Publications by authors named "Csaba Váradi"

17 Publications

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The Analysis of Human Serum N-Glycosylation in Patients with Primary and Metastatic Brain Tumors.

Life (Basel) 2021 Jan 6;11(1). Epub 2021 Jan 6.

Institute of Chemistry, Faculty of Materials Science and Engineering, University of Miskolc, 3515 Miskolc, Hungary.

The identification of patients with different brain tumors is solely built on imaging diagnostics, indicating the need for novel methods to facilitate disease recognition. Glycosylation is a chemical modification of proteins, reportedly altered in several inflammatory and malignant diseases, providing a potential alternative route for disease detection. In this paper, we report the quantitative analysis of serum N-glycosylation of patients diagnosed with primary and metastatic brain tumors. PNGase-F-digested and procainamide-labeled serum glycans were purified by magnetic nanoparticles, followed by quantitative liquid chromatographic analysis. The glycan structures were identified by the combination of single quad mass spectrometric detection and exoglycosidase digestions. Linear discriminant analysis provided a clear separation of different disease groups and healthy controls based on their N-glycome pattern. Altered distribution of biantennary neutral, sialylated but nonfucosylated, and sialylated-fucosylated structures were found to be the most significant changes. Our results demonstrate that serum glycosylation monitoring could improve the detection of malignancy.
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http://dx.doi.org/10.3390/life11010029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825111PMC
January 2021

Clinical Features of Parkinson's Disease: The Evolution of Critical Symptoms.

Authors:
Csaba Váradi

Biology (Basel) 2020 May 19;9(5). Epub 2020 May 19.

Institute of Chemistry, Faculty of Materials Science and Engineering, University of Miskolc, 3515 Miskolc, Hungary.

Parkinson's disease (PD) is a multi-attribute neurodegenerative disorder combining motor and nonmotor symptoms without well-defined diagnostic clinical markers. The presence of primary motor features (bradykinesia, rest tremor, rigidity and loss of postural reflexes) are the most characteristic signs of PD that are also utilized to identify patients in current clinical practice. The successful implementation of levodopa treatment revealed that nonmotor features are the main contributors of patient disability in PD, and their occurrence might be earlier than motor symptoms during disease progression. Targeted detection of prodromal PD symptoms can open up new possibilities in the identification of PD patients and provide potential patient populations for developing novel neuroprotective therapies. In this review, the evolution of critical features in PD diagnosis is described with special attention to nonmotor symptoms and their possible detection.
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http://dx.doi.org/10.3390/biology9050103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285080PMC
May 2020

Analysis of cetuximab N-Glycosylation using multiple fractionation methods and capillary electrophoresis mass spectrometry.

J Pharm Biomed Anal 2020 Feb 7;180:113035. Epub 2019 Dec 7.

Characterisation and Comparability Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Blackrock, Co. Dublin, A94 X099, Ireland; School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4, D04 V1W8, Ireland.

Site-specific glycosylation of Cetuximab was characterized in this study using multiple fractionation methods and capillary electrophoresis coupled to mass spectrometry (CE-MS) based glycomics. IdeS digested Cetuximab with subsequent reduction was fractionated using reversed-phase chromatography resulting in 3 fragments; Fd, Lc and Fc/2. Glycan release of the different fragments was performed in O enriched water providing the possible quantification of site occupancy. 2-AA labelled glycan structures were annotated by CE-MS profiling in combination with exoglycosidase sequencing, revealing potential structures with terminal α-galactose and N-glycolyl-neuraminic acid (NGNA) mainly originating from the Fd fragment. Glycosylation analysis was also performed on different charge variants of Cetuximab that were separated using pH gradient cation-exchange chromatography to investigate the impact of glycosylation on the net charge of the protein.
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http://dx.doi.org/10.1016/j.jpba.2019.113035DOI Listing
February 2020

Purification of Fluorescently Derivatized N-Glycans by Magnetic Iron Nanoparticles.

Nanomaterials (Basel) 2019 Oct 17;9(10). Epub 2019 Oct 17.

Faculty of Materials Science and Engineering, Institute of Chemistry, University of Miskolc, 3515 Miskolc, Hungary.

A novel glycoanalytical approach was developed in this study for the purification of fluorescently derivatized N-glycans. Polyethylene glycol (PEG) modified iron-nanoparticles were synthetized by the combination of sonochemical treatment and combustion method. The prepared nanomaterials were applied for a systematic clean-up optimization to maximize purification efficiency of 2-AA labelled glycans. PEG 1000 modified iron-oxalate was found to be the most effective for the selective enrichment of serum N-glycans providing high reproducibility. Different acetonitrile percentages for binding and washing steps were also tested to ensure the same relative peak areas compared to the unpurified sample. The generated novel clean-up strategy provides a potential route to use in-house synthetized magnetic nanoparticles for glycan sample preparation.
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http://dx.doi.org/10.3390/nano9101480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6835309PMC
October 2019

Serum N-Glycosylation in Parkinson's Disease: A Novel Approach for Potential Alterations.

Molecules 2019 Jun 13;24(12). Epub 2019 Jun 13.

Characterisation and Comparability Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Blackrock, Co., Dublin A94 X099, Ireland.

In this study, we present the application of a novel capillary electrophoresis (CE) method in combination with label-free quantitation and support vector machine-based feature selection (support vector machine-estimated recursive feature elimination or SVM-RFE) to identify potential glycan alterations in Parkinson's disease. Specific focus was placed on the use of neutral coated capillaries, by a dynamic capillary coating strategy, to ensure stable and repeatable separations without the need of non-mass spectrometry (MS) friendly additives within the separation electrolyte. The developed online dynamic coating strategy was applied to identify serum N-glycosylation by CE-MS/MS in combination with exoglycosidase sequencing. The annotated structures were quantified in 15 controls and 15 Parkinson's disease patients by label-free quantitation. Lower sialylation and increased fucosylation were found in Parkinson's disease patients on tri-antennary glycans with 2 and 3 terminal sialic acids. The set of potential glycan alterations was narrowed by a recursive feature elimination algorithm resulting in the efficient classification of male patients.
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http://dx.doi.org/10.3390/molecules24122220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6630595PMC
June 2019

Multi-site N-Glycan mapping study 2: UHPLC.

Electrophoresis 2018 04 6;39(7):998-1005. Epub 2018 Feb 6.

Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen, Hungary.

In the first part of this publication, the results from an international study evaluating the precision (i.e., repeatability and reproducibility) of N-glycosylation analysis using capillary electrophoresis of APTS-labeled N-glycans were presented. The corresponding results from ultra-high performance liquid chromatography (UHPLC) with fluorescence detection are presented here from 12 participating sites. All participants used the same lot of samples, reagents, and columns to perform the assays. Elution time, peak area and peak area percent values were determined for all peaks ≥0.1% peak area, and statistical analysis was performed following ISO 5725-2 guideline principles. The results demonstrated adequate reproducibility, within any given site as well across all sites, indicating that standard UHPLC-based N-glycan analysis platforms are appropriate for general use.
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http://dx.doi.org/10.1002/elps.201700463DOI Listing
April 2018

Stable Isotope Quantitative N-Glycan Analysis by Liquid Separation Techniques and Mass Spectrometry.

Methods Mol Biol 2017 ;1606:353-366

NIBRT-The National Institute for Bioprocessing Research & Training, Foster Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland.

Liquid phase separation analysis and subsequent quantitation remains a challenging task for protein-derived oligosaccharides due to their inherent structural complexity and diversity. Incomplete resolution or co-detection of multiple glycan species complicates peak area-based quantitation and associated statistical analysis when optical detection methods are used. The approach outlined herein describes the utilization of stable isotope variants of commonly used fluorescent tags that allow for mass-based glycan identification and relative quantitation following separation by liquid chromatography (LC) or capillary electrophoresis (CE). Comparability assessment of glycoprotein-derived oligosaccharides is performed by derivatization with commercially available isotope variants of 2-aminobenzoic acid or aniline and analysis by LC- and CE-mass spectrometry. Quantitative information is attained from the extracted ion chromatogram/electropherogram ratios generated from the light and heavy isotope clusters.
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http://dx.doi.org/10.1007/978-1-4939-6990-6_23DOI Listing
February 2018

Quantitative glycomics using liquid phase separations coupled to mass spectrometry.

Analyst 2017 Feb;142(5):700-720

National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Dublin, A94 X099, Ireland. and School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4, D04 V1 W8, Ireland.

Post-translational modification of proteins by the attachment of glycans is governed by a variety of highly specific enzymes and is associated with fundamental impacts on the parent protein's physical, chemical and biological properties. The inherent connection between cellular physiology and specific glycosylation patterns has been shown to offer potential for diagnostic and prognostic monitoring of altered glycosylation in the disease state. Conversely, glycoprotein based biopharmaceuticals have emerged as dominant therapeutic strategies in the treatment of intricate diseases. Glycosylation present on these biopharmaceuticals represents a major critical quality attribute with impacts on both pharmacokinetics and pharmacodynamics. The structural variety of glycans, based upon their non-template driven assembly, poses a significant analytical challenge for both qualitative and quantitative analysis. Labile monosaccharide constituents, isomeric species and often low sample availability from biological sources necessitates meticulous sample handling, ultra-high-resolution analytical separation and sensitive detection techniques, respectively. In this article a critical review of analytical quantitation approaches using liquid phase separations coupled to mass spectrometry for released glycans of biopharmaceutical and biomedical significance is presented. Considerations associated with sample derivatisation strategies, ionisation, relative quantitation through isotopic as well as isobaric labelling, metabolic/enzymatic incorporation and targeted analysis are all thoroughly discussed.
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http://dx.doi.org/10.1039/c6an02715fDOI Listing
February 2017

Quantitative twoplex glycan analysis using C and C stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry.

Anal Bioanal Chem 2016 Dec 23;408(30):8691-8700. Epub 2016 Sep 23.

Characterisation and Comparability Laboratory, NIBRT-The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland.

Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available C stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for C 'light' and C 'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance.
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http://dx.doi.org/10.1007/s00216-016-9935-8DOI Listing
December 2016

Multi-Site N-glycan mapping study 1: Capillary electrophoresis - laser induced fluorescence.

MAbs 2016 14;8(1):56-64. Epub 2015 Oct 14.

a Horváth Laboratory of Bioseparation Sciences; University of Debrecen ; Debrecen , H-4032 , Hungary.

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established.
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http://dx.doi.org/10.1080/19420862.2015.1107687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966509PMC
September 2016

Comparative glycoprofiling of HIV gp120 immunogens by capillary electrophoresis and MALDI mass spectrometry.

Electrophoresis 2015 Jun 15;36(11-12):1305-13. Epub 2015 May 15.

Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen, Hungary.

The human immunodeficiency virus (HIV) envelope glycoprotein (Env) is the primary antigenic feature on the surface of the virus and is of key importance in HIV vaccinology. Vaccine trials with the gp120 subunit of Env are ongoing, with the recent RV144 trial showing moderate efficacy. gp120 is densely covered with N-linked glycans that are thought to help evade the host's humoral immune response. To assess how the global glycosylation patterns vary between gp120 constructs, the glycan profiles of several gp120s were examined by CE with LIF detection and MALDI-MS. The glycosylation profiles were found to be similar for chronic versus transmitter/founder isolates and only varied moderately between gp120s from different clades. This study revealed that the addition of specific tags, such as the herpes simplex virus glycoprotein D tag used in the RV144 trial, had significant effects on the overall glycosylation patterns. Such effects are likely to influence the immunogenicity of various Env immunogens and should be considered for future vaccine strategies, emphasizing the importance of the glycosylation analysis approach described in this paper.
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http://dx.doi.org/10.1002/elps.201500054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4544863PMC
June 2015

Sample preparation for N-glycosylation analysis of therapeutic monoclonal antibodies by electrophoresis.

Methods Mol Biol 2015 ;1274:183-95

Horváth Laboratory of Bioseparation Sciences, University of Debrecen, Nagyerdei krt 98, Elméleti tömb, 1/111, 4032, Debrecen, Hungary.

There are a considerable number of biopharmaceuticals that have been approved for clinical use in the past decade. Over half of these new generation drugs are glycoproteins, such as monoclonal antibodies or other recombinant glycoproteins, which are mostly produced in mammalian cell lines. The linked carbohydrate moieties affect not only their physicochemical properties and thermal stability but also crucial features like receptor-binding activity, circulating half-life, as well as immunogenicity. The structural diversity of these attached glycans can be manifested in altered monosaccharide composition and linkages/positions among the monosaccharide building blocks. In addition, as more and more biosimilar products hit the market, understanding the effects of their glycosylation modification has become a recent target in efficacy and safety issues. To ensure consistent quality of these products, glycosylation profiles have to be monitored and controlled in all steps of the manufacturing process, i.e., from clone selection to lot release. In this paper, we describe some of the recently introduced and commonly used sample preparation techniques for capillary electrophoresis (CE)-based profiling and structural elucidation of N-glycans. The presented protocols include protein A affinity partitioning of monoclonal antibodies (mAbs), enzymatic release of the N-linked glycans, labeling of the liberated carbohydrates, reaction mixture purification techniques to remove the excess labeling reagent, and high-resolution and rapid capillary electrophoresis-laser-induced fluorescence (CE-LIF)-based profiling of the labeled and purified N-glycans.
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http://dx.doi.org/10.1007/978-1-4939-2353-3_16DOI Listing
November 2015

Combination of IgG N-glycomics and corresponding transcriptomics data to identify anti-TNF-α treatment responders in inflammatory diseases.

Electrophoresis 2015 Jun 15;36(11-12):1330-5. Epub 2015 Apr 15.

Horváth Laboratory of Bioseparation Sciences, School of Medicine, MMKK, University of Debrecen, Debrecen, Hungary.

Prediction of responsiveness in biological therapies is an important and challenging issue in different diseases. Analyzing glycosylation pattern changes of key serum glycoproteins is one of the possible avenues to follow disease remission. The aim of this study was to investigate the changes of serum IgG glycoforms in Crohn's disease (CD) and rheumatoid arthritis patients in response to antitumor necrosis factor alpha (anti-TNF-α) treatment. IgG was isolated from patient serum samples using Protein A affinity pull-down, followed by the release of N-glycans with peptide-N-glycosidase F. The released glycans were fluorescently tagged with 8-aminopyrene-1,3,6-trisulfonate and analyzed by CGE with laser-induced fluorescent detection. Significant alterations were detected between responders and nonresponders in both disease groups. In CD patients, disease-specific alteration was found in response to anti-TNF-α therapy, which was also confirmed by transcriptomics data analysis of the corresponding glycosyltransferases and glycosidases.
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http://dx.doi.org/10.1002/elps.201400575DOI Listing
June 2015

Rapid magnetic bead based sample preparation for automated and high throughput N-glycan analysis of therapeutic antibodies.

Anal Chem 2014 Jun 9;86(12):5682-7. Epub 2014 Jun 9.

Horváth Laboratory of Bioseparation Sciences, University of Debrecen, Nagyerdei krt 98, Elméleti tömb, 1/111 H-4032, Debrecen, Hungary.

Full automation to enable high throughput N-glycosylation profiling and sequencing with good reproducibility is vital to fulfill the contemporary needs of the biopharmaceutical industry and requirements of national regulatory agencies. The most prevalently used glycoanalytical methods of capillary electrophoresis and hydrophilic interaction liquid chromatography, while very efficient, both necessitate extensive sample preparation and cleanup, including glycoprotein capture, N-glycan release, fluorescent derivatization, purification, and preconcentration steps during the process. Currently used protocols to fulfill these tasks require multiple centrifugation and vacuum-centrifugation steps, making liquid handling robot mediated automated sample preparation difficult and expensive. In this paper we report on a rapid magnetic bead based sample preparation approach that enables full automation including all the process phases just in a couple of hours without requiring any centrifugation and/or vacuum centrifugation steps. This novel protocol has been compared to conventional glycan sample preparation strategies using standard glycoproteins (IgG, fetuin, and RNase B) and featured rapid processing time, high release and labeling efficiency, good reproducibility, and the potential of easy automation.
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http://dx.doi.org/10.1021/ac501573gDOI Listing
June 2014

Analysis of haptoglobin N-glycome alterations in inflammatory and malignant lung diseases by capillary electrophoresis.

Electrophoresis 2013 Aug 12;34(16):2287-94. Epub 2013 Jul 12.

Horváth Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen, Hungary.

A CE-based method was introduced to compare the N-glycosylation profile of haptoglobin in normal and pathologic conditions. To assess the biomarker potential of glycosylation changes in various lung diseases, haptoglobin was isolated from plasma samples of healthy, pneumonia, chronic obstructive pulmonary disease, and lung cancer patients by means of two haptoglobin-specific monoclonal antibodies. Haptoglobin N-glycans were then enzymatically released, fluorescently labeled, and profiled by CE. Disease-associated changes of core and antennary fucosylation were identified by targeted exoglycosidase digestions and their levels were compared in the different patient groups. Terms such as core- and arm-fucosylation degree, as well as branching degree, were introduced for easier characterization of the changes and statistical analysis was used to examine which structures were responsible for the observed differences. Increased level of α1-6 fucosylated tri-antennary glycans was found in all disease groups compared to the control. Elevated amounts of core- and arm-fucosylation on tetra-antennary glycans were detected in the lung cancer group compared to the chronic obstructive pulmonary disease group. A larger scale study is necessary to confirm and validate these preliminary findings in the glycosylation changes of haptoglobin, so could then be used as biomarkers in the diagnosis of malignant and inflammatory lung diseases.
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http://dx.doi.org/10.1002/elps.201300041DOI Listing
August 2013

Peripheral blood gene expression and IgG glycosylation profiles as markers of tocilizumab treatment in rheumatoid arthritis.

J Rheumatol 2012 May 1;39(5):916-28. Epub 2012 Apr 1.

Department of Biochemistry and Molecular Biology, Apoptosis and Genomics Research Group, Hungarian Academy of Sciences, Budapest, Hungary.

Objective: Tocilizumab, a humanized anti-interleukin-6 receptor monoclonal antibody, has recently been approved as a biological therapy for rheumatoid arthritis (RA) and other diseases. It is not known if there are characteristic changes in gene expression and immunoglobulin G glycosylation during therapy or in response to treatment.

Methods: Global gene expression profiles from peripheral blood mononuclear cells of 13 patients with RA and active disease at Week 0 (baseline) and Week 4 following treatment were obtained together with clinical measures, serum cytokine levels using ELISA, and the degree of galactosylation of the IgG N-glycan chains. Gene sets separating responders and nonresponders were tested using canonical variates analysis. This approach also revealed important gene groups and pathways that differentiate responders from nonresponders.

Results: Fifty-nine genes showed significant differences between baseline and Week 4 and thus correlated with treatment. Significantly, 4 genes determined responders after correction for multiple testing. Ten of the 12 genes with the most significant changes were validated using real-time quantitative polymerase chain reaction. An increase in the terminal galactose content of N-linked glycans of IgG was observed in responders versus nonresponders, as well as in treated samples versus samples obtained at baseline.

Conclusion: As a preliminary report, gene expression changes as a result of tocilizumab therapy in RA were examined, and gene sets discriminating between responders and nonresponders were found and validated. A significant increase in the degree of galactosylation of IgG N-glycans in patients with RA treated with tocilizumab was documented.
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http://dx.doi.org/10.3899/jrheum.110961DOI Listing
May 2012

[Our experience with patients operated after neoadjuvant therapy].

Magy Seb 2004 Dec;57(6):332-5

Fovárosi Onkormányzat Bajcsy-Zsilinszky Kórház es Rendelointézet Altalános Sebészet, Mellkassebészeti es Ersebészeti Osztály, Budapest.

The authors performed thoracotomies on 47 patients because of NSCLC between 1 January 2000 and 31 December 2003. All patients had neoadjuvant therapy which was indicated by IIIA stage NSCLC with N2 nodal status. After the neoadjuvant therapy almost all tumors (92.7%) became resectable. The combinations of therapy types, the operations type and the surgical complications are analysed. Long term outcome proves, that multimodal therapy of NSCLC (in IIIA stage) is an effective treatment method.
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December 2004