Specific BRCA and immune configurations determine optimal response to platinum-based chemotherapy in triple negative breast and ovarian carcinomas

Loss of homologous recombination repair (HRR) via germline and somatic BRCA1 or BRCA2 gene mutations and via BRCA1 promoter methylation has been associated with better response to platinum agents and PARP inhibitors, in both triple negative breast cancer (TNBC) and ovarian carcinoma (OvCa). A major conundrum arising from recent clinical studies is why cancers with BRCA1 promoter methylation (BRCA1meth) respond more poorly as compared to those bearing mutations in BRCA1 and BRCA2 (BRCAmut), given the biologically equivalent HRR deficiency in both states. We dissected this problem through detailed genomic analyses of primary TNBC and OvCa cohorts, as well as experimentation with patient-derived xenograft (PDX) models and genetically engineered cell lines. Using the precise genomic scar of the tandem duplicator phenotype as a precise genomic indicator of BRCA1 deficiency, we found that, in all cohorts, BRCA1mut and BRCA1meth cancers share an equivalent degree of BRCA1-linked genomic rearrangements. Nonetheless, we consistently found that patients with BRCAmut cancers, but not those with BRCA1meth cancers, had significantly better response outcomes when compared to those with BRCA proficient cancers. When fully promoter methylated BRCA1 PDX TNBCs were exposed to a single short course of platinum chemotherapy an unmethylated BRCA1 promoter allele emerged in resultant tumors associated with an increase in BRCA1 expression. A separate analysis of PDXs derived from treatment naive TNBCs featured complete methylation of the BRCA1 promoter, whereas those derived from post-chemotherapy TNBCs invariably had only partial methylation. PDXs with partial methylation were significantly associated with lower response rates to in vivo platinum-based therapy compared to those with complete promoter methylation. Using single cell clonal expansions from a partially BRCA1meth PDX, we confirmed that the reduced level of methylation was due to the demethylation of one of the BRCA1 promoter alleles and not to the outgrowth of a nonmethylated clone. Clinically, analysis of primary OvCas confirmed that high levels of BRCA1 methylation were significantly associated with reduced BRCA1 gene expression whereas cancers with lower levels of BRCA1 methylation had expression levels approaching those found in BRCA1 proficient cancers. These data suggest that unlike BRCAmut cancers, where HRR deficiency is achieved via mutations that are genetically 'fixed', BRCA1meth cancers are highly adaptive to genotoxin exposure and more likely to recover BRCA1 expression, which may explain their poorer therapeutic response. We further found that an increased immune transcriptional signal, especially an elevated M1 macrophage signature, is associated with enhanced response to platinum-based chemotherapy only in patients with BRCA proficient cancers, in both TNBC and OvCa cohorts underscoring the importance of characterizing molecular heterogeneity to enhance predictive precision in assigning response probabilities in TNBC and OvCa.

Peripheral Blood BRCA1 Methylation Positively Correlates with Major Alzheimer’s Disease Risk Factors

BACKGROUND: Recent biomarker studies demonstrated that the central nervous system (CNS) environment can be observed from peripherally-derived samples. In a previous study, we demonstrated significant hypomethylation of the BRCA1 promoter region in neuronal cells from post-mortem brains of Alzheimer’s disease patients through neuron-specific methylome analysis. Thus, we investigate the methylation changes in the BRCA1 promoter region in the blood samples. OBJECTIVES: To analyze the methylation level of the BRCA1 promoter in peripheral blood from AD patients and normal controls. DESIGN, SETTING, PARTICIPANTS: Genomic DNA samples from peripheral blood were obtained from the J-ADNI repository, and their biomarker data were obtained J-ADNI from the National Bioscience Database Center. Genomic DNA samples from an independent cohort for validation was obtained from Niigata University Hospital (Niigata, Japan). Amyloid positivity was defied by visual inspection of amyloid PET or a CSF Aβ42 value ≤ 333 pg/mL at the baseline. MEASUREMENTS: Methylation level of the BRCA1 promoter was analyzed by pyrosequencing. RESULTS: Compared to normal controls, methylation of the BRCA1 promoter in AD patients was not significantly changed; however, in AD patients, it showed a positive correlation with AD risk factors. CONCLUSIONS: Our data confirmed the importance of cell-type specific methylome analysis and also suggested that environmental changes in the CNS can be detected by observing the peripheral blood, implying that the peripheral BRCA1 methylation level can be a surrogate for AD.

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

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.

BRCA1 Promoter Hypermethylation is Associated with Good Prognosis and Chemosensitivity in Triple-Negative Breast Cancer

The aberrant hypermethylation of BRCA1 promoter CpG islands induces the decreased expression of BRCA1 (Breast Cancer 1) protein. It can be detected in sporadic breast cancer without BRCA1 pathogenic variants, particularly in triple-negative breast cancers (TNBC). We investigated BRCA1 hypermethylation status (by methylation-specific polymerase chain reaction (MS-PCR) and MassARRAY® assays), and BRCA1 protein expression using immunohistochemistry (IHC), and their clinicopathological significance in 248 chemotherapy-naïve TNBC samples. Fifty-five tumors (22%) exhibited BRCA1 promoter hypermethylation, with a high concordance rate between MS-PCR and MassARRAY® results. Promoter hypermethylation was associated with reduced IHC BRCA1 protein expression (p = 0.005), and expression of Programmed death-ligand 1 protein (PD-L1) by tumor and immune cells (p = 0.03 and 0.011, respectively). A trend was found between promoter hypermethylation and basal marker staining (p = 0.058), and between BRCA1 expression and a basal-like phenotype. In multivariate analysis, relapse-free survival was significantly associated with N stage, adjuvant chemotherapy, and histological subtype. Overall survival was significantly associated with T and N stage, histology, and adjuvant chemotherapy. In addition, patients with tumors harboring BRCA1 promoter hypermethylation derived the most benefit from adjuvant chemotherapy. In conclusion, BRCA1 promoter hypermethylation is associated with TNBC sensitivity to adjuvant chemotherapy, basal-like features and PD-L1 expression. BRCA1 IHC expression is not a good surrogate marker for promoter hypermethylation and is not independently associated with prognosis. Association between promoter hypermethylation and sensitivity to Poly(ADP-ribose) polymerase PARP inhibitors needs to be evaluated in a specific series of patients.