Acute Exacerbations of Chronic Obstructive Pulmonary Disease Identification of Biologic Clusters and Their Biomarkers Mona Bafadhel1,2, Susan McKenna1, Sarah Terry1, Vijay Mistry1,2, Carlene Reid1, Pranabashis Haldar2, Margaret McCormick3, Koirobi Haldar2, Tatiana Kebadze4, Annelyse Duvoix5, Kerstin Lindblad6, Hemu Patel7, Paul Rugman3, Paul Dodson3, Martin Jenkins3, Michael Saunders3, Paul Newbold3, Ruth H. Green1, Per Venge6, David A. Lomas5, Michael R. Barer2,7, Sebastian L. Johnston4, Ian D. Pavord1, and Christopher E. Brightling1,2 1

Institute for Lung Health, and 2Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom; AstraZeneca R&D Charnwood, Loughborough, Leicestershire, United Kingdom; 4Department of Respiratory Medicine, National Heart and Lung Institute, Centre for Respiratory Infections, Imperial College London, United Kingdom; 5Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; 6Department of Medical Sciences, Clinical Chemistry, University of Uppsala, Uppsala, Sweden; and 7 Department of Clinical Microbiology, University Hospitals of Leicester NHS Trust, Leicester, United Kingdom 3

Rationale: Exacerbations of chronic obstructive pulmonary disease (COPD) are heterogeneous with respect to inflammation and etiology. Objectives: Investigate biomarker expression in COPD exacerbations to identify biologic clusters and determine biomarkers that recognize clinical COPD exacerbation phenotypes, namely those associated with bacteria, viruses, or eosinophilic airway inflammation. Methods: Patients with COPD were observed for 1 year at stable and exacerbation visits. Biomarkers were measured in sputum and serum. Viruses and selected bacteria were assessed in sputum by polymerase chain reaction and routine diagnostic bacterial culture. Biologic phenotypes were explored using unbiased cluster analysis and biomarkers that differentiated clinical exacerbation phenotypes were investigated. Measurements and Main Results: A total of 145 patients (101 men and 44 women) entered the study. A total of 182 exacerbations were captured from 86 patients. Four distinct biologic exacerbation clusters were identified. These were bacterial-, viral-, or eosinophilicpredominant, and a fourth associated with limited changes in the inflammatory profile termed “pauciinflammatory.” Of all exacerbations, 55%, 29%, and 28% were associated with bacteria, virus, or a

(Received in original form April 2, 2011; accepted in final form June 15, 2011) Supported by the Medical Research Council (United Kingdom) and AstraZeneca jointly as a “Biomarker Call Project”; C.E.B. is a Wellcome Trust Senior Clinical Fellow, and GlaxoSmithKline supported the measurement of surfactant protein D. The research was performed in laboratories partly funded by the European Regional Development Fund (ERDF 05567). The Medical Research Council, Wellcome Trust, and the European Regional Development Fund had no involvement in the design of the study, data collection, analysis and interpretation of the data, in the writing of the manuscript, or in the decision to submit the manuscript. Author Contributions: S.M. and S.T. were involved in the recruitment of volunteers and in data collection. C.R., V.M., K.H., H.P., A.D., and K.L. were involved in data collection and interpretation. M.M., P.R., P.D., P.N., M.J., and M.S. were involved in study design, data collection, and interpretation. R.H.G. and P.H. were involved in study design and data interpretation. M.R.B., D.A.L., S.L.J., P.V., and I.D.P. were involved in the design of the study, data collection, and interpretation. M.B. and C.E.B. were involved in the study design, volunteer recruitment, data collection, data interpretation, and data analysis, and had full access to the data and are responsible for the integrity of the data and final decision to submit. All authors contributed to the writing of the manuscript and have approved the final version for submission. Correspondence and requests for reprints should be addressed to Christopher E. Brightling, M.B.B.S., B.Sc. (Hons.), Ph.D., Institute for Lung Health, Clinical Sciences Wing, University Hospitals of Leicester, Leicester, LE3 9QP, UK. E-mail: [email protected] 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 184. pp 662–671, 2011 Originally Published in Press as DOI: 10.1164/rccm.201104-0597OC on June 16, 2011 Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

Exacerbations of chronic obstructive pulmonary disease (COPD) are a major health burden worldwide, and affect a vulnerable population at risk of significant comorbidities. COPD exacerbations are heterogeneous with respect to etiology and inflammation and biomarkers are required to phenotype this heterogeneity. What This Study Adds to the Field

We have shown that there are biologic COPD exacerbation clusters that are clinically indistinguishable, and that biomarkers can be used to identify specific clinical phenotypes during exacerbations of COPD (specifically those associated with bacteria, virus, and sputum eosinophilia). Bacterial and eosinophilic clinical exacerbation phenotypes can be identified from stable state. Our data further delineate the heterogeneity during COPD exacerbations and may identify populations that appropriately require corticosteroids and antibiotics at the onset of an exacerbation.

sputum eosinophilia. The biomarkers that best identified these clinical phenotypes were sputum IL-1b, 0.89 (area under receiver operating characteristic curve) (95% confidence interval [CI], 0.83–0.95); serum CXCL10, 0.83 (95% CI, 0.70–0.96); and percentage peripheral eosinophils, 0.85 (95% CI, 0.78–0.93), respectively. Conclusions: The heterogeneity of the biologic response of COPD exacerbations can be defined. Sputum IL-1b, serum CXCL10, and peripheral eosinophils are biomarkers of bacteria-, virus-, or eosinophilassociated exacerbations of COPD. Whether phenotype-specific biomarkers can be applied to direct therapy warrants further investigation. Keywords: chronic obstructive pulmonary disease; phenotypes; exacerbations; airway inflammation; infection

Acute exacerbations of chronic obstructive pulmonary disease (COPD) are associated with substantial morbidity and mortality (1, 2). Exacerbations are typically associated with increased neutrophilic and to a lesser extent eosinophilic airway inflammation (3, 4). Respiratory viral and bacterial infections have been implicated in causing most exacerbations (5–7), but how these infections alter lower airway inflammation and relate to treatment

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response is not completely understood. This heterogeneity translates that at present clinicians have limited tools to phenotype exacerbations. During stable state a sputum eosinophilia is associated with corticosteroid responsiveness (8–10), whereas the presence of a high bacterial load and sputum purulence has favorable outcomes with antibiotics (11–15). These findings suggest that it is possible to identify clinically important COPD exacerbation phenotypes. This is crucial because systemic corticosteroids and antibiotics have marginal efficacy (16–21) and the potential to cause adverse events in an already vulnerable population. We hypothesize that approaches aimed at the identification of COPD exacerbation phenotypes may allow for better prognostic, therapeutic, and mechanistic applications (22–24). In this study we investigated whether during exacerbations of COPD there are (1) definable biologic phenotypes using unbiased mathematical tools (namely factor and cluster analysis); (2) identifiable biomarkers associated with clinical phenotypes, specifically those associated with bacteria, viruses, or sputum eosinophilia; and (3) exacerbation phenotypes that can be predicted from stable state.

METHODS Patients Patients with a physician diagnosis of COPD and a post-bronchodilator FEV1/FVC ratio of less than 0.7 as per global initiative for chronic obstructive lung disease (GOLD) criteria (1) were recruited from the Glenfield Hospital, Leicester, United Kingdom, and through local advertising. All patients fulfilled the inclusion criteria of age greater than 40 years, GOLD stage I–IV, and greater than or equal to one exacerbation in the preceding 12 months defined as the requirement of emergency health care (25). Patients were excluded if there was a documented inability to produce sputum after the induced sputum procedure, a current or previous history of asthma, currently active pulmonary tuberculosis, or any other clinically relevant lung disease other than COPD. The presence of comorbidity, reported atopy to common aeroallergens, or reversibility on lung function testing was not an exclusion criterion. All patients gave informed written consent and the study was approved by the local ethics committee.

Study Design This was a prospective observational study. Patients were seen at stable state and during exacerbations for the duration of 1 year. Stable visits including the baseline visits were 8 weeks free from an exacerbation. All patients were given daily diary cards to complete, and asked to contact the research team if there was an increase in symptoms of breathlessness, sputum volume, and purulence. Exacerbations were defined according to Anthonisen criteria (14) and health care use (25). Exacerbation data recording and sampling were only performed in patients who had not received prior oral corticosteroids or antibiotics. Patients were all clinically assessed (including chest radiograph, temperature recording, and blood gas analysis if clinically necessary) to exclude other causes of breathlessness. Patients with an exacerbation of COPD were then treated according to guidelines (2).

Measurements At all visits, patients underwent pre– and post–400-mg albuterol bronchodilator spirometry (Vitalograph, Buckingham, Buckinghamshire, UK); induced or spontaneous sputum collection (26); and measurements of symptoms and health quality assessments using the Visual Analog Scale (27) and the Chronic Respiratory Disease Interviewer-Administered Standardized Questionnaire (CRQ) (28). Sputum was collected and analyzed for bacteria (29–31) (using standard routine culture, CFU, and real-time quantitative polymerase chain reaction [qPCR]), for viruses by PCR (32), and processed to produce cytospins for cell differential and supernatant for fluid phase measurements (33). A broad panel of serum and sputum biomarkers were measured using the Meso-Scale-Discovery (MSD, Gaithersburg, MD) platform standard preprepared plates (MSD, MD)

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and single ELISA at stable and exacerbation visits (see Table E1 in the online supplement).

Definition of Bacteria-, Virus-, and Sputum Eosinophil–associated Exacerbations of COPD Bacteria-associated exacerbations were defined as a positive bacterial pathogen on routine culture (Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Staphylococcus aureus, or Pseudomonas aeruginosa) or a total aerobic CFU count greater than or equal to 107 cells (12, 15). qPCR bacterial detection methods were not used to define bacteria-associated exacerbations in this study. A virus-associated exacerbation was defined as one that had a positive sputum viral PCR, whether in isolation or in combination with a positive bacterial pathogen on routine culture. A sputum eosinophil–associated exacerbation was defined as the presence of more than 3% nonsquamous cells.

Statistical Analyses Statistical analyses were performed using PRISM version 4 (GraphPAD PRISM, La Jolla, CA) and SPSS version 16 (IBM, Chicago, IL). Parametric and nonparametric data are presented as mean (SEM) and median (interquartile range). Log transformed data are presented as geometric mean (95% confidence interval [CI]). Multivariate modeling using principal component analysis in sputum biomarkers was used to explore biomarker pattern expression at exacerbations. No adjustments for multiple comparisons have been made across biomarkers. Factor analysis, a mathematical method that discovers patterns of relationships within large datasets, was used to identify factors in sputum mediators at exacerbations thereby demonstrating biologic factors independent of each other and of any clinical expression. This method using unsupervised principal component analysis demonstrated three factors accounting for 75% of the total variance (see Table E2). Cluster analysis, an unbiased mathematical method, allows one to classify groups on similar chosen characteristics alone. Thus, after demonstrating three biologic factors, we used hierarchical cluster analysis to generate four biologic clusters for exacerbation events and cases. Clinical characteristics for all exacerbation events were tabulated for each biologic cluster. Oneway analysis of variance, Kruskal-Wallis test, and the chi-square test were used to compare the clinical characteristics between cluster groups. For comparison of clinical and biomarker changes between baseline and exacerbation visits the paired t test or Wilcoxon matched pairs test was used. For comparison of exacerbations associated with or without bacteria, virus, and sputum eosinophilia the t test and Mann-Whitney test were used, respectively. To determine suitable biomarkers, the receiver operating characteristic curves were plotted for (1) exacerbation versus stable state; (2) bacteria- versus nonbacteria-associated exacerbations; (3) virus- versus nonvirus-associated exacerbations; and (4) sputum eosinophilia– (. 3% nonsquamous cells) versus nonsputum eosinophilia–associated exacerbations. Validation of the identified biomarkers for bacteria-, virus-, and sputum eosinophil–associated exacerbation was performed in a further 89 exacerbation events from an independent cohort of subjects with COPD. These subjects with COPD were recruited to enter a prospective study with identical inclusion and exclusion criteria and study design as the current study. Stable and exacerbation visits were treated in accordance to the main study. A P value of less than 0.05 was taken as the threshold of statistical significance.

RESULTS One hundred fifty-six patients were enrolled; 145 (101 men and 44 women) completed the first visit and 115 completed 12 months (Figure 1; see Figure E1). At baseline 3%, 48%, 30%, and 19% had GOLD I, II, III, and IV, respectively. Most patients recruited were current or exsmokers (142 of 145), with a mean (range) pack-year history of 49 (10–153) with an absolute and percentage mean (SEM) reversibility to inhaled bronchodilator on study entry of 47 ml (11 patients) and

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Figure 1. Subject enrolment flow diagram for 12-month observational period.

4% (one patient), respectively. Skin prick testing or serumspecific IgE to a wide panel of aeroallergens confirmed that 20% were atopic. Bacterial colonization, defined as the presence of a potentially pathogenic microorganism (H. influenzae, M. catarrhalis, S. pneumoniae, S. aureus, or P. aeruginosa) in a standard culture technique (29), was present in 28% of patients at baseline. Using qPCR a bacterial pathogen (H. influenzae, M. catarrhalis, S. pneumoniae, or S. aureus) was detected in 86% of patients at the baseline stable visit. A virus was detected in 5% of subjects at study entry, whereas eosinophilic airway inflammation (. 3% nonsquamous cells) was present in 27% of patients. Baseline and exacerbation clinical characteristics are shown in Table 1 (see Table E3). Exacerbations

A total of 182 exacerbation events were captured from 86 patients; of these 21 exacerbations warranted hospitalization. There was a reduction in the FEV1 and CRQ from baseline to exacerbation (FEV1 [L] 1.33 vs. 1.10; mean difference 0.24; 95% CI, 0.12–0.36; P , 0.001) (CRQ [units] 4.11 vs. 3.12; mean difference 0.99; 95% CI, 0.74–1.23; P , 0.001). The magnitude of these changes was independent of smoking status, sex,

GOLD severity (1), or Anthonisen criteria (14). Hospitalized exacerbations were associated with a greater decline in lung function compared with exacerbations that were not hospitalized (DFEV1 [ml] 2355 vs. 2131; mean difference 224; 95% CI of difference, 2356 to 292; P , 0.001), but not health status decline (DCRQ [units] 21.25 vs. 20.91; mean difference 0.34; 95% CI of difference, 20.83 to 0.15; P ¼ 0.18). Serum and sputum mediator data were available in 148 exacerbation events from 75 patients. Serum biomarkers that increased during an exacerbation were IL-6, tumor necrosis factor (TNF) receptors I and II, serum amyloid-A, C-reactive protein (CRP), procalcitonin, and serum eosinophil cationic protein (Table E4A). Sputum biomarkers that increased were IL-1b, TNF-a, TNFRI, TNFRII, IL6, CCL5, and CCL4 (Table E4B). No single biomarker had a receiver operating curve area under the curve greater than 0.70 in determining an exacerbation from stable state (Figure E2). Of all sputum and serum biomarkers measured there was a significantly increased level of serum TNF-a and CRP in patients who were hospitalized (CRP median [IQR] 56 (102) vs. 8 (14); P ¼ 0.002) (serum TNF-a geometric mean 4.3 [95% CI, 3.4–5.4] vs. 3.4 [95% CI, 3.2–3.6]; P ¼ 0.02).

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TABLE 1. CLINICAL CHARACTERISTICS OF ALL PATIENTS AT ENTRY INTO THE STUDY AND CLINICAL FEATURES AT EXACERBATION Study Entry Male, n (%) 101 (70) Age* 69 (43–88) Age at diagnosis* 62 (38–83) Current smokers, n (%) 42 (29) Exsmokers, n (%) 100 (69) Pack-year history* 49 (10–153) Exacerbation rate in previous 12 mo 3 (0.2) Maintenance prednisolone, n (%) 9 (6) Prednisolone dosage, mg* 6 (4–10) Inhaled corticosteroid use, n (%) 125 (86) Inhaled corticosteroid dose, mgx 1,540 (59) Inhaled long-acting b agonist use, n (%) 110 (76)

Study Entry †

FEV1,L FEV1% predicted† Reversibility, ml FEV1/FVC ratio, %† CRQTOTAL, units VASTOTAL, mm Peripheral leukocyte count (3109 cells/L)‡ Peripheral neutrophil count (3109 cells/L)‡ Peripheral eosinophil count (3109 cells/L)‡ Total sputum cell count (3106 cells/g sputum)‡ Sputum neutrophil count, % Sputum eosinophil count, %‡

1.33 52 47 52 4.11 142 8.2 5.3 0.21 3.8 68 1.2

(0.05) (2) (11) (2) (0.10) (6) (7.9–8.6) (5–5.6) (0.18–0.23) (3.1–4.7) (2) (1–1.6)

Exacerbation 1.10 42 37 50 3.12 239 9.3 6.3 0.19 6.4 74 1.1

(0.04) (1) (11) (1) (0.08) (6) (8.9–9.8) (6–6.7) (0.17–0.22) (5.2–7.8) (2) (0.9–1.5)

P Value ,0.001 ,0.001 0.50 0.65 ,0.001 ,0.001 ,0.001 ,0.001 0.84 ,0.001 0.02 0.58

Definition of abbreviations: CRQ ¼ Chronic Respiratory Disease Questionnaire score; VAS ¼ visual analog score. Data presented as mean (SEM) unless stated. * Mean (range). y Post-bronchodilator. z Geometric mean (95% confidence interval). x Beclomethasone dipropionate equivalent.

Factor and Cluster Analysis

Factor analysis identified three biologic factors at exacerbation representing proinflammatory, Th1, and Th2 factors as determined by their cytokine expression profiles (Table E3). Cluster analysis using the highest loading biomarker from each factor (TNFRII, CXCL11, and CCL17) revealed four biologic cluster populations for exacerbation events. Three clusters were termed as “bacteria-predominant,” “eosinophil-predominant,” and “viruspredominant.” A fourth cluster demonstrated low sputum mediator concentrations and had fewer events associated with known etiology and was termed “pauciinflammatory.” Factor mean scores were plotted for each cluster (Figures 2A, and E3A). Biologic cluster ellipsoids were calculated and plotted for all exacerbation events to schematically represent biologic clusters of COPD exacerbations in three dimensions (Figure 2B, Figure E3B). Exacerbation event characteristics of these biologic clusters are presented in Table 2 (Table E5). The baseline characteristics for each subject within each biologic cluster are shown in Tables 2 and E5. Cluster membership was determined using either a patient’s first exacerbation event or the dominant cluster in patients with multiple exacerbations. The intraclass correlation coefficient of the biologic clusters for patients with repeated exacerbations was 0.73. Each biologic cluster was found to be differentially related to inflammation and etiology, but was otherwise clinically indistinguishable. Exacerbations Associated with Bacteria

Fifty-five percent of exacerbations were bacteria-associated exacerbations (positive bacterial pathogen on routine culture or CFU > 107). Blood and sputum neutrophils were increased. Total bacterial load (16S) was higher in patients with a bacteriaassociated exacerbation than those without (geometric mean 7.67 [95% CI, 4.27 to 1.48] vs. 2.88 [95% CI, 1.78 to 4.78]; P ¼ 0.001). There was no difference in the 16S signal across exacerbations of Anthonisen type (analysis of variance; P ¼ 0.64). Using qPCR, acquisition of a new species occurred in 15% of exacerbations. Clinical assessments of change in FEV1, symptoms of sputum production, and sputum purulence had an area under the receiver operating characteristic curve of 0.45 (95% CI, 0.35–0.55), 0.50 (95% CI, 0.40–0.60), and 0.58 (95% CI, 0.48–0.68), respectively. The most suitable biomarker for determining bacteria-associated exacerbations was sputum IL-1b with an area under the receiver operating characteristic curve of 0.89 (95% CI, 0.83–0.95). A cutoff of 125 pg/ml had a

sensitivity of 90% and a specificity of 80% (Figures 3A and E4A). The best serum biomarker was CRP with an area under the receiver operating characteristic curve of 0.65 (95% CI, 0.57– 0.74). A serum CRP cutoff of 10 mg/L had a sensitivity of 60% and specificity of 70%. Exacerbations Associated with Virus

Twenty-nine percent of exacerbations were associated with a virus, most commonly rhinovirus. Virus-associated exacerbations had a larger fall in % FEV1 compared with nonvirus-associated exacerbations (217% vs. 29%; mean difference 28%; 95% CI, 216 to 21; P ¼ 0.04). Clinical assessments of change in FEV1, symptoms of cough and breathlessness, had an area under the receiver operating characteristic curve of 0.43 (95% CI, 0.32– 0.53), 0.62 (95% CI, 0.52–0.72), and 0.51 (95% CI, 0.41–0.62), respectively. The best marker for distinguishing the presence of a virus at exacerbation was serum CXCL10 (IP-10), with an area under the receiver operating characteristic curve of 0.76 (95% CI, 0.67–0.86). A serum CXCL10 cut off of 56 pg/ml gave a sensitivity of 75% and specificity of 65% (Figures 3B and E4B). For exacerbations associated with virus alone the area under the receiver operating characteristic curve for serum CXCL10 improved to 0.83 (95% CI, 0.70–0.96). Exacerbations Associated with Sputum Eosinophilia

A sputum eosinophilia was observed in 28% of exacerbations. The most sensitive and specific measure to determine a sputum eosinophilia at exacerbation was the percentage peripheral blood eosinophil count with an area under the receiver operating characteristic curve of 0.85 (95% CI, 0.78–0.93). A cutoff of 2% peripheral blood eosinophils had a sensitivity of 90% and specificity of 60% for identifying a sputum eosinophilia of greater than 3% at exacerbation (Figure 3C, Figure E4C). In summary, the etiologic and inflammatory causes of exacerbation events were as follows: bacteria alone 37%, virus alone 10%, sputum eosinophilia alone 17%, bacteria plus virus 12%, bacteria plus sputum eosinophilia 6%, virus plus sputum eosinophilia 3%, bacteria plus virus plus sputum eosinophilia 1%, and none 14%. Multivariate modeling using combinations of two or three biomarkers for the detection of bacteria-, virus-, and eosinophilassociated exacerbations did not significantly improve on the single mediators alone (data not shown). Differential clinical and biomarker expression for exacerbations associated with bacteria, virus, and sputum eosinophilia are shown in Tables E4–E6.

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Figure 2. (A) Bar chart representing the mean factor scores for the three identified biologic factors (proinflammatory, Th1, and Th 2) categorized according to the four biologic clusters. (B) Proportional representation of biologic chronic obstructive pulmonary disease exacerbation clusters in three-dimensional ellipsoids. Cluster 1 is termed “bacteriapredominant” and is outlined in blue, cluster 2 is termed “eosinophil-predominant” and is outlined in green, cluster 3 is termed “virus-predominant” and is outlined in red, and cluster 4 is termed “pauciinflammatory” and is outlined in gray.

Predicting Bacteria-, Virus-, or Sputum-associated Exacerbations

The odds ratio for a bacteria or an eosinophil-associated exacerbation was 4.9 (95% CI, 2.4–9.9; P , 0.001) or 2.7 (95% CI, 1.3–5.7; P ¼ 0.01) if the patient had a bacterial pathogen on diagnostic routine culture or a sputum eosinophilia on greater than or equal to one occasion at stable state. The odds ratio for a virus-associated exacerbation if the patient had a virus at stable state was 0.5 (95% CI, 0.1–3.9; P ¼ 0.67). Validation of the Biomarkers Peripheral Blood Eosinophils, Sputum IL-1b, Serum CRP, and Serum CXCL10

In an independent study of COPD exacerbations, 89 subjects (57 men and 32 women) with a mean (range) age of 68 (46–86) years

and mean (SEM) FEV1% predicted of 46 (2) percent sputum IL-1b and serum CXCL10 was measured using a commercial ELISA (R&D Systems, Abingdon, UK). The area under the receiver operating characteristic curve for percentage blood eosinophils to identify a sputum eosinophil–associated exacerbation was 0.95 (95% CI, 0.87–1.00) with a cutoff of 2% having a sensitivity and specificity of 90% and 60%. The area under the receiver operating characteristic curve for sputum IL-1b and serum CRP to identify a bacteria-associated exacerbation was 0.73 (95% CI, 0.61–0.85) and 0.70 (95% CI, 0.59–0.82); a sputum IL-1b cutoff of 130 pg/ml had a sensitivity and specificity of 80% and 60%, and a serum CRP cutoff of 10 mg/L had sensitivity and specificity of 65%. The area under the receiver operating characteristic curve for serum CXCL10 to identify a virus-associated exacerbation was 0.65 (95% CI,

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TABLE 2. BIOLOGIC CLUSTERS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE EXACERBATIONS, WITH CLINICAL EXACERBATION AND BASELINE CHARACTERISTICS

Exacerbation characteristics Number (%) Sputum TNFRII (pg/ml)* Sputum CXCL11 (pg/ml)* Sputum CCL17 (pg/ml)* Bacterial exacerbation, % (95% CI) Viral exacerbation, % (95% CI) Eosinophilic exacerbation, % (95% CI) D FEV1, ml† D CRQ, units † D VASTOTAL, mm† Baseline characteristics Number, (%) Male, n (%) Age, yrs‡ Current smokers, n (%) Pack-years smoked‡ Exacerbation rate in previous 12 mo Exacerbation rate during study Inhaled corticosteroid dose, mgx Residual volume, % TLCO % predicted FEV1% predicted, baseline FEV1/FVC ratio (%) CRQTOTAL , units VASTOTAL , mm Total sputum cell count (3106 cells/g)* Sputum neutrophil count, % Sputum eosinophil count, %* Bacterial colonization, % (95% CI)

Cluster 1: Bacteria-predominant

Cluster 2: Eosinophil-predominant

Cluster 3: Virus-predominant

Cluster 4: Pauciinflammatory

52 1,722 3.1 5.5 86 22 6 2132 20.9 79

(35) (1,402–2,117) (2.2–4.3) (4.5–6.7) (73–92) (13–35) (1–16) (2251 to 235) (21.2 to 20.6) (42–116)

44 353 10.9 34.8 29 10 60 2110 20.9 80

(30) (287–433) (7.7–15.5) (27.3–44.5) (18–45) (3–23) (45–74) (2230 to 231) (21.3 to 20.5) (41–119)

36 1,254 799 23.5 44 57 28 2232 20.9 120

(24) (969–1,623) (415–1,539) (16.2–34.1) (28–61) (39–73) (16–44) (2340 to 2124) (21.4 to 20.4) (86–154)

16 77 17.3 4.7 31 30 27 2280 21 73

(11) (41–147) (5.6–53.1) (3.5–6.3) (12–58) (10–61) (10–52) (2524 to 236) (21.9 to 20.1) (38–108)

28 18 69 8 44 3.8 3.8 1,507 134 56 53 51 4.14 178 8.3 75 1 63

(37) (64) (52–84) (29) (10–122) (0.5) (0.3) (147) (8) (5) (3) (2) (0.20) (15) (5.5–12.5) (5) (0.6–1.6) (48–77)

19 14 68 8 50 4.3 3.6 1,567 150 59 51 47 3.90 142 2.3 53 3.1 27

(25) (74) (45–88) (42) (20–106) (0.5) (0.4) (133) (9) (5) (5) (2) (0.22) (18) (1.6–3.2) (4) (1.4–6.6) (15–43)

20 14 70 4 47 4 3.2 1,470 120 57 53 50 4.10 124 2.5 68 1 11

(27) (70) (49–84) (20) (10–134) (0.7) (0.3) (160) (8) (6) (5) (3) (0.26) (18) (1.2–5.3) (4) (0.5–1.9) (3–29)

8 7 69 3 72 4.9 3.1 1,150 146 46 40 47 3.66 147 3.5 81 0.5 38

(11) (88) (61–85) (38) (23–120) (1.2) (0.5) (188) (23) (7) (7) (5) (0.50) (37) (1.2–10.7) (6) (0.2–1) (18–61)

P Value — ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 0.32 0.99 0.39 — 0.63 0.62 0.48 0.11 0.58 0.64 0.55 0.11 0.62 0.34 0.67 0.74 0.14 0.002 0.003 0.012 0.001

Definition of abbreviations: CCL ¼ ; CI ¼ confidence interval; CRQ ¼ Chronic Respiratory Disease Questionnaire score; CXCL ¼ ; TLCO ¼ carbon monoxide transfer factor; TNF ¼ tumor necrosis factor; VAS ¼ visual analog score. Data presented as mean (SEM), unless stated. * Geometric mean (95% CI). y Mean change (95% CI) between exacerbation and baseline. z Mean (range). x Beclomethasone dipropionate equivalent.

0.52–0.78) with a cutoff of 145 pg/ml having a sensitivity and specificity of 70% and 60%. Further details and results are available in the online supplement.

DISCUSSION In this study we have used two methods to investigate biomarkers in COPD. The first using unbiased statistical tools, free from bias and independent of clinical expression, identified biologic COPD exacerbation phenotypes and characterized exacerbations into four biologic clusters, The second method used current clinical exacerbation phenotypes of COPD related to potential etiology and inflammation, namely exacerbations that are associated with bacteria, virus, or a sputum eosinophilia. Interestingly, we were unable to define biomarkers for exacerbations per se, despite a generalized increase in systemic and airway inflammation (34–36). The biologic exacerbation clusters were bacterial-, viral-, or eosinophilic-predominant, and a fourth was associated with limited changes in the inflammatory profile and was termed “pauciinflammatory.” These clusters were remarkably similar to our clinical exacerbation phenotypes. We identified biomarkers for our clinical exacerbation phenotypes that were then validated in an independent cohort. The bacteriaand sputum eosinophil–associated exacerbations rarely coexisted, and were reliably predicted from stable state suggesting fundamental differences in their immunopathogenesis. Therefore, in addition to identifying potential biomarkers to direct therapy,

these exacerbation clinical phenotypes are likely to represent distinct pathophysiologic entities with specific biomarker signatures. Biomarker profiling in COPD exacerbations has the potential to further the understanding of disease mechanisms (22), whereas phenotypic approaches lend to prognostic and therapeutic strategies (37, 38). Using factor and cluster analysis, a novel approach of characterizing COPD and exacerbations (23), we were able to reduce an extensive panel of measured sputum biomarkers into three factors, from which we determined four biologic clusters. This analytic strategy is free from investigator bias. These biologic clusters could not be distinguished clinically or by Anthonisen criteria (14) and the exacerbation severity was similar across the clusters. Importantly, using factor analysis we have shown differential inflammatory profiles between the bacteria-predominant, eosinophil-predominant, virus-predominant, and pauciinflammatory clusters. In patients with multiple exacerbations the biologic clusters were repeatable, and exacerbations associated with bacteria or a sputum eosinophilia but not viruses could be predicted from stable state. Therefore, our data are consistent with the view that bacterial and eosinophilic exacerbations may reflect instability within a complex and inherently unstable system, whereas viral exacerbations are more likely to represent acquisition of a new pathogen. It is likely both of these mechanisms drive exacerbations, but critically we have determined biologic clusters and clinical phenotypes that may respond to different management strategies, which can potentially be identified using biomarker profiles.

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Figure 3. Receiver operating characteristic curve with area under the curve (95% confidence interval) illustrating biomarkers that positively predict (A) bacteria-, (B) virus-, and (C) eosinophil-associated exacerbations. Area under the curve (95% confidence interval) is shown in the parentheses. CCL ¼ ; CRP ¼ C-reactive protein; CXCL ¼ ; TNF ¼ tumor necrosis factor.

Bafadhel, McKenna, Terry, et al.: Biomarkers in COPD Exacerbations

The inflammatory profile of a COPD exacerbation is typically neutrophilic, but eosinophilic airway inflammation also exists, and is associated with a favorable response to corticosteroid therapy (8–10). Eosinophilia in inflammatory airways disease is associated with increased all-cause mortality (39, 40) and may highlight different genetic, biologic, and pathologic disease processes. Importantly, the sputum differential rather than total eosinophil count has consistently been shown to be associated with important clinical outcomes (9, 10). We found that the peripheral percentage eosinophil count was a sensitive biomarker of a sputum eosinophilia. Current guidelines recommend the use of systemic corticosteroids for COPD exacerbations, although the magnitude of the benefit is marginal and their use associated with significant side effects (18). Our findings raise the possibility that targeting corticosteroid therapy in a subgroup of exacerbations dependent on the peripheral eosinophil count may reduce inappropriate use of systemic corticosteroids. Bacteria are considered to play a role in up to 50% of exacerbations (7). Current guidelines propose sputum purulence to guide antibiotic therapy (13). Sputum purulence is sensitive for detecting bacterial culture or high bacterial yields at exacerbation in COPD (12). However, the use of sputum purulence alone is confounded by its presence at stable state and chronic bacterial colonization (41), possibly as a consequence of poor bacterial clearance (42). Furthermore, the change in sputum purulence or sputum production symptoms in our cohort was not sensitive or specific for identifying a bacteria-associated exacerbation. The most sensitive and specific assay for determining bacteria-associated exacerbations was sputum IL-1b. This extends previous findings that bronchoalveolar lavage IL1b was a good biomarker for ventilator-associated pneumonia (43), and suggests that this airway marker may suitably determine bacterial infections, above that of serum CRP or procalcitonin whose use could not be demonstrated in this study or in others (34, 35). Sputum IL-1b could thus be used as a biomarker to correctly identify bacteria-associated exacerbations but would require the development of a rapid near patient test to be of use in clinical practice. Viruses have been implicated as a major cause of COPD exacerbations and are detected in approximately half of severe COPD exacerbations (5, 6). The total sputum eosinophil count has been proposed as a potential biomarker of a viral exacerbation (5). Here we also found that the total absolute sputum eosinophil count was increased in virus-associated exacerbations, but not the differential sputum eosinophil count, suggesting the association was largely a consequence of a change in the totalcell count. The application of clinical symptoms in combination with serum CXCL10 (IP-10) has been proposed as a possible biomarker for rhinovirus infection at exacerbation (44). This study confirms that serum CXCL10 levels as a potential predictor of a virus-associated exacerbation, independent of a requirement for symptom evaluation. Novel antiviral approaches are in development and CXCL10 is thus a promising biomarker to direct future antiviral therapy. One potential criticism is that this is a single-center study and therefore our findings need to be replicated across multiple centers, and validated prospectively to identify the biologic clusters and our proposed biomarkers for the clinical exacerbation phenotypes; nonetheless, this approach may represent a new paradigm in the management of COPD exacerbations. Importantly, we have replicated the biomarkers peripheral blood eosinophils, sputum IL-1b, and serum CXCL10 in a validation cohort. Peripheral blood eosinophils remained a strong marker of a sputum eosinophilia. Sputum IL-1b and serum CXCL10 were measured using a different platform but remained significant albeit weaker predictive markers of identifying a bacteria- or virus-associated

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exacerbation. In our study a statistical analytic limitation was that we did not correct for repeated measures and assessed changes in biomarkers in paired or unpaired tests; however, we examined two methods to investigate biomarkers in COPD exacerbations, using unbiased statistical tools to demonstrate four biologic clusters and analysis of biomarkers to look at predefined clinical exacerbation subgroups, and further used multivariate analysis to determine that combinations of markers did not improve our predictive model. The presence of coinfection with virus and bacteria in our study was lower than that previously reported (5), but may reflect differences in the severity of exacerbations. The relationship between virus and bacterial infection in exacerbations, however, remains poorly understood (5, 45). We chose to define a bacteria-associated exacerbation based on a positive routine culture or a high bacterial load; however, the causal links between the presence of bacteria and exacerbations has not been rigorously confirmed, and evidence for efficacy of antibiotics in treatment is conflicting (13, 19–21, 46). Developments in molecular bacterial identification of bacteria (47) and emerging microbiomics (48) are beginning to redefine the microbiota of the airway in health and disease and will likely change the view of what defines a bacterial infection. Improvements in viral detection and the identification of new respiratory viruses are also changing the understanding of the associations between exacerbations and these agents. Further work is required before therapeutic implications and interpretative criteria can be established for these sensitive detection methods. Whether identification of a pauciinflammatory biologic cluster and a proportion of subjects without clear evidence of a cause for their exacerbation reflect the insensitivity of our chosen cutoffs for definitions or a real entity requires further clarifications. In conclusion, COPD exacerbations are heterogeneous. This phenotypic heterogeneity can be defined. Using unbiased statistical tools we have determined four biologic exacerbation clusters that relate to identifiable patterns of inflammation and potential causative pathogens. We have defined sensitive and specific biomarkers to identify predefined clinical exacerbation phenotypes, which need to be tested in randomized prospective studies of targeted therapy. These subgroups are independent and suggest that the mechanisms driving their exacerbations are distinct and may be amenable to more specific interventions, potentially moving the management of COPD exacerbations toward the realization of phenotype-specific management. Author Disclosure: M.B. received grant support from the Medical Research Council (MRC). S.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. V.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.M. is employed by and owns stocks in AstraZeneca (AZ). K.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.R., P.D., and M.J. are employed by and own stocks in AZ. M.S. is employed by AZ. P.N. was an employee of AZ at the time of conducting this research and preparation of the manuscript. He is now an employee of MedImmune LLC, which is a subsidiary of AZ and owns stocks in AZ. R.H.G. was a consultant for Nycomed and received travel accommodations from Chiesi. P.V. received institutional grant support from the Swedish Research Council. D.A.L. received institutional grant support and was a consultant for GlaxoSmithKline (GSK). He is on the Advisory Board and received honorarium from GSK. M.R.B. received institutional grant support form the MRC. S.L.J. was a consultant for AZ, Centocor, Sanofi-Pasteur, Synairgen, GSK, and Chiesi. He received

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institutional grant support from AZ, Centocor, Sanofi-Pasteur, Synairgen, and AZ. He received lecture fees from AZ, owns stocks in Synairgen, and received travel accommodations from Pfizer. I.D.P. received institutional grant support from the MRC and received honorarium from GSK, AZ, Merck, and Novartis. He received travel accommodations from Boehringer Ingelheim (BI). C.E.B. received institutional grant support from the MRC, AZ, MedImmune, and Roche. He received support for the development of SPD assays in Cambridge from GSK. He is on the Advisory Board of GSK, AZ, Roche, Novartis, Genentech, and MedImmune and was a consultant for MedImmune and Novartis. He received travel accommodations from BI. Acknowledgment: The authors thank all the research volunteers who participated in the study, and also the following people for their valuable assistance throughout the study: J. Aniscenko, M. Bourne, R. Braithwaite, D. Burke, J. Footitt, E. Goldie, J. Goldie, N. Goodman, S. Gupta, B. Hargadon, I. Rushby, M. Shelley, A. Singapuri, D. Vara, R. Walton, and S. Winpress.

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