甲状腺乳头状癌淋巴结转移灶的全外显子组测序研究

发布时间：2019-06-24 12:32

【摘要】：背景甲状腺乳头状癌是十分常见的甲状腺恶性肿瘤。其特点为发病率高但预后较好。甲状腺乳头状癌易发生颈部淋巴结转移,但由于转移灶经常较小,难以被术前超声或术中触诊发现。因此为了降低疾病复发的可能风险,相当部分的术前未发现淋巴结转移的甲状腺乳头状癌患者接受了预防性的中央组淋巴结清扫术。但该手术会增加喉返神经损伤以及甲状旁腺损伤的风险。一个新的判断甲状腺乳头状癌患者是否存在淋巴结转移的检查可以帮助检查为阴性的患者避免进行中央组淋巴结清扫术,从而降低手术相关并发症的发生率。近年来对甲状腺乳头状癌的研究发现了多个与疾病发生相关的突变,但淋巴结转移的机制和驱动基因尚未被发现。全外显子组测序技术可以发现与疾病相关的大量单核苷酸多态性(Single Nucleotide Polymorphism, SNP)位点以及可能的基因突变。对淋巴结转移灶标本进行全外显子组测序,既可以帮助阐明转移的机制,又可以指导基于疾病相关SNP的诊断工具的开发。因此本研究的目的包括：(1)通过对甲状腺乳头状癌原发灶及转移灶的全外显子组测序获得与肿瘤转移相关的突变与SNP；(2)解释甲状腺乳头状癌发生淋巴结转移的可能机制。方法本研究纳入通过术前超声、术中触诊和术后病理检查证实存在多个淋巴结转移的甲状腺乳头状癌患者,并通过手术留取肿瘤原发灶和转移灶的标本。通过DNA提取、纯化、杂交、捕获等步骤,对样品进行全外显子组测序。对测序获得的结果进行严格的质量控制。最终获得的与甲状腺癌转移相关的SNP位点经过多种生物信息学方法的筛选以及同已知的与甲状腺乳头状癌发病相关的SNP数据库进行比对分析,获得最终可能对转移有重要意义的SNP和突变。结果共获得来自3个患者的淋巴结及原发灶样本共9份。外显子组测序平均深度为122×,每个样品获得SNP总数超过40000个。经过筛选以及与已知数据库对比分析后,获得可能与转移相关的SNP位点53个。进一步筛选得到出现频率较高的SNP位点14个。对测序结果的分析表明,BRAF突变在淋巴结的转移中可能不起主要作用。而CHEK2突变可能对该过程有重要的影响。此外,涉及PDE4DIP, SPTB, TBC1D32等多个基因的SNP改变均可能与甲状腺乳头状癌的转移相关。结论甲状腺癌原发灶和淋巴结转移灶均含有大量SNP,与淋巴结转移相关的SNP超过50个。CHEK2可能在淋巴结转移的过程中起到重要作用。TBC1D32、 PDE4DIP、SPTB等基因的SNP可能与淋巴结转移密切相关。
[Abstract]:Background papilla thyroid carcinoma is a very common malignant tumor of thyroid gland. It is characterized by high incidence but good prognosis. Papilla thyroid carcinoma is prone to cervical lymph node metastasis, but because the metastasis is often small, it is difficult to be detected by preoperative ultrasound or intraoperative palpation. Therefore, in order to reduce the possible risk of recurrence, a considerable number of patients with papilla thyroid carcinoma who did not find lymph node metastasis before operation underwent prophylactic central group lymph node dissection. But the operation increases the risk of recurrent laryngeal nerve injury and parathyroid injury. A new examination to determine whether there is lymph node metastasis in patients with papilla thyroid cancer can help patients with negative examination avoid central group lymph node dissection, thus reducing the incidence of operation-related complications. In recent years, a number of mutations related to the occurrence of thyroid papilla carcinoma have been found, but the mechanism of lymph node metastasis and driving genes have not yet been found. A large number of single nucleotides polymorphism (Single Nucleotide Polymorphism, SNP) loci and possible gene mutations related to disease can be found by total exocrine group sequencing. The sequencing of lymph node metastases can not only help to clarify the mechanism of metastasis, but also guide the development of diagnostic tools based on disease-related SNP. Therefore, the purpose of this study is as follows: (1) the mutation related to tumor metastasis and SNP; (2) explain the possible mechanism of lymph node metastasis in papilla thyroid carcinoma by sequencing the total exocrine group of primary and metastases of papilla thyroid carcinoma. Methods thyroid papilla carcinoma with multiple lymph node metastasis was confirmed by preoperative ultrasound, intraoperative palpation and postoperative pathological examination, and the specimens of primary tumor and metastasis were retained by operation. The samples were sequenced by DNA extraction, purification, hybridization and capture. Strict quality control of sequencing results is carried out. Finally, the SNP loci related to metastasis of thyroid carcinoma were screened by various bioinformatics methods and compared with the known SNP database related to the pathogenesis of thyroid papilla cancer, and the SNP and mutation which may be of great significance to metastasis were obtained. Results A total of 9 samples of lymph nodes and primary foci were obtained from 3 patients. The average depth of sequencing was 122 脳, and the total number of SNP obtained from each sample was more than 40000. After screening and comparative analysis with known databases, 53 SNP loci which may be related to metastasis were obtained. 14 SNP loci with high frequency were obtained by further screening. The analysis of sequencing results showed that BRAF mutation might not play a major role in lymph node metastasis. CHEK2 mutation may have an important effect on this process. In addition, SNP changes involving PDE4DIP, SPTB, TBC1D32 and other genes may be related to the metastasis of papilla thyroid carcinoma. Conclusion there are more than 50 SNP associated with SNP, in both primary and lymph node metastases of thyroid carcinoma. CHEK2 may play an important role in the process of lymph node metastasis. SNP of TBC1D32, PDE4DIP,SPTB and other genes may be closely related to lymph node metastasis.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R736.1

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