JAK2V617F-negative Polycythemia Vera Research Articles

JAK2 exon 12 mutations in cases with JAK2V617F-negative polycythemia vera and primary myelofibrosis.

Molecular detection of JAK2 mutation (V617F or exon 12) is included as a major diagnostic criterion for polycythemia vera (PV) by the WHO 2016 guidelines. JAK2 exon 12 mutations are seen in about 2-5% of JAK2V617F-negative cases of PV. Mutations in JAK2 cause constitutive activation of JAK-STAT pathway which results in variable phenotypes. PV patients with exon 12 mutations in JAK2 present characteristically with erythrocytosis. There are limited reports describing the spectrum of JAK2 exon12 mutations in myeloproliferative neoplasms (MPNs). Here, we describe the characteristics of a series of MPN patients with mutations in exon 12 of JAK2 of which two were novel variants associated with polycythemia. Interestingly, we noted two patients presenting as myelofibrosis having JAK2 exon 12 mutations.

A Rare Hemoglobinopathy as a Cause of Erythrocytosis in a Patient with Suspected JAK2V617F Negative Polycythemia Vera: A Case Report

Introduction: Differential diagnosis of polyglobulia/erythrocytosis which is caused by deregulated erythropoiesis with an overproduction of red blood cells resulting in elevated hemoglobin and hematocrit levels is a diagnostic challenge. Case Report: A 31-year-old man was referred to us with a suspected diagnosis of polycythemia vera (a clonal myeloproliferative neoplasm) because of erythrocytosis. One year before, he suffered from an ischemic central retinal vein occlusion of unknown cause. Aspirin treatment was initiated. His mother was diagnosed with a JAK2V617F negative polycythemia vera years earlier and treated with phlebotomies as well as aspirin. Apart from erythrocytosis, laboratory analysis showed a normal white blood cell and platelet counts. The differential blood picture and lactate dehydrogenase were within the normal ranges. Molecular testing for BCR-ABL, JAK2V617F and Calreticulin gene mutations by PCR was negative. Bone marrow biopsy was normal without signs for a myeloproliferative neoplasm. As almost all patients with polycythemia vera carry the phenotype-driver mutation JAK2V617F, further genetic testing for congenital causes of erythrocytosis was conducted. Mutations in the EPO-receptor gene were not found, but a very rare heterozygous point mutation in the beta-globin-chain [exon 2 (c.119A>C) leading to a change in codon 40 (CAG>CCG)] was detected by next generation sequencing. This rare variant belongs to the high oxygen affinity hemoglobinopathies leading to a reduction of oxygen supply in tissues and an increase in red cell production. Conclusion: The diagnosis of a JAK2 mutation negative polycythemia vera must always be questioned and congenital causes of erythrocytosis excluded as the therapeutic and prognostic consequences are immense. Rare variants of hemoglobinopathies, particularly those with high oxygen affinity need to be excluded by molecular testing.

Detection of JAK2 Exon 12 Mutations in JAK2 V617F-Negative Polycythemia Vera Patients by Cloning Technique

Introduction: The identification of mutations of the JAK2 gene is a useful marker in the diagnosis of polycythemia vera (PV) patients. We studied the frequency of JAK2 mutations in a group of PV patients because data are still very limited regarding this subject in Polish patients. Methods: The JAK2 V617F mutation was examined using the amplification refractory mutation system (ARMS)-PCR method. Direct sequencing and a cloning technique were performed to determine alternations in exon 12 of the JAK2 gene. Results: A group of 90 consecutive patients with a suspected diagnosis of polycythemia vera were investigated. In 91% of the cases, the JAK2 V617F mutation was identified. The remaining JAK2 V617F-negative patients were subjected to examination for JAK2 exon 12 by direct PCR product sequencing and the cloning technique. The following mutations were identified: H538-K539delinsL, E543-D544del and N542-E543del. These exon 12 mutants constituted 50% of PV JAK2 V617F-negative group and 4.4% (out of 90) of all PV patients (JAK2 V617F-positive and JAK2 V617F-negative). Conclusion: Our results demonstrate the prevalence of JAK2 mutations (V617F and in exon 12) in PV cases. Moreover, the data show that direct sequencing is not an adequate technique for exon 12 mutation identification; therefore, appropriate methodology should be considered for using this molecular marker in the process of diagnosis.

MPL p.S204P Is a Recurrent Mutation in Essential Thrombocythemia

Introduction: The JAK2 p.V617F, MPL p.W515K/L and CALR indels occur in a mutually exclusive pattern in 80-90% of cases with Essential Thrombocythemia (ET), but the driver mutations are unknown in the remaining 10-20%. In this study we aimed to identify driver mutations in the latter group of triple negative (TN) ET by exome sequencing of 10 such cases.Results: We found 27 somatic variants, including indels, in 6 out of 10 TN ET patients (range: 1-10 mutations/case; mean: 2,7 mutations/case), none of which were recurrent. In one case, we found a MPL c.610T>C (p.S204P) mutation, which is located in the extracellular domain of the MPLreceptor. By Sanger sequencing of MPL exon 4 in 20 additional TN ET cases, an additional patient with the MPL S204P mutation was identified. Moreover, this mutation was previously reported in one case with idiopathic myelofibrosis1.In order to study the effect of this mutation on the function of MPL, we produced stable Ba/F3 cell lines expressing MPL S204P, MPL W515K or MPL WT, and assessed the dependence of their growth on exogenous thrombopoietin (TPO). Only MPL W515K transduced Ba/F3 cells proliferated in the absence of TPO, but growth of MPL S204P Ba/F3 and of MPL WT Ba/F3 could be rescued by exogenous TPO, indicating the proper surface expression and the functionality of the transduced receptors. The levels of phospho-JAK2 and phospho-STAT5 were low in cytokine-deprived MPL S204P cells but increased upon TPO stimulation. In contrast, phospho-JAK2 and phospho-STAT5 were detectable in MPL W515K transduced Ba/F3 in the absence of cytokines as assessed by Western blotting. Culture of MPL S204P transduced Ba/F3 in the presence of TPO over a range of concentrations (0,01-10 ng/ml) yielded growth curves comparable with MPL WT transduced Ba/F3.Using flow cytometry, we also explored cell surface marker expression on peripheral blood platelets from the two MPL S204P ET patients. Data were compared with healthy donors or ET patients with JAK2 or CALR mutations. MPL S204P ET platelets displayed higher expression of CD61 than platelets from healthy donors or from JAK2 or CALR mutated ET (p<0,01). In addition, there was a trend for higher expression of KIT, CD36 and CD42b on platelets from the MPL S204P ET cases. Moreover, following platelet activation through the protease activated receptor 1, the degranulation response of platelets from MPL S204P ET was decreased in comparison with JAK2 or CALR mutated ET.Conclusion: The MPL S204P mutation is a recurrent mutation in TN ET, with a frequency of 7% (2/30) in this series, but this mutation does not induce TPO-independent growth nor increased TPO-sensitivity in Ba/F3 cells. However, preliminary phenotypic and functional evidence supports the notion that MPL S204P platelets display specific characteristics as compared with JAK2 or CALR mutated ET. The mechanisms by which the MPL S204P mutation influences megakaryopoiesis and platelet function remain to be elucidated.1. Williams DM, et al. Phenotypic variations and new mutations in JAK2 V617F-negative polycythemia vera, erythrocytosis, and idiopathic myelofibrosis. Exp Hematol 2007; 35: 1641. DisclosuresGraux:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Presence of Calreticulin Mutations in JAK2-Negative Polycythemia Vera

Calreticulin (CALR) mutations have recently been reported in JAK2- and MPL-negative Myeloproliferative Neoplasms (MPN), particularly essential thrombocythemia (ET) and primary myelofibrosis (PMF).The clinical course of sporadic CALR-mutated patients seems to be more indolent than that of JAK2-mutated patients. In contrast, no CALR mutation has been found in the 647 published cases of Polycythemia Vera (PV) patients tested. Consequently, CALR mutations were considered exclusive to JAK2 and MPL mutations. Since 98% of PV patients harbor a JAK2 mutation (mostly the V617F mutation in exon 14 and more rarely, in exon 12), the absence of CALR mutations in PV seemed logical. Here, we describe two JAK2V617F-negative PV patients who presented with a CALR mutation at the time of diagnosis.Patient # 1 had hemoglobin at 168 g/L, hematocrit at 51.3%, and increased red cell mass (RCM) at 128% associated with a normal erythropoietin level. The bone marrow biopsy showed hypercellularity for age, panmyelosis associated with normal megakaryocytes and rare isolated abnormal enlarged forms. Using reticulin stain, no myelofibrosis was noted. Patient # 2 had hemoglobin at 194 g/L, hematocrit at 53% and low erythropoietin level without any dehydration. Both had moderately elevated platelet counts (658 and 575 x109/L respectively) with normal leukocyte counts. They were negative for BCR-ABL. No mutation was found in JAK2 exons 12, 13 and 14 by HRM and allele-specific real-time PCR or in MPL exon 10. Using HRM analysis, CALR mutations were suspected in both patients and confirmed using Sanger sequencing and product sizing analyses: CALR mutations were in both patients type 1 deletions (52-bp deletion; c.1092_1143del). To complete genomic tests made on peripheral blood granulocytes, we performed colony assays in methylcellulose and in collagen, picked single BFU-E colonies grown after 14 days in the presence of erythropoietin, and genotyped each colony individually for CALR. Of the 27 colonies genotyped, 6 had no PCR amplification and 21 harboured the same CALR mutation observed in peripheral blood granulocytes, i.e 52-bp deletion; c.1092_1143del. BFU-E were found heterozygous for CALR, with a mean allele burden of 49%. To our knowledge, these patients are the first cases of CALR-mutated PV to be reported. However, since a biclonalJAK2V617F and CALR MPN case recently reported, we cannot rule out the possibility of a biclonal disease involving a yet unknown mutation associated with a CALR mutation. On the other hand, the presence of a CALR mutation both in peripheral granulocytes and in BFU-E suggests that the CALR mutation plays a role in the polycythemia phenotype. Our observations highlight the fact that in the absence of JAK2 mutation, CALR mutations can also be associated with PV.In conclusion, our data indicate that testing JAK2-negative PV patients for CALR mutations may be useful. DisclosuresNo relevant conflicts of interest to declare.

High-Resolution Melting (HRM) Curve Analysis Is a Reliable and Relevant Tool In JAK2V617F-Negative Myeloproliferative Neoplasms

IntroductionThe JAK2V617F mutation is observed in more than 95% of patients with Polycythemia Vera (PV), and 50–60% of patients with Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF) and Refractory Anaemia with ringed Sideroblasts with marked Thrombocytosis (RARS-T). As a consequence, for JAK2V617F-negative cases it is worth looking for other mutations in the JAK2 and MPL genes as they are an important diagnostic aid for the myeloproliferative neoplasms (MPN). For example, half of JAK2V617F-negative PV patients carry mutations in the exon 12 of JAK2. To date, 40 different JAK2 exon 12 mutants have been reported. All are located between aa536 and aa547 and include deletions, insertions, and duplications. In the same way, nearly 5% and 10% of JAK2V617F-negative ET and PMF carry mutations in exon 10 of MPL, mainly affecting codons S505 and W515. Techniques such as allele-specific polymerase chain reaction (AS-PCR) are inappropriate for screening and sequencing, including Next Generation Sequencing (NGS), is time consuming as a first screening. High-resolution melting (HRM) curve analysis allows a rapid and whole exon mutation scanning approach associated with excellent sensitivity. The goal of our study was to evaluate the relevance of HRM in JAK2V617F-negative MPN and RARS-T in 3 French hospitals. MethodsHRM is based on the analysis of fusion curves of a short amplification product of PCR. This procedure, which derives PCR in real time and uses a fluorescent agent intercalated DNA, makes it possible to obtain a good resolution and to detect different types of mutations. The analyses were carried out on DNA from purified blood granulocytes. Diagnosis of PV, ET, PMF and RARS-T was established according to the 2008 WHO criteria. All patients were tested for their JAK2V617F status. To evaluate the efficiency of HRM in a routine diagnostic setting, we investigated 1997 patients with suspicion for a MPN from 3 French university hospitals. Samples with abnormal HRM curves were sequenced in order to confirm and characterize the mutations. ResultsFor 37/881 (4.2%) JAK2V617F-negative IE and PV patients, the HRM curve raised suspicion of the presence of a mutation in JAK2 exon 12, all confirmed by sequencing: the most frequent mutations were H538Q K539L (n=6), R541 E543 delins K (n=6), N542 E543 del (n=5), F537–I546 dup 10_F547L (n=4). For 2 additional PV cases, an abnormal HRM curve was obtained for JAK2 exon 14. Sequencing analysis revealed a V617F mutation with additional mutation of base 1831 (1831T>G), changing leucine 611 for a valine (L611V). For the second PV patient, a silent mutation corresponding to a substitution of a cytosine by a thymine in position 1848 of exon 14 of JAK2 was observed (C616), also associated with a V617F mutation. In both cases the JAK2V617F mutation had not been detected because the primers of the JAK2V617F AS-qPCR assay covered the sequence coding for L611 and C616. In addition, mutations in JAK2 exons 13 and 15 were tested on 33 PV-suspected samples: only one PV with a JAK2 exon 15 mutation was noted. Regarding the detection of mutations in exon 10 of MPL, an abnormal HRM curve was noted for 59/1116 (5.3%) MPN suspected. The mutations found were W515L (31 ET, 4 PMF), W515K (8 ET, 3 PMF), W515A (2 ET), W515R (1 ET, 1 PMF), W515S (1 ET), S505N (4 ET, 2 PMF, 1 RARS-T), V501A (1 ET). ConclusionIn ET and PMF, the HRM approach offers the advantage of quickly and reliably assessing patients for W515 and S505 mutations. In JAK2V617F-negative PV, due to the large number of possible mutations, HRM is a highly potent screening technique and allows a rapid scan of the whole exon 12, leading secondarily to sequence only a small number of HRM positive cases. Moreover, in 2 PV, HRM analysis of JAK2 exon 14 allowed to detect a V617F mutation hidden by a second mutation that inhibited primer hybridization and subsequent DNA amplification in the routine AS-qPCR assay. In summary, HRM is a highly relevant technique, cheaper than NGS, for routine molecular diagnosis of JAK2V617F-negative MPN, which typically carry a large variety of mutations, expressed at low levels. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Red Cell Mass (RCM) Value In Polycythaemia Vera Disease In The JAK2 V617F Era

IntroductionIn Polycythaemia Vera (PV), the RBC lineage is involved with increased haemoglobin, RBC count and haematocrit. WHO diagnostic criteria for PV are JAK2 V617F mutation and elevated red cell mass (RCM) > 25% of mean normal value. In addition, tests of marrow hypercellularity, blood erythropoietin and colony formation, are minor criteria. However, the diagnostic role of RCM test is still controversial and requires clarification. In this work, PV patients who had both an RCM study and JAK2 V617F mutation test, and routine laboratory tests, are evaluated to check if RCM was essential in the diagnostic work up for PV. MethodsOver 2 years, 75 patients with abnormal haematocrit (men ≥ 0.50, women ≥ 0.45) had RCM and JAK2 V617F mutation tests (except JAK2 exon 12 mutation). All subjects consented to the study approved by the ethics committee. RCM was done by Cr-51 RBC radiolabeling method (no prior venesection at least 1 month). Statistical analysis involved descriptive statistics and chi-square test. ResultsThere were 71 males and 4 females, mean age 46 y (range 17-75 y). Increased RCM was found in 41/75 (55%). Positive JAK2 V617F was found in 13/75 patients (17%), who also had RCM above the mean normal predicted value, however, when the WHO RCM criteria were applied, only 7/13 (54%) could be considered as having “truly” increased RCM. In the patient group with negative JAK2 V617F test, 12/28 (43%) had RCM results as per WHO criteria. There was no statistical association between presence of JAK2 V617F and the RCM values. ConclusionIn patients with negative JAK2 V617F but with high clinical suspicion for PV and all other causes of secondary and idiopathic erythrocytosis excluded, an increase in RCM would support the diagnosis of PV (about 10 % PV cases). In patients with JAK2 positive mutation and high haematocrit but RCM below the WHO cut-off level, an increased RCM would still count to confirm the diagnosis as the current standard level seems too stringent. ReferencesJames C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C et al. A unique clonal JAK2 mutation leadingto constitutive signaling causes polycythaemia vera. Nature 2005; 434(7037): 1144-8.Kralovics R, Passamonti F, Buser AS, Soon-Siong T, Tiedt R, Passweg JR, et al. A Gain-of-Function Mutation of JAK2 in Myeloproliferative Disorders. Merck Manual of Diagnosis and Therapy. 16th Edition, 1992McMullin MF, Bareford D, Campbell P, Green AR, Claire Harrison C, Hunt B, Oscier D, et al. Guidelines for the diagnosis, investigation and management of polycythaemia/erythrocytosis. British Journal of Haematology 2005;130(2): 174-95.Scott LM, Tong W, Levine RL, et al. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. NEngl J Med. 2007;356:459-468.Pardanani A, Lasho TL, Finke C, et al. Prevalence and clinicopathologic correlates of JAK2 exon 12 mutations in JAK2V617F-negative polycythemia vera. Leukemia. 2007;21:1960-1963.Pancrazzi A, Guglielmelli P, Ponziani V, et al. A sensitive detection method for MPLW515L or MPLW515K mutation in chronic myeloproliferative disorders with locked nucleic acid-modified probes and real-time polymerase chain reaction. J Mol Diagn. 2008;10:435-441. Disclosures:No relevant conflicts of interest to declare.

Integrated Genetic Profiling Of JAK2 Wildtype Chronic-Phase Myeloproliferative Neoplasms

BackgroundThe Myeloproliferative Neoplasms (MPNs), including Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF) are clonal hematopoietic disorders. JAK2V617F mutations are observed in approximately 90-95% of PV cases, but only 40-50% of ET and PMF cases. Although JAK2 exon 12 and LNK mutations are observed in the majority of JAK2V617F-negative PV patients, candidate gene and exome sequencing studies to date have not identified activating oncogenes in the majority of JAK2V617F-negative ET/PMF patients. Thus, further genetic investigations are needed to define the mutational architecture of these JAK2 wildtype MPNs in order to gain insight into the biology of these diseases, the clinical implications of genetic events that do occur, and the elucidation of potential therapeutic targets. AimsTo characterize the spectrum of genetic alterations in JAK2 wildtype chronic-phase myeloproliferative neoplasms. MethodsWe identified 32 patients with a confirmed diagnosis of an MPN (per 2008 WHO criteria), including MF, PV and ET, who were negative for JAK2V617F using a CLIA-certified allele specific assay for the JAK2 disease allele. Genomic DNA and total RNA was isolated from formalin fixed paraffin embedded (FFPE) tissue, blood and bone marrow aspirates. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >590X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. ResultsHigh coverage sequencing allowed us to identify JAK2V617F mutations in two patients (allele burden 3-5%) that were below the limit of detection of the CLIA assay. The most common mutations observed in JAK2V617F-negative MPN were in ASXL1 (22% of patients) and in TET2 (9%). Taken together, mutations in known epigenetic modifiers (ASXL1, TET2, DNMT3A, EZH2, MLL) were observed in 43% of samples, including a MLL-PTD mutation in one patient with PMF. We identified mutations in spliceosome components (SRSF2, U2AF1), in a subset of patients, consistent with previous reports, and in each case mutations in spliceosome components were mutually exclusive.We identified mutations in the JAK-STAT pathway (MPL, TYK2) and the RAS pathway components (KRAS, NF1) in 9% of this patient cohort, suggesting that there are alternate disease alleles that activate signaling in JAK2V617F-negative MPN. RNA-sequencing identified a ETV6-ABL1 fusion in one patient, and we identified amplification of PIK3CA in one patient in our cohort; these data suggest fusion genes and amplifications activate signaling in a subset of patients with JAK2V617F-negative MPN. We also identified novel mutations in MPN patients which have not been reported to date, including mutations in DNA repair genes (ATM and BRCA) in 25% of cases and mutations in the Notch signaling pathway (NOTCH1-4) in 31% of cases. The functional implications of these novel mutations remain to be elucidated.In univariate analysis, ASXL1 mutations were found to associate with impaired overall survival (Figure 1, p=0.049). These findings are consistent with data demonstrating an impaired survival in patients with MDS and PMF, and suggest that ASXL1 mutations represent an important biomarker for adverse outcome in JAK2V617F-negative MPN. [Display omitted] ConclusionsThese data demonstrate that the mutational spectra of JAK2V617F-negative MPN includes genes implicated in epigenetic regulation, novel mutations which activate gene signaling, and fusion genes/copy number alterations which provide a novel mechanism of oncogenic activation not previously reported in MPN. ASXL1 mutations occur frequently in JAK2 wildtype Philadelphia-Chromosome negative MPNs, and are associated with impaired overall survival. Collectively, these findings support the importance of ASXL1 mutations in predicting outcome in JAK2V617F-negative MPN, demonstrate that mutations in signaling effectors and in epigenetic regulators are common in MPN, and illustrate the genetic heterogeneity of JAK2V617F-negative MPN. Disclosures:Rampal:Foundation Medicine: Consultancy. Levine:Foundation Medicine, Inc: Consultancy.

TheJAK2gene as a protagonist in chronic myeloproliferative neoplasms

The identification of the association of a JAK2 gene mutation with chronic myeloproliferative neoplasms (cMPN) negative for BCR-ABL(1,2) has allowed significant advances in the understanding of this group of hematologic diseases. The JAK2 gene, located on chromosome 9p24, encodes the JAK2 protein which is a cytoplasmic tyrosine kinase that plays an important role in the signal transduction of various hematopoietic growth factors. The JAK2V617F mutation results in the substitution of the amino acid valine for phenylalanine in the pseudokinase domain (JH2) causing constitutive activation of the kinase domain (JH1) and hypersensitivity to protein growth factors. Among the BCR-ABL negative cMPN cases, the JAK2617F mutation occurs at a frequency of 96% in polycythemia vera (PV), and 50% of essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (MF) patients(2). The association of this mutation with BCR-ABL negative cMPN has contributed to improve the diagnosis, classification and treatment of patients, in particular in respect to PV. Tefferi and Pardanani(3) suggested that an investigation of the JAK2V617F mutation in the peripheral blood should be integrated into the initial assessment of patients with suspected diagnosis of PV and of those of thrombocytosis of unknown cause, with thrombotic complications, including cerebral or abdominal thrombosis, and other clinical manifestations of myeloproliferative diseases. Other less frequent mutations were also found in the JAK2 gene in JAK2V617F-negative PV patients, as well as in other myeloproliferative neoplasms. Several studies report deletions, point mutations and duplications(4) mainly affecting the seven highly conserved amino acid residues (F537-F547) in the JAK2 protein. PV patients positive for these mutations are often heterozygous for the mutation and are characterized by the predominance of myelopoesis, serum erythropoietin levels below normal and lower age at diagnosis(3-5). The clinical evolution of these patients is similar to JAK2V617F-positive PV patients(5). The V617F mutation of the JAK2 gene triggers three clinical manifestations and there is evidence that genetic and additional epigenetic events contribute to the pathogenesis(6). In addition, other genes, such as the MPL, TET2 and ASXL1 genes, may also be mutated with the accumulation of mutations possibly explaining the different phenotypes observed in MPN. The MPL gene, located on chromosome 1p34, encodes the thrombopoietin receptor (cMPL). Its expression is important for growth and survival of megakaryocytes. Some mutations in this gene result in gain of function and have been associated with thrombocytosis, splenomegaly, myelofibrosis and an increased risk of thrombosis(7). Mutations in the transmembrane domain of cMPL were observed in nine patients negative for the JAK2V617F mutation (MPLW515L and MPLW515K); mutations were also detected in JAK2V617F-positive patients. The TET2 (4q24) gene has many mutations (frameshift, missense, nonsense) that are observed in JAK2V617F cMPN-positive (17%) and JAK2V617F-negative patients (7%), with mutational frequencies of approximately 16% in PV, 5% in ET, 17% in MF, 14% in post-PV MF, 14% in post-TE MF and 17% in blast-phase MPN(8). The main function of the TET2 protein is the conversion of 5-methyl-cytosine to 5-hydroxymethyl cytosine; it eventually affects the epigenetic regulation of transcription. The ASXL1 gene maps to chromosome 20q11.1 and belongs to the enhancer of trithorax and polycomb gene family. The function of this gene is believed to include dual activator/suppressor activity toward transcription and includes repression of retinoic acid receptor-mediated transcription. Mutations in this gene are associated with myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CML)(9). In a recent study of 300 patients with a spectrum of non-MPN myeloid malignancies, ASXL1 gene mutations were found in 62 patients (~21%): ~7% in MDS without excess blasts, 11-17% in MDS with ring sideroblasts, 31% in MDS with excess blasts, 23% in post-MDS acute myeloid leukemia (AML), 33% in CML and 30% in primary AML. It was observed that mutations of the ASXL1 gene occur in MPNs in both chronic and blast phases. In a study of 64 patients with ET (n = 35), MF (n = 11), PV (n = 10), blast-phase MPN (n = 5) and unclassifiable MPNs (n = 3), mutations of this gene in heterozygosis were identified in five JAK2V617F-negative patients (~ 8%; 3 MF, 1 ET and 1 blast-phase ET)(10). There are other reports in the literature associating gene mutations to BCR-ABL-negative cMPN. The knowledge about genotype-phenotype interaction could elucidate the molecular mechanisms and contribute to improvements in diagnosis, in the status and in the treatment of patients with ET, MF and PV.

Cytogenetics, JAK2 and MPL mutations in polycythemia vera, primary myelofibrosis and essential thrombocythemia

BackgroundThe detection of molecular and cytogenetic alterations is important for the diagnosis, prognosis and classification of myeloproliferative neoplasms.ObjectivesThe aim of this study was to detect the following mutations: JAK2 V617F, JAK2 exon 12 and MPL W515K/L, besides chromosomal abnormalities. Furthermore, molecular and cytogenetic alterations were correlated with the leukocyte and platelet counts, hemoglobin levels and age in all patients and with the degree of fibrosis in primary myelofibrosis cases.MethodsTwenty cases of polycythemia vera, 17 of essential thrombocythemia and 21 of primary myelofibrosis were selected in the Hematology Department of the Universidade Federal de São Paulo (UNIFESP) between February 2008 and December 2009. The JAK2 V617F, JAK2 exon 12 mutations, MPL W515K and MPL W515L mutations were investigated by real-time PCR and direct sequencing. G-band karyotyping and fluorescence in situ hybridization were used to detect chromosomal abnormalities.ResultsChromosomal abnormalities were observed only in polycythemia vera (11.8%) and primary myelofibrosis cases (17.6%), without correlation to clinical data. Chromosomal abnormalities were not detected by fluorescence in situ hybridization. The JAK2 V617F mutation was observed in polycythemia vera (90%), primary myelofibrosis (42.8%) and essential thrombocythemia (47%). Patients with JAK2 V617F-negative polycythemia vera had lower platelet and leukocyte counts compared to V617F-positive polycythemia vera (p-value = 0.0001 and p-value = 0.023, respectively). JAK2 V617F-positive and MPL W515L-positive primary myelofibrosis cases had a higher degree of fibrosis than V617F-negative cases (p-value = 0.022). JAK2 exon 12 mutations were not detected in polycythemia vera patients. The MPL W515L mutation was observed in one case of primary myelofibrosis and in one of essential thrombocythemia. The MPL W515K mutation was not found in patients with essential thrombocythemia or primary myelofibrosis. The MPL W515L-positive patient with primary myelofibrosis had more severe anemia than other patients with primary myelofibrosis.ConclusionsThis study demonstrates that karyotyping for JAK2 and MPL mutations is useful in the diagnosis of myeloproliferative neoplasms. The precise pathogenetic contribution of these alterations is still unclear. However, this study adds more information about the pathophysiology of polycythemia vera, essential thrombocythemia and primary myelofibrosis.

The importance of cytogenetics in polycythemia vera, primary myelofibrosis and essential thrombocythemia

The importance of cytogenetics in polycythemia vera, primary myelofibrosis and essential thrombocythemia

In BCR-ABL1 Negative Myeloproliferative Neoplasms the Detection of JAK2exon12, MPLW515, CBL, KITD816V, FIP1L1-PDGFRA Mutations Are Closely Linked to Specific Entities, Whereas the JAK2V617F, TET2, or EZH2 Mutations Demonstrate a Broader Diversity: Patterns From Diagnostic Reports of 18,547 Patients Analyzed

AbstractAbstract 458The first mutation detected in BCR-ABL1 negative myeloproliferative neoplasms (MPN) was JAK2V617F that revolutionized diagnostics of MPN during the last five years. However, although this genetic marker is useful to discriminate MPN from reactive disorders, it is not specific for one entity. In addition, approximately 5% of all polycythemia vera (PV) and 50% of essential thrombocytosis (ET) and primary myelofibrosis (PMF) are not JAK2V617F mutated. In these entities other activating mutations, e.g. MPLW515 mutations or JAK2exon12 mutations, cover additional small proportions of patients without JAK2V617F mutation. To further improve the molecular genetic characterization of MPN research focuses on the identification of novel mutations and, recently, CBL, TET2, and EZH2 genes were identified to be mutated in MPN. We here report on our single centre experience in applying these markers in a daily diagnostic work flow comprizing a total cohort of 18,547 cases with suspected MPN that were investigated between 8/2005 und 8/2010 with individual patient specific combinations of these markers as soon as published. Thus, the most frequently tested marker was JAK2V617F that was applied in 17,027 pts. In 6,622/17,027 (38.9%) a definite diagnosis of MPN could be made or confirmed on the basis of the detection of JAK2V617F mutation. More detailed, the percentage of JAK2V617F positive cases varied depending on the suspected diagnoses: In patients with cytomorphologically confirmed or suspected ET 581/891 (65.2%) were JAK2V617F positive, in PMF: 168/290 (57.9%), in PV: 800/942 (84.9%), in MPN-U: 51/212 (24.0%), in CMML: 38/383 (9.9%), in “MPN” not further specified by the referring physician: 4741/11249 (42.1%), and in those with unexplained leukocytosis/thrombocytosis/splenomegaly or suspected hematologic malignancy: 139/2492 (5.6%). Many of the before mentioned cases were suspected MPN and therefore analyzed for both JAK2V617F and BCR-ABL1. Thus, in 9,924 pts BCR-ABL1 and JAK2V617F testing were performed in parallel. As such, in 541/9,924 (5.5%) analyses BCR-ABL1 positive CML was identified and 3,558 cases were JAK2V617F mutated (35.9%). Only 8 pts were BCR-ABL1/JAK2V617F double positive (0.08%), thus this is a very rare event. In cases with JAK2V617F negative PV in a second step JAK2exon12 mutation was analyzed and 27/147 (18.3%) were tested positive. JAK2V617F negative ET or PMF were analyzed in a second step for MPLW515 mutations. In ET 24/258 (9.3%) and in PMF 14/164 (8.5%) cases were tested positive. JAK2exon12 or MPLW515 were never concomitantly detected with JAK2V617 in our cohort (parallel assessments: n=3,769). PCR for detection of FIP1L1-PDGFRA was performed in 1,086 cases with suspected HES/CEL or unclear eosinophilia but only 26 (2.4%) were tested positive and a CEL could be diagnosed. However, in 36/130 (27.7%) FIP1L1-PDGFRA negative cases a KITD816V mutation was detected and thus a diagnosis of mastocytosis could be established. In addition, confirmation of mastocytosis was achieved in further 326/731 (44.6%) pts with suspected mastocytosis, three of these pts had a JAK2V617F mutation in addition. Further analyses were recently done on selected well characterized cohorts of MPN: CBL mutations were analyzed in 623 cases and tested positive in 54 (8.7%): 26/199 CMML (13.0%), 1/25 PMF, 27/293 MPN-U (9.2%), but never were detected in ET (n=61) or PV (n=45). TET2 sequencing detected mutations in 56/191 (29.3%) of pts analyzed: ET: 6/28 (21.4%), PMF: 4/12 (33.3%), PV: 10/31 (32.3%), CMML: 17/22 (77.3%) cases, MPN-U: 17/86: (19.8%), HES: 1/9 cases, Mastocytosis: 1/3 cases. Thus, TET2 mutations are widely spread in different entities and were frequently associated with other mutations: JAK2V617F: n=16, JAK2exon12: n=1, MPLW515: n=2, CBL: n=5, FIP1L1-PDGFRA: n=1, KITD816V: n=1, and EZH2: n=2. Finally, EZH2 sequence analysis detected mutations in 4/68 (5.9%) cases (1/16 PV, 2/11 PMF, 1/17 MPN-U, 0/20 ET, 0/4 CEL). In conclusion, these data show that the analysis of molecular mutations greatly improved the diagnostic work up of MPN in the last 5 years. The detection of some mutations (JAK2exon12, MPLW515, CBL) are useful to further subclassify MPNs. Others (JAK2V617F, TET2, EZH2) are widely distributed and are helpful for classification and also to discriminate MPN from reactive disorders. The individual power of each marker for prognostication in MPN remains to be defined in future studies. Disclosures:Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Gene Expression Profiling Distinguishes Essential Thrombocythemia from Polycythemia Vera Patients and Identifies a Common Expressed Set of Genes in Relation to JAK2V617F Status

Introduction. The existence of the JAK2V617F mutation in a high proportion of Myeloproliferative Disorders (MPD) BCR-ABL-negative has provided important insight into the pathogenesis of these diseases. However, much of the molecular abnormalities associated to BCR-ABL-negative MPD remain unknown, specially in those which do not display JAK2V617F. In a previous study, we performed gene expression analysis by using the microarray technique in 20 essential thrombocythemia (ET) patients (44K whole human genome oligo microarrays, Agilent Technologies) and the results were confirmed in 40 ET patients by using TaqMan® Low Density Arrays Arrays (LDA, Applied Biosystems). In our previous experience the results showed different gene expression patterns in ET and a supervised clustering of the data identified genes differentially expressed between JAK2V617F-negative and JAK2V617F-positive ET patients, and a characteristic gene expression profile for JAK2V617F-negative patients (Puigdecanet et al.,2008).Aim. Our aim was to confirm the ET gene expression profile in an extended group of patients and to explore the differences and similarities in polycythemia vera (PV) and reactive thrombocytosis (RT) patients by real-time quantitative RT-PCR (RQ-PCR) technique using the LDA platform. In addition, we wanted to analyze the relationship between gene expression data and JAK2V617F status.Patients and Methods. The following patients were included in the study: 58 ET (23 JAK2V617F-negative, 34 JAK2V617F in heterozygosity and one JAK2V617F in homozygosity) and 41 PV (7 JAK2V617F-negative, 25 JAK2V617F in heterozygosity and 9 JAK2V617F in homozygosity) patients, diagnosed according to the WHO criteria (2001) and who had never received cytoreductive treatment, and six patients with RT. Based on the previous results, we designed a new LDA platform containing 96 assays in duplicate, which included the most expressed genes in ET in relation to healthy controls and the most differentially expressed genes between JAK2V617F-negative and JAK2V617F-positive ET patients. The RQ-PCR analysis was performed in RNA from peripheral blood granulocytes and the relative gene expression quantification was achieved using GAPDH as the endogenous control and a pool of 10 healthy individuals as the calibrator.Results. ETvsPV: The majority of the genes studied presented significant higher expression in ET than in PV patients. Interestingly, FOSB was one of the most differentially expressed gene (FC= 8.3), and CISH and C13orf18 did not distinguish between the two groups.ET: We confirmed the differentially expression of the majority of the genes previously detected between JAK2V617F-negative and JAK2V617F-positive ET patients and we extended the set of genes. Among them, we highlight the differential expression of CISH, FOSB and C13orf18 genes (p<0.01).PV: Supervised analysis showed that CD44, BATF and CISH clearly distinguish JAK2V617F-negative PV patients from the JAK2V617F-positive.ET and PVvsRT: Some differentially expressed genes between ET and RT patients were detected, but the most significant gene was TNF (p<0.001), which presented a higher expression in RT (FC=5.9). The same difference was observed between PV and RT.Conclusions. We have detected a different gene expression pattern in ET and in PV patients. However, we also identified a set of genes which expression was related to JAK2V617F status, both in ET and PV patients. These findings would be interesting to identify other signal transduction pathways besides JAK-STAT involved in the pathogenesis of ET and PV.

JAK2 and MPL mutations in myeloproliferative neoplasms: discovery and science

Although it has long been known that the myeloproliferative neoplasms (MPN) polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are clonal hematopoietic stem-cell disorders, for many years the genetic basis for these disorders was elusive. A new era in MPN biology began in 2005 with the discovery of a somatic point mutation in JAK2 tyrosine kinase (JAK2V617F), which was identified in a significant proportion of patients with PV, ET and PMF. Based on the hypothesis that JAK-STAT signaling is central to the pathogenesis of JAK2V617F-negative MPN, genomic studies have identified JAK2 exon 12 mutations in JAK2V617F-negative PV and activating mutations in MPL in patients with JAK2V617F-negative ET and PMF. In this review, we will discuss the role of these mutant alleles in the pathogenesis of PV, ET and PMF, the potential therapeutic implications of these discoveries, and the implications of these discoveries for genomic studies of hematopoietic malignancies.

Differential Expression of miRNAs in Polycythemia Vera Peripheral Blood Cells.

Polycythemia vera (PV) is a clonal myeloproliferative disorder caused by somatic mutation of a hematopoietic multipotent cell. PV hematopoiesis is characterized by an accumulation of phenotypically normal erythrocytes with overproduction of leukocytes and platelets. A somatic JAK2 V617F point mutation occurs in the majority (>95%) of PV patients. However, this mutation is also found in ∼50% of patients with either essential thrombocythemia (ET) or idiopathic myelofibrosis (MF). The non-specificity of this mutation, the presence of JAK2 V617F-negative PV patients, JAK2 V617F-negative and positive relatives, and evidence of both JAK2 V617F-negative and positive PV clones demonstrate that the JAK2 V617F mutation is not the initial and sole somatic event for the pathogenesis of PV. MicroRNAs (miRNAs) have been shown to be important regulators of hematopoiesis. To identify deregulated miRNAs involved in PV pathogenesis, we studied gene expression of miRNAs of in vitro expanded erythroid progenitors (EPs), peripheral blood mononuclear cells (MNCs), granulocytes, reticulocytes, and platelets from PV patients and healthy controls. Initially, we performed gene expression profiling in 5 PV patients and 5 control cells using CombiMatrix MicroRNA CustomArray with 326 probes. The array data were analyzed by Genesis software to determine differentially expressed miRNAs in PV. These miRNAs were tested in a larger set of samples (n=18) by qRT-PCR and their expression was correlated to the JAK2 V617F mutational level. Hierarchical clustering analysis defined miRNA expression profiles of particular cell lineage for normal and PV cells. Further, ANOVA identified 31 miRNAs differently (P<0.05) expressed in at least some PV lineage cells. Of these miRNAs, we confirmed downregulation of miR-150 in expanded EPs of all stages of maturation, downregulation of let7a and upregulation of miR-182 in PV granulocytes; upregulation of miR-143, miR-145 and miR-223 in PV MNCs; and down-regulation of miR-30b, miR-30c and miR-150 in PV reticulocytes by qRT-PCR. Correlation analysis of miRNA expression with JAK2 V617F mutational level showed a positive correlation of miR-143 (r=0.68) and a negative correlation of let7a (r =−0.63), miR-30c (r=−0.74), miR-342 (r=−0.66) and miR-150 (r=−0.89). To validate PV specificity, we compared expression levels of these miRNAs to other MPD disorders (MF and ET) in which the JAK2 V617F mutation occurs. Putative miRNA targets were predicted by TargetScan 4.0 and PicTar software, and transcript levels of selected target genes are being analyzed to determine their potential deregulation at the mRNA level. Downregulated miR-150 is predicted to the target MYB oncogene that plays an important role in erythropoiesis by maintaining proliferation at the early stages. The most potential target of let7a is HGMA2, whose aberrant expression may contribute to clonal hematopoiesis in PNH. The verification of these predictions is in process by use of miRNA inhibitors, protein levels, and functional studies of the progenitors. Our study demonstrates that the specific signatures of miRNAs define particular peripheral blood cell lineages. Furthermore, the deregulated miRNAs, whose expressions correlate with the JAK2 mutational level, may be associated with the PV phenotype. Understanding the role of deregulated miRNAs in PV should provide insight into the pathogenesis of PV and may lead to novel therapeutic strategies.

JAK2 Mutations at Exon 12 and 14 in Polycythemia Vera and Idiopathic Erythrocytosis: Incidence and Correlation with Clinical Characteristics.

Introduction. Exon 12 mutations of the JAK2 gene have been described in polycythemia vera (PV) and idiopathic erythrocytosis (IE) patients. These patients display a clinical phenotype different to that observed in V617F-positive PV patients, but no information is available on their clinical outcome.Patients and methods. Twenty JAK2 V617F-negative PV or IE patients and 86 V617F-positive PV patients were assessed for exon 12 and exon 14 mutations. Analysis of cell lineage mutational status was assessed following cell sorting. Aim. To analyze the presence of JAK2 mutations at exon 12 and 14 in a cohort of V617F-negative PV and IE patients and to correlate them with the patient clinicohematological and evolutive data.Results. Exon 12 mutations were detected in 4 (20%) V617F-negative patients (K539L, two cases, N542_E543del and H538_K539delinsL, one case each). In 16 patients, no mutations were found in either exon 12 or exon 14. Additional mutations in exon 14 were found in two V617F-positive patients. Two patients with exon 12 mutations developed thrombosis after diagnosis, with the probability of thrombosis-free survival being 75% at 5 years. Another patient with exon 12 mutation developed myelofibrosis at 20 years of PV diagnosis. JAK2 mutations were present in granulocytes, platelets and monocytes, but not in lymphocytes or NK cells.Conclusions. Patients harboring exon 12 mutations represent a subset of JAK2 V617F-negative PV or IE patients. These patients have initial hematological data different from V617F-positive patients, but they do not differ with regard to thrombosis development and myelofibrotic transformation.Table 1:JAK2 exon 12 and exon 14 mutations in blood cell subpopulations.MutationGranulocytesPlateletsCD14+CD3+CD19+CD56+Patient 1*K539L (AAA →TTA)++−−−−Patient 2*K539L (AAA →CTA)+++−−−Patient 3*H538_K539delinsL++ND−−NDPatient 4*N542_E543del+++−−−Patient 5#V617F (GTC →TTT)++−−−NDC618R (TGT →CGT)++−−−NDPatient 6#V617F (GTC →TTC)+++−−NDC616C (TGT →TGC)+++−−ND* exon 12; # exon 14; + indicates the presence of the mutation; − indicates no detectable mutation ; ND not determined

Phenotypic variations and new mutations in JAK2 V617F–negative polycythemia vera, erythrocytosis, and idiopathic myelofibrosis

The chronic myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis, and idiopathic myelofibrosis (IMF), are characterized by a spectrum of clinical features and linked by common genetic lesions in JAK2 and MPL. However, the clinical phenotypes in genetically undefined MPD patients are similar to those patients with JAK2 and MPL lesions. We, therefore, sought to determine whether there were JAK2 or MPL lesions in a well-defined, JAK2 V617F-negative MPD cohort, and to determine if clinical associations could be identified based on variations identified in these genes. We examined the JAK2 and MPL genes in JAK2 V617F-negative PV, IMF, and idiopathic erythrocytosis patients for sequence variations. We identified two previously unrecognized JAK2 mutations and three previously unrecognized MPL mutations in JAK2 V617F-negative PV, erythrocytosis, and IMF patients. We identified JAK2 exon 12 lesions in 30% of JAK2 V617F-negative PV patients, and either JAK2 V617F or JAK2 exon 12 lesions in 9% of erythrocytosis patients. In IMF, in addition to the MPL gene mutation, W515K, we identified three additional mutations: 204P and two intervening sequence transitions, IVS 11/12 and 10/11. While the clinical phenotype of JAK2 exon 12 lesions in the MPD was predominantly erythroid, there was significant disease spectrum overlap between JAK2 V617F and JAK2 exon 12 mutations. By contrast, MPL gene mutations were not associated with erythrocytosis, but segregated primarily with the phenotypes of thrombocytosis, extramedullary disease, myelofibrosis, and osteosclerosis.

Mpl Genotype and Phenotype Correlations in the Chronic Myeloproliferative Disorders: Relationship of Mpl Mutations, JAK2V617F Status and Mpl Protein Expression.

The chronic myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis (ET) and idiopathic myelofibrosis (IMF), are linked by a common genetic lesion, JAK2V617F, and abnormalities of the thrombopoietin receptor, Mpl. JAK2 is an obligate Mpl chaperone and is responsible for its cell surface expression. We observed a reciprocal relationship between neutrophil JAK2V617F allele percentage and platelet Mpl expression in JAK2V617F-positive PV, IMF and ET patients. However, severely impaired platelet Mpl expression was also present in the majority of JAK2V617F-negative PV and IMF patients, with the most severe reductions in IMF. We previously identified a constitutional variation in the Mpl gene in the distal N-terminal domain (K39N) associated with thrombocytosis, and an Mpl splice variant involving the same region that was increased in PV and IMF patients compared to controls. Given the association of Mpl genetic lesions and protein expression abnormalities in the MPD, we hypothesized that other lesions in Mpl may be present in the MPD. To test this, we sequenced all of the exons, the flanking intervening sequences and the 5′-proximal untranslated region of the Mpl gene in 1 ET, 3 PV and 4 IMF patients, all of whom were JAK2V617F-negative and all of whom had markedly reduced (<5%) platelet Mpl protein. In one IMF patient, we identified a two base substitution at nucleotide 1543 of the Mpl gene which results in a tryptophan to lysine substitution at amino acid 515 (W515K); the wild-type sequence was not identified from either peripheral blood CD34+ cells or neutrophils isolated from this patient. The patient developed IMF at the age of 26, and had the disease 23 years before succumbing to complications of extensive extramedullary hematopoiesis and anemia. In a second male patient, we identified a heterozygous nucleotide transition in the intervening sequence distal to exon 10 close to the splice donor site; the mutation was also detected in buccal cell DNA. This patient developed IMF at age 35 and has had the disease for 31 years characterized by anemia and extramedullary hematopoiesis. To test whether this or other lesions in exon 10 of the Mpl gene were present, we directly sequenced exon 10 in 9 patients with JAK2V617F-negative PV, 21 with JAK2V617F-negative IMF, and 13 with JAK2V617F-positive IMF. Neither these nor other lesions in exon 10 were identified in these 43 MPD patients; no other unreported SNPs were identified either. We did not identify the recently described MplW515L reported as present in 4/44 JAK2V617F-negative IMF patients (PLoS Med. 2006; 3:e270) in our 21 JAK2V617F-negative IMF patients. These data indicate that, in contrast to other cytokine receptors, genetic and epigenetic variations of Mpl have unique roles in the pathogenesis of the chronic MPD. We conclude that significant disruptions of Mpl, whether acquired or constitutional, at the genomic level as described above or at the proteomic level as we have previously described, associate with a chronic MPD characterized by significant extramedullary hematopoiesis.

Molecular Mimicry in the Chronic Myeloproliferative Disorders: Reciprocity between JAK2 V617F Genotype and Mpl Expression.

Polycythemia vera (PV), idiopathic myelofibrosis (IMF) and essential thrombocytosis (ET) are clonal myeloproliferative disorders (MPD) that share in common increased blood production, megakaryocyte dysplasia, growth factor-independent colony formation and similar cytogenetic defects. Until recently, a dearth of molecular markers hindered analysis of the interrelationship of PV, IMF and ET. Mpl protein expression is decreased in most PV patients and the degree of impairment correlated with disease duration and extent. Impaired Mpl expression was also present in many IMF and ET patients. The recent discovery of the JAK2 V6217F mutation in PV recapitulated this behavior since JAK2 V617F was present in many classical IMF and ET patients. Importantly, JAK2 is an obligate Mpl chaperone required for Mpl surface expression (JBC 280: 27251, 2005). Since JAK2 V617F is constitutively active, we hypothesized that both its presence and gene dosage could influence Mpl expression. Therefore, we examined the concordance of the JAK2 V617F genotype and platelet Mpl protein expression in 78 PV, 13 IMF and 82 ET patients, using allele-specific, real-time PCR screening of granulocyte DNA for JAK2 V617F and quantitative densitometry for platelet Mpl with a cutoff of ≤ 60% for reduced and ≥ 120% for overexpression. JAK2 V617F prevalence was 91% in PV (40% homozygous), 46% in IMF (66% homozygous) and 41% in ET (none homozygous). JAK2 V617F-negative patients (7 PV, 7 IMF and 58 ET) were indistinguishable clinically from JAK2 V617F-positive patients. Mean disease duration was similar for homozygous JAK2 V617F (11 years, range 1–25) and heterozygous JAK2 V617F (8 years, range 1–22) PV patients. Mean disease duration was also similar for JAK2 V617F-positive (7.5 years, range 1–18) and JAK2 V617F-negative (6 years, range 1–22) ET patients. Mean Mpl expression was 31% in PV patients (range 1–227, reduced in 89%), 7% in IMF (range 1–22, reduced in 100%) and 72% in ET (range 1–246, reduced in 52%). Of the JAK2 V617F-positive patients, mean platelet Mpl was 27% in PV (range 1–86%, reduced in 92%), 5% in IMF (range 1–11%, reduced in 100%) and 38% in ET (range 1–127%, reduced in 81%). JAK2 V617F genotype significantly inversely correlated (p<0.004) with platelet Mpl expression: in JAK2 V617F homozygotes, mean platelet Mpl expression was 12% (range 1–44, 100% reduced); in JAK2 V617F heterozygotes, mean Mpl expression was 35% (range 1–127, 86% reduced), while in JAK2 V617F-negative patients, mean Mpl was 82% (range 1–246%, 44% reduced). JAK2 V617F-negative PV and IMF patients had either markedly reduced Mpl (8 patients, mean 6%) or markedly elevated Mpl (2 patients, mean 200%). In ET, JAK2 V617F-positive patients had reduced platelet Mpl (38% of normal, range 1–127%) compared to JAK2 V617F-negative patients (92% of normal, range 1–246%) (p=0.004). However, JAK2 V617F-negative ET patients were heterogeneous; 42% had reduced, 24% normal and 33% elevated platelet Mpl expression. These data indicate that JAK2 V617F expression and gene dosage directly influence platelet Mpl expression in PV, IMF and ET. Less commonly however, impaired Mpl expression, often marked, also occurred in the JAK2 V617F-negative setting, suggesting that other genetic or epigenetic lesions can create the same molecular abnormality. We conclude that Mpl protein expression abnormalities, whether associated with JAK2 V617F or not, provide a common denominator for the MPD phenotype. JAK2 V617F genotype does not necessarily predict MPD phenotype, but the degree of Mpl reduction does.