Loss of nuclear BRCA1 protein staining in normal tissue cells derived from BRCA1 and BRCA2 mutation carriers

Enhanced genomic instability has been recently reported in normal cells derived from BRCA1/2 mutation carriers when placed in vitro in non-physiological stress conditions. We present here original data which help to explain the observed genomic instability. Leucocytes from BRCA1/2 mutation carriers, sporadic breast cancer patients and controls were prepared for BRCA1 immunocytochemistry. We show that BRCA1 containing nuclear dot like structures are detectable in about 80% of the leucocytes from controls and sporadic breast cancer patients, but are absent in the majority of normal cells from BRCA1 as well as BRCA2 mutation carriers (also in their normal breast cells). Our results thus indicate that the genomic instability observed in normal cells from BRCA1 and BRCA2 mutation carriers is associated with a down-regulation of nuclear BRCA1 protein accumulation in the dot like structures. These results suggest in addition that immunocytochemical or alternative molecular screening strategies might help to identify women with a high risk for breast (ovarian) cancer even when the underlying genetic defect remains undetectable.     

Assessing Associations between the AURKA-HMMR-TPX2-TUBG1 Functional Module and Breast Cancer Risk in BRCA1/2 Mutation Carriers

While interplay between BRCA1 and AURKA-RHAMM-TPX2-TUBG1 regulates mammary epithelial polarization, common genetic variation in HMMR (gene product RHAMM) may be associated with risk of breast cancer in BRCA1 mutation carriers. Following on these observations, we further assessed the link between the AURKA-HMMR-TPX2-TUBG1 functional module and risk of breast cancer in BRCA1 or BRCA2 mutation carriers. Forty-one single nucleotide polymorphisms (SNPs) were genotyped in 15,252 BRCA1 and 8,211 BRCA2 mutation carriers and subsequently analyzed using a retrospective likelihood approach. The association of HMMR rs299290 with breast cancer risk in BRCA1 mutation carriers was confirmed: per-allele hazard ratio (HR) = 1.10, 95% confidence interval (CI) 1.04 – 1.15, p = 1.9 x 10⁻⁴ (false discovery rate (FDR)-adjusted p = 0.043). Variation in CSTF1, located next to AURKA, was also found to be associated with breast cancer risk in BRCA2 mutation carriers: rs2426618 per-allele HR = 1.10, 95% CI 1.03 – 1.16, p = 0.005 (FDR-adjusted p = 0.045). Assessment of pairwise interactions provided suggestions (FDR-adjusted p_interaction values > 0.05) for deviations from the multiplicative model for rs299290 and CSTF1 rs6064391, and rs299290 and TUBG1 rs11649877 in both BRCA1 and BRCA2 mutation carriers. Following these suggestions, the expression of HMMR and AURKA or TUBG1 in sporadic breast tumors was found to potentially interact, influencing patients’ survival. Together, the results of this study support the hypothesis of a causative link between altered function of AURKA-HMMR-TPX2-TUBG1 and breast carcinogenesis in BRCA1/2 mutation carriers.     

BRCA1-hapoinsufficiency: Unraveling the molecular and cellular basis for tissue-specific cancer

Over the past 20 years tremendous progress has been made in understanding the function of BRCA1 gene products. Yet one question still remains: why is mutation of BRCA1 typically associated with preferential development of breast and ovarian cancers and not tumors in other tissues? Here we discuss recent evidence documenting the effect of BRCA1-haploinsufficiency in different cells and tissues and synthesize a model for how mutations in a single BRCA1 allele in human cells might preferentially confer increased cancer risk in breast epithelial cells.     

DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03–1.16), p = 2.7×10⁻³) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03–1.21, p = 4.8×10⁻³). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.     

CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, EGFR, MUC1 and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response.     

Synergistic anticancer action of quercetin and curcumin against triple-negative breast cancer cell lines

Women with the breast cancer type 1 susceptibility protein (BRCA1) mutation and loss of BRCA1 expression are reported to have an increased risk of triple-negative breast cancer (TNBC). Targeting BRCA1 modulation might offer a therapeutic option to treat TNBC patients. Our studies detected that BRCA1 is poorly expressed in TNBC cell lines and highly expressed in ER⁺ breast cancer cell lines. To modulate BRCA1 expression, we tested two different dietary components to find out if any would induce tumor suppressor genes. We detected that quercetin and curcumin dose-dependently enhanced the BRCA1 expression. Further, a synergistic action of quercetin and curcumin was observed in modulating the BRCA1 level and in inhibiting the cell survival and migration of TNBC cell lines. Quercetin and curcumin appeared to induce BRCA1 promoter histone acetylation. Furthermore, BRCA1 knockdown induced cell survival and cell migration in ER ⁺ cells were significantly decreased by the combined treatment of quercetin and curcumin. Our present study concluded that the combination treatment of quercetin and curcumin acts synergistically to induce anticancer activity against TNBC cells by modulating tumor suppressor genes.     

Telomere Length Shows No Association with BRCA1 and BRCA2 Mutation Status

This study aimed to determine whether telomere length (TL) is a marker of cancer risk or genetic status amongst two cohorts of BRCA1 and BRCA2 mutation carriers and controls. The first group was a prospective set of 665 male BRCA1/2 mutation carriers and controls (mean age 53 years), all healthy at time of enrolment and blood donation, 21 of whom have developed prostate cancer whilst on study. The second group consisted of 283 female BRCA1/2 mutation carriers and controls (mean age 48 years), half of whom had been diagnosed with breast cancer prior to enrolment. TL was quantified by qPCR from DNA extracted from peripheral blood lymphocytes. Weighted and unweighted Cox regressions and linear regression analyses were used to assess whether TL was associated with BRCA1/2 mutation status or cancer risk. We found no evidence for association between developing cancer or being a BRCA1 or BRCA2 mutation carrier and telomere length. It is the first study investigating TL in a cohort of genetically predisposed males and although TL and BRCA status was previously studied in females our results don''t support the previous finding of association between hereditary breast cancer and shorter TL.     

Clinical and pathological characteristics of Chinese patients with BRCA related breast cancer

Breast cancers related to BRCA mutations are associated with particular biological features. Here we report the clinical and pathological characteristics of breast cancer in Chinese women with and without BRCA mutations and of carriers of BRCA1 mutations compared to BRCA2 mutations. Two hundred and 26 high-risk Hong Kong Chinese women were tested for BRCA mutations, medical information was obtained from medical records, and risk and demographic information was obtained from personal interviews. In this cohort, 28 (12.4%) women were BRCA mutation carriers and among these carriers, 39.3% were BRCA1 and 60.7% were BRCA2 mutations. Mutation carriers were more likely to have a familial history of breast and ovarian cancer, high-grade cancers, and triple negative (TN) cancers. Prevalence of TN was 48.3% in BRCA carriers and 25.6% in non-carriers and was 67.7% in BRCA1 and 35.3% in BRCA2 carriers. Estrogen receptor (ER) negative cancer was significantly associated with BRCA1 mutations, especially in those under 40 years of age. BRCA-related breast cancer in this Chinese population is associated with family history and adverse pathological/prognostic features, with BRCA2 mutations being more prevalent but BRCA1 carriers having more aggressive and TN cancers. Compared to Caucasian populations, prevalence of BRCA2 mutations and TN cancer in BRCA2 mutation carriers in Chinese population are elevated.     

Worse Breast Cancer Prognosis of BRCA1/BRCA2 Mutation Carriers: What's the Evidence? A Systematic Review with Meta-Analysis

Objective

Conflicting conclusions have been published regarding breast cancer survival of BRCA1/2 mutation carriers. Here we provide an evidence-based systematic literature review.

Methods

Eligible publications were observational studies assessing the survival of breast cancer patients carrying a BRCA1/2 mutation compared to non-carriers or the general breast cancer population. We performed meta-analyses and best-evidence syntheses for survival outcomes taking into account study quality assessed by selection bias, misclassification bias and confounding.

Results

Sixty-six relevant studies were identified. Moderate evidence for a worse unadjusted recurrence-free survival for BRCA1 mutation carriers was found. For BRCA1 and BRCA2 there was a tendency towards a worse breast cancer-specific and overall survival, however, results were heterogeneous and the evidence was judged to be indecisive. Surprisingly, only 8 studies considered adjuvant treatment as a confounder or effect modifier while only two studies took prophylactic surgery into account. Adjustment for tumour characteristics tended to shift the observed risk estimates towards a relatively more favourable survival.

Conclusions

In contrast to currently held beliefs of some oncologists, current evidence does not support worse breast cancer survival of BRCA1/2 mutation carriers in the adjuvant setting; differences if any are likely to be small. More well-designed studies are awaited.     

Germline BRCA1 mutations predispose to pancreatic adenocarcinoma

Although the association of germline BRCA2 mutations with pancreatic adenocarcinoma is well established, the role of BRCA1 mutations is less clear. We hypothesized that the loss of heterozygosity at the BRCA1 locus occurs in pancreatic cancers of germline BRCA1 mutation carriers, acting as a “second-hit” event contributing to pancreatic tumorigenesis. Seven germline BRCA1 mutation carriers with pancreatic adenocarcinoma and nine patients with sporadic pancreatic cancer were identified from clinic- and population-based registries. DNA was extracted from paraffin-embedded tumor and nontumor samples. Three polymorphic microsatellite markers for the BRCA1 gene, and an internal control marker on chromosome 16p, were selected to test for the loss of heterozygosity. Tumor DNA demonstrating loss of heterozygosity in BRCA1 mutation carriers was sequenced to identify the retained allele. The loss of heterozygosity rate for the control marker was 20%, an expected baseline frequency. Loss of heterozygosity at the BRCA1 locus was 5/7 (71%) in BRCA1 mutation carriers; tumor DNA was available for sequencing in 4/5 cases, and three demonstrated loss of the wild-type allele. Only 1/9 (11%) sporadic cases demonstrated loss of heterozygosity at the BRCA1 locus. Loss of heterozygosity occurs frequently in pancreatic cancers of germline BRCA1 mutation carriers, with loss of the wild-type allele, and infrequently in sporadic cancer cases. Therefore, BRCA1 germline mutations likely predispose to the development of pancreatic cancer, and individuals with these mutations may be considered for pancreatic cancer-screening programs.     

<Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis> germline mutation analysis among Indian women from south India: identification of four novel mutations and high-frequency occurrence of 185delAG mutation

Mutations in the BRCA1 and BRCA2 genes profoundly increase the risk of developing breast and/or ovarian cancer among women. To explore the contribution of BRCA1 and BRCA2 mutations in the development of hereditary breast cancer among Indian women, we carried out mutation analysis of the BRCA1 and BRCA2 genes in 61 breast or ovarian cancer patients from south India with a positive family history of breast and/or ovarian cancer. Mutation analysis was carried out using conformation-sensitive gel electrophoresis (CSGE) followed by sequencing. Mutations were identified in 17 patients (28.0%); 15 (24.6%) had BRCA1 mutations and two (3.28%) had BRCA2 mutations. While no specific association between BRCA1 or BRCA2 mutations with cancer type was seen, mutations were more often seen in families with ovarian cancer. While 40% (4/10) and 30.8% (4/12) of families with ovarian or breast and ovarian cancer had mutations, only 23.1% (9/39) of families with breast cancer carried mutations in the BRCA1 and BRCA2 genes. In addition, while BRCA1 mutations were found in all age groups, BRCA2 mutations were found only in the age group of ≤40 years. Of the BRCA1 mutations, there were three novel mutations (295delCA; 4213T→A; 5267T→G) and three mutations that have been reported earlier. Interestingly, 185delAG, a BRCA1 mutation which occurs at a very high frequency in Ashkenazi Jews, was found at a frequency of 16.4% (10/61). There was one novel mutation (4866insT) and one reported mutation in BRCA2. Thus, our study emphasizes the importance of mutation screening in familial breast and/or ovarian cancers, and the potential implications of these findings in genetic counselling and preventive therapy.     

RAD51 135G-->C modifies breast cancer risk among BRCA2 mutation carriers: results from a combined analysis of 19 studies

RAD51 is an important component of double-stranded DNA–repair mechanisms that interacts with both BRCA1 and BRCA2. A single-nucleotide polymorphism (SNP) in the 5′ untranslated region (UTR) of RAD51, 135G→C, has been suggested as a possible modifier of breast cancer risk in BRCA1 and BRCA2 mutation carriers. We pooled genotype data for 8,512 female mutation carriers from 19 studies for the RAD51 135G→C SNP. We found evidence of an increased breast cancer risk in CC homozygotes (hazard ratio [HR] 1.92 [95% confidence interval {CI} 1.25–2.94) but not in heterozygotes (HR 0.95 [95% CI 0.83–1.07]; P=.002, by heterogeneity test with 2 degrees of freedom [df]). When BRCA1 and BRCA2 mutation carriers were analyzed separately, the increased risk was statistically significant only among BRCA2 mutation carriers, in whom we observed HRs of 1.17 (95% CI 0.91–1.51) among heterozygotes and 3.18 (95% CI 1.39–7.27) among rare homozygotes (P=.0007, by heterogeneity test with 2 df). In addition, we determined that the 135G→C variant affects RAD51 splicing within the 5′ UTR. Thus, 135G→C may modify the risk of breast cancer in BRCA2 mutation carriers by altering the expression of RAD51. RAD51 is the first gene to be reliably identified as a modifier of risk among BRCA1/2 mutation carriers.     

Genome-Wide Association Study in BRCA1 Mutation Carriers Identifies Novel Loci Associated with Breast and Ovarian Cancer Risk

BRCA1-associated breast and ovarian cancer risks can be modified by common genetic variants. To identify further cancer risk-modifying loci, we performed a multi-stage GWAS of 11,705 BRCA1 carriers (of whom 5,920 were diagnosed with breast and 1,839 were diagnosed with ovarian cancer), with a further replication in an additional sample of 2,646 BRCA1 carriers. We identified a novel breast cancer risk modifier locus at 1q32 for BRCA1 carriers (rs2290854, P = 2.7×10⁻⁸, HR = 1.14, 95% CI: 1.09–1.20). In addition, we identified two novel ovarian cancer risk modifier loci: 17q21.31 (rs17631303, P = 1.4×10⁻⁸, HR = 1.27, 95% CI: 1.17–1.38) and 4q32.3 (rs4691139, P = 3.4×10⁻⁸, HR = 1.20, 95% CI: 1.17–1.38). The 4q32.3 locus was not associated with ovarian cancer risk in the general population or BRCA2 carriers, suggesting a BRCA1-specific association. The 17q21.31 locus was also associated with ovarian cancer risk in 8,211 BRCA2 carriers (P = 2×10⁻⁴). These loci may lead to an improved understanding of the etiology of breast and ovarian tumors in BRCA1 carriers. Based on the joint distribution of the known BRCA1 breast cancer risk-modifying loci, we estimated that the breast cancer lifetime risks for the 5% of BRCA1 carriers at lowest risk are 28%–50% compared to 81%–100% for the 5% at highest risk. Similarly, based on the known ovarian cancer risk-modifying loci, the 5% of BRCA1 carriers at lowest risk have an estimated lifetime risk of developing ovarian cancer of 28% or lower, whereas the 5% at highest risk will have a risk of 63% or higher. Such differences in risk may have important implications for risk prediction and clinical management for BRCA1 carriers.     

Reproductive factors and breast cancer risk among BRCA1 or BRCA2 mutation carriers: Results from ten studies

Although reproductive factors are among the most well-established risk factors for breast cancer in the general population, it is still a matter for debate whether these factors act as risk modifiers among BRCA1 or BRCA2 mutation carriers. This meta-analysis is the first to be performed to determine the relationship between reproductive factors and breast cancer risk among BRCA1 and BRCA2 mutation carriers. We searched the PubMed database up to February 2013. A total of ten studies met the inclusion criteria. The results showed that the reproductive factors may be associated with breast cancer risk only among BRCA1 mutation carriers. No association was found between parity and breast cancer risk. Compared with women at the youngest age in the first-birth category, women in the oldest age category were at a 38% lower risk of breast cancer (RR = 0.62, 95%CI = 0.45–0.85). Breastfeeding for at least 1 or 2 years was associated with a 37% reduction in breast cancer risk (RR = 0.63, 95%CI = 0.46–0.86). Women at the oldest age in the menarche category were at a 34% lower risk of breast cancer (RR = 0.66, 95%CI = 0.53–0.81) than women in the youngest age category. However, none of the reproductive factors were associated with breast cancer risk among BRCA2 mutation carriers. In conclusion, late age at first birth, breastfeeding, and late age at menarche protect against breast cancer in BRCA1 mutation carriers only. Further studies are needed to explore the mechanisms.     

BRCA1 and BRCA2 missense variants of high and low clinical significance influence lymphoblastoid cell line post-irradiation gene expression

The functional consequences of missense variants in disease genes are difficult to predict. We assessed if gene expression profiles could distinguish between BRCA1 or BRCA2 pathogenic truncating and missense mutation carriers and familial breast cancer cases whose disease was not attributable to BRCA1 or BRCA2 mutations (BRCAX cases). 72 cell lines from affected women in high-risk breast ovarian families were assayed after exposure to ionising irradiation, including 23 BRCA1 carriers, 22 BRCA2 carriers, and 27 BRCAX individuals. A subset of 10 BRCAX individuals carried rare BRCA1/2 sequence variants considered to be of low clinical significance (LCS). BRCA1 and BRCA2 mutation carriers had similar expression profiles, with some subclustering of missense mutation carriers. The majority of BRCAX individuals formed a distinct cluster, but BRCAX individuals with LCS variants had expression profiles similar to BRCA1/2 mutation carriers. Gaussian Process Classifier predicted BRCA1, BRCA2 and BRCAX status, with a maximum of 62% accuracy, and prediction accuracy decreased with inclusion of BRCAX samples carrying an LCS variant, and inclusion of pathogenic missense carriers. Similarly, prediction of mutation status with gene lists derived using Support Vector Machines was good for BRCAX samples without an LCS variant (82–94%), poor for BRCAX with an LCS (40–50%), and improved for pathogenic BRCA1/2 mutation carriers when the gene list used for prediction was appropriate to mutation effect being tested (71–100%). This study indicates that mutation effect, and presence of rare variants possibly associated with a low risk of cancer, must be considered in the development of array-based assays of variant pathogenicity.     

Common breast cancer-predisposition alleles are associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers

Germline mutations in BRCA1 and BRCA2 confer high risks of breast cancer. However, evidence suggests that these risks are modified by other genetic or environmental factors that cluster in families. A recent genome-wide association study has shown that common alleles at single nucleotide polymorphisms (SNPs) in FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) are associated with increased breast cancer risks in the general population. To investigate whether these loci are also associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers, we genotyped these SNPs in a sample of 10,358 mutation carriers from 23 studies. The minor alleles of SNP rs2981582 and rs889312 were each associated with increased breast cancer risk in BRCA2 mutation carriers (per-allele hazard ratio [HR] = 1.32, 95% CI: 1.20-1.45, p(trend) = 1.7 x 10(-8) and HR = 1.12, 95% CI: 1.02-1.24, p(trend) = 0.02) but not in BRCA1 carriers. rs3803662 was associated with increased breast cancer risk in both BRCA1 and BRCA2 mutation carriers (per-allele HR = 1.13, 95% CI: 1.06-1.20, p(trend) = 5 x 10(-5) in BRCA1 and BRCA2 combined). These loci appear to interact multiplicatively on breast cancer risk in BRCA2 mutation carriers. The differences in the effects of the FGFR2 and MAP3K1 SNPs between BRCA1 and BRCA2 carriers point to differences in the biology of BRCA1 and BRCA2 breast cancer tumors and confirm the distinct nature of breast cancer in BRCA1 mutation carriers.