【作者】 谷晓鸿；

【导师】 刘惜时； 马端； 卢媛；

【作者基本信息】 复旦大学 ， 妇科肿瘤， 2008， 博士

【摘要】 卵巢恶性肿瘤是死亡率最高的女性生殖系统肿瘤,其中90%以上为上皮性卵巢癌(epithelial ovarian cancer,EOC)。由于缺乏有效的早期诊断方法,70%的患者得以诊断时已达Ⅲ期或Ⅳ期,五年生存率仅为15%-20%,因此发现新的既灵敏又特异的肿瘤标志物,对卵巢癌的早期诊断,提高患者的生存率具有重要意义。不仅不同期别和病理类型的上皮性卵巢癌患者的预后差异极大;相同期别和病理类型的患者也常常对相同的治疗反应不一,预后相差也很大。产生这种异质性的根本原因很可能是由于肿瘤细胞分子生物学的特性不同。因此,建立肿瘤的分子分型,更好地指导个体化治疗,这对提高患者的生存率,准确估计预后都具有重要意义。DNA甲基化是表观遗传的主要方式之一,协助调控基因表达。近年的研究发现,DNA异常甲基化与肿瘤,包括卵巢肿瘤的发生发展有着密切的联系。DNA异常甲基化是肿瘤发生的早期事件,可早于基因及其表达产物的改变。而且肿瘤患者血清DNA含量明显升高,并具有与肿瘤DNA类似的遗传学和表观遗传学改变。因此DNA异常甲基化包括血清DNA的异常甲基化在肿瘤发病机制的研究、早期诊断和指导治疗方面有着广泛的应用前景。本研究首先使用基于芯片技术的差异甲基化杂交(differential methylationhybridization,DMH),在全基因组范围建立上皮性卵巢癌的异常甲基化模式,探讨基于该模式的分子分型在辅助预后中的应用价值。之后用Taqman实时荧光定量PCR(MethyLight)对DMH结果进行大样本的组织学验证,寻找新的卵巢癌特异性的肿瘤标志物,为探索可用于上皮性卵巢癌早期诊断的新方法奠定基础。最后探讨血清DNA定量及甲基化标志物检测在卵巢癌诊断中的应用价值。第一部分:目的:检测人上皮性卵巢癌的DNA异常甲基化模式,探讨其在分子分型及发现新的癌相关基因中的应用价值。方法:用激光显微切割(laser microdissection,LMD)技术从20例冻存的原发性上皮性卵巢癌组织中获取肿瘤细胞作实验组,用原代培养的5例正常卵巢上皮细胞(human normal ovarian surface epithelium,HOSE)作对照组,用基于芯片技术的DMH检测人上皮性卵巢癌的DNA异常甲基化模式。用层次聚类法建立基于该甲基化模式的分子分型,用COX回归模型分析该分子分型及患者的各项临床指标与生存预后的相关性。序列比对分析各异常DNA甲基化位点与临近基因启动子的位置关系。结果:182个过甲基化位点和64个低甲基化位点(阳性率25%以上的点分别有18个和31个)组成了人上皮性卵巢癌的DNA异常甲基化模式。层次聚类法建立基于全部过/低甲基化位点、低甲基化位点、过甲基化位点的三种分子分型,COX回归模型分析发现除病理类型、病理分级、手术病理分期之外,基于过甲基化的分子分型也是患者预后的影响因子,其影响患者预后的风险系数为13.459(P=0.022)。DNA序列比对分析发现有15个异常甲基化位点位于某些基因启动子区CpG岛(CpG island,CGI),提示这些基因可能是通过启动子区的异常甲基化而参与卵巢癌的发生发展。结论:基于芯片技术的差异甲基化杂交是一种高通量的DNA异常甲基化模式的筛查方法,人上皮性卵巢癌的DNA异常甲基化模式在分子分型及发现新的癌相关基因中都具有很好的应用价值。第二部分:目的:检测上皮性卵巢癌患者肿瘤组织DNA多个基因启动子区CGI的甲基化状态,验证前期DMH芯片结果,寻找新的卵巢癌特异性的肿瘤标志物,为探索可用于上皮性卵巢癌早期诊断的新方法奠定基础。方法:选择7个DMH结果显示在上皮性卵巢癌中低甲基化的基因启动子CGI,用MethyLight检测其在87例上皮性卵巢癌(含20例前期DMH检测的原发病例)和42例卵巢良性病变患者肿瘤组织中的甲基化状态。结果:前期DMH检测的20例原发性上皮性卵巢癌患者肿瘤组织DNA中,7个CGI均呈不同程度的低甲基化。上皮性卵巢癌和卵巢良性病变患者组织DNA中,基因LSM2、EGFLAM和CDKN2A的甲基化率依次为11%(10/87)和33%(14/42)、8%(7/87)和21%(9/42)、9%(8/87)和31%(13/42),与卵巢良性病变患者相比,上皮性卵巢癌患者三个基因甲基化程度均有显著下降(P＜0.05,χ~2检验)。三个CGI组合的甲基化率(三个CGI中至少有一个出现甲基化),全部上皮性卵巢癌患者(19/87,22%)和Ⅰ期上皮性卵巢癌患者(10/33,30%)均比良性卵巢病变患者(23/42,55%)显著降低(P＜0.05,χ~2检验)。结论:大样本肿瘤组织MethyLight检测很好的验证了DMH芯片结果,基因EGFLAM、CDKN2A和LSM2启动子区CGI有可能成为新的上皮性卵巢癌特异性的低甲基化肿瘤标志物,联合检测三个CGI的甲基化程度可能有助于卵巢癌的早期诊断。第三部分:目的:检测上皮性卵巢癌患者血清DNA水平,及基因EGFLAM、CDKN2A和LSM2启动子区CGI的甲基化程度,探索可用于上皮性卵巢癌诊断的新方法。方法:用微量基因组DNA抽提试剂盒提取30例原发性上皮性卵巢癌、15例卵巢良性病变患者血清DNA,用SYBR Green I荧光染色法测定其含量,用MethyLight检测基因EGFLAM、CDKN2A和LSM2启动子区CGI甲基化程度。结果:30例原发性上皮性卵巢癌患者血清DNA含量(59.69±88.43ng/ml)显著高于15例卵巢良性病变患者(21.35±10.02ng/ml,P＜0.05)。血清DNA中基因EGFLAM、CDKN2A和LSM2启动子区CGI的甲基化率,上皮性卵巢癌患者(依次为17%(5/30)、10%(3/30)、33%(10/30))比卵巢良性病变患者(依次为33%(5/15)、20%(3/15)、60%(9/15))降低,但差异无显著性(P＞0.05,χ~2检验)。血清DNA前述三个CGI组合的甲基化率(三个CGI中至少有一个出现甲基化),上皮性卵巢癌患者(53%,16/30)比卵巢良性病变患者(80%,12/15)降低,但差异无显著性(P=0.074,χ~2检验);而晚期上皮性卵巢癌患者(40%,8/20)比卵巢良性病变患者(80%,12/15)显著降低(P=0.015,χ~2检验)。将血清DNA含量(33ng/ml以上)及其甲基化状态(三个CGI均未出现甲基化者诊断为上皮性卵巢癌)两项指标结合用于上皮性卵巢癌的诊断,总的敏感性和特异性分别是70%和73.3%。结论:上皮性卵巢癌患者血清DNA定量及其甲基化标志物EGFLAM、CDKN2A和LSM2基因启动子区CGI的甲基化状态检测有可能成为辅助上皮性卵巢癌诊断的新方法。更多还原

【Abstract】 Ovarian cancer has the highest mortality rate of the female reproductive malignancies,the majority of which(＞90%) are believed to derive from the ovarian surface epithelium. Asymptomatic and absent of efficient diagnostic approaches in its early stages,most ovarian cancers are diagnosed at the late stages ofⅢandⅣ,which makes 5-year survival for patients markedly down to less than 20%.Therefore it is very important for patients’ survival to find novel tumor markers with high sensitivity and specificity in early diagnosis.Besides,patients with epithelial ovarian cancer have greatly different survival among those in divergent as well as identical clinical stages and pathologic types who reacting differently to same therapies.Distinct biomolecular characteristics of tumor cells are hypothesized underlying the heterogeneity.Therefore it is very important for improve and estimate patients’ survival to establish tumor molecular classification,guiding individual treatment better.As one of the most frequent epigenetic event,DNA methylation aids in regulating genes’ expression.Recent studies have shown that aberrant DNA methylation has strong association with the development and progression of tumor,including ovarian cancer.Being early events in tumorigenesis,aberrant DNA methylation could occur before the alteration of gene and its expression products.In addition,serum DNA of tumor patients would increase obviously in the level and exhibit genetic and epigenetic characteristics identical with tumor DNA.So aberrant DNA methylation including that occurred in serum DNA has intensive perspective in tumorigenesis,early diagnosis and treatment guidance.In the present study,a method called DMH(differential methylation hybridization) based on microarray technology was used to investigate genomic DNA methylation pattern in tumor cells. Molecular classification concerning this methylation profile was then interrogated for its merit in prognosis evaluation.Then large-scale ovarian cancer tissues was tested by fluorescence Taqman real-time quantitative PCR(MethyLight) to confirm the differential methylation detected using DMH and to search for novel cancer-specific tumor markers,laying the foundation for future application research of new method in early diagnosis of human epithelial ovarian cancer.Finally, the application value of serum DNA quantification and its methylation markers detection in diagnosis of ovarian cancer was discussed.The first part:Objective To profile methylation alterations of CpG islands in human epithelial ovarian cancer and interrogate its applications in molecular classification and finding new cancer related genes.Methods Cancer cells were obtained by laser microdissection from 20 frozen-preserved human epithelial ovarian tumors.Epithelial ceils secured from 5 normal ovaries were primary cultured.Differential methylation hybridization(DMH) based on microarray assay was conducted using the DNAs of the two groups of cells mentioned above to construct the aberrant DNA methylation pattern of human epithelial ovarian cancer.The correlation between the patients’ survival and their molecular classification derived from the methylation pattern via hierarchical clustering and other clinical indexes was analyzed by COX regression.Sequences of the aberrant methylated DNA loci and the nearby genes were compared. Results The aberrant DNA methylation pattern of human epithelial ovarian cancer includes 182 hypermethylated loci and 64 hypomethylated loci(18 and 31 loci,individually,were positive in more than 25%arrays).The 20 patients were classified via hierarchical clustering on the basis of hyper-,hypo- and entire hyper-/hypo- methylated loci,respectively.COX regression analysis revealed that the impact factors of patients’ survival were comprised of pathological type, pathological grade,operation-pathological stage and molecular type based on hyper-methylation, and the risk ratio of the latter was 13.459(P=0.022).15 loci located in CpG islands(CGI) of some genes’ promoters,indicating that these genes may participate in the development and progression of ovarian cancer through aberrant promoter methylation.Conclusions Differential methylation hybridization is a high throughput method which could screen aberrant DNA methylation patterns of many diseases.The pattern of human epithelial ovarian cancer could be applied to molecular classification and finding new cancer related genes.The second part:Objective To investigate DNA methylation alterations of several promoter CpG islands in human epithelial ovarian cancer tissues,verifying the differential methylation detected by DMH in our previous study.To identify novel candidate cancer-specific epigenetic markers,laying the foundation for future application research of new method in early diagnosis of human epithelial ovarian cancer.Methods MethyLight was conducted to verify the methylation status of 7 hypomethylated promoter CGIs detected by DMH in tumor tissues of 87 patients with ovarian cancer(including the 20 primary ovarian cancer tissues used in our previous DMH assay) and 42 patients with benign ovarian diseases.Results The 7 CGIs were hypomethylated in different degrees in the 20 primary epithelial ovarian cancer tissues tested by our previous DMH assay.The methylation ratio of gene LSM2, EGFLAM and CDKN2A in tissue DNA of patients with epithelial ovarian cancer and benign ovarian diseases was 11%(10/87) versus 33%(14/42),8%(7/87) versus 21%(9/42),9%(8/87) versus 31%(13/42),respectively.The methylation degree of all the three gene was significantly decreased in patients with ovarian cancer compared to the patients with benign ovarian diseases (P＜0.05,χ~2 test).The methylation ratio of 3 CGI panel(at least one CGI methylated) was significantly decreased in all ovarian cancer patients(19/87,22%) and patients in I stage(10/33, 30%) compared to the patients with benign ovarian diseases(23/42,55%,P＜0.05,χ~2 test).Conclusions Differential methylation in tumor cells determined by DMH was well confirmed by MethyLight using large-scale tumor tissues,identifying three promoter CGIs of gene LSM2, EGFLAM and CDKN2A as novel candidate cancer-specific hypomethylated tumor markers.The total methylation status of the 3 CGIs may help in early diagnosis of ovarian cancer.The third part:Objective To quantify serum DNA of patients with epithelial ovarian cancer and to detect the methylation status of the promoter CpG islands(CGIs) of gene EGFLAM、CDKN2A and LSM2, searching for novel diagnosis methods for human epithelial ovarian cancer.Methods The preoperative serum DNA of 30 patients with primary epithelial ovarian cancer and 15 patients with benign ovarian diseases was extracted using micro-genomic DNA extraction kit and quantified by SYBR greenⅠfluorescent staining.The methylation status of the promoter CGIs of gene EGFLAM、CDKN2A and LSM2 was evaluated by Taqman fluorescent quantitative real-time PCR(MethyLight). Results The serum DNA concentration of 30 patients with primary epithelial ovarian cancer (59.69±88.43ng/ml) was significantly higher than that of 15 patients with benign ovarian diseases (21.35±10.02ng/ml,P＜0.05).In serum DNA,the methylation ratio of the promoter CGIs in gene EGFLAM、CDKN2A and LSM2 of patients with epithelial ovarian cancer(17%(5/30),10% (3/30),33%(10/30),separately) was lower,but not significantly(P＞0.05,χ~2 test),than that of patients with benign ovarian diseases(33%(5/15),20%(3/15),60%(9/15),separately).The methylation ratio of the CGI panel in serum DNA(at least one of the above three CGIs methylated) of patients with epithelial ovarian cancer(53%,16/30) was lower,but not significantly(P=0.074,χ~2 test),than that of patients with benign ovarian diseases(80%,12/15).While the CGI panel’s methylation ratio of late-stage patients(40%,8/20) was significantly lower(P=0.015,χ~2 test) than that of patients with benign ovarian diseases(80%,12/15).The total diagnostic sensitivity and specificity of the serum DNA level(＞33ng/ml) and its methylation status(none of the three CGIs methylated) were 70%and 73.3%,respectively.Conclusions Quantification of serum DNA of patients with epithelial ovarian cancer and its methylation markers of the promoter CGIs in gene EGFLAM、CDKN2A and LSM2 might be novel auxiliary methods for the diagnosis of epithelial ovarian cancer.更多还原