Background The aim of this study was to identify the presence and frequency of human papillomavirus (HPV) nucleic acid in p16‐positive oral squamous cell carcinomas (OSCCs), to assess whether the virus was transcriptionally active and to assess the utility of p16 overexpression as a surrogate marker for HPV in OSCC. Methods Forty‐six OSCC patients treated between 2007 and 2011 with available formalin‐fixed paraffin‐embedded (FFPE) specimens were included. Twenty‐three patients were positive for p16 by immunohistochemistry (IHC) and these were matched with 23 patients with p16‐negative tumours. Laser capture microdissection of the FFPE OSCC tissues was undertaken to isolate invasive tumour tissue. DNA was extracted and tested for high‐risk HPV types using a PCR‐ELISA method based on the L1 SPF10 consensus primers, and a real‐time PCR method targeting HPV‐16 and HPV‐18 E6 region. Genotyping of HPV‐positive cases was performed using a reverse line blot hybridization assay (Inno‐LiPA). RNAScope® (a chromogenic RNA in situ hybridization assay) was utilized to detect E6/E7 mRNA of known high‐risk HPV types for detection of transcriptionally active virus. Results HPV DNA was found in 3 OSCC cases, all of which were p16 IHC‐positive. Two cases were genotyped as HPV‐16 and one as HPV‐33. Only one of the HPV‐16 cases was confirmed to harbour transcriptionally active virus via HPV RNA ISH. Conclusion We have shown that the presence of transcriptionally active HPV rarely occurs in OSCC and that p16 is not an appropriate surrogate marker for HPV in OSCC cases. We propose that non‐viral mechanisms are responsible for the majority of IHC p16 overexpression in OSCC.

Extracellular vesicles (EVs) are secreted from most cell types and utilized in a complex network of near and distant cell-to-cell communication. Insight into this complex nanoscopic interaction in the development, progression and treatment of oral squamous cell carcinoma (OSCC) and precancerous oral mucosal disorders, termed oral potentially malignant disorders (OPMDs), remains of interest. In this review, we comprehensively present the current state of knowledge of EVs in OSCC and OPMDs. A systematic literature search strategy was developed and updated to December 17, 2019. Fifty-five articles were identified addressing EVs in OSCC and OPMDs with all but two articles published from 2015, highlighting the novelty of this research area. Themes included the impact of OSCC-derived EVs on phenotypic changes, lymph-angiogenesis, stromal immune response, mechanisms of therapeutic resistance as well as utility of EVs for drug delivery in OSCC and OPMD. Interest and progress of knowledge of EVs in OSCC and OPMD has been expanding on several fronts. The oral cavity presents a unique and accessible microenvironment for nanoparticle study that could present important models for other solid tumours.

Background Green tea is heavily consumed on a global basis for its health benefits. The active ingredient, (‐)‐epigallocatechin gallate (EGCG), is a major polyphenol demonstrated to inhibit the growth of various non‐oral cancer cell lines and interfere with the carcinogenic process, including downregulation of the epidermal growth factor receptor (EGFR). Our aim was to determine the phenotypic changes of oral cancer cells treated with EGCG and concurrently assess the effect on EGFR expression and activation. Methods Oral cancer cells (H400 and H357) were treated with 10 µg/mL and 20 µg/mL of EGCG for up to 72 hours. Phenotypic changes were assessed by performing cell proliferation analysis and cell migration (Transwell) assays. Expression of EGFR and its phosphorylated form (p‐EGFR) was determined by Western blotting. Results Cell proliferation of both cell lines was significantly reduced at 48hrs when treated with 20 µg/mL EGCG. However, after 72 hours of treatment the effect of EGCG on cell proliferation ceased. Treatment of both cell lines with 10 µg/mL and 20 µg/mL of EGCG resulted in significant reduction in cell migration. Mechanistically, EGFR expression did not change significantly after treatment with EGCG; however, there was a reduction in its phosphorylated form. Conclusion EGCG transiently inhibits both cell proliferation and migration of oral cavity cancer cells. This effect is associated with a decrease in the expression of phosphorylated EGFR. It is possible that more frequent bursts of EGCG could result in a persistent and sustained cancer inhibition, but this requires further research for clarification.

A distinct clinical subgroup of nonsmoking (NS) and nondrinking (ND) patients with oral squamous cell carcinoma (OSCC) has been identified. The objective of the study was to assess this cohort for molecular variations in the disease process and if these could be attributed to clinical or epidemiological characteristics. One hundred and twenty-nine consecutive patients (71 males, 58 females) treated for OSCC were assessed at the Royal Melbourne Hospital between January 2007 and July 2010. Formalin-fixed paraffin embedded (FFPE) sections were stained for p53, p16, cyclin D1, and epidermal growth factor receptor (EGFR). Biomarker overexpression was observed in 72 (56%) cases for p53, 23 (18%) for p16, 45 (35%) for cyclin D1, and 72 (56%) for EGFR. Multiple logistic regression analysis revealed that tongue tumors (p ¼ 0.012) and late stage cancers (p ¼ 0.031) were more likely to have cyclin D1 overexpression. Further, older patients significantly more often had cyclin D1 overexpression (p ¼ 0.008) and NSND patients had more p16 expression (p ¼ 0.043). In contrast, smokers were more likely to have EGFR overexpression (p ¼ 0.033). Concurrent overexpression of p53 and cyclin D1 were observed (p ¼ 0.030). Smoking, site, and stage of OSCC can influence biomarker expression, with p16 overexpression specifically observed in NSND, indicating fundamental differences in the mechanisms of oral carcinogenesis among different patient cohorts.

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