Single-cell transcriptomic analysis of HPV-driven cellular and molecular mechanisms in oropharyngeal cancers

Abstract

HPV-positive oropharynx squamous cell carcinoma is now the most common type of mucosal Head and Neck cancer in the U.K. The vogue in contemporary clinical research is to de-escalate treatment paradigms with the aim reducing morbidity from treatment related toxicities. However, roughly 15% of HPV-positive disease remains treatment refractory. Furthermore, the clinical tests used to assess HPV involvement can be discordant and do not always reflect the underlying biology. Examining HPV-driven cellular and molecular mechanisms can help us understand why some patients still have poor outcomes and help develop novel biomarkers to aid treatment decisions. Tumour biopsies and matched normal tissue from 10 patients with both HPV-positive and negative oropharynx cancer underwent single cell RNA sequencing. The resulting dataset consists of 77,807 cells, making it one of the largest of its type in the current literature. Of key interest was confirmation of the existence of epithelial cells within HPV-positive tumours that do not express any HPV genes (termed “HPVoff” as opposed to “HPVon”). These cells have been characterised as senescent and therefore potentially immune evasive in other datasets. These data suggests that these HPVoff cells have a strong keratinization program and that features previously interpreted as senescence may instead reflect epithelial differentiation. HPVon cells have a proliferative phenotype in keeping with known HPV oncogenic mechanisms. Spatial transcriptomics localised HPVoff cells to regions of terminally keratinocyte differentiation, supported by keratinisation module scoring and GRHL-associated transcriptional programs. These findings indicate that HPV transcriptional state is tightly linked to epithelial differentiation, with HPVoff cells representing a more differentiated epithelial phenotype rather than a distinct immune evasive phenotype.

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Identifiers

ORCID for Tim Fenton: orcid.org/0000-0002-4737-8233

ORCID for Ian Reddin: orcid.org/0000-0001-5478-7855

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