Systemic Soluble Receptor for Advanced Glycation ... - ATS Journals

Systemic Soluble Receptor for Advanced Glycation Endproducts Is a Biomarker of Emphysema and Associated with AGER Genetic Variants in Patients with Chronic Obstructive Pulmonary Disease Donavan T. Cheng1,2*, Deog Kyeom Kim3,4*, Debra A. Cockayne1,5, Anton Belousov6, Hans Bitter1,7, Michael H. Cho4, Annelyse Duvoix8,9, Lisa D. Edwards10, David A. Lomas8,9, Bruce E. Miller11, Niki Reynaert12, Ruth Tal-Singer11, Emiel F. M. Wouters12, Alvar Agustı´13, Leonardo M. Fabbri14, Alex Rames15, Sudha Visvanathan1,16, Stephen I. Rennard17, Paul Jones18, Harsukh Parmar1,19, William MacNee20, Gerhard Wolff1,21, Edwin K. Silverman4, Ruth J. Mayer11y, and Sreekumar G. Pillai1yz; on behalf of the TESRA and ECLIPSE Investigators 1

Hoffmann-La Roche Inc., Nutley, New Jersey; 2Memorial Sloan-Kettering Cancer, New York, New York; 3Division of Pulmonary and Critical Care Medicine, Seoul National University Boramae Medical Center, Seoul, South Korea; 4Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; 5Boston Scientific, San Jose, California; 6Hoffmann-La Roche, Penzberg, Germany; 7 Novartis Institutes for Biomedical Research, Cambridge, Massachusetts; 8Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, United Kingdom; 9Division of Medicine, Faculty of Medical Sciences University College London, London, United Kingdom; 10GlaxoSmithKline Research and Development, Research Triangle Park, North Carolina; 11GlaxoSmithKline Research and Development, King of Prussia, Pennsylvania; 12Department of Respiratory Medicine, Maastricht University Medical Centre, Maastricht, the Netherlands; 13Institut del To`rax, Hospital Clı´nic, IDIBAPS, Universitat de Barcelona, Barcelona, Spain; 14Department of Respiratory Diseases, University of Modena and Reggio Emilia, Modena, Italy; 15Hoffmann-La Roche, Basel, Switzerland; 16Boehringer Ingelheim, Ridgefield, Connecticut; 17University of Nebraska Medical Center, Omaha, Nebraska; 18St George’s, University of London, London, United Kingdom; 19EMD Serono, Billerica, Massachusetts; 20 Queen’s Medical Research Institute, Edinburgh, United Kingdom; and 21Mitsubishi Tanabe, Warren, New Jersey

Rationale: Emphysema in chronic obstructive pulmonary disease (COPD) can be characterized by high-resolution chest computed tomography (HRCT); however, the repeated use of HRCT is limited because of concerns regarding radiation exposure and cost. Objectives: To evaluate biomarkers associated with emphysema and COPD-related clinical characteristics, and to assess the relationships of soluble receptor for advanced glycation endproducts (sRAGE), a candidate systemic biomarker identified in this study, with single-nucleotide polymorphisms (SNPs) in the gene coding for RAGE (AGER locus) and with clinical characteristics. Methods: Circulating levels of 111 biomarkers were analyzed for association with clinical characteristics in 410 patients with COPD enrolled in the TESRA study. sRAGE was also measured in the ECLIPSE cohort in 1,847 patients with COPD, 298 smokers and 204 nonsmokers. The association between 21 SNPs in the AGER locus with sRAGE levels and clinical characteristics was also investigated. Measurements and Main Results: sRAGE was identified as a biomarker of diffusing capacity of carbon monoxide and lung density in the TESRA cohort. In the ECLIPSE cohort, lower sRAGE levels were associated with increased emphysema, increased Global Initiative for Chronic Obstructive Lung Disease stage, and COPD disease status. The associations with emphysema in both cohorts remained significant after covariate adjustment (P , 0.0001). One SNP in the AGER

(Received in original form February 27, 2013; accepted in final form July 23, 2013) * These authors contributed equally. y z

These authors contributed equally. Current address: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN.

The ECLIPSE study was supported by GlaxoSmithKline and the TESRA study was supported by Roche Pharmaceuticals. Correspondence and requests for reprints should be addressed to Sreekumar G. Pillai, Ph.D., Eli Lilly and Company, Lilly Corporate Center, DC1522, Indianapolis, IN 46285. E-mail: [email protected]

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

Patients with chronic obstructive pulmonary disease often have a combination of emphysema and small airway disease, both of which make independent contributions to airflow obstruction. Emphysema can be characterized by chest computed tomography, but no easily measureable biomarker for emphysema is available. What This Study Adds to the Field

The current study using two independent large cohorts clearly establishes systemic soluble receptor for advanced glycation endproducts as a biomarker for chronic obstructive pulmonary disease that is specifically associated with emphysema. The study also demonstrates that genetic polymorphisms in the AGER locus were associated with soluble receptor for advanced glycation endproducts levels in patients with chronic obstructive pulmonary disease.

locus, rs2070600, was associated with circulating sRAGE levels both in TESRA (P ¼ 0.0014) and ECLIPSE (7.07 3 10216), which exceeded genome-wide significance threshold. Another SNP (rs2071288) was also associated with sRAGE levels (P ¼ 0.01) and diffusing capacity of carbon monoxide (P ¼ 0.01) in the TESRA study. Conclusions: Lower circulating sRAGE levels are associated with emphysema severity and genetic polymorphisms in the AGER locus are associated with systemic sRAGE levels. Clinical trial registered with www.clinicaltrials.gov (NCT 00413205 and NCT 00292552). Keywords: single-nucleotide polymorphism; lung density; DLCO

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 188, Iss. 8, pp 948–957, Oct 15, 2013 Copyright ª 2013 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201302-0247OC on August 15, 2013 Internet address: www.atsjournals.org

Chronic obstructive pulmonary disease (COPD) is a disorder with great phenotypic heterogeneity, characterized by persistent airflow limitation and chronic airway inflammation. Patients with

Cheng, Kim, Cockayne, et al.: AGER Variants Influence the COPD Biomarker sRAGE

COPD often have a combination of emphysema and small airway disease, both of which make independent contributions to airflow limitation. Emphysema is characterized by permanent expansion of airspaces distal to the terminal bronchioles and can be characterized by high-resolution computed tomography (HRCT) of the chest. Emphysema has been shown to be associated with increased mortality and risk of lung cancer (1–4) in patients with COPD. However, the use of HRCT in routine clinical practice and in clinical trials is limited because of concerns regarding radiation exposure and cost of measurement. Consequently, identifying systemic biomarkers for emphysema could be of significant value in the diagnosis and management of patients with COPD. The receptor for advanced glycation endproducts (RAGE) is a member of the immunoglobulin superfamily that is highly expressed in normal human lung (5). It senses hyperglycemia, hypoxia, and oxidative stress by binding a wide range of ligands that are considered to be damage-associated molecular patterns including nonenzymatically glycated adducts (advanced glycation endproducts); b-amyloid fibrils; proinflammatory mediators of the S100/calgranulin family; and amphoterin or high-mobility group box 1 (HMGB1), a nuclear protein found in the extracellular matrix (6). Soluble RAGE (sRAGE) is generated either as a splice variant of AGER or by proteolysis of the receptor from the cell surface, and can act as a decoy receptor that binds to RAGE ligands and prevents signaling at the cell surface receptor (7). The RAGE pathway has been shown to be associated with several inflammatory diseases including arthritis, diabetes mellitus, cardiovascular, peripheral vascular, and respiratory diseases (8–14). More specifically, sRAGE protein levels were significantly lower in COPD relative to control subjects in recent reports (15–18). These studies reported an association between sRAGE levels and lung function in patients with COPD. The AGER (Human Genome Organization approved name for RAGE) locus has in turn been associated with lung function in population studies (19, 20), but there has been no reported investigation of the relationship of AGER variants with circulating sRAGE levels in COPD.

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This study sought to evaluate biomarkers associated with emphysema and other COPD-related clinical characteristics; and assess the relationships of sRAGE, a candidate systemic biomarker identified in this study, with clinical characteristics and with single-nucleotide polymorphisms (SNPs) in the gene coding for RAGE (AGER locus). To achieve these goals we used two large and well-characterized cohorts of patients with COPD recruited in the TESRA (Treatment of Emphysema with a Selective Retinoid Agonist) and ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) studies.

METHODS Patients TESRA. Patients (n ¼ 410 at baseline) from a Phase 2, double-blind, placebo-controlled, randomized, multicenter study, assessing the safety and efficacy of 5 mg/day palovarotene were included in this analysis. The study design and clinical outcomes are reported elsewhere (21) and briefly summarized in the online supplement. Emphysema was assessed by low-dose spiral CT and single breath diffusing capacity of carbon monoxide (DLCO) (22). ECLIPSE. ECLIPSE is a longitudinal observational study conducted at 46 clinical centers in 12 countries. All consented subjects with COPD underwent spirometry and a low-dose CT scan of the chest. The evaluation of the outcome measures is described elsewhere (23). In this analysis, 2,349 subjects with available serum sRAGE levels were included (1,847 COPD subjects with Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 2 or greater, 298 smoking control subjects, and 204 nonsmoking control subjects).

HRCT Analyses TESRA. Two CT scans were performed at each visit, one scan after a breath-hold at TLC followed immediately by a second scan at FRC. Volume-adjusted 15th percentile of the lung density histogram (LP15A) was computed as per published methods (24). See the online supplement for more details.

TABLE 1. SUBJECT CHARACTERISTICS IN THE TESRA AND ECLIPSE STUDY POPULATIONS ECLIPSE (n ¼ 2,349) TESRA (n ¼ 410) COPD Cases

COPD Cases (n ¼ 1,847)

Smoking Control Subjects (n ¼ 298)

Nonsmoking Control Subjects (n ¼ 204)

Age, yr 66.7 6 8.1 (61.0 to 72.0) 63.6 6 6.9 (59 to 69) 55.0 6 8.8 (48 to 61) 53.8 6 8.9 (46.5 to 60.5) Males 273 (66.6%) 1211 (65.6%) 168 (56.4%) 74 (36.3%) Body mass index 26.0 6 5.1 (23.2 to 28.4) 26.5 6 5.6 (22.7 to 29.4) 26.8 6 4.6 (23.8 to 29.0) 27.3 6 5.4 (23.7 to 29.9) Height, cm 169.6 6 9.1 (163.0 to 176.0) 169.6 6 9.0 (163 to 176) 172.0 6 9.2 (165 to 178) 168.2 6 9.2 (162 to 175) Pack-years 47.7 6 25.2 (30.0 to 60.0) 48.4 6 27.1 (30 to 58) 31.3 6 21.8 (18 to 38) 0.0 6 0.0 GOLD stage II 183 (44.6%) 844 (45.7%) — — III 225 (54.9%) 764 (41.4%) — — IV 2 (0.5%) 239 (12.9%) — — FEV1, ml 1324.5 6 365.9 (1032.5 to 1575.0) 1242.5 6 487.8 (858.0 to 1548.0) 3212.3 6 733.9 (2685 to 3766) 3247.0 6 775.5 (2,643 to 3,716) FEV1 % predicted 49.1 6 9.1 (42.1 to 55.9) 48.9 6 15.7 (36.5 to 61.8) 108.4 6 11.7 (100.1 to 116.0) 116.0 6 13.8 (105.0 to 124.9) post-bronchodilation 0.43 6 0.08 (0.37 to 0.48) 0.45 6 0.12 (0.36 to 0.54) 0.79 6 0.05 (0.76 to 0.82) 0.81 6 0.05 (0.77 to 0.85) FEV1/FVC ratio DLCO (ml/min/mm Hg) 12.1 6 3.9 (9.0 to 14.7) N/A N/A N/A 48.5 6 13.1 (38.8 to 59.2) N/A N/A N/A DLCO %predicted post-bronchodilation LP15A: TLC lung density 2937.0 6 28.5 (2958.9 to 2919.9) P15 (HU) (TESRA)* FracVol less than 2950 17.5 6 12.1 (7.9 to 25.1)* 2.5 6 3.2 (0.4 to 3.2)* 4.0 6 4.2 (1.2 to 5.1)* HU (ECLIPSE)*

Definition of abbreviations: DLCO ¼ diffusing capacity of carbon monoxide; GOLD ¼ Global Initiative for Chronic Obstructive Lung Disease. Values indicate mean 6 SD (25th–75th percentile). * LP15A: TLC lung density P15 (HU) (TESRA)/FracVol less than 2950 HU (ECLIPSE). Note that higher LP15A is associated with less severe emphysema, whereas higher FracVol less than 2950 is associated with more severe emphysema.

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ECLIPSE. Subjects underwent low-dose volumetric CT scan (120 kV peak, 40 mA, and 1.00- or 1.25-mm slice thickness) at full inspiration. The extent of radiographic emphysema was measured in two ways. First, each CT scan was reviewed by two radiologists and assigned an ordinal score. Second, the extent of emphysema was assessed using density mask analysis, using the percentage of lung voxels with attenuation less than 2950 HU (FracVol , 2950) (25). Note that higher LP15A is associated with less severe emphysema, whereas higher FracVol less than 2950 is associated with more severe emphysema (directionality of these two methods of representing emphysema severity are opposite).

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Index (modified Medical Research Council scale). A multivariate model was used to test COPD-related clinical characteristics significant in univariate analysis for colinearity in contribution to log10 sRAGE levels (age, sex, and BMI included as covariates). Association analyses of SNPs with plasma sRAGE and clinical outcomes were performed using allelic and genotypic tests using PLINK (26). Only white subjects were analyzed in TESRA and ECLIPSE data were adjusted using the principal components from the GWAS data to account for population stratification. Regional association of an interesting index SNP with remaining SNPs was plotted using LocusZoom (27). Displayed linkage disequilibrium estimates were obtained from HapMap phase 2 (whites).

Sample Collection and Analysis TESRA. Concentrations of 111 protein biomarkers in ethylenediaminetetraacetic acid plasma were measured (in duplicate) at Rules Based Medicine (Austin, TX) and Quest Diagnostics (Valencia, CA). The selection of biomarkers was based on the relationship to the disease pathology and availability of sensitive measurement methods. A list of all the biomarkers measured and their characteristics are provided in Table E1 in the online supplement. ECLIPSE. sRAGE levels in the serum samples were assessed at Visit 4 (12 mo after recruitment to the study) in all of the ECLIPSE samples using the Quantikine human RAGE ELISA kit (R&D Systems, Minneapolis, MN) with an antibody that detects the extracellular domain of RAGE. Reproducibility of the sRAGE measurement was demonstrated by remeasuring the levels in a subgroup of 189 individuals with COPD 18 months after the initial assessment. The correlation coefficient between the two measures was 0.86, with evenly distributed variability.

Analysis of SNPs TESRA. Twenty-one SNPs (Illumina Omni-1M) in the AGER locus (5kb upstream/downstream of transcription start site/39UTR) were selected for genetic association analysis. Analysis was performed on a subset of 335 white subjects; age, sex, and body mass index (BMI) were included as covariates in the model. ECLIPSE. Genetic analyses were done in two steps. (1) Chromosome 6 analyses: All genotyped SNPs in ECLIPSE or imputed SNPs using HapMap2 from chromosome 6 were analyzed for association with sRAGE levels. Twenty genome-wide significant SNPs with P value less than 5.0 3 1028 were identified. Four of these SNPs had directly genotyped data from the ECLIPSE samples (Illumina HAPMAP 550) and these were also tested for association with clinical parameters. (2) AGER locus analysis: Among the 20 genome-wide significant SNPs, two genotyped SNPs (rs2070600 and rs204993) and one imputed SNP (rs2022059) overlapped with the SNPs in the AGER locus tested in TESRA.

Data Analysis Proteins with significant univariate associations with baseline clinical characteristics in TESRA were identified using a linear model, adjusting for age, sex, and BMI as covariates (P value for association , 0.05, corrected for multiple testing [Benjamini-Hochberg]). Five-fold crossvalidation was performed using 10 random seeds for a total of 50 runs; to ensure the comparability of the test and validation data, stratification for sex and clinical site was applied. Analytes selected in at least 80% of the 50 runs constituted the final multivariate predictor set for each clinical disease score. Log10 sRAGE levels were tested for univariate association with other COPD-related clinical characteristics in TESRA and ECLIPSE cohorts by Pearson correlation. Analysis was performed using R version 2.13.2 (TESRA, http://www.R-project.org) and SAS 9.2 (ECLIPSE, SAS Inc., Cary, NC). The contribution of log10 sRAGE levels in explaining variability in COPD-related clinical characteristics was assessed using linear regression model, where age, sex, and BMI were included as covariates. The contribution of sRAGE levels was measured in terms of the difference in R2 between models including covariates only and models including covariates with sRAGE. The following COPD-related clinical characteristics were tested: FEV1 (milliliters); FEV1 % predicted; FEV1/FVC; DLCO % predicted (TESRA only); LP15A (TESRA)/FracVol less than 2950 (ECLIPSE); square root of airway wall area of a hypothetical 10-mm internal perimeter airway (Pi10, ECLIPSE only); 6-minutewalk distance; St. Georges Respiratory Questionnaire; and Dyspnea

RESULTS The baseline demographic characteristics of the two cohorts, TESRA and ECLIPSE, are shown in Table 1. The TESRA cohort consisted of moderate to severe COPD (GOLD 2–4) patients with CT-defined emphysema, whereas the ECLIPSE cohort included patients with COPD (GOLD 2–4) and smoking control subjects and nonsmoking control subjects. Clinical characteristics of the subjects in different GOLD categories are shown in Table E2. sRAGE Levels Are Associated with Emphysema Measures in Patients with COPD

A total of 111 plasma protein biomarkers were measured at baseline in TESRA. Univariate regression was used to determine the biomarkers significantly associated with baseline lung density values as measured by LP15A (P , 0.05; false discovery rate (FDR)-corrected for multiple testing) or with DLCO % predicted (Hb corrected) (Table 2). Nine biomarkers were significantly associated with DLCO; of these biomarkers, sRAGE showed the strongest association (R2 ¼ 0.057; P , 0.001; FDR-corrected). In both TABLE 2. UNIVARIATE ASSOCIATIONS OF BIOMARKER LEVELS WITH BASELINE LUNG DENSITY (LP15A) AND DLCO IN THE TESRA COHORT All Data Analyte LP15A sRAGE ICAM1 EGFR IL2RA IL1A IL7 IL16 IL3 MIP3A TS1 IL2 DLCO sRAGE MIP1A MMP10 TS1 EGFR MDC IL12P40 APOA1 Fibrinogen

R

2

Training

P FDR

R

0.079 0.055 0.035 0.036 0.033 0.03 0.025 0.025 0.026 0.025 0.023

,0.001 ,0.001 0.005 0.005 0.009 0.01 0.02 0.02 0.02 0.02 0.027

0.057 0.036 0.034 0.032 0.03 0.024 0.023 0.02 0.02

,0.001 0.004 0.005 0.007 0.008 0.03 0.03 0.04 0.04

2

Test 2

P FDR

R

P FDR

0.057 0.047 0.028 0.022 0.038 0.035 0.02 0.022 0.023 0.031 0.029

0.03 0.07 0.22 0.28 0.12 0.14 0.31 0.28 0.27 0.19 0.19

0.056 0.043 0.03 0.024 0.039 0.035 0.015 0.018 0.024 0.034 0.03

0.04 0.09 0.21 0.26 0.12 0.14 0.43 0.34 0.27 0.17 0.19

0.057 0.038 0.031 0.034 0.028 0.026 0.025 0.022 0.024

0.03 0.11 0.16 0.14 0.2 0.23 0.23 0.29 0.25

0.061 0.038 0.039 0.035 0.037 0.025 0.026 0.023 0.02

0.02 0.11 0.1 0.13 0.11 0.24 0.22 0.25 0.31

Definition of abbreviations: DLCO ¼ diffusing capacity of carbon monoxide; FDR ¼ false discovery rate. P values corrected for multiple testing using Benjamini-Hochberg procedure. The robustness of the identified associations was assessed by performing stratified fivefold cross-validation 50 times. Briefly, within each cross-validation run and for each analyte, data from 80% of the subjects (training subset) were used to train a linear model associating LP15A with the analyte; the trained model was then used to predict LP15A values in the test subset.


the lung density analysis and the DLCO analysis, only sRAGE showed significant association in the test sets (P , 0.05; FDRcorrected) by cross-validation. The association of sRAGE levels with lung density was evaluated in both TESRA and ECLIPSE. In both cohorts, subjects were grouped based on sRAGE quartiles and lung density distributions were compared across quartiles. In TESRA, the most severe emphysema (lowest volume-adjusted lung density) was in the bottom log10 sRAGE quartile (lower quartile, 2946.42; upper quartile, 2928.17; P , 0.001) (Figure 1A). Moreover, the lowest sRAGE quartile also had the lowest DLCO % predicted (lower quartile, 45.1%; upper quartile, 52.1%; P , 0.001) (Figure 1B). In ECLIPSE, the most severe emphysema (highest FracVol , 2950) was similarly found in the lowest log10 sRAGE quartile: lower quartile, 22.5 6 13.0; upper quartile, 13.06 6 9.9; P , 0.05) (Figure 1C). Radiologist’s score of emphysema was also available in ECLIPSE and subjects with the most severe emphysema on radiologist’s assessment (.50% emphysema) had lower levels of sRAGE compared with those with mild/trivial emphysema less than 5% emphysema (1136.3 6 523.2 pg/ml for subjects with severe emphysema vs. 1485.6 6 650.5 pg/ml for subjects with mild/trivial emphysema; P , 0.001) (Figure 1D).

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sRAGE Levels Are Associated with COPD and COPD Severity

Data from ECLIPSE were analyzed to evaluate the association between sRAGE levels and COPD affection status and severity. sRAGE levels were significantly lower in patients with COPD compared with smoking and nonsmoking control subjects (sRAGE, 1351.1 6 627.3 pg/ml for COPD cases; 1736.6 6 675.1 pg/ml for smoking control subjects; 1797.3 6 639.7 pg/ ml for nonsmoking control subjects) (Figure 2A). There was no significant difference in sRAGE levels between smoking and nonsmoking control subjects. Because the TESRA cohort did not have control subjects, this analysis was conducted solely in ECLIPSE. sRAGE levels were also significantly associated with GOLD stage in ECLIPSE, decreasing with increasing disease severity (sRAGE, 1442.1 6 630.4 pg/ml for GOLD 2 patients; 1311.2 6 629.0 pg/ml for GOLD 3; 1157.7 6 551.6 pg/ml for GOLD 4; P , 0.001) (Figure 2B). There was no significant difference in sRAGE levels between GOLD 2 and GOLD 3 subjects in TESRA. To understand the relationship of sRAGE and emphysema in the context of COPD severity (GOLD stage), we plotted the three-dimensional graph of sRAGE, GOLD stage, and quantitative emphysema (Figure 3). This visualization suggests that sRAGE levels are associated

Figure 1. Association of soluble receptor for advanced glycation endproducts (sRAGE) with emphysema and diffusing capacity in the TESRA and ECLIPSE cohorts. (A) LP15A values (mean 6 SD) plotted for subjects in the TESRA cohort stratified by sRAGE quartile, after adjustment for age, sex, and body mass index (Q1 vs. Q4, P , 0.001) . (B) Percent predicted DLCO (mean 6 SD) plotted for subjects in the TESRA cohort stratified by sRAGE quartile after adjustment for age, sex, and body mass index (Q1 vs. Q4, P , 0.001). (C) FracVol , 2950 (mean 6 SD) plotted for subjects in the ECLIPSE cohort, stratified by sRAGE quartile (Q1 vs. Q4, P , 0.05). (D) Serum sRAGE plotted for the ECLIPSE cohort stratified by emphysema severity based on radiology score (emphysema , 5% vs. .50%, P , 0.001). The graph in A differs in direction because higher LP15A is associated with less severe emphysema, whereas higher FracVol , 2950 is associated with more severe emphysema.

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Figure 2. Association of soluble receptor for advanced glycation endproducts (sRAGE) levels with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage in the ECLIPSE cohort. (A) Serum sRAGE levels compared between chronic obstructive pulmonary disease (COPD) and control subjects in the ECLIPSE cohort. *P , 0.001. (B) Serum sRAGE levels compared across GOLD stage 2–4 in the ECLIPSE cohort. *P , 0.001. NS ¼ nonsmoker; S ¼ smoker.

with emphysema irrespective of GOLD status. We analyzed the sRAGE levels in patients with COPD with radiologist-defined emphysema (.5% emphysema on visual examination of CT scans) versus no emphysema (,5% emphysema) in ECLIPSE and the data are shown in Figure E1. sRAGE levels are significantly lower in patients with emphysema in GOLD 2 and 3. The levels were numerically lower in GOLD 4 also, but did not reach statistical significance because of small sample size. Association of sRAGE with Additional Clinical Characteristics

Because sRAGE was significantly associated with COPD affection status and COPD severity, we evaluated the relationships of sRAGE with other clinical parameters in COPD. By univariate analysis, sRAGE levels were significantly correlated with several additional clinical characteristics of different aspects of COPD in both cohorts (Table 3 and Table E3 for different GOLD categories). Associations of sRAGE levels with several of these measures remained significant after adjustment for demographic covariates (age, sex, and BMI) (Table 4). sRAGE levels were not associated with pack-years of smoking in both cohorts and there was no association with current smoking status in ECLIPSE. In TESRA, sRAGE levels explained a significant fraction of variability in DLCO and lung density, whereas more modest associations were observed for FEV1/FVC and St. Georges Respiratory Questionnaire. Significant associations with similar effect size were observed

in ECLIPSE for lung density and FEV1/FVC; associations with other COPD-related traits were weak (R2 , 0.03) but statistically significant. sRAGE Levels and Emphysema after Covariate Adjustment

We used a multivariate model to test COPD-related traits significant in univariate analysis (FEV1/FVC ratio, 6-min-walk distance) for their contribution to log10 sRAGE levels (Table 5). Multivariate regression analysis showed that, in TESRA, age was the only significant demographic covariate. Both DLCO and quantitative emphysema remained significantly correlated with sRAGE levels after adjustment (see Table E4). In ECLIPSE, age, sex, and height had significant associations with sRAGE levels, but similar to TESRA, the severity of quantitative emphysema remained significantly inversely correlated with sRAGE after adjustment for covariates. SNPs in the AGER Locus Are Associated with Plasma sRAGE Levels

We also sought to determine in both cohorts whether there was an association between sRAGE protein levels and SNP genotypes in the AGER locus on chromosome 6. We considered SNPs 5 kb upstream of the transcription start site from AGER and 5 kb downstream of the 39 UTR region that were polymorphic in TESRA, yielding a total of 21 SNPs. To limit potential

Figure 3. Three-dimensional representation of relationship of soluble receptor for advanced glycation endproducts (sRAGE) with FEV1 (Global Initiative for Chronic Obstructive Lung Disease [GOLD]) and emphysema severity. (A) TESRA patients with chronic obstructive pulmonary disease: three-dimensional plot of quantitative emphysema (LP15A) quartiles, GOLD stage, and log10 sRAGE. Because the number of GOLD 4 patients is limited, GOLD 3 and 4 were combined as one category. (B) ECLIPSE study patients with chronic obstructive pulmonary disease: three-dimensional plot of quantitative emphysema (FracVol950) quartiles, GOLD stage, and log10 sRAGE.

Cheng, Kim, Cockayne, et al.: AGER Variants Influence the COPD Biomarker sRAGE TABLE 3. PEARSON CORRELATION COEFFICIENTS FOR sRAGE (LOG10) VERSUS COPD-RELATED CLINICAL CHARACTERISTICS IN THE TESRA AND ECLIPSE COHORTS TESRA COPD Cases Clinical Characteristics FEV1, ml FEV1 % predicted FEV1/FVC DLCO % predicted LP15A* FracVol less than 2950 HU* Airway wall thickness (Pi10) 6-min walk distance SGRQ Dyspnea index

ECLIPSE COPD Cases

Rho

P

Rho

P

20.02 0.06 0.16 0.24 0.23 N/A N/A 20.11 0.08 0.03

0.72 0.26 ,0.001 ,0.001 ,0.001 N/A N/A 0.03 0.10 0.58

0.11 0.17 0.21 N/A N/A 20.28 0.04 0.07 20.09 20.09

,0.0001 ,0.0001 ,0.0001 N/A N/A ,0.0001 0.12 0.002 ,0.0001 0.0003

Definition of abbreviations: COPD ¼ chronic obstructive pulmonary disease; DLCO ¼ diffusing capacity of carbon monoxide; SGRQ ¼ St. Georges Respiratory Questionnaire. * Note that higher LP15A (above) is associated with less severe emphysema, whereas higher FracVol less than 2950 is associated with more severe emphysema, but lower sRAGE is correlated with more severe disease.

population stratification in TESRA, we performed our analysis in the subset of 335 white subjects. Of the selected SNPs, rs2071288 showed an association with plasma sRAGE levels (Table 6) and DLCO % predicted. After adjustment for age, sex, and BMI, subjects with the CT genotype had a plasma sRAGE level of 1,772 6 1,945 pg/ml, compared with 2,681 6 2,117 pg/ml for subjects with the CC genotype. Consistent with our previous observation that lower plasma sRAGE levels are associated with decreased DLCO % predicted and LP15A, subjects with the rs2071288 CT genotype also displayed lower DLCO % predicted values (40.2 6 3.5%), compared with subjects with the CC genotype (49.1 6 0.7%; P ¼ 0.01) Subjects with the CT genotype had a trend toward lower LP15A than subjects with the CC genotype (2950.36 vs. 2937.65 HU); however, this difference was not statistically significant. rs2070600 was significantly associated with plasma sRAGE levels, albeit with weaker effect than rs2071288 (Figure 4A) (sRAGE levels were 1,615 6 1,945 pg/ml for subjects with the CT genotype compared with 2,686 6 2,110 pg/ml for subjects with the CC genotype). Unlike rs2071288, rs2070600 was not significantly associated with DLCO % predicted. In ECLIPSE, three SNPs from the same region showed significant association with sRAGE levels (Table 6). An association plot of the SNPs analyzed in ECLIPSE is shown in Figure 4B. Replication results for rs2071288 were not available because it

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had not been genotyped in ECLIPSE and was not included in the imputation panel for HapMap2. Association of rs2070600 with sRAGE levels was replicated in ECLIPSE (P ¼ 7.07 3 10216). The results for rs2070600 in ECLIPSE were similar in direction to TESRA (Figure 4A) (sRAGE, 453.5 6 251.0 pg/ml, 1041.0 6 527.7 pg/ml, and 1381.0 6 624.7 pg/ml for TT, CT, and CC genotypes, respectively). Similarly to TESRA, rs2070600 was not significantly associated with lung density in ECLIPSE. Other SNPs in the AGER locus were also evaluated for association with a number of clinical characteristics in the TESRA and ECLIPSE cohorts (see Table E5); however, AGER genetic associations with clinical variables were weak in general and none were significant after multiple testing corrections.

DISCUSSION In the current study, a broad evaluation of potential systemic biomarkers was performed in the TESRA cohort and one biomarker, sRAGE, was significantly associated with both CT-defined emphysema and DLCO. This initial observation was extended into a second cohort and also into an evaluation of relationships between sRAGE, COPD clinical characteristics, and genetic variants at the AGER locus. The association of lower sRAGE with emphysema was robust. We also conducted multivariate analyses, and sRAGE levels were significantly associated with DLCO and emphysema even after correcting for spirometric and demographic variables. These results strongly suggest that the association of sRAGE with emphysema is independent of the COPD severity/GOLD status. Low sRAGE levels have also been reported in three small studies of individuals with COPD (15–17) and an association between sRAGE, emphysema, and cor pulmonale in 200 individuals with COPD has recently been reported (18). By contrast, we used two independent, large, wellcharacterized COPD cohorts where we were able to clearly demonstrate the association of systemic sRAGE levels with airflow limitation and CT-defined emphysema and DL CO , after adjusting for relevant covariates. In addition to the association of sRAGE with emphysema, two SNPs in the AGER locus were significantly associated with systemic sRAGE levels. The SNP rs2071288 was associated with both D LCO and sRAGE level in the TESRA study; however, these SNP data were not available in the ECLIPSE study. The SNP rs2070600 was associated with sRAGE levels in both cohorts. The association of SNP rs2070600 with systemic sRAGE levels was previously reported in subjects with normal glucose metabolism, impaired glucose metabolism, and type 2 diabetes mellitus (28). rs2070600 has been shown to be associated with a wide variety of diseases (29–34) and lung function in the

TABLE 4. MULTIVARIATE ASSOCIATIONS OF sRAGE TO COPD CLINICAL VARIABLES IN TESRA AND ECLIPSE TESRA COPD CASES Clinical Measure FEV1, ml FEV1 % predicted FEV1/FVC DLCO % predicted LP15A FracVol less than 2950 HU Airway wall thickness (Pi10) 6-min walk distance, m SGRQ Dyspnea Index, mMRC

2

ECLIPSE COPD CASES 2

R (%)

Coefficient Estimate

Coefficient SE

Coefficient P

R (%)

Coefficient Estimate

Coefficient SE

Coefficient P

0.18 0.02 3.43 5.24 7.79

51.9 0.40 0.05 10.06 27.02

48.6 1.49 0.01 1.94 4.54

0.29 0.78 ,0.0001 ,0.0001 ,0.0001

2.18 2.60 4.03

374.2 13.16 0.12

54.4 1.84 0.013

,0.0001 ,0.0001 ,0.0001

7.60 0.22 0.92 0.79 0.92

217.65 0.050 59.22 29.34 20.52

1.52 0.026 14.1 2.48 0.13

,0.0001 0.047 ,0.0001 0.0002 ,0.0001

0.46 1.43 0.08

226.12 6.77 0.067

18.4 2.76 0.118

0.15 0.01 0.58

Definition of abbreviations: COPD ¼ chronic obstructive pulmonary disease; DLCO ¼ diffusing capacity of carbon monoxide; mMRC ¼ modified Medical Research Council; SGRQ ¼ St. Georges Respiratory Questionnaire. COPD-related clinical characteristics were the outcomes in these multivariate models, which were adjusted for age, sex, and body mass index.

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TABLE 5. MULTIVARIATE ANALYSES OF SERUM OR PLASMA sRAGE* (LOG10) LEVELS WITH CLINICAL PARAMETERS AND LUNG DENSITY TESRA

Age Sex Body mass index Height FEV1/FVC ratio 6-min-walk distance LP15A† FRACVOL , 2950 HU†

ECLIPSE

Estimate

SE

t Value

P Value

Estimate

SE

t Value

P Value

0.011 20.014 20.0049 0.00089 0.18 20.00014 0.0028

0.0018 0.039 0.0031 0.0020 0.214 0.00013 0.00065

6.27 20.36 21.60 0.422 0.851 21.14 4.34

,0.001 0.72 0.11 0.67 0.39 0.25 1.86 3 1025

0.004 0.035 20.001 0.001 0.001 0.00004

0.0006 0.013 0.001 0.0007 0.0005 0.00004

5.04 2.71 21.01 2.14 1.80 0.85

,0.0001 0.007 0.31 0.03 0.07 0.39

20.004

0.0005

27.92

,0.0001

* Note that sRAGE circulating levels were the outcome in these multivariate models. y Note that higher LP15A (above) signifies less severe emphysema, whereas higher FracVol less than 2950 signifies more severe emphysema; in both cohorts, lower sRAGE is correlated with more severe disease.

general population (19, 20). The SNP rs2070600 is a nonsynonymous coding variant (Gly82Ser) in the ligand binding domain and is likely to be functional. This SNP was associated with systemic sRAGE levels in both the TESRA and ECLIPSE COPD populations. However, this SNP did not show significant association with emphysema or other COPD-related clinical characteristics, except for a nominal association with 6-minutewalk distance in ECLIPSE. Because rs2070600 is a relatively uncommon variant, the lack of association of this SNP with specific clinical characteristics may be caused by the sample size limitations (minor allele frequency: TESRA 3.9%, ECLIPSE 3.4%). In the published genome-wide association studies on lung function, the T allele of rs2070600 was shown to be associated with FEV1/FVC ratio; however, the association of this SNP with FEV1 was not significant (19, 20, 35). AGER maps to the HLA region on chromosome 6, and Soler and coworkers (35) reported a novel of association with FEV1/FVC at SNP rs2857595 at this locus that is independent of rs2070600. Therefore, the genetic association of this locus with lung function and COPD may be complex. Moreover, the T allele of rs2070600 is associated with increased FEV1/ FVC ratio and protection from COPD (36). Young and coworkers (37, 38) reported that the T allele of the same SNP is associated with protection from COPD in a cohort of normal smokers and smokers with COPD defined by prebronchodilator spirometry. This allele has been consistently negatively associated with circulating sRAGE levels, which is robustly associated with emphysema in our study. Thus, it is highly plausible that the AGER locus may be associated with both lung function and emphysema and different SNPs may be driving these associations. Very recently, additional information on the role of RAGE in lung development and maintenance has been reported. It has been shown that when human RAGE is overexpressed in mouse lung, RAGE has an important role in alveolar morphogenesis

and lung homeostasis and overexpression in the adult lungs leads to emphysema (39, 40). Therefore, it is possible that sRAGE, which is a decoy receptor for RAGE, has different roles during alveolar morphogenesis and in adult lungs. The effect of sRAGE in alveolar morphogenesis may have an impact on the lung during development and subsequently on lung function in adults. Alternatively, low sRAGE that develops in adults may result in a higher extent of RAGE signaling and result in alveolar destruction and emphysema. RAGE expression has been shown to be increased in the lungs of patients with COPD (41, 42), and recent data from ECLIPSE has shown that baseline sRAGE levels are associated with progression in emphysema in COPD (43). These speculations need to be assessed using experimental studies. Because sRAGE has been correlated previously with neutrophilic inflammation or other diseases, such as cardiovascular disease, we considered these as explanations of the observed correlations in this study. It is possible that the association observed between the sRAGE and emphysema may be related to neutrophilic inflammation as suggested for sRAGE in neutrophilic asthma, COPD (44), and cystic fibrosis (45). We assessed whether the sRAGE levels were correlated with markers of inflammation measured in our studies. The results indicated that there were no significant correlations between systemic sRAGE levels and the inflammatory biomarkers in the blood measured in our studies (see Table E6). Because coronary artery disease is a comorbidity in COPD and sRAGE has been shown to be associated with coronary artery disease (34), we analyzed the CT scans from the ECLIPSE study and measured the coronary artery calcification (Agatston score) to understand this relationship. A total of 581 subjects had both Agaston score and sRAGE measurements available. Spearman correlation with sRAGE and Agatston score was found to be 20.0005 (P ¼ 0.912). Thus, we have not found any evidence in our study that

TABLE 6. RESULTS OF THE ASSOCIATION ANALYSES OF THE SERUM SRAGE WITH GENETIC POLYMORPHISMS AT THE AGER LOCUS ON CHROMOSOME 6 ECLIPSE SNP rs2070600 rs2022059 rs204993 rs2071288

MAF 0.044 0.047 0.270 N/A

Beta 20.1364 20.1290 20.0454 N/A

SE 0.0167 0.0161 0.0081 N/A

TESRA P 216

7.07 3 10 2.53 3 10215 2.08 3 1028 N/A

MAF

Beta

SE

P

0.020 0.031 0.236 0.019

20.201 20.101 20.013 20.261

0.080 0.061 0.027 0.081

0.01 0.10 0.63 0.0014

Definition of abbreviation: SNP ¼ single-nucleotide polymorphism. A total of 21 SNPs 5 kb upstream/downstream of the transcription start site /39 UTR of the AGER locus were selected for analysis in the TESRA study. Of the genome-wide significant SNPs on chromosome 6 from the ECLIPSE cohort, three were mapped to the AGER locus. Genotyped or imputed (with underline) SNPs overlapped in both cohorts at the AGER locus are shown.


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Figure 4. Association plot of the soluble receptor for advanced glycation endproducts (sRAGE) locus and association of rs2070600 with bloodstream sRAGE levels in the ECLIPSE and TESRA cohorts. (A) Circulating sRAGE levels in patients with chronic obstructive pulmonary disease with different genotypes of rs2070600. Bars indicate predicted values, holding age, sex, and body mass index constant at their median values. Error bars indicate standard error of the mean (CC vs. CT, P ¼ 0.01). (B) Association plot showing single-nucleotide polymorphisms (SNPs) in the AGER locus (ECLIPSE study) created using LocusZoom (CC vs. TT, P , 0.001).

the association of sRAGE with emphysema is driven by neutrophilic inflammation or cardiovascular comorbidity. The nature of the specific mechanistic relationship between the AGER variants, sRAGE, and development of emphysema is unclear. RAGE levels are increased in the alveolar wall (41) and airway epithelium (42) in patients with COPD. Reduced circulating sRAGE may reflect a change in receptor shedding caused by either ligand engagement or functional receptor changes,

transcriptional regulation of soluble form expression, or a combination. HMGB1, one of several ligands for RAGE, also was reported to be increased in the bronchoalveolar lavage of patients with COPD suggesting that the receptor could be engaged in disease (42) but no correlation between serum HMGB1 and sRAGE was observed in our study (see Table E6). As discussed previously, a role in inflammation or “danger response” would be consistent with reported relationships to inflammation in other

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diseases and could reflect a complex relationship between surface receptor, ligands, and circulating sRAGE. AGER SNPs may be related directly to clinical characteristics, but we did not find evidence for significant associations, potentially because of lower power. Alternatively, the AGER SNPs may influence the levels or function of sRAGE in the context of disease. For example, ligand interactions with the variant surface receptor could modulate the function or the shedding, or ligand binding to sRAGE variant could be affected. Thus, the interaction between AGER variants, control of sRAGE levels, and ligand binding could influence the development of COPD. This study has some limitations. The sRAGE levels were measured in plasma in TESRA and serum in ECLIPSE. However, the levels were similar in both cohorts and are also similar to published reports. Although the associations of systemic sRAGE levels with airflow obstruction and emphysema are very strong, the associations with other clinical parameters are inconsistent in direction and less significant; these results are presented for completeness. A true association of sRAGE with these additional clinical parameters is uncertain. In addition, the association of AGER SNPs with sRAGE levels is robust, but the association of the SNPs with the clinical parameters is weak at best. Analysis of the association of these SNPs in large COPD populations would be needed to establish whether there is a significant genetic relationship with clinical parameters. Conclusions

In two large well-characterized cohorts, this study demonstrates that circulating levels of sRAGE are associated with emphysema in patients with COPD, suggesting that sRAGE may be a useful biomarker for patient stratification in clinical development programs.Further studies are requiredto precisely define the relationship of AGER SNPs to specific clinical COPD characteristics. Author disclosures are available with the text of this article at www.atsjournals.org. Principal Investigators and Centers Participating in ECLIPSE: Bulgaria: Yavor Ivanov, Pleven; Kosta Kostov, Sofia. Canada: Jean Bourbeau, Montreal, QUE; Mark Fitzgerald, Vancouver, BC; Paul Hernandez, Halifax, NS; Kieran Killian, Hamilton, ON; Robert Levy, Vancouver, BC; Francois Maltais, Montreal, QUE; Denis O’Donnell, Kingston, ON. Czech Republic: Jan Krepelka, Praha. Denmark: JØrgen Vestbo, Hvidovre. Netherlands: Emiel Wouters, Horn. New Zealand: Dean Quinn, Wellington. Norway: Per Bakke, Bergen. Slovenia: Mitja Kosnik, Golnik. Spain: Alvar Agustı´, Jaume Sauleda, Palma de Mallorca. Ukraine: Yuri Feschenko, Kiev; Vladamir Gavrisyuk, Kiev; Lyudmila Yashina, Kiev; Nadezhda Monogarova, Donetsk. United Kingdom: Peter Calverley, Liverpool; David Lomas, Cambridge; William MacNee, Edinburgh; David Singh, Manchester; Jadwiga Wedzicha, London. United States: Antonio Anzueto, San Antonio, TX; Sidney Braman, Providence, RI; Richard Casaburi, Torrance, CA; Bart Celli, Boston, MA; Glenn Giessel, Richmond, VA; Mark Gotfried, Phoenix, AZ; Gary Greenwald, Rancho Mirage, CA; Nicola Hanania, Houston, TX; Don Mahler, Lebanon, NH; Barry Make, Denver, CO; Stephen Rennard, Omaha, NE; Carolyn Rochester, New Haven, CT; Paul Scanlon, Rochester, MN; Dan Schuller, Omaha, NE; Frank Sciurba, Pittsburgh, PA; Amir Sharafkhaneh, Houston, TX; Thomas Siler, St. Charles, MO; Edwin Silverman, Boston, MA; Adam Wanner, Miami, FL; Robert Wise, Baltimore, MD; Richard ZuWallack, Hartford, CT. Steering Committee: Harvey Coxson (Canada), Lisa Edwards (GlaxoSmithKline, US), David Lomas (UK), William MacNee (UK), Edwin Silverman (US), Ruth Tal-Singer (Co-chair, GlaxoSmithKline, US), Jørgen Vestbo (Co-chair, Denmark), Julie Yates (GlaxoSmithKline, US). Scientific Committee: Alvar Agustı´ (Spain), Peter Calverley (UK), Bartolome Celli (US), Courtney Crim (GlaxoSmithKline, US), Gerry Hagan (GlaxoSmithKline, UK), William MacNee (Chair, UK), Bruce Miller (GlaxoSmithKline, US), Stephen Rennard (US), Ruth Tal-Singer (GlaxoSmithKline, US), Emiel Wouters (The Netherlands), Julie Yates (GlaxoSmithKline, US). TESRA Investigators: Ognian Georgiev, Sofia, Bulgaria; Dimitar Popov, Sofia, Bulgaria; Hristo Metev, Ruse, Bulgaria; Vasil Dimitrov, Sofia, Bulgaria; Yavor Ivanov, St. Pleven, Bulgaria; Libor Fila, Praha, Czech Republic; Vladimir Zindr Vitezna, Karlovy Vary, Czech Republic; Kamil Klenha, Tabor, Czech Republic; Jiri Votruba, Praha, Czech Republic; Jaromir Roubec, Ostrava, Czech Republic; Barna Szima, Szombathely, Hungary; Zsuzsanna Mark, Torokbalint, Hungary; Zoltan Baliko, Pecs Hungary; Zoltan Bartfai, Budapest, Hungary; Katalin Gomori, Balassagyarmat, Hungary; Andres Sigvaldason, Reykjavik, Iceland; Mordechai Kramer, Petach Tikva, Israel; Gershon Ya Fink, Rehovot, Israel; Zeev Weiler, Ashkelon, Israel; Joel Greif, Tel Aviv, Israel; Issahar Ben-Dov, Ramat Gan, Israel; Mordechai Yigla, Haifa,

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Israel; Leonardo Fabbri, Modena, Italy; Pierluigi Paggiaro, Pisa, Italy; Giorgio Canonica, Genova, Italy; Isa Cerveri, Pavia, Italy; Antra Bekere, Riga, Latvia; Aurika Babjoniseva, Riga, Latvia; Alvil Krams, Stopinu Pagasts, Rigas Rajons, Latvia; Wladyslaw Pierzchala, Katowice, Poland; Dariusz Nowak, Lodz, Poland; Robert Mroz, Bialystok, Poland; Hanna Szelerska-Twardosz, Poznan Poland; Malgorzata Rzymkowska, Poznan, Poland; Ismail Abdullah, Durban, South Africa; Christo Van Dyk, Western Cape, Worcester, South Africa; Nyda Fourie, Bloemfontein, South Africa; John O’Brien, Cape Town, South Africa; J. Joubert, Bellville-Cape Province, South Africa; Abdool Gafar, Kwa-Zulu Natal, Amanzimtoti, South Africa; Mary Bateman, Cape Town, South Africa; Hannes Van Rensburg, Centurion, South Africa; Lyudmila Yashina, Kiev, Ukraine; Nadezda Monogarova, Donetsk, Ukraine; Oleksandr Dzyublik, Kiev, Ukraine; Volodymyr Gavrysyuk, Kiev, Ukraine; Yuriy Feshchenko, Kiev, Ukraine; David Parr, Coventry, United Kingdom, Stephen Rennard, Omaha, NE; Richard Casaburi, Torrance, CA; Gerard Criner, Philadelphia, PA; Mark Dransfield, Birmingham, AL; Charles Fogarty, Spartanburg, SC; Nicola Hanania, Houston, TX; Carl Griffin, Oklahoma City, OK; Kathi Mcdavid, Oklahoma City, OK; Paul Kvale, Detroit, MI; Barry Make, Denver, CO; Joe Ramsdell, San Diego, CA; Michael D. Roth, Los Angeles, CA; Amir Harafkhaneh, Houston, TX; Peter Sporn, Chicago, IL. Steering Committee: Alvar Agustı´ (Spain), Peter Calverley (UK), Leonardo Fabbr (Italy), Klaus F. Rabe (Netherlands), Nicolas Roche (France), Michael Roth (US), Jorgen Vestbo (Denmark), Stephen Rennard (US).

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