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Michael A. Goldfarb, MD, FACS†
Louis J. Zinterhofer, MD*
*Department of Pathology
†Department of Surgery
An Affiliate of the Saint Barnabas Health Care System
300
KEY WORDS: colorectal carcinoma, p53 protein, apoptosis
ABSTRACT
Background: Wild p53 protein is believed to play a role in the regulation of cell proliferation. Various studies report that overexpression of mutated p53 protein in colorectal cancer correlates with poor prognosis. However, other reports in the literature correlate low expression of this protein and adverse outcome. The study goal was to investigate whether a relationship between levels of p53 and survival exists in our patients.
Methods: Archival samples of consecutive colorectal tumors from 29 patients with Duke’s C colon carcinoma underwent immunohistochemical staining with a monoclonal antibody against p53. The same physician performed all analyses. The immunoreactivity of the p53 protein in the tumor was compared with clinicopathologic findings in each case. Prognosis was evaluated by univariate analysis. The pathologists were blinded to outcome or life span of the patient.
Results: Statistical analysis (MedCalc) with a Kaplan-Meier survival curve was performed, graphing the level of p53 staining in tumor tissue versus survival in months. In patients with absent or low expression of p53, median survival was 5.3 months versus at least 122 months in patients with a high expression of p53 (P < 0.0005).
Conclusions: These data suggest that absent p53 in colorectal tumors is a significant risk factor. The deletion of both p53 alleles at a genomic level may be responsible for poor survival in some patients. Our findings suggest that lack of p53 expression is more relevant to survival than overexpression. This article discusses pathogenic mechanism for lack of this expression and poor survival.
Introduction
The p53 gene (also known at TP53) is a tumor suppressor gene located on the short arm of chromosome 17 p13.1 which plays an important role in cellular growth control.1–10 Mutations of the p53 gene have been reported in numerous human malignancies.11–13 There are 2 types of p53 proteins: 1) normal or wild type and 2) mutant type. The wild type (normal) p53 protein is located in the nucleus, and it functions primarily by controlling the transcription of several other genes. It has a short half-life of only 20 minutes.
Wild type p53 is believed to play a role in the regulation of cell proliferation and acts as a tumor suppressor by the following mechanism: when DNA is damaged by irradiation, UV light or mutagenic chemicals, wild p53 protein levels increase in the cell. The accumulated wild p53 binds to DNA, stimulates transcription of several genes, and mediates 2 major effects on the cell. One effect is cell cycle arrest in G1 phase, which allows the damaged DNA to repair genes. If the DNA repair has occurred successfully, the level of wild p53 decreases with the help of mdm2 gene products. The cell begins to form new cells without any defects. The other effect occurs when the DNA damage cannot be successfully repaired. In that situation, p53 initiates the cell death (apoptosis) command, and cells die with the help of cell death genes (bax and IgF-BP3).
If the DNA damage goes unrepaired and the cells do not
die, mutations become fixed in the dividing cells and malignant transformation
of cells occurs. These malignant cells start to accumulate the mutated
p53 protein, which has the ability to induce tumor.14 Mutant p53 protein
can be detected using specific antibodies.15,16
Detection of p53 protein in the cell indicates the presence of a mutation
in the p53 gene, and absence of this protein indicates deletion of
both 17 p13.1 alleles at the genomic level.17,18
Overexpression of p53 protein has been documented in a number of human malignancies and shown to be associated with poor prognosis in patients with breast, gastric, and colorectal cancers.19–21 However, controversies exist in the literature about the relationship of overexpression of mutated p53 protein and survival in colorectal cancer. Some reports in the literature have suggested that overexpression of the mutated p53 protein correlates with poor survival.22,23 Additionally, a few scattered reports indicate that absence of this protein is associated with poor survival.24,25 The goal of this study was to see if overexpression or absent expression were related to different survival rates in our patients.
Methods
Patients
Between 1987 and 1995, 29 consecutive patients underwent colon surgery for Duke’s C colon cancer, and the tumors were evaluated for overexpression or absence of p53 protein. Follow-up information was available on all patients through the tumor registry. The same surgeon performed all of the surgeries. Patients included 17 women and 12 men, aged 42 to 86 years (mean, 69 years). All patients underwent standard colon resection. At the time of surgery, no patients had known distant metastases.
Histopathologic Examination
The resected specimens
were fixed in 10% buffered formalin. All tumors were cut into
5-mm slices after formalin fixation. The slices were embedded in paraffin,
and serial sections (4 mm thick) were prepared and stained with hematoxylin
and eosin for histopathologic diagnosis. Pathologic evaluations were
based on the guidelines for clinical and pathologic studies of carcinoma
of the colon as proposed by Duke’s classification.
Immunohistochemical Staining for p53
Archival tissue was obtained, and immunohistochemical
staining was performed by the avidin-biotin-peroxidase complex method
as described by Hsu et al.26 Primary
antibodies included monoclonal antibodies raised against p53 (DO-7,
Dako,
Two independent pathologists who did not know any details of patient survival analyzed samples from 29 patients under a light microscope. p53 was scored in the area in which the highest nuclear staining of p53 was observed. Approximately 1,000 cells were counted from the most aggressive area of the tumor. Staining was identified by the presence of a brown reaction in the nuclei. The specimens were graded as negative (0), weak (+), and intense (++) immunostaining with diffuse or focal distribution. Specimens in which less than 10% of the cancer cells were immunostained with p53 were classified as negative, and the rest were classified as positive, as described by Jeng et al.27
Results
Clinical and Histopathologic Features
Table 1 shows clinical and histopathological findings in 29 cases of Duke’s C colorectal cancer. Fourteen patients had adenocarcinoma of the right colon and 15 patients had left colon cancer. Seven patients received chemotherapy postoperatively. Thirteen tumors had negative or low expression of p53, and 16 had high expression of p53. Nine patients who had tumors with a high expression of p53 are currently alive, with an average survival of 144 months. One patient who had a tumor with negative p53 expression is currently alive 60 months after the diagnosis. Survival rates were calculated using the Kaplan-Meier method, comparing level of p53 staining with survival in months. Patients with absent or low expression of p53 had a median survival of 5.3 months as opposed to at least 122 months for patient with a high expression of p53 (P > 0.0005) (Figure 1).
Figure 2 shows case 21. The patient in this case is
a 52-year-old man with a diagnosis of adenocarcinoma of the left colon.
The tumor in this case showed 70% immunostaining of the tumor cell
nuclei with p53-specific monoclonal antibody (clone DO-7, Dako,
Discussion
Traditionally, the prognosis for colorectal cancer has been largely determined by the histologic stage at the time of surgery. It has been proposed that genotype alterations acquired during tumor progression may convey additional prognostic information. p53 mutations and allelic losses on 17p have been shown to provide prognostic information in colorectal cancer and other malignancies.28,29 Correlation between poor survival and overexpression of p53 mutant protein has been reported in colorectal cancer. However, this finding has not been consistent in all reported studies and, in fact, scattered reports in the literature indicate that the absence of p53 protein in colorectal cancer is associated with poor survival.24,25
Researchers have proposed that different tumorigenic mechanisms are involved in carcinoma of the proximal and distal colon, based on studies of allelic losses, c-myc overexpression, and microsatellite instability.30,31 Forster et al31 noted that sporadic carcinoma of the proximal colon involves only microsatellite instability (MIN) and negative p53 expression, suggesting good prognosis. They attributed these results to the fact that MIN is an earlier event in colonic carcinogenesis than other genomic changes.
Our study shows that absent or low expression of p53 is associated with a shorter survival rate (5.3) months and overexpression is associated with a longer survival rate (122 months) in Duke’s C colorectal cancer. These findings mirror reports by other researchers,24,25 who also showed that overexpression of p53 was associated with favorable prognosis and lack of p53 was a significant risk factor in Duke’s C colon cancer survival. Nathonson et al24 also reported that lack of p53 expression might also predict adverse outcome in colorectal cancer.
Donehower et al32 reported that wild type p53-deficient mice develop spontaneous tumors. They proposed that these mice develop a different range of tumor types according to their genetic background. They also demonstrated that a loss of wild type p53 function predisposed animals to spontaneous tumor development.
There appear to be 2 mechanisms to explain absent or
low p53 expression in our patients. First, there may be a deletion
of both alleles of chromosome 17p close to p53 locus resulting in
no detectable p53 protein. Second, MIN may have occurred with no mutational
changes of p53 locus.31
We believe that in our patient population, absent p53
expression is probably related to deletion of both p53 alleles. We
hypothesize that lack of p53 protein may perhaps have contributed
to shorter patient survival because it signifies losing the tumor-suppressor
effect of the gene. However, one patient with negative p53 expression,
who is still alive at the writing of this manuscript
may have a different molecular mechanism for the development of colorectal
cancer.30
Conclusions
In summary, p53,
which has been known as a “guardian of the human genome”33 in response
to DNA damage, is absent or low in some patients with Duke’s C colorectal
cancer. These patients have a poorer prognosis, and patients who overexpress
the mutated form of p53 protein may experience a longer survival.
In addition, lack of p53 expression in patients without mutation of
p53 gene may represent a different molecular mechanism of tumorigenesis
to explain their longevity. A further study with a larger number of
patients is required to elucidate this finding.
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Figure 1. Kaplan-Meier survival curves for patients
with colorectal carcinoma, classed by p53 immunoreactivity. p53 negative
tumors are associated with significantly poorer prognosis than tumors
with overexpression of p53 (P < 0.0005).
Figure 2. Case 21 with immunostaining of a tumor
with p53-specific monoclonal antibody (clone DO-7). The nuclei of
p53 positive cells (70%) were stained a brownish color. This tumor
was classified as p53 positive (original magnification, X 300).
Figure 3. Case 5 with immunostaining of a tumor
with a p53 specific monoclonal antibody (clone DO-7). The nuclei of
the tumor cells were negative (< 10%). This tumor was classified
as p53 negative (original magnification X 300).
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