The Genomics of Oral and Oropharyngeal Squamous Cell Carcinoma
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2021-05-30. This item is currently available to University of Melbourne staff and students only, login required.
© 2019 Dr. Kendrick Koo
Head and neck squamous carcinomas (HNSCCs) are a diverse group of squamous cell cancers in the upper aerodigestive tract. Whilst tobacco, alcohol, and the human papilloma virus (HPV) have been implicated in HNSCC oncogenesis, there is an incomplete understanding of how these factors impact upon molecular characteristics. In this study, we aim to better understand the genomic characteristics of HNSCC and the role of these risk factors in tumour development. Data for 528 patients from the Cancer Genome Atlas (TCGA) was used for in silico analyses to delineate the role of HPV in oral cavity cancers, where evidence for viral-induced oncogenesis remains contentious. A clear mutational signature for viral oncogenesis was identified in oral cavity carcinomas, as well as corresponding signatures in gene expression and DNA methylation, indicative of a role for HPV in the formation of oral cavity cancers. TCGA patients with and without tobacco and alcohol risk factors - non-smokers/non-drinkers (NSND) – were evaluated. Unfortunately, the proportion of NSND patients in the TCGA was insufficient for adequate analysis. Targeted next-generation sequencing for 69 genes as well as 4 HPV subtypes was therefore carried out on 186 patients recruited from our hospital. As frozen tissue was not available for all patients, a workflow to sequence and analyse formalin fixed paraffin embedded (FFPE) samples was developed. Differential mutation of 5 different genes was identified in the NSND group, more than for any other clinicopathologic variable. A high rate of mutations in the extra-cellular domain of NOTCH1 was noted in the mutational data. The role of these putatively inactivating NOTCH1 mutations in HNSCC remains uncertain. NOTCH1 knockouts were induced in HNSCC cell lines using the CRISPR/Cas9 gene editing system to assess the role of these mutations. No phenotypical differences could be identified using in vitro assays. Transcriptomic analysis of the edited cell lines identified alterations in gene expression in cellular adhesion pathways, consistent with epithelial-mesenchymal transition and the known activity of the Notch pathway. Overall, it appears that inactivating mutations in NOTCH1 may result in a paradoxical activation of the Notch pathway. In conclusion, we have established that differing exposures to risk factors for HSNCC result in molecular differences. These differences require validation and in-depth exploration. HPV has also been implicated in the development of oral cavity carcinomas, and novel treatment protocols for HPV-positive oropharyngeal cancers should also be considered for patients with these cancers. The prominence of the Notch pathway in HNSCC is notable, and the apparent paradoxical role of NOTCH1 mutations on pathway function deserves further follow up.
Keywordsgenomics; oral cancer; oropharyngeal cancer; head and neck cancer; notch pathway; human papilloma virus; personalised medicine; bioinformatics
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