title: Dissecting Genomic Aberrations in Myeloproliferative Neoplasms by Multiplex-PCR and Next Generation Sequencing.
abstract: In order to assess the feasibility of amplicon-based parallel next generation sequencing (NGS) for the diagnosis of myeloproliferative neoplasms (MPN), we investigated multiplex-PCR of 212 amplicons covering genomic mutational hotspots in 48 cancer-related genes. Samples from 64 patients with MPN and five controls as well as seven (myeloid) cell lines were analyzed. Healthy donor and reactive erythrocytosis samples showed several frequent single-nucleotide polymorphisms (SNPs) but no known pathogenic mutation. Sequencing of the cell lines confirmed the presence of the known mutations. In the patient samples, JAK2 V617F was present in all PV, 4 of 10 ET, and 16 of 19 MF patients. The JAK2 V617F allele burden was different in the three groups (ET, 33+/-22%; PV 48+/-28% and MF 68+/- 29%). Further analysis detected both previously described and undescribed mutations (i.e., G12V NRAS, IDH1 R132H, E255G ABL, and V125G IDH1 mutations). One patient with lymphoid BC/Ph+ ALL who harbored a T315I ABL mutation and was treated with ponatinib was found to have developed a newly acquired V216M TP53 mutation (12% of transcripts) when becoming resistant to ponatinib. Ponatinib led to a decrease of ABL T315I positive transcripts from 47% before ponatinib treatment to 16% at the time of ponatinib resistance in this patient, suggesting that both TP53 and ABL mutations were present in the same clone and that the newly acquired TP53 mutation might have caused ponatinib resistance in this patient. In conclusion, amplicon-sequencing-based NGS allows simultaneous analysis of multiple MPN associated genes for diagnosis and during treatment and measurement of the mutant allele burden.
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University, Aachen, Germany.
authors.forename: Martin M J
authors.initials: MM
authors.lastname: Kirschner
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University, Aachen, Germany.
authors.forename: Mirle
authors.initials: M
authors.lastname: Schemionek
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University, Aachen, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Schubert
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University, Aachen, Germany.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Chatain
authors.affiliation: Institute for Biomedical Engineering-Cell Biology, RWTH Aachen University, Aachen, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Sontag
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University, Aachen, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Isfort
authors.affiliation: Institute for Human Genetics, Medical Faculty, RWTH Aachen University, Aachen, Germany.
authors.forename: Nadina
authors.initials: N
authors.lastname: Ortiz-Brüchle
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University, Aachen, Germany.
authors.forename: Karla
authors.initials: K
authors.lastname: Schmitt
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University, Aachen, Germany.
authors.forename: Luisa
authors.initials: L
authors.lastname: Krüger
authors.affiliation: Institute for Human Genetics, Medical Faculty, RWTH Aachen University, Aachen, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Zerres
authors.affiliation: Institute for Biomedical Engineering-Cell Biology, RWTH Aachen University, Aachen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Zenke
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University, Aachen, Germany.
authors.forename: Tim H
authors.initials: TH
authors.lastname: Brümmendorf
authors.affiliation: Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University, Aachen, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Koschmieder
mesh_terms: D000483:Alleles; D019046:Bone Marrow Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D005500:Follow-Up Studies; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009154:Mutation / Q000235:genetics; D009196:Myeloproliferative Disorders / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011786:Quality Control
chemical_list: D053614:Janus Kinase 2