【作者】 缪珑昇；

【导师】 沈镇宙； 相加庆； 张亚伟；

【作者基本信息】 复旦大学 ， 肿瘤学， 2009， 博士

【摘要】 食管鳞癌是我国常见的恶性肿瘤之一,其发生是一个多阶段,涉及遗传学及表观遗传学的复杂过程,近年来表观遗传学改变在恶性肿瘤的发生、发展中的作用越来越受到重视,其中抑癌基因启动子甲基化作为影响基因表达的新机制被广泛研究,已陆续有文献报道其在预后方面的价值,而食管鳞癌中有关抑癌基因甲基化与临床病理特征及预后之间关系的研究相对较少。微卫星不稳定则是近年来恶性肿瘤研究的又一热点,微卫星不稳定是基因组不稳定的表现,导致细胞增殖及分化异常,促进肿瘤的发生,许多肿瘤中都发现了微卫星不稳定,而食管鳞癌中有关微卫星不稳定的研究结果差异较大。有关微卫星不稳定与hMLH1基因甲基化之间的关系尚无定论,国内也未见相关报道。因此本研究选取p16^INK4a、E-cadherin及hMLH1三个抑癌基因,探讨抑癌基因甲基化在食管鳞癌发生发展中的作用,并对微卫星不稳定在食管鳞癌中的发生情况及与hMLH1基因异常之间的关系做初步的研究。现对上述研究内容分别报告如下:第一部分食管鳞癌hMLH1、E-cadherin、p16^INK4a基因启动子甲基化的研究目的:1.检测食管鳞癌癌组织及配对正常组织中hMLH1、E-cadherin及p16^INK4a基因启动子甲基化的发生情况。2.评价上述三基因甲基化在食管鳞癌发生发展中所起到的作用。方法:收集整理105例食管鳞癌患者的临床病理资料,采用酚—氯仿法提取这105例食管鳞癌癌组织及配对癌旁正常组织的基因组DNA,运用甲基化特异PCR（Methylation Specific PCR,MSP）的方法对所提DNA分别进行hMLH1、E-cadherin及p16^INK4a基因甲基化检测,抽选MSP产物进行普通测序或克隆测序,验证PCR扩增结果。采用Envision二步法对癌组织中hMLH1、E-cadherin及p16^INK4a基因蛋白表达进行免疫组化检测。分析上述三个抑癌基因甲基化在食管鳞癌及配对正常组织中的发生情况及与临床病理特征的关系。结果:1.癌组织E-cadherin、hMLH1及p16^INK4a。基因启动子甲基化的阳性率分别为57.1%（60/105）、20.9%（22/105）和50.5%（53/105）,正常食管粘膜相应三个基因甲基化阳性率分别为10.5%（11/105）、1.9%（2/105）和7.6%（8/105）,E-cadherin（P=0.002）、hMLH1（P=0.042）及p16^INK4a（P=0.004）基因在癌组织中的甲基化率均显著高于正常食管粘膜。2.食管鳞癌E-cadherin、hMLH1及p16`（INK4a）蛋白表达阳性率分别为51.4%（54/105）、78.1%（82/105）、28.6%（30/105）,E-cadherin（P=0.021）及p16<sup>INK4a</sup>（P=0.026）基因启动子甲基化与相应蛋白失表达显著相关,而hMLH1基因甲基化与蛋白表达无相关性。3.E-cadherin基因启动子甲基化的食管鳞癌中淋巴结转移更多见（P=0.016）,p16<sup>INK4a</sup>基因启动子甲基化的食管鳞癌中低分化癌更多见（P=0.024）,hMLH1基因启动子甲基化与各项临床病理特征无显著关联。结论:1.食管鳞癌中p16<sup>INK4a</sup>及E-cadherin基因启动子甲基化较常见,癌组织的甲基化率均远高于癌旁正常食管组织,并且导致蛋白失表达,这两个甲基化位点与食管鳞癌密切相关。2.p16`（INK4a）基因启动子甲基化与肿瘤低分化有关,但与肿瘤的浸润深度及淋巴结转移等无关,提示这一变化可能出现在食管鳞癌发生的早期阶段。E-cadherin基因启动子甲基化与淋巴结转移有关,提示其在食管鳞癌的恶性进展中可能起到一定的作用。3.食管鳞癌中有一定频率的hMLH1基因启动子甲基化,但与蛋白表达无关,与各项临床病理特征也无关,可能并不直接参与食管鳞癌的发生发展。第二部分食管鳞癌术后复发危险因素的研究目的:1.分析食管鳞癌术后两年内复发转移的发生情况及与临床病理因素的关系。2.评价hMLH1、E-cadherin及p16^INK4a基因甲基化对于复发的预测价值。方法:回顾分析了2006年1月至2007年2月在复旦大学附属肿瘤医院接受了食管癌二野清扫根治手术的患者105例,总结术后两年内复发情况,结合第一部分甲基化的检测结果,应用Pearson卡方检验或精确概率法比较复发率的差异,应用Logistic回归分析判定肿瘤复发的危险因素。结果:1.2年内41例（39%）患者复发。局部-区域性复发占21例（51.2%）,其中纵隔淋巴结复发11例（52.4%）,颈部淋巴结复发5例（23.8%）。血行转移占17例（41.5%）,以肺（6例）、骨骼（5例）和肝（3例）为主,共占血行转移的82.3%,局部-区域性复发与血行转移同时发生3例（7.3%）。2.单因素分析显示淋巴结转移数目（P＜0.001）、浸润深度（P=0.002）及淋巴管、血管侵犯（P=0.001）与复发密切相关,其中淋巴结转移数目≥3个的患者术后两年复发率为69%。3.多因素Logistic回归分析显示T3期（P=0.023）,淋巴结转移数目（P=0.001）是术后复发的独立危险因子,淋巴管、血管侵犯不是独立危险因素。4.无淋巴结转移食管鳞癌患者中,E-cadherin基因甲基化是唯一与复发相关的因素,有甲基化患者的复发率显著高于无甲基化患者（33.3%VS4.2%,P=0.031）。结论:1.食管癌二野清扫术后两年内39%的患者出现复发,纵隔淋巴结、颈部淋巴结、肺、骨及肝是主要的复发部位,术后应重点对这些部位定期复查。2.T3及淋巴结转移数目是食管鳞癌术后复发的危险因素,其中淋巴结转移数目≥3枚的患者术后两年复发比例很高,应考虑采取更积极的治疗方法。3.E-cadherin基因启动子甲基化与无淋巴结转移食管鳞癌的术后复发显著相关,可以考虑将其作为新的分子标志物用于复发的预测。第三部分食管鳞癌微卫星不稳定及与hMLH1基因改变之间关系的研究目的:1.检测食管鳞癌中微卫星不稳定及hMLH1基因内的D3S1611位点杂合性缺失的发生情况。2.探讨微卫星不稳定在食管鳞癌中的作用及与hMLH1基因改变之间的关系。方法:以第一部分提取的105对癌及癌旁正常组织的基因组DNA为模板,选取BAT-26、D16s265、D9S1748及位于hMLH1基因内的D3S1611共四个微卫星位点进行检测,使用荧光标记引物扩增微卫星片段,产物进行毛细管电泳并由DNA序列检测仪自动收集荧光扫描结果,Genemapper软件进行数据收集及分析,记录并分析上述四个位点的微卫星不稳定发生情况及D3S1611位点杂合性缺失情况。结果:1.食管鳞癌MSI发生率是19%（20/105）,其中D3s1611、D9s1748及D16s265位点分别有8例、5例和7例。BAT-26位点未发现微卫星不稳定,也未发现两个位点同时有MSI（MSI-H）的病例,20例均为MSI-L。2.MSI-L与各项临床病理因素均无显著关联。3.hMLH1基因内D3s1611位点的杂合性缺失率为58.6%,但与hMLH1蛋白失表达及hMLH1基因甲基化均无显著关系。4.MSI-L与hMLH1基因甲基化、杂合性缺失及蛋白失表达均无显著关系。结论:1.食管鳞癌中仅存在一定比例的低度微卫星不稳定（MSI-L）,未发现高度微卫星不稳定（MSI-H）。2.MSI-L与各项临床病理因素均无关,在食管鳞癌的发生发展中可能不起关键作用。3.hMLH1基因异常可能不是MSI-L发生的原因。3.hMLH1基因内D3s1611位点有较高的杂合性缺失率,hMLH1基因可能是食管鳞癌基因改变的靶点,但基因缺失与甲基化及蛋白表达均无关,是否存在突变等其他异常有待进一步研究。更多还原

【Abstract】 Study of tumor suppressor gene promoter hypermethylation and microsatellite instability in esophageal squamous cell carcinoma Esophageal squamou cell carcinoma is one of the most common cancer in China.Esophageal carcinogenesis is a multistep process involving genetic and epigenetic alterations.Epigenetic alterations attracts more and more attention recently.Promoter methylation of suppressor gene,another mechanism of gene inactivation,was studied widely.Its prognostic value has been suggested in several reports.But study of the correlation between methylation and clinicopathological features or prognosis is relatively less in esophageal squamou cell carcinoma.Microsatellite instability is another hot research topic in cancer which reflect genomic instability and play an important role in tumorigenesis.It was explored in various tumor.In ESCC,the role of MSI is not clear,less reports focus on the relationship between MSI and hypermethylation of hMLH1 gene in domestic.The current research explored the role of these genetic and epigenetic changes in ESCC tumorigenesis by detected the frequency of promoter hypermethylation in hMLH1、E-cadherin and p16^INK4a gene and the incidence of MSI.The current research is comprised of the following three parts.Part 1 Study of promoter hypermethylation of hMLH1,E-cadherin,and p16^INK4a in esophageal squamous cell carcinoma.Objectives:1.To detect the frequency of hypermethylation in the promoter region of hMLH1、E-cadherin and p16^INK4A gene.2.To explore the role of these epigenetic changes in ESCC tumorigenesis.Methods:Review the clinical data of 105 patients with squamous cell carcinoma of esophagus.The genomic DNA from these patients was obtained from the frozen tumor specimens and matched normal mucosal tissue specimens using phenol-chloroform extraction.Methylation changes in the promoter region of hMLH1,E-cadherin,and p16^INK4a genes were determined by using methylation-specific PCR（MSP）.The results of MSP were confirmed by using general sequencing or clone sequencing of selected PCR products. Immunohistochemical staining of hMLH1,E-cadherin,and p16^INK4a protein was performed in the tumor tissues using Envision two-step method.The frequency of hypermethylation of hMLH1、E-cadherin and p16^INK4a in tumor tissues and normal mucosal tissue and its relationship with clinicopathological features was explored.Results:1.The positive rates of genes promoter hypermethylation of E-cadherin,hMLH1,and p16^INK4a were 57.1%（60/105）,20.9%（22/105）和50.5% （53/105） in tumor tissues respectively,which was 10.5%（11/105）、1.9% （2/105）和7.6%（8/105） in normal mucosal tissue respectively.The positive rates were significantly higher in tumor tissues than in normal mucosal tissue for each of three genes.2.The positive rates of protein expression of E-cadherin,hMLH1,and p16^INK4a were 51.4%（54/105）, 78.1%（82/105）,28.6%（30/105） respectively.Promoter hypermethylation of E-cadherin（P=0.021） or p16（INK4a）（P=0.026）gene was remarkably associated with the loss of protein.HMLH1 promoter hypermethylation was not significantly correlated with the loss of protein.3.ESCC with E-cadherin gene promoter hypermethylation was significantly correlated with lymph node metastasis（P=0.016）,p16^INK4a gene promoter hypermethylation was more likely to happen in poorly-differentiated squamous cell carcinoma （P=0.024）,hMLH1 promoter hypermethylation was not associated with any of the clinicopathological features.Conclusions:1.Promoter hypermethylation of p16^INK4a and E-cadherin genes was common in ESCC and occurs more frequent in tumor tissues than in normal mucosal tissues.They were associated with loss of protein,which might be involved in tumorigenic process of ESCC.2.P16^INK4a promoter hypermethylation was only correlated with poorly-differentiation.It may occur early in tumorigenic process of ESCC.E-cadherin promoter hypermethylation was correlated with expression deletion and lymph node metastasis,which may play an significant role in pathogenesis of ESCC.3.There were some hMLH1 promoter hypermethylation cases in ESCC,but not associated with expression deletion and clinicopathological features.It might not be involved in tumorigenesis of ESCC.Part 2 Study of risk factors for postoperative recurrence in esophageal squamous cell carcinomaObjectives:To analyze the recurrence pattern of esophageal squamous cell carcinoma and the correlation between recurrence and clinicopathological features.To explore the value of promoter hypermethylation of hMLH1、E-cadherin and p16^INK4a used as recurrence-associated prognostic indicators.Methods:Clinical data of 105 patients with squamous cell carcinoma of esophagus,who underwent esophagectomy with two-field lymph node dis section from Jan.2006 through Feb.2007,were reviewed.The recurrence pattern was investigated,along with promoter hypermethylation of hMLH1、E-cadherin and p16^INK4a.X² test（or the Fisher’s exact test） were used to analyze the difference of recurrence rate.Multivariate logistic regression analysis was carried out in order to investigate the relationship between the development of recurrence and each of independent variables.Results:1.Recurrence was recognized in 41（39%） patients within 2 years after operation.Of the 41 cases of recurrence,21（51.2%） were locoregional,including 11 cases（52.4%） of mediastinal lymph node metastasis,and 5 cases（23.8%） of cervical lymph node metastasis,17 （41.5%） were hematogenous,and mainly located at lung,bone,and liver（14,82.4%）.3 cases（17.3%）was simultaneous locoregional and hematogenous recurrence.2.Univariate analysis showed that greater lymph node metastasis（P＜0.001）、depth of invasion（P=0.002） and lymphatic vessel invasion（P=0.001） significantly predicted recurrence.The two year recurrence rate of patients with number of positive lymph node≥3 was 69%.3.Logistic analysis showed that T3（P=0.023） and number of lymph node metastasis（P=0.001） independently predicted recurrence,but lymphatic vessel invasion was not an independent risk factor.4.E-cadherin promoter hypermethylation was the only recurrence-associated prognostic indicator in patients with lymph node-negative esophageal squamous cell carcinoma.Conclusions:1.About 39%patients would develop recurrent disease within 2 years after esophagectomy with two-field lymph node dissection.The main sites of recurrence were mediastinal lymph node,cervical lymph node, lung,bone,and liver,to which much attention should be paid after operation.2.T3 and number of lymph node metastasis were the important risk factors influencing recurrence.The two year recurrence rate of patients with lymph node metastases≥3 were very high,more aggressive therpy would be helpful for them.3.E-cadherin promoter hypermethylation was significantly associated with recurrence in patients with lymph node-negative esophageal squamous cell carcinoma.Our study suggests that E-cadherin promoter methylation may be a new prognostic biomarker in lymph node-negative esophageal squamous cell carcinoma.Part 3 Study of MSI and its correlation with abnormality of hMLH1 gene in esophageal squamous cell carcinomaObjective:1.To detect the frequency of MSI and LOH of D3S1611（an introgenic marker of hMLH1 gene） in ESCC.2.To explore the role of microsatellite instability（MSI） in carcinogenesis and the relationship between MSI and abnormality of hMLH1 gene in ESCC.Methods:The genomic DNA of tumor and matched normal mucosal were obtained from the first part of study.Four microsatellite loci were selected to test microsatellite alterations,which involved BAT-26,D16s265,D9S1748,and D3S1611（an introgenic marker of hMLH1 gene）. Fluorescence polymerasechain reaction was used to amplify the microsatellite.Automatic capillary array elec-trophoresis DNA analysis system and Genemapper software was used to do the test.MSI of the four microsatellite loci and LOH of D3S1611 loci were recorded.Results:1.The incidence of MSI in esophageal squamous cellcarcinoma was 19%（20/105）.MSI in D3s1611,D9s1748 and D16s265 were detected in 8,5,and 7cases,respectively.MSI was not detected in BAT-26 loci.All MSI positive cases were MSI-L,none of MSI-H was found.2.MSI-L was not correlated with any clinicopathological characteristics.3.LOH in D3S1611,a in trogenic marker of hMLH1,was observed in 58.6%of informative cases,but it did not correlate with expression deletion or promoter hypermethylation.4.MSI-L was not associated with LOH,expression deletion,and promoter hypermethylation of hMLH1 gene.Conclusions:1.MSI-H was rare in ESCC.Relatively low-levels of MSI-L was found in ESCC.2.MSI-L was not correlated with any clinicopathological characteristics.In ESCC,MSI-L was not considered as a major event in its tumorigenesis.3.MSI-L might not correlate with the abnormality of hMLH1 gene.4.LOH of hMLH1 was common in ESCC,but it was not correlated with expression deletion or promoter hypermethylation.HMLH1 may be a target gene,but it need further reaserach.更多还原