Survival of BRCA1 negative ovarian cancer patients based on family history.

OBJECTIVE: To compare survival of ovarian cancer patients with and without a family history of breast or ovarian cancer who are known to be without mutations in BRCA1. METHODS: Patients with ovarian cancer were tested for germline mutations in BRCA1 by polymerase chain reaction amplification of DNA for single-strand conformation polymorphism and direct sequencing analysis to examine the 22 coding exons of BRCA1 in fresh and archived tumor specimens. Demographic and survival data were collected for statistical analysis. Survival data were calculated by the method of Kaplan and Meier and compared by the log-rank test. RESULTS: Of the 110 patients tested at our institution, 100 were noted to be negative for BRCA1 mutations. After exclusion of nonepithelial histologies, benign tumors, primary peritoneal carcinoma, and incomplete staging, 87 patients remained for analysis, of which 37 demonstrated a family history of breast or ovarian cancer. The two groups showed similar age at diagnosis, stage, grade, residual disease, and type of chemotherapy. Median actuarial survival was 75 months for those patients with a family history versus 70 months for those without (P = 0.73). Evaluation of patients with two or more relatives with breast or ovarian cancer also revealed no differences in survival. CONCLUSIONS: Family history of breast or ovarian cancer does not affect survival of patients with ovarian cancer in the absence of mutations in BRCA1. Previously described differences in survival among patients with BRCA1 mutations may be more related to genetic factors than to biases introduced by the presence of family history.

Development of a yeast stop codon assay readily and generally applicable to human genes.

We established a yeast-based method to screen chain-terminating mutations that is readily applicable to any gene of interest. Based on the finding that 18- to 24-base-long homologous sequences are sufficient for gap repair in vivo in yeast, we used a strategy to amplify a test-gene fragment with addition of 24-bp sequences homologous to both cut-ends of a yeast expression vector, pMT18. After co-transformation with the amplified fragment and the linearized pMT18, each yeast (Saccharomyces cerevisiae) cell automatically forms a single-copy circular plasmid (because of CEN/ARS), which expresses a test-gene::ADE2 chimera protein. When the reading frame of the test-gene contains a nonsense or frameshift mutation, truncation of the chimera protein results in lack of ADE2 activity, leading to formation of a red colony. By using a nested polymerase chain reaction using proofreading Pfu polymerase to ensure specificity of the product, the assay achieved a low background (false positivity). We applied the assay to BRCA1, APC, hMSH6, and E-cadherin genes, and successfully detected mutations in mRNA and genomic DNA. Because this method--universal stop codon assay--requires only 4 to 5 days to screen a number of samples for any target gene, it may serve as a high-throughput screening system of general utility for chain-terminating mutations that are most prevalent in human genetic diseases.

Risk-reduction mastectomy: clinical issues and research needs.

Risk-reduction mastectomy (RRM), also known as bilateral prophylactic mastectomy, is a controversial clinical option for women who are at increased risk of breast cancer. High-risk women, including women with a strong family history of breast cancer and BRCA1/2 mutation carriers, have several clinical options: risk-reduction surgery (bilateral mastectomy and bilateral oophorectomy), surveillance (mammography, clinical breast examination, and breast self-examination), and chemoprevention (tamoxifen). We review research in a number of areas central to our understanding of RRM, including recent data on 1) the effectiveness of RRM in reducing breast cancer risk, 2) the perception of RRM among women at increased risk and health-care providers, 3) the decision-making process for follow-up care of women at high risk, and 4) satisfaction and psychological status after surgery. We suggest areas of future research to better guide high-risk women and their health-care providers in the decision-making process.

Functional analysis of CpG methylation in the BRCA1 promoter region.

Understanding the role for DNA methylation in tumorigenesis has evolved from defining the location and extent of methylation in a variety of cancer-related genes to clarifying the functional and site-specific effects of aberrant methylation on gene expression. Our objectives were to characterize the functional effects of DNA methylation in the BRCA1 promoter and to clarify the functional status of the BRCA1 CRE (cAMP response element) motif. Luciferase reporter assays confirm that an intact CRE is important for BRCA1 expression in transient transfections. Luciferase activities were decreased in constructs where the CRE recognition sequence was altered and when constructs were methylated in vitro. Gel mobility shift and competition assays identified a DNA-protein complex recognizing the CRE motif that we were able to supershift using CREB-specific antibody. Furthermore this CRE is methylation sensitive, and we localized this methylation effect to a CpG dinucleotide within the BRCA1 CRE motif. The consequences of aberrant DNA methylation at specific transcription factor motifs, along with the multiple mutational events that can occur in a variety of essential genes such as BRCA1, paint a complex picture where both genetic and epigenetic changes contribute to tumour formation.

Uptake of screening and prevention in women at very high risk of breast cancer.

Management of women at high lifetime risk of familial breast cancer is hampered because of limited data concerning the appropriateness of treatment options. Over the past 8 years women at very high (>40%) lifetime risk of breast cancer have had the option of entering two chemoprevention treatment trials, a magnetic resonance imaging (MRI) breast screening study, or a risk-reducing mastectomy (RRM) study. Only 10% of eligible women have entered one of the chemotherapy trials with a similar proportion opting for RRM (>50% in mutation carriers) compared with 60% opting for MRI screening. Future chemotherapy trials will have to be designed to address this poor recruitment.

Profiling of protein kinases in the neoplastic transformation of human ovarian surface epithelium.

OBJECTIVE: The aim of this study was to study the pattern of protein kinase expression in a culture model of epithelial ovarian carcinogenesis. METHODS: Cultures of normal human ovarian surface epithelium (OSE), OSE from women with BRCA1 mutations, a cell culture model of preneoplastic (SV40 T-antigen-immortalized, nontumorigenic) and neoplastic (SV40-E-cadherin transfected, tumorigenic) OSE, and three ovarian cancer cell lines were used to represent OSE phenotypes of different genetic backgrounds and at different, progressive stages of neoplastic transformation. The protein kinase network signaling was studied by Western blotting, simultaneously using multiple antibodies for specific protein kinases. RESULTS: High levels of cGMP-dependent protein kinase were found in normal and preneoplastic OSE, but were absent in neoplastic OSE. In contrast, expression of MEK6 was detected exclusively in neoplastic OSE. The expressions of casein kinase II (CK2), p38 mitogen-activated protein kinase (MAPK), cyclin-dependent kinase, and the phosphatidylinositol 3-kinase (PI3K) effectors Akt2 and p70 S6 kinase (S6K) were several-fold higher in neoplastic OSE than in normal OSE, whereas the expressions of the MAPKs extracellular signal-regulated kinases ERK1 and -2 were unchanged. Importantly, constitutive phosphorylation of Akt2 and p70 S6K, as found in neoplastic OSE, was also observed in overtly normal OSE from women with predisposing BRCA1 gene mutations. CONCLUSIONS: These data demonstrate that different repertoires of downstream signaling proteins, particularly those of the MEK6-p38 MAPK-CK2 pathway and the PI3K pathway, are correlated with phenotypic manifestations of a cell culture model of OSE at progressive stages in the development of ovarian cancer. Changes in PI3K effectors are already found in overtly normal OSE from women with BRCA1 mutations.

Mutations of the BRCA1 and BRCA2 genes in patients with bilateral breast cancer.

Mutations of the BRCA1 or BRCA2 genes have been shown to strongly predispose towards the development of contralateral breast cancer in patients from large multi-case families. In order to test the hypothesis that BRCA1 and BRCA2 mutations are more frequent in patients with bilateral breast cancer, we have investigated a hospital-based series of 75 consecutive patients with bilateral breast cancer and a comparison group of 75 patients with unilateral breast cancer, pairwise matched by age and family history, for mutations in the BRCA1 and BRCA2 genes. Five frameshift deletions (517delGT in BRCA1; 4772delA, 5946delCT, 6174delT and 8138del5 in BRCA2) were identified in patients with bilateral disease. No further mutations, apart from polymorphisms and 3 rare unclassified variants, were found after scanning the whole BRCA1 and BRCA2 coding sequence. Three pathogenic BRCA1 mutations (Cys61Gly, 3814del5, 5382insC) were identified in the group of patients with unilateral breast cancer. The frequencies of common BRCA1 and BRCA2 missense variants were not different between the 2 groups. In summary, we did not find a significantly increased prevalence of BRCA1 and BRCA2 mutations in a hospital-based cohort of German patients with bilateral breast cancer. We conclude that bilaterality of breast cancer on its own is not strongly associated with BRCA1 and BRCA2 mutations when adjusted for age and family history. The high frequency of bilateral disease in multi-case breast cancer families may be due to a familial aggregation of additional susceptibility factors modifying the penetrance of BRCA1 and BRCA2 mutations.

A founder mutation of the BRCA1 gene in Western Sweden associated with a high incidence of breast and ovarian cancer.

The aim of this study was to describe and characterise a founder mutation of the BRCA1 gene in western Sweden. Of 62 families screened for BRCA mutations, 24 had BRCA1 mutations and two had BRCA2 mutations. Tumours that occurred in family members were histologically reviewed and mutational status was analysed using archival paraffin-embedded tissues. The same BRCA1 mutation, 3171ins5, was found in 16 families who were clustered along the western coast of Sweden. Mutation analysis revealed a maternal linkage in 13 families and a paternal linkage in 3. There was complete agreement between mutation analysis results obtained from blood and archival tissues. The penetrance of breast or ovarian cancer by age 70 years was estimated to be between 59 and 93%. There were no differences in survivals between breast or ovarian cancer patients with the mutation and age-matched controls. Thus, a predominant BRCA1 gene founder mutation associated with a high risk of breast and ovarian cancer has been identified and found to occur in a restricted geographical area, thereby allowing timely and cost-effective mutation screening using blood samples or archival histological material.

Family history of breast and ovarian cancers and BRCA1 and BRCA2 mutations in a population-based series of early-onset breast cancer.

BACKGROUND: BRCA1 and BRCA2 are the two major susceptibility genes involved in hereditary breast cancer. This study was undertaken to provide reliable population-based estimates of genetic influence and to characterize the nature and prevalence of BRCA1 and BRCA2 germline mutations in early-onset breast cancer. METHODS: In a series comprising all women diagnosed with breast cancer under the age of 41 years in southern Sweden during 1990 through 1995 (n = 262), family history of cancer was evaluated in 95% (n = 250) of the case subjects and germline mutations in BRCA1 and BRCA2 were analyzed in 89% (n = 234). All statistical tests were two-sided. RESULTS: A total of 97 case subjects had at least one first- or second-degree relative with breast or ovarian cancer; 34 (14%; 95% confidence interval [CI] = 9.6% to 18%) cases had at least two first- or second-degree relatives, 22 (8.8%; 95%CI = 5.3% to 12%) had one first-degree relative, and 41 (16%; 95% CI = 12% to 21%) had one second-degree relative with either cancer. If two females affected with breast or ovarian cancer who were related through an unaffected male were also defined as first-degree relatives, then a higher number of case subjects, 120 (48%; 95% CI = 42% to 54%), had at least one first-degree or second-degree relative with breast or ovarian cancer. Sixteen (6.8%; 95% CI = 4.0% to 11%) BRCA1 mutation carriers and five (2.1%; 95% CI = 0.70% to 4.9%) BRCA2 mutation carriers were identified. Among case subjects with one first- or more than one first- or second-degree relative with breast or ovarian cancer, BRCA mutations were more frequent (P<.001) than among the case subjects without this degree of family history. BRCA mutations were also statistically significantly more common among women with bilateral breast cancer than among women with unilateral breast cancer (P =.002). BRCA mutations were more common among younger case subjects than among older ones (P =.0027). CONCLUSIONS: Almost half (48%) of women in southern Sweden with early-onset breast cancer have some family history of breast or ovarian cancer, and 9.0% of early-onset breast cancer cases are associated with a germline mutation in BRCA1 or BRCA2. Mutation carriers were more prevalent among young women, women with at least one first- or second-degree relative with breast or ovarian cancer, and women with bilateral breast cancer.

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.

The incidence of breast cancer from screening women according to predicted family history risk: Does annual clinical examination add to mammography?

In breast cancer, mutations of predisposition genes such as BRCA-1/2 and other genes as yet uncharacterised are manifest in up to 10% of cases. Although the prior probability of the presence of a breast cancer predisposing gene can be calculated for individual women, there is no published evidence to justify predicted risk as a selection criteria for screening. This study aims to define which patient groups with a significant family history should be screened, and whether clinical examination is necessary in addition to mammography. The Claus model was used to predict breast cancer risk in women with a family history. Women were divided into two groups according to their predicted risk: group I consisted of women at standard risk (lifetime risk less than 1:6) and group II with moderate/high risk (lifetime risk greater than or equal to 1:6). Women were cancer-free at the point of entry, and screening consisted of annual clinical examination and mammography from the age of 35 years. This study consisted of 1500 women in group I and 1078 in group II. The period of observation was 5902.0 and 4327.8 women years, respectively. A total of 31 cancers were detected, 12 in group I and 19 in group II. The median age at diagnosis in group II was 45 years (range 26-66 years) compared with 54.5 years (range 38-63 years) in group I (P=0.03). The relative risk of developing breast cancer in group II was 2.6 (95% confidence interval (CI) 1.2-5.8). When compared with breast cancer incidence in the normal population, the standardised incidence ratio in group II was significantly higher at 2.8 (95% CI: 1.7-4.2). The standardised incidence ratio of women in group I was similar to that of the general population (1.1 (95% CI: 0.6-1.8)). A total of 26/31 (84%) cancers detected were palpable, of which 14 (54%) were not visible on mammography. Approximately one-third of all palpable cancers were detected at routine follow-up. Mammography correctly identified 17/31 cancers (55%), but 29% of these were not palpable. Family history screening programmes are effective and women should be selected for screening according to predicted risk. The younger age of diagnosis in group II justifies screening from an earlier age using both annual clinical examination and mammography.

[Medical management of women with inherited predisposition to breast cancer: indications and procedures for mammographic screening]. / Prise en charge des femmes à risque héréditaire de cancer du sein: indications et modalités de dépistage par mammographie.

Women identified or suspected as carriers of mutations in BRCA1 or BRCA2 susceptibility genes have a high risk to develop an early breast cancer and thus, require appropriate management. Some consensus guidelines were provided for women at hereditary risk and two possible strategies of prevention are suggested: breast cancer screening and prophylactic surgery. We present the French recommendations for breast cancer surveillance and discuss the justification, indications and modalities of mammographic screening. Screening by annual mammography is recommended from age 30 years in experienced centers, in association with semi-annual clinical breast examination from age 20 years. These recommendations apply to women who were identified as carriers of a cancer-predisposing mutation of BRCA1 or BRCA2 genes. In families for whom any mutation of the two genes could be identified, the same modalities apply also to women with a higher probability than 25% of being a carrier. We present here an illustration of the calculation of such probabilities from two example-pedigrees.

MR imaging in screening women at increased risk for breast cancer.

Hereditary breast cancer accounts for 5% to 10% of the total breast cancer burden. Screening of this group of women has been done by palpation and conventional mammography until recently, but because of the age group, mammography has a limited value. MR mammography has been demonstrated to be a reliable imaging modality in this group of patients.

Comparison of breast magnetic resonance imaging, mammography, and ultrasound for surveillance of women at high risk for hereditary breast cancer.

PURPOSE: Recommended surveillance for BRCA1 and BRCA2 mutation carriers includes regular mammography and clinical breast examination, although the effectiveness of these screening techniques in mutation carriers has not been established. The purpose of the present study was to compare breast magnetic resonance imaging (MRI) with ultrasound, mammography, and physical examination in women at high risk for hereditary breast cancer. PATIENTS AND METHODS: A total of 196 women, aged 26 to 59 years, with proven BRCA1 or BRCA2 mutations or strong family histories of breast or ovarian cancer underwent mammography, ultrasound, MRI, and clinical breast examination on a single day. A biopsy was performed when any of the four investigations was judged to be suspicious for malignancy. RESULTS: Six invasive breast cancers and one noninvasive breast cancer were detected among the 196 high-risk women. Five of the invasive cancers occurred in mutation carriers, and the sixth occurred in a woman with a previous history of breast cancer. The prevalence of invasive or noninvasive breast cancer in the 96 mutation carriers was 6.2%. All six invasive cancers were detected by MRI, all were 1.0 cm or less in diameter, and all were node-negative. In contrast, only three invasive cancers were detected by ultrasound, two by mammography, and two by physical examination. The addition of MRI to the more commonly available triad of mammography, ultrasound, and breast examination identified two additional invasive breast cancers that would otherwise have been missed. CONCLUSION: Breast MRI may be superior to mammography and ultrasound for the screening of women at high risk for hereditary breast cancer.

Breast cancer mortality among Ashkenazi Jewish women in São Paulo and Porto Alegre, Brazil.

BACKGROUND: Increased BRCA1 and BRCA2 germline mutation rates have been reported in Ashkenazi Jewish women in North America, Europe and Israel, and have been mentioned as possibly related to a higher incidence of breast and ovarian cancer among these communities. The present study was carried out with the aim of obtaining evidence on the magnitude of breast cancer as a cause of death among Ashkenazi women in Brazil. METHODS: We reviewed all death certificates archived in the Jewish Burial Societies of São Paulo (1971-1997) and Porto Alegre (1948-1997), two of the main and oldest Jewish communities in Brazil. Breast cancer observed deaths were compared with expected deaths according to breast cancer mortality in the general population. RESULTS: The observed ratios were approximately quite close to unity, suggesting a similar breast cancer mortality pattern among the Ashkenazi population and the general population in both cities. These results maintain similar behavior regardless of whether analyzed before or after the mid-1980s, when mammography came to be increasingly performed in Brazil. Cancer proportional mortality ratios were 1.04 (0.83-1.29) in São Paulo and 1.16 (0.84-1.57) in Porto Alegre before 1985, and 1.17 (1.00-1.44) and 1.21 (0.81-1.79), respectively, between 1985 and 1997. Some evidence of the maintenance of protective risk factors such as high parity has been observed among Ashkenazi women in São Paulo. CONCLUSION: A quite similar breast cancer mortality pattern was observed between Ashkenazi Jewish women and the general population in São Paulo and Porto Alegre, Brazil. These results may suggest an environmental role on germ mutation expression reported in this ethnic group.

Genetic counseling impacts decision for prophylactic surgery for patients perceived to be at high risk for breast cancer.

BACKGROUND: This study addresses the impact a comprehensive genetic counseling program had on women considering prophylactic mastectomy or oophorectomy. METHODS: Sixty patients underwent detailed family evaluation and risk was estimated. Recommendations were made regarding testing. Ramifications of testing were discussed in detail. RESULTS: Thirty-one women (37%) were considering prophylactic surgery believing themselves to be at high risk. Of these, 23 had testing recommended. Seven patients proceeded with prophylactic surgery based solely on high-risk assessment. Ten women were tested; five were positive. Three patients proceeded with prophylactic surgery despite a negative test. One decided against surgery despite a positive test. After counseling, prophylactic surgery was performed in just over half the initial candidates. CONCLUSIONS: Breast cancer risk estimation and genetic evaluation can be complex. Comprehensive genetic risk assessment programs can play a significant role in the management of patients considering prophylactic surgery for perceived high risk.