To the editor:
The JAK2V617F mutation is found in most patients with polycythemia vera (PV) and 50% to 60% of those with essential thrombocythemia (ET). JAK2V617F-homozygous precursors arise through mitotic recombination, form larger clones in PV compared with ET,1 and may play a causal role in PV phenotypes.2,3 However, acquisition of homozygosity is not sufficient to cause PV, because many ET patients also harbor homozygous-mutant clones1 and several studies have suggested that JAK2V617F, especially when homozygous, may not confer an advantage to hematopoietic stem cells (HSCs).3-5 Moreover, many PV and ET patients have multiple independently acquired homozygous-mutant clones, with most remaining small.1 PV is distinguished from ET by expansion of 1 dominant JAK2V617F-homozygous subclone. Here we investigated whether subclone expansion reflects acquisition of additional genetic lesions conferring a clonal advantage.
We studied 2 patients with chronic-phase PV (see supplemental Table 1 on the Blood Web site) and large JAK2V617F-homozygous clones.1 Genotyping of burst-forming unit-erythroid (BFU-E) colonies for JAK2V167F and microsatellite markers (as previously described1 ) demonstrated that both patients had 3 detectable JAK2V617F-homozygous subclones, 1 of which was 9 to 15 times larger than minor subclones in the same patient (Figure 1A). Serial assays confirmed that these subclones persisted for approximately 2 years and that the dominance of 1 subclone remains stable (Figure 1B). To investigate whether this dominance arose in HSCs or later progenitors, we isolated highly purified hematopoietic progenitors from patient PV1 (supplemental Methods). Colony genotyping demonstrated that the major JAK2V617F-homozygous subclone in BFU-Es also predominated in HSCs, common myeloid progenitors, granulocyte-monocyte progenitors, and megakaryocyte-erythroid progenitors (Figure 1C), suggesting that this dominance arose early in hematopoiesis.
Exome sequencing (supplemental Methods) was next performed to search for mutations present in major, but not minor, JAK2V617F-homozygous subclones. High tumor-burden samples were used to maximize mutation detection. Variant validation and colony genotyping were performed by capillary sequencing. For PV1, granulocyte DNA (JAK2V617F allele burden 99%) was used for exome sequencing and validation of somatic mutations in 8 genes plus JAK2V617F (Figure 1D; supplemental Table 2). Of these, the exact nonsense variant in transcriptional repressor GATAD2B has been identified in 3 patients with acute myeloid leukemia, whereas none of the other genes is recurrently mutated in myeloid malignancies.6 The GATAD2B mutation was detected in the major JAK2V617F-homozygous subclone but also in 1 minor JAK2V617F-homozygous subclone (C), together with a subset of JAK2V617F-heterozygous colonies (Figure 1D).
For PV2, granulocyte DNA had a low tumor burden (JAK2V617F allele, 18.1%), reducing the sensitivity of exome sequencing. To circumvent this issue, we pooled BFU-E colonies from subclones A and B and performed independent exome sequencing on these subclones. Variants identified in both (supplemental Table 3) were not pursued. Of the variants in subclone A alone, 5 mutations (plus JAK2V617F) were validated by capillary sequencing (Figure 1E-F; supplemental Table 2). Of these genes, BCOR shows recurrent frameshift mutations in acute myeloid leukemia and myelodysplasias,7 whereas the others are not recurrently mutated in hematological malignancies.6 The hemizygous BCOR mutation was detected in the dominant subclone A but also in a subset of colonies from minor subclone B (Figure 1E-F).
In summary, the dominant JAK2V617F-homozygous subclone in both PV patients harbored a mutation in an additional gene that is mutated in myeloid malignancies. Interestingly, both GATAD2B and BCOR are transcriptional repressors associated with the Mi-2/NuRD complex,8,9 raising the possibility that aberrant histone deacetylation is advantageous to JAK2V617F-homozygous cells. However, both mutations were also present in a minor JAK2V617F-homozygous subclone and cannot account for the dominance of the larger clones.
Our results therefore indicate the absence of known “driver” mutations specific for the dominant JAK2V617F-homozygous subclone. Expansion of the latter may nonetheless reflect genetic differences between dominant and minor subclones. For example, we cannot exclude additional mutations in regions poorly covered by exome sequencing, epigenetic differences, or disadvantageous mutations in the minor subclones. Alternatively, other mutations restricted to the dominant subclones may represent rarely mutated cancer genes, or extension of 9p LOH could provide an advantage in certain cases (eg, PV1). However our data raise the alternative possibility that in at least some patients, nongenetic stochastic mechanisms may favor individual subclones by chance and establish long-term subclone dominance. In this scenario, subclone expansion would not require a genetic advantage but instead may reflect the unique combination of environmental inputs experienced by a particular stem cell.10
The online version of this article contains a data supplement.
Acknowledgments: The authors thank the core facility of the Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, Cambridge, and the flow cytometry facility of the Cancer Research UK Cambridge Institute for technical assistance. Samples were provided by the Cambridge Blood and Stem Cell Biobank, which is supported by the Cambridge National Institute for Health Research Biomedical Research Centre and the Cambridge Experimental Cancer Medicine Centre, Cambridge, United Kingdom. The work in A.R.G.’s laboratory is supported by Leukemia and Lymphoma Research, Cancer Research UK, the Wellcome Trust, the Medical Research Council, the Kay Kendall Leukaemia Fund, the Cambridge National Institute for Health Research Biomedical Research Center, the Cambridge Experimental Cancer Medicine Centre, and the Leukemia and Lymphoma Society of America. This work was supported by the Kay Kendall Leukaemia Fund (A.L.G., J.N.); and a postdoctoral fellowship from the Canadian Institutes of Health Research and a Lady Tata Memorial Trust International Award for Research in Leukaemia (D.G.K.). P.J.C. is a Wellcome Trust senior clinical fellow.
Contribution: A.L.G. performed the research and wrote the paper; J.N., C.E.M., E.P., and P.J.C. performed exome sequencing and data analysis; E.J.B. prepared samples and performed quantitative polymerase chain reactions and quality control; D.G.K. performed fluorescence-activated cell sorting experiments; A.R.G. directed the research and wrote the paper; and all authors had the opportunity to review the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: A. R. Green, Cambridge Institute for Medical Research, Hills Rd, Cambridge, United Kingdom; e-mail: email@example.com.