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大腸直腸腺瘤及腺癌之p53蛋白表現在台灣：兩家醫院之研究
曾志恩 陳榮達1 何 霖1
佛教大林慈濟綜合醫院病理科 台中榮民總醫院病理部1
摘要
目的：1.探討大腸直腸腺瘤及腺癌之p53蛋白表現在台灣的現狀及與其他族群是否不同？2.p53蛋白可否當做大腸纖維鏡術的一種輔助性檢查？材料與方法：收集福馬林固定處理後之腺瘤110個，源發自腺瘤之腺癌46個及腺癌62個，進行常規p53蛋白之免疫組織化學染色並統計分析其結果。結果：腺瘤、源發自腺瘤之腺癌及腺癌之平均年齡分別為63.3歲、67.1歲和69.3歲；其p53蛋白陽性率分別為6.4%，28.3%/32.6%和71.0%。腺瘤之纖毛型態及異生嚴重度與p53蛋白表現無正相關。結論：1.大腸直腸腺瘤及腺癌之p53蛋白表現在台灣與其他族群並無不同。2.腺瘤的絨毛和異生嚴重度與p53蛋白表現不相稱。3.從腺瘤至腺癌，p53蛋白表現逐漸增加，支持前人腺瘤－腺癌系列之假說。從腺瘤至長成腺癌約需6年。4.對低度異生或絨毛組成較少但p53蛋白過度表現之切除不完全腺瘤，應積極的進一步完全切除。(慈濟醫學2003;
15:305-310)
關鍵語：p53蛋白，大腸直腸，腺瘤，腺癌
收文日期：92年4月3日，修改日期：92年5月19日，接受日期：92年6月16日
抽印本索取及聯絡地址：嘉義縣大林鎮民生路2號 佛教大林慈濟綜合醫院病理科 曾志恩醫師
P53 Protein Presentation of Colorectal Adenomas and Carcinomas in Taiwan:
Experience of Two Hospitals
Jeh-En Tzeng, Jung-Ta Chen1, William L. Ho1
Department of Pathology, Buddhist Dalin Tzu Chi General Hospital, Chiayi,
Taiwan; Department of Pathology1,Taichung Veterans General Hospital,
Taichung, Taiwan
ABSTRACT
Objective: We first attempted to determine if the p53 protein presentation
of colorectal adenomas and carcinomas showed any difference between
Taiwanese and other populations. Second, as incompletely polypectomized or
biopsied adenomas of the colorectum are a troublesome issue in routine
practice, we sought to determine if p53 protein is a helpful indicator for
clinicians in deciding the subsequent therapeutic mode for incompletely
removed tumors. Materials and Methods: We studied formalin-fixed tissue,
including 110 adenomas, 46 adenocarcinomas arising from adenomas, and 62
frank adenocarcinomas with routine immunohistochemical staining and
statistical analysis. Results: The average ages of patients with adenomas,
adenocarcinomas arising from adenomas, and frank adenocarcinomas were 63.3,
67.1, and 69.3 years. The p53 protein-positive rates of these 3 groups were
6.4%, 28.3%/32.6% (adenomatous part/adenocarcinomatous part), and 71.0%,
respectively. Conclusions: First, there was no statistical difference in
colorectal p53 protein presentation between Taiwanese and other populations.
Second, histologically, the villous morphology and dysplastic severity of
the adenoma were not correlated with p53 protein expression. Third, the
sequential increase in p53 protein overexpression from adenomas to frank
adenocarcinomas supports the hypothesis of an adenoma-carcinoma sequence in
polypoid colorectal tumors. The transformation duration from adenomas to
carcinomas is about 6 years. Fourth, an incompletely polypectomized adenoma,
which shows low dysplasia or a small villous component but p53 protein
overexpression, should be aggressively completely resected. (Tzu Chi Med J
2003; 15:305-310)
Key words: p53 protein, colorectum, adenoma, adenocarcinoma
Received: April 3, 2003, Revised: May 19, 2003, Accepted: June 16, 2003
Address reprint requests and correspondence to: Dr. Jeh-En Tzeng, Department
of Pathology, Buddhist Dalin Tzu Chi General Hospital, 2, Min Sheng Road,
Dalin, Chiayi, Taiwan
INTRODUCTION
Colorectal cancer is a globally important cause of cancer-related death.
Abnormal cell growth and differ-entiation, associated with the accumulation
of genetic alternations over a long time, are the etiology of colo-rectal
cancer, and a genetic model for colorectal tumorigenesis is well established
[1]. The p53 gene is located on the short arm of chromosome 17, which
encodes a 53-kD phosphoprotein. This protein might play a certain role in
the regulatory control of normal cell prolif-eration. The wild-type p53
protein usually does not accumulate in amounts detectable by
immunohistochemistry because of its short half-life of 6-20 minutes.
However, the mutant type has a half-life of up to 6 hours [2]. This
functionally inactive and stabilized p53 protein can be detected in nuclei
of cells. By using this nature of the p53 protein, many researchers focusing
on the relationship of p53 protein or the p53 gene with colorectal cancer
have obtained significant results [3-11]. But, there is still limited study
of p53 in Taiwan to our knowledge. The first aim of this study was to
demonstrate colorectal p53 protein expression in Taiwan and to determine if
any differences exist with other popula-tions.
Clinically, an incompletely polypectomized ade-noma or incompletely biopsied
adenoma is occasionally encountered when colonoscopy is performed. In those
situations, what is the best suggestion for clinicians? An indicator, which
could help distinguish high-risk adenomas and which is easily detected, is
necessary. Now that the p53 gene is known to play a key role in the
adenoma-carcinoma sequence [1], it seems reasonable to use the p53 protein
as an indicator for this purpose. The secondary purpose of this study was to
determine whether the p53 protein, detected by an immunohistochemical
method, is a good adjuvant indicator for colonoscopy.
MATERIALS AND METHODS
Archival formalin-fixed, paraffin wax-embedded tissues from the Buddhist
Dalin Tzu Chi General Hospital and Taichung Veterans General Hospital of
Taiwan were used. During August 1999 to February 2002, 110 adenomas from 89
patients were studied after colofiberoscopic biopsies or polypectomies.
Dysplasia of the adenomas was graded on a 2-tier scale as either
mild/moderate or severe as per the WHO classification. The villous
morphology of the adenoma was classified into 3 groups: less than 25%,
between 25% and 75%, and more than 75% of the mucosal surface. Sixty-two
frank adenocarcinomas, which were defined as adenocarcinomas with tumors
invading beyond the mucosa muscularis of the colorectal wall, from 60
patients including 13 cancers via colonoscopic biopsies and 49 via
excisions, were collected. A single block representative of the tumor was
selected in each case. Forty-six adenocarcinomas arising from adenomas
including surgically removed specimens and colonoscopic biopsies were
obtained from 45 patients.
For the immunohistochemical staining of p53 protein, formalin-fixed, parafin-embedded
tissue was used. Three-micrometer-thick sections were deparafin-ized in
xylene, re-hydrated in a series of graded alcohol, and later exposed to 3%
hydrogen peroxidase to drive off the endogenous peroxidase. Sections were
immersed in cuvettes with citrate buffer (pH 6.0), which were placed in a
bowl containing 1500 mL tap water. The bowl was transferred into a microwave
oven (National, Tou-Yan, Taiwan) with an automatic rotating plate, and
irradiated at 720 W for 10 minutes for antigen retrieval [12]. After
microwave treatment, sections were pre-incubated in PBS. Monoclonal antibody
DO-7 (Dako, Denmark, code M7001), which reacts with wild-type and
mutant-type human p53 protein, was applied to the sections (dilution 1:50;
incubation 30 minutes). Biotinylated rabbit anti-mouse antibody (Dako, code
K0672 bottle 2; incubation 10 minutes) was used as the secondary antibody.
The immunoreaction was visualized using the avidin/biotin complex (Dako,
code K0672 bottle 3; incubation 10 minutes) with hydrogen peroxide as the
substrate and N,N-dimethylformamide (DMF) as a chromogen. Counterstaining
was performed with Mayer's hematoxylin. Positive control was performed by
using colonic carcinomatous sections with high p53 overexpression. Normal
mucosa was stained as a negative control.
Sections from each tumor were examined at X40 to X400 magnification. The
ratio of the p53 protein-positive nuclei was blindly calculated by 2
pathologists. The presence of more than 10% p53 protein-positive nuclei was
regarded as being a p53 protein-positive presenta-tion, and a consensus was
reached when a discrepancy was encountered between the 2 pathologists. p53
protein-positive nuclei were visualized as having dispersed or compact
patterns [13]. The former is spottily distributed and usually only involves
a small proportion of glandular nuclei. The latter shows a contiguous area
of p53-reactive nuclei and usually involves more-glandular nuclei.
RESULTS
Seven adenomas (6.4%) of these 110 adenomas had evidence of p53 protein
overexpression. The mean age of the 89 cases (54 males and 35 females) was
63.3 years (Table 1). The p53 protein reaction was always localized in the
nuclei, and all positive cases had the compacted pattern (Fig. 1). Twelve
(11.7%) of the 103 negative adenomas had less than 10% p53 protein-positive
nuclei but with the compacted pattern. Fourteen of the 110 adenomas had less
than 25% villous part, 82 ade-nomas had 25%-75% villous part, and 14
adenomas had more than 75% villous part. Ninety-five of the 110 ade-nomas
had mild to moderate dysplasia, and 15 adenomas had severe dysplasia. All 7
p53 positive-cases had 25%-75% villous parts and showed mild to moderate
dysplasia. Neither the amount of the villous part nor the severity of
dysplasia was correlated to p53 protein overexpression.
Sixty patients with 62 frank adenocarcinomas, which included 13 cancers
retrived via colonoscopic biopsies and 49 via excisions, were studied.
Forty- four adenocarcinomas (71.0%), which included 8 (61.5%) of the 13
biopsied cancers and 36 (73.5%) of the 49 excised cancers, had p53 protein
overexpression. The mean age of these 60 cases (39 males and 21 females) was
69.3 years (Table 1). All p53-positive cases showed the compacted pattern
and only 1 of the negative adenocarcinomas focally demonstrated the
compacted pattern. Another case of a negative adenocarcinoma showed a
uniform dispersed pattern.
In the group of 46 adenocarcinomas arising from adenomas, the mean age of
the 45 cases (29 males and 16 females) was 67.1 years (Table 1). Eleven
(23.9%) of the 46 tumors had p53 protein overexpression in both the
adenomatous parts and cancerous parts (Fig. 2). Twenty-nine (63.0%) of the
46 tumors showed no p53 reaction in either the adenomatous part or the
cancerous part. Four tumors showed a positive p53 reaction in the cancerous
part, but were negative in the adenomatous part. The last 2 tumors
demonstrated a positive p53 reaction in the adenomatous part, but were
negative in the cancerous part (Table 2).
DISCUSSION
Nearly all p53-positive stains were presented within nuclei. The cytoplasma
either negatively stained in most cases or very weakly stained in some cases
in our study. Nuclear accumulation of the p53 protein, as previously
reported [13], was visualized as following 2 distinct patterns: compact or
dispersed. These 2 distinct patterns were correlated to the rate of positive
nuclei. When the rate of nuclear staining was greater than 10% of tumor
nuclei, the majority of positively stained nuclei had the compact pattern.
However, when the p53 protein-positive rate was less than 10% of tumor
nuclei, positively stained nuclei always showed the dispersed pattern. The
correlation between positively stained nuclear rates and their distributive
patterns indicated that the 10% cut-off value for p53-positive staining is
reasonable.
Twelve cases (11.6%) of adenomas showed less than a 10% p53 protein-positive
presentation but had the focally compact positive p53 nuclear pattern. This
phenomenon might have been due to focally reactive changes or an early
sequential transformation from an adenoma to an invasive carcinoma [13].
Seven cases (6.4%) of adenomas had positive p53 expression. This finding was
similar to results of Kikuchi et al [14] and Van et al [15], both of which
showed 8% with positive p53 expression. Histologically, neither the villous
ratio nor the severity of dysplasia of the adenomas was correlated to p53
protein accumulation. These findings are similar to those of previous
reports [3,11,16,17], but differ from Kikuchi et al.'s results [14]. The
latter showed an increasing p53 mutation rate of from 0%, 8%, 15%, to 40% in
adenomas with moderate dys-plasia, adenomas with severe dysplasia,
intramucosal carcinomas, and invasive carcinomas, respectively. The reason
why we failed to demonstrate a relationship between p53 expression and the
degree of dysplasia is unclear. But, it might have been due to a focally
small number of p53-positive glands not being found because of a lack of
series cuts to the entire adenoma [16] or to missense mutations at the
genetic level [18].
Forty-four tumors (71%) of 62 frank adenocarcinomas showed p53
overexpression. There was no obvious difference in p53 protein
overexpression (p > 0.05) between biopsied specimens (8/13; 61.5%) and
excised specimens (36/49; 73.5%). This ratio was similar to well-documented
reports of p53 protein [3,5,9,11,19,20] and gene [21,22] presentations of
colorectal adenocarcino-mas. Those studies showed the p53 protein positive
rate to be within 59.6% to 72%, and, the rate of the p53 genetic abnormality
to be 60% to 75% in colorectal cancers. This finding, in our study,
demonstrates that p53 protein overexpression in Taiwanese is similar to that
seen in other populations.
In the group of adenocarcinomas arising from ade-nomas (Table 2), the p53
protein-positive rate of the adenomatous part was 28.3% (13/46) and of the
adenocarcinomatous part was 32.6% (15/46). There was no statistical
difference in p53 protein presentation between the adenomatous part and the
adenocarinomatous part in this group (p > 0.05). This group's result was
between the pure adenomatous group, which showed a 6.4% p53 protein-positive
rate, and the frank adenocar-cinomatous group, which showed a 71.0% p53
protein-positive rate, in this study. Obviously, there was a sequential
increase in p53 protein overexpression from adenomas to adenocarcinomas
arising from adenomas, and then to frank adenocarcinomas. This result is
similar to previous reports of Auvinen et al [5] and Vogel-stein et al [21],
and supports the hypothesis of the ade-enoma-carcinoma sequence. It was
interesting to find out its next change in a p53 protein-positive adenoma.
At the genetic level, these p53-positive adenomas had either a point
mutation or allelic deletion [2,7,14,19,21-24], and these changes were
irreversible. Due to loss of normal regulatory function of cellular
proliferation of the defective p53 gene, it is reasonable to predict that
the p53 protein-positive focus of an adenoma would expend and transform to a
malignancy. This transformation process had no return, and the p53 protein
showed sequentially increasing accumulation. This phenomenon makes the p53
protein an adjuvant indicator for clinicians to decide on a more-aggressive
therapeutic policy in an incompletely polypectomized or biopsied
p53-positive adenoma.
The 2 adenocarcinomas arising from adenomas which were p53-postive in the
adenomatous parts but p53-negative in the adenocarcinomatous parts had
smaller malignant foci within relatively larger adenomas. About 5% of the
nuclei of these 2 malignant foci stained p53-postive, but their adenomatous
counterparts had more than 10% p53-positive nuclei. The exact reason for
this phenomenon is unclear. But, it might have been due to a bias of small
foci with early malignant changes or another p53 gene mutation coding for a
stop code and inducing no products of p53 protein during the cancerous
transformation.
The mean ages of these 3 groups also indicated sequential changes: 63.3,
67.1, and 69.3 years, respectively. Only the age difference between the
adenomatous group and the frankly adenocarcinomatous group reached
statistical significance (p < 0.05). However, this finding roughly predicts
the transformation from adenomas to frank adenocarcinomas to be about 6
years and supports Muller et al's study [25] which showed that a colonoscopy
maintained a protective influence for 6 years.
CONCLUSIONS
Colorectal p53 protein overexpression in Taiwanese is similar to those of
related reports for other populations. Histologically, the villous
morphology and dysplastic severity of the adenoma were not correlated with
p53 overexpression. The sequential increase in p53 protein overexpression
from adenomas to frank carcinomas supports the previous hypothesis of an
adeno-ma-carcinoma sequence in polypoid colorectal tumors. An incompletely
polypectomized adenoma, which shows low dysplasia or a small villous
component but p53 protein overexpression, should be aggressively completely
resected.
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Fig. 1. A p53 protein-positive adenoma showing p53-reactive nuclei and the
compacted nuclear pattern (right); a p53 protein-negative adenoma
demonstrating the dispersed pattern of scattered spotty p53-reactive nuclei
(left). (200X)
Fig. 2. Adenocarcinoma arising from an adenoma showing positive p53
reactivity in both (right) the adenocar-cinomatous part and (left) the
adenomatous part. (400X)
Table 1. Basic Data and p53 Protein Presentations of Adenomas,
Adenocarcinomas Arising from Adenomas and Frank Adenocarcinomas
|
| |
Adenomas |
#Ca from adenomas |
Adenocarcinomas |
|
|
No. of tumor |
110 |
46 |
62 |
|
No. of cases |
89 |
45 |
60 |
|
M:F |
54:35 |
29:16 |
39:21 |
|
Mean age (years) |
63.3 |
67.1 |
69.3 |
|
p53+ |
6.4%(7/110) |
*28.3%/32.6% |
71.0%(44/62) |
|
#: Ca from adenomas: Adenocarcinomas arising from adenomas; *: Adenomatous
part/adenocarcinomatous part
Table 2. Immunohistochemical Presentation of p53 Protein in 46 Colorectal
Adenocarcinomas Arising from Adenomas
|
| Ap |
Acp
|
| p53 positive |
p53 negative |
Total |
|
|
p53 positive |
11 (23.9%) |
2 ( 4.4%) |
13 (28.3%) |
|
p53 negative |
4 ( 8.7%) |
29 (63.0%) |
33 (71.7%) |
| Total |
15 (32.6%) |
31 (67.4%) |
46 (100%) |
|
Acp: adenocarcinomatous part; Ap: adenomatous part |