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

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.

为评估基于扩增子的平行二代测序（next generation sequencing, NGS）用于诊断骨髓增殖性肿瘤（myeloproliferative neoplasms, MPN）的可行性，本研究针对48个癌症相关基因的基因组突变热点区域设计了212个扩增子，并开展多重聚合酶链式反应（multiplex-PCR）分析。本研究共纳入64例MPN患者、5例对照样本以及7株髓系细胞系作为研究对象。健康供者及反应性红细胞增多症样本中检出多种常见单核苷酸多态性（single-nucleotide polymorphisms, SNPs），但未发现已知致病性突变。对细胞系的测序结果验证了已知突变的存在。在患者样本中，JAK2 V617F突变检出于所有真性红细胞增多症（polycythemia vera, PV）患者、10例原发性血小板增多症（essential thrombocythemia, ET）患者中的4例，以及19例骨髓纤维化（myelofibrosis, MF）患者中的16例。三组患者的JAK2 V617F等位基因负荷存在显著差异：ET组为33%±22%，PV组为48%±28%，MF组为68%±29%。进一步分析还检出了既往已报道及未报道的突变，包括G12V NRAS、IDH1 R132H、E255G ABL及V125G IDH1突变。1例携带T315I ABL突变的淋巴母细胞危象/费城染色体阳性急性淋巴细胞白血病（lymphoid BC/Ph+ ALL）患者，在接受普纳替尼（ponatinib）治疗后出现耐药，此时检出新发的V216M TP53突变（占转录本的12%）。该患者中，普纳替尼治疗前ABL T315I阳性转录本占比为47%，至耐药时降至16%，这提示TP53与ABL突变共存于同一克隆，且新发TP53突变可能导致了该患者对普纳替尼的耐药。综上，基于扩增子测序的NGS可同时分析多种MPN相关基因，用于诊断、治疗监测及突变等位基因负荷的定量检测。