Comprehensive Profiling of 8P11-12 Amplification in Breast Cancer
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Comprehensive Profiling of 8p11-12 Amplification in Breast Cancer Ve´ronique Gelsi-Boyer,1,3 Be´atrice Orsetti,4 Nathalie Cervera,1 Pascal Finetti,1 Fabrice Sircoulomb,1 Carole Rouge´,4 Laurence Lasorsa,4 Anne Letessier,1 Christophe Ginestier,1 Florence Monville,1 Se´verine Esteyrie`s,1 Jose´Ade´laı¨de,1 Benjamin Esterni,2 Catherine Henry,6 Stephen P. Ethier,7 Fre´de´ric Bibeau,5 Marie-Joe¨lle Mozziconacci,1,3 Emmanuelle Charafe-Jauffret,1,3 Jocelyne Jacquemier,1,3 Franc¸ois Bertucci,1 Daniel Birnbaum,1 Charles Theillet,4 and Max Chaffanet1 1Marseilles Cancer Institute, Department of Molecular Oncology, UMR599 Institut National de la Sante et de la Recherche Medicale and Institut Paoli-Calmettes; 2Department of Biostatistics, UMR599 and Institut Paoli-Calmettes; 3Department of BioPathology, Institut Paoli-Calmettes, Marseilles, France; 4EMI229 Institut National de la Sante et de la Recherche Medicale and 5Department of Pathology, CRLC Val d’Aurelle-Paul Lamarque, Montpellier, France; 6Laboratory of Cytogenetics, Centre Hospitalier Universitaire of Rennes, Rennes, France; and 7Karmanos Institute, Detroit, Michigan Abstract region just telomeric to and associated with the In human carcinomas, especially breast cancer, amplification. Finally, we show that 8p11-12 chromosome arm 8p is frequently involved in amplification has a pejorative effect on survival in complex chromosomal rearrangements that combine breast cancer. (Mol Cancer Res 2005;3(12):655–67) amplification at 8p11-12, break in the 8p12-21 region, and loss of 8p21-ter. Several studies have identified Introduction putative oncogenes in the 8p11-12 amplicon. The short arm of chromosome 8 is a frequent target of genetic However, discrepancies and the lack of knowledge alterations in a wide variety of human cancers (1, 2). on the structure of this amplification lead us to think In breast cancer, the 8p12-21 region often displays complex that the actual identity of the oncogenes is not combinations of chromosomal breaks, losses, and DNA definitively established. We present here a amplification (3-11).8,9 Loss-of-heterozygosity studies have comprehensive study combining genomic, expression, identified at least two regions of loss (7, 11), whereas recurrent and chromosome break analyses of the 8p11-12 chromosome breaks are found in 6% of breast cancers at the region in breast cell lines and primary breast tumors. NRG1 locus (12, 13). Amplification of 8p11-12 occurs in 10% We show the existence of four amplicons at to 15% of breast cancers (14-17) and is often visible 8p11-12 using array comparative genomic cytogenetically as homogeneously staining region (9, 18-20). hybridization. Gene expression analysis of 123 The frequency of 8p11-12 alterations contrasts with our limited samples using DNA microarrays identified 14 genes knowledge of the genes involved, although several candidates significantly overexpressed in relation to have been suggested. No oncogene from the 8p11-12 amplifi- amplification. Using fluorescence in situ hybridization cation unit (amplicon) has been identified with certainty. The analysis on tissue microarrays, we show the role of the FGFR1 gene, which codes for a tyrosine kinase existence of a cluster of breakpoints spanning a receptor, has been evoked (17), but normal levels of FGFR1 expression in some amplified tumors have suggested that it may not be the actual or sole oncogene involved (21). Due to its importance in breast cancer, several recent studies Received 8/8/05; revised 10/26/05; accepted 11/14/05. have aimed at a better definition of the 8p11-12 amplification. Grant support: Institut National de la Sante et de la Recherche Medicale, Institut Paoli-Calmettes, Association pour la Recherche sur le Cancer (2002-2003), Ligue Ray et al. characterized the 8p11-12 amplicon in three breast Nationale Contre le Cancer (Label 2003-2006), and Ministries of Health and tumor cell lines (21). Several potential oncogenes other than Research (Cance´ropoˆles PACA and GSO); Ligue Nationale Contre le Cancer fellowship (A. Letessier) and Ministry of Research fellowship (C. Ginestier, F. FGFR1 were suggested. Reyal et al. analyzed transcriptome data Monville, F. Sircoulomb, and S. Esteyrie`s). of 130 invasive breast carcinomas using an approach based on The costs of publication of this article were defrayed in part by the payment of the calculation, for each gene, of the correlation between page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. its expression profile and that of its neighbors (22). A list of Note: V. Gelsi-Boyer and B. Orsetti have equally contributed to this work. candidate oncogenes has been established, including some Supplementary data for this article are available at Molecular Cancer Research proposed previously (21). However, this approach may have Online (http://mcr.aacrjournals.org/). Requests for reprints: Daniel Birnbaum, UMR599 Institut National de la Sante et de la Recherche Medicale, 27 Bd. Leı¨Roure, 13009 Marseilles, France. Phone: 33-4-91-75-84-07; Fax: 33-4-91-26-03-64. E-mail: [email protected] Copyright D 2005 American Association for Cancer Research. 8 http://amba.charite.de/~ksch/cghdatabase/index.htm. doi:10.1158/1541-7786.MCR-05-0128 9 http://cgap.nci.nih.gov/Chromosomes/RecurrentAberrations. Mol Cancer Res 2005;3(12). December 2005 655 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. 656 Gelsi-Boyer et al. Mol Cancer Res 2005;3(12). December 2005 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Profiling of 8p11-12 Amplification in Breast Cancer 657 excluded isolated candidate genes and did not inform clearly on our series with 87.1% of the tumors and 100% of the cell lines the structure of the amplicon. The most extensive study of 8p showing either gains or losses. Tumors and cell lines showed amplification to date has been reported by Garcia et al. (23). similar aCGH profiles. Losses were prevalent from telomere to These authors studied 80 breast and ovarian tumors and cell lines 8p12 and gains in the 8p12-11 region. Transitions between gains by using both high-resolution array comparative genomic and losses were concentrated in a region located at the 8p12- hybridization (aCGH) and gene expression analyses. They 8p21 boundary. Individual aCGH profiles showed wavy defined a 1-Mb region of common amplification. This patterns, suggesting the existence of several peaks of amplifi- region excluded most previously proposed candidate genes, cation within this region of gain. Compilation of gains and including FGFR1, and contained four potential oncogenes, losses data from all samples further supported this idea FLJ14299, SPFH domain family, member 2, related to stomatin, (Supplementary Fig. S2A). Alignment of the regions of gains a component of lipid rafts (SPFH2/C8orf2), BRF2,and showed three recurrent interruptions and four subregions of RAB11FIP1, which showed good correlation between amplifi- amplification (Fig. 1A). The latter were designated amplicons cation and overexpression. A novel amplicon resulting from A1 to A4, from telomere to centromere. NRG1 rearrangements and associated with poor prognosis was The boundaries of the amplified subregions were positioned recently described (24). This small amplicon is included in the immediate nonamplified neighbor BAC clones. In in the 1-Mb minimal amplicon defined by Garcia et al. (23) addition, to ascertain that the subregion selected corresponded and contains two previously proposed candidate genes (22, 23). strictly to an amplification common to several tumor samples, we The four studies provided very valuable information. However, determined its core by using a stringent cutoff and considering the structure of the amplification can be refined further, only BAC clones whose log2 ratio exceeded 0.75 (Fig. 1B). especially with regard to its centromeric part and its association Amplicons and their cores were then characterized. Locations, with breaks and to the number and identity of potential BAC and gene contents, and sizes and cores of the amplicons are oncogenes it contains. detailed in Fig. 2 and Table 1. Although we defined amplicon A1 We report here a detailed genomic analysis of the 8p11-12 as a single core, aCGH profiles indicated the existence of two region in a series of breast cell lines and primary breast tumors. subpeaks, a main one centered f37.3 Mb and a second at 37.8 Using aCGH, we show that the 8p11-12 amplification can Mb, which we designated A1V(Fig. 1B). Profiles of the other be divided in four amplicons. Correlation analysis between amplicons did not show clear-cut subpeaks. According to amplification and gene expression identified several candidate National Center for Biotechnology Information Build 35 of the oncogenes. Using fluorescence in situ hybridization (FISH) Human Genome Sequence, amplicons A1 to A4 contained 17, on primary breast tumors organized in tissue microarrays, we 25, 8, and 10 genes or part of genes, respectively (Table 1; Fig. identified a cluster of breakpoints just upstream of the 2B and C). The four amplicons showed separate cores but amplification. Finally, we show the clinical significance of overlapped at their extremities: A1/A2, A2/A3, and A3/A4 8p12 amplicons in breast cancers. overlaps included 11, 2, and 3 genes, respectively (Table 1; Fig. 2B and C). The amplicons could be observed independently in Results some samples (Fig. 1A). Thus, DNA amplification at 8p11-12 aCGH Profiles Showed Multiphasic DNA Amplification presented a complex structure, suggesting multiple causal at 8p12 Associated to Loss of Distal 8p elements. We studied genomic alterations at 8p in 37 breast cell lines and 62 primary breast tumors by using a custom-made BAC Gene Expression Profiling Revealed Overexpression of array that included a near tiling-path coverage from centromere 8p11-12 Genes (43.2 Mb) to 8p21 (26.1 Mb).