Diagnostic Values For Club Cell Secretory Protein (CC16) in Serum of Patients of Combined Pulmonary Fibrosis and Emphysema.

Combined pulmonary fibrosis and emphysema (CPFE) is an under-recognized syndrome for which the diagnostic use of serum biomarkers is an attractive possibility. We hypothesized that CC16 and/or TGF-β1 or combinations with other biomarkers are useful for diagnosing CPFE. Patients with respiratory symptoms and a smoking history, with or without chronic obstructive pulmonary disease, were divided into the following three groups according to findings of high-resolution computed tomography of the chest: controls without either emphysema or fibrosis, patients with emphysema alone, and patients compatible with the diagnosis of CPFE. Serum concentrations of CC16, TGF-β1, SP-D, and KL-6 were measured in patients whose condition was stable for at least 3 months. To investigate changes in biomarkers of lung fibrosis in patients with a life-long smoking history, additional measurements were performed on the patients with idiopathic pulmonary fibrosis (IPF) of smoking history. The mean age of the first three groups was 68.0 years, whereas that of the IPF group was 71.8 years, and the groups contained 36, 115, 27, and 10 individuals, respectively. The serum concentration of CC16 in the four groups was 5.67 ± 0.42, 5.66 ± 0.35, 9.38 ± 1.04 and 22.15 ± 4.64 ng/ml, respectively, indicating that those patients with lung fibrosis had a significantly higher concentration. The combined use of CC16, SP-D, and KL-6 provided supportive diagnosis in conjunction with radiological imaging in diagnosis of CPFE. We conclude that a combination of biomarkers including CC16 could provide useful information to screen and predict the possible diagnosis of CPFE.


Introduction
Historically, the presence of excess fi brosis has been thought to preclude the diagnosis of emphysema (1). In histopathological analysis, obvious fi brosis has been excluded from the diagnostic defi nition of emphysema, but increased collagen content in emphysemic lungs has been reported by some studies (2)(3)(4)(5)(6). Th ese data suggest that emphysema may be accompanied with fi brosis to some extent, despite appearing without obvious fi brosis under a light microscope.
In 2005, Cottin and associates reported a new clinical entity: combined pulmonary fi brosis and emphysema (CPFE), which is typically characterized by upper lobe emphysema and pulmonary fi brosis of the lower lungs (7). Subsequently, several groups made a series of reports that support CPFE as a distinct syndrome (8)(9)(10). Although a similar concept was reported earlier (11), the notion of CPFE opened up a new perspective on chronic obstructive pulmonary disease (COPD) and interstitial lung disease, particularly idiopathic pulmonary fi brosis (IPF), which is being actively studied in terms of new therapeutic regimens (12) or new genetic associations (13). CPFE syndrome has characteristic imaging features and consists of a combination of distinct signs and symptoms, which include severe dyspnea; physiological testing reveals normal lung volume indices with markedly impaired diff usion capacity, hypoxemia during exercise (14) and, occasionally, pulmonary hypertension (15,16). CPFE, however, has not yet been recognized widely, and more studies are needed to determine the entire clinical and basic features of CPFE (17). Currently, serum biomarkers for lung diseases are an active area of research, but fi nding a biomarker useful for diagnosis or prognosis is a major challenge. Club cell secretory protein (CC16) and/or transforming growth factor β1 (TGF-β1) show promise as serum markers for assessing the cellular integrity or permeability of the lung structure (18,19). Accordingly, we aimed to fi nd specifi c biomarkers that ideally would diff erentiate CPFE from uncomplicated emphysema in daily practice. We tested the hypothesis that CC16, TGF-β1, and combinations with other promising biomarkers are useful for diff erential diagnosis of CPFE.

Study design and patients
We enrolled 410 consecutive patients with long-term smoking history who had visited the Respiratory Care Clinic, Nippon Medical School, Tokyo, Japan, from November 2003 to March 2008 regarding expectoration or chronic cough and/or dyspnea during exercise. Detailed information on eligibility and exclusion criteria is available in the Supplementary Material found online.
To investigate changes in CC16 in lung fi brosis with life-long smoking history, 10 smokers with idiopathic pulmonary fi brosis (IPF) who visited the university hospital, Nippon Medical School, Tokyo, Japan, were recruited. Th e diagnosis of IPF was confi rmed after detailed examinations in accordance with the criteria of ATS/ERS/JRS/ ALAT statement (20).
Th is study was conducted in accordance with the Declaration of Helsinki, and all patients provided written informed consent. Th e study protocol was approved by the Institutional Review Board of Nippon Medical School, Tokyo, Japan.

Study procedures (Detailed information is available in the Supplementary Material)
At fi rst visit, the patients underwent examinations as follows: post-bronchodilator pulmonary function tests, diff usion capacity, arterial blood gas with room air breathing, and 6-minute walking tests (6MWT). Highresolution computed tomography (HRCT) scanning of the chest was performed, followed by a quantitative assessment of the extent of emphysema (%LAA) using software. Echocardiography was performed by a technician, under the supervision of a qualifi ed cardiologist to assess pulmonary hypertension or left ventricular heart failure.

Subgroups and biomarkers
Severity classifi cation of COPD was based on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria (21). We defi ned CPFE according to the original report by Cottin and colleagues (7) as a syndrome characterized by upper lobe emphysema and pulmonary fi brosis of the lower lungs according to HRCT of the chest. Th e latter images were reviewed separately by a radiologist and two of the authors who did not have access to any information on the clinical status of the study patients. A fourth opinion was sought if there was no clear consensus.
According to the chest HRCT fi ndings, all individuals were divided into three groups: control smokers who had neither emphysema nor fi brosis; patients who had emphysema alone; and patients with CPFE who had upperlobe-dominant emphysema and lower-lobe-dominant fi brosis. A fi brotic score was determined according to the method described by Kazerooni and colleagues (22).
Serum samples for biomarker measurements were stored at -80°C until biomarker assays. Analysis of serum concentrations of transforming growth factor β1 (TGF-β1), surfactant protein D (SP-D), club cell secretory protein 16 (CC16), and Krebs von den Lungen 6 (KL-6) was conducted in the three groups and 10 patients with IPF as described in the Online www.copdjournal.com Supplement. Th e data for each biomarker were compared among the three groups, and the same procedure was followed for combinations of 2-3 biomarkers. In order to exclude the eff ects of renal insuffi ciency, creatinine concentration was measured in all serum samples; we excluded samples in which the creatinine concentration exceeded the reference range.

Statistical analysis
Comparison among the three groups was carried out by means of analyses of variance (ANOVA) or Kruskal-Walls tests as appropriate; t-tests based on the appropriate linear combinations of the random eff ects, and their standard errors were used to compare the means. When p values were less than 0.05, the diff erences were considered statistically signifi cant. Adjustments were made for age, sex, smoking status, and blood creatinine levels. Th e ability to classify a group was assessed using the C statistic (23). Th e overall C statistic is defi ned as the probability of concordance among groups that can be compared. Th e biomarkers can be compared if it can be determined which one is suitable for detecting CPFE.
Th e C statistic was calculated as the sum of concordance values divided by the number of comparable pairs among all or several biomarkers. In addition, Receiver Operator Characteristic (ROC) curves were plotted for the diagnostic models of the biomarkers. All analyses were conducted using the JMP software, version 9.0.3 for Windows (SAS Institute Inc.). Additional details about the statistics can be found in the Online Supplement.

Results
A fl ow diagram of the study design and the selection process for eligible patients is shown in Figure 1. Ultimately, 178 individuals were divided into the following three groups: smoker control, emphysema alone, and CPFE, consisting of 36, 115, and 27 individuals, respectively.
Th e baseline characteristics of the patients, which include the data on severity of COPD according to the GOLD criteria, are shown in Table 1 and Table 2 (only key parameters are shown here, and detailed data that include various other measurements are available in the Online Supplement in ETables 1-3). Th e CPFE group was signifi cantly older (p < 0.0001), showing greater prevalence of a smoking history (p = 0.0005), a smaller distance in 6MWT with signifi cant arterial desaturation (p < 0.0001) compared with the smoker control or emphysema alone groups.
With regard to the pulmonary function, the total lung capacity was the lowest in the CPFE group (p = 0.0015), whereas the emphysema alone group showed greater airfl ow obstruction. Th e mean %LAA in the upper lung fi eld was similar in the emphysema alone group, whereas in the CPFE group %LAA was signifi cantly larger than that of the smoker control group (p < 0.0001). Th e mean fi brotic score in the CPFE group was 8.9 ± 5.7 (Kazerooni's score range: no lung fi brosis to maximum, 0-30). Serum creatinine levels were within normal limits and closely correlated with the serum concentration of CC16 for all individuals (p < 0.00001); however, no association was found between the CC16 concentration and carbon monoxide in blood air.
Th e serum concentration of CC16 in the smoker control, emphysema alone, and CPFE groups was 5.67 ± 0.42,  5.66 ± 0.35, and 9.38 ± 1.04 ng/mL, respectively, and it was highest in the CPFE group (p = 0.0009, Figure 2). No significant diff erence was observed in serum concentrations of TGF-β1 among the three groups; the concentrations were 37.5 ± 1.9, 42.6 ± 2.4, and 37.2 ± 2.6 ng/ml in the smoker control, emphysema alone, and CPFE groups, respectively. Furthermore, signifi cant diff erences were observed among the groups in serum concentrations of KL-6, SP-D, and CC16; a signifi cant association was found between CC16 and SP-D after logarithmic transformation (p = 0.01) for all individuals.
In addition, we investigated the association between these biomarkers and various clinical parameters in the CPFE group. Th e percent predicted forced expiratory volume in 1 second (FEV 1    The AUC for each combination was as follows: KL-6 and CC16 (Fig 3A), SP-D and CC16 (Fig 3B), KL-6, SP-D, and CC16 (Fig 3C)  www.copdjournal.com Th e mean age of the IPF group was 71.8 years. Th e results of the pulmonary function tests in this group showed a mild restrictive disorder and severe diff usion disturbance. Th e mean fi brotic score in these patients with IPF was higher than that of the CPFE patients (12.7 ± 5.0 vs. 8.9 ± 5.7). Th e serum concentrations of CC16, KL-6, and SP-D were 22.15 ± 4.64 ng/ml, 1128.8 ± 556.3 U/ml, and 134.9 ± 68 ng/ml, respectively, in the patients with IPF, and the CC16 concentration was higher in the IPF group than in the CPFE group.
Detailed data that include various other measurements are available in ETable 5 in the Online Supplement.

Discussion
In the present study, we have shown that the serum concentration of CC16 increases in patients with CPFE and that combined testing for KL-6 and CC16 can eff ectively diff erentiate CPFE from emphysema alone. Th e diagnosis of usual or classical interstitial pneumonia (UIP) is made using a required histopathological assessment since the seminal report by Liebow and Smith in 1968 (24). More recently, imaging analysis such as highresolution computed tomography (HRCT) has shown remarkable development, and UIP can now be detected in HRCT images (12). Nonetheless, some cases are diffi cult to diagnose even using both histopathological evaluation and an advanced imaging technique; therefore, a diff erent (simpler) diagnostic method is needed.

Increased concentration of CC16
Th e 15.8 kDa club cell protein (CC16) is the major protein secreted by club cells and one of the main secretory proteins in the lung. CC16 occurs at high concentrations in the epithelial lining fl uid where it appears to play an antioxidant/anti-infl ammatory role mostly by modulating the production and/or activity of phospholipase A2, interferon-γ, and tumor necrosis factor α (18,25,26). A previous report has shown that serum CC16 levels are signifi cantly elevated in idiopathic interstitial pneumonia (27), which is consistent with the data in the present study.
Similarly, in cases of scleroderma or sarcoidosis, which is associated with interstitial pneumonia, the extent of fi brosis signifi cantly correlates with the level of CC16 (28)(29)(30). Th e results of the present study suggest that the increased level of CC16 in CPFE might refl ect the degree of lung infl ammation and/or fi brosis. It is unknown whether the elevated CC16 concentration is a transient phenomenon in CPFE or whether it is related to transient infl ammation in the lung tissue.
Lakind and colleagues (31) examined the relationship between serum CC16 and asthma etiology and exacerbations, and they found that acute exposure to certain pulmonary irritants or localized pulmonary infl ammation can cause a transient increase in the serum CC16 level. Th ey also showed that a transient increase in serum CC16 is not associated with detectable pulmonary damage or impairment of pulmonary function (31). In this regard, changes in the serum CC16 concentration during follow-up for CPFE, if they exist, might be not only a diagnostic predictor but also a therapeutic biomarker; further research concerning this use of CC16 is needed.

Effects of smoking on CC16
Th e data on CC16 in the smoker control and emphysema alone groups are also interesting. Chronic smoking reduces the serum concentration of CC16 in a dose-dependent manner; this eff ect is associated with The data were analyzed using ANOVA and are presented in the table as mean ± SD (range). FVC: forced vital capacity, FEV 1 : forced expiratory volume in one second, DLCO: diffusing capacity of the lung, PaO 2 : partial pressure of oxygen in arterial blood, PaCO 2 : partial pressure of carbon dioxide in arterial blood, AaDO 2 : Alveolar-arterial oxygen difference, 6MWT: 6-minute walking test, LVEF: left ventricular ejection fraction, PAP: pulmonary artery pressure, #: assessed by echocardiogram and NS: not signifi cant.
a concomitant reduction of CC16 in lung lavage and a progressive decline of club cell numbers (18). Th is observation also suggests that cessation of smoking restores the serum concentration of CC16 (18). In one followup study of COPD patients, the serum concentration of CC16 at baseline correlated with a slower rate of decline of FEV 1 (32).
Lomas and coworkers showed that the median serum CC16 level is signifi cantly lower for current and former smokers with COPD than for current and former smokers with no airfl ow obstruction in a post hoc analysis of the ECLIPSE study (33). Th e smoking status should aff ect the concentration of CC16, but this concentration appears to be close to normal in the smoker control group, including current and ex-smokers. Th ere are several possible explanations. Data on the smoking duration for each patient in the present study was obtained from self-reports (which can be inaccurate), and smoking status and/or duration might aff ect the serum CC16 concentration after smoking cessation; thus, prospective research is useful and necessary in this regard.
We hypothesize that club cell activity decreases in emphysemic lungs because of the loss of bronchioles in the pathogenesis of COPD (34), whereas the activity of club cells might increase when emphysema coexists with fibrosis in a different part of the same lung (as in CPFE), in keeping with the findings outlined above. Although it is intriguing that the activity of club cells in emphysema and lung fibrosis is different and may change in opposite directions, further investigation is needed to clarify the physiological basis for this phenomenon.

Other serum biomarkers in lung fi brosis
Krebs von den Lungen 6 (KL-6) shows promise as a diagnostic marker of interstitial lung diseases (26). Serum KL-6 is elevated in the majority of patients with interstitial lung diseases relative to patients with bacterial pneumonia and healthy individuals (35). KL-6 levels depend on the number of regenerating type II epithelial cells and the integrity of the alveolar-capillary membrane (36). Because KL-6 is chemotactic for human fi broblasts, this protein may also play a functional role in fi brosis (37).
Th e levels of KL-6 were signifi cantly diff erent ( p = 0.02) between smoker control and emphysema alone groups. Th is observation was consistent with a previous report showing that the serum concentration of KL-6 was higher in patients with emphysema versus healthy control individuals (38). Th e present data show that the combination of biomarkers CC16 and KL-6 was the best predictor for CPFE (AUC = 0.828); however, the combination of three promising biomarkers (CC16, KL-6, and SP-D) was the second best (AUC = 0.813). Th e two values are close, suggesting that the triple combination should not be discarded and can still be considered useful for the diagnosis of CPFE; this possibility needs further research in a large cohort study.
In order to investigate changes in CC16 levels in patients with the combined eff ects of lung fi brosis and life-long smoking, we studied an additional ten patients with IPF. Th e data show that the serum concentration of CC16 in the IPF group was signifi cantly higher that of the CPFE group. We had hoped to be able to compare the data of patients with IPF and CPFE as a case control study, ideally in patients with similar fi brotic scores; however, we were unable to recruit such patients for the present study.
Despite this defi cit, the present data provide useful information for further understanding role of CC16 in the CPFE and IPF groups. A higher serum concentration of CC16 in the IPF group might simply be a refl ection of fi brotic changes per se, similarly that in the CPFE group. If this speculation were correct, the pathogenesis of lung fi brosis in CPFE and IPF would be similar as has been previously reported (39). Further studies are needed to confi rm this hypothesis.
CPFE is defi ned as a clinical diagnosis based mainly on characteristic radiological fi ndings (7). However, the CC16 data in this study might provide additional diagnostically relevant information, since serum concentrations of CC16 are decreased in patients with chronic lung damage caused by tobacco smoke and other air pollutants as a consequence of the destruction of club cells.

Characteristics of the study design and limitations
Among the enrolled cases in the present study, we carefully selected cases compatible with the criteria described in the original report by Cottin et al. (7). In CPFE patients in the present study, the mean age was 74.9 versus 65.2 years in the previous study (7); never-smokers were not examined in both studies. Also not examined in either study were HRCT imaging for pulmonary interstitial shadows, which are consistent with idiopathic pulmonary fi brosis (IPF), and data on pulmonary function during mild restrictive and obstructive ventilator disturbance.
A decrease in walking distance during a 6-minute walking test (and slight arterial desaturation during the test) as well as an increase in mean pulmonary arterial pressure in echocardiograms were observed in both studies; our data are consistent with those by Cottin et al. (7) and others (9,10,(14)(15)(16)(17). By defi nition, CPFE is emphysema in the upper lobe and interstitial pneumonia in the lower lobe (7), but there are many cases with comorbidities and a mixed clinical picture, and the degree of pathological changes that are diagnostic of CPFE have yet to be clearly defi ned. Moreover, interstitial pneumonia is diverse; the degree of disease activity at the tissue level in the same lung is not uniform. Under such circumstances, in the CPFE cases where fi brosis and emphysema are mixed to various degrees, for clinical diagnosis, physicians can assume that combinations including CC16 are reliable.
Th e fi brotic score of CPFE in the present study was suggestive of mild fi brosis, which might result in a mild increase in serum concentrations of both KL-6 and SP-D, whereas the emphysema score, which was assessed by %LAA, indicated moderate emphysema.
www.copdjournal.com It would be interesting to conduct a study that compares diagnostic power for CPFE between chest HRCT and biomarkers of other types of interstitial fi brosis. We did not evaluate the infl uence of medication in this study, such as inhaled corticosteroids (this infl uence needs to be assessed in future research).
Although the present study addressed only the diagnostic role of serum biomarkers among the patients with CPFE alone, which were compatible with the criteria of the original report (7), these new data on CC16 in CPFE might shed some light on the pathogenesis of this disease.

Conclusions
Combinations of biomarkers including CC16 can provide useful information for screening and predicting CPFE, in addition to lung imaging data.