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2067
Expression of p21 (waf1/cip1/sdi1), but Not p53
Protein, Is a Factor in the Survival of Patients with
Advanced Gastric Carcinoma
Yoshihito Gomyo, M.D.1,2
Mitsuyuki Ikeda, M.D.1
Mitsuhiko Osaki, Ph.D.1
Shigeru Tatebe, M.D.1
Shunichi Tsujitani, M.D.2
Masahide Ikeguchi, M.D.2
Nobuaki Kaibara, M.D.2
Hisao Ito, M.D.1
BACKGROUND. The authors examined whether expression of p21 (waf1/cip1/sdi1)
and p53 protein was related to survival rates in patients with advanced gastric
carcinoma.
METHODS. The expression of p21 and p53 protein was analyzed by immunohisto-
1
First Department of Pathology, Faculty of Medicine, Tottori University, Yonago/Tottori, Japan.
2
First Department of Surgery, Faculty of Medicine, Tottori University, Yonago/Tottori, Japan.
chemistry in 93 patients with advanced gastric carcinoma with serosal invasion and
lymph node metastasis. All patients underwent curative surgery. The probability of
survival was calculated by the Kaplan-Meier method and compared by the generalized Wilcoxon test.
RESULTS. Various levels of p21 and p53 immunoreactivities in carcinoma cells
were detected in 30 (32%) and 60 (65%), respectively, of 93 samples. There was no
correlation between p21 and p53 expression. The 5-year survival rate of patients
with p21 expression was 69.4%, which was significantly better than that of patients
without p21 expression (38.1%; P õ 0.05). However, p53 protein expression did
not correlate with patient survival.
CONCLUSIONS. Expression of p21 protein may be a better prognostic factor than
p53 protein expression in patients with advanced gastric carcinoma. Cancer
1997;79:2067–72. q 1997 American Cancer Society.
KEYWORDS: p21, p53, gastric carcinoma, immunohistochemistry, prognosis.
T
This work was supported by a Grants-in-Aid for
Cancer Research from the Ministry of Education, Science, Sports, and Culture of Japan.
The authors would like to thank Mr. N. Itaki for
his skillful technical assistance.
Address for reprints: Hisao Ito, M.D., First Department of Pathology, Faculty of Medicine, Tottori University, Nishi-machi 86, Yonago/Tottori,
683 Japan.
Received May 31, 1996; revisions received October 24, 1996, and December 31, 1996; accepted December 31, 1996.
he p53 tumor suppressor gene localized to the short arm of human
chromosome 17 (17p13.1)1 is mutated in a variety of human neoplasms. In nonneoplastic cells, p53 modulates cell proliferation and
differentiation by regulating the transcription of several gene products. Wild type p53 induces p21 (waf1/cip1/sdi1) expression, which
acts as a regulator of the cell cycle at the G1 check point.2,3 Moreover,
p53 plays a crucial role in repairing damaged DNA through the induction of GADD45 (growth arrest and DNA damage [inducible]).4 p53
induces the bax gene, which is followed by apoptosis, in contrast to
Bcl-2.5 Therefore, the action of wild type p53 is considered to be the
‘‘guardian of the genome.’’6
The level of wild type p53 protein is extremely low and because
of its short half-life, it is undetectable by standard immunohistochemical staining in normal cells and tissues. Conversely, the mutated
p53 protein accumulates in the nucleus through binding to other
oncogenic proteins or by prolonging its half-life.7,8 p53 protein overexpression detected by immunohisitochemistry is a prognostic factor
in many human neoplasms, such as gastric,9 – 13 colon,10,14 esophageal,15 and lung16 carcinomas.
p21 is one of the inhibitors of the phosphorylation of the cyclin-
q 1997 American Cancer Society
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CANCER June 1, 1997 / Volume 79 / Number 11
FIGURE 1. Nuclear immunostaining for (A) p53 and (B) p21 in a moderately differentiated gastric carcinoma (original magnification 1200).
cdk complex.2 In fact, p21 inhibits the growth of human tumor cell lines and normal diploid fibroblasts
when introduced via transfection,2 and induces arrest
of the G1 phase. p21 is induced not only by p53 but
also by other growth factors.17 Moreover, senescent
cells express p21.18 Thus, the function of p21 as a tumor growth suppressor may be retained after p53 mutation19 and p21 expression may reflect the long term
survival rate.
In this study, the authors analyzed the expression
of p21 and p53 in gastric carcinomas and their relationship to patient survival rates.
MATERIALS AND METHODS
Clinical Data
A total of 93 patients (56 males and 37 females; mean
age, 63.9 years [range, 32 – 91 years]) with primary advanced gastric adenocarcinomas underwent curative
surgery between 1983 and 1992 in the First Department of Surgery, Faculty of Medicine, Tottori University. None of the patients had received preoperative
adjuvant therapy (radiotherapy or chemotherapy). All
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93 tumors had invaded the gastric serosa and metastasized to regional lymph nodes histologically. All specimens were fixed in 10% formalin and embedded in
paraffin wax. Two or three representative blocks were
selected (mainly including the greatest dimension of
the tumors) and serial sections were examined by immunohistochemistry.
Immunohistochemistry
Immunohistochemistry was performed using the avidin-biotin-peroxidase complex described by Hsu et
al.,20 using primarily monoclonal antibodies raised
against p53 (BP53-12, diluted 1:100; Novocastra Laboratories Ltd., Newcastle, United Kingdom) and p21
(p21, diluted 1:1000; Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA). Briefly, dewaxed 4-mm sections were
heated in a microwave oven for 12 minutes to retrieve
antigens.21 Endogenous peroxidase was blocked using
3% hydrogen peroxide in methanol. Tissue sections
were preblocked for 20 minutes with 10% rabbit serum, after which the primary antibody described earlier was added for 60 minutes. After washing, the sec-
W: Cancer
Expression of p21 in Gastric Carcinomas/Gomyo et al.
2069
TABLE 1
Expression of p21 Protein and Histopathologic Characteristics of Patients with Advanced Gastric Carcinoma
Expression of p21
Variable
Gender
Female
Male
Age (yrs)
õ 59
¢ 59
Tumor size (cm)
õ7
¢7
Location
Cardia
Midbody
Antrum
Histology
Intestinal
Diffuse
Negative
Weak
Strong
Chi-square
P value
27
36
10
15
0
5
3.55
0.17
24
39
3
22
3
2
7.44
0.02
32
31
13
12
4
1
0.55
0.76
11
22
30
6
10
9
1
3
1
2.15
0.71
21
42
9
16
4
1
3.82
0.15
TABLE 2
Expression of p53 Protein and Histopathologic Characteristics of Patients with Advanced Gastric Carcinoma
Expression of p53
Variable
Gender
Female
Male
Age (yrs)
õ 59
¢ 59
Tumor size (cm)
õ7
¢7
Location
Cardia
Midbody
Antrum
Histology
Intestinal
Diffuse
Negative
Weak
Strong
Chi-square
P value
14
19
9
12
14
25
0.42
0.81
10
23
7
14
13
26
0.08
0.96
12
21
7
14
16
23
0.38
0.83
8
10
15
5
7
9
5
18
16
2.89
0.58
13
20
7
14
14
25
0.22
0.90
TABLE 3
Correlation between p21 and p53 Protein Expression in Gastric Carcinoma Patients
Expression of p21
Expression of p53
Negative
Weak
Strong
Chi-square
P value
Negative
Weak
Strong
21
18
24
9
3
13
3
0
2
5.1
0.27
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CANCER June 1, 1997 / Volume 79 / Number 11
FIGURE 2. Kaplan–Meier plot for cumulative survival in 93 patients with
advanced gastric carcinoma, comparing p53 negative and p53 positive
tumors.
cinoma cells were detected in 1 – 20%); and (//):
strong expression (positive carcinoma cells detected
in ú20%). This classification was based on reported
findings that demonstrated the presence of p53 positive cells, even in nonneoplastic mucosa of the gallbladder22 and colon,23 in a scattered manner. The authors have also detected a few scattered p53 positive
cells in some cases of gastric intestinal metaplasia
glands.24 However, diffuse or numerous p53 positive
cells have not been reported in nonneoplastic mucosa.
Moreover, some authors have indicated that a diffuse
pattern of p53 positive cells could be categorized as
overexpression of p53 protein and a marker for carcinoma.23,25 The cases with ú20% of p53 positive carcinoma cells were considered to be a diffuse pattern.
Classification of p21 positive cells was followed by that
of p53 positive cells.
Statistics
Survival data and causes of death were available for
all patients. Corrected survival rates were used (i.e.,
only deaths caused by gastric adenocarcinoma were
taken as outcome events, whereas all others were considered censored events). The probability of survival
was calculated using the Kaplan – Meier method, and
the significance of the difference between pairs of
Kaplan – Meier curves was calculated using the generalized Wilcoxon test. All P values were calculated using
the chi-square test, and those õ0.05 were considered
significant.
FIGURE 3. Kaplan–Meier plot for cumlative survival in 93 patients with
advanced gastric carcinoma, comparing p21 negative and p21 positive
tumors.
tions were incubated with the secondary antimouse
immunoglobulin (Ig) conjugated with biotin (Nichirei,
Tokyo, Japan) for 30 minutes at room temperature,
followed by incubation with streptavidin-peroxidase
complex (Nichirei) for 30 minutes. The reaction products of peroxidase were visualized by incubation with
0.05 M Tris-HCl buffer (pH 7.6) containing 20 mg 3,3*diaminobendizine (Nichirei) and 100 mL 5% hydrogen
peroxide per 100 mL. Finally, the slides were counterstained for nuclei by methyl green stain.
To examine the specificity of immunostaining, the
primary antibody was replaced by mouse normal IgG
at a 1:100 dilution and Tris-buffered saline. Control
slides were invariably negative for immunostaining.
To obtain constant immunohistochemical findings,
appropriate control slides of gastric carcinoma were
stained at the same time.
The staining pattern was classified as follows: (0):
negative or equivocal staining; (/): weak (positive car-
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RESULTS
p21 and p53 immunoreactivities were not detected in
adjacent nonneoplastic gastric mucosa, except for one
sample in which the intestinal metaplasia contained
focally p53 positive cells. Both p21 and p53 immunoreactivities were localized in the nuclei of carcinoma
cells (Fig. 1).
A total of 63 (68%) and 33 (36%) of the 93 tumor
specimens showed no expression of p21 and p53, respectively. p21 expression was weak in 25 of 93 cases
(27%) and strong in 5 of 93 cases (5%). Conversely,
p53 expression was weak in 21 cases (23%) and strong
in 39 cases (42%). The expression intensity of p21 protein did not correlate with gender, histologic type, or
tumor dimension, whereas expression was more frequently detected in patients older than 59 years than
in those younger than 59 years (Table 1). Table 2 shows
no significant relationship between p53 expression
and clinicopathologic findings in terms of gender, age,
tumor size, location, and histology. No apparent correlation was noted between p21 and p53 protein expression (Table 3).
Strong and weak expression of p53 did not predict
W: Cancer
Expression of p21 in Gastric Carcinomas/Gomyo et al.
a long survival period (Fig. 2). The 5-year survival rates
were 39.6% for strong and weak p53 positive tumors
and 63.4% in negative samples (P Å 0.11). In the former group, 39 cases were classified as strong p53-expressing tumors, with a survival rate of 49.2% (P Å
0.29). Conversely, strong and weak expression of p21
tended to predict a long survival period (Fig. 3). The
5-year survival rates were 69.4% for strong and weak
p21 positive tumors, compared with 38.1% in negative
samples (P Å 0.011).
DISCUSSION
The tumor stage at diagnosis and grade of differentiation are considered to be prognostic indicators of gastric carcinoma. Several investigations have analyzed
the occurrence of p53 mutations and p53 protein overexpression in gastric carcinoma.9 – 13 Most showed a
positive correlation between p53 positive expression
and poor prognosis. This might reflect the fact that
p53 positive tumors occur frequently in patients with
advanced gastric carcinoma. In this study, the authors
selected 93 patients with advanced gastric carcinomas
with serosal invasion and regional lymph node metastasis to analyze the correlation between the expression
of p21 and p53 and patient prognosis, regardless of
tumor stage. The prognosis of patients with advanced
gastric carcinomas tended to be related to p53 expression, but it was not significant (P Å 0.11). This may
be because the authors selected tumors at the same
stage. Of note is the belief that p53 expression is not
necessarily consistent with p53 gene mutation.26 – 28 Silent p53 mutations, such as frame shift and stop codon
mutations, do not produce p53 mutated protein. Expression of p53 protein by immunostaining does not
necessarily indicate a prognostic factor.
p21 function has been studied by some groups in
cultured cell lines.2,3 In irradiated cells, p21 regulates
the cell cycle and arrests the cell at the G1 checkpoint.
p21 positive cells proceed to apoptosis or DNA repair
in cell lines.2,29,30 Moreover, a direct effect of p21 as an
inhibitor of cell proliferation has been demonstrated
by its transient expression in tumor cells31 and in normal fibroblasts.32 However, p21 expression in gastric
carcinoma in vivo has not been elucidated. The authors found p21 positive tumor cells in 30 of 93 advanced gastric carcinomas (32%). Immunoreactive p21
protein might be the wild, nonmutated type, because
a recent study failed to detect p21 gene mutations in
a large series of various human tumors,33 including
brain tumors.34
The biologic significance of p21 overexpression
has not been elucidated sufficiently. Recent studies
have demonstrated a correlation with poor prognosis
in breast carcinoma patients.35,36 To the authors’
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2071
knowledge, this is the first study reporting that p21
expression is related to prognosis in patients with advanced gastric carcinoma.
The current study detected no relationship between p53 and p21 expression. Barboule et al.19 also
reported that the absence of p21 expression was not
correlated with p53 mutation in ovarian carcinoma
specimens. p21 expression might be regulated not only
by p53 expression (p53-dependent) but also by other
various factors (p53-independent) in vivo.17,18,37 DiGiuseppe et al.38 also indicated p21 expression might be
induced by a p53-independent pathway in human
pancreatic carcinomas.
In summary, p21, but not p53 expression, is related to a favorable prognosis in patients with advanced gastric carcinoma. Therefore, the identification of p21 expression provides useful information for
the postoperative management of these patients.
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