Kategorie: Myeloproliferativní nemoci
Téma: Myeloproliferative neoplasms – Clinical
Číslo abstraktu: P267
Autoři: Niels Pallisgaard; MD Hans Hasselbalch; PharmD Bruno Cassinat, PhD; Dr. Alessandro Pancrazzi; Dr. Lasse Kjaer; Marta Vorland; Frank Dicker; Beatriz Bellosillo Paricio; Thomas Kielsgaard Kristensen, Ph.D.; Morten Andersen; MD Eric Lippert, PhD; MUDr. Jiří Schwarz, CSc.; Dr. Jane Bryon; Dr. Dina Naguib; Josep F. Nomdedéu; Anni Aggerholm ; Marzena Wojtaszewska; MD Hajnalka Andrikovics, PhD.; Maruska Marusic; MD Rosa Ayala, Ph.D.; Elisabeth Oppliger Leibundgut, PharmD; Emma Samuelson; Laurence Lodé; MD Francois Girodon, PhD; Melanie Percy, PhD, FRCPath; MD Sylvie Hermouet, PhD
Analysis for the 1849G>T mutation in JAK2 (encoding JAK2- V617F) is routine in the diagnosis of myeloproliferative neoplasms (MPNs). The quantification of the allelic burden in JAK2-V617F positive patients is increasingly used to monitor treatment response of new targeted therapies as well as in transplanted patients.
Across Europe the quantitative JAK2-V617F analysis is performed using a number of different assays and analysis platforms and calibration is therefore needed in order to standardise the results.
Blood samples from 10 JAK2-V617F positive patients were aliquoted (1 ml) and sent out with overnight courier together with 4 reference DNA samples, a JAK2- V617F real time quantitative PCR (qPCR) reference assay and 4 DNA samples (blood samples were collected after informed consent according to the guidelines of the Danish Regional Science Ethics Committee). The reference DNAs were made from a 80 mL pool of normal donor DNA from blood samples by spiking four 10 fold dilutions of a 650 bp PCR product containing the JAK2-V617F mutation into 20 mL aliquots. The copy number of JAK2- V617F and JAK2 wild type in the 4 DNA pools were determined by qPCR and by digital PCR and the allelic ratios of JAK2-V617F were calculated to 75%, 23%, 2.9% and 0.2%, respectively. The qPCR reference assay (Larsen et al BJH 2007) that is recommended by the ELN for quantitative PCR (Jovanovic et al submitted) was prepared as 10 fold concentrated wild type and V617F mutant primer/probe mixtures. The 4 DNA samples contained either wild type DNA (normal donor DNA), V617F positive DNA (HEL cell line DNA), water (negative control) or normal donor DNA spiked with 1% JAK2-V617F. Two local JAK2-V617F positive DNA samples were also included and these were analysed as both non-diluted and ten fold diluted in order to identify a potential inhibition.
Twenty four laboratories from 13 European countries participated in the study. DNA from the 10 patient blood samples was purified according to local protocols. All samples were run in triplicates (or in duplicates in a few labs) with both the local JAK2-V617F assay and the supplied reference assay (384 qPCR wells per lab). Protocol information, Ct (PCR cycle) values and calculated copy numbers from the local assay were sent to Vejle for analysis (approximately 14,000 data points). Although the reported copy numbers in the 10 patient samples varied between labs the percentage of JAK2-V617F alleles was rather consistent for both the local assay and the reference assay. All labs were able to indentify the 1% JAK2 V617F sample as positive when using the reference assay. However, in several labs the allelic burden of the 1% sample was not significantly different from the normal wild type DNA sample when using the local JAK2 assay indicating limited specificity due to nonspecific amplification
Summary / Conclusion:
In 24 labs across Europe the detection and quantification of the JAK2-V617F mutation was relatively consistent in patient samples with an allelic burden above 1%. For values below 1% the specificity and thereby the sensitivity of the analysis varied between labs and this was related to the JAK2-V617F assay used.
Datum přednesení příspěvku: 14. 6. 2013