Serum Krebs von den Lungen-6 Level as a Reflecting Biomarker in Patients with Interstitial Lung Abnormalities
Article information
Abstract
Background
Research on the relationship between the progression of interstitial lung abnormalities (ILA) and serum biomarkers, including white blood cell differential counts and Krebs von den Lungen-6 (KL-6), is limited. This study aimed to examine the clinical characteristics of patients with ILA and evaluate the association between disease progression and serum biomarkers.
Methods
This retrospective cohort study analyzed data from 159 patients (63 with ILA and 74 with interstitial lung diseases) between October 2021 and September 2022. Data collected included clinical characteristics, pulmonary function tests, chest computed tomography (CT), complete blood cell counts, and KL-6 levels. In 52 of these patients who had previously undergone chest CT, the utility of serum biomarkers in reflecting radiologic progression was assessed using receiver operating characteristic curve analysis.
Results
Patients with ILA exhibited clinical characteristics similar to those with idiopathic pulmonary fibrosis. Serum KL-6 levels did not correlate with forced vital capacity or diffusing capacity of the lung for carbon monoxide in patients with ILA. Among the 52 patients with ILA, 13 demonstrated radiologic progression. Serum KL-6 displayed moderate predictive performance, with area under the curves ranging from 0.57 to 0.89 (p=0.014) for radiologic progression. Levels of KL-6 greater than 400 U/mL were more frequently observed in patients with radiologic progression (61.5% vs. 20.5%, p=0.006). In multivariate analysis, age and KL-6 were independently associated with radiologic progression in patients with ILA.
Conclusion
Serum KL-6 levels may serve as a potential indicator of ILA progression in asymptomatic patients. Those with KL-6 levels exceeding 400 U/mL should be closely monitored for radiologic progression.
Introduction
Interstitial lung disease (ILD) is a diverse group of inflammatory and fibrotic conditions, with fibrotic ILD representing a significant subset that poses a major global health challenge [1]. Several fibrotic ILDs, including idiopathic pulmonary fibrosis (IPF), can lead to progressive declines in lung function, respiratory symptoms, and overall quality of life [2]. Fibrosis is characterized by recurrent, subclinical epithelial injury combined with accelerated epithelial aging, resulting in architectural distortion, irreversible loss of function, and early mortality [3]. Interstitial lung abnormalities (ILA) are defined as non-dependent findings on chest computed tomography (CT) that may indicate ILD in patients who do not have a clinical suspicion of the condition [4]. They are common incidental findings on CT, with a prevalence of 2% to 10% [4-6] and a progression rate of 20% to 72% in imaging [7-9]. According to imaging patterns, ILA is classified into nonsubpleural, subpleural nonfibrotic, and subpleural fibrotic types [4]. Though advanced age and copies of the mucin 5B (MUC5B) genotype are associated with imaging progression [7], little is known about the relationship between imaging progression and serum biomarkers, including white blood cell differential counts and Krebs von den Lungen-6 (KL-6), in ILA. Serum KL-6 is a circulating, high molecular weight glycoprotein expressed by type II pneumocytes. Its levels increase during the regeneration of type II pneumocytes and the destruction of the air-blood barrier in affected lungs [10]. Serum KL-6 has been reported as a sensitive marker for diagnosing and predicting the prognosis of ILD [11-13]. Although the pathophysiology of fibrotic ILD is not yet fully understood, various immune cells, such as monocytes, neutrophils, and lymphocytes, have been linked to its pathogenesis [14]. Several studies have demonstrated that serum monocyte count is a simple and cost-effective prognostic biomarker in patients with ILD [14-16]. However, few studies have examined serum biomarkers in patients with ILA. In this study, we aimed to explore the clinical characteristics of ILA in comparison to ILDs. Given that ILA may be associated with the early stages of progressive pulmonary fibrosis, including IPF, we also sought to determine whether serum biomarkers, such as KL-6 and white blood differential cell counts, correlate with imaging progression in patients with ILA.
Materials and Methods
1. Patients
In this observational cohort study, we retrospectively reviewed the medical records of 159 patients who underwent blood tests, including KL-6, and chest CT at a teaching hospital from October 2021 to September 2022. After excluding seven patients with unilateral abnormalities on chest CT and pneumonia, we analyzed the records of 64 patients with ILA and 88 patients with ILD. All patients with ILA presented to the hospital for a medical checkup with CT abnormalities, but without any symptoms or signs. Based on imaging patterns, the 64 patients with ILA were categorized into nonsubpleural, subpleural nonfibrotic, and subpleural fibrotic patterns [4]. Previous studies have indicated a higher risk of radiologic progression in subpleural ILA [17]. Consequently, we included 32 patients with subpleural nonfibrotic ILA and 31 patients with subpleural fibrotic ILA. Out of 88 patients with ILD, 14 were excluded due to small sample sizes. This group included eight patients with idiopathic interstitial pneumonia with autoimmune features, three with unclassifiable idiopathic interstitial pneumonia, one with cryptogenic organizing pneumonia, one with chronic hypersensitivity pneumonitis, and one with pleuroparenchymal fibroelastosis. Ultimately, 54 patients with IPF and 20 with connective tissue disease-associated ILD (CTD-ILD) were included (Figure 1).
Study population. KL-6: Krebs von den Lungen-6; ILA: interstitial lung abnormality; ILD: interstitial lung disease; IPAF: interstitial pneumonia with autoimmune feature; IIP: idiopathic interstitial pneumonia; COP: cryptogenic organizing pneumonia; HP: hypersensitivity pneumonitis. PPFE: pleuroparenchymal fibroelastosis; IPF: idiopathic pulmonary fibrosis; CTD-ILD: connective tissue disease-associated interstitial lung disease.
Patient medical records were reviewed to gather data on medical conditions, pulmonary function tests, laboratory findings, and chest CT images. All patients included in the analysis underwent blood tests, including complete blood cell counts and KL-6, as well as a chest CT. Among the 63 patients with ILA, 53 had previously undergone chest CT more than 12 months prior and were enrolled to assess radiologic progression, as all patients had chest CT scans at the time of enrollment. Although the specific duration of radiologic progression in ILA is not clearly defined, the criteria for progressive pulmonary fibrosis typically involve a 1-year timeframe [18]. Due to the retrospective nature of this study, chest CT scans performed exactly 1 year after diagnosis were not always available. Therefore, we included patients who had follow-up CT scans obtained within 18 months. One patient who had undergone a chest CT more than 36 months prior was excluded. Since none of the patients without radiologic progression showed improvements, all patients with chest CT scans taken more than 1 year apart were included. For patients with radiologic progression, we used the chest CT obtained within 12 months (up to a maximum of 18 months). For those without progression, we selected the most recent CT scan taken approximately 12 months after diagnosis. In total, 52 patients were analyzed for radiologic progression in this study.
The Institutional Review Board of Inje University Ilsan Paik Hospital approved this study, including the review and publication of information obtained from patient records (IRB no. 2022-09-044). The requirement for informed consent was waived for the use of patient medical data, as all patient information was anonymized and de-identified prior to analysis.
2. Measurement
Demographic features, smoking history, and comorbidities were obtained by reviewing the available medical records on the day the patients underwent laboratory tests. Diagnoses of IPF and CTD-ILD were established through a multidisciplinary approach in accordance with diagnostic guidelines [18,19]. Comorbidities were reviewed, including hypertension, diabetes mellitus, ischemic heart disease, chronic obstructive pulmonary disease (COPD), and emphysema. Emphysema was confirmed through chest CT.
CT scans were obtained using a 320-detector row CT scanner (Aquilion ONE/PRIME SP; Canon Medical Systems, Otawara, Japan). ILA are defined by incidental CT findings of non-dependent abnormalities, according to the Fleischner position paper [4]. These findings include ground-glass or reticular abnormalities, lung distortion, traction bronchiectasis, honeycombing, and non-emphysematous cysts [4]. ILA was categorized into subpleural nonfibrotic and fibrotic ILA based on the presence of pulmonary fibrosis, characterized by architectural distortion, traction bronchiectasis, or honeycombing [4]. Radiologic progression was defined as an increased extent of fibrotic features—such as traction bronchiectasis, ground-glass opacity, reticulation, and honeycombing— on chest CT imaging obtained at enrollment compared to previous chest CT imaging [18].
In our analysis, we did not include features like non-dependent ground-glass opacities or centrilobular nodularity in the definition of progression. This progression was assessed independently by one pulmonologist and one radiologist. We evaluated inter-observer agreement for key radiologic features using the kappa statistic. In cases of disagreement, both readers reviewed the images together to reach a consensus.
Spirometry was performed following the official guidelines of the American Thoracic Society [20]. The absolute values of forced vital capacity (FVC) and diffusing capacity of the lung for carbon monoxide (DLCO) were obtained, and the percentage of predicted values (% predicted) for both FVC and DLCO was calculated using equations derived from a representative Korean sample [21].
Medical records of blood tests, including complete blood count and KL-6 levels, were collected. We included only patients who had both chest CT and serum biomarker measurements available within a 2-week window. Neutrophil, lymphocyte, and monocyte counts, as well as the neutrophil-lymphocyte ratio (NLR), were calculated from the complete blood count results. The threshold for stratifying patients by monocyte count was set at 600 cells/μL [14]. Serum KL-6 levels were measured using the Nanopia KL-6 assay (Sekisui Diagnostics, Tokyo, Japan). While the manufacturers recommend a cutoff value of 500 U/mL for the KL-6 assay, we used 400 U/mL based on the results of the receiver operating characteristic (ROC) curves conducted in this study.
3. Statistical analysis
Data are presented as medians and interquartile ranges (IQRs) for continuous variables, and as counts (%) for categorical variables. Continuous variables were compared using the Mann–Whitney U test, while categorical variables were analyzed using either Pearson’s chi-square test or Fisher’s exact test. To evaluate the relationship between serum biomarkers and FVC in patients with ILA and IPF, Spearman’s correlation and a linear model were employed. ROC curves were generated for KL-6, neutrophils, lymphocytes, monocytes, and NLR to assess each biomarker's ability to discriminate radiologic progression, with varying thresholds for each variable. The areas under the ROC curves (AUC) were calculated and compared. Additionally, multivariate logistic regression analysis was conducted to identify independent factors associated with radiologic progression in patients with ILAs. A statistical p-value of <0.05 was deemed significant. All analyses were performed using IBM SPSS Statistics for Windows version 22.0 (IBM Corp., Armonk, NY, USA).
Results
1. Baseline characteristics of study patients compared with IPF
The clinical characteristics of the study participants are summarized in Table 1. Among the 63 patients with ILA, 60 (95.2%) were men, 11 (17.5%) were lifetime nonsmokers, and the median age was 74 years (IQR, 69 to 78). Of these patients, 22 (34.9%) had been diagnosed with ischemic heart disease, and 18 (29%) had COPD. In the ILA cohort, age, sex, smoking history, and comorbidities were significantly different compared to patients with CTD-ILD, but not with those with IPF (Supplementary Table S1).
Baseline characteristics of patients with ILAs and IPF
For patients with ILA, the median FVC (% predicted) and DLCO (% predicted) were 94% (IQR, 85% to 104%) and 85% (IQR, 74% to 99%), respectively. The median white blood cell count (cells/μL), neutrophil count (cells/μL), monocyte count (cells/μL), and KL-6 level (U/mL) were 6,790/μL (IQR, 5,555 to 7,500), 3,762/μL (IQR, 2,981 to 4,510), 518/μL (IQR, 420 to 604), and 331.0 U/mL (IQR, 251.7 to 431.1), respectively. These values were significantly lower than those observed in patients with IPF (Figure 2). Monocyte counts greater than 600/μL were less frequent in patients with ILA compared to those with IPF. Serum biomarkers were not associated with FVC or DLCO in patients with ILA, while KL-6 exhibited significant negative correlations with both measures in IPF, and no meaningful correlations were observed for other biomarkers (Supplementary Table S2).
Comparison of serum biomarkers between interstitial lung abnormality (ILA) and idiopathic pulmonary fibrosis (IPF). (A) Blood cell counts including white blood cells (WBCs), neutrophil, lymphocyte, and monocyte. (B) Serum Krebs von den Lungen-6 (KL-6). NEU: neutrophil; LYM: lymphocyte; MO: monocyte; IQR: interquartile range.
2. Characteristics of ILA according to radiologic progression
A total of 52 patients with ILA were divided into two groups: those with radiologic progression (n=13) and those without (n=39). The baseline characteristics, pulmonary functions, complete blood cell counts, and ILA types were not significantly different between the two groups (Table 2). Notably, elevated KL-6 levels (>400 U/mL) were significantly more common in patients with radiologic progression (61.5% vs. 20.5%, p=0.006).
Baseline characteristics of 52 patients with ILAs according to radiologic progression
3. Factors associated with radiologic progression of ILAs
Candidate variables for logistic regression analysis included DLCO (% predicted), KL-6 >400 U/mL, and subpleural fibrotic ILA. These objective variables had p<0.1 in the comparison between ILA patients with and without radiologic progression, along with age, sex, and smoking status (Table 3). In the multivariate analysis, age (adjusted odds ratio [aOR], 1.16; 95% confidence interval [CI], 1.01 to 1.34; p=0.039) and KL-6 >400 U/mL (aOR, 8.32; 95% CI, 1.46 to 47.32; p=0.017) were independently associated with radiologic progression.
Factors associated with radiologic progression of ILAs
4. The performance of biomarkers in reflecting the radiologic progression of ILA
The overall performance of biomarkers, including KL-6, NLR, lymphocytes, neutrophils, and monocytes, in reflecting radiologic progression over 12 to 18 months was evaluated by constructing ROC curves (Figure 3). The AUC of the ROC curves were as follows: 0.730 for KL-6 (95% CI, 0.571 to 0.888; p=0.014), 0.560 for NLR (95% CI, 0.359 to 0.762; p=0.519), 0.394 for lymphocytes (95% CI, 0.208 to 0.581; p=0.258), 0.473 for neutrophils (95% CI, 0.275 to 0.672; p=0.775), and 0.458 for monocytes (95% CI, 0.227 to 0.638; p=0.650). When the threshold value from the ROC analysis was set at 400 U/mL for serum KL-6, the sensitivity, specificity, positive predictive value, and negative predictive value were 61.5%, 79.5%, 50.0%, and 86.1%, respectively.
Receiver operating characteristic (ROC) curves of radiologic progression and biomarkers in patients with interstitial lung abnormalities. KL-6: Krebs von den Lungen-6; AUC: area under the ROC curve; CI: confidence interval.
Discussion
In the present study, we found that patients with ILAs exhibited clinical characteristics similar to those seen in IPF, rather than in CTD-ILD. We also demonstrated that serum KL-6 levels were associated with radiologic progression over 12 to 18 months in asymptomatic patients with ILA, but were not correlated with pulmonary function in these patients, which contrasts with findings in IPF and CTD-ILD. Furthermore, we found that KL-6 levels greater than 400 U/mL were linked to radiologic progression in patients with ILA. White blood cell differential counts—including neutrophils, lymphocytes, the NLR, and monocytes—were not significantly associated with pulmonary function or radiologic progression in patients with ILA. ILA is defined as an incidental CT finding of non-dependent abnormalities occurring in the absence of clinical respiratory symptoms [4]. Identifying the early stages of an undiagnosed form of progressive ILD is an important challenge in asymptomatic patients with ILA [22]. A cohort study has shown that the histopathologic features of ILA represent an early stage and mild form of pulmonary fibrosis in some cases. Specifically, ILA with subpleural radiologic features is associated with histopathological findings of usual interstitial pneumonia [23]. Additionally, the subpleural fibrotic pattern is linked to the highest risk of progression [7]. In our study, while subpleural fibrotic ILA is not significantly associated with radiologic progression, a tendency was observed linking subpleural fibrotic ILA to radiologic progression.
Serum KL-6 is classified as a human MUC1 mucin protein, with regenerating type 2 pneumocytes serving as the primary cellular source of KL-6/MUC1 in the lungs of patients with ILD [24]. It is significantly elevated in patients with ILD due to increased expression by regenerating type 2 pneumocytes and heightened permeability following damage to the alveolocapillary barriers in the lungs [25]. Previous studies have shown that serum KL-6 correlates with lung function and radiologic parameters, including the extent of fibrotic abnormalities in patients with ILD [26,27]. Additionally, they demonstrated that the serum KL-6 level at the time of diagnosis predicts the progression and acute exacerbation of ILD [28-31].
In this study, our data from patients with ILA revealed that serum KL-6 levels were not linked to pulmonary function but were associated with radiologic progression. Specifically, we found that KL-6 levels exceeding 400 U/mL correlated with radiologic progression within 12 to 18 months. The lack of association between serum KL-6 and pulmonary function may be attributed to our focus on asymptomatic patients with ILA, who generally exhibited relatively normal lung function compared to those with IPF and CTD-ILD. Nevertheless, our findings provide significant insights into the radiologic progression of ILA.
Previous studies have shown that immune-related cells, such as monocytes, neutrophils, and lymphocytes, may serve as potential cellular biomarkers for disease progression. This aligns with existing knowledge about the roles of monocytes and other immune cells in the development of ILD [14,16,32,33].
Our study found that white blood cell differential counts—specifically neutrophils, lymphocytes, and monocytes—were not associated with radiologic progression in patients with idiopathic lung disease (ILA). The white blood cell count serves as a nonspecific marker of inflammation or immune response. Asymptomatic patients with ILA may not experience disease progression due to minimal inflammation. The strength of our study lies in the evaluation of serum biomarkers, including KL-6 and white blood cell differential counts, as prognostic factors in asymptomatic patients with ILA, an approach previously explored only in patients with ILD. Additionally, we consistently assessed chest CT imaging using uniform criteria at a single institution. Although the Fleischner Society [4] defined the criteria for ILA, a skilled physician specializing in this area conducted a thorough review of the images using standardized criteria. This aspect can yield more reliable and consistent data.
The present study has several limitations. First, due to its retrospective observational design, there is a possibility that selection bias or confounding factors may have influenced our findings. This design also restricts our ability to assess serum biomarkers as prognostic indicators. Second, we were unable to evaluate a healthy control group without ILA or ILD. Future prospective studies comparing patients with ILA to healthy controls would provide valuable insights into the utility of KL-6 in this population. Nonetheless, our study does highlight differences in the clinical use of serum KL-6 between patients with ILA and those with ILD. Third, the small sample size limits our ability to identify risk factors for radiologic progression. Despite this limitation, we were able to demonstrate a significant association between KL-6 levels and radiologic progression. Finally, since blood samples were not collected for this study, we could not assess other systemic biomarkers, such as matrix metalloproteinase 7, surfactant protein D, and chemokine ligand 18. Further research exploring the relationships between these systemic biomarkers and radiologic progression would be beneficial for identifying risk factors associated with progression.
In conclusion, this study demonstrated that serum KL-6 may serve as a predictive marker for imaging progression within 12 to 18 months in patients with ILA. Additionally, the ROC-derived threshold value of KL-6 for radiologic progression was found to be 400 U/mL. These results highlight the importance of careful monitoring, as a KL-6 level exceeding 400 in asymptomatic patients with ILA.
Notes
Authors’ Contributions
Conceptualization: Kang HK, Shin JW. Methodology: Kang HK, Koo HK, Jung JW, Choi JC, Kim JY, Shin JW. Formal analysis: Kang HK, Shin JW. Data curation: Kang HK, Chung SJ, Kang J, Koo HK, Lee SS, Shin JW. Project administration: Kang HK, Koo HK, Jung JW, Choi JC, Kim JY, Shin JW. Software: Kang HK. Validation: Kang HK, Koo HK, Shin JW. Investigation: Kang HK. Writing - original draft preparation: Kang HK. Writing - review and editing: all authors. Approval of final manuscript: all authors.
Conflicts of Interest
No potential conflict of interest relevant to this article was reported.
Funding
This work was supported by a grant from research year of Inje University in 2022 (20220021).
Supplementary Material
Supplementary material can be found in the journal homepage (http://www.e-trd.org).
Baseline characteristics of patients with ILAs and CTD-ILD.
Association between serum biomarkers and pulmonary function parameters in patients with ILAs and IPF.