Essential thrombocytosis (ET), polycythemia vera (PV), and myelofibrosis (MF; post ETMF, post PVMF and primary MF) share the JAK2V617F mutation, but differ with regard to clinical phenotype, rate of disease progression, and risk of transformation. Variation in the JAK2V617F neutrophil allele burden does not account for these observed differences in clinical behavior or natural history. We therefore investigated the JAK2V617F burden and JAK2 genotype composition in the hematopoietic stem cell (HSC) population of MPN patients.


We studied 47 JAK2V617F-positive MPN patients during 51 distinct disease phases. Circulating CD34+ cells were flow-sorted based on the stem cell markers CD34, CD38 and aldehyde dehydrogenase (ALDH). CD34+ CD38- ALDH+ HSC were sorted into 96 well plates and single cell JAK2 genotypes (average 40 single cells genotyped/patient with >1000 total single cells genotyped) were obtained using a nested PCR assay. Additional genomic lesions and chromosomal copy number variation were investigated in the sorted, single cell fractions in informative patients by FISH or multiplex single cell PCR. Distribution of JAK2V617F stem cell genotypes were correlated with disease phenotype, neutrophil JAK2V617F allele burden, splenomegaly, white cell count, chemotherapy requirement and disease evolution.


In all MPN cases, regardless of disease class, the JAK2V617F mutation was detected in the CD34+ CD38- ALDH+ fraction - the same population in which normal HSC reside. All ET and MF patients, and the majority of PV patients, had three JAK2 genotypes coexisting in their respective HSC populations. ET was characterized by a high percentage of JAK2WT stem cells (>75%) despite the concomitant presence of JAK2V617F homozygous clones and disease durations >15 years. Importantly, in the ET patients where JAK2WT clones fell to less than 50%, a PV phase followed. MF was characterized by a relatively low percentage of JAK2WT stem cells (median 24%), regardless of disease duration. PV had the most variable JAK2 genotypes with a wide range of JAK2WT stem cells (4%-92%) and a wide range of JAK2V617F homozygous stem cells (2-100%), and in 5/16 PV cases, only JAK2WT and JAK2V617F homozygous stem cells were identified.

PV patients with JAK2V617F homozygous clones comprising more than 50% of their stem cells, regardless of disease duration, had higher white cell counts, higher neutrophil allele burdens, larger spleens and higher prevalence of chemotherapy compared to PV patients who had less than 50% JAK2V617F homozygous HSCs. The percentage of JAK2V617F homozygous HSC did not correlate with disease duration: some PV patients with a disease duration of >18 years had less than 10 % JAK2V617F homozygous HSC.

A JAK2V617F - positive PV patient with a high JAK2V617F HSC burden and a high neutrophil JAK2V617F burden transformed to a JAK2V617F-negative chronic myelomonocytic leukemia (CMMoL); at the time of HSC analysis, the neutrophil JAK2V617F allele burden was 0% (previously 90%) and the HSC JAK2V617F homozygous percentage fell to 3% (previously 60%). While this patient's CMMoL was molecularly undefined, lesions identified in other JAK2V617F-positive patients (including mutations of ASXL1, TET2, deletion of 5q, 7q and 11q, trisomy 8 and 9), were also found in the CD34+ CD38- ALDH+ HSCs using single cell techniques, sometime coexistent with JAK2V617F-positive HSC, and sometimes in JAK2WT HSC.


Driver and progression lesions in the JAK2V617F-positive MPN are acquired at the primitive HSC level. Despite decades of disease, the HSC pool in the MPN is mosaic for acquired lesions and also retains JAK2WT clones. Dominance of a particular JAK2 genotype at the primitive HSC level is variable, and distinguishes ET, where JAK2WT stem cells outnumber JAK2V617F-positive HSC, from MF, where JAK2WT HSC are the minority. PV is the most variable of the three MPN with regard to JAK2 genotype mosaicism. The allelic burden of HSC JAK2V617F in PV correlates with clinical disease burden. However, neither time nor JAK2V617F genotype determines the HSC burden in ET and PV, indicating that an undefined factor is a modifier of this important disease-defining process. Understanding the biology of HSC JAK2V617F homozygous clonal dominance may define an exploitable target to control disease burden, and to mitigate disease progression and evolution.


Moliterno:incyte: Membership on an entity's Board of Directors or advisory committees. Spivak:Incyte: Membership on an entity's Board of Directors or advisory committees.

Author notes


Asterisk with author names denotes non-ASH members.