基于深度测序技术的急性髓细胞白血病（AML）分子分型研究

Acute myeloid leukemia (AML) is a group of heterogeneous diseases with variable causes and clinical outcomes. Molecular diagnosis of patients with AML is pivotal for the classification of disease subtypes, the prediction of disease outcome and the development of personalized therapeutic interventions. Recently, a large number of previously unidentified low-frequency mutations, structural variations and fusion genes have been found in AML patients by whole-genome sequencing. However, the mechanism underlying these newly identified molecular markers and their potential relationship with therapeutic outcome remain unclear and await further investigation. In this study, we will select a large variety of molecular markers including gene mutations, fusion genes, aberrantly expressed genes based on the currently available datasets of whole-genome sequencing and perform a large-scale clinical screening by deep resequencing. We will further investigate the pathogenic mechanisms, cross-correlation and the prognosis relationship among different molecular markers. Through this study, we will establish a comprehensive and objective molecular genetics abnormal platform in Chinese AML patients which will be of great help to optimize the prognostic stratification and establish a practical AML molecular diagnostic system based on clinical significance. This study will also provide a theoretical basis and a preliminary practice for the application of high-throughput molecular diagnosis and the development of personalized targeted therapy.

急性髓系白血病（AML）是一类高度异质性的疾病群，针对个体基因型的分子检测能为区分疾病亚型、提供治疗策略和判断疾病预后提供重要依据。全基因组测序技术从广度上发现了大量以前从未报道的低频突变、结构变异和融合基因，然而这些分子标志与AML发病及治疗机制的关系尚不明了。 本课题拟采用大规模AML样本，应用高通量测序Ion Torrent技术，以全基因组测序鉴别到的候选基因为研究对象，通过覆盖目标基因全范围的深度测序，揭示不同分子标志在疾病发生发展机制中的作用、彼此之间的交叉相关性、与临床预后危险度之间的关系等一系列科学问题。通过本研究将建立全面客观的AML分子遗传学异常谱，优化细化临床预后危险度分层，以期建立有临床指导意义的AML分子分型体系，评价疗效和判断预后，并为高通量分子诊断的临床开发应用与药物靶点研究提供理论依据和初步实践。

在此项研究中，本课题组首先通过筛选公共数据库及文献整理，建立了较全面的AML分子标志检测位点数据库。应用Ion PGM平台和Ion Ampliseq技术，以全基因组测序鉴别到的候选基因为研究对象，自主研发设计覆盖56个AML相关分子突变候选基因的AML深度靶向NGS突变检测panel。通过临床验证，体现出高效、敏感、准确、便捷的临床应用前景。..其次，本课题组应用上述检测panel，开展临床大样本深度测序筛查，完成了中国人群正常核型急性髓细胞白血病（CN-AML）病例的分子遗传学突变图谱分析，阐明中国人群突变分布特点（CEBPAbi突变率高、DNMT3A、NPM1和FLT3-ITD突变率低）；结合临床预后相关性分析，提出CN-AML 中CEBPAmo和WT1突变的独立预后指示意义。结合NCCN指南进行预后分层优化，以FLT3-ITD、TP53、DNMT3A、CEBPAmo及WT1基因突变为高危组分子标志，能显著区分出CN-AML中预后不良的病例，对不同预后患者的精确区分效果优于现有的NCCN国际指南。进一步对CN-AML中CEBPA基因伴随突变模式的分析研究结果显示：CEBPA单端突变比双端突变伴随有更多的不良预后附加突变，可能与CEBPA基因双端突变与单端突变两种突变模式的预后差异相关。这一系列研究建立了全面客观的CN-AML分子遗传学异常谱，实现了优化细化现有AML分子分型体系的目标，具有重要的临床治疗策略指导价值。同时，为AML高通量深度测序分子诊断的临床开发应用了提供理论依据和初步实践。.综合应用全转录组、全外显子测序技术，本课题组还完成了大样本的T-ALL病例的转录组学分析，发现了一批新分子标志（融合基因、基因突变、基因表达聚类等），建立了新型的T-ALL基因表达谱亚型，描绘了全景式的T-ALL分子遗传学图谱，对深入理解成人与儿童T-ALL发病机制内在差异提供了重要线索。.本课题实施以来，已发表SCI论文2篇，中文核心期刊论文1篇，中文核心期刊综述2篇，均已标注课题编号。