JAK2-V617F mutation in a patient with Philadelphia-chromosome-positive chronic myeloid leukaemia.

In July, 2000, a 50-year-old man presented with leukocytosis and splenomegaly (21 cm). Leucocyte concentration was 93×10/L, haemoglobin 150 g/L, and platelets 345×10/L (fi gure 1). A diff erential blood count showed 54% neutrophils, 2% lymphocytes, 13% myelocytes, 7% metamyelocytes, 2% promyelocytes, 1% blasts, and 7% basophils. Lactate dehydrogenase (LDH) concentration was increased at 484 U/L. 17 months previously, the blood count had been in the normal range. Bonemarrow aspiration was dry and bone-marrow biopsy showed marked myeloid hyperplasia, increased megakaryopoiesis, and the beginning of fi brosis. Cytogenetic analysis revealed a chromosome translocation (t[9;22][q34;q11]) in all ten metaphases examined. Expression of B3A2 BCR-ABL mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in peripheral-blood leucocytes. A diagnosis of BCR-ABLpositive chronic myeloid leukaemia (CML) was made and treatment with hydroxycarbamide was started, resulting in rapid normalisation of peripheral-blood leucocytes. After 3 weeks, treatment was changed to imatinib (400 mg/day). Within 1 month, a complete haem atological response was recorded. Cytogenetic and interphase fl uorescence in-situ hybridisation at 6 months after the start of imatinib showed a complete cytogenetic response. The BCR-ABL:ABL ratio dropped from 90% at diagnosis to 0·021% after 6 months. 16 months after starting imatinib, BCR-ABL transcripts became undetectable by real-time and nested RT-PCR (fi gure 2). Repeated cytogenetic and quantitative BCRABL RT-PCR analyses showed a continuing complete cytogenetic and molecular response, as shown in fi gure 2. However, in 2003, an unexpected decrease in platelet count and a continuous increase in serum LDH concentration was noted (fi gure 1). In 2004, immature myeloid cells began to appear in the peripheral blood and a bone-marrow biopsy in May, 2004, revealed advanced fi brosis. In 2005, leucocyte concentration continually rose above the upper normal limit. The spleen was enlarged to 18 cm. Cytogenetic analysis identifi ed a normal karyotype and BCR-ABL transcripts were still undetectable. Therefore, a JAK2-V617F mutational analysis was done, which highlighted the presence of a heterozygous JAK2-V617F mutation, leading to the diagnosis of idiopathic myelofi brosis (IMF) according to the WHO diagnostic criteria. For JAK2 genotyping, DNA was amplifi ed and PCR products were directly sequenced using the PCR primers JAK2-1F (5’-TGCTGAAAGTAGGAGAAAGTGCAT-3’) and JAK21R (5’-TCCTACAGTGTTTTCAGTTTCAA-3’). The proportion of mutant JAK2-V617F alleles was quantifi ed using pyrosequencing as described. Additionally, the allelic ratio of JAK2-V617F was confi rmed by a newly established quantitative real-time (RQ) PCR assay based on LightCycler technology (Roche Diagnostics, Mannheim, Germany). DNA was extracted from peripheral blood leucocytes after red-cell lysis by standard procedures. Total JAK2 was established using a forward primer, 5’-AGGCTACATCCATCTACCTCAG-3’, a reverse primer, 5’-CCTAGCTGTGATCCTGAAACTG-3’ (MWG Biotech, Ebersberg, Germany), and the hybridisation probes 5’-ACAGGCTTGA CCCATAACACCT GAAATA GAG-3’ and 5’-GAGTGGTACAGGAATCATGAATAATGCCAGTCA-3’ (Tib Molbiol, Berlin, Germany). Mutant JAK2-V617F alleles were quantifi ed using the same forward primer and probes in combination with a mutation-specifi c reverse primer, 5’-TTTTACTTACTCTCGTCTCCACAGAA-3’ (MWG Bio tech, Ebersberg, Germany). The allele copy number was identifi ed using a plasmid standard curve. JAK2-V617F positivity was expressed as the ratio between mutant JAK2-V617F and total JAK2. Dilution experiments showed an assay sensitivity of 1%. Retrospective analysis of frozen samples of peripheralblood leucocytes showed that the JAK2-V617F mutation was already present at initial diagnosis of BCR-ABLpositive CML (fi gure 3). Consistent with the presence of an acquired somatic mutation, the JAK2-V617F mutation was absent in purifi ed CD3+ T lymphocytes (fi gure 3) Lancet Oncol 2007; 8: 658–60