Value of p53 protein expression and its relationship with short-term prognosis in colorectal cancer.

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Annals of Saudi Medicine, Vol 22, Nos 5-6, 2002
377




VALUE OF P53 PROTEIN EXPRESSION AND ITS RELATIONSHIP WITH
SHORT-TERM PROGNOSIS IN COLORECTAL CANCER

Yamaç Erhan, MD; Mustafa Ali Korkut, MD; Eray Kara, MD;
Hasan Aydede, MD; Aslan Sakarya, MD; Özer Ilkgül, MD


P53 mutations are the most frequently detected genetic
alteration in human cancer.1,2 The p53 gene is located on
the short (p) arm of chromosome 17, and 17p deletions are
found in 6%-25% of colonic adenomas and in as many as
75% of colonic carcinomas.3,4 Mutations of p53, often
accompanied by loss of the wild-type allele, typically occur
when an in situ neoplasm develops into invasive
malignancy.3 Wild-type p53 acts to regulate transcription,
and when cells are damaged by UV light, irradiation or
chemotherapeutic agents, p53 accumulates and helps to
stop cells in the G1-phase of cell cycle.5 Wild-type p53
binds to double-stranded DNA at specific sequences as a
tetramer. In some cases where the presence of a mutant p53
allele and loss of the wild-type allele result in tumor
formation due to the loss of the growth-suppressive
function of p53, it acts as a tumor suppressor gene.
Consistent with this finding, the growth of some cancer cell
lines having only a mutant p53 allele can be suppressed by
transfection of the wild-type p53 gene into these cells.6
Mutant p53 protein could act in a dominant-negative
fashion to inhibit the function of wild-type p53, resulting in
a growth advantage for these clones of cells. Once point
mutation occurs on one allele, the other allele is rapidly
lost, resulting in the high incidence of concomitant p53
mutation plus chromosomal loss.6 Mutant p53 expression is
found in as many as 70% of colorectal carcinomas.7
Previous studies have shown that allelic loss of 17p,
indicative of p53 mutation, is associated with a likelihood
of distant metastasis, poor prognosis, and DNA
aneuploidy.8
The mutant p53 gene product has a prolonged half life
and is, therefore, detectable by immunohistochemistry
which seems to be a valid test for p53 mutation in
colorectal cancer.9 Immunohistochemical studies have also
demonstrated that nuclear p53 protein over-expression in
the primary colorectal carcinoma is associated with
decreased survival in both patients with positive lymph


From the Departments of Surgery (Drs. Erhan, Kara, Aydede and
Sakarya), Faculty of Medicine, Celal Bayar University, and Faculty of
Medicine (Dr. Ilkgül), Ege University, Izmir, Turkey.
Address reprint requests and correspondence to Dr. Aydede: 89 sok.
No. 15/1 Kat:1 Faikbey, Izmir 35290, Turkey.
Accepted for publication 20 June 2002. Received 08 October 2001.

FIGURE 1. A case of moderately differentiated adenocarcinoma of the
colon showing p53 positivity diffusely present in the nuclei of the
pleomorphic tumor cells (anti p53, 100x).

FIGURE 2. A case of well-differentiated adenocarcinoma of the colon
showing p53 positivity in tumor cells. p53 staining is not seen in normal
cells (anti p53, 200x).

nodes10 and those associated with advanced hepatic
metastasis.11 This study investigates the relation between
expression and percentage of p53 positive cells and eight
clinicopathologic variables, and the potential role of p53
molecule as a valuable prognostic marker in colorectal
cancer.
Materials and Methods

Twenty-eight patients, comprising 12 women and 16
men (aged 43-84, median 65 years) with colorectal cancer
were included in this study which took place from May to
November 1997. Surgical resection was performed in all
patients. Samples of tumor and adjacent normal mucosa
after surgical resection were removed from the specimen
and later used for immunohistochemical examination.
Dukes’ stage was used for staging these patients at the
time of resection and specimens were reviewed by the same
pathologist for histological grade, lymph node metastasis
and immunohistochemistry. Postoperative Dukes’ stage
distribution was: 1 patient (4%) with Dukes’ A; 13 patients
(46%) with Dukes’ B; 13 patients (46%) with Dukes’ C;
and 1 patient (4%) with Dukes’ D. Fifteen (54%), 6 (21%),
6 (21%), and 1 (4%) tumors were located in the rectum,
ascending colon, descending colon and transverse colon,
respectively. Continuous follow-up was done for 24 of 28
cases (mean follow-up = 32.9 months, range 10-43
months). Data on degree of differentiations, tumor volume,
lymph node metastasis, recurrence or metastasis were
evaluated.

Immunohistochemical Method for p53 Expression
Twenty-eight consecutive colorectal cancer resection
specimens obtained from 28 patients were examined.
Samples of tumor and adjacent normal mucosa were fixed
in 10% neutral-buffered formalin embedded in paraffin, and
5 µm consecutive sections were cut. The avidin-biotin

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ERHAN ET AL
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Annals of Saudi Medicine, Vol 22, Nos 5-6, 2002
peroxidase complex (ABC) method was used for
immunostaining. Sections obtained from formalin-fixed
material were cut, dewaxed and treated with 0.3% hydrogen
peroxide (H2O2) in methanol for 30 minutes to quench the
endogenous peroxidase activity. Slides were rinsed in
distilled water and incubated for 10 minutes at 750W in 10
mM citrate buffer in a thermoresistant container. Distilled
water and buffer were added periodically to the container to
prevent drying during the incubation process. The slides
were cooled in buffer for 20 minutes to room temperature,
washed in distilled water and rinsed in phosphate-buffered
saline (PBS). The primary antibody was applied to the
sections and incubated for 30 minutes at room temperature.
This was followed by incubation with a 1/100 dilution of
biotin-labeled anti-mouse secondary antibody for 15
minutes and ABC for 15 minutes. Careful rinses with PBS
between each step of the procedure were performed. The
color was developed with diaminobenzidine (DAB)
solution and the sections were lightly counterstained with
hematoxylin, dehydrated, and mounted. For
immunohistochemical staining of p53, the primary antibody
used was p53 protein clone-7, diluted 1:100 (Dako,
Copenhagen, Denmark). Protein expression was determined
by nuclear staining of tumor cells. In each tumor,
percentage of p53 positive cells was calculated. A tumor

TABLE 1. The distribution of p53 expression (%), the percentage of
p53 (+), cells (%) and their relations with common clinicopathologic
variables.

Clinicopathologic variables (n)
Sex
Female
Male
16
Age
<65 (years)
>65 (years) 15
Tumor location
Ascendant
Descendant
Rectum, transverse
15
Tumor size
<4 cm
>4 cm 12
Tumor grade differentiated
Poorly
Other 21
Stage
Dukes A + B
Dukes C + D
14
Lymph node metastasis
Negative
Positive 10
Recurrence and/or metastasis
Negative
Positive
Out of follow-up 4
*p<0.05.






Cases p53
positivity (%)

75
87

69
93

67
50
100*

87
75

71
86

82
88

89
70

77
90
p53(+)
cells (%)

12

34
35

13

28
50

6
6

22
31
40

16


7

30
37

14

39
45

18

36
33

14
10

26
54*
19
TABLE 2. Actual number of cells used for calculating the p53
percentage in each patient.
Patient no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28

with less than 5% tumor cell nuclei showing p53 staining
were scored as p53 negative. All other tumors showing p53
immunoreactivity were considered to be positive.

Statistical Analysis
The frequency of p53 positive tumors was compared for
each variable by using chi-squared analysis and Fisher’s
exact test. The method of One-way Anova was used for
percentage of p53 positive cells. Values of P<0.05 were
considered to be significant in all analyses.

Results

P53 staining was positive in 23 of 28 patients (82.1%)
with adenocarcinomas. Staining was confined to malignant
nuclei and was never found in adjacent normal mucosa. The
relationship between p53 expression and several
clinicopathological variables are summarized in Table 1.
No correlation was found between p53 staining with sex,
age, tumor volume, tumor grade, tumor stage, lymph node
metastasis and recurrence and/or metastatic cancer.
Interestingly, carcinoma of the rectum showed much more
p53 over-expression than carcinomas of the colon (P<0.05).
In the study, the percentages of p53 positive cells were
also compared with several clinocopathologic variables.
The results are summarized in Tables 1 and 2. There was no
correlation between the percentage of p53 positive cells and
sex, age, tumor site, tumor volume, tumor grade, tumor
stage and lymph node metastasis. However, recurrent
and/or metastatic cancer showed higher percentage of p53
Number cells (%)
80
18
40
–
80
61
30
55
74
8
20
24
–
20
20
15
20
50
48
40
–
70
80
–
–
15
20
70

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p53 PROTEIN EXPRESSION

Annals of Saudi Medicine, Vol 22, Nos 5-6, 2002
379
positive cells than non-metastatic or non-recurrent cancers
(P<0.05)

Discussion

The disease stage which was assessed by serosal
invasion and metastases of lymph node, liver and
peritoneum, is the most important prognostic factor
reflecting a patient’s five-year survival. These parameters
predicting the disease stage as well as other parameters
such as DNA ploidy, cell proliferative activity and
protooncogene products have been studied frequently. In
colorectal carcinoma, there are many reports that couple
protooncogene p53 products with the malignant
potential.12,13 Alteration of this gene has been associated
with postoperative outcome and poor prognosis.14 This may
be due to loss of wild type p53 tumor suppressor function,
radioresistance or chemoresistance, or acquisition of
oncogenic properties.
A review of the literature shows that over-expression of
p53 can be an independent significant predictor for
survival,15 but some authors failed to show the independent
prognostic value of p53 in colorectal cancer.16,17 In this
study, the relationship between p53 over-expression and
several clinocopathologic variables in 28 colorectal cancer
patients were examined. Regarding the relationship
between p53 immunoreactivity and some clinicopathologic
variables, no significant correlation was found between p53
expression and sex, age, tumor volume, tumor grade, tumor
stage, lymph node metastasis, recurrence and or metastatic
cancer. But there was a significant correlation between p53
expression and rectal carcinomas (P<0.05). The percentage
of p53 positive cells were also compared with these
clinicopathologic variables in this study. No correlation
were found between the percentage of p53 positive cells
and sex, age, tumor site, tumor volume, tumor grade, tumor
stage and lymph node metastasis. However, there was
significant correlation between the percentage of p53
positive cells and recurrent and/or metastatic cancer
(P<0.05).
Although the present study had a small number of cases
with short-term follow-up, the immunohistochemical
assessment of p53 and, more importantly, the percentage of
p53 positive cells may be valuable in predicting the risk of
recurrence and metastasis after curative surgery of
colorectal cancer. Since the conventional clinico-
pathological prognostic factors cannot be assessed in the
preoperative evaluation of colorectal carcinoma, assessment
of the percentage of p53 positive cells on samples obtained
during diagnostic endoscopic biopsies can be important in
the preoperative evaluation of colorectal carcinomas.

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