BRCA1 Promoter Methylation Research Articles

Molecular and clinical determinants of response and resistance to rucaparib for recurrent ovarian cancer treatment in ARIEL2 (Parts 1 and 2)

ARIEL2 (NCT01891344) is a single-arm, open-label phase 2 study of the PARP inhibitor (PARPi) rucaparib in relapsed high-grade ovarian carcinoma. In this post hoc exploratory biomarker analysis of pre- and post-platinum ARIEL2 samples, RAD51C and RAD51D mutations and high-level BRCA1 promoter methylation predict response to rucaparib, similar to BRCA1/BRCA2 mutations. BRCA1 methylation loss may be a major cross-resistance mechanism to platinum and PARPi. Genomic scars associated with homologous recombination deficiency are irreversible, persisting even as platinum resistance develops, and therefore are predictive of rucaparib response only in platinum-sensitive disease. The RAS, AKT, and cell cycle pathways may be additional modulators of PARPi sensitivity.

9P BRCA1 promoter methylation confers a more favorable prognosis to systemically untreated young triple-negative breast cancer patients than tumour BRCA1 mutation

The prognoses of systemically treated, triple-negative breast cancer (TNBC) patients with a pathogenic tumor BRCA1 mutation (tBRCA1m) or BRCA1 promoter methylation (BRCA1 PM) have been widely studied. However, the prognosis for systemically untreated women remains unknown. This study investigates the prognosis of systemically untreated young N0 TNBC patients according to tumor BRCA1 status.

Examination of ATM, BRCA1, and BRCA2 promoter methylation in patients with pancreatic cancer

BackgroundPancreatic cancer is a lethal disease with a poor 5-year survival rate. Pathogenic germline variants in the coding regions of ATM, BRCA1, and BRCA2 are found in up to 4.8% of pancreatic cancer patients. Germline promoter methylation and gene silencing arising from a germline variant or through other mechanisms have been described as a cause of tumor suppressor gene inactivation. MethodsWe measured the level of promoter methylation of the ATM, BRCA1, and BRCA2 genes in peripheral blood lymphocytes from 655 patients with pancreatic cancer using real-time PCR. ResultsNo evidence of germline promoter methylation of any of these genes was found. Promoter methylation levels were minimal with no patient having promoter methylation greater than 3.4%, 3.3%, and 7.6% for ATM, BRCA1 and BRCA2, respectively, well below levels found in patients who have inherited promoter methylation (∼50%). ConclusionsWe found no evidence of germline promoter methylation for the pancreatic susceptibility genes ATM, BRCA1 and BRCA2 in patients with pancreatic cancer. This study reveals that constitutive germline methylation of promoter CpG islands is rare in pancreatic cancer.

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.

Modification of Homologous Recombination Deficiency Score Threshold and Association with Long-Term Survival in Epithelial Ovarian Cancer.

Simple SummaryKnowledge of ovarian cancer molecular characteristics is increasingly crucial to individualizing and optimizing treatment strategies for this heterogeneous disease. Molecular features such as germline or somatic mutations, homologous recombination deficiency (HRD) status, microsatellite instability, and tumor mutational burden are associated with increased susceptibility to poly-ADP ribose polymerase inhibitors (PARPi) or immunotherapy. Our aim was to characterize these molecular features among ovarian cancer patients and determine their association with survival. Two different HRD score thresholds were evaluated: one currently used in clinics (≥42) and another proposed as a new threshold (≥33). An HRD score ≥33 was associated with improved overall survival in ovarian cancer. As HRD assays are increasingly used for treatment planning, future studies should evaluate an HRD score threshold of ≥33 compared to the currently used threshold of ≥42 for PARPi use.New therapies, such as poly-ADP ribose polymerase inhibitors (PARPi), and immunotherapy treatments have generated great interest in enhancing individualized molecular profiling of epithelial ovarian cancer (EOC) to improve management of the disease. In EOC patients, putative biomarkers for homologous recombination deficiency (HRD), microsatellite instability (MSI), and tumor mutational burden (TMB) were characterized and correlated with survival outcomes. A series of 300 consecutive EOC patients were enrolled. Patients underwent neoadjuvant chemotherapy (n = 172) or primary cytoreductive surgery (n = 128). Molecular profiling and survival analyses were restricted to the primary cytoreductive surgery cohort due to tissue availability. All patients underwent germline testing for HRD- and MSI-related gene mutations. When sufficient tissue was available, screening for somatic BRCA1/2 mutations, BRCA1 promoter methylation, HRD score (a measure of genomic instability), MSI, and TMB testing were performed. HRD score ≥33 was associated with improved overall survival on multivariable analysis. In the era of biomarker-driven clinical care, HRD score ≥33 may be a useful adjunctive prognostic tool and should be evaluated in future studies to predict PARPi benefits.

Clinicopathological Correlates of γδ T Cell Infiltration in Triple-Negative Breast Cancer.

Simple SummaryThe prognostic impact of the different tumor-infiltrating lymphocyte (TIL) subpopulations remains debated in solid cancers. We investigated the clinicopathological correlates and prognostic impact of TILs, particularly of γδ T cells, in 162 triple-negative breast cancer (TNBC) patients. A high γδ T cell density was significantly associated with younger age, higher tumor histological grade, adjuvant chemotherapy, BRCA1 promoter methylation, TIL density, and PD-L1 and PD-1 expression. In multivariate analyses, γδ T cell infiltration was an independent prognostic factor. However, this prognostic impact varied according to the tumor PIK3CA mutational status. High γδ T cell infiltration was associated with better survival in patients with PIK3CA wild-type tumors, without significant difference in the PIK3CA-mutated tumor subgroup. Altogether, these data suggest that high γδ T cell infiltrate is correlated with immune infiltration and might represent a prognostic tool in TNBC patients.The prognostic impact of the different tumor-infiltrating lymphocyte (TIL) subpopulations in solid cancers is still debated. Here, we investigated the clinicopathological correlates and prognostic impact of TILs, particularly of γδ T cells, in 162 patients with triple-negative breast cancer (TNBC). A high γδ T cell density (>6.625 γδ T cells/mm2) was associated with younger age (p = 0.008), higher tumor histological grade (p = 0.002), adjuvant chemotherapy (p = 0.010), BRCA1 promoter methylation (p = 0.010), TIL density (p < 0.001), and PD-L1 (p < 0.001) and PD-1 expression (p = 0.040). In multivariate analyses, γδ T cell infiltration (cutoff = 6.625 γδ T cells/mm2) was an independent prognostic factor (5-year relapse-free survival: 63.3% vs. 89.8%, p = 0.027; 5-year overall survival: 73.8% vs. 89.9%, p = 0.031, for low vs. high infiltration). This prognostic impact varied according to the tumor PIK3CA mutational status. High γδ T cell infiltration was associated with better survival in patients with PIK3CA wild-type tumors, but the difference was not significant in the subgroup with PIK3CA-mutated tumors. Altogether, these data suggest that high γδ T cell infiltrate is correlated with immune infiltration and might represent a candidate prognostic tool in patients with TNBC.

Peripheral blood BRCA1 methylation profiling to predict familial ovarian cancer.

ObjectiveFamilial cancer appears at a young age and its incidence is increasing. About 12% of familial ovarian cancer cases are associated with BRCA1/2 mutations (BRCAm). In this study, we investigated BRCA1 methylation may predict ovarian cancer in those with a family history of cancer (FHC) but without BRCA1/2 mutations (BRCAwt).MethodsUsing peripheral blood DNA from 55 subjects without a history of cancer [cancer(−)] and 52 ovarian cancer patients, we examined BRCA1 promoter methylation through bisulfite sequencing of the promoter and expressed the results as the cumulative methylation index. Then, we evaluated the BRCA1 promoter methylation according to BRCA1/2 germline mutations.ResultsBRCA1 methylation was more prevalent in the BRCAm cancer(−) group than in the BRCAwt cancer(−) group and ovarian cancer patients (p=0.031 and p=0.019, respectively). In the BRCAwt cancer(−) group, BRCA1 methylation was more prevalent in those with an FHC than in those without one and in the BRCAm cancer(−) group with an FHC (p=0.001 and p<0.001, respectively).ConclusionOur data suggest a predictive role of BRCA1 methylation profile for ovarian cancer in those without a history of cancer but with an FHC. BRCA1 methylation has important implications for diagnostic and predictive testing of those with BRCAwt cancer(−) status with FHC.

DNA Methylation in Ovarian Cancer Susceptibility.

Simple SummaryIt is well established that ovarian cancer “runs in families”, where ovarian and other cancers (commonly breast cancer) occur at early ages at onset and in multiple generations. After decades of genetic studies, rare high-risk genetic mutations in cancer susceptibility genes and over 40 common genetic variants with much smaller risks have been identified. However, based on familial studies, we know that additional heritable genetic risk factors exist. It is possible that epigenetic variation—differences in how DNA is read, and which genes are actively expressed (or not) —also contributes to ovarian cancer susceptibility. This review summarizes the current collection of epidemiological studies that have investigated the role of DNA methylation—one type of epigenetic mechanism—in the risk of ovarian cancer.Epigenetic alterations are somatically acquired over the lifetime and during neoplastic transformation but may also be inherited as widespread ‘constitutional’ alterations in normal tissues that can cause cancer predisposition. Epithelial ovarian cancer (EOC) has an established genetic susceptibility and mounting epidemiological evidence demonstrates that DNA methylation (DNAm) intermediates as well as independently contributes to risk. Targeted studies of known EOC susceptibility genes (CSGs) indicate rare, constitutional BRCA1 promoter methylation increases familial and sporadic EOC risk. Blood-based epigenome-wide association studies (EWAS) for EOC have detected a total of 2846 differentially methylated probes (DMPs) with 71 genes replicated across studies despite significant heterogeneity. While EWAS detect both symptomatic and etiologic DMPs, adjustments and analytic techniques may enrich risk associations, as evidenced by the detection of dysregulated methylation of BNC2—a known CSG identified by genome-wide associations studies (GWAS). Integrative genetic–epigenetic approaches have mapped methylation quantitative trait loci (meQTL) to EOC risk, revealing DNAm variations that are associated with nine GWAS loci and, further, one novel risk locus. Increasing efforts to mapping epigenome variation across populations and cell types will be key to decoding both the genomic and epigenomic causal pathways to EOC.

BRCA Methylation Testing Identifies a Subset of Ovarian Carcinomas without Germline Variants That Can Benefit from PARP Inhibitor.

Homologous Recombination Deficiency (HRD) is a frequent feature of high-grade epithelial ovarian carcinoma (EOC), associated with sensitivity to PARP-inhibitors (PARPi). The best characterized causes of HRD in EOCs are germline or somatic mutations in BRCA1 and BRCA2 genes. Although promoter methylation is a well-known mechanism of gene transcriptional repression, few data have been published about BRCA gene methylation in EOCs. In this retrospective study, we quantitatively analyzed by pyrosequencing a selected series of 90 formalin-fixed (FFPE) primary EOCs without BRCA germline mutations. We identified 20/88 (22.7%) EOCs showing BRCA promoter methylation, including 17/88 (19.3%) in BRCA1 and 4/86 (4.6%) in BRCA2 promoters, one of which showing concomitant BRCA1 methylation. Mean methylation levels were 49.6% and 45.8% for BRCA1 and BRCA2, respectively, with methylation levels ≥50% in 10/20 methylated EOCs. Constitutive BRCA methylation was excluded by testing blood-derived DNA. In conclusion, pyrosequencing methylation analysis of BRCA genes is a robust, quantitative and sensitive assay applicable to FFPE samples. Remarkably, a considerable subset of germline BRCA-negative EOCs showed somatic methylation and, likely, HRD. A subpopulation of women with BRCA methylation, even without BRCA mutations, could potentially benefit from PARP-inhibitors; further clinical studies are needed to clarify the predictive role of somatic BRCA methylation of PARP-therapy response.

Assessment of tumor suppressor promoter methylation in healthy individuals

BackgroundThe number of tumor suppressor genes for which germline mutations have been linked to cancer risk is steadily increasing. However, while recent reports have linked constitutional normal tissue promoter methylation of BRCA1 and MLH1 to ovarian and colon cancer risk, the role of epigenetic alterations as cancer risk factors remains largely unknown, presenting an important area for future research. Currently, we lack fast and sensitive methods for assessment of promoter methylation status across known tumor suppressor genes.ResultsIn this paper, we present a novel NGS-based approach assessing promoter methylation status across a large panel of defined tumor suppressor genes to base-pair resolution. The method omits the limitations related to commonly used array-approaches. Our panel includes 565 target regions covering the promoters of 283 defined tumor suppressors, selected by pre-specified criteria, and was applied for rapid targeted methylation-specific NGS. The feasibility of the method was assessed by analyzing normal tissue DNA (white blood cells, WBC) samples from 34 healthy postmenopausal women and by performing preliminary assessment of the methylation landscape of tumor suppressors in these individuals. The mean target coverage was 189.6x providing a sensitivity of 0.53%, sufficient for promoter methylation assessment of low-level methylated genes like BRCA1. Within this limited test-set, we detected 206 regions located in the promoters of 149 genes to be differentially methylated (hyper- or hypo-) at > 99% confidence level. Seven target regions in gene promoters (CIITA, RASSF1, CHN1, PDCD1LG2, GSTP1, XPA, and ZNF668) were found to be hyper-methylated in a minority of individuals, with a > 20 percent point difference in mean methylation across the region between individuals. In an exploratory hierarchical clustering analysis, we found that the individuals analyzed may be grouped into two main groups based on their WBC methylation profile across the 283 tumor suppressor gene promoters.ConclusionsMethylation-specific NGS of our tumor suppressor panel, with detailed assessment of differential methylation in healthy individuals, presents a feasible method for identification of novel epigenetic risk factors for cancer.

Promoter Hypermethylation and Expression Changes of BRCA1 Gene in a Cohort of Sporadic Breast Cancer Cases among Pakistani Population.

Objective:The purpose of our study was to determine the frequency of BRCA1 promoter hypermethylation and its association with expression changes of BRCA1 and main morphological features in sporadic breast cancer. Methods:A retrospective review of cases was performed to select those with specific morphological features suggestive of breast cancer. BRCA1 promoter hypermethylation and changes in protein expression were evaluated in 30 cancerous and 30 non-cancerous tissue samples. A tissue microarray containing samples from normal and tumor tissue was prepared and stained for BRCA1 protein expression using a commercially available monoclonal antibody against BRCA1 (Ab-1) clone MS110 (mAb). DNA was extracted using modified protocol of Qiagen minikit. DNA was modified using a Bisulfite conversion kit and BRCA1 hypermethylation was detected using a methylation specific PCR. Results:Promoter hypermethylation was negative in 30 non-cancerous samples with retained BRCA1 protein expression. Methylation was positive in 82.6% (n=19/23) of the sporadic cancer samples that had loss of BRCA1 expression and 50% (n=2/4) of the samples with equivocal protein expression. Methylation was negative in all the sporadic breast cancer samples (n=3/3) with retained protein expression. Chi-square analysis showed significant association of BRCA1 promoter methylation with decreased protein expression (P=0.016) and co-existence of loss of BRCA1 and Her2neu at chromosome 17 (P=0.026) respectively. There was no significant association of BRCA1 methylation with morphological features excluding necrosis (P=0.035). Promoter hypermethylation was found to be most common (68.75%) among Triple Negative Breast Cancer (TNBC) females less than 45 years old. Conclusion:Our study suggests that BRCA1 promoter hypermethylation has significant contribution in sporadic breast carcinogenesis. This was our preliminary study in Pakistan. Further studies aimed to determine the in-depth mechanisms of BRCA1 epigenetics in TNBC. BRCAness enriched phenotype in TNBC might be used as a biomarker for the exploitation of therapeutic and clinical implications.

Abstract IA02: DNA repair gene promoter methylation patterns adapt and influence PARP inhibitor response

Abstract PARP inhibitor (PARPi) resistance in high-grade serous ovarian carcinoma (HGSOC) can be acquired as a result of restored homologous recombination (HR) due to secondary or reversion mutations in HR genes, such as BRCA1, BRCA2, and RAD51C, or due to loss of BRCA1 promoter methylation (meBRCA1). We have demonstrated that homozygous meBRCA1 can be lost or reverted to heterozygous methylation following treatment with platinum-based chemotherapy, resulting in HR-competent PARPi-resistant tumors. RAD51C promoter methylation (meRAD51C) is detected in approximately 2% of HGSOC cases and, as with meBRCA1, is associated with gene silencing and HR deficiency. We are exploring PARPi response in meRAD51C preclinical models to determine clinical relevance. Two patient-derived xenograft (PDX) models of HGSOC with RAD51C gene silencing caused by meRAD51C have distinct meRAD51C profiles (measured by methylation-specific high-resolution melt analysis and targeted bisulfite next-generation sequencing), and different responses to PARPi treatment pressure. PDX PH039 loses methylation and regains RAD51C expression after only 2 cycles of PARPi retreatment (niraparib), resulting in PARPi-refractory tumors by cycle 3-4. Illumina EPIC methylation array analysis of PH039 revealed increasing global methylation losses following each round of PARPi treatment. Lack of meRAD51C stability and rapid development of PARPi resistance in PH039 may be due to the high degree of meRAD51C heterogeneity within the tumor favoring selection of pre-existing HR-competent clones under PARPi pressure. In contrast, PDX 183 has a relatively homogeneous and stable meRAD51C profile. We have multiple examples of using unique PDX models to demonstrate various important features of PARPi resistance, including loss of promoter methylation for BRCA1 vs. RAD51C. Thus, meRAD51C confers response to PARPi in HGSOC, but PARPi treatment pressure can cause loss of methylation and drug resistance in some tumors. The contrasting PARPi responses of these PDX provide a platform for the study of meRAD51C stability in vivo and may present therapeutic opportunities to improve meRAD51C durability and PARPi responses in patients. Citation Format: Kasenija Nesic, Rachel M Hurley, Cordelia McGehee, Olga Kondrashova, Maria I. Harrell, Giada V. Zapparoli, Ashan Musafer, Ming E. Wong, John Weroha, Xiaonan Hou, Hu Li, Vivian Negron, Kevin Peterson, Paula Schneider, Elizabeth M. Swisher, Melissa Southey, Alexander Dobrovic, Matthew Wakefield, Scott H. Kaufmann, Clare L. Scott. DNA repair gene promoter methylation patterns adapt and influence PARP inhibitor response [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA02.

Abstract A33: Loss of ZC3H18 disrupts homologous recombination repair and sensitizes ovarian cancer cells to PARP inhibitors and DNA cross-linking agents

Abstract About 50% of high-grade serous ovarian cancers (HGSOC) have defects in homologous recombination (HR) DNA repair. Intriguingly, HR-defective tumors are highly susceptible to poly(ADP-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapy. Mutations in BRCA1/2 or epigenetic silencing of BRCA1 via promoter hypermethylation contributes to ~30% of the HR defects, but other genetic alterations that cause HR deficiency in HGSOC remain unclear. To identify novel genes that may regulate HR, we studied Zinc Finger CCCH-Type Containing 18 (ZC3H18), encoded by ZC3H18. Notably, ZC3H18 lies within 16q24.2, a region with frequent copy number loss in HGSOC. Here we demonstrate that BRCA1 is transcriptionally activated by a previously unknown function of ZC3H18. We show that ZC3H18 interacts with an E2F site in the BRCA1 promoter where it facilitates recruitment of E2F4 to an adjacent E2F site to promote BRCA1 transcription. Consistent with the role of ZC3H18 in promoting BRCA1 expression, ZC3H18 depletion induces BRCA1 promoter methylation, reduces BRCA1 expression, disrupts HR, and sensitizes ovarian cancer cells to PARP inhibitors and platinum agents. Moreover, in patient-derived xenografts and primary HGSOC tumors, ZC3H18 mRNA levels were positively correlated with BRCA1 mRNA levels, further supporting the role of ZC3H18 in regulating BRCA1. Together, these findings suggest that loss and/or reduced ZC3H18 expression may help identify HGSOC patients most likely to benefit from PARP inhibitor and platinum-based therapies. Summary of the Unpublished Data/Significance/Innovation: These studies are innovative on several fronts. First, although BRCA1 methylation is a frequent event that mediates tumorigenesis and therapeutic responses in HGSOC, how BRCA1 methylation is regulated remains unknown. These studies will be the first to provide a mechanistic basis for how BRCA1 is methylated in HGSOC. Second, our studies are the first to show a role for ZC3H18 in any cancer. Third, we show that ZC3H18 regulates HR and that its depletion sensitizes ovarian cancer cells to PARP inhibitors and platinum therapy. Thus, these studies will provide important basis for developing ZC3H18 loss/decreased abundance as a novel biomarker for ovarian cancers that may respond to PARP inhibitors and platinum agents. Citation Format: Arun Kanakkanthara, Catherine J. Huntoon, Xiaonan Hou, Minzhi Zhang, Ethan P Heinzen, Ann L. Oberg, John S. Weroha, Scott H. Kaufmann, Larry M. Karnitz. Loss of ZC3H18 disrupts homologous recombination repair and sensitizes ovarian cancer cells to PARP inhibitors and DNA cross-linking agents [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A33.

BRCA1 Promoter Methylation and Clinical Outcomes in Ovarian Cancer: An Individual Patient Data Meta-Analysis.

BRCA1 methylation has been associated with homologous recombination deficiency, a biomarker of platinum sensitivity. Studies evaluating BRCA1-methylated tubal and ovarian cancer (OC) do not consistently support improved survival following platinum chemotherapy. We examine the characteristics of BRCA1-methylated OC in a meta-analysis of individual participant data. Data of 2636 participants across 15 studies were analyzed. BRCA1-methylated tumors were defined according to their original study. Associations between BRCA1 methylation and clinicopathological characteristics were evaluated. The effects of methylation on overall survival (OS) and progression-free survival (PFS) were examined using mixed-effects models. All statistical tests were 2-sided. 430 (16.3%) tumors were BRCA1-methylated. BRCA1 methylation was associated with younger age and advanced-stage, high-grade serous OC. There were no survival differences between BRCA1-methylated and non-BRCA1-methylated OC (median PFS = 20.0 vs 18.5 months, hazard ratio [HR] = 1.01, 95% CI = 0.87 to 1.16; P = .98; median OS = 46.6 vs 48.0 months, HR = 1.02, 95% CI = 0.87 to 1.18; P = .96). Where BRCA1/2 mutations were evaluated (n = 1248), BRCA1 methylation displayed no survival advantage over BRCA1/2-intact (BRCA1/2 wild-type non-BRCA1-methylated) OC. Studies used different methods to define BRCA1 methylation. Where BRCA1 methylation was determined using methylation-specific polymerase chain reaction and gel electrophoresis (n = 834), it was associated with improved survival (PFS: HR = 0.80, 95% CI = 0.66 to 0.97; P = .02; OS: HR = 0.80, 95% CI = 0.63 to 1.00; P = .05) on mixed-effects modeling. BRCA1-methylated OC displays similar clinicopathological features to BRCA1-mutated OC but is not associated with survival. Heterogeneity within BRCA1 methylation assays influences associations. Refining these assays may better identify cases with silenced BRCA1 function and improved patient outcomes.

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...

Analysis of Epigenetic Alterations in Homologous Recombination DNA Repair Genes in Male Breast Cancer.

Background: Male breast cancer (BC) is a distinct neoplasm with low but rising incidence, frequently diagnosed as advanced stage disease. Considering the relevance of altered homologous recombination repair (HRR) in male BC, we aimed to explore the biomarker potential of aberrant promoter methylation of ATM, BRCA1, PALB2, RAD51B, and XRCC3. Methods: Formalin-fixed paraffin-embedded (FFPE) tissue samples from 128 male BC patients, paired adjacent normal tissue and 19 gynecomastia cases were collected and assessed by quantitative methylation-specific PCR (qMSP). Non-parametric tests were used to compare methylation levels between tumor and non-tumor samples and to seek for associations with clinicopathological variables. Results: Only RAD51B and XRCC3 disclosed significant differences between tumor and gynecomastia (p < 0.0001 and p = 0.020, respectively). Assembled in a panel, RAD51B and XRCC3 promoter methylation discriminated male BC from gynecomastia with 91.5% sensitivity, 89.5% specificity, and 91.2% accuracy. Moreover, promoter methylation levels were lower in paired non-tumor tissues, comparing to tumor samples. No associations were found between epigenetic alterations and clinicopathological features, as well as with RAD51 and XRCC3 immunoexpression and methylation levels. Conclusion: Quantitative promoter methylation of RAD51B and XRCC3 constitutes a promising and accurate biomarker for male BC. Validation in larger series and in liquid biopsies is warranted to confirm its usefulness in detection and monitoring settings.

Analysis of BRCA1 and RAD51C Promoter Methylation in Italian Families at High-Risk of Breast and Ovarian Cancer.

Previous studies on breast and ovarian carcinoma (BC and OC) revealed constitutional BRCA1 and RAD51C promoter hypermethylation as epigenetic alterations leading to tumor predisposition. Nevertheless, the impact of epimutations at these genes is still debated. One hundred and eight women affected by BC, OC, or both and considered at very high risk of carrying BRCA1 germline mutations were studied. All samples were negative for pathogenic variants or variants of uncertain significance at BRCA testing. Quantitative BRCA1 and RAD51C promoter methylation analyses were performed by Epityper mass spectrometry on peripheral blood samples and results were compared with those in controls. All the 108 analyzed cases showed methylation levels at the BRCA1/RAD51C promoter comparable with controls. Mean methylation levels (± stdev) at the BRCA1 promoter were 4.3% (± 1.4%) and 4.4% (± 1.4%) in controls and patients, respectively (p > 0.05; t-test); mean methylation levels (± stdev) at the RAD51C promoter were 4.3% (± 0.9%) and 3.7% (± 0.9%) in controls and patients, respectively (p > 0.05; t-test). Based on these observations; the analysis of constitutional methylation at promoters of these genes does not seem to substantially improve the definition of cancer risks in patients. These data support the idea that epimutations represent a very rare event in high-risk BC/OC populations.

The association between the methylation frequency of BRCA1/2 gene promoter and occurrence and prognosis of breast carcinoma: A meta-analysis.

Objectives:Breast cancer susceptibility gene 1/2 (BRCA1/2) is a promising tumor marker in many types of cancer. However, the methylation frequency of BRCA1/2 gene with occurrence risk and survival benefit of patients with breast carcinoma remains controversy. The aim of the present study was to assess the relationship between BRCA1/2 gene promoter methylation and the occurrence and prognosis in breast carcinoma based on a meta-analysis, meanwhile, this article explored the differential expression levels of BRCA1/2 gene promoter methylation in peripheral blood and tumor tissues of breast cancer patients.Methods:Electronic databases (PubMed, Medline, Cochrane Library, and CNKI) were searched up to June 2019. The number of BRCA1/2 promoter methylation-positive and -negative patients in breast carcinoma patients were measured, and hazard ratio (HR) with 95% confidence interval (CI) for the association between BRCA1/2 gene promoter methylation and the prognosis of breast carcinoma patients. Primary end points were presence of breast cancer, overall survival (OS), disease-free survival (DFS). Statistical analysis was performed with STATA 12.0.Results and conclusions:Fifty-eight articles including 19,084 individuals met full eligibility criteria. We observed that the frequency of BRCA1 gene promoter methylation was higher in breast cancer tissues compared with normal tissues, and the prognostic analysis suggested that BRCA1 gene promoter methylation was significantly associated with poor overall survival and poor disease-free survival. This study also verified that there was no statistically significant difference in the methylation frequency of BRCA1 gene promoter between peripheral blood and tumor tissues in breast cancer patients, which suggests that the detection of BRCA1 promoter methylation in peripheral blood may be a non-invasive and rapid way to monitor the occurrence breast cancer.

Abstract P1-10-15: BRCA1 alterations with additional defects in DNA damage repair genes may confer chemoresistance to BRCA-like breast cancers treated with neoadjuvant chemotherapy

Abstract The BRCA-like phenotype is a feature that some sporadic breast cancers share with those occurring in germinal BRCA1- or BRCA2-mutation carriers. Since tumors with the phenotype have defects in the DNA damage repair pathway, which may increase sensitivity to drugs such as DNA cross-linking agents and PARP inhibitors, a method to identify this phenotype is important. The prediction of chemoresistance, which frequently develops in these tumors, is also crucial for improving therapy. We examined genomic aberrations and BRCA1 promoter methylation in tumors of 73 breast cancer (20 HR-/HER2- and 53 HR+/HER2-) patients, who underwent diagnostic core-needle biopsy and received neoadjuvant chemotherapy with anthracycline, cyclophosphamide, and taxane, using SNP array CGH and quantitative PCR. They had a tumor size of T1, T2, T3, or T4, a lymph node status of N0, N1, N2, or N3, and no distant metastasis (M0) at diagnosis, and were enrolled as subjects in this study. The methylation and/or loss or UPD of BRCA1 (termed BRCA1 alterations) and the loss or UPD of BRCA2 (termed BRCA2 alterations) were detected in 27 (37%) and 21 (29%), respectively, out of 73 tumors. Tumors with BRCA1 or BRCA2 alterations were associated with a higher number of genomic aberrations (P&amp;lt;0.001 and P&amp;lt;0.001) and higher percentage of TP53 alterations (the loss or UPD and/or mutations) than those without (P&amp;lt;0.001 and P&amp;lt;0.001). Overall survival (OS) rates were similar between patients with or without BRCA1 or BRCA2 alterations. However, when 27 patients with BRCA1-altered tumors were classified into those with or without the loss or UPD of PALB2, PAGR1, RAD51B, FANCM, MLL4 or ERCC1/2 in tumors, patients with additional defects in DNA damage repair genes had worse OS (P=0.037, 0.045, 0.038, 0.044, 0.041, or 0.019) than those without. These defects may confer chemoresistance and predict poor outcomes in patients with BRCA1-altered breast cancer. Citation Format: Mamoru Takada, Yasuhiko Kaneko, Masayuki Haruta, Shigenori Nagai, Kenichi Inoue, Katsunori Toduka, Masafumi Kurozumi, Hiroyuki Takei, Seishi Ogawa, Takeshi Nagashima, Takafumi Sangai, Hiroshi Fujimoto, Junta Sakakibara, Ryotaro Teranaka. BRCA1 alterations with additional defects in DNA damage repair genes may confer chemoresistance to BRCA-like breast cancers treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-15.

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.