Multiple myeloma (MM) is a terminal differentiated B-cell chronic lymphoproliferative disorder, characterized by the latent accumulation of plasma cells with a low proliferative index and an extended lifespan in bone marrow or extramedullary tissues.1, 2 Studies on neoplastic cells isolated directly from MM patients or on MM-derived cell lines demonstrated that interleukin (IL)-6 is a major growth and survival factor for the initiation of signaling in MM cells in an autocrine or paracrine manner.3 However, some MM cells independent of on extracellular signaling such as IL-6 have been reported, suggesting other mechanisms may be involved in signal transduction pathways as well as pathogenesis in MM cells (Huang et al., Blood. 2000; 96: 3241, abstract).4 Accumulating evidence showed that IL-6 induces intracellular signaling through members of the signal transducers and activators of transcription (STAT) family of proteins by activating the Janus tyrosine kinase (JAK) family of protein tyrosine kinases, which subsequently phosphorylate and activate cytoplasmic STAT proteins. Activated STAT proteins dimerize and translocate to the nucleus, where they bind to specific DNA response elements and induce expression of STAT-regulated genes.2, 3 Previous studies demonstrated that one of the STAT family proteins, STAT3, is being constitutively activated in the majority of MM patients, suggesting constitutively activated JAK-2 kinase activities upon extracellular IL-6 stimulation.3 However, other mechanisms may also be responsible for constitutive JAK2 activation in MM cells, in particular in IL-6-independent MM cells (Huang et al. Blood. 2000; 96: 3241, abstract).4, 5 Recently, an aquired somatic mutation in exon 12 of JAK2 gene has been described in higher frequency of centain myeloproliferative diseases: Philadelphia chromosome negative as polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis.6, 7 More recently this mutation has also been identified in lower frequency in acute myeloid leukemia, myelodysplastic syndrome and in atypical chronic myeloid leukemia (BCR-ABL negative).8 This point mutation results in a substitution of valine for phenylalanine at position 617 (V617F) in the JH2 domain of the gene, and leads to the constitutive tyrosine phosphorylation and cytokine hypersensitivity.6, 7, 8 Neoplastic cells carrying this mutation can be heterozygous or hemizygous, if they have loss of heterozygosity of 9p chromosome by mitotic recombination, where JAK2 is located. The point mutation yields constitutive activation of JAK-2 kinase activity in the presence or absence of extracellular signaling and may result in the pathogenesis of those disorders. Furthermore, previous study showed that V617F JAK2 is a subtle mutation that induces a low gain of function in JAK2. There is increasing evidence that its activity requires the presence of cytokine receptors to induce signaling.5 In this study, we determine whether JAK-2 (Val617Phe) point mutation was present in MM patients by using an allele-specific polymerase chain reaction followed by separation and detection with capillary electrophoresis. The test has a detection sensitivity of up to 200 pg mutated DNA. Bone marrow aspirates with various involvements of MM (range: 10–80% of marrow cellularity) were selected, and all cases have been verified by morphologic examination, immunophenotypic analysis either by flow cytometry or immunohistochemistry. All cases demonstrated to have positive immunoglobulin heavy or κ light gene rearrangements. Negative and positive controls containing homozygous and heterozygous mutations of V617F were included. A total 59 bone marrow specimens from MM patients were tested for JAK2 (Val617Phe) mutation. All the tested specimens displayed a wild-type JAK-2 gene genotype, and none of the MM cells with the specific point mutation was identified (Figure 1). Selected cases were verified by direct DNA sequencing. The results indicated that JAK2 (Val617Phe) mutation is very infrequently, if any, in MM cells, despite the fact that constitutional activation of JAK2 kinase as well as STAT3 phosphorylation is frequently seen in MM patients. The study suggests that JAK2 mutation V617F does not play a significant role in the pathogenesis of MM and that other yet unknown mechanisms may also involve constitutive JAK2 activation, particularly in IL-6-independent MM cells.
Note: During the preparation of this manuscript for submission, we noticed a report of similar study from Dr Fiorini et al.9 Our results are consistent with their results and confirm the conclusion that JAK2 V617F mutation is very infrequent in human MM despite the fact that constitutional activation of JAK2 kinase as well as STAT3 phosphorylation is frequently seen in MM patients. So other mechanisms, different from JAK2 V617F mutation, could explain the pathogenesis of MM.
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Leukemia Research (2009)