Role of serum erythropoietin, erythropoietin-independent erythroid colony formation, and bone marrow assessment in the diagnosis of polycythemia vera in patients with JAK2 V617F mutation

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 7085-7085
Author(s):  
J. M. MacKenzie-Feder ◽  
C. Eaves ◽  
K. Lambie ◽  
A. Abdi-Ali ◽  
K. M. Ramadan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Polycythemia Vera ◽  
Jak2 V617f ◽  
World Health ◽  
Jak2 V617f Mutation ◽  
Number Of Patients ◽  
V617f Mutation ◽  
Minor Criteria ◽  
Mutant Cells ◽  
Serum Erythropoietin

7085 Background: The discovery of the JAK2 V617F mutation in over 95% of polycythemia vera (PV) patients has led to the development of the new 2008 World Health Organization diagnostic criteria for PV. These specify a requirement for an elevated hemoglobin (Hb, males: >185 g/L, females: >165 g/L) and either evidence of JAK2-mutant cells plus any one of the following minor criteria: a below normal level of serum erythropoietin (Epo), detectable Epo-independent erythroid colony (EEC) formation in vitro, or panmyelosis in the bone marrow, or two of the latter in the absence of detectable JAK2-mutant cells. However, in patients with an elevated Hb and JAK2V617F positivity, there are few data to determine whether any of the 3 minor criteria actually add an independent contribution to the diagnosis of PV. Methods: We performed a retrospective chart review of 77 patients who had an elevated Hb and were positive for the JAK2 V617F mutation and who also had a test for at least one of the 3 minor criteria (serum Epo level: n = 53; EEC formation: n = 66; bone marrow examination: n = 16) to determine the frequency of cases who might lack one or more of these. Results: Although the number of patients with a complete set of data was limited, the results, nevertheless, were sufficient to show that all 3 minor criteria were highly represented and all 77 of the patients analyzed (100%) were positive for at least one of them; i.e., 47 of 53 tested (89%) had a reduced serum Epo level; 65 of 66 tested (98%) had EECs and 15 of 16 tested (94%) had evidence of bone marrow panmyelosis. Conclusions: Neither the Epo level, nor the presence of EECs nor evidence of bone marrow panmyelosis provided additional diagnostic specificity in a population of 77 patients with both JAK2 V617F-positive cells and an elevated Hb. Consideration should be given to limiting serum Epo, EEC, and bone marrow assessments to JAK2 mutant-negative patients. No significant financial relationships to disclose.

High Frequency of Concurrent JAK2 V617F Mutation and BCR/ABL Fussion Gene in a Cohort (18/142) of Mexican Patients with MPD

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1766-1766 ◽  
Author(s):  
Rosa Maria Arana Trejo ◽  
Veronica A Gonzalez ◽  
Israel Saldivar ◽  
Beatriz Sðnchez ◽  
Yolanda Lugo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
High Frequency ◽  
Kinase Inhibitor ◽  
Fusion Gene ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Number Of Patients ◽  
V617f Mutation

Abstract Abstract 1766 The MPD are characterized by clonal neoplastic proliferation of one or more of the myeloid lineage in the bone marrow. The BCR/ABL fusion gene is a key genetic marker for CML an the mutation JAK2 V617F is present by over 95% of patients with PV and more than 50% of patients with ET and IMF. Both JAK2 V617F and BCR/ABL result in perturbation of tyrosine kinases and their downstream signaling pathway. The coexistence of BCR/ABL fusion gene and JAK2 V617F mutation have been described in some cases. In the differential diagnosis of MPD vs CML we performed both determinations BCR/ABL fusion gene and JAK2 V617F mutation and observed high frequency of the coexistence. Material and Methods: We performed 350 JAK2V617F and 1500 BCR-ABL PCR assays in LAOH, between January 2011 and July 2012, for the patients with suspected MPD vs CML that were sent from diferents hospitals. This cases without definitive diagnosis were maked both test and the cohort was of 142 patients. Peripheral blood or bone marrow samples were included of 56 cases with CML, 48 cases of isolated thrombocytosis, 34 cases in which total blood count disclosed elevation of ≥2 myeloid cell types and 4 cases with PV diagnosis. RNA and DNA were extracted using Trizol® reagent and Quiagen® commercial kit, the RT-PCR from b3a2/b2a2 BCR-ABL transcript was performed according to protocols standarized. For JAK2 V617F we using the allele-specific PCR method and the restiction enzyme method with BsaXI enzyme wich restriction site is present in exon 14 of JAK2. To validate the specificity of our result we sequenced the PCR products in 10 cases and two normal controls. Results. Of the 142 patients evaluated for both JAK2 V617F and BCR-ABL fusion gene, 18 patients were positive for both (12.7%). Ten with CML de novo, seven cases with ET, and one case in follow-up with imatinib. The average age of these JAK2 V617F mutation and BCR-ABL positive patients (n=18) was 63.5 (ranging from 45–82) and in patients JAK2 V617F mutation and BCR-ABL negative (n=124) were 58 years old (rank 18–85). The patient with CML in therapy with imatinib by one year, was present increase WBC; and his molecular analysis for JAK2 V617F was positive and also BCR/ABL+. The patient′s treatment regimen was modified, with initiation of hydroxyurea and imatinib; the patient achieved complete hematological response. Discussion. The others cases reports in the literature are most often a JAK2 V617F+ MPN developed in the setting of a previously diagnosed CML undergoing treatment with a tyrosine kinase inhibitor, or alternately, CML developed months to years after patients had been diagnosed with a JAK2 V617F+ MPN. In this report the most of our cases the BCR-ABL and JAK2 V617F positive cells were detected before the treatment. This studies have suggested two possibilities to account for expression of BCR-ABL and JAK2 V617F anomalies concomitant in a single patient. The first is that there are two clones each having BCR-ABL and JAK2 V617F mutation and another possibility is a single clone concurrently possesses both BCR-ABL and JAK2 V617F. It remains to be seen whether or not this patients would benefit from combined treatment with tyrosine kinase inhibitor and interferon or hydroxyurea. The clinical following up of this patients help us to response this questions. We have the highest frequency of concurrent JAK2 V617F mutation and BCR-ABL fusion gene reported, but is neccessary determined the true incidence in a greater number of patients with CML and MPD. Disclosures: No relevant conflicts of interest to declare.

Molecularly Confirmed Polycythemia Vera with Elevated Endogenous Serum Erythropoietin Level: Diagnostic Algorithms Revisited.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4964-4964
Author(s):  
Paul J. Thurmes ◽  
David P. Steensma
Keyword(s):  
Bone Marrow ◽  
Polycythemia Vera ◽  
Bone Marrow Biopsy ◽  
Arterial Oxygen Saturation ◽  
Jak2 V617f ◽  
The Body ◽  
Jak2 V617f Mutation ◽  
Arterial Oxygen ◽  
V617f Mutation ◽  
Dissociation Curves

Abstract Background: It is widely accepted that an elevated serum endogenous erythropoietin (EPO) level in a patient presenting with erythrocytosis and a genuinely increased red cell mass makes a diagnosis of polycythemia vera (PV) extremely unlikely (Mossuz et al Haematologica2004:1194; Tefferi and Gilliland Mayo Clin Proc2005:947.) However, in the absence of a definitive molecular marker for PV, there is often uncertainty, especially when PV-associated clinical features are present. Here we describe 4 patients presenting with Budd-Chiari syndrome (BCS) and erythrocytosis who had EPO levels well above the normal reference range (Levy et al Hepatology1985:858), yet all had the newly described JAK2 V617F point mutation in myeloid cells, confirming PV. Methods and Results: A 30-year-old man presented in March 2005 with ascites, mild splenomegaly, abnormal liver tests, and erythrocytosis (hemoglobin (Hb) 18.6 g/dL) with a normal platelet count (187 x 10(9)/L). BCS was diagnosed by Doppler ultrasonography, transjugular venogram, and liver biopsy. While bone marrow examination showed hypercellularity and megakaryocyte clustering consistent with chronic myeloproliferative disorder (CMPD), his EPO level was elevated to 32 U/L (normal 4–16 U/L), suggesting secondary erythrocytosis. Arterial oxygen saturation, Hb oxygen-dissociation curves (P50), molecular studies of the globin loci, Hb chromatography, and CT scans of the body were all normal. Granulocyte DNA demonstrated the JAK2 V617F mutation (heterozygous/mixed clonality), absent in buccal cells, confirming PV. Karyotype was normal and BCR/ABL was not present. The patient was treated with phlebotomy, warfarin, and hydroxyurea. One month later, his EPO level was markedly reduced at 13 U/L. We hypothesized that since hepatocytes express low levels of EPO, a remnant of fetal erythropoiesis, acute necrosis from BCS could lead to transient EPO surge even in the presence of chronic EPO suppression from PV. We then reviewed clinical data from all 33 patients at our institution since 1995 with BCS that was ultimately believed to be associated with PV. We identified 13 such patients in whom endogenous EPO levels were measured. Most had a low EPO level at the time of diagnosis (range of <1.5 to 9 U/L), but 3 of these patients were found to have an elevated EPO level. DNA was extracted from archival samples from these 3 patients; JAK2 V617F mutation was detected in all 3. Clinical presentations were as follows: Patient 1: 21-year-old woman with BCS and pulmonary embolus, Hb 15.9 mg/dL, EPO 28 U/L. Bone marrow biopsy was consistent with PV. Subsequent EPO level was 19U/L in the setting of progressive liver failure. Patient 2: 48-year-old woman with known PV receiving intermittent phlebotomy presented with BCS, Hb 17.2 mg/dL, EPO 25 U/L. Marrow was consistent with CMPD. Patient 3: 33-year-old woman presented with BCS, Hb 18.8 mg/dL, EPO 53 U/L. Bone marrow biopsy showed early CMPD. Conclusion: Elevated EPO levels do not exclude the diagnosis of PV, especially in the presence of BCS with hepatic necrosis. These cases highlight the continuing challenges in accurate PV and CMPD diagnosis and emphasize the importance of complementing clinical assessment with molecular genetic testing, including JAK2 mutation analysis.

V617F JAK2 Mutation and Bone Marrow Fibrosis Define Subgroups Of Patients With Polycythemia Vera and Essential Thrombocythemia With Shared Clinicobiological Profiles

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5268-5268
Author(s):  
Panagiotis Baliakas ◽  
Vassiliki Douka ◽  
Michalis Iskas ◽  
Tasoula Touloumenidou ◽  
Angeliki Paleta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Jak2 V617f ◽  
Age At Diagnosis ◽  
Advanced Age ◽  
Jak2 V617f Mutation ◽  
Hydroxyurea Treatment ◽  
V617f Mutation ◽  
Wbc Count

Abstract The JAK2 V617F mutation is of high diagnostic value in the evaluation of myeloproliferative neoplasms (MPN) as it helps to document clonality; in addition, it may also predict for response to hydroxyurea treatment. According to recent studies, the presence of bone marrow (BM) fibrosis at diagnosis may be associated with the clinical evolution of MPNs, in particular development of secondary acute myeloid leukemia (AML) or transformation to myelofibrosis (MF), however the underlying mechanisms remain unknown. In this study we characterized in detail subgroups of patients with Polycythemia Vera (PV) and Essential Thrombocythemia (ET) carrying the JAK2 V617F mutation (M-JAK2) or displaying BM fibrosis at diagnosis with the ultimate aim of identifying potential associations and/or overlapping phenotypes. The present single-institution patient cohort included 118 cases diagnosed according to WHO 2008 criteria. Patient characteristics were as follows: (i) Diagnosis: PV/ET, 37/82; (ii) Gender: male/female, 58/60; (iii) median age at diagnosis: 59.8 years (range, 25-90). M-JAK2 was detected in 86/118 (72.9%) cases [PV: 32/37 (86.5%) - ΕΤ: 54/82 (65%)]. BM fibrosis was observed in 28/112 (25%) cases [PV: 10/34 (29.4%), ΕΤ: 18/78 (23%)], grade I in 24/28 (85%) cases and grade II in 4 cases (all with ΕΤ). Thirteen patients without BM fibrosis at diagnosis underwent a second BM biopsy at a median time of 4.7 years (range, 1-10): BM fibrosis was observed in 5/13 (38.4%), 4 carrying M-JAK2, of whom only one had received anagrelide before the second BM biopsy. With a median follow up of 6 years (range 1-10), one of these five patients developed AML. There was no statistically significant association between M-JAK2 and BM fibrosis at diagnosis, neither in the entire cohort, nor in each MPN (ET or PV) separately. In PV: (i) M-JAK2 was significantly (p<0.05) associated with advanced age at diagnosis, increased hemoglobin levels (Hb) and white blood cell (WBC) count at diagnosis; (ii) the presence of BM fibrosis demonstrated a strong trend for correlation with increased platelet counts at diagnosis (p=0.08). In ΕΤ: (i) M-JAK2 was significantly (p<0.05) associated with advanced age at diagnosis, splenomegaly, increased WBC count and Hb levels at diagnosis, and increased incidence of thrombotic events (12/54 versus 1/28); (ii) BM fibrosis was correlated with increased WBC and platelet count at diagnosis. Neither M-JAK2 nor BM fibrosis were correlated with increased incidence of hemorrhagic events, development of secondary AML or the presence of other concurrent malignancy. Furthermore, neither of these two parameters had any impact on overall survival, it has to be noted though that patients were not treated uniformly. In conclusion, the present analysis did not document a statistically significant correlation between M-JAK2 and BM fibrosis. Nonetheless, the clinicobiological similarities of patient subgroups defined by either of these parameters, as well as the increased incidence of BM fibrosis in sequential BM samples amongst M-JAK2 patients are suggestive of common pathogenetic mechanisms. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The study of Jak2 V617F mutation in polycythemia vera with zebrafish model

Cell Research ◽  
2008 ◽  
Vol 18 (S1) ◽  
pp. S141-S141
Author(s):  
Alvin CH Ma ◽  
Alice MS Cheung ◽  
Alister C Ward ◽  
Wing-Yan Au ◽  
Yok-Lam Kwong ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Zebrafish Model ◽  
V617f Mutation

Relation between JAK2 (V617F) mutation status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral blood in myeloproliferative disorders

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3676-3682 ◽  
Author(s):  
Francesco Passamonti ◽  
Elisa Rumi ◽  
Daniela Pietra ◽  
Matteo G. Della Porta ◽  
Emanuela Boveri ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Gene Dosage ◽  
Jak2 V617f ◽  
Myeloproliferative Disorders ◽  
Jak2 V617f Mutation ◽  
Disease Category ◽  
Mutation Status ◽  
Cell Counts ◽  
Cd34 Cells ◽  
V617f Mutation

We studied the relationship between granulocyte JAK2 (V617F) mutation status, circulating CD34+ cells, and granulocyte activation in myeloproliferative disorders. Quantitative allele-specific polymerase chain reaction (PCR) showed significant differences between various disorders with respect to either the proportion of positive patients (53%-100%) or that of mutant alleles, which overall ranged from 1% to 100%. In polycythemia vera, JAK2 (V617F) was detected in 23 of 25 subjects at diagnosis and in 16 of 16 patients whose disease had evolved into myelofibrosis; median percentages of mutant alleles in these subgroups were significantly different (32% versus 95%, P < .001). Circulating CD34+ cell counts were variably elevated and associated with disease category and JAK2 (V617F) mutation status. Most patients had granulocyte activation patterns similar to those induced by administration of granulocyte colony-stimulating factor. A JAK2 (V617F) gene dosage effect on both CD34+ cell counts and granulocyte activation was clearly demonstrated in polycythemia vera, where abnormal patterns were mainly found in patients carrying more than 50% mutant alleles. These observations suggest that JAK2 (V617F) may constitutively activate granulocytes and by this means mobilize CD34+ cells. This exemplifies a novel paradigm in which a somatic gain-of-function mutation is initially responsible for clonal expansion of hematopoietic cells and later for their abnormal trafficking via an activated cell progeny.

scholarly journals JAK1 and Tyk2 Activation by the Homologous Polycythemia Vera JAK2 V617F Mutation

2005 ◽  
Vol 280 (51) ◽  
pp. 41893-41899 ◽  
Author(s):  
Judith Staerk ◽  
Anders Kallin ◽  
Jean-Baptiste Demoulin ◽  
William Vainchenker ◽  
Stefan N. Constantinescu
Keyword(s):  
Polycythemia Vera ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
V617f Mutation

Relationship between JAK2 V617F Mutation Status, Granulocyte CD177 mRNA Expression and CD177 Soluble Protein Level in Patients with Polycythemia Vera.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2578-2578
Author(s):  
Daniela Pietra ◽  
Alessandra Balduini ◽  
Carmela Marseglia ◽  
Matteo G. Della Porta ◽  
Luca Malcovati ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Polycythemia Vera ◽  
Protein Level ◽  
Mononuclear Cells ◽  
Jak2 V617f ◽  
Myeloproliferative Disorders ◽  
Jak2 V617f Mutation ◽  
Serum Samples ◽  
Close Relationship ◽  
V617f Mutation

Abstract A unique gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been recently described in patients with polycythemia vera (PV), essential thrombocythemia and chronic idiopathic myelofibrosis [N Engl J Med. 2005 Apr 28;352(17):1779–90]. Although the currently available data clearly demonstrate that the JAK2 V617F mutation participates in the pathogenesis of myeloproliferative disorders, the mutation’s precise place in the hierarchical order of pathogenetic events remains to be established. We have recently reported that altered gene expression in myeloproliferative disorders correlates with activation of signaling by the V617F mutation of JAK2 (Blood. 2005 Aug 4; Epub ahead of print). Granulocyte CD177 (PRV1) mRNA overexpression has been initially reported as a potential marker of PV but later shown by us to rather be a marker of neutrophil activation [Br J Haematol. 2004 Sep;126(5):650–6]. In this study, we analyzed the relationship between JAK2 V617F mutation status, granulocyte CD177 mRNA expression and CD177 soluble protein level in 72 patients with PV. We also investigated the ontogeny of CD177 expression by hematopoietic cells with the aim of defining the stage of mRNA expression during myeloid, erythroid and megakaryocytic cell differentiation. Finally we studied the effect of soluble CD177 protein on hematopoietic cell proliferation and differentiation. Granulocyte CD177 mRNA expression and percentage of JAK2 V617F alleles were evaluated by quantitative Real Time PCR (qRT-PCR), while serum CD177 protein level was measured by a flow cytometry-based competitive antibody-binding assay. Liquid cultures were performed by culturing peripheral blood mononuclear cells obtained from healthy individuals and PV patients in the presence of high CD177-expressing, low CD177-expressing or CD177-depleted sera. After 12 days of culture, cells were collected, counted and evaluated for colony growth, and for flow cytometry analysis of myeloid, erythroid, megakaryocytic and CD34-positive cell subpopulations. qRT-PCR studies showed a close relationship between CD177 mRNA level and percentage of JAK2 V617F alleles (r=0.412, P&lt;0.001). CD177 mRNA expression was almost undetectable in cell populations other than granulocytes. Studies of CFU-GM growth and differentiation indicated that CD177 mRNA expression is a late event restricted to the neutrophil stage of differentiation. Analysis of serum samples showed variable values for mean fluorescence intensity (MFI), indicating variable levels of the soluble CD177 protein in the patients studied. A very close relationship was found between granulocyte CD177 mRNA expression and soluble CD177 protein level (r=0.56, P=0.02). Incubation of mononuclear cells with serum samples showing high levels of soluble CD177 protein resulted in increased numbers of CD34-positive cells (P&lt;0.02) and of erythroid progenitors (P&lt;0.03). This effect was not detectable when low CD177-expressing or CD177-depleted sera were employed. These observations clearly indicate that the JAK2 V617F mutation is associated with enhanced granulocyte CD177 mRNA expression, and that this latter results in high levels of soluble CD177 protein. These elevated levels might contribute to the increased red cell production that characterizes polycythemia vera.

In Vitro Expansion of Polycythemia Vera Progenitors Favors Expansion of Erythroid Precursors without JAK2 V617F Mutation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3506-3506 ◽  
Author(s):  
Josef T. Prchal ◽  
Ko-Tung Chang ◽  
Jaroslav Jelinek ◽  
Yongli Guan ◽  
Amos Gaikwad ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Polycythemia Vera ◽  
Peripheral Blood ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Erythroid Progenitors ◽  
Erythroid Precursors ◽  
In Vitro Expansion ◽  
V617f Mutation

Abstract A single acquired point mutation of JAK2 1849G&gt;T (V617F), a tyrosine kinase with a key role in signal transduction from growth factor receptors, is found in 70%–97% of patients with polycythemia vera (PV). In the studies of tyrosine kinase inhibitors on JAK2 1849G&gt;T (see Gaikwad et all abstract at this meeting) we decided to study the possible therapeutic effect of these agents using native in vitro expanded cells from peripheral blood. To our surprise, the in vitro expansion of PV progenitors preferentially augmented cells without JAK2 1849G&gt;T mutation. We used a 3 step procedure to amplify erythroid precursors in different stages of differentiation from the peripheral blood of 5 PV patients previously found to be homozygous or heterozygous for the JAK2 1849G&gt;T mutation. In the first step (days 1–7), 106/ml MNCs were cultured in the presence of Flt-3 (50 ng/ml), Tpo (100 ng/ml), and SCF (100 ng/ml). In the second step (days 8–14), the cells obtained on day 7 were re-suspended at 106/ml in the same medium with SCF (50 ng/ml), IGF-1 (50 ng/ml), and 3 units/ml Epo. In the third step, the cells collected on day 14 were re-suspended at 106/ml and cultured for two more days in the presence of the same cytokine mixture as in the step 2 but without SCF. The cultures were incubated at 37oC in 5% CO2/95% air atmosphere and the medium renewed every three days to ensure good cell proliferation. The expanded cells were stained with phycoerythrin-conjugated anti-CD235A (glycophorin) and fluorescein isothiocyanate-conjugated anti-human-CD71 (transferrin receptor) monoclonal antibodies and analyzed by flow cytometry. The cells were divided by their differential expression of these antigens into 5 subgroups ranging from primitive erythroid progenitors (BFU-Es and CFU-Es) to polychromatophilic and orthochromatophilic erythroblasts; over 70% of harvested cells were early and late basophilic erythroblasts. The proportion of JAK2 1849G&gt;T mutation in clonal PV granulocytes (GNC) before in vitro expansion and in expanded erythroid precursors was quantitated by pyrosequencing (Jelinek, Blood in press) and is depicted in the Table. These data indicate that in vitro expansion of PV progenitors favors expansion of erythroid precursors without JAK2 V617F mutation. Since three PV samples were from females with clonal granulocytes, erythrocytes, and platelets, experiments were underway to determine if the in vitro expanded erythroid cells were clonal PV cells without JAK2 V617F mutation, or derived from polyclonal rare circulating normal hematopoietic progenitors. The Proportion of JAK2 T Allele Patients GNC T Allele (%) Expanded Cells T Allele (%) PV1 (Female) 81 10 PV2 (Male) 77 28 PV3 (Male) 44 42 PV4 (Female) 78 19 PV5 (Female) 78 28

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