Quantification of JAK2V617F mutation by next‐generation sequencing technology
Authors: Emna Abdelhamid, Martin Figeac, Aline Renneville, Sabine Quief, Céline Villenet, Thomas Boyer, Olivier Nibourel, Valerie Coiteux, Bruno Cassinat, Eric Lippert, Nathalie Helevaut, Zohra Soua, Claude Preudhomme
Journal: American Journal of Hematology (2013)

In 2005, different groups discovered a recurrent mutation in JAK2 gene, V617F, in varying proportions of patients with myeloproliferative neoplasms (MPN): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) 1. The aim of the present study was to evaluate the feasibility and the performances of a home-made NGS assay using the Ion Torrent platform to quantify the JAK2V617F mutation in MPN patients. This retrospective study included 4 healthy subjects and 33 adult patients with MPN collected in 3 different French centers and analyzed with Ion Torrent technologies in Lille laboratory. Blood samples from MPN patients were selected because the JAK2 mutation was found to be present with a mutated JAK2 V617F / total JAK2 allelic ratio upper than 0.1%,(using the Ipsogen JAK2 MutaQuant™ kit (Ipsogen, Marseille, France), suitable for showing possible differences in results deriving from the use of the two laboratory techniques. To investigate the linearity and the sensitivity of our NGS assay, we used the JAK2V617F mutated UKE1 cell line that was serially diluted in a JAK2 wild-type control samples (D) collected from a healthy subject. For NGS technologies, a fragment of 81 base pairs (bp) was amplified by PCR from genomic DNA using the following primers: forward primer: 5′-TCTCACAAGCATTTGGTT-3′ in exon 14, and reverse primer: 5′-GGCATTAGAAAGCCTGTA-3′ in intron 14. The sequencing was performed on 314 chips using the PGM (Ion Torrent, Life Technologies) with the 100-bp sequencing kit. Raw flow sequences were analyzed by Torrent Server 2.0 and transformed to raw nucleotide sequences.The data and statistical analysis were based on the 11-bp fragment including the c.1849G>T substitution (GTATGTGTCTG) was considered to establish the number of mutated and wild-type reads. As the mutated allele generates a homopolymer (TTT), one sequencing error in this region was allowed. The probability that JAK2 V617F/JAK2 allelic ratio was positive was computed with Fisher exact test. A P value ≤ 0.05 was considered to indicate statistical significance. First, the four samples collected from healthy subjects with theoretically no JAK2V617F mutation showed three reads with the mutation in a total of 352,476 reads by NGS. The computed allelic ratios from the diluted UKE1 showed a straight linear correlation with theoretical dilution (r = 0.983 Pearson correlation, P value = 1.100 e −08) (Fig. 1D). All diluted samples but one were below the theoretical values. Last but not least, all diluted samples showed positive allelic ratio values with a P value < 2.5−5. In a second step, DNA samples from 33 patients with JAK2V617F-positive MPN were quantified by NGS and the results were compared to those previously obtained with the JAK2 MutaQuant assay. All samples showed linear correlation with NGS results (r = 0.98 Pearson correlation, P value = 0.2 e−07; r = 0.95 Pearson correlation, P value = 17.8 e −07and r = 0.98 Pearson correlation, P value = 0.1 e −07, respectively) (Fig. 1(A,B,C)). The high number of reads provided by our NGS assay allowed a better assessment of the percentage of JAK2V617F mutant allele especially for samples with less than 0.1% of mutated allele. In our study, measurement of JAK2V617F mutation by NGS technology in patients with JAK2V617F-positive MPN allowed encouraging results to use it as a new tool for MRD monitoring and assessment of therapeutic response, especially with the current development of new drugs inhibiting the JAK2 pathway 2 but also MRD assessment based on gene mutations in various hematological malignancies. EA performed the research and wrote the paper, MF interpreted the results and revised the manuscript, CV and SQ performed the research, VC, BC, and EL supplied patient samples data, AR and ON supervised molecular analysis, TB performed the research and submitted the manuscript, AR and CP revised the manuscript, ZS and CP supervised the project. This work was supported by the Northwest Canceropole and the SIRIC ONCOLILLE. Emna Abdelhamid1,2Martin Figeac3,4Aline Renneville2,3,5Sabine Quief4,5Céline Villenet3,4Thomas Boyer2Olivier Nibourel2,3,5Valerie Coiteux2Bruno Cassinat6Eric Lippert7Nathalie Helevaut2,5Zohra Soua1Claude Preudhomme2,3,5 1U.R. “Biologie Moléculaire des leucémies et lymphomes”, Faculté de Médecine de Sousse, Université de Sousse, Tunisia; 2Laboratoire d'Hématologie, Centre de Biologie-Pathologie, CHRU de Lille, Lille, France; 3Université de Lille Nord de France, Lille, France; 4Plate-frome de génomique fonctionnelle et structurale, UDSL, Lille France; 5Inserm, U837, Team 3, Institut de Recherche sur le Cancer de Lille, Lille, France; 6Unité de Biologie Cellulaire, Hoˇpital Saint-Louis, Paris, France; 7Laboratoire d'Hématologie, CHU de Bordeaux, U1035, Université Bordeaux, Bordeaux, France