N-ras and p53 gene mutations in Japanese patients with myeloproliferative disorders

Alterations of the N-ras oncogene and p53 tumor suppressor gene have been demonstrated to play an important role in pathogenesis of hematological malignancies. We simultaneously investigated genetic lesions of both genes in bone marrow cells from 64 Japanese patients with myeloproliferative disorders (MPD), including polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (MF), by direct sequencing analysis. No mutations of the N-ras gene were detected in any cases. Two patients, one with chronic neutrophilic leukemia derived from PV and one with acute mylogenous leukemia derived from ET, exhibited three mutations of the p53 gene. Among them, two were missense mutations in exon 5 or 7 and one was a deletion in exon 5. All samples in chronic phase or from MF were devoid of mutations in both genes. These data suggested that disruptions of both genes are extremely rare in MPD in chronic phase and that loss of functions in the p53 gene could be involved in progression of MPD such as PV and ET.     

Increased platelet activation and abnormal membrane glycoprotein content and redistribution in myeloproliferative disorders

Chronic myeloproliferative disorders (MPDs) are characterized by a high incidence of thrombohaemorrhagic complications, possibly caused by platelet dysfunction. In an attempt to define platelet functional abnormalities, we assessed the expression of activation-dependent membrane proteins in unstimulated and agonist [ADP and thrombin receptor-activating peptide (TRAP)]-stimulated platelets using quantitative whole blood flow cytometry in samples from 50 MPD patients and 30 controls. The receptor densities of activation markers and glycoproteins (GPs) were quantified using standardized fluorescent beads. Compared with controls, the mean percentage of P-selectin-positive (15.3% vs. 7.2%; P < 0.001) and thrombospondin (TSP)-positive (6.6% vs. 3.7%; P = 0.003) platelets was increased in unstimulated platelets from patients. Patients having experienced a thrombotic event had a higher mean percentage of TSP-positive non-stimulated platelets than patients without a history of thrombosis (9.0% vs. 4.6%; P = 0.02) and a higher GPIV molecules of equivalent fluorochrome (MEF) value (33113 vs. 24471 MEF; P = 0.02). Mean MEF values of monoclonal antibodies (mAbs) against GPIb (34055 vs. 38945 MEF; P < 0.001) and GPIIb/IIIa (1416 vs. 1648 MEF; P < 0. 001) were significantly reduced among patients, whereas surface expression of GPIV was increased in patients (28273 vs. 16258 MEF; P < 0.001). In TRAP (10 micromol/l) stimulated whole blood, the MEF of P-selectin (9611 vs. 13293 MEF; P = 0.004) and CD63 (2385 vs. 5177 MEF; P < 0.001) and the ratio of PAC-1/GPIIb/IIIa MEF (0.98 vs. 2. 00; P < 0.001) was reduced in patients, indicating either a reduced granule GP content or an intrinsic cellular defect in receptor-mediated granule secretion and activation of the GPIIb/IIIa complex. Expressed as the relative change of MEF compared with unstimulated platelets, TRAP induced decrease of GPIb (7.8% vs. 45%; P < 0.001) and increase of GPIIb/IIIa (49.1% vs. 95.7%; P < 0.001) and GPIV expression (17.8% vs. 55.2%; P < 0.001) was attenuated in patients.     

Use of whole blood platelet lumi-aggregometry to optimize anti-platelet therapy in patients with chronic myeloproliferative disorders

Twenty-seven patients with chronic myeloproliferative disorders and in vitro evidence of platelet hyperactivity on whole blood platelet lumi-aggregometry were commenced on anti-platelet therapy comprising aspirin, clopidogrel, and/or odorless garlic and the studies were repeated to assess the efficacy of the therapeutic agent(s). Only 8 patients showed clear evidence of anti-platelet effect while receiving the standard low-dose (100 mg/day) aspirin therapy. Thirteen patients required a higher dosage of aspirin and/or an additional anti-platelet agent to achieve therapeutic adequacy. Lumi-aggregometry also proved useful to optimize therapy in the 6 patients who received clopidogrel or odorless garlic because of aspirin intolerance.     

Thrombosis and bleeding in myeloproliferative disorders: identification of at-risk patients with whole blood platelet aggregation studies

Seventy-five patients with chronic myeloproliferative disorders were studied to investigate platelet function by simultaneous measurement of platelet aggregation by the impedance method and ATP dense granule release using a whole blood platelet lumi-aggregometer, in an attempt to identify patients at risk for thrombosis and bleeding. Thirty-nine patients had at least one abnormal result indicating platelet hyperactivity (i.e. impedance or release with one agonist being above the reference range); 16 patients had platelet hypoactivity (i.e. at least one result was below the reference range), whilst 14 had co-existence of hyper- and hypoactivity. Six patients had normal results. 20/53 patients with platelet hyperactivity (alone or mixed) had a positive history of venous and/or arterial thrombosis; in comparison, only two of the other 22 patients had a positive history. During a median follow-up of 33 months, nine patients with and one patient without platelet hyperactivity respectively developed new thrombotic events before the addition of specific therapy. A total of 50 patients with and eight patients without platelet hyperactivity respectively received specific treatment including aspirin and/or cytotoxic therapy. All but one elderly patient with platelet hyperactivity have remained free of new thrombotic events on specific therapy. Two of the 17 patients with platelet hypoactivity had major clinical bleeding. These observations highlight the need to test platelets for hyper- as well as hypo-function and suggest a useful role for routine whole blood platelet aggregation studies to identify the patients at risk for thrombosis or bleeding.     

Changes in distribution of platelet membrane glycoproteins in patients with myeloproliferative disorders

Glycoproteins have been discovered to be important to platelet function both in normal and pathological states. We have studied membrane glycoprotein patterns in 16 patients with various myeloproliferative disorders. There was an abnormal ratio of glycoprotein I glycoprotein IV in patients with myeloproliferative disease compared with controls. There was no discernible correlation between glycoprotein pattern and aggregation response or platelet count, but patients with megathrombocytes had higher values for glycoprotein IV than those without megathrombocytes. These experiments suggest that patients with myeloproliferative disorders may have alterations in membrane glycoproteins that could alter platelet function.     

Phospholipid determination in platelets,plasma and red cells of patients with chronic myeloproliferative disorders

Abstract: Red cell phospholipids (PLs) were assessed in 11 patients with essential thrombocythemia and 5 patients with polycythemia vera. Platelet and plasma PLs were also determined in 10 of these patients, and the results were compared with studies performed in 16 healthy volunteers. The amount of platelet PLs in patients was similar to controls (556 ± 90 nmol/10⁹cells, versus 481 ± 91 nmol/10⁹cells), as was the percentage of the main specimens of these compounds, including phosphatidylserine (11.1 ± 0.8%), which is relevant for platelet procoagulant activity. We did not find differences between red cell PLs of patients (300 ± 60 nmol/10⁹cells), versus controls (289 ± 71 nmol/10⁹cells), and the sphingomyelin/phosphatidylcholine ratio in these cells was the same in both groups (0.75 ± 0.1). Finally, we did not detect any alteration in the amount of plasma PLs specimens.     

Increased platelet CD36 constitutes a common marker in myeloproliferative disorders

Summary. The distribution of the major platelet membrane glycoproteins (GP), Ib, IX, IIb-IIIa and IV (or CD36), which play important roles as receptors for adhesive molecules in haemostasis and thrombosis, was studied in 34 patients with myeloproliferative disorders (MPD): 13 had essential thrombocythaemia (ET), 12 had polycythaemia vera (PV) and nine had chronic myelogenous leukaemia (CML). Only occasionally were modifications of the numbers of GPIb or GPIIb-IIIa measured using the binding of specific radiolabeled antibodies to platelets. In contrast, 2-3-fold increases of the total CD36 content and the surface CD36 expression were measured in almost all patients studied, using a radioimmunoassay and the direct binding of the radiolabelled antibody, FA6-152, to the platelet surface, respectively. These results indicate that the abnormality affected both the external and internal CD36 pools. Therefore platelet CD36 may be a useful tool for the diagnosis and the follow-up of MPD patients.
Surface CD36 has been proposed as a platelet receptor for thrombospondin, an adhesive glycoprotein that is released from platelets upon activation and promotes aggregate formation. Despite a 2-fold increase of CD36 molecules, resting and thrombin-activated platelets from ET patients expressed the same amount of thrombospondin as normal platelets, suggesting that there is not a direct correlation between the CD36 expression and thrombospondin binding either spontaneously or after activation.     

Concomitant neutrophil JAK2 mutation screening and PRV-1 expression analysis in myeloproliferative disorders and secondary polycythaemia

Polycythaemia vera (PV) is closely associated with both an acquired activating mutation of the JAK2 tyrosine kinase (JAK2(V617F)) in granulocyte-derived DNA and increased granulocyte polycythaemia rubra vera-1 (PRV-1) expression. In order to explore the correlation between these two biological markers and compare their diagnostic utility, mutation analysis for JAK2(V617F) and quantitative measurement of granulocyte PRV-1 expression were performed on the same study sample from 100 participants: 38 with PV, 22 with essential thrombocythaemia (ET), 10 with agnogenic myeloid metaplasia (AMM), 19 with secondary polycythaemia (SP) and 11 healthy volunteers. The respective overall (homozygous) JAK2(V617F) mutational frequencies were 95% (26%), 55% (0%), 30% (0%), 0% and 0%. The corresponding figures for increased PRV-1 expression were 89%, 18%, 20%, 21% and 9%. In patients with either ET or AMM, the likelihood of detecting JAK2(V617F) was significantly higher in the presence of an increased PRV-1 expression (83% vs. 38%; P = 0.05). Similarly, in patients with PV, homozygous as compared with heterozygous JAK2(V617F) correlated with higher levels of PRV-1 expression (P = 0.11). The present study suggests an allele dose-dependent effect of JAK2(V617F) on granulocyte PRV-1 expression. However, compared with the PRV-1 assay, mutation screening for JAK2(V617F) displayed greater accuracy in distinguishing PV from SP.     

Proto-oncogene c-mpl is involved in spontaneous megakaryocytopoiesis in myeloproliferative disorders

Spontaneous megakaryocytic colonies (CFU-MK) formation without the addition of Meg-CSA in myeloproliferative disorders (MPD) has been reported by many laboratories. The mechanism by which this occurs is still unknown. In our previous work we have found that the spontaneous colonies persisted in serum-free agar culture although the colony cells were smaller and the colony numbers fewer than in plasma clot culture and that monoclonal antibodies against IL3, IL6 and GM-CSF had no inhibitory effect on spontaneous CFU-MK in both semi-solid cultures. Recently, proto-oncogene c-mpl and c-mpl ligand, thrombopoietin (TPO), have been shown to specifically participate in the regulation of normal human megakaryocytopoiesis. In order to test the hypothesis that c-mpl c-mpl ligand pathway is involved in the spontaneous growth of megakaryocyte progenitors, we investigated mRNA expressions of c-mpl and TPO in cells grown in serum-free liquid culture using RT-PCR. The c-mpl expression was detected in the cultured cells from all nine patients (six with ET, two with PV, one with PMF) who had spontaneous CFU-MK in clonal assays. However, none of the patients expressed TPO mRNA in these cells. Pre-incubation of nonadherent mononuclear cells with thioester-modified antisense oligodeoxynucleotide to c-mpl at a concentration of 6μ M significantly decreased the cloning efficiency of spontaneous megakaryocyte growth by 42.5% ( P <0.05) in plasma clot assay (seven with ET, one with PV) and 69.6% ( P <0.05) in serum-free agar culture (six with ET, one with PV). In control experiments the introduction of a scrambled oligomer to antisense oligodeoxynucleotide had no such effect on spontaneous colony formation. These results indicate that c-mpl exerts an important effect in the growth of spontaneous megakaryocytopoiesis in MPD.     

Proto-oncogene c-mpl is involved in spontaneous megakaryocytopoiesis in myeloproliferative disorders

Spontaneous megakaryocytic colonies (CFU-MK) formation without the addition of Meg-CSA in myeloproliferative disorders (MPD) has been reported by many laboratories. The mechanism by which this occurs is still unknown. In our previous work we have found that the spontaneous colonies persisted in serum-free agar culture although the colony cells were smaller and the colony numbers fewer than in plasma clot culture and that monoclonal antibodies against IL3, IL6 and GM-CSF had no inhibitory effect on spontaneous CFU-MK in both semi-solid cultures. Recently, proto-oncogene c-mpl and c-mpl ligand, thrombopoietin (TPO), have been shown to specifically participate in the regulation of normal human megakaryocytopoiesis. In order to test the hypothesis that c-mplc-mpl ligand pathway is involved in the spontaneous growth of megakaryocyte progenitors, we investigated mRNA expressions of c-mpl and TPO in cells grown in serum-free liquid culture using RT-PCR. The c-mpl expression was detected in the cultured cells from all nine patients (six with ET, two with PV, one with PMF) who had spontaneous CFU-MK in clonal assays. However, none of the patients expressed TPO mRNA in these cells. Pre-incubation of nonadherent mononuclear cells with thioester-modified antisense oligodeoxynucleotide to c-mpl at a concentration of 6μM significantly decreased the cloning efficiency of spontaneous megakaryocyte growth by 42.5% (P<0.05) in plasma clot assay (seven with ET, one with PV) and 69.6% (P<0.05) in serum-free agar culture (six with ET, one with PV). In control experiments the introduction of a scrambled oligomer to antisense oligodeoxynucleotide had no such effect on spontaneous colony formation. These results indicate that c-mpl exerts an important effect in the growth of spontaneous megakaryocytopoiesis in MPD.     

c-kit point mutation of extracellular domain in patients with myeloproliferative disorders

Summary. c-kit is a tyrosine kinase receptor whose ligand is stem cell factor (SCF). Gene alteration of the c-kit extracellular domain was analysed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) in 25 patients with myeloproliferative disorders (MPD). In the N-terminal part of the domain, mobility shifts indicating sequence alteration were detected in three of the patients, two primary myelofibrosis (PMF) and one chronic myelogenous leukaemia (CML). The subsequent sequencing revealed the same point mutations at codon 52 causing amino acid substitution (Asp → Asn). To our knowledge this is the first report with a c-kit point mutation found in human fresh tumour cells.     

Platelet P-selectin expression in patients with sickle cell disease who undergo apheresis

Activated platelets have been identified in patients with sickle cell disease. However, the association of platelet P-selectin expression and automated red cell exchange procedures in these patients is not well known. We hypothesized that altered whole platelet P-selectin expression is associated with automated red cell exchange. Flow cytometric quantification of platelet P-selectin expression was carried out in 23 patients with sickle cell disease before and after automated red cell exchange. P-selectin expression was quantified as a binding index for platelet P-selectin (the percentage of positive platelets multiplied by the mean fluorescence of positive platelets). The patients were divided into two groups: individuals with painful vaso-occlusive crises (four women and five men; group 1) and those in a steady state (six women and eight men; group 2). The 33 exchange procedures were evaluated prospectively and used acid-citrate-dextrose A solution (whole blood to anticoagulant ratio = 14:1). Platelet P-selectin expression did not significantly change after automated red cell exchange. Clinical factors such as the volume of replacement fluid and the citrate infusion rate did not correlate with postapheresis platelet P-selectin expression. In addition, the association of platelet P-selectin expression and automated red cell exchange was independent of other laboratory factors (hematocrit level, hemoglobin S level, platelet count, and nitric oxide level). Finally, the difference between the study groups regarding platelet P-selectin expression before and after apheresis was insignificant. In conclusion, automated red cell exchange procedures do not induce platelet P-selectin expression in patients with sickle cell disease in the steady state or in vaso-occlusive crisis.     

氧化型脂蛋白(a)对人脐静脉血管内皮细胞P选择素表达的影响

目的探讨氧化修饰的脂蛋白(a)[Ox-Lp(a)]能否诱导人脐静脉内皮细胞表达P选择素,以阐明其在动脉粥样硬化发生中的作用.方法在内皮细胞培养基中分别加入天然脂蛋白(a)[n-Lp(a)]与ox-Lp(a)及天然与氧化的低密度脂蛋白(n-LDL、ox-LDL).用细胞酶联免疫检测P选择素蛋白的表达,用单核细胞黏附率试验检测单核细胞黏附,并用Northern杂交检测P选择素mRNA的表达.结果ox-Lp(a)呈剂量依赖性增加内皮细胞P选择素蛋白表达,10nmol/L及20nmol/LOx-Lp(a)分别使P选择素表达明显增加到(145±10)%及(202±22)%.同时观察到Ox-Lp(a)促使单核细胞黏附率增加,并且黏附率与加入的Ox-Lp(a)浓度呈正相关.四种脂蛋白的作用进行比较显示Ox-Lp(a)的作用最强,10nmol/Ln-LDL、n-Lp(a)、ox-LDL、ox-Lp(a)分别使P选择素表达增加到(105±7)%、(127±9)%、(176±19)%、(245±22)%.Northernblot结果显示Ox-Lp(a)能促使P选择素mRNA表达明显增加.结论Ox-Lp(a)能诱导内皮细胞表达P选择素增强,这可能与脂蛋白(a)致动脉粥样硬化作用机制有关.     

Analysis of JAK2(V617F) mutation in Chinese patients with myeloproliferative disorders

It is now well-recognized that the activating JAK2(V617F) mutation occurs in the majority of patients with polycythemia vera (PV) and approximately half of those with either essential thrombocythemia (ET) or myelofibrosis with myeloid metaplasia (MMM). Here we analyzed JAK2(V617F) mutation in 137 Chinese patients with myeloproliferative disorders by allele-specific polymerase chain reaction (PCR). DNA was extracted from methanol/acetic acid-fixed cells that had been routinely prepared for cytogenetic analysis. A single point mutation (Val617Phe) was identified in JAK2 in 42 (73.7%) of 57 patients with PV, 40 (58.8%) of 68 with ET, and eight (66.7%) of 12 with MMM.     

Soluble angiogenic factors: implications for chronic myeloproliferative disorders

The role of angiogenesis for the progressive growth and metastatic process of tumours is well established. What is not clear, though, is the clinical prognostic significance of the angiogenic factors in malignant haematological diseases. In this study, we have assessed the plasma and serum levels of two major angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) in 55 patients affected by chronic myeloproliferative disorders (CMD). This series included 25 patients with essential thrombocythemia (ET), 10 patients with chronic myelocytic leukaemia (CML), 14 patients with polycythemia vera (PV), and 6 patients with primary myelofibrosis (MF), and they were compared to 20 healthy control subjects. In all patients the plasma VEGF concentration was significantly increased to the healthy control group (P < 0.004). The highest concentrations were found in the patients with ET (178.25 +/- 125.22 pg/ml). The VEGF levels were significantly higher in CMD patients with vascular complications than those in CMD patients without complications (P < 0.01). The b-FGF serum levels also appeared to be significantly higher in almost all the CMD patients compared to the control group (P < 0.07). A significant correlation was found between the VEGF levels and the platelet count in the ET patients and the spleen index in the CML patients. VEGF level, in this study, is associated with increased risk of thrombotic complications. There is evidence of increased levels of soluble angiogenic factors in malignant haematological disorders, but their contribution to the progression of diseases is yet unclear.     

高脂蛋白血症患者血浆P-选择素表达的研究

目的　观察高脂蛋白血症患者血浆P 选择素表达及普伐他汀的作用。方法　应用酶联免疫吸附法检测 31例高脂蛋白血症患者服用普伐他汀前后血浆P 选择素水平 ,并与 35例血脂正常者对照。结果　高脂蛋白血症组血清总胆固醇、低密度脂蛋白胆固醇及血浆P 选择素水平均显著高于正常对照组 (P <0 .0 1) ,普伐他汀治疗后血浆P 选择素水平显著下降 (P <0 .0 1)。结论　高脂蛋白血症患者血浆P -选择素表达异常可能参与高脂蛋白血症的致动脉粥样硬化作用。     

Megakaryocyte c-Mpl expression in chronic myeloproliferative disorders and the myelodysplastic syndrome: immunoperoxidase staining patterns and clinical correlates

The objectives of this study were to expand on recent observations that have suggested decreased thrombopoietin receptor (c-Mpl) expression in megakaryocytes of patients with polycythemia vera (PV) and agnogenic myeloid metaplasia (AMM). We applied an immunoperoxidase method with anti-c-Mpl antibody to 55 bone marrow sections from previously untreated patients with chronic myeloproliferative disorder (CMPD) or myelodysplastic syndrome (MDS). These included 8 patients with PV, 15 with AMM, 9 with essential thrombocythemia, 5 with chronic myelocytic leukemia, 9 with the 5q-syndrome and 9 with MDS with fibrosis. The findings were compared with those in four patients with reactive erythrocytosis (RE), six with immune thrombocytopenic purpura (ITP) and five normal controls. Staining intensity (SI) was moderate to strong both in normal controls and in patients with RE or ITP. In contrast, SI was weak in variable proportions of the megakaryocytes in every one of the aforementioned clonal myeloid disorders. The staining pattern (SP) was relatively uniform in MDS and heterogeneous in CMPD. Neither SI nor SP was significantly correlated with certain clinical or laboratory parameters. We concluded that altered megakaryocyte c-Mpl expression may be a nonspecific phenomenon in various subtypes of both CMPD and MDS. However, the characteristic staining patterns may complement the morphological distinction between clonal and reactive myeloproliferation.     

Flow cytometric analysis of megakaryocyte ploidy in chronic myeloproliferative disorders and reactive thrombocytosis

Abstract: Megakaryocyte (MK) ploidy patterns were analysed by flow cytometry in 29 newly diagnosed and previously untreated patients with chronic myeloproliferative disorders (MPD) and concomitant thrombocytosis, in 9 patients with reactive thrombocytosis (RT) and in 12 healthy individuals. Unfractionated bone marrow from routine aspirates was used. MKs were identified with a fluorescein labelled monoclonal antibody specific for glycoprotein IIIa (GPIIIa) and DNA was stained with propidium iodide. For the 12 healthy volunteers the mean modal ploidy number was 16 N; the 9 patients with RT displayed an identical MK ploidy pattern. The frequency of MKs with a ploidy ≥32 N was 45% among the patients with essential thrombocythaemia (ET) compared to 32% among the healthy volunteers (p < 0.001). MKs with ploidy number ≥ 64 N, comprising approximately 13% of the total number of MKs, was a characteristic finding in the patients with ET. Similar findings were present in 8 patients with polycythaemia vera (PV). In patients with PV 34% and 6% of the MKs displayed ploidies ≥32 N and ≥64 N, respectively. In contrast, a distinct shift towards lower ploidy number, with 63% of MKs ≤ 8 N, was found among the 4 patients with chronic myeloid leukaemia (CML). The present results indicate that by using flow cytometric analysis of MK ploidy distribution in patients with thrombocytosis, those with a reactive cause are likely to be discriminated from patients with myeloproliferative thrombocytosis, i.e. PV and ET on one hand and CML on the other hand. The distinction between ET and PV, however, has to be made on other grounds.