Human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HPV-positive HNSCC) has distinct biological characteristics from HPV-negative HNSCC. Using an AI-based analytical platform on meta cohorts, we profiled expression patterns of viral transcripts and HPV viral genome integration, and classified the tumor microenvironment (TME). Unsupervised clustering analysis revealed five distinct and novel TME subtypes across patients (immune-enriched, highly immune and B-cell enriched, fibrotic, immune-desert, and immune-enriched luminal). These TME subtypes were highly correlated with patient prognosis. In order to understand specific factors associated with prognosis, we used the unsupervised clustering of an HPV-positive HNSCC cohort from The Cancer Genome Atlas (TCGA) (n = 53) based on HPV transcript expression, and identified four HPV-related subtypes (E2/E5, E6/E7, E1/E4 and L1/L2). Utilizing both viral transcript and TME subtypes, we found that the E2/E5 HPV subtype was associated with an immune-enriched TME and had a higher overall survival rate compared to other subtypes. The E2/E5 subtype was also enriched for samples without HPV-genome integration, suggesting that the episomal HPV status and E2/E5 expression pattern may be associated with an inflamed microenvironment and improved prognosis. In contrast, E6/E7 subtype samples were associated with the fibrotic and immune-desert TME subtypes, with lower values of T-cell and B-cell gene expression signatures and lower overall survival. Both E1/E4 and L1/L2 subtypes were associated with the immune-enriched luminal subtype. Our results suggest that HPV-transcript expression patterns may drive the modulation of the TME, and thereby impact prognosis.

Human papillomavirus (HPV)-associated Head and Neck Squamous Cell Carcinoma (HPV+HNSCC) is now the most common HPV-associated malignancy in the United States. Current treatments can be associated with severe side-effects or lack of efficacy yet prognostic biomarkers are limited, slowing efforts to personalize treatment in HPV+HNSCC. Here, we describe the use of a transcriptomic-based analytical platform to analyze expression patterns of viral transcripts, the tumor microenvironment (TME), and viral genome integration, and associate these features with overall survival. Functional gene expression signatures were analyzed on publicly available HPV+HNSCC expression data (n=266). Unsupervised clustering analysis revealed 5 distinct and novel TME types across patients (immune-enriched non-fibrotic, immune-enriched fibrotic, fibrotic, immune-desert, immune-enriched luminal). These microenvironment subtypes were highly correlated with both overall survival and patient prognosis. Tumors with an immune-enriched microenvironment showed the highest survival rates, whereas fibrotic TME types were associated with poor survival (p < 0.05). Unsupervised clustering of a HPV+HNSCC cohort from The Cancer Genome Atlas (TCGA) (n=53), based on HPV transcript expression, revealed 4 HPV-related subtypes. Each subtype was enriched for distinct viral transcripts: E2/E5, E6/E7, E1/E4 and L1/L2. We then validated TME and HPV transcript-related classifications on an independent HPV+HNSCC cohort (n=132). Utilizing both viral transcript and TME subtypes, we found that the E2/E5 HPV subtype was associated with an immune-enriched TME and had a higher overall survival compared to the other subtypes. The E2/E5 subtype was also enriched for samples without HPV-genome integration, suggesting that HPV episomal DNA status and E2/E5 expression pattern may drive an inflamed microenvironment and improved prognosis. In contrast, E6/E7 subtype samples were associated with the fibrotic and depleted TME types, with lower values of T-cell and B-cell gene expression signatures and a lower survival rate. Both E1/E4 and L1/L2 subtypes were associated with the immune-enriched luminal TME types. These findings suggest that HPV-transcript expression patterns may drive modulation of the TME, and hence impact prognosis. Further validation of the relationships between viral gene expression, TME, and prognosis is warranted to understand if such subtypes could aid in the development of prognostic biomarkers for treatment selection. Citation Format: Daria Kiriy, Dmitry Tychinin, Nikita Kotlov, Olga Kudryashova, Anastasia Nikitina, Andrey Tyshevich, Naira Samarina, Ksenia Demina, Sandrine Degryse, Susan Raju Paul, Mark Poznansky, Krystle Lang Kuhs, James S. Lewis, Robert L. Ferris, Xiaowei Wang, Alexander Bagaev, Nathan Fowler, Lori Wirth, Daniel Faden. Viral transcript and tumor immune microenvironment-based transcriptomic profiling of HPV-associated head and neck cancer identifies subtypes associated with prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3823.