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Baseline serum Krebs von den Lungen-6 as a biomarker for the disease progression in idiopathic pulmonary fibrosis

Disease progression (DP) is an important parameter for the prognosis of idiopathic pulmonary fibrosis (IPF). This study aimed to evaluate the baseline serum biomarkers for predicting the DP in IPF. Seventy-four patients who were diagnosed with IPF and had their serum Krebs von den Lungen-6 (KL-6) and monocyte count, which might be associated with prognosis of IPF, checked more than twice were included. KL-6 ≥ 1000 U/mL and monocyte ≥ 600/μL were arbitrarily set as the cut-off values for DP. The DP was defined as a 10% reduction in forced vital capacity, a 15% reduction in diffusing capacity of the lung for carbon monoxide relative to the baseline, or disease-related mortality. Of the 74 patients, 18 (24.3%) were defined as having DP. The baseline KL-6 level was significantly increased in the DP group compared to the stable disease group (median, 1228.0 U/mL vs. 605.5 U/mL, P = 0.019). Multivariate Cox analyses demonstrated that a high KL-6 level (KL-6 ≥ 1000 U/mL; hazard ratio, 2.761 or 2.845; P = 0.040 or 0.045) was independently associated with DP in each model. The baseline serum KL-6 level might be a useful biomarker for DP in IPF.


Risk factors for disease progression.
Univariate analysis demonstrated that a high KL-6 level (≥ 1000 U/ mL) was significantly associated with DP, while a high monocyte count (≥ 600/μL) was not. Multivariate analysis demonstrated that a high KL-6 level was independently associated with DP (hazard ratio, 2.761; 95% confidence interval, 1.048-7.273; P = 0.040), after adjusting for BMI. When including clinically significant variables, multivariate analysis demonstrated that a high KL-6 level was independently associated with DP (hazard ratio, 2.845; 95% confidence interval, 1.023-7.912; P = 0.045), after adjusting for age, sex, BMI, forced expiratory volume at 1 s (FEV1), FVC, and DL CO ( Table 3). The Kaplan-Meier curve demonstrated that the high KL-6 group showed a higher risk of DP than the low KL-6 group (P = 0.032, Fig. 2). However, the high monocyte count group showed no significant differences from the low monocyte count group (P = 0.440).
We also compared lung function between the increased KL-6 group and the non-increased KL-6 group to determine whether the interval change of KL-6 level is associated with DP. The increased KL-6 group (n = 34) and the non-increased KL-6 group (n = 26) showed no significant differences in the proportion of DP (P = 0.817, Table 4). Moreover, the serially measured lung functions at the 3rd, 6th, and 9th months from baseline were not significantly different between the groups (FVC; P = 0.590, DLco; P = 0.964, Fig. 3).

Discussion
In the current study, the baseline serum KL-6 levels increased in the DP group compared to the SD group in patients with IPF. The baseline serum KL-6 level was independently associated with DP in multivariate analysis. These findings suggest that the baseline serum KL-6 level might be a useful predictive marker for the DP in IPF.
Previous studies have reported that several factors are associated with mortality in IPF, including age, sex, smoking status, dyspnea, BMI, pulmonary hypertension, PFT, 6MWT, and serum and bronchoalveolar lavage fluid biomarkers 4,22 . Among these factors, declined lung function is one of the best surrogate markers for diseaserelated mortality in IPF 23 . In particular, FVC and DL CO are the only physiologic variables in the IPF staging system GAP index 24 and they are also used for defining the DP in IPF 22 . Thus, FVC and DL CO are considered to be good indicators of lung function and prognosis in IPF. As a result, reducing the decline of FVC is an important goal in IPF treatment and is regarded as the primary outcome of studies on anti-fibrotic drugs 7,8 . However, few studies have focused on the predictors of DP in IPF, especially using biomarkers.
Biomarkers might have advantages in reproducibility because they are less influenced by patients' general conditions or examiners' variations. Many potential biomarkers have been studied, including KL-6, SP-A, SP-D, MMPs, and CCLs 9-13 . Among them, KL-6 is well established and used in clinical practice [14][15][16][17] . KL-6 is a mucinlike glycoprotein and is strongly expressed on the surface of type II pneumocytes, which are regenerated over the alveolar basement membrane after the death of type I pneumocytes during the lung injury in interstitial lung disease 25 . Patients with IPF with an elevated level of KL-6 (KL-6 > 1000 U/mL) are known to have increased mortality compared to patients with a KL-6 level within the normal range 17,26 . In this study, baseline KL-6 ≥ 1000 U/mL was significantly associated with the DP in IPF, suggesting that KL-6 might act as a surrogate marker for DP in IPF.
There are conflicting studies regarding the values of serial changes in serum KL-6 levels for the prognosis in IPF. Wakamatsu et al. reported that patients with increased serum KL-6 levels during follow-up had significant decline in FVC compared to those with no KL-6 increase 15 . d' Alessandro et al. also showed that patients with persistently elevated KL-6 had decreased FVC, whereas patients with decreased KL-6 had preserved FVC 27 . However, Ishii et al. suggested that a reduction in serum KL-6 level with untreated IPF does not indicate stable IPF status, but DP in IPF with a natural decline in serum KL-6 level 28 . It is possible that the serum KL-6 level reflects the extent of damaged alveolar epithelium 25 rather than the degree of disease activity. In this study, the increased KL-6 group showed no significant difference in DP and lung function compared to the non-increased KL-6 group. Therefore, our results suggest that patients with high level of baseline serum KL-6 should be considered to have the higher possibility of DP regardless of the follow-up serum KL-6 level. However, it should be considered that patients with severely deteriorated lung function, including deceased, could not perform follow-up lung function test, and therefore may not have been included in the analysis. Further studies will be needed for the serial measurement of KL-6 level.   www.nature.com/scientificreports/     21 .
Considering the results of previous studies, and the fact that only six patients (8%) had a monocyte count ≥ 950/ μL in our study, we arbitrarily set a monocyte count ≥ 600/μL as the cut-off value. However, the high monocyte count was not associated with the DP in IPF in our data, possibly because our sample size was small to validate it or because of the influence of other situations, including other co-incidental infections. This study has some limitations. First, this was a single-center, retrospective, nonrandomized study which might induce a selection bias. Second, the monocyte count was calculated by white blood cell differential on complete blood count (CBC) rather than flow cytometry, so the calculated monocyte count may differ from the real value. However, previous studies checked the monocyte count calculated by white blood cell differential on CBC and showed the associations between the monocyte count and the prognosis in IPF 20,21 . Third, the followup periods were relatively short, with a median follow-up of 9 months. However, approximately one-fourth of patients were classified as having DP, which was sufficient compared to previous studies 14,17 . Fourth, subgroup analysis according to the anti-fibrotic medication profile could not be performed due to our small sample. Despite these limitations, our study revealed the clinical role of KL-6 in predicting DP of patients with IPF.
This real-world population analysis suggests that elevated baseline serum KL-6 level might predict the DP in IPF and it could be a useful biomarker in clinical practice.

Methods
Study population. Seventy-four patients who were diagnosed with IPF and had their KL-6 and monocyte count checked more than twice between January 2020 and June 2021 at Asan Medical Center, Seoul, Republic of Korea were included in this study. All patients underwent thorough systemic history taking. Patients with definite connective tissue disease or an exposure history in relation to the possible causes of interstitial lung disease were excluded. IPF diagnosis was performed by a multidisciplinary approach, using the diagnostic criteria set by the American Thoracic Society (ATS)/European Respiratory Society (ERS)/Japanese Respiratory Society/ Latin American Thoracic Association in 2018 1 . All available baseline clinical parameters were obtained within 3 months of the initial blood sampling time. The study protocol was approved by the Institutional Review Board of Asan Medical Center (approval number: 2021-0787). The requirement for informed consent was waived because of the retrospective study design. The present study complied with the Declaration of Helsinki and all methods were performed in accordance with the relevant guidelines and regulations. Serum biomarkers. As biomarkers, serum KL-6 and monocyte count were evaluated in this study. The serum KL-6 concentration was measured using a Nanopia KL-6 assay (SEKISUI MEDICAL, Tokyo) using a latex-enhanced immunoturbidimetric assay method. The monocyte count was calculated using white blood cell differential on CBC. Serum KL-6 ≥ 1000 U/mL is well known to predict poor prognosis in IPF, and, as such, was set as the cut-off value in this study 15,17,26 . However, the optimal cut-off value of monocytes has not yet been validated. Therefore, we arbitrarily set monocytes ≥ 600/μL as a cut-off value based on recent studies 20,21 . Moreover, to determine whether the interval change in KL-6 level is related to DP, we divided patients into two groups according to the KL-6 level at the 3rd month from the baseline. Patients who had increased KL-6 levels at the 3rd month compared to baseline level were classified as the increased KL-6 group, and other patients were classified as the non-increased KL-6 group.