Hemizygous/homozygous and heterozygous <i>JAK2</i> mutation detected in plasma of patients with myeloproliferative diseases: correlation with clinical behaviour (2006)

Point mutation of the JAK2 gene has recently been identified in patients with myeloproliferative disease (MPD) including nearly all of those examined who were diagnosed with polycythemia vera (PV), and significant fractions of those diagnosed with idiopathic myelofibrosis (IMF) and essential thrombocythemia (ET) (Baxter et al, 2005; James et al, 2005; Kralovics et al, 2005; Levine et al, 2005). The mutation results in substitution of phenylalanine for valine at amino acid 617 (V617F) (Baxter et al, 2005; James et al, 2005; Kralovics et al, 2005; Levine et al, 2005). The majority of the MPD patients examined were heterozygous for V617F, however, many were hemizygous (one mutant and deleted wild-type gene), or homozygous (two mutant genes). Any residual normal non-malignant cells in the patient samples could confound the designation of patients as heterozygous (Kralovics et al, 2005). Based on our findings, that the plasma from patients with leukaemia was enriched in tumour-specific proteins and nucleic acids (Albitar et al, 2004; Rogers et al, 2004), we rationalised that testing plasma rather than cells from patients with MPD might more effectively detect hemizygous/homozygous V617F JAK2 mutations. In an Institutional Review Board-approved study, we used bi-directional direct sequencing of RNA isolated from peripheral blood plasma to detect the V617F JAK2 mutations. When a peak for the mutant allele (G) was present, and no significant signal for the wild-type nucleotide (T) was detected, patients were considered to be homozygous/hemizygous. Heterozygous samples almost always showed similar intensity peaks. Paired cell and plasma samples from 61 patients with MPD were first compared. Except for one sample, in which V617F mutation was detected in plasma and not in cells, we obtained identical results in detecting the presence or absence of V617F mutation. However, hemizygosity/homozygosity mutations were more evident in plasma sequences, whereas cells of the same patients displayed both mutant and wild-type alleles (Fig 1A). Testing was bi-directional and all duplicates showed identical results. We found no change in results when 25 samples were retested after being kept at room temperature for 72 h. Mixing normal plasma with lysates from the HEL cell line, which contains only the V617F allele (hemizygous/homozygous), showed that the V617F could be detected in a mixture containing as few as five cells in 1 mL of plasma. (A) Detection of the V617F JAK2 mutation in cell and plasma samples. The control sample shows the wild-type codon GTC, encoding valine. The plasma sample shows a substitution of T for G (flanked by black lines), resulting in the codon TTC for phenylalanine. A peak corresponding to G in this position is not evident in the plasma sample. In the cells sample from the same patient, a mixture of peaks for G and T are evident, indicating that a mixture of wild-type and mutant alleles are present in the sample. (B). The survival of patients with myeloproliferative disease who had <20% blasts and were heterozygous (Het, n = 23) was significantly longer than those without mutation. The hemizygous/homozygous (hemi/homozygous; n = 19) for V617F had a survival that was not significantly different from those with wild type. (C) Patients who had myeloproliferative disease not otherwise classified (MPD-NC) and carried the V617F mutation (heterozygous or hemizygous/homozygous; n = 6) had significantly better survival (P = 0·05) than those who were wild-type (n = 17). Testing 86 stored plasma samples from patients with chronic MPD and long-term follow-up showed that 56% (22/39) of IMF patients had the V617F mutation and 41% of the mutated cases were hemizygous/homozygous. In patients with PV, 88% (14/16) had the V617F mutation, of which 50% were hemizygous/homozygous. In ET patients, 25% (2/8) had the mutation and 50% were hemizygous/homozygous. About 26% (6/23) patients with MPD not otherwise classified (MPD-NC) had the V617F mutation of which 33% were hemizygous/homozygous. The V617F mutation was not detected in 31 control plasma samples from normal individuals. When all patients were considered, those hemizygous/homozygous for the mutant JAK2 had significant enlargement of the spleen (P = 0·0001), a greater percentage of monocytes (P = 0·03), a higher white blood cell count (P = 0·001), higher levels of bilirubin (P = 0·02), and were more likely to have a history of prior malignancy. In patients with MPD in the chronic phase (<20% blasts; 84 patients), heterozygous patients for V617F had significantly greater survival than patients with wild type (P = 0·04; Fig 1B), whereas the survival of hemizygous/homozygous patients did not differ significantly from the wild-type group (P = 0·5). In patients with MPD-NC, the presence of the V617F mutation, whether heterozygous, hemizygous/homozygous, was associated with better survival (P = 0·05; Fig 1C). In summary, direct sequencing of plasma samples reliably distinguishes hemizygosity/homozygosity for V617F from heterozygosity. Although the number is small and larger studies are needed, patients with MPD who were heterozygous for V617F as detected using plasma had features of less aggressive MPD disease and should be distinguished from hemizygous/homozygous or unmutated patients.