PROJECT SUMMARY/ABSTRACT Oral and oropharyngeal cancers result in over 10,000 deaths each year in the United States. Although oral squamous cell carcinoma (OSCC) patients with localized disease have survival rates of up to 80%, about two-thirds present clinically with regional and distant metastases associated with five-year survival rates of 50% and 35%, respectively. Despite advances in immunotherapy, the five-year mortality rate for OSCC has remained constant over the last several decades, underscoring the importance of early detection and intervention. The majority of OSCCs arise from pre-cancerous lesions called oral epithelial dysplasias (OED), only some of which will progress to invasive cancers. Patients with OEDs that will progress would likely benefit from more aggressive treatment early on; however, the morbidities associated with aggressive treatment are significant, preventing their broad use in all patients. While this general outlook is similar across many cancers, the accessibility of oral cavity lesions also provides a unique opportunity for detailed analysis to understand the biological processes that contribute to or protect against progression into invasive and malignant cancer. We will test the hypothesis that the immune response to OED regulates the risk of progression. The immune system responds to disruptions and danger in tissues. Significant evidence supports the important role of the immune system in responding to early lesions in the oral cavity, including an abundance of immune cells infiltrating these tissues, elevated risk in immunosuppressed individuals, and loss of MHC class I antigen presentation machinery in many OSCC tumors. However, features of the immune response are not currently utilized to define treatment strategies or to stratify risk in OED or OSCC patients, presenting an unmet opportunity. The recent development of multiplexed ion beam imaging (MIBI) enables unprecedented detailed analysis of archival pathological tissues. This technology, which we recently implemented with the help of an NIH Instrumentation Grant, uses antibodies conjugated to heavy-metal reporter ions to quantify up to 50 proteins simultaneously at subcellular (400nm) resolution in formalin-fixed paraffin-embedded tissues. We have collated a substantial number of archival tissues from OED patients with detailed clinical and follow up data, including progression to OSCC. Here, we will leverage MIBI to conduct a detailed analysis of immune responses in these tumors, providing new insight into the immunological mechanisms and cellular interactions in these microenvironments. In Aim 1, we will test the hypothesis that the types of immune cells present and their activation states are distinct between OEDs that went on to progress versus those that have not. In Aim 2, we will test the hypothesis that the architecture and cellular neighborhoods within the tissue are distinct in OEDs that progressed to OSCC. In Aim 3, we will use these da...