Oral human papillomavirus (HPV) infection and its association with head and neck cancers (HNCs) have been established by many studies. The characteristics of HPV-associated HNCs are distinguishable from those of non HPV-associated HNCs. HPV-associated HNCs are related to sexual behaviour, particularly the lifetime number of oral sex partners. The oral and oropharyngeal HPV epidemiology in South African men has not yet been researched. The objective of this study was to determine the oral and oropharyngeal HPV strain prevalence and associated factors in a selected male population in Pretoria, South Africa. Male factory workers were recruited on a voluntary basis to be part of this study. Oral rinse and gargle samples were tested for 37 HPV types using the HPV linear array genotyping kit (Roche Molecular System). A questionnaire was utilised to obtain information regarding age, medical conditions, substance and alcohol use and sexual behaviour. HIV testing was optional. The HPV prevalence was 5.6% among the men (n=125) aged 17-64 years. High risk HPV (hrHPV) types 16 and 68 were found in two men. Amongst the majority of the participants oral sex seemed to be an uncommon practice however, those participants with hrHPV did practice oral sex. A statistically significant association between HPV infection and an increased number of sexual partners (p=0.027) was seen but not between substance use, HIVstatus or clinical mucosal pathology. Considering the oral and oropharyngeal HPV prevalence found in this study compared to those reported in other countries. It is therefore proposed that a larger nationwide study be conducted to give a more representative view of the burden of oral and oropharyngeal HPV infection in South Africa. / Dissertation (MSc)--University of Pretoria, 2014. / lk2014 / Community Dentistry / MSc / Unrestricted

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Only a few individual cells within less than 5% of all primary tumors form the cell lines commonly used in cancer research. These growth bottlenecks result in cell lines that are often poor models of primary tumors. Co-culture of primary tumor-derived cells with an irradiated mouse fibroblast feeder layer and ROCK inhibitor, known as the Georgetown Method, offers a way to culture over 80% of tumor-derived cells in vitro to create more representative tumor cell models. In our studies, we optimized the Georgetown Method to culture head and neck cancer cells, including oropharyngeal squamous cell carcinoma, and investigated its mechanism of conditionally immortalizing cells in culture. Differential trypsinization and regular feeder layer replacement were found to significantly improve the efficacy of immortalizing co-cultured cells at both atmospheric and physiological oxygen levels. Medium conditioned by irradiated fibroblasts can also substitute for direct co-culture with a feeder layer. The Georgetown Method was found to maintain low levels of p16 in co-cultured cells, suggesting a potential mechanism by which the Georgetown Method prevents differentiation and senescence. Our ability to culture over 80% of primary tumor-derived cells allows us to test the translational value of tumor-derived cell cultures and xenografts using BH3 profiling. Conditioned medium simplifies maintenance of cell cultures and will also allow us to perform high-throughput screens without the need to separate tumor-derived cells from the fibroblast feeder layer. The Georgetown Method provides opportunities to expand small tissue specimens for future diagnostics, therapeutics, and biobanking.

Les carcinomes épidermoïdes des voies aéro-digestives supérieures (VADS) se situent au sixième rang des cancers les plus fréquents dans le monde. Ces tumeurs sont liés à deux facteurs de risque : l’intoxication éthylo-tabagique (80% des cas) et l’infection de l’épithélium des VADS par les papillomavirus humain (HPV) à haut risque oncogène (20% des cas). Ces derniers définissent une sous-population de patients de meilleur pronostic. Une des hypothèses actuellement étudiées, afin d’expliquer la survie améliorée des patients HPV positifs, serait une hypoxie moindre dans ces tumeurs. En effet, les tumeurs des VADS sont fréquemment hypoxiques, et l’hypoxie intratumorale est un facteur de mauvais pronostic. Dans une première partie de cette thèse, nous avons entrepris une caractérisation moléculaire de l’hypoxie intratumorale dans les tumeurs humaines oropharyngées en fonction du statut HPV. Il apparaît que les tumeurs HPV positives présentent un statut hypoxique moindre comparées aux tumeurs HPV négatives. Ces tumeurs se caractérisent également par une abondante vascularisation intratumorale, qui pourrait être à l’origine de ce statut hypoxique moindre. Dans une deuxième partie, nous avons étudié l’adaptation à l’hypoxie de la lignée cellulaire HPV négative SQ20B et la lignée cellulaire HPV positive SCC90. De plus, des modèles de xénogreffes ont été établis à partir de ces mêmes lignées cellulaires et ont été analysés du point de vue de l’hypoxie intratumorale. De façon comparable aux tumeurs HPV positives, les xénogreffes obtenus à partir de la lignée SCC90 montre un statut hypoxique réduit comparés aux xénogreffes SQ20B. Les deux lignées cellulaires s’adaptent également différemment en hypoxie in vitro. La réponse à l’hypoxie dans la lignée SCC90 semble plus dynamique. En effet, la lignée SCC90 tente de s’adapter et de répondre à cet environnement hypoxique en induisant de fort niveau d’expression de gènes comparée à la lignée SQ20B. / Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common malignancy worldwide. The major risk factors for HNSCC identified are tobacco use and alcohol consumption (80% of all HNSCC), which seem to have a synergistic effect. A subgroup of HNSCCs (20% of cases), particularly those of the oropharynx, is caused by infection with high-risk types of human papillomavirus (HPV). Human papillomavirus HPV-related oropharyngeal squamous cell carcinoma defines a distinct clinical subgroup of head and neck cancer patients with improved prognosis. Currently, one of the several hypothesis studied to account for their improved survival outcomes could be a distinct hypoxia status compared to their HPV-negative counterpart. Indeed, tumour hypoxia is common in solid tumours including head and neck tumours, and hypoxia is a well-known poor prognosis factor. In first part of this thesis, we have performed a molecular characterisation of tumor hypoxia on cohort of oropharyngeal tumours according to HPV status of the patients. The results support the hypothesis that HPV-related tumours display a lesser hypoxia status compared to HPV-negative oropharyngeal tumours. These HPV-related tumours also characterize by an abundant tumour vascularisation, which could be responsible for a lesser hypoxia status. In a second part, we have studied the ability of the adaptation to hypoxia of the HPV-positive SCC90 cell line and HPV-negative SQ20B cell line. Furthermore, HPV-positive and HPV-negative HNSCC xenograft models have been established and have been analysed about tumor hypoxia. Similar to HPV-related HNSCC, tumours-derived HPV positive cell lines display a reduced hypoxic status compared to tumours-derived HPV negative cell lines. The two cell lines adapt also differently to in vitro hypoxia. In the HPV-positive cell line, the hypoxia response pathways could be more dynamics. Indeed, SCC90 cell lines attempt to adapt and to reply to hypoxic environment inducing highly expression of all of the hypoxia related genes compared to SQ20B cell lines.

Detection of p16 through immunohistochemistry (IHC) is the standard for determining the HPV status of the tumor according the TNM eighth edition released in 2017 and has become crucial for determining the HPV status of oropharyngeal squamous cell carcinomas (OPSCC) with direct impact on staging and prognostication. In recent years, detection of HPV DNA in mouthwashes has been proposed as a noninvasive alternative, both for OPSCCs and for other head and neck squamous cell carcinomas (HNSCCs). However, the prospect of using the mouthwashes to monitor the response to therapy is unclear. To evaluate the effect of curative therapy on the detection of HPV DNA, we performed a prospective study comparing the detection frequency of high-risk HPV DNA (HR-HPV-DNA) in pre- and post-therapy mouthwashes. We collected 137 mouthwashes from 88 pathologically confirmed HNSCC patients for DNA isolation and HPV genotyping with the Inno- LiPA assay. We show that HPV DNA in pretherapeutic mouthwashes can detect HPV-driven HNSCCs with a sensitivity of 50.0% and specificity of 85.4%, alongside a high negative predictive value of 79.5% and an accuracy of 74.5%. Furthermore, we observed a notable decrease in the detection frequency of HR-HPV-DNA after successful treatment (pre-therapy 50.0% (9/18) versus post-therapy 9.7% (3/28)). However, the comparatively low sensitivity regarding detection of HPV-driven OPSCC argues against its use in clinical routine.