The Investigation of JAK2 V617F Mutation in Chinese Patients with Hematological Malignancies.

Abstract Myeloproliferative Diseases (MPD) are a spectrum of pathogenetically related disorders of varying clinical manifestations, characterized by neoplastic expansion of relatively mature granulocyte, erythroid, megakaryocyte, or monocyte and eosinocyte lineage cells. Recently a novel point mutation affecting the Janus tyrosine kinase 2 (JAK2 V617F) was identified as pathogenetically mechanisms by multiple competing groups. To investigate its prevalence and clinical significance in Chinese patients with hematological malignancies, we introduced Allele-specific PCR (AS-PCR) combined with sequence analysis to screen JAK2 V617F mutation. A total of 98 Chinese MPD patients and 120 additional hematological malignancies including AML, ALL, MDS were analyzed for the JAK2 V617F mutation. 98 MPD patients were referred for PV (n=57), ET (n=18), IMF (n=12), HES (n=2) and CML (n=9). 2 ml peripheral blood samples of MPD and 5–10 ml bone marrow samples of AML, ALL, MDS at the time of initial diagnosis were obtained with informed consent and genomic DNA was isolated presently. In addition, peripheral blood samples from 20 healthy donors were also collected as control. All samples were first screened by AS-PCR. The positive samples were subsequently confirmed by sequence analysis. The results showed that JAK2 V617F mutation was detected in 43 of 57 PV patients (75.4%), 7 of 18 ET patients (38.9%) and 5 of 12 IMF patients (41.7%). None of the AML, ALL, MDS, CML was found JAK2 V617F. There is no statistical difference of JAK2 V617F positive ratio between PV, ET and IMF. Furthermore, the mutation was not detected in the HES patient and 20 healthy controls. There is no other mutations and polymorphisms throughout exon 12 of JAK2. To our knowledge, this is the first report of JAK2 V617F mutation in a number of Chinese patients with hematological malignancies especially BCR/ABL-negative MPD. The incidence of JAK2 V617F of our study is a little lower compared with other publications especially in PV patients. The main reason may be firstly attributed to ethnic difference. In addition, with more MPD patients introduced and more sensitive methods such as ARMS-PCR or Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) applied, more JAK2 V617F mutation will be identified.

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In Vitro Expansion of Polycythemia Vera Progenitors Favors Expansion of Erythroid Precursors without JAK2 V617F Mutation.

Blood ◽

10.1182/blood.v106.11.3506.3506 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3506-3506 ◽

Cited By ~ 1

Author(s):

Josef T. Prchal ◽

Ko-Tung Chang ◽

Jaroslav Jelinek ◽

Yongli Guan ◽

Amos Gaikwad ◽

...

Keyword(s):

Tyrosine Kinase ◽

Polycythemia Vera ◽

Peripheral Blood ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Erythroid Progenitors ◽

Erythroid Precursors ◽

In Vitro Expansion ◽

V617f Mutation

Abstract A single acquired point mutation of JAK2 1849G>T (V617F), a tyrosine kinase with a key role in signal transduction from growth factor receptors, is found in 70%–97% of patients with polycythemia vera (PV). In the studies of tyrosine kinase inhibitors on JAK2 1849G>T (see Gaikwad et all abstract at this meeting) we decided to study the possible therapeutic effect of these agents using native in vitro expanded cells from peripheral blood. To our surprise, the in vitro expansion of PV progenitors preferentially augmented cells without JAK2 1849G>T mutation. We used a 3 step procedure to amplify erythroid precursors in different stages of differentiation from the peripheral blood of 5 PV patients previously found to be homozygous or heterozygous for the JAK2 1849G>T mutation. In the first step (days 1–7), 106/ml MNCs were cultured in the presence of Flt-3 (50 ng/ml), Tpo (100 ng/ml), and SCF (100 ng/ml). In the second step (days 8–14), the cells obtained on day 7 were re-suspended at 106/ml in the same medium with SCF (50 ng/ml), IGF-1 (50 ng/ml), and 3 units/ml Epo. In the third step, the cells collected on day 14 were re-suspended at 106/ml and cultured for two more days in the presence of the same cytokine mixture as in the step 2 but without SCF. The cultures were incubated at 37oC in 5% CO2/95% air atmosphere and the medium renewed every three days to ensure good cell proliferation. The expanded cells were stained with phycoerythrin-conjugated anti-CD235A (glycophorin) and fluorescein isothiocyanate-conjugated anti-human-CD71 (transferrin receptor) monoclonal antibodies and analyzed by flow cytometry. The cells were divided by their differential expression of these antigens into 5 subgroups ranging from primitive erythroid progenitors (BFU-Es and CFU-Es) to polychromatophilic and orthochromatophilic erythroblasts; over 70% of harvested cells were early and late basophilic erythroblasts. The proportion of JAK2 1849G>T mutation in clonal PV granulocytes (GNC) before in vitro expansion and in expanded erythroid precursors was quantitated by pyrosequencing (Jelinek, Blood in press) and is depicted in the Table. These data indicate that in vitro expansion of PV progenitors favors expansion of erythroid precursors without JAK2 V617F mutation. Since three PV samples were from females with clonal granulocytes, erythrocytes, and platelets, experiments were underway to determine if the in vitro expanded erythroid cells were clonal PV cells without JAK2 V617F mutation, or derived from polyclonal rare circulating normal hematopoietic progenitors. The Proportion of JAK2 T Allele Patients GNC T Allele (%) Expanded Cells T Allele (%) PV1 (Female) 81 10 PV2 (Male) 77 28 PV3 (Male) 44 42 PV4 (Female) 78 19 PV5 (Female) 78 28

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WP1066 Inhibits Growth of Human Cells Carrying the JAK2 V617F Mutation.

Blood ◽

10.1182/blood.v108.11.4885.4885 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4885-4885

Author(s):

Taghi Manshouri ◽

Zeev Estrov ◽

Alfonso Quintas-Cardama ◽

Jorge Cortes ◽

Francis Giles ◽

...

Keyword(s):

Cell Line ◽

Anticancer Agents ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Jak2 V617f ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Dependent Manner ◽

Hematopoietic Stem ◽

V617f Mutation

Abstract Myeloproliferative disorders (MPDs) are characterized by proliferation of one or more myeloid cell lineages in bone marrow and peripheral blood, with relatively preserved differentiation. Recent discovery of a dominant gain-of-function mutation in the Janus kinase 2 (JAK2) gene in patients with MPDs, involving the substitution of valine for phenylalanine at position 617 of the JAK2 protein (JAK2 V617F), represents the first acquired somatic mutation in hematopoietic stem cells described in these disorders. This discovery has opened new avenues for the development of targeted therapies for MPDs. WP1066 is a small molecule, a member of a novel class of anticancer agents whose development was based upon the backbone of AG490, a tyrphostin with activity against JAK2 V617F-expressing cell lines but limited in vivo activity. We investigated the inhibitory activity of the WP1066 against the JAK2 V617F-mutant expressing erythroid leukemia HEL cell line and peripheral blood mononuclear cells from patients with polycythemia vera (PV). WP1066 significantly inhibited the phosphorylation of JAK2 and downstream signal transduction proteins STAT3, STAT5, and ERK1/2 in a dose- and time-dependent manner. It induced a time- and dose-dependent antiproliferative and pro-apoptotic effects (activation of caspase 3, release of cytochrome c, and cleavage of PARP) in the JAK2 V617F-bearing HEL cell line in the low micromolar range. Pretreatment of cells with pan-caspase inhibitor Z-VAD abolished WP1066-induced apoptosis. The expression of apoptosis related proteins bcl-2, bax, and XIAP, however, was not changed. More important, WP1066 was effective in inhibiting cell growth in clonogenic assays of mononuclear cells harboring the JAK2 V617F mutation obtained from peripheral blood of patients with PV. We conclude that WP1066 is active both in vitro and ex vivo against cells carrying the JAK2 V617F mutation and represents a solid candidate for the treatment of JAK2 V617V-expressing MPDs.

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LY2784544, a Novel JAK2 Inhibitor, Decreases In Vitro Growth of Hematopoietic Human Progenitors From JAK2 V617F Positive Polycythemia Vera Patients

Blood ◽

10.1182/blood.v116.21.5054.5054 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5054-5054 ◽

Cited By ~ 1

Author(s):

Lourdes Florensa ◽

Beatriz Bellosillo ◽

Leonor Arenillas ◽

Liandong Ma ◽

Richard Walgren ◽

...

Keyword(s):

Polycythemia Vera ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Dependent Manner ◽

In Vitro Growth ◽

Vitro Assay ◽

V617f Mutation

Abstract Abstract 5054 Introduction: The discovery of JAK2 V617F mutation in patients with myeloproliferative disorders (MPD) has opened new perspectives for the development of targeted therapies. We have studied the efficacy of a novel molecule LY2784544 with JAK2 inhibitory activity in the in vitro growth of myeloid progenitors from JAK2 V617F-positive polycythemia vera (PV) patients. Objectives: To investigate the efficacy of LY2784544 in the inhibition of endogenous(e)BFU-E and CFU-GM growth in PV patients. Methods: In vitro cultures in semisolid media were performed from peripheral blood mononuclear cells (PBMC) of 6 PV patients who had never received cytoreductive treatment (4 patients with homozygous JAK2 V617F and 2 patients with heterozygous JAK2 V617F). PBMC were suspended in methylcellulose (Methocult. StemCell, Vancouver, Canada) without the addition of EPO and containing 0–30.0 μM LY2784544 drug. Concurrent plates containing EPO were plated as control cultures. The medium was distributed in multidishes and they were incubated at 37° with 5% CO2 and 95% humidity. Hemoglobinized colonies and granulomonocytic colonies were counted on day 14 by standard criteria (BFU-E defined by an aggregate of >50 hemoglobinized cells or three or more erythroid subcolonies and CFU-GM was defined by an aggregate of >50 cells). Each in vitro assay was performed in duplicate. DNA was obtained from peripheral blood granulocytes from each patient to quantify the JAK2 V617F allele burden at the time of culture assay. Results: LY2784544, at concentrations ranging from 0.03–30.0 μM, inhibited growth of unselected peripheral blood eBFU-E and CFU-GM from PV patients carrying the JAK2 V617F mutation in a dose-dependent manner, although without achieving complete inhibition of all colonies (fig.1). Conclusions: In vitro studies show that LY2784544 decreases the eBFU-E and CFU-GM growth in therapy-naive JAK2 V617F positive PV patients. Our data suggest that LY2784544 may be a candidate for the treatment of MPD carrying the JAK2 V617F mutation. Disclosures: Ma: Eli Lilly and Company: Employment. Walgren:Eli Lilly and Company: Employment.

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Differential Expression of miRNAs in Polycythemia Vera Peripheral Blood Cells.

Blood ◽

10.1182/blood.v110.11.262.262 ◽

2007 ◽

Vol 110 (11) ◽

pp. 262-262

Author(s):

Hana Bruchova ◽

Michaela Merkerova ◽

Eva Otahalova ◽

Josef T. Prchal

Keyword(s):

Gene Expression ◽

Polycythemia Vera ◽

Mirna Expression ◽

Peripheral Blood ◽

Expression Profiles ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Aberrant Expression ◽

Qrt Pcr ◽

V617f Mutation

Abstract Polycythemia vera (PV) is a clonal myeloproliferative disorder caused by somatic mutation of a hematopoietic multipotent cell. PV hematopoiesis is characterized by an accumulation of phenotypically normal erythrocytes with overproduction of leukocytes and platelets. A somatic JAK2 V617F point mutation occurs in the majority (>95%) of PV patients. However, this mutation is also found in ∼50% of patients with either essential thrombocythemia (ET) or idiopathic myelofibrosis (MF). The non-specificity of this mutation, the presence of JAK2 V617F-negative PV patients, JAK2 V617F-negative and positive relatives, and evidence of both JAK2 V617F-negative and positive PV clones demonstrate that the JAK2 V617F mutation is not the initial and sole somatic event for the pathogenesis of PV. MicroRNAs (miRNAs) have been shown to be important regulators of hematopoiesis. To identify deregulated miRNAs involved in PV pathogenesis, we studied gene expression of miRNAs of in vitro expanded erythroid progenitors (EPs), peripheral blood mononuclear cells (MNCs), granulocytes, reticulocytes, and platelets from PV patients and healthy controls. Initially, we performed gene expression profiling in 5 PV patients and 5 control cells using CombiMatrix MicroRNA CustomArray with 326 probes. The array data were analyzed by Genesis software to determine differentially expressed miRNAs in PV. These miRNAs were tested in a larger set of samples (n=18) by qRT-PCR and their expression was correlated to the JAK2 V617F mutational level. Hierarchical clustering analysis defined miRNA expression profiles of particular cell lineage for normal and PV cells. Further, ANOVA identified 31 miRNAs differently (P<0.05) expressed in at least some PV lineage cells. Of these miRNAs, we confirmed downregulation of miR-150 in expanded EPs of all stages of maturation, downregulation of let7a and upregulation of miR-182 in PV granulocytes; upregulation of miR-143, miR-145 and miR-223 in PV MNCs; and down-regulation of miR-30b, miR-30c and miR-150 in PV reticulocytes by qRT-PCR. Correlation analysis of miRNA expression with JAK2 V617F mutational level showed a positive correlation of miR-143 (r=0.68) and a negative correlation of let7a (r =−0.63), miR-30c (r=−0.74), miR-342 (r=−0.66) and miR-150 (r=−0.89). To validate PV specificity, we compared expression levels of these miRNAs to other MPD disorders (MF and ET) in which the JAK2 V617F mutation occurs. Putative miRNA targets were predicted by TargetScan 4.0 and PicTar software, and transcript levels of selected target genes are being analyzed to determine their potential deregulation at the mRNA level. Downregulated miR-150 is predicted to the target MYB oncogene that plays an important role in erythropoiesis by maintaining proliferation at the early stages. The most potential target of let7a is HGMA2, whose aberrant expression may contribute to clonal hematopoiesis in PNH. The verification of these predictions is in process by use of miRNA inhibitors, protein levels, and functional studies of the progenitors. Our study demonstrates that the specific signatures of miRNAs define particular peripheral blood cell lineages. Furthermore, the deregulated miRNAs, whose expressions correlate with the JAK2 mutational level, may be associated with the PV phenotype. Understanding the role of deregulated miRNAs in PV should provide insight into the pathogenesis of PV and may lead to novel therapeutic strategies.

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Comparative Analysis of Proinflammatory Cytokine Levels in Peripheral Blood and Bone Marrow of Patients with Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2018-99-115600 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4995-4995

Author(s):

Pu Chen ◽

Boting Wu ◽

Xia Shao ◽

Chanjuan Liu ◽

Zhenglin Yu ◽

...

Keyword(s):

Bone Marrow ◽

Proinflammatory Cytokines ◽

Peripheral Blood ◽

Proinflammatory Cytokine ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Cytokine Levels ◽

V617f Mutation ◽

Calr Mutations

Abstract Background Myeloproliferative neoplasms (MPNs) are characterized by marker somatic mutations involving JAK2, MPL, and CALR, which lead to constitutive activation of tyrosine kinase signaling cascades, subsequently dysregulated proliferative of myeloid linages, and eventually myelofibrosis or leukemic transformation. Recently, it has been argued that proinflammatory processes are crucial to the pathogenesis and progression of MPNs. A number of proinflammatory cytokines including lipocalin, IL-1, IL-2R, IL-6, IL-8, IL-12, IL-15, and IP-10 have been found elevated in the peripheral blood (PB) of MPN patients. However, there has been limited data on the levels of proinflammatory cytokines in the bone marrow (BM) of MPN patients. The present study determined and compared 40 proinflammatory cytokine levels in the PB and BM plasma of MPN patients with unequivocal molecular background, thus intending to illustrate the proinflammatory features of BM microenvironment as well as to evaluate the credibility of PB cytokine profiles. Methods Newly diagnosed MPN patients (n=12, 8 had JAK2 V617F mutation, 4 had CALR mutations) were included in the present study. PB samples were taken within 48 hours of BM samples. Paired PB and BM plasma cytokine profiles were measured as well as 10 health control PB plasma samples in a single procedure by Quantibody Human Inflammatory Array 3 (RayBiotech, Norcross, GA) which permitted detection of 40 inflammation-associated cytokines. Results Among 12 MPN patients, 8 had JAK2 V617 mutation (6 males, median age 61.5 years), and 4 had CALR mutations (3 males, median age 53.5 years). Positive linear correlations between PB and BM levels were found in 12 proinflammatory cytokines including BLC (r=0.613, p=0.034), I-309 (r=0.872, p<0.001), IL-1α (r=0.666, p=0.018), IL-1β (r=0.929, p<0.001), IL-12p40 (r=0.642, p=0.024), IL-15 (r=0.608, p=0.036), M-CSF (r=0.906, p<0.001), MIG (r=0.596, p=0.041), MIP-1α (r=0.787, p=0.002), MIP-1δ (r=0.648, p=0.023), sTNFRI (r=0.827, p=0.001), and sTNFRII (r=0.644, p=0.024). Compared to health controls, BM levels of G-CSF, IL-2, IL-4, IL-6R, IL-7, IL-8, IL-10, IL-13, MIP-1β, PDGF-BB, RANTES, and TIMP-1 were markedly elevated (all p<0.01), among which IL-2, IL-4, and IL-7 levels were even higher in patients with CALR mutations than those with classic JAK2 V617F mutation (all p<0.05). Conclusions Optimal linear correlation could only be found in limited species of proinflammatory cytokines, especially I-309, IL-1β, M-CSF, and sTNFRI, between PB and BM plasma of MPN patients. Therefore, caution should be recommended during attempts to illustrate the status of inflammation in MPN patients by circulating cytokine markers. A stronger inflammatory component might exist in MPN patients with CALR mutations than those with classic JAK2 V617F mutation. Disclosures No relevant conflicts of interest to declare.

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The V617F JAK2 Mutation Is Uncommon in Cancers and Mutations in STAT5A, STAT5B and the JAK Family Genes Do Not Account for V617F Negative Myeloproliferative Disorders.

Blood ◽

10.1182/blood.v106.11.2598.2598 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2598-2598

Author(s):

Joanna E. Baxter ◽

Linda M. Scott ◽

Peter J. Campbell ◽

Tony Todd ◽

Philip Stephens ◽

...

Keyword(s):

Signaling Pathways ◽

Cell Lines ◽

Hematological Malignancies ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Jak2 V617f Mutation ◽

Hematological Cancers ◽

History Of ◽

V617f Mutation ◽

Stat Pathway

Abstract The JAK2 V617F mutation is present in 97% patients with polycythemia vera (PV) and approximately 50% of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). JAK2 is widely expressed and is a critical component of signaling pathways in multiple cell types. It remains unclear to what extent the V617F mutation contributes to the pathogenesis of non-hematological cancers and hematological malignancies other than the classical myeloproliferative disorders. To determine the frequency with which the V617F mutation occurs in a broad range of human malignancies, we sequenced 486 cell lines derived from over 30 different histological types of solid tumor, together with 132 cell lines derived from hematological malignancies. Only the HEL cell line, which was derived from an erythroleukemia, was positive for the mutation. We then screened a further 211 primary hematological malignancies and found the mutation in 5/90 AML patients, 1/20 myelodysplastic syndrome (MDS) and 1/91 chronic myeloid leukemia (CML) patients. The five V617F positive AML patients were male, significantly older than wild-type AML patients and did not have FLT3-ITD mutation, or the t(8;21), inv(16) or t(15;17) rearrangements. In addition, none of the five patients had a history of a prior overt myeloproliferative disorder (MPD). The V617F positive MDS patient was unusual in that he had trephine features of myelodysplasia with fibrosis, consistent with a myelodysplasia/myeloproliferative overlap syndrome. The V617F-positive CML patient had a twelve-year history of BCR/ABL-negative thrombocytosis prior to presenting with CML. The molecular basis of the V617F-negative MPDs remains obscure. We therefore screened samples from 24 MPD patients without the V617F allele for alternative JAK2 mutations, and for mutations in other components of the JAK/STAT pathway. All 128 coding exons of JAK1, JAK2, JAK3, TYK2, STAT5A and STAT5B were sequenced in peripheral blood granulocyte DNA from 19 ET and 5 IMF patients that lack the V617F allele. No mutations were detected in these individuals. Collectively, these data suggest that the JAK2 V617F mutation does not occur in non-haematological cancers, that this mutation is uncommon in myeloid malignancies other than the classic BCR/ABL-negative MPDs, and that the V617F-negative MPDs are likely to reflect mutations in other molecules that modulate the JAK/STAT pathway, or mutations in different signaling pathways.

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Relationship between JAK2 V617F Mutation Status and Constitutive Mobilization of CD34-Positive Cells into Peripheral Blood in Patients with Chronic Myeloproliferative Disorder.

Blood ◽

10.1182/blood.v106.11.2586.2586 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2586-2586

Author(s):

Francesco Passamonti ◽

Elisa Rumi ◽

Emanuela Boveri ◽

Daniela Pietra ◽

Laura Vanelli ◽

...

Keyword(s):

Bone Marrow ◽

Cell Count ◽

Peripheral Blood ◽

Jak2 V617f ◽

Myeloproliferative Disorder ◽

Jak2 V617f Mutation ◽

Jak2 Mutation ◽

Mutation Status ◽

Cell Counts ◽

V617f Mutation

Abstract A gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been recently reported in patients with polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF) [N Engl J Med. 2005 Apr 28;352(17):1779–90]. Abnormal trafficking of CD34-positive cells with increased numbers in the peripheral blood is found in CIMF and in advanced stages of other myeloproliferative disorders. To determine whether the unique JAK2 V617F mutation affects the mobilization of CD34-positive cells into peripheral blood, we studied the relationship between JAK2 mutation status, bone marrow and circulating CD34-positive cells in 72 patients diagnosed according to the WHO criteria. A quantitative real-time polymerase chain reaction (PCR)-based allelic discrimination assay was used for the quantitative detection of the JAK2 V617F alleles in circulating granulocytes. Bone marrow CD34-positive cells were quantitatively assed on paraffin immunostained sections, while circulating CD34-positive cells were enumerated by flow cytometry using a single-platform assay. Overall, 57% of the patients studied carried the JAK2 V617F mutation. Within these patients, median values for JAK2 V617F alleles in circulating granulocytes were as follows: 29% in PV, 4% in ET, 12% in prefibrotic CIMF, 27% in fibrotic CIMF, and 99% in post-PV myelofibrosis. The vast majority of circulating granulocytes were homozygous for the mutation in all but one of patients with post-PV myelofibrosis. Decreased numbers of bone marrow CD34-positive cells and increased counts of circulating CD34-positive cells were detected in patients with fibrotic bone marrow. The higher the degree of fibrosis, the higher the circulating CD34-positive cell count (P<0.001) and the lower the bone marrow CD34-positive cell count (P<0.01). All patients with PV, ET and prefibrotic CIMF, and 7 out of 21 patients with fibrotic CIMF had circulating CD34-positive cell counts lower than 10 x 106/L. Conversely, all patients with post-PV myelofibrosis had counts higher than 10 x 106/L. In univariate analysis, there was an inverse relationship between percentage of JAK2 V617F alleles and bone marrow CD34-positive cells (r=−0.35, P<0.01), and a direct relationship between percentage of JAK2 mutant alleles and circulating CD34-positive cells (r=0.46, P=0.001). Multivariate analysis showed that disease category (P=0.0008) and percentage of JAK2 V617F alleles (P=0.03) were independently related to circulating CD34-positive cell counts. These observations suggest that the JAK2 V617F mutation might be involved in the constitutive mobilization of CD34-positive cells into peripheral blood that is found in patients with myeloproliferative disorder. Nonetheless, constitutive mobilization is present in a considerable portion of patients who do not carry the JAK2 mutation, pointing to additional pathogenetic mechanisms. Findings on patients with PV suggest that transition form heterozygosity to homozygosity for JAK2 V617F may represent an important step in the progression of PV to myelofibrosis. Thus, sequential evaluation of the percentage of JAK2 mutant alleles and enumeration of circulating CD34-positive cells may be useful for disease monitoring in PV.

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Lineage Distribution of JAK2 Exon12 Mutations and JAK2-V617F in Patients with Polycythemia Vera

Blood ◽

10.1182/blood.v110.11.1527.1527 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1527-1527

Author(s):

Sai Li ◽

Robert Kralovics ◽

Gennaro De Libero ◽

Andre Tichelli ◽

Radek C. Skoda

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Polycythemia Vera ◽

Peripheral Blood ◽

Jak2 V617f ◽

Lymphoid Cells ◽

Jak2 V617f Mutation ◽

V617f Mutation ◽

Lineage Distribution

Abstract Mutations in exon 12 of the JAK2 gene have been identified in a minority of patients with myeloproliferative disorders (MPD) and are associated with a selective increase in erythropoiesis resulting in polycythemia vera (PV) or idiopathic erythrocytosis. We compared the lineage distribution of JAK2 mutations in the peripheral blood of 8 PV patients with mutations in exon 12 and 21 PV patients with the JAK2-V617F mutation. Five different exon 12 mutations were observed in the 8 patients studied. Peripheral blood cells were fractionated to obtain granulocytes, platelets and mononuclear cells, which were further separated by FACS into T cells, B cells, NK cells and monocytes. Using a sensitive and quantitative assay to assess the percentages of chromosomes carrying exon 12 mutations, we detected exon 12 mutations in purified granulocytes, monocytes and platelets of all patients studied. A similar distribution was also found for the JAK2-V617F mutation. Exon 12 mutation was absent in sorted lymphoid cells of 5/8 patients, in 2/8 patients only NK cells were positive and in 1/8 patients also B cells carried the mutation. Exon 12 mutations were absent in T cells of all patients studied and JAK2-V617F was present in T cells of only 1/21 patients. Thus, inter-individual differences are notable in the involvement of lymphoid lineages for both exon 12 and JAK2-V617F mutations. To determine the presence of the JAK2 mutations in the erythroid lineage, we performed colony assays in methylcellulose, picked single erythroid colonies grown in the presence or absence of Epo and determined the allelic ratios for each individual colony. In 4/8 patients an exon 12 mutation (E543-D544del) was present in all EECs examined and similarly, in 8/12 patients the JAK2-V617F mutation was found in all EECs. In the remaining patients, we detected some EECs with only the wild type JAK2, suggesting that additional clonal events may also be present in some patients with exon 12 mutations. Interestingly, one patient carried exon 12 and JAK2-V617F mutations. None of the erythroid colonies in this patient carried both mutations simultaneously, indicating that the exon 12 mutation and JAK2-V617F represent two separate clones. One patient displayed erythroid colonies homozygous for the exon 12 mutation and an allelic ratio greater that 50% in granulocytes, indicating that progression to homozygosity can occur in some patients. The lineage distributions of exon 12 mutations and JAK2-V617F are similar and do not explain why exon 12 mutations are associated solely with PV phenotype, whereas JAK2-V617F can also cause essential thrombocythemia or primary myelofibrosis.

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