BRCA1 Promoter Hypermethylation Research Articles

Validation and Performance of Quantitative BRCA1 and RAD51C Promoter Hypermethylation Testing in Breast and Ovarian Cancers.

Poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors represent a significant advance in the treatment of epithelial ovarian cancer, triple negative breast cancer, pancreatic and castrate-resistant prostate cancer and they are poised to improve treatment in an increasing number of other cancer types. PARP inhibitor efficacy as monotherapy has been primarily observed in tumors with deleterious genetic variants in genes involved in the homologous recombination repair pathway, but tumors without these variants have also been shown to respond, notably those with hypermethylation at all alleles of the BRCA1 or RAD51C promoter can respond to PARP inhibitors. These epigenetic biomarkers therefore represent a patient population that may also benefit from this targeted therapy. However, there has not been a robust test to identify these biomarkers in routine clinical specimens that is amenable to implementation for decentralized testing. This study describes the analytical and clinical validation of a BRCA1 and RAD51C promoter methylation test that can be run with a single-day library preparation workflow for sequencing on any NGS platform. The results demonstrate that this test can accurately quantitate the level of promoter methylation at the BRCA1 and RAD51C genes using FFPE samples, even when the extracted DNA is extremely degraded or the input amount is limited. This test increases the precision of diagnostic tests aimed at identifying patients who are likely and unlikely to respond to PARP inhibitor therapy.

Increased Serum Levels of Inflammatory Markers and Promoter Hypermethylation of BRCA1 and BRCA2 in Oral Contraceptive Users

Background: Oral contraceptives (OCs) are widely used to prevent pregnancy, particularly among reproductive-age women. Although undesired physiological consequences, such as increased susceptibility to cancer, have been suggested, the exact molecular mechanism is not well elucidated. Thereby, the current study aimed to assess the effects of OCs on the inflammatory markers and BRCA1 and BRCA2 gene-specific DNA methylation in the serum of OCs-exposed women. Methods: The current cross-sectional study involved 70 adult women, 35 of whom had used oral contraceptive pills (OCP, 0.03 mg ethinyl estradiol, and 0.15 mg levonorgestrel) to prevent pregnancy, and 35 of whom had used condoms. The promoter methylation status of the two mentioned tumor suppressor genes was assessed by methylation-specific PCR. Moreover, serum levels of IL-1, IL-6, and TNF-α were evaluated using the ELISA method. Results: The findings revealed a significant difference in cytokines between groups (p <0.001). However, no significant differences were revealed regarding TNF-α between the two groups. Additionally, the frequency of promoter hypermethylation of BRCA1 and BRCA2 in OCP users was significantly higher (p <0.05). Conclusion: The current findings suggested that OCP usage could increase serum levels of inflammatory markers and promote the hypermethylation of two suppressor genes. Hence, further studies are encouraged to reveal the association between OCP usage and cancer through hypermethylation of BRCA1 and BRCA2 and induction of inflammation.

Association of HRD ovarian cancer by RAD51 with hot immune microenvironment: Translational analyses of MITO 16A/MaNGO-OV2 trial.

5527 Background: Homologous recombination repair (HRR)-deficient Ovarian Cancer (OvC) is sensitive to PARP inhibitors and platinum salts. Genomic HRR-deficiency (HRD) tests (i.e. Myriad MyChoice) are currently used to guide treatment selection in the maintenance setting. However, they are showing a limited predictive value, they are expensive and static, i.e. unable to capture the restoration of the HRR functionality, as a mechanism of drug resistance. Methods: We performed the functional and dynamic RAD51 test on 164 patients with high-grade serious OvC, enrolled in the MITO16A/MANGO OV2 trial and treated with first-line therapy containing carboplatin, paclitaxel, and bevacizumab. The RAD51 assay included the immunofluorescence of gH2AX (as a marker of Double Strand Breaks DNA damage), BRCA1 and RAD51 nuclear foci. Stromal Tumor Infiltrating Lymphocytes (TIL) by Immunohistochemistry, and gene expression profile were performed on the same samples. Results: RAD51 assay was informative on 70% (115/164) of the samples. On evaluable samples, median gH2AX score was 57%. BRCA1 foci by immunofluorescence were detected on 55% (49/112) of the samples; 45% (63/112) of the tumors did not show BRCA1 foci likely due to germline or somatic BRCA1 mutations, or BRCA1 promoter hypermethylation. RAD51 identified 69% (79/115) of all tumors as HRD, with a median RAD51 score of 8.8%. HRD tumors presented a statistically significantly higher TIL content than HRR-proficient ones (p = 0.03). Conclusions: Our preliminary results confirmed the feasibility of the RAD51 functional assay on primary untreated OvC. The association between HRD by RAD51 and TIL content encouraged to investigate the efficacy, in clinical trials, of the combination of PARPi and immune-checkpoint inhibitors on selected patients (i.e. HRD by RAD51 and TIL high). Gene expression profile and comparison of the HRD status by RAD51 and genomic HRD (both Myriad MyChoice and academic) and correlations with clinical outcomes will be available for the meeting.

Evaluation of promoter hypermethylation of tumor suppressor gene BRCA1 in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is a serious gynecological issue worldwide and its late detection is the major encumbrance in treatment procedures. Hypermethylation-mediated BRCA1 gene silencing results in failure of the repair system of damaged DNA playing an important role in ovarian carcinogenesis. BRCA1 gene hypermethylation can serve as a safe and highly specific clinical marker for EOC. The present study was conducted to evaluate the promoter hypermethylation of BRCA1 gene in EOC patients. This hospital-based case-control study carried out in the tertiary care hospital in New Delhi. Subjects and Methods: Promoter hypermethylation of BRCA1 gene was examined in 30 EOC diagnosed untreated cases confirmed by histopathological examinations and compared with 30 normal healthy controls matched for age using methylation specific-polymerase chain reaction. We found significantly higher BRCA1 promoter hypermethylation in the serum of EOC cases as compared to controls with P < 0.0001. BRCA1 gene methylation was found to have 70% sensitivity for the diagnosis of EOC with 100% specificity. A significant difference was observed in the range of CA125 levels, B12 and Folate levels between EOC cases and controls. We conclude that BRCA1 gene is significantly hypermethylated in EOC patients and thus can prove to be a noninvasive diagnostic tool. Our results provide prefatory evidence that epithelial ovarian epigenome can be influenced by dietary nutrients.

Pan-cancer analysis of genomic scar patterns caused by homologous repair deficiency (HRD)

Homologous repair deficiency (HRD) is present in many cancer types at variable prevalence and can indicate response to platinum-based chemotherapy and PARP inhibition. We developed a tumor classification system based on the loss of function of genes in the homologous recombination repair (HRR) pathway. To this end, somatic and germline alterations in BRCA1/2 and 140 other HRR genes were included and assessed for the impact on gene function. Additionally, information on the allelic hit type and on BRCA1 promoter hypermethylation was included. The HRDsum score including LOH, LST, and TAI was calculated for 8847 tumors of the TCGA cohort starting from genotyping data and for the subcohort of ovarian cancer also starting from WES data. Pan-cancer, deleterious BRCA1/2 alterations were detected in 4% of the tumors, while 18% of the tumors were HRD-positive (HRDsum ≥ 42). Across 33 cancer types, both BRCA1/2 alterations and HRD-positivity were most prevalent in ovarian cancer (20% and 69%). Pan-cancer, tumors with biallelic deleterious alterations in BRCA1/2 were separated strongly from tumors without relevant alterations (AUC = 0.89), while separation for tumors with monoallelic deleterious BRCA1/2 alterations was weak (AUC = 0.53). Tumors with biallelic deleterious alterations in other HHR genes were separated moderately from tumors without relevant alterations (AUC = 0.63), while separation for tumors with such monoallelic alterations was weaker (AUC = 0.57). In ovarian cancer, HRDsum scores calculated from WES data correlated strongly with HRDsum scores calculated from genotyping data (R = 0.87) and were slightly (4%) higher. We comprehensively analyzed HRD scores and their association with mutations in HRR genes in common cancer types. Our study identifies important parameters influencing HRD measurement and argues for an integration of HRDsum score with specific mutational profiles.

DNA comethylation analysis reveals a functional association between BRCA1 and sperm DNA fragmentation

To identify the DNA comethylation patterns associated with sperm DNA fragmentation (SDF) and to explore the potential associations of hub genes with SDF. Prospective study. University-affiliated reproductive medicine center. A total of 300 male patients consulting for couple infertility were recruited from the First Affiliated Hospital of Wenzhou Medical University. None. Comethylation network analysis based on the genome-wide methylation profile of spermatozoal DNA from 20 men was performed to identify hub modules and genes involved in SDF. Human spermatozoa were used for targeted bisulfite amplicon sequencing (267 men) or droplet digital polymerase chain reaction (45 men). The potential role of Brca1 in DNA damage was explored in mouse GC2 spermatocyte cells. Oxidative damage to spermatocytes was modeled by incubating GC2 cells with H2O2 (25 mM) for 90 minutes. BRCA1 was identified as a hub gene in SDF. Promoter hypermethylation of BRCA1 was observed in those samples with a high DNA fragmentation index (DFI) compared to those with a low DFI. Concomitantly, BRCA1 mRNA expression was lower in samples with a high DFI than with a low DFI. In the GC2 cell model, Brca1 knockdown reduced cell proliferation and increased sensitivity to oxidative stress. Moreover, it increased double-strand breaks and decreased the protein levels of the DNA repair genes MRE11 and RAD51. A prominent cluster of comethylated patterns associated with SDF was identified. BRCA1 may be the hub gene involved in sperm DNA damage.

BRCA1 and RAD51C promotor methylation in human resectable pancreatic adenocarcinoma

BackgroundHomozygous Recombination Deficiency (HRD) is associated with sensitivity to PARP-inhibitors (PARPi) in different cancer types. In pancreatic adenocarcinoma (PA) the main cause of HRD is BRCA1/2 germline mutation and patients with mutations in BRCA1/2 may benefit from PARPi. Recently other mechanisms leading to HRD were described in different cancer types, including gene mutations and epigenetic changes such as promoter hypermethylation. In PA, BRCA1 promoter hypermethylation, a known mechanism of gene silencing, was recently described. However, results are discordant between North American studies (0.7% of PA) and Asian ones (up to 60% of PA) and the association with HRD is not clear. MethodsHere, we developed 2 quantifications methods to explore BRCA1 and RAD51C promoter methylation in a series of 121 Formalin Fixed-Paraffin-Embedded (FFPE) specimens from resected PA without neoadjuvant treatment. The methylation-specific PCR was done with 2 different methods after DNA bisulfite conversion: a digital droplet PCR, and a PCR followed by capillary electrophoresis, to score the methylated / non methylated ratios in tumor samples. Methods were validated for specificity and sensibility using 100, 20, 10, 5 and 0% methylated commercial DNA for fragment analysis with a detection cutoff of 5–10%. Limit of blank was defined as 5 dropplets/20µL for RAD51C and 1 dropplet/20µL for BRCA1 for ddPCR. Samples were reviewed by a pathologist, macrodissected before DNA extraction to obtain 50–60% of tumoral cells. DNAs were treated for bisulfite conversion and analyzed using both methods in parallel to known positive and negative controls in each run. Results and conclusionNo methylation at BRCA1 or RAD51C was found in this series of PA suggesting that HRD gene promoter methylation is a rare event in European patients.

Value of the loss of heterozygosity to BRCA1 variant classification

At least 10% of the BRCA1/2 tests identify variants of uncertain significance (VUS) while the distinction between pathogenic variants (PV) and benign variants (BV) remains particularly challenging. As a typical tumor suppressor gene, the inactivation of the second wild-type (WT) BRCA1 allele is expected to trigger cancer initiation. Loss of heterozygosity (LOH) of the WT allele is the most frequent mechanism for the BRCA1 biallelic inactivation. To evaluate if LOH can be an effective predictor of BRCA1 variant pathogenicity, we carried out LOH analysis on DNA extracted from 90 breast and seven ovary tumors diagnosed in 27 benign and 55 pathogenic variant carriers. Further analyses were conducted in tumors with PVs yet without loss of the WT allele: BRCA1 promoter hypermethylation, next-generation sequencing (NGS) of BRCA1/2, and BRCAness score. Ninety-seven tumor samples were analyzed from 26 different BRCA1 variants. A relatively stable pattern of LOH (65.4%) of WT allele for PV tumors was observed, while the allelic balance (63%) or loss of variant allele (15%) was generally seen for carriers of BV. LOH data is a useful complementary argument for BRCA1 variant classification.

BRCA1 promoter hypermethylation on circulating tumor DNA correlates with improved survival of patients with ovarian cancer.

Methylation of the BRCA1 promoter is an epigenetic gene expression regulator and is frequently observed in ovarian cancer; however, conversion of methylation status is thought to drive disease recurrence. Therefore, longitudinal monitoring of methylation status by liquid biopsy in cell‐free DNA may be a predictive marker. In total, 135 plasma samples were collected from 69 ovarian cancer patients before and during systemic treatment. Our liquid biopsy assay could detect down to a single molecule of methylated DNA in a high background of normal DNA (0.03%) with perfect specificity in control samples. We found that 60% of the cancer patients exhibited BRCA1 promoter hypermethylation at one point, although 24% lost hypermethylation during treatment. Multivariate survival analyses indicate that relapses are independent events and that hypermethylation and methylation conversion are independently correlated to longer relapse‐free survival. We present a highly sensitive and specific methylation‐specific quantitative PCR‐based liquid biopsy assay. BRCA1 promoter hypermethylation is frequently found in ovarian cancer and is often reversed upon recurrence, indicating the selection of therapy‐resistant clones and unfavorable clinical outcome.

Assessment of Promoter hypermethylation of APC and BRCA1 in endometrial cancer.

Introduction: Endometrial cancer is one of the most common cancers in women worldwide. Theunderlying cause of endometrial tumorigenesis remains elusive. Several genetic and epigeneticalterations are known to be involved in the carcinogenesis of endometrial carcinoma. One importantand early epigenetic alteration that is attributed to endometrial carcinoma is the aberrant promoterhypermethylation of gene promoters. In this study, we have assessed the aberrant promoterhypermethylation of APC and BRCA1 in 78 endometrial cancer samples. Methods: Histologicallyconfirmed tumour tissue samples were obtained post-surgery and DNA was extracted. The DNA wassubjected to sodium bisulfite conversion and used as a template for a polymerase chain reaction.The PCR was performed using a nested PCR followed by methylation specific PCR. Results: A33.33% and 46.15% methylation frequency was observed for APC and BRCA1 genes respectively. Ahigher percentage of methylation was observed in stage IV for APC (66.66%) and in stage II forBRCA1 (88.88%). Conclusion: Aberrant promoter hypermethylation is an early event inendometrial carcinoma and can serve as a useful molecular marker for diagnosis and prognosis ofthe disease along with existing screening modalities.

Prevalence of BRCA1 and BRCA2 genes promoter hypermethylation in breast cancer tissue.

Recent advances in the treatment of breast cancer (BC) have been related to the personalization of therapy. The methylation status of the promoter regions of tumor suppressor genes such as BRCA1and BRCA2is supposed to be useful as a prognostic factor in BC patients. To investigate the frequency of hypermethylation in the promoter regions of BRCA1and BRCA2genes in tumor tissue of BC patients, and the relation of hypermethylation to the clinical course of the disease. Molecular genetic studies were performed on 50BC tissue samples in order to determine the methylation status of the promoter regions of the BRCA1and BRCA2genes. Hypermethylation of the BRCA1promoter region was detected in 34% of BC cases, hypermethylation of the BRCA2promoter region- in 50% of cases, and hypermethylation of the promoter region of both genes- in 20% of cases. A significant increase in the incidence of hypermethylation of the BRCA2promoter region was found in the group of patients older than 56years, mainly in patients with triple-negative breast cancer and without family history of BC. The high frequency of hypermethylation in the promoter regions of BRCA1and BRCA2genes, as well as their co-methylation in tumor tissue of BC patients has been detected.

The RECAP Test Rapidly and Reliably Identifies Homologous Recombination-Deficient Ovarian Carcinomas.

Simple SummaryThe sensitivity to PARP inhibitors (PARPi) is related to tumor-specific defects in homologous recombination (HR) and extends beyond BRCA1/2-related deficiencies. A robust method to identify HR-deficient (HRD) carcinomas is therefore of utmost clinical importance. In this study, we evaluated the use of a functional test (the RECAP test) for the identification of HRD ovarian carcinomas. Forty-nine epithelial ovarian carcinomas (EOC) were analyzed by the RECAP test. Thirty-nine of these tumors were of the high-grade serous (HGSOC) histologic subtype. Ten out of these 39 HGSOC specimens showed HRD (26%), whereas ovarian carcinomas of other histologic subtypes (n = 10) were all HR-proficient (HRP). Eight out of 9 sequenced HRD tumors showed pathogenic BRCA1/2 variants or BRCA1 promoter hypermethylation. This study shows that the RECAP test is a reliable and rapid test to identify functional deficiencies in HR and a good alternative to DNA-based HRD tests.Recent studies have shown that the efficacy of PARP inhibitors in epithelial ovarian carcinoma (EOC) is related to tumor-specific defects in homologous recombination (HR) and extends beyond BRCA1/2 deficient EOC. A robust method with which to identify HR-deficient (HRD) carcinomas is therefore of utmost clinical importance. In this study, we investigated the proficiency of a functional HR assay based on the detection of RAD51 foci, the REcombination CAPacity (RECAP) test, in identifying HRD tumors in a cohort of prospectively collected epithelial ovarian carcinomas (EOCs). Of the 39 high-grade serous ovarian carcinomas (HGSOC), the RECAP test detected 26% (10/39) to be HRD, whereas ovarian carcinomas of other histologic subtypes (n = 10) were all HR-proficient (HRP). Of the HRD tumors that could be sequenced, 8/9 showed pathogenic BRCA1/2 variants or BRCA1 promoter hypermethylation, indicating that the RECAP test reliably identifies HRD, including but not limited to tumors related to BRCA1/2 deficiency. Furthermore, we found a trend towards better overall survival (OS) of HGSOC patients with RECAP-identified HRD tumors compared to patients with HRP tumors. This study shows that the RECAP test is an attractive alternative to DNA-based HRD tests, and further development of a clinical grade RECAP test is clearly warranted.

A Survey of Rare Epigenetic Variation in 23,116 Human Genomes Identifies Disease-Relevant Epivariations and CGG Expansions

There is growing recognition that epivariations, most often recognized as promoter hypermethylation events that lead to gene silencing, are associated with a number of human diseases. However, little information exists on the prevalence and distribution of rare epigenetic variation in the human population. In order to address this, we performed a survey of methylation profiles from 23,116 individuals using the Illumina 450k array. Using a robust outlier approach, we identified 4,452 unique autosomal epivariations, including potentially inactivating promoter methylation events at 384 genes linked to human disease. For example, we observed promoter hypermethylation of BRCA1 and LDLR at population frequencies of ∼1 in 3,000 and ∼1 in 6,000, respectively, suggesting that epivariations may underlie a fraction of human disease which would be missed by purely sequence-based approaches. Using expression data, we confirmed that many epivariations are associated with outlier gene expression. Analysis of variation data and monozygous twin pairs suggests that approximately two-thirds of epivariations segregate in the population secondary to underlying sequence mutations, while one-third are likely sporadic events that occur post-zygotically. We identified 25 loci where rare hypermethylation coincided with the presence of an unstable CGG tandem repeat, validated the presence of CGG expansions at several loci, and identified the putative molecular defect underlying most of the known folate-sensitive fragile sites in the genome. Our study provides a catalog of rare epigenetic changes in the human genome, gives insight into the underlying origins and consequences of epivariations, and identifies many hypermethylated CGG repeat expansions.

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.

Comprehensive molecular comparison of BRCA1 hypermethylated and BRCA1 mutated triple negative breast cancers

Homologous recombination deficiency (HRD) is a defining characteristic in BRCA-deficient breast tumors caused by genetic or epigenetic alterations in key pathway genes. We investigated the frequency of BRCA1 promoter hypermethylation in 237 triple-negative breast cancers (TNBCs) from a population-based study using reported whole genome and RNA sequencing data, complemented with analyses of genetic, epigenetic, transcriptomic and immune infiltration phenotypes. We demonstrate that BRCA1 promoter hypermethylation is twice as frequent as BRCA1 pathogenic variants in early-stage TNBC and that hypermethylated and mutated cases have similarly improved prognosis after adjuvant chemotherapy. BRCA1 hypermethylation confers an HRD, immune cell type, genome-wide DNA methylation, and transcriptional phenotype similar to TNBC tumors with BRCA1-inactivating variants, and it can be observed in matched peripheral blood of patients with tumor hypermethylation. Hypermethylation may be an early event in tumor development that progress along a common pathway with BRCA1-mutated disease, representing a promising DNA-based biomarker for early-stage TNBC.

Genetic and epigenetic profiling of BRCA1/2 in ovarian tumors reveals additive diagnostic yield and evidence of a genomic BRCA1/2 DNA methylation signature

Poly-ADP-ribose-polymerase inhibitor (PARPi) treatment is indicated for advanced-stage ovarian tumors with BRCA1/2 deficiency. The “BRCAness” status is thought to be attributed to a tumor phenotype associated with a specific epigenomic DNA methylation profile. Here, we examined the diagnostic impact of combined BRCA1/2 sequence, copy number, and promoter DNA methylation analysis, and evaluated whether genomic DNA methylation patterns can predict the BRCAness in ovarian tumors. DNA sequencing of 172 human tissue samples of advanced-stage ovarian adenocarcinoma identified 36 samples with a clinically significant tier 1/2 sequence variants (point mutations and in/dels) and 9 samples with a CNV causing a loss of function in BRCA1/2. DNA methylation analysis of the promoter of BRCA1/2 identified promoter hypermethylation of BRCA1 in two mutation-negative samples. Computational modeling of genome-wide methylation markers, measured using Infinium EPIC arrays, resulted in a total accuracy of 0.75, sensitivity: 0.83, specificity: 0.64, positive predictive value: 0.76, negative predictive value: 0.74, and area under the receiver’s operating curve (AUC): 0.77, in classifying tumors harboring a BRCA1/2 defect from the rest. These findings indicate that the assessment of CNV and promoter DNA methylation in BRCA1/2 increases the cumulative diagnostic yield by 10%, compared with the 20% yield achieved by sequence variant analysis alone. Genomic DNA methylation data can partially predict BRCAness in ovarian tumors; however, further investigation in expanded BRCA1/2 cohorts is needed, and the effect of other double strand DNA repair gene defects in these tumors warrants further investigations.

An integrative molecular framework to predict homologous recombination deficiency.

e15664 Background: Homologous recombination deficiency (HRD) is the primary biomarker for sensitivity to PARP inhibitors, but identifying the genetic and transcriptomic characteristics that fully capture all HRD patients has remained difficult. For example, DNA-based approaches are limited to patients with pathogenic mutations and loss of heterozygosity (LOH) events, and can fail to properly classify patients with variants of unknown significance. To capture more dynamic cellular processes that arise immediately upon induction of HRD through silencing or loss of BRCA 1/2, a more integrated approach that includes both RNA and DNA based models is necessary. Methods: Using DNA sequencing we developed a genome-wide LOH score that combines pathogenic mutation status and LOH at the BRCA1/2 loci, and the proportion of bases sequenced in the Tempus xT panel that undergo LOH. We also developed three independent RNA-based models to predict BRCA deficiency: 1) An elastic net transcriptome model to predict DNA-based HRD scores derived from exome and SNP array data for each tumor type represented in TCGA; 2) A logistic model to detect BRCA1 promoter hypermethylation from the transcriptome in TCGA data; 3) A model that leveraged the mSigDB annotated gene sets to conduct single sample gene set enrichment analysis (ssGSEA) on Tempus-sequenced patients, selecting over a hundred gene sets that were predictive of BRCA-deficiency. These 4 features were combined to develop a stacked, linear-regression model to distinguish BRCA-intact from BRCA-deficient patients. Results: We found that the genome-wide LOH score alone is predictive of BRCA deficiency. However, our integrated model was highly accurate at distinguishing between BRCA-intact and BRCA-deficient patients and outperformed any single RNA- or DNA-based model. Using this model, we identified many patients that are likely to respond to PARP inhibitors that would have been overlooked using RNA or DNA-based inferences alone. Conclusions: Our approach highlights the strength of integrating diverse molecular features to refine diagnosis and enable oncologists to deliver the most effective therapies to patients.

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.

Aberrant Promoter Hypermethylation of RASSF1a and BRCA1 in Circulating Cell-Free Tumor DNA Serves as a Biomarker of Ovarian Carcinoma.

Objective:Ovarian cancer is one of the leading causes of cancer deaths in women. Ovarian cancer is diagnosed at the late stages and generally relapses within 12-14 months of cytoreductive surgery. This is attributed to lack of precise molecular detection methodologies to detect and track the disease. Epigenetic alteration such as aberrant promoter hypermethylation is an important early event that occurs during cancer development and progression. This study focuses on development of a minimally invasive methylation marker that could be used for detection and prognosis of ovarian cancer patients. Methods:Aberrant promoter hypermethylation of RASSF1a and BRCA1 was assessed in circulating DNA of 72 EOC patients using methylation-specific PCR. The findings were correlated with various clinicopathological parameters. Statistical analysis was done using the Fisher exact test and chi-square test. Results:The aberrant methylation patterns of RASSF1a and BRCA1 was identified to be present in the cancerous samples. A total of 31.9 % and 56.9% methylation was observed for RASSF1a and BRCA1 respectively. A striking 50% methylation of BRCA1 was identified in the benign sample cohort, which marks the significance of assessing the hypermethylation pattern to detect cancer at its early stages. Methylation of the two tumor suppressor genes was evident across various stages and grades of ovarian tumors suggesting that this could also help as a prognostic marker. Conclusion:The results of the current study hold significance since the hypermethylation patterns can be identified in the cell-free circulating tumor DNA from a small volume of blood plasma and is a simple and minimally-invasive method. Assessment of hypermethylation patterns of a panel of TSG along with the existing screening markers could aid in better diagnosis and management of the disease. It could also aid in designing specifically tailored treatment strategies to fight the disease.

Whole-genome sequencing of triple-negative breast cancers in a population-based clinical study.

SummaryWhole genome sequencing (WGS) brings comprehensive insights to cancer genome interpretation. To explore clinical value of WGS, we sequenced 254 triple negative breast cancers (TNBC) with associated treatment and outcome data collected between 2010-2015 via the population-based Sweden Cancerome Analysis Network-Breast (SCAN-B) project (ClinicalTrials.gov ID:NCT02306096). Applying the HRDetect mutational-signature-based algorithm to classify tumors, 59% were predicted to have Homologous-recombination-repair deficiency (HRDetect-high): 67% explained by germline/somatic mutations of BRCA1/BRCA2, BRCA1 promoter hypermethylation, RAD51C hypermethylation or biallelic loss of PALB2. A novel mechanism of BRCA1 abrogation was discovered via germline SINE-VNTR-Alu retrotransposition. HRDetect provided independent prognostic information, with HRDetect-high patients having better outcome on adjuvant chemotherapy for invasive disease-free survival (Hazard Ratio, HR=0.42, 95% confidence interval, CI=0.2-0.87), and distant relapse-free interval (HR=0.31, CI=0.13-0.76) compared to HRDetect-low, regardless of whether a genetic/epigenetic cause was identified. HRDetect-intermediate, some possessing potentially targetable biological abnormalities, had poorest outcomes. HRDetect-low cancers also had inadequate outcomes: ~4.7% were mismatch-repair-deficient - another targetable defect, not typically sought; and was enriched for (but not restricted to) PIK3CA/AKT1 pathway abnormalities. New treatment options need to be considered for now-discernible HRDetect-intermediate and HRDetect-low categories. This population-based study advocates for WGS of TNBC to better inform trial stratification and improve clinical decision-making.