BRCA1 promoter methylation in peripheral blood cells is associated with increased risk of breast cancer with BRCA1 promoter methylation

BRCA1 promoter methylation reportedly plays an important part in the pathogenesis of human breast cancer. In the present study, we investigated whether or not BRCA1 promoter methylation in peripheral blood cells (PBCs) can serve as a risk factor for developing breast cancer. The association of BRCA1 promoter methylation in PBCs with breast cancer risk was examined in a case–control study (200 breast cancer patients and 200 controls). BRCA1 promoter methylation in PBCs and breast tumors was determined with a methylation-specific quantitative PCR assay. BRCA1 promoter methylation in PBCs was seen in 43 (21.5%) of the breast cancer patients and in 27 (13.5%) of the controls. The odds ratio for breast cancer adjusted for other epidemiological risk factors was 1.73 (1.01–2.96) and was statistically significant (P = 0.045). When breast tumors were classified into those with and without BRCA1 promoter methylation, the odds ratio was 0.84 (0.43–1.64) (P = 0.61) for BRCA1 promoter methylation-negative and 17.78 (6.71–47.13) (P < 0.001) for BRCA1 promoter methylation-positive breast tumors. BRCA1 promoter methylation in PBCs is significantly associated with risk of breast cancer with BRCA1 promoter methylation. This seems to indicate that BRCA1 promoter methylation in PBCs may constitute a novel risk factor for breast cancer with BRCA1 promoter methylation.     

Methylation of the BRCA1 promoter region in sporadic breast and ovarian cancer: correlation with disease characteristics

Reduced expression of BRCA1 has been reported in sporadic breast cancer, although the mechanisms underlying this phenomenon remain unclear. Abnormal methylation leading to silencing of tumour suppressor genes has been implicated in tumorigenesis in a wide range of sporadic cancers. Therefore, we sought to determine the frequency of methylation within the BRCA1 promoter region in a large group of sporadic invasive breast (n =96) and ovarian (n = 43) carcinomas using Southern analyses. Overall, methylation was detected in 11% of breast cancer cases and in 5% of ovarian tumours. Methylation of the BRCA1 promoter region was strongly correlated with lack of estrogen and progesterone receptor expression. It is clear from the frequency of abnormal methylation of the BRCA1 promoter region, that this cannot be the sole mechanism mediating the reduced expression of BRCA1 that has previously been reported to occur in the majority of invasive sporadic breast tumours. Nevertheless this study suggests that abnormal methylation of the BRCA1 promoter may be important in tumorigenesis in a subset of sporadic breast and ovarian cancers.     

BRCA1 promoter methylation of normal breast epithelial cells as a possible precursor for BRCA1‐methylated breast cancer

The breast cancer susceptibility gene 1 (BRCA1) and glutathione S‐transferase P1 (GSTP1) promoters are reportedly often methylated in breast cancer tissues. Their methylation status in surrounding normal breast tissues has not been examined thoroughly although this may well be important for a better understanding of breast carcinogenesis. In this study, BRCA1 and GSTP1 promoter methylation was examined by methylation‐specific PCR assay. Patients with BRCA1‐methylated (n = 15) or BRCA1‐unmethylated (n = 15) tumors and those with GSTP1‐methylated (n = 9) or GSTP1‐unmethylated (n = 11) tumors were included in the present study. Methylation status of manually micro‐dissected normal epithelial cells from the formalin‐fixed paraffin‐embedded sections of normal breast tissues adjacent to and distant from the tumors was examined at multiple sites (n = 1–5). Of the 15 patients with BRCA1‐methylated tumors, 9 harbored BRCA1 promoter methylation in at least one site of the normal breast tissues. However, no BRCA1 promoter methylation was observed at any site of the normal tissues of the 15 patients with BRCA1‐unmethylated tumors. No GSTP1 promoter methylation was observed in the normal tissues regardless of the methylation status of the tumors. The presence of BRCA1 promoter methylation in the normal tissues was confirmed in the epithelial cells enriched with the magnetic‐activated cell sorting method. Our findings suggest that a small proportion of normal breast epithelial cells with BRCA1 promoter methylation can be precursor cells from which BRCA1‐methylated breast tumors may originate. This does not apply to GSTP1 promoter methylation.     

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.     

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。     

散发性乳腺癌组织中BRCA1基因启动子甲基化与蛋白表达的相关性

目的：探讨BRCA1基因启动子甲基化对BRCA1蛋白表达的影响,及与散发性乳腺癌发病之间的关系。方法：甲基化特异性PCR（MSP）法检测51例散发性乳腺浸润导管癌和10例乳腺良性组织的BRCA1基因启动子甲基化状态,SP法检测BRCA1蛋白表达水平。结果：10例乳腺良性组织中均未检测到BRCA1启动子的异常甲基化,BRCA1蛋白在细胞核均阳性表达,其中70％（7／10）为强阳性表达。在51例散发性乳腺浸润性导管癌组织中检测到7例（13．73％）BRCAl启动子发生异常甲基化,其余44例（86．27％）未检测到BRCA1启动子甲基化。7例BRCA1启动子甲基化的组织均未见BRCA1蛋白细胞核阳性表达（0／7）,仅有1例BRCA1蛋白细胞质表达;44例未检测到BRCA1启动子甲基化的浸润性导管癌中,30例（68．18％）BRCA1蛋白在细胞核阳性表达,14例（31．82％）BRCA1蛋白在细胞核阴性表达。BRCA1启动子甲基化与BRCA1蛋白核低表达密切相关,X^2=11．9591,P=0．0005;BRCA1蛋白的表达在乳腺良性组织与癌组织之间差异有统计学意义,X^2=4．5879,P=0．032。结论：乳腺癌易感基因BRCA1启动子甲基化可抑制BRCA1蛋白表达,并影响其参与散发性乳腺癌的发生发展过程。     

Constitutional methylation of the BRCA1 promoter is specifically associated with BRCA1 mutation-associated pathology in early-onset breast cancer

Women carrying germline mutations in BRCA1 are at a substantially elevated risk of breast cancer and their tumors typically have distinctive morphologic features. We hypothesized that constitutional methylation of the BRCA1 promoter region could give rise to such breast cancers in women. We selected 255 women diagnosed with breast cancer before the age of 40 years for whom BRCA1 germline mutations had not been identified. Of them, 52 had five or more of nine BRCA1 mutation-associated morphologic features (group 1), 39 had four (group 2), and 164 had three or less (group 3). The prevalence of detectable BRCA1 promoter methylation in peripheral blood DNA decreased from 31% to 10% to 5% across groups 1-3, respectively (P = 0.000002), and was significantly greater than the 4% frequency in unaffected controls (P = 0.004). Peripheral blood methylation was associated with a 3.5-fold (95% CI, 1.4-10.5) increased risk of having early onset breast cancer. Methylation was consistently mosaic in the peripheral blood where the estimated allelic frequency of BRCA1 promoter methylation ranged from 0.1% to 17%. Group 1 women, but not group 3 women, with detectable methylation of peripheral blood DNA had high levels of BRCA1 promoter methylation of their tumor DNA, indicating that constitutional BRCA1 methylation strongly predisposes toward the development of BRCA1 methylated tumors that then have features resembling BRCA1 mutated tumors. Screening peripheral blood for BRCA1 promoter methylation might thus predict early-onset breast cancers. This raises the possibility of chemoprevention or other intervention to diminish the risk of developing breast cancer in these women.     

BRCA1 promoter methylation in peripheral blood DNA of mutation negative familial breast cancer patients with a BRCA1 tumour phenotype

Introduction

Individuals with germline mutations in the BRCA1 gene have an elevated risk of developing breast cancer, and often display characteristic clinicopathological features. We hypothesised that inactivation of BRCA1 by promoter methylation could occur as a germline or an early somatic event that predisposes to breast cancer with the phenotype normally associated with BRCA1 germline mutation.

Methods

We examined seven cases from breast-ovarian cancer families with tumours that showed BRCA1-like pathology but did not have detectable BRCA1 or BRCA2 germline mutations present. Methylation levels were tested by several quantitative techniques including MethyLight, methylation-sensitive high resolution melting (MS-HRM) and a newly developed digital MS-HRM assay.

Results

In one patient, methylation of 10% of the BRCA1 alleles was detected in the peripheral blood DNA, consistent with 20% of cells having one methylated allele. Buccal mucosa DNA from this individual displayed approximately 5% BRCA1 methylation. In two other patients, methylation of BRCA1 was detected in the peripheral blood at significantly lower but still readily detectable levels (approximately 1%). Tumour DNAs from these three patients were heavily methylated at BRCA1. The other patients had no detectable BRCA1 methylation in their peripheral blood. One of seven age-matched controls showed extremely low levels of methylation in their peripheral blood (approximately 0.1%).

Conclusion

These results demonstrate that in some cases of breast cancer, low-level promoter methylation of BRCA1 occurs in normal tissues of the body and is associated with the development of BRCA1-like breast cancer.     

BRCA1 in breast and ovarian cancer predisposition

Women carrying one mutated BRCA1 allele are at increased risk of developing breast and ovarian cancer but tumor initiation requires the loss of the wild-type allele indicating that it is a tumor suppressor gene. In the 10 years since the cloning of BRCA1, a function for the gene product in the DNA damage response has been established. However, identifying the exact biochemical activities of BRCA1 has been a more difficult task. Our current understanding suggests that the molecular functions mediated by the terminal ends of BRCA1, which include an E3 ubiquitin ligase activity at the N-terminus and a protein-protein interaction surface at the C-terminus, are critical to the function of this protein in the response to DNA damage.     

BRCA1 promoter region hypermethylation in ovarian carcinoma: a population-based study

There is a clear association between germ-line BRCA1 mutations and inherited ovarian cancer; however, the association between BRCA1 mutations and sporadic ovarian cancer remains ambiguous. The frequency of BRCA1 promoter hypermethylation as an epigenetic means of BRCA1 inactivation was determined for a large, population-based cohort of ovarian cancer patients. BRCA1 promoter hypermethylation was determined by methylation-specific restriction digestion of tumor DNA, followed by Southern blot analysis and confirmed by methylation-specific PCR. BRCA1 promoter hypermethylation was observed in 12 of 98 ovarian tumors. BRCA1 methylation status of the primary tumor was conserved in six recurrent tumors after interim chemotherapy. None of the 12 tumors with BRCA1 promoter hypermethylation demonstrated BRCA1 protein expression by immunohistochemistry. BRCA1 methylation was only seen in ovarian cancer patients without a family history suggestive of a breast/ ovarian cancer syndrome. Therefore, the 12 BRCA1 methylated tumors represented 15% (12 of 81) of the sporadic cancers analyzed in this study. Although the clinical significance of BRCA1 promoter hypermethylation is yet to be determined, promoter hypermethylation may be an alternative to mutation in causing the inactivation of the BRCA1 tumor suppressor gene in sporadic ovarian cancer.     

BRCA1 promoter methylation is associated with increased mortality among women with breast cancer

Promoter-CpG island hypermethylation has been proposed as an alternative mechanism to inactivate BRCA1 in the breast where somatic mutations of BRCA1 are rare. To better understand breast cancer etiology and progression, we explored the association between BRCA1 promoter methylation status and prognostic factors as well as survival among women with breast cancer. Promoter methylation of BRCA1 was assessed in 851 archived tumor tissues collected from a population-based study of women diagnosed with invasive or in situ breast cancer in 1996–1997, and who were followed for vital status through the end of 2002. About 59% of the tumors were methylated at the promoter of BRCA1. The BRCA1 promoter methylation was more frequent in invasive cancers (P = 0.02) and among premenopausal cases (P = 0.05). BRCA1 promoter methylation was associated with increased risk of breast cancer-specific mortality (age-adjusted HR 1.71; 95% CI: 1.05–2.78) and all-cause mortality (age-adjusted HR 1.49; 95% CI: 1.02–2.18). Neither dietary methyl intakes in the year prior to the baseline interview nor the functional polymorphisms in one-carbon metabolism were associated with BRCA1 methylation status. Our study is the first epidemiological investigation on the prognostic value of BRCA1 promoter methylation in a large population-based cohort of breast cancer patients. Our results indicate that BRCA1 promoter methylation is an important factor to consider in predicting breast cancer survival. This work was supported by grants from the National Institutes of Health (CA109753 to JC; DK55865 to SZ) and in part by grants from Department of Defense (BC031746), National Cancer Institute and the National Institutes of Environmental Health and Sciences (UO1CA/ES66572, UO1CA66572, P30CA013696, P30ES09089 and P30ES10126); and by the University of North Carolina Clinical Nutrition Research Unit (DK56350) and Center for Environmental Health and Susceptibility (ES10126). Xu, X. is a recipient of the Predoctoral Traineeship Award (W81XWH-06-1-0298) of Department of Defense Breast Cancer Research Program.     

BRCA1 and BRCA2 genes: role in hereditary breast and ovarian cancer in Italy.

The heritable defects of BRCA1 and BRCA2 genes have been shown to predispose to breast and ovarian cancers. In a previous report, we analyzed 46 Italian families with breast and/or ovarian cancer for BRCA1 mutations. In the present study, those families and 11 others were screened for BRCA2 mutations; the newly enrolled families were also analyzed for the BRCA1 gene. The coding region and splice boundaries of BRCA2 and BRCA1 genes were assessed by the protein-truncation test and single-strand conformational polymorphism. A total of 20 different mutations were found in 21 families (37%). A total of 9 families (16%) showed mutations in the BRCA1 gene, including the one new mutation identified in this study (5382insC), and 12 families (21%) presented mutations in the BRCA2 gene. BRCA2-mutated families presented breast and ovarian cancers or breast cancers only, whereas most BRCA1-mutated families presented ovarian cancer alone or in association with breast cancer. All the BRCA2 mutations led to a truncated protein: 6 were frameshift mutations, 4 were non-sense mutations and 2 involved the intronic invariant region leading to splice variants. Therefore, in the Italian population, the cumulative proportion of BRCA1 and BRCA2 mutations was within the range observed in other studies (37%), with higher involvement of BRCA2 than of BRCA1. Many families in which no mutations were found presented a very high incidence of breast and/or ovarian cancer. Among the 36 BRCA1 and BRCA2 wild-type families, 24 presented at least 4 cancer cases, indicating the existence of other important predisposing genes.     

BRCA1 promoter methylation in peripheral blood is associated with the risk of triple-negative breast cancer

Methylation of the promoter of the BRCA1 gene in DNA derived from peripheral blood cells is a possible risk factor for breast cancer. It is not clear if this association is restricted to certain types of breast cancer or is a general phenomenon. We evaluated BRCA1 methylation status in peripheral blood cells from 942 breast cancer patients and from 500 controls. We also assessed methylation status in 262 paraffin-embedded breast cancer tissues. Methylation status was assessed using methylation-sensitive high-resolution melting and was categorized as positive or negative. BRCA1 methylation in peripheral blood cells was strongly associated with the risk of triple-negative breast cancer (TNBC) (odds ratio [OR] 4.70; 95% confidence interval [CI]: 3.13–7.07; p < 0.001), but not of estrogen-receptor positive breast cancer (OR 0.80; 95% CI: 0.46–1.42; p = 0.46). Methylation was also overrepresented among patients with high-grade cancers (OR 4.53; 95% CI: 2.91–7.05; p < 0.001) and medullary cancers (OR 3.08; 95% CI: 1.38–6.88; p = 0.006). Moreover, we detected a significant concordance of BRCA1 promoter methylation in peripheral blood and paired tumor tissue (p < 0.001). We found that BRCA1 promoter methylation in peripheral blood cells is associated with approximately five times greater risk of TNBC. We propose that BRCA1 methylation in blood-derived DNA could be a novel biomarker of increased breast cancer susceptibility, in particular for triple-negative tumors.     

BRCA1 promoter methylation status does not predict response to tamoxifen in sporadic breast cancer patients

The purpose of this study is to investigate whether BRCA1 promoter methylation is associated with poorer outcome in sporadic breast cancer cases treated with tamoxifen. BRCA1 promoter methylation was determined by bisulfite pyrosequencing in two groups of sporadic breast cancer patients, systemically untreated (N = 497) and treated with adjuvant tamoxifen (N = 497). Associations of BRCA1 promoter methylation with clinopathological characteristics and the effect of BRCA1 promoter methylation on time to first recurrence (TTR) and overall survival (OS) were examined. No significant differences were observed between BRCA1 promoter methylation and clinopathological characteristics in untreated and tamoxifen-treated groups. Cut point analysis did not find any promising cut point for BRCA1 promoter methylation that would differentially influence TTR and OS in untreated and tamoxifen-treated group. Using the median (2.53 %) and an arbitrary value of 10 % as a cut point for methylation, we still found no significant effect of BRCA1 promoter methylation on TTR and OS in untreated and tamoxifen-treated group. Despite data suggesting that BRCA1 levels impact estrogen receptor response to tamoxifen, our results indicate that BRCA1 promoter methylation is not associated with poorer outcome in sporadic breast cancer cases treated with tamoxifen.     

Risk of ovarian cancer in BRCA1 and BRCA2 mutation-negative hereditary breast cancer families

Women from site-specific hereditary breast cancer families who carry a BRCA1 or BRCA2 mutation are at increased risk for ovarian cancer. It is less clear, however, whether individuals from hereditary breast cancer families who do not carry such a mutation are also at increased ovarian cancer risk. To determine whether women from BRCA mutation-negative hereditary breast cancer families are at increased risk for ovarian cancer, 199 probands from BRCA mutation-negative, site-specific breast cancer kindreds who consented to prospective follow-up at the time of genetic testing were identified. The incidence of new breast and ovarian cancers in probands and their families since receipt of their genetic test results was determined by questionnaire. The expected number of cancers and standardized incidence ratios (SIRs) were determined from age-specific cancer incidence rates from the Surveillance, Epidemiology, and End Results (SEER) program by using the method of Byar. All statistical tests were two-sided. During 2534 women-years of follow-up in 165 kindreds, 19 new cases of breast cancer were diagnosed, whereas only 6.07 were expected (SIR = 3.13, 95% confidence interval [CI] = 1.88 to 4.89; P < .001), and one case of ovarian cancer was diagnosed, whereas only 0.66 was expected (SIR = 1.52, 95% CI = 0.02 to 8.46; P = .48). These results suggest that women from BRCA mutation-negative, site-specific breast cancer families are not at increased risk for ovarian cancer.     

Outcome of ovarian cancer after breast cancer in BRCA1 and BRCA2 mutation carriers

Background:

It is unknown whether a history of breast cancer (BC) affects the outcome of BRCA1/2-associated epithelial ovarian cancer (EOC). This was investigated in the current analysis.

Methods:

We included 386 BRCA1/2-associated EOC patients diagnosed between 1980 and 2015. Progression-free survival (PFS), progression-free interval (PFI), overall survival (OS) and ovarian cancer-specific survival (OCSS) were compared between EOC patients with and without previous BC.

Results:

BRCA-associated EOC patients with, vs without, a BC history had a significantly worse PFS and PFI (multivariate hazard ratio (HR_mult) 1.47; 95% confidence interval (CI) 1.03–2.08 and HR_mult 1.43; 95% CI 1.01–2.03), and a non-significantly worse OS (HR_mult 1.15; 95% CI 0.84–1.57) and OCSS (HR_mult 1.18; 95% CI 0.85–1.62). Ovarian cancer-specific survival was significantly worse for the subgroup treated with adjuvant chemotherapy for BC (HR_mult 1.99; 95% CI 1.21–3.31).

Conclusions:

Our results suggest that BRCA1/2-associated EOC patients with a previous BC have a worse outcome than EOC patients without BC, especially when treated with adjuvant chemotherapy.     

BRCA1-related breast cancer in Austrian breast and ovarian cancer families: Specific BRCA1 mutations and pathological characteristics

We identified 17 BRCA1mutations in 86 Austrian breast and ovarian cancer families (20%) that were screened for mutations by denaturing high-performance liquid chromatography (DHPLC) and the protein<0B> <0R>truncation test (PTT). Eleven distinct mutations were detected, 4 of them (962del4, 2795del4, 3135del4 and L3376stop) not previously reported in families of non-Austrian origin. In addition, 6 rare missense mutations (allele frequency < 1%) with unknown biological effects were identified. Four mutations occurred more than once in the Austrian population: 2795del4 (3 times), Cys61Gly (3 times) 5382insC (2 times) and Q1806stop (2 times). Haplotype analysis of the 4 recurrent mutations suggested a common ancestor for each of these. Thirty-four breast cancer cases from 17 families with BRCA1 mutations were further analyzed. We observed a low median age of onset (39.5 years). Sixty-eight percent of all BRCA1 breast cancer cases had negative axillary lymph nodes. This group showed a significant prevalence of a negative estrogen and progesterone receptor status and stage I tumors compared with an age-related, node-negative control group. The prevalence of grade III tumors was marginally significant . Survival analysis either with a control group matched for age (within 5 years), grade, histologic subtype and estrogen receptor status, or with an age-related, node-negative comparison group, showed no statistical difference. Int. J. Cancer 77:354–360, 1998. © 1998 Wiley-Liss, Inc.