JAK2 Mutations Are Present in All Cases of Polycythemia Vera.

The JAK2^V617F point mutation has been described in 65 to 97% of patients with polycythemia vera (PV). Previously, 17 phenotypical cases of PV from a large group of patients from three institutions were initially reported as JAK2^V617F negative. These cases were re-analyzed in detail to determine the cause, if any, of the JAK2^V617F negativity. Reasons for this initial negativity included inaccurate clinical diagnosis, relatively insensitive laboratory techniques, insufficient material for re-testing, and possible treatment effect. It was predicted that when tested appropriately, a JAK2^V617F mutation would be found in all new cases of PV. We developed a highly sensitive amplification refractory mutation system assay (ARMS) for the detection of JAK2^V617F. The analytic sensitivity of the assay is 0.05-0.1% as defined by mixing JAK2^V617F-containing HEL cells with normal peripheral blood mononuclear cells. At this level of sensitivity, the assay has a false positive rate of less than 1% (we found 1 positive case in 300 individuals without myeloid disorders). Pyrosequencing involves synthetic nucleotide extension by DNA polymerase. Unlike ARMS, the PCR pyrosequencing method reliably detects JAK2^V617F only when the mutant allele is more than 5%. The sensitivity of the ARMS assay is therefore approximately 50 times greater than the pyrosequencing method. Of 105 PV cases diagnosed by Polycythemia Vera Study Group criteria, pyrosequencing detected the mutation in 96 cases. The ARMS technique detected JAK2^V617F alleles in 8 of the remaining 9 cases. Of the 8, 2 had been treated by phlebotomy only, 4 had been on interferon for a median of 13 years, and 2 on imatinib for 3 years. In the last patient, who was negative by both assays, the JAK2 exon 12 deletion (E543-D544) was detected. This patient had been on imatinib for 3 years and remains in complete remission. We conclude that a sensitive assay is required to detect the JAK2 mutations in patients with polycythemia vera, especially in patients whose JAK2 mutation load might be affected by treatment.