Tumour-specific distribution of BRCA1 promoter region methylation supports a pathogenetic role in breast and ovarian cancer

The role of BRCA1 in sporadic breast and ovarian cancers remains elusive. Direct involvement of BRCA1 in the development of breast and ovarian cancer is suggested by the finding that the BRCA1 promoter region CpG island is methylated in a proportion of breast and ovarian cancers. The aim of this study was to compare the incidence of BRCA1 promoter region methylation in tumours in which loss of BRCA1 has been shown to play a role in pathogenesis (breast and ovarian carcinomas) with the incidence in tumours in which BRCA1 is unlikely to play a role in pathogenesis. Promoter region hypermethylation was significantly more common (P < 0.008) in breast and ovarian cancer (6/38 tumours methylated) than in colon cancer (0/35 tumours methylated) or in leukaemias (0/19 samples methylated). The restriction of BRCA1 promoter region hypermethylation to breast and ovarian cancer is consistent with a pathogenetic role of BRCA1 promoter methylation in these tumours. We suggest that the rarity of observed BRCA1 mutations in sporadic breast and ovarian cancer is due to the greater likelihood of BRCA1 inactivation by non-mutational mechanisms such as methylation.     

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。     

Frequent inactivation of PTEN by promoter hypermethylation in microsatellite instability-high sporadic colorectal cancers

Loss of PTEN tumor suppressor function is observed in tumors of breast, prostate, thyroid, and endometrial origin. Allelic losses in the proximity of the PTEN locus (10q23) also occur in sporadic colorectal cancers (CRCs), but biallelic inactivation of this site has not been frequently demonstrated. We hypothesized that alternative mechanisms of PTEN allelic inactivation, such as promoter hypermethylation, might be operative in CRC and that PTEN inactivation may be related to recognized forms of genomic instability. We characterized a cohort of 273 sporadic CRCs by determining their microsatellite instability (MSI) status. Of these, 146 cancers were examined for PTEN promoter methylation by methylation-specific PCR. Mutations at the poly(A)6 repeat sequences in PTEN exons 7 and 8 and deletions at the 10q23 locus were also identified using microsatellite analysis. The presence of PTEN protein was determined by immunostaining, and the results were correlated with the promoter methylation status. We observed that PTEN promoter hypermethylation was a frequent occurrence in MSI-high (MSI-H) tumors (19.1% of MSI-H versus 2.2% of MSI-low/microsatellite stable tumors; P = 0.002). A PTEN mutation or a deletion event was present in 60% of the tumors with promoter region hypermethylation. Hypermethylation of the PTEN promoter correlated significantly with either decreased or complete loss of PTEN protein expression (P = 0.004). This is the first demonstration of PTEN inactivation as a result of promoter hypermethylation in MSI-H sporadic CRCs. These data suggest that this silencing mechanism plays a major role in PTEN inactivation and, in colon cancer, may be more important than either allelic losses or inactivating mutations. The significant correlation of PTEN hypermethylation with MSI-H tumors further suggests that PTEN is an additional important "target" of methylation along with the hMLH1 gene in the evolution of MSI-H CRCs and also confers the "second hit" in the biallelic inactivation mechanism for some proportion of tumors.     

Aberrant Promoter Hypermethylation of RASSF1a and BRCA1 in Circulating Cell-Free Tumor DNA Serves as a Biomarker of Ovarian Carcinoma

Objective: Ovarian cancer is one of the leading causes of cancer deaths in women. Ovarian cancer is diagnosed at the late stages and generally relapses within 12-14 months of cytoreductive surgery. This is attributed to lack of precise molecular detection methodologies to detect and track the disease. Epigenetic alteration such as aberrant promoter hypermethylation is an important early event that occurs during cancer development and progression. This study focuses on development of a minimally invasive methylation marker that could be used for detection and prognosis of ovarian cancer patients. Methods: Aberrant promoter hypermethylation of RASSF1a and BRCA1 was assessed in circulating DNA of 72 EOC patients using methylation-specific PCR. The findings were correlated with various clinicopathological parameters. Statistical analysis was done using the Fisher exact test and chi-square test. Results: The aberrant methylation patterns of RASSF1a and BRCA1 was identified to be present in the cancerous samples. A total of 31.9 % and 56.9% methylation was observed for RASSF1a and BRCA1 respectively. A striking 50% methylation of BRCA1 was identified in the benign sample cohort, which marks the significance of assessing the hypermethylation pattern to detect cancer at its early stages. Methylation of the two tumor suppressor genes was evident across various stages and grades of ovarian tumors suggesting that this could also help as a prognostic marker. Conclusion: The results of the current study hold significance since the hypermethylation patterns can be identified in the cell-free circulating tumor DNA from a small volume of blood plasma and is a simple and minimally-invasive method. Assessment of hypermethylation patterns of a panel of TSG along with the existing screening markers could aid in better diagnosis and management of the disease. It could also aid in designing specifically tailored treatment strategies to fight the disease.     

Methylation not a frequent “second hit” in tumors with germline BRCA mutations

Mutations in tumor suppressor genes BRCA1 and BRCA2 confer an increased lifetime risk of breast and ovarian cancer. Loss of heterozygosity (LOH) of the wildtype allele has been observed in ~80% of tumors from BRCA1 carriers and 70% of tumors from BRCA2 carriers and accounts for the majority of the “second-hits” occurring in BRCA-related tumors. Few sporadic tumors have been reported to have mutations in BRCA. Some sporadic tumors do show LOH of BRCA1 and BRCA2. BRCA1 promoter methylation has also been observed in sporadic ovarian and breast tumors; however, BRCA2 promoter methylation has not been reported in sporadic tumors. The relationship between BRCA LOH and BRCA promoter methylation has not been well characterized in tumors from BRCA germline mutation carriers. The goal of this study was to determine if BRCA1 and BRCA2 promoter hypermethylation serves as a “second-hit” in tumors from mutation carriers that do not show LOH. We studied 38 tumors from BRCA1 carriers and 23 tumors from BRCA2 carriers for LOH. To determine if BRCA1 and BRCA2 promoter hypermethylation serves as a “second-hit” in tumors with germline mutations, we tested 15 tumors lacking LOH and nine tumors with LOH for BRCA1 or BRCA2 promoter methylation. We identified seven BRCA1 tumors and nine BRCA2 tumors lacking LOH. Of these, only one tumor with a BRCA2 mutation showed promoter methylation. These data indicate that promoter methylation is a not a frequent “second-hit” in tumors from BRCA1 or BRCA2 carriers.     

BRCA1 promoter hypermethylation and protein expression in ovarian carcinoma—an Indian study

Mounting evidences suggest that aberrant methylation of CpG islands is a major pathway leading to the inactivation of tumour suppressor genes and the development of cancer. The aim of the current study was to examine the prevalence of the promoter hypermethylation and protein expression of the BRCA1 gene in epithelial ovarian carcinoma (EOC) to understand the role of epigenetic silencing in ovarian carcinogenesis. We studied the promoter methylation of the BRCA1 gene by methylation-specific PCR in a cohort of 88 patients with EOC, 14 low malignant potential (LMP) tumours and 20 patients with benign tumours of the ovary. The expression of the BRCA1 protein by immunohistochemical analysis was carried out in a subset of 64 EOCs, 10 LMP tumours, 10 benign tumours and 5 normal ovarian tissues. The frequencies of methylation in EOCs and LMP tumours were 51.2 and 57 %, respectively, significantly higher (p?=?0.000 and p?=?0.001) in comparison to benign tumours and normal ovarian tissue where no methylation was seen. Expression of BRCA1 was significantly lower in EOCs (p?=?0.003). Lack of protein expression correlated with tumour grade and type. The methylation status correlated well with downregulation of BRCA1 expression. Our results clearly demonstrate that hypermethylation of BRCA1 promoter is a frequent event in ovarian cancer. These data support the hypothesis that BRCA1 promoter methylation plays an important role in the functional inactivation of BRCA1. Follow-up clinical data will reveal the impact of BRCA1 methylation on survival.     

High Frequency of Genes’ Promoter Methylation, but Lack of BRAF V600E Mutation among Iranian Colorectal Cancer Patients

Gene silencing due to DNA hypermethylation is a major mechanism for loss of tumor suppressor genes function in colorectal cancer. Activating V600E mutation in BRAF gene has been linked with widespread methylation of CpG islands in sporadic colorectal cancers. The aim of the present study was to evaluate the methylation status of three cancer-related genes, APC2, p14ARF, and ECAD in colorectal carcinogenesis and their association with the mutational status of BRAF and KRAS among Iranian colorectal cancer patients. DNA from 110 unselected series of sporadic colorectal cancer patients was examined for BRAF V600E mutation by PCR-RFLP. Promoter methylation of genes in tumors was determined by methylation specific PCR. The frequency of APC2, E-CAD, and p14 methylation was 92.6%, 40.4% and 16.7%, respectively. But, no V600E mutation was identified in the BRAF gene in any sample. No association was found in cases showing epigenetic APC, ECAD, and p14 abnormality with the clinicopathological parameters under study. The association between KRAS mutations and the so called methylator phenotype was previously reported. Therefore, we also analyzed the association between the hot spot KRAS gene mutations in codons of 12 and 13 with genes' promoter hypermethylation in a subset of this group of patients. Out of 86 tumors, KRAS was mutated in 24 (28%) of tumors, the majority occurring in codon 12. KRAS mutations were not associated with genes' methylation in this tumor series. These findings suggest a distinct molecular pathway for methylation of APC2, p14, and ECAD genes from those previously described for colorectal cancers with BRAF or KRAS mutations.     

Distinction of hereditary nonpolyposis colorectal cancer and sporadic microsatellite-unstable colorectal cancer through quantification of MLH1 methylation by real-time PCR.

PURPOSE: Promoter hypermethylation occurs frequently in tumors and leads to silencing of tumor-relevant genes like tumor suppressor genes. In a subset of sporadic colorectal cancers (CRC), inactivation of the mismatch repair gene MLH1 due to promoter methylation causes high level of microsatellite instability (MSI-H). MSI-H is also a hallmark of hereditary nonpolyposis colorectal cancer (HNPCC) in which mismatch repair inactivation results from germ-line mutations. For differentiation of sporadic and hereditary MSI-H tumor patients, MLH1 promoter methylation analysis is a promising tool but is not yet used in daily diagnostics because only qualitative techniques without standardization are available. The aim of this study is to establish a reliable and quantitative MLH1 methylation analysis technique and to define valid MLH1 methylation cutoff values for HNPCC diagnostics. EXPERIMENTAL DESIGN: We developed a new real-time PCR-based technique to detect and quantify methylation of both proximal and distal hMLH1 promoter regions. We established and validated this technique in a cohort of 108 CRCs [94 MSI-H and 16 microsatellite stable (MSS) cases] comprising a reference (n = 58) and a tester tumor group (n = 50). RESULTS: The reference tumor group contained 28 HNPCC with proven germ-line mutations or positive Amsterdam I criteria (median age, 37 years) and loss of MLH1 expression, 14 sporadic MSI-H CRC tumors with loss of MLH1 expression and BRAF V600E mutation (median age, 80.5 years), and 16 sporadic MSS CRC (median age, 76.5 years). No MLH1 promoter methylation could be found in any MSS tumors. HNPCC patients showed no or low level of MLH1 promoter methylation. A cutoff value of 18% methylation extent could be determined in this study to define MLH1 hypermethylation specific for sporadic MSI-H cases. Methylation could also be verified qualitatively by melting point analysis. BRAF V600E mutations were not detected in any HNPCC patients (n = 22 informative cases). CONCLUSION: According to the present data, quantitative MLH1 methylation analysis in MSI-H CRC is a valuable molecular tool to distinguish between HNPCC and sporadic MSI-H CRC. The detection of a BRAF V600E mutation further supports the exclusion of HNPCC.     

A hereditary nonpolyposis colorectal carcinoma case associated with hypermethylation of the MLH1 gene in normal tissue and loss of heterozygosity of the unmethylated allele in the resulting microsatellite instability-high tumor

Fourteen suspected hereditary nonpolyposis colorectal carcinoma cases with microsatellite unstable(microsatellite instability-high; MSI-H) tumors but no germ-line MSH2, MSH6, or MLH1 mutations were examined for hypermethylation of CpG sites in the critical promoter region of MLH1. The methylation patterns were determined using methylation-specific PCR and by sequence analysis of sodium bisulfite-treated genomic DNA. In one case, DNA hypermethylation of one allele was detected in DNA isolated from blood. In the MSI-H tumor from this case, the unmethylated MLH1 allele was eliminated by loss of heterozygosity, and the methylated allele was retained. This biallelic inactivation resulted in loss of expression of MLH1 in the tumor as confirmed by immunohistochemistry. These results suggest a novel mode of germ-line inactivation of a cancer susceptibility gene.     

Methylation profiles of the BRCA1 promoter in hereditary and sporadic breast cancer among Han Chinese

The development of breast cancer is a multistep process associated with complex changes in host gene expression patterns including inactivation of tumor suppressor genes and activation of oncogenes. Critically, hereditary predisposition plays a significant role in cancer susceptibility. However, mutation of the BRCA1 gene is found only in the minority of hereditary breast cancer, which indicates that there might be alternative, novel mechanisms contributing to inactivation of the BRCA1 gene. Studies have shown that aberrant methylation of genomic DNA plays an important role in carcinogenesis. The aim of this study was to investigate whether DNA methylation may be an alternative mechanism for the inactivation of BRCA1 as an epigenetic modification of the genome and whether hereditary breast cancer has a different BRCA1 methylation phenotype pattern than sporadic breast cancer. The pattern of CpG island methylation within the promoter region of BRCA1 was assessed by bisulfite sequencing DNA from peripheral blood cells of 72 patients with hereditary predisposition but without BRCA1 mutations and 30 sporadic breast cancer controls. The overall methylation level in patients with hereditary predisposition was significantly lower than that in the sporadic control group. However, patients with hereditary predisposition showed a significantly higher methylation susceptibility for the sites -518 when compared to controls. These results suggest that there might be different BRCA1 promoter methylation levels and patterns in sporadic and hereditary breast cancer in peripheral blood DNA. These findings may facilitate the early diagnosis of hereditary breast cancer.     

Tumor cell-specific BRCA1 and RASSF1A hypermethylation in serum, plasma, and peritoneal fluid from ovarian cancer patients

Because existing surgical and management methods can consistently cure only early-stage ovarian cancer, novel strategies for early detection are required. Silencing of tumor suppressor genes such as p16INK4a, VHL, and hMLH1 have established promoter hypermethylation as a common mechanism for tumor suppressor inactivation in human cancer and as a promising target for molecular detection in bodily fluids. Using sensitive methylation-specific PCR, we screened matched tumor, preoperative serum or plasma, and peritoneal fluid (washes or ascites) DNA obtained from 50 patients with ovarian or primary peritoneal tumors for hypermethylation status of the normally unmethylated BRCA1 and RAS association domain family protein 1A tumor suppressor genes. Hypermethylation of one or both genes was found in 34 tumor DNA (68%). Additional examination of one or more of the adenomatous polyposis coli, p14ARF, p16INK4a, or death associated protein-kinase tumor suppressor genes revealed hypermethylation in each of the remaining 16 tumor DNA, which extended diagnostic coverage to 100%. Hypermethylation was observed in all histologic cell types, grades, and stages of ovarian tumor examined. An identical pattern of gene hypermethylation was found in the matched serum DNA from 41 of 50 patients (82% sensitivity), including 13 of 17 cases of stage I disease. Hypermethylation was detected in 28 of 30 peritoneal fluid DNA from stage IC-IV patients, including 3 cases with negative or atypical cytology. In contrast, no hypermethylation was observed in nonneoplastic tissue, peritoneal fluid, or serum from 40 control women (100% specificity). We conclude that promoter hypermethylation is a common and relatively early event in ovarian tumorigenesis that can be detected in the serum DNA from patients with ovary-confined (stage IA or B) tumors and in cytologically negative peritoneal fluid. Analysis of tumor-specific hypermethylation in serum DNA may enhance early detection of ovarian cancer.     

Promoter Hypermethylation and Expression Changes of BRCA1 Gene in a Cohort of Sporadic Breast Cancer Cases among Pakistani Population

Objective: The purpose of our study was to determine the frequency of BRCA1 promoter hypermethylation and its association with expression changes of BRCA1 and main morphological features in sporadic breast cancer. Methods: A retrospective review of cases was performed to select those with specific morphological features suggestive of breast cancer. BRCA1 promoter hypermethylation and changes in protein expression were evaluated in 30 cancerous and 30 non-cancerous tissue samples. A tissue microarray containing samples from normal and tumor tissue was prepared and stained for BRCA1 protein expression using a commercially available monoclonal antibody against BRCA1 (Ab-1) clone MS110 (mAb). DNA was extracted using modified protocol of Qiagen minikit. DNA was modified using a Bisulfite conversion kit and BRCA1 hypermethylation was detected using a methylation specific PCR. Results: Promoter hypermethylation was negative in 30 non-cancerous samples with retained BRCA1 protein expression. Methylation was positive in 82.6% (n=19/23) of the sporadic cancer samples that had loss of BRCA1 expression and 50% (n=2/4) of the samples with equivocal protein expression. Methylation was negative in all the sporadic breast cancer samples (n=3/3) with retained protein expression. Chi-square analysis showed significant association of BRCA1 promoter methylation with decreased protein expression (P=0.016) and co-existence of loss of BRCA1 and Her2neu at chromosome 17 (P=0.026) respectively. There was no significant association of BRCA1 methylation with morphological features excluding necrosis (P=0.035). Promoter hypermethylation was found to be most common (68.75%) among Triple Negative Breast Cancer (TNBC) females less than 45 years old. Conclusion: Our study suggests that BRCA1 promoter hypermethylation has significant contribution in sporadic breast carcinogenesis. This was our preliminary study in Pakistan. Further studies aimed to determine the in-depth mechanisms of BRCA1 epigenetics in TNBC. BRCAness enriched phenotype in TNBC might be used as a biomarker for the exploitation of therapeutic and clinical implications.     

Inactivation of the Fanconi anemia/BRCA pathway in lung and oral cancers: implications for treatment and survival

Inactivation of the FANC-BRCA pathway via promoter methylation of the FANCF gene renders cells sensitive to DNA crosslinking agents, and has been identified in ovarian cancer cell lines and sporadic primary tumor tissues. We investigated epigenetic alterations in the FANC-BRCA pathway in head and neck squamous cell carcinomas (HNSCC) and non-small-cell lung cancers (NSCLC) using methylation-specific PCR. Promoter methylation of FANCF occurred in 15% (13/89) of HNSCCs and 14% (22/158) of NSCLCs. Methylation of BRCA1 occurred only in 6/158 NSCLC, and was limited to adenocarcinomas and large-cell carcinomas of the lung. No methylation of BRCA2 was detected. FANCF methylation was associated with a shorter duration of tobacco use (P=0.03) and a younger age of starting smoking (P=0.06) in NSCLC, and with a greater number of years of alcohol drinking (P=0.02) in HNSCC. In adenocarcinomas of the lung, FANCF promoter methylation was a significant predictor of poor survival with a hazard ratio of 3.1 (95% CI 1.2-7.9). This study demonstrates that inactivation of the FANC-BRCA pathway is relatively common in solid tumors and may be related to tobacco and alcohol exposure and survival of these patients.