转移性乳腺癌患者循环肿瘤DNA个体化检测新技术的建立及临床应用研究

The management of metastatic breast cancer requires monitoring of the tumor burden to determine the response to treatment. Following this process in patients is challenging, as repeated biopsies of tumor tissue involves invasive procedures and tumor heterogeneity confounds interpretation of analyses. Fortunately, rapid developments in the technology and protocols required for analyzing circulating DNA in peripheral blood are making it easier to obtain the necessary samples without the need for physical biopsies. Circulating DNA, representing a liquid biopsy, have become increasingly recognized as potential biomarkers for the clinical monitoring of various human cancers. The general clinical value of circulating total cell-free DNA, however, is controversial because non-specific elevations are seen in patients with benign diseases. Recent studies have shown that circulating tumor DNA (ctDNA) represents a more informative, inherently tumor specific, and highly sensitive biomarker of metastatic breast cancer burden than circulating total DNA. Thus, a sensitive and accurate method for reliably isolating and analyzing ctDNA in patients with metastatic disease is especially necessary. Here we start with identifying ctDNA genomic alternations using whole-genome sequencing technology and designing personalized assays, which then goes through carrying out a sequence specific DNA capture technology and a BEAMing method to isolate specific ctDNA targets and measure the ctDNA incorporating identified specific genomic alterations in plasma. It will be a feasible, accurate and sensitive method for relatively non-invasive detection of the change of levels and mutation status of ctDNA. This study will soon prove valuable as a diagnostic technique for making informed treatment decisions, as well as for tracking the occurrence of resistance mutations during and after treatment, so that medication can be adjusted accordingly to maximize therapy effectiveness.

构建高特异高灵敏高通量的循环肿瘤DNA(Circulating Tumor DNA, ctDNA)分离检测新技术是解决当前国际上通过监测肿瘤负荷以确定转移性乳腺癌治疗反应难点的关键环节。本项目针对循环总DNA与肿瘤负荷变化相关性弱影响因素多的缺陷，利用ctDNA与肿瘤负荷变化相关性强特异性高的优点，基于国际上最前沿的利用分子标记来分离ctDNA的原理及课题组前期科研成果，采用高通量测序技术对ctDNA特异性分子标记进行个体化识别，进而通过基因组靶序列捕获技术和BEAMing技术进行ctDNA的个体化分离和定量检测，建立一个具有国际水平、自主创新的癌症患者ctDNA个体化检测新技术。本研究可解决目前ctDNA分离检测的关键技术问题，不仅能为今后的ctDNA个体化检测服务产业化提供关键参数；并且可通过对治疗压力选择下ctDNA遗传状态和浓度水平变化的连续监测，为更精准的个体化治疗提供科学依据。

背景：构建高特异高灵敏高通量的循环肿瘤DNA(ctDNA)分离检测新技术是解决当前国际上通过监测肿瘤负荷以确定转移性乳腺癌治疗反应难点的关键环节。.研究内容：本项目以乳腺癌为案例，首先，基于癌症基因组图谱库（TCGA）和肿瘤体细胞突变目录（COSMIC）数据库，采用生物信息学和数据挖掘技术筛选出与乳腺癌高度相关的目标基因区域，并组合成“靶向区域（Selector）”，为后续的靶向捕获测序提供靶标，减少盲目性，节约实验成本，使检测技术的针对性更强，适用性更广；其次，以转移性乳腺癌患者为研究对象，基于Digital PCR技术平台定量检测外周血游离DNA（cfDNA）水平的变化规律，评估血浆cfDNA在转移性乳腺癌患者病情监测、疗效评价、预后转归预测中的应用价值；最后，在第一部分和第二部分研究成果的基础上，建立了一种超灵敏高覆盖的ctDNA定量检测技术，并系统评价了治疗前后ctDNA与癌抗原CA 15-3及cfDNA在转移性乳腺癌患者随访监测、治疗效果评价、预后转归评价中应用价值的优劣。.重要结果：（1）“Selector”共计包含了834个乳腺癌相关基因，961个外显子，23982049个单核苷酸变异(SNVs)，134个Indels，52个Fusions，长度为118.24kb，遍布在所有编号染色体上。“Selector”在训练阶段可以覆盖100%的乳腺癌患者，预测集验证阶段亦可以覆盖高达88.7%的乳腺癌患者，平均识别每位患者2个及以上SNVs。40例独立转移性乳腺癌患者不同治疗阶段的72份生物标本验证结果显示，该区域可以覆盖100%的患者和生物标本。（2）ctDNA在转移性乳腺癌患者的检测灵敏度优于CA15-3和cfDNA异常升高检出率，并且与治疗过程中肿瘤负荷的动态变化高度相关。（3） 转移性乳腺癌患者首程化疗后血浆中ctDNA水平检测对患者短期疗效转归具有良好的预测价值。（4）与治疗前相比首程化疗后血浆ctDNA升高者和首程化疗后ctDNA高于183GE/mL者疾病在短期内进展的风险显著升高。（5）与cfDNA相比，ctDNA的动态变化能够更灵敏地反映转移性乳腺癌患者病情的进展和缓解。.科学意义：建立了一种对转移性乳腺癌患者血浆ctDNA高覆盖超灵敏的定量的新技术，为转移性乳腺癌患者治疗过程中的疗效监测提供了一种可行的“液体活检”方案。