Exosomes in NSCLC: Analysis of its cargo as a source of biomarkers

Potential Non-Invasive Biomarkers for Early Diagnosis of Oral Squamous Cell Carcinoma

This study aimed to investigate the role of a panel of salivary cytokines as biomarkers for early detection oral squamous cell carcinoma (OSCC), comparing their levels among healthy individuals, patients with oral leukoplakia (OL), and malignant lesions. Cytokine profiling analysis performed in a minimally invasive sample was correlated with clinicopathological variables in our patient cohorts. Unstimulated saliva was obtained from subjects with OSCC at early (n = 33) and advanced (n = 33) disease, OL with homogeneous (n = 33) and proliferative verrucous (n = 33) clinical presentations, and healthy controls (n = 25). Salivary IL-1α, IL-6, IL-8, IP-10, MCP-1, TNF-α, HCC-1, and PF-4 levels we…

Impact of DLK1-DIO3 imprinted cluster hypomethylation in smoker patients with lung cancer.

// Sonia Molina-Pinelo 1, 2 , Ana Salinas 1 , Nicolas Moreno-Mata 2 , Irene Ferrer 1, 2 , Rocio Suarez 1, 2 , Eduardo Andres-Leon 1 , Manuel Rodriguez-Paredes 4, 5 , Julian Gutekunst 4 , Eloisa Jantus-Lewintre 6, 7 , Carlos Camps 8, 9 , Amancio Carnero 1 , Luis Paz-Ares 1, 2 1 Instituto de Biomedicina de Sevilla (IBIS) (HUVR, CSIC, Universidad de Sevilla), Sevilla, Spain 2 Medical Oncology Department, Hospital Universitario Doce de Octubre & Centro Nacional de Investigaciones Oncologicas (CNIO), Madrid, Spain 3 Thoracic Surgery Department, Hospital Universitario Virgen del Rocio, Sevilla, Spain 4 Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany…

Prognostic value of CTCs in advanced NSCLC patients treated with platinum-based chemotherapy

Abstract Background Lung cancer is the leading cause of cancer-related mortality worldwide. At diagnosis, 70% of patients present advanced disease being chemotherapy the standard of care. Although, specific tumour biomarkers that allow a better treatment selection and monitoring is absent at the moment. Enumeration and characterization of circulating tumor cells (CTCs) have the potential to perform as a prognostic biomarker for a precision medicine approach to lung cancer care. The present study was conducted to validate the characterization CTCs from patients with advanced Non-small cell lung cancer (NSCLC) as a valuable tool for anticipating the disease evolution and the therapy response.…

Biofabrication of 3D tumor models in cancer research

Abstract Tumors are complex tissues in which cancer cells are interwoven with fibroblasts, endothelial cells, pericytes, and inflammatory cells; these cells and the extracellular matrix constitute the tumor microenvironment (TME). The TME can modulate the behavior of tumor cells in terms of capacity to invade neighboring or distant tissues and drug resistance, by secreting tumor-promoting growth factors and cytokines. The poor efficacy of many anticancer drugs in clinical trials can be partly justified by the lack of predictive preclinical models. Prior to in vivo testing, biofabrication of tools for investigation in three-dimensional (3D) could be useful. Indeed, cells grown in 3D matrices…

Abstract 4010: Prognostic role of miRNAs in resectable non-small cell lung cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA BACKGROUND MicroRNAs (miRNA) are a class of small RNAs that regulate gene expression, and are implicated in several diseases including cancer. The aim of this study was to analyze the prognostic implications of deregulated miRNAs identified by NGS in a previous study performed on resectable-NSCLC tumor samples. MATERIAL AND METHODS Fresh-frozen tumor and normal adjacent lung tissue samples (N = 195) were obtained by surgical resection from NSCLC patients and storage in RNAlater® at -80°C until analysis. RNA was isolated biological specimens by Trizol based-methods. 22 deregulated miRNA were analyzed in paired t…

Investigation of Complement Activation Product C4d as a Diagnostic and Prognostic Biomarker for Lung Cancer

[EN] Background There is a medical need for diagnostic biomarkers in lung cancer. We evaluated the diagnostic performance of complement activation fragments. Methods We assessed complement activation in four bronchial epithelial and seven lung cancer cell lines. C4d, a degradation product of complement activation, was determined in 90 primary lung tumors; bronchoalveolar lavage supernatants from patients with lung cancer (n = 50) and nonmalignant respiratory diseases (n = 22); and plasma samples from advanced (n = 50) and early lung cancer patients (n = 84) subjects with inflammatory lung diseases (n = 133), and asymptomatic individuals enrolled in a lung cancer computed tomography screenin…

Carcinoma of unknown origin: Deep genomic profile helps to achieve an accurate diagnosis

Clinical utility of plasma-based digital next-generation sequencing (NGS) in patients with advance-stage lung adenocarcinomas with insufficient tumor samples for tissue genotyping.

9101Background: Approximately 20 % of tumor biopsies from patients with advanced-stage lung adenocarcinomas yield insufficient tissue for successful molecular subtyping. In this study, we have anal...

CD5 and CD6 as immunoregulatory biomarkers in non-small cell lung cancer

Background: The study of immune surveillance in the tumour microenvironment is leading to the development of new biomarkers and therapies. The present research focuses on the expression of CD5 and CD6-two lymphocyte surface markers involved in the fine tuning of TCR signaling-as potential prognostic biomarkers in resectable stages of non-small cell lung cancer (NSCLC).; Methods: CD5 and CD6 gene expression was analysed by reverse transcription quantitative polymerase chain reaction (RTqPCR) in 186 paired fresh frozen tumour and normal tissue samples of resected NSCLC.; Results: Patients with higher CD5 expression had significantly increased overall survival (OS, 49.63 vs. 99.90 months, P=0.…

Tumor expression levels of CSC markers in resectable non-small cell lung cancer.

7562 Background: Cancer stem cells (CSCs) have been proposed as the driving force of tumorigenesis and the seeds of metastases. However, their existence and role remain a topic of intense debate. T...

Lung tumorspheres reveal cancer stem cell-like properties and a score with prognostic impact in resected non-small-cell lung cancer

AbstractThe high resistance against current therapies found in non-small-cell lung cancer (NSCLC) has been associated to cancer stem-like cells (CSCs), a population for which the identification of targets and biomarkers is still under development. In this study, primary cultures from early-stage NSCLC patients were established, using sphere-forming assays for CSC enrichment and adherent conditions for the control counterparts. Patient-derived tumorspheres showed self-renewal and unlimited exponential growth potentials, resistance against chemotherapeutic agents, invasion and differentiation capacities in vitro, and superior tumorigenic potential in vivo. Using quantitative PCR, gene express…

MicroRNA profiling associated with non-small cell lung cancer: next generation sequencing detection, experimental validation, and prognostic value

[EN] Background: The average five-year survival for non-small cell lung cancer (NSCLC) patients is approximately 15%. Emerging evidence indicates that microRNAs (miRNAs) constitute a new class of gene regulators in humans that may play an important role in tumorigenesis. Hence, there is growing interest in studying their role as possible new biomarkers whose expression is aberrant in cancer. Therefore, in this study we identified dysregulated miRNAs by next generation sequencing (NGS) and analyzed their prognostic value. Methods: Sequencing by oligo ligation detection technology was used to identify dysregulated miRNAs in a training cohort comprising paired tumor/normal tissue samples (N = …

Immune Checkpoints Score and CD8+ T Cells Infiltration Are Independent Prognostic Biomarkers in Resected Nsclc

Differential expression of tumorspheres in CSC-markers and signaling pathways from non-small cell lung cancer.

e23276Background: Chemoresistance, tumor progression and metastasis have made of lung cancer the first cause of death cancer-related worldwide. Cancer stem cells (CSCs) are small subpopulations of ...

MA04.09 Study of Exosomes in NSCLC for Biomarkers Searching

Clinical utility of plasma-based digital next-generation sequencing in patients with advance-stage lung adenocarcinomas with insufficient tumor samples for tissue genotyping

[Background] Approximately 30% of tumor biopsies from patients with advanced-stage lung adenocarcinomas yield insufficient tissue for successful molecular subtyping. We have analyzed the clinical utility of next-generation sequencing (NGS) of cell-free circulating tumor DNA (ctDNA) in patients with inadequate tumor samples for tissue genotyping. [Patients and methods] We conducted the study in a multi-institutional prospective cohort of clinically unselected patients with advanced-stage lung adenocarcinomas with insufficient tissue for EGFR, ALK or ROS1 genotyping across 12 Spanish institutions (n = 93). ctDNA NGS was carried out by Guardant Health (Guardant360, Redwood City, CA), using a h…

MiRNA profiling by NGS in resectable non-small cell lung cancer: Prognostic implications.

7559 Background: MicroRNAs (miRNA) regulate gene expression, and are implicated in several processes like tumorogenesis. Here, we applied a multiplexed NGS approach to study differentially expressed miRNAs (tumor/ normal) in a cohort of resectable NSCLC patients and its correlation with clinical outcome. Methods: RNA was isolated from frozen lung specimens (tumor/ normal) from 33 patients. High-quality samples were analyzed and enriched in the miRNA fraction. Libraries were prepared according to manufactured instruction (SOLID), and miRNAs were sequenced. Data analysis was carried out using CLCbio software. First, raw data were annotated using miRBase and normalized by total reads followed …

Upregulated Mirna 21 in Kras Mutated Patients is Related to Prognosis in Resectable Non-Small Cell Lung Cancer

CD5 and CD6: Evaluation of their role as prognostic biomarkers in resectable non-small cell lung cancer

EpCAM duality becomes this molecule in a new Dr. Jekyll and Mr. Hyde tale.

EpCAM, known as an epithelial cell adhesion molecule, plays an essential role in cell adhesion, migration, metastasis and cell signalling. Rather than acting as an apoptosis antagonist, it induces cellular proliferation that impacts the cell cycle, and as a signalling transducer it uses and enhances the Wnt pathway, which is significantly relevant in cell renewal and cancer. EpCAM has become a marker of circulating tumour cells (CTCs) in lung cancer due to its specificity, and its high and stable expression level. Recent findings have allowed us to relearn and discover EpCAM again as a CSCs marker by demonstrating its role in human epithelial cancer progression. In line with this, the focus…

P1.05-013 Lung Tumorspheres as a Platform for Testing New Therapeutic Strategies in Non-Small Cell Lung Cancer

Profile of the Roche cobas® EGFR mutation test v2 for non-small cell lung cancer

Abstract: Introduction: The discovery of driver mutations in non-small cell lung cancer (NSCLC) has led to the development of genome-based personalized medicine. Fifteen to 20% of adenocarcinomas harbor an epidermal growth factor receptor (EGFR) activating mutation associated with responses to EGFR tyrosine kinase inhibitors (TKIs). Individual laboratories' expertise and the availability of appropriate equipment are valuable assets in predictive molecular pathology, although the choice of methods should be determined by the nature of the samples to be tested and whether the detection of only well-characterized EGFR mutations or rather, of all detectable mutations, is required.Areas covered:…

Oral microbiome in Proliferative Verrucous Leukoplakia exhibits loss of diversity and enrichment of pathogens.

Abstract Objectives Oral microbiome plays an important role in oral diseases. Among them, proliferative verrucous leucoplakia (PVL) is an uncommon form of progressive multifocal leukoplakia with a worryingly rate of malignant transformation. Here, we aimed to characterize the oral microbiome of PVL patients and compare it with those of healthy controls. Material and methods Oral biopsies from ten PVL patients and five healthy individuals were obtained and used to compare their microbial communities. The sequence of the V3-V4 region of 16S rRNA gene was used as the taxonomic basis to estimate and analyze the composition and diversity of bacterial populations present in the samples. Results O…

ANGIOMET: Analysis of the correlations between angiogenic markers and outcome in patients (p) with advanced nonsquamous NSCLC (NS-NSCLC) treated with carboplatin, paclitaxel, and bevacizumab (CPB).

e19014 Background: In NS-NSCLC CPB achieved median OS > 1 y and supported use of B. A broad range of predictive/prognostic markers explored for B use. In VEGF pathway ligands and receptors play an ...

Abstract 4424: Cancer stem cells-related microRNAs have prognostic implications in non-small cell lung cancer

Abstract Background: Lung cancer stem cells (CSCs) are a small population of stem-like cells that remains largely unknown. MicroRNAs (miRNAs) regulation of CSCs gene expression has been reported, constituting a promising therapeutic target. The aim of this study was to isolate and analyze differential expression of a set of miRNAs in tumorspheres from lung cancer cell lines and tumor tissue from resectable non-small cell lung cancer (NSCLC) patients and to determine the prognostic implications of these miRNAs in a cohort of resected-NSCLC patients. Methods: Lung CSC-related miRNAs (miR-145-5p, miR-188-5p, miR-218-5p, miR-34a-5p, miR-21-5p and miR-125a-5p) were analyzed in cells from seven N…

BCL6: somatic mutations and expression in early-stage chronic lymphocytic leukemia

BCL6 somatic mutations affect normal and tumoral post germinal center B-lymphocytes. Our objective was to analyse expression, mutations and polymorphisms in the BCL6 gene and to correlate those variables with the clinical outcome in early-stage chronic lymphocytic leukemia (CLL). CLL samples were used for characterisation of the mutational status of BCL6/ immunoglobulin variable heavy chain (IGHV) genes, and expression of BCL6 was determined by real time PCR and immunoblot. Out of 68 cases, 29% show somatic mutations on BCL6 which occur exclusively in IGHV mutated cases. They are single nucleotide substitutions located mainly in two short mutational hot spots. CLL cells express different le…

NGS‐based liquid biopsy profiling identifies mechanisms of resistance to ALK inhibitors: a step toward personalized NSCLC treatment

Despite impressive and durable responses, nonsmall cell lung cancer (NSCLC) patients treated with anaplastic lymphoma kinase (ALK) inhibitors (ALK‐Is) ultimately progress due to development of resistance. Here, we have evaluated the clinical utility of circulating tumor DNA (ctDNA) profiling by next‐generation sequencing (NGS) upon disease progression. We collected 26 plasma and two cerebrospinal fluid samples from 24 advanced ALK‐positive NSCLC patients at disease progression to an ALK‐I. These samples were analyzed by NGS and digital PCR. A tool to retrieve variants at the ALK locus was developed (VALK tool). We identified at least one resistance mutation in the ALK locus in ten (38.5%) p…

An expression signature characterizes cancer stem cells from lung adenocarcinoma patients

Analysis of immunoregulatory biomarkers in early stages of non-small cell lung cancer

Molecular subtyping of colon cancer (CC) based on mutational status of RAS, BRAF, and DNA mismatch repair (MMR) proteins. Prognostic value.

e15094Background: CC is a heterogeneuous disease with clinical, pathological and biological variability. Molecular classification could indentify prognostic subtypes. Methods: 105 patients with sta...

EGFR mutant cfDNA and CTC detection as biomarkers in patients diagnosed with advanced non-small cell lung cancer.

e23039Background: One of the most promising developments in translational cancer has been the emergence of liquid biopsy as a non-invasive biomarker. CTCs and cfDNA offer valuable prognostic and pr...

Influence of different complement sources in apoptosis of B-CLL cells with rituximab

Abstract Introduction: Rituximab (Rtx) is an anti-CD20 monoclonal antibody that is clinically active in B-cell chronic lymphocytic leukemia (B-CLL). In vitro and in vivo data indicate that Rtx mediates its biologic effect through multiple mechanisms including apoptosis, complement dependent cytotoxicity (CDC), and antibody dependent cellular cytotoxicity (ADCC). Objetive: To study in vitro sensitivity to apoptosis by Rtx of isolated cells obtained from B-CLL patients Binet stage A, in the presence of complement from different sources and correlate with CD20 and CD59 molecules expression. Methods: PBMCs were isolated from peripheral blood samples by centrifugation on a Ficoll/Hypaque gradien…

Clinical utility of plasma-based digital next-generation sequencing in oncogene-driven non-small-cell lung cancer patients with tyrosine kinase inhibitor resistance

[Objectives] Resistance to tyrosine-kinase inhibitors (TKIs) is a clinical challenge in patients with oncogene-driven non-small-cell lung cancers (NSCLC). We have analyzed the utility of next-generation sequencing (NGS) of cell-free circulating tumor DNA (ctDNA) to impact the clinical care of patients with TKI resistance.

Characterization of the Proteomic and Genomic Profiles of Chronic Lymphocytic Leukemia Patients with Distinct Clinical Prognosis According to the Mutational Status of the IgVH and BCL6 and Expression Level of CD38 and ZAP70.

Abstract Introduction: In recent years several molecular prognostic factors have been identified in Chronic Lymphocytic Leukemia B (B-CLL). These include mutations in the variable region of the immunoglobulin genes (IgVH), somatic mutations in the BCL6 gene (Leukemia (2004) 18, 743–746) and the expression level of CD38 and ZAP70. However its biological significance is not clear. In order to identify novel molecular markers with prognostic and therapeutic value we have analyzed the proteomic and genomic profile of 40 B-CLL patients (Binet stage A). Material and methods: 100 μg of total PBMC proteins were used for IEF followed by 2D electrophoresis. Image analysis of scanned gels was used to …

Complement activation product C4d in oral and oropharyngeal squamous cell carcinoma

OBJECTIVE: Complement C4d-containing fragments have been proposed as diagnostic markers for lung cancer. The purpose of this study was to evaluate the presence of C4d in oropharyngeal (OPSCC) and oral (OSCC) squamous cell carcinomas. SUBJECTS AND METHODS: C4d staining was analyzed by immunohistochemistry in 244 OPSCC surgical specimens. C4d levels were quantified by ELISA in resting saliva samples from 48 patients with oral leukoplakia and 62 with OSCC. Plasma samples from 21 patients with leukoplakia and 30 with oral carcinoma were also studied. RESULTS: C4d staining in OPSCC specimens was associated with nodal invasion (P = 0.001), histopathologic grade (P = 0.014), disease stage (P = 0.0…

Analysis of immunosuppressive factors produced by tumorspheres in NSCLC: Prognostic value of galectin-3 in adenocarcinoma

Abstract Background Cancer stem cells (CSCs) are targets poorly recognized by the immune surveillance system given that they induce an immunosuppressive microenvironment. The aim of this work is to study the interactions between CSCs and the immune microenvironment in NSCLC. Methods Tumor cells from 8 resected NSCLC patients and 12 cell lines were established as tumorspheres enriched in CSCs and as monolayers. The gene expression of IL-4, IL-10, IL6, IL8 and LGALS-3 was analyzed by RTqPCR. Results were validated at a protein level by a sensitivity bead-based multiplex immunoassay using the Millipore kit for Luminex 100/200. The prognostic value of these factors in silico was determined in a…

Applicability of ctDNA at diagnosis and during the monitoring of EGFR-mutated patients

Analysis of the immune microenvironment in resected non-small cell lung cancer: the prognostic value of different T lymphocyte markers

[EN] The prognosis of non-small cell lung cancer (NSCLC) remains poor and heterogeneous and new biomarkers are needed. As the immune system plays a pivotal role in cancer, the study of immune-related markers may provide valuable prognostic information of NSCLC. In 122 formalin-fixed, paraffin-embedded tumor tissue samples from early-stage NSCLC, tumor and tumor-near stromal areas were microdissected and gene expression levels of conventional and regulatory T cell markers were assessed by quantitative polymerase chain reaction. Also, the presence of infiltrating CD4+, CD8+, and FOXP3+ cells in tumor samples was assessed by immunohistochemistry. The relative proportion of conventional and reg…

Abstract 3354: Characterization of lung tumorspheres by gene expression and flow cytometry: differential expression in CSC-related markers and signaling pathways

Abstract Chemoresistance, progression and metastasis have made of lung cancer the first cause of cancer mortality. These features were linked to a subpopulation of cells, named cancer stem cells (CSCs), which remain largely unknown. The aim of this study was to isolate and characterize CSCs from lung cancer cell-lines and tumor-tissue from resectable non-small cell lung cancer (NSCLC). Methods: Tumor cells from resected NSCLC and cell lines (H1650, H1993, A549, and PC9) were grown in monolayer and as spheroids. RTqPCR was performed to analyze the mRNA expression of CSCs-related genes: CSC-markers (EPCAM1, ALDH1A1, CD166, ABCG2, CD44, CD133); pluripotency genes (KLF4, OCT4, NANOG, SOX2, MYC,…

Lung tumorspheres as a drug screening platform against cancer stem cells.

Treatment resistance and metastasis are linked to cancer stem cells (CSCs). This population represents a promising target, but remains unexplored in lung cancer. The main objective of this study was to characterize lung CSCs and discover new therapeutic strategies.

MicroRNAs: Promising New Antiangiogenic Targets in Cancer

[EN] MicroRNAs are one class of small, endogenous, non-coding RNAs that are approximately 22 nucleotides in length; they are very numerous, have been phylogenetically conserved, and involved in biological processes such as development, differentiation, cell proliferation, and apoptosis. MicroRNAs contribute to modulating the expression levels of specific proteins based on sequence complementarity with their target mRNA molecules and so they play a key role in both health and disease. Angiogenesis is the process of new blood vessel formation from preexisting ones, which is particularly relevant to cancer and its progression. Over the last few years, microRNAs have emerged as critical regulat…

Prognostic role of immune checkpoint-related genes in resectable lung adenocarcinomas.

11085 Background: Immune checkpoints blockade has demonstrated promising clinical results in NSCLC patients. In this study we have investigated the prognostic role of immune checkpoint markers (CTLA4, PD1 and PDL1) in resectable lung adenocarcinoma (ADC). Methods: RNA was isolated from fresh-frozen lung specimens (tumor and normal lung) from resectable ADC patients (n=92). RT-PCR was performed to analyze the expression of CTLA4, PD1 and PDL1 by the use of hydrolysis probes. Relative gene expression was assessed by Pfaffl formula and normalized by CDKN1B and ACTB as endogenous genes (selected by geNorm algorithm). Statistical analyses were considered significant at p<0.05. Results: Patient’s…

Stemness characterization of tumorspheres from non-small cell lung cancer: Differential expression in CSC-related markers and signaling pathways

Soluble biomarker signature to predict outcome of patients with non-small-cell lung cancer (NSCLC) treated with anti-PD1/PDL1 monoclonal antibodies.

e20685 Background: Immunotherapy with anti-PD1/PDL1 monoclonal antibodies has become the second line standard treatment for most patients diagnosed of advanced Non-Small-Cell lung cancer (NSCLC). The aim of this study is to assess the utility of circulating biomarkers such as sPDL1, sPDL2, sCD137, sIDO, sTIM3, sCD28, sCD27, sCTLA4, sHVEM, sLAG3, sCD80 and sGITR for predicting efficacy of immunotherapy with anti-PD1/PDL1 therapies. Methods: Blood samples were collected before treatment from 50 NSCLC patients who received anti PD1/PDL1 therapies (second line). Plasma biomarkers´ levels were measured by Multiplex bead-based assays. Continuous variables were categorized using the median as a c…

Interactions of cancer stem cell and immune microenvironment in non-small cell lung cancer (NSCLC)

Comprehensive cross-platform comparison of methodologies for noninvasive EGFR mutation testing: Results of the RING observational trial.

e21518 Background: Several platforms for non-invasive EGFR testing are currently used in the clinical setting, with sensitivities ranging from 30 to 100%. Comparison studies in prospective cohorts remain limited and reports evaluating mutant allelic fractions (MAFs) are particularly scarce. The RING observational trial (ClinicalTrials.gov identifier NCT03363139) was designed to comprehensively analyze the concordance between methodologies for EGFR mutation detection in blood. Methods: Seventy-two EGFR mutant NSCLC patients were enrolled in the trial. Plasma samples were prospectively collected at progression to first line Tyrosine Kinase Inhibitor and tested for EGFR mutations by 7 methodo…

Exosomes in NSCLC as a source of biomarkers

Abstract Background Exosomes are small membranous vesicles (around 40-130 nm), that have been detected in different biological samples, that play a key role in NSCLC and being relevant in stem cell differentiation as well. The main objective of this study was to analyze the exosomes cargo from NSCLC cell cultures growth in monolayer (2D) and suspension conditions (3D, lung tumorespheres). Methods Cultures were established from NSCLC resected patients and cell lines. Exosomes isolation was performed by ultracentrifugation. Characterization was carried out by NTA, electron microscopy, immunoblot and flow cytometry. Mutational status of EGFR and RAS genes was analyzed by BEAMing dPCR. Transcri…

Analysis of gut microbiota in advanced non-small cell lung cancer (NSCLC) patients treated with immune-checkpoints blockers

Abstract Background The microbiota community is considered as an organ of the human body. Recent studies have found that dysbiosis may have an impact on the interaction between immune regulation and tumor treatment. The main objective of this study is to characterize the gut microbiota in patients with non-small cell lung cancer (NSCLC) in advanced stages, and its relationship with obesity, smoking habits, clinical pathological features and response to treatment with immune-checkpoints blockers (ICB). Methods 16S rRNA gene sequencing was performed on 48 stool samples from advanced NSCLC patients prior to treatment with ICB (PD-1 inhibitor and PD-L1 inhibitor). The database used for the taxo…

Prognostic Impact of Vegfa in Resectable Non-Small-Cell Lung Cancer

ABSTRACT Aim: Angiogenesis is a main process which happens in tumors and that promotes its growth, invasive capacity and metastasis. Host genetic variability within VEGF pathway may affect angiogenic signaling and alter patient's sensitivity to anti-angiogenic therapies and therefore the prognostic. The goal in the present study is to analyze the prognostic value of several SNPs in angiogenic genes and the relative expression of those genes, using a cohort of patients diagnosed with resectable non-small cell lung cancer. Methods: This study included 127 resectable (I-IIIA) NSCLC patients. RNA and DNA extractions from tissues were performed using Trizol®. 20 ng of DNA were used for studies o…

Lung tumorspheres as a platform for testing new therapeutic strategies in lung cancer.

e23177Background: Resistance to treatment is one of the causes influencing the high mortality of lung cancer. This feature seems to be dependent of a small subset of special characteristics of self...

Snps in Angiogenic Factors As Predictive Markers for Outcome in Patients (P) with Advanced Non-Squamous Nsclc (NS-NSCLC) Treated with Carboplatin, Paclitaxel (CP) and Bevacizumab (BEV). Final Results of Angiomet Spanish Lung Cancer Group Trial

Analysis of Exosomal Cargo Provides Accurate Clinical, Histologic and Mutational Information in Non-Small Cell Lung Cancer

Simple Summary Non-small cell lung cancer (NSCLC) is the second most commonly diagnosed cancer and the leading cause of cancer-related death worldwide. Clinical decision-making depends on the histological classification; however, tissue biopsy is frequently not technically feasible due to tumor location or limited tissue samples. Therefore, we propose to find clinical, molecular and histological biomarkers using a minimally invasive approach based on the analysis of the cargo of the blood extracellular vesicles. Exosomes are membranous vesicles present in several biological fluids, which carry biological information to distant tissues, regulating several tumor processes. This study aims to …

Prospective multicenter real-world RAS mutation comparison between OncoBEAM-based liquid biopsy and tissue analysis in metastatic colorectal cancer

[EN] BACKGROUND: Liquid biopsy offers a minimally invasive alternative to tissue-based evaluation of mutational status in cancer. The goal of the present study was to evaluate the aggregate performance of OncoBEAM RAS mutation analysis in plasma of colorectal cancer (CRC) patients at 10 hospital laboratories in Spain where this technology is routinely implemented. METHODS: Circulating cell-free DNA from plasma was examined for RAS mutations using the OncoBEAM platform at each hospital laboratory. Results were then compared to those obtained from DNA extracted from tumour tissue from the same patient. RESULTS: The overall percentage agreement between plasma-based and tissue-based RAS mutatio…

Abstract 4330: Immune checkpoint expression score is an independent prognostic biomarker in resectable non-small cell lung cancer

Abstract BACKGROUND Immune checkpoints blockade, which activate antitumor immunity, has demonstrated promising clinical results in NSCLC. In this study we have investigated the prognostic role of immune checkpoint expression markers and its correlation with immune-cells infiltration and clinico-pathological characteristics in a cohort of resectable NSCLC patients. MATERIAL AND METHODS RNA was isolated from fresh-frozen lung specimens (tumor and normal lung) (n = 178). RTqPCR was performed to analyze the expression of CTLA-4, PD-1 and PD-L1 by the use of hydrolysis probes. Relative gene expression was assessed by Pfaffl formula and normalized by the use of CDKN1B, GUS and ACTB as endogenous …

miRNA detection methods and clinical implications in lung cancer

[EN] Lung cancer is the leading cause of cancer death worldwide. Therefore, advances in the diagnosis and treatment of the disease are urgently needed. miRNAs are a family of small, noncoding RNAs that regulate gene expression at the transcriptional level. miRNAs have been reported to be deregulated and to play a critical role in different types of cancer, including lung cancer. Thus, miRNA profiling in lung cancer patients has become the core of several investigations. To this end, the development of a multitude of platforms for miRNA profiling analysis has been essential. This article focuses on the different technologies available for assessing miRNAs and the most important results obtai…

Prognostic Impact of Immune Microenvironment Markers in Tumor and Stroma in Resectable Nsclc

ABSTRACT Aim: Tumor-infiltrating immune cells have been proved to play an important role in tumor progression. In this study we have analyzed the expression of 11 immune related genes in both tumor and stroma samples of resectable NSCLC patients. Methods: In this retrospective study FFPE samples from 117 early-stage NSCLC patients of primary tumor tissue were used. The most representative areas of tumor and tumor stroma of each sample were carefully micro-dissected. RT-PCR using hydrolysis probes (TaqMan, Applied Biosystems) was performed to assess the expression of Treg markers such as: CD127, CD25, FOXP3, CTLA-4, IL-10, TGFB-1, LAG-3, GITR and TNFA as well as CD4 and CD8. Relative gene ex…

Abstract 1918: Gene characterization of lung-tumorspheres for their usage as an in vitro screening platform for testing new therapeutic strategies

Abstract Background: Lung cancer features like treatment resistance or tumor relapse have been linked to cancer stem cells (CSCs), a population of cells with self-renewal properties, and the ability to grow forming tumorspheres in non-adherent conditions. The aim of this study was to isolate and characterize tumorspheres from lung cancer cell lines and tumor tissue from resectable non-small cell lung cancer (NSCLC) patients and to use them as an in vitro platform for drug screening. Methods: This study was performed on cells from seven NSCLC tumor samples and five cell lines (H1650, H1993, H1395, A549 and PC9) grown in monolayer and as spheroids. The expression of 60 genes, including CSC-ma…

Analysis of the Gut Microbiota: An Emerging Source of Biomarkers for Immune Checkpoint Blockade Therapy in Non-Small Cell Lung Cancer

Simple Summary The immune checkpoint blockade (ICB), and concretely the blockade of the PD1/PDL1 axis, has opened up a new standard of treatment for non-small cell lung cancer (NSCLC). However, despite substantial advances in clinical care, many patients still remain refractory to these therapies. Biomarkers such as PD-L1 expression and tumor mutational burden have been associated with ICB efficacy, but the mechanisms underlying variable responses are not yet fully understood. Recently, the differential composition of the gut microbiota was studied as one of the variables accounting for interpatient heterogeneity in ICB responses. To better understand the potential role of the gut microbiot…

EP912 Characterization of the tumour microenvironment in high-grade serous ovarian cancer (HGSOC): prognostic value of the lymphocytic infiltration patterns and immune-related genes

Introduction/Background Lymphocytic infiltration areas (immunoreactive), frequently found in high-grade serous ovarian cancer (HGSOC), are associated with a better prognosis and increased survival. The cross-talk between tumour cells and lymphocytes conditions the capacity of the immune system (IS) to cope with the tumour in the so-called immune-checkpoints. Therefore, assessing IS-related genes and infiltration patters might provide valuable prognostic biomarkers. Methodology This retrospective study includes 57 samples from patients with HGSOC who underwent cytoreductive surgery at Hospital General (Valencia). Clinical variables, the features of the lymphocytic infiltration (pattern, loca…

New insights in non-small-cell lung cancer: circulating tumor cells and cell-free DNA

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…

MA04.03 Lung Tumorspheres Characterization Reveals Cancer Stem-Like Cells Potential Targets and Prognostic Markers in Non-Small Cell Lung Cancer

New generation of cancer stem cells inhibitors in non-small cell lung cancer.

e20545Background: Lung cancer is the commonest tumor worldwide of which the most frequent type is non-small cell lung cancer (NSCLC) that represents a large proportion of cancer deaths and is chara...

Facilitated Anion Transport Induces Hyperpolarization of the Cell Membrane That Triggers Differentiation and Cell Death in Cancer Stem Cells

Facilitated anion transport potentially represents a powerful tool to modulate various cellular functions. However, research into the biological effects of small molecule anionophores is still at an early stage. Here we have used two potent anionophore molecules inspired in the structure of marine metabolites tambjamines to gain insight into the effect induced by these compounds at the cellular level. We show how active anionophores, capable of facilitating the transmembrane transport of chloride and bicarbonate in model phospholipid liposomes, induce acidification of the cytosol and hyperpolarization of plasma cell membranes. We demonstrate how this combined effect can be used against canc…

3D printing novel in vitro cancer cell culture model systems for lung cancer stem cell study

Two-dimensional (2D) in vitro cell cultures and laboratory animals have been used traditionally as the gold-standard preclinical cancer model systems. However, for cancer stem cell (CSC) studies, they exhibit notable limitations on simulating native environment, which depreciate their translatability for clinical development purposes. In this study, different three-dimensional (3D) printing platforms were used to establish novel 3D cell cultures enriched in CSCs from non-small cell lung cancer (NSCLC) patients and cell lines. Rigid scaffolds with an elevated compressive modulus and uniform pores and channels were produced using different filaments. Hydrogel-based scaffolds were printed with…

Comprehensive cross-platform comparison of methods for non-invasive EGFR mutation testing : results of the RING observational trial

Plasma samples from 72 EGFR‐mutant advanced NSCLC patients, collected upon progression to first‐line TKI, were analyzed by seven methodologies (two NGS‐based methods, three high‐sensitivity PCR‐based platforms, and two FDA‐approved methods). Our study demonstrates a good to excellent agreement between methodologies and supports the use of liquid biopsies for therapy decision‐making.

Serum metabolomic profiling facilitates the non-invasive identification of metabolic biomarkers associated with the onset and progression of non-small cell lung cancer

Lung cancer (LC) is responsible for most cancer deaths. One of the main factors contributing to the lethality of this disease is the fact that a large proportion of patients are diagnosed at advanced stages when a clinical intervention is unlikely to succeed. In this study, we evaluated the potential of metabolomics by H-1-NMR to facilitate the identification of accurate and reliable biomarkers to support the early diagnosis and prognosis of non-small cell lung cancer (NSCLC). We found that the metabolic profile of NSCLC patients, compared with healthy individuals, is characterized by statistically significant changes in the concentration of 18 metabolites representing different amino acids…

Role of RAS mutation status as a prognostic factor for patients with advanced colorectal cancer treated with first-line chemotherapy based on fluoropyrimidines and oxaliplatin, with or without bevavizumab: A retrospective analysis

The role of Kirsten rat sarcoma viral oncogene homolog (KRAS) and neuroblastoma RAS viral oncogene homolog (NRAS) mutations as negative predictors for anti-epidermal growth factor receptor (EGFR) therapies in metastatic colorectal cancer (CRC) has been firmly established. However, whether the RAS mutation status plays a role as a biomarker for anti-vascular endothelial growth factor (VEGF) treatment remains controversial. Data from 93 CRC patients who received first-line cytotoxic chemotherapy with fluoropyrimidines and oxaliplatin, with or without bevacizumab, were analyzed. We investigated the association between the RAS mutation status and clinical outcomes in terms of response rate, pro…

Characterization of lung tumourspheres reveals cancer stem-like cells potential targets and prognostic markers in non-small cell lung cancer

Abstract Background Non-small cell lung cancer (NSCLC) is the first cause of death cancer-related worldwide mainly due to high therapeutic resistance. This resistance is related to cancer stem-like cells (CSCs), for which the identification of targets and markers is still ongoing. Methods Primary cultures from 8 NSCLC patients were established as tumorspheres and as monolayers. CSCs properties were tested for both conditions in vitro and in vivo. The expression of 50 CSCs-related genes was assessed by RTqPCR and proteins of significantly overexpressed genes were examined by immunoblot and immunofluorescence. The prognostic role of these genes was analyzed in a cohort of 661 NSCLC patients f…

Analysis of the prognostic impact of Treg-related genes in tumor and stroma in resectable NSCLC

11073 Background: Immunosuppressive regulatory T lymphocytes (Tregs) have been proved to play a critical role in immune tolerance to tumor. In this study we have analyzed the expression of 11 genes related to Tregs in both tumor and stroma samples of resectable NSCLC patients. Methods: Primary tumor tissues of FFPE samples from 125 early-stage NSCLC patients were used in this retrospective study. The most representative areas of tumor cells and tumor stroma of each sample were carefully micro-dissected. RTqPCR using hydrolysis probes (TaqMan, Applied Biosystems) was performed to assess the expression of Treg markers such as: CD127, CD25, FOXP3, CTLA-4, IL-10, TGFB-1, LAG-3, GITR and TNF-a …

Extracellular vesicles as miRNA nano-shuttles : dual role in tumor progression

[EN] Tumor-derived extracellular vesicles (EVs) have a pleiotropic role in cancer, interacting with target cells of the tumor microenvironment, such as fibroblasts, immune and endothelial cells. EVs can modulate tumor progression, angiogenic switch, metastasis, and immune escape. These vesicles are nano-shuttles containing a wide spectrum of miRNAs that contribute to tumor progression. MiRNAs contained in extracellular vesicles (EV-miRNAs) are disseminated in the extracellular space and are able to influence the expression of target genes with either tumor suppressor or oncogenic functions, depending on both parental and target cells. Metastatic cancer cells can balance their oncogenic pote…

Update on biomarkers for the detection of lung cancer

Patients at risk for lung cancer may have subclinical disease for years before presentation. The diagnosis of this disease is primarily based on symptoms, and detection often occurs after curative intervention is no longer possible. At present, no lung cancer early-detection biomarker is clinically available. This study reviews the most recent advances in early detection and molecular diagnostic biomarkers for the detection of lung cancer. This review includes an overview of the various biological specimens and matrices in which these biomarkers could be analyzed, as well as the diverse strategies and approaches for identifying new biomarkers that are currently being explored. Several novel…

Genomic profiling in advanced stage non-small-cell lung cancer patients with platinum-based chemotherapy identifies germline variants with prognostic value in SMYD2

Abstract Objective The aim of the study was to investigate the relationship between germline variations as a prognosis biomarker in patients with advanced Non-Small-Cell-Lung-Cancer (NSCLC) subjected to first-line platinum-based treatment. Materials and Methods We carried out a two-stage genome-wide-association study in non-small-cell lung cancer patients with platinum-based chemotherapy in an exploratory sample of 181 NSCLC patients from Caucasian origin, followed by a validation on 356 NSCLC patients from the same ancestry (Valencia, Spain). Results We identified germline variants in SMYD2 as a prognostic factor for survival in patients with advanced NSCLC receiving chemotherapy. SMYD2 al…

Differentially methylated genes in proliferative verrucous leukoplakia reveal potential malignant biomarkers for oral squamous cell carcinoma

Objectives: To explore the pathophysiology of proliferative verrucous leucoplakia (PVL) through a methylated DNA immunoprecipitation and high-throughput sequencing (MeDIP-seq) case-control study. Materials and & nbsp; Methods: Oral biopsies from ten PVL patients and five healthy individuals were obtained and used to compare their epigenetic patterns. Network biology methods and integrative analyses of MeDIP-seq and RNAseq data were applied to investigate functional relations among differentially methylated genes (DMGs). The value of selected genes as malignant biomarkers was evaluated in a large cohort of oral squamous cell carcinoma (OSCC) patients from TCGA.& nbsp; Results: A total of 464…

Analysis of the prognostic role of an immune checkpoint score in resected non-small cell lung cancer patients

[EN] Tumors develop mechanisms to recruit tolerogenic immune cells and to induce the expression of molecules that act as immune checkpoints. This regulation of the immune microenvironment favors immune tolerance to the neoplastic cells. In this study, we have investigated the prognostic role of immune-checkpoint expression markers in a cohort of resectable non-small cell lung cancer (NSCLC) patients. RNA was isolated from fresh-frozen lung specimens (tumor and normal lung) (n = 178). RTqPCR was performed to analyze the relative expression of 20 immune-related genes that were normalized by the use of endogenous genes selected by GeNorm algorithm. Patients with higher expression levels of IL2…

Functional FLT1 genetic variation is a prognostic factor for recurrence in stage I-III non-small cell lung cancer

Background: We propose that single-nucleotide polymorphisms (SNPs) in genes of the vascular endothelial growth factor pathway of angiogenesis will associate with survival in non-small-cell lung cancer (NSCLC) patients. Methods: Fifty-three SNPs in vascular endothelial growth factor-pathway genes were genotyped in 150 European stage I-III NSCLC patients and tested for associations with patient survival. Replication was performed in an independent cohort of 142 European stage I-III patients. Reporter gene assays were used to assess the effects of SNPs on transcriptional activity. Results: In the initial cohort, five SNPs associated (q < 0.05) with relapse-free survival (RFS). The minor allele…

Abstract 3056: Lung tumor spheres as in vitro platform for testing new therapeutic strategies against cancer stem cells

Abstract Background: Treatment resistance is related to cancer stem cells (CSCs), a highly tumorigenic subpopulation of cells capable of growing and forming tumor spheres under non-adherent conditions. This study aimed to isolate and characterise CSCs from resected non-small cell lung cancer (NSCLC) patients' tumor-tissue and cell lines like tumor spheres and to use them as an in vitro platform for drug screening. Methods: The study was performed on tumour cells from 8 resected NSCLC patients and 12 NSCLC cell lines grown in monolayer and as spheres. The expression of 60 genes, including CSC-markers, pluripotency inducers, cell cycle regulators and components of the Notch, Wnt and Hedgehog …

TMPRSS4: a novel tumor prognostic indicator for the stratification of stage IA tumors and a liquid biopsy biomarker for NSCLC patients

Relapse rates in surgically resected non-small-cell lung cancer (NSCLC) patients are between 30% and 45% within five years of diagnosis, which shows the clinical need to identify those patients at high risk of recurrence. The eighth TNM staging system recently refined the classification of NSCLC patients and their associated prognosis, but molecular biomarkers could improve the heterogeneous outcomes found within each stage. Here, using two independent cohorts (MDA and CIMA-CUN) and the eighth TNM classification, we show that TMPRSS4 protein expression is an independent prognostic factor in NSCLC, particularly for patients at stage I: relapse-free survival (RFS) HR, 2.42 (95% CI, 1.47&ndash

A gene expression signature to characterize human lung adenocarcinoma cancer stem cells.

e20547Background: Treatment resistance is linked to cancer stem cells (CSCs), a highly tumorigenic subpopulation of cells with the ability to grow as spheres in non-adherent conditions. The aim of ...

Isolation and characterization of lung cancer sphere-forming cells as platforms for the development of new therapeutic strategies

e22190 Background: Resistance to treatment is one of major causes influencing the high mortality of lung cancer. This feature seems to be dependent of a minority of cancer cells with special charac...

Ratios between VEGF ligands and receptors in tumor and stroma have impact on the outcome in resectable NSCLC.

e22147 Background: In tumor angiogenesis there is a complex interplay between endothelial, stromal and tumor cells. Some key regulators of this process are the members of the vascular endothelial growth factor (VEGF) family of ligands and receptors and the neuropilins (NRP). This study analyzes the correlations between the expression of these angiogenic factors in tumor cells and tumor stroma, and their prognostic role in tissue samples from resected non-small cell lung cancer (NSCLC) patients. Methods: Representative tumor and stroma areas from FFPE tissue samples of 125 early-stage NSCLC patients were carefully micro-dissected. RNA isolated from the samples was retrotranscripted and prea…

CCDC 1414524: Experimental Crystal Structure Determination

Related Article: Vanessa Soto-Cerrato, Pilar Manuel-Manresa, Elsa Hernando, Silvia Calabuig-Fariñas, Alicia Martínez-Romero, Víctor Fernández-Dueñas, Kristoffer Sahlholm, Thomas Knöpfel, María García-Valverde, Ananda M. Rodilla, Eloisa Jantus-Lewintre, Rosa Farràs, Francisco Ciruela, Ricardo Pérez-Tomás, and Roberto Quesada|2015|J.Am.Chem.Soc.|137|15892|doi:10.1021/jacs.5b09970

CCDC 1414523: Experimental Crystal Structure Determination

Related Article: Vanessa Soto-Cerrato, Pilar Manuel-Manresa, Elsa Hernando, Silvia Calabuig-Fariñas, Alicia Martínez-Romero, Víctor Fernández-Dueñas, Kristoffer Sahlholm, Thomas Knöpfel, María García-Valverde, Ananda M. Rodilla, Eloisa Jantus-Lewintre, Rosa Farràs, Francisco Ciruela, Ricardo Pérez-Tomás, and Roberto Quesada|2015|J.Am.Chem.Soc.|137|15892|doi:10.1021/jacs.5b09970