scholarly journals Effects of the Sympathicomimetic Agonist Mirabegron on Disease Course, Mutant Allele Burden, Marrow Fibrosis, and Nestin Positive Stem Cell Niche in Patients with JAK2-Mutated Myeloproliferative Neoplasms. a Prospective Multicenter Phase II Trial SAKK 33/14

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3108-3108 ◽  
Author(s):  
Beatrice Drexler ◽  
Jakob Passweg ◽  
Martin Bigler ◽  
Alexandre PA Theocharides ◽  
Nathan Cantoni ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Phase Ii ◽  
Mutant Allele ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Nerve Fibers ◽  
Allele Burden ◽  
Sympathetic Nerve Fibers ◽  
Bone Marrow Histology ◽  
Cell Niche

Abstract Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders characterized by aberrant proliferation of erythroid, megakaryocytic and myeloid lineages. They are associated with decreased survival, thromboembolic complications, hemorrhage and leukemic transformation. MPN can be subdivided into polycythemiavera(PV), essentialthrombocythemia(ET) and primary myelofibrosis (PMF). The JAK2-V617F mutation is present in 70-80% of all MPN patients. MPN is initiated and maintained by mutated hematopoietic stem and progenitor cells (HSPC). Bone marrow mesenchymal stem cells expressing the intermediate filament proteinnestin(nestin+ MSCs) that are innervated by sympathetic nerve fibers constitute an important component of the stem cell niche and regulate normal HSCs. Thesenestin+ MSCs are strongly reduced in bone marrow of JAK2-V617F positive MPN patients and in mice expressing JAK2-V617F due to damage of the sympathetic nerve fibers triggered by cytokines from the mutant cells. In a JAK2-V617F mouse model of MPN, treatment with a beta-3sympathicomimeticagonist corrected the damage inflicted by the MPN clones on their niches and ameliorated the MPN phenotype. To test the potentially beneficial effect on disease-control by modulating bone marrow niche cells in patients with MPN, we performed a phase II trial with the beta-3sympathicomimeticagonistmirabegron. Patients and Methods: The trial consisted ofmirabegrontreatment with 25 mg daily during the first week, followed by 50 mg daily for at least 24 weeks. Patients with acytohistologicallyconfirmed diagnosis of MPN and a JAK2-V617F allele burden >20% in granulocytes at study entry were eligible, if not treated with JAK2 inhibitors or interferon. Reduction of the JAK2-V617F mutant allele burden ³50% in granulocytes was defined as the primary end point. Secondary end points included changes in blood counts or MPN related symptoms. As a side study, bone marrow biopsies were quantified fornestin+ MSCs, fibrosis and CD34+ HSPCs. N=39 patients have been accrued in 10 institutions in Switzerland. Eight (21%) had ET, 22 (56%) PV, and 9 (23%) PMF. N=27 (69%) were male, the median age was 62 (Q1-Q3 53-72) years. Median mutated allele burden at study onset was 52% (Q1-Q3 33-73%). All patients had prior treatment, N=28 (72%) patients hadcytoreductivetreatment, the remaining patients hadantiaggregation, anticoagulation or phlebotomy. Results: No patient reached the primary endpoint of 50% reduction in allele burden, one patient achieved a 25% reduction by 24 weeks of treatment. Adverse events were mostly grade I or II on the CTCAE scale. Three patients had grade III events: two were considered to be at least possibly related to study medication. In the side study, 24 patients agreed to bone marrow biopsy prior to and at the end ofmirabegrontreatment and for 20 patients both measurements are available. In these patients an increase in thenestin+ MSCs cells from a median of 1.09 (Q1-Q3 0.38-3.27)/mm2 to 3.95 (Q1-Q3 1.98-8.79)/mm2 (p<0.0001, Wilcoxon signed-rank test) and a slight decrease of myelofibrosis from a median grade of 1.00 (Q1-Q3 0.50-3.00) to 0.75 (Q1-Q3 0.50-2.00) (p=0.02), were observed. The mean change in thenestin+ cells from baseline to week 24 was 3.52 (95% confidence interval 1.65-5.39)/mm2. Morphometric changes in thenestin+ MSCs were significant for PV (n=13, p=0.007) and PMF (n=5, p=0.04). Bone marrow CD34+ cells slightly increased from a median 2.50 (Q1-Q3 2.00-3.25) to 3.00 (Q1-Q3 2.00-3.75) (p=0.06). Conclusion: In this prospective phase II clinical trial treatment with the beta-3-sympathicomimetic agonistmirabegronfor 24 weeks failed to achieve the primary endpoint to reduce the JAK2-V617F mutant allele burden >50% in patients with MPN. However, an increase in thenestin+ MSCs in bone marrow and a slight decrease of myelofibrosis were found, which will be further investigated. Figure 1 Bone marrow histology of a patient before (week 0) and at the end ofmirabegron treatment (week 24). Upper panel,reticulin fibers are stained black by silver impregnation (Gomori). Lower panel, immunohistochemistry staining with antibodies against humannestin protein (brown staining). Note decrease inreticulin fibrosis and increase innestin+ cells after 24 weeks of treatment. Magnification: 200x. Figure 1. Bone marrow histology of a patient before (week 0) and at the end ofmirabegron treatment (week 24). Upper panel,reticulin fibers are stained black by silver impregnation (Gomori). Lower panel, immunohistochemistry staining with antibodies against humannestin protein (brown staining). Note decrease inreticulin fibrosis and increase innestin+ cells after 24 weeks of treatment. Magnification: 200x. Disclosures Theocharides: Novartis: Consultancy, Honoraria. Rüfer:Novartis: Consultancy, Speakers Bureau. Benz:Celgene: Consultancy. Tzankov:Novartis: Speakers Bureau; Abbott: Speakers Bureau. Skoda:Novartis: Consultancy, Speakers Bureau; Baxalta: Speakers Bureau; Shire: Consultancy, Speakers Bureau.

scholarly journals Single Centre Analysis of JAK2 V617F and Exon 12 Negative Idiopathic Erythrocytosis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4620-4620
Author(s):  
Kushani Ediriwickrema ◽  
Andrew J Wilson ◽  
Jenny O'Nions ◽  
Mallika Sekhar ◽  
Syeda Ahmed ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mutational Analysis ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Full Blood Count ◽  
Polycythaemia Vera ◽  
Normal Range ◽  
High Affinity ◽  
Bone Marrow Histology ◽  
Idiopathic Erythrocytosis

Abstract Introduction The molecular categorisation of myeloproliferative neoplasms (MPNs) has changed the landscape of diagnosis and treatment. Polycythaemia vera (PV) is characterised by red blood cell proliferation and JAK2 V617F or Exon 12 mutations in up to 98% of patients 1. However, some patients without such mutations have an arduous diagnostic course with varying management and prognostic outcomes 2. We present our experience in managing this challenging cohort and aim to illuminate a potential diagnostic pathway for patients. Method We searched electronic records of patients attending haematology clinics over the last 20 years at University College Hospital with a prior diagnosis of PV / erythrocytosis (presenting with raised Haemoglobin (Hb) &/ Haematocrit (Hct)) with no evidence of JAK2 exon 12 or 14 mutations on bone marrow or peripheral blood molecular analysis (multiplex PCR sensitivity 0.1%). We reviewed their diagnostic workup, which included full blood count & where available, bone marrow myeloid (Illumina TruSight Myeloid) & erythroid next generation sequencing (NGS) panels. Results 37 patients with JAK2 V617F & Exon 12 mutation negative erythrocytosis were identified. Patients were categorised in to 3 groups 2; idiopathic erythrocytosis (IE), secondary polycythaemia (SP) & high affinity haemoglobinopathies (HAH); patient characteristics are summarised in Table 1. The median age of IE & HAH was younger, their presenting Hb/Hct levels was higher compared to SP, with a male predominance. Constitutional symptoms were only reported in the IE cohort. Erythropoietin (EPO) was elevated in HAH & IE patients but within normal range in SP. Thrombotic events occurred in all cohorts, most frequently in IE. Splenomegaly was reported in 4/21 IE, 1/13 SP, but was not a feature in HAH patients. When performed, IE red cell mass (RCM) studies were raised but within normal range in SP patients. Table 2 details IE cohort erythroid mutations. Myeloid NGS only identified MPL and BCOR mutations of pathogenic significance and multiple single nucleotide polymorphisms of no known significance. No abnormalities were demonstrated in 15% of SP patients that underwent bone marrow myeloid mutational analysis. Venesections (VS) were instigated in 95% of the IE cohort, antiplatelets (AP) in 52%, anticoagulation (AC) in 14% and cytoreductive therapy (CT) in 19% due to intolerance/failure of VS. VS programme was instigated in 46% of SP patients, AP in 7% and AC in 47%. Discussion The median age of our IE cohort was 48 yrs with a 19% incidence of thrombosis. Where performed, bone marrow histology demonstrated hypercellularity but was not consistent with MPN diagnostic criteria. Myeloid NGS panel mutations such as BCOR may represent clonal haematopoieis of indeterminate potential. Heterozygous VHL C598T & C376A mutations, in keeping with Chuvash polycythaemia, were demonstrated. Mutations in EGLN1 & BPGM,were detected in our patients, however there was an absence of correlating haematological parameters or family history to support a diagnosis of congenital erythrocytosis (2). Variants of unknown significance were also detected in SH2B3, BMP6 & EGLN3 gene duplication. SP patients were older (median age 68 years) and where performed had normal RCM and no myeloid mutations identified. The initial approach adopted at our centre for diagnosing and managing JAK2 V617F & Exon 12 negative erythocytosis begins with clinical evaluation for secondary causes. This is followed by assessment of EPO level, RCM study, extended molecular mutational analysis involving screening for high affinity haemoglobins, congenital erythrocytosis and bone marrow histology. Where patients are symptomatic or considered at high risk for thrombosis, we venesect to a personalised target and patients are offered AP and/or AC. CT is instigated where VS is not tolerated or ineffective, however this approach is not corroborated in the literature. Our experience highlights the clinical heterogeneity of JAK2 negative erythrocytosis and the need to develop a robust and systematic diagnostic and treatment algorithm with further clarification of the role of molecular profiling. 1. William W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017 Feb; 129(6):667-679 2. McMullin MF. Idiopathic erythrocytosis: a disappearing entity. Hematology Am Soc Educ Program. 2009; 2009(1):629-635 Figure 1 Figure 1. Disclosures Sekhar: Novartis: Consultancy, Research Funding.

scholarly journals Prevalence of the JAK2 V617F Mutation in Patients with Peripheral Arterial Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3165-3165
Author(s):  
Elena Kinz ◽  
Klaus Gasser ◽  
Axel Muendlein ◽  
Andreas Leiherer ◽  
Michael Steurer ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Arterial Disease ◽  
Jak2 V617f Mutation ◽  
Allele Burden ◽  
Cell Counts ◽  
V617f Mutation ◽  
Artery Disease ◽  
Peripheral Arterial

Abstract Introduction: The acquired JAK2 V617F mutation is common in patients with myeloproliferative neoplasms and increases thrombotic risk. We previously showed that JAK2 V617F is also found in healthy subjects as well as in patients with coronary artery disease (0.6% and 1.3%, respectively). Peripheral arterial disease (PAD) is an important manifestation of diffuse atherosclerosis and PAD patients are at exceptionally high risk for cardiovascular events, showing a worse prognosis than that of patients with coronary artery disease Due to the close relation of the JAK2 V617F mutation to thrombotic events we hypothesized that this mutation may play an important role in the risk management of PAD patients. However, prevalence of JAK2 V617F or of occult myeloproliferative neoplasms is unknown in PAD patients. Methods: In the present study we determined the prevalence of JAK2 V617F in a cohort of 287 patients with sonographically proven PAD. JAK2 mutational status from 997 age-matched healthy people was available from a previous study. JAK2 V617F screening and quantification of allele burden in both cohorts was performed with allele-specific quantitative real-time PCR. Results: From a total of 287 PAD patients samples, 9 (3.1%) were tested positive for JAK2 V617F mutation corresponding to a 5-fold, highly significant increase compared with healthy people (p<0.001). Mutant allele burden of JAK2 V617F positive samples was ranging between 0.2% and 96.2% (median=0.75%). Generally, our study showed no significant association of the JAK2 V617F mutation with abnormal blood cell counts. However, the patient with the highest mutant allele burden showed elevated hemoglobin values (> 18.5 g/dL) indicating polycythemia vera (PV). Conclusion: We conclude that the prevalence of JAK2 V617F mutation is significantly increased in PAD patients compared to the general population. For this reason mutation analysis should be considered in PAD patients with abnormal blood cell counts to identify occult myeloproliferative neoplasms and to adjust therapeutic treatment, possibly reducing the risk of future vascular complications. Disclosures No relevant conflicts of interest to declare.

Clonal Analysis of SF3B1, JAK2 and MPL in Refractory Anemia with Ring Sideroblasts Associated with Marked Thrombocytosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 172-172
Author(s):  
Ilaria Ambaglio ◽  
Anna Gallì ◽  
Daniela Pietra ◽  
Matteo G Della Porta ◽  
Marta Ubezio ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Refractory Anemia ◽  
Wild Type ◽  
Mutation Status ◽  
Allele Burden ◽  
Ring Sideroblasts ◽  
Exon 10

Abstract Abstract 172 Somatic mutations of the RNA splicing machinery have been recently identified in patients with myelodysplastic syndrome (MDS). In particular, a strong association has been found between SF3B1 mutation and the MDS subtype defined as refractory anemia with ring sideroblasts (RARS). Similarly, within myelodysplastic/myeloproliferative neoplasms (MDS/MPN) a high prevalence of SF3B1 mutations has been reported in the provisional entity defined as refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T). These findings strongly support a causal relationship between SF3B1 mutations and ring sideroblasts. Interestingly, a high proportion of RARS-T patients also harbor JAK2 and/or MPL mutations. The available evidence suggests that somatic mutations of SF3B1 might be an early pathogenetic event determining myelodysplastic features, and that subsequent occurrence of JAK2 and/or MPL mutations may cause the myeloproliferative phenotype. In this work, we studied the mutation status of SF3B1, JAK2 and MPL in circulating granulocytes and bone marrow cells from RARS-T patients. We also studied the in vitro growth of hematopoietic progenitors (BFU-E, CFU-GM), and genotyped individual colonies to examine the mutation status of the above genes. The coding exons of SF3B1 were screened using massively parallel pyrosequencing. A real time PCR-based allelic discrimination assay was used for the detection of JAK2 (V617F), while Sanger sequencing was employed for JAK2 exon 12 and MPL exon 10 mutation analysis. Twenty-eight patients affected with RARS-T were assessed for SF3B1, JAK2 and MPL exon 10 mutation status. Eighteen patients (64%) showed somatically acquired mutation of SF3B1. The median mutant allele burden was 43%, consistent with the presence in the majority of patients of clonal hematopoiesis characterized by a dominant clone carrying a heterozygous SF3B1 mutation. Fourteen patients carried the JAK2 (V617F) mutation (median allele burden 6.5%, range 0.4–29.5%), while one had a JAK2 exon 12 mutation. In 13 cases, the JAK2 mutation was detected at the time of diagnosis, whereas in 2 patients, who had a typical RARS phenotype and were negative for JAK2 mutations at clinical onset, JAK2 (V617F) was detected 18 and 32 months after diagnosis, respectively, and concomitantly with a progressive increase in platelet count. Four patients, two of whom were JAK2 (V617F)-positive, carried the MPL (W515L) mutation (median allele burden 27.5%, range 25–50%). Concomitant mutations of SF3B1 and JAK2 or MPL were observed in 8 cases. Seven patients carried an SF3B1 mutation and JAK2 (V617F), while one carried SF3B1 (K700E), JAK2 (V617F), and MPL (W515L). In all these cases, the SF3B1 mutant allele burden was higher than that of JAK2 or MPL, indicating the existence of an SF3B1-mutated dominant clone with minority JAK2- or MPL-mutated clones. We genotyped individual colonies from peripheral blood in 2 patients with concomitant mutations. In a patient with granulocyte SF3B1 and JAK2 mutant allele burdens equal to 45% and 8%, respectively, SF3B1 (H662Q) was detected in 9 of 11 colonies, three of which also carried JAK2 (V617F); the remaining two colonies had wild type SF3B1 and JAK2. These data are consistent with the existence of a dominant hematopoietic clone carrying the SF3B1 mutation and the subsequent emergence of a JAK2-mutated subclone. The other patient, who was initially SF3B1- mutated and JAK2 wild type, at the time of colony assay had a mutant allele burden equal to 50% and 1% for SF3B1 (K700E) and JAK2 (V617F), respectively. Forty-three of 45 colonies were heterozygous for SF3B1 (K700E) and wild type for JAK2. The opposite pattern was observed in the remaining 2 colonies, which carried just JAK2 (V617F). These data indicate the coexistence of two distinct clones, a dominant one carrying the SF3B1 mutation and a minority one carrying JAK2 (V617F). In summary, these observations suggest that the occurrence of an SF3B1 mutation represents an early event in patients with RARS-T, likely causing mitochondrial iron overload, ring sideroblasts, ineffective erythropoiesis and anemia, typical myelodysplastic features. The subsequent occurrence of a somatic mutation of JAK2 or MPL involves the emergence of minority clones and the acquisition of myeloproliferative features. JAK2- mutated clones may emerge as subclones of the dominant SF3B1-mutated clone or as independent clones. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Using the Minor Variant Finder software to identify and quantify the allelic burden level of somatic mutations in oncohematologic diseases

2020 ◽  
Vol 15 (2) ◽  
pp. 85-91
Author(s):  
T. N. Subbotina ◽  
I. E. Maslyukova ◽  
A. A. Faleeva ◽  
P. A. Nikolaeva ◽  
A. S. Khazieva ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Sanger Sequencing ◽  
Quantitative Assessment ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Mutant Gene ◽  
Positive Sample ◽  
Allele Burden ◽  
Minor Variant ◽  
Allelic Burden

Background. There are problems related to both quantitative assessment of an allele burden level of a mutant gene and interpretation of results in DNA samples with the burden level of the mutant allele less than 15–20 %, when using Sanger sequencing for analyzing somatic mutations. Applied Biosystems (USA) has developed new software Minor Variant Finder, which allows determining mutations with the allele burden level from 5 %.The objective: to determine the allele burden level and identification of minor variants of somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene using Minor Variant Finder software in patients with myeloproliferative neoplasms.Materials and methods. The level of mutant allele burden for 15 patients with myeloproliferative neoplasms was determined by the identified mutations using the Minor Variant Finder software, after analysis of point somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene by Sanger sequencing.Results. The allele burden level in all 5 ASXL1-positive samples and BCR-ABL-positive sample was determined as higher than 20 % using the Minor Variant Finder software. The allele burden level in 2 cases was higher than 20 % and in 7 cases lower than 20 %, when we analyzed 9 JAK2-positive samples.Conclusion. Minor Variant Finder software can be used to estimate the allele burden level and to identify minor variants of somatic mutations in the ASXL, JAK2 and BCR-ABL genes.

Abstract 12726: Crucial Role of Jak2 V617f-positive Myeloproliferative Neoplasm in the Development of Aortic Aneurysm

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Tetsuro Yokokawa ◽  
Tomofumi Misaka ◽  
Yusuke KIMISHIMA ◽  
Kento Wada ◽  
Keiji Minakawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Angiotensin Ii ◽  
Aortic Aneurysm ◽  
Mrna Expression ◽  
Abdominal Aorta ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Vascular Complication ◽  
The Impact

Objective: To investigate the impact of hematopoietic JAK2V617F, which causes myeloproliferative neoplasms (MPNs), on the development of aortic aneurysm (AA). Approach and Results: We applied a bone marrow transplantation (BMT) strategy using the donor cells from Jak2 V617F transgenic (JAK2 V617F ) mice into the lethally irradiated apolipoprotein E-deficient mice. To induce the AA formation and progression, the recipient mice (BMT mice) were subjected to continuous angiotensin II infusion. Abdominal aortic diameter in JAK2 V617F -BMT mice was significantly enlarged compared to the control wild-type (WT)-BMT mice in response to angiotensin II. The incidence of abdominal AA was significantly higher in JAK2 V617F -BMT mice than in WT-BMT mice. Hematopoietic JAK2V617F accelerated aortic elastic lamina degradation as well as activation of matrix metalloproteinase (MMP)-2 and MMP-9 in the abdominal aorta. The numbers of CD68 + macrophages and Ly6B.2 + neutrophils and cytokine expressions such as Ccl6 and Tgfb1 were significantly increased in the abdominal aorta of JAK2 V617F -BMT mice accompanied by STAT3 activation. Bone marrow-derived macrophages carrying JAK2V617F showed elevations of both Mmp2 and Mmp9 mRNA expression. Finally, we found that 23% of MPN patients with JAK2 V617F mutation showed the presence of AA and increases in TGFB3 and IL-8 mRNA expression of the peripheral leukocytes. Conclusions: Hematopoietic JAK2V617F was involved in the development of AA through increases in the infiltration of inflammatory cells and MMP activation. Our findings provide a novel feature of vascular complication of AA in MPNs due to constitutive activation of the hematopoietic JAK-STAT pathway.

Identification of oncostatin M as a JAK2 V617F‐dependent amplifier of cytokine production and bone marrow remodeling in myeloproliferative neoplasms

2011 ◽  
Vol 26 (2) ◽  
pp. 894-906 ◽  
Author(s):  
Gregor Hoermann ◽  
Sabine Cerny‐Reiterer ◽  
Harald Herrmann ◽  
Katharina Blatt ◽  
Martin Bilban ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cytokine Production ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Oncostatin M

Cultivation of Megakaryocytes On a Collagen Matrix: A More Sensitive and Reproducible Test for the Diagnosis of Patients with Essential Thrombocythaemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4970-4970
Author(s):  
Adrian Emanuel Schmidt ◽  
Patricia Darlington ◽  
Lucie Kopfstein ◽  
Elisabeth Ischi ◽  
Elisabeth Oppliger Leibundgut ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Negative Predictive Value ◽  
Predictive Value ◽  
Healthy Subjects ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Essential Thrombocythaemia ◽  
Jak2 Mutation ◽  
Spontaneous Proliferation

Abstract Abstract 4970 Background Essential thrombocythaemia (ET) is one of the chronic myeloproliferative neoplasms (MPN), along with polycythaemia vera (PV), primary myelofibrosis (PMF) and chronic myeloid leukaemia (CML). Their common feature is excessive proliferation of a certain stem or progenitor cell in the bone marrow; in the case of ET, the megakaryocytic lineage is affected. Clinical manifestations include thrombotic events and haemorrhage. Diagnosis of ET according to new WHO-criteria requires a sustained high platelet count, bone marrow biopsy showing proliferation of the megakaryocytic lineage with large and mature morphology, demonstration of JAK2 V617F (although only present in about 50% of patients with ET) or another clonal marker and explicit exclusion of other myeloid and myeloproliferative neoplasms as well as signs of reactive thrombocytosis. Additionally, spontaneous proliferation of megakaryocytes obtained from peripheral blood can be detected in in vitro culture assays. Presently, we use agar as a matrix for megakaryocyte cultivation, although this assay has never been validated in connection with ET. The identification of megakaryocytic colonies grown on agar can sometimes be quite difficult. Our aims were therefore to technically evaluate the use of a collagen based matrix and to investigate its suitability to identify patients with ET. Patients and Methods We have examined 63 patients (26 with ET, 21 with PV, 8 with myelofibrosis [MF; including PMF and post-ET/PV-MF], 6 with secondary or idiopathic erythrocytosis and 2 with secondary thrombocytosis; mean age=59.8, male=33, female=30, mean platelet count 457 G/l) and 5 healthy subjects. Following informed consent, both clinical and laboratory data was collected. Medication intake, phlebotomies, smoking habits and regular haemogram results were noted in order to recognise possible confounding factors influencing laboratory results. Results of megakaryocyte cultivation on both agar and collagen matrixes were recorded, considering both spontaneous growth and growth stimulated by megakaryocyte derived growth factor (MDGF). Results Based on our collagen culture results we were able to define 2 or more spontaneously grown megakaryocyte colonies as the most optimal cut-off for the identification of patients with MPN (sensitivity 71%, specificity 100% with positive and negative predictive values of 100% and 45%, respectively). Compared to the agar culture results (where a specificity and a positive predictive value of 100% were demonstrated at a cut-off value of ≥ 10 CFU-Mega) we found a higher accuracy and better reproducibility. In addition, we observed an improved negative predictive value (45% with collagen versus 25% with agar cultures) reducing false negative results. Healthy subjects and patients with secondary thrombocytosis showed no significant spontaneous megakaryocyte proliferation. In patients with MF, we observed strong spontaneous and MDGF-stimulated growth of megakaryocytic colonies. At a cut-off value of ≥ 50 CFU-Mega (after stimulation with MDGF), the collagen assay showed a sensitivity of 100% and a specifity of 70% for this special form of MPN, resulting in a negative predictive value of 100%. We found no confounding clinical or laboratory parameters such as medication intake (particularly cytoreductive treatment with hydroxyurea) or phlebotomies influencing our culture results, and no significant effect of the Jak2-V617F mutation on the growth behaviour of megakaryocytic colonies. Conclusion The results of this ongoing study imply that the collagen based assay is more sensitive, specific, time efficient and user friendly regarding the detection of spontaneous proliferation of megakaryocytes than the currently used agar based culture assay. In addition, the collagen based assay also has the great advantage that it allows isolation of single megakaryocytic colonies for further analyses, for example PCR-based identification of a JAK2 mutation. Furthermore, the collagen based assay facilitates the diagnosis of patients with MPN, especially in cases where conventional diagnostic criteria are lacking, such as in ET without a JAK2 mutation. Ultimately, the new assay may well be able to detect transformation from PV/ET to MF. Disclosures No relevant conflicts of interest to declare.

Direct Activation of STAT5 by TEL-Lyn Fusion Protein Promotes Induction of Myeloproliferative Neoplasms with Myelofibrosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4114-4114
Author(s):  
Yusuke Takeda ◽  
Chiaki Nakaseko ◽  
Hiroaki Tanaka ◽  
Masahiro Takeuchi ◽  
Makiko Yui ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Signaling Molecules ◽  
Janus Kinase ◽  
Idiopathic Myelofibrosis ◽  
Lyn Kinase

Abstract Abstract 4114 Background Myeloproliferative neoplasms (MPN), a group of hematopoietic stem cell (HSC) disorders, are often accompanied by myelofibrosis. The V617F somatic mutation in the Janus kinase 2 (JAK2) gene has recently been found in the majority of patients with polycythemia vera (PV) and more than half of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). The expression of JAK2 V617F causes a PV-like disease with myelofibrosis in a murine bone marrow (BM) transplant model. In addition, a gain-of-function c-MPL W515 mutation was described in nearly 10% of patients with JAK2 V617F-negative IMF. However, the mechanism responsible for MPD and the formation of myelofibrosis in patients without JAK2 or c-MPL mutations is still unclear. We previously identified the fusion of the TEL gene to the Lyn gene (TEL-Lyn) in idiopathic myelofibrosis with ins(12;8)(p13;q11q21). The introduction of TEL-Lyn into HSCs resulted in fatal MPN with massive myelofibrosis in mice, implicating the rearranged Lyn kinase in the pathogenesis of MPN with myelofibrosis. However, the signaling molecules directly downstream from and activated by TEL-Lyn remain unknown. Design and Methods We examined the signaling pathways activated by TEL-Lyn by Western blotting, immunoprecipitation, and in vitro kinase assay using a TEL-Lyn kinase-dead mutant as a control. We further characterized the functional properties of Stat5-deficient HSCs transduced with TEL-Lyn by colony-forming assay and bone marrow transplantation to evaluate the role of STAT5 in TEL-Lyn-induced MPN. Results TEL-Lyn was demonstrated to be constitutively active as a kinase through autophosphorylation. In TEL-Lyn-expressing cells, STAT5, STAT3, and Akt were constitutively activated. Among these signaling molecules, STAT5 was activated most prominently and this occurred without the activation of Jak2, the major kinase for STAT5. TEL-Lyn was co-immunoprecipitated with STAT5, and STAT5 was phosphorylated when incubated with TEL-Lyn, but not with TEL-Lyn kinase-dead mutant. These results indicate that TEL-Lyn interacts with STAT5 and directly activates STAT5 both in vitro and in vivo. Of note, the capacity of TEL-Lyn to support the formation of hematopoietic colonies under cytokine-free conditions in vitro and to induce MPN with myelofibrosis in vivo was profoundly attenuated in a Stat5-null background. Conclusions In this study, we clearly showed that TEL-Lyn directly activates STAT5 and the capacity of TEL-Lyn to induce MPN with myelofibrosis was profoundly attenuated in the absence of STAT5. Our findings of TEL-Lyn in this study support the role of the Src family kinases in the regulation of STAT pathways and implicate active Lyn in the alternative pathway for STAT activation in pathological cytokine signaling. Our mouse model of MPD with myelofibrosis would be beneficial for the analysis of therapeutic approaches for myelofibrosis. Disclosures: No relevant conflicts of interest to declare.

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