Soluble and EV-bound CD27 act as antagonistic biomarkers in patients with solid tumors undergoing immunotherapy

Abstract

Background: The major breakthrough in cancer therapy with immune checkpoint inhibitors (ICIs) has highlighted the important role of immune checkpoints in antitumoral immunity. However, most patients do not achieve durable responses, making biomarker research in this setting essential. CD27 is a well known costimulatory molecule, however the impact of its soluble form in ICI is poorly investigated. Therefore, we aimed at testing circulating concentrations of soluble CD27 (sCD27) and CD27 bound to extracellular vesicles (EVs) as potential biomarkers to predict response and overall survival (OS) in patients undergoing ICI.

Methods: Serum and plasma levels of sCD27 were assessed by immunoassay in three patient cohorts (n = 187) with advanced solid malignancies including longitudinal samples (n = 126): a training (n = 84, 210 specimens, Aachen ICI) and validation cohort (n = 70, 70 specimens, Hamburg ICI), both treated with ICI therapy, and a second independent validation cohort (n = 33, 33 specimens, Hamburg non-ICI) undergoing systemic therapy without any ICI. In a subset (n = 36, 36 baseline and 108 longitudinal specimens), EV-bound CD27 from serum was measured, while EV characterization studies were conducted on a fourth cohort (n = 45).

Results: In the Aachen and Hamburg ICI cohorts, patients with lower circulating sCD27 levels before and during ICI therapy had a significantly longer progression-free survival (PFS) and OS compared to patients with higher levels, a finding that was confirmed by multivariate analysis (MVA) (Aachen ICI: p_PFS = 0.012, p_OS = 0.001; Hamburg ICI: p_PFS = 0.040, p_OS = 0.004) and after randomly splitting both cohorts into training and validation. This phenomenon was not observed in the Hamburg non-ICI cohort, providing a rationale for the predictive biomarker role of sCD27 in immune checkpoint blockade. Remarkably, EV-bound CD27 baseline levels and dynamics during ICI therapy also emerged as potent predictive biomarkers, acting however antagonistically to soluble sCD27, i.e. higher levels were associated with PFS and OS benefit. Combining both molecules ("multi-CD27" score) enhanced the predictive ability (HR_PFS: 17.21 with p < 0.001, HR_OS: 6.47 with p = 0.011).

Conclusion: Soluble and EV-bound CD27 appear to have opposing immunomodulatory functions and may represent easily measurable, non-invasive prognostic markers to predict response and survival in patients undergoing ICI therapy.

Keywords: Biomarker; Immunotherapy; Liquid biopsy; PD-1; Prognosis; Soluble immune checkpoints.

Fig. 1

sCD27 concentrations are significantly elevated in cancer patients compared to cancer-free controls across three different cohorts and predict best response in patients undergoing immune checkpoint blockade (A-C) sCD27 concentrations in cancer patients and healthy controls: ICI treated Aachen cohort (n = 84 cancer patients, n = 35 healthy controls) (A), ICI-treated Hamburg 1 cohort (n = 70 cancer patients, n = 32 controls) (B), non-ICI treated Hamburg 2 cohort (n = 33 cancer patients, n = 32 controls) (C). (D-E) sCD27 concentrations according to best response in the ICI-treated Aachen (D) and Hamburg ICI (E) cohorts, as well as the Hamburg non-ICI cohort (F). *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001

Fig. 2

Baseline concentrations of sCD27 predict progression-free and overall survival in patients under ICI therapy. Kaplan Meier curves for PFS (A, C, E) and OS (B, D, F) stratified by ideal baseline cut-off of soluble CD27 levels calculated for PFS in the ICI-treated Aachen cohort (A-B) and Hamburg 1 cohort (C-D), as well as non-ICI treated Hamburg 2 cohort (E–F)

Fig. 3

Longitudinal abundance and dynamic changes of EV-bound CD27 predict response, PFS and OS in patients undergoing ICI antagonistically to soluble CD27. A Levels of CD27 across serum, small extracellular vesicles (sEV), microparticles (MP), and EV-depleted serum (n = 45)). (B-C) EV-bound CD27 concentrations stratified by best response (B) and response at 3 months (C) over time. (D-G) Kaplan Meier curves for PFS (D, F) and OS (E, G) stratified by ideal baseline cut-off (D-E) and delta between 6 week and baseline (F-G) of EV-bound CD27 levels. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001

Fig. 4

A combined immunological MCD27 score comprising sCD27 and EV-bound CD27 baseline concentrations in peripheral blood is a predictor of progression-free and overall survival in patients undergoing ICI therapy for advanced solid malignancies. Kaplan Meier curves for PFS (A-B) and OS (C-D) stratified by number of risk factors: 0 vs. 2 (A, C) and 0–2 (B, D). Risk factors are defined as either sCD27 above ideal baseline cut-off or, EV-CD27 below ideal baseline cut-off (each 1 risk factor)