ABSTRACT
Cancer represents the phenotypic end point of multiple genetic lesions that endow cells with a full range of biological properties required for tumorigenesis. Among the hallmark features of the cancer genome are recurrent regional gains and losses that, upon detailed characterization, have provided highly productive discovery paths for new oncogenes and tumor suppressor genes. In this study, we describe the use of an oligonucleotide-based microarray platform and development of requisite assay conditions and bioinformatic mining tools that permits high-resolution genome-wide array-comparative genome hybridization profiling of human and mouse tumors. Using a commercially available 60-mer oligonucleotide microarray, we demonstrate that this platform provides sufficient sensitivity to detect single-copy difference in gene dosage of full complexity genomic DNA while offering high resolution. The commercial availability of the microarrays and associated reagents, along with the technical protocols and analytical tools described in this report, should provide investigators with the immediate capacity to perform DNA analysis of normal and diseased genomes in a global and detailed manner.
Subject(s)
Chromosome Aberrations , Computational Biology/methods , Gene Expression Profiling/methods , Neoplasms/genetics , Oligonucleotide Array Sequence Analysis/methods , Animals , Cell Line, Tumor , DNA, Neoplasm/genetics , Female , Gene Dosage , Humans , Male , Mice , Neoplasms, Experimental/genetics , Nucleic Acid Hybridization , Polymerase Chain Reaction/methodsABSTRACT
The pancreatic adenocarcinoma genome harbors multiple amplifications and deletions, pointing to the existence of numerous oncogenes and tumor suppressor genes driving the genesis and progression of this lethal cancer. Here, array comparative genomic hybridization on a cDNA microarray platform and informatics tools have been used to define the copy number alterations in a panel of 24 pancreatic adenocarcinoma cell lines and 13 primary tumor specimens. This high-resolution genomic analysis has identified all known regional gains and losses as well as many previously uncharacterized highly recurrent copy number alterations. A systematic prioritization scheme has selected 64 focal minimal common regions (MCRs) of recurrent copy number change. These MCRs possess a median size of 2.7 megabases (Mb), with 21 (33%) MCRs spanning 1 Mb or less (median of 0.33 Mb) and possessing an average of 15 annotated genes. Furthermore, complementary expression profile analysis of a significant fraction of the genes residing within these 64 prioritized MCRs has enabled the identification of a subset of candidates with statistically significant association between gene dosage and mRNA expression. Thus, the integration of DNA and RNA profiles provides a highly productive entry point for the discovery of genes involved in the pathogenesis of pancreatic adenocarcinoma.
Subject(s)
Adenocarcinoma/genetics , Pancreatic Neoplasms/genetics , Animals , Cell Line, Tumor , Chromosomes/genetics , Chromosomes, Human, Pair 17 , Computational Biology/methods , Cyclin-Dependent Kinase Inhibitor p16/genetics , Gene Deletion , Gene Dosage , Gene Expression , Genome , Homozygote , Humans , Nucleic Acid Hybridization/methods , Oligonucleotide Array Sequence AnalysisABSTRACT
Chromosomal numerical aberrations (CNAs), particularly regional amplifications and deletions, are a hallmark of solid tumor genomes. These genomic alterations carry the potential to convey etiologic and clinical significance by virtue of their clonality within a tumor cell population, their distinctive patterns in relation to tumor staging, and their recurrence across different tumor types. In this study, we showed that array-based comparative genomic hybridization (CGH) analysis of genome-wide CNAs can classify tumors on the basis of differing etiologies and provide mechanistic insights to specific biological processes. In a RAS-induced p19(Arf-/-) mouse model that experienced accelerated melanoma formation after UV exposure, array-CGH analysis was effective in distinguishing phenotypically identical melanomas that differed solely by previous UV exposure. Moreover, classification by array-CGH identified key CNAs unique to each class, including amplification of cyclin-dependent kinase 6 in UV-treated cohort, a finding consistent with our recent report that UVB targets components of the p16(INK4a)-cyclin-dependent kinase-RB pathway in melanoma genesis (K. Kannan, et al., Proc. Natl. Acad. Sci. USA, 21: 2003). These results are the first to establish the utility of array-CGH as a means of etiology-based tumor classification in genetically defined cancer-prone models.
