靶向深度测序检测循环肿瘤DNA在早期预警肝癌转移复发及疗效监测中的应用

Currently, there is an urgent need for a reliable method that integrates the prediction and surveillance of metastasis and recurrence, and monitoring antitumor treatment response of hepatocellular carcinoma (HCC) in a real-time manner. Temporal and spatial heterogeneities of malignant tumor are proposed to cause this dilemma. As the representative of liquid biopsy, circulating tumor DNA (ctDNA) is the mutant DNA fragment released to blood by tumor cells, which could dynamically and comprehensively reflect the whole genomic landscape of cancer and break through the restriction of tumor heterogeneity. Up to now, few studies on HCC ctDNA have been reported. Our former work found circulating DNA could predict postoperative recurrence of HCC patients, and ctDNA was detectable in HCC patients whereas single mutation rate was very low and the positive rate was even lower by detecting the single-site mutation. This study aims to explore the possibility of detecting ctDNA to overcome heterogeneity of HCC by comparing the mutational profile of HCC tumor tissues with that of ctDNA through whole exome sequencing. Then, ctDNA specific mutation panel will be created to customize a DNA fragment capture chip for detecting ctDNA with targeted deep sequencing at multiple time points from HCC patients undergoing tumor resection or sorafenib therapy, respectively. Subsequently, metastasis and recurrence-associated or drug resistance-associated ctDNA profiles are constructed to search for differentially expressed mutant genes, and the biological functions and molecular mechanisms are further explored in vitro and in vivo. Conclusively, this study focuses on ctDNA to establish the foundation for deciphering the molecular mechanisms involved in HCC metastasis/recurrence and drug resistance, and provide a novel method for the prediction and surveillance of HCC metastasis/recurrence and treatment response.

目前临床缺乏早期预警肝癌转移复发及疗效监测有效手段，肿瘤时间和空间异质性是造成该困局主因。作为“液体活检”代表，循环肿瘤DNA（circulating tumor DNA, ctDNA）是肿瘤释放到外周血的突变DNA，能克服肿瘤异质性局限，而肝癌ctDNA研究罕有报道。我们前期研究发现血浆DNA能预测肝癌复发；肝癌患者血浆存在ctDNA，但单基因突变频率低，单位点突变检测阳性率更低。本课题拟通过全外显子测序比较肝癌患者肿瘤及ctDNA突变信息，探讨ctDNA反映肿瘤异质性的可靠性并绘制肝癌ctDNA特征谱；进一步构建DNA捕获芯片、靶向深度测序检测肝癌切除和索拉菲尼治疗患者不同时间点ctDNA，建立转移复发和耐药相关ctDNA特征谱，探寻转移复发及耐药关键基因，并进行体内外验证。本研究将从ctDNA这个新视点为揭示肝癌转移复发及耐药机制奠定基础，为早期预警肝癌转移复发及疗效监测提供新思路。

肝癌转移复发和靶向治疗耐药是困扰手术疗效的难题。课题组从肿瘤内异质性和循环肿瘤DNA（ctDNA）液体活检入手，取得如下成果：（1）系统研究肝癌的肿瘤内异质性与测序深度、测序样本数量的相关性，提出靶向深度测序是减低肝癌异质性对发现基因变异影响的关键措施。在统一测序深度的情况下，进一步增加检测样本并不会改变异质性程度，表明肝癌的异质性具有“饱和性”，单一肿瘤样本的靶向深度测序发现的肝癌基因组特征可以满足临床需求。cfDNA在靶向深度测序的情况下仅反应约50%的突变位点，因此其临床价值可能作为组织测序的补充、动态监测以及远处转移而无法手术患者的肿瘤基因组突变信息获取。（2）证实ctDNA反映肿瘤异质性、动态监测的临床价值，建立肝癌ctDNA检测的高灵敏度平台，并发现索拉非尼耐药潜在标志物。通过采用数个肝癌相关高频突变位点，利用高灵敏度的ddPCR系统，首次在肝癌患者血浆DNA中检测到突变DNA，且能够反映肝癌的肿瘤内异质性；首次证实了血浆cfDNA完整度对于肝癌转移复发早期预警及动态监测的价值；对比肝癌手术前后ctDNA的动态改变，发现术后ctDNA消失或者突变频率降低与OS和DFS延长显著相关，可作为肝癌动态监测的标志物。此外，通过对索拉非尼靶向治疗的肝癌患者进行动态监测，发现CUL3基因突变可能和肝癌患者索拉非尼耐药相关。（3）构建国际首个肝癌特异性靶向深度测序方案。（4）课题组基于临床实际需求，建立了预测索拉非尼用药敏感性的方法。通过本课题的研究，为肝癌转移复发研究提供了新的思路，为肝癌靶向治疗耐药提供了新的证据，具有一定临床转化价值。