Abstract 1554: Identification of BRCA1 and BRCA2 somatic mutations in breast cancer tumors with loss of BRCA1 nuclear expression

Abstract

Abstract Breast cancer is the first cause of death by cancer in women in Chile as in many countries around the world. Until today, BRCA1 and BRCA2 are the most relevant genes for breast cancer high risk. Moreover, different studies have shown that BRCA1 is under expressed in breast tumors. Our group has found that in a high percentage of hereditary tumors BRCA1 protein have a diminished or null expression, or is mislocalized to the cytoplasm (unpublished data). In this regard, it has become a goal for us to identify if mislocalization and/or loss of expression of BRCA1 and BRCA2 could be related to somatic mutations in the tumors. In this work, we sequenced BRCA1 and BRCA2 in DNA extracted from 14 fresh/frozen tumors. DNA was isolated from each tumor and amplified by PCR in 5 multiplexes (a total of 93 amplicons) considering all exons and intron/exon junctions with approximately 30bp of intronic sequence each side. Multiplexes were then pooled for each patient and processed for sequencing in the GS Junior Roche. All reads were processed with AVA software to detect nucleotide changes in BRCA1 and BRCA2 sequences and analyzed manually to discard misinterpretations. All mutations identified were confirmed by Sanger. From the same tumors we obtained a second section that was formalin fixed and paraffin embedded. This FFPE fragment was cut in 4 μm sections and analyzed by immunohistochemistry and immunofluorescence to assess BRCA1 expression and localization. We found a total of 5 mutations, 3 in BRCA1 and 2 in BRCA2, in 6 tumors. Interestingly, one BRCA1 mutation present in 7 to 20% of reads, was found in 5 tumors. Among those, two tumors presented two different mutations in BRCA2, in addition. The last tumor presented one extra mutation in BRCA1. BRCA1 expression analysis was coherent between immunohistochemistry and immunofluorescence assays. In general 9 tumors showed weak or negative BRCA1 nuclear expression and 5 mainly moderate nuclear staining. In addition 6 tumors presented moderate to strong cytoplasmic expression of the protein, considered abnormal. Considering tumors with weak or negative expression of BRCA1, 40% presents BRCA1 or BRCA2 mutations. The relevance of the absence of BRCA1 expression, or function impairment of BRCA1 and/or BRCA2 due to somatic mutations, relays on the possible treatment with PARP1 inhibitors. Supported by FONDECYT 1120200. Citation Format: Carolina Alvarez, David Wiener, Patricia Gajardo, Wanda Fernandez, Valeria Cornejo, Jorge Gamboa, Pilar Carvallo. Identification of BRCA1 and BRCA2 somatic mutations in breast cancer tumors with loss of BRCA1 nuclear expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1554. doi:10.1158/1538-7445.AM2014-1554