RESEARCH ARTICLES Characterization of Amplicons in Neuroblastoma: High-Resolution Mapping Using DNA Microarrays, Relationship with Outcome, and Identification of Overexpressed Genes Anne Fix, 1 Carlo Lucchesi, 1 Agne ` s Ribeiro, 2 Delphine Lequin, 1 Gae ¨ lle Pierron, 2 Gudrun Schleiermacher, 1,3 Olivier Delattre, 1,2 and Isabelle Janoueix-Lerosey 1 * 1 INSERM U830, Laboratoire de G en etique et Biologie des Cancers, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France 2 Institut Curie,Unit e de G en etique Somatique, 26 rue d’Ulm, 75248 Paris Cedex 05, France 3 Institut Curie, D epartement de P ediatrie, 26 rue d’Ulm, 75248 Paris Cedex 05, France Somatically acquired chromosomal imbalances are a key feature of neuroblastoma, a heterogeneous pediatric solid tumor. Among these alterations, genomic amplification targeting the MYCN oncogene and observed in about 25–30% of the cases, strongly correlates with advanced stage and poor outcome. In this work, we have used BAC and SNP arrays as well as gene expression arrays to characterize amplifications in neuroblastoma. Eighty-eight distinct BACs defining high-level amplification events were identified in 65 samples, including 43 tumors and 22 cell lines. Although the highest recurrence was observed on chromosome 2, clones on chromosomes 8, 12, 16, and 17 also revealed genomic amplification in several samples. A detailed analysis of the 2p22-2p25 MYCN containing region indicated highly complex patterns in a number of cases. Coamplifications involving MYCN and other regions were explored by FISH in three cell lines. High-resolution arrays then allowed us to further refine the mapping of 25 amplicons in 19 samples, either reducing the size of a single continuous amplicon or increasing the complexity by highlighting multiple noncontiguous regions of amplification. Combined analysis of gene expression profiling and array-CGH data indicated that 12 to 25% of the genes that are targeted by genomic amplification are actually over-expressed in tumorcells, several of them having already been implicated in cancer. Finally, our results suggest that the presence of ampli- cons localized outside of chromosome 2, in addition to MYCN amplification, may be linked to a particularly severe outcome in neuroblastoma patients. V V C 2008 Wiley-Liss, Inc. INTRODUCTION Neuroblastoma (NB), the most common extrac- ranial tumor of childhood is characterized by a great clinical and genetic heterogeneity (Brodeur, 2003; Maris et al., 2007). Somatically acquired chromosomal imbalances are a key feature of this pathology: they occur as genomic amplifications as well as whole or segmental chromosome imbalan- ces. Amplification of the MYCN oncogene at chro- mosome band 2p24 is observed in about one-third of NB cases and is associated with advanced-stage disease and rapid tumor progression. It represents a strong well-defined prognostic factor, routinely used in clinical practice to assign therapeutic inten- sity (Maris and Matthay, 1999). MYCN amplifica- tion is observed cytogenetically as acentric autono- mously replicating double min chromosomes (dmins) or as intrachromosomally homogeneously staining regions (hsrs). Other regional amplifica- tions targeting various sites including 1p34, 1p36, 2p14, 5q33, 8q24, 11q13, 12q13-14, and 16q22 have been described in NB (Jinbo et al., 1989; Molenaar et al., 2003; Fix et al., 2004; Su et al., 2004; Mosse et al., 2005; Van Roy et al., 2006; Carr et al., 2007; Mosse et al., 2007). Segmental aberra- tions include 17q gain, 1p, 3p, 4p, 11q, 14q, 16p, and 19q deletions (Brodeur, 2003). It has been demonstrated that distinct genomic profiles deter- mined according to the observed aberrations in the tumor DNA may be associated with particular prognostic characteristics of the corresponding *Correspondence to: Isabelle Janoueix-Lerosey, INSERM U830, Laboratoire de Ge ´ne ´tique et Biologie des Cancers, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France. E-mail: janoueix@curie.fr Additional Supporting Information may be found in the online version of this article. Supported by: Institut National de la Sante ´ et de la Recherche Me ´ dicale; Ligue Nationale contre le Cancer (Equipe labellise ´e); Carte d’Identite ´ des Tumeurs/Program of the Ligue Nationale Contre le Cancer. Received 21 December 2007; Accepted 14 May 2008 DOI 10.1002/gcc.20583 Published online 13 June 2008 in Wiley InterScience (www.interscience.wiley.com). V V C 2008 Wiley-Liss, Inc. GENES, CHROMOSOMES & CANCER 47:819–834 (2008)