Molecular features of the basal-like breast cancer subtype based on BRCA1 mutation status Aleix Prat, Cristina Cruz, Katherine A. Hoadley, Orland Díez, Charles M. Perou & Judith Balmaña Breast Cancer Research and Treatment ISSN 0167-6806 Breast Cancer Res Treat DOI 10.1007/s10549-014-3056-x 1 23 Your article is published under the Creative Commons Attribution Non-Commercial license which allows users to read, copy, distribute and make derivative works for noncommercial purposes from the material, as long as the author of the original work is cited. All commercial rights are exclusively held by Springer Science + Business Media. You may self-archive this article on your own website, an institutional repository or funder’s repository and make it publicly available immediately. 1 23 Breast Cancer Res Treat DOI 10.1007/s10549-014-3056-x BRIEF REPORT Molecular features of the basal-like breast cancer subtype based on BRCA1 mutation status Aleix Prat • Cristina Cruz • Katherine A. Hoadley • Orland Dı´ez • Charles M. Perou • Judith Balman˜a Received: 23 January 2014 / Accepted: 5 July 2014 Ó The Author(s) 2014. This article is published with open access at Springerlink.com Abstract BRCA1-mutated breast cancer is associated miRNA expression, and DNA methylation variation, with basal-like disease; however, it is currently unclear if according to BRCA1 status (either germinal or somatic). the presence of a BRCA1 mutation depicts a different entity However, there were significant differences according to within this subgroup. In this study, we compared the average number of mutations and DNA copy-number molecular features among basal-like tumors with and aberrations, and four amplified regions (2q32.2, 3q29, without BRCA1 mutations. Fourteen patients with BRCA1- 6p22.3, and 22q12.2), which are characteristic in high- mutated (nine germline and five somatic) tumors and basal- grade serous ovarian carcinomas, were observed in both like disease, and 79 patients with BRCA1 non-mutated germline and somatic BRCA1-mutated breast tumors. tumors and basal-like disease, were identified from the These results suggest that minor, but potentially relevant, cancer genome atlas dataset. The following molecular data baseline molecular features exist among basal-like tumors types were evaluated: global gene expression, selected according to BRCA1 status. Additional studies are needed protein and phospho-protein expression, global miRNA to better clarify if BRCA1 genetic status is an independent expression, global DNA methylation, total number of prognostic feature, and more importantly, if BRCA1 somatic mutations, TP53 and PIK3CA somatic mutations, mutation status is a predictive biomarker of benefit from and global DNA copy-number aberrations. For intrinsic DNA-damaging agents among basal-like disease. subtype identification, we used the PAM50 subtype pre- dictor. Within the basal-like disease, we observed minor Keywords Basal-like Á BRCA1 Á Intrinsic subtype Á molecular differences in terms of gene, protein, and Breast cancer Abbreviations Aleix Prat and Cristina Cruz have contributed equally to this work. TN Triple-negative BRCA1 Breast cancer 1 early onset Electronic supplementary material The online version of this article (doi:10.1007/s10549-014-3056-x) contains supplementary material, which is available to authorized users. A. Prat (&) K. A. Hoadley Á C. M. Perou Translational Genomics Group, Vall d´Hebron Institute of Lineberger Comprehensive Cancer Center, University of North Oncology (VHIO), Pg Vall d´Hebron, 119-129, 08035 Barcelona, Carolina at Chapel Hill, Chapel Hill, NC 27514, USA Spain e-mail: [email protected] e-mail: [email protected] C. M. Perou e-mail: [email protected] C. Cruz Á J. Balman˜a High Risk and Cancer Prevention Group, Vall d´Hebron Institute O. Dı´ez of Oncology (VHIO), 08035 Barcelona, Spain Oncogenetics Group, Hospital Universitari de la Vall d´Hebron, e-mail: [email protected] Vall d´Hebron Institute of Oncology (VHIO), 08035 Barcelona, J. Balman˜a Spain e-mail: [email protected] e-mail: [email protected] 123 Breast Cancer Res Treat ID4 Inhibitor of DNA binding 4, dominant negative methylation variation: ‘‘BRCA.methylation.27 k.450 k.txt.’’ helix-loop-helix protein For microarray DNA copy-number aberration data: PIK3CA Phosphatidylinositol-4, 5-bisphosphate ‘‘brca_scna_all_thresholded.by_genes.txt.’’ For intrinsic 3-kinase, catalytic subunit alpha subtype identification, we used the PAM50 subtype calls as provided in the TCGA portal. Independent dataset Introduction We evaluated an independent and publicly available microarray-based gene expression dataset (GSE40115) that Studies based on gene expression data have identified and includes breast tumors from 32 patients with basal-like characterized four main intrinsic subtypes of breast cancer disease (20 with BRCA1 germline mutations and 12 with (luminal A, luminal B, HER2-enriched, and basal-like) [1, sporadic tumors [i.e. unknown BRCA1 status]). The file 2]. Among them, the basal-like subtype is associated with ‘‘GSE40115-GPL15931_series_matrix.txt’’ with the nor- young age, BRCA1 germline and somatic mutations [1, 3, malized log2 ratios (Cy5 sample/Cy3 control) of probes 4] and an overall poor prognosis despite that a subgroup of was used. Probes mapping to the same gene (Entrez ID as patients with these tumors has an excellent outcome when defined by the manufacturer) were averaged to generate treated with chemotherapy [5]. In the clinical setting, basal- independent expression estimates. like tumors are usually identified by the lack of expression of hormone receptors by immunohistochemistry (IHC) and Seven-TN subtype classification lack of overexpression of HER2 by IHC and/or FISH (the so called triple-negative [TN] status) [1, 2, 6]. Although the To identify the 7-TN subtypes described by Lehmann et al. TN definition enriches for basal-like disease, considerable [16], (i.e., basal one, basal two, immunomodulatory, discordance exists [2, 6]. luminal androgen receptor, mesenchymal, mesenchymal BRCA1 mutations and other associated molecular traits stem cell, and unstable), we submitted the raw gene might confer sensitivity to specific therapeutic agents [7– expression data of each individual dataset of basal-like 10]. Nevertheless, it is unclear how different, from a bio- disease to the TNBC type online predictor (http://cbc.mc. logical perspective, BRCA1-mutated basal-like tumors are vanderbilt.edu/tnbc/) [17]. from BRCA1 non-mutated basal-like tumors, and whether BRCA1 mutation is an independent prognostic and/or pre- Statistical analysis dictive biomarker when the intrinsic subtype is taken into account [11–15]. This line of thought directed us to for- All multiple-testing comparisons were done using an unpaired mulate the question of how much the biology of basal-like two-class significance analysis of microarrays (SAM, http:// tumors with BRCA1 mutations differs from the biology of www-stat.stanford.edu/*tibs/SAM/). The mutation rates of basal-like tumors without BRCA1 mutations. To address TP53 and PIK3CA genes between two groups, the 7-TN this question, we interrogated The Cancer Genome Atlas subtype distribution between BRCA1-mutated and non- (TCGA) breast cancer project which provides various types mutated basal-like tumors, and the amplification rates of ID4 of molecular data coming from DNA, RNAs, and proteins between two groups, were compared using the Chi square and [1]. Fisher’s exact tests. The total number of somatic mutations between two groups was compared using a Student’s t test. All statistical computations were performed in R v.2.15.1 (http:// Methods cran.r-project.org). The Cancer Genome Atlas dataset Results and discussion In this study, we evaluated TCGA breast cancer dataset and all data were obtained from the TCGA breast cancer online portal From TCGA breast cancer dataset, we identified 12 tumor (https://tcga-data.nci.nih.gov/docs/publications/brca_2012/). samples with BRCA1 germline mutations (all classified as The following files were used. For microarray gene expres- deleterious), seven tumor samples with somatic BRCA1 sion data: ‘‘BRCA.exp.547.med.txt.’’ For reverse-phase mutations, and one tumor sample with both BRCA1 protein array (RPPA) expression data: ‘‘rppaData- germline and somatic mutations (Supplemental Material). 403Samp-171Ab-Trimmed.txt.’’ For sequencing miRNA As expected, 70 % of BRCA1 mutated tumors where of the expression: ‘‘BRCA.780.mimat.txt.’’ For microarray DNA basal-like intrinsic subtype (nine germline and five 123 Breast Cancer Res Treat Fig. 1 Intrinsic profile of BRCA1-mutated breast tumors. Hierarchical clustering of 509 breast samples of the cancer genome atlas (TCGA) project using the *1,900 intrinsic gene list [30]. PAM50 intrinsic subtype calls [30] and BRCA1 mutation status is shown below the array tree Table 1 Significant molecular Total biomarkers evaluated Type of Comparison Significant Percentage differences between basal-like evaluation (more expressed biomarkers of altered BRCA1-mutated tumors or amplified) identified biomarkers (%) (n = 14) and basal-like BRCA1 (FDR = 0%) non-mutated tumors (n = 79) 17,786 (unique genes) Expression BRCA1MUT 0 0 BRCA1WT 0 171 (unique proteins or Expression BRCA1MUT 0 0.6 phospho-proteins by BRCA1WT 1 RPPA) 1,222 (mature/star miRNA Expression BRCA1MUT 3 0.2 strands) BRCA1WT 0 530 (unique genes) Methylation BRCA1MUT 0 1.1 RPPA reverse-phase protein BRCA1WT 6 arrays, FDR false discovery rate 19,613 (unique genes) DNA BRCA1MUT 250 1.3 BRCA1WT BRCA1 wild-type, amplification BRCA1WT 0 BRCA1MUT BRCA1 mutated somatic), but luminal A (two germline, one germline/ somatic, and one somatic), luminal B (one germline), and HER2-enriched (one somatic) tumors were also identified (Fig. 1). Similarly, 66.7 % of BRCA1 mutated tumors were TN. Within basal-like disease, we observed minor molecular differences (0–1.1 %) in terms of gene expression, protein expression, miRNA expression, and DNA methylation variation according to BRCA1 status (Table 1 and Sup- plemental Material). Indeed, no genes among 17,876 genes were found differentially expressed between basal-like BRCA1-mutated tumors versus basal-like BRCA1 non- mutated tumors (Table 1), including the BRCA1 mRNA transcript (Fig.