Autonomous Megakaryocyte Growth in Essential Thrombocythemia and Idiopathic Myelofibrosis Is Not Related to a c-mpl Mutation or to an Autocrine Stimulation by Mpl-L

Abstract

Essential thrombocythemia (ET) and idiopathic myelofibrosis (PMF) are two myeloproliferative diseases characterized by a marked megakaryocytic (MK) involvement. The pathogenesis of these two diseases is unknown. Recently it has been shown that overexpression of Mpl-ligand (Mpl-L) in mice induces thrombocytosis and myelofibrosis. In this study, we investigated whether Mpl-L was responsible for the pathogenesis of ET and PMF. Using in vitro cultures of blood or marrow CD34+ cells, we investigated whether MK growth was abnormal in these two diseases. Spontaneous MK growth involving only a fraction (20%) of the MK progenitors, as compared with growth in the presence of pegylated recombinant human megakaryocyte growth and development factor (PEG-rhuMGDF), was found in both diseases (21ET and 14PMF) using serum-free semisolid and liquid cultures, including cultures at one cell per well. We first searched for ac-mpl mutation/deletion by sequencing the entire coding region of the gene by polymerase chain reaction (PCR) in nine ET patients and five PMF patients, but no mutation was found. We subsequently investigated whether an autocrine stimulation by Mpl-L could explain the autonomous MK growth. Addition of different preparations of soluble Mpl receptor (sMpl) containing a Fc domain of IgG1 (sMpl-Fc) markedly inhibited MK spontaneous growth in both ET and PMF patients. This effect was specific for sMpl because a control soluble receptor (s4-1BB-Fc) had no inhibitory effect and an sMpl devoid of the Fc fragment had the same inhibitory efficacy as the sMpl-Fc. This inhibition was reversed by addition of PEG-rhuMGDF or a combination of cytokines. The sMpl-Fc markedly altered the entry into cell cycle of the CD34+ cells and increased the apoptosis that occurs in most patient CD34+ cells in the absence of exogenous cytokine, suggesting an autocrine stimulation. In contrast, a neutralizing antibody against Mpl-L did not alter the spontaneous MK growth, whereas it totally abolished the effects of 10 ng/mL PEG-rhuMGDF on patient or normal CD34+ cells. Mpl-L transcripts were detected at a very low level in the patient CD34+cells and MK and only when a highly sensitive fluorescent PCR technique was used. By quantitative reverse-transcription (RT)-PCR, the number of Mpl-L transcripts per actin transcripts was lower than detected in human Mpl-L–dependent cell lines, suggesting that this synthesis of Mpl-L was not biologically significant. In favor of this hypothesis, the Mpl-L protein was not detected in culture supernatants using either an enzyme-linked immunosorbent assay (ELISA) or a biological (Ba/F3huc-mpl) assay, except in one PMF patient. Investigation of Mpl-L signaling showed an absence of constitutive activation of STATs in spontaneously growing patient MKs. Addition of PEG-rhuMGDF to these MKs activated STATs 3 and 5. This result further suggests that spontaneous growth is neither related to a stimulation by Mpl-L nor to ac-mpl mutation. In conclusion, our results show that Mpl-L or Mpl are not directly implicated in the abnormal proliferation of MK cells from ET and PMF. The mechanisms by which the sMpl mediates a growth inhibition will require further experiments.

Essential thrombocythaemia in children

“…Thrombopoietin concentrations in peripheral blood and bone marrow as well as thrombopoietin mRNA in marrow stromal cells from patients with ET were found to be comparable to those of normal control subjects (Hirayama et al, 1998). In patients with non-familial ET, thrombopoietin transcript and protein concentrations are low in CD34 cells, megakaryocytes and in supernatants from in vitro cultures of blood or marrow CD34 () cells (Taksin et al, 1999). Moreover, a neutralizing antibody against thrombopoietin does not alter autonomous megakaryocyte growth (Taksin et al, 1999).…”

“…In patients with non-familial ET, thrombopoietin transcript and protein concentrations are low in CD34 cells, megakaryocytes and in supernatants from in vitro cultures of blood or marrow CD34 () cells (Taksin et al, 1999). Moreover, a neutralizing antibody against thrombopoietin does not alter autonomous megakaryocyte growth (Taksin et al, 1999). Thrombopoietin levels tested in three reported paediatric ET cases by biological assay using mice were normal (Eyster et al, 1986;Kapoor et al, 1996).…”

Features of essential thrombocythaemia in childhood: a study of five children

“…In fact, TPO levels were normal in all the cases tested. Therefore, studies are needed to identify such possibilities, even if they do not seem to apply to adult ET (Taksin et al, 1999).…”

Primary and secondary thrombocytosis in childhood

“…Megakaryopoiesis can be additionally stimulated in primary thrombocytosis by an increased sensitivity of CFU‐Meg to Tpo (Mi et al , 2001). While the proliferation of megakaryopoietic progenitor cells cannot be inhibited by antibodies directed against IL‐3, IL‐6, GM‐CSF, or Tpo, blocking of c‐mpl reduces the proliferation rate of megakaryopoietic progenitor cells in vivo (Kimura et al , 1998; Taksin et al , 1999). This phenomenon has been discussed as a paradox, Tpo‐independent, but c‐mpl‐dependent modulation of megakaryopoietic progenitors in primary thrombocytosis (Tefferi, 2001).…”