Page 547 - Withrow and MacEwen's Small Animal Clinical Oncology, 6th Edition

CHAPTER 25 Tumors of the Skeletal System 525

A distribution favoring bone marrow volume was seen for dogs 21 spontaneously arising OSAs failed to identify gross RB gene
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exposed to strontium-90. OSA is a rare, late complication of alterations by Southern blotting, and protein expressions of RB
were identified in all OSA samples evaluated.
radiation therapy (RT) in dogs, with an incidence of less than
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VetBooks.ir 5%. 39,41,43–45 samples, the observed translational normalcy does not exclude
Despite normal protein expression of RB in canine OSA
Genetic Factors
Ample evidence exists implicating the involvement of genetic the possibility for allelic deletion of the RB gene as prior studies
in human OSA samples have demonstrated that LOH at the RB
and heritable factors for the development of OSA in dogs. Cur- gene locus does not absolutely correlate with inactivation of the
rently, the most thoroughly described gene mutation that con- RB gene at the protein level. Substantiating the possibility that
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tributes to OSA formation and/or progression in dogs is p53, RB gene may have allelic deletion in spontaneously arising canine
which is supported by in vitro and in vivo studies. 48–55 In cell OSA, analysis of 38 OSA samples with comparative genomic
culture, missense point mutations within the DNA-binding hybridization techniques identified copy number loss in 29% of
domain of p53 have been identified in canine OSA cell lines cases, resulting in a correlative reduction or absence of RB pro-
with consequent loss of p53 functionality demonstrated by tein expression in 62% of OSA samples tested. Most recently,
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inappropriate downstream target gene transcription (p21 and the importance of RB functionality in OSA biologic behavior has
MDM2) after genotoxic insult. Similarly, p53 mutations have been explored with aberrant RB-E2F transcriptional regulation
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been identified in dogs with spontaneously arising OSA through contributing to aggressive OSA biologic behavior and worse clini-
various genetic techniques including single-strand conforma- cal outcomes. Collectively, these findings support the functional
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tional polymorphism (SSCP), polymerase chain reaction (PCR), role of the RB-E2F signaling pathway in OSA biology. Based
or Southern blotting, followed by nucleotide sequence analysis, upon these investigative findings, it is likely that aberrations in the
whereby missense mutations involving exons 4 to 8 of p53 have RB gene or transcriptional activities participate in sporadic OSA
been identified in 24% to 47% of all spontaneously arising OSA formation and/or progression in dogs.
samples. 48,52,54,55 In addition to exons 4 to 8, the entire gene In addition to p53 and RB gene abnormalities, the phospha-
sequence of p53 has also been assessed by PCR and SSCP from tase and tensin homolog (PTEN) tumor suppressor gene is sus-
59 spontaneously arising appendicular and axial OSA samples pected to participate in the genetic pathogenesis of canine OSA.
with missense point mutations affecting p53 identified as the Original in vitro studies conducted with canine OSA cell lines
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most common gene abnormality. Finally, through the imple- demonstrated that most cell lines (60%) harbored mutations in
mentation of targeted microarray-based comparative genomic PTEN. Corroborating the cell line findings, expression of PTEN
hybridization analysis of 38 canine OSA cases, similar recur- was either absent (n = 6) or variable (n = 4) in 15 spontaneously
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rent cytogenetic aberrations classically present in human OSA arising OSA samples. Further support for the loss of PTEN
samples were also identified in OSA specimens collected from gene in canine OSA pathogenesis has been the identification of
dogs, including loss of heterozygosity (LOH) of the p53 gene in specific recurrent chromosome copy number aberrations through
18% of tumors. 56 targeted microarray-based comparative genomic hybridization
Substantiation for the presence of p53 mutations in spo- studies, with the identification of chromosomal region deletions
radic canine OSA has also been documented by immuno- and high recurrent copy number losses encompassing the PTEN
histochemical studies, as a hallmark of many p53 mutations gene locus. 56,62 In addition to the PTEN gene, other genes identi-
is enhanced protein stability of this normally labile protein, fied by high resolution comparative genome hybridization studies
enabling detection of protein with methodologies such as potentially involved in the genetic pathogenesis of OSA included
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immunohistochemistry (IHC). In one study of 106 osteo- overexpression of RHOC and RUNX2 and under expression of
genic tumors, a greater percentage of appendicular (84%) TUSC3. Most recently, through a comparative approach uti-
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OSAs overexpressed p53 protein in comparison with OSAs lizing high resolution oligonucleotide array comparative genomic
arising from the axial skeleton (56%) and other non-OSA bone hybridization, additional disease-associated DNA copy number
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tumors (20%). Analogously, in a complementary study focus- aberrations have been identified as an ever expanding list of gene
ing on 103 OSA samples, 67% stained positively for p53 pro- candidates that may have functional significance in the develop-
tein and associated staining intensity was significantly greater ment of OSA, including ADAM15, CTC1, MEN1, and CDK7. 63
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in OSAs derived from the appendicular versus axial skeleton. A growing body of evidence in dogs supports breed-associated
Recently, a homolog within the p53 family of proteins, specifi- inheritance of OSA, especially in Scottish Deerhounds, Rottwei-
cally ΔNp63, has been identified to be involved in canine OSA lers, Greyhounds, Great Danes, Saint Bernards, and Irish Wolf-
cellular survival and metastasis. The functionality of ΔNp63 hounds. 26,64–69 The presumed hereditary basis for OSA in specific
in canine OSA cell lines contributed to apoptosis resistance canine breeds has been affirmed through genome-wide analyses
through perturbations in the balance of proapoptotic Bcl-2 whereby 33 genetic loci were identified to contribute to heritable
family members, Puma and Noxa, as well as STAT3 phosphor- OSA in Greyhounds, Rottweilers, and Irish Wolfhounds. These
ylation and modulation of angiogenesis and invasion through findings are highly significant and point to a polygenic spectrum
VEGF-A and IL-8 expressions. 58 of germline risk factors for OSA development in these three breeds
Another tumor suppressor gene likely contributing to OSA with putative genes involved in OSA genesis connected with bone
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development is retinoblastoma (RB). Based upon investigations differentiation and growth pathways. Another breed at risk is
using five tumorigenic immortalized canine OSA cell lines, the the Scottish Deerhound with a 15% incidence of OSA 65–67 and a
RB gene signaling pathway was dysregulated with the persistence narrow heritability of 0.69, which indicates that 69% of the cause
of hyperphosphorylated RB protein in the absence of mito- for OSA development in Scottish Deerhounds is due to heritable
gen stimulation. Despite apparent aberrant RB gene signaling, trait, likely a Mendelian major gene with dominant expression.
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reduction in RB protein was only identified in one of five cells A confirmatory study of familial OSA development in Scottish
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lines. Corroborating these in vitro findings, the evaluation of Deerhound litters further supports that the inheritance of OSA in