Date of Award

8-9-2021

Degree Type

Thesis

Degree Name

PhD in Health Sciences

First Advisor

Dr Rozina Karmaliani

Second Advisor

Dr Adnan Jabbar

Third Advisor

Dr Shahid Pervez

Department

Biological and Biomedical Sciences

Abstract

Introduction: Oral cancer (OC) is the most common cancer in Pakistani males and the second most common in Pakistani females. The major risk factor includes peculiar chewing habits. However, for this largely preventable cancer, which commonly presents as a non-healing ulcer or mass, little insight is available regarding its molecular pathogenesis and its association with inflammation. Therefore, this study was undertaken to determine the association, if any, of inflammatory biomarkers like NFκB and COX-2 along with EGFR expression, miRNAs, and high-risk human papillomavirus (HRHPV).
Methodology: In Phase 1, a total of 186 patients diagnosed and treated for Oral squamous cell carcinoma (OSCC) were recruited. All relevant information regarding clinicopathological features, socioeconomic status (SES –education, occupation, and income), and chewing habits were obtained on a structured proforma with patients’ consent. Formalin-fixed paraffin-embedded (FFPE) biopsy specimens of all 186 samples were tested for HR-HPV type 16/18 by PCR followed by p16 immunostaining in a subset of cases (n=50). In phase 2, a subset of 50 samples was analysed for expression of EGFR, NFκB, and COX-2 by qPCR. In addition, EGFR gene amplification was tested by FISH, while COX-2 protein expression was tested by immunohistochemistry (IHC). For miRNA analysis, array profiling of ~2500miRNAs was performed on nine samples. From this profiling data, five miRNAs were selected for validation in 50 samples by qPCR on the basis of highly significant p-values or if associated with EGFR/COX-2 in the NetAffyx database. Appropriate statistical tests were applied, and significance criteria were set as p< 0.05 with 95% CI. >< 0.05 with 95% CI.
Results: Mean age of all 186 OSCC patients was 47.6±12.18 years, with a median of 48years and Male:Female ratio of 4:1. Most patients belonged to low SES (68.3%), and 77.4% were chronic habitual chewers. Male gender and SES were significantly associated with chewing habits (p< 0.05). The odds of developing OSCC involving buccal mucosa in chewers of any substance were higher than non-chewers. HR-HPV presence was seen in only seven cases out of 186, i.e., 3.8%. Out of these seven cases, five were positive for HPV16, whereas two were positive for both HPV16/18. Of the 50 samples analysed, transcriptional expression of EGFR was observed in 13 (26%), NFκB in 11 (22%), and COX-2 in 17 (34%). Expression of these genes was more common in chewers than non-chewers. FISH analysis of the EGFR gene showed the amplified copy number in ten of qPCR EGFR positive cases. All these three genes were found to be significantly associated with each other (Fischer Exact test, p>< 0.001). EGFR mRNA and COX-mRNA expression was significantly associated with the chewing of gutka (the most carcinogenic form of chewing substance). COX-2 protein expression was also found to be associated with all forms of chewing substances (p>< 0.05). A total of 281 miRNAs were dysregulated in nine cases: three cases of chewers, three nonchewers, and three HR-HPV+ve in miRNA profiling. No significant association of five miRNAs chosen for further validation was seen with chewing status or HR-HPV; however, miR 222-3p was found to be significantly downregulated in chewers expressing EGFR, NFκB, and COX-2 compared to non-chewers (p>< 0.05). >< 0.05). The odds of developing OSCC involving buccal mucosa in chewers of any substance were higher than non-chewers. HR-HPV presence was seen in only seven cases out of 186, i.e., 3.8%. Out of these seven cases, five were positive for HPV16, whereas two were positive for both HPV16/18. Of the 50 samples analysed, transcriptional expression of EGFR was observed in 13 (26%), NFκB in 11 (22%), and COX-2 in 17 (34%). Expression of these genes was more common in chewers than non-chewers. FISH analysis of the EGFR gene showed the amplified copy number in ten of qPCR EGFR positive cases. All these three genes were found to be significantly associated with each other (Fischer Exact test, p< 0.001). EGFR mRNA and COX-mRNA expression was significantly associated with the chewing of gutka (the most carcinogenic form of chewing substance). COX-2 protein expression was also found to be associated with all forms of chewing substances (p>< 0.05). >< 0.001). EGFR mRNA and COX-mRNA expression was significantly associated with the chewing of gutka (the most carcinogenic form of chewing substance). COX-2 protein expression was also found to be associated with all forms of chewing substances (p< 0.05). >< 0.05). A total of 281 miRNAs were dysregulated in nine cases: three cases of chewers, three nonchewers, and three HR-HPV+ve in miRNA profiling. No significant association of five miRNAs chosen for further validation was seen with chewing status or HR-HPV; however, miR 222-3p was found to be significantly downregulated in chewers expressing EGFR, NFκB, and COX-2 compared to non-chewers (p< 0.05). >< 0.05).
Conclusion: It was concluded that in Pakistan, OC has a very strong association with chewing habits as ~80% of patients had a history of chronic chewing on consecutive sampling, making it a largely preventable cancer. The prevalence of HR-HPV in OSCC was very low. A significant association of gutka chewing was seen with EGFR and COX-2 expression. In addition, the molecular pathogenesis of OSCC suggests the significant interplay of inflammatory biomarkers (NFκB and COX-2) with EGFR and miRNAs in chronic chewers.

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Last Page

173

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