中期肝癌突变及病理特征异质性的临床意义研究

发布时间：2018-02-10 05:19

本文关键词： 肝细胞癌中期液态活检循环肿瘤细胞循环肿瘤DNA 肿瘤异质性　出处：《第二军医大学》2016年硕士论文　论文类型：学位论文

【摘要】：肝细胞癌(HCC)是人类常见的致命性恶性肿瘤之一,其瘤Qg异质性明显,五年生存率低,缺乏有效的非手术治疗手段和预后监测手段。中期肝癌(BCLC B期)为肝癌临床分期中异质性极为明显的一类患者群体,其瘤Qg异质性和患者群体在治疗、预后方面的差异极为明显。在多个实体肿瘤中都可发现肿瘤异质性现象,导致肿瘤构成上的复杂、时间上的变化,影响患者的肿瘤分型、治疗效果、耐药改变以及预后情况等。然而,传统的活检取材难以代表肿瘤的整体特征。近年,液态活检的概念被提出,并获得较大发展,已可以实现在循环肿瘤DNA(cfDNA)和循环肿瘤细胞(CTC)层面进行高深度、高精准的测序研究,为液态活检测序分析体现整体肿瘤变异特征打下基础。鉴于中期肝癌的瘤Qg异质性和在治疗、预后方面的明显差异,我们设计研究对这两个方面进行探讨。第一部分:利用ctDNA-NGS技术评估中期肝癌癌内分子异质性的可行性研究目的:通过对瘤体较大的中期肝癌患者的多点组织样品和血液样品进行高深度的特定区域捕获测序,评估肝癌肿瘤组织内部的基因变异异质性,探讨液态活检和传统活检取材在体现肿瘤突变特征方面的表现。方法:选择肿瘤最大直径超过5cm的中期肝癌患者5例,对肿瘤组织的主要肿瘤、周边癌灶等进行多点取材,同时取材血浆和血细胞,利用特定区域捕获测序方法进行高深度的测序。测序检测不同样本的体细胞变异,分析变异在不同样品中的分布,并对变异基因进行相关通路的分析。结果:5例中期肝癌患者中,3例患者循环肿瘤细胞计数为2个,1例为1个,1例未抽取到;5例患者cfDNA样品捕获测序深度平均为5711×,体细胞突变位点的平均等位基因突变频率(MAF)为28.34%(四分位数间距:6.89%-39.49%)。5例患者中,1例未检出体细胞变异,此外4例分别检测出3、4、6和12个体细胞变异,而在此4位患者的12个肿瘤组织样品中,有50%(6/12)的样品不能检测出其患者的全部突变,然而,组织中检测到的所有变异位点,都可在cfDNA中检测到,体细胞变异检出率达100%。体细胞突变在17个基因的外显子区存在分布,皆导致编码氨基酸序列的改变,其中分布有=2个体细胞变异的基因有TP53(3患者中检出4个突变)、APC(2患者中检出2个突变)和AXIN1(2患者中检出2个突变)。结论:较之传统活检的单点组织取材,液体活检取材,特别是循环肿瘤DNA作为检材,更能体现肿瘤的整体突变特征。因此,液体活检技术在评估肿瘤异质性,进行耐药检测和预测预后方面存在极大应用潜力。第二部分:通过Bolondi再分期模型探讨中期肝癌癌间病理异质性对肝切除术后生存的影响目的:探讨巴塞罗那(BCLC)中期肝癌再分期模型对肝切除术后总体生存的预测作用。方法:2008年至2010年东方肝胆外科医院施行肝切除治疗的343例中期肝癌患者。全组患者按Bolondi再分期模型分为四个亚期。末次随访时间为2014年2月,总体生存为终点事件。用Kaplan-Meier生存曲线和Log-rank检验比较各亚期患者间总体生存的差异,Cox风险模型分析总体生存的独立危险因素。结果:全组病人分为B1亚期143例,B2亚期183例,B3亚期12例和B4亚期5例,其术后5年生存率分别为45.5%,30.4%,13.0%和0%,中位生存时间分别为55.1,35.1,14.4和4.7个月(P0.001)。术前白蛋白(ALB)≤35 g/L,血红蛋白≤正常下限,手术切缘肿瘤侵犯,微血管侵犯(MVI)和肿瘤直径5 cm是B1期和B2期患者肝切除术后总体生存的独立风险因素。结论:再分期模型对中期肝癌病人肝切除术后生存具有良好的预测作用。B1和B2亚期肝癌病人肝切除后可获得较好的远期生存。
[Abstract]:Hepatocellular carcinoma (HCC) is one of the most common human fatal malignant tumor, the tumor Qg marked heterogeneity, the five year survival rate is low, the lack of effective means of non-surgical treatment and prognosis monitoring. (BCLC B) - liver cancer liver cancer clinical staging heterogeneity in extremely patient group significantly. The Qg tumor heterogeneity and patients in the treatment group, the difference of prognosis is extremely obvious. In a number of solid tumors are found tumor heterogeneity phenomenon, leading to tumor composition complex, change in time, affect the therapeutic effect in patients with tumor type, drug resistance, change and prognosis. However the traditional, biopsy is difficult to represent the overall characteristics of the tumor. In recent years, the concept of liquid biopsy is put forward, and obtained great development, already can be achieved in circulating tumor DNA (cfDNA) and circulating tumor cells (CTC) level in advanced degrees, sequencing of high precision, Lay the foundation for reflecting the overall variability of tumor biopsy liquid sequencing. In view of the interim liver Qg tumor heterogeneity and in treatment, significant differences in prognosis, we design and study of the two aspects. The first part: the mid-term evaluation of the feasibility study of hepatocellular carcinoma in the molecular heterogeneity to capture sequencing using ctDNA-NGS technique the specific area of tumor larger stage liver cancer patients of multiple tissue samples and blood samples were high in depth, to assess gene mutation heterogeneity within tumor tissue, to investigate the liquid biopsy and biopsy in traditional tumor mutation characteristics of performance. Methods: the patients with hepatocellular carcinoma interim maximum tumor diameter of more than 5 of 5cm the main tumor cases, the tumor tissue, the surrounding tumors were multiple biopsy, and were sacrificed in plasma and blood cells capture sequencing method using a specific area High depth of sequencing. Somatic variation of different sample sequencing, variation analysis in different samples, and analyzed the related pathway on gene mutation. Results: 5 cases of mid HCC patients, 3 cases of circulating tumor cells in patients with count for 2, 1 cases 1, 1 cases were not selected to; 5 cases of patients with cfDNA sample capture sequencing depth averaged 5711 *, somatic mutations of the average allele mutation frequency (MAF) was 28.34% (four percentile interval: 6.89%-39.49%).5 patients, 1 cases were not detected in somatic cell variation, in addition to the 4 cases were detected by 3,4,6 and 12 individual cell variants, and in the 4 patients with 12 tumor tissue samples, 50% (6/12) samples cannot detect all mutations, the patients however, all mutations were detected in the tissue, can be detected in cfDNA, somaclonal variation detection rate of 100%. somatic mutations in 17 A gene exon region distribution, encoding the amino acid sequence of all lead to the change of the distribution of =2 gene TP53 mutation of individual cells (4 mutations were detected in 3 patients (2), APC mutation were detected in 2 patients (2) and AXIN1 mutations were detected in 2 patients). Conclusion: single compared with the traditional tissue biopsy, liquid biopsy, especially circulating tumor DNA as samples, the overall mutation characteristics can reflect the tumor. Therefore, liquid biopsy in evaluation of tumor heterogeneity, for drug resistance detection and prognosis have great application potential. The second part: through Bolondi model to discuss the stage the middle hepatic carcinoma pathological heterogeneity effect on survival after hepatectomy (BCLC) objective: To investigate the Barcelona stage liver cancer restaging model predictive effect on overall survival after hepatectomy. Methods: from 2008 to 2010, Oriental Hospital Department of Hepatobiliary Surgery The implementation of 343 cases of patients with hepatocellular carcinoma interim hepatic resection. All patients according to Bolondi staging model is divided into four sub periods. At the end of the follow-up time is February 2014, the overall survival end point by Kaplan-Meier. The survival curve and Log-rank test to compare each sub period between patients with overall survival difference, Cox risk model analysis of independent risk the factors of overall survival. Results: all patients were divided into B1 sub period 143 cases, B2 183 cases, 12 cases of stage B3 and B4 sub sub period in 5 cases, the postoperative 5 year survival rates were 45.5%, 30.4%, 13% and 0%, the median survival time was 4.7 months and 55.1,35.1,14.4 (P0.001). Preoperative albumin (ALB) less than 35 g/L, less than the lower limit of normal hemoglobin, surgical margin of tumor invasion, vascular invasion (MVI) and tumor diameter of 5 cm is an independent risk factor for the overall survival of patients with liver B1 and B2 after surgery. Conclusion: the model of interim restaging patients with hepatocellular carcinoma Survival after hepatectomy has a good predictive effect for.B1 and B2 subphase liver cancer patients with better long-term survival after hepatectomy.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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