Methylation of BRCA1 Promoter Region Is Associated with Unfavorable Prognosis in Women with Early-Stage Breast Cancer

BRCA1-associated breast cancers are associated with particular features such as early onset, poor histological differentiation, and hormone receptor negativity. Previous studies conducted in Taiwanese population showed that the mutation of BRCA1 gene does not play a significant role in the occurrence of breast cancer. The present study explored methylation of BRCA1 promoter and its relationship to clinical features and outcome in Taiwanese breast cancer patients. Tumor specimens from a cohort of 139 early-stage breast cancer patients were obtained during surgery before adjuvant treatment for DNA extraction. Methylation of BRCA1 promoter region was determined by methylation-specific PCR and the results were related to clinical features and outcome of patients using statistical analysis. Methylation of the BRCA1 promoter was detected in 78 (56%) of the 139 tumors. Chi-square analysis indicated that BRCA1 promoter methylation correlated significantly with triple-negative (ER-/PR-/HER2-) status of breast cancer patients (p = 0.041). The Kaplan-Meier method showed that BRCA1 promoter methylation was significantly associated with poor overall survival (p = 0.026) and disease-free survival (p = 0.001). Multivariate analysis which incorporated variables of patients' age, tumor size, grade, and lymph node metastasis revealed that BRCA1 promoter methylation was associated with overall survival (p = 0.27; hazard ratio, 16.38) and disease-free survival (p = 0.003; hazard ratio, 12.19). Our findings underscore the clinical relevance of the methylation of BRCA1 promoter in Taiwanese patients with early-stage breast cancer.


Introduction
Breast cancer is the most common type of cancer and ranked fourth in cancer mortality in Taiwan [1]. Incidence of breast cancer in Taiwan has been increasing steadily and the rise can be largely attributed to the rise of newly diagnosed young patients [2]. It has been speculated that the early onset of breast cancer in Taiwanese women may be explained by the combination of Westernized diet, sedentary lifestyle, delay childbearing, and exposure to environmental chemicals.
BRCA1 is a major cancer predisposition gene that has been the subject of intense investigation since it was identified and cloned in 1994 [3]. Breast tumors arising from BRCA1 mutation acquired distinct pathological and gene expression profiles [4]. The spectrum of BRCA1 gene mutations in breast cancer patients in various populations has been investigated [5][6][7][8][9][10]. Studies conducted in Taiwan had suggested that the mutation of BRCA1 contributes little to the occurrence of breast cancer [11,12].
Despite germ-line mutations in BRCA1 can account for a significant fraction of familial breast cancer cases, a large proportion of familial breast cancer are not related to mutations in BRCA1 or BRCA2 [13][14][15][16]. Hypermethylation of cytosine residues in CpG islands within the promoter of many tumor suppressor genes is strongly correlated with the absence of gene expression. The epigenetic transcriptional silencing provides an alternative mechanism for the loss of function of tumor suppressor gene during cancer development [17,18]. Hypermethylation of the BRCA1 promoter has been reported in breast cancer with links to down-regulated mRNA and protein level in tumors and cell lines [19][20][21][22][23]. Aberrant BRCA1 promoter methylation has also been found to be associated with particular biological and clinicopathological features [24][25][26][27]. However these reports did not result in similar conclusion as the limited population examined and the high heterogeneity among breast cancer patients in these studies might lead to the inconclusive finding. The aim of the current study was to examine the prevalence and clinical relevance of BRCA1 promoter methylation in Taiwanese women with breast cancer. Our results indicated that promoter region of BRCA1 gene is frequently methylated in Taiwanese patients with early-stage breast cancer. BRCA1-methylated tumor exhibited poorer survival outcome.

Ethics Statement
The study was approved by the institutional review board of Kaohsiung Medical University Hospital, Taiwan. Written informed consent was obtained from all participants.

Study subjects
Surgically resected specimens from a cohort of one hundred thirty-nine patients with early-stage breast cancer (stages I and IIA) were collected at Kaohsiung Medical University Hospital between October 2007 and July 2009. Tissues were frozen at 280uC until the extraction of DNA. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions.

Methylation-specific PCR
One mg of genomic DNA was modified using a CpGenomet DNA modification kit (Chemicon, Temecula, CA) according to the manufacturer's protocol and resuspended in TE buffer. Modified DNA was amplified in a total volume of 10 mL solution containing 1x PCR buffer, 1.5 mM MgCl 2 , 200 ng of each primer, 0.2 mM of each dNTP and 1 U Platinum Taq Polymerase (Life Technologies, Carlsbad, CA). Primers and conditions used for the methylation-specific PCR were first developed by Esteller et al [19]. Primer sequences for unmethylated PCR were 59-TTG GTT TTT GTG GTA ATG GAA AAG TGT-3' and 5'-CAA AAA ATC TCA ACA AAC TCA CAC CA-3' and primers for methylated reaction were 5'-TCG TGG TAA CGG AAA AGC GC-3' and 5'-AAA TCT CAA CGA ACT CAC GCC G-3'. The sense primers of the unmethylated PCR and methylated PCR begin at nucleotide position 1536 and 1543 bp of BRCA1 (GenBank sequence U37574), respectively. This region crosses the major transcription start site of BRCA1 gene [28].The amplified product of unmethylated PCR is 86 bp and that of methylated product is 75 bp.
For methylated genes, SssI treated DNA (MDA-MB-231 cells) was used as a positive control and DNA from normal lymphocytes was used as a negative control. After amplification, PCR products were then loaded and electrophoresed on 2% agarose gels, stained with ethidium bromide and visualized under UV illumination. The presence of a product in the methylated reaction indicated the presence of methylated BRCA1 genes. Tumors that were positive for both methylated and unmethylated reactions were classified as having methylated BRCA1 genes.

Immunohistochemical staining
Immunohistochemical staining was performed on representative samples. The selected paraffin-embedded sections were cut and mounted on poly-l-lysine-coated slides. Staining was carried out with a mouse monoclonal BRCA1 antibody (Clone MS110, Biocare Medical, Concord, CA) at 1:50 dilution in a Leica Bondmax automated immunostainer (Leica Microsystems, Newcastle, UK), according to manufacturer's protocol.

Statistical analysis
Association between BRCA1 methylation status and clinicopathological characteristics was analyzed by use of Pearson chisquare test. Survival rate and disease recurrence were calculated using Kaplan-Meier analysis and compared by the Cochran-Mantel-Haenszel test. Overall survival (OS) was calculated from the time of initial diagnosis to death of any cause. Disease-free survival (DFS) was defined as the time between initial diagnosis and diseases recurrence. Patients alive or disease free at the end of the follow-up period were censored. Data from medical charts were retrieved, and the patients' outcomes were followed until January, 2012 or date of death, whichever occurred first. Potential confounding factors were adjusted and analyzed by use of the Cox proportional hazards regression model. The variables in the model included age, tumor size, lymph node metastasis, and histological grade. All statistical calculations were done using SPSS version 17.0 for windows (SPSS, Inc., Chicago, IL). A p ,0.05 was considered significant.

Results
The clinical characteristics of the 139 early-stage breast cancer patients at the time of surgery are summarized in Table 1. Among these patients, the medium age was 49 years (ranging from 24 to 71 years). Histologically, all cases were invasive ductal carcinomas. Methylation of the BRCA1 promoter was detected in 78 (56%) of the 139 tumors examined in which 28 (20%) tumors were positive only for the methylated reaction and 50 (36%) were positive for both unmethylated and methylated reactions. Figure 1A shows representative methylation status of BRCA1 promoter by methylation-specific PCR. To evaluate the effect of DNA methylation on protein expression, immunohistochemical staining of BRCA1 was carried out. Consistent with the methylation status of BRCA1 promoter, the expression levels of BRCA1 protein were reduced in tumors with methylated genes ( Figure 1B).
Chi-square analysis revealed that despite there was no significant difference between status of BRCA1 promoter methylation in age, tumor size, lymph node status, stage, histologic grade, ER, PR, and HER-2 positivity. A statistically significant association was found between BRCA1 promoter methylation and triplenegative breast cancer (ER-/PR-/HER2-) in this cohort of patients (p = 0.041). In Cox proportional hazards analysis, BRCA1 promoter methylation status was analyzed with tumor size, lymph node metastasis, histological grade, and age for their impact on OS and DFS. Adjusted for all these covariates, BRCA1 promoter methylation was identified as an independent predictor for OS (p = 0.027; hazard ratio = 16.38) and DFS (p = 0.003; hazard ratio = 12.19) ( Table 2). No other clinical variable was found to be independently associated with death in this set of early-stage breast cancer patients. Kaplan-Meier survival curves showed that methylated promoter of the BRCA1 gene was associated with poor OS (p = 0.026; Figure 2A) and DFS (p = 0.001; Figure 2B).

Discussion
The incidence of breast cancer in Taiwan has increased at a significantly faster pace than western countries over the last two decades [2] and stage 0 to IIA breast cancer currently comprises more than half of all newly diagnosed cases.
BRCA1 promoter hypermethylation has been implicated as one of the mechanisms of loss of gene expression and has been identified in 9-32% of unselected sporadic breast cancer [19,25,[28][29][30][31]. We report here that hypermethylation of the BRCA1 gene promoter is present in 56% (78 of 139) of Taiwanese women with early-stage sporadic breast carcinomas, which is significantly higher than previously reported frequencies for this  alteration in unselected sporadic breast tumors. The incidence of BRCA1 methylation has previously been reported to be higher in breast tumors of infiltrating ductal type [31]. Since all our patients are of the infiltrating ductal type, this finding was somewhat comparable to that of current literature. BRCA1 protein expression was found to be absent or markedly decreased in the majority of the BRCA1 methylated tumors, suggesting epigenetic gene silencing in these tumors [31]. Breast cancers with BRCA1 promoter methylation also showed decreased expression of ER [25,29,31] and basal-like phenotype [25]. Our results indicated that BRCA1 promoter methylation correlated significantly with triple-negative breast cancer. (p = 0.041).
More than half of patients with BRCA1 mutation have triplenegative breast cancer, and share common clinical and pathological features [10,32,33]. However, a significant portion of triplenegative breast cancer patients do not carry BRCA1 mutations. Alteration in the function of the BRCA1 gene product has also been identified as an alternative route which also results in impaired BRCA1 function in triple-negative breast cancer [34]. Our finding suggests that BRCA1 promoter methylation may also have an etiological role on the development of triple-negative phenotype and underlie triple-negative breast cancer.
Given that BRCA1-associated breast cancers are more likely to be of high grade [35], or estrogen-receptor negative [35], and p53 positive [36], it has been speculated that BRCA1-related breast cancer is more aggressive than sporadic breast cancer. However, most studies conducted in specific populations suggest that survival for women with BRCA1 mutations is similar to that of women without the mutations [37,38].
The Kaplan-Meier survival curves of the early-stage breast cancer patients plotted into 2 groups according methylation of BRCA1 promoter region showed that patients with methylated BRCA1 promoter had a significantly shorter OS and DFS than patients with unmethylated gene. (p = 0.026 and p = 0.001, respectively). Multivariate analysis adjusted for age, lymph node metastasis, size and grade of tumor also revealed that methylation of the BRCA1 promoter was an independent prognostic marker for survival in early-stage breast cancer (OS, p = 0.027; DFS, p = 0.003). Our findings suggests that BRCA1 promoter methylation is a better predictor of recurrence and survival than factors such as tumor size, lymph node metastasis, histological grade, and age in early-stage breast cancer.
In conclusion, this study highlights the frequent promoter methylation of BRCA1 and its prognostic significance, irrespective of BRCA1 gene mutation in Taiwanese patients with early-stage breast cancer. BRCA1 methylated tumors showed a statistically significant association with triple-negative status and exhibited poor outcome in terms of survival.
Genetic differences among different ethnicities/races may account for disparities in breast cancer susceptibility [3,39]. Aberrant gene promoter methylation has also been shown to be affected by ethnicity in breast cancer [40]. Increased understanding of the genetic/epigenetic abnormality together with the ethnic factors/differences involved in the pathogenesis of breast cancer is crucial and may provide a basis for detection and treatment.
Whether the phenomenon we observed in this study are due to ethnicity or etiology remained to be determine in larger studies that include breast cancer patients of different ethnicities/races.

Author Contributions
Conceived and designed the experiments: FC MH. Performed the experiments: NCH YH PC. Analyzed the data: NCH YH KKY. Wrote the paper: NCH.