Genome profiling of ERBB2-amplified breast cancers

Fabrice Sircoulomb 0 Ismahane Bekhouche 0 Pascal Finetti 0 Jos Adlade 0 Azza Ben Hamida 0 Julien Bonansea 0 Stphane Raynaud 0 Charlne Innocenti 0 Emmanuelle Charafe-Jauffret 0 2 4 Carole Tarpin 1 Farhat Ben Ayed 3 Patrice Viens 1 4 Jocelyne Jacquemier 0 2 Franois Bertucci 0 1 4 Daniel Birnbaum 0 Max Chaffanet 0 0 Marseille Cancer Research Center, UMR891 Inserm, Institut Paoli-Calmettes, Department of Molecular Oncology , Marseille , France 1 Department of Medical Oncology, Institut Paoli-Calmettes , Marseille , France 2 Department of BioPathology, Institut Paoli-Calmettes , Marseille , France 3 Department of Medical Oncology, Salah Azaiz Institute , Tunis , Tunisia 4 Universite de la Mediterranee , Marseille , France Background: Around 20% of breast cancers (BC) show ERBB2 gene amplification and overexpression of the ERBB2 tyrosine kinase receptor. They are associated with a poor prognosis but can benefit from targeted therapy. A better knowledge of these BCs, genomically and biologically heterogeneous, may help understand their behavior and design new therapeutic strategies. Methods: We defined the high resolution genome and gene expression profiles of 54 ERBB2-amplified BCs using 244K oligonucleotide array-comparative genomic hybridization and whole-genome DNA microarrays. Expression of ERBB2, phosphorylated ERBB2, EGFR, IGF1R and FOXA1 proteins was assessed by immunohistochemistry to evaluate the functional ERBB2 status and identify co-expressions. Results: First, we identified the ERBB2-C17orf37-GRB7 genomic segment as the minimal common 17q12-q21 amplicon, and CRKRS and IKZF3 as the most frequent centromeric and telomeric amplicon borders, respectively. Second, GISTIC analysis identified 17 other genome regions affected by copy number aberration (CNA) (amplifications, gains, losses). The expression of 37 genes of these regions was deregulated. Third, two types of heterogeneity were observed in ERBB2-amplified BCs. The genomic profiles of estrogen receptor-postive (ER+) and negative (ER-) ERBB2-amplified BCs were different. The WNT/b-catenin signaling pathway was involved in ERERBB2-amplified BCs, and PVT1 and TRPS1 were candidate oncogenes associated with ER+ ERBB2-amplified BCs. The size of the ERBB2 amplicon was different in inflammatory (IBC) and non-inflammatory BCs. ERBB2-amplified IBCs were characterized by the downregulated and upregulated mRNA expression of ten and two genes in proportion to CNA, respectively. IHC results showed (i) a linear relationship between ERBB2 gene amplification and its gene and protein expressions with a good correlation between ERBB2 expression and phosphorylation status; (ii) a potential signaling cross-talk between EGFR or IGF1R and ERBB2, which could influence response of ERBB2-positive BCs to inhibitors. FOXA1 was frequently coexpressed with ERBB2 but its expression did not impact on the outcome of patients with ERBB2-amplified tumors. Conclusion: We have shown that ER+ and ER- ERBB2-amplified BCs are different, distinguished ERBB2 amplicons in IBC and non-IBC, and identified genomic features that may be useful in the design of alternative therapeutical strategies. - Background Gene amplification is a frequent alteration in breast cancer (BC) that affects multiple genomic regions [1-3]. One of the most studied amplifications is located in chromosomal region 17q12 and involves the ERBB2 gene. ERBB2 encodes a transmembrane tyrosine kinase receptor of the ERBB/EGFR family. ERBB2 is amplified in around 20% of BCs. The receptor is overexpressed in most amplified cases and in some non-amplified cases as well. This alteration is associated with a poor clinical outcome. BCs with ERBB2 overexpression can benefit from a targeted therapy that uses the humanized monoclonal antibody trastuzumab or the ERBB kinase inhibitor lapatinib [4,5]. However, resistance to trastuzumab is frequent [6] and its mechanisms are poorly understood [7], although ERBB2 phosphorylation [8], PTEN and PIK3CA status [9] seem important factors. ERBB2 gene amplification can be detected by various methods including fluorescence in situ hybridization (FISH) or quantitative PCR [10]. Overexpression of the receptor is detected by immunohistochemistry (IHC) by using the standardized Dako Herceptest. Gene expression studies have shown that BCs with a specific gene expression signature including ERBB2-overexpression constitute a separate molecular subtype [11-13]. However, a substantial number of breast tumors assigned to the ERBB2 subtype lacks ERBB2 protein expression and/ or ERBB2 gene amplification [14,15] and ERBB2-positive cancers that express estrogen receptor (ER) fall into the luminal subtypes [11,13,16]. Several 17q12-q21 genes are variably coamplified and coexpressed with ERBB2 [17]. This could influence the response to trastuzumab and/or constitute accessory targets for synergistic treatment [18-21]. A better knowledge of ERBB2-amplified BCs may thus help design new therapeutic strategies. To better characterize this particular group of BCs, we used high-resolution array-comparative genomic hybridization (aCGH) and whole-genome DNA microarrays to define the genome and gene expression profiles of 54 BCs with ERBB2 amplification. Methods Breast cancer samples Tumor tissues were collected from 340 patients with invasive adenocarcinoma who underwent initial surgery at the Institut Paoli-Calmettes (Marseille, France) between 1987 and 2007 (from a cohort of 2,175 patients with frozen tumor sample) and from a series of 91 Tunisian T4d tumors (TNM, UICC) treated between 1994 and 1998 at the Salah Azaiz Institute (Tunis, Tunisia). Each patient gave informed consent and the study was approved by our institutional review board (also called COS). Samples were macrodissected and frozen in liquid nitrogen within 30 minutes of removal. The panel was not made of consecutive tumors but enriched in various forms of BCs. These include inflammatory BCs (IBCs), non-inflammatory BCs (NIBC), very young women BCs (YWBCs, 35 years) and older women BCs (OWBCs, 45 years). The 340 tumors were analyzed by high resolution aCGH 244K in our previous studies [[3,22], unpublished data]. In all these cases, profiled samples were always collected before any systemic therapy (chemotherapy, hormone or trastuzumab therapy). They corresponded to a tumor surgically removed for NIBCs, and to the diagnostic surgical biopsy for IBCs. A total of 54 (16%) cases presented amplification of the ERBB2 locus. Features of these ERBB2-amplified tumors are reported in Additionnal file 1-Table S1. All specimens contained >60% of tumor cells (as assessed before RNA extraction using frozen sections adjacent to the profiled samples). IHC data included status for estrogen and progesterone receptors (ER and PR), P53 (positivity cut-off value of 1%), ERBB2 (0-3+ score, DAKO HercepTest kit scoring guidelines, defined as positive with 3+ and 2+ controled by FISH according to ASCO guidelines), and Ki67 (posit (...truncated)