Genotypes of Human Papilloma Virus and Identification of Prospective Protein Biomarkers in Oral Rinse from O Ral Squamous Cell Carcinoma

Abstract

Background: Oral cancer is the second preeminent malignancy in Pakistan after breast cancer, ascribed to widespread use of numerous perilous chewable tobacco formulations. The Human Papilloma Virus (HPV) has come forward as a new malefactor of malignant and pre malignant oral lesions. HPV related oral squamous cell carcinoma (OSCC) constitute an epidemiological, molecular and clinical distinctive subset of oral cancer. Regardless of the HPV status being related with molecular and clinical differences, all oral cancers are managed equally. Proteomic studies may help to understand the differences between HPV+VE and HPV-VE OSCC and let us to develop biomarkers for early detection, recurrence and prognosis leading to identification of therapeutic targets which will further initiate precisional treatment based on the biology of tumors. This study was designed to determine the association of HPV high-risk genotypes 16/18 in oral mucosa of chewable tobacco users and OSCC as well as identification of proteins in Oral rinse of OSCC patients with and without HPV with major focus on search for prospective tumor biomarker for HPV related OSCC. Methods: A case control study was designed with 100 OSCC patients and 200 controls. Persons addicted to chewable tobacco formulations such as gutka, pan, betel nut and naswar with or without oral lesions, having no delirious conditions were included. DNA from oral rinse of 300 subjects was taken. Samples were analysed by both conventional and real time PCR using “HPV consensus Gp5+/Gp6+ and HPV 16, 18 specific primers”. After PCR analysis, a random subset of 75 subjects was selected: 25 each of HPV +IVE OSCC, HPV –IVE OSCC and non- tobacco chewers. The peptides were separated by nanoflow liquid chromatograph system coupled online to LTQ-Orbitrap Velose mass spectrometer using a nanoelectrospray ion source (Thermo Scientific, Schwerte, Germany). Enrichment and protein–protein interaction (PPI) network analysis of the identified proteins was performed using FunRich: Functional Enrichment analysis tool version 3.1.3. HPV rates and types were compared between controls and OSCC and oral habits associated and non-associated OSCC samples by Chi-square test. Odds ratios (ORs) and 95% Confidence intervals for HPV and types were obtained by univariate and multivariate analysis. Posterior error probability (PEP) was calculated using Bayesian statistics as a probability of false hit using the peptide identification score (s) and length of peptide. Gene ontology (GO) functional categories, significant interactions and pathways associated with datasets were identified by using the hypergeometric test and pvalue correction with the BH and Bonferroni tests. In all statistical analysis, only p-value <0.05 was considered significant. Results: Out of 300 persons, 74/300 (25%) were found to be infected with HPV: “46/100(46%) from cases and 28/200(14%) from controls”. 26(35%) were infected by “both HPV 16/18 (23(50%) from cases and 3(12%) from controls”. Persons who were infected with HPV 16&18 had higher chances to develop OSCC as compared to those who didn‟t have HPV 16/18 (AOR: 21.4, 95% CI: 5.73 – 80.8). A total of 1995 proteins from HPV +ive OSCC (995), HPV –ive OSCC (816) and control samples (184) respectively were identified. Pairwise comparison revealed 37% of HPV +ive OSCC proteins were also present in HPV –ive OSCC samples whereas HPV-ive and HPV +ive OSCC share 18.6% and 17.1% of control proteins respectively. The 7-10 differentially expressed proteins from 74 secretory proteins in HPV +IVE OSCC were observed associated with 10 fold enriched pathways related to viral mRNA translation. The ribosomal proteins (RPS20, RPLP1, RLP0, RPS26, RPL12, RPS28 and RPL3) and glycosylated proteins were related to this pathway. Conclusion: The exposure to high risk strains of Human papilloma virus (16/18) in combination (p < 0.001, Adjusted odds ratio; 21.42) can be cause of development of OSCC. Chewing tobacco may be the cause of HPV transmission in the oral squamous cells through rough mucosa (p < 0.0001, Adjusted odds ratio; 11.85). To best of our knowledge, identification and interaction of secretory proteins of HPV +IVE OSCC are reported for the first time. The extensive ribosomal protein variations and their interaction in viral mRNA translation pathway may designate them as the potential biomarker for HPV +IVE OSCC. The protein level expression of RPLP1 and its involvement in OSCC may have been explored for the first time.