Application of exosome CA125 biomarker in evaluation of ovarian malignant tumors

Abstract

The invention provides application of an exosome CA125 biomarker in evaluation of ovarian malignant tumors. The invention relates to an application of a detection reagent of a biomarker from an exosome in preparation of a composition or a kit for evaluating, diagnosing and / or monitoring ovarian cancer of a subject, and the biomarker from the exosome comprises the exosome CA125. 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 exosome marker provided by the invention can be used for conveniently and quickly evaluating, diagnosing and identifying the ovarian cancer with better performance.

Description

Technical Field

[0001] The present invention generally relates to the field of evaluation, diagnosis, and / or monitoring of malignant tumors such as 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 tumors are 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. 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 BRCA1 / 2 germline mutations. Ovarian malignant tumors include multiple 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] Many 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 △N10, 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 CA125, transferrin, haptoglobin, ApoA1, 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 domain 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), transforming protein containing phosphorylated Src homology 2 domain 1 (Shc), E (She) containing phosphorylated Src homology 2 domain, 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 examination (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 antigens and related to epithelial ovarian cancer antigen. In women with epithelial ovarian cancer, more than 80% of patients have a CA125 concentration > 35 U / mL critical value. 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, and among this group, 5% have benign diseases and 28% have non-gynecological system tumors. In addition, patients with heart failure, cirrhosis, and chronic active hepatitis may also show 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, one-fifth of ovarian cancer patients may be missed. Some benign ovarian diseases can also lead to elevated CA125 levels, resulting in false positives. In addition, the serum biomarker HE4 is another biomarker for ovarian cancer diagnosis. 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.

[0007] 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 the 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.

[0008] 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, and more and more evidence shows that exosomes play an important role in promoting tumor growth, participating 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 healthy people. Exosomes secreted in the human body have high specificity, and the information contained in 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 the selection and ranking of 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, and 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, and selecting a suitable model to evaluate the risk of malignant ovarian cancer). CN113718031A discloses blood sample collection, extraction of cfDNA and genomic DNA (genomic DNA), protein marker detection (detecting 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 verification 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 multi-index combined diagnosis of epithelial ovarian cancer, and the clinical verification of multi-index combined detection of ovarian cancer.

[0009] However, since exosomes originate from the invagination of the cell membrane to form early endosomes containing membrane proteins, 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 it 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 to diagnose the cancer by detecting the biomarker of exosomes, thus bringing difficulties to the evaluation of cancer using exosome biomarkers.

[0010] 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

[0011] In some embodiments, the exosomal CA125 biomarker provided by the present invention is a convenient, rapid, and better-performing diagnostic tool for assisting in the diagnosis and differentiation of ovarian cancer.

[0012] In some embodiments, the present invention provides a method for evaluating, diagnosing, and / or monitoring ovarian cancer in a subject, wherein exosomal CA125 is used as an ovarian cancer biomarker. It has been identified that exosomal CA125 in ovarian cancer patients can be used as a biomarker for ovarian cancer. 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.

[0013] In some embodiments, the present invention has surprisingly found that exosomal CA125 exhibits excellent sensitivity and specificity in the detection of ovarian cancer. In some embodiments, the ovarian cancer biomarker of the present invention may include exosomal CA125 alone or a combination of exosomal CA125 with other markers, such as a combination with markers from serum (such as CEA) and / or other markers of exosomes. In some embodiments, exosomal CA125 as an ovarian cancer biomarker can be detected in combination with other known ovarian cancer markers, such as known serum protein markers, serum microRNA markers, exosomal protein markers, exosomal microRNA markers, etc. In some embodiments, exosomal CA125 as an ovarian cancer biomarker can be combined with one, two or all of exosomes or blood HE4, C5a and PLG. However, in some embodiments, when exosomal CA125 is combined with other markers, C5a may not be included in the combination of the markers. In some embodiments, when exosomal CA125 is combined with other markers, one, two or all of exosomal HE4, C5a and PLG may not be included in the combination of the markers. In some embodiments, when exosomal CA125 is combined with other markers, one, two or all of blood HE4, C5a and PLG may not be included in the combination of the markers. In some embodiments, exosomal CA125 as an ovarian cancer biomarker can be used alone. In some embodiments, it is very advantageous to use fewer biomarkers because, compared to using a combination 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 has surprisingly found that the detection of ovarian cancer can be achieved by exosomal CA125, and it shows at least comparable or even significantly improved sensitivity and / or specificity. In some embodiments, the present invention has surprisingly found that the combination of exosomal CA125 with other markers, such as markers 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 marker is larger than that of the combination of exosomal CA125, HE4 and C5a markers, which is 0.922 (95% confidence interval: 0.882 - 0.962, P < 0.0005), which is completely unexpected. In contrast, exosomal CA125 achieves a comparable or even more excellent tumor assessment effect than using other markers or combinations of markers.

[0014] Thus, in some embodiments, the present invention relates to the use of biomarkers from exosomes (exosomal CA125), which can be used alone or in combination with other biomarkers, such as in combination with other blood (including serum or plasma, etc.) biomarkers and / or other exosomal biomarkers. In some embodiments, exosomal CA125 is not used in combination with any of c5a, HE4, and PLG from exosomes. In some embodiments, the biomarker used consists of exosomal CA125. 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 exosomal CA125 or a combination thereof compared to a control (such as the use of a combination of more biomarkers, or the use of 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 by 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 exosomal CA125 compared to a control (such as serum biomarkers or other exosomal biomarkers or a combination thereof different from the present invention), such as by 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 exosomal CA125 compared to a control (such as serum biomarkers or other exosomal biomarkers or a combination thereof different from the present invention), such as by 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.

[0015] In some embodiments, the methods of the present invention include determining biomarkers from exosomes. Methods for detecting biomarkers are known in the art, and 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, immunoassay (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, the detection reagents for detecting the biomarkers of the present invention (such as exosomal CA125) include antibodies (such as monoclonal antibodies). In some embodiments, reagents such as antibodies (such as monoclonal antibodies) for detecting the biomarkers of the present invention (such as exosomal CA125) can be conjugated to a solid support, a detectable label, or a binding partner. For example, direct or indirect conjugation can be performed. 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 not limited to metal particles, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, immuno-labels, 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.

[0016] 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, spliced 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 exosomal CA125 in a biological sample (such as a biological sample from exosomes) from a subject suspected of having ovarian cancer.

[0017] 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.

[0018] 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 early ovarian cancer screening and diagnosis. Exosomes (extracellular vesicles, exosomes) are a type of exocytic vesicle that exchange information between cells by transporting proteins, nucleic acids, etc. in the mother cells. Almost all cells secrete exosomes, and the exosomes secreted by cells carry a lot of biological information from the mother 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, diseased cells 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.

[0019] In some embodiments, the exosomes are derived from body fluids, such as 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 derived from cultures, solutions such as lavage fluids, for example, cell and organ culture media, including cell or organ conditioned media, that are in contact with a subject or a sample from a subject. In some embodiments, the exosomal CA125 protein of the present invention can be derived from exosomal proteins in samples such as whole blood, plasma, serum, ascites, lymph fluid, etc.

[0020] 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 the biomarker in exosomes from a subject.

[0021] 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 beads, 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 broadly include, for example, 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 against exosome CA125 and / or primers, probes for 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. In order to release exosome contents, the related exosome extraction and / or isolation and / or analysis reagents of 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.

[0022] 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 a 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 the expression of the biomarker. 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, antibody derivative, or 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 the protein or its 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.

[0023] 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 (for example, using a kit to detect) the presence and / or concentration of the biomarker in the exosomes. Compared with a control group of patients without ovarian cancer, a change in the level or evaluation of the biomarker indicates that the patient has ovarian cancer.

[0024] The present invention generally relates to cancer biomarkers, and particularly to biomarkers associated with ovarian cancer. The present invention provides methods for predicting, evaluating, 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.

[0025] The present invention relates to the biomarker exosomal CA125. The present invention relates to the sources of biomarkers including exosomes.

[0026] 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; they also include fluids in the pleura, pericardium, peritoneum, abdomen, and other body cavities. Biological fluids may also include liquid solutions, lavage fluids, etc. that are in contact with an object or biological source (e.g., cell and organ culture media, including cell or organ conditioned media).

[0027] The present invention includes a kit 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.

[0028] 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, significantly reducing 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.

[0029] In some embodiments, the present invention finds that the biomarker exosomal CA125 can achieve excellent sensitivity and specificity for cancer diagnosis. However, in some embodiments, the biomarker exosomal CA125 can also be combined with known biomarkers for cancer diagnosis and prognosis analysis.

[0030] In some embodiments, the present invention includes using exosomes extracted from body fluid samples to replace body fluid samples as the detection object. 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, treatment, etc. In some embodiments, it can be applied to one or more aspects such as ovarian cancer (risk) assessment, diagnosis, detection, monitoring, prognosis, treatment, etc. through exosomal CA125.

[0031] In some embodiments, the biomarker level in a patient is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more times higher or lower than that in a biomarker without having 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, related metabolite level, etc.) can be used alone for the evaluation, diagnosis and / or prognosis of ovarian cancer.

[0032] In some embodiments, the present invention provides the use of an exosomal CA125 biomarker in the evaluation of ovarian malignancies. In some embodiments, the present invention determines the risk of ovarian cancer by detecting the concentration of exosomal protein CA125.

[0033] In some embodiments, the present invention may include the following steps:

[0034] S1: Sample collection

[0035] Collect samples from a certain number of patients with ovarian malignancies and patients with ovarian benign tumors.

[0036] S2: Exosomal protein extraction

[0037] This operation extracts exosomes in the sample by methods such as precipitation method or immunomagnetic bead method, and then lyses the extracted exosomes to obtain exosomal proteins.

[0038] S2: Detection of exosomal CA125 protein

[0039] This operation detects exosomal CA125 protein in the sample by the double antibody sandwich method.

[0040] S3: Result analysis

[0041] Compare the concentration of the detected exosomal CA125 protein with the cutoff value. If it is greater than or equal to the cutoff value, it indicates that the patient has a higher risk of ovarian cancer. If it is less than the cutoff value, it indicates that the patient has a lower risk of ovarian cancer.

[0042] 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 possibility 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.

[0043] The present invention advantageously has one or more of the following advantages:

[0044] The present invention proposes to use the concentration of exosomal CA125 protein to determine whether a patient has ovarian cancer. Currently, there is no report on using exosomal CA125 protein to diagnose ovarian cancer.

[0045] The present invention proposes a method for detecting the concentration of exosomal CA125 protein. Currently, there is no report on detecting exosomal CA125.

[0046] The exosomal CA125 protein proposed by the present invention can be exosomal CA125 protein from samples such as whole blood, plasma, serum, ascites, lymph fluid, etc. Clinical collection is relatively easy and the patient compliance is relatively high.

[0047] The detection result of the present invention is not affected by factors such as menstrual cycle, etc. It is not necessary 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 concentration of exosomal protein CA125.

[0048] 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 condition of patients with ovarian epithelial cancer, the diagnosis of ovarian cancer by the present invention has higher specificity and effectively reduces the risk of delayed treatment for patients.

[0049] This operation is simpler and the detection cost is lower. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 : Receiver operating characteristic curve (ROC) generated from the results of detecting exosomal CA125 extracted by the protein precipitation method and the pathological diagnosis results.

[0051] Figure 2 : Receiver operating characteristic curve (ROC) generated from the results of detecting exosomal CA125 extracted by the immunomagnetic bead method and the pathological diagnosis results.

[0052] Figure 3a and Figure 3b : Receiver operating characteristic curve (ROC) generated from the results of detecting the comparison between the exosomal CA125 biomarker and the combined biomarker and the pathological diagnosis results.

[0053] Figure 4a and Figure 4b : Receiver operating characteristic curve (ROC) generated from the results of detecting the combination of exosomal CA125 biomarkers and the pathological diagnosis results.

[0054] Figure 5a and Figure 5b: Receiver operating characteristic curve (ROC) generated from the results of serum CEA detection and pathological diagnosis results. Detailed implementation

[0055] The exosome CA125 biomarker for diagnosing ovarian cancer provided by the present invention is significantly different from existing methods. The detection method provided in the present invention has higher sensitivity and specificity, is more convenient to operate, has lower detection costs, and the biomarker for diagnosing ovarian cancer in the present invention is exosomal protein. The application examples of the exosome biomarker CA125 in the evaluation of ovarian malignancies are as follows. Unless otherwise specified, the reagents used in the examples are all commercially available reagents. Among them, exosomal protein extraction, CA125, HE4, and C5a protein detection are carried out using kits (Shanghai 3D Medicines Inc., product number: China Medical Device Registration No. 20243400138), and serum CEA detection is carried out using the carcinoembryonic antigen (CEA) detection kit (chemiluminescence method) (Shanghai Medical Device Registration No. 20232400201) produced by Shanghai 3D Medicines Inc.

[0056] Example 1 Application of an exosome CA125 biomarker in the evaluation of ovarian malignancies:

[0057] S1 Collection of ovarian tumor samples:

[0058] A total of 168 serum samples from patients with adnexal masses who underwent surgical treatment were collected. Among them, 97 patients with epithelial ovarian malignancies after postoperative pathological diagnosis were used as the positive group, and 71 patients with benign masses were used as the negative control group.

[0059] The screening criteria for 168 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.

[0060] S2 Extraction of exosome CA125 protein by precipitation method:

[0061] 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. Place the centrifuge tube on a shaker to shake and mix the sample with the precipitation reagent. 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 connected, 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 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.

[0062] Detection of CA125 protein in S3 exosomes:

[0063] Take 30 μL of the filtered exosome sample and detect the exosome sample on an automatic chemiluminescence analyzer.

[0064] The detection results are as follows.

[0065] S4: Result analysis

[0066] Generate a receiver operating characteristic curve (ROC) from the detection results and the pathological diagnosis results, as Figure 1 shown.

[0067] According to the generated curve, the most appropriate cutoff value is selected as 8.95 U / mL. At this time, the area under the ROC curve (AUC) is maximized at 0.922 (95% confidence interval: 0.882 - 0.962, P < 0.0005). An AUC between 0.7 and 0.9 indicates a certain degree of diagnostic accuracy, and the cutoff at this time has practical significance. At this cutoff value, the sensitivity of exosomal CA125 concentration for ovarian cancer diagnosis is 81.4%, and the specificity is 97.2%.

[0068] Example 2 Application of an exosomal CA125 biomarker in the evaluation of ovarian malignancies:

[0069] S1 Collection of ovarian tumor samples: [[ID=2*]]

[0070] The samples collected are the same as those in Example 1, that is, the serum samples in S1 of Example 1 are used for subsequent detection and analysis.

[0071] S2 Extraction of exosomal CA125 protein by immunomagnetic bead method (Exosome Extraction and Nucleic Acid Purification Reagent (Magnetic Bead Method) (Shanghai 3D Medicines Inc., Product Number: Shanghai Min Equipment 20210060)):

[0072] Centrifuge the sample at 12000g for 10 min and take the supernatant. Add 1 mg of CD63 and CD9 immunomagnetic beads to 1 mL of the sample, incubate with shaking at 37 °C for 30 min, discard the supernatant after magnetic separation, add 1 mL of PBS (10 mM) for washing once, resuspend the immunomagnetic beads with 200 μL of PBS (10 mM) after magnetic separation again, and add 10 μL of a trypsin aqueous solution with a concentration of 2.5 mg / mL, keep in a constant temperature water bath for 3 min (37 °C). Then add 10 μL of a soybean trypsin inhibitor with a concentration of 2.5 mg / mL to the mixture solution and mix well to terminate digestion. Collect the supernatant after magnetic separation, and the obtained supernatant is the exosome solution eluted from the immunomagnetic beads. Then add a certain amount of exosome lysis reagent to the supernatant and shake on a shaker for at least 30 minutes. Filter the shaken and well-mixed solution, and the filtrate can be used for subsequent protein detection.

[0073] S3 Detection of exosomal CA125 protein:

[0074] Take 30 μL of the filtered exosome sample and detect the exosome sample on a fully automatic chemiluminescence analyzer by the double antibody sandwich method.

[0075] The detection results are as follows.

[0076] S4: Result analysis

[0077] Generate a receiver operating characteristic curve (ROC) from the detection results and the pathological diagnosis results, as Figure 2 shown.

[0078] According to the generated curve, select the optimal cutoff value of 9.02 U / mL. At this time, the area under the ROC curve (AUC) is maximally 0.917 (95% confidence interval: 0.875 - 0.958, P < 0.0005). An AUC between 0.7 and 0.9 indicates a certain degree of diagnostic accuracy, and the cutoff at this time has practical significance. At this cutoff value, the sensitivity of exosomal CA125 concentration for ovarian cancer diagnosis is 84.4%, and the specificity is 95.8%. The sensitivity and specificity of exosomal CA125 protein extracted by the immunomagnetic bead method and the precipitation method for ovarian cancer diagnosis are basically the same.

[0079] Comparative Example 1: Detection method of serum CA125 for diagnosing ovarian cancer

[0080] Use the serum CA125 protein concentration, which is widely used clinically at present, to diagnose ovarian cancer, and compare the analysis results with those of Example 1.

[0081] S1 Collection of ovarian tumor samples:

[0082] The collected samples are the same as those in Example 1, that is, the serum samples in S1 of Example 1 are used for subsequent detection and analysis.

[0083] Detection of CA125 protein in S2 serum:

[0084] The CA125 concentration in the serum sample is detected using the CA125 detection kit (electrochemiluminescence method, registered in China for import 20153401561) of Roche Diagnostic Corporation.

[0085] S3: Result analysis

[0086] The cutoff value of serum CA125 is 35 U / mL. The sensitivity of serum CA125 concentration for ovarian cancer diagnosis is 65.81%, and the specificity is 59.46%. The sensitivity and specificity of exosomal CA125 for ovarian cancer diagnosis are significantly improved compared with serum CA125.

[0087] Comparative Example 2: Comparison between exosomal CA125 biomarker and the combination of exosomal CA125, HE4, and C5a biomarkers: [[ID=1,7]]

[0088] Collection of S1 ovarian tumor samples:

[0089] A total of 168 serum samples of patients with adnexal masses who underwent surgical treatment were collected. Among them, 97 patients with epithelial ovarian malignant tumors after postoperative pathological diagnosis were used as the positive group, and 71 patients with benign masses were used as the negative control group.

[0090] The screening criteria for 168 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.

[0091] Extraction of exosomal CA125, HE4, and C5a proteins:

[0092] First, take 300 μL of serum sample into a centrifuge tube, and add 75 μL of exosome 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 put the centrifuge tube into a centrifuge and centrifuge at 4700×g at 4°C for 10 minutes. Remove the supernatant. The precipitate at the bottom of the centrifuge tube is the extracted protein connected, embedded, and coated with phospholipid bilayers. Then add 300 μL of exosome 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.

[0093] Detection of exosomal CA125, HE4, and C5a proteins

[0094] Take 30 μL of the filtered exosome sample and detect the exosome sample on a fully automated chemiluminescence analyzer using the double antibody sandwich method.

[0095] S4: Result analysis

[0096] Generate a receiver operating characteristic curve (ROC) from the test results and the pathological diagnosis results, as Figure 3a 、 Figure 3b shown.

[0097] According to the generated curve, the area under the curve (AUC) of the exosomal CA125 biomarker is larger than that of the combination of exosomal CA125, HE4, and C5a biomarkers, which is 0.922 (95% confidence interval: 0.882 - 0.962, P < 0.0005). An AUC greater than 0.9 indicates high diagnostic accuracy, and the cutoff value at this time has practical significance.

[0098] Example 3 Combination of exosomal CA125 biomarker and other biomarkers

[0099] S1 Collection of ovarian tumor samples:

[0100] A total of 168 serum samples from patients with adnexal masses who underwent surgical treatment were collected. Among them, 97 patients with epithelial ovarian malignant tumors diagnosed by postoperative pathology were used as the positive group, and 71 patients with benign masses were used as the negative control group.

[0101] The screening criteria for the 168 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. All the enrolled samples were collected before surgery.

[0102] S2 Extraction of exosomal CA125 protein:

[0103] First, take 300 μL of the sample in a centrifuge tube and add 75 μL of the exosome precipitation reagent from the exosome protein extraction and purification kit (the same as in Example 1). Place the centrifuge tube on a shaker to shake and mix the sample with the precipitation reagent. 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 connected, embedded, and coated with phospholipid bilayers. Then add 300 μL of the exosome lysis reagent from 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 after filtration can be used for subsequent protein detection.

[0104] S3 Detection of exosomal CA125 protein:

[0105] Take 30 μL of the filtered exosome sample and detect the exosome sample on an automatic chemiluminescence analyzer using the double antibody sandwich method.

[0106] CEA protein detection in S4 serum:

[0107] Use the carcinoembryonic antigen (CEA) detection kit (chemiluminescence method) (Shanghai Medical Device Approval No. 20232400201) produced by Shanghai 3D Medicines Inc. to detect the CEA concentration in serum samples.

[0108] S4: Result analysis

[0109] Generate a receiver operating characteristic curve (ROC) from the test results and the pathological diagnosis results, as Figure 4a shown (in which a diagonal line segment generated from the results is shown).

[0110] Generate the area under the receiver operating characteristic curve from the test results and the pathological diagnosis results, as Figure 4b shown.

[0111] According to the generated curve, the combination of exosomal CA125 and serum CEA markers has a larger area under the ROC curve (AUC) of 0.919 (95% confidence interval: 0.879 - 0.960, P < 0.0005) compared to the combination of exosomal CA125, HE4, and C5a markers. An AUC greater than 0.9 indicates high diagnostic accuracy, and the cutoff value at this time has practical significance.

[0112] Comparative Example 3: Detection method of serum CEA for diagnosing ovarian cancer

[0113] S1 Collection of ovarian tumor samples: <{

[0114] A total of 168 serum samples from patients with adnexal masses who underwent surgical treatment were collected. Among them, 97 patients with epithelial ovarian malignant tumors diagnosed by postoperative pathology were used as the positive group, and 71 patients with benign masses were used as the negative control group.

[0115] The screening criteria for 168 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. All the enrolled samples were collected before surgery.

[0116] S2 Serum CEA protein detection:

[0117] Use the carcinoembryonic antigen (CEA) detection kit (chemiluminescence method) (Shanghai Medical Device Approval No. 20232400201) produced by Shanghai 3D Medicines Inc. to detect the CEA concentration in serum samples.

[0118] S3: Result analysis

[0119] Generate a receiver operating characteristic curve (ROC) from the test results and the pathological diagnosis results, as Figure 5a shown (in which a diagonal line segment generated from the results is shown).

[0120] The test results and the pathological diagnosis results generate the area under the receiver operating characteristic curve, as Figure 5b shown.

[0121] According to the generated curve, the area under the ROC curve (AUC) of the serum CEA marker is 0.533 (95% confidence interval: 0.443 - 0.624, P < 0.0005). The AUC has a relatively low accuracy in 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 evaluating, diagnosing, and / or monitoring ovarian cancer in a subject, wherein the exosome-derived biomarker comprises exosomal CA125, and wherein evaluating, 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.

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