|Year : 2020 | Volume
| Issue : 3 | Page : 569-574
Comparative evaluation of survivin expression in leukoplakia, lichen planus, and oral squamous cell carcinoma: An immunohistochemical study
D Angelin1, Bindu J Nair2
1 Department of Oral Pathology, Sree Mookambika Institute of Dental Sciences, Kulasekharam, Tamil Nadu, India
2 Dr. Vivek's Dental Clinic, Thiruvananthapuram, Kerala, India
|Date of Submission||16-Jun-2019|
|Date of Decision||13-Oct-2019|
|Date of Acceptance||01-Dec-2019|
|Date of Web Publication||18-Jul-2020|
Department of Oral Pathology, Sree Mookambika Institute of Dental Sciences, Kanayakumari, Kulase kharam - 629 161, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Screening for oral squamous cell carcinomas (OSCCs) and oral premalignant lesions may decrease the devastating morbidity and mortality associated with the disease. This has led to widespread research for the identification of molecular-based biomarkers. Among them, survivin is a recently characterized protein which is a member of the inhibitor of apoptosis family. The aim of this study is evaluating the expression of survivin in oral leukoplakia, oral lichen planus, and OSCC compared with normal mucosa.
Materials and Methods: The retrospective study consisted of twenty cases of oral leukoplakia, oral lichen planus, and OSCC in the age group of 20–70 years. Twenty cases of normal mucosa made up the control group. Immunohistochemical staining was performed with the use of survivin polyclonal antibody. Grades of expression of survivin were evaluated. Kruskal–Wallis test was used for statistical analysis.
Results: The expression of survivin was higher in OSCC (80%) when compared to oral leukoplakia (70%), oral lichen planus (45%), and normal mucosa (35%). The variation in the expression of survivin between the samples was statistically significant with P = 0.015 (Kruskal–Wallis test significant at 0.01 level).
Conclusion: It is concluded that survivin can be identified as a useful tool for the identification of potentially malignant disorders at higher risk for progression into invasive carcinoma.
Keywords: Apoptosis, biomarkers, immunohistochemistry, squamous cell carcinoma
|How to cite this article:|
Angelin D, Nair BJ. Comparative evaluation of survivin expression in leukoplakia, lichen planus, and oral squamous cell carcinoma: An immunohistochemical study. J Can Res Ther 2020;16:569-74
|How to cite this URL:|
Angelin D, Nair BJ. Comparative evaluation of survivin expression in leukoplakia, lichen planus, and oral squamous cell carcinoma: An immunohistochemical study. J Can Res Ther [serial online] 2020 [cited 2020 Aug 7];16:569-74. Available from: http://www.cancerjournal.net/text.asp?2020/16/3/569/289972
| > Introduction|| |
Oral squamous cell carcinoma (OSCC) is the most frequent and aggressive malignant tumor of the oral cavity with high mortality and morbidity, which commonly occurs in middle-aged males and older individuals.,, The OSCC has been strongly correlated with specific risk factors, such as the use of tobacco and alcohol. In spite of the therapeutic advances, the 5-year survival time remains at about 55%. OSCC can arise de novo or from potentially malignant disorders, such as verrucous hyperplasia, leukoplakia, submucous fibrosis, and lichen planus.,,, Previous studies have shown that between 16% and 62% of OSCCs are associated with oral leukoplakia, the best known oral precancerous lesion.
Oral epithelial dysplasia, not associated with any specific clinical appearance, is a term assigned to the histopathological changes associated with increased risk of malignant transformation. Lichen planus is a cell-mediated immune condition of unknown etiology, in which T-lymphocytes accumulate beneath the epithelium of the oral mucosa and increase the rate of differentiation of stratified squamous epithelium, resulting in hyperkeratosis and erythema with or without ulceration. There is considerable controversy as to the potentially malignant nature of this condition, while some opinion leaders have stated that lichen planus carries an unequivocal malignant potential and an unspecified risk.,,,,
Although up to a third of oral precancerous lesions will evolve into invasive OSCC over a 10-year interval, no reliable histopathological parameters have been identified that predict their potential for subsequent transformation. A surgical management is often impractical, especially in patients with multiple and extensive precancerous lesions. Novel molecular predictors of malignant progression are needed to identify oral precancerous lesions at greater risk of invasive transformation., Much importance has been given to apoptotic markers which are expressed in both premalignant and malignant lesions. Among them, survivin is a recently characterized protein which is found expressed in solid and hematological malignancies. Survivin, a unique member in the inhibitor of apoptosis protein (IAP) family, is undetectable in most normal adult tissues but is highly expressed in cancer. It is cell cycle regulated and is involved in both control of apoptosis and regulation of cell division.,,,
Further investigation of survivin and other apoptotic inhibitors during tumor growth and progression may yield important therapeutic strategies for combating cancer. Lo Muzio et al. and Jinbu et al. have done studies on survivin expression in potentially malignant and malignant oral lesions and have opined about its usefulness in the prediction of tumor progression of the oral mucosa. The present study is, therefore, designed to analyze the immunohistochemical expression of survivin in oral leukoplakia, oral lichen planus, and OSCC.
| > Materials and Methods|| |
The retrospective study was carried out in the department of oral pathology and microbiology. Formalin-fixed paraffin-embedded tissue blocks were obtained from the archives, and those fulfilling inclusion and exclusion criteria were selected for the study. Tissue blocks of clinically diagnosed and histopathologically confirmed cases in the age group of 20–70 years were included, whereas tissue blocks from which proper antigen retrieval could not be done adequately and tissues from nonoropharyngeal site were not considered.
The study group comprised twenty cases each of oral leukoplakia, oral lichen planus, and OSCC. OSCC cases were graded for differentiation according to the Broder's grading system, whereas cases of oral leukoplakia were graded for dysplasia according to the WHO 2005 criteria. Accordingly, in our study, there were 5 well-differentiated and 15 moderately differentiated cases of OSCC, whereas there were 16 mild, 3 moderate, and 1 severe cases of epithelial dysplasia. Oral lichen planus was diagnosed according to the histological criteria set by Eisenberg. Twenty biopsies of noninflamed normal mucosa from the retromolar region from those patients undergoing extraction of third molar were obtained with their consent, which constituted the control group. From all the obtained eighty blocks, two sections of each were taken at 3–4-μ thickness. One set of sections was subjected to the routine hematoxylin and eosin staining to confirm the diagnosis, whereas others were stained for immunohistochemical staining for survivin. The study was approved by the institutional ethical committee, and informed consent was obtained from all the patients.
Tissue sections were mounted on 3-aminopropyltriethoxysilane-coated slides, and immunohistochemical staining was performed using horseradish peroxidase (HRP) technique. Antigen retrieval was performed by keeping in Tris-EDTA buffer and heating for 15–20 min using a pressure cooker. Hydrogen peroxide block was added to prevent endogenous peroxidase activity. Slides were then incubated with rabbit polyclonal antibody to survivin (Abcam, ab15527; Cambridge, UK) for 30 min at room temperature. Slides were then treated with PolyExcel target binder for 15 min, followed by PolyExcel HRP (Thermo Fisher; Massachusetts, USA) for 15 min. Freshly prepared 3,3'-Diaminobenzidine tetrahydrochloride (DAB) substrate chromogen was added and kept for 2–5 min, followed by counterstaining with Harris hematoxylin. The expression of survivin in normal stomach mucosa served as the positive control, whereas section not stained with survivin was taken as a negative control.
Slides were observed under binocular light microscope (Labomed LX200; USA), and survivin expression in the cytoplasm of epithelial cells was graded according to the grading criteria, followed by Jinbu et al. Grading was done concurrently by two oral pathologists to whom the clinical data were unknown. All of the cases were graded into three groups, namely Grade 0 (negative expression), Grade 1+ (weak expression), and Grade 2+ (strong expression) based on the degree of survivin staining.
The data were analyzed by the Statistical Package for the Social Services (SPSS 16.0 version, IBM Corporation, New York, USA). Kruskal–Wallis test was applied to find statistical significance between the groups, and P value at 0.01 level was considered as statistically significant.
| > Results|| |
In the present study, all the study samples were in the age range of 20–70 years. Oral leukoplakia samples had a mean age of 54.2 years and a male predilection in the ratio of 3:1. The OSCC showed a mean age of 59 years and male predominance with a male-to-female ratio of 3:2. The documentation of the associated habits and common site of the lesion showed tobacco and betel chewing as the most prevalent cause and buccal mucosa as the most favorable site for both oral leukoplakia and OSCC. Similarly, the oral lichen planus showed a mean age of 45.8 years, female predominance with a male-to-female ratio of 1:3, and buccal mucosa as the most favorable site.
In the present study, of the 20 cases of oral leukoplakia studied, 14 were positive and 6 were negative for survivin staining. Among the positive ones, two cases showed a strong expression, whereas 12 cases showed a weak expression. Similarly, among the 20 cases of oral lichen planus in our study, 9 of them showed positivity for survivin expression with 2 and 11 cases among them showing strong and weak expressions, respectively. Eleven cases of oral lichen planus had a negative expression. In OSCC cases, 16 of them showed positivity, whereas 4 cases had a negative expression for survivin staining. Among the positive cases, six and ten cases had strong and weak expressions, respectively. Survivin positivity in normal mucosa was noted in seven cases with weak expression and the remaining were negative for survivin [Table 1].
There was no statistical significance when the expression of survivin in oral leukoplakia and oral lichen planus was compared with normal mucosa. However, when the expression of survivin in OSCC was compared with that of normal mucosa, a statistical significance was obtained at 0.01 levels. A comparison of survivin expression among all the four groups obtained a statistical significance with P = 0.015 [Table 2] and [Graph 1]. There was no statistical significance in relation to age, sex, site, and survivin expression in oral leukoplakia, oral lichen planus, and OSCC.
|Table 2: Multiple comparisons of percentage positivity between the groups|
Click here to view
| > Discussion|| |
OSCC is one of the most common malignant tumors in humans, the development of which is due to a number of malfunctions in gene regulation such as activation of oncogenes and inhibition of tumor suppressor genes. Studies by Thompson et al. suggested that the deregulation of apoptosis plays a critical role in the onset and progression of cancer. Singh et al. in their study showed that deregulated Bcl-2 in severely dysplastic oral epithelial lesions was linked to the progression of OSCC. A similar paradigm has also been suggested for other solid malignancies, thus prompting the search for additional molecular markers potentially influencing the cell death/cell viability balance in cancer. In this context, recent studies identified survivin, an inhibitor of apoptosis family protein to be overexpressed in most human cancers but downregulated in normal tissues. Differently from Bcl-2 family molecules, IAP proteins are thought to block a highly evolutionarily conserved step in cell death by binding and inhibiting terminal effector caspases-3 and 7, thus providing a separate pathway of cell viability in cancer.
Studies in India by Gupta et al. in 1980 have reported that oral leukoplakia has a high malignant transformation rate of 2.2%. The presence of severe epithelial dysplasia characterized by enlarged nuclei and eosinophilic nucleoli, hyperchromatism, dyskeratosis, and aberrant mitoses is suggestive of malignant transformation. Oral lichen planus is another potentially malignant lesion with a low malignant transformation rate of 0.4%–5%. Studies comparing the expression of survivin in OSCC and oral leukoplakia have been conducted earlier, but no studies have till now been done comparing the expression of survivin in OSCC, oral leukoplakia, and oral lichen planus.
Lo Muzio et al. have shown that survivin is upregulated early during malignant transformation of the oral cavity and that its upregulation is overwhelmingly associated with precancerous lesions that evolved into full-blown invasive carcinomas. According to the study by Tanaka et al., one-third of the oral premalignant lesions examined had survivin protein expression. Considerable evidence suggests that the elevated expression of survivin may promote tumorigenesis, and in fact, survivin is highly expressed in human common cancers.
In our study, we investigated the immunohistochemical expression of survivin in twenty cases each of oral leukoplakia, oral lichen planus, and OSCC and compared it with its expression in normal mucosa. In the study by Tanaka et al., 37% of oral leukoplakia showed survivin positivity, and they suggested that survivin protein accumulation might be an early event during oral carcinogenesis. Lo Muzio et al. in their study on oral precancers showed a survivin positivity of 33% in dysplasia that did not progress into malignancy and 94% positivity in dysplasias that evolved into OSCC. In the present study, the overall percentage of survivin positivity in oral leukoplakia was 70% [Figure 1]. The percentage of survivin positivity was greater than that reported by Lo Muzio et al. and Tanaka et al. We did not find a significant correlation between the grades of dysplasia and survivin expression. In our study, one case of severe dysplasia showed a negative survivin expression. Lo Muzio et al. in their study has reported that 50% of severe dysplasia cases showed a negative survivin expression and those cases did not progress into OSCC. However, the follow-up of patients with oral leukoplakia was beyond the scope of this study.,
|Figure 1: Microphotograph showing Grade 2 survivin expression in oral leukoplakia (IHC staining; ×400)|
Click here to view
Oral lichen planus is a T-cell-mediated autoimmune disease in which the cytotoxic CD8+ T-cells trigger the apoptosis of oral epithelial cells. Oluwadara et al. in their study showed a 64.3% positivity in survivin expression in biopsies from oral lichen planus patients. According to Fukuda and Pelus, survivin is expressed in thymocytes, splenic T-cells, and human adult peripheral blood T-lymphocytes. Li et al. proposed that survivin is not essential for T-cell apoptosis control but is crucial for T-cell maturation and proliferation at multiple stages. The total survivin positivity in our study in oral lichen planus was 45% which was much less than the percentage of positivity reported by Oluwadara et al. (64.3%). Some cases showed positivity for the lymphocytes present subepithelially which was consistent with the review by Fukuda and Pelus that survivin is expressed in T-lymphocytes. Oluwadara et al. explained that the high percentage of survivin positivity in their study suggests that these lesions may represent a heightened risk for transformation into OSCC. They further proposed that oral lichen planus lesions that are in the process of transforming into OSCC may histopathologically represent as oral lichen planus but possess a certain molecular signature that represents the specific factors that drive progression to cancer. Even though there are still controversies regarding the malignant potential of oral lichen planus, studies have indeed proven it and the WHO in 2005 has recognized oral lichen planus as a potentially malignant disorder. However, the overall survivin protein expression in oral lichen planus in our study was much lower than that in oral leukoplakia [Figure 2].
|Figure 2: Microphotograph showing Grade 2 survivin expression in oral lichen planus (IHC staining; ×100)|
Click here to view
Kim et al. in their study obtained a 100% survivin positivity in OSCC cell lines in hamster oral carcinogenesis model and showed that OSCC patients with high survivin expression in the tumor mass had a shorter overall survival. Lo Muzio et al. observed an 80% survivin expression in OSCC. In another study by the same investigators in OSCC and precancerous lesions, a score of 82.7% positivity was seen in OSCC. Studies by Tanaka et al. and Jinbu et al. both revealed a 58% survivin-positive immunostaining in OSCC cases., The overall survivin positivity in our study in OSCC was 80% [Figure 3]. This was consistent with the observations of Lo Muzio et al. In our study, we observed that the grades of survivin expression increased from well-differentiated to moderately differentiated OSCC even though the results were not statistically significant.
|Figure 3: Microphotograph showing Grade 2 survivin expression in oral squamous cell carcinoma (IHC staining; ×100)|
Click here to view
Earlier studies by Lo Muzio et al. have reported a mild expression of survivin in the epithelial cells of normal mucosa. In the study by Tanaka et al., all the normal oral mucosa examined showed the absence or significant downregulation of survivin expression and were considered negative. Fukuda and Pelus in their review on survivin stated that although survivin is expressed and regulated in normal tissues characterized by self-renewal and proliferation, its expression is significantly lower than in transformed cells. The total positivity rate in normal mucosa in our study was 35% and only weak staining was observed [Figure 4]. The results in this study were in accordance with the studies reported by Lo Muzio et al. and Tanaka et al. Normal oral mucosa showed a low survivin expression in comparison with oral leukoplakia, oral lichen planus, and OSCC.
|Figure 4: Microphotograph showing Grade 1 survivin expression in normal mucosa (IHC staining; ×100)|
Click here to view
In the present study, the survivin expression levels in oral leukoplakia and OSCC were significantly higher than that in oral lichen planus and normal oral tissues. As the data presented here suggest, survivin expression might provide a strong advantage for tumor progression by protecting tumor cells from broad apoptosis-inducing stimuli and by maintaining proper mitotic progression of the proliferating and metastatic population.
| > Conclusion|| |
Based on the results, it can be concluded that survivin which is an IAP can be identified as a useful tool for the identification of potentially malignant disorders at higher risk for progression into invasive carcinoma. Further studies on a larger series of patients with follow-up may provide more accurate information about the involvement of survivin in the development and progression of oral carcinogenesis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
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.
Daniel FI, Fava M, Hoffmann RR, Compos MM, Yurgel LS. Main molecular markers of oral squamous cell carcinoma. Appl Cancer Res 2010;30:279-88.
Neville BW, Day TA. Oral cancer and precancerous lesions. CA Cancer J Clin 2002;52:195-215.
Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74-108.
Banerjee AG, Bhattacharyya I, Vishwanatha JK. Identification of genes and molecular pathways involved in the progression of premalignant oral epithelia. Mol Cancer Ther 2005;4:865-75.
Reibel J. Prognosis of oral pre-malignant lesions: Significance of clinical, histopathological, and molecular biological characteristics. Crit Rev Oral Biol Med 2003;14:47-62.
Chen YK, Hsuen SS, Lin LM. Expression of inducible nitric oxide synthase in human oral premalignant epithelial lesions. Arch Oral Biol 2002;47:387-92.
Pindborg JJ, Murti PR, Bhonsle RB, Gupta PC, Daftary DK, Mehta FS. Oral submucous fibrosis as a precancerous condition. Scand J Dent Res 1984;92:224-9.
Krutchkoff DJ, Cutler L, Laskowski S. Oral lichen planus: The evidence regarding potential malignant transformation. J Oral Pathol 1978;7:1-7.
Hsue SS, Wang WC, Chen CH, Lin CC, Chen YK, Lin LM. Malignant transformation in 1458 patients with potentially malignant oral mucosal disorders: A follow-up study based in a Taiwanese hospital. J Oral Pathol Med 2007;36:25-9.
Warnakulasuriya S, Johnson NW, van der Waal I. Nomenclature and classification of potentially malignant disorders of the oral mucosa. J Oral Pathol Med 2007;36:575-80.
Epstein JB, Wan LS, Gorsky M, Zhang L. Oral lichen planus: Progress in understanding its malignant potential and the implications for clinical management. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:32-7.
van der Meij EH, Schepman KP, van der Waal I. The possible premalignant character of oral lichen planus and oral lichenoid lesions: A prospective study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:164-71.
Silverman S Jr. Oral lichen planus: A potentially premalignant lesion. J Oral Maxillofac Surg 2000;58:1286-8.
Scully C, Carrozzo M. Oral mucosal disease: Lichen planus. Br J Oral Maxillofac Surg 2008;46:15-21.
Li F. Role of survivin and its splice variants in tumorigenesis. Br J Cancer 2005;92:212-6.
Chiou SK, Jones MK, Tarnawski AS. Survivin-an anti-apoptosis protein: Its biological roles and implications for cancer and beyond. Med Sci Monit 2003;9:PI25-9.
Dallaglio K, Marconi A, Pincelli C. Survivin: A dual player in healthy and diseased skin. J Invest Dermatol 2012;132:18-27.
Fukuda S, Pelus LM. Survivin, a cancer target with an emerging role in normal adult tissues. Mol Cancer Ther 2006;5:1087-98.
Jinbu Y, Tsukinoki K, Miyagi N, Senna T, Obi Y, Matsumoto K, et al
. Expression of survivin in oral squamous cell carcinoma. Oral Med Pathol 2006;11:41-4.
Akinyamoju AO, Adeyemi BF, Kolude B, Adisa AO. Histological grading of oral squamous cell carcinoma in Ibadan using Bryne's and Broder's grading systems – A comparative study. AFR J Med Sci 2013;42:333-7.
Barnes L, Eveson J W, Reichart P, SidranskyD. World Health Organisation Classification of Tumors. Pathology and Genetics of Head and Neck Tumors. Lyon: IARC Press; 2005.
Eisenberg E. Oral lichen planus: A benign lesion. J Oral Maxillofac Surg 2000;58:1278-85.
Thompson C. Apoptosis in the pathogenesis and treatment of disease. Science 1995;267:1456-62.
Singh BB, Chandler FW Jr., Whitaker SB, Forbes-Nelson AE. Immunohistochemical evaluation of bcl-2 oncoprotein in oral dysplasia and carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:692-8.
Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med 1997;3:917-21.
Tanaka C, Uzawa K, Shibahara T, Yokoe H, Noma H, Tanzawa H. Expression of an inhibitor of apoptosis, survivin, in oral carcinogenesis. J Dent Res 2003;82:607-11.
Gupta PC, Mehta FS, Daftary DK, Pindborg JJ, Bhonsle RB, Jalnawalla PN, et al
. Incidence rates of oral cancer and natural history of oral precancerous lesions in a 10-year follow-up study of Indian villagers. Community Dent Oral Epidemiol 1980;8:283-333.
Rajendran R, Sivapathasundaram B. Diseases of Skin, Shafer's Textbook of Oral Pathology. 6th
ed. India: Elsevier; 2009. p. 799-803.
Oluwadara O, Giacomelli L, Christensen R, Kossan G, Avezova R, Chiappelli F. LCK, survivin and PI-3K in the molecular biomarker profiling of oral lichen planus and oral squamous cell carcinoma. Bioinformation 2009;4:249-57.
Li F, Yang J, Ramnath N, Javle MM, Tan D. Nuclear or cytoplasmic expression of survivin: What is the significance? Int J Cancer 2005;114:509-12.
Kim YH, Kim SM, Kim YK, Hong SP, Kim MJ, Myoung H. Evaluation of survivin as a prognostic marker in oral squamous cell carcinoma. J Oral Pathol Med 2010;39:368-75.
Muzio LL, Pannone G, Staibano S, Mignogna MD, Rubini C, Mariggiò MA, et al
. Survivin expression in oral squamous cell carcinoma. Br J Cancer 2003;89:2244-8.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]