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RESEARCH PRODUCT

Analysis of the prognostic impact of Treg-related genes in tumor and stroma in resectable NSCLC

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11073 Background: Immunosuppressive regulatory T lymphocytes (Tregs) have been proved to play a critical role in immune tolerance to tumor. In this study we have analyzed the expression of 11 genes related to Tregs in both tumor and stroma samples of resectable NSCLC patients. Methods: Primary tumor tissues of FFPE samples from 125 early-stage NSCLC patients were used in this retrospective study. The most representative areas of tumor cells and tumor stroma of each sample were carefully micro-dissected. RTqPCR 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 TNF-a 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 analyses were considered significant at p< 0.05. Results: In both tumor and stroma, we found over-expression of CD25 (5.40X and 7.95X, respectively) and down-expression of CD127 (0.28X and 0.37X, respectively). There was a tendency toward higher expression of FOXP3 (1.67X and 2.01X, respectively) and CTLA-4 (1.92X and 1.76X, respectively) as well. Paired Wilcoxon test showed significant gene expression differences between tumor and stroma in FOXP3 (p=0.006), CD25 (p<0.0001), CD4 (p<0.0001), CD8 (p=0.028), IL-10 (p<0.0001) and TGFB-1 (p<0.0001). Kruskal-Wallis test showed that FOXP3 expression was higher in adenocarcinoma than in SCC samples (2.56X vs 1.79X, p=0.002). Survival analyses revealed that patients with a “Treg profile” (↑CD25 and ↓CD127) had a reduced overall survival (OS) (median 29.90 vs 74.33 months, p=0.003). We also found that those patients with higher levels of the ratio FOXP3 stroma/tumor had worse time to progression (TTP) (median 32.50 vs NR, p=0.04). Conclusions: Treg markers in the 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.