血浆ctDNA多基因检测panel在肝癌早期诊断治疗中的应用研究

HCC is highly invasive, poor prognosis, so early diagnosis and treatment of liver cancer is of particular importance. Currently, the lack of molecular indicator and the difficult to get the live specimen made early diagnosis and treatment of liver cancer poorly. Therefore, it is necessary to identify the tumor markers with high specificity and sensitivity and the detection method with high usefulness for early diagnosis and treatment of liver cancer. In this study, HCV-related HCC patient data were selected and analyzed deeply through the bioinformatics technical, we aimed to identify high frequently mutation genes for multiple-genes test panels, and their exon regions were set as the target sequencing selectors (Onco-panel), based on Onco-panel, cancer personalized profiling by deep sequencing (CAPP-Seq) were performed with normal liver and cancer liver samples respectively, as well as the plasma CtDNAs; After that, personalized high frequency mutation genetic markers and noninvasive detection technology were identified for early diagnosis and treatment of liver cancer, and multi-plexed liquichip assay for liver tumor markers was lastly established. This study will provide new research methods to identify molecular biomarker for noninvasive assessment in liver cancer early diagnosis treatment and monitoring, thereby laying a foundation for early diagnosis and treatment for liver cancer patients and facilitating personalized cancer therapy.

肝癌是一种恶性程度高、进展快、预后差、侵袭性及转移能力极强的肿瘤。目前导致肝癌早期诊断差的因素主要为肝癌早期诊断分子指标的缺失及肝癌患者活体标本的获得。因此，寻找特异、敏感的肿瘤标志物及检测方法是肝癌早期诊断的关键及希望所在。本项目基于已鉴定的乙肝肝癌患者高频突变基因作为多基因检测Panel，拟定制其外显子区段作为靶向测序筛选器（Onco-Selector)对10例肝癌患者肿瘤组织及癌旁正常组织进行个体化深度测序（CAPP-Seq），并结合10例肝癌患者血浆CtDNA个体化深度测序 （CAPP-Seq）分析结果，筛选肝癌患者分子早期诊断相关的高频突变基因及变异，鉴定个体化高频突变基因标志物用于肝癌早期诊断治疗的无创检测技术，研发肝癌早期诊断治疗检测芯片。本研究将为肝癌患者早期诊断治疗及检测提供新的研究方法，为实现肝癌患者精准早期诊断治疗奠定基础。

肝癌是一种恶性程度高、进展快、预后差、侵袭性及转移能力极强的肿瘤。目前导致肝癌早期诊断差的因素主要为肝癌早期诊断分子指标的缺失及肝癌患者活体标本的获得。因此，寻找特异、敏感的肿瘤标志物及检测方法是肝癌早期诊断的关键及希望所在。本项目基于已鉴定的乙肝肝癌患者高频突变基因作为多基因检测Panel，拟定制其外显子区段作为靶向测序筛选器（Onco-Selector)对90例肝癌患者CtDNA样本，并结合10例正常健康人血浆CtDNA进行个体化深度测序（CAPP-Seq），筛选肝癌患者分子早期诊断相关的高频突变基因及变异，鉴定个体化高频突变基因标志物用于肝癌早期诊断治疗的无创检测技术，研发肝癌早期诊断治疗检测芯片。研究结果表明：CtDNA检测panel可以检测出98.89％（90名患者中的89名）患者样本的突变。另外，我们研究发现突变主要存在于10个关键基因中，包括TP53, ACVR2A, MUC16, TBCK, ADGRV1, PCLO, COL11A1, TTN, DNAH9 and GABBR1 等基因。基因互作现象广泛存在于突变基因中，研究发现TTN和TP53，TTN和DNAH9基因的突变在肝癌患者中存在着互作现象。我们进一步发现19个关键点突变位于10个肝癌相关基因(ACVR2A, PCLO, TBCK, ADGRV1, COL1A1, GABBR1, MUC16, MAGEC1, FASLG and JAK1)中，检出率为96.7％（90位患者中的87位）。最重要的是，我们发现这些变异中的大多数由插入（Frame_Shift_Ins）和缺失（Frame_Shift_Del和In_Frame_Del）组成，例如ACVR2A，PCLO和TBCK中的插入变异，这些变异几乎可以检测到95％的患者。我们提出，与错义突变相比，变异的插入和缺失可能在肝癌中起重要的调节作用。本研究将为肝癌患者早期诊断治疗及检测提供新的研究方法，为实现肝癌患者精准早期诊断治疗奠定基础。

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