OBJECTIVES/GOALS: The objective of our research is to define unique molecular and immune markers in benign breast tissue to better identify women at risk of node-positive breast cancer (BC). The goal of the work is to improve individualized risk assessment, to guide targeted prevention and screening recommendations, and to reduce disease incidence and mortality. METHODS/STUDY POPULATION: From the Mayo Clinic’s Benign Breast Disease (BBD) cohort, we matched women who developed node-positive breast cancer after a BBD biopsy (cases; n=42) with women who remained cancer-free (controls; n=37), considering patient age and biopsy date. We used NanoString gene expression profiling to identify differentially expressed genes (DEGs) between cases and controls. We optimized a multiplex immunofluorescence (mIF) approach to simultaneously detect multiple markers within single FFPE tissue slides to correlate cells expressing DEGs in relation to innate and adaptive immune effectors. We used tissue segmentation, cell phenotyping, and spatial relationships to define molecular and immune differences between cases and controls. RESULTS/ANTICIPATED RESULTS: We discovered higher gene expression levels of IRF8 (interferon regulatory factor 8, a factor involved in immune cell differentiation) in controls as compared to cases (p = 0.0024) and found that IRF8 expression is also associated with longer cancer onset times among cases (p = 0.0012). Our pilot mIF experiments revealed higher frequencies of CD4+, CD8+, CD68+, CD20+ and CD11c+ cells in controls compared to cases. We predict that higher IRF8 expression and increased frequencies of immune cells in BBD biopsies indicate a proactive immune environment that may act to prevent cancer development. Furthermore, we predict that our analyses of the spatial localization of these markers by mIF may offer further predictive insight. DISCUSSION/SIGNIFICANCE: Deciphering the relationship between immune alterations in BBD and risk of node positive BC has the potential to improve individualized risk prediction. These insights will foster improved surveillance and informed screening and prevention, ultimately reducing BC incidence and mortality.