基于全基因组测序的多灶性甲状腺乳头状癌克隆相关关系研究

发布时间：2017-12-28 03:20

本文关键词：基于全基因组测序的多灶性甲状腺乳头状癌克隆相关关系研究　出处：《第二军医大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景和目的:近年来,包括我国在内的多个国家和地区流行病数据均显示甲状腺癌发病率不断攀升,为广大患者以及医疗体系带来了严重的负担。甲状腺癌中最常见的亚型为甲状腺乳头状癌(papillary thyroid carcinoma,PTC),约占80%-85%左右。据文献报导PTC常表现为多灶性,即同一个甲状腺内可见多个肿瘤病灶。然而,针对这一常见的临床现象背后不同病灶之间的克隆相关关系目前尚无令人满意的研究方法。新一代测序技术的出现和普及将肿瘤发生发展、耐药机制以及预后监控等各个方面的研究都推进到了前所未有的一个层面。全基因组测序(whole genome sequencing,WGS)是新一代测序技术中代表性的一种应用。随着技术的发展,完成单个样本全基因组测序所需的时间和成本逐年降低,使其具有用于科研甚至推广至临床检测的可行性。本研究旨在(1)对本大学附属第一医院甲乳外科近2年来甲状腺乳头状癌患者病历资料进行统计分析,总结出多灶发生率以及多灶性甲状腺乳头状癌(multifocal papillary thyroid carcinoma,m PTC)所伴随的临床特点;(2)通过收集m PTC病例手术切除标本并分别对不同病灶进行全基因组测序,利用生物信息学分析方法分析点突变、插入缺失突变、拷贝数变异和结构变异等信息,判断不同病灶间克隆相关关系。研究方法:收集2013年1月至2015年5月期间就诊于本大学附属第一医院甲乳外科PTC患者的临床信息,对患者年龄、性别、肿瘤病灶数量、主要病灶最大直径、病灶位置、合并其他病症以及转移情况等临床特征进行综合统计和分析,以筛选出男、女性患者中m PTC独特的临床特点。经患者知情同意后,收集m PTC患者术前外周血以及手术切除甲状腺癌灶及癌旁组织,经病理确认病灶性质后对各癌灶进行基因组DNA提取,经过建库和质控后利用Hiseq X Ten进行测序。在基本掌握WGS数据生物信息分析方法的基础上,从质控、过滤、比对、体细胞突变、驱动突变以及异质性和克隆进化等方面完成对测序数据的分析,从而对病灶间克隆相关关系进行分析。此外,采用Sanger测序和免疫组化对驱动突变BRAF V600E进行确证。研究结果:第一部分研究中,共统计分析了2013年1月至2015年5月期间920名PTC患者的临床信息,其中636名女性患者、284名男性患者,平均年龄为45.69±12.68岁。在所有这920名患者中,323名患者(35.1%)经病理确诊为多灶患者,即甲状腺内存在至少2个癌灶。经统计分析发现,男性和女性患者m PTC患者中发生淋巴结转移的比例显著高于单灶PTC(男性中47.8%vs.25.8%,P0.001;女性中31.3%vs.22.1%,P=0.01),女性患者中m PTC比单灶PTC的主要肿瘤直径要大(P0.001),此外,女性患者单灶PTC中伴随非癌结节比例要显著高于m PTC(P0.001)。第二部分研究中,共计完成对3名m PTC患者(编号为m PTC_P1-P3)的8个癌灶以及对应3个生殖系对照进行了WGS(肿瘤样本目标测序深度为50X;对照样本目标测序深度为30X),共计获得1897 Gbases的测序数据。经过全基因组范围克隆级别单核苷酸变异(single nucleotide variant,SNV)位点和频率的比较,发现m PTC_P1和m PTC_P2不同病灶间克隆关系为克隆独立型(即不同癌灶独立成瘤),而m PTC_P3不同病灶间克隆关系为克隆同源型(即不同癌灶是腺内转移形成)。WGS、Sanger测序和免疫组化均显示着8个癌灶有BRAF V600E突变。对于克隆同源型的m PTC_P3,其中的淋巴结转移灶与原发灶相比,不仅共有3个外显子区域突变,而且还携带2个独有的外显子区域突变。在m PTC_P3的3个癌灶均可见22号染色体短臂存在拷贝数缺失变异。突变特征分析还发现与AID/APOBEC活性有关的13号特征和2号特征存在于m PTC_P3的2个病灶中。对克隆突变分发现5/8个癌灶中可见瘤内异质性。克隆演化分析发现m PTC_P3的淋巴结转移的发生属于早期事件。研究结论:(1)PTC发生多灶的情况较为常见,本研究中920名PTC病例的多灶发生率为35.1%。(2)m PTC具有独特的临床表现特征,包括比单灶甲状腺乳头组癌(s PTC)具有更高的淋巴结转移发生率(男性中47.8%vs.25.8%,P0.001;女性中31.3%vs.22.1%,P=0.01);女性患者中m PTC比s PTC的主要肿瘤直径要大(P0.001),此外,女性患者s PTC中伴随非癌结节比例要显著高于m PTC(P0.001)。(3)WGS成功解析了本研究中8个PTC癌灶的克隆相关关系,其中5个为克隆独立型,另外3个为克隆同源型。(4)非外显子区域突变信息可为肿瘤克隆相关关系的分析提供重要参考信息。(5)WGS不仅在分析克隆相关关系方面比传统分析方法具有更广的适用性和更高的可靠性,还能提供肿瘤异质性、克隆进化以及分子遗传特征等诸多重要信息。
[Abstract]:Background and purpose: in recent years, epidemiological data in many countries and regions including China show that the incidence of thyroid cancer is increasing, which has brought a serious burden to the majority of patients and the medical system. The most common subtype of thyroid carcinoma is thyroid papillary carcinoma (papillary thyroid carcinoma, PTC), accounting for about 80%-85%. According to the literature, it is reported that PTC is often multifocal, that is, multiple tumor lesions can be seen in the same thyroid. However, there is no satisfactory research method for the clones related to the different lesions behind this common clinical phenomenon. The advent and popularization of next-generation sequencing technology has promoted all aspects of tumor development, drug resistance mechanism and prognosis monitoring to an unprecedented level. Whole genome sequencing (WGS) is one of the representative applications of the new generation sequencing technology. With the development of technology, the time and cost of completing the whole genome sequencing of a single sample are decreasing year by year, making it feasible for scientific research and even extending to clinical detection. The purpose of this study was to (1) of the First Affiliated Hospital of the University of thyroid and breast surgery statistical analysis of patients with papillary thyroid carcinoma medical records in recent 2 years, summarized the incidence of multifocal and multifocal papillary thyroid carcinoma (multifocal papillary thyroid carcinoma, m PTC) with clinical characteristics; (2) through the collection m PTC cases of resected specimens were performed whole genome sequencing of different lesions, using bioinformatics analysis of point mutation, insertion and deletion, copy number variation and variation information, determine the relationship between different lesions humancloning. Methods: from January 2013 to May 2015 during the visit to the University of the First Affiliated Hospital of the clinical information of thyroid and breast surgery in patients with PTC, patients' age, gender and tumor number of lesions, mainly including maximal diameter, location, and other clinical features of disease and metastasis of the comprehensive statistics and analysis, to screen out the male and female patients m PTC unique clinical characteristics. After informed consent of patients, we collected peripheral blood from patients with m PTC before operation and surgical resection of thyroid cancer and adjacent tissues. After pathological confirmation of the nature of the lesions, genomic DNA was extracted from all the carcinomas. After construction and quality control, Hiseq X Ten was sequenced. On the basis of basic understanding of WGS data and bioinformatics analysis methods, we completed the analysis of sequencing data from quality control, filtration, comparison, somatic mutation, drive mutation, heterogeneity and clonal evolution, so as to analyze the correlation between two clones. In addition, Sanger sequencing and immunohistochemistry were used to confirm the driving mutation of BRAF V600E. Results: in the first part, the clinical information of 920 PTC patients from January 2013 to May 2015 was analyzed, including 636 female patients and 284 male patients, with an average age of 45.69 + 12.68 years. Of all 920 patients, 323 patients (35.1%) were pathologically diagnosed with multifocal patients, that is, there were at least 2 cancer foci in the thyroid gland. The statistical analysis showed that PTC was significantly higher than that of single foci of lymph node metastasis ratio of m in male and female PTC patients (male 47.8%vs.25.8%, female P0.001; 31.3%vs.22.1%, P=0.01, m) in female patients with PTC is larger than the main tumor foci of PTC single diameter (P0.001), in addition, female patients with single lesion PTC along with the proportion of non cancerous nodules was significantly higher than that of M PTC (P0.001). In the second part, a total of 3 cancer patients with m PTC (m PTC_P1-P3) and 8 corresponding 3 reproductive systems were completed. WGS (the depth of tumor target sequencing was 50X, the target depth of control sample was 30X), and 1897 Gbases sequencing data were obtained. After the complete genome clone of single nucleotide variants (single nucleotide level variant, SNV) and frequency comparison sites, m PTC_P1 and m PTC_P2 found that different lesions between clone to clone independent type (i.e. different cancers, and independent tumor) m PTC_P3 between different lesions clone for homologous cloning (i.e. different tumor type is the gland metastasis formation). WGS, Sanger sequencing and immunohistochemical staining showed that 8 cancer foci were BRAF V600E mutations. For the cloned m PTC_P3, the lymph node metastasis has 3 exon region mutations and 2 unique exon region mutations compared with the primary one. There was a copy number deletion mutation in the short arm of chromosome 22 in all 3 cancers of M PTC_P3. The analysis of mutation characteristics also found that No. 13 and 2 characteristics associated with AID/APOBEC activity were found in 2 lesions of M PTC_P3. The intratumoral heterogeneity was found in 5/8 cancer foci on the cloned mutation. Cloning and evolution analysis found that the occurrence of lymph node metastases of M PTC_P3 is an early event. Conclusions: (1) the incidence of multifocal in PTC is more common. In this study, the incidence of multifocal in 920 cases of PTC was 35.1%. (2) m PTC has unique clinical features, including papillary thyroid carcinoma than single lesion group (s PTC) has a higher incidence of lymph node metastasis (male 47.8%vs.25.8%, female P0.001; 31.3%vs.22.1%, P=0.01; m) in female patients with PTC than the main tumor diameter to s PTC (P0.001 in addition, s), female patients in PTC with non cancer nodules ratio was significantly higher than that of M PTC (P0.001). (3) WGS successfully analyzed the clone correlation of 8 PTC carcinomas in this study, of which 5 were clone independent, and the other 3 were cloned homologous. (4) the mutation information of the non exon region can provide important reference information for the analysis of the correlation of tumor cloning. (5) WGS not only has wider applicability and higher reliability than traditional methods in the analysis of clonal correlation, but also provides many important letters such as tumor heterogeneity, clonal evolution and molecular genetic characteristics.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R736.1

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