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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 8  |  Issue : 3  |  Page : 87-92

Study of expression of p53 and Ki-67 in Benign, premalignant, and malignant lesions of the gallbladder


1 Department of Pathology, North Delhi Municipal Corporation Medical College and Hindu Rao Hospital, Delhi, India
2 Division of Cytopathology, ICMR-National Institute of Cancer Prevention and Research, Noida, Uttar Pradesh, India

Date of Submission30-Mar-2020
Date of Decision03-Feb-2021
Date of Acceptance08-Feb-2021
Date of Web Publication01-Sep-2021

Correspondence Address:
Dr. Shakti Kumar Yadav
Department of Pathology, North Delhi Municipal Corporation Medical Collegeand Hindu Rao Hospital, New Delhi - 110 007
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JCRP.JCRP_7_21

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  Abstract 


Background: Gallbladder cancer (GBC) has a distinctly higher incidence in certain demographic groups and areas. Chronic cholecystitis and epithelial changes such as metaplasia/dysplasia are associated with a higher incidence of gallbladder carcinoma. Progression from chronic cholecystitis to metaplasia/dysplasia to carcinoma is multifactorial. Ki-67 and p53 are involved at various stages of the cell cycle, and altered expressions of Ki-67 and p53 have been implicated in the carcinogenesis of various malignant tumors. Materials and Methods: Eighty gallbladder specimens were included in the study. Cases were grouped as chronic cholecystitis (Group 1), metaplasia-dysplasia (Group 2), and GBC (Group 3). P53 and Ki-67 immunoexpressions were determined and results were compared between groups. Results: The p53 expression score was highest in Group 3 (4.35 ± 1.72) and lowest in Group 1 (0.73 ± 0.98). The difference in the mean level of p53 expression was significantly (P = 0.0001) different among the groups. Ki-67 index was highest in Group 3 (47.85 ± 17.46) and lowest in Group 1 (6.50 ± 3.88). The mean Ki-67 index was significantly higher in Group 3 compared to Groups 1 and 2. P53 overexpression and Ki-67 expression were significantly associated with the presence of GBC (P = 0.0001). There was a positive correlation (r2 = 0.37, P = 0.001) between the expressions of p53 and Ki-67. Conclusion: P53 overexpression and Ki-67 index were significantly higher in the patients with GBC compared to those with chronic cholecystitis. This supports the theory of progression from chronic cholecystitis to metaplasia/dysplasia to carcinoma in the gallbladder. The expressions of p53 and Ki-67 in the metaplasia and dysplasia group were between the GBC and chronic cholecystitis groups.

Keywords: Carcinoma, cholecystitis, dysplasia, gallbladder, Ki-67, metaplasia, p53


How to cite this article:
Kumar R, Yadav SK, Singh G, Gupta R, Singh S. Study of expression of p53 and Ki-67 in Benign, premalignant, and malignant lesions of the gallbladder. J Cancer Res Pract 2021;8:87-92

How to cite this URL:
Kumar R, Yadav SK, Singh G, Gupta R, Singh S. Study of expression of p53 and Ki-67 in Benign, premalignant, and malignant lesions of the gallbladder. J Cancer Res Pract [serial online] 2021 [cited 2021 Dec 5];8:87-92. Available from: https://www.ejcrp.org/text.asp?2021/8/3/87/324926




  Introduction Top


Gallbladder cancer (GBC) is a rare tumor with variations in geographic distribution, ethnicity, and gender. It frequently occurs in the northern part of India, especially in regions alongside the Gangetic belt.[1] The high prevalence in this region is due to the presence of heavy metals including nickel, chromium, and cadmium in the water and the presence of high levels of dichlorodiphenyltrichloroethane, an organochlorine pesticide, in the soil.[2] GBC is one of the most lethal malignancies and is more prevalent in females.[3]

GBC has a distinctly higher incidence in certain demographic groups and areas. Women are affected three times more often than men, and the vast majority of patients with GBC are older than 40 years of age. A high incidence has been reported in women in countries such as Chile, Poland, India, Israel, Pakistan, Ecuador, South Korea, and Japan, whereas GBC is considered a rare neoplasm in most Western countries and the United States.[4]

Cancer development is as multistep genetic process, which involves activation of proto-oncogenes and tumor suppressor gene inactivation.[5] P53 is an important tumor suppressor gene which is located on the short arm of chromosome 17. It encodes for a 53-kD nuclear phosphoprotein, which inhibits cellular proliferation.[6],[7] P53 has been described as “the guardian of the genome” due to its ability to prevent genome mutation.[8] If there is a mutation in the p53 gene, the tumor suppression ability is severely diminished. Chemical exposure, radiation, or viruses can also damage the p53 gene, increasing the likelihood of uncontrolled cellular division. Mutations or deletions of the p53 gene are seen in more than 50% of human tumors.[9]

Several studies have shown that the immunohistochemical expression of p53 nuclear protein is well correlated with its level of genetic expression.[10],[11],[12] However, some studies have suggested that this correlation is weak in some tumors and that it does not indicate the underlying genetic mutation.[13] Wistuba et al. used the term “overexpression” to refer to the accumulation of immunohistochemically detectable p53 protein due to increased production.[14]

Cell cycle activity and kinetics are important indicators of the growth and behavior of several human tumors.[15],[16],[17] Ki-67 nucleoprotein is a marker for cellular proliferation. The highest concentrations of Ki-67 are achieved in G2 and M phases, while it is not expressed in the G0 phase or early G1 phase. Immunohistochemical detection of Ki-67 has been accepted as an indicator of cell proliferation.[16]

Metaplastic alterations such as pyloric and intestinal metaplasia are associated with GBC. Metaplasia is observed in the tumor area; however, it is more frequently seen in the area surrounding the tumor. Dysplasia is also present in the area surrounding the tumor, and this may be a stage of progression from metaplasia to cancer.[18],[19] The present study aimed to study the immunoexpressions of p53 and Ki-67 in patients with GBC.


  Materials and Methods Top


Ethical and scientific approval were obtained from the Institutional Ethics Committee of Hindu Rao Hospital, Delhi-07, through No HRH/2014/1 on Jan. 22th, 2014. Written informed consent was obtained from all patients included in the study.

All consecutive patients regardless of gender with chronic cholecystitis or metaplasia or dysplasia or GBC were included in this study (n = 80). After a detailed gross examination of the specimen, representative sections were taken, and the tissue was routinely processed and stained by hematoxylin and eosin. The cases were divided into three groups as: Group 1, chronic cholecystitis; Group 2, metaplasia and/or dysplasia; and Group 3, GBC. For Group 3 cases, tumor size, histological type, grade, and TNM stage were determined. Immunostaining was done using anti-p53 (BioGenex, USA) and anti-Ki-67 (BioGenex, USA) antibodies.

The degree of p53 overexpression was scored using a semiquantitative method evaluating the intensity and incidence of positively stained cells, as described by Wee et al.[20] The intensity was graded as absent (0), mild (1), moderate (2), or intense (3), and the incidence was categorized as absent (0), <10% (1), 10–50% (2), or >50% positive cells (3). All evaluations were carried out by two independent histopathologists, and discordant cases were considered again to achieve consensus. A final staining score for each case was obtained (range 0–6) by adding both variables (intensity and incidence). In this semiquantitative method, a score of ≥3 was considered to be positive for overexpression. In all specimens, only nuclear staining was regarded as specific staining [Figure 1].
Figure 1: Microsections showing p53 expression in the chronic cholecystitis (a: DAB, ×100), metaplasia (b: DAB, ×100), and gallbladder carcinoma groups (c: DAB, ×400)

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The Ki-67 index was calculated as the percentage of positively stained tumor cell nuclei out of the total tumor cells counted. A percentage of >20% stained cells was considered to be positive, regardless of the intensity of staining [Figure 2].
Figure 2: Microsections showing Ki-67 expression in the chronic cholecystitis (a: DAB, ×100), metaplasia (b: DAB, ×40), and gallbladder carcinoma groups (c: DAB, ×40)

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The results were presented as mean ± standard deviation and percentages. The Chi-square test was used to compare categorical/dichotomous variables among groups. ANOVA followed by t-tests was used to compare continuous variables among groups. P < 0.05 was considered to be statistically significant. All analyses were carried out using IBM SPSS version 23.0 (Armonk, USA).


  Results Top


The majority of the cases in Group 2 had pyloric metaplasia (73.3%). Most of the patients in Group 3 had moderately differentiated carcinoma (45%), followed by well-differentiated carcinoma (30%) [Table 1]. Most of the cases in Group 3 were in Stage T2 (45%), followed by T3 (40%) and T1 (15%) [Table 1].
Table 1: Distribution of cases among groups

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Of the total 80 cases, about one-third (32.5%) were >50 years of age. The mean age of the cases was 45.12 (±14.10) years, and the majority were female (87.5%). The age and gender distribution of all cases according to group are described in [Table 2].
Table 2: Age and sex distribution of all the patients

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The mean p53 expression score was highest in Group 3 (4.35 ± 1.72) and lowest in Group 1 (0.73 ± 0.98). The difference in the mean level of p53 expression was significantly (P = 0.0001) different among the groups [Table 3]. P53 overexpression was defined as a score of ≥3, and it was identified in 8 (26.7%) cases in Group 2 and 17 (85%) cases in Group 3, but no cases in Group 1. P53 overexpression was significantly associated with the presence of GBC [P = 0.0001, [Table 4]].
Table 3: P53 Expression score according to the type of lesions

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Table 4: Comparison of overexpression of p53 (overexpression defined as score≥3) among the groups

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The Ki-67 index was highest in Group 3 (47.85 ± 17.46) and lowest in Group 1 (6.50 ± 3.88). The mean Ki-67 index was significantly higher in Group 3 compared to Groups 1 and 2 [both P = 0.0001, [Table 5]]. Almost all of the cases in Group 1 (100%) and Group 2 (96.7%) were negative for Ki-67 expression. However, 19 of the 20 cases (95%) in Group 3 were positive for Ki-67 expression. We found that the Ki-67 expression was significantly associated with GBC [P = 0.0001, [Table 6]]. In addition, there was a positive correlation (r2 = 0.37) between the expressions of p53 and Ki-67 [P = 0.001, [Figure 3]].
Table 5: Ki-67 expression (Ki-67 index) according to the type of lesions

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Table 6: Comparison of expression of Ki-67 among the groups

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Figure 3: Scatter diagram showing the correlation between Ki-67 and p53 expression

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  Discussion Top


In this study, the overexpression of p53 was not found in any of the patients with chronic cholecystitis and in 26.7% of those with metaplasia and dysplasia. The highest level of p53 overexpression was observed in the patients with GBC (85%), and the level was significantly (P = 0.0001) different from the metaplasia and dysplasia group. Doval (2014) and Ghosh et al.[21] reported the overexpression of p53 in 54% and 56.3% of their patients with GBC, respectively. Ghosh et al.[21] also found no overexpression of p53 in the chronic cholecystitis and control groups, similar to their previous study.[22] There was no association between different types of GBC and metaplasia and dysplasia with the overexpression of p53 in this study. In addition, the expression of p53 in the patients with GBC was significantly higher than in those with other gallbladder lesions, which is in agreement with the study by Ghosh et al.[21] Many previously published reports have reported the overexpression of p53, with rates ranging from 20% to 58.7%.[21],[23],[24]

It is well known that the p53 gene can undergo nonsense mutations or methylation changes resulting in null p53 expression. These were not included in this study, and this may lead to a lower degree of p53 expression in comparison to the studies using molecular methods.

Hidalgo et al. reported a slightly higher p53 expression in moderately to poorly differentiated adenocarcinoma. However, in the present study, a similar expression was noted according to the degree of differentiation of the tumor.[25]

In this study, the level of p53 expression score was significantly (P = 0.0001) different among the groups. The t-test showed that there was a significant (P = 0.0001) difference in the level of p53 expression between each group. To the best of our knowledge, no previous study has reported average p53 expressions, so comparison could not be done.

Ki-67 is a nuclear and nucleolar protein expressed during the G1, S, G2, and M phases of the cell cycle while not being present in resting cells (G0 phase). Therefore, it can provide an index of cell growth fraction. Ki-67 is a proliferative marker, and in rapidly dividing cells (such as in carcinoma), it shows higher activity than in normally dividing cells. In the present study, Ki-67 expression was higher in moderately differentiated compared to poorly differentiated GBC.[26],[27]

A previous study by Hui et al.[28] concluded that a high Ki-67 expression was correlated with lymphovascular invasion and that it could predict early recurrence after surgery.

In the present study, the expression of Ki-67 was not found in any of the cases with chronic cholecystitis. In addition, the expression of Ki-67 was higher in the patients with GBC (95%) than in those with metaplasia and dysplasia (3.3%). Xuan et al.[29] reported an increased expression of Ki-67 in gallbladder carcinomas, and Stancu et al.[30] reported that the expression of Ki-67 was present in all cases with chronic cholecystitis and carcinoma.

In this study, the level of Ki-67 expression was significantly (P = 0.0001) different among the groups. Post hoc analysis showed that there was a significant (P = 0.0001) difference in the level of Ki-67 expression between the patients with chronic cholecystitis (6.50 ± 3.88) and those with GBC (47.85 ± 17.46) as well as those with metaplasia and dysplasia (8.10 ± 7.54) and those with GBC. Parul et al. reported an average level of Ki-67 expression of 28.20 ± 7.83% in the neoplastic group.[31]


  Conclusion Top


The overexpression of p53 and expression of Ki-67 were significantly higher in the patients with GBC compared to those with chronic cholecystitis. The expressions of p53 and Ki-67 in the metaplasia and dysplasia group were between the GBC and chronic cholecystitis groups. This supports the theory of progression from chronic cholecystitis to metaplasia/dysplasia to carcinoma in the gallbladder.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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