Isolation, Expansion and Characterization of BM-Mesenchimal Cells (MSCs) in Patients Affected By Chronic Myeloproliferative neoplasm (MPN)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5142-5142
Author(s):  
Patrizia Chiusolo ◽  
Francesca Annunziata ◽  
Elena Rossi ◽  
Silvia Betti ◽  
Silvia Bellesi ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Gene Mutations ◽  
Jak2 V617f ◽  
Cell Types ◽  
Erythropoietin Receptor ◽  
Jak2 V617f Mutation ◽  
Hematopoietic Stem ◽  
V617f Mutation

Abstract Introduction MSCs constitute a pivotal cell type capable of shaping both the architecture of the microenvironment and modulating communication between the various cell types through effects on the extracellular matrix (ECM) and by secretion of various growth factors and cytokines. MSCs and hematopoietic stem cells are thought to share the same mesenchymal origin. Some data confirm that MSCs express a functional erythropoietin receptor and JAK2-transduction pathway, but their role in the development and evolution of MPN is still not well known so our aim was the isolation, expansion and characterization of MSCs in patients affected by MPN. Some data indicates that BM-MSCs of patients affected by MPN do not carry the JAK2-V617F mutation. We studied 20 patients affected by MPN with the following characteristics: M/F 12/8, median age 53years, 8 affected by PV, 8 by ET and 4 by PMF. 15 patients were positive for JAK2 V617F mutation, 1 pts for cMPL and 2 were CARL mutations carriers. Methods: MSC were isolated by bone marrow fraction by gradient separation on Lympholyte cell separation media and expanded in culture with a specific medium (MesenCult) in plastic-adherent cultures up to the second passage. DNA was extracted from MSC using QIAmp DNA Mini kit and the study of recurrent alterations (JAK2, MPL and CARL gene mutations was performed). Results: MCS were expanded in 14 on 21 patients. Flow citometry analysis confirmed the standard MSC phenotype (CD45 negative, CD73 positive, CD90 positive and CD105 positive). The molecular analysis of JAKV617F, cMPL and CARL mutations resulted negative in all analyzed samples both in patients carriers of mutations and in wild type ones. Conclusions: We conclude that common mutations markers of MPN neoplasm are absent in the mesenchymal compartment of bone marrows of patients affected by MPN and are restricted to the neoplastic clone. This research project was supported by a grant from Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.) Disclosures No relevant conflicts of interest to declare.

The Decrease of JAK2 V617F Allele Burden in Leukemia Transformation of An Elderly Patient with Myelofibrosis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4990-4990
Author(s):  
Su-Jiang Zhang ◽  
Jianyong Li ◽  
Wei Xu
Keyword(s):  
Conflicts Of Interest ◽  
Molecular Genetic ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Leukemia Cells ◽  
Elderly Aml ◽  
Genetic Abnormalities ◽  
V617f Mutation

Abstract Abstract 4990 Recently there were two different model about clone genesis of acute myeloid leukemia (AML) transformed from pre-existing JAK2 V617F positive myeloproliferative neoplasm (MPN). One model showed the leukemia cells were come from JAK2 V617F negative clone, however, the other indicated that the leukemia cells were still developed from JAK2 V617F positive clone. Here, we report a elderly AML patient who was developed from pre-existing myelofibrosis (MF) with homozygous JAK2 V617F mutation. In leuekmic transformation phase, heterozygous JAK2 V617F mutation was identified, supported the idea that the leukemia cells may be come from JAK2 V617F negative clone. Moreover, no other cytogenetic and molecular genetic abnormalities were further found. After one course of CAG regimen, complete remission was achieved. Further follow-up is still in progress. Disclosures No relevant conflicts of interest to declare.

JAK2 (V617F) Mutation and Cerebral Venous Thrombosis,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3339-3339
Author(s):  
Ida Martinelli ◽  
Serena Maria Passamonti ◽  
Eugenia Biguzzi ◽  
Franca Franchi ◽  
Francesca Gianniello ◽  
...  
Keyword(s):  
Risk Factors ◽  
Venous Thrombosis ◽  
Whole Blood ◽  
Cerebral Venous Thrombosis ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
V617f Mutation

Abstract Abstract 3339 Background. Whether or not cerebral venous thrombosis, such as splanchnic venous thrombosis, can be the first manifestation of an underlying myeloproliferative neoplasm is currently unclear. Methods. Patients with cerebral venous thrombosis were tested for the JAK2 (V617F) mutation within one year from the onset of thrombosis and were followed until the development of a myeloproliferative neoplasm or censored at the end of follow-up. Results. Ten of 152 patients (6.6%) carried the JAK2 (V617F) mutation. Three of them had known acquired risk factors for thrombosis and 5 had thrombophilia. The median duration of follow-up was 7.8 years (6 months to 21.3 years). Six patients met the diagnostic criteria for myeloproliferative neoplasm at the time of cerebral venous thrombosis, while three additional patients developed the disease during the follow-up, for an annual incidence of 0.26% patient-years (95% CI 0.05–0.64). The last patient has no evidence of disease after three years of follow-up. Patients without the JAK2 (V617F) mutation at the time of cerebral venous thrombosis were re-tested at the end of the follow-up and remained negative, with normal whole blood counts [log-rank test c2: 159 (p<0.0001)]. Hence, a myeloproliferative neoplasm was diagnosed in 90% of patients with the JAK2 (V617F) mutation and in none of those without (Fisher's exact test p<0.0001). Conclusions. Cerebral venous thrombosis can be the first symptom of a myeloproliferative neoplasm. Thus, patients with cerebral venous thrombosis should be tested for the JAK2 (V617F) mutation, irrespective of whole blood counts and the presence of other risk factors for thrombosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Frequency of Calr and MPL Gene Mutations in Jak2 V617F - Positive Chronic Myeloproliferative Neoplasms in Russia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5400-5400
Author(s):  
Tatiana V Makarik ◽  
Adhamjon O Abdullaev ◽  
Sergei M. Kulikov ◽  
Elena E Nikulina ◽  
Svetlana A Treglazova ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Cell Clone ◽  
Myeloproliferative Neoplasms ◽  
Gene Mutations ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Allele Burden ◽  
Chronic Myeloproliferative Neoplasms ◽  
V617f Mutation ◽  
Calr Mutations

Background. Ph-negative chronic myeloproliferative neoplasms (MPNs) are characterized by proliferation of one or more myeloid cell lineages and include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Somatic Jak2, MPL and CALR gene mutations are responsible for more than 90% of NPM cases. These mutations affect sequential stages of prolipherative signal transduction and therefore after the emergence of one type of mutation another types basically should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Aim. To evaluate frequencies of MPL and CALR mutations in Jak2 positive MPN cases for Russian cohort of patients. Methods. Archival DNA samples from MPN patients followed up at the National Research Center for Hematology between 2014 and 2019 included into retrospective study. DNAs and RNAs were extracted from blood using reagent kit from Interlabservice (Russia). Jak2 V617F mutation was quantified by real-time PCR kit from Syntol (Russia) according to manufacturers instructions. CALR exon 9 deletions/insertions were analyzed by fragment analysis (sensitivity >= 3%). MPL W515L/K mutations were assessed by in-house allele specific PCR. All cases were tested for phi-negativity using BCR-ABl p210 PCR kit from Interlabservice (Russia). Results. At least one of the mutations was found in 3863 cases. Jak2 V617F mutation - 3385 cases (87.6%); CALR insertion or deletion - 471 case (12.2%); MPLW515L/K mutation - 31 case (0.8%). We have found 28 cases (0.7%) with Jak2 and CALR mutations combined and 3 cases (0.1%) with Jak2 and MPL mutations in the cohort studied. Matched measures were obtained at least twice at different time points during the course of disease for these cases. No cases with simultaneous CALR and MPL mutations were detected. In 23 from 31 (74%) cases with combined mutations Jak2 V617F allele burden was lower than 3%. Among cases with combined mutations 5 were diagnosed with PV, 8 - with ET, 8 - with PMF and 10 with unclassified MPN. No correlations between diagnosis, mutation combination or allele burden were found. Conclusions. Based on the data, obtained on retrospective DNA samples we cannot state whether combined mutations are present in different clones of myeloid cells or in one. Indirectly, the fact that more often mutations in CALR and MPL genes were found in the cases with a low Jak2 V617F allele burden may indicate that additional mutations occur in the "competing" cell clone. Further prospective studies with mutation monitoring over the therapy are required to assess the value of combined mutations for MPN pathogenesis. Disclosures No relevant conflicts of interest to declare.

Concomitant detection of BCR-ABL translocation and JAK2 V617F mutation in five patients with myeloproliferative neoplasm at diagnosis

2012 ◽  
Vol 35 (1) ◽  
pp. e4-e5 ◽  
Author(s):  
M. Cappetta ◽  
V. Pérez ◽  
M. N. Zubillaga ◽  
V. Elizondo ◽  
G. Manrique ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
V617f Mutation

WP1066 Inhibits Growth of Human Cells Carrying the JAK2 V617F Mutation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4885-4885
Author(s):  
Taghi Manshouri ◽  
Zeev Estrov ◽  
Alfonso Quintas-Cardama ◽  
Jorge Cortes ◽  
Francis Giles ◽  
...  
Keyword(s):  
Cell Line ◽  
Anticancer Agents ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Jak2 V617f ◽  
Janus Kinase ◽  
Jak2 V617f Mutation ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
V617f Mutation

Abstract Myeloproliferative disorders (MPDs) are characterized by proliferation of one or more myeloid cell lineages in bone marrow and peripheral blood, with relatively preserved differentiation. Recent discovery of a dominant gain-of-function mutation in the Janus kinase 2 (JAK2) gene in patients with MPDs, involving the substitution of valine for phenylalanine at position 617 of the JAK2 protein (JAK2 V617F), represents the first acquired somatic mutation in hematopoietic stem cells described in these disorders. This discovery has opened new avenues for the development of targeted therapies for MPDs. WP1066 is a small molecule, a member of a novel class of anticancer agents whose development was based upon the backbone of AG490, a tyrphostin with activity against JAK2 V617F-expressing cell lines but limited in vivo activity. We investigated the inhibitory activity of the WP1066 against the JAK2 V617F-mutant expressing erythroid leukemia HEL cell line and peripheral blood mononuclear cells from patients with polycythemia vera (PV). WP1066 significantly inhibited the phosphorylation of JAK2 and downstream signal transduction proteins STAT3, STAT5, and ERK1/2 in a dose- and time-dependent manner. It induced a time- and dose-dependent antiproliferative and pro-apoptotic effects (activation of caspase 3, release of cytochrome c, and cleavage of PARP) in the JAK2 V617F-bearing HEL cell line in the low micromolar range. Pretreatment of cells with pan-caspase inhibitor Z-VAD abolished WP1066-induced apoptosis. The expression of apoptosis related proteins bcl-2, bax, and XIAP, however, was not changed. More important, WP1066 was effective in inhibiting cell growth in clonogenic assays of mononuclear cells harboring the JAK2 V617F mutation obtained from peripheral blood of patients with PV. We conclude that WP1066 is active both in vitro and ex vivo against cells carrying the JAK2 V617F mutation and represents a solid candidate for the treatment of JAK2 V617V-expressing MPDs.

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.

JAK2 Signaling Specifies Phenotypic Pleiotropy In Myeloproliferative Neoplasms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1608-1608
Author(s):  
Lily Huang ◽  
Huiyu Yao ◽  
Yue Ma
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Phenotypic Diversity ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Wild Type ◽  
Gain Of Function ◽  
Hematopoietic Stem ◽  
Phenotypic Differences ◽  
V617f Mutation

Abstract Myeloproliferative neoplasms (MPNs) are a phenotypically diverse group of pre-leukemic diseases characterized by overproduction of one or more of the myeloid cell lineages. Gain-of -function mutations in the Janus tyrosine kinase 2 (JAK2) are major determinants in MPNs, These include the V617F mutation and mutations in exon 12. Interestingly, MPN phenotype in patients with exon 12 mutations is distinct from that of patients with the V617F mutation. Mechanisms underlying the phenotypic differences are not well understood. We performed an unbiased screen for residues essential for JAK2 auto-inhibition, and identified a panel of novel gain-of-function mutations. Interestingly, three of them with similar kinase activities in vitro elicited distinctive hematopoietic abnormalities in mice. Specifically, JAK2(K539I) results primarily in erythrocytosis, JAK2(N622I) predominantly granulocytosis, and JAK2(V617F) in both. These phenotypes are consistent with clinical data showing that patients with the V617F mutation exhibit erythrocytosis and granulocytosis, whereas those with mutations in exon 12 (where K539 resides) exhibit erythrocytosis only. To determine the mechanisms underlying the phenotypic differences by different JAK2 mutants, we characterized hematopoietic progenitors and precursor subsets in these mice for their proliferation, apoptosis and differentiation. Quantification of the hematopoietic stem and progenitor population showed an increased percentage of granulocyte-monocyte progenitors (GMP) and skewing of differentiation towards the granulocytic lineage in JAK2(V617F) and JAK2(N622I) mice compared to JAK2(K539I) or wild-type JAK2 mice. Because no difference was observed in the proliferation or apoptosis of bone marrow progenitors from JAK2 mutant mice, differentiation of the common myeloid progenitors (CMP) was likely skewed towards GMP by JAK2(V617F) and JAK2(N622I). Consistent with this hypothesis, similar results were observed in colony forming assays from sorted CMP populations. In the spleen, a decrease in GMP apoptosis and an increase in apoptosis of the megakaryocyte-erythrocyte progenitors (MEP) also contributed to the skewing towards the granulocytic lineage in JAK2(N622I) mice. Similar to MPN patients, mice expressing JAK2 mutants exhibited splenomegaly. We found that JAK2 mutants caused redistribution of hematopoietic stem and progenitors from the bone marrow to spleen. As a result, more differentiated precursors were expanded in the spleens of JAK2 mutants mice compared to mice expressing wild-type JAK2. Consistent with their phenotypes, the percentage of Annexin V＋7AAD－erythroblasts in JAK2(K539I) and JAK2(V617F) mice was significantly less than in JAK2(N622I) or wild-type JAK2 mice. On the other hand, both proliferation and apoptosis contribute to the differential degrees of granulocytosis among mice expressing different JAK2 mutants. In line with the different effects elicited by different JAK2 mutants in progenitor and precursor cells, signal transduction pathways were differentially activated downstream of different JAK2 mutants. In summary, our results showed that JAK2 mutants differentially skew differentiation in early stem and progenitor compartments, and also regulate apoptosis and proliferation of distinct precursor subsets to cause erythrocytosis or granulocytosis in mice. These results provide the mechanistic basis for the phenotypic diversity observed in MPNs with different JAK2 mutants. Disclosures: No relevant conflicts of interest to declare.

The Hematopoietic Stem Cell Compartment Is Different in Polycythemia Vera and Idiopathic Myelofibrosis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 259-259 ◽  
Author(s):  
Chloe James ◽  
Frederic Mazurier ◽  
Ronan Chaligne ◽  
Sabrina Dupont ◽  
Francois Delhommeau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
B Lymphocytes ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
V617f Mutation ◽  
Myeloid Progenitors

Abstract X-linked clonality studies showed that myeloproliferative disorders derive from an abnormal hematopoietic stem cell (HSC). This has been recently confirmed by studies showing that the JAK2 V617F mutation was present in multipotent cells from patients with Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primitive Myelofibrosis (PMF). How one unique mutation could give rise to three different diseases remains unexplained and we hypothesized that the HSC compartment may be different in PV and PMF. To investigate whether the V617F mutation occurs in HSCs in PV and PMF, and whether the HSC compartments are different in these 2 diseases, we performed simultaneously two types of experiments: Myeloid, B, and NK in vitro differentiation assays and Repopulation of immune-deficient NOD/SCID mice with human JAK2 V617F CD34+ cells followed by analysis of the frequency of JAK2 V617F clones. We first confirmed that the JAK2 V617F mutation is present in HSCs, enabling long-term (15 weeks) in vivo hematopoietic reconstitution, both in PV and PMF patients. Nevertheless, we found marked differences between PV and PMF samples. Indeed, the frequency of JAK2 V617F lympho-myeloid progenitors was much higher in MF (6 PMF and one post PV-MF) than in PV patients (n = 9) (87.2% +/− 31.3 vs 21% +/− 21.1 respectively). Similarly, most of the human myeloid progenitors present in mice transplanted with CD34+ cells from MF patients (n = 7) 15 weeks post-transplantation were JAK2 V617F. On the contrary, human myeloid progenitors were predominantly JAK2 WT after transplantation of PV CD34+ cells (9 patients). To determine if the mutation was present in HSC able to differentiate into B-lymphocytes in PV and MF, we sorted and genotyped the fraction of B-lymphocytes (CD45+CD19+) that differentiated in the bone marrow of mice. In 5 mice transplanted with PV CD34+ cells, the fraction of B-cells was always JAK2 WT whereas in 2 out of 3 mice transplanted with MF CD34+ cells, B-lymphocytes were JAK2 V617F. To determine if the mutation was present in HSC capable of very long-term reconstitution in PV and PMF, we looked for the presence of long-term culture-initiating cells (LTC-IC) among human CD34+ cells isolated from the bone marrow of NOD/SCID mice (2 PV, 3 MF) 15 weeks after transplantation. In 2 mice transplanted with PMF CD34+ cells, the majority of LTC-ICs (70/70 and 95/166) were JAK2 V617F. On the contrary, in 3 mice reconstituted with PV CD34+ cells, most of the LTC-ICs were JAK2 WT (23/23, 4/4 and 126/168) although we could find some LTC-ICs that were JAK2 V617F, demonstrating that in PV also, the JAK2 V617F mutation is present in Long Term-HSC. Taken together these results demonstrate that the JAK2 V617F mutation is present in a small subset of HSCs in PV patients, whereas in MF, the vast majority of HSCs is JAK2 V617F. This suggests that these two diseases are two stages of the same pathology and that in MF the JAK2 V617F HSCs have acquired a proliferative advantage on JAK2 WT HSCs and thus have invaded the hematopoietic system.

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