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2324
Altered p53 Is Associated with Aggressive Behavior
of Chondrosarcoma
A Long Term Follow-Up Study
Yumi Oshiro, M.D.1
Vijaya Chaturvedi, M.A.1
Dorothy Hayden, M.D.1
Tipu Nazeer, M.D.1
Mark Johnson, M.D.2
Dennis A. Johnston, Ph.D.3
Nelson G. Ordóñez, M.D.1
Alberto G. Ayala, M.D.1
Bogdan Czerniak, M.D.1
1
Department of Pathology, The University of Texas
M. D. Anderson Cancer Center, Houston, Texas.
2
Department of Surgical Oncology, The University
of Texas M. D. Anderson Cancer Center, Houston,
Texas.
3
Department of Biomathematics, The University of
Texas M. D. Anderson Cancer Center, Houston,
Texas.
BACKGROUND. p53 is a major tumor suppressor gene that has been implicated in
the biology of a variety of human neoplasms, including some that affect the
skeleton. Recent studies based on small numbers of cases have shown that overexpression or alteration of the p53 gene is frequently present in high grade,
clinically aggressive chondrosarcomas of bone. In this study, the authors addressed
the relation between overexpression and alteration of the p53 gene and the clinical
aggressiveness of chondrosarcoma in a large series of patients for whom long term
follow-up data were available.
METHODS. The authors analyzed the expression and/or alteration of the p53 gene
in 158 cases of chondrosarcoma of bone using immunohistochemistry, singlestrand conformation polymorphism, and direct sequencing. They then related the
findings to various clinicopathologic parameters and long term follow-up data.
RESULTS. The presence of overexpression and/or structural alterations of the p53
gene was documented in 38.1% of chondrosarcomas of bone. A statistically significant correlation was observed between overexpression or alteration of the p53
gene and both the histologic grade of the tumor and the presence of metastasis.
The probability of local recurrence free, metastasis free, and overall survival was
significantly higher for patients with no overexpression or alteration of p53 than for
patients with p53 overexpression or alteration.
CONCLUSIONS. Overexpression or alteration of the p53 gene is an important predictor of aggressive clinical behavior in chondrosarcoma of bone. Cancer 1998;83:
2324 –34. © 1998 American Cancer Society.
KEYWORDS: chondrosarcoma, p53, immunohistochemistry, sequencing, clinical behavior.
The authors thank Donna Sprabary for secretarial
assistance. This work was supported in part by
NIH grant CA66723-04 to Bogdan Czerniak.
Dr. Oshiro’s current address: Yumi Oshiro, M.D.,
2nd Dept. of Pathology, Kyushu University, 3-1-1
Maidashi, Higashi-ku, Fukuoka, 812 Japan.
Presented in part at the 1998 International Academy of Pathology meeting, Boston, Massachusetts, February 28 –March 6, 1998.
Address for reprints: Bogdan Czerniak, M.D., Department of Pathology, The University of Texas
M. D. Anderson Cancer Center, Box 085, 1515
Holcombe Blvd., Houston, TX 77030.
Received January 14, 1998; revision received April
27, 1998; accepted April 27, 1998.
© 1998 American Cancer Society
C
hondrosarcoma is the second most common malignant tumor of
bone, accounting for approximately 25% of all primary sarcomas
of bone.1 The incidence rate varies in different age groups, from 0.2 to
0.9 per 100,000 persons, and shows a gradual age-related increase that
peaks among patients older than 50 years.1 The overall 5-year survival
rate is about 70%, but the behavior of individual tumors varies widely,
ranging from locally aggressive, nonmetastasizing tumors to high
grade malignancies with a high propensity for metastasis. The behavior of these tumors can be predicted to some extent from their
histologic grade.2– 6 Nonmetastasizing, locally aggressive lesions are
typically of low histologic grade (Grade 1). Virtually all Grade 3 tumors
are highly aggressive and exhibit a high propensity for distant metastasis. Grade 2 tumors represent an intermediate group. Some Grade 2
chondrosarcomas behave like locally aggressive lesions, whereas others may metastasize. In addition to histologic grade, other factors,
Altered p53 in Chondrosarcoma of Bone/Oshiro et al.
such as DNA ploidy, tumor location, and tumor resectability, also affect prognosis.7–13
Little is known about the molecular alterations
involved in the pathogenesis of chondrosarcoma. On
the chromosomal level, chondrosarcomas exhibit
complex alterations with nonreciprocal translocations
and deletions involving numerous chromosomes.14,15
It appears that alterations of chromosomes 1p, 4, 5, 9,
and 20 are nonrandom and may play a role in the
biology of these neoplasms.14,15 The presence of amplifications and deletions of genetic material on multiple chromosomes was recently confirmed by comparative genomic hybridization studies.16 Alterations
of putative tumor suppressor genes (CDKN2 and
MTS-2) located on chromosome 9 were identified in
chondrosarcoma cell lines.17 Amplification of the cmyc protooncogene without any apparent correlation
to histologic grade was found in another small series
of chondrosarcoma cases.18 More recently, evidence
of frequent loss of heterozygosity on 17p and the presence of multiple chromosomal alterations were correlated with high histologic grade19 –23 and aggressive
clinical behavior21 in a few small series of chondrosarcoma of bone. Several recent studies suggest that,
similar to many other human tumors, a major tumor
suppressor gene located on 17p (the p53 gene) may
play an important role in the pathogenesis of chondrosarcoma.9,19,21–23
In this study, we report alterations of the p53 gene
in a series of 158 cases of chondrosarcoma of bone
and relate the findings to clinicopathologic parameters, such as histologic grade, tumor extension, tumor
location, and type of surgical procedure, as well as
long term follow-up data.
PATIENTS AND METHODS
Patients
One hundred fifty-eight patients with chondrosarcoma of bone were selected from the case files of the
Department of Pathology at the University of Texas
M. D. Anderson Cancer Center in Houston, Texas. All
cases were reviewed to verify the diagnosis and tumor
grade and to select a representative paraffin block for
the study. In 124 of these cases, radiologic and long
term follow-up data were available for analysis. In
nine patients, secondary chondrosarcoma developed
in association with multiple exostoses; in three patients, it was associated with Ollier’s disease. Three
patients with chondrosarcoma had solitary osteochondroma in sites distinct from that of the chondrosarcoma. Cases of dedifferentiated, mesenchymal, and
clear cell chondrosarcoma were excluded from the
study. All lesions were unifocal chondrosarcomas involving one bone. Tumors were histologically classi-
2325
fied using a three-tier grading system. Grade 2 tumors
with predominantly myxoid matrix were distinguished
from tumors with predominantly hyaline cartilaginous
matrix. None of the patients received chemotherapy
or radiation therapy before surgery.
Immunohistochemistry
Tissue sections were cut 4 ␮m thick, deparaffinized in
xylene, and rehydrated in alcohol. They were then
immersed in 3% hydrogen peroxide in methanol for 30
minutes to block endogenous peroxidase activity. After several washes in distilled water and phosphatebuffered saline, sections were incubated with normal
horse serum to minimize background staining. This
was followed by overnight incubation at 4°C with the
DO-1 mouse monoclonal antibody to p53 (Oncogene
Science, Uniondale, NY; 1:80 dilution). The immunoperoxidase staining was done using an ABC Elite kit
(Vector Laboratories, Burlingame, CA) with 0.05%
3,3´-diaminobenzidine in Tris-HCl buffer containing
0.01% hydrogen peroxide (pH 7.6). Sections were
counterstained with 0.01% toluidine blue and
mounted in permount. Tumors that had 25% or more
of positive tumor cell nuclei were defined as positive
for overexpression of the p53 gene product.
Single-Strand Conformation Polymorphism and
Sequencing
Single-strand conformation polymorphism (SSCP)
and sequencing of exons 5 through 9 of the p53 gene
were performed using template DNA extracted from
formalin fixed, paraffin embedded tumor tissues. Tissue sections (50 ␮m thick) were deparaffinized in xylene and alcohol, and genomic DNA was extracted by
using the proteinase K–phenol chloroform procedure.
SSCP analysis of the amplified gene fragments was
performed using the following primers flanking coding sequences of exons 5 through 9 of the p53 gene:
exon 5:5´-TTCCTCTTCCTGCAGTACTC-3´
5´-ACCCTGGGCAACCAGCCCTGT-3´
exon 6:5´-ACAGGGCTGGTTGCCCATGGGT-3´
5´-AGTTGCAAACCAGACCTAT-3´
exon 7:5´-GTGTTGTCTCCTAGGTTGGC-3´
5´-GTCAGAGGCAAGCAGAGGCT-3´
exon 8:5´-TATCCTGAGTAGTGGTAATC-3´
5´-AAGTGAATCTGAGGCATAAC-3´
exon 9:5´-GCAGTTATGCCTCAGATTCAC-3´
5´-AAGACTTAGTACCTGAAGGGT-3´
Amplifications of the gene fragments were performed by polymerase chain reaction (PCR) using 150
ng of genomic DNA in a 10-␮L volume containing 1
␮M of each primer, 200 ␮M of dNTPs, 2.5 ␮Ci of
[alpha-32P]dCTP, and 0.5 U of Ampli-taq DNA poly-
2326
CANCER December 1, 1998 / Volume 83 / Number 11
FIGURE 1.
Distributions of chondrosarcoma are shown by age and anatomic site.
merase (Perkin Elmer Cetus, Norwalk, CT). PCR was
carried out using 33 cycles (1 minute at 94°C, 1 minute
at 55°C, and 2 minutes at 72°C) on a programmable
thermal cycler (Perkin Elmer Cetus). Samples were
heat denatured at 95°C for 5 minutes and then loaded
on a 6% polyacrylamide gel. A nondenatured sample
from each case was loaded simultaneously. Electrophoresis was performed at room temperature with a
constant power of 3 W for 16 –20 hours. Autoradiography was carried out for 18 –24 hours at – 80°C.
PCR-amplified exons that showed band shifts on
SSCP analysis suggestive of an altered p53 gene were
sequenced. Direct sequencing of PCR-generated gene
fragments was performed using the Sequenase PCR
Product Sequencing kit (United States Biochemical
Corporation, Cleveland, OH), according to the protocol supplied by the manufacturer. PCR was carried out
as described above, but without the isotope. Briefly,
30 – 40 ng of the amplified PCR product was treated
with 1 U of exonuclease 1 and 2 U of shrimp alkaline
phosphatase at 37°C for 15 minutes to remove excess
primers and dNTPs. Following heat inactivation of the
added enzymes, the PCR product was annealed with
40 ng of the appropriate primer and subsequently
labeled with [alpha-35S] dATP. Samples were heated
to 80°C, loaded (3.5 ␮L) onto a 6% polyacrylamide
urea gel, and run at 1500 V, 55 W for 1.5– 4 hours.
Autoradiographs were developed after 5– 8 days of ex-
posure without intensifying screens at room temperature.
Data Analysis
The correlations between overexpression and alteration of the p53 gene and various clinicopathologic
parameters were analyzed using chi-square statistics.
The length of survival was tested by Kaplan–Meier
analysis using both the Gehan–Wilcoxon and Peto log
rank tests. To determine which of the clinical parameters would predict p53 status, a logistic multiparameter regression analysis was performed.
RESULTS
Clinicopathologic Data
The patients’ ages at diagnosis ranged from 10 to 91
years (median age, 50 years). The highest incidence of
chondrosarcoma occurred in the fifth and sixth decades of life. Seventy-eight of the patients were men,
71 were women, and 9 were of unknown gender. The
skeletal and age-related distributions of the tumors
are summarized in Figure 1. The most common site
was the pelvis, followed by the femur and humerus.
Eighty-three patients complained of pain related to
the lesion. Five patients had pathologic fracture at
presentation. Twenty-six tumors were treated by intralesional resection, 27 by marginal resection, 54 by
wide resection, and 5 by radical resection. The exact
Altered p53 in Chondrosarcoma of Bone/Oshiro et al.
2327
TABLE 1
Survival Rates and Development of Metastases in Chondrosarcoma
by Histologic Grade
Histologic grade
Survival/metastases
1
2
2-myxoid
3
5-yr survival (%)
10-yr survival (%)
Metastases (%)
91
91
5
66
66
30
68
61
42
60
60
50
evident that overall survival and metastasis free rates
for patients with Grade 2–3 chondrosarcoma were
lower than those for patients with Grade 1 chondrosarcoma. Survival rates for patients with Grade 1
chondrosarcoma after 5 and 10 years were similar and
were within the range of 90% (Table 1). The survival
rates for patients with higher grade chondrosarcomas
(Grades 2–3) ranged from 60% to 68% after 5 and 10
years. The survival rates for patients who had chondrosarcoma involving trunk bones were significantly
lower than the rates for those whose chondrosarcomas involved other anatomic sites (Fig. 2C).
Alterations of p53
FIGURE 2. Kaplan–Meier plots of overall survival (A) and metastasis free
survival (B) by histologic grade, as well as overall survival by tumor location (C),
are shown for 158 cases of chondrosarcoma of bone.
type of surgical procedure performed on 46 patients
was unclear. Tumor size ranged from 2 to 42 cm
(median size, 10 cm). Distribution by histologic grade
was Grade 1 in 80 cases, Grade 2 in 37 cases, Grade
2-myxoid in 32 cases, and Grade 3 in 9 cases.
The overall survival and metastasis free curves for
patients with chondrosarcoma are shown by histologic grade in Figures 2A and 2B, respectively. It is
Examples of p53 protein expression and alterations of
the p53 gene documented by immunohistochemistry,
SSCP analysis, and direct sequencing are shown in
Figure 3. Complete immunohistochemical and SSCP
data were available for 118 cases. The correlation between immunohistochemical and SSCP data is summarized in Table 2. The immunohistochemical analysis documented the presence of overexpression of the
p53 gene product in 27 cases of chondrosarcoma
(22.9%). In 17 of these cases the SSCP data suggested
the presence of the altered p53 gene. Overall, the SSCP
analysis suggested the presence of alterations of the
p53 gene in 29.6% of the cases. In 11 cases, direct
sequencing of the PCR-amplified fragments confirmed the presence of alterations in the p53 gene
(Table 3). However, for technical reasons, we were
able to confirm the presence of structural alterations
of the p53 gene by sequencing in only 31.4% of the
cases in which SSCP suggested the presence of an
altered p53 gene. In the remaining cases, the quality
and/or amount of DNA extracted from paraffin blocks
was not adequate for successful sequencing of the
PCR-amplified gene fragments. Theoretically, DNA
that is used for SSCP could be subjected to sequence
analysis. However, in paraffin embedded material,
sometimes it is possible to generate readable SSCP
radiograms, but the sequencing from the same sample
may be unsuccessful, mainly because of insufficient
2328
CANCER December 1, 1998 / Volume 83 / Number 11
FIGURE 3. Alterations of p53 in chondrosarcoma. (A) Immunohistochemical overexpression of p53 protein. Intense nuclear staining is present in the tumor cells
of Grade 1 (top), Grade 2-myxoid (middle), and Grade 3 (bottom) chondrosarcoma (original magnification ⫻200). (B) Single-strand conformation polymorphism
analysis of exons 7 (top) and 8 (bottom) of p53. Arrows indicate the abnormal bands. N: nondenatured sample; D: denatured sample. (C) Sequence analysis of exon
7 (same case as in top panel of B) demonstrates the G deletion at codon 249.
amplification and template contamination with degraded DNA fragments. Seventeen tumors (14.4%) exhibited immunohistochemical overexpression of p53
protein and band shifts suggestive of altered p53 gene
by SSCP. In 73 cases (61.9%), no immunohistochemical overexpression or altered p53 gene by SSCP could
be documented. Thus, the overall concordance rate
between immunohistochemical and SSCP data was
76.3%.
In 10 cases (8.5%), overexpression of the p53 protein with no evidence of an altered p53 gene by SSCP
was present. In 18 cases (15.2%), no immunohistochemical overexpression of the p53 protein was documented, whereas SSCP showed band shifts sugges-
Altered p53 in Chondrosarcoma of Bone/Oshiro et al.
TABLE 2
Corelation Between Immunohistochemical Evidence of p53 Protein
Expression and Alteration of the p53 Gene Documented by SSCP in
118 Cases of Chondrosarcoma
Alteration of p53
by SSCP
Immunohistochemistry
Negative
Positive
Total
Negative or positive in ⬍25% of cells
Positive in ⬎25% of cells
Total
73 (61.9)a
10 (8.5)
83 (70.4)
18 (15.2)
17 (14.4)
35 (29.6)
91 (77.1)
27 (22.9)
118
SSCP: single-strand conformation polymorphism.
a
No. of cases/% of cases within the group.
tive of an altered gene. In 11 of these cases, the
presence of structural alteration in the gene was confirmed by direct sequencing. Six of these cases showed
alterations caused by frame shifts and point mutations
resulting in amino acid substitutions that could be
documented in four cases. A nucleotide insertion
causing a stop signal sequence was found in one case.
In one remaining case, a nucleotide substitution was
located outside of the coding sequence.
In summary, the absence of overexpression of the
p53 protein and the presence of the normal p53 gene
was suggested in 61.9% of cases. In 14.4% of cases, the
presence of an overexpressed protein and altered gene
was documented. In 15.2% of cases, the presence of an
altered gene was documented by SSCP and sequencing with no evidence of immunohistochemical overexpression. The overexpression of an apparently normal p53 protein was present in 8.5% of chondrosarcomas. Finally, combined results from immunohistochemical analysis, SSCP, and direct sequencing suggested the overexpression and/or alteration of the p53
gene in 38.1% of chondrosarcomas.
The correlation between the overexpression or alteration of the p53 gene and clinicopathologic features
of chondrosarcoma of bone are summarized in Table
4. Strong, statistically significant correlations between
the overexpression or alteration of the p53 gene and
histologic grade (P ⫽ 0.003) and the presence of metastases (P ⫽ 0.034) were found. No statistically significant correlations were found between the overexpression or alteration of p53 and age, gender, presence or
absence of extension into soft tissue, tumor size, or the
presence or absence of local recurrence. However, the
percentage of tumors with overexpression or alteration of the p53 gene was consistently slightly higher
in association with the advanced age of patients, larger
tumor size, and presence of local recurrence. Grade
2-myxoid and Grade 3 chondrosarcomas showed
2329
TABLE 3
Sequencing Analysis of Cases with Alteration of the p53 Gene
Documented by SSCP
Case no.
Exon
Codon
Nucleotide change
Amino acid alteration
2
9
10
10
10
41
51
79
101
104
110
124
128
128
7
6
7
7
8
6
6
8
8
7
7
6
6
6
240
212
246
249
274
212
220
276
279
Intron
241
Intron
190
192
T3A
G ins.
G ins.
G del.
T3C
G ins.
CG ins.
T ins.
T ins.
a ins.
C3G
G3A
T3G
C3G
Ser 3 Arg
Frame shift
Frame shift
Frame shift
Silent
Frame shift
Frame shift
Frame shift
Nonsense
No change
Ser 3 Cys
Splice site mutation
Silent
Gln 3 Glu
SSCP: single-strand conformation polymorphism; del: deletion; ins: insertion.
overexpression or alteration of p53 in 60% and 100% of
the cases, respectively.
The survival rates of patients with overexpression
or alteration of the p53 gene were significantly lower
than the survival rates of patients who had no evidence of p53 overexpression or alteration (Fig. 4A).
Similarly, the probability of longer local recurrence
free and metastasis free survival was significantly
greater for patients who had no evidence of p53 overexpression or alteration, compared with the same
probability for patients whose tumors exhibited overexpression or alteration of the p53 gene (Fig. 4B and
4C). In patients with evidence of p53 overexpression
or alteration, virtually all local recurrences and distant
metastases occurred within 3 years. On the other
hand, patients whose tumors showed no evidence of
p53 overexpression or alteration had gradually declining relapse free rates, and their tumors continued to
recur as long as 10 years after the primary diagnosis.
Because the outcome of patients might be dependent
on the type of surgical procedure, i.e., incomplete
resection, positive margins, contamination by ruptured tumor, etc., all cases were divided into two
groups: 1) cases treated with intralesional excision,
marginal excision, or any other type of incomplete
excision; and 2) cases treated with wide local or radical
complete excision.
There was a highly significant correlation between
overall survival, local recurrence free and metastasis
free survival, and p53 status for patients with chondrosarcoma who underwent complete excision. Completely excised lesions that exhibited overexpression
or alteration of the p53 gene were associated with
significantly lower overall survival, local recurrence
2330
CANCER December 1, 1998 / Volume 83 / Number 11
TABLE 4
Overexpression/Alteration of p53 Gene by Clinicopathologic Parameters in 118 Cases of Chondrosarcoma
Clinicopathologic features
No. of cases examined
Positive cases (%)
All patients
Age
⬍50 yrs
ⱖ50 yrs
Gender
Male
Female
Tumor extension
Confined to bone
Invasion of soft tissue
Tumor size
⬍10 cm
ⱖ10 cm
Local recurrence
No
Yes
Distant metastasis
No
Yes
Histologic grade
1
2
2-myxoid
3
118
45 (38.1)
60
38
20 (33.3)
17 (50.0)
0.257
50
53
16 (32.0)
22 (41.5)
0.318
27
38
9 (33.3)
18 (47.4)
0.258
27
29
10 (37.0)
15 (48.4)
0.269
49
33
18 (36.7)
14 (42.4)
0.605
65
14
22 (33.8)
9 (64.3)
0.034
60
28
25
5
14 (23.3)
11 (39.3)
15 (60.0)
5 (100.0)
0.003
free survival, and metastasis free survival rates (Fig. 5).
Conversely, for patients who underwent incomplete
excision, there was no significant correlation between
overexpression or alteration of the p53 gene and overall survival, local recurrence free survival, or metastasis free survival rates (data not shown). An analysis of
the correlation between the p53 status restricted to the
combined group of Grade 1 and 2 chondrosarcomas
and treatment by complete excision showed that overexpression or alteration of p53 had a similar strong
association with a high propensity for local recurrence
and distant metastases as well as an association with
lower overall survival rates (Fig. 6). A similar separate
analysis of p53 status in Grade 1, 2, and 3 chondrosarcomas was not possible because of the insufficient
number of tumors treated with complete excision or
the inappropriate stratification of cases with different
p53 status within these groups. A small proportion of
Grade 1 tumors showed evidence of overexpression or
alteration of p53, and all Grade 3 tumors exhibited
evidence of p53 alteration or overexpression. The
number of Grade 2 lesions treated with complete excision was too small for the analysis to be performed.
It is noteworthy that the analysis of local recurrence
free time according to the histologic grade of completely excised tumors disclosed an increased propensity for local recurrence of Grade 2 and 3 tumors as
compared with Grade 1 lesions, but the differences
Univariative analysis (P value)
were not statistically significant (Fig. 7). Therefore, the
propensity for local recurrence is unlikely to be
strongly associated with histologic grade if the tumor
is completely excised. This observation, together with
a strong correlation of local recurrence rate, propensity for metastasis, and overall status of survival to the
p53 altered status of low-to-intermediate-grade tumors, suggests that this correlation might be independent of histologic grade.
To determine which of the clinical parameters
would predict p53 status, a logistic multiparameter
regression analysis was performed that included individual clinical parameters. As expected from the univariate analysis, histologic grade was the single best
univariate parameter from the list (log likelihood [LL]
⫽ ⫺69.34; P ⬍0.001). An unexpected finding was that
histologic grade and local advancement of the tumor
(tumor with extension into soft tissue) was the best
pair of clinical parameters (LL ⫽ ⫺37.23; P ⬍0.001), as
opposed to tumor grade and distant metastasis (LL
⫽ ⫺43.28; P ⬍0.001). The three best clinical parameters associated with the overexpression or alteration of
p53 were high histologic grade (Grades 2 and 3), the
presence of distant metastasis, and tumor size ⬎10 cm
(LL ⫽ ⫺29.04; P ⬍0.001). The four best were high
histologic grade, the presence of distant metastases,
large tumor size (⬎10 cm), and extension into soft
tissue (LL ⫽ ⫺26.66; partial P ⫽ 0.017). The combina-
Altered p53 in Chondrosarcoma of Bone/Oshiro et al.
FIGURE 4. Kaplan–Meier plots of local recurrence free (A), metastasis free
(B), and overall survival (C) are shown in relation to p53 status. Analysis is
based on follow-up data of 118 cases of chondrosarcoma of bone, regardless
of complete or incomplete excision of the primary tumor. p53⫹: presence of
overexpression and/or alteration of the p53 gene; p53-: absence of overexpression and alteration of the p53 gene.
tion of all five parameters (the four just cited and
tumor site) was not significantly better than the best
four (LL ⫽ ⫺25.63; partial P ⫽ 0.30).
DISCUSSION
In this study, we demonstrate the presence of overexpression and/or structural alterations of the p53 gene
2331
FIGURE 5. Kaplan–Meier plots of local recurrence free survival (A), metastasis free survival (B), and overall survival (C) are shown in relation to the p53
status of 59 patients treated by complete local or radical excision. The analysis
of patients treated by intralesional, marginal, or in any other way incomplete
excision showed no statistical significance of local recurrence free and metastasis free survival or of overall survival in relation to p53 status (data not
shown). p53⫹: presence of overexpression and/or alteration of the p53 gene;
p53-: absence of overexpression and alteration of the p53 gene.
in 38.1% of chondrosarcomas of bone. The presence of
an overexpressed, structurally altered gene product
was documented in 14.4% of the cases. An apparently
normal overexpressed p53 protein was found in 8.5%
of the cases, and an altered gene without overexpres-
2332
CANCER December 1, 1998 / Volume 83 / Number 11
FIGURE 7. A Kaplan–Meier plot shows local recurrence free survival by
histologic grade for 59 patients who underwent complete local or radical
excision. Although patients with Grade 2 or 3 tumors showed decreased rates
of freedom from local recurrence as compared with patients who had Grade 1
tumors, the difference was not statistically significant.
FIGURE 6. Kaplan–Meier plots of local recurrence free survival (A), metastasis free survival (B), and overall survival (C) are shown according to the p53
status of 55 patients with Grade 1 or 2 chondrosarcoma who underwent
complete local or radical excision. Analysis within other individual histologic
grades of chondrosarcoma was not possible because of an insufficient number
of cases and stratification of p53- versus p53⫹ groups. p53⫹: presence of
overexpression and/or alteration of the p53 gene; p53-: absence of overexpression and alteration of the p53 gene.
sion of its protein was present in 15.2% of the cases. It
should be mentioned, however, that the tests and
laboratory procedures used in this study were not
capable of identifying all conceivable alterations of the
p53 gene. Hence, for cases with immunohistochemical evidence of p53 overexpression but no evidence of
p53 alterations by SSCP or direct sequencing, the presence of a structurally altered gene could not be completely ruled out.24 In such cases, the alterations might
have been present outside of the screened sequences.
Nonmutational stabilization of the wild p53 protein
should also be considered in these cases. Moreover,
some genes upstream or downstream from the p53
gene that are involved in cell-cycle regulation may be
altered and indirectly cause p53 overexpression without evidence of structural gene alterations. The absence of overexpressed p53 protein in cases with a
documented altered p53 gene can be explained by the
presence of nonsense mutations, insertions, or deletions that lead to premature cessation of protein synthesis and/or generate a gene product that has a different antigenicity and cannot be identified by the
antibody used in this study. In fact, our sequencing
data invariably showed deletions and/or insertions
that resulted in frame shifts in some cases without
overexpression of the p53 protein. Previous reports
based on limited numbers of cases have shown that
the dominant type of point mutation within the p53
gene in chondrosarcoma results in amino acid substitution.19,23,25,26 On the other hand, a comparative
analysis of p53 alterations has demonstrated that sarcomas in general have a higher rate of nonsense mutations that result in frame shift or larger deletions
than epithelial malignancies have.26 Our results are in
keeping with these observations and indicate that the
p53 gene in chondrosarcoma of bone predominantly
shows nonsense substitutions or insertions that result
in frame shifts, as compared with mutations that
cause amino acid change.
Altered p53 in Chondrosarcoma of Bone/Oshiro et al.
In our study, the concordance rate between gene
alteration and protein overexpression was 76.3%. The
reported concordance rates between immunohistochemical evidence of p53 overexpression and the
presence of an altered p53 gene varied in different
studies, from 55% up to 100%.27–32
The analysis of our data showed a strong correlation between overexpression or alteration of p53 and
high histologic grade as well as the propensity for
distant metastasis in chondrosarcoma of bone. In univariate analysis, no correlation was found between
overexpression or alteration of p53 and age, gender,
soft tissue extension, tumor size and location, or the
presence of local recurrence. Analysis of our data has
shown that overexpression or alteration of the p53
gene in chondrosarcoma of bone is strongly associated with a higher propensity for local recurrence and
metastasis and overall shorter survival times, meaning
that it signifies a clinically aggressive variant of the
tumor. The association of the altered p53 status with a
high propensity for local recurrence and distant metastasis as well as overall poor survival in a group of
low and intermediate grade chondrosarcoma (Grades
1 and 2) suggests that the p53 status may represent a
predictor of aggressive behavior of chondrosarcoma
that is independent of grade. However, more detailed
analysis of p53 status independent of histologic grade,
i.e., with each histologic grade individually, was not
possible in the cohort enrolled in this study.
Multivariate analysis by logistic regression has
shown that histologic grade is the best parameter to
predict p53 status, because high grade (Grade 3) chondrosarcomas of bone often exhibit overexpression
and/or alteration of the p53 gene. The three best univariate parameters associated with overexpression or
alteration of the p53 gene were high histologic grade,
presence of distant metastasis, and tumor size ⬎10
cm. In multivariate analysis, the association of p53
overexpression or alteration with larger tumor size
(⬎10 cm) and higher stage (soft tissue extension) suggests that the alterations of this gene may represent a
later phenomenon in the development of chondrosarcoma of bone.
The published data on chondrosarcoma of bone
are based on small series of cases without long term
follow-up, and they suggest that p53 is predominantly
altered in high grade lesions.9,19 –23,33 The correlation
between immunohistochemical evidence of p53 overexpression and a lower 5-year survival rate was documented in a series of 29 cases of chondrosarcoma of
bone.21 All dedifferentiated chondrosarcomas have
been reported to exhibit immunohistochemical evidence of overexpression of the p53 protein.9,22 The
presence of a mutated gene, as shown by SSCP and
2333
direct sequencing, was documented in several cases of
Grade 3 and dedifferentiated chondrosarcomas based
on the analysis of 2 small series consisting of 14 and 22
cases of chondrosarcomas of bone, respectively.19,23
Loss of heterozygosity on chromosome 17p in the
region corresponding to the p53 locus was shown to
be present in approximately 25% of chondrosarcomas
of bone.23 This percentage is in the same range as the
evidence of overexpression or alterations of p53 provided by our study and suggests that in chondrosarcoma of bone altered p53 frequently acts in a homozygotic state. In several reports, all immunohistochemically analyzed enchondromas were shown to be
negative for p53 protein.9,20,21,33 Therefore, immunohistochemical evidence of p53 overexpression and/or
the identification of an altered p53 gene in neoplastic
cartilage lesions of bone signifies aggressiveness of the
lesion.
In summary, this study documents the frequent
presence of p53 overexpression or alteration in chondrosarcoma of bone and establishes the usefulness of
the gene as an important prognostic factor in the
workup of patients with this cancer.
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