Dissecting Genomic Aberrations in Myeloproliferative Neoplasms by Multiplex-PCR and Next Generation Sequencing.

Martin M J Kirschner,Susanne Isfort,Nadina Ortiz-Brüchle,Luisa Krüger,Tim H Brümmendorf,Mirle Schemionek,Nicolas Chatain,Claudia Schubert,Stephanie Sontag,Martin Zenke,Klaus Zerres,Karla Schmitt,Steffen Koschmieder,Ken Mills

doi:10.1371/journal.pone.0123476

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.

Highlights

Myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic disorders which arise from genetically altered myeloid stem or progenitor cells
The coverage and sensitivity of amplicon-based sequencing are dependent on the number of samples that are analyzed within the same approach, with both parameters being adversely affected by too few or too many samples used for one analysis
Further to the two heterozygous KIT mutations V560G (51%) and D816V(52%) that are known to be present in the mastocytosis cell line HMC-1.2, we found an acquired TP53 C277F mutation in 16% of sequencing reads, suggesting that TP53 mutations might develop during passages of KIT mutated cells

Summary

Introduction

Myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic disorders which arise from genetically altered myeloid stem or progenitor cells. Sanger sequencing, having dominated the field for decades, is complemented by “ generation sequencing” (NGS). These methods allow for a fast, sensitive, and cost-efficient high-throughput screening of genomic aberrations and their application has already fundamentally improved our understanding of how genetic alterations affect health and disease. Using a whole genome sequencing approach, Klampfl et al and Nagalia et al identified calreticulin (CALR) mutations in the majority of ET and PMF patients that are negative for JAK2 or MPL alterations [3,4]. In systemic mastocytosis (SM), a gain-of-function mutation in the tyrosine kinase receptor (KIT D816V) contributes to cell-autonomous proliferation of atypical mast cells

Methods

Results

Conclusion