Canine array comparative genomic hybridization (aCGH)
Many human and canine cancers exhibit highly comparable clinical presentation and progression, and as with many human cancers, canine tumors also demonstrate recurrent chromosome aberrations. In both species, it has been observed that even in tumors presenting with apparently similar histopathology, the pattern of chromosome aberrations may be distinctly different. This indicates that the tumors represent different genetic subgroups of the same cancer, and may also demonstrate disparity in their subsequent clinical behavior. A detailed knowledge of the pattern of chromosome aberrations in genetically heterogeneous tumors may therefore improve existing methods for diagnosis, prognosis and the selection of appropriate therapy.
Patterns of recurrent chromosome aberrations may be characterized using a range of fluorescence in-situ hybridization (FISH) based techniques. Amongst these, comparative genomic hybridization (CGH) analysis allows a genome-wide survey of chromosome imbalances in a single experiment. In human medicine, these experiments are now typically performed using microarray analysis, since array-based CGH enables genomic abnormalities to be detected more efficiently and at higher resolution than conventional chromosome-based techniques.
Generation of genomic microarrays is a multistage process, and a range of approaches have been described. In our chosen method, large-insert genomic (BAC) clones from the species of interest are first amplified individually by degenerate PCR. Then a secondary round of linker PCR is performed to attach an amino-molecule that allows these products to be bound covalently onto amine-binding glass slides. In the hybridization procedure, tumor DNA is labeled with a fluorochrome and combined with differentially-labeled DNA from a normal reference individual. The probe mixture is then hybridized onto the microarray, in the presence of competitor DNA to block highly repetitive DNA sequences. The relative ratio of fluorescence intensities at each locus indicates the relative copy number of that locus in the tumor (image- top left). This in turn demonstrates whether the corresponding chromosome region shows a normal copy number in the tumor (i.e. a 1:1 fluorescence ratio), or whether it is over- or under-represented compared to the normal control (image- top right).
Following recent advances made in human genomics, we have developed a series of DNA microarrays for the domestic dog, which are being used in CGH analysis for the detection and characterization of DNA copy number changes in canine tumors. Our main interests lie in the study of canine multicentric lymphoma, leukemia, central nervous system tumors, osteosarcoma and soft-tissue sarcomas.
In transferring this technology for the first time to a canine system, we have developed a series of canine arrays culminating in the deveopment of a microarray that surveys the canine genme at 1Mb intervals (1). Each clone on this array has a defined location within the canine genome assmebly and has been verfied to have a unique cytogenetic location, using FISH analysis. Each BAC cone on the array is thus available also for direct evauation of tumor cells.
- Thomas, R., Duke, S.E., Karlsson, E.K., Evans, A., Ellis, P., Lindblad-Toh, K., Langford, C.F. and Breen, M. (2008). A genome assembly-integrated dog 1Mb BAC microarray: a cytogenetic resource for canine cancer studies and comparative genomic analysis. Cytogenetics and Genome Research 122:110-121. [PUBMED LINK]
- Wellcome Trust Sanger Institute, UK (Dr. Cordelia Langford and Dr. Peter Ellis)
- Broad Institute, MA (Dr. Kerstin-Lindblad-Toh and Dr. Elinor Karlsson)