Application of exosome HE4 biomarker in evaluation of ovarian malignant tumors

Abstract

The invention provides application of an exosome HE4 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 exosome 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 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 assessment, diagnosis and / or monitoring of malignant tumors such as ovarian cancer. The present invention relates to the use of biomarkers derived from exosomes for ovarian cancer assessment, and provides related analytical reagents, kits and related applications. Background Art

[0002] Ovarian cancer is a common malignant tumor of the female reproductive system. Its annual incidence ranks third among female reproductive system tumors, after cervical cancer and uterine corpus malignancies, and its mortality rate is increasing annually. It ranks first among female reproductive tract malignancies, making it a serious threat to women's health. The incidence of ovarian cancer in my country is 7.48 per 100,000 population, estimating nearly 50,000 new cases and 35,000 deaths annually. Because the ovaries are located deep within the pelvis, early-stage tumors can be difficult to detect during pelvic examinations. Consequently, 70% of ovarian cancers are diagnosed at an advanced stage, with a survival rate of less than five years for 70% of patients. Even after recovery, approximately 70% of patients will experience a recurrence within three years. Due to its insidious nature in its early stages and its potent lethality, ovarian cancer is often referred to as the "silent killer." Therefore, the key to preventing and treating ovarian cancer lies in proactive treatment. Early detection can increase the five-year survival rate of ovarian cancer patients from 20%-30% to 60%-70%, with some patients potentially being curable.

[0003] Most ovarian cancers are sporadic, while hereditary ovarian cancer accounts for approximately 15% of all ovarian cancer cases. Currently, germline mutations in more than ten tumor suppressor genes have been linked to hereditary ovarian cancer, with over 80% of hereditary ovarian cancers linked to germline mutations in BRCA1 / 2. Ovarian malignancies encompass a variety of pathological types, the most common of which is epithelial carcinoma, accounting for approximately 80% of ovarian malignancies, followed by malignant germ cell tumors and sex cord-stromal tumors, accounting for approximately 10% and 5%, respectively.

[0004] Numerous studies have been conducted on biomarkers for ovarian cancer. For example, WO2005034732 discloses DNA sequences related to various specific genes as marker genes for ovarian cancer. WO2005098447 mentions Apo A1, modified Apo A1, transthyretin ΔN10, native transthyretin, cysteinylated transthyretin, sulfonated transthyretin, CysGly-modified transthyretin, glutathionylated transthyretin, IAIH4 fragment No. 1, IAIH4 fragment No. 2, and IAIH4 fragment No. 3, and combinations thereof, as biomarkers for determining ovarian cancer status. WO2007002264 discloses CTAP3-related proteins and HE4, transferrin, haptoglobin, ApoA1, transthyretin, ITIH4 internal fragment, beta 2-microglobulin, hepcidin, prostatin, osteopontin, esoinophil-derived neurotoxins, leptin, prolactin, IGF-II, hemoglobin and its modified forms, HE4 II, CA15-3, CA19-9, CA72-4, CA 195. Tumor-associated trypsin inhibitor (TATI), CEA, placental alkaline phosphatase (PLAP), sialic acid (Sialyl) TN, galactosyltransferase, macrophage colony-stimulating factor (M-CSF, CSF-1), lysophosphatidic acid (LPA), the 110KD component of the extracellular segment of the epidermal growth factor receptor (p110EGFR), tissue kallikrein, 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 peptide antibody (TPA), SMRP, osteopontin and haptoglobin, insulin-like growth factor I and insulin-like growth factor II, etc. are used to identify ovarian cancer status.WO2015042115 mentions that examples of biomarkers for ovarian cancer assessment include: aldehyde dehydrogenase 1 (ALDH1), ApoCI, ApoAII, ApoCII, β-hemoglobin, calcium cycle protein, calgranulin A, calgranulin C, tight junction protein (claudin)-3, connective tissue growth factor (CTGF), eosinophil-derived neurotoxin, fibroblast growth factor 2 (basic) (FGF2), folate receptor 1 (FOLRI), reproductive glycoprotein (glycodelin), GPCR49, glutathione S-transferase θ1 (GSTT1), hepsin, hepcidin, insulin-like growth factor-II, inter-α-trypsin inhibitor heavy chain H4 (inter-α-trypsin inhibitor heavy chain The antibodies included leptin, macrophage inhibitory factor, mucin-16 (HE4), osteopontin, prolactin, proteaseserine 8 (PRSS8), protein C inhibitor, solute carrier family 39 (zinc transporter) member 4 (SLC39A4), small MBL-associated protein C-terminal fragment, stratum corneum chymotrytic enzyme, transferrin, transthyretin, WAP four-disulfide core domain 2 (HE4), transforming protein 1 containing phosphorylated Src homology region 2 domain (Shc), E containing phosphorylated Src homology region 2 domain (She), and autoantibodies specific to casein kinase 1ε.

[0005] Currently, auxiliary diagnosis of pelvic masses relies primarily on two testing methods. One is transvaginal ultrasound (TVS), which is considered an important indicator for surgical indications for pelvic masses. This imaging method can be used to examine the female reproductive organs, including the uterus, ovaries, cervix, and vagina. Despite its widespread use, it cannot completely accurately determine whether a mass is benign or malignant. Furthermore, this method requires experienced clinicians to interpret the test results. Another common test is the serum biomarker CA125, which is widely used in clinical practice to diagnose ovarian cancer, assess treatment efficacy, chemotherapy sensitivity, and tumor recurrence after treatment. However, CA125 has low specificity and sensitivity, making it prone to false negatives and false positives. Approximately 20% of patients with ovarian tumors do not have elevated CA125 levels, meaning that one in five ovarian cancer patients may be missed. Certain benign ovarian diseases can also cause 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 CA125 and HE4 detection kits approved by the National Medical Products Administration (NMPA) are intended for use only as an auxiliary monitoring tool for treatment efficacy, recurrence, and progression of epithelial ovarian cancer patients and cannot be used for the differential diagnosis of ovarian cancer. Therefore, there is an urgent need for a better diagnostic tool to assist in the diagnosis and differentiation of ovarian cancer in clinical practice.

[0006] HE4 is primarily distributed in reproductive system epithelia, such as the epididymis, seminiferous tubules, vas deferens, fallopian tube epithelium, and endometrium. Among malignant tumors, HE4 expression levels are highest in ovarian serous carcinoma, followed by lung adenocarcinoma, while lung squamous cell carcinoma has lower expression. Endometrial carcinoma, transitional cell carcinoma, breast cancer, and pancreatic cancer all have moderate expression, though some have expression levels comparable to ovarian serous carcinoma. Colon, gastric, liver, and prostate cancers mostly show low expression.

[0007] Currently, most commercially available diagnostic reagents directly detect changes in a specific indicator (protein, nucleic acid, etc.) in body fluids (blood, urine, etc.) to draw conclusions. However, body fluids typically contain a large number of highly abundant proteins. Taking serum as an example, the top ten most abundant proteins account for more than 90% of the total serum protein. During disease progression, due to the small size of the lesions, mild symptoms, and limitations of detection instruments and reagents, the changes in the cells at the lesions and the amount of biological information secreted and released into the body fluids can easily be obscured by these highly abundant proteins. It is very difficult to detect biomarkers directly from body fluids to detect early-stage disease progression.

[0008] Exosomes are extracellular vesicles released by all cells, with a diameter of 30 to 150 nm. Exosomes contain DNA, RNA, and proteins, and are secreted by cells into the bloodstream. Exosomes can be detected in the tumor microenvironment, and growing evidence suggests that exosomes play a crucial role in promoting tumor growth, participating in activities such as angiogenesis, immune responses, and tumor metastasis. Studies using ELISA to detect, characterize, and quantify exosomes have found that some proteins expressed in exosomes from patients are significantly higher than those in healthy controls. Exosomes secreted by the human body are highly specific, and the information contained in exosomes secreted from body fluids may be more representative than that contained in body fluids. Therefore, traditional serum tumor markers can also be extracted and detected from exosomes, and the physiological information obtained has greater clinical value in assisting tumor diagnosis. For example, CN108841954B discloses the selection and ranking of candidate markers (using a proteomics method to perform a full scan analysis of proteins in the sample to screen markers); exosome extraction (adding an exosome extraction reagent to a serum or plasma sample, mixing thoroughly, 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 to select an appropriate model to assess the risk of malignant ovarian cancer). CN113718031A discloses blood sample collection, extraction of cfDNA and genomic DNA, protein marker detection (using Roche Cobas The e411 detection system performs plasma protein marker HE4 detection, high-throughput sequencing of CfDNA TP53, identification of somatic mutations, construction of a predictive model, and model validation. CN106248940A discloses the preparation of fusion proteins SUMO-C1D, SUMO-CCL18, SUMO-CXCL1, SUMO-TM4SF1, SUMO-FXR1, and SUMO-TIZ, the preparation of a liquid suspension chip for multi-indicator combined diagnosis of epithelial ovarian cancer, and the clinical validation of multi-indicator combined detection of ovarian cancer.

[0009] However, because exosomes originate from the invagination of the cell membrane, forming early endosomes containing membrane proteins, the endosomes further inwardly encapsulate proteins and RNAs in the cytoplasm, forming multivesicles within the cytoplasm. These multivesicles fuse with the cell membrane and release the exosomes into the extracellular matrix. Therefore, given the source of the exosomes, it is difficult to predict which proteins or nucleic acids will be included in the exosome formation process and further released into the extracellular matrix. Therefore, even if a known cancer marker is present in the patient's exosomes, it is not necessarily present in the patient's exosomes. Furthermore, it is difficult to determine whether such a known cancer marker can be obtained from the exosomes of the cancer patient, whether it has the same specificity and sensitivity as a tumor marker, and whether the cancer can be diagnosed by detecting such markers in exosomes, thus making it difficult to assess cancer using exosome biomarkers.

[0010] Therefore, despite the related 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 HE4 marker provided by the present invention is a convenient, rapid, and better-performing diagnostic tool to assist in the diagnosis and identification of ovarian cancer.

[0012] In some embodiments, the present invention provides methods for assessing, diagnosing, and / or monitoring ovarian cancer in a subject, wherein exosomal HE4 is used as an ovarian cancer biomarker. Exosomal HE4 has been identified in ovarian cancer patients and can be used as a biomarker for ovarian cancer. In some embodiments, the method for assessing, diagnosing, and / or monitoring ovarian cancer in a subject comprises the steps of: 1) obtaining exosomes from the subject, and 2) determining the presence and / or level of a biomarker in the exosomes from the subject.

[0013] In some embodiments, the present invention has surprisingly discovered that exosomal HE4 exhibits excellent sensitivity and specificity in detecting ovarian cancer. In some embodiments, the ovarian cancer biomarkers of the present invention may include exosomal HE4 alone or in combination with other markers, such as serum markers (e.g., CEA) and / or other exosomal markers. In some embodiments, exosomal HE4 as an ovarian cancer biomarker may 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 HE4 as an ovarian cancer biomarker may be combined with one, two, or all of exosomal or blood CA125, C5a, and PLG. However, in some embodiments, when exosomal HE4 is combined with other markers, C5a may not be included in the combination of markers. In some embodiments, when exosomal HE4 is combined with other markers, the combination may not include one, two, or all of exosomal CA125, C5a, and PLG. In some embodiments, when exosomal HE4 is combined with other markers, the combination may not include one, two, or all of blood CA125, C5a, and PLG. In some embodiments, exosomal HE4 can be used alone as an ovarian cancer biomarker. In some embodiments, using fewer biomarkers is highly advantageous because, compared to using a combination of more biomarkers, using fewer biomarkers for tumor assessment and / or diagnosis not only saves costs but also provides a more convenient, rapid, and high-performance diagnostic tool. Therefore, the present invention surprisingly discovered that exosomal HE4 can be used to detect ovarian cancer with at least comparable, or even significantly improved, sensitivity and / or specificity. In some embodiments, the present invention surprisingly discovered that combining exosomal HE4 with other markers, such as serum markers (e.g., CEA), can achieve synergistic effects in ovarian cancer detection, such as synergistically improved sensitivity and specificity. For example, using ROC curve analysis, it was found that the exosomal HE4 marker combination had a greater area under the ROC curve (AUC) of 0.926 (95% confidence interval: 0.891-0.962, P < 0.0005) than the exosomal CA125, HE4, and C5a marker combination, which was completely unexpected. In contrast, exosomal HE4 achieved comparable or even superior tumor assessment results compared to other markers or marker combinations.

[0014] Therefore, in some embodiments, the present invention relates to the use of an exosome-derived biomarker (exosomal HE4), which can be used alone or in combination with other biomarkers, such as other blood (including serum or plasma) biomarkers and / or other exosomal biomarkers. In some embodiments, exosomal HE4 is not used in combination with any of the exosome-derived c5a, CA125, and PLG. In some embodiments, the biomarker used consists solely of exosomal HE4. In some embodiments, the biomarkers of the present invention improve the sensitivity and / or specificity of ovarian cancer analysis, or provide at least comparable or even significantly improved sensitivity and / or specificity of ovarian cancer analysis compared to a combination of more biomarkers by reducing the number of markers in the marker combination. In some embodiments, comparable sensitivity and / or specificity means a difference of less than 5% in sensitivity and / or specificity compared to a control (e.g., a combination of more biomarkers, or other biomarkers). In some embodiments, improved or increased sensitivity and / or specificity means an increase of 5% or more compared to a control. In some embodiments, the sensitivity and / or specificity of ovarian cancer analysis that is at least equivalent to that of a control (e.g., a combination of more biomarkers, or other biomarkers) can be obtained by using a biomarker of the present invention including exosomal CA125 or a combination thereof. In some embodiments, the sensitivity and / or specificity of ovarian cancer analysis is improved by using a biomarker of the present invention by, for example, 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 more. In some embodiments, the sensitivity of ovarian cancer detection is improved by the biomarkers of the invention, including exosomal HE4, compared to a control (e.g., serum biomarkers or other exosomal biomarkers other than the markers of the invention, or a combination thereof), for example, 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 more. In some embodiments, the specificity of ovarian cancer detection is improved by a biomarker of the invention comprising exosomal HE4 compared to a control (e.g., serum biomarkers or other exosomal biomarkers other than the markers of the invention, or a combination thereof), for example, 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 more.

[0015] In some embodiments, the methods of the present invention include measuring 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, radioimmunoassay, chemiluminescence, real-time PCR, nucleic acid hybridization methods, Western blot analysis, immunoassays (such as immunoprecipitation and / or immunofluorescence), Southern hybridization, and the like 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 biomarkers of the present invention (e.g., exosomal HE and / or additional biomarkers) include antibodies (e.g., monoclonal antibodies). In some embodiments, reagents for detecting biomarkers of the invention (e.g., exosomal HE and / or additional biomarkers), such as antibodies (e.g., monoclonal antibodies), can be conjugated to a solid support, a detectable label, or a binding partner, for example, directly or indirectly, for example, where the solid support comprises a microtiter plate, plastic, a membrane such as a nitrocellulose membrane, glass, magnetic beads, or a metal support, for example, where the detectable label can include, but is not limited to, metal particles, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, immunolabels, and radiolabels, or enzyme labels, for example, colloidal gold, radioisotopes, fluorophores, spin labels, or phage labels, for example, rhodamine, fluorescein, acridinium esters, luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase, glucose oxidase, galactose oxidase, or glucose-6-phosphate dehydrogenase labels, for example, where the binding partner comprises biotin, streptavidin, or avidin, etc.

[0016] In some embodiments, a biomarker refers to a molecule associated with a disease (e.g., ovarian cancer), for example, a molecule associated with the presence, stage, prognosis, predicted treatment response, etc. of ovarian cancer. Ovarian cancer biomarkers can include proteins (e.g., full-length polypeptides, splice variants, post-translationally modified polypeptides, etc.) that are differentially expressed in subjects with ovarian cancer, as well as 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 measuring exosomal HE4 in a biological sample (e.g., a biological sample derived from exosomes) from a subject suspected of having ovarian cancer.

[0017] In some embodiments, the biomarker itself and / or a product of the biomarker, such as a metabolite of the biomarker and / or other products that directly associate with the biomarker, can be detected.

[0018] In some embodiments, the methods of the present invention include the step of measuring biomarkers from exosomes. In some embodiments, it has been found that detecting biomarkers in exosome contents can improve the sensitivity, specificity, and positive predictive value of early ovarian cancer screening and diagnosis. Exosomes (extracellular vesicles, exosomes) are small exocytic vesicles that exchange information between cells by transporting proteins, nucleic acids, and other substances from parent cells. Almost all cells secrete exosomes, which carry a wealth of biological information from the parent cells. They have been found to be important potential biomarkers. Due to a large number of immune responses, inflammatory responses, coagulation reactions, apoptosis, and autophagy, cells in diseased states release more exosomes, and the content of exosomes changes significantly in both quantity and quality. Compared to detecting biomarkers directly from blood, detecting biomarkers (such as the biomarkers described herein) in exosome contents has been found to be a method for concentrating information and is particularly useful for the assessment 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, breast 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, that have been in contact with a subject or a sample from a subject, such as cell and organ culture media, including cell or organ conditioned media. In some embodiments, the exosomal HE4 protein described herein can be derived from samples such as whole blood, plasma, serum, ascites, lymph, and the like.

[0020] In some embodiments, the present invention provides the use of biomarker detection reagents in the preparation of a composition or kit for evaluating, diagnosing and / or monitoring ovarian cancer in a subject. In some embodiments, the present invention provides the use of a composition and / or kit for preparing a composition and / or kit for evaluating, diagnosing and / or monitoring ovarian cancer in a subject by the methods of the present invention. In some embodiments, the composition or kit comprises 1) a reagent for extracting exosomes (e.g., an exosome-specific antibody) and / or a device (e.g., a test tube, a filter, an immunomagnetic bead, etc. for isolating exosomes), and 2) a reagent for determining the presence and / or level of a biomarker in exosomes from a subject (e.g., an antibody against the biomarker).

[0021] In some embodiments, the detection reagents for biomarkers derived from exosomes of the present invention may include exosome extraction reagents. In some embodiments, the detection reagents for biomarkers derived from exosomes of the present invention may also include exosome lysis reagents. Methods for isolating exosomes 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 sedimentation, filtration, immunomagnetic bead method, chromatography, and comprehensive methods (methods that use different methods for crude extraction and purification of exosomes). In some embodiments, exosome extraction reagents may include, for example, reagents for exosome sedimentation, such as PEG or other hydrophilic reagents. In some embodiments, the biomarker detection reagents and / or exosome extraction reagents of the present invention can broadly include, for example, related devices for biomarker analysis and / or exosome isolation, such as centrifuges, test tubes for centrifugation, filters for isolating exosomes (such as large-pore filters for filtering cells, small-pore filters for filtering protein impurities, and filters with a pore size of approximately 0.1 μm for isolating exosomes), filter paper, membranes (such as nitrocellulose membranes), gels, immunomagnetic beads, etc. In some embodiments, the exosome extraction reagents include antibodies, such as monoclonal antibodies, directed against exosome-specific biomarkers. In some embodiments, the exosome extraction reagents can include various exosome extraction and / or analysis kits provided by manufacturers (such as SBI, Invitrogen, etc.). In some embodiments, the biomarker detection reagents and / or exosome extraction reagents can include various buffers, such as PBS and electrophoresis buffer, used for biomarker analysis and / or exosome isolation and / or analysis. In some embodiments, biomarker detection reagents and / or exosome extraction reagents may include antibodies and / or primers or probes targeting specific biomarkers, such as antibodies targeting exosomal HE4 and / or primers or probes targeting 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 reagents for exosome-derived biomarkers of the present invention may also include an exosome lysis reagent. As known to those skilled in the art, exosomes have a phospholipid bilayer similar to a cell membrane. To release the exosome contents, the relevant exosome extraction, separation, and / or analysis reagents described herein may include an exosome lysis reagent such as RIPA lysis buffer. In some embodiments, the detection reagents for exosome-derived biomarkers of the present invention may also include a protein inhibitor. In some embodiments, direct analysis of exosome contents can be performed without the use of specialized lysis reagents, for example, by loading the exosome suspension directly onto a gel for electrophoresis analysis.In some embodiments, biomarker detection reagents and / or exosome extraction reagents may include protein analysis reagents such as Western Blot, ELISA analysis reagents, proteomic analysis reagents such as protein spectrum and antibody chip, etc. In some embodiments, biomarker detection reagents and / or exosome extraction reagents may include a tracing reagent, for example, to link the biomarker and / or exosome to a visible marker (such as a PKH26 tag, etc.). In some embodiments, biomarker detection reagents and / or exosome extraction reagents may include sequence analysis reagents such as high-throughput sequencing reagents.

[0022] In some embodiments, the present invention provides compositions and / or kits for assessing, diagnosing, and / or monitoring ovarian cancer in a subject using the methods of the present invention, wherein the composition or kit comprises a detection reagent for the biomarker. In some embodiments, the detection reagent may comprise a protein and / or nucleic acid detection reagent. In some embodiments, the detection reagent may comprise an immunoassay reagent. In some embodiments, the kit comprises reagents for determining biomarker expression. In some embodiments, the kit comprises multiple probes that specifically bind to a biomarker corresponding to the present invention. In some embodiments, the kit comprises multiple antibodies that specifically bind to a biomarker according to the present invention. In some embodiments, the kit comprises antibodies, antibody derivatives, or antibody fragments that specifically bind to a biomarker protein or protein fragment. In some embodiments, the kit comprises multiple antibodies, antibody derivatives, or antibody fragments that specifically bind to a biomarker protein or fragment thereof. In some embodiments, the kit comprises a capture reagent that binds to a biomarker according to the present invention and a container comprising at least one biomarker. In some embodiments, the capture reagent may bind to multiple biomarkers or may bind to at least one known biomarker. In some embodiments, the kit may further comprise a second or more capture reagents. In some embodiments, the kit comprises a buffer. In some embodiments, the kit comprises instructions for use. In some embodiments, the kit comprises a chip or microarray.In some embodiments, the kit comprises one or more matrices to which an adsorbent is attached.

[0023] In some embodiments, the present invention provides a method for assessing, diagnosing and / or monitoring the status of ovarian cancer, comprising the steps of extracting exosomes from a biological fluid sample from the patient, and detecting (e.g., using a kit) the presence and / or concentration of a biomarker in the exosomes, wherein an alteration in the level or evaluation of the biomarker compared to a control group of patients without ovarian cancer indicates that the patient has ovarian cancer.

[0024] The present invention generally relates to cancer biomarkers, and in particular 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 a reagent set or array for evaluating the expression levels of biomarkers associated with ovarian cancer.

[0025] The present invention relates to a biomarker exosome HE4. The source of the biomarker of the present invention includes exosomes.

[0026] In the present invention, sources of exosomes 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, breast milk, tears, and ascites; and also include fluids from the pleura, pericardium, peritoneum, abdomen, and other body cavities. Biological fluids may also include liquid solutions, lavage fluids, and the like that come into contact with a subject or biological source (e.g., cell and organ culture media, including cell or organ conditioned media).

[0027] The present invention includes kits for detecting the above-mentioned biomarkers using immunological methods. In some embodiments, the kits include 1) reagents and / or devices for extracting exosomes, and 2) reagents for determining the presence and / or level of the biomarkers in exosomes from a subject.

[0028] Unlike existing techniques that typically analyze biomarkers directly in body fluid samples, this method isolates specific exosomes to concentrate biological information at the lesion site, significantly reducing interference from other information in the body fluid and thereby amplifying the information at the lesion site. This allows for more accurate early-stage disease diagnosis within the current sensitivity of testing instruments and reagents.

[0029] In some embodiments, the present invention has found that the marker exosomal HE4 can achieve excellent sensitivity and specificity in cancer diagnosis. However, in some embodiments, the marker exosomal HE4 can also be combined with known markers for cancer diagnosis and prognosis analysis.

[0030] In some embodiments, the present invention includes the use of exosomes extracted from body fluid samples as a test subject instead of body fluid samples. These have been found to be effective in one or more aspects of ovarian cancer (risk) assessment, diagnosis, detection, monitoring, prognosis, and treatment. In some embodiments, exosomal HE4 can be used in one or more aspects of ovarian cancer (risk) assessment, diagnosis, detection, monitoring, prognosis, and treatment.

[0031] In some embodiments, the biomarker level of the patient is 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times or more higher or lower than the biomarker level of the patient who does not have 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 the biomarker (e.g., protein level, protein activity level, related metabolite level, etc.) can be used alone for the assessment, diagnosis and / or prognosis of ovarian cancer.

[0032] In some embodiments, the present invention provides a use of an exosomal HE4 biomarker in the assessment of ovarian malignancies. In some embodiments, the present invention determines the risk of ovarian cancer by detecting the concentration of the exosomal protein HE4.

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

[0034] S1: Sample Collection

[0035] A certain number of samples were collected from patients with ovarian malignant tumors and benign ovarian tumors.

[0036] S2: Exosome protein extraction

[0037] This procedure involves extracting exosomes from samples using precipitation or immunomagnetic bead methods, and then lysing the extracted exosomes to obtain exosomal proteins.

[0038] S2: Detection of exosomal HE4 protein

[0039] This procedure uses a double antibody sandwich method to detect exosomal HE4 protein in samples.

[0040] S3: Result Analysis

[0041] The concentration of the detected exosomal HE4 protein is compared with the cutoff value. If it is greater than or equal to the cutoff value, it means that the patient has a higher risk of ovarian cancer. If it is less than the cutoff value, it means that the patient has a lower risk of ovarian cancer.

[0042] The methods, compositions, and kits of the present invention can be used to assess whether a subject has ovarian cancer; assess the stage of ovarian cancer in a subject; assess the grade of ovarian cancer in a subject; assess the benign or malignant nature of ovarian cancer in a subject; assess the metastatic potential of ovarian cancer in a subject; assess the presence of ovarian cancer cells; assess the effect of one or more candidate compounds in inhibiting ovarian cancer in a subject; assess 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; assess the carcinogenicity 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 HE4 protein to determine whether a patient has ovarian cancer. Currently, there are no reports on the use of exosomal HE4 protein to diagnose ovarian cancer.

[0045] The present invention proposes a method for detecting the concentration of exosome HE4 protein. Currently, there are no reports on detecting exosome HE4.

[0046] The exosomal HE4 protein proposed in the present invention can be exosomal HE4 protein from samples such as whole blood, plasma, serum, ascites, lymph fluid, etc., which is relatively easy to collect clinically and has high patient compliance.

[0047] The detection results of the present invention are not affected by factors such as the menstrual cycle, and do not need to be combined with other factors to judge the patient's ovarian cancer risk. The patient's ovarian cancer risk can be directly judged based on the concentration of the exosomal protein HE4.

[0048] Compared with the products currently on the market that assist in monitoring the treatment effects of ovarian epithelial cancer patients and assist in monitoring the recurrence and progression of the disease in ovarian epithelial cancer patients, the present invention has a higher specificity in diagnosing ovarian cancer 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 (ROC) curves were generated based on the results of HE4 detection of exosomes extracted by protein precipitation and the pathological diagnosis results.

[0051] Figure 2 : Receiver operating characteristic (ROC) curve generated by the results of HE4 detection of exosomes extracted by immunomagnetic beads and the pathological diagnosis results.

[0052] Figure 3a and Figure 3b : Receiver operating characteristic (ROC) curves generated by comparing the detection results of exosomal HE4 biomarkers with combined biomarkers and pathological diagnosis results.

[0053] Figure 4a and Figure 4b : Receiver operating characteristic (ROC) curve generated by the detection results of the exosomal HE4 biomarker combination and the pathological diagnosis results.

[0054] Figure 5a and Figure 5b : Receiver operating characteristic (ROC) curve generated by serum CEA test results and pathological diagnosis results. DETAILED DESCRIPTION

[0055] The exosomal HE4 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 simpler to operate, and has lower detection costs. In addition, the marker for diagnosing ovarian cancer of the present invention is an exosomal protein. The application examples of the exosomal biomarker HE4 of the present invention in the assessment of ovarian malignant tumors are as follows. Unless otherwise specified, the reagents used in the examples are all commercially available reagents, among which exosomal protein extraction is performed using exosomal protein extraction, CA125, HE4, and C5a protein detection is performed using (Shanghai Sili Di Biomedical Technology Co., Ltd., Article No.: National Medical Device Registration No. 20243400138), and serum CEA detection is performed using the carcinoembryonic antigen (CEA) detection kit (chemiluminescence method) (Shanghai Medical Device Registration No. 20232400201) produced by Shanghai Sili Di Biomedical Technology Co., Ltd.

[0056] Example 1 Application of an exosomal HE4 biomarker in the assessment of ovarian malignant tumors:

[0057] S1 Ovarian Tumor Sample Collection:

[0058] A total of 185 serum samples were collected from patients with adnexal masses who underwent surgical treatment. Among them, 117 patients with epithelial ovarian malignant tumors diagnosed by postoperative pathology served as the positive group, and 68 patients with benign masses served as the negative control group.

[0059] 185 patients with adnexal masses undergoing surgical treatment were screened for inclusion. The criteria were: ≥18 years of age, adnexal masses requiring surgery, no history of malignancy, no history of chemoradiation or radiotherapy, and no pregnancy. All samples were collected preoperatively.

[0060] Extraction of HE4 protein from S2 exosomes by precipitation method:

[0061] First, transfer 300 μL of sample to a centrifuge tube and add 75 μL of the exosome precipitation reagent from the exosome protein extraction and purification kit. Place the tube on a shaker to mix the sample and precipitation reagent thoroughly. Once mixed, let it sit at room temperature for 30 minutes. Then, place the tube in a centrifuge and centrifuge at 4700 × g for 10 minutes at 4°C. Remove the supernatant. The pellet at the bottom of the tube represents the extracted protein bound to, encapsulated with, or bound to the phospholipid bimolecule. Add 300 μL of the exosome lysis reagent from the exosome protein extraction and purification kit to the pellet and shake on a shaker for at least 30 minutes. Filter the thoroughly mixed solution. The filtrate can be used for subsequent protein analysis.

[0062] S3 exosome HE4 protein detection:

[0063] Take 30uL of the filtered exosome sample and detect it on a fully automatic chemiluminescence instrument using the double antibody sandwich method.

[0064] The test results are shown below.

[0065] S4: Results Analysis

[0066] The test results and pathological diagnosis results are used to generate a receiver operating characteristic (ROC) curve, such as Figure 1 shown.

[0067] Based on the generated curve, the optimal cutoff value was selected as 3.81 U / mL, at which the area under the receiver operating characteristic (ROC) curve (AUC) reached a maximum of 0.926 (95% confidence interval: 0.891-0.962, P < 0.0005). An AUC greater than 0.9 indicates high diagnostic accuracy, and this cutoff value is meaningful. At this cutoff value, the sensitivity of exosomal HE4 concentration for ovarian cancer diagnosis was 82.1% and the specificity was 91.17%.

[0068] Example 2 Application of an exosomal HE4 biomarker in the assessment of ovarian malignant tumors:

[0069] S1 Ovarian Tumor Sample Collection:

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

[0071] S2 exosome HE4 protein immunomagnetic bead method (Exosome extraction and nucleic acid purification reagent (magnetic bead method) (Shanghai Silidi Biomedical Technology Co., Ltd., catalog number: Huminjibei 20210060)) extraction:

[0072] The sample was centrifuged at 12,000 g for 10 minutes, and the supernatant was collected. 1 mg of CD63 and CD9 immunomagnetic beads was added to 1 mL of sample, and the mixture was incubated at 37°C with shaking for 30 minutes. After magnetic separation, the supernatant was discarded and washed once with 1 mL of 10 mM PBS. After magnetic separation again, the immunomagnetic beads were resuspended in 200 μL of 10 mM PBS and 10 μL of 2.5 mg / mL trypsin solution was added. The mixture was incubated in a constant temperature water bath at 37°C for 3 minutes. Then, 10 μL of 2.5 mg / mL soybean trypsin inhibitor was added to the mixture and mixed to terminate the digestion. After magnetic separation, the supernatant was collected. This supernatant is the exosome solution eluted from the immunomagnetic beads. A quantitative exosome lysis reagent was then added to the supernatant, and the mixture was shaken on a shaker for at least 30 minutes. The shaken and mixed solution was filtered, and the filtrate was used for subsequent protein analysis.

[0073] S3 exosome HE4 protein detection:

[0074] Take 30uL of the filtered exosome sample and detect it on a fully automatic chemiluminescence instrument using the double antibody sandwich method.

[0075] The test results are shown below.

[0076] S4: Results Analysis

[0077] The test results and pathological diagnosis results are used to generate a receiver operating characteristic (ROC) curve, such as Figure 2 shown.

[0078] Based on the generated curve, the optimal cutoff value was selected as 3.81 U / mL, at which the area under the receiver operating characteristic (ROC) curve (AUC) reached a maximum of 0.919 (95% confidence interval: 0.881-0.957, P < 0.0005). An AUC greater than 0.9 indicates high diagnostic accuracy, and this cutoff value is meaningful. At this cutoff value, the sensitivity of exosomal HE4 concentration for ovarian cancer diagnosis was 82.1% and the specificity was 89.7%. The sensitivity and specificity of exosomal HE4 protein extracted by the immunomagnetic bead method and the precipitation method for ovarian cancer diagnosis were essentially the same.

[0079] Comparative Example 1: Detection Method for Serum HE4 for Diagnosis of Ovarian Cancer

[0080] The serum HE4 protein concentration currently widely used in clinical practice was used to diagnose ovarian cancer, and the analysis results were compared with those in Example 1.

[0081] S1 Ovarian Tumor Sample Collection:

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

[0083] S2 serum HE4 protein detection:

[0084] The HE4 concentration in serum samples was detected using the human epididymis protein 4 detection kit (electrochemiluminescence method, National Medical Device Registration No. 20153403726) from Roche Diagnostics.

[0085] S3: Result Analysis

[0086] Using a serum HE4 cutoff of 92.1 pmol / L for premenopausal women and 121 pmol / L for postmenopausal women, the sensitivity of serum HE4 for diagnosing ovarian cancer in premenopausal women was 41.18%, and the specificity was 48.48%. In postmenopausal women, the sensitivity and specificity of serum HE4 for diagnosing ovarian cancer were both lower than those of exosomal HE4.

[0087] Comparative Example 2: Comparison of exosomal HE4 biomarker and a combination of exosomal CA125, HE4 and C5a biomarkers

[0088] S1 Ovarian Tumor Sample Collection:

[0089] A total of 185 serum samples were collected from patients with adnexal masses who underwent surgical treatment. Among them, 117 patients with epithelial ovarian malignant tumors diagnosed by postoperative pathology served as the positive group, and 68 patients with benign masses served as the negative control group.

[0090] 185 patients with adnexal masses undergoing surgical treatment were screened for inclusion. The criteria were: ≥18 years of age, adnexal masses requiring surgery, no history of malignancy, no history of chemoradiation or radiotherapy, and no pregnancy. All samples were collected preoperatively.

[0091] S2 exosome CA125, HE4 and C5a protein extraction:

[0092] First, place 300 μL of serum sample in a centrifuge tube and add 75 μL of exosome precipitation reagent. Place the tube on a shaker to mix the sample and precipitation reagent thoroughly. Once mixed, let it sit at room temperature for 30 minutes. Then, place the tube in a centrifuge and centrifuge at 4700 × g for 10 minutes at 4°C. Remove the supernatant. The precipitate at the bottom of the tube is the extracted protein bound to, encapsulated by, or linked to the phospholipid bimolecule. Then, add 300 μL of exosome lysis reagent to the precipitate and shake on a shaker for at least 30 minutes. Filter the thoroughly mixed solution. The filtrate can be used for subsequent protein analysis.

[0093] S3 exosome CA125, HE4 and C5a protein detection:

[0094] Take 30uL of the filtered exosome sample and detect it on a fully automatic chemiluminescence instrument using the double antibody sandwich method.

[0095] S4: Results Analysis

[0096] The test results and pathological diagnosis results are used to generate a receiver operating characteristic (ROC) curve, such as Figure 3a 、 Figure 3b shown.

[0097] The generated curve showed that the exosomal HE4 marker combination had a higher area under the receiver operating characteristic (ROC) curve (AUC) of 0.926 (95% confidence interval: 0.891-0.962, P < 0.0005) than the exosomal CA125, HE4, and C5a marker combination. An AUC greater than 0.9 indicates high diagnostic accuracy, and the cutoff value at this point is meaningful.

[0098] Example 3 Combination of exosomal HE biomarkers with other biomarkers

[0099] S1 Ovarian Tumor Sample Collection:

[0100] A total of 185 serum samples were collected from patients with adnexal masses who underwent surgical treatment. Among them, 117 patients with epithelial ovarian malignant tumors diagnosed by postoperative pathology served as the positive group, and 68 patients with benign masses served as the negative control group.

[0101] 185 patients with adnexal masses undergoing surgical treatment were screened for inclusion. The criteria were: ≥18 years of age, adnexal masses requiring surgery, no history of malignancy, no history of chemoradiation or radiotherapy, and no pregnancy. All samples were collected preoperatively.

[0102] S2 exosome HE4 protein extraction:

[0103] First, take 300uL of sample into a centrifuge tube and add 75uL of the exosome precipitation reagent from the exosome protein extraction and purification kit (same as in Example 1). Place the centrifuge tube on a shaker to mix the sample and 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. The precipitate at the bottom of the centrifuge tube is the extracted protein connected, chimeric, and coated with the phospholipid bimolecule. Then, add 300uL 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 evenly mixed solution by suction. The filtrate can be used for subsequent protein detection.

[0104] S3 exosome HE4 protein detection:

[0105] Take 30uL of the filtered exosome sample and detect it on a fully automatic chemiluminescence instrument.

[0106] S4 Serum CEA protein test:

[0107] The carcinoembryonic antigen (CEA) concentration in serum samples was detected using the carcinoembryonic antigen (CEA) detection kit (chemiluminescence method) (Shanghai Medical Device Registration No. 20232400201) produced by Shanghai Silidi Biomedical Technology Co., Ltd.

[0108] S5: Results Analysis

[0109] The test results and pathological diagnosis results are used to generate a receiver operating characteristic (ROC) curve as follows Figure 4a As shown (where the diagonal segments generated by the results are shown).

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

[0111] The generated curve showed that the area under the receiver operating characteristic (ROC) curve (AUC) for the combination of exosomal HE4 and serum CEA was significantly higher than that for the combination of exosomal CA125, HE4, and C5a, at 0.916 (95% confidence interval: 0.878-0.955, P < 0.0005). An AUC greater than 0.9 indicates high diagnostic accuracy, and the cutoff value at this point is meaningful.

[0112] Comparative Example 3: Detection Method for Diagnosing Ovarian Cancer Using Serum CEA

[0113] S1 Ovarian Tumor Sample Collection:

[0114] A total of 185 serum samples were collected from patients with adnexal masses who underwent surgical treatment. Among them, 117 patients with epithelial ovarian malignant tumors diagnosed by postoperative pathology served as the positive group, and 68 patients with benign masses served as the negative control group.

[0115] 185 patients with adnexal masses undergoing surgical treatment were screened for inclusion. The criteria were: ≥18 years of age, adnexal masses requiring surgery, no history of malignancy, no history of chemoradiation or radiotherapy, and no pregnancy. All samples were collected preoperatively.

[0116] S2 Serum CEA protein test:

[0117] The carcinoembryonic antigen (CEA) concentration in serum samples was detected using the carcinoembryonic antigen (CEA) detection kit (chemiluminescence method) (Shanghai Medical Device Registration No. 20232400201) produced by Shanghai Silidi Biomedical Technology Co., Ltd.

[0118] S3: Result Analysis

[0119] The test results and pathological diagnosis results are used to generate a receiver operating characteristic (ROC) curve, such as Figure 5a As shown (where the diagonal segments generated by the results are shown).

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

[0121] According to the generated curve, the area under the ROC curve (AUC) of serum CEA marker was 0.532 (95% confidence interval: 0.441-0.622, P < 0.0005). AUC in the range of 0.5-0.7 has a lower accuracy.

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 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 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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