scholarly journals Comparative Analysis of Proinflammatory Cytokine Levels in Peripheral Blood and Bone Marrow of Patients with Myeloproliferative Neoplasms

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4995-4995
Author(s):  
Pu Chen ◽  
Boting Wu ◽  
Xia Shao ◽  
Chanjuan Liu ◽  
Zhenglin Yu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Proinflammatory Cytokines ◽  
Peripheral Blood ◽  
Proinflammatory Cytokine ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Cytokine Levels ◽  
V617f Mutation ◽  
Calr Mutations

Abstract Background Myeloproliferative neoplasms (MPNs) are characterized by marker somatic mutations involving JAK2, MPL, and CALR, which lead to constitutive activation of tyrosine kinase signaling cascades, subsequently dysregulated proliferative of myeloid linages, and eventually myelofibrosis or leukemic transformation. Recently, it has been argued that proinflammatory processes are crucial to the pathogenesis and progression of MPNs. A number of proinflammatory cytokines including lipocalin, IL-1, IL-2R, IL-6, IL-8, IL-12, IL-15, and IP-10 have been found elevated in the peripheral blood (PB) of MPN patients. However, there has been limited data on the levels of proinflammatory cytokines in the bone marrow (BM) of MPN patients. The present study determined and compared 40 proinflammatory cytokine levels in the PB and BM plasma of MPN patients with unequivocal molecular background, thus intending to illustrate the proinflammatory features of BM microenvironment as well as to evaluate the credibility of PB cytokine profiles. Methods Newly diagnosed MPN patients (n=12, 8 had JAK2 V617F mutation, 4 had CALR mutations) were included in the present study. PB samples were taken within 48 hours of BM samples. Paired PB and BM plasma cytokine profiles were measured as well as 10 health control PB plasma samples in a single procedure by Quantibody Human Inflammatory Array 3 (RayBiotech, Norcross, GA) which permitted detection of 40 inflammation-associated cytokines. Results Among 12 MPN patients, 8 had JAK2 V617 mutation (6 males, median age 61.5 years), and 4 had CALR mutations (3 males, median age 53.5 years). Positive linear correlations between PB and BM levels were found in 12 proinflammatory cytokines including BLC (r=0.613, p=0.034), I-309 (r=0.872, p<0.001), IL-1α (r=0.666, p=0.018), IL-1β (r=0.929, p<0.001), IL-12p40 (r=0.642, p=0.024), IL-15 (r=0.608, p=0.036), M-CSF (r=0.906, p<0.001), MIG (r=0.596, p=0.041), MIP-1α (r=0.787, p=0.002), MIP-1δ (r=0.648, p=0.023), sTNFRI (r=0.827, p=0.001), and sTNFRII (r=0.644, p=0.024). Compared to health controls, BM levels of G-CSF, IL-2, IL-4, IL-6R, IL-7, IL-8, IL-10, IL-13, MIP-1β, PDGF-BB, RANTES, and TIMP-1 were markedly elevated (all p<0.01), among which IL-2, IL-4, and IL-7 levels were even higher in patients with CALR mutations than those with classic JAK2 V617F mutation (all p<0.05). Conclusions Optimal linear correlation could only be found in limited species of proinflammatory cytokines, especially I-309, IL-1β, M-CSF, and sTNFRI, between PB and BM plasma of MPN patients. Therefore, caution should be recommended during attempts to illustrate the status of inflammation in MPN patients by circulating cytokine markers. A stronger inflammatory component might exist in MPN patients with CALR mutations than those with classic JAK2 V617F mutation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Megakaryocytic morphology in Janus kinase 2 V617F positive myeloproliferative neoplasm

2017 ◽  
Vol 06 (02) ◽  
pp. 075-078
Author(s):  
Shuchi Ghai ◽  
Sharada Rai
Keyword(s):  
Bone Marrow ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Janus Kinase ◽  
Jak2 V617f Mutation ◽  
Fisher Exact Test ◽  
Janus Kinase 2 ◽  
Bone Marrow Biopsies ◽  
V617f Mutation

Abstract Context: Alterations in megakaryocyte morphology are the hallmark of myeloproliferative neoplasms (MPNs). These neoplasm are also associated with Janus kinase 2 (JAK2) V617F mutation in nearly 95% patients with polycythemia vera (PV), 40% patients of essential thrombocythemia (ET) and 50% patients of myelofibrosis (MF). The utility of megakaryocyte morphology in these disorders in correlation with JAK2 V617F remains unresolved. Aims: The aim of the study was to assess the morphology of megakaryocytes in bone marrow aspirates (BMAs) and bone marrow biopsies of patients of BCR-ABL negative MPNs with JAK2 V617F mutation. Settings and Design: This study was a retrospective and prospective, hospital-based study undertaken for a period ranging from January 2011 to April 2015. Subjects and Methods: Assessment of morphological features of megakaryocytes in 15 BMAs and their respective biopsies which included seven cases of PV, three cases of ET, and five cases of MF with JAK2 V617F mutation. Statistical Analysis Used: Chi-square test and Fisher exact test were used to compare the different features of megakaryocytes. Software version SPSS 13.0 was used. Results: Megakaryocytes in ET were found to have characteristically large size with staghorn multinucleated nuclei and exhibiting large amount of cytoplasm. MF showed dense clustering of megakaryocytes with staghorn nucleus along with sinusoidal dilatation and intrasinusoidal hematopoiesis. PV showed loose and dense clustering of megakaryocytes with a predominance of cloud-like nuclei. Few of the megakaryocytic morphologic features showed overlap between MF and PV and between ET and early MF. Conclusions: Megakaryocytic morphology can aid in the accurate diagnosis of the different subcategories of MPNs. This would help in categorization of clinically suspicious patients of JAK2 V617F negative patients.

Relationship between JAK2 V617F Mutation Status and Constitutive Mobilization of CD34-Positive Cells into Peripheral Blood in Patients with Chronic Myeloproliferative Disorder.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2586-2586
Author(s):  
Francesco Passamonti ◽  
Elisa Rumi ◽  
Emanuela Boveri ◽  
Daniela Pietra ◽  
Laura Vanelli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Count ◽  
Peripheral Blood ◽  
Jak2 V617f ◽  
Myeloproliferative Disorder ◽  
Jak2 V617f Mutation ◽  
Jak2 Mutation ◽  
Mutation Status ◽  
Cell Counts ◽  
V617f Mutation

Abstract A gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been recently reported in patients with polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF) [N Engl J Med. 2005 Apr 28;352(17):1779–90]. Abnormal trafficking of CD34-positive cells with increased numbers in the peripheral blood is found in CIMF and in advanced stages of other myeloproliferative disorders. To determine whether the unique JAK2 V617F mutation affects the mobilization of CD34-positive cells into peripheral blood, we studied the relationship between JAK2 mutation status, bone marrow and circulating CD34-positive cells in 72 patients diagnosed according to the WHO criteria. A quantitative real-time polymerase chain reaction (PCR)-based allelic discrimination assay was used for the quantitative detection of the JAK2 V617F alleles in circulating granulocytes. Bone marrow CD34-positive cells were quantitatively assed on paraffin immunostained sections, while circulating CD34-positive cells were enumerated by flow cytometry using a single-platform assay. Overall, 57% of the patients studied carried the JAK2 V617F mutation. Within these patients, median values for JAK2 V617F alleles in circulating granulocytes were as follows: 29% in PV, 4% in ET, 12% in prefibrotic CIMF, 27% in fibrotic CIMF, and 99% in post-PV myelofibrosis. The vast majority of circulating granulocytes were homozygous for the mutation in all but one of patients with post-PV myelofibrosis. Decreased numbers of bone marrow CD34-positive cells and increased counts of circulating CD34-positive cells were detected in patients with fibrotic bone marrow. The higher the degree of fibrosis, the higher the circulating CD34-positive cell count (P&lt;0.001) and the lower the bone marrow CD34-positive cell count (P&lt;0.01). All patients with PV, ET and prefibrotic CIMF, and 7 out of 21 patients with fibrotic CIMF had circulating CD34-positive cell counts lower than 10 x 106/L. Conversely, all patients with post-PV myelofibrosis had counts higher than 10 x 106/L. In univariate analysis, there was an inverse relationship between percentage of JAK2 V617F alleles and bone marrow CD34-positive cells (r=−0.35, P&lt;0.01), and a direct relationship between percentage of JAK2 mutant alleles and circulating CD34-positive cells (r=0.46, P=0.001). Multivariate analysis showed that disease category (P=0.0008) and percentage of JAK2 V617F alleles (P=0.03) were independently related to circulating CD34-positive cell counts. These observations suggest that the JAK2 V617F mutation might be involved in the constitutive mobilization of CD34-positive cells into peripheral blood that is found in patients with myeloproliferative disorder. Nonetheless, constitutive mobilization is present in a considerable portion of patients who do not carry the JAK2 mutation, pointing to additional pathogenetic mechanisms. Findings on patients with PV suggest that transition form heterozygosity to homozygosity for JAK2 V617F may represent an important step in the progression of PV to myelofibrosis. Thus, sequential evaluation of the percentage of JAK2 mutant alleles and enumeration of circulating CD34-positive cells may be useful for disease monitoring in PV.

scholarly journals Myeloproliferative neoplasms with concurrent BCR–ABL1 translocation and JAK2 V617F mutation: a multi-institutional study from the bone marrow pathology group

2018 ◽  
Vol 31 (5) ◽  
pp. 690-704 ◽  
Author(s):  
Craig R Soderquist ◽  
Mark D Ewalt ◽  
David R Czuchlewski ◽  
Julia T Geyer ◽  
Heesun J Rogers ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
V617f Mutation

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.

scholarly journals CRISPR/Cas12a-Based Ultrasensitive and Rapid Detection of JAK2 V617F Somatic Mutation in Myeloproliferative Neoplasms

Biosensors ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 247
Author(s):  
Miaomiao Chen ◽  
Chunhua Zhang ◽  
Zhiqing Hu ◽  
Zhuo Li ◽  
Menglin Li ◽  
...  
Keyword(s):  
Detection System ◽  
Myeloproliferative Neoplasms ◽  
Cost Effective ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Minimum Detectable Concentration ◽  
Lateral Flow Strip ◽  
Whole Process ◽  
Detectable Concentration ◽  
V617f Mutation

The JAK2 V617F mutation is a major diagnostic, therapeutic, and monitoring molecular target of Philadelphia-negative myeloproliferative neoplasms (MPNs). To date, numerous methods of detecting the JAK2 V617F mutation have been reported, but there is no gold-standard diagnostic method for clinical applications. Here, we developed and validated an efficient Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 12a (Cas12a)-based assay to detect the JAK2 V617F mutation. Our results showed that the sensitivity of the JAK2 V617F/Cas12a fluorescence detection system was as high as 0.01%, and the JAK2 V617F/Cas12a lateral flow strip assay could unambiguously detect as low as 0.5% of the JAK2 V617F mutation, which was much higher than the sensitivity required for clinical application. The minimum detectable concentration of genomic DNA achieved was 0.01 ng/μL (~5 aM, ~3 copies/μL). In addition, the whole process only took about 1.5 h, and the cost of an individual test was much lower than that of the current assays. Thus, our methods can be applied to detect the JAK2 V617F mutation, and they are highly sensitive, rapid, cost-effective, and convenient.

Evaluation of the JAK2 V617F gene mutation in myeloproliferative neoplasms cases: a one-center study from Eastern Anatolia

2019 ◽  
Vol 44 (4) ◽  
pp. 492-498
Author(s):  
Gonca Gulbay ◽  
Elif Yesilada ◽  
Mehmet Ali Erkurt ◽  
Harika Gozukara Bag ◽  
Irfan Kuku ◽  
...  
Keyword(s):  
Blood Cell ◽  
Essential Thrombocythemia ◽  
Myeloproliferative Neoplasms ◽  
Hematological Parameters ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Myeloproliferative Disorders ◽  
Jak2 V617f Mutation ◽  
V617f Mutation ◽  
The Difference

AbstractObjectiveDetection ofJAK2V617F in myeloproliferative neoplasms (MPNs) is very important in both diagnosis and disease progression. In our study, we investigated the frequency ofJAK2V617F mutation in patients with myeloproliferative disorders.MethodsWe retrospectively reviewed the records of 720 patients (174 females and 546 males) who were tested for JAK2 V617F mutation from January 2007 to December 2017.ResultsIn our patients were determined 22.6%JAK2V617F mutation. 33.3% in women, 19.2% in men have been positive forJAK2V617F mutation. In our studyJAK2V617F present in 48.6% of essential thrombocythemia, 80.5% of polycythemia rubra vera (PV), 47.5% of primary myelofibrosis, 10% of MPNs, unclassifiable, 0.8% of others. We also investigated the difference in hematological parameters [white blood cell, hemoglobin (Hb), hematocrit (HCT), red blood cell distribution widths (RDW) and platelets count (PLT)] betweenJAK2V617F positive andJAK2V617F negative patients.ConclusionsInvestigation of the JAK2 V617F mutation is very important in cases of MPNs. In our study JAK2 V617F mutation was higher in PV, essential thrombocythemia, and primary myelofibrosis patients. However, there were significant differences in Hb, HCT, RDW and PLT levels in mutation-positive patients.

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

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

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

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.

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