CLONALITY ASSAYS AND MEGAKARYOCYTE CULTURE TECHNIQUES IN ESSENTIAL THROMBOCYTHEMIA

The development of techniques permitting in vitro growth of human mega karyocytes progenitors and more recently identification of the proto o ncogene c-mpl (Mpl-R) and its ligand (Mpl-L) have created new opportun ities for studying pathophysiology of E.T. Plasma or serum of E.T. pat ients was unable to overestimulate MK colony formation by normal bone marrow cells. Significant increases in circulating CFU MK in E.T. pati ents have been repeatedly observed while in E.T. marrow, due to inappr opriate sampling, colony number was not significantly different from n ormal. Spontaneous colony formation is observed in approximately 100% bone marrow and 85% blood from E.T. patients. Spontaneous colony forma tion persisted in plasma clot assay without added plasma or serum and in serum free agar cultures but only at a slightly lower rate than in plasma clot. Spontaneous colony formation in culture condition without plasma and serum were never observed with normal bone marrow and bloo d. Spontaneous MK growth was observed in a higher proportion of E.T. p atients than erythroid colony formation but both phenomenon can occur in about 50% of the patients. CFU MK colony formation disappeared in s erum free cultures using highly purified CD 34 cells. MK development i s not completely independent of regular control. An hypersensitivity o f E.T. MK progenitors to growth factors known to stimulate normal hema topoiesis (IL3, IL6, GM CSF, has been shown as well as a decreased sen sitivity to negative regulators (TGF beta), has been suggested. The nu mber of spontaneous MK colonies was not significantly decreased by add ed anti IL3, IL6 or anti GM CSF, antibodies in culture medium. Pre inc ubation of blood non adherent mononuclear cells of E.T. patients with antisense oligonucleotides to c-mpl significantly decreased the clonin g efficiency of spontaneous megakaryocyte growth as compared to the in troduction of scrambled oligomers. Finally m RNA expression of the Mpl -L (TPO) was not formed in MK spontaneously grown in serum free liquid cultures after 12 days. These results suggest that human c-mpl proto oncogene may be implicated in the pathway of spontaneous megakaryocyto poiesis in MPD but an absence of autocrine-stimulation by TPO of spont aneous growth in MPD. Analysis of peripheral blood cell clonality was performed in 55 E.T. patients using either the DNA methylation pattern of the androgen receptor (A R) gene or m RNA transcripts of G6PD or I DS genes. 51 out of 55 patients were informative. Non random X inactiv ation was found on unfractioned blood in 73% as compared with 23% in n ormal females (skewed Lyonisation). In 12 patients monoclonality of he matopoiesis was definitely confirmed by recording polyclonality of the mononuclear fraction or of T lymphocytes. In 4 patients monoclonal he matopoiesis was limited to platelets, 7 patients remained polyclonal i n whole blood and all cellular fractions studied. MK colony formation (provided that the serum free agar culture system is clearly standardi sed) and clonality studies on whole blood or granulocyte, T lymphocyte and platelet fractions may be proposed as positive criteria for diagn osis of E.T.