Myeloproliferative Disorders Patients Research Articles

Risk of Unemployment Among Chronic Myeloproliferative Disorders Patients

Overall survival of chronic myeloproliferative disorders (CMPD) such as Policytemia Vera (PV), Essential Thrombocitemia (ET), Myelofibrosis (MF), Chronic Myeloid Leukemia (CML) and Myelodysplastic Syndrome (MDS) is often long term because of their natural history and therapeutic improvement (ie: TKI, RH-EPO, JAK2 inhibitors). On the other hand, quality of life may be often compromised by persistent symptomatologic burden and chronic therapy (TX) that may worsen work ability. Although the highest incidence falls between 60 and 70 years old, prevalence studies show that nearly 50% of such patients (PTS) are less than 70 YO, hence in their working age. To the best of our knowledge no data have been collected to explore the rate of loss of employment among this group of PTS. Aim of this study is to collect data to estimate among CMPD PTS: rate of long term loss of employment, impact of diagnostic work-up, correlation between loss of employment and diagnosis. METHODS From febbruary 01 until april 30, 2022 all outpatients complying with the following criteria were recruited: CML, PV, TE, MDS, MF diagnosis; life expectancy of more than 5 years based on currently used prognostic scores (CML: ELTS, MF: IPSS, MDS: R-IPSS, PV: IWG-PV score, TE: IPSET); age > 18 years old, ≤ 70 years old. Loss of employment was defined as any modality of abandonement of work after disease diagnosis (retirement, dismissal, abandonment). Demographic and basic disease data were collected. A 29 items questionnaire (QST) mainly based on the "Work Ability Index" was prepared for the purpose of this study. After informed consent acquisition, the QST was administered with psyco-oncology support to PTS planned for routine control. PTS missing control for any reason, were contacted by phone and QST was subsequently administered after informed consent reading. RESULTS 104 PTS have been recruited (M/F: 47/57); mean age at diagnosis was 52,1 for M (range 24-69) and 50,2 for F (range 18-69). PTS diagnosis were: 30 CML, 26 MF, 24 ET, 9 PV, 7 Low-Risk MDS, 4 other CMPD, 4 NA. 68/104 PTS were on active specific TX for their disease (66 on oral TX, 2 on parenteral TX). At diagnosis 58/104 PTS (55,8%) were employed 32 M (55,2%) and 26 F (44,8%). 29 had a permanent employment, 12 had a fixed term employment, 8 flexible employment. Only 8/58 (13.8%) suspended their job during work up (sick leave). 28 of 105 pts (26.7%) (retired, employed, unemployed and housewives) reduced their daily activity and 14 of them haven't recovered yet 1 to 13 YY after diagnosis. 15 of 58 PTS (25,9%) left their employment definitively: 7 could retire because of age; 8 (13,8%), 4M and 4F, left their employment earlier after diagnosis of CMPD (4 LMC, 3 MF, 1 ET). Their jobs were distributed as follows: blue collars (3 F/2 M), driver (1M), white collars (1M/1F). Main reasons for loss of employment were: desease symptoms (10/15), TX side effects (6/15), job characteristics (6/15), anxiety/depression (5/15). Loss of employment caused depression in 9/15 PTS, and in 6/15 PTS echonomic issues and loss of social well being. We could not find any correlation between loss of employment and diagnosis probably due to the low numeber of PTS recruited. CONCLUSIONS In our cohort of 105 retrospectively analyzed good prognosis CMPD PTS, loss of employment rate was quite high soon after diagnosis (25,9%); 13,8% could retire because of age. Surprisingly, also CML PTS left their employment despite the well known prognostic improvement. Most of the early retired PTS were blue collars or drivers (requiring higher phisical commitment). Depression, echonomic issues and loss of social well being were the main consenquences of loss of employment. The high rate of loss of employment may be related to overestimation of disease severity and limited social assistance support. A careful early multidisciplinary approach (hematologist/occupational doctor/psyco-oncologist) could probably help to avoid unnecessary unemployment, improve psycho-social well-being and reduce echonomical anxiety. Further studies are warranted to increase population sample and address methodology biases that limit our study. A specific questionnaire should be validated.

Role of Endoglin Expression in Acute Myeloblastic Leukemia

AbstractBackground: Endoglin (CD105), a cell membrane glyco-protein of approximately 180kDa, has been described as a proliferation-associated antigen of leukemia and endothelial cells, and it represents a powerful marker to quantify tumor angiogenesis. CD105 expression is higher on leukemic cells with immature morphological and phenotypic characteristics compared to normal hematopoietic progenitors. Higher amounts of sCD105 were measured in AML and Chronic Myeloproliferative Disorders (CMD) patients than in healthy subjects. CD105 is involved in the control of vascular en-dothelial cell proliferation, adhesion and migration. CD105 might have a direct involvement in cancer by increase of tumor mass. CD105 levels may be useful as an indicator for disease progression and to identify patients at risk of recurrence and metastasis.Aim of Study: Our aim is to evaluate the role of endoglin (CD 105) expression in patients with AML to clarify its role as a prognostic marker.Material and Methods: This study was carried out on 50 patients with newly diagnosed acute myeloid leukemia who attended the Hematology/Oncology Unit of Tanta University Hospitals. The patients were selected for the study on the basis of standard clinical, hematological and immune pheno-typic criteria for diagnosis of AML. CD105 expression was measured in bone marrow aspirate of AML patients using RD SYSTEMS kits and BD FACS calibur.Subjects included in this study were divided into two groups according to CD 105 exppression; positive and negative groups and classified according to FAB subtypes and immu-nophenotypic features.Results: There was a significant difference in distribution of CD105 expression in different FAB subtypes. There was a significant positive correlation between CD 105 expression and Hb concentration, WBCs count and blast count in periph-eral blood and BM. There was no significant correlation between CD105 expression and platelets count and serum LDH level. Kaplan-Meier survival curve show significant higher overall survival and disease free survival in negative CD105 expression group than in positive CD105 expression group.Conclusions: Positive CD105 expression levels are asso-ciated with a bad outcome in AML patients and its expression can be easy determined in routine flow cytometric analysis. Therefore, it should be regularly investigated as a bad prog-nostic factor for assessment of AML patients.

Frequency of <i>L1721W</i> Polymorphism in <i>TET2</i> Gene Among a Cohort of Sudanese Patients with Myeloproliferative Disorders: Possible Roles in Pathogenicity and Leukemic Transformation

Transformation of myeloproliferative disorders (MPDs) to acute leukemia is an evitable event that represents a stumbling block in the management of patients. The Janus Kinase-2 JAK2V617F mutation of MDP does not clarify the phenotypic variability observed in this disorder. But, a mutations in Ten-eleven-translocation-2 (TET2), a putative tumor suppressor gene, was recently implicated in MPDs and other hematologic malignancies. TET-2 is believed to play a role in leukemic transformation. This study aimed to determine the frequency of L1721W polymorphism in TET2 gene in a cohort of Sudanese patients with MPDs. Following informed consent, 25 (25/50, 50%) patients with polycythemia rubra vera (PRV), thirteen patients (13/50, 26%) with essential thrombocythemia (ET), eleven patients (11/50, 22%) with chronic myeloid leukemia (CML), and one patient (1/50, 2%) with primary myelofibrosis (PMF) were enrolled. None of the patients was in the transformation phase. Patients were diagnosed based on clinical picture, hematological parameters and JAK2V617F and BCR_ABL molecular aberrations. JAK2V617F was detected in Ph-negative-MPDs cases as (24/25, 96%) in PRV, (10/13, 76%) in ET, and (1/1, 100%) in PMF. BCR_ABL fusion was detected in all (11/11, 100%) cases of CML. DNA was extracted using the guanidine chloride method, followed by (PCR-RFLP) analysis. Only one patient showed the presence of L1721W polymorphism of the TET2. It was inferred that the low frequency of this transformation within the study cohort [all in chronic phase] probably indicates that it plays a minor role in MPD pathogenesis, while its role in blast transformation needs further studies in MPD patients.

JAK2V617F Promotes Atherosclerosis

Myeloproliferative disorder (MPD) patients are at high risk of atherothrombotic events including myocardial infarction and stroke. The somatic mutation (JAK2V617F) is commonly found in MPD patients and is associated with increased vascular risk. Moreover, the prevalence of JAK2V617F mutation is also significantly increased in non-MPD coronary artery disease (CAD) and peripheral artery disease (PAD) patients compared to the general population. Our goal was to investigate potential mechanisms of accelerated atherothrombosis using a JAK2V617F mouse model.Subletahlly irradiated Ldlr-/- mice were transplanted with bone marrow cells from wild type (WT) or Mx1-Cre/Jak2V617F (Jak2VF) mice and fed a high fat high cholesterol Western type diet (WTD). After 7 weeks of WTD Jak2VFmice had sustained neutrophilia, monocytosis and erythrocytosis; despite lower plasma cholesterol levels they showed ~1.6 fold larger atherosclerotic lesions in the aortic root (p=0.003) and accelerated carotid artery thrombosis (FeCl3 injury). After 12 weeks of WTD feeding, lesion area was only increased 1.3-fold compared to controls but showed features of plaque instability (increased necrotic core area). Early lesions (7 weeks) displayed significantly increased neutrophils (p=0.05, n=7) but not Mac 3+ macrophages. The increased neutrophil infiltration in Jak2VF mice correlated with atherosclerotic lesion size (p=0.001, r=0.78), suggesting a critical role of neutrophils in early atherogenesis. Advanced lesions (12 weeks) also showed significantly increased neutrophil (p=0.001) and macrophage (p=0.05) infiltration. No difference in CD3+ T cells or CD41+ platelet infiltration was observed at either time-point. In addition, five out of nine Jak2VF mice had prominent iron deposition (Prussian blue) and RBC marker Ter-119 staining in advanced atherosclerotic lesions (p<0.05). RBC marker Ter-119 co-localized with Mac-3(+) macrophages surrounding necrotic cores, indicating erythrophagocytosis and plaque instability.In summary, the Jak2V617F mutation promotes early atherosclerotic lesion development and plaque instability in advanced lesions. Neutrophil infiltration appears to be an important contributor to both early lesion development and necrotic core formation. These studies also suggest a novel role for RBC infiltration, iron deposition and erythrophagocytosis in advanced lesional necrotic core formation in JAK2V617F mice. DisclosuresLevine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees. Tall:Amgen: Consultancy; CSL Behring: Consultancy.

FAS/FASL gene polymorphisms in Turkish patients with chronic myeloproliferative disorders.

IntroductionChronic myeloproliferative disorders (CMPD) are chronic myeloid hematological disorders, characterized by increased myeloid cell proliferation and fibrosis. Impaired apoptotic mechanisms, increased cell proliferation, uncontrolled hematopoietic cell proliferation and myeloaccumulation may contribute to the pathogenesis of CMPD. The aim of our study was to show the possible role of FAS/FASL gene polymorphisms in CMPD pathogenesis and investigate the association with clinical parameters and susceptibility to disease.Material and methodsWe included 101 (34 polycythemia vera (PV), 23 primary myelofibrosis (PMF), 44 essential thrombocythemia (ET)) CMPD patients diagnosed according to the WHO classification criteria and 95 healthy controls in this study. All the patients and the controls were investigated for FAS/FASL gene expression, allele frequencies and phenotype features, and also FAS mRNA levels were analyzed.ResultsChronic myeloproliferative disorders patients showed increased FAS-670AG + GG genotype distribution compared with the control group (p < 0.05). While the A allele was more frequent in both groups, AG genotype was more frequent in CMPD patients. There was no association between FAS-670A>G gene polymorphism and some clinical parameters such as splenomegaly and thrombosis (p > 0.05). No statistically significant difference in FASL+843C>T genotype or allele frequency was found between groups (p > 0.05). Moreover, no statistically significant difference was detected in FASL and JAK2V617F mutations (p > 0.05). FAS mRNA expression was 1.5-fold reduced in patients compared to healthy subjects.ConclusionsAccording to our findings, FAS/FASL gene expression may contribute to the molecular and immunological pathogenesis of CMPD. More investigations are needed to support these data.

Fasudil, a clinically safe ROCK inhibitor, decreases disease burden in a Cbl/Cbl-b deficiency-driven murine model of myeloproliferative disorders

Objectives: Mutations in Cbl or Cbl-b gene occur in 10% of myeloproliferative disorder (MPD) patients and are associated with poor prognosis. Hematopoietic Cbl/Cbl-b double knockout (DKO) leads to a disease in mice phenotypically similar to human MPDs. The aim of this study was to evaluate the anti-MPD activity of a clinically safe drug, Fasudil, identified in an in vitro kinase inhibitor as an inhibitor of proliferation of DKO mouse hematopoietic stem/progenitor cells (HSPCs).Methods: Fasudil exhibited relatively selective anti-proliferative activity against Cbl/Cbl-b DKO vs. control murine bone marrow HSPCs. We established a mouse model with uniform time of MPD onset by transplanting Cbl/Cbl-b DKO HSPCs into busulfan-conditioned NOD/SCID/gamma chain-deficient mice. Four weeks post-transplant, mice were treated with 100 mg/kg fasudil (13 mice) or water (control, 8 mice) daily by oral gavage, followed by blood cell count every 2 weeks.Results: By 2 weeks of treatment, total white cell and monocyte counts were significantly lower in mice treated with fasudil. We observed a trend towards improved survival in fasudil-treated mice that did not reach statistical significance. Notably, prolonged survival beyond 27 weeks was observed in two fasudil-treated mice, nearly twice the 16-week average life-span in the Cbl/Cbl-b DKO MPD model.Conclusions: Our results suggest a therapeutic potential for fasudil, a clinically safe drug with promising results in vascular diseases, in the treatment of MPDs or other mutant Cbl-driven myeloid disorders.

The Polymorphisms In LNK Gene Correlated To The Clinical Type Of Myeloproliferative Disorders

ObjectiveLNK (lymphocyte adaptor protein) is an adapter protein negatively regulating JAK/STAT cell signaling pathway. In this study, we observed the effects of variations in LNK gene on the clinical type of myeloproliferative disorders (MPD). MethodsA total of 285 MPD cases were recruited, including essential thrombocythemia (ET) 154 cases, polycythemia vera (PV) 76 cases, idiopathic myelofibrosis (IMF) 19 cases, and chronic myeloid leukemia (CML) 36 cases. Ninety-three healthy individuals were used as normal controls. V617F mutation in JAK2 was searched by allele-specific primers method, RT-PCR was used for the detection of BCR/ABL1 fusion gene, and mutations and variations in exons and their flanking sequences of LNK gene were examined by PCR-sequencing. Count data were analyzed by χ2 or Fisher exact test. ResultsThe genotypes of 4 SNPs (rs3184504, rs111340708, rs78894077and rs7973120) could be identified. Missense mutations of A300V, V402M, and R415H in LNK were found in 8 patients, of whom 3 ET patients had homozygous A300V mutation combined with JAK2V617F mutation. The genotypes and allele types of the 3 SNPs (rs3184504, rs111340708 and rs78894077) correlated to the clinical type of MPD. For rs3184504, the allele frequencies of C/T were 0.5/0.5 in normal controls, 0.117/0.883 in ET, 0.132/0.868 in PV, 0.211/0.789 in IMF, and 0.944/0.056 in CML; T allele (p.262Trp) was significantly higher in ET, IMF and PV (P<0.01), and C allele (p.262Arg) was significantly higher in CML (P<0.01), as compared with the controls. For rs78894077, the allele frequencies of C/T were 0.097/0.903 in normal controls, 0.045/0.955 in ET, 0.086/0.914 in PV, 0.053/0.947 in IMF, and 0.167/0.833 in CML; T allele (p.242Ser) was higher in ET (P<0.05) than in normal controls. For rs111340708 which has 2 alleles of repeated sequences (TGGGG x5/TGGGG x4), the allele frequencies of TGGGG x5/TGGGG x4 were 0.462/0.538 in normal controls, 0.719/0.281 in ET, 0.533/0.467 in PV, 0.789/0.211 in IMF, and 0.639/0.361 in CML; TGGGG x4 allele was significantly lower in ET, IMF and CML than in normal controls (P<0.01). However, the allele frequencies of rs3184504 and rs111340708 were unrelated to the presence of JAK2V617F mutation and BCR/ABL1 fusion gene in MPD patients. ConclusionThe polymorphisms of SNPs in LNK gene correlated to the clinical type of MPD. Disclosures:No relevant conflicts of interest to declare.

Increased reactive oxygen species production and p47phox phosphorylation in neutrophils from myeloproliferative disorders patients with JAK2 (V617F) mutation

Myeloproliferative disorders are associated with increased risk of thrombosis and vascular complications. The pathogenesis of these complications is not completely known. Reactive oxygen species produced by the neutrophil NADPH oxidase could have a role in this process. The aim of this study was to evaluate reactive oxygen species production by neutrophils of myeloproliferative disorder patients. Patients with or without the JAK2 V617F mutation were characterized. Reactive oxygen species production was assessed by chemiluminescence, and phosphorylation of the NADPH oxidase subunit p47phox was analyzed by Western blots. In a comparison of controls and myeloproliferative disorder patients without the JAK2 V617F mutation, reactive oxygen species production by neutrophils from patients with the JAK2 V617F mutation was dramatically increased in non-stimulated and in stimulated conditions. This increase was associated with increased phosphorylation of the p47phox on Ser345 and of the uspstream kinase ERK1/2. In neutrophils from healthy donors, JAK2 can be activated by GM-CSF. GM-CSF-induced p47phox phosphorylation and priming of reactive oxygen species production are inhibited by the selective JAK2 inhibitors AG490 and lestaurtinib (CEP-701), supporting a role for JAK2 in the upregulation of NADPH oxidase activation. These findings show an increase in reactive oxygen species production and p47phox phosphorylation in neutrophils from myeloproliferative disorder patients with the JAK2 V617F mutation, and demonstrate that JAK2 is involved in GM-CSF-induced NADPH oxidase hyperactivation. As neutrophil hyperactivation could be implicated in the thrombophilic status of patients with myeloproliferative disorders, aberrant activation of JAK2 V617F, leading to excessive neutrophil reactive oxygen species production might play a role in this setting.

Dysfunction of the PI3 kinase/Rap1/integrin αIIbβ3 pathway underlies ex vivo platelet hypoactivity in essential thrombocythemia

AbstractPatients with myeloproliferative disorders (MPDs), such as essential thrombocythemia (ET) have increased risk of thrombosis and bleeding, which are major sources of morbidity and mortality. Most MPD patients have a gain of function mutation in Janus kinase 2 (JAK2V617F), but little is known how JAK2V617F affects platelet function. Here, we demonstrate that platelets from ET patients have impaired SFLLRN-mediated fibrinogen binding and have lost the potentiating effect of thrombopoietin (which couples to JAK2) on this pathway. In contrast, SFLLRN-mediated P-selectin expression, ATP secretion, phosphorylation of the PKC substrate pleckstrin, and Ca2+ mobilization were unaffected in JAK2V617F positive platelets. In addition, thrombopoietin-mediated JAK2 phosphorylation was unchanged, suggesting that signaling pathways activated downstream of JAK2 are impaired. Indeed, we found that platelets from JAK2V617F-positive ET patients have significantly reduced phosphorylation of the PI3 kinase substrate Akt, and have reduced activation of Rap1 in response to thrombopoietin, IGF-1, ADP, SFLLRN, and thrombin. This effect was independent of Giα P2Y12 purinergic receptor function as ADP-mediated inhibition of VASP phosphorylation was unchanged. These results demonstrate that the PI3 kinase/Rap1 pathway is intrinsically impaired in platelets from JAK2V617F-positive ET patients, resulting in diminished thrombin and thrombopoietin-mediated integrin αIIbβ3 activation.

Amino acid residue E543 in JAK2 C618R is a potential therapeutic target for myeloproliferative disorders caused by JAK2 C618R mutation

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays a key role in the hematopoietic and immune responses. The acquired JAK2 C618R somatic mutation is detected in a subset of myeloproliferative disorders (MPDs) patients and presumed to be a biomarker for MPDs. However, how JAK2 C618R mutation causes MPDs is still unclear. Our results indicate that the amino acid residue E543 in JAK2 C618R is indispensable for its constitutive activation. When the glutamic acid at this position was mutated to alanine (E543A) in the JAK2 C618R, its activity significantly decreased. However when the glutamic acid was mutated to the acidic amino acid, aspartic acid, JAK2 C618R activity changed little. These results suggest that there is an interaction between the amino acid residue R618 and E543, and that this interaction is crucial to sustain the constitutive activation of JAK2 C618R. More importantly, the E543 single mutation had no effects on the function of wild type JAK2 (WT JAK2). This study suggests that the amino acid residue E543 might be a potential target for specific inhibitors to treat MPDs caused by the JAK2 C618R mutation.

Bone Marrow Fibrosis in Chronic myeloid leukemia (CML) and other Myeloproliferative Disorders Evaluated by Using Special Histochemical Stains for Collagen.

Background: It is still difficult to give a final diagnosis in chronic myeloproliferative disorders (CMPDs) because of the overlap of the common pathological and clinical features of these disorders like bone marrow fibrosis which is considered important because it affects the normal function of the bone marrow. The collagen fibers are of different types, but in the bone marrow, the two main types are: collagen I, which is the most abundant type and collagen III (reticular) which is often associated with type I.Objectives:To study bone marrow fibrosis (BMF) in samples of bone marrow biopsies (BMB) of chronic myeloid leukemia (CML) and other chronic myeloproliferative disorders using histochemical stains to establish the grade of fibrosis and enabling a correct differentiation between chronic myeloid leukemia, essential thrombocythemia (ET), polycythemia rubra vera (PRV), and idiopathic marrow fibrosis (IMF) as subtypes of myeloproliferative disorders.Patients and methods: This retrospective study included collection of previously preserved formalin fixed- paraffin embedded bone marrow trephine biopsies of patients with chronic myeloproliferative disorders from January 2003 through December 2008 .The relevant clinical data of patients were retrieved from the stored case sheets. Applied histochemical stains (Reticulin stain, Van Gieson stain, and trichrome stain) with Haematoxylin and Eosin (H&amp;E) stain on sections from these specimens. These stains were used to detect the presence and the degree of pathological marrow fibrosis by the most recent grading system, the European Consensus 2005(EC2005) originally described by Thiele at 2003. Using Trichrome stain for collagen type I and reticulin stain for reticulin fibers (collagen type III) and by using a special marrow fibrosis grading system as a routine work with H&amp;E is valuable in determining the degree of marrow fibrosis on bone marrow biopsy examination and simplifies the diagnosis.Results: Sixty eight percent of chronic myeloproliferative disorders patients had no marrow fibrosis when diagnosed by H&amp;E, while only 30% of chronic myeloproliferative disorders patients had no marrow fibrosis when the diagnosis was made by special stains and marrow fibrosis grading system. There is rare marrow fibrosis in essential thrombocythemia, polycythemia rubra vera, but present in chronic myeloid leukemia and almost always in marrow fibrosis. Some patients really have myelofibrosis of different grades and the histological findings by using histochemical stains are crucial to distinguish between myeloproliferative diseasesConclusion: Patients with chronic myeloid leukemia and other chronic myeloproliferative disorders had marrow fibrosis of different grades, which is confirmed by using histochemical stains for different collagen fibers and special grading system for marrow fibrosis (EC2005) that has to be applied. It can be used routinely to avoid misdiagnosis of the primary disease or its conversion and transition to another chronic myeloproliferative disorders type, in which the clinical and laboratory features overlap, but the prognosis and therapeutic implications are significantly different.

Low prevalence of JAK2 V617F mutation in patients with first unprovoked venous thromboembolism

Low prevalence of <i>JAK2</i> V617F mutation in patients with first unprovoked venous thromboembolism

Polymorphonuclear neutrophils from JAK2V617F positive MPD patients do not support hypercoagulability: A study with calibrated automated thrombography (CAT)

Essential thrombocythemia and polycythemia vera are myeloproliferative disorders (MPD) with an elevated thrombotic risk. Leukocytosis has recently emerged as a new risk factor and there is increasing evidence that polymorphonuclear neutrophils (PMN) are involved. Procoagulant activity (PCA) of PMN in MPD has not yet been investigated. PCA of PMN from 22 patients with JAK2V617F positive MPD and 26 healthy subjects was studied using calibrated automated thrombography: in vitro thrombin generation induced with 1 pM tissue factor in the presence of added procoagulant phospholipids. There were no differences between patients and controls regarding the ability of PMN to increase thrombin generation. More surprisingly, basal thrombin generation in acellular MPD-plasma was found decreased for as yet unknown reasons. The presence of an active protein C pathway or platelets might provide a better insight into the coagulation phenotype in MPD.

Analysis of the JAK2 Gene cDNA Full-Length In One Chinese Familial Myeloproliferative Disorders

AbstractAbstract 5056 Introduction:JAK2V617F point mutation have been confirmed to be one of the major molecular mechanism of BCR/ABL negative myeloproliferative disorders(MPD). Besides, some other gene mutations such as JAK2 exon12, MPL W515L/K, c-mpl and EPOR have extended the scope of the research in this field. Most of the MPD patients are sporadic and there are seldom reports in Chinese familial MPD. 2008 ASH metting we have reported in a Chinese family of MPD's findings, the two brothers in our hospital diagnosis for MPD (one is a PV, another is ET), then we investigated the 15 members of the family. We discovered that there were three male members carried the JAK2V617F mutation in this family, including the two MPD patients and their father, which affected in two generations. All the family members were confirmed as BCR/ABL, MPL W515L/K, c-mpl, and EPOR negative. Subsequently, in order to understand the existence of family members in addition to the gene JAK2 V617F mutation, the existence of JAK2 gene mutations in other parts of the? if other mutations in existence and the high incidence of family members of MPD? We focus on the cDNA full-length of JAK2 gene to provide some theory basis on the pathogenesis in MPD. Methods:A total of 15 family members were enrolled in our study, including 2 brothers of MPD patients (the older one was thrombocythemia (ET), and another is polycythemia vera (PV)) and the other members in the same family. The mRNA of mononuclear cells from peripheral blood sample was extracted according to the manufacturer's instruction (TAKARA). RT-PCR and DNA sequencing have been used to analyze the cDNA full-length of the JAK2 gene. Results:All of the samples can be analyzed for JAK2 cDNA full-length. 3 members carried the JAK2V617F mutation (1849G®T) in this family, including the two MPD patients and their father. And the older brother was homozygous mutation and the other two were heterozygous mutation. All of the 15 samples were JAK2 exon12 gene mutation negative. 2 persons who were the male ET patient's children had a heterozygous mutation (380G®A) in JAK2 exon 3, caused a glycine-to-asparticacid substitution at position 127. Besides, 13 persons had 489C®T mutation in exon 4 and 14 persons had 2490G→A mutation in exon 17 in this family, But they were both same-sense mutation. Conclusion:It is necessary to do routine analysis of blood and other related inspection for MPD patient's family members, so as to make diagnosis earlier. However, we are not sure that the sequencing results are unique to all the familial MPD and need to be confirmed by more cases. We still do not determine the current discovery point mutations have biological significance, still need to be further explored. Disclosures:No relevant conflicts of interest to declare.

Pharmacologic Restoration of PP2A Activity and Interference with the SET-PP2A Interplay by FTY720 and Its Non-Immunosuppressive Derivative as a Novel and Efficient Therapy for Ph-Negative Myeloproliferative Disorders

AbstractAbstract 775We have shown (Oaks JJ et al. ASH 2009) that the tumor suppressor Protein Phosphatase 2A (PP2A) is functionally inactivated by Jak2V617F in cell line models of Jak2V617F myeloproliferative disorders (MPD) and Jak2V617F-transduced primary mouse bone marrow cells. Inhibition of Jak2 (600 nM Jak Inhibitor I; 50 μM AG490; 10h) or treatment with the PP2A activator FTY720 (2.5μM, 24 hours) restored PP2A activity that caused loss of Jak2V617F protein/activity, impaired Jak2V617F-driven colony formation, and induced apoptosis of Jak2V617F+ but not normal myeloid cells. Notably, FTY720 is a sphingosine analog suggested by the FDA to treat patients with Multiple Sclerosis due to its immunosuppressive activity when phosphorylated by sphingosine kinase 2 (SPHK2).Here we show that FTY720 treatment of CD34+ primary bone marrow cells from JakV617F+ PV patients (n=3) also rescued PP2A activity, induced Jak2 downregulation and significantly impaired cytokine-dependent clonogenic potential. Thus, FTY720 could be used as an alternative to Jak2 inhibitors, as in vitro and in animal assays showed that FTY720 (2.5μM) is not toxic against normal human myeloid progenitors while decreasing survival of CD34+ progenitors from MPD patients.To find out whether FTY720 uses the same mechanism to exert its immunosuppressive and anti-leukemic activities, we determined if the conversion of FTY720 into its phosphorylated form is important for rescuing PP2A activity in Jak2V617F-expressing cells. Impaired FTY720-P conversion by exposure to the SPHK inhibitor dimethylsphingosine (2.5μM, 6 hours) did not affect the ability of FTY720 to activate PP2A. Also, a synthetically phosphorylated FTY720 (FTY720-P, 2.5μM, 6 hours) was unable to activate PP2A or exert any anti-leukemic activity, suggesting that the anti-proliferative and pro-apoptotic effects of FTY720 are independent of its phosphorylation and interaction with the S1PR1 receptor. We found that activation of S1PR1 through the specific agonist SEW2871 (10μM), FTY720-P (2.5μM), or sphingosine-1-phosphate (100nM) markedly suppresses (~60% inhibition) rather than activates PP2A in normal myeloid progenitors. As expected, knockdown of S1PR1 had no effect on FTY720-mediated PP2A activation in Jak2V617F-transformed cells.Mechanistically we found that Jak2V617F and PP2Ac were found in a ternary complex with the PP2A inhibitor SET. SET knockdown by shRNA restored PP2A activity in Jak2V617F+ Ba/F3 cells to levels similar to those found in non-transformed cells, and led to an 84% decrease in Jak2V617F+-driven colony formation. In addition, co-immunoprecipitation assays revealed that FTY720 (10μM) disrupts Jak2-PP2A, PP2A-SET and Jak2-SET interactions, suggesting that SET may be the target of FTY720. Consistently, affinity chromatography showed that FTY720 efficiently interferes with the ability of C6-ceramide (10μM) to bind SET as the amount of SET eluted from the biotin-labeled C6-ceramide was significantly reduced by exposure of the cell lysate to FTY720. As well, lentiviral-mediated expression of wild type or K209D SET mutant (ceramide binding deficient) in Ba/F3 cells impaired PP2A activity (≥80% decrease), which could be totally rescued by FTY720 only in cells transduced with wild type but not K209D SET. The formal demonstration that FTY720 activates PP2A by displacing SET came when we found SET in anti-NBD immunoprecipitates from Jak2V617F-expressing Ba/F3 cells treated with FTY720-phenoxy-NBD (10μM; 30 min).Together, our data show that FTY720 has the potential to be an effective therapeutic agent for MPD patients by virtue of its low toxicity and ability to activate PP2A by displacing SET; however, FTY720 still retains the ability to become phosphorylated and inhibit, at least in part, PP2A. Thus, we developed non-phosphorylatable FTY720 derivatives and assessed them for their ability to: activate PP2A; induce downregulation/inactivation of targeted kinases (e.g. Jak2, BCR-ABL1, Akt); act as anti-proliferative and pro-apoptotic agent to leukemic but not normal myeloid/lymphoid progenitors; do not interact with S1PR1; and show no in vivo effects on B220+/CD19+ and CD4 or CD8 cellular compartments. These FTY720 derivatives were found to be not immunosuppressive but able to mirror FTY720 in terms of inducing Jak2V617F downregulation and cell killing while retaining the parent compound's minimal toxicity towards untransformed cells. Disclosures:Verstovsek:Incyte Corporation: Research Funding.

Evaluation of platelet function and pharmacological platelet inhibition in patients with myeloproliferative disorders using multiple electrode aggregometry

Background The aim of this study was to describe platelet aggregation characteristics by multiple electrode aggregometry (MEA) and to evaluate MEA for its potential to detect platelet dysfunction and response to anti-aggregatory drugs in patients with myeloproliferative disorders (MPD). Methods We compared the platelet response to arachidonic acid (ASPI test), adenosine diphosphate (ADP test) and thrombin receptor activating peptide (TRAP test) in hirudin-anticoagulated blood of 55 patients with polycythaemia vera and essential thrombocythaemia and 75 controls. Results Comparing MPD patients and controls no statistically significant difference indicative of platelet dysfunction was found in MPD patients. Analysis of covariance revealed platelet- and leukocyte count as a significant influencing factor on MEA function. Furthermore we could demonstrate that ASA and clopidogrel treatment results in a statistically significant lower ASPI (Controls: p < 0.0001, MPD: p < 0.0001) and ADPtest value (MPD: p = 0.00125) compared to untreated patients thereby validating the method for monitoring of anti-aggregatory therapy. Conclusion In this study MEA was confirmed as a valid method for monitoring of ASA and clopidogrel treatment in patients with MPD and normal control subjects. The platelet and leukocyte count were identified as major influencing factors on MEA aggregation tests both in MPD patients and controls. No functional platelet abnormalities were detected in MPD patients.

AT9283, a potent inhibitor of the Aurora kinases and Jak2, has therapeutic potential in myeloproliferative disorders

Constitutive activation of Janus kinase (Jak) 2 is the most prevalent pathogenic event observed in the myeloproliferative disorders (MPD), suggesting that inhibitors of Jak2 may prove valuable in their management. Inhibition of the Aurora kinases has also proven to be an effective therapeutic strategy in a number of haematological malignancies. AT9283 is a multi-targeted kinase inhibitor with potent activity against Jak2 and Aurora kinases A and B, and is currently being evaluated in clinical trials. To investigate the therapeutic potential of AT9283 in the MPD we studied its activity in a number of Jak2-dependent systems. AT9283 potently inhibited proliferation and Jak2-related signalling in Jak2-dependent cell lines as well as inhibiting the formation of erythroid colonies from haematopoietic progenitors isolated from MPD patients with Jak2 mutations. The compound also demonstrated significant therapeutic potential in vivo in an ETV6-JAK2 (TEL-JAK2) murine leukaemia model. Inhibition of both Jak2 and Aurora B was observed in the model systems used, indicating a dual mechanism of action. Our results suggest that AT9283 may be a valuable therapy in patients with MPD and that the dual inhibition of Jak2 and the Aurora kinases may potentially offer combinatorial efficacy in the treatment of these diseases.

JAK2-V617F mutation in Moroccan patients with myeloproliferative disorders: Contribution, diagnosis and therapeutic prospects

Aims The JAK2 V617F is a recent discovery. The implication of this mutation in the pathogenesis of the myeloproliferative disorders (MPDs) is currently confirmed. Our study is the first to be interested in the status of the JAK2 V617F mutation among myeloproliferative disorders patients in Morocco. Patients and methods Our study focused on 70 non-CML MPD patients, attending several departments of hematology and internal medicine across Morocco. The mutation was detected by allele-specific PCR (AS-PCR). Results The V617F JAK2 mutation incidence in polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis are respectively 89.47%, 62.5% and 33.33%. The V617F JAK2 mutation was absent within the patients with secondary erythrocytosis or thrombocytosis. We also found that the patients carrying the mutation displayed a leucocytosis and higher levels of haemoglobin and hematocrit than mutation-negative patients. Conclusion Our study is the first to assess the status of the JAK2 V617F mutation in patients with MPDs in Morocco. However, our data seem to confirm that the JAK2 V617F mutation is rather uncommon in myeloid malignancies other than the classical BCR/ABL MPD negative.

Interferon-Alpha Preferentially Targets JAK2V617F-Positive Rather Than Wild-Type Early Progenitor Cells in Myeloproliferative Disorders.

Abstract 436Polycythemia vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF) are myeloproliferative disorders (MPDs) without curative treatment, unless hematopoietic stem cell (HSC) transplantation is performed. However, for several years the use of interferon-alpha (IFNα) has provided an efficient therapeutic alternative for MPD patients. IFNα was shown to induce complete long-term hematologic and molecular remissions in JAK2V617F-positive MPD patients, suggesting a possible curative effect of IFNα.In order to better understand mechanisms of action of this drug, experiments were perfomred on cell lines, patient cells and mice cells harboring a JAK2V617F mutation. We hypothesized that IFNα may target directly MPD cells through binding to its specific receptor, in addition to the potential immunological effect of this molecule and could induce cell cycle entry of the pathological quiescent HSCs.Human cell lines harboring JAK2 mutation or BCR-ABL oncogene were treated with increasing doses of IFNα. A significant anti-proliferative effect at low concentrations (100 IU/ml) on the JAK2V617F-positive HEL cell line was observed. On the contrary, at this low dose IFNα did not influence growth of the BCR-ABL-positive K562 and the non-mutated UT-7 cell lines. This result supported a direct effect of IFNα in JAK2V617F cells. Suppressor of cytokine signaling (SOCS) are potent inhibitors of the JAK-STAT pathway by proceeding to a classic negative regulation loop proteins. Following IFNα stimulation of HEL cells, SOCS1 and SOCS3 mRNAs expression were induced (p=0.00036 and p=0.0012, respectively) and efficient knock-down of either SOCS1 or SOCS3 by shRNAs expression in HEL cells was able to counteract the anti-proliferative effect of IFNα (p=0.028 and p=0.031, respectively). We concluded that SOCS1 and SOCS3 are involved, in IFNα proliferative inhibition activity of HEL cells.To determine whether JAK2V617F confer hypersensitivity to IFNα inhibitory effect, proliferation and genotyping of CD34+ progenitors isolated from the bone marrow of JAK2V617F–positive MPD patients (n=5) and control subjects (n=4) were plated at one cell per well in 96-well plates and counted and genotyped at Day 10-12. A significant decrease of the patients progenitors clonogenicity was observed when exposed to IFNα (10 000 IU/ml, p<0.05). On the contrary, normal progenitors were not sensitive to the anti-proliferative effect of IFNα (p=0.2). Patients colonies were genotyped for JAK2V617F. After IFNα exposure, the amount of homozygous JAK2V617F clones decreased in 3 over 5 patients in favor of the heterozygous JAK2V617F and/or wild-type clone(s) confirming the preferential action of IFNα on JAK2V617F progenitors. This inhibitory effect was more drastic on progenitors carrying the JAK2V617F mutation at the homozygous state.Lastly, in order to explain the long term molecular responses observed in PV patients treated by IFNα, we investigated the effect of IFNα on the cell cycle in more immature cells. BrdU assay on JAK2V617F Knock-in (KI) mice was performed. Five-months old JAK2V617F KI and wild-type mice received or not 10,000 UI of murine IFNα during 3 days. Sixteen hours before analysis, BrdU was injected i.p. in the animals. Bone marrow Lin-Sca+cKit+cells (LSK) were stained with CD150 and CD48 antibodies before BrdU labelling was analyzed by flow cytometry. Analysis of BrdU postive cells confirmed that i-JAK2V617F induces LSK CD150+CD48- to enter cell cycle ( 8.55+/-3.12% for WT cells versus 19.92+/-3,19% for JAK2V617F KI cells respectively (p = 0.04)) and ii- That IFNα induces CD150+CD48- LSK to enter cell cycle whatever the JAK2 WT (8.55+/-3.12% for non treated animals versus 15.43+/-3.19% for IFNα receiving mice (p=0.02)) or mutated status but this induction was more statistically significant in JAK2V617F mice (19.92+/-1.82% versus 25.23+/-0.57% respectively( p=0.02)).In conclusion, we gave rise to a double effect of IFNα in MPD cells: A direct preferential anti-proliferative effect of IFNα on JAK2V617F–positive MPD progenitor cells, possibly through the induction of SOCS over-expressions and a direct cell cycling effect on JAK2V617F stem cells suggesting that IFNa containing treatments could be of interest for JAK2V617F patients cure. Disclosures:No relevant conflicts of interest to declare.

Elevated Circulating Platelet-Derived Microparticles in Ph1 Negative Chronic Myeloproliferative Disorders.

Abstract 1906Poster Board I-929Microparticles (MP) are plasma membrane vesicles bearing potent procoagulant proteins and released into the circulation by various blood and endothelial cells during cellular activation and apoptosis. Their concentration has been shown markedly increased in vascular and thromboembolic diseases and in several types of cancer. Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary Myelofibrosis (PM) are Bcr-Abl-negative chronic myeloproliferative disorders (MPD) with common biological and clinical features, especially thrombocytosis and more or less effective megakaryocytic proliferation. ET, PV and PMF are also characterized by an increased risk of thrombotic complications. Given the involvement of megacaryocytopoiesis in this group of diseases, we chose to focus on circulating platelet-derived microparticles (PMP) whose count can be established by flow cytometry. Our goals were to look for an increased release of PMP in MPD and if so, try to establish a correlation between thrombotic events recorded retrospectively and PMP level. Patients and methods:Plasma samples were collected from 85 patients with MPD (40 ET, 28 PV, 17 PMF) at any time of the disease course except blastic phase and 31 healthy age-paired controls ; the quantity of PMP (positive for the platelet marker CD41 and the microparticle marker AnnexinV) was measured by flow cytometric analysis using the FC500 cytometer from Beckmann-Coulter™. Pre-analytic and testing procedures complied with the recommendations of the ISTH Standardization Subcommittee and besides, our laboratory participates in a multicenter program to standardize the enumeration of cellular microparticles. Results:The number of PMP is significantly higher in ET, PV and MFP patients (median: 4862 MPP/mL, 3289 MPP/mL and 6114 MPP/mL respectively) than in controls (median 1310 MPP/mL) [p=0.000018, p=0.0013, p=0.0009]. Neither in MPD patients nor in control group PMP correlates with platelet count and appears thus as an independent parameter. ROC method allows to fix a cut-off at 3121 MPP/mL to discriminate MPD from controls. This value has a sensibility of 75 % and a specificity of 94%. Interestingly, the total number of PMP is not different between untreated patients (mean=7246 MPP/mL) and those who received myelosuppressive therapy (mean=6320 MPP/mL). Regarding thrombotic events, we failed to demonstrate any significant difference between patients with previous thrombosis and those without; however, the small size of the series and the retrospective assessment are possible bias. Conclusion:Patients with Phi negative MPDs have a high number of circulating PMP, irrelevant to platelet count or treatment by Hydroxy-urea or aspirin. A role for microparticles in the development of these diseases and in the occurrence of thrombotic complications can be hypothesized but requires further studies -;preferably prospective- on larger cohorts of patients. Disclosures:Charpentier:Schering-Plough: Research Funding.