Use of cucurbitacin as a marker in the preparation of a product for the differential diagnosis or diagnosis of adrenocortical carcinoma

Abstract

The present application relates to the use of cucurbitacin as a marker in the preparation of products for identifying or diagnosing adrenocortical carcinoma. The present application finds that cucurbitacin is closely related to the occurrence of adrenocortical carcinoma, and therefore first proposes to use cucurbitacin as a marker in the preparation of related products for identifying, diagnosing, and screening adrenocortical carcinoma. By using this compound as a marker for predicting the occurrence of adrenocortical carcinoma, the early onset of adrenocortical carcinoma can be accurately and sensitively predicted and identified, thereby reducing the technical difficulty of diagnosing adrenocortical carcinoma, and further providing a brand-new objective index for the identification, diagnosis, screening, or drug efficacy evaluation of adrenocortical carcinoma.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to the application of cucurbitacin as a biomarker in the preparation of products for the identification or diagnosis of adrenocortical carcinoma. Background Technology

[0002] Adrenocortical carcinoma (ACC) is a malignant tumor that occurs in the adrenal cortex, with a clinical incidence of 0.5-2 per million people, classifying it as an extremely rare disease. ACC is characterized by high malignancy, aggressiveness, and a high rate of metastasis, resulting in a poor prognosis. Most patients with ACC survive for 4-30 months, with a 5-year overall survival rate of 16%-47%, while the 5-year survival rate for patients with advanced-stage ACC is only 5%-10%. Recent studies have shown a significant increasing incidence of ACC, accounting for approximately 2.4% of adrenal neoplasia and 68.8% of adrenal malignancies. Surgery is the most effective treatment for ACC, and complete tumor resection is currently the only potentially curative method, especially suitable for patients without extensive metastasis. However, due to the high malignancy and rapid progression of ACC, many patients present with tumor infiltration into surrounding tissues or distant metastasis, thus losing the opportunity for surgery. Early diagnosis and complete surgical resection of the tumor remain crucial for the treatment of adrenocortical carcinoma. Therefore, early and accurate diagnosis is particularly important for the treatment and prognosis of adrenocortical carcinoma. However, some adrenocortical adenomas (ACA) and adrenocortical carcinomas with difficult-to-define characteristics pose challenges to current conventional endocrine, imaging, and pathological diagnostic methods. The search for and discovery of novel biomarkers to assist clinicians in diagnosing adrenocortical carcinoma, assessing its severity, treatment effectiveness, and prognosis is also an urgent problem to be solved in the field of adrenocortical carcinoma diagnosis and treatment. This application aims to find a small molecule compound to achieve rapid identification of adrenocortical carcinoma. Summary of the Invention

[0003] To address the aforementioned technical problems, some embodiments of this application provide a biomarker for the identification, diagnosis, screening, or efficacy evaluation of adrenocortical carcinoma and its application.

[0004] The technical solution is as follows:

[0005] A biomarker for the identification, diagnosis, screening, or efficacy evaluation of adrenocortical carcinoma, wherein the biomarker is cucurbitacin.

[0006] Application of cucurbitacin as a biomarker in the preparation of products for the identification, diagnosis and / or auxiliary diagnosis of adrenocortical carcinoma.

[0007] Using cucurbitacin as a biomarker for adrenocortical carcinoma provides an easily detectable and objective indicator for the identification, diagnosis, and / or auxiliary diagnosis of adrenocortical carcinoma, characterized by high targeting, stability, and sensitivity.

[0008] Application of cucurbitacin as a biomarker in the preparation of products for screening and / or assisting in the screening of adrenocortical carcinoma.

[0009] Using cucurbitacin as a biomarker associated with adrenocortical carcinoma, and applying it in the screening and / or adjuvant screening of adrenocortical carcinoma, can relatively accurately predict whether adrenocortical carcinoma will occur and its progression, providing a convenient and non-invasive screening method for adrenocortical carcinoma.

[0010] Application of cucurbitacin as a biomarker in the preparation of products for evaluating the efficacy of adrenocortical carcinoma treatment.

[0011] The product uses cucurbitacin as a marker to detect the level of cucurbitacin in a sample, thereby identifying, diagnosing, screening, or evaluating the treatment efficacy of adrenocortical carcinoma.

[0012] In one embodiment, the product is a diagnostic reagent.

[0013] In one embodiment, the diagnostic reagent includes one or more of test strips, kits, and chips.

[0014] In one embodiment, the product includes one or more of test strips, reagent kits, chips, and devices.

[0015] In one embodiment, the product includes a medical device.

[0016] In one embodiment, the product includes a reagent for detecting the content of the marker in a sample.

[0017] In one embodiment, the sample includes serum, plasma, or whole blood.

[0018] In one embodiment, the sample is plasma.

[0019] In one embodiment, patients with adrenocortical adenoma had lower levels of cucurbitacin compared to patients with adrenocortical adenoma or healthy individuals.

[0020] In one embodiment, the reagent for detecting the content of the marker in the sample includes one or more detection reagents selected from chromatography, spectroscopy, mass spectrometry, nuclear magnetic resonance, and chemical analysis.

[0021] In one embodiment, the reagent for detecting the content of the marker in the sample includes reagents for liquid chromatography-mass spectrometry analysis.

[0022] A product for the identification, diagnosis, screening, or efficacy evaluation of adrenocortical carcinoma, the kit comprising reagents for detecting biomarkers in samples, said biomarkers including cucurbitacin.

[0023] In one embodiment, the product includes a medical device. The medical device operates by using cucurbitacin as a marker to detect the level of cucurbitacin in a sample, thereby identifying, diagnosing, screening, or evaluating the treatment efficacy of adrenocortical carcinoma. Using this medical device allows for convenient and non-invasive diagnosis or treatment evaluation of adrenocortical carcinoma with high sensitivity.

[0024] A computer-aided diagnostic or screening system or device for adrenocortical carcinoma, comprising:

[0025] The result determination unit is used to analyze the level of markers in the detected samples and output the results, wherein the markers include cucurbitacin.

[0026] Optionally, the sample is selected from serum, plasma, or whole blood.

[0027] Optionally, it also includes a detection unit for detecting the level of markers in the sample.

[0028] Optionally, the result determination unit includes an input section for inputting the level of the marker.

[0029] Optionally, the result determination unit further includes an analysis section for analyzing whether the subject is a patient with adrenocortical carcinoma based on the level of the biomarker.

[0030] Optionally, the result determination unit further includes an output section for outputting the analysis results of the analysis section.

[0031] A computer-readable storage medium includes a computer program that is executed by a processor and, when executed by the processor, performs the functions of different units in the computer diagnostic or screening system or device.

[0032] Compared with traditional technologies, this application has the following advantages:

[0033] This study found a close correlation between cucurbitacin and the occurrence of adrenocortical carcinoma. Based on blood samples from three groups—a healthy control group, an adrenocortical adenoma group, and an adrenocortical carcinoma group—it was found that cucurbitacin levels were significantly lower in patients with adrenocortical adenoma, and further lower in patients with adrenocortical carcinoma. Significant differences in cucurbitacin levels were observed among the three groups. Therefore, this study proposes for the first time to use cucurbitacin as a biomarker in the preparation of products for the identification, diagnosis, and screening of adrenocortical carcinoma. Using this compound as a biomarker to predict the occurrence of adrenocortical carcinoma can accurately and sensitively predict and identify the early onset and malignancy of adrenocortical carcinoma, thereby reducing the technical difficulty of diagnosing adrenocortical carcinoma. This provides a new objective indicator for the identification, diagnosis, screening, and drug efficacy evaluation of adrenocortical carcinoma, offering a new approach to assessing the severity, treatment effectiveness, and prognosis of adrenocortical carcinoma. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 PCA was used to visualize the differences among the three groups for comparative analysis of the metabolomics profiles among the three groups.

[0036] Figure 2 Heatmap of differentially metabolites in the plasma of patients with adrenocortical carcinoma.

[0037] Figure 3 This is a comparison of cucurbitacin levels in the plasma of patients with adrenocortical carcinoma, adrenocortical adenoma, and healthy individuals in the examples (***P<0.001).

[0038] Figure 4 This is a ROC curve of plasma cucurbitacin levels in the diagnosis of adrenocortical carcinoma patients and healthy controls.

[0039] Figure 5 This is a ROC curve of plasma cucurbitacin levels in the diagnosis of adrenocortical carcinoma and adrenocortical adenoma. Detailed Implementation

[0040] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided below. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0042] In this document, the term "and / or" includes any and all combinations of one or more of the related listed items.

[0043] In order to find novel biomarkers for adrenocortical carcinoma, the inventors of this application used non-targeted metabolomics technology to analyze the levels of 488 small metabolic molecules in the plasma of patients with adrenocortical carcinoma, patients with adrenocortical adenoma, and age- and sex-matched healthy controls. They found that the levels of various metabolites in patients with adrenocortical carcinoma changed significantly. Among these differentially expressed metabolites, the small molecule metabolite cucurbitacin showed a significant difference between patients with adrenocortical carcinoma, patients with adrenocortical adenoma, and healthy controls. This suggests that cucurbitacin is closely related to the occurrence of adrenocortical carcinoma and can be used as a biomarker for the identification, diagnosis, auxiliary diagnosis, screening, and / or adjuvant therapy of adrenocortical carcinoma.

[0044] Cucurbitacin, with the molecular formula C12H20O3 and a molecular weight of 212.29, is a fatty acyl substance. Studies on plasma cucurbitacin levels and disease development are scarce. To date, no literature has reported a correlation between cucurbitacin levels and adrenocortical carcinoma. The structural formula of cucurbitacin is as follows:

[0045]

[0046] Based on this, one embodiment of this application provides the use of cucurbitacin as a marker in the preparation of products for the identification, diagnosis and / or auxiliary diagnosis of adrenocortical carcinoma.

[0047] One embodiment of this application also provides the use of cucurbitacin as a marker in the preparation of products for screening and / or assisting in the screening of adrenocortical carcinoma.

[0048] This application proposes for the first time the application of cucurbitacin as a biomarker in the diagnosis of adrenocortical carcinoma and related fields. For example, cucurbitacin can be used as a biomarker in the preparation of products for diagnosing adrenocortical carcinoma, thereby reducing the difficulty of diagnosis. Furthermore, using cucurbitacin as a biomarker for adrenocortical carcinoma provides an easily detectable and objective indicator for the identification, diagnosis, and screening of this disease, exhibiting high targeting, stability, and sensitivity.

[0049] In a specific example, the product mentioned above for the identification, diagnosis, and screening of adrenocortical carcinoma is a diagnostic reagent, which may include diagnostic test strips, reagent kits, and diagnostic chips.

[0050] In one specific example, the test sample for the above-mentioned diagnostic reagent is plasma, and the diagnostic reagent is used to detect the level of cucurbitacin in the plasma.

[0051] It is understood that products that detect cucurbitacin levels in plasma can be used to prepare products for the identification, diagnosis, and / or auxiliary diagnosis of adrenocortical carcinoma; or, they can be used to prepare products for screening and / or auxiliary screening of adrenocortical carcinoma. Therefore, the application of products that detect cucurbitacin levels in plasma in the identification, diagnosis, and screening of adrenocortical carcinoma also falls within the scope of protection of this application.

[0052] Similarly, cucurbitacin can be used not only in the preparation of products for the identification, diagnosis, and screening of adrenocortical carcinoma, but also in the evaluation of the treatment efficacy of adrenocortical carcinoma, to assess the therapeutic effects of related drugs. Therefore, the application of cucurbitacin as a biomarker in this regard also falls within the scope of protection of this application.

[0053] One embodiment of this application provides the following product:

[0054] a. Products used for the identification, diagnosis, and / or auxiliary diagnosis of adrenocortical carcinoma.

[0055] b. Products used for screening and / or assisting in the screening of adrenocortical carcinoma.

[0056] c. Products used for evaluating the efficacy of drugs for adrenocortical carcinoma.

[0057] Among them, the identification, diagnosis and / or auxiliary diagnosis of adrenocortical carcinoma refers to the identification, diagnosis and / or auxiliary diagnosis of whether adrenocortical carcinoma has occurred and whether it is in the early or advanced stage by the level of cucurbitacin in the plasma.

[0058] This application also provides a medical device for the identification, diagnosis, screening, or efficacy evaluation of adrenocortical carcinoma, which uses cucurbitacin as a marker to detect the level of cucurbitacin in a sample.

[0059] In a specific example, the aforementioned medical devices used for the identification, diagnosis, screening, or efficacy evaluation of adrenocortical carcinoma include diagnostic test strips, reagent kits, diagnostic chips, and related testing equipment.

[0060] In one specific example, the test sample for the aforementioned medical device is plasma.

[0061] This application is the first to propose the use of cucurbitacin as a biomarker in the preparation of products for the identification, diagnosis, screening, or efficacy evaluation of adrenocortical carcinoma. It can be understood that any reagents capable of detecting cucurbitacin concentration or level can be used to prepare products for the identification, diagnosis, screening, or efficacy evaluation of adrenocortical carcinoma, and are therefore within the scope of protection of this application. Furthermore, any product used for the identification, diagnosis, screening, or efficacy evaluation of adrenocortical carcinoma that uses cucurbitacin as a biomarker and detects the level of cucurbitacin in the sample, based on this application, falls within the scope of protection of this application.

[0062] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0063] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0064] Example 1

[0065] This study used healthy individuals as controls (HC) and patients with adrenocortical carcinoma (ACC) and adrenocortical adenoma (ACA) as subjects to verify the close relationship between plasma cucurbitacin levels and adrenocortical carcinoma.

[0066] 1. Subjects:

[0067] Subject population: Subjects were patients treated at Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, and recruited healthy individuals.

[0068] Inclusion and exclusion criteria for patients with adrenocortical carcinoma and adrenocortical adenoma: (1) Inclusion criteria: 1) Age ≥ 18 years. 2) Inpatients with a confirmed diagnosis of adrenocortical carcinoma or adrenocortical adenoma, with the diagnosis based on pathological results. (2) Exclusion criteria: 1) Postoperative patients. 2) Patients treated with mitotane, ketoconazole or mifepristone.

[0069] A total of 42 patients with adrenocortical carcinoma, 56 patients with adrenocortical adenoma, and 54 healthy controls were selected (matched by age, sex, and disease group). All subjects signed informed consent forms.

[0070] 2. Preparation of blood samples

[0071] (1) Collection and storage of blood samples: Venous blood was collected from fasting patients and healthy controls. Ethylenediamine tetraacetic acid (EDTA) was used as an anticoagulant, and the blood was centrifuged at 5000 r / min for 10 minutes at 4°C within 30 minutes of collection. The supernatant was transferred to cryovials, rapidly frozen in liquid nitrogen, and then stored at -80°C. All sample transport was performed on dry ice to ensure that the samples were not subjected to repeated freeze-thaw cycles before testing.

[0072] (2) Thaw the sample on an ice bath, transfer 50 μL to a 1.5 mL tube, add 150 μL of ultrapure water, and vortex for 30 seconds to mix. Add 400 μL of acetonitrile and vortex again for 30 seconds to mix. Centrifuge the sample at 14000 g for 10 minutes to remove the protein precipitate. Transfer the supernatant to a 10 mL glass tube and evaporate to dryness using a container evaporator at room temperature. Dissolve the residue in 200 μL of a mixed solution (2% acetonitrile), filter the solution using an ultracentrifuge filter to remove molecules with a molecular weight greater than 10 kDa, collect the supernatant, and analyze it using liquid chromatography-mass spectrometry (LC-MS).

[0073] 3. Analytical conditions for liquid chromatography-mass spectrometry:

[0074] (1) Chromatographic conditions:

[0075] Chromatographic column: Waters HSS C18 column (3.0×100mm, 1.7μm); Mobile phase: Phase A: water (containing 0.1% formic acid); Phase B: acetonitrile; Gradient elution program: 0-1 min, 2% solvent B; 1-3 min, 2%-15% solvent B; 3-6 min, 15%-50% solvent B; 6-9 min, 50%-95% solvent B; 9-9.1 min, 95%-100% solvent B; 9.1-12 min, 100% solvent B; 12-12.1 min, 100%-2% solvent B; and 12-17 min, 2% solvent B. Flow rate: 0.5 mL / min; Column temperature: 45℃; Injection volume: 5 μL; Injector temperature: 4℃.

[0076] (2) Mass spectrometry conditions

[0077] MS and MS / MS spectra were acquired using a data-dependent acquisition mode. The 10 most abundant ions were fragmented by MS / MS at collision energies of 35 ± 15 eV.

[0078] 4. Statistical Analysis:

[0079] PCA was used to visualize the differences among the three groups for comparative analysis of the metabolomics profiles among the three groups.

[0080] Using the LC-MS detection results from step 3, software was used to analyze and plot a graph comparing plasma cucurbitacin levels in adrenocortical carcinoma patients and healthy controls (HC), as well as an ROC curve for diagnosing adrenocortical carcinoma based on plasma cucurbitacin levels.

[0081] 5. Results

[0082] like Figure 1 The results show that PCA was used to visualize the differences among the three groups for comparative analysis of their metabolomics profiles. Each group exhibited significantly different clusters, indicating that the metabolomics characteristics of the three groups were clearly different.

[0083] like Figure 2 This image shows a heatmap of differentially expressed metabolites in the plasma of patients with adrenocortical carcinoma, reflecting the overall distribution of small molecules in the plasma. Columns represent samples, rows represent metabolites, the clustering tree on the left is the differentially expressed metabolite clustering tree, and the top is the sample clustering tree. Gradient colors indicate quantitative values; redder colors indicate higher expression levels, and bluer colors indicate lower expression levels. As shown in the image, the small molecule cucurbitacin exhibits significant changes in the plasma of patients with adrenocortical carcinoma.

[0084] like Figure 3 The box plots show the logarithmic and distributional differences in plasma cucurbitacin concentrations among the healthy control group, the adrenocortical adenoma group, and the adrenocortical carcinoma patient group, with each point representing one sample. As can be seen from the figure, the plasma cucurbitacin concentration in ACC patients was significantly lower than that in the adrenocortical carcinoma group and the healthy population. The statistical p-value was less than 0.001 (generally considered less than 0.05 to indicate statistical significance), further demonstrating the reliability and high reproducibility of the results.

[0085] like Figure 4 The ROC curves of plasma cucurbitacin levels in the diagnosis of adrenocortical carcinoma in patients and healthy controls are shown, serving as a comprehensive indicator of continuous variables reflecting sensitivity and specificity. Plotting sensitivity (true positive rate) on the Y-axis and 1-specificity (false positive rate) on the X-axis, the area under the ROC curve (AUC) was 0.987, greater than 0.5, indicating good diagnostic efficacy; the P-value was <0.0001, indicating good statistical significance; the optimal cutoff value showed a sensitivity of 0.982 and a specificity of 1.

[0086] like Figure 5 The ROC curves of plasma cucurbitacin levels in the diagnosis of adrenocortical carcinoma and adrenocortical adenoma showed that the area under the ROC curve (AUC) was 0.752, which is greater than 0.5, indicating good diagnostic efficacy; the P value was <0.0001, indicating good statistical significance; the sensitivity of the optimal cutoff value was 0.691, and the specificity was 0.977.

[0087] The study results indicate that plasma cucurbitacin levels can serve as a biomarker for the clinical diagnosis of adrenocortical carcinoma.

[0088] In summary, the research results of this application found that cucurbitacin is closely related to the occurrence of adrenocortical carcinoma. For the first time, it is proposed to use cucurbitacin as a biomarker in the preparation of products for the identification, diagnosis, and screening of adrenocortical carcinoma. This provides a new objective indicator for the identification, diagnosis, screening, or drug efficacy evaluation of adrenocortical carcinoma, and offers a new approach to assessing the severity, treatment effect, and prognosis of adrenocortical carcinoma.

[0089] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0090] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. The use of cucurbitacin as a biomarker in the preparation of products for the identification, diagnosis and / or auxiliary diagnosis of adrenocortical carcinoma, with samples including serum, plasma or whole blood.

2. The use of cucurbitacin as a biomarker in the preparation of products for screening and / or assisting in the screening of adrenocortical carcinoma, with samples including serum, plasma or whole blood.

3. The application according to claim 1 or 2, characterized in that, The products include one or more of test strips, reagent kits, and chips.

4. The application according to claim 1 or 2, characterized in that, The products include medical devices.

5. The application according to claim 1 or 2, characterized in that, The product includes reagents for detecting the content of the markers in a sample.

6. The application according to claim 1 or 2, characterized in that, Compared to patients with adrenocortical adenoma or healthy individuals, patients with adrenocortical carcinoma have lower levels of cucurbitacin.

7. The application according to claim 5, characterized in that, The reagents used to detect the content of the marker in the sample include one or more reagents selected from chromatography, spectroscopy, mass spectrometry, nuclear magnetic resonance, and chemical analysis.

8. The application according to claim 5, characterized in that, The reagents used to detect the content of the markers in the sample include reagents for liquid chromatography-mass spectrometry analysis.

9. A computer-aided diagnostic or screening system for adrenocortical carcinoma, characterized in that, include: The result determination unit is used to analyze the level of the markers in the detected samples and output the results, wherein the markers include cucurbitacin. The result determination unit includes an input section for inputting the level of the marker; The result determination unit also includes an analysis section, which is used to analyze whether the subject is a patient with adrenocortical carcinoma based on the level of the biomarker; The result determination unit also includes an output section for outputting the analysis results of the analysis section; It also includes a detection unit for detecting the level of markers in the sample; The sample is selected from serum, plasma or whole blood.

10. A computer-readable storage medium, characterized in that, The computer-readable storage medium includes a computer program that is executed by a processor and, when executed by the processor, performs the functions of different units in the computer diagnostic or screening system of claim 9.

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