Acute Exacerbations of Chronic ObstructivePulmonary DiseaseIdentiﬁcation of Biologic Clusters and Their BiomarkersMona 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. Brightling1,21 Institute for Lung Health, and 2Department of Infection, Immunity and Inﬂammation, University of Leicester, Leicester, United Kingdom;3 AstraZeneca R&D Charnwood, Loughborough, Leicestershire, United Kingdom; 4Department of Respiratory Medicine, National Heart and LungInstitute, Centre for Respiratory Infections, Imperial College London, United Kingdom; 5Cambridge Institute for Medical Research, University ofCambridge, Cambridge, United Kingdom; 6Department of Medical Sciences, Clinical Chemistry, University of Uppsala, Uppsala, Sweden; and7 Department of Clinical Microbiology, University Hospitals of Leicester NHS Trust, Leicester, United KingdomRationale: Exacerbations of chronic obstructive pulmonary disease(COPD) are heterogeneous with respect to inﬂammation and etiology. AT A GLANCE COMMENTARYObjectives: Investigate biomarker expression in COPD exacerbationsto identify biologic clusters and determine biomarkers that recog- Scientiﬁc Knowledge on the Subjectnize clinical COPD exacerbation phenotypes, namely those associ- Exacerbations of chronic obstructive pulmonary diseaseated with bacteria, viruses, or eosinophilic airway inﬂammation. (COPD) are a major health burden worldwide, and affectMethods: Patients with COPD were observed for 1 year at stable and a vulnerable population at risk of signiﬁcant comorbidities.exacerbation visits. Biomarkers were measured in sputum and COPD exacerbations are heterogeneous with respect toserum. Viruses and selected bacteria were assessed in sputum by etiology and inﬂammation and biomarkers are required topolymerase chain reaction and routine diagnostic bacterial culture. phenotype this heterogeneity.Biologic phenotypes were explored using unbiased cluster analysisand biomarkers that differentiated clinical exacerbation phenotypeswere investigated. What This Study Adds to the FieldMeasurements and Main Results: A total of 145 patients (101 menand 44 women) entered the study. A total of 182 exacerbations We have shown that there are biologic COPD exacerbationwere captured from 86 patients. Four distinct biologic exacerbation clusters that are clinically indistinguishable, and that bio-clusters were identiﬁed. These were bacterial-, viral-, or eosinophilic- markers can be used to identify speciﬁc clinical phenotypespredominant, and a fourth associated with limited changes in the during exacerbations of COPD (speciﬁcally those associ-inﬂammatory proﬁle termed “pauciinﬂammatory.” Of all exacerba- ated with bacteria, virus, and sputum eosinophilia). Bac-tions, 55%, 29%, and 28% were associated with bacteria, virus, or a terial and eosinophilic clinical exacerbation phenotypes can be identiﬁed from stable state. Our data further delineate the heterogeneity during COPD exacerbations and may(Received in original form April 2, 2011; accepted in ﬁnal form June 15, 2011) identify populations that appropriately require cortico-Supported by the Medical Research Council (United Kingdom) and AstraZeneca steroids and antibiotics at the onset of an exacerbation.jointly as a “Biomarker Call Project”; C.E.B. is a Wellcome Trust Senior ClinicalFellow, and GlaxoSmithKline supported the measurement of surfactant proteinD. The research was performed in laboratories partly funded by the EuropeanRegional Development Fund (ERDF 05567). The Medical Research Council, Well-come Trust, and the European Regional Development Fund had no involvement sputum eosinophilia. The biomarkers that best identiﬁed these clinicalin the design of the study, data collection, analysis and interpretation of the data, phenotypes were sputum IL-1b, 0.89 (area under receiver operatingin the writing of the manuscript, or in the decision to submit the manuscript. characteristic curve) (95% conﬁdence interval [CI], 0.83–0.95); serumAuthor Contributions: S.M. and S.T. were involved in the recruitment of volunteers CXCL10, 0.83 (95% CI, 0.70–0.96); and percentage peripheral eosino-and in data collection. C.R., V.M., K.H., H.P., A.D., and K.L. were involved in data phils, 0.85 (95% CI, 0.78–0.93), respectively.collection and interpretation. M.M., P.R., P.D., P.N., M.J., and M.S. were involved Conclusions: The heterogeneity of the biologic response of COPDin study design, data collection, and interpretation. R.H.G. and P.H. were in- exacerbations can be deﬁned. Sputum IL-1b, serum CXCL10, andvolved in study design and data interpretation. M.R.B., D.A.L., S.L.J., P.V., andI.D.P. were involved in the design of the study, data collection, and interpreta- peripheral eosinophils are biomarkers of bacteria-, virus-, or eosinophil-tion. M.B. and C.E.B. were involved in the study design, volunteer recruitment, associated exacerbations of COPD. Whether phenotype-speciﬁc bio-data collection, data interpretation, and data analysis, and had full access to the markers can be applied to direct therapy warrants further investigation.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 Keywords: chronic obstructive pulmonary disease; phenotypes; exac-ﬁnal version for submission. erbations; airway inﬂammation; infectionCorrespondence and requests for reprints should be addressed to ChristopherE. Brightling, M.B.B.S., B.Sc. (Hons.), Ph.D., Institute for Lung Health, Clinical Acute exacerbations of chronic obstructive pulmonary diseaseSciences Wing, University Hospitals of Leicester, Leicester, LE3 9QP, UK. E-mail: (COPD) are associated with substantial morbidity and firstname.lastname@example.org (1, 2). Exacerbations are typically associated with increased neu-This article has an online supplement, which is accessible from this issue’s table of trophilic and to a lesser extent eosinophilic airway inﬂammationcontents at www.atsjournals.org (3, 4). Respiratory viral and bacterial infections have been impli-Am J Respir Crit Care Med Vol 184. pp 662–671, 2011Originally Published in Press as DOI: 10.1164/rccm.201104-0597OC on June 16, 2011 cated in causing most exacerbations (5–7), but how these infec-Internet address: www.atsjournals.org tions alter lower airway inﬂammation and relate to treatment
Bafadhel, McKenna, Terry, et al.: Biomarkers in COPD Exacerbations 663response is not completely understood. This heterogeneity trans- and single ELISA at stable and exacerbation visits (see Table E1 in thelates that at present clinicians have limited tools to phenotype online supplement).exacerbations. During stable state a sputum eosinophilia is asso-ciated with corticosteroid responsiveness (8–10), whereas the Deﬁnition of Bacteria-, Virus-, and Sputumpresence of a high bacterial load and sputum purulence has favor- Eosinophil–associated Exacerbations of COPDable outcomes with antibiotics (11–15). These ﬁndings suggest Bacteria-associated exacerbations were deﬁned as a positive bacterialthat it is possible to identify clinically important COPD exacer- pathogen on routine culture (Haemophilus inﬂuenzae, Moraxella catar-bation phenotypes. This is crucial because systemic corticosteroids rhalis, Streptococcus pneumoniae, Staphylococcus aureus, or Pseudo-and antibiotics have marginal efﬁcacy (16–21) and the potential to monas aeruginosa) or a total aerobic CFU count greater than orcause adverse events in an already vulnerable population. equal to 107 cells (12, 15). qPCR bacterial detection methods were We hypothesize that approaches aimed at the identiﬁcation of not used to deﬁne bacteria-associated exacerbations in this study.COPD exacerbation phenotypes may allow for better prognostic, A virus-associated exacerbation was deﬁned as one that had a positivetherapeutic, and mechanistic applications (22–24). In this study sputum viral PCR, whether in isolation or in combination with a positive bacterial pathogen on routine culture. A sputum eosinophil–associatedwe investigated whether during exacerbations of COPD there are exacerbation was deﬁned as the presence of more than 3% nonsquamous(1) deﬁnable biologic phenotypes using unbiased mathematical cells.tools (namely factor and cluster analysis); (2) identiﬁable bio-markers associated with clinical phenotypes, speciﬁcally those Statistical Analysesassociated with bacteria, viruses, or sputum eosinophilia; and(3) exacerbation phenotypes that can be predicted from stable Statistical analyses were performed using PRISM version 4 (Graph-state. PAD 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 areMETHODS presented as geometric mean (95% conﬁdence interval [CI]). Multi- variate modeling using principal component analysis in sputum bio-Patients markers was used to explore biomarker pattern expression atPatients with a physician diagnosis of COPD and a post-bronchodilator exacerbations. No adjustments for multiple comparisons have beenFEV1/FVC ratio of less than 0.7 as per global initiative for chronic made across biomarkers.obstructive lung disease (GOLD) criteria (1) were recruited from the Factor analysis, a mathematical method that discovers patterns ofGlenﬁeld Hospital, Leicester, United Kingdom, and through local ad- relationships within large datasets, was used to identify factors in sputumvertising. All patients fulﬁlled the inclusion criteria of age greater than mediators at exacerbations thereby demonstrating biologic factors inde-40 years, GOLD stage I–IV, and greater than or equal to one exacer- pendent of each other and of any clinical expression. This method usingbation in the preceding 12 months deﬁned as the requirement of emer- unsupervised principal component analysis demonstrated three factorsgency health care (25). Patients were excluded if there was a documented accounting for 75% of the total variance (see Table E2). Cluster analysis,inability to produce sputum after the induced sputum procedure, a cur- an unbiased mathematical method, allows one to classify groups onrent or previous history of asthma, currently active pulmonary tubercu- similar chosen characteristics alone. Thus, after demonstrating three bi-losis, or any other clinically relevant lung disease other than COPD. The ologic factors, we used hierarchical cluster analysis to generate four bi-presence of comorbidity, reported atopy to common aeroallergens, or ologic clusters for exacerbation events and cases. Clinical characteristicsreversibility on lung function testing was not an exclusion criterion. All for all exacerbation events were tabulated for each biologic cluster. One-patients gave informed written consent and the study was approved by way analysis of variance, Kruskal-Wallis test, and the chi-square testthe local ethics committee. 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 testStudy Design was used. For comparison of exacerbations associated with or withoutThis was a prospective observational study. Patients were seen at stable bacteria, virus, and sputum eosinophilia the t test and Mann-Whitneystate and during exacerbations for the duration of 1 year. Stable visits test were used, respectively. To determine suitable biomarkers, theincluding the baseline visits were 8 weeks free from an exacerbation. All receiver operating characteristic curves were plotted for (1) exacerba-patients were given daily diary cards to complete, and asked to contact tion versus stable state; (2) bacteria- versus nonbacteria-associatedthe research team if there was an increase in symptoms of breathless- exacerbations; (3) virus- versus nonvirus-associated exacerbations;ness, sputum volume, and purulence. Exacerbations were deﬁned and (4) sputum eosinophilia– (. 3% nonsquamous cells) versus non-according to Anthonisen criteria (14) and health care use (25). Exac- sputum eosinophilia–associated exacerbations.erbation data recording and sampling were only performed in patients Validation of the identiﬁed biomarkers for bacteria-, virus-, and spu-who had not received prior oral corticosteroids or antibiotics. Patients tum eosinophil–associated exacerbation was performed in a further 89were all clinically assessed (including chest radiograph, temperature exacerbation events from an independent cohort of subjects withrecording, and blood gas analysis if clinically necessary) to exclude COPD. These subjects with COPD were recruited to enter a prospec-other causes of breathlessness. Patients with an exacerbation of COPD tive study with identical inclusion and exclusion criteria and study de-were then treated according to guidelines (2). sign 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 statisticalMeasurements signiﬁcance.At all visits, patients underwent pre– and post–400-mg albuterol broncho-dilator spirometry (Vitalograph, Buckingham, Buckinghamshire, UK); in- RESULTSduced or spontaneous sputum collection (26); and measurements ofsymptoms and health quality assessments using the Visual Analog Scale One hundred ﬁfty-six patients were enrolled; 145 (101 men and(27) and the Chronic Respiratory Disease Interviewer-Administered Stan- 44 women) completed the ﬁrst visit and 115 completed 12dardized Questionnaire (CRQ) (28). Sputum was collected and analyzed months (Figure 1; see Figure E1). At baseline 3%, 48%,for bacteria (29–31) (using standard routine culture, CFU, and real-time 30%, and 19% had GOLD I, II, III, and IV, respectively. Mostquantitative polymerase chain reaction [qPCR]), for viruses by PCR (32),and processed to produce cytospins for cell differential and supernatant patients recruited were current or exsmokers (142 of 145),for ﬂuid phase measurements (33). A broad panel of serum and sputum with a mean (range) pack-year history of 49 (10–153) withbiomarkers were measured using the Meso-Scale-Discovery (MSD, an absolute and percentage mean (SEM) reversibility to in-Gaithersburg, MD) platform standard preprepared plates (MSD, MD) haled bronchodilator on study entry of 47 ml (11 patients) and
664 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 184 2011 Figure 1. Subject enrolment ﬂow diagram for 12-month observational period.4% (one patient), respectively. Skin prick testing or serum- GOLD severity (1), or Anthonisen criteria (14). Hospitalizedspeciﬁc IgE to a wide panel of aeroallergens conﬁrmed that exacerbations were associated with a greater decline in lung20% were atopic. Bacterial colonization, deﬁned as the pres- function compared with exacerbations that were not hospital-ence of a potentially pathogenic microorganism (H. inﬂuen- ized (DFEV1 [ml] 2355 vs. 2131; mean difference 224; 95% CIzae, M. catarrhalis, S. pneumoniae, S. aureus, or P. aeruginosa) of difference, 2356 to 292; P , 0.001), but not health statusin a standard culture technique (29), was present in 28% of decline (DCRQ [units] 21.25 vs. 20.91; mean difference 0.34;patients at baseline. Using qPCR a bacterial pathogen (H. 95% CI of difference, 20.83 to 0.15; P ¼ 0.18).inﬂuenzae, M. catarrhalis, S. pneumoniae, or S. aureus) was Serum and sputum mediator data were available in 148 ex-detected in 86% of patients at the baseline stable visit. A virus acerbation events from 75 patients. Serum biomarkers that in-was detected in 5% of subjects at study entry, whereas eosin- creased during an exacerbation were IL-6, tumor necrosisophilic airway inﬂammation (. 3% nonsquamous cells) was factor (TNF) receptors I and II, serum amyloid-A, C-reactive pro-present in 27% of patients. Baseline and exacerbation clinical tein (CRP), procalcitonin, and serum eosinophil cationic proteincharacteristics are shown in Table 1 (see Table E3). (Table E4A). Sputum biomarkers that increased were IL-1b, TNF-a, TNFRI, TNFRII, IL6, CCL5, and CCL4 (Table E4B).Exacerbations No single biomarker had a receiver operating curve area underA total of 182 exacerbation events were captured from 86 the curve greater than 0.70 in determining an exacerbationpatients; of these 21 exacerbations warranted hospitalization. from stable state (Figure E2). Of all sputum and serum bio-There was a reduction in the FEV1 and CRQ from baseline markers measured there was a signiﬁcantly increased level ofto exacerbation (FEV1 [L] 1.33 vs. 1.10; mean difference 0.24; serum TNF-a and CRP in patients who were hospitalized95% CI, 0.12–0.36; P , 0.001) (CRQ [units] 4.11 vs. 3.12; mean (CRP median [IQR] 56 (102) vs. 8 (14); P ¼ 0.002) (serumdifference 0.99; 95% CI, 0.74–1.23; P , 0.001). The magnitude TNF-a geometric mean 4.3 [95% CI, 3.4–5.4] vs. 3.4 [95%of these changes was independent of smoking status, sex, CI, 3.2–3.6]; P ¼ 0.02).
Bafadhel, McKenna, Terry, et al.: Biomarkers in COPD Exacerbations 665TABLE 1. CLINICAL CHARACTERISTICS OF ALL PATIENTS AT ENTRY INTO THE STUDY AND CLINICAL FEATURES AT EXACERBATION Study Entry Study Entry Exacerbation P ValueMale, n (%) 101 (70) FEV1,L† 1.33 (0.05) 1.10 (0.04) ,0.001Age* 69 (43–88) FEV1% predicted† 52 (2) 42 (1) ,0.001Age at diagnosis* 62 (38–83) Reversibility, ml 47 (11) 37 (11) 0.50Current smokers, n (%) 42 (29) FEV1/FVC ratio, %† 52 (2) 50 (1) 0.65Exsmokers, n (%) 100 (69) CRQTOTAL, units 4.11 (0.10) 3.12 (0.08) ,0.001Pack-year history* 49 (10–153) VASTOTAL, mm 142 (6) 239 (6) ,0.001Exacerbation rate in previous 12 mo 3 (0.2) Peripheral leukocyte count (3109 cells/L)‡ 8.2 (7.9–8.6) 9.3 (8.9–9.8) ,0.001Maintenance prednisolone, n (%) 9 (6) Peripheral neutrophil count (3109 cells/L)‡ 5.3 (5–5.6) 6.3 (6–6.7) ,0.001Prednisolone dosage, mg* 6 (4–10) Peripheral eosinophil count (3109 cells/L)‡ 0.21 (0.18–0.23) 0.19 (0.17–0.22) 0.84Inhaled corticosteroid use, n (%) 125 (86) Total sputum cell count (3106 cells/g sputum)‡ 3.8 (3.1–4.7) 6.4 (5.2–7.8) ,0.001Inhaled corticosteroid dose, mgx 1,540 (59) Sputum neutrophil count, % 68 (2) 74 (2) 0.02Inhaled long-acting b agonist use, n (%) 110 (76) Sputum eosinophil count, %‡ 1.2 (1–1.6) 1.1 (0.9–1.5) 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 sensitivity of 90% and a speciﬁcity of 80% (Figures 3A andFactor analysis identiﬁed three biologic factors at exacerbation E4A). The best serum biomarker was CRP with an area underrepresenting proinﬂammatory, Th1, and Th2 factors as deter- the receiver operating characteristic curve of 0.65 (95% CI, 0.57–mined by their cytokine expression proﬁles (Table E3). Cluster 0.74). A serum CRP cutoff of 10 mg/L had a sensitivity of 60%analysis using the highest loading biomarker from each factor and speciﬁcity of 70%.(TNFRII, CXCL11, and CCL17) revealed four biologic clusterpopulations for exacerbation events. Three clusters were termed Exacerbations Associated with Virusas “bacteria-predominant,” “eosinophil-predominant,” and “virus- Twenty-nine percent of exacerbations were associated with a vi-predominant.” A fourth cluster demonstrated low sputum medi- rus, most commonly rhinovirus. Virus-associated exacerbationsator concentrations and had fewer events associated with had a larger fall in % FEV1 compared with nonvirus-associatedknown etiology and was termed “pauciinﬂammatory.” Factor exacerbations (217% vs. 29%; mean difference 28%; 95% CI,mean scores were plotted for each cluster (Figures 2A, and 216 to 21; P ¼ 0.04). Clinical assessments of change in FEV1,E3A). Biologic cluster ellipsoids were calculated and plotted symptoms of cough and breathlessness, had an area under thefor all exacerbation events to schematically represent biologic receiver operating characteristic curve of 0.43 (95% CI, 0.32–clusters of COPD exacerbations in three dimensions (Figure 0.53), 0.62 (95% CI, 0.52–0.72), and 0.51 (95% CI, 0.41–0.62),2B, Figure E3B). Exacerbation event characteristics of these respectively. The best marker for distinguishing the presence ofbiologic clusters are presented in Table 2 (Table E5). The base- a virus at exacerbation was serum CXCL10 (IP-10), with anline characteristics for each subject within each biologic cluster area under the receiver operating characteristic curve of 0.76are shown in Tables 2 and E5. Cluster membership was deter- (95% CI, 0.67–0.86). A serum CXCL10 cut off of 56 pg/ml gavemined using either a patient’s ﬁrst exacerbation event or the a sensitivity of 75% and speciﬁcity of 65% (Figures 3B anddominant cluster in patients with multiple exacerbations. The E4B). For exacerbations associated with virus alone the areaintraclass correlation coefﬁcient of the biologic clusters for under the receiver operating characteristic curve for serumpatients with repeated exacerbations was 0.73. Each biologic CXCL10 improved to 0.83 (95% CI, 0.70–0.96).cluster was found to be differentially related to inﬂammationand etiology, but was otherwise clinically indistinguishable. Exacerbations Associated with Sputum Eosinophilia A sputum eosinophilia was observed in 28% of exacerbations.Exacerbations Associated with Bacteria The most sensitive and speciﬁc measure to determine a sputumFifty-ﬁve percent of exacerbations were bacteria-associated eosinophilia at exacerbation was the percentage peripheralexacerbations (positive bacterial pathogen on routine culture blood eosinophil count with an area under the receiver operatingor CFU > 107). Blood and sputum neutrophils were increased. characteristic curve of 0.85 (95% CI, 0.78–0.93). A cutoff of 2%Total bacterial load (16S) was higher in patients with a bacteria- peripheral blood eosinophils had a sensitivity of 90% and spec-associated exacerbation than those without (geometric mean iﬁcity of 60% for identifying a sputum eosinophilia of greater7.67 [95% CI, 4.27 to 1.48] vs. 2.88 [95% CI, 1.78 to 4.78]; P ¼ than 3% at exacerbation (Figure 3C, Figure E4C).0.001). There was no difference in the 16S signal across exacer- In summary, the etiologic and inﬂammatory causes of exacerba-bations of Anthonisen type (analysis of variance; P ¼ 0.64). tion events were as follows: bacteria alone 37%, virus alone 10%,Using qPCR, acquisition of a new species occurred in 15% of sputum eosinophilia alone 17%, bacteria plus virus 12%, bacteriaexacerbations. Clinical assessments of change in FEV1, symp- plus sputum eosinophilia 6%, virus plus sputum eosinophilia 3%,toms of sputum production, and sputum purulence had an area bacteria plus virus plus sputum eosinophilia 1%, and none 14%.under the receiver operating characteristic curve of 0.45 (95% Multivariate modeling using combinations of two or threeCI, 0.35–0.55), 0.50 (95% CI, 0.40–0.60), and 0.58 (95% CI, biomarkers for the detection of bacteria-, virus-, and eosinophil-0.48–0.68), respectively. The most suitable biomarker for deter- associated exacerbations did not signiﬁcantly improve on the singlemining bacteria-associated exacerbations was sputum IL-1b mediators alone (data not shown). Differential clinical and bio-with an area under the receiver operating characteristic curve marker expression for exacerbations associated with bacteria,of 0.89 (95% CI, 0.83–0.95). A cutoff of 125 pg/ml had a virus, and sputum eosinophilia are shown in Tables E4–E6.
666 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 184 2011 Figure 2. (A) Bar chart representing the mean factor scores for the three identi- ﬁed biologic factors (proinﬂammatory, Th1, and Th 2) categorized according to the four biologic clusters. (B) Propor- tional representation of biologic chronic obstructive pulmonary disease exacerba- tion clusters in three-dimensional ellip- soids. Cluster 1 is termed “bacteria- predominant” and is outlined in blue, cluster 2 is termed “eosinophil-predomi- nant” and is outlined in green, cluster 3 is termed “virus-predominant” and is out- lined in red, and cluster 4 is termed “pau- ciinﬂammatory” and is outlined in gray.Predicting Bacteria-, Virus-, or and mean (SEM) FEV1% predicted of 46 (2) percent sputumSputum-associated Exacerbations IL-1b and serum CXCL10 was measured using a commercialThe odds ratio for a bacteria or an eosinophil-associated exac- ELISA (R&D Systems, Abingdon, UK). The area under theerbation was 4.9 (95% CI, 2.4–9.9; P , 0.001) or 2.7 (95% CI, receiver operating characteristic curve for percentage blood1.3–5.7; P ¼ 0.01) if the patient had a bacterial pathogen on eosinophils to identify a sputum eosinophil–associated exacer-diagnostic routine culture or a sputum eosinophilia on greater bation was 0.95 (95% CI, 0.87–1.00) with a cutoff of 2% havingthan or equal to one occasion at stable state. The odds ratio for a sensitivity and speciﬁcity of 90% and 60%. The area undera virus-associated exacerbation if the patient had a virus at the receiver operating characteristic curve for sputum IL-1bstable state was 0.5 (95% CI, 0.1–3.9; P ¼ 0.67). 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 spec-Validation of the Biomarkers Peripheral Blood Eosinophils, iﬁcity of 80% and 60%, and a serum CRP cutoff of 10 mg/LSputum IL-1b, Serum CRP, and Serum CXCL10 had sensitivity and speciﬁcity of 65%. The area under the re-In an independent study of COPD exacerbations, 89 subjects (57 ceiver operating characteristic curve for serum CXCL10 tomen and 32 women) with a mean (range) age of 68 (46–86) years identify a virus-associated exacerbation was 0.65 (95% CI,
Bafadhel, McKenna, Terry, et al.: Biomarkers in COPD Exacerbations 667TABLE 2. BIOLOGIC CLUSTERS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE EXACERBATIONS, WITH CLINICALEXACERBATION AND BASELINE CHARACTERISTICS Cluster 1: Cluster 2: Cluster 3: Cluster 4: Bacteria-predominant Eosinophil-predominant Virus-predominant Pauciinﬂammatory P ValueExacerbation characteristicsNumber (%) 52 (35) 44 (30) 36 (24) 16 (11) —Sputum TNFRII (pg/ml)* 1,722 (1,402–2,117) 353 (287–433) 1,254 (969–1,623) 77 (41–147) ,0.0001Sputum CXCL11 (pg/ml)* 3.1 (2.2–4.3) 10.9 (7.7–15.5) 799 (415–1,539) 17.3 (5.6–53.1) ,0.0001Sputum CCL17 (pg/ml)* 5.5 (4.5–6.7) 34.8 (27.3–44.5) 23.5 (16.2–34.1) 4.7 (3.5–6.3) ,0.0001Bacterial exacerbation, % (95% CI) 86 (73–92) 29 (18–45) 44 (28–61) 31 (12–58) ,0.0001Viral exacerbation, % (95% CI) 22 (13–35) 10 (3–23) 57 (39–73) 30 (10–61) ,0.0001Eosinophilic exacerbation, % (95% CI) 6 (1–16) 60 (45–74) 28 (16–44) 27 (10–52) ,0.0001D FEV1, ml† 2132 (2251 to 235) 2110 (2230 to 231) 2232 (2340 to 2124) 2280 (2524 to 236) 0.32D CRQ, units † 20.9 (21.2 to 20.6) 20.9 (21.3 to 20.5) 20.9 (21.4 to 20.4) 21 (21.9 to 20.1) 0.99D VASTOTAL, mm† 79 (42–116) 80 (41–119) 120 (86–154) 73 (38–108) 0.39Baseline characteristicsNumber, (%) 28 (37) 19 (25) 20 (27) 8 (11) —Male, n (%) 18 (64) 14 (74) 14 (70) 7 (88) 0.63Age, yrs‡ 69 (52–84) 68 (45–88) 70 (49–84) 69 (61–85) 0.62Current smokers, n (%) 8 (29) 8 (42) 4 (20) 3 (38) 0.48Pack-years smoked‡ 44 (10–122) 50 (20–106) 47 (10–134) 72 (23–120) 0.11Exacerbation rate in previous 12 mo 3.8 (0.5) 4.3 (0.5) 4 (0.7) 4.9 (1.2) 0.58Exacerbation rate during study 3.8 (0.3) 3.6 (0.4) 3.2 (0.3) 3.1 (0.5) 0.64Inhaled corticosteroid dose, mgx 1,507 (147) 1,567 (133) 1,470 (160) 1,150 (188) 0.55Residual volume, % 134 (8) 150 (9) 120 (8) 146 (23) 0.11TLCO % predicted 56 (5) 59 (5) 57 (6) 46 (7) 0.62FEV1% predicted, baseline 53 (3) 51 (5) 53 (5) 40 (7) 0.34FEV1/FVC ratio (%) 51 (2) 47 (2) 50 (3) 47 (5) 0.67CRQTOTAL , units 4.14 (0.20) 3.90 (0.22) 4.10 (0.26) 3.66 (0.50) 0.74VASTOTAL , mm 178 (15) 142 (18) 124 (18) 147 (37) 0.14Total sputum cell count (3106 cells/g)* 8.3 (5.5–12.5) 2.3 (1.6–3.2) 2.5 (1.2–5.3) 3.5 (1.2–10.7) 0.002Sputum neutrophil count, % 75 (5) 53 (4) 68 (4) 81 (6) 0.003Sputum eosinophil count, %* 1 (0.6–1.6) 3.1 (1.4–6.6) 1 (0.5–1.9) 0.5 (0.2–1) 0.012Bacterial colonization, % (95% CI) 63 (48–77) 27 (15–43) 11 (3–29) 38 (18–61) 0.001 Deﬁnition of abbreviations: CCL ¼ ; CI ¼ conﬁdence interval; CRQ ¼ Chronic Respiratory Disease Questionnaire score; CXCL ¼ ; TLCO ¼ carbon monoxide transferfactor; 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 these exacerbation clinical phenotypes are likely to represent dis-speciﬁcity of 70% and 60%. tinct pathophysiologic entities with speciﬁc biomarker signatures. Further details and results are available in the online supplement. Biomarker proﬁling in COPD exacerbations has the potential to further the understanding of disease mechanisms (22), whereas phenotypic approaches lend to prognostic and thera-DISCUSSION peutic strategies (37, 38). Using factor and cluster analysis,In this study we have used two methods to investigate bio- a novel approach of characterizing COPD and exacerbationsmarkers in COPD. The ﬁrst using unbiased statistical tools, free (23), we were able to reduce an extensive panel of measuredfrom bias and independent of clinical expression, identiﬁed bi- sputum biomarkers into three factors, from which we deter-ologic COPD exacerbation phenotypes and characterized exac- mined four biologic clusters. This analytic strategy is free fromerbations into four biologic clusters, The second method used investigator bias. These biologic clusters could not be distin-current clinical exacerbation phenotypes of COPD related to po- guished clinically or by Anthonisen criteria (14) and the exac-tential etiology and inﬂammation, namely exacerbations that are erbation severity was similar across the clusters. Importantly,associated with bacteria, virus, or a sputum eosinophilia. Inter- using factor analysis we have shown differential inﬂammatoryestingly, we were unable to deﬁne biomarkers for exacerbations proﬁles between the bacteria-predominant, eosinophil-predominant,per se, despite a generalized increase in systemic and airway in- virus-predominant, and pauciinﬂammatory clusters. In patientsﬂammation (34–36). The biologic exacerbation clusters were bac- with multiple exacerbations the biologic clusters were repeat-terial-, viral-, or eosinophilic-predominant, and a fourth was able, and exacerbations associated with bacteria or a sputumassociated with limited changes in the inﬂammatory proﬁle and eosinophilia but not viruses could be predicted from stable state.was termed “pauciinﬂammatory.” These clusters were remark- Therefore, our data are consistent with the view that bacterialably similar to our clinical exacerbation phenotypes. We identi- and eosinophilic exacerbations may reﬂect instability withinﬁed biomarkers for our clinical exacerbation phenotypes that a complex and inherently unstable system, whereas viral exacer-were then validated in an independent cohort. The bacteria- bations are more likely to represent acquisition of a new patho-and sputum eosinophil–associated exacerbations rarely coexisted, gen. It is likely both of these mechanisms drive exacerbations, butand were reliably predicted from stable state suggesting funda- critically we have determined biologic clusters and clinical phe-mental differences in their immunopathogenesis. Therefore, in notypes that may respond to different management strategies,addition to identifying potential biomarkers to direct therapy, which can potentially be identiﬁed using biomarker proﬁles.
668 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 184 2011 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 paren- theses. CCL ¼ ; CRP ¼ C-reactive protein; CXCL ¼ ; TNF ¼ tumor necrosis factor.
Bafadhel, McKenna, Terry, et al.: Biomarkers in COPD Exacerbations 669 The inﬂammatory proﬁle of a COPD exacerbation is typically exacerbation. In our study a statistical analytic limitation wasneutrophilic, but eosinophilic airway inﬂammation also exists, that we did not correct for repeated measures and assessedand is associated with a favorable response to corticosteroid ther- changes in biomarkers in paired or unpaired tests; however,apy (8–10). Eosinophilia in inﬂammatory airways disease is as- we examined two methods to investigate biomarkers in COPDsociated with increased all-cause mortality (39, 40) and may exacerbations, using unbiased statistical tools to demonstratehighlight different genetic, biologic, and pathologic disease pro- four biologic clusters and analysis of biomarkers to look at pre-cesses. Importantly, the sputum differential rather than total deﬁned clinical exacerbation subgroups, and further used multi-eosinophil count has consistently been shown to be associated variate analysis to determine that combinations of markers didwith important clinical outcomes (9, 10). We found that the not improve our predictive model. The presence of coinfectionperipheral percentage eosinophil count was a sensitive biomarker with virus and bacteria in our study was lower than that previ-of a sputum eosinophilia. Current guidelines recommend the use ously reported (5), but may reﬂect differences in the severityof systemic corticosteroids for COPD exacerbations, although the of exacerbations. The relationship between virus and bacterialmagnitude of the beneﬁt is marginal and their use associated with infection in exacerbations, however, remains poorly understoodsigniﬁcant side effects (18). Our ﬁndings raise the possibility that (5, 45). We chose to deﬁne a bacteria-associated exacerbationtargeting corticosteroid therapy in a subgroup of exacerbations based on a positive routine culture or a high bacterial load;dependent on the peripheral eosinophil count may reduce inap- however, the causal links between the presence of bacteriapropriate use of systemic corticosteroids. and exacerbations has not been rigorously conﬁrmed, and evi- Bacteria are considered to play a role in up to 50% of exac- dence for efﬁcacy of antibiotics in treatment is conﬂicting (13,erbations (7). Current guidelines propose sputum purulence 19–21, 46). Developments in molecular bacterial identiﬁcationto guide antibiotic therapy (13). Sputum purulence is sensitive of bacteria (47) and emerging microbiomics (48) are beginningfor detecting bacterial culture or high bacterial yields at exac- to redeﬁne the microbiota of the airway in health and diseaseerbation in COPD (12). However, the use of sputum purulence and will likely change the view of what deﬁnes a bacterial in-alone is confounded by its presence at stable state and chronic fection. Improvements in viral detection and the identiﬁcationbacterial colonization (41), possibly as a consequence of poor of new respiratory viruses are also changing the understandingbacterial clearance (42). Furthermore, the change in sputum of the associations between exacerbations and these agents.purulence or sputum production symptoms in our cohort was Further work is required before therapeutic implications andnot sensitive or speciﬁc for identifying a bacteria-associated interpretative criteria can be established for these sensitive de-exacerbation. The most sensitive and speciﬁc assay for deter- tection methods. Whether identiﬁcation of a pauciinﬂammatorymining bacteria-associated exacerbations was sputum IL-1b. biologic cluster and a proportion of subjects without clear evi-This extends previous ﬁndings that bronchoalveolar lavage IL- dence of a cause for their exacerbation reﬂect the insensitivity1b was a good biomarker for ventilator-associated pneumonia of our chosen cutoffs for deﬁnitions or a real entity requires(43), and suggests that this airway marker may suitably deter- further clariﬁcations.mine bacterial infections, above that of serum CRP or procalci- In conclusion, COPD exacerbations are heterogeneous. Thistonin whose use could not be demonstrated in this study or in phenotypic heterogeneity can be deﬁned. Using unbiased statis-others (34, 35). Sputum IL-1b could thus be used as a biomarker tical tools we have determined four biologic exacerbation clus-to correctly identify bacteria-associated exacerbations but ters that relate to identiﬁable patterns of inﬂammation andwould require the development of a rapid near patient test to potential causative pathogens. We have deﬁned sensitive andbe of use in clinical practice. speciﬁc biomarkers to identify predeﬁned clinical exacerbation Viruses have been implicated as a major cause of COPD exac- phenotypes, which need to be tested in randomized prospectiveerbations and are detected in approximately half of severe studies of targeted therapy. These subgroups are independentCOPD exacerbations (5, 6). The total sputum eosinophil counthas been proposed as a potential biomarker of a viral exacerbation and suggest that the mechanisms driving their exacerbations(5). Here we also found that the total absolute sputum eosino- are distinct and may be amenable to more speciﬁc interventions,phil count was increased in virus-associated exacerbations, but potentially moving the management of COPD exacerbations to-not the differential sputum eosinophil count, suggesting the ward the realization of phenotype-speciﬁc management.association was largely a consequence of a change in the total- Author Disclosure: M.B. received grant support from the Medical Research Coun-cell count. The application of clinical symptoms in combination cil (MRC). S.M. does not have a ﬁnancial relationship with a commercial entitywith serum CXCL10 (IP-10) has been proposed as a possible 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 thisbiomarker for rhinovirus infection at exacerbation (44). This manuscript. V.M. does not have a ﬁnancial relationship with a commercial entitystudy conﬁrms that serum CXCL10 levels as a potential pre- that has an interest in the subject of this manuscript. C.R. does not have a ﬁnancialdictor of a virus-associated exacerbation, independent of a re- 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 entityquirement for symptom evaluation. Novel antiviral approaches that has an interest in the subject of this manuscript. M.M. is employed by andare in development and CXCL10 is thus a promising biomarker owns stocks in AstraZeneca (AZ). K.H. does not have a ﬁnancial relationship withto direct future antiviral therapy. 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 in- One potential criticism is that this is a single-center study and terest in the subject of this manuscript. A.D. does not have a ﬁnancial relationshiptherefore our ﬁndings need to be replicated across multiple cen- with a commercial entity that has an interest in the subject of this manuscript.ters, and validated prospectively to identify the biologic clusters 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 relation-and our proposed biomarkers for the clinical exacerbation phe- ship with a commercial entity that has an interest in the subject of this manu-notypes; nonetheless, this approach may represent a new para- script. P.R., P.D., and M.J. are employed by and own stocks in AZ. M.S. isdigm in the management of COPD exacerbations. Importantly, employed by AZ. P.N. was an employee of AZ at the time of conducting thiswe have replicated the biomarkers peripheral blood eosinophils, research and preparation of the manuscript. He is now an employee of MedI- mmune LLC, which is a subsidiary of AZ and owns stocks in AZ. R.H.G. wassputum IL-1b, and serum CXCL10 in a validation cohort. Pe- a consultant for Nycomed and received travel accommodations from Chiesi.ripheral blood eosinophils remained a strong marker of a sputum P.V. received institutional grant support from the Swedish Research Council.eosinophilia. Sputum IL-1b and serum CXCL10 were measured D.A.L. received institutional grant support and was a consultant for GlaxoSmithK- line (GSK). He is on the Advisory Board and received honorarium from GSK.using a different platform but remained signiﬁcant albeit weaker M.R.B. received institutional grant support form the MRC. S.L.J. was a consultantpredictive markers of identifying a bacteria- or virus-associated for AZ, Centocor, Sanoﬁ-Pasteur, Synairgen, GSK, and Chiesi. He received
670 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 184 2011institutional grant support from AZ, Centocor, Sanoﬁ-Pasteur, Synairgen, and AZ. pulmonary disease: a prospective randomised controlled trial. LancetHe received lecture fees from AZ, owns stocks in Synairgen, and received travel 1999;354:456–460.accommodations from Pﬁzer. I.D.P. received institutional grant support from the 18. Niewoehner DE, Erbland ML, Deupree RH, Collins D, Gross NJ, LightMRC and received honorarium from GSK, AZ, Merck, and Novartis. He receivedtravel accommodations from Boehringer Ingelheim (BI). C.E.B. received institu- RW, Anderson P, Morgan NA. Effect of systemic glucocorticoids ontional grant support from the MRC, AZ, MedImmune, and Roche. He received exacerbations of chronic obstructive pulmonary disease. Departmentsupport for the development of SPD assays in Cambridge from GSK. He is on the of Veterans Affairs Cooperative Study Group. N Engl J Med 1999;Advisory Board of GSK, AZ, Roche, Novartis, Genentech, and MedImmune and 340:1941–1947.was a consultant for MedImmune and Novartis. He received travel accommoda- 19. Puhan MA, Vollenweider D, Latshang T, Steurer J, Steurer-Stey C.tions from BI. Exacerbations of chronic obstructive pulmonary disease: when areAcknowledgment: The authors thank all the research volunteers who participated antibiotics indicated? A systematic review. Respir Res 2007;8:30.in the study, and also the following people for their valuable assistance throughout 20. Puhan MA, Vollenweider D, Steurer J, Bossuyt PM, Ter RG. Where isthe study: J. Aniscenko, M. Bourne, R. Braithwaite, D. Burke, J. Footitt, E. Goldie, the supporting evidence for treating mild to moderate chronic ob-J. Goldie, N. Goodman, S. Gupta, B. Hargadon, I. Rushby, M. Shelley, A. Singapuri, structive pulmonary disease exacerbations with antibiotics? A sys-D. Vara, R. Walton, and S. Winpress. tematic review. BMC Med 2008;6:28. 21. 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