Cancer diagnosis

Abstract

The present disclosure relates to the field of cancer. More specifically, the present invention relates to methods of diagnosing and treating cancer, including determining its cancer type. These methods involve detecting markers in an exosome sample of a subject.

Description

[0001] Related applications

[0002] This application claims priority to Australian Patent Application No. 2019904005, filed on 24 October 2019, the contents and elements of which are incorporated herein by reference for all purposes. Technical Field

[0003] This application relates to cancer. More specifically, the present invention relates to methods for diagnosing cancer, including determining the type of cancer. Background Technology

[0004] Despite advances in screening and treatment, the global cancer burden continues to rise steadily, with one in three men and one in four women developing cancer in their lifetime. Furthermore, one in eight men and one in eleven women will die from incurable cancer progression, making cancer one of the leading causes of death worldwide. Significant unmet clinical needs exist for identifying patients at early stages and developing new therapies to reduce cancer mortality. Early detection of cancer is crucial because most localized cancers can be cured with surgery alone. Therefore, improved methods for diagnosing cancer in patients via minimally invasive sampling, such as blood tests, are needed. Cancer blood tests should be highly sensitive and specific, and can be implemented as routine population screening to avoid excessive false positives and false negatives. Summary of the Invention

[0005] This invention broadly relates to determining the expression levels of one or more exosomal proteins in a subject as diagnostic markers for cancer, including specific cancer types. In some aspects, the invention also broadly relates to prognostic methods and cancer therapies that utilize such exosomal proteins to inform treatment selection and / or decision-making.

[0006] In a first aspect, the present invention provides a method for diagnosing cancer or cancer recurrence in a subject, the method comprising the step of determining the expression level of one or more biomarkers in an exosome sample of the subject, wherein the biomarkers are selected from the group consisting of: apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentraxin 3, testican-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, disaccharide proteoglycans, and any combination thereof, and the expression level of one or more biomarkers indicates or is associated with the diagnosis or recurrence of cancer.

[0007] In some implementations, the method of this aspect also includes a step of determining the type of cancer in a subject diagnosed with cancer.

[0008] In some implementations, a relatively increased expression level of one or more biomarkers is used to diagnose cancer or cancer recurrence in a subject.

[0009] In a second aspect, the present invention relates to a method for determining the type of cancer in a subject with cancer, the method comprising the step of determining the expression level of one or more biomarkers in an exosome sample of the subject, wherein the biomarkers are selected from the group consisting of: apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentraxin 3, testican-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, disaccharide proteoglycan, and any combination thereof, and the expression level of one or more biomarkers indicates or is associated with the type of cancer.

[0010] For the methods of the first and second aspects, the one or more biomarkers are suitably selected from the group consisting of: multifunctional protein core protein, nestin-1, pentamericin 3, platelet-reactive protein-1, and any combination thereof. In one specific embodiment, the one or more biomarkers include multifunctional protein core protein, nestin-1, pentamericin 3, and platelet-reactive protein-1.

[0011] In a third aspect, the present invention relates to a method for determining the invasiveness of cancer in a subject, the method comprising the step of determining the expression level of one or more biomarkers in an exosome sample of the subject, wherein the biomarkers are selected from the group consisting of: apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentamin 3, testis proteoglycan-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, disaccharide proteoglycan, and any combination thereof, and the expression level of one or more biomarkers indicates or is associated with the level of invasiveness of cancer.

[0012] In a fourth aspect, the present invention provides a method for determining cancer prognosis in a subject, the method comprising the step of determining the expression level of one or more biomarkers in an exosome sample of the subject, wherein the biomarkers are selected from the group consisting of: apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentamin 3, testis proteoglycan-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, disaccharide proteoglycans, and any combination thereof, and the expression level of one or more biomarkers indicates a poor or good prognosis of the cancer or is associated with a poor or good prognosis of the cancer.

[0013] In specific embodiments of the above two aspects, a relatively decreased expression level of one or more biomarkers indicates a better prognosis and / or less aggressive cancer or is associated with a better prognosis and / or less aggressive cancer; and / or a relatively increased expression level of one or more biomarkers indicates a poorer prognosis and / or highly aggressive cancer or is associated with a poorer prognosis and / or highly aggressive cancer.

[0014] In some embodiments of the foregoing, the method further includes the step of diagnosing a subject who has: (i) highly aggressive cancer or less aggressive cancer; and / or (ii) a poor or good prognosis.

[0015] In a fifth aspect, the present invention relates to a method for predicting and / or determining the responsiveness of a subject to anticancer therapy, the method comprising the step of determining the expression level of one or more biomarkers in an exosome sample of the subject, wherein the biomarkers are selected from the group consisting of: apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentamin 3, testis proteoglycan-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, disaccharide proteoglycans, and any combination thereof, and the altered or regulated expression level of one or more biomarkers indicates or is associated with a relative increase or decrease in responsiveness of the cancer to anticancer therapy.

[0016] The methods described above appropriately include further steps of treating the subject's cancer.

[0017] In a sixth aspect, the present invention relates to a method for treating cancer in a subject, the method comprising the step of determining the expression level of one or more biomarkers in an exosome sample of the subject, wherein the biomarkers are selected from the group consisting of: apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentamin 3, testis proteoglycan-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, disaccharide proteoglycans, and any combination thereof, and based on the determination made, initiating, continuing, modifying or stopping anticancer treatment.

[0018] In some implementations, the methods of the third, fourth, fifth, and sixth aspects include the further step of determining the type of cancer in subjects diagnosed with cancer.

[0019] Regarding the fifth and sixth aspects, anticancer treatment appropriately includes administering a therapeutically effective amount of an anticancer agent to the subject, said anticancer agent reducing the expression and / or activity of one or more biomarkers.

[0020] For the fifth and sixth aspects, the method may include the further step of administering a therapeutically effective amount of anticancer treatment or anticancer agent to the subject.

[0021] In an implementation of the above-mentioned aspect, the method further includes the step of obtaining an exosome sample from the subject.

[0022] In a specific embodiment of the above aspects, the method further includes comparing the expression levels of one or more markers in an exosome sample with the reference exosome expression levels of the corresponding one or more markers.

[0023] Suitablely, the cancers and / or cancer types mentioned above are selected from the group consisting of: lung cancer, such as NSCLC and SCLC, breast cancer, colorectal cancer, prostate cancer, stomach cancer, skin cancer, such as melanoma, brain cancer, such as glioblastoma multiforme (GBM), ovarian cancer, esophageal cancer, and any combination thereof.

[0024] In a seventh aspect, the present invention relates to a method for identifying or generating a reagent for treating cancer in a subject, the method comprising the following steps:

[0025] (a) Contacting cells expressing one or more biomarkers with a candidate reagent, said biomarkers being selected from the group consisting of: apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentamin 3, testis proteoglycan-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, disaccharide proteoglycans, and any combination thereof; and

[0026] (b) Determine whether the candidate reagent modulates the expression and / or activity of one or more biomarkers.

[0027] In some embodiments, the candidate reagent at least partially reduces, eliminates, blocks, or inhibits the expression and / or activity of the biomarker.

[0028] Suitablely, for the methods of the third, fourth, fifth, sixth and seventh aspects, one or more markers are selected from the group consisting of: multifunctional protein core protein, nestin-1, pentamericin 3, platelet-reactive protein-1 and any combination thereof.

[0029] In some embodiments of the third, fourth, fifth, sixth, and seventh aspects, the method further includes the step of determining the expression level of one or more other markers, such as platelet-reactive protein-1, in the exosome sample of the subject.

[0030] In an eighth aspect, the present invention provides a reagent identified or generated by the method of the seventh aspect for use in the method according to the fifth or sixth aspect.

[0031] Appropriately, the subjects in the above-mentioned aspects are mammals, preferably humans.

[0032] In another aspect, the present invention provides a composition comprising an exosome sample from a subject who has or is suspected of having cancer, and a reagent for determining the expression level of one or more of VCAN, NID1, PTX3, and THBS1.

[0033] In some embodiments, the exosome sample contains reagents in a single composition for determining the levels of VCAN, NID1, PTX3, and THBS1. In some alternative embodiments, the exosome sample contains reagents in a separate composition for determining the levels of VCAN, NID1, PTX3, and THBS1.

[0034] In another aspect, a diagnostic kit or testing device comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more specific binding members and one or more reagents for detecting one or more specific binding members, or one or more reagents for detecting and / or quantifying complexes formed by said specific binding members and said biomarkers, each of said specific binding members selectively binding to biomarkers selected from the group consisting of: APOE, PRSS23, VCAN, HAPLN3, COL4A1, NID1, CTGF, COL4A2, CPD, CCBE1, PTX3, SPOCK1, AIMP1, THBS1, and BGN.

[0035] In some implementations, the diagnostic kit or testing device is used in methods for diagnosing cancer or cancer recurrence in a subject, or in methods for determining the type of cancer in a subject.

[0036] In some implementations, the markers include VCAN, NID1, PTX3, and THBS1.

[0037] In another aspect, the present invention provides the use of one or more of the following as biomarkers for determining whether a subject is sensitive to chemotherapy: APOE, PRSS23, VCAN, HAPLN3, COL4A1, NID1, CTGF, COL4A2, CPD, CCBE1, PTX3, SPOCK1, AIMP1, THBS1, and BGN, optionally wherein the use is as a biomarker in any of the methods described above or elsewhere herein.

[0038] Unless the context requires otherwise, the term inclusion (“comprise”, “comprises”, and “comprising”) or similar terms are intended to indicate non-exclusive inclusion, such that the listed elements or features do not include only those that are stated or listed, but may include other elements or features that are not listed or stated.

[0039] The indefinite articles “a” and “an” are used here to refer to or include singular or plural elements or features, and should not be considered as meaning or defining “a” or “single” element or feature. For example, “an” cell includes one cell, one or more cells, or multiple cells. Attached Figure Description

[0040] Figure 1 ： Oncogenic-induced alterations in the protein composition of cell-derived exosomes. (A) Morphology of isolated exosomes was assessed using transmission electron microscopy. Images of normal and transformed HBEC-derived exosomes (size bar 200 nm). (B) Nanoparticle analysis using tunable resistive pulse sensing (TRPS) of exosomes isolated from HBECs showed that most exosomes were between 30 and 150 nm in size, and that this transformation did not lead to increased exosome secretion. (C) Western blot analysis of exosomes from HBECs showed the presence of exosomal proteins HSP70 and CD63, but the absence of the cellular marker calcinin. (D) Quantitative mass spectrometry identified 15 proteins that were significantly upregulated on the extracellular surface of exosomes derived from transformed HBECs (FDR < 0.02). (E) Mass spectrometry results were confirmed by ELISA of normal and transformed HBECs using THBS1, NID1, PTX3, and VCAN. (F) Compared with the expression levels of normal HBEC exosomes, exosomes from 22 cell lines including NSCLC, GBM, CRC, BCa, PCa, MEL, ECa, and OVA showed significantly increased expression of THBS1, NID1, PTX3, and VCAN.

[0041] Figure 2 ： Oncogenic exosome markers were used to diagnose cancer in patient plasma. (A) Compared with healthy controls, cancer patients showed increased expression levels of THBS1, NID1, PTX3, and VCAN. (B) Logistic regression demonstrated the excellent diagnostic capability of the 4-protein exosome group, with an AUC of 0.96. (C) The sensitivity of the diagnostic exosome markers for each cancer was assessed at a fixed specificity of 95%. Error bars represent 95% confidence intervals.

[0042] Figure 3 ： The individual diagnostic capabilities of (A) THBS1, (B) NID1, (C) PTX3, and (D) VCAN were assessed by ELISA, demonstrating a range of diagnostic capabilities for each exosome protein in non-small cell lung cancer, glioblastoma, colorectal cancer, prostate cancer, melanoma, gastric cancer, esophageal cancer, and small cell lung cancer, as assessed by recipient operating characteristic (ROC) curves.

[0043] Figure 4 ：Sensitivity of diagnostic exosome markers for tumor staging. A. In NSCLC, esophageal cancer, and gastric cancer, diagnostic exosome markers can detect early-stage I patients and advanced-stage II-IV patients with 95% specificity. Error bars represent 95% confidence intervals.

[0044] Figure 5 ： Machine learning can help identify cancer types. The percentage of patients correctly or incorrectly identified with their cancer type suggests that exosome labeling can help identify the type of cancer present. Detailed Implementation

[0045] This invention is based, at least in part, on the surprising discovery that upregulated exosomal proteins from normal human bronchial epithelial cells (HBECs) transformed with tumorigenic mutations, identified in vitro, are accurate diagnostic biomarkers for a variety of cancers. By extension, these exosomal biomarkers can also serve as biomarkers for cancer progression and invasiveness, as well as for patient responses to anticancer therapies.

[0046] In a broader sense, this invention relates to identifying or measuring one or more markers, such as..., in a subject's exosome sample. Figure 1 The expression levels provided in the text are methods for diagnosing or detecting cancer, including cancer recurrence, in subjects.

[0047] Therefore, in one aspect, the present invention provides a method for diagnosing cancer or cancer recurrence in a subject, the method comprising the step of determining the expression level of one or more biomarkers in an exosome sample of the subject, wherein the biomarkers are selected from the group consisting of: apolipoprotein E (APOE; Uniprot accession number: P02649), serine protease 23 (PRSS23; Uniprot accession number: O95084), multifunctional protein core protein (VCAN; Uniprot accession number: P13611), hyaluronic acid and proteoglycan connexin 3 (HAPLN3; Uniprot accession number: Q96S86), type IV collagen α1 chain (COL4A1; Uniprot accession number: P02462), nestin-1 (NID1; Uniprot accession number: P14543), connective tissue growth factor (CTGF; Uniprot accession number: P29279), type IV collagen The expression levels of one or more of the following biomarkers indicate or are associated with the diagnosis or recurrence of cancer: α2 chain (COL4A2; Uniprot accession number: P08572), carboxypeptidase D (CPD; Uniprot accession number: O75976), protein 1 containing collagen and calcium-binding EGF domains (CCBE1; Uniprot accession number: Q6UXH8), pentamericin 3 (PTX3; Uniprot accession number: P26022), testis proteoglycan-1 (SPOCK1; Uniprot accession number: Q08629), multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex (AIMP1; Uniprot accession number: Q12904), platelet-reactive protein-1 (THBS1; Uniprot accession number: P07996), disaccharide proteoglycan (BGN; Uniprot accession number: P21810), and any combination thereof. The proteins defined by these expression numbers are typically those with human wild-type amino acid sequences; however, the genes or proteins mentioned may include sequences from other mammals (e.g., mammalian homologs).

[0048] In some implementations, the method of this aspect also includes a step of determining the type of cancer in a subject diagnosed with cancer.

[0049] In a related aspect, the present invention relates to a method for determining or diagnosing cancer type in a subject with cancer, the method comprising the step of determining the expression level of one or more biomarkers in an exosome sample of the subject, wherein the biomarkers are selected from the group consisting of: apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentamin 3, testis proteoglycan-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, disaccharide proteoglycans, and any combination thereof, and the expression level of one or more biomarkers indicates or is associated with cancer type.

[0050] The terms "diagnosis," "diagnosing," and "diagnostic" refer to methods by which a technician assesses and / or determines whether a patient has a given disease or condition, such as cancer or a specific type of cancer. Skilled technicians often make diagnoses based on one or more diagnostic indicators (e.g., exosome markers), the presence, absence, amount, or change in amount of which can indicate the presence, severity, or absence of a condition. It should also be understood that the term "diagnosis" does not refer to the ability to determine with 100% accuracy whether a particular disease, such as cancer, is present, or even to the ability to determine that a given course or outcome is more likely to occur. Rather, those skilled in the art will understand that the term "diagnosis" means an increased likelihood in a subject of the presence of a disease, symptom, or condition, such as cancer or a specific type of cancer.

[0051] Therefore, in a particular implementation, a positive diagnosis of cancer or cancer type indicates that the chance or probability of having cancer or cancer type in the subject is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (or any range thereof). The term "about" in this context means + / - 2%.

[0052] As used herein, the terms “cancer,” “tumor,” “malignant,” and “malignant tumor” refer to a disease or condition, or cells or tissues associated with a disease or condition, characterized by aberrant or abnormal cell proliferation, differentiation, and / or migration, often accompanied by aberrant or abnormal molecular phenotypes, including one or more gene mutations or other genetic changes associated with tumorigenesis, tumor marker expression, loss of expression or activity of tumor suppressor factors, and / or aberrant or abnormal expression of cell surface markers.

[0053] Cancer can include any invasive or potentially invasive cancer, tumor, or other malignant tumor, such as those listed in the NCI Cancer Index at http: / / www.cancer.gov / cancertopics / alphalist, including all major forms of cancer such as sarcoma, carcinoma, lymphoma, leukemia, and germ cell tumor, but is not limited to these. These can include breast cancer, lung cancer (including lung adenocarcinoma and mesothelioma), reproductive system cancers (including ovarian cancer, cervical cancer, uterine cancer, testicular cancer, and prostate cancer), brain and nervous system cancers, head and neck cancers, gastrointestinal cancers including colon cancer, colorectal cancer, esophageal cancer, and stomach cancer, liver cancer, bladder cancer, kidney cancer, skin cancers such as melanoma and skin cancer, blood cell cancers including lymphoma and myeloma, endocrine system cancers such as pancreatic cancer, adrenal cancer, and pituitary cancer, and musculoskeletal cancers including bone cancer and soft tissue cancer, but are not limited to these.

[0054] In a particular implementation, the cancers and / or cancer types described herein are selected from the group consisting of: lung cancer, such as non-small cell carcinoma (NSCLC) (i.e., squamous cell carcinoma, adenocarcinoma, and large cell carcinoma), small cell carcinoma (SCLC) and mesothelioma, breast cancer, colorectal cancer, prostate cancer, gastric cancer, skin cancer such as melanoma, brain cancer such as glioblastoma multiforme (GBM), ovarian cancer, esophageal cancer, and any combination thereof.

[0055] As used generally herein, the terms “recurrence” and “cancer recurrence” refer to the reappearance of signs and symptoms of cancer after a period of improvement or remission. In some implementations, this recurrence occurs after a period when the cancer is undetectable, after at least partial surgical removal, or after therapeutic treatment has suppressed cancer growth. Cancer can recur or return in the same location as the original (primary) tumor, or, for example, in other parts of the body via metastatic recurrence.

[0056] As used in this article, "metastasis" or "metastatic" refers to the migration or metastasis of malignant tumor cells or growths through the circulatory or lymphatic system or through natural body cavities, typically from the primary lesion of the tumor, cancer, or growth to distant sites of the body, and the subsequent development of one or more secondary tumors or colonies at one or more new locations. "Metastasis" refers to secondary tumors or colonies resulting from metastasis, including micrometastases and regional metastases, including lymph node and distant metastases.

[0057] In a particular implementation, the methods of this aspect can be used to diagnose any minimal residual disease of cancer. In this regard, the term "minimal residual disease" refers to a small number of cancer cells remaining in a subject during or after treatment, when the subject is in remission and generally does not exhibit symptoms or signs of cancer.

[0058] As used in this article, the term “cancer type” refers to a type of cancer that is determined by the type of tissue from which the cancer originated (histological type), or the primary site, or the body site where the cancer first formed, or the type of cells from which the cancer originated, and the appearance of the cancer cells.

[0059] Interestingly, the applicant has determined that the expression profiles or identifiers of one or more exosome biomarkers described herein can be used not only to diagnose or detect cancer in subjects, but also to classify or identify specific types and / or subtypes of cancer once diagnosed. Therefore, clinicians can subsequently use these exosome biomarkers to provide indications such as cancer aggressiveness, survival prognosis, treatment options, and response to treatment, which are specific to the particular type of cancer diagnosed in the subjects.

[0060] As used in this article, the term "cancer subtype" refers to a secondary classification of cancer types, belonging to the category of cancer. It can be referred to as the molecular classification of cancer. In particular, cancer subtypes can be associated with molecular alterations, cancer survival, distinct clinical and pathological features, specific gene expression markers, and dysregulated signaling pathways.

[0061] Determining the type of cancer may include one or more of the following steps:

[0062] (a) Calculate or obtain the expression profile or identifier of one or more exosome biomarkers described herein from biological samples such as exosome samples from subjects;

[0063] (b) Compare the expression profiles of biological samples from the subject with one or more reference expression profiles corresponding to one or more tested cancer types or reference cancer types; and

[0064] (c) Assigning the expression profile of a biological sample to a cancer type, wherein the cancer type is identified as the test cancer type corresponding to the closest or most recent test cancer type in the comparison reference expression profile compared to the expression profile of the subject's biological sample.

[0065] In some implementations, the cancer diagnosis or cancer type is used at least in part to determine whether a subject will benefit from cancer treatment. For example, a patient diagnosed with a less aggressive cancer or cancer type is less likely to suffer rapid local progression and / or metastasis of the cancer and may be exempt from more aggressive monitoring and / or treatment.

[0066] In another implementation, a treatment strategy is developed for the subject using at least part of the cancer diagnosis and / or cancer type. Therefore, in a particular implementation, the diagnostic methods described above are combined with an appropriate treatment plan for the cancer and / or cancer type diagnosed in the subject.

[0067] Technicians should understand that exosomes are small (i.e., typically 30-150 nm) membrane vesicles derived from endocytosis within the cell. They may contain lipids, nucleic acids, and proteins and are released into the extracellular environment after fusing with the plasma membrane. Typically, exosomes are characterized by the presence of marker proteins, including CD63, CD9, HSP70, Flotillin-1, and TSG101, as well as their morphology and size.

[0068] According to the method of the present invention, exosome samples containing one or more exosomes can comprise or be obtained from most biological fluids, including but not limited to blood, serum, plasma, ascites, cystic fluid, pleural fluid, peritoneal fluid, cerebrospinal fluid, tears, urine, saliva, sputum, nipple aspiration fluid, lymph, respiratory tract, intestinal and genitourinary tract fluids, breast milk, intra-organ system fluids, or combinations thereof. For this purpose, exosome samples, such as those provided above, can be isolated or purified from biological fluids or samples to remove contaminating proteins, lipoproteins, etc.

[0069] Therefore, exosomes or exosome samples can be separated by any method known in the art, such as, but not limited to, ultracentrifugation, size exclusion chromatography, exosome precipitation (e.g., ExoQuick from System Biosciences), affinity-based exosome capture (e.g., affinity purification using antibodies against CD63, CD81, CD82, CD9, Alix, annexin, EpCAM, and Rab5), and any combination thereof.

[0070] Suitablely, one or more exosomal markers of apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentamin 3, testis proteoglycan-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, and / or disaccharide proteoglycans may be used as a single biomarker, or as any combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of one or more biomarkers (e.g., as an expression profile or marker).

[0071] In one specific embodiment, the one or more biomarkers are suitably selected from the group consisting of: multifunctional protein core protein, nestin-1, pentamericin 3, platelet-reactive protein-1, and any combination thereof. Therefore, in one specific embodiment, the one or more biomarkers include multifunctional protein core protein, nestin-1, pentamericin 3, and platelet-reactive protein-1.

[0072] In a particular implementation, the method described above includes the further step of determining the expression level of one or more additional biomarkers or biomarkers in a subject's biological sample, such as a blood sample.

[0073] Envisioning additional biomarkers could be any biomarkers known in the art that can be used to diagnose or detect cancer in a subject and / or determine the type of cancer. For example, additional biomarkers could include one or more of the eight circulating protein biomarkers (e.g., cancer antigen 125 (CA-125), carcinoembryonic antigen (CEA), cancer antigen 19-9 (CA19-9), prolactin (PRL), hepatocyte growth factor (HGF), osteopontin (OPN), myeloperoxidase (MPO), and tissue inhibitor of metalloproteinases 1 (TIMP-1)) and / or tumor-specific mutations in circulating DNA as described in the CancerSEEK test (see, for example, Cohen, Science 2018).

[0074] Unless otherwise stated, as used generally herein, one or more such as Figure 1 The expression level of an upregulated exosome marker protein identified in the study can refer to the expression level of the nucleic acid encoding the protein (e.g., RNA, mRNA, and cDNA), the expression level of the protein itself, or both.

[0075] For the purposes of this invention, "isolated" means a substance that has been removed from its natural state or otherwise artificially manipulated. An isolated substance may be substantially or substantially free of the components that normally accompany it in its natural state, or may be manipulated to be in an artificial state together with the components that normally accompany it in its natural state. An isolated substance may be in natural, chemically synthesized, or recombinant form.

[0076] “Protein” means a polymer of amino acids. Amino acids can be natural or non-natural amino acids, D- or L-amino acids, as well known in the art. As those skilled in the art will understand, the term “protein” also includes, within its scope, phosphorylated forms of proteins (i.e., phosphoproteins) and / or glycosylated forms of proteins (i.e., glycoproteins). “Peptide” is a protein having no more than fifty (50) amino acids. “Polypeptide” is a protein having more than fifty (50) amino acids.

[0077] Protein “variants” of one or more markers provided herein are also provided, such as naturally occurring variants (e.g., allelic variants) and orthologs or isotypes, for example... Figure 1Those listed above. Preferably, the protein variant shares at least 70% or 75%, more preferably at least 80% or 85%, or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with one or more markers disclosed herein or known in the art. For this purpose, reference Registry numbers for examples of protein sequences of the cited protein markers, which are well known in the art, are described above and are incorporated herein by reference.

[0078] Protein fragments are also provided, including peptide fragments containing less than 100% of the complete amino acid sequence. In a particular embodiment, the protein fragment may contain, for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, and 1200 consecutive amino acids of the protein.

[0079] As used herein, a "gene" is a nucleic acid, which is a structural genetic unit of the genome and may include, but is not limited to, nucleotide sequences encoding one or more amino acids and one or more non-coding nucleotide sequences, including promoters and other 5' untranslated sequences, introns, polyadenylated sequences and other 3' untranslated sequences. In most cellular organisms, genes are nucleic acids that contain double-stranded DNA.

[0080] As used herein, the term "nucleic acid" refers to single-stranded or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA, and autocatalytic RNA. Nucleic acids can also be DNA-RNA hybrids. Nucleic acids comprise a nucleotide sequence that typically includes nucleotides containing A, G, C, T, or U bases. However, the nucleotide sequence may include, but is not limited to, other bases such as inosine, methylcytosine, methylinosine, methyladenosine, and / or thiouridine.

[0081] It also includes “variant” nucleic acids, which comprise nucleic acids containing naturally occurring nucleotide sequence (e.g., allele) variants and orthologs (e.g., from different species) of nucleic acids respectively encoding one or more markers provided herein. Preferably, the nucleic acid variant shares at least 70% or 75%, more preferably at least 80% or 85%, or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequences disclosed herein.

[0082] This also includes nucleic acid fragments. A “fragment” is a segment, domain, part, or region of a nucleic acid that constitutes less than 100% of the nucleotide sequence. Non-limiting examples are amplification products or primers or probes. In a particular embodiment, the nucleic acid fragment may comprise, for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, and 7500 consecutive nucleotides of the nucleic acid.

[0083] As used herein, a “polynucleotide” is a nucleic acid having eighty (80) or more consecutive nucleotides, while an “oligonucleotide” has fewer than eighty (80) consecutive nucleotides. A “probe” can be a single-stranded or double-stranded oligonucleotide or polynucleotide, for example, appropriately labeled for detecting complementary sequences in Northern or Southern blots. A “primer” is typically a single-stranded oligonucleotide, preferably having 15-50 consecutive nucleotides, capable of annealing to a complementary nucleic acid “template” and extending in a template-dependent manner by the action of a DNA polymerase, such as Taq polymerase, RNA-dependent DNA polymerase, or Sequenase. TM "Template" nucleic acid is the nucleic acid used for nucleic acid amplification.

[0084] As those skilled in the art will understand, the expression levels of one or more biomarkers of genes and / or proteins provided herein may be relatively (i) higher, increased, or elevated; or (ii) lower, decreased, or reduced, when compared to expression levels in control or reference samples or to threshold expression levels. In some embodiments, if the expression level exceeds the mean and / or median expression level of a reference population, it may be classified as higher, increased, or elevated. In some embodiments, if the expression level is lower than the mean and / or median expression level of a reference population, it may be classified as lower, decreased, or reduced. In this regard, the reference population may be a group of subjects who have the same cancer type, subgroup, stage, and / or grade as the mammal from which the expression level is determined. In other embodiments, the reference population may be a group of healthy subjects who have been determined not to have or not have cancer. In yet another embodiment, the control sample is obtained from the subject in question prior to testing the subject in question to diagnose cancer or cancer type.

[0085] As used herein, the terms “higher,” “increased,” or “elevated” refer to an increase in the amount or level of nucleic acids and / or proteins in an exosome sample compared to a control or reference level or amount. The expression levels of one or more markers of nucleic acids and / or proteins can be relative or absolute. In some embodiments, the expression level of one or more biomarkers’ genes and / or proteins is higher, increased, or elevated if the expression level of the corresponding or appropriate gene and / or protein in a control or reference level or amount is about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, or at least about 500%.

[0086] As used herein, the terms “lower,” “reduced,” or “decreased” refer to a decrease in the amount or level of nucleic acids and / or proteins in an exosome sample, for example, compared to a control or reference level or amount. The expression levels of one or more biomarkers of nucleic acids and / or proteins provided herein may be relative or absolute. In some embodiments, the gene and / or protein expression of one or more biomarkers is considered lower, reduced, or decreased if the expression level of the gene and / or protein of one or more biomarkers is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the corresponding gene and / or protein expression level or amount in a control or reference level or amount, or even less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, or 0.0001%.

[0087] The term "control sample" generally refers to a biological sample, such as an exosome sample, from a (healthy) non-disease-free individual who does not have cancer. In some embodiments, the control sample may be from a subject known to be cancer-free or a sample obtained from the subject at an earlier time point. Alternatively, the control sample may be from a subject in cancer remission. Control samples can be pooled, averaged, or individual samples. An internal control is a biomarker from the same biological sample (e.g., an exosome sample) being tested.

[0088] As used herein, gene and / or protein expression levels can be absolute or relative. Therefore, in some embodiments, the gene and / or protein expression levels of one or more biomarkers provided herein are compared to control expression levels, such as the expression levels of one or more “housekeeper” genes and / or proteins in a subject’s exosome sample.

[0089] In another embodiment, the gene and / or protein expression levels of one or more biomarkers are compared to an expression threshold level, such as the gene and / or protein expression level in an exosome sample. The expression threshold level is typically a quantitative level of gene and / or protein expression of one or more biomarkers of the present invention. Generally, gene and / or protein expression levels of one or more biomarkers in an exosome sample above or below the expression threshold level predict a specific disease state or outcome. The nature and numerical value (if any) of the expression threshold level typically vary depending on the chosen method for determining the expression of one or more genes or their products used to determine, for example, a diagnosis of cancer, cancer type, prognosis, and / or response to anticancer therapy in a subject.

[0090] Using any method known in the art for measuring gene or protein expression, such as those described herein, those skilled in the art will be able to determine a threshold level (e.g., a predetermined threshold) of gene and / or protein expression in an exosome sample that can be used to determine, for example, a diagnosis of cancer, cancer type, prognosis, and / or response to anticancer therapies. In some embodiments, the threshold level is the mean and / or median gene and / or protein expression level (median or absolute value) of one or more markers in a reference population, such as the subject whose expression level is determined, having the same cancer type, subgroup, stage, and / or grade. Furthermore, the concept of a threshold level of expression should not be limited to a single value or outcome. In this respect, the threshold level of expression can encompass multiple threshold expression levels, which can represent, for example, a high, moderate, or low probability of cancer metastasis in a subject.

[0091] As used herein, the term "predetermined threshold" refers to a value that is higher or lower than a value indicating the responsiveness of a disease to treatment or the general prognosis of the disease. For example, for the purposes of this invention, a predetermined threshold may represent the level or activity of a protein in a sample from an appropriate control subject, or the expression level of a nucleic acid encoding the protein, such as a subject known to have cancer, or a subject with a predisposition to cancer recurrence, or a median or average of multiple control subjects. Thus, as taught herein, activity or expression levels above or below the threshold indicate the likelihood of the presence of cancer in the subject, or the likelihood of cancer recurrence in the subject, or the likelihood of a tumor response to anticancer therapy. In other examples, a predetermined threshold may represent a value greater than or less than a level determined for a control subject in order to further incorporate confidence that levels or ratios above or below the predetermined threshold indicate the presence of cancer in the subject, or cancer recurrence in the subject, or a tumor response to anticancer therapy. For example, a predetermined threshold may represent the mean or median activity level of a biomarker disclosed herein in a group of control subjects, plus or minus one, two, three, or more standard deviations. Those skilled in the art can readily determine appropriate predetermined thresholds based on the analysis of biological samples from suitable control subjects.

[0092] In some implementations, a relatively increased expression level of one or more biomarkers is used to diagnose cancer or cancer recurrence in a subject. In related implementations, a relatively decreased or relatively unchanged expression level of one or more biomarkers is used to diagnose subjects who do not have cancer or cancer recurrence.

[0093] The terms “determine,” “measure,” “evaluate,” “estimate,” and “determine” are used interchangeably herein and may include any form of measurement known in the art, such as those described below.

[0094] Protein levels of one or more exosomal proteins can be determined, estimated, evaluated, determined, or measured using any technique known in the art, capable of detecting such proteins, whether expressed on the surface or inside exosomes, or proteins isolated, extracted, or otherwise obtained from exosomal samples of a subject. These techniques include, but are not limited to, antibody-based detection using one or more antibodies that bind to the protein, electrophoresis, isoelectric focusing, protein sequencing, chromatography, and mass spectrometry, as well as combinations thereof. Antibody-based detection may include, but is not limited to, flow cytometry using fluorescently labeled antibodies, ELISA, Western blotting, immunoprecipitation, radioimmunoassay (RIA), and immunocytochemistry using, but are not limited to, these techniques.

[0095] The corresponding nucleic acids, such as RNA, mRNA, and cDNA, of one or more biomarkers provided herein can be identified, estimated, evaluated, determined, or measured using any techniques known in the art. These can include techniques such as nucleic acid sequence amplification, nucleic acid hybridization, nucleotide sequencing, mass spectrometry, and any combination of these techniques.

[0096] Nucleic acid amplification techniques typically involve annealing one or more primers to a "template" nucleotide sequence under appropriate conditions and using a polymerase to synthesize a complementary nucleotide sequence to the target, thereby "amplifying" the target nucleotide sequence. Nucleic acid amplification techniques are well known to those skilled in the art and include, but are not limited to, polymerase chain reaction (PCR); strand displacement amplification (SDA); rolling circle replication (RCR); sequence-based amplification (NASBA); Q-β replicase amplification; helicase-dependent amplification (HAD); loop-mediated isothermal amplification (LAMP); nicking enzyme amplification reaction (NEAR); and recombinase polymerase amplification (RPA), but are not limited thereto. As used generally herein, "amplification product" refers to a nucleic acid product produced by nucleic acid amplification techniques.

[0097] PCR includes quantitative and semi-quantitative PCR, real-time PCR, allele-specific PCR, methylation-specific PCR, asymmetric PCR, nested PCR, multiplex PCR, falling PCR, digital PCR, and other variations and modifications of "basic" PCR amplification.

[0098] Nucleic acid amplification techniques can be performed using DNA or RNA extracted, isolated, or otherwise obtained from cell or tissue sources. In other embodiments, nucleic acid amplification can be performed directly on appropriately treated cell or tissue samples.

[0099] Nucleic acid hybridization typically involves hybridizing a nucleotide sequence (usually in the form of a probe) with a target nucleotide sequence under appropriate conditions, thereby subsequently detecting the hybridized probe-target nucleotide sequence. Non-limiting examples include, but are not limited to, Northern blotting, slot-blotting, in situ hybridization, and fluorescence resonance energy transfer (FRET) detection. Nucleic acid hybridization can be performed using DNA or RNA extracted, isolated, amplified, or otherwise obtained from cell or tissue sources, or directly on appropriately treated cell or tissue samples.

[0100] It should also be understood that a combination of nucleic acid amplification and nucleic acid hybridization can be used.

[0101] It should be understood that determining the expression of one or more biomarkers provided herein may include determining their nucleic acid levels (e.g., by nucleic acid amplification and / or nucleic acid hybridization) and their protein levels. Therefore, detecting and / or measuring the expression of one or more biomarkers from a subject's exosome sample can be performed by any of those methods described herein or a combination thereof (e.g., measuring mRNA levels or amplified cDNA copies and / or by measuring their protein products), but is not limited thereto.

[0102] In light of the foregoing, it should be further understood that the expression level of one or more markers provided herein can be the absolute or relative amount of an expressed gene or its gene product, including nucleic acids such as RNA, mRNA and cDNA and / or proteins.

[0103] In another aspect, the present invention relates to a method for determining the invasiveness of cancer in a subject, the method comprising the step of determining the expression level of one or more biomarkers in an exosome sample of the subject, wherein the biomarkers are selected from the group consisting of: apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentamin 3, testis proteoglycan-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, disaccharide proteoglycans, and any combination thereof, and the expression level of one or more biomarkers indicates or is associated with the level of invasiveness of cancer.

[0104] In another aspect, the present invention provides a method for determining cancer prognosis in a subject, the method comprising the step of determining the expression level of one or more biomarkers in an exosome sample of the subject, wherein the biomarkers are selected from the group consisting of: apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentamin 3, testis proteoglycan-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, disaccharide proteoglycans, and any combination thereof, and the expression level of one or more biomarkers indicates a poor or good prognosis of the cancer or is associated with a poor or good prognosis of the cancer.

[0105] In some embodiments of the foregoing, the method further includes diagnosing the subject with: (i) highly aggressive cancer or less aggressive cancer; and / or (ii) a poor or good prognosis.

[0106] "Invasiveness" and "aggressiveness" refer to the nature or tendency of cancer to have a relatively poor prognosis due to one or more of a combination of the following characteristics or factors, including, but not limited to: at least partial resistance to therapies available for cancer treatment; invasiveness; metastatic potential; recurrence after treatment; and a low probability of patient survival.

[0107] In certain implementations, the proteins provided herein, such as Figure 1 The proteins provided herein can predict aggressive disease, particularly by shortening the time to pathological recurrence and / or reducing patient survival. In another embodiment, the proteins provided herein, such as... Figure 1 The proteins provided in the text are associated with or indicate metastatic cancer.

[0108] The terms “prognosis” and “outcome” are used herein to include making a prognosis that can provide predictions of clinical outcomes (with or without drug treatment), selection of an appropriate treatment regimen (or effectiveness of treatment), and / or monitoring of current treatment and potential changes in treatment. This may be based, at least in part, on determining the gene and / or protein expression levels of one or more biomarkers by the methods of the present invention, which may be combined with determining the expression levels of additional protein and / or other nucleic acid biomarkers, such as thromboretin-1 or those described above for CancerSEEK. Prognosis may also include the prediction, indication, or expectation of any ongoing or persistent physical or psychological effects of the cancer suffered by the subject after the cancer has been successfully treated or otherwise resolved. In addition, prognosis may include one or more of the following: determining metastatic potential or occurrence, treatment responsiveness, implementation of an appropriate treatment regimen, determining the probability, likelihood, or potential for cancer recurrence after treatment, and predicting resistance to established therapies (e.g., chemotherapy). It should be understood that a positive prognosis typically refers to a beneficial clinical outcome or prospect, such as long-term survival without recurrence of the cancer in the subject, while a negative prognosis typically refers to a negative clinical outcome or prospect, such as cancer recurrence or progression.

[0109] In some embodiments of the methods in the two aforementioned aspects, a relatively decreased or relatively unchanged expression level of one or more biomarkers indicates a better prognosis and / or less aggressive cancer or is associated with a better prognosis and / or less aggressive cancer; and / or a relatively increased expression level of one or more biomarkers indicates a poorer prognosis and / or highly aggressive cancer or is associated with a poorer prognosis and / or highly aggressive cancer.

[0110] In one particular implementation, cancer prognosis or invasiveness is used at least in part to determine the likelihood of cancer metastasis in the subject.

[0111] Suitablely, a relatively decreased or unchanged expression level of one or more biomarkers indicates a reduced likelihood of cancer metastasis or is associated with a reduced likelihood of cancer metastasis; and / or a relatively increased expression level of one or more biomarkers indicates an increased likelihood of cancer metastasis or is associated with an increased likelihood of cancer metastasis.

[0112] In some implementations, cancer prognosis or invasiveness is used at least in part to determine whether the subject will benefit from cancer treatment. For example, patients with a good prognosis and / or low cancer invasiveness are less likely to suffer rapid local progression and / or metastasis of cancer and may be spared more aggressive monitoring and / or treatment.

[0113] In another implementation, cancer prognosis or invasiveness is used at least in part to develop treatment strategies for the subjects.

[0114] In some implementations, cancer prognosis or aggressiveness is used at least in part to determine minimal residual disease, disease progression, or recurrence in subjects.

[0115] In some implementations, cancer prognosis or invasiveness is used at least in part to determine estimated survival time.

[0116] Suitablely, the methods of the foregoing aspects also include diagnosing the subject as having: (i) highly aggressive cancer or less aggressive cancer; and / or (ii) a poor or good prognosis.

[0117] In some embodiments, relatively low expression levels of the genes and / or proteins of one or more biomarkers provided herein indicate or are associated with a relatively increased responsiveness to anticancer therapy in cancer. In alternative embodiments, relatively low expression levels of the genes and / or proteins of one or more biomarkers provided herein indicate or are associated with a relatively decreased responsiveness to anticancer therapy in cancer.

[0118] Appropriately, cancer is the type described above, but not limited to it.

[0119] In another aspect, the present invention relates to a method for predicting and / or determining the responsiveness of cancer to anticancer therapy in a subject, the method comprising the step of determining the expression level of one or more biomarkers in an exosome sample of the subject, wherein the biomarkers are selected from the group consisting of: apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentamin 3, testis proteoglycan-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, disaccharide proteoglycans, and any combination thereof, and the altered or regulated expression level of one or more biomarkers indicates or is associated with a relative increase or decrease in responsiveness of cancer to anticancer therapy.

[0120] As those skilled in the art will understand, when an expression level is higher / increased or lower / decreased compared to a control or reference sample or expression level (e.g., a threshold level), the expression level of a gene or protein can be considered "altered" or "regulated." In some embodiments, an expression level greater than the mean and / or median relative expression level of a reference population can be classified as high, and an expression level lower than the mean and / or median expression level of the reference population can be classified as low. In this regard, the reference population can be a group of subjects who share the same cancer type, subgroup, stage, and / or grade as the mammal from which the expression level is determined. Furthermore, the expression level can be relative or absolute.

[0121] In some embodiments, one or more biomarkers are selected from the group consisting of: multifunctional protein core protein, nestin-1, pentamericin 3, thromboretin-1, and any combination thereof. More specifically, one or more biomarkers suitably include multifunctional protein core protein, nestin-1, and pentamericin 3, and optionally also include the additional biomarker, thromboretin-1.

[0122] In some embodiments, relatively higher expression levels of one or more biomarkers indicate or are associated with a relatively increased responsiveness to anticancer therapy. In alternative embodiments, relatively higher expression levels of one or more biomarkers indicate or are associated with a relatively decreased responsiveness to anticancer therapy.

[0123] In some implementations, the expression level of one or more biomarkers, such as their relatively high expression level, indicates the persistence of cancer (e.g., minimal residual disease), progression or recurrence, or the lack of or reduced responsiveness of cancer to anticancer treatment, or is associated with the persistence of cancer (e.g., minimal residual disease), progression or recurrence, or the lack of or reduced responsiveness of cancer to anticancer treatment.

[0124] In some embodiments, the method of the present invention includes assessing the activity, expression, or level of one or more biomarkers in a subject or in a sample (e.g., an exosome sample) to obtain a sample profile of multiple or more biomarkers; and making predictions based on the sample profile of one or more biomarkers. Optionally, predictions are made by comparing the sample profile with a control profile. For example, suitable control profiles that may be used are: (i) obtained from a control subject population with cancer; or (ii) obtained from a control subject or control subject population with cancer recurrent; (iii) a predetermined profile of “mean, median, or average” or “standard range” values ​​of biomarker expression, activity, or amount obtained from a control subject or control subject population with cancer; (iv) obtained from a control sample having a known value indicating a “mean, median, or average” value of a biomarker in a control subject or control subject population with cancer; (v) a predetermined profile of “threshold” values ​​of biomarker expression, activity, or amount obtained from a control subject or control subject population with cancer; or (vi) obtained from a control sample having a known value indicating a “threshold” value of a biomarker in a control subject or control subject population with cancer.

[0125] Regarding the invention described above, the method suitably includes further steps of treating cancer in a subject.

[0126] Other aspects of the invention relate to treating cancer in a subject.

[0127] In one specific aspect, cancer treatment is conducted in conjunction with determining the expression levels of one or more biomarkers in the exosome samples of the subjects, wherein the biomarkers are selected from the group consisting of: apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentamin 3, testis proteoglycan-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, disaccharide proteoglycans, and any combination thereof, and based on the determination made, cancer treatment is initiated, continued, modified, or stopped.

[0128] In one specific implementation, one or more biomarkers are selected from the group consisting of: multifunctional protein core protein, nestin-1, pentamericin 3, thromboretin-1, and any combination thereof. More specifically, one or more biomarkers suitably include multifunctional protein core protein, nestin-1, and pentamericin 3, and optionally also include the additional biomarker, thromboretin-1.

[0129] In this regard, it should be understood that the methods described herein for predicting and / or determining cancer responsiveness to anticancer agents may further include the step of administering a therapeutically effective amount of an anticancer treatment, such as an anticancer agent, to a mammal. In a preferred embodiment, anticancer treatment is administered when the gene and / or protein expression levels of one or more of the markers described herein indicate or are associated with a relatively increased cancer responsiveness to anticancer agents.

[0130] In other embodiments, anticancer treatment may be modified or discontinued when the gene and / or protein expression levels of one or more of the markers described herein indicate the persistence of cancer (e.g., minimal residual disease), progression or recurrence, or a lack of or reduced responsiveness to anticancer treatment, or when such behavior is associated with the persistence of cancer (e.g., minimal residual disease), progression or recurrence, or a lack of or reduced responsiveness to anticancer treatment.

[0131] Suitablely, the reagent (one or more) is administered to a subject as a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, or excipient. In this respect, any dosage form and route of administration, such as those provided herein, can be used to administer the compositions of the present invention to a subject.

[0132] Cancer treatment may include, but is not limited to, drug therapy (small organic or inorganic molecules, chemotherapy, antibodies, nucleic acids and other biomolecular therapies, radiation therapy, surgery, nutritional therapy, relaxation or meditation therapy, and other natural or holistic therapies. Generally, drugs (such as small organic or inorganic molecules), biomolecules (such as antibodies, inhibitory nucleic acids such as siRNA), or chemotherapeutic agents are referred to herein as “anticancer therapeutic agents” or “anti-cancer agents”.

[0133] Approaches to treating cancer can be preventative, therapeutic, or preventative, and are applicable to the treatment of cancers in mammals, particularly humans. As used herein, “treating,” “treat,” or “treatment” refers to a therapeutic intervention, process, or program that at least improves cancer symptoms after cancer and / or its symptoms have at least begun to occur. As used herein, “preventing,” “prevent,” or “prevention” refers to a therapeutic intervention, process, or program that begins before the onset of cancer and / or cancer symptoms in order to prevent, inhibit, or delay the development or progression of cancer or its symptoms.

[0134] The term "therapeuticly effective amount" describes an amount of a specific agent sufficient to achieve the desired effect in a subject treated with the agent. For example, this could be an amount of chemotherapeutic agent necessary to reduce, improve, and / or prevent cancer or cancer-related diseases, symptoms, or conditions. In some embodiments, a "therapeuticly effective amount" is sufficient to reduce or eliminate symptoms of cancer. In other embodiments, a "therapeuticly effective amount" is an amount sufficient to achieve the desired biological effect, such as an amount that effectively reduces or prevents cancer growth and / or metastasis.

[0135] Ideally, the therapeutically effective amount of an agent is an amount sufficient to induce the desired outcome in a subject without causing substantial cytotoxicity. The effective amount of an agent that can be used to reduce, improve, and / or prevent cancer will depend on the subject being treated, the type and severity of any associated disease, condition, and / or symptom (e.g., the number and location of any associated metastases), and the manner in which the therapeutic composition is administered.

[0136] Suitable, the anticancer therapeutic agent is administered as a pharmaceutical composition to mammals, said pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient.

[0137] "Pharmaceutically acceptable carriers, diluents, or excipients" refers to solid or liquid fillers, diluents, or encapsulating substances that are safe for systemic administration. Depending on the specific route of administration, a variety of carriers well known in the art may be used. These carriers may be selected from the group consisting of: sugars, starches, cellulose and their derivatives, malt, gelatin, talc, calcium sulfate, liposomes and other lipid-based carriers, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsifiers, isotonic saline solutions, and salts such as inorganic acid salts, including hydrochlorides, bromides, and sulfates, organic acids such as acetates, propionates, and malonates, and pyrogen-free water.

[0138] A useful reference describing pharmaceutically acceptable carriers, diluents, and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ USA, 1991), which is incorporated herein by reference.

[0139] The compositions of the present invention can be administered to patients via any safe route of administration. For example, they can be administered orally, rectally, parenterally, sublingually, orally, intravenously, intra-articularly, intramuscularly, intradermally, subcutaneously, by inhalation, intraocularly, intraperitoneally, intraventricularly, or percutaneously. Intramuscular and subcutaneous injections are suitable for administration, for example, of immunotherapeutic compositions, protein vaccines, and nucleic acid vaccines.

[0140] Dosage forms include tablets, dispersants, suspensions, injections, solutions, syrups, lozenges, capsules, suppositories, aerosols, transdermal patches, etc. These dosage forms may also include injection or implantable controlled-release devices or other modified forms of implants specifically designed for this purpose to additionally function in this manner. Controlled release of the therapeutic agent can be achieved, for example, by coating the therapeutic agent with a hydrophobic polymer, including acrylic resins, waxes, higher fatty alcohols, polylactic acid and polyglycolic acid, and certain cellulose derivatives such as hydroxypropyl methylcellulose. Furthermore, controlled release can be achieved by using other polymer matrices, liposomes, and / or microspheres.

[0141] The compositions of the present invention suitable for oral or parenteral administration can be in discrete unit form, such as capsules, sachets, or tablets, each containing a predetermined amount of one or more therapeutic agents of the present invention, as a powder or granules, or as a solution, or as a suspension in an aqueous liquid, non-aqueous liquid, oil-in-water emulsion, or water-in-oil emulsion. Such compositions can be prepared by any pharmaceutical method, but all methods include the step of combining one or more of the pharmaceutical agents as described above with a carrier constituting one or more essential components. Typically, the compositions are prepared by uniformly and tightly mixing the pharmaceutical agents of the present invention with a liquid carrier or a finely divided solid carrier, or both, and then, if desired, shaping the product into a desired appearance.

[0142] The above-described compositions can be administered in a dosage form compatible and in a pharmaceutically effective amount. In the context of this invention, the dose administered to the patient should be sufficient to produce a beneficial response in the patient over an appropriate time period. The amount of the agent (one or more) to be administered may depend on the subject to be treated, including their age, sex, weight, and general health condition, which will depend on the practitioner's judgment.

[0143] In specific implementation schemes, anticancer treatments and / or agents may target the inhibition of one or more biomarkers and / or the reduction of the expression of one or more biomarkers.

[0144] In other implementations, anticancer treatments and / or agents may be targeted at preventing or inhibiting cancer metastasis.

[0145] In alternative embodiments, the anticancer treatment and / or agent may target genes or gene products other than one or more biomarkers of the present invention. For example, the anticancer treatment may target genes or gene products that are known to interact directly or indirectly with one or more biomarkers.

[0146] In specific implementations, the present invention provides “companion diagnostics” for cancer treatment, thereby providing clinicians and others with information on the expression levels of one or more biomarkers of the present invention for the safe and / or effective administration of said cancer treatment.

[0147] Appropriately, cancer is the type described above, but not limited to it.

[0148] In a specific implementation, the method further includes comparing the expression levels of one or more biomarkers in an exosome sample with the corresponding reference or control exosome expression levels of one or more biomarkers, as described above.

[0149] Referring to the foregoing aspects, the method suitably includes the initial step of obtaining an exosome sample from the subject, for example from those biological samples and / or the isolation methods described above.

[0150] In some implementations, the methods described above further include the step of determining the subject's cancer type. In this way, the cancer type can be used to determine, for example, the aggressiveness of the cancer in the subject, prognosis, treatment options, and / or responsiveness to cancer treatment.

[0151] In another aspect, the present invention provides a method for identifying or generating a reagent for treating cancer in a subject, comprising the following steps:

[0152] (a) Contacting cells expressing a biomarker with a candidate reagent, said biomarker being selected from the group consisting of: apolipoprotein E, serine protease 23, multifunctional protein core protein, hyaluronic acid and proteoglycan connexin 3, type IV collagen α1 chain, nestin-1, connective tissue growth factor, type IV collagen α2 chain, carboxypeptidase D, protein 1 containing collagen and calcium-binding EGF domains, pentamin 3, testis proteoglycan-1, multifunctional protein 1 interacting with aminoacyl-tRNA synthetase complex, platelet-reactive protein-1, disaccharide proteoglycans, and any combination thereof; and

[0153] (b) Determine whether the candidate reagent modulates the expression and / or activity of the biomarker.

[0154] In some implementations, the candidate reagent at least partially reduces, eliminates, blocks, or inhibits the expression and / or activity of the biomarker.

[0155] Appropriately, the reagent has or shows little or no significant off-target and / or nonspecific effects.

[0156] Preferably, the reagent is an antibody or a small molecule.

[0157] Appropriately, the markers are selected from the group consisting of: multifunctional core protein, nestin-1, pentamericin-3, platelet-reactive protein-1, and any combination thereof.

[0158] In implementation schemes involving antibody inhibitors, the antibody may be polyclonal or monoclonal, natural or recombinant. Well-known protocols suitable for antibody production, purification, and use can be found, for example, in Chapter 2 of *Coligan et al., CURRENTPROTOCOLS IN IMMUNOLOGY* (John Wiley & Sons NY, 1991–1994) and *Harlow, E. & Lane, D. Antibodies: A Laboratory Manual*, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988, both of which are incorporated herein by reference.

[0159] Typically, the antibodies of this invention bind to or conjugate isolated proteins, fragments, variants, or derivatives of the marker. For example, the antibody may be a polyclonal antibody. Such antibodies can be prepared, for example, by injecting isolated proteins, fragments, variants, or derivatives of the marker protein product into a production species, which may include mice or rabbits, to obtain polyclonal antiserum. Methods for producing polyclonal antibodies are well known to those skilled in the art. Exemplary methods that can be used are described, for example, in *Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY* (ibid.) and *Harlow & Lane, 1988* (ibid.).

[0160] Monoclonal antibodies can be produced using standard methods, such as immortalizing spleens or other antibody-producing cells derived from a producing species that has been inoculated with one or more isolated marker protein products and / or fragments, variants, and / or derivatives thereof. The description is in Milstein's 1975 Nature 256, 495 article, which is incorporated herein by reference or by its most recent modifications, such as those described, for example, in Coligan et al. Current Protocols Inimulogy (ibid.).

[0161] Typically, the inhibitory activity of candidate inhibitor antibodies can be assessed by in vitro and / or in vivo assays that detect or measure the expression level and / or activity of the biomarker protein in the presence of the antibody.

[0162] In some implementations, modulators, such as inhibitors, can be rationally designed. These methods may include structural analysis of markers and the design and / or construction of molecules that bind to, interact with, or otherwise modulate the activity of markers. These methods may specifically include computer-aided three-dimensional modeling of the interactions between candidate modulators and markers.

[0163] In other embodiments, modulators, such as small organic molecule inhibitors, may involve screening large libraries of compounds, numbered in the hundreds of thousands to millions, of candidate inhibitors (chemical compounds, including small synthetic organic molecules or natural products, such as inhibitory peptides or proteins), where the bioactivity of said candidate inhibitors can be screened or tested on any of hundreds of molecular targets to find potential new drugs or lead compounds. Screening methods may include, but are not limited to, computer-based ("in silico") screening and in vitro assays based on high-throughput screening.

[0164] Typically, the active compounds, or "hit" compounds, from this initial screening process are then sequentially tested through a series of additional in vitro and / or in vivo tests to further characterize the active compounds. At each stage, a decreasing number of "successful" compounds are selected for subsequent testing, ultimately resulting in one or more selected drug candidates that continue to be tested in human clinical trials.

[0165] At the clinical level, screening candidate reagents may involve obtaining samples from test subjects before and after exposure to the test compound. The levels of biomarker proteins in the samples, such as exosomes, can then be measured and analyzed to determine whether the levels and / or activities of the biomarker proteins have changed after exposure to the candidate reagent. For example, the levels of protein products in the samples can be determined by mass spectrometry, Western blotting, ELISA, electrochemistry, and / or any other suitable method known to those skilled in the art.

[0166] In this regard, candidate agents identified as capable of reducing, eliminating, blocking, or inhibiting the expression level and / or activity of biomarkers can then be administered to patients with cancer. For example, if increased activity of a biomarker at least partially contributes to cancer progression and / or onset, the administration of candidate agents that inhibit or reduce the activity and / or expression of the biomarker can treat the cancer and / or reduce the risk of cancer.

[0167] In a final aspect, the present invention provides a reagent identified or generated by the foregoing aspects for use in accordance with the methods described herein.

[0168] For the purposes of the foregoing, the term "subject" includes, but is not limited to, mammals, including humans, performing animals (such as horses, camels, and greyhounds), livestock (such as cattle, sheep, and horses), and companion animals (such as cats and dogs). Preferably, the subject is a human.

[0169] All computer programs, algorithms, patents, and scientific literature mentioned in this article are incorporated herein by reference.

[0170] For the purposes of this invention, the database accession numbers or unique identifiers of genes or proteins provided herein, as well as one or more gene and / or protein sequences associated therewith, are incorporated herein by reference.

[0171] To fully understand and practice the preferred embodiments of the present invention, reference is made to the following non-limiting examples.

[0172] Example

[0173] Small extracellular vesicles, called exosomes, have recently been shown to be a potential non-invasive method for identifying cancer. Exosomes are small membrane-bound vesicles (30-150 nm in diameter) released by all cells, including cancer cells. The protein content of exosomes depends on their cellular origin, and it is now suggested that exosomes represent a viable source of material for diagnostic and prognostic purposes. However, there is currently a lack of available evidence for specific biomarkers to differentiate exosomes derived from cancer cells from normal cells. Identifying cancer-specific exosomal biomarkers could allow for the identification of patients with cancer and could potentially lead to improved survival rates.

[0174] Numerous attempts have been made to determine the optimal analysis of human biological fluids for identifying the presence of cancer, including the analysis of tumor-secreting factors and circulating tumor DNA (ctDNA). Because the increased half-life of exosomes and the active processes of exosome secretion provide the sustained presence of cancer antigens or biomarkers, cancer-derived exosomes can serve as better liquid biomarkers compared to ctDNA. In this embodiment, we describe a comprehensive clinical assay for cancer diagnosis utilizing cancer-derived exosomal proteins. This assay exhibits high sensitivity and specificity in clinical presentation, capable of detecting cancer as early as stage 1, thus providing opportunities for early detection and improved survival rates for numerous cancer patients.

[0175] Cell culture

[0176] Cell line identification was performed using short tandem repeat profiles. This was achieved using p53 knockdown and Kras v12 overexpression (30KT). p53 / KRAS Transformed syngeneically immortalized normal human bronchial epithelial cells (HBEC30KT) are a gift from Dr. Jill Larsen. 22,27 HBECs were cultured in serum-free keratinocyte medium (KSFM) supplemented with EGF (5 ng / mL) and bovine pituitary extract (50 mg / L) at 37°C and 5% CO2. All other cell lines were maintained in DMEM or RPMI supplemented with 5% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin and incubated at 37°C with 5% CO2. Cell conditioned medium (CCM) was collected from cells cultured in serum-free medium. The culture was carried out by culturing 100,000 g of HBECs in serum-free medium.avg CCM was collected from HBEC cells in KSFM depleted of bovine exosomes by overnight centrifugation.

[0177] Exosome isolation and analysis

[0178] As mentioned above 8,28 Exosomes were isolated and analyzed. For exosomes used in mass spectrometry analysis, CCM was centrifuged at 300g for 10 min at 4°C and filtered through a 0.22μm filter to remove floating cells and large extracellular vesicles. The clarified CCM was then concentrated to 500μL, coated on a discontinuous iodixanol density gradient, and centrifuged at 100,000g at 4°C. avg Centrifuge for 16 hours. Dilute the fraction containing exosomes to 20 mL in PBS and centrifuge at 100,000 g at 4°C. avg Centrifuge for 2 hours. Resuspend the resulting precipitate in PBS and store at -80°C until use. Similarly, clarify and concentrate all other exosome isolates from in vitro CCM as described above, and then purify using size exclusion chromatography. To isolate exosomes from human plasma, thaw the plasma at room temperature and remove residual platelets and large vesicles by centrifugation at 1,500 g and 10,000 g for 10 min and 20 min, respectively. Coat the prepared plasma onto a size exclusion column and elute with PBS. The micon was then concentrated in a size-exclusion centrifugal filter unit with a nominal molecular weight of kDa and stored at -80°C until use. As previously stated. 8 Exosomes isolated from cell cultures and human plasma were identified by Western blotting, tunable resistive pulse sensing (TRPS), and transmission electron microscopy.

[0179] Antibodies and reagents

[0180] The following antibodies were used for Western blotting: Calnexin (Cell Signaling Technology, 2679S), CD63 (Abcam, ab8219), and HSP70 (Transduction Laboratories, 610608). Horseradish peroxidase (HRP) conjugated secondary antibodies were purchased from Thermo Scientific. THBS1, NID1, and PTX3 ELISA DuoSets were purchased from R&D Systems, and the VCAN ELISA kit was purchased from Novus Biologicals. qEV columns were purchased from Izon and stored at 4°C in PBS (0.1% sodium azide).

[0181] Western blot analysis

[0182] As mentioned above1,2 Western blotting was performed. In short, proteins were separated by SDS-PAGE, transferred to a polyvinylidene fluoride membrane, blocked in PBS-T (0.5% Tween-20) containing 5% skim milk powder, and detected with antibody probes. Protein bands were detected using an enhanced chemiluminescence reagent (Amersham ECL Select).

[0183] Mass spectrometry

[0184] Exosome preparations were reduced by adding 10 mM dithiothreitol (4°C, 1 h; 22°C, 2 h) in the presence of 2% SDS, a protease inhibitor (Sigma-Aldrich, P8340), and 50 mM Tris.HCl at pH 8.8. The samples were then alkylated by adding iodoacetamide to 25 mM (22°C, 1 h) and methanol, and co-precipitated with trypsin overnight at -20°C (enzyme:substrate 1:100). The precipitate was resuspended in 10% acetonitrile and 40 mM ammonium bicarbonate and digested at 37°C for 8 h, followed by further addition of trypsin (enzyme:substrate 1:100) 2 h later.

[0185] The acidified digest (trifluoroacetic acid) was analyzed by LCMS using a NanoAcquity UPLC (Waters) connected before the Elite Orbitrap ETD mass spectrometer (Thermo Fisher Scientific). Two μg of digest was loaded onto a 20 mm x 180 μm Symmetry C18 trap (Waters) and separated on a 200 mm x 75 μm, BEH130 1.7 μm column (Waters) using the following linear gradient (buffer A: 0.1% formic acid aqueous solution; buffer B: acetonitrile solution containing 0.1% formic acid) for 120 min: 2% B to 5% B, 5 min; 30% B, 75 min; 50% B, 10 min; 95% B, 5 min; and held for 6 min before reequilibration in 2% B. The eluent from the column was introduced into the mass spectrometer via a 10 μm P200P-coated silica emitter (New Objective) and a Nanospray-Flex source (Proxeon Biosystems A / S). The source voltage was 1.8 kV, the capillary temperature was 275 °C, and the first 15 positions were acquired using the first 15 positions method. MS was acquired in the 120,000 resolution orbital trap AGC1E6, and MS2 in the ion trap AGC1E4, with a maximum injection time of 50 ms. A lock mass of 445.120024 was used for MS1.

[0186] Using MaxQuant (version 1.4.1.2) 3MaxQuant was used for protein identification and label-free quantification. It was employed to extract peak lists from Xcalibur raw files (Thermo Fisher Scientific, Germany) and included an embedded database search engine, Andromeda. 4 Peptide profiling matching (PSM) was performed. The searched database was the complete Homo sapiens proteome (88,378 canonical sequences downloaded from www.uniprot.org in August 2013). Reverse sequences and the MaxQuant contamination database were also searched. Label-free quantification was performed with the instrument type set to Orbitrap, precursor mass tolerance set to 20 ppm for the first search and 4.5 ppm for the main search, fragment ion mass tolerance set to 0.5 Da, enzyme specificity set to trypsin / P, allowing a maximum of two missed cleavages, carbamoylcysteine ​​specified as a fixed modification, and N-terminal acetylation, asparagine / glutamine deamidation, and methionine oxidation specified as variable modifications. A second peptide search and matching was performed between runs using default settings. For identification, the PSM and protein level FDR were set to 0.01. Default settings were applied to all other parameters as previously stated. 5 Protein inference and label-free quantification are performed using spectral counting (including normalization).

[0187] patient groups

[0188] A retrospective analysis was conducted on patient cohorts for whom plasma / serum samples were collected between 2001 and 2019.

[0189] Statistical analysis

[0190] GraphPad Prism version 6.0, EdgeR version 2.6.10, MedCalc version 16.8.4, and IPA were used for all calculations. An unpaired Student's t-test was used to calculate differences in protein expression values ​​from exosomes. Subcellular localization of proteins was generated via IPA (QIAGENInc). A negative binomial exact test was used to assess mass spectrometry-derived spectral counts, with Benjamini-Hochberg adjustment applied to control for FDR. Differences with p-values ​​less than 0.05 were considered significant (*p<0.05, **p<0.01, ***p<0.001), except for those with an FDR threshold of 0.001.

[0191] result

[0192] Generation of cancer exosome protein markers

[0193] We hypothesize that HBECs with oncogenic mutations secrete exosomes with different exosomal protein profiles. We isolated normal HBECs and transformed HBECs.(p53 / KRAS) Secreted exosomes. TEM, nanoparticle tracking, and Western blotting showed that the exosomes exhibited a typical size distribution and contained canonical exosome markers (…). Figure 1 AC). The proteomes of normal and transformed HBECs were then assessed using mass spectrometry. Label-free quantification by spectral counting identified 15 extracellular proteins upregulated in transformed HBECs compared to normal HBECs. Figure 1 D). THBS1, NID1, PTX3, and VCAN were selected for further evaluation and confirmed by ELISA in HBEC. Figure 1 E).

[0194] Currently, the clinical management of all solid malignancies is guided by the histopathological and / or molecular characteristics of the primary tumor. However, the expression of biomarkers used for tumor classification can be highly variable, even within a single tumor. During cancer progression, cancer cells can exhibit a wide range of phenotypes, some of which are caused by epigenetic alterations, oncogenic transformation, or even changes in environmental cues. Even within specific cancers (lung cancer, brain cancer, melanoma), tumors are highly heterogeneous, reflected in various clinical and molecular classifications. Given this, we evaluated the expression of these four proteins in other cancer cell lines to determine whether these proteins are generally upregulated across various cancer lines or specific to particular cancer cell subpopulations. To address this issue, we isolated exosomes from a total of 22 cell lines, including: non-small cell lung cancer (NSCLC), glioblastoma (GBM), colorectal cancer (CRC), breast cancer (BCa), prostate cancer (PCa), melanoma (MEL), esophageal cancer (ECa), and ovarian cancer (OVA). Interestingly, we found that all four markers, especially NID1, were upregulated in cancer cell-derived exosomes compared to normal HBEC exosomes, regardless of cancer type.

[0195] Assessment of exosomal protein markers in cancer patients

[0196] We then hypothesized that carcinogenic-induced exosome changes could serve as a diagnostic biomarker for the presence of disease in cancer patients. Exosomes were isolated from the serum / plasma of 250 healthy controls and 497 cancer patients diagnosed with lung cancer, brain cancer, colorectal cancer, prostate cancer, melanoma, gastric cancer, and esophageal cancer. The median age at diagnosis for both healthy controls and patients was 65.5 years.

[0197] Interestingly, compared with healthy controls, exosomes derived from cancer subjects showed an increase in the levels of four protein exosome markers (THBS1, NID1, PTX3, and VCAN). Figure 2A). As assessed by receiver operating characteristic (ROC) curves, each protein identified by exosomes possesses a range of diagnostic capabilities across different cancer groups. Figure 3 Interestingly, using a combination of label proteins utilizing logistic regression, we were able to separate healthy individuals from cancer patients quite well, with an area under the curve (AUC) of 0.96. Figure 2 B). Importantly, with a fixed specificity of 95%, the median sensitivity of diagnostic exosome markers was 77.6% across eight cancer types (95% CI: 72.0%, 82.3%). This ranged from 44% for prostate cancer to 100% for gastric cancer. Figure 2 C).

[0198] Next, we wanted to evaluate the ability of diagnostic exosome markers in identifying patients with early-stage cancer. For liquid biopsy to be most effective, it needs to identify patients as early as possible to significantly improve overall cancer survival. We were able to assess the sensitivity of exosome biomarkers in NSCLC, esophageal cancer, and gastric cancer at stage I compared to stages II-IV. Importantly, the sensitivity at 95% specificity was comparable to that at advanced stages in all three cancers. Figure 4 This indicates that diagnostic exosome markers can identify patients with early-stage cancer.

[0199] Recently, CancerSEEK (REF) demonstrated that liquid biopsies can not only be used to identify the presence of cancer, but also to identify tumor types using machine learning in clinical follow-up. Therefore, we investigated whether our exosome markers could also accurately identify the type of cancer a patient had. The accuracy of the tests varied greatly, with NSCLC showing the highest accuracy and gastric cancer the lowest. Figure 5 ).

[0200] References

[0201] 1.Lobb,RJ,et al.Optimized exosome isolation protocol for cellculture supernatant and human plasma.J Extracell Vesicles 4,27031(2015).

[0202] 2.Lobb,R.J.,et al.Exosomes derived from mesenchymal non-small celllung cancer cells promote chemoresistance.International journal of cancer141,614-620(2017).

[0203] 3.Cox,J.&Mann,M.MaxQuant enables high peptide identification rates,individualized p.p.b.-range mass accuracies and proteome-wide proteinquantification.Nature biotechnology 26,1367-1372(2008).

[0204] 4.Cox,J.,et al.Andromeda:a peptide search engine integrated into theMaxQuant environment.Journal of proteome research 10,1794-1805(2011).

[0205] 5.Dave,K.A.,et al.A comprehensive proteomic view of responses of A549type II alveolar epithelial cells to human respiratory syncytial virusinfection.Molecular&cellular proteomics:MCP 13,3250-3269(2014).

Claims

1. Use of a plurality of markers in an exosome sample in the manufacture of a diagnostic test kit for diagnosing cancer or recurrence of cancer in a subject, wherein the markers are selected from the group consisting of: VCAN, NID1, PTX3 and THBS1, and any combination thereof, wherein the expression level of the plurality of markers is indicative of or associated with the diagnosis or recurrence of the cancer.

2. Use of a plurality of markers in an exosome sample in the manufacture of a diagnostic test kit for diagnosing a cancer type, wherein the markers are selected from the group consisting of: VCAN, NID1, PTX3 and THBS1, and any combination thereof, wherein the expression level of the plurality of markers is indicative of or associated with a cancer type.

3. Use of a plurality of markers in an exosome sample in the manufacture of a diagnostic test kit for determining cancer aggressiveness, wherein the markers are selected from the group consisting of: VCAN, NID1, PTX3 and THBS1, and any combination thereof, wherein the expression level of the plurality of markers is indicative of or associated with the cancer aggressiveness level.

4. The use according to claim 3, wherein a relative decreased or unchanged expression level of the plurality of markers is indicative of or associated with a better prognosis and / or a less aggressive cancer; and / or a relative increased expression level of the plurality of markers is indicative of or associated with a worse prognosis and / or a highly aggressive cancer.

5. The use according to any one of claims 1-4, wherein the diagnostic test kit is used in a method comprising the step of diagnosing a subject as having: (i) a highly aggressive cancer or a less aggressive cancer; and / or (ii) a worse prognosis or a better prognosis.

6. The use according to any one of claims 1-4, wherein the diagnostic test kit is used in a method comprising the step of comparing the expression level of the plurality of markers in the exosome sample with a reference exosome expression level of the corresponding plurality of markers.

7. The use according to any one of claims 1-4, wherein the cancer and / or cancer type is selected from the group consisting of: lung cancer, breast cancer, colorectal cancer, prostate cancer, gastric cancer, skin cancer, brain cancer, ovarian cancer, esophageal cancer, and any combination thereof.

8. The use according to claim 7, wherein the lung cancer is NSCLC and SCLC.

9. The use according to claim 7, wherein the skin cancer is melanoma.

10. The use according to claim 7, wherein the brain cancer is glioblastoma multiforme.

11. A composition comprising an exosome sample from a subject having or suspected of having cancer, and reagents for determining the expression level of VCAN, NID1, PTX3 and THBS1.

12. The composition according to claim 11, wherein the exosome sample comprises reagents for determining the level of each of VCAN, NID1, PTX3 and THBS1 in a single composition.

13. The composition according to claim 11 or claim 12, wherein the exosome sample comprises reagents for determining the respective levels of VCAN, NID1, PTX3 and THBS1 in separate compositions.

14. A diagnostic kit or test device comprising a plurality of specific binding members, and a plurality of reagents for detecting the specific binding members or a plurality of reagents for detecting and / or quantifying the formation of complexes formed by the specific binding members and markers, wherein the plurality of specific binding members each selectively bind to a marker selected from the group consisting of: VCAN, NID1, PTX3 and THBS1.

15. The diagnostic kit or test device according to claim 14, for use in a method of diagnosing the type of cancer in a subject.

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