食管癌组织与外周血RASSF 1A甲基化的检测及其临床意义

目的:通过对食管癌组织及同一患者对应的术前外周血中RASSF1A基因甲基化的检测,加深食管癌变分子机制的了解并为食管癌早期诊断和高危人群预警提供候选指标.方法:采用甲基化特异性PCR方法,分别检测来自食管癌高发区的30例食管癌患者血浆、肿瘤组织及癌旁正常组织中RASSF1A基因启动子甲基化状况.结果:食管癌组织RASSF1A甲基化阳性率为40%(12/30),而这12例癌组织甲基化阳性的患者其外周血甲基化阳性共7例,癌组织和外周血RASSF1A甲基化阳性一致率为58%(7/12),18例癌组织甲基化阴性的患者其外周血也均为阴性,阴性一致率为100%(18/18).癌旁正常食管组织甲基化率为13%(4/30),明显低于癌组织(40%,12/30),P<0.05.7例外周血甲基化阳性的患者中,淋巴结转移阳性5例(55%,5/9),阴性2例(10%,2/21),两者差异有统计学意义,P<0.05.低分化鳞癌的RASSF1A甲基化阳性率(83%,5/6)明显高于中分化鳞癌(24%,5/21),P<0.05.结论:外周血RASSF1A甲基化可以反映同一个体食管鳞癌组织中RASSF1A基因甲基化的状态,可能是高危人群筛查重要候选分子标志之一.     

MSP法检测胃癌组织中RASSF1A基因启动子区甲基化改变

目的:探讨抑癌基因RASSF1A启动子区CpG岛甲基化与胃癌及临床病理特征的关系.方法:采用甲基化特异性PCR(methylation-specific PCR,MSP)法检测60例胃癌组织及相应癌旁组织和30例对照组织中RASSF1A基因启动子区甲基化状态.结果:胃癌组织中RASSF1A基因启动子区CpG岛甲基化率为65.0%(39/60),显著高于癌旁组织6.7%(4/60),及对照组0%(0/30)(P＜0.01).胃癌组织中不同年龄、性别、分化程度及淋巴结转移与否的RASSF1A基因甲基化率的差异均无统计学意义.结论:胃癌中RASSF1A基因启动子区的高甲基化提示其与胃癌的发生密切相关,MSP法对RASSF1A基因启动子区甲基化的检测有望成为胃癌早期监测的重要方法.     

宫颈癌组织中RASSF1A基因启动子甲基化对其表达水平的影响及临床意义

目的 研究宫颈癌组织中RAS相关区域家族1A基因(RASSF1A)启动子甲基化水平和RASSF1A基因mRNA表达水平,分析其与宫颈癌临床病理参数的关系及临床意义.方法 收集40例宫颈癌组织及相应癌旁组织,采用巢式特异性甲基化方法检测RASSF1A基因启动子甲基化水平,采用实时荧光定量PCR(qRT-PCR)检测宫颈癌和癌旁组织中RASSF1A基因mRNA表达水平.结果 宫颈癌组织中RASSF1A基因mRNA表达水平(0.26±0.05)显著低于癌旁组织(0.28±0.03),差异有统计学意义(t=2.27,P=0.026);宫颈癌组织中RASSF1A基因启动子区的甲基化率(0.71%±0.04%)显著高于癌旁组织(0.66%±0.03%),差异有统计学意义(t=6.78,P=0.000);RASSF1A基因mRNA表达水平与病理分化程度(t=3.31,P=0.002)、国际妇产科联合会(FIGO)分期(t=2.13,P=0.040)、淋巴结转移(t=2.56,P=0.015)、浸润深度(t=2.93,P=0.006)有关;RASSF1A基因启动子区的甲基化水平与病理分化程度(t=2.08,P=0.045)、FIGO分期(t=2.66,P=0.011)、淋巴转移(t=2.22,P=0.033)、浸润深度(t=2.12,P=0.041)有关.结论 宫颈癌组织中RASSF1A基因启动子甲基化和RASSF1A基因mRNA的表达水平与宫颈癌的恶性程度相关,RASSF1A基因甲基化水平有望成为宫颈癌转移风险的重要指标.     

MSP法检测胃癌组织中RASSF1A基因启动子区甲基化改变

目的：探讨抑癌基因RASSF1A启动子区CpG岛甲基化与胃癌及临床病理特征的关系。方法：采用甲基化特异性PCR（methylation—specific PCR,MSP）法检测60例胃癌组织及相应癌旁组织和30例对照组织中RASSF1A基因启动子区甲基化状态。结果：胃癌组织中RASSF1A基因启动子区CpG岛甲基化率为65．0％（39／60）,艋著高于癌旁组织6．7％（4／60）,及对照组0％（0／30）（P〈0．01）。胃癌组织中不同年龄、性别、分化程度及淋巴结转移与否的RASSF1A基因甲基化率的差异均无统计学意义。结论：胃癌中RASSF1A基因启动子区的高甲基化提示其与胃癌的发生密切相关,MSP法对RASSF1A基因启动子区甲基化的检测有望成为胃癌早期监测的重要方法。     

RAS相关家族1A基因在胃癌中的表达和启动子甲基化

目的分析散发性胃癌中RAS相关家族1A基因(RASSF1A基因)的表达、突变和启动子甲基化状况,探讨RASSF1A基因在胃癌发生、发展中的意义.方法采用定量PCR、RT-PCR和SSCP的检测90例胃癌组织和30例相应癌旁正常组织中RASSF1A基因表达水平以及基因突变的情况;采用甲基化特异性PCR (MSP)方法检测RASSF1A基因启动子甲基化情况.结果 90例胃癌中有52例(57.8%)RASSF1A无表达或表达低下.RASSF1A无表达或表达低下和肿瘤细胞的分化(P<0.05)以及分期(P<0.001)相关,但是和肿瘤的浸润深度以及淋巴结转移不相关(P>0.05).90例胃癌中52例启动子发生甲基化(57.8%),其中90.3%(47/52)的RASSF1A无表达或表达低下组织中检测到RASSF1A基因启动子的甲基化,然而癌旁正常组织未发现有RASSF1A基因启动子的甲基化,SSCP没有发现任何突变.结论胃癌中存在较多的启动子异常甲基化造成RASSF1A基因失活,这可能是胃癌发生发展的因素之一.     

散发性乳腺癌患者血浆BRCA1基因启动子异常甲基化的检测

目的:探讨散发性乳腺癌组织及外周血浆中BRCA1基因启动子异常甲基化状况及其在散发性乳腺癌诊断中的价值。方法:用甲基化特异PCR方法对散发性乳腺癌患者癌组织、癌旁组织及相应血浆进行BRCA1异常甲基化检测。结果:93例散发性乳腺癌组织中,BRCA1基因启动子异常甲基化率为29%(27/93),相应血浆中BRCA1的甲基化检出率为24.7%(23/93),而癌旁组织、正常对照血浆未检出甲基化,只检出未甲基化的BRCA1。血浆中甲基化改变与肿瘤组织甲基化状况显著相关(P<0.05);BRCA1异常甲基化与髓样癌和粘液腺癌的组织分型显著相关(P<0.05),也与肿瘤淋巴结转移显著相关(P<0.005);但与散发性乳腺癌患者年龄(绝经与否)及肿瘤分级无显著的相关性(P>0.05)。结论:血浆BRCA1基因异常甲基化改变的检测在散发性乳腺癌的特异诊断、组织分型和淋巴结恶性转移等方面有一定的应用价值。     

非小细胞肺癌RASSF1A启动子甲基化状态临床研究

目的 探讨非小细胞肺癌(NSCLC)中RASSF1A启动子的甲基化状态及临床意义.方法 采用甲基化特异的PCR(MSP)检测150例NSCLC、34例癌旁正常组织和20例肺部良性病变中RASSF1A启动子甲基化状态.结果 150例NSCLC中58例发现RASSF1A启动子存在甲基化(38.7%),34例癌旁正常组织和20例肺部良性病变无一例发现RASSF1A启动子甲基化.RASSF1A启动子甲基化与年龄、性别、吸烟指数、组织类型、TNM临床分期均无关(P＞0.05),与开始吸烟年龄、肿瘤分化程度相关(P＜0.05).结论 RASSF1A启动子甲基化状态可以作为NSCLC诊断的一个候选分子标志.     

宫颈鳞癌组织中RASSF1A基因甲基化状态及其临床意义

背景与目的:Ras相关区域家族1A(ras associated domain family 1A,RASSF1A)基因是一种肿瘤候选抑痛基因,其启动子区高甲基化与多种人类上皮性肿瘤的发生有关.本研究通过检测宫颈鳞癌(cervical squamous cell carcinoma,CSCC)组织中RASSF1A基因启动子区甲基化状态及其蛋白表达情况,旨在分析RASSF1A基因甲基化与基因失活的相关性,并探讨RASSF1A基因甲基化与CSCC发生的关系.方法:分别采用甲基化特异性PCR(methylation-specific PCR,MSP)及免疫组织化学方法检测15例正常宫颈组织和46例CSCC组织中RASSF1A基因的甲基化程度及RASSF1A蛋白的表达情况,并分析RASSF1A基因甲基化与RASSF1A蛋白表达及其与CSCC临床病理特征之间的关系.结果:正常宫颈组织中未见RASSF1A基因甲基化,而CSCC组织中RASSF1A基因甲基化率为43.5%(20/46),两者比较差异具有统计学意义(P＜0.05).CSCC中临床Ⅰ期、Ⅱ期、Ⅲ～Ⅳ期的甲基化阳性率呈逐渐增高趋势(19.0%→58.8%→75.0%,P＜0.05),不同组织学分级、有无淋巴结转移与RASSF1A基因甲基化间未见明显相关性(P＞0.05).RASSF1A基因甲基化与RASSF1A蛋白的表达呈负相关(r=-0.469,P＜0.05).结论:RASSF1A基因启动子区甲基化是导致RASSF1A蛋白低表达的机制之一,可能与CSCC的发生发展有关.     

结直肠癌中 RASSF1A 启动子甲基化与Cyclin D1和P53表达的关系

目的： 检测结直肠癌组织中Ras相关区域家族1A（ras-association domain family 1A, RASSF1A )基因启动子甲基化以及Cyclin D1和P53蛋白的表达,分析它们与结直肠癌临床病理特征的关系。 方法： 收集2008年8月至2009年8月长海医院病理科37例结直肠癌组织和14例癌旁组织(癌灶边缘5 cm以外组织）标本。甲基化特异性PCR（methylation-specific PCR,MSP）检测结直肠癌组织中 RASSF1A 基因启动子的甲基化,免疫组织化学法检测结直肠癌组织中 Cyclin D1和P53蛋白的表达,分析 RASSF1A 启动子甲基化、Cyclin D1和P53表达的关联性以及三者与结直肠癌临床病理特征的相关性。 结果： 37例结直肠癌组织中 RASSF1A 启动子甲基化有23例（62.16%）,14例癌旁组织中 RASSF1A 启动子甲基化有12例（85.71%）;37例癌组织中Cyclin D1阳性14例（37.84%）、P53阳性15例（40.54%）,14例癌旁组织中Cyclin D1和p53表达均为阴性。直肠癌组织中 RASSF1A 启动子甲基化阳性率高于结肠癌（P<0.05）。结直肠癌患者年龄与Cyclin D1表达呈负相关（P<005）,淋巴结转移与P53表达呈正相关（P<0.05）。结直肠癌组织中 RASSF1A 启动子甲基化与Cyclin D1和P53的表达无相关性。 结论： RASSF1A 启动子甲基化、Cyclin D1和P53蛋白表达三者的联合检测对研究结直肠癌的发生、发展有一定意义。     

Methylation Profiles of BRCA1, RASSF1A and GSTP1 in Vietnamese Women with Breast Cancer

Objective: This study investigated the DNA promoter methylation profiles of BRCA1, RASSF1A and GSTP1 genes,both individually and in an integrative manner in order to clarify their correlation with clinicopathological parameters ofbreast cancer from Vietnamese patients, and establish new potential integrative methylation biomarkers for breast cancerdetection. Material and methods: The methylation frequencies of BRCA1, RASSF1A and GSTP1 were analyzed bymethylation specific polymerase chain reaction (MSP) in 70 specimens of breast carcinomas and 79 pairs of tumor andmatched adjacent normal tissues from breast cancer patients. Results: All the three analyzed genes showed a concordanceconcerning their promoter methylation in tumor and adjacent normal tissue. The methylation of BRCA1, RASSF1Aand GSTP1 was found in 58.23 %, 74.68 % and 59.49 % of tumor tissues and 51.90 %, 63.29 % and 35.44 % ofcorresponding adjacent tissues, respectively. When each gene was assessed individually, only the methylation ofGSTP1 was significantly associated with tumor tissues (p=0.003). However, the methylation frequency of at least one ofthe three genes and the methylation frequency of all the three genes both showed significant association with tumor(p=0.008 and p=0.04, respectively). The methylation of BRCA1 was found to be significantly associated with tumorgrade (p=0.01). Conclusion: This study emphasized that the panel of the three genes BRCA1, RASSF1A and GSTP1can be further developed as potential biomarkers in diagnosis and classification of breast cancer in Vietnamese women.     

Promoter hypermethylation in benign breast epithelium in relation to predicted breast cancer risk.

INTRODUCTION: The tumor suppressor genes RASSF1A, APC, H-cadherin, RARbeta2, and cyclin D2 are methylated more frequently in breast cancer than in adjacent benign tissue. However, it is unclear whether promoter methylation of tumor suppressor genes in benign breast tissue is associated with an increased risk for breast cancer. METHODS: Promoter hypermethylation was measured in benign and malignant breast samples obtained by fine needle aspiration biopsy from 27 breast cancer patients and 55 unaffected women whose risk of breast cancer had been defined using the Gail, Claus, and BRCAPRO models. RESULTS: Cyclin D2 methylation occurred in 57% of tumor samples but not in corresponding benign breast samples and in only one sample from an unaffected patient (P < 0.0001). RARbeta2 methylation occurred in 32% of benign breast samples from cancer patients but only 9% of similar samples from unaffected women (P = 0.002). Promoter methylation of RASSF1A and APC occurred more frequently (70% and 56%, respectively) in unaffected women at high-risk for breast cancer as defined by the Gail model than in low/intermediate risk women (29% and 20%, P = 0.04 and P = 0.03). Of the Gail model risk factors, only number of prior breast biopsies was highly correlated with APC and RASSF1A methylation (P = 0.0001 and 0.02, respectively). CONCLUSIONS: Since cyclin D2 promoter methylation occurs almost exclusively in tumors, it may be possible to exploit it for the early detection of breast cancer. Promoter methylation of APC, RARbeta2, and RASSF1A in benign breast epithelium is associated with epidemiologic markers of increased breast cancer risk.     

非小细胞肺癌患者血清Ras相关区域家族1A基因启动子异常甲基化检测及其临床意义

目的 研究非小细胞肺癌(NSCLC)患者血清Ras相关区域家族1A(RASSF1A)基因启动子区域的甲基化状态及其临床意义.方法 采用甲基化特异性聚合酶链反应(MSP)技术,检测75例NSCLC患者血清RASSF1A基因启动子区域的甲基化状态,并分析其与临床病理参数之间的相关性.结果75例NSCLC患者血清RASSF1A基因启动子区域异常甲基化检出率为30.7%(23/75),而35例肺部良性疾病患者和15例健康志愿者中检出率均为0,差异有统计学意义(P＜0.001).RASSF1A启动子异常甲基化与NSCLC患者的年龄、性别、病理类型无显著相关性,但在晚期及肿瘤分化程度较低的患者中检出率较高(P＜0.05).结论 RASSF1A启动子异常甲基化在NSCLC的发生、发展中起重要作用,有望成为NSCLC辅助诊断和预后判断的分子标记.     

Expression and promoter methylation of the RASSF1A gene in sporadic breast cancers in Chinese women

The novel tumor suppressor RASSF1A is frequently inactivated during human tumorigenesis by promoter methylation. In this study, we detected the RASSF1A promoter methylation by methylated-specific PCR and investigated RASSF1A gene expression by semi-quantitative RT-PCR and immunohistochemical staining in 36 cases of breast cancer and their adjacent normal tissues in Chinese women. The promoter methylation of the RASSF1A gene was found to be a frequent event in the breast cancers (61.1%). RASSF1A methylation was not found in the matched adjacent normal tissues. The loss frequency of RASSF1A mRNA was 33.3% and that of the RASSF1A protein was 44.4% in breast cancers. RASSF1A mRNA and protein were all expressed in adjacent normal tissues. The mRNA and protein expression level of RASSF1A was significantly lower in breast cancer than in adjacent normal tissue. However, the promoter methylation of the RASSF1A gene in breast cancers were not correlated with clinical parameters, such as ages, histological types, TNM stages and lymph node metastases. Thus, the promoter methylation of RASSF1A was one reason for the low level of RASSF1A mRNA and protein expression and was a frequent event in primary sporadic breast tumorigenesis in Chinese women.     

外周血PTEN、RASSF1A甲基化及其mRNA表达与结节性甲状腺疾病的关系

目的 探讨PTEN、RASSF1A基因启动子甲基化及其mRNA表达与结节性甲状腺疾病之间的关系。方法 选取46例结节性甲状腺肿、34例甲状腺腺瘤、24例甲状腺乳头状癌患者（甲癌）和30例健康体检者,利用甲基化特异性PCR检测外周血中PTEN、RASSF1A基因启动子区甲基化状态及利用反转录—多聚酶链反应法（RT-PCR）检测mRNA表达情况。结果 （1）腺瘤组和甲癌组中PTEN、RASSF1A甲基化率分别与对照组比较, 差异均有统计学意义（均P<0.05）。（2）腺瘤组和甲癌组中PTEN、RASSF1A基因mRNA表达阳性率分别与对照组比较, 差异均有统计学意义（均P<0.05）。（3）甲癌组中PTEN和RASSF1A基因甲基化率、mRNA表达与淋巴结转移有关（均P<0.05）,与年龄、性别、结节大小无关。结论 甲状腺肿瘤外周血中存在抑癌基因PTEN、RASSF1A高甲基化改变进而导致其功能失活,从而在甲状腺肿瘤的发生发展过程中发挥重要作用。     

Analysis of RASSF1A promoter hypermethylation in serum DNA of non-small cell lung cancer

OBJECTIVE: To detect the hypermethylation status of RASSF1A promoter in serum DNA of non-small cell lung cancer (NSCLC) patient and evaluate its correlation with clinicopathological parameters. METHODS: Serum DNA was extracted from the peripheral blood of 75 NSCLC patients and another 35 patients with benign pulmonary disease and 15 healthy donors. The methylation status of RASSF1A promoter was determined using methylation-specific PCR (MSP), and the correlation of methylation profiles with clinicopathological parameters was statistically analyzed. RESULTS: Aberrant methylation of RASSF1A was detected in 23 of 75 (30.7%) cancer patients, but in none of patients with benign pulmonary disease or in healthy donors (P <0.001). RASSF1A hypermethylation status was found to be correlated with late stage and poor differentiation (P < 0.05), but not with gender, age or histopathology in NSCLC patients. CONCLUSION: Hypermethylated RASSF1A promoter is frequently found in the serum DNA of non-small cell lung cancer patient, and RASSF1A may become a promising novel biomarker for diagnosis and prognosis prediction in lung cancer.     

Promoter Methylation of BRCA1, DAPK1 and RASSF1A is Associated with Increased Mortality among Indian Women with Breast Cancer

Background: Promoter methylation has been observed for several genes in association with cancer development and progression. Hypermethylation mediated-silencing of tumor suppressor genes (TSGs) may contribute to breast cancer pathogenesis. The present study was conducted to investigate the promoter methylation status of BRCA1, DAPK1 and RASSF1A genes in Indian women with breast cancer. Materials and Methods: Promoter methylation was evaluated in DNA extracted from mononuclear cells (MNCs) in peripheral blood samples of 60 histopathologically confirmed newly diagnosed, untreated cases of breast cancer as well as 60 age and sex matched healthy controls using MS-PCR. Association of promoter methylation with breast cancer-specific mortality was analyzed with Cox proportional hazards models. Kaplan-Meier survival analysis was performed for overall survival of the breast cancer patients. Results: We observed a significant increase of BRCA1, DAPK1 and RASSF1A promoter methylation levels by 51.7% (P <0.001), 55.0% (P <0.001) and 46.6% (P <0.001), respectively, when compared to healthy controls. A strong correlation was noted between hypermethylation of the tumor suppressor genes BRCA1 (P= 0.009), DAPK1 (P= 0.008) and RASSF1A (P= 0.02)) with early and advanced stages of breast cancer patients. We also found that breast cancer-specific mortality was significantly associated with promoter methylation of BRCA1 [HR and 95% CI: 3.25 (1.448-7.317)] and DAPK1 [HR and 95% CI: 2.32 (1.05-5.11)], whereas limited significant link was evident with RASSF1A [HR and 95% CI: 1.54 (0.697-3.413]. Conclusion: Our results suggest that promoter methylation of BRCA1, DAPK1 and RASSF1A genes may be associated with disease progression and poor overall survival of Indian women with breast cancer.     

RASSF1A基因甲基化与非小细胞肺癌预后的相关性研究

背景与目的研究发现在很多肿瘤中均存在RASSF1A基因启动子区域高甲基化状态导致基因表达失活的现象,本研究就RASSF1A基因启动子的甲基化状态与非小细胞肺癌预后的关系进行探讨。方法采用甲基化特异的PCR检测150例非小细胞肺癌和20例肺部良性病变RASSF1A启动子甲基化状态。结果150例非小细胞肺癌中58例发现RASSF1A启动子存在甲基化(58/158,38.7%),20例肺部良性病变中无一例发现RASSF1A启动子甲基化。存在RASSF1A启动子高甲基化的病例预后较未发现RASSF1A甲基化的病例差(P=0.004),Cox回归分析显示RASSF1A启动子的甲基化状态是非小细胞肺癌术后的一个预后相关因素(RR=1.584,95%CI:1.040-2.411,P=0.032)。结论MSP法检测RASSF1A启动子甲基化状态可以作为非小细胞肺癌术后的一个预后评价指标。     

Detection of RASSF1A aberrant promoter hypermethylation in sputum from chronic smokers and ductal carcinoma in situ from breast cancer patients

The newly identified 3p21.3 tumour suppressor gene RASSF1A is methylated in the majority of primary lung tumours, lung tumour cell lines and in a variable percentage of breast tumours. To determine the extent of RASSF1A promoter hypermethylation in early lung tumorigenesis, we analysed sputum samples from lung cancer patients and from current and former smokers using a sensitive methylation-specific PCR (MSP) technique. We also analysed RASSF1A promoter region hypermethylation in trios of normal breast/invasive ductal breast carcinoma/ductal carcinoma in situ (DCIS) from breast cancer patients and DCIS without invasive cancer. We found that 50% of small cell lung cancer (SCLC) and 21% of non-small cell lung cancer (NSCLC) patients had RASSF1A methylation, while one of two former smokers and four of 13 current smokers demonstrated RASSF1A methylation in sputum. Furthermore, two of the four current smokers and one former smoker showing RASSF1A methylation in their sputum developed cancer within 12-14 months of bronchoscopy. In our breast cancer trios, RASSF1A promoter hypermethylation was detected in 65% of invasive cancers, in 42% of corresponding DCIS but in none of the normal breast samples. In addition, we found that three out of 10 DCIS without invasive breast cancer also underwent RASSF1A promoter hypermethylation. Our findings suggest that RASSF1A promoter region hypermethylation may be a useful molecular marker for early detection of lung cancer. Furthermore, since RASSF1A promoter hypermethylation was detected in ductal carcinoma in situ, inactivation of RASSF1A may be an early event in breast tumorigenesis.