High-Resolution Detection and Mapping of Genomic DNA Alterations in Neuroblastoma Yael P. Mosse, 1 Joel Greshock, 2 Adam Margolin, 2 Tara Naylor, 2 Kristina Cole, 1 Deepa Khazi, 1 George Hii, 1 Cynthia Winter, 1 Syed Shahzad, 2 Muhammad Usman Asziz, 2 Jaclyn A. Biegel, 3 Barbara L. Weber, 2 and John M. Maris 1,2 * 1 Division of Oncology, Children’s Hospital of Philadelphia and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 2 Abramson Family Cancer Research Institute,University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 3 Division of Human Genetics, Children’s Hospital of Philadelphia and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania We used array-based comparative genomic hybridization (aCGH) to measure genomic copy number alterations (CNAs) in 42 neuroblastoma cell lines with known 1p36.3, 2p24 (MYCN), 11q23, and 17q23 allelic status. All cell lines showed CNAs, with an average of 22.0% of the genome of each sample showing evidence of gain (11.6%) or loss (10.4%). MYCN amplifica- tion was detected in 81% of cell lines, but other regions with high-level genomic amplification were observed only rarely. Gain of 17q material was present in 75% of the samples, and four discrete genomic regions at 17q23.2–17q25.3 were defined. Novel regions of gain were identified, including a 2.6-Mb subtelomeric region at 5p that includes the telomerase reverse transcriptase gene (TERT), which was found in 45% of the cell lines. Hemizygous deletions were noted at 1p36.23– 1p36.32 and 11q23.3–11q25 in 60% and 36%, respectively, of the samples, with other frequent (>25%) regions of deletion localized to 1p32.1, 3p21.31–3p22.1, 5q35.2–5q35.3, 7q31.2, 7q34, 9q22.3–9q24.1, 10q26.11–10q26.12, 16q23.1–16q24.3, 18q21.32–18q23, and 20p11.21–20p11.23. A smallest region of overlap (SRO) for CNAs was mapped across all experiments and in each case was consistent with or refined the published data. A single cell line showed a homozygous deletion at 3p22.3, which was verified, and this location was refined by FISH and PCR. There was outstanding concordance of aCGH with PCR-based CNA detection methods. Several potential cooperating loci were identified, including deletion of 11q23– 25, which was highly associated with both regional gain and loss at multiple chromosomal loci but was inversely correlated with the deletion of 1p36. Taking all of this together indicates that aCGH can accurately measure CNAs in the neuroblas- toma genome and facilitate gene discovery efforts by high-throughput refinement of candidate loci. Supplementary material for this article can be found on the Genes, Chromosomes and Cancer website at http://www.interscience.wiley.com/jpages/1045- 2257/suppmat/index.html. V V C 2005 Wiley-Liss, Inc. INTRODUCTION Detailed knowledge of the cancer genome will be instrumental in understanding tumorigenesis, predicting patient outcome, and discovering genes targeted by chromosomal rearrangement. Each of the standard techniques for the detection of chro- mosomal imbalances has limitations. For example, PCR-based methodologies for the detection of loss of heterozygosity are extremely sensitive to the amount of normal DNA contaminating the tumor sample, a problem inherent in any human cancer molecular diagnostic setting. Metaphase CGH also has been commonly used but has lim- ited resolution (<10 Mb). Recently developed SNP-based methods for high-throughput detec- tion of CNAs across the genome are hampered by the requirement of having PCR amplification beyond the linear range, which ultimately dilutes tumor-specific differences because of continued amplification of normal DNA alleles. Array-based comparative genomic hybridization (aCGH), developed for genomewide analysis of DNA sequence copy number in a single experiment, is extremely well suited to high-throughput detec- tion of chromosomal gain and loss at high resolu- tion. Array CGH is particularly amenable to analy- sis of cancer cells because it is relatively insensi- tive to normal cell contamination (Hodgson et al., 2001), and high-quality probes may be prepared from small amounts of archival material (Jain et al., 2001). It is clear that aCGH can detect high-level amplifications and homozygous dele- tions using both cDNA- and BAC-based array *Correspondence to: John M. Maris, MD, Division of Oncology, Children’s Hospital of Philadelphia, Abramson Pediatric Research Center 902A, 3615 Civic Center Blvd., Philadelphia, PA 19104-4318. E-mail: maris@email.chop.edu Supported by: American Society of Clinical Oncology (to Y.P.M.). Caitlin Robb Foundation (to Y.P.M.), National Institutes of Health; Grant numbers: R01-CA78545 and R01-CA87847 (to J.M.M.). Abramson Family Cancer Research Institute (to J.M.M. and B.L.W.). Received 29 December 2004; Revised 8 March 2005; Accepted 18 March 2005 DOI 10.1002/gcc.20198 Published online 12 May 2005 in Wiley InterScience (www.interscience.wiley.com). V V C 2005 Wiley-Liss, Inc. GENES, CHROMOSOMES & CANCER 43:390–403 (2005)