1、论文独创性声明83英文缩略词注释缩略词英文全称中文全称CRCcolorectal cancer结直肠癌CEAcarcinoembryonic antigen癌胚抗原ctDNAcirculating tumor DNA循环肿瘤核酸cfNAcell free nucleic acid无细胞的核酸cfDNAcirculating free DNA循环游离核酸PCRpolymerase chain reaction聚合酶链式反响RT-PCRreverse transcription PCR逆转录PCRdPCRdigital PCR数字PCRARMSamplification refractory mu
2、tation system突变扩增阻滞系统BEAMingbeads，emulsions，amplification and magnetics流式技术的磁珠乳液扩增方法ddPCRdroplet digital PCR数字液滴PCRNGSnext generation sequencing第二代测序技术TAM-Seqtagged-amplicon deep sequending标记扩增深度测序技术CAPP-Seqcancer personalized profiling by deep sequencing肿瘤个体化分析深度测序法EGFRepidermal growth factor recep
3、tor表皮生长因子受体KRASkirsten rat sarcoma viral oncogene鼠类肉瘤病毒癌基因NCCNNational Comprehensive Cancer Network 美国国立综合癌症网络mCRCmetastatic colorectal cancer转移性结直肠癌NRASNeuroblastoma RAS viral oncogene homolog神经母细胞瘤RAS病毒癌基因同源物BRAFv-raf murine sarcoma viral oncogene homolog B1鼠类肉瘤病毒癌基因同源物B1PIK3CAPhosphotylinosital 3
4、 kinase磷酸肌醇3激酶AJCCAmerican Joint Committee On Cancer 美国抗癌联合会TNMtumor；node；metastasis肿瘤分期模型qPCRquantitative real-time PCR实时定量PCRMRDminimal residual disease最小残留病灶RFSrelapse free survival无复发生存期CTCcirculating tumor cells循环肿瘤细胞EMTepithelial-mesenchymal transition上皮间质转变EpCAMepithelial cell adhesion molecu
5、le上皮细胞黏附分子FDAfood and drug Administration食品药品监督管理局PFSprogression-free survival无进展生存期OSoverall survival总生存期DFSdisease-free survival无病生存期PET-CTpositron emission tomography-computed tomography正电子发射计算机断层显像LARClocal advanced rectal cancer局部进展期直肠癌中文研究目的：结直肠癌colorectal cancer，CRC早期、精准诊疗是临床面临的新挑战。传统上，肿瘤诊断是以
6、组织标本提供的病理学为金标准，但在获取组织活检时存在有创操作的风险、无法获取肿瘤组织的困难。而以循环肿瘤DNAcirculating tumor DNA，ctDNA为代表的液体活检给临床诊治带来了新的希望。ctDNA的检测具有易获取、可重复采集、相对无创、敏感度及特异度较高的优点。目前主要的检测途径分为两大类，一类为基于PCR的途径，另一类是基于基因测序的途径。本研究我们通过使用基于PCR和基因测序的ctDNA检测技术，寻找CRC患者外周血ctDNA中KRAS突变规律与临床病理特征的相关性，以及与肿瘤组织中KRAS突变的一致性。进一步了解CRC伴肝转移患者ctDNA基因表达情况及其临床意义。研
7、究方法：1. 用基于qPCR的super-ARMS方法对95例AJCC分期为I-IV期的CRC患者ctDNA进行检测，评价组织突变、外周血ctDNA突变与临床病理特征之间的相关性，并通过评价组织中与ctDNA中KARS基因突变的一致性，来探索ctDNA在CRC患者中的应用价值。2. 通过基于二代测序的方法，对20例结直肠癌伴肝转移的患者的组织标本，术前术后的ctDNA进行检测和分析，探讨组织和ctDNA基因突变的表达情况和相关的临床意义。研究结果：1. 组织KRAS基因突变与临床特征如年龄、性别、肿瘤家族史、是否吸烟、部位、血清CEA、TNM分期等因素无明显相关性。2. 血浆ctDNA的KRA
8、S基因突变和M分期相关，说明有远处转移患者突变率更高。3. ctDNA与组织的KRAS一致性检验提示两者的总体；进一步亚组分析中可以看到，年龄6个月进行分析发现，术前和术后ctDNA突变状态与患者影像学复发无明显相关性。进一步比拟cfDNA和ctDNA含量提示，术后复发与无复发患者其含量在术前、术后检测均无统计学差异。研究结论：1. 在CRC患者中应用super-ARMS法检测ctDNA是简便、可行的。ctDNA检测突变具有相对较高的特异度和阳性预测值。在年龄60岁组，分期相对较晚的亚群中，原发肿瘤组织和ctDNA中KRAS基因突变的一致性尚可接受。对于晚期CRC患者，在选择抗EGFR靶向药物
9、治疗前， ctDNA的检测可用于对KRAS基因突变情况进行初筛，防止不必要的治疗。2. 基于二代测序的ctDNA的分子检测与组织基因检测突变的一致性较高，在检测治疗反响和耐药性方面，具有替代肿瘤组织基因检测的潜在效用。特别是对于无法取活检的肿瘤患者如转移性癌症患者，或者当组织学样本的质量太差无法用于分子检测时。3. 术前、后ctDNA突变状态和ctDNA、cfDNA含量与患者影像学复发无统计学相关性。但术前血、组织与术后血突变状态一致性可能与术后是否出现肿瘤复发的风险有关，其识别复发敏感性可能早于肿瘤指标的动态演变。关键词：结直肠癌，循环肿瘤DNA，循环游离DNA，二代测序AbstractOb
10、jectives:Colorectal cancer (CRC) is the most common malignancy in the digestive system and seriously jeopardizes public health. Early and accurate diagnosis and treatment of CRC are new clinical challenges. Traditionally, tumor diagnosis is based on the pathological criteria provided by tissue speci
11、mens, but there is a risk of invasive procedures in obtaining tissue biopsy and difficulties in obtaining tumor tissue. The liquid biopsy represented by circulating tumor DNA (ctDNA) brings new hope to clinical diagnosis and treatment. The detection of ctDNA has the advantages of easy access, reprod
12、ucible acquisition, relative non-invasiveness, and high sensitivity and specificity. At present, the main detection methods are divided into two types, one is a PCR-based method, and the other is a method based on gene sequencing. In this study, we used PCR-based and gene-sequencing ctDNA detection
13、techniques to look for correlations between KRAS mutations and clinical pathological features in peripheral blood ctDNA of patients with CRC, as well as consistency with KRAS mutations in tumor tissues. Furthermore, we attempeted to validate the ctDNA mutations in CRC patients with liver metastases and their clinical significance.Methods:1. The qPCR-based super-ARMS method was used to detect ctDNA in peripheral blood of 95 c
