Nuclear Factor-Kappa B Expression in Oral Leukoplakia and Squamous Cell Carcinoma: An Immunohistochemical Study

INTRODUCTION: Oral cancer constitutes the sixth most common cancer worldwide and accounts for approximately 5% of all malignant tumours worldwide.1 In India and South East Asia, Oral Squamous Cell Carcinoma (OSCC) is the third most common malignancy constituting 50% of all malignant tumours. OSCC is the most common type of oral cancer and is often preceded by or associated with potentially malignant lesions or conditions such as leukoplakia and oral submucous fibrosis. Oral leukoplakia (OL) is “a white patch or plaque that cannot be characterized clinically or pathologically as any other disease”. It is the most common potentially malignant lesion of the oral mucosa. Tobacco and areca nut chewing (alone or in combination such as paan) are the habits that are positively associated with oral leukoplakia. Chewing areca nut containing betel quid without tobacco is an independent risk factor for developing oral cancer. When betel quid with tobacco is consumed with alcohol and smoking the relative risk increases eleven fold. The malignant transformation rate for leukoplakia ranges from 5-20% and is particularly correlated with the degree of dysplasia. The grading of epithelial dysplasia remains subjective as it relies on cellular atypia and architectural disturbances. It is important to identify markers that could help us to ascertain the malignant transformation of potentially malignant lesions to target them for aggressive treatment. NF-κ B is a family of nuclear transcription factors identified by Sen and Baltimore in 1986. It is involved in immune response and comprises of hetero or homo dimers of 5 different subunits NF-κB1, NF-κB2, REL A, REL B, cREL. NF-κB’s are transiently activated in response to infection or injury and are aberrantly activated in cancers contributing to their pathogenesis and therapeutic resistance. NF-κB is bound to inhibitor -kappa B (IκB) and is found in the cytoplasm of cells in an inactive form known as the canonical or classical form of NF-κB. In response to stimulus by growth factors or cytokines, IκB is phosphorylated, ubiquitinated and degraded by proteosome resulting in the activation of NF-κB which translocates to the nucleus and regulates target genes involved in immunoregulation, inflammation, proliferation and apoptosis. NF-κB is involved in the regulation of cell-cycle and apoptosis, thus playing an important role in cell proliferation and survival. Inappropriate NF-κB activation can mediate oncogenesis and tumor progression. It is known to inhibit apoptosis through the induction of anti-apoptotic proteins, and to suppress the apoptotic potential of chemotherapeutic agents, leading to chemoresistance. The present study was done to evaluate and compare the expression of NF-κB in formalin fixed paraffin embedded tissues of oral leukoplakia, oral squamous cell carcinoma and normal oral mucosa. AIM OF THE STUDY: To assess NF-κB expression in Oral squamous cell carcinoma, leukoplakia and normal buccal mucosa. OBJECTIVES OF THE STUDY: 1. To evaluate the expression of NF-κB in formalin fixed paraffin embedded tissues of normal buccal mucosa by immunohistochemistry. 2. To evaluate the expression of NF-κB in formalin fixed paraffin embedded tissue of oral squamous cell carcinoma from patients by immunohistochemistry. a) With areca chewing habit. b) With tobacco chewing habit. c) With both areca and tobacco chewing. d) With both smoking and chewing. 3. To evaluate the expression of NF-κB in formalin fixed paraffin embedded tissue of oral leukoplakia (epithelial dysplasia) by immunohistochemistry. 4. To compare the expression of NF-κB n oral squamous cell carcinoma, epithelial dysplasia and normal buccal mucosa. HYPOTHESIS (NULL): There is no difference in the expression of NF-κB in oral squamous cell carcinoma and leukoplakia when compared with normal buccal mucosa. MATERIALS AND METHODS: Study Design: A retrospective study was conducted in Department of Oral and Maxillofacial Pathology, Ragas Dental College & Hospital, Chennai, using archival paraffin embedded tissues. Study samples: The study material comprised of 80 formalin fixed, paraffin embedded tissue specimens (archival blocks). • Forty (n=40) histopathologically confirmed OSCC tissue specimens. • Twenty (n=20) histopathologically confirmed epithelial dysplasia tissue specimens • Twenty (n=20) normal buccal mucosa tissue specimens Study subjects The study comprised of 3 groups: Group 1 – (CASES) - Forty tissue blocks from patients diagnosed with OSCC clinically and confirmed histopathologically. Group 2- (CASES) - Twenty tissue blocks from patients diagnosed with leukoplakia clinically and confirmed histopathologically as epithelial dysplasia. Group 3- (CONTROLS) - Twenty patients who had clinically normal buccal mucosa, reporting to the outpatient department of oral and maxillofacial surgery for removal of impacted third molar constituted the control group. Inclusion Criteria: • They had no habit of smoking, alcohol consumption or chewing areca nut. • They were apparently healthy with no systemic disorders. • They were not on any medications for systemic diseases like hypertension, diabetes. METHODOLOGY: 1. A detailed case history including age, sex and occupation, past medical and dental history, history of habits, drugs and trauma were recorded. 2. General examination and intraoral examination was done. 3. Biopsy was done in both cases and controls. 4. The tissue taken was immediately transferred to 10% buffered formalin. 5. After adequate fixation, tissues were embedded in paraffim. 6. From the paraffim embedded blocks 4 micron thick, sections were cut and used, for routine hematoxylin and eosin (H&E) staining and Immunohistochemical (IHC) staining. 7. This project was approved by Institutional Review Board (IRB) of Ragas Dental College & Hospital, Chennai 8. Patient consent was taken for those patients from whom normal mucosa was obtained. Statistical Analysis: Statistical analysis was done using SPSS TM software (version 10.0.5). p value ≤0.05 was considered to be statistically significant. 1. Pearson’s Chi-square test was done to compare mean age, the distribution of gender and habits, tissue localization of stain, cellular location, nature of stain, intensity of stain and the percentage of cells stained among the three study groups. 2. The inter-observer variability for the intensity of stain was assessed by 2 investigators and their reports were confirmed with kappa statistics. 3. The mean labeling index between the groups was analysed by Mann Whitney U test. SUMMARY: 1. The study group comprised of Group I (OSCC n=40), Group II (leukoplakia n=20) and Group III (Normal n=20). 2. In OSCC, there was 98% positivity for NF – κB with 35% of cytoplasmic expression and 65% of nuclear expression. 3. In leukoplakia; there was 100% positivity for NF – κB expression; with 20% of cytoplasmic expression and 80% of nuclear staining. 4. In normal, there was 80% of positivity for NF – κB expression within 65% of cytoplasmic expression and 35% of nuclear expression. 5. In OSCC and leukoplakia when the epithelial staining intensity was compared with the connective tissue staining intensity there was a statistically significant correlation (p=0.001) and (p=0.043) respectively. 6. Nuclear expression of NF – κB exhibited difference (p=0.011) between the groups. CONCLUSION: In conclusion, our results show that there is increased expression of NF – κ B in OSCC and leukoplakia when compared to normal. Although the mean labeling index did not show any significant difference between OSCC and leukoplakia, further studies on a larger sample will help in ascertaining the exact role of NF – κ B expression in leukoplakia samples