Characterisation of the human papillomavirus genome and p53 mutations in head and neck squamous cell carcinomas

[摘要] High-risk human papillomaviruses (HR-HPV) are ubiquitous, sexually transmitted, aetiologic agents of head and neck cancer (HNC). To date, no large-scale South African studies report on HPV type distribution and prevalence associated with head and neck cancer. In a previous study from our research group, HR-HPV was detected in biopsy samples from histologically confirmed head and neck squamous cell carcinoma (HNSCC) patients in a South African cohort. This study went on to determine genetic changes that accumulate within HR-HPV genomes, other than the well-researched HPV 16, that confer differences in oncogenicity. Unlike cervical carcinomas, it is unknown whether HPV variant research in HNSCC translates into clinical application.The first complete genomes of HPV 18 and HPV 31 from HNC were amplified and subjected to deep sequencing analysis. VBD 17/15, the South African HPV 18 isolate, clustered in lineage A1. Mutations were identified in the E2 and long control region (LCR) that might lead to differences in oncogenicity. Evidence of how papillomaviruses evolved is shown in this study, in a phenomenon known as linkage disequilibrium. A novel mutation of the South African isolate is described and further investigations in a larger cohort will determine whether this is a single nucleotide polymorphism unique to variants that preferentially infect the head and neck region. Although geographic and ethnic associations have been described for HPV 16 and 18, this study supports the use of alphanumeric nomenclature. Having obtained the first complete genome of HPV 31, deep sequencing analysis showed that this laryngeal carcinoma was co-infected with a closely related viral variant. HPV quasispecies has recently been described in cervical carcinoma and this is the first evidence in the head and neck region. The quasispecies described belonged to HPV 31 lineage B2. A unique deletion within the E5 gene needs to be investigated further to determine what this deletion represents to viral fitness. Polymorphisms in the LCR were investigated with pBlue-Topo® vector, a reporter gene system. Increased β-galactosidase expression was observed in the mutant that possessed a single nucleotide change within the YY1 binding site. This study provides evidence of sequence variation within HPV 31 LCR having a functional effect on viral p97 promoter activity. HPV-HNSCC is complicated by the synergistic interaction with the host. The human tumour suppressor gene, p53 was investigated for mutations in a subset of HNSCC samples. As the p53 gene is frequently mutated in most cancers, it has been proposed as a biomarker to deintensify treatment of HPV-HNSCC patients. The involvement of high-risk HPV in HNSCC is an alternative mechanism to inactivate the p53 protein function. Evidence of p53 mutations was shown, with a predominance of substitution patterns that are induced by a carcinogen from tobacco smoke. Immunostaining of p16 as a biomarker of HPV infection did not correlate with HPV detection by PCR. It is unknown whether the genomes of participants of African descent are too diverse from the reference genome used in this study to accurately use frequency and functional data of known mutations. All of the mutations within this study were detected within intron 5. Whether there may be mutations lying outside of the area investigated or whether other cancer driver genes are involved in tumourigenesis of this cohort of HNSCC samples is still to be determined. Sequence data for South African isolates from patients with HNSCC adds to the global understanding of this virus-related epidemic and contributes to elucidating the underlying molecular mechanisms of HPV infection in HNC in sub-Saharan Africa, especially in light of high HPV burden in the cervix.

[发布机构] University of the Free State