Abstract Background: Human papillomaviruses are a family of DNA viruses that infect the epithelium leading to the formation of lesions with the ability to progress into carcinoma in many forms. Research into causality of head and neck cancers has found a link to HPV infections affiliated with better prognosis. In addition, advances in immuno-oncology have brought immunotherapy to the forefront of cancer treatment across cancer types. Here we present data that demonstrate a combined diagnostic approach to quantify two markers important in the management of head and neck cancer. Methods: Swabs were collected from patients of Institut Gustave Roussy with lesions of the oral pharynx. Swabs were collected and placed into a collection vial with a proprietary fixation/permeabilization solution (IncellCollect, IncellDx, Inc.) and shipped overnight on cold packs for processing. Upon receipt, samples were passed through a 35 µM filter to remove aggregates. Cells were labeled with CD45 antibodies to separate epithelial from immune cells, RNA in situ hybridization with E6, E7 mRNA probes (HPV OncoTect) was performed, and these cells were labeled with PD-L1 Ab (clone 28-8) prior to analysis on the flow cytometer (CytoFlex, Beckman Coulter, Inc). Samples were also stained with DAPI to identify single nucleated cells, and to analyze cell cycle. Results: We analyzed samples from 8 patients with oral cancer with the combined E6, E7 mRNA/PD-L1 protein assay by flow cytometry. The percentage of tumor cells expressing PD-L1 averaged 9.1% in all of the samples. Of the HPV DNA positive tumor samples, the percentage of cells overexpressing HPV E6, E7 mRNA was 4.8%. Interestingly, the percentage of cells overexpressing E6, E7 mRNA that also expressed PD-L1 was statistically lower (1.4%) than the percentage of all tumor cells that expressed PD-L1 (P=0.01). Conclusions: We report a novel flow cytometric assay to quantify both HPV E6, E7 mRNA and PD-L1 simultaneously in single cells. Further, we found the expression of PD-L1 in tumor cells with HPV E6, E7 mRNA to be significantly lower than the expression of PD-L1 in tumor cells not expressing HPV E6, E7 mRNA. These findings may influence prognosis and treatment strategies. Note: This abstract was not presented at the conference. Citation Format: Rian J. Morgan, Amanda N. Chargin, Haitham Mirghani, Bruce K. Patterson. Simultaneous quantification of HPV oncogene (E6, E7 mRNA) and PD-L1 protein expression in oral cancer samples using flow cytometry [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 49.