Visualization of c-Mpl Expression in Ph-Negative Myeloproliferative Diseases and So-Called Essential Thrombocythemia with Ringed Sideroblasts by Confocal Laser Scanning Microscopy.

Abstract

c-Mpl acts as a primary regulator of megakaryocytopoiesis and platelet production and is essential for the maintenance of normal hematopoiesis in humans. An impaired posttranslational processing of Mpl with underglycosylation has been proposed as a molecular defect of polycythemia vera (PV) and chronic idiopathic myelofibrosis (CIMF; Molinterno and Spivak, Blood 1999; 94: 2555).To visualize the subcellular localization of megakaryocyte c-Mpl protein in myeloproliferative diseases and myelodysplastic/myeloproliferative disorders we performed an imaging study by confocal laser scanning microscopy. Bone marrow trephines from normal bone marrow donors (n=14), patients with essential thrombocythemia (ET, n=15), PV (n=18), CIMF (n=15), myelodysplastic syndromes (MDS) including refractory anemia with ringed sideroblasts (RS; n=12) and so-called essential thrombocythemia with RS (ET/RS; n=67) were examined. Immunohistochemistry was performed using a rabbit polyclonal antibody recognizing glycosylated and unglycosylated c-Mpl and semiquantitatively evaluated by scoring of plasmalemmal vs. cytoplasmic labeling (score 1–3).Our results provide evidence that normal megakaryocytes show a pronounced cell surface expression of c-Mpl. In PV and CIMF, c-Mpl suface expression was significantly reduced (p<0.001), while cytoplasmic expression was often strongly accentuated. In ET, heterogeneous patterns of membraneous labeling were observed ranging from normal to decreased. In MDS, plasmalemmal immunoreacativity was generally down-regulated. In ET/RS c-Mpl imaging revealed a heterogeneous positivity with different degrees of plasmalemmal vs. cytoplasmic immunoreactivity in keeping either with CIMF, true ET or MDS.The role of c-Mpl as a biomarker of Ph-negative MPDs is controversely discussed. A weak megakaryocyte c-Mpl staining which may complement morphologic distinction has been observed in PV (Tefferi et al., Blood 2000; 96: 771). Our findings suggest that the strikingly reduced cell surface c-Mpl expression despite equivalent or increased mRNA levels (Bock et al., J Pathol 2004; 203: 609) may result in impaired receptor domains for TPO in PV and CIMF and is a characteristic epigenetic abnormality of theses entities. Upon consideration of all our clinico-pathological data, so-called ET/RS is now fracturing in clearly defined haematological diseases which could be categorized as CIMF/RS (38/67), true ET/RS (10/67) or different MDS categories (n=19). The heterogeneous patterns of c-Mpl expression further contribute to define positive criteria for the discrimination of the distinct entities within in the ET/RS group.