JAK2 Mutations in Myeloproliferative Disorders
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《新英格兰医药杂志》
To the Editor: An activating somatic mutation involving the JH2 pseudokinase domain of Janus kinase 2 (JAK2 ) has been associated with myeloproliferative disorders. The mutation is detectable in 65 percent1 to 97 percent2 of cases of polycythemia vera. Kralovics and colleagues (April 28 issue)1 report a significant association between homozygosity for the JAK2 (V617F) mutation, which occurred in approximately one quarter of the patients with polycythemia vera, and increased duration of disease in polycythemia vera, essential thrombocythemia, and myelofibrosis with myeloid metaplasia. A similar observation was made by others,3 raising the possibility that homozygosity for the mutant allele is a time-dependent clonal evolution.
We tested this hypothesis by performing mutational analysis in archived bone marrow cells from patients showing homozygosity in DNA derived from peripheral-blood granulocytes. Among 220 patients with either polycythemia vera or myelofibrosis with myeloid metaplasia who were seen at the Mayo Clinic and not included in previous publications,3 granulocyte-based mutation screening identified 21 patients who were homozygous for JAK2 (V617F) — 13 who had polycythemia vera and 8 who had myelofibrosis with myeloid metaplasia. In the case of 5 of these 21 patients, including 2 with polycythemia vera, the study was performed at the time of diagnosis. However, laboratory records in the two patients with polycythemia vera revealed a preexisting increase in the hematocrit from baseline that had been unrecognized for at least two years. Stored bone marrow from six patients, collected 1.5 to 9.5 years before the current analysis, showed variable degrees of heterozygosity in four patients at different times during their clinical course (Figure 1). The pattern of change over time, especially as depicted in Patient 3, favors a time-dependent increase in clonal dominance rather than a two-step molecular event. This possibility is consistent with the occurrence of a mixed-clonality pattern in purified CD34+ cell fractions in patients whose granulocytes show homozygosity4 and the in vitro demonstration of a JAK2 (V617F)–induced proliferative advantage in cell lines.1,5 Furthermore, as noted above, the occurrence of subclinical clonal myelopoiesis might partly explain why heterozygosity is not always documented at the time of clinical diagnosis.
Figure 1. Mutational Analysis of JAK2 (V617F) in Serial Bone Marrow Specimens from Six Patients with Myeloproliferative Disorders.
Bone marrow from Patients 1, 2, 3, and 6 shows variable degrees of homozygosity during their clinical course. Arrows indicate the nucleotide substitution of T for G.
Ayalew Tefferi, M.D.
Terra L. Lasho, M.T.
Mayo Clinic
Rochester, MN 55905
tefferi.ayalew@mayo.edu
Gary Gilliland, M.D., Ph.D.
Harvard Medical School
Boston, MA 02115
References
Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352:1779-1790.
Baxter EJ, Hochhaus A, Bolufer P, et al. The t(4;22)(q12;q11) in atypical chronic myeloid leukaemia fuses BCR to PDGFRA. Hum Mol Genet 2002;11:1391-1397.
Levine RL, Wadleigh M, Cools J, 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.
Lasho TL, Mesa R, Gilliland DG, Tefferi A. Mutation studies in CD3, CD19 and CD34 cell fractions in myeloproliferative disorders with homozygous JAK2 in granulocytes. Br J Haematol 2005;130:797-799.
James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005;434:1144-1148.
The authors reply: We proposed a two-step model for the role of JAK2 (V617F) in the clonal evolution of myeloproliferative disorders. The first step consists of a G-to-T mutation in one allele of the JAK2 gene that is acquired as a somatic mutation in a hematopoietic progenitor cell or stem cell. This cell gives rise to a clone that is heterozygous for JAK2 (V617F) and expands to replace hematopoietic cells without the JAK2 mutation. The second step consists of a mitotic recombination in one of the progenitor cells or stem cells heterozygous for the JAK2 mutation that generates uniparental disomy and homozygosity for JAK2 (V617F) in one of the two daughter cells. This daughter cell gives rise to a clone that is homozygous for JAK2 (V617F) and expands to replace heterozygous hematopoietic cells. The results of the test described by Tefferi and colleagues provide evidence that confirms the predictions of our model. A strict two-step process applies at the level of individual cells (i.e., each cell can be either heterozygous or homozygous, provided that gene amplification at the JAK2 locus is not involved). In contrast, when cell populations are analyzed, the transition from heterozygosity to homozygosity will be a continuous process, since the proportion of homozygous cells in this mixed population of cells will gradually increase until the homozygous cells fully dominate hematopoiesis. Analyses of mixed-cell populations, such as those of bone marrow or blood, taken during this transition period are expected to show a time-dependent increase of clonal dominance rather than a two-step transition. Thus, the data from Tefferi and colleagues do not contradict our model but instead confirm its predictions. We have applied a quantitative allele-specific polymerase-chain-reaction technique to determine the ratios of wild-type and mutant JAK2 alleles, and we obtained very similar results to those reported here by Tefferi and colleagues.
Robert Kralovics, Ph.D.
University Hospital Basel
4031 Basel, Switzerland
Mario Cazzola, M.D.
University of Pavia Medical School
27100 Pavia, Italy
Radek C. Skoda, M.D.
University Hospital Basel
4031 Basel, Switzerland
radek.skoda@unibas.ch
We tested this hypothesis by performing mutational analysis in archived bone marrow cells from patients showing homozygosity in DNA derived from peripheral-blood granulocytes. Among 220 patients with either polycythemia vera or myelofibrosis with myeloid metaplasia who were seen at the Mayo Clinic and not included in previous publications,3 granulocyte-based mutation screening identified 21 patients who were homozygous for JAK2 (V617F) — 13 who had polycythemia vera and 8 who had myelofibrosis with myeloid metaplasia. In the case of 5 of these 21 patients, including 2 with polycythemia vera, the study was performed at the time of diagnosis. However, laboratory records in the two patients with polycythemia vera revealed a preexisting increase in the hematocrit from baseline that had been unrecognized for at least two years. Stored bone marrow from six patients, collected 1.5 to 9.5 years before the current analysis, showed variable degrees of heterozygosity in four patients at different times during their clinical course (Figure 1). The pattern of change over time, especially as depicted in Patient 3, favors a time-dependent increase in clonal dominance rather than a two-step molecular event. This possibility is consistent with the occurrence of a mixed-clonality pattern in purified CD34+ cell fractions in patients whose granulocytes show homozygosity4 and the in vitro demonstration of a JAK2 (V617F)–induced proliferative advantage in cell lines.1,5 Furthermore, as noted above, the occurrence of subclinical clonal myelopoiesis might partly explain why heterozygosity is not always documented at the time of clinical diagnosis.
Figure 1. Mutational Analysis of JAK2 (V617F) in Serial Bone Marrow Specimens from Six Patients with Myeloproliferative Disorders.
Bone marrow from Patients 1, 2, 3, and 6 shows variable degrees of homozygosity during their clinical course. Arrows indicate the nucleotide substitution of T for G.
Ayalew Tefferi, M.D.
Terra L. Lasho, M.T.
Mayo Clinic
Rochester, MN 55905
tefferi.ayalew@mayo.edu
Gary Gilliland, M.D., Ph.D.
Harvard Medical School
Boston, MA 02115
References
Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352:1779-1790.
Baxter EJ, Hochhaus A, Bolufer P, et al. The t(4;22)(q12;q11) in atypical chronic myeloid leukaemia fuses BCR to PDGFRA. Hum Mol Genet 2002;11:1391-1397.
Levine RL, Wadleigh M, Cools J, 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.
Lasho TL, Mesa R, Gilliland DG, Tefferi A. Mutation studies in CD3, CD19 and CD34 cell fractions in myeloproliferative disorders with homozygous JAK2 in granulocytes. Br J Haematol 2005;130:797-799.
James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005;434:1144-1148.
The authors reply: We proposed a two-step model for the role of JAK2 (V617F) in the clonal evolution of myeloproliferative disorders. The first step consists of a G-to-T mutation in one allele of the JAK2 gene that is acquired as a somatic mutation in a hematopoietic progenitor cell or stem cell. This cell gives rise to a clone that is heterozygous for JAK2 (V617F) and expands to replace hematopoietic cells without the JAK2 mutation. The second step consists of a mitotic recombination in one of the progenitor cells or stem cells heterozygous for the JAK2 mutation that generates uniparental disomy and homozygosity for JAK2 (V617F) in one of the two daughter cells. This daughter cell gives rise to a clone that is homozygous for JAK2 (V617F) and expands to replace heterozygous hematopoietic cells. The results of the test described by Tefferi and colleagues provide evidence that confirms the predictions of our model. A strict two-step process applies at the level of individual cells (i.e., each cell can be either heterozygous or homozygous, provided that gene amplification at the JAK2 locus is not involved). In contrast, when cell populations are analyzed, the transition from heterozygosity to homozygosity will be a continuous process, since the proportion of homozygous cells in this mixed population of cells will gradually increase until the homozygous cells fully dominate hematopoiesis. Analyses of mixed-cell populations, such as those of bone marrow or blood, taken during this transition period are expected to show a time-dependent increase of clonal dominance rather than a two-step transition. Thus, the data from Tefferi and colleagues do not contradict our model but instead confirm its predictions. We have applied a quantitative allele-specific polymerase-chain-reaction technique to determine the ratios of wild-type and mutant JAK2 alleles, and we obtained very similar results to those reported here by Tefferi and colleagues.
Robert Kralovics, Ph.D.
University Hospital Basel
4031 Basel, Switzerland
Mario Cazzola, M.D.
University of Pavia Medical School
27100 Pavia, Italy
Radek C. Skoda, M.D.
University Hospital Basel
4031 Basel, Switzerland
radek.skoda@unibas.ch