Abstract
The EvA study is a European Union-funded project under the Seventh Framework Programme (FP7), which aims at defining new markers for chronic obstructive pulmonary disease (COPD) and its subtypes. The acronym is derived from emphysema versus airway disease, indicating that the project targets these two main phenotypes of the disease. The EvA study is based on the concept that emphysema and airway disease are governed by different pathophysiological processes, are driven by different genes and have differential gene expression in the lung. To define these genes, patients and non-COPD controls are recruited for clinical examination, lung function analysis and computed tomography (CT) of the lung. CT scans are used to define the phenotypes based on lung density and airway wall thickness. This is followed by bronchoscopy in order to obtain samples from the airways and the alveoli. These tissue samples, along with blood samples, are then subjected to genome-wide expression and association analysis and markers linked to the phenotypes are identified. The population of the EvA study is different from other COPD study populations, since patients with current oral glucocorticoids, antibiotics and exacerbations or current smokers are excluded, such that the signals detected in the molecular analysis are due to the distinct inflammatory process of emphysema and airway disease in COPD.
Chronic obstructive pulmonary disease (COPD) is characterised by an airflow limitation that is not fully reversible [1]. COPD is an inflammatory disease that shows gradual progression over time and periods of acute deterioration (exacerbations) will lead to further decline in lung function [2]. Inflammatory changes occur in the proximal and distal airways and in the lung parenchyma where increased numbers of leukocytes such as neutrophils, macrophages and CD8 lymphocytes can be found [3]. These leukocytes and the mediators they release (e.g. cytokines and proteases) lead to tissue remodelling and destruction of airways and parenchyma [4].
The disease is caused by smoke inhalation either from cigarettes [5, 6] or from exposure to other noxious particles, such as those from open fires used for cooking or heating in poorly ventilated homes [7, 8]. In addition to these environmental triggers, the development of the disease is controlled by genes as suggested by the finding that only ∼20% of smokers succumb to COPD [9] and by the observation that the disease runs in families [10, 11].
In the quest to define the genes involved, large numbers of patients have been studied for the association of COPD with genetic markers as defined by single-nucleotide polymorphism (SNP) genotyping using blood leukocyte DNA. These studies have revealed association with several candidate loci such as chromosome 4q31 locus (HHIP), chromosome 4q22 locus (FAM13A) and chromosome 15q25 locus (CHRNA3/CHRNA5/IREB2), as summarised by Silverman et al. [12], but it remains unclear whether and how the respective genes encoded in these loci are involved in the pathophysiology of the disease.
One of the difficulties in identifying relevant markers associated with COPD is that COPD is not one entity but is a heterogeneous disease with different phenotypes. While fixed airway obstruction, as assessed by post-bronchodilator forced expiratory volume in 1 s (FEV1), is the hallmark of COPD, this airway obstruction can arise by different mechanisms. One is due to inflammatory destruction of the alveoli and elastic fibres leading to emphysema. Here the airways collapse because they are not properly embedded and are not kept suspended by the surrounding lung tissue. Another mechanism is more direct and is due to narrowing of the airway lumen because of inflammatory thickening of the airway walls, a process termed bronchitis or airway disease.
The EvA project is based on the concept that many patients with COPD show both emphysema and airway disease but others present with a predominance of either emphysema (E) versus airway disease (A). These phenotypes may have different clinical courses, they may respond differently to therapy and, importantly, they may be controlled by different genes. Therefore it is important to define emphysema and airway disease by robust technology.
Computed tomography (CT) of the lung has emerged as one such technology. The use of lung CT for assessment of emphysema was pioneered in the study by Hayhurst et al. [13]. Here the degree of emphysema is reflected in the reduction of lung density. Digital image analysis of whole-lung scans is now available to express this in Hounsfield units. However, the degree of airway disease is reflected in a thickening of the airway wall and this can be determined as a percentage of the airway wall area relative to the entire airway area from appropriate CT scans. It is the obstruction of the small airways (<2 mm internal diameter) that is relevant to the increased airway resistance in COPD [14, 15], but assessment of the dimensions for these small airways is beyond the resolution of CT.
However, Nakano et al. [16] demonstrated that the dimension of proximal airways as assessed by CT correlates with the dimensions of small airways as assessed in histology, indicating that small airway disease is reflected in the dimensions of the larger proximal airways. In addition, airway dimensions assessed by CT were shown to correlate with crucial lung function parameters such as FEV1 in several reports [17, 18]. Importantly, Nakano et al. [17] have demonstrated that CT image analysis for both airway dimensions and for lung density can be used for the description of COPD phenotypes.
This approach of CT phenotyping is currently being used in several large studies in order to discover markers associated with the emphysema and airway disease phenotypes [19, 20]. Genetic markers expressed by the crucial lung cells, i.e. the alveolar macrophages and bronchial epithelial cells, are, however, not being investigated in the above studies. The EvA study uses the CT approach for definition of the patient population and performs bronchoscopy in order to recover these cells by lavage and bronchial brush. Genes expressed in these tissues are then tested for association with the E and A phenotypes of COPD in a quest for new targets for diagnosis and therapy.
AIMS
The aim of the EvA project is to identify novel markers for COPD and its main phenotypes, i.e. emphysema and airway disease, by studying gene expression in bronchial and alveolar samples from patient lungs compared to matched controls.
CONSORTIUM STRUCTURE
The EvA study is a European Union-funded project which brings together 10 clinical centres in five European countries, four diagnostic centres including one small-to-medium-sized enterprise and the European Respiratory Society. The EvA-Coordinator is L. Ziegler-Heitbrock (Helmholtz-Zentrum Muenchen and Asklepios Hospital, Muenchen Gauting, Germany). The project is managed by a steering committee that reports to the entire consortium which takes final decisions at its annual meeting.
OVERALL DESIGN
Recruitment of cases and controls to the study was performed over a 3-yr period (February 2009 to March 2012). The study aimed to perform bronchoscopy in 300 controls and CT and bronchoscopy in 500 cases. Participants were invited to visit the clinical centres on three occasions ideally within a period of 4–6 weeks. The following investigations are performed. Visit one: detailed history, clinical examination, 6-min walking distance (6MWD) test, bioimpedance clinical laboratory, ECG and spirometry before and after bronchodilation. Visit two: CT scans. Visit three: bronchoscopy with brushing of the airways and bronchoalveolar lavage of the alveoli.
Gene expression in the two types of lung tissue, i.e. bronchial epithelial cells and alveolar macrophages, is then compared to CT-defined emphysema and airway disease phenotypes. The details of this study are given below.
Recruitment
Recruitment of cases and controls is initiated by using small newspaper advert campaigns. Candidates are preselected by telephone interview and detailed information on study protocol and consent forms sent by post. In addition, a multi-language webpage (www.eva-copd.eu/eva/english/) informs candidates about the details of the study. When invited to the study centre participants are reminded not to take their respiratory medication on that day. Recruitment to this type of study requires quite some effort since volunteers will be subjected to extensive investigations including radiation exposure, but most importantly they need to agree to undergo the invasive procedure of bronchoscopy. Also, we have to take into account that there will be a fraction of participants who decide late in the study not to proceed with bronchoscopy.
Initial examination at visit one
During visit one candidates willing to participate have the opportunity to discuss all aspects and informed consent is obtained in line with the Declaration of Helsinki and based on approval by the local ethics committees.
A clinical history, including smoking pack-years, is taken and Medical Research Council (MRC) dyspnoea score is determined. Lung function is performed using body plethysmography before and after maximal bronchodilation using four inhalations of 100 μg salbutamol each time. Lung function parameters that are assessed include: oxygen tension; carbon dioxide tension; transfer factor of the lung for carbon monoxide/alveolar volume (TL,CO/VA); total lung capacity (TLC); inspiratory capacity; residual volume; FEV1; forced vital capacity (FVC) before bronchodilation and FEV1; and FVC and resistance after bronchodilation. Lung function analysis will exclude many patients because of a strong asthmatic component (ΔFEV1 before and after bronchodilation >400 mL) or because their post-bronchodilator FEV1/FVC ratio is ≥0.7 such that they do not qualify as COPD. Also, stage IV cases and those with a FEV1 <1 L are excluded.
The history covers general medical aspects and details of smoking habits (cigarettes only, cigar and pipe smoking is ignored) and respiratory disease. Bioimpedance measurement is taken for body composition, 6MWD test is performed as part of the body mass index, airflow obstruction, dyspnoea and exercise capacity (BODE) score and after inhalation of isotonic saline solution-induced sputum is recovered.
A detailed clinical examination is performed and blood is taken for extensive clinical chemistry in order to document comorbidities and to identify diseases that might affect fitness to participate. ECG is performed to exclude any cardiac disease that might preclude bronchoscopy.
Exclusion criteria
While many other studies into COPD can include any stage of the disease, the EvA group is different because of the invasive bronchoscopy procedure, such that most severe forms of COPD with very low FEV1 and oxygen levels and patients at older ages are excluded from participation (table 1). Also, since the inflammatory process in the lung is studied in EvA we exclude current smokers in order to avoid smoke-induced inflammation. Patients with frequent exacerbations leading to hospitalisation are excluded as well, since the sequelae of such acute events may still impact on the inflammatory response at the time of bronchoscopy. With this approach we want to ensure that the markers, which we will identify in the COPD lung, are due to COPD and not to any other inflammatory process. We also excluded patients on oral glucocorticoids since these drugs may substantially reduce inflammation in the lung.
In order to focus on prototypic smoke-induced COPD we exclude patients with low smoke exposure (<5 pack-yrs), excessive smoke exposure (>80 pack-yrs) and young age. Also, other diseases that lead to lung destruction, such as α1-antitrypsin deficiency and immunoglobulin G deficiency, are excluded. Patients with a pronounced asthmatic component (>400 mL improvement of FEV1 post-bronchodilation) are excluded since there is the possibility that the fixed obstruction in an ex-smoker is not due to COPD but is the result of chronic asthma. For control donors the same exclusion criteria apply, but asthma is allowed for.
CT at visit two
CT scans on COPD patients and a limited number of healthy controls (n=30) will be done in 10 clinical centres using either General Electric (Sterling Heights, MI, USA) or Siemens (Erlangen, Germany) scanners. The settings and the recons are predefined such that there is minimal deviation among the different scanners.
The whole-lung EvA-E scans cover the entire lung from the diaphragm recesses to the apex. This scan is used for assessment of lung density. The limited EvA-A scan includes the region from 2 cm below the carina to the top of the aorta. The limited scan will be used to determine dimensions of third-generation airways like S1. All scans are performed at maximum inspiration with breath holding. The recording is taken from base to apex, which is important for the whole-lung scan in order to avoid motion artefacts at the diaphragm at the end of the 20-s breath holding. Before the procedure the patients inhale 4×100 μg of salbutamol using a spacer and after 15 min they are positioned in the scanner. A phantom containing three rods of different density is mounted to the chest in the sternum area.
The scans are recorded with a rotation time of 0.5 s, a tube voltage of 120 kV, and a tube current of 40 (EvA-E) or 50 mA (EvA-A); this dose is kept fixed for all patients [21]. The EvA-E protocol is optimal for density resolution at the expense of spatial resolution. Conversely, for the EvA-A protocol, pitch and speed are lower, which allows for a better spatial resolution for the airway analysis. This, however, goes along with a higher radiation dose. In order to keep the radiation exposure low the EvA-A scan covers a limited part of the lung (including the right S1 segmental bronchus), such that this protocol is available at a dose of 0.42 mSv (millisievert) only. The total radiation exposure for both scans is 2.1 mSv. Approval is obtained from the relevant radiation protection authorities.
CTs are inspected for clinical reporting by the clinical radiologist on duty. Patients with nodules >10 mm will not go forward for EvA bronchoscopy but will be referred for further diagnostics and therapy for potential malignancy. When these findings have been investigated then the patients can be re-entered into the EvA study. Cases with nodules <10 mm can proceed to EvA bronchoscopy and will be referred for further management thereafter.
The whole lung EvA-E scans are analysed for lung density using the PULMO CMS software (Medis Specials, Leiden, the Netherlands) and expressed as Perc15 (HU), which is the density in Hounsfield units of the lower 15th percentile of the lung. Additional readouts will be the percentage of lung tissue with different lung density cut-offs (e.g. -950 HU) and the different regions of the lung will be analysed. Correction of lung density for inspiratory level will be performed using TLC derived from body plethysmography based on the “sponge model” [22].
In the limited EvA-A scans the S1 bronchus is studied for airway wall area using Emphylx-J (University of British Columbia, Vancouver, BC, Canada). The right S1 runs almost perpendicular to the CT slices and therefore its inner and outer diameter can be assessed directly without adjustment for oblique angles. The area of the airway wall is then expressed as percentage of the entire airway area (WA %). In order to assess airways of any orientation three-dimensional software packages will be used to study volumetric airway wall dimensions for a series of airways down to the 6th generation.
For normalisation, a set of phantoms, which provide different densities and different airway wall areas, is used. The Warwick phantom was put together at the University of Coventry and Warwickshire, Coventry, UK, and consists of nine cores of polyurethane foam with different degrees of expansion held in a solid water frame. The airway phantom was generated at the University of Leicester, Leicester, UK, and contains nine teflon tubings of different sizes in a polystyrene block. A commercial phantom (CTP674, Catphan®; The Phantom Laboratory, Salem, NY, USA) also contains tubings of different dimensions. These three phantoms, plus the density rods mentioned above, are scanned with the EvA-E and EvA-A protocols on all 10 scanners and the readings are used to determine correction factors for both Perc15 HU and WA %.
The values Perc15 HU and WA % will be used to define patients with pronounced emphysema and little or no airway wall thickening (emphysema dominant) and patients with little or no emphysema and pronounced airway wall thickening (airway disease dominant) according to the approach taken by Nakano et al. [17] with the cut-off values to be defined based on the distribution of the values. The definition of the phenotypes will be assisted by testing additional relevant parameters such as TL,CO/VA. The phenotypes derived from the CT scan analysis will be analysed for associations with the DNA, RNA and protein markers generated within the EvA study.
Bronchoscopy at visit three
For bronchoscopy, patients are asked not to use inhaled drugs on the day. The patients are placed in a semi-recumbent position and receive a mild sedation via venous access. The flexible endoscope is inserted via a nostril and the airways are inspected. Brush and lavage samples are taken from defined areas of the lung as shown in figure 1. In order to ensure a uniform approach by the bronchoscopists a video of the procedure is produced and can be viewed by all partners on the EvA data centre platform.
Schematic representation of the airways with the relevant segmental bronchi numbered. For the right lung the arrows show the sites for brushing. For the left lung the ring indicates the preferred segment for lavage. BAL: bronchoalveolar lavage.
Brushing is performed in the right lung with sheathed brushes with a diameter of 5 mm at bristle level (#BC-202D 5010; Olympus, Hamburg, Germany). After three strokes the brush is rotated in the airway five times and then the brush is resheathed and recovered. Three brushings are taken from the subsegments of S1 in the right upper lobe and are pooled as one sample and three brushings from the right lower lobe from subsegments of S8, S9 and/or S10 are pooled as one sample. The brushes are transferred into RNAprotect (#76526; Qiagen, Hilden, Germany) immediately. The lavage is performed in the left upper lobe or lingula with a total volume of 150 mL of sterile, pyrogen-free, 37 °C saline, which is instilled in 20 mL fractions typically into the left S1 segment with aspiration of the volume with 20 mL syringes. The lavage fluid is spun immediately and the cells are counted and transferred into RNAprotect. Patients are monitored until completely recovered and are discharged after medical examination.
The approach taken allows for an outpatient bronchoscopy since there is no general anaesthesia and the volume for lavage is very low, thereby reducing any impairment of gas exchange. At the same time it will provide two types of lung samples, i.e. bronchial epithelial cells and bronchoalveolar lavage that is composed predominantly of alveolar macrophages.
DATA STORAGE AND QUALITY CONTROL
Data are all entered into a web-based central storage, based on a Cold Fusion/Java Interface system that is linked to a H2 relational database and all entries are logged in separate files. Data management includes plausibility, and range checks and protocol violations are flagged. Time-stamped data backup as well as a mirror copy of the whole system is run every night onto a second server. Data access is restricted to users identified by password while the database holds only unique IDs for the study probands.
MARKER ANALYSIS
The coded cell samples from blood and from the lung are stored at -20°C and batches are processed for DNA and RNA isolation. Quality will be assessed based on amount and on integrity of RNA of the samples. Using state-of-the-art technology, the transcriptome will be determined with the aim of identifying transcripts that show a differential expression between the COPD phenotypes; DNA is used for SNP analysis and for whole-genome sequencing in order to assess the number and nature of acquired mutations. Also, SNPs will be tested for association with gene expression data. Fluids are analysed for proteins encoded by genes that show differential expression between COPD and controls and between the phenotypes of COPD. In addition, the candidate proteins eosinophil cationic protein, eosinophil protein X and myeloperoxidase, human neutrophil lipocalin, lysozyme, human phospholipase B-II and sCEACAM8, as well as neutrophil α-defensins and the cathelicidin hCAP18/LL-37 are determined in the fluids from blood and the lung.
Markers determined at the DNA, mRNA and protein level are then tested for association with COPD and with the COPD phenotypes E and A. For the final data analysis a SQL export is being prepared and imported into a R statistical analysis system where further datasets from DNA and RNA sequencing are being merged. R 2.12 is run under R Studio 09.4 including numerous libraries including R Bioconductor. For the main analysis we will perform a final classification of COPD cases and controls while for the main COPD subtype analysis emphysema cases will be tested against airway disease cases. Following baseline characteristics of cases and controls we will compute crude odds ratios for different categories of risk factors from these two 2×2 tables, while for an advanced analysis of indicators of previous disease we will use multivariate linear and logistic regression models, also taking into account confounders like current treatment. More explorative analysis of bronchial and alveolar gene expression will use principal component analysis and other clustering methods to define unique profiles and biological pathways that are relevant to COPD subtypes.
DISCUSSION
Many studies into the genetics of COPD have, in the past, investigated the disease as one entity such that genes linked to subphenotypes have evaded detection. Therefore, in the EvA study we define the COPD phenotypes emphysema and airway disease using CT image analysis and search for markers associated with these, as is being done in other ongoing studies into COPD [18, 19]. In addition to looking at associations with DNA markers in blood we take samples from alveoli and airways in order to study the transcriptome in alveolar macrophages and bronchial epithelial cells, respectively.
Recruitment to this type of study is obviously a challenge because of the invasive procedure, such that patients and controls who are willing to participate in a COPD study are reluctant to be included when it comes to bronchoscopy. Therefore, for an invasive diagnostic study like EvA it is difficult to predict whether a target for numbers of bronchoscopy can really be achieved.
The population of the EvA study is unique since there are several exclusion criteria, which are meant to: 1) focus on bona fide COPD; 2) avoid effects of current smoking and immunosuppression on gene expression; and 3) prevent any harm to participants during bronchoscopy. The latter involves excluding patients with stage IV COPD, FEV1 <1 L and long-term oxygen therapy, since very low FEV1 and respiratory failure carry a higher risk for a decrease in blood oxygen levels during bronchoscopy, and inclusion of such patients cannot be ethically justified when there is no medical indication for the procedure. In addition, comorbidities such as symptomatic coronary artery disease, arrhythmias (e.g. atrial fibrillation), uncontrolled hypertension and severe liver and kidney diseases lead to exclusion.
Current smoking would introduce many artefacts including inflammation in the airways. Also, active smokers have increased numbers of alveolar macrophages that are loaded with tar and have impaired function [23]. When we want to study genes that are regulated by the emphysema or airway disease processes in COPD, the interference by current cigarette smoking will very likely blur the pattern. Therefore all study participants have to be ex-smokers for more than a year.
It may be argued that exclusion of late-stage patients with severe disease, i.e. low FEV1 and requirement of oxygen therapy, does not give the full account of the COPD spectrum. While patients with a rapid progression of disease have been discussed to be a separate phenotype, many stage IV patients have started with mild disease and have gradually progressed. Hence they are COPD patients that are studied at a later point in time. Analysis of the transcriptome in stage IV patients may even be misleading in that hypoxia, the restricted mobility and the use of oxygen therapy may impact on gene expression and mimic a unique phenotype.
The unique composition of the patient population will always have to be taken into account when data obtained under EvA is compared to other studies which allow for factors that modulate inflammation and which use a noninvasive approach. Therefore, findings of the EvA study will have to be confirmed for the entire spectrum of COPD including stage IV patients, current smokers and those aged >75 yrs.
A problem with the selection of appropriate patients is the differentiation of COPD from asthma. Fabbri et al. [24] observed that the fixed airway obstruction in adults with a history of asthma can be very similar to those seen in patients with a history of COPD. A population of ex-smokers with a fixed airway obstruction may contain cases that have always been told they have COPD because they were smokers. Such cases may be identifiable based on high eosinophil count in sputum [22] and this parameter will be used for post hoc exclusion in the EvA study.
One challenge of the project is that scans obtained with different scanners (General Electric and Siemens) and different models are used by the EvA partners and it is important to standardise these with the use of phantoms. For this, standardisation against air, standard density rods and a series of newly generated phantoms are used. We expect this strategy to substantially improve data interpretation in this multicentre study. For determination of the degree of emphysema and airway wall thickening the study protocol calls for two different dedicated CT scans. While the scan for airway wall area provides a higher resolution, it is also possible to determine dimensions of multiple airways using the lower resolution scans of the whole lung, i.e. this scan might be used for assessment of both emphysema and airway disease.
The bronchoscopy will be performed with a flexible instrument and with mild sedation such that participants need not stay in hospital overnight. This is a safe routine procedure; however, it carries, as with any invasive procedure, the risk of infection and bleeding plus, as a rare event, pneumothorax. Therefore, not only ethics approval but also insurance cover is required for the study. The brushing to obtain airway epithelial cells will target the right lung and here specifically the S1 bronchus with its subsegments (fig. 1). This type of sample is expected to be highly informative since it is taken from the very site for which we determine the airway wall thickness in CT image analysis. It will be exciting to correlate the thickness of the airways in this area with the gene expression by the epithelial cells obtained from S1.
The EvA study is designed such that samples from the lung are collected over a 3-yr period. Only when the collection is complete will the molecular analysis be done in large batches. The specific platform and the depth of analysis will be decided at the time based on the technology available.
The EvA study is taking approaches that are distinct to other ongoing studies. ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points) is a longitudinal study in a group of over 2,000 patients that aims to identify markers and parameters that correlate with clinically relevant COPD subtypes, including CT phenotypes, and predict disease progression [19]. Biomarkers are assessed in blood, urine, sputum and exhaled breath. In comparison, the EvA study with 500 patients is cross-sectional and, in addition to sampling of blood and sputum, a bronchoscopy is performed and airway brush samples and bronchoalveolar lavage samples are recovered. Therefore, the EvA study is closer to the main organ affected by COPD and it aims at identifying markers expressed in the lung tissue at the DNA, RNA and protein level.
COPDGene (Genetic epidemiology of COPD) is a genome-wide association study aiming for the participation of 10,000 patients [20]. Similar to EvA it will determine the degree of emphysema and airway wall thickening using CT scans. DNA extracted from blood is then used to run SNP arrays and associations are determined with parameters of emphysema and airway wall thickening taken from the CT scan analysis. By contrast, in the EvA study, association with SNPs is only a secondary aim and will be restricted to confirmation of known associations. COPDGene is restricted to analysis of blood while EvA focuses on gene expression in the lung. The number of patients enrolled into EvA is low compared to the previously mentioned studies. This may be counterbalanced by the stringent exclusion criteria (table 1), which result in a more clearly defined patient population with, for instance, little contamination by asthma cases. In addition, power calculations have shown that the study is sufficiently powered to detect a 20% difference between E and A (two-tailed at 5% α-error is 94.6%).
Taken together EvA is a Europe-wide case–control study into COPD that aims at defining COPD phenotypes by CT image analysis and which puts an emphasis on the analysis of gene expression in tissue samples taken from the lung. We expect this approach to provide exciting new data on novel markers that can be used for diagnosis of and as target for therapy of COPD phenotypes.
Acknowledgments
We are indebted to the European Respiratory Society for their support in launching the project.
Author affiliations are as follows. L. Ziegler-Heitbrock, I. Heimbeck and D. Burggraf: EvA Study Center, Helmholtz Zentrum Muenchen, Gauting, Germany. M. Frankenberger: Comprehensive Pneumonology Center, Helmholtz Zentrum Muenchen, Muenchen, Germany. M. Wjst: Institute for Lung Biology and Disease, Helmholtz Zentrum Muenchen, Muenchen, Germany. K. Häussinger: Asklepios Hospital Muenchen-Gauting, Gauting, Germany. C. Brightling and S. Gupta: Institute of Lung Health, University of Leicester, Leicester, UK. D. Parr and D Subramanian: Dept of Respiratory Medicine, University Hospitals of Coventry and Warwickshire, Coventry, UK. D. Singh and U. Kolsum: University of Manchester, Medicines Evaluation Unit and University Hospital of South Manchester Foundation Trust, Manchester, UK. P. Boschetto and A. Potena: Dept of Clinical and Experimental Medicine, University of Ferrara, Ferrara, Italy. D. Gorecka and A. Nowinski: Second Dept of Respiratory Medicine, National Tuberculosis and Lung Diseases Research Institute, Warsaw, Poland. I. Barta: Dept of Pathophysiology, National Koranyi Institute for Pulmonology, Budapest, Hungary. B. Döme: Dept of Tumour Biology, National Koranyi Institute for Pulmonology, Budapest, Hungary. J. Strausz: National Koranyi Institute for Pulmonology Budapest, Hungary, and Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria. T. Greulich and C. Vogelmeier: University Hospital of Giessen and Marburg, Dept of Respiratory Medicine, Marburg, Germany. R. Bals: Division of Pulmonary Diseases, University Hospitals Saarland, Homburg, Germany. J.M. Hohlfeld: Dept of Clinical Airway Research, Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany. T. Welte: Dept of Respiratory Medicine, Hannover Medical School, Hannover, Germany. P. Venge: Dept of Medical Sciences, Uppsala University, Uppsala, Sweden. I. Gut: Centre Nacional D'Anàlisi Genòmica, Parc Cientific de Barcelona, Barcelona, Spain. A. Boland, R. Olaso and J Hager: CEA Genomics Institute, National Genotyping Center, Evry, France. P. Hiemstra: Dept of Pulmonology, Leiden University Medical Center, Leiden, the Netherlands. K.F. Rabe: Center for Pulmonology and Thoracic Surgery, University Kiel and Krankenhaus Grosshansdorf, Grosshansdorf, Germany. M. Unmüßig, J. Müller-Quernheim and A. Prasse: Dept of Pneumology, University Medical Center Freiburg, Freiburg, Germany.
Footnotes
Support Statement
The EvA study is an EU-funded project (# 200506) under the Seventh Framework Programme (FP7).
Statement of Interest
Statements of interest for K. Ha¨ussinger, C. Brightling, D. Parr, D. Singh, J.M. Hohlfeld, P. Venge, P. Hiemstra, K.F. Rabe and A. Prasse can be found at www.erj.ersjournals.com/site/misc/statements.xhtml
- Received August 19, 2011.
- Accepted December 23, 2011.
- ©ERS 2012
