Estrogen receptor-alpha promoter methylation in sporadic basal-like breast cancer of Chinese women

Basal-like breast cancer (BLBC) appears to be characterized by a relatively unfavorable prognosis and lack of a specific therapeutic target. Estrogen receptor-alpha (ERα) has been widely accepted as a prognostic marker and a predictor for endocrine therapy response of breast cancer. This study aimed to clarify the correlation of ERα methylation with the pathogenesis and clinicopathological significance of sporadic BLBC of Chinese women without a family history of the cancer. The methylation of ERα promoter was investigated in genomic DNA of 60 sporadic BLBC with 108 cases of non-BLBC as control by methylation-specific polymerase chain reaction. We also investigated the expression of p53, breast cancer gene (BRCA)-1, and BRCA-2 by immunohistochemistry and analyzed the correlation between ERα methylation and clinicopathological features of BLBC. ERα methylation was observed in 48 of 60 (80.0%) sporadic BLBC, which was significantly higher than in sporadic non-BLBC cancer (47/108, 43.5%; χ2 = 20.89, p < 0.01). No correlation was found between the ERα methylation and age and menopausal status, while it was significantly associated with lymph node metastasis, tumor stage, nuclear p53 accumulation, and BRCA-1 and BRCA-2 expression in sporadic BLBC. The ERα methylation status in basal-like breast cancer was significantly higher than in sporadic non-basal-like breast cancer. It was associated with the lymph node metastasis, tumor stage, p53 nuclear accumulation, and BRCA-1 and BRCA-2 expression in BLBC. It may play an important role in BLBC pathogenesis.     

BRCA1 promoter methylation in sporadic breast tumors: relationship to gene expression profiles

BRCA1 is a tumor suppressor gene that functions in DNA repair. Basal-like tumors are a distinctive subtype of breast cancer defined by gene expression profiles. Hereditary BRCA1 breast tumors and basal-like sporadic tumors have a similar phenotype and gene expression signature, suggesting involvement of BRCA1 in the pathogenesis of sporadic basal-like cancer. This study evaluates the role of BRCA1 in sporadic breast tumorigenesis. BRCA1 protein expression and promoter methylation are compared to tumor histopathology and gene expression profiles. We find BRCA1 protein expression correlates with tumor mitotic rate, consistent with normal cell-cycle regulation of the BRCA1 gene. Methylation is found in 21% of tumors and is associated with lower BRCA1 protein, but not with specific pathologic features. Basal-like tumors, defined by hierarchical clustering of gene expression, have infrequent BRCA1 methylation and high levels of BRCA1 protein expression consistent with their high mitotic rate. Tumors with BRCA1 promoter methylation are present in all expression clusters; however, a subgroup of ER-positive high-grade tumors has a significantly greater number of BRCA1 methylated tumors. Absence of BRCA1 promoter methylation and high levels of BRCA1 expression in basal-like sporadic tumors suggest alternate explanations for the phenotypic similarities of these tumors to hereditary BRCA1 tumors.     

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.     

BRCA1 expression status in relation to DNA methylation of the BRCA1 promoter region in sporadic breast cancers.

To understand the biological role of BRCA1 in sporadic breast cancers, the relationship between DNA methylation of the BRCA1 promoter region and BRCA1 expression was studied using molecular biological and immunohistochemical methods. Furthermore, BRCA1 expression was compared with the expression of various cell cycle regulatory proteins and the morphological nuclear grade of cancer cells. Of 32 sporadic breast cancers investigated in this study, 10 (31%) revealed DNA methylation of the BRCA1 promoter region. The expression of BRCA1 was observed in the nuclei of cancer cells and 18 (56%) of 32 cancers were positive for BRCA1 immunoreactivity. Breast cancers with BRCA1 methylation lacked BRCA1 expression, except for only three cancers, and there was a significant inverse relationship between BRCA1 methylation and its expression in sporadic breast cancers (P = 0.043). Compared with the expression of various cell cycle regulatory proteins, breast cancers with BRCA1 methylation showed decreased expression of estrogen receptor (P = 0. 016) and p27 (P = 0.018) and increased expression of p21 (P = 0.011). Furthermore, breast cancers without BRCA1 expression or with BRCA1 methylation had a tendency to contain nuclei with higher grade. These findings indicate that BRCA1 methylation might greatly influence its expression and BRCA1 expression might play an important role in cell cycle regulation and influence the grade of malignancy of sporadic breast cancers.     

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。     

Modest promoter methylation of E-cadherin gene in sporadic colorectal cancers: a quantitative analysis

Although E-cadherin expression is frequently reduced in colorectal cancers (CRCs), this does not appear to be due to gene mutation or allele loss. We investigated the hypothesis that promoter methylation could be responsible for suppression of E-cadherin expression in 142 pairs of sporadic CRCs and respective normal mucosae. E-cadherin expression was examined by Western blot. E-cadherin methylation at two promoter regions was quantitatively measured by methylation specific real time PCR (MethyLight). We found that E-cadherin protein levels were significantly lower in CRCs, even in Dukes' A tumors, compared to normal mucosae. Decreased E-cadherin protein expression in CRCs was an independent poor prognostic factor in multivariate disease-free survival analysis. However, the extent of DNA methylation was extremely modest at both regions of the E-cadherin promoter. There was no correlation between DNA methylation and E-cadherin protein levels in either tumors or matched normal tissues. These findings suggested that suppression of E-cadherin expression in CRCs is a significant event and is possibly involved in both carcinoma development and progression. However, our data did not support a crucial role of promoter methylation of the E-cadherin gene in the remarkable downregulation of E-cadherin expression in CRCs. Methylated E-cadherin gene as a CRC biomarker therefore needs further validation.     

Frequent RASSF1A tumour suppressor gene promoter methylation in Wilms' tumour and colorectal cancer

The 3p21.3 tumour suppressor gene (TSG) RASSF1A is inactivated predominantly by promoter methylation and rarely by somatic mutations. Recently we demonstrated that epigenetic inactivation of RASSF1A is frequent in both clear cell and papillary adult renal cell carcinomas (even though 3p21.3 allele loss is rare in papillary tumours). Wilms' tumour is the most common childhood kidney tumour, but relatively little is known about its molecular pathogenesis. Thus TSGs such as WT1, p16(CDKN2a) and p53 are inactivated in only a minority of cases. In view of the involvement of RASSF1A in adult renal cancers we investigated RASSF1A as a candidate Wilms' TSG. We detected RASSF1A hypermethylation in 21 of 39 (54%) primary Wilms' tumours. 3p21.3 allele loss was not detected in nine informative Wilms' tumours (five with RASSF1A methylation). In contrast to RASSF1A, only a minority (10.3%) of Wilms' tumours demonstrated p16 promoter methylation. As chromosome 3p allele loss is frequent in colorectal cancer, we proceeded to investigate RASSF1A promoter methylation in colorectal cancer and detected RASSF1A methylation in 80% (4/5) colorectal cancer cell lines and 45% (13/29) primary colorectal cancers. There was no correlation between RASSF1A and p16 methylation in colorectal cancer. We have demonstrated that RASSF1A inactivation is the most frequent genetic or epigenetic event yet reported in Wilms' tumourigenesis and that allelotyping studies may fail to identify regions containing important TSGs.     

K-ras mutations and RASSF1A promoter methylation in colorectal cancer

Human cancer is characterized by genetic and epigenetic alterations. In this study we provide evidence for the interruption of Ras signaling in sporadic colorectal cancer (CRC) by either genetic activation of the K-ras oncogene or epigenetic silencing of the putative tumor suppressor gene RASSF1A. Paraffin embedded tumor tissue samples from 222 sporadic CRC patients were analysed for K-ras codon 12 and codon 13 activating mutations and RASSF1A promoter hypermethylation. Overall, K-ras mutations were observed in 87 of 222 (39%) and RASSF1A methylation was observed in 45 of 222 (20%) of CRCs. Mutation of K-ras alone was detected in 76 of 222 (34%) CRCs. RASSF1A promoter methylation with wild-type K-ras was observed in 34 of 222 (15%) CRCs. In 101 of 222 (46%) CRCs neither K-ras mutations nor RASSF1A methylation was observed and 11 of 222 (5%) CRCs showed both K-ras mutations and RASSF1A methylation. These data show that the majority of the studied CRCs with K-ras mutations lack RASSF1A promoter methylation, an event which occurs predominantly in K-ras wild-type CRCs (P=0.023, Chi-square test).     

女性散发性基底细胞型乳腺癌ERα启动子甲基化的研究

目的:探讨ERα甲基化与基底细胞型乳腺癌发生发展的相关性。方法:用甲基化PCR研究60例散发性基底型乳腺癌ERα启动子甲基化情况,并研究其与临床病因素之间的关系。结果:女性散发性基底细胞型乳腺癌组织中ERα基因启动子甲基化发生率为80.0%(48/60)。ERα基因启动子甲基化与淋巴结转移情况、肿瘤分期、p53蛋白、BRCA-1蛋白和BRCA-2蛋白表达情况有相关性,与患者年龄及绝经状态无关。结论:ERα基因启动子甲基化在基底细胞型乳腺癌发生发展中可能起重要作用。     

Somatic mutations in the BRCA1 gene in Chinese women with sporadic breast cancer

Recent studies suggested that breast cancer patients who carry a BRCA1 germline mutation benefit from poly (ADP-ribose) polymerase (PARP) inhibitors; therefore, it would be of great interest to detect BRCA1 somatic mutations in sporadic breast cancers. In this study, we detected BRCA1 somatic mutations in tumor cDNA from 144 Chinese women with sporadic breast cancer by using polymerase chain reaction (PCR)-direct sequencing assay. In total, eight BRCA1 alterations (three nonsense mutations and five missense mutations) were identified in this cohort of 144 sporadic breast cancers. We further confirmed that 5 out of 144(3.5%) sporadic breast cancer cases carried a BRCA1 somatic mutation, including two novel nonsense mutations (c.191_212del22 and c.2963C>G) resulting in a truncated protein and three missense mutations (c.114G>T, c.925A>C, and c.824G>A). The two cases with BRCA1 somatic truncating mutations also contained a TP53 somatic mutation in the tumors. Our study suggested that a small subset of sporadic breast cancers do harbor BRCA1 somatic mutations; these patients who carry a BRCA1 somatic mutation may be potential candidates for treatment with PARP inhibitors.     

RASSF1A在胃癌中的甲基化检测及表达

目的:探讨RASSF1A基因在胃癌及癌前病变组织中的表达及与启动子区甲基化的关系.方法: RT-PCR方法检测40例胃癌、20例中-重度慢性萎缩性胃炎伴肠上皮化生和不典型增生组织及20例癌旁正常组织RASSF1A mRNA表达,甲基化特异性PCR方法检测RASSF1A启动子区CpG岛甲基化状态;Western blot方法检测RASSF1A蛋白表达.结果: RASSF1AmRNA和蛋白表达水平在胃腺癌组织中明显低于中-重度慢性萎缩性胃炎伴肠上皮化生和不典型增生组及癌旁正常组织;RASSF1A在胃癌组织、中-重度慢性萎缩性胃炎伴肠上皮化生和不典型增生组织和正常组织中的甲基化频率分别为80%、25%和5%, 差异有显著性 (P<0.01);在胃癌组织中,RASSF1A mRNA阳性表达组甲基化明显低于表达缺失组.结论: 胃癌组织RASSF1A mRNA和蛋白表达缺失或低下与其启动子甲基化程度增高有关,启动子甲基化参与了胃癌的发生发展.     

RASSF1A基因在胶质瘤组织中甲基化研究及临床意义

目的：探讨胶质瘤组织及脑正常组织中抑癌基因RASSF1A启动子区甲基化程度及与临床特征的关系。方法：采用甲基化特异性聚合酶链反应（MS-PCR）方法检测46例脑胶质瘤（其中星形细胞瘤19例,室管膜瘤16例,胶质母细胞瘤11例）及6例脑正常组织中RASSF1A基因启动子区甲基化状态。并对RASSF1A基因启动子区甲基化发生情况与临床各因素之间的关系进行分析。结果：（1）46例脑胶质瘤组织DNA标本中RASSF1A基因启动子区甲基化发生率为65．2％（30／46）;6例脑正常组织中RASSF1A基因未发生甲基化;RASSF1A在胶质瘤和脑正常组织之间发生甲基化率比较差异有显著性（P=0.017）。在46例脑胶质瘤中RASSF1A有30例发生甲基化,其中低级别组14例（14／26）,高级别组16例（16／20）,两组之间甲基化率比较无明显差异（P〉0．05）。其中星形细胞瘤、室管膜瘤、胶质母细胞瘤中甲基化发生率分别为63．2％（12／19）、68、8％（11／16）、63．6％（7／11）,各组之间甲基化发生率比较均无统计学意义（P=0．211）。（2）RASSFIA基因启动子区甲基化程度与脑胶质瘤病理分型、肿瘤大小之间无显著相关性。结论：RASSF1A在胶质瘤中有甲基化发生,在脑正常组织中未发生甲基化,检测胶质瘤中RASSF1A基因启动子区甲基化情况对临床判断肿瘤发生发展有指导意义。     

BRCA1基因启动子甲基化在三阴性乳腺癌中的表达及其临床意义

目的:评估三阴性乳腺癌（TNBC）患者中乳腺癌易感基因1（BRCA1）甲基化的表达及与患者预后的关系。方法:收集在我院甲乳科治疗的乳腺癌患者手术切除标本524例,应用联合亚硫酸氢钠限制性内切酶分析法,观察BRCA1启动子甲基化状态。应用定量逆转录聚合酶链反应评估BRCA1 mRNA表达,应用免疫组织化学法评估BRCA1蛋白表达。结果:共有157（30.0%）例TNBC患者,有25（4.77%）例存在BRCA1启动子甲基化,所有BRCA1启动子甲基化的肿瘤均为TNBC。TNBC患者中,BRCA1启动子甲基化患者的BRCA1 mRNA水平显著低于BRCA1启动子未甲基化患者［（0.019±0.005） vs （0.095±0.013）,P<0.001］,免疫组化分析未在BRCA1启动子甲基化患者中检测出BRCA1蛋白表达。BRCA1启动子甲基化患者的总生存率（OS）显著低于非甲基化患者（logrank P=0.038）。BRCA1蛋白低表达患者的OS与RFS均低于高表达组,但差异没有统计学意义（分别logrank P=0.526,P=0.467）。结论:BRCA1启动子甲基化导致了BRCA1表达的下降,并与TNBC患者较差预后相关。BRCA1启动子甲基化是一种促成BRCA1功能丧失的重要机制。     

上皮性卵巢肿瘤RASSF1A基因甲基化与蛋白表达的关系及意义

目的：检测上皮性卵巢肿瘤中RASSF1A基因启动子CpG岛的甲基化状态,并探讨基因异常甲基化与蛋白表达的关系及其意义。方法：运用甲基化特异性PCR（MSP）方法对62例上皮性卵巢癌、21例交界性囊腺瘤及30例良性囊腺瘤的RASSF1A基因启动子甲基化状态进行检测,免疫组化S-P法检测上述标本中RASSF1A蛋白表达,并结合肿瘤生物学行为进行分析。结果：上皮性卵巢癌组织、卵巢交界性囊腺瘤组织中RASSF1A基因启动子区甲基化率（58.06%、42.85%）显著高于卵巢良性囊腺瘤组织（13.33%）,差异有统计学意义（P〈0.05）。RASSF1A基因启动子区甲基化与上皮性卵巢癌的细胞分化程度和临床分期密切相关（P〈0.05）而与组织类型和患者年龄及绝经与否无相关性（P〉0.05）;RASSF1A基因甲基化与其蛋白表达下降一致。结论：卵巢癌组织中存在RASSF1A基因启动子CpG岛的异常甲基化,可能和该蛋白表达缺失的主要原因,是导致该基因失活的重要机制之一。     

上皮性卵巢肿瘤RASSF1A基因甲基化与蛋白表达的关系及意义

目的:检测上皮性卵巢肿瘤中RASSF1A基因启动子CpG岛的甲基化状态,并探讨基因异常甲基化与蛋白表达的关系及其意义。方法:运用甲基化特异性PCR(MSP)方法对62例上皮性卵巢癌、21例交界性囊腺瘤及30例良性囊腺瘤的RASSF1A基因启动子甲基化状态进行检测,免疫组化S-P法检测上述标本中RASSF1A蛋白表达,并结合肿瘤生物学行为进行分析。结果:上皮性卵巢癌组织、卵巢交界性囊腺瘤组织中RASSF1A基因启动子区甲基化率(58.06%、42.85%)显著高于卵巢良性囊腺瘤组织(13.33%),差异有统计学意义(P<0.05)。RASSF1A基因启动子区甲基化与上皮性卵巢癌的细胞分化程度和临床分期密切相关(P<0.05)而与组织类型和患者年龄及绝经与否无相关性(P>0.05);RASSF1A基因甲基化与其蛋白表达下降一致。结论:卵巢癌组织中存在RASSF1A基因启动子CpG岛的异常甲基化,可能和该蛋白表达缺失的主要原因,是导致该基因失活的重要机制之一。     

散发性乳腺癌中MTA1表达与ERα甲基化关系的研究

目的:通过甲基化特异性PCR和免疫组化研究女性散发性乳腺癌中ERα基因启动子区甲基化和MTA1蛋白表达的相关性。方法:甲基化PCR研究102例散发性乳腺癌ERα启动子甲基化情况,免疫组化研究其MTA1蛋白表达情况。结果:乳腺癌组织中ERα启动子甲基化率为37.3%,乳腺癌组织中MTA1表达率为29.4%,高于其在癌旁正常乳腺组织中的表达(P<0.05),MTA1表达和ERα启动子甲基化均与乳腺肿瘤大小、TNM分期及淋巴结转移相关(P<0.05),且乳腺癌中ERα启动子甲基化与MTA1阳性表达呈正相关(P<0.05)。结论:女性散发性乳腺癌中ERα基因启动子甲基化与MTA1表达升高密切相关,其在乳腺癌发展过程中可能起重要作用。     

RASSF1A基因启动子外周血和组织甲基化在乳腺癌中的意义

目的通过对乳腺癌、癌旁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,加深对乳腺癌发病分子机制的了解并为乳腺癌早期诊断和预后判断提供候选指标。方法采用甲基化特异性PCR方法,分别检测156例乳腺癌患者血浆、肿瘤组织及癌旁正常组织和39例乳腺良性病变血浆及其正常组织中RASSF1A基因启动子甲基化状况。结果早期乳腺癌组织RASSF1A基因启动子甲基化发生率为62.2%(23/37),同一患者外周血甲基化发生率为56.7%(21/37),Kappa值为0.6553(P<0.001),40例血浆RASSF1A甲基化的患者中,37例发生淋巴结转移92.5%(37/40),3例未发生淋巴结转移7.5%(3/40),差异有显著性(P=0.0003);乳腺癌组织和外周血中的RASSF1A甲基化水平与乳腺癌的年龄、家族史、分型、分期、ER、PR、CerbB-2无关;晚期癌复发的86例中,其中癌组织甲基化病例的复发率为25.5%(13/51),癌旁组织甲基化的复发率为75.7%(53/70),两组差异有显著性(P<0.0001)。结论乳腺癌血浆RASSF1A甲基化与组织中的变化较为一致,可作为早期病例筛查和判断淋巴结转移的候选指标之一;晚期乳腺癌癌旁RASSF1A甲基化可能参与肿瘤复发,可作为预测晚期病例预后的候选指标之一。