Gallbladder carcinoma: the role of p53 protein overexpression and Ki-67 antigen expression as prognostic markers

Abstract

Background

The overexpression of p53 protein and the expression of Ki-67 antigen may affect the survival of patients with gallbladder carcinoma. This association has been tested in a series of 41 patients with relatively early carcinoma of the gallbladder.

Methods

Forty-one surgical specimens from patients with a postoperative histological diagnosis of gallbladder carcinoma were studied. All patients were operated by simple cholecystectomy only because the tumours were not advanced and/or their general condition was poor. Patients submitted to radical operations were excluded. p53 expression was calculated from nuclear staining according to the intensity and extent of positive cells, as graded on a scale from 1 to 3; a combined score of >3 was considered as overexpression. Ki-67 expression was calculated by the MIB-I index: the percentage of positively stained tumour cell nuclei out of the total tumour cells counted (n = 1000); >20% of stained cells was considered positive.

Results

Twenty-nine gallbladder carcinomas (71%) overexpressed p53 protein in the cell nuclei. No significant differences were found in relation to cell differentiation on the level of tumour infiltration through the gallbladder wall. Five-year survival of patients with gallbladder carcinoma overexpressing p53 was 17.2%, while survival of patients without p53 overexpression was 30% (not significant). Twenty-four cases (58.5%) were considered positive for the MIB-I index. There were no differences between the grade of cell differentiation and wall infiltration. Five-year survival of the patients with a MIB-I positive index was 9.2% as opposed to 27.7% for those with a negative index (not significant).

Conclusions

p53 protein nuclear overexpression and Ki-67 protein expression in gallbladder carcinoma were not related to histological differentiation, level of gallbladder wall invasion or patient survival.

Introduction

Gallbladder carcinoma is a relatively uncommon disease, accounting for about 0.3–0.7% of all cancer cases and having a peak incidence among women aged between 70 and 79 years ¹. The prognosis is poor, related to diagnostic delay and biological behaviour of the tumour ². The pathogenic mechanisms of gallbladder carcinoma are only partially known, but genetic and environmental risk factors have been identified ¹,³.

The development of human cancer is related to multi-step genetic changes, which include activation of cellular protooncogenes and inactivation of tumour suppressor genes ⁴. The p53 tumour suppressor gene, located on the short arm of chromosome 17, encodes a 53-kD nuclear phosphoprotein, which acts as an inhibitor of cell proliferation ⁵,⁶. Point mutations of the p53 gene are the most common single change in human cancer ⁷ and are especially high in carcinomas of the lung ⁸, colon ⁹ and urinary bladder ¹⁰. These mutations may lead to a loss of the production of the protein and the synthesis of a mutated protein that exhibits an increased half-life, tends to accumulate in the cell nuclei and can be detected by immunohistochemistry ⁶. In several studies, nuclear p53 protein detected by immunohistochemical staining has been correlated with the presence of p53 gene mutation ⁸,¹¹,¹²,¹³,¹⁴. Although in some tumours this correlation has been reported to be poor and the immunohistochemical detection of p53 protein may not indicate an underlying gene mutation, this correlation may be a useful indicator of an altered p53 protein function ¹⁵. To obviate all these problems, the term overexpression, used by Wistuba and colleagues ¹⁶, refers to the accumulation of increased immunohistochemichally detectable p53 protein, whatever the mechanisms of nuclear accumulation.

Cell cycle activity and cell kinetics are important indicators of growth and behaviour of several human tumours ¹⁷,¹⁸,¹⁹,²⁰,²¹,²². A gene on chromosome 10 encodes a nuclear protein of 345–395 kD that is recognised by the antibody to the Ki-67 antigen. Peak concentrations of Ki-67 in cycling cells are achieved in G2 and M phases, while there is no expression in GO phase and early Gl phase. Ki-67 expression detected by immunohistochemistry has been accepted as a good indicator of cell proliferation in histological material ²².

Several studies of p53 protein overexpression in gallbladder carcinoma and its precursor lesions have suggested an important role for p53 genetic abnormalities in the pathogenesis of that tumour ¹⁶,²³,²⁴,²⁵. The Ki-67 expression has been found to be a good prognostic indicator for gallbladder carcinoma ²²,²⁶. The present study was carried out to determine if there is a relationship between both p53 overexpression and Ki-67 expression and the grade of cell differentiation, level of gallbladder wall invasion and survival in patients affected by gallbladder carcinoma.

Materials and Methods

Patients

Forty-one patients with a definitive postoperative histological diagnosis of gallbladder carcinoma attending this department were included in this study. The mean age of the group was 74–8 years (SD 9.8 years); 33 patients were women and 8 men. The surgical procedure consisted of a simple cholecystectomy or only tumour biopsies when local spread precluded resection. More aggressive operations were not considered because either the tumours were not advanced (Tis, T1 and T2) or the general condition of the patients was very poor (age >80 years and/or intercurrent disease). Patients who received radical surgery within this period were excluded.

Tissue specimens and histological analysis

Surgical specimens were fixed in 10% buffered formalin and embedded in paraffin wax. Slides stained with haematoxylineosin were evaluated for diagnosis, which was based on light microscopy study. Carcinomas were classified according to criteria established previously ²⁷; 38 cases were intestinal-type adenocarcinomas and 3 were papillary adenocarcinomas. The grade of cell differentiation, according to WHO criteria ²⁷, was as follows: 18 cases were well differentiated, 13 moderately differentiated and 10 poorly differentiated.

Immunohistochemical method

Immunostaining was performed using a biotin-streptavi-din-alkaline phosphatase method. Two adjacent sections (2–4 µm thick) were used, one for immunostaining and one as a negative control. A positive control was simultaneously performed. The sections were deparaffinised in xylene and rehydrated with graded alcohol, followed by two washes in deionised water. Pretreatment in a wet-heat sterilising device for 40 min in a citrate buffer (0.01 M, pH 6) was used to facilitate antigen retrieval. Primary anti-p53 antibody (Dako, Clone DO-7) and MIB-1 (Ki-67 antibody, Biogenex) were applied to the sections at a dilution of 1:50 and were incubated for 2 h at room temperature, followed by incubation for 20 min with biotinylated anti-mouse/anti-rabbit secondary antibody and 20 min with alkaline phosphatase-conjugated streptavidin (Alkaline Phosphatase Kit, Biogenex). The primary antibody was substituted with phosphate buffer saline (PBS) in sections used as negative controls. The staining was visualised with Fast Red chromogen. The sections were counterstained with Harris haematoxylin, and coverslipped with aqueous mounting media.

Analysis of p53 immunostaining

The degree of p53 overexpression was scored by a semi-quantitative method evaluating the intensity and incidence of positive stained cells, as previously described by Wee and colleagues ²⁴. The intensity was graded as absent (0), mild (1), moderate (2) or intense (3); the incidence was categorised as absent (0), <10% (1), 10–50% (2) or >50% of positive cells (3). The evaluation was carried out by two independent histopathologists, and discordant cases were considered again to achieve consensus. A staining score for each case was obtained (range 0–6) by adding both variables, the intensity and incidence. In this semi-quantitative method, a score >3 was considered as positive for overexpression. In all specimens, only nuclear staining was regarded as specific staining.

Analysis of Ki-67 immunostaining

The MIB-1 index was calculated as the percentage of positively stained tumour cell nuclei out of the total tumour cells counted (n = 1000). A percentage >20% of stained cells was considered positive, regardless of the intensity of staining.

Tumour staging and survival

The depth of invasion of the gallbladder wall (T) in the TNM classification ²⁸ was used for tumour staging, while lymph node invasion by gallbladder carcinoma (N) was not considered. Postoperative clinical follow-up allowed a calculation of survival by the Kaplan–Meier curves.

Statistical analysis

Statistical analysis was performed to compare cases with p53 overexpression or Ki-67 expression with those that were negative for either marker. The χ test or two-tailed Fisher's exact test (when expected frequencies were <5) were used to compare the grade of cell differentiation and the level of gallbladder wall invasion according to p53 overexpression and Ki-67 expression; the criterion for significance was p < 0.05. The log rank test was used for statistical comparison of Kaplan–Meier curves.

Results

p53 overexpression

Twenty-nine (70.7%) gallbladder carcinomas overex-pressed p53 protein in the cell nuclei after immunohistochemical procedures. Twenty-five of 35 intestinal type adenocarcinomas overexpressed p53 protein (71.4%) and one of the three papillary adenocarcinomas (33.3%).

The distribution of p53 overexpression according to cell differentiation is described in Table 1. p53 over-expression predominated in moderately differentiated tumours, but no significant differences were found between the three types of cell differentiation. The same result was obtained when the infiltration level of the gallbladder wall and p53 overexpression were compared, although p53 overexpression predominated in T3 tumours.

Table 1. p53 overexpression in 41 patinets with Gallbladde Carcinoma.

	p53 overexpression No. positive cases/ total number of cases
Gallbladder adenocarcinoma	No. positive cases/ total number of cases	%
Differentiation grade
Well differentiated	11/18	61.1
Moderately differentiated	11/13	84.6 p = ns
Poorly differentiated	7/10	70
Parietal infiltration
Tis	2/3	66.6
Tl	2/4	50
T2	3/5	60 p = ns
T3	19/23	82.6
T4	3/6	50

The survival curves are shown in Figure 1. The 5-year survival of patients with gallbladder carcinoma over-expressing p53 was 17.2%, while survival of patients with non-overexpressing gallbladder carcinoma was 30%. Statistical analysis showed no differences between groups.

Figure 1. .

survival and p53 overexpression in gallbladder carcinoma.

Ki-67 expression

The results of the MIB-1 index are shown in Table 2. The MIB-1 index ranged between 0 and 70%, with a mean of 25.8% (SD= 16.4); 24 cases expressed MIB-1 index >20% and were considered positive (58.5%). There were no differences between the grade of cell differentiation and parietal infiltration when the mean percentage of stained cells was considered. The same situation was found when cases with a positive MIB-1 index were compared against those with a negative MIB-1 index in relation to cell differentiation. However, the three cases of Tis tumour had a negative MIB-1 index, and thus make for a significant difference when parietal infiltration was considered (p < 0.05).

Table 2. Ki-67 expression patients with gallbladder.

Gallbladder adenocarcinoma	Mean MIB-1 index	No. positive/total cases	%
Differentiation grade
Well differentiated	25.0% (SD± 17.0%)	10/18	55.5
Moderately differentiated	30.7% (SD± 16.8%) p = ns	9/13	69.2 p = ns
Poorly differentiated	21.2% (SD± 14.4%)	5/10	50
Parietal infiltration
Tis	12.0% (SD± 5.9)	0/3	0
Tl	34.8% (SD ± 23.9)	3/4	75
T2	33.8% (SD± 20.1) p = ns	4/5	80 p = 0.05
T3	27.5% (SD± 14.7)	15/23	65
T4	I3.9%(SD±9.5)	2/4	50

Figure 2 shows the Kaplan–Meier survival curves. The 5-year survival of patients with a MIB-1 positive index was 9.2% as opposed to 27.7% for those with a negative index (not significant).

Figure 2. .

Cumulative survival and Ki-67 expression in gallbladder carcinoma.

Discussion

Our results suggest that: 1) there is a high incidence of p53 overexpression in gallbladder carcinoma, 2) neither gallbladder wall infiltration nor cell differentiation are influenced by p53 overexpression or Ki-67 expression, and 3) tumour p53 overexpression and Ki-67 expression are not prognostic factors for survival in patients with gallbladder carcinoma.

The rates of p53 protein nuclear overexpression in gallbladder carcinoma range from 39.6% ²⁹ to 92% of cases ²⁴,²⁵. Our results (70.7%) are similar to those obtained by other workers ¹⁶,³⁰,³¹. This variability of results could depend on the different methods used to analyse the p53 nuclear expression, although all these authors evaluated their results by a semi-quantitative method including both the number of cells stained and the intensity of staining. The studies that consider epithelial dysplasia, carcinoma in situ and invasive carcinoma have shown a progressively increasing incidence of p53 overexpression from premalignant lesions to invasive tumour ¹⁶,²³,²⁴, which supports the view that abnormalities in p53 protein are an early event in the carcinogenesis and are probably the usual route for gallbladder carcinoma development ³¹,³². Interestingly, Itoi and colleagues ³³ suggested that there are two distinct genetic pathways in gallbladder carcinogenesis: de novo carcinoma develops from a predominant p53 alteration with low K-ras mutation, while carcinoma-in-pyloric gland-type adenoma develops from p53, K-ras and APC gene-unrelated alteration. Hanada and associates have shown that in stage I gallbladder carcinoma p53 mutations may play a role in the carcinogenesis of flat-type carcinomas but not in the polypoid type, which probably follows a different pathway ³⁴,³⁵.

Several studies conclude that high-grade (poorly differentiated) gallbladder carcinomas tend to overexpress p53 protein more than low-grade tumours (well differentiated) ²,¹⁶,²³,²⁴,²⁵,³⁰,³⁶,³⁷,³⁸,³⁹, but most of these series are too small for statistical analysis. In the present study, although the rate of moderately differentiated (80%) and poorly differentiated gallbladder carcinoma (72%) is higher than the rate of well-differentiated gallbladder carcinoma (58.8%), there is no statistical significance when both groups are compared, in line with the results obtained elsewere ³¹. Kamel and associates ²³ raised the hypothesis that, accepting that p53 is an early event in the carcinogenesis of a group of gallbladder carcinoma, p53-positive dys-plasias evolve to a more aggressive type of tumour, which would be indicated by association with high tumour grade and p53 positivity. Roa and associates ⁴⁰, studied 53 cases of advanced gallbladder carcinoma and found that in subserosal tumours p53 expression was significantly lower than that of tumours reaching the serosa. The results of our study corroborate others in which p53 overexpression was not related to the level of gallbladder wall invasion by the tumour ¹⁶,²⁹,³¹.

The expression of p53 protein has been used as a prognostic factor in several tumours. A tendency for shorter survival times has been found in patients with colon carcinoma who expressed p53 protein ⁴¹ and a correlation between more aggressive behaviour of urinary bladder carcinoma and p53 protein expression has been demostrated ⁴². In the present study, gallbladder carcinomas that overexpressed p53 protein did not show better survival then gallbladder carcinoma without such overexpression, in line with other series ²⁹,³¹,³⁷. However, a decreased patient survival in poorly differentiated gallbladder carcinoma that expressed p53 protein was found in one study ³⁶.

Immunoreactivity for Ki-67 antigen is low in benign lesions such as chronic cholecystitis or hyperplasia when compared with gallbladder carcinoma ²²,⁴³, suggesting that epithelial hyperplasia with increased cellular proliferative activity plays an important role in carcinogenesis ⁴⁴. But recently, Yanagisawa and associates have suggested the possibility of unknown cofactors causing genomic damage in gallbladder carcinoma, because in their study background mucosa of gallbladder carcinoma showed similar Ki-67 expression to that in chronic cholecystitis ⁴⁵.

The finding that Ki-67 expression is higher in moderately differentiated than in poorly differentiated gallbladder carcinoma ²²,²⁶,⁴⁶ is not corroborated by our data. Nevertheless, the mean percentage expressions in our study are similar to those reported previously ²²,²⁶. In gallbladder carcinoma in situ, the mean Ki-67 expression was low compared with lesions showing more advanced gallbladder wall infiltration. The percentage of positive cases was significantly lower in Tis tumours, a fact that might be related to a lower cellular proliferative activity, but the sample size (three specimens) is too small to allow a definite conclusion.

Shrestha and colleagues ²⁶ found that patients with gallbladder carcinoma that expressed high Ki-67 immunoreactivity had a worse postoperative prognosis than those that did not, but there was no relationship between p53-positive tumours, Ki-67 immunoreactivity and patient survival.

In conclusion, p53 protein nuclear overexpression and Ki-67 protein expression were not found to be related to the degree of histological differentiation, the level of gallbladder wall invasion or patient survival in gallbladder carcinoma.