Essential thrombocytosis (ET), polycythemia vera (PV), and myelofibrosis (MF) share the JAK2V617F mutation, but differ with regard to clinical phenotype, rate of disease progression, and risk of leukemic transformation. Variation in the JAK2V617F neutrophil allele burden does not account for these observed differences in clinical behavior. We therefore investigated JAK2V617Fallele burden and genotype in the stem/progenitor populations of MPN patients in chronic and leukemic phases.


We studied 39 JAK2V617Fpositive MPN patients evaluated between 2005 and 2013, including 8 patients at leukemic transformation. CD34+ cells isolated from peripheral blood were flow sorted based on CD34, CD38, and the stem cell marker aldehyde dehydrogenase (ALDH). JAK2V617F allele percentages were calculated using an allele specific real time PCR assay. Cells were sorted into 96 well plates and single cell JAK2V617Fgenotypes were obtained using a nested PCR assay. Additional genomic lesions and chromosomal copy number variation were investigated in the sorted fractions when applicable.


In all MPN cases, the JAK2V617F mutation was detected in the CD34+CD38-ALDHhigh fraction – the same population in which the normal hematopoietic stem cell resides. Quantitative JAK2V617F allele burdens in this fraction were highest in MF > PV > ET. Single cell JAK2V617F genotyping revealed a higher proportion of JAK2V617F-/- cells in ET and PV than in MF, but JAK2V617F-/- cells were detectable in the CD34+CD38-ALDHhigh fraction of all cases. In most cases of PV and MF, this fraction contained a mixture of JAK2V617F-/-, JAK2V617F-/+, and JAK2V617F+/+ cells. Additional chronic phase lesions (including mutations of ASXL1 & TET2) were also found in the CD34+CD38-ALDHhighfraction.

Two patterns of leukemic transformation were observed. The first pattern (in 7/8 patients) was identical to that of de novo AML (Gerber JM, Blood 2012), with emergence of a unique CD34+CD38-ALDHint fraction, which was clonal by JAK2V617F genotype and contained leukemia-specific lesions (e.g., 5q deletion). In contrast, the residual CD34+CD38-ALDHhigh population lacked the leukemic abnormalities and was oligoclonal with respect to JAK2V617F. In 3 of these AML cases, the CD34+CD38-ALDHint fraction was JAK2V617F-/-, while the JAK2V617F mutation remained detectable in the CD34+CD38-ALDHhigh fraction. Single cell genotyping performed during the leukemic phase of a PV patient revealed only JAK2V617F-/- CD34+CD38-ALDHint cells but identified JAK2V617F-/-, JAK2V617F-/+, and JAK2V617F+/+ CD34+CD38-ALDHhigh cells; JAK2V617F levels were barely detectable in the progenitors and neutrophils during this leukemic phase. Upon achievement of complete remission from AML, high JAK2V617F allele burdens were then found in the progenitors and neutrophils, as well as in the CD34+CD38-ALDHhigh fraction. A second pattern of leukemic transformation was seen in one patient, in whom no CD34+CD38-ALDHint population was present. The CD34+CD38-ALDHhigh population was expanded in this case and harbored JAK2V617F+/+ positive cells with the leukemia-specific lesion.


Unlike CML, in which the BCR/ABL oncogene is typically present in the majority of CD34+CD38-ALDHhigh cells at diagnosis (Gerber JM, Am J Hematol 2011), the JAK2V617F mutation was present in only a minority of CD34+CD38-ALDHhigh cells in JAK2V617F positive ET and PV. Moreover JAK2V617F-/- cells were detected even in longstanding, advanced phase PV and MF. Lower JAK2V617F clonal burdens in the primitive CD34+CD38-ALDHhigh compartment as compared to neutrophils in most cases of MPN suggest that the JAK2V617F mutation does not confer a significant advantage at the stem cell level and that other genetic lesions may drive expansion of this population. JAK2V617F negative leukemias occur in about 35% of PV patients, apparently arising from the residual JAK2V617F negative CD34+CD38-ALDHhigh reservoir. We conclude that primitive stem/progenitor cells are mosaic with regard to JAK2V617F mutation status in the majority of MPN patients. Furthermore, acquisition of JAK2V617F, development of JAK2V617F homozygosity, and accrual of other acquired lesions in chronic phase MPN all occur in the primitive CD34+CD38-ALDHhigh compartment. Lesions specific to post MPN AML segregate to a distinct CD34+CD38-ALDHint population.


Jones:Cytomedix: Patent holder for Aldefluor reagent, which is licensed by Cytomedix. This relationship is managed by the Johns Hopkins Office of Policy Coordination., Patent holder for Aldefluor reagent, which is licensed by Cytomedix. This relationship is managed by the Johns Hopkins Office of Policy Coordination. Patents & Royalties.

Author notes


Asterisk with author names denotes non-ASH members.