Prognostic Impact of Immune Microenvironment Markers in Tumor and Stroma in Resectable Nsclc

Abstract

Aim: Tumor-infiltrating immune cells have been proved to play an important role in tumor progression. In this study we have analyzed the expression of 11 immune related genes in both tumor and stroma samples of resectable NSCLC patients. Methods: In this retrospective study FFPE samples from 117 early-stage NSCLC patients of primary tumor tissue were used. The most representative areas of tumor and tumor stroma of each sample were carefully micro-dissected. RT-PCR using hydrolysis probes (TaqMan, Applied Biosystems) was performed to assess the expression of Treg markers such as: CD127, CD25, FOXP3, CTLA-4, IL-10, TGFB-1, LAG-3, GITR and TNFA as well as CD4 and CD8. Relative gene expression was assessed using GAPDH and CDKN1B as endogenous controls and results were normalized against a human cDNA (Clontech) as a reference. All statistical analysis were considered significant at p< 0.05. Results: In both tumor and stroma, we found over-expression of CD25 (19.46X and 11.59X, respectively), FOXP3 (4.96X and 4.08X, respectively), CTLA-4 (2.89X and 3.02, respectively) and TGBF-1 (3.24X and 2.11X, respectively) and down-expression of TNFA (0.39X and 0.27X, respectively). Survival analyses revealed that patients with lower expression levels of CD8 in the tumor have worse overall survival (OS) (median 37.20 vs 81.23 months, p = 0.001) and progression free survival (PFS) (median 19.43 vs 81.23 months, p = 0.002). Lower expression levels were also associated with worse OS for CD4 (42.90 vs 81.23 months, p= 0.026) and LAG-3 (36.20 vs 69 months, p = 0.017) in tumor. Furthermore, patients with a "Treg profile" (↑CD25 and ↓CD127) in stroma had worse PFS (median 12.7 vs 35.36 months, p = 0.004). We also found that those patients with higher levels of the ratio FOXP3 stroma/FOXP3 tumor had worse OS (median 42.9 vs NR, p = 0.002) and PFS (19.43 vs NR, p = 0.001), and the same was observed for the ratio FOXP3 stroma/CD4 tumor (for OS, median 46.66 vs 81.23 months, p = 0.025 and for PFS, median 22.13 vs 37.8 months, p= 0.027) and for ratio FOXP3 stroma/ CD8 tumor (for OS, median 46.43 vs 74.33 months, p = 0.017 and for PFS, median 22.13 vs 37.8 months, p= 0.022). Conclusions: Immune biomarkers in tumor microenvironment seem to play an important prognostic role in early-stage NSCLC patients. Supported by grants PS09-01149 and RD06/0020/1024 from ISCIII. Disclosure: All authors have declared no conflicts of interest.