Detection of the activating JAK2 V617F mutation in paraffin-embedded trephine bone marrow biopsies of patients with chronic myeloproliferative diseases

Abstract

The discovery of the activating V617F mutation in the JAK2 tyrosine kinase in a high proportion of patients with Ph- chronic myeloproliferative diseases (CMPD) represents a diagnostic breakthrough for these disorders. Trephine bone marrow biopsy is an essential part of the diagnostic workup of CMPD and represents a valuable archival source of DNA. Therefore, we studied 152 paraffin-embedded trephines with CMPD and related disorders for the presence of the V617F mutation, using both allele-specific polymerase chain reaction (PCR) and nested PCR with subsequent digestion with BsaXI. Only 6 of 152 (4%) samples were not evaluable because of poor DNA quality. The V617F mutation was detected in 27 of 28 (96%) cases of polycythemia vera, 17 of 23 (74%) cases of essential thrombocythemia, 28 of 45 (62%) cases of chronic idiopathic myelofibrosis, six of eight (75%) cases of CMPD unclassified, and two of four (50%) cases of myelodysplastic/myeloproliferative syndrome. Ph+ chronic myelogenous leukemia (four cases), reactive (secondary) erythrocytosis (14 cases), and thrombocytosis (one case) as well as normal controls (19 cases) all lacked the V617F mutation. Based on results of BsaXI digestion and sequencing, 24 of 54 (44%) evaluable V617F+ cases were considered homozygously mutated. Thus, detection of the V617F JAK2 mutation is feasible in paraffin-embedded trephine biopsies and represents a major advance in the diagnostic evaluation of CMPD.

Figure 1

Detection of the JAK2 V617F mutation in paraffin-embedded BM trephine biopsies. A: Allele-specific multiplex PCR. The upper band of 364 bp confirms the presence of amplifiable DNA. A second band of 203 bp of varying intensity is clearly visible in cases of Ph− CMPD showing the mutation. Lanes 1, 5, and 9: PV. Lanes 2 and 3:Reactive erythrocytosis. Lanes 4, 6, 8, and 10:CIMF. Lane 7:ET. C indicates the negative control with DNA derived from a normal donor, N is a no template control. B: The same 10 cases analyzed by nested PCR of JAK2 exon 12 with subsequent digestion with BsaXI. Cases 2, 3, and 6 negative for V617F by allele-specific PCR show a lack of undigested PCR product, confirming the absence of the mutation. The ratio of the undigested upper band versus the digested fragments gives an estimate for the relative abundance of mutated DNA in the template.

Figure 2

Comparison of BsaXI digestion (A) with sequencing chromatograms (B) derived from the same PCR products. Left lane and top chromatogram:CIMF without evidence of the mutation and normal wild-type DNA sequence. Middle lane:Heterozygous case of ET with predominance of digested PCR product and predominance of the wild-type allele (G>T) in sequencing. Right lane and bottom chromatogram:Homozygous case of PV with a majority of undigested PCR product and almost complete dominance of the mutated allele in the chromatogram.