【作者】 王玉龙；

【导师】 吴毅； 嵇庆海； 王久存； 李端树； 周晓燕；

【作者基本信息】 复旦大学 ， 肿瘤学， 2009， 博士

【摘要】 目的探索中国人乳头状甲状腺癌RET基因重排、BRAF突变、H4-PTEN基因重排的发生情况及其临床病理相关性,研究DNA双链断裂修复基因XRCC3的多态性与乳头状甲状腺癌的遗传易感性和基因重排发生的相关性。材料与方法第一部分143例甲状腺肿瘤组织、瘤旁正常组织的RNA抽提及逆转录合成cDNA,其中包括6例结节性甲状腺肿,2例滤泡性腺瘤,7例甲状腺髓样癌和126例乳头状甲状腺癌。以cDNA为模板进行RET基因重排方式RET/PTC-1、RET/PTC-2、RET/PTC-3、ELKS/RET的PCR扩增和测序鉴定。对RET重排阳性和阴性的病人的临床病理特点进行分析。第二部分125例乳头状甲状腺癌组织、癌旁正常组织RNA和基因组DNA的抽提,以RNA为模板合成cDNA。以DNA为模板进行BRAF外显子PCR扩增及测序鉴定,比对癌及癌旁正常组织的序列,进行BRAF的突变分析。以cDNA为模板进行H4与PTEN基因重排产物的PCR扩增,测序鉴定,根据H4/PTEN的重排方式,设计PTEN/H4引物进行回复性重排的PCR扩增和测序鉴定。综合分析RET重排、H4-PTEN重排、BRAF突变在乳头状甲状腺癌的发生情况及其与临床病理之间的相关性和生物学意义。第三部分173例乳头状甲状腺癌病人以及120名正常对照群体血样基因组DNA的抽提,以DNA为模板进行DNA双链断裂修复基因XRCC3外显子的PCR扩增。运用DGGE（变性梯度凝胶电泳,Denatured Gradient Gel Electrophoresis）对PCR产物进行突变分析。根据DGGE结果,对阳性的外显子PCR产物进行测序鉴定。基于病人和正常人的XRCC3多态性的病例-正常对照分析,进行XRCC3多态性与乳头状甲状腺癌的遗传易感性的相关性研究。基于基因重排阳性病人和基因重排阴性病人的XRCC3基因多态性差异的病例之间对照分析,进行DNA双链断裂修复基因与基因重排的相关性分析。结果第一部分在126例乳头状甲状腺癌组织中,发现了18例（14.3%）RET基因重排,包括12例RET/PTC-1和6例RET/PTC-3,癌旁正常甲状腺组织、非乳头状甲状腺癌肿瘤组织未发现RET重排,所有组织没有发现RET/PTC-2、ELKS/RET重排。RET重排在年龄＜20岁、20-40岁和≥40岁三组中的概率分别为:50%、14.6%和11.1%,年龄＜20岁的群体的RET重排的概率最高（P=0.033）。RET/PTC-1阳性的病例更容易在良性甲状腺疾病的基础上伴随乳头状甲状腺癌（P=0.021）,尤其是甲状腺炎的基础上;RET/PTC-3阳性的病例具有高的甲状腺外侵犯的特点（P=0.005）,同时其分期也相对较晚（P=0.007）。通过多因素的回归分析,RET重排（OR=8.703,95%CI 1.690-44.818）,男性（OR=3.875,95%CI 1.405-10.685）,年龄（OR=0.964,95%CI 0.933-0.996）,多灶性（OR=3.543,95%CI 1.333-9.416）和晚期的T分类（OR=7.318,95%CI2.910-18.400）是乳头状甲状腺癌颈侧区（Ⅱ-Ⅴ区）淋巴结转移的高危因素。第二部分在125例乳头状甲状腺癌中,58例发现了BRAF突变V600E（46.4%）,12例为RET/PTC-1（9.6%）,6例为RET/PTC-3（4.8%）,6例H4/PTEN重排（4.8%）,7例发现了PTEN/H4重排（5.6%）。在BRAF突变阳性的病人中,11例（19.0%）同时检测出了一种或者多种的重排,同时在RET/PTC和H4-PTEN阳性的样本中,发生另外一种突变的概率分别为38.9%（7/18）和75%（9/12）,多重分子改变群体比单种分子病理改变和没有分子病理改变的病人发病年龄要轻（P=0.019）。在发生重排的四种基因里,H4基因（CCDC4）,也就是形成RET/PTC-1和H4-PTEN的基因,是最常见的涉及重排的基因（17.6%,22/125）。基因重排阳性的乳头状甲状腺癌的病人的发病年龄比那些没有基因重排的病人要年轻（36.81±12.416 VS 43.53±13.890,P=0.025）,在年龄小于20岁的病人中,拥有最高的基因重排的发生率（50%）,在年龄≥60岁的病人中,BRAF突变是唯一一个检测出的分子改变（81.8%）。基因重排的发生率随着年龄的增大而减少,而BRAF突变的发生率随着年龄的增大而增大。重排阳性的病人比阴性的病人具有更高的淋巴结转移的可能（92.6%VS 70.4%,P=0.018）。第三部分根据XRCC3（AF037222）的序列,共发现了6种多态性位点6390G＞T（位于5’-UTR）、6410A＞G（dSNP登录号:rs56377012）、14242G＞A（Val165Ile）、14304C＞T、14362T＞C、18440C＞T,6410A＞G和14304C＞T的杂合度（Heterozygosity）相对较高,在正常人群均为2.5%,正常人群中野生型等位基因的基因频率均为98.8%,其它4种多态性均为少见突变。6410A＞G和14304C＞T所显示的基因频率和基因型频率符合Hardy-Weinburg Equilibrium（P＞0.05）。6410A＞G的G等位基因与乳头状甲状腺癌的发病相关（基因频率在病例和对照分别为4.3%和1.3%）,P值为0.033,OR值为3.580（95%CI,1.025-12.505）。由6410A＞G和14304C＞T两个多态性位点构成的单倍型（haplotype） A⁶⁴¹⁰C¹⁴³⁰⁴为常见单倍型,在对照和病例中的发生率为97.5%与93.1%,与少见单倍型(A⁶⁴¹⁰T¹⁴³⁰⁴,G⁶⁴¹⁰C¹⁴³⁰⁴,G⁶⁴¹⁰T¹⁴³⁰⁴)相比,A⁶⁴¹⁰C¹⁴³⁰⁴单倍型对乳头状甲状腺癌的发生具有明显的保护性作用,P值为0.017（OR 2.907,95%CI 1.170-7.224）。XRCC3 6410A＞G的A/G和GG型个体所患甲状腺癌组织的RET重排率高于AA型个体的RET重排发生率（25%V.S.14%）,但差异没有统计学意义（P=0.335）。结论第一部分本研究中中国人乳头状甲状腺癌仅存在RET/PTC-1和RET/PTC-3两种重排方式,在年龄＜20岁的病人群体,RET重排的概率是50%,明显高于其它的年龄群体;RET重排同乳头状甲状腺癌Ⅱ-Ⅴ区淋巴结密切相关,RET/PTC、男性、发病年龄早、多发病灶、晚期的T分类被证实为乳头状甲状腺癌侧颈部淋巴结转移的高危险因素,应当对RET/PTC阳性乳头状甲状腺癌的病人的侧颈部进行更详细的术前评估和术后随访。我们推测RET/PTC-1和RET/PTC-3在甲状腺癌的演进方面具有不同的作用,RET/PTC-1可能在乳头状甲状腺癌与甲状腺炎的相关性方面具有一定的作用。第二部分基因重排的发生率随着年龄的增大而减少,而BRAF突变的发生率随着年龄的增大而增大,是年龄≥60岁的病人中唯一被检测出的分子改变（81.8%）。RET重排,BRAF突变和H4-PTEN重排可以发生在同一个乳头状甲状腺癌的标本,这种多重分子病理改变容易在年轻的病人中发生。联合检测RET/PTC、BRAF突变、H4-PTEN和其他的涉及乳头状甲状腺癌分子改变的危险因素,将会提高分子标记作为一种诊断指标和靶向治疗指标的预测意义。第三部分XRCC3基因6410A＞G（rs56377012）G等位基因与乳头状甲状腺癌的发病相关,与另外一个SNP 14304C＞T进行的单倍型分析,发现常见单倍型A⁶⁴¹⁰C¹⁴³⁰⁴与其他少见单倍型相比,对乳头状甲状腺癌的发生具有明显的保护性作用。6410A＞G的A/G和GG型个体发生RET基因重排的概率高于AA型个体的重排发生率。这些基因多态性对乳头状甲状腺癌发病的影响机制,可能是由于多态性自身作为致病因素,或者可能是由于该多态性位点与另外一个致病性多态性位点处于连锁不平衡的结果,尚需进一步实验来证实。更多还原

【Abstract】 ObjectivesTo investigate the prevalance and clinicopathological significance of RET rearrangement,BRAF mutation,H4-PTEN gene rearrangement in papillary thyroid carcinoma,the association of XRCC3 gene（one of DNA double strand break repair genes） polymorphism and predisposition to papillary thyroid carcinoma or with gene rearrangement.Subjects and MethodsPARTⅠOne hundred and fourty three thyroid tumors were enrolled in this study which included 6 nodular goiter,2 folicular tumor,7 medullary thyroid carcinoma and 126 papillary thyroid carcinoma.Total RNA were isolated from the tumor tissues and para-tumor tissues and reversely transcribed to cDNA.RET/PTC-1,RET/PTC-2,RET/PTC-3 and ELKS/RET fusion genes were amplified by PCR and confirmed by sequencing.The clinicopathological significance of RET rearrangement was analyzed.PARTⅡTotal RNA and DNA of tumor and para-tumor tissues of 125 papillary thyroid carcinoma were isolated.BRAF gene coding sequence were amplified by PCR with DNA as template and sequenced.H4 and PTEN rearrangement,H4/PTEN,was amplified by PCR with cDNA as template and sequenced.According to the sequencing results of H4/PTEN,new primers were designed to amplify PTEN/H4,the reciprocal rearrangement of H4/PTEN,and the PCR products were sequenced to confirm the rearrangement style.The interaction,exclusivity and clinicopathological significance of RET rearrangement,BRAF mutation and H4-PTEN rearrangement were analyzed.PARTⅢThe genomic DNA of 173 papillary thyroid carcinoma patients and 120 normal controls were isolated and used as the template to amplify the DNA double strand break repair gene XRCC3 exons.The PCR products were electrophoresed on DGGE（Denatured Gradient Gel Electrophoresis） system to detect the mutaions.The mutation positive exons were reamplified and sequenced from both the cases and controls to get the mutation allele. Association study of XRCC3 polymorphism and papillary thyroid carcinoma predisposition were analyzed by case-control study including the patients and normal controls.The XRCC3 polymorphisms were compared between the gene rearrangement positive patients and negative patients to get the relationship of DNA double strand break repair gene polymorphisms and gene rearrangement.ResultsPARTⅠRET rearrangement,which included 12 RET/PTC-1 and 6 RET/PTC-3,was only detected in 14.3%（18 of 126） of PTC and was not observed in non-PTC thyroid tumor and para-tumor normal tissues.No RET/PTC-2 and ELKS/RET were detected in this serial study.The patient group of age＜20 years had the highest frequency（50%） of RET rearrangement among the groups of age＜20 years,20-40 years and≥40 years（P = 0.033）.RET/PTC-1 positive patients were more easily to suffer from another benign thyroid disease simultaneously（P=0.021）,especially thyroiditis.RET/PTC-3 positive patients had a higher frequency of extrathyroidal extension（P =0.005） and advanced T classification（P = 0.007）.RET rearrangement （OR = 8.703,95%CI 1.690-44.818）,male（OR = 3.875,95%CI 1.405-10.685）, age（OR = 0.964,95%CI 0.933-0.996）,multifocality（OR = 3.543,95%CI 1.333-9.416） and advanced T classification（OR = 7.318,95%CI 2.910-18.400） were all identified as risk factors of levelⅡ-Ⅴlymph node involvement in the multivariate analysis.PARTⅡAmong 125 cases of papillary thyroid carcinoma,58 cases of BRAF mutation（46.4%）,12 cases of RET/PTC-1（9.6%）,6 cases of RET/PTC-3（4.8%）,6 cases of H4/PTEN（4.8%） and 7 cases of PTEN/H4（5.6%） were dectected.Eleven cases（19.0%） of BRAF mutation positive patients were found with another genetic aberration and the frequency of multi-genetic aberrantions of RET rearrangement and H4-PTEN rearrangement were 38.9%（7/18）and 75%（9/12）,respectively.H4 gene （CCDC4）,the fusion parter of RET/PTC-1 and H4-PTEN,is the most frequently involved gene in rearrangement（17.6%,22/125）.The age of gene rearrangement positive patients was younger than that of the negative patients（36.81±12.416 VS 43.53±13.890,P= 0.025）,and the highest frequency of gene rearrangement were detected in patients with age＜20 years（50%）.BRAF was the only genetic aberration in patients of age≥60 years（81.8%）.The frequency of gene rearrangement was decreasing with the age while the trendency of BRAF mutation was reverse.Rearrangment positive patients had a higher lymph node involvement than negative patients（92.6%VS 70.4%,P=0.018）.PARTⅢAccording to the sequence of XRCC3（AF037222）,6 SNPs were detected,which included 6390G＞T（located at 5’ -UTR）,6410A＞G（dSNP number:rs56377012）,14242G＞A（Val165Ile）,14304C＞T,14362T＞C and 18440C＞T.The heterozygosity of both 6410A＞G and 14304C＞T of normal controls were 2.5%,and their wild type allele frequcncy were 98.8%, while other four polymorphisms were incidental mutation with lower heterozygosity.The allelic and genotypic frequency of 6410A＞G and 14304C＞T were tested with Hardy-Weinburg Equilibrium（P＞0.05）.6410A＞G allele G was associated with papillary thyroid carcinoma（the allele frequency of case and control were 4.3%and 1.3%,respectively）,P =0.033（OR 3.580,95%CI 1.025-12.505）.Haplotylpe analysis of 6410A＞G and 14304C＞T showed that the frequency of haplotype A⁶⁴¹⁰C¹⁴³⁰⁴ at case and control were 97.5%and 93.1%,and haplotype A⁶⁴¹⁰C¹⁴³⁰⁴ had a protective effect on papillary thyroid carcinoma genetic predisposition（P=0.017,OR 2.907,95%CI 1.170-7.224） when compared with the less frequent haplotypes (A⁶⁴¹⁰T¹⁴³⁰⁴,G⁶⁴¹⁰C¹⁴³⁰⁴,G⁶⁴¹⁰T¹⁴³⁰⁴).The frequency of RET rearrangement of tumor samples of XRCC3 6410A＞G A/G and GG cases were higer than the XRCC3 6410A＞G AA cases（25%V.S.14%,P=0.335）.ConclusionPARTⅠOnly two RET rearrangements,RET/PTC-1 and RET/PTC-3,were detected in Chinese patients with papillary thyroid carcinoma in the present study.The patients with age＜20 years have the highest frequency of RET rearrangement among the four age groups（50%）.RET rearrangement is associated with LevelⅡ-Ⅴlymph node metastasis.Male,young age, multifocality,advanced T classification and RET rearrangement were indentified as the risk factors of lateral neck lymph node metastasis. Serious pre-operative scanning and intensive post-operative followup should be performed to the patients with the risk factors.RET/PTC-1 and RET/PTC-3 have different role in the progression of throid cancer,and RET/PTC-1 may be associated with thyroiditis.PARTⅡThe frequency of gene rearrangement was decreasing with the increasing age.While BRAF mutation was the only genetic aberration of papillary thyroid carcinoma in patients of age≥60 years（81.8%） and have a reverse trendency.RET rearrangement,BRAF mutation and H4-PTEN rearrangement can be detected in one tumor sample simultaneously, especially in patients with young age.The predictive value of molecular changes as a diagnostic and target therapeutic markers will be improved by screening RET rearrangement,BRAF mutation,H4-PTEN rearrangement and other possibly involved genes together.PARTⅢXRCC3 gene 6410A＞G（rs56377012）G allele is associated with papillary thyroid carcinoma risk.Haplotylpe analysis of 6410A＞G and 14304C＞T shows that haplotype A⁶⁴¹⁰C¹⁴³⁰⁴ has a protective effect on papillary thyroid carcinoma genetic predisposition compared with the less frequent haplotype(A⁶⁴¹⁰T¹⁴³⁰⁴,G⁶⁴¹⁰C¹⁴³⁰⁴,G⁶⁴¹⁰T¹⁴³⁰⁴).The frequency of RET rearrangement of tumor samples of XRCC3 6410A＞G A/G and GG cases is higer than that of XRCC3 6410A＞G AA cases.These SNPs may affect the papillary thyroid carcinoma risk as a potential etiological factor or just becase of linkage disequilibrium with another etiological SNP.Futher experiments need to be carried out to confirm these associations.更多还原