Influence of genetic background on tumor karyotypes: evidence for breed-associated cytogenetic aberrations in canine appendicular osteosarcoma

Abstract

Recurrent chromosomal aberrations in solid tumors can reveal the genetic pathways involved in the evolution of a malignancy and in some cases predict biological behavior. However, the role of individual genetic backgrounds in shaping karyotypes of sporadic tumors is unknown. The genetic structure of purebred dog breeds, coupled with their susceptibility to spontaneous cancers, provides a robust model with which to address this question. We tested the hypothesis that there is an association between breed and the distribution of genomic copy number imbalances in naturally occurring canine tumors through assessment of a cohort of Golden Retrievers and Rottweilers diagnosed with spontaneous appendicular osteosarcoma. Our findings reveal significant correlations between breed and tumor karyotypes that are independent of gender, age at diagnosis, and histological classification. These data indicate for the first time that individual genetic backgrounds, as defined by breed in dogs, influence tumor karyotypes in a cancer with extensive genomic instability.

Figure 1

Summary of highly-recurrent genomic imbalances in dog OSA. Genomic location (chromosome and assembly position) of loci that were aberrant in ≥ 30% of the cohort are shown on the x-axis, along with their cytogenetic location (Breen et al. 1999) and their Mb position within the dog genome sequence assembly (Lindblad-Toh et al. 2005; Thomas et al. in press). The y-axis indicates the percentage of the OSA cohort that showed copy number gain (green bar above the x-axis) or loss (red bar below the x-axis) of that locus. Regions harboring known cancer-associated genes are indicated above the corresponding bar.

Figure 2

Copy number imbalances for 11 cancer-associated genes within the OSA cohort. Cytogenetic and genome locations are indicated above each gene name

Figure 3

aCGH-directed FISH analysis of dog OSA. a) and b) show multicolor FISH analysis of 10 BAC clones on metaphase chromosomes (left) and interphase nuclei (right) from a case of osteoblastic osteosarcoma in a female Rottweiler. Numerous aberrant bi-armed chromosomes are apparent, resulting from fusion events between acrocentric chromosomes. FISH analysis supported genomic copy number aberrations identified in prior aCGH-analysis, involving a series of known oncogenes and tumor suppressor genes, including homozygous deletion of PTEN and CDKN2A, and high-level amplification of MYC. Additionally, targeted FISH analysis revealed structural chromosome aberrations that are intractable to aCGH analysis. For example, a) shows a metaphase spread with three copies of the TP53 marker (CFA 5q21, red probe). One hybridization signal was located on each arm of a bicentric chromosome structure (white arrowhead), suggestive of an isochromosome of CFA 5q. The third TP53 signal was located on another biarmed structure, consistent with CFA 5 fused with another chromosome. The extent of CFA 5 DNA sequence represented by these aberrant chromosomes was then investigated by hybridization of a panel of six BAC clones selected at intervals along the full length of the CFA 5 sequence assembly. These six clones are located on CFA 5 at 3Mb (yellow signal); 24Mb (green signal); 36Mb (TP53, red signal), 53Mb (purple signal), 74Mb (aqua signal) and 92Mb (red signal) (Thomas et al. 2007). The resulting pattern of probe hybridization (c) supported the presence of an isochromosome of CFA 5 (left homologue). d) Summary of enumeration data for selected BAC probes in a) and b) within ≥ 30 cells from the OSA case. These data also show the log₂ ratios from the BAC array for these loci and indicate that the FISH probe enumeration data and aCGH data are mutually supportive. Scale bars in a) and b) represent 10μm and the scale bar in c) represents 5μm.

Figure 4

Breed-associated chromosome copy number changes in OSA. Eleven genomic regions showed a significant association between copy number status and the breed of the patient. Regions harboring known cancer-associated genes are indicated in the corresponding bar. The p-value denoting the significance of breed-association is listed against each locus.