基于二代测序的急性髓系白血病突变谱及预后分层研究

发布时间：2019-05-06 07:19

【摘要】：急性髓系白血病(Acute Myeloid Leukemia,AML)是不同的致癌因子之间复杂的相互作用产生的一类遗传异质性疾病,基因组学的研究不但使人们发现了AML的分子发病机制,并得以发现各亚型之间巨大的多样性。近年来,NCCN指南加入了几个常见的基因突变来指导预后(NPM1、CEBPA、FLT3-ITD、TP53、KIT),但仍不足以将占比60%以上的中危核型AML(Intermediate-risk AML,IR-AML)很好地分层,尚需纳入新的基因及突变的组合来进一步分层。此外,突变位点往往不止一个,多种基因突变亦常伴随发生,单一突变及位点已不足以指导临床预后。基于此,传统的一代测序已无法满足需求,亟需新的技术手段来深入AML分子机制的研究及协助多突变联合分析用于临床。本研究我们借助二代测序(Next Generation Sequencing,NGS)平台在220例AML中,研究了恶性血液病相关的111个基因的突变情况,并结合临床分析探讨对表型及预后分层的指导意义。首先,通过测序数据分析过滤及验证,得到中国人特有的AML突变频率及突变谱。然后,从两个方面对AML发生的分子机制及突变与临床的关系进行研究。一方面通过单突变分析不同突变之间,突变与临床表型、疗效及预后的关系,证实了不同生物学功能突变之间的协作和互斥模式,基因型和临床表型(年龄、核型、初诊白细胞计数、免疫表型等)之间的内在既定关系,以及突变对临床疗效(缓解率和生存期)的影响。另一方面通过整合多突变分析,证实NPM1、FLT3-ITD和DNMT3A突变三者通常共发生,且与高龄、初诊高白细胞数、髓单核细胞形态、化疗未缓解及不良预后相关;而NPM1、IDH1/2突变同时伴FLT3-ITD阴性的AML则与较低的初诊白细胞数和特定的免疫表型相关,并对化疗敏感。此外,根据3种不同的突变组合:NPM1或bi-CEBPA突变同时不伴有其他突变,FLT3-ITD、DNMT3A、ASXL1、TET2、TP53或PFH6突变同时不伴有CEBPA突变,及其他基因型,将中危AML进一步分为低、中、高危三个不同的预后组,使真正的中危AML患者减少三分之一。在AML的分子发病机制和生物学特征方面,本研究进行了深入分析,并证实可通过基于NGS的多种突变的不同组合将中危AML进一步预后分层,为将来制定新的危险度分层系统及实现AML的个体化精准治疗奠定基础。
[Abstract]:Acute myeloid leukemia (Acute Myeloid Leukemia,AML) is a class of heterogeneous diseases caused by complex interactions between different carcinogenic factors. Genomics studies have not only led to the discovery of the molecular pathogenesis of AML. Great diversity among subtypes was found. In recent years, several common gene mutations have been added to the NCCN guidelines to guide prognosis (NPM1,CEBPA,FLT3-ITD,TP53,KIT), but still not enough to delaminate the moderately endangered karyotype AML (Intermediate-risk AML,IR-AML), which accounts for more than 60% of the total. A combination of new genes and mutations needs to be incorporated to further delaminate. In addition, more than one mutation site is often associated with multiple gene mutations, and a single mutation or locus is no longer sufficient to guide the clinical prognosis. Therefore, the traditional generation sequencing can no longer meet the demand. It is urgent for new technical means to deepen the study of molecular mechanism of AML and to assist in multi-mutation joint analysis for clinical application. In this study, we used the second generation sequencing (Next Generation Sequencing,NGS) platform to study the mutations of 111 genes associated with malignant hematological diseases in 220 cases of AML, and discussed the guiding significance for phenotypic and prognostic stratification in combination with clinical analysis. Firstly, the Chinese-specific mutation frequency and spectrum of AML were obtained by filtering and validating the sequencing data. Then, the molecular mechanism of AML occurrence and the relationship between mutation and clinic were studied from two aspects. On the one hand, the relationship between mutations and clinical phenotype, efficacy and prognosis was analyzed by single mutation analysis, which confirmed the collaboration and mutual exclusion pattern, genotype and clinical phenotype (age, karyotype, age, karyotype) among different biological function mutations. The relationship between leukocyte count, immunophenotype, etc., as well as the effect of mutation on clinical efficacy (remission rate and survival time). On the other hand, it was confirmed that the mutation of NPM1,FLT3-ITD and DNMT3A were usually co-occurring by integrated mutagenesis analysis, and were associated with advanced age, high white blood cell count, morphology of myelomonocyte, unrelieved chemotherapy and poor prognosis. AML with NPM1,IDH1/2 mutation combined with FLT3-ITD negative was associated with lower number of newly diagnosed leukocytes and specific immunophenotypes and was sensitive to chemotherapy. In addition, according to three different mutation combinations: NPM1 or bi-CEBPA mutation with no other mutation, FLT3-ITD,DNMT3A,ASXL1,TET2,TP53 or PFH6 mutation with no CEBPA mutation, and other genotypes, the medium-risk AML was further classified as low, medium. The high-risk three different pre-treatment groups reduced the number of true moderate-risk AML patients by 1/3. In terms of molecular pathogenesis and biological characteristics of AML, this study has carried out in-depth analysis, and confirmed that the prognosis of intermediate-risk AML can be further stratified by different combinations of NGS-based mutations. It lays a foundation for the development of a new risk stratification system and the realization of individualized and accurate treatment for AML in the future.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.71

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