Introduction
Chronic obstructive pulmonary disease (COPD) is a phenotypically heterogeneous condition characterized by airflow limitation that is not fully reversible [
1]. Some, but not all, COPD subjects have emphysema,
i.e., airspace enlargement distal to the terminal bronchioles [
2]. Determining the presence of emphysema is important, as it has been independently associated with increased respiratory symptoms, more rapid decline in lung function, increased risk of lung cancer, higher rates of cardiovascular disease and increased mortality risk [
3]-[
6]. Surprisingly, there are some subjects with significant smoking history that have emphysema, but no airflow limitation [
7]. Understanding the molecular signatures underlying emphysema may shed light on the pathogenesis of emphysema and its systemic complications.
The best current non-invasive method of detecting emphysema is high-resolution computed tomography (HRCT) [
8],[
9]. The drawbacks to HRCT include cost, radiation exposure, and a high rate of false positive clinical significant findings (
e.g. benign nodules); however, HRCT can provide significant information relevant to lung pathology. For instance, lung attenuation area (LAA) at -950 or -910 Hounsfield units (HU) and the mean lung attenuation value at the 15
th percentile (LP15A) on the lung attenuation curve are density-based measurements that correlate with emphysema [
8],[
10],[
11]. Although the optimal method and normal values for describing radiologic emphysema have not been fully validated, it has been shown that control smokers without COPD have percent LAA ≤ -950 HU of <5% [
9].
The first reported blood biomarker of emphysema was α1-antitrypsin (AAT); however, AAT deficiency accounts for only 1-2% of COPD [
12]. Another recently reported independent biomarker of emphysema is soluble RAGE or advanced glycosylation end product receptor (AGER) [
13]. Peripheral blood adiponectin and bronchoalveolar lavage fluid eotaxin levels have also correlated with radiologic emphysema [
14],[
15]. There are other reports of peripheral blood biomarkers of airflow limitation such as interleukin-6, surfactant protein D and C-reactive protein [
16],[
17]. Therefore, the presence of systemic biomarkers in peripheral blood, which can be easily measured and offer information regarding COPD phenotypes, may provide another method of significant value in diagnosing and managing individuals with emphysema [
18]. In addition, a biomarker signature of emphysematous phenotypes may provide insight to the pathogenesis of disease. Limitations of some previous emphysema biomarker studies include small sample size and lack of replication. With this in mind, using one of the largest studies to date, we sought to determine a peripheral blood biomarker signature of emphysema, independent of other clinical variables, in current and former cigarette smokers with normal lung function and with COPD, and relate the biomarker signature to different methods of defining radiologic emphysema. Key findings were validated in an independent COPD cohort.
Discussion
COPD is a phenotypically heterogeneous disease, with the presence of emphysema having implications for risk stratification and management [
3]-[
5],[
18]. In this study, we successfully identified and replicated a panel of peripheral blood biomarkers that was associated with emphysema independent of age, smoking status, body mass index, airflow limitation, and gender. These biomarkers (AGER, ICAM1 and CCL20) were associated with emphysema regardless of quantification technique (%LAA ≥ -950 and ≥ -910 HU and LP15A) and were replicated in an independent COPD cohort (TESRA), thus strengthening their potential utility for defining clinically relevant emphysema.
Our study reports lower RAGE levels in peripheral blood as a biomarker of increased emphysema percentage in the lungs independent of gender, age, airflow limitation, body mass index and current smoking status. RAGE (advanced glycosylation end-product receptor or AGER) is an immunoglobulin family member that is highly expressed in human lung [
25]. The RAGE pathway and soluble RAGE (sRAGE), a splice variant or proteolytic cleavage product of RAGE, have been associated with several inflammatory conditions such as diabetes mellitus, vascular disease and arthritis [
26],[
27]. The sRAGE molecule binds damaged ligands preventing these from binding to cell surface receptors and activating cell signaling pathways [
28]. RAGE is active in damage-related conditions such as hyperglycemia, hypoxia, inflammation and oxidative stress [
29]. While fasting blood glucose measurements were not available, 66 individuals reported a history of diabetes mellitus in the COPDGene biomarker study and there was no association between RAGE levels and self-reported history of diabetes mellitus (p = 0.88). Lower levels of sRAGE have been described in individuals with airflow limitation [
30],[
31]. Other studies have found lower sRAGE levels associated with CT-assessed emphysema severity and cor pulmonale [
32] and with CT-assessed emphysema and lower diffusing capacity of carbon monoxide using the TESRA data described in this study in combination with the ECLIPSE investigators [
13]. Some studies suggest that sRAGE is increased in the lungs of patients with COPD and high levels of sRAGE may be associated with progression of emphysema [
33]. Interestingly, animal studies suggest RAGE/sRAGE plays a role in alveolar development and overexpression in mouse lung leads to the development of emphysema [
34]. This suggests that sRAGE, by acting as a decoy molecule, may have a different role in the developing lung and the adult lung or low sRAGE levels in COPD may result in increased inflammatory signaling in the lung.
In the present study, we found decreased ICAM1 levels correlate with increased severity of emphysema on CT scan, independent of smoking status, FEV
1 and other covariates. ICAM1 is expressed on vascular endothelial and immune cells and mediates cell transmigration and adhesion [
35]. ICAM1 plays a role in the recruitment of inflammatory cells to the lung. There is currently quite limited information about the association of ICAM1 to COPD and emphysema. Higher serum levels of soluble ICAM1 have been demonstrated in COPD, where it correlated with the severity of airflow limitation, arterial hypoxemia and hypercarbia [
36],[
37]. Other studies relate ICAM1 levels to active smoking [
38] and preliminary analysis from The MESA Lung Study demonstrated that ICAM1 predicted 0.15%/year increase in CT-assessed emphysema, suggesting a role for this molecule as a biomarker of emphysema and that it may play a role in emphysema pathogenesis [
39].
CCL20 or macrophage inhibitory protein 3a, a chemokine receptor ligand, is involved in the recruitment of inflammatory cells through chemokine receptor 6 (CCR6), its only known receptor [
40]. In both the COPDGene study and the TESRA study, CCL20 levels were inversely and significantly associated with emphysema although methodological considerations prevented a meta-analysis. Lower CCL20 levels have been described in bronchoalveolar lavage fluid of smokers [
41]. The CCR6/CCL20 complex is one of the most potent regulators of dendritic cell migration to the lung and CCR6 knockout mice may be partially protected against cigarette smoke-induced emphysema due to reduced recruitment of inflammatory cells to the lung [
42]. These data suggest that increased activity of the CCL20/CCR6 pathway may increase the susceptibility to emphysema.
CDH1 was negatively correlated with radiologic emphysema across all emphysema outcome measurements. CDH1 or E cadherin is an epithelial cell adhesion molecule that regulates cell differentiation and morphogenesis, and is associated with lung fibrosis and cancer [
43]. CDH1 may be a marker of epithelial cell injury and epithelial to mesenchymal transition that is believed to play a role in small airway remodeling in COPD [
44]. Genetic polymorphisms in CDH1 have been associated with development of COPD and decline in lung function [
45]. CDH13 or H cadherin is another adhesion molecule that may influence surfactant protein D levels and serum adiponectin levels, both implicated in the pathogenesis of COPD; however, CDH13 itself has not been associated with quantitative emphysema to date [
46],[
47]. We found higher levels of CDH13 to be associated with CT-assessed emphysema in the COPDGene cohort, but these were not available for validation in the TESRA cohort. Higher SERPINA7 levels were also associated with more radiologic emphysema. SERPINA7 does not have protease inhibitor capabilities and is also known as thyroid binding globulin. This study represents a new association for SERPINA7 with COPD.
With regard to the ability of biomarkers to predict the presence of any emphysema compared to no emphysema, ROC curves demonstrated a small contribution of plasma biomarkers separate to the covariates alone. This is likely because when individuals with more severe levels of emphysema are included, covariates alone, especially FEV
1, are highly predictive of emphysema in their own right. However, the biomarkers are more useful for predicting the presence of emphysema in those that do not already have severe airflow limitation, because the covariates alone were not as good at predicting emphysema in this group and biomarkers combined with covariates increased the area under the curve. This may be useful clinically since determining the presence of underlying emphysema at this early stage in those that do not yet have severe airflow limitation may have outcome benefits for the individuals [
3]-[
6].
This COPDGene biomarker study is one of the largest emphysema biomarker studies to date on carefully phenotyped individuals with COPD. The TESRA cohort provides validation of a number of the findings. The study confirms a previously identified association between radiologic emphysema and sRAGE, builds on data suggesting a role for ICAM1 as a biomarker, in addition to discovering previously not identified biomarkers associated with emphysema such as CCL20, cadherin 1, cadherin 13 and SERPINA7. The study also highlights the potential usefulness of a panel of biomarkers to predict the presence of emphysema compared to using clinical data alone, especially in those who do not yet have severe abnormalities in lung function. However there are limitations; the TESRA cohort was different from the COPDGene cohort in that its population was comprised of ex-smokers with at least mild COPD and did not include control subjects and only 2 of the 3 quantitative emphysema measurements were made (-910 HU and LP15A). Since emphysema can occur in smokers without COPD and emphysema measurements are highly co-linear, these limitations may be of minor importance. Other limitations include the fact that the majority of subjects in both cohorts were non-Hispanic white, thus, the generalizability of these findings to other populations remains unknown and emphysema measurements from both COPDGene and TESRA were cross sectional; therefore, the significance of these biomarkers for emphysema progression remains unknown. A final limitation of this study, an in many biomarker studies, is the magnitude of association between the change in biomarker levels and the change in emphysema severity. While the biomarker associations are highly statistically significant, and validation suggests the associations are real, further studies are needed to evaluate the role of these biomarkers in disease pathogenesis and as markers of disease presence and progression [
48],[
49].
Competing interests
Dr. Carolan: The author has received lecture fees from Novartis. Dr. Hersh: The author is a consultant for CSL Behring and receives lecture fees from Novartis. Dr. Rennard: Entities with which SIR currently has relationships are as follows: GlaxoSmithKline, Boehringer Ingelheim, Forest, AstraZeneca, Chiesi, CME Incite, Takeda, Regeneron, Pearl, CIPLA, CSA, American Board of Internal Medicine, Merck, Medimmune, Synapse, Nycomed, Dalichi Sankyo, Novartis, Johnson and Johnson, Quadrant, Gerson Lehman, Able Associates, CSL Behring, CTS Carmel, Decision Resources, FirstWord, Gilead, Guidepoint Global, Pulmatrix, Saatchi and Saatchi, Schlesinger Associates, Cory Paeth, Frankel Group, Medical Knowledge, Pro Ed Communication, LEX Consulting. Dr. Belloni: The author is an employee of Genentech and receives stock or stock options from Genentech/Roche. Dr. Comellas: The author is a consultant for VIDA diagnostics. The other authors declare that they have no competing interests.
Authors' contributions
BJC: Contributed to the data acquisition, analysis, and interpretation, manuscript drafting and critical review for intellectual content and final approval of the manuscript. BJC had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. GH: Contributed to the data analysis and interpretation, critical review for important intellectual content and final approval of the manuscript. JM: Contributed to the TESRA data analysis and interpretation. CPH: Contributed to TESRA data acquisition, analysis and critical review for the important intellectual content, and final approval of the manuscript. WKO: Critical review for the important intellectual content and final approval of the manuscript. SR: Critical review for the important intellectual content and final approval of the manuscript. SGP: Contributed to TESRA data acquisition, analysis and critical review for the important intellectual content, and final approval of the manuscript. PB: Contributed to TESRA data acquisition, analysis and critical review for the important intellectual content, and final approval of the manuscript. DAC: Contributed to TESRA data acquisition, analysis and critical review for the important intellectual content, and final approval of the manuscript. APC: Contributed to data acquisition, analysis and critical review for the important intellectual content and final approval of the manuscript. MH: Critical review for the important intellectual content and final approval of the manuscript. RLZ: Critical review for important intellectual content and final approval of the manuscript KK: Contributed to the data analysis and interpretation and drafting, critical review for important intellectual content, and final approval of the manuscript. RPB: Contributed to the study conception and design; data analysis and interpretation; manuscript drafting, critical review for the important intellectual content, and final approval of the manuscript. All authors read and approved the final manuscript.