Abstract B30: Digital multiplex immunofluorescence analysis identifies immune profiles in the tumor stroma associated with clinical outcome

Background: The recent successes of immunotherapy in non-small cell lung cancer (NSCLC) raise the interest to the deeper understanding of the diversity of the immune microenvironment. Meticulous characterization of the immune cell infiltrates is crucial for patients’ prognosis and the prediction therapeutic effects. The aim of this study was to analyze expression profiles of the immune cells and their linkage to clinical parameters in NSCLC patients. Methods: A tissue microarray of the NSCLC cohort comprised of 55 cases was used in the study. The staining was performed with the antibodies against CD8, CD20, CD4, FoxP3, CD45RO and pan-cytokeratin together with immuno-fluorescent markers (Opal, Perkin Elmer) and digital images acquired by a multispectral imaging system (Vectra 3, PerkinElmer). Marker expression pattern was evaluated on a cell level in total sample area, or in stroma and tumor area separately. Conventional immunohistochemical techniques and RNAseq data were used to validate the method. Results: Marker expression intensities were used to identify 5 non-epithelial cell classes, which express CD8, CD20, CD4, FoxP3, CD45RO. Relative abundance of marker-positive cells in individual cases were subjected to hierarchical clustering analysis, for the discovery of distinct immune cell infiltration patterns. The resulted clusters were exploratively analyzed with regard to survival associations. Patients with specific immune profile with high infiltration of FoxP3+ cells, moderate infiltration of CD4+ and CD8+ cells and low quantity of CD20+ and CD45RO+ cells in the stroma were associated with longer survival (median 87 vs. 54 month, log-rank p=0.050). Conclusion: Multi-marker analysis on single-cell levels with the fluorescence multiplexed IHC technique provides a valuable basic immune profile of cancer patients. Clustering analysis of the expression of immune markers identified a group of patients with improved survival in NSCLC independent of histology. Note: This abstract was not presented at the conference. Citation Format: Artur Mezheyeuski, Christian Bergsland, Max Backman, Tobias Sjoblom, Ragnhild A. Lothe, Jarle Bruun, Patrick Micke. Digital multiplex immunofluorescence analysis identifies immune profiles in the tumor stroma associated with clinical outcome [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B30.