Development and Validation of New Molecular Diagnostic Assays for the Jak2 V617F Screening and Quantification. O. Biglia¹, J.P. LeCouedic², S. Hermouet³, F. Hermitte¹, N. Maroc¹ 1) Ipsogen, Marseille, France; 2) INSERM U790-Institut Gustave Roussy, Villejuif, France; 3) Laboratoire d'Hématologie & INSERM U601, CHU Nantes, France.

   JAK2 V617F is an acquired mutation found in > 95% of patients with polycythemia vera (PV), and in > 50% of patients with essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF). The discovery of this mutation has profoundly modified the diagnosis of Ph- (BCR-ABL negative) Myeloproliferative Disorders (MPD). Using different technologies, often multi step and time consuming, variable incidences of this mutation in each pathology subtypes have been reported. Those variations are mainly due to a great heterogeneity in the technologies used and sometimes to their poor sensitivity, highlighting the needs for a simple, standardized, accurate and sensitive assay. We developed two assays: Jak2 MutaScreen assay, based on TaqMan allelic discrimination, is dedicated to Jak2 mutation screening on DNA samples at the time of diagnosis, while Jak2 MutaQuant assay is an allele specific RQ-PCR and is dedicated to mutation load quantification on follow-up samples. We performed a multi centre performance evaluation of our Jak2 MutaScreen genotyping assay. Multi centre study in 14 labs on 7 different apparatus (ABI-prism, LightCycler, iCycler) on 296 MPD samples demonstrated 98.65% correlation with technologies used at diagnosis. Parallel internal validation on 142 samples allowed identifying 13% more mutated cases than direct sequencing method. We will also present analytical validation of both assays, inter and intra-laboratory reproducibility, and technology performance comparison (sequencing versus Jak2 MutaScreen, Jak2 MutaScreen + Reference Scale versus Jak2 MutaQuant). Sensitivity, dynamic range and inter platform capability of Jak2 MutaScreen assay are compatible with a wide use for highly accurate and sensitive detection of JAK2 V617F mutation at diagnosis. Jak2 MutaQuant provides a tool for the monitoring of minimal residual disease in clinical research studies; clinical utility of this test will have to be addressed in multicentric prospective clinical trials.