Molecular profiles of BRCA1-mutated and matched sporadic breast tumours: relation with clinico-pathological features

About 5-10% of breast cancers are hereditary; a genetically and clinically heterogeneous disease in which several susceptibility genes, including BRCA1, have been identified. While distinct tumour features can be used to estimate the likelihood that a breast tumour is caused by a BRCA1 germline mutation it is not yet possible to categorize a BRCA1 mutated tumour. The aim of the present study is to molecularly classify BRCA1 mutated breast cancers by resolving gene expression patterns of BRCA1 and matched sporadic surgical breast tumour specimens. The expression profiles of 6 frozen breast tumour tissues with a proven BRCA1 gene mutation were weighed against those from 12 patients without a known family history but who had similar clinico-pathological characteristics. In addition two fibroblast cultures, the breast cancer cell-line HCC1937 and its corresponding B-lymphoblastoid cell line (heterozygous for mutation BRCA1 5382insC) and an epithelial ovarian cancer cell line (A2780) were studied. Using a high density membrane based array for screening of RNA isolated from these samples and standard algorithms and software, we were able to distinguish subgroups of sporadic cases and a group consisting mainly of BRCA1-mutated breast tumours. Furthermore this pilot analysis revealed a gene cluster that differentially expressed genes related to cell substrate formation, adhesion, migration and cell organization in BRCA1-mutated tumours compared to sporadic breast tumours.     

A role for BRCA1 in sporadic breast cancer

To test the hypothesis that altered expression of BRCA1 protein may play an important role in sporadic breast cancer development, 50 randomly selected primary breast cancers (frozen sections, 5 years' median follow-up) were immunolabelled with two monoclonal BRCA1 antibodies (MS110 and MS13). MS110 labelling was exclusively nuclear showing no relation to outcome or tumour pathology. Western blotting demonstrated crossreactivity, suggesting antibody nonspecificity. MS13 labelling was predominantly cytoplasmic. Intense labelling predicted decreased overall survival (P=0.012), disease-free survival (P=0.029), oestrogen receptor negativity (P=0.0004) and c-erbB-2 overexpression (P=0.006). Western blotting detected a 110 kDa molecule consistent with BRCA1 delta11b splice variant. BRCA1 protein is postulated to function as a tumour suppressor. We demonstrate cytoplasmic localisation in sporadic breast cancer suggesting excess delta11b splice variant production, reduced production of full-length BRCA1 and thus postulate reduced tumour suppressor activity. BRCA1 protein appears to have a significant role in both sporadic and hereditary breast cancers.     

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.     

Prognostic value of maspin mRNA expression in ER alpha-positive postmenopausal breast carcinomas

Maspin, a member of the serpin family, has a role in cell migration, angiogenesis and apoptosis. Little is known of the clinical significance of maspin gene expression in human cancers. We developed a real-time quantitative RT-PCR assay to quantify the full range of maspin mRNA copy numbers in a series of 10 ER alpha-positive and 10 ER alpha-negative breast tumours. We observed a statistical link between low maspin mRNA levels and positive oestrogen status (P=0.0012). In consequence, to better assess the prognostic value of maspin gene expression in breast cancer, we then quantified maspin mRNA content in an additional independent well-defined cohort of 105 ER alpha-positive postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone. Maspin expression varied widely in tumour tissues (by nearly four orders of magnitude), being underexpressed in 33 out of 105 tumours (31.4%) and overexpressed in 24 out of 105 tumours (22.9%) relative to normal breast tissues. Immunohistochemical studies demonstrated that maspin protein was strictly expressed in myoepithelial cells of normal breast tissue and in tumour epithelial cells, exclusively in maspin-overexpressing tumours. Patients with tumours overexpressing the maspin gene had significantly shorter relapse-free survival after surgery than patients whose tumours normally expressed or underexpressed maspin (P=0.0011). The prognostic significance of maspin overexpression persisted in Cox multivariate regression analysis (P=0.0024). These findings show that the maspin mRNA level can have important prognostic significance in human breast cancer, and point to the maspin gene as a putative molecular predictor of hormone responsiveness in breast cancer.     

Epigenetic factors controlling the BRCA1 and BRCA2 genes in sporadic ovarian cancer

Hypermethylation of the BRCA1 promoter has previously been shown to cause reduced mRNA expression in both breast and ovarian cancers. Nothing is yet known of the expression pattern or methylation status of the promoter region of BRCA2 in sporadic ovarian cancer. Whereas our analysis of 30 sporadic ovarian carcinomas showed a statistically significant reduction of BRCA1 mRNA expression (P = 0.001), it also showed, in contrast, overexpression of BRCA2 mRNA (P = 0.002) in tumor compared with nontumor. Hypermethylation of the BRCA1 promoter highly correlated with decreased BRCA1 expression (P = 0.017). Methylated CpGs at the BRCA2 promoter were either absent or at very low levels in tumor DNA, whereas they were present at high levels in nontumor DNA. Such hypomethylation also correlated with elevated levels of BRCA2 mRNA (P = 0.043) and showed a statistically significant correlation with tumor stage (P = 0.037). This supports the role of methylation in BRCA2 contributing to the pathogenesis of sporadic ovarian cancer. Furthermore, 14 (58.2%) and 9 (56.3%) of all of the cases with aberrant BRCA mRNA expression and methylation patterns, respectively, demonstrated opposing mRNA expression and methylation patterns of the BRCA1 and BRCA2 genes within the same cases. Our findings suggest that both genes may be involved in the development of sporadic ovarian cancer.     

Mammary intraepithelial neoplasia outgrowths (MINO): MINO and human ductal carcinoma <Emphasis Type="Italic">in situ</Emphasis>

Germ-line mutations in the BRCA1 tumour suppressor gene contribute to familial breast tumour formation, but there is no evidence for direct mutation of the BRCA1 gene in the sporadic form of the disease. In contrast, decreased expression of the BRCA1 gene has been shown to be common in sporadic tumours, and the magnitude of the decrease correlates with disease progression. BRCA1 expression is also tightly regulated during normal breast development. Determining how these developmental regulators of BRCA1 expression are co-opted during breast tumourigenesis could lead to a better understanding of sporadic breast cancer aetiology and the generation of novel therapeutic strategies aimed at preventing sporadic breast tumour progression.     

The DNA damage signalling kinase ATM is aberrantly reduced or lost in BRCA1/BRCA2-deficient and ER/PR/ERBB2-triple-negative breast cancer

The ataxia-telangiectasia-mutated (ATM) kinase is a key transducer of DNA damage signals within the genome maintenance machinery and a tumour suppressor whose germline mutations predispose to familial breast cancer. ATM signalling is constitutively activated in early stages of diverse types of human malignancies and cell culture models in response to oncogene-induced DNA damage providing a barrier against tumour progression. As BRCA1 and BRCA2 are also components of the genome maintenance network and their mutations predispose to breast cancer, we have examined the ATM expression in human breast carcinomas of BRCA1/2 mutation carriers, sporadic cases and familial non-BRCA1/2 patients. Our results show that ATM protein expression is aberrantly reduced more frequently among BRCA1 (33%; P=0.0003) and BRCA2 (30%; P=0.0009) tumours than in non-BRCA1/2 tumours (10.7%). Furthermore, the non-BRCA1/2 tumours with reduced ATM expression were more often estrogen receptor (ER) negative (P=0.0002), progesterone receptor (PR) negative (P=0.004) and were of higher grade (P=0.0004). In our series of 1013 non-BRCA1/2 cases, ATM was more commonly deficient (20%; P=0.0006) and p53 was overabundant (47%; P<0.0000000001) among the difficult-to-treat ER/PR/ERBB2-triple-negative subset of tumours compared with cases that expressed at least one of these receptors (10 and 16% of aberrant ATM and p53, respectively). We propose a model of 'conditional haploinsufficiency' for BRCA1/2 under conditions of enhanced DNA damage in precancerous lesions resulting in more robust activation and hence increased selection for inactivation or loss of ATM in tumours of BRCA1/2 mutation carriers, with implications for genomic instability and curability of diverse subsets of human breast cancer.     

Clinical impact of detection of loss of heterozygosity of BRCA1 and BRCA2 markers in sporadic breast cancer.

The development of familial and sporadic breast cancer is based on genetic alterations of tumour-suppressor genes, for which loss of heterozygosity (LOH) is one mechanism of gene inactivation. To investigate LOH of BRCA1 (17q21) and BRCA2 (13-q12-13) in sporadic breast cancer, polymerase chain reaction (PCR)-based fluorescent DNA technology for detection of microsatellite polymorphisms was applied. A total of 137 breast cancer and 15 benign breast specimens with matched normal tissue were examined. Fluorescent-labelled PCR products were analysed in an automated DNA sequencer (ALFTM Pharmacia). Losses at both loci were correlated with different histological types, age, tumour size, lymph node status, grading and steroid hormone receptor expression, [SHR: oestrogen receptor (ER), progesterone receptor (PgR)]. For BRCA1 (D17S855, THRA1, D17S579) losses could be detected in invasive ductal carcinoma (IDC; n = 108) in 32-38%, invasive lobular carcinoma (ILC; n = 19) in 21-42% depending on the marker applied, but not in benign breast tumours (n = 15). Losses of BRCA1 markers correlated with larger tumour size, higher grade, and PgR expression. For BRCA2 (D13S260, D13S267, D13S171) losses could be detected in 108 IDCs in 30-38%, in 19 ILCs in 17-39% depending on the marker applied, but not in benign breast tumours. Losses of BRCA2 markers correlated only with higher grade. Microsatellite analyses combined with detection of fluorescent-labelled PCR products by an automated laser DNA sequencer can be used for routine determination of LOH. In sporadic breast cancer, LOH of BRCA1 of BRCA2 does not add decisive prognostic value as stated for familial breast cancer.     

Epigenetic inactivation of BRCA1 is associated with aberrant expression of CTCF and DNA methyltransferase (DNMT3B) in some sporadic breast tumours

We assessed expression of the BRCA1, CTCF and DNMT3b methyltransferase genes along with BRCA1 promoter methylation to better define the epigenetic events involved in BRCA1 inactivation in sporadic breast cancer. These gene expression patterns were determined in 54 sporadic breast tumours by immunohistochemistry and the methylation status of the BRCA1 promoter was evaluated using methylation-specific PCR. We observed significant DNMT3b expression in 80% of the tumours and that 43% of tumours exhibited novel cytoplasmic CTCF expression. Pairwise analyses of gene expression patterns showed that 28/32 tumours lacked BRCA1 expression and also exhibited cytoplasmic CTCF staining, while 24/32 of these tumours also overexpressed DNMT3b. Furthermore, 86% of the BRCA1 low-expressing tumours were methylated at the BRCA1 promoter and a subset of these tumours displayed both cytoplasmic CTCF and increased DNMT3b expression. Thus, tumour subsets exist that display concurrent decreased BRCA1 expression, BRCA1 promoter methylation, cytoplasmic CTCF expression and with DNMT3b over-expression. We suggest that these altered CTCF and DNMT3b expression patterns represent (a) critical events responsible for the epigenetic inactivation of BRCA1 and (b) a diagnostic signature for epigenetic inactivation of other tumour suppressor genes in sporadic breast tumours.     

Regulation of BRCA1 expression and its relationship to sporadic breast cancer

Germ-line mutations in the BRCA1 tumour suppressor gene contribute to familial breast tumour formation, but there is no evidence for direct mutation of the BRCA1 gene in the sporadic form of the disease. In contrast, decreased expression of the BRCA1 gene has been shown to be common in sporadic tumours, and the magnitude of the decrease correlates with disease progression. BRCA1 expression is also tightly regulated during normal breast development. Determining how these developmental regulators of BRCA1 expression are co-opted during breast tumourigenesis could lead to a better understanding of sporadic breast cancer aetiology and the generation of novel therapeutic strategies aimed at preventing sporadic breast tumour progression.     

Frequent somatic loss of BRCA1 in breast tumours from BRCA2 germ-line mutation carriers and vice versa.

Breast cancer susceptibility genes BRCA1 and BRCA2 are tumour suppressor genes the alleles of which have to be inactivated before tumour development occurs. Hereditary breast cancers linked to germ-line mutations of BRCA1 and BRCA2 genes almost invariably show allelic imbalance (AI) at the respective loci. BRCA1 and BRCA2 are believed to take part in a common pathway in maintenance of genomic integrity in cells. We carried out AI and fluorescence in situ hybridization (FISH) analyses of BRCA2 in breast tumours from germ-line BRCA1 mutation carriers and vice versa. For comparison, 14 sporadic breast tumours were also studied. 8 of the 11 (73%) informative BRCA1 mutation tumours showed AI at the BRCA2 locus. 53% of these tumours showed a copy number loss of the BRCA2 gene by FISH. 5 of the 6 (83%) informative BRCA2 mutation tumours showed AI at the BRCA1 locus. Half of the tumours (4/8) showed a physical deletion of the BRCA1 gene by FISH. Combined allelic loss of both BRCA1 and BRCA2 gene was seen in 12 of the 17 (71%) informative hereditary tumours, whereas copy number losses of both BRCA genes was seen in only 4/14 (29%) sporadic control tumours studied by FISH. In conclusion, the high prevalence of AI at BRCA1 in BRCA2 mutation tumours and vice versa suggests that somatic events occurring at the other breast cancer susceptibility gene locus may be selected in the cancer development. The mechanism resulting in AI at these loci seems more complex than a physical deletion.     

散发性乳腺癌中影响BRCA1蛋白失表达的参数分析

目的: 探讨影响散发性乳腺癌中乳腺癌易感基因(BRCA1)蛋白表达的因素与临床病理指标的相关性。 方法: 免疫组织化学SP法检测乳腺良性病变组织和散发性乳腺癌组织中BRCA1、ERα和HER-2的表达,收集患者临床资料,通过Spearman相关分析和校正的Pearsonχ²检验进行统计分析。 结果: 在良性乳腺病变组织中,BRCA1和ERα蛋白在细胞核的阳性表达率分别为100.0%(20/20)和80.0%(16/20),HER-2在细胞膜阳性表达率为15.0%(3/20);在散发性乳腺癌组织中,BRCA1和ERα蛋白在细胞核阳性表达率分别为57.5%(88/153)和64.1%(98/153),HER-2在细胞膜阳性表达率为34.0%(52/153);与良性乳腺病变组织比较,乳腺癌组织中BRCA1阳性表达率明显降低,差异显著(P<0.05),ERα和HER-2表达率差异不显著(P>0.05)。蛋白表达相关分析及患者临床资料分析表明:BRCA1失表达与ERα失表达相关、与HER-2过表达相关,ERα失表达与HER-2过表达相关;在HER-2过表达、ERα失表达的癌组织中BRCA1失表达率最高;BRCA1失表达与患者高发病年龄、腋淋巴结转移相关,而与乳腺癌病理类型无明显相关。 结论: 在散发性乳腺癌组织中,BRCA1蛋白失表达与ERα失表达、HER-2过表达、患者年龄较高及伴有腋淋巴结转移等相关,BRCA1蛋白失表达参与散发性乳腺癌的发生发展。     

Allelic loss at chromosome 13q12-q13 is associated with poor prognosis in familial and sporadic breast cancer.

Loss of heterozygosity (LOH) was analysed in 84 primary tumours from sporadic, familial and hereditary breast cancer using five microsatellite markers spanning the chromosomal region 13q12-q13 which harbours the BRCA2 breast cancer susceptibility gene, and using one other marker located within the RBI tumour-suppressor gene at 13q14. LOH at the BRCA2 region was found in 34% and at RBI in 27% of the tumours. Selective LOH at BRCA2 occurred in 7% of the tumours, whereas selective LOH at RBI was observed in another 7%. Moreover, a few tumours demonstrated a restricted deletion pattern, suggesting the presence of additional tumour-suppressor genes both proximal and distal of BRCA2. LOH at BRCA2 was significantly correlated to high S-phase values, low oestrogen and progesterone receptor content and DNA non-diploidy. LOH at BRCA2 was also associated, albeit non-significantly, with large tumour size and the ductal and medullar histological types. No correlation was found with lymph node status, patient age or a family history of breast cancer. A highly significant and independent correlation existed between LOH at BRCA2 and early recurrence and death. LOH at RBI was not associated with the above mentioned factors or prognosis. The present study does not provide conclusive evidence that BRCA2 is the sole target for deletions at 13q12-q13 in breast tumours. However, the results suggest that inactivation of one or several tumour-suppressor genes in the 13q12-q13 region confer a strong tumour growth potential and poor prognosis in both familial and sporadic breast cancer.     

Epigenetic silencing and deletion of the BRCA1 gene in sporadic breast cancer

Introduction  

BRCA1 or BRCA2 germline mutations increase the risk of developing breast cancer. Tumour cells from germline mutation carriers have frequently lost the wild-type allele. This is predicted to result in genomic instability where cell survival depends upon dysfunctional checkpoint mechanisms. Tumorigenic potential could then be acquired through further genomic alterations. Surprisingly, somatic BRCA mutations are not found in sporadic breast tumours. BRCA1 methylation has been shown to occur in sporadic breast tumours and to be associated with reduced gene expression. We examined the frequency of BRCA1 methylation in 143 primary sporadic breast tumours along with BRCA1 copy number alterations and tumour phenotype.     

Prognostic value of loss of heterozygosity at BRCA2 in human breast carcinoma.

To confirm several recent studies pointing to loss of heterozygosity (LOH) at BRCA2 as a prognostic factor in sporadic breast cancer, we examined this genetic alteration in a large series of human primary breast tumours for which long-term patient outcomes were known. LOH at BRCA2 correlated only with low oestrogen and progesterone receptor content. Univariate analysis of metastasis-free survival and overall survival (log-rank test) showed no link with BRCA2 status (P = 0.34, P = 0.29 respectively). LOH at BRCA2 does not therefore appear to be a major prognostic marker in sporadic breast cancer.     

Reduction of BRCA1 protein expression in Japanese sporadic breast carcinomas and its frequent loss in BRCA1-associated cases.

BRCA1 is a tumor suppressor gene that is responsible for hereditary breast and ovarian cancer syndrome. To clarify the possible involvement of the BRCA1 protein in mammary carcinogenesis in sporadic and hereditary forms, we have analyzed the BRCA1 protein expression pattern in five breast epithelial cell lines, including a BRCA1-deficient cell line, and 162 breast cancer tissue samples [including 108 sporadic, 35 hereditary (BRCA1 status unknown), and 19 BRCA1-associated cases] from Japanese women. Twelve anti-BRCA1 antibodies were tested by fixation conditions, in which nuclear localization of BRCA1 protein was preserved, and by specificity of the antibodies, which was evaluated in BRCA1-deficient cancer cells. Using monoclonal antibodies applicable to immunohistochemical analysis of paraffin-embedded tissue sections, we found high-level expression of BRCA1 protein in normal mammary epithelium and various degrees of reduced expression in breast cancer cells. Of the 19 BRCA1-associated breast cancer tissues, 15 (79%) showed reduction (8 cases) or complete loss (7 cases) of nuclear expression. Thirty (28%) of 108 sporadic and 6 (17%) of 35 hereditary carcinomas showed reduced BRCA1 protein expression. Reduction of BRCA1 protein expression in sporadic carcinomas was associated with solid-tubular phenotype, with poor tubular differentiation, and with an overexpression of c-erbB-2 protein, which is one of the prognostic factors in breast cancer. Our data suggest that reduced expression of BRCA1 protein may play an important role in mammary carcinogenesis, not only in BRCA1-associated breast carcinomas, but also in sporadic carcinomas, and also suggest that mechanisms other than mutation may be involved in its reduced expression.