BackgroundLimited studies examine the immune landscape in Esophageal Adenocarcinoma (EAC). We aim to identify novel associations, which may inform immunotherapy treatment stratification.MethodsThree hundred twenty-nine EAC cases were available in Tissue Microarrays (TMA) format. A discovery cohort of 166 EAC cases were stained immunohistochemically for range of adaptive immune (CD3, CD4, CD8 and CD45RO) and immune checkpoint biomarkers (ICOS, IDO-1, PD-L1, PD-1). A validation cohort of 163 EAC cases was also accessed. A digital pathology analysis approach was used to quantify biomarker density.ResultsCD3, CD4, CD8, CD45RO, ICOS and PD-1 were individually predictive of better overall survival (OS) (Log rank p = < 0.001; p = 0.014; p = 0.001; p = < 0.001; p = 0.008 and p = 0.026 respectively). Correlation and multivariate analysis identified high CD45RO/ICOS patients with significantly improved OS which was independently prognostic (HR = 0.445, (0.223–0.886), p = 0.021). Assessment of CD45RO and ICOS high cases in the validation cohort revealed an associated with improved OS (HR = 0.601 (0.363–0.996), p = 0.048). Multiplex IHC identified cellular co-expression of high CD45RO/ICOS. High CD45RO/ICOS patients have significantly improved OS.ConclusionsMultiplexing identifies true cellular co-expression. These data demonstrate that co-expression of immune biomarkers are associated with better outcome in EAC and may provide evidence for immunotherapy treatment stratification.