Blood-based miRNA, exosomal PD-L1, and immune profiling analysis as predictive biomarkers for immune checkpoint inhibitor treatment response in urothelial cancer.

Abstract

e16559 Background: Predicting response to immune checkpoint inhibitor (ICI) is difficult and adequate biomarkers are lacking. PD-L1 expression of tumor cells (TC) and tumor-infiltrating immune cells (IC) has limitations. We hypothesized that peripheral blood analysis would provide systemic insights about the immune responses induced by therapies blocking the PD-1/PD-L1 pathway. Methods: First, plasma samples from urothelial cancers (N = 30) with matched paired samples (N = 27) at baseline, after 1 week of PD-L1 inhibitor administration, and at response evaluation were analyzed. Exosomes were extracted and the expression of plasma-derived exosomal PD-L1 (exo PD-L1) and cytokine levels (IFN-γ, IL-10, TGF-ß, granzyme B) were determined with ELISA. Peripheral blood mononuclear cells (PBMCs) were analyzed with FACS. Samples were stained with anti-CD3, CD8, CD69, and PD1. Secondly, small RNA-sequencing was done using plasma samples from 8 patients and samples from 4 normal ones to identify differentially expressed miRNAs (DE-miRNAs) between treatment responders and non-responders. Expressions of selected DE-miRNAs were validated in a separate patient cohort of 54 patients. Results: In all of patients’ samples tested, exoPD-L1 was detected. Pretreatment level of exoPD-L1 was significantly higher in patients with objective tumor response (P = 0.010). For both clinical responders and non-responders, the level of exoPD-L1 expression increased at treatment day 8. A lower fold change (< 18) in plasma exoPD-L1 was associated with a better rate of response to the anti-PD-L1 therapy (P = 0.032). Compared to non-responder group, the PD1+CD8+T-cell fraction was significantly lower at baseline (p = 0.005) and increased at treatment day 8 in the responder group (p = 0.018). At baseline, the IL-10 level was significantly higher in the responder group (p = 0.005). Decreased levels of immune-suppressive cytokines, TGF-beta1/2 and IL-10, at post-treatment day 8 indicated better response (p = 0.005 and 0.020, respectively). From small RNA sequencing analysis, two upregulated (miR-451a and let-7i-5p) and eight downregulated (miR-1290, miR-151a-3p, miR-10b-5p, miR-148a-3p, miR-10a-5p, miR-1246, miR-34c-3p, let-7b-3p) differentially expressed miRNA in responders were identified. Conclusions: Our findings suggested that the higher expression of exoPD-L1, early peripheral PD1+CD8+T-cell response, and decrease of immunosuppressive cytokines could serve as blood-based predictive biomarkers for responses to ICI treatment and that miRNAs could act as regulators in these processes.