| Abstract|| |
Background: Clinical significance of survivin (antiapoptosis protein) in gallbladder cancer is not yet established. Aims: This study was performed to assess the expression pattern of survivin in benign and malignant gallbladder lesions using immunohistochemistry (IHC), and to assess its clinicopathological significance. Settings and Design: Prospective study from July 2012 to July 2014 was performed as a part of intramural research project. Materials and Methods: Tissue samples from resected gallbladder for cholelithiasis (n = 27) and carcinoma gallbladder (n= 24) were evaluated for survivin expression by IHC using a scoring system. Their expression was correlated with different clinicopathological parameters. Statistical Analysis: Fisher's exact test, Student's t-test, and Chi-square test were used as appropriate for data analysis. Kaplan–Meier methods were used to calculate overall and disease-free survival rates among different groups. Two-sided P< 0.05 was considered as significant. Results: Benign group (19 females, age [mean ± standard deviation [SD] 45 ± 14 years) and malignant group (20 females, age [mean ± SD] 48.9 ± 13.4 years) were comparable with respect to menopausal status, presence, size and types of stones. However, survivin expression was significantly higher (66.7%, 95% confidence interval [CI] 24–75) in gallbladder cancer than in cholelithiasis group (33%, CI 46–83), P= 0.025). Its expression did not correlate with gender, age, menopausal status, presence of gallstones or their size, number and type, tumor differentiation, and tumor stage. Conclusions: Significantly higher expression of survivin protein in gallbladder cancer as compared to cholelithiasis group suggests its role in gallbladder carcinogenesis though it may not have prognostic value.
Keywords: Apoptosis, biliary tract cancer, cholelithiasis
|How to cite this article:|
Gupta V, Goel MM, Chandra A, Gupta P, Kumar S, Nigam J. Expression and clinicopathological significance of antiapoptotis protein survivin in gallbladder cancer. Indian J Pathol Microbiol 2016;59:143-7
|How to cite this URL:|
Gupta V, Goel MM, Chandra A, Gupta P, Kumar S, Nigam J. Expression and clinicopathological significance of antiapoptotis protein survivin in gallbladder cancer. Indian J Pathol Microbiol [serial online] 2016 [cited 2019 Dec 11];59:143-7. Available from: http://www.ijpmonline.org/text.asp?2016/59/2/143/182035
| Introduction|| |
Incidence of gallbladder cancer is very high in North India, and it remains the most common digestive tract cancer in females. Chronic gallbladder inflammation, mostly due to cholelithiasis, is thought to be the most important underlying causative factor for gallbladder carcinogenesis. At molecular level, however, actual genetic mechanisms involved in gallbladder carcinogenesis are still being explored.
Apoptosis that is important for organ homeostasis has been thought to play a role in cancer onset and progression and may also affect treatment outcome (e.g., decreased tumor cell sensitivity to chemotherapy). In this context, the study of the molecules possibly involved in the modulation of apoptosis seems to be particularly attractive. Survivin, a newly described protein, belongs to a family of proteins known as the inhibitor of apoptosis proteins (IAPs). These IAPs are crucial regulator of the molecular mechanisms involved in apoptosis. Survivin is expressed almost exclusively during development, in proliferating cells and in variety of cancers, but is not expressed in terminally differentiated, nonproliferating tissues. Overexpression of survivin has oncogenic potential and has prognostic significance also. Its high expression has been associated with more aggressive tumor and poor prognosis in certain cancers such as neuroblastoma, oral squamous cell carcinoma, colorectal cancer, and breast cancer. Although expression of survivin has been assessed by immunohistochemistry (IHC) in many gastrointestinal cancers such as esophageal, pancreatic, hepatocellular carcinoma, and colorectal cancers, its expression by IHC has not been reported in gallbladder cancer in English literature so far.
The aim of this study was to assess the expression of survivin protein in benign and malignant gallbladder lesions (chronic cholecystitis and gallbladder cancer) by IHC and correlate its expression with different clinicopathological parameters.
| Materials and Methods|| |
This prospective study was conducted from July 2012 to July 2014. The work was initiated after obtaining ethical clearance from Institutional Ethical Committee of the university. Patients diagnosed as cases of symptomatic gallbladder stone disease based on clinical evaluation and confirmed by abdominal ultrasound examination were recruited for the collection of the benign gallbladder tissues. Patients suspected of having gallbladder cancer based on clinical evaluation and imaging (ultrasonography, computed tomography) were recruited for the collection of the malignant gallbladder tissues.
Benign gallbladder tissue samples were collected from patients who underwent simple laparoscopic or open cholecystectomy for symptomatic gallstone disease. Malignant gallbladder tissues were collected from patients who underwent exploration for primary gallbladder cancer followed by either radical cholecystectomy or biopsy of metastatic lesions found during exploration (open exploration or staging laparoscopy). All tissue samples were collected in 10% buffered formalin and processed for routine histopathological examination. Five micrometers thick sections from formalin fixed paraffin embedded (FFPE) blocks were cut and stained with hematoxylin and eosin for histopathological diagnosis. After histological confirmation of the diagnosis, (i.e. chronic cholecystitis and primary adenocarcinoma of the gallbladder) presence of metaplasia (in chronic cholecystitis), tumor grade, depth of tumor infiltration, and nodal involvement were assessed in gallbladder carcinoma group, and final tumor-node-metastasis staging was done as per American Joint Committee on Cancer 2010.
For IHC staining, 5 µm thick sections from FFPE tissues were taken on silanized slides. Heat-induced antigen retrieval was done in EZ Antigen Retriever System (BioGenex, USA) in citrate buffer pH 6.0 followed by immunostaining using primary monoclonal antibody survivin, Clone 12 C4 (DAKO, Denmark), and secondary antibody Novolink Min Polymer Novocastra (Leica Biosystems, New Castle, USA). Expression of survivin protein in the cells was observed both in the nucleus and cytoplasm [Figure 1].
|Figure 1: Chronic cholecystitis (a) H and E, ×10, (b) Positive staining for Survivin protein on immunohistochemistry (×20) Carcinoma gallbladder (c) H and E, ×10 (d) Positive staining for Survivin protein on immunohistochemistry (×10)|
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Scoring of immunohistochemistry
The level of expression was assessed by semi-quantitative scoring which included the overall percentage area of the lesion stained positive (0–100%) and the staining intensity (0–3). Grading for percentage area positivity was done as follows: ≤10% = 0, 11–25% = 1, 26–50% = 2, 51–75% = 3, 76–100% = 4. The intensity grading was done as: 0 = none, 1 = weak, 2 = moderate, 3 = strong staining. The percentage area positivity score (0–4) was multiplied by the intensity score (0–3) and a final score was assigned: 0–4 as negative staining, 5–12 as positive staining. Survivin expression was compared between the benign and malignant groups and was correlated with different clinical and pathological parameters.
Statistical analysis was performed usingSPSS version 12 software (SPSS IBM SPSS Statistics, IBM Corporation, Armonk, NY). Continuous variables were expressed as means (±standard deviation, range). Fisher's exact test, Student's t-test, and Chi-square test were used as appropriate for data analysis. Graph Pad Prism 6 software (GraphPad Software, Inc, USA) was used for survival analysis. Kaplan–Meier methods were used to calculate overall and disease-free survival rates among different groups. These groups were then compared using log-rank methods. Two-sided P < 0.05 was considered as significant.
| Results|| |
A total of 60 patients (30 in each group) were recruited. On immunostaining, 51 patients (27 benign group, 24 malignant group) were found suitable for IHC analysis and scoring. Thus, overall results are based on these 51 patients.
There were 27 patients in this group with 70.4% females. All patients presented with pain (duration 355 ± 371 days), and 57.8% females were premenopausal [Table 1]. All patients had histologically proven chronic cholecystitis with hyperplasia present in 3 (11%) patients and metaplasia present in 1 patient (3.7%, gastric antral metaplasia). Most of the patients (20, 74%) had multiple stones in the gallbladder having a mean size of 10.93 ± 4.61 mm with mixed as predominant stone type (66.6%).
|Table 1: Clinicopathological profile of patients with benign and malignant gallbladder lesions|
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There were 24 patients in this group with 83.3% females. All patients presented with pain (duration 163 ± 379 days) and 40% females were premenopausal. All patients had histologically proven primary adenocarcinoma of the gallbladder. Associated cholelithiasis was present in 54.1% patients. Most of the patients (8/13, 61.5%) had multiple stones, and predominant stone type was mixed (92.3%).
Survivin expression on immunohistochemistry
Overall, on IHC, positive survivin protein expression was observed in 49% (25/51) cases. Significantly higher expression was noted in gallbladder cancer than in chronic cholecystitis (66.7%, 16/24 vs. 33.3%, 9/27, P = 0.025). Nuclear expression alone was seen in 9; cytoplasmic expression alone in 6, and 10 patients had both cytoplasmic and nuclear expression of survivin protein.
Survivin expression and clinicopathological correlation
Overall, survivin expression was significantly higher in the malignant group as compared to the benign group. However, its expression did not correlate with age, sex, menopausal status, tumor grading, tumor stage, and presence of hyperplasia in chronic cholecystitis (P = 1.00), presence of cholelithiasis, and stone size or type ([Table 2]). In addition, no significant difference in survivin expression was noted with respect to nodal status or distant metastases.
|Table 2: Correlation of survivin expression with different clinicopathological parameters|
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Similarly, its expression did not correlate with poor overall and disease-free survival (positive expression vs. negative expression: Median disease-free survival 14 vs. 12.5 months, P = 0.671, hazard ratio 1.40, 95% confidence interval of ratio 0.28–7.44).
| Discussion|| |
Evasion of apoptosis and subsequent malignant transformation may result from disrupted balance between proapoptotic and antiapoptotic factors, impaired death receptor signaling, or reduced caspase function. Increased expression of IAP is one of the mechanisms of dysregulated balance between proapoptotic and antiapoptotic factors. Survivin (BIRC5) is one of eight IAPs. This protein has been investigated in various cancers and found to be overexpressed in pancreatic adenocarcinoma,, esophageal squamous cell carcinoma, colorectal cancer, hepatocellular carcinoma, and cholangiocarcinoma  as well as in nondigestive cancers, for example, breast cancer, squamous cell carcinoma of the tongue, nonsmall cell lung cancer, and urinary bladder cancer. Though many digestive tract cancers have been investigated for survivin expression, its expression in gallbladder cancer has only been reported recently.,, However, in these studies, survivin mRNA has been assessed in cancer tissues and in blood, and authors have found significantly higher expression of survivin mRNA in gallbladder cancer (2.9-fold) and cholelithiasis (1.85-fold) as compared with control.
In this study, survivin expression was assessed with IHC. To the best of our knowledge, this is the first study that has evaluated survivin expression in gallbladder cancer with IHC. We have observed survivin overexpression in 66% of gallbladder adenocarcinoma (predominantly nuclear, 43.5%) as compared to 33% cases of chronic cholecystitis (predominantly both nuclear and cytoplasmic, 66.7%). On the other hand, a varied range of survivin overexpression has been observed in various digestive tract cancers: 54% (cytoplasmic) in extrahepatic cholangiocarcinoma, 53–61% of colorectal carcinomas,, 50–88% in gastric cancer,, 80% (nuclear) in esophageal squamous cell carcinoma, and 88–94% (cytoplasmic) in pancreatic adenocarcinoma., Intrinsic differences in tumor biology and higher affinity of the polyclonal antibody as compared to monoclonal antibody may explain such wide variation in the prevalence of survivin expression in different malignancies. In this study, monoclonal antibody was used and may also explain the lower rate of expression (66% in gallbladder cancer).
Survivin gene generally remains quiescent in terminally differentiated tissues with limited expression. Its expression in normal digestive tract is seen in the mucosal surface epithelial cells of the stomach where it may a role in maintaining gastric mucosal integrity and regulation of cell cycle mucosa. The presence of stones may stimulate epithelial proliferation in gallbladder as suggested by increased mitotic rate. In the presence of inflammation, such proliferation may be more intense, and survivin protein is expected to express in such proliferating epithelium. In this study, however, only one-third cases with chronic calculous cholecystitis showed overexpression of survivin. However, epithelial hyperplasia was noted in only three cases with no significant correlation survivin expression with epithelial hyperplasia (P = 1.00).
The clinical significance of survivin expression in different malignancies has been unfolded in recent time. However, such clinical significance in gallbladder cancer is still unknown given very limited literature.,, It has been evaluated as a diagnostic and prognostic marker as well as a marker for chemo and/or radiotherapy resistance.
It has been evaluated as a diagnostic marker in oral squamous cell carcinoma and was found to be expressed in 33% of precancerous oral lesions, and all tumors that transformed from these precancerous lesions preserved 100% of their survivin positivity. In this study, 33% of chronic cholecystitis cases showed expression of survivin though no malignancy was detected on histopathology. Whether survivin expression in these cases suggests potential malignant transformation in future had the gallbladder in these cases not been removed remains an unanswered question.
Survivin has been evaluated as a prognostic marker in various malignancies. It has been found as a poor prognostic indicator in hepatocellular carcinoma, urinary bladder carcinoma, gastric cancer, colorectal cancers, esophageal cancer, and breast cancer. Nigam et al. have also shown poorer survival in patients with higher survivin mRNA and protein expression. In this study, however, no correlation was observed between survivin expression and survival.
In addition, in this study, no correlation was also noted between survivin expression and any of the examined clinical or pathological characteristics of the gallbladder cancer (e.g. age, sex, menopausal status, tumor grading, tumor stage, nodal involvement, and presence of hyperplasia or metaplasia in chronic cholecystitis, presence of cholelithiasis, and stone size or type). However, Nigam et al. has showed significantly higher expression of survivin in poorly differentiated (as compared to well-differentiated) and Stage III (as compared to Stage II) gallbladder cancer. On the other hand, such lack of correlation has been noted in other digestive tract cancers, for example, pancreas, gastric, and colorectal cancers. Differences in these observed results may be due the small samples size of the study population and differences in the methodology of survivin expression assessment (IHC vs. mRNA detection by polymerase chain reaction). Study with large sample size is imperative to know the true impact of survivin expression in gallbladder cancer.
Overexpression of survivin has been shown to induce resistance to chemotherapy and radiotherapy in pancreatic, colorectal, and prostatic cancers. We have not studied the effect of survivin expression on chemotherapy and radiotherapy sensitivity, and there is a lack of literature on this aspect in gallbladder cancer. Given the lack of effective chemotherapy in gallbladder cancer, studying the role of survivin and its inactivation in an attempt to improve chemosensitivity might be the scope of future research.
| Conclusions|| |
Significantly higher expression of survivin in gallbladder cancer may suggest its role in gallbladder carcinogenesis. However, its expression was not found to serve as a poor prognostic factor for gallbladder cancer in our study. Its expression in one-third of cholelithiasis patients might be related to chronic inflammation of the gallbladder. Future research is imperative to understand its true diagnostic and prognostic role in gallbladder cancer.
Financial support and sponsorship
This study was conducted as a part of an intramural research project with grant (58 E.C.M. IIB/P12) from the Research Cell, King George's Medical University, Lucknow, Uttar Pradesh, India.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Dhir V, Mohandas KM. Epidemiology of digestive tract cancers in India IV. Gall bladder and pancreas. Indian J Gastroenterol 1999;18:24-8.
Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995;267:1456-62.
Adida C, Berrebi D, Peuchmaur M, Reyes-Mugica M, Altieri DC. Anti-apoptosis gene, survivin, and prognosis of neuroblastoma. Lancet 1998;351:882-3.
Lo Muzio L, Staibano S, Pannone G, Mignogna MD, Mariggiò A, Salvatore G, et al.
Expression of the apoptosis inhibitor survivin in aggressive squamous cell carcinoma. Exp Mol Pathol 2001;70:249-54.
Kawasaki H, Altieri DC, Lu CD, Toyoda M, Tenjo T, Tanigawa N. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res 1998;58:5071-4.
Tanaka K, Iwamoto S, Gon G, Nohara T, Iwamoto M, Tanigawa N. Expression of survivin and its relationship to loss of apoptosis in breast carcinomas. Clin Cancer Res 2000;6:127-34.
Grabowski P, Kühnel T, Mühr-Wilkenshoff F, Heine B, Stein H, Höpfner M, et al.
Prognostic value of nuclear survivin expression in oesophageal squamous cell carcinoma. Br J Cancer 2003;88:115-9.
Lee MA, Park GS, Lee HJ, Jung JH, Kang JH, Hong YS, et al.
Survivin expression and its clinical significance in pancreatic cancer. BMC Cancer 2005;5:127.
Fields AC, Cotsonis G, Sexton D, Santoianni R, Cohen C. Survivin expression in hepatocellular carcinoma: Correlation with proliferation, prognostic parameters, and outcome. Mod Pathol 2004;17:1378-85.
Sabin LH. TNM Classification of Malignant Tumours. 7th
ed. London: Wiley-Blackwell; 2010.
Cohen C, Lohmann CM, Cotsonis G, Lawson D, Santoianni R. Survivin expression in ovarian carcinoma: Correlation with apoptotic markers and prognosis. Mod Pathol 2003;16:574-83.
Wong RS. Apoptosis in cancer: From pathogenesis to treatment. J Exp Clin Cancer Res 2011;30:87.
Sarela AI, Verbeke CS, Ramsdale J, Davies CL, Markham AF, Guillou PJ. Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma. Br J Cancer 2002;86:886-92.
Ikeguchi M, Kaibara N. survivin messenger RNA expression is a good prognostic biomarker for oesophageal carcinoma. Br J Cancer 2002;87:883-7.
Huang YJ, Qi WX, He AN, Sun YJ, Shen Z, Yao Y. The prognostic value of survivin expression in patients with colorectal carcinoma: A meta-analysis. Jpn J Clin Oncol 2013;43:988-95.
Liu JL, Zhang XJ, Zhang Z, Zhang AH, Wang W, Dong JH. Meta-analysis: Prognostic value of survivin in patients with hepatocellular carcinoma. PLoS One 2013;8:e83350.
Javle MM, Tan D, Yu J, LeVea CM, Li F, Kuvshinoff BW, et al.
Nuclear survivin expression predicts poor outcome in cholangiocarcinoma. Hepatogastroenterology 2004;51:1653-7.
Song J, Su H, Zhou YY, Guo LL. Prognostic value of survivin expression in breast cancer patients: A meta-analysis. Tumour Biol 2013;34:2053-62.
Dogan M, Çagli S, Yüce I, Bayram A, Somdas MA, Karatas D, et al.
Survivin expression correlates with nodal metastasis in T1-T2 squamous cell carcinoma of the tongue. Eur Arch Otorhinolaryngol 2015;272:689-94.
Krepela E, Dankova P, Moravcikova E, Krepelova A, Prochazka J, Cermak J, et al.
Increased expression of inhibitor of apoptosis proteins, survivin and XIAP, in non-small cell lung carcinoma. Int J Oncol 2009;35:1449-62.
Jeon C, Kim M, Kwak C, Kim HH, Ku JH. Prognostic role of survivin in bladder cancer: A systematic review and meta-analysis. PLoS One 2013;8:e76719.
Nigam J, Chandra A, Kazmi HR, Parmar D, Singh D, Gupta V, et al.
Expression of survivin mRNA in gallbladder cancer: A diagnostic and prognostic marker? Tumour Biol 2014;35:9241-6.
Nigam J, Chandra A, Kazmi HR, Singh A, Gupta V, Parmar D, et al.
Expression of serum survivin protein in diagnosis and prognosis of gallbladder cancer: A comparative study. Med Oncol 2014;31:167.
Nigam J, Chandra A, Kazmi HR, Parmar D, Singh D, Gupta V. Prognostic significance of survivin in resected gallbladder cancer. J Surg Res 2015;194:57-62.
Sarela AI, Scott N, Ramsdale J, Markham AF, Guillou PJ. Immunohistochemical detection of the anti-apoptosis protein, survivin, predicts survival after curative resection of stage II colorectal carcinomas. Ann Surg Oncol 2001;8:305-10.
Lee GH, Joo YE, Koh YS, Chung IJ, Park YK, Lee JH, et al.
Expression of survivin in gastric cancer and its relationship with tumor angiogenesis. Eur J Gastroenterol Hepatol 2006;18:957-63.
Okada E, Murai Y, Matsui K, Isizawa S, Cheng C, Masuda M, et al.
Survivin expression in tumor cell nuclei is predictive of a favorable prognosis in gastric cancer patients. Cancer Lett 2001;163:109-16.
Chiou SK, Moon WS, Jones MK, Tarnawski AS. Survivin expression in the stomach: Implications for mucosal integrity and protection. Biochem Biophys Res Commun 2003;305:374-9.
Lamote J, Willems G. DNA synthesis, cell proliferation index in normal and abnormal gallbladder epithelium. Microsc Res Tech 1997;38:609-15.
Lo Muzio L, Pannone G, Leonardi R, Staibano S, Mignogna MD, De Rosa G, et al.
Survivin, a potential early predictor of tumor progression in the oral mucosa. J Dent Res 2003;82:923-8.
Liu JL, Gao W, Kang QM, Zhang XJ, Yang SG. Prognostic value of survivin in patients with gastric cancer: A Systematic review with meta-analysis. PLoS One 2013;8:e71930.
Lu CD, Altieri DC, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res 1998;58:1808-12.
Department of Surgical Gastroenterology, King George's Medical University, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]