Rapid Molecular Diagnosis Of JAK2V617F Negative MPN By Targeted Deep Sequencing Using The Ion Torrent PGM

Myeloproliferative neoplasms (MPN) are a heterogeneous group of blood disorders characterized by excess production of mature blood cells, increased risk of thrombotic complications and slow progression to myelofibrosis or, less often, leukemia. Activation of the JAK-STAT signaling pathway is a common underlying feature of these diseases and JAK kinase inhibitors are efficacious in the more advanced forms of disease. Most cases of polycythemia vera (PV) and approximately 60% of essential thrombocythemia (ET) and primary myelofibrosis (MF) harbor a point mutation in JAK2 (V617F) which leads to constitutive JAK-STAT signaling and factor independent cell growth. The remaining 40% of cases of MF and ET harbor a broad range of mutations in many genes including those involved in cytokine receptor signaling, other components or the JAK-STAT pathway or epigenetic regulators. This poses a challenge for rapid molecular diagnosis. Also, since ET is essentially a diagnosis of exclusion of reactive causes of thrombocytosis, many cases of chronic ‘ET’ may not be clonal hematological neoplasms but reactive conditions.

We have developed a rapid deep sequencing pipeline to detect mutations in 65 genes which have been implicated in MPN through previous reports of human mutations, mouse models of MPN, or other known components of hematopoietic cytokine receptor signaling. We used 10ng of DNA from blood to amplify and sequence all the exons of these 65 genes using Ampliseq and Ion Torrent PGM. Using 318 PGM chips and 8-fold multiplexing we achieved on average 200 fold coverage of the target exome. The bioinformatics of SNP validation and rapid generation of reports will be presented. From a pilot study of 30 cases referred for molecular diagnosis, we have detected the likely causative mutation in approximately 80% of ET and MF where JAK2 is wild type. Many of these mutations are known to be causative in MPN, including those in MPL, ASXL1, SET2, SH2B3 (LNK), EZH2, CBL, DNMT3A and other genes. We have identified a novel inherited mutation in a family with MPN and validated it in BAF3 factor-independency assays. We have identified further novel mutations in JAK3, EED, DNMT3A, APC and two phosphatases involved in silencing activated JAK-STAT pathway components. The biological significance of these is under investigation and progress will be reported. In many cases we find evidence for clonal evolution involving secondary mutations in epigenetic modifying proteins on top of driver mutations in the JAK-STAT pathway.

In short, targeted exome re-sequencing using Ampliseq and Ion Torrent PGM provides a rapid and relatively cheap method for molecular diagnosis and characterization of most cases of MPN.