scholarly journals Myeloproliferative disorders

Blood ◽  
2008 ◽  
Vol 112 (6) ◽  
pp. 2190-2198 ◽  
Author(s):  
Ross L. Levine ◽  
D. Gary Gilliland
Keyword(s):  
Potential Role ◽  
Significant Proportion ◽  
Primary Myelofibrosis ◽  
Myeloproliferative Disorders ◽  
Nonreceptor Tyrosine Kinase ◽  
Activating Mutation ◽  
Genetic Events ◽  
Improve Clinical Outcome ◽  
Stat Pathway

Abstract In 1951 William Dameshek classified polycythemia vera (PV), essential thombocytosis (ET), and primary myelofibrosis (PMF) as pathogenetically related myeloproliferative disorders (MPD). Subsequent studies demonstrated that PV, ET, and PMF are clonal disorders of multipotent hematopoietic progenitors. In 2005, a somatic activating mutation in the JAK2 nonreceptor tyrosine kinase (JAK2V617F) was identified in most patients with PV and in a significant proportion of patients with ET and PMF. Subsequent studies identified additional mutations in the JAK-STAT pathway in some patients with JAK2V617F− MPD, suggesting that constitutive activation of this signaling pathway is a unifying feature of these disorders. Although the discovery of mutations in the JAK-STAT pathway is important from a pathogenetic and diagnostic perspective, important questions remain regarding the role of this single disease allele in 3 related but clinically distinct disorders, and the role of additional genetic events in MPD disease pathogenesis. In addition, these observations provide a foundation for development of small molecule inhibitors of JAK2 that are currently being tested in clinical trials. This review will discuss our understanding of the pathogenesis of PV, ET, and PMF, the potential role of JAK2-targeted therapy, and the important unanswered questions that need to be addressed to improve clinical outcome.

scholarly journals Targeting Signal Transducer and Activator of Transcription Signaling Pathway in Leukemias

2009 ◽  
Vol 27 (26) ◽  
pp. 4422-4432 ◽  
Author(s):  
Mustafa Benekli ◽  
Heinz Baumann ◽  
Meir Wetzler
Keyword(s):  
Potential Role ◽  
Cellular Transformation ◽  
Therapeutic Strategies ◽  
Signal Transducer ◽  
Stat Proteins ◽  
Constitutive Activation ◽  
Targeting Signal ◽  
Novel Therapeutic Strategies ◽  
Stat Pathway

Signal transducer and activator of transcription (STAT) proteins comprise a seven-member family of latent cytoplasmic transcription factors that are activated through tyrosine phosphorylation by a variety of cytokines and growth factors. Aberrant activation of STATs accompanies malignant cellular transformation with resultant leukemogenesis. Constitutive activation of STATs has been demonstrated in various leukemias. A better understanding of the mechanisms of dysregulation of the STAT pathway and understanding of the cause and effect relationship in leukemogenesis may serve as a basis for designing novel therapeutic strategies directed against STATs. Mechanisms of STAT activation, the potential role of STAT signaling in leukemogenesis, and recent advances in drug discovery targeting the STAT pathway are the focus of this review.

scholarly journals Conditional expression of heterozygous or homozygous Jak2V617F from its endogenous promoter induces a polycythemia vera–like disease

Blood ◽  
2010 ◽  
Vol 115 (17) ◽  
pp. 3589-3597 ◽  
Author(s):  
Hajime Akada ◽  
Dongqing Yan ◽  
Haiying Zou ◽  
Steven Fiering ◽  
Robert E. Hutchison ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Gene Dosage ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Erythroid Progenitors ◽  
Conditional Expression ◽  
Biochemical Analyses ◽  
Genetic Events ◽  
Serum Erythropoietin

Abstract A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in a majority of patients with polycythemia vera (PV), essential thrombocythemia, and primary myelofibrosis. However, contribution of the JAK2V617F mutation in these 3 clinically distinct myeloproliferative neoplasms (MPNs) remained unclear. To investigate the role of JAK2V617F in the pathogenesis of these MPNs, we generated an inducible Jak2V617F knock-in mouse, in which the expression of Jak2V617F is under control of the endogenous Jak2 promoter. Expression of heterozygous mouse Jak2V617F evoked all major features of human polycythemia vera (PV), which included marked increase in hemoglobin and hematocrit, increased red blood cells, leukocytosis, thrombocytosis, splenomegaly, reduced serum erythropoietin (Epo) levels and Epo-independent erythroid colonies. Homozygous Jak2V617F expression also resulted in a PV-like disease associated with significantly greater reticulocytosis, leukocytosis, neutrophilia and thrombocytosis, marked expansion of erythroid progenitors and Epo-independent erythroid colonies, larger spleen size, and accelerated bone marrow fibrosis compared with heterozygous Jak2V617F expression. Biochemical analyses revealed Jak2V617F gene dosage-dependent activation of Stat5, Akt, and Erk signaling pathways. Our conditional Jak2V617F knock-in mice provide an excellent model that can be used to further understand the molecular pathogenesis of MPNs and to identify additional genetic events that cooperate with Jak2V617F in different MPNs.

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.

scholarly journals Targeting the Interleukin-6/Jak/Stat Pathway in Human Malignancies

2012 ◽  
Vol 30 (9) ◽  
pp. 1005-1014 ◽  
Author(s):  
Pasquale Sansone ◽  
Jacqueline Bromberg
Keyword(s):  
Interleukin 6 ◽  
Potential Role ◽  
Tumor Formation ◽  
Janus Kinase ◽  
Cytokine Signaling ◽  
Metastatic Progression ◽  
And Function ◽  
Principal Pathway ◽  
Stat Pathway

The Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway was discovered 20 years ago as a mediator of cytokine signaling. Since this time, more than 2,500 articles have been published demonstrating the importance of this pathway in virtually all malignancies. Although there are dozens of cytokines and cytokine receptors, four Jaks, and seven Stats, it seems that interleukin-6–mediated activation of Stat3 is a principal pathway implicated in promoting tumorigenesis. This transcription factor regulates the expression of numerous critical mediators of tumor formation and metastatic progression. This review will examine the relative importance and function of this pathway in nonmalignant conditions as well as malignancies (including tumor intrinsic and extrinsic), the influence of other Stats, the development of inhibitors to this pathway, and the potential role of inhibitors in controlling or eradicating cancers.

New Advances in the Pathogenesis and Therapy of Essential Thrombocythemia

Hematology ◽  
2008 ◽  
Vol 2008 (1) ◽  
pp. 76-82 ◽  
Author(s):  
Ross L. Levine ◽  
Mark Heaney
Keyword(s):  
Essential Thrombocythemia ◽  
Myeloid Leukemia ◽  
Significant Proportion ◽  
Myeloproliferative Neoplasm ◽  
Primary Myelofibrosis ◽  
Clinical Syndrome ◽  
Activating Mutations ◽  
Thrombopoietin Receptor ◽  
Increased Risk

Abstract Essential thrombocythemia (ET) is a hematopoietic disorder that manifests clinically as thrombocytosis, and patients with ET are at increased risk for developing thrombosis, myelofibrosis, and transformation to acute myeloid leukemia. Although ET was recognized as a distinct clinical syndrome more than 6 decades ago and was classified as a myeloproliferative neoplasm (MPN) by William Dameshek in 1951, the molecular pathogenesis of ET remained unknown until 2005, when activating mutations in the JAK2 tyrosine kinase (JAK2V617F) were identified in a significant proportion of patients with ET, polycythemia vera (PV) and primary myelofibrosis (PMF). In addition, subsequent studies have identified gain-of-function mutations in the thrombopoietin receptor (MPL) in a subset of patients with JAK2V617F-negative ET, suggesting that JAK2 activation by distinct mechanisms contributes to the pathogenesis of ET. Despite these important observations, important questions remain regarding the role of JAK2/MPL mutations in ET pathogenesis, the etiology of JAK2/MPL negative ET, the factors that distinguish ET from other MPNs with the JAK2V617F mutation, and the role of JAK2-targeted therapies for the treatment of these MPNs.

Thrombocytosis and Thrombosis

Hematology ◽  
2007 ◽  
Vol 2007 (1) ◽  
pp. 363-370 ◽  
Author(s):  
Alessandro M. Vannucchi ◽  
Tiziano Barbui
Keyword(s):  
Management Strategies ◽  
Primary Myelofibrosis ◽  
Myeloproliferative Disorders ◽  
Platelet Counts ◽  
Molecular Abnormalities ◽  
Bone Marrow Histology ◽  
Potential Risk Factors ◽  
Diagnostic Approaches

Abstract The aim of this review is to discuss current diagnostic approaches to, and classification of, patients presenting with thrombocytosis, in light of novel information derived from the discovery of specific molecular abnormalities in chronic myeloproliferative disorders (CMPD), which represent the most common cause of primary thrombocytosis. The JAK2V617F and the MPLW515L/K mutations have been found in patients with essential thrombocythemia, polycythemia vera, and primary myelofibrosis, and less frequently in other myeloproliferative disorders complicated by thrombocytosis. However, neither mutation is disease specific nor is it universally present in patients with elevated platelet counts due to a CMPD; therefore, distinguishing between reactive and primary forms of thrombocytosis, as well as among the different clinical entities that constitute the CMPD, still requires a multifaceted diagnostic approach that includes as a key step the accurate evaluation of bone marrow histology. The role of elevated platelet counts in thrombosis, which represent the predominant complication of CMPD,significantly affecting prognosis and quality of life as well as, paradoxically, in the pathogenesis of the hemorrhagic manifestations, will be discussed. Established and novel potential risk factors for thrombosis, including the clinical relevance of the JAK2V617F mutation, and current management strategies for thrombocytosis are also briefly discussed.

scholarly journals Differential Expression of Genes Associated with Oncogene-Induced Senescence and Senescence Associated Secretory Phenotype in the Absence of Differential Expression of High Molecular Risk Genes and Genes Associated with JAK-STAT Pathway in Sorted Cells of Patients with Polycythemia Vera and Primary Myelofibrosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4283-4283
Author(s):  
Chieh Lee Wong ◽  
Andrew Innes ◽  
Baoshan Ma ◽  
Gareth Gerrard ◽  
Zainul Abidin Norziha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differential Expression ◽  
Primary Myelofibrosis ◽  
Cell Types ◽  
Differentially Expressed ◽  
Cell Type ◽  
Expression Of Genes ◽  
A Cell ◽  
Stat Pathway

Abstract Introduction Despite significant progress in the understanding of the molecular pathogenesis of myeloproliferative neoplasms (MPN) and the identification of high molecular risk (HMR) genes (i.e. ASXL1, EZH2, IDH1 and IDH2 genes), the mechanisms by which different cell types predominate in the different disease subtypes and their implications for prognosis remain uncertain. Given the recently described association of senescence and fibrosis in a number of pathologies by Menoz-Espin et al, we hypothesized that genes implicated in oncogene-induced senescence (OIS) and senescence associated secretory phenotype (SASP) may contribute to the pathogenesis of these neoplastic bone marrow disorders that frequently show evidence of fibrosis. Specifically, we were interested in the gene expression levels in different disease subtypes, at a cell-type level, and whether these patterns of differential expression were distinct from the transforming JAK-STAT pathway and the HMR genes. Aim To elucidate the role of OIS and SASP genes in the pathogenesis of MPN subtypes by determining the differential expression of the genes in specific cell types in patients with MPN. Methods We performed gene expression profiling on normal controls (NC) and patients with MPN who were diagnosed with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) according to the 2008 WHO diagnostic criteria. Two cohorts of patients, the patient and validation cohorts, from 3 tertiary-level hospitals were recruited prospectively over 3 years. Peripheral blood samples were taken and sorted into polymorphonuclear cells (PMN), mononuclear cells (MNC) and T cells. RNA was extracted from each cell population. Gene expression profiling of the human transcriptome was performed using microarray and RNA sequencing on the patient and validation cohorts respectively. Gene expression analyses (GEA) were performed on 4 sets of genes derived from publicly available or custom derived gene set enrichment analysis: 92 OIS genes, 88 SASP genes (Gil et al), 4 HMR genes, and 126 genes associated with JAK-STAT pathway. Gene expression levels for each cell type in each disease were compared with NC to obtain the differential expression of the genes. RNA-seq analysis of samples from the validation cohort was used to validate the microarray results from the patient cohort. Results Twenty-eight patients (10 ET, 11 PV and 7 PMF) and 11 NC were recruited into the patient cohort. Twelve patients (4 ET, 4 PV and 4 PMF) and 4 NC were recruited into the validation cohort. After combination of the microarray and RNA-seq datasets, GEA of the OIS genes revealed the differential expressions of MCTP1 and SULT1B1 genes by PMN in PV but of none in PMF. In contrast, the BEX1 gene was identified as differentially expressed by MNC in PMF but none in PV. GEA of the SASP genes revealed differential expression of THBS1 gene by MNC in PMF but of none in PV. None of the SASP genes were differentially expressed by PMN in either PV or PMF. No differentially expressed genes were identified by PMN or MNC in ET, or by T cells in any of the diseases. Notably, GEA of the HMR genes and genes associated with the JAK-STAT pathways did not show any differential expression in any disease subtype by any cell type. Conclusions We have found strikingly distinct patterns of differential expression of senescence associated genes by PMN (in PV) and MNC (in PMF). These results provide a novel insight into the mechanisms underlying the different phenotype of the MPN subtypes and also to the cells responsible for mediating the differences. The lack of differential expression of OIS and SASP genes in ET may reflect the milder clinical phenotype of the disease. Although mutations in the HMR genes are associated with poor prognosis in PMF, the lack of differential expression in these genes and genes associated with the JAK-STAT pathway is in keeping with their mutated status and suggests that they give rise to the disease phenotypes via altering downstream expression of genes associated in other pathways such as the senescence pathways studied here. Further studies are warranted to investigate the role of these genes and the pathways involved in senescence at a cell-type specific level in order to gain further insight into how they can potentially give rise to the various disease phenotypes in MPN and unmask potential therapeutic targets. Disclosures Aitman: Illumina: Honoraria.

MPLW515L/K and JAK2V617F Mutations: Single Colony Studies, Lineage Restriction, and Chronology of Clonal Emergence.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 116-116
Author(s):  
Animesh Pardanani ◽  
Terra Lasho ◽  
Christy Finke ◽  
Ruben Mesa ◽  
William Hogan ◽  
...  
Keyword(s):  
B Lymphocytes ◽  
Low Frequency ◽  
Myeloproliferative Disorders ◽  
Myeloid Metaplasia ◽  
Clonal Hematopoiesis ◽  
Agnogenic Myeloid Metaplasia ◽  
Frequency Occurrence ◽  
Endogenous Colonies ◽  
Genetic Events

Abstract Background: We recently reported on MPLW515L/K mutations in 1182 patients with myeloid disorders (Blood, First Edition July 25, 2006) and found low frequency occurrence in in agnogenic myeloid metaplasia (AMM) (~5%) and essential thrombocythemia (ET) (~1%), but not polycythemia vera (PV). Of the 20 MPLW515L/K-positive patients in that series, 6 (30%) also carried a low burden of the JAK2V617F mutation. Studies of mutation prevalence in individuals with clonal granulopoiesis as well as familial myeloproliferative disorders (MPD) studies suggest that these mutations (JAK2V617F) may be acquired as secondary genetic events. Aim: to gain additional insight regarding the aforementioned two mutations and MPD pathogenesis by determining (i) concordance between mutation expression and clonal hematopoiesis, (ii) the instructive role of each mutation in determining lineage fate, iii) differences in pattern and lineage restriction of these mutations between various MPD, and (iv) chronology of clonal emergence and clonal dominance for the 2 mutations. Methods: Sixteen MPD patients were studied (13 JAK2V617F+ and 3 MPLW515L/K+); 9 had PV (all JAK2V617F+), 2 ET (both JAK2V617F+), 1 CMML (JAK2V617F+), and 4 AMM (2 MPLW515K+, and one each with MPLW515L and JAK2V617F). CD34+ progenitor cells were plated in methylcellulose and resulting hematopoietic colonies were scored on day 10–12. Colonies were obtained with (all patients) and without (10 patients; 5 with PV, 1 ET, and 4 AMM) cytokine support. Individual colonies were screened for presence of either mutation by DNA sequencing. In addition, MPLW515L/K analysis was performed in T and B lymphocytes in 3 informative cases. Finally, serially stored bone marrow was examined in 3 patients with co-expression of MPL515 and JAK2V617F mutations. Results: A total of 552 hematopoietic colonies from 16 MPD patients were genotyped. We found cohabitation of both mutation-positive and mutation-negative endogenous colonies in PV as well as other MPD. For JAK2V617F+ patients, the majority (75–100%) of erythropoietin-independent erythroid colonies (EEC) but not endogenous granulocyte colonies (20–70%) were mutation-positive. In contrast, no EEC were obtained from progenitors from the 3 MPLW515L/K+ patients. For 2 of the 3 patients, the endogenous myeloid colonies were predominantly mutation-positive (~90%). A mixed allele pattern (homozygous and/or heterozygous) was seen with both mutations regardless of MPD phenotype, but the overall mutant colony burden was higher with MPLW515L/K mutations relative to JAK2V617F. When such testing was possible, MPL515 mutations were detected in T-lymphocytes (2 of 3 patients) as well as B-lymphocytes (2 of 2 patients). Finally, among 3 patients carrying both mutations, serial marrow studies (25 time points over 4–8 years) revealed the occurrence of both mutations at diagnosis and persistent dominance of MPLW515L/K over JAK2V617F over time. Conclusions: Our observations provide direct evidence for clonal hematopoiesis that antedates acquisition of either MPLW515L/K or JAK2V617F that bias towards myeloid and erythroid fates, respectively. Furthermore, both mutations appear to occur early in the disease course and lineage studies suggest a hierarchically earlier stem cell origin for MPLW515L/K as compared to JAK2V617F.

Double Edge Sword Behavior of Carbendazim: A Potent Fungicide With Anticancer Therapeutic Properties

2018 ◽  
Vol 18 (1) ◽  
pp. 38-45 ◽  
Author(s):  
Karan Goyal ◽  
Ajay Sharma ◽  
Ridhima Arya ◽  
Rohit Sharma ◽  
Girish K. Gupta ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Potential Role ◽  
Anticancer Agent ◽  
Significant Proportion ◽  
Dual Role ◽  
Pharmacokinetic Studies ◽  
Phase I Clinical Trials ◽  
Discovery Research ◽  
Drug Discovery Research

Background: A number of benzimidazole derivatives such as benomyl and carbendazim have been known for their potential role as agricultural fungicides. Simultaneously carbendazim has also been found to inhibit proliferation of mammalian tumor cells specifically drug and multidrug resistant cell lines. Objective: To understand the dual role of Carbendazim as a fungicide and an anticancer agent, the study has been planned referring to the earlier studies in literature. Results: Studies carried out with fungal and mammalian cells have highlighted the potential role of carbendazim in inhibiting proliferation of cells, thereby exhibiting therapeutic implications against cancer. Because of its promising preclinical antitumor activity, Carbendazim had undergone phase I clinical trials and is under further clinical investigations for the treatment of cancer. A number of theoretical interactions have been pinpointed. There are many anticancer drugs in the market, but their usefulness is limited because of drug resistance in a significant proportion of patients. The hunger for newer drugs drives anticancer drug discovery research on a global platform and requires innovations to ensure a sustainable pipeline of lead compounds. Conclusion: Current review highlights the dual role of carbendazim as a fungicide and an anticancer agent. Further, the harmful effects of carbendazim and emphasis upon the need for more pharmacokinetic studies and pharmacovigilance data to ascertain its clinical significance, have also been discussed.

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.

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