Comparison of HPV E6,E7 mRNA/PD-L1 assay using flow cytometry and p16 IHC expression in oral cancer samples.

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114 Background: Research into causality of head and neck squamous cell carcinoma (HNSCC) has found a link to HPV infections affiliated with better survival than tobacco associated HNSCC. Currently, p16 immunohistochemistry is used as a predictive biomarker for HPV infection in HNSCC. We were interested in looking at an additional biomarker, HPV E6,E7 mRNA overexpression by flow cytometry, to see if it correlated with p16 status. Each test looks at a different marker of HPV infection, p16 as a surrogate of E7 activity, and E6,E7 mRNA overexpression as a marker of transcriptionally active and integrated virus. Currently p16 positive samples are confirmed with an ISH assay. Additionally, we looked at the PD-L1 expression in these tumors to see if it correlated with HPV mRNA overexpression. Advances in immuno-oncology have brought immunotherapy to the forefront of cancer treatment including HNSCC. Methods: Swabs were collected from Institut Gustave Roussy patients with lesions of the oral pharynx. Swabs were placed into a vial with a proprietary fixation solution and shipped overnight for processing. Upon receipt, samples were passed through a 35 uM filter to remove aggregates. Cells underwent in situ hybridization with E6, E7 mRNA probes (HPV OncoTect), were labeled with PD-L1 Ab, and then stained with a cell cycle dye identify single nucleated cells prior to analysis on the flow cytometer. FFPE biopsy tissue of the lesion was tested with p16 IHC. Positive samples were confirmed by ISH. Results: We analyzed samples from 27 patients with oral cancer with the combined E6, E7 mRNA/PD-L1 assay by flow cytometry and p16/ISH. Concordance between HPV E6,E7 mRNA positive results and p16 positive confirmed by ISH was 74%. Interestingly, PD-L1 expression was seen only in samples without HPV infection (according to HPV E6,E7 mRNA flow result). Samples are still being accrued and updated data will be presented at the meeting. Conclusions: Here we report a novel assay to quantify both HPV E6, E7 mRNA and PD-L1 simultaneously in single cells from head and neck squamous cell carcinoma. In addition, the ability to characterize both E6,E7 mRNA expression and PD-L1 in one test can provide clinicians with insight into treatment options.