本文關(guān)鍵詞：基于全基因組測序的多灶性甲狀腺乳頭狀癌克隆相關(guān)關(guān)系研究　出處：《第二軍醫(yī)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 甲狀腺乳頭狀癌 多灶性 全基因組測序 突變 克隆相關(guān)關(guān)系

【摘要】：研究背景和目的:近年來,包括我國在內(nèi)的多個(gè)國家和地區(qū)流行病數(shù)據(jù)均顯示甲狀腺癌發(fā)病率不斷攀升,為廣大患者以及醫(yī)療體系帶來了嚴(yán)重的負(fù)擔(dān)。甲狀腺癌中最常見的亞型為甲狀腺乳頭狀癌(papillary thyroid carcinoma,PTC),約占80%-85%左右。據(jù)文獻(xiàn)報(bào)導(dǎo)PTC常表現(xiàn)為多灶性,即同一個(gè)甲狀腺內(nèi)可見多個(gè)腫瘤病灶。然而,針對這一常見的臨床現(xiàn)象背后不同病灶之間的克隆相關(guān)關(guān)系目前尚無令人滿意的研究方法。新一代測序技術(shù)的出現(xiàn)和普及將腫瘤發(fā)生發(fā)展、耐藥機(jī)制以及預(yù)后監(jiān)控等各個(gè)方面的研究都推進(jìn)到了前所未有的一個(gè)層面。全基因組測序(whole genome sequencing,WGS)是新一代測序技術(shù)中代表性的一種應(yīng)用。隨著技術(shù)的發(fā)展,完成單個(gè)樣本全基因組測序所需的時(shí)間和成本逐年降低,使其具有用于科研甚至推廣至臨床檢測的可行性。本研究旨在(1)對本大學(xué)附屬第一醫(yī)院甲乳外科近2年來甲狀腺乳頭狀癌患者病歷資料進(jìn)行統(tǒng)計(jì)分析,總結(jié)出多灶發(fā)生率以及多灶性甲狀腺乳頭狀癌(multifocal papillary thyroid carcinoma,m PTC)所伴隨的臨床特點(diǎn);(2)通過收集m PTC病例手術(shù)切除標(biāo)本并分別對不同病灶進(jìn)行全基因組測序,利用生物信息學(xué)分析方法分析點(diǎn)突變、插入缺失突變、拷貝數(shù)變異和結(jié)構(gòu)變異等信息,判斷不同病灶間克隆相關(guān)關(guān)系。研究方法:收集2013年1月至2015年5月期間就診于本大學(xué)附屬第一醫(yī)院甲乳外科PTC患者的臨床信息,對患者年齡、性別、腫瘤病灶數(shù)量、主要病灶最大直徑、病灶位置、合并其他病癥以及轉(zhuǎn)移情況等臨床特征進(jìn)行綜合統(tǒng)計(jì)和分析,以篩選出男、女性患者中m PTC獨(dú)特的臨床特點(diǎn)。經(jīng)患者知情同意后,收集m PTC患者術(shù)前外周血以及手術(shù)切除甲狀腺癌灶及癌旁組織,經(jīng)病理確認(rèn)病灶性質(zhì)后對各癌灶進(jìn)行基因組DNA提取,經(jīng)過建庫和質(zhì)控后利用Hiseq X Ten進(jìn)行測序。在基本掌握WGS數(shù)據(jù)生物信息分析方法的基礎(chǔ)上,從質(zhì)控、過濾、比對、體細(xì)胞突變、驅(qū)動(dòng)突變以及異質(zhì)性和克隆進(jìn)化等方面完成對測序數(shù)據(jù)的分析,從而對病灶間克隆相關(guān)關(guān)系進(jìn)行分析。此外,采用Sanger測序和免疫組化對驅(qū)動(dòng)突變BRAF V600E進(jìn)行確證。研究結(jié)果:第一部分研究中,共統(tǒng)計(jì)分析了2013年1月至2015年5月期間920名PTC患者的臨床信息,其中636名女性患者、284名男性患者,平均年齡為45.69±12.68歲。在所有這920名患者中,323名患者(35.1%)經(jīng)病理確診為多灶患者,即甲狀腺內(nèi)存在至少2個(gè)癌灶。經(jīng)統(tǒng)計(jì)分析發(fā)現(xiàn),男性和女性患者m PTC患者中發(fā)生淋巴結(jié)轉(zhuǎn)移的比例顯著高于單灶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比單灶PTC的主要腫瘤直徑要大(P0.001),此外,女性患者單灶PTC中伴隨非癌結(jié)節(jié)比例要顯著高于m PTC(P0.001)。第二部分研究中,共計(jì)完成對3名m PTC患者(編號(hào)為m PTC_P1-P3)的8個(gè)癌灶以及對應(yīng)3個(gè)生殖系對照進(jìn)行了WGS(腫瘤樣本目標(biāo)測序深度為50X;對照樣本目標(biāo)測序深度為30X),共計(jì)獲得1897 Gbases的測序數(shù)據(jù)。經(jīng)過全基因組范圍克隆級(jí)別單核苷酸變異(single nucleotide variant,SNV)位點(diǎn)和頻率的比較,發(fā)現(xiàn)m PTC_P1和m PTC_P2不同病灶間克隆關(guān)系為克隆獨(dú)立型(即不同癌灶獨(dú)立成瘤),而m PTC_P3不同病灶間克隆關(guān)系為克隆同源型(即不同癌灶是腺內(nèi)轉(zhuǎn)移形成)。WGS、Sanger測序和免疫組化均顯示著8個(gè)癌灶有BRAF V600E突變。對于克隆同源型的m PTC_P3,其中的淋巴結(jié)轉(zhuǎn)移灶與原發(fā)灶相比,不僅共有3個(gè)外顯子區(qū)域突變,而且還攜帶2個(gè)獨(dú)有的外顯子區(qū)域突變。在m PTC_P3的3個(gè)癌灶均可見22號(hào)染色體短臂存在拷貝數(shù)缺失變異。突變特征分析還發(fā)現(xiàn)與AID/APOBEC活性有關(guān)的13號(hào)特征和2號(hào)特征存在于m PTC_P3的2個(gè)病灶中。對克隆突變分發(fā)現(xiàn)5/8個(gè)癌灶中可見瘤內(nèi)異質(zhì)性�？寺⊙莼治霭l(fā)現(xiàn)m PTC_P3的淋巴結(jié)轉(zhuǎn)移的發(fā)生屬于早期事件。研究結(jié)論:(1)PTC發(fā)生多灶的情況較為常見,本研究中920名PTC病例的多灶發(fā)生率為35.1%。(2)m PTC具有獨(dú)特的臨床表現(xiàn)特征,包括比單灶甲狀腺乳頭組癌(s PTC)具有更高的淋巴結(jié)轉(zhuǎn)移發(fā)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的主要腫瘤直徑要大(P0.001),此外,女性患者s PTC中伴隨非癌結(jié)節(jié)比例要顯著高于m PTC(P0.001)。(3)WGS成功解析了本研究中8個(gè)PTC癌灶的克隆相關(guān)關(guān)系,其中5個(gè)為克隆獨(dú)立型,另外3個(gè)為克隆同源型。(4)非外顯子區(qū)域突變信息可為腫瘤克隆相關(guān)關(guān)系的分析提供重要參考信息。(5)WGS不僅在分析克隆相關(guān)關(guān)系方面比傳統(tǒng)分析方法具有更廣的適用性和更高的可靠性,還能提供腫瘤異質(zhì)性、克隆進(jìn)化以及分子遺傳特征等諸多重要信息。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R736.1

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