Identifying Allelic Loss and Homozygous Deletions in Pancreatic Cancer without Matched Normals Using High-Density Single-Nucleotide Polymorphism Arrays Eric S. Calhoun, 1 Tomas Hucl, 1 Eike Gallmeier, 1 Kristen M. West, 3 Dan E. Arking, 3 Anirban Maitra, 1,2 Christine A. Iacobuzio-Donahue, 2 Aravinda Chakravarti, 3 Ralph H. Hruban, 1,2 and Scott E. Kern 1,2 Departments of 1 Oncology and 2 Pathology, The Sol Goldman Pancreatic Cancer Research Center at the Sidney Kimmel Comprehensive Cancer Center, and 3 Department of Genetic Medicine at the Mckusick-Nathans Institute of Genetic Medicine, Johns Hopkins Medical Institutes, Baltimore, Maryland Abstract Recent advances in oligonucleotide arrays and whole-genome complexity reduction data analysis now permit the evaluation of tens of thousands of single-nucleotide polymorphisms simultaneously for a genome-wide analysis of allelic status. Using these arrays, we created high-resolution allelotype maps of 26 pancreatic cancer cell lines. The areas of heterozygosity implicitly served to reveal regions of allelic loss. The array- derived maps were verified by a panel of 317 microsatellite markers used in a subset of seven samples, showing a 97.1% concordance between heterozygous calls. Three matched tumor/normal pairs were used to estimate the false-negative and potential false-positive rates for identifying loss of heterozygosity: 3.6 regions (average minimal region of loss, 720,228 bp) and 2.3 regions (average heterozygous gap distance, 4,434,994 bp) per genome, respectively. Genomic fractional allelic loss calculations showed that cumulative levels of allelic loss ranged widely from 17.1% to 79.9% of the haploid genome length. Regional increases in ‘‘NoCall’’ frequencies combined with copy number loss estimates were used to identify 41 homozygous deletions (19 first reports), implicating an additional 13 regions disrupted in pancreatic cancer. Unexpectedly, 23 of these occurred in just two lines (BxPc3 and MiaPaCa2), suggesting the existence of at least two subclasses of chromosomal instability (CIN) patterns, distin- guished here by allelic loss and copy number changes (original CIN) and those also highly enriched in the genomic ‘‘holes’’ of homozygous deletions (holey CIN). This study provides previously unavailable high-resolution allelotype and deletion breakpoint maps in widely shared pancreatic cancer cell lines and effectively eliminates the need for matched normal tissue to define informative loci. (Cancer Res 2006; 66(16): 7920-8) Introduction A greater efficiency in analyzing loss of heterozygosity (LOH) in cancer genomes is needed. Long-standing problems have ham- peredeffortstocharacterizeLOH,particularlyintumortypessuch as adenocarcinoma of the pancreas, which contain as few as 10% neoplastic cells (1). Primary tissues are also inherently limited resources, which eventually deplete through analysis. Access is often limited to select individuals/institutions where patients are seen, limiting the ability of the scientific community to validate published results. By establishing in vitro and/or xenografted culturesofcarcinomacells,manyoftheproblemsassociatedwith primary tissues can be overcome. From a genetic perspective, early-passagecelllinesrepresentanalmostidealformoftissuefree of contaminating normal cells. With unlimited expandability, as well as portability, cell lines have the potential to be shared freely between investigators and/or established in commercial cell-line banks. Unfortunately, in vitro cultures generally lack a matched normal tissue. In the past, this impaired the ability to discern between hemizygosity and homozygosity as well as between polymorphic (germ line) variants and true somatic mutations. The efficiency of analyzing allelic loss is also dependent on technological limitations. The analysis of microsatellite markers has been the gold standard for estimating allelic loss because the rateofheterozygosityateachlocusiscomparablyhigherthanthat seen for diallelic single-nucleotide polymorphisms (SNP; refs. 2–4). Such low heterozygosity frequencies have been one reason why analyses based on SNPs have found limited use in genetic studies (5).Technicaldifficultiesinthescalabilityformicrosatellitemarker studies, however, limit the number of samples and/or the number of markers used, effectively ensuring a low genomic coverage and poor resolution of LOH breakpoints (6). Recent advances in detectingchromosomalcopynumber(e.g.,bacterialartificialchro- mosome array–based comparative genomic hybridization and representational oligonucleotide microarray analysis; refs. 7–11) have provided a solution to some of these problems but fail to identify many cases of LOH, as when uniparental disomy or polysomy is present. Additionally, genomic coverage remains somewhat limited by the number of probes arrayed (recently f5,400 probes per array; ref. 10). New high-throughput technologies, such as the Affymetrix genotyping arrays which evaluate >100,000 genotypic-variant loci simultaneously,effectivelyallowforahigh-resolutiongenome-wide analysis of allelic status on large sample sets (3, 12). The reduced informativeness of diallelic markers is overcome by an increase in the number of evaluated loci and subsequently the number of informative calls. For example, Matsuzaki et al. (3) reported a heterozygous frequency rate of 30.41% using the 100K SNP arrays, which would yield nearly 30,000 informative calls in diploid individuals.Thequalityoftheresults,however,isdependentonthe purity of the starting tissue. Allelic loss studies are dramatically Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Eric S. Calhoun, Department of Oncology, Johns Hopkins MedicalInstitutes,CRB1,Room464,1650OrleansStreet,Baltimore,MD21231.Phone: 410-614-3314; Fax: 410-614-9705; E-mail: ecalhou2@jhmi.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-0721 Cancer Res 2006; 66: (16). August 15, 2006 7920 www.aacrjournals.org Research Article