We genotyped a total of 6495 BFU-E colonies from 77 patients: 30 with JAK2V617F
-positive PV, 29 with JAK2V617F
-positive ET, and 18 with JAK2
exon 12-mutated PV. Homozygous-mutant precursors were present in all 3 disease groups (). Patients with JAK2V617F
-positive PV () showed the highest proportions of homozygous-mutant precursors, consistent with previous reports.10,14,15,20
Homozygosity was undetectable in 6 patients with JAK2V617F
-positive PV (20%), despite assessment of a large number of colonies and the use of low erythropoietin conditions that select for JAK2V617F
-homozygous precursors. Acquired TET2
mutations were reported in 3 of 5 such “heterozygous-only” patients14,21
but were not found in our 6 patients, indicating that many such patients do not carry TET2
Proportions of JAK2 genotypes in BFU-Es from patients with JAK2-mutated PV and ET
-positive ET and JAK2
exon 12-mutated PV, JAK2V617F
-homozygous colonies were identified in a surprisingly large percentage of patients (52% and 44%, respectively). Homozygous clone sizes were small; and in exon 12-mutated PV, homozygosity was associated with both K539L
-type and E543del
-type mutations (). The relative proportions of heterozygous and homozygous-mutant colonies were stable over time in 16 patients tested on 2 separate occasions (; and data not shown). Homozygous-mutant BFU-E are therefore a persistent feature in many patients with JAK2V617F
-positive PV, JAK2V617F
-positive ET, and JAK2
exon 12-mutated PV, and are more frequent than previously recognized in the latter 2 disorders.5,20,22,23
Comparison of patients with and without detectable homozygosity did not reveal any differences in blood counts at diagnosis, presence of palpable splenomegaly, or thrombotic history (Mann-Whitney U
test/Fisher exact tests, P
> .05 for each disease subgroup).
To determine whether JAK2V617F
-homozygous colonies were part of a single clone or reflected recurrent acquisition of loss of heterozygosity (LOH), breakpoints for chromosome 9p LOH were mapped using fluorescence microsatellite PCR in 576 homozygous-mutant colonies from 10 patients (8 PV and 2 ET). Results for 1 PV patient and 1 ET patient are shown in , with the others summarized in . At least 2 distinct homozygous subclones were identified in 5 of 8 PV patients and both ET patients, indicating that independent homozygous-mutant clones arise frequently in both PV and ET. Importantly, the resolution of breakpoint mapping was limited (2.3-14.2 MB), so the number of distinct subclones may be an underestimate. The high prevalence of homozygous-mutant clones may reflect a role for JAK2V617F in homologous recombination24
and/or inappropriate survival of cells after DNA damage.25
There was no obvious relationship between the presence of multiple homozygous-mutant subclones and patient age, disease duration, or therapy (supplemental Table 3). Homozygous-mutant colonies did not arise from short-lived progenitors because distinct subclones persist over time. Patient PV24 had 2 subclones whose relative proportions remained unchanged over 10 months (supplemental Figure 1), indicating that they arose from early stem/progenitor cells.
Microsatellite mapping of 9p LOH in JAK2V617F-homozygous colonies
Importantly, patients with PV and ET differed in that the former harbored a major homozygous-mutant clone that was 8-85 times the size of minor subclones in the same patient (). This observation demonstrates that the large numbers of homozygous-mutant colonies present in most PV patients do not reflect accumulation of numerous independent subclones but rather the expansion of 1 dominant clone. Given the circumstantial evidence that JAK2V617F homozygosity enhances erythropoiesis, it seems probable that the dominant clone present in many PV patients is causally related to the development of erythrocytosis, with other mechanisms operating in the minority of patients with small or undetectable homozygous-mutant clones.
There are at least 2 explanations for the development of a dominant homozygous-mutant clone in PV patients. First, the dominant subclone might derive from a preexisting minor subclone after a second more centromeric mitotic recombination event, with the selective advantage reflecting extension of the region of LOH. However, there was no region of LOH that was common to the dominant subclones and absent from minor subclones (). Moreover, in some patients, the breakpoint in the dominant subclone was clearly telomeric to that of a minor subclone (eg, PV24, PV26). We therefore favor the alternative explanation that minor and dominant subclones arose independently, with the selective advantage of the latter reflecting acquisition of additional genetic or epigenetic changes.