The Val617Phe mutation in JAK2 (Janus kinase) has been reported in approximately 90% of patients with polycythemia vera (PV) and in almost 40% of patients with essential thrombocythemia (ET) or idiopathic myelofibrosis (IMF). JAK2 is a cytoplasmic tyrosine kinase that binds to growth factor receptors and phosphorylates several downstream proteins that are involved in cell proliferation and growth. The Val617Phe mutation can be detected in circulating granulocytes. Homozygous as well as hemizygous (deletion of 1 allele and mutation in the second) mutations have been reported. The clinical importance of hemi/homozygous as compared to heterozygous mutation is not known. Furthermore, the presence of normal lymphoid cells or non-neoplastic cells in samples makes it difficult to be certain whether the mutation is homozygous in a subpopulation of cells, or heterozygous. Since we have shown that the plasma of patients with leukemia is enriched with leukemia-specific DNA, RNA, and protein, we tested the possibility of detecting JAK2 mutations in patients with chronic myeloproliferative diseases using RNA from peripheral blood (PB) plasma rather than cells. In this study, PB plasma was obtained from 39 patients with IMF, 16 with PV, 8 with ET, and 23 with other chronic myeloproliferative disease not otherwise classified (MPD-NC). Bi-directional direct sequencing of RNA extracted from plasma was used to detect mutations in the JAK2 transcript. The Val617Phe mutation was detected in 88% of PV patients, of whom 50% (44% of all samples) were hemi/homozygous. This mutation was detected in 56% of IMF patients, 41% of whom were hemi/homozygous. The number of patients with ET was small, but 25% (2 patients) had hemi/homozygous mutation. 26% of the patients with MPD-NC had JAK2 mutation; of these, 33% had hemi/homozygous mutation. The plasma of 31 normal control individuals showed no evidence of mutation and mixing studies showed that the Val617Phe mutation was easily detectable when lysate from HEL cell line (homozygous for the mutation) were mixed with plasma down to 5 cell/1ml plasma. Patients with hemi/homozygous mutation had significantly larger spleens (P=0.0001), increased white cell counts (P=0.001), and increased monocyte counts in bone marrow (P=0.03). We cannot demonstrate statistically significant difference in survival between hemi/homozygous and heterozygous, however, the number of cases is too small. The presence of mutation (hemi/homozygous or heterozygous) was associated with better overall survival in patients <65 years of age irrespective of the type of MPD (n=54, P=0.05). Patients with MPD-NC had more aggressive disease than other patients (ET, PV and IMF) (P=0.02), but presence of the Val617Phe mutation was associated with longer survival in the MPD-NC group (P=0.05). This does not only confirm that PB plasma is a reliable source for detecting JAK2 mutations, but also show that this mutation is often hemi/homozygous. The presence of JAK2 mutation as detected in plasma of patients with various types of chronic myeloproliferative diseases is associated with longer survival in patients <65 years and is also a favorable prognostic factor in patients with MPD-NC.

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