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Use of the 46/1 haplotype to model JAK2V617F clonal architecture in PV patients: clonal evolution and impact of IFNα treatment

V617F mutation of the protein kinase JAK2 is the most prevalent genetic abnormality in the three myeloproliferative neoplasms (MPNs), namely polycythemia vera (PV, 95%), essential thrombocythemia (ET, 60%) and myelofibrosis (MF, 60%).1, 2, 3, 4 This mutation, which usually affects only one of the JAK2 gene alleles in ET, frequently becomes homozygous in PV and MF. This homozygosity is related to chromosome 9p uniparental disomy (UPD 9p) due to mitotic homologous recombination. It was recently shown that the 46/1 haplotype, defined by four single-nucleotide polymorphisms (SNPs: rs3780367, rs10974944, rs12343867 and rs1159782), was strongly associated with the cis acquisition of JAK2V617F mutation with complete linkage disequilibrium.5, 6, 7 As UPD 9p involves most of the chromosome 9, JAK2V617F and the SNPs that tag 46/1 are converted into homozygosity simultaneously, and the 46/1 SNP allele burden reflects the percentage of JAK2V617F/V617F cells in 46/1 heterozygous PV patients. The JAK2V617F allele burden depends on the fraction of cells with a wild type, heterozygous and homozygous genotype. These fractions are usually determined by single-cell analyses. Here, we report that the combined measurement of 46/1 SNPs and JAK2V617F allele burdens in hematopoietic progenitors and differentiated myeloid compartments allows dissecting the clonal architecture in each cell compartment. This approach can be used to explore the impact of treatments, as demonstrated in patients exposed to alpha-interferon (IFNα).

Modeling JAK2V617F clonal architecture in PV patients

We first explored three patients with hemochromatosis, wild-type JAK2 and rs12343867 heterozygosity. Genotyping of 500 single-cell colonies established from sorted CD34+CD38 and CD34+CD38+ progenitors did not identify any rs12343867 homozygous colony (Supplementary Figure S1), indicating that recombination of the 46/1 haplotype was not a common event.

We selected 9 PV patients heterozygous for rs12343867 allele and JAK2V617F negative in CD3+ cells using Taqman allelic discrimination PCR. In all the patients, the rs12343867 and JAK2V617F allele burdens were measured in sorted CD34+ cells by both Taqman allelic discrimination PCR and next-generation sequencing (NGS), allowing calculation of the percentage of JAK2V617F cells. The percentage of cells with a homozygous JAK2 mutation (JAK2V617F/V617F) was calculated from the formula: (rs12343867 allele burden−50%) × 2, the percentage of cells with a heterozygous JAK2 mutation (JAK2V617F/WT) by the formula: (% of JAK2V617F allele burden−% of JAK2V617F/V617F cells) × 2, and the percentage of JAK2wt/wt cells by the formula: 100−(% of JAK2V617F/V617F+% of JAK2V617F/WT cells) described in the Supplementary Methods. In parallel, colonies obtained by CD34+CD38+ culture were genotyped individually using allelic discrimination PCR. In seven out of nine patients, we observed a good correlation between the expected (calculated from PCR and from NGS data) and the observed (in single-cell-derived colonies) values of homozygous JAK2V617F/V617F and heterozygous JAK2V617F/WT cell fractions. Of note, one patient (#1) had only JAK2V617F/V617F cells and another (#9) had a majority of WT cells. Patient #3 displayed a mixture of WT and heterozygous cells, patients #6 and #8 had a mixture of WT and homozygous cells, and patient #4 of WT, heterozygous and homozygous cells (Figure 1). Interestingly, NGS gave quite similar values than those with allele-specific PCR. Surprisingly, analysis of single-cell-derived colonies of patients #5 and #7 progenitors showed more wild-type cells than expected from our PCR- and NGS-based calculations, that is, the JAK2V617F burden obtained by genotyping CD34+-derived colonies was much lower than measured in the CD34+ cell population. This result was not related to an artifact because experiments could be reproduced at least three times for each patient. One possibility is that a secondary genetic event affects self-renewal capacities of in vitro growth of heterozygous and homozygous JAK2V617F progenitors and prevents them from growing in short-term culture. This strongly suggests that in vitro cell growth induces a bias in clonal architecture analysis. Nevertheless, our results validate the use of the 46/1 haplotype to construct the JAK2V617F clonal architecture in CD34+ cells of PV patients.

Figure 1

Modeling JAK2V617F clonal architecture in PV patients. CD34+ cells from nine PV patients were cloned and cultured for 14 days in serum-free medium in the presence of cytokines. DNA was extracted from each colony (an average of 100 colonies were genotyped per patient) and was subjected either to JAK2 or rs12343867 allele discrimination (observed values). Alternatively, quantification of JAK2 mutation and rs12343867 variant was obtained by allelic discrimination ‘genotyping’ or NGS both in global CD34+ and granulocyte cells from 9 PV patients. The expected and the observed values of WT, JAK2V617F/WT and JAK2V617F/V617F clones are represented in the figure. In none of the nine patients studied, the 9pLOH precedes acquisition of JAK2V617F mutation.

JAK2V617F clonal amplification pattern in hematopoietic stem cells and myeloid compartments

Some evidence suggests that, in MPN patients, the JAK2V617F clone expands during myeloid differentiation.8, 9, 10 In mouse models, Jak2V617F also stimulates the proliferation of hematopoietic stem cells (HSCs) or early progenitors.11, 12 Calculations obtained from allele-specific PCR and NGS were used with the rs12343867 and JAK2V617F allele burdens of granulocytes of nine PV patients. Results clearly showed that JAK2V617F/V617F clone preferentially expands during myeloid differentiation at the expense of WT and JAK2V617F/WT clones (Figure 1). When JAK2V617F/V617F clone was absent (patients #3 and #9), JAK2V617F/WT had no or very little proliferative advantage (Figure 1). Moreover, we investigated JAK2V617F clonal proportions in the HSC compartments in six patients. Colonies were genotyped from CD34+CD38+, CD34+CD38 and CD90+CD34+CD38 cells and revealed no proliferative advantage of JAK2V617F/V617F or JAK2V617F/WT (Supplementary Figures S2 and S3). These results are in accordance with previous reports showing that JAK2V617F/V617F clones expand with myeloid differentiation, but their size remains limited in the HSC compartment.8, 9, 10

Effect of IFNα treatment on JAK2V617F clonal structure

We used our model to assess the therapeutic potential of pegylated IFNα in MPN treatment. We tested 4 molecular nonresponding and 11 responding PV patients according to the criteria of Barosi et al.13 using NGS or allele-specific PCR to determine rs12343867 and JAK2V617F allele burdens in granulocytes. We observed that the homozygous cells were in all cases targeted by IFNα (P1, P2, P3, P6, P12, P14, P15), whereas heterozygous cells were nonresponders in 53% of patients (P5, P10, P11, P13, P1, P3, P6, P15). Of note, the four nonresponding patients displayed initially only a heterozygous clone (Figure 2a). The percentage of allele burdens decreased more dramatically and faster in homozygous patients than in heterozygous responding patients (73% to 4% in 3.25 years versus 27% to 2% in 5.1 years in average; Figure 2b). Moreover, in patients with mixed cells (JAK2V617F/V617F, JAK2V617F/WT, JAK2WT/WT), IFNα preferentially targeted homozygous cells during the follow-up (P1, P2, P14). The preferential targeting of homozygous cells by IFNα is also exhibited by appearance of heterozygous cells during the treatment of patients at the time of eradication of homozygous cells (P1, P3, P6, P15). In addition, the JAK2V617F burden increased at 48% after 2 years of IFNα interruption in patient #6 and this relapse was mainly related to the JAK2V617F/V617F clone. Finally, the screening for mutations in DNMT3A, TET2, ASXL1, SUZ12, EZH2, ZRSR2, SF3B1, RUNX1, IDH1/2, U2AF1 genes revealed additional mutations in few patients (Supplementary Figure S4), which did not clearly affect the response to IFNα treatment contrary to what was suggested.14

Figure 2

Modeling JAK2V617F clonal architecture in PV patients treated with IFNα. (a) The calculated values of WT, JAK2V617F/WT and JAK2V617F/V617F clones were obtained after quantifying JAK2V617F and rs12343867 variant by NGS or allele-specific PCR in global granulocytes from 15 IFNα-treated patients over time. Patients were considered as responders except patients P5, P10, P11, P13 as previously established.13 (b) The percentage of JAK2V617F allele burdens were calculated in pure homozygous or pure heterozygous patients before the treatment with IFNα and after molecular response. The duration of the molecular response is also indicated in years (**P<0.01; ***P<0.001).

These results demonstrate that this simple modeling can be useful to follow the efficacy and specificity of treatment on JAK2V617F clones in MPNs, without absolutely requiring investigation at the unicellular level. In addition, it suggests that IFNα treatment more specifically target the JAK2V617F/V617F clone in responding patients, as suggested by in vitro clonal studies on progenitors from PV.15 These results suggest a link between the levels of JAK2 signaling and IFNα response. This may be important to determine the mechanism of action of IFNα on JAK2V617F stem cell.


  1. 1

    Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054–1061.

    CAS  Article  Google Scholar 

  2. 2

    James C, Ugo V, Casadevall N, Constantinescu SN, Vainchenker W . A JAK2 mutation in myeloproliferative disorders: pathogenesis and therapeutic and scientific prospects. Trends Mol Med 2005; 11: 546–554.

    CAS  Article  Google Scholar 

  3. 3

    Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779–1790.

    CAS  Article  Google Scholar 

  4. 4

    Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005; 7: 387–397.

    CAS  Article  Google Scholar 

  5. 5

    Jones AV, Chase A, Silver RT, Oscier D, Zoi K, Wang YL et al. JAK2 haplotype is a major risk factor for the development of myeloproliferative neoplasms. Nat Genet 2009; 41: 446–449.

    CAS  Article  Google Scholar 

  6. 6

    Kilpivaara O, Mukherjee S, Schram AM, Wadleigh M, Mullally A, Ebert BL et al. A germline JAK2 SNP is associated with predisposition to the development of JAK2(V617F)-positive myeloproliferative neoplasms. Nat Genet 2009; 41: 455–459.

    CAS  Article  Google Scholar 

  7. 7

    Olcaydu D, Harutyunyan A, Jager R, Berg T, Gisslinger B, Pabinger I et al. A common JAK2 haplotype confers susceptibility to myeloproliferative neoplasms. Nat Genet 2009; 41: 450–454.

    CAS  Article  Google Scholar 

  8. 8

    Anand S, Stedham F, Beer P, Gudgin E, Ortmann CA, Bench A et al. Effects of the JAK2 mutation on the hematopoietic stem and progenitor compartment in human myeloproliferative neoplasms. Blood 2011; 118: 177–181.

    CAS  Article  Google Scholar 

  9. 9

    Dupont S, Masse A, James C, Teyssandier I, Lecluse Y, Larbret F et al. The JAK2 617V&gt;F mutation triggers erythropoietin hypersensitivity and terminal erythroid amplification in primary cells from patients with polycythemia vera. Blood 2007; 110: 1013–1021.

    CAS  Article  Google Scholar 

  10. 10

    Godfrey AL, Chen E, Pagano F, Ortmann CA, Silber Y, Bellosillo B et al. JAK2V617F homozygosity arises commonly and recurrently in PV and ET, but PV is characterized by expansion of a dominant homozygous subclone. Blood 2012; 120: 2704–2707.

    CAS  Article  Google Scholar 

  11. 11

    Akada H, Yan D, Zou H, Fiering S, Hutchison RE, Mohi MG . Conditional expression of heterozygous or homozygous Jak2V617F from its endogenous promoter induces a polycythemia vera-like disease. Blood 2010; 115: 3589–3597.

    CAS  Article  Google Scholar 

  12. 12

    Mullally A, Poveromo L, Schneider RK, Al-Shahrour F, Lane SW, Ebert BL . Distinct roles for long-term hematopoietic stem cells and erythroid precursor cells in a murine model of Jak2V617F-mediated polycythemia vera. Blood 2012; 120: 166–172.

    CAS  Article  Google Scholar 

  13. 13

    Barosi G, Birgegard G, Finazzi G, Griesshammer M, Harrison C, Hasselbalch HC et al. Response criteria for essential thrombocythemia and polycythemia vera: result of a European LeukemiaNet consensus conference. Blood 2009; 113: 4829–4833.

    CAS  Article  Google Scholar 

  14. 14

    Quintas-Cardama A, Abdel-Wahab O, Manshouri T, Kilpivaara O, Cortes J, Roupie AL et al. Molecular analysis of patients with polycythemia vera or essential thrombocythemia receiving pegylated interferon alpha-2a. Blood 2013; 122: 893–901.

    CAS  Article  Google Scholar 

  15. 15

    Lu M, Zhang W, Li Y, Berenzon D, Wang X, Wang J et al. Interferon-alpha targets JAK2V617F-positive hematopoietic progenitor cells and acts through the p38 MAPK pathway. Exp Hematol 2010; 38: 472–480.

    CAS  Article  Google Scholar 

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We are grateful to Françoise Wendling and Caroline Marty for improving the manuscript. We thank Yann Lecluse and Philippe Rameau from the Imaging Platform for their help in cytometry analysis and cell sorting, and Yannis Dufourd and Noemie Pata-Merci from the Genomic Platform for NGS analysis. This work was supported by grants from the Agence Nationale de la Recherche (Epigenome 2010, Thrombocytosis 2011), the Foundation ARC (Association pour la Recherche contre le Cancer, IP), the Ligue Nationale contre le Cancer (WV, ES) and the “Investissements d’avenir” programme (Labex GR-Ex; IP, WV). HS, FF and BM-M were supported by fellowships from la Ligue Nationale Contre le Cancer, the Foundation ARC, and the Fondation pour la Recherche Médicale, respectively. WV is a recipient of a research fellowship from Gustave Roussy and INSERM (contrat d’interface). IP is supported by grants from Foundation ARC (projet libre 2012).

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Correspondence to I Plo.

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Hasan, S., Cassinat, B., Droin, N. et al. Use of the 46/1 haplotype to model JAK2V617F clonal architecture in PV patients: clonal evolution and impact of IFNα treatment. Leukemia 28, 460–463 (2014).

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