Discussion
The present study provided two novel findings with clinical relevance. First, serum KL-6 levels could predict the clinical outcomes of Japanese patients with COVID-19 in this large multicentre study; additionally, we successfully determined the optimal cut-off value of serum KL-6 levels to predict such critical outcomes. It has already been reported that serum KL-6 effectively predicts the severity of COVID-19 or fibrotic change in patients with severe COVID-19.40 41 However, in a large patient population including patients with mild disease, for the first time in this study, KL-6 was stratified according to disease severity and was shown to be associated with hard endpoints. The present study also allowed us to perform a multivariate analysis where we showed an association between KL-6 levels and clinical outcomes using confounding factors, including biomarkers other than serum KL-6 and known risk factors.34–39 Moreover, the results showed that serum KL-6 levels were related to complications during hospitalisation. Therefore, the serum KL-6 levels of patients with COVID-19 may be a clinical indicator for providing more aggressive treatment. Second, the MUC1 variant (rs4072037) was related to serum KL-6 levels but not to the critical outcomes of patients with COVID-19. To the best of our knowledge, this is the first study to examine the association of MUC1 polymorphism with serum KL-6 levels and COVID-19 clinical outcomes.
The present study provides strong evidence that KL-6 levels can predict the critical outcomes of COVID-19 through significant multicentre validation. However, the efficacy of several serological biomarkers, such as those related to the inflammatory response and coagulation predisposition, in predicting COVID-19 critical outcomes has been previously reported.37–39 Notably, our results also showed that KL-6 was independently associated with critical outcomes, even when adding CRP, ferritin and D-dimer, which are known serological biomarkers of COVID-19, to the multivariable analysis. This suggests that serum KL-6 levels can be a complementary measurement of these biomarkers (online supplemental figure 4).37 38 Serum KL-6 is a known biomarker of lung injury and fibrosis, reflecting damage or regeneration of type 2 pneumocytes,42 based on its previously reported association with the severity and prognosis of ILDs and acute respiratory distress syndrome (ARDS).43–45 In the present study, KL-6 levels were also associated with the degree of COVID-19-derived pneumonia on imaging evaluation. A possible mechanism underlying the increased serum KL-6 levels in severe COVID-19 cases is the increased production of type 2 pneumocytes due to direct viral infection and leakage into the blood due to disruption of the alveolar–capillary barrier.40 Patients with severe COVID-19 often end up having ARDS, and deaths due to COVID-19 are associated with a higher incidence of ARDS than that in COVID-19 survivors. It has been reported that autopsy samples of the lungs of patients with COVID-19 show a pattern of diffuse alveolar damage (DAD), a pathology of ARDS.46 47 Therefore, it can be inferred that the primary mechanism underlying the increase in serum KL-6 levels is the leakage of KL-6 from the lungs into the blood due to virus proliferation and destruction of the alveolar epithelium and basement membrane with DAD.40 In fact, not only blood but also epithelial lining fluid KL-6 levels are increased in patients with ARDS,48 and high levels of this biomarker have also been reported in the bronchoalveolar lavage fluid of patients with severe COVID-19.49 In the present study, KL-6 levels were associated with bilateral shadows on imaging findings, consistent with the results of previous studies.16 50
The KL-6 cut-off value for predicting critical outcomes was 304 U/mL. This value was lower than the cut-off values found for diagnosing ILDs and poor prognostic factors for ARDS and ILDs.45 51 Furthermore, the KL-6 cut-off value was lower than that previously reported for predicting the severity of COVID-19.12 14 41 We hypothesise that this may be because previous studies had fewer participants but included more patients with relatively severe disease compared with that in the present study. The alveolar–capillary permeability in COVID-19 is elevated in the acute phase and may further increase with lung fibrosis.12 A previous report on COVID-19 suggested that peak serum KL-6 levels during hospitalisation are more helpful than those at diagnosis in predicting clinical outcomes.12 Therefore, further evaluation of serum KL-6 levels over time should be explored in future studies.
The present study also revealed the relationships between the MUC1 variant and serum KL-6 levels in Japanese patients with COVID-19. The results obtained here are consistent with those of previous reports demonstrating that the MUC1 variant affects KL-6 levels in ILDs and sarcoidosis.21 22 MUC1 is an extracellular protein anchored to the epithelial surface and involved in morphogenetic signalling.21 22 52 The presence of the rs4072037 SNP (single nucleotide polymorphism) causes alternative splicing of the exon regions under its regulation, leading to abnormal transcription.53 MUC1 has been demonstrated to be a critical innate immunity modulator,54 acting as an important and necessary anti-inflammatory agent during airway infection.25 55 It is controversial whether the MUC1 variant affects the severity of ILDs. In support of this hypothesis, it has been reported that the MUC1 variant affects the severity of pulmonary alveolar proteinosis.24 However, some studies showed that rs4072037 may be involved in disease susceptibility to ILDs but is not related to the severity of the disease.23 For example, high frequency of the rs4072037 C allele and serum KL-6 levels have been reported in patients with antisynthetase syndrome compared with those in healthy control subjects.23 It has also been reported that in patients with systemic sclerosis-ILD, the rs4072037 mutation is significantly associated with increased serum KL-6 levels but is not a predictor of a lower diffusing capacity for carbon monoxide.23 In the present study, the MUC1 variant did not correlate with COVID-19 severity. Moreover, it has been reported that the optimal cut-off value of serum KL-6 levels discriminating patients with ILD from healthy control subjects differs following the rs4072037 genotype.21 The results of the present study suggest that high serum KL-6 levels may be due not only to severe COVID-19, but also to the MUC1 variant, which may result in false-positive serum KL-6 in patients with severe COVID-19. Although it is not possible to measure MUC1 variants in actual clinical practice at this time, caution should be exercised in interpreting serum KL-6 in patients with COVID-19.
Recently, it has been recognised that COVID-19 symptoms can persist after the disease, which is referred to as long COVID or postacute COVID-19 syndrome.56 57 It is estimated that 22.9% of patients with COVID-19 have residual shortness of breath.58 It has also been reported that within 1–6 months of discharge from the hospital, 55.7% of patients have residual abnormal findings on CT scans and 44.3% have residual abnormal pulmonary function tests.59 Furthermore, 56% of patients treated with mechanical ventilation have been reported to have reduced pulmonary diffusion capacity 6 months after discharge.60 Serum KL-6 levels may also help predict and diagnose long COVID. An association between residual CT shadows and KL-6 levels at 3 months has been reported.17 Future studies should examine the relationship among serum KL-6 levels, changes on imaging and long COVID on admission, during hospitalisation and after discharge.
There are several limitations to the present study. First, image scoring was not available. Previous studies have reported a correlation between CT quantitative pneumonia range and serum KL-6 levels.61 62 In this large, multicentre study, CT could only be evaluated qualitatively; future comparisons with quantitative evaluations are desirable. Second, serum KL-6 levels were not measured over time. Serum KL-6 levels continue to rise during hospitalisation and peak later in severe cases than in mild cases62 63; moreover, peak serum KL-6 levels have been reported to be more useful in predicting COVID-19 severity and death than serum KL-6 levels on admission.12 41 64 Although measuring peak levels may improve sensitivity and specificity, the fact that serum KL-6 levels on admission were predictive of outcome is both convenient and clinically useful. Third, critical outcomes are significantly higher in patients whose serum KL-6 levels were measured on admission than in those who did not. It is speculated that this result might be because KL-6 was aggressively measured in patients with more severe COVID-19 on admission to assess the extent of lung disease. This selection bias may affect the prediction values of serum KL-6 values for critical outcomes. Fourth, in this study, we were unable to show the prevalence and severity of ARDS with serum KL-6 levels because we could not assess PaO2 (partial pressure of arterial oxygen) levels via arterial blood gas analysis on admission. Severe COVID-19 is associated with ARDS, but the effect of ARDS on serum KL-6 levels of patients with COVID-19 cannot be determined from this study. However, we revealed that serum KL-6 levels were higher in patients with more severe disease on admission or those with rapid deterioration. These findings support that serum KL-6 is a predictive marker in patients with ARDS-like severe COVID-19 or rapidly deteriorating COVID-19 on early admission.
In conclusion, the levels of serum KL-6 within 48 hours of admission were associated with critical outcomes in a large Japanese multicentre study involving patients with COVID-19. Further, the MUC1 variant was associated with serum KL-6 levels within 48 hours of admission but not with critical outcomes. Thus, serum KL-6 levels on admission may be a useful biomarker of critical COVID-19 outcomes.