When considering family history, hereditary breast cancer (HBC) syndromes correspond to nearly 5-10% of all breast cancer (BC) cases. However, pathogenic variants in known moderate- and high-risk BC genes explain only ~30% of familial BC cases. Recent advances in sequencing technology resulted in an increasing number of BC genes being revealed in previously negative families by using extended gene panels and whole-exome sequencing (WES). WES offers the opportunity to concomitantly investigate several rare risk genes as well as to identify new BC-predisposing genes. Thus, in this study we used WES to disclose variants contributing to BC increased risk in patients that were negative for mutations in three major BC genes (BRCA1/2 and TP53) and met stringent clinical criteria indicating a genetic predisposition to BC. We selected 17 women (two of them sisters) who fulfilled one or more of the following criteria: early onset BC (<36 years); bilateral BC; breast and other primary related tumor (ovary, fallopian tube, or primary peritoneal tumors). Firstly, we used WES data to search for rare variants in 27 well-established and emerging HBC predisposing genes. This analysis identified two patients harboring truncating mutations, one in ATM (p.Tyr2334Glnfs*4 – pathogenic in ClinVar database) and one in BARD1 (Tyr739Leufs*2 – not described, probably pathogenic according to ACMG guidelines). Additionally, we identified nine variants of unknown clinical significance in these genes, occurring in seven patients. For the remaining 15 patients with no putative pathogenic mutations in HBC genes, we first applied quality filters to exclude false-positive results (included variants: variant base coverage ≥5X, variant frequency ≥25%, QD>2, FS<6). Remaining variants were excluded if present in population databases with a minor allele frequency (MAF) > 0.01 or present in five BRCA1 mutated patients sequenced in the same platforms. Next, using a functional-based variant prioritization, we selected candidate variants according to the predicted impact in the protein function, the affected gene, and segregation with the phenotype (in the family-based study of the two sisters). Candidate variants included all loss-of-function variants as well as missense and in-frame indels occurring in a specific list of 651 genes of interest (DNA repair and cancer-related genes) and if predicted to be damaging by at least 3/6 prediction software. The resulting 244 selected variants were submitted for technical validation by targeted NGS and, of these, 220 were validated (89%). Using the same custom panel, we evaluated 25 control samples of healthy Brazilian women for filtering common polymorphisms in our population, resulting in a final 139 variants in 129 genes (89 LOF and 50 missense variants). For two families---one with WES of two affected sisters and target NGS of one affected aunt; one with WES of the affected daughter and target NGS of the affected mother---we were able to perform cosegregation analysis in other family members and 11 out of 23 variants were found to be segregating with the disease. These 11 distinct genes as well as 82 genes harboring LOF variants were evaluated in an independent cohort of 34 WES of high-risk BC patients. Several rare, possibly damaging variants were identified in this cohort, providing additional evidence of the potential role in BC predisposition of some new genes. Of those, we highlight ATRX, FANCC, TET2, ERCC1, PTPRD, and ROS1 genes, which are involved in DNA repair and other tumorigenic pathways. Overall, our results provide a set of putative BC-predisposing genes and underpin WES as a useful tool for assessing the complex landscape of HBC predisposition. The discovery and validation of new HBC genes in different populations will continue to provide insights into disease mechanisms, eventually leading to the development of more effective therapies and improved management of affected families. Citation Format: Giovana T. Torrezan, Fernanda GSR Almeida, Marcia CP Figueiredo, Bruna DF Barros, Claudia A. Paula, Renan Valieris, Jorge ES Souza, Elisa N. Ferreira, Felipe CC Silva, Amanda F. Nobrega, Maria Isabel Achatz, Edenir I. Palmero, Rodrigo F. Ramalho, Sandro J. Souza, Dirce M. Carraro. Complex landscape of germline variants in hereditary and early-onset breast cancer ascertained through whole exome sequencing [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A37.

Background: Triple-negative breast cancer (TNBC) is a breast cancer subtype that accounts for 15-20% of all breast cancer cases. TNBC treatment is challenging because it does not respond to conventional hormonal and available target therapies, which are effective in other subtypes, making systemic chemotherapy the mainstay treatment. Moreover, TNBC displays higher aggressiveness and distinct metastatic pattern compared to other breast tumors, resulting in worse prognosis and survival. We and others have identified high prevalence of BRCA1 germline mutation in TNBC. However, BRCA1 deficiency may also be caused by somatic gene promoter methylation among other mechanisms. BRCA1/2 deficiency may lead to impairment of DNA repair and tumor development. Hence, understanding the mechanisms of BRCA deficiency in driving this tumor subtype, in both hereditary and sporadic scenery, is of great clinical and biologic interest. Methods: We included in this study TNBC samples unselected for age or family history and attending A. C. Camargo Cancer Center. Tumor tissues were screened for point mutation in BRCA1/2 using next-generation sequencing (NGS). Sanger sequencing was performed in both tumor and normal tissue/leucocyte for categorizing the pathogenic mutations in germline or somatic. Multiplex Ligation-dependent Probe Amplification (MLPA) was used to investigate BRCA1 copy number alterations (CNA) resulting from large rearrangements in tumor and leucocyte. Additionally, for BRCA1 gene silencing investigation a customized bisulfite NGS approach was performed to assess the BRCA1 promoter methylation in tumor tissue. Results: Point mutations were screened in 131 TNBC tumor samples, detecting a total of 18 pathogenic mutations (13.7%)---16 (88.8%) in BRCA1 and 2 (11.2%) in BRCA2. No large rearrangement was detected by MLPA in tumor tissue or leucocyte. Mutations classified as germline accounted for the majority of the pathogenic mutations detected in tumor tissue (93.75% - 15/16)---81.25% (13/16) in BRCA1 and 12.5% (2/16) in BRCA2. Only one somatic mutation was detected (6.25% - 1/16) in BRCA1 gene. Considering early onset TNBC (≤40 year of age) the germline mutation rate increased to 25% (8/32), mainly in BRCA1 gene (22% - 7/32). BRCA1 promoter hypermethylation in tumor was detected in 20.6%, all in sporadic TNBC, with a slight increase to 28% in early onset cases. Ultimately, BRCA impairment by either constitutive or somatic events was identified in 34% (45/131) of the cases and was significantly more frequent in young women (56% in ≤40; 33% in 41-50; 23% in >50 year of age; p=0.007) and associated with better overall and disease-free survival rates in this group of patients. Conclusions: We observed that a high number of the TNBC cases are hereditary and BRCA1-related, especially in young women. For the sporadic cases, BRCA1 gene silencing was also a recurrent event. Taken together, our data showed that BRCA1 impairment (either by germline mutation or somatic promoter hypermethylation) was present at high frequency in the early-onset cases and was associated with better survival for these patients. With the new treatment modalities including Poly (ADP-ribose) polymerase (PARP) inhibitors being investigated, our results shed light that a significant proportion of young women with TNBC might benefit from PARP-inhibitor and future investigation in this subject is warranted. Citation Format: Rafael Canfield Brianese, Kivvi D. M. Nakamura, Fernanda G. S. R. Almeida, Rodrigo F. Ramalho, Elisa N. Ferreira, Bruna D. F. Barros, Maria N. C. Formiga, Victor P. Andrade, Vladmir C. C. Lima, Dirce M. Carraro. BRCA1 deficiency is a recurrent event in early onset triple-negative breast cancer (TNBC): A comprehensive analysis of germline mutations and somatic promoter methylation [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A49.