Expression and clinicopathological correlation of Ki-67 in gallbladder carcinoma

Amit Gupta¹, Sweety Gupta², Deepak Rajput¹, Prashant Durgapal³, Jaine John Chennatt¹, Sanjeev Kishore³, Shalinee Rao³, Puneet Dhar⁴, Manoj Gupta², Ravi Kant^5
1 Department of Surgery, All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India
² Department of Radiation Oncology, All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India
³ Department of Pathology, All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India
⁴ Department of Surgical Gastroenterology, All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India
⁵ All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India

Correspondence Address:
Amit Gupta
Department of Surgery, All India Institute Of Medical Sciences, Rishikesh, 249 203, Uttarakhand
India.

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcar.JCar_9_21

Abstract
INTRODUCTION: Gallbladder cancer is an aggressive cancer with short median survival from the time of diagnosis. Improved understanding of the pathological molecular mechanisms of gallbladder carcinogenesis is important to refine the diagnosis, prognosis, and also to develop novel targeted therapies for patients with advanced Gallbladder cancer (GBC) malignancy. Ki-67 is a marker of cell proliferation and its detection by immunohistochemistry is considered to be an effective method for the detection of prognosis in several tumors. In the present study, we have analyzed expression of immunohistochemical marker Ki-67 in gallbladder carcinoma and its correlation with clinicopathological and radiological parameters.
MATERIALS AND METHODS: This prospective observational study was conducted from December 2017 to July 2020. The patients of newly diagnosed gallbladder cancer were enrolled as per the inclusion and exclusion criteria defined in the study protocol. Contrast-enhanced computer tomography of the chest and abdomen and serum tumor markers such as carbohydrate antigen (CA)-19.9, carcinoembryonic antigen, and CA 125 were done. Immunohistochemical expression of Ki-67 was evaluated on biopsy tissue from the gallbladder mass.
RESULTS: Fifty newly diagnosed patients of carcinoma gallbladder were included in the present study. The correlation was studied between clinicodemographic parameters and Ki-67, but no association was found with age, gender, and symptoms. There was a weak positive correlation between Ki-67 and direct bilirubin, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, and alkaline phosphatase (P = 0.094; 0.126; 0.542; and 0.328, respectively). There was a weak positive correlation between body mass index (Kg/m²) and Ki-67, but this correlation was not statistically significant (P = 0.304).
CONCLUSIONS: Ki-67 is a marker of proliferation and it correlated with histological differentiation, jaundice and liver function tests, presence of stones, and location of metastases but did not correlate with stage and extent of disease.

Keywords: Expression, gallbladder carcinoma, immunohistochemistry, Ki-67.

Introduction
The most common malignancy of the biliary system is gallbladder cancer, and it ranks sixth among gastrointestinal cancers. It is a notoriously lethal malignancy with marked variation in the ethnic group and geographical distributions.^[1] In the biliary system, gallbladder cancer is the most aggressive cancer with the shortest median survival from the time of diagnosis.^[2] Gallbladder cancer exhibits striking variability in the global rates, reaching epidemic levels for some regions and ethnicities. The basis of its variability resides in differences in environmental exposure and intrinsic genetic predisposition to carcinogenesis.^[3] The exact etiology is unknown, but the association of gallstones with an increased risk for GBC has been reported. Risk factors for the development of GBC also include infection and the presence of an anomalous pancreaticobiliary ductal junction. Histopathologically, more than 90% of cases of adenocarcinoma and the remaining are squamous cell carcinoma and others. Less than 10% of tumors are resectable at the time of diagnosis and the role of systemic chemotherapy is not clear. Treatment of GBC remains a therapeutic challenge despite the available treatment modalities. Therefore, an improved understanding of the pathological molecular mechanisms of gallbladder carcinogenesis is important to refining the diagnosis, prognosis, and also to develop novel targeted therapies for patients with advanced GBC malignancy.^[4] Ki-67 is a marker of cell proliferation first described in 1983. The name was derived from Kiel, Germany, and the number 67 came from the clone number in the 96-well plate. The gene encoding Ki-67 is located on chromosome 10q25-ter and is comprised 15 exons with sizes ranging from 67 to 6845 bp and 14 introns with sizes ranging from 87 to 3569 bp.^[5] It is a labile, nonhistone nuclear protein expressed in active phases G1, S, G2, and M phases of cell cycle, then rapidly catabolized at end of M phase so the level decreases and is not detectable in G0 and early G1 phases.^[6] Ki-67 is associated with proliferation of malignant tumor cells and so is considered a a marker for aggressiveness.^[7] The prognostic value of pKi-67 has been investigated as a reliable marker in cancers of the breast, soft tissue, lung, prostate, cervix, and central nervous system in certain studies.^{[8],[9],[10],[11]} Ki-67 detection by immunohistochemistry is considered to be an effective method for the detection of prognosis in several tumors.^[12] Expression of Ki-67 is evaluated by immunostaining and this is considered the gold standard, and cutoff levels between 10% and 14% positively-stained cells are defined as high risk in terms of prognosis. Ki-67 has been studied in GBC and biliary tract cancer and a significant association has been identified with histological grade.^[13],[14] In the present study, we have analyzed the expression of immunohistochemical marker Ki-67 in gallbladder carcinoma and its correlation with clinicopathological parameters.

Materials and Methods
This prospective observational study was conducted from December 2017 to July 2020. The patients of newly diagnosed gallbladder cancer presenting to the hepatopancreatobiliary clinic of the department of surgery and oncology outpatient department (OPD) were enrolled as per the inclusion and exclusion criteria defined in the protocol. Institutional ethical committee clearance was obtained and informed consent was obtained from all the patients.

Inclusion criteria

• Aged ≥18 years
• Recently diagnosed and biopsy-proven patients of gallbladder carcinoma.

Exclusion criteria

• Presence of synchronous/metachronous second malignancy.

A complete history and physical examination of patients of diagnosed GBC coming to the department of surgery and oncology OPD was documented. Contrast-enhanced computer tomography of the chest and abdomen and serum tumor markers such as carbohydrate antigen (CA)-19.9, carcinoembryonic antigen (CEA), and CA 125 were done for all the patients. After diagnostic workup, staging was done on the basis of American Joint Committee on Cancer 8^th edition. Surgically resectable patients underwent radical cholecystectomy and surgically unresectable or metastatic disease patients were considered for ultrasound-guided biopsy from gallbladder mass lesions and then referred for chemotherapy.

Immunohistochemistry procedure

Immunohistochemistry was performed using commercially available antibodies for Ki-67 (MIB1) (Ki-67 [Clone 1A6] Mouse monoclonal Antibody).

Immunostaining methods

Serial 4 μ thick sections were cut from the selected representative paraffin-embedded tissue blocks and overlaid on poly-L-lysine-coated slides which were used for immunohistochemistry. These were deparaffinized (2 changes of xylene for 5 min each and 1 change of acetone for 1 min) followed by rehydration in decreasing concentration of alcohol (95% ethanol for 3 min, 70% ethanol for 3 min, distilled water for 1 min).

Antigen retrieval

Antigen retrieval was done by heating the sections immersed in citrate buffer inside a 600-watt microwave oven at full power for 30 min.

Steps for immunostaining

To diminish the nonspecific immunostaining (endogenous peroxidase activity), each slide was treated with methanol containing 4% hydrogen peroxide for 30 min. After brief rinsing, the sections were placed in 0.05M Tris-HCL buffer at a pH of 7.4 for 10 min. Excess buffer was then tapped off, followed by careful wiping around the specimen. Sections were then overlaid with an adequate amount of appropriately diluted primary antibody, followed by overnight incubation at 4°C in a humid chamber.

Slides were rinsed with 3 changes of Tris-HCL buffer for 5 min each. Sections were then covered with substrate chromogen solution freshly prepared by dissolving 50 μl of diaminobenzidine (DAB) chromogen to 1 ml of DAB substrate buffer. The slides were incubated at room temperature for few minutes under microscopic control till the optimum development of brown-colored peroxidase reactant product. After rinsing in distilled water, the sections were counterstained with hematoxylin for 5 s, the color is controlled by microscopic evaluation, followed by mounting with DPX as mounting media.

The MIB-1 index (Ki-67 labeling index [LI]) was calculated as the percentage of positively stained tumor cell nuclei out of the total tumor cells counted (n = 1000). A percentage >20% of stained cells was considered positive.

The positive marker analyzed was correlated with the clinicopathological staging. Statistical analysis was performed using IBM SPSS version 25.0 statistical software (SPSS Inc., Chicago, IL, United States).

Parametric data were calculated by mean and standard deviation (SD) and nonparametric data by median and interquartile range (IQR). Fisher’s test was done for categorical variables and P < 0.05 was considered significant. Nonparametric tests (Wilcoxon-Mann–Whitney U test) were used to make group comparisons.

Results
Fifty newly diagnosed patients of carcinoma gallbladder were included in the present study. Age ranged from 29 to 69 years (mean 53.42). There were 31 females and 19 males, a ratio of 1.6:1. Blood Group A + was the most common in 50% of patients. The body mass index (BMI) ranged from 17.3 to 34.93, mean 22.37 ± 3.97. Six percent of the participants had Stage I, 4% of the participants had Stage IIA, 2% had Stage IIB. 24.4% Stage had IIIA, 9.6% had Stage IIIB, 24.4% had Stage IVA, and 29.6% had Stage IVB. Six out of fifty patients were resectable and underwent radical cholecystectomy. The histopathology for all resectable specimens was adenocarcinoma. Ki-67 expression the total bilirubin (mg/dL) ranged from 0.2 to 31.4 (4.65 ± 8.13). Red cell distribution width (RDW) ranged from 11.23 to 19.3 (14.88 ± 1.98). Biochemical profile of GBC patients is mentioned in [Table 1]. Ki-67 immunopositivity ranged from 15 to 95 [Figure 1]. The correlation was studied between clinicodemographic parameters and Ki-67, but no association was found with age, gender, blood group, jaundice, pain, and fever. [Table 2] shows the association between clinicodemographic profile and Ki-67. [Figure 2] scatterplot depicts the correlation between BMI (Kg/m²) and Ki-67. Nonparametric tests (Spearman correlation) were used to explore the correlation between the two variables. There was a weak positive correlation between BMI (Kg/m²) and Ki-67, but this correlation was not statistically significant (rho = 0.24, P = 0.304).

Figure 1: Immunohistochemistry Ki67 staining in GBC Click here to view

Figure 2: Correlation between body mass index (Kg/m2) and Ki 67 Click here to view

Table 1: Biochemical profile of gallbladder cancer patients Click here to view

Table 2: Association between Ki 67 and clinicodemographic parameters Click here to view

Nonparametric tests (Spearman correlation) were used to explore the correlation between the two liver function tests and Ki-67 variables. There was a moderate positive correlation between total bilirubin (mg/dL) and Ki-67, and this correlation was statistically significant (rho = 0.36, P = 0.023). For every 1 unit increase in total bilirubin (mg/dL), the Ki-67 increased by 0.81 units. There was a weak positive correlation between Ki-67 and direct bilirubin, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, and alkaline phosphatase (P = 0.094; 0.126; 0.542; and 0.328, respectively). There was a weak positive correlation between RDW (%) and Ki-67, and this correlation was not statistically significant (rho = 0.28, P = 0.199) [Figure 3]. Correlation of Ki-67 expression was done between resectable (74.17 ± 20.60) and unresectable (50.92 ± 21.49) gallbladder cancer patients, but it was statistically insignificant (P = 0.124). A correlation was also done between Ki-67 and pathological and radiological parameters. It was high in patients with associated stones 57.86 ± 23.54 compared to those who did not have stones 46.79 ± 17.28. The values of Ki-67 increased with grades and higher being for poorly differentiated tumors. There was no statistically significant difference between the groups in terms of Ki-67 (χ^[2] = 3.687, P = 0.158). The median (IQR) of Ki-67 in the involved lymph node was 70 (60–80). The median (IQR) of Ki-67 in the no lymph node involvement was 55 (45–80) [Table 3].

Figure 3: Scatter plot showing a correlation between red cell distribution width (%) and Ki 67 Click here to view

Table 3: Association between Ki 67 and radiological and pathological parameters Click here to view

Discussion
Gallbladder cancer is the most common biliary tract malignancy. It is the sixth most common malignancy in the digestive system. It has an unremarkably higher frequency in certain ethnic groups and geographic regions. Notwithstanding the tremendous enhancement in diagnosis and surgical skills, gallbladder cancer prognosis is still very poor. The 5-year survival for all stages of this malignancy is approximately 5%.

Ki-67 is considered a marker of proliferation.^[15] It is a nuclear protein present on chromosome 10. Ki-67 expression is firmly connected with cell proliferation. It is communicated during the cell cycle’s busy period, such as G-1, S, G-2, and mitosis. However, it lacks the resting phase of the cell cycle (G0).^[16] Ki-67 scoring is done by calculating the percentage of tumor cells with nuclear staining. Proliferating cells’ growth fraction correlates with the activity of Ki-67.^[17] Ki LI is used for scoring, and >20% is considered positive. One patient had Ki LI <20% out of 50 cases.

The present study has shown a weak negative correlation between age and Ki-67, and this correlation was not statistically significant (P = 0.308). For the patient below the age group of below 40 years, the mean of Ki-67 expression was 65 compared to above 40 years, 52 females have a mean expression of 57.7 compared to males 48.2, yet this distinction was not huge (P = 0.095). Like the Doval et al. study, which has shown Ki-67 expression was higher in patients of age group <40 years and patients with poorly differentiated tumor,^[18] the present study has shown that Ki-67 has higher expression <40 years of age and more in females, but the results were not statically significant (P = 0.308). Ki-67 did not provide any correlation with the biomarker expression CEA, CA-125. Interestingly in this study, there was a moderate negative correlation between CA-19.9 (IU/L) and Ki-67, and this relationship was significant (P = 0.023). For each 1-unit increment in Ki-67, CA-19.9 (IU/L) decreased by 35.18. Hui et al. found that a higher Ki-67 index was significantly correlated with tumor lymphatic invasion.^[19] The mean (SD) of Ki-67 in the lymph nodes of involved group was 70.00 (28.28) and not involved group was 61.33 (21.25) with (P = 0.764). In the present study, the mean (SD) of Ki-67 in the N Stage of N2 group was 63.33 (37.86), which was higher than N0 group 55.71 (20.27). Nevertheless, there was no statistically significant difference (P = 0.575). There was no difference between grades of differentiation and gallbladder wall infiltration similar to Grau et al.’s study (2004), also no such association with histological grading (P = 0.125). The present study has shown gallbladder cancer patients with high Ki-67 expression and cholelithiasis (P = 0.035). We need further studies with control as a gallstone disease. Toledo et al. and Grau et al. observed that the mean Ki-67 expression was low in early cancer than in the advanced stage, which contradicts our findings.^[20] The present study has shown higher expression in early cancer (P = 0.024). The mean (SD) of Ki-67 in the Stage I/II and Stage III/IV was 74.17 and 51.36, respectively. Dabbs^[21] reported that Ki-67 immunopositivity varied with grade of dysplasia and was higher for higher grades.

Conclusions
Ki-67 is a marker of proliferation and it correlated with histological differentiation, jaundice and liver function tests, presence of stones, and location of metastases but did not correlate with stage and extent of disease.

Acknowledgment

None.

Financial support and sponsorship

This study was financially supported by the Uttarakhand Council of Science and Technology (UCOST), Dehradun, India, for providing funding to conduct this study in the form of an extramural research grant.

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]