Evidence That the MPL 515L and 515K Mutations Occur in a Lympho-Myeloid Progenitor in Idiopathic Myelofibrosis and Are Associated with TPO Hypersensitivity.

The MPL 515L mutation has recently been described in 10% of Idiopathic Myelofibrosis (MMM) as an essential oncogenic event. Here we report another MPL mutation in the same codon of MPL (MPL515K). The MPL515L mutation has been detected in granulocytes but has not been yet detected in patient hematopietic stem cells. This raises the question whether this molecular event occurs in a true stem cell (ie: lymphoid/myeloid progenitor cell), as it has already been shown for JAK2 V617F occurrence in MMM and PV.

The aim of this work was to study the presence of the mutation and another MPL mutation (MPL 515K) in both myeloid and lymphoid lineages in JAK2 V617F negative MMM. We therefore looked for the mutation first in mature myeloid and lymphoid cells and second in lymphoid/myeloid progenitors progeny after CD34+ and CD34+CD38- cell isolation from peripheral blood. Three IMF, patients harbouring MPL mutations were enrolled in this study after informed consent. Peripheral blood granulocytes and platelets were purified by standard methods and B, T, NK cells and monocytes were solated by combined immunomagnetic and flow cytometric procedures. The same techniques were used to sort CD34+ and CD34+CD38- subpopulations from peripheral blood. Clonal B/NK/Myeloid differentiation from CD34+CD38- cells and T cell differentiation from CD34+ cells were performed respectively onto a MS5 layer in the presence of SCF, FLT3L, IL2, IL3, IL7, IL15, TPO and in Fetal Thymic Organ Cultures (FTOC). Genotyping of mature cell populations, B/NK/Myeloid clones and CD34+ derived T cells were performed by direct sequencing. Moreover, CD34+ cells were cultured in a one cell per well experiment to determine the sensitivilty of these patient’s cells to low dose TPO.

The MPL mutations were present in granulocytes and platelets from all patients, and in monocytes. We detected the mutation in B and NK cells in all three cases. The MPL 515L and MPL 515K mutations could be subsequently detected in CD34+ cells and in B/NK/Myeloid and/or NK/myeloid CD34+CD38- derived clones from all IMF patients. Interestingly, MPL 515L homozygous clones were detected in B/NK/Myeloid and/or NK/Myeloid clones from 1 MMM patient. Using the FTOC assay, the mutation was also detected in all T cell fractions derived from CD34+ cells from 2 MMM patients. At least we found that MPL mutations induce a spontaneous megakaryocytic growth in cell culture but also a hypersensitivity to low dose TPO.

These results demonstrate that the mutation occur in a lymphoid/myeloid progenitor cell in MPL 515L or K MMM and give rise to a hypersensitivity to TPO. Thus, these MPD take their origin in a true lymphoid/myeloid progenitor cell. In accordance with mouse model, this represent a good argument for a causative role of MPL mutations in MMM.