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Breen, Matthew, PhD. C.Biol. M.I.Biol
Associate Professor of Genomics
Canine array- CGH
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. 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.
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, central nervous system tumors, osteosarcoma and soft-tissue sarcomas.
In transferring this technology for the first time to a canine system, we developed a series of small-scale canine arrays. Our first generation array focused predominately upon a collection of canine orthologs of human proto-oncogenes and tumor suppressor genes that have been implicated in the development of a number of cancer types (1). This allows us to detect chromosome copy number aberrations that affect the genomic representation of these loci in any tumor of interest. The array also contains a panel of clones from those dog chromosomes we have previously shown to be most commonly under- or over-represented in canine tumors (2). We demonstrated the value of this technique by the application of a small set of canine multicentric lymphoma samples to the array, which showed abnormalities that had not previously been detected by conventional metaphase-based CGH analysis (3).
Our second generation array expanded upon the first by the addition of a panel of additional canine BAC clones that map to unique locations on each of the 38 canine autosomes and the sex chromosomes. This array comprises a total of 226 BAC clones and is being used to detect gross levels of aneuploidy in canine tumors, prior to undertaking more focused investigations.
Our longer-term goal is the development of a genome-wide microarray comprising clones spanning the canine genome at 1Mb intervals. This is possible due to the generation of an integrated genome map for the dog (4) and the availability of a complete genome sequence. We have recently completed the generation of an array with approximately 3Mb resolution, comprising 1250 BAC clones. The majority of these clones have been chromosomally assigned and ordered using fluorescence in-situ hybridization analysis and radiation-hybrid mapping analysis, and all are integrated within the current canine genome assembly. This array will give us a complete view of the pattern of chromosome imbalances in any tumor of interest. It will also be used to establish the precise structural composition of abnormal chromosomes and for improving our knowledge of the evolutionary relationships between the human and canine genomes.
(1) Thomas R, Bridge W, Benke K and Breen M. Isolation and chromosomal assignment of canine genomic BAC clones representing 25 cancer-related genes. Cytogenetic and Genome Research102 249-253 (2003).
(2) Thomas R, Smith KC, Ostrander EA, Galibert F and Breen M. Chromosome aberrations in canine multicentric lymphomas detected with comparative genomic hybridisation and a panel of single locus probes. British Journal of Cancer 89 1530-1537 (2003).
(3) Thomas R, Fiegler F, Ostrander EA, Galibert F, Carter NP and Breen M. A canine cancer-gene microarray for CGH analysis of canine tumours. Cytogenetic and Genome Research 102 254-250 (2003).
(4) Breen, M., Hitte, C., Lorentzen, T., Thomas, R., Cadieu, E., Sabacan, L., Scott, A., Evanno, G., Parker, H.G., Kirkness, E., Hudson, R., Guyon, R., Mahairas, G.G., Gelfenbeybn, B., Fraser, C.M., André, C., Galibert, F., Ostrander, E.A. (2004). An Integrated 4300 Marker FISH/RH Map of the Canine Genome. BMC Genomics5:65, 1-11.
THIS WORK IS GENEROUSLY SUPPORTED BY FUNDING FROM THE AMERICAN KENNEL CLUB CANINE HEALTH FOUNDATION
Wellcome Trust Sanger Institute, UK (Dr. Cordelia Langford)
NIH (Dr. Elaine Ostrander)
CNRS, Rennes, France (Dr. Francis Galibert)
AMC Cancer Research Center, Denver CO (Dr. Jaime Modiano)
Broad Institute, MA (Dr. Kerstin-Lindblad-Toh)
An example of the co-hybridization of differentially-labeled test (tumor- green fluorochrome) and reference (normal- red fluorochrome) genomic DNA probes onto our second generation canine BAC array. Where the copy number of a locus is normal in both probes (1:1 fluorescent ratio), that BAC clone appears yellow. Over-representation of a locus in the tumor generates a skew towards the green fluorochrome, whilst under-representation causes the spotted BAC clone DNA to appear red. The relative fluorescent ratios are quantified and compared using a series of computer algorithms, and then plotted.
A simplified array CGH profile of tumor (male) vs. reference (male) hybridization. This tumor case was a low grade, B-cell multicentric lymphoma of the centroblastic-centrocytic form, in a 10-year old male Golden Retriever. Mean, normalized, background-subtracted fluorescence ratio data are presented. Error bars show the limited standard deviation in fluorescence ratios for triplicate spots representing the same locus. Clones that lie above the green 1.15:1 ratio bar represent copy number gains, and those below the red 0.85:1 bar represent genomic losses.
Previously, metaphase CGH of this case demonstrated gain of CFA 13 and loss of CFA 14. Array analysis confirmed these data. The under-representation of all CFA Y clones also implies deletion of this chromosome in the tumor. This is also consistent with observations from prior metaphase CGH analysis. Importantly, however, array analysis detected a copy number gain for the genomic regions representing the FES, TSC2 and HRAS genes, which were not detected by conventional CGH, most likely due to the superior resolution of the array technique.