Marco Giallombardo

Exosomes isolation and characterization in serum is feasible in non-small cell lung cancer patients: critical analysis of evidence and potential role in clinical practice

Exosomes are nano-sized vesicles of endolysosomal origin, released by several cytotypes in physiological and pathological conditions. Tumor derived exosomes, interacting with other cells of the tumor microenvironment, modulate tumor progression, angiogenic switch, metastasis, and immune escape. Recently, extracellular vesicles were proposed as excellent biomarkers for disease monitoring and prognosis in cancer patients. Non-small cell lung cancer (NSCLC) has a poor 5-year survival rate due to the delay in the detection of the disease. The majority of patients are diagnosed in an advanced disease stage. Exosomes might be promising beneficial tools as biomarker candidates in the scenario of NSCLC, since they contain both, proteins and miRNAs. The clinical case reported in this manuscript is a proof of concept revealing that NSCLC exosomes and sorted miRNAs might constitute, in a near future, novel biomarkers. This review summarizes the role of exosomes in NSCLC, focusing on the importance of exosomal microRNAs in lung cancer diagnosis and prognosis.

Entrectinib: a potent new TRK, ROS1, and ALK inhibitor

Introduction: Receptor tyrosine kinases (RTKs) and their signaling pathways, control normal cellular processes; however, their deregulation play important roles in malignant transformation. In advanced non-small cell lung cancer (NSCLC), the recognition of oncogenic activation of specific RTKs, has led to the development of molecularly targeted agents that only benefit roughly 20% of patients. Entrectinib is a pan-TRK, ROS1 and ALK inhibitor that has shown potent anti-neoplastic activity and tolerability in various neoplastic conditions, particularly NSCLC. Areas covered: This review outlines the pharmacokinetics, pharmacodynamics, mechanism of action, safety, tolerability, pre-clinical studies and clinical trials of entrectinib, a promising novel agent for the treatment of advanced solid tumors with molecular alterations of Trk-A, B and C, ROS1 or ALK. Expert opinion: Among the several experimental drugs under clinical development, entrectinib is emerging as an innovative and promising targeted agent. The encouraging antitumor activity reported in the Phase 1 studies, together with the acceptable toxicity profile, suggest that entrectinib, thanks to its peculiar mechanism of action, could play an important role in the treatment-strategies of multiple TRK-A, B, C, ROS1, and ALK- dependent solid tumors, including NSCLC and colorectal cancer. That being said, further evidence for its clinical use is still needed.

Exosomal microRNAs in liquid biopsies: future biomarkers for prostate cancer

Prostate cancer is the second most diagnosed cancer in males in the world. Plasma quantification of prostate-specific antigen substantially improved the early detection of prostate cancer, but still lacks the required specificity. Clinical management of prostate cancer needs advances in the development of new non-invasive biomarkers, ameliorating current diagnosis and prognosis and guiding therapeutic decisions. microRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. These miRNAs are expressed in the cells and are also present in cell-derived extracellular vesicles such as exosomes. Exosomes have been shown to act as mediators for cell to cell communication because of the regulatory functions of their content. High levels of exosomes are found in several body fluids from cancer patients and could be a potential source of non-invasive biomarkers. In this review, we summarize the diagnostic and prognostic utility of exosomal miRNAs in prostate cancer.

Curcumin modulates chronic myelogenous leukemia exosomes composition and affects angiogenic phenotype, via exosomal miR-21

Tumor derived exosomes are vesicles which contain proteins and microRNAs that mediate cell-cell communication and are involved in angiogenesis and tumor progression. Curcumin derived from the plant Curcuma longa, shows anticancer effects. Exosomes released by CML cells treated with Curcumin contain a high amount of miR-21 that is shuttled into the endothelial cells in a biologically active form. The treatment of HUVECs with CML Curcu-exosomes reduced RhoB expression and negatively modulated endothelial cells motility. We showed that the addition of CML control exosomes to HUVECs caused an increase in IL8 and VCAM1 levels, but Curcu-exosomes reversed these effects thus attenuating their angiogenic properties. This antiangiogenic effect was confirmed with in vitro and in vivo vascular network formation assays. SWATH analysis of the proteomic profile of Curcu-exosomes revealed that Curcumin treatment deeply changes their molecular properties, in particular, Curcumin induces a release of exosomes depleted in pro-angiogenic proteins and enriched in proteins endowed with anti-angiogenic activity. Among the proteins differential expressed we focused on MARCKS, since it was the most modulated protein and a target of miR-21. Taken together our data indicated that also Curcumin attenuates the exosomes ability to promote the angiogenic phenotype and to modulate the endothelial barrier organization.

The potential of neurotrophic tyrosine kinase (NTRK) inhibitors for treating lung cancer

ABSTRACT Introduction: Molecular alterations in neurotrophic tyrosine kinase (NTRK) genes have been identified in several solid tumors including lung cancer. Pre-clinical and clinical evidence suggested their potential role as oncogenic drivers and predictive biomarkers for targeted inhibition, leading to the clinical development of a new class of compounds blocking the NTRK molecular pathway, which are currently undner early clinical investigation. Area covered: This review describes the biology of the NTRK pathway and its molecular alterations in lung cancer. It focuses on the pre-clinical and clinical development of emerging NTRK inhibitors, which have shown very promising activity in early phase I studies. Expert opinion: Among the several NTRK-inhibitors, entrectinib and LOXO-101 are those in more advanced stage of clinical development. Both agents have shown encouraging activity along with a tolerable safety profile in patients with different solid tumors harboring NTRK-fusions, emerging as new promising therapeutic options for molecularly selected patients with advanced Trk-driven lung cancers. Results from ongoing phase II basket trials are eagerly awaited.

Amphiregulin contained in NSCLC-exosomes induces osteoclast differentiation through the activation of EGFR pathway

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths worldwide. The majority of patients are diagnosed in advanced disease stage. Bone metastasis is the most frequent complication in NSCLC resulting in osteolytic lesions. The perfect balance between bone-resorbing osteoclasts and bone-forming osteoblasts activity is lost in bone metastasis, inducing osteoclastogenesis. In NSCLC, the epidermal growth factor receptor (EGFR) pathway is constitutively activated. EGFR binds Amphiregulin (AREG) that is overexpressed in several cancers such as colon, breast and lung. Its levels in plasma of NSCLC patients correlate with poor prognosis and AREG was recently found as a signaling molecule in exosomes derived from cancer cell lines. Exosomes have a key role in the cell-cell communication and they were recently indicated as important actors in metastatic niche preparation. In the present work, we hypothesize a role of AREG carried by exosomes derived from NSCLC in bone metastasis induction. We observed that NSCLC-exosomes, containing AREG, induce EGFR pathway activation in pre-osteoclasts that in turn causes an increased expression of RANKL. RANKL is able to induce the expression of proteolytic enzymes, well-known markers of osteoclastogenesis, triggering a vicious cycle in osteolytic bone metastasis.

Nintedanib in non-small cell lung cancer: from preclinical to approval

Angiogenesis is a driving force of a tumor’s development. Targeting this process is an attractive option, as this is a feature shared by most of the solid tumors. A lot of antiangiogenic drugs have been developed following this path, including bevacizumab, sorafenib, sunitinib, vandetanib, ramucirumab, motesanib and many others. The latest drug of this class to be approved for patients with non-small cell lung cancer (NSCLC) was nintedanib, a triple angiokinase inhibitor. This molecule targets vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) pathways, avoiding the tumor’s switch to normal escape mechanisms. The pharmacokinetic, pharmacodynamic and toxicity profiles of nintedanib have been tested in several studies. These trials revealed it to be very interesting, as this agent did not lead to the classical adverse events of other tyrosine kinase inhibitors. A phase III clinical trial that recently concluded provided us with relevant information in patients with NSCLC of adenocarcinoma histology. Here we present a short overview of the tumor angiogenesis pathways and antiangiogenic drugs. In particular, we will focus on nintedanib, from the preclinical studies to the latest phase III clinical trial that allowed this new agent to be approved by the European Medicines Agency as a second-line treatment option in association with docetaxel for NSCLC patients with adenocarcinoma histology.

Exosome-mediated drug resistance in cancer: the near future is here

Drug resistance exerts a crucial role in several cancer treatments. Understanding the resistance mechanisms against different therapeutic agents can be helpful to determine the prognosis, but remains a tricky task. In this context, tumor-derived exosomes (TDEs) may give crucial answers about these resistance mechanisms. Exosomes are biological nanovesicles with an average size around 30–100 nm of diameter (Figure 1) that originate from the endocytic pathway by the inward budding of multivesicular bodies (MVB), and they function as cell-free messengers, involved in the cell–cell communication [Kowal et al. 2014]. It has been demonstrated that both cells in physiological and pathological conditions release exosomes and that exosomes are easily detected in several body fluids, such as plasma, serum, urine, saliva, etc. [Rolfo et al. 2014]. It has been demonstrated that TDEs contain different proteins, lipids, mRNAs and miRNAs and have pleiotropic functions in the tumor microenvironment, tumor growth and progression, immune escape, angiogenesis, invasion, and drug resistance [Kowal et al. 2014; Rolfo et al. 2014; Fontana et al. 2013].

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool.

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2(-) (ΔΔct) (13). Control values were used as baseline and results are shown in logarithmic scale.

BRAF mutations in non-small cell lung cancer: has finally Janus opened the door?

B-Raf mutations occur in about 1-2% of non-small cell lung cancers (NSCLC). These mutations generate a permanent activation of the mitogen activated protein kinase (MAPK) pathway, which promotes tumor growth and proliferation. In the present review, we discuss B-Raf mutation epidemiology, diagnostic methods to detect B-Raf mutations, the role of B-Raf as a driver mutation and a potential therapeutic target in NSCLC. The results of clinical trials involving B-Raf or MAPK pathway inhibitors for the treatment of NSCLC are also discussed. Clinical trials evaluating B-Raf inhibitors in BRAF mutated NSCLC patients have shown promising results, and larger prospective studies are warranted to validate these findings. Enrollment of these patients in clinical trials is an interesting strategy to offer a potentially more effective and less toxic targeted therapy.