肿瘤基因突变高通量测序检测游离DNA参考物质的研究

Analysis of tumor-associated genetic mutations via circulating cell-free DNA (cfDNA), which is referred to as “liquid biopsy”, is increasingly valuable in clinical practices. By far, reference materials for circulating tumor DNA (ctDNA) are not yet established due to the lack of positive clinical samples. Therefore simulated samples are now being considered to be alternatives. Ideal cfDNA reference materials are required to mimic the size distribution of cfDNA fragments corresponding to nucleosomes. Specially, they should comprise both normal genomic DNA and edited mutation-containing genomic DNA for better applications in next-generation sequencing (NGS) experiments. In the previous study, we have prepared cfDNA reference materials for non-invasive prenatal testing for the first time, and constructed the NGS reference materials for tumor-specific mutations testing using CRISPR/Cas9 technology and synthetic DNA spike-in materials. This study aims to establish cell-line as well as plasma-derived cfDNA reference materials for tumor-linked genetic alterations by means of CRISPR/Cas9 technology, micrococcal nuclease (MNase) treatment, site-directed mutagenesis, digital PCR and NGS. Established references materials will be quantified and evaluated for their feasibilities in different platforms. Our study will establish standardized processes for preparations of cfDNA reference material, which are believed to greatly promote the standardizations of tumor-specific mutations cfDNA testing in clinical practices.

通过血浆游离DNA进行肿瘤基因突变检测具有重要临床应用价值，也称为“液体活检”。由于临床样本不易获得，目前尚没有肿瘤基因突变游离DNA检测的参考物质, 只能采用模拟样本来建立。模拟样本中含有的片段化DNA应具有血浆游离DNA核小体单体大小为主的片段分布特征，并包括人基因组DNA和以此模板编辑的突变人基因组DNA，以适用于高通量测序（NGS）方法。在前期研究中，我们首次建立了无创产前检测游离DNA参考物质，并采用CRISPR/Cas9编辑细胞系和合成DNA片段建立肿瘤基因突变NGS参考物质。本研究拟在现有基础上，采用CRISPR/Cas9、微球菌核酸酶消化、定点诱变PCR、数字PCR和NGS等技术，分别建立基于细胞系和血浆的肿瘤基因突变游离DNA参考物质，并进行量值测定和不同方法适用性的验证。本研究将为游离DNA参考物质的制备提供平台，推动肿瘤基因突变游离DNA检测工作的标准化和规范化。

通过血浆游离DNA进行肿瘤基因突变检测具有重要临床应用价值，也称为“液体活检”。由于临床样本不易获得，目前尚没有肿瘤基因突变游离DNA检测的参考物质,只能采用模拟样本来建立。模拟样本中含有的片段化DNA应具有血浆游离DNA核小体单体大小为主的片段分布特征，并包括人基因组DNA和以此模板编辑的突变人基因组DNA，以适用于高通量测序方法。本项目采用CRISPR/Cas9结合微球菌核酸酶消化，以及定点诱变PCR 结合超声打断两种技术方案，构建了基于核小体特异切割位点的肿瘤游离DNA（ctDNA）基因突变检测参考物质，建立四种最常见的肿瘤基因突变类型（包括点突变、短片段缺失、插入和融合基因）游离DNA参考物质的制备平台。结果表明基于微球菌核酸酶作用于核小体特异的切割位点的机制，制备的参考物质具有肿瘤游离DNA核小体单体大小为主的片段分布特征，与临床样本生物特征一致，并具有方法广泛适用性。基于该参考物质，开展了我国和澳大利亚两国的肿瘤游离DNA检测室间质量评价。研究成果发表在临床实验室诊断领域最有影响力的Clinical Chemistry杂志，申请国家发明专利，并完成科研成果转化。本研究解决了ctDNA基因突变检测试剂方法建立、性能确认、临床实验室检测室内质量控制、室间质量评价参考物质来源问题，对保证ctDNA实验室检测质量和患者健康具有重要意义。

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