Biomarker for diagnosing related liver injury in treatment of patient with primary liver cancer and application of biomarker

Abstract

The invention relates to the technical field of biomedical detection, and particularly discloses a biomarker for diagnosing treatment-related liver injury of a primary liver cancer patient in a combined treatment process based on an immune checkpoint inhibitor and application of the biomarker. The biomarker is a collagen XII type alpha 1 chain (COL12A1) and a voltage dependent calcium channel alpha 2 / delta 1 subunit (CACNA2D1). By detecting the expression level of COL12A1 and / or CACNA2D1 protein in a blood sample of a subject and comparing the expression level with a normal level, specific diagnosis for treating related liver injury can be realized. ROC curve analysis verifies that the diagnosis AUC values of the two markers on liver injury are both greater than 0.85, and the two markers have excellent diagnosis efficiency. The invention also relates to a kit for detecting the biomarker.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to biomarkers for the diagnosis of liver injury related to the treatment of patients with primary liver cancer and their applications. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a malignant tumor with high incidence and mortality rates. Due to its insidious onset, most patients are diagnosed at an intermediate or advanced stage, requiring systemic anti-tumor therapy. Combination therapy based on immune checkpoint inhibitors (ICIs), including dual immunotherapy or targeted immunotherapy, has become an important treatment for intermediate and advanced HCC. However, compared to ICI monotherapy, it significantly increases the incidence of treatment-related liver injury.

[0003] Currently, research on biomarkers for treatment-related adverse events (AEs) largely focuses on pan-cancer samples. However, hepatocellular carcinoma patients often have underlying chronic liver disease, and their symptoms can easily overlap with or worsen treatment-related liver injury, leading to difficulties in clinical differential diagnosis. For example, liver injury symptoms may lack specificity or be confused with symptoms of toxicity in other organs. Existing studies suffer from problems such as small sample sizes and a lack of large-scale cohort validation, and there are currently no universally recognized biomarkers that can specifically diagnose liver injury in the complex context of systemic treatment for hepatocellular carcinoma.

[0004] Therefore, there is an urgent need in this field to find biomarkers that can accurately and specifically identify liver injury caused by ICI-based combination therapy in order to achieve early intervention and optimize the treatment benefit-risk ratio for patients.

[0005] Therefore, this invention proposes biomarkers for the diagnosis of treatment-related liver injury in patients with primary liver cancer and their applications. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing biomarkers for the diagnosis of liver injury related to the treatment of primary liver cancer patients and their applications.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: Biomarkers for the diagnosis of treatment-related liver injury in patients with primary liver cancer, wherein the biomarkers are COL12A1 protein and / or CACNA2D1 protein.

[0008] Preferably, the liver injury is treatment-related liver injury induced by combination therapy based on immune checkpoint inhibitors.

[0009] A kit for diagnosing treatment-related liver injury in patients with primary liver cancer, the kit comprising reagents for detecting the expression levels of biomarkers COL12A1 protein and / or CACNA2D1 protein.

[0010] Preferably, the detection is a protein level detection.

[0011] Preferably, the detection is based on mass spectrometry or immunological detection techniques.

[0012] Preferably, the mass spectrometry technique is a data-independent acquisition mass spectrometry technique.

[0013] The use of a non-diagnostic detection method for COL12A1 and / or CACNA2D1 proteins in the preparation of products for diagnosing treatment-related liver injury in patients with primary liver cancer.

[0014] Preferably, the detection is performed by analyzing a blood sample from the subject.

[0015] The beneficial effects of this invention are as follows: High specificity: This invention is the first to screen COL12A1 and CACNA2D1 as specific biomarkers using proteomics methods. These biomarkers can effectively distinguish between patients with liver injury caused by combination therapy based on immune checkpoint inhibitors and those without liver injury. This solves the clinical diagnostic challenge of liver injury symptoms lacking specificity and easily overlapping with other adverse events in liver cancer patients with chronic liver disease.

[0016] Excellent diagnostic performance: Validated by receiver operating characteristic (ROC) curves, the areas under the diagnostic curves for candidate biomarkers COL12A1 and CACNA2D1 were both greater than 0.85, demonstrating extremely high diagnostic accuracy and reliability, and can be used for the early identification of liver injury.

[0017] Based on cohort analysis: This study adopted a nested case-control method and conducted systematic screening of differentially expressed proteins (e.g., 62 differentially expressed proteins were screened between the liver injury and non-injury groups, and 42 differentially expressed proteins were screened between the liver injury and other injury groups). Finally, candidate biomarkers were determined through joint analysis. The screening process was scientific and rigorous, and the results were more convincing.

[0018] Providing an early intervention window: By monitoring changes in COL12A1 and CACNA2D1 protein levels in blood samples during treatment, it is possible to achieve early diagnosis of treatment-related liver injury before clinical symptoms become apparent. This provides valuable time for clinicians to adjust treatment plans and conduct early interventions, thereby optimizing the benefit-risk balance for patients. Attached Figure Description

[0019] Figure 1 : Comparison of expression levels of candidate biomarker COL12A1 (Q99715); Figure 2 : Comparison of expression levels of candidate biomarker CACNA2D1 (P54289); Figure 3Hierarchical clustering heatmap of differentially expressed proteins; Figure 4 Gene Ontology (GO) enrichment analysis diagram of differentially expressed proteins; Figure 5 : Disease enrichment analysis of differentially expressed proteins; Figure 6 KEGG enrichment analysis of differentially expressed proteins; Figure 7 ROC curve of biomarker COL12A1 (Q99715) in diagnosing liver injury; Figure 8 ROC curve of biomarker CACNA2D1 (P54289) for diagnosing liver injury. Detailed Implementation

[0020] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0021] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0022] Example 1: Sample Collection and Processing Sample source: Twenty-two patients with primary liver cancer who received ICI-based combination therapy were included. Blood samples were collected from their baseline status, before the first five cycles of combination therapy, and at the time of adverse events. Among them, 4 patients experienced liver function impairment, 10 patients experienced toxicity to other organs, and 8 patients did not experience any adverse events.

[0023] Protein extraction: Blood samples were processed using the Thermo Fisher TOP14 de-abundance protein kit. 10 μL of sample was added to a column equilibrated to room temperature, vortexed, and then mixed vertically for 10 minutes by inverting the column. After centrifugation at 1000g for 2 minutes, the eluent was collected as the de-abundance protein solution.

[0024] Protein quantification: Dilute the sample to the standard curve range with lysis buffer. Take 10 μL of the diluted sample and react it with 300 μL of protein quantification dye in the dark for 10 minutes. Measure the absorbance at 595 nm using a microplate reader. Calculate the sample concentration based on the BSA standard curve.

[0025] Enzymatic digestion: Add an appropriate amount of protein solution to an octet containing MMB magnetic beads and react at 37°C for 30 minutes. Add binding buffer and incubate at room temperature with shaking for 15 minutes. Discard the supernatant and wash the magnetic beads three times with washing buffer. Resuspend the magnetic beads in enzyme working solution and incubate at 37°C for at least 4 hours. Add stop solution to terminate the digestion and lyophilize.

[0026] Example 2: Mass Spectrometry Detection and Data Analysis HPLC-MS detection: The lyophilized powder was dissolved in 10 μL of mobile phase A (100% water, 0.1% formic acid), centrifuged at 14000g for 20 minutes, and the supernatant was injected. HPLC gradient: Mobile phase B (80% acetonitrile, 0.1% formic acid) was set at 8% at 0 min, increasing to 30% within 35 min, and then to 95% within 10 min, maintaining this level for 15 min. Mass spectrometry employed a data-independent acquisition mode with a full scan range of m / z 350-1500, a first-order resolution of 120,000, and a second-order resolution of 30,000.

[0027] Database search: The generated raw mass spectrometry data were analyzed using the Diann software.

[0028] Data preprocessing: The search results are normalized to eliminate experimental errors and missing values ​​are filled.

[0029] Differential protein screening: T-tests were performed on protein expression data, with p-value < 0.05 and FoldChange > 1.5 used as the screening criteria. The results are as follows: Sixty-two differentially expressed proteins were screened between the liver injury group and the non-injury group (21 upregulated and 41 downregulated).

[0030] Forty-two differentially expressed proteins were screened between the liver injury group and other types of injury groups (eight were upregulated and 34 were downregulated).

[0031] Through joint analysis, COL12A1 and CACNA2D1 were ultimately identified as candidate biomarkers that can specifically distinguish liver injury.

[0032] Example 3: Biomarker Validation Expression level comparison: Quantitative comparison using violin plots showed that the expression levels of COL12A1 and CACNA2D1 in the liver injury group were significantly different from those in other groups.

[0033] Diagnostic efficacy verification: Receiver operating characteristic (ROC) curve analysis of COL12A1 and CACNA2D1 showed that the area under the curve for both was greater than 0.85, confirming their good diagnostic specificity and sensitivity.

[0034] In conclusion, COL12A1 and CACNA2D1 can serve as effective biomarkers for diagnosing liver injury induced by ICI-based combination therapy in patients with primary liver cancer.

[0035] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A biomarker for diagnosing treatment-related liver injury in patients with primary liver cancer, characterized in that, The biomarkers are COL12A1 protein and / or CACNA2D1 protein.

2. The biomarker for diagnosing treatment-related liver injury in patients with primary liver cancer according to claim 1, characterized in that, The liver injury referred to is treatment-related liver injury resulting from combination therapy based on immune checkpoint inhibitors.

3. A kit for diagnosing treatment-related liver injury in patients with primary liver cancer, characterized in that, The kit contains reagents for detecting the expression levels of the biomarkers COL12A1 protein and / or CACNA2D1 protein as described in claim 1 or 2.

4. The kit for diagnosing treatment-related liver injury in patients with primary liver cancer according to claim 3, characterized in that, The test is for protein levels.

5. The kit for diagnosing treatment-related liver injury in patients with primary liver cancer according to claim 4, characterized in that, The detection is based on mass spectrometry or immunological detection techniques.

6. The kit for diagnosing treatment-related liver injury in patients with primary liver cancer according to claim 5, characterized in that, Mass spectrometry is a data-independent mass spectrometry technique.

7. The use of a non-diagnostic detection method for COL12A1 protein and / or CACNA2D1 protein in the preparation of a product for diagnosing treatment-related liver injury in patients with primary liver cancer.

8. The application according to claim 7, characterized in that, The test is conducted by analyzing blood samples from the test subjects.

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