PMF Patients Research Articles

Preferential Nuclear Accumulation of JAK2V617F In CD34+ but Not Granulocytic, Megakaryocytic or Erythroid Cells of Patients with Philadelphia-Negative Myeloproliferative Neoplasia

AbstractAbstract 4089Constitutive activation of JAK2 by chromosomal translocations or a point mutation is a frequent event in hematological malignancies particularly in Philadelphia-negative MPN. Recently, Dawson et al. identified a novel nuclear role of JAK2 in the phosphorylation of Tyr 41 of histone H3 leading to chromatin displacement of HP1a in hematopoietic cell lines and in the CD34+ cells collected from the peripheral blood of one PMF patient with JAK2V617F mutation. To determine whether the V617F mutation observed in MPN patients affects the sub-cellular localization of JAK2, we first analyzed by confocal immunofluorescence (CIF) microscopy and Western Blot (WB), K562 cells stably transfected with pMSCV-puroJAK2V617F or pMSCV-puroJAK2. The results confirm the nuclear and cytoplasmic localization of JAK2 in K562 as reported by Dawson et al. However, we consistently observed a much stronger nuclear signal in the cells expressing JAK2V617F than in those carrying wt JAK2 suggesting that the mutation leads to a preferential accumulation of JAK2V617F in the nucleus. To determine whether there is a preferential nuclear translocation of JAK2V617F in vivo, we analyzed by CIF microscopy and WB the total BM cells of 10 JAK2V617F-positive MPN patients (ET n=4, PV n=3, PMF n=3, allele burden median: 56%, 70%, 72% respectively) and of 5 MPN patients with wt JAK2 (PMF n= 2, ET n=3). We found a strong nuclear signal in mononucleated cells of 10 of 10 JAK2V617F-positive patients but not in those with wt JAK2. The JAK2 signal was observed almost exclusively in the nucleus suggesting a predominantly nuclear homing of JAK2V617F. To identify the phenotype of these cells, we used fluorescence activated cell sorting (FACS) to isolate CD34+, CD15+, CD41+ and CD71+ fractions from the BM of three JAK2V617F-positive MPN patients (1 ET, 1 PV, 1 early PMF). We found nuclear JAK2 in CD34+ positive cells collected from BM only in V617F mutated patients. No obvious nuclear signal was detected in differentiated granulocytic, megakaryocytic and erythroid cells obtained from the patients (n=15). To determine whether the block of JAK2 activity could interfere with nuclear localization of JAK2, we incubated JAK2V617F and JAK2 expressing K562 with the selective JAK2 inhibitor AG490. At the IC50 dose (25 uM) and after 3 h of incubation, CIF images showed the JAK2 redistribution in the vast majority of V617F expressing K562 and the replacement in the cytoplasm but not in wt cells. By QRT-PCR we demonstrated that the V617F mutation strongly up-regulates LMO2 expression in K562 and in CD34+ cells. In our assay, the addiction of AG490 progressively and completely restore LMO2 levels in V617F expressing K562. Our data corroborate recently published results of a nuclear localization of JAK2 in hematopoietic cells and they also extend these findings by showing that in all subtypes of MPN patients JAK2V617F accumulates in the nucleus of progenitor CD34+ cells while remains mostly in the cytoplasm of their differentiated progeny. The chromatin alterations due to the preferential accumulation of JAK2V617F in the nucleus correlates with a significant increase in LMO2 expression in cell lines and in sorted CD34+ cells. The selective JAK2 inhibitor AG490 is able to revert nuclear JAK2 and normalize LMO2 levels in vitro, suggesting how the block in JAK2 nuclear translocation could be a new treatment strategy for JAK2 mutated patients. Disclosures:No relevant conflicts of interest to declare.

The Effect of CXCL12 Processing on CD34+ Cell Migration in Myeloproliferative Neoplasms

Primary myelofibrosis (PMF) and polycythemia vera (PV) are chronic myeloproliferative neoplasms. PMF and, to a lesser degree, PV are characterized by constitutive mobilization of hematopoietic stem cells (HSC) and progenitor cells (HPC) into the peripheral blood (PB). The interaction between the chemokine CXCL12 and its receptor CXCR4 plays a pivotal role in determining the trafficking of CD34(+) cells between the bone marrow (BM) and the PB. PMF, but not PV, is associated with downregulation of CXCR4 by CD34(+) cells due to epigenetic events. Both PV and PMF patients have elevated levels of immunoreactive forms of CXCL12 in the BM and PB. Using electrospray mass spectrometry, the PB and BM plasma of PV and PMF patients was shown to contain reduced amounts of intact CXCL12 but significant amounts of several truncated forms of CXCL12, which are lacking in normal PB and BM plasma. These truncated forms of CXCL12 are the product of the action of several serine proteases, including dipeptidyl peptidase-IV, neutrophil elastase, matrix metalloproteinase-2 (MMP-2), MMP-9, and cathepsin G. Unlike CXCL12, these truncates either lack the ability to act as a chemoattractant for CD34(+) cells and/or act as an antagonist to the action of CXCL12. These data suggest that proteolytic degradation of CXCL12 is characteristic of both PV and PMF and that the resulting truncated forms of CXCL12, in addition to the reduced expression of CXCR4 by CD34(+) cells, lead to a profound mobilization of HSC/HPC in PMF.

Clinical utility of routine MPL exon 10 analysis in the diagnosis of essential thrombocythaemia and primary myelofibrosis

Approximately 50% of essential thrombocythaemia and primary myelo-fibrosis patients do not have a JAK2 V617F mutation. Up to 5% of these are reported to have a MPL exon 10 mutation but testing for MPL is not routine as there are multiple mutation types. The ability to routinely assess both JAK2 and MPL mutations would be beneficial in the differential diagnosis of unexplained thrombocytosis or myelofibrosis. We developed and applied a high resolution melt (HRM) assay, capable of detecting all known MPL mutations in a single analysis, for the detection of MPL exon 10 mutations. We assessed 175 ET and PMF patients, including 67 that were JAK2 V617F-negative by real time polymerase chain reaction (PCR). Overall, 19/175 (11%) patients had a MPL exon 10 mutation, of whom 16 were JAK2 V617F-negative (16/67; 24%). MPL mutation types were W515L (11), W515K (4), W515R (2) and W515A (1). One patient had both W515L and S505N MPL mutations and these were present in the same haemopoietic colonies. Real time PCR for JAK2 V617F analysis and HRM for MPL exon 10 status identified one or more clonal marker in 71% of patients. This combined genetic approach increases the sensitivity of meeting the World Health Organization diagnostic criteria for these myeloproliferative neoplasms.

Epigenetic Modifications: New Therapeutic Targets in Primary Myelofibrosis

Primary Myelofibrosis previously also known as Agnogenic Myeloid Metaplasia or Idiopathic Myelofibrosis is a complex myeloproliferative disease. Although the initial genetic insult that causes uncontrolled proliferation of the defective Hematopoietic Stem Cell/Hematopoietic Progenitor Cell is still elusive, literature is being enriched with reports on the molecular mechanisms that provide growth advantage to the mutant clone and the secondary events that lead to stem cell mobilization, bone marrow fibrosis, osteosclerosis and angiogenesis. Identification of these mechanisms reveals dys-regulation of genes. Dys-regulation of genes that causes silencing of the tumor suppressors is of common occurrence in various cancers. Treatment methods have been targeted against the causative chromatin modifying agents such as DNA methyl transferases and Histone Deacetylases. Ensued success with inhibitors of these agents in the treatment of various cancers has gained precedent in the treatment of Primary Myelofibrosis. In vitro experiments with DNA methyl transferase inhibitors and HDAC inhibitors on patient samples provide promising results and clinical trials for the treatments of PMF patients are under way. Positive outcomes of such clinical trials could pave way for better treatment strategies for this complex disorder and improve the quality of life of these patients.

Abnormal P-Selectin Localization During Megakaryocyte Development Determines Thrombosis in the Gata1low Model of Myelofibrosis.

Abstract 1907Poster Board I-930Primary myelofibrosis (PMF) is characterized by abnormalities of megakaryocyte maturation, bone marrow fibrosis, increased angiogenesis, bone formation and extramedullary hematopoiesis. Patients are often anemic and thrombocytopenic and, as patients with other myeloproliferative disorders, have increased risk for bleeding and thrombosis. It is debated whether propensity to thrombosis is due to increased numbers of platelet microparticles (PMP) and/or to pathological platelet-neutrophil interactions. The megakaryocytes of these patients, in fact, express normal levels of P-selectin (P-sel, an adhesion receptor that mediates interaction with neutrophils) that remains abnormally localized to the demarcation membrane system (DMS) rather than being assembled into the a-granules. Mice carrying the hypomorphic Gata1low mutation (deletion of the regulatory sequences that drive Gata1 expression in megakaryocytes) express the same megakaryocyte abnormalities presented by PMF patients, including abnormal P-sel localization to the DMS and develop with age myelofibrosis, a disease that closely resembles PMF. Whether these mice would also develop thrombosis has not been investigated as yet. The aim of this study was to determine whether Gata1low mice would develop thrombosis with age and, in this case, the role played by P-sel in the development of the trait. To this aim, Gata1low mice were crossed with P-selnull mice (kindly provided by dr. P. Frenette) according to standard genetic protocols and Gata1lowP-selWT, Gata1lowP-selnull and Gata1WTP-selnull or Gata1WTP-selWT (as controls) littermates obtained. The hematocrit (Hct), blood platelet counts (ptl) and PMP counts were compared among the four experimental groups (Table 1). [Display omitted] There were no significant differences among the four groups of mice with respect of Hct (Kruskal-Wallis nonparametric analysis of variance, 2-sided p=0.89) although there were differences among these groups with respect to platelet levels (Kruskal-Wallis 2-sided, p= 0.002). In particular, Gata1WT mice had significantly greater platelet levels than Gata1low mice regardless of P-sel (Wilcoxon rank test, p ≤0.0001, no adjustment for multiple comparisons). PMP were found to be reduced in Gata1low mice compared to Gata1WT littermates (Table I). Gata1WTP-selnull and Gata1low/P-selnull littermates had also reduced numbers of PMP. P-sel was only expressed by PMP from Gata1WTP-selWT mice. On the other hand, not only adult and old Gata1lowP-selWT mice but also Gata1WT/P-selnull and Gata1low/P-selnull littermates had bleeding times longer (<600 sec) than those observed with Gata1WT/P-selWT mice (181±69 sec). Ethical limitations in bleeding time estimates prevented assessment of eventual differences between the bleeding times of mice carrying the different mutations. The presence of thrombosis in heart and kidney of mice carrying the different mutations was determined according to standard histological criteria. In these experiments, mice were divided into young (2 months), adult (5-9 months) and old (10-16 months) age group (n=3 in each group). No thrombosis was found in Gata1WTP-selWT, Gata1WT/P-selnull or Gata1low/P-selnull mice of any age group or in young Gata1lowP-selWT mice. However, in adult and old Gata1lowP-selWT mice thrombosis was found in 50% of the organs investigated. The majority of the thrombi were found in adult mice (67% vs 33% of organs affected).Therefore, the maturation defect induced in megakaryocytes by the Gata1low mutation leads to a pro-thrombotic state detectable from 5 months of age. The presence of the P-selnull mutation rescues the thrombotic phenotype but not the ptl, PMP or bleeding time deficiency induced by the Gata1low mutation. Thus, abnormal P-sel localization to the DMS, rather then altered PMP numbers, appears to be responsible for the thrombogenicity induced by the Gata1low mutation. These results suggest P-sel as possible target for therapeutic prevention of thrombosis in PMF. Disclosures:No relevant conflicts of interest to declare.

Reconciling Phenotype with Genotype in the MPN: Impact of SNP Array-Based Chromosomal Analysis.

Abstract 1893Poster Board I-916The diagnosis and accurate prognostication of the myeloproliferative neoplasms (MPN) is complicated by phenotypic mimicry and variable rates of disease evolution. The somatic mutation of JAK2 (JAK2V617F), identified in more than 90% of PV and in 50% of ET and PMF patients, is associated with acquired uniparental disomy (aUPD) on chromosome 9p, generating copy number-neutral loss of heterozygosity (CN-LOH) and results in homozygosity of JAK2V617F in roughly one third of JAK2V617F-positive patients. The molecular events upstream of the mitotic recombination that leads to somatic CN-LOH are essentially unknown. Intriguingly, JAK2V617F itself has been implicated in the generation of genomic instability (Plo et al 2008). Although these observations provide a rationale for how a single mutation could give rise to different clinical pathologies and downstream genomic instability in MPN, the JAK2V617F allele burden (AB) has not been systematically correlated with high resolution evaluation of amplifications, deletions, and CN-LOH in a large MPN cohort. We performed single nucleotide polymorphism arrays (SNP-A), a powerful karyotyping tool with the unique ability to detect CN-LOH on neutrophil DNA from 90 MPN patients. Published copy number variants (CNVs) and those identified in our internal cohort of 995 healthy controls were excluded as well as germline, non-clonal CN-LOH regions based on size criteria. Genomic results were correlated with the quantitative JAK2V617F AB, clinical phenotypes within and between disease classes, and in 21 paired longitudinal samples. SNP-A detected aUPD and/or chromosomal gains or losses in 81% of MPN patients. The prevalence of genomic lesions was lowest in ET compared to PV or PMF (40% of ET cases were lesion free), and lower still in JAK2V617F-negative ET (62% lesion free). aUPD was the most common genomic lesion, occurring in 59% of the MPN cases, and involving every chromosome except chromosomes 18 and 23. As expected, aUPD most commonly involved 9p (38 cases), encompassing JAK2. aUPD encompassing the TET2 gene at 4q24, recently implicated in MPN and other myeloid disorders, was seen in 3 of the 90 cases, with deletions spanning this gene in 2 additional cases. Five cases of aUPD at 2q were observed, and other recurrent regions were limited to 3 cases or fewer. The prevalence of chromosomal losses alone was far lower than UPD (22%), most commonly involved chromosome regions 20q, 13q and 17q, and was a feature primarily of PMF. The prevalence of aUPD in our MPN series of 59% was higher than in MDS (20%, p<0.001), MDS/MPN (35%, p=0.0074), and sAML (23%, p=0.0004) using the same arrays and analytical methods. Independent of MPN classification category, JAK2V617F positive patients had a higher prevalence of UPD than did JAK2V617F negative patients, although when excluding 9p UPD (24/59 vs 11/31) this was not significant. However, homozygous JAK2V617F patients had twice the prevalence of aUPD (again excluding 9P UPD) than did the heterozygous patients (8/25 vs 5/33), and twice the prevalence of multiple aUPD within individual cases. Of the 21 patients studied longitudinally over 5 years (range 2–9), 3 patients with chromosomal lesions present on the first sample were not present in the second: 9p UPD resolved in one PV patient who transformed to PMF, 1p UPD resolved in a JAK2V617F-negative ET patient, and a 20q deletion resolved in a heterozygous JAK2V617F-positive ET patient who transitioned to PV. Additional UPD regions occurred in only 1/8 heterozygous JAK2V617F positive MPN patients, while 5/11 JAK2V617F homozygous patients developed additional aUPD. In contrast, the acquisition of deletions occurred primarily in JAK2V617F heterozygous ET patients, all of whom had transitioned to PV or PMF. We conclude that aUPD is increased in the MPN compared to other myeloid disorders, and associates with the presence of JAK2 V617F in a gene dose-dependent manner. We hypothesize that aberrant JAK2 signaling contributes to the development of UPD but not chromosomal losses or gains. Furthermore, comparison of deletion, amplification, and UPD events in ET and PV relative to PMF suggest that PMF bears a closer genomic resemblance to MDS than to ET or PV. Thus SNP-A provides unique insight into MPN disease classification (PMF), disease progression, and genomic instability mechanisms. Disclosures:No relevant conflicts of interest to declare.

High-Resolution SNP 6.0 Array Profiling Discloses Novel Genomic Aberrations in Polycythemia Vera and Essential Thrombocythemia.

Abstract 1898Poster Board I-921 Introduction:JAK2V617F mutation is detected in more than 90% of cases of polycythemia vera (PV) and in about 50% of cases of essential thrombocythemia (ET). Recently, JAK2-exon 12 and MPL mutations have been reported in myeloproliferative neoplasms (MPN). All these three mutations have a disease causing potential. There are still 50% of ET and PMF patients negative for JAK2V617F mutation. Thus, further genetic analysis to identify novel disease-related aberrations in MPN is required. Methods:We performed whole genome analysis on granulocytic DNA of 45 MPN patients (19 PV and 26 ET) using the single nucleotide polymorphism (SNP) Array 6.0 platform [Affymetrix 6.0]. Genotypes were analyzed using Genotyping Console 3.0.2. Data were normalized against a commercial and an own set of reference samples. All patients had JAK2V617F screening performed. Clinical and analytical data and results of cytogenetics study performed at diagnosis of MPN were colected from clinical reports. Results:From 45 MPN patients, 41 had normal cytogenetics at diagnosis; there were no data concerning cytogenetics study from the rest 4 patients. Using SNP-A 6.0 platform, we detected aberrations (gain or loss of the molecular material) in the following regions: 1q12, 9p1, 17q21, 4q, 3q26 and 8p12. Aberrations in a region 1q12 (n=14) were presented in 8 of PV (gain in 3 and loss in 5) and in 6 of ET (gain in 2 and loss in 4) patients. Aberrations in a region 9p1 (n=27) were observed in 11 of PV (gain in 6 and loss in 5) and in 16 of ET (gain in 10 and loss in 6) patients. Aberrations in a region 17q21 (n=26) were presented in 11 of PV (gain in 3 and loss in 8) and in 15 of ET (gain in 9 and loss in 6) patients. Aberrations in the region 4q (n=20) were detected in 8 of PV (gain in 5 and loss in 3) and in 12 of ET (gain in 9 and loss in 3) patients. Interestingly, only the gain of the molecular material was detected in a region 3q26 (n=12; 5 PV and 7 ET patients). In case of aberration in a region 8p12 (n=20), all 12 ET patients presented gain of the molecular material, whereas PV patients had gain (n=3) or loss (n=3) of the molecular material. There were no relation between the presence of these aberrations and the status of the JAK2V617F mutation, analytical data or clinical outcome of the patients. Conclusions:1. As we know, patients with ET have low frequency of cytogenetics aberrations. Nevertheless, using SNP-A 6.0 platform [Affymetrix 6.0], it is possible to detect new genomic aberrations in these group of patients. 2. According to our results, gain in 8p12 region is especially related to ET patients. Recently has been reported that, in a 8p12 region there is localized gen INDOL1 that, may be involved in the inhibition of immune response to tumours. 3. Gain in a 3q26 region can be related with both PV and ET. In this region, there are localized two microRNA: hsa-mir-1263 and has-mir-720. 4. SNP-A 6.0 technology should not replace conventional cytogenetics in study of MPN patients. However, SNP-A 6.0 platform, as a high resolution assay, can be useful in identification of new genomic abnormalities that may be relevant for pathogenesis of MPN. Disclosures:No relevant conflicts of interest to declare.

Role of JAK2V617F during In Vitro PV Erythroid Progenitor Differentiation.

The dominant gain-of-function mutation in the tyrosine kinase JAK2 (1849 G to T) has been detected in over 80% PV patients and in approximately half of ET or PMF patients. The mutation results in a non-synonymous amino acid substitution at position 617 (valine to phenylalanine) in the JH2 pseudo-kinase auto-inhibitory domain. Current models predict that expression of mutant JAK2V617F in patient hematopoietic stem cells leads to activation of signaling pathways important for proliferation and survival. Furthermore, experimental evidence using murine models, in which JAK2V617F is retrovirally over-expressed, display a phenotype that closely resembles PV, including progression to myelofibrosis. Previous reports have indicated that “in vitro” expansion of PV progenitors favors erythroid precursors with decreased JAK2V617F mutational burden (Exp. Hematol . ( 2007) 35(4): 587–95). Investigators in these studies, however, did not report changes in JAK2V617F frequency in sorted live erythroid progenitors after “in vitro” expansion. Furthermore, CD34+cells from PV patients cultured in the presence of 20% serum indicated an inverse relationship between JAK2V617F frequency and Epo dose (Blood ( 2006) 108(9):3128–34). However, these reports failed to correlate Epo dose, and JAK2V617F frequency, with live “in vitro” expanded purified erythroid progenitor cells. Using our published procedure (Exp. Hematol . ( 2007) 35(4): 587–95), we expanded erythroid progenitors from healthy volunteers and JAK2V617F positive PV patients (mutational burden ranging from 20–90%) in serum-free media supplemented with SCF, Tpo, and Flt3L (100 ng/mL each - week 1) followed by SCF (50 ng/mL), IGF1 (50 ng/mL), and Epo (0, 0.003, 0.03, 0.3, and 3 Units/mL - week 2). Progression of progenitor expansion, erythroid lineage commitment, live/dead cells, and JAK2 mutation frequency was followed by flow cytometry and real-time PCR. At the end of week 2, cells were harvested, stained with anti-CD71 (transferrin receptor), anti-CD235A (glycophorin A), and the Aqua Live/Dead cell reagent. Stained cells were fixed in 2% paraformaldehyde, analyzed, and sorted on a FACSAria flow cytometer. Live, expanded progenitor cells were sorted into CD235A positive and negative populations, and subsequently used for estimation of JAK2 frequency by quantitative real-time PCR (Exp. Hematol . ( 2007) 35(1): 32–38). Our results indicate expansion and erythroid commitment of PV progenitor cells in the absence of Epo correlates with the patients' intrinsic JAK2V617F burden. Furthermore, irrespective of JAK2V617F frequency in purified peripheral blood granulocytes, an inverse correlation between Epo dose and mutation frequency in “in vitro” expanded purified erythroid progenitors was not observed in all patients examined.

Relation between JAK2 V617F Mutation and Chronic Myeloproliferative Disorders.

The chronic myeloproliferative disorders (cMPDs) are a group of clonal malignant tumors of the hematological system, and derived from the pluripotential hemopoietic stem cells, manifested as the proliferation of one or more series of cells in the bone marrow as well as the occurrence of excessive mature or naive cells in the peripheral blood. It was reported by several research groups that there was the acquired JAK2 V617F mutation in the majority of the PV patients and in a part of the ET or PMF patients, which provided the new ideas for the investigation of the pathogenesis of BCR/ABL- cMPDs. In the present study, the JAK2 V617F mutation was detected in a larger collection of Chinese cMPD patients, to give a picture of the incidence of JAK2 V617F mutation in the Asian pollutions. A total of 523 patients with the cMPDs, including 278 males and 245 females, were analyzed. Their median age was 50 years old (7 to 83 years old). According to the WHO diagnostic criteria, among the 523 patients were 88 cases of CML at the chronic phase (including 59 males and 29 females with a median age of 40 years old), 25 of CML complicated with myelofibrosis, 116 of PV (64 males and 52 females; a median age of 53; among them were six cases of PV with the secondary myelofibrosis), 153 of ET (63 males and 91 females; a median age of 50; 15 cases of ET with the secondary myelofibrosis), 142 of PMF (71 males and 71 females; a median age of 53.5), four of unclassified CMPD (CMPD-U) (2 males and 2 females; a median age of 60), seven of high eosinophil syndrome (HES) (6 males and 1 female; a median age of 32), and 13 of chronic eosinophilic leukemia (13 males; a median age of 34). In addition, 140 of healthy adults were included in the control group. Allele-specific PCR (ASP) was applied to identify JAK2 V617F mutation, the mutation status was analyzed by PCR-RFLP, and the results were confirmed by sequence analysis. The mutation load was calculated by the ratio of T/G. Then explore the correlation between the allele load and the clinical, hematologic features. To those without JAK2 V617F, MPL W515L mutation was analyzed. JAK2 V617F was detected in 66%(346/523) of all patients (94%(109/116) in PV, 79%(122/153) in ET, 78%(111/142) in PMF, 75%(3/4) in CMPD-U and 14%(1/7) in HES).Majority of patients carried JAK2 V617F mutation were heterozygous, homozygote was found in only 5 cases (4 in PV and 1 in ET). The mutation load in majority patients (71.5%) was low, PV>ET>MF when compared with mutation load (p=0.003). Hemoglobin level was significantly related to high mutation load in PV (p=0.033, r=0.203). Bone marrow megakaryocyte counts were found to be marked increased in ET with high JAK2 V617F loads (P=0.024, r=0.205), and hepatomegaly in PMF was also significiently associated with high JAK2 V617F mution load (p=0.003, 0.001)(p=0.001, r=0.315). Oue data showed that1.Majority of cMPD patients, especialy with PV, carried JAK2 V617F mutation, but JAK2 V617F was absent in CML;2.98% of JAK2 V617F mutation occurs in a heterozygous status, only 4 patients with PV and 1 with ET were homozygouse.;3.PV> ET> MF when compared with mutation load. High JAK2 V617F loads were found to be significantly associated with higher hemoglobin level in PV and higher bone marrow megakaryocyte counts in ET;4.The correlation between hepatomegaly and JAK2 V617F mutation load were also found in PMF.

Clinical Evolution to Primary Myelofibrosis - Blast Phase: An International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) Collaborative Retrospective Analysis.

Background: We sought to retrospectively analyze the clinical differences, at diagnosis and throughout the disease course, between patients with primary myeloifbrosis whom undergo blastic transformation (PMF-BP) and those whom expire from PMF complications without undergoing transformation.Methods: An international collaborative database of patients with PMF which progressed to PMF BP, and a control group of individuals with PMF who expired secondary to PMF was created. Data regarding clinical course, bone marrow, karyotypic, quantitative JAK2V617F analysis (when able), laboratory values, and therapy at the diagnosis of PMF, PMF BP and up to 4 return visits (R1-4) in between these milestones was abstracted.Results: COMPARISON AT DIAGNOSIS OF PMF: 136 cases of PMF who eventually underwent PMF-BP were included, with a control group of 42 PMF patients, were analyzed. Both groups of patients had similar demographic (median age at diagnosis 59 years (range 23–83)/ 59.1 (15–93); and disease parameters (median hemoglobin 10 g/dL (range 2–15.7) /9.8 (4.9–14.6); median platelet count 181 x 109/L (range 7–1400)/168 (16–1916)), and Lille PMF prognostic scores (p = n.s.) for PMF-BP vs. PMF control group respectively. Additionally, blasts (peripheral blood /marrow) were a median of 0% (range 0–18%)/ 2% (range 0–16%) for the study group versus 0.25% (range 0–5%)/ 3% (range 0–4%) (p = n.s. for both) for the control group, respectively. Both groups had similar rates of requiring therapy (86%; 93%) and survival from diagnosis (median 34 months (range 2–441); 29 months (1–236)) (p = n.s.). However, lactate dehydrogenase (LDH) (although high in both groups) was higher at diagnosis (median 802 IU/L (range 83–10353) vs. 416 IU/L (124–2197)) (p=0.04), as well as an abnormal marrow karyotype (85% vs. 25%; p<0.001) amongst the PMF BP group.SUBSEQUENT CLINICAL COURSE: Analyzing the clinical evolution between groups demonstrated persistently higher LDH and worsening thrombocytopenia in the PMF-BP cohort (see figure). [Display omitted] Peripheral blood blasts increased in both groups, but values above 10% were unique to the PMF BP group. Additionally marrow karyotype displayed clonal evolution developed in 56% of the PMF-BP patients as opposed to 14% of controls (p<0.001). Initial and serial quantitative JAK2V617F mutation analysis (available from 20 /19 patients from the PMF-BP and control group at diagnosis, with 11 / 12 serial samples) showed no difference between the groups, nor a pattern of mutation burden elevation with transformation to leukemia in the study group.Conclusions: Increasing LDH, peripheral blood blast percentages >10%, and clonal evolution are features commonly seen in PMF patients whom evolve to blast phase and may be a harbinger of movement towards PMF BP.

Autonomous Megakaryocyte Growth in Essential Thrombocythemia and Idiopathic Myelofibrosis Is Not Related to a c-mpl Mutation or to an Autocrine Stimulation by Mpl-L

AbstractEssential thrombocythemia (ET) and idiopathic myelofibrosis (PMF) are two myeloproliferative diseases characterized by a marked megakaryocytic (MK) involvement. The pathogenesis of these two diseases is unknown. Recently it has been shown that overexpression of Mpl-ligand (Mpl-L) in mice induces thrombocytosis and myelofibrosis. In this study, we investigated whether Mpl-L was responsible for the pathogenesis of ET and PMF. Using in vitro cultures of blood or marrow CD34+ cells, we investigated whether MK growth was abnormal in these two diseases. Spontaneous MK growth involving only a fraction (20%) of the MK progenitors, as compared with growth in the presence of pegylated recombinant human megakaryocyte growth and development factor (PEG-rhuMGDF), was found in both diseases (21ET and 14PMF) using serum-free semisolid and liquid cultures, including cultures at one cell per well. We first searched for ac-mpl mutation/deletion by sequencing the entire coding region of the gene by polymerase chain reaction (PCR) in nine ET patients and five PMF patients, but no mutation was found. We subsequently investigated whether an autocrine stimulation by Mpl-L could explain the autonomous MK growth. Addition of different preparations of soluble Mpl receptor (sMpl) containing a Fc domain of IgG1 (sMpl-Fc) markedly inhibited MK spontaneous growth in both ET and PMF patients. This effect was specific for sMpl because a control soluble receptor (s4-1BB-Fc) had no inhibitory effect and an sMpl devoid of the Fc fragment had the same inhibitory efficacy as the sMpl-Fc. This inhibition was reversed by addition of PEG-rhuMGDF or a combination of cytokines. The sMpl-Fc markedly altered the entry into cell cycle of the CD34+ cells and increased the apoptosis that occurs in most patient CD34+ cells in the absence of exogenous cytokine, suggesting an autocrine stimulation. In contrast, a neutralizing antibody against Mpl-L did not alter the spontaneous MK growth, whereas it totally abolished the effects of 10 ng/mL PEG-rhuMGDF on patient or normal CD34+ cells. Mpl-L transcripts were detected at a very low level in the patient CD34+cells and MK and only when a highly sensitive fluorescent PCR technique was used. By quantitative reverse-transcription (RT)-PCR, the number of Mpl-L transcripts per actin transcripts was lower than detected in human Mpl-L–dependent cell lines, suggesting that this synthesis of Mpl-L was not biologically significant. In favor of this hypothesis, the Mpl-L protein was not detected in culture supernatants using either an enzyme-linked immunosorbent assay (ELISA) or a biological (Ba/F3huc-mpl) assay, except in one PMF patient. Investigation of Mpl-L signaling showed an absence of constitutive activation of STATs in spontaneously growing patient MKs. Addition of PEG-rhuMGDF to these MKs activated STATs 3 and 5. This result further suggests that spontaneous growth is neither related to a stimulation by Mpl-L nor to ac-mpl mutation. In conclusion, our results show that Mpl-L or Mpl are not directly implicated in the abnormal proliferation of MK cells from ET and PMF. The mechanisms by which the sMpl mediates a growth inhibition will require further experiments.

Autonomous Megakaryocyte Growth in Essential Thrombocythemia and Idiopathic Myelofibrosis Is Not Related to a c-mpl Mutation or to an Autocrine Stimulation by Mpl-L

Essential thrombocythemia (ET) and idiopathic myelofibrosis (PMF) are two myeloproliferative diseases characterized by a marked megakaryocytic (MK) involvement. The pathogenesis of these two diseases is unknown. Recently it has been shown that overexpression of Mpl-ligand (Mpl-L) in mice induces thrombocytosis and myelofibrosis. In this study, we investigated whether Mpl-L was responsible for the pathogenesis of ET and PMF. Using in vitro cultures of blood or marrow CD34+ cells, we investigated whether MK growth was abnormal in these two diseases. Spontaneous MK growth involving only a fraction (20%) of the MK progenitors, as compared with growth in the presence of pegylated recombinant human megakaryocyte growth and development factor (PEG-rhuMGDF), was found in both diseases (21ET and 14PMF) using serum-free semisolid and liquid cultures, including cultures at one cell per well. We first searched for ac-mpl mutation/deletion by sequencing the entire coding region of the gene by polymerase chain reaction (PCR) in nine ET patients and five PMF patients, but no mutation was found. We subsequently investigated whether an autocrine stimulation by Mpl-L could explain the autonomous MK growth. Addition of different preparations of soluble Mpl receptor (sMpl) containing a Fc domain of IgG1 (sMpl-Fc) markedly inhibited MK spontaneous growth in both ET and PMF patients. This effect was specific for sMpl because a control soluble receptor (s4-1BB-Fc) had no inhibitory effect and an sMpl devoid of the Fc fragment had the same inhibitory efficacy as the sMpl-Fc. This inhibition was reversed by addition of PEG-rhuMGDF or a combination of cytokines. The sMpl-Fc markedly altered the entry into cell cycle of the CD34+ cells and increased the apoptosis that occurs in most patient CD34+ cells in the absence of exogenous cytokine, suggesting an autocrine stimulation. In contrast, a neutralizing antibody against Mpl-L did not alter the spontaneous MK growth, whereas it totally abolished the effects of 10 ng/mL PEG-rhuMGDF on patient or normal CD34+ cells. Mpl-L transcripts were detected at a very low level in the patient CD34+cells and MK and only when a highly sensitive fluorescent PCR technique was used. By quantitative reverse-transcription (RT)-PCR, the number of Mpl-L transcripts per actin transcripts was lower than detected in human Mpl-L–dependent cell lines, suggesting that this synthesis of Mpl-L was not biologically significant. In favor of this hypothesis, the Mpl-L protein was not detected in culture supernatants using either an enzyme-linked immunosorbent assay (ELISA) or a biological (Ba/F3huc-mpl) assay, except in one PMF patient. Investigation of Mpl-L signaling showed an absence of constitutive activation of STATs in spontaneously growing patient MKs. Addition of PEG-rhuMGDF to these MKs activated STATs 3 and 5. This result further suggests that spontaneous growth is neither related to a stimulation by Mpl-L nor to ac-mpl mutation. In conclusion, our results show that Mpl-L or Mpl are not directly implicated in the abnormal proliferation of MK cells from ET and PMF. The mechanisms by which the sMpl mediates a growth inhibition will require further experiments.