Immune checkpoint blockade (ICB) therapies have emerged as an essential treatment modality in lung cancer. While ICB therapies are approved in several indications for stage IV and metastatic non-small cell lung cancers (NSCLC), approvals for early but invasive tumors have been limited. To investigate the molecular landscape potentially underlying this difference, we assessed levels of known ICB biomarkers and transcriptome-wide pathway activity between stage III and IV NSCLC primary tumors across a real-world cohort.

Following exome-capture bulk RNA sequencing of tumors with non-squamous NSCLC histology, data underwent quality control and normalization. We employed a linear regression approach adjusted for age, sex, and tumor purity to identify hundreds of differentially expressed (DE) genes by stage (false discovery rate=5%). Correlations were evaluated by Pearson’s coefficient, and differences in means were assessed by Mann-Whitney U test and Student’s t-test, as appropriate.

From KEGG gene set enrichment analysis of the DE genes, we found metabolic pathways were significantly enriched in stage IV tumors. In contrast, several immune-related pathways were relatively depleted in stage IV tumors, including pathways associated with NF-κB signaling, IL-17 signaling, TNF signaling, Th1 and Th2 differentiation, and Th17 differentiation. Consistent with this depletion of immune signaling pathways in stage IV tumors, several important ICB biomarkers including CTLA4 and PDCD1 (PD-1) had increased expression in stage III tumors relative to stage IV. Notably, CD274 (PD-L1) expression did not differ by stage, nor did tumor cell PD-L1 protein levels, as measured by IHC. In addition, PD-L1 gene expression and protein levels were concordant across both stages (P<2.2e-16). Based on these results, we next examined differences in overall level of immune infiltration using estimated immune proportions derived from gene expression data. While overall immune proportion did not differ significantly between stage III and IV tumors, stage III tumors had a significantly higher estimated proportion of CD4+ T cells relative to stage IV, and stage IV tumors had a significantly higher proportion of macrophages relative to stage III.

These results demonstrate enrichment of immune activation gene expression pathways and paradoxically, increased expression of immune checkpoint biomarkers in stage III tumors relative to stage IV tumors, suggesting that evolution of metastasis in NSCLC tumors is accompanied by consequential changes in the tumor-immune microenvironment. This comprehensive assessment of the expression of immune checkpoint genes, and the transcriptome more broadly, may help to inform the development of anti-cancer immune modulating agents or use of existing ICB in early-stage NSCLC.

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