血清P53抗体与BRAF基因突变检测在PTC诊疗中的决策作用

发布时间：2018-03-02 15:18

本文选题：血清p53抗体　切入点：BRAFV600E基因突变　出处：《吉林大学》2016年博士论文　论文类型：学位论文

【摘要】：近年来,甲状腺癌作为最常见的内分泌系统肿瘤,在全球范围内呈快速增长的趋势。而占甲状腺癌80-90%的甲状腺乳头状癌(PTC)发病率迅速升高,在其中起到主要作用。探讨甲状腺乳头状癌的精准化诊治策略已经逐渐成为甲状腺癌的诊疗的核心问题。目前,临床常规的诊断技术,包括超声或者超声影像学检查及超声引导下的细针穿刺抽吸病理学检查(fine needle aspiration biopsy,FNAB),可鉴别甲状腺结节性质指导手术治疗方案。但临床应用过程中,仍然存在超声及穿刺细胞学出现可疑结果而导致无法确诊,分子学检测手段被认为是主要的解决方案,但国内针对PTC的相关血清标志物及分子学检测手段刚刚起步,未在临床推广。因此,探讨及规范甲状腺癌相关分子标志物,将其应用与临床,进行甲状腺恶性肿瘤的临床诊断、术后风险评估及预后评估意义重大。在基因标志物中,BRAF V600E基因突变作为甲状腺乳头状癌和一些未分化癌中常见的基因突变类型,约45%的甲状腺乳头状癌中存在该突变基因,通过对细针穿刺样本进行BRAF基因突变检测正作为重要术前分子检测技术在临床中推广。而另一相关基因P53基因作为甲状腺未分化癌及低分化癌中的重要突变基因,同时也分布于甲状腺乳头状癌,且对其产生失分化起重要的作用。两种基因突变在各甲状腺癌类型上的分布十分相似,且同为甲状腺癌发生发展及肿瘤失分化的重要促进因子,具有一定联系。但在临床,术前检测P53基因突变存在局限性,而另一方面与其密切相关的血清P53抗体检测受到越来越多的关注。血清P53抗体作为一种重要的肿瘤患者血清学标志物,与P53基因突变及蛋白积累有一定相关性。而在噬菌体检测技术的介入,可进一步提升血清P53抗体的检出率。基于噬菌体技术的血清P53抗体检测和无负压吸引(fine needle capillary cytology,FNCC)细针穿刺标本检测BRAFV600E基因突变是否能够为术前评估两种基因突变情况提供支持。两种检测是否可以辅助甲状腺乳头状癌术前诊断、手术方案指导及预后判断是本研究关注的重点。针对以上问题,本论文以甲状腺乳头状癌患者为研究对象,系统探讨了如何提高甲状腺癌患者血清p53抗体检测效率,并优化BRAFV600E检测方法以判断两种基因检测的相关性,以及血清学p53抗体和BRAFV600E基因突变与在甲状腺乳头状癌术前诊断、手术指导及预后分析中的应用等问题。针对如何进行P53抗体检测方法的优化以达到应用于甲状腺良恶性疾病鉴别的问题,本研究首先利用合成P53蛋白、并将P53蛋白N端表位SS多肽及SP多肽展示于噬菌体,分别对53例甲状腺良性疾病及139例PTC患者进行血清p53抗体ELISA检测研究。经过统计结果显示,噬菌体phage-SP在三种抗原中,其检出率最高,良恶性疾病中检出率有明显差异。同时,可选择ROC曲线最佳工作值为切点值用于检测,同时可将Phage-SP与P53蛋白联合检测,可提高灵敏度和准确度。同时,进一步研究发现,P53抗体与甲状腺自身免疫抗体无明显相关性,而与甲状腺肿瘤内P53蛋白的积累正相关性。上述结果表明,血清p53抗体可辅助应用于甲状腺良恶性疾病的临床诊断。针对如何优化BRAFV600E检测方法的问题,随后本实验通过对甲状腺癌中BRAFV600E检测方法中的金标准直接测序法(Sanger法测序),临床常用试剂盒Realtime PCR荧光探针试剂盒检测法,及免疫组化法(BRAFV600E单克隆抗体VE1)进行对比,选择有效的检测手段,并探讨BRAF基因检测在临床诊疗中的应用。本研究对44例PTC患者及12例良性患者的石蜡病理组织进行以上三种检测方法。结果显示,对比金标准直接测序法,另外两种方法均可检出PTC肿瘤组织中的BRAFV600E基因突变,且荧光PCR法的特异性更高,而免疫组化法的定位性更好。上述结果表明三种方法均可有效检测BRAFV600E基因突变,但直接测序法检出率上更占优势。针对血清学p53抗体和BRAFV600E基因突变在术前诊断、手术指导及预后分析中的应用的问题。本研究对227例PTC患者及85例良性患者进行两种检测手段的对比研究。结果显示血清学P53抗体检测可作为临床重要的肿瘤筛查标记物,同时FNCC联合BRAF基因突变检测,因其较高的敏感性,阳性预测值及准确性可作为PTC术前诊断的重要手段。而血清P53抗体与BRAF基因突变与PTC不良术后病理组织特征有密切关系,且呈一定的互补性,两种检测同为阳性需要更积极的治疗手段。结论证实两种检测手段可作为PTC术前诊断及手术方案指导的重要依据。
[Abstract]:In recent years, thyroid cancer as the most common endocrine tumors, the trend of rapid growth in the global scope. While accounting for the thyroid papillary carcinoma of thyroid cancer 80-90% (PTC) incidence rate increased rapidly, which plays an important role. To explore the strategy of accurate diagnosis and treatment of papillary thyroid carcinoma has gradually become the core problem of diagnosis and treatment thyroid cancer. At present, the conventional clinical diagnosis technology, including the examination of needle aspiration pathology ultrasound or ultrasound imaging and ultrasound guided (fine needle aspiration biopsy, FNAB), can distinguish thyroid nodules to guide surgical treatment. But the clinical application process, there are still ultrasound and aspiration cytology suspicious result cannot be confirmed, the molecular detection method is considered to be the main solution, but the domestic relevant serum markers for PTC and molecular detection Measuring means has just started, not in clinical application. Therefore, to explore and standardize the markers of thyroid cancer related molecules, its application and clinical, clinical diagnosis of thyroid malignant tumor, postoperative assessment of risk assessment and prognosis of great significance. In genetic markers, BRAF and V600E gene mutation in papillary thyroid carcinoma and some undifferentiated carcinoma type common gene mutations, the mutation of thyroid papillary carcinoma about 45%, through the detection of BRAF gene mutation is important as a generalization of preoperative molecular detection technology in clinical on fine needle aspiration samples. And the other related gene P53 gene as an important gene mutation of undifferentiated thyroid carcinoma and low the differentiation of cancer, but also distributed in papillary thyroid carcinoma, and loss of differentiation plays an important role in it. The two mutations distributed in various types of thyroid cancer is very similar, and An important promoting factor with thyroid cancer development and tumor dedifferentiation, has some relationship. But in clinical, preoperative detection of P53 gene mutation limitations, and serum P53 antibody detection on the other hand, closely related to received more and more attention. The serum P53 antibody as an important marker of serum tumor patients with P53, there is a certain correlation between the gene mutation and protein accumulation. In the phage detection technology involved, can further improve the detection rate of serum P53 antibody. The detection of serum P53 antibody phage display technology and non negative pressure (fine needle capillary based on cytology, FNCC) is able to assess support two gene mutations before operation fine needle biopsy specimens of mutation detection of BRAFV600E gene. Two kinds of detection can be diagnosis of papillary thyroid carcinoma before operation, operation scheme and prognosis of the study guide The focus of attention. To solve the above problems, this paper in papillary thyroid carcinoma patients as the research object, discusses how to improve the efficiency of detection of thyroid cancer patients serum p53 antibody, and optimized the BRAFV600E detection method to determine the correlation between the two kinds of genetic testing, and serum p53 antibody and BRAFV600E gene mutation in thyroid cancer papillary before diagnosis, the application of analysis and prognosis of surgery. In view of how to optimize the methods for detection of P53 antibody to applied in the identification of benign and malignant thyroid diseases, this study firstly in the synthesis of P53 protein, and P53 protein N terminal epitope SS peptide and SP peptide displayed on phage, respectively. Study on the detection of serum p53 antibody ELISA in 53 cases of benign thyroid diseases and 139 cases of PTC patients. The statistical results showed that phage phage-SP in three kinds of antigen, the detection rate of the High detection rate of benign and malignant diseases have obvious difference. At the same time, can choose the best value for the detection of ROC curve for cutoff values, while the combined detection of Phage-SP and P53 protein, can improve the sensitivity and accuracy. At the same time, further study found that P53 antibody and thyroid autoimmune antibodies had no significant correlation with P53 protein accumulation in thyroid tumor positive correlation. The results showed that the serum p53 antibody can be used in clinical diagnosis of benign and malignant thyroid diseases. In order to optimize the detection methods for BRAFV600E problems, then this experiment through the gold standard of BRAFV600E in thyroid carcinoma in the detection method of direct sequencing (Sanger sequencing), clinical the commonly used kit Realtime PCR Fluorescent Probe Kit assay and immunohistochemical method (BRAFV600E monoclonal antibody VE1) were compared to select effective means of detection, and discusses the BRAF based Due to the application of detection in the clinical diagnosis and treatment. In this study, 44 patients with PTC and 12 patients with benign pathological paraffin tissue more than three kinds of detection methods. The results showed that compared with the gold standard of direct sequencing, two other methods can be detected BRAFV600E gene PTC mutation in tumor tissue, and specific fluorescent PCR method the higher, and immunohistochemical method. The results show that better positioning of three methods can effectively detect the BRAFV600E gene mutation, but the detection rate of direct sequencing was more dominant. The serological p53 antibody and BRAFV600E gene mutation in the preoperative diagnosis, analysis and prognosis of surgical guidance in the comparative study of this problem. Study of two kinds of detection methods of 227 PTC patients and 85 cases of benign patients. The results of serological detection of P53 antibody can be used as a clinical marker of cancer screening, and FNCC combined with BRAF gene mutation Change detection, due to its high sensitivity, positive predictive value and accuracy can be used as an important means for preoperative diagnosis of PTC. Serum P53 antibody and BRAF gene mutation is closely related with the pathological features of PTC adverse postoperative, and are complementary to each other, two with positive detection require more aggressive treatment. An important basis for the conclusion that two detection methods can be used as the diagnosis and surgical plan PTC operation guidance.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R736.1

【相似文献】