Abstract 1146: Three-color array CGH differentiates constitutional from acquired CNVs in cancer genomes

Abstract

Abstract In recent years microarray-based comparative genomic hybridisation (CGH) has become the assay of choice for high-resolution screening of genetic aberrations in research and clinical diagnostics, and has revealed a plethora of small, constitutional copy number variations (CNVs) in healthy individuals. The appearance of these benign CNVs on the array profile affects the ability to identify CNVs that are acquired and pathogenic, in particular if these are also small in size. It is possible to eliminate constitutional CNVs by using a matched self-reference for each sample, but this compromises ability to compare array profiles between different individuals. In addition, the ability to link specific constitutional CNVs or combinations of these to disease susceptibility is lost. We have developed a new 3-color microarray CGH assay which allows identification of constitutional CNVs while retaining the ability to compare between different samples by simultaneous application of two distinctly labelled reference samples. The new 3-color microarray CGH assay uses three spectrally distinct fluorescent dyes to label the test sample, matched self-reference, and generic reference, for simultaneous hybridization to the same array platform. We have applied this new protocol to the analysis of samples from healthy individuals, leukemia patients, and the leukemia-derived cell line K562, and performed pair-wise comparison of 26 array-CGH profiles from the new 3-color array with corresponding profiles from conventional 2-color arrays. Our results demonstrate that introducing the third dye has no adverse effect on array assay noise, as measured by paired ttest of the standard deviation of the log2 ratio of all clones on the 2-color and 3-color array profile. Copy number calls from the new 3-color array assays show a consistency of up to 97% with the corresponding conventional 2-color array assays, equivalent to the level of consistency that was achieved when repeating array-CGH on the same sample using the same protocol. The prevalence-adjusted Cohen Kappa test indicates almost perfect agreement between the new 3-color assay and conventional 2-color assay. Application of the new 3-color microarray assay has enabled us to identify 44 constitutional CNVs in 57% of our leukemia samples. At least one third of these CNVs have been reported as sites of normal copy number variation and are listed in The Human Genome Variation Database. Introducing a third sample into the microarray assay is a new and more economical approach for high resolution genome-wide comparison of DNA, and therefore potentially amenable to uptake in both research and diagnostics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1146.