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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 (-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 (-value=0.009-0.02) and a low amount of miR-106b predicted longer OS (=0.069). In addition, it was observed that high levels of miR-106b and miR-451a are indicative of a high number of metastases (=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.

INTRODUCTION

METHODS

RESULTS

DISCUSSION

引言

循环细胞外囊泡（cEVs）可能成为肿瘤诊断和预后的新型非侵入性生物标志物。在非小细胞肺癌（NSCLC）免疫治疗背景下，迫切需要新型预测和预后生物标志物。本研究旨在分析诊断时PD-L1表达≥50%的晚期NSCLC患者在一线帕博利珠单抗治疗前血清中的cEVs微小RNA，以评估其作为免疫治疗反应预测和预后潜在生物标志物的可能作用。

方法

从健康受试者和诊断时的NSCLC患者血清中分离cEVs。所有患者肿瘤PD-L1≥50%，且在一线帕博利珠单抗治疗前提取cEVs。然后对cEVs的形态、完整性、浓度、大小和蛋白质污染物进行表征。随后，通过数字PCR研究微小RNA含量（miR-10a、miR-21、miR-22、miR-30a、miR-34a、miR-106b、miR-125b、miR-150、miR-155、miR-181a、miR-181b、miR-451a）。此外，评估了微小RNA靶标及其作用。所有数据均与免疫治疗反应、无进展生存期（PFS）、总生存期（OS）、东部肿瘤协作组体能状态（ECOG-PS）和转移情况相关。

结果

首次在特定队列的转移性晚期NSCLC患者血清cEVs中发现了12种与NSCLC相关的微小RNA。通过功能分析，发现这些微小RNA相互关联，并参与NSCLC的病理过程，特别是在IGF/P53/VEGF/NOTCH/PI3K途径、细胞因子/白细胞介素信号传导和免疫系统中。具体而言，我们证明与有反应的患者相比，无反应患者中cEV miR-106b、miR-451a、miR-181和miR-就已经能够高精度预测治疗反应。此外，所有这些微小RNA水平较低预示着PFS改善（P值=0.009-0.02），而miR-106b含量较低预示着OS更长（P=0.069）。此外，观察到miR-106b和miR-451a水平较高分别表明转移数量较多（P值分别为0.05/0.04）和ECOG-PS=0。