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Vet Pathol 40:237-248 (2003)
© 2003 American College of Veterinary Pathologists

Clinical and Pathologic Relevance of p53 Index in Canine Osseous Tumors

P. Loukopoulos, J. R. Thornton and W. F. Robinson

School of Veterinary Science, The University of Queensland, Brisbane, Queensland, Australia


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The clinicopathologic value of the immunohistochemical (IHC) expression of p53 protein was evaluated in 167 canine osseous tumors. p53 staining frequency and intensity in tumor cells was expressed as a p53 index. p53 index was significantly higher in osteosarcomas than in other sarcomas, chondrosarcoma, multilobular tumor of bone, and tumors initially misdiagnosed as osteosarcomas as well as in appendicular versus axial and in distal versus proximal osteosarcomas. A strong correlation is demonstrated between the p53 index and a range of clinicopathologic parameters in osteosarcoma, including the tumor site, histologic grade and score, mitotic index, degree of tumor necrosis, and pleomorphism. Chondroblastic osteosarcomas had significantly higher and telangiectatic osteosarcomas significantly lower p53 index than did osteosarcomas belonging to other histopathologic subtypes, a fact that tends to reinforce the perception of these osteosarcomas as distinct clinicopathologic entities. Entire males had higher p53 index than did neutered males. p53 index was higher in Rottweilers than in Great Danes and Terriers, confirming breed susceptibilities to osteosarcoma. p53 index showed no association with age, primary or secondary site status, or the presence of metastases or other tumor types. Biopsy samples had a higher p53 index than did postmortem samples, either because of differences in sample processing or the possibility that p53 overexpression is more evident at the earlier stages of osteosarcoma pathogenesis, presumably represented by the biopsy material. IHC examination for p53 and the derived index has the potential to be used as an additional diagnostic tool and prognostic indicator for osseous tumors.

Key words: Bone diseases; dogs; immunohistochemistry; neoplasms; osseous tumors; osteosarcoma; p53.

The p53 gene encodes a nuclear phosphoprotein (p53) that normally acts as a tumor-suppressor gene.10 p53 guards cells against replication when their genome is damaged or abnormal, by initially arresting the cell cycle and then by either activating and regulating DNA repair or by inducing programmed cell death (apoptosis) if genomic damage is excessive.28,57,65

Absence of a functional p53 gene, p53 protein, or p53 pathways may remove mechanisms that normally arrest the proliferation of transformed cells, disrupting the apoptotic response that may ensue and therefore contributing to the development and progression of neoplastic clones.93 Because of this central role in cell cycle and in carcinogenesis,28 p53 is the most frequently altered gene in human tumors;37,39 p53 gene mutations have been found in 40–45% of cases of the 10 most frequent cancers in humans.99

Wild-type p53 is present in all cell types, usually in low quantities,78 and has a short half-life that makes it difficult to detect by immunohistochemical (IHC) techniques.53 However, abnormal, nonfunctional p53, either caused by mutations of p53 gene40 or by the formation of p53 protein complexes with viral or endogenous proteins87 such as a large T antigen of simian virus 40 or MDM2 (murine double minute-2),58 may result in the synthesis of an abnormal protein that is metabolically stable26,35 and has a 10- to 20-fold longer half-life than wild-type p53,26 accumulating in the cell and reaching concentration levels detectable by immunohistochemistry.44 IHC detection of p53 indicates, therefore, alterations in the p53 gene or p53 product and demonstrates a progression toward a tumor-associated phenotype.60,105 p53 protein overexpression has been detected at high levels in a large number of sporadic tumors and in virally and chemically transformed cell lines from mice and humans.3,41,44,78 Furthermore, p53 is a highly conserved gene in evolution, with regions of high homology in mammals, birds, fish, and amphibians;77 most mutations occur within the highly conserved regions of p53.40

Demonstration of the presence of p53 abnormalities is predictive of biological behavior in a wide spectrum of human malignancies,18,86,117 including osteosarcoma,64 synovial sarcoma,94 breast cancer,80 ovarian carcinoma,20,96 prostate carcinoma,11,12,115 renal adenocarcinoma,62 gastric cancer,43,95 adenoma and carcinoma of the ampulla of Vater,85 colorectal adenoma54 and carcinoma,32,34,48,52,55 gliomas,83 astrocytic tumors,23 tumors of the urinary bladder,24,25,97 cutaneous68 and head and neck squamous cell carcinomas,6,45,66,67,121 and advanced head and neck cancer treated with chemo/radiotherapy.86,117

The canine p53 gene has been assigned to chromosome 5,36 and the complete wild-type canine p53 cDNA has been isolated and sequenced14,112 and the full length canine p53 protein characterized.112 A high level of sequence homology was demonstrated with human sequences and other higher vertebrates,112 with the conserved domains II, III, IV, and V being identical to those of humans.82

Alterations in the p53 gene have been reported in a variety of canine cancers, including osteosarcoma. Mutations were found, depending on the study, in 7 of 15 appendicular osteosarcomas,50 4 of 17,110 and 8 of 2175 primary osteosarcomas screened, as well as in osteosarcoma cell lines.59 The locations and types of mutations were nearly identical to those reported in human cancer. Van Leeuwen et al.111 also showed the presence of missense mutations accompanied by loss of the wild-type allele in three of nine canine mammary tumor cell lines and in two of the corresponding tumor tissues of origin analyzed. Again, all mutations were located in one of the four regions that are frequently affected in human cancers. Somatic mutations, accompanied in some cases by a spectrum of chromosomal aberrations, have also been reported in other canine benign79 or malignant14,69–71,79,114 mammary tumors, thyroid carcinoma,19 skin tumors,71 an adenoma of the circumanal gland,73 lymphosarcoma,81 and lymphoma,113 as well as in keratoconjunctivitis sicca30 but are not present in all cases and tumor types in dogs.72 Germ-line mutations have been reported in one case each of canine lymphoma113 and mammary cancer.114

Tumors in bone are not infrequent in the dog,8,17 the main type being osteosarcoma, followed by chondrosarcoma,106 fibrosarcoma, haemangiosarcoma, and multilobular tumor of bone. In contrast with their counterparts in humans where metastasis to bone of carcinomas, in particular, is common, they are mainly primary tumors.118 Osteosarcoma, in particular, accounts for the majority of these tumors7–9,61,63 and is one of the most malignant and aggressive tumors in dogs, with a predilection in large and giant breeds.109 The majority of cases that involve the appendicular skeleton often have lung metastases before the primary tumor is diagnosed.84 Despite advances in its management, there are few established or proposed prognostic indicators for canine osteosarcoma, including age,100 body weight,38,90,109 breed,15,90 tumor location,38,76 serum alkaline phosphatase levels,21,31 completeness of surgical excision,38 tumor grade,56 tumor diameter and volume,27,76 tumor subtype,38,76 tumor microvessel density,16 and tumor necrosis after chemotherapy.89

The aim of the present study was to characterize osseous tumors according to both their p53 protein IHC expression and their clinical and histopathologic characteristics as a basis for developing a prognostic evaluation protocol of these tumors in clinical patients.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
One hundred sixty-seven archival cases of osseous tumors were retrieved and their clinical and pathologic features examined in detail. Histologic slides were examined by all investigators. The initial diagnosis of osteosarcoma was rejected in 32 cases (Table 1). The diagnoses offered are given in Table 1.


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  		Table 1. Tumor types examined and their p53 index and FPN values.

 
Osteosarcomas were classified into histologic subtypes, as described previously,98 on the basis of the quality and quantity of the extracellular matrix (ECM) produced (osteoblastic productive, minimally productive, chondroblastic and fibroblastic subtypes); the degree of differentiation (poorly differentiated subtype); the abundant presence of giant cells (giant cell subtype); and the dominant presence of blood-filled cystic lesions (telangiectatic subtype). Osteosarcomas were classified as simple if osteoid or bone was the only matrix or as compound if there were more than one type of ECM produced by the malignant cells.

The mitotic index, the degree of nuclear pleomorphism, and the degree of tumor necrosis of osteosarcomas were assessed to classify them into grades, as described previously.102 The mitotic index (number of mitoses per 10 fields at 400x) was estimated and scored in the following way: 1–10 mitoses = score 1, 11–20 = score 2, 21–30 = score 3, 31 or more = score 4. The degree of tumor necrosis was scored in the following way: 0% = score 0, 1–15% = score 1, 16–50% = score 2, 51–100% = score 3. The degree of nuclear pleomorphism was estimated as mild, moderate, or marked (score 1, 2, or 3). The three scores obtained were added to calculate the histologic score (HS) of the tumors (2–10), which was then used to classify the tumors into grades in the following way: HS 1–5 = grade I, HS 6–8 = grade II, and HS 9 or 10 = grade III.

Each tissue sample had been fixed in 10% neutral buffered formalin for a varying period of time, routinely processed, and embedded in paraffin blocks. Five-micrometer-thick serial sections were prepared from each archival tissue block, mounted on silane-coated slides, and used for IHC staining. Tissue sections were similarly stained with hematoxylin and eosin for review of the initial morphologic diagnosis and for evaluation of nuclear pleomorphism, mitotic index, and the extent of necrosis.

Immunohistochemistry

An IHC protocol was developed and optimized for the demonstration of p53 protein in canine osseous tumors. In preliminary experiments, canine osteosarcomas and known p53-positive human ovarian carcinomas were examined with serially diluted primary antibodies to decide a suitable concentration of antibody (1:75). Microwaving was compared and preferred to boiling as a means of antigen retrieval.88

Two human ovarian carcinomas known to be p53 positive from previous studies20,119 were kindly provided by Mr. M. D. Walsh (Department of Pathology, University of Queensland) and used as positive controls (Fig. 1). For negative controls, the primary antibody was replaced with Tris-buffered saline (TBS), pH 7.6. Nontumorous areas of the positive control slides served as internal negative tissue controls.



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  		Fig. 1. Human ovarian carcinoma. Positive control. IHC localization of p53 protein: intense nuclear staining of tumor cells. Streptavidin–biotin–peroxidase method, Mayer's hematoxylin counterstain. Bar = 60 µm.
Fig. 2. Canine osteosarcoma. IHC localization of p53 protein: intense nuclear staining of tumor cells. Streptavidin–biotin–peroxidase method, Mayer's hematoxylin counterstain. Bar = 60 µm.

 
Sections were deparaffinized in 100% xylene (3 x 2 minutes) and rehydrated through 100% ethanol (3 x 2 minutes). Sections were then subjected to heat for antigen retrieval by microwaving in preheated citrate buffer (pH 6.0) at 95 C for 10 minutes. Sections were allowed to cool for 20 minutes and rinsed with TBS. Sections were treated for 15 minutes with 3% (v/v) H2O2 in distilled water to inhibit endogenous peroxidase activity. Nonspecific antibody binding was reduced by incubating the sections with 4% commercial nonfat skim milk powder in TBS for 25 minutes. Sections were then incubated with primary antibody (diluted 1:75) overnight at 4 C. The antibody used was the polyclonal rabbit anti-human antibody CM-1 (Signet Laboratories, Dedham, MA), which recognizes both wild and mutant types of p53 protein. The sections were incubated with a rabbit biotin-labeled secondary antibody and then a streptavidin–biotin–peroxidase complex (both Dako, Carpinteria, CA) at room temperature.

After washing, 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Company, St. Louis, MO) was used as chromogen with H2O2 as substrate. The sections were counterstained lightly with Mayer's hematoxylin (Sigma), dehydrated, cleared in xylene, and mounted on cover slips with Permount (Fisher Scientific, Fairlawn, NJ).

All sections were evaluated independently by two observers (P. Loukopoulos and W. F. Robinson), and variations in the interpretation of results were resolved using a dual-headed microscope. The results of the IHC analysis were interpreted without knowledge of clinical data. Negative and positive control slides were compared for nonspecific and background staining or cross-reactivity.2 Only those tumors containing cells with a distinct nuclear staining for p53 were considered to be positive. The fraction of positively stained nuclei (FPN) for p53 was scored in the following way: 0% = score 0 (negative tumors), 1–10% = score 1, 11–25% = score 2, 26–50% = score 3, 51–100% = score 4 (positive tumors).

Simultaneously, the mean nuclear staining intensity (NSI) was estimated and expressed as weak, moderate, or strong (score 1, 2, or 3). To express the staining semiquantitatively, a p53 index was calculated by multiplying the fraction of stained nuclei score (0, 1, 2, 3, or 4) by the nuclear staining intensity (1, 2, or 3). Cell staining patterns, regional variation, staining specificity, and agreement between multiple tumor sections were also noted.

Statistical analysis

The p53 index, NSI, and FPN were related to clinicopathologic parameters and compared between tumor types. To assess the statistical significance of intergroup differences in quantitative data, the Kruskal-Wallis one-way analysis of variance (ANOVA) on Ranks or the Mann-Whitney rank sum test were performed. Data grouped in contingency tables were analyzed statistically using the chi-square test or Fisher exact test for comparison of proportions.

Statistical significance was set at 0.05. The collected data were analyzed using the Jandel Sigmastat® Statistical Software (version 2.0, SPSS Science, Chicago, IL).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
A streptavidin–biotin IHC protocol for the detection of p53 protein in canine osseous tumors was developed and optimized. One hundred sixty-nine canine tumors were evaluated (Fig. 2). The number of positive cases for every tumor type is given in Table 1. FPN was significantly associated with NSI (P < 0.001).

Osteosarcomas showed significantly higher p53 index, FPN, NSI, and ratio of positive cases when compared with all other tumors examined as a group as well as when compared with the group of other osseous tumors, multilobular tumors of bone (MTB), and benign tumors as a group (Table 2). Osteosarcomas also had significantly higher p53 index and FPN than did osseous tumors that were initially diagnosed as osteosarcomas, the diagnosis of which was revised (misdiagnosed tumors), other sarcomas of bone as a group, and chondrosarcomas (Table 3).


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  		Table 2. P53 index and FPN values for selected osteosarcoma groups.

 

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  		Table 3. P values for p53 parameters resulting from the comparison of osteosarcoma with other tumor types.*

 
There were no significant differences in p53 index, FPN, or NSI when tumors other than osteosarcomas were compared pairwise. All benign tumors (n = 8), including four cases of MTB and a case each of osteoma, cartilaginous exostosis, bone cyst, and cementoblastoma, were p53 negative.

p53 index, FPN, and NSI were significantly associated with the histopathologic grade of osteosarcomas (Kruskal-Wallis one-way ANOVA on ranks, P < 0.001 for all three parameters), the histologic score (one-way ANOVA on ranks, P < 0.001 for all parameters), the mitotic index (P = 0.004 for p53 index and FPN, P < 0.001 for NSI), the degree of tumor necrosis (P = 0.02 for p53 index, P = 0.028 for FPN, P = 0.032 for NSI), and the degree of nuclear pleomorphism (P < 0.001 for all parameters). Osteosarcomas with a high tumor grade, mitotic index, or degree of pleomorphism had significantly higher p53 index and FPN than did osteosarcomas where such parameters were low (Table 5). Tables 4 and 5 show the interrelationships between tumor grade, mitotic index, necrotic score, and degree of pleomorphism groups of osteosarcoma when compared pairwise for p53 index, FPN, and NSI values.


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  		Table 5. P values for p53 parameters for pathologic data relating to tumor grade in canine osteosarcomas.*{dagger}

 

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  		Table 4. Histopathologic data relating to tumor grade for osteosarcoma cases.*

 
Chondroblastic osteosarcomas had significantly higher p53 index (P = 0.043), FPN (P = 0.050), and NSI (P = 0.019) than telangiectatic osteosarcomas. Osteoblastic productive and osteoblastic minimally productive osteosarcomas had significantly higher NSI (P = 0.012 and 0.038, respectively) but not p53 index (P = 0.082 and 0.084, respectively) or FPN (P = 0.084 and 0.099, respectively), compared with telangiectatic osteosarcomas. Telangiectatic osteosarcomas also had significantly lower p53 index (P = 0.050) and NSI (P = 0.009) but not FPN (P = 0.058) than the group of all other osteosarcoma subtypes. Compound osteosarcomas did not have a significantly higher p53 index than did simple ones (P = 0.32).

Appendicular osteosarcomas showed significantly higher p53 index (P = 0.028), NSI (P = 0.006) and ratio of positive cases (58/84 versus 16/38, P = 0.009) but not FPN (P = 0.086), compared with axial osteosarcomas but not with extraskeletal ones (n = 4). There was no significant difference in p53 parameters between appendicular and axial osteosarcoma metastases.

The ratio of p53-positive cases was significantly higher in appendicular osteosarcomas that arose in distal (25/28 or 89%) compared with proximal metaphyses (23/36 or 63%) (chi-square test, P = 0.042).

In relation to tumor location, antebrachial osteosarcomas had the highest p53 index, whereas cranial ones had the lowest. p53 index was significantly lower in nongnathic cranial osteosarcomas compared with femoral (P = 0.049), tibial (P = 0.032), and antebrachial (P = 0.026) osteosarcomas but not with humeral (P = 0.09) or hind limb osteosarcomas (n = 38, P = 0.08).

There was no statistical difference in p53 index in the primaries of metastatic osteosarcomas (n = 28) compared with osteosarcomas without demonstrable metastases at the time of biopsy or necropsy (n = 38), in osteosarcoma metastasies (n = 24) compared with the primaries of metastatic or nonmetastatic osteosarcomas, in osteosarcoma cases with documented parallel presence of other tumors (n = 9) compared with osteosarcoma cases without it (n = 49), or in forelimb (n = 44) compared with hind limb osteosarcomas (n = 38).

p53 parameters were not associated with age or sex alone but were associated with the neutered or entire status of the animal alone or in combination with the sex of the animal. Entire animals, whether male or female, had significantly higher FPN than did neutered animals (P = 0.042). Entire males had higher p53 index, FPN, and NSI than did neutered females, entire females, and neutered males, in descending order. Entire males had significantly higher p53 index (P = 0.009) and FPN (P = 0.003) but not NSI (P = 0.058) than did neutered males. Entire females had significantly higher FPN (P = 0.025) but not p53 index (P = 0.065) or NSI (P = 0.145) than did neutered males. Neutered females tend to have higher p53 index than do neutered males but the difference was not significant (P = 0.066).

p53 expression was associated with the breed of the animal. Rottweilers (n = 23) had higher p53 index (4.8 ± 4.5) than did Great Danes (1.8 ± 2.9, n = 10)(P = 0.040) or Terriers as a group (1.0 ± 1.6, n = 6) (P = 0.038).

Samples taken as surgical specimens and biopsy samples, or biopsy samples alone, had a higher p53 index than did samples taken during postmortem examinations (P = 0.031 and 0.01, respectively).

Cytoplasmic staining was observed in 21 tumors (12%) including 15 osteosarcomas, 3 MTB, 2 chondrosarcomas, and 1 plasmocytic sarcoma. These tumors were registered as positive only if nuclear staining was present, as was the case for 17 cases, including all osteosarcomas. In all but three cases, cytoplasmic staining was moderate or intense and, where applicable, as intense as nuclear staining, a fact that sometimes created difficulties in the interpretation of the results, especially when complicated by background staining.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study, p53 overexpression is demonstrated in a number of canine osseous tumors, and evidence is provided for the clinical and pathologic relevance of p53 index in these tumors, with particular emphasis on osteosarcoma.

IHC overexpression of p53 in canine tumors has so far been reported in osteosarcomas and MTB,92 benign and malignant mammary tumors,46,91 epithelial colorectal tumors,74,120 cutaneous mast cell tumors,33,49 skin papillomas,108 seminomas and Sertoli-cell tumors but not in Leydig-cell tumors,47 squamous cell carcinomas,107 nasal adenocarcinomas,29 and a small number of other canine tumors.29,51 In one study on mammary tumors, mammary osteosarcomas were the only tumor type that exhibited p53 immunoreactivity.46

In the present study, p53 protein expression was detected immunohistochemically in 10 osseous tumor types, in most of them for the first time. Our results are in accordance with a previous study,92 in that p53 protein is overexpressed in most of the canine osteosarcomas and that the prevalence of overall positivity is higher in osteosarcoma compared with MTB and in appendicular compared with axial osteosarcomas. Furthermore, in our study using the derived term, p53 index, osteosarcomas showed significantly higher p53 index compared with chondrosarcomas, benign tumors, other osseous tumors, and other osseous sarcomas, confirming the observation that p53 expression correlates with highly aggressive tumor behavior. Interestingly, osteosarcomas also had higher p53 index than did misdiagnosed tumors that were initially diagnosed as osteosarcomas. This fact, in conjunction with the above, leads us to suggest that the addition of IHC examination of canine osseous tumors for p53 may prove to be a valuable tool in their sometimes challenging differential diagnosis, particularly regarding cartilage-forming tumors, such as chondrosarcoma and MTB, which were misdiagnosed more frequently than other tumor types.

Previous studies suggested that p53 expression is not useful prognostically, in association with survival time, in canine epithelial colorectal tumors120 or cutaneous mast cell tumors.33,49 In one of the latter studies on cutaneous mast cell tumors, p53 expression was associated with tumor grade and site but not with breed predisposition, tumor size, grade, and differentiation.33

In the present study, a direct correlation is demonstrated between the p53 index and a range of clinicopathologic parameters, including the histologic grade and score of osteosarcomas, the mitotic index, degree of tumor necrosis, degree of nuclear pleomorphism within the tumor, histopathologic subtype, appendicular or axial location of the tumor, tumor site and proximity to the body axis, breed, sex, and entire or neutered status.

The question of the association of survival time and the p53 index for osteosarcoma remains to be determined. However, recent studies have shown that the histologic grading of osteosarcomas is of prognostic significance56 and is currently used routinely to make decisions on the clinical management of the tumors,102 particularly regarding chemotherapy protocols.4,103,104 p53 index may therefore be useful as an additional indicator to those traditionally used in the prognostic evaluation of osteosarcoma cases.

The relevance of classifying osteosarcomas into subtypes has been questioned in the past by some authors and the issue remains somewhat unresolved.76,101 The significant difference in p53 index between the various subtypes in our study tends to support the view that subtypes represent distinct clinicopathologic entities. Whether certain subtypes have any association with increased or decreased survival times remains to be answered. We consider, however, that familiarity with the different histopathologic presentations of osteosarcoma may be helpful in establishing an accurate diagnosis and, consequently, a more suitable therapeutic approach, having an indirect positive effect on survival time. The higher p53 index observed in chondroblastic osteosarcomas may be caused by their, yet to be confirmed, more aggressive biological behavior and higher rate of unrestrained cell proliferation. Alternatively, our findings may suggest that p53 overexpression is not exclusively associated with malignancy but may in part be attributed to the increased rate of matrix turnover and tissue remodeling within chondroblastic osteosarcomas and the recently proposed role p53 may play in these processes in conjunction with matrix metalloproteinases.13

Similarly, the lower p53 index observed in telangiectatic osteosarcomas may be attributed to their possibly less malignant phenotype or to the possibility that the formation of blood-filled cystic lesions, which characterizes telangiectatic osteosarcomas,98 is a early event in their pathogenesis, unlike, perhaps, the inactivation of the p53 gene or p53 protein.

The presence of metastases or other tumors did not generally appear to influence p53 expression, either because p53 index alone truly does not reflect metastatic potential or because the retrospective nature of the study resulted in cases with possibly incomplete necropsies being included in the nonmetastatic group.

Body weight90 and size109 are established negative prognostic indicators in canine osteosarcoma. The higher p53 index observed in distal compared with proximal osteosarcomas may be attributed to the greater effect body weight has on the distal compared with the proximal metaphyses of long bones.

Interestingly, p53 index in osteosarcoma was higher in Rottweilers than in Great Danes and Terriers, a finding that may be related to selective breeding for desired traits in certain breeds of dogs. Knowledge of the relative frequency of the breeds within the canine population and the overall risk of osteosarcoma and other tumors for every breed would help create a more complete picture. In any case, further epidemiologic and genetic studies are needed to elucidate potential breed susceptibilities and we tend to agree with Veldhoen et al.114 in that acquiring information about p53 inactivation in canine tumors could prove invaluable in the successful outbreeding of inherited predisposition to cancer in the dog.

An interesting finding of the study was the fact that entire males had higher p53 index than did neutered males. This suggests a role for androgenic hormones in the pathogenesis of canine osteosarcoma that requires further investigation.

The origin of the sample has a clear effect on p53 immunoreactivity. The reduced immunoreactivity of postmortem samples may be caused by their sometimes prolonged fixation time, the longer interval before fixation or the larger size of the samples compared with biopsy material, or it may be caused by the possibility that p53 overexpression is more evident at the earlier stages of osteosarcoma pathogenesis, presumably represented by the biopsy material. Cytoplasmic staining was detected in 21 (12%) of the tumors examined. Most previous reports have concentrated on nuclear immunolocalization of p53 protein, whereas only a few have reported or discussed its cytoplasmic localization,42,116 including that in human osteosarcomas,5 canine intestinal adenocarcinomas,29 and testicular tumors.42,116 Accumulation of p53 protein in the cytoplasm may be a result of an abnormal nuclear localization signal or may be caused by cytoplasmic factors, such as a 70-kDa heat shock protein (HSP70) that binds to the mutant p53 protein, causing its cytoplasmic localization.42,26 Although p53 positivity is usually expressed as FPN, the nuclear staining intensity was associated with a number of clinicopathologic parameters in our study, in some cases in the absence of such association of these parameters with FPN, prompting us to include it in our analysis. p53 indices (positivity x staining intensity) have also been used in assessing p53 expression in other, human, malignancies.1,22,95 The present study is, to our knowledge, the only one to describe a close relationship between the p53 index of a nonhuman tumor and its clinicopathologic parameters. It is also one of few studies to describe a relationship between the p53 index of any tumor and certain pathologic parameters such as the tumor subtype. It is evident that there is a fundamental abnormality in the function of the p53 tumor-supressor gene in canine osseous tumors and osteosarcomas in particular. This is clearly central not only to the development of the disease but, apparently, also to the differentiation of osteosarcomas into particular histopathologic subtypes. From this study, we consider that the addition of an IHC examination for p53 and the derived index has the potential to be used as an additional prognostic indicator for canine osseous tumors. A strategic approach incorporating other indicators will provide avenues to understanding the etiology of canine osteosarcoma and will help develop a broader-based, and therefore more accurate, diagnostic and prognostic index and therapeutic protocol for canine osteosarcoma patients.


   Acknowledgments
 
This study was supported by a grant from the Australian Companion Animal Health Foundation. Mr. Loukopoulos was a University of Queensland Sister Janet Mylonas Memorial Scholarship holder. We thank Ms. J. Priest for statistical advice, Mr. C. Cazier for technical support, and the members of the Veterinary Teaching Hospital and the Departments of Companion Animal Sciences and Veterinary Pathology and Anatomy, University of Queensland, for providing the clinical and archival material. Preliminary results from this study were presented at the WSAVA/FECAVA World Congress 2000.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Backe J, Gassel AM, Hauber K: p53 protein in endometrial cancer is related to proliferative activity and prognosis but not to expression of p21 protein. Int J Gynecol Pathol 16:361-368, 1997[ISI][Medline]
  2. Barrett J: Immunohistochemical study of cytoskeletal filaments in spindle cell tumors of the dog (Canis familiaris). MVSc Thesis, University of Queensland, Brisbane, Australia 1998
  3. Bartek J, Iggo R, Gannon J: Genetic and immunochemical analysis of mutant p53 in human breast cancer cell lines. Oncogene 5:893-899, 1990[ISI][Medline]
  4. Bergman PJ, MacEwen EG, Kurzman ID: Amputation and carboplatin for treatment of dogs with osteosarcoma: 48 cases (1991 to 1993). J Vet Intern Med 10:76-81, 1996[ISI][Medline]
  5. Bodey B, Groger AM, Bodey BJ: Immunohistochemical detection of p53 protein overexpression in primary human osteosarcomas. Anticancer Res 17:493-498, 1999
  6. Brennan JA, Mao L, Hruban RH: Molecular assessment of histopathological staging in squamous-cell carcinoma of the head and neck. N Engl J Med 332:429-435, 1995[Abstract/Free Full Text]
  7. Brodey RS, McGrath JT, Reynolds H: A clinical and radiological study of canine bone neoplasms: part I. J Am Vet Med Assoc 134:53-71, 1959[Medline]
  8. Brodey RS, Riser WH: Canine osteosarcoma. A clinicopathologic study of 194 cases. Clin Orthop 62:54-64, 1969[Medline]
  9. Brodey RS, Sauer RM, Medway W: Canine bone neoplasms. J Am Vet Med Assoc 143:471-495, 1963[ISI][Medline]
  10. Carson DA, Lois A: Cancer progression and p53. Lancet 346:1009-1011, 1995[CrossRef][ISI][Medline]
  11. Cheng L, Leibovich BC, Bostwick DG: p53 alteration in regional lymph node metastases from prostate carcinoma: a marker of disease progression. Cancer 85:2455, 1999[CrossRef][ISI][Medline]
  12. Cheng L, Sebo TJ, Bostwick DG: p53 protein overexpression is associated with increased cell proliferation in patients with locally recurrent prostate carcinoma after radiation therapy. Cancer 85:1293, 1999[CrossRef][ISI][Medline]
  13. Chiarugi V, Magnelli L, Cinelli M: Oncogenes, p53, and tumor angiogenesis. J Cancer Res Clin Oncol 124:523-525, 1998[CrossRef][ISI][Medline]
  14. Chu LL, Rutteman GR, Kong JM: Genomic organization of the canine p53 gene and its mutational status in canine mammary neoplasia. Breast Cancer Res Treat 50:11-25, 1998[CrossRef][ISI][Medline]
  15. Cohen D, Reif JS, Brodey RS: Epidemiological analysis of the most prevalent sites and types of canine neoplasia observed in a veterinary hospital. Cancer Res 34:2859-2868, 1974[Abstract/Free Full Text]
  16. Coomber BL, Denton J, Sylvestre A: Blood vessel density in canine osteosarcoma. Can J Vet Res 62:199-204, 1998[ISI][Medline]
  17. Cotchin E: Spontaneous sarcomas of bone in dogs. Br Vet J 109:248-257, 1953
  18. de Krijger RR, van der Harst E, Bosman FT: Prognostic value of p53, bcl-2 and c-erbB-2 protein expression. J Pathol 188:51, 1999[CrossRef][ISI][Medline]
  19. Devilee P, Van Leeuwen IS, Voesten A: The canine p53 gene is subject to somatic mutations in thyroid carcinoma. Anticancer Res 14:2039-2046, 1994[ISI][Medline]
  20. Dong Y, Walsh MD, McGuckin MA: Reduced expression of retinoblastoma gene product (pRB) and high expression of p53 are associated with poor prognosis in ovarian cancer. Int J Cancer 74:407-415, 1997[CrossRef][ISI][Medline]
  21. Ehrhart N, Dernell WS, Hoffmann WE: Prognostic importance of alkaline phosphatase activity in serum from dogs with appendicular osteosarcoma: 75 cases (1990–1996). J Am Vet Med Assoc 213:1002-1006, 1998[ISI][Medline]
  22. Ellis PA, Lonning PE, Borresen-Dale A: Absence of p21 expression is associated with abnormal p53 in human breast carcinomas. Br J Cancer 76:480-485, 1997[ISI][Medline]
  23. Ellison DW, Steart PV, Bateman AC: Prognostic indicators in a range of astrocytic tumors: an immunohistochemical study with Ki-67 and p53 antibodies. J Neurol Neurosurg Psychiatry 59:413-419, 1995[Abstract]
  24. Esrig D, Elmajian D, Groshen S: Accumulation of nuclear p53 and progression in bladder cancer. N Engl J Med 331:1259-1264, 1994[Abstract/Free Full Text]
  25. Esrig D, Spruck CH, Nichols PW: p53 nuclear protein accumulation correlates with mutations in the p53 gene, tumor grade, and stage in bladder cancer. Am J Pathol 143:1389-1397, 1993[Abstract]
  26. Finlay CA, Hinds PW, Tan TH: Activating mutations for transformation by p53 produce a gene product that forms an hsc-70-p53 complex with an altered half life. Mol Cell Biol 8:531-539, 1988[Abstract/Free Full Text]
  27. Forrest LJ, Dodge RK, Page RL: Relationship between quantitative tumor scintigraphy and time to metastasis in dogs with osteosarcoma. J Nucl Med 33:1542-1547, 1992[Abstract/Free Full Text]
  28. Furihata M, Sonobe H, Ohtsuki Y: The aberrant p53 protein. Int J Oncol 6:1209-1226, 1995 [Review]
  29. Gamblin RM, Sagartz JE, Couto CG: Overexpression of p53 tumor suppressor protein in spontaneously arising neoplasms of dogs. Am J Vet Res 58:857-863, 1997[ISI][Medline]
  30. Gao J, Schwalb TA, Addeo JV: The role of apoptosis in the pathogenesis of canine keratoconjunctivitis sicca: the effect of topical cyclosporin a therapy. Cornea 17:654-663, 1998[CrossRef][ISI][Medline]
  31. Garzotto CK, Berg J, Hoffmann WE: Prognostic significance of serum alkaline phosphatase activity in canine appendicular osteosarcoma. J Vet Intern Med 14:587-592, 2000[CrossRef][ISI][Medline]
  32. Giatromanolaki A, Stathopoulos GP, Koukourakis MI: Combined role of tumor angiogensis, bcl-2, and p53 expression in the prognosis of patients with colorectal carcinoma. Cancer 86:1421, 1999[CrossRef][ISI][Medline]
  33. Ginn PE, Fox LE, Brower JC: Immunohistochemical detection of p53 tumor-suppressor protein is a poor indicator of prognosis for canine cutaneous mast cell tumors. Vet Pathol 37:33-39, 2000[Abstract/Free Full Text]
  34. Girlando S, Slomp P, Caffo O: p21 expression in colorectal carcinomas: a study on 103 cases with analysis of p53 gene mutation/expression and clinic-pathological correlations. Virchows Arch 435:559-565, 1999[CrossRef][ISI][Medline]
  35. Greenblatt M, Bennett W, Hollstein M: Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 54:4855-4878, 1994[Free Full Text]
  36. Guevara-Fujita M, Loechel R, Venta P: Chromosomal assignment of seven genes on canine chromosomes by fluorescence in situ hybridization. Mamm Genome 7:268-270, 1996[CrossRef][ISI][Medline]
  37. Hainaut P, Hernandez T, Robinson A: IARC Database of p53 gene mutations in human tumors and cell lines: updated compilation, revised formats and new visualisation tools. Nucl Acids Res 26:1998, 1998
  38. Hammer AS, Weeren FR, Weisbrode SE: Prognostic factors in dogs with osteosarcomas of the flat or irregular bones. J Am Anim Hosp Assoc 31:321-326, 1995[Abstract]
  39. Hernandez T, Hainaut P, Montesano R: The IARC Database of somatic p53 mutations in human tumors and cell lines. http://www.iarc.fr/p53/, International Agency for Research on Cancer 2000
  40. Hollstein M, Sidransky D, Vogelstein B: p53 mutations in human cancers. Science 253:49-53, 1991[Abstract/Free Full Text]
  41. Hurlimann J, Chaubert P, Benhattar J: p53 gene alterations and p53 protein accumulation in infiltrating ductal breast carcinomas: correlation between immunohistochemical and molecular biology techniques. Mod Pathol 7:423-428, 1994[ISI][Medline]
  42. Hwang J-K, Lin C-T: Co-localization of endogenous and exogenous p53 proteins in nasopharyngeal carcinoma cells. J Histochem Cytochem 45:991-1003, 1997[Abstract/Free Full Text]
  43. Igarashi N, Takahashi M, Fujimoto S: Predictive value of Ki-67, p53 protein, and DNA content in the diagnosis of gastric carcinoma. Cancer 86:1449, 1999[CrossRef][ISI][Medline]
  44. Iggo R, Gatter K, Bartek J: Increased expression of mutant forms of p53 oncogene in primary lung cancer. Lancet 335:675-679, 1990[CrossRef][ISI][Medline]
  45. Ikeda G, Isaji S, Kawarada Y: Prognostic significance of biologic factors in squamous cell carcinoma of the esophagus. Cancer 86:1396, 1999[CrossRef][ISI][Medline]
  46. Inoue M, Shiramizu K: Immunohistochemical detection of p53 and c-myc proteins in canine mammary tumors. J Comp Pathol 120:169-175, 1999[CrossRef][ISI][Medline]
  47. Inoue M, Wada N: Immunohistochemical detection of p53 and p21 proteins in canine testicular tumors. Vet Rec 146:370-372, 2000[Abstract/Free Full Text]
  48. Ioachim E, Goussia A, Agnantis NJ: Glycoprotein CD44 expression in colorectal neoplasms. An immuno-histochemical study including correlation with cathepsin D, extracellular matrix components, p53, Rb, bcl-2, c-erbB-2, EGFR and proliferation indices. Virchows Arch 434:45-50, 1999[CrossRef][ISI][Medline]
  49. Jaffe MH, Hosgood G, Taylor HW: Immunohistochemical and clinical evaluation of p53 in canine cutaneous mast cell tumors. Vet Pathol 37:40-46, 2000[Abstract/Free Full Text]
  50. Johnson SA, Couto CG, Weghorst CM: Mutation of the p53 tumor suppressor gene in spontaneously occurring osteosarcomas of the dog. Carcinogenesis 19:213-217, 1998[Abstract/Free Full Text]
  51. Johnston HM, Thompson H, Pirie HM: p53 immunohistochemistry in domestic animal tumors. Eur J Vet Pathol 2:135-140, 1996
  52. Kahlenberg MS, Stoler DL, Petrelli NJ: p53 tumor suppressor gene mutations predict decreased survival of patients with sporadic colorectal carcinoma. Cancer 88:1814, 2000[CrossRef][ISI][Medline]
  53. Kaklamanis L, Gatter KC, Mortensen N: p53 expression in colorectal adenomas. Am J Pathol 142:87-93, 1993[Abstract]
  54. Karamitopoulou E, Perentes E, Diamantis I: p53 protein expression in colorectal adenomas: an immunohistochemical study using an antigen retrieval system. Histopathology 27:517-523, 1995[ISI][Medline]
  55. Kaserer K, Schmaus J, Wrba F: Staining patterns of p53 immunohistochemistry and their biological significance in colorectal cancer. J Pathol 190:450, 2000[CrossRef][ISI][Medline]
  56. Kirpensteijn J: Clinical and pathogenetic studies in canine osteosarcoma. PhD Thesis, Universiteit Utrecht, Utrecht, The Netherlands 1999
  57. Lane DP: P53, guardian of the genome. Nature 358:15-16, 1992[CrossRef][Medline]
  58. Lane DP, Crawford LV: T antigen is bound to a host protein in SV 40-transformed cells. Nature 278:261-263, 1979[CrossRef][Medline]
  59. Levine RA, Fleischli MA: Inactivation of p53 and retinoblastoma family pathways in canine osteosarcoma cell lines. Vet Pathol 37:54-61, 2000[Abstract/Free Full Text]
  60. Liang S-B, Ohtsuki Y, Furihata M: Sun-exposure and aging-dependent p53 protein accumulation results in growth advantage for tumor cells in carcinogenesis of nonmelanocytic skin cancer. Virchows Arch 434:193-199, 1999[CrossRef][ISI][Medline]
  61. Ling GV, Morgan JP, Pool RR: Primary bone tumors in the dog: a combined clinical, radiographic, and histologic approach to early diagnosis. J Am Vet Med Assoc 165:55-67, 1974[ISI][Medline]
  62. Lipponen P, Eskelinen M, Hietala K: Expression of proliferating cell nuclear antigen (PC10), p53 protein and c-erbb-2 in renal adenocarcinoma. Int J Cancer 57:275-280, 1994[ISI][Medline]
  63. Liu S-K, Dorfman H, Hurvitz A: Primary and secondary bone tumors in the dog. J Small Anim Pract 18:313-326, 1977[ISI][Medline]
  64. Lonardo V, Takafumi U, Huvos AG: p53 and MDM2 alterations in osteosarcomas: correlation with clinicopathologic features and proliferative rate. Cancer 79:1541-1547, 1997[CrossRef][ISI][Medline]
  65. Luo J, Su F, Chen D: Deacetylation of p53 modulates its effect on cell growth and apoptosis. Nature 408:377-381, 2000[CrossRef][Medline]
  66. Ma L, Ronai A, Riede U: Clinical implication of screening p53 gene mutations in head and neck squamous cell carcinomas. J Cancer Res Clin Oncol 124:389-396, 1998[CrossRef][ISI][Medline]
  67. Malof FR: P53 presentation and other prognostic indicators in oral squamous cell carcinoma. MDSc Thesis, University of Sydney, Sydney, Australia 1996
  68. Mansoor A, McKee PH, Simpson JA: Prognostic significance of Ki-67 and p53 immunoreactivity in cutaneous squamous cell carcinomas. Am J Dermatopathol 18:351-357, 1996[CrossRef][ISI][Medline]
  69. Mayr B, Dressler A, Reifinger M: Cytogenetic alterations in eight mammary tumors and tumor-suppressor gene p53 mutation in one mammary tumor from dogs. Am J Vet Res 59:69-78, 1998[ISI][Medline]
  70. Mayr B, Loupal G, Schleger W: Genetic alterations in four cases of canine mammary tumors: two trisomies, translocations and one tumor suppressor gene p53 mutation. Arch Zootec 45:315-321, 1996
  71. Mayr B, Reifinger M, Alton K: Novel canine tumor suppressor gene p53 mutations in cases of skin and mammary neoplasms. Vet Res Commun 23:285-291, 1999[CrossRef][ISI][Medline]
  72. Mayr B, Reifinger M, Brem G: Cytogenetic, ras, and p 53: studies in cases of canine neoplasms (hemangiopericytoma, mastocytoma, histiocytoma, chloroma). J Hered 90:124-128, 1999[Abstract/Free Full Text]
  73. Mayr B, Schaffner W, Botto I: Canine tumor suppressor gene p53—mutation in a case of adenoma of circumanal glands. Vet Res Commun 21:369-373, 1997[CrossRef][ISI][Medline]
  74. McEntee MF, Brenneman KA: Dysregulation of beta-catenin is common in canine sporadic colorectal tumors. Vet Pathol 36:228-236, 1999[Abstract]
  75. Mendoza S, Konishi T, Dernell WS: Status of the p53, Rb and MDM2 genes in canine osteosarcoma. Anticancer Res 18:4449-4453, 1998[ISI][Medline]
  76. Misdorp W, Hart AA: Some prognostic and epidemiologic factors in canine osteosarcoma. J Natl Cancer Inst 62:537-545, 1979
  77. Montenarh M: Biochemical properties of the growth suppressor/oncoprotein p53. Oncogene 7:1673-1680, 1992[ISI][Medline]
  78. Mukhopadhyay T, Maxwell SA, Roth JA: p53 Suppressor Gene, 1st ed. pp 1-129, Springer-Verlag, New York 1995
  79. Muto T, Wakui S, Takahashi H: p53 gene mutations occurring in spontaneous benign and malignant mammary tumors of the dog. Vet Pathol 37:248-253, 2000[Abstract/Free Full Text]
  80. Nakopoulou LL, Alexiadou A, Theodoropoulos GE: Prognostic significance of the co-expression of p53 and c-erbB-2 proteins in breast cancer. J Pathol 179:31-38, 1996[CrossRef][ISI][Medline]
  81. Nasir L, Argyle DJ: Mutational analysis of the tumor suppressor gene p53 in lymphosarcoma in two bull mastiffs. Vet Rec 145:23-24, 1999[Free Full Text]
  82. Nasir L, Argyle DJ, McFarlane ST: Nucleotide sequence of a highly conserved region of the canine p53 tumor suppressor gene. DNA Seq 8:83-86, 1997[ISI][Medline]
  83. Nieder C, Petersen S, Thames HD: The challenge of p53 as prognostic and predictive factor in gliomas. Cancer Treat Rev 26:67, 2000[CrossRef][ISI][Medline]
  84. Palmer N: X. Neoplastic and tumorous conditions of bones. In: Pathology of Domestic Animals, ed. Jubb KVF, Kennedy PC, Palmer N, 4th ed., pp 125-138, Academic Press, San Diego, CA 1993
  85. Park SH, Kim YI, Park YH: Clinicopathologic correlation of p53 protein overexpression in adenoma and carcinoma of the ampulla of Vater. World J Surg 24:54-59, 2000[CrossRef][ISI][Medline]
  86. Peller S: Clinical implications of p53: effect on prognosis, tumor progression and chemotherapy response. Semin Cancer Biol 8:379, 1998[CrossRef][ISI][Medline]
  87. Pietenpol JA, Vogelstein B: No room at the p53 inn. Nature 365:17-18, 1993[CrossRef][Medline]
  88. Pileri S, Roncador G, Ceccarelli C: Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 183:116-123, 1997[CrossRef][ISI][Medline]
  89. Powers BE, Withrow SJ, Thrall DE: Percent tumor necrosis as a predictor of treatment response in canine osteosarcoma. Cancer 67:126-134, 1991[CrossRef][ISI][Medline]
  90. Ru G, Terracini B, Glickman L: Host related risk factors for canine osteosarcoma. Vet J 156:31-39, 1998[CrossRef][ISI][Medline]
  91. Rungsipipat A, Tateyama S, Yamaguchi R: Immunohistochemical analysis of c-yes and c-erbB-2 oncogene products and p53 tumor suppressor protein in canine mammary tumors. J Vet Med Sci 61:27-32, 1999[CrossRef][ISI][Medline]
  92. Sagartz JE, Bodley WL, Gamblin RM: p53 tumor suppressor protein overexpression in osteogenic tumors of dogs. Vet Pathol 33:213-221, 1996[Abstract]
  93. Salnikow K, Costa M, Blagosklonny MV: Hyperinducibility of hypoxia-responsive genes without p53/p21 dependent checkpoint in aggressive prostate cancer. Cancer Res 60:5630, 2000[Abstract/Free Full Text]
  94. Schneider-Stock R, Onnasch D, Haeckel C: Prognostic significance of p53 gene mutations and p53 protein expression in synovial sarcomas. Virchows Arch 435:407-412, 1999[CrossRef][ISI][Medline]
  95. Setala L, Kosma V-M, Lipponen P: Clinical relevance of p53 index and expression of proliferating cell nuclear antigen and Ki-67 in gastric cancer. J Cancer Res Clin Oncol 124:497-502, 1998[CrossRef][ISI][Medline]
  96. Shahin MS, Hughes JH, Buller RE: The prognostic significance of p53 tumor suppressor gene alterations in ovarian carcinoma. Cancer 89:2006, 2000[CrossRef][ISI][Medline]
  97. Shiina H, Igawa M, Nagasaki M: Clinical significance of mdm2 and p53 expression in bladder cancer. Oncology 56:239, 1999[CrossRef][ISI][Medline]
  98. Slayter MV, Boosinger TR, Pool RR: Histological Classification of Bone and Joint Tumors of Domestic Animals, vol. 1. pp 1-50, Armed Forces Institute of Pathology, Washington, DC 1994
  99. Soussi T, Legros Y, Lubin R: Multifactorial analysis of p53 alteration in human cancer. Int J Cancer 57:1-9, 1994 [Review][ISI][Medline]
  100. Spodnick GJ, Berg J, Rand WM: Prognosis for dogs with appendicular osteosarcoma treated by amputation alone: 162 cases (1978–1988). J Am Vet Med Assoc 200:995-999, 1992[ISI][Medline]
  101. Straw RC: Bone and joint tumors. In: Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat, ed. Ettinger SJ, Feldman EC, 5th ed., pp 535-540, WB Saunders, Philadelphia, PA 2000
  102. Straw RC, Powers BE, Klausner J: Canine mandibular osteosarcoma: 51 cases (1980–1992). J Am Anim Hosp Assoc 32:257-262, 1996[Abstract]
  103. Straw RC, Withrow SJ, Powers BE: Management of canine appendicular osteosarcoma. Vet Clin North Am Small Anim Pract 20:1141-1161, 1990[ISI][Medline]
  104. Straw RC, Withrow SJ, Richter SL: Amputation and cisplatin for treatment of canine osteosarcoma. J Vet Intern Med 5:205-210, 1991[ISI][Medline]
  105. Sun Y, Nakamura K, Wendel E: Progression toward tumor cell phenotype is enhanced by overexpression of a mutant p53 tumor suppressor gene isolated from nasopharyngeal carcinoma. Proc Natl Acad Sci USA 90:2827-2831, 1993[Abstract/Free Full Text]
  106. Sylvestre A, Brash M, Atilola M: A case series of 25 dogs with chondrosarcoma. Vet Comp Orthop Traumatol 5:13-17, 1992
  107. Teifke JP, Löhr CV: Immunohistochemical detection of p53 overexpression in paraffin wax-embedded squamous cell carcinomas of cattle, horses, cats and dogs. J Comp Pathol 114:205-210, 1996[CrossRef][ISI][Medline]
  108. Teifke JP, Löhr CV, Shirasawa H: Detection of canine oral papillomavirus-DNA in canine oral squamous cell carcinomas and p53 overexpressing skin papillomas of the dog using the polymerase chain reaction and non-radioactive in situ hybridization. Vet Microbiol 60:119-130, 1998[CrossRef][ISI][Medline]
  109. Tjalma RA: Canine bone sarcoma: estimation of relative risk as a function of body size. J Natl Cancer Inst 36:1137-1150, 1966
  110. Van Leeuwen IS, Cornelisse CJ, Misdorp W: P53 gene mutations in osteosarcomas in the dog. Cancer Lett 111:173-178, 1997[CrossRef][ISI][Medline]
  111. Van Leeuwen IS, Hellmen E, Cornelisse CJ: p53 mutations in mammary tumor cell lines and corresponding tumor tissues in the dog. Anticancer Res 16:3737-3740, 1996[ISI][Medline]
  112. Veldhoen N, Milner J: Isolation of canine p53 cDNA and detailed characterization of the full length canine p53 protein. Oncogene 16:1077-1084, 1998[CrossRef][ISI][Medline]
  113. Veldhoen N, Stewart J, Brown R, Milner J: Mutations of the p53 gene in canine lymphoma and evidence for germ line p53 mutations in the dog. Oncogene 16:249-255, 1998[CrossRef][ISI][Medline]
  114. Veldhoen N, Watterson J, Brash M: Identification of tumor-associated and germ line p53 mutations in canine mammary cancer. Br J Cancer 81:409-415, 1999[CrossRef][ISI][Medline]
  115. Vesalainen SLB, Lipponen PK, Talja MT: Proliferating cell nuclear antigen and p53 expression as prognostic factors in T1–2m0 prostatic adenocarcinoma. Int J Cancer 58:303-308, 1994[ISI][Medline]
  116. Vitellozzi G, Mariotti F, Ricci G: Immunohistochemical expression of the p53 protein in testicular tumors in the dog. Eur J Vet Pathol 4:61-65, 1998
  117. Warnakulasuriya S, Jia C, Houghton J: p53 and P-glycoprotein expression are significant prognostic markers in advanced head and neck cancer treated with chemo/radiotherapy. J Pathol 191:33, 2000[CrossRef][ISI][Medline]
  118. Waters DJ, Cooley DM: Skeletal neoplasms. In: Cancer in Dogs and Cats. Medical and Surgical Management, ed. Morrison WB, 1st ed., pp 639-654, Williams and Wilkins, Baltimore, MD 1998
  119. Webb PM, Green A, Cummings MC: Relationship between number of ovulatory cycles and accumulation of mutant p53 in epithelial ovarian cancer. J Natl Cancer Inst 90:1729-1734, 1998[Abstract/Free Full Text]
  120. Wolf JC, Ginn PE, Homer B: Immunohistochemical detection of p53 tumor suppressor gene protein in canine epithelial colorectal tumors. Vet Pathol 34:394-404, 1997[Abstract]
  121. Xie X, Clausen OPF, Boysen M: The prognostic value of spontaneous apoptosis, Bax, Bcl-2 and p53 in oral squamous cell carcinoma of the tongue. Cancer 86:913, 1999[CrossRef][ISI][Medline]
Request reprints from Dr. P. Loukopoulos, Pathology Division, National Cancer Center Research Institute, 5–1–1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan). E-mail: ploukopo{at}gan2.res.ncc.go.jp.





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