Published ahead of print on June 23, 2006, doi:10.1164/rccm.200604-506OC
© 2006 American Thoracic Society doi: 10.1164/rccm.200604-506OC
Use of Plasma Biomarkers at Exacerbation of Chronic Obstructive Pulmonary DiseaseAcademic Unit of Respiratory Medicine, Royal Free and University College Medical School, London; GlaxoSmithKline Research and Development, Greenford, Middlesex, United Kingdom; and GlaxoSmithKline Research and Development, Upper Merion, Pennsylvania, and Research Triangle Park, North Carolina Correspondence and requests for reprints should be addressed to Jadwiga A. Wedzicha, M.D., Academic Unit of Respiratory Medicine, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK. E-mail: j.a.wedzicha{at}medsch.ucl.ac.uk
Impact: This study explores the use of measuring plasma biomarkers at exacerbation of chronic obstructive pulmonary disease (COPD), providing insight into the underlying pathogenesis of these important events. Rationale: The use of measuring C-reactive protein (CRP) to confirm exacerbation, or to assess exacerbation severity, in COPD is unclear. Furthermore, it is not known whether there may be more useful systemic biomarkers. Objective: To assess the use of plasma biomarkers in confirming exacerbation and predicting exacerbation severity. Methods: We assessed 36 biomarkers in 90 paired baseline and exacerbation plasma samples from 90 patients with COPD. The diagnosis of exacerbation fulfilled both health care use and symptom-based criteria. Biomarker concentrations were related to clinical indices of exacerbation severity. Interrelationships between biomarkers were examined to gain information on mechanisms of systemic inflammation at exacerbation of COPD. Measurements and Main Results: To confirm the diagnosis of exacerbation, the most selective biomarker was CRP. However, this was neither sufficiently sensitive nor specific alone (area under the receiver operating characteristic curve [AUC], 0.73; 95% confidence interval, 0.66–0.80). The combination of CRP with any one increased major exacerbation symptom recorded by the patient on that day (dyspnea, sputum volume, or sputum purulence) significantly increased the AUC to 0.88 (95% confidence interval, 0.82–0.93; p < 0.0001). There were no significant relationships between biomarker concentrations and clinical indices of exacerbation severity. Interrelationships between biomarkers suggest that the acute-phase response is related, separately, to monocytic and lymphocytic–neutrophilic pathways. Conclusions: Plasma CRP concentration, in the presence of a major exacerbation symptom, is useful in the confirmation of COPD exacerbation. Systemic biomarkers were not helpful in predicting exacerbation severity. The acute-phase response at exacerbation was most strongly related to indices of monocyte function.
Key Words: chronic obstructive pulmonary disease • exacerbation • inflammation The C-reactive protein (CRP) assay is widely performed in the assessment of many acute medical conditions including exacerbations of chronic obstructive pulmonary disease (COPD). However, CRP concentrations are elevated in most infective, inflammatory, and neoplastic pathologies (1), and the relevance in a particular disease of a high CRP level is often less well defined. This is particularly true for conditions such as COPD, where much of the morbidity, mortality, and health care costs are associated with acute-on-chronic deteriorations, with the presence of elevated systemic inflammation even in the stable state (2). Acute-on-chronic deteriorations of respiratory health in COPD are termed exacerbations. Exacerbations are important events in the natural history of COPD, contributing to the decline in lung function (3) and impairment to health status (4), and responsible for many of the hospital admissions (5) and therefore health care costs and mortality (6) associated with the disease. It is now accepted that respiratory viruses and pathogenic bacteria cause most exacerbations and the presence of bronchial infection would be expected to cause a rise in CRP. However, as noted above, COPD is associated with raised systemic inflammation in the stable state (2) and therefore the relevance of an elevated CRP concentration at exacerbation is unclear. The definition of exacerbation in COPD remains controversial (7): whether reliant solely on symptoms or also requiring an element of health care use, criteria remain subjective, either on the part of the patient or the physician. This creates difficulty, both in clinical practice and research, in reliably distinguishing exacerbations from day-to-day symptom variation. There is therefore the need for a "biomarker" to provide objective confirmation of exacerbation. Furthermore, a useful biomarker might not only differentiate stable disease from exacerbation, but may also predict the severity of such events. CRP is widely measured at exacerbation but the use of CRP as an exacerbation biomarker is unclear. There are no previous studies of other systemic biomarkers in this context. The primary aim of this study was to assess the use of plasma biomarkers in the confirmation of COPD exacerbation. We have performed the largest and most comprehensive study of plasma biomarkers at exacerbation of COPD ever reported. Thirty-six candidate molecules were assessed in paired baseline and exacerbation plasma samples from 90 patients, tested against a "gold standard" definition of exacerbation that met both health care use and prospective symptom-based criteria. We also examined relationships between biomarker concentrations and exacerbation severity, and investigated interrelationships between exacerbation biomarkers to gain information about the underlying pathogenic mechanisms of systemic inflammation during such events. Some of the results of this study have been previously published in the form of abstracts (8, 9).
The following is an abridged version of METHODS; see the online supplement for the full version.
Study Subjects and Protocol Plasma samples were obtained from all 90 patients at baseline and exacerbation. Each patient contributed one baseline and one exacerbation sample. The patients were trained to record daily, on diary cards, their morning peak expiratory flow (PEF) rate and any change in respiratory symptoms. At the time of any change in symptoms, patients were instructed to contact the study team and attend the research clinic for clinical assessment, sampling, and confirmation of exacerbation, using the criteria described below. Plasma samples at exacerbation were therefore obtained early in the time course of the event, before the initiation of any additional treatment. At baseline visits the diary cards were carefully examined to exclude the presence of exacerbation, and to assess the degree of day-to-day symptom variation, also as described below. The study had approval from the local research ethics committee, and all participants provided written, informed consent.
Definitions of Exacerbation The attending physician judged that antibiotics and/or corticosteroids were required in all 90 exacerbations and therefore all 90 exacerbations met both symptom-based and health care use definitions of exacerbation. All 90 patients at baseline visits were judged by the attending physician to be in a stable state and not to require additional therapy, and although individual symptoms may have been recorded at these visits (reflecting baseline symptom variability in COPD), these did not meet our definition of exacerbation.
Exacerbation Severity
Plasma Samples and Analysis of Plasma Proteins
Statistical Analysis
Role of Funding Source
Baseline Patient Characteristics Baseline clinical characteristics of the 90 patients included in this analysis are reported in Table 1, which demonstrates that the cohort has moderately severe COPD with a median (IQR) FEV1 of 1.00 (0.74–1.33) L or 43.9 (27.5–56.8)% predicted. Most of the patients were taking inhaled corticosteroids with a median beclomethasone-equivalent dose of 1,000 (800–2,000) µg/d. There were no significant relationships between the dose of inhaled steroid and the concentrations of inflammatory markers at baseline. Four percent of the patients were taking long-term oral prednisolone, too small for a meaningful comparison of subjects who were and were not taking such therapy.
Changes in Plasma Markers between Baseline and Exacerbation Concentrations of the 36 plasma biomarkers, at baseline and exacerbation, and the magnitude of change between these two clinical states, are reported in Table 2. The significant differences observed between baseline and exacerbation for CRP, IL-6, myeloid progenitor inhibitory factor-1 (MPIF-1), pulmonary and activation-regulated chemokine (PARC), adiponectin (ACRP-30), and soluble intercellular adhesion molecule-1 (s-ICAM-1) are illustrated in Figure 1 (using p < 0.01, as described above).
Use of Plasma Biomarkers or Symptoms in the Diagnosis of Exacerbation All 90 exacerbations fulfilled both our prospective, validated symptom-based definition (3) and the need for additional therapy. Judged against either of these standards, using the area under the curve (AUC) and 95% confidence intervals (CI) of ROC analysis, the three best performing biomarkers were CRP (AUC, 0.73; 95% CI, 0.66–0.80), IL-6 (AUC, 0.67; 95% CI, 0.59–0.74), and MPIF-1 (AUC, 0.64; 95% CI, 0.56–0.72), all less than the accepted standard of AUC  0.8 (16). The AUC for CRP was significantly greater than that for MPIF-1 (p = 0.03), but not for IL-6 (p = 0.11). Illustrating the clinical implications of these data, the widely employed clinical CRP cutoff at 5 mg/L would be 74.4% sensitive and 57.5% specific for confirmation of exacerbation. Furthermore, to achieve 90% sensitivity, a CRP concentration of 2.3 mg/L would be only 29% specific, whereas at 90% specificity a CRP of 27.6 mg/L would be only 40% sensitive. Moreover, assessed by AUC, assay of these biomarkers in isolation would not provide a more useful confirmation of exacerbation than the presence of an increase in any one of the major exacerbation symptoms alone on that day (AUC, 0.83; 95% CI, 0.77–0.89). This is illustrated in Figure 2.
Regarding the ability of combinations of biomarkers to confirm exacerbation, of the 630 biomarker pairs only the AUC for combinations of CRP with matrix metalloproteinase-9 (MMP-9), MPIF-1, and vascular endothelial growth factor (VEGF) were greater than that for CRP alone (respectively: AUC, 0.73; 95% CI, 0.66–0.81; AUC, 0.73; 95% CI, 0.60–0.81; and AUC, 0.73; 95% CI, 0.65–0.80). These results were not significantly different from CRP alone (p > 0.05), and still not greater than the commonly employed cutoff at AUC 0.8 (16). Similarly, the best combination of three biomarkers (CRP, MMP-9, and MPIF-1) resulted in an AUC of 0.75 (95% CI, 0.67–0.82), also not significantly better than CRP alone.
Combinations of Biomarkers and Symptoms in Diagnosis of Exacerbation
Plasma Biomarkers and Exacerbation Severity We examined relationships between the concentration of systemic biomarkers at exacerbation, and the change in concentration from baseline to exacerbation, with the four indices of exacerbation severity: the magnitude of change at onset, and recovery time in PEF and Symptom Count. When corrected for multiple comparisons, there were no statistically significant relationships for any of the 36 analytes.
Interrelationships between Plasma Biomarkers at Exacerbation of COPD
Regarding changes in the concentrations of markers between baseline and exacerbation, only the changes in MPIF-1 and IL-1Ra remained significantly associated with the change in IL-6 (rho = 0.78, p < 0.0001, and rho = 0.53, p < 0.0001, respectively). As expected, the increase in IL-6 remained significantly associated with the increase in CRP (rho = 0.63, p < 0.0001). Relationships between the changes in concentrations of biomarkers between baseline and exacerbation are illustrated in Figure 4. The dendrogram is constructed to graphically present similarities in the change in concentration with exacerbation of the 36 biomarkers. Conceptually, the position of each biomarker is defined by its correlation with the other biomarkers: the closest or most highly correlated are joined first, and this connection has the shortest horizontal distance on the dendrogram. Once joined, the average position of the pair is used in linking with the next most closely related biomarker. This process is repeated until all the biomarkers are linked. Thus, for example, the changes in concentration at exacerbation for IL-6, MPIF-1, and CRP were closely correlated, as were the changes in myeloperoxidase and MMP-9, but the changes observed between these two groups were not closely related.
This study has demonstrated that plasma CRP, in the presence of one or more recorded major exacerbation symptoms, is able to reliably differentiate exacerbation of COPD from day-to-day symptom variation, judged against either a validated symptom-based definition or the need for additional exacerbation therapy. CRP (or any other of the plasma analytes alone) was neither sufficiently sensitive nor specific to be a useful biomarker in the absence of symptom assessment. CRP was the most useful of the 36 biomarkers assessed. Plasma biomarkers were not useful in predicting the clinical severity of exacerbation. Interrelationships between plasma biomarkers at exacerbation suggest that the acute-phase response is related, separately, to both monocytic and granulocytic–lymphocytic indices, with the strongest relationship for the monocyte marker MPIF-1. There is ongoing debate about how exacerbation of COPD should be defined (7). In practical terms, an exacerbation represents an acute deterioration in symptoms that is beyond the patient's usual day-to-day variation. However, this definition requires subjective assessment by both the patient and the physician, and there is a need in both the clinic and the context of clinical trials for an objective method of confirming exacerbation: a "biomarker." A useful biomarker might also reflect exacerbation severity or etiology. We have previously developed a symptom-based definition of exacerbation that has been validated against important outcome measures in COPD including airway inflammatory markers (15), quality of life (4), and rate of decline in FEV1 (3). This method requires daily recording of changes in symptoms by patients, and careful interpretation of data, such that it may be regarded as rather complex and expensive. However, data collected on the basis of this definition have allowed us to determine that up to 50% of exacerbations go unreported to health care professionals, and therefore that definitions of exacerbation based solely on health care use criteria underestimate the true impact of these events (4).
Because of these difficulties in objectively assessing symptom changes, there has been great interest in the development of a biomarker that is present at different concentration during exacerbations from that present in stable disease. Accepted definitions of COPD now include reference to the presence of airway inflammation, but airway inflammatory markers are difficult to assess. In general, bronchoalveolar lavage and endobronchial biopsy are too invasive, and sputum and exhaled breath condensate are too complex to analyze, producing results that are inconsistent across studies. Both approaches are therefore impractical in the clinical setting. Attention has consequently turned to systemic markers and, indeed, it is now widely accepted that COPD is associated with increased systemic inflammation compared with control subjects (2), and that there is further up-regulation of systemic inflammation at the time of exacerbation. Markers reported to be higher in blood during exacerbation compared with the baseline state include CRP (12, 18–21), IL-8 (19), tumor necrosis factor- Clearly, there is the need for a plasma biomarker in COPD that will not only aid in distinguishing stable disease from exacerbation, but might also have prognostic value. Fortuitously, given the ready availability of CRP assay, CRP was the most useful biomarker of the 36 candidate molecules assessed. In part, this may reflect previous findings that CRP is an exceptionally stable analyte (1). There is an extensive literature reporting associations of CRP with clinical parameters in stable COPD including the severity of FEV1 impairment (28), impaired energy metabolism, decreased exercise tolerance and poorer quality of life (29), and the presence of a sputum bacterial pathogen (10). However, evidence to support the use of CRP in confirming or excluding exacerbation in COPD is minimal. One study, examining the use of CRP to identify exacerbations associated with a sputum bacterial pathogen, observed that CRP was not elevated in 16% of patients with hospitalized exacerbations (30). This may have reflected the difficulty arising from raised CRP levels in the stable state, and although the change in CRP concentration may be more useful, few patients will have baseline levels immediately available at the time exacerbation diagnosis is required. In this study, CRP was the most effective biomarker for differentiating exacerbation from day-to-day symptom variation. CRP is an acute-phase protein produced by the liver in response to IL-6 stimulation (1). CRP is raised in most conditions associated with infection, inflammation, or tissue damage, for which it is a sensitive marker (1). Evidence suggests that CRP may also be implicated in the pathophysiology of COPD: many deaths at exacerbation are due to cardiovascular disease (31) and an elevated CRP concentration is associated with increased cardiovascular morbidity (17). CRP may even have a direct role in the instability of atheromatous plaques, having been identified in such lesions ex vivo (32). Furthermore, by binding to the low-density lipoproteins present in plaques, CRP may activate the complement cascade (33). CRP and IL-6 could therefore provide a link between airway inflammation, systemic inflammation, and cardiovascular disease in COPD that is worthy of further study. Moreover, therapies that reduce CRP in COPD could impact on this excess cardiovascular morbidity. This study is the first to report on the use of systemic biomarkers, with and without symptoms, in the confirmation of exacerbation in COPD. The strengths of the present analysis are the assessment of 36 candidate biomarkers, in 90 exacerbations from 90 separate patients, with exacerbations meeting both health care use and symptom-based definitions. Moreover, individual symptom changes were also recorded at baseline (allowing us to exclude intercurrent respiratory illnesses) and both the patients and the exacerbations were comprehensively assessed, enabling an analysis of the relationships between plasma markers and clinical indices of exacerbation severity. The completion of daily diary cards enabled us to precisely define the onset and therefore duration of changes in symptoms and PEF. Relationships between plasma inflammatory indices and the clinical severity of exacerbation were absent, consistent with previous studies (30), and this may reflect the heterogeneity of such events. Our patients had a range of disease severity, and we believe these patients to be representative of a wider population with COPD. However, most of the patients were undergoing inhaled corticosteroid therapy, which is known to reduce CRP in COPD (34), and the results may therefore not be applicable to a steroid-naive population. It is important to remark that the methodology employed did not allow us to assess the use of biomarkers in distinguishing exacerbations from other causes of symptom deteriorations in patients with COPD, such as cardiac failure or pneumonia, diagnoses that were excluded in our patients clinically and, where appropriate, with investigations involving such techniques as electrocardiography and chest radiography. Investigating interrelationships between biomarkers has the potential to yield information about the underlying mechanisms of exacerbation and therefore to aid development of novel therapeutic strategies. We have particularly focused on relationships involving acute-phase proteins given the recognized associations between systemic inflammation and cardiovascular risk (17), and the ready availability of CRP assay in many health care settings. As expected, we demonstrated relationships between the concentrations of plasma IL-6 and the hepatic acute-phase proteins CRP and IL-1Ra. However, we have also demonstrated that IL-6 is associated with markers from both monocytic and, separately, neutrophilic–lymphocytic pathways. The former relationship remained statistically significant when assessing correlations between the changes in biomarker concentration at exacerbation. Such findings suggest that strategies aimed at damping monocyte-macrophage–mediated inflammation may be the most successful in reducing cardiovascular risk in the periexacerbation period. Regarding interrelationships between inflammatory markers, MPIF-1, a newly identified CC chemokine, appeared to be the most closely associated with plasma IL-6 concentration. So named from a suppressive effect on hematopoiesis, MPIF-1 has chemotactic activity on resting (but not activated) T lymphocytes and monocytes, but is less active on neutrophils (35). To our knowledge, this is the first report of an MPIF-1 assay performed on patients with COPD. In summary, in patients with COPD presenting with increased symptoms, CRP was the most effective of 36 biomarkers studied to confirm exacerbation. The CRP cutoff may be chosen to maximize sensitivity or specificity, as required. CRP alone was neither sufficiently sensitive nor specific to confirm exacerbation, and the magnitude or rise in CRP concentration did not reflect the clinical severity of the event. Finally, interrelationships between biomarkers at exacerbation suggest that monocytic pathways may be an important target for future research, with the aim of reducing the considerable cardiovascular morbidity associated with this prevalent disease.
Supported by GlaxoSmithKline. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200604-506OC on June 23, 2006 Conflict of Interest Statement: J.R.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.C.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. W.R.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.M.A.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.A.B. at the time of this study was a full-time employee of GlaxoSmithKline (GSK) and is no longer an employee. He has about 5,000 stock options remaining (granted at about $47). G.W.H. is an employee of GSK and holds stock options with GSK, as part of the common GSK employee remuneration. R.S.V. is an employee of GSK and has stock options. J.A.W. has received honoraria for lectures at meetings and/or attendance at advisory boards from the following companies: GSK, Boehringer Ingelheim, AstraZeneca, Bayer, Novartis, and Aventis Pasteur. She has received research grants totaling approximately $300,000 from GSK for studies of various aspects of COPD exacerbations. She has received a grant of approximately $450,000 for a study of tiotropium in COPD and $300,000 from Aventis Pasteur for a study of viral epidemiology of COPD. Received in original form April 11, 2006; accepted in final form June 23, 2006
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