Array-based comparative genomic hybridization identifies localized DNA amplifications and homozygous deletions in pancreatic cancer

Abstract

Pancreatic cancer, the fourth leading cause of cancer death in the United States, is frequently associated with the amplification and deletion of specific oncogenes and tumor-suppressor genes (TSGs), respectively. To identify such novel alterations and to discover the underlying genes, we performed comparative genomic hybridization on a set of 22 human pancreatic cancer cell lines, using cDNA microarrays measuring approximately 26,000 human genes (thereby providing an average mapping resolution of <60 kb). To define the subset of amplified and deleted genes with correspondingly altered expression, we also profiled mRNA levels in parallel using the same cDNA microarray platform. In total, we identified 14 high-level amplifications (38-4934 kb in size) and 15 homozygous deletions (46-725 kb). We discovered novel localized amplicons, suggesting previously unrecognized candidate oncogenes at 6p21, 7q21 (SMURF1, TRRAP), 11q22 (BIRC2, BIRC3), 12p12, 14q24 (TGFB3), 17q12, and 19q13. Likewise, we identified novel polymerase chain reaction-validated homozygous deletions indicating new candidate TSGs at 6q25, 8p23, 8p22 (TUSC3), 9q33 (TNC, TNFSF15), 10q22, 10q24 (CHUK), 11p15 (DKK3), 16q23, 18q23, 21q22 (PRDM15, ANKRD3), and Xp11. Our findings suggest candidate genes and pathways, which may contribute to the development or progression of pancreatic cancer.

Figure 1

Array CGH identifies localized DNA amplifications in pancreatic cancer. (A and B) Graphic displays of DNA copy number alteration for selected localized amplifications identified in pancreatic cancer cell lines. Test/reference fluorescence ratios are plotted on a log₂ scale according to chromosome nucleotide (Megabase) position. Shaded regions highlight localized high-level amplifications. Insets display genes within highlighted amplicons, ordered by map position and color-coded according to mean-centered expression levels (log₂ ratio scale indicated). (A) 7q21 amplicon in AsPC-1. (B) 11q22 amplicon in Colo-357. Complete genomewide profiles of DNA copy number alteration for the 22 pancreatic cancer cell lines are viewable in Figure W2. (C) FISH validation of 7q21 amplification in AsPC-1. Spectrum Orange chromosome 7 centromere probe detects three signals, whereas Spectrum Green 7q21 locus probe identifies multiple signal clusters indicative of DNA amplification. Nonamplified cell line BxPC-3 (triploid for chromosome 7) is shown for comparison.

Figure 2

Array CGH identifies localized homozygous DNA deletions in pancreatic cancer. (A and B) Test/reference fluorescence ratios are plotted on a log₂ scale according to chromosome nucleotide (Megabase) position. Shaded regions highlight localized homozygous deletions. Insets display genes within highlighted deletions, ordered by map position and color-coded according to mean-centered expression levels (log₂ ratio scale provided; unfilled green bar indicates measured intensity less than background). (A) 11p15 deletion in BxPC-3. (B) 21q22 deletion in BxPC-3 and Panc 02.13. (C) PCR validation of homozygous deletion at 11p15. DKK3, located within the homozygous deletion, is PCR-amplified in control cell line Panc 02.13 but not in BxPC-3. TEAD1, a control gene flanking the deletion, is PCR-amplified in both cell lines. (D) PCR validation of homozygous deletion at 21q22. PRDM15, located within the homozygous deletion, is PCR-amplified in control cell lines (MiaPaCa2 and PL8) but not in BxPC-3 and Panc 02.13. HMGN1, a control gene flanking the deletion, is PCR-amplified in all four cell lines.