Modulated Expression of BCL-xL and GATA-1 Genes Is a Common Feature in Myeloproliferative Disorders (MPD) Both in JAK2-V617F Positive and Negative Patients

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1533-1533
Author(s):  
Sarah Pozzi ◽  
Francesca Bertolotti ◽  
Silvio Parodi ◽  
Raffaella Ponassi ◽  
Barbara Biasotti ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Statistical Significance ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Myeloproliferative Disorders ◽  
Expression Level ◽  
Jak2 V617f Mutation ◽  
Mutational Status ◽  
V617f Mutation

Abstract JAK2-V617F mutation has been identified in more than 90% of patients with Polycythemia Vera (PV) and in 50 to 60% of patients with Essential Thrombocythemia (ET) or Primary Myelofibrosis (PMF). The mutation has reinforced the original idea that ET, PV, and PMF have a common background; however, some key questions remain open: why, in JAK2-V617F patients, only a proportion of progenitors is bearing the mutation and the other is wild type (wt); why patients with the same mutation have a different disease; what have in common patients JAK2-V617F positive (mutated) and wt with the same disease. We observed that a new synthetic peptide (072RB) able to bind Bcl-xL molecule, exerting an apoptotic effect, inhibited the in vitro cord blood (CB) mononuclear cells (MNC) growth. Moreover, this effect correlated with a high expression level of Bcl-xL messenger (RQ-PCR). Since Bcl-xL was involved in erythropoiesis, we extended the expression studies to bone marrow (BM) MNC from16 PV (13/16 mutated),15 ET (9/15 mutated) and peripheral blood (PB) MNC from 18 PMF (9/18 mutated). JAK2 mutational status was assigned by allele-specific-PCR (AS-PCR). MNC from PV-BM and PMF-PB showed a Bcl-xL level of expression significantly higher than in MNC from healthy donors (NBM and NPB) both in mutated and in wt patients (PV: p=0.01 and p=0.004; PMF: p=0.005 and p=0.05 respectively). In ET, the expression level of Bcl-xL tended to be elevated compared to controls but did not reach the statistical significance. Since other factors can modulate Bcl-xL expression independently from the constitutive activation of JAK2/STATs pathway induced by JAK2-mutated, we analysed GATA-1 gene, a transcription factor that binds the Bcl-xL promoter and that is involved in erythropoiesis and megakariocytopoiesis. We observed that GATA-1 was highly expressed in PV-BM and PMF-PB MNC both in mutated and in wt patients (PV: p=0.01 and p=0.05; PMF: p=0.001 and p=0.03). In ET-BM MNC, GATA-1 followed the Bcl-xL pattern of expression. The highest messengers levels were observed PMF-PB MNC and CB MNC that, after in vitro 072RB peptide treatments, showed a 25%–50% of cells growth inhibition with respect to untreated controls. The protein expression was confirmed by cytofluorimetric analyses. Our finding may indeed be compatible with reduced apoptosis both in mutated and wt patients. Thus, the elevated expression levels of Bcl-xL and GATA-1 genes may represent a common feature in MPD, independent from the presence of the JAK2-V617F mutation and supports the hypothesis of a “phenotypic continuum” in MPD. It is noteworthy that in patients bearing the mutation, a variable proportion of hematopoietic progenitors in PV, ET and PMF have been documented to be wt. In this context, our findings may explain why wt hematopoiesis is not overtaken by the mutated counterpart. These results could open a new insight in the field of MPD molecular characterization and may lead to new therapeutic approaches.

Evaluation of the JAK2 V617F gene mutation in myeloproliferative neoplasms cases: a one-center study from Eastern Anatolia

2019 ◽  
Vol 44 (4) ◽  
pp. 492-498
Author(s):  
Gonca Gulbay ◽  
Elif Yesilada ◽  
Mehmet Ali Erkurt ◽  
Harika Gozukara Bag ◽  
Irfan Kuku ◽  
...  
Keyword(s):  
Blood Cell ◽  
Essential Thrombocythemia ◽  
Myeloproliferative Neoplasms ◽  
Hematological Parameters ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Myeloproliferative Disorders ◽  
Jak2 V617f Mutation ◽  
V617f Mutation ◽  
The Difference

AbstractObjectiveDetection ofJAK2V617F in myeloproliferative neoplasms (MPNs) is very important in both diagnosis and disease progression. In our study, we investigated the frequency ofJAK2V617F mutation in patients with myeloproliferative disorders.MethodsWe retrospectively reviewed the records of 720 patients (174 females and 546 males) who were tested for JAK2 V617F mutation from January 2007 to December 2017.ResultsIn our patients were determined 22.6%JAK2V617F mutation. 33.3% in women, 19.2% in men have been positive forJAK2V617F mutation. In our studyJAK2V617F present in 48.6% of essential thrombocythemia, 80.5% of polycythemia rubra vera (PV), 47.5% of primary myelofibrosis, 10% of MPNs, unclassifiable, 0.8% of others. We also investigated the difference in hematological parameters [white blood cell, hemoglobin (Hb), hematocrit (HCT), red blood cell distribution widths (RDW) and platelets count (PLT)] betweenJAK2V617F positive andJAK2V617F negative patients.ConclusionsInvestigation of the JAK2 V617F mutation is very important in cases of MPNs. In our study JAK2 V617F mutation was higher in PV, essential thrombocythemia, and primary myelofibrosis patients. However, there were significant differences in Hb, HCT, RDW and PLT levels in mutation-positive patients.

Relationship between JAK2 V617F Mutation Status, Granulocyte CD177 mRNA Expression and CD177 Soluble Protein Level in Patients with Polycythemia Vera.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2578-2578
Author(s):  
Daniela Pietra ◽  
Alessandra Balduini ◽  
Carmela Marseglia ◽  
Matteo G. Della Porta ◽  
Luca Malcovati ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Polycythemia Vera ◽  
Protein Level ◽  
Mononuclear Cells ◽  
Jak2 V617f ◽  
Myeloproliferative Disorders ◽  
Jak2 V617f Mutation ◽  
Serum Samples ◽  
Close Relationship ◽  
V617f Mutation

Abstract A unique gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been recently described in patients with polycythemia vera (PV), essential thrombocythemia and chronic idiopathic myelofibrosis [N Engl J Med. 2005 Apr 28;352(17):1779–90]. Although the currently available data clearly demonstrate that the JAK2 V617F mutation participates in the pathogenesis of myeloproliferative disorders, the mutation’s precise place in the hierarchical order of pathogenetic events remains to be established. We have recently reported that altered gene expression in myeloproliferative disorders correlates with activation of signaling by the V617F mutation of JAK2 (Blood. 2005 Aug 4; Epub ahead of print). Granulocyte CD177 (PRV1) mRNA overexpression has been initially reported as a potential marker of PV but later shown by us to rather be a marker of neutrophil activation [Br J Haematol. 2004 Sep;126(5):650–6]. In this study, we analyzed the relationship between JAK2 V617F mutation status, granulocyte CD177 mRNA expression and CD177 soluble protein level in 72 patients with PV. We also investigated the ontogeny of CD177 expression by hematopoietic cells with the aim of defining the stage of mRNA expression during myeloid, erythroid and megakaryocytic cell differentiation. Finally we studied the effect of soluble CD177 protein on hematopoietic cell proliferation and differentiation. Granulocyte CD177 mRNA expression and percentage of JAK2 V617F alleles were evaluated by quantitative Real Time PCR (qRT-PCR), while serum CD177 protein level was measured by a flow cytometry-based competitive antibody-binding assay. Liquid cultures were performed by culturing peripheral blood mononuclear cells obtained from healthy individuals and PV patients in the presence of high CD177-expressing, low CD177-expressing or CD177-depleted sera. After 12 days of culture, cells were collected, counted and evaluated for colony growth, and for flow cytometry analysis of myeloid, erythroid, megakaryocytic and CD34-positive cell subpopulations. qRT-PCR studies showed a close relationship between CD177 mRNA level and percentage of JAK2 V617F alleles (r=0.412, P<0.001). CD177 mRNA expression was almost undetectable in cell populations other than granulocytes. Studies of CFU-GM growth and differentiation indicated that CD177 mRNA expression is a late event restricted to the neutrophil stage of differentiation. Analysis of serum samples showed variable values for mean fluorescence intensity (MFI), indicating variable levels of the soluble CD177 protein in the patients studied. A very close relationship was found between granulocyte CD177 mRNA expression and soluble CD177 protein level (r=0.56, P=0.02). Incubation of mononuclear cells with serum samples showing high levels of soluble CD177 protein resulted in increased numbers of CD34-positive cells (P<0.02) and of erythroid progenitors (P<0.03). This effect was not detectable when low CD177-expressing or CD177-depleted sera were employed. These observations clearly indicate that the JAK2 V617F mutation is associated with enhanced granulocyte CD177 mRNA expression, and that this latter results in high levels of soluble CD177 protein. These elevated levels might contribute to the increased red cell production that characterizes polycythemia vera.

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5054-5054 ◽  
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.

A High Sensitivity Detection Technique Reveals JAK2-V617F Mutation in Additional 20% of Patients with Essential Thrombocytemia, but Not in Patients with Primary Myelofibrosis, Considered Negative with a Conventional ASO-PCR

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2801-2801
Author(s):  
Francesca Bertolotti ◽  
Sarah Pozzi ◽  
Massimo Ulivi ◽  
Marina Podestà ◽  
Davide Imperiale ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
High Sensitivity ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Myeloproliferative Disorders ◽  
Jak2 V617f Mutation ◽  
The Real ◽  
V617f Mutation

Abstract Polycytemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF) are defined as the major Philadelphia-negative myeloproliferative disorders (MPD). The JAK2-V617F gene mutation is a common feature of MPD but it is present in a variable proportion in patients: 95% of PV, 23 to 57% of ET and 30 to 57% of PMF patients. At the present, the most important role of JAK2-V617F mutation testing, over all during the initial evaluation of MPD patients, is that it can definitively confirm the diagnosis. In fact, its specificity permits to discriminate the considerable clinical overlap between reactive cellular proliferations and clonal myeloproliferative disorders. Moreover, because V617F is an acquired mutation that can be present in a small proportion of granulocytic populations, especially for ET, a highly sensitive detection method is essential. In fact, phenotypic effects ascribed to the presence of the JAK2-V617F mutation have been reported over all in studies with ET patients. Different sensitivity of various assays methods partially accounts for the wide range of mutation frequencies reported in literature for ET and PMF. Thus, to try to increase the sensitivity of JAK2 mutational status detection, we developed a Real-Time PCR technique that enhances allele discrimination between mutant and wild-type sequence. A Locked Nucleic Acid (LNA) clamping oligomer was added to the PCR reaction solution. In this macromolecules, the ribose moiety is modified with an extra bridge connecting the 2′ and 4′ carbons. The bridge “locks” the ribose in the 3′-end structural conformation. It binds preferentially the JAK2 wild type sequence preventing from polymerase elongation. The melting curve was used to analyse amplification products, avoiding post-PCR processing and supplying the diagnostic information immediately at the end of the amplification. 236 genomic DNA samples from healthy donors, ET and PMF patients were tested for JAK2-V617F mutation detection with a conventional allelespecific PCR (ASO-PCR, sensitivity: 1–3%). All samples were re-evaluated with a seminested PCR protocol and Real-Time PCR based method in order to improve the sensitivity. The cell line HEL DNA dilutions were used to assess the semi-nested PCR and the Real-Time PCR assay sensitivity level (0,1%). The frequency of JAK2-V617F point mutation increased from 52 to 72% in 168 ET samples. All results obtained with the new technique were confirmed by the semi-nested PCR protocol. In 58 PMF samples tested, 50% of patients were positive for the mutation in ASO-PCR while 55% resulted positive with the semi-nested protocol confirmed by the Real-Time PCR test. The high sensitivity in the JAK2-V617F mutation detection obtained with the semi-nested and the Real-Time PCR revealed that 20% of patients affected by ET and considered JAK2-V617F negative with a conventional ASO-PCR, were effectively positive. The comparison between three different analytical methods revealed that in ET but not in PMF patients, the mutation can be present only in a small proportion of granulocytic populations. Thus, since the prognostic relevance of V617F allele in ET, unlike in PMF, seems to be relevant, our high sensitive detection protocol can be effective for a correct molecular characterization and a diagnostic classification. The finding that a large proportion of ET patients bears a very small amount of JAK2-V617F mutated hematopoiesis further emphasizes the problem of the role of this small clone and warrants longitudinal analysis to understand whether this proportion remains stable or expands over time.

JAK2 V617F mutational status predicts progression to large splenomegaly and leukemic transformation in primary myelofibrosis

Blood ◽  
2007 ◽  
Vol 110 (12) ◽  
pp. 4030-4036 ◽  
Author(s):  
Giovanni Barosi ◽  
Gaetano Bergamaschi ◽  
Monia Marchetti ◽  
Alessandro M. Vannucchi ◽  
Paola Guglielmelli ◽  
...  
Keyword(s):  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Jak2 V617f Mutation ◽  
Confirmatory Test ◽  
Progression Rate ◽  
Homozygous Mutation ◽  
Leukemic Transformation ◽  
Jak2 Mutation ◽  
Mutational Status ◽  
V617f Mutation

Abstract Few investigators have evaluated the usefulness of the JAK2 V617F mutation for explaining the phenotypic variations and for predicting the risk of major clinical events in primary myelofibrosis (PMF). In a transversal survey we assayed by allele-specific polymerase chain reaction (PCR) the JAK2 V617F mutational status in 304 patients with PMF. Multiple DNA samples were collected prospectively from 64 patients, and a highly sensitive quantitative PCR was used as a confirmatory test. In a longitudinal prospective study we determined the progression rate to clinically relevant outcomes in 174 patients who had JAK2 mutation determined at diagnosis. JAK2 V617F was identified in 63.4% of patients. None of the V617F-negative patients who were sequentially genotyped progressed to become V617F positive, whereas progression rate from heterozygous to homozygous mutation was 10 per 100 patient-years. JAK2 V617F mutation contributed to hemoglobin, aquagenic pruritus, and platelet count variability, whereas homozygous mutation was independently associated with higher white blood cell count, larger spleen size, and greater need for cytoreductive therapies. Adjusting for conventional risk factors, V617F mutation independently predicted the evolution toward large splenomegaly, need of splenectomy, and leukemic transformation. We conclude that JAK2 V617F genotype should be considered in any future risk stratification of patients with PMF.

Prevalence of the JAK2-V617F mutation in Taiwanese patients with chronic myeloproliferative disorders

2008 ◽  
Vol 38 (6a) ◽  
pp. 422-426 ◽  
Author(s):  
C.-H. Lieu ◽  
H.-S. Wu ◽  
Y.-C. Hon ◽  
W.-H. Tsai ◽  
C.-F. Yang ◽  
...  
Keyword(s):  
Jak2 V617f ◽  
Myeloproliferative Disorders ◽  
Jak2 V617f Mutation ◽  
Chronic Myeloproliferative Disorders ◽  
V617f Mutation

Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3676-3682 ◽  
Author(s):  
Francesco Passamonti ◽  
Elisa Rumi ◽  
Daniela Pietra ◽  
Matteo G. Della Porta ◽  
Emanuela Boveri ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Gene Dosage ◽  
Jak2 V617f ◽  
Myeloproliferative Disorders ◽  
Jak2 V617f Mutation ◽  
Disease Category ◽  
Mutation Status ◽  
Cell Counts ◽  
Cd34 Cells ◽  
V617f Mutation

We studied the relationship between granulocyte JAK2 (V617F) mutation status, circulating CD34+ cells, and granulocyte activation in myeloproliferative disorders. Quantitative allele-specific polymerase chain reaction (PCR) showed significant differences between various disorders with respect to either the proportion of positive patients (53%-100%) or that of mutant alleles, which overall ranged from 1% to 100%. In polycythemia vera, JAK2 (V617F) was detected in 23 of 25 subjects at diagnosis and in 16 of 16 patients whose disease had evolved into myelofibrosis; median percentages of mutant alleles in these subgroups were significantly different (32% versus 95%, P < .001). Circulating CD34+ cell counts were variably elevated and associated with disease category and JAK2 (V617F) mutation status. Most patients had granulocyte activation patterns similar to those induced by administration of granulocyte colony-stimulating factor. A JAK2 (V617F) gene dosage effect on both CD34+ cell counts and granulocyte activation was clearly demonstrated in polycythemia vera, where abnormal patterns were mainly found in patients carrying more than 50% mutant alleles. These observations suggest that JAK2 (V617F) may constitutively activate granulocytes and by this means mobilize CD34+ cells. This exemplifies a novel paradigm in which a somatic gain-of-function mutation is initially responsible for clonal expansion of hematopoietic cells and later for their abnormal trafficking via an activated cell progeny.

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