|Year : 2017 | Volume
| Issue : 1 | Page : 39-44
Immunohistochemical expression of Survivin in oral leukoplakia and oral squamous cell carcinoma
Cherukuri Gayathri1, Guttikonda V Rao2
1 Department of Oral Pathology and Microbiology, Panineeya Mahavidyalaya Institute of Dental Sciences, Hyderabad, Telangana, India
2 Department of Oral Pathology and Microbiology, Mamata Dental College, Khammam, Telangana, India
|Date of Web Publication||20-Mar-2017|
Panineeya Mahavidyalaya Institute of Dental Sciences, Hyderabad, Telangana
Source of Support: None, Conflict of Interest: None
Background and Objectives: Survivin is an inhibitor of apoptosis protein. It is involved in the modulation of the cell death and cell division process. Alterations in the expression of Survivin have been reported in several inflammatory, premalignant, and malignant lesions. Hence, the objectives of the present study were to compare the expression of Survivin with respect to the degree of dysplasia in oral leukoplakia (OL) and to evaluate the expression of Survivin with respect to different histopathological grades of oral squamous cell carcinoma (OSCC).
Materials and Methods: Neutral-buffered, formalin-fixed and paraffin-embedded biopsy specimens from 30 patients, each with OL and OSCC, were included in this study. The expression of Survivin was detected by immunohistochemistry. The scores obtained were subjected to Chi-square test.
Results: Survivin expression was detected in all grades of dysplasia and OSCC. Expression of Survivin was significant when compared with different degrees of dysplasia and different histopathological grades of OSCC (P < 0.05). A significant correlation was not found in immunostaining between dysplasia and OSCC groups.
Conclusion: Higher immunohistochemical scores were obtained with increased histopathological grades of OL and OSCC. High expression of Survivin may be related to malignant transformation in OL and poor prognosis in OSCC.
Keywords: Oral leukoplakia, oral squamous cell carcinoma, Survivin
|How to cite this article:|
Gayathri C, Rao GV. Immunohistochemical expression of Survivin in oral leukoplakia and oral squamous cell carcinoma. J NTR Univ Health Sci 2017;6:39-44
|How to cite this URL:|
Gayathri C, Rao GV. Immunohistochemical expression of Survivin in oral leukoplakia and oral squamous cell carcinoma. J NTR Univ Health Sci [serial online] 2017 [cited 2019 Aug 22];6:39-44. Available from: http://www.jdrntruhs.org/text.asp?2017/6/1/39/202585
| Introduction|| |
Survivin is a recently characterized smallest member of the Inhibitor of Apoptosis (IAP) family of proteins, a cluster of genes playing an important role in apoptosis regulation, involved both in the cell death regulation and the different aspects of cell division. The gene encoding Survivin is located on chromosome 17q25 in humans. This single gene contains four exons and three introns and gives rise to alternatively spliced transcripts. Among the IAP family members, Survivin has certain unique characteristics. The first is its structure which contains 142 amino acids, approximately 16.5 kDa, with a single baculovirus IAP repeat domain and lack of zinc binding C-terminal Really Interesting New Gene (RING) finger domain and a caspase recruitment domain. The second is its dual role as a bifunctional protein.
Biological functions of Survivin include inhibition of apoptosis through several pathways and proper execution of mitosis and cell division. Survivin can directly interact with caspases leading to the inhibition of caspase activity, or Survivin can inhibit the function of a pro-apoptotic complex called second mitochondria-derived activator of caspase (Smac)/direct IAP binding protein with low isoelectricpoint (DIABLO). In addition to its anti-apoptotic function, Survivin can form a complex with Aurora B kinase and the inner centromere protein, called INCENP, during the Gap2/Mitotic phase (G2/M) of the cell cycle to help in the proper segregation of chromosomes. Several signalling pathways have been involved in the regulation of Survivin expression such as transforming growth factor β (TGF-β) and p53 signalling pathways.
In addition to being a well-known inhibitor of apoptosis, Survivin also plays a role in promoting cell proliferation and angiogenesis. Unlike other IAP proteins, Survivin is expressed during embryonic and fetal development and may be important in tissue homeostasis and differentiation. However, Survivin is completely downregulated and undetectable in normal, terminally differentiated adult tissues, and becomes prominently expressed in most of the common human cancers, including cancer of the lung, colon, stomach, esophagus, uterus, ovary, pancreas, prostate, breast, and melanoma skin cancer. High Survivin expression in tumors correlates with more aggressive and invasive clinical phenotype.,
In recent years, the number of molecular-based assays has increased, however, histopathology remains the gold standard for most diagnostic and therapeutic decisions. Immunohistochemical staining is becoming a globally available tool that complements histopathological analysis by detecting gene expression at the protein level.
The present study was conducted to observe the expression of Survivin in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) and to compare the intensity of immunohistochemical staining with respect to different histological grades of the same.
| Materials and Methods|| |
Thirty neutral-buffered, formalin-fixed, paraffin-embedded tissues of OL and OSCC was retrieved from the Department of Oral Pathology and Microbiology, Mamata Dental College, Khammam for the purpose of this study. The histological grading of OL was done based on the criteria given by the World Health Organization. Cases of OSCC were graded histopathologically using the Broders' (1927) grading system.
A 4-µ thick section of each paraffin-embedded case was prepared. All tissue sections were eparaffinised and dehydrated with a descending series of alcohol. Antigen retrieval was performed in tris buffered saline using microwave oven at 100°C for 5 min for 4 times. All the slides were allowed to cool to room temperature. All the reagents stored in the refrigerator were bought to room temperature (24–28°C) prior to immunostaining. All the incubations were performed at room temperature using a humidifying chamber. At no time the tissue sections were allowed to dry during the staining procedure. The slides were washed gently with PBS three times for 2 min each. After tapping off the excess buffer from the slide, the sections were covered with 3% hydrogen peroxide for 15–20 minutes and then washed gently with PBS three times for 2 min each.
After the Power Block was tapped off, the sections were covered completely with pre-diluted Survivin primary antibody (Biogenex super sensitive detection system, USA) in 1:100 dilution, except the negative control. The slides were incubated for 1 h at 21°C in a humidifying chamber. Super Enhancer was applied and left for 30 min and then was washed gently with PBS three times for 2 min each. After tapping off the excess buffer, the sections were incubated with secondary antibody for 30 min followed by the tissue sections were completely covered with freshly prepared substrate chromogen solution using Pasteur pipette for 10 min. The sections were gently washed in distilled water and immersed in Mayer's hematoxylin followed by placement in alcohol in descending series. The sections were kept immersed in xylene bath and mounted with DPX.
Presence of a brown-colored end product at the site of target antigen was indicative of positive immunoreactivity. The negative control tissue demonstrated no staining. Transitional cell carcinoma of urinary bladder tissue [Figure 1]a was taken as positive control with each batch of staining and normal oral mucosal tissue [Figure 1]b as negative control. The evaluation of study cases was done subsequently in a similar manner and was graded as positive or negative. In cases with staining heterogeneity, the expression was grouped according to the predominant staining pattern. It was graded as: 0 – negative staining, 1 – mild staining, 2 – moderate staining, and 3 – intense staining. All these observations were carried out by two observers to eliminate interobserver bias. The results were analyzed statistically using Chi-square test.
|Figure 1: (a) Transitional cell carcinoma of urinary bladder tissue as positive control (×400 view). (b) Normal oral mucosal tissue as negative control (×400 view)|
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| Results|| |
Out of a total of 30 cases in the dysplasia group, the intensity of staining was found to be 0 in 4 cases (13.33%), 1 in 10 cases (33.33%), 2 in 11 cases (36.67%), and 3 in 5 cases (16.67%). Similarly, in the carcinoma group, out of a total of 30 cases, 2 cases (6.67%) had negative staining, 13 cases (43.33%) had score 1; 11 cases (36.67%) had score 2, and 4 cases (13.33%) had score 3. When a comparison was made with respect to the intensity of staining between the dysplasia and carcinoma groups, the results were found to be statistically insignificant with a P value of 0.7604 [Table 1].
|TABLE 1: COMPARISON OF CARCINOMA AND DYSPLASIA GROUPS FOR SURVIVIN EXPRESSION WITH RESPECT TO THE INTENSITY OF STAINING|
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Out of the 12 cases of mild dysplasia [Figure 2]a, the intensity of staining was observed to be 0 in 4 cases, 1 in 7 cases, and 2 in 1 case. In 13 cases of moderate dysplasia [Figure 2]b, the intensity of staining was 1 in 3 cases and 2 in 10 cases. Out of 5 cases of severe dysplasia [Figure 2]c, the intensity of staining was observed to be 3 in all cases. A statistically significant difference was observed between various histological grades of dysplasia with respect to immunohistochemistry scores with a P value of 0.0000 [Table 2].
|Figure 2: Immunohistochemical expression of Survivin. (a) Mild dysplasias. (b) moderate dysplasias. (c) severe dysplasias (×400 view)|
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|TABLE 2: COMPARISON OF VARIOUS HISTOLOGICAL GRADES OF DYSPLASIAS (MILD, MODERATE, SEVERE) FOR SURVIVIN EXPRESSION WITH RESPECT TO THE INTENSITY OF STAINING|
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Out of 11 Grade I [Figure 3]a cases of OSCC, the intensity of staining was observed to be 0 in 2 cases and 1 in 9 cases. In 14 cases of Grade II [Figure 3]b, the intensity of staining was 1 in 4 cases and 2 in 10 cases. Out of 5 cases of Grade III [Figure 3]c, the intensity of staining was observed to be 2 in 1 case and 3 in 4 cases. A statistically significant difference was observed between various histological grades of OSCC with respect to immunohistochemistry scores with a P value of 0.0000 [Table 3].
|Figure 3: Immunohistochemical expression of Survivin. (a) Grade I OSCC. (b) Grade II OSCC. (c) Grade III OSCC (×400 view)|
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|TABLE 3: COMPARISON OF VARIOUS HISTOLOGICAL GRADES (I, II, III) OF OSCC WITH RESPECT TO THE INTENSITY OF STAINING|
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| Discussion|| |
Survivin, also called baculoviral inhibitor of apoptosis repeat-containing 5 or BIRC5, is a protein that, in humans, is encoded by the BIRC5 gene. A member of the IAP family, Survivin functions to inhibit caspase activation, thereby leading to the negative regulation of apoptosis or programmed cell death. This has been shown by the disruption of Survivin induction pathways leading to an increase in apoptosis and decrease in tumor growth.
Survivin seems to exist in two subcellular pools, cytoplasmic and nuclear. This is consistent with its function in the regulation of both cell viability and cell division. One possibility is that the nuclear pool of Survivin is involved in promoting cell proliferation in most cases, whereas the cytoplasmic pool of Survivin may participate in controlling cell survival but not cell proliferation. In many immunohistochemical studies, nuclear expression of Survivin is an unfavorable factor for prognosis.,,
A sharp differential expression in cancer versus normal tissues is one of the most intriguing features of Survivin, and is unlike any other IAPs. Survivin is strongly and broadly expressed in embryonic and fetal organs, however, becomes undetectable in most terminally differentiated normal tissues, perhaps with the exception of thymocytes, CD34+ stem cells, and basal colonic epithelial cells.
Survivin expression has been shown in various preneoplastic and benign lesions including polyps of the colon, breast adenomas, nearly all cases of Bowen's disease, cervical dysplasia, and hypertrophic actinic keratosis suggesting that expression of Survivin may occur early during malignant transformation.
Aberrations in apoptotic programmes are a hallmark of perhaps all cancers. Dramatic overexpression of Survivin has been demonstrated in prostate cancer, rectal cancer, esophageal squamous cell carcinoma, colorectal carcinoma, breast cancer, laryngeal squamous cell carcinoma, hepatocellular carcinoma, ovarian carcinoma, non-small cell lung carcinoma, glioblastoma, and pancreatic cancer which signal more aggressive and disseminated disease and unfavorable clinical outcome.,
Among the head and neck cancers, Survivin was expressed in brain tumours, OSCC,,,, B-cell lymphomas,, pheochromocytoma, astrocytic tumors,, salivary gland cancer, and soft tissue sarcomas.,, Survivin was also found to be expressed in oral premalignant lesions and conditions as well as in odontogenic cysts.,,,,,
Its overexpression in premalignancy suggests an early event during step-wise malignant transformation, and in head and neck cancers reflects the biologic aggressiveness of these tumours. Survivin has a definite role in tumor progression in OSCC and may provide prognostic information.
In the present study, an attempt was made to evaluate the expression of Survivin immunohistochemically with respect to different histological grades of oral dysplasia and OSCC. The results of the present study showed an increased expression of Survivin with respect to varying histological grades of oral epithelial dysplasia and OSCC.
It was hypothesized that Survivin protein accumulation might be an early event during oral carcinogenesis because one-third of oral premalignant lesions that were examined had protein expression. This hypothesis was further supported by Chen et al. who stated that epigenetic and genetic pathways were associated with IAP expression in OSCC.
When Survivin mRNA levels in normal mucosa, oral leukoplakia, and oral cancer were measured by real time reverse transcriptase–polymerase chain reaction (RT-PCR) technique, a significant difference between cancer and normal mucosa and between cancer and potentially malignant lesions was found, whereas the comparison between normal mucosa and potentially malignant samples showed no statistically significant difference. However, in the present study, no statistically significant difference between the expression of Survivin in oral leukoplakia and OSCC was found immunohistochemically.
It has been reported that there was a widespread Survivin positivity in 33% of oral precancerous lesions without malignant progression and in 94% of the lesions that evolved into SCC. In the present study, statistically significant results (P < 0.05) were obtained when the intensity of Survivin staining was compared with various histological grades of dysplasia.
When the expression of Survivin was related with the clinicopathological characteristics in OSCC, it was found that the percentage of Survivin-positivity in the T1, T2 group was significantly higher than that in the T3, T4 group, and the percentage of Survivin positivity in the N0 group was also significantly higher than in the N+ group.
When 110 cases of OSCC together with lymph node metastasis were analyzed for expression of Survivin by immunohistochemistry and Western blotting, it was found that Survivin expression was increased in poorly-differentiated tumors, and patients with low Survivin expression had better survival rates than the groups with medium and high Survivin expression. These findings were similar with the present study where Survivin stained intense in Grade III cases compared to Grades II and I cases and statistically significant results (P < 0.05) were obtained when intensity of immunostaining was compared with various histological grades of OSCC.
Further, studies on Survivin expression in oral leukoplakia and high-grade OSCC among Indian tobacco chewers were carried out by Jane et al., in which 65% of oral leukoplakia cases showed cytoplasmic Survivin positivity, and 18% of poorly-differentiated OSCC showed nuclear positivity. Based on these findings, we can say that Survivin stains both nucleus and cytoplasm. In our study, all cases of OSCC and cases of moderate-to-severe dysplasia showed nuclear positivity and cytoplasmic positivity was observed only in few cases of mild dysplasia.
Expression of Survivin has also been observed in salivary gland cancer by Stenner et al., Positive cytoplasmic expression in both mucoepidermoid carcinoma and adenocarcinoma was observed which was related to poor overall survival. In adenoid cystic carcinoma, both nuclear and cytoplasmic Survivin was highly expressed with respect to tumor size.
Effects of Survivin were investigated on the tumorigenesis and development of oral cancer and its correlation to angiogenesis. Results showed that Survivin expression was found to be significantly stronger in OSCC than in normal mucosa and dysplastic leukoplakia. The expression of Survivin was significantly stronger in moderately and poorly-differentiated OSCC than in well-differentiated OSCC. Similar findings were found in our study, where the intensity of immunostaining was stronger in moderately and poorly-differentiated OSCC than in well-differentiated cases of OSCC and statistically significant results (P < 0.05) were obtained when a comparison between intensity of staining and various histological grades was made.
When the prognostic significance of Survivin protein expression was evaluated and correlated with clinicopathologic features in OSCC by Liping et al., it was found that immunostaining of Survivin protein was significantly stronger in OSCC tissues with poorer tumor differentiation, higher clinical stage, and the presence of lymphnode metastasis. However, in the present study, the intensity of staining was compared only with various histological grades of oral epithelial dysplasia and OSCC.
| Conclusions|| |
The following conclusions were drawn from the study
- Expression of Survivin varied with different histological grades of dysplasia and OSCC indicating its role as a diagnostic marker
- Higher immunohistochemical scores were obtained with increasing grades of dysplasia and OSCC, indicating its role as a prognostic marker
- The results also suggest no statistical significance in the expression of Survivin when a comparison was done between the dysplasia and OSCC groups
- Comparison of histological grades with respect to intensity of staining in both dysplasia and OSCC was found to be statistically significant.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
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.
Chaiyarit P, Jintakanon D, Klanrit P, Siritapetawee M, Thongprasom K. Immunohistochemical analyses of Survivin and Heat Shock Protein 90 expression in patients with Oral Lichen planus. J Oral Pathol Med 2009;38:55-62.
Andric M, Dozic B, Popovic B, Stefanovic D, Basta-jovanovic G, Djogo N, et al
., Survivin expression in Odontogenic Keratocysts and Correlation with Cytomegalovirus infection. Oral Dis 2010;16:156-9.
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.
Oliveira LR, Ribeiro-Silva A. Prognostic significance of immunohistochemical biomarkers in Oral Squamous Cell Carcinoma. Int J Oral Maxillofac surg 2011;40:298-307.
Lodi G, Franchini R, Bez C, Sardella A, Moneghini L, Pellegrini C, et al
. Detection of Survivin mRNA in healthy Oral mucosa, Oral Leukoplakia and Cancer. Oral Dis 2010;16:61-7.
Yamamoto H, Ngan CY, Monden M. Cancer cells survive with Survivin. Cancer Sci 2008;99:1709-14.
Johnson ME, Howerth EW. Survivin: A Bifunctional Inhibitor of Apoptosis Protein. Vet Pathol 2004;41:599-607.
Li F, Jie Yang, Ramnath N, Javle MM, Tan D. Nuclear or Cytoplasmic expression of Survivin: What is the significance? Int J Cancer 2005;114:509-12.
Stauber RH, Wolf Mann, Knauer SK. Nuclear and Cytoplasmic Survivin: Molecular Mechanism, Prognostic, and Therapeutic Potential. Cancer Res 2007;67:5999-6002.
Altieri DC. Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene 2003;22:8581-9.
Jane C, Nerurkar AV, Shirsat NV, Deshpande RB, Amrapurkar AD, Karjodkar FR. Increased Survivin expression in high-grade Oral Squamous Cell Carcinoma: A study in Indian tobacco chewers. J Oral Pathol Medicine 2006;35:595-601.
Muzio L, Pannone G, Leonardi R, Staibano S, Mignogna MD, De Rosa, et al
. Survivin, a Potential Early Predictor of Tumor Progression in the Oral Mucosa. J Dent Res 2003;82:923.
Ko YH, Roh SY, Won HS, Jeon EK, Hong SH, Lee MA, et al
. Prognostic significance of Nuclear Survivin Expression in Resected Adenoid Cystic Carcinoma of the Head and Neck. Head Neck Oncol 2010;2:30.
Muzio L, Pannone G, Staibano S, Mignogna MD, Rubini C, Mariggio MA, et al
. Survivin expression in Oral Squamous Cell Carcinoma. Br J Cancer 2003;89:2244-8.
Liping S, Ying W, Mingzhen X, Yuan L, Lei Y, Xi. Up-regulation of Survivin in Oral Squamous Cell Carcinoma correlates with poor prognosis and chemoresistance. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;110:484-91.
Adida C, Haioun C, Gaulard P, Lepage E, Morel P, Josette Briere J, et al
. Prognostic significance of Survivin expression in Diffuse large B-cell lymphomas. Blood 2000;96:1921-25.
Schlette EJ, Medeiros LJ, Goy A, Lai R, Rassidakis GJ. Survivin expression predicts poorer prognosis in Anaplastic Large-Cell Lymphoma. J Clin Oncol 2004;22:1682-8.
Koch CA, Vortmeyer AO, Diallo R, Poremba C, Giordano TJ, Sanders D, et al
. Survivin: A novel neuroendocrine marker for Pheochromocytoma. Eur J Endocrinol 2002;146:381-8.
Kajiwara Y, Yamasaki F, Hama S, Yahara K, Yoshioka H, Sugiyama K, et al
. Expression of Survivin in Astrocytic Tumors. Cancer 2003;97:1077-83.
Huang Y, Chen X, Chen N, Nie L, Xu M, Zhou Q. Expression and prognostic significance of Survivin splice variants in diffusely infiltrating astrocytoma. J Clin Pathol 2011;64:953-9.
Stenner M, Weinell A, Ponert T, Hardt A, Hahn M, Preuss SF, et al
. Cytoplasmic expression of Survivin is an independent predictor of poor prognosis in patients with salivary gland cancer. Histopathology 2010;57:699-706.
Kappler M, Kotzsch M, Bartel F, Fussel S, Lautenschlager C, Schmidt U, et al
. Elevated expression level of Survivin protein in soft-tissue sarcomas is a strong independent predictor of survival. Clin Cancer Res 2003;9:1098-104.
Taubert H, Heidenreich C, Holzhausen HJ, Schulz A, Bache M, Kappler M, et al
. Expression of Survivin detected by immunohistochemistry in the cytoplasm and in the nucleus is associated with prognosis of leiomyosarcoma and synovial sarcoma patients. BMC Cancer 2010;10:1-7.
Lechler P, Renkawitz T, Campean V, Balakrishnan S, Tingart M, Grifka J, et al
. The antiapoptotic gene Survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro.
BMC Cancer 2011;11:120.
Oluwadara O, Giacomelli L, Christensen R, Kossan G, Avezova R, Chiappelli F. LCK, Survivin and PI -3K in the molecular biomarker profiling of Oral Lichenplanus and Oral Squamous Cell Carcinoma. Bioinformation 2009;4:249-57.
Zhou S, Qu X, Yu Z, Zhong L, Ruan M, Ma C, et al
. Survivin as a potential early marker in the carcinogenesis of Oral Submucous Fibrosis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:575-81.
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.
Chen YK, Huse SS, Lin LM. Expression of inhibitor of apoptosis family proteins in human Oral Squamous Cell Carcinogenesis. Head Neck 2011;33:985-98.
Liu YM, Huang JH, Feng DY, Guo XC. Expression of Survivin and its correlation to angiogenesis in Oral Squamous Cell Carcinoma. Ai Zheng 2005;24:1354-7.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]