Abstract
OBJECTIVES: To determine the prognostic significance of spatial proximity of lung cancer cells and specific immune cells in the tumor microenvironment. MATERIALS AND METHODS: We probed formalin-fixed, paraffin-embedded (FFPE) tissue microarrays using a novel tyramide signal amplification multiplexing technique labelling CD8, CD4, Foxp3, and CD68+ cells. Each multiplex stained immunohistochemistry slide was digitally processed by Vectra INFORMS software, and an X- and Y-coordinate assigned to each labeled cell type. The abundance and spatial location of each cell type and their proximity to one another was analyzed using a novel application of the G-cross spatial distance distribution method which computes the probability of finding at least one immune cell of any given type within a rμm radius of a tumor cell. Cox proportional hazards multiple regression was used for multivariate analysis of the influence of proximity of lymphocyte types. RESULTS: Pathologic tumor specimens from 120 NSCLC patients with pathologic tumor stage I-III disease were analyzed. On univariate analysis, age (P = .0007) and number of positive nodes (P = .0014) were associated with overall survival. Greater area under the curve (AUC) of the G-cross function for tumor cell-Treg interactions was significantly associated with worse survival adjusting for age and number of positive nodes (HR 1.52 (1.11-2.07), P = .009). Greater G-cross AUC for T-reg-CD8 was significantly associated with better survival adjusting for age and number of positive lymph nodes (HR 0.96 (0.92-0.99), P = .042). CONCLUSION: Increased infiltration of regulatory T cells into core tumor regions is an independent predictor of worse overall survival in NSCLC. However, increased infiltration of CD8+ cytotoxic T cells among regulatory T cells seems to mitigate this effect and was significantly associated with better survival. Validation of the G-cross method of measuring spatial proximity between tumor and immune cell types and exploration of its use as a prognostic factor in lung cancer treatment is warranted.