BRCA1 deficiency was recurrent in early-onset triple-negative breast cancer in Brazilian patients and associated with improved survival. With the new treatment modalities being investigated, including poly (ADP-ribose)-polymerase (PARP) inhibitor therapy, our results suggest that a significant proportion of young women with this subtype of tumor might benefit from PARP inhibitor treatment, which warrants further investigation.

Circular RNAs (circRNAs) are a class of long non-coding RNAs that have the ability to sponge RNA-Binding Proteins (RBPs). Triple-negative breast cancer (TNBC) has very aggressive behavior and poor prognosis for the patient. Here, we aimed to characterize the global expression profile of circRNAs in TNBC, in order to identify potential risk biomarkers. For that, we obtained RNA-Seq data from TNBC and control samples and performed validation experiments using FFPE and frozen tissues of TNBC patients and controls, followed by in silico analyses to explore circRNA-RBP interactions. We found 16 differentially expressed circRNAs between TNBC patients and controls. Next, we mapped the RBPs that interact with the top five downregulated circRNAs (hsa_circ_0072309, circ_0004365, circ_0006677, circ_0008599, and circ_0009043) and hsa_circ_0000479, resulting in a total of 16 RBPs, most of them being enriched to pathways related to cancer and gene regulation (e.g., AGO1/2, EIF4A3, ELAVL1, and PTBP1). Among the six circRNAs, hsa_circ_0072309 was the one that presented the most confidence results, being able to distinguish TNBC patients from controls with an AUC of 0.78 and 0.81, respectively. This circRNA may be interacting with some RBPs involved in important cancer-related pathways and is a novel potential risk biomarker of TNBC.

Triple-negative breast cancer (TNBC), characterized by lack of expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), is associated with a higher prevalence of germline BRCA1 mutations, especially in younger women. TNBC comprises approximately 15% of breast cancer cases and is considered one of the most aggressive subtypes. BRCA1 protein plays a pivotal role in DNA damage repair, cell cycle control and transcriptional regulation. In this context, we aimed to investigate the transcriptional difference of paired tumor and adjacent normal tissue from young patients in two TNBC groups - with or without BRCA1 germline mutation. Fourteen early-onset patients (≤41 years), with TNBC previously evaluated for BRCA1 and 2 germline mutation, were included. From these, nine patients are BRCA1/2 wild-type and five patients were BRCA1 germline mutation carriers. RNASeq libraries were constructed from rRNA depleted total RNA extracted from paired tumor and normal adjacent samples and sequenced on Illumina NextSeq 500 platform. Approximately 20 million reads aligned to human genome were generated per sample, revealing about 14,500 expressed genes with at least 10 copies per sample. Expression values were normalized by library-size read counts followed by a gene-wise normalization. To explore similarity level between samples we used the full matrix of the log2-transformed gene expression values and calculated sample-to-sample distances for each tumor compared to each normal sample, based on the Euclidian distances between samples considering all available genes. We observed that 80% of the BRCA1-mutated and 67% of the BRCA1/2 wild-type samples were clustered together. Also, we detected that BRCA1-mutated TNBC showed, in general, less difference in transcriptional pattern related to normal breast tissue than the BRCA1/2-wild type TNBC. Moreover, we compared differential expression between normal and tumor samples of each group (BRCA1-mutated and BRCA1/2-wild-type samples) using a linear-based model in DESeq2 program. Based on the criteria of fold-change ≥ │4│and FDR ≤ 0.01 we identified 2,809 DEGs, in the group of BRCA1-mutated samples, being 722 up regulated and 2,087 down regulated in the tumor and 904 DEGs in the group of BRCA1/2- wild-type samples, 399 were upregulated and 505 downregulated in the tumor. Interestingly, both lists of upregulated genes in TNBC of BRCA1-mutated and BRCA1/2-wild type were significantly enriched in the same molecular pathway involved with the cell cycle and mitosis, according to Reactome Pathways software. Overall, our first observation based on this preliminary data was that, in terms of general gene expression analysis, BRCA1-mutated TNBC has less transcriptional modification in comparison to the normal tissue than the BRCA1/2-wild type and that the tumorigenic process of both groups are similar in terms of defective biological pathway. Citation Format: Kivvi Duarte Nakamura, Elisa Ferreira, Rodrigo Ramalho, Rafael Brianese, Dirce Carraro. Transcriptoma analyses in triple-negative breast cancer with BRCA1 germline mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1453. doi:10.1158/1538-7445.AM2017-1453

Intra-tumor genetic heterogeneity is a possible cause of therapy resistance and has consequences in the disease progression and patient survival. Triple-negative breast cancer (TNBC) is associated with rapid growth, early metastasis, and worse prognosis than other breast cancer subtypes. Close association has been identified between TNBC and germline loss-of-function mutations in BRCA1 gene which trigger the tumorigenic process in hereditary TNBC. Although at genomic level, TNBC was previously characterized as more heterogeneous than other breast cancer subtypes, the difference in genomic, epigenomic, and transcriptional variability between two groups of TNBC—sporadic (BRCA1-proficent) and hereditary (BRCA1-associated)—remains unclear. Here, we investigated both sporadic and hereditary TNBC, by using data from copy-number alterations (CNAs) array, methylation array, and RNA-Seq. A total of 80 samples, 40 TNBC BRCA1-proficient (classified as sporadic), 18 TNBC with BRCA1 germline mutation (classifed as hereditary) and 22 normal breast tissues used as controls, were used. Data from methylation and CNA arrays were analyzed with the RnBeads and snapCGH softwares, respectively. RNA-seq data was analyzed mainly with the STAR aligner, Bioconductor-GenomicFeatures and DESeq2 softwares. To assess the intragroup genomic, transcriptional, and epigenomic variability we used the inter-quantile difference between patients of the same group. Our results clearly demonstrated that the TNBC BRCA1-proficient group has a higher intragroup heterogeneity than the BRCA1-mutated group regarding (i) global pattern of gene expression, (ii) number and intensity of CNAs (copy number alterations), and (iii) level of promoter methylation. Moreover, normal breast samples showed the lowest intra-group heterogeneity for the last two features. Then, our results show higher genomic, transcriptional, and epigenomic variability in sporadic than hereditary TNBC and motivates further molecular subtyping of the sporadic TNBCs, which may reveal new targets to improve TNBC treatment. Citation Format: Kivvi Duarte de Mello Nakamura, Rodrigo Fernandes Ramalho, Rafael Canfield Brianese, Ana Krepischi, Bruna D. F. Barros, Dirce Maria Carraro. Intragroup genomic, transcriptional, and epigenomic variability is higher in sporadic than in BRCA1-associated hereditary triple-negative breast tumors [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 A25.