Breast Cancer BRCA2 Research Articles

Abstract A23: Plasma osteoprotegerin and breast cancer risk in BRCA1 and BRCA2 mutation carriers

Abstract Background: There is emerging evidence to suggest that progesterone-mediated upregulation of the receptor activator of nuclear factor κ β (RANK)/RANK ligand (RANKL) signaling pathway plays a critical role in mammary gland epithelial cell proliferation, mammary stem cell expansion and carcinogenesis. Of relevance for women at a high risk of developing breast cancer due to an inherited BRCA mutation, are recent findings showing that circulating levels of osteoprotegerin (OPG) (an endogenous decoy receptor for RANKL and thus inhibitor of RANK/RANKL-mediated signaling) are lower in women with a BRCA1 or BRCA2 mutation compared to non-carriers. Whether low OPG concentrations contribute to the high breast cancer risk in this population is unknown. If so, a therapeutic intervention that mimics the action of OPG might be used for primary prevention. We evaluated the relationship between plasma OPG and breast cancer risk among women with a BRCA1 or BRCA2 mutation in a prospective study. Methods: Baseline blood samples were available from 206 BRCA mutation carriers with no previous history of cancer. Plasma OPG concentrations were measured using a commercial enzyme-linked immunosorbent assay (ELISA) and categorized dichotomously as high vs. low based on the median of the entire cohort. The cumulative incidence of breast cancer by baseline plasma OPG concentration was estimated using Kaplan-Meier survival analysis. Results: Over a mean follow-up period of 6.5 years (range 0.1-18.8 years), 18 incident cases of primary invasive breast cancer were observed in the cohort. Women who developed breast cancer had significantly lower mean baseline OPG concentrations (90.59 pg/ml [range 4.2-205.7 pg/ml]) compared to the OPG concentrations of women who did not develop breast cancer ((117.9 pg/ml [7.4-547.7]) (P = 0.04). BRCA mutation carriers with low baseline OPG concentrations (< 95 pg/ml) had a significantly higher risk of developing breast cancer compared to those with high baseline OPG concentrations (≥95 pg/ml). After ten years of follow-up, the cumulative incidence of breast cancer among women with low OPG concentrations was 21% compared to 9% among women with high OPG concentrations (P-log rank test = 0.046). There was no evidence of effect modification by menopausal status or BRCA mutation type. After multivariate adjustment, women with high plasma OPG had a significantly decreased risk of developing breast cancer, compared to women with low OPG (HR = 0.25; 95%CI 0.08-0.78; P = 0.02). Conclusions: Our preliminary data suggest that low OPG concentrations are associated with an increased risk of breast cancer in BRCA1 and BRCA2 mutation carriers. These data support the potential for targeting of the RANKL pathway as a plausible cancer prevention strategy among women with germline BRCA mutations. Additional analyses with a larger sample size are underway and may help inform strategies of personalized prevention. These findings will not only further our understanding of the progesterone/OPG/RANKL pathway in breast cancer development, but will improve our identification of high-risk populations that can be targeted by prevention options that are currently available (i.e., denosumab) to simultaneously prevent breast cancer development and maintain bone health (particularly after salpingo-oophorectomy). Citation Format: Leonardo Salmena, Lovisa Oden, Shana Kim, Mohammad Akbari, Ping Sun, Steven Narod, Joanne Kotsopoulos. Plasma osteoprotegerin and breast cancer risk in BRCA1 and BRCA2 mutation carriers. [abstract]. In: Proceedings of the AACR Special Conference: Improving Cancer Risk Prediction for Prevention and Early Detection; Nov 16-19, 2016; Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(5 Suppl):Abstract nr A23.

Associations Between Cancer Predisposition Testing Panel Genes and Breast Cancer

Germline pathogenic variants in BRCA1 and BRCA2 predispose to an increased lifetime risk of breast cancer. However, the relevance of germline variants in other genes from multigene hereditary cancer testing panels is not well defined. To determine the risks of breast cancer associated with germline variants in cancer predisposition genes. A study population of 65 057 patients with breast cancer receiving germline genetic testing of cancer predisposition genes with hereditary cancer multigene panels. Associations between pathogenic variants in non-BRCA1 and non-BRCA2 predisposition genes and breast cancer risk were estimated in a case-control analysis of patients with breast cancer and Exome Aggregation Consortium reference controls. The women underwent testing between March 15, 2012, and June 30, 2016. Breast cancer risk conferred by pathogenic variants in non-BRCA1 and non-BRCA2 predisposition genes. The mean (SD) age at diagnosis for the 65 057 women included in the analysis was 48.5 (11.1) years. The frequency of pathogenic variants in 21 panel genes identified in 41 611 consecutively tested white women with breast cancer was estimated at 10.2%. After exclusion of BRCA1, BRCA2, and syndromic breast cancer genes (CDH1, PTEN, and TP53), observed pathogenic variants in 5 of 16 genes were associated with high or moderately increased risks of breast cancer: ATM (OR, 2.78; 95% CI, 2.22-3.62), BARD1 (OR, 2.16; 95% CI, 1.31-3.63), CHEK2 (OR, 1.48; 95% CI, 1.31-1.67), PALB2 (OR, 7.46; 95% CI, 5.12-11.19), and RAD51D (OR, 3.07; 95% CI, 1.21-7.88). Conversely, variants in the BRIP1 and RAD51C ovarian cancer risk genes; the MRE11A, RAD50, and NBN MRN complex genes; the MLH1 and PMS2 mismatch repair genes; and NF1 were not associated with increased risks of breast cancer. This study establishes several panel genes as high- and moderate-risk breast cancer genes and provides estimates of breast cancer risk associated with pathogenic variants in these genes among individuals qualifying for clinical genetic testing.

Abstract B36: Targeting BRCA1 through natural HSP90 inhibitors to reverse platinum resistance in TNBC

Abstract Background: Triple-negative breast cancers (TNBCs) are highly associated with an aggressive clinical course, resistance to chemotherapy, and poor prognosis compared to other breast cancer subtypes. However, breast cancers arising in BRCA1 mutation carriers appear to be particularly sensitive to platinum-based chemotherapy agents. The tumor suppressor BRCA1 (breast cancer type 1 susceptibility protein) is part of a protein complex that repairs DNA damage by homologous recombination. Recent studies have shown that tumor cells expressing high levels of BRCA1 are resistant to both ionizing radiation (IR) and platinum-based chemotherapy agents, and that ablation of BRCA1 expression can restore sensitivity to these agents. We have demonstrated that the chaperone protein heat shock protein 90 (HSP90) is required for BRCA1 stability. However, established HSP90 inhibitors exhibit a high level of toxicity unrelated to HSP90. Recently, we observed that the natural compounds gedunin and celastrol modulate HSP90 activity without inducing side effects. We believe that combinatory use of these compounds can further reduce BRCA1 expression beyond their individual effect to overcome BRCA1-mediated resistance to platinum-based chemotherapies. Study Design: The goal of these experiments is to demonstrate that combination treatment with the HSP90 inhibitors gedunin and celastrol will maximize BRCA1 degradation (via inhibition of HSP90), impair the DNA repair system, and allow carboplatin to effectively eradicate TNBC cells. We will confirm that this is a HSP90 mediated event that results in the impairment of DNA repair. Using in vivo models of breast cancer, we will demonstrate that natural compounds could sensitize BRCA1-expressing TNBC cells to carboplatin, inhibiting breast cancer progression and tumor growth. Results: Using Western blot analysis, we observed that the combination of gedunin and celastrol attenuates HSP90 activity, leading to a dose-dependent reduction in BRCA1 degradation in HCC1937BRCA1 TNBC cells. Additionally, we the combination facilitated carboplatin-mediated inhibition of proliferation in both BT-20 and HCC1937BRCA1 cells. Immunofluorescence staining for g-H2AX and RAD51 demonstrated that the two HSP90 inhibitors prevent BRCA1-mediated recruitment of DNA repair complexes following ionizing radiation-induced DNA damage. To test the effects of celastrol and gedunin in vivo, we generated mouse xenografts by performing fat pad injections of HCC1937BRCA1 cells. Mice were dosed I.P. every 5 days with celastrol (5 mg/kg), gedunin (5 mg/kg), and carboplatin (25 mg/kg) for 20 days. Treatment with the HSP90 inhibitors along with carboplatin resulted in significant reduction in both tumor size and weight. In contrast treatment with carboplatin alone resulted in tumors of size similar to vehicle-treated animals. Additionally, phospho-H3 and cleaved caspase 3 immunofluorescence staining indicated a corresponding decrease in cell proliferation and increased apoptosis in tumors from celastrol+gedunin+carboplatin -treated animals compared to either vehicle or carboplatin alone-treated. Future studies will focus on examining the effects of this combination treatment in TNBC xenografts created with patient specimens, and in defining the interaction between HSP90 and BRCA1. Significance: In order to increase survival rates in TNBC patients, it is essential that resistance to chemotherapy be overcome. Successful completion of these studies demonstrating that natural compounds can act as HSP90 inhibitors to reverse platinum resistance without added toxicity would facilitate their rapid incorporation into clinical trials and provide a critically needed therapy for women with TNBC. Citation Format: Kelli Valdez, Prabhu Ramamoorthy, Shrikant Anant, Roy Jensen. Targeting BRCA1 through natural HSP90 inhibitors to reverse platinum resistance in TNBC [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B36.

Abstract B37: PARP inhibitor olaparib induces genomic instability in normal mammalian cells

Abstract Poly(ADP-ribose) polymerases (PARPs) are the first proteins involved in cellular DNA damage response pathways to be targeted by specific inhibitors for clinical benefit. Tumors with defects in homologous recombination (HR) are hypersensitive to PARP inhibitors (PARPi), and early phase clinical trials have been promising in patients with advanced BRCA1 and BRCA2-associated breast, ovary and prostate cancer. Unlike HR-defective cells, HR-proficient cells manifest low cytotoxicity when exposed to PARPi. Nonetheless, they mount a DNA damage response and show a genomic instability phenotype as demonstrated by an increased frequency of sister chromatid exchange. To study molecular mechanisms underlying PARPi-generated genomic instability, we took advantage of olaparib an agent in clinical use. Our results in mouse embryonic stem cells show that olaparib increases HR leading to copy number alterations and induces a mutator phenotype. Further, using a genome wide approach we finally demonstrate that PARPi treatment increases DNA double-strand breaks genome-wide which can lead to translocations and other genome aberrations. Our findings have important implications for therapies with regard to sustained genotoxicity to normal cells. Genomic instability arising from PARPi warrants consideration in prevention strategies or for non-oncologic indications. Citation Format: Fabio Vanoli, Shuhei Ito, Richard L. Frock, Frederick W. Alt, Mary Ellen Moynahan, Maria Jasin. PARP inhibitor olaparib induces genomic instability in normal mammalian cells [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B37.

Stimulation of lactate receptor (HCAR1) affects cellular DNA repair capacity

Numerous G-protein coupled receptors have been reported to enhance cancer cell survival and resistance to clinically used chemotherapeutics. Recently, hydroxycarboxylic acid receptor 1 (HCAR1) was shown to drive lactate-dependent enhancement of cell survival and metastasis in pancreatic and breast cancers. Furthermore, our previous study confirmed the involvement of HCAR1 in lactate-related enhancement of DNA repair in cervical cancer cells. In the present study, we examined the possible mechanisms of HCAR1-mediated enhancement of DNA repair capacity. We observed that the HCAR1 agonist dihydroxybenzoic acid (DHBA) up-regulated BRCA1 (breast cancer type 1 susceptibility protein) and NBS1 (Nijmegen breakage syndrome 1) expression in HeLa cells. Moreover, HCAR1 silencing decreased mRNA and protein levels of BRCA1 by 30% and 20%, respectively. Immunocytochemical analyses of BRCA1, nibrin and DNA-PKcs indicated an increased accumulation of these proteins in cell nuclei after DHBA stimulation. Subsequently, these changes in the DNA repair protein levels translated into an enhanced DNA repair rate after doxorubicin treatment, as shown by γ-H2AX and comet assay experiments. In contrast, the down-regulation of HCAR1 decreased the efficiency of DNA repair. Finally, we observed the abrogation of DHBA-driven BRCA1 protein up-regulation and enhanced DNA repair following the preincubation of cells with the PKC inhibitor Gö6983. Taken together, our data indicate that lactate receptor/HCAR1 expression in cervical carcinoma cells may contribute to the modulation of cellular DNA repair mechanisms.

Abstract P2-03-01: Mutational landscape of breast cancers from PALB2 germline mutation carriers

Abstract Background: The PALB2 gene encodes the partner and localizer of the BRCA2 protein, which participates in homologous recombination during DNA repair via an interaction with BRCA1 and BRCA2. Germline mutations in PALB2 are associated with an increased risk of breast cancer, with a cumulative risk of 35% by age 70 in female PALB2 mutant carriers. The aims of this project were to characterize the genomic landscape of PALB2 breast cancers and define the differences in the repertoire of somatic genetic alterations and mutational signatures between PALB2, BRCA1 and BRCA2 breast cancers. Methods: Representative samples from fourteen breast cancers from patients with known PALB2 germline mutations (seven frame-shift (2 H1170fs, 3 K346fs, 1 T841fs and 1 L531fs), five truncating (3 W1038* and 2 Q775*) and two missense (W1140G and L35P)) were microdissected to ensure a tumor cell content of >70%. DNA samples from microdissected tumors and their matched normal counterparts were subjected to whole exome sequencing on an Illumina HiSeq2000 to a median depth of 118x (range 33-193x). Somatic single nucleotide variations were detected using MuTect, and small insertions and deletions were identified using Strelka and Varscan2. Using ABSOLUTE and FACETS, we investigated the presence of loss of heterozygosity (LOH) of the PALB2 wild-type allele in these tumors. In addition, the mutational signatures and large scale state transitions (LSTs) were defined. The repertoire of somatic mutations identified in PALB2 breast cancers was compared to that of breast cancers from BRCA1 (n=11) and BRCA2 (n=10) germline mutation carriers from The Cancer Genome Atlas study. Results: PALB2 breast cancers were found to harbor a median of 80 somatic mutations (range 22-286) and one somatic mutation (range 0-13) affecting known cancer genes. Somatic loss of the PALB2 wild-type allele was found in five cases, and in three additional cases, a second PALB2 somatic mutation likely constituted the second 'hit' (two with truncating mutations, Q479* and Q61*, and one with a Q921fs frameshift mutation). Six PALB2 breast cancers displayed the BRCA mutations signature; of these, five had PALB2 bi-allelic inactivation (three LOH of the wild-type allele and two a second PALB2 somatic mutation). 71% of the samples were found to have LSTs, including all cases with a BRCA mutational signature. A significant association between PALB2 bi-allelic inactivation and concurrent BRCA signature and high LST was observed (p=0.015). Breast cancers from PALB2 mutation carriers had fewer somatic TP53 mutations than BRCA1 breast cancers (3/14, 21% vs 9/11, 82%, p=0.004), but no difference in the repertoire of somatic mutations compared to that of BRCA2 breast cancers. Conclusions: PALB2 breast cancers were found to harbor pathogenic mutations in driver genes, including TP53, PIK3CA, NF1 and NCOR1, however lacked highly recurrent somatic mutations. Unlike BRCA1/2 breast cancers, the majority of breast cancers from PALB2 germline mutation carriers lacked LOH of the PALB2 wild-type allele. Importantly, however, an association between PALB2 bi-allelic inactivation and the BRCA mutational signature and LSTs was observed, providing additional evidence for a homologous recombination-deficient phenotype at least in a subset of PALB2 cancers. Citation Format: Burke KA, Berman S, Geyer FC, Piscuoglio S, Ng CK, Wen YH, Mannermaa A, Peterlongo P, Tondini C, Janatova M, Soo Hwang T, Ng P-S, Looi LM, Chenevix-Trench G, Southey MC, Weigelt B, Foulkes W, Tischkowitz M, Reis-Filho JS, PALB2 Interest Group. Mutational landscape of breast cancers from PALB2 germline mutation carriers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-03-01.

Abstract S2-02: The landscape of somatic genetic alterations in BRCA1 and BRCA2 breast cancers

Abstract Introduction: Women carrying BRCA1 or BRCA2 germline mutations have a 45-80% lifetime risk of developing breast cancer (BC). BRCA1 and BRCA2 are perceived as bona fide tumor suppressor genes, whereby bi-allelic inactivation in tumor cells is required for tumorigenesis. Recent studies have indicated that loss of heterozygosity (LOH) of the wild-type allele of BRCA1 may be heterogeneous and constitute a late event. Therefore, additional somatic events prior to full BRCA1/2 inactivation may be required for tumorigenesis. Given that the somatic events that result in the development of BRCA1/2-BCs and their chronology are not understood, here we sought to define the genomic landscape of BRCA1/2-BCs and whether LOH of BRCA1/2 wild-type allele and/or mutations affecting additional tumor suppressor genes would be clonal or subclonal in these cancers. Methods: We retrieved 29 BRCA1-BCs and 10 BRCA2-BCs from the Pathology Departments of the authors' institutions. DNA extracted from microdissected tumor and normal breast samples was subjected to targeted capture massively parallel sequencing using either the MSK-IMPACT assay or an assay targeting all exons of 254 genes recurrently mutated in BC or related to DNA repair. Somatic single nucleotide variants, small insertions and deletions and copy number alterations affecting genes present in both sequencing assays (111 genes) were defined using state-of-the-art bioinformatics algorithms. ABSOLUTE and FACETS were employed to define clonal (i.e. present virtually in 100% of the cancer cells of a given case) and subclonal mutations and the presence of LOH of the BRCA1 and BRCA2 wild-type alleles. Results: Our analysis revealed bi-allelic inactivation of BRCA1 in 28 of 29 BRCA1-BCs (93% harbored LOH of the BRCA1 wild-type allele and 3% harbored a second somatic BRCA1 pathogenic mutation). The only BRCA1-BC lacking bi-allelic inactivation of BRCA1 was an estrogen receptor-positive lobular carcinoma, lacking genomic features consistent with homologous recombination DNA repair defects, diagnosed at 62 years of age. Bi-allelic inactivation of BRCA2 was found in all cases (100% of harbored LOH of the BRCA2 wild-type allele). A clonal somatic 'second hit' resulting in bi-allelic inactivation of BRCA1 or BRCA2 was detected in 76% and 100% of BRCA1-BCs and BRCA2-BCs, respectively. In BRCA1-BCs, TP53 mutations were detected in 76% of cases, and these mutations were found to be clonal in 58% of cases. The repertoire of somatic mutations affecting BRCA1-BCs included clonal somatic mutations or homozygous deletions of known tumor suppressor genes, such as PTEN, RB1, CDKN2A and NF1. In contrast, only 10% of the BRCA2-BCs harbored TP53 somatic mutations. Though clonal somatic mutations in several cancer genes were detected, 40% of BRCA2-BCs had no mutations affecting the cancer genes analyzed. Conclusions: Bi-allelic inactivation of BRCA1 and BRCA2 are frequent events in BRCA1-BCs and BRCA2-BCs, respectively. In a subset of BRCA1-BCs, however, the second 'hit' appeared to be subclonal, whereas mutations affecting TP53 and other tumor suppressor genes were clonal, supporting the notion that at least in a subset of these tumors, loss of the wild-type allele of BRCA1 may be preceded by inactivation of another tumor suppressor gene. Citation Format: Geyer FC, Burke KA, Macedo GS, Piscuoglio S, Ng CK, Martelotto LG, Papanastatiou AD, De Filippo MR, Schultheis AM, Brogi E, Robson M, Wen YH, Weigelt B, Schnitt SJ, Tung N, Reis-Filho JS. The landscape of somatic genetic alterations in BRCA1 and BRCA2 breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S2-02.

Abstract OI-1: OI-1 Decoding breast cancer predisposition genes

Abstract Women with one or more first-degree female relatives with a history of breast cancer have a two-fold increased risk of developing breast cancer. This risk increases with the number of affected family members, particularly for those diagnosed at a young age or with bilateral disease. Segregation analyses of breast cancer families led to the identification of inactivating mutations in the BRCA1 and BRCA2 breast cancer susceptibility genes. Pathogenic variants resulting in truncation of these proteins, along with a small number of pathogenic missense variants, have been associated with 55% to 65% lifetime risk of breast cancer in the general population and up to 85% lifetime risks among women with a strong family history of the disease. In contrast, the influence on cancer risk of many rare variants of uncertain significance (VUS) is not known. Mutations in several genes in addition to BRCA1 and BRCA2 have been implicated in predisposition to breast cancer through family segregation studies. These include moderate risk mutations (relative risk between 2.0 and 5.0) in CHEK2 and ATM, high-risk mutations (relative risk greater than 5.0) in PALB2, and high-risk mutations in CDH1, PTEN, STK11, and TP53 that are associated with hereditary diffuse gastric cancer, Cowden, Peutz-Jeghers, and Li-Fraumeni syndromes. However, risks of breast and other cancers associated with mutations in these and other genes that have been suggested to predispose to cancer have not been established or have very broad confidence intervals. Despite the absence of accurate risk estimates clinical testing with gene panels for hereditary cancer gene mutations has become an integral component of medical management for high-risk families and individuals with triple negative breast cancer. To estimate breast cancer risks several large studies of breast cancer patients from the general population and from high-risk families are underway. These studies are expected to identify the subset of panel genes that are associated with increased risk of breast cancer or breast cancer subtypes and to establish more accurate risks of breast and other cancers. In addition, classification of the clinical relevance of VUS in BRCA1, BRCA2, and other predisposition genes remains a significant problem, with patients unable to benefit from enhanced risk assessment, prevention, and clinical management. Thousands of VUS in BRCA1 and BRCA2 have already been identified through clinical testing and many more in BRCA1, BRCA2, and other predisposition genes are being identified through panel-based genetic testing and tumor sequencing studies. Efforts to classify VUS as neutral or pathogenic have focused on large case-control studies and quantitative multifactorial models based on family history of cancer and pathology. However, more recent studies have established the utility of validated functional assays for VUS assessment. Hundreds of BRCA1 and BRCA2 VUS have been reclassified by commercial testing laboratories or by the ENIGMA consortium that serves as the expert review panel for variants in these genes for the ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and BRCA Exchange (http://brcaexchange.org) publically accessible websites. Common genetic variants associated with breast cancer risk have also been identified through genome-wide association studies (GWAS). The magnitude of these associations have ranged from 1.03 to 1.29 in women of European ancestry. While the majority of the 179 known breast cancer variants are associated with estrogen receptor (ER) positive breast cancer, 21 of these show specific associations with ER negative or triple negative (ER negative, PR negative, HER2 negative) breast cancer. The variants in risk loci account for approximately 19% of the familial relative risk of breast cancer. Several studies have recently established that polygenic risk scores derived from these risk variants improve personalized risk assessment by identifying women at increased or reduced risk of breast cancer in the general population and by modifying risks of breast cancer among BRCA1 and BRCA2 mutation carriers. In summary, germline genetic studies have substantially changed the landscape of breast cancer, identifying mutations associated with moderate and high risks of breast cancer in up to 5% of population-based breast cancer cases and 35% of high-risk families and providing personalization of underlying breast cancer risk for all women. Citation Format: Couch FJ. OI-1 Decoding breast cancer predisposition genes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OI-1.

Abstract P5-09-06: Ovarian reserve and response to controlled ovarian hyperstimulation (COH) in breast cancer women with and without BRCA mutation

Abstract Background: 6300 new cases of breast cancer arise in young women under 40 each year in France. Some of them are BRCA 1 or 2 mutation carriers. Most of them receive a potentially gonadotoxic chemotherapy while they have not yet completed their family. Since 2011, a systematic proposal of ovarian reserve follow-up and fertility preservation by oocyte freezing is provided to each young early breast cancer (BC) patients (pts) of our program (NCT 01614704). Preliminary results were presented at the SABCS in 2013. We now investigate the impact of BRCA mutation on the ovarian reserve and the ovarian response to simulation. Methods: 115 young BC pts were systematically referred to a reproductive medicine centre before starting chemotherapy. Inclusion criteria were age 18 to 38, histologically confirmed invasive breast carcinoma, absence of metastases. According to their personal and familial history, genetic counselling was performed and if the patient met the criteria and agreed, BRCA genes were analysed. Pts in an adjuvant setting and who were asking for fertility preservation underwent COH during the interval between complete surgery and start of adjuvant chemotherapy. Ovarian stimulation protocol consisted in a conventional antagonist protocol with recombinant FSH starting on day 2 of the menstrual cycles. The GnRh antagonist was started on day of the COH and the final oocyte maturation was achieved by an injection of triptorelin 0.2 mg when at least 3 follicles reached 18 mm of diameter. All pts gave their informed consent for COH, egg/embryo freezing and follow-up. Results: 115 pts achieved pre-treatment AMH and AFC assessment. 60 (52,1%) were eligible for COH in order to cryopreserve egg or embryos. BRCA analysis was performed in 83 pts. 23 did not meet the criteria or refused. 9 analyses are still in process. 17 (20.4%) pts were positive for BRCA mutation (BRCA1: 13; BRCA2: 4) and 66 were not. In the mutation carriers group (n=17), median age was 32 years (Range 25-37). Median initial AMH levels and AFC were 23 pmol/l (5.1–223) and 20 (6-100), respectively. Eight pts underwent COH. Median duration of stimulation was 9,5 days (8-13) with a median cumulative dose of gonadotropins of 2875 UI (1200-5450). The median number of vitrified oocytes was 5,5 (0-15). Two patients chose frozen embryo preservation (1 and 2 eggs respectively). In the non-carriers group (n=66), median age was 31 years (24-37). Median initial AMH levels and AFC were 23.4 pmol/l (0.8-136) and 24 (1-68). 27 pts underwent COH. Median duration of stimulation was 10 days (7-14). Dose of gonadotropins was 2700 UI (1365-5600). The median number of vitrified oocytes was 6 (0-18). The 3 patients chose eggs preservation (0.0 and 3 eggs respectively). There was no significant difference in the two groups. Discussion/ Conclusion: Few studies stated that BRCA1 mutation may be associated with reduced ovarian reserve in healthy BRCA mutation carriers. Meirow and al concluded that both healthy and BC BRCA mutation carriers demonstrated normal ovarian response in vitro fertilization cycles. Our results show that ovarian reserve of BRCA 1/2 mutations BC carriers do not differ from that of non-carriers. Response to COH seems similar in both groups too. Citation Format: Mailliez A, Keller L, Menu-Hespel S, Plouvier P, D'Orazio E, Basson L, Pigny P, Bonneterre J, Decanter C. Ovarian reserve and response to controlled ovarian hyperstimulation (COH) in breast cancer women with and without BRCA mutation [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-09-06.

Hsa-miR-520d-5p promotes survival in human dermal fibroblasts exposed to a lethal dose of UV irradiation.

We previously reported that hsa-miR-520d-5p is functionally involved in the induction of the epithelial–mesenchymal transition and stemness-mediated processes in normal cells and cancer cells, respectively. On the basis of the synergistic effect of p53 upregulation and demethylation induced by 520d-5p, the current study investigated the effect of this miRNA on apoptotic induction by ultraviolet B (UVB) light in normal human dermal fibroblast (NHDF) cells. 520d-5p was lentivirally transfected into NHDF cells either before or after a lethal dose of UVB irradiation (302 nm) to assess its preventive or therapeutic effects, respectively. The methylation level, gene expression, production of type I collagen and cell cycle distribution were estimated in UV-irradiated cells. NHDF cells transfected with 520d-5p prior to UVB irradiation had apoptotic characteristics, and the transfection exerted no preventive effects. However, transfection with 520d-5p into NHDF cells after UVB exposure resulted in the induction of reprogramming in damaged fibroblasts, the survival of CD105-positive cells, an extended cell lifespan and prevention of cellular damage or malfunction; these outcomes were similar to the effects observed in 520d-5p-transfected NHDF cells (520d/NHDF). The gene expression of c-Abl (Abelson murine leukemia viral oncogene homolog 1), ATR (ataxia telangiectasia and Rad3-related protein), and BRCA1 (breast cancer susceptibility gene I) in transfectants was transcriptionally upregulated in order. These mechanistic findings indicate that ATR-dependent DNA damage repair was activated under this stressor. In conclusion, 520d-5p exerted a therapeutic effect on cells damaged by UVB and restored them to a normal senescent state following functional restoration via survival of CD105-positive cells through c-Abl-ATR-BRCA1 pathway activation, p53 upregulation, and demethylation.

Can we prevent BRCA1-associated breast cancer by RANKL inhibition?

BRCA1 mutation carriers face a high lifetime risk of breast cancer, estimated at 60% compared to 10% in the general population. BRCA1 breast cancers typically have an aggressive course (i.e., high-grade, triple-negative) and are associated with a poor prognosis. At present, primary prevention is limited to prophylactic removal of the unaffected breasts. Effective chemopreventive strategies are not yet available. Emerging evidence suggests that BRCA1 mutation carriers have high circulating levels of progesterone which may play a role in their susceptibility to breast cancer. Recently, the RANK/RANKL system was found to be dysregulated in women with a BRCA1 mutation. Mutation carriers had significantly lower endogenous levels of osteoprotegerin (OPG) than women without a BRCA1 mutation. OPG is an endogenous decoy receptor for RANKL and inhibits RANKL-mediated signaling. RANKL binds to RANK on mammary epithelial cells and stimulates their proliferation and maturation. Low OPG levels may contribute to mammary tumorigenesis through increased proliferation and may explain in part the increased breast cancer risk in BRCA1 mutation carriers. Denosumab is an anti-RANKL monoclonal antibody which is approved to treat osteoporosis and to prevent skeletal damage caused by bone metastases. The emerging role of aberrant RANK-signaling in BRCA1 tumorigenesis suggests that targeting of RANKL may prevent breast cancer among women with germline BRCA1 mutations. Clinical investigations of denosumab are warranted and may lead to a novel chemopreventive approach for breast cancer for high-risk women.

Outcome of ovarian cancer after breast cancer in BRCA1 and BRCA2 mutation carriers.

Background:It is unknown whether a history of breast cancer (BC) affects the outcome of BRCA1/2-associated epithelial ovarian cancer (EOC). This was investigated in the current analysis.Methods:We included 386 BRCA1/2-associated EOC patients diagnosed between 1980 and 2015. Progression-free survival (PFS), progression-free interval (PFI), overall survival (OS) and ovarian cancer-specific survival (OCSS) were compared between EOC patients with and without previous BC.Results:BRCA-associated EOC patients with, vs without, a BC history had a significantly worse PFS and PFI (multivariate hazard ratio (HRmult) 1.47; 95% confidence interval (CI) 1.03–2.08 and HRmult 1.43; 95% CI 1.01–2.03), and a non-significantly worse OS (HRmult 1.15; 95% CI 0.84–1.57) and OCSS (HRmult 1.18; 95% CI 0.85–1.62). Ovarian cancer-specific survival was significantly worse for the subgroup treated with adjuvant chemotherapy for BC (HRmult 1.99; 95% CI 1.21–3.31).Conclusions:Our results suggest that BRCA1/2-associated EOC patients with a previous BC have a worse outcome than EOC patients without BC, especially when treated with adjuvant chemotherapy.

Relationships, Intimacy, & Body Image After Breast Cancer

Relationships, Intimacy, & Body Image After Breast Cancer

Reappearance from Obscurity: Mammalian Rad52 in Homologous Recombination.

Homologous recombination (HR) plays an important role in maintaining genomic integrity. It is responsible for repair of the most harmful DNA lesions, DNA double-strand breaks and inter-strand DNA cross-links. HR function is also essential for proper segregation of homologous chromosomes in meiosis, maintenance of telomeres, and resolving stalled replication forks. Defects in HR often lead to genetic diseases and cancer. Rad52 is one of the key HR proteins, which is evolutionarily conserved from yeast to humans. In yeast, Rad52 is important for most HR events; Rad52 mutations disrupt repair of DNA double-strand breaks and targeted DNA integration. Surprisingly, in mammals, Rad52 knockouts showed no significant DNA repair or recombination phenotype. However, recent work demonstrated that mutations in human RAD52 are synthetically lethal with mutations in several other HR proteins including BRCA1 and BRCA2. These new findings indicate an important backup role for Rad52, which complements the main HR mechanism in mammals. In this review, we focus on the Rad52 activities and functions in HR and the possibility of using human RAD52 as therapeutic target in BRCA1 and BRCA2-deficient familial breast cancer and ovarian cancer.

Bilateral Oophorectomy and Breast Cancer Risk in BRCA1 and BRCA2 Mutation Carriers.

Whether oophorectomy reduces breast cancer risk among BRCA mutation carriers is a matter of debate. We undertook a prospective analysis of bilateral oophorectomy and breast cancer risk in BRCA mutation carriers. Subjects had no history of cancer, had both breasts intact, and had information on oophorectomy status (n = 3722). Women were followed until breast cancer diagnosis, prophylactic bilateral mastectomy, or death. A Cox regression model was used to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) of breast cancer associated with oophorectomy (coded as a time-dependent variable). All statistical tests were two-sided. Over a mean follow-up of 5.6 years, 350 new breast cancers were diagnosed. Among women with a BRCA1 or BRCA2 mutation, oophorectomy was not associated with breast cancer risk compared with women who did not undergo an oophorectomy. The age-adjusted hazard ratio associated with oophorectomy was 0.96 (95% CI = 0.73 to 1.26, P = 76) for BRCA1 and was 0.65 (95% CI = 0.37 to 1.16, P = 14) for BRCA2 mutation carriers. In stratified analyses, the effect of oophorectomy was statistically significant for breast cancer in BRCA2 mutation carriers diagnosed prior to age 50 years (age-adjusted HR = 0.18, 95% CI = 0.05 to 0.63, P = 007). Oophorectomy was not associated with risk of breast cancer prior to age 50 years among BRCA1 mutation carriers (age-adjusted HR = 0.79, 95% CI = 0.55 to 1.13, P = 51). Findings from this large prospective study support a role of oophorectomy for the prevention of premenopausal breast cancer in BRCA2, but not BRCA1 mutation carriers. These findings warrant further evaluation.

Cirugía profiláctica en síndrome de cáncer hereditario de mama

IntroductionApproximately 5-10% of global cancer has a genetic base. Nowadays it is possible to identify those who have a genetic predisposition for breast cancer, so they can be treated in short term. ObjectivesEvaluate how useful is prophylactic surgery on Hereditary Breast cancer Syndrome. Materials and methodsThis investigation is a literature review of different research papers from electronic databases such as Scientific Electronic Library Online (SciELO), MedLine-PubMed and UpToDate. The research was made with clinical queries and MeSH terms, specially focused on Hereditary Breast cancer Syndrome. ResultsThis research shows that prophylactic bilateral mastectomy (PBM) decreases 90% risk of developing breast cancer in BRCA1 and BRCA2 carriers. On the other hand, Salpyngo-oophoferectomy (SO) technique decreases around 53% chances of breast cancer, depending on the associated mutation. ConclusionProphylactic surgery has shown an important role decreasing the risk on Hereditary Breast cancer Syndrome. So on, in cases of BRCA1 and BRCA2 carriers we suggest to perform prophylactic surgery.

PARP Inhibitors in Clinical Use Induce Genomic Instability in Normal Human Cells.

Poly(ADP-ribose) polymerases (PARPs) are the first proteins involved in cellular DNA repair pathways to be targeted by specific inhibitors for clinical benefit. Tumors harboring genetic defects in homologous recombination (HR), a DNA double-strand break (DSB) repair pathway, are hypersensitive to PARP inhibitors (PARPi). Early phase clinical trials with PARPi have been promising in patients with advanced BRCA1 or BRCA2-associated breast, ovary and prostate cancer and have led to limited approval for treatment of BRCA-deficient ovary cancer. Unlike HR-defective cells, HR-proficient cells manifest very low cytotoxicity when exposed to PARPi, although they mount a DNA damage response. However, the genotoxic effects on normal human cells when agents including PARPi disturb proficient cellular repair processes have not been substantially investigated. We quantified cytogenetic alterations of human cells, including primary lymphoid cells and non-tumorigenic and tumorigenic epithelial cell lines, exposed to PARPi at clinically relevant doses by both sister chromatid exchange (SCE) assays and chromosome spreading. As expected, both olaparib and veliparib effectively inhibited poly-ADP-ribosylation (PAR), and caused marked hypersensitivity in HR-deficient cells. Significant dose-dependent increases in SCEs were observed in normal and non-tumorigenic cells with minimal residual PAR activity. Clinically relevant doses of the FDA-approved olaparib led to a marked increase of SCEs (5-10-fold) and chromatid aberrations (2-6-fold). Furthermore, olaparib potentiated SCE induction by cisplatin in normal human cells. Our data have important implications for therapies with regard to sustained genotoxicity to normal cells. Genomic instability arising from PARPi warrants consideration, especially if these agents will be used in people with early stage cancers, in prevention strategies or for non-oncologic indications.

Abstract 100: The landscape of somatic genetic alterations in BRCA1 and BRCA2 breast cancers

Abstract Background: BRCA1 or BRCA2 germline mutations account for a substantial proportion of hereditary breast cancers. The majority of BRCA1-associated breast cancers are of triple-negative phenotype (estrogen receptor (ER)-, progesterone receptor (PR)- and HER2-negative) and harbor TP53 somatic mutations. BRCA2-associated cancers are less homogeneous and often ER-positive. Systematic analyses of the profiles of somatic genetic alterations in BRCA1- and BRCA2-breast cancers have yet to be reported. Here we sought to determine the repertoire of somatic mutations and copy number alterations (CNAs) in breast cancers occurring in patients with BRCA1 or BRCA2 germline mutations.. Methods: Eleven BRCA1- and five BRCA2-associated breast cancers were microdissected. DNA was extracted from microdissected frozen tumor-normal pairs and subjected to targeted capture massively parallel sequencing using the MSK-IMPACT platform, which includes all exons and selected introns and regulatory regions of 410 key cancer genes. Somatic single nucleotide variants, small insertions and deletions, CNAs and the cancer cell fraction (CCF) of each alteration were defined using state-of-the-art bioinformatics algorithms. Results: 82% (9/11) and 18% (2/11) of BRCA1-breast cancers were triple-negative and ER-positive/HER2-negative, respectively. All BRCA2-cancers were ER-positive, of which one was HER2-positive. Sequencing analysis revealed a median of four (2-11) and two (0-6) non-synonymous somatic mutations in BRCA1- and BRCA2-breast cancers, respectively. Within BRCA1-breast cancers all but one (10/11, 91%) harbored TP53 clonal somatic mutations, and clonal somatic loss of the BRCA1 wild-type allele was found in 8 of these cases. The BRCA1-breast cancer lacking these alterations was ER-positive and the only case harboring a PIK3CA mutation. Additional clonal mutations identified in BRCA1-breast cancers included somatic mutations affecting EGFR and RB1. Subclonal mutations in known cancer genes (e.g. GATA3 and PTEN) were also identified, suggesting intra-tumor genetic heterogeneity. All BRCA2-breast cancers analyzed displayed clonal loss of the wild-type of BRCA2, but no gene was found to be recurrently mutated. Conclusions: BRCA1- and BRCA2-breast cancers are both characterized by clonal inactivation of the BRCA1 and BRCA2 wild-type alleles, respectively, which likely constitute early genetic events. Within BRCA1-breast cancers, TP53 mutations are highly recurrent and clonal, and may precede somatic loss of the BRCA1 wild-type allele, as the latter was subclonal in one case harboring a clonal TP53 somatic mutation. Citation Format: Gabriel S. Macedo, Kathleen A. Burke, Salvatore Piscuoglio, Charlotte K. Y. Ng, Felipe C. Geyer, Luciano G. Martelotto, Anastasios D. Papanastatiou, Maria R. De Filippo, Anne M. Schultheis, Edi Brogi, Mark E. Robson, Hannah Y. Wen, Britta Weigelt, Jorge S. Reis-Filho. The landscape of somatic genetic alterations in BRCA1 and BRCA2 breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 100.

Abstract 134: Mutational landscape of breast cancers from PALB2 germline mutation carriers

Abstract Introduction: The PALB2 gene encodes the partner and localizer of BRCA2 protein, which interacts with BRCA1/2 and is involved in homologous recombination DNA repair. Germline mutations in PALB2 are associated with an increased risk of breast cancer, with a cumulative risk of 35% by age 70 in female PALB2 mutation carriers. Whether the PALB2 wild-type allele is lost in the development of PALB2 breast cancers has yet to be defined. Further, the repertoire of somatic genetic alterations in these tumors is currently unknown. In this study we sought to characterize the genomic landscape of PALB2 breast cancers and to define the differences in the repertoire of somatic genetic alterations and mutational signatures between PALB2 and BRCA1 and BRCA2 breast cancers. Material and Methods: Representative samples from nine breast cancers from patients with known PALB2 germline mutations were microdissected. DNA samples from microdissected tumors and matched normal counterparts were subjected to whole exome sequencing on an Illumina HiSeq2000. Somatic mutations were defined using MuTect and insertions and deletions using Strelka and Varscan2. Driver mutations were defined by state-of-the-art bioinformatics methods. Mutational signatures were defined using non-negative matrix factorization. Copy number alterations (CNAs) and regions with loss of heterozygosity were determined using FACETS. The mutational frequency of breast cancers from PALB2 germline mutation carriers was compared to that of breast cancers from BRCA1 (n = 11) and BRCA2 (n = 10) germline mutation carriers from The Cancer Genome Atlas study. Results: Three patients harbored germline frame-shift PALB2 mutations (2 S1169fs, 1 T841fs), five displayed truncating mutations (3 W1038* and 2 Q775*) and 1 harbored a missense mutation (W1140G, of uncertain significance). Somatic loss of the PALB2 wild-type allele was found in 3 cases, in 2 of which the loss was caused by CNAs and in 1 case it was caused by a somatic PALB2 Q479* mutation. A median of 65 somatic mutations (range 45-223) and a median of 1 driver mutation (range 0-3) were identified per tumor. Cancer genes mutated in PALB2 breast cancers included TP53 (n = 2), PIK3CA (n = 2), NF1 (n = 1) and NCOR1 (n = 1). Six cases displayed mutational signatures consistent with the aging process; the BRCA signature was not found in any of the cases analyzed. Breast cancers from PALB2 mutation carriers had fewer somatic TP53 mutations than BRCA1 breast cancers (2/9, 22% vs 9/11, 82%, p = 0.02). No difference in the repertoire of somatic mutations between PALB2 and BRCA2 breast cancers was observed. Conclusion: Unlike breast cancers from BRCA1 and BRCA2 mutation carriers, the majority of breast cancers from PALB2 mutation carriers lacked somatic loss of the wild-type allele and none displayed a BRCA mutational signature. No highly recurrently mutated gene was identified, but pathogenic mutations in driver genes (TP53, PIK3CA, NF1 and NCOR1) were found. Citation Format: Salvatore Piscuoglio, Charlotte KY Ng, Y Hannah Wen, Arto Mannermaa, Paolo Peterlongo, Carlo Tondini, Marketa Janatova, Teo Soo Hwang, Pei-Sze Ng, Lai-Meng Looi, William Foulkes, Georgia Chenevix-Trench, Britta Weigelt, Melissa C. Southey, Marc Tischkowitz, Jorge S. Reis-Filho, PALB2 Interest Group. Mutational landscape of breast cancers from PALB2 germline mutation carriers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 134.

Abstract CT051: Carboplatin and talazoparib combination therapy results in differential efficacy and hematologic toxicity in BRCA-mutated patients

Abstract Background: Talazoparib is a novel PARP inhibitor (PARPi) in clinical development. Synergistic anti-tumor effects of PARPi and chemotherapy have been observed in preclinical models. This Phase 1 trial evaluates the tolerability, dose limiting toxicities (DLT) and efficacy of talazoparib in combination with carboplatin in patients with or without germ line DNA repair mutations. We hypothesize that talazoparib may overcome carboplatin resistance but induce greater toxicity in patients with DNA repair defects. Methods: Talazoparib and carboplatin pharmacokinetics (PK), safety and anti-tumor activity were evaluated in a 3+3 dose escalation design. Genetic testing, PK, pharmacodynamic effects (PD), biomarkers, and alternate dose modeling were evaluated to better understand the interaction of carboplatin and talazoparib. Results: 24 patients (median age 59y) were enrolled in 4 cohorts and treated with talazoparib 0.75 or 0.1 mg/day and carboplatin AUC 1 or 1.5 for 3/3 or 2/3 weeks. Tumor types included: breast (n = 11), prostate (n = 5), cholangiocarcinoma (n = 2), ovarian (n = 2), bladder (n = 1), adenoid cystic carcinoma (n = 1) and adenocarcinoma of unknown origin (n = 2). Germline mutations were noted in BRCA1 (n = 3), BRCA2 (n = 3), BRIP1 (n = 1), and MSH6 (n = 1) and germ line variants of uncertain significance in BRIP1 (n = 1) and BRCA2 (n = 1). Somatic mutations were found in BRCA2 (n = 1), BAP1 (n = 2) and PALB2 (n = 1). DLTs included fatigue and thrombocytopenia; other non-DLT grade 3/4 toxicities included fatigue (13%), neutropenia (33%), thrombocytopenia (33%), and anemia (58%). Post cycle 1 hematological toxicities required dose delays and reductions in almost all patients. One complete response occurred in a patient with germline BRCA1 (gBRCA1) breast cancer and a partial response (PR) in a gBRCA2 bladder cancer patient; 11 patients (pts) had stable disease (SD) for ?3 months. 5 pts had progressive disease (PD) and 6 pts are not yet evaluable. Of those with PD, 83% had prior platinum therapy, whereas in those with SD or disease response, 46% had prior platinum therapy. Effects of talazoparib/carboplatin on platelet (-11.4% vs. -1.1% P<0.001) and neutrophil counts (-9.1% vs. -3.8% P<0.001) were significantly more profound in gBRCA carriers. Simulations using this model suggest that q4-weekly carboplatin dosing in patients with gBRCA or DNA repair defects and every other week dosing in non-carriers would be more tolerable for the combination. Conclusions: Mutation carriers in BRCA or other DNA repair genes responded better to this combination than non-carriers. Progression with prior platinum or PARPi did not exclude response or clinical benefit. Talazoparib and carboplatin showed significant hematologic toxicity in those with gBRCA or other germ line DNA repair mutations. Lower dosing frequencies of carboplatin when given with PARPi may be required in gBRCA mutation carriers. Citation Format: Mallika S. Dhawan, Rahul Aggarwal, Imke Bartelink, Jim Leng, Scott Thomas, Nela Pawlowska, Laurie Stevenson, Amy Jo Chien, Robin Kate Kelley, Pamela N. Munster. Carboplatin and talazoparib combination therapy results in differential efficacy and hematologic toxicity in BRCA-mutated patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT051.