Khola Ahmad Khan1 BDS
Ahmad Waqas Javed2 BDS
Mariyah Javed3 BDS
OBJECTIVE: The aim of the study was to observe the expression of survivin in buccal mucosa of oral submucous fibrosis in betel nut and tobacco users.
METHODOLOGY: Forty patients were evaluated as per inclusion and exclusion criteria and informed consent was taken. Diagnosed Oral Submucous fibrosis patients inclusive of all age groups and both gender were included. Punch biopsy was performed under local anesthesia, and the sample size measuring 5mm was taken from the buccal mucosa. Tissue specimens were fixed and after tissue processing, staining with Hematoxylin and eosin (H&E) along with immunohistochemistry (IHC) was done.
RESULTS: Chi-square test was applied to observe the relation between immunoreactive score (IRS) score and Age groups, suggesting significant relation showing the value (p=.031). Relation between IRS levels and gender, suggesting no significant relation (p=0.243) Also insignificant association with respect to IRS and duration of smoking (p=0.155) was seen. Association between IRS and frequency of smoking daily (p=0.276) and IRS and the frequency of betel nut use (p=0.101) suggested insignificant results. However significant relation was seen between IRS levels and duration of betel nut usage (p=0.027).
STATISTICAL ANALYSIS: Data was entered in statistical package for social sciences (SPSS version 20) and all quantitative and qualitative variables were analyzed by using the software. Chi square test was applied to observe the association of age and gender with tobacco and betal nut chewing by observing the expression of survivin.
CONCLUSION: The purpose of an early diagnosis of OSF provides good quality of life to the patient by improving oral hygiene and oral intake quality and at the same time to obtain a sufficient mouth opening. The results showed that those who had history of betal nut chewing are more prone to malignant transformation.
HOW TO CITE: Khan KA, Javed AW, Javed M. Correlation of smoking and betel nut with the effect of survivin expression in oral Submucous fibrosis in local population. J Pak Dent Assoc 2015; 24(3):121-128.
Received: August 4 2015, Accepted: October 26 2015
Oral Squamous Cell Carcinoma (OSCC) is among the first eight most common cancers of the world.1 In 2005 a cancer research survey reported that Pakistan has an increased risk for oral cancer and graded as the second most common cancer reporting country as 8.8% in both men and women.2 Another global survey in 2005 on OSCC reported that more than one third of oral cancer was found in India and Pakistan.3 In 2000 Merchant and his coworkers observed that In Pakistan, females are more prone to oral cancers as compared to males.4
OSCC is a malignancy associated with multifactorial causative agent, combination of various factors like environmental, viral infections and genetic alteration which altogether results in a malignant lesion. Commonly OSCC occur in the presence of common premalignant conditions as Oral Leukoplakia, Oral Lichen Planus and Oral Submucous Fibrosis.1 Other contributing factors are alcohol and tobacco usage associated with viruses like papilloma viruses, Epstein-Barr virus, herpes simplex virus.5
Squamous cell carcinoma (SCC), common head and neck malignancy, with reported 30,000 patients including oral and pharyngeal cancer diagnosed annually in the United States alone. Globally there are approximately 200,000 deaths annually, with minimal improvement in survival rates despite advances in surgery, radiation and chemotherapy.6
OSF is a chronic progressive premalignant condition and commonly reported in Taiwan, Bangladaish, Pakistan, Mainland China and India.1 OSF is rarely seen in the United States and is seen among immigrants residing there, especially from South Asian population. Globally, OSF is found in 2.5 million people mostly from southern India and ranges from 0.2-2.3% in males and 1.2-4.57% in females.7 OSF has high morbidity because it causes inability to open mouth, trouble eating and eventually leading to nutritional deficits. Significant death rate since it can transform into OSCC.8 OSF as a precancerious condition has been proved by Pindborg and many other researchers and presented five characteristic points for its malignant tendency. Malignant transformation rate of OSF was found in the range of 7-13% depending on various studies mainly conducted in INDIA and Taiwan. The OSF geographic distribution is obvious as betal quid chewing habit (areca nut, slaked limeand others). So incidence of betal quid is directly propotional to severity of OSF that is an alarming situation in future.1 Another main contributing factor in the incidence and progression of OSF is tobacco use and according to Brundtland study in 2000, worldwide approximately 5 million deaths have been recorded annually because of tobacco and its associated products.9 WHO released a report indicating these deaths would be double in next ten years since the use of tobacco products will peak 10 million yearly.10
Survivin is a recently characterized dual functioning protein that plays an important role in apoptosis suppression, cell division, blood vessel growth, immune regulation and tumor metastasis.
Survivin expression can be used to predict the prognosis of cancer, since its expression is increased in malignant neoplasms. Various pre -clinical trials have shown its resistance to anti-cancer drugs and ionizing radiations given to the patient. Survivin is weakly expressed in the areas where there is increase in apoptosis, radiations given to cancerous tissues, and sensitized neoplastic cells to chemotherapy.11,12 Survivin expression is lowered in normal tissues and characterizes by selfrenewal and proliferation. Research revealed intense expression of survivin in solid neoplasms and blood cancers.13
Some studies detected the high expression of survivin protein in OSCC, which showed the important role of surviving in the progression of oral cancer.14-16 So the main idea behind this study was to observe the expression of survivin with relation to betal quid and tobacco usage and its tendency to transform OSF condition into OSCC. Survivin expression assessed in present study described the importance of immunohistochemistry and it can be used as a tool for the detection of early carcinogenesis in OSF. Therefore finding the key molecule in OSF malignant transformation is urgent and may contribute to add on current knowledge on the prevention, diagnosis and therapy of this disease.
All patients were evaluated as per inclusion and exclusion criteria. Tumors, OSF and healthy mucosa were taken at the time of surgical resection at Punjab Dental Hospital Lahore. Written informed consent was taken from all enrolled individuals. Proforma was provided, filled and signed by all enrolled subjects. The complete procedure was thoroughly explained to the patients. The clinical diagnosis and pathological stage of OSF is determined in terms of Pingborg criteria by the Department of Oral Pathology, Xiangya Hospital,
Central South University.17,18 OSF was classified into three stages:
- Stage I with mouth opening 45 mm
- Stage II Restricted with mouth opening 20-44 mm3. Stage III – Mouth opening < 20 mm Forty diagnosed OSF cases were selected without OSCC or neoplastic disease. Punch biopsy was performed under local anesthesia, and the sample size measuring 5mm was taken from the affected buccal mucosa. Tissue Processing
Cautious resection of OSF tissue was taken from buccal mucosa and processed in an automated processor.
After 24 hours, specimens were fixed in 4% buffered formalin solution. Three sections each of 3 to 4 microns thickness were cut from each selected block using a rotary microtome. Two respective slides were made from one tissue specimen. One of them was made for Haematoxylin and Eosin staining and the other one was
Figure 1: Showing no expression of surviving in the epithelial cells.
Figure 2: Showing intense expression of surviving in the epithelial cells in which nucleus and cytoplasm showed positivity in brown and dense granules.
Figure 3: Showing Dense Underlying Connective Tissue on H&E
made for immunohistochemistry with survivin. Sections were taken on the charged slides and were processed for respective staining.20
The prepared slides were seen under light microscope for the diagnosis of OSF and the findings noted down in proforma. Cytological findings
The findings in epithelium like presence of keratinization, thickness, dysplastic changes, inflammatory cells, pleomophishm, mitotic figures and hyperchromatism were recorded.
Apart from these the presence of fibrous tissue, hyalinization and inflammatory cells in the connective tissue was also noted and entered in the proforma. The readings were then entered onto the proforma of each subject, previously having their bio data.
An antibody will specifically bind to an antigen in the cells of tissue sections to produce an exclusive antibody-antigen complex that can be used for the
detection of specific molecule.21
Immunohistochemical staining of survivin antibody was performed as follows
- The de-paraffinized tissues segments treated with heat induced epitope retrieval (HIER) earlier to immunohistochemistry staining processes. Water bath temperature was sustained at 95-99ºC and the tissue sections were placed in pre heated buffer solution for HIER.
- HIER was done for recommended 40 minutes at 95-99ºC.
- Once the sections in the buffer were cool down for 20 minutes at room temperature after the heat treatment, the slides were washed with buffer water. Salinized slides (code S3003) for better fixing of tissue sections along with target retrieving solution (code S1700) were suggested.
- Sections were cut in 3 to 4 £gm thickness and placed on poly-lysine coated slide. The slides were fixed in the oven at 58ºC to 60ºC for 50 to 60 minutes.
- The sections were brought to water (de wax in xylene) and hydration through a descending alcohol series followed by a through wash in running tap water.
Carcinoma of intestine and stomach were used as Positive controls for survivin and also stained as per manufactured advice. Statistical Analysis:
Statistical analysis was calculated with Statistical Package for Social Sciences (SPSS) software, version 20.00. Chi square test was applied to observe the association of age and gender with tobacco and betal nut chewing by observing the expression of survivin.
A total number of 40 samples from the buccal mucosa of the patients with oral submucous fibrosis were taken. Age range of the subjects was divided into two groups i.e. 18-30 years (26 patients) and 31-45 years (14 patients). Both genders were included with males (24 patients) and females (16 patients).
One sample from each subject was taken and 2 slides were prepared of a single subject with a sub total of 80 slides. Slides were stained with H&E and immunohistochemistry was done. After completing the proformas the results were compared with the variables.
Table 1: IRS ASSOCIATIONS
Table1.1: Association of survivin expression with age groups
Chi square was applied to observe the relation between survivin expression and Age groups, suggesting there is significant relation between the two variables. Results are given in the table 1. smoking daily
Table 2: Association of survivin expression with duration of smoking
Chi square test was applied to calculate level of significance with respect to duration of smoking and insignificant results were obtained, as shown in table 2.
Table 3: Association of survivin expression with frequency of smoking daily
Another comparison between survivin expression and frequency of smoking daily gave insignificant results when chi square test was applied. The results are shown in the table 3.
Table 4: Association of survivin expression with duration of betel nut (years) use
Association between survivin expression and duration of betel nut usage daily gave significant results and suggested that increasing the duration of betel nut use increases the IRS Levels i.e. both the percentage and intensity of survivin is increased. The results are shown in the table 4.
Table 5: Association of survivin expression with frequency of betel nut use daily
However no significant relation was seen between the survivin expression and the frequency of betel nut use. The results are given in the table 5.
Oral squamous cell carcinoma (OSCC) is the most dominant carcinoma, accounting for more than eighty percent of head and neck cancers.22
In Pakistan, among all cancers, oral cancer is ranked as second prevalent cancer in females and third prevalent cancer in males.4
Oral premalignant lesions and OSCC are mainly treated on the basis of histological features, site involved, and stage of the disease. However, Oral health personnel should be aware of all the recent advances so the accurate, less time consuming diagnosis of oral premalignant lesions or OSCC can be made and eventually beneficial to the patient.23
Hence OSF is considered as a pre malignant condition usually associated with betel quid chewing and tobacco intake. Microscopically it is characterized by epithelial atrophy, basal cell layer hyperplasia, excessive deposition of collagen fibers in connective tissue and surface keratinization. Malignant transformation rate of oral sub mucous fibrosis into squamous cell carcinoma is 7.6% .24
Pre-cancerous conditions (33%) have a tendency to convert into OSCC. No absolute confirmation can be done histologically about their malignant transformation; therefore the need of new markers should be emphasized.16
Survivin an IAP which is expressed in tumors and also blood related malignancies.25 Apoptosis has been revolutionary in cancer research and new cancer advances. It is the programmed cell death which is controlled genetically.26 Survivin inhibits caspase 3 activity. Cytoplasmic survivin suppresses apoptosis whereas nuclear survivin controls cell division.27
Inhibition of apoptosis results in tumour progression and expression of survivin can be related to its antiapoptotic activity induced by FAS/TNF legation, proapoptotic Bax, effector caspases, and many chemotherapeutic drugs.28
L Lo Muzio in 2013, conducted a research in which the expression of survivin was seen both by IHC and western blotting in OSCC cases. A total number of 110 cases were taken for the study out of which 91 cases were strongly positive for survivin however the controlled group in which the normal oral mucosa was taken none of the case showed any positivity since survivin is expressed only in tumoral tissue and not in the normal mucosa. Tobacco used in two forms that is smoked tobacco in the form of cigarettes and smokeless tobacco which is chewed. A study was done by Merchant A in 2000 in Pakistan, in which 79 cases of OSCC were taken. Sixty eight percent were men, 22 years old (youngest) and 80 years old (eldest) with an average of 49 years. Patients having OSF were 19.1 times more expected to develop oral cancer than those with no OSF.4
The relationship of survivin expression with age, gender, size of tumor and involvement of nodes was insignificant.29
About 20 of 40 (50%) OSF cases showed phosphorylation of survivin. Three/10 (30%) of early stage (mean score 0.231, SD 0.169) and 7/15 (46.7%) of moderately advanced stage of OSF were found to show weak survivin (mean score 0.515, SD 0.102). Out of the 15 advanced stage of OSF cases, 10 (66.7%) showed moderate survivin (mean score 1.687, SD 0.304). No statistically significant correlation was found between the survivin and the pathological stages of OSF (P>0.05).
Punch biopsy can be considered a safe, easy and valid procedure to take biopsies from oral mucosa for the diagnosis of lesions.30
Chi-square test was applied to observe the relation between IRS score and Age groups, suggesting statistical significant (p=.031), however association between IRS score and gender, (p=.243) IRS and duration of smoking (p=.155), IRS and frequency of smoking daily and IRS and the frequency of betel nut use (p=.101), gave insignificant results (p=.276). IRS and duration of betel nut usage daily (p=.027).
A study conducted by Lo Muzio L in 2003, analyzed survivin expression in 10/30 cases (33%) of oral precancerous lesions without any malignant transformation, and 15/16 cases (94%) of oral precancerous lesions transformed into squamous cell carcinoma. Tumors originated from precancerous lesions, showed strong survivin positivity (100%). No significant relation was found between survivin expression and degree of dysplasia.16
In study by Ding YP in 2010, he investigated the role of survivin and caspase -3 in the development of oral cancer.17 OSCC and 28 cases of oral leukoplakia with dysplastic changes were seen. Immunohistochemical staining showed positivity with survivin in OSCC (21.89 ± 10.45) %. Caspase 3 was down regulated in dysplasia concluding survivin and caspase-3 are associated with carcinogenesis of the oral mucosa and survivin may restrain cell apoptosis by inhibiting caspase3.31
Tanaka C in 2003 conducted a study in which expression of survivin and its role in carcinogenesis was seen by immunohistochemistry .Premalignant lesions (37%) and malignancies (58%) showed strong positivity suggesting its role in the process of carcinogenesis.14
In the present study, association between Gender and history of smoking, duration of smoking, duration of betel nut use, frequency of smoking daily, frequency of betel nut use daily showed insignificant results (p>.05).
In contrast to the present study, the relationship of survivin expression with age, gender, size of tumor and involvement of nodes was also found insignificant by Millane and Ward.29
The male to female ratio of oral sub mucous fibrosis may differ with area, but females have a gender predilection. A study from Durban, South Africa, a female predilection was observed, with a male-to-female ratio of 1:13.32
It was confirmed by other studies, with a male-tofemale ratio of 1:733. In Pakistan, studies suggested a male-to-female ratio of 1:2.3.8
Patients with both OSF and OSCC were young males in a study by Chaturvedi with a history of chewing betel nut had better prognosis, less metastases to nodes and well differentiated34, however in the present study no significant association was seen between gender and dysplastic changes.
Considering age, Chi-square test was applied in the present study to observe the relation between Age groups and history of smoking, duration of smoking, frequency of smoking daily, duration of betel nut usage daily and frequency of betel nut use suggesting there is statistical significant relation between the age and history (p=.043), age and duration (p=.004), age and frequency of smoking
(p=.043), age and duration (p<.001), age and frequency of betel nut (p=.002).
In a research conducted in Saipan, 8.8% of teens (16.3± 1.5 y) diagnosed with oral sub mucous fibrosis .35 Most patient ages are ranged between 45-54 years with a history of betel nuts intake 5 times per day8 similar results showed by Santosh Patil in his study in 2014, that Betel nut was the main etiological factor with 73.3% and tobacco being the second one with 69.2% in 18-56 years old patients.36
Sarode observed in his study that the malignant transformation rate of oral sub mucous fibrosis into squamous cell carcinoma is 7.6%.24
A study was done by Merchant in 2000 in Pakistan, in which 79 cases of OSCC were taken.68% were men, age range 22 – 80 years with an average of 49 years. Patients with OSF were 19.1 times more prone to get squamous carcinoma than those with no OSF.4
The purpose of an early and more aggressive approach towards OSF is to provide good quality of life to the patient by improving oral hygiene and oral intake quality and at the same time to obtain a sufficient mouth opening. Immunoreactive (IRS) score changes assessed by our study depict the importance of immunohistochemistry and its expression can be used as a tool for the detection of early carcinogenesis in oral sub mucous fibrosis in those patients who are betal nut users or using tobacco. The individual mechanisms operating at various stages of the disease need further study in order to propose appropriate therapeutic interventions.
Disclosure: All authors contributed equally towards the final manuscript
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- Bhurgri Y. Cancer of the oral cavity – trends in KarachiSouth (1995-2002). Asian Pac J Cancer Prev. 2005;6:2226.
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1. Research Assistant (Oral Pathology) Department of Post Graduate Medical Institute, Lahore,Pakistan.
2. Research Assistant (Oral and Maxillofacial Surgery) Demondmerency College, Department of Maxillofacial Surgery, Lahore, Pakistan.
3.Research Assistant (Oral Pathology) Department of Oral Health Sciences, Shaikh Zayed Medical Complex, Lahore, Pakistan.
Corresponding author: “Dr. Khola Ahmad Khan ” < firstname.lastname@example.org >