correspondence
The V617F mutation in Jak2 is not found in childhood acute lymphoblastic leukaemia
Members of the Jak kinase family, including Tyk2, Jak1, Jak2 and Jak3, play an important role in mediating cellular signals between cytokine/hormone receptors and downstream effector proteins by virtue of their tyrosine phosphorylation activities. They share structural homology and have two kinase domains, JH1 and JH2. While only the JH1 domain has kinase functionality, JH2 plays an important negative-regulatory role on JH1 activity. Recently a somatic, gain of function, mutation has been identified in JAK2 in a range of Philadelphia chromosomenegative myeloproliferative diseases including polycythaemia vera, essential thrombocythaemia and chronic idiopathic myelofibrosis (Baxter et al, 2005; Kralovics et al, 2005; Levine et al, 2005). A point mutation in exon 12 of the JAK2 gene results in substitution of valine for phenylalanine at amino acid position 617 in the JH2 domain, leading to constitutive tyrosine phosphorylation activity and cytokine hypersensitivity. Neoplastic cells can be heterozygous for the mutation or hemizygous if they are associated with loss of heterozygosity (LOH) of 9p, where JAK2 is situated. The explanation for V617F yielding different chronic myeloproliferative disease phenotypes is not clear but may relate to the target cell for transformation or the presence of other co-operating genetic events. Jak2 activation is also implicated in the pathology of childhood acute lymphoblastic leukaemia (ALL). Two studies showed constitutive activation of Jak2 in both primary samples and leukaemic cell lines, with inhibition blocking leukaemic cell growth selectively, in vitro and in vivo, by inducing programmed cell death (Meydan et al, 1996). In addition, LOH of 9p is a relatively common event, with JAK2 invariably being present in the minimally deleted region. One known mechanism of JAK2 activation in ALL is chromosomal translocation with the t(9;12)(p24;p13) yielding a chimaeric Tel–Jak2 fusion protein that has deregulated Jak2 kinase activity, confers cytokine-independent proliferation and results in the formation of ALL in a mouse model system (Lacronique M
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et al, 1997; Peeters et al, 1997; Carron et al, 2000). However, this is a rare occurrence. Taken together, these observations provide a rationale for investigating the presence of V617F in childhood ALL. To detect the V617 mutation, we performed allele-specific polymerase chain reaction using genomic DNA isolated from bone marrow samples from children with ALL treated at our institution (Baxter et al, 2005). Eighty-six samples were collected during diagnosis and 42 samples during relapse. All samples were collected with appropriate consent. Amplicons were size fractioned by standard agarose gel electrophoresis. DNA from HEL92.1.7, an erythroleukaemic cell line, served as a positive control. None of the 128 ALL samples examined were positive for the 203-bp product, indicative of V617F mutation, but all were positive for the internal control (representative gel shown in Fig 1). Using HEL92.1.7 cells spiked into normal peripheral blood, the sensitivity of the assay was determined to be between 1% and 5%. Thus the V617F mutation is absent in childhood ALL and this finding concords with a mutational screening study of the JH2 domain in a small cohort of ALL patients (Cools et al, 1999). Given the importance of the Jak2 activation in primary ALL, a comprehensive mutational screen of all of its coding exons is warranted. Sarina Sulong1 Marian Case1 Lynne Minto1 Bridget Wilkins2 Andy Hall1 Julie Irving1 1
Northern Institute for Cancer Research, and
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Royal Victoria Infirmary, Newcastle upon Tyne, Tyne, UK.
E-mail.
[email protected]
References
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Fig 1. Allele-specific polymerase chain reaction (PCR) for the V617F mutation. The 364-bp band serves as an internal control. The 203-bp band indicates the presence of the mutation. Lanes 1–7, acute lymphoblastic leukaemia samples; lane 8, HEL92.1.7 positive control; lane 9, PCR negative control with no DNA template; M, marker lanes.
doi:10.1111/j.1365-2141.2005.05697.x
Baxter, E.J., Scott, L.M., Campbell, P.J., East, C., Fourouclas, N., Swanton, S., Vassiliou, G.S., Bench, A.J., Boyd, E.M., Curtin, N., Scott, M.A., Erber, W.N. & Green, A.R. (2005) Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet, 365, 1054–1061. Carron, C., Cormier, F., Janin, A., Lacronique, V., Giovannini, M., Daniel, M.T., Bernard, O. & Ghysdael, J. (2000) TEL-JAK2 transgenic mice develop T-cell leukemia. Blood, 95, 3891–3899.
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 964–972
Correspondence Cools, J., Peeters, P., Voet, T., Aventin, A., Mecucci, C., Grandchamp, B. & Marynen, P. (1999) Genomic organization of human JAK2 and mutation analysis of its JH2-domain in leukemia. Cytogenetics and Cell Genetics, 85, 260–266. Kralovics, R., Passamonti, F., Buser, A.S., Teo, S.S., Tiedt, R., Passweg, J.R., Tichelli, A., Cazzola, M. & Skoda, R.C. (2005) A gain-offunction mutation of JAK2 in myeloproliferative disorders. New England Journal of Medicine, 352, 1779–1790. Lacronique, V., Boureux, A., Valle, V.D., Poirel, H., Quang, C.T., Mauchauffe, M., Berthou, C., Lessard, M., Berger, R., Ghysdael, J. & Bernard, O.A. (1997) A TEL-JAK2 fusion protein with constitutive kinase activity in human leukemia. Science, 278, 1309–1312. Levine, R.L., Wadleigh, M., Cools, J., Ebert, B.L., Wernig, G., Huntly, B.J., Boggon, T.J., Wlodarska, I., Clark, J.J., Moore, S., Adelsperger, J., Koo, S., Lee, J.C., Gabriel, S., Mercher, T., D’Andrea, A., Frohling, S., Dohner, K., Marynen, P., Vandenberghe, P., Mesa, R.A., Tefferi, A., Griffin, J.D., Eck, M.J., Sellers, W.R., Meyerson, M., Golub, T.R.,
Lee, S.J. & Gilliland, D.G. (2005) Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell, 7, 387–397. Meydan, N., Grunberger, T., Dadi, H., Shahar, M., Arpaia, E., Lapidot, Z., Leeder, J.S., Freedman, M., Cohen, A., Gazit, A., Levitzki, A. & Roifman, C.M. (1996) Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nature, 379, 645–648. Peeters, P., Raynaud, S.D., Cools, J., Wlodarska, I., Grosgeorge, J., Philip, P., Monpoux, F., Van Rompaey, L., Baens, M., Van den Berghe, H. & Marynen, P. (1997) Fusion of TEL, the ETS-variant gene 6 (ETV6), to the receptor-associated kinase JAK2 as a result of t(9;12) in a lymphoid and t(9;15;12) in a myeloid leukemia. Blood, 90, 2535–2540.
Keywords: childhood acute lymphoblastic leukaemia, Jak2, mutation.
Risk factors for highly unstable response to oral anticoagulation
Palareti et al (2005) have investigated risk factors associated with a highly unstable response to oral anticoagulation. However, the small number of patients from 35 Italian anticoagulant clinics, which comprise the unstable case group, is so highly selected as to be of very questionable practical relevance. I have previously shown in a large number of unselected patients that severe over-anticoagulation [International Normalised Ratio (INR) ‡8], with high risk of major bleeding, is most likely to occur in the early stages of oral anticoagulation (Murphy et al, 1998). Yet, among other selection criteria, patients included in the unstable case group had to be on oral anticoagulation for over 8 months, not suffer from cancer and not be hospitalised during the previous 6 months. Palareti et al (2005) also reported that the unstable case group were more likely to be over-anticoagulated (INR >4Æ5) than controls. However, information on the frequency of more severe over-anticoagulation (INR >6) might have been of more practical use as outpatients with INR >6 face a significant short-term risk of major haemorrhage (Hylek et al, 2000). It is also unfortunate that despite the large number of factors that the authors were able to compare statistically between the unstable case group and stable controls, there was no evaluation of bleeding and thrombotic complications between the two groups because of the ‘limited sample size’. Given the highly select nature of the unstable case group and the fact that these patients were, apparently, compliant enough to attend well organised and well equipped, computerised anticoagulant clinics at their appointed times, I doubt that patients in the unstable case group were at any statistically higher risk of
major haemorrhage or thrombosis than the stable controls. It seems likely that Palareti et al (2005) excluded anticoagulated patients at highest risk of complications from their study and that the risk factors for highly unstable response to oral anticoagulation in their unstable case group are of limited practical benefit in dealing with truly high-risk anticoagulated patients. Philip T. Murphy Department of Haematology, Beaumont Hospital, Dublin, Ireland. E-mail:
[email protected]
References Hylek, E.M., Chang, Y.C., Skates, S.J., Hughes, R.A. & Singer, D.E. (2000) Prospective study of the outcomes of ambulatory patients with excessive warfarin anticoagulation. Archives of Internal Medicine, 160, 1612–1617. Murphy, P.T., Casey, M.C. & Abrams, K. (1998) Audit of patients on oral anticoagulants with international normalised ratios of eight or above. Clinical and Laboratory Haematology, 20, 253–257. Palareti, G., Legnani, C., Guazzaloca, G., Lelia, V., Cosmi, B., Lunghi, B., Marchetti, G., Poli, D. & Pengo, V. (2005) Risk factors for highly unstable response to oral anticoagulation: a case–control study. British Journal of Haematology, 129, 72–78.
Keywords: oral anticoagulation, bleeding, thrombosis. doi:10.1111/j.1365-2141.2005.05707.x
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