|Year : 2021 | Volume
| Issue : 3 | Page : 87-92
Study of expression of p53 and Ki-67 in Benign, premalignant, and malignant lesions of the gallbladder
Ranjeet Kumar1, Shakti Kumar Yadav1, Garima Singh1, Ruchika Gupta2, Sompal Singh1
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 Submission||30-Mar-2020|
|Date of Decision||03-Feb-2021|
|Date of Acceptance||08-Feb-2021|
|Date of Web Publication||01-Sep-2021|
Dr. Shakti Kumar Yadav
Department of Pathology, North Delhi Municipal Corporation Medical Collegeand Hindu Rao Hospital, New Delhi - 110 007
Source of Support: None, Conflict of Interest: None
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 Oct 15];8:87-92. Available from: https://www.ejcrp.org/text.asp?2021/8/3/87/324926
| Introduction|| |
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. 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. GBC is one of the most lethal malignancies and is more prevalent in females.
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.
Cancer development is as multistep genetic process, which involves activation of proto-oncogenes and tumor suppressor gene inactivation. 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., P53 has been described as “the guardian of the genome” due to its ability to prevent genome mutation. 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.
Several studies have shown that the immunohistochemical expression of p53 nuclear protein is well correlated with its level of genetic expression.,, However, some studies have suggested that this correlation is weak in some tumors and that it does not indicate the underlying genetic mutation. Wistuba et al. used the term “overexpression” to refer to the accumulation of immunohistochemically detectable p53 protein due to increased production.
Cell cycle activity and kinetics are important indicators of the growth and behavior of several human tumors.,, 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.
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., The present study aimed to study the immunoexpressions of p53 and Ki-67 in patients with GBC.
| Materials and Methods|| |
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. 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|| |
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].
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].
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 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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|Figure 3: Scatter diagram showing the correlation between Ki-67 and p53 expression|
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| Discussion|| |
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. reported the overexpression of p53 in 54% and 56.3% of their patients with GBC, respectively. Ghosh et al. also found no overexpression of p53 in the chronic cholecystitis and control groups, similar to their previous study. 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. Many previously published reports have reported the overexpression of p53, with rates ranging from 20% to 58.7%.,,
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.
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.,
A previous study by Hui et al. 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. reported an increased expression of Ki-67 in gallbladder carcinomas, and Stancu et al. 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.
| Conclusion|| |
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.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Pandey M, Shukla VK. Diet and gallbladder cancer: A case-control study. Eur J Cancer Prev 2002;11:365-8.
Unisa S, Jagannath P, Dhir V, Khandelwal C, Sarangi L, Roy TK. Population-based study to estimate prevalence and determine risk factors of gallbladder diseases in the rural Gangetic basin of North India. HPB (Oxford) 2011;13:117-25.
Katoch V. Three-Year Report of Population Based Cancer Registries 2012–2014. Bengaluru, India: National Centre for Disease Informatics and Research – National Cancer Registry Programme; 2016.
Kai K, Aishima S, Miyazaki K. Gallbladder cancer: Clinical and pathological approach. World J Clin Cases 2014;2:515-21.
Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759-67.
Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations in the p53 tumor suppressor gene: Clues to cancer etiology and molecular pathogenesis. Cancer Res 1994;54:4855-78.
Vogelstein B, Kinzler KW. p53 function and dysfunction. Cell 1992;70:523-6.
Diamantis I, Karamitopoulou E, Perentes E, Zimmermann A. p53 protein immunoreactivity in extrahepatic bile duct and gallbladder cancer: Correlation with tumor grade and survival. Hepatology 1995;22:774-9.
Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991;253:49-53.
Bártková J, Bártek J, Lukás J, Vojtĕsek B, Stasková Z, Rejthar A, et al
. p53 protein alterations in human testicular cancer including pre-invasive intratubular germ-cell neoplasia. Int J Cancer 1991;49:196-202.
Cunningham J, Lust JA, Schaid DJ, Bren GD, Carpenter HA, Rizza E, et al
. Expression of p53 and 17p allelic loss in colorectal carcinoma. Cancer Res 1992;52:1974-80.
Iggo R, Gatter K, Bartek J, Lane D, Harris AL. Increased expression of mutant forms of p53 oncogene in primary lung cancer. Lancet 1990;335:675-9.
Sheu LF, Chen A, Tseng HH, Leu FJ, Lin JK, Ho KC, et al
. Assessment of p53 expression in nasopharyngeal carcinoma. Hum Pathol 1995;26:380-6.
Wistuba II, Gazdar AF, Roa I, Albores-Saavedra J. p53 protein overexpression in gallbladder carcinoma and its precursor lesions: An immunohistochemical study. Hum Pathol 1996;27:360-5.
Garcia RL, Coltrera MD, Gown AM. Analysis of proliferative grade using anti-PCNA/cyclin monoclonal antibodies in fixed, embedded tissues. Comparison with flow cytometric analysis. Am J Pathol 1989;134:733-9.
Hall PA, Levison DA. Review: assessment of cell proliferation in histological material. J Clin Pathol 1990;43:184-92.
Meyer JS. Cell kinetic measurements of human tumors. Hum Pathol 1982;13:874-7.
Kijima H, Watanabe H, Iwafuchi M, Ishihara N. Histogenesis of gallbladder carcinoma from investigation of early carcinoma and microcarcinoma. Acta Pathol Jpn 1989;39:235-44.
Roa I, de Aretxabala X, Araya JC, Roa J. Preneoplastic lesions in gallbladder cancer. J Surg Oncol 2006;93:615-23.
Wee A, Teh M, Raju GC. Clinical importance of p53 protein in gall bladder carcinoma and its precursor lesions. J Clin Pathol 1994;47:453-6.
Ghosh M, Sakhuja P, Singh S, Agarwal AK. p53 and beta-catenin expression in gallbladder tissues and correlation with tumor progression in gallbladder cancer. Saudi J Gastroenterol 2013;19:34-9.
] [Full text]
Ghosh M, Sakhuja P, Agarwal AK. Xanthogranulomatous cholecystitis: A premalignant condition? Hepatobiliary Pancreat Dis Int 2011;10:179-84.
Chaube A, Tewari M, Garbyal RS, Singh U, Shukla HS. Preliminary study of p53 and c-erbB-2 expression in gallbladder cancer in Indian patients manuscript id: 8962091628764582. BMC Cancer 2006;6:126.
Jarnagin WR, Klimstra DS, Hezel M, Gonen M, Fong Y, Roggin K, et al
. Differential cell cycle-regulatory protein expression in biliary tract adenocarcinoma: Correlation with anatomic site, pathologic variables, and clinical outcome. J Clin Oncol 2006;24:1152-60.
Hidalgo Grau LA, Badia JM, Salvador CA, Monsó TS, Canaleta JF, Nogués JM, et al
. Gallbladder carcinoma: The role of p53 protein overexpression and Ki-67 antigen expression as prognostic markers. HPB (Oxford) 2004;6:174-80.
Lee CS. Differences in cell proliferation and prognostic significance of proliferating cell nuclear antigen and Ki-67 antigen immunoreactivity in in situ
and invasive carcinomas of the extrahepatic biliary tract. Cancer 1996;78:1881-7.
Shrestha ML, Miyake H, Kikutsuji T, Tashiro S. Prognostic significance of Ki-67 and p53 antigen expression in carcinomas of bile duct and gallbladder. J Med Invest 1998;45:95-102.
Hui AM, Shi YZ, Li X, Sun L, Guido T, Takayama T, et al
. Proliferative marker Ki-67 in gallbladder carcinomas: High expression level predicts early recurrence after surgical resection. Cancer Lett 2002;176:191-8.
Xuan YH, Choi YL, Shin YK, Kook MC, Chae SW, Park SM, et al
. An immunohistochemical study of the expression of cell-cycle-regulated proteins p53, cyclin D1, RB, p27, Ki67 and MSH2 in gallbladder carcinoma and its precursor lesions. Histol Histopathol 2005;20:59-66.
Stancu M, Căruntu ID, Sajin M, Giuşcă S, Bădescu A, Dobrescu G. Immunohistochemical markers in the study of gallbladder premalignant lesions and cancer. Rev Med Chir Soc Med Nat Iasi 2007;111:734-43.
Parul G, Nirupma L, Aqwam S, Nahid S, Osman M. Assessment of Morphometric analysis, AgNOR Score & IHC expression of Ki67 in Gallbladder carcinoma. Int J Adv Res 2016;4:312-26.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]