JAK2(V617F) negatively regulates p53 stabilization by enhancing MDM2 via La expression in myeloproliferative neoplasms.

JAK2(V617F) is a gain of function mutation that promotes cytokine-independent growth of myeloid cells and accounts for a majority of myeloproliferative neoplasms (MPN). Mutations in p53 are rarely found in these diseases before acute leukemia transformation, but this does not rule out a role for p53 deregulation in disease progression. Using Ba/F3-EPOR cells and ex vivo cultured CD34(+) cells from MPN patients, we demonstrate that expression of JAK2(V617F) affected the p53 response to DNA damage. We show that E3 ubiquitin ligase MDM2 accumulated in these cells, due to an increased translation of MDM2 mRNA. Accumulation of the La autoantigen, which interacts with MDM2 mRNA and promotes its translation, was responsible for the increase in MDM2 protein level and the subsequent degradation of p53 after DNA damage. Downregulation of La protein or cell treatment with nutlin-3, a MDM2 antagonist, restored the p53 response to DNA damage and the cytokine-dependence of Ba/F3-EPOR-JAK2(V617F) cells. Altogether, these data indicate that the JAK2(V617F) mutation affects p53 response to DNA damage through the upregulation of La antigen and accumulation of MDM2. They also suggest that p53 functional inactivation accounts for the cytokine hypersensitivity of JAK2(V617F) MPN and might have a role in disease progression.

The first international meeting on V617F JAK2 mutation and its relevance in Philadelphia-negative myeloproliferative disorders.

The first international meeting on V617F JAK2 mutation in myeloproliferative disorders (MPD) was held by the PV-Nord group on behalf of the French Society of Hematology and Paris 13 University on November 18, 2005, in Paris (France). Twelve speakers, including representatives of the three European groups who discovered the V617F JAK2 mutation and international experts in the field of Philadelphia-negative MPD, presented original biological and clinical data that allow better insight in the relevance of V617F JAK2 mutation in the pathogenesis and management of those diseases. The role of V617F JAK2 in cytokine receptors trafficking and signaling was described. Follow-up of transgenic mice expressing V617F JAK2 showed that they develop typical features of myelofibrosis. Comparisons of JAK2 mutational status to clonality of hematopoiesis in essential thrombocythemia on the one hand, and to activation of transcription factors in myelofibrosis with myeloid metaplasia on the other hand, suggest that JAK2 mutation could be a second genetic event in a subset of patients. Alternatively, other gene mutation(s) have to be found to explain the development of V617F-negative MPD. In large series of MPD patients presented, clinical characteristics of mutated and non-mutated patients were found different. Finally, the place of V617F JAK2 testing in the diagnosis and management of MPD was discussed.

Oncogenic mechanisms in myeloproliferative disorders.

Myeloproliferative disorders (MPDs) are clonal haematopoietic malignancies involving the abnormal proliferation of myeloid lineages. The World Health Organisation (WHO) classification of haematopoietic malignancies distinguishes MPDs from myelodysplastic/ myeloproliferative disorders and systemic mastocytosis. These malignancies frequently involve constitutive tyrosine kinase activity, resulting from either oncogenic fusion protein production or from point mutations. Chronic myelogenous leukaemia is the model used for studies of the consequences of such molecular defects. However, the heterogeneity of the clinical course of MPDs should be seen in a more rationale conceptual framework, including the many molecular events associated with these diseases. This review focuses on the various tyrosine kinase-related molecular mechanisms underlying both MPDs and rare diseases with myeloproliferative features. We pay particular attention to the newly identified JAK2 V617F mutation in polycythaemia vera, essential thrombocythaemia and idiopathic myelofibrosis and deal with disease heterogeneity and putative additional molecular mechanisms.

Common 4q24 deletion in four cases of hematopoietic malignancy: early stem cell involvement?

We determined bone marrow karyotype at diagnosis in four female acute myeloid leukemia (AML) or myelodysplasia patients, aged between 52 and 56 years. In each case, we observed chromosome rearrangement involving the same 4q24 band. Three patients had a balanced reciprocal translocation as the sole abnormality - t(3;4)(q26;q24), t(4;5)(q24;p16) and t(4;7)(q24;q21) - and the fourth had del(4)(q23q24), +4. We used a set of 4q BAC probes for fluorescent in situ hybridization (FISH) in these four cases. We found a 4q24 submicroscopic deletion in all three translocations, with a common deletion of approximately 0.5 Mb. In three cases, we concluded that rearrangement occurred in an early hematopoietic stem cell, as it was detected, in mosaic with a normal karyotype, in a fraction of remission bone marrow cells, peripheral T and B lymphocytes, malignant lymph node T-lymphoma cells in one case and B-lymphoblastoid cell lines established in two cases. Moreover, one of 10 additional AML patients tested by FISH had a normal karyotype and deletion of one of the commonly deleted probe sequences. A tumor suppressor gene may therefore be involved, especially as two patients developed malignant lymphoma at the same time as myeloid proliferation.

[Antibodies against human recombinant erythropoietin: an unusual cause of erythropoietin resistance]. / Anticorps anti-érythropoïétine humaine recombinante: une cause exceptionnelle de résistance à l'érythropoïétine.

In a 70 year old man with primary glomerulonephritis, severe anemia occurred after 4 years on hemodialysis and rHu-EPO. The usual mechanisms of EPO-resistance were excluded. A bone marrow sample showed red all aplasia. No circulating EPO could be detected; the serum inhibited the growth of erythroid precursors in bone marrow cultures. Immunoprecipitation identified an IgG anti-EPO, still active against deglycosylated EPO, i.e. directed against the peptidic matrix. Its high neutralising capacity and the absence of any immune abnormality rule out an auto-antibody. Anti-rHu EPO immunisation is a very rare occurrence, made severe by transfusion-dependence and the risk of hemosiderosis. An immuno-modulating treatment can therefore be justified.

Autoimmune disorders of erythropoiesis.

Immune-mediated disorders of erythropoiesis can result in acquired severe anemia, low reticulocyte counts, and bone marrow exhibiting pure red cell aplasia or ineffective erythropoiesis. Erythropoiesis can be suppressed or impaired by humoral or cellular mechanisms. In vitro inhibition of erythroid colony growth by immunoglobulins or lymphocytes can be a strong argument for the immune origin of the disease. Classical etiologies are thymoma and hematologic malignancies such as chronic lymphocytic leukemia (CLL). Clonal proliferation of T cells has been incriminated. Recently, acquired circulating autoantibodies directed against erythropoietin have been detected in a case of pure red cell aplasia. Autoimmune mechanisms have also been detected or suggested in synartesis and in Fas-associated dyserythropoiesis, two distinct syndromes recently described where morphologic abnormalities specific to the erythroid lineage illustrate ineffective erythropoiesis.

Factor V Leiden related Budd-Chiari syndrome.

BACKGROUND: The role of factor V Leiden as a cause of Budd-Chiari syndrome has only recently been described. AIMS: To assess the specific features of factor V Leiden related Budd-Chiari syndrome. PATIENTS: Sixty three consecutive patients with hepatic vein or terminal inferior vena cava thrombosis. METHODS: Standardised chart review. RESULTS: Factor V Leiden was found in 20 patients (31% (95% CI 20-43)). In the subgroup of patients with, compared with the subgroup without, factor V Leiden, a combination of prothrombotic states was more common (70% (95% CI 50-90) v 14% (95% CI 3-24)); inferior vena cava thrombosis was more frequent (40% (95% CI 19-61) v 7% (95% CI 0-14)); and distribution of initial alanine aminotransferase values was bimodal (almost normal or extremely increased) versus unimodal (p=0.003). Factor V Leiden accounted for four of five cases of massive ischaemic necrosis (transaminases >50-fold the upper limit of normal values) (p=0.014), and also for all three cases developing during pregnancy. Patients with and without factor V Leiden did not differ with regard to mortality, portosytemic shunting, or listing for liver transplantation. Hepatocellular carcinoma developed in two patients; both had factor V Leiden and indolent obstruction of the inferior vena cava. CONCLUSIONS: In patients with Budd-Chiari syndrome, factor V Leiden (a) is common; (b) precipitates thrombosis mostly when combined with another risk factor; (c) is associated with one of two contrasting clinical pictures: indolent thrombosis-particularly of the inferior vena cava-or massive ischaemic necrosis; and (d) is a major cofactor of Budd-Chiari syndrome developing during pregnancy.

[Erythropoiesis disorders and other central autoimmune anemias]. / Erythroblastopénie et autres anémies centrales auto-immunes.

Immune-mediated acquired disorders of erythropoiesis can result in pure red cell aplasia or ineffective erythropoiesis. Erythropoiesis can be suppressed or impaired by humoral or cellular mechanisms. In vitro inhibition of erythroid colony growth by humoral factors or lymphocytes is a strong argument for the immune origin of the disease. Classical aetiologies are thymoma and hematological malignancies such as chronic lymphocytic leukaemia. Clonal proliferation of T cells has also been incriminated. Recently, acquired circulating autoantibodies directed against erythropoietin have been detected in a case of pure red cell aplasia. Autoimmune mechanisms have also been suggested in two dyserythropoietic syndromes recently described.

Therapy-related acute myeloid leukaemia after successful therapy for acute promyelocytic leukaemia with t(15;17): a report of two cases and a review of the literature.

We describe two patients with positive t(15;17) acute promyelocytic leukaemia (APL) that developed into a therapy-related myelodysplasia 2-2.5 years after complete remission (CR) and then evolved into therapy-related acute myeloid leukaemia (t-AML). Both patients received anthracyclines as potential leukaemogenic drugs. In both cases, cytogenetic changes usually occurring after use of alkylating agents were noticed: monosomy 7 associated with monosomy 5 or 5q- chromosome. A review of the literature on t-AML occurring after successful therapy for APL showed only one report similar to these two cases. These observations suggest that anthracyclines can cause t-AML similar to that induced by alkylating agents.

Successful treatment of thymoma-associated pure red cell aplasia with intravenous immunoglobulins.

Repeated cycles of intravenous immunoglobulins (IVIG) have been reported to be successful in a few patients with idiopathic pure red cell aplasia (PRCA) or associated with another pathology. The efficacy of this treatment for PRCA with thymoma has not been reported previously. We describe here the case of a 75-yr-old man who presented with PRCA associated with a benign thymoma. After failure of thymectomy, corticosteroids and octreotide, a complete durable remission was obtained after a single 5-d cycle of IVIG.

The immunophenotype of 177 adults with acute myeloid leukemia: proposal of a prognostic score.

In acute myeloid leukemia (AML) patients, a variety of clinical and biologic parameters, including phenotype, have been examined for potential value in predicting treatment response and survival. The European Group for the Immunological Classification of Leukaemias (EGIL) has proposed that AML be defined immunologically by the expression of 2 or more of the following myeloid markers: myeloperoxidase, CD13, CD33, CDw65, and CD117. With regard to this classification, the prognostic significance of 21 antigens taken separately and with immunophenotypic subgroups were evaluated and compared with other clinical and biological variables in 177 adult AML patients. None of the antigens tested were associated with treatment outcome. In contrast, patients with blasts disclosing a full expression of panmyeloid phenotype (defined by the expression of all 5 myeloid markers) had a higher complete remission rate (P <. 0001) and differed significantly in disease-free survival (P =.02) and overall survival (P =.008) than patients whose cells expressed fewer than 5 of these markers. In multivariate analysis, only age, panmyeloid phenotype, performance status, and permeability glycoprotein activity influence treatment outcome. Cytogenetics was significant in univariate analysis but not in multivariate analysis, most likely because of the redundancy with panmyeloid phenotype and a higher sensitivity of immunophenotyping. Patients whose cells exhibit the panmyeloid phenotype appear to define a relatively homogeneous biological subset of AML. The 4 independent prognostic factors were used to create a prognostic score, defined by the number of factors present. This score permitted a stratification of patients with AML, thereby allowing for the consideration of innovative therapies to improve outcome in the poorer outcome groups.

Cause of portal or hepatic venous thrombosis in adults: the role of multiple concurrent factors.

According to a recent hypothesis, venous thrombosis results from the concurrence of several factors. This hypothesis was assessed in patients with portal or hepatic venous thrombosis by simultaneously investigating most of the currently identified prothrombotic disorders, local precipitating factors, and other risk factors such as oral contraceptive use. Patients with a tumorous obstruction and patients with cirrhosis with portal vein thrombosis were excluded. The prothrombotic disorders that were investigated included classical and occult myeloproliferative disorders; antiphospholipid syndrome; protein C; protein S and antithrombin deficiency; factor V Leiden; factor II; and methylene-tetrahydrofolate-reductase gene mutations. We found 1 or several prothrombotic disorders and a local precipitating factor in 26 and 10 of the 36 patients with portal vein thrombosis, respectively; and in 28 and none of the 32 patients with hepatic vein thrombosis, respectively. We found a combination of prothrombotic disorders in 5 and 9 patients with portal and hepatic vein thrombosis, respectively, whereas such a combination is expected in less than 1% of asymptomatic subjects. Of the 10 patients with a local precipitating factor, 8 had a prothrombotic disorder. Of the 13 patients who use oral contraceptives, 10 had a prothrombotic disorder. We conclude that portal or hepatic venous thrombosis should be regarded as an index for 1 or several prothrombotic disorders, whether or not local precipitating factors or oral contraceptive use are found. Concurrence of prothrombotic disorders is more common than expected. Extensive investigation of prothrombotic disorders and anticoagulation should be considered in patients with portal or hepatic venous thrombosis.

Erythroblastic synartesis: an auto-immune dyserythropoiesis.

Erythroblastic synartesis is a rare form of acquired dyserythropoiesis, first described by Breton-Gorius et al in 1973. This syndrome is characterized by the presence of septate-like membrane junctions and "glove finger" invaginations between erythroblasts, which are very tightly linked together. This phenomenon, responsible for ineffective erythropoiesis, leads to an isolated severe anemia with reticulocytopenia. In the following report, we describe 3 new cases of erythroblastic synartesis associated with dysimmunity and monoclonal gammapathy. In all cases, the diagnosis was suggested by characteristic morphological appearance of bone marrow smears, and further confirmed by electron microscopy. Ultrastructural examination of abnormal erythroblast clusters showed that these cells were closely approximated with characteristic intercellular membrane junctions. The pathogenesis of the dyserythropoiesis was modeled in vitro using crossed erythroblast cultures and immunoelectron microscopy: when cultured in the presence of autologous serum, the erythroblasts from the patients displayed synartesis, whereas these disappeared when cultured in normal serum. Moreover, synartesis of normal erythroblasts were induced by the patient IgG fraction. Immunogold labeling showed that the monoclonal IgG were detected in, and restricted to, the synartesis. A discrete monoclonal plasmacytosis was also found in the patient bone marrow. The adhesion receptor CD36 appeared to be concentrated in the junctions, suggesting that it might be involved in the synartesis. These experiments indicated that a monoclonal serum immunoglobulin (IgG in the present cases) directed at erythroblast membrane antigen was responsible for the erythroblast abnormalities. Specific therapy of the underlying lymphoproliferation was followed by complete remission of the anemia in these cases.

Contribution of D-Dimer determination in the exclusion of deep venous thrombosis in spinal cord injury patients.

UNLABELLED: Deep vein thrombosis (DVT) is a common complication of paraplegia despite prophylactic anticoagulant therapy. The diagnosis relies primarily on ultrasonography or phlebography; these investigations are difficult, expensive and can be time-consuming in paraplegic patients. STUDY DESIGN: To evaluate the usefulness of coagulation activation markers in excluding a diagnosis of DVT, D-Dimers, thrombin-antithrombin complexes, prothrombin fragments (F1+2) and activated factor VIIa. OBJECTIVES: To improve the diagnosis of deep venous thrombosis in paraplegic patients. SETTING: This collaborative work was done at Raymond Poincaré Hospital, Garches, France. METHODS: To evaluate the usefulness of coagulation activation markers in excluding a diagnosis of DVT, D-Dimers (D-Di), thrombin-antithrombin (TAT) complexes, prothrombin fragments (F1+2) and activated factor VIIa (FVIIa), were determined in a prospective study of 67 consecutive patients with paraplegia or tetraplegia. Doppler ultrasonography and/or phlebography of the lower limbs and D-Di, TAT, F1+2 level determination were systematically done in each patient at admission to our rehabilitation unit. RESULTS: Despite prophylactic low molecular weight heparin therapy, six of the 67 patients developed DVT diagnosed by radiologic explorations. D-Di levels measured by a reference ELISA (Asserachrom D-Di, Diagnostica Stago) or a new rapid automated turbidimetric test (STA-Liatest D-Di) were greater than 500 ng/ml in all DVT patients and in 40 non-DVT patients, of whom most had urinary tract infections, osteomas, or pressure sores. D-Di values were normal in only 21/67 patients (31%). The negative predictive value of D-Di in our study was 100% since all DVT patients had D-Di values greater than 500 ng/ml. TAT and F1+2 levels were not correlated with D-Di levels but also had a negative predictive value of 100%. Comparison of D-Di levels obtained using the two tests showed that results of the reference ELISA were closely correlated to those of the new rapid automated turbidimetric. TAT, F1+2, and factor VIIa are not useful for measuring hypercoagulability in paraplegic or tetraplegic patients since no rapid tests for determining these parameters are available. CONCLUSION: D-Di levels determined using an ELISA or a new rapid automated turbidimetric test have a good negative predictive value for DVT in paraplegic or tetraplegic patients and may reduce the need for Doppler ultrasonography and/or phlebography by 31%.

Aplastic anemia responsive to cyclosporine complicating the evolution of polycythemia vera.

We report here a very unusual patient with Polycythemia vera treated with Pipobroman who developed severe aplastic anemia following administration of the drug. Six months later, because of lack of response, cyclosporine therapy was given there was rapid and complete hematological recovery, highly suggestive of an immune-mediated mechanism, in this case.