Quantitative measurement of JAK2 V617F mutation in myeloproliferative disorders. Validation and qualification of a new RQPCR diagnostic assay.

A4 JAK2 V617F is a mutation found in the majority of patients with polycythemia vera (PV), and a significant number of patients with essential thrombocytopemia (ET) and chronic idiopathic myelofibrosis (CIMF).The discovery of this mutation has profoundly modified the diagnosis of Ph- MyeloProliferative Disorders (MPD), but its clinical significance still remains undefined.Using different technologies, characterized by various sensitivity and sensibility, it has been reported that the incidence of this mutation is ranging from 65% to 97% in PV, 25 to 57% in ET and 35 to 57% in CIMF. Those variations are mainly due to the heterogeneity and to the poor sensitivity of the assay used, highlighting the needs of a standardized, accurate and sensitive assay. The aim of this study was to validate the performances of a new allele specific RQPCR genotyping assay and to compare with the traditional PCR sequencing strategy.This assay benefit from the plasmid technology which allows the precise calibration and normalisation of RQ-PCR results and is highly reproducible. By generating standard curves based on the known concentration of plasmid dilutions, this technology allows precise measurement of the JAK2 wild type (WT) and V617F alleles copy number in human cell samples and, without the need of subsequent handling such as capillary electrophoresis or melting curve analysis, directly provides a normalized value which is independent of the cell count in each assayed sample. Normalized results can be compared between RQ-PCR systems and testing sites.For accuracy testing and determination of detection limit we used serial dilutions of V617F homozygous HEL cell line and genomic DNA mix into non mutated K562 cell or genomic DNA. Our data show a reproducible baseline threshold of 512 V617F homozygous cells in 5 10 -6 WT cells (10 -4 ) and 6.4 pg of positive genomic DNA (2.5 10 -4 , 48 copies) with very good correlations toward serial dilution in each case (R² = 0.99).The greater sensitivity of this assay allowed identifying 17% more mutated patients as compared with PCR sequencing. Healthy subjects and secondary polyglobuly patient samples always resulted WT (>99.9%). In patient samples VF allele range from 5 to 96% in positive cases and is always inferior to 0.3% in negative. No significant differences were observed in JAK2 V617F % range between the different diagnostic groups, but our results show differences in median value ranging from 80% for PV to 25% for ET and IMF. Results obtained on shared samples (n=32) in two different labs show a very high correlation (R²=0.98).The sensitivity and the dynamic range of the JAK2 V617F assay are compatible with its use for highly precise and sensitive quantification at diagnosis and during MRD follow-up. Validation of an equivalent assay for the quantification of the mutation on RNA is ongoing.