Serial KL-6 measurements in COVID-19 patients

SARS-CoV2-induced direct cytopathic effects against type II pneumocytes are suspected to play a role in mediating and perpetuating lung damage. The aim of this study was to evaluate serum KL-6 behavior in COVID-19 patients to investigate its potential role in predicting clinical course. Sixty patients (median age IQR, 65 (52–69), 43 males), hospitalized for COVID-19 at Siena COVID Unit University Hospital, were prospectively enrolled. Twenty-six patients were selected (median age IQR, 63 (55–71), 16 males); all of them underwent follow-up evaluations, including clinical, radiological, functional, and serum KL-6 assessments, after 6 (t1) and 9 (t2) months from hospital discharge. At t0, KL-6 concentrations were significantly higher than those at t1 (760 (311–1218) vs. 309 (210–408) p = 0.0208) and t2 (760 (311–1218) vs 324 (279–458), p = 0.0365). At t0, KL-6 concentrations were increased in patients with fibrotic lung alterations than in non-fibrotic group (755 (370–1023) vs. 305 (225–608), p = 0.0225). Area under the receiver operating curve (AUROC) analysis showed that basal KL-6 levels showed good accuracy in discriminating patients with fibrotic sequelae radiologically documented (AUC 85%, p = 0.0404). KL-6 concentrations in patients with fibrotic involvement were significantly reduced at t1 (755 (370–1023) vs. 290 (197–521), p = 0.0366) and t2 (755 (370–1023) vs. 318 (173–435), p = 0.0490). Serum concentrations of KL-6 in hospitalized COVID-19 patients may contribute to identify severe patients requiring mechanical ventilation and to predict those who will develop pulmonary fibrotic sequelae in the follow-up. Supplementary Information The online version contains supplementary material available at 10.1007/s11739-020-02614-7.


Introduction
Krebs von den Lungen-6 (KL-6) is a serum high molecular weight glycoprotein, increased in many interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis and hypersensitivity pneumonitis [1][2][3]. It is mainly produced by damaged or regenerating alveolar type II pneumocytes and its serum concentrations are regarded as biomarker of lung epithelial damage (reff). The prognostic value of peripheral KL-6 in ILD has been validated as well as its promising value in predicting the response to antifibrotic therapies [4,5]. Moreover, KL-6 has been also proposed as bioindicator of acute respiratory distress syndrome (ARDS) and infective pneumonia (ref). Since the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, KL-6 has also been proposed as a prognostic marker for this disease [6][7][8][9]. The pathogenesis of COVID-19 (as the lung disease caused by SARS-CoV-2 is defined) is not entirely clear [10], although it is postulated that elevated serum concentrations of proinflammatory cytokines and oxidative stress mediators contribute to lung injury, facilitating the onset of an acute respiratory syndrome (similar to ARDS) [11]. Host susceptibility and virus-induced direct cytopathic effects against type I and II pneumocytes are suspected to play a crucial role in mediating and perpetuating lung damage [12][13][14].
Our research group first reported elevated serum concentrations of KL-6 in critical COVID-19 patients and our results were soon confirmed by two other papers [8,9]. However, no data are currently available on KL-6 role in the follow-up of COVID-19 patients or its potential predictive value for fibrotic lung alteration development.
The aim of this study was to evaluate serum KL-6 behavior in a population of COVID-19 hospitalized patients along the 9-month follow-up of our Centre, to investigate its potential role in predicting clinical course of disease.

Study population
Sixty patients (median IQR, 65 (52-69), 43 males), all hospitalized for COVID-19 at Siena COVID Unit University Hospital, were prospectively enrolled. Fifteen of these patients need intensive care unit (ICU) and mechanical ventilation. Our sixty patients underwent serum sampling specifically for KL-6 assessment on admission (t0). Patients who did not give informed consent to the study or who had a previous diagnosis of interstitial lung disease or chronic obstructive pulmonary disease were excluded.
After 6 (t1) and 9 (t2) months from hospital discharge, twenty-six patients (median age (IQR) 63 (55-71) years, 16 males) underwent follow-up evaluations including physical examination, lung function tests, diffusing capacity of the lung for carbon monoxide (DLCO), blood gas analysis and high-resolution computed tomography (HRCT) of the chest. CT features (fibrotic interstitial lung abnormalities, ground-glass opacities, and air-trapping) were evaluated by on-site radiologists, experienced in interstitial lung diseases. All patients gave their written informed consent to the study for clinical data collection. The study was approved by our local ethics committee (C.E.A.V.S.E Markerlung 17,431).

KL-6 assay
Serum samples were obtained from all patients on admission, before any biological treatment or intravenous infusion of high-dose steroids or invasive ventilation, and from 26 patients at follow-up, 6 and 9 months after discharge from hospital. Serum concentrations of Krebs von den Lungen-6 were measured by KL-6 reagent assay (Fujirebio Europe, UK), as previously reported [2,3,9,15,16]. The principle of the assay is agglutination of sialylated carbohydrate antigen with KL-6 mAb by antigen-antibody reaction. The change in absorbance was measured to determine serum concentrations of KL-6, which were expressed in U/ml. A cut-off value of 465 U/ml was applied, as previously reported [17].

Lung function tests
Lung function tests were performed according to ATS/ERS standards [18] using a Jaeger body plethysmograph with corrections for temperature and barometric pressure. Forced expiratory volume in the first second (FEV1), forced vital capacity (FVC) and diffusing capacity of the lung for carbon monoxide (DLCO) were recorded.

Statistical analysis
The data did not show a normal distribution. Results were reported as median (IQR), unless otherwise stated. Oneway ANOVA nonparametric test (Kruskal-Wallis test) and Dunn's post test were used for multiple comparisons. The Mann-Whitney test was used to compare pairs of variables. Wilcoxon matched pairs signed rank test was used to compare variables of the same patient at t0 and t1. The Chisquared test was used for categorical variables as appropriate. Statistical analysis and graphic representation of the data were performed with GraphPad Prism 8.0 software. Table 1 shows the main characteristics of our COVID-19 population at t0 (hospital admission). Serum concentrations of KL-6, PFT and blood gas analysis parameters were collected (Table1).

Results
At each sampling time, the study population did not differ significantly in terms of sex, age or comorbidities, whereas serum KL-6 concentrations were higher at hospitalization (t0) than at t1 (760 (311-1218) vs 309 (210-408) p = 0.0208) and t2 (760 (311-1218) vs 324 (279-458), p = 0.0365). About PFT parameters, we did not collect these data at t0 in hospitalized patients affected by COVID19. According to CT radiological features, patients were classified according to the evidence of fibrotic lung alterations (including ground-glass opacities, linear thickening, or organizing pneumonia areas).
Concerning respiratory function, no significant differences in FVC and/or DLCO percentages were found between patients with and without fibrotic lung alterations at CT scan.

Discussion
This report first described KL-6 peripheral concentrations in a population of COVID-19 patients after 6 and 9 months from hospitalization discharge together with radiological and functional parameters. Serum concentrations of KL-6 at hospital admission were reported to be significantly increased in severe patients admitted to intensive care unit and requiring intubation with mechanical ventilation, but not in mild-moderate patients with less severe respiratory impairment [9]. This mucin protein has been widely studied in idiopathic pulmonary fibrosis and ARDS patients, but limited data are available on its prognostic potential in infectious viral pneumonia [11][12][13]. Our interest aroused from the observation that KL-6 was associated with prognosis in ILD and ARDS, reflecting type I and type II alveolar pneumocyte damage. SARS-CoV-2 is known to have specific tropism for alveolar epithelial cells, causing interstitial lung damage, epithelial lung alterations, and regenerative processes, mainly in the acute phase [9]. A significant increase in serum concentrations of KL-6 was demonstrated in patients critically ill with COVID-19 [8,9] and was further confirmed in our study. Interestingly, our results demonstrated for the first time a normalization of serum concentrations of KL-6 (< 465U/ ml) 6 and 9 months after discharge from hospital. Lung damage due to COVID-19 seemed not to be necessarily progressive, unlike in idiopathic pulmonary fibrosis, where KL-6 tends to increase with IPF progression [1,4,5]. Moreover, in the majority of patients with fibrotic sequelae, the decrease and substantial normalization of serum KL-6 concentrations may reflect the amelioration of the clinical status, suggesting that fibrotic interstitial lung abnormalities related to COVID-19 are not associated with persistent active epithelial damage and consequent aberrant fibrogenesis. On the contrary, those patients who developed severe persistent fibrotic lung sequelae at HRCT showed persistent high levels of KL-6 in the follow-up, implicating that this biomarker may be useful in the mid-long-term management of these patients. However, these data will be further evaluated in a longer follow-up to be confirmed. Moreover, in our population, lung volumes were almost preserved at follow-up as well as DLCO percentages, differently from a recent follow-up study reporting functional alterations in more than 25% of patients, albeit after a shorter follow-up of 3 months [19], and this aspect may surely influence the heterogeneity of our population.
Thus, this study contributes to the definition of the natural course of COVID-19 as the normalization of peripheral KL-6 concentrations, recorded 6 and 9 months after the acute phase of SARS-CoV-2 infection, suggesting a non-progressive fibrotic lung involvement in the majority of patients.
Serum concentrations of KL-6 seemed to reflect lung involvement in COVID-19 patients as reflected by HRCT features at 6-month follow-up.
In conclusion, increased serum concentrations of KL-6 in hospitalized COVID-19 patients may help to early discriminate severe patients requiring mechanical ventilation and predict those developing fibrotic lung sequelae in the follow-up.
Author contributions MD, LB, PC, and EB conceived the study and supervised all aspects. MD, PC, GC, LR, FB, and MAM collected data and built the database. MD, PC, LB, EB, and MAM oversaw data analysis and interpretation of results. All authors drafted and revised the paper.
Funding Open Access funding provided by Università degli Studi di Siena. The study was performed at Siena University without funding or sponsors.

Compliance with ethical standards
Conflict of interest All the authors have no conflicts of interest to declare.

Statement of human and animal rights
The research was approved by the local ethics committee (MARKERLUNG n° 17431, C.E.A.V.S.E n° 18712).
Informed consent All subjects gave their written informed consent to the study.
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