【作者】 刘晓莉；

【导师】 孙辉； 邢明照；

【作者基本信息】 吉林大学 ， 普通外科学， 2013， 博士

【摘要】 端粒酶是一种保持染色体末端端粒长度的核糖核蛋白复合体，在维持细胞永生化和肿瘤发病机制中发挥关键作用。端粒酶逆转录酶（TERT）是端粒酶的催化亚基。TERT基因启动子突变位于5号染色体，最近在黑色素瘤被报道。其中2个启动子突变，1295228C>T和1295250C>T（分别称为C228T和C250T）尤为常见。他们位于ATG翻译起始位点上游-124C>T和-146C>T，TERT基因启动子区域。这2个突变都可以形成11个碱基的核苷酸伸展5’-CCCCTTCCGGG-3’，其中包含与ETS转录因子结合位点GGAA，ETS因子靶向作用于MAPK信号转导通路。在甲状腺乳头状癌（PTC）致癌与肿瘤进展中，BRAFV600E激活MAPK信号转导通路发挥关键作用。因此，很有可能TERT启动子突变涉及MAPK信号转导通路，进一步参与甲状腺癌发病机制。通过对大样本美国人群甲状腺癌患者及细胞系基因组DNA测序，结果发现TERT启动子C228T突变在甲状腺良性肿瘤中发生率为0.0％（0/85），在甲状腺乳头状癌中发生率为11.7％（30/257），在甲状腺滤泡癌（FTC）中发生率为11.4％（9/79）,在低分化甲状腺癌（PDTC）中发生率为37.5％（3/8），在甲状腺未分化癌（ATC）中发生率为42.6％（23/54），在甲状腺癌的细胞系中发生率为66.7％（8/12）。TERT启动子C250T突变相对罕见，与C228T突变呈现相互排斥现象，统计2个突变总的发生率，在甲状腺滤泡癌中为13.9％（11/79），在甲状腺未分化癌中发生率为46.3％（25/54），在甲状腺癌的细胞系中发生率为91.7％（11/12）。在甲状腺乳头状癌亚型分析，TERT启动子C228T突变发生率在甲状腺高柱状细胞癌（TCPTC）中为30.8％（4/13），在经典甲状腺乳头状癌（CPTC）中为12.3％（23/187），在滤泡型甲状腺乳头状癌（FVPTC）中为3.6％（2/56）。16例甲状腺髓样癌中未见TERT启动子突变。在甲状腺乳头状癌中TERT启动子C228T突变与BRAF V600E突变存在相关性，BRAFV600E突变阳性的甲状腺乳头状癌（PTC）患者中，C228T突变发生率为18.3％（19/104）,BRAF V600E突变阴性的甲状腺乳头状癌患者中，C228T突变发生率仅为7.2％（11/153），两者差异显著（P=0.0094）。同样，TERT启动子C228T突变阳性的甲状腺乳头状癌患者中，BRAFV600E突变发生率为63.3％（19/30）,明显多于C228T突变阴性的甲状腺乳头状癌患者中BRAF V600E突变发生率37.4％（85/227）。在中国人群中，TERT启动子C228T突变在甲状腺乳头状癌中发生率为9.6%(39/408)， C250T突变发生率为1.7%(7/408),总计TERT启动子突变发生率为11.3%(46/408)。甲状腺滤泡癌中，C228T突变发生率为31.8%(7/22)，C250T突变发生率为4.6%(1/22),总计TERT启动子突变发生率为36.4%(8/22)。44例甲状腺良性肿瘤中，未见TERT突变阳性患者。TERT启动子突变在BRAF突变阴性患者中发生率为3.8%(6/158) vs. BRAF突变阳性患者中发生率16.0%(40/250)，P值为5.87×10-4,提示TERT启动子突变与BRAF突变密切相关。与甲状腺癌临床特点相关性分析显示，与甲状腺乳头状癌患者年龄及肿瘤大小、腺外浸润、高风险TNM分期相关。本研究在国际上首次报告甲状腺癌TERT启动子突变筛查，尤其普遍存在于高侵袭性甲状腺癌和BRAF V600E突变阳性甲状腺乳头状癌中。首次对在不同类型及亚型的甲状腺癌TERT启动子突变进行筛查与分析，提示TERT启动子突变在甲状腺癌失分化与肿瘤进展等方面起到重要作用。首次对中国人群甲状腺癌TERT启动子突变的筛查，研究样本量是目前甲状腺癌TERT启动子突变筛查中最大的，并与美国人群甲状腺癌TERT启动子突变结果进行多种人群验证分析，进一步揭示了TERT启动子突变与甲状腺癌高风险因素及临床预后密切相关和进行TERT启动子突变联合BRAF V600E突变诊断的潜在优势，为甲状腺癌分子遗传学研究开启新的篇章。更多还原

【Abstract】 Telomerase, a ribonucleoprotein complex that maintains telomerelength at the end of chromosomes, plays a key role in cellular immortalityand tumorigenesis. Its catalytic subunit is telomerase reverse transcriptase(TERT). Promoter mutations in the TERT gene on chromosome5haverecently been reported in melanomas. Two TERT promoter mutations,1295228C>T and1295250C>T (termed C228T and C250T hererespectively), are particularly common. They represent nucleotidechanges of-124C>T and-146C>T (where K1is the base just upstreamof the A of the ATG translation start site) respectively in the TERTpromoter. Both the mutations create an11-base nucleotide stretch5’-CCCCTTCCGGG-3’, which contains a consensus binding site, GGAA(in reverse complement), for ETS transcription factors, suggestingpotentially important biological relevance of these mutations. In fact, thetwo mutations have been demonstrated to confer increased transcriptionalactivity on the TERT promoter. These mutations are not found in normalhuman subjects and in the public genetic databases and are, therefore,cancer specific somatic genetic alterations, further supporting theirpotentially important role in human tumorigenesis. This is consistent withthe previously observed increased telomerase activities in some humancancers. Thus, TERT promoter mutations, by promoting the expression ofthe catalytic subunit of telomerase in response to ETS transcription factors, probably represent a novel mechanism by which telomerase playsan important role in human tumorigenesis. Melanomas and follicularcell-derived thyroid cancer share considerably similar geneticbackgrounds; for example, they both harbor the BRAF V600E mutationwith a high prevalence. We were, therefore, prompted to explore TERTpromoter mutations in thyroid cancers in the present study.Follicular cell-derived thyroid cancer is a common endocrinemalignancy the incidence of which, similar to that of melanoma, has beenrising rapidly globally in recent years. Follicular cell-derived thyroidcancer can be classified into several histological types, among which themost common types are papillary thyroid cancer (PTC) and follicularthyroid cancer (FTC), which account for85–90%and10–15%of all thethyroid cancers respectively. PTC can be further classified into a fewsubtypes or variants, the most common of which include conventionalPTC (CPTC), follicular variant PTC (FVPTC), and tall-cell PTC(TCPTC). Other subtypes of PTC, such as the columnar variant, are rare.Unlike the rare but rapidly aggressive undifferentiated anaplastic thyroidcancer (ATC), PTC and FTC are indolent differentiated thyroid cancers(DTCs). There is also poorly differentiated thyroid cancer (PDTC), whichhas aggressiveness between that of DTC and ATC. ParafollicularC-cell-derived medullary thyroid cancer (MTC) is uncommon. Benignthyroid tumors are far more common than thyroid cancers. Variousgenetic alterations have been identified in thyroid cancers, which, byaberrantly driving various signaling pathways, play a fundamental role inthyroid tumorigenesis. In the present study, we examined TERT promotermutations in various thyroid tumors to explore novel genetic alterations inthyroid tumorigenesis. Mutations1295228C>T and1295250C>T (termed C228T andC250T respectively),corresponding to-124C>T and-146C>T from thetranslation start site in the promoter of the telomerase reversetranscriptase (TERT) gene, have recently been reported in human cancers,but not in thyroid cancers yet.We explored these mutations in thyroid cancers by genomicsequencing of a large number of primary tumor samples. We found theC228T mutation in0of85(0.0%) benign thyroid tumors,30of257(11.7%) papillary thyroid cancers (PTC),9of79(11.4%) follicularthyroid cancers (FTC),3of8(37.5%) poorly differentiated thyroidcancers (PDTC),23of54(42.6%) anaplastic thyroid cancers (ATC), and8of12(66.7%) thyroid cancer cell lines. The C250T mutation wasuncommon, but mutually exclusive with the C228T mutation, and the twomutations were collectively found in11of79(13.9%) FTC,25of54(46.3%) ATC, and11of12(91.7%) thyroid cancer cell lines. Among PTCvariants, the C228T mutation was found in4of13(30.8%) tall-cell PTC(TCPTC),23of187(12.3%) conventional PTC, and2of56(3.6%)follicular variant PTC samples. No TERT mutation was found in16medullary thyroid cancer samples. The C228T mutation was associatedwith the BRAF V600E mutation in PTC, being present in19of104(18.3%) BRAF mutation-positive PTC vs.11of153(7.2%) the BRAFmutation-negative PTC samples (P=0.0094). Conversely, BRAF mutationwas found in19of30(63.3%) C228T mutation-positive PTC vs.85of227(37.4%) C228T mutation-negative PTC samples (P=0.0094).The C228T mutation was found in9.6%(39/408) of papillarythyroid cancer (PTC) and C250T in1.7%(7/408) of PTC, and they werecollectively found in11.3%(46/408) of PTC. C228T was found in 31.8%(7/22) and C250T in4.6%(1/22) of follicular thyroid cancer(FTC), and they were collectively found in36.4%(8/22) of FTC. NoTERT mutation was found in44benign thyroid tumors. The twomutations were found in3.8%(6/158) of BRAF mutation-negative PTCvs.16.0%(40/250) of BRAF mutation-positive PTC (P=5.87×10-4),demonstrating their association with BRAF mutation. Unlike BRAFmutation, TERT promoter mutations were not associated with high iodineintake, but were associated with older patient age, larger tumor size,extrathyroidal invasion, and advanced stages III/IV of PTC.In Chinese Cohort,The C228T mutation was found in9.6%(39/408)of papillary thyroid cancer (PTC) and C250T in1.7%(7/408) of PTC,and they were collectively found in11.3%(46/408) of PTC. C228T wasfound in31.8%(7/22) and C250T in4.6%(1/22) of follicular thyroidcancer (FTC), and they were collectively found in36.4%(8/22) of FTC.No TERT mutation was found in44benign thyroid tumors.The twomutations were found in3.8%(6/158) of BRAF mutation-negative PTCvs.16.0%(40/250) of BRAF mutation-positive PTC (P=5.87×10-4),demonstrating their association with BRAF mutation and associated witholder patient age, larger tumor size, extrathyroidal invasion, and advancedstages III/IV of PTC.We thus for the first time, to our knowledge, demonstrate TERTpromoter mutations in thyroid cancers, that are particularly prevalent inthe aggressive thyroid cancers TCPTC, PDTC, ATC and BRAFmutation-positive PTC, revealing a novel genetic background for thyroidcancers.更多还原