Chromosome aberrations in canine multicentric lymphomas detected with comparative genomic hybridisation and a panel of single locus probes

Abstract

Recurrent chromosome aberrations are frequently observed in human neoplastic cells and often correlate with other clinical and histopathological parameters of a given tumour type. The clinical presentation, histology and biology of many canine cancers closely parallels those of human malignancies. Since humans and dogs demonstrate extensive genome homology and share the same environment, it is expected that many canine cancers will also be associated with recurrent chromosome aberrations. To investigate this, we have performed molecular cytogenetic analyses on 25 cases of canine multicentric lymphoma. Comparative genomic hybridisation analysis demonstrated between one and 12 separate regions of chromosomal gain or loss within each case, involving 32 of the 38 canine autosomes. Genomic gains were almost twice as common as losses. Gain of dog chromosome (CFA) 13 was the most common aberration observed (12 of 25 cases), followed by gain of CFA 31 (eight cases) and loss of CFA 14 (five cases). Cytogenetic and histopathological data for each case are presented, and cytogenetic similarities with human non-Hodgkin's lymphoma are discussed. We have also assembled a panel of 41 canine chromosome-specific BAC probes that may be used for accurate and efficient chromosome identification in future studies of this nature.

Figure 1

Cytogenetic distribution of 41 canine BAC clones representing a panel of chromosome-specific single locus FISH probes. In this example, probes are labelled with either Spectrum Red (presented as red signal), Spectrum Green (presented as green signal), Spectrum Gold (yellow), DEAC (blue) or biotin-Cy5 (pink). Each canine chromosome can be identified unequivocally using this panel of probes on the basis of the size of the chromosome, the cytogenetic location of the BAC clone and the fluorochrome with which it is labelled.

Figure 2

(A) Detection of unbalanced chromosome aberrations in canine lymphoma case 4138/00 using CGH analysis. Cohybridisation of male test (green) and female reference (red) probes onto a normal male metaphase spread is demonstrated. The sex-mismatch results in a 2 : 1 ratio of red : green for the X chromosome, which thus appears red. The Y chromosome appears green due to the 0 : 1 ratio of red:green for this chromosome. A number of autosomes appear to deviate from the expected 1 : 1 fluorochrome ratio. The complex microenvironment of the hybridisation reaction may produce apparent variation in hybridisation characteristics, as is evident in this cell for the CFA 31 homologues. However, profiling of 10–15 metaphase preparations serves to neutralise such differences. (B) Verification of chromosome identity using chromosome-specific single-locus probes. In this example, a total of 11 chromosome-specific single-locus probes were cohybridised in two successive reactions to the metaphase spread shown in (A) to facilitate accurate chromosome identification for CGH profiling. In the first reaction, the SLP for CFA 14 and CFA 30 were labelled with DEAC (presented as blue signal), CFA 26, CFA 28 and CFA 33 were labelled with Spectrum Orange (presented as green signal) and CFA 20 and CFA 32 were labelled with biotin-16-dUTP and detected with Cy5 (presented as pink signals). Following image acquisition, these probes were stripped from the chromosomes. The slides were then reprobed with a second group of SLPs, in which CFA 27 and CFA 31 were labelled in Spectrum Orange (presented as yellow signal) and CFA 25 and CFA 38 were labeled with DEAC (presented as orange signal). In this figure, the data from the two successive rounds of FISH have been overlaid to generate a composite metaphase spread showing all 11 SLPs.

Figure 3

Composite of CGH profiles from 25 canine lymphoma cases. The DAPI-banded ideogram of Breen et al (1999a) is displayed. For each case, genomic gains and losses are shown as green and red bars to the right and left of each chromosome, respectively. Each vertical bar represents a site of genomic imbalance in a single case (cases are identified at the top or bottom of red/green bars), and demonstrates the physical extent of the chromosome over which the aberration was detected. The evolutionarily conserved chromosome segments shared with the human karyotype (taken from Breen et al, 2001) are identified with coloured bars to the far left of each chromosome.