Human papillomavirus and the landscape of secondary genetic alterations in oral cancers.

Human papillomavirus (HPV) is a necessary but insufficient cause of a subset of oral squamous cell carcinomas (OSCCs) that is increasing markedly in frequency. To identify contributory, secondary genetic alterations in these cancers, we used comprehensive genomics methods to compare 149 HPV-positive and 335 HPV-negative OSCC tumor/normal pairs. Different behavioral risk factors underlying the two OSCC types were reflected in distinctive genomic mutational signatures. In HPV-positive OSCCs, the signatures of APOBEC cytosine deaminase editing, associated with anti-viral immunity, were strongly linked to overall mutational burden. In contrast, in HPV-negative OSCCs, T>C substitutions in the sequence context 5'-ATN-3' correlated with tobacco exposure. Universal expression of HPV E6*1 and E7 oncogenes was a sine qua non of HPV-positive OSCCs. Significant enrichment of somatic mutations was confirmed or newly identified in PIK3CA, KMT2D, FGFR3, FBXW7, DDX3X, PTEN, TRAF3, RB1, CYLD, RIPK4, ZNF750, EP300, CASZ1, TAF5, RBL1, IFNGR1, and NFKBIA Of these, many affect host pathways already targeted by HPV oncoproteins, including the p53 and pRB pathways, or disrupt host defenses against viral infections, including interferon (IFN) and nuclear factor kappa B signaling. Frequent copy number changes were associated with concordant changes in gene expression. Chr 11q (including CCND1) and 14q (including DICER1 and AKT1) were recurrently lost in HPV-positive OSCCs, in contrast to their gains in HPV-negative OSCCs. High-ranking variant allele fractions implicated ZNF750, PIK3CA, and EP300 mutations as candidate driver events in HPV-positive cancers. We conclude that virus-host interactions cooperatively shape the unique genetic features of these cancers, distinguishing them from their HPV-negative counterparts.

The unveiled reality of human papillomavirus as risk factor for oral cavity squamous cell carcinoma.

The prognostic impact of human papillomavirus (HPV) in oropharyngeal cancer is generally acknowledged, and HPV-status is assessed routinely in clinical practice. Paradoxically, while the oral cavity seems the predilection site for productive HPV-infections, figures on HPV-attribution in oral cavity squamous cell carcinoma (OCSCC) differ widely, and prognostic impact is uncertain. Major obstacles are the lack of reproducible assays to detect HPV in nonoropharyngeal cancers, the relatively small cohorts studied and consequently the shortfall of convincing data. In our study, we used a validated, nucleic acid-based workflow to assess HPV-prevalence in a consecutive cohort of 1016 OCSCCs, and investigated its prognostic impact. In parallel, we analyzed p16-immunohistochemistry (p16-IHC) as surrogate marker for transforming HPV-infection and independent prognosticator. All OCSCC-patients diagnosed between 2008 and 2014 at two Dutch university medical centers were included (N = 1069). Formalin-fixed, paraffin-embedded (FFPE)-samples of 1016 OCSCCs could be retrieved. Punch biopsies were taken from the tumor area in the FFPE-blocks and tested for HPV. P16-IHC was performed on 580 OCSCCs, including all HPV-positive tumors. From 940 samples (92.5%), nucleic acids were of sufficient quality for HPV-testing. In total, 21 (2.2%) OCSCCs were HPV DNA-positive. All HPV DNA-positive tumors were E6 mRNA-positive and considered as true HPV-positive. There was no difference in survival between HPV-positive and HPV-negative OCSCCs. In total, 46 of 580 (7.9%) OCSCCs were p16-immunopositive, including all HPV-positive tumors. Survival was comparable in p16-positive and p16-negative OCSCCs. To conclude, HPV-prevalence is very low in OCSCC and neither HPV-status nor p16-status affects outcome. Based on these data, determining HPV-status in OCSCC seems irrelevant for clinical management.

Endogenous APOBEC3B overexpression characterizes HPV-positive and HPV-negative oral epithelial dysplasias and head and neck cancers.

The DNA cytosine deaminase APOBEC3B (A3B) is a newly recognized endogenous source of mutations in a range of human tumors, including head/neck cancer. A3B inflicts C-to-T and C-to-G base substitutions in 5'-TCA/T trinucleotide motifs, contributes to accelerated rates of tumor development, and affects clinical outcomes in a variety of cancer types. High-risk human papillomavirus (HPV) infection causes A3B overexpression, and HPV-positive cervical and head/neck cancers are among tumor types with the highest degree of APOBEC signature mutations. A3B overexpression in HPV-positive tumor types is caused by the viral E6/E7 oncoproteins and may be an early off-to-on switch in tumorigenesis. In comparison, less is known about the molecular mechanisms responsible for A3B overexpression in HPV-negative head/neck cancers. Here, we utilize an immunohistochemical approach to determine whether A3B is turned from off-to-on or if it undergoes a more gradual transition to overexpression in HPV-negative head/neck cancers. As positive controls, almost all HPV-positive oral epithelial dysplasias and oropharyngeal cancers showed high levels of nuclear A3B staining regardless of diagnosis. As negative controls, A3B levels were low in phenotypically normal epithelium adjacent to cancer and oral epithelial hyperplasias. Interestingly, HPV-negative and low-grade oral epithelial dysplasias showed intermediate A3B levels, while high-grade oral dysplasias showed high A3B levels similar to oral squamous cell carcinomas. A3B levels were highest in grade 2 and grade 3 oral squamous cell carcinomas. In addition, a strong positive association was found between nuclear A3B and Ki67 scores suggesting a linkage to the cell cycle. Overall, these results support a model in which gradual activation of A3B expression occurs during HPV-negative tumor development and suggest that A3B overexpression may provide a marker for advanced grade oral dysplasia and cancer.

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.

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.

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.

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.

Notch Signaling and Human Papillomavirus-Associated Oral Tumorigenesis.

The NOTCH pathway is critical for the development of many cell types including the squamous epithelium lining of cutaneous and mucosal surfaces. In genetically engineered mouse models, Notch1 acts as one of the first steps to commit basal keratinocytes to terminally differentiate. Similarly, in human head and neck squamous cell cancers (HNSCCs), NOTCH1 is often lost consistent with its essential tumor-suppressive role for initiating keratinocyte differentiation. However, constitutive NOTCH1 activity in the epithelium results in expansion of the spinous keratinocyte layers and impaired terminal differentiation is consistent with the role of NOTCH1 as an oncogene in other cancers, especially in T-cell acute lymphoblastic leukemia. We have previously observed that NOTCH1 plays a dual role as both a tumor suppressor and oncogene, depending on the mutational context of the tumor. Namely, gain or loss or NOTCH1 activity promotes the development of human papillomavirus (HPV)-associated cancers. The additional HPV oncogenes likely disrupt the tumor-suppressive activities of NOTCH and enable the oncogenic pathways activated by NOTCH to promote tumor growth. In this review, we detail the role of NOTCH pathway in head and neck cancers with a focus on HPV-associated cancers.

The prevalence of oral high-risk HPV infection in Indonesian oral squamous cell carcinoma patients.

OBJECTIVES: The objective of this study was to report the integrated observations of high-risk HPV-related oral squamous carcinoma (OSCC) at our national referral center for cancer, the Dharmais National Cancer Hospital (DNCH), Jakarta, from 2003 to 2013. MATERIALS AND METHODS: Seventy-eight formalin-fixed paraffin-embedded specimens obtained from OSCC cases were collected from 2003 to 2013 DNCH archives and were included in this high-risk HPV (HR-HPV) study. Seventy-nine DNA samples from the normal oral mucosa of healthy individuals were obtained from the Oral Biology Laboratory DNA archives from 2001 to 2005. Glyceraldehyde 3-phosphate dehydrogenase was used as a control to ensure the DNA integrity for the subsequent HPV DNA PCR detection. High-risk HPV16/18 DNA amplification was conducted by nested PCR using two pairs of primers that were designed specifically to identify the region of gene L1 HPV16 and the HPV16/18 region. RESULTS AND CONCLUSIONS: A high prevalence of HPV16/18 was detected in OSCC cases (17.9%). HPV18 occurred more often than HPV16 (86%) among OSCC patients who were HPV positive. This result supports high HPV18 prevalence among Indonesian cervical cancer patients studied in 1995 and 2006. The prevalence of high-risk HPV remains low in the normal Indonesian population (3.8%), but HPV16 is consistently more frequently detected in non-cancer populations.

Multisite HPV infections in the United States (NHANES 2003-2014): An overview and synthesis.

HPV is the most common sexually transmitted infection in the U.S., infecting both anogenital and oral sites. Nationally representative data are collected through the National Health and Nutrition Examination Survey (NHANES). However, changing designations of HPV genotypes as high or low risk and varying underlying populations as new results are reported have made direct comparison of results difficult. We reanalyzed HPV data from NHANES derived from self-collected cervicovaginal swabs (women ages 18-59, 2003-14), penile swabs (men ages 18-59, 2013-14), and oral rinses (men and women ages 18-69, 2009-14), using consistent populations and definitions across NHANES cycles. These data strengthen our understanding of age trends in HPV prevalence: cervicovaginal prevalence decreases with age, penile prevalence increases with age, and oral prevalence is bimodal but with an earlier first peak in women. There is strong evidence for reduced prevalence of vaccine genotypes (6, 11, 16, 18) in vaccinated men and women (ages 18-24) at both genital (RR 0.2 (0.1-0.3) in women and 0.7 (0.1-5.4) in men) and oral sites (RR 0.1 (0.0-1.3) in women; no infections detected in vaccinated men). A more complete picture of the burden of HPV in the U.S. is emerging, including evidence for reduced HPV genital and oral prevalence in vaccinated individuals.

Novel Herpesvirus Associated With Oropharyngeal Squamous Cell Carcinoma in Smooth Green Snakes (Opheodrys vernalis).

During a 19-month period, 5 smooth green snakes (Opheodrys vernalis) maintained as an ex situ conservation colony presented with rapid clinical progression of locally invasive oropharyngeal squamous cell carcinoma. All 5 originated from the same wild source and were housed together or in close proximity. An infectious cause was considered likely, and nested conventional polymerase chain reaction (PCR) and in situ hybridization confirmed the presence of a novel alphaherpesvirus, Opheodrys herpesvirus 1, in the neoplastic tissue in 4 of the 5 snakes. Retrospective screening of previously submitted smooth green snakes by in situ hybridization did not detect virus in prior submissions from the colony. This report documents molecular characterization of an ophidian herpesvirus as well as colocalization of its viral nucleic acid with neoplastic transformation in snakes.

Exploring lay public and dental professional knowledge around HPV transmission via oral sex and oral cancer development.

BACKGROUND: Human papillomavirus (HPV) has been associated with certain types of oropharyngeal cancers and yet, the level of knowledge that dental professionals and the lay public have in terms of HPV transmission, oral sexual activities, and oral cancer development needs exploration. The aim of this study was to assess the knowledge held by practicing dental professionals as well as the lay public regarding Human Papillomavirus (HPV) transmission through oral sex and subsequent oropharyngeal cancer development. METHODS: Textual data were collected from a public forum with dental professionals in. Vancouver, who discussed the HPV-oral sex-oral cancer triad, and from survey data gathered from 212 lay public participants (also in Vancouver) who answered a 13-item questionnaire on the perceived risks of oral sex in terms of HPV infection and oropharyngeal cancer development. The data were analyzed statistically by age group, gender, and sexual orientation using descriptive statistics, while an ANOVA test was used to compare variation in the responses to the survey (p-value = 0.05). RESULTS: The forum engaged 46 health care professionals, many of whom were aware of the potential risks for head and neck cancer development due to HPV infection, while also questioning "how to effectively talk about HPV with patients." The survey revealed that 34.5% of the participants believed that oral sex is an activity with no or low risk for the transmission of HPV, while 84% of participants believed the same sexual practices were of low or no-risk for HIV (Human Immunodeficiency Virus) transmission. Most participants (82%) never discussed oral sexual activities with their physicians or dentists/dental hygienists. CONCLUSIONS: The general public remains mostly unaware of the potential links between HPV infection and oropharyngeal cancer. Physicians and dental providers should discuss oral sexual practice with their patients to raise awareness.

Epstein-Barr Virus-Positive Diffuse Large B-Cell Lymphoma, Not Otherwise Specified, in the Oral Cavity.

Lymphomas of the oral cavity are rare and the most frequent type is diffuse large B-cell lymphoma (DLBCL). Epstein-Barr virus (EBV) is known to be associated with the development of different lymphomas. In 2008, the World Health Organization provisionally included the EBV-positive DLBCL of the elderly in the classification of hematopoietic and lymphoid tumors as a lymphoma occurring in older individuals without any known immunodeficiency. However, it has since been recognized that this entity may occur in younger individuals and present similar clinical parameters in both age groups. As a result, the 2017 revision has declined the term elderly and modified it to EBV-positive DLBCL, not otherwise specified (NOS). In this report, we describe a rare case of EBV-positive DLBCL, NOS, presenting as a painless swelling in the oral cavity. This entity shows a more aggressive clinical course than EBV-negative DLBCL, and other lymphoproliferative disorders should be considered in the differential diagnosis.

Human Papillomavirus-Related Oral Cancer: Knowledge and Awareness Among Spanish Dental Students.

Human papillomavirus (HPV) infections are responsible for a significant part of the global burden of cancer. Epidemiologic studies have shown increasing trends of HPV-related oral cancers worldwide. Dental professionals need comprehensive up-to-date HPV-related information to be able to provide correct advice to their patients. The aim of this paper is to describe knowledge and awareness levels of dental students from Spain on HPV infection, HPV vaccination, oral cancer prevention, and HPV-related oropharyngeal cancer. A survey was distributed to 240 dental students, of which 158 returned it. Most students reported not been vaccinated against HPV (n = 81, 51.3%) and believed that HPV infection was linked to oropharyngeal cancer (75%). Overall, advanced students showed better knowledge, attitudes, and perceptions regarding this issue while novice students showed relevant shortcomings. However, their attitudes in relation to the diagnosis of oral cancer were adequate. These results suggest that there are important HPV-related knowledge deficits among Spanish dental students, which hinders their interventions in oropharyngeal primary prevention efforts. Findings of this study suggest the inclusion and standardization of HPV-related educational information to the dental curriculum.

Susceptibilidad genética frente al cáncer bucal por infección del virus del papiloma humano.

Environmental carcinogens and human papillomavirus (HPV) are the main responsible factors for oral cancer. Susceptibility factors in the human genome play a risk-modulating role; however not all individuals exposed to these carcinogens suffer from cancer. The purpose of the present review is to describe the main factors of genetic susceptibility to oral cancer due to HPV infection. A systematic search was carried out in three databases in English, with only 7 articles meeting the selection criteria. Genetic polymorphisms are shown in three categories, which are related to HPV and participate in oncogenesis. Three articles related to deregulation of cell cycle control mechanisms were identified, as well as one referring to mutations in the apoptosis pathway and three about polymorphisms in inflammatory and immune response genes. The association of polymorphisms for the development of oral cancer by HPV is evident, although it remains under study. Oral neoplasms' oncogenesis pattern is not always associated with HPV, but with other environmental or epigenetic factors.

Los carcinógenos ambientales y el virus del papiloma humano (VPH) son los principales responsables del cáncer bucal. Los factores de susceptibilidad en el genoma humano desempeñan un papel modulador del riesgo, sin embargo, no todos los individuos expuestos a los carcinógenos padecen cáncer. El objetivo de la presente revisión es describir los principales factores de susceptibilidad genética para cáncer bucal en individuos con infección por VPH. Se realizó una búsqueda sistemática en tres bases de datos en inglés; solo siete artículos cumplieron con los criterios de selección. Se registraron polimorfismos genéticos en tres categorías relacionados con el VPH y que participan en la oncogénesis. Se identificaron tres artículos relativos a la desregulación de los mecanismos de control del ciclo celular, uno relativo a mutaciones en la vía de la apoptosis y tres a polimorfismos en genes de respuesta inflamatoria e inmune. La asociación entre polimorfismos para el desarrollo de cáncer bucal y VPH es evidente, aunque continúa en estudio ya que no siempre el patrón de oncogénesis de las neoplasias bucales está relacionado con el VPH, sino con otros factores ambientales o epigenéticos.

Association of DFNA5, SYK, and NELL1 variants along with HPV infection in oral cancer among the prolonged tobacco-chewers.

Southeast Asia, especially India, is well known for the highest use of smokeless tobacco. These products are known to induce oral squamous cell carcinoma. However, not all long-term tobacco-chewers develop oral squamous cell carcinoma. In addition, germline variants play a crucial role in susceptibility, prognosis, development, and progression of the disease. These prompted us to study the genetic susceptibility to oral squamous cell carcinoma among the long-term tobacco-chewers. Here, we presented a retrospective study on prolonged tobacco-chewers of Northeast India to identify the potential protective or risk-associated germline variants in tobacco-related oral squamous cell carcinoma along with HPV infection. Targeted re-sequencing (n = 60) of 170 genetic regions from 75 genes was carried out in Ion-PGM™ and validation (n = 116) of the observed variants was done using Sequenom iPLEX MassARRAY™ platform followed by polymerase chain reaction-based HPV genotyping and p16-immunohistochemistry study. Subsequently, estimation of population structure, different statistical and in silico approaches were undertaken. We identified one nonsense-mediated mRNA decay transcript variant in the DFNA5 region (rs2237306), associated with Benzo(a)pyrene, as a protective factor (odds ratio = 0.33; p = 0.009) and four harmful (odds ratio > 2.5; p < 0.05) intronic variants, rs182361, rs290974, and rs169724 in SYK and rs1670661 in NELL1 region, involved in genetic susceptibility to tobacco- and HPV-mediated oral oncogenesis. Among the oral squamous cell carcinoma patients, 12.6% (11/87) were HPV positive, out of which 45.5% (5/11) were HPV16-infected, 27.3% (3/11) were HPV18-infected, and 27.3% (3/11) had an infection of both subtypes. Multifactor dimensionality reduction analysis showed that the interactions among HPV and NELL1 variant rs1670661 with age and gender augmented the risk of both non-tobacco- and tobacco-related oral squamous cell carcinoma, respectively. These suggest that HPV infection may be one of the important risk factors for oral squamous cell carcinoma in this population. Finally, we newly report a DFNA5 variant probably conferring protection via nonsense-mediated mRNA decay pathway against tobacco-related oral squamous cell carcinoma. Thus, the analytical approach used here can be useful in predicting the population-specific significant variants associated with oral squamous cell carcinoma in any heterogeneous population.

High-Risk Human Papillomavirus in Oral Cancer: Clinical Implications.

Oral cancer is the eleventh most common cancer globally, with well-established major risk factors of tobacco, areca nut, alcohol, and high-risk human papillomavirus (HR-HPV) types 16 and 18. HR-HPV16/18 are the etiologic agents of cervical cancers and a proportion of oropharyngeal cancers. HPV-associated oropharyngeal and oral cancers show better prognosis and response to therapy. However, the picture of HR-HPV16/18 and the clinical implications of oral cancers are not clear with the majority of reports combining oral cancer data with head and neck cancers. The current review compiles the global prevalence of HR-HPV16/18 in oral cancers, highlighting the unique clinical and molecular pathologic features, prognosis and therapeutic strategies in the prevention and management of HPV-positive oral cancers. The potential for the use of de-intensified therapy and prophylactic prevention in HPV-positive oral cancer patients is highlighted.

Differences in LC3B expression and prognostic implications in oropharyngeal and oral cavity squamous cell carcinoma patients.

BACKGROUND: This study examined the prognostic significance of microtubule-associated protein light chain 3B (LC3B) expression in oropharyngeal and oral cavity squamous cell carcinoma (SCC). The prognostic significance of LC3B expression in relation to human papillomavirus (HPV) status in oropharyngeal SCC was also examined. METHODS: Tissue microarrays (TMAs) were constructed from formalin-fixed, paraffin-embedded oropharyngeal (n = 47) and oral cavity (n = 95) SCC tissue blocks from patients with long-term recurrence and overall survival data (median = 47 months). LC3B expression on tumour was assessed by immunohistochemistry and evaluated for associations with clinicopathological variables. LC3B expression was stratified into high and low expression cohorts using ROC curves with Manhattan distance minimisation, followed by Kaplan-Meier and multivariable survival analyses. Interaction terms between HPV status and LC3B expression in oropharyngeal SCC patients were also examined by joint-effects and stratified analyses. RESULTS: Kaplan-Meier survival and univariate analyses revealed that high LC3B expression was correlated with poor overall survival in oropharyngeal SCC patients (p = 0.007 and HR = 3.18, 95% CI 1.31-7.71, p = 0.01 respectively). High LC3B expression was also an independent prognostic factor for poor overall survival in oropharyngeal SCC patients (HR = 4.02, 95% CI 1.38-11.47, p = 0.011). In contrast, in oral cavity SCC, only disease-free survival remained statistically significant after univariate analysis (HR = 2.36, 95% CI 1.19-4.67, p = 0.014), although Kaplan-Meier survival analysis showed that high LC3B expression correlated with poor overall and disease-free survival (p = 0.046 and 0.011 respectively). Furthermore, oropharyngeal SCC patients with HPV-negative/high LC3B expression were correlated with poor overall survival in both joint-effects and stratified presentations (p = 0.024 and 0.032 respectively). CONCLUSIONS: High LC3B expression correlates with poor prognosis in oropharyngeal and oral cavity SCC, which highlights the importance of autophagy in these malignancies. High LC3B expression appears to be an independent prognostic marker for oropharyngeal SCC but not for oral cavity SCC patients. The difference in the prognostic significance of LC3B between oropharyngeal and oral cavity SCCs further supports the biological differences between these malignancies. The possibility that oropharyngeal SCC patients with negative HPV status and high LC3B expression were at particular risk of a poor outcome warrants further investigation in prospective studies with larger numbers.

Salivary extracellular vesicle-associated miRNAs as potential biomarkers in oral squamous cell carcinoma.

BACKGROUND: Several studies in the past have investigated the expression of micro RNAs (miRNAs) in saliva as potential biomarkers. Since miRNAs associated with extracellular vesicles (EVs) are known to be protected from enzymatic degradation, we evaluated whether salivary EVs from patients with oral squamous cell carcinoma (OSCC) were enriched with specific subsets of miRNAs. METHODS: OSCC patients and controls were matched with regards to age, gender and risk factors. Total RNA was extracted from salivary EVs and the differential expression of miRNAs was evaluated by qRT-PCR array and qRT-PCR. The discrimination power of up-regulated miRNAs as biomarkers in OSCC patients versus controls was evaluated by the Receiver Operating Characteristic (ROC) curves. RESULTS: A preliminary qRT-PCR array was performed on samples from 5 OSCC patients and 5 healthy controls whereby a subset of miRNAs were identified that were differentially expressed. On the basis of these results, a cohort of additional 16 patients and 6 controls were analyzed to further confirm the miRNAs that were up-regulated or selectively expressed in the previous pilot study. The following miRNAs: miR-302b-3p and miR-517b-3p were expressed only in EVs from OSCC patients and miR-512-3p and miR-412-3p were up-regulated in salivary EVs from OSCC patients compared to controls with the ROC curve showing a good discrimination power for OSCC diagnosis. The Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis suggested the possible involvement of the miRNAs identified in pathways activated in OSCC. CONCLUSIONS: In this work, we suggest that salivary EVs isolated by a simple charge-based precipitation technique can be exploited as a non-invasive source of miRNAs for OSCC diagnosis. Moreover, we have identified a subset of miRNAs selectively enriched in EVs of OSCC patients that could be potential biomarkers.

Quantitative polymerase chain reaction-based detection of HPV 16 E6 and E7 DNA in oral squamous cell carcinoma.

BACKGROUND: Although human papillomavirus (HPV) is considered as a causative factor in oral squamous cell carcinoma (OSCCs), its pathogenetic role is not well established. Moreover, a limited number of studies have compared the techniques of detecting the HPV infection in OSCC. This study aimed at the detection of HPV 16 E6 and E7 DNA in OSCC by quantitative polymerase chain reaction (qPCR) technique. METHODOLOGY: This retrospective study included 297 tissue sections obtained from histopathologically confirmed OSCC patients. The classification of tumors as poorly differentiated, moderately differentiated and well differentiated was performed by H&E staining following the WHO criteria for OSCC. The presence of HPV infection was detected by p16INK4A expression, conventional PCR technique, HPV 16 E6, and E7 by qPCR and flow cytometry. All statistical analysis was performed using MedCalc software v.16.4.3. P < 0.05 is considered as statistically significant. RESULTS: Of 297 samples, 128 samples were found to be HPV-positive by p16. Of total 128 HPV-positive samples, PCR, E6, and E7 qPCR were positive in 19, 97, and 98 samples, respectively. qPCR techniques were found highly significant in the detection of moderately differentiated (P < 0.0001) and widely differentiated (P < 0.0001) cases. The positivity of E6 qPCR increased as the p16 expression increased. A significant variation in E6 DNA copies was observed in different grades of p16 expression (P < 0.0001). However, overall E7 (5.4 × 105 copies/µL) DNA copies were higher than E6 (7.7 × 103 copies/µL). CONCLUSION: qPCR detection of HPV infection is a fast, reliable, and accurate technique gives valuable information about the infection status in terms of viral load.