Increased interleukin-6/C-reactive protein levels are associated with the upregulation of the adenosine pathway and serve as potential markers of therapeutic resistance to immune checkpoint inhibitor-based therapies in non-small cell lung cancer

Abstract

Background: Systemic immune activation, hallmarked by C-reactive protein (CRP) and interleukin-6 (IL-6), can modulate antitumor immune responses. In this study, we evaluated the role of IL-6 and CRP in the stratification of patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs). We also interrogated the underlying immunosuppressive mechanisms driven by the IL-6/CRP axis.

Methods: In cohort A (n=308), we estimated the association of baseline CRP with objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) in patients with NSCLC treated with ICIs alone or with chemo-immunotherapy (Chemo-ICI). Baseline tumor bulk RNA sequencing (RNA-seq) of lung adenocarcinomas (LUADs) treated with pembrolizumab (cohort B, n=59) was used to evaluate differential expression of purine metabolism, as well as correlate IL-6 expression with PFS. CODEFACS approach was applied to deconvolve cohort B to characterize the tumor microenvironment by reconstructing the cell-type-specific transcriptome from bulk expression. Using the LUAD cohort from The Cancer Genome Atlas (TCGA) we explored the correlation between IL-6 expression and adenosine gene signatures. In a third cohort (cohort C, n=18), plasma concentrations of CRP, adenosine 2a receptor (A2aR), and IL-6 were measured using ELISA.

Results: In cohort A, 67.2% of patients had a baseline CRP≥10 mg/L (CRP-H). Patients with CRP-H achieved shorter OS (8.6 vs 14.8 months; p=0.006), shorter PFS (3.3 vs 6.6 months; p=0.013), and lower ORR (24.7% vs 46.3%; p=0.015). After adjusting for relevant clinical variables, CRP-H was confirmed as an independent predictor of increased risk of death (HR 1.51, 95% CI: 1.09 to 2.11) and lower probability of achieving disease response (OR 0.34, 95% CI: 0.13 to 0.89). In cohort B, RNA-seq analysis demonstrated higher IL-6 expression on tumor cells of non-responders, along with a shorter PFS (p<0.05) and enrichment of the purinergic pathway. Within the TCGA LUAD cohort, tumor IL-6 expression strongly correlated with the adenosine signature (R=0.65; p<2.2e-16). Plasma analysis in cohort C demonstrated that CRP-H patients had a greater median baseline level of A2aR (6.0 ng/mL vs 1.3 ng/mL; p=0.01).

Conclusions: This study demonstrates CRP as a readily available blood-based prognostic biomarker in ICI-treated NSCLC. Additionally, we elucidate a potential link of the CRP/IL-6 axis with the immunosuppressive adenosine signature pathway that could drive inferior outcomes to ICIs in NSCLC and also offer novel therapeutic avenues.

Keywords: Adenosine; Immune Checkpoint Inhibitors; Inflammation; Lung Neoplasms; Tumor Microenvironment.

Figure 1

Kaplan-Meier survival estimates according to the CRP category among the whole study population. (A) Overall survival (OS). The median OS of patients with CRP-high was 8.6 months (95% CI: 6.6 to 11.0; 137 events), while the median OS of patients with CRP-low was 14.8 months (95% CI: 9.8 to 27.2; 52 events) (p=0.006) HR=1.52 (95% CI: 1.12 to 2.03). (B) Progression-free survival (PFS). The median PFS of patients with CRP-high was 3.3 months (95% CI: 3.3 to 4.7; 168 events) while the median PFS of CRP-low patients was 6.6 months (95% CI: 5.3 to 8.7; 71 events) (p=0.013) HR=1.42 (95% CI: 1.07 to 1.87). (C) Objective response rate (ORR) analysis according to the CRP category among the whole study population and the ICI alone, Chemo-ICI cohorts separately. For the overall population, among the evaluable CRP-low patients, the ORR was 46.3% (95% CI: 27.9 to 73.5), while among CRP-high was 24.7% (95% CI: 15.3 to 37.7); p=0.015. Among the ICI alone cohort, patients with CRP-low experienced higher ORR in comparison to patients with CRP-high (42.1% vs 25.0%, p=0.060). Similarly, among the Chemo-ICI cohort patients with CRP-low achieved an ORR compared with patients with CRP-high (100% vs 20%, p=0.040). CRP, C-reactive protein; Chemo-ICI: chemotherapy and immune checkpoint inhibitor.

Figure 2

Data from cohort B comprising lung adenocarcinoma tumors. (A) Boxplot showing that the IL-6 expression in cancer cell is significantly higher in non-responders (blue; n=44) versus responders (red; n=15) with Wilcoxon rank-sum p<0.08, whereas the IL-6 expression in T-cell (B) shows the opposite trend (Wilcoxon rank-sum p<0.08). Non-responders (NR) included SD/PD and responders (R(included tumors with PR/CR. (C) Kaplan-Meier plot of the progression-free survival of patients with ICT-treated lung cancer with high IL-6 expression (transcripts per million; TPM) in cancer cells (blue; n=24) versus low IL-6 expression in cancer (yellow; n=35). The median survival difference was 81 days, p=0.039. (D) (Korean cohort) Pathway enrichment analysis of the genes upregulated in high IL-6 expression (in cancer cells) versus low IL-6 expression (in cancer cells). The pathways are listed in the vertical axis, and the enrichment p values are denoted in the horizontal axis (−log(P)). Large number on the horizontal axis denotes a more significant enrichment. (E) (TCGA analysis) X-axis shows IL-6 expression and Y-axis shows the adenosine pathway signature from a study by Araki et al. The Spearman rank correlation coefficient and the associated p value is noted at the top of the figure. CR, complete response; IL, interleukin; MHC, major histocompatibility complex; PD, progressive disease; PR, partial response; RSEM, RNA-seq by expectation-maximization; SD, stable disease; TCGA, The Cancer Genome Atlas.

Figure 3

(A) Correlation of baseline IL-6 in the plasma with the blood CRP in n=18 patients in the East Carolina University cohort demonstrating a correlation coefficient (r) of 0.76. (B) Median levels of A2aR in the plasma were significantly higher for CRP-high versus CRP-low as well as in (C) patients with above and below median levels of IL-6 in the plasma. A2aR, adenosine 2a receptor; CRP, C-reactive protein; IL, interleukin.