First Achievements of MPN&MPNr-EuroNet (COST Action BM0902), a New European Network Dedicated to the Diagnosis of Myeloproliferative Neoplasms and Hereditary Erythrocytosis and Thrombocytosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2809-2809
Author(s):  
Sylvie Hermouet ◽  
Eric Lippert ◽  
Niels Pallisgaard ◽  
Jiri Schwarz ◽  
Mary Frances McMullin ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Hereditary Diseases ◽  
Diagnostic Tools ◽  
Training School ◽  
Scientific Cooperation ◽  
Jak2 Mutation ◽  
Cost Action ◽  
New Genes ◽  
Training Schools

Abstract Abstract 2809 Background: The MPN&MPNr-EuroNet network, created in November 2009, is supported by the European program COST (CoOperation in Science and Technology). It is open to all colleagues active in the fields of myeloprolifeative neoplasms (MPN) and related hereditary diseases (MPNr: hereditary erythrocytosis and thrombocytosis). AIMS: To facilitate, improve and innovate in the diagnosis of MPN and hereditary erythrocytosis and thrombocytosis in Europe. Methods: MPN&MPNr-EuroNet has formed 4 working groups (WG): WG 1 focuses on JAK2 -mutated MPN; WG 2 is dedicated to thrombocythemia and myelofibroses without mutation of JAK2 and includes subgroups specialized in hereditary thrombocytosis (HT) and in histopathology; WG 3 is dedicated to hereditary erythrocytosis (HE); WG 4 is responsible for scientific cooperation and the diffusion of scientific knowledge. Results: During the first 18 months of MPN&MPNr-EuroNet activity, 77 colleagues from 19 countries (16 European countries plus Israel, Turkey, and the USA), joined the network and participated in the four WG, resulting in the achievements listed below. WG 1: 1) determination of the best JAK2 V617F assays, a joint MPN&MPNr-EuroNet/European LeukemiaNet project; 2) on-going study of MPN cases with low JAK2 V617F burden; 3) on-going study of MPN cases with multiple JAK2 mutation. WG 2: 1) list of laboratories responsible for the diagnosis of MPL and THPO mutations in Europe; 2) first international quality test of the detection of MPL mutations; 3) on-going study of new THPO and MPL mutations in HT cases; 4) on-going study of the histopathology of MPN without JAK2 mutation. WG 3: 1) list of laboratories responsible for the diagnosis of HE in Europe; 2) consensus on a diagnostic algorithm for the diagnosis of HE; 3) close interaction with COST Action TD0901 (HypoxiaNet) to facilitate the discovery of new genes of interest for the diagnosis of HE; 4) exchange of positive control samples for the main mutations responsible for HE; 5) study of idiopathic erythrocytosis. WG 4: 1) MPN&MPNr-EuroNet website: www.mpneuronet.eu; 2) organization of bi-annual meetings (5th meeting: March 7–9, 2012, Belfast, United Kingdom); 3) organization of annual training schools: a May training school dedicated to the molecular detection of JAK2 and MPL mutations (in Nîmes, France), and an October training school dedicated to hereditary erythrocytosis (in Coimbra, Portugal); 4) financial support for short term scientific missions for exchange and collaborative studies between participating institutions. Conclusion: MPN&MPNr-EuroNet will enable European researchers, biologists and clinicians to define new diagnostic tools and exchange technologies. MPN&MPNr-EuroNet is open to all interested physicians and scientists and we invite new members, including those from outside Europe, to join. Scholarships are available to finance participation in meetings and training schools, and to facilitate exchanges between participating institutions. For detailed information on all MPN&MPNr-EuroNet activities, see www.mpneuronet.eu. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Vannucchi:Novartis: Honoraria.

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.

Analysis of JAK2 V617F Mutation and Its Clinical Significance in Patients with Thrombocythemia and Other Myeloproliferative Neoplasms in Chinese

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4687-4687
Author(s):  
Yue Xu ◽  
Changxin Yin ◽  
Han He ◽  
Lingling Shu ◽  
Fuqun Wu ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Jak2 Mutation ◽  
Western Countries ◽  
Arms Pcr ◽  
V617f Mutation

Abstract Abstract 4687 JAK2 mutation is commonly found in Philadelphia-negative myeloproliferative neoplasms (MPNs). In Western countries, this mutation is found in approximately 96 percent of people with polycythemia vera, half of individuals with essential thrombocythemia or primary myelofibrosis. We used the method of amplification refractory mutation PCR (ARMS-PCR) to investigate MPN patients in China. We focused our study on patients with essential thrombocythemia (ET). ARMS-PCR was used to detect JAK2 V617F mutation in the bone barrow (BM) or peripheral blood of 37 MPN patients, which consisting of 7 ET, 5 polycythemia vera (PV), 5 chronic myeloid leukemia (CML), 5 chronic idiopathic myelofibrosis (CIMF), as well as 15 suspected MPNs. 17 cases of JAK2 V617F mutation (45.9%) were found in 37 patients, including 4 ET (57.1%), 4 PV (80.0%), 3 CIMF (60.0%), 6 suspected MPNs (40.0%). We did not find JAK2 V617F in the patients with CML. Our results indicated that the frequency of JAK2 V617F mutation in bcr/abl-negative MPNs in Chinese is similar to that in MPN patients in Western countries. At the same time, ARMS-PCR can distinguish the mutation is heterozygous or homozygous. Most patients were heterozygous for JAK2 but only a few were homozygous. In conclusion, our study showed that JAK2 V617F mutation frequency in Chinese MPN patients is similar to that in patients with this disorder in the West. It is the major molecular genetic abnormality in bcr-abl negative MPN and it can be used for diagnosis of MPN in China. Disclosures: No relevant conflicts of interest to declare.

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

2017 ◽  
Vol 44 (3-4) ◽  
pp. 97-104 ◽  
Author(s):  
Matthias Lamy ◽  
Paola Palazzo ◽  
Pierre Agius ◽  
Jean Claude Chomel ◽  
Jonathan Ciron ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Cerebral Venous Thrombosis ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Janus Kinase ◽  
Jak2 V617f Mutation ◽  
Janus Kinase 2 ◽  
Jak2 Mutation ◽  
Venous Thromboembolic Events ◽  
V617f Mutation

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

scholarly journals JAK2 V617F Mutational Burden and Clinical Findings Relationship in Myeloproliferative Neoplasms

2021 ◽  
Author(s):  
Cigdem Yuce Kahraman ◽  
Gulden Sincan ◽  
Abdulgani Tatar
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Spleen Size ◽  
Jak2 Mutation ◽  
Laboratory Findings ◽  
Treatment Protocols ◽  
Mutational Burden ◽  
Mutation Burden ◽  
Significant Difference

Abstract Background: Philadelphia-negative chronic myeloproliferative neoplasms(MPN) are associated with various genetic abnormalities. JAK2 V617F mutation is the most common one and important for diagnosis. We aimed to evaluate JAK2 mutation status and clinical parameters relationship of the MPN patients referred to our clinic.Methods and Results: We evaluate 143 JAK-2 positive patients diagnosed with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). JAK2 mutational burden was higher in PV and PMF than ET. Laboratory findings were different in MPN groups and higher –lower JAK2 mutational burden groups. JAK2 mutational burden was correlated with spleen size and LDH level, particularly in PMF. There was no significant difference in age, gender, jak2 mutation burden and laboratory findings in patients with and without thrombosis and bleeding. Common treatment protocols were acetylsalicylic acid (ASA) + hydroxyurea, ASA and ASA + phlebotomy and others respectively. JAK2 mutational burden, mean age and LDH level were higher significantly in the patients treated with ASA+ hydroxyurea than the patients treated with ASA.Conclusion: We speculate that if the spleen size in MPN is as large as the massive splenomegaly and the LDH level is high, the JAK2 mutation burden may tend to be higher. This relationship is more pronounced for PMF.

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

2010 ◽  
Vol 138 (9-10) ◽  
pp. 614-618
Author(s):  
Vesna Spasovski ◽  
Natasa Tosic ◽  
Tatjana Kostic ◽  
Sonja Pavlovic ◽  
Milica Colovic
Keyword(s):  
Cell Proliferation ◽  
Myeloproliferative Neoplasms ◽  
Myeloid Cell ◽  
Jak2 V617f ◽  
Signalling Pathway ◽  
Janus Kinase ◽  
Jak2 V617f Mutation ◽  
Polycythaemia Vera ◽  
Jak2 Mutation ◽  
V617f Mutation

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

The JAK2 46/1 Haplotype Analysis In Essential Thrombocythaemia and Polycythaemia Rubra Vera Reveals That CC Genotype Is Associated with a Higher JAK2V617F and c-MPL W515 Allele Burden

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1977-1977
Author(s):  
Shameem Mahmood ◽  
Louise Mellish ◽  
Nicholas Lea ◽  
Austin G Kulasekararaj ◽  
Atiyeh Abdallah ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Association Studies ◽  
Statistical Significance ◽  
Jak2 V617f ◽  
Tagging Snp ◽  
Jak2 Mutation ◽  
Allele Burden ◽  
Significant Difference ◽  
Jak2v617f Allele Burden

Abstract Abstract 1977 First 2 authors contributed equally. Background: Genomic-wide association studies have identified the germline 46/1 haplotype as a predisposing allele associated with JAK2V617F positive myeloproliferative neoplasms (MPN). The present study analysed data on 856 JAK2V617F positive patients, 326 of which had complete clinical data. Aims: To evaluate the JAK2 46/1 haplotype frequencies, JAK2V617F allele burden, c-MPL 515 mutation and risk of transformation. Methods: Genomic DNA from whole peripheral blood or bone marrow patient samples was analysed as follows: JAK2V617F allele burden by Q-PCR, JAK2 exon 12 mutations by Q-PCR and PCR fragment analysis, MPL W515 L and K mutations by allele specific PCR. The 46/1 JAK2 mutation susceptibility haplotype (46/1) tagging SNP rs12343867 (susceptibility allele C) were analysed by pyrosequencing. Results: The allele frequency for the 46/1 tag SNP rs1234867 in the 856 patients was calculated for the total JAK2V617F cohort (0.48) and the clinical entities ET (0.34) and PRV (0.44) confirming that the 46/1 haplotype is greatly over represented in JAK2V617F MPD patients as compared to published the control population (Wellcome Trust Case Control Consortium (WTCCC) (0.24). The Analysis of the 856 patients demonstrated that JAK2V617F and c-MPL W515L/K mutations co-existed in 16 patients(1.9%), the incidence of c-MPL W515L being twice as common as the c-MPL W515K mutations. There was no correlation between these mutations and age or 46/1 haplotype status. The JAK2V617F allele burden (AB) was lower in the c-MPL mutant patients, the average JAK2AB 31%. 3 out 4 c-MPL patients for which clinical information was available had a diagnosis of ET. No JAK2 exon 12 mutations were found in any of the 859 JAK2V617F positive samples suggesting that co-existing JAK2 exon 14 and exon 12 mutations are extremely rare. The genotypic data in ET patients showed: C/C 12%, C/T 44%, T/T 44% and their respective JAK2V617 allele burden (AB) were 46%, 32%, 29%. The genotype data in PRV patients: C/C 18%, C/T 53%, T/T 28.6% and their respective AB were 47%, 31% and 39%. The median AB was 32% (n=121) for ET and 37% (n=103) for PRV. Within a cohort of 255 patients (ET=138, PRV=117) 4% of ET and 6% of PRV patients transformed to acute myeloid leukaemia or myelofibrosis with no predominant haplotype association. In the ET patients, the median AB was 35%, there was no significant difference in the JAK2 V617F AB between those who transformed or not (p=0.45). Interestingly, on the whole ET group C/C genotype patients were more likely to have an allele burden >50% (p=0.058). In the PRV patients, the median AB was 48%. Again, the C/C genotype, PRV patients were more likely to have an AB>50% (p=0.06), although not reaching statistical significance. Conclusions: The 46/1 haplotype in both clinical entities ET and PRV demonstrated a higher allele burden in the C/C genotype in comparison to the other genotypes. No predominant haplotype predicted the risk of transformation to a more aggressive disease such as MF or AML. The analysis also showed that c-MPL W515K/L mutations can co-exist with JAK2V617F. The c-MPL W515K/L mutations did not exhibit a positive correlation with a preferential 46/1, but was associated with a lower allele burden. No co-existing exon 12 and exon 14 mutations were found, suggesting the rarity of this occurrence. Disclosures: No relevant conflicts of interest to declare.

Effects of the JAK2 mutation on the hematopoietic stem and progenitor compartment in human myeloproliferative neoplasms

Blood ◽  
2011 ◽  
Vol 118 (1) ◽  
pp. 177-181 ◽  
Author(s):  
Shubha Anand ◽  
Frances Stedham ◽  
Philip Beer ◽  
Emma Gudgin ◽  
Christina A. Ortmann ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Myeloid Differentiation ◽  
Jak2 Mutation ◽  
Hematopoietic Stem ◽  
Progenitor Compartment ◽  
Proliferative Advantage

Abstract The JAK2 V617F mutation is present in the majority of patients with a myeloproliferative neoplasm (MPN) and is sufficient to recapitulate an MPN in murine models. However, the consequences of JAK2 mutations for myeloid differentiation are poorly understood. After systematic analyses of a large cohort of JAK2-mutated MPN patients, we demonstrate in vivo that JAK2 mutations do not alter hematopoietic stem and progenitor cell com-partment size or in vitro behavior but generate expansion of later myeloid differentiation compartments, where homozygous expression of the mutation confers an added proliferative advantage at the single-cell level. In addition, we demonstrate that these findings may be partially explained by the expression pattern of JAK2, which markedly increases on myeloid differentiation. Our findings have potential clinical relevance, as they predict that JAK2 inhibitors may control myeloproliferation, but may have limited efficacy in eradicating the leukemic stem cells that sustain the human MPN.

scholarly journals Myeloproliferative Neoplasm or Reactive Process? A Rare Case of Acute Myeloid Leukemia and Transient Posttreatment Megakaryocytic Hyperplasia with JAK-2 Mutation

2016 ◽  
Vol 2016 ◽  
pp. 1-5
Author(s):  
Steven Wang ◽  
Jie Yan ◽  
Guangde Zhou ◽  
Rebecca Heintzelman ◽  
J. Steve Hou
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Rare Case ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Cell Transplant ◽  
Jak2 Mutation ◽  
V617f Mutation ◽  
Acute Myeloid

Myeloproliferative neoplasms (MPNs) are hematopoietic malignancies characterized by unchecked proliferation of differentiated myeloid cells. The most common BCR-ABL1-negative MPNs are polycythemia vera, essential thrombocythemia, and primary myelofibrosis. The discovery of JAK2 V617F mutation has improved our understanding of the molecular basis of MPN. The high frequency of JAK2 mutation in MPN makes JAK2 mutation testing an essential diagnostic tool and potential therapeutic target for MPN. Here, we present a rare case of a 34-year-old patient who was initially diagnosed with acute myeloid leukemia (AML) with mutated NPM1. After chemotherapy treatment followed by granulocyte colony stimulating factor administration, the patient achieved complete remission of AML. However, the bone marrow showed hypercellularity with granulocytic hyperplasia, markedly increased atypical megakaryocytes (50.2/HPF) with focal clustering, and reticulin fibrosis (3/4). JAK2 V617F mutation was also detected. Considering the possibility of AML transformed from a previous undiagnosed MPN, patient underwent peripheral blood allogenic stem cell transplant. This case illustrates the diagnostic challenges of firmly establishing a diagnosis between similar, but distinct, disease entities and an accurate clinicopathological differentiation is crucial.

Single Center Experience with Screening for JAK2 V617F, cMPL W515L, cMPL W515K, and JAK2 L611V Mutations in MPN. Rarity of JAK2 L611V Does Not Justify Its Routine Screening

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5167-5167
Author(s):  
Soo Jin Kim ◽  
Sabina Swierczek ◽  
Jonathan Schumacher ◽  
Todd W. Kelley ◽  
Sherrie L. Perkins ◽  
...  
Keyword(s):  
Somatic Mutation ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Routine Screening ◽  
Locked Nucleic Acid ◽  
Diagnostic Tools ◽  
Wild Type ◽  
Allelic Discrimination ◽  
Clinical Diagnoses

Abstract Abstract 5167 The myeloproliferative neoplasms (MPNs) polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF) are caused by an unregulated clonal proliferation of cells derived from a pluripotent stem cell. The exon 14 JAK2 V617F somatic mutation and cMPL mutations in codon 515 of exon 10 have provided excellent diagnostic tools to distinguish MPNs from other diseases with similar clinical features. JAK2 V617F mutations are present in ∼95% of PV patients and ∼50% of ET or PMF patients. Other somatic mutations have also been found in JAK2 exon 12 in patients with PV. Recently, a new somatic mutation of JAK2 in exon 14, L611V, was reported in 3 out of 168 PV patients in cis with JAK2 V617F (Cleyrat Leukemia 2010). Two cMPL mutations, cMPL W515L and cMPL W515K, have been identified in MPNs. The cMPL W515L mutation is reported to be present in ET and PMF patients, while cMPL W515K in some PMF patients. We designed and validated a new rapid and sensitive allele-specific quantitative PCR assay (AS-qPCR) for determination of the JAK2 L611V mutation. In our design, we included a locked nucleic acid and a mismatch in the allelic specific primers in order to enhance allelic discrimination in the PCR reaction. The assay was sensitive to mutant frequencies of >2% in the background of wild-type alleles. Allelic discrimination of the assay is 14 cycles for wild type and 11 cycles for mutant alleles. We studied 584 patients referred with clinical diagnoses of MPN belonging to two different groups: 1) 390 patients, who, based on clinical and laboratory evaluations, had diagnoses of PV (n=74), ET (n=60), MF (n=30), other unspecified MPNs (n=189), and other non-PV patients with erythrocytosis (n=37); 2) DNA from patients referred to ARUP Laboratories with otherwise unspecified clinical diagnoses of MPN who were found to be 196 JAK2 V617F-positive but appropriate clinical and other testing was not available for classification. The first group was tested for the JAK2 V617F, JAK2 L611V, cMPL W515L and W515K mutations. Out of 390 patients, 37% (148/390) were JAK2 V617F positive (over >90% of PV patients), 9 (ET or PMF) patients carried the cMPL W515L mutation, and 1 PMF patient had 93% allelic burden for the cMPL W515K mutation. While cMPL W515L mutation was not previously reported in PV, one PV patient was positive for both the cMPL W515L and JAK2 V617F mutations. Of the 10 cMPL W515L mutations, two MF patients carried JAK2 V617F mutations concurrently. No JAK2 L611V mutation was detected in this cohort. The additional 196 JAK2 V617F-positive samples from the patients with unspecified MPNs were also screened for the JAK2 L611V mutation and none were positive. We conclude that the rarity of the JAK2 L611V mutation does not justify its routine screening. We suggest that those patients who fulfill the WHO criteria for PV should be further evaluated by more productive laboratory studies such as screening for JAK2 exon 12 mutations, clonality studies in females and if negative assays for germline mutations such as EPOR and VHL gene mutations. Disclosures: No relevant conflicts of interest to declare.

Mutations of Calreticulin in Myeloproliferative Neoplasms Contribute to Disease Progression Not through Inhibition of Phagocytosis, but through Enhanced Proliferation Production of Myeloid Myelo-Erythroid Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4594-4594
Author(s):  
Shinya Daitoku ◽  
Katsuto Takenaka ◽  
Takuji Yamauchi ◽  
Koichi Akashi
Keyword(s):  
Progenitor Cells ◽  
Myeloproliferative Neoplasms ◽  
Hematopoietic Cells ◽  
Jak2 V617f ◽  
Jak2 Mutation ◽  
Hematopoietic Stem ◽  
Calr Mutations ◽  
Phagocytosis Index ◽  
Calr Mutation

Abstract Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell disorders characterized by overproduction of mature myeloid cells. Recently, somatic mutation of calreticulin (CALR) was frequently found in MPN patients who do not have JAK2 mutation. The CALR mutation in MPN patients usually resulted in loss-of-function of CALR, which may induce impairment of physiological phagocytotic pathway, because surface CALR plays a critical role for macrophages in recognition of low-density lipoprotein receptor-related protein 1 (LRP1) on the targets, mediating pro-phagocytic signals. We hypothesized that the non-functional CALR mutation renders cells resistant to phagocytosis, and impairs the “programmed cell removal” of progenitors or mature blood cells, resulting in accumulation of hematopoietic cells in MPNs. In 135 Japanese MPNs patients enrolled in this study, including polycythemia vera (PV), essential thrombocytosis (ET) or primary myelofibrosis (PMF), 34 patients (25.2%) had CALR mutations, and 80 (59.3%) patients had JAK2 V617F mutation, respectively. CALR mutations were heterozygous in all 34 patients (27 patients with ET, 7 with PMF). On the other hand, JAK2 V617F mutations were found in 26 patients with PV, 39 with ET, and 15 with PMF. The expression levels of pro-phagocytotic CALR were normal in these MPN patients. We then performed in vitro phagocytosis assay to test whether the heterozygous CALR mutation affects engulfment of blood cells by macrophages. Hematopoietic stem cells (HSCs), progenitor cell populations such as common myeloid progenitors (CMPs), megakaryocyte/erythroid progenitors (MEPs) and granulocyte/monocyte progenitors (GMPs), and mature myeloid cells were isolated and opsonized, and were co-cultured with activated macrophages for 2 hours. After the culture, we enumerate macrophages and engulfed cells to analyze phagocytosis index (number of engulfed cells/number of macrophages) (Kuriyama et al. Blood 2012). However, the phagocytosis index was not changed in any of purified hematopoietic cells, irrespective of the presence of CALR or JAK2 mutation. These results strongly suggest that heterozygous, non-functional CALR mutation, and gain-of-function JAK2 mutations should not affect the engulfment process for hematopoietic cells by macrophages. We then investigated the effect of CALR or JAK2 mutations on differentiation and proliferation of stem or progenitor cells in MPNs. We performed colony-forming cell assay of multipotent cells, such as HSCs and CMPs, and evaluated clonal burden of CALR and JAK2 mutations in colonies derived from these stem and progenitor cells. In vitro culture showed that HSCs and CMPs with CALR and JAK2 mutations gave rise to granulocyte/monocyte (GM) or megakaryocyte/erythroid (MegE)-related colonies, whose frequencies were almost identical to those in wild-type controls, suggesting that these mutations do not affect myelo-erythroid lineage commitment at the multipotent stem or progenitor stages. In contrast, when we cultured GMPs and MEPs, frequencies of colonies with CALR or JAK2 mutations were significantly higher as compared to those in HSCs or CMPs (P<0.05); In patients with CALR mutation, 32.5% of HSC-derived colonies had CALR mutations, whereas in MEPs and GMPs, CALR mutations were found in 51.0% and 70%, respectively. In JAK2 mutated MPNs, 17.2% of HSC-derived colonies had JAK2 mutation, whereas 64.7% of MEP- and 87.9% of GMP-derived colonies had this mutation. These results indicate that clones with CALR or JAK2 mutations could contribute more robustly to maintain MEPs and GMPs, and these committed progenitors with mutations might produce higher amounts of mature myelo-erythroid cells, leading to progression of MPNs. Thus, CALR mutation contributes to progression of MPN, not through inhibition of phagocytic clearance, but presumably through enhanced production of myelo-erythroid lineage cells, as JAK2 mutation does. It is important to investigate the mechanism on which the CALR mutation causes overproduction of myelo-erythroid cells in future study. Disclosures No relevant conflicts of interest to declare.

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