The Advantage of Targeted Next-Generation Sequencing over qPCR in Testing for Druggable Variants in Non-Small-Cell Lung Cancer.

The emergence of targeted therapies in non-small-cell lung cancer (NSCLC), including inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase, has increased the need for robust companion diagnostic tests. Nowadays, detection of actionable variants in exons 18-21 of the gene by qPCR and direct DNA sequencing is often replaced by next-generation sequencing (NGS). In this study, we evaluated the diagnostic usefulness of targeted NGS for druggable variants testing in clinical NSCLC material previously analyzed by the IVD-certified qPCR test with respect to DNA reference material.

Targeted Next-Generation Sequencing of Thymic Epithelial Tumours Revealed Pathogenic Variants in , , , and in 30% of Thymic Carcinomas.

A better understanding of the molecular pathogenesis of thymic epithelial tumours (TETs) could revolutionise their treatment. We evaluated thymomas and thymic carcinomas by next-generation sequencing (NGS) of somatic or germline single nucleotide variants (SNVs) in genes commonly mutated in solid tumours. In total, 19 thymomas and 34 thymic carcinomas were analysed for nonsynonymous SNVs in 15 genes by targeted NGS (reference genome: hg19/GRCh37).

Detection of EGFR mutations in liquid biopsy samples using allele-specific quantitative PCR: A comparative real-world evaluation of two popular diagnostic systems.

Purpose: The detection of epidermal growth factor receptor (EGFR) mutations in plasma cell-free DNA (cfDNA) is an auxiliary tool for the molecular diagnosis of non-small cell lung cancer (NSCLC), especially when an adequate tumor tissue specimen cannot be obtained. We compared the diagnostic accuracy of two commonly used in vitro diagnostic-certified allele-specific quantitative PCR assays for detecting plasma cfDNA EGFR mutations.

Methods: We analyzed EGFR mutations in plasma cfDNA from 90 NSCLC patients (stages I-IV) before treatment (n ​= ​60) and after clinical progression on EGFR tyrosine kinase inhibitors (n ​= ​30) using the cobas EGFR mutation test v2 (Roche Molecular Systems, Inc.

miR-625-3p and lncRNA GAS5 in Liquid Biopsies for Predicting the Outcome of Malignant Pleural Mesothelioma Patients Treated with Neo-Adjuvant Chemotherapy and Surgery.

Combining neo-adjuvant chemotherapy and surgery is part of multimodality treatment of malignant pleural mesothelioma (MPM), but not all patients benefit from this approach. In this exploratory analysis, we investigated the prognostic value of circulating miR-625-3p and lncRNA GAS5 after neo-adjuvant chemotherapy. 36 MPM patients from the SAKK 17/04 trial (NCT00334594), whose blood was available before and after chemotherapy were investigated.

The expression of circulating miR-504 in plasma is associated with EGFR mutation status in non-small-cell lung carcinoma patients.

MicroRNAs (miRNAs), key regulators of gene expression at the post-transcriptional level, are grossly misregulated in some human cancers, including non-small-cell lung carcinoma (NSCLC). The aberrant expression of specific miRNAs results in the abnormal regulation of key components of signalling pathways in tumour cells. MiRNA levels and the activity of the gene targets, including oncogenes and tumour suppressors, produce feedback that changes miRNA expression levels and indicates the cell's genetic activity.

miRNAs as Biomarkers and Therapeutic Targets in Non-Small Cell Lung Cancer: Current Perspectives.

Lung cancer is the most common cancer worldwide. Up to 85% of lung cancer cases are diagnosed as non-small cell lung cancer (NSCLC). The effectiveness of NSCLC treatment is expected to be improved through the implementation of robust and specific biomarkers.

Plasma cell-free DNA levels and integrity in patients with chest radiological findings: NSCLC versus benign lung nodules.

Effective discrimination between lung cancer and benign tumours is a common clinical problem in the differential diagnosis of solitary pulmonary nodules. The analysis of cell-free DNA (cfDNA) in blood may greatly aid the early detection of lung cancer by evaluating cancer-related alterations. The plasma cfDNA levels and integrity were analysed in 65 non-small cell lung cancer (NSCLC) patients, 28 subjects with benign lung tumours, and 16 healthy controls using real-time PCR.

Coexistence of Borrelia burgdorferi s.l. genospecies within Ixodes ricinus ticks from central and eastern Poland.

The purpose of the study was to assess the prevalence and coinfection rates of Borrelia burgdorferi sensu lato genotypes in Ixodes ricinus (L.) ticks sampled from diverse localities in central and eastern regions of Poland. In years 2009-2011, questing nymphs and adults of I.

Studies of hepatocyte growth factor in bronchoalveolar lavage fluid in chronic interstitial lung diseases.

Introduction:  Previous studies have suggested that hepatocyte growth factor (HGF) inhibits lung fibrosis as an antagonist of transforming growth factor β (TGF‑β).

Objectives:  We assessed HGF expression levels in the lower airways of patients with selected interstitial lung diseases.

Patients And Methods:  HGF levels were examined by an enzyme‑linked immunosorbent assay in bronchoalveolar lavage (BAL) fluid supernatants from patients with pulmonary sarcoidosis (PS, n = 52), idiopathic pulmonary fibrosis (IPF, n = 23), nonspecific interstitial pneumonia (NSIP, n = 14), extrinsic allergic alveolitis (EAA, n = 6), bronchiolitis obliterans organizing pneumonia (BOOP, n = 8), chronic eosinophilic pneumonia (EP, n = 6), and in control subjects (n = 13).

[Current methods to detect EGFR gene mutations as predictive factor for targeted therapies in non-small cell lung cancer - is there a "golden standard" in diagnostics?].

According to current Polish and international recommendations, detection of EGFR gene somatic mutations is the essential part of routine diagnostic algorithm in advanced NSCLC patients considered for tyrosine kinase inhibitor therapy. Molecular heterogeneity of tumor tissue and cytology materials used for molecular diagnostics is challenging for classic methods of genetic analysis, such as Sanger sequencing, driving the development and implementation of specialized, highly sensitive techniques for mutations detection. Constant, dynamic progress in molecular biology techniques, particularly development of next-generation sequencing, should enable clinical implementation of simultaneous multiple therapeutic biomarkers analysis as well as non-invasive EGFR mutations diagnostic based on free-circulating DNA isolated from blood of non-small cell lung cancer patients.

[Higher incidence of alveolar lymphocytes (AL) apoptosis in smokers depends on BCL-2 expression and specific response to tumor necrosis factor alpha (TNFalpha). Bronchoalveolar lavage (BAL) material analysis from selected interstitial lung diseases (ILD) and healthy controls].

Background: We have previously described the increased apoptosis rate in smokers alveolar lymphocytes (AL) that was independent from the FASL/ FAS system activation. Consequently, the role of intrinsic apoptosis pathway and other ligand/death receptor pairs as TNFalpha/TNFR1 and TRAIL/DR4 important for apoptosis regulation should be considered in this phenomenon. The purpose of the study was to evaluate the impact of tobacco consumption on expression of selected BCL-2 family members and ligand/receptors pairs in bronchoalveolar lavage (BAL) harvested from patients with pulmonary sarcoidosis (PS), idiopathic pulmonary fibrosis (IPF) and healthy volunteers.

Reliable detection of rare mutations in EGFR gene codon L858 by PNA-LNA PCR clamp in non-small cell lung cancer.

PNA-LNA PCR clamp real-time PCR method represents allele-specific approach to mutation analysis of EGFR gene in NSCLC. Due to its unique design, it is characterized by exceptionally high specificity and sensitivity but also allows detection of rare or not specifically-targeted EGFR mutations within examined exons, otherwise undetectable by other mutation-specific fluorescent probes. We herein present two cases of rare mutations revealed by PNA-LNA PCR clamping of NSCLC samples referred for routine EGFR gene molecular diagnostics.

Simple flow cytometric protocol of CD4+/CD8+ lymphocyte ratio assessment in bronchoalveolar lavage fluids from patients with interstitial lung diseases.

Objective: To validate the fast and accurate flow cytometric (FCM) protocol using blood-standardized antibodies for alveolar lymphocyte subtyping with respect to standard immunocytochemistry (IC).

Study Design: FCM and IC were applied to immunophenotype T cell subsets in bronchoalveolar lavage (BAL) fluids from patients with interstitial lung diseases. Diagnostic BAL specimens from 50 patients with suspected sarcoidosis, idiopathic pulmonary fibrosis, and hypersensitivity pneumonitis were evaluated by both IC and FCM.

Quantitative analysis of free-circulating DNA in plasma of patients with resectable NSCLC.

Objective: Minute amounts of free-circulating DNA are present in plasma of healthy individuals, whereas its increased concentration was observed in patients with malignant tumors including non-small cell lung cancer (NSCLC). This study aimed at demonstrating the potential usefulness of plasma DNA concentration monitoring in NSCLC patients for therapy effectiveness assessment throughout the treatment and follow-up period.

Methods: Plasma DNA concentration was assessed in 50 NSCLC patients (stage I - IIIA) prior and following the radical treatment using real-time quantitative PCR method.

Molecular evidence of Anaplasma phagocytophilum and Babesia microti co-infections in Ixodes ricinus ticks in central-eastern region of Poland.

The aim of the study was to elucidate the distribution of Anaplasma phagocytophilum and Babesia microti co-infection in Ixodes ricinus populations within the central-eastern region of Poland. The prevalence of analysed tick-borne human pathogens in single and polymicrobial infections in I. ricinus ticks were analysed using the conventional and nested PCR techniques.

Enhanced expression of Fas Ligand (FasL) in the lower airways of patients with fibrotic interstitial lung diseases (ILDs).

The exact role of FasL, and particularly its soluble and membrane-bound forms, in the development of chronic ILDs and lung fibrosis has not been extensively explored. We aimed at analyzing membrane-bound FasL expression on alveolar macrophages (AM) and lymphocytes (AL) as well as soluble FasL (sFasL) levels in bronchoalveolar lavage (BAL) from ILDs patients, incl. pulmonary sarcoidosis (PS), hypersensitivity pneumonitis (HP), silicosis, asbestosis, idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), and healthy subjects (n = 89, 12, 7, 8, 23, 6, 17, respectively).

[Molecular diagnostics of alpha-1-antitrypsin deficiency in clinical practice].

The deficiency of serine protease inhibitor, alpha-1-antitrypsin (AATD), is genetically determined defect that increases the risk of lung and liver disease development. The results of recent epidemiological studies indicate the overwhelming majority of individuals with alpha-1-antitrypsin deficiency still remain undiagnosed. The complete laboratory diagnosis of AATD is based on combination of quantitative and qualitative methods.

The impact of genetic markers on the diagnosis of lung cancer: a current perspective.

Lung cancer is the leading worldwide source of cancer-related death. It is acknowledged that prognosis and treatment outcomes in lung cancer might be improved by increasing the effectiveness of early-stage diagnosis. Several recently published studies have produced intriguing results regarding the detection of biomarkers in tumor samples, but also in easily accessible specimens such as sputum, plasma, and exhaled breath condensate.

Expression of insulin-like growth factor-I (IGF-I) in alveolar macrophages and lymphocytes obtained by bronchoalveolar lavage (BAL) in interstitial lung diseases (ILD). Assessment of IGF-I as a potential local mitogen and antiapoptotic cytokine.

Little is known about IGF-I expression in the alveolar lymphocytes (AL), and about local role of IGF-I in physiological conditions and in interstitial lung diseases. Bronchoalveolar lavage was carried out in patients with silicosis, asbestosis, idiopathic pulmonary fibrosis (IPF) and sarcoidosis, as well as in control subjects (n = 13, 9, 12, 56, 15, resp). Alveolar macrophages (AM) and lymphocytes (AL) were studied for (1) IGF-I, BCL-2, Fas and Fas Ligand expression and (2) cell cycle (incl.

[Aptamers in clinical diagnostics].

Aptamers are single-stranded DNA or RNA oligonucleotides selected in vitro from combinatorial libraries in a process called SELEX (Systematic Evolution of Ligands by EXponential Enrichment). Aptamers play a role of artificial nucleic acid ligands that can recognize and bind to various organic or inorganic target molecules with high specificity and affinity. They can discriminate even between closely related targets and can be easily chemically modified for radioactive, fluorescent and enzymatic labeling or biostability improvement.

[Studies on insulin-like growth factor-I expression in human lower airways].

Unlabelled: Antisense techniques that inhibit intracellular expression of insulin-like growth factor-I (IGF-I) were efficient in gene therapy of some tumor diseases. IGF-I in the airways is considered to induce lung fibrosis in interstitial lung diseases, inhibit apoptosis of epithelial cells and participate in local carcinogenesis.

Aim Of The Study: The aim of the study was--by examining the IGF-I expression in the lower airways--to evaluate preliminary the efficacy of anti-IGF-I antisense technicques in the treatment of airways diseases.