MPLW515L/K and JAK2V617F Mutations: Single Colony Studies, Lineage Restriction, and Chronology of Clonal Emergence.

Background: We recently reported on MPL W515L/K mutations in 1182 patients with myeloid disorders ( Blood, First Edition July 25, 2006 ) and found low frequency occurrence in in agnogenic myeloid metaplasia (AMM) (~5%) and essential thrombocythemia (ET) (~1%), but not polycythemia vera (PV). Of the 20 MPL W515L/K- positive patients in that series, 6 (30%) also carried a low burden of the JAK2 V617F mutation. Studies of mutation prevalence in individuals with clonal granulopoiesis as well as familial myeloproliferative disorders (MPD) studies suggest that these mutations ( JAK2 V617F) may be acquired as secondary genetic events. Aim: to gain additional insight regarding the aforementioned two mutations and MPD pathogenesis by determining (i) concordance between mutation expression and clonal hematopoiesis, (ii) the instructive role of each mutation in determining lineage fate, iii) differences in pattern and lineage restriction of these mutations between various MPD, and (iv) chronology of clonal emergence and clonal dominance for the 2 mutations. Methods: Sixteen MPD patients were studied (13 JAK2 V617F+ and 3 MPL W515L/K+); 9 had PV (all JAK2 V617F+), 2 ET (both JAK2 V617F+), 1 CMML ( JAK2 V617F+), and 4 AMM (2 MPL W515K+, and one each with MPL W515L and JAK2 V617F). CD34+ progenitor cells were plated in methylcellulose and resulting hematopoietic colonies were scored on day 10–12. Colonies were obtained with (all patients) and without (10 patients; 5 with PV, 1 ET, and 4 AMM) cytokine support. Individual colonies were screened for presence of either mutation by DNA sequencing. In addition, MPL W515L/K analysis was performed in T and B lymphocytes in 3 informative cases. Finally, serially stored bone marrow was examined in 3 patients with co-expression of MPL 515 and JAK2 V617F mutations. Results: A total of 552 hematopoietic colonies from 16 MPD patients were genotyped. We found cohabitation of both mutation-positive and mutation-negative endogenous colonies in PV as well as other MPD. For JAK2 V617F+ patients, the majority (75–100%) of erythropoietin-independent erythroid colonies (EEC) but not endogenous granulocyte colonies (20–70%) were mutation-positive. In contrast, no EEC were obtained from progenitors from the 3 MPL W515L/K+ patients. For 2 of the 3 patients, the endogenous myeloid colonies were predominantly mutation-positive (~90%). A mixed allele pattern (homozygous and/or heterozygous) was seen with both mutations regardless of MPD phenotype, but the overall mutant colony burden was higher with MPL W515L/K mutations relative to JAK2 V617F. When such testing was possible, MPL 515 mutations were detected in T-lymphocytes (2 of 3 patients) as well as B-lymphocytes (2 of 2 patients). Finally, among 3 patients carrying both mutations, serial marrow studies (25 time points over 4–8 years) revealed the occurrence of both mutations at diagnosis and persistent dominance of MPL W515L/K over JAK2 V617F over time. Conclusions: Our observations provide direct evidence for clonal hematopoiesis that antedates acquisition of either MPL W515L/K or JAK2 V617F that bias towards myeloid and erythroid fates, respectively. Furthermore, both mutations appear to occur early in the disease course and lineage studies suggest a hierarchically earlier stem cell origin for MPL W515L/K as compared to JAK2 V617F.