Abstract
The JAK2 V617F mutation is present in most patients with polycythemia vera (PV) and half with essential thrombocythemia (ET). Using real-time quantitative PCR, we analyzed the levels of JAK2 V617F in granulocytes and/or bone marrow mononuclear cells from 159 PV and 149 ET patients. High JAK2 V617F levels were correlated with higher leukocyte, granulocyte, hemoglobin values and higher endogenous erythroid colony formation. Thus, the phenotype of PV and ET may be closely linked to the JAK2 V617F level, which may reflect the clonal genotypic pattern of hematopoietic progenitor cells. It is thought that the occurrence of the mitotic recombination, which generates homozygous JAK2 V671F clones, is a key molecular event for the onset of PV. In this work, we aimed to study the consequences of the clonal JAK2 V617F genotype on the amplification properties and erythropoietin (EPO) hypersensitivity of PV (n=14) and ET (n=6) progenitors.Analysis of clonal genotypic patterns shows that ET patients harbor a mix of heterozygous and normal progenitors. Interestingly, we distinguish pure heterozygous PV profiles (3/14 patients) with no homozygous progenitors from homozygous PV profiles (11/14 patients) with normal, heterozygous and homozygous progenitors. Similar low frequencies of mutated immature progenitors, comprising long-term culture-initiating cells and lympho-myeloid progenitors, are found in ET and PV. In contrast, PV patients with pure heterozygous PV profiles have striking higher proportions (>90%) of mutated committed progenitors than other PV and ET patients. This result suggests a selective amplification of heterozygous cells in the early phases of hematopoiesis. Furthermore, by using increasing concentrations of EPO, homozygous mutated erythroid progenitors are demonstrated to be more sensitive to EPO than heterozygous ones, a majority of the former (69,5%) being EPO independent. Moreover, we demonstrate a two to three fold increase in in vitro amplification of ET and PV progenitor cells when compared to normal ones in serum free liquid culture containing IL3, Stem Cell Factor, Dexamethasone and 1 IU/mL EPO. In addition, the quantification of the mutated allele in immature CD34+CD38- cells, in CD34+CD38+ committed progenitor cells, in mature erythroblasts (GPA+) and in granulocytes shows a marked in vivo selective advantage of mutated cells in late stages of hematopoiesis.These results suggest that in PV, erythrocytosis results from two distinct mechanisms: a terminal erythroid amplification advantage triggered by homozygosity or a two-step process including the upstream amplification of heterozygous cells that may involve additional molecular event(s).
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