A "Lymphocyte MicroRNA Signature" as Predictive Biomarker of Immunotherapy Response and Plasma PD-1/PD-L1 Expression Levels in Patients with Metastatic Renal Cell Carcinoma: Pointing towards Epigenetic Reprogramming

Abstract

Introduction of checkpoint inhibitors resulted in durable responses and improvements in overall survival in advanced RCC patients, but the treatment efficacy is widely variable, and a considerable number of patients are resistant to PD-1/PD-L1 inhibition. This variability of clinical response makes necessary the discovery of predictive biomarkers for patient selection. Previous findings showed that the epigenetic modifications, including an extensive microRNA-mediated regulation of tumor suppressor genes, are key features of RCC. Based on this biological background, we hypothesized that a miRNA expression profile directly identified in the peripheral lymphocytes of the patients before and after the nivolumab administration could represent a step toward a real-time monitoring of the dynamic changes during cancer evolution and treatment. Interestingly, we found a specific subset of miRNAs, called "lymphocyte miRNA signature", specifically induced in long-responder patients (CR, PR, or SD to nivolumab >18 months). Focusing on the clinical translational potential of miRNAs in controlling the expression of immune checkpoints, we identified the association between the plasma levels of soluble PD-1/PD-L1 and expression of some lymphocyte miRNAs. These findings could help the development of novel dynamic predictive biomarkers urgently needed to predict the potential response to immunotherapy and to guide clinical decision-making in RCC patients.

Keywords: PD-1; PD-L1; miRNA; microRNA; predictive biomarkers; renal cell carcinoma; soluble immune checkpoints.

Figure 1

Microarray analysis of 377 miRNAs from 23 mRCC patients, analyzed before nivolumab treatment (T0) and after a 4-weeks period (T1) with nivolumab treatment (2 cycles of nivolumab administration), showing 66 differentially expressed miRNAs in peripheral lymphocytes at T1 vs. T0. Sixty-four miRNAs are downregulated and 2 are upregulated. The heat map of differentially expressed miRNAs was generated from microarray data reflecting mean expression values in 23 clear cell RCC (mccRCC) patients before treatment with nivolumab (T0) and 4 weeks after treatment (T1). Only upregulated miRNAs with fold change >2 and downregulated miRNAs with fold change < 0.3 were considered (p < 0.05). Each row represents the mean expression levels for a single miRNA tested at baseline and after treatment. Each column shows the mean expression levels for all miRNAs tested for a single patient group. The absolute expression value of each miRNA is derived from the mean Ct value calculated for each patient group. The color scale bar on the top represents signal intensity variations ranging from green (low mean Ct value and highly expressed miRNAs) to red (high mean Ct value and poorly expressed or unexpressed miRNAs). Black boxes indicate intermediate expression values. Data were obtained by TaqMan^® Low Density Array A Human MicroRNA using RNU48 as an endogenous control.

Figure 2

(a) Twenty-eight miRNAs were specifically induced by Nivolumab treatment in mRCC patients with PR/CR/SD as the best response; (b) miRNAs versus pathways heat map (clustering based on significance levels). The dendrograms placed on both axes depict hierarchical clustering results for miRNAs and pathways, respectively. On the miRNA axis, we can identify clustered miRNAs by exhibiting similar pathway targeting patterns. Eight miRNAs were specifically induced by Nivolumab treatment in long-responders (>12 months) mRCC patients. Hierarchical clustering was realized using DIANA-miRPath v3.0.

Figure 3

Validation of microarray expression data of 8 specific miRNA in a cohort of 8 mccRCC patients by quantitative Real-Time PCR analysis. The expression of each miRNA was analyzed before nivolumab treatment (T0) and 4 weeks after treatment (T1). Data are presented as Ct values ± SDs. Undetermined values of Ct were estimated between 35 and 40 Ct (the last cycle of the reactions), whereas a cut off <35 Ct was used to select reliably quantifiable miRNAs. RNU48 was used as an endogenous control. * p value < 0.0001.

Figure 4

(a) We investigated the association between plasma levels of soluble PD-1/PD-L1 and lymphocyte miRNA expression profile in long-responder mccRCC patients. (b) miR-22 and miR-24 levels were inversely associated with plasma PD-1 and PD-L1 levels in long-responder patients to nivolumab treatment. At baseline, high sPD-1/sPD-L1 levels were observed, whereas the expression of lymphocyte miR-22/miR-24 was silenced. After 4 weeks from starting nivolumab, sPD-1/sPD-L1 levels were strongly reduced and the expression of miR-22/miR-24 was restored only in patients with PR/CR/SD to nivolumab >18 months.

Figure 5

Correlation between expression of miRNAs 22/24 and plasma levels of soluble PD-1/PD-L1 in a group of 9 long-responder patients. (a) Pie chart representation of 23 patients receiving nivolumab treatment divided by PFS: 2 patients with PFS < 6 months (group A); 12 patients with PFS between 12−18 months (group B) and 9 patients with PFS > 18 months (group C). (b) Comparison between plasma levels of PD-1/PD-L1 and miRNA 22/24 expression before nivolumab treatment (T0) vs. 4 weeks later (T1). The histograms of PD-1 and PD-L1 correlate the expression level of immune checkpoints with time. The histograms of microRNA 22/24 correlates the value of fold change in relation to time. * p value < 0.01 (c) Tables represent the concentrations of immune checkpoints PD-1 and PD-L1 in 9 patients at T0 e T1.