Prevalence of BRCA mutations in an unselected population of triple-negative breast cancer.

BACKGROUND: This study assessed BRCA1 and BRCA2 mutation prevalence in an unselected cohort of patients with triple-negative breast cancer (BC). METHODS: One hundred ninety-nine patients were enrolled. Triple negativity was defined as <1% estrogen and progesterone staining by immunohistochemistry and HER-2/neu not overexpressed by fluorescence in situ hybridization. Having given consent, patients had BRCA1 and BRCA2 full sequencing and large rearrangement analysis. Mutation prevalence was assessed among the triple-negative BC patients and the subset of patients without a family history of breast/ovarian cancer. Independent pathological review was completed on 50 patients. RESULTS: Twenty-one deleterious BRCA mutations were identified--13 in BRCA1 and 8 in BRCA2 (prevalence, 10.6%). In 153 patients (76.9%) without significant family history (first-degree or second-degree relatives with BC aged <50 years or ovarian cancer at any age), 8 (5.2%) mutations were found. By using prior National Comprehensive Cancer Network (NCCN) guidelines recommending testing for triple-negative BC patients aged <45 years, 4 of 21 mutations (19%) would have been missed. Two of 21 mutations (10%) would have been missed using updated NCCN guidelines recommending testing for triple-negative BC patients aged <60 years. CONCLUSIONS: The observed mutation rate was significantly higher (P = .0005) than expected based on previously established prevalence tables among patients unselected for pathology. BRCA1 mutation prevalence was lower, and BRCA2 mutation prevalence was higher, than previously described. Additional mutation carriers would have met new NCCN testing guidelines, underscoring the value of the updated criteria. Study data suggest that by increasing the age limit to 65 years, all carriers would have been identified.

BRCA1 and BRCA2 mutations in women of different ethnicities undergoing testing for hereditary breast-ovarian cancer.

BACKGROUND: In women at increased risk for breast and ovarian cancer, the identification of a mutation in breast cancer gene 1 (BRCA1) and BRCA2 has important implications for screening and prevention counseling. Uncertainty regarding the role of BRCA1 and BRCA2 testing in high-risk women from diverse ancestral backgrounds exists because of variability in prevalence estimates of deleterious (disease-associated) mutations in non-white populations. In this study, the authors examined the prevalence of BRCA1 and BRCA2 mutations in an ethnically diverse group of women who were referred for genetic testing. METHODS: In this cross-sectional analysis, the prevalence of BRCA1 and BRCA2 mutations was assessed in a group of non-Ashkenazi Jewish women who underwent genetic testing. RESULTS: From 1996 to 2006, 46,276 women who met study criteria underwent DNA full-sequence analysis of the BRCA1 and BRCA2 genes. Deleterious mutations were identified in 12.5% of women, and recurrent deleterious mutations (prevalence >2%) were identified in all ancestral groups. Women of non-European descent were younger (mean age, 45.9 years; standard deviation [SD], 11.6 years) than European women (mean age, 50 years; SD, 11.9 years; P < .001). Women of African (15.6%; odds ratio [OR], 1.3 [95% confidence interval (95% CI), 1.1-1.5]) and Latin American (14.8%; OR, 1.2 [95% CI, 1.1-1.4]) ancestries had a significantly higher prevalence of deleterious BRCA1 and BRCA2 mutations compared with women of Western European ancestry (12.1%), primarily because of an increased prevalence of BRCA1 mutations in those 2 groups. Non-European ethnicity was associated strongly with having a variant of uncertain significance; however, reclassification decreased variant reporting (from 12.8%-->5.9%), and women of African ancestry experienced the largest decline (58%). CONCLUSIONS: Mutation prevalence was found to be high among women who were referred for clinical BRCA1 and BRCA2 testing, and the risk was similar across diverse ethnicities. BRCA1 and BRCA2 testing is integral to cancer risk assessment in all high-risk women.

Functional assays for classification of BRCA2 variants of uncertain significance.

The assessment of the influence of many rare BRCA2 missense mutations on cancer risk has proved difficult. A multifactorial likelihood model that predicts the odds of cancer causality for missense variants is effective, but is limited by the availability of family data. As an alternative, we developed functional assays that measure the influence of missense mutations on the ability of BRCA2 to repair DNA damage by homologous recombination and to control centriole amplification. We evaluated 22 missense mutations from the BRCA2 DNA binding domain (DBD) that were identified in multiple breast cancer families using these assays and compared the results with those from the likelihood model. Thirteen variants inactivated BRCA2 function in at least one assay; two others truncated BRCA2 by aberrant splicing; and seven had no effect on BRCA2 function. Of 10 variants with odds in favor of causality in the likelihood model of 50:1 or more and a posterior probability of pathogenicity of 0.99, eight inactivated BRCA2 function and the other two caused splicing defects. Four variants and four controls displaying odds in favor of neutrality of 50:1 and posterior probabilities of pathogenicity of at least 1 x 10(-3) had no effect on function in either assay. The strong correlation between the functional assays and likelihood model data suggests that these functional assays are an excellent method for identifying inactivating missense mutations in the BRCA2 DBD and that the assays may be a useful addition to models that predict the likelihood of cancer in carriers of missense mutations.

Multiple rare nonsynonymous variants in the adenomatous polyposis coli gene predispose to colorectal adenomas.

It has been proposed that multiple rare variants in numerous genes collectively account for a substantial proportion of multifactorial inherited predisposition to a variety of diseases, including colorectal adenomas (CRA). We have studied this hypothesis by sequencing the adenomatous polyposis coli (APC) gene in 691 unrelated North American patients with CRAs and 969 matched healthy controls. Rare inherited nonsynonymous variants of APC were significantly overrepresented in patients who did not carry conventional pathogenic mutations in the APC or MutY homologue genes [non-familial adenomatous polyposis (FAP) non-MUTYH-associated polyposis (MAP) patients; 81 of 480, 16.9%] compared with patients with FAP or MAP (20 of 211, 9.5%, P = 0.0113), and this overrepresentation was highest in those non-FAP non-MAP patients with 11 to 99 CRAs (30 of 161, 18.6%, P = 0.0103). Furthermore, significantly more non-FAP non-MAP patients carried rare nonsynonymous variants in the functionally important beta-catenin down-regulating domain compared with healthy controls (32 of 480 versus 37 of 969, P = 0.0166). In silico analyses predicted that approximately 46% of the 61 different variants identified were likely to affect function, and upon testing, 7 of 16 nonsynonymous variants were shown to alter beta-catenin-regulated transcription in vitro. These data suggest that multiple rare nonsynonymous variants in APC play a significant role in predisposing to CRAs.

A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes.

Mutation screening of the breast and ovarian cancer-predisposition genes BRCA1 and BRCA2 is becoming an increasingly important part of clinical practice. Classification of rare nontruncating sequence variants in these genes is problematic, because it is not known whether these subtle changes alter function sufficiently to predispose cells to cancer development. Using data from the Myriad Genetic Laboratories database of nearly 70,000 full-sequence tests, we assessed the clinical significance of 1,433 sequence variants of unknown significance (VUSs) in the BRCA genes. Three independent measures were employed in the assessment: co-occurrence in trans of a VUS with known deleterious mutations; detailed analysis, by logistic regression, of personal and family history of cancer in VUS-carrying probands; and, in a subset of probands, an analysis of cosegregation with disease in pedigrees. For each of these factors, a likelihood ratio was computed under the hypothesis that the VUSs were equivalent to an "average" deleterious mutation, compared with neutral, with respect to risk. The likelihood ratios derived from each component were combined to provide an overall assessment for each VUS. A total of 133 VUSs had odds of at least 100 : 1 in favor of neutrality with respect to risk, whereas 43 had odds of at least 20 : 1 in favor of being deleterious. VUSs with evidence in favor of causality were those that were predicted to affect splicing, fell at positions that are highly conserved among BRCA orthologs, and were more likely to be located in specific domains of the proteins. In addition to their utility for improved genetics counseling of patients and their families, the global assessment reported here will be invaluable for validation of functional assays, structural models, and in silico analyses.

Three-dimensional adipose tissue model using low shear bioreactors.

Presented here are techniques developed to culture and analyze three-dimensional (3-D) adipose-like tissues as a means to bridge the gap between current limitations in culturing preadipocytes (PAs) and that of providing clinically relevant volumes of adipose tissue useful for soft tissue engineering strategies in reconstructive surgery. Pilot studies were performed to determine techniques to visualize and analyze 3-D PA-like tissues as well as to develop successful strategies to culture 3T3-L1 cells in a high aspect ratio vessel rotating-wall bioreactor both with and without microcarriers. Next, a series of cultures were accessed to verify these techniques as well as to compare the culture of the cells with and without microcarriers. Finally, a perfused rotating-wall bioreactor was used to further investigate the nature of the aggregates or tissues being generated. The aggregates that formed in the perfused system were analyzed via histology and in vivo animal studies. PA-like tissues as large as 4-5 mm in diameter without microcarriers that were capable of lipid-loading and composed of viable cells were achieved. We have successfully demonstrated that large tissue aggregates can be grown in bioreactor culture systems.

Microvascular endothelial cells sustain preadipocyte viability under hypoxic conditions.

Obesity, soft tissue wound healing, adipose tissue engineering, lipomas, and other physiological and pathophysiological conditions necessitate a clear understanding of the interactions between adipocytes and endothelial cells. Adipogenesis and angiogenesis are intimately integrated, despite not being in direct apposition with one another. However, underlying mechanisms have not been elucidated. In this study, the interactions of preadipocytes (PAs) and microvascular endothelial cells are investigated under varying defined O2 conditions, using a coculture system. Results clearly demonstrate that endothelial cells release a soluble factor that sustains PAs viability under hypoxic conditions. Vascular endothelial cell growth factor is not the potential soluble factor (data not shown).

Isolation and culture of rat microvascular endothelial cells.

The purpose of this study is to identify the separation techniques that result in pure cultures of rat microvascular endothelial cells (MECs). A multistep process is used to optimize the separation of the cells from rat epididymal fat pads, obtaining as pure a culture as possible within a relatively short processing time. The process initially employs the digestion, filtration, and density gradient separation steps. We further describe the use of an attachment phase that allows the differential adherence of contaminating cell types. Immunomagnetic purification is the final step in the process and is performed using anti-PECAM-1 (CD31) monoclonal antibody-labeled DynaBeads.