JAK2-V617F mutation in a patient with Philadelphia - Mannheimer ...

Case ReportJAK2-V617F mutation in a patient with Philadelphiachromosome-positivechronic myeloid leukaemiaAlwin Krämer, Andreas Reiter, Jens Kruth, Philipp Erben, Andreas Hochhaus, Martin Müller, Nicholas C P Cross, Amy V Jones, Anthony D Ho,Manfred HenselLancet Oncol 2007; 8: 658–60Department of InternalMedicine V, University ofHeidelberg, HeidelbergGermany (Prof A Krämer MD,Prof AD Ho MD, M Hensel MD);Department of InternalMedicine III, Faculty of ClinicalMedicine Mannheim, Universityof Heidelberg, Mannheim,Germany (Prof A Reiter MD,J Kruth MD, P Erben MD,Prof A Hochhaus MD,M Müller MD); Wessex RegionalGenetics Laboratory, Salisbury,UK (Prof NCP Cross PhD,AV Jones PhD)Correspondence to:Prof Alwin Krämer, ClinicalCooperation Unit MolecularHaematology/Oncology, GermanCancer Research Centre andDepartment of Internal MedicineV, University of Heidelberg,69120 Heidelberg, Germanya.kraemer@dkfz-heidelberg.deLeucocytes (/nL) and metamyelocytes in peripheral blood (%)100806040200Spleen 21·3 cmIn July, 2000, a 50-year-old man presented with leukocytosisand splenomegaly (21 cm). Leucocyte concentrationwas 93×10 9 /L, haemoglobin 150 g/L, and platelets345×10 9 /L (figure 1). A differential blood count showed54% neutrophils, 2% lymphocytes, 13% myelocytes, 7%metamyelocytes, 2% promyelocytes, 1% blasts, and7% basophils. Lactate dehydrogenase (LDH) concentrationwas increased at 484 U/L. 17 months previously,the blood count had been in the normal range. Bonemarrowaspiration was dry and bone-marrow biopsyshowed marked myeloid hyperplasia, increased megakaryopoiesis,and the beginning of fibrosis. Cytogeneticanalysis revealed a chromosome translocation(t[9;22][q34;q11]) in all ten metaphases examined. Expressionof B3A2 BCR-ABL mRNA was detected by reversetranscriptase polymerase chain reaction (RT-PCR) inperipheral-blood leucocytes. A diagnosis of BCR-ABLpositivechronic myeloid leukaemia (CML) was madeand treatment with hydroxycarbamide was started,resulting in rapid normalisation of peripheral-bloodleucocytes. After 3 weeks, treatment was changed toimatinib (400 mg/day). Within 1 month, a completehaem atological response was recorded. Cytogenetic andinterphase fluorescence in-situ hybridisation at6 months after the start of imatinib showed a completecytogenetic response. The BCR-ABL:ABL ratio droppedfrom 90% at diagnosis to 0·021% after 6 months.16 months after starting imatinib, BCR-ABL transcriptsbecame undetectable by real-time and nested RT-PCRLeucocytesMetamyelocytes and myelocytesin peripheral bloodPlateletsLactate dehydrogenaseSpleen 17·7 cm7/00 8/00 10/00 7/01 6/02 12/03 3/04 8/05 11/05 5/06 8/06Time (month/year)Figure 1: Course of peripheral-blood parameters during treatment with imatinib in a patient with BCR-ABLpositivechronic myeloid leukaemia8006004002000Platelets (/nL) and lactate dehydrogenase (U/L)(figure 2). 1 Repeated cytogenetic and quantitative BCR-ABL RT-PCR analyses showed a continuing completecytogenetic and molecular response, as shown infigure 2. However, in 2003, an unexpected decrease inplatelet count and a continuous increase in serum LDHconcentration was noted (figure 1). In 2004, immaturemyeloid cells began to appear in the peripheral bloodand a bone-marrow biopsy in May, 2004, revealedadvanced fibrosis. In 2005, leucocyte concentrationcontinually rose above the upper normal limit. Thespleen was enlarged to 18 cm. Cytogenetic analysisidentified a normal karyotype and BCR-ABL transcriptswere still undetectable. Therefore, a JAK2-V617F mutationalanalysis was done, which highlighted the presenceof a heterozygous JAK2-V617F mutation, leading to thediagnosis of idiopathic myelofibrosis (IMF) accordingto the WHO diagnostic criteria. For JAK2 genotyping,DNA was amplified and PCR products were directlysequenced using the PCR primers JAK2-1F(5’-TGCTGAAAGTAGGAGAAAGTGCAT-3’) and JAK2-1R (5’-TCCTACAGTGTTTTCAGTTTCAA-3’).The proportion of mutant JAK2-V617F alleles wasquantified using pyrosequencing as described. 2 Additionally,the allelic ratio of JAK2-V617F was confirmed bya newly established quantitative real-time (RQ) PCRassay based on LightCycler technology (Roche Diagnostics,Mannheim, Germany). DNA was extracted fromperipheral blood leucocytes after red-cell lysis by standardprocedures. Total JAK2 was established using aforward primer, 5’-AGGCTACATCCATCTACCTCAG-3’,a reverse primer, 5’-CCTAGCTGTGATCCTGAAACTG-3’(MWG Biotech, Ebersberg, Germany), and thehybridisation probes 5’-ACAGGCTTGA CCCATAA-CACCT GAAATA GAG-3’ and 5’-GAGTGGTACAGGAA-TCATGAATAATGCCAGTCA-3’ (Tib Molbiol, Berlin,Germany). Mutant JAK2-V617F alleles were quantifiedusing the same forward primer and probes incombination with a mutation-specific reverse primer,5’-TTTTACTTACTCTCGTCTCCACAGAA-3’ (MWG Bio -tech, Ebersberg, Germany). The allele copy number wasidentified using a plasmid standard curve. JAK2-V617Fpositivity was expressed as the ratio between mutantJAK2-V617F and total JAK2. Dilution experimentsshowed an assay sensitivity of 1%.Retrospective analysis of frozen samples of peripheralbloodleucocytes showed that the JAK2-V617F mutationwas already present at initial diagnosis of BCR-ABLpositiveCML (figure 3). Consistent with the presence ofan acquired somatic mutation, the JAK2-V617F mutationwas absent in purified CD3+ T lymphocytes (figure 3)658 http://oncology.thelancet.com Vol 8 July 2007

Case Reportand buccal cells (data not shown). As demonstrated bytwo independent methods—JAK2-V617F pyrosequencingand PCR—the proportion of the mutant allele stayedroughly constant from diagnosis of CML in July, 2000, toFebruary, 2006 (figure 2). Repeated cytogenetic analysisin May, 2006 again showed a normal karyotype.JAK2 is a tyrosine kinase that has an important role inthe signalling pathways of many haemopoietic growthfactorreceptors. The single acquired point mutationV617F (1849G>T) in JAK2 occurs in 50–97% of patientswith IMF, essential thrombocytosis, and polycythemiavera. 2–6 By contrast, the JAK2-V617F mutation has neverbeen identified in a patient with BCR-ABL-positiveCML. 7 This is the first description of a patient, to ourknowledge, in whom the BCR-ABL fusion gene and anacquired somatic JAK2-V617F mutation could bedetected contemporaneously, with IMF becomingclinically relevant after successful treatment of CML byimatinib.Several conclusions can be drawn from these data.First, bone-marrow fibrosis was not a consequence ofprogressive CML. Rather, myelofibrosis developed as asecond myeloproliferative disorder (MPD) duringcomplete cytogenetic and molecular remission of CML.Although the BCR-ABL fusion gene might represent theinitiating lesion in most cases of CML, clinicalmanifestations of the disease can be variable. Specifically,bone-marrow fibrosis can occur, sometimes varyinggreatly during the course of the disease, and has beenused to categorise patients into different groups withdistinctive survival caracter istics. 8 Fibrogenesis in MPDis generally assumed to be mediated by the abnormalrelease of transforming growth factor-β and plateletderivedgrowth factor (PDGF). Imatinib, a selectiveinhibitor of ABL, KIT, and PDGF receptor tyrosinekinases, and which is not active against JAK2, has beenseen to reduce the content of bone-marrow fibre inpatients with CML. 9 Regression of myelofibrosis byimatinib is thought to be caused either by a direct PDGFantagonisticeffect or by normalisation of megakaryopoiesisfrom which abnormal concentrations of PDGFare released. How ever, expression of JAK2-V617F inmurine haematopoietic cells leads to MPD associatedwith myelofibrosis. 10 Moreover, expression of a BCR-JAK2fusion gene has been described in a patient with atypicalCML and bone-marrow fibrosis, 11 and the PCM1-JAK2fusion is associated with the development of myelofibrosisin patients with chronic and acute leukaemias. 12 Thesefindings suggest that JAK2-V617F contributes to thedevelopment of myelofibrosis in MPD, 3,10 and might havebeen responsible for the induction of myelofibrosis inour patient which, consequently, was independent of thepresence of BCR-ABL and did not respond to imatinibtreatment. This assumption is supported by the absenceof clinical and molecular responses of patients with IMFand polycythemia vera treated with imatinib. 13,14 Iftreatment with imatinib had not been initiated, theclinical course of the patient could only have beenspeculated upon. Treatment and response to imatinibmight have accelerated the outgrowth of the IMF.Second, our data suggest that in our patient, the JAK2-V617F mutation had occurred before the acquisition ofthe Philadelphia (Ph)-chromosome. Development of aPh-positive CML has rarely been reported in patientswith MPD. 15 In the few cases that have been reported,ACPercentage of tumour cells100958075605540352015Figure 2: BCR-ABL:ABL ratio and JAK2-V617F allele frequency during treatment with imatinib in a patientwith BCR-ABL-positive chronic myeloid leukaemiaParameters established by pyrosequencing and PCR.A0T G TA T G TTGTGT C T GT C T GTime (month/year)BDA T G T G T C T GA T G TBCR-ABL:ABLJAK2/V617F-pyrosequencingJAK2/V617F-PCR7/00 7/01 7/02 7/03 7/04 7/05 7/06TGT C T GFigure 3: Presence of the JAK2-V617F mutation in a patient with BCR-ABL-positive chronic myeloid leukaemiaA heterozygous G→T transversion in JAK2 (arrows) is present in DNA from leucocytes (A,C,D) at different timepoints (A: July, 2000; C: November, 2001; D: May, 2006) during the course of the disease, but absent in T cells(B; May, 2006), consistent with the existence of an acquired somatic origin of the mutation.http://oncology.thelancet.com Vol 8 July 2007 659

Case Reportthere is a debate as to whether MPD and CML ariseseparately from each other, representing independenttransformation of a normal stem cell, or whether the Phchromosomearises in a stem cell that was part of theMPD clone. In our patient, JAK2-V617F was detectable atan almost constant allele frequency in all peripheralbloodsamples examined (figure 2). The most likelyexplanation for this finding is the presence of aheterozygous JAK2-V617F mutation in most of theperipheral-blood leucocytes, implicating the simultaneouspresence of the BCR-ABL fusion gene and the JAK2-V617F mutation in white blood cells. Because BCR-ABLbecame undetectable after treatment with imatinib, butthe JAK2-V617F allele frequency remained virtuallyunchanged, the conclusion can be drawn that the Phchromosomewas acquired from a haematopoietic cellcarrying the JAK2-V617F mutation. Several findingssuggest that MPD are caused by a mutation in an, as yet,unknown gene that precedes the acquisition of either aJAK2-V617F mutation or a BCR-ABL fusion gene. 3,16 Themost reasonable explanation for the findings presentedhere is that BCR-ABL can, in rare cases, occur on thebackground of the JAK2-V617F mutation, possiblyaugmented by an elusive initial mutation predisposing tothe acquisition of both a JAK2-V617F mutation and aBCR-ABL translocation.Conflicts of interestThe authors declared no conflicts of interest.AcknowledgmentsWe thank Brigitte Schreiter for excellent technical assistance. This workwas supported by a grant from the German José Carreras LeukemiaFoundation (DJCLS R 06/04).References1 Emig M, Saussele S, Wittor H, et al. Accurate and rapid analysis ofresidual disease in patients with CML using specific fluorescenthybridization probes for real time quantitative RT-PCR. Leukemia1999; 13: 1825–32.2 Jones AV, Kreil S, Zoi K, et al. Widespread occurrence of the JAK2V617F mutation in chronic myeloproliferative disorders. Blood 2005;106: 2162–68.3 Kralovics R, Passamonti F, Buser AS, et al. A gain-of-functionmutation of JAK2 in myeloproliferative disorders. N Engl J Med2005; 352: 1779–90.4 Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of thetyrosine kinase JAK2 in human myeloproliferative disorders.Lancet 2005; 365: 1054–61.5 James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2mutation leading to constitutive signalling causes polycythaemiavera. Nature 2005; 434: 1144–48.6 Levine RL, Wadleigh M, Cools J, et al. Activating mutation in thetyrosine kinase JAK2 in polycythemia vera, essentialthrombocythemia, and myeloid metaplasia with myelofibrosis.Cancer Cell 2005; 7: 387–97.7 Jelinek J, Oki Y, Gharibyan V, et al. JAK2 mutation 1849G>T is rarein acute leukemias but can be found in CMML, Philadelphiachromosome-negative CML, and megakaryocytic leukemia.Blood 2005; 106: 3370–73.8 Kvasnicka HM, Thiele J, Schmitt-Graeff A, et al. Bone marrowfeatures improve prognostic efficiency in multivariate riskclassification of chronic-phase Ph(1+) chronic myelogenousleukemia: a multicenter trial. J Clin Oncol 2001; 19: 2994–3009.9 Beham-Schmid C, Apfelbeck U, Sill H, et al. Treatment of chronicmyelogenous leukemia with the tyrosine kinase inhibitor STI571results in marked regression of bone marrow fibrosis. Blood 2002;99: 381–83.10 Lacout C, Pisani DF, Tulliez M, Moreau Gachelin F, Vainchenker W,Villeval JL. JAK2V617F expression in murine hematopoietic cellsleads to MPD mimicking human PV with secondary myelofibrosis.Blood 2006; 108: 1652–60.11 Griesinger F, Hennig H, Hillmer F, et al. A BCR-JAK2 fusion geneas the result of a t(9;22)(p24;q11.2) translocation in a patient with aclinically typical chronic myeloid leukemia. Genes ChromosomesCancer 2005; 44: 329–33.12 Reiter A, Walz C, Watmore A, et al. The t(8;9)(p22;p24) is arecurrent abnormality in chronic and acute leukemia that fusesPCM1 to JAK2. Cancer Res 2005; 65: 2662–67.13 Tefferi A, Mesa RA, Gray LA, et al. Phase 2 trial of imatinibmesylate in myelofibrosis with myeloid metaplasia. Blood 2002;99: 3854–56.14 Jones AV, Silver RT, Waghorn K, et al. Minimal molecular responsein polycythemia vera patients treated with imatinib or interferonalpha. Blood 2006; 107: 3339–41.15 Curtin NJ, Campbell PJ, Green AR. The Philadelphia translocationand pre-existing myeloproliferative disorders. Br J Haematol 2005;128: 734–36.16 Kralovics R, Teo SS, Li S, et al. Acquisition of the V617F mutationof JAK2 is a late genetic event in a subset of patients withmyeloproliferative disorders. Blood 2006; 108: 1377–80.660 http://oncology.thelancet.com Vol 8 July 2007