Interstitial lung disease (ILD) refers to a heterogenous group of diseases that mainly affect the alveolar–interstitial space and that share common features, such as the presence of lung fibrosis.1 Diagnosis is based on clinical, radiological and, if necessary, histological findings. Follow-up consists mainly of clinical evaluation, pulmonary function tests (PFTs), and chest high-resolution computed tomography (HRCT). However, there is a growing interest in serum biomarkers as a non-invasive means of early detection and follow-up of patients with ILD.

The therapeutic and prognostic impact of ILD in connective tissue disease (CTD) is especially important given the high associated morbidity and mortality.2–4 In fact, for all patients with systemic sclerosis, a recent expert consensus5 recommends screening for ILD that includes HRCT, PFTs, and chest auscultation. Early detection is crucial since ILD may already be at an advanced stage by the time symptoms appear or PFT results are altered.

Krebs von den Lungen 6 (KL-6) is a mucin-1 glycoprotein produced by type II pneumocytes. High levels of alveolar KL-66 and blood KL-67 may be found in patients with lung fibrosis due to epithelial damage, pneumocyte regenerating activity, and increased vascular permeability.

KL-6 has been extensively studied in Asia,8 mainly in the Japanese population, where values are used in current clinical practice to diagnose9 and especially to evaluate ILD progression. KL-6 implications for idiopathic pulmonary fibrosis (IPF) diagnosis and prognosis10 has also been explored for fibrosing lung diseases associated with CTD,11,12 including systemic sclerosis.13

Nonetheless, there is a lack of information regarding KL-6 cut-off points, diagnostic and prognostic value for the European population. Therefore, the aim of this study was to establish the cut-off point for serum KL-6 associated with the presence of ILD in the Spanish population.

A total of 179 patients were included, 102 with ILD. Mean (SD) age was 65.4 (12.9) years and 58% (n=104) were women. The main characteristics of the ILD and non-ILD groups are summarized in Table 1. There were statistically significant differences between the ILD and non-ILD groups regarding age and sex, but not smoking history.

Diagnoses (after multidisciplinary committee discussion) were as follows: IPF, 7 (6.9%); fibrotic non-specific interstitial pneumonitis (NSIP), 9 (8.9%); and fibrotic hypersensitivity pneumonitis, 9 (8.9%). Systemic sclerosis and Sjögren syndrome were the most prevalent CTDs in the ILD group patients: 16 (15.7%) and 14 (13.7%), respectively (see all ILD etiologies/diagnoses in Supplementary material Table 1).

The main radiological pattern was NSIP in 77 patients (43%), followed by usual interstitial pneumonia (UIP) in 57 (32%). Sub-analysis by radiological pattern showed statistically significant differences between the UIP and non-UIP groups for FVC <70%, DLCO, sex, and age but not for baseline KL-6 (see Supplementary material Table 2).

Mean (SD) FVC was 91.8% (21.7%) and mean (SD) DLCO was 72.7% (21%). Statistically significant difference was observed for FVC and DLCO between the ILD group and the non-ILD group (p<0.01). Group sub-analysis according to FVC >70% vs <70% revealed differences in KL-6, DLCO, UIP vs non-UIP radiology patterns, and sex. No differences were found with respect to age or smoking history (Table 2).

KL-6 Values and ROC Analysis

Mean (SD) serum KL-6 values overall were 762 (704)U/mL, and were 1080 (787)U/mL for the ILD group vs 340 (152)U/mL for the non-ILD group (p<0.0001).

Fig. 1 depicts results of the ROC analysis using AUC, showing greater specificity (86%) and sensitivity (82%) for KL-6 ≥465U/mL for patients with ILD. ROC analysis to determine the optimal cut-off value for screening purposes showed 95% sensitivity for KL-6 ≥307U/mL. Fig. 2 depicts sensitivity and specificity values according to KL-6 values and the chosen cut-off points.

Fig. 1.

ROC analysis using area under curve.

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Fig. 2.

Evolution of sensitivity and specificity depending on KL-6 values.

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In order to establish a better correlation between KL-6 values and PFT we performed a secondary ROC analysis to identify ILD based on FVC and DLCO ranges. An optimal KL-6 cut-off point was 449U/mL for FVC greater than 80% group (sensitivity 83%, specificity 81%) and 942U/mL for FVC between 50 and 80%. As for DLCO, the optimal cut-off was 424U/mL for DLCO greater than 80% (sensitivity 82%, specificity 74%), 469U/mL for DLCO between 50 and 80% and 489U/mL for DLCO <50% (see Supplementary material Figs. 1 and 2).

Sub-analyses of ILD, ILD-CTD, non-ILD, and non-ILD-CTD groups revealed no statistically significant differences in KL-6 values between the ILD and ILD-CTD groups, or between the non-ILD and non-ILD-CTD groups.

The multivariate logistic regression model pointed to male sex, higher KL-6 values, lower FVC and low DLCO values as independent factors associated with ILD (Table 3).

Table 3.

Prognostic Factors for Interstitial Lung Disease.

N=152	Logistic Multivariate Model
	OR	95% CILower Limit	95% CIUpper Limit	Wald Testp-Value
Gender (male vs female)	2.57	1.37	4.83	0.0032
FVC (%)	0.94	0.90	0.98	0.0036
DLCO (%)	0.96	0.93	0.99	0.0169
KL-6 (for each increase of 100 units)	2.33	1.57	3.46	<0.0001

OR: odds ratio; CI: confidence interval; DLCO: lung diffusing capacity for carbon monoxide; FVC: forced vital capacity; KL-6: Krebs von den Lungen.

Finally, Fig. 3 depicts the conditional inference tree model to facilitate ILD prediction/identification, starting with an initial KL-6 value of 364U/mL and then including sex and a second KL-6 value of 650U/mL. In men compared to women, the probability of ILD increased by 40% when KL-6 was below 364U/mL. Considering only the KL-6 value, the probability increased by 95% from 650U/mL onwards.

Fig. 3.

Decision tree model.

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The serum KL-6 cut-off value for ILD diagnosis in the European population has not been elucidated. In this prospective study, we found that 465U/mL was the optimal KL-6 cut-off for the presence of ILD, while a KL-6 cut-off of 307U/mL showed greater sensitivity for screening purpose. As reported in other series,7,21 serum KL-6 levels are independently correlated with the presence of ILD. Our population showed a wide range of KL-6 values. This variability might be attributed to the diverse ILD etiologies and the varying degrees of disease extension and activity at the time of inclusion.

High variability in KL-6 cut-off values for ILD diagnostic purposes has been observed in different populations. In Asian cohorts, mainly Japanese, the optimal cut-off is around 500U/mL,22–25 a value that differs from those reported in the few studies of KL-6 that exist for European populations. A small-scale retrospective Irish study with 34 patients26 reported an optimal KL-6 cut-off of 459U/mL, similar to that of our cohort. In contrast, for a retrospective French series of 75 patients, aimed at detecting ILD in patients with systemic sclerosis, a higher KL-6 value of 872U/mL was reported, with specificity of 95%.27 Horimasu et al.28 performed a landmark large-scale study (453 subjects) that compared serum KL-6 levels in German and Japanese ILD patients and healthy subjects, and also evaluated MUC1 polymorphisms that could affect KL-6 levels; the authors reported an optimal cut-off value of 659U/mL for the German subjects and 461U/mL for the Japanese subjects, concluding, after a genotype analysis, that this cut-off difference was partially due to different distributions of the MUC1 rs4072037 genotypes. Moreover, and as was the case in our study, KL-6 was found to be an independent factor for ILD presence and was not correlated with age or smoking.

The findings of Horimasu et al.28 reinforce the hypothesis of variations between populations and also point to differences reflecting different genetic bases. This would suggest that KL-6 values are not absolute and that KL-6 cut-off values may need to be established and validated for specific populations.

Another finding for our cohort was an optimal KL-6 cut-off for ILD screening purposes of 307U/mL with up to 95% sensitivity. This high sensitivity was kept in a subgroup analysis with preserve PFT, which highlights KL-6 utility for early stages of disease. Early diagnosis of ILD is currently a challenge, particularly for ILD-CTD, as diagnostic delay29–31 has a significant impact on survival.32 Further investigations are needed to facilitate early detection aimed at changing the natural course of this disease.

Drawing on the logistic regression analysis, we created a conditional inference tree based on KL-6 values and sex, as potentially useful to predict ILD. A recent study also used a decision tree to predict ILD, but based on smoking history and age as well as KL-6 levels.33 We suggest that our simple screening tool, based on KL-6 and sex, may be useful in clinical practice for ILD screening purposes.

Regarding radiology findings, NSIP was the main pattern (43%) followed by UIP (32%). In a subgroup analysis comparing the UIP and non-UIP groups, no statistically significant differences in serum KL-6 values were evident, a finding similar to other studies that found no KL-6 level differences between patients with UIP and NSIP.34,35

Our subgroup analysis for FVC pointed to higher KL-6 levels in patients with FVC <70%, corroborating the study by Horimasu et al.,28 who reported that serum KL-6 levels were inversely correlated with FVC and DLCO values. A correlation between KL-6 baseline levels and the annual changes of PFTs has also been found in another recent study.36

The main limitations of our study are its single-centre nature and the relatively small number of included patients. Furthermore, although radiology patterns were established, the degree of fibrosis was not quantified in the HRCT scans. As a strength, our study was prospective and included a control group.

Our data show that serum KL-6 values greater than 465U/mL have excellent sensitivity and specificity for detecting ILD. KL-6 values could be used to screen for ILD risk for instance in patients with CTD. Multicentre studies are needed to validate our results.

PMB, DC and IC conceived the idea and designed the methods. DC, SB and PMB collected the data. LMM and AM performed the KL-6 analysis. TF and MA contributed to the analysis and interpretation of data. All the authors reviewed the results and approved the final manuscript.

This study was supported by Fujirebio (product supply).

D. Castillo reports personal fees and non-financial support from Roche, grants, personal fees and non-financial support from Boehringer-Ingelheim, grants from Fujirebio, personal fees from Veracyte, outside the submitted work. D. Castillo is part of the Editorial board of Archivos de Bronconeumologia and declare that they have remained outside the evaluation and decision-making process in relation to this article.

P. Millán-Billi, I. Castellví, L. Martinez-Martinez, A. Mariscal, S. Barril, Miriana D’Alessandro and T. Franquet declare no conflicts of interest.