Veterinary Surgery 37:454–460, 2008
TP53 Gene Mutations in Canine Osteosarcoma JOLLE KIRPENSTEIJN,
Prof., DVM, PhD, Diplomate ACVS and ECVS,
ECVIM-CA,
MARJA KIK, DVM, PhD, ERIK TESKE, and GERARD R. RUTTEMAN, DVM, PhD, Diplomate ECVIM-CA
DVM, PhD, Diplomate
Objective—To investigate mutations of the TP53 gene in canine osteosarcoma (OS). Study Design—Clinical historic cohort study. Animals—Client-owned dogs. Methods—OS (n ¼ 59) were screened for mutations of the complete TP53 gene using polymerase chain reaction and the mutation was analyzed by single-strand conformational polymorphism. Clinical outcome of dogs with TP53-mutated OS were compared with dogs with OS without a mutation after complete surgical excision of the primary tumor. Results—TP53 gene mutations were observed in 24 of 59 (40.7%) OS; 3 mutated OS had 2 mutations. The alterations consisted mainly of point mutations (74%). Dogs with mutated OS had a significantly shorter survival time (ST) after surgery than dogs with normal tumor TP53 gene expression (P ¼ .03). Other significant prognosticators for ST and disease-free interval included elevated serum alkaline phosphatase (Po.01) and tumor grade (P ¼ .01). Conclusion—TP53 genetic mutations are common in canine OS and may have a prognostic value. Clinical Relevance—Mutations of the TP53 gene may influence survival and should be considered when evaluating canine OS. r Copyright 2008 by The American College of Veterinary Surgeons
42%.13–16 Alterations, varying from point mutations, deletions, and gross rearrangements have been described in canine and feline OS.17–20 The incidence of TP53 mutations in canine OS varies from 24% to 47%. Most mutations are located in the highly conserved domains and were nearly identical to those reported in human OS.18,20 Both studies evaluated a relatively small group of dogs and only screened the highly conserved domains (exons 4–8). Mutations that may have occurred outside this area were not evaluated. Because of this, and also the limited number of cases, the true incidence of mutations may have been underrepresented.15 The effect of TP53 alterations on survival outcome, as described in humans,21,22 has not been reported for dogs. We evaluated the entire TP53 gene for alterations in a large group of canine OS. Survival data of dogs that had OS with TP53 mutations were compared with dogs that had tumors with a wild-type TP53 gene.
INTRODUCTION
O
STEOSARCOMA (OS), the most common bone tumor in dogs, is observed more frequently in males and in large breeds. OS is often located in the metaphyseal area of the appendicular skeleton and has an aggressive biological behavior with local invasion of normal tissues and rapid metastatic spread.1–4 The cause of OS is unknown, but it has been speculated that genetic factors such as the TP53 suppressor gene may play a role. The TP53 gene encodes for a protein that serves as a regulator in cellular proliferation, DNA repair, and programmed cell death (for a review, see Greenblatt et al5). Changes in the genetic makeup of the TP53 gene have been observed in many canine tumors.6–12 Alterations of the TP53 gene are common in human OS. The incidence of these alterations varies from 15% to
From the Department of Clinical Sciences of Companion Animals and Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. Address reprint requests to J. Kirpensteijn Prof., DVM, PhD, Diplomate ACVS and ECVS, Department of Clinical Sciences of Companion Animals and Pathobiology, Faculty of Veterinary Medicine, Utrecht University, PO Box 80.154, NL-3508 TD, Utrecht, The Netherlands. E-mail:
[email protected]. Submitted June 2007; Accepted April 2008 r Copyright 2008 by The American College of Veterinary Surgeons 0161-3499/08 doi:10.1111/j.1532-950X.2008.00407.x
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455
KIRPENSTEIJN ET AL Table 1. Classification Schedule for Determination of Tumor Grade Tumor Cells in Vessels 0 (no) 3 (yes)
Pleomorphism
Mitoses
Tumor Matrix
Tumor Cells
Necrosis
1 (o25%) 2 (25–50%) 3 (450%)
0 (0) 2 (1) 3 (41)
1 (450%) 2 (25–50%) 3 (o25%)
1 (o25%) 2 (25–50%) 3 (450%)
1 (o25%) 2 (25–50%) 3 (450%)
The tumor grade for each animal was calculated by adding the individual scores of each histologic variable (range, 4–18).
MATERIALS AND METHODS Animals Dogs eligible for this study consisted of 2 groups: dogs admitted with OS from 1994 to 1998 and dogs admitted from 1983 to 1997 that had archived frozen tumor material. Dogs that had been previously administered cytostatic or cytotoxic therapy were excluded from analysis.
Clinical and Survival Data
1–3 (1, o25% tumor cells; 2, 25–50% tumor cells; 3, 450% tumor cells). Tumor necrosis was evaluated on a scale of 1–3 (1, o25% necrosis; 2, 25–50% necrosis; 3, 450% necrosis). Number of multinucleated giant cells (MNGCs) was estimated on a scale of 0–3 (0, no MNGC; 1, minimal number of MNGC; 2, moderate amount of MNGC; 3, large number of MNGC). Whirl formation was estimated on a scale of 0–3 (0, no whirl formation; 1, minimal whirl formation; 2, moderate whirl formation; 3, maximal whirl formation). Tumor grade for each dog was calculated by adding the individual scores of each histologic variable (Table 1).
The clinical data evaluated were breed, sex, age, weight, affected bone, location of the tumor, serum alkaline phosphatase (AP) concentration, and treatment protocol. Dogs were staged minimally by 2-projection thoracic radiography. Survival data consisted of disease-free interval (DFI), survival time (ST), location of metastasis, whether the dog was alive at last evaluation, and whether metastasis or recurrence of the tumor had occurred. Dogs were examined for metastases or recurrence every 3 months for 1 year and after that twice yearly or when clinical signs occurred. DFI was defined as time from surgery to either recurrence or metastasis and ST was defined as time from surgery until death (recorded as caused by disease or unrelated).
DNA from frozen normal tissue or histologically examined paraffin-embedded normal tissues of dogs in which tumor tissue contained TP53 mutations was prepared and analyzed as described.6,9 The sequence of the 10 coding exons of TP53 were amplified by polymerase chain reaction and analyzed by single-strand conformational polymorphism (SSCP) as described by Chu et al.6 If TP53 mutations were observed, DNA was prepared from frozen normal leukocytes, muscle, or from histologically examined paraffin-embedded normal tissues of the same dog and analyzed as described previously.
Biopsy Specimens and Histologic Analysis
Statistical Analysis
Tumor tissue samples were obtained at surgery or necropsy from spontaneously occurring lesions with radiographic appearance of OS. Additionally, normal tissue or blood was obtained from all dogs. Tissue samples were snap-frozen in liquid nitrogen immediately after removal and stored at 701C. Adjacent parts of all specimens were fixed in a 10% formalin solution and processed for histologic evaluation. Using a standard classification system,23 all available tissue samples were reexamined by a certified pathologist (M.K.). Invasion of tumor in the vessels was evaluated throughout the biopsy specimen (0, no signs of tumor growth into the vessels; 3, tumor invasion into blood vessels). Tumor cell pleomorphism (TCP) was evaluated on a scale of 1–3 (1, o25% TCP; 2, 25–50% TCP; 3, 450% TCP). Number of mitoses was calculated by adding the number of cells in mitosis of 3 randomly selected high-power fields ( 400) and evaluated on a scale of 0–3 (0, no mitosis; 2, single mitosis; 3, 41 mitosis). The amount of tumor matrix was evaluated on a scale of 1–3 (1, 450% tumor matrix; 2, 25–50% tumor matrix; 3, o25% tumor matrix). Tumor cell density was evaluated on a scale of
Pertinent clinical and histologic data were compared between tumors with and without TP53 mutations. Frequency distributions were calculated and categorical data (sex, location of tumor, treatment protocol, and presence of metastasis in vessels) were compared using w2 analysis. A Fisher’s exact test was performed if 425% of cells had expected counts o5. Normally distributed, continuous, and interval categorical data (age, weight, serum AP levels, tumor grade, amount of pleomorphism, number of mitoses, number of tumor cells, amount of necrosis, whirl formation, and number of MNGC) were analyzed using ANOVA. Logarithmic transformation was performed on variables that were not normally distributed. A Kaplan–Meier product limit method was performed to estimate the median DFI and ST of dogs with OS (Fig 1). Dogs that had died from unrelated causes or were still alive at the time of follow-up were considered censored. The effect of TP53 mutation on DFI and ST was compared using the log rank test.24 Hazard ratios (HRs) for survival data were calculated with a univariate Cox proportional hazard analysis. Po.05 was considered significant.
DNA Preparation
456
TP53 GENE MUTATIONS IN CANINE OSTEOSARCOMA
Histologic Data
1.0
p53 mutation no yes censored censored
Survival percentage
0.8
0.6
0.4
0.2
OS was histologically confirmed in all dogs. Nine dogs with OS had concurrent analyses of their metastases (6 dogs, 1 metastasis; 1 dog, 2 metastases; 1 dog, 4 metastases; and 1 dog, 8 metastases). One dog with a primary OS developed metastatic mammary carcinoma after surgical resection of the primary tumor. All other metastases were histologically confirmed OS. Mean ( SEM) score of amount of matrix in TP53mutated OS (2.00 0.12) was significantly greater (P ¼ .02) than non-mutated OS (1.63 0.10). All other mean histologic scores did not significantly differ between the two groups.
TP53 Mutation Analysis
0.0 0
500
1000 1500 Survival time (days)
2000
2500
Fig 1. Kaplan–Meier survival curve of dogs with osteosarcoma containing TP53 mutations (solid line) and wild-type TP53 (dotted line). Censored cases are depicted by þ .
RESULTS Clinical Data Tissue was obtained from OS in 59 dogs (mean [ SD] weight, 37.4 10.7 kg; range, 8–70 kg; mean age, 7.3 2.8 years; range, 1.2–12.0 years). Age and weight were not significantly different among dogs with OS containing a mutated or a non-mutated TP53 gene. Twentynine dogs were male (7 castrated) and 30 dogs were female (13 spayed). OS originated from the appendicular skeleton in 47 dogs, the axial skeleton in 11 dogs, and was extraskeletal (eye) in 1 dog. Subtypes were 24 osteoblastic and 35 mixed, of which the osteoblastic–fibroblastic subtype (14) was most common. Twelve dogs were euthanatized directly before the histologic specimen was obtained, 7 had no further therapy, 3 had a marginal excision of the tumor, and 37 had complete tumoral excision (margins 43 cm). All dogs included in the survival analysis did not have metastases at the time of initial evaluation, whereas 4 dogs that were euthanatized had metastasis in the lungs at the time of euthanasia. Twenty dogs with complete tumoral excision were administered 2–4 doses of lobaplatin (35 mg/m2 intravenously). None of the dogs with marginal excisions were administered adjunctive therapy. There was no significant difference in treatment protocols between dogs with and without p53 mutations.
TP53 mutations were present in 24 OS. If we evaluate the OS with clinical and histologic data, 19 of 47 (40.4%) appendicular and 5 of 11 (45.5%) axial OS had mutations of the TP53 gene. The ocular extraskeletal OS did not have a TP53 mutation. No significant differences were observed between dogs that had OS with TP53 gene mutations and dogs that did not for the variables sex, tumor side (left versus right), tumor location, and tumor subtype. The cumulative tumor grade did not differ significantly between OS with a TP53 gene that was mutated and those without mutations. Serum AP concentration was significantly higher in dogs with OS with a TP53 gene mutation compared with dogs without (P ¼ .037). In the 24 OS tumors with TP53 alterations, 27 mutations were observed. Three OS had 2 TP53 mutations occurring concurrently whereas the remaining 21 each had only 1 TP53 mutation. Most TP53 gene abnormalities were located in exons 4 and 5 (Table 2). Two TP53 mutations were located on the non-coding part of the TP53 gene, and 1 mutation was observed in exon 9. Of the 27 alterations, we observed 20 (74%) point mutations and 7 (26%) deletions. Nineteen of the 20 point mutations resulted in an amino acid substitution. Mutations were present in 21 different locations. The canine codons that corresponded to human codon 120 were mutated in 5 tumors, codon 174 in 4 tumors, and codon 214 in 2 tumors. All other codons had a mutation in 1 individual tumor. Five of 9 metastatic OS contained an identical TP53 gene alteration in the metastases compared with the primary tumor. One primary tumor (OS50) did not have a TP53 mutation, whereas the corresponding metastasis did. None of OS metastases had a double mutation. All other metastases had normal TP53 genes. Evaluation of normal tissues of dogs that had tumors with TP53 alterations did not reveal any mutations.
457
KIRPENSTEIJN ET AL Table 2. Sequencing Data for OS with TP53 Mutations Tumor Cells (%)
Primary (P) Metastasis (M)
Normal Sequence
Mutated Sequence
Tumor ID
Codon
OS4
34 (34)
9
P
OS16
50 (51)
18
P
OS18 OS18M OS33 OS37 OS52 GRTO51
107 (120)
100 NS 60 33 25 NS
P M P P P P
GRTO50
52 (53)
50
P
OS13 OS13M
135 (148)
100 NS
P M
OS16 OS21 OS25 GRTO56
161 (174)
100 100 100 100
P P P P
. . . GTGCGGCGCTGCC . . . . . . ValArgArgCys . . .
OS51
163 (176)
67
P
OS24
17
P
OS39
119–120 (132–133) 155 (168)
100
P
GRTO-4
144 (158)
100
P
GRTO-56
67
P
OS12
143–144 (156–157) 202 (214)
100
P
OS21
202 (214)
33
P
GRTO-8
178 (190)
50
P
... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
GRTO-9.1 GRTO-9.2
Donor splice site
33 NS
P M
. . . TCCAGgtag . . . . . . SerArgSTOP . . .
. . . TCCAGatag . . . . . . SerArgSTOP . . .
GRTO-55
226 (238)
67
P
OS22
266 (278)
60
P
... ... ... ...
... ... ... ...
OS47 OS47-1 OS47-3 OS47-4 OS47-5 OS47-6 OS47-7 OS47-8 OS47-9 OS47.10
278–280 (290–292)
100 NS NS NS NS NS NS NS NS NS
P M M M M M M M M M
OS50M
261 (273)
63
M
GRTO-51
258 (270)
25
P
GRTO-6.1 GRTO-6.2 GRTO-6.3 GRTO-6.4 GRTO-6.5
308 (320)
NS 83 NS NS NS
P M M M M
. . . AAGAAGAAG . . . . . . LysLysLys . . .
AAGTAGAAG . . . . . . LysSTOP
100
P
. . . ccccagctt . . . . . . ProGlnLeu . . .
. . . cccctgctt . . . . . . ProLeuLeu . . .
OS10
Intron
... ... ... ...
TCTTCGGAG . . . SerSerGlu . . . GTCGTGAAC . . . ValValAsn . . .
... ... ... ... ... ...
TCTTAGGAG . . . SerSTOP GTCATGAAC . . . ValMetAsn . . . GCCAGGTCT . . . AlaArgSer . . .
... ... ... ...
AACCGCCTA . . . AsnArgLeu . . . GTCGGCTCC . . . ValGlySer . . .
. . . GCCAAGTCT . . . . . . AlaLysSer . . . ... ... ... ...
AACTGGCTA . . . AsnTrpLeu . . . GTCAGCTCC . . . ValSerSer . . . .
CGCTGCCCC . . . ArgCysPro . . . AACAAGTTGTTTTGC . . . AsnLysLeuPheCys . . . GAGTTCGTC . . . GluPheVal . . . GTCCGCGCT . . . ValArgAla . . . ACCTGTGTCCGCG . . . ThrCysValArg . . . CGACACAGTGTG . . . ArgHisSerVal . . . CGACACAGT . . . ArgHisSer . . . GCCCCTCCT . . . . AlaProPro . . .
ATGTGTAAC . . . MetCysAsn . . . TGTCCCGGG . . . CysProGly . . .
. . . TTCCACAAGAAGGGGG . . . . . . PheHisLysLysGly . . .
... ... ... ...
GTACGCGTT . . . ValArgVal . . . AGCTTTGAG . . . SerPheGlu . . .
Location of mutation on canine TP53 gene (corresponding number on human gene). NS, not sequenced.
. . . GTGCGCGCTGCC . . . . . . ValArgAlaAla . . .
... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...
CGCTTCCCC . . . ArgPhePro . . . AACTTTTGC . . . AsnPheCys . . . GAGTACGTC . . . GluTyrVal . . . GTCCACGCT . . . ValHisAla . . . ACCTGCGCG . . . ThrCysAla . . . CCACAAGTG . . . ArgGlnVal . . . CGACGCAGT . . . . ArgArgSer . . . GCCCTTCCT . . . AlaLeuPro . . .
ATGTATAAC . . . MetTyrAsn . . . TGTCGCGGG . . . CysArgGly . . .
. . . TTCCGGGGG . . . . . . PheArgGly . . .
... ... ... ...
GTACACGTT . . . ValGlnVal . . . AGCTCTGAG . . . SerSerGlu . . .
458
TP53 GENE MUTATIONS IN CANINE OSTEOSARCOMA
Survival Data ST was available for 47 dogs and DFI for 37 dogs. Of these 37 dogs, 34 OS were removed with wide margins and 3 with narrow (clean) margins (2 ulnectomies, 1 rib resection). The remaining 10 dogs had only a biopsy performed. Five dogs were alive at final evaluation, 9 dogs were censored. All dogs that were alive had no signs of metastases or recurrent disease. The number of dogs studied was too small to allow multivariate analysis. Dogs with tumors that contained mutations for TP53 had a significantly shorter median ST (81 days, 95% confidence interval [CI] 73–89 days) than dogs that did not (256 days, 95% CI 119–392 days; P ¼ .026). DFI was not significantly different between the 2 groups (P ¼ .08). An HR of 2.05 was calculated for OS with TP53 mutations (P ¼ .03; 95% CI, 1.07–3.90). If the data were stratified for chemotherapy, an HR ratio of 1.97 was observed (P ¼ .044; 95% CI, 1.02–3.81). Other significant variables included serum AP level (HR ¼ 1.003; P ¼ .008, 95% CI, 1.01–1.05) and tumor grade (HR ¼ 1.29; P ¼ .005; 95% CI, 1.080–1.540) for ST and serum AP level (HR ¼ 1.004; P ¼ .009; 95% CI, 1.001–1.007) and tumor grade (HR ¼ 1.288; P ¼ .012; 95% CI, 1.057–1.571) for DFI. DISCUSSION Progression from normal bone to OS in dogs and humans is most likely caused, in part, by molecular abnormalities such as alterations in tumor suppressor genes. Of these, the TP53 gene is the most commonly implicated gene that affects bone tumor development.13,17,18,20,25–27 Studies evaluating TP53 alterations in canine OS have used small sample sizes and have only screened the highly conserved domains of the TP53 gene (exons 4–8).17,18,20 In contrast, we evaluated 59 OS and the entire intron/ exon sequence of the TP53 gene. An expected small increase in the number of TP53 mutations was observed compared with the percentages reported by Mendoza et al17 and van Leeuwen et al20 because a larger section of the TP53 gene was examined. Our study confirmed that most mutations occur in exons 4–8. The high incidence of TP53 alterations observed by Johnson et al18 is most likely caused by their small sample size. Our data do not explain why most (72%) canine OS have an increased expression TP53 immunohistochemically.26,28 Explanations for the discrepancy between the high incidence of TP53 overexpression and the actual number of alterations in the gene could be the restricted analysis of specific areas of the TP53 gene in previous studies or an increased rate of non-neoplastic matrix turnover.28 Our study shows that other reasons, such as the limitations of the SSCP techniques, which may obscure up to 15% of mutations present, and stabilization of the TP53 protein
through fixation with unknown products, may be the cause for this difference.29 Also, selective detection of mutant TP53 protein, using newer monoclonal antibodies, may decrease the difference between our results and other reports.30 The incidence of TP53 mutations in our dogs is higher than that reported in humans. Although loss of heterozygosity affecting chromosome 17p has been described in 70–80% of human OS,31 only 20–30% of tumors contain small alterations of the TP53 gene.13,14,32–34 The high frequency of large alterations of the TP53 gene in human OS15,35,36 is not observed in canine OS.17 Large alterations of TP53 are more commonly observed in younger human patients, whereas small alterations, such as mutations, were more common in older patients.29,37 The higher incidence of small alterations in dogs may be explained by the fact that OS is primarily a disease of the older dog, in contrast to man, where it occurs most frequently during adolescence.38–40 If we compare our data with previously reported TP53 alterations in various canine tumors, we observe identical mutations in (human-corresponding) codons 158,17 174,6 176,8 190,20 218,20 and 238.20 A CGA–CAA mutation in codon 273 was reported previously in an OS in the cat19 and is comparable to the CGC–CAC mutation in 1 of our dogs (OS50 M). All other mutations identified in our study have not been reported in dogs or cats before. By comparison with human sarcomas, 3 codons had identical mutations: 176,41 214,42 and 273.15 Codons 158,14,43 209,44 and 27313,41,44 also contained mutations, but of a different kind. Metastatic lesions often contain the same TP53 mutation as the primary tumor,35 and this was not different in our study. Only 1 of the metastases had a TP53 mutation that could not be detected in the primary tumor. It is possible that the metastasis developed from a subset of cells of the primary tumor that was different from the cells analyzed. Differences between the primary and metastatic tumor were described previously in human but not in canine OS.32 Within the limits of our study, dogs harboring TP53 mutations in their OS seemed to have a decreased ST compared with dogs that did not have TP53 alterations. This is the first study in dogs that evaluates the prognostic significance of TP53 mutations in a larger group of patients with sufficient follow-up data. An earlier study also concluded that an increased IHC TP53 index was associated with more aggressive OS behavior.28 Conflicting conclusions can be drawn when you consider reported data in humans. TP53 gene alterations and TP53 protein overexpression have been associated with more progressive disease and a poor prognosis in OS and Ewing’s sarcoma.21,43,45 Other studies, however, have not found any prognostic value of TP53 alterations.15,29,32,46,47
KIRPENSTEIJN ET AL
A recent human metaanalysis of 499 OS showed that TP53 status was associated with decreased survival but not with histologic response to chemotherapy.48 Failure to detect a significant difference in DFI between the 2 groups may be caused by the small number of dogs and the lack of regular (e.g., monthly) radiographic evaluations for signs of early metastatic disease. Multivariate analysis in a larger patient group is warranted to further evaluate the effect of TP53 mutational changes on DFI and ST in canine OS. We conclude that TP53 mutations are common and may play a role in the tumorigenesis of canine OS. Screening the entire TP53 gene in a sufficient number of dogs is warranted if an accurate evaluation of the spectrum and the effect on prognosis of TP53 gene mutation is desired.
ACKNOWLEDGMENTS The authors thank Daniel J. Compton for critically reviewing the article and L.L. Chu and Dr. J. Pelletier of the Department of Biochemistry of McGill University, Montreal, Quebec, Canada for their help in processing the samples.
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