Abstract A49: BRCA1 deficiency is a recurrent event in early onset triple-negative breast cancer (TNBC): A comprehensive analysis of germline mutations and somatic promoter methylation

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