Enumeration of Circulating Platelet-Derived Microparticles Clearly Separates PV and ET From Secondary Polycythemia or Thrombocytosis

Abstract Abstract 1745 Rationale: The diagnosis of Polycythemia Vera (PV) and Essential Thrombocytemia (ET) relies on a set of criteria gathered in the 2008 WHO classification; however, it may occasionnaly be difficult, especially in the absence of molecular markers such as Jak2V617→F or Mpl mutations. Having previously reported a significant increase in circulating platelets microparticles (PMP) in patients bearing Myeloproliferative neoplasms versus healthy donors (V.Tintillier-Colin, ASH Annual Meeting 2009, Abstr. 1906), our purpose has been to assess the usefulness of blood PMP count as an additional criteria in dubious cases of ET and PV. Patients and methods: We performed PMP counts in 100 newly diagnosed and still untreated patients (55 ET and 45 PV in accordance to WHO criteria), 40 secondary or reactive states (erythrocytosis and thrombocytosis, 20 cases each) and 75 controls matched for age and sex. The PMP were characterized by their size and co-expression of Annexin V and CD41; their concentration was measured on plasma extracts using the FC500 flow-cytometer (Beckman-Coulter™). Pre-analytical and testing procedures complied with the recommendations of the ISTH Standardization Sub-committee with each test performed in duplicate. Results: The 3 groups were homogeneous regarding age and sex-ratio. Mean platelet counts were 703.109/l in the TE group, 652.109/l in the cohort with secondary thrombocytosis and 247.109/l in the control group. Mean Hb level was 18.0g/dl in the PV group, 18.1g/dl in the secondary erythrocytosis group and 15g/dl in controls. The median value of PMP concentration is much higher (p < 0.05, Krusall-Wallis test) in the TE group (5900/μl) than in the reactive thombocytosis group (1283/μl), this latter being no different from the control group (996/μl). In the ET group, PMP count is similarly increased whether patients beared Jak2V617→F mutation or not. Using the ROC statistical method, we defined a threshold value of 3400 PMP/μl able to discriminate ET from reactive thrombocytosis with 93% specificity and 67% sensitivity. Similarly, the median value of PMP count was neatly higher in the PV group (3258/μl) than in secondary erythocytosis (671/μl) and healthy controls (996/μl) (p < 10−5, Krusall-Wallis test). The ROC method fixed a 1300/μl threshold separating PV from secondary erythrocytosis with 89% sensibility and 63% specificity. Conclusion: We report an evaluation of the blood PMP count in a quite large series of newly diagnosed untreated ET and PV patients compared to patients with secondary thrombocytosis or erythrocytosis and healthy volunteers; this study emphasizes the interest of the circulating PMP count in order to differentiate MPN neoplasms from normal subjects as well as patients with reactive conditions. This test, quick, reproducible and cheap, especially shows its value as an additional diagnostic criteria in ET or PV when clonal markers are lacking. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Phenotypic Profile of Circulating Microparticles Is Different in Polycythemia Vera and Secondary Erythrocytosis

Blood ◽

10.1182/blood.v118.21.5166.5166 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5166-5166

Author(s):

Agnès Charpentier ◽

Stéphanie Devaux ◽

Judith Bruge-Debreu ◽

Nathalie Cambier ◽

Christian Rose ◽

...

Keyword(s):

Endothelial Cells ◽

Polycythemia Vera ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Annexin V ◽

Blood Stream ◽

Phenotypic Profile ◽

Testing Procedures ◽

Differentiated Cells ◽

Circulating Microparticles

Abstract Abstract 5166 Rationale: In Ph1 myeloproliferative neoplasms (MPN), the blood stream carries a variety of microparticles and exosomes released by cells from myeloid lineages and endothelium; we had already demonstrated an excess of platelet microparticles (PMP) in ET and PV patients (ASH Annual Meeting 2009, Abstr. 1906). A further step has been to characterize five categories of MP depending on their origin. Then we used this method to compare the sub-populations found in Polycythemia vera (PV) and secondary erythrocytosis (SE). Patients and methods: We analyzed plasma samples from 20 PV diagnosed in accordance to WHO classification and 20 SE patients. SE was defined by elevated hematocrit, normal or increased serum EPO, Jak2V617F negative and a presumable cause, mainly respiratory. The 2 groups were comparable for age and sex-ratio. Mean Hb level was 18.0g/dl in the PV group and 18.1g/dl in the SE group. The concentration of MP was measured by flow cytometry using a FC500 cytometer (BeckmanCoulter™). All the MP subtypes express Annexin V thus defining the total MP. Additional co-expression of either CD41, CD144, CD14, CD11b or GPA identify MP populations originating respectively from platelets (PMP), endothelial cells (EMP), monocytes (MoMP), granulocytes (GrMP) and red cells (EryMP). Pre-analytical and testing procedures complied with the recommendations of the ISTH Standardization Sub-committee. Results: MP from endothelial cells (EMP) and leukocytes (GrMP and MoMP) are minor populations whose proportions are similar in both groups of patients (7%, 4% and 2% respectively). Conversely, EryMP and PMP are significantly different (p<0.001 Kursall-Wallis test) in PV and SE as shown in the following table. So, in PV most of the MP are PMP while EryMP widely predominate in SE; in an attempt to explain such unexpected results we postulate that the cells generating MP are more or less mature depending on the physiopathology: clonal hematopoietic progenitors in MPN and well-differentiated cells in reactive conditions. However, even in SE, the EryMP concentration does not correlate with RBC count, haemoglobin or hematocrit. Conclusion: This study demonstrates a different phenotypic profile of circulating MP in PV and SE, possibly helpful to precise the origin of an elevated hematocrit, especially when molecular markers are negative. The significance of the MP subtype pattern observed in PV and SE remains unclear, calling for further studies. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Markers of Metabolic Syndrome in Patients with Pituitary Adenoma: A Case Series of 303 Patients

Hormone and Metabolic Research ◽

10.1055/a-1020-3992 ◽

2019 ◽

Vol 51 (11) ◽

pp. 709-713 ◽

Cited By ~ 2

Author(s):

Seher Çetinkaya Altuntaş ◽

Mehtap Evran ◽

Murat Sert ◽

Tamer Tetiker

Keyword(s):

Metabolic Syndrome ◽

Pituitary Adenoma ◽

Waist Circumference ◽

Case Series ◽

Low Density Lipoproteins ◽

Control Group ◽

Newly Diagnosed ◽

Patient Groups ◽

Relationship Of ◽

Age And Sex

AbstractTo assess the demographic characteristics and hormonal status of patients who presented to our clinic with pituitary adenoma and to demonstrate the presence, prevalence, and relationship of metabolic syndrome parameters in these patients. The study included 303 patients with known or newly diagnosed pituitary adenoma and 52 age- and sex-matched healthy controls. The patients were classified into 3 groups; acromegaly (ACRO) (n=54),prolactinoma (PRLoma) (n=163), and non-functional adenoma (NFA) (n=86). in 55.6% (n=172) and 52% (n=163) of the patients, respectively. The waist circumference of all patients (p<0.001) and body mass index (BMI) of patients with PRLoma (p=0.03) and ACRO (p<0.001) were found to be significantly higher than in the controls. The HbA1c, insulin and HOMA-IR values were significantly higher in the ACRO and PRLoma groups, whereas the insulin and HOMA-IR values were significantly higher in the NFA group compared with the control group (p<0.001 and p<0.001, respectively). When the 3 patient groups were compared, waist circumference and BMI were significantly higher in the ACRO group than in the PRLoma group (p=0.04 and p=0.03, respectively). In patients developing pituitary failure after treatment, age, waist circumference, plasma glucose, low-density lipoproteins and triglyceride values were significantly increased when compared with those without pituitary failure after treatment (p<0.001). In our study, it was found that there was increased metabolic and cardiovascular risk in functional pituitary adenoma and NFA.

Download Full-text

In vivo and in vitro effects of statins on lymphocytes in patients with Hashimoto’s thyroiditis

Acta Endocrinologica ◽

10.1530/eje.1.01941 ◽

2005 ◽

Vol 153 (1) ◽

pp. 41-48 ◽

Cited By ~ 18

Author(s):

Sevim Gullu ◽

Rifat Emral ◽

Mehmet Bastemir ◽

Arthur B Parkes ◽

John H Lazarus

Keyword(s):

Thyroid Function ◽

Hashimoto’S Thyroiditis ◽

Annexin V ◽

Normal Subjects ◽

Hashimoto's Thyroiditis ◽

Control Group ◽

Cd4 Cells ◽

Apoptotic Effects

Background: Statins have apoptotic effects on many cell types. Hashimoto’s thyroiditis (HT) is an autoimmune disease in which cell-mediated autoimmune mechanisms are pathogenetically involved. Objective: The aim of this study was to evaluate the in vivo effects of Simvastatin on thyroid function, lymphocyte subtypes and also to investigate the apoptotic effects of Simvastatin, Mevastatin, Pravastatin and Cerivastatin on lymphocytes from patients with HT. Methods: In the first part of the study, 11 patients with HT and subclinical hypothyroidism (SH) were given Simvastatin (20 mg/day) for 8 weeks. Ten patients with SH and HT served as the control group. No treatment was given to controls. Thyroid function, C-reactive protein (CRP) levels and lymphocyte subtypes of both groups were determined before the study and after 8 weeks. In the second part of the study, the apoptotic effects of statins on lymphocytes were evaluated in patients with HT (n = 10) and normal subjects (n = 10) in vitro. Apoptosis was investigated by using Annexin-V and propidium iodide. Lymphocytes from patients and controls were incubated with different concentrations of Simvastatin, Cerivastatin, Mevastatin and Pravastatin. Results: An increase in serum free tri-iodothyronine and free thyroxine levels and a decrease in TSH levels were observed (P < 0.05) with Simvastatin treatment. CD4 + cells and B lymphocytes increased whilst CD8 + cells, natural killer cells and activated T lymphocytes decreased significantly in the treatment group (P < 0.05). The CRP level of the group also decreased with Simvastatin but it did not reach significance (P = 0.057). None of parameters was found to be different from the baseline in the control group. In in vitro experiments, apoptosis was observed in CD3 + (both in CD8 + and CD4 + cells) with all statins in both patient and control samples. Mevalonate, which was used in experiments, reversed apoptosis in some but not all samples. Conclusions: The results of this study suggested that Simvastatin is an immune modulatory agent and improves thyroid function in patients with HT. This effect is probably mediated via lymphocyte apoptosis as demonstrated with in vitro experiments and is not confined to Simvastatin since Mevastatin, Pravastatin and Cerivastatin also induced apoptosis in lymphocytes.

Download Full-text

Ineffective Erythropoiesis With Increased Neutrophils In Old Mice Overexpressing Hmga2 cDNA

Blood ◽

10.1182/blood.v122.21.2469.2469 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2469-2469

Author(s):

Kazuhiko Ikeda ◽

Philip J. Mason ◽

Kayo Shirado Harada ◽

Kazuei Ogawa ◽

Monica Bessler ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Annexin V ◽

Primary Myelofibrosis ◽

Erythroid Cells ◽

Ineffective Erythropoiesis ◽

Hematopoietic Stem ◽

Cell Counts ◽

Young Mice

Abstract HMGA2 is frequently overexpressed in hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) and primary myelofibrosis (PMF), and occasionally in myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN). We recently reported that transgenic mice overexpressing truncated Hmga2 cDNA (ΔHmga2 mice) showed increased numbers in all lineages of peripheral blood (PB) cells, hypercellular bone marrow (BM), and splenomegaly in young adults of 3 months old (Ikeda et al, Blood, 2011). ΔHmga2 mice also showed growth advantage of hematopoietic stem cells (HSCs) in serial BM transplants and of committed progenitors in colony-replating assays. Thus, overexpression of HMGA2 may contribute to clonal expansion in PNH or myeloproliferation in PMF and MDS/MPN. Although these disorders show disease progression and development of leukemia in the long term, consequences of long-term overexpression of HMGA2 in hematopoiesis is unknown. Therefore, in this study, we investigated hematopoiesis in old ΔHmga2 mice at 15 months old. We found an increase in PB neutrophils in 16 old ΔHmga2 mice compared with 8 old wild-type (WT) mice (mean ± SD; 4.3 ± 1.7 vs. 2.4 ± 0.6 x109/L, p<0.01) as well as young mice. Strikingly, in contrast to young mice, we found a decrease in PB red blood cells of older ΔHmga2 mice compared with WT mice (8.5 ± 0.5 vs. 9.3 ± 0.4 x1012/L, p<0.01), which was associated with an increase in the mean corpuscular volume (MCV, 48.5 ± 3.7 vs. 42.1 ± 1.8 fL, p<0.01). PB total leukocyte and platelet counts were similar between old ΔHmga2 mice and old WT mice. Total BM cell counts were higher in 11 old ΔHmga2 mice compared with 12 WT mice (2.1 ± 0.4 vs. 1.4 ± 0.5 x107/femur, p<0.01). Spleens were 2-fold heavier in old ΔHmga2 mice (n=11) compared with old WT mice (n=12, p<0.01). Histology showed hypercellular BM with erythroid dysplasia, but neither fibrosis nor leukemia, in 5 old ΔHmga2 mice. Absolute numbers of BM lineage-Sca-1+c-kit+ HSCs were higher in 5 old ΔHmga2 mice compared with 3 old WT mice (73 ± 31 vs. 23 ± 10 x103/femur, p=0.036). In addition, proportions of CD71+Ter119+ erythroblasts were higher in 3 old ΔHmga2 mice compared with 3 WT mice (11.8 ± 1.2 vs. 5.1 ± 1.1%, p<0.01), although absolute number of megakaryocyte-erythrocyte progenitors did not show a statistical difference (137 ± 36 vs. 96 ± 18 x103/femur, p=0.1). Progenitor assay also showed increased numbers of BFU-E colonies in 3 old ΔHmga2 mice compared with 3 WT mice (25 ± 2 vs. 13 ± 2, p<0.01), but new BFU-E colonies did not grow from replating of these colonies of old ΔHmga2 mice, in contrast to young ΔHmga2 mice. Annexin V staining showed increased spontaneous apoptosis after 24-hour incubation in Ter119+CD71+ cells of 3 old ΔHmga2 mice compared with 3 old WT mice (26.6 ± 2.7 vs. 14.3 ± 7.0%, p=0.033). In MACS-sorted Ter119+ erythroid cells, expression of anti-apoptotic Bcl-xl mRNA in old ΔHmga2 mice was lower compared with old WT mice (3.3 ± 1.2 vs. 5.3 ± 0.3, p=0.016), while that in young ΔHmga2 mice were higher than young WT mice (6.9 ± 0.4 vs. 3.5 ± 0.3, p<0.01), suggesting the possibility that some changes, including expression of Bcl-xl affected sensitivity of erythroid cells to apoptosis during aging, resulted in ineffective erythropoiesis. In conclusion, long-term expression of HMGA2 may lead to neutrophilia with ineffective erythropoiesis, which may mimic MDS/MPN rather than myelofibrosis, possibly through impairment of Bcl-xl in erythroid cells during a long course. Ineffective erythropoiesis may also in part explain BM failure in PNH. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Initial Analysis of Lipid Metabolomic Profile Reveals Differential Expression Features in Myeloid Malignancies

Blood ◽

10.1182/blood.v124.21.5958.5958 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5958-5958

Author(s):

Adriana Ramos Oliveira ◽

Ismael Silva ◽

Edson LoTurco ◽

Helio Martins ◽

Maria L. Chauffaille

Keyword(s):

Likelihood Ratio ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Positive Likelihood Ratio ◽

Negative Likelihood Ratio ◽

Ethyl Esters ◽

Control Group ◽

Metabolomic Profile ◽

Hematopoietic Stem ◽

Myeloid Neoplasms

Abstract Introduction: Lipids are molecules that stand out among the different cellular metabolites by their enormous molecular diversity. Their functions were initially related to the composition of biological membranes and energy storage, but currently, these molecules have been analyzed considering different functions and regulatory signaling (Loizides-Mangold, 2013). It is known that lipid membranes and lipid mediators constitute specific phenotypes, including tumors (Hilvo et al, 2011). Lipid metabolism in cancer had been studied predominantly at the genetic level has recently gained further interest. Lipidomics studies show a powerful means of investigating pathophysiological issues and involvement of lipids in pathological states for both diseases in which lipids are known to play a role, but also for those which role is not well characterized (Roberts et al., 2008). Myeloid neoplasms are clonal diseases of the hematopoietic stem cell which can be present in the bone marrow and/or peripheral blood. Over the past two decades, mass spectrometry (MS) has emerged as the main method used in lipidomics analysis, which allows the structural characterization and quantification of complex lipids and their metabolites (MURPHY et al, 2005). Due to the importance of this field we have considered the use of the lipidomic innovative platform to identify differences in the plasma lipid metabolomic profile of hematological patients with Myeloid Neoplasms. Methods: Untargeted Shotgun MS/MS Analysis was performed on an independent service at the AB-Sciex Laboratory located in Sao Paulo, SP, Brazil on a 5600 Triple TOF mass spectrometer (ABSciex) instrument with an acquisition scan rate of 100 spectra/sec and stable mass accuracy of ~2 ppm. Plasma samples from 153 participants were analyzed being, 90 of the Control Group, 43 Myeloproliferative Neoplasms (MPN), 11 Myelodysplastic Syndromes (MDS) and 9 Acute Myeloid Leukemias (AML). Data were acquired using the AB-Sciex Analyst TF, processed using the AB-Sciex LipidViewTM and the web-based analytical pipeline MetaboAnalyst 2.0 (www.metaboanalyst.ca) (Xia et al, 2012). Results: Untargeted analysis identified in negative and positive-modes a total of 658 features at 2 ppm resolution. PCA and PLS-DA analysis revealed clear discrimination among groups, in particular for AML patients. Main lipid groups differentially expressed were: Monoacylglycerols (MAG), Glucosylceramide E (GlcdE), Ethyl Esters (EE), Lysophosphatidic acid (LPA), Sulfoquinovosil diacylglycerols (SQDG), Monoglycerols (MG), Methyl Ethanolamines (ME), Lysophosphatidylcholines (LPC), Dimethyl Phosfatidyletanilamines (DMPE), Monometylphosphatidiletanolamines (MMPE), Ceramide-1-phosphate (CerP), Glicerophosphoglycerols (GP), Lysomonomethyl Glycerophosphocholine (LMMPE), Phosphatidic Acids (PA), Ergosterols (ERG), Glycerophosphoserine (PS), Diacylglycerols (DAG), Hexocylceramides (HexCer) and Lanosterol (Lan). ROC Curve Analysis revealed Total LMMPE as the strongest discriminating marker between Controls from Patients with MDS or AML (Sensitivity= 0.95 (0.824-1); Specificity= 0.8941 (0.847-0953); Positive Likelihood Ratio= 8.972 and Negative Likelihood Ratio =0.05592 and T Test= 7.576E-12). In addition these lipids were also able to differentiate MDS and AML from NMP (Sensitivity= 0.9118 (0.824-1), Specificity= 0.95 (0.85-1), Positive Likelihood Ratio= 18.2 and Negative Likelihood Ratio= 0.05592). Conclusions: The Myeloproliferative Neoplasms from the point of view of global plasma lipidomics are accompanied by several modifications. In particular the Lysomonomethyl-Phosphatidylethanolamines (LMMPE) seems to play important differentiating roles among them. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Depletion of Neoplastic CD11b Positive Cells in Jak2V617F Mutant Mice Reduced Fibrocytes in Bone Marrow and Improved Myelofibrosis

Blood ◽

10.1182/blood-2019-130042 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 310-310

Author(s):

Yoshinori Ozono ◽

Kotaro Shide ◽

Takuro Kameda ◽

Ayako Kamiunten ◽

Yuki Tahira ◽

...

Keyword(s):

Bone Marrow ◽

Blood Cells ◽

Diphtheria Toxin ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

The Other ◽

Immunofluorescent Staining ◽

Control Group ◽

Other Hand

Myelofibrosis (MF) associated with myeloproliferative neoplasms (MPN) has been considered to be a reactive phenomenon caused by mesenchymal stromal cells (MSCs) stimulated by cytokines such as TGFb-1 overproduced by neoplastic megakaryocytes (MKs) and platelets. TGFb-1 stimulates non-neoplastic mesenchymal cells to produce collagen and fibronectin and to induces bone marrow (BM) fibrosis. However, the involvement of neoplastic fibrocyte in MF has recently been reported (Verstovsek et al. JEM 2016), and among blood cells, monocytes in particular are considered to be the main source of neoplastic fibrocytes. In this study, we assesed the role of neoplastic fibrocytes using a mouse model of MPN induced by Jak2V617F (Shide et al. Leukemia 2008). First, the distribution of neoplastic fibrocyte in the BM of Jak2V617F transgenic (TG) mice was examined. We transplanted wild-type (WT) or Jak2V617F TG cells (B6-CD45.2), together with WT BM cells (B6-CD45.1) into irradiated WT recipient mice (B6-CD45.1). Only recipient mice transplanted with a mixture of Jak2V617F cells and WT cells developed BM fibrosis. In immunofluorescent staining of fibrotic BM, cells expressing the fibrocyte marker CD45/Collagen-1(Col-1) were observed much more than cells expressing the fibroblast marker CD90(usually positive for MSCs)/Col-1. As for CD45/Col-1 positive cells, cells expressing CD45.2/Col-1 were much more than cells expressing CD45.1/Col-1, clearly indicating that these cells were derived from Jak2V617F mutant blood cells. On the other hand, in the BM of recipient mice transplanted with control WT cells, few cells expressing CD45/Col-1 or CD90/Col-1 were present. To examine the differentiation ability of Jak2V617F blood cells to fibrocytes directly, peripheral blood (PB) mononuclear cells (MNC) of Jak2V617F mice or WT mice were cultured in vitro. After 5 days of culture, PB MNCs from Jak2V617F mice differentiated into mature fibrocytes exhibiting a long spindle shape with Col-1 expression. On the other hand, there were very few fibrocytes differentiated from PB MNC from WT mice. Next, we depleted monocytes, the main source of fibrocytes, and observed its effects on BM fibrosis in vivo. Jak2V617F TG mice were mated with CD11b-diphtheria toxin receptor (DTR) TG mice (Duffield et al. JCI 2005) to obtain Jak2V617F/CD11b-DTR double TG mice. Mice transplanted with BM cells from Jak2V617F/CD11b-DTR double TG mice (hereinafter called Jak2V617F/CD11b-DTR mice) exhibit leukocytosis, thrombocytosis, anemia, splenomegaly, and BM fibrosis with increased megakaryocytes. Jak2V617F/CD11b-DTR mice was administered diphtheria toxin (DT) intraperitoneally to deplete monocytes. One day after DT administration, the number of PB monocytes (CD11b+/F4/80+) drastically decreased in Jak2V617F/CD11b-DTR mice, and the reduction of monocyte was maintained by every-other-day DT administration. After 8 weeks DT treatment, mice were sacrificed and analyzed. As a control group, Jak2V617F/CD11b-DTR mice treated with PBS were examined. DT treatment drastically decreased the number of neoplastic fibrocytes expressing CD45.2/Col-1 in BM and spleen of Jak2V617F/CD11b-DTR mice compared with control mice treated with PBS. Consistently, reticulin fibers were eliminated almost completely and collagen fibers almost fully disappeared in BM, which led to a reversal of the decrease in BM cellularity, although the number of MKs was not affected. Similar findings were observed in the spleen, although not completely. Plasma TGF-b1 level were about 2-fold higher in Jak2V617F/CD11b-DTR mice than in WT mice. Neoplastic monocyte depletion significantly decreased TGF-b1 level. Since MK numbers did not change, this indicates that fibrocytes are one of the main sources of TGF-b1. In other features of MF in Jak2V617F/CD11b-DTR mice, splenomegaly was ameliorated by DT treatment. Microscopic analysis revealed an improvement in the damaged spleen architecture and the disappearance of splenic fibrosis. In summary, most collagen-producing cells in BM were neoplastic fibrocytes in Jak2V617F-induced MPN, indicating that neoplastic fibrocytes played an essential role and mesenchymal fibroblasts had a minor contribution in fibrosis in MPN. Depletion of neoplastic monocytes also improved splenomegaly as well as BM fibrosis in mice, and this cell fraction could be a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Defective Functional Activity of Platelets in Children with Severe Chronic Immune Thrombocytopenia and Its Correction By Romiplostim Treatment

Blood ◽

10.1182/blood.v124.21.1466.1466 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1466-1466

Author(s):

Irina Demina ◽

Elena Suntsova ◽

Alexey Maschan ◽

Michael Maschan ◽

Galina Novichkova ◽

...

Keyword(s):

Platelet Count ◽

Functional Activity ◽

Conflicts Of Interest ◽

Annexin V ◽

Dense Granule ◽

Control Group ◽

Platelet Counts ◽

Chronic Itp ◽

Increased Risk ◽

Granule Release

Abstract BACKGROUND: Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by antibody-mediated platelet destruction, which together with suboptimal platelet production lead to thrombocytopenia. Children with low platelet counts due to ITP are at an increased risk of bleeding, which is not always directly correlated to the severity of thrombocytopenia. Relationship of platelet functional activity and bleeding risk in ITP is poorly investigated. There are conflicting reports on whether platelets in adult ITP patients differ from normal ones, and no data for children are available. Information about the thrombopoietin receptor agonists (eltrombopag and romiplostim, which are increasingly used to treat ITP) effect on platelet quality is also scarce. Data for eltrombopag are inconclusive, and no studies for romiplostim are known. OBJECTIVES: To investigate platelet functional activity before and during the romiplostim treatment of ITP in children. METHODS: The study group consisted of patients from 4 to 11 years old (n=8) with chronic ITP; the control group included healthy donors (n=12). All patients initially had low platelet counts (<15*109/L) and did not respond to the first- and second- line therapy. Blood samples were collected before treatment with romiplastim and then monthly until the onset of clinical remission. Platelets in whole blood were either left intact or activated with collagen-related peptide (0.18 mkg/ml) and thrombin receptor activating peptide (12.5 mkM), labelled and analysed by flow cytometry. To characterize platelet functions, we used fluorescencently labeled antibodies against glycoprotein Ib (CD42b), total and active integrin αIIbβ3 (CD61 and PAC-1), and P-selectin (CD62P); dense granule release was assessed using platelet loading with mepacrine, and procoagulant activity was determined with a phosphatidylserine marker annexin V. RESULTS: All investigated parameters of platelet function in children with severe chronic ITP before treatment were greatly impaired. CD42b and CD61 were up to several-fold lower the normal values, while phosphatidylserine exposure, dense granule release and integrin activation upon activation were decreased by at least an order of magnitude. Romiplostim treatment improved platelet parameters, although not always to the normal level. Five patients out of eight responded partially or completely to the romiplostim therapy. The clinical response (relief of hemorrhagic manifestations) correlated well with improvement of the functional state of platelets, in one case, even without significant platelet count increase. CONCLUSIONS. Our data suggest essential revision of the pathophysiology of severe chronic ITP in children: their platelets are not only small in their number, but also appear to have severe defects of all major functions. The mechanism of action of romiplostim in these patients might also be in some need of revision, as it seems to greatly improve not only quantity, but also quality of platelets. Our data demonstrate the important role of monitoring platelet functional activity in addition to platelet count. Disclosures No relevant conflicts of interest to declare.

Download Full-text

DECREASED ACTIVITY of ADAMTS13 IN PATIENTS with DEEP Vein Thrombosis.

Blood ◽

10.1182/blood.v114.22.3991.3991 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3991-3991

Author(s):

Bruna de Moraes Mazetto ◽

Fernanda A. Orsi ◽

Silmara Aparecida Lima Montalvao ◽

Tayana B Mello ◽

Erich Vinicius de Paula ◽

...

Keyword(s):

Deep Vein Thrombosis ◽

Conflicts Of Interest ◽

Inverse Correlation ◽

Normal Subjects ◽

Categorical Variables ◽

Control Group ◽

Continuous Variables ◽

Adamts13 Activity ◽

Vein Thrombosis ◽

Deep Vein

Abstract Abstract 3991 Poster Board III-927 Introduction Several risk factors, including increased levels of factor VIII (FVIII) and von Willebrand factor (VWF), have already been identified in patients with deep vein thrombosis (DVT). Recently we published a study showing that in a Brazilian population, plasma levels of FVIII over 180U/dl and VWF over 165U/dl were associated with the occurrence of venous thrombosis (odds ratio = 4.1 and 3.8 respectively) (Mello TB et al, 2009). The level of VWF in plasma and consequently FVIII is the result of genetic and acquired factors. ADAMTS13 (ADisintegrin And Metalloprotease with Thrombospondin type 1 repeats) is an enzyme responsible for cleavage of VWF, and its activity could contribute to VWF and FVIII plasmatic levels in patients with DVT. Objective To evaluate the activity of ADAMTS13 in patients with DVT associated with an increase of VWF and FVIII. Patients and methods: Fifty-six patients with FVIII > 180U/dl or FVW>165U/dl were selected from a cohort of 175 patients with DVT from the study mentioned above. Fifty-four normal subjects were selected as controls. The activity of ADAMTS 13 was performed by binding of residual VWF to collagen; VWF activity was measured by collagen binding, VWF antigen was determined by ELISA and FVIII was measured by a one-stage coagulation assay. Continuous variables were analyzed by Mann-whitney test and categorical variables by the Chi-square test. Results The demographic distribution of patients and controls were similar. Among the 56 patients the median age was 37.5 years, 39 were women, 46 had blood typing “non-O”, 34 had DVT caused by transient risk factor, especially the use of oral contraceptives, 10 patients had a hereditary thrombophilia and 3 were carriers of antiphospholipid antibodies. No patient had renal, hepatic or malignant disease. The median ADAMTS 13 activity was significantly lower in patients (112.9%, 44.4 - 327.6%) when compared to controls (142.9%, 76.7 - 323.6%), P = 0.001. The VWF activity was also higher in patients (109.7%, 26.8 - 422.6%) when compared to controls, (79.1%, 45.5 - 203.8%), P=0.038. The median level of VWF antigen was significantly higher in the group of patients when compared to the control group (178.1U/dl versus 111.9 U/dl respectively, P <0.0001). There was an inverse correlation between ADAMTS13 activity and VWF activity. Conclusion: This study suggests that the increased VWF and FVIII activity in patients with DVT can be a result of decreased ADAMTS13 activity. The decreased activity of ADAMTS13 may be influenced by the action of cytokines in inflammatory processes, even after acute period. Future studies will be important to determine the correlation between activity of ADAMTS 13, VWF, and inflammatory markers in the pathogenesis of DVT. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Centrosomal Disruption As Early Event in Myeloma Development

Blood ◽

10.1182/blood.v118.21.1806.1806 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1806-1806

Author(s):

Sabina Sevcikova ◽

Fedor Kryukov ◽

Elena Dementyeva ◽

Kubiczkova Lenka ◽

Pavel Nemec ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Genomic Instability ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Centrosome Duplication ◽

Early Event ◽

Newly Diagnosed ◽

Long Term Study ◽

Healthy Donors

Abstract Abstract 1806 Background: Multiple myeloma (MM) is a malignancy of differentiated B-lymphocytes characterized by accumulation of clonal plasma cells (PCs) in the bone marrow. Genome of malignant PCs is extremely unstable and characterized by complex combination of structural and numerical abnormalities. The basis of genomic instability underlying MM is still unclear. Centrosome amplification (CA) is present in about a third of MM cases and may represent a mechanism leading to genomic instability in myeloma. We have previously shown that CA is present in pre-plasma cells stages of B-cell development. Aims: The objective of our study was to evaluate changes in expression of chosen key genes involved in centrosome duplication process in both B and PCs, including their possible changes during MM progression. Materials and Methods: In total, 56 patients were evaluated for this study. Patients' characteristics were as follows: males/females 38/37, median age 69 years (range 43–83 years). Newly diagnosed (27/56) and relapsed (29/56) patients were included in this study; most of them had advanced stage of MM (DS III n=48; ISS II/III n=45). qRT-PCR (51 PCs and 32 B cell samples) was performed on a chosen set of mitotic genes, according to their role in normal centrosome duplication process. Results: Expression of genes involving in centrosome duplication process was studied in B cells of MM patients and healthy donors. Group of MM patients showed significant increase in relative quantification coefficient R in the following genes: AURKA, AURKB, CCNB1, CCNB2, HMMR, PLK4, TACC3 and TUBG1. It is notable that TUBG1 was approximately 330-times overexpressed in MM B cells vs healthy donors. Expression of studied genes was significantly different in B and PCs populations of MM patients: AURKB and TACC3 were upregulated in B cells while CENT2 and TUBG1 were upregulated in PCs. Analyses of gene expression in newly diagnosed and relapsed patients showed significant changes of the following genes: AURKA and TACC3 were 2-times upregulated in PCs of relapsed patients; no differences were detected in population of B cells. Conclusion: Considering revealed changes in B cells of myeloma patients, we suspect early damage in cell-cycle regulating mechanism. This hypothesis correlates with our previous findings of CA in MM B cells. We anticipate that dynamic accumulation of showed changes plays a role in final transformation of malignant PCs. We assume that centrosomal disruption likely contributes to accumulation of genomic abnormalities in tumor cells during MM progression which can be proven in future long-term study. Acknowledgment: This study was supported by grants NT11154, NS10207, MSM0021622434 Disclosures: No relevant conflicts of interest to declare.

Download Full-text

JAK2 Mutation Analysis and Function of Bone Marrow Mesenchymal Stem Cells in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v118.21.5179.5179 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5179-5179

Author(s):

Hong Tian ◽

Yang Xu ◽

Guanghua Chen ◽

Man Qiao ◽

Wu Depei

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Jak2v617f Mutation ◽

Primary Study ◽

Jak2 Mutation ◽

Healthy Donors ◽

Marrow Mesenchymal Stem Cells

Abstract Abstract 5179 Background: JAK2V617F and JAK2 exon12 mutations in haematopoietic cells were partially responsible for the pathogenesis of myeloproliferative neoplasms (MPN).But it was still unclear whether bone marrow mesenchymal stem cells (BMSCs), the significant component of hemopoiesis microenvironment, were participated in the pathogenesis of MPN. Objective: To study the physiopathology characteristics and analyze JAK2 mutation in BMSCs from MPN patients. Methods: By searched for the JAK2V617F mutation and exon 12 mutation in 135 MPN patients' blood /bone marrow samples, 20 patients with JAK2V617F mutation, 10 patients with JAK2 exon 12 mutation, 5 JAK2-mutation-negetive patients and 10 healthy donors were recruited. The phenotype, mesenchymal differentiation capacity, expression of hematopoietic and immune molecules and JAK2 mutation of isolated bone marrow BMSCs were detected. Results: BMSCs derived from the four groups were found to be similar in morphology, differentiation ability and expression of hematopoietic and immune molecules. Primary study indicated that the isolated BMSCs from patients groups were not able to harbor JAK2 mutation in spite of positive or negative JAK2 mutation in blood /bone marrow samples. Conclusion: BMSCs from MPN patients had similar biological characteristics to healthy donors, and BMSCs were not likely involved in pathogenesis of MPN. Disclosures: No relevant conflicts of interest to declare.

Download Full-text