Epithelial-Mesenchymal Transition Is Associated with a Distinct Tumor Microenvironment Including Elevation of Inflammatory Signals and Multiple Immune Checkpoints in Lung Adenocarcinoma

Abstract

Purpose: Promising results in the treatment of non-small cell lung cancer (NSCLC) have been seen with agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed death ligand-1 (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune checkpoint blockade is critical to successful clinical translation of these agents.

Methods: We conducted an integrated analysis of three independent large datasets, including The Cancer Genome Atlas of lung adenocarcinoma and two datasets from MD Anderson Cancer Center (Houston, TX), Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax (named PROSPECT) and Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (named BATTLE-1). Comprehensive analysis of mRNA gene expression, reverse-phase protein array, IHC, and correlation with clinical data were performed.

Results: Epithelial-mesenchymal transition (EMT) is highly associated with an inflammatory tumor microenvironment in lung adenocarcinoma, independent of tumor mutational burden. We found immune activation coexistent with elevation of multiple targetable immune checkpoint molecules, including PD-L1, PD-L2, PD-1, TIM-3, B7-H3, BTLA, and CTLA-4, along with increases in tumor infiltration by CD4(+)Foxp3(+) regulatory T cells in lung adenocarcinomas that displayed an EMT phenotype. Furthermore, we identify B7-H3 as a prognostic marker for NSCLC.

Conclusions: The strong association between EMT status and an inflammatory tumor microenvironment with elevation of multiple targetable immune checkpoint molecules warrants further investigation of using EMT as a predictive biomarker for immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly a broad range of other cancers. Clin Cancer Res; 22(14); 3630-42. ©2016 AACRSee related commentary by Datar and Schalper, p. 3422.

Figure 1. Elevated PD-1:PD-L1/PD-L2 axis in “mesenchymal” versus “epithelial” lung adenocarcinoma

“Epithelial” lung adenocarcinoma is defined by EMT scores ≤ lowest 1/3 as described in methods and represented as E. Similarly, “mesenchymal” lung adenocarcinoma is defined by EMT scores ≥ highest 1/3 and represented as M. Gene expression levels of PD-1: PD-L1/PD-L2 axis in TCGA dataset (A), PROSPECT dataset (B). PD-L1 (C) and PD-1 expression (D) by IHC in tumors from PROSPECT dataset are shown. 35 and 33 tumor tissues with “mesenchymal” or “epithelial” lung adenocarcinomas were used for the IHC study. Five random regions (1 mm²) in the core of each tumor in each group were analyzed. Unpaired T test was performed. 200 µm scale bar is shown in each representative IHC picture. PD-L1 RPPA correlated to PD-L1 mRNA or EMT score, RPPA in “epithelial” versus “mesenchymal” lung adenocarcinomas, and versus E-cadherin RPPA in TCGA (E) and PROSPECT (F) samples.

Figure 2. Elevation of multiple immune checkpoint molecules in “mesenchymal” as compared to “epithelial” lung adenocarcinoma

Supervised cluster heatmap of immune related molecules in “epithelial” lung adenocarcinoma versus “mesenchymal” lung adenocarcinoma from TCGA (A), and PROSPECT (B) respectively. Expression levels of immune checkpoint molecules in “epithelial” lung adenocarcinoma versus “mesenchymal” lung adenocarcinoma in tumor tissues from TCGA (C) and PROSPECT (D) respectively.

Figure 3. Increased tumor infiltrating CD3⁺ T cells and CD4⁺FOXP3⁺ regulatory T cells, but not CD8⁺ T cells and macrophages in “mesenchymal” versus “epithelial” lung adenocarcinoma

IHC staining and scoring of CD3 (A), CD4 (B), FOXP3 (C), CD8 (D) and CD68 (E) were performed in tumors from PROSPECT. 35 and 33 tumor tissues with “mesenchymal” or “epithelial” lung adenocarcinomas were used for the IHC study, respectively. The staining and density of each marker were analyzed using the cell membrane staining algorithm, except for FOXP3, which used a nuclear staining algorithm. Five random regions (1 mm²) in the core of each tumor at the same region in each group were analyzed. 200 µm scale bar is shown in each representative IHC picture.

Figure 4. Evidence of immune activation in “mesenchymal” compared to “epithelial” adenocarcinoma

Gene expression levels of multiple immune stimulatory molecules including CD80, CD86, OX40L, 4-1BB, ICOS, CD127 were analyzed in “mesenchymal” lung adenocarcinoma versus “epithelial” lung adenocarcinoma in tumor tissues from (A) TCGA, (B) PROSPECT. Gene expression of IFN-γ and IFN-γ inducible genes including CXCL10 and IDO were also analyzed in “mesenchymal” in comparison to “epithelial” lung adenocarcinoma in tumor tissues from (C) TCGA, (D) PROSPECT.

Figure 5. EMT is not associated with tumor mutational burden

Non-silent mutational rate, transversion rate and TP53 mutation of TCGA lung adenocarcinoma samples were included in the analysis. (A) Spearman correlation between non-silent mutational rate per Mb and EMT score. (B) Non-silent mutational rate in “epithelial” versus “mesenchymal” lung adenocarcinoma. (C) Non-silent mutational rate in TP53 mutant versus TP53 wild-type lung adenocarcinoma. (D) Non-silent mutational rate in TP53 mutant versus wild-type lung adenocarcinoma further separated by molecular smoking exposure via transversion high and low. (E) Non-silent mutational rate in “epithelial” versus “mesenchymal” lung adenocarcinoma further separated by molecular smoking exposure via transversion high and low.

Figure 6. B7-H3 is associated with poor OS and RFS in lung adenocarcinoma

The probability of overall survival and recurrence free survival of patients from PROSPECT were analyzed by dividing the patients into either high or low group based on the expression levels of each immune checkpoint molecule or EMT score. Patients with gene expression levels higher or lower than average expression level of each gene or EMT score are considered as high or low respectively. (A) Overall survival, (B) Recurrence free survival. Both are from PROSPECT.