Clonality Assays and Megakaryocyte Culture Techniques in Essential Thrombocythemia

Abstract

The development of techniques permitting in vitro growth of human megakaryocytes progenitors and more recently identification of the proto oncogene c-mpl (Mpl-R) and its ligand (Mpl-L) have created new opportunities for studying pathophysiology of E.T. Plasma or serum of E.T. patients was unable to overestimulate MK colony formation by normal bone marrow cells. Significant increases in circulating CFU MK in E.T. patients have been repeatedly observed while in E.T. marrow, due to inappropriate sampling, colony number was not significantly different from normal. Spontaneous colony formation is observed in approximately 100% bone marrow and 85% blood from E.T. patients. Spontaneous colony formation 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 blood. Spontaneous MK growth was observed in a higher proportion of E.T. patients than erythroid colony formation but both phenomenon can occur in about 50% of the patients. CFU MK colony formation disappeared in serum free cultures using highly purified CD 34 cells. MK development is not completely independent of regular control. An hypersensitivity of E.T. MK progenitors to growth factors known to stimulate normal hematopoiesis (IL3. IL6, GM CSF, has been shown as well as a decreased sensitivity to negative regulators (TGF β), has been suggested. The number of spontaneous MK colonies was not significantly decreased by added anti IL3, IL6 or anti GM CSF, antibodies in culture medium. Pre incubation of blood non adherent mononuclear cells of E.T. patients with antisense oligonucleotides to c-mpl significantly decreased the cloning efficiency of spontaneous megakaryocyte growth as compared to the introduction of scrambled oligomers. Finally m RNA expression of the MpI-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 megakaryocytopoiesis in MPD but an absence of autocrine-stimulation by TPO of spontaneous 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 IDS genes. 51 out of 55 patients were informative. Non random X inactivation was found on unfractioned blood in 73% as compared with 23% in normal females (skewed Lyonisation). In 12 patients monoclonality of hematopoiesis was definitely confirmed by recording polyclonality of the mononuclear fraction or of T lymphocytes. In 4 patients monoclonal hematopoiesis was limited to platelets, 7 patients remained polyclonal in whole blood and all cellular fractions studied.

MK colony formation (provided that the serum free agar culture system is clearly standardised) and clonality studies on whole blood or granulocyte, T lymphocyte and platelet fractions may be proposed as positive criteria for diagnosis of E.T.

Key Words:

• Clonality assays
• megakarocyte cultures
• essential thrombocythemia