Promoter hypermethylation of BRCA1 correlates with absence of ...

[Epigenetics 3:3, 157-163; May/June 2008]; ©2008 Landes Bioscience

Research Paper

Promoter hypermethylation of BRCA1 correlates with absence of expression in hereditary breast cancer tumors Teresa Tapia,1 Susan V. Smalley,1 Paulina Kohen,4 Alex Muñoz,4 Luisa M. Solis,2 Alejandro Corvalan,2 Paola Faundez,1 Luigi Devoto,4 Mauricio Camus,3 Manuel Alvarez3 and Pilar Carvallo1,* 1Department

of Cell and Molecular Biology; Faculty of Biological Sciences; 2Department of Pathology; 3Centro de Cáncer; Faculty of Medicine; Pontificia Universidad Católica de Chile; Santiago, Chile and 4Instituto de Investigaciones Materno Infantil (IDIMI); Faculty of Medicine; Universidad de Chile; Santiago, Chile

Key words: BRCA1 promoter hypermethylation, BRCA1 protein expression, hereditary breast cancer, breast cancer biopsies, estrogen progesterone receptors

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utation in BRCA1.2 Breast tumors from germline mutation carriers m on BRCA1 frequently present a genomic deletion of the normal allele, in accordance with Knudson hypothesis.3 As BRCA1 is a tumor suppressor gene, the loss of expression of both alleles is necessary for function failure. The most prominent feature of BRCA1 deficient cells is the inability to repair DNA cross-links and DNA double strand breaks by error-free homologous recombination.4 Due to its important role in genome stability, the complete loss of function of BRCA1 in breast epithelial cells has been suggested as an accelerator of proliferation and tumor progression.4 Different mechanisms, as promoter hypermethylation and somatic mutations, have been proposed to explain the loss of expression of tumor suppressor genes in cancer biopsies, besides LOH. The abnormal methylation of various tumor suppressor gene promoters has been extensively described, as a relevant mechanism of gene silencing in carcinogenesis.5,6 In relation to sporadic breast cancer, several reports describe the aberrant methylation of the BRCA1 gene promoter in primary breast tumors.7-9 In addition, a low or absent BRCA1 expression has been detected in breast tumors from sporadic cases, indicating that loss of BRCA1 function is involved in the pathogenesis of breast cancer.10-12 No somatic mutations in BRCA1 have been described in sporadic breast tumors, that could explain the decrease or absence of its expression. In a more extended study, Wei et al.13 analyzed the methylation status of the BRCA1 promoter in sporadic tumors finding a 29.8% of cases methylated. In addition, when microdissected tumor cells were analyzed only methylated products were obtained in some tumors and in others a decrease in BRCA1 copy number alleles have been encountered. They proposed that the methylation of the first BRCA1 allele may serve as a first hit for carcinogenesis, followed by reduced BRCA1 copy number in some cases.13 In hereditary cases, germline mutations in BRCA1 are the most common causes described until today for breast cancer, followed by the loss of the second allele frequently through genomic deletion. Only one report has described BRCA1 promoter hypermethylation in 21/50 (42%) tumors without germline mutation in BRCA1 or BRCA2.14 The authors describe a high heterogeneity among these tumors, as it has been stated by others, demonstrating in addition the loss of one BRCA1 allele in 37% of methylated cases. Also, tumors presenting

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Germline mutations in BRCA1 account for a low proportion of hereditary cases in diverse populations. Several efforts have been made to find new genes involved in the inheritance of breast cancer with no success until today. The participation of BRCA1 in the development of breast cancer has been proposed in several studies where hypermethylation of its promoter and a decrease in expression has been reported for sporadic cases and one study on familial cases. To explore the participation of BRCA1 in hereditary carcinogenesis through a different mechanism than the inheritance of germline mutations, we studied the methylation status of its promoter in breast tumors, from patients previously screened for BRCA1/BRCA2 germline mutations. We also determined the presence of the BRCA1 protein in these tumors and correlated both events with tumor grade, hormone receptors and ERBB2 presence. Promoter hypermethylation of the BRCA1 gene was detected in 51% of our biopsies, among which 67% did not express the respective protein. This result leads us to suggest that hypermethylation could be considered as an inactivating mechanism for BRCA1 expression, either as a first or second hit. Moreover, a number of biopsies with absence of expression on BRCA1 showed negative detection of estrogen and progesterone receptors, a similar phenotype to BRCA1 mutated breast tumors.

Introduction BRCA1 is one of the two major susceptibility genes involved in hereditary breast cancer.1 Since the discovery of BRCA1, several reports have described germline mutations accounting for a proportion of hereditary breast cancer cases, that varies among populations. We have previously screened the BRCA1 gene in 54 breast cancer families from Chile and found a 7.4% presenting a germline *Correspondence to: Pilar Carvallo; Departamento de Biología Celular y Molecular; Facultad de Ciencias Biológicas; Pontificia Universidad Católica de Chile; Casilla 114-D; Santiago, Chile; Tel.: +56.2.686.2705; Fax: +56.2.686.2693; Email: [email protected] Submitted: 06/03/08; Accepted: 06/05/08 Previously published online as an Epigenetics E-publication: http://www.landesbioscience.com/journals/epigenetics/article/6387 www.landesbioscience.com

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BRCA1 hypermethylation and silencing in breast cancer

Figure 1. BRCA1 gene promoter with each CpG site located proportionally to the DNA sequence. Location of the region analyzed with MS-PCR primers (nt 1536–1621) (Genbank accession # U37574).

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BRCA1 promoter hypermethylation and LOH show a concordant basal-like phenotype.14 All these data support the hypothesis that, in breast carcinogenesis, the loss of function of BRCA1 is caused by germline mutations, and also through the epigenetic silencing of its promoter. We report here the methylation status of the BRCA1 promoter and the correlation with its expression through immunohistochemistry in breast cancer biopsies from hereditary cases, previously screened for BRCA1 and BRCA2 mutations.

Figure 2. (A) MS-PCR products of BRCA1. Primer sets are designated M for methylated alleles and UM for unm-

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Hypermethylation and expres- ethylated alleles. Electrophoresis in NuSieve-agarose (2:1) 3% gel. Molecular weight marker pBR322/HinfI (A) sion of BRCA1. The promoter MS-PCR Amplification of the BRCA1 gene. (B) DNA sequence profile of an of MS-PCR promoter fragment, with region of the BRCA1 gene analyzed primers that amplify BRCA1 methylated (M) and modified sequences. for methylation status, shown in Figure 1, spans from position -38 to +37 including the +1 transcrip- of hormonal receptors, ERBB2 and p53; the detection of BRCA1 or tion initiation site, corresponding to position nt 1543 to 1617 of BRCA2 germline mutations; the methylation status of the BRCA1 Genbank accession # U37574.18 MS-PCR was performed in a total promoter and the respective expression of the protein. As shown in of 49 biopsies, obtaining amplification products in 47 of them. Table 1, biopsies with promoter hypermethylation were 19/35 IDC We were not able to obtain MS-PCR products for two remaining (54%); 1/6 DCIS (17%); 2/3 ILC (66%) and 2/3 LCIS (66%). Even biopsies, probably due to the low quality DNA isolated from these though there is no apparent correlation between the histological type, samples. As shown in Figure 2A, 44/47 samples gave methylated and age at diagnosis and grade of each tumor with the status of promoter unmethylated products, probably due to the admixture of normal methylation, 60% of p53 positive biopsies showed hypermethylation and tumor cells, or the methylation of only one allele. In three of the BRCA1 promoter. No correlation between methylation of the biopsies that showed no expression of BRCA1 (T32, T35 and T45) BRCA1 promoter and the presence or absence of hormonal receptors only the methylated product was obtained, revealing that methyla- have been found, either independently (ER, p value = 0.7702; PR, tion of both alleles may have occurred. MS-PCR fragments of the p value = 0.7661) or together (ER-/PR-; ER+/PR+, p value = BRCA1 promoter from three tumors were cloned and sequenced to 1.0000). confirm the methylation and modification status of DNA (Fig. 2B). The absence of BRCA1 expression determined through IHC, All sequenced clones showed the expected C > T changes in sites shown in Figure 3, correlates with the promoter hypermethylation other than CpG dinucleotides, and conserved a cytosine in at least in 16/24 (67%) samples (p value = 0.0199). Figure 3A shows a 8/9 CpG sites included in the MS-PCR fragment of 75 bp. Among positive BRCA1 nuclear staining in epithelial cells from a normal the 47 tumor samples, 24 (51%) biopsies showed hypermethylation breast used as a positive control, and in panel D an example of posiof the BRCA1 promoter and 23 (49%) were unmethylated. Table 1 tive staining in a breast tumor (T23); in contrast with a negative shows the histology and grade of each tumor; the presence or absence detection in panel C corresponding to tumor T29. The twenty three 158

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Table 1 Histopathological characteristics of 49 hereditary breast cancers evaluated for the methylation status of the BRCA1 promoter

IDC, invasive ductal carcinoma; DCIS, ductal invasive carcinoma in situ; ILC, invasive lobulillar carcinoma; LCIS, lobulillar carcinoma in situ. Grade I, well differentiated; II, moderately differentiated; III, poorly differentiated. (+) presence and (–) absence; (n/d), not determined; M, methylated; UM, unmethylated.

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Figure 3. Immunohistochemical staining of BRCA1 in breast tissue. (A) Positive control of a normal breast lobule with strong nuclear staining. (B) Negative control, normal breast with no antibody added. (C) Invasive ductal carcinoma (T29) without protein immunoreactivity. (D) Invasive ductal carcinoma (T23) showing protein immunoreactivity. Black bar: scale 40 μm.

nonmethylated biopsies include 16 (70%) expressing BRCA1 and 7 (30%) with negative expression. In order to explain the lack of expression in some biopsies we verified the possible loss of one BRCA1 allele in a parallel array-CGH performed in microdissected cells, in the same group of biopsies (manuscript submitted). We detected three tumors (T21, T35 and T38), with no germline mutation in BRCA1 and no expression of BRCA1, presenting a deletion of one BRCA1 allele. Two of these samples T21 and T35, gave methylated products in MS-PCR of the BRCA1 promoter in concordance with the hypothesis of two hits: one allele methylated, the second allele 160

deleted. The third biopsy (T38) did not show hypermethylation of the BRCA1 promoter, so the loss of expression could be explained by a somatic truncating mutation in the second allele. Two biopsies with germline mutation in BRCA2 (T5: c.5374_5377delTATG and T44: c.4970_4971insTG) have a nuclear expression of BRCA1. Even though tumor T5 show methylation products for BRCA1 promoter, these two biopsies do not present deletion of BRCA1 alleles in the array-CGH analyses, suggesting that only one allele in T5 may be silenced through epigenetic modification. In relation to biopsies with germline BRCA1 mutations, we found that tumors T24 and

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Methylation of the BRCA1 promoter as a mechanism of gene silencing in hereditary breast tumors. We found an important fraction of the tumors with methylation of the BRCA1 promoter in which the protein was not detected. For these tumors we could suggest that methylation is at least one of the events that silence the expression of the gene. In three tumors with no BRCA1 germline mutation, only methylated products were obtained through MS-PCR of the BRCA1 promoter, revealing a high proportion of methylated alleles. In sporadic breast cancer cases, the methylation of both alleles has been reported by Wei et al.13 in microdissected cells from fresh frozen tumors. It is widely accepted that the percentage of BRCA1 mutated tumors that retain the second allele (not mutated) is low, making difficult to study methylation as a second hit in these tumors. One report in which different tumor suppressor genes were analyzed in hereditary breast cancer, show one BRCA1 mutated tumor in which methylation of the non mutated allele occurred.19 We have among our samples three BRCA1 mutated tumors, two of them methylated in at least one allele. In one BRCA1 mutated tumor (c.3936C > T), with methylation, we found a positive detection of the protein in the nucleus. Since this mutation is the cause for breast cancer in three Chilean families from our study, we suggest that methylation is occurring in the not mutated allele, as a second hit. We could explain the positive detection of BRCA1, because the monoclonal antibody used in IHC recognizes the amino terminal portion of the protein and, therefore it is possible that the truncated BRCA1 is not degraded in the cell and gives a positive signal. The two patients (sisters) sharing the same BRCA1 mutation (c.3450_3453delCAAG) lost the mutated allele in the tumor, as we detected through SSCP (Single Strand Conformation Polymorfism) analysis, what lead us to think in a third hit to explain the absence of the BRCA1 protein. One of these tumors (T25) is methylated, so methylation could be considered as a mechanism of inactivation of the second allele in BRCA1 mutated tumors, when the mutated allele is deleted. Correlation between methylation of the BRCA1 promoter and ER/PR status. We did not find a significant correlation between the methylated status of the BRCA1 promoter and the presence or absence of hormone receptors, as also has been described by others in hereditary14 or sporadic tumors.18,24 In contrast, three reports on sporadic cases found a correlation between methylation and ER-/ PR- tumors.13,20,21 As we discuss below we detected a good correlation of ER-/PR- tumors with the absence of the BRCA1 protein, so we could explain the controversial data regarding the correlation between methylation and hormone receptors status, by differences in the concordance of methylation and gene silencing in the different studies. Therefore methylation of BRCA1 promoter may represent a partial gene inactivation in some biopsies,13 constituting a first hit that do not lead into total gene silencing until the second hit occurs. We conclude that methylation by itself may not imply absence of the protein unless both alleles are affected, but it still could be considered as a genetic marker of tumor progression. The absence of the BRCA1 protein and its correlation with ER-/PR- tumors. The absence of BRCA1 is a clearly relevant phenotype in hereditary breast cancer, thus the lack of detection of the protein in non mutated BRCA1 tumors may represent an important signal of the involvement of BRCA1 in hereditary breast carcinogenesis. Since other mechanisms besides methylation may be involved in BRCA1 gene silencing, we considered in our analyses all biopsies with BRCA1 negative expression, either methylated or not

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T25 from two sisters sharing the c.3450_3453delCAAG mutation presented the deletion of the mutated allele, detected through SSCP (Single Strand Conformer Polymorphism) analysis. Both biopsies did not express BRCA1, nevertheless only T25 has promoter hypermethylation (Table 1). The fact that T24, has no promoter hypermethylation, suggests the occurrence of another type of somatic alteration for the second allele. Apparently, different events are responsible for the loss of the second allele expression in these biopsies. A third patient with a germline BRCA1 truncating mutation (c.3936C > T), has promoter hypermethylation in the tumor (T49), and shows apparently normal expression in the nucleus. Even though we cannot define which allele has promoter hypermethylation, a possible explanation for the immunodetection of BRCA1 is that the truncated protein, not active as the wild type, is not degraded in the cell and localizes in the nucleus. In summary, we detected 24 biopsies having promoter hypermethylation, among which 16 do not express BRCA1 and eight show a positive expression. The lack of expression is probably due to the loss of the second allele through a different or equivalent mechanism, and therefore we attribute the presence of BRCA1 in the eight biopsies, to the maintenance of expression of the second allele. Concerning the expression of estrogen receptor (ER), progesterone receptor (PR) and ERBB2, we found that 24% of BRCA1 hypermethylated biopsies were triple negative (ER-/PR-/ERBB2-) and had no expression of BRCA1 (Table 1). Since methylation of BRCA1 promoter may occur in only one allele, it is expected that only a proportion of methylated tumors do not express the protein. In contrast, we found seven tumors not methylated with no expression of BRCA1, in which the silencing of both alleles could involve other mechanisms besides methylation (somatic mutation, genomic deletion). We also studied the correlation of BRCA1 expression with the presence or absence of hormone receptors, and found that among 25 BRCA1 negative tumors, 12 (48%) are ER-, and 10 (40%) were also PR-. In addition, 63% of ER- tumors do not express BRCA1, and 53% are ER-/PR-/ BRCA1- therefore, the lack of ER is in high correlation with the absence of PR and BRCA1. In contrast, only 16% of ER- biopsies are negative for PR and show a positive expression of BRCA1 (ER-/PR-/BRCA1+).

Promoter hypermethylation of BRCA1 has been previously reported in 42% of 50 hereditary tumor biopsies with no mutations in BRCA1 or BRCA2.14 In our sample we found BRCA1 promoter hypermethylation in 51%, 24 out of 47 tumors, which is not significantly different from the previous report,14 considering the number of cases. In comparison to sporadic cases, in which 11 to 30% show BRCA1 promoter methylation,18-21 hereditary cases have shown a higher proportion of tumors with this condition.14 Interestingly, hypermethylation of BRCA1 has been found mostly in breast and ovarian cancer, and not seen in other types of cancers such as colon and liver.18,22,23 As described by others,21,24,25 in this study an important proportion (67%) of breast tumors with BRCA1 promoter hypermethylation showed a decrease or no expression of the protein. In addition, we found that 7 unmethylated tumors were negative for BRCA1 expression. These findings suggests that, in hereditary breast cancer, other mechanisms besides allele methylation and germline mutations, as somatic mutations or genomic deletions, may be involved in BRCA1 gene silencing. www.landesbioscience.com

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Materials and Methods

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Tumor samples. A total of 49 formalin-fixed paraffin embedded (FFPE) biopsies from women with hereditary breast cancer were analyzed. These patients have been previously screened for BRCA1 and BRCA2 germline mutations.2 Families and patients were selected using standard criteria for hereditary breast cancer.2 All patients signed an informed consent, and this protocol was approved by the Ethics Committee of the Faculty of Medicine, Pontificia Universidad Católica de Chile. The histological type and grade of the tumors were classified according to the World Health Organization criteria and paraffin sections were processed according to standards protocols. A total of 49 biopsies were analyzed for expression of ER, PR, p53 and ERBB2 by immunohistochemistry (IHC). DNA isolation and modification. DNA was extracted following the standard proteinase K protocol and chemically modified with sodium bisulfite as described previously.15 Chemical modification converts all unmethylated cytosines to uracils and leaves methylcytosines unaltered. Briefly, DNA (2.5 μg) in a volume of 50 μl was denatured by NaOH (final concentration 0.2 M) for 10 min at 37°C. Hydroquinone (final concentration 2 mM) and sodium bisulfite at pH 5.0 (final concentration 2.6 M) was added and samples were incubated at 55°C for 4 hrs.16,17 Modified DNA was purified using the Wizard DNA purification resin (Promega Cat. # A7280) and eluted with 50 μl of TE PCR (0.1 mM EDTA, 10 mM Tris-HCl). Modification was completed by NaOH treatment (final concentration 0.3 M) for 5 min at room temperature followed by ethanol precipitation. DNA was resuspended in water and stored at -20°C. Normal lymphocyte DNA was methylated in vitro with SssI methyltransferase (New England Biolabs) and modified, and used as a positive control for methylated BRCA1 alleles. Modified normal lymphocyte DNA was used as a positive control for unmethylated alleles. The quality and integrity of non modified tumoral DNA was seen in an agarose 1% gel. Unmodified and modified DNA were measured by spectrophotometry using Nanodrop technology. MS-PCR amplification. Methylation analysis of a fraction of the BRCA1 promoter was done using a methylation specific PCR-based (MS-PCR) approach as previously described.16 MS-PCR was performed in 25 μl reaction volumes containing 1 x PCR Buffer (20 mM Tris HCl, pH 8.4, 50 mM KCl), 0.02 mM each deoxynucleoside triphosphates, 2.6 mM MgCl2, 0.24 μM of each primer and 1 Unit of Platinium® Taq DNA Polymerase (Invitrogen). The promoter of gene BRCA1 was amplified with 2 set of primers (M and UM) (Fig. 1). The methylated primers (M) are localized -38 bp from the +1 transcriptional start site and its amplicon size is 75 bp. The unmethylated primers (UM) are localized -45 bp and amplify 86 bp (Genbank accession # U37574).18 The amplified fragments were gel purified (Wizard Gel purification system) and cloned into the vector pGEMT (Promega Inc.,). Individual clones were automatically sequenced. Immunohistochemistry for BRCA1. Forty nine breast tumor biopsies were formalin fixed and paraffin embedded. For each sample, morphological assessment was carried out on a 5 μm tissue section stained with Haematoxylin-Eosin and the following sections were used for immunostaining with the avidin-biotin immunoperoxidase technique (Polyvalent immunoperoxidase kit; Omnitag, Lipshaw, MI, USA). Before deparaffinising and re-hydrating, the slides were heated at 70°C for 40 min. Endogenous

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methylated. We found that among the 25 BRCA1(-) tumors 48% were ER- and 40% were also PR-. It has been well established that a high proportion of BRCA1 mutant breast tumors correspond to the called triple negative group, which do not express the estrogen and progesterone receptors, as well as ERBB2.26,27 In this sense, 10 BRCA1(-) biopsies from our study, one of these with a BRCA1 germline mutation, display a similar phenotype than BRCA1 mutants in relation to hormone receptors expression. These results are concordant with the ones described for sporadic biopsies, in which there is concordance between the absence of expression of BRCA1, the negativity of both hormone receptors and the high grade.13,21,25 BRCA1 methylation, expression and correlation with tumor grade and ER/PR status. We detected among ten tumors ER-/PR-/ BRCA1-, eight IDC grade III, one IDC grade II and one LCIS, 60% being also methylated in BRCA1. In contrast, tumors ER+/PR+/ BRCA1+ are lower grade being six IDC grade II, one IDC grade I, and interestingly the six ductal carcinoma in situ (DCIS) fall in this category. In this study, tumors with negative ER/PR are mainly high grade IDC, and tumors with positive ER/PR are IDC grade II and I, as well as DCIS. It is interesting to note that among the seven IDC grade I, six were methylated and four of them have no expression of BRCA1. Also we would like to enhance that only two of these seven tumors are ER-, and none of them are ER-/PR-, a phenotype highly present in grade III. Something similar has been reported by Foulkes et al.26 showing that the proportion of cancers ER+ decrease as the histological grade increase. According to this result, in our sample grade I IDC tumors are positive for both hormone receptors (4/7) or for one of them (3/7). Also methylation of BRCA1 is present in grade I tumors concordant with the absence of BRCA1, so we propose that methylation may occur early in tumor development. The relation of BRCA1 expression with the presence and function of ER has been discussed by others, describing an inhibition of estrogen transcriptional activity,28 through the interaction of BRCA1 with ERα.29 Moreover, a mechanism for explaining the absence of ER in BRCA1 tumors has been proposed by Hosey et al.30 demonstrating that BRCA1 modulates estrogen receptor transcription interacting with its promoter through the transcription factor Oct-1. Also a relation of ER activity and PR expression has been described, revealing that ER is a known activator of PR expression.31 All these data are in agreement with the negative expression of ER and PR in BRCA1 negative tumors, and positive expression of ER and PR in tumors expressing BRCA1, even though BRCA1 is not the only regulator of ER expression. This is the second report on promoter hypermethylation in hereditary breast cancer and the first in which correlation with protein expression was determined. This finding supports the hypothesis that BRCA1 undergoes epigenetic silencing, and participates this way in the progression of hereditary breast cancer, most probably in early stages. For specific tumor suppressor genes, detection of their promoter methylation has been achieved in the serum of patients with different types of cancer, as breast21 and colorectal.32 These studies describe that the promoter methylation of tumor suppressor genes detected is concordant with the methylation status of the same genes in the tumor, proposing a potential utility of this screening in serum for the early detection of cancer.21 In this regard, the detection of BRCA1 promoter hypermethylation in serum may be considered as an early and non invasive diagnosis for hereditary breast cancer in women at high risk. 162

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Acknowledgements

15. Frommer M, McDonald L, Millar D, Collis C, Watt F, Grigg G, et al. A genomic sequencing protocol that yields a positive display of 5-methylcitosine residues in individual DNA strands. Proc Natl Acad Sci 1992; 89:1827-31. 16. Herman J, Graft J, Myöhänen S, Nelkin B, Baylin S. Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci 1996; 93:9821-6. 17. Clarke S, Statham A, Stirzaker C, Molloy P, Frommer M. DNA methylation:bisulphite modification and analysis. Nat Protoc 2006; 1:2353-64. 18. Esteller M, Silva J, Dominguez G, Bonilla F, Matias-Guiu X, Lerma Enrique, et al. Promoter Hypermethylation and BRCA1 Inactivation in Sporadic Breast and Ovarian Tumors. J Natl Cancer Inst 2000; 92:564-9. 19. Esteller M, Fraga M, Guo M, Garcia-Foncillas J, Hedenfalk I, Godwin A, et al. DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis. Hum Mol Genet 2001; 10:3001-7. 20. Catteau A, Harris W, Xu C, Solomon E. Methylation of the BRCA1 promoter region in sporadic breast and ovarian cancer: correlation with disease characteristics. Oncogene 1999; 11:1957-65. 21. Mirza S, Sharma G, Prasad C, Parshad R, Shrivastava A, Gupta S, et al. Promoter hypermethylation of TMS1, BRCA1, ERalpha and PRB in serum and tumor DNA of invasive ductal breast carcinoma patients. Life Sci 2007; 81:280-7. 22. Bianco T, Chenevix-Trench G, Walsh DC, Cooper JE, Dobrovic A. Tumour-specific distribution of BRCA1 promoter region methylation supports a pathogenetic role in breast and ovarian cancer. Carcinogenesis 2000; 21:147-51. 23. Baldwin RL, Nemeth E, Tran H, Shvartsman H, Cass I, Narod S, et al. BRCA1 promoter region hypermethylation in ovarian carcinoma: a population-based study. Cancer Res 2000; 60:5329-33. 24. Matros E, Wang ZC, Lodeiro G, Miron A, Iglehart JD, Richardson AL. BRCA1 promoter methylation in sporadic breast tumors: relationship to gene expression profiles. Breast Cancer Res Treat 2005; 91:179-86. 25. Birgisdottir V, Stefansson OA, Bodvarsdottir SK, Hilmarsdottir H, Jonasson JG, Eyfjord JE. Epigenetic silencing and deletion of the BRCA1 gene in sporadic breast cancer. Breast Cancer Res 2006; 8:38. 26. Foulkes WD, Metcalfe K, Sun P, Hanna WM, Lynch HT, Ghadirian P, et al. Estrogen receptor status in BRCA1- and BRCA2-related breast cancer: the influence of age, grade and histological type. Clin Cancer Res 2004; 10:2029-34. 27. Lakhani SR, Reis-Filho JS, Fulford L, Penault-Llorca F, van der Vijver M, Parry S, et al. Prediction of BRCA1 status in patients with breast cancer using estrogen receptor and basal phenotype. Clin Cancer Res 2005; 11:5175-80. 28. Fan S, Wang J, Yuan R, Ma Y, Meng Q, Erdos MR, et al. BRCA1 inhibition of estrogen receptor signaling in transfected cells. Science 1999; 284:1354-6. 29. Fan S, Yuan R, Ma YX, Meng Q, Goldberg ID, Rosen EM. Mutant BRCA1 genes antagonize phenotype of wild-type BRCA1. Oncogene 2001; 20:8215-35. 30. Hosey AM, Gorski JJ, Murray MM, Quinn JE, Chung WY, Stewart GE, et al. Molecular basis for estrogen receptor alpha deficiency in BRCA1-linked breast cancer. J Natl Cancer Inst 2007; 99:1683-94. 31. Read LD, Snider CE, Miller JS, Greene GL, Katzenellenbogen BS. Ligand-modulated regulation of progesterone receptor messenger ribonucleic acid and protein in human breast cancer cell lines. Mol Endocrinol 1988; 2:263-71. 32. Leung W, To K, Man E, Chan M, Bai A, Hui A, et al. Quantitative detection of promoter hypermethylation in multiple genes in the serum of patients with colorectal cancer. Am J Gastroenterol 2005; 100:2274-9.

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peroxidase was inactivated by incubation with 3% H2O2/methanol for 15 min. Each slide was treated with an antigen unmasking solution (Sodium Citrate 10 mM and Tween 20) in a microwave oven for 10 min. The slides were cooled in the same solution and washed three times in phosphate buffer saline-PBS 1% (3 min each time). After blocking for one hour the non specific protein binding with protein blocking agent-PBA, the slides were washed in PBS 1%-BSA (bovine serum albumin) three times, 3 min each. Then, they were incubated overnight with the primary monoclonal antibody BRCA1 (Calbiochem Ab-1, dilution 1:50), raised against the first 200 aminoacids of the protein. The antibody was diluted in PBS 0.1%-BSA. After PBS-BSA 1% washing, slides were incubated with a universal secondary biotinylated polyvalent antibody for one hour at room temperature. Slides were then washed and incubated with peroxidase streptavidin for 30 min at room temperature. The chromogenic reaction was developed by incubation with freshly prepared solution 3-amino-9-ethilcarbazol (AEC) and counterstained with Haematoxylin before mounting of slides. In sections used as negative controls, the primary antibody was omitted. Non-cancer breast samples from healthy women undergoing reduction mammoplasties were used as positive controls. The interpretation of the slides was done in an independent manner by two pathologists. Positive was scored as protein detection in more than 10% of the examined area. Statistical analysis. All comparisons for statistical significance were performed by use of Fisher’s exact test, representing two tailed test and statistically significant at p ≤ 0.05 (GraphPad software).

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We are grateful to FONDECYT grant 1040779 for the financial support of this study.

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References

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