Plasma Immune Proteins and Circulating Tumor DNA Predict the Clinical Outcome for Non-Small-Cell Lung Cancer Treated with an Immune Checkpoint Inhibitor

Abstract

Immunotherapy has altered the therapeutic landscape for patients with non-small-cell lung cancer (NSCLC). The immune checkpoint inhibitor pembrolizumab targets the PD-1/PD-L1 signaling axis and produces durable clinical responses, but reliable biomarkers are lacking. Using 115 plasma samples from 42 pembrolizumab-treated patients with NSCLC, we were able to identify predictive biomarkers. In the plasma samples, we quantified the level of 92 proteins using the Olink proximity extension assay and circulating tumor DNA (ctDNA) using targeted next-generation sequencing. Patients with an above-median progression-free survival (PFS) had significantly higher expressions of Fas ligand (FASLG) and inducible T-cell co-stimulator ligand (ICOSLG) at baseline than patients with a PFS below the median. A Kaplan-Meier analysis demonstrated that high levels of FASLG and ICOSLG were predictive of longer PFS and overall survival (OS) (PFS: 10.83 vs. 4.49 months, OS: 27.13 vs. 18.0 months). Furthermore, we identified a subgroup with high expressions of FASLG and ICOSLG who also had no detectable ctDNA mutations after treatment initiation. This subgroup had significantly longer PFS and OS rates compared to the rest of the patients (PFS: 25.71 vs. 4.52 months, OS: 34.62 vs. 18.0 months). These findings suggest that the expressions of FASLG and ICOSLG at baseline and the absence of ctDNA mutations after the start of treatment have the potential to predict clinical outcomes.

Keywords: biomarkers; circulating tumor DNA; immune checkpoint inhibitor; immuno-oncology; immunotherapy; non-small-cell lung cancer.

Figure 1

Volcano plots showing log2 fold change in plasma protein expression between patients with above-median PFS and patients with below-median PFS at three different time points. Proteins with a positive log2 FC (the right part of the volcano plot) are upregulated in patients with above-median PFS, while proteins with a negative log2 FC (the left part of the volcano plot) are upregulated in patients with below-median PFS. (A) Differences in protein expression before treatment initiation, (B) after one and (C) two cycles of treatment. Vertical dashed lines represent 2-fold change, while the horizontal dashed line represent a p-value of 0.05. Proteins with expressions below the limit of detection were excluded from the analysis. FC, fold change; PFS, progression-free survival; T₀, pre-treatment; T₁, after one cycle of therapy; T₂, after two cycles of therapy.

Figure 2

Kaplan–Meier survival analyses for PFS and OS. (A) PFS according to median expression of ICOSLG. (B) OS according to median expression of ICOSLG. (C) PFS according to median expression of FASLG. (D) OS according to median expression of FASLG. (E) PFS according to high expression of FASLG (expression in the upper quartile). (F) OS according to high expression of FASLG. FASLG, Fas ligand; ICOSLG, inducible T-cell co-stimulator ligand; OS, overall survival; PFS, progression-free survival.

Figure 3

Expressions of FASLG and ICOSLG. (A) NPX values at T₀. (B) Correlation between FASLG and ICOSLG NPX values at T₀. The dotted lines represent the 95% confidence interval. (C) Dynamics in FASLG expression during the initial weeks of treatment. (D) Dynamics in ICOSLG expression during the initial weeks of treatment. FASLG, Fas ligand; ICOSLG, inducible T-cell co-stimulator ligand; NPX, normalized protein expression; T₀, pre-treatment; T₁, after one cycle of therapy; T₂, after two cycles of therapy; ns, not significant; *, p < 0.05; ***, p < 0.001.

Figure 4

Combining expressions of FASLG and ICOSLG before treatment initiation. (A) PFS according to FASLG and ICOSLG expressions. Patients were stratified into the high-expression subgroup if they had an above-median expression of either FASLG and/or ICOSLG. Patients with low expressions of both proteins were stratified into the low-expression subgroup. (B) OS according to FASLG and ICOSLG expressions. (C) Forest plot showing HR and 95% CI for single and combined protein-expression subgroups for PFS. (D) Forest plot showing HR and 95% CI for single and combined protein-expression subgroups for OS. CI, confidence interval; FASLG, Fas ligand; ICOSLG, inducible T-cell co-stimulator ligand; OS, overall survival; PFS, progression-free survival.

Figure 5

Combining circulating biomarkers. (A) Proportion of patients in each FASLG and ICOSLG expression subgroup having a presence or absence of ctDNA mutations after treatment initiation. (B) PFS according to presence or absence of ctDNA mutations after treatment initiation in the high-expression FASLG and ICOSLG subgroups. (C) OS according to the presence or absence of ctDNA mutations after treatment initiation in the high FASLG and ICOSLG expression subgroup. (D) PFS according to a combined biomarker profile, i.e., patients in the high-expression FASLG and ICOSLG subgroups with an absence of ctDNA mutations, compared to the rest of the patients. (E) OS according to a combined biomarker profile. FASLG, Fas ligand; ICOSLG, inducible T-cell co-stimulator ligand; ns, not significant; OS, overall survival; PFS, progression-free survival.