【作者】 陈晨；

【导师】 尹邦良；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 胸腺上皮肿瘤是前纵隔常见的肿瘤之一,其在生物学、肿瘤学和组织学上特性极为复杂,病理分型和分期一直是近年来外科学和病理学领域极具争议的难题之一。Masaoka病理分期根据术中和镜下肿瘤侵犯范围对胸腺上皮肿瘤进行分期,被认为是判断疾病预后的有效指标之一。但由于该方法没有考虑组织学因素,因而存在一定局限性。1999年WHO推行的胸腺上皮肿瘤组织学分型标准,按照淋巴细胞/上皮细胞比例和上皮细胞形态将胸腺上皮肿瘤分为:A型、AB型、B1型、B2型、B3型和C型,从组织病理学角度对胸腺上皮肿瘤进行较为详细的分类,为疾病诊断和预后判断提供了较好的组织病理学标准。尽管目前在胸腺上皮肿瘤的病理分型、分期、诊断和治疗方法研究方面取得了一定进展,但对于其具体的发病分子机理仍不清楚。近年来研究发现肿瘤当中某些肿瘤抑制基因失活,但其DNA序列完整,并未有突变、缺失等变化,用经典的遗传调控理论难以解释其失活的现象,因而逐渐认识到表观遗传调控在肿瘤发生和发展当中的作用。表观遗传调控主要包括DNA甲基化和组蛋白修饰,其中DNA甲基化是目前表观遗传学领域研究的热点。近年来研究认为DNA甲基化是肿瘤抑制基因失活的主要原因之一,在某些情况下甚至可能为调控基因转录的唯一的机制。研究认为肿瘤发生时肿瘤抑制基因启动子区高甲基化失活的同时,通常伴随有基因组DNA整体的低甲基化和DNA甲基化转移酶的高表达,这三种异常的甲基化状态被称为“甲基化失衡”。目前在肺癌、结肠癌、口腔粘膜上皮癌中均观察到这种典型改变,但在胸腺上皮肿瘤中仍缺乏相应报道。为此,我们第一部分根据胸腺上皮肿瘤病例临床、病理和随访资料,进行统计学分析,寻找胸腺上皮肿瘤预后相关因素;第二部分首先采用实时定量RT-PCR技术检测胸腺上皮肿瘤中肿瘤抑制基因mRNA表达情况,再采用巢式MSP技术检测其启动子区甲基化情况,并与临床病理资料进行分析;采用免疫组织化学法和ELISA法检测胸腺上皮肿瘤石蜡标本和新鲜组织标本中基因组DNA总体甲基化情况,探讨其与临床病理之间的关系;最后采用实时定量RT-PCR技术检测DNA甲基化转移酶mRNA表达情况,并与临床病理资料进行分析,系统观察胸腺上皮肿瘤中DNA甲基化失衡情况,以期进一步探讨与疾病相关的发病机制和可能的生物学标志物。第一部分胸腺上皮肿瘤病理分型分期及预后影响因素分析目的通过对胸腺上皮肿瘤患者临床、病理及随访资料进行分析,探寻与疾病预后相关的影响因素。方法本院心胸外科1997年6月至2007年9月胸腺上皮肿瘤病例137例,记录患者临床资料、病理记录,并于术后进行随访。应用Kaplan-Meier法、COX回归模型进行生存分析并判断疾病预后影响因素。结果所有病例中124例（90.5%）行全切手术,9例（6.6%）行姑息性切除,4例（2.9%）行活检术。MasaokaⅠ期、Ⅱ期病例中手术完全切除率为100%,明显高于Ⅲ期、Ⅳ期病例的67.9%和63.6%（P＜0.001）。137例病例中总的5年、10年总生存率为71.4%和50.1%。MasaokaⅠ/Ⅱ期病例5年、10年生存率为86.8%和62.5%,明显高于Ⅲ/Ⅳ期病例的28.4%和17.0%（P＜0.001）;按照WHO分型,A/AB/B1型组5年、10年生存率为88.6%和67.0%,明显高于B2/B3/C型组病例的40.5%和19.3%（P＜0.001）;完全切除组病例术后5年、10年生存率为76.8%和53.3%,明显高于不完全切除和活检组（P＜0.001）。COX回归分析发现,Masaoka病理分期,WHO组织学分型,肿瘤完全切除及手术时年龄与预后相关,合并重症肌无力及性别与疾病预后无关。结论Masaoka病理分期,WHO组织学分型,肿瘤完全切除及手术时患者年龄是胸腺上皮肿瘤患者重要的预后相关因素。第二部分胸腺上皮肿瘤中DNA甲基化失衡状态的实验研究第一节胸腺上皮肿瘤中肿瘤抑制基因表达失活及启动子区甲基化情况研究目的:观察多种肿瘤抑制基因在胸腺上皮肿瘤中表达情况,并检测其启动子区DNA甲基化状态,探讨胸腺上皮肿瘤中肿瘤抑制基因失活的可能机制。方法:采用实时定量RT-PCR技术从mRNA水平检测胸腺上皮肿瘤组织中肿瘤抑制基因APC1A,RARβ,E-cad,hMLH1,RASSF1A,FHIT,P16^INK4a,MGMT和DAPK表达情况;采用巢式MSP技术检测胸腺上皮肿瘤组织中多种肿瘤抑制基因启动子区甲基化情况,并分析其与临床病理参数之间的关系。结果:按照WHO组织学分型,与A/AB/B1组相比,APC1A,FHIT,RARβ,E-cad,hMLH1和RASSF1A mRNA表达水平在B2/B3/C组中明显下降（APC1A,p=0.002;FHIT,p=0.035;RARβ,p=0.002;E-cad,p=0.036;hMLH1,p=0.024;RASSF1A,p=0.042）,而MGMT,P161^INK4a和DAPK则没有表现出明显的差异(MGMT,p=0.083;P16^INK4a,p=0.41;DAPK,p=0.49)。按照Masaoka病理分期,Ⅲ/Ⅳ期与Ⅰ/Ⅱ期相比,APC1A,FHIT,RARβ,E-cad和hMLH1 mRNA表达明显下调（APC1A,p=0.005;FHIT,p=0.015;RARβ,p=0.047;E-cad,p=0.003;hMLH1,p=0.003）。而DAPK,P16^INK4a,RASSF1A和MGMT则没有表现出明显差异(DAPK,p=0.632;P16^INK4a,p=0.248;RASSF1A,p=0.114;MGMT,p=0.136)。在所有65例病例中,A型病例中无一例病例检测到肿瘤抑制基因启动子区高甲基化改变,在AB型和B1型病例当中,分别约有60%和66.7%的病例检测到至少一种基因启动子区存在高甲基化。而在B2型、B3型和C型病例中,几乎所有的病例均能检测到多个肿瘤抑制基因启动子区高甲基化。单个基因的甲基化频率最高的为FHIT（46.2%）,最低的为RASSF1A（15.4%）。按照Masaoka分期,Ⅲ/Ⅳ期病例中肿瘤抑制基因高甲基化率明显高于Ⅰ/Ⅱ期（p＜0.05）。DAPK高甲基化状态与性别存在一定关系（P=0.034）;P16^INK4a基因高甲基化状态与手术时年龄相关联（P=0.011）。重症肌无力与肿瘤抑制基因的启动子区高甲基化无关（P＞0.05）。结论:胸腺上皮肿瘤中存在多种肿瘤抑制基因mRNA表达下调,启动子区高甲基化可能是其失活的重要机制,且这一过程与WHO组织学分型和Masaoka病理分期密切相关。第二节胸腺上皮肿瘤中基因组DNA总体甲基化改变及其临床意义的研究目的:检测胸腺上皮肿瘤细胞中基因组DNA总体甲基化水平,并分析其与临床病理资料间的关系,探寻基因组DNA总体甲基化状态与胸腺上皮肿瘤发生的关系。方法:应用5-甲基胞嘧啶（5-MC）特异性抗体,采用免疫组织化学法和ELISA法,观察胸腺上皮肿瘤石蜡标本和新鲜组织标本中肿瘤上皮细胞中基因组DNA总体甲基化状态,并分析其与Masaoka病理分期、WHO组织学分型等临床病理资料间的关系。结果:按照Masaoka病理分期,肿瘤早期（Ⅰ/Ⅱ期）5-MC染色明显高于晚期（Ⅲ/Ⅳ期）病例（p＜0.01）。按照WHO组织学分型,肿瘤上皮细胞分化越差,其5-MC染色越浅。我们分别对比了A、AB、B1、B2、B3和C型六种亚型间染色差异,并参考组织病理学将A和AB型归为A组,B1、B2和B3型归为B组,C型作为C组,比较三组之间的染色差异;最后按照A/AB/B1组和B2/B3/C组比较染色差异。结果发现AB型与B1型之间5-MC染色无明显差异（p=0.455）,B3型与C型之间5-MC染色无明显差异（p=0.779）,其余各亚型之间染色差异均具有统计学意义。按照A、B和C三组,三组之间5-MC染色均具有明显差异（A和B,p＜0.01;A和C,p＜0.01;B和C,p＜0.01）。按照A/AB/B1组和B2/B3/C组,B2/B3/C组5-MC染色明显低于A/AB/B1组（p＜0.01）。ELISA法检测新鲜组织中基因组DNA甲基化情况与石蜡标本结果相似。基因组DNA总体甲基化情况与性别和合并MG无明显关系。结论:在胸腺上皮肿瘤细胞当中基因组DNA总体甲基化水平低下,随着肿瘤恶性程度的增高和肿瘤的进展,基因组DNA总体甲基化程度越来越低,且与WHO组织学分型和Masaoka病理分期关系密切。第三节胸腺上皮肿瘤中DNA甲基化转移酶表达及其临床意义的研究目的:观察DNA甲基化转移酶（DNMT）在胸腺上皮肿瘤中表达情况,并与临床病理资料进行分析,探讨DNA甲基化转移酶在胸腺上皮肿瘤异常甲基化过程中的作用。方法:采用实时定量RT-PCR技术从mRNA水平检测胸腺上皮肿瘤组织中DNMT1、DNMT3a和DNMT3b表达情况,并分析与临床、病理间的关系。结果:在胸腺上皮肿瘤组织中DNMT1、DNMT3a和DNMT3bmRNA表达随着肿瘤恶性程度和肿瘤进展呈现上升趋势。在B2/B3/C组中,DNMT1、DNMT3a和DNMT3b mRNA表达均较A/AB/B1组明显升高（DNMT1,p＜0.05;DNMT3a,p＜0.05;DNMT3b,p＜0.01）。按照Masaoka病理分期,在肿瘤晚期（Ⅲ/Ⅳ期）,DNMT的表达也明显高于肿瘤早期（Ⅰ/Ⅱ期）（DNMT1,p＜0.01;DNMT3a,p＜0.01:DNMT3b,p＜0.05）。DNMT1,DNMT3a和DNMT3b mRNA表达之间存在明显关联（DNMT1与DNMT3a,r=0.592,p=0.001;DNMT1与DNMT3b,r=0.419,p=0.033;DNMT3a与DNMT3b,r=0.777,p=0.001）。Pearson’s相关分析显示三种DNMT中仅DNMT3b高表达与基因组DNA总体低甲基化呈负相关(（DNMT1,r=-0.215,p=0.291;DNMT3a,r=-0.337,p=0.057;DNMT3b,r=-0.522,p=0.006）,DNMT1、DNMT3a和DNMT3b mRNA表达与性别和合并MG均无明显关系。结论:胸腺上皮肿瘤中三种DNMT呈高表达状态,且与Masaoka分期、WHO分型相关;三种DNMT mRNA表达上调之间相互关联,其中DNMT3b的高表达与基因组DNA总体低甲基化的关系密切。更多还原

【Abstract】 Thymic epithelial tumors（TETs） are primary neoplasms of the anterior mediastinum with highly variable histopathological characteristics.This heterogeneity has long hindered the development of a standardized protocol for diagnostic and prognostic evaluation of TETs. The modified Masaoka staging system,is currently the most commonly employed scoring method,but its value is limited by the fact that it assigns scores only for specific macroscopic surgical,but not histological, characteristics,making it difficult to reliably assess tumor severity in patients.In 1999,the World Health Organization（WHO） published histologic criteria of TETs that was based on the morphology of epithelial cells and on the ratio of lymphocyte-to-epithelial cell,which stratified TETs into six entities:types A,AB,B1,B2,B3 and C,and provided a new means of predicting states of malignancy of TETs.However,its factual accuracy is disputed.The insufficiencies of current staging systems are confounded by the fact that no genetic or epigenetic prognostic indicators have been included.Previous studies showed that multiple genetic alterations involved in many kinds of carcinomas,but the molecular mechanisms of TETs are poorly understood.Recent data suggests that carcinomas appear to be a process that is caused by genetic alterations and by epigenetic mechanism.It mainly involves DNA methylation and histone modifications.DNA methylation is one of the major mechanisms of epigenetics.Aberrant methylation of promoter regions resulting in inactivation of human tumor suppressor gene expression has been proposed to be an important mechanism in cancer. Cancer cell DNA is frequently characterized by global DNA hypomethylation,localized gene-specific hypermethylation,and overexpression of DNA methyltransferases（DNMTs）,which were so called "methylation imbalance",have already been reported in many kinds of tumors,such as lung cancer,colon cancer and oral carcinoma. Although multiple genetic and epigenetic changes have been detected in many kinds of cancers,the precise molecular mechanisms in the development and/or progression of TETs still remain unknown.In this study,we firstly evaluated the prognostic factors of TETs based on the clinic and pathologic data of the patients,then detected the expression and the methylation pattern of TSGs in TETs by using the real-time RT-PCR and the nested MSP,and the expressions of DNMTs were also measured by real-time RT-PCR.Finaly the global DNA methylation status in TETs were measured by Immunohistologic analysis and ELISA. PartⅠRetrospective study of prognostic factors of patients with thymic epithelial tumorsObjective:To investigate the prognostic factors of patients with thymic epithelial tumors（TETs）.Methods:137 patients with TETs were surgically treated at our hospital from June 1997 and September 2007.The data of patients including age,gender,symptoms,histological types,stage and grade, pathological findings and operative reports were recorded.Follow-up was obtained via telephone and postal mail.The patients were divided into MasaokaⅠ/Ⅱgroup andⅢ/Ⅳgroup,and WHO A/AB/B1group and B2/B3/C group,and the Kaplan-Meier method,log-rank test and COX regression model were performed to analyze the prognostic factors of TETs.Results:Among the 137 patients,124（90.5%） received complete resection,9（6.6%） incomplete resection,and 4（2.9%） surgicat biopsy. The rate of complete resection was significantly higher in Masaoka stagesⅠ/Ⅱthan that in stagesⅢ/Ⅳ（P＜0.001）.The overall survival rate of 5-year and 10-year were 71.4%and 50.1%,respectively.Patients in stagesⅠ/Ⅱhad a better long-term survival than those in stagesⅢ/Ⅳ（P＜0.001）.According to WHO histologic criteria,the rate of 5-year and 10-year survival in patients with type A/AB/B1 TETs were significantly higher than those in patients with type B2/B3/C TETs（P＜0.001）.The 5-year and 10-year survival rate in complete resection group were significantly better than those in incomplete resection and biopsy group （P＜0.001）.Cox regression analysis showed that the prognosis of patients with TETs was related to the Masaoka stage,WHO histologic criteria, extent of resection and the age at operation.Conclusions:The Masaoka stage,WHO histologic criteria,extent of resection and the age at operation were the important prognostic factors in patients with TETs. PartⅡAbnormal DNA methylation and its clinical significance in TETsPart A The mRNA expression levels and promoter methylation patterns of TSGs in TETsObjective:To investigate the expression and the promoter hypermethylation of TSGs and potential clinical significance in patients with TETs.Methods:Real-time RT-PCR and methylation-specific PCR（MSP） were performed for evaluated the expression and promoter region methylation patterns of TSGs in TETs.The correlation between TSGs mRNA,TSGs methylation and clinicopathological data were analyzed.Results:According to WHO criteria,The mRNA levels of six of these genes,E-cad,RARβ,hMLH1,RASSF1A,APC1A and FHIT, were significantly reduced in WHO type B2/B3/C tumors relative to WHO type A/AB/B1 tumors（E-cad,p=0.036;RARβ,p=0.002;hMLH1, p=0.024;RASSF1A,p=0.042;APC1A,p=0.02;FHIT,p=0.035）.The mRNA levels of APC1A,FHIT,RARβ,E-cad and hMLH1 were significantly reduced in stageⅢ/Ⅳrelative to stageⅠ/Ⅱ（APC1A,p= 0.005;FHIT,p=0.015;RARβ,p=0.047;E-cad,p=0.003;hMLH1, p=0.003）.Of all 65 TETs samples,the promoter region of all 9 genes in all type A group samples displayed an unmethylated phenotype,whereas 60%of type AB samples,66.7%of type B1 samples,and all of WHO type B2/B3/C samples showed promoter hypermethylation in at least one gene.The frequency of hypermethylation of individual genes varied between 15.4%（RASSF1A） and 46.2%（FHIT）.With the exception of RASSF1A and P16^INK4a,the frequency of promoter hypermethylation for each gene correlated with Masaoka stage.DAPK methylation linked to gender（p=0.034） and P16^INK4a promoter methylation correlated with the age of the patient when operated（p=0.011）.No correlations were found between TSG promoter hypermethylation and the occurrence of myasthenia gravis（MG） symptoms（p＞0.05）.Conclusion:Our findings suggest that epigenetic silencing of TSGs expression by promoter hypermethylation could play an important role in TETs,and may closely related to the Masaoka stage and WHO criteria.Part B Global DNA methylation status and its clinic significance in TETsObjective:To evaluate the global DNA methylation status in patients with TETs.Methods:Immunohistochemistry and ELISA with an antibody against 5-methylcytosine（5-MC） were performed to observe the global DNA methylation status in TETs.The results were compared with the clinicopathological data.Results:Tumors from primary stage（stageⅠ/Ⅱ） were more darkly stained compared to tumors from advanced stage（stageⅢ/Ⅳ）（p＜0.05）. We compared the stain intensity amongst different subgroups.In WHO type A,AB,B1,B2,B3 and C,significant differences were detected amongst all the six groups,except between AB and B1（p=0.455）,B3 and C（p=0.779）.We then set WHO type A and AB to group A,WHO type B1,B2 and B3 to group B,WHO type C to group C.Statistic difference were found among these three groups（group A and B,p＜0.01; group A and C,p＜0.01;group B and C,p＜0.01）.According to group A/AB/B1 and group B2/B3/C,the tumors from A/AB/B1 group were more darkly stained than that from B2/B3/C group（p=0.001）.The similar results in fresh frozen TETs tissue were found by ELISA test. Correlations between global DNA methylation levels and gender or MG symptoms were also evaluated,but no statistically significant differences were found.Conclusion:Global DNA hypomethylation is associated with increasing severity and may be a determining factor in the development of TETs. Part C The expressions of DNMTs and clinic significance in TETsObjective:To investigate the expression of DNMTs gene and its potential clinical significance in TETs.Methods:The real-time RT-PCR was performed to evaluate the expression of the DNMTs.The correlations between DNMTs mRNA and clinicopathological data were analyzed.Results:All three DNMTs were found to be significantly up-regulated in WHO type B2/B3/C tumors relative to type A/AB/B1 （DNMT1,p＜0.05;DNMT3a,p＜0.05;DNMT3b,p＜0.01）.Similarly,the expression of each DNMT was significantly increased in Masaoka stageⅢ/Ⅳtumors relative to stageⅠ/Ⅱtumors（DNMT1,p＜0.01;DNMT3a, p＜0.01;DNMT3b,p＜0.05）.Moreover,a significant negative correlation was found between DNMT3b mRNA levels and the OD value of global methylation levels（r=-0.522,p=0.006）.This correlation did not occur between OD values and DNMT1 or DNMT3a expression levels（r=-0.215, p=0.291,for DNMT1;r=-0.337,p=0.057,for DNMT3a）,although the expression of the three genes correlated with each other across all TETs samples（DNMT1 vs.DNMT3a,r=0.592,p=0.001;DNMT1 vs. DNMT3b,r=0.419,p=0.033;DNMT3a vs.DNMT3b,r=0.777,p=0.001）.Conclusion:DNMTs genes were overexpressed in TETs tissue,and overexpression of DNMTs was correlated with Masaoka pathologic system and WHO histologic criteria.更多还原