The JAK2 V617F mutation is present in a minority of human hematopoietic stem cells from patients with myeloproliferative disorders and does not modify their self-renewal properties Dr. C. James, Bordeaux, France    - Biography English - 2007-06-09 Topic: Myeloproliferative disorders
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THE JAK2 V617F MUTATION IS PRESENT IN A MINORITY OF HUMAN HEMATOPOIETIC STEM CELLS FROM PATIENTS WITH MYELOPROLIFERATIVE DISORDERS AND DOES NOT MODIFY THEIR SELF-RENEWAL PROPERTIES

Chloé James, Fréderic Mazurier, Ronan Chaligne, Isabelle Lamrissi-Garcia, Sabrina Dupont, Stéphane Giraudier, François Delhommeau, Eric Lippert, François-Xavier Mahon, Jean-Max Pasquet, Hubert de Verneuil, William Vainchenker.

BACKGROUND: The JAK2 V617F mutation is the most common genetic defect observed in hematological malignancies since it is observed in essential thrombocythemia (ET), polycythemia vera (PV) and primitive myelofibrosis (PMF). Whether it is the first genetic defect in these 3 myeloproliferative disorders (MPD) remains a matter of debate. However, all these malignancies are considered to arise from a defect in a hematopoietic stem cell (HSC). AIMS: To investigate whether the V617F mutation occurs in a true stem cell and modifies its self-renewal and differentiation properties, we repopulated immune-deficient mice with human JAK2 V617F cells.

METHODS: After informed consent was obtained, CD34+ cells isolated from the blood or bone marrow (BM) of 28 patients with JAK2 V617F-positive MPDs (14 PV, 9 PMF, 4 post PV-MF, 1 ET) were intravenously injected into sublethally irradiated and anti-CD122 antibody-treated NOD/SCID mice. 3, 6, and 12 weeks after transplant, immunophenotypic analysis on BM aspirates was performed to characterize human cell engraftment. 15 weeks after transplant, mice were sacrificed and hematopoietic cells were used for secondary transplants. Before transplant and 3, 6, 12, 15 weeks after, cells were seeded in methylcellulose for colony assays and each harvested colony was genotyped to assess the presence of JAK2 wild type (WT) and JAK2 V617F alleles.

RESULTS: 105 to 106 CD34+ cells were injected in one to 3 mice per patient’s sample. Three weeks after transplant, the mean percentage of human cells (including CD45+, CD45-/CD36+, CD45-/CD36-/GpA+) was 22 ± 21% but decreased to 6 ± 6% at week 6. This decrease of engraftment was observed in all mice. Analysis of long-term reconstitution (12 and 15 weeks post-transplant) distinguished 2 groups of mice: 1) most of them (25 patients’ samples) had a progressive reduction in human cell engraftment, with a majority of CD19+ cells, such as observed in mice repopulated with normal CD34+ cells. No engraftment was detected in secondary transplant. 2) On the contrary, a small number of mice (2 repopulated with PMF cells and 1 with PV cells) developed a MPD-like syndrome characterized by an increase in human cell engraftment, a majority of CD33+ cells and even some GpA positive cells. Genotypic analysis of human colonies demonstrated that the first group of mice contained a majority of JAK2 WT colonies. However, in this group, we could observe some heterozygous colonies at 12 and 15 weeks after transplant, in 2 mice reconstituted with cells from 2 PV patients, confirming that the JAK2 V617F mutation is indeed present in HSCs. Genotyping of the second group of mice is still under investigation and results will be presented.

CONCLUSION: Using NOD/SCID mice we demonstrated that the JAK2 V617F mutation is present in only a minority of HSCs in MPDs and that it does not induce self-renewal advantage in human HSCs. We can hypothesize that the HSCs engrafted in mice that develop a MPD-like syndrome probably have an additional genetic event that modifies their stem cell properties.