Evidence for MPL W515L/K mutations in hematopoietic stem cells in primitive myelofibrosis

Abstract

IntroductionThe BCR-ABL-negative chronic myeloproliferative disorders (MPDs) include polycythemia vera (PV), essential thrombocytosis (ET), and primitive myelofibrosis (PMF). 1 These disorders are hematologic malignancies characterized by a clonal proliferation of one or several myeloid lineages. 2 PV, ET, PMF, and chronic myeloid leukemia (CML), defined as the classical MPDs, are considered to arise from the transformation of a multipotent hematopoietic stem cell. [3][4][5][6] However, the stem cell origin of the malignant clone had been demonstrated only in CML by the detection of either the characteristic t(9;22) translocation or the BCR/ABL transcript in all hematopoietic lineages. 7 More recently, detection of the JAK2 V617F mutation in a lympho/myeloid progenitor has been shown in some PV and PMF. 8 The JAK2 V617F mutation is found in almost all patients with PV, in 35% to 70% with ET, and in 50% with PMF. 9-13 Recently, we have detected the JAK2 V617F mutation in B and natural killer (NK) cells in approximately half of the PMF patients and in a minority of PV patients. Using the fetal thymus organ culture (FTOC) and B/NK/myeloid assays, we also have demonstrated that immature lympho/myeloid progenitors from PV and PMF carry the JAK2 V617F. 8 In 2006, 2 novel MPL somatic mutations (MPL W515L and MPL W515K) have been discovered in 5% and 1%, respectively, of JAK2 V617F-negative PMFs. The MPL W515L mutation confers to hematopoietic cells a cytokine-independent proliferation capacity, and results in a constitutive activation of JAK-STAT signaling. 14 The MPL W515K was later identified by sequencing granulocytes from 1182 MPD patients, but its precise effects on signaling are presently unknown. 15 W515 is the key amino acid located in a unique amphipathic domain that prevents spontaneous activation of MPL. 16 The substitution of W515 to an alanine or a serine has been shown to induce a factor-independent growth of the Ba/F3 cell line. 14,16 Therefore, it is expected that other amino acid changes in 515, such as the W515K, may induce a spontaneous MPL signaling.The growth factor-independent cellular proliferation of the megakaryocytic lineage was suggested to be based on the spontaneous homodimerization of MPL. [17][18][19][20][21][22] The viral oncogenic form of MPL, v-MPL, is characterized by the deletion of its extracellular domain. Its homodimerization results from the fusion of the truncated MPL sequence with the virus envelope. 18,23 Furthermore, introduction of a cysteine residue at codon 368 leads to a covalent disulfide-bonded homodimerization 19 and ligand-independent receptor activation. Similarly, the forced dimerization of MPL using monoclonal antibodies or the FK506-binding protein FKPB12 induces factor-independent proliferation. 24 W515 seems to play an important role in the prevention of basal MPL homodimerization. Indeed, the amphipathic domain of MPL, more precisely W515, prevents 2 receptors from coming close to each other in the absence of thrombopoietin (TPO). 16 In in vivo models, transpla...

Screening and monitoring of MPL W515L mutation with real-time PCR in patients with myelofibrosis undergoing allogeneic-SCT

“…Chaligne et al 19 have shown that MPLW515K mutations are already present in early hematopoietic stem and progenitor cells, including SCID-repopulating cells. For this reason and based on the experience with the JAK2V617F, it might well be expected that MPLW515L would become positive in the case of malignant relapse.…”

“…17,18 To detect MPLW515L/K mutations, genotyping techniques using allele-specific PCR were first used with qualitative or semiquantitative results and a 3-5% sensitivity level. 6,19 Pyrosequencing is a quantitative technique resulting in a sensitivity of only 5%. 20,21 Melting curve analysis using LightCycler assay was also reported to be sensitive for mutant allele burden of at least 5%.…”

Involvement of mast cells by the malignant process in patients with Philadelphia chromosome negative myeloproliferative neoplasms

“…1,[19][20][21][22][23][24][25][26] Using cell clonality assays including X-chromosome-linked polymorphisms, FISH and specific mutations (Ras, JAK2V617F and MplW515L), several myeloid lineages (granulocytes, erythrocytes, platelets and monocytes) have been shown to be involved by the malignant process in the majority of patients with PV, PMF and ET; and the lymphoid cells in a subpopulation of these patients. [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] Although MCs originate from the same progenitor cells, as granulocytes, erythrocytes, platelets and monocytes, whether MCs are also involved by the malignant process in PV, PMF and ET has not been previously explored. In this study, we established MC cultures from CD34 þ cells purified from PB of patients with PV and PMF, and examined the presence of JAK2V617F and/or MplW515L mutations as well as chromosomal abnormalities in the HCMCs.…”

“…27 The JAK2V617F mutation is present in the CD34 þ cells as well as cells belonging to several myeloid lineages (granulocytes, erythrocytes and monocytes) in the majority of patients with PV and the lymphoid cells of a sub-population of PV patients. [28][29][30][31] Hu et al 32 and Chaligné et al 33 have reported that the MplW515L/K mutations are present in granulocytes, monocytes, platelets, natural killer cells as well as B-and Tlymphocytes in patients with PMF, indicating that the MplW515L/K mutations originate in a progenitor cell common to both myeloid and lymphoid cells. 32,33 Despite being one of the myeloid lineages, the involvement of MCs by the malignant process in PV, ET and PMF has not been reported earlier.…”

New mutations of MPL in primitive myelofibrosis: only the MPL W515 mutations promote a G1/S-phase transition

“…1 MPDs are hematological diseases characterized by a clonal proliferation of one or several myeloid lineages 2 arising from the transformation of a multipotent hematopoietic stem cell by an oncogenic event such as Bcr-abl fusion protein kinase, mutated Janus kinase JAK2 V617F or thrombopoietin receptor (TPO-R) MPL W515L that induces constitutive signaling. [3][4][5] However, approximately 40% of patients with ET and PMF do not present such mutations, [6][7][8][9][10][11] and genetic analysis is still under progress to elucidate the responsible oncogenic events.…”