Dog models of naturally occurring cancer

Abstract

Studies using dogs provide an ideal solution to the gap in animal models for natural disease and translational medicine. This is evidenced by approximately 400 inherited disorders being characterized in domesticated dogs, most of which are relevant to humans. There are several hundred isolated populations of dogs (breeds) and each has a vastly reduced genetic variation compared with humans; this simplifies disease mapping and pharmacogenomics. Dogs age five- to eight-fold faster than do humans, share environments with their owners, are usually kept until old age and receive a high level of health care. Farseeing investigators recognized this potential and, over the past decade, have developed the necessary tools and infrastructure to utilize this powerful model of human disease, including the sequencing of the dog genome in 2005. Here, we review the nascent convergence of genetic and translational canine models of spontaneous disease, focusing on cancer.

Figure 1. Dog cancer genetics

(A) Protein sequence conservation in dogs. (i) Phylogenetic tree of the mammalian c-Met receptor. The branching pattern corresponds well with the organismal relationships. For example, the Boreoeutheria clade comprises two sister taxa which include primates, rodents, rabbits and a taxa including carnivorans and most hoofed animals. Although mouse and human c-Met branch together, the branch length of mouse c-Met shows that the protein sequence is more divergent than human and dog (scale bar shows amino acid changes per site). (ii) Dog proteins are more similar to those of humans than are mouse proteins. Phylogenetic treeing analysis of a composite of 10 cancer proteins branches human and dog proteins apart from mouse with a bootstrap value of 100. The following proteins were included: MYC, ERBB2, KIT, ret proto-oncogene (RET), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), PTEN, RB1, CDKN2A, breast cancer 1, early onset (BRCA1), TP53. [Neighbor-Joining trees shown (500-replicate bootstrap values); Maximum Parsimony topology is the same]. (B) Examples of breed-specific germ line variation with potential cancer relevance. (i) Common missense variant in Rottweiler c-Met receptor. WebLogo analysis shows a close-up of the consensus amino acid sequence of c-Met from 23 mammals. Letter height corresponds to the frequency of a given amino acid at each position, with the highest letters signifying complete conservation. 70% of Rottweiler’s have a missense variant at Gly 966, which is located in the extracellular region and could thus affect ligand binding or receptor signaling [71]. (ii) More than 60% of Rottweilers have a 273-kb copy number variant (CNV) in an intron of CSMD1, but it has not been observed in diverse other breeds (UCSC Browser; human gene transcribed right to left) [72]. (iii) Close-up of one of several non-coding conserved elements within the CSMD1 CNV (Vista Browser, conservation with human >60% shown by red coloring). The most conserved region within this area contains three candidate binding sites for the tumor suppressor transcription factor E2A (another conserved element contains TP53 binding sites [72]). The conservation (which is absent in chicken) is reduced in mouse in comparison to more distantly related mammals, horse and dog. (C) Somatic genome alterations in canine cancer. Kisseberth et al. isolated the OSW T cell lymphoma cell line and identified several genomic alterations [73]. A single two copy loss was found, and it affects the CDKN2A tumor suppressor gene. Subsequent analysis of OSW by high resolution tiling ologinucleotide-array CGH revealed many additional alterations, including focal two-copy deletions affecting as few as a single gene [72]. (i) Whole genome display of CGH analysis of OSW [72]. The midline shows a 1:1 DNA ratio to the reference genome of a boxer. Deletion CNVs are segments below the midline, and gains are above the midline (log 2 scale). “Un” denotes unmapped contigs and is highly enriched for repetitive sequences; the Y chromosome is absent from the canFam2 genome assembly. (ii, iii) Close-up of CGH analysis of chromosomes 11 and 22. Both chromosomes have 2-copy microdeletions. One confirms complete deletion of the tumor suppressor, p16/CDKN2A. The other spans a single active gene, SLITRTK1, which was previously implicated in malignant hematopoiesis [74]. This illustrates how dogs can be used as translational models of known human cancer genetics, as well as for discovery of novel genes in the same genetic pathways. (D) Second generation genotyping technology allows the integration of single nucleotide polymorphism and CNV maps. CNVs from two Greyhounds are shown. This 170k oligonucleotide array enables simultaneous SNP genotyping and DNA copy number determination (Illumina CanineHD). For each pair, the top window (i) shows DNA copy number as Log 2 R ratios, with the midline generally corresponding to copy number of 2. The bottom windows (ii) show allele frequencies. A copy number gain is detected as an upward shift on Log R ratio, and as a shift from B allele ratios of 1:1 (left and right segments) to 1:2 and 2:1 allele ratios (center segment). A copy number loss is detected as a downward shift in Log R ratio, and as a shift from allele ratios of 1:1 (left, right segments) to an allele ratio of 1:0 (or loss of heterozygosity; center).

Fig. 2. An example of the clinical relevance of dogs for cancer treatments

Canines are increasingly being used in clinical cancer drug trials to determine the efficacy of treatment given how closely many of the cancer they develop recapitulate the human cancer. (A) A picture of a Boston terrier, a breed predisposed to the development of Mast cell tumors. (B) London et al. conducted a clinical trial of an oral receptor tyrosine kinase inhibitor, Palladia on dogs with recurrent mast cell tumors. Shown here is a Kaplan-Meier survival analysis demonstrating time to tumor progression in placebo-treated and Palladia-treated dogs with Mast Cell Tumors [75]. (C) A breakdown of the clinical trial of Palladia, including the demonstrated advantages of dogs as models of pharmacologic cancer intervention. Reproduced with Permission, from [75].

Fig. 3. Prevalence of B and T-cell lymphoma in Dog breeds

A varying excess of T and B-cell lymphoma, in a breed specific manner, has been noted. Presented here is the observed percentage of T vs B-cell lymphoma by breed: Irish wolfhounds (100:0 Siberian huskies (88.9:11.1), Shih Tzus (81:19), Airedale terriers (80:20), Cavalier King Charles spaniels (80:20), and Yorkshire terriers (80:20). By contrast, the breeds with an excessive occurrence of B-cell compared to T-cell lymphomas were cocker spaniels (93.2:6.8) and basset hounds (94.4:5.6) [59]. Photo sources follow: http://www.dublinirishfestival.org/animals/irishwolfhound.php; http://blogneffy.blogspot.com/2010/06/wanted-shih-tzu-breeders-in-davao-city.html; http://sentinelkennels.com/images/airedale.jpg; http://www.justdogbreeds.com/images/breeds/cavalier-king-charles-spaniel.jpg; http://www.petsflick.com/images/yorkshire-terrier.jpg; http://tidyyourdog.com/wp-content/uploads/2009/04/siberian-husky.jpg; http://www.petside.com/breeds/chinese-shar-pei.php; http://www.fordogtrainers.com/ProductImages/dog-breeds-muzzles/Australian-Shepherd-muzzle-Australian-Shepherd.jpg; http://www.breederretriever.com/photopost/pindex/516/; http://retrieverman.files.wordpress.com/2009/01/white-golden-retriever-wikipedia.jpg; http://www.dogtastic.org/dogtastic/images/BreedPics/cocker%20spaniel.jpg; http://a1.cdnsters.com/static/images/dogster/breeds/basset_hound.jpg.

Fig. 4. Translational potential of tumor bearing dogs

On the bottom is the typical course of human drug research and development. There is no established paradigm for the drug research and development in dogs and other companion animals [6]. Although our schematic mirrors the same process in pets, most drugs used on patient animals are taken from human drug development or are approved human drugs used off-label. Indeed, few regulations exist for Phase I/II/III clinical trials before drugs are used in pets.