分子特征在识别碘难治性甲状腺癌中的意义

发布时间：2018-08-02 12:33

【摘要】：该博士论文由相互联系的三部分组成。第一部分远处转移性甲状腺癌(DM-DTC)TERT及BRAF突变与碘难治关系研究端粒酶反转录酶(telomerase reverse transcriptase,TERT)启动子突变在一些研究中报道与分化型甲状腺癌(differentiated thyroid cancer,DTC)的侵袭性特征如复发、死亡等相关。然而其与远处转移性DTC(distant metastatic DTC,DM-DTC)远处转移病灶的摄1311特征及1311治疗反应之间的关系尚未可知。本论文第一部分的研究目的为在中国DM-DTC患者人群中检测TERT启动子的突变率,并与BRAF突变相比较,评价其与远处转移病灶的摄131I特征及1311治疗反应之间的关系。本研究回顾性收集了 66例DM-DTC患者的手术原发病灶石蜡切片组织,提取基因组DNA,采用PCR扩增及Sanger测序技术进行TERT启动子及BRAF突变检测,中位随访时间为46.5个月(四分位数间距:29个月～70.5个月)。在随访终点,根据随访过程中的血清学反应[刺激性甲状腺球蛋白(stimulated thyroglobulin,sTg)水平的变化]、1311全身显像所示摄碘特征(摄1311或不摄1311)及其他影像学证据,将1311治疗反应分为:①碘难治,②碘治疗反应佳。对其中的36例患者的131I全身显像摄碘情况进行了半定量分析,得到肿瘤病灶摄取量/本底摄取量(tumor/background,T/B)。本研究结果显示:TERT启动子突变在DM-DTC中的突变率为22.73%(15/66),其中C228T位点的突变率(13/15)比C250T位点(2/15)更高。TERT启动子突变的患者有93.33%(14/15)在131I治疗后出现sTg水平升高20%。而BRAF与TERT启动子均不突变的患者有78.12%(25/32)在1311治疗后出现sTg水平下降20%。TERT启动子突变与DM-DTC患者1311治疗反应差密切相关(P0.001),全部15例TERT启动子突变的DM-DTC在随访终点均出现了碘难治,阳性预测值高达100%。TERT启动子突变与DM-DTC的侵袭性特征如诊断时年龄大(P0.001)、肿瘤直径大(P=0.013)、合并BRAF突变(P=0.044)显著相关。对36例DM-DTC患者的1311全身显像半定量分析提示:TERT启动子突变与远处转移病灶的摄碘差显著相关,其T/B水平明显低于TERT野生组(P0.001)。另外,TERT启动子突变的DM-DTC患者比BRAF突变的患者出现碘难治的时间更早(8/8vs.5/11,Fisher exact检验,P=0.018)。据此得出结论:TERT启动子突变与DM-DTC远处转移病灶的不摄碘特征密切相关;且与BRAF相比,其对1311摄取特征的负性影响更加严重。TERT启动子突变可作为识别碘难治性甲状腺癌(radioiodine-refractory DTC,RAIR-DTC)的指标之一。第二部分miR-125a-3p与碘难治性甲状腺癌(RAIR-DTC)关系研究目前国内外关于DTC病灶1311摄取的相关小RNA(microRNA,miRNA)研究尚局限于体外细胞水平,缺乏RAIR-DTC肿瘤组织的miRNA表达谱研究。本论文第二部分研究的主要目的在于寻找DM-DTC远处转移病灶不同1311治疗反应的患者,其手术原发病灶组织的miRNA表达谱的差异,以期为RAIR-DTC的识别提供分子依据。本研究回顾性筛选了接受至少两次131I治疗的DM-DTC患者99例。初始治疗(甲状腺全切术后+1311治疗)后的中位随访时间为51.2个月。在随访终点,将所有患者根据1311治疗影像学及血清学反应分为两组:①碘难治,②碘治疗反应佳。收集这些患者手术原发病灶石蜡包埋组织,提取总RNA。对质量鉴定合格的88例进行后续实验,其中56例进行miRNA表达谱芯片杂交,另外32例作为独立样本对芯片筛选的2个可能靶向调控钠碘同向转运体(sodium iodide symporter,NIS)mRNA表达的miRNA进行实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)验证,最终验证得到1个在RAIR-DTC中高表达且可能靶向调控 NIS mRNA 的 miRNA:hsa-miR-125a-3p。在DTC细胞株B-CPAP中转染miR-125a-3p的mimic、inhibitor及其各自阴性对照,结果显示:转染miR-125a-3p mimic较其阴性对照可显著下调NIS mRNA及蛋白的表达水平;可显著下调促甲状腺激素受体(thyroid stimulating hormone receptor,TSHR)mRNA的表达水平,但对TSHR蛋白的影响不显著;对甲状腺球蛋白(thyroglobulin,TG)及甲状腺过氧化物酶(thyroidperoxidase,TPO)mRNA及蛋白的影响不显著;B-CPAP细胞的1311摄取率较其阴性对照显著下降。转染miR-125a-3p inhibitor较其阴性对照可显著上调NIS mRNA及蛋白的表达水平;可显著上调TSHR mRNA的表达水平,但对TSHR蛋白的影响不显著;对TG及TPO mRNA及蛋白的影响不显著;B-CPAP细胞的1311摄取率较其阴性对照显著升高。据此得出结论:hsa-miR-125a-3p可通过抑制NIS的表达导致DTC细胞的131I摄取率下降,与RAIR-DTC的发生相关。第三部分1311治疗前刺激性甲状腺球蛋白可预测分化型甲状腺癌治疗反应研究提示sTg在1311治疗后可有效监测DTC的复发及疾病持续状态,但其在1311治疗前评估中的作用尚未可知。有研究显示1311治疗前刺激性甲状腺球蛋白(preablative sTg,ps-Tg)水平与DTC的远处转移相关,然而ps-Tg与131I治疗反应的关系尚未可知。本论文第三部分的目的为研究ps-Tg与2015年美国甲状腺协会(AmericanThyroidAssociation,ATA)指南提出的131I治疗反应之间的关系,并计算可预测1311治疗影像学反应不佳的ps-Tg临界值。本研究回顾性研究了在北京协和医院核医学科甲状腺癌全切术后接受131I治疗的患者。除去甲状腺球蛋白抗体(anti-Tgantibody,TgAb)阳性的患者,将452例DTC 患者按照 ps-Tg 水平分为 3 组:1 组,0～1 ng/ml(n=82);2 组,1～10ng/ml(n=173);3组,≥10ng/ml(n=197)。中位随访38个月后,按照2015年ATA指南治疗反应分级,将治疗反应分为疗效满意(excellentresponse,ER)、疗效不确切(indeterminate response,IDR)、血清学反应欠佳(biochemical incomplete response,BIR)和影像学反应欠佳(strctural incomplete response,SIR)。其中,SIR 为反应差。Ps-Tg水平可明显区分不同的治疗反应。在随访终点,1组、2组、3组分别有0%、1.73%、42.74%患者的治疗反应为SIR(x2=123.037,P0.001)。通过受试者工作特征曲线(receiver operating characteristic,ROC)计算得到区分SIR与其他治疗反应(ER+IDR+BIR)的 ps-Tg 临界值为 26.75ng/ml,曲线下面积(area under curve,AUC)为 0.947,阴性预测值(negative predictive value,NPV)为 96.99%。Ps-Tg为预测SIR的独立预测因子(OR:42.312,P0.001)。Ps-Tg,尤其是ps-Tg高水平,可有效预测不同的1311治疗反应,为1311治疗决策的制定提供增益价值。
[Abstract]:The doctoral thesis consists of three interrelated parts. Part 1 the relationship between TERT and BRAF mutations and iodine refractory mutations in distant metastatic thyroid carcinoma (DM-DTC) study of the promoter mutation of the telomerase reverse transcriptase (telomerase reverse transcriptase, TERT) promoter in some studies and differentiated thyroid cancer (differentiated thyroid cancer, DTC) Invasive characteristics such as recurrence and death are related. However, the relationship between the 1311 features of distant metastatic DTC (distant metastatic DTC, DM-DTC) and the 1311 treatment response is not known. The first part of this thesis is to detect the mutation rate of the TERT promoter in the Chinese population of DM-DTC patients and to be with BRAF process. A retrospective study was made to evaluate the relationship between the 131I feature and the 1311 treatment response. In this study, the paraffin section of the primary lesions of 66 patients with DM-DTC was collected, the genomic DNA was extracted and the TERT promoter and BRAF mutation were detected by PCR amplification and Sanger sequencing. The median follow-up time was 46.5. A month (four division interval: 29 months to 70.5 months). At the end of the follow-up, according to the serological response [stimulating thyroid globulin (stimulated thyroglobulin, sTg) levels in the follow-up process], 1311 whole body imaging showed the iodine characteristics (1311 or no 1311) and other imaging evidence, and the 1311 treatment reactions were divided into: 1. Iodine refractory, A semi quantitative analysis of iodine uptake in 36 cases of 131I was carried out and the uptake of tumor lesions / tumor/background (T/B) was obtained. The results of this study showed that the mutation rate of the TERT promoter mutation in DM-DTC was 22.73% (15/66), and the mutation rate of the C228T locus (13/15) was more than the C250T site. (2/15) 93.33% (14/15) in patients with higher.TERT promoter mutation (14/15) increased sTg level after 131I and 78.12% (25/32) in patients with BRAF and TERT promoter without mutation (25/32), the decrease of sTg level after 1311 treatment was closely related to the poor response of 1311 of DM-DTC patients. The mutation of DM-DTC appeared at the end point of the follow-up. The positive predictive value was up to the 100%.TERT promoter mutation and the invasive characteristics of DM-DTC, such as the age of diagnosis (P0.001), the large diameter of the tumor (P=0.013), and the combined BRAF mutation (P=0.044). A semi quantitative analysis of the 1311 body imaging of 36 cases of DM-DTC patients: TERT promoter mutation The T/B level was significantly lower than that in the TERT wild group (P0.001). In addition, the DM-DTC patients with TERT promoter mutation appeared earlier than those with BRAF mutations (8/8vs.5/11, Fisher exact test, P=0.018). Accordingly, the conclusion was that the TERT promoter mutation and the distant metastasis of the DM-DTC were not taken. Iodine characteristics are closely related; and compared with BRAF, the negative effect on the 1311 uptake characteristics is more serious..TERT promoter mutation can be used as one of the indicators for identifying iodine refractory thyroid carcinoma (radioiodine-refractory DTC, RAIR-DTC). The relationship between the second part of miR-125a-3p and iodized thyroid adenocarcinoma (RAIR-DTC) is a study of DTC disease at home and abroad. The study of small RNA (microRNA, miRNA) uptake of focal 1311 is still limited to the level of in vitro cells and the lack of miRNA expression profiles in RAIR-DTC tumor tissues. The main purpose of the second part of this study is to find the differences in the miRNA expression profiles of the primary foci in the patients with distant metastatic lesions of DM-DTC. This study provides a molecular basis for RAIR-DTC identification. This study reviewed 99 cases of DM-DTC patients receiving at least two 131I treatments. The median follow-up time after initial treatment (+1311 after total thyroidectomy) was 51.2 months. All patients were divided into two groups according to the 1311 treatment imaging and serological response at the end of the follow-up: 1. Treatment, iodine therapy was better. Collect the paraffin embedded tissues of the primary foci of these patients, and extract the total RNA. for 88 cases of quality identification. 56 of them were hybridized with miRNA expression spectrum chip, and the other 32 were selected as independent samples to regulate the sodium iodide symporte (iodide symporte). R, NIS) mRNA expressed miRNA was verified by real-time quantitative polymerase chain reaction (quantitative real-time polymerase chain reaction, qRT-PCR). Negative control, the results showed that transfection of miR-125a-3p mimic significantly down the expression level of NIS mRNA and protein compared with the negative control, and significantly down regulation of the expression level of thyroid stimulating hormone receptor (thyroid stimulating hormone receptor, TSHR) mRNA, but no significant effect on TSHR protein; The effect of thyroidperoxidase (TPO) mRNA and protein was not significant. The 1311 uptake rate of B-CPAP cells was significantly lower than that of the negative control. The expression level of NIS mRNA and protein could be significantly up-regulated by transfection of miR-125a-3p inhibitor compared with the negative control, and the expression level of TSHR mRNA was significantly up, but the effect on TSHR protein was not obvious. The effect on TG and TPO mRNA and protein was not significant; the 1311 uptake rate of B-CPAP cells was significantly higher than that of the negative control. Accordingly, it was concluded that hsa-miR-125a-3p could decrease the 131I uptake rate of DTC cells by inhibiting the expression of NIS and related to the occurrence of RAIR-DTC. Third part of the stimulative thyroglobulin can be predicted before the treatment of part 1311. The study of therapeutic response to thyroid cancer suggests that sTg can effectively monitor the recurrence and disease status of DTC after 1311 treatment, but its role in the assessment before 1311 is not known. Studies have shown that the level of preablative sTg (ps-Tg) before 1311 treatment is related to the distant metastasis of DTC, but ps-Tg and 131I therapy The third part of this paper aims to study the relationship between ps-Tg and the AmericanThyroidAssociation (ATA) guidelines of the American Thyroid Association (AmericanThyroidAssociation, ATA) in 2015, and to calculate the critical value of ps-Tg for predicting the poor response of the 1311 treatment imaging. This study reviewed the Peking Union Medical College Hospital in a retrospective study. The patients who received 131I after total thyroidectomy in the nuclear medicine department were divided into 3 groups according to ps-Tg level, excluding the positive of thyroid globulin antibody (anti-Tgantibody, TgAb). The 1 group, 0~1 ng/ml (n=82), the 2 group, 1 to 10ng/ml (n=173), 3 groups, and more 10ng/ml (n=197) were followed up for 38 months after the median follow-up, according to the ATA guide of 2015. The treatment reaction classification was divided into excellentresponse (ER), the curative effect was not accurate (indeterminate response, IDR), the serological response was not good (biochemical incomplete response, BIR) and the imaging reaction was not good (strctural incomplete response). The therapeutic response was SIR (x2=123.037, P0.001) in 0%, 1.73%, and 42.74% patients in the 1 and 2 groups at the end point of follow-up. The ps-Tg critical value of the SIR and other therapeutic reactions (ER+ IDR+BIR) was calculated by the work characteristic curve of the subjects (receiver operating characteristic, ROC). Urve, AUC) is 0.947, and the negative predictive value (negative predictive value, NPV) is 96.99%.Ps-Tg as the independent predictor of SIR (OR:42.312, P0.001).Ps-Tg, especially the ps-Tg high level, which can effectively predict different 1311 treatment responses and provide gain value for the formulation of 1311 treatment decisions.
【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】：2017
【分类号】：R736.1

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