316, P316


Niels Pallisgaard1,*, Julia Asp2, Hans Karl Hasselbalch3, Bruno Cassinat4, Alessandro Pancrazzi5, Lasse Kjær3, Marta Vorland6, Frank Dicker7, Beatriz Bellosillo8, Thomas Kielsgaard Kristensen9, Morten Tolstrup Andersen10, Eric Lippert11, Jiri Schwarz12, Jane Bryon13, Dina Naguib14, Josep Nomdedéu15, Anni Aggerholm16, Krzysztof Lewandowski17, Hajnalka Andrikovics18, Maruska Marusic19, Rosa Ayala20, Elisabeth Oppliger Leibundgut21, Emma Samuelson2, Laurence Lodé22, François Girodon23, Melanie Percy24, Sylvie Hermouet25

1Clinical Biochemistry, Vejle Hospital, Vejle, Denmark, 2Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden, 3Hematology, Roskilde Hospital, Roskilde, Denmark, 4Service de Biologie Cellulaire, Hôpital Saint-Louis, Paris, France, 5Sezione di Ematologia, Università degli Studi, Firenze, Italy, 6Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway, 7MLL Münchner Leukämielabor GmbH, Munich, Germany, 8Laboratori de Biologia Molecular, Hospital del Mar, Barcelona, Spain, 9Pathology, Odense University Hospital, Odense, 10Clinical Genetics, Rigshospitalet, Copenhagen, Denmark, 11Laboratoire Hématopoïèse Leucémique, Bordeaux University Hospital, Bordeaux, France, 12Institute of Hematology & Blood Transfusion, Prague, Czech Republic, 13Genetics Laboratory, Birmingham Womens Hospital, Birmingham, United Kingdom, 14Laboratoire d'Hématologie, CHU Côte de Nacre, Caen, France, 15Hospital de Sant Pau, Barcelona, Spain, 16Hæmodiagnostisk Laboratorium, Aarhus University Hospital, Aarhus, Denmark, 17Medical University in Poznan, Poznan, Poland, 18National Blood Transfusion Service, Budapest, Hungary, 19Molecular Diagnostics and Genetics, Dubrava University Hospital, Zagreb, Croatia, 20Hopital 12 de Octubre Madrid, Madrid, Spain, 21Hematology, Inselspital, University Hospital Bern, Bern, Switzerland, 22Laboratoire d'Hématologie, CHU Nantes , Nantes, 23Laboratoire d'Hématologie, CHU Dijon, Dijon, France, 24Belfast City Hospital, Belfast, United Kingdom, 25University of Nantes, Nantes, France

Introduction: Analysis for the 1849G>T mutation leading to the V617F substitution in JAK2 is routine in the diagnosis of myeloproliferative neoplasms. 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.

Objectives: The objective was to standardize quantification of JAK2-V617F across Europe.

Methods: Blood samples from JAK2-V617F positive patients were collected after informed consent according to the guidelines of the Danish Regional Science Ethics Committee. Ten blood samples, DNA controls, reference DNA and primer/probe mixes for the wild type and V617F mutant Larsen reference assay that is recommended by the ELN for quantitative PCR were distributed to 23 European laboratories with overnight courier. Copy numbers and the allelic ratios of JAK2-V617F and JAK2 wild type was determined by the participating laboratories using both the reference and in-house assay. Protocol information and results were sent for analysis in Vejle and Gothenburg.

Results: Twenty three laboratories from 13 European countries participated in the study. Results were compared both for the Larsen reference assay and for the in-house assays. In addition, for the ten patient samples the results were compared to allelic ratios determined by digital PCR.

Although the reported copy numbers in the samples varied between laboratories the % JAK2-V617F alleles was rather consistent. The Larsen reference assay showed a good overall agreement. Also in-house assays compared well, particularly for samples with low % JAK2-V617F. To monitor performance in coming QC trials, a scoring system is being adopted from the data in the study.

Conclusion: In 23 laboratories the detection and quantification of the JAK2-V617F mutation was relatively consistent in patient samples with an allelic burden above 1%. This was particularly seen when calibrating to a common reference material.

Keywords: Biomarker, Cancer, DNA