Application of exosome biomarker CA125 and HE4 combination in evaluation of ovarian malignant tumors and auxiliary diagnosis algorithm for evaluating benign and malignant ovarian cancers

Abstract

The invention provides an application of an exosome CA125 and HE4 combined biomarker in evaluation of ovarian malignant tumors. The invention relates to application of a detection reagent of a biomarker from exosomes to preparation of a composition or a kit for evaluating, diagnosing and / or monitoring ovarian cancer of a subject, and the biomarker from the exosomes comprises exosomes CA125 and HE4. Wherein assessing, diagnosing and / or monitoring ovarian cancer in a subject comprises 1) obtaining exosomes from the subject, and 2) determining the presence and / or level of biomarkers in the exosomes from the subject. The invention further provides an auxiliary diagnosis algorithm for evaluating benign and malignant ovarian cancer. According to the exosome marker and the algorithm provided by the invention, the ovarian cancer can be conveniently and quickly evaluated, diagnosed and identified with better performance.

Description

Technical Field

[0001] The present invention generally relates to the fields of evaluation, diagnosis, and / or monitoring of malignant tumors such as ovarian cancer, and an auxiliary diagnosis algorithm for evaluating the benign and malignant nature of ovarian cancer. The present invention relates to the use of biomarkers from exosomes for the evaluation of ovarian cancer, and provides related analytical reagents, kits, and related applications. Background Art

[0002] Ovarian malignant tumor is one of the common malignant tumors in the female reproductive system. The annual incidence rate ranks the 3rd among female reproductive system tumors, after cervical cancer and uterine body malignant tumors, showing an increasing trend year by year. The fatality rate ranks the first among female genital tract malignant tumors, and it is a malignant tumor that seriously threatens women's health. The incidence rate of ovarian cancer in China is 7.48 / 100,000. It is estimated that there are nearly 50,000 new cases in China every year, and about 35,000 deaths. Since the ovaries are located deep in the pelvic cavity, early tumors are difficult to detect during pelvic examinations, resulting in 70% of ovarian cancers being diagnosed at an advanced stage; the survival rate of 70% of patients is less than 5 years; for patients after cure, about 70% of them will relapse within 3 years. Due to the concealment at the initial stage of onset and its powerful "killing power", ovarian cancer is also known as the "silent killer". Therefore, the key to the prevention and treatment of ovarian cancer lies in seizing the opportunity. If detected early, the 5-year survival rate of ovarian cancer patients can be increased from 20%-30% to 60%-70%, and some may be cured.

[0003] Most ovarian cancers are sporadic, and hereditary ovarian cancer accounts for about 15% of all ovarian cancer patients. Currently, germline mutations in more than a dozen tumor suppressor genes have been found to be related to the incidence of hereditary ovarian cancer, and more than 80% of hereditary ovarian cancers are related to germline mutations in BRCA1 / 2. Ovarian malignant tumors include various pathological types, among which the most common is epithelial carcinoma, accounting for about 80% of ovarian malignant tumors, followed by malignant germ cell tumors and sex cord-stromal tumors, accounting for about 10% and 5% respectively.

[0004] Numerous studies have been conducted on biomarkers for ovarian cancer. For example, WO2005034732 discloses DNA involving various specific sequences as marker genes for ovarian cancer. WO2005098447 mentions Apo A1, modified Apo A1, transthyretin ΔN10, native transthyretin, cysteinylated transthyretin, sulfated transthyretin, CysGly-modified transthyretin, glutathioneylated transthyretin, IAIH4 fragment No.1, IAIH4 fragment No.2, and IAIH4 fragment No.3 and their compositions as biomarkers for determining the status of ovarian cancer. WO2007002264 discloses CTAP3-related proteins and CAl25, transferrin, haptoglobin, ApoAl, transthyretin, internal fragments of ITIH4, beta 2-microglobulin, hepcidin, prostatin, osteopontin, eosinophil-derived neurotoxin, leptin, prolactin, IGF-II, hemoglobin and its modified forms, CA125 II, CA15-3, CA19-9, CA72-4, CA 195, tumor-associated trypsin inhibitor (TATI), CEA, placental alkaline phosphatase (PLAP), sialyl TN, galactosyltransferase, macrophage colony-stimulating factor (M-CSF, CSF-1), lysophosphatidic acid (LPA), 110KD component of the extracellular segment of epidermal growth factor receptor (p110EGFR), tissue kallikreinogen, such as kallikrein 6 and kallikrein 10 (NES-1), serine protease (prostasin), HE4, creatine kinase B (CKB), LASA, HER-2 / neu, urinary gonadotropin peptide, Dianon NB 70 / K, tissue polypeptide antibody (TPA), SMRP, osteopontin and haptoglobin, insulin-like growth factor I, and insulin-like growth factor II, etc. for identifying the status of ovarian cancer.Examples of biomarkers mentioned in WO2015042115 for ovarian cancer assessment include: aldehyde dehydrogenase 1 (ALDH1), ApoCI, ApoAII, ApoCII, β-hemoglobin, calcyclin, calgranulin A, calgranulin C, claudin-3, connective tissue growth factor (CTGF), eosinophil-derived neurotoxin, fibroblast growth factor 2 (basic) (FGF2), folate receptor 1 (FOLR1), glycodelin, GPCR49, glutathione S-transferase theta 1 (GSTT1), hepsin, hepcidin, insulin-like growth factor-II, inter-α-trypsin inhibitor heavy chain H4, kallikrein-related peptidase 6 (KLK6 / 7), kallikrein 10, leptin, macrophage inhibitory factor, mucin-16 (CA125), osteopontin, prolactin, protease serine 8 (PRSS8), protein C inhibitor, solute carrier family 39 (zinc transporter) member 4 (SLC39A4), small MBL-associated protein C-terminal fragment, stratum corneum chymotryptic enzyme, transferrin, transthyretin, WAP four-disulfide core domain 2 (HE4), SH2 domain-containing transforming protein 1 (Shc), SH2 domain-containing E (She), and autoantibody specific to casein kinase 1ε, etc.

[0005] Currently, the auxiliary diagnosis of pelvic masses mainly relies on two detection methods. One is transvaginal ultrasound (TVS), which is regarded as an important indicator for the surgical indication of pelvic masses. This imaging method can be used to examine the reproductive organs of women, including the uterus, ovaries, cervix, and vagina. Although it is widely used, it cannot completely and accurately determine whether the mass is benign or malignant. In addition, this method also requires experienced clinicians to interpret the test results. Another conventional detection method is to detect the serum biomarker CA125, which is widely used clinically to diagnose ovarian cancer, evaluate the treatment effect of ovarian cancer, chemotherapy sensitivity, and whether the tumor recurs after treatment, etc.

[0006] CA125 is a glycoprotein with a molecular weight greater than 200 KD, belonging to surface antigen and related to epithelial ovarian cancer antigen. Among women with epithelial ovarian cancer, more than 80% of patients have a CA125 concentration > the critical value of 35 U / mL. Therefore, the detection of CA125 has great guiding significance for the diagnosis and treatment of ovarian cancer. However, 1% - 2% of the healthy population also have elevated CA125 values. Among this part of the population, 5% have benign diseases and 28% have non-gynecological system tumors. In addition, patients with heart failure, cirrhosis, and chronic active hepatitis may also have elevated CA125. However, the specificity and sensitivity of CA125 are relatively low, and false negatives or false positives are likely to occur. Approximately 20% of ovarian tumor patients do not have an elevated CA125 level. That is to say, one-fifth of ovarian cancer patients may be missed. Some benign ovarian diseases can also lead to elevated CA125 levels, resulting in false positives.

[0007] Serum biomarker HE4 is also another biomarker for ovarian cancer diagnosis. HE4 is mainly distributed in the epithelium of the reproductive system, such as the epididymis, seminiferous tubules, vas deferens epithelium, fallopian tube epithelium, and endometrium. In malignant tumors, the highest expression level of HE4 is in ovarian serous carcinoma, followed by lung adenocarcinoma, while the expression in lung squamous carcinoma is relatively low. Endometrial cancer, transitional cell carcinoma, breast cancer, and pancreatic cancer all have medium-level expressions, but there are also individual cases with expression levels equivalent to those of ovarian serous carcinoma among them. Most cases in colon cancer, gastric cancer, liver cancer, and prostate cancer show low-level expressions. Compared with CA125, HE4 has higher sensitivity and stronger specificity. However, the commercially available CA125 and HE4 detection kits approved by NMPA are only intended to assist in monitoring the treatment effect of ovarian epithelial cancer patients and assist in monitoring the recurrence and progression of the disease in ovarian epithelial cancer patients, and cannot be used for the differential diagnosis of ovarian cancer.

[0008] Currently, most commercially available diagnostic reagents directly detect the changes in a certain index (such as protein, nucleic acid, etc.) in body fluids (blood, urine, etc.) to draw conclusions. However, since body fluids usually contain a large amount of high-abundance proteins, taking serum as an example, the top ten abundant proteins account for more than 90% of the total serum proteins. During the progression of the disease, due to the small size of the lesion, mild symptoms, and limitations of detection instruments and reagents, the changes in cells at the lesion site and the biological information released into body fluids are easily masked by these high-abundance proteins. It is very difficult to directly detect biomarkers from body fluids to discover early-stage progressive diseases.

[0009] Exosomes are extracellular vesicles released by all cells, with a diameter of 30 - 150 nm. Exosomes contain contents such as DNA, RNA, and proteins, and can be secreted by cells into the blood circulation. Exosomes can be detected in the tumor microenvironment. Increasing evidence indicates that exosomes play an important role in promoting tumor growth and are involved in activities such as angiogenesis, immune response, and tumor metastasis. Some studies have used ELISA to detect, characterize, and quantify exosomes and found that some proteins in the exosomes of patients are expressed at much higher levels than those in the healthy population. Exosomes secreted in the human body have high specificity, and the information contained in the exosomes secreted in body fluids may be more representative than the information contained in body fluids. Therefore, for traditional serum tumor markers, they can also be extracted and detected from exosomes, and the physiological information obtained has higher clinical value for the auxiliary diagnosis of tumors. For example, CN108841954B discloses selecting and ranking candidate markers (screening markers by performing a full-scan analysis of proteins in a sample through proteomics methods); exosome extraction (adding exosome extraction reagents to a serum or plasma sample, mixing well, then adding Roche lysis buffer to lyse and extract exosomes), and model establishment and analysis (analyzing and comparing the serum ROMA model and the exosome ABD model to select an appropriate model to evaluate the risk of malignant ovarian cancer). CN113718031A discloses blood sample collection, extraction of cfDNA and genomic DNA (genomic DNA), detection of protein markers (detecting the plasma protein marker CA125 using the Roche Cobas e411 detection system), high-throughput sequencing of CfDNATP53 and identification of somatic mutations, construction of a prediction model, and validation of the model. CN106248940A discloses the preparation of fusion proteins SUMO-C1D, SUMO-CCL18, SUMO-CXCL1, SUMO-TM4SF1, SUMO-FXR1, SUMO-TIZ, the preparation of a liquid suspension chip for the combined detection of multiple indicators for epithelial ovarian cancer, and the clinical validation of the combined detection of multiple indicators for ovarian cancer.

[0010] However, since exosomes originate from the invagination of the cell membrane, forming early endosomes containing membrane proteins, and the endosomes further invaginate inward to enclose proteins and RNAs in the cytoplasm, forming multivesicular bodies in the cytoplasm, and the multivesicular bodies fuse with the cell membrane to release exosomes into the extracellular matrix. Therefore, from the source of exosomes, it is difficult to predict which proteins or nucleic acids will be included and further released into the extracellular matrix during exosome formation. Thus, even if a certain protein or nucleic acid is known as a biomarker for a certain cancer, it does not necessarily exist in the exosomes of patients. Furthermore, it is difficult to determine whether the known cancer biomarker can be obtained from the exosomes of cancer patients, whether it has the corresponding specificity and sensitivity of a tumor biomarker, and diagnose the cancer by detecting the biomarker in exosomes, thus bringing difficulties to the evaluation of cancer using exosome biomarkers.

[0011] Therefore, despite relevant research, there is still a need in the art for favorable biomarkers that can be effectively used for ovarian cancer screening and diagnosis. Summary of the Invention

[0012] In some embodiments, the biomarker comprising the combination of exosomal CA125 and HE4 provided by the present invention is a convenient, rapid and better-performing diagnostic tool for assisting in the diagnosis and differentiation of ovarian cancer.

[0013] In some embodiments, the present invention provides a method for evaluating, diagnosing and / or monitoring ovarian cancer in a subject, wherein a biomarker comprising the combination of exosomal CA125 and HE4 is used as an ovarian cancer biomarker. The biomarker comprising the combination of exosomal CA125 and HE4 in ovarian cancer patients has been identified and can be used as an ovarian cancer biomarker. In some embodiments, the method for evaluating, diagnosing and / or monitoring ovarian cancer in a subject comprises the following steps: 1) obtaining exosomes from the subject, and 2) determining the presence and / or level of the biomarker in the exosomes from the subject.

[0014] In some embodiments, the present invention has surprisingly found that a biomarker combination including exosomal CA125 and HE4 shows excellent sensitivity and specificity in the detection of ovarian cancer. In some embodiments, the ovarian cancer biomarkers of the present invention may include exosomal CA125 and HE4 or a combination thereof with other biomarkers, such as a combination with biomarkers from serum (such as CEA) and / or other biomarkers of exosomes. In some embodiments, exosomal CA125 and HE4 as ovarian cancer biomarkers can be detected in combination with other known ovarian cancer biomarkers, such as known serum protein biomarkers, serum microRNA biomarkers, exosomal protein biomarkers, exosomal microRNA biomarkers, etc. In some embodiments, exosomal CA125 and HE4 as ovarian cancer biomarkers can be combined with one or both of blood or exosomal C5a and PLG. However, in some embodiments, when exosomal CA125 and HE4 are combined with other biomarkers, C5a may not be included in the combination of the biomarkers. In some embodiments, when exosomal CA125 and HE4 are combined with other biomarkers, one or both of blood or exosomal C5a and PLG may not be included in the combination of the biomarkers. In some embodiments, it is very advantageous to use fewer biomarkers because, compared with combinations of more biomarkers, using fewer biomarkers for tumor assessment and / or diagnosis can not only save costs but also provide a more convenient, rapid, and better-performing diagnostic tool. Therefore, the present invention surprisingly finds that the detection of ovarian cancer can be achieved through a biomarker combination including exosomal CA125 and HE4, and shows at least comparable or even significantly improved sensitivity and / or specificity. In some embodiments, the present invention surprisingly finds that a combination of exosomal CA125 and HE4 with other biomarkers, such as biomarkers from serum (such as CEA), can achieve a synergistic effect in the detection of ovarian cancer, such as synergistically improved sensitivity and specificity. For example, it has been found that using ROC curve analysis, the area under the ROC curve (AUC) of the exosomal CA125 and HE4 biomarker combination is larger at 0.906 (95% confidence interval: 0.861 - 0.952, P < 0.0005) compared to the exosomal CA125, HE4, and C5a biomarker combination, which is completely unexpected. In contrast, the exosomal CA125 and HE4 combination achieves a comparable or even more excellent tumor assessment effect compared to using other biomarkers or combinations of biomarkers.

[0015] Thus, in some embodiments, the present invention relates to the use of a combination of two biomarkers from exosomes (exosomal CA125 and HE4 combination), which can be further combined with other biomarkers, such as in combination with other blood (including serum or plasma, etc.) biomarkers and / or other exosomal biomarkers. In some embodiments, the exosomal CA125 and HE4 combination is not used in combination with any of c5a and PLG from exosomes. In some embodiments, the biomarkers used consist of the exosomal CA125 and HE4 combination. In some embodiments, the sensitivity and / or specificity of ovarian cancer analysis is improved by the biomarkers of the present invention, or at least comparable or even significantly improved sensitivity and / or specificity of ovarian cancer analysis is provided by reducing the number of biomarkers in the biomarker combination compared to the use of a combination of more biomarkers. In some embodiments, comparable sensitivity and / or specificity means that the difference between the sensitivity and / or specificity compared to a control (such as a combination of more biomarkers, or other biomarkers) is less than 5%. In some embodiments, improved or enhanced sensitivity and / or specificity means an increase of 5% or more compared to a control. In some embodiments, at least comparable sensitivity and / or specificity of ovarian cancer analysis can be obtained by the biomarkers of the present invention including the exosomal CA125 and HE4 combination compared to a control (such as a combination of more biomarkers, or other biomarkers). In some embodiments, the sensitivity and / or specificity of ovarian cancer analysis is increased by the biomarkers of the present invention, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold or higher. In some embodiments, the sensitivity of ovarian cancer detection is increased by the biomarkers of the present invention including the exosomal CA125 and HE4 combination compared to a control (such as a serum biomarker or other exosomal biomarkers different from the biomarkers of the present invention or a combination thereof), such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold or higher. In some embodiments, the specificity of ovarian cancer detection is increased by the biomarkers of the present invention including the exosomal CA125 and HE4 combination compared to a control (such as a serum biomarker or other exosomal biomarkers different from the biomarkers of the present invention or a combination thereof), such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold or higher.

[0016] In some embodiments, the methods of the present invention include determining biomarkers from exosomes. Methods for detecting biomarkers are known in the art. For example, methods including but not limited to enzyme-linked immunosorbent assay (ELISA), mass spectrometry, radioimmunity, chemiluminescence, real-time PCR, nucleic acid hybridization methods, Western blot analysis, immunodetection (such as immunoprecipitation and / or immunofluorescence), Southern hybridization, etc. can be used to detect biomarkers. For example, monoclonal antibodies can be used as detection reagents for biomarkers. In some embodiments, detection reagents for detecting the biomarkers of the present invention (such as exosomal CA125, HE4, and / or additional biomarkers) include antibodies (such as monoclonal antibodies). In some embodiments, reagents for detecting the biomarkers of the present invention (such as exosomal CA125, HE4, and / or additional biomarkers), such as antibodies (such as monoclonal antibodies), can be conjugated to a solid support, a detectable label, or a binding partner. For example, conjugation can be performed directly or indirectly. For example, the solid support includes microtiter plates, plastics, membranes such as nitrocellulose membranes, glass, magnetic beads, or metal supports. For example, the detectable label can include but is not limited to metal particles, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, immunolabels, and radioactive labels, or enzyme labels. For example, it can be colloidal gold, radioisotopes, fluorophores, spin labels, or phage labels. For example, it can be rhodamine, fluorescein, acridinium ester, luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, glucose oxidase, galactose oxidase, or glucose-6-phosphate dehydrogenase labels. For example, the binding partner includes biotin, streptavidin, or avidin, etc.

[0017] In some embodiments, a biomarker refers to a molecule associated with a disease (such as ovarian cancer), for example, a molecule associated with the presence, stage, prognosis, prediction of treatment response, etc. of ovarian cancer. Ovarian cancer biomarkers can include proteins that are differentially expressed in ovarian cancer subjects (such as full-length polypeptides, splicing variants, post-translationally modified polypeptides, etc.) and fragments of gene products and corresponding polynucleotide sequences, such as mRNA, DNA, etc. In some embodiments, the methods of the present invention include the step of determining the combination of exosomal CA125 and HE4 in a biological sample (such as a biological sample from exosomes) from a subject suspected of having ovarian cancer.

[0018] In some embodiments, the biomarker itself and / or the product of the biomarker can be detected, such as the metabolite of the biomarker and / or other products directly associated with the biomarker.

[0019] In some embodiments, the method of the present invention includes the step of determining biomarkers from exosomes. In some embodiments, it has been found that detecting biomarkers in exosomal contents can improve the sensitivity, specificity, and positive predictive value in the early ovarian cancer screening and diagnosis. Exosomes (extracellular vesicles) are a type of exocytic vesicle that exchange information between cells by transporting proteins, nucleic acids, etc. in the parent cells. Almost all cells secrete exosomes, and the exosomes secreted by cells carry a lot of biological information from the parent cells. It has been found to be an important potential biomarker. Due to a large number of immune responses, inflammatory responses, coagulation responses, apoptosis, and autophagy, cells in the disease state release more exosomes, and the contents of exosomes have changed significantly both quantitatively and qualitatively. Compared with directly detecting biomarkers in blood, it has been found that detecting biomarkers in exosomal contents (such as the biomarkers described herein) is a method of concentrating information, especially for the evaluation and analysis of ovarian cancer.

[0020] In some embodiments, the exosomes are from body fluids, such as from blood, serum, serous fluid, plasma, lymph, urine, cerebrospinal fluid, saliva, mucosal secretions of secretory tissues and organs, vaginal secretions, milk, tears, ascites, such as fluids from the pleura, pericardium, peritoneum, abdomen, or other body cavities. In some embodiments, the exosomes are from cultures, solutions such as lavage fluids that are in contact with the subject or in contact with samples from the subject, for example, cell and organ culture media, including cell or organ conditioned media. In some embodiments, the exosomal CA125 and HE4 proteins of the present invention can be from exosomal proteins in samples such as whole blood, plasma, serum, ascites, lymph fluid, etc.

[0021] In some embodiments, the present invention provides the use of biomarker detection reagents in the preparation of compositions or kits for evaluating, diagnosing, and / or monitoring ovarian cancer in a subject. In some embodiments, the present invention provides the use of preparing compositions and / or kits for evaluating, diagnosing, and / or monitoring ovarian cancer in a subject by the method of the present invention. In some embodiments, the composition or kit includes 1) reagents (such as exosome-specific antibodies) and / or devices (such as tubes, filters, immunomagnetic beads, etc. for separating exosomes) for extracting exosomes, and 2) reagents (such as antibodies against the biomarker) for determining the presence and / or level of biomarkers in exosomes from a subject.

[0022] In some embodiments, the detection reagent for biomarkers from exosomes in the present invention may include an exosome extraction reagent. In some embodiments, the detection reagent for biomarkers from exosomes in the present invention may further include an exosome lysis reagent. Methods for exosome isolation are known to those skilled in the art. For example, exosome isolation methods include ultracentrifugation, density gradient centrifugation (such as sucrose density gradient centrifugation), exosome precipitation, filtration, immunomagnetic bead method, chromatography, comprehensive methods (methods for crude extraction and purification of exosomes using different methods), etc. In some embodiments, the exosome extraction reagent may include, for example, a reagent for exosome precipitation, such as PEG or other hydrophilic reagents. In some embodiments, the detection reagent for biomarkers and / or the exosome extraction reagent of the present invention may, in a broad sense, include related devices for biomarker analysis and / or exosome isolation, such as centrifuges, test tubes for centrifugation, filters for exosome separation (such as large pore filters for filtering cells, small pore filters for filtering protein impurities, and filters with a pore size of about 0.1 μm for separating exosomes), filter paper, membranes (such as nitrocellulose membranes), gels, immunomagnetic beads, etc. In some embodiments, the exosome extraction reagent includes antibodies specific for exosome biomarkers, such as monoclonal antibodies. In some embodiments, the exosome extraction reagent may include various exosome extraction and / or analysis kits provided by manufacturers (such as SBI, Invitrogen, etc.). In some embodiments, the detection reagent for biomarkers and / or the exosome extraction reagent may include various buffers for biomarker analysis and / or exosome isolation and / or analysis, such as PBS, electrophoresis buffer, etc. In some embodiments, the detection reagent for biomarkers and / or the exosome extraction reagent may include antibodies and / or primers, probes specific for specific biomarkers, such as antibodies and / or primers, probes for the combination of exosome CA125 and HE4 and related nucleic acid sequences. Such antibodies, primers, and / or probes can be prepared by methods known in the art or purchased from manufacturers. In some embodiments, the detection reagent for biomarkers from exosomes in the present invention may further include an exosome lysis reagent. As known to those skilled in the art, exosomes have a phospholipid bilayer similar to the cell membrane. To release exosome contents, the relevant exosome extraction and / or isolation and / or analysis reagents described in the present invention may include an exosome lysis reagent such as RIPA lysis buffer. In some embodiments, the detection reagent for biomarkers from exosomes in the present invention may further include a protein inhibitor. In some embodiments, it is possible to directly analyze exosome contents without using a dedicated lysis reagent. For example, the exosome suspension can be directly loaded onto a gel for electrophoresis analysis.In some embodiments, the detection reagent for biomarkers and / or the exosome extraction reagent may include protein analysis reagents such as Western Blot, ELISA analysis reagents, proteomic analysis reagents such as protein mass spectrometry and antibody chips, etc. In some embodiments, the detection reagent for biomarkers and / or the exosome extraction reagent may include a tracer reagent, for example, linking the biomarker and / or exosome with a visual label (such as PKH26 label, etc.). In some embodiments, the detection reagent for biomarkers and / or the exosome extraction reagent may include sequence analysis reagents such as high-throughput sequencing reagents.

[0023] In some embodiments, the present invention provides a composition and / or kit for evaluating, diagnosing, and / or monitoring ovarian cancer in a subject by the method of the present invention, wherein the composition or kit includes the detection reagent for the biomarker. In some embodiments, the detection reagent may include protein and / or nucleic acid detection reagents. In some embodiments, the detection reagent may include immunoassay reagents. In some embodiments, the kit contains reagents for determining biomarker expression. In some embodiments, the kit contains a plurality of probes that specifically bind to the biomarker corresponding to the present invention. In some embodiments, the kit contains a plurality of antibodies that specifically bind to the biomarker of the present invention. In some embodiments, the kit includes an antibody, an antibody derivative, or an antibody fragment that specifically binds to the biomarker protein or protein fragment. In some embodiments, the kit may include a plurality of antibodies, antibody derivatives, or antibody fragments that specifically bind to the biomarker and its protein or fragment. In some embodiments, the kit includes a capture reagent that binds to the biomarker of the present invention and a container containing at least one biomarker. In some embodiments, the capture reagent may bind to multiple biomarkers or may also bind to at least one known biomarker. In some embodiments, the kit may further include a second or more capture reagents. In some embodiments, the kit includes a buffer. In some embodiments, the kit includes an instruction manual. In some embodiments, the kit includes a chip or microarray. In some embodiments, the kit includes one or more matrices attached with adsorbents.

[0024] In some embodiments, the present invention provides a method for evaluating, diagnosing, and / or monitoring the status of ovarian cancer, which includes the following steps: extracting exosomes from a biological fluid sample of the patient and detecting (such as detecting using a kit) the presence and / or concentration of biomarkers in the exosomes. Compared with control patients without ovarian cancer, a change in the level or evaluation of the biomarker indicates that the patient has ovarian cancer.

[0025] The present invention generally relates to cancer biomarkers and, in particular, to biomarkers associated with ovarian cancer. The present invention provides methods for predicting, assessing, diagnosing, and monitoring cancer, particularly ovarian cancer, by measuring certain biomarkers, and also provides reagent sets or arrays for evaluating the expression levels of biomarkers associated with ovarian cancer.

[0026] The present invention relates to the combination of biomarker exosomal CA125 and HE4. The present invention relates to the sources of biomarkers including exosomes.

[0027] The sources of exosomes in the present invention include body fluids. Body fluids include blood, serum, serous fluid, plasma, lymph, urine, cerebrospinal fluid, saliva, mucosal secretions of secretory tissues and organs, vaginal secretions, milk, tears, and ascites; and also include fluids in the pleura, pericardium, peritoneum, abdomen, and other body cavities. Biological fluids may also include liquid solutions, lavage fluids, etc. in contact with an object or biological source (e.g., cell and organ culture media, including cell or organ conditioned media).

[0028] The present invention includes kits for detecting the above-mentioned biomarkers using immunological methods. In some embodiments, the kit includes 1) reagents and / or devices for extracting exosomes, and 2) reagents for determining the presence and / or level of biomarkers in exosomes from a subject.

[0029] Different from the prior art which usually directly analyzes biomarkers in body fluid samples, the present invention can directionally concentrate the biological information at the lesion site by separating specific exosomes, which can significantly reduce the interference of other information in the body fluid, thereby amplifying the information at the lesion site. This makes the diagnosis of early diseases more accurate under the sensitivity of current test instruments and reagents.

[0030] In some embodiments, the present invention finds that the combination of biomarker exosomal CA125 and HE4 can obtain excellent sensitivity and specificity for cancer diagnosis. However, in some embodiments, the combination of biomarker exosomal CA125 and HE4 can also be further combined with known biomarkers for cancer diagnosis and prognosis analysis.

[0031] In some embodiments, the present invention includes using exosomes extracted from body fluid samples to replace body fluid samples as detection objects. It has been found that it can be effectively used in one or more aspects such as ovarian cancer (risk) assessment, diagnosis, detection, monitoring, prognosis, and treatment. In some embodiments, it can be applied to one or more aspects such as ovarian cancer (risk) assessment, diagnosis, detection, monitoring, prognosis, and treatment through the combination of exosomal CA125 and HE4.

[0032] In some embodiments, the biomarker level of the patient is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more higher or lower than that of a biomarker of a patient without the cancer. In some embodiments, the present invention provides a combination of biomarkers, which includes a combination of two or more biomarkers of the present invention. In some embodiments, the level of a biomarker (such as protein level, protein activity level, level of related metabolites, etc.) can be used alone for the evaluation, diagnosis, and / or prognosis of ovarian cancer.

[0033] In some embodiments, the present invention provides the use of a biomarker including a combination of exosomal CA125 and HE4 in the evaluation of ovarian malignancies. In some embodiments, the present invention determines the risk of ovarian cancer by detecting the concentration of a combination of exosomal proteins CA125 and HE4.

[0034] In some embodiments, the present invention may include the following steps:

[0035] S1: Sample collection

[0036] Collect samples from a certain number of patients with ovarian malignancies and patients with ovarian benign tumors.

[0037] S2: Exosomal protein extraction

[0038] This operation extracts exosomes from the sample by methods such as precipitation or immunomagnetic bead method, and then lyses the extracted exosomes to obtain exosomal proteins.

[0039] S2: Detection of exosomal CA125 and HE4 proteins

[0040] This operation uses the double antibody sandwich method to detect exosomal CA125 and HE4 proteins in the sample.

[0041] S3: Result analysis

[0042] Compare the detected concentrations of exosomal CA125 and HE4 proteins with the cutoff value. If it is greater than or equal to the cutoff value, it indicates a higher risk of ovarian cancer in the patient; if it is less than the cutoff value, it indicates a lower risk of ovarian cancer in the patient.

[0043] The methods, compositions, and kits of the present invention can be used to evaluate whether a subject has ovarian cancer; evaluate the stage of ovarian cancer in a subject; evaluate the grade of ovarian cancer in a subject; evaluate the benign or malignant nature of ovarian cancer in a subject; evaluate the likelihood of metastasis of ovarian cancer in a subject; evaluate the presence of ovarian cancer cells; evaluate the effect of one or more candidate compounds in inhibiting ovarian cancer in a subject; evaluate the effect of a treatment method; monitor the progression of ovarian cancer in a subject; screen for compositions or treatment methods that inhibit ovarian cancer in a subject; evaluate the ovarian cancer-inducing ability of a test compound; and prevent the onset of ovarian cancer in a subject at risk of developing ovarian cancer.

[0044] In some embodiments, based on the analysis of experimental data, the present invention further provides an auxiliary diagnostic algorithm for evaluating the benign and malignant nature of ovarian cancer by combining the exosomal biomarkers CA125 and HE4, wherein:

[0045] a) Use multiple protein indicators (CA125 / HE4) contained in serum-derived exosomes as biological markers:

[0046] b) Calculate the multiple indicators into a single indicator through the algorithm to further improve the specificity and sensitivity, and can effectively make an auxiliary differential diagnosis of ovarian malignant tumors.

[0047] In some embodiments, the auxiliary diagnostic evaluation formula for the benign and malignant nature of ovarian cancer is:

[0048] OCS = ey / (1 + ey),

[0049] where y = Ax ln[HE4] + B x ln[CA125] - y0, y0 = 4.08,

[0050] where OCS is the evaluation score for a subject suffering from epithelial ovarian cancer, and the benign and malignant nature of the patient's tumor is indicated according to the OCS value. HE4 is the concentration of exosomal HE4 protein, CA125 is the concentration of exosomal CA125 protein, the value range of A is between 0.304 and 1.216, preferably 1.279, and the value range of B is between 0.6395 and 2.558, preferably 0.608.

[0051] In some embodiments, the auxiliary diagnostic evaluation formula for the benign and malignant nature of ovarian cancer is:

[0052] OCS = ey / (1 + ey),

[0053] where y = 1.279x ln[HE4] + 0.608x ln[CA125] - y0, y0 = 4.08.

[0054] In some embodiments, the present invention provides a computer device for the auxiliary diagnosis of ovarian cancer, comprising a processor and a memory storing instructions thereon, which when executed by the processor cause the processor to execute the ovarian cancer auxiliary diagnosis algorithm of the present invention.

[0055] In some embodiments, the present invention provides a computer-readable storage medium storing instructions, which when executed by a processor cause the processor to execute the ovarian cancer auxiliary diagnosis algorithm of the present invention.

[0056] In some embodiments, one or more steps of the algorithms described herein can be carried out using a computer program. In some embodiments, the present invention includes steps performed by a computer program. In some embodiments, the present invention includes a computer-readable storage medium storing executable instructions, which when executed by one or more processors can cause the one or more processors to perform one or more steps of the method of the present invention.

[0057] The present invention advantageously has one or more of the following advantages:

[0058] The present invention proposes to use the concentrations of exosomal CA125 and HE4 proteins to determine whether a patient has ovarian cancer, and there is currently no report on using exosomal CA125 and HE4 proteins to diagnose ovarian cancer.

[0059] The present invention proposes a method for detecting the concentrations of exosomal CA125 and HE4 proteins, and there is currently no report on detecting the combination of exosomal CA125 and HE4.

[0060] The present invention further provides an auxiliary diagnosis algorithm for evaluating the benign and malignant nature of ovarian cancer using the combination of exosomal biomarkers CA125 and HE4.

[0061] The exosomal CA125 and HE4 proteins proposed by the present invention can be exosomal CA125 and HE4 proteins from samples such as whole blood, plasma, serum, ascites, lymph fluid, etc., which are relatively easy to collect clinically and have a relatively high patient compliance.

[0062] The test results of the present invention are not affected by factors such as the menstrual cycle, and there is no need to combine other factors to judge the risk of ovarian cancer in patients. The risk of ovarian cancer in patients can be directly judged according to the concentrations of the combination of exosomal proteins CA125 and HE4.

[0063] Compared with the currently marketed products for assisting in monitoring the treatment effect of patients with ovarian epithelial cancer and assisting in monitoring the recurrence and progression of the disease in patients with ovarian epithelial cancer, the present invention has a higher specificity in diagnosing ovarian cancer and effectively reduces the risk of delayed treatment for patients.

[0064] This operation is more simple and the detection cost is lower. Brief Description of the Drawings

[0065] Figure 1 : Receiver operating characteristic curve (ROC) generated from the results of exosome biomarker detection and pathological diagnosis results.

[0066] Figure 2a and Figure 2b : Receiver operating characteristic curve (ROC) generated from the results of the detection of comparison of exosome combined biomarkers and pathological diagnosis results.

[0067] Figure 3a and Figure 3b : Receiver operating characteristic curve (ROC) of comparison of the auxiliary diagnosis algorithm for evaluating the benign and malignant nature of ovarian cancer using the exosome biomarker combination of the present invention.

[0068] Figure 4 : Receiver operating characteristic curve (ROC) of comparison of the auxiliary diagnosis algorithm for evaluating the benign and malignant nature of ovarian cancer using the exosome biomarker combination of the present invention.

[0069] Figure 5 : Receiver operating characteristic curve (ROC) of comparison of the auxiliary diagnosis algorithm for evaluating the benign and malignant nature of ovarian cancer using the exosome biomarker combination of the present invention.

[0070] Figure 6a and Figure 6b : Receiver operating characteristic curve (ROC) generated from the results of the detection of the combination of exosome biomarkers CA125 and HE4 with other biomarkers and pathological diagnosis results.

[0071] Figure 7a and Figure 7b : Receiver operating characteristic curve (ROC) generated from the results of the detection of serum CEA and pathological diagnosis results. Detailed Description of the Invention

[0072] The combination of exosomal CA125 and HE4 biomarkers provided by the present invention for diagnosing ovarian cancer is significantly different from existing methods. The detection method provided in the present invention has higher sensitivity and specificity, is easier to operate, has lower detection costs, and the biomarkers for diagnosing ovarian cancer in the present invention are exosomal proteins. The application examples of the combination of exosomal biomarkers CA125 and HE4 in the evaluation of ovarian malignancies are shown below. Unless otherwise specified, the reagents used in the examples are commercially available reagents. Among them, exosomal protein extraction, CA125, HE4, and C5a protein detection are carried out using an external kit (Shanghai Sino-US Biopharmaceutical Co., Ltd., product number: National Medical Device Approval No. 20243400138), and serum CEA detection is carried out using a carcinoembryonic antigen (CEA) detection kit (chemiluminescence method) (Shanghai Medical Device Approval No. 20232400201) produced by Shanghai Sino-US Biopharmaceutical Co., Ltd.

[0073] Example 1 Application of a combination of exosomal biomarkers CA125 and HE4 in the evaluation of ovarian malignancies:

[0074] S1 Collection of ovarian tumor samples:

[0075] A total of 172 serum samples from patients with adnexal masses who underwent surgical treatment were collected. Among them, 98 patients with epithelial ovarian malignancies diagnosed by postoperative pathology were used as the positive group, and 74 patients with benign masses were used as the negative control group.

[0076] The screening criteria for 172 patients with adnexal masses who underwent surgical treatment were: ≥18 years old, those with adnexal masses also needed to undergo surgery, no history of malignant tumors, no history of radiotherapy or chemotherapy, and no pregnancy. The enrolled samples were all preoperative collected samples.

[0077] S2 Extraction of exosomal CA125 protein and HE4 protein:

[0078] It is carried out using an exosomal protein extraction and purification kit. First, take 300 μL of serum sample in a centrifuge tube and add 75 μL of exosomal precipitation reagent. Place the centrifuge tube on a shaker to shake to mix the sample and the precipitation reagent evenly. After mixing, let it stand at room temperature for 30 minutes. Then place the centrifuge tube in a centrifuge and centrifuge at 4700×g at 4°C for 10 minutes. Remove the supernatant, and the precipitate at the bottom of the centrifuge tube is the extracted protein linked, embedded, and coated with phospholipid bilayers. Then add 300 μL of exosomal lysis reagent to the precipitate at the bottom of the centrifuge tube and shake on a shaker for at least 30 minutes. Filter the shaken and mixed solution, and the filtrate can be used for subsequent protein detection.

[0079] S3 Detection of exosomal CA125 protein and HE4 protein:

[0080] Take 30 μL of the filtered exosome sample and detect the CA125 protein and HE4 protein. Detect the exosome sample on a fully automated chemiluminescence analyzer.

[0081] S4: Result analysis

[0082] Generate a receiver operating characteristic curve (ROC) from the test results and the pathological diagnosis results, as Figure 1 shown.

[0083] According to the generated curve, the maximum area under the ROC curve (AUC) is 0.906 (95% confidence interval: 0.861 - 0.952, P < 0.0005). An AUC greater than 0.9 indicates high diagnostic accuracy, and the cutoff value at this time has practical significance.

[0084] Compare with the detection method of serum CA125 for diagnosing ovarian cancer in Example 1:

[0085] Use the serum CA125 protein concentration widely used clinically at present to diagnose ovarian cancer, and compare the analysis results with those in Example 1.

[0086] S1 Collection of ovarian tumor samples:

[0087] The collected samples are the same as those in Example 1, that is, use the serum samples in S1 of Example 1 for subsequent test analysis.

[0088] S2 Detection of serum CA125 protein:

[0089] Use the carbohydrate antigen 125 detection kit (electrochemiluminescence method, national medical device registration number 20153401561) of Roche Diagnostics to detect the CA125 concentration of the serum sample.

[0090] S3: Result analysis

[0091] Taking the serum CA125 concentration of 35 U / mL as the cutoff value, the sensitivity of the serum CA125 concentration for diagnosing ovarian cancer is 69.39%, and the specificity is 59.46%.

[0092] Compare with the detection method of serum HE4 for diagnosing ovarian cancer in Example 2:

[0093] Use the serum HE4 protein concentration widely used clinically at present to diagnose ovarian cancer, and compare the analysis results with those in Example 1.

[0094] S1 Collection of ovarian tumor samples:

[0095] The collected samples are the same as those in Example 1, that is, use the serum samples with menopause information in S1 of Example 1 for subsequent test analysis.

[0096] S2 Serum HE4 Protein Detection:

[0097] Use the reagent of the human epididymis protein 4 detection kit (electrochemiluminescence method, registered in China for import of medical devices No. 20153403726) from Roche Diagnostics to detect the HE4 concentration in serum samples.

[0098] S3: Result Analysis

[0099] For premenopausal women, the cutoff value of serum HE4 is 92.1 pmol / L, and for postmenopausal women, the cutoff value of serum HE4 is 121 pmol / L. The sensitivity of serum HE4 concentration for the diagnosis of ovarian cancer in premenopausal women is 41.18%, and the specificity is 48.48%. The sensitivity of serum HE4 concentration for the diagnosis of ovarian cancer in postmenopausal women is 40.58%, and the specificity is 68.75%.

[0100] Comparison of the combination of exosomal biomarkers CA125 and HE4 in Example 3 with the results of serum CA125 and serum HE4

[0101] Use the algorithm (ROMA) that combines serum CA125 and HE4 proteins, which is widely used clinically at present, to evaluate the risk of ovarian malignancy to diagnose ovarian cancer, and compare the analysis results with those in Example 1.

[0102] S1 Collection of Ovarian Tumor Samples:

[0103] The collected samples are the same as those in Example 1, that is, use the serum samples with menopause information in S1 of Example 1 for subsequent detection and analysis.

[0104] S2 Serum HE4 Protein Detection:

[0105] Use the reagent of the carbohydrate antigen 125 detection kit (electrochemiluminescence method, registered in China for import of medical devices No. 20153401561) and the human epididymis protein 4 detection kit (electrochemiluminescence method, registered in China for import of medical devices No. 20153403726) from Roche Diagnostics to detect the CA125 and HE4 concentrations in serum samples.

[0106] S3: Result Analysis

[0107] For premenopausal women, the cutoff value of ROMA is 11.4%, and for postmenopausal women, the cutoff value of ROMA is 29.9%. The sensitivity of serum ROMA for the diagnosis of ovarian cancer in premenopausal women is 70.00%, and the specificity is 3.23%. The sensitivity of serum ROMA for the diagnosis of ovarian cancer in postmenopausal women is 80.00%, and the specificity is 35.48%.

[0108] Comparison of the combination of exosomal biomarkers CA125 and HE4 in Example 4 with the results of the combination of exosomal biomarkers CA125, HE4 and c5a

[0109] S1 Ovarian tumor sample collection:

[0110] A total of 172 serum samples were collected from patients with adnexal masses who underwent surgical treatment. Among them, 98 patients with epithelial ovarian malignant tumors diagnosed by postoperative pathology were used as the positive group, and 74 patients with benign masses were used as the negative control group.

[0111] The screening criteria for the 172 patients with adnexal masses who underwent surgical treatment were as follows: ≥18 years old, those with adnexal masses also needed to undergo surgery, no history of malignant tumors, no history of radiotherapy and chemotherapy, and no pregnancy. The enrolled samples were all preoperative collected samples.

[0112] S2 Extraction of exosomal CA125, HE4 and C5a proteins:

[0113] It was carried out by an exosomal protein extraction and purification kit. First, 300 uL of serum sample was taken into a centrifuge tube, and 75 uL of exosome precipitation reagent was added. The centrifuge tube was placed on a shaker to shake to mix the sample and the precipitation reagent evenly. After mixing, it was left to stand at room temperature for 30 minutes. Then the centrifuge tube was put into a centrifuge and centrifuged at 4700×g at 4°C for 10 minutes. The supernatant was removed, and the precipitate at the bottom of the centrifuge tube was the extracted protein linked, embedded and coated with phospholipid bilayers. Then 300 uL of exosome lysis reagent was added to the precipitate at the bottom of the centrifuge tube and shaken on a shaker for no less than 30 minutes. The shaken and evenly mixed solution was filtered, and the filtrate could be used for subsequent protein detection.

[0114] S3 Detection of exosomal CA125, HE4 and C5a proteins:

[0115] Take 30 uL of the filtered exosomal sample and detect the exosomal sample on an automatic chemiluminescence analyzer by the double antibody sandwich method.

[0116] S4: Result analysis

[0117] The detection results and the pathological diagnosis results were used to generate a receiver operating characteristic curve (ROC), as Figure 2a and Figure 2b shown.

[0118] According to the generated curve, the area under the curve (AUC) of the combination of exosomal CA125 and HE4 markers was larger than that of the combination of exosomal CA125, HE4 and C5a markers (OCS), which was 0.906 (95% confidence interval: 0.861 - 0.952, P < 0.0005). An AUC greater than 0.9 indicates a high diagnostic accuracy, and the cutoff at this time has practical significance.

[0119] In some embodiments, the present invention provides an auxiliary diagnostic algorithm for evaluating the benign and malignant nature of ovarian cancer by combining the exosomal biomarkers CA125 and HE4, wherein:

[0120] a) Use multiple protein indicators (CA125 / HE4) contained in serum-derived exosomes as biological markers;

[0121] b) Calculate multiple indicators into a single indicator through an algorithm to further improve specificity and sensitivity, and can effectively perform auxiliary differential diagnosis on ovarian malignant tumors.

[0122] In some embodiments, the auxiliary diagnostic evaluation formula for the benign and malignant nature of ovarian cancer is:

[0123] OCS = ey / (1 + ey),

[0124] where y = Ax ln[HE4] + B x ln[CA125] - y0, y0 = 4.08,

[0125] where the value range of A is between 0.304 and 1.216, preferably 1.279, and the value range of B is between 0.6395 and 2.558, preferably 0.608.

[0126] In some embodiments, the auxiliary diagnostic evaluation formula for the benign and malignant nature of ovarian cancer is:

[0127] OCS = ey / (1 + ey),

[0128] where y = 1.279x ln[HE4] + 0.608 x ln[CA125] - y0, y0 = 4.08.

[0129] Example 2

[0130] 2.1 Analysis and selection of reference population

[0131] The selection of the experimental population was set according to the methodology and results of domestic and foreign studies on clinical markers for the differential diagnosis of ovarian cancer. First, the population range was women with pelvic masses detected by ultrasound, which is a common feature of ovarian cancer and other benign ovarian diseases; second, approximately 90% of ovarian cancers are of epithelial origin, so in the malignant ovarian cancer patient group, the proportion of epithelial ovarian cancer is not less than 90%. Finally, in the benign ovarian disease patient group, patients with benign diseases that can cause pelvic masses and whose mass characteristics under ultrasound imaging are similar to those of ovarian cancer were included in the patient samples according to the actual outpatient reception situation.

[0132] 2.2 Samples

[0133] According to the requirements of the reference population, a total of 172 serum samples from patients with adnexal masses who underwent surgical treatment were collected in this experiment. After postoperative pathological diagnosis, there were 98 patients with epithelial ovarian malignant tumors and 74 patients with benign masses.

[0134] 2.3 Experimental reagents and instruments

[0135] Reagent kits: Exosome protein extraction, CA125, HE4, and C5a protein detection reagent kits from Shanghai Sino-Diagnostic Biomedical Technology Co., Ltd. (Product number: National Medical Device Registration No. 20243400138)

[0136] Instruments used:

[0137] Automatic chemiluminescence analyzer, Chongqing Cosmed Biotechnology Co., Ltd., Smart 6500.

[0138] 2.4 Detection

[0139] Exosome proteins were extracted from the 172 serum samples using protein, polypeptide extraction or purification reagents, and detected using CA125, HE4, and C5a antibodies.

[0140] 2.5 Statistics

[0141] Formula determination

[0142] For single-index comparison, non-parametric comparison (Mann-Whitney-U) was used. When constructing the index model, since the distribution of the index did not conform to the typical Gaussian distribution (normal distribution), after natural logarithm transformation of the index, the natural logarithm of the index was used to calculate the predicted probability through logistic regression.

[0143] Obtained formula

[0144]

[0145] Among them: y = 1.279×ln[HE4] + 0.608×ln[CA125] - 4.080, where ln = natural logarithm.

[0146] Statistical analysis of measurement results

[0147] Analysis and statistics were performed using IBM SPSS software. The predicted probability was used for the receiver operating characteristic curve (ROC curve). The area under the ROC curve (AUC) was better than that of serum CA125 and HE4 (see Figure 3a and Figure 3b ).

[0148] Example 3

[0149] 3.1 Analysis and Selection of the Reference Population

[0150] The selection of the experimental population was set according to the methodology and results of domestic and foreign studies on clinical markers for the differential diagnosis of ovarian cancer. First, the population scope was women with pelvic masses detected by ultrasound, which is a common feature of ovarian cancer and other benign ovarian diseases. Second, about 90% of ovarian cancers are of epithelial origin. Therefore, in the group of malignant ovarian cancer patients, the proportion of epithelial ovarian cancer is not less than 90%. Finally, in the group of patients with benign ovarian diseases, patients with benign diseases that can cause pelvic masses and whose mass characteristics under ultrasound imaging are similar to those of ovarian cancer were included in the patient samples according to the actual outpatient reception situation.

[0151] 3.2 Samples

[0152] According to the requirements of the reference population, a total of 172 serum samples from patients with adnexal masses who underwent surgical treatment were collected in this experiment. Among them, 98 were patients with epithelial ovarian malignancies and 74 were patients with benign masses after postoperative pathological diagnosis.

[0153] 3.3 Reagents and Instruments for the Experiment

[0154] Reagent Kits

[0155] Exosome protein extraction, CA125, HE4, and C5a protein detection reagent kits from Shanghai 3D Medicines Inc. (Product number: National Medical Device Registration No. 20243400138)

[0156] Instruments Used:

[0157] Automatic chemiluminescence analyzer, Chongqing Cosmed Biotechnology Co., Ltd., Smart 6500.

[0158] 3.4 Detection

[0159] Exosome proteins from these 172 serum samples were extracted using protein, polypeptide extraction or purification reagents and detected with CA125, HE4, and C5a antibodies.

[0160] 3.5 Statistics

[0161] The HE4 parameter B was adjusted to 2.558 and 0.6395, and the area under the ROC curve (AUC) was better than that of serum CA125 and HE4.

[0162]

[0163] Among them: y = 0.608 × ln[CA125] + B × ln[HE4] - 4.080, where ln = natural logarithm.

[0164] See Figure 4 、 Figure 5 。

[0165] Combination of Exosomal Biomarkers CA125 and HE4 with Other Biomarkers in Example 4

[0166] S1 Collection of Ovarian Tumor Samples:

[0167] A total of 172 serum samples were collected from patients with adnexal masses who underwent surgical treatment. Among them, 98 patients with epithelial ovarian malignant tumors diagnosed by postoperative pathology were used as the positive group, and 74 patients with benign masses were used as the negative control group.

[0168] The screening criteria for 172 patients with adnexal masses who underwent surgical treatment were as follows: ≥18 years old, those with adnexal masses also needed to undergo surgery, no history of malignant tumors, no history of radiotherapy or chemotherapy, and no pregnancy. All the enrolled samples were preoperative samples.

[0169] S2 Extraction of Exosomal CA125, HE4 and C5a Proteins:

[0170] First, take 300 μL of the sample in a centrifuge tube, and add 75 μL of the exosome precipitation reagent in the exosome protein extraction and purification kit (same as Example 1). Place the centrifuge tube on a shaker to mix the sample and the precipitation reagent evenly, and let it stand at room temperature for 30 minutes after mixing. Then place the centrifuge tube in a centrifuge and centrifuge at 4700×g at 4°C for 10 minutes. Remove the supernatant, and the precipitate at the bottom of the centrifuge tube is the extracted protein linked, embedded, and coated with phospholipid bilayers. Then add 300 μL of the exosome lysis reagent in the exosome protein extraction and purification kit to the precipitate at the bottom of the centrifuge tube. Shake on a shaker for at least 30 minutes. Filter the shaken and mixed solution, and the filtrate can be used for subsequent protein detection.

[0171] S3 Detection of Exosomal CA125, HE4 and C5a Proteins:

[0172] Take 30 μL of the filtered exosome sample and detect CA125, HE4, and C5a. Detect the exosome sample on an automatic chemiluminescence analyzer.

[0173] S4 Detection of Serum CEA Protein:

[0174] Use the carcinoembryonic antigen (CEA) detection kit (chemiluminescence method) (Shanghai Medical Device Registration No. 20232400201) produced by Shanghai 3D Medicines Inc. to detect the CEA concentration of serum samples.

[0175] S5: Result Analysis

[0176] The results of the detection and the pathological diagnosis results were used to generate a receiver operating characteristic curve (ROC), as Figure 6a shown (where the diagonal line segment generated from the results is shown).

[0177] The results of the detection and the pathological diagnosis results were used to generate the area under the receiver operating characteristic working curve, as Figure 6b shown.

[0178] According to the generated curve, the combination of exosomal CA125, HE4 and serum CEA markers had a larger area under the ROC curve (AUC) compared to the combination of exosomal CA125, HE4 and C5a markers, which was 0.914 (95% confidence interval: 0.860 - 0.949, P < 0.0005). An AUC greater than 0.9 indicates high diagnostic accuracy, and the cutoff value at this time has practical significance.

[0179] Comparative Example 5: Detection method of serum CEA for diagnosing ovarian cancer

[0180] S1 Collection of ovarian tumor samples:

[0181] A total of 185 serum samples from patients with adnexal masses who underwent surgical treatment were collected. Among them, 117 patients with epithelial ovarian malignant tumors diagnosed by postoperative pathology were used as the positive group, and 68 patients with benign masses were used as the negative control group.

[0182] The screening criteria for 185 patients with adnexal masses who underwent surgical treatment were: ≥18 years old, those with adnexal masses also needed to undergo surgery, no history of malignant tumors, no history of radiotherapy and chemotherapy, and no pregnancy. The enrolled samples were all preoperative collected samples.

[0183] S2 Detection of serum CEA protein:

[0184] The CEA concentration of the serum samples was detected using a carcinoembryonic antigen (CEA) detection kit (chemiluminescence method) (Shanghai Meddevice Approval No. 20232400201) produced by Shanghai 3D Medicines Inc.

[0185] S3: Result analysis

[0186] The results of the detection and the pathological diagnosis results were used to generate a receiver operating characteristic curve (ROC), as Figure 7a shown (where the diagonal line segment generated from the results is shown).

[0187] The results of the detection and the pathological diagnosis results were used to generate the area under the receiver operating characteristic working curve, as Figure 7b shown.

[0188] Based on the generated curve, the area under the ROC curve (AUC) for the serum CEA marker was 0.532 (95% confidence interval: 0.441 - 0.622, P < 0.0005). The AUC had a relatively low accuracy within the range of 0.5 - 0.7.

Claims

1. Use of a detection reagent for an exosome-derived biomarker in the preparation of a composition or kit for assessing, diagnosing, and / or monitoring ovarian cancer in a subject, wherein the exosome-derived biomarker comprises exosomal CA125 and HE4, and wherein assessing, diagnosing, and / or monitoring ovarian cancer in a subject comprises 1) obtaining exosomes from the subject, and 2) determining the presence and / or level of the biomarker in the exosomes from the subject.

2. The method of claim 1, wherein the biomarker does not include exosomal C5a.

3. The use according to claim 1 or 2, wherein the composition or kit further comprises a marker from serum.

4. The use according to claim 1 or 2, wherein the detection reagent for the biomarker from exosomes comprises a monoclonal antibody.

5. The use according to claim 1 or 2, wherein the exosomes are derived from body fluids.

6. The use according to claim 1 or 2, wherein the exosomes are derived from blood, serum, serous fluid, plasma, lymph, urine, cerebrospinal fluid, saliva, vaginal secretions, milk, tears or ascites.

7. The use according to claim 1 or 2, wherein the exosomes are derived from mucosal secretions of secretory tissues and organs.

8. The use according to claim 1 or 2, wherein the exosomes are derived from the fluid of the pleura, pericardium, or peritoneum.

9. A composition or kit for evaluating, diagnosing and / or monitoring ovarian cancer in a subject, wherein the composition or kit comprises a detection reagent for a biomarker as defined in any one of claims 1 to 8, wherein the composition or kit comprises 1) a reagent and / or device for extracting exosomes, and 2) a reagent for determining the presence and / or level of the biomarker in exosomes from the subject.

10. The composition or kit of claim 9, wherein the detection reagent for the biomarker from exosomes comprises an immunodetection reagent.

11. A computer system comprising a processor and a storage device storing computer executable code, wherein when the computer executable code is executed at the processor, it is configured to: Using the combination of biomarkers CA125 and HE4 in exosomes from the subject as an index, the index was calculated into a single index using the following algorithm: OCS=ey / (1+ey), in, y=A x ln[HE4]+B x ln[CA125]-y0, y0=4.08, The value range of A is between 0.304 and 1.216, and the value range of B is between 0.6395 and 2.

558.

12. The computer system of claim 11, wherein the indicator is calculated as a single indicator by the following algorithm: OCS=ey / (1+ey), in, y=1.279x ln[HE4]+0.608x ln[CA125]-y0, y0=4.

08.

13. A computer-readable medium having instructions stored thereon, which, when executed by a processor, cause the processor to execute the computer-executable code defined in claim 11 or 12.

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