by means of magnetic sorting (MiltenyiBiotech). Peripheral-blood mononuclear cells (PBMCs) were preparedwith the use of Ficoll gradient centrifugation. Buccal mucosalcells were obtained with cytologic brushes, and hair-follicleDNA was prepared from plucked hair. Genomic DNA was isolatedwith the use of the QIAmp DNA Blood Mini Kit (Qiagen).
Detection of LOH
LOH was detected by means of fluorescence microsatellite polymerase-chain-reaction(PCR) analysis with the use of primer sequences from publicdatabases (listed in the Supplementary Appendix, available withthe complete text of this article at www.nejm.org). The sampleswere analyzed on a DNA genetic analyzer (model 3100, AppliedBiosystems). LOH was considered to be present if one alleleshowed a reduction in the peak fluorescence intensity of morethan 90 percent in granulocytes, but two alleles were presentin nonclonal tissues (T cells, PBMCs, buccal mucosa, or hair-folliclecells) from the same patient.
Analysis of the Number of Gene Copies
The number of copies of chromosome 9p in granulocyte DNA wasdetermined with the use of quantitative PCR by comparing twosingle-copy loci: one in exon 14 of JAK2 and an uncharacterizedgene on chromosome 13. The primers and details of these assaysare provided in the Supplementary Appendix.
JAK2 Sequencing
We sequenced the JAK2 complementary DNA (cDNA) with reverse-transcriptase(RT) PCR using seven pairs of overlapping primers describedin the Supplementary Appendix (sequence information is availableon request).
DNA Constructs
The mutant JAK2 cDNA was amplified by RT-PCR with the use ofgranulocyte RNA from a patient who was homozygous for the mutation.The Supplementary Appendix gives details of the primers andcloning into vectors.
Proliferation Assays
The mouse interleukin 3–dependent cell line BaF3 and thehuman thrombopoietin-dependent cell line UT-7/TPO (kindly providedby Dr. N. Komatsu) were transfected by electroporation withthe wild-type JAK2 (plasmid murine stem-cell virus [pMSCV]–Jak2)and mutant JAK2 (pMSCV-V617F-Jak2) constructs. The Supplementary Appendixgives details of the assays for proliferation and cellviability.
Immunoprecipitation and Western Blotting
BaF3 cells were incubated in a culture medium (RPMI) containing10 percent fetal-calf serum in the absence of interleukin-3for 12 hours at 37°C, whereupon various concentrations ofinterleukin-3 were added for 15 minutes. Cell lysates were preparedas previously described.32 Immunoprecipitation and immunoblottingwere carried out with the use of polyclonal antibodies againstJAK2 (Upstate) and STAT5 (Santa Cruz) and the phosphotyrosine-specificmouse monoclonal antibody 4G10 (Upstate).
Statistical Analysis
We used the chi-square or Fisher's exact test where appropriateto compare categorical variables among the groups, which werecategorized according to mutational status (heterozygous, homozygous,or wild type), and the Mann–Whitney U test or Kruskal–Wallistest to compare continuous variables among the groups. For someanalyses, the heterozygous and the homozygous groups of patientswere pooled and compared with patients without JAK2 mutations.
Results
Fine Mapping of the Common 9pLOH Region
Using 10 microsatellite markers covering chromosome 9p, we found9pLOH in granulocytes from 51 of 244 patients with myeloproliferativedisorders (21 percent), but not in those from any of the controlsubjects: 41 healthy subjects, 9 patients with CML, and 11 patientswith secondary erythrocytosis. The frequency of 9pLOH was 34percent among patients with polycythemia vera, 22 percent amongpatients with idiopathic myelofibrosis, and 3 percent amongpatients with essential thrombocythemia. The size of the chromosomalregion showing LOH varied, but the telomeric region of chromosome9p was always involved (Figure 1A). By aligning the LOH regionsof all 51 patients with 9pLOH, we identified a 6.2-Mbp intervalcommon to all patients that extended from the telomere to markerD9S1852 and contained the gene for the tyrosine kinase JAK2(Figure 1B). Since JAK2 mediates signaling through several hematopoieticcytokine receptors, we considered JAK2 an attractive candidategene.
Figure 1. Mapping of the Minimal 9pLOH Region in Patients with Myeloproliferative Disorders.
Panel A shows the mapping results for 51 patients with 9pLOH. Solid squares indicate LOH in granulocytes detected by the corresponding microsatellite marker; open squares represent the absence of LOH. Vertical lines represent individual patients. The patients' results are arranged from left to right in the order of increasing size of the LOH region. The minimal LOH region is delineated by the gray background color. For clarity, markers that were uninformative have been omitted. Panel B shows a physical map of genes within the common 9pLOH region (not drawn to scale). The positions of microsatellite markers used to identify the common LOH region (gray zone) are shown as vertical lines. Numbers indicate the physical distance from the chromosome 9p telomere in megabase pairs (Mbp). Black boxes represent genes. The results of microsatellite mapping in four patients with the shortest LOH region are shown below the map. Solid squares indicate LOH, and open squares represent the absence of LOH. The mitotic recombination breakpoint in these patients occurred in the 0.9-Mbp region between the markers D9S1681 and D9S1852.
9pLOH and a GT Mutation in the Coding Region of JAK2
The coding region of JAK2 in all 51 patients with 9pLOH hada GT transversion that changed a valine to a phenylalanine atposition 617 (V617F; GenBank accession number AY973037
[GenBank]
) (Table 1and Figure 2). All patients with 9pLOH are expected to behomozygous (both alleles mutated) or hemizygous (one allelemutated and the other absent) for the V617F mutation. However,in eight patients, the findings were compatible with the presenceof heterozygosity (one allele was mutated, and the other waswild type) (Table 1). Our definition of LOH allows 10 percentof granulocytes without 9pLOH to be present in a given sampleand the ratios of G and T peak intensities in sequencing chromatogramscannot be used to quantify allelic ratios. It is therefore likelythat an admixture of granulocytes with wild-type or heterozygousV617F genotypes caused the apparent heterozygosity in theseeight patients. Of the remaining 193 patients without 9pLOH,34 percent were heterozygous for V617F and 66 percent were homozygousfor the wild-type allele; none were homozygous for V617F (Table 1).The V617F mutation was absent in 71 healthy controls, 11patients with secondary erythrocytosis, and 9 patients withCML (Table 1).
Figure 2. The JAK2 Mutation in Patients with Myeloproliferative Disorders.
Panel A shows a homozygous GT transversion in JAK2 (arrow) in a patient with 9pLOH (left) and a heterozygous mutation in a patient without 9pLOH (right). The mutation was present in DNA from granulocytes, but absent in T cells, consistent with the existence of an acquired somatic origin of the mutation. Panel B shows the domain structure of the JAK2 protein. Numbers indicate the amino acid position within the protein. The arrow indicates the position of the V617F mutation. The alignment of sequences of the JAK family of proteins is shown below. Panel C shows the somatic origin of the JAK2 mutation. DNA from granulocytes and buccal mucosa cells showed the GT transversion, but DNA from hair follicles demonstrated absence of the mutation. FERM denotes band 4.1(f), ezrin, radixin, and moesin; and SH2 SRC homology 2.
The mutation was present in 65 percent of patients with polycythemiavera, as compared with 57 percent of those with idiopathic myelofibrosis(P=0.72) and 23 percent of those with essential thrombocythemia(P<0.001) (Table 1). Homozygosity for V617F was underrepresentedamong patients with essential thrombocythemia, as compared withpatients with polycythemia vera and patients with idiopathicmyelofibrosis, and was linked to the presence of 9pLOH.
The median duration of disease was significantly longer amongpatients with myeloproliferative disorders who were homozygousfor the V617F mutation than among those who were heterozygousfor the mutation (48 months [range, 0 to 576] vs. 23 months[range, 0 to 252], P<0.02 by the Mann–Whitney U test).This difference is compatible with the existence of a two-stepprocess to acquire homozygosity. Interestingly, patients withmyeloproliferative disorders who had wild-type JAK2 had theshortest duration of disease (15 months; range, 0 to 330; P=0.05for the comparison with heterozygous patients).
Identification of a Somatic Mutation in Myeloproliferative Disorders
We sequenced DNA from T cells, PBMCs, or nonhematopoietic tissuesfrom 89 patients with the V617F mutation. We could not detectthe mutant JAK2 allele in these control tissues, a result consistentwith the existence of a somatic mutation. In two patients, wedetected the V617F mutation in buccal mucosa cells, but sincesamples of buccal mucosa can be contaminated with blood, weanalyzed hair-follicle cells from these patients and found onlythe wild-type JAK2 sequence (Figure 2C).
Cause of 9pLOH
The 9pLOH in myeloproliferative disorders could result fromdeletions of the telomeric portions of chromosome 9p or mitoticrecombination between chromatids of homologous 9p chromosomes(Figure 3A). In the case of a deletion, we would expect to findonly one copy of DNA for the deleted region, whereas two copiesshould be expected in the event of mitotic recombination (Figure 3A).To evaluate these possibilities, we used quantitative PCRto determine the number of copies of JAK2 and, as a control,the number of copies of a single-copy gene on chromosome 13.Of 33 patients with 9pLOH, all had two copies of chromosome9p (Figure 3B). This result argues against deletions as thecause of 9pLOH and makes mitotic recombination the most likelymechanism. We also analyzed parental chromosomes of two patientswith 9pLOH. In one (Patient 50), the maternally derived chromosome9p was lost, whereas in the other (Patient 116), the paternalchromosome 9p was missing (Figure 3C). Thus, either maternalor paternal chromosome 9p can be lost, suggesting that genomicimprinting is not involved in the expansion of cells with 9pLOH.
Two alternative models are presented in Panel A. The chromosome 9 with the wild-type JAK2 sequence (G) is depicted in white, and the chromosome 9 with the GT transversion (T) is shown in red. Circles symbolize the nuclei of the cells. Deletion of the telomeric part of wild-type chromosome 9p as a potential mechanism for 9pLOH is shown on the left. Alternatively, mitotic recombination could also result in 9pLOH, shown on the right. The events during mitosis and the resulting cell progeny after mitotic recombination of chromosome 9p are also shown. Panel B shows the number of copies of JAK2 among 33 patients with 9pLOH, 12 healthy controls, and 3 archival tumor samples from patients with monosomy 9. Panel C shows the parental origin of the lost chromosome in two patients with 9pLOH. In one (Patient 50), the maternally derived chromosome 9p was lost, whereas in the other (Patient 116), the paternal chromosome 9p was missing. Data obtained by real-time polymerase chain reaction comparing the abundance of JAK2 and a single copy gene on chromosome 13 are shown (CT).
Proliferative and Survival Advantage Afforded by V617F
To investigate whether the V617F mutation has functional consequences,we analyzed data on endogenous erythroid colonies in 180 patientswith myeloproliferative disorders. Endogenous erythroid colonieswere present in 77 of 87 patients with the V617F mutation, whereasonly 57 of 93 patients without the mutation had them (P<0.001by Fisher's exact test). Next, we expressed the JAK2 V617F proteinin the mouse interleukin-3–dependent cell line BaF3 (Figure 4).These transfected cells were hypersensitive to low concentrationsof interleukin-3 and were more numerous (elevated baseline value)than control cells in the absence of interleukin-3 (Figure 4A).The same results were obtained in four independent experimentsand in analogous experiments with BaF3 cells that were doublytransfected with the erythropoietin receptor gene and JAK2 andin V617F-transfected thrombopoietin-dependent UT-7/TPO humancells (data not shown).
Figure 4. Functional Effects of the JAK2 V617F Mutation in Stably Transfected Murine Interleukin-3–Dependent BaF3 Cells.
Panel A shows the proliferation of BaF3 cells transfected with the V617F mutant JAK2, the wild-type JAK2, or the empty vector in the absence of interleukin-3 (a concentration of 0) and the presence of increasing concentrations of interleukin-3, as determined by the tetrazolium salt (XTT) assay. The mean (±SD) of triplicate results is shown (in some cases, the error bars are hidden behind the symbols). Increased optical density (OD) of the XTT dye corresponds to increased numbers of cells. Panel B shows the survival of stably transfected BaF3 cells in the absence of interleukin-3. The percentage of viable cells was determined by the exclusion of the dye trypan blue from the cells. The mean (±SD) of triplicate results is shown. Panel C shows stably transfected BaF3 cells maintained in the absence of interleukin-3 for 10 days. BaF3 cells transfected with JAK2 V617F are viable, whereas almost all cells transfected with the wild-type JAK2 or the empty vector are dead. Panel D shows the activation of JAK2 and STAT5 in response to interleukin-3. BaF3 cells transfected with the empty vector (V), the wild-type JAK2 (W), or the JAK2 V617F mutant (M) were incubated for 12 hours without interleukin-3 but with 10 percent fetal-calf serum and were then stimulated for 15 minutes with increasing concentrations of interleukin-3, as indicated. Immunoprecipitation (IP) was carried out with the phosphotyrosine-specific mouse monoclonal antibody 4G10 (pTyr) followed by Western blotting (WB) with the use of antibodies against JAK2 or STAT5, as indicated. Levels of expression of JAK2 and STAT5 proteins in the lysates used for immunoprecipitation were visualized by immunoblotting with the use of the corresponding antibodies and are shown at the bottom.
Interleukin-3, erythropoietin, and thrombopoietin signalingis dependent on JAK2.33,34 Differences in cell viability wereapparent when the transfected BaF3 cell lines were maintainedin serum-containing medium in the absence of interleukin-3 (Figure 4B).This result suggests that the transfected JAK2 mutant increasedsurvival of the cells in the absence of the cytokine. Sincethe proliferation assay measures the numbers of cells afterthree days of culture, the results obtained in the absence ofinterleukin-3 in Figure 4A can be compared with those of theviability curves on day 3 in Figure 4B: both show approximatelytwice the number of viable mutant cells as control cells. Whenkept under the same conditions for more than 10 days, BaF3 cellstransfected with the JAK2 V617F survived, and their numbersincreased by a factor of 100, whereas cells transfected withthe wild-type JAK2 or the vector control died (Figure 4C).
We also examined the phosphorylation of JAK2 and STAT5 proteinsunder the same conditions as in the proliferation assays (Figure 4D).JAK2 phosphorylation was only slightly increased in theabsence of or at low concentrations of interleukin-3. A morepronounced difference was detected in the phosphorylation ofSTAT5, one of the principal protein substrates of JAK2 (Figure 4D).At higher interleukin-3 concentrations, these differencesdisappeared. The differences in signaling mirrored the differencesin growth and survival observed at the same cytokine concentrations.Thus, the presence of JAK2 V617F conferred a proliferative andsurvival advantage by rendering the cells more sensitive toincoming stimulatory signals.
Correlations with Clinical Data
Possible associations between the V617F mutation and clinicalfeatures were retrospectively analyzed in 244 patients. Theonly significant differences between patients who were heterozygousand those who were homozygous for the V617F mutation were theabove-mentioned increase in the median duration of disease andthe correlation between 9pLOH and homozygosity for the mutation.Therefore, we limited our comparison to patients with the V617Fmutation and those without the mutation (Table 2). Significantlymore patients with the mutation than without the mutation hadcomplications (secondary fibrosis, hemorrhage, and thrombosis)and had received cytoreductive treatment. Of five patients withleukemic transformation, four had the V617F mutation (P=0.20).
Table 2. Characteristics Associated with the JAK2 V617F Mutation.
Discussion
Fine mapping of the 9pLOH region in patients with myeloproliferativedisorders identified a 6.2-Mbp genomic interval that containsthe gene for the tyrosine kinase JAK2 (Figure 1). This gene,JAK2, was a strong candidate for causing clonal expansion ofhematopoietic progenitors in myeloproliferative disorders becauseof its essential function in hematopoiesis.33,34 We found aGT transversion that results in a change in a single amino acid,V617F, in the pseudokinase domain of JAK2 (Figure 2 and Table 1).V617F is not a polymorphism, since it was absent from 142chromosomes of healthy people and is not recorded in the databasesof single-nucleotide polymorphisms. Moreover, we did not detectthe V617F mutation in 11 patients with secondary erythrocytosisor 9 patients with CML.
We found conclusive evidence that V617F is a somatic mutationin hematopoietic cells. We cannot exclude the possibility ofgerm-line mutations in some cases of familial myeloproliferativedisorders, although chromosome 9p was previously excluded bylinkage analysis in four families with polycythemia vera.26The frequency of the V617F mutation was highest among patientswith polycythemia vera and lowest among patients with essentialthrombocythemia (Table 1). Essential thrombocythemia is themost heterogeneous myeloproliferative disorder, with a substantialproportion of patients showing polyclonal hematopoiesis.4,6,35The low frequency of 9pLOH in this disease might reflect thelow mitotic activity in the essential-thrombocythemia progenitoror stem-cell pool.
It is remarkable that we found an identical somatic GT transversionin JAK2 in 117 unrelated patients, or 48 percent of the patientswith myeloproliferative disorders (Table 1). The V617F mutationis located in the pseudokinase domain of JAK2 (Figure 2B), aregion that inhibits JAK2 kinase activity.36,37 Mutations affectingthis domain are associated with malignant transformation ofhematopoietic cells in drosophila.38
BaF3 and UT-7/TPO cells that were transfected with the mutantJAK2 gene were hypersensitive to low concentrations of interleukin-3and erythropoietin and thrombopoietin, respectively. In thepresence of serum, transfected BaF3 cells showed increased survival,proliferation, and phosphorylation of JAK2 and STAT5, even inthe absence of interleukin-3 (Figure 4). This result suggeststhat other cytokines in serum at low concentrations are mostlikely sufficient to maintain the viability and proliferationof cells expressing JAK2 V617F. Similarly, the growth of endogenouserythroid colonies in myeloproliferative disorders also dependson the presence of serum.39
The effect of the JAK2 V617F mutation on the proliferation ofBaF3 cells was less dramatic than that of the TEL-JAK2 fusionprotein found in acute lymphoblastic leukemias.40 The relativelysubtle effect of the V617F mutation fits well with the mildand indolent nature of the clonal proliferation observed inmyeloproliferative disorders. Hypersensitivity to insulin-likegrowth factor, thrombopoietin, interleukin-3, and other cytokineshas been described in hematopoietic progenitor cells from patientswith myeloproliferative disorders.39,41,42,43 How the V617Fmutation sustains the increased phosphorylation of JAK2 in thepresence of serum, but in the absence of interleukin-3, andthe factor or factors in serum that mediate this effect remainunknown. Nevertheless, the functional relevance of the V617Fmutation is supported by the finding that endogenous erythroidcolonies were present in 89 percent of patients with V617F andthe close association between homozygosity for the V617F mutationand 9pLOH, which demonstrates the survival advantage of suchhomozygous hematopoietic cells.
The gene-dosage analysis suggests that mitotic recombinationbetween chromatids of homologous chromosomes 9p is the mostlikely mechanism leading to 9pLOH (Figure 3). The absence ofhomozygosity for the V617F mutation in patients with myeloproliferativedisorders who do not have 9pLOH suggests that a transition fromheterozygosity to homozygosity by means of a mechanism thatis independent of mitotic recombination is rare. Mitotic recombinationis a frequent genetic mechanism in the inactivation of tumor-suppressorgenes in solid tumors,44,45,46 but not in malignant diseasesof the hematopoietic system.47
Our data suggest two possible pathogenetic mechanisms of theJAK2 V617F mutation. In one (model A in Figure 5), the heterozygousJAK2 V617F mutation alone or in combination with one or morepreexisting somatic mutations causes the myeloproliferative-disorderphenotype in approximately 50 percent of patients. A somaticmutation in an as yet unknown gene would be responsible forthe phenotype in the other patients (not shown). This modeldoes not explain the presence of the V617F mutation in severalmyeloproliferative disorders. Perhaps the genetic backgroundor additional somatic mutations in hematopoietic cells alterthe effects of the V617F mutation on the phenotype. In modelB, JAK2 V617F occurs after the appearance of the myeloproliferative-disorderphenotype as a mutation associated with disease progressionbut is not necessary or sufficient to cause the phenotype (Figure 5).In this model, patients with myeloproliferative disorderswithout the JAK2 mutation have an early stage of the disease.One prediction of this model is that the duration of diseaseshould be shortest in patients without the JAK2 mutation andlongest in those who are homozygous for the V617F mutation,as we observed. In both models, the transition from heterozygosityto homozygosity for the mutation represents clonal evolution.Our data indicate that mitotic recombination in patients with9pLOH produces a daughter cell that is homozygous for the V617Fmutation and can outcompete the parental cells that are heterozygous(Figure 3A and Figure 5).
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.
Evaluation of the clinical data (Table 2) revealed significantcorrelations between the presence of the V617F mutation andthe frequency of complications (secondary fibrosis, hemorrhage,and thrombosis). These correlations could be a consequence ofthe longer duration of disease in patients with the JAK2 mutationor could be linked to a more aggressive phenotype, perhaps owingto an increased responsiveness to cytokines. Patients with theV617F mutation were older than those without the mutation, anda higher proportion had received cytoreductive therapy at thetime of sample collection. The increased frequency of cytoreductivetherapy does not necessarily imply the presence of a more aggressivedisease and could be based on age alone, according to widelyaccepted therapeutic guidelines.
Taken together, our data suggest that the V617F mutation inJAK2 is a dominant gain-of-function mutation that contributesto the expansion of the myeloproliferative-disorder clone. TheV617F mutation could form the basis for a new molecular classificationof myeloproliferative disorders. Furthermore, in view of theinvariant nature of the JAK2 mutation, small molecules couldbe developed that specifically target the mutated protein inpatients with myeloproliferative disorders.
Supported in part by a grant (3100-066 949.01) from the SwissNational Science Foundation, grants (OCS-01163-09-2001 and OCS-01411-08-2003)from the Swiss Cancer League, and a grant from the Stiftungfür Hämatologische Forschung (all to Dr. Skoda); bya grant from the Krebsliga beider Basel (to Dr. Kralovics);and by a grant from the Associazione Italiana per la Ricercasul Cancro, Milan (to Dr. Cazzola).
We are indebted to the patients and their physicians for contributingto the study, to Gary Gilliland for sharing data before publication,to Guido Sauter for providing cells with monosomy 9, and toAlois Gratwohl, Jürg Schwaller, Markus Heim, and Ed Palmerfor helpful comments on the manuscript.
Source Information
From the Department of Research, Experimental Hematology (R.K., S.-S.T., R.T., R.C.S.), and the Departments of Hematology (A.S.B., J.R.P.) and Laboratory Medicine (A.T.), University Hospital Basel, Basel, Switzerland; and the Division of Hematology, University of Pavia Medical School and Istituto di Ricovero e Cura a Carattere Scientifico, Policlinico San Matteo, Pavia, Italy (F.P., M.C.).
Address reprint requests to Dr. Skoda at the Department of Research, University Hospital Basel, Hebelstr. 20, CH-4031 Basel, Switzerland, or at radek.skoda{at}unibas.ch.
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(2008). Downregulation of Signal Transducer and Activator of Transcription 5 (STAT5) in CD34+ Cells Promotes Megakaryocytic Development, Whereas Activation of STAT5 Drives Erythropoiesis. Stem Cells
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(2008). Somatic Mutations of JAK1 and JAK3 in Acute Leukemias and Solid Cancers. Clin. Cancer Res.
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(2008). Lestaurtinib (CEP701) is a JAK2 inhibitor that suppresses JAK2/STAT5 signaling and the proliferation of primary erythroid cells from patients with myeloproliferative disorders. Blood
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(2008). The impact of JAK2 and MPL mutations on diagnosis and prognosis of splanchnic vein thrombosis: a report on 241 cases. Blood
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(2008). LS104, a non-ATP-competitive small-molecule inhibitor of JAK2, is potently inducing apoptosis in JAK2V617F-positive cells. Molecular Cancer Therapeutics
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Xiang, Z., Zhao, Y., Mitaksov, V., Fremont, D. H., Kasai, Y., Molitoris, A., Ries, R. E., Miner, T. L., McLellan, M. D., DiPersio, J. F., Link, D. C., Payton, J. E., Graubert, T. A., Watson, M., Shannon, W., Heath, S. E., Nagarajan, R., Mardis, E. R., Wilson, R. K., Ley, T. J., Tomasson, M. H.
(2008). Identification of somatic JAK1 mutations in patients with acute myeloid leukemia. Blood
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Grebien, F., Kerenyi, M. A., Kovacic, B., Kolbe, T., Becker, V., Dolznig, H., Pfeffer, K., Klingmuller, U., Muller, M., Beug, H., Mullner, E. W., Moriggl, R.
(2008). Stat5 activation enables erythropoiesis in the absence of EpoR and Jak2. Blood
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Loriaux, M. M., Levine, R. L., Tyner, J. W., Frohling, S., Scholl, C., Stoffregen, E. P., Wernig, G., Erickson, H., Eide, C. A., Berger, R., Bernard, O. A., Griffin, J. D., Stone, R. M., Lee, B., Meyerson, M., Heinrich, M. C., Deininger, M. W., Gilliland, D. G., Druker, B. J.
(2008). High-throughput sequence analysis of the tyrosine kinome in acute myeloid leukemia. Blood
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(2008). Somatic mutations and germline sequence variants in the expressed tyrosine kinase genes of patients with de novo acute myeloid leukemia. Blood
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(2008). Ratio of mutant JAK2-V617F to wild-type Jak2 determines the MPD phenotypes in transgenic mice. Blood
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(2008). Clonal heterogeneity in polycythemia vera patients with JAK2 exon12 and JAK2-V617F mutations. Blood
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Wernig, G., Gonneville, J. R., Crowley, B. J., Rodrigues, M. S., Reddy, M. M., Hudon, H. E., Walz, C., Reiter, A., Podar, K., Royer, Y., Constantinescu, S. N., Tomasson, M. H., Griffin, J. D., Gilliland, D. G., Sattler, M.
(2008). The Jak2V617F oncogene associated with myeloproliferative diseases requires a functional FERM domain for transformation and for expression of the Myc and Pim proto-oncogenes. Blood
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(2008). Prevention of thrombosis in polycythemia vera and essential thrombocythemia. haematol
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Lu, X., Huang, L. J.-S., Lodish, H. F.
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(2008). Response: Mutations of JAK2, JAK3 and GATA1 in acute megakaryoblastic leukemia of Down syndrome. Blood
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Teofili, L., Foa, R., Giona, F., Larocca, L. M.
(2008). Childhood polycythemia vera and essential thrombocythemia: does their pathogenesis overlap with that of adult patients?. haematol
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(2008). WP1066, a Novel JAK2 Inhibitor, Suppresses Proliferation and Induces Apoptosis in Erythroid Human Cells Carrying the JAK2 V617F Mutation. Clin. Cancer Res.
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(2008). Chromosomal lesions and uniparental disomy detected by SNP arrays in MDS, MDS/MPD, and MDS-derived AML. Blood
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(2008). Somatic mutations of JAK2 exon 12 in patients with JAK2 (V617F)-negative myeloproliferative disorders. Blood
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Kawamata, N., Ogawa, S., Zimmermann, M., Kato, M., Sanada, M., Hemminki, K., Yamatomo, G., Nannya, Y., Koehler, R., Flohr, T., Miller, C. W., Harbott, J., Ludwig, W.-D., Stanulla, M., Schrappe, M., Bartram, C. R., Koeffler, H. P.
(2008). Molecular allelokaryotyping of pediatric acute lymphoblastic leukemias by high-resolution single nucleotide polymorphism oligonucleotide genomic microarray. Blood
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(2008). Highly penetrant myeloproliferative disease in the Ts65Dn mouse model of Down syndrome. Blood
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(2008). Postsurgery outcomes in patients with polycythemia vera and essential thrombocythemia: a retrospective survey. Blood
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(2008). JAK2V617F mutation status identifies subtypes of refractory anemia with ringed sideroblasts associated with marked thrombocytosis. haematol
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Antonioli, E., Guglielmelli, P., Poli, G., Bogani, C., Pancrazzi, A., Longo, G., Ponziani, V., Tozzi, L., Pieri, L., Santini, V., Bosi, A., Vannucchi, A. M., for the Myeloproliferative Disorders Research Cons,
(2008). Influence of JAK2V617F allele burden on phenotype in essential thrombocythemia. haematol
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(2008). Systemic Mastocytosis Associated with Chronic Idiopathic Myelofibrosis: A Distinct Subtype of Systemic Mastocytosis-Associated Clonal Hematological Nonmast Cell Lineage Disorder Carrying the Activating Point Mutations KITD816V and JAK2V617F. J. Mol. Diagn.
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(2008). Integration of Histology and Genetics in the Diagnosis and Classification of Myeloproliferative Neoplasms. Am Soc Clin Oncol Ed Book
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(2008). Bone marrow pathology in essential thrombocythemia: interobserver reliability and utility for identifying disease subtypes. Blood
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Rumi, E., Passamonti, F., Della Porta, M. G., Elena, C., Arcaini, L., Vanelli, L., Del Curto, C., Pietra, D., Boveri, E., Pascutto, C., Cazzola, M., Lazzarino, M.
(2007). Familial Chronic Myeloproliferative Disorders: Clinical Phenotype and Evidence of Disease Anticipation. JCO
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(2007). The frequency of JAK2 exon 12 mutations in idiopathic erythrocytosis patients with low serum erythropoietin levels. haematol
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(2007). JAK2 V617F mutational status predicts progression to large splenomegaly and leukemic transformation in primary myelofibrosis. Blood
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(2007). Evidence for MPL W515L/K mutations in hematopoietic stem cells in primitive myelofibrosis. Blood
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(2007). The revised WHO diagnostic criteria for Ph-negative myeloproliferative diseases are not appropriate for the diagnostic screening of childhood polycythemia vera and essential thrombocythemia. Blood
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[Abstract][Full Text]
Ma, A. C. H., Ward, A. C., Liang, R., Leung, A. Y. H.
(2007). The role of jak2a in zebrafish hematopoiesis. Blood
110: 1824-1830
[Abstract][Full Text]
Spittle, C., Ward, M. R., Nathanson, K. L., Gimotty, P. A., Rappaport, E., Brose, M. S., Medina, A., Letrero, R., Herlyn, M., Edwards, R. H.
(2007). Application of a BRAF Pyrosequencing Assay for Mutation Detection and Copy Number Analysis in Malignant Melanoma. J. Mol. Diagn.
9: 464-471
[Abstract][Full Text]
Stoj, A., Rudzki, Z., Stachura, J., Sacha, T., Czekalska, S.
(2007). The JAK2 V617F mutation in Philadelphia-negative chronic myeloproliferative disorders. J. Clin. Pathol.
60: 1070-1071
[Full Text]
Chen, G. L., Prchal, J. T.
(2007). X-linked clonality testing: interpretation and limitations. Blood
110: 1411-1419
[Abstract][Full Text]
Vickers, M. A.
(2007). JAK2 617V>F positive polycythemia rubra vera maintained by approximately 18 stochastic stem-cell divisions per year, explaining age of onset by a single rate-limiting mutation. Blood
110: 1675-1680
[Abstract][Full Text]
Tefferi, A., Thiele, J., Orazi, A., Kvasnicka, H. M., Barbui, T., Hanson, C. A., Barosi, G., Verstovsek, S., Birgegard, G., Mesa, R., Reilly, J. T., Gisslinger, H., Vannucchi, A. M., Cervantes, F., Finazzi, G., Hoffman, R., Gilliland, D. G., Bloomfield, C. D., Vardiman, J. W.
(2007). Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel. Blood
110: 1092-1097
T transversion, causing phenylalanine to be substituted for valine at position 617 of JAK2 (V617F). All 51 patients with 9pLOH had the V617F mutation. Of 193 patients without 9pLOH, 66 were heterozygous for V617F and 127 did not have the mutation. The frequency of V617F was 65 percent among patients with polycythemia vera (83 of 128), 57 percent among patients with idiopathic myelofibrosis (13 of 23), and 23 percent among patients with essential thrombocythemia (21 of 93). V617F is a somatic mutation present in hematopoietic cells. Mitotic recombination probably causes both 9pLOH and the transition from heterozygosity to homozygosity for V617F. Genetic evidence and in vitro functional studies indicate that V617F gives hematopoietic precursors proliferative and survival advantages. Patients with the V617F mutation had a significantly longer duration of disease and a higher rate of complications (fibrosis, hemorrhage, and thrombosis) and treatment with cytoreductive therapy than patients with wild-type JAK2. 
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