Serum extracellular vesicle microRNAs as potential biomarkers to predict pembrolizumab response and prognosis in metastatic non-small cell lung cancer patients

Abstract

Introduction: Circulating Extracellular Vesicles (cEVs) could represent new non-invasive biomarkers for diagnosis and prognosis in tumors. In the context of Non-Small Cell Lung Cancer (NSCLC) immunotherapy there's a great need for novel predictive and prognostic biomarkers. This study aims to analyze cEVs microRNAs in serum of advanced stage NSCLC patients with PD-L1 expression ≥50% at diagnosis, before first-line pembrolizumab, to evaluate their possible role as potential biomarkers for immunotherapy response prediction and outcomes.

Methods: cEVs were isolated from serum of healthy subjects and NSCLC patients at diagnosis. All patients had tumor PD-L1≥50% and cEVs were extracted before first-line pembrolizumab treatment. cEVs were then characterized for morphology, integrity, concentration, size and protein contaminants. Subsequently, microRNA content (miR-10a, miR-21, miR-22, miR-30a, miR-34a, miR-106b, miR-125b, miR-150, miR-155, miR-181a, miR-181b, miR-451a) was investigated by digital PCR. Additionally, miRNA-targets and their roles were evaluated. All data were associated with immunotherapy response, Progression Free Survival (PFS), Overall Survival (OS), Eastern Cooperative Oncology Group Performance Status (ECOG-PS) and metastases.

Results: Twelve NSCLC-related microRNAs have been found, for the first time, in serum cEVs from a specific cohort of metastatic advanced stage NSCLC patients. Through a functional analysis, these microRNAs are found to be connected to each other and involved in the pathology of NSCLC, particularly in IGF/P53/VEGF/NOTCH/PI3K pathways, in cytokine/interleukin signaling and in the immune system. Specifically, we demonstrated that cEV miR-106b, miR-451a, miR-181 and miR-10a were significantly up-regulated in non-responder patients compared to responder ones (p-value=0.08-0.1) predicting with high accuracy, already at diagnosis, treatment response. Furthermore, a low level of all these microRNAs predicted improved PFS (p-value=0.009-0.02) and a low amount of miR-106b predicted longer OS (p=0.069). In addition, it was observed that high levels of miR-106b and miR-451a are indicative of a high number of metastases (p=0.05/0.04, respectively) and of ECOG-PS=0.

Discussion: This is the first study that investigated specific potential serum cEV miRNAs to predict with high accuracy immunotherapy response and prognosis in specific metastatic NSCLC patients, already at diagnosis. Collectively, our cEV miRNA analysis identifies novel circulating biomarkers that are easily accessible and non-invasive, offering a potential blood-based tool to guide personalized medicine in NSCLC.

Keywords: NSCLC; biomarkers; extracellular vesicles; immunotherapy; liquid biopsy; microRNAs; prognosis; response prediction.

Figure 1

Characterization of cEVs from healthy subjects (HS) and advanced stage NSCLC patients. (A) Representative TEM photos of cEVs derived from HS and Advanced NSCLC patient (bars indicate 50 nm). (B) Flow cytometer CFSE-H/SSC-H dot plots of a representative unstained and stained HS EVs and stained NSCLC EVs. (C) NTA dot plot of cEV concentration (n. EVs/ml) comparison between n=21 HS vs n=21 advanced NSCLC patients. Each symbol represents single sample and horizontal bars represent median values. Statistically significant analyses are indicated by asterisks: **p < 0.01. (D) Three representative NTA size distribution profile of cEVs from one HS and two NSCLC patients. (E) NTA dot plots of cEV mean, mode, D10 and D90 of comparison between n=21 HS vs n=21 Advanced NSCLC patients. Each symbol represents single sample and horizontal bars represent median values. Statistically significant analyses are indicated by asterisks: *p < 0.05. (F) Column bar of lipoproteins (VLDL, LDL and HDL), apolipoproteins (APO A1 and APO B), total proteins and Albumin in sera, EV samples and supernatants of n=3 representative HS and NSCLC patients. The EV column bar is zero. The bar-graphs represent mean + SD from three independent experiments.

Figure 2

cEV miRNA detection in advanced NSCLC patients and systematic analysis of their targets. (A) Dot plots of miR-10a, miR-21, miR-22, miR-30a, miR-34a, miR-106b, miR-125b, miR-150, miR-155, miR-181a, miR-181b and miR-451a expressions in cEVs (n. copies/µl) from n=21 advanced stage NSCLC patients. Each symbol represents single sample and horizontal bars indicate the median values with range. (B) Concentric network layout obtained from miRTargetLink 2.0 indicating only strong-validated target-genes of microRNAs: miR-10a, miR-21, miR-30a, miR-34a, miR-106b, miR-125b, miR-150, miR-155, miR-181a, miR-181b and miR-451a. The target genes of six miRNAs are indicated in the red circle, the target genes of four miRNAs are indicated in the green circles and in blue circles the target genes of three miRNAs. (C) Pathway analysis of miRNA target-genes using KEGG, Panther, Reactome and WikiPathways databases.

Figure 3

Association of miRNA expression with immunotherapy response and survival outcomes in advanced NSCLC patients. (A) Dot plot comparison and ROC curve of miR-181b expression (n. copies/µl) in responder (R) patients vs non-responder (NR) ones. (B) Dot plots comparison of miR-106b and miR-451a expression (n. copies/µl) in R vs NR. The horizontal bars indicate the median values with range. AUC and statistical analysis (p-value and/or asterisks *p<0.05) are indicated on graphs. (C) Kaplan-Meier curves for PFS of cEV miR-181b, miR-106b and miR-451a in advanced NSCLC patients. Median expression values classified patients into low/high expression groups. Log-rank (Mantle-Cox) test was used to compare two curves. (D) Dot plot comparison of cEV concentration (EVs/ml) in advanced NSCLC patients divided for ECOG PS scale. Horizontal bars represent median values. Statistically significant analyses are indicated by asterisks: *p < 0.05.

Figure 4

Analysis of miRNA expression in R vs NR advanced adenocarcinoma NSCLC patients. Dot plot comparison and ROC curve of (A) miR-181b (B) miR-106b, miR-451a, miR-10a and miR-181a expression (n. copies/µl) in R vs NR adenocarcinoma NSCLC patients. The horizontal bars indicate the median values with range. AUC and statistical analysis (p-value and/or asterisks *p<0.05) are indicated on graphs.

Figure 5

Association of miRNA expression with survival outcomes in advanced stage NSCLC patients. Kaplan-Meier curves for (A) PFS according to the expression of cEV miR-181b, miR-106b, miR-451a, miR-10a, miR-22 and miR-34a, and for (B) OS according to the expression of cEV miR-106b in adenocarcinoma NSCLC patients. Median expression values classified patients into low/high expression groups. Log-rank (Mantle-Cox) test was used to compare two curves.

Figure 6

Association of miRNA expressions and metastases. (A) Dot plot comparisons and relative ROC curves of miR-106b and miR-451a expression (n. copies/µl) in patients with n=1/2 metastatic sites vs patients with n=3/4 metastatic sites. (B) Dot plots of miR-106b and miR-451a expression in different metastatic sites (lung, liver, brain, bone, lymph nodes and others). The horizontal bars indicate the median values with range. AUC and statistical analysis (p-value and/or asterisks *p<0.05) are indicated on graphs.

Figure 7

Liquid biopsy in advanced stage NSCLC patients (with PD-L1 expression ≥50%): role of serum cEVs and of their associated miRNAs. Circulating EVs (cEVs) were isolated from serum of metastatic advanced stage NSCLC patients at diagnosis, before first-line immunotherapy treatment. cEV concentration was associated to NSCLC diagnosis and ECOG PS scale; cEV miRNA levels were associated to treatment response, Progression Free Survival (PFS), Overall Survival (OS) and number of metastases. This figure was created with BioRender.com.