Using digital spatial profiling to analyse onco-immune-related gene expression within oropharyngeal tumours in relation to HPV and p16 status

Human Papilloma virus (HPV)-mediated oropharyngeal cancer (OPC) has significantly better prognosis compared with HPV-negative, stimulating interest in treatment de-intensification approaches to reduce long-term sequelae. Routine clinical testing frequently utilises immunohistochemistry to detect upregulation of p16 protein as a surrogate marker of HPV-mediation. However, this does not detect discordant HPV+/p16-cases and incorrectly assigns HPV-/p16+ cases, which, given their inferior prognosis compared to HPV+/p16+, may have important clinical implications. The biology underlying poorer prognosis of HPV/p16 discordant OPC requires exploration. Here, we utilised digital spatial profiling to compare the expression patterns of selected immuno-oncology-related genes within the tumour and stromal compartments of HPV+/p16+, HPV-/p16+ and HPV-/p16-OPC tumour tissues (n=12). KRT and HIF1A were upregulated in HPV-/p16+ and HPV-/p16-tumours relative to HPV+/p16+. Conversely, multiple genes associated with antitumour immune responses (such as CXCL9, CXCL10, CD74 ) were upregulated in HPV+/p16+ tumours. Of note, HPV-/p16+ and HPV-/p16-tumours displayed highly similar gene expression profiles, with only CXCL9 showing differential expression in stromal regions. These results are consistent with described prognostic patterns (HPV+p16+ > HPV-/p16+ > HPV-/p16-) and underline the need for dual HPV and p16 testing to guide clinical decision making.