【作者】 张凌；

【导师】 嵇庆海；

【作者基本信息】 复旦大学 ， 肿瘤学， 2014， 博士

【摘要】 第一部分：促甲状腺激素是甲状腺结节病患者发生甲状腺乳头状癌的高危因素回顾性分析2007～2009年间在复旦大学附属肿瘤医院进行手术治疗的4639例甲状腺结节病患者临床病理资料。术后石蜡病理学提示：2581例患者为良性甲状腺肿瘤,2058例患者为乳头状甲状腺癌。采用1ogistic回归分析术前血清TSH水平以及TgAb水平是否是甲状腺结节病患者中发生乳头状甲状腺癌危险因素。发现术前血清TSH水平是甲状腺结节病患者发生乳头状甲状腺癌的独立危险因素。采用多因素Logistic回归分析来预测甲状腺乳头状癌患者的淋巴结转移风险并进行相关建模。Hosmer-Lemeshow检验用来评估所建模型是否与临床病理学变量匹配。采用受试者工作特征曲线(ROC)对于TSH和+TGAb基础上的数学预测模型进行了评价。我们观察到基于TSH数学预测模型的曲线下面积(AUC)为0.834(95%CI,0.823-0.846),而基于TSH+ TGAB模型数学预测模型的曲线下面积(AUC)为0.805(95%cI,0.792-0.817)。通过医学经济学比较,认为基于TSH数学预测模型能够给临床医生评估甲状腺结节病患者是否需要进行穿刺提供参考。此外术前血清TSH水平升高对于甲状腺结节病患者发生乳头状甲状腺癌的风险有着很强的正相关性。促甲状腺激素抑制治疗可能在血清TSH水平较高的患者甲状腺结节病被使用,以降低甲状腺乳头状癌的发生概率。第二部分：促甲状腺激素介导桥本氏甲状腺炎背景中甲状腺结节发生乳头状甲状腺癌为了识别患者的桥本甲状腺炎(HT)共存的甲状腺乳头状癌(PTC)患者的临床病理特点,并分析其独有的临床特征,为此类病人的诊断提供帮助。该研究纳入2008-2010年,3年间,6109例初次在复旦大学肿瘤医院头颈外科治疗的甲状腺肿瘤患者。所有患者根据他们的最终石蜡病理学诊断均分类。并收集下列,几个临床病理因素,如年龄,性别,原发肿瘤大小,原发肿瘤浸润状态,中央区淋巴结转移情况和术前血清促甲状腺激素(TSH)水平,各组患者之间进行比较。有653例患者最终诊断为桥本氏甲状腺炎。而在甲状腺乳头状癌患者中,桥本氏甲状腺炎更多伴发乳头状甲状腺癌状(381/653或58.3%),明显高于无桥本氏甲状腺炎背景的患者(2416/5456或44.3%,P<0.05)。在桥本氏甲状腺炎及乳头状甲状腺癌共同罹患的患者中,其特征为年轻患者,女性患者,原发肿瘤较小,原发灶为多发性,并有较高的血清TSH水平。多因素分析表明,伴发桥本氏甲状腺炎以及较高的血清TSH水平为乳头状甲状腺癌发生的危险因素。进一步分析在乳头状癌的患者中,桥本氏甲状腺炎可能使得乳头状甲状腺癌的淋巴结转移率降低,但血清TSH水平与淋巴结转移却没有关联。我们假设,长期桥本氏甲状腺炎引起升高的血清促甲状腺激素,从而导致桥本氏甲状腺炎诱发乳头状甲状腺癌的假说。第三部分：FOXE1基因多态性在TSH-cAMP通路中诱发乳头状甲状腺癌发生的重要作用甲状腺癌是女性第7位常见的恶性肿瘤,乳头状甲状腺癌占其中的90%以上。近期研究发现,在甲状腺结节患者中,血清促甲状腺激素(TSH)升高是乳头状甲状腺癌的危险因素,但TSH-cAMP通路如何诱导甲状腺滤泡细胞恶变的分子机制并不清楚。我们的研究发现,受TSH调控的FOXE1基因是乳头状甲状腺癌易感基因,而FOXE1是调控甲状腺滤泡细胞分化、增殖的潜在的关键因子。我们发现FOXE1基因的多态性：rs1867277(A>G),rs907577(C>T)以及rs3758248(C>T),在乳头状甲状腺癌,甲状腺良性肿瘤,正常人群中呈动态变化,并且,在rs3758248C,rs1867277G患者组中,随着血清TSH上升,患者发生乳头状甲状腺的风险是增加的,这对于临床上,对于良性甲状腺结节何时采用TSH抑制治疗,对于乳头状甲状腺癌患者术后如何分层进行TSH抑制治疗,提供了分子生物学参考。我们通过大样本汉族人群的基因多态性分析,发现受TSH调控的FOXE1基因多态性,在升高TSH刺激下可能使得乳头状甲状腺癌发生。第四部分：表观遗传学调节FOXE1基因在甲状腺乳头状癌中的异常表达通过第三部分研究发现,FOXE1在乳头状甲状腺癌发生过程中起着十分重要的作用。进而,我们通过评估FOXE1在乳头状甲状腺癌组织和细胞系的甲基化状态及表达情况,进一步明确其在乳头状甲状腺癌发生中的机制。本研究采用人乳头状癌细胞系W3, TPC1, IHH-4和正常的甲状腺滤泡上皮细胞系Nthy-ori3-1细胞系；79例乳头状甲状腺癌和癌旁组织,32例良性甲状腺肿瘤和肿瘤旁组织。FOXE1启动子CpG位点高甲基化是在正常甲状腺细胞株和正常甲状腺组织标本中观察到,而低甲基化则是在3个乳头状甲状腺癌细胞系中观察到。乳头状甲状腺癌组织中的FOXE1表达明显高于与配对的乳头状甲状腺癌旁正常甲状腺组织(P<0.001)；同样FOXE1在良性甲状腺肿瘤组织中的表达与比较良性甲状腺肿瘤组织旁正常甲状腺组织高(P=0.023)。但是FOXE1的甲基化状态和BRAF突变之间无显着差异。由于FOXE1甲基化状态在乳头状甲状腺癌与癌旁正常组织之间的明显不同,因此,FOXE1基因的表达可被用作生物标记物来诊断乳头状甲状腺癌。通过进一步研究,FOXE1与其他基因的甲基化状态组合,可以早期诊断乳头状甲状腺癌,判断其预后。更多还原

【Abstract】 Part Ⅰ:The Predictor Value of Preoperative Serum Thyroid Stimulating Hormone for Papillary Thyroid Cancer in Nodular Thyroid DiseaseWe conducted a systemic retrospective study of 4639 patients from 2007 to 2009, including 2581 patients with benign nodule,2058 with PTC. The independence of serum TSH risk and thyroidglobulin antibody (TgAb) risk was evaluated by logistic regression. Calibration of the risks was evaluated using the Hosmer-Lemeshow test. The predictor models based on TSH and TSH+TgAb were evaluated using receiver operating characteristic curves. Preoperative serum TSH is a independent factor on risk of PTC in NTD. Meanwhile, serum TgAb is a risk fact for the PTC. We observed an AUC of 0.834 (95% CI,0.823-0.846) for the TSH+TgAb model risk compared with AUC of 0.805 (95% CI,0.792-0.817) for TSH model. There is a strong positive correlation effect of preoperative serum TSH on probability of PTC in NTD. The TSH suppress therapy might be used in the NTD patients whose serum TSH are high. The model based on TSH has a remarkable value in predicting the risk of PTC. Multicenter research is recommended to verify the predictor model based on TSH.Part Ⅱ:The clinical features of papillary thyroid cancer in Hashimoto’s thyroiditis patients from an area with a high prevalence of Hashimoto’s diseaseTo identify the clinicopathological factors of co-existing papillary thyroid cancer (PTC) in patients with Hashimoto’s thyroiditis (HT) and provide information to aid in the diagnosis of such patients. This study included 6109 patients treated in a university-based tertiary care cancer hospital over a 3-year period. All the patients were categorized based on their final diagnosis. Several clinicopathological factors, such as age, gender, nodular size, invasive status, central compartment lymph node metastasis (CLNM) and serum thyroid stimulating hormone (TSH) level, were compared between the various groups of patients. There were 653 patients with a final diagnosis of HT. More PTC was found in those with HT (381 out of 653 or 58.3%) than in those without HT (2416 out of 5456 or 44.3%, p< 0.05). The HT patients with co-occurring PTC were more likely to be younger, female, have smaller nodules and have higher TSH levels than those without PTC. A multivariate analysis demonstrated that the presence of HT and higher TSH levels were risk factors for a diagnosis of PTC. In the PTC patients, moreover, the presence of HT or other benign nodule was a protective factor for CLNM, while the TSH level had no significant associations.Part III:The Polymorphisms of FOXE1 in TSH-cAMP-FOXE1 pathway in the occurence of papillary thyroid cancerThyoid cancer is the most popular endocrine malignant, which is the 7th cancer in women, and about 90% thyoid cancer is papillary thyroid cancer. Recently reasearches show high level thyroid stimulating hormone (TSH) causes higher risk of thyroid cancer. In our research, we confirm this phenomenon in Chinese patients. Unfortunately, the molecular mechanism of how TSH-cAMP pathway casused thyroid cancer was unclear, we find FOXE1 gene:rs1867277(A>G),rs907577(C>T) and rs3758248(C>T) are the suspective SNP of papillary thyroid cancer. TSH could cause the risk higher in these patients. In future study, we will analysis the genetic variant of FOXE1 under the stimulation of TSH, and furthermore we will investigate the differentiation and proliferation of thyroid follicle cell under TSH stiumlation.Part IV:Aberrant hypermethylation and increased expression of the FOXE1 gene in papillary thyroid cancerThe methylation status of FOXE1 was analyzed by bisulfite-sequencing PCR (BSP) in W3, TPC1, IHH-4 and Nthy-ori 3-1 cell lines. In total,111 paired clinical thyroid tumors (79 PTC and 32 benign thyroid tumors) and adjacent normal thyroid samples were analyzed by methylation-specific PCR (MSP). FOXE1 mRNA expression levels were analyzed by real-time polymerase chain reaction (qRT-PCR). The genotype of BRAF V600E was detected in PTC samples by direct sequencing. We noted a significant difference in the methylation level of FOXE1 in PTC (5.06%) and benign thyroid tumors compared with paired adjacent normal thyroid tissues (16.46%) (p=0.040). Hypomethylation in the promoter region of FOXE1 was significantly associated with its increased expression in PTC (p<0.050). In addition, the methylation status of FOXE1 was significantly associated with Hashimoto’s thyroiditis (HT) (p=0.003), the T stage of patients (p<0.001) and the capsular invasion (CI) of PTC (p<0.001). No statistically significant association was identified between the BRAF V600E mutation and the DN A methylation pattern in FOXE1. Epigenetic inactivation of the FOXE1 gene plays a potentially important role in PTC, and it might be a prospective biomarker for the diagnosis of PTC.更多还原