BRCA1 Promoter Methylation Status Research Articles

587P Sequential approach to determine the HRD status with BRCA1 promotor methylation status and shallow whole genome sequencing (sWGS)

High-grade serous ovarian cancer (HGSOC) is now characterized by the status deficient (HRD) or proficient (HRP) of homologous recombination repair (HR). This status is currently determined by the screening of tumoral BRCA1/2 mutation and the Genomic Instability score (GIS) first developed by Myriad. Our goal is to test and develop other simpler and less expensive approach to determine the HR status with BRCA1 promoter methylation status and shallow whole genome sequencing (sWGS) profile.

MicroRNA-126 expression in the peripheral white blood cells of patients with breast and ovarian cancer is a potential biomarker for the early prediction of cancer risk in the carriers of methylated BRCA1.

Constitutive breast cancer type 1 gene (BRCA1) promoter methylation is associated with increased cancer risk, but its role in cancer-free (CF) female carriers is incompletely understood. MicroRNA (miR) is modulated during early tumorigenesis. The present study assessed the modulation of miR-126 expression in the peripheral white blood cells (WBC) of patients with breast cancer (BC) and ovarian cancer (OC) as a biomarker of cancer risk in BRCA1 methylation carriers. A total of 1,114 female subjects [502 patients with BC, 187 patients with OC and 425 CF volunteers] were involved. Screening for BRCA1 promoter methylation in WBC was performed using the methylation–specific polymerase chain reaction (PCR) assay, BRCA1 mRNA was analyzed using a reverse transcription-quantitative PCR assay and miR-126 expression was analyzed using a stem-loop RT-qPCR assay. WBC BRCA1 promoter methylation status was significantly associated with OC (P=0.0266), early-onset BC (P=0.0003) and triple-negative BC (P=0.0066). Notably, 9.4% of the CF group exhibited WBC BRCA1 promoter methylation. In addition, high levels of miR-126 in WBCs were detected in all three groups. The increased level of miR-126 was significantly associated with a lower risk of distant metastasis (P=0.045) in BC, but a higher risk of disease progression and death (P=0.0029) in OC. There was a positive correlation between BRCA1 mRNA and miR-126 levels in the WBCs of all three groups, regardless of BRCA1 promoter methylation status. Notably, circulating miR-126 level was decreased in the BC and OC groups, but not in the CF group. Together, these results suggest the likely involvement of miR-126 in the constitutional methylation of BRCA1 promoter-related malignancies. Therefore, miR-126 may be a candidate biomarker for the early prediction of BC and OC risk in CF BRCA1 methylation carriers.

Loss of BRCA1 expression and morphological features associated with BRCA1 promoter methylation status in triple-negative breast cancer.

Aberrant DNA methylation in the BRCA1 promoter region causes epigenetic silencing of BRCA1 gene expression, which is critical for breast cancer development. However, how BRCA1 promoter methylation status alters histological features remains poorly understood. Here, we investigated the possibility to predict BRCA1 promoter methylation status based on the morphological and immunohistochemical features of triple-negative breast cancers (TNBCs). The morphological features of 53 TNBCs were evaluated with hematoxylin-eosin staining, with immunohistochemical staining of BRCA1, androgen receptor, p53, cytokeratin 5/6, and epidermal growth factor receptor. BRCA1 promoter methylation status was used to distinguish BRCA1 promoter-methylated tumors (BPMTs) from BRCA1 promoter-unmethylated tumors (BPUTs) dependent on pathological characteristics. BPMTs comprised approximately 26% of the TNBCs. Immunohistochemical analysis found that BRCA1 protein expression was significantly lower in BPMT compared with BPUT (p = 0.016). Morphologically, BPMTs were associated with high mitotic index (p = 0.017), pushing margin (p = 0.017), a circumscribed growth pattern (p = 0.014), and a syncytial growth pattern (p = 0.034) compared with BPUTs. We then assessed the potential of predicting BRCA1 promoter methylation status by using published score systems based on these morphological characteristics. A receiver operating characteristic analysis showed an area under the curve of 0.80. This study found that BRCA1 promoter methylation status could be derived from morphological features and lower BRCA1 expression of TNBCs, which may help identify suitable cases for target treatment with PARP inhibitors.

Neoantigen load and HLA-class I expression identify a subgroup of tumors with a T-cell-inflamed phenotype and favorable prognosis in homologous recombination-proficient high-grade serous ovarian carcinoma

BackgroundThere is increasing evidence for the benefit of poly ADP ribose polymerase (PARP) inhibitors in a subset of high-grade serous ovarian carcinoma (HGSC) patients, especially those with homologous recombination (HR)-deficient...

BRCA1 Promoter Methylation Status in 1031 Primary Breast Cancers Predicts Favorable Outcomes Following Chemotherapy.

BackgroundBreast Cancer 1 gene (BRCA1) is known to be inactivated in breast tumors by promoter methylation. Tumor cells in patients carrying a germline mutation in BRCA1 are sensitive to cytotoxic drugs that cause DNA double strand breaks. However, very little is known on whether patients with BRCA1 promoter methylated tumors are similarly sensitive to cytotoxic drugs. In this study, we address this by making use of extensive follow-up data on patients treated with cyclophosphamide, methotrexate, and fluorouracil in Iceland between 1976 and 2007.MethodsWe analyzed BRCA1 promoter methylation by pyrosequencing DNA from tumor samples from 1031 patients with primary breast cancer. Of those, 965 were sporadic cases, 61 were BRCA2, and five were BRCA1 germline mutation carriers. All cases were examined with respect to clinicopathological parameters and breast cancer–specific survival in patients treated with cytotoxic drugs. Information on chemotherapy treatment in noncarriers was available for 26 BRCA1 methylated tumors and 857 unmethylated tumors.Results BRCA1 was promoter methylated in 29 sporadic tumors or in 3.0% of cases (29 of 965), whereas none of the tumors derived from BRCA germline mutation carriers were promoter methylated. Important to note, patients with BRCA1 promoter methylation receiving chemotherapeutic drug treatment show highly improved breast cancer–specific survival compared with unmethylated controls (hazard ratio = 0.10, 95% confidence interval = 0.01 to 0.75, two-sided P = .02).Conclusions BRCA1 promoter methylation is predictive of improved disease outcome in patients receiving cyclophosphamide, methotrexate, and fluorouracil drug treatment. Our results support the use of markers indicative of “BRCAness” in sporadic breast cancers to identify patients that are likely to benefit from the use of DNA-damaging agents.

Genomic Profile and BRCA-1 Promoter Methylation Status in BRCA Mutated Ovarian Cancer: New Insights in Predictive Biomarkers of Olaparib Response.

Objective: We assessed the genomic profile of four representative BRCA-mutated ovarian cancer (OC) patients treated with olaparib to investigate the relationship between intratumor heterogeneity and response to olaparib treatment. The main aim is to identify possible predictive biomarkers of response to olaparib through the analysis of HRD or not HRD genes and the definition of BRCA1 promoter methylation status.Methods: DNA, isolated from formalin-fixed, paraffin-embedded (FFPE) diagnostic OC tissues, was analyzed by FoundationOneCDx™. This assay detects alterations in a total panel of 324 genes, using the Illumina® HiSeq 4000 platform. Methylation analysis of the BRCA gene promoter was carried out by pyrosequencing with PyroMark Q24 platform (Qiagen), an in vitro nucleic acid sequence-based detection test based on pyrosequencing technology for quantitative measurements of methylation status.Results: Case #1 and #2 were defined Long-term responders since they received olaparib for 27 and 36 months, respectively. These remarkable results could be explained, at least in part, by the presence of somatic IDH1 mutation in case #1 and PI3K and SOX2 amplification in the case #2. In case #3, the somatic NF1 mutation appeared to be related to the short duration of response. In the case #4, in which the patients is on olaparib from 1 year achieving a stable disease, a somatic mutation of BRCA1 was recorded. Moreover, in all cases, levels of BRCA1 promoter were strictly related to olaparib response.Conclusions: Based on our experience, genomic analysis of tumor tissue at diagnosis might help to determine the future response to olaparib in advanced OC setting, revealing predictive biomarkers beyond BRCA 1-2 and HRD status.

Candidate biomarkers of PARP inhibitor sensitivity in ovarian cancer beyond the BRCA genes

BackgroundOlaparib (Lynparza™) is a PARP inhibitor approved for advanced BRCA-mutated (BRCAm) ovarian cancer. PARP inhibitors may benefit patients whose tumours are dysfunctional in DNA repair mechanisms unrelated to BRCA1/2. We report exploratory analyses, including the long-term outcome of candidate biomarkers of sensitivity to olaparib in BRCA wild-type (BRCAwt) tumours.MethodsTumour samples from an olaparib maintenance monotherapy trial (Study 19, D0810C00019; NCT00753545) were analysed. Analyses included classification of mutations in genes involved in homologous recombination repair (HRR), BRCA1 promoter methylation status, measurement of BRCA1 protein and Myriad HRD score.ResultsPatients with BRCAm tumours gained most benefit from olaparib; a similar treatment benefit was also observed in 21/95 patients whose tumours were BRCAwt but had loss-of-function HRR mutations compared to patients with no detectable HRR mutations (58/95). A higher median Myriad MyChoice® HRD score was observed in BRCAm and BRCAwt tumours with BRCA1 methylation. Patients without BRCAm tumours derived benefit from olaparib treatment vs placebo although to a lesser extent than BRCAm patients.ConclusionsOvarian cancer patients with tumours harbouring loss-of-function mutations in HRR genes other than BRCA1/2 may constitute a small, molecularly identifiable and clinically relevant population who derive treatment benefit from olaparib similar to patients with BRCAm.

Abstract 3307: BRCA1 promoter methylation analyzed in 1032 primary breast cancers predicts favorable response to chemotherapy independently of p53 mutations

Abstract Individuals with germline mutations in the breast cancer susceptibility gene, BRCA1, are at increased risk of developing breast- and ovarian cancer. BRCA1 is involved in DNA double strand break repair (DSB) by homologous recombination (HR), an error-free DNA repair pathway that uses an intact sister chromatid for the repair. BRCA1 mutated tumors are therefore HR deficient and sensitive to drugs that stall DNA replication forks, resulting in DSBs. Tumor cells lacking the protein are characterized by genomic instability and a pattern of specific genome-wide mutation signature as a consequence of dependence on alternative error prone repair pathways. BRCA1 can also be inactivated epigenetically by CpG promoter hypermethylation. These BRCA1 epigenetically silenced tumors are more likely to be of the basal-like subtype and are strongly associated with this pattern of mutational signature characteristic for individuals with germline mutations in BRCA1. This suggests that the tumorigenesis in the sporadic cancer cases is similar to tumors where germline mutations are present, highlighting the importance of epigenetic silencing of BRCA1. The aim of the study was to examine the clinical significance of epigenetic silencing of BRCA1 in primary breast cancer, focusing on the response to chemotherapy. DNA methylation analysis of the BRCA1 promoter was performed by pyrosequencing tumor samples from 1032 patients with primary breast cancer of which 966 were sporadic, 61 were BRCA2 germline mutation carriers and 5 were BRCA1 mutation carriers. The p53 mutational status was also studied in the same primary tumors by DNA sequencing. We analysed the methylation status of BRCA1 in tumors derived from 1032 patients and found BRCA1 to be promoter hypermethylated in 29 sporadic tumors (29 out of 966, 3.0 %) whereas none of the tumors derived from BRCA mutation carriers were BRCA1 promoter hypermethylated. Patients with BRCA1 promoter hypermethylation receiving chemotherapeutic drug treatment show significantly better breast cancer specific survival (P=0.0192). The trend is opposite in patients not receiving chemotherapeutic treatment, were patients with BRCA1 hypermethylated tumors show worse survival (P=0.074). The impact of BRCA1 methylation with respect to survival in chemotherapy treated patients was found to be independent of p53 mutational status, ER status, tumorsize, lymph node status, year- and age at diagnosis (HR = 0.108; 95% CI = 0.015-0.794; P = 0.0288). BRCA1 promoter hypermethylation predicts improved disease outcome in anti-cancer drug treated patients. Therefore, BRCA1 promoter methylation status in primary breast cancer is an important factor to be considered when deciding treatment options. Citation Format: Olafur A. Stefansson, Holmfridur Hilmarsdottir, Kristrun Olafsdottir, Laufey Tryggvadottir, Asgerdur Sverrisdottir, Jon G. Jonasson, Jorunn E. Eyfjord, Stefan Sigurdsson. BRCA1 promoter methylation analyzed in 1032 primary breast cancers predicts favorable response to chemotherapy independently of p53 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3307.

Abstract 5721: Neoantigen load and HLA-class I expression characterize a subset of HR-proficient high-grade serous ovarian carcinomas with favorable prognosis and T cell-inflamed phenotype

Abstract Aim: Around 50% of high-grade serous ovarian carcinomas (HGSC) have deficiency in homologous recombination (HR) pathways. There has been increased evidence of benefit for the use of poly ADP ribose polymerase (PARP) inhibitors in a group of HGSC patients who especially have HR-deficient tumors. Therefore, it is needed to develop a new treatment strategy for the rest, an HR-proficient tumor. The aim of this study is to investigate immunological background of HR-proficient HGSC by integrated molecular analysis to explore the subset that would become candidates for immunotherapy such as immune checkpoint inhibitors. Methods: In total, 80 cases of HGSC were analyzed in this study. Exome and RNA sequencing were performed to identify germline and somatic mutations. Methylation arrays were also carried out to evaluate BRCA1 and RAD51C promoter methylation status. Predicted neoantigens derived from mutations were identified based on the MHC class I binding prediction algorithm NetMHCpan 2.8. Immune profile in the tumor was assessed by differential gene expression analysis and gene set enrichment analysis (GSEA) using RNA sequencing data. Neoantigen load, antigen presentation, immune profile and their relevance to clinical outcomes were also investigated. Results: Either BRCA1/2, RAD51C/D mutations, or BRCA1, RAD51C promoter methylation was defined as HR-deficient. A total of 34 (42.5%) and 46 (57.5%) patients were classified as having HR-deficient and HR-proficient tumors, respectively. As expected, the numbers of predicted neoantigens were lower in HR-proficient than HR-deficient tumors (p<0.01). However, 40% of the patients with HR-proficient tumors still had high numbers of neoantigens and displayed better survival trend than those who had lower numbers of neoantigens. Incorporation of HLA-class I expression status in the analysis revealed that the patients who had both high neoantigen number and high HLA-class I expression showed improved survival in HR-proficient HGSC (p=0.02). GSEA demonstrated that the gene sets for effector memory CD8, TH1, and type I and type II interferon (IFN) responses were enriched in those patients. Conclusions: The number of neoantigens and HLA-class I expression characterized a subset of HR-proficient HGSC with improved prognosis and immunologically hot phenotype. This subset might be a candidate target for immune checkpoint inhibitors rather than PARP inhibitors. Citation Format: Hirokazu Matsushita, Kosei Hasegawa, Katsutoshi Oda, Shogo Yamamoto, Kayo Asada, Akira Yabuno, Akira Nishijima, Takahiro Karasaki, Yuji Ikeda, Keiichi Fujiwara, Hiroyuki Aburatani, Kakimi Kazuhiro. Neoantigen load and HLA-class I expression characterize a subset of HR-proficient high-grade serous ovarian carcinomas with favorable prognosis and T cell-inflamed phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5721.

White Blood Cell BRCA1 Promoter Methylation Status and Ovarian Cancer Risk.

The role of normal tissue gene promoter methylation in cancer risk is poorly understood. To assess associations between normal tissue BRCA1 methylation and ovarian cancer risk. 2 case-control (initial and validation) studies. 2 hospitals in Norway (patients) and a population-based study (control participants). 934 patients and 1698 control participants in the initial study; 607 patients and 1984 control participants in the validation study. All patients had their blood sampled before chemotherapy. White blood cell (WBC) BRCA1 promoter methylation was determined by using methylation-specific quantitative polymerase chain reaction, and the percentage of methylation-positive samples was compared between population control participants and patients with ovarian cancer, including the subgroup with high-grade serous ovarian cancer (HGSOC). In the initial study, BRCA1 methylation was more frequent in patients with ovarian cancer than control participants (6.4% vs. 4.2%; age-adjusted odds ratio [OR], 1.83 [95% CI, 1.27 to 2.63]). Elevated methylation, however, was restricted to patients with HGSOC (9.6%; OR, 2.91 [CI, 1.85 to 4.56]), in contrast to 5.1% and 4.0% of patients with nonserous and low-grade serous ovarian cancer (LGSOC), respectively. These findings were replicated in the validation study (methylation-positive status in 9.1% of patients with HGSOC vs. 4.3% of control participants-OR, 2.22 [CI 1.40 to 3.52]-4.1% of patients with nonserous ovarian cancer, and 2.7% of those with LGSOC). The results were not influenced by tumor burden, storage time, or WBC subfractions. In separate analyses of young women and newborns, BRCA1 methylation was detected in 4.1% (CI, 1.8% to 6.4%) and 7.0% (CI, 5.0% to 9.1%), respectively. Patients with ovarian cancer were recruited at the time of diagnosis in a hospital setting. Constitutively normal tissue BRCA1 promoter methylation is positively associated with risk for HGSOC. Norwegian Cancer Society.

BRCA1 deficiency is a recurrent event in early-onset triple-negative breast cancer: a comprehensive analysis of germline mutations and somatic promoter methylation.

BRCA1 germline mutation is closely associated with triple-negative breast cancer. BRCA deficiency leads to impaired DNA repair and tumor development, and understanding this deficiency, in both hereditary and sporadic scenarios, is of great clinical and biological interest. Here, we investigated germline or somatic events that might lead to BRCA1 impairment in triple-negative breast cancer. We also analyzed the clinical implications associated with BRCA deficiency. Next-generation sequencing for the BRCA1/2 genes and multiplex ligation-dependent probe amplification (MLPA) for the BRCA1 gene were performed for mutation screening. A customized bisulfite next-generation sequencing approach was used for assessing BRCA1 promoter methylation status in tumor tissue. A total of 131 triple-negative cases were assessed, and germline pathogenic variants were detected in 13.0% of all cases and in 26% of cases diagnosed in young women. Most germline pathogenic variants (88.2%) occurred in the BRCA1 gene. BRCA1 promoter hypermethylation was detected in 20.6% of tumors; none of these tumors were in BRCA1/2 pathogenic variant carriers. BRCA1 impairment by either germline or somatic events was significantly more frequent in young women (55% in those≤40years; 33% in those 41-50years; 22% in those>50years of age) and associated with better overall and disease-free survival rates in this group of patients. BRCA1 deficiency was recurrent in early-onset triple-negative breast cancer in Brazilian patients and associated with improved survival. With the new treatment modalities being investigated, including poly (ADP-ribose)-polymerase (PARP) inhibitor therapy, our results suggest that a significant proportion of young women with this subtype of tumor might benefit from PARP inhibitor treatment, which warrants further investigation.

Loss of BRCA1 promotor hypermethylation in recurrent high-grade ovarian cancer.

BackgroundApproximately 20-25% of ovarian cancers are attributable to germline or somatic BRCA1/2 mutations, resulting in defects in the homologous recombination pathway. Inactivation of these genes can also be mediated by epigenetic changes, e.g., hypermethylation of CpG islands in the promoter regions. In such homologous recombination deficient tumors, platinum based chemotherapy is in general effective, however, loss of hypermethylation might lead to refractory disease. The aim of this study was to evaluate the stability of BRCA1 promoter hypermethylation in recurrent disease after platinum based chemotherapy.MethodsTumor tissue from 76 patients with primary and 48 patients with platinum-sensitive recurrent high-grade ovarian cancer was collected. In a subgroup of 12 patients, ‘paired’ tumor tissue from primary and recurrent surgery was available. BRCA1 promoter methylation status was assessed using methylation specific polymerase chain reaction and was verified by Sanger Sequencing.Results73.7% (56/76) of primary and 20.8% (10/48) of recurrent tumors displayed BRCA1 promoter hypermethylation. BRCA1 promoter methylation status was not associated with progression-free- or overall survival. In the paired subgroup 83.3% (10/12) of the primary vs. 16.7% (2/12) of the recurrent tumors showed hypermethylation. In eight patients loss of BRCA1 hypermethylation was observed, whereas two patients had stable methylation status.ConclusionsLoss of BRCA1 promoter methylation may be a mechanism to restore BRCA1 function in recurrent disease. However, currently the clinical significance is still unclear and should be evaluated in prospective clinical trials.

Abstract A27: The genomic profile of BRCA1-associated estrogen-receptor positive breast cancer does not resemble BRCA1-associated triple negative cancers, but is more similar to BRCA2-associated breast cancer

Abstract Background: Most breast cancers arising in BRCA1-mutation carriers are triple negative (TN). Therefore, it has been suggested that TN status is intrinsic to BRCA1-mutated tumors. However, 10 to 20% of BRCA1-associated breast cancers are estrogen receptor positive (ER+). As these tumors arise more often in older patients, and have less aggressive tumor characteristics, it has been suggested that these tumors are ‘sporadic’ and not related to BRCA1 deficiency. With the introduction of targeted treatments specific for tumors with a non-functioning BRCA1 or BRCA2 gene (i.e. PARP-inhibitors), the question whether the BRCA genes are impaired in the tumor, is highly relevant. Therefore, we performed a genomic analysis of a series of BRCA1-associated ER+ tumors, and compared them with BRCA1-associated TN tumors, as well as BRCA2-associated and sporadic breast cancers. Material and Methods: Genomic profiling of 18 BRCA1-associated ER+ tumors was performed using Nimblegen 135K arrays. We previously developed a BRCA1-like and BRCA2-like genomic profile, containing specific copy number alterations for respectively BRCA1-associated and BRCA2-associated breast cancer. These genomic profiles were applied on the current series. In addition, the BRCA1 promoter methylation status was determined, and LOH analysis was performed to assess if the wildtype or mutant allele was lost in the tumor. Results were compared with BRCA1 associated ER- tumors, BRCA2 associated tumors, and sporadic ER+ and TN tumors. Results: Only 2 out of 18 ER+ BRCA1 associated tumors showed a BRCA1-like genomic profile, while 90% of all ER- BRCA1-associated tumors show this genomic profile. Interestingly, 11 out of 18 ER+ BRCA1-associated tumors showed a BRCA2-like genomic profile. BRCA1 promoter methylation was absent in ER+ BRCA1-associated tumors, similar to TN BRCA1-associated tumors. LOH analysis was possible in 12 tumors, loss of the wildtype BRCA1 allele was shown in 10 out of 12 ER+ BRCA1-mutated tumors. One sample showed loss of the mutant allele, while another sample did not show loss at the BRCA1 locus. The genomic profiles of ER+ BRCA1-associated tumors were more similar to BRCA2-associated breast cancers and sporadic ER+ breast tumors than to BRCA1-associated TN tumors. Conclusion: The majority of BRCA1-associated ER+ tumors did not show a BRCA1-like genomic profile, even though LOH analysis indicated that loss of the wildtype BRCA1 allele was a frequent event. Therefore, it is likely that the complete loss of the BRCA1 gene plays a crucial role in tumorgenesis in this group of breast tumors. Remarkably, a BRCA2-like genomic profile was observed in the majority of ER+ BRCA1 associated tumors in this series. A clinical consequence of our findings is that ER+ BRCA1-associated breast tumors are probably highly sensitive to PARP inhibitors, similar to TN BRCA1-associated breast cancers. However, as ER+ BRCA1-mutated tumors clearly have different tumor characteristics compared to TN BRCA1-mutated tumors, they should be considered as a special group, and response to therapies exploiting the BRCA1 gene defect should be specifically monitored in this subgroup. Citation Format: Esther H. Lips, Rashmie Debipersad, Esther Scheerman, Lennart Mulder, Lizet E. van der Kolk, Jelle Wesseling, Frans B.L. Hogervorst, Petra M. Nederlof. The genomic profile of BRCA1-associated estrogen-receptor positive breast cancer does not resemble BRCA1-associated triple negative cancers, but is more similar to BRCA2-associated breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A27.

Abstract A05: BRCA1 promoter hypermethylation loss in recurrent high-grade serous carcinoma.

Abstract Objective: BRCA1 germline mutations are associated with improved ovarian cancer survival, yet secondary revertant mutations that impair chemo-sensitivity can develop after platinum-based therapy. BRCA1 promoter hypermethylation, present in ~10% of all high-grade serous carcinomas (HGSCs), is associated with reduced gene and protein expression. We hypothesize that BRCA1 promoter hypermethylation decreases in recurrent HGSCs compared with matched primary samples. Methods: Primary, untreated HGSCs with known BRCA1 promoter methylation status and matched recurrent samples were macrodissected from archival slides. After DNA was extracted and bisulfite treated, pyrosequencing was performed across 11 CpG sites within the BRCA1 promoter to quantify methylation using a CLIA-approved assay. The methylation percentage was calculated from allele frequencies and 10% or greater is considered hypermethylated. Validation data were obtained from The Cancer Genome Atlas (TCGA) substudy of paired primary and recurrent HGSCs. Results: Eleven patients with matched primary and recurrent tumors were evaluated. Six patients had hypermethylated primary tumors, with methylation percentages ranging from 35-69%. We found that BRCA1 promoter methylation decreased 16-95% in five recurrent samples compared to their primary tumors. One patient with a demethylated recurrent tumor had platinum refractory disease clinically and died 12 months after diagnosis. In contrast, the only patient with an increase (74%) in promoter methylation between primary and recurrent specimens is a long-term survivor alive with disease 14 years after diagnosis. There was negligible change in promoter methylation between primary and recurrent cases for the five unmethylated samples. Three of 17 paired samples from the ovarian TCGA substudy had BRCA1 promoter hypermethylation and showed a 20-50% decrease between primary and recurrent samples with a corresponding increase in BRCA1 gene expression. Conclusion: BRCA1 promoter hypermethylation generally decreases between primary and recurrent HGSCs and can be associated with remarkable clinical outcomes. BRCA1 promoter methylation may be a marker of platinum response in the recurrent disease setting and can be assessed in prospective clinical trials. In addition to BRCA1 somatic revertant mutations, loss of BRCA1 promoter methylation may be another mechanism to restore BRCA1 function in recurrent disease. Citation Format: Maria Bisogna, Fanny Dao, Narciso Olvera, Ruben Bacares, Liying Zhang, Douglas A. Levine. BRCA1 promoter hypermethylation loss in recurrent high-grade serous carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A05.

Evaluation of BRCA1-related molecular features and microRNAs as prognostic factors for triple negative breast cancers.

BackgroundThe BRCA1 gene plays a key role in triple negative breast cancers (TNBCs), in which its expression can be lost by multiple mechanisms: germinal mutation followed by deletion of the second allele; negative regulation by promoter methylation; or miRNA-mediated silencing. This study aimed to establish a correlation among the BRCA1-related molecular parameters, tumor characteristics and clinical follow-up of patients to find new prognostic factors.MethodsBRCA1 protein and mRNA expression was quantified in situ in the TNBCs of 69 patients. BRCA1 promoter methylation status was checked, as well as cytokeratin 5/6 expression. Maintenance of expressed BRCA1 protein interaction with BARD1 was quantified, as a marker of BRCA1 functionality, and the tumor expression profiles of 27 microRNAs were determined.ResultsmiR-548c-5p was emphasized as a new independent prognostic factor in TNBC. A combination of the tumoral expression of miR-548c and three other known prognostic parameters (tumor size, lymph node invasion and CK 5/6 expression status) allowed for relapse prediction by logistic regression with an area under the curve (AUC) = 0.96.BRCA1 mRNA and protein in situ expression, as well as the amount of BRCA1 ligated to BARD1 in the tumor, lacked any associations with patient outcomes, likely due to high intratumoral heterogeneity, and thus could not be used for clinical purposes.ConclusionsIn situ BRCA1-related expression parameters could be used for clinical purposes at the time of diagnosis. In contrast, miR-548c-5p showed a promising potential as a prognostic factor in TNBC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1740-9) contains supplementary material, which is available to authorized users.

Epigenetic Inactivation of BRCA1 Through Promoter Hypermethylation and Its Clinical Importance in Triple-Negative Breast Cancer

BackgroundTriple-negative breast cancer (TNBC) has many similarities with basal-like breast cancer. Additionally, TNBCs are associated with Breast cancer susceptibility gene I (BRCA1) functional loss, which leads to impaired homologous recombination-mediated DNA repair. Although somatic mutations in BRCA1 rarely occur in sporadic breast cancer, lower than normal rates of expression of BRCA1 is reported to be an important factor that contributes to tumorigenesis in sporadic tumors. The epigenetic inactivation of BRCA1 expression might thus play an important role in sporadic breast cancer cases. Patients and MethodsBreast cancer specimens were obtained from 69 TNBC and 161 non-TNBC patients who underwent surgery without neoadjuvant systemic therapy. BRCA1 promoter methylation status was investigated using combined bisulfite and restriction analysis. BRCA1 mRNA expression was evaluated using quantitative reverse transcriptase polymerase chain reaction and BRCA1 protein expression was assessed using immunohistochemistry. ResultsBRCA1 promoter methylation was found in 11 tumors and all of these were in TNBC cases (P < .0001). BRCA1 promoter methylation was significantly associated with lymphovessel invasion (P = .02), high nuclear grade (P = .05), low BRCA1 mRNA expression (P < .0001), and loss of BRCA1 protein expression (P = .0015). BRCA1 promoter methylation was significantly associated with shorter overall survival (P = .038). ConclusionBRCA1 promotor methylation was found only in TNBC cases and the methylated cases account for 16% of TNBC. BRCA1 promoter methylation was significantly associated with reduced BRCA1 expression, aggressive phenotype, and poor prognosis. BRCA1 promoter methylation is an important mechanism that leads to functional loss of BRCA1.

Abstract P5-06-01: Homologous recombination deficiency (HRD) score predicts response to standard neoadjuvant chemotherapy in patients with triple negative or BRCA1/2 mutation-associated breast cancer

Abstract Background: Defects in the homologous recombination (HR) DNA repair pathway sensitize tumors to therapeutics that target this pathway. A significant proportion of triple negative breast cancers (TNBC) carry HR defects. Recently three DNA-based measures (LOH, Abkevich et al.; TAI, Birkbak et al; LST, Popova et al.) have been developed and shown to be highly associated with BRCA1/2 mutation status and sensitivity to platinum-based chemotherapy in TNBC. Standard chemotherapy consists of some combination of an anthracycline and cyclophosphamide, with or without a taxane. This study assesses the association of LOH, TAI, LST and the sum of these measures, the HRD Score, with response to standard neoadjuvant chemotherapy in patients with TNBC. Methods: Tumor samples were retrospectively obtained from 45 TNBC patients and 2 BRCA1/2 mutation positive hormone receptor-positive/HER2-negative breast cancer patients who received anthracycline-based neoadjuvant chemotherapy at Stanford University Medical Center or Cedars-Sinai Medical Center under IRB approved protocols. Measures of LOH, TAI, LST were obtained; tumor BRCA1/2 mutation analysis was conducted; and BRCA1 promoter methylation status was determined. Response was categorized by the residual cancer burden (RCB) score with responders defined as RCB 0 or 1, and non-responders as RCB 2 or 3. Data were also analyzed using the outcome of pathologic complete response (RCB 0). BRCA1/2 deficiency was defined as either BRCA1/2 germline or somatic mutant, or BRCA1 methylated with loss of the second allele in the tumor confirmed by LOH at the affected gene. Associations of LOH, TAI, LST, HRD Score, BRCA1/2 deficiency and mutation status with response to neoadjuvant therapy were evaluated with univariate logistic regression models. Results: All three metrics showed significant association with response (LOH: p=0.0018; TAI: p=0.0057; LST: p=0.0043) and pCR (LOH: p=0.0061; TAI: p=0.0044; LST: p=0.047). The sum of the scores, HRD Score, was also significantly associated with response (p=0.0021) and pCR (0.011). Neither BRCA1/2 mutation status (p=0.78 and p=0.31 respectively) nor BRCA1/2 deficiency (p=0.12 and p=0.34 respectively) were significantly associated with response or pCR in this cohort. When the analysis was restricted to BRCA1/2 intact samples (n=29) all scores were still significantly associated with pCR (LOH: p=0.019; TAI: p=0.0046; LST: p=0.013; HRD: p=0.0067), while only the sum was significant for response (p=0.042). Conclusions:All three measures of HR deficiency, LOH, TAI and LST and the sum of these three metrics, the HRD Score, are significantly associated with response to standard neoadjuvant chemotherapy in TNBC. This observation is consistent with the mechanisms of action of doxorubicin and cyclophosphamide as DNA damaging agents, though we have yet to examine cohorts of patients treated with non-anthracycline-containing standard chemotherapy regimens, such as docetaxel and cyclophosphamide. The HRD Score could be used clinically to identify patients with increased sensitivity to DNA damaging therapeutics. Citation Format: Melinda L Telli, William Audeh, Kirstin C Jensen, Shikha Bose, Kirsten Timms, Alexander Gutin, Victor Abkevich, Jerry Lanchbury, Chris Neff, Elisha Hughes, Zaina Sangale, Joshua Jones, Richard Wenstrup, Anne-Renee Hartman, Pei-Jen Chang, Shaveta Vinayak, James M Ford. Homologous recombination deficiency (HRD) score predicts response to standard neoadjuvant chemotherapy in patients with triple negative or BRCA1/2 mutation-associated breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-06-01.

BRCA1 gene promoter methylation status in high-grade serous ovarian cancer patients – A study of the tumour Bank ovarian cancer (TOC) and ovarian cancer diagnosis consortium (OVCAD)

BackgroundMutations in BRCA1/2 genes are involved in the pathogenesis of breast and ovarian cancer. Inactivation of these genes can also be mediated by hypermethylation of CpGs in the promoter regions. Aim of this study was to analyse the clinical impact of BRCA1 promoter gene methylation status in a homogenous cohort of high-grade serous ovarian cancer (HGSOC) patients. MethodsThe cohort included 257 primary HGSOC patients treated by cytoreduction and platinum-based chemotherapy. DNA was extracted from fresh frozen tissue samples. BRCA1 gene promoter methylation rate was assessed using polymerase chain reaction (PCR). Results14.8% of patients presented hypermethylation within a selected region of the BRCA1 promoter. The rate of hypermethylation was significantly higher in younger patients (20.8% hypermethylation in the age group ⩽58years versus 8.7% hypermethylation in the age group >58years; p=0.008). Optimal tumour debulking could be reached in 63% of patients, without significant differences in the extent of residual disease with respect to the methylation status. No impact of BRCA1 gene promoter methylation status on progression free- and overall-survival rates was found. No significant differences within BRCA1 promoter methylation status between primary and metastatic tissue could be observed. These results on BRCA1 promoter methylation status were also confirmed in a subgroup of 107 patients found negative for BRCA1 exon 11 mutations. ConclusionsOur data suggest that BRCA1 methylation determines the earlier onset of HGSOC. Furthermore our study supports the idea that BRCAness is not only due to mutations but also to epigenetic changes in BRCA1 promoter gene.

Abstract P1-08-40: BRCA-1 promotor methylation and p53 mutation in triple-negative breast cancer patients refrectory to taxane-based neoadjuvant chemotherapy

Abstract Introduction Triple-negative breast cancer (TNBC) without pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) is associated with high risk of disease recurrence. In ABCSG trials 14 and 24, concomitant epirubicin plus docetaxel (+/- capecitabine) was used as NAC regimen; patients (pts) without pCR routinely received another 4-8 cycles of adjuvant CMF. Tumours harbouring BRCA-1 germline mutations are apparently less responsive to taxane-based chemotherapy, while sensitivity to DNA-damaging agents is retained. In sporadic TNBC, BRCA-1 promotor methylation is frequently observed; this may also result in an impaired activity of genetic repair mechanisms. Therefore, we investigated the effect of adjuvant CMF as salvage therapy in pts with or without BRCA-1 promotor methylation who did not achieve pCR to taxane-based (cyclophosphamide-free) NAC. Moreover, the predictive role of TP53 mutations was investigated in these tumours. Methods All pts with TNBC refractory to taxane-based NAC who received adjuvant CMF were included. DNA was extracted from formalin-fixed paraffin-embedded tissue samples with the Qiagen DNA FFPE Tissue Kit® and purified. For determining BRCA1 promoter methylation status, DNA was bisulfite-treated; the TaqMan® assay was used in order to perform a quantitative methylation-specific PCR. DNA quantity was normalized using Actin-b. For the investigation of TP53 mutations in exons 4-9, purified DNA was PCR amplified, sequenced by Sanger sequencing and results were analyzed with SeqScape® software version 2.7. Mutations were validated using the IARC p53 database. Results Twenty-four pts, median age 47 years, were available for this analysis. In 10/24 pts (41.7%), a BRCA-1 promotor methylation was detected; TP53 mutations were observed in 16/24 pts (66.7%). At a median follow-up of 27.5 months, 2/10 pts (20%) with BRCA-1 promotor metyhlation had a disease-free survival (DFS) event, as compared to 9/14 (64.3%) in the non-methylated group (p = 0.0472; Fisher's exact test). Kaplan Meier estimation of disease-free survival (DFS) in the non-methylated group was 16 months (95% CI 0.0-41.73) and was not reached in the methylated group (n.s.). TP53 mutation was neither associated with increased risk of disease recurrence nor with DFS. No correlation was observed between BRCA-1 promotor methylation and TP53 mutation; still, all methylated samples were exclusively affected be missense and nonsense mutations. Conclusions Adjuvant CMF is of limited activity in TNBC refractory to taxane-based NAC. Still, in breast cancer harbouring BRCA-1 promotor methylation, a significant decrease of DFS events was observed. TP53 mutation status on the other hand, was not associated with outcome; while no correlation was found between BRCA-1 promotor methylation and TP53 mutation status, we only observed missense and non-sense mutations in methylated samples. In conclusion, tumours harbouring BRCA-1 promotor methylation are apparently sensitive to DNA damage caused by cyclophosphamide. Further clinical validation of this concept is warranted. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-40.

Abstract 1770: Frequent BRCA1 promoter methylation and low 53BP1 status in sporadic triple-negative breast cancer.

Abstract Purpose: BRCA1 plays a key role in DNA repair and cellular stress response. Inhibitors of PARP show promising clinical activity in metastatic, triple-negative BRCA-mutated breast cancer. P53 binding protein 1 (53BP1) is a protein involved in DNA damage checkpoint activation and DNA repair that is involved in both non homologous end-joining and homology-mediated repair of double-strand DNA breaks. When both BRCA1 and 53BP1 are lost, homology-mediated repair appears to be restored and the sensitivity to DNA damaging agents is reduced. Loss of 53BP1 leads to resistance to cis-platinum and PARP inhibitors in BRCA1-deficient cells, suggesting that 53BP1 is a critical modulator of repair choice in BRCA1 mutant, and possibly epigenetically inactivated cells. Reduced 53BP1 expression has been reported in sporadic basal-like and triple-negative and BRCA-associated breast cancers. It thus appears important to simultaneously evaluate 53BP1 status altogether with BRCA1 mutation/methylation in order to estimate the homology-mediated functionality in breast cancer tumors. Methods: We investigated cytosolic 53BP1 levels and BRCA1 gene promoter hypermethylation profile in 151 surgical tumor samples from patients treated in our institution between January 2006 and November 2009 and evaluated their statistical association with classical breast cancer predictive and prognostic factors. DNA methylation patterns in the CpG islands of BRCA1 gene promoter were determined by a methylation specific PCR distinguishing unmethylated from methylated alleles in a given gene on the basis of sequence changes produced following bisulfite treatment of DNA. Cytosolic 53BP1 level was evaluated using a commercially available assay kit. Results: Median age was 54 years (range 29-75 years). Median 53BP1 protein level (pg/mg of cytosolic protein) was 9.5. Using this cut-off, low 53BP1 levels were significantly associated with negative ER and PR status, triple-negative profile, low cytosolic PARP activity, BRCA1 promoter hypermethylation and absence of nodal metastases. The hypermethylation status was significantly associated with UPA and PAI-1 levels, SBR grade, SBR Mitotic count score, ER, PR and HER-2 negative status, and then conversely triple negative (ER, PR and HER-2 negative) profile. Twenty nine percent of the triple negative tumors presented a hypermethylated status, comparing to 5 and 2% for HR+/HER-2- and HER-2+ tumors respectively. In the triple negative population (N=48), only 2 of the 13 tumors with BRCA1 promoter hypermethylation, presented high (&amp;gt;9.5 pg/mg) 53BP1 protein levels Conclusion: Triple negative breast cancers harboring simultaneously a high level of 53BP1 and BRCA1 promoter hypermethylation are a putative target population for development of drugs targeting DNA repair. However, this population appears to be restricted to a small subgroup of triple negative breast cancers. Citation Format: William Jacot, Simon Thezenas, Anne-Claire Laberenne, Romain Senal, Cathy Viglianti, Frédéric Bibeau, Jean-Pierre Bleuse, Gilles Romieu, Pierre-Jean Lamy. Frequent BRCA1 promoter methylation and low 53BP1 status in sporadic triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1770. doi:10.1158/1538-7445.AM2013-1770