Characterization of the Immune Landscape of EGFR-Mutant NSCLC Identifies CD73/Adenosine Pathway as a Potential Therapeutic Target

Abstract

Introduction: Lung adenocarcinomas harboring EGFR mutations do not respond to immune checkpoint blockade therapy and their EGFR wildtype counterpart. The mechanisms underlying this lack of clinical response have been investigated but remain incompletely understood.

Methods: We analyzed three cohorts of resected lung adenocarcinomas (Profiling of Resistance Patterns of Oncogenic Signaling Pathways in Evaluation of Cancer of Thorax, Immune Genomic Profiling of NSCLC, and The Cancer Genome Atlas) and compared tumor immune microenvironment of EGFR-mutant tumors to EGFR wildtype tumors, to identify actionable regulators to target and potentially enhance the treatment response.

Results: EGFR-mutant NSCLC exhibited low programmed death-ligand 1, low tumor mutational burden, decreased number of cytotoxic T cells, and low T cell receptor clonality, consistent with an immune-inert phenotype, though T cell expansion ex vivo was preserved. In an analysis of 75 immune checkpoint genes, the top up-regulated genes in the EGFR-mutant tumors (NT5E and ADORA1) belonged to the CD73/adenosine pathway. Single-cell analysis revealed that the tumor cell population expressed CD73, both in the treatment-naive and resistant tumors. Using coculture systems with EGFR-mutant NSCLC cells, T regulatory cell proportion was decreased with CD73 knockdown. In an immune-competent mouse model of EGFR-mutant lung cancer, the CD73/adenosine pathway was markedly up-regulated and CD73 blockade significantly inhibited tumor growth.

Conclusions: Our work revealed that EGFR-mutant NSCLC has an immune-inert phenotype. We identified the CD73/adenosine pathway as a potential therapeutic target for EGFR-mutant NSCLC.

Keywords: Adenosine; CD73; EGFR-mutant lung cancer; Immune microenvironment; T cells.

Figure 1.

EGFR-mutant tumors had lower PD-L1, TMB and CD8+ T cells. (A) PD-L1 by IHC H score in PROSPECT and density in ICON, (B) Tumor mutational burden by mutations per megabase (mut/Mb) in PROSPECT, ICON, TCGA, (C) CD3+ T cells by IHC in PROSPECT, density by multiplex immunofluorescent (mIF) in ICON, and the percent of inferred T cells using MCP Counter in TCGA, (D) CD8+ T cells by IHC in PROSPECT, density by immunofluorescent in ICON, and the percent of inferred CD8+ T cells using MCP Counter in TCGA, (E-F) representative mIF images from EGFR-mutant and WT tumor sections. Color keys are at the right of the images. White arrows and arrowhead indicate CD3+CD8+ T cells.

Figure 2.

EGFR-mutant tumors had decreased TCR clonality but intact ex vivo T cell expansion capacity. (A) TCR analysis from ICON cohort for clonality, after adjusting for TMB difference between EGFR-mutant and WT tumors (only cases with total non-synonymous mutations less than 100 were used for comparison). (B) TCR analysis from ICON cohort for richness and density, after adjusting for TMB difference between EGFR-mutant and WT tumors. (C) T cell ex vivo expansion capacity, including success rate, final cell count after successful expansion, and days to successful expansion.

Figure 3.

EGFR-mutant tumors had decreased INF-gamma signaling. (A-F) GZMA, CD8A, INFG, STAT1, IRF1 and CCL5 RNA expression levels were compared between EGFR-mutant and WT tumors in PROSPECT, ICON and TCGA. Fold-change was calculated comparing expression of EGFR-mutant tumors to WT, using the lower expressed as the denominator. A positive fold-change value indicates overexpression in EGFR-mutant tumors and a negative value indicates decreased expression in EGFR-mutant tumors. The univariant analysis was used for p-value. (G) gene expression fold change in the TCGA dataset. The genes with a fold change (EGFR/WT) >1.2 or < −1.2 were included.

Figure 4.

CD73/adenosine pathway was upregulated in EGFR-mutant tumors. (A-D) NT5E (CD73), CD38, ADORA1 and ADORA2A RNA expression levels were compared between EGFR-mutant and WT tumors in PROSPECT, ICON and TCGA. Fold-change was calculated comparing expression of EGFR-mutant tumors to WT, using the lower expressed as the denominator. A positive fold-change value indicates overexpression in EGFR-mutant tumors and a negative value indicates decreased expression in EGFR-mutant tumors. The univariant analysis was used for p-value. (E) Feature plots showing EPCAM+ cells from the 3 single-cell RNAseq samples (right panel: cells by sample; middle panel: cells by malignant versus normal; left panel, the expression levels of NT5E in each cell) (F). Porportion of NT5E expression cells in each sample. (G) phosphorylated EGFR, CD73 and CD38 protein levels were compared between EGFR-mutant and WT tumors in the ICON cohort, from the reverse-phase protein array (RPPA).

Figure 5.

CD73 blockade modulates T cell composition and function in EGFR-mutant non-small cell lung cancer cells. (A) CD73 cell surface expression on H1975 cells was assessed by flow cytometry with or without control and CD73 siRNAs. (B) Healthy donor PBMCs were incubated with conditioned median collected from H1975 cells after siRNA treatment. T regulatory cell population was assessed in each condition by flow cytometry using CD4+ and FoxP3+ as markers. (C) Healthy donor PBMCs were incubated with conditioned median collected from H1975 cells or H1975 after recombinant CD73 overexpression. T regulatory cell population was assessed by flow cytometry using CD4+ and FoxP3+ as markers. (D) Healthy donor PBMCs were co-cultured with H1975 cells with anti-CD73 and anti-PD-1 treatment, alone or in combination. Percent of tumor cell lysis was assessed using cytotoxicity assay. (E) Healthy donor PBMCs were co-cultured with H1975 cells with anti-CD73 and anti-PD-1 treatment, alone or in combination. The cell medium was collected and assessed for interferon release by ELISA INF-gamma assay. Student t-test was used, statistical significance: * indicates p value less than 0.05, ** indicates p value less than 0.005, *** indicates p value less than 0.0005, and **** indicates p value less than 0.0001.

Figure 6.

Anti-CD73 therapy-induced tumor reduction in EGFR-mutant murine lung cancers. (A) NT5E (CD73) and ADORA1 RNA expression levels in EGFR-mutant tumors and normal lung. (B) adenosine and AMP metabolite levels in EGFR-mutant tumors and normal lung. (C) CD73 immunohistochemistry for EGFR-mutant tumors and adjacent normal lung. (D) The change of tumor sizes with 2 weeks of treatment was documented and compared between vehicle-treated and anti-CD73 treated groups.