Abstract 2347: Multiplexed quantitative assessment and prognostic value of TIL subtypes in non-small cell lung cancer

Abstract

Abstract Background: The presence of high levels of tumor-infiltrating lymphocytes (TILs) is associated with favorable outcome in diverse human neoplasms including non-small cell lung cancer (NSCLC). Previous studies suggest that different TIL subtypes have distinct biological roles in cancer progression and their contribution to prognosis remains unclear. Here, we simultaneously quantified TIL subtypes using 2 different automated, objective methods in human NSCLC and determined their association with tumor characteristics and outcome. Methods: Using a sequential multiplexed immunofluorescence staining protocol with tyramide signal amplification and heat-inactivation steps, we simultaneously visualized all cell nuclei (DAPI), epithelial cells (Cytokeratin AE1/AE3, Dako), T helper cells (CD4-368, Leica), Cytotoxic T cells (CD8, C8/144B, Dako), B-lymphocytes (CD20, L26, Leica) and Regulatory T cells [Tregs], (Foxp3, D608R, CST) in 202 stages I-IV NSCLC represented in tissue microarray format. The preparations were digitalized and the number of each TIL subpopulation was counted using multispectral fluorescence analysis and a trainable tissue/cell image segmentation algorithm with the inForm software (PerkinElmer). Markers were also measured using the AQUA method of quantitative immunofluorescence (QIF). Results: NSCLC samples show a highly variable TIL content ranging from 0 to 2717 lymphocytes per tumor histospot. The median TIL content was 23.9 T-helper cells, 55.8 Cytotoxic T cells, 59.7 B-lymphocytes and 6.1 Tregs. Although the amount of CD4+, CD8+ and CD20+ cells was comparable between the histological NSCLC subtypes, the average number of Tregs (CD4+/Foxp3+ cells) was significantly higher in squamous cell carcinomas than in adenocarcinomas (10.4 and 4.9 cells per histospot, respectively, P = 0.03). Increased numbers of CD8+ or CD20+ TILs were significantly associated with longer overall survival (log-rank P<0.05), but the amount of CD4+ and Foxp3+ cells were not. Correlations were seen between multiplexed counting methods and QIF. Conclusions: This strategy allows simultaneous and objective quantification of diverse TIL subpopulations in lung tumor samples. Our results indicate that increased Cytotoxic T cells and B-lymphocytes, but not T helper cells or Tregs have prognostic value in NSCLC. Accurate quantification of TIL subpopulations in the tumor microenvironment could be useful to predict response and study the local effects of novel anti-cancer immunostimulatory therapies. Citation Format: Kurt A. Schalper, Cliff Hoyt, Chichung Wang, David Rimm. Multiplexed quantitative assessment and prognostic value of TIL subtypes in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2347. doi:10.1158/1538-7445.AM2015-2347