食管鳞状细胞癌及癌前病变组织中p16基因甲基化的研究

目的探讨食管鳞状细胞癌及癌前病变组织p16基因CpG岛的甲基化情况,及其在食管癌发生和早期诊断中的价值。方法采用MSP方法,对食管癌前病变不同阶段、鳞状细胞原位癌和浸润癌的病变组织进行了甲基化检测,并与其相应的正常组织和慢性食管炎组织的甲基化情况进行对比分析。结果轻、中、重度不典型增生、鳞状细胞原位癌和浸润癌的甲基化频率分别为22.73%(5/22)、46.15%(6/13)、77.78%(7/9)、78.57%(11/14)和64.86%(24/37)。67例正常对照组织中3例(4.48%)p16基因甲基化,与病变组织相比,差异有统计学意义,P=0.000。10例慢性食管炎组织中1例(10%)p16基因甲基化。结论p16基因甲基化可能是食管癌发生的最早期事件之一。     

血浆p16和FHIT基因甲基化在食管癌及癌前病变检测的临床意义

目的:研究食管癌及癌前病变患者血浆中p16和FHIT基因甲基化情况,探讨其对食管早期癌和癌前病变诊断的临床应用价值。方法:应用甲基化特异性聚合酶链反应(methylation-specific PCR,MSP)方法,对食管癌及癌前病变、慢性食管炎患者组织及血浆标本进行了p16和FHIT基因甲基化检测。结果:在44例癌前病变、14例原位癌、37例浸润癌和10例慢性食管炎共105例患者组织DNA中,分别发现18例、11例、24例和1例p16基因甲基化,15例、9例、25例和0例FHIT基因甲基化。癌前病变和慢性食管炎患者血浆中未发现两基因甲基化。51例食管癌患者血浆中共检出14例p16基因和16例FHIT基因甲基化,其中2例为原位癌。结论:p16及FHIT基因甲基化检测有望将食管癌的筛查提前到原位癌阶段,为食管癌的早期发现提供帮助。     

食管鳞状细胞癌及癌前病变组织中p16基因甲基化的研究

目的：探讨食管鳞状细胞癌及癌前病变纽织p16基因CpG岛的甲基化情况．及其在食管癌发生和早期诊断中的价值。方法：采用MSP方法，对食管癌前病变不同阶段、鳞状细胞原位癌和浸润癌的病变组织进行了甲基化检测．并与其相应的正常组织和慢性食菅炎组织的甲基化情况进行对比分析。结果：轻、中、重度不典型增生、鳞状细胞原位癌和浸润癌的甲基化频率分别为22．73％(5/27)、46．15％(6/13)、77．78％(7/9)、78．57％(11/14)和64．86％(24/37)。67例正常对照组织中3例(4．48％)p16基因甲基化，与病变组织相比，差异有统计学意义，P=0．000。10例慢性食管炎组织中1例(10％)p16基因甲基化。结论：p16基因甲基化可能是食管癌发生的最早期事件之一。     

FHIT和p16基因甲基化与肺癌的发生

背景与目的:探讨FHIT和p16基因甲基化与肺癌发生之间的关系. 材料与方法:采用甲基化特异性PCR检测59例原发性肺癌组织,相对应的32例正常组织及11例支气管鳞状化生组织中FHIT基因、p16基因启动子区CpG岛甲基化状况.结果:肺癌组织和正常肺组织中的FHIT基因甲基化率分别为37.3%(22/59)、0.0%(0/32),两组间的差异有统计学意义(P<0.01);支气管鳞状化生组织FHIT基因甲基化阳性率为18.1%(2/11).肺癌组织和正常肺组织中的p16基因甲基化率分别为50.8%(30/59)、0.0%(0/32),两组间的差异具有统计学意义(P<0.01);支气管鳞状化生组织p16基因甲基化阳性率为18.1%(2/11).肺癌患者吸烟组中FHIT、p16基因甲基化联合检测的阳性率为90.6%(29/32),与非吸烟组(33.3%.9/27)相比较,其差异具有统计学意义(P<0.05).结论: FHIT基因和p16基因启动子区甲基化可能与肺癌的发生有关.     

食管鳞状细胞癌中MGMT和p16基因启动子甲基化关系分析

背景与目的:探讨食管鳞状细胞癌中06-甲基鸟嘌呤-DNA甲基转移酶(O6-methylguanine DNA methyltransferase,MGMT)基因和p16基因启动子的甲基化情况,了解这两个基因启动子甲基化与食管癌发生的关系. 材料与方法:采用甲基化特异性PCR(MSP)方法分别对44例食管鳞状细胞癌组织进行MGMT基因和p16基因的甲基化检测,并对其甲基化频率分别进行差异性检验和关联性分析.结果:在44例食管鳞癌组织中,有18例(40.9%)出现MGMT基因启动子甲基化,23例(52.3%)出现p16基因启动子甲基化,7例(14.9%)两基因均出现甲基化,10例(22.7%)两基因均未出现甲基化.MGMT和p16基因启动子甲基化率差异无统计学意义(P＞0.05).MGMT和p16基因启动子甲基化无明显相关关系(P＞0.05).结论:MGMT和p16基因启动子甲基化均可能与食管鳞状细胞癌发生、发展过程密切相关,但二者是相互独立进行的.     

食管鳞癌中hMLH1、E-cadherin、p16^INK4a基因启动子甲基化及其意义

背景与目的:目前认为抑癌基因启动子甲基化导致转录抑制是恶性肿瘤发生的重要机制之一,hgLH1、E-cadherin及p16INK4a基因在多种恶性肿瘤中都已被证实存在较高频率的甲基化.本研究通过检测食管鳞癌组织及癌旁组织中hMLH1、E-cadherin,p16INK4a基因启动子甲基化的发生情况,探讨hMLH1、E-cadherin、p16INK4a基因启动子甲基化在食管鳞癌发生发展中的作用.方法:采用酚-氯仿法提取105例食管鳞癌组织及癌旁组织的基因组DNA,应用甲基化特异性PCR对所提DNA进行hMLH1、E-cadherin、p16INK4a基因甲基化检测.采用EnVison免疫组织化学二步法对癌组织中上述3种基因蛋白表达进行检测.结果:癌组织中E-cadherin、hMLH1、p16INK4a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),而癌旁食管组织中相应的3个基因的甲基化率分别为10.5%(11/105)、1.9%(2/105)和7.6%(8/105),均显著低于癌组织.E-cadherin(P=0.021)及p16INK4a(P=0.026)基因甲基化与蛋白表达缺失密切相关,而hMLH1基因甲基化与蛋白表达无显著相关性.E-cadherin基因启动子甲基化与淋巴结转移有关(P=0.016),p16INK4a基因启动子甲基化与低分化癌有关性(P=0.024).hMLH1基因甲基化与各项临床病理特征均无关.结论:食管鳞癌中p16INK4a基因启动子甲基化与相应蛋白表达缺失密切相关,且在低分化癌中更多见;E-cadherin基因启动子甲基化与相应蛋白质表达缺失有相关性,并且有淋巴结转移多见的显著特征,这2个基因的甲基化位点与食管鳞癌密切相关.hMLH1基因甲基化可能并不直接参与食管鳞癌的发生、发展.     

FHIT基因在食管鳞状细胞癌及癌旁组织中的表达

目的 :探讨FHIT基因在食管鳞状细胞癌及癌旁组织中的表达及意义。方法 :应用免疫组织化学S P法对 6 4例食管鳞状细胞癌组织标本及癌旁组织进行FHIT基因的表达水平检测 ,分析其与临床病理分期、细胞分化程度及淋巴转移等的关系。结果 :食管鳞状细胞癌组织中FHIT基因阳性表达率为 2 8 1% (18/6 4 ) ,癌旁组织中阳性表达率为 87 5 % (5 6 /6 4 ) ,二者差异有显著统计学意义 (χ2 =4 6 3,P <0 0 1) ,FHIT基因表达下降与患者PTNM分期、细胞分化程度、淋巴结转移、性别、年龄、吸烟史、饮酒史及病史长短均无明显关系 (P >0 0 5 )。结论 :FHIT基因的表达下降可能与食管鳞状细胞癌的发生、发展相关 ,与临床病理分期、细胞分化程度及淋巴结转移无关。     

食管鳞癌中p16基因启动子区甲基化及其表达

 目的探讨食管鳞癌（ESCC）p16基因甲基化的状况及其表达与食管鳞癌临床病理特征之间的关系。方法采用甲基化特异性PCR方法（MSP）分别检测75例食管癌组织、癌旁组织和切缘组织p16基因启动子区域CpG岛甲基化状态。采用Envision免疫组化法检测食管癌组织及癌旁组织的p16蛋白的表达。结果75例标本中，食管癌组织、癌旁组织和切缘细织p16基因甲基化率分别为41．3％（31／75）、13．3％（10／75）和6．67％（5／75）。癌组织和癌旁组织P16蛋白的阳性表达率分别为29．3％（22／75）和56．7％0（17／30）。31例癌组织p16基因甲基化阳性标本中有2例（6．4％）检测到P16蛋白的表达，而44例癌组织p16基因甲基化阴性标本中有20例（45．5％）检测到P16蛋白的表达。食管癌组织p16基因甲基化率显著高于癌旁组织和切缘组织（P〈0.01），P16蛋白表达与p16基因甲基化呈负相关。p16基因启动子区甲基化与食管癌的组织学分级、肿瘤部位无明显相关，与临床分期、淋巴转移密切相关。结论p16基因甲基化在食管癌发生发展中起着重要作用，食管鳞癌的分期和淋巴结转移与p16基因甲基化之间有密切关系。     

食管鳞状细胞癌中MGMT基因异常甲基化与MTHFR C677T基因多态的关系

背景与目的:作为基因失活的主要原因之一,肿瘤相关基因启动子区异常甲基化正日益受到关注,但是在食管癌方面研究尚十分有限.本研究的目的在于探讨DNA损伤修复基因MGMT异常甲基化与食管鳞状细胞痛临床特征和叶酸代谢酶基因MTHFR C677T多态之间的联系.方法:选择2005年1月至2006年3月间新发的、经病理学检查确诊并在江苏省扬中市人民医院进行手术治疗的食管鳞癌患者开展流行病学问卷调查,并采集其外周静脉血标本、癌组织和癌旁正常组织标本.应用甲基化特异性PcR法检测MGMT基因启动子区CpG岛甲基化状态.应用限制性片断长度多态(restriction fragment length polymorphism,RFLP)技术检测叶酸代谢酶基因MTHFR C677T多态.并分析和探讨食管粘膜组织中MGMT基因甲基化分布规律及其与MTHFR C677T基因多态间的联系.结果:125例食管鳞状细胞癌组织中MGMT基因甲基化频率达27.2%,癌旁组织甲基化频率11.2%,10例正常成人健康对照的食管粘膜均呈去甲基化状态.患者淋巴结转移与否与DNA甲基化频率有关,存在淋巴结转移的患者癌组织中MGMT基因甲基化频率(37.3%)高于无淋巴结转移的患者(18.2%).患者性别、年龄、吸烟、饮酒、饮茶等因素与DNA异常甲基化之间的相关性无统计学意义(P>0.05).在调整了相关潜在混杂因素后,携带MTHFR变异基因型CT和TT者食管癌组织中MGMT基因甲基化频率增高,与野生基因型CC比较,比值比OR分别为3.34(95%Cl:1.07～10.39)和3.83(95%CI:1.13～12.94).结论:食管鳞状细胞癌中MGMT基因启动子区异常甲基化与MTHFR基因多态有关,携带MTHFRC677基因变异基因型CT与TT的癌组织中MGMT基因异常甲基化频率更高.     

膜联蛋白-1在食管鳞状细胞癌发生发展中的表达差异

目的　检测膜联蛋白 -1(annexin -1)在食管鳞状细胞癌及癌前病变中的表达差异。方法　应用免疫组化S -P方法检测 13 5例食管鳞状细胞癌及其癌旁正常鳞状上皮、原位癌及其不典型增生病变中annexin -1的表达情况。结果　annexin -1在 12 9例 (95 .6% )癌旁正常食管鳞状上皮呈强阳性表达 ;在 3 5例 (2 5 .9% )鳞状细胞癌呈阳性表达 ,60例 (4 4 .4% )为弱阳性 ,40例(2 9.6% )为阴性 ,统计学处理有极显著性差异 (P <0 .0 1)。食管鳞状细胞癌annexin -1表达下降与患者的年龄、性别、肿瘤分化程度及淋巴结转移无关 (P >0 .0 5 )。annexin -1在原位癌和不典型增生病变的表达亦呈下降趋势 ,与正常鳞状上皮比较有非常显著性差异 (P <0 .0 1)。结论　annexin -1蛋白在食管鳞状细胞癌和癌前病变中有不同程度的丢失 ,它很可能成为食管癌早期诊断以及预测食管癌前病变癌变风险的具有重要应用价值的指标     

DNA methylation and loss of protein expression in esophageal squamous cell carcinogenesis of high-risk area

DNA methylation is an important mechanism for gene silence. The purpose of this study was to investigate aberrant promoter methylation of the p16 and FHIT genes in tissues and plasma and loss of protein expression in esophageal precancerous conditions (EPC) and esophageal squamous cell carcinoma (ESCC) of high-risk area. Methylation-specific PCR(MSP) was employed to examine the DNA methylation in the plasma and tissues of 95 patients of EPC, ESCC and 10 chronic esophagitis (CE). Loss of protein expression of p16 and FHIT was detected immunohistochemically. In total 95 lesion tissues of EPC and ESCC, p16 methylation was found in 53 (55.79%) cases, and 41 of 53 (77.36%) cases were demonstrated deletion of the p16 protein immunohistochemically. FHIT methylation was found in 49 (51.58%) cases, and 40 of 49 (81.63%) were demonstrated deletion of the FHIT protein. Only 1 (10%) case of 10 CE p16 methylation was found in the tissues. In the plasma of total 105 samples, 2 of 23 high grade intraepithelial neoplasia (HGIN) and 12 of 37 ESCC were detected p16 methylation, and 2 of 23 HGIN and 14 of 37 ESCC were detected FHIT methylation. These results indicate that p16 and FHIT methylation may be one of the earliest events and an important mechanism for gene silencing in esophageal squamous cell carcinogenesis. This study may be helpful for screening the candidate molecular markers for early diagnosis of ESCC in high-risk area.     

5' CpG island methylation of the FHIT gene is correlated with loss of gene expression in lung and breast cancer

Allele loss and loss of expression of fragile histidine triad (FHIT), a putative tumor suppressor gene located in chromosome region 3p14.2, are frequent in several types of cancers. Tumor-acquired methylation of promoter region CpG islands is one method for silencing tumor suppressor genes. We investigated 5' CpG island methylation of the FHIT gene in 107 primary non-small cell lung cancer (NSCLC) samples and corresponding nonmalignant lung tissues, 39 primary breast carcinomas, as well as in 49 lung and 22 breast cancer cell lines by a methylation-specific PCR assay. In addition, we analyzed brushes from the bronchial epithelium of 35 heavy smokers without cancer. FHIT methylation was detected in 37% of primary NSCLCs, 31% of primary breast cancers, and 65% of lung and 86% of breast cancer cell lines. The frequency of methylation in small cell and NSCLC cell lines were identical. Methylation was found in 9% of the corresponding nonmalignant lung tissues and in 17% of bronchial brushes from heavy cigarette smokers. FHIT methylation was significantly correlated with loss of FHIT mRNA expression by Northern blot analysis in lung cancer cell lines and with loss of Fhit expression in NSCLC and breast tumors by immunostaining. We conclude that methylation of FHIT is a frequent event in NSCLC and breast cancers and is an important mechanism for loss of expression of this gene. Methylation of FHIT commences during lung cancer pathogenesis and may represent a marker for risk assessment.     

胃癌E-cadherin基因启动子CpG岛甲基化的研究

背景与目的:目前认为CpG岛甲基化导致转录抑制是恶性肿瘤发生的重要机制之一。E-cadherin能抑制肿瘤细胞的浸润和转移,被公认为是浸润、转移抑制基因。低分化胃癌常表现有E-cadherin基因失活。我们通过检测胃癌及癌旁组织中E-cadherin基因启动子CpG岛甲基化水平,初步探讨胃癌的发病机制。方法:采用特异性甲基化PCR(MSP)法检测51例胃癌组织及37例癌旁组织中的E-cadherin基因启动子CpG岛甲基化水平,采用免疫组织化学染色法检测E-cadherin表达。结果:健康对照组织中未检测到CpG岛甲基化,4例(10.8%)癌旁组织检测到CpG岛甲基化,32例(62.7%)胃癌组织中检测到CpG岛甲基化。低分化腺癌(72.2%,26/36)CpG岛甲基化显著高于高分化腺癌(33.3%,6/15)(P<0.01),T1/T2期(55.6%,10/18)与T3/T4期胃癌(66.7%,22/33)的CpG岛甲基化率无显著性差异(P>0.05)。32例CpG岛甲基化胃癌中27例(84.5%)E-cadherin表达下调,19例非甲基化胃癌中5例(26.3%)E-cadherin表达下调。未发现E-cadherin基因CpG岛甲基化与幽门螺杆菌感染有关。结论:胃癌、尤其是低分化腺癌广泛存在E-cadherin基因启动子CpG岛甲基化,启动子CpG岛甲基化可能参与胃癌的早期过程,E-cadherin基因CpG岛甲基化与幽门螺杆菌感染无相关关系。     

Aberrant methylation of the FHIT gene in chronic smokers with early stage squamous cell carcinoma of the lung

Fragile histidine triad (FHIT) gene plays an important role in the pathogenesis of lung cancer. However, the clinicopathological significance of CpG island hypermethylation of FHIT gene in non-small cell lung cancer (NSCLC) remains to be elucidated. We studied FHIT methylation in 254 NSCLCs in order to further understand the clinicopathological and prognostic significance of FHIT methylation in NSCLC. Methylation status of the FHIT gene was examined using Methylation-Specific PCR. All statistical analyses were two-sided, with a 5% type I error rate. Hypermethylation of the FHIT gene occurred more frequently in squamous cell carcinoma than adenocarcinoma. For 93 adenocarcinomas there was no statistically significant association between FHIT methylation and age, gender, smoking history, pathologic stage and p16 methylation. However, FHIT methylation in 125 squamous cell carcinomas was associated with exposure to tobacco smoke and p16 methylation, but not with age, gender and pathologic stage. Hypermethylation of FHIT in squamous cell carcinomas occurred more frequently in current smokers (45%) than in never-smokers (13%). FHIT methylation was significantly associated with p16 methylation in current- and ex-smokers (P = 0.02 and P = 0.01, respectively) with squamous cell carcinoma and in patients with pathologic stage I squamous cell carcinoma (P = 0.001). Patients with p16 methylation were 3.74 times [95% confidence interval (CI) = 1.62 - 7.95; P = 0.001] more likely to have FHIT methylation in squamous cell carcinoma. FHIT methylation in squamous cell carcinoma occurred at a 4.62 times (95% CI = 1.26 - 34.97; P = 0.02) higher prevalence in current smokers than in never-smokers. No prognostic effect of FHIT methylation was observed in stage I and stage II NSCLCs. In conclusion, hypermethylation of the FHIT gene did not have a prognostic significance in early stage NSCLCs. The FHIT methylation is associated with the p16 methylation and smoking in squamous cell carcinoma, suggesting that FHIT may cooperate with p16 for the development of squamous cell carcinoma of lung in individuals exposed to tobacco smoke.     

Aberrant DNA methylation profiles of non-small cell lung cancers in a Korean population

We performed this study to investigate the aberrant methylation profile of the cancer-related genes in Korean non-small cell lung cancer (NSCLC) that previously exhibited high frequencies of methylation in Western populations. The aberrant promoter methylation of eight genes (GSTP1, p16, FHIT, APC, RASSF1A, hMLH1, hMSH2, AGT) was determined by MSP in 99 surgically resected NSCLCs and their corresponding nonmalignant lung tissues. Methylation in the tumor samples was detected at 15% for GSTP1, 22% for p16, 34% for FHIT1, 48% for APC, 40% for RASSF1A, 18% for hMLH1, 8% for hMSH2 and 21% for AGT, whereas it occurred at lower frequencies in the corresponding nonmalignant lung tissues, particularly in the p16 (1%) and RASSF1A (1%) genes. These results suggest that the methylation profiles of NSCLCs in a Korean population are similar to those in Western populations.     

FHIT和p16基因甲基化与胃癌的关系研究

目的探讨FHIT和p16基因甲基化在胃癌发生发展中的作用。方法应用甲基化特异性PCR（MSP）,检测胃癌组织和正常胃黏膜组织中FHIT基因和p16基因的启动子CpG岛的甲基化状态。结果胃癌组织中FHIT和p16基因甲基化阳性率分别为51.4%和56.8%,两者的甲基化均与胃癌患者年龄、性别、Lauren分型、Borrmann分型、淋巴结转移以及TNM分期无关（P〉0.05）,且两者的甲基化不存在明显正相关关系（P〉0.05,γ=0.17）。结论 FHIT基因和p16基因的甲基化修饰在胃癌发生发展中均起重要作用,两者可能是胃癌发生的早期事件。     

食管癌中DNA修复基因MGMT启动子区CpG岛过甲基化研究

目的：探索O~0-甲基鸟嘌呤-DNA甲基转移酶(O~6-methylguanine-DNA methyltransferase,MGMT)基因启动子区过甲基化状态与食管癌的关系。方法：采用甲基化特异性聚合酶链反应(methylation-specific PCR,MSP)分析食管癌、癌旁和正常食管黏膜细胞中MGMT基因启动子区过甲基化状态。用免疫组织化学SP法检测上述组织中MGMT蛋白表达情况。结果：76例食管癌组织中有26例(34.2%)MGMT基因启动子过甲基化,相应的癌旁组织中有8例发生甲基化,而正常食管黏膜细胞中均无甲基化。所有甲基化的癌组织中均无MGMT蛋白表达,而所有非甲基化的癌组织、相应癌旁组织和8例正常组织中均有MGMT蛋白表达。结论：MGMT基因启动子区过甲基化而使其蛋白表达缺失,可能是食管癌发生的重要因素。     

子宫内膜癌中p16基因缺失及CpG岛甲基化的研究

目的　研究子宫内膜癌中 p16基因缺失及CpG岛甲基化情况。 方法　用PCR技术检测 p16基因在子宫内膜癌中的纯合性缺失及第一外显子异常甲基化。结果　 36例癌组织中 9例发生基因缺失 ,12例发生甲基化 ,未发现基因缺失与甲基化同时存在的病例。结论　 1.p16基因缺失与子宫内膜癌组织学分级严重程度和临床分期呈正相关 ;2 .5’CpG甲基化可能是 p16基因失活的重要机理 ,参与子宫内膜癌发生、发展。