Telomerase and Telomeres in Polycythemia Vera and Essential Thrombocythemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4948-4948
Author(s):  
Andrea Gallamini ◽  
Anna Maria Ferraris ◽  
Daniele Mattei ◽  
Natalija Pujic ◽  
Rosa Mangerini ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Mononuclear Cells ◽  
Chronic Phase ◽  
Extended Period ◽  
Myeloproliferative Disorders ◽  
Telomerase Activity ◽  
Prognostic Indicators ◽  
Immature Myeloid Cells ◽  
Clonal Development

Abstract Polycythemia Vera (PV) and Essential Thrombocythemia (ET) are chronic myeloproliferative disorders (CMPD) with a relatively favorable prognosis and a long natural history. They originate from a multipotent progenitor cell with dominance of the transformed clone over normal hematopoiesis. However, the heterogeneity of these diseases with respect to clonal development from a common progenitor has been established, and no specific molecular or cytogenetic markers are so far considered useful prognostic indicators. In the current study we correlate telomerase activity (TA) and telomeres [terminal restriction fragment (TRF)] lengths in granulocytes (PMN) and mononuclear cells (MNC), with the results of the clonality status, as determined by investigation of X chromosome inactivation patterns (XCIPs), from 22 informative female patients with ET and PV. All patients were in chronic phase without immature myeloid cells in the peripheral blood. After testing for heterozygosity at the HUMARA locus PMN expressed a monoclonal XCIP in 9 out of 13 subjects with ET (69%), and in 6 out of 9 with PV (67%). MNC were always polyclonal. The amount of TA for each reaction was expressed in Total Product Generated (TPG) units. ΔTRF length was calculated by subtracting the telomer length of PMNs from that of MNCs and expressed as kilobases. TA and ΔTRF results were blindly compared with those from XCIP analysis. Monoclonal PMN displayed a TA ranging between 47.6 and 212.5 TPG units (mean 118.2 ± 45.5 SD), whereas polyclonal PMN had a TA between 1.0 and 33.3 TPG units (mean 14.4 ± 11.9 SD) (p<0.001). MNC had a TA similar to that of polyclonal PMN. Mean ΔTRF of clonal PMNs was 4.6 kilobases, while that of polyclonal PMNs was 0.44 kilobases (p<0.001). PMNs from normal control subjects had a mean ΔTRF of 0.1 kilobases, similar to that of polyclonal PMNs (p= n.s.). The results of our study clearly show the existence of two distinct subgroups of CMPD, with polyclonal or monoclonal granulopoiesis, each of them correlating with a peculiar pattern of TA and telomere lengths. Although none of the patients analyzed showed any difference in clinical characteristics with respect to the parameters considered, we believe that TA and ΔTRF analysis and their relationship with clonality status may represent an useful tool for further investigation, to predict survival in larger cohorts of patients with PV and ET, over an extended period of follow up.

High telomerase activity in granulocytes from clonal polycythemia vera and essential thrombocythemia

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 2138-2140 ◽  
Author(s):  
Anna Maria Ferraris ◽  
Rosa Mangerini ◽  
Natalija Pujic ◽  
Omar Racchi ◽  
Davide Rapezzi ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Progenitor Cell ◽  
Significant Positive Correlation ◽  
Mononuclear Cells ◽  
Myeloproliferative Disorders ◽  
Telomerase Activity ◽  
Prognostic Indicators ◽  
Multiple Tumor ◽  
Clonal Development

Abstract Essential thrombocythemia (ET) and polycythemia vera (PV) are chronic myeloproliferative disorders that share the involvement of a multipotent progenitor cell and dominance of the transformed clone over normal hematopoiesis. On the other hand, the heterogeneity of these diseases with respect to clonal development from a common progenitor has been well established. To identify useful prognostic indicators, we analyzed telomerase activity (TA), a known marker of neoplastic proliferation, in granulocytes (PMNs) and mononuclear cells (MNCs) from 22 female patients with ET and PV. Clonality status was determined by investigation of X chromosome inactivation patterns (XCIPs). We found a statistically significant positive correlation between high TA and monoclonal pattern of XCIP. Therefore, our data suggest that the use of multiple tumor markers may contribute to a better understanding of the deregulated physiology of these disorders and provide useful prognostic factors.

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&lt;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&lt;0.02) and of erythroid progenitors (P&lt;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.

JAK2 Mutation Status in Granulocytes, Platelets and Erythrocytes Differs Between Polycythemia Vera and Essential Thrombocythemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5228-5228
Author(s):  
Kohtaro Toyama ◽  
Norifumi Tsukamoto ◽  
Akio Saito ◽  
Hirotaka Nakahashi ◽  
Yoko Hashimoto ◽  
...  
Keyword(s):  
T Cells ◽  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Mononuclear Cells ◽  
Rna Extraction ◽  
Jak2 V617f ◽  
Janus Kinase ◽  
Jak2 V617f Mutation ◽  
Mutation Status ◽  
V617f Mutation

Abstract Background The gain-of-function point mutation in Janus kinase 2 exon 14 gene (JAK2-V617F) influences the diagnosis of bcr/abl-negative chronic myeloproliferative disorders (CMPDs). We previously reported that analyzing platelets is advantageous in detecting the JAK2-V617F mutation, particularly in essential thrombocythemia (ET), when compared to granulocytes. However, there have been few reports analyzing the JAK2-V617F mutation in erythroid lineage cells, and comparing the mutation status in all three lineages. Method Study protocols were approved by the Institutional Review Board of Gunma University Hospital, and written informed consent was obtained from all the patients. Heparinized peripheral blood was obtained from 113 patients with CMPDs (82 with ET, 25 with polycythemia vera (PV), and 6 with primary myelofibrosis (PMF). After centrifugation, platelets were collected from the upper plasma layer. Remaining blood was mixed with Hank’s Balanced Salt Solution and was subjected to Ficoll-Hypaque density gradient centrifugation. Granulocytes were obtained from the pellet. Mononuclear cells were resuspended in RPMI 1640 medium; 5 × 105 cells were plated in duplicate in 1 ml of methylcellulose medium and cultured in a humidified atmosphere of 5 % of carbon dioxide at 37°C for 14 days in the presence of erythropoietin to obtain erythroid colonies (BFU-E). T-cells were obtained from the remaining mononuclear cells using anti-CD3 immunoconjugated magnetic beads. After extraction of DNA from granulocytes, T-cells and BFU-E, and RNA extraction from granulocytes and platelets, PCR amplification and sequencing of exon 14 of the Jak2 gene was performed to confirm the presence of JAK2-V617F mutations. To confirm the mutation status of granulocytes, T-cells and BFU-E, allele-specific PCR (AS-PCR) was performed. Results For ET, 57 out of 82 patients (69.5%) had the JAK2-V617F mutation. In the 57 patients with the JAK2-V617F mutation, 38 (67%) had the mutation in all three lineages, 5 had the mutation in granulocytes and platelets, 2 had the mutation in platelets and BFU-E, 10 patients had the mutation only in platelets and 2 patients had the mutation only in BFU-E. In contrast, for PV, 22/25 patients (88%) had the JAK2-V617F mutation. Of note, in 22 patients having JAK2-V617F mutation, 20 (91%) were JAK2-V617F mutation-positive in all three lineages; the remaining two patients had the mutation in either platelets or BFU-E. The frequency of JAK2-V617F in all three lineages was significantly higher in PV than in ET (p &lt; 0.05). For PMF, 5 of 6 patients had the mutation in granulocytes, and 3 of these had it in all three lineages. Conclusion Among JAK2-V617F mutation-positive CMPDs, most PV patients had the JAK2-V617F mutation in all three lineages, thus suggesting that the JAK2-V617F mutation occurs in progenitor cell(s) common to granulocytes, platelets and erythrocytes. In contrast, only 67% of ET patients had the JAK2-V617F mutation in three lineages; in the remaining cases, not all of the three lineages have the mutation. This difference in lineages showing the JAK2-V617F mutation between the ET and PV may be related to the pathophysiological differences in ET and PV. Furthermore, the heterogeneous mutation status in ET may be related to its heterogeneous clinical manifestation.

The comparison of clinical features between JAK2V617F positive and negative subgroups of essential thrombocythemia

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 17523-17523
Author(s):  
S. Zhang ◽  
J. Li ◽  
M. Lu
Keyword(s):  
Sequence Analysis ◽  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Important Significance

17523 Background: JAK2V617F is a novel molecular mechanisms of BCR/ABL-negative MPD especially polycythemia vera. However, its role may not be clearly identified in essential thrombocythemia (ET). Methods: To investigate its prevalence and clinical significance in Chinese ET patients, Allele-Specific PCR (AS-PCR) in combination with sequence analysis were introduced to screen JAK2V617F mutation in our research. Genomic DNA of 40 ET patients was extracted from peripheral blood mononuclear cells, and AS-PCR was firstly adopted to amplify the exon 12 of JAK2 gene which harbored V617F mutation. The positive samples with JAK2V617F mutation were further confirmed by sequence analysis. In addition, the features of JAK2V617F positive and negative subgroup were compared according to laboratory examination. Results: The JAK2V617F mutation was identified in 18 ET patients (45%, 18/40) by AS-PCR and sequence analysis. Its presence is significantly associated with a higher hemoglobin, hematocrit and neutrophilic granulocyte percentage in ET patients. Conclusions: AS-PCR is a sensitive and accurate technique in identification of JAK2V617F mutation. JAK2V617F mutation is of important significance in Chinese ET patients. Moreover, this ET subgroup may be prone to transform to polycythemia vera. No significant financial relationships to disclose.

scholarly journals Transgenic expression of JAK2V617F causes myeloproliferative disorders in mice

Blood ◽  
2008 ◽  
Vol 111 (10) ◽  
pp. 5109-5117 ◽  
Author(s):  
Shu Xing ◽  
Tina Ho Wanting ◽  
Wanming Zhao ◽  
Junfeng Ma ◽  
Shaofeng Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Gene Promoter ◽  
Primary Myelofibrosis ◽  
Myeloproliferative Disorders ◽  
Transgenic Expression ◽  
Cell Counts

Abstract The JAK2V617F mutation was found in most patients with myeloproliferative disorders (MPDs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We have generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The mice are viable and fertile. One line of the transgenic mice, which expressed a lower level of JAK2V617F, showed moderate elevations of blood cell counts, whereas another line with a higher level of JAK2V617F expression displayed marked increases in blood counts and developed phenotypes that closely resembled human essential thrombocythemia and polycythemia vera. The latter line of mice also developed primary myelofibrosis-like symptoms as they aged. The transgenic mice showed erythroid, megakaryocytic, and granulocytic hyperplasia in the bone marrow and spleen, displayed splenomegaly, and had reduced levels of plasma erythropoietin and thrombopoietin. They possessed an increased number of hematopoietic progenitor cells in peripheral blood, spleen, and bone marrow, and these cells formed autonomous colonies in the absence of growth factors and cytokines. The data show that JAK2V617F can cause MPDs in mice. Our study thus provides a mouse model to study the pathologic role of JAK2V617F and to develop treatment for MPDs.

Methylation of Progesterone Receptor Promoter-Associated CpG Island in Polycythemia Vera and Related Myeloproliferative Disorders.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3507-3507
Author(s):  
Jaroslav Jelinek ◽  
Srdan Verstovsek ◽  
Carlos E. Bueso-Ramos ◽  
Josef T. Prchal ◽  
Jean-Pierre J. Issa
Keyword(s):  
Progesterone Receptor ◽  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Cpg Island ◽  
Cpg Islands ◽  
Myeloproliferative Disorders ◽  
Receptor Expression ◽  
Quantitative Detection ◽  
Jak2 Mutation ◽  
A Genome

Abstract Polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF) are clonal myeloproliferative disorders (MPD). A recently discovered activating mutation of JAK2 tyrosine kinase has been found in most patients with polycythemia vera (PV), in about half of those with essential thrombocythemia (ET) and myelofibrosis (MF), and in 10–20% patients with chronic myelomonocytic leukemia, Philadelphia-negative CML, atypical or unclassified MPD and megakaryocytic leukemia. It is not known what other factors determine the disease phenotype of PV, MF, and other MPD, and what factors other than JAK2 lead to disease progression. Very little is known about epigenetic changes in PV. DNA methylation of promoter-associated CpG islands is a well-recognized mechanism of epigenetic silencing used by tumors for evasion from regulatory mechanisms, and it is an alternative to genetic lesions in cancer causation. Using a genome-wide screen for differentially methylated CpG islands, we found methylation of progesterone receptor promoter region (PGR) in PV granulocytes. We then developed pyrosequencing assays for quantitative detection of PGR methylation in bisulfite-treated PCR-amplified DNA. The PGR methylation above normal control levels was observed in ET (2/12 patients, 17%), PV (10/22 patients, 45%), MF (8/12 patients, 67%), and patients with acute myeloid leukemia and antecedent PV (6/7 patients, 86%). We compared the levels of PGR methylation in MPD with the mutation status of JAK2. The 1849G&gt;T JAK2 mutation was present in 16/27 (59%) MPD patients with unmethylated PGR and 21/26 (80%) patients with methylated PGR; the difference not statistically significant; p=0.135. The role of progesterone receptor signaling in hematopoiesis is not known. Using real time quantitative RT-PCR assay for progesterone receptor expression we found detectable levels in granulocytes from 4/5 normal individuals while the expression in granulocytes from 5/5 PV patients was not detectable. To assess the functional significance of progesterone receptor silencing, we explored the effect of mifepristone, a progesterone receptor antagonist, on the response of BFU-E progenitors to erythropoietin. Mifepristone increased the sensitivity of BFU-E progenitors from normal blood to low concentrations of erythropoietin (60–250 mU/ml) suggesting that disabling of progesterone receptor may increase the response of hematopoietic cells to proliferative stimuli. In conclusion, our data show that PGR methylation is present in half of PV patients and it is even more frequent in MF and PV transformed to AML. Silencing of progesterone receptor by methylation may be an epigenetic change contributing to MPD phenotype and transformation to leukemia.

Rethinking Disease Definitions and Therapeutic Strategies in Essential Thrombocythemia and Polycythemia Vera

Hematology ◽  
2010 ◽  
Vol 2010 (1) ◽  
pp. 129-134 ◽  
Author(s):  
Claire Harrison
Keyword(s):  
Data Collection ◽  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Low Frequency ◽  
Myeloproliferative Disorders ◽  
Therapeutic Strategies ◽  
Secondary Myelofibrosis ◽  
The Impact ◽  
Current Terminology

AbstractThe seminal discovery of the JAK2V617F mutation, which is highly prevalent in Philadelphia-negative myeloproliferative disorders, now renamed neoplasms, triggered an almost unprecedented explosion of interest and data in the field. Descriptions of additional mutations in exon 12 of JAK2, at position 515 in MPL, and a number of other mutations at low frequency followed these discoveries. These advances in our understanding of molecular pathogenesis of these conditions coincided with the publication of results from two major clinical studies, ECLAP and PT-1, which contributed important clinical insights and facilitated significant correlative data collection. This article, focusing mainly upon essential thrombocythemia and polycythemia vera, reviews four major themes: the impact upon classification of these disorders considering a radical review of current terminology, and then three areas pertinent to clinical management: the indications for cytoreductive therapy in which the key targets are to reduce thrombohemorrhagic complications, relieve disease-related symptoms, and minimize the risk of transformation to secondary myeloid malignancy such as myelodysplasia, leukemia, and secondary myelofibrosis; and second reviewing current and, last, future therapeutic options, in particular interferon and JAK2 inhibitors.

scholarly journals Management of Polycythemia Vera and Essential Thrombocythemia

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 201-208 ◽  
Author(s):  
Peter J. Campbell ◽  
Anthony R. Green
Keyword(s):  
Clinical Trials ◽  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Randomized Clinical Trials ◽  
Randomized Study ◽  
Myeloproliferative Disorders ◽  
Optimal Management ◽  
Jak2 Mutation ◽  
The Past ◽  
Therapeutic Decisions

Abstract The optimal management of patients with polycythemia vera (PV) and essential thrombocythemia (ET) continues to be controversial. Both diseases present diagnostic challenges and there is a paucity of data from randomized clinical trials to guide therapeutic decisions. However, the past two years have seen major advances in our understanding of these myeloproliferative disorders (MPD). First, the ECLAP study demonstrated the anti-thrombotic efficacy of aspirin in patients with PV.1 Second, the PT-1 trial, the largest randomized study of any MPD, has provided much needed guidance on the optimal management of patients with ET.2 Third, the identification of a single JAK2 mutation in most patients with PV, and in some of those with ET, illuminates the pathogenesis of these diseases and raises questions about the boundary between them.3–7 For the purpose of management decisions, it remains appropriate to consider them as separate entities for the time being. However, as we learn more about the clinical significance of the JAK2 mutation, it seems likely that the coming years will see major changes in the way we classify and manage these disorders.

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