WO2022095227A1

WO2022095227A1 - Plasma-soluble urokinase plasminogen activator receptor and application thereof

Info

Publication number: WO2022095227A1
Application number: PCT/CN2020/136279
Authority: WO
Inventors: 朱海红; 王云云; 吴凤天; 陈超; 陈智
Original assignee: 浙江大学
Priority date: 2020-11-03
Filing date: 2020-12-14
Publication date: 2022-05-12
Also published as: CN112630442A

Abstract

The present invention pertains to the field of clinical medicine, and relates to a soluble urokinase plasminogen activator receptor (suPAR), which can be used as an independent biomarker for clinical outcomes of patients with acute-on-chronic liver failure. Provided are a plasma-soluble urokinase plasminogen activator receptor and an application thereof. Further provided is an application of a reagent of the soluble urokinase plasminogen activator receptor in the preparation of an early diagnosis kit for liver cancer.

Description

A plasma-soluble urokinase-type plasminogen activator receptor and its application

technical field

The invention belongs to the field of clinical medicine, and relates to the fact that soluble urokinase plasminogen activator receptor (suPAR) can be used as an independent biomarker for clinical outcome of patients with acute-on-chronic liver failure.

Background technique

Acute-on-chronic liver failure (ACLF) is a complex syndrome defined by the onset of acute liver failure in patients with pre-existing chronic liver disease. It is characterized by high short-term mortality, organ failure and excessive systemic inflammation. Excessive systemic inflammation is thought to be a major driver of ACLF development. Traditional scoring systems for end-stage liver disease (MELD), MELD-Na and Child-Turcotte-Pugh (CTP) scoring models cannot accurately predict mortality and multiple organ failure (MOF) in ACLF. More recently, the CANONIC study developed the CLIF Concomitant Organ Failure (CLIF-C) score, which proved to be more useful in predicting ACLF outcomes than conventional scoring systems. However, the scoring process is a bit complicated and may hinder timely understanding of the patient's condition. Therefore, there is a need to discover new biomarkers with good predictive value.

The urokinase-type plasminogen activator receptor (uPAR) is part of the plasminogen activator (PA) system. This system is involved in many physiological and pathological processes, including thrombosis, inflammation, tissue remodeling, and tumorigenesis. suPAR is a stable protein released from the cleavage of the urokinase plasminogen activator receptor (uPAR, CD87) during inflammation. uPAR is predominantly expressed on the membranes of circulating immune cells such as monocytes and neutrophils and is closely associated with immune functions such as cell attachment, motility, migration, proliferation and fibrinolysis. suPAR retains most of the functionality of uPAR. Furthermore, suPAR may bind to podocyte β3 integrin to cause kidney disease and enhance lipopolysaccharide-induced neutrophil activation. It is elevated in various infections such as HIV infection, malaria, tuberculosis and sepsis, suggesting that it may predict these disease outcomes. This predictive ability may also be useful in ACLF, but few studies have looked at suPAR levels in ACLF patients. Using commercial ELISA kits, the determination of suPAR content is simple and fast. Therefore, we explored whether suPAR is a suitable biomarker to predict the outcome of patients with ACLF.

SUMMARY OF THE INVENTION

In view of the above technical problems existing in the prior art, the present invention provides an independent biomarker for clinical outcome of patients with acute-on-chronic liver failure.

An independent biomarker for clinical outcome of patients with acute-on-chronic liver failure, the marker is soluble urokinase-type plasminogen activator receptor.

In addition the present invention provides new uses of the reagents for said independent biomarkers.

The application of the reagent for soluble urokinase-type plasminogen activator receptor in the preparation of an early diagnosis kit for liver cancer.

The detection method was: according to ELISA, plasma samples (25 μL) were used to measure suPAR by ELISA (suiro) (ViroGates, Denmark).

The present invention provides an independent biomarker for clinical outcome of patients with acute-on-chronic liver failure.

Description of drawings

Figure 1 Comparison of plasma suPAR concentrations in different disease groups.

(a) Plasma suPAR concentrations in healthy controls (n=14), chronic hepatitis B patients (n=14) and acute-on-chronic liver failure patients (n=42).

(b) Comparison of suPAR levels in patients with acute-on-chronic liver failure with and without clinical complications. The horizontal line represents the median.

ns, no statistical difference; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001.

Figure 2 Comparison of K-M survival curves between patients with acute-on-chronic liver failure with or without cirrhosis and with or without hepatic encephalopathy. Cumulative 90-day survival between groups was compared using the log-rank test.

Detailed ways

In order to describe the present invention more specifically, the technical solutions of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

1. Cases, materials and methods

(1) Case enrollment

From December 10, 2016 to March 10, 2018, adult patients with suspected ACLF were enrolled at the First Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang Youth Hospital and Ningbo Yinzhou Second Hospital. ACLF diagnosis is based on the Asia Pacific Association for Studies (APASL) criteria: "Acute liver injury manifested by jaundice (bilirubin ≥ 5 mg/dl) and coagulopathy (INR > 1.5) in patients with previously diagnosed or undiagnosed chronic liver disease, Concurrent ascites and/or encephalopathy within 4 weeks." Cirrhosis was diagnosed by previous liver biopsy, endoscopy, radiological evidence, or clinical manifestations of liver decompensation. Hepatorenal syndrome (HRS), spontaneous bacterial peritonitis (SBP), and ascites were diagnosed using the criteria established by the International Ascites Club and the American Association for the Study of Liver Disease, respectively. ACLF patients were then further classified as acute multiple hepatic failure (ACLF-MOF) based on the presence of two or more extrahepatic organ failures, the others ACLF, and were classified as ACLF. Exclusion criteria were pregnancy, a diagnosis of acquired immunodeficiency syndrome (AIDS), any type of malignancy, or liver transplantation. Chronic hepatitis B (CHB) is defined as patients with chronic hepatitis B by histological, radiographic, laboratory or clinical or evidence of cirrhosis or fibrosis or chronic liver inflammation and positive serum HBsAg for more than six months diagnosis. Healthy controls (HC) had no past or current medical history or clinical evidence and were negative for serum HBsAg. CHB and HC were sex- and age-matched with ACLF patients. The study conformed to the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine. The study cohort was followed for 90 days after enrollment, and the endpoint was death or liver transplantation.

(2) suPAR and cytokine measurement

Volunteer whole blood samples were collected and plasma obtained after centrifugation and immediately stored at -80°C. Plasma samples (25 μL) were used for the measurement of suPAR by ELISA (suiro) (ViroGates, Denmark) according to ELISA, and plasma samples (20 μL) were used for various cytokine assays (Bio-Rad, Hercules, CA).

2. Results

(1) Patient characteristics:

A total of 282 ACLF patients who met the inclusion and exclusion criteria were enrolled. Table 1 shows a comparison of the baseline characteristics of these patients with and without MOF. suPAR and baseline characteristics were measured in all participants. Patients with MOF had significantly higher plasma suPAR on admission than those without MOF (11.9 (9.1-15.5) ng.mL and 16.4 (11.5-24.0) ng/mL; p<0.001, Table 1). Certain clinical events (eg, HRS and hepatic encephalopathy (HE)), laboratory data (eg, white blood cell count (WBC), international normalized ratio (INR), and total bilirubin (Tbil)) were also found between the two groups. There are significant differences, as well as prognostic scoring systems (eg CTP, MELD and SOFA scores). All patients were followed up at the end of this point.

(2) Baseline plasma suPAR levels and their relationship with ACLF disease progression:

The determination of plasma suPAR in HC, CHB and ACLF patients showed that the level of suPAR in ACLF patients was significantly higher than that in HC and CHB patients. (Fig. 1a, 12.16(7.61-17.57) vs. 2.3(2.00-2.89) vs 2.7(2.16-4.00) ng/L; p<0.001). However, there is no difference between HC and CHB. Then, plasma suPAR levels were further compared in ACLF patients with and without complications at admission or during follow-up. On admission, patients with HE, HRS, UGBI, or infection had higher suPAR levels than those without these complications. The differences in plasma suPAR levels were most pronounced in HRS patients (30.15 (13.57–36.35) ng/L vs 12.30 (9.33–16.46) ng/L; p<0.001). Patients who exhibited circulatory failure during follow-up also showed significantly higher levels of suPAR than those without circulatory failure (Fig. 1b).

In addition to clinical characteristics, clinical laboratory data also found significant associations with plasma suPAR, and prognostic scores were associated with mortality (Table 2). All three prognostic scores correlated with suPAR levels. The strongest correlation was with MELD (r=0.421, p<0.001) (Table 2). In laboratory data, various infection- and immune-related data were shown to be positively correlated with suPAR levels, including bacterial or fungal infections, the incidence of WBC, and PCT. Strong correlations were found between two of the 27 cytokines, MIP1beta and IL-8, and plasma suPAR levels in 40 patients (r=0.453, p=0.007; r=0.448, p = 0.003) (Table 2). There were also significant correlations between suPAR and liver-related data, kidney-related data, and thyroid-related data, but not heart-related data (Table 2).

(3) Survival analysis:

During the 30-day follow-up, 62 patients (22.0%) died and 34 patients (12.1%) received liver transplantation. During the 90-day follow-up period, 82 patients (29.1%) died and 41 patients (14.5%) received liver transplantation. During the 90-day follow-up period, patients who died or underwent transplantation (n=123) had higher baseline plasma suPAR than those who survived without liver transplantation (n=159) (16.03 (11.65–23.70) vs. 11.14 (8.41– 14.14) ng/L, p<0.001; Fig. 1b).

The optimal cut-off point for plasma suPAR to predict 90-day mortality was 14.7 ng/mL according to the Youden index. Based on this optimal cut-off point, Kaplan-Meier curves significantly indicated 30-day and 90-day mortality in ACLF patients. Interestingly, the effect of suPAR levels on mortality was significantly greater in patients without cirrhosis or with HE (Figure 2). The effect of high suPAR levels on 30-day mortality in patients with ACLF and cirrhosis was less than that on 90-day mortality (Figure 2). During the 90-day follow-up period, patients with high suPAR (suPAR ≥ 14.7 ng/mL) and HE had the highest mortality rate, while ACLF patients with low suPAR and no HE had the lowest mortality rate (Figure 2).

Multivariate analysis using Cox regression determined that suPAR ≥ 14.7 ng/mL and WBC ≥ 6.6 x 10 ⁹ , and MELD ≥ 23.1 SOFA ≥ 9.5, were independent predictors of 90-day and 30-day mortality (Table 3).

Furthermore, analysis of the area under the receiver operating characteristic curve (ROC-AUC) suggested that suPAR may be a useful predictor of 30- and 90-day mortality in ACLF patients (0.751 and 0.742, respectively) (Table 4).

Table 1. Baseline Characteristics of ACLF Patients

ACLF: acute-on-chronic liver failure; ACLF-MOF: ACLF-multiple organ failure; CHB: chronic hepatitis B; HC: healthy control group; UGIB: upper gastrointestinal bleeding; HRS: hepatorenal syndrome; HE: hepatic Encephalopathy; SBP: Spontaneous Bacterial Peritonitis; WBC: White Blood Cell Count; INR: International Normalized Ratio; CTP: Child-Turcotte-Pugh; MELD: Model of End-Stage Liver Disease; SOFA: Sequential Organ Failure Assessment.

Table 2 Correlation of clinical parameters and prognostic scoring system containing suPAR concentrations

HBcAb: anti-HBV core antibody; PCT: procalcitonin; CRP: c-reactive protein; MIP1beta: macrophage inflammatory protein 1beta; INR: international normalized ratio; MAP: mean arterial pressure; hsTnI: highly sensitive troponin I ; GFR: glomerular filtration rate; T3: total triiodothyronine; FT3: free triiodothyronine; CTP: Child-Turcotte-Pugh; MELD: model of end-stage liver disease; SOFA: sequential organ failure assessment ; UGIB: upper gastrointestinal bleeding; WBC: white blood cell count. *p<0.05, **p<0.01, ***p<0.001

Table 3. Multivariate Cox regression model for short-term mortality in patients with acute-on-chronic liver failure

Table 4. ROC area of suPAR for predicting mortality in ACLF patients

Claims

An independent biomarker for clinical outcome of patients with acute-on-chronic liver failure, the marker is soluble urokinase-type plasminogen activator receptor.
Application of the reagent for detecting the independent biomarker of claim 1 in the preparation of a kit for early diagnosis of liver cancer.