Lung cancer is one of the highest incidence cancer types worldwide and one with the lowest 5-year survival rate of all cancer types. Despite recent insights into lung cancer pathobiology, including novel biomarker-targeted therapies and immunotherapies, most of lung patients are diagnosed at late stages with limited and ineffective treatments. Therefore, more approaches are needed to eradicate lung cancer. In the last years, small extracellular vesicles (EVs) secreted by tumor cells have been gaining relevance. These intercellular signal mediators, called exosomes, contain a huge range of biological elements, including lipids, nucleic acids and miRNAs, among others, that carry relevant information. The role of exosomes in cancer progression is dependent on cancer type, molecular characteristics and stage. MicroRNAs molecules are a big part of the content of exosomes cargo and probably the most studied ones. Due to the regulatory role in gene expression, miRNAs may provide information of the molecular characteristics of the tumor and be also able to reprogram distant target cells. Exosomal miRNAs can modulate different biological processes in cancer such as growth, progression, invasion, angiogenesis, metastasis and drug resistance; playing a critical role in modifying the microenvironment of non-small cell lung cancer (NSCLC). Therefore, they can act by regulating tumor resistance and also be useful to monitoring the response/relapse to targeted therapies.In this work, we summarize the relevant advances on the potential role of exosomal miRNAs in NSCLC pathobiogenesis, highlighting the clinical utility of exosomal microRNAs as biomarkers for the NSCLC diagnosis, prognosis, drug resistance and therapeutic strategies.

Lung cancer is the second most frequent tumor and the leading cause of death by cancer in both men and women. Increasing knowledge about the cancer genome and tumor environment has led to a new setting in which morphological and molecular characterization is needed to treat patients in the most personalized way in order to achieve better outcomes. Since tumor products can be detected in body fluids, the liquid biopsy, particularly, peripheral blood, has emerged as a new source for lung cancer biomarker's analysis. A variety of tumor components can be used for this purpose. Among them, circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) should be especially considered. Different detection methods for both CTCs and ctDNA have been and are being developed to improve the sensitivity and specificity of these tests. This would lead to better characterization and would solve some clinical doubts at different disease evolution times, e.g., intratumoral or temporal heterogeneity, difficulty in the obtaining a tumor sample, etc., and would also avoid the side effects of very expensive and complicated tumor obtaining interventions. CTCs and ctDNA are useful in different lung cancer settings. Their value has been shown for the early diagnosis, prognosis, prediction of treatment efficacy, monitoring responses and early detection of lung cancer relapse. CTCs have still not been validated for use in clinical settings in non-small-cell lung cancer (NSCLC), while ctDNA has been approved by the Food and Drug Administration (FDA) and European Medical Association (EMA), and the main clinical guidelines used for detect different epidermal growth factor receptor (EGFR) mutations and the monitoring and treatment choice of mutated patients with tyrosine kinase inhibitors (TKIs). This review, describes how ctDNA seem to be winning the race against CTCs from the laboratory bench to clinical practice due to easier obtaining methods, manipulation and its implementation into clinical practice.

Lung cancer is a malignant disease with high mortality and poor prognosis, frequently diagnosed at advanced stages. Nowadays, immense progress in treatment has been achieved. However, the present scenario continues to be critical, and a full comprehension of tumor progression mechanisms is required, with exosomes being potentially relevant players. Exosomes are membranous vesicles that contain biological information, which can be transported cell-to-cell and modulate relevant processes in the hallmarks of cancer. The present research aims to characterize the exosomes’ cargo and study their role in NSCLC to identify biomarkers. We analyzed exosomes secreted by primary cultures and cell lines, grown in monolayer and tumorsphere formations. Exosomal DNA content showed molecular alterations, whereas RNA high-throughput analysis resulted in a pattern of differentially expressed genes depending on histology. The most significant differences were found in XAGE1B, CABYR, NKX2-1, SEPP1, CAPRIN1, and RIOK3 genes when samples from two independent cohorts of resected NSCLC patients were analyzed. We identified and validated biomarkers for adenocarcinoma and squamous cell carcinoma. Our results could represent a relevant contribution concerning exosomes in clinical practice, allowing for the identification of biomarkers that provide information regarding tumor features, prognosis and clinical behavior of the disease.

Introduction: Immune checkpoint inhibitors (ICIs) have demonstrated clinical efficacy in non-small cell lung cancer (NSCLC) patients (p). However, ICIs can also trigger a self-reactive response in the adjacent healthy lung tissue that can eventually lead to life-threatening immune-related adverse events (irAEs), like checkpoint inhibitor pneumonitis (CIP). We hypothesized that a pre-treatment state of chronic inflammation or immune system imbalance could predict which p are at higher risk of developing CIP. Methodology: We retrospectively collected pre-ICI-treatment FFPE tumor tissue and matching plasma samples from 17 CIP- and 24 non-CIP lung cancer p. An additional 40 plasma samples, including 3 CIP and 37 non-CIP p were used as a validation cohort. The miRCURY exosome isolation kit (Qiagen) was used for extracellular vesicle (EV) enrichment from 500 μL of plasma and RNA was extracted using TRI-reagent. EV-mRNA was then pre-amplified (10 cycles) using the Low RNA Input Amplification kit (NanoString Technologies). FFPE mRNA was extracted using the High Pure FFPET RNA Isolation Kit (Roche). Gene expression analysis was performed on tissue and EV-derived mRNA using the NanoString nCounter platform with the Human PanCancer IO360 panel, which targets 770 genes related to tumor biology, immune response and microenvironment. Differential expression (DE) analysis was carried out based on the development of CIP. Finally, a classifier was created using a bioinformatic recursive feature elimination and a leave-one-out cross validation algorithm to predict which combination of genes is most effective to predict CIP development. Results: DE analysis revealed 54 differentially expressed genes (DEGs) in pre-treatment tissue of CIP vs. non-CIP p. An 8-gene CIP mRNA signature was able to distinguish between the two cohorts with areas under the ROC curve (AUC) of 0.81-0.95. When analyzing plasma EV samples, we found 57 DEGs. The tissue CIP signature was not translatable to EVs, yielding AUCs of only 0.53-0.54. Therefore, we developed a new 4-gene EV-based mRNA signature that could differentiate CIP vs. non-CIP developing p with AUCs of 0.82-0.90 and an overall accuracy of 89.9%. The negative- and positive predictive values (NPV and PPV) were 92.7% and 78.6%, respectively with a Youden´s index of 0.67. The 4 genes included in the EV signature were upregulated in CIP p and were found to be involved in T-cell activation and immune cell localization to the tumor. Conclusions: We have created a 4-gene EV-mRNA signature that associates with CIP development upon ICI treatment. Our results also indicate that plasma EV-mRNA was non-inferior to invasive tissue biopsy analysis in predicting CIP development. Validation studies in larger patient cohorts are ongoing. Citation Format: Jillian Wilhelmina Bracht, Santiago Viteri-Ramirez, Andrés Aguilar, Silvia Calabuig-Fariñas, Juan José García-Mosquera, Chung-Ying Huang, Elena Duréndez-Sáez, Nicolas Potie, Erika Aldeguer, Ana Gimenez-Capitán, Sonia Rodriguez, Ruth Roman, Cristina Aguado, Sarah Warren, Carlos Camps, Rafael Rosell, Eloisa Jantus-Lewintre, Miguel-Angel Molina-Vila, Maria González-Cao. A pre-treatment plasma extracellular vesicle-mRNA signature associates with checkpoint inhibitor pneumonitis in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 409.