Analysis of the immune microenvironment in resected non-small cell lung cancer: the prognostic value of different T lymphocyte markers

Abstract

The prognosis of non-small cell lung cancer (NSCLC) remains poor and heterogeneous and new biomarkers are needed. As the immune system plays a pivotal role in cancer, the study of immune-related markers may provide valuable prognostic information of NSCLC. In 122 formalin-fixed, paraffin-embedded tumor tissue samples from early-stage NSCLC, tumor and tumor-near stromal areas were microdissected and gene expression levels of conventional and regulatory T cell markers were assessed by quantitative polymerase chain reaction. Also, the presence of infiltrating CD4+, CD8+, and FOXP3+ cells in tumor samples was assessed by immunohistochemistry. The relative proportion of conventional and regulatory T cells present in the tumor environment was assessed and found to be key to understand the importance that the immune system analysis has in the prognostics of NSCLC patients. The presence of CD8+ cells in the tumor compartment was associated with better outcome, whereas the presence of FOXP3+ cells was associated with worse overall survival. The negative prognostic value of combined biomarkers, indicating high levels of FOXP3 in the stroma and low levels of CD4 or CD8 in tumors, was observed at mRNA level and was validated by immunohistochemistry.In conclusion, the proportion of T helper and cytotoxic cells vs. regulatory T cells in different locations of the tumor microenvironment have opposite prognostic impacts in resected NSCLC.

Keywords: NSCLC; immune-biomarker; prognostic; tumor compartment; tumor stroma; Autophagy.

Figure 1. Representative immunohistochemical staining of FOXP3, CD4 and CD8 in tumor and stroma compartments

Original magnification X200. A. Low infiltration of FOXP3⁺ lymphocytes in tumor compartment, B. high infiltration of FOXP3⁺ lymphocytes in tumor compartment, C. low infiltration of FOXP3⁺ lymphocytes in stroma compartment, D. high infiltration of FOXP3⁺ lymphocytes in stroma compartment, E. low infiltration of CD4⁺ lymphocytes in tumor compartment, F. high infiltration of CD4⁺ lymphocytes in tumor compartment, G. low infiltration of CD4⁺ lymphocytes in stroma compartment and, H. high infiltration of CD4⁺ lymphocytes in stroma compartment, I. Low infiltration of CD8⁺ lymphocytes in tumor compartment, J. high infiltration of CD8⁺ lymphocytes in tumor compartment, K. low infiltration of CD8⁺ lymphocytes in stroma compartment, L. high infiltration of CD8⁺ lymphocytes in stroma compartment.

Figure 2. Kaplan-Meier plots for progression free survival and overall survival according to immune cells infiltration

A., B. Infiltration of CD8⁺ cells in tumor compartment and, C., D. infiltration of FOXP3⁺ cells in the stroma compartment. Densities were dichotomized as high and low infiltration according to the median of positive lymphocytes..

Figure 3. Kaplan-Meier plots for progression free survival and overall survival according to the ratios for conventional and regulatory gene expression markers

A., B. FOXP3 stroma/ CD4 tumor expression ratio and, C., D. FOXP3 stroma/ CD8 tumor expression ratio. Gene expression levels were dichotomized according to the median.

Figure 4. Kaplan-Meier plots for progression free survival and overall survival according to the FOXP3 cells proportion to conventional T cells markers

A., B. Combination of FOXP3⁺ cells in the stroma/CD4⁺ cells in the tumor, and C., D. combination of FOXP3⁺ cells in the stroma/CD8⁺ cells in the tumor.