The repertoire of mutational signatures in tobacco- and non-tobacco-induced oral cancer

The use of tobacco products is one of the established contributors toward the development and spread of oral cancer. Additionally, recent research has indicated oral microbiome, infections with Human papilloma virus (HPV), Epstein–Barr virus (EBV), Candida as significant contributing factors to this disease along with lifestyle habits. Deregulation of cellular pathways envisaging metabolism, transcription, translation, and epigenetics caused by these risk factors either individually or in unison is manifold, resulting in the increased risk of oral cancer. Globally, this cancer continues to exist as one of the major causes of cancer-related mortalities; the numbers in the developing South Asian countries clearly indicate yearly escalation. This review encompasses the variety of genetic modifications, including adduct formation, mutation (duplication, deletion, and translocation), and epigenetic changes evident in oral squamous cell carcinoma (OSCC). In addition, it highlights the interference caused by tobacco products in Wnt signaling, PI3K/Akt/mTOR, JAK-STAT, and other important pathways. The information provided also ensures a comprehensive and critical revisit to non-tobacco-induced OSCC. Extensive literature survey and analysis has been conducted to generate the chromosome maps specifically highlighting OSCC-related mutations with the potential to act as spectacles for the early diagnosis and targeted treatment of this disease cancer (AU)

Epstein-Barr Virus Detection and LMP-1 Expression in Thai Patients with Oral Squamous Cell Carcinoma.

BACKGROUND: To date, high-risk human papillomavirus (HPV) is primarily linked to oropharyngeal carcinoma, but only a small subset of oral squamous cell carcinoma (OSCC) is truly driven by high-risk HPV. In addition, Epstein-Barr virus (EBV) is another potential oncogenic virus for OSCC development. This study aims to investigate the role of EBV infection in Thai patients with OSCC. METHODS: Forty-seven formalin-fixed paraffin-embedded specimens of OSCC were obtained. EBV DNA was detected by polymerase chain reaction analysis using primers for LMP-1 region of EBV. EBV-positive OSCC cases were subjected to LMP-1 immunohistochemical analysis and EBV-encoded small RNA (EBER) in situ hybridization to determine EBV cellular localization in OSCC. LMP-1 immunohistochemical analysis was also performed in all EBV-negative OSCC cases. RESULTS: Of the 47 OSCC specimens, ten (21%) exhibited EBV DNA by PCR analysis. Seven of ten (70%) EBV-positive specimens showed high-grade LMP-1 expression by immunohistochemistry. However, no EBER expression was detected in all EBV-positive OSCC specimens. In EBV-negative specimens, LMP-1 was also negative except in 3 specimens which showed low grade expression of LMP-1. CONCLUSION: The prevalence of EBV infection in OSCC in this group of Thai patients was 21%. Most of EBV-positive OSCC cases showed LMP-1 expression but a lack of EBER expression. From our findings, we presume that EBV may take some roles in OSCC development in this group of participants.

Demographic Profile of p16 Immunopositive and HPV DNA PCR Positive Oral Squamous Cell Carcinoma in a Large Cohort of Indian Patients.

BACKGROUND: The Indian subcontinent has the highest incidence of oral cavity squamous cell carcinoma in the world. The high incidence of tobacco chewing habit with or without smoking has been found to be the chief culprit. However in a minor subset of patients Human Papilloma Virus may play a role. MATERIALS AND METHODS: A total of 800 cases of Oral squamous cell carcinoma were included in the study. The patients were given a questionnaire comprising of questions about demographic details and habits. The biopsy samples were routinely processed for immunohistochemistry for p16 (E6H4 clone, CINtec histology, Roche diagnostics). Cases with 2+/3+ positive nuclear staining with more than 75% cells immunopositive were taken as p16 immunopositive as per the AJCC criteria and were further subjected to HPV DNA PCR for which DNA was extracted from the formalin fixed paraffin embedded tissue. RESULTS: Out of 800 OSCC cases 139 (17.37%) showed p16 immunopositivity by AJCC criteria. Out of these, 104 (104/139, 74.8%) cases were positive by HPV DNA PCR for HPV-16/18. Following patient characteristics were associated with a higher proportion of p16 and HPV DNA positivity-urban residence, vegetarian diet, illiteracy, graduate or higher education. No correlation was noted with gender, tobacco smoking or chewing habits, religion, occupation or site of tumor. The p16 immunopositivity was higher in the younger age group with no tobacco habits. CONCLUSION: A significant proportion of OSCC cases in India are associated with HPV infection. A higher percentage of p16 immunopositivity amongst younger patients with no tobacco habits points towards a distinct subset of patients in whom HPV may be the chief culprit and not just playing a supporting role.

Exome Sequencing with Validations and Expression of p16/CDKN2A Shows no Association with HPV in Oral Cancers.

PURPOSE: Despite many studies attributing HPV infection to oropharyngeal tumorigenesis, its involvement in non-oropharyngeal cancers is ambiguous. We have evaluated the mutation profile of p16 along with protein expression and correlated it with the HPV status in oral cancers. METHODS: Somatic mutations in p16 were studied by exome sequencing (n=25) and validated by Sequenom Mass spectrometry (n=50).  Expression of p16 was studied by immunohistochemistry (IHC) and correlated with HPV16/18 status evaluated by PCR, and IHC (n=221) in oral cancers. RESULTS: Out of 25 oral cancer patients' samples sequenced by Exome sequencing, p16 mutations were found in 4 samples (16%). All the p16 mutations were identified in patients with cancers in the site of gingivobuccal complex and not tongue subsite. All the 4 patients with p16 mutations had failed treatment, and showed a significantly poor disease-free survival. Insilico analysis of the types of p16 mutations showed mutated, truncated p16 protein having an increased intrinsic disorder, and all the mutations involved truncation post arginine. Validation of the p16 mutations by mass spectrometry showed 8/50 (16%) of patients harbouring pArg80Ter mutation, of which 7/8 (87.5%) had failed treatment.  Overexpression of p16 in >70% of the tumour cells was found in 21.4% (26/121) OSCC patients, 6.75% (5/74) OPML patients and p16 expression was significantly correlated (p=0.001; χ2 = 25.601) to the grade. All the samples were studied for HPV presence by PCR and IHC. We found that none of the p16 positive tumours showing expression in >70% of the tumour cells harbored HPV both by PCR as well as IHC. CONCLUSION: Our study emphasises the importance of p16 in oral cancers, and shows that oral cancer is not HPV associated and p16 expression is not a surrogate marker for HPV.

Prevalence of Merkel Cell Polyomavirus (MCPyV) in the Oral Cavity Biopsies in Northern Iran.

OBJECTIVE: Infection with human tumor viruses is one of the hypothesized causes of cancer. The current investigation aimed to explore the presence and quantitative analysis of a new human tumor virus, Merkel cell polyomavirus (MCPyV) in tissue samples of 114 patients with oral cavity lesions including oral squamous cell carcinoma (OSCC), oral lichen planus (OLP), Dysplasia and oral irritation fibroma (OIF) in Northern Iran. METHODS: From 114 formalin fixed paraffin embedded samples; 35 with SCC, 29 with OLP, 14 with dysplasia and 36 with OIF were cut, deparaffinized and DNA was extracted. Quantitative detection of MCPyV large T antigen was performed by absolute quantitative Real-Time PCR. RESULT: MCPyV DNA was detected in 30.6% (n: 11/36) of IF, 24.1% (n; 7/29) of OLP, 21.4% (n:3/14) of dysplasia and 20% (n;7/35) of OSCC samples. The mean MCPyV DNA copy number was 2.32×10-2 ± 3.97 ×10-2, 2.02×10-2 (SD=3.13×10-2), 2.69×10-4 (SD=2.51×10-4), and 2.56×10-4 (SD=6.73×10-4) per cell in OSCC, dysplasia and both of OLP and OIF samples, respectively (P=0.76). CONCLUSION: This study provides the first data from Iran regarding the presence of MCPyV genome in oral cavity lesions and oral cancer. These results also emphasize that MCPyV has an active role in the occurrence of oral lesions and progression to cancer. Further studies should be carried out to clarify the role of MCPyV in oral cavity lesions.

Double Positivity for HPV DNA/P16INK4a Does Not Influence Survival of Patients With Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: We investigated the prevalence of human papillomavirus (HPV) in a prospective cohort of patients with squamous cell carcinoma of the oral cavity (OSCC) using both p16INK4a and HPV DNA, i.e., double positivity, as a definition criterion. Additionally, we examined the association of HPV with survival. PATIENTS AND METHODS: Samples from 280 OSCC patients were analyzed for HPV-positivity using p16INK4a immunohistochemistry (IHC) and in situ hybridization (ISH)/LCD arrays, for HPV low and high-risk types. Only patients positive for both p16INK4a and HPV DNA were considered as HPV-positive. Survival probabilities and 95% confidence intervals were estimated using the Kaplan-Meier method. Cox proportional hazards models were used to assess HPV association with disease-free survival (DFS), cause-specific survival (CSS) and overall survival (OS) in a competing risks scenario. RESULTS: Specimen from 30 (10.7%) patients were p16+ and HPV DNA+, while 31 (11.0%) were either p16+ or HPV DNA+ only. OS probabilities at five years for HPV-positive and -negative groups were 50.9% (35.4%-73.1%) and 52.9% (47.0%-59.5%), respectively. HPV double positivity influenced neither OS, CSS nor DFS: HR=0.84 (0.43-1.63), 1.64 (0.76-3.54) and 1.13 (0.55-2.35), respectively. CONCLUSION: In contrast to oropharyngeal cancer, the prevalence of HPV in OSCC is low and the presence of HPV does not influence survival outcomes. Hence, there is no evidence to support a parallel transfer of therapy regimen for HPV-positive OPC to OSCC, in terms of therapy de-escalation and/or vaccination.

Detection of human papillomavirus infection in oral cancers reported at dental facility: assessing the utility of FFPE tissues.

Incidence of human papillomavirus (HPV)-associated oral cancers is on the rise. However, epidemiological data of this subset of cancers are limited. Dental hospital poses a unique advantage in detection of HPV-positive oral malignancies. We assessed the utility of formalin-fixed paraffin-embedded (FFPE) tissues, which are readily available, for evaluation of high-risk HPV infection in oral cancer. For protocol standardization, we used 20 prospectively collected paired FFPE and fresh tissues of histopathologically confirmed oral cancer cases reported in Oral Medicine department of a dental hospital for comparative study. Only short PCRs (~ 200 bp) of DNA isolated using a modified xylene-free method displayed a concordant HPV result. For HPV analysis, we used additional 30 retrospectively collected FFPE tissues. DNA isolated from these specimens showed an overall 23.4% (11/47) HPV positivity with detection of HPV18. Comparison of HPV positivity from dental hospital FFPE specimens with overall HPV positivity of freshly collected oral cancer specimens (n = 55) from three cancer care hospitals of the same region showed notable difference (12.7%; 7/55). Further, cancer hospital specimens showed HPV16 positivity and displayed a characteristic difference in reported sub-sites and patient spectrum. Overall, using a xylene-free FFPE DNA isolation method clubbed with short amplicon PCR, we showed detection of HPV-positive oral cancer in dental hospitals.

Impact of SARS-CoV-2 infection on oral carcinoma patients.

Coronavirus-related Severe Acute Respiratory Syndrome (SARS-CoV) in 2002/2003, Middle-East Respiratory Syndrome (MERS-Cov) in 2012/2013, and especially the current 2019/2020 Severe Acute Respiratory Syndrome-2 (SARS-CoV-2) tested the national health systems' endurance worldwide. In order to fight this emergency situation, a variety of pharmaceutical companies focused on the design and development of efficient vaccines that are considered necessary for providing a level of normalization in totally affected human social-economical activity worldwide. COVID-19 led to an increased uncertainty in the field of oncological patients' management disrupting the normal conditions of therapeutic and monitoring procedures. In the current article, we explored the impact of SARS-CoV-2 infection on oral carcinoma patients. We observed COVD-19 pandemic negatively affects the normality regarding early diagnosis and optimal management (surgical operation, post-operational follow up/monitoring) in HNSCC/OSCC patients. Understanding the involvement of SARS-CoV-2 in the progression of malignancies is the first critical step for targeting the virus by efficient monoclonal antibodies and vaccines.

Overview of Candida albicans and Human Papillomavirus (HPV) Infection Agents and their Biomolecular Mechanisms in Promoting Oral Cancer in Pediatric Patients.

Oral carcinoma represents one of the most common malignancies worldwide. Oral squamous cell carcinomas (OSCCs) account over 90% of all oral malignant tumors and are characterized by high mortality in the advanced stages. Early diagnosis is often a challenge for its ambiguous appearance in early stages. Mucosal infection by the human papillomavirus (HPV) is responsible for a growing number of malignancies, particularly cervical cancer and oropharyngeal carcinomas. In addition, Candida albicans (C. albicans), which is the principal fungi involved in the oral cancer development, may induce carcinogenesis through several mechanisms, mainly promoting inflammation. Medical knowledge and research on adolescent/pediatric patients' management and prevention are in continuous evolution. Besides, microbiota can play an important role in maintaining oral health and therefore all human health. The aim of this review is to evaluate epidemiological and pathophysiological characteristics of the several biochemical pathways involved during HPV and C. albicans infections in pediatric dentistry.

Germline determinants of humoral immune response to HPV-16 protect against oropharyngeal cancer.

Although several oropharyngeal cancer (OPC) susceptibility loci have been identified, most previous studies lacked detailed information on human papillomavirus (HPV) status. We conduct a genome-wide analysis by HPV16 serology status in 4,002 oral cancer cases (OPC and oral cavity cancer (OCC)) and 5,256 controls. We detect four susceptibility loci pointing to a distinct genetic predisposition by HPV status. Our most notable finding in the HLA region, that is now confirmed to be specific of HPV(+)OPC risk, reveal two independent loci with strong protective effects, one refining the previously reported HLA class II haplotype association. Antibody levels against HPV16 viral proteins strongly implicate the protective HLA variants as major determinants of humoral response against L1 capsid protein or E6 oncoprotein suggesting a natural immune response against HPV(+)OPC promoted by HLA variants. This indicates that therapeutic vaccines that target E6 and attenuate viral response after established HPV infections might protect against HPV(+)OPC.

Cell-free DNA and circulating tumor cell kinetics in a pre-clinical head and neck Cancer model undergoing radiation therapy.

BACKGROUND: Monitoring circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), known as liquid biopsies, continue to be developed as diagnostic and prognostic markers for a wide variety of cancer indications, mainly due to their minimally invasive nature and ability to offer a wide range of phenotypic and genetic information. While liquid biopsies maintain significant promising benefits, there is still limited information regarding the kinetics of ctDNA and CTCs following radiation therapy which remains a vital treatment modality in head and neck cancers. This study aims to describe the kinetics of ctDNA and CTCs following radiation exposure in a preclinical rabbit model with VX2 induced buccal carcinoma. METHODS: Seven rabbits were inoculated with VX2 cells in the buccal mucosa and subjected to radiation. At selected time points, blood sampling was performed to monitor differing levels of ctDNA and CTC. Plasma ctDNA was measured with quantitative PCR for papillomavirus E6 while CTCs were quantified using an immunomagnetic nanoparticles within a microfluidic device. Comparisons of CTC detection with EpCAM compared to multiple surface markers (EGFR, HER2 and PSMA) was evaluated and correlated with the tumor size. RESULTS: Plasma ctDNA reflects the overall tumor burden within the animal model. Analysis of correlations between ctDNA with tumor and lymph node volumes showed a positive correlation (R = 0.452 and R = 0.433 [p < 0.05]), respectively. Over the course of treatment, ctDNA levels declined and quickly becomes undetectable following tumor eradication. While during the course of treatment, ctDNA levels were noted to rise particularly upon initiation of radiation following scheduled treatment breaks. Levels of CTCs were observed to increase 1 week following inoculation of tumor to the primary site. For CTC detection, the use of multiple surface markers showed a greater sensitivity when compared to detection using only EpCAM. Plasma CTC levels remained elevated following radiation therapy which may account for an increased shedding of CTCs following radiation. CONCLUSION: This study demonstrates the utility of ctDNA and CTCs detection in response to radiation treatment in a preclinical head and neck model, allowing for better understanding of liquid biopsy applications in both clinical practice and research development.

Salivary detection of high-risk human papillomavirus 16 in oral squamous cell carcinoma using polymerase chain reaction in the South Indian population.

INTRODUCTION: Human papilloma virus (HPV) has been associated with oral squamous cell carcinoma (OSCC) as a potential carcinogen. There are several types of HPV, of which type 16 has been strongly implicated in carcinogenesis. HPV16 in saliva can potentially facilitate early detection of subclinical cases that may warrant further diagnosis, monitoring and intervention. AIM: The aim of this study was to evaluate the presence of HPV 16 in saliva and lesional tissue of OSCC and to determine the use of saliva as an alternative non invasive diagnostic tool in HPV16 identification. MATERIALS AND METHODS: 30 cases of Histopathologically confirmed OSCC with HPV positive on ELISA were taken up for the study. The tumour tissue and saliva sample of each patient were obtained to detect the presence of specific HPV16 genotype by polymerase chain reaction (PCR). The data was subjected to statistical analysis using Student t-test. RESULTS: In our study we found 28/30, 26/30 positive for HPV 16 in tissue and saliva samples respectively on PCR analysis. The P value was statistically significant (0.00). CONCLUSION: The study revealed significant prevalence of HPV 16 among both tissue and salivary specimens of OSCC patients in south Indian population. Though, the yielded content was relatively less in saliva, it can be concluded that, saliva being a non invasive tool proved to be as useful as tissue specimen and can be used as an alternative indicator of HPV16 positivity in OSCC.

The Effect of Prophylactic HPV Vaccines on Oral and Oropharyngeal HPV Infection-A Systematic Review.

Human papillomavirus (HPV) imposes an increased risk of developing cervical, anal and oropharyngeal cancer. In the Western world, HPV infection is currently the major cause of oropharyngeal cancer. The effectiveness of HPV vaccines for oral or oropharyngeal HPV infection is yet to be determined. This study conducted a systematic literature search in Pubmed and Embase. Studies investigating the impact of HPV vaccines on oral or oropharyngeal HPV infection were enrolled. This review reports the relative prevention percentage (RPP), including a risk of bias assessment as well as a quality assessment study. Nine studies were included (48,777 participants): five cross-sectional studies; one randomized community trial study (RCT); one longitudinal cohort study; and two case-control studies. A significant mean RPP of 83.9% (66.6-97.8%) was calculated from the cross-sectional studies, 82.4% in the included RCT and 83% in the longitudinal cohort study. Further, two case-control studies that measured antibody response in participants immunized with HPV vaccines were included. Respectively, 100% and 93.2% of participants developed HPV-16 Immunoglobulin G (IgG) antibodies in oral fluids post-vaccination. Analysis of the studies identified a significant decrease in vaccine-type oral or oropharyngeal HPV infections in study participants immunized with HPV vaccines across study designs and heterogenous populations. Further, a significant percentage of participants developed IgG antibodies in oral fluid post-vaccination.

Conversion from epithelial to partial-EMT phenotype by Fusobacterium nucleatum infection promotes invasion of oral cancer cells.

The ability of cancer cells to undergo partial-epithelial mesenchymal transition (p-EMT), rather than complete EMT, poses a higher metastatic risk. Although Fusobacterium nucleatum mainly inhabits in oral cavity, attention has been focused on the F. nucleatum involvement in colorectal cancer development. Here we examined the p-EMT regulation by F. nucleatum in oral squamous cell carcinoma (OSCC) cells. We cultured OSCC cells with epithelial, p-EMT or EMT phenotype with live or heat-inactivated F. nucleatum. Expression of the genes involved in epithelial differentiation, p-EMT and EMT were examined in OSCC cells after co-culture with F. nucleatum by qPCR. Cell growth and invasion of OSCC cells were also examined. Both live and heat-inactivated F. nucleatum upregulated the expression of p-EMT-related genes in OSCC cells with epithelial phenotype, but not with p-EMT or EMT phenotype. Moreover, F. nucleatum promoted invasion of OSCC cells with epithelial phenotype. Co-culture with other strains of bacteria other than Porphyromonas gingivalis did not alter p-EMT-related genes in OSCC cells with epithelial phenotype. F. nucleatum infection may convert epithelial to p-EMT phenotype via altering gene expression in OSCC. Oral hygiene managements against F. nucleatum infection may contribute to reduce the risk for an increase in metastatic ability of OSCC.

Reciprocal transactivation of Merkel cell polyomavirus and high-risk human papillomavirus promoter activities and increased expression of their oncoproteins.

BACKGROUND: Approximately 15% of human cancers are attributed to viruses. Numerous studies have shown that high-risk human polyomaviruses (HR-HPV) and Merkel cell polyomavirus (MCPyV) are two human tumor viruses associated with anogenetal and oropharyngeal cancers, and with Merkel cell carcinoma, respectively. MCPyV has been found in HR-HPV positive anogenetal and oropharyngeal tumors, suggesting that MCPyV can act as a co-factor in HR-HPV induced oncogenesis. This prompted us to investigate whether the oncoproteins large T-antigen (LT) and small antigen (sT) of MCPyV could affect the transcriptional activity HPV16 and HPV18 and vice versa whether HPV16 and HPV18 E6 and E7 oncoproteins affected the expression of MCPyV LT and sT. Reciprocal stimulation of these viral oncoproteinscould enhance the oncogenic processes triggered by these tumor viruses. METHODS: Transient co-transfection studies using a luciferase reporter plasmid with the long control region of HPV16 or HPV18, or the early or late promoter of MCPyV and expression plasmids for LT and sT, or E6 and E7, respectively were performed in the HPV-negative cervical cancer cell line C33A, in the keratinocyte cell line HaCaT, and in the oral squamous cell carcinoma cell line HSC-3. Transfections were also performed with deletion mutants of all these promoters and with mutants of all four oncoproteins. Finally, the effect of E6 and E7 on LT and sT expression in the MCPyV-positive Merkel cell carcinoma cell line WaGa and the effect of LT and sT on the expression of E6 and E7 was monitored by Western blotting. RESULTS: LT and sT stimulated the transcriptional activity of the HPV16 and HPV18 LCR and v.v. E6 and E7 potentiated the MCPyV early and late promoter in all cell lines. Induction by E6 and E7 was p53- and pRb-independent, and transactivation by LT did not require DNA binding, nuclear localization and HSC70/pRb interaction, whereas sT stimulated the HPV16/18 LCR activity in a PP2A- and DnaJ-independent manner. CONCLUSIONS: These results indicate that the co-infection of MCPyV may act as a co-factor in the initiation and/or progression of HPV-induced cancers.

Salivary High-Risk Human Papillomavirus (HPV) DNA as a Biomarker for HPV-Driven Head and Neck Cancers.

High-risk human papillomavirus (HR-HPV) infection is a major risk factor of head and neck cancers (HNCs). Despite the rising prevalence of HPV-driven HNC (HPV-HNC), biomarkers for detection, prognostication, and disease monitoring are lacking. To evaluate the capacity of salivary HR-HPV DNA as a biomarker of HPV-HNC, the salivary HR-HPV statuses of 491 and 10 patients with primary and recurrent HNC, respectively, were determined at diagnosis, using quantitative real-time PCR and MassARRAY. Tumor cyclin-dependent kinase inhibitor 2A (p16) expression was determined by IHC analysis. Patients with oropharyngeal cancer (OPC) (n = 215) were followed up for ≤5 years. Survival characteristics were evaluated in terms of event-free and cause-specific survival. Of the primary-HNC cohort, 43.2% were positive for salivary HR-HPV DNA, with most having OPC. Salivary HR-HPV DNA was detected in 81.4% of tumor p16-positive OPC patients at diagnosis. Prognosis in salivary HR-HPV-positive OPC patients was favorable compared with that in salivary HR-HPV-negative patients (event-free survival, hazard ratio = 0.42 [95% CI, 0.21-0.81, P = 0.010]; cause-specific survival, hazard ratio = 0.39 [95% CI, 0.18-0.86, P = 0.019]). In the recurrent-HNC cohort, salivary HR-HPV DNA was detected in 83.3% of those who previously had tumor p16-positive HNC. These findings indicate that this liquid biopsy-based, noninvasive biomarker can play an essential role in the detection and management of HPV-HNC.

Prevalence of human papillomavirus (HPV) in oral mucosal lesions in Iran: A systematic review and meta-analysis.

The role of human papillomavirus (HPV) in the development of oral lesions is controversial. There has been no comprehensive study about HPV prevalence in Iran. This systematic review and meta-analysis were aimed at finding HPV prevalence of oral lesions and normal oral mucosa in Iran. International (PubMed, Web of Science, and Scopus) and national (Iranmedex, Irandoc, and SID) databases were searched systematically until October 2020. Studies that examined the prevalence of HPV in oral lesions by polymerase chain reaction method were included. The heterogeneity of articles was assessed with the Cochran test and I-Square statistics. The prevalence rate of HPV was calculated using a random-effect model. Of 3729 initially searched articles, 29 articles were eligible for inclusion. The overall prevalence of HPV in oral lesions was 21%. The prevalence was the highest in Rasht (50%) city. Lip lesions had the highest HPV prevalence (40%). According to the classification of lesions, the highest prevalence was of precancerous lesions (29%) and the lowest in normal mucosa (8%). Well-differentiated tumors showed a higher prevalence than poorly-differentiated ones. The highest prevalence of HPV was hairy leukoplakia (70%) and the lowest was of pyogenic granuloma (6%). Also, the prevalence was 31% in oral squamous cell carcinoma. There are differences between HPV prevalence according to the geographical area, intraoral location, type of lesion, and grading. As HPV prevalence was fairly high, further attention to vaccination and treatment for HPV in Iran, as a potential risk factor for oral precancerous and cancerous lesions is recommended.

Application of the Bethesda system of reporting for cervical cytology to evaluate human papilloma virus induced changes in oral leukoplakia, oral squamous cell carcinoma, and oropharyngeal squamous cell carcinoma: A cytomorphological and genetic study.

BACKGROUND: Human papilloma virus (HPV) has a well-established carcinogenic role in certain head and neck cancers. These HPV associated cancers possess unique clinicopathological behavior and exhibits better prognosis than their negative counterparts. Detection through polymerase chain reaction (PCR) has been considered as the "gold standard" but imposes burden in low resource settings. Therefore, in the present study, we assessed the validity of cytomorphological features for the detection of HPV in oral leukoplakia (OL), oral squamous cell carcinoma (OSCC), and oropharyngeal squamous cell carcinoma (OPSCC). METHODOLOGY: This study included 63 subjects comprising of 25 OL, 26 OSCC, and 12 OPSCC cases. Exfoliated cells were collected and processed for PCR followed by Papanicolaou staining and subsequent grading. Additionally the non-classical signs were evaluated and statistical analysis included Chi-square and Spearman's test. RESULT: 23/63 (36.5%) cases showed PCR positivity for HPV16. Most of the cytomorphological features showed significant correlation for the presence of HPV. A greater sensitivity and specificity was observed in the Bethesda system for reporting cervical cytology (TBS) than the Papanicolaou grading system. CONCLUSION: We conclude that the non-classic cytological features could be employed in the detection of HPV in low resource settings with improved sensitivity. Liquid based cytology graded using TBS could be suitable for oral cytology in the detection of early atypical changes.

Co-expression of low-risk HPV E6/E7 and EBV LMP-1 leads to precancerous lesions by DNA damage.

BACKGROUND: Low-risk human papillomavirus (HPV), such as types 6 and 11, is considered non-oncogenic, but these types have been detected in oral cancer tissue samples, suggesting their possible involvement in oral carcinogenesis. Because double infection of high-risk HPV and Epstein-Barr virus (EBV) is known to be involved in oral carcinogenesis, we hypothesized that low-risk HPV and EBV co-infection can transform the oral cells. To verify our hypothesis, we evaluated the transformation activity of cell lines expressing both low-risk HPV E6/E7 and EBV LMP-1. METHODS: We transduced HPV6, 11 and 16 E6/E7 genes and EBV LMP-1 gene into primary mouse embryonic fibroblasts. The cell lines were examined for indices of transformation activity such as proliferation, induction of DNA damage, resistance to apoptosis, anchorage-independent growth, and tumor formation in nude mice. To evaluate the signaling pathways involved in transformation, NF-κB and p53 activities were analyzed. We also assessed adhesion signaling molecules associated with anchorage-independent growth such as MMP-2, paxillin and Cat-1. RESULTS: Co-expression of low-risk HPV6 E6 and EBV LMP-1 showed increased cell proliferation, elevated NF-κB activity and reduced p53 induction. Moreover, co-expression of low-risk HPV6 E6 and EBV LMP-1 induced DNA damage, escaped from apoptosis under genotoxic condition and suppression of DNA damage response (DDR). Co-expression of low-risk HPV11 E6/E7 and EBV LMP-1 demonstrated similar results. However, it led to no malignant characteristics such as anchorage-independent growth, invasiveness and tumor formation in nude mice. Compared with the cells co-expressing high-risk HPV16 E6 and EBV LMP-1 that induce transformation, co-expression of low-risk HPV6 E6 and EBV LMP-1 was associated with low MMP-2, paxillin and Cat-1 expression. CONCLUSIONS: The co-expression of low-risk HPV E6/E7 and EBV LMP-1 does not induce malignant transformation, but it allows accumulation of somatic mutations secondary to increased DNA damage and suppression of DDR. Thus, double infection of low-risk HPV and EBV could lead to precancerous lesions.

Molecular and Phylogenetic Characterization of Novel Papillomaviruses Isolated from Oral and Anogenital Neoplasms of Japanese Macaques (Macaca fuscata).

Papillomaviruses (PVs) are a diverse group of host species-specific DNA viruses, etiologically linked with various benign and malignant neoplasms of cutaneous and mucosal epithelia. Here, we describe the detection and characterization of the first two PVs naturally infecting Japanese macaques (Macaca fuscata), including the determination of their etiological association(s) with the development of original neoplasms. The molecular and phylogenetic analyses were performed on complete genome sequences of Macaca fuscata PV types 1 (MfuPV1) and 2 (MfuPV2), which were completely sequenced in samples of a malignant oral tumor and benign anogenital neoplasm of Japanese macaques, respectively. Subsequently, two type-specific quantitative real-time PCRs were developed to estimate viral loads of MfuPV1 and MfuPV2 and to evaluate their etiological roles. The in silico molecular analyses revealed that both viral genomes encode characteristic PV proteins with conserved functional domains and have a non-coding genomic region with regulatory sequences to regulate and complete the viral life cycle. However, additional experimental evidence is needed to finally confirm the presence and biological functionality of the molecular features of both novel PVs. While MfuPV1, together with PVs identified in other macaques, is classified into the Alphapapillomavirus (Alpha-PV) species 12, MfuPV2 is most likely a representative of the novel viral species within the Alpha-PV genus. Their relatively high viral loads suggest that both PVs are etiologically linked with the development of the original neoplasms.