B7-h3 molecular marker and use thereof in in vitro assessment of risk of poor prognosis in hepatitis e virus-associated acute liver failure

Abstract

The application discloses a B7-H3 molecular marker and application thereof in in-vitro evaluation of a poor prognosis risk of hepatitis E virus (HEV)-associated acute liver failure (ALF), and belongs to the technical field of biomedical detection. The application can identify the short-term poor prognosis risk of an HEV-ALF patient, can not only make up for the defects of insufficient sensitivity and specificity of a prognosis evaluation method in the prior art, but also has high clinical application potential and popularization value, and provides a new technical means for high-risk patient identification, risk stratification and clinical auxiliary decision-making.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically involving the detection of B7-H3 molecular markers and their application in in vitro assessment of poor prognostic risk in hepatitis E-related acute liver failure. Background Technology

[0002] Hepatitis E virus (HEV) infection is a significant cause of acute viral hepatitis, carrying a high disease burden globally. Most HEV-infected individuals present with self-limiting hepatitis; however, in certain populations, particularly those with compromised immune status, underlying liver disease, or certain metabolic or inflammatory backgrounds, the disease can rapidly progress to severe liver damage, even developing into acute liver failure. Hepatitis E-related acute liver failure (HEV-ALF) is characterized by rapid onset, rapid progression, and highly heterogeneous clinical outcomes. Some patients may experience deterioration of coagulation function, persistently elevated bilirubin, hepatic encephalopathy, renal impairment, and even multiple organ failure within a short period, resulting in a high short-term mortality rate. Therefore, identifying high-risk patients in the early stages of the disease, achieving effective risk stratification, and supporting clinical decision-making have become crucial clinical challenges in this field.

[0003] Currently, risk assessment for patients with acute liver failure or severe liver injury primarily relies on comprehensive scoring systems such as serum total bilirubin (TBIL), prothrombin time (PT), international normalized ratio (INR), creatinine (Cr), and MELD. While these indicators have some value in reflecting organ function impairment and disease severity, they are essentially phenotypic indicators of end-stage organ decompensation, often reflecting existing damage rather than earlier changes in biological risk. In other words, although existing indicators can describe the severity of the condition, they are insufficient for determining whether a patient will worsen or belong to a high-risk group for short-term death. Especially among patients with similar baseline laboratory indicators, clinical outcomes may still show significant differences, suggesting that relying solely on traditional biochemical indicators and comprehensive scoring systems is insufficient to meet the needs for refined prognostic stratification of HEV-ALF patients.

[0004] From a pathophysiological perspective, the development of HEV-ALF is not a single hepatocellular injury event, but rather the result of the combined effects of virus-related damage, host immune activation, amplified inflammation, imbalanced liver regeneration, and multi-system stress responses. Therefore, ideal prognostic biomarkers should not only reflect disease severity but also, to some extent, integrate or map host immune status, the intensity of inflammatory regulation, and the tendency of disease progression. This would provide more sensitive and earlier risk signals for short-term outcome assessment before the disease fully enters the irreversible decompensated stage.

[0005] B7-H3 (CD276) belongs to the B7 family of immune-regulating molecules and plays an important biological role in immune response regulation, inflammatory microenvironment formation, tissue damage response, and disease progression. Current research suggests that B7-H3 can participate in the interaction between innate and adaptive immunity and is associated with the degree of inflammation, tissue damage status, and adverse clinical outcomes. However, current technologies mainly focus on basic or translational research on B7-H3 in areas such as tumors, infectious diseases, and organ damage. A systematic technical approach is still lacking to establish its dynamic changes in HEV-ALF, its correlation with short-term mortality outcomes, and its application value as an in vitro prognostic biomarker.

[0006] In particular, there is currently no mature in vitro assessment method for short-term adverse prognostic risk in HEV-ALF patients based on B7-H3 and in vitro blood sample testing results. Furthermore, there is a lack of an application system built around this biomarker that simultaneously provides evidence from cohort studies, internal validation, and external validation support. Therefore, developing an in vitro assessment method based on B7-H3 to identify short-term adverse prognostic risk in HEV-ALF patients would not only compensate for the deficiencies in sensitivity and specificity of existing prognostic assessment methods but also have high clinical application potential and widespread value.

[0007] Based on this, the inventors conducted a systematic study on the serum B7-H3 level of HEV-ALF patients, and intend to establish an in vitro application scheme suitable for short-term adverse prognostic risk assessment of such patients, in order to provide new technical means for high-risk patient identification, risk stratification and clinical auxiliary decision-making. Summary of the Invention

[0008] Technical problem solved: To address the above-mentioned technical problems, this invention provides the B7-H3 molecular marker and its application in in vitro assessment of adverse prognostic risk in hepatitis E-related acute liver failure. It can identify the short-term adverse prognostic risk in HEV-ALF patients, which not only makes up for the lack of sensitivity and specificity of prognostic assessment methods in the prior art, but also has high clinical application potential and promotion value, providing a new technical means for high-risk patient identification, risk stratification and clinical decision support.

[0009] Technical solution: B7-H3, a molecular marker for poor prognosis risk in hepatitis E-related acute liver failure.

[0010] Application of reagents for detecting B7-H3 levels in the preparation of in vitro assessment products for poor prognostic risk of hepatitis E-related acute liver failure.

[0011] Preferably, the adverse prognostic risk is the risk of short-term mortality.

[0012] Furthermore, the short-term mortality risk is the 28-day mortality risk.

[0013] Preferably, the reagent is used to detect the level of B7-H3 in the peripheral blood sample of the subject.

[0014] Furthermore, the ex vivo peripheral blood sample is serum and / or plasma.

[0015] Preferably, the level of B7-H3 is detected by immunological quantitative detection methods, including enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, fluorescence immunoassay, immunoturbidimetry, and mass spectrometry.

[0016] Beneficial effects: (1) This invention focuses on HEV-ALF, a severe liver disease with highly heterogeneous clinical outcomes and a high short-term mortality rate, and proposes and establishes an in vitro risk assessment scheme based on B7-H3. Compared with existing assessment strategies that mainly rely on bilirubin, coagulation function, creatinine, or comprehensive scoring systems, the technical solution provided by this invention does not simply repeat traditional organ dysfunction decompensation indicators, but introduces molecular markers that are more closely related to host immune regulation and disease outcome, thus providing a new technical path for identifying the short-term adverse prognostic risk of HEV-ALF.

[0017] (2) This invention is the first to systematically demonstrate that B7-H3 shows an increasing trend in healthy controls, patients with hepatitis E, and patients with hepatitis E-related acute liver failure, and further confirms that it is closely related to 28-day mortality in HEV-ALF patients. These results indicate that B7-H3 is not only associated with the disease, but also reflects the risk characteristics of the disease evolving from a general infection state to a severe decompensated state, and has clear clinical translational significance.

[0018] (3) The B7-H3 described in this invention has high discriminative ability and stability. In the training cohort, the AUC of B7-H3 in predicting 28-day mortality risk reached 0.97; after internal validation via bootstrap, the AUC remained at 0.96; and in the independent external validation cohort, the AUC still reached 0.90. The above results show that this invention does not only perform well in a single research cohort, but also has strong support in terms of internal stability and external generalization ability, demonstrating high reliability in terms of technical effectiveness.

[0019] Furthermore, multivariate logistic regression analysis showed that B7-H3 remained statistically significant after incorporating other clinical variables, indicating that it can provide relatively independent prognostic information. In other words, the biomarker described in this invention is not a simple repetition of traditional indicators, but rather provides a new and supplementary source of risk information outside of existing assessment systems. This characteristic makes it more suitable for early identification and refined prognostic stratification of high-risk patients.

[0020] (4) This invention is based on the detection of peripheral blood samples outside the body. The sample acquisition method is mature, minimally invasive, and convenient, and has high compatibility with existing clinical laboratory testing procedures. The detection methods used include common platforms such as ELISA and chemiluminescence, with moderate technical thresholds and strong feasibility, making it easy to be transferred and implemented in medical institutions of different levels.

[0021] Furthermore, the present invention provides a threshold stratification example based on B7-H3 in specific embodiments, showing that under certain detection systems and study population conditions, when B7-H3 reaches or exceeds a certain level, the short-term mortality risk of patients increases significantly. This result demonstrates that the present invention not only has discriminative value at the continuous variable level, but also can further derive clinically understandable and implementable risk stratification methods, thereby enhancing the application depth and practicality of the present invention.

[0022] (5) The technical solution established by this invention has strong scalability. On the one hand, its core lies in the discovery of a new use for B7-H3 as an in vitro biomarker for assessing short-term poor prognosis in HEV-ALF; on the other hand, the solution can be appropriately adjusted according to different detection platforms, sample systems and application scenarios without departing from the technical essence of this invention. Therefore, this invention not only has clear technical effects, but also has good prospects for industrialization and subsequent application development. Attached Figure Description

[0023] Figure 1 A graph comparing serum B7-H3 levels in a healthy control group, a hepatitis E group, and a hepatitis E-related acute liver failure group; Figure 2A graph comparing serum B7-H3 levels between the 28-day survival group and the death group in patients with hepatitis E-related acute liver failure. Figure 3 The maximum selection rank statistic plot for determining the optimal risk stratification threshold for B7-H3; Figure 4 This is a 28-day Kaplan-Meier survival curve after grouping according to the B7-H3 level; Figure 5 The graph shows the correlation between B7-H3 and clinical indicators. Figure 6 ROC curve for predicting 28-day mortality risk in patients with hepatitis E-related acute liver failure using B7-H3; Figure 7 Bootstrap internal validation ROC curve for predicting 28-day mortality risk for B7-H3; Figure 8 ROC curves comparing the prediction performance of B7-H3 with MELD, TBIL, Cr, and INR; Figure 9 ROC curve for predicting 28-day mortality risk for B7-H3 in an external validation cohort; Figure 10 The ROC curve for bootstrap validation of B7-H3 in the external validation queue. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Example 1

[0025] This invention first provides the application of reagents for detecting B7-H3 levels in the preparation of products for in vitro assessment of the risk of poor prognosis in patients with hepatitis E-related acute liver failure.

[0026] In one embodiment of the present invention, the adverse prognostic risk is the short-term mortality risk; preferably, the short-term mortality risk is the 28-day mortality risk. The above risk assessment is not based on direct diagnostic procedures performed on living human subjects, but rather on information obtained from the B7-H3 levels in ex vivo samples of the subjects, to assess the likelihood of short-term adverse outcomes in vitro.

[0027] In a further embodiment of the present invention, the product achieves risk assessment by detecting the B7-H3 level in a subject's ex vivo peripheral blood sample. The ex vivo peripheral blood sample is preferably serum and / or plasma. Because peripheral blood samples are easy to obtain, have good clinical compliance, and can be collected repeatedly, this facilitates the widespread application of the method of the present invention in practical clinical settings.

[0028] In a preferred embodiment of the present invention, the B7-H3 level is detected using an immunological quantitative detection method, including but not limited to enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, fluorescence immunoassay, immunoturbidimetry, and mass spectrometry. All of the above detection platforms can achieve quantitative or semi-quantitative detection of the B7-H3 level. Those skilled in the art can choose an appropriate method to implement the present invention based on specific application scenarios, experimental conditions, and detection throughput requirements.

[0029] In the course of research for this invention, the inventors compared and analyzed samples from healthy controls, hepatitis E patients, and hepatitis E-related acute liver failure (HEV-ALF) patients, and found that B7-H3 levels differed significantly among different disease states, and were significantly elevated in HEV-ALF patients (see [link to invention]). Figure 1 Further comparison of 28-day survival and death outcomes within HEV-ALF patients revealed that B7-H3 levels were significantly higher in deceased patients than in surviving patients (see [link to study]). Figure 2 Based on this, the present invention establishes the feasibility basis for B7-H3 as a biomarker for assessing the short-term adverse prognostic risk of HEV-ALF.

[0030] In one specific embodiment of the present invention, the optimal risk stratification threshold for B7-H3 is determined by the maximum selection rank statistic (see results). Figure 3 Furthermore, 28-day Kaplan-Meier survival curves were plotted according to the B7-H3 level to assess their risk stratification ability (see results). Figure 4 Meanwhile, multivariate logistic regression analysis was used to examine the relationship between B7-H3 and 28-day mortality risk after incorporating other clinical variables (results are shown in [link to results]). Figure 5 The results showed that B7-H3 remained statistically significant in the multivariate model, suggesting that it is not merely a simple replacement for traditional biochemical indicators, but can provide prognostic information with independent significance.

[0031] In a further embodiment of the present invention, the discriminant performance of B7-H3 on the short-term mortality risk of HEV-ALF patients was evaluated by ROC curve analysis (see results). Figure 6 ), and further internal validation was performed using bootstrap resampling (see results). Figure 7 To assess model stability, B7-H3 was compared with MELD, TBIL, CR, and INR under the same conditions for in vitro assessment of short-term adverse prognostic risk in HEV-ALF patients. The predictive performance comparison ROC curves are shown below. Figure 8 The results showed that B7-H3 had a more significant warning effect on poor short-term prognosis.

[0032] Furthermore, this invention also establishes an independent external validation queue to verify the external repeatability and generalization ability of B7-H3. The ROC curve results of the external validation queue can be found in [reference needed]. Figure 9 The corresponding bootstrap validation ROC curve results can be found in [link to relevant documentation]. Figure 10 The results indicate that B7-H3 exhibits high discriminative power in the training queue, internal validation, and external validation.

[0033] In a preferred embodiment of the present invention, stratified threshold analysis of B7-H3 can be performed based on a specific research cohort and detection system to obtain a risk stratification critical value applicable to the implementation conditions. It should be noted that the specific critical value may vary depending on the detection platform, calibration system, sample source, and study population, but this does not affect the core technical essence of the present invention: using B7-H3 as an in vitro biomarker for assessing the adverse prognostic risk of HEV-ALF.

[0034] In summary, this invention establishes a technical pathway of "ex vivo sample detection - B7-H3 quantification - risk assessment," thereby enabling in vitro assessment of the short-term adverse prognostic risk in HEV-ALF patients. This constitutes a complete, feasible, and clinically valuable technical solution.

Claims

1. B7-H3, a molecular marker for poor prognostic risk of acute liver failure associated with hepatitis E.

2. Application of reagents for detecting B7-H3 levels in the preparation of in vitro assessment products for poor prognostic risk of hepatitis E-related acute liver failure.

3. The application of the reagent for detecting B7-H3 levels according to claim 2 in the preparation of an in vitro assessment product for poor prognostic risk of hepatitis E-related acute liver failure, characterized in that... The adverse prognostic risk mentioned refers to the risk of short-term mortality.

4. The application of the reagent for detecting B7-H3 levels according to claim 3 in the preparation of an in vitro assessment product for the poor prognostic risk of hepatitis E-related acute liver failure, characterized in that... The short-term mortality risk mentioned refers to the 28-day mortality risk.

5. The application of the reagent for detecting B7-H3 levels according to claim 2 in the preparation of an in vitro assessment product for poor prognostic risk of hepatitis E-related acute liver failure, characterized in that... The reagent is used to detect the level of B7-H3 in peripheral blood samples taken from subjects outside the body.

6. The application of the reagent for detecting B7-H3 levels according to claim 5 in the preparation of an in vitro assessment product for poor prognostic risk of hepatitis E-related acute liver failure, characterized in that, The ex vivo peripheral blood sample is serum and / or plasma.

7. The application of the reagent for detecting B7-H3 levels according to claim 2 in the preparation of an in vitro assessment product for poor prognostic risk of hepatitis E-related acute liver failure, characterized in that... The level of B7-H3 was detected by immunological quantitative detection methods, including enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, fluorescence immunoassay, immunoturbidimetry, and mass spectrometry.

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