key: cord-0702861-zctwrzjq authors: Deng, Kai; Fan, Qinghong; Yang, Yanhong; Deng, Xizi; He, Ruiying; Tan, Yizhou; Lan, Yun; Deng, Xilong; Pan, Yuejun; Wang, Yaping; Guan, Yujuan; Liu, Huiyuan; Chen, Fengjuan; Mo, Xiaoneng; Tan, Xinghua; Luo, Chun; Wen, Xueliang; Liu, Ying; Liu, Jinxin; Zhang, Lieguang; Tang, Xiaoping; Hu, Fengyu; Li, Feng title: Prognostic roles of KL‐6 in disease severity and lung injury in COVID‐19 patients: A longitudinal retrospective analysis date: 2021-01-22 journal: J Med Virol DOI: 10.1002/jmv.26793 sha: af5f0bf58aed63661e2c112f1d9d04be7969157c doc_id: 702861 cord_uid: zctwrzjq To investigate the dynamic changes of Krebs von den Lungen‐6 (KL‐6) among patients with coronavirus disease 2019 (COVID‐19) and the role of KL‐6 as a noninvasive biomarker for predicting long‐term lung injury, the clinical information and laboratory tests of 166 COVID‐19 patients were collected, and a correlation analysis between KL‐6 and other parameters was conducted. There were 17 (10.2%, 17/166) severe/critical and 149 (89.8%, 149/166) mild COVID‐19 patients in our cohort. Serum KL‐6 was significantly higher in severe/critical COVID‐19 patients than in mild patients (median 898.0 vs. 451.2 U/ml, p < .001). KL‐6 was next confirmed to be a sensitive and specific biomarker for distinguishing mild and severe/critical patients and correlate to computed tomography lung lesions areas. Serum KL‐6 concentration during the follow‐up period (>100 days postonset) was well correlated to those concentrations within 10 days postonset (Pearson r = .867, p < .001), indicating the prognostic value of KL‐6 levels in predicting lung injury after discharge. Finally, elevated KL‐6 was found to be significantly correlated to coagulation disorders, and T cells subsets dysfunctions. In summary, serum KL‐6 is a biomarker for assessing COVID‐19 severity and predicting the prognosis of lung injury of discharged patients. of tons of immune cells in the lung inevitably produce cytokines and chemical mediators, which evoke the "cytokine storm" affecting other organs especially in severe/critical patients. 6 Notably, coagulation abnormalities, such as elevated D-dimer and lactate dehydrogenase (LDH), prolonged prothrombin time (PT) 7 are identified to be associated with poor prognosis and high mortality. 8 However, easy and affordable biomarkers indirectly reflecting lung injury are rarely employed to evaluate the long-term outcome, and the relationship between lung injury and coagulation abnormalities in COVID-19 patients remains uninvestigated. Krebs von den Lungen-6 (KL-6) is a glycoprotein mainly produced by damaged or regenerating lung type II pneumocytes. Elevated serum KL-6 concentrations have been utilized as a surrogate of interstitial lung diseases for more than 2 decades. [9] [10] [11] Recently KL-6 was reported to be associated with COVID-19 severity. 12 One hundred sixty-six COVID- 19 Five-hundred microliters of peripheral blood mononuclear cells were isolated from 10 ml of whole blood of patients and processed with flow cytometry with a panel of antibodies 12 including CD3-Pacific-Blue anti-human antibody (B286012; Biolegend), CD4-APC/cy7 antihuman antibody (B299289; Biolegend), CD8-BV510 anti-human antibody (B303256; Biolegend), CD161-FITC anti-human antibody (B302548; Biolegend), CD25-APC anti-human antibody (B294860; Biolegend), CD127-PE/cy7 anti-human antibody (B286366; Biolegend), CXCR5-Perp/cy5.5 anti-human antibody (B295344; Biolegend), and PD-1-PE anti-human antibody (B304891; Biolegend). Data were analyzed using FlowJo software (BD-Biosciences). Continuous variables were expressed as medians (interquartile) or mean ± SEM. Categorical variables were summarized as the counts and percentages in each category. Student's t tests were applied to continuous variables, the Mann-Whitney U test and χ 2 test were used for categorical variables as appropriate. Areas In total, 166 COVID-19 patients were enrolled in this study (Table 1) . A total of 149 (89.8%) patients were diagnosed as mild and 17 (10.2%) were as severe/critical. To get the base level of KL-6 level in the whole population, we measured the samples from 59 healthy volunteers (n = 59) and found that the median value of serum KL-6 was 180.9 U/ml ( Figure 1 and Table S1 ). Patients with COVID-19 had significantly higher levels of KL-6 compare to healthy volunteers (p < .001, Figure 1 ). The severe/ critical patients had even significantly higher levels of serum KL-6 than the mild (median 898.0 vs. 452.1 U/ml, p < .001, Table 1 and Figure 1 ). We found that KL-6 increased from symptom onset, reached a peak approximately within a month, and then gradually decreased ( Figure S1 ). How long it requires to reach KL-6 peak will be critical to reflect the extent and the speed of lung injury in patients with COVID-19. Here, we defined the day with the highest KL-6 among more than three detections within 1 month postonset as the peak day. We found a delayed Figure S2 ). Besides, we observed that it took 17.5 ± 1.9 (mean ± SEM) days for mild and 10.6 ± 1.8 (mean ± SEM) days for severe/critical patients to reach a higher level of KL-6 (cutoff = 600 U/ml, p = .011, Figure S3 ). Altogether, our results indicated that lung injury deteriorated rapidly and that continuous and progressive lung injury among severe/critical COVID-19 patients. Recently serum KL-6 as well as other parameters, for example, D-dimer and LDH, [15] [16] [17] [18] were suggested as a biomarker of COVID-19 severity, but the conclusion was drawn from a small cohort (n = 22 or 2). 12, 13 In our cohort (n = 166), ROC analysis supported the use of serum KL-6 distinguishing mild and severe/ critical patients (Table S2 and Figure S4 ). 3.2 | Early serum KL-6 predicted the prognosis of lung injury of discharged COVID-19 patients Figure 2 ). Since the hospital stay of COVID-19 was usually less than a month (Table 1 ), these data indicated that early serum KL-6 could be a potent predictor for the prognosis of lung injury of discharged patients. For those patients conducting computed tomography (CT) examination, we also explored whether there was an association between CT lung lesions areas and the KL-6 values within the previous week (−7 days) and within the next week (+7 days). We found that KL-6 values within the previous week had no significant correlations with lung lesions areas (p > .05, Figure 3A ). In contrast, KL-6 values within the next week were linearly correlated to CT left lung, right lung, and left plus right lung lesions areas (p < .001, Figure 3B ). Therefore, the late serum KL-6 was correlated to CT lung lesions areas based on our findings. Coagulation dysfunctions have been reported in severe COVID-19 patients. 8, 15, 19, 20 In our cohort, compared to the mild, the coagulation dysfunctions, for example, significantly elevated D-dimer (p = .003) and decreased prothrombin time activity (PTA, p < .001), were observed in severe/critical patients (Table 1) (Table S3) . Actually, for most mild patients, the coagulation indexes were normal during most of the hospital stay, as shown in Figure S5 . (Table S3 ). In contrast to mild patients, an elevated KL-6 was usually accompanied by coagulation dysfunctions among severe/critical patients, as shown in Figure 4 , which shows the dynamic profile of KL-6 and coagulation indexes of a severe/critical case. In all, these data suggested a correlation between elevated KL-6 and coagulation dysfunctions among patients with COVID-19. For all patients with COVID-19, the KL-6 level was negatively correlated to CD3+ T cell counts (Table S3) (Table S3) . For mild patients, a weak correlation between KL-6 and CD4+PD-1+ T cells (r = .273, p = .003), and CD3+ T cells (r = −.325, p < .001) was found (Table S3 ). Excessive activation of the immune system and production of inflammatory cytokines, termed as "cytokine storm," was considered to be one of the main causes of lung injury in severe COVID-19 patients. 21, 22 Here, we showed that among severe/cri- In this study, we showed a complete dynamic profile of serum KL-6 among COVID-19 patients from disease onset to postdischarge. In addition, we found that serum KL-6 of discharged COVID-19 patients could be predicted by its value at an early stage (Figure 2) , rendering Undoubtedly, pulmonary function testings would provide a more comprehensive assessment of the recovery of discharged patients. One of the most important questions in controlling COVID-19 is to identify the risk factors of severe illness or death. 23 Here, we confirmed that serum KL-6 was associated with COVID-19 severity in a cohort including 17 severe/critical patients. In addition, we also found a linear correlation between serum KL-6 and CT lung lesions areas, which links to COVID-19 progress. 24 Therefore, COVID-19 patients with an elevated KL-6 level on admission deserve a close observation in case of progression. Coagulation dysfunctions like high D-dimer levels on admission have been proved to be associated with poor overall survival. 25, 26 Innate immune response to SARS-CoV-2, dysfunctional ACE2, and inflammation activation may participate in the coagulation dysfunctions, 27 but the specific mechanism is unknown. Here, we demonstrated that KL-6 was correlated to coagulation indexes, for example, among severe/critical patients, KL-6 was strongly correlated to D-dimer (r = .692, p < .001) and FDP (r = .641, p = .001, Table S3 ), both of which have been suggested as risk factors of COVID-19 severity. 28 It is worth noting that for mild patients, KL-6 was weakly correlated to some coagulation indexes (Table S3) , which were normal during most of the hospital stay ( Figure S5 ), and serum KL-6 of some mild patients remained low from symptom onset ( Figure S1 ). So the interpretation of these correlations in mild patients should be careful. The role of host immunity in COVID-19-associated morbidity and mortality remains controversial. It is generally considered that overactivation of the immune system termed as "cytokine storm" mediates lung injury, 6 34 which was similar to that in our study. By now the cumulative number of locally confirmed cases in Guangzhou was 377 according to National Health Commission Report. 35 Thus, collecting lots of severe/critical patients in this region seems difficult. In summary, our findings support KL-6 as a biomarker of COVID-19 severity, and also a predictor of the prognosis of lung injury of discharged patients. The dynamic profile of KL-6 was closely correlated to coagulation disorder and immune dysfunction, especially among severe/critical patients, highlighting the possibility that coagulation disorder and immune dysfunction may be a contributor to lung injury of COVID-19, and the underlying mechanism requires further research. This study was supported by Guangdong Provincial Department of Science and Technology Fund (No. 2020B1111330002). The authors would like to thank Guangzhou Kangrun Biotech Co. for their technical assistance. The authors declare that there are no conflict of interests. Xiaoping Tang Qinghong Fan analyzed the data and prepared the manuscript. 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