JAK2 V617F Mutation Is Infrequent in “5q-Syndrome” and 5q-Associated MDS.

Abstract Background: V617F mutation in Janus Kinase 2 (JAK2) gene has been found in chronic myeloproliferative disorders (MPD) including polycythemia vera (90%), essential thrombocythemia and chronic idiopathic myelofibrosis (30–50%), and occasionally in myelodysplastic syndromes (MDS). “5q- Syndrome” is a MDS that shares features with MPD and characterized by an atypical megakaryocytic hyperplasia in bone marrow and usually thrombocytosis in peripheral blood. The most common deleted region for this syndrome is 5q13.3q33.1. An interstitial deletion with variable proximal (5q12-14) and distal (5q31-33) breakpoints has been found in other MDS with/without additional chromosomal abnormalities beyond “5q- Syndrome”. To date, JAK2 mutation was detected in 6/97(6.2%) of patients having diagnosis of MDS with “5q- Syndrome”. Design: In our study 21 MDS patients (10 with “5q- Syndrome” and 11 MDS with isolated or complex 5q-) whose diagnosis by both bone marrow aspiration/biopsy and conventional chromosomal analysis were confirmed. Materials and Method: Primers were created to amplify a 460 bp fragment containing the site of JAK2 V617F mutation. Forty-five cycles of PCR were performed at an annealing temperature of 57°C. Resulting PCR product was digested with 2 U BsaXI for 16 hours and with an additional 2 U BsaXI for another 16 hours at 37°C, then analyzed on a 2% agarose gel. The mutant allele remained undigested whereas the wild-type allele was digested into 241 bp, 189 bp and 30 bp fragments. All experiments included a positive (HEL cells) and negative (K562 cells) control. Results: PCR results showed clear wild type PCR patterns in all 21 cases. Conclusion: No JAK2 mutations were detected in 21 patients either with “5q- Syndrome” or other 5q- associated MDS suggesting that JAK2 mutations are infrequent in these MDS patients.

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Coexistence of a Heterozygous JAK2(V617F) Mutation and a Secondary BCR-ABL Translocation within the Compartment of Committed Myeloid Progenitors in Chronic Idiopathic Myelofibrosis.

Blood ◽

10.1182/blood.v108.11.4871.4871 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4871-4871

Author(s):

Martin Bornhaeuser ◽

Brigitte Mohr ◽

Uta Oelschlaegel ◽

Peter Bornhauser ◽

Swen Jacki ◽

...

Keyword(s):

Bone Marrow ◽

Philadelphia Chromosome ◽

Jak2 V617f ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Idiopathic Myelofibrosis ◽

Jak2 Mutation ◽

V617f Mutation ◽

Chronic Idiopathic Myelofibrosis ◽

Philadelphia Chromosome Positive

Abstract Myeloproliferative disorders such as polycythemia vera (PV), essential thrombocytosis (ET) and chronic idiopathic myelofibrosis (CIMF) are clonal hematopoietic diseases with clinical similarities including the risk of transformation into acute myelogeneous leukemia. By definition, these diseases have been separated from Philadelphia chromosome positive (Ph+) CML requiring negativity for the BCR-ABL transcript in PCR studies of bone marrow or peripheral blood. Several groups independently discovered a gain of function mutation of the Janus kinase 2 (JAK2) gene in Ph-negative myeloproliferative diseases. This mutation has been associated with the proliferation of clonogenic progenitors independently of exogenous cytokine stimulation. A sixty-six year old male patient presented with moderate splenomegaly (3 cm under the costal marigin), mild anemia (11.3 g/dl), elevated lactate deyhdrogenase, an increased count of circulating CD34+ cells and a dry bone marrow aspirate. Marrow histology confirmed a prefibrotic stage of chronic idiopathic myelofibrosis (CIMF). Metaphase cytogenetics as well as BCR-ABL FISH were performed on samples from bone marrow, blood and sorted CD34+, CD3+, CD19+ and CD14+ cells from a steady-state back-up leukapheresis. The JAK2(V617F) mutation was confirmed by an allele-specific PCR assay. A screen for BCR-ABL was performed by FISH and PCR in sorted cells as well as in individual colonies (CFU-GM and CFU-E). Four Philadelphia-chromosome positive metaphases could be detected out of 86 derived from the autologous leukapheresis product harvested and cryopreserved as back-up shortly after diagnosis. The BCR-ABL translocation could be detected by fluorescence in-situ hybridisation (FISH) in 2/16 (12.5%) isolated granulocyte/macrophage colonies only whereas all erythroid colonies were negative. The JAK2 mutation was detectable in all clones and was enriched in CD34+ selected cells. The patient experienced progressive splenomegaly despite the achievement of a molecular response measured by quantitative BCR-ABL PCR after treatment with imatinib mesylate. Our in-vitro investigations suggest that the secondary BCR-ABL translocation within the myeloid compartment was of minor pathophysiological relevance in this patient with CIMF harbouring a heterozygous JAK2 mutation.

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JAK2 (V617F) Mutation Status and Neutrophil Apoptotic Resistance in Myelofibrosis with Myeloid Metaplasia (MMM): Correlation and Potential Identification through Phosphorylation Status of STAT3.

Blood ◽

10.1182/blood.v106.11.3503.3503 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3503-3503

Author(s):

Ruben A. Mesa ◽

Ayalew Tefferi ◽

Heather Powell ◽

Terra Lasho ◽

David Loegering ◽

...

Keyword(s):

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Neutrophil Apoptosis ◽

Wild Type ◽

Jak2 Mutation ◽

Myeloid Metaplasia ◽

Mutation Status ◽

Stat3 Phosphorylation ◽

V617f Mutation ◽

Myelofibrosis With Myeloid Metaplasia

Abstract Background: We have previously described a resistance to the normal process of apoptosis in neutrophils of patients with myelofibrosis with myeloid metaplasia (MMM) (Blood2003;102:11). Most recently, an activating mutation of JAK2 (V617F) has been described in approximately half of the patients with MMM as well as in variable proportion of patients with other myeloproliferative disorders (MPD). In the current study, we investigated the correlation between JAK2 V617F mutation status and neutrophil apoptosis in MMM. Methods: Neutrophils were isolated by density centrifugation from patients with MMM, other MPDs, and normal controls and assessed for apoptosis at baseline and after 24 hours in culture (IMDM with 20% sterilized fetal calf serum to simulate spontaneous apoptosis). Apoptosis was quantified using three-color flow cytometry using CD45 (to confirm leukocyte presence), annexin V (AN) (marker of apoptosis; detects aberrant externalization of phosphatidylserine during apoptosis), and propidium iodide (PI) (marker of dead cells). Mutation analysis for JAK2 V617F was performed in DNA derived from the isolated neutrophils using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Apoptotic rates after 24 hours in culture were correlated between patients and controls for both JAK2 mutation status and clinical parameters. Immunoblotting was performed on a subset of patients for correlation of JAK2 mutation status and downstream phosphorylation of the JAK2 target, STAT3, which transcriptionally activates several antiapoptotic genes. Results: Spontaneous neutrophil apoptosis was significantly decreased in MMM patients (n=50; median % apoptotic cells at 41%) compared to both healthy volunteers (n=9; 66%) and patients with other MPD (n=11; 53%) (p=0.002). Resistance to apoptosis in MMM correlated with both anemia (p=0.01) and the presence of the JAK2 V617F mutation (p=0.01). Furthermore, the specific abnormality was more pronounced in patients with homozygous JAK2 V617F; median % apoptotic cells of 47% for patients with wild-type allele (n=22) vs. 39% for heterozygotes (n=23) vs. 22% for homozygotes (n=5; p=0.008). The JAK2 mutation status did not appear dependent on other peripheral blood or clinical features. Neutrophils from 14 MMM patients were assessed simultaneously for both JAK2 mutation and STAT3 phosphorylation status by immunoblotting. Strong expression of phosphorylation of STAT3 was seen in all 3 homozygotes and 4 of 5 heterozygotes, but only 1 of 6 with wild-type allele (p=0.026). Conclusions: Impaired neutrophil apoptosis in patients with MMM correlates with the functional presence of JAK2 V617F in an allele-dose dependent manner and STAT3 phosphorylation. The current observation supports a pathogenetic role for the specific mutation in sustaining clonal myeloproliferation.

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Detection of the JAK2 V617F Mutation by Real Time PCR in Granulocytes and Platelets of ET Patients.

Blood ◽

10.1182/blood.v108.11.4877.4877 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4877-4877

Author(s):

Beatriz Bellosillo ◽

Eva Gimeno ◽

Raquel Longaron ◽

Lourdes Florensa ◽

Antonio Salar ◽

...

Keyword(s):

Real Time ◽

Direct Sequencing ◽

Specific Treatment ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Wild Type ◽

Rt Pcr ◽

Jak2 Mutation ◽

Allele Specific ◽

V617f Mutation

Abstract Introduction. The JAK2 V617F mutation has been detected in 23%–57% of ET patients by direct sequencing or allele-specific (AS) PCR. It remains unknown, however, if the mutation detected in the granulocyte population, may be equally detected in platelets from these patients. Objective. To compare the detection of the JAK2V617F mutation in granulocytes and platelets from ET patients by real time AS RT-PCR. Patients and methods. Platelets and granulocytes from 50 ET patients from a single institution were studied. Patients were diagnosed according to the WHO criteria. At the time when JAK2 mutation was analyzed 16/50 patients were receiving platelet-lowering therapy ± ASA, 14/50 patients only received ASA and 20/50 received no specific treatment. JAK2 mutation was analyzed by real-time AS RT-PCR with probes specific for the mutated and the wild type form. Results. The V617F JAK2 mutation was detected in 18 out of 50 patients in both granulocytes and platelets by real time AS RT-PCR, and was negative in both cell populations in the remaining 32 patients. In the V617F JAK2 positive cases, the mean Ct(V617FJAK2)/Ct(wild type JAK2) ratio was 1.074±0.062 for granulocytes and 1.038±0.039 for platelets (p=0.048). These values corresponded to a 17.79 ±7.4% of mutated population when granulocytes were analyzed, whereas, a significantly higher percentage of mutated population was observed, 23.45±7.78 %, when platelets were analyzed (p=0.032). Conclusions. The results of V617FJAK2 mutation detection by AS RT-PCR were the same in granulocytes and platelets (either positive or negative). The percentage of clonal population detected in ET patients was significantly higher in platelets than in granulocytes.

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Homologous Recombination of Wild TYPE JAK2, A NOVEL EARLY STEP In the DEVELOPMENT of Myeloproliferative Neoplasm

Blood ◽

10.1182/blood.v118.21.2805.2805 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2805-2805

Author(s):

Mathias Vilaine ◽

Damla Olcaydu ◽

Ashot Harutyunyan ◽

Jonathan Bergeman ◽

Tiab Mourad ◽

...

Keyword(s):

Homologous Recombination ◽

Snp Array ◽

Myeloproliferative Neoplasm ◽

Jak2 V617f ◽

Jak2 V617f Mutation ◽

Wild Type ◽

Jak2 Mutation ◽

Chromosome 9P ◽

V617f Mutation ◽

Array Analyses

Abstract Abstract 2805 Background: Adequate expression and function of Jak2 in hematopoietic progenitors is critical for normal myelopoiesis. The JAK2 46/1 (GGCC) haplotype, a congenital particularity, predisposes to myeloproliferative neoplasm (MPN) both independently and through mutation of the JAK2 gene. The JAK2 V617F mutation and acquired homozygous status for JAK2 V617F are frequent in MPN. JAK2 V617F homozygosity is currently explained acquisition of the JAK2 V617F mutation followed by mitotic homologous recombination (HR) of JAK2 occurred between wild-type and mutant alleles, leading to uniparental disomy (UPD) of chromosome 9p (9pUPD). Here we report the cases of 2 PV patients (Na1061 and Na1253) with acquired homozygous status for the JAK2 46/1 haplotype yet their granulocytes carried <20% JAK2 V617F. Aim: To determine whether HR of JAK2 can precede the V617F mutation in MPN. Methods: Granulocyte DNA and CD3+ lymphocyte DNA were examined in parallel with qPCR assays specific for the wild type and 46/1 haplotypes using rs12343867, a JAK2 intron 14 marker, as well as 4 other single nucleotide polymorphisms (SNP) on chromosome 9p. 9pUPD clonality and length were determined using SNP array studies. Results: For both patients, lymphocytes were heterozygous for the 46/1 haplotype, confirming that granulocyte 46/1 homozygosity was acquired. Direct sequencing of the JAK2 and GNE genes and SNP array analyses revealed homologous recombination of part of the JAK2 gene (exons 6–19, patient Na1061) and of the complete 46/1 JAK2 haplotype (patient Na1253). Furthermore, for both patients, full length sequencing of JAK2 cDNA revealed no additional mutation. In both cases, HR of wild-type JAK2 was associated with growth advantage and high expression of recombined JAK2. For both patients, further SNP array analyses revealed partial 9pUPD concerning <30% cells, which correlated with %JAK2 V617F and was consistent with 9pUPD having occurred after JAK2 V617F (Figure 1). The distortion of SNP allelic differences was higher at the telomeric end than in the centromeric region of chr. 9p. This indicated 2 distinct partial 9pUPDs for Na1061 and 1 partial 9pUPD for Na1253. Conclusion: Homologous recombination involving wild type JAK2 can precede JAK2 mutation and 9pUPD in MPN. Thus multiple paths and diverse alterations of the JAK2 gene can lead to MPN in individuals carrying the JAK2 GGCC haplotype. We propose a new model with JAK2 HR as early event, followed or not by JAK2 mutation, or/and JAK2 mutation(s) facilitating subsequent recombination resulting in 9pUPD and JAK2 V617F homozygosity. Disclosures: No relevant conflicts of interest to declare.

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Should We Screen for Janus Kinase 2 V617F Mutation in Cerebral Venous Thrombosis?

Cerebrovascular Diseases ◽

10.1159/000471891 ◽

2017 ◽

Vol 44 (3-4) ◽

pp. 97-104 ◽

Cited By ~ 5

Author(s):

Matthias Lamy ◽

Paola Palazzo ◽

Pierre Agius ◽

Jean Claude Chomel ◽

Jonathan Ciron ◽

...

Keyword(s):

Venous Thrombosis ◽

Cerebral Venous Thrombosis ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Janus Kinase 2 ◽

Jak2 Mutation ◽

Venous Thromboembolic Events ◽

V617f Mutation

Background: The presence of Janus Kinase 2 (JAK2) V617F mutation represents a major diagnostic criterion for detecting myeloproliferative neoplasms (MPN) and even in the absence of overt MPN, JAK2 V617F mutation is associated with splanchnic vein thrombosis. However, the actual prevalence and diagnostic value of the JAK2 V617F mutation in patients with cerebral venous thrombosis (CVT) are not known. The aims of this study were to assess the prevalence of JAK2 V617F mutation in a large group of consecutive CVT patients, to detect clinical, biological, and radiological features associated with the mutation, and to determine the long-term venous thrombosis recurrence rate in CVT patients with JAK2 mutation but without overt MPN in order to recommend the best preventive treatment. Methods: This was a prospective study conducted on consecutive patients with a first-ever radiologically confirmed CVT. JAK2 V617F mutation analysis was assessed in all the study subjects. JAK2 V617F-positive patients were followed up to detect new venous thrombotic events. Results: Of the 125 included subjects, 7 were found to have JAK2 V617F mutation (5.6%; 95% CI 2.3-11.2). Older age (p = 0.039) and higher platelet count (p = 0.004) were independently associated with JAK2 V617F positivity in patients without overt MPN. During a mean follow-up period of 59 (SD 46) months, 2 JAK2 V617F-positive patients presented with 4 new venous thromboembolic events. Conclusions: Screening for the JAK2 V617F mutation in CVT patients seems to be useful even in the absence of overt MPN and/or in the presence of other risk factors for CVT because of its relatively high prevalence and the risk of thrombosis recurrence.

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The mortality outcomes and survival pattern of patients of Myeloproliferative Neoplasms in Malaysia

10.21203/rs.3.rs-195078/v1 ◽

2021 ◽

Author(s):

yeeyee yap ◽

Jameela Sathar ◽

Kian Boon Law ◽

MPN registry working group

Keyword(s):

Diabetes Mellitus ◽

Bone Marrow ◽

Overall Survival ◽

Jak2 V617f ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Bone Marrow Fibrosis ◽

Survival Pattern ◽

V617f Mutation ◽

Marrow Fibrosis

Abstract Background: The prognostication of myeloproliferative neoplasm (MPN) has always been challenging even in the advent of Janus kinase 2 (JAK2 V617F) molecular studies. The survival pattern of MPN in a developing country such as Malaysia is still undetermined.Materials and Methods: This was a retrospective study using information from 774 patients from the National MPN Registry conducted from the year 2009 to 2015 in Malaysia. Patients with the diagnosis of essential thrombocythaemia (ET), polycythaemia vera (PV), primary myelofibrosis (PMF) and unclassified MPN (MPN-U) were included. Survival data were traced until December 2018. Results: The cohort consisted of 42.0% ET, 41.0% PV, 8.9% PMF and 8.1% MPN-U, with 48.8% Malay, 39.1% Chinese, 7.1% Indian, 5.0% Others. The subtypes analysis revealed that male MPNs was more than female MPNs except in ET. The Chinese ethnicity was associated with the highest incidence of ET. The mortality rate was the highest in PMF followed by MPN-U then PV and ET (p<0.0001). Survival analysis revealed that the overall survival differed significantly according to characteristics such as sex, sub-types, JAK2 V617F mutation, bone marrow fibrosis, presence of splenomegaly, diabetes mellitus, hypertension, and bleeding manifestation. Cox regression analysis identified age, haemoglobin level, sex, and subtype as a significant risk factor for mortality outcome. Conclusion: Patients with ET had the slightly better OS while PMF had the worst OS. This is in conjunction with low haemoglobin, worsening bone marrow fibrosis, splenomegaly, diabetes mellitus, hypertension and bleeding. JAK2 V617F mutation was seemingly resulting in inferior overall survival especially in ET and PMF. The survival outcome of the MPN registry is instrumental for future policy development of effective healthcare in Malaysia.

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JAK2-V617F mutation in patients with myeloproliferative neoplasms: Association with FLT3-ITD mutation

Srpski arhiv za celokupno lekarstvo ◽

10.2298/sarh1010614s ◽

2010 ◽

Vol 138 (9-10) ◽

pp. 614-618

Author(s):

Vesna Spasovski ◽

Natasa Tosic ◽

Tatjana Kostic ◽

Sonja Pavlovic ◽

Milica Colovic

Keyword(s):

Cell Proliferation ◽

Myeloproliferative Neoplasms ◽

Myeloid Cell ◽

Jak2 V617f ◽

Signalling Pathway ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Polycythaemia Vera ◽

Jak2 Mutation ◽

V617f Mutation

Introduction. An acquired somatic mutation V617F in Janus kinase 2 gene (JAK2) is the cause of uncontrolled proliferation in patients with myeloproliferative neoplasms. It is known that uncontrolled myeloid cell proliferation is also provoked by alteration in other genes, e.g. mutations in receptor tyrosine kinase FLT3 gene. FLT3 represents the most frequently mutated gene in acute myeloid leukaemia. Interestingly, mutated FLT3- ITD (internal tandem duplication) protein is a member of the same signalling pathway as JAK2 protein, the STAT5 signalling pathway. STAT5 activation is recognized as important for selfrenewal of haematopoetic stem cells. Objective. The aim of this study was the detection of JAK2- V617F mutation in patients with myeloproliferative neoplasms. Additionally, we investigated the presence of FLT3-ITD mutation in JAK2-V617F-positive patients in order to shed the light on the hypothesis of a similar role of these two molecular markers in haematological malignancies. Methods. Using allele-specific PCR, 61 patients with known or suspected diagnosis of myeloproliferative neoplasms were tested for the presence of JAK2-V617F mutation. Samples that were positive for JAK2 mutation were subsequently tested for the presence of FLT3-ITD mutation by PCR. Results. Eighteen of 61 analysed patients were positive for JAK2-V617F mutation. Among them, 8/18 samples were diagnosed as polycythaemia vera, and 10/18 as essential thrombocythaemia. None of JAK2-V617F-positive patient was positive for FLT3-ITD mutation. Conclusion. This study suggests that one activating mutation is sufficient for aberrant cell proliferation leading to malignant transformation of haematopoetic stem cell.

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Megakaryocytic morphology in Janus kinase 2 V617F positive myeloproliferative neoplasm

South Asian Journal of Cancer ◽

10.4103/2278-330x.208854 ◽

2017 ◽

Vol 06 (02) ◽

pp. 075-078

Author(s):

Shuchi Ghai ◽

Sharada Rai

Keyword(s):

Bone Marrow ◽

Myeloproliferative Neoplasms ◽

Myeloproliferative Neoplasm ◽

Jak2 V617f ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Fisher Exact Test ◽

Janus Kinase 2 ◽

Bone Marrow Biopsies ◽

V617f Mutation

Abstract Context: Alterations in megakaryocyte morphology are the hallmark of myeloproliferative neoplasms (MPNs). These neoplasm are also associated with Janus kinase 2 (JAK2) V617F mutation in nearly 95% patients with polycythemia vera (PV), 40% patients of essential thrombocythemia (ET) and 50% patients of myelofibrosis (MF). The utility of megakaryocyte morphology in these disorders in correlation with JAK2 V617F remains unresolved. Aims: The aim of the study was to assess the morphology of megakaryocytes in bone marrow aspirates (BMAs) and bone marrow biopsies of patients of BCR-ABL negative MPNs with JAK2 V617F mutation. Settings and Design: This study was a retrospective and prospective, hospital-based study undertaken for a period ranging from January 2011 to April 2015. Subjects and Methods: Assessment of morphological features of megakaryocytes in 15 BMAs and their respective biopsies which included seven cases of PV, three cases of ET, and five cases of MF with JAK2 V617F mutation. Statistical Analysis Used: Chi-square test and Fisher exact test were used to compare the different features of megakaryocytes. Software version SPSS 13.0 was used. Results: Megakaryocytes in ET were found to have characteristically large size with staghorn multinucleated nuclei and exhibiting large amount of cytoplasm. MF showed dense clustering of megakaryocytes with staghorn nucleus along with sinusoidal dilatation and intrasinusoidal hematopoiesis. PV showed loose and dense clustering of megakaryocytes with a predominance of cloud-like nuclei. Few of the megakaryocytic morphologic features showed overlap between MF and PV and between ET and early MF. Conclusions: Megakaryocytic morphology can aid in the accurate diagnosis of the different subcategories of MPNs. This would help in categorization of clinically suspicious patients of JAK2 V617F negative patients.

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Relationship between JAK2 V617F Mutation Status and Constitutive Mobilization of CD34-Positive Cells into Peripheral Blood in Patients with Chronic Myeloproliferative Disorder.

Blood ◽

10.1182/blood.v106.11.2586.2586 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2586-2586

Author(s):

Francesco Passamonti ◽

Elisa Rumi ◽

Emanuela Boveri ◽

Daniela Pietra ◽

Laura Vanelli ◽

...

Keyword(s):

Bone Marrow ◽

Cell Count ◽

Peripheral Blood ◽

Jak2 V617f ◽

Myeloproliferative Disorder ◽

Jak2 V617f Mutation ◽

Jak2 Mutation ◽

Mutation Status ◽

Cell Counts ◽

V617f Mutation

Abstract A gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been recently reported in patients with polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF) [N Engl J Med. 2005 Apr 28;352(17):1779–90]. Abnormal trafficking of CD34-positive cells with increased numbers in the peripheral blood is found in CIMF and in advanced stages of other myeloproliferative disorders. To determine whether the unique JAK2 V617F mutation affects the mobilization of CD34-positive cells into peripheral blood, we studied the relationship between JAK2 mutation status, bone marrow and circulating CD34-positive cells in 72 patients diagnosed according to the WHO criteria. A quantitative real-time polymerase chain reaction (PCR)-based allelic discrimination assay was used for the quantitative detection of the JAK2 V617F alleles in circulating granulocytes. Bone marrow CD34-positive cells were quantitatively assed on paraffin immunostained sections, while circulating CD34-positive cells were enumerated by flow cytometry using a single-platform assay. Overall, 57% of the patients studied carried the JAK2 V617F mutation. Within these patients, median values for JAK2 V617F alleles in circulating granulocytes were as follows: 29% in PV, 4% in ET, 12% in prefibrotic CIMF, 27% in fibrotic CIMF, and 99% in post-PV myelofibrosis. The vast majority of circulating granulocytes were homozygous for the mutation in all but one of patients with post-PV myelofibrosis. Decreased numbers of bone marrow CD34-positive cells and increased counts of circulating CD34-positive cells were detected in patients with fibrotic bone marrow. The higher the degree of fibrosis, the higher the circulating CD34-positive cell count (P<0.001) and the lower the bone marrow CD34-positive cell count (P<0.01). All patients with PV, ET and prefibrotic CIMF, and 7 out of 21 patients with fibrotic CIMF had circulating CD34-positive cell counts lower than 10 x 106/L. Conversely, all patients with post-PV myelofibrosis had counts higher than 10 x 106/L. In univariate analysis, there was an inverse relationship between percentage of JAK2 V617F alleles and bone marrow CD34-positive cells (r=−0.35, P<0.01), and a direct relationship between percentage of JAK2 mutant alleles and circulating CD34-positive cells (r=0.46, P=0.001). Multivariate analysis showed that disease category (P=0.0008) and percentage of JAK2 V617F alleles (P=0.03) were independently related to circulating CD34-positive cell counts. These observations suggest that the JAK2 V617F mutation might be involved in the constitutive mobilization of CD34-positive cells into peripheral blood that is found in patients with myeloproliferative disorder. Nonetheless, constitutive mobilization is present in a considerable portion of patients who do not carry the JAK2 mutation, pointing to additional pathogenetic mechanisms. Findings on patients with PV suggest that transition form heterozygosity to homozygosity for JAK2 V617F may represent an important step in the progression of PV to myelofibrosis. Thus, sequential evaluation of the percentage of JAK2 mutant alleles and enumeration of circulating CD34-positive cells may be useful for disease monitoring in PV.

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The Jak2-Inhibitor AG409 Affects Survival of BFU-E and CFU-GM of Myeloproliferative Disorder Cells Carrying the JAK2 V617F Mutation.

Blood ◽

10.1182/blood.v108.11.4907.4907 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4907-4907

Author(s):

Adrian Duek ◽

Ina Lichman ◽

Mordejai Shtalrid ◽

Alain Berrebi

Keyword(s):

Growth Factors ◽

Therapeutic Potential ◽

Dominant Role ◽

Jak2 V617f ◽

Myeloproliferative Disorder ◽

Growth Patterns ◽

Janus Kinase ◽

Jak2 V617f Mutation ◽

Jak2 Mutation ◽

V617f Mutation

Abstract In the last few years, point mutation of the Janus Kinase 2 (JAK2) gene (V617F) was found in the majority of patient with Polycythemia Vera and two related diseases ET and MMM. This mutation initiates constitutive activation of JAK2 transduction gene, inducing growth factor- independent hemopoiesis and plays a dominant role in origin of myeloproliferative disorder. Inactivation of this mutated gene may restore the normal hemopoiesis. Based on the evidence that the inhibition of the Jak-Stat axis by AG490 induced apoptosis in leukemic compared with normal cell, we attempt to analyzed the AG490 effect on the survival of MPD cells carrying JAK2 mutation in cultures. Peripheral blood (PB) samples were collected from 49 MPD patients (PV n=31, ET n=14, MMM n=4) at the time of diagnosis or during the follow up. The JAK2-V617F mutation tested by an allele-specific, semi-quantitative PCR was detected in 35 patients. MNPBC’s from four patients homozygous for the JAK2 mutation and from two normal volunteers were cultured in methylcellulose with 50 ng/ml SCF, 10ng/ml GM-CSF, 10ng/ml IL-3 and 3U/ml EPO, and in the same medium without growth factors. On the sixth day, 50 micromole of AG490 were added to the cultures and dishes with no addition of AG490 were kept for control. Colonies were counted on 6th day and on 14th day, and we checked the growth patterns on 14th day. Spontaneous colonies without growth factors were found only in the four MPD patients. Although, the number of the colonies was not significantly decreased after the addition of the AG490 inhibitor, we observed an obvious change in the normal growth pattern. The appearance of the colonies became darker reflecting an apoptotic process and death. Such a phenomenon was not seen in normal controls or in MPD cells without addition of the inhibitor. In conclusion our experiment clearly demonstrated preferential inhibition of MPD cells with JAK2 mutation by AG490, probably by induction of apoptosis. Our results suggest a therapeutic potential of inhibitors of JAK2 for the future.

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