Biomarker for ovarian cancer diagnosis and application and device thereof
By detecting the extracellular vesicle subpopulations carrying DNA and CA125 protein, the problem of insufficient sensitivity and specificity in early diagnosis of ovarian cancer is solved, efficient and accurate diagnosis of ovarian cancer is achieved, detection process is simplified, and cost is reduced.
Patent Information
- Application Number
- CN202510407006.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-07-04
Smart Images
Figure CN120254259A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and particularly to a biomarker for ovarian cancer diagnosis, its application and device. Background Art
[0002] Ovarian cancer (OC), as a common malignant tumor of the female reproductive system, often has relatively hidden clinical manifestations in its early stage.
[0003] Currently, the accurate staging of ovarian cancer highly depends on the pathological examination of ovarian masses, which needs to be carried out after surgery, so there is a lag in treatment. Clinically, in the face of suspected ovarian cancer cases, especially when a female is diagnosed with an adnexal mass, doctors often choose to directly perform an ovarian adnexectomy for pathological analysis. However, this approach has led to a large number of unnecessary surgical treatments, not only increasing the physical and mental burden of patients, but also causing a waste of medical resources.
[0004] To improve this situation, the development of non-invasive diagnostic techniques is particularly important. Although imaging techniques such as transvaginal ultrasound and computed tomography can provide intuitive image information to help doctors initially judge the presence and morphology of masses, their accuracy in differentiating between benign and malignant masses still has limitations. In addition, serum carbohydrate antigen 125 (CA125), as a biomarker traditionally used for the auxiliary detection of ovarian cancer, its sensitivity and specificity have not reached the ideal level. Specifically, the serum CA125 levels of about one-third of early ovarian cancer patients do not increase significantly, and ovarian benign diseases and some inflammatory diseases may also cause abnormal increases in serum CA125 levels, resulting in false negative and false positive results, further limiting its application value in early diagnosis.
[0005] To improve the detection effectiveness, researchers have tried to combine the detection of CA125 with other biomarkers, such as human epididymis protein 4 (HE4), in order to improve the accuracy and specificity of diagnosis. However, although this combined detection strategy has improved the diagnostic effect to a certain extent, it has also increased the complexity and cost of the test, which is not conducive to wide promotion in clinical practice.
[0006] In summary, the early diagnosis of ovarian cancer currently still faces many challenges, including but not limited to the lack of non-invasive diagnostic techniques with high sensitivity and specificity, the over-reliance on pathological examination, and unnecessary surgical treatments, etc. Therefore, developing a new biomarker that is accurate, sensitive and can be used for the early diagnosis of ovarian cancer is of great significance for improving the early diagnosis rate of ovarian cancer, optimizing the clinical decision-making process and improving the prognosis of patients. Summary of the Invention
[0007] In view of the defects and deficiencies in the prior art, the present application provides a biomarker for ovarian cancer diagnosis, its applications and devices, which can significantly improve the accuracy of ovarian cancer diagnosis, optimize the early diagnosis process of ovarian cancer, improve the sensitivity and specificity of the biomarker, and demonstrate potential clinical application value.
[0008] To achieve the above object, the present application adopts the following technical methods:
[0009] In the first aspect, the present application provides a biomarker for ovarian cancer diagnosis, and the biomarker is a subset of extracellular vesicles carrying DNA and CA125 protein.
[0010] As an implementation manner, the extracellular vesicles are derived from body fluids.
[0011] As a specific implementation manner, the body fluids include blood, plasma / serum, urine, ascites, etc.
[0012] As an implementation manner, the ovarian cancer is early ovarian cancer or advanced ovarian cancer, where early ovarian cancer includes stage I and stage II ovarian cancer, and advanced ovarian cancer includes stage III and stage IV ovarian cancer.
[0013] As an implementation manner, the positive detection rate of the subset of extracellular vesicles is positively correlated with the progression of ovarian cancer.
[0014] In the second aspect, the present application provides the use of the biomarker for ovarian cancer diagnosis described in the first aspect in the preparation of products for screening diagnosis, molecular typing or treatment evaluation of ovarian cancer.
[0015] As an implementation manner, the products contain reagents for detecting the level of the subset of extracellular vesicles carrying CA125 protein and reagents for detecting the level of the subset of extracellular vesicles carrying DNA.
[0016] As an implementation manner, the ovarian cancer is selected from early ovarian cancer or advanced ovarian cancer, where early ovarian cancer includes stage I and stage II ovarian cancer, and advanced ovarian cancer includes stage III and stage IV ovarian cancer.
[0017] In the third aspect, the present application provides a device for screening diagnosis, molecular typing or treatment evaluation of ovarian cancer in an object, and the device contains reagents for detecting the levels of the subset of extracellular vesicles carrying DNA and CA125 protein in the object.
[0018] As an implementation manner, the reagent includes a first reagent subgroup, a second reagent subgroup, and a third reagent subgroup. The first reagent subgroup includes a first antibody against CA125 and a second antibody that binds to the first antibody and is labeled with a fluorescent marker; the second reagent subgroup includes a lysis buffer for lysing the cell membrane of extracellular vesicles; the third reagent subgroup includes a MDA amplification reaction mixture for performing a MDA amplification reaction on the DNA carried by extracellular vesicles and a fluorescent dye EvaGreen.
[0019] As an implementation manner, the extracellular vesicles are from the body fluids of the subject.
[0020] As a specific implementation manner, the body fluids include blood, plasma / serum, urine, ascites, and the like.
[0021] As described above, the biomarker for ovarian cancer diagnosis, its application, and device of the present application have the following beneficial effects:
[0022] The biomarker for ovarian cancer diagnosis provided by the present application is a subgroup of extracellular vesicles carrying DNA and CA125 protein, which can accurately distinguish healthy, benign, early, and late malignant ovarian cancer subjects. The overall accuracy rate is as high as 93.3%, which is much higher than the detection of a single biomarker, providing strong support for the early diagnosis of ovarian cancer, significantly improving the accuracy of ovarian cancer diagnosis, optimizing the early diagnosis process of ovarian cancer, improving the sensitivity and specificity of the biomarker, and demonstrating potential clinical application value. Description of the Drawings
[0023] Figure 1 Shows the P(DNA + EV) values measured in EV samples in the plasma of healthy, benign, early ovarian cancer, and late ovarian cancer patients.
[0024] Figure 2 Shows the P(CA125 + EV) values measured in EV samples in the plasma of healthy, benign, early ovarian cancer, and late ovarian cancer patients.
[0025] Figure 3 Shows the P(DNA + CA125 + EV) values measured in EV samples in the plasma of healthy, benign, early ovarian cancer, and late ovarian cancer patients.
[0026] Figure 4 Shows the confusion matrix diagram for classifying four groups of healthy, benign, early ovarian cancer, and late ovarian cancer patients based on linear discriminant analysis.
[0027] Figure 5Shown as P(DNA + EV), P(CA125 + EV), P(DNA + CA125 + EV), serum CA125 and the linear discriminant scores of three parameters were used to distinguish between benign and early ovarian cancer patients in the ROC curve graph.
[0028] Figure 6 Shown as P(DNA + EV), P(CA125 + EV), P(DNA + CA125 + EV) values measured in plasma EV samples of patients with endometriosis and early ovarian cancer.
[0029] Figure 7 Shown as P(DNA + EV), P(CA125 + EV), P(DNA + CA125 + EV) values were used to distinguish between patients with endometriosis and early ovarian cancer in the ROC curve graph. Detailed implementation manners
[0030] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.
[0031] For those not specified in the examples regarding specific technologies or conditions, they shall be in accordance with the technologies or conditions described in the literature in this field, or in accordance with the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through regular channels.
[0032] Circulating free DNA (cfDNA), as a widely recognized biomarker for non-invasive cancer screening, has shown application potential in the early detection of ovarian cancer. However, the diagnosis of cfDNA mainly relies on expensive next-generation sequencing technology, which is not only time-consuming but also costly. In contrast, extracellular vesicles (EVs), these extremely low-density nanoscale vesicular structures with sizes ranging from 30 nanometers to 1000 nanometers released by almost all cells, can provide another perspective.
[0033] EVs secreted by cells carry abundant bioactive information. They are like tiny information repositories, carrying diverse biological information of the source cells. Particularly importantly, the DNA encapsulated in EVs can be protected from degradation by exogenous factors due to its unique double-membrane structure and maintain long-term stability in body fluids. Notably, compared with normal cells, EVs derived from tumor cells often show a higher DNA positive rate, which greatly enhances the value of EV-DNA as a potential molecular biomarker.
[0034] In addition, EV proteins derived from tumor cells, such as EV-CA125 (i.e., CA125 in extracellular vesicles), are increasingly attracting attention in the fields of cancer monitoring and evaluation of treatment effects. Compared with traditional serum CA125 detection, the detection level of EV-CA125 is more significant, thus greatly improving the sensitivity of ovarian cancer diagnosis. By jointly analyzing multiple biomarkers, more-dimensional biological information can be obtained, thereby enhancing the predictability and accuracy of diagnosis.
[0035] Therefore, the comprehensive analysis of multiple biomolecules in extracellular vesicles undoubtedly opens up new ideas and methods for the early diagnosis and clinical application of ovarian cancer. This innovative diagnostic strategy is expected to provide more comprehensive and in-depth information support for the precision medicine of ovarian cancer.
[0036] (I) Experimental methods
[0037] 1.1 Clinical sample collection
[0038] During the experiment, 4 mL of blood samples were collected from each participant into EDTA anticoagulant tubes. After inverting and mixing at room temperature, they were centrifuged twice at 4°C for 15 minutes each time at a speed of 2500 g within 2 hours. Finally, the cell-free plasma supernatant was collected and stored at -80°C for subsequent extracellular vesicle isolation experiments.
[0039] 1.2 Isolation of EVs
[0040] Total Exosome Isolation Kits (Thermo Fisher Scientific) were used to extract and purify extracellular vesicles from the collected cell-free plasma supernatant samples for subsequent characterization. The purified EVs samples were resuspended in PBS and stored at -80°C for subsequent use.
[0041] 1.3 Microfluidic device
[0042] An AutoCAD software was used to design a PDMS microfluidic chip with a size of 5 mm (W) × 15 mm (L) × 20 μm (H), with 6 chambers set inside, and it was fabricated using standard soft lithography technology.
[0043] 1.4 Joint detection of DNA and protein in single EV
[0044] Rabbit anti-CA125 antibody was used to label the surface protein of EVs. Four-arm polyethylene glycol acrylate (M W = 10000) (Seebio Biotech) and bis-mercapto polyethylene glycol (HS-PEG-SH, M W = 3400) (Seebio Biotech) (the molar ratio of four-arm polyethylene glycol acrylate to HS-PEG-HS was 1:(1-4)) were dissolved in phosphate buffered saline (PBS) and used to dilute EVs. The above mixture was used to generate hydrogel droplets containing EVs in mineral oil using a portable tip capillary device. After the hydrogel droplets containing EVs were gelated at 37 °C for 1 h, they were loaded into a microfluidic device (i.e., a microfluidic chip). A coverslip was placed on the chamber of the microfluidic chip to force the hydrogel droplets containing EVs to form a monolayer. The oil phase was removed by washing with isopropanol and PBS to form hydrogel microspheres. AF594-labeled goat anti-rabbit secondary antibody (2 mg / ml, diluted 1:100 - 1:2000) was added to the chip and incubated at room temperature for 1 h, followed by washing three times with PBS for 5 min each. CA125 immunofluorescence detection was performed.
[0045] After CA125 imaging, the microfluidic chip was placed in lysis buffer (10 mM KCl, 30 mM Tris-HCl and 1% Triton-X100) and incubated at room temperature for 30 minutes to disrupt the EVs membrane. After lysis, it was washed 3 times with PBS for 5 min each. A 20 μL MDA amplification reaction mixture was prepared using 2 μL Phi29 DNA polymerase (10 U / μL), 2 μL 10×Phi29 polymerase buffer, 1 μL random primer (1 mM), 2 μL dNTP (10 mM), 1 μL 20×EvaGreen and 12 μL nuclease-free ddH2O. The mixture was loaded into the microfluidic chip and reacted at 30 °C for 8 h and at 65 °C for 10 min. The DNA clusters were identified using the EvaGreen fluorescent dye contained in the MDA amplification reaction mixture.
[0046] 1.5 Statistical analysis
[0047] Bright-field and fluorescence detections were performed using a Nikon ECLIPSE Ti2 inverted microscope. For each field of view, three images were acquired along the optical axis direction for focus stacking to obtain fluorescence images. For each clinical sample, the positive percentage of EVs was detected at least 3 times. Image processing software was used to analyze the bright-field and fluorescence images, and the data were expressed as mean ± standard deviation (Mean±SD). Mean box plots were used to describe the distribution within each group. The two-tailed Mann-Whitney U test was used for inter-group comparison.
[0048] (2) Combined detection of CA125 and DNA in single EVs for the diagnosis of ovarian cancer
[0049] The research results showed that compared with the healthy control group and the benign group, the ovarian tumor group showed a significant increasing trend in multiple indicators. Specifically, as Figures 1 to 3 shown, the levels of extracellular vesicle subsets carrying DNA (P(DNA + EV)), extracellular vesicle subsets carrying CA125 protein (P(CA125 + EV)), and extracellular vesicle subsets carrying both DNA and CA125 protein (P(CA125 + DNA + EV)) were all significantly increased in the ovarian tumor group. As Figure 4 shown, in order to effectively distinguish healthy individuals, patients with benign lesions, and patients with early and advanced ovarian cancer, the linear discriminant analysis (LDA) method was used, and the optimal discriminant combination was successfully determined, with an overall classification accuracy rate as high as 93.3%.
[0050] Given that the early detection of ovarian cancer is crucial for improving the prognosis, accurately differentiating benign ovarian diseases from ovarian cancer plays a crucial role in the early diagnosis of ovarian cancer.
[0051] The above method was further applied to the study of benign samples and early ovarian cancer samples. The results showed that whether it was the level of P(DNA + EV), the level of P(CA125 + EV), or the level of P(CA125 + DNA + EV), all showed a significant upward trend. As Figure 5 and Table 1 shown, according to the receiver operating characteristic (ROC) analysis, the AUC value of P(CA125 + EV) was 0.755 (95% confidence interval: 0.545 - 0.964), the AUC value of P(DNA + EV) was 0.823 (95% confidence interval: 0.630 - 1.000), while the AUC value of P(CA125 +DNA + The AUC value of EV) was even as high as 0.955 (95% confidence interval: 0.861 - 1.000). This result strongly suggests that the malignancy of ovarian cancer is related to CA125 + DNA + There is a close association between the biogenesis of EV. Notably, when these three parameters are used in combination, their detection performance is further improved, with an AUC value as high as 0.973 (95% confidence interval: 0.910 - 1.000) and relatively high sensitivity and specificity.
[0052] Therefore, the subset of extracellular vesicles carrying DNA and CA125 protein has great potential to become a novel biomarker, showing great value in accurately detecting early ovarian cancer in clinical applications.
[0053] Table 1
[0054]
[0055] (III) Distinguishing patients with endometriosis by combined detection of CA125 and DNA in single EV
[0056] In addition to ovarian tumors, elevated CA125 levels can also originate from a series of non - neoplastic diseases, and endometriosis is a typical example. To explore this phenomenon in depth, the aforementioned research methods were applied to the sample analysis of endometriosis and early ovarian cancer (OC).
[0057] As Figure 6 shown, the average P(CA125 + EV), P(DNA + EV), and P(CA125 + DNA + EV) values in early OC samples were 65%, 114%, and 190% higher, respectively, compared with the endometriosis group. However, no significant difference was observed between the two groups in blood CA125 detection. Notably, the abundance of the subset of extracellular vesicles (EVs) carrying DNA showed a significant increasing trend, which makes it a specific marker for distinguishing cancer from non - cancer diseases.
[0058] As Figure 7 shown, based on P(DNA +The receiver operating characteristic curve (AUC) value of endometriosis and early OC was as high as 0.983 (95% confidence interval: 0.932-1.000). In addition, when the extracellular vesicle subpopulation indicators carrying DNA and CA125 were analyzed, the AUC value reached 1.000 (95% confidence interval: 1.000-1.000).
[0059] Given the high correlation between EV-DNA and cancer, EV-DNA measured at the level of single EVs shows great potential in distinguishing non-tumor-related diseases with high CA125 levels from early ovarian cancer. This high specificity will play a vital role in the diagnosis of early ovarian cancer and provide strong support for early detection and treatment of the disease.
[0060] The technical solution proposed in this application aims to provide an innovative biomarker for the early diagnosis of ovarian cancer. The core advantages of this application are reflected in its non-invasiveness, high precision, high sensitivity and high specificity, which can achieve accurate identification and quantification of EVs (extracellular vesicles) biomarkers in clinical samples. Compared with other similar technologies, this application has significant ease of operation and does not require tedious and complicated operations such as sequencing. By analyzing the indicators of extracellular vesicle subpopulations carrying DNA and CA125 protein, this application provides strong support for the early diagnosis of ovarian cancer.
[0061] In addition, the technical solution of this application has broad application prospects. It is not only limited to the early diagnosis of ovarian cancer, but is also expected to play an important role in the treatment monitoring, efficacy evaluation and prognosis judgment of ovarian cancer. It is expected to open up new paths for precision medicine of ovarian cancer and bring substantial improvements to the health and quality of life of patients.
[0062] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.
Claims
1. A biomarker for ovarian cancer diagnosis, characterized in that, The biomarker is a subset of extracellular vesicles carrying DNA and CA125 protein.
2. The biomarker according to claim 1, wherein Extracellular vesicles are derived from body fluids.
3. The biomarker according to claim 1, wherein The ovarian cancer is early-stage ovarian cancer or late-stage ovarian cancer, where early-stage ovarian cancer includes stage I and stage II ovarian cancer, and late-stage ovarian cancer includes stage III and stage IV ovarian cancer.
4. The biomarker according to claim 1, wherein The positive detection rate of the subset of extracellular vesicles is positively correlated with the progression of ovarian cancer.
5. Use of the biomarker according to any one of claims 1 to 4 in the preparation of a product for screening diagnosis, molecular typing or treatment evaluation of ovarian cancer.
6. The use according to claim 5, characterized in that, The product contains reagents for detecting the level of the subset of extracellular vesicles carrying CA125 protein and reagents for detecting the level of the subset of extracellular vesicles carrying DNA.
7. The use according to claim 5, characterized in that, The ovarian cancer is selected from early-stage ovarian cancer or late-stage ovarian cancer, where early-stage ovarian cancer includes stage I and stage II ovarian cancer, and late-stage ovarian cancer includes stage III and stage IV ovarian cancer.
8. A device for screening, diagnosing, molecular typing, or therapeutic evaluation of ovarian cancer in a subject, characterized in that, The device contains reagents for detecting the level of the subset of extracellular vesicles carrying DNA and CA125 protein in the subject.
9. The device according to claim 8, characterized in that, The reagents include a first reagent subgroup, a second reagent subgroup and a third reagent subgroup. The first reagent subgroup contains a first antibody against CA125 and a second antibody that binds to the first antibody and is labeled with a fluorescent marker; the second reagent subgroup contains a lysis buffer for lysing the cell membrane of extracellular vesicles; the third reagent subgroup contains a MDA amplification reaction mixture for performing MDA amplification reaction on the DNA carried by extracellular vesicles and a fluorescent dye EvaGreen.
10. The device according to claim 8, characterized in that, Extracellular vesicles are from the body fluids of the subject.