卵巢上皮性肿瘤中Skp2、p27kipl、E-cad的表达

目的检测Skp2、p27kipl和E-cad在卵巢上皮性肿瘤中的表达情况及其相互关系。方法采用免疫组化SP法，检测99例卵巢上皮性肿瘤组织中Skp2、p27kipl及E-cad的表达。结果p27kipl在卵巢良性肿瘤、交界性肿瘤的表达率分别为76．5％、70．6％高于卵巢癌29．2％（P〈0．05），在早期癌中的表达高于晚期癌（P〈0．05）。Skp2在卵巢良性肿瘤、交界性肿瘤的表达率分别为0、23．5％，均低于卵巢癌50．8％（P〈0．05），在早期癌的表达低于晚期癌（P〈0．05）。卵巢癌中skp2表达与组织分化有关，在低分化癌的阳性表达率高于高分化癌（P〈0．05），在组织学类型方面，浆液性癌中skp2的阳性表达率高于非浆液性癌（P〈0．05）。E—cad在良性肿瘤、交界性肿瘤和卵巢癌表达率分别为88．2％、82．4％、55．4％，恶性组低于良性组（P〈0．05）。E—cad在早期癌的表达率高于晚期癌（P〈0．05）。Skp2表达与p27kipl表达呈负相关（P〈0．05），E-cad表达与p27kipl表达呈正相关（P〈0．05），而E-cad表达与Skp2表达则呈负相关（P〈0．05）。结论skp2过度表达与p27kipl表达减少可能与卵巢上皮性肿瘤的发生发展密切相关，而E-cad在晚期卵巢癌中表达降低可能反映了卵巢癌分化程度的降低。     

CDH1基因启动子甲基化在上皮性卵巢癌转移方面的研究

目的探讨CDH1基因启动子甲基化对上皮性卵巢癌转移的影响。方法采用免疫组织化学方法检测38例正常卵巢上皮和80例上皮性卵巢癌组织中E-钙黏附素（E-cadherin）表达；应用甲基化特异的PCR（MSP）检测上述组织中CDH1基因启动子区甲基化；应用5-氮-2＇-脱氧胞苷（5-aza-CdR）使SKOV3细胞去甲基化，观察SKOV3细胞体外侵袭性的改变，并通过RT-PCR检测CDH1基因的改变。结果E-cadherin在正常卵巢组织中表达明显高于上皮性卵巢癌（P〈0．05）。34例CDH1基因启动子区甲基化全部出现在卵巢癌组织中，有淋巴结转移组织中甲基化明显高于无淋巴结转移者（P〈0．05），而CDH1基因启动子区有甲基化的卵巢癌组织中E-cadherin表达明显降低（P〈0．05）。经5-Aza-CdR处理后的SKOV3细胞体外侵袭性降低（P〈0．01），CDH1基因的表达明显上调（P〈0．01）。结论E-cadherin表达降低与上皮性卵巢癌转移关系密切，CDH1基因启动子区甲基化可能是导致E-cadherin蛋白表达减低的重要原因之一，因此启动子区甲基化与上皮性卵巢癌转移有关。     

BARD1剪切变异体在卵巢癌组织中的表达及临床意义

目的探讨卵巢癌肿瘤抗原新基因OCY-142即BARD1的剪切变异体在卵巢癌中的表达及临床意义。方法用RT—PCR的方法检测OCY-142在卵巢癌组织和正常卵巢组织中的表达。结果在28例卵巢癌组织中,有10例（35．7％）明显表达OCY-142抗原基因,而30例正常卵巢组织中均没有OCY-142抗原基因的表达,两者之间有显著性差异（P〈0．05）。OCY-142的表达与卵巢癌的病理类型有关,在浆液性乳头状囊腺癌患者中有较高的特异性（P〈0．05）,但OCY-142的表达与卵巢癌的临床分期和组织分化程度没有相关性（P〉0．05）。结论卵巢癌肿瘤抗原基因OCY-142是BARD1基因的1个剪切变异体,其有可能成为卵巢恶性肿瘤免疫治疗的新靶点。OCY-142在卵巢浆液性囊腺癌中有较高的表达,其临床意义值得进行更深入的研究。     

应用荧光原位杂交技术检测卵巢浆液性囊腺癌中ZNF217基因拷贝数

目的探讨锌指蛋白217（zinc finger protein 217，ZNF217）基因在卵巢浆液性囊腺癌中20号染色体基因拷贝数改变情况及其临床意义。方法应用荧光原位杂交技术及LSI ZNF217探针对2种卵巢癌细胞株SKOV3、HO-8910和23例卵巢浆液性囊腺癌、10例浆液性囊腺瘤及7份正常卵巢组织进行检测。结果两种卵巢癌细胞株及12例卵巢癌出现ZNF217基因扩增，浆液性囊腺瘤有1例发生了基因的扩增，其余的及正常卵巢组织未出现基因扩增，卵巢癌与卵巢浆液性囊腺瘤、正常卵巢细胞相比较，ZNF217基因拷贝数改变差异有统计学意义（P〈0．05），而低分化的卵巢癌比高分化发生ZNF217基因扩增的几率明显增高（P〈0．05），Ⅲ～Ⅳ期比Ⅰ期的卵巢癌ZNF217基因拷贝数明显增多（P〈0．05）。结论ZNF217基因很可能是卵巢癌发生的促进因子之一，并与卵巢癌分化及预后不良有关。     

卵巢浆液性和黏液性肿瘤MUC1、MUC2的表达及其意义

目的探讨卵巢浆液性和黏液性肿瘤中黏蛋白MUC1、MUC2的表达与临床病理特征的相关性。方法免疫组化S—P法检测90例卵巢浆液性和黏液性肿瘤的黏蛋白MUC1、MUC2的表达,并对其中50例恶性病例作生存分析。结果（1）交界性与恶性卵巢肿瘤中黏蛋白MUC1的表达阳性率明显高于良性肿瘤,差异有显著性（P〈0．001）;黏蛋白MUC1与WHO病理分级、FIGO临床分期、大网膜转移显著相关（P〈0．05）。（2）黏蛋白MUC2与组织学类型、WHO病理分级相关（P〈0．05）。（3）黏蛋白MUC1与MUC2呈负相关（P〈0．05）。（4）对50例恶性浆液性和黏液性肿瘤进行的生存分析中,单因素分析显示：WHO病理分级、FIGO临床分期、大网膜转移、MUC1表达程度与预后相关（P〈0．05）,而多因素分析中只有FIGO临床分期、MUC1表达程度具有独立的预后意义（P〈0．05）,Kaplan—Meier生存曲线分析显示,Ⅲ、Ⅳ期较Ⅰ、Ⅱ期生存率差异有显著（P〈0．01）,MUC1阳性组和阴性组生存率差异有显著性（P〈0．01）。结论黏蛋白MUC1、MUC2与恶性卵巢浆液性和黏液性肿瘤的浸润、转移相关,Ⅲ、Ⅳ期肿瘤、MUC1强表达可作为恶性卵巢浆液性和黏液性肿瘤预后不良的可行性指标。     

血清YKL-40和CA125联合检测在卵巢癌诊断中的应用

目的探讨血清YKL-40和CA125联合检测在卵巢癌诊断中的应用价值。方法分别用酶免分析法（EIA）和微粒子酶免分析法（MEIA）测定39例卵巢癌、27例卵巢良性肿瘤及40例健康对照妇女血清YKL-40和CA125水平,YKL-40以健康对照组95％可信区间的上限值为阳性,比较YKL-40和CA125在三组间、卵巢癌病人不同临床分期和手术治疗前后水平和阳性率的差异。结果正常对照组血清YKL-40水平的95％可信区间的上限值为71．9ng／mL;卵巢癌患者血清YKL-40水平及阳性率显著高于卵巢良性肿瘤组和对照组（t〉4．83,P〈0．01）,而卵巢良性肿瘤组和对照组之间差异无统计学意义（t=0．96,P〉0．05）。Ⅲ／Ⅳ期卵巢癌患者血清YKL-40水平显著高于Ⅰ／Ⅱ期患者（167．9ng／mL vs 87．7ng／mL,t=1．86,P〈0．01）。卵巢癌患者术后第3天和第7天血清YKL-40水平显著低于术前水平（t〉2．92,P〈0．01）;未能手术切除的患者血清YKL-40水平显著高于手术切除者（t=4．06,P〈0．01）。YKL-40联合CA125诊断卵巢癌的灵敏度为87．2％,特异性为91．0％,阳性预测值为85．0％,阴性预测值为92.4％;YKL-40与CA125联合诊断早期卵巢癌的灵敏度从CA125单指标的33．3％提高到66．7％。结论YKL-40是一种新的诊断卵巢癌的肿瘤标志物,联合YKL-40与CA125检测可提高对早期卵巢癌诊断的灵敏度。     

髓母细胞瘤与幕上原始神经外胚叶肿瘤中RASSF1A基因的甲基化差异

目的通过研究髓母细胞瘤与幕上原始神经外胚叶肿瘤（SPNET）中RASSF1A基因的甲基化改变,探讨颅内原始神经外胚叶肿瘤（PNET）的不同亚型中该基因的表遗传学差异及其意义。方法收集25例原发髓母细胞瘤,9例原发SPNET,3株髓母细胞瘤细胞系和2株SPNET细胞系。采用甲基化特异性聚合酶链反应（MSP）检测RASSF1A基因启动子区的甲基化状态。应用去甲基化试剂5-aza-2’deoxycytidine处理存在基因表达缺失的细胞系,探讨基因表达与甲基化之间的关系。结果100％（25／25）的原发髓母细胞瘤、6／9的原发SPNET及全部PNET细胞系中均检测到RASSFIA基因的甲基化。相反,该基因甲基化在全部正常组织（包括2例小脑,5例大脑）中均未检测到。并且,RASSF1A在SPNET中的甲基化率明显低于髓母细胞瘤（Fisher精确检验,P=0．014）。在经去甲基化试剂处理的PNET细胞中,该基因表达得以恢复,证明甲基化与该基因沉默相关。结论RASSF1A甲基化是肿瘤特异性的,RASSF1A甲基化与PNET的发生有一定关联,不同亚型的PNET之间RASSF1A基因的不同甲基化状态提示髓母细胞瘤和SPNET是表遗传学上存在差异的两类肿瘤。     

肿瘤抑制基因DNA甲基化与卵巢癌的研究进展

卵巢癌是妇科常见的恶性肿瘤,严重威胁着女性的生命和健康。研究发现,DNA异常甲基化,尤其是基因启动子Cp G岛高甲基化,使肿瘤抑制基因RASSF1A、BRCA1等失活,在卵巢癌发生、发展中起重要作用。综述肿瘤抑制基因DNA异常甲基化在卵巢癌中的研究进展,可为卵巢癌发病机制、早期诊断、治疗及判断预后提供新的方法。     

PTEN蛋白和FHIT蛋白在上皮性卵巢癌中的表达及临床病理意义

目的检测抑癌基因蛋白PTEN、FHIT表达在卵巢癌发展中的作用及相关病理机制。方法采用免疫组化法检测45例卵巢癌组织、10例卵巢交界性肿瘤、10例卵巢良性肿瘤、5例正常卵巢组织中PTEN和FHIT蛋白表达，并比较这两种蛋白表达与临床病理特征的关系。结果卵巢癌和交界性卵巢肿瘤组织中PTEN蛋白和FHIT蛋白表达显著低于卵巢囊肿和正常卵巢（P〈0．05），在上皮性卵巢癌中这两种蛋白表达与组织分化程度正相关（P〈0．05），它们表达降低或失活与卵巢癌淋巴结转移相关（P〈0．005），但与组织学类型无显著相关性。PTEN蛋白表达与恶性肿瘤临床分期负相关．FHIT蛋白表达与临床分期无显著相关性。这两种蛋白在上皮性卵巢癌中表达的相关性有统计学意义（P=0．0343）。结论在卵巢癌发生过程中PTEN蛋白表达降低起到重要作用，预示着卵巢癌的不良预后；FHIT蛋白缺失是恶性上皮性卵巢肿瘤的特征，其表达缺失在卵巢肿瘤的发生发展中起一定作用，可能与上皮性卵巢癌的恶性进展及转移有关．这两种蛋白可能协同促进卵巢癌的发生、发展、浸润及转移。     

p73基因在卵巢上皮性肿瘤中的表达及甲基化研究

目的 探讨卵巢上皮性肿瘤中p73蛋白的表达和基因启动子的甲基化情况,并观察其与临床病理学特征的关系.方法 制备包括68例卵巢癌、37例卵巢交界性肿瘤和21例卵巢良性肿瘤的组织芯片,用免疫组织化学EnVision法检测上述组织中p73蛋白表达情况,用亚硫酸氧盐修饰后测序法检测13例新鲜卵巢癌组织及5例新鲜卵巢交界性肿瘤组织的p73基因启动子甲基化情况.结果 92.6％ (63/68)的卵巢癌表达p73,p73蛋白总体表达率均值为32％(p73表达率指p73阳性细胞数所占的百分比),其中浆液性癌( 26/26)的表达率均值为40％,高于其他组织类型的癌(P=0.006).按照卵巢癌发病模式区分,Ⅱ型卵巢癌p73表达率均值(40％)高于Ⅰ型卵巢癌(24％),P=0.010.卵巢癌中p73的表达与临床分期及组织学分级无相关性(均P＞0.05).卵巢交界性肿瘤组(30/37)和良性肿瘤组(12/21)p73的总体表达率均值分别为16％和15％,该两组肿瘤中浆液性肿瘤表达率均值均高于黏液性肿瘤(P-0.003,P=0.026).卵巢癌组的p73阳性表达率均值明显高于交界性肿瘤组和良性肿瘤组(均P ＜0.05),交界性肿瘤组与良性肿瘤组比较差异无统计学意义(P＞0.05).浆液性肿瘤( 49/53)中,卵巢癌组(26/26) p73阳性表达率均值明显高于交界性肿瘤组(12/14)和良性肿瘤组(11/13;P =0.024和P=0.002),而卵巢交界性肿瘤组和良性肿瘤组比较差异无统计学意义(P=0.428).黏液性肿瘤(15/27)中,卵巢癌组(6/7)p73阳性表达率均值高于良性肿瘤组( 1/8;p=0.032),而卵巢癌组与卵巢交界性肿瘤组(8/12)、交界性肿瘤组与良性肿瘤组比较,差异均无统计学意义(P=0.234和P=0.201).p73启动子的甲基化结果显示,13例卵巢癌有8例发生甲基化,但每例样本甲基化频率有所不同,总体甲基化频率均值为8.0％.5例交界性肿瘤有2例发生甲基化,总体甲基化频率均值为9.0％,两组比较差异无统计学意义(P＞0.05).卵巢癌组p73甲基化额率与组织类型、发病模式、组织学分级及临床分期均无相关性(均P＞0.05).结论 卵巢上皮性肿瘤多数表达p73,卵巢癌p73的表达率均值明显高于交界性肿瘤和良性肿瘤,浆液性肿瘤高于其他组织类型;p73蛋白表达率与p73基因甲基化程度不存在简单线性相关关系.     

RASSF1基因转录本A和C的表达与卵巢癌关系的研究

目的探讨RASSF1基因转录本A和C在多个卵巢癌细胞系和卵巢癌组织中所起的作用。方法应用逆转录聚合酶链反应(RT PCR)和激光捕获显微切割技术检测3个卵巢癌细胞系和80例人原发卵巢上皮性恶性肿瘤组织中RASSF1A和RASSF1CmRNA的表达。结果RASSF1A mRNA在卵巢癌SK OV3细胞中表达缺失;RASSF1A和RASSF1C在人卵巢癌组织中的表达率分别为40.0%(32/80)和91.3%(73/80)。RASSF1AmRNA的表达在浆液性癌、黏液性癌和内膜样癌组织中分别是41.2%(20/48),38.1%(8/21)和36.4%(4/11),P>0.05;临床Ⅰ期和Ⅱ期分别为71.4%和75.0%(10/14,9/12),明显高于临床Ⅲ期和Ⅳ期(26.7%、12/45和14.1%,1/9),P<0.05;高和中分化组分别为58.6%和50.0%(17/29,10/20),明显高于低分化组(16.1%,5/31),P<0.05。结论卵巢癌细胞和人原发卵巢癌组织中存在RASSF1AmRNA表达的缺失,RASSF1A的表达与卵巢癌的临床分期和组织学分级有关,可能作为一种新的抑癌基因在卵巢癌的发生和发展过程中起重要作用,RASSF1AmRNA的缺失提示预后不良。     

Aberrant hypermethylation of RASSF1A promoter in ovarian borderline tumors and carcinomas

The newly identified 3p21.3 tumor suppressor gene RASSF1A is inactivated by hypermethylation in variable solid tumors, including those of the lung, breast, prostate, kidney, and ovary. The purpose of this study was to evaluate the methylation status of RASSF1A in various types and stages of ovarian epithelial tumors. We analyzed the DNA methylation status of ovarian tumors using methylation-specific polymerase chain reaction in 54 frozen ovarian tumor tissues and in 97 cases of archival ovarian serous epithelial tumors using a microdissection procedure. Hypermethylation statuses were examined vs clinicopathologic findings. RASSF1A promoter methylation rates in the various types of fresh ovarian tissues were as follows: serous cystadenoma (1/5), serous tumor of borderline malignancy (2/7), serous adenocarcinoma (4/10), mucinous cystadenoma (0/5), mucinous tumor of borderline malignancy (2/7), mucinous adenocarcinoma (3/6), transitional-cell carcinoma (1/3), clear-cell carcinoma (3/3), and malignant müllerian mixed tumor (3/3). In archived serous tumor tissues, RASSF1A promoter hypermethylation was detected in serous cystadenoma (1/6, 16.6%), serous tumor of borderline malignancy (20/41, 48.8%), and in serous adenocarcinoma (25/50, 50%). The status of RASSF1A hypermethylation in borderline tumors was found to correlate statistically with the presence of microinvasion (p=0.002), peritoneal implant (p<0.001), and bilaterality (p=0.019). The RASSF1A promoter hypermethylation was frequently found in borderline tumors and carcinomas, suggesting that RASSF1A promoter hypermethylation may be a useful molecular marker for the early detection of ovarian tumors.     

Dysregulation of the epigenome in triple-negative breast cancers: Basal-like and claudin-low breast cancers express aberrant DNA hypermethylation

A subset of human breast cancer cell lines exhibits aberrant DNA hypermethylation that is characterized by hyperactivity of the DNA methyltransferase enzymes, overexpression of DNMT3b, and concurrent methylation-dependent silencing of numerous epigenetic biomarker genes. The objective of this study was to determine if this aberrant DNA hypermethylation (i) is found in primary breast cancers, (ii) is associated with specific breast cancer molecular subtypes, and (iii) influences patient outcomes. Analysis of epigenetic biomarker genes (CDH1, CEACAM6, CST6, ESR1, GNA11, MUC1, MYB, SCNN1A, and TFF3) identified a gene expression signature characterized by reduced expression levels or loss of expression among a cohort of primary breast cancers. The breast cancers that express this gene expression signature are enriched for triple-negative subtypes — basal-like and claudin-low breast cancers. Methylation analysis of primary breast cancers showed extensive promoter hypermethylation of epigenetic biomarker genes among triple-negative breast cancers, compared to other breast cancer subclasses where promoter hypermethylation events were less frequent. Furthermore, triple-negative breast cancers either did not express or expressed significantly reduced levels of protein corresponding to methylation-sensitive biomarker gene products. Together, these findings suggest strongly that loss of epigenetic biomarker gene expression is frequently associated with gene promoter hypermethylation events. We propose that aberrant DNA hypermethylation is a common characteristic of triple-negative breast cancers and may represent a fundamental biological property of basal-like and claudin-low breast cancers. Kaplan–Meier analysis of relapse-free survival revealed a survival disadvantage for patients with breast cancers that exhibit aberrant DNA hypermethylation. Identification of this distinguishing trait among triple-negative breast cancers forms the basis for development of new rational therapies that target the epigenome in patients with basal-like and claudin-low breast cancers.     

High frequency of promoter hypermethylation of RASSF1A in tumorous and non-tumourous tissue of breast cancer

AIM: To determine the presence of RASSF1A promoter methylation in tumorous and non-tumorous tissues of breast cancer. METHODS: Methylation-specific PCR was used to detect RASSF1A methylation in DNA extracted from tumorous and paired non-tumorous tissues of 40 breast cancer patients. The associations of RASSF1A hypermethylation with clinicopathological characteristics in tumorous and non-tumorous breast tissues were analysed. RESULTS: RASSF1A promoter hypermethylation was detected in 38 of the 40 breast cancer tissues (95%) and 37 of the paired non-tumorous tissues (92.5%). When compared with the non-tumorous tissues, aberrant methylation was detected to be higher in 24 of the tumorous tissues (60%). The latter was found to be associated with lower histological grade tumours (p=0.048). CONCLUSION: RASSF1A promoter hypermethylation occurred at a high frequency in breast cancer tumorous and non-tumorous tissues; the majority of tumours have a higher level of methylation status when compared with non-tumorous tissues. This supports the notion that RASSF1A methylation is an early and premalignant alteration.     

Methylation profiles of hereditary and sporadic ovarian cancer

Bol G M, Suijkerbuijk K P M, Bart J, Vooijs M, van der Wall E & van Diest P J
(2010) Histopathology 57, 363–370
Methylation profiles of hereditary and sporadic ovarian cancer Aims: Tumour suppressor gene silencing through promoter hypermethylation plays an important role in oncogenesis. Carcinogenesis of hereditary cancers usually differs from that of their sporadic counterparts, but methylation has hardly been studied in hereditary ovarian cancer. The aim of this study was to investigate promoter methylation of a set of common tumour suppressor genes in BRCA1‐related ovarian cancer in comparison with sporadic ovarian cancer. Methods and results: Methylation‐specific multiplex ligation‐dependent probe amplification was used to assess the extent of promoter methylation of 24 different tumour suppressor genes in BRCA1‐associated (n = 25) and matched sporadic ovarian tumours (n = 50). A cumulative methylation index (CMI) was calculated and differences between individual genes were analysed. There was no significant difference in cumulative methylation between BRCA1‐associated and sporadic ovarian carcinomas (median CMI 108; CMI 110; P = 0.86). Also, methylation patterns of individual genes did not show distinct differences after correction for multiple comparisons. CDH13, GSTP1 and RASSF1 were frequently methylated in both sporadic and hereditary ovarian cancers. BRCA1 methylation occurred in 14% of sporadic tumours, but was not detected in BRCA1‐associated tumours. Conclusions: CDH13, GSTP1 and RASSF1 are frequently methylated in both sporadic and BRCA1‐associated ovarian cancers. Interestingly, methylation of BRCA1, while frequent in sporadic ovarian cancer, never occurred in the hereditary group. BRCA1‐associated ovarian cancers mimic their sporadic counterparts in extent and pattern of promoter methylation of several common tumour suppressor genes. This finding could have implications for future chemotherapy regimens based on epigenetic changes.     

Methylation of tumor associated genes in tissue and plasma samples from liver disease patients

To investigate whether aberrant hypermethylation in plasma DNA could be used as diagnosis makers for hepatocellular carcinoma (HCC), we performed methylation-specific PCR (MSP) to check the methylation status of five tumor associated genes in 36 cases of tissue and 42 cases of plasma samples from HCC and liver cirrhosis patients, respectively. The hypermethylation frequency of GSTP1 and RASSF1A showed significant difference between HCCs and liver cirrhosis with or without HBV infection (P < 0.05), but differences of the hypermethylation status of APC, E-cadherin, and P16 were not statistically significant. There were no significant differences in the hypermethylation status of five genes between the groups of cirrhosis with and without HBV infection. The significant differences of E-cadherin, GSTP1, P16, and RASSF1A in methylation between HCCs and liver cirrhosis were not observed in the plasma samples. Furthermore, the inconsistent results of MSP and real-time quantitative PCR for the paired samples of tissue and plasma suggested that plasma DNA could not fully stand for tissue DNA. In conclusion, hypermethylation of some specific, but not all, tumor associated genes may be involved in hepatocarcinogenesis; examination of the methylation status of E-cadherin, GSTP1, P16, and RASSF1A in the plasma samples might have limited usage for HCC diagnosis.