BRCA1 plays a critical role in homology-directed repair (HDR) of DNA double strand breaks, and the repair defect of BRCA1-mutant cancer cells is being targeted with platinum drugs and poly (ADP-ribose) polymerase (PARP) inhibitors. We have employed relatively simple and sensitive assays to determine the function of BRCA1 variants or mutants in two HDR mechanisms, homologous recombination (HR) and single strand annealing (SSA), and in conferring resistance to cisplatin and olaparib in human cancer cells. Our results define the functionality of the top 22 patient-derived BRCA1 missense variants and the contribution of different domains of BRCA1 and its E3 ubiquitin ligase activity to HDR and drug resistance. Importantly, our results also demonstrate that the BRCA1-PALB2 interaction dictates the choice between HR and SSA. These studies establish functional and mutational landscapes of BRCA1 for HDR and therapy resistance, while revealing novel insights into BRCA1 regulatory mechanisms and HDR pathway choice.
Mitotic homologous recombination promotes genome stability through the precise repair of DNA double-strand breaks and other lesions that are encountered during normal cellular metabolism and from exogenous insults. As a result, homologous recombination repair is essential during proliferative stages in development and during somatic cell renewal in adults to protect against cell death and mutagenic outcomes from DNA damage. Mutations in mammalian genes encoding homologous recombination proteins, including BRCA1, BRCA2 and PALB2, are associated with developmental abnormalities and tumorigenesis. Recent advances have provided a clearer understanding of the connections between these proteins and of the key steps of homologous recombination and DNA strand exchange.
The BRCA2 tumor suppressor has been implicated in the maintenance of chromosomal stability through a function in DNA repair. In this report, we examine human and mouse cell lines containing different BRCA2 mutations for their ability to repair chromosomal breaks by homologous recombination. Using the I-SceI endonuclease to introduce a double-strand break at a specific chromosomal locus, we find that BRCA2 mutant cell lines are recombination deficient, such that homology-directed repair is reduced 6- to >100-fold, depending on the cell line. Thus, BRCA2 is essential for efficient homology-directed repair, presumably in conjunction with the Rad51 recombinase. We propose that impaired homology-directed repair caused by BRCA2 deficiency leads to chromosomal instability and, possibly, tumorigenesis, through lack of repair or misrepair of DNA damage.
Purpose Patients with hormone receptor–negative breast cancer generally do not benefit from endocrine-targeted therapies. However, a subset with androgen receptor (AR) expression is predicted to respond to antiandrogen therapies. This phase II study explored bicalutamide in AR-positive, estrogen receptor (ER), and progesterone receptor (PgR)-negative metastatic breast cancer. Experimental Design Tumors from patients with ER/PgR-negative advanced breast cancer were tested centrally for AR [immunohistochemistry (IHC) > 10% nuclear staining considered positive]. If either the primary or a metastatic site was positive, patients were eligible to receive the AR antagonist bicalutamide at a dose of 150 mg daily. Clinical benefit rate (CBR), the primary endpoint, was defined as the total number of patients who show a complete response (CR), partial response (PR), or stable disease (SD) > 6 months; secondary endpoints included progression-free survival (PFS) and toxicity. Correlative studies included measurement of circulating endocrine markers and IHC surrogates for basal-like breast cancer. Results Of 424 patients with ER/PgR-negative breast cancer, 12% tested AR-positive. The 6-month CBR was19%[95% confidence interval (CI), 7%–39%]for bicalutamide. The median PFS was 12 weeks (95% CI, 11–22 weeks). Bicalutamide was well-tolerated with no grade 4/5 treatment-related adverse events observed. Conclusion AR was expressed in 12% of patients with ER/PgR-negative breast cancer screened for this trial. The CBR of 19% observed with bicalutamide shows proof of principle for the efficacy of minimally toxic androgen blockade in a select group of patients with ER/PgR-negative, AR-positive breast cancer.
IMPORTANCE Estrogen receptor α (ESR1) mutations found in metastatic breast cancer (MBC) promote ligand-independent receptor activation and resistance to estrogen-deprivation therapy in laboratory models. The prevalence of these mutations and their potential impact on clinical outcomes has not been established. OBJECTIVE To determine the prevalence of ESR1 mutations (Y537S and D538G) in estrogen receptor (ER)-positive MBC and determine whether mutation is associated with inferior outcomes. DESIGN, SETTING, AND PARTICIPANTS From December 16, 2014, to August 26, 2015, we analyzed cell-free DNA (cfDNA) from baseline plasma samples from participants in the BOLERO-2 double-blind phase 3 study that randomized patients from 189 centers in 24 countries with MBC to exemestane plus placebo or exemestane plus everolimus. The study enrolled postmenopausal women with a diagnosis of MBC and prior exposure to an aromatase inhibitor. Baseline plasma samples were available from 541 of 724 patients (74.7%). We assessed the effect of mutation on overall survival of the population and the effect of mutation on progression-free survival (PFS) by treatment arm. INTERVENTIONS Patients were randomized to treatment with exemestane (25 mg oral daily) together with everolimus (10 mg oral daily) or with placebo. MAIN OUTCOMES AND MEASURES The 2 most frequent mutations in ESR1 (Y537S and D538G) were analyzed from cfDNA using droplet digital polymerase chain reaction and samples scored as wild-type, D538G, Y537S, or double mutant. Cox-proportional hazards model was used to assess PFS in patient subgroups defined by mutations, and the effect of each mutation on overall survival. RESULTS Of 541 evaluable patients, 156 (28.8%) had ESR1 mutation D538G (21.1%) and/or Y537S (13.3%), and 30 had both. These mutations were associated with shorter overall survival (wild-type, 32.1 months [95%CI, 28.09–36.40 months]; D538G, 25.99 months [95% CI, 19.19–32.36 months]; Y537S, 19.98 months [13.01–29.31 months]; both mutations, 15.15 months [95%CI, 10.87–27.43 months]). The D538G group (hazard ratio, 0.34 [95%CI, 0.02–0.57]) derived a similar PFS benefit as wild type from addition of everolimus to exemestane. CONCLUSIONS AND RELEVANCE ESR1 mutations are prevalent in ER-positive aromatase inhibitor-treated MBC. Both Y537S and D538G mutations are associated with more aggressive disease biology. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00863655
Purpose: In breast cancer, somatic mutations in the PIK3CA gene are common. The prognostic implication of these activating mutations remains uncertain as moderately sized studies have yielded variable outcomes. Our aim was to determine the prognostic implications of PIK3CA mutations in breast cancer. Experimental Design: Archival formalin-fixed paraffin-embedded primary breast tumors, from 590 patients selected for known vital status with a median follow-up of 12.8 years and a tumor >1 cm, were genotyped for PIK3CA mutations. Mutation rates and associations between mutation site and clinicopathologic characteristics were assessed. Progression-free survival, overall survival, and breast cancer-specific survival were examined using Kaplan-Meier or competing risk methodology. Results: PIK3CA mutation is identified in 32.5% of breast cancers. PIK3CA mutation significantly associates with older age at diagnosis, hormone receptor positivity, HER2 negativity, lower tumor grade and stage, and lymph node negativity. Patients with PIK3CA mutated tumors have significant improvement in overall survival (P = 0.03) and breast cancer-specific survival (P = 0.004). Analysis for PIK3CA mutation site-specific associations reveals that the H1047R kinase domain mutation highly associates with node negativity (P = 0.007), whereas helical domain hotspot mutations associate with older age at diagnosis (P = 0.004). Conclusion: This study defines the positive prognostic significance of PIK3CA mutations. This work is clinically relevant, as it will significantly affect the design of clinical trials planned for phosphatidylinositol 3-kinase-targeted therapy. Future work may define a population of older age breast cancer patients in whom therapy can be minimized. (Clin Cancer Res 2009;15(16):5049-59)
To directly determine whether recombinational repair of double-strand breaks (DSBs) can occur between heterologous chromosomes and lead to chromosomal rearrangements in mammalian cells, we employed an ES cell system to analyze recombination between repeats on heterologous chromosomes. We found that recombination is induced at least 1000-fold following the introduction of a DSB in one repeat. Most (98%) recombinants repaired the DSB by gene conversion in which a small amount of sequence information was transferred from the unbroken chromosome onto the broken chromosome. The remaining recombinants transferred a larger amount of information, but still no chromosomal aberrations were apparent. Thus, mammalian cells are capable of searching genome-wide for sequences that are suitable for DSB repair. The lack of crossover events that would have led to translocations supports a model in which recombination is coupled to replication.
Weekly trastuzumab and paclitaxel therapy is active in women with metastatic breast cancer. Therapy was relatively well tolerated; however, attention to cardiac function is necessary.
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