Molecular assays for detecting human papillomavirus in head and neck squamous cell carcinoma
Sekee, Tumelo Robert
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Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide and is traditionally associated with alcohol and tobacco. However over the past few decades there has been a decrease in smoking and drinking but still an increase in incidence of HNSCC with reports across South America, Europe and Asia. The increase in incidence is now attributed to human papillomavirus (HPV), an etiological agent of cervical cancer. HPV belongs to the Papillomaviridae family and over 150 HPV types have been identified. HPVs can be grouped into three groups based on the association with cancer; high risk HPV (HR-HPV) types which are associated with cancer, low risk HPV (LR-HPV) types which are not associated with cancer and possible cancer causing HPV types. Little is known about the association between HPV and HNSCC in South Africa (SA) with few studies conducted in Northern Transvaal and to our knowledge none in the Free State. Additionally, there is no standardized method that can be used for the detection of HPV in HNSCC. Therefore the aims of this study were to investigate molecular assays that can be used for detection of HPV types circulating in the Free State province, SA and to develop a method that can be used to determine transcriptionally active HPV. Three molecular assays were compared by screening for HPV DNA in a total of 74 tissue biopsies from patients with confirmed head and neck tumours. A nested polymerase chain reaction (PCR) that targets part of the L1 region, an E6 multiplex hemi-nested type specific PCR using type specific primers for HPV types -6,-11, -16, -18, -31, -33, -45, -58 and -84 that target the E6 region and the Roche linear array (LA) that target part of the L1 region. To investigate the performance of the Roche LA assay, the PCR targeting the L1 region was repeated on selected samples using modified primers PGMY11/09 and GP5+/6+ (nested PCR). A total of 4/74 (5.4%) samples tested positive for HPV DNA by nested PCR and sequencing analysis revealed HPV types -11, -16, -18 and -31. A total of 5/74 (6.8%) samples tested positive by the E6 multiplex hemi-nested type specific PCR which included the four HPV types already genotyped by nested PCR and an additional HPV type -45. Using the LA assay 60/74 (81.1%) samples tested positive for HPV DNA; 57/60 samples were positive for HPV type -84, one sample positive for HPV type -45 and two samples were positive for co-infections (-16/84 and -18/84). A conventional PCR was used to screen 10/57 samples that tested positive for HPV type -84 and all the 10 samples tested negative. Due to the fact that HR-HPV types are known to be carcinogenic, four samples from this study that tested positive for HR-HPV types -16, -18, -31 and -45 were tested for transcriptionally active HPV infection by developing an E6 HnRT-PCR. All samples tested negative for HPV E6mRNA using the E6 HnRT-PCR. In conclusion the E6 multiplex hemi-nested type specific PCR detected all five HPV types in the study whereas nested PCR did not detect HPV type -45 and the Roche LA did not detect HPV types -11 and -31. Therefore the E6 multiplex hemi-nested type specific PCR will be used to screen additional samples for HPV DNA in tissue biopsies from head and neck tumours in our laboratory. However there is a limitation that needs to be kept in mind when working with the E6 multiplex hemi-nested type specific PCR and expanding the primers to include other HR-HPV types would be applicable.