Concordance of Assays Designed for the Quantitation of JAK2 1849G>T (V617F): A Multi-Centre Study.

Studies of myeloproliferative disorders (MPDs) aiming to evaluate the fraction of the clone bearing the JAK2 1849T mutation occasionally report discordant findings. One reason could be different sensitivity and accuracy of the various assays designed for the detection and quantitation of JAK2 1849G>T. We studied the concordance of 10 published JAK2 1849G>T assays. 29 samples of genomic DNA were distributed to 14 laboratories in France, USA, Australia, Germany, Holland, Italy and Switzerland for blinded assessment of JAK2 1849T levels. DNA was extracted from granulocytes of patients diagnosed with MPD, eosinophilia or secondary polycythemia. The 10 assays tested included 5 TaqMan assays with specificity based on primers (4) or competing probes (1) and allele-specific PCR (AS-PCR) (3), pyrosequencing (1) and FRET/melting curve (1) assays. Standards used for calibration were dilutions of DNA from plasmids (3 sets), cell lines or patient granulocytes. Results were expressed as %1849T allele/total JAK2 (13 centres) or as %1849T allele/control gene (1 centre). One centre had one false negative result; there were no false positive results. PCR equipment did not significantly affect the quantitation of 1849T: after adaptation of the technique, one centre tested one AS-PCR assay on 2 apparatus, 5 other centres tested one TaqMan assay on 3 apparatus; comparable results were obtained in the 6 centres. For 6 assays (10 centres), quantitation in the 26 positive samples, ranging from 1% to 96%, did not differ significantly. Overall variation was 30%; concordance improved with increasing mutational load (18% variation for samples with >8% 1849T). Three TaqMan and 1 AS-PCR assays gave significantly different results, 2 with overall low quantitation. For 3 assays, discordance was explained by an incorrect estimation of 1849T content in the standards. For the 4^th discordant assay, expressing results as % 1849T/control gene, values tended to be higher proportional to the consensus. Interestingly, results were consistent with the presence of >2 copies of JAK2 per cell in 4 samples. The study underlined the importance of using defined standards when analysing JAK2 1849T levels. After adaptation to the equipment and with the use of correct standards, all assays gave comparable quantitation of JAK2 1849T, with a sensitivity <1%. Finally, quantitation of a second gene, in order to detect additional copies of JAK2 (>2/cell), should be considered.