Background: The molecular drivers of human papillomavirus-related head and neck squamous cell carcinoma (HPV + HNSCC) are not entirely understood. This study evaluated the relationship between HPV integration, expression of E6/E7, and patient outcomes in p16+ HNSCCs. Methods: HPV type was determined by HPV PCR-MassArray, and integration was called using detection of integrated papillomavirus sequences polymerase chain reaction (PCR). We investigated whether fusion transcripts were produced by reverse transcriptase polymerase chain reaction (RT-PCR). E6/E7 expression was assessed by quantitative RT-PCR. We assessed if there was a relationship between integration and E6/E7 expression, clinical variables, or patient outcomes. Results: Most samples demonstrated HPV integration, which sometimes resulted in a fusion transcript. HPV integration was positively correlated with age at diagnosis and E6/E7 expression. There was a significant difference in survival between patients with vs without integration. Conclusions: Contrary to previous reports, HPV integration was associated with improved patient survival. Therefore, HPV integration may act as a molecular marker of good prognosis. K E Y W O R D S head and neck squamous cell carcinoma (HNSCC), human papillomavirus (HPV), integration, oropharynx, survival

Human papillomavirus (HPV) genomic integration is frequently seen in oropharyngeal squamous cell carcinoma (OPSCC). Integration into cellular genes can cause the generation of HPV-host fusion transcripts, which have been associated with altered gene expression of critical cellular pathways and worse survival in OPSCC patients. The mechanism of these transcriptomic alterations by fusion transcripts and how they contribute to poor outcome are unknown. We began our analysis by investigating whether our cell lines models of HPV+ OPSCC recapitulate these gene expression alterations using a qRT-PCR profiling system. We compared the expression of 128 genes in relevant pathways in four fusion transcript positive cell lines versus two fusion transcript negative cell lines and found a number of genes that were differentially expressed between the two groups. This suggests that these cell lines are appropriate models to study the mechanism of fusion transcript-dependent transcriptional regulation. To confirm our results in another system, we created fusion transcript positive/negative cell lines by cloning two pairs of either an HPV-human fusion transcript or an HPV only transcript into normal keratinocytes. These cloned cells were then run through the same qRT-PCR profiling system, and we saw differentially expressed genes between the two groups. There was an overlapping set of gene pathways between the HPV+ OPSCC cell lines and cloned keratinocytes, suggesting HPV-human fusion transcripts are sufficient to alter cellular gene expression. Subsequent in-depth molecular characterization and in vitro studies will help clarify the mechanism of these changes and their effects on cancer formation and metastasis. These studies will advance our understanding of how fusion transcripts lead to worse patient survival and help develop new therapies for these patients. Citation Format: Lisa M. Pinatti, Hana Sinha, Chad Brenner, Heather M. Walline, Thomas E. Carey. Transcriptomic alterations by HPV-human fusion transcripts in HPV+ HNSCC cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4896.

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