Abstract Background: Head and neck cancer remains a challenging disease, with the majority of patients diagnosed at an advanced stage when five-year survival is approximately 50%. The main risk factors include tobacco use, excessive alcohol use, betel quid use, and oncogenic HPV virus infection. The incidence of HPV-related oropharyngeal cancer is increasing and differs from traditional oropharyngeal cancer in that it tends to affect younger patients, has distinct biologic characteristics, and a better prognosis. In this study we determine whether a surrogate marker for oncogenic HPV cancer, p16, has potential to enhance detection of oral and oropharyngeal cancer when combined with a CD44 and total protein-based laboratory test, used in some countries as an aid in diagnosis. Methods: We used residual archived oral rinses from a prior frequency-matched case:control study. Controls were frequency matched to cases for age, race, gender, and tobacco and alcohol habits and were therefore at high risk. Samples included 91 oral cavity and oropharyngeal cases and 71 controls. An additional group of 2 oropharyngeal cases and 16 normal healthy volunteers was also included. We performed an ELISA test for CD44, BCA assay for total protein, and ELISA test for p16 to determine whether the addition of p16 improved accuracy. Results: Mean CD44, total protein, and p16 levels were significantly elevated in cases compared to controls (CD44: cases= 2.944ng/ml, controls= 2.048ng/ml, p<0.005; total protein: cases= 0.522mg/ml, controls= 0.435mg/ml, p< 0.05; p16: cases= 270ng/ml, controls= 172ng/ml, p< 0.05). Using established cutpoints of 1.65ng/ml for CD44 and 0.320 mg/ml for protein resulted in a sensitivity of 82% and specificity of 25%. We developed receiver operator curves to establish new preliminary cutpoints for the combinations of CD44 (2.2ng/ml), TP (.700mg/ml), and p16 (.270ng/ml). With these cutpoints the sensitivity for 81% and the specificity was 51%. Conclusion: This feasibility study suggests that addition of a p16 assay to a CD44 and total protein laboratory test may improve specificity nearly 2-fold with a minimal decrease in sensitivity. Since CD44 and total protein are poor prognostic markers and p16 is a marker of good prognosis, the p16 assay may broaden range of cancers detected. Further study is warranted. Disclosures: Michael Donovan is a consultant and Chief Medical Officer for Vigilant Biosciences. The University of Miami and Dr. Franzmann hold intellectual property used in the study and have the potential for financial benefit from its future commercialization. Dr. Franzmann is the Chief Scientific Officer, consultant, and an equity holder in Vigilant Biosciences, licensee of the IP used in the study. Citation Format: Drew Smith, Elizabeth Franzmann, Michael Donovan. CD44, total protein, and p16 marker expression in oral rinses from oral and oropharyngeal cancer patients and controls [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A05.