BARD1 Autoantibody Blood Test for Early Detection of Ovarian Cancer.

BACKGROUND: Ovarian cancer (OC) is the most lethal gynaecological cancer. It is often diagnosed at an advanced stage with poor chances for successful treatment. An accurate blood test for the early detection of OC could reduce the mortality of this disease. METHODS: Autoantibody reactivity to 20 epitopes of BARD1 and concentration of cancer antigen 125 (CA125) were assessed in 480 serum samples of OC patients and healthy controls. Autoantibody reactivity and CA125 were also tested for 261 plasma samples of OC with or without mutations in BRCA1/2, BARD1, or other predisposing genes, and healthy controls. Lasso statistic regression was applied to measurements to develop an algorithm for discrimination between OC and controls. Findings and interpretation: Measurement of autoantibody binding to a number of BARD1 epitopes combined with CA125 could distinguish OC from healthy controls with high accuracy. This BARD1-CA125 test was more accurate than measurements of BARD1 autoantibody or CA125 alone for all OC stages and menopausal status. A BARD1-CA125-based test is expected to work equally well for average-risk women and high-risk women with hereditary breast and ovarian cancer syndrome (HBOC). Although these results are promising, further data on well-characterised clinical samples shall be used to confirm the potential of the BARD1-CA125 test for ovarian cancer screening.

BARD1 serum autoantibodies for the detection of lung cancer.

PURPOSE: Currently the screening for lung cancer for risk groups is based on Computed Tomography (CT) or low dose CT (LDCT); however, the lung cancer death rate has not decreased significantly with people undergoing LDCT. We aimed to develop a simple reliable blood test for early detection of all types of lung cancer based on the immunogenicity of aberrant forms of BARD1 that are specifically upregulated in lung cancer. METHODS: ELISA assays were performed with a panel of BARD1 epitopes to detect serum levels of antibodies against BARD1 epitopes. We tested 194 blood samples from healthy donors and lung cancer patients with a panel of 40 BARD1 antigens. Using fitted Lasso logistic regression we determined the optimal combination of BARD1 antigens to be used in ELISA for discriminating lung cancer from healthy controls. Random selection of samples for training sets or validations sets was applied to validate the accuracy of our test. RESULTS: Fitted Lasso logistic regression models predict high accuracy of the BARD1 autoimmune antibody test with an AUC = 0.96. Validation in independent samples provided and AUC = 0.86 and identical AUCs were obtained for combined stages 1-3 and late stage 4 lung cancers. The BARD1 antibody test is highly specific for lung cancer and not breast or ovarian cancer. CONCLUSION: The BARD1 lung cancer test shows higher sensitivity and specificity than previously published blood tests for lung cancer detection and/or diagnosis or CT scans, and it could detect all types and all stages of lung cancer. This BARD1 lung cancer test could therefore be further developed as i) screening test for early detection of lung cancers in high-risk groups, and ii) diagnostic aid in complementing CT scan.

Antagonizing functions of BARD1 and its alternatively spliced variant BARD1δ in telomere stability.

Previous reports have shown that expression of BARD1δ, a deletion-bearing isoform of BARD1, correlates with tumor aggressiveness and progression. We show that expression of BARD1δ induces cell cycle arrest in vitro and in vivo in non-malignant cells. We investigated the mechanism that leads to proliferation arrest and found that BARD1δ overexpression induced mitotic arrest with chromosome and telomere aberrations in cell cultures, in transgenic mice, and in cells from human breast and ovarian cancer patients with BARD1 mutations. BARD1δ binds more efficiently than BARD1 to telomere binding proteins and causes their depletion from telomeres, leading to telomere and chromosomal instability. While this induces cell cycle arrest, cancer cells lacking G2/M checkpoint controls might continue to proliferate despite the BARD1δ-induced chromosomal instability. These features of BARD1δ may make it a genome permutator and a driver of continuous uncontrolled proliferation of cancer cells.

BARD1 splice variants display mislocalization in breast cancer cells and can alter the apoptotic response to cisplatin.

We previously showed that BARD1 is a shuttling protein with pro-apoptotic activity in MCF-7 breast cancer cells. BARD1 is expressed as splice variant isoforms in breast cancer. Here we characterized YFP-tagged BARD1 splice variants (beta, omega, phi, ΔRIN, epsilon) for subcellular localization and apoptotic efficacy. We found that loss of nuclear localization (NLS) or export (NES) sequences influenced cellular distribution. The beta and omega variants (+NLS/-NES) shifted exclusively to the nucleus. In contrast, BARD1-epsilon (-NLS/+NES) was mostly cytoplasmic. Variants that lacked both NLS and NES were evenly distributed. Interestingly, the more nuclear isoforms (omega and beta) were least apoptotic in MCF-7 cells as measured by FACS. The cytoplasmic localization of BARD1 isoforms correlated with increased apoptosis. This relationship held in cells exposed to low dose (5 µM) of cisplatin. At 20 µM cisplatin, the main observation was a protective effect by the omega isoform. Similar analyses of HCC1937 cells revealed less pronounced changes but a significant protective influence by BARD1-epsilon. Thus BARD1 variants differ in localization and apoptotic ability, and their expression profile may aid prediction of drug efficacy in breast cancer.

New concepts on BARD1: Regulator of BRCA pathways and beyond.

For nearly two decades most research on BARD1 was closely linked to research on BRCA1, the breast cancer predisposition gene. The co-expression of BARD1 and BRCA1 genes in most tissues, the nearly identical phenotype of Bard1 and Brca1 knock-out mice, and the fact that BRCA1 and BARD1 proteins form a stable complex, led to the general assumption that BARD1 acts as an accessory to BRCA1. More recent research on both proteins showed that BRCA1 and BARD1 might have common as well as separate functions. This review is an overview of how BARD1 functions and controls BRCA1. It highlights also experimental evidence for dominant negative, tumor promoting, functions of aberrant isoforms of BARD1 that are associated with and drivers of various types of cancer.

Cancer predisposing BARD1 mutations affect exon skipping and are associated with overexpression of specific BARD1 isoforms.

BARD1 is the main binding partner of BRCA1 and is required for its stability and tumor-suppressor functions. In breast cancer and other epithelial cell carcinomas, alternatively spliced isoforms of BARD1 are highly upregulated and correlated with poor outcome. Recent data indicate that germline mutations of BARD1 may predispose to breast and/or ovarian cancer. To evaluate the role of BARD1 germline mutations in predisposition to ovarian cancer we scanned a cohort of 255 patients for the presence of previously reported mutations located in exons 5, 8 and 10 using high-resolution melting analysis. Within this group we identified single-patients carrying mutation in exon 8 (c.1690C>T, p.Gln564Ter), two different variants in exon 10 (c.1972C>T, p.Arg658Tyr; c.1977A>G, p.=) and a carrier of novel missense mutation located in exon 5 (c.1361C>T, p.Pro454Leu). Three out of four identified mutations alter exonic splicing enhancing motives and result in expression of incorrect splicing skipping of exons 5, 8, and 2-9, respectively. Our data indicate that BARD1 variants may predispose to ovarian cancer in limited number of patients although based on actual data it is difficult to estimate its actual penetrance.

BARD1 mediates TGF-ß signaling in pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a rapid progressive fibro-proliferative disorder with poor prognosis similar to lung cancer. The pathogenesis of IPF is uncertain, but loss of epithelial cells and fibroblast proliferation are thought to be central processes. Previous reports have shown that BARD1 expression is upregulated in response to hypoxia and associated with TGF-ß signaling, both recognized factors driving lung fibrosis. Differentially spliced BARD1 isoforms, in particular BARD1ß, are oncogenic drivers of proliferation in cancers of various origins. We therefore hypothesized that BARD1 and/or its isoforms might play a role in lung fibrosis. METHODS: We investigated BARD1 expression as a function of TGF-ß in cultured cells, in mice with experimentally induced lung fibrosis, and in lung biopsies from pulmonary fibrosis patients. RESULTS: FL BARD1 and BARD1ß were upregulated in response to TGF-ß in epithelial cells and fibroblasts in vitro and in vivo. Protein and mRNA expression studies showed very low expression in healthy lung tissues, but upregulated expression of full length (FL) BARD1 and BARD1ß in fibrotic tissues. CONCLUSION: Our data suggest that FL BARD1 and BARD1ß might be mediators of pleiotropic effects of TGF-ß. In particular BARD1ß might be a driver of proliferation and of pulmonary fibrosis pathogenesis and progression and represent a target for treatment.

Analysis of large mutations in BARD1 in patients with breast and/or ovarian cancer: the Polish population as an example.

Only approximately 50% of all familial breast cancers can be explained by known genetic factors, including mutations in BRCA1 and BRCA2. One of the most extensively studied candidates for breast and/or ovarian cancer susceptibility is BARD1. Although it was suggested that large mutations may contribute substantially to the deleterious variants of BARD1, no systematic study of the large mutations in BARD1 has been performed. To further elucidate the role of large mutations in BARD1, we designed a multiplex ligation-dependent probe amplification (MLPA) assay and performed an analysis of 504 women with a familial breast and/or ovarian cancer and 313 patients with ovarian cancer. The investigation did not reveal any large mutations in the BARD1 gene. Although the analysis was not focused on identification of small mutations, we detected seven deleterious or potentially deleterious point mutations, which contribute substantially to the total number of BARD1 mutations detected so far. In conclusion, although we cannot exclude the presence of large mutations in BARD1, our study indicates that such mutations do not contribute substantially to the risk of breast and/or ovarian cancer. However, it has to be noted that our results may be specific to the Polish population.

Long non-coding RNA and microRNAs might act in regulating the expression of BARD1 mRNAs.

Long non-coding RNAs (lncRNAs) are ubiquitously expressed RNA molecules of more than 200 nucleotides without substantial ORFs. LncRNAs could act as epigenetic regulators of gene expression affecting transcription, mRNA stability and transport, and translation, although, precise functions have been attributed to only few of them. Competing endogenous RNAs (ceRNAs) represent one recently emerged type of functional lncRNAs that share microRNA recognition sequences with mRNAs and may compete for microRNA binding and thus affect regulation and function of target mRNAs. We studied the epigenetic regulation of the BARD1 gene. The BARD1 protein acts as tumor suppressor with BRCA1. In cancer, mRNAs encoding the tumor suppressor full length BARD1 are often down-regulated while the expression of oncogenic truncated isoforms is boosted. We found that the BARD1 3'UTR is almost 3000nt long and harbors a large number of microRNA binding elements. In addition we discovered a novel lncRNA, BARD1 9'L, which is transcribed from an alternative promoter in intron 9 of the BARD1 gene and shares part of the 3'UTR with the protein coding BARD1 mRNAs. We demonstrate with the example of two microRNAs, miR-203 and miR-101, that they down-regulate the expression of FL BARD1 and cancer-associated BARD1 mRNAs, and that BARD1 9'L counteracts the effect of miR-203 and miR-101, As BARD1 9'L is abnormally over-expressed in human cancers, we suggest it might be a tumor promoting factor and treatment target. This article is part of a Directed Issue entitled: The Non-coding RNA Revolution.