BRCA1 Promoter Research Articles

Abstract P1-06-06: Direct ex vivo observation of homologous recombination defect reversal after DNA damaging chemotherapy in metastatic breast cancer patients

Abstract Introduction Better predictive biomarkers for response to Poly ADP-Ribose inhibitors (PARPi) are required, since on the one hand evidence is emerging that PARPi are also effective beyond germline BRCA mutated (gBRCAm) cancers and on the other hand gBRCAm cancers can become resistant to PARPi. Therefore, we previously developed a functional homologous recombination (HR) assay exploiting the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh primary breast cancer (BrC) tissue (n=148): the REpair CAPacity (RECAP) test. The aim of the current study is to molecularly characterize real-time HR deficient (HRD) tumors and explore the utility of RECAP as a predictive biomarker for PARPi treatment in metastatic BrCs. Material and method Patients with advanced or recurrent BrC with easily accessible metastases were eligible. Fresh tissue biopsies from metastatic BrC lesions were collected in customized medium, irradiated with 5 Gy and cultured for 2 hours. Molecular characterization of functional HRD biopsies as well as platinum/PARP resistant biopsies was performed by targeted sequencing (BRCA1/2, TP53, CHEK2, PALB2), BRCA1 promoter methylation analysis and multiplex ligation-dependent probe amplification (MLPA) analysis of BRCA1 and BRCA2 to identify large rearrangements. Results 41 biopsies were derived from 38 patients with recurrent or metastatic BrC. The RECAP test had a high success rate (93%) when performed on core needle or punch biopsies, and test results were available within 1 week. HRD was detected in 13 out of 41 biopsies (32%), among which 5 were gBRCAm, indicating that the RECAP test identifies more patients who may benefit from PARPi treatment than gBRCA analysis only. Among the 8 non-gBRCAm HRD tumors was one tumor with a germline PALB2 mutation, one with BRCA1 promoter hypermethylation and two with somatic variants of unkown significance (VUSes) in BRCA2. In three gBRCAm patients BRCA reversion was detected, as the HRD tumors became HR proficient (HRP) after showing in vivo progressive disease (PD) on cisplatin/PARPi treatment. One of these patients obtained a secondary BRCA1 mutation that restored the open reading frame and led to production of full-length BRCA1 protein, while the causative molecular event in the other patients is still elusive. Conclusion The RECAP test is a robust and reproducible HRD test which identifies approximately 60% more potential candidates for PARPi treatment, as 40% of HRD tumors were caused by gBRCAm. Due to its functional character, the RECAP test reflects the real-time HR status regardless of BRCA mutational status and therefore detects HR reversal upon therapy resistance. Thus, RECAP shows great potential as a predictive biomarker for PARPi treatment of metastatic BrC. Citation Format: Meijer TG, Verkaik NS, Van Deurzen CH, Dubbink HJ, Den Toom TD, Dinjens WN, Kanaar R, Van Gent DC, Jager A. Direct ex vivo observation of homologous recombination defect reversal after DNA damaging chemotherapy in metastatic breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-06-06.

Abstract P3-11-06: HRD (homologous recombination deficiency) score and its clinicopathological features in neoadjuvant chemotherapy treated sporadic breast cancers

Abstract [Purpose]Homologous Recombination Deficiency (HRD) score has been developed to evaluate the DNA double-strand break repair function. BRCA1/2 germline mutation-associated breast cancers are known to show a high HRD score and a high sensitivity to platinum-based chemotherapy as well as PARP inhibitor. HRD can be theoretically induced by not only the dysfunction of BRA1/2 but also the dysfunction of the other molecules involved in homologous recombination, and, actually, high HRD score is reported to be seen in a significant proportion of sporadic breast tumors without BRCA1/2 mutation. The aim of the present study was to elucidate the clinicopathological characteristics of sporadic breast tumors with high HRD score as well as their sensitivity to sequential paclitaxel and FEC chemotherapy (P-FEC). [Methods]Tumor tissues obtained by Mammotome from 132 sporadic breast cancer patients (stage II-III) before neoadjuvant chemotherapy with P-FEC were subjected to assay for HRD score using Oncoscan CNV kit®. HRD score was a simple sum of NtAI, LOH, and LST (cutoff, 42), and were also subjected to the gene expression analysis using the Affymetrix microarray (U133 plus 2.0). BRCA1 promoter methylation was assayed by methylation specific real-time PCR. [Result]Of the 132 breast tumors, 32% showed a high HRD score which were significantly associated with high histological grade (P=0.001), negative progesterone receptor (P=0.023) and high Ki67 index (P=0.004). Triple negative breast cancer (TNBC)(n=19) showed a significantly (P<0.001) higher HRD score than the other subtypes (HR+/HER2-(n=73), HR+/HER2+(n=12), HR-/HER2+(n=18)). BRCA1 promoter methylation was significantly associated with a high HRD score. There was no significant correlation between HRD score and pCR to P-FEC when all tumors were considered but tumors with a high HRD score showed a significantly (P=0.020) lower pCR rate when only TNBC were considered. In TNBC, majority of tumors showed a high HRD score in five subtypes (BL1, BL2, IM, M, MSL) but no tumor showed a high HRD score in LAR (luminal androgen receptor) subtype. [Conclusion]Approximately one third of sporadic breast tumors show a high HRD score, indicating the presence of homologous recombination dysfunction, and they are most often seen in TNBC with BRCA1 promoter methylation and never in LAR subtype. Interestingly, breast tumors with a high HRD score were less sensitive to P-FEC. Citation Format: Imanishi S, Naoi Y, Shimazu K, Shimoda M, Kagara N, Tanei T, Miyake T, Kim SJ, Noguchi S. HRD (homologous recombination deficiency) score and its clinicopathological features in neoadjuvant chemotherapy treated sporadic breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-11-06.

Constitutive BRCA1 Promoter Hypermethylation Can Be a Predisposing Event in Isolated Early-Onset Breast Cancer.

Early age at onset of breast cancer (eoBC) is suggestive of an increased genetic risk. Although genetic testing is offered to all eoBC-affected women, in isolated cases the detection rate of pathogenic variants is <10%. This study aimed at assessing the role of constitutive promoter methylation at BC-associated loci as an underlying predisposing event in women with eoBC and negative family history. Promoter methylation at 12 loci was assessed by the MassARRAY technology in blood from 154 BRCA1/2 negative patients with eoBC and negative family history, and 60 healthy controls. Hypermethylation was determined, within each promoter, by comparing the patient’s mean methylation value with thresholds based on one-sided 95% bootstrap confidence interval of the controls’ mean. Three patients had hypermethylated results, two at BRCA1 and one at RAD51C. Analyses on tumor tissue from the patient exceeding the highest threshold at BRCA1 revealed a mean methylation >60% and loss of heterozygosity at chromosome 17q. The patient hypermethylated at RAD51C showed low methylation in the tumor sample, ruling out a role for methylation-induced silencing in tumor development. In isolated eoBC patients, BRCA1 constitutive promoter methylation may be a predisposing event. Further studies are required to define the impact of methylation changes occurring at BC-predisposing genes and their role in tumorigenesis.

Clinicopathological analysis of homologous recombination-deficient breast cancers with special reference to response to neoadjuvant paclitaxel followed by FEC.

This study aimed to elucidate the clinicopathological characteristics of breast tumors with homologous recombination deficiency (HRD) and the sensitivity to neoadjuvant paclitaxel followed by fluorouracil, epirubicin, and cyclophosphamide (P-FEC). Tumor biopsy samples obtained before P-FEC from 141 patients with stages II-III breast cancer including the luminal (n = 76), luminal-HER2 (n = 13), HER2 (n = 17), and triple-negative (TNBC, n = 35) subtypes were subjected to assay for HRD score using the OncoScan CNV FFPE Assay Kit. HRD score was a simple sum of NtAI, LOH, and LST (cutoff, 42). TNBCs were also subjected to the gene expression assay using the Affymetrix microarray (U133 plus 2.0) and to the BRCA1 promoter methylation assay using the methylation-specific real-time PCR. Of the 141 breast tumors, 45 samples (32%) had high HRD scores and were associated with high histological grade (P = 0.001), negative progesterone receptor (P = 0.018), high Ki67 index (P = 0.032), and BRCA1 promoter methylation (P = 3.6e-07). The proportion of tumors with high HRD scores was significantly higher in the TNBC subtype than the others (P = 0.006). In the TNBC subtype, but not the others, high HRD scores were significantly (P = 0.001) associated with a low pathological complete response rate to P-FEC. Among the molecular TNBC subtypes, a majority of tumors belonging to the basal-like 1, immunomodulatory, mesenchymal, mesenchymal stem-like, but not luminal androgen receptor (LAR), subtypes had high HRD scores. Approximately one-third of sporadic breast tumors show a high HRD score, indicating the presence of homologous recombination dysfunction, and they are characterized by biologically aggressive phenotypes, most commonly in the TNBC subtype, and less sensitive to P-FEC.

Combined Targeted Resequencing of Cytosine DNA Methylation and Mutations of DNA Repair Genes with Potential Use for Poly(ADP-Ribose) Polymerase 1 Inhibitor Sensitivity Testing

Current molecular tumor diagnostics encompass panel sequencing to detect mutations, copy number alterations, and rearrangements. However, tumor suppressor genes can also be inactivated by methylation within their promoter region. These epigenetic alterations are so far rarely assessed in the clinical setting. Therefore, we established the AllCap protocol facilitating the combined detection of mutations and DNA methylation at the coding and promoter regions of 342 DNA repair genes in one experiment. We demonstrate the use of the protocol by applying it to ovarian cancer cell lines with different responsiveness to poly(ADP-ribose) polymerase inhibition. BRCA1, ATM, ATR, and EP300 mutations and methylation of the BRCA1 promoter were detected as potential predictors for therapy response. The required amount of input DNA was optimized, and the application to formalin-fixed, paraffin-embedded tissue samples was verified to improve the clinical applicability. Thus, by adding DNA methylation values to panel resequencings, the AllCap assay will add another important level of information to clinical tests and will improve stratification of patients for systemic therapies.

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.

Genome-wide DNA methylation assessment of ‘BRCA1-like’ early-onset breast cancer: Data from the Australian Breast Cancer Family Registry

Breast cancers arising in women carrying a germline mutation in BRCA1 are typically high-grade, early-onset and have distinct morphological features (BRCA1-like). However, the majority of early-onset breast cancers of this morphological type are not associated with germline BRCA1 mutations or constitutional BRCA1 promoter methylation. We aimed to assess DNA methylation across the genome for associations with the “BRCA1-like” morphology. Genome-wide methylation in blood-derived DNA was measured using the Infinium HumanMethylation450K BeadChip assay for women under the age of 40 years participating in the Australian Breast Cancer Family Study (ABCFS) diagnosed with: i) BRCA1-like breast cancer (n = 30); and ii) breast cancer without BRCA1-like morphological features (non BRCA1-like; n = 30), and age-matched unaffected women (controls; n = 30). Corresponding tumour-derived DNA from 43 of the affected women was also assessed. Methylation of blood-derived DNA was found to be elevated across 17 consecutive marks in the BRCA1 promoter region and decreased at several other genomic regions (including TWIST2 and CTBP1) for 7 women (23%) diagnosed with BRCA1-like breast cancer compared with women in the other groups. Corresponding tumour-derived DNA available from 5 of these 7 women had elevated methylation within the BRCA1 and SPHK2 promoter region and decreased methylation within the ADAP1, IGF2BP3 and SPATA13 promoter region when compared with the other breast tumours. These methylation marks could be biomarkers of risk for BRCA1-like breast cancer, and could be responsible in part for their distinctive morphological features and biology. As such, they may assist with prevention and targeted therapies for this cancer subtype.

Methylation pattern and mutational status of BRCA1 in canine mammary tumors in a Brazilian population

In female dogs, mammary tumors are the most common neoplasia representing about 50% of the tumors affecting this species. In women, the importance of mutations in BRCA1 and mammary tumors development is well established. However, little information is available on the molecular mechanisms that contribute to canine mammary tumors. In this work, we evaluated the mutational and methylation status of the BRCA1 gene, in tumoral and non-tumoral tissues of canine mammary glands in order to characterize its influence in mammary carcinogenesis on this species. Samples of 16 animals were collected and two hotspot regions (intron 8-exon 9 and 5′UTR) were sequenced. For methylation analysis, the bisulfite sequencing PCR approach was used. No evidence of hypermethylation was observed in the BRCA1 promoter region, suggesting this mechanism may not be involved in BRCA1 silencing in canine mammary tumorigenesis. No alteration was observed in intron 8-exon 9 region. On the other hand, two polymorphisms in the 5′UTR region were observed: a transition (T > C) that has not been previously described in the literature, and observed in one patient with an unfavorable prognosis, and the previously described transversion (C > G). We suggest that methylation is not the main BRCA1 inactivation mechanism in sporadic CMTs. Regarding the genetic alterations, two variations were detected in our population, and we were able to detect regional allele frequency differences in our population.

A RAD51 assay feasible in routine tumor samples calls PARP inhibitor response beyond BRCA mutation

Poly(ADP‐ribose) polymerase (PARP) inhibitors (PARPi) are effective in cancers with defective homologous recombination DNA repair (HRR), including BRCA1/2‐related cancers. A test to identify additional HRR‐deficient tumors will help to extend their use in new indications. We evaluated the activity of the PARPi olaparib in patient‐derived tumor xenografts (PDXs) from breast cancer (BC) patients and investigated mechanisms of sensitivity through exome sequencing, BRCA1 promoter methylation analysis, and immunostaining of HRR proteins, including RAD51 nuclear foci. In an independent BC PDX panel, the predictive capacity of the RAD51 score and the homologous recombination deficiency (HRD) score were compared. To examine the clinical feasibility of the RAD51 assay, we scored archival breast tumor samples, including PALB2‐related hereditary cancers. The RAD51 score was highly discriminative of PARPi sensitivity versus PARPi resistance in BC PDXs and outperformed the genomic test. In clinical samples, all PALB2‐related tumors were classified as HRR‐deficient by the RAD51 score. The functional biomarker RAD51 enables the identification of PARPi‐sensitive BC and broadens the population who may benefit from this therapy beyond BRCA1/2‐related cancers.

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.

Evaluation of the BRCAness phenotype and its correlations with clinicopathological features in triple-negative breast cancers

Some sporadic triple-negative breast cancers (TNBCs) share similar clinicopathological and molecular characteristics to BRCA1/2-mutant breast cancers, a phenotype described as "BRCAness." Identifying BRCAness in TNBCs could expand the target group for platinum salts and poly(adenosine diphosphate-ribose) polymerase inhibitors. The aims of our study were to assess the clinical validity of BRCA1/2 promoter methylation and BRCA1-like genomic profile to identify BRCAness and to evaluate its correlations with clinicopathological features in TNBCs. Formalin-fixed, paraffin-embedded tissues and fresh tissues of 151 primary invasive TNBCs were collected. BRCA1/2 promoter methylation and BRCA1-like genomic profile were detected using methylation-specific multiplex ligation-dependent probe amplification and multiplex ligation-dependent probe amplification assay, respectively. BRCA1/2 messenger RNA expression was evaluated by quantitative reverse-transcription polymerase chain reaction. Of the 151 patients, 38 (25.2%) showed BRCA1 promoter methylation. Of the 124, 52 (41.9%) had a BRCA1-like multiplex ligation-dependent probe amplification profile. The frequency of BRCAness phenotype was 54.8% (68/124). BRCA1 germline mutation and BRCA1 promoter methylation were mutually exclusive (P = .002). The BRCAness phenotype was significantly associated with large tumor size (>2 cm, P = .009), positive lymph nodes (P = .008), grade 3 tumor (P = .0001), high Ki-67 levels (P = .001), and basal-like breast cancers (P = .0001). Combined detections of BRCA1 promoter methylation and BRCA1-like genomic profile could identify more BRCAness cases in TNBCs. BRCA1 promoter methylation might rule out BRCA1 germline mutation in TNBCs. BRCAness was associated with aggressive clinicopathological features. These findings might have clinical values in hereditary breast cancer screening and target treatment of TNBCs.

BRCA1 and BRCA2 5' noncoding region variants identified in breast cancer patients alter promoter activity and protein binding.

The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early‐onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5′ noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency < 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.‐287C>T and PAX5 binding to BRCA2:c.‐296C>T. Clinical classification of variants affecting promoter activity, using existing prediction models, found no evidence to suggest that these variants confer a high risk of disease. Further studies are required to determine if such variation may be associated with a moderate or low risk of BC.

A Dominantly Inherited 5′ UTR Variant Causing Methylation-Associated Silencing of BRCA1 as a Cause of Breast and Ovarian Cancer

Pathogenic variants in BRCA1 or BRCA2 are identified in ∼20% of families with multiple individuals affected by early-onset breast and/or ovarian cancer. Extensive searches for additional highly penetrant genes or alternative mutational mechanisms altering BRCA1 or BRCA2 have not explained the missing heritability. Here, we report a dominantly inherited 5′ UTR variant associated with epigenetic BRCA1 silencing due to promoter hypermethylation in two families affected by breast and ovarian cancer. BRCA1 promoter methylation of ten CpG dinucleotides in families who are affected by breast and/or ovarian cancer but do not have germline BRCA1 or BRCA2 pathogenic variants was assessed by pyrosequencing and clonal bisulfite sequencing. RNA and DNA sequencing of BRCA1 from lymphocytes was undertaken to establish allelic expression and the presence of germline variants. BRCA1 promoter hypermethylation was identified in 2 of 49 families in which multiple women are affected by grade 3 breast cancer or high-grade serous ovarian cancer. Soma-wide BRCA1 promoter hypermethylation was confirmed in blood, buccal mucosa, and hair follicles. Pyrosequencing showed that DNA was ∼50% methylated, consistent with the silencing of one allele, which was confirmed by clonal bisulfite sequencing. RNA sequencing revealed the allelic loss of BRCA1 expression in both families and that this loss of expression segregated with the heterozygous variant c.−107A>T in the BRCA1 5′ UTR. Our results establish a mechanism whereby familial breast and ovarian cancer is caused by an in cis 5′ UTR variant associated with epigenetic silencing of the BRCA1 promoter in two independent families. We propose that methylation analyses be undertaken to establish the frequency of this mechanism in families affected by early-onset breast and/or ovarian cancer without a BRCA1 or BRCA2 pathogenic variant.

Methylation of BRCA1 and MGMT genes in white blood cells are transmitted from mothers to daughters

BackgroundConstitutive methylation of tumor suppressor genes are associated with increased cancer risk. However, to date, the question of epimutational transmission of these genes remains unresolved. Here, we studied the potential transmission of BRCA1 and MGMT promoter methylations in mother-newborn pairs.MethodsA total of 1014 female subjects (cancer-free women, n = 268; delivering women, n = 295; newborn females, n = 302; breast cancer patients, n = 67; ovarian cancer patients, n = 82) were screened for methylation status in white blood cells (WBC) using methylation-specific PCR and bisulfite pyrosequencing assays. In addition, BRCA1 gene expression levels were analyzed by quantitative real-time PCR.ResultsWe found similar methylation frequencies in newborn and adults for both BRCA1 (9.9 and 9.3%) and MGMT (12.3 and 13.1%). Of the 290 mother-newborn pairs analyzed for promoter methylation, 20 mothers were found to be positive for BRCA1 and 29 for MGMT. Four mother-newborn pairs were positive for methylated BRCA1 (20%) and nine pairs were positive for methylated MGMT (31%). Intriguingly, the delivering women had 26% lower BRCA1 and MGMT methylation frequencies than those of the cancer-free female subjects. BRCA1 was downregulated in both cancer-free woman carriers and breast cancer patients but not in newborn carriers. There was a statistically significant association between the MGMT promoter methylation and late-onset breast cancers.ConclusionsOur study demonstrates that BRCA1and MGMT epimutations are present from the early life of the carriers. We show the transmission of BRCA1 and MGMT epimutations from mother to daughter. Our data also point at the possible demethylation of BRCA1and MGMT during pregnancy.

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&amp;lt;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.

Abstract 1385: Galangin is an epigenetic modulator or BRCA1 and induces estrogen receptor alpha in triple negative breast cancer cells

Abstract The aryl hydrocarbon receptor (AhR) is a driver of breast cancer progression and its overexpression is often observed in triple negative breast cancer (TNBC). Activation of AhR results in epigenetic silencing of the breast cancer 1 gene (BRCA1), a necessary factor in the transcriptional activation of estrogen receptor-alpha (ERα). The purpose of this study was to examine the capacity for galangin (GAL; 3,5,7-trihydroxy-2-phenylchromen-4-one), a natural AhR antagonist, to mimic the effects of synthetic AhR antagonists alpha-naphthoflavone (αNF, 2-phenylbenzo[h]chromen-4-one) and 3'-methoxy-4'-nitroflavone (MNF; 2-(3-methoxy-4-nitrophenyl)chromen-4-one) in regards to counteracting epigenetic silencing of BRCA1 and restoring ERα protein expression. We utilized TNBC cells with hypermethylated BRCA1 and constitutively active AhR (HCC38 cells) for these experiments. After 72h in phenol-red free media, cells were treated for up to 72h against a range of GAL, αNF, and MNF concentrations in the presence or absence of estradiol (E2). Transcript levels of AhR and its downstream target genes CYP1A1 and CYP1B1 were determined by real-time polymerase chain reaction (RT-PCR). Protein levels of BRCA1, ERα, and AhR were determined by Western blot. Treatment with GAL results in stabilization of BRCA1 and ERα protein levels, similarly to αNF and MNF. To determine potential mechanisms of these observations, we are currently conducting experiments to analyze changes in BRCA1 promoter methylation using methylation-specific RT-PCR (MSP) and DNA methyltransferase 1 (DNMT1) protein levels by Western blot. These data suggest the naturally occurring flavanoid GAL may be an effective compound for modulating epigenetic repression of BRCA1 and restoring ERα levels, which may provide options for anti-hormonal therapy in ERα-negative breast cancers. Citation Format: Micah G. Donovan, Ornella I. Selmin, Donato F. Romagnolo. Galangin is an epigenetic modulator or BRCA1 and induces estrogen receptor alpha in triple negative breast cancer cells [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 1385.

Validation and Implementation of BRCA1/2 Variant Screening in Ovarian Tumor Tissue

BRCA1/2 variant analysis in tumor tissue could streamline the referral of patients with epithelial ovarian, fallopian tube, or primary peritoneal cancer to genetic counselors and select patients who benefit most from targeted treatment. We investigated the sensitivity of BRCA1/2 variant analysis in formalin-fixed, paraffin-embedded tumor tissue using a combination of next-generation sequencing and copy number variant multiplex ligation-dependent probe amplification. After optimization using a training cohort of known BRCA1/2 mutation carriers, validation was performed in a prospective cohort in which screening of BRCA1/2 tumor DNA and leukocyte germline DNA was performed in parallel. BRCA1 promoter hypermethylation and pedigree analysis were also performed. In the training cohort, 45 of 46 germline BRCA1/2 variants were detected (sensitivity, 98%). In the prospective cohort (n=62), all six germline variants were identified (sensitivity, 100%), together with five somatic BRCA1/2 variants and eight cases with BRCA1 promoter hypermethylation. In four BRCA1/2 variant-negative patients, surveillance or prophylactic management options were offered on the basis of positive family histories. We conclude that BRCA1/2 formalin-fixed, paraffin-embedded tumor tissue analysis reliably detects BRCA1/2 variants. When taking family history of BRCA1/2 variant-negative patients into account, tumor BRCA1/2 variant screening allows more efficient selection of epithelial ovarian cancer patients for genetic counseling and simultaneously selects patients who benefit most from targeted treatment.

Prognostic value of androgen receptor and FOXA1 co-expression in non-metastatic triple negative breast cancer and correlation with other biomarkers

BackgroundIn luminal androgen receptor (AR) tumours, FOXA1 may direct AR to sites occupied by ER in luminal tumours, thus stimulating proliferation.MethodsAR and FOXA1 expression were evaluated by immunohistochemistry in 333 non-metastatic triple-negative breast cancers (TNBC). Positivity threshold was set at ≥ 1% staining. Lymphocytic infiltration, PD-L1expression, PIK3CA mutations, PTEN defects and BRCA1 promoter methylation were assessed.ResultsAR + /FOXA1 + tumours (42.4%) were more frequently: found in older patients, lobular, of lower nuclear grade, with more frequently PIK3CA mutations; exhibited less frequently BRCA1 promoter methylation, defects of PTEN and PD-L1 expression than others. Recurrence-free and overall survivals were significantly lower for AR + /FOXA1 + TNBC (median follow-up: 7.8 years).ConclusionsAR + /FOXA1 + expression defines a luminal-like TNBC subgroup affected with a worse outcome compared to other TNBC and a higher risk of late recurrences. This subgroup appears enriched in PIK3CA mutations, suggesting a role for PI3K inhibitors in this subgroup.

Genetic dissection of the BRCA2 promoter and transcriptional impact of DNA variants.

Promoter mutations may affect transcription and can be associated with human diseases. However, the promoters of the breast cancer (BC) genes are not regularly screened. Our goal was to investigate the BRCA2 promoter in order to study a possible correlation between impaired transcription and disease. The proximal and core promoter of the BRCA2 gene was sequenced in 95 high-risk BC patients. A BRCA2-promoter insert [-938 to + 312 from the transcription start site (TSS)] was generated and cloned into the firefly luciferase vector pGL4.10. Promoter variants and deletions were introduced by site-directed mutagenesis and quantified by Dual-Luciferase assays and semi-quantitative RT-PCR. Three different variants were detected in high-risk BC patients: rs3092989, rs206118, and rs563971900. Functional mapping of 13 overlapping deletions revealed four down-regulating segments (TSS positions): -59_-10del/µdel3 (16% of activity of the wild-type construct), -104_-55del/µdel4 (62%), -239_-190del/µdel7 (39%), -464_-415/µdel12 (78%), suggesting the presence therein of putative transcriptional activator motifs. Additionally, six microdeletions rendered luciferase overexpression: +32_+81del/µdel1 (356%), -14_+36del/µdel2 (180%), -194_-145del/µdel6 (154%), -284_-235del/µdel8 (168%), -329_-280del/µdel9 (111%), and -509_-460del/µdel13 (139%), which is indicative of repressor elements. Functional assays of 15 promoter variants (including those detected in patients) showed that ten of them significantly altered expression with seven up-regulating (113-163%) and three down-regulating (rs551887850_G, rs570548398_T, rs55880202_T; 72-83%) SNPs. Eight of them were located in an ENCODE-DNase Hypersensitive Cluster (TSS - 185 to + 105) where most active transcriptional motifs are known to be placed. BRCA2 expression is highly sensitive to promoter variations as most of them induced relevant changes. Moreover, we mapped critical regions of the BRCA2 promoter that may constitute potential targets for regulatory variants. Three SNPs moderately decreased luciferase activity, but confirmation of its potential pathogenicity requires further analysis. These data reinforce the need to screen the promoter regions of breast cancer genes with a view to discovering novel deleterious mutations.