RASSF1A methylation and K-ras and B-raf mutations and recurrent endometrial cancer.

BACKGROUND: Aberrations in mediators of Ras signaling may increase the risk of developing recurrent endometrial carcinoma. PATIENTS AND METHODS: Primary tumors of patients with (n = 44) and without (n = 44) recurrent stage I endometrioid endometrial carcinoma were compared regarding the presence of K-ras mutations (codons 12 and 13), B-raf mutations (V599), and RASSF1A gene promoter methylation. RESULTS: K-ras mutations were present in 18% of the patients independent of recurrent disease. No B-raf mutations were found. RASSF1A methylation was demonstrated in 85% of endometrial carcinomas, independent of recurrence. The presence of K-ras mutations and RASSF1A promoter methylation were not related, either directly or inversely. Analysis in premenopausal endometrial carcinomas demonstrated K-ras mutations in 40%, no B-raf mutations, and RASSF1A promoter methylation in 70% of the cases. RASSF1A methylation was also observed in samples of cyclic (n = 14), hyperplastic (n = 8), and atrophic (n = 13) endometrial tissues in 21%, 50% and 38%, respectively. CONCLUSIONS: RASSF1A methylation was observed in a high frequency in endometrioid endometrial carcinoma whereas K-ras and B-raf mutations were observed in a low frequency. No association was observed with the development of recurrent disease. High-frequency RASSF1A methylation in premenopausal carcinomas and an increased frequency in endometrial hyperplasia indicate that this may be an early event in endometrial carcinogenesis.     

Frequent RASSF1A promoter hypermethylation and K-ras mutations in pancreatic carcinoma

Recently, we have characterized the Ras association domain family 1A gene (RASSF1A) at the segment 3p21.3, which is frequently lost in variety of human cancers and epigenetically inactivated in many types of primary tumors, such as lung, breast, kidney, prostate and thyroid carcinomas. Here, we investigated the methylation status of the RASSF1A CpG island promoter in the pathogenesis of pancreatic cancer. RASSF1A hypermethylation was detected in 29 out of 45 (64%) primary adenocarcinomas, in 10 out of 12 (83%) endocrine tumors and in eight out of 18 (44%) pancreatitis samples. In seven out of eight pancreas cancer cell lines, RASSF1A was silenced and was retranscribed after treatment with 5-aza-2'-deoxycytidine. Additionally, we analysed the aberrant methylation frequency of cell cycle inhibitor p16(INK4a) and K-ras gene mutations in the pancreatic samples. p16 inactivation was detected in 43% of adenocarcinomas, in 17% of neuroendocrine tumors, in 18% of pancreatitis and in 63% of pancreas cancer cell lines. K-ras mutations were detected in 16 out of 45 (36%) primary adenocarcinomas. Pancreatic adenocarcinomas with K-ras mutation have significantly less RASSF1A methylation and vice versa (P=0.001, chi(2) test). In conclusion, our data indicate that inactivation of the RASSF1A gene is a frequent event in pancreatic cancer and suggest an inverse correlation between RASSF1A silencing and K-ras activation.     

RASSF1A promoter methylation and Kras2 mutations in non small cell lung cancer

In the present studies, we investigated the correlation between RASSF1A promoter methylation status and Kras2 mutations in 65 primary non small cell lung cancer (NSCLC) including 33 adenocarcinomas, 12 large cell carcinomas, and 20 squamous cell carcinomas. Mutational analysis of Kras2 showed: 30% (10 of 33) of adenocarcinomas, 25% (3 of 12) of large cell carcinomas, and only 5% (1 of 20) of squamous cell carcinomas contained activated Kras2 mutation at codon 12 or 13. RASSF1A promoter region CpG island methylation was detected in adenocarcinomas (55%), large cell carcinomas (25%), and squamous cell carcinomas (25%). Interestingly, combined RASSF1A methylation and Kras2 mutation data show that only - 7% adenocarcinomas/large cell carcinomas exhibited both KRASSF1A promoter methylation and Kras2 mutation, whereas 24% adenocarcinomas, 50% large cell carcinomas, and 70% squamous cell carcinomas showed neither Kras2 mutation nor RASSF1A promoter methylation. These results showed that the majority of the primary NSCLCs with Kras2 mutations lack RASSF1A inactivation, and both RASSF1A inactivation and Kras2 mutation events occur frequently in adenocarcinomas and large cell carcinomas. Our results indicate a trend of inverse relationship between Kras2 activation and RASSF1A promoter methylation in the majority of human lung adenocarcinomas and large cell carcinomas.     

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.     

Correlation between hypermethylation of the RASSF2A promoter and K-ras/BRAF mutations in microsatellite-stable colorectal cancers

Recently, RASSF2A was identified as a potential tumor suppressor epigenetically inactivated in human cancers. Here, we evaluated the methylation status of RASSF2A in colorectal cancer (CRC) and analyzed its correlation with K-ras/BRAF mutations, microsatellite instability status and other clinicopathological features. Using methylation-specific PCR and bisulfite sequencing, we analyzed the methylation status in primary CRC, adenomas and corresponding normal tissues and then compared it with the presence of K-ras and BRAF mutations. We also examined the expression and methylation status of RASSF2A in CRC cell lines. We found that aberrant methylation of RASSF2A promoter regions is associated with gene silencing in CRC cell lines. In primary CRC, the frequency of RASSF2A methylation was 72.6%, and it was found in 16 of 16 (100%) adenomas. In addition, there was a positive correlation between K-ras/BRAF mutations and RASSF2A methylation in primary CRC. Furthermore, a significant positive correlation between K-ras/BRAF mutations and RASSF2A methylation was also observed in microsatellite-stable (p = 0.033) and distal CRC (p = 0.025). These results show that RASSF2A methylation is a frequent event in colorectal tumorigenesis and positively correlates with K-ras/BRAF mutation in microsatellite-stable or distal CRC.     

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.     

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.     

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。     

Association of RASSF1A promoter methylation with gastric cancer risk: a meta-analysis

Ras-association domain family 1A (RASSF1A), a candidate tumor suppressor gene, is frequently silenced and inactivated by hypermethylation of its promoter region in several human tumors. However, the association between RASSF1A promoter methylation and gastric cancer risk remains conflicting. The aim of this study was to assess the association of RASSF1A promoter methylation with gastric cancer risk by a comprehensive meta-analysis. Relevant studies were identified by searches of PubMed and Web of Science databases with no restrictions. Combined odds ratio (OR) and 95 % confidence interval (CI) were used to assess the strength of the association between RASSF1A promoter methylation and gastric cancer risk. A chi-square-based Q test and sensitivity analyses were performed to test the between-study heterogeneity and the contributions of single studies to the final results, respectively. Funnel plots were carried out to evaluate publication bias. Overall, a significant association was observed between RASSF1A promoter methylation and gastric cancer risk (OR, 12.67; 95 % CI, 8.12–19.78; p?<?0.001) with no between-study heterogeneity. Subgroup analyses further revealed that gastric cancer risk was increased for individuals carrying the methylated RASSF1A compared with those with unmethylated RASSF1A. In addition, no publication bias was detected in the overall and subgroup analyses. This study identified a strong association between RASSF1A promoter methylation and risk of gastric cancer and highlighted a promising potential for RASSF1A promoter methylation in gastric cancer risk prediction.     

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.     

结直肠癌不同病变阶段K-ras基因突变的研究

目的:研究结直肠癌不同病变阶段K-ras基因变化及其对结直肠癌演变的影响.方法:提取20例结直肠癌患者的原发灶、淋巴结转移灶、远处转移灶、结直肠腺瘤和相应正常结直肠组织的DNA各20 份,经PCR扩增,对产物进行基因序列分析.结果: 正常结直肠组织均表达野生型K-ras基因,结直肠腺瘤、原发灶、淋巴结转移灶和远处转移灶的K-ras突变率分别为20.0%(4/20)、30.0%(6/20)、25.0%(5/20)和30.0%(6/20),有3例远处转移灶出现复合突变.在发生K-ras突变的标本中,结直肠腺瘤、淋巴结转移灶、远处转移灶的突变类型与原发灶的一致性分别为0%(0/4)、40.0%(2/5)和50.0%(3/6).结论:结直肠腺瘤、淋巴结转移灶和远处转移灶不宜作为临床检测K-ras突变的常规标本,但在无法获得原发灶标本时,淋巴结转移灶和远处转移灶的检测结果也有一定的参考价值;结直肠癌发生、发展的过程中,K-ras基因可能在多阶段发生多次不同的突变.     

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。     

The association between RASSF1A promoter methylation and prostate cancer: evidence from 19 published studies

Ras-associated domain family 1A (RASSF1A) is a putative tumor suppressor gene located at 3p21.3, and the epigenetic inactivation of RASSF1A by hypermethylation of CpG islands within the promoter region has been observed in various cancer types, including prostate cancer (PCa). However, results from published studies on the association between RASSF1A promoter methylation and PCa risk are conflicting and inconclusive. Hence, we conducted a meta-analysis of 19 eligible studies with odds ratio (OR) and its corresponding 95% confidence intervals (95% CI) in order to investigate the strength of relationship of RASSF1A promoter methylation with PCa risk and its clinicopathological variables. Overall, the RASSF1A promoter methylation was significantly associated with PCa risk (OR?=?9.58, 95% CI 5.64–16.88, P heterogeneity <0.001) and Gleason score (GS) (OR?=?2.58, 95% CI 1.64–4.04, P heterogeneity?=?0.019). In addition, subgroup analysis by testing material demonstrated the significant association between RASSF1A methylation and GS (OR?=?3.09, 95% CI 1.92–4.97, P heterogeneity =0.042), PSA level (OR?=?2.75, 95% CI 1.67–4.52, P heterogeneity?=?0.639), and tumor stage (OR?=?1.74, 95% CI 1.05–2.87, P heterogeneity?=?0.026) in tissue rather than urine samples. In conclusion, this meta-analysis suggested that RASSF1A promoter methylation was significantly associated with an increased risk for PCa; furthermore, the RASSF1A methylation status in tissue rather than urine was positively correlated with GS, serum PSA level, and tumor stage, which can be utilized for the early detection and prognosis prediction of PCa.     

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。     

Concomitant RASSF1A hypermethylation and KRAS/BRAF mutations occur preferentially in MSI sporadic colorectal cancer

Methylation-associated inactivation of RASSF1A has frequently been observed in several human malignancies including sporadic colorectal and gastric cancer. However, nothing is known about the RASSF1A methylation status in the setting of MMR-deficient gastrointestinal tumours. In this study, we analysed systematically alterations in KRAS, BRAF and RASSF1A, in order to define the frequency and the pattern of these genetic/epigenetic alterations in three distinct subsets of MSI gastrointestinal tumours. Further, an association study was performed between RASSF1A methylation and the clinicopathological parameters in order to determine the profile of tumours harbouring this epigenetic event. A total of 56 MSI sporadic gastrointestinal tumours (31 colorectal and 25 gastric) and 20 MSI HNPCC analysed for KRAS/BRAF were analysed for RASSF1A promoter hypermethylation by MSP and DNA sequencing. No significant differences were found between the frequency of RASSF1A methylation in sporadic MSI colorectal and gastric carcinomas and HNPCC carcinomas (P=0.31). Methylation of RASSF1A was present in 16 of 31 (52%) sporadic MSI colorectal and 11 of 25 (44%) MSI gastric carcinomas, and in six of 20 (30%) HNPCC carcinomas. Nearly 36% of MSI sporadic colorectal carcinomas (CRCs) had RASSF1A methylation and activating mutations at KRAS and/or BRAF. In contrast, only 10 and 8% of HNPCC and sporadic gastric carcinomas, respectively, had concomitant KRAS mutations and RASSF1A methylation. The MSI sporadic gastric and CRCs with RASSF1A methylation were preferentially poorly differentiated (P=0.03, 0.05, respectively). We show that the profile of alterations RASSF1A, KRAS/BRAF is different among the three groups of MSI gastrointestinal tumours. Further, we demonstrate that MSI sporadic CRCs accumulated significantly more epigenetic/genetic alterations in RASSF1A, KRAS/BRAF than MSI sporadic gastric or HNPCC carcinomas (P=0.016). These results are likely to have therapeutic implications in the near future, due to the possibilities of using specific kinase inhibitors alone or in association with demethylating agents in MSI tumour types harbouring KRAS or BRAF mutations and RASSF1A methylation.     

Promoter methylation of RASSF1A and DAPK and mutations of K-ras,p53, and EGFR in lung tumors from smokers and never-smokers

Background  

Epidemiological studies indicate that some characteristics of lung cancer among never-smokers significantly differ from those of smokers. Aberrant promoter methylation and mutations in some oncogenes and tumor suppressor genes are frequent in lung tumors from smokers but rare in those from never-smokers. In this study, we analyzed promoter methylation in the ras-association domain isoform A (RASSF1A) and the death-associated protein kinase (DAPK) genes in lung tumors from patients with primarily non-small cell lung cancer (NSCLC) from the Western Pennsylvania region. We compare the results with the smoking status of the patients and the mutation status of the K- ras, p53, and EGFR genes determined previously on these same lung tumors.     

Applicability of HIN-1, MGMT and RASSF1A promoter methylation as biomarkers for detecting field cancerization in breast cancer

Introduction

It has been shown in some articles that genetic and epigenetic abnormalities cannot only be found in tumor tissues but also in adjacent regions that appear histologically normal. This phenomenon is metaphorically called field cancerization or field defect. Field cancerization is regarded as clinically significant because it is assumed to be an important factor in local recurrence of cancer. As the field showing these molecular abnormalities may not be removed completely by surgery, these changes might lead to neoplasms and subsequent transformation to a tumor. We aimed to investigate the applicability of the methylation status of six tumor suppressor genes as biomarkers for detecting field cancerization in breast cancer.

Methods

The promoter methylation status of CCND2, DAPK1, GSTP1, HIN-1, MGMT and RASSF1A was determined by methylation-sensitive high-resolution melting (MS-HRM) analysis. MS-HRM methods for CCND2, MGMT and RASSF1A were developed in-house, primer sequences for DAPK1, GSTP1 and HIN-1 have already been published. Biopsy samples were taken from tumor, tumor-adjacent and tumor-distant tissue from 17 breast cancer patients. Normal breast tissues of four healthy women served as controls.

Results

All MS-HRM methods proved to be very sensitive. LODs were in the range from 0.1 to 1.5 %, LOQs ranged from 0.3 to 5.3 %. A total of 94 %, 82 % and 65 % of the tumors showed methylation of RASSF1A, HIN-1 and MGMT promoters, respectively. The methylation status of these promoters was significantly lower in tumor-distant tissues than in tumor tissues. Tumor-adjacent tissues showed higher methylation status of RASSF1A, HIN-1 and MGMT promoters than tumor-distant tissues, indicating field cancerization. The methylation status of the HIN-1 promoter in tumor-adjacent tissues was found to correlate strongly with that in the corresponding tumors (r = 0.785, p < 0.001), but not with that in the corresponding tumor-distant tissues (r = 0.312, p = 0.239).

Conclusions

Among the gene promoters investigated, the methylation status of the HIN-1 promoter can be considered the best suitable biomarker for detecting field cancerization. Further investigation is needed to test whether it can be used for defining surgical margins in order to prevent future recurrence of breast cancer.