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

essential thrombocythemia (ET) and idiopathic myelofibrosis (PMF) are two m yeloproliferative diseases characterized by a marked megakaryocytic (MK) in volvement. The pathogenesis of these two diseases is unknown. Recently it h as been shown that overexpression of Mpl-ligand (Mpl-L) in mice induces thr ombocytosis and myelofibrosis. In this study, we investigated whether Mpl-L was responsible for the pathogenesis of ET and PMF, Using in vitro culture s of blood or marrow CD34(+) cells, we investigated whether MK growth was a bnormal in these two diseases. Spontaneous MK growth involving only a fract ion (20%) of the MK progenitors, as compared with growth in the presence of pegylated recombinant human megakaryocyte growth and development factor (P EG-rhuMGDF), was found in both diseases (21ET and 14PMF) using serum-free s emisolid and liquid cultures, including cultures at one cell per well. We f irst searched for a c-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 investiga ted whether an autocrine stimulation by Mpl-L could explain the autonomous MK growth. Addition of different preparations of soluble Mpl receptor (sMpl ) containing a Fe domain of IgG1 (sMpl-Fc) markedly inhibited MK spontaneou s growth in both FT and PMF patients. This effect was specific for sMpl bec ause a control soluble receptor (s4-1BB-Fc) had no inhibitory effect and an sMpl devoid of the Fe fragment had the same inhibitory efficacy as the sMp l-Fc. This inhibition was reversed by addition of PEG-rhuMGDF or a combinat ion 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 al ter 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 we re detected at a very low level in the patient CD34(+) cells and MK and onl y when a highly sensitive fluorescent PCR technique was used. By quantitati ve reverse-transcription (RT)-PCR, the number of Mpl-L transcripts per acti n transcripts was lower than detected in human Mpl-L-dependent cell lines, suggesting that this synthesis of Mpl-L was not biologically significant. I n favor of this hypothesis, the Mpl-L protein was not detected in culture s upernatants using either an enzyme-linked immunosorbent assay (ELISA) or a biological (Ba/F3hu c-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 ac tivated STATs 3 and 5. This result further suggests that spontaneous growth is neither related to a stimulation by Mpl-L nor to a c-mpl mutation. In c onclusion, our results show that Mpl-L or Mpl are not directly implicated i n the abnormal proliferation of MK cells from ET and PMF. The mechanisms by which the sMpl mediates a growth inhibition will require further experimen ts. (C) 1999 by The American Society of Hematology.