The Hematopoietic Stem Cell Compartment Is Different in Polycythemia Vera and Idiopathic Myelofibrosis.

X-linked clonality studies showed that myeloproliferative disorders derive from an abnormal hematopoietic stem cell (HSC). This has been recently confirmed by studies showing that the JAK2 V617F mutation was present in multipotent cells from patients with Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primitive Myelofibrosis (PMF). How one unique mutation could give rise to three different diseases remains unexplained and we hypothesized that the HSC compartment may be different in PV and PMF. To investigate whether the V617F mutation occurs in HSCs in PV and PMF, and whether the HSC compartments are different in these 2 diseases, we performed simultaneously two types of experiments:

• Myeloid, B, and NK in vitro differentiation assays and

• Repopulation of immune-deficient NOD/SCID mice with human JAK2 V617F CD34+ cells followed by analysis of the frequency of JAK2 V617F clones.

We first confirmed that the JAK2 V617F mutation is present in HSCs, enabling long-term (15 weeks) in vivo hematopoietic reconstitution, both in PV and PMF patients. Nevertheless, we found marked differences between PV and PMF samples. Indeed, the frequency of JAK2 V617F lympho-myeloid progenitors was much higher in MF (6 PMF and one post PV-MF) than in PV patients (n = 9) (87.2% +/− 31.3 vs 21% +/− 21.1 respectively). Similarly, most of the human myeloid progenitors present in mice transplanted with CD34+ cells from MF patients (n = 7) 15 weeks post-transplantation were JAK2 V617F. On the contrary, human myeloid progenitors were predominantly JAK2 WT after transplantation of PV CD34+ cells (9 patients). To determine if the mutation was present in HSC able to differentiate into B-lymphocytes in PV and MF, we sorted and genotyped the fraction of B-lymphocytes (CD45+CD19+) that differentiated in the bone marrow of mice. In 5 mice transplanted with PV CD34+ cells, the fraction of B-cells was always JAK2 WT whereas in 2 out of 3 mice transplanted with MF CD34+ cells, B-lymphocytes were JAK2 V617F. To determine if the mutation was present in HSC capable of very long-term reconstitution in PV and PMF, we looked for the presence of long-term culture-initiating cells (LTC-IC) among human CD34+ cells isolated from the bone marrow of NOD/SCID mice (2 PV, 3 MF) 15 weeks after transplantation. In 2 mice transplanted with PMF CD34+ cells, the majority of LTC-ICs (70/70 and 95/166) were JAK2 V617F. On the contrary, in 3 mice reconstituted with PV CD34+ cells, most of the LTC-ICs were JAK2 WT (23/23, 4/4 and 126/168) although we could find some LTC-ICs that were JAK2 V617F, demonstrating that in PV also, the JAK2 V617F mutation is present in Long Term-HSC. Taken together these results demonstrate that the JAK2 V617F mutation is present in a small subset of HSCs in PV patients, whereas in MF, the vast majority of HSCs is JAK2 V617F. This suggests that these two diseases are two stages of the same pathology and that in MF the JAK2 V617F HSCs have acquired a proliferative advantage on JAK2 WT HSCs and thus have invaded the hematopoietic system.