JAK2 V617F-positive Myeloproliferative Disorders Research Articles

Study of Tumor Necrosis Factor-Alpha-Induced Protein 3 Gene Single-Nucleotide Variants in JAK2 V617F-Positive Myeloproliferative Disorders: A Case–Control Study

Background and Objectives: Myeloproliferative neoplasms (MPNs) are Philadelphia negative disorders involving polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF). Although JAK2 mutation is almost involved, other several mutations are linked to MPNs risk and prognosis. TNFAIP3 genetic mutations are related to several cancers and autoimmune diseases. Our study aimed to demonstrate the effects of rs2230926_T/G & rs5029939_C/G SNVs of TNFAIP3 gene on the risk and prognosis of JAK2 V617F positive MPNs.
 Methods: Matched 2 groups in age, sex and race were enrolled in our research; 80 MPNs cases group and 130 normal healthy controls group with follow up of MPNs cases for 3 years. Taqman assay probes involved in real time polymerase chain reaction (PCR) were utilized for variants analysis.
 Results: The rs2230926 & rs5029939 SNVs were in modest linkage disequilibrium (LD) in MPNs cases. The observed frequencies of G allele and its genotypes of both variants were more prevalent in MPNs patients than normal controls. The bleeding symptoms and the presence of splenomegaly were more existent in the heterozygous genotype and the combined G involved genotypes respectively. The overall survival (OS) was lower in G containing genotypes of both variants but the progression free survival (PFS) was affected only in rs5029939 SNV.
 Conclusion: Our study revealed the association of G containing genotypes of both rs2230926 & rs5029939 SNVs to the increased MPNs incidence as well to poor clinical course and prognosis of JAK2 V617F positive MPNs disorders in Egyptian ethnic.

The Leukemic Stem Cell in Polycythemia Vera and Primary Myelofibrosis Is Distinct From the Initiating JAK2 V617F-Positive Hematopoiertic Stem Cell

Abstract 613The chronic myeloproliferative disorders (MPD), polycythemia vera (PV) and primary myelofibrosis (PMF) are clonal disorders involving a multipotent hematopoietic stem cell (HSC) but the identity of the involved HSC is a matter of controversy. For example, involvement of lymphoid cells in the MPD clone as determined by JAK2 V617F expression implies that these disorders arise in a pluripotent HSC. However, evidence has been presented that PV CD34+ HSC expressing JAK2 V617F are predisposed to erythroid differentiation, while mathematical modeling of JAK2 V617F-positive MPD suggests that these disorders arise in a hematopoietic progenitor cell that acquires JAK2 V617F, and then a mutation conferring self-renewal. Recently, a high level of aldehyde dehydrogenase (ALDHhigh) activity was used to distinguish normal CD34+CD38− HSC from committed hematopoietic progenitor cells, which lack ALDH activity, and also to separate leukemic stem cells (LSC) from CD34+/CD38−(ALDHhigh) HSC due to the presence of lower ALDH activity(ALDHint) in the LSC. We, therefore, sought to determine whether ALDH activity could be used to define the HSC involved in JAK2 V617F-positive PV and PMF, and whether leukemic transformation in these disorders was associated with a change in ALDH activity in the involved stem cell. To this end, we studied the immunophenotypic characteristics and ALDH activity of circulating CD34+ cells from 6 PV, 6 PMF (3 PMF and 3 post PV/MF) and 3 post MPD acute myelogenous leukemia (AML) patients (2 PV and 1PMF). CD34+ cells were isolated from peripheral blood using immunomagnetic bead technology with a purity of 95 % and a viability of 98 %, and analyzed for CD34 and CD38 expression and ALDH activity by flow cytometry. Cell sorting was carried out for measurement of the JAK2 V617F allele burden in discrete cell populations using purified genomic DNA and either a quantitative allele-specific assay or pyrosequencing. Where informative, sorted cell populations were analyzed by FISH for chromosomal abnormalities. As a per cent of total leukocytes, the median CD34+ cell fraction was 0.07 (range 0.01–0.2) for PV; 0.5 (range 0.4–1.3) for PV/MF and 2.3 (range 0.2–2.9) for PMF. The CD34+CD38− fraction was 17.8% of total PV CD34+ cells, 20.7% of total PV/MF CD34+ cells (p = 0.69) and 51.6 % of total PMF CD34+ cells (p= 0.003 and p= 0.007 respectively), indicating greater expansion of the PMF CD34+CD38− cell population, even though there was not a significant difference in disease duration between the three groups. ALDH activity was high in the CD34+CD38− cell population of all three groups. In 9/9 patients studied, the JAK2 V617F mutation was present in the CD34+CD38−(ALDHhigh) cell population at approximately 80 % of the patients’ neutrophil JAK2 V617F allele burden, substantiating that the JAK2 V617F mutation was present in primitive PV and PMF HSC. We next analyzed the CD34+CD38− cell population for ALDH activity in 1 PMF and 2 PV patients who had transformed to AML. In addition to an CD34+/CD38−(ALDHhigh) cell population, all three patients had an additional CD34+/CD38− population with lower ALDH activity (ALDHint), identical to that of LSC. Importantly, this latter CD34+/CD38−(ALDHint) cell population was not present before AML transformation in any patient. In 2 of 2 patients studied, the CD34+/CD38−(ALDHint) cell population expressed JAK2 V617F with an allele burden comparable to the CD34+/CD38−(ALDHhigh) population. Significantly, in one of the patients, who had acquired a 5q- deletion, the chromosomal abnormality was present only in the CD34+CD38−(ALDHint) cell population. In conclusion, these data support the contention that in PV and PMF, JAK2 V617F is acquired in a primitive CD34+CD38−(ALDHhigh) HSC. In addition, in contrast to PV and PV/MF, expansion of total CD34+ cells in PMF was also accompanied by differential expansion of this primitive CD34+CD38−(ALDHhigh) population. Furthermore, AML transformation in PV and PMF appeared to occur in an LSC, which had an aberrant ALDH activity pattern (ALDHint) identical to that seen in de novo AML LSC. The 5q- deletion, a molecular marker characteristic of AML, also tracked with the LSC cell population but not with the CD34+CD38−(ALDHhigh) cell population, while JAK2 V617F was equivalently expressed by both cell populations. This observation supports the contention that with respect to AML transformation in PV and PMF, JAK2 V617F is essentially a passenger lesion. Disclosures:No relevant conflicts of interest to declare.

A CK2-dependent mechanism for activation of the JAK-STAT signaling pathway

AbstractJAK-STAT signaling is involved in the regulation of cell survival, proliferation, and differentiation. JAK tyrosine kinases can be transiently activated by cytokines or growth factors in normal cells, whereas they become constitutively activated as a result of mutations that affect their function in tumors. Specifically, the JAK2V617F mutation is present in the majority of patients with myeloproliferative disorders (MPDs) and is implicated in the pathogenesis of these diseases. In the present study, we report that the kinase CK2 is a novel interaction partner of JAKs and is essential for JAK-STAT activation. We demonstrate that cytokine-induced activation of JAKs and STATs and the expression of suppressor of cytokine signaling 3 (SOCS-3), a downstream target, are inhibited by CK2 small interfering RNAs or pharmacologic inhibitors. Endogenous CK2 is associated with JAK2 and JAK1 and phosphorylates JAK2 in vitro. To extend these findings, we demonstrate that CK2 interacts with JAK2V617F and that CK2 inhibitors suppress JAK2V617F autophosphorylation and downstream signaling in HEL92.1.7 cells (HEL) and primary cells from polycythemia vera (PV) patients. Furthermore, CK2 inhibitors potently induce apoptosis of HEL cells and PV cells. Our data provide evidence for novel cross-talk between CK2 and JAK-STAT signaling, with implications for therapeutic intervention in JAK2V617F-positive MPDs.

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.

Gender Differences In the Prevalence of Thrombosis In the JAK2 V617F-Positive Myeloproliferative Disorders

AbstractAbstract 3080 Introduction:Thrombotic events are prevalent and morbid conditions in essential thrombocytosis (ET), polycythemia vera (PV), and primary myelofibrosis (PMF), but the exact mechanisms aside from hyperviscosity and dysfunctional platelet behavior are largely undefined. Since ET, PV and PMF are chronic, latent disorders of middle and older age, we examined the influence of traditional thrombotic risk factors on the tendency toward thrombotic events. To this end, we retrospectively analyzed a cohort of 270 JAK2 V617F-positive patients from the Johns Hopkins Center for Myeloproliferative Disorders. Methods:Thrombotic events within 5 years preceding or at any time following an official diagnosis of ET (n=59), PV (n=166), or MF (post-ETMF, post-PVMF, and PMF) (n=45) were captured by retrospective review of the medical record; the events were characterized as microvascular or macrovascular, arterial or venous. Traditional thrombotic risk factors were recorded, including hypertension, diabetes, dyslipidemia, and history of smoking) and since there were gender differences in diagnostic class (ET and PV were more common in women), age at diagnosis, smoking history, dyslipidemia, and leukocyte count, these variables were incorporated into a multivariable logistic regression model. Result:Of the entire MPD cohort, 67 patients (25%) had 79 microvascular or macrovascular thrombotic events, including 14 (24%) with ET (18 events), 45 (27%) with PV (51 events), and 8 (18%) with MF (10 events). The thrombosis prevalence did not differ statistically by disease class. However, thrombosis prevalence did differ by gender: women had a higher prevalence of thrombosis compared to men (29% vs. 18%; p=0.035) and the odds ratio remained significant after multivariable adjustment (OR 1.98, p=0.04). There were no gender differences with respect to treatment (aspirin or cytoreductive agents), nor were there differences due to age (median 56 years) or disease duration at the time of thrombosis. The frequency of JAK2 V617F homozygosity also did not differ by gender. In men, 9 of 13 (69%) thrombotic events occurred at diagnosis or in the preceding 5 years; however, in women, thrombotic events during follow-up were as likely since only 20 of 45 (44%) thrombotic events occurred at diagnosis or in the preceding 5 years. Nine patients experienced multiple thrombotic events, of these, 6 were women; in 8 cases, these thrombotic events involved different vascular beds, suggesting a general proclivity for thrombosis. Overall, a dependence between sex and the type of thrombosis could not be identified, possibly due to inadequate power (p=0.36). However, there was an indication of gender differences in some thrombotic subgroups: for example, of 20 abdominal venous thromboses (AVT, including hepatic vein, portal vein, mesenteric vein and splenic vein thrombosis), 16 (80%) occurred in women (p=0.07). AVT was the most common event in PV, occurring in 23% of cases, and of these, 11 of 12 were reported in women. Further, in 7 of these women, an AVT was the presenting manifestation of their MPD. AVT was also the most common type of thrombosis in MF, occurring in 50% of cases; of these, 4 of 5 also occurred in women. In contrast to PV, rather than manifesting at diagnosis, 3 of the 5 events (60%) complicated splenectomy. Neurologic events also appeared to be more frequent in women: 6 of 7 CVA's (86%) occurred in women (p=0.14), and of 11 TIA's, 8 (73%) occurred in women (p=0.4). Conclusion:In this retrospective analysis of 270 cases, thrombotic events were equally prevalent in ET, PV, and MF, though importantly more common in women in this patient cohort. There was also a trend suggesting differences in thrombosis type between women and men, since TIA, CVA, and especially, AVT appeared to be more common in women. Indeed, in women, AVT was often the presenting feature leading to a diagnosis of PV. This study indicates unique disease and gender specific interactions in the generation of thrombosis in the MPD, and suggests that women are paradoxically at higher risk for thrombosis in the MPD. Though a mechanism is not yet defined, this analysis contributes to a growing literature emphasizing gender differences in all three MPD, which already includes differences in the JAK2 V617F allele burden and disease class. Further, this analysis supports the important impact of individual and host variation on the clinical manifestations of the MPD. Disclosures:No relevant conflicts of interest to declare.

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.

Gender Is a Phenotypic Modifier in the JAK2 V617F-Positive MPD.

Introduction: The myeloproliferative disorders (MPD), essential thrombocytosis (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) share the same acquired genetic lesion, JAK2V617F, but differ with respect to epidemiology, natural history and gender. We have shown that the JAK2V617F allele burden at both the neutrophil and CD34+ cell level varies amongst MPD patients, and is a consequence of both the degree of dominance of the JAK2V617F clone and the specific JAK2V617F genotype (heterozygous versus homozygous) of the disease clones. The variability of the JAK2V617F expression, which in some female ET patients is restricted to platelets only, is a major modifier of MPD disease class and within PV, a modifier of white cell count and extramedullary disease. We hypothesized that gender may be an important modifier of the JAK2V617F burden and, therefore, the disease phenotype, and examined the relationship between gender, disease phenotype and the quantitative JAK2V617F allele burden in both CD34+ cells and neutrophils in JAK2V617F -positive MPD patients.Materials and Methods: Serial blood samples were obtained prospectively between May of 2005 and July of 2008 from MPD patients evaluated and treated at the Johns Hopkins Center for Chronic Myeloproliferative Disorders. Clinical parameters including the white blood cell count, chemotherapy, and splenomegaly were extracted from the medical record. The JAK2V617F allele burden was measured by allele-specific quantitative PCR using genomic DNA from purified neutrophils and peripheral blood CD34+ cells.Results: We evaluated 248 consecutive JAK2 V617F -positive patients, the majority of which (62%) were females. Of the entire cohort, 55 (22%) patients had ET, 158 (64%) PV, and 35 (14%) PMF. When stratified by gender, the distribution of the disease phenotypes significantly differed as 25%, 67% and 8% of females had ET, PV and PMF, respectively, compared to 18%, 59% and 23% of males (p=0.005). Median age at diagnosis was significantly lower in females (50 years) compared to males (55 years) (p=0.028). When stratified by gender, JAK2 V617F -positive PV females had a lower median neutrophil allele burden (NAB) than males (64% vs. 76%) (p=0.007). These differences were independent of age at diagnosis or chemotherapy use, but there was an effect of disease duration. At diagnosis, female and male PV patients had a similar median NAB (60 vs. 59%, p= 0.915). However, while the NAB did not vary over time from diagnosis in females (60% within year of diagnosis versus 62.5 % at 6 years or more (p=0.142)), in males, the median NAB increased over time (59% at diagnosis versus 82% at 6 years or more from diagnosis) (p=0.003)). These cross-sectional observations were confirmed by prospective analysis of 32 females and 13 males with PV. Similar to the findings in NAB, females had lower JAK2V617F CD34+ cell AB (49% vs. 57%) compared to males. 22% of the PV patients had antecedent ET, and of this group, 71% were women; in contrast, 23% of the PMF patients had antecedent ET, and 75% were men (p=0.037).Conclusion: Gender is a significant modifier of JAK2V617F -positive MPD. Overall, JAK2V617F positive disease was more common in females compared to males, and was manifest earlier. Females with JAK2V617F positive MPD have lower allele burdens both at the neutrophil and CD34+ cell level compared to males, accounting for their relative increase in ET and relative decrease in IMF compared to males with JAK2V617F positive disease. Lower allele burdens in females may be due to a lower frequency of mitotic recombination events resulting in fewer homozygous JAK2V617F clones. Our findings suggest that in the genotype-phenotype discrepancy amongst the JAK2V617F -positive MPD, gender is an important modifier of the JAK2V617F allele burden, and should be taken into account when evaluating MPD patients with regard to the diagnosis, prognosis, disease complications and the evaluation of response to molecularly targeted therapies.

An Open-Label Study of CEP-701 in Patients with JAK2 V617F-Positive Polycythemia Vera and Essential Thrombocytosis

Polycythemia vera (PV) and essential thrombocytosis (ET) are clonal hematopoietic stem cell disorders characterized by the over production of phenotypically normal circulating blood cells. Most PV and approximately half of ET patients harbor the activating mutation JAK2 V617F. CEP-701 is an orally available, potent low nanomolar inhibitor of both the wild-type and mutated JAK2 tyrosine kinase, with its inhibitory effect demonstrated both in enzymatic and cellular assays and in vivo, where CEP-701 significantly inhibited the growth of JAK2 V617F-positive HEL.92 xenografts in mice. These findings suggest that CEP-701 is an attractive candidate for clinical evaluation in JAK2 V617F-positive myeloproliferative disorders. The purpose of this study is to test the safety and efficacy of CEP-701 administration in JAK2 V617F positive ET and PV patients. The primary endpoint is reduction in JAK2 V617F neutrophil allele burden; secondary endpoints are reduction in phlebotomy rates, improvement in hemoglobin, white cell and platelet counts, reduction in hydroxyurea (HU) dose, and reduction in spleen size. The secondary endpoints include the pharmacokinetics and pharmacodynamics of CEP-701 and the safety of CEP-701 treatment in patients with JAK2 V617F-positive PV and ET. This is a multicenter study with an anticipated enrollment of 40 PV and ET patients. Inclusion criteria include JAK2 V617F-positive PV and ET patients; patients with PV either have a neutrophil count greater than 7,000/ul or are receiving HU, while ET patients are receiving concomitant HU. Other inclusion criteria include an ECOG performance status of 0, 1 or 2, and 18 years of age or older. Exclusion criteria include the active use of anegrelide or interferon, or a recent history of venous or arterial thrombosis. This is an 18 week trial with an optional 1 year extension period; doses will escalate from 80 mg twice daily to a maximum of 120 mg twice daily. To date, 20 subjects, 11 PV and 9 ET, comprised of 11 females and 9 males, ages 34 to 74, have enrolled. Approximately 27% of the PV patients were taking HU. The most common adverse events have been gastrointestinal (GI) and constitutional in nature. No related serious adverse events have been observed. Five patients have discontinued study participation, all for adverse events: 1 due to disease progression, 1 leg cramps, and 3 GI. To date, 7 patients have completed 18 weeks of therapy and 6 of these patients will continue to receive CEP-701 on the extension phase of the trial. Five of 8 subjects with splenomegaly have responded with reductions in spleen size evident within 6 weeks of therapy initiation. Updated results on current and future patients will be presented at the meeting.

Elevated Leukocyte Alkaline Phosphatase Scores Induced by Jak2 V617F Mutation

Leukocyte alkaline phosphatase (LAP) enzymatic activity is a marker of the last stages of myeloid differentiation. The level of LAP is quantitated as the LAP score. Estimation of the LAP score has been useful for distinguishing chronic myelogenous leukemia (CML) from BCR-ABL–negative chronic myeloproliferative disorders (MPDs) and neutrophilic reactions in severe infections. CML patients usually have a low LAP score, whereas elevated LAP scores are seen in patients with polycythemia vera (PV), primary myelofibrosis (PMF), and leukocytosis caused by infections. An acquired Jak2 V617F mutation is seen in approximately 95% of patients with PV and in about 50% of patients with essential thrombocythemia or PMF. It has been shown that Jak2 V617F mutation induced constitutive activation of the JAK-STAT signaling pathway. We speculated that an elevated LAP score might be caused due to activation of JAK-STAT signaling through a Jak2 V617F mutation, and conducted this study to address this question. We analyzed the LAP scores in Jak2 V617F-positive and -negative MPD patients. Jak2 V617F-positive MPD patients had higher LAP scores than Jak2 V617F-negative patients. Moreover, patients carrying homozygous mutations had higher LAP scores than patients with heterozygous mutations. AG490, the Jak2 inhibitor, was shown to significantly decrease the LAP expression in neutrophils of Jak2 V617F-positive patients. We lentivirally transfected the acute promyelocytic leukemia cell line NB4 with the Jak2 V617F mutation and wild-type Jak2 V617F. The expression level of Jak2 was not significantly different between the Jak2 V617F mutation and wild-type Jak2 V617F. We then examined the LAP scores of transfected NB4 cells after these cells were differentiated by all-trans retinoic acid and granulocyte colony stimulating factor. It was observed that the Jak2 V617F mutation and not the wild-type Jak2 induced elevated LAP scores. Furthermore, we showed that Jak2 followed the MAP kinase pathway and not the PI3 kinase pathway, as a downstream signaling pathway to elevate the LAP scores using MEK 1/2 (U0126) and PI3 kinase (LY294002) inhibitors. In conclusion, we obtained direct evidence that Jak2 V617F mutation induces elevated LAP scores via the MAP kinase pathway.

The hematopoietic stem cell compartment of JAK2V617F-positive myeloproliferative disorders is a reflection of disease heterogeneity

AbstractThe JAK2V617F somatic point mutation has been described in patients with myeloproliferative disorders (MPDs). Despite this progress, it remains unknown how a single JAK2 mutation causes 3 different MPD phenotypes, polycythemia vera (PV), essential thrombocythemia, and primitive myelofibrosis (PMF). Using an in vivo xenotransplantation assay in nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice, we tested whether disease heterogeneity was associated with quantitative or qualitative differences in the hematopoietic stem cell (HSC) compartment. We show that the HSC compartment of PV and PMF patients contains JAK2V617F-positive long-term, multipotent, and self-renewing cells. However, the proportion of JAK2V617F and JAK2 wild-type SCID repopulating cells was dramatically different in these diseases, without major modifications of the self-renewal and proliferation capacities for JAK2V617F SCID repopulating cells. These experiments provide new insights into the pathogenesis of JAK2V617F MPD and demonstrate that a JAK2 inhibitor needs to target the HSC compartment for optimal disease control in classical MPD.

Methylation of the suppressor of cytokine signaling 3 gene (SOCS3) in myeloproliferative disorders

The JAK2 V617F mutation can be found in patients with polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. Mutation or methylation of other components of JAK/STAT signaling, such as the negative regulators suppressor of cytokine signaling 1 (SOCS1) and SOCS3, may contribute to the pathogenesis of both JAK2 V617F positive and negative myeloproliferative disorders. A cohort of patients with myeloproliferative disorders was assessed for acquired mutations, aberrant expression and/or CpG island hypermethylation of SOCS1 and SOCS3. No mutations were identified within the coding region of either gene in 73 patients with myeloproliferative disorders. No disease-specific CpG island methylation of SOCS1 was observed. SOCS1 expression was raised in myeloproliferative disorder granulocytes but the level was independent of JAK2 V617F status. Hypermethylation of the SOCS3 promoter was identified in 16 of 50 (32%) patients with idiopathic myelofibrosis but not in patients with essential thrombocythemia, polycythemia vera or myelofibrosis preceded by another myeloproliferative disorder. Confirmation of methylation status was validated by nested polymerase chain reaction and/or bisulphite sequencing. SOCS3 transcript levels were highest in patients with polycythemia vera and other JAK2 V617F positive myeloproliferative disorders, consistent with SOCS3 being a target gene of JAK2/STAT5 signaling. There was a trend towards an association between SOCS3 methylation and lower SOCS3 expression in JAK2 V617F negative patients with idiopathic myelofibrosis but not in JAK2 V617F positive ones. Finally, SOCS3 methylation was not significantly correlated with survival or other clinical variables. SOCS3 promoter methylation was detected in 32% of patients with idiopathic myelofibrosis suggesting a possible role for SOCS3 methylation in this disorder. The pathogenetic consequences of SOCS3 methylation in idiopathic myelofibrosis remain to be fully elucidated.

Phenotypic variability within the JAK2 V617F-positive MPD: Roles of progenitor cell and neutrophil allele burdens

The myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), differ phenotypically, but share the same JAK2(V617F) mutation. We examined the relationship of the quantitative JAK2(V617F) allele burden to MPD disease phenotype among the three MPD classes and within PV. We measured the JAK2(V617F) allele percentage in genomic DNA from neutrophils, CD34(+) cells, and cloned progenitors in 212 JAK2(V617F)-positive MPD patients and correlated the allele burdens to both disease class and disease features. In ET and PV, mean CD34(+) cell JAK2(V617F) allele burdens were lower than the corresponding neutrophil allele burdens, but these were equivalent in PMF. JAK2(WT) progenitors were present in ET and PV when the CD34(+) JAK2(V617F) allele burden was lower than the neutrophil allele burden, but not in PV and PMF subjects in whom the CD34(+) cell and neutrophil allele burdens were similar. CD34(+) cell JAK2(V617F) clonal dominance, defined as coherence between the CD34(+) cell and neutrophil JAK2(V617F) allele burdens, was present in 24% of ET, 56% of PV, and 93% of PMF patients, and was independent of the CD34(+) cell JAK2(V617F) genotype. Clonally dominant PV patients had significantly longer disease durations, higher white cell counts, and larger spleens than nondominant PV patients. We conclude that the extent of JAK2(V617F) CD34(+) cell clonal dominance is associated with disease phenotype within the MPD and, in PV, is associated with extramedullary disease, leukocytosis, and disease duration.

LS104, a non-ATP-competitive small-molecule inhibitor of JAK2, is potently inducing apoptosis in JAK2V617F-positive cells

The activating JAK2V617F mutation has been described in the majority of patients with BCR-ABL-negative myeloproliferative disorders (MPD). In this report, we characterize the small-molecule LS104 as a novel non-ATP-competitive JAK2 inhibitor: Treatment of JAK2V617F-positive cells with LS104 resulted in dose-dependent induction of apoptosis and inhibition of JAK2 autophosphorylation and of downstream targets. Activation of these targets by JAK2 was confirmed in experiments using small interfering RNA. LS104 inhibited JAK2 kinase activity in vitro. This effect was not reversible using elevated ATP concentrations, whereas variation of the kinase substrate peptide led to modulation of the IC50 value for LS104. In line with these data, combination treatment using LS104 plus an ATP-competitive JAK2 inhibitor (JAK inhibitor I) led to synergistically increased apoptosis in JAK2V617F-positive cells. Furthermore, LS104 strongly inhibited cytokine-independent growth of endogenous erythroid colonies isolated from patients with JAK2V617F-positive MPD in vitro, whereas there was no significant effect on growth of myeloid colonies obtained from normal controls. Based on these data, we have recently started a phase I clinical trial of LS104 for patients with JAK2V617F-positive MPDs. To the best of our knowledge, this is the first report on a non-ATP-competitive kinase inhibitor being tested in a clinical trial.

HLA-G turns off erythropoietin receptor signaling through JAK2 and JAK2 V617F dephosphorylation: clinical relevance in polycythemia vera

HLA-G5 is secreted by erythroblasts in all hematopoietic organs, suggesting a role for this protein in erythropoiesis. To examine this, we analyzed whether HLA-G5 affects the proliferation of UT7/EPO and HEL erythroleukemia cells and characterized the mechanism by which HLA-G5 influences erythropoietin receptor (EPOR) signaling. We show that HLA-G5 inhibits the proliferation of UT7/EPO cells, the EPOR signaling of which is similar to that of normal erythroid progenitors. HLA-G5-mediated inhibition was associated with reduced phosphorylation of JAK2 kinase and that of the downstream signaling proteins STAT-5 and STAT-3. Involvement of JAK2 in erythroid cell proliferation has been highlighted by the role of JAK2 V617F mutation in polycythemia vera (PV), a myeloproliferative disorder characterized by erythroid lineage overproduction. We demonstrate that HLA-G5 downregulates EPOR constitutive signaling of JAK2 V617F-expressing HEL cells, leading to inhibition of cell proliferation through G1 cell cycle arrest. Combination of HLA-G5 with JAK inhibitor I further decreases HEL cell growth. Clinical relevance is provided by analysis of PV patients who carry JAK2 V617F mutation, showing that HLA-G5 inhibits the formation of erythropoietin-independent erythroid colonies. Such HLA-G5-mediated inhibition constitutes a new parameter to be considered in the design of future approaches aimed at treating JAK2 V617F-positive myeloproliferative disorders.

Clonal Dominance at the Progenitor Cell Level Defines Clinical Phenotype More Accurately Than the Neutrophil Allele Burden in JAK2V617F-Positive MPD Patients.

The chronic myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis (ET) and idiopathic myelofibrosis (IMF) differ phenotypically but share the same mutation, JAK2V617F. To determine how the same mutation yields three different phenotypes, we examined the relationship between disease phenotype and the quantitative JAK2V617F allele burden in both CD34+ cells and neutrophils. The study population consisted of 126 PV, 41 ET and 18 IMF JAK2V617F-positive patients with a median disease duration of 8 years. The JAK2V617F allele burden was determined by allele-specific quantitative PCR using genomic DNA from neutrophils and circulating purified CD34+ cells; in 6 ET, 6 IMF and 13 PV patients, erythroid colonies cloned from CD34+ cells were genotyped for JAK2V617F. As we previously reported, the mean neutrophil JAK2V617F allele burdens were lower in ET compared to PV and IMF (38% vs 66%; p<.001). In PV but not in ET, the neutrophil JAK2V617F allele burden correlated directly with the reticulocyte count (R=.452, p<.001), the white cell count (R=.519; p<.001) and splenomegaly (R=0.378; p<.001) and inversely with the platelet count (R=0.323; p=0.004). In both ET and PV, the median CD34+ cell JAK2V617F allele burdens were lower than the corresponding neutrophil allele burdens (25% vs 39% in ET, p=.04, and 56% vs 64% in PV, p=.01), but these were equivalent in IMF (77% and 77%). The median CD34+ cell allele burden was lower in ET patients compared to PV or IMF patients (p<.001). In contrast to neutrophils in which there was no correlation between allele burden and disease duration (R = 0.067; p = ns), in PV the CD34+ cell allele burden correlated with disease duration (R=0.358; p = 0.035), and more strongly correlated with the white cell count (R=.725; p<0.001) and spleen size (R=0.635; p<0.001) than the neutrophil allele burden. BFU-e clonogenic assays identified JAK2 wild-type progenitors in ET and PV patients in whom the CD34+ JAK2V617F allele burden was lower than the neutrophil allele burden, and did not identify wild-type progenitors in PV and IMF subjects in whom the CD34+ cell and neutrophil allele burdens were similar. Based on this observation, we defined clonal dominance at the progenitor level as a difference of 10 or less between the neutrophil and CD34+ allele burdens. Clonal dominance was present in 22% of ET, 53% of PV and 90% of IMF patients, and was independent of the clonal genotype as clonal dominance was present with heterozgous only (ET, PV and IMF), homozygous only (PV or IMF) or mixtures of heterozygous and homozygous clones (PV, IMF). Within PV, clonally dominant subjects had significantly longer disease durations (11 vs 5.5 years, p=.01), higher white cell counts (18.2 vs 11.1, p=.02), larger spleens (4 vs 0 cm below the costal margin, p=.003), and were more commonly receiving chemotherapy than the nonclonally dominant PV patients (46% vs 22%, p=.02). Importantly, the neutrophil allele burden did not differ between clonally dominant and nondominant PV patients (mean 72.8 vs 68.7%, p=0.506). We conclude that the extent of JAK2V617F CD34+ cell clonal dominance is a major determinant of disease class within the MPD, and within PV, is a major determinant of extramedullary disease and leukocytosis. The extent of clonal dominance at the progenitor level cannot be determined from the neutrophil allele burden alone, and thus limits the utility of the neutrophil allele burden as a disease marker.

Leukemic blasts in transformed JAK2-V617F–positive myeloproliferative disorders are frequently negative for the JAK2-V617F mutation.

To study the role of the JAK2-V617F mutation in leukemic transformation, we examined 27 patients with myeloproliferative disorders (MPDs) who transformed to acute myeloid leukemia (AML). At MPD diagnosis, JAK2-V617F was detectable in 17 of 27 patients. Surprisingly, only 5 of 17 patients developed JAK2-V617F-positive AML, whereas 9 of 17 patients transformed to JAK2-V617F-negative AML. Microsatellite analysis in a female patient showed that mitotic recombination was not responsible for the transition from JAK2-V617F-positive MPD to JAK2-V617F-negative AML, and clonality determined by the MPP1 polymorphism demonstrated that the granulocytes and leukemic blasts inactivated the same parental X chromosome. In a second patient positive for JAK2-V617F at transformation, but with JAK2-V617F-negative leukemic blasts, we found del(11q) in all cells examined, suggesting a common clonal origin of MPD and AML. We conclude that JAK2-V617F-positive MPD frequently yields JAK2-V617F-negative AML, and transformation of a common JAK2-V617F-negative ancestor represents a possible mechanism.

Myeloid Blasts in Transformed JAK2-V617F Positive Myeloproliferative Disorders Are Frequently Negative for the JAK2-V617F Mutation.

Acute myeloid leukemia (AML) is a common complication of myeloproliferative disorders (MPDs). The role of the JAK2-V617F mutation in this process is unknown. We performed a retrospective analysis of DNA samples from MPD patients with secondary AML. We analysed DNA samples taken at the time of transformation to AML from 54 MPD patients (24 PV, 21 ET, 9 IMF). In addition, DNA samples taken at diagnosis of MPD were obtained in 21 of these patients. DNA was extracted from bone marrow or peripheral blood films, purified granulocytes or frozen cells. FACS sorting of blast cells, T cells and neutrophils was performed in some of the samples. The allelic ratio of JAK2-V617F was determined by allele-specific quantitative PCR (AS-PCR). We obtained AS-PCR data on 52/54 samples taken at the time of transformation (96%), whereas 2 samples did not yield PCR products: 24/52 samples were negative for JAK2-V617F (46%) and 28/52 were positive (54%). For 14/24 negative patients (58%) we had additional DNA samples taken at the time of MPD diagnosis and interestingly, 5 of these 14 patients (36%) were positive for JAK2-V617F at this earlier time point before AML transformation. This suggests that in these patients the JAK2-V617F positive clone was lost during the evolution to AML. Furthermore, comparison of the JAK2-V617F allelic ratios with the percentage of blast cells in patient samples positive at transformation revealed 8/28 cases where the JAK2-V617F allelic ratio was markedly lower than the percentage of blasts, e.g. 8%T-allele and 52% myeloid blast cells. In these patients a JAK2-V617F negative AML clone most likely co-exists with a JAK2-V617F positive MPD clone. To address the question whether the AML clone arose independently from the JAK2-V617F clone, we analyzed loss of heterozygosity on chromosome 9p (9pLOH) in one informative patient who displayed a high allelic ratio of mutant JAK2 at diagnosis (94%T). The CD15+ cells from this patient showed 9pLOH at diagnosis, as demonstrated with two independent microsatellite markers. In contrast, the FACS sorted blast cells at the time of transformation contained both parental alleles in the 9p region and were JAK2-V617F negative by AS-PCR. This excludes the possibility that the AML clone lost the JAK2V617F in the process of undergoing mitotic recombination at a stage heterozygous for JAK2-V617F. Analysis of additional patients is under way. In summary, we found in a cohort of 54 MPD patients, 13 patients initially positive for JAK2-V617F that transformed into JAK2-V617F negative AML. Although not confirmed in the one patient analyzed, we cannot exclude that other patients the JAK2-V617F positive MPD clone lost the JAK2 mutation during the process of transformation. Alternatively, the AML clone could have developed de novo from a JAK2-V617F negative progenitor or stem cell. The latter model has difficulties explaining the high incidence of de novo AML (8/54 patients), unless the JAK2-V617F negative progenitor already carried an as yet unknown mutation and was part of the MPD clone.

The 2001 World Health Organization and Updated European Clinical and Pathological Criteria for the Diagnosis, Classification, and Staging of the Philadelphia Chromosome-Negative Chronic Myeloproliferative Disorders

The clinical criteria according to the Polycythemia Vera Study Group (PVSG) do not distinguish between essential thrombocythemia (ET), thrombocythemia associated with early-stage polycythemia vera (PV) and prefibrotic chronic idiopathic myelofibrosis (CIMF). The criteria only classify the advanced stage of PV with increased red cell mass. The classification of myeloproliferative disorders (MPDs), proposed by the World Health Organization (WHO) in 2001, is a compromise of the clinical PVSG and WHO bone marrow criteria, and excludes early stages of ET and PV. The updated European clinical and pathological criteria combine the WHO bone marrow criteria with established and new clinical, laboratory, biological, and molecular MPD markers. This allows clinicians and pathologists to diagnose early-stage MPD and to differentiate ET, PV, and prefibrotic chronic idiopathic myelofibrosis (CIMF). Depending on laboratory tests and diagnostic criteria used, the population of the MPD patients defined as ET, PV, and CIMF are heterogeneous at the clinical, laboratory, and biological and pathological levels. The recent discovery of the JAK2 V617F mutation, which is the cause of a distinct trilinear MPD in its manifold clinical manifestations during long-term follow-up, increases the specificity of a positive JAK2 V617F polymerase chain reaction (PCR) test for the diagnosis of MPD (near 100%), but only half of the ET and CIMF patients according to the PVSG (sensitivity 50%) and the majority of PV patients (sensitivity 95%) are JAK2 V617F positive. A comparison of the laboratory features of JAK2 V617-positive and JAK2 wild-type ET patients clearly showed that JAK2 V617-positive ET is characterized by higher values for hemoglobin, hematocrit, and neutrophil counts; lower values for serum erythropoietin (EPO) levels, serum ferritin, and mean corpuscular volume; and by increased cellularity of the bone marrow in biopsy material. This indicates that JAK2 V617-positive ET patients, diagnosed according to the PVSG criteria, represent a "forme fruste of PV" consistent with early PV mimicking ET (JAK2 V617F trilinear MPD). In contrast, the JAK2 wild-type ET patients had significantly higher platelet counts and usually had a clinical picture of ET with normal serum EPO levels, PRV-1 expression, and leukocyte alkaline phosphatase score, and a typical WHO ET bone marrow picture. The clinical and pathological data on JAK2 V617F-positive MPD patients suggest that the JAK2 V617F mutation defines one disease entity with several sequential steps of ET, PV, and secondary myelofibrosis during long-term follow-up, and that the wild-type JAK2 MPDs may represent another distinct entity with a related but different molecular etiology. MPD-specific markers such as serum EPO, endogenous erythroid colony formation (EEC), and JAK2 V617F have high specificities, but the sensitivities are not high enough to detect the early stages of the MPDs, ET, PV, and prefibrotic CIMF. Bone marrow histopathology in addition to clinical, laboratory, biological, and molecular markers, including the JAK2 V617 PCR test, serum EPO, PRV-1, EEC, LAP score, peripheral blood parameters, and spleen size on echogram will detect the early stages of MPD and allows diagnostic differentiation of the three primary MPDs (ET, PV, and CIMF) in both JAK2 V617F-positive and JAK2 wild-type MPD patients.