Cancer diagnosis

Abstract

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

Description

[Technical Field]

[0001] [Related Applications] This application claims priority from Australian Patent Application No. 2019904005, filed on October 24, 2019, the contents and material of which are incorporated herein by reference for all purposes. FIELD OF THE INVENTION This application relates to cancer. More particularly, the present invention relates to methods for diagnosing cancer, including determining cancer type. [Background technology]

[0002] Despite advances in screening and treatment, the global burden of cancer is constantly increasing, with one in three men and one in four women developing cancer during their lifetime. Furthermore, one in eight men and one in 11 women die from intractable cancer progression, making cancer one of the leading causes of death worldwide. To reduce cancer mortality, there is a significant unmet clinical need for early patient identification and the development of novel treatments. Early cancer detection is crucial because most localized cancers can be cured with surgery alone. Therefore, improved methods for diagnosing cancer in patients through minimally invasive sampling, such as blood tests, are needed. Cancer blood tests should be highly sensitive and specific to be used for routine population screening, avoiding excessive false positives and false negatives. Summary of the Invention

[0003] The present invention broadly relates to determining the expression level of one or more exosomal proteins as diagnostic markers for cancer (including specific cancer types) in a subject. In some aspects, the present invention also broadly relates to cancer prognosis methods and treatments using such exosomal proteins to inform treatment selection and / or decision-making.

[0004] In a first aspect, the present invention provides a method of diagnosing cancer or cancer recurrence in a subject, the method comprising determining the expression level of one or more markers in an exosome sample from the subject, wherein the markers are selected from the group consisting of apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, biglycan, and any combination thereof, and wherein the expression level of the one or more markers is indicative of or correlates with the diagnosis or recurrence of cancer.

[0005] In some embodiments, the methods of this aspect further comprise determining the type of cancer in a subject diagnosed with cancer.

[0006] In some embodiments, a relatively high expression level of one or more markers is diagnostic of cancer or recurrence of cancer in the subject.

[0007] In a second aspect, the present invention relates to a method for determining the cancer type of a subject having cancer, comprising the step of determining the expression level of one or more markers in an exosome sample from the subject, wherein the markers are selected from the group consisting of apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl-tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, biglycan, and any combination thereof, and wherein the expression level of the one or more markers is indicative of or correlates with the cancer type.

[0008] With respect to the methods of the first and second aspects, the one or more markers are preferably selected from the group consisting of versican core protein, nidogen-1, pentraxin 3, thrombospondin-1, and any combination thereof. In one particular embodiment, the one or more markers comprise versican core protein, nidogen-1, pentraxin 3, and thrombospondin-1.

[0009] In a third aspect, the present invention relates to a method for determining the aggressiveness of cancer in a subject, comprising determining the expression level of one or more markers in an exosome sample from the subject, wherein the markers are selected from the group consisting of apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl-tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, biglycan, and any combination thereof, and wherein the expression level of the one or more markers is indicative of or correlates with the level of aggressiveness of the cancer.

[0010] In a fourth aspect, the present invention provides a method of determining the prognosis of cancer in a subject, the method comprising determining in an exosome sample from the subject the expression level of one or more markers selected from the group consisting of apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl-tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, biglycan, and any combination thereof, wherein the expression level of the one or more markers is indicative of or correlates with a less favorable or more favorable prognosis for the cancer.

[0011] In particular embodiments of the two aforementioned aspects, a relatively low expression level of one or more markers is indicative of or correlates with a better prognosis and / or a less aggressive cancer; and / or a relatively high expression level of one or more markers is indicative of or correlates with a poor prognosis and / or a more aggressive cancer.

[0012] In certain embodiments of the above aspects, the method further comprises the step of diagnosing the subject as having (i) a highly aggressive cancer or a less aggressive cancer, and / or (ii) a poor prognosis or a more favorable prognosis.

[0013] In a fifth aspect, the present invention relates to a method of predicting and / or determining the responsiveness of a cancer to anti-cancer treatment in a subject, comprising determining the expression level of one or more markers in an exosome sample from the subject, wherein the markers are selected from the group consisting of apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl-tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, biglycan, and any combination thereof, and wherein a change or modulation in the expression level of the one or more markers is indicative of or correlates with a relative increase or decrease in the responsiveness of the cancer to the anti-cancer treatment.

[0014] The methods of the foregoing aspects preferably include the further step of treating cancer in the subject.

[0015] In a sixth aspect, the present invention relates to a method of treating cancer in a subject, the method comprising the steps of determining the expression level of one or more markers in an exosome sample from the subject, and initiating, continuing, modifying, or discontinuing anti-cancer treatment based on the determination made, wherein the markers are selected from the group consisting of apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl-tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, biglycan, and any combination thereof.

[0016] In certain embodiments, the methods of the third, fourth, fifth, and sixth aspects comprise the further step of determining the cancer type of a subject diagnosed with cancer.

[0017] With respect to the fifth and sixth aspects, the anti-cancer treatment suitably comprises administering to the subject a therapeutically effective amount of an anti-cancer agent that reduces the expression and / or activity of one or more markers.

[0018] With respect to the fifth and sixth aspects, the method may comprise the further step of administering to the subject a therapeutically effective amount of an anti-cancer treatment or agent.

[0019] In embodiments of the foregoing aspects, the method further comprises obtaining an exosome sample from the subject.

[0020] In certain embodiments of the above aspects, the method further comprises comparing the expression level of the one or more markers in the exosome sample to a reference exosomal expression level of each of the one or more markers.

[0021] Suitably, the cancer and / or cancer type in the above aspects is selected from the group consisting of lung cancer such as NSCLC and SCLC, 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.

[0022] In a seventh aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) contacting a candidate agent with cells expressing one or more markers selected from the group consisting of apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl-tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, biglycan, and any combination thereof; (b) determining whether the candidate agent modulates the expression and / or activity of one or more markers; The present invention relates to a method of identifying or manufacturing an agent for use in treating cancer in a subject, comprising:

[0023] In some embodiments, the candidate agent at least partially reduces, eliminates, suppresses, or inhibits the expression and / or activity of the marker.

[0024] Preferably, for the third, fourth, fifth, sixth and seventh aspects, the one or more markers are selected from the group consisting of versican core protein, nidogen-1, pentraxin 3, thrombospondin-1, and any combination thereof.

[0025] In some embodiments of the third, fourth, fifth, sixth, and seventh aspects, the method further comprises determining the expression level of one or more additional markers (e.g., thrombospondin-1) in the exosome sample from the subject.

[0026] In an eighth aspect, the present invention provides an agent identified or produced by the method of the seventh aspect for use in accordance with the method of the fifth or sixth aspect.

[0027] Suitably, the subject of the above aspects is a mammal, preferably a human.

[0028] In another aspect, the present invention provides a composition comprising an exosome sample from a subject having or suspected of having cancer and a reagent for determining the expression level of one or more of VCAN, NID1, PTX3, and THBS1.

[0029] In some embodiments, the exosome sample comprises reagents for determining the levels of each of VCAN, NID1, PTX3, and THBS1 in a single composition. In some alternative embodiments, the exosome sample comprises reagents for determining the levels of each of VCAN, NID1, PTX3, and THBS1 in separate compositions.

[0030] In yet another aspect, a diagnostic kit or test device comprising one, two, three, four, five, six, seven, eight, nine, ten or more specific binding members, each of which selectively binds to a marker selected from the group consisting of APOE, PRSS23, VCAN, HAPLN3, COL4A1, NID1, CTGF, COL4A2, CPD, CCBE1, PTX3, SPOCK1, AIMP1, THBS1, and BGN, and one or more reagents for detecting the one or more specific binding members, or one or more reagents for detecting and / or quantifying the formation of a complex formed by the specific binding member and the marker.

[0031] In some embodiments, the diagnostic kit or test device is used in a method for diagnosing cancer or cancer recurrence in a subject, or for determining the type of cancer in a subject.

[0032] In some embodiments, the markers include VCAN, NID1, PTX3, and THBS1.

[0033] In yet another aspect, the present invention provides use of one or more of APOE, PRSS23, VCAN, HAPLN3, COL4A1, NID1, CTGF, COL4A2, CPD, CCBE1, PTX3, SPOCK1, AIMP1, THBS1, and BGN as markers for determining whether a subject is sensitive to treatment with a chemotherapeutic agent, optionally wherein the use is as a marker in a method according to any one of the aspects described above or elsewhere herein.

[0034] Unless the context requires otherwise, the terms "comprise," "comprises," and "comprising," or similar terms, are intended to mean a non-exclusive inclusion, such that an enumerated list of elements or features does not include only those stated or listed elements, but may also include other elements or features that are not listed or stated.

[0035] The indefinite articles "a" and "an" are used herein to refer to or include singular or plural elements or features and should not be construed to mean or define "one" or "single" element or feature. For example, "a" cell includes one cell, one or more cells, and plural cells. [Brief explanation of the drawings]

[0036] [Figure 1]Carcinogenesis-induced changes in the protein composition of cell-derived exosomes. (A) The morphology of isolated exosomes was assessed using transmission electron microscopy. Images of exosomes from normal HBECs and transformed HBECs (size bar 200 nm). (B) Nanoparticle analysis of exosomes isolated from HBECs using tunable resistive pulse sensing (TRPS) demonstrates that the majority of exosomes are in the 30-150 nm size range and that transformation does not increase exosome secretion. (C) Western blot of exosomes from HBECs showing the presence of exosomal proteins HSP70 and CD63 and the absence of the cellular marker calnexin. (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 using ELISA for THBS1, NID1, PTX3, and VCAN in normal and transformed HBECs. (F) Exosomes from 22 cell lines from NSCLC, GBM, CRC, BCa, PCa, MEL, ECa, and OVA show a clear increase in the expression of THBS1, NID1, PTX3, and VCAN relative to the expression levels of normal HBEC exosomes. [Figure 2] Oncogenic exosome signatures diagnose cancer in patient plasma. (A) Expression levels of THBS1, NID1, PTX3, and VCAN are increased in cancer patients compared to healthy controls. (B) Logistic regression demonstrates the excellent diagnostic ability of the four-protein exosome panel with an AUC of 0.96. (C) The sensitivity of each cancer diagnostic exosome signature was evaluated at a fixed specificity of 95%. Error bars represent 95% confidence intervals. [Figure 3-1] Evaluation of the individual diagnostic capabilities of (A) THBS1, (B) NID1, (C) PTX3, and (D) VCAN by ELISA shows that each exosomal protein has different diagnostic capabilities in non-small cell lung cancer, glioblastoma, colorectal cancer, prostate cancer, melanoma, gastric cancer, esophageal cancer, and small cell lung cancer, as assessed by receiver operating characteristic (ROC) curves. [Figure 3-2] Evaluation of the individual diagnostic capabilities of (A) THBS1, (B) NID1, (C) PTX3, and (D) VCAN by ELISA shows that each exosomal protein has different diagnostic capabilities in non-small cell lung cancer, glioblastoma, colorectal cancer, prostate cancer, melanoma, gastric cancer, esophageal cancer, and small cell lung cancer, as assessed by receiver operating characteristic (ROC) curves. [Figure 4] Sensitivity of diagnostic exosome signatures by tumor stage. A, The diagnostic exosome signature can detect early stage I and late stage II-IV NSCLC, esophageal, and gastric cancer patients with 95% specificity. Error bars represent 95% confidence intervals. [Figure 5] Machine learning can help distinguish cancer types. The percentage of patients correctly or incorrectly identified as to cancer type indicates that exosome signatures can help distinguish the type of cancer present. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention is based, at least in part, on the surprising discovery that exosomal proteins identified as upregulated in vitro from normal human bronchial epithelial cells (HBECs) transformed with oncogenic mutations are accurate diagnostic biomarkers for a wide range of cancers. Furthermore, these exosomal markers may also serve as biomarkers of cancer progression and aggressiveness, as well as response to anti-cancer treatment in patients.

[0038] In a broad aspect, the present invention relates to a method of diagnosing or detecting cancer (including recurrence of cancer) in a subject by determining or measuring the expression level of one or more markers, such as those shown in FIG. 1, in an exosome sample from the subject.

[0039]

[0013] Accordingly, in one aspect, the present invention provides a method of diagnosing cancer or cancer recurrence in a subject, comprising determining the expression level of one or more markers in an exosome sample from the subject, wherein the markers are apolipoprotein E (APOE; Uniprot Accession No: P02649), serine protease 23 (PRSS23; Uniprot Accession No: O95084), versican core protein (VCAN; Uniprot Accession No: P13611), hyaluronan and proteoglycan link protein 3 (HAPLN3; Uniprot Accession No: Q96S86), type IV collagen alpha 1 chain (COL4A1; Uniprot Accession No: P02462), nidogen-1 (NID1; Uniprot Accession No: P14543), connective tissue growth factor (CTGF; Uniprot Accession No: P29279), type IV collagen alpha 2 chain (COL4A2; Uniprot Accession No: P14544), and / or IL-1. No: P08572), carboxypeptidase D (CPD; Uniprot Accession No: O75976), collagen- and calcium-binding EGF domain-containing protein 1 (CCBE1; Uniprot Accession No: Q6UXH8), pentraxin 3 (PTX3; Uniprot Accession No: P26022), testican-1 (SPOCK1; Uniprot Accession No: Q08629), aminoacyl-tRNA synthase complex-interacting multifunctional protein 1 (AIMP1; Uniprot Accession No: Q12904), thrombospondin-1 (THBS1; Uniprot Accession No: P07996), biglycan (BGN; Uniprot Accession No: P21810), and any combination thereof, wherein the expression level of one or more markers is indicative of or correlates with a diagnosis or recurrence of cancer.The proteins defined by these expression numbers are typically of human wild-type amino acid sequences, although reference to a gene or protein may include other mammalian sequences (e.g., mammalian homologs).

[0040] In some embodiments, the methods of this aspect further comprise determining the type of cancer in a subject diagnosed with cancer.

[0041] In a related aspect, the invention relates to a method of determining or diagnosing a cancer type in a subject having cancer, comprising determining the expression level of one or more markers in an exosome sample from the subject, wherein the markers are selected from the group consisting of apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl-tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, biglycan, and any combination thereof, and wherein the expression level of the one or more markers is indicative of or correlates with the cancer type.

[0042] The terms "diagnosis," "diagnosing," and "diagnostic" refer to methods by which one skilled in the art can estimate and / or determine whether a patient is afflicted with a given disease or condition, e.g., cancer or a specific cancer type. Those skilled in the art often make a diagnosis based on one or more diagnostic indicators (e.g., exosomal markers) whose presence, absence, amount, or change in amount can indicate the presence, severity, or absence of a condition. It will also be recognized that the term "diagnosis" does not refer to the ability to determine with 100% accuracy the presence or absence of a particular disease, such as cancer, or to an increased likelihood of a given course or outcome compared to an undiagnosed case. Instead, those skilled in the art will understand that the term "diagnosis" refers to an increased likelihood that a particular disease, disorder, or condition, e.g., cancer or a specific cancer type, is present in a subject.

[0043] Thus, in certain embodiments, a positive diagnosis of a cancer or cancer type indicates at least about a 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) probability or likelihood that the cancer or cancer type is present in the subject. The term "about" in this context refers to ±2%.

[0044] As generally used herein, the terms "cancer," "tumor," "malignant," and "malignant" refer to a disease or condition, or to cells or tissues associated with a disease or condition, characterized by aberrant or abnormal cell proliferation, differentiation, and / or migration, often accompanied by an aberrant or abnormal molecular phenotype, including one or more genetic mutations or other genetic alterations associated with tumor formation, expression of tumor markers, loss of tumor suppressor expression or activity, and / or aberrant or abnormal cell surface marker expression.

[0045] Cancer can include any aggressive or potentially aggressive cancer, tumor, or other malignancy, such as those listed in the NCI Cancer Index at http: / / www.cancer.gov / cancertopics / alphalist, including all major forms of cancer, such as sarcomas, carcinomas, lymphomas, leukemias, and blastomas, including, but not limited to, breast cancer, lung cancer, such as lung adenocarcinoma and mesothelioma, cancers of the reproductive system, such as ovarian cancer, cervical cancer, uterine cancer, testicular cancer, and prostate cancer, cancers of the brain and nervous system, head and neck cancer, cancers of the digestive system, such as colon cancer, colorectal cancer, esophageal cancer, and stomach cancer, liver cancer, bladder cancer, kidney cancer, skin cancer, such as melanoma and skin cancer, cancers of blood cells, such as lymphocytic cancer and myelomonocytic cancer, cancers of the endocrine system, such as pancreatic cancer, adrenal cancer, and pituitary cancer, and musculoskeletal cancer, such as bone and soft tissue cancer.

[0046] In certain embodiments, the cancer and / or cancer type described herein is selected from the group consisting of lung cancer (e.g., squamous cell carcinoma, adenocarcinoma, and large cell carcinoma), such as non-small cell lung cancer (NSCLC), 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.

[0047] As generally used herein, the terms "recurrence" and "cancer recurrence" refer to the return of signs and symptoms of cancer after a period of improvement or remission. In some embodiments, cancer has recurred after a period in which the cancer could not be detected, or has recurred after the cancer has been at least partially removed by surgery, or after the growth of the cancer has been inhibited by therapeutic treatment. Cancer may recur or return to the same location as the original (primary) tumor, or may recur or return to another location in the body, for example, by metastatic recurrence.

[0048] As used herein, "metastasis" or "metastatic" refers to the migration or movement of malignant tumor cells or neoplasms, typically from the primary tumor, cancer, or neoplasm, to a distant site in the body via the circulatory or lymphatic system or via a natural body cavity, and the subsequent development of one or more secondary tumors or colonies thereof at one or more new locations. "Metastasis" refers to secondary tumors or colonies formed as a result of metastasis, and also encompasses micrometastases and regional metastases, such as lymph nodes, and distant metastases.

[0049] In certain embodiments, the methods of this aspect may be used to diagnose any minimal residual disease of cancer. In this context, the term "minimal residual disease" refers to the small number of cancer cells that remain in a subject during or after treatment when the subject is in remission and typically does not exhibit symptoms or signs of cancer.

[0050] As used herein, the term "cancer type" means the type of cancer determined by the type of tissue in which the cancer originates (histological type), or by the primary site, or location in the body where the cancer first begins, or the type of cell from which the cancer originates, and the appearance of the cancer cells.

[0051] Interestingly, applicants have determined that the expression profile or signature of one or more exosomal markers described herein may be used not only to diagnose or detect cancer in a subject, but also to classify or identify the cancer as diagnosed into a particular type and / or subtype of cancer. Accordingly, these exosomal markers may then be used by clinicians to provide indications of, for example, cancer aggressiveness, survival prognosis, treatment regimens, response to treatment, etc., specific to the particular cancer type diagnosed in a subject.

[0052] As used herein, the term "cancer subtype" refers to a subclassification of cancer types and is included within cancer types. This term may refer to a molecular classification of cancer. In particular, cancer subtypes may be associated with molecular alterations, cancer survival, distinct clinical and pathological features, specific gene expression signatures, and deregulated signaling pathways.

[0053] With regard to determining the cancer type, this may include one or more of the following: (a) calculating or obtaining an expression profile or signature from one or more exosomal markers described herein in a biological sample, such as an exosomal sample, from a subject; (b) comparing the expression profile of the biological sample from the subject to one or more reference expression profiles corresponding to one or more test cancer types or reference cancer types; and (c) assigning the expression profile of the biological sample to a cancer type, which cancer type is identified as the test cancer type corresponding to the reference expression profile that most closely matches or matches the test cancer type when compared with the expression profile of the biological sample from the subject.

[0054] In some embodiments, the cancer diagnosis or cancer type is used, at least in part, to determine whether a subject will benefit from cancer treatment. By way of example, a patient diagnosed with a less aggressive cancer or cancer type may be less likely to suffer from rapid local progression and / or metastasis of the cancer and may be spared active monitoring and / or treatment.

[0055] In another embodiment, the cancer diagnosis and / or cancer type is used, at least in part, to develop a treatment strategy for the subject. Thus, in certain embodiments, the diagnostic methods of the foregoing aspects are combined with a treatment plan appropriate for the subject's diagnosed cancer and / or cancer type.

[0056] Those skilled in the art will recognize that exosomes are small (i.e., typically 30-150 nm) cell-derived membrane vesicles of endocytic origin. Exosomes may contain lipids, nucleic acids, and proteins and are released into the extracellular environment upon fusion with the plasma membrane. Exosomes are generally characterized by the presence of marker proteins, such as CD63, CD9, HSP70, flotillin-1, and TSG101, as well as their morphology and size.

[0057] In accordance with the methods of the present invention, an exosome sample containing one or more exosomes can comprise or be obtained from most bodily fluids, including, but not limited to, blood, serum, plasma, ascites, cyst fluid, pleural fluid, peritoneal fluid, cerebrospinal fluid, tears, urine, saliva, sputum, nipple aspirate, lymphatic fluid, respiratory tract, intestinal tract, urinary tract fluid, breast milk, visceral system fluid, or a combination thereof. To this end, the exosome sample can be isolated or purified from bodily fluids or biological samples, such as those set forth above, to facilitate the removal of contaminating proteins, lipoproteins, and the like.

[0058] To this end, exosomes or exosome samples may be isolated by any means known in the art, including, but not limited to, ultracentrifugation, size exclusion chromatography, exosome precipitation (e.g., ExoQuick from System Biosciences), affinity-based capture of exosomes (e.g., affinity purification with antibodies against CD63, CD81, CD82, CD9, Alix, annexin, EpCAM, and Rab5), and any combination thereof.

[0059] Suitably, one or more of the exosomal markers apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl-tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, and / or biglycan may be utilized as a single biomarker, or any combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., or more of the one or more biomarkers may be utilized (e.g., as an expression profile or signature).

[0060] In a particular embodiment, the one or more markers are preferably selected from the group consisting of versican core protein, nidogen-1, pentraxin 3, thrombospondin-1, and any combination thereof. Thus, in a particular embodiment, the one or more markers include versican core protein, nidogen-1, pentraxin 3, and thrombospondin-1.

[0061] In certain embodiments, the methods of the above aspects comprise the further step of determining the expression level of one or more additional markers or biomarkers in a biological sample, such as a blood sample, from the subject.

[0062] It is anticipated that the additional biomarkers may be any known in the art that may be utilized in diagnosing or detecting cancer and / or determining cancer type in a subject. By way of example, the additional biomarkers may 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 described in connection with the CancerSEEK study (see, e.g., Cohen, Science 2018).

[0063] As generally used herein, the expression level of one or more exosome marker proteins identified in FIG. 1 as being upregulated can refer to the expression level of nucleic acid (e.g., RNA, mRNA, and cDNA) encoding the protein, the protein itself, or both, unless otherwise specified.

[0064] For purposes of this invention, "isolated" means material that has been removed from the natural state or that has otherwise been subjected to human manipulation. An isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or it may have been manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in natural, chemically synthesized, or recombinant form.

[0065] "Protein" refers to a polymer of amino acids. The amino acids may be natural or unnatural amino acids, D- or L-amino acids, as is well understood in the art. As will be recognized by those of skill in the art, the term "protein" also includes within its scope phosphorylated forms of proteins (i.e., phosphoproteins) and / or glycosylated forms of proteins (i.e., glycoproteins). A "peptide" refers to a protein having 50 or fewer amino acids. A "polypeptide" refers to a protein having more than 50 amino acids.

[0066] Also provided are protein "variants," e.g., naturally occurring variants (e.g., allelic variants) and orthologs or isoforms of one or more markers provided herein, e.g., those listed in Figure 1. Preferably, protein variants share at least 70% or 75%, 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 amino acid sequence of one or more markers disclosed herein or known in the art. To this end, Accession Numbers referencing exemplary protein sequences of the listed protein markers are set forth above and are incorporated herein by reference, as is well understood in the art.

[0067] Also provided are protein fragments, including peptide fragments that comprise less than 100% of the entire amino acid sequence. In certain embodiments, a protein 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, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, and 1200 consecutive amino acids of the protein.

[0068] As used herein, a "gene" refers to a nucleic acid that is a structural, hereditary unit of the genome, which may contain one or more amino acid-encoding nucleotide sequences and one or more non-coding nucleotide sequences, such as, but not limited to, promoters and other 5' untranslated sequences, introns, polyadenylation sequences and other 3' untranslated sequences. In most cellular organisms, genes are nucleic acids that comprise double-stranded DNA.

[0069] The term "nucleic acid" as used herein 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 include nucleotide sequences that typically include nucleotides containing A, G, C, T, or U bases. However, nucleotide sequences can include other bases, such as, but not limited to, inosine, methylcytosine, methylinosine, methyladenosine, and / or thiouridine.

[0070] Also included are "variant" nucleic acids, such as nucleic acids comprising the nucleotide sequences of naturally occurring (e.g., allelic) variants and orthologs (e.g., from different species) of the nucleic acids encoding one or more markers, respectively, provided herein. Preferably, nucleic acid variants share at least 70% or 75%, 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.

[0071] Also included are nucleic acid fragments. A "fragment" is a segment, domain, portion, or region of a nucleic acid, each comprising less than 100% of the nucleotide sequence. Non-limiting examples are amplification products, primers, or probes. In certain embodiments, a 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.

[0072] As used herein, a "polynucleotide" refers to a nucleic acid having 80 or more consecutive nucleotides, while an "oligonucleotide" refers to a nucleic acid having fewer than 80 consecutive nucleotides. A "probe" may be a single- or double-stranded oligonucleotide or polynucleotide, appropriately labeled for the purpose of detecting complementary sequences, for example, in Northern or Southern blotting. A "primer" is typically a single-stranded oligonucleotide, preferably having 15 to 50 consecutive nucleotides, that can anneal to a complementary nucleic acid "template" and be extended in a template-dependent manner by the action of a DNA polymerase, such as Taq polymerase, RNA-dependent DNA polymerase, or Sequenase™. A "template" nucleic acid refers to a nucleic acid that is subjected to nucleic acid amplification.

[0073] As will be understood by those skilled in the art, the gene and / or protein expression levels of one or more markers provided herein may be relatively (i) high, increased, or greater; or (ii) low, decreased, or less when compared to the expression level in a control or reference sample or compared to a threshold expression level. In some embodiments, an expression level may be classified as higher, increased, or greater if it exceeds the mean and / or median expression level of a reference population. In some embodiments, an expression level may be classified as lower, decreased, or less if it is lower than the mean and / or median expression level of a reference population. In this regard, the reference population may be a group of subjects having the same cancer type, subgroup, stage, and / or grade as the mammal for which the expression level is determined. In other embodiments, the reference population may be a group of healthy controls who do not have cancer or who are documented not to have cancer. In further embodiments, the control sample is obtained from the subject in question prior to undergoing a diagnostic test for cancer or cancer type.

[0074] Terms such as "higher," "increasing," and "greater," as used herein, refer to an increase in the amount or level of a nucleic acid and / or protein, for example, in an exosome sample, compared to a control or reference level or amount. The nucleic acid and / or protein expression levels of one or more markers can be relative or absolute. In some embodiments, gene and / or protein expression of one or more markers is higher, increased, or greater if the level of expression 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% greater than the level of gene and / or protein expression of the respective or corresponding gene and / or protein at a control or reference level or amount.

[0075] The terms "lower," "small," and "reduced," as used herein, refer to a decrease in the amount or level of a nucleic acid and / or protein, for example, in an exosome sample, compared to a control or reference level or amount. The nucleic acid and / or protein expression levels of one or more markers provided herein can be relative or absolute. In some embodiments, gene and / or protein expression of one or more markers is lower, smaller, or reduced if the level of expression is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%, or even less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, or 0.0001%, of the gene and / or protein expression level or amount of the respective or corresponding gene and / or protein at the control or reference level or amount.

[0076] The term "control sample" typically refers to a biological sample, such as an exosome sample, from a non-diseased (healthy) individual without cancer. In some embodiments, the control sample may be from a subject known to be cancer-free or may be a sample obtained from this subject at an earlier time point. Alternatively, the control sample may be from a subject in cancer remission. The control sample may be a pooled sample, an average sample, or an individual sample. An internal control is a marker from the same biological sample (e.g., exosome sample) being tested.

[0077] As used herein, gene and / or protein expression levels may refer to absolute or relative amounts. Thus, in some embodiments, the gene and / or protein expression levels of one or more markers provided herein are compared to a control level of expression, such as the gene and / or protein expression levels of one or more "housekeeping" genes and / or proteins in a subject's exosome sample.

[0078] In further embodiments, the gene and / or protein expression levels of one or more markers are compared to a threshold level of expression, such as the level of gene and / or protein expression in an exosome sample. A threshold level of expression generally refers to a quantified level of gene and / or protein expression of one or more markers of the invention. Typically, gene and / or protein expression levels of one or more markers in an exosome sample that are above or below the threshold level of expression predict a particular disease state or outcome. The nature and value (if any) of the threshold level of expression typically varies based on the method selected to determine the expression of one or more genes or their products used in, for example, determining the diagnosis of a subject's cancer, cancer type, prognosis, and / or response to anti-cancer treatment.

[0079] Using any method for measuring gene or protein expression known in the art, such as those described herein, one skilled in the art may determine a threshold level (e.g., a predetermined threshold) of gene and / or protein expression in an exosome sample that can be used, for example, in determining the diagnosis of cancer, cancer type, prognosis, and / or response to anti-cancer treatment. In some embodiments, the threshold level refers to, for example, the average and / or median gene and / or protein expression level (median or absolute value) of one or more markers in a reference population having the same cancer type, subgroup, stage, and / or grade as the subject whose expression level is being determined. Additionally, the concept of a threshold level of expression should not be limited to a single value or outcome. In this regard, a threshold level of expression can encompass multiple threshold expression levels, which may indicate, for example, a high, moderate, or low probability of metastasis of the subject's cancer.

[0080] As used herein, the term "predetermined threshold" refers to a value above or below which indicates the responsiveness of a disease to treatment or the general prognosis of a disease. For example, for purposes of the present invention, a predetermined threshold may represent the level or activity of a protein, or the expression level of a nucleic acid encoding the protein, in a sample from an appropriate control subject, e.g., a subject known to have cancer or a subject susceptible to cancer recurrence, or from multiple control subjects, or in a median or mean value across multiple control subjects. Thus, activity or expression levels above or below the threshold indicate the likelihood of cancer being present in the subject, or the likelihood of cancer recurrence in the subject, or the likelihood of a tumor responding to anti-cancer treatment, as taught herein. In other examples, a predetermined threshold may represent a value greater or less than the level determined for a control subject, to incorporate additional confidence that a level or ratio above or below the predetermining threshold indicates the presence of cancer in the subject, or the likelihood of cancer recurrence in the subject, or the tumor responding to anti-cancer treatment. For example, a predetermined threshold can represent the mean or median activity level of a marker disclosed herein in a group of control subjects, together with ±1, 2, 3, or more standard deviations. One of skill in the art can readily determine an appropriate predetermined threshold based on analysis of biological samples from appropriate control subjects.

[0081] In some embodiments, a relatively high expression level of one or more markers is diagnostic of cancer or cancer recurrence in the subject, hi related embodiments, a relatively low or relatively unchanged expression level of one or more markers is diagnostic of the subject not having cancer or not having cancer recurrence.

[0082] The terms "determining," "measuring," "evaluating," "assessing," and "assaying" are used interchangeably herein and can include any form of measurement known in the art, such as those described below.

[0083] Determining, assessing, evaluating, assaying, or measuring the protein level of one or more exosomal proteins can be performed by any technique known in the art that can detect such proteins, whether expressed on the surface or within exosomes, or isolated, extracted, or otherwise obtained from a subject's exosome sample. 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, chromatographic techniques, and mass spectrometry, as well as combinations thereof. Antibody-based detection can include, but is not limited to, flow cytometry using fluorescently labeled antibodies, ELISA, immunoblotting, immunoprecipitation, radioimmunoassay (RIA), and immunocytochemistry.

[0084] Determining, assessing, evaluating, assaying, or measuring the nucleic acids, e.g., RNA, mRNA, and cDNA, corresponding to one or more markers provided herein can be performed by any technique known in the art, including nucleic acid sequence amplification, nucleic acid hybridization, nucleotide sequencing, mass spectrometry, and any combination thereof.

[0085] Nucleic acid amplification techniques typically involve repeated cycles of annealing one or more primers to a "template" nucleotide sequence under appropriate conditions and using a polymerase to synthesize a nucleotide sequence complementary to the target, thereby "amplifying" the target nucleotide sequence. Nucleic acid amplification techniques are 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); nucleic acid sequence-based amplification (NASBA); Q-beta replicase amplification; helicase-dependent amplification (HAD); loop-mediated isothermal amplification (LAMP); nicking enzyme amplification reaction (NEAR); and recombinase polymerase amplification (RPA). As generally used herein, "amplification product" refers to a nucleic acid product produced by a nucleic acid amplification technique.

[0086] PCR includes quantitative and semi-quantitative PCR, real-time PCR, allele-specific PCR, methylation-specific PCR, asymmetric PCR, nested PCR, multiplex PCR, touchdown PCR, digital PCR, and other variations and modifications to the "basic" PCR amplification.

[0087] Nucleic acid amplification techniques may be performed using DNA or RNA extracted, isolated, or otherwise obtained from cell or tissue sources. In other embodiments, nucleic acid amplification may be performed directly on appropriately processed cell or tissue samples.

[0088] Nucleic acid hybridization involves hybridizing a nucleotide sequence, typically in the form of a probe, to a target nucleotide sequence under appropriate conditions, followed by detection of 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 may be performed using DNA or RNA extracted, isolated, amplified, or otherwise obtained from a cell or tissue source, or may be performed directly with appropriately processed cell or tissue samples.

[0089] It will also be appreciated that a combination of nucleic acid amplification and nucleic acid hybridization may be employed.

[0090] It will be appreciated that determining the expression of one or more markers provided herein can include both determining their nucleic acid levels, such as by nucleic acid amplification and / or nucleic acid hybridization, and determining their protein levels. Thus, detecting and / or measuring the expression of one or more markers from a subject's exosome sample can be performed by any of these methods described herein or a combination thereof (e.g., by measuring mRNA levels or amplified cDNA copies thereof, and / or by measuring their protein products), but is not limited to these methods.

[0091] In light of the foregoing, it will be further appreciated that the expression level of one or more markers provided herein can be the absolute or relative amount of the expressed gene or its gene product, including nucleic acids such as RNA, mRNA, and cDNA, and / or protein.

[0092] In a further aspect, the present invention relates to a method for determining the aggressiveness of cancer in a subject, comprising determining the expression level of one or more markers in an exosome sample from the subject, wherein the markers are selected from the group consisting of apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl-tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, biglycan, and any combination thereof, and wherein the expression level of the one or more markers is indicative of or correlates with the level of aggressiveness of the cancer.

[0093] In another aspect, the present invention provides a method of determining the prognosis of cancer in a subject, comprising determining the expression level of one or more markers in an exosome sample from the subject, wherein the markers are selected from the group consisting of apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl-tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, biglycan, and any combination thereof, and wherein the expression level of the one or more markers is indicative of or correlates with a poor or better prognosis for the cancer.

[0094] In certain embodiments of the above aspects, the method further comprises the step of diagnosing the subject as having (i) a highly aggressive cancer or a less aggressive cancer, and / or (ii) a poor prognosis or a more favorable prognosis.

[0095] "Aggressiveness" and "aggressive" refer to the quality or tendency of a cancer to have a relatively poor prognosis due to one or more of the following characteristics or a combination of factors, including, but not limited to, at least partial resistance to available therapies for treating the cancer; invasiveness; metastatic potential; recurrence after treatment; and a low likelihood of patient survival.

[0096] In certain embodiments, the proteins provided herein, e.g., those provided in Figure 1, are prognostic for aggressive disease, and in particular are prognostic for shorter time to pathological recurrence and / or shorter patient survival. In further embodiments, the proteins provided herein, e.g., those provided in Figure 1, correlate with or are indicative of metastatic cancer.

[0097] The terms "prognosis" and "prognostic" are used herein to include determining a prognosis, which may provide for predicting clinical outcome (with or without medical treatment), selecting an appropriate course of treatment (or whether treatment will be effective), and / or monitoring current treatment to potentially modify said treatment. This may be based at least in part on determining the gene and / or protein expression levels of one or more markers by the methods of the invention, which may be combined with determining the expression levels of additional protein and / or other nucleic acid biomarkers, e.g., thrombospondin-1, or those described below with respect to CancerSEEK. Prognosis may also include the prediction, forecast, or expectation of any lasting or permanent physical or psychological effects of a cancer suffered by a subject after the cancer has been successfully treated or otherwise resolved. Furthermore, prognosis may include one or more of determining the likelihood or occurrence of metastasis, therapeutic responsiveness, implementation of an appropriate treatment regimen, determining the probability, likelihood, or potential for cancer recurrence after treatment, and predicting the development of resistance to established treatments (e.g., chemotherapy). It will be appreciated that a good prognosis typically refers to a favorable clinical outcome or outlook, such as long-term survival without recurrence of the subject's cancer, and a poor prognosis typically refers to a negative clinical outcome or outlook, such as recurrence or progression of the cancer.

[0098] In some embodiments of the methods of the two foregoing aspects, a relatively low or relatively unchanged expression level of one or more markers is indicative of or correlates with a better prognosis and / or a less aggressive cancer; and / or a relatively high expression level of one or more markers is indicative of or correlates with a poor prognosis and / or a more aggressive cancer.

[0099] In one particular embodiment, the prognosis or aggressiveness of the cancer is used, at least in part, to determine the likelihood of the cancer metastasizing in said subject.

[0100] Suitably, a relatively low or unchanged expression level of one or more markers indicates or correlates with a low likelihood of metastasis of said cancer; and / or a relatively high expression level of one or more markers indicates or correlates with a high likelihood of metastasis of said cancer.

[0101] In some embodiments, the prognosis or aggressiveness of the cancer is used, at least in part, to determine whether a subject will benefit from treatment for the cancer. By way of example, patients with a good prognosis and / or a less aggressive cancer may be less likely to suffer from rapid local progression of the cancer and / or metastases and may be spared more aggressive monitoring and / or treatment.

[0102] In another embodiment, the prognosis or aggressiveness of the cancer is used, at least in part, to develop a treatment strategy for the subject.

[0103] In some embodiments, the cancer prognosis or aggressiveness is used, at least in part, to determine minimal residual disease, disease progression, or recurrence in a subject.

[0104] In some embodiments, the prognosis or aggressiveness of the cancer is used, at least in part, to determine an estimated survival time.

[0105] Preferably, the method of the above aspect further comprises the step of diagnosing said subject (i) as having a highly aggressive cancer or a less aggressive cancer, and / or (ii) as having a poor prognosis or a more favorable prognosis.

[0106] In some embodiments, relatively low gene and / or protein expression levels of one or more markers provided herein are indicative of or correlate with a relatively high responsiveness of the cancer to anti-cancer treatment. In alternative embodiments, relatively low gene and / or protein expression levels of one or more markers provided herein are indicative of or correlate with a relatively low responsiveness of the cancer to anti-cancer treatment.

[0107] Preferably, but not limited to, the cancer is of the types described below.

[0108] In a further aspect, the present invention pertains to a method of predicting and / or determining the responsiveness of a cancer to anti-cancer treatment in a subject, comprising the step of determining the expression level of one or more markers in an exosome sample from the subject, wherein the markers are selected from the group consisting of apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl-tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, biglycan, and any combination thereof, and wherein a change or modulation in the expression level of the one or more markers is indicative of or correlates with a relative increase or decrease in the responsiveness of the cancer to the anti-cancer treatment.

[0109] As will be understood by those skilled in the art, the expression level of a gene or protein may be determined to be "altered" or "modulated" if the expression level is higher / increased or lower / decreased when compared to a control or reference sample or expression level, e.g., a threshold level. In some embodiments, an expression level may be classified as high if it is greater than the mean and / or median relative expression level of a reference population, and an expression level may be classified as low if it is less than the mean and / or median expression level of the reference population. In this regard, the reference population may be a group of subjects having the same cancer type, subgroup, stage, and / or grade as the mammal for which the expression level is being determined. Furthermore, the expression level may be relative or absolute.

[0110] In some embodiments, the one or more markers are selected from the group consisting of versican core protein, nidogen-1, pentraxin 3, thrombospondin-1, and any combination thereof. More specifically, the one or more markers preferably include versican core protein, nidogen-1, and pentraxin 3, and optionally further include an additional biomarker for thrombospondin-1.

[0111] In some embodiments, a relatively high expression level of one or more markers indicates or correlates with a relatively high responsiveness of the cancer to anti-cancer treatment. In alternative embodiments, a relatively high expression level of one or more markers indicates or correlates with a relatively low responsiveness of the cancer to anti-cancer treatment.

[0112] In some embodiments, the expression level of one or more markers, e.g., a relatively high expression level thereof, is indicative of or correlates with the continued presence (e.g., minimal residual disease), progression or recurrence of cancer, or lack or poor responsiveness of the cancer to anti-cancer treatment.

[0113] In some embodiments, the methods of the invention comprise assessing the activity, expression, or amount of one or more biomarkers in a subject or in a sample (e.g., an exosome sample) derived from the subject to obtain a sample profile of more biomarkers; and making a prediction based on the sample profile of the one or more biomarkers. Optionally, the prediction is made by comparing the sample profile with a control profile. For example, suitable control profiles that may be used are (i) obtained from a population of control subjects with cancer; or (ii) obtained from a control subject or population of control subjects with cancer that is a recurrent cancer; (iii) a predetermined profile of the "mean, median, or average" or "normal range" of biomarker expression, activity, or quantity values ​​obtained from a control subject or population of control subjects with cancer; (iv) obtained from a control sample with a known "mean, median, or average" value of the biomarker indicative of a control subject or population of control subjects with cancer; (v) a predetermined profile of a "threshold" of biomarker expression, activity, or quantity obtained from a control subject or population of control subjects with cancer; or (vi) obtained from a control sample with a known "threshold" of the biomarker indicative of a control subject or population of control subjects with cancer.

[0114] With respect to the aforementioned aspects of the invention, the method preferably comprises the further step of treating cancer in the subject.

[0115] A further aspect of the invention relates to treating cancer in a subject.

[0116] In one particular embodiment, the treatment for cancer is performed in conjunction with determining the expression level of one or more markers in an exosome sample from the subject, the markers being selected from the group consisting of apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl-tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, biglycan, and any combination thereof, and the treatment for cancer is initiated, continued, modified, or discontinued based on the determination made.

[0117] In a specific embodiment, the one or more markers are selected from the group consisting of versican core protein, nidogen-1, pentraxin 3, thrombospondin-1, and any combination thereof. More specifically, the one or more markers preferably include versican core protein, nidogen-1, and pentraxin 3, and optionally further include an additional biomarker of thrombospondin-1.

[0118] In this regard, it will be appreciated that the methods described herein for predicting and / or determining the responsiveness of a cancer to an anti-cancer drug may further comprise the step of administering to the mammal an anti-cancer treatment, e.g., a therapeutically effective amount of an anti-cancer drug. In a preferred embodiment, the anti-cancer treatment is administered if the gene and / or protein expression levels of one or more markers described herein are indicative of or correlate with a relatively high responsiveness of the cancer to the anti-cancer drug.

[0119] In other embodiments, the anti-cancer treatment is altered or discontinued if the gene and / or protein expression levels of one or more markers described herein are indicative of or correlate with the continued presence (e.g., minimal residual disease), progression or recurrence of the cancer, or lack or decreased responsiveness of the cancer to the anti-cancer treatment.

[0120] Preferably, the agent(s) are administered to the subject as a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, or excipient. In this regard, any dosage form and route of administration, such as those described herein, may be used to administer the compositions of the present invention to a subject.

[0121] Cancer treatments may include, but are not limited to, drug therapies, such as 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 (e.g., small organic or inorganic molecules), biomolecules (e.g., antibodies, inhibitory nucleic acids such as siRNA), or chemotherapeutic agents are referred to herein as "anti-cancer therapeutic agents" or "anti-cancer agents."

[0122] Methods of treating cancer may be prophylactic, preventative, or therapeutic, and may be suitable for treating cancer in mammals, particularly humans. As used herein, "treating," "treat," or "treatment" refers to a therapeutic intervention, course of action, or protocol that at least alleviates the symptoms of cancer after the cancer and / or its symptoms have at least begun to develop. As used herein, "preventing," "prevent," or "prevention" refers to a therapeutic intervention, course of action, or protocol that is initiated prior to the onset of cancer and / or symptoms of cancer in order to prevent, inhibit, or delay the onset or progression of the cancer or symptoms of cancer.

[0123] The term "therapeutically effective amount" describes the amount of a particular agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this may be the amount of a chemotherapeutic agent necessary to alleviate, relieve, and / or prevent cancer or a cancer-related disease, disorder, or condition. In some embodiments, a "therapeutically effective amount" is sufficient to reduce or eliminate the symptoms of cancer. In other embodiments, a "therapeutically effective amount" is an amount sufficient to achieve a desired biological effect, e.g., an amount effective to reduce or prevent cancer growth and / or metastasis.

[0124] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce a desired result without causing substantial cytotoxic effects in the subject. The effective amount of an agent useful for reducing, alleviating, and / or preventing cancer will depend on the subject being treated, the type and severity of any associated disease, disorder, and / or condition (e.g., the number and location of any associated metastases), and the method of administration of the therapeutic composition.

[0125] Preferably, the anti-cancer therapeutic agent is administered to the mammal as a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, or excipient.

[0126] "Pharmaceutically acceptable carrier, diluent, or excipient" refers to a solid or liquid filler, diluent, or encapsulating substance that can be safely used in systemic administration. A variety of carriers known in the art can be used depending on the particular route of administration. These carriers can be selected from the group consisting of sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, liposomes and other lipid-based carriers, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifiers, isotonic saline, and salts, such as mineral acid salts, e.g., hydrochlorides, bromides, and sulfates, organic acids, e.g., acetates, propionates, and malonates, and pyrogen-free water.

[0127] A useful reference describing pharmaceutically acceptable carriers, diluents, and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991), which is incorporated herein by reference.

[0128] Any safe route of administration may be used to administer the compositions of the present invention to a patient, including oral, rectal, parenteral, sublingual, buccal, intravenous, intraarticular, intramuscular, intradermal, subcutaneous, inhalation, intraocular, intraperitoneal, intracerebroventricular, transdermal, etc. Intramuscular and subcutaneous injections are suitable, for example, for administering immunotherapeutic compositions, proteinaceous vaccines, and nucleic acid vaccines.

[0129] Dosage forms include tablets, dispersions, suspensions, injectables, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches, and the like. These dosage forms may also include injecting or implanting controlled-release devices specifically designed for this purpose, or other forms of implants modified to further act in this manner. Controlled release of therapeutic agents may be achieved, for example, by coating the therapeutic agent with hydrophobic polymers such as acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids, and certain cellulose derivatives such as hydroxypropylmethylcellulose. Additionally, controlled release may be achieved using other polymer matrices, liposomes, and / or microspheres.

[0130] Compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units 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 suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy, but all methods include the step of bringing into association one or more of the above-described agents with the carrier, which constitutes one or more necessary ingredients. Generally, the compositions are prepared by uniformly and intimately admixing an agent of the present invention with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired presentation.

[0131] The compositions can be administered in a manner compatible with the dosage form and in an amount that is pharmaceutically effective.The dosage administered to a patient in the context of the present invention should be sufficient to bring about a beneficial response in the patient over a reasonable period of time.The amount of drug(s) administered can depend on the subject being treated, such as the age, sex, weight, and general health of the subject, factors that depend on the judgment of a physician.

[0132] In certain embodiments, anti-cancer treatments and / or anti-cancer agents may be aimed at inhibiting the action of and / or reducing the expression of one or more markers.

[0133] In other embodiments, the anti-cancer treatment and / or anti-cancer agent may be aimed at preventing or inhibiting metastasis of the cancer.

[0134] In alternative embodiments, anti-cancer treatments and / or agents may be directed at genes or gene products other than one or more markers of the invention. By way of example, anti-cancer treatments may target genes or gene products known to interact directly or indirectly with one or more markers.

[0135] In certain embodiments, the present invention provides a "companion diagnostic" in connection with a cancer treatment, whereby the expression level of one or more markers of the present invention provides clinicians and others with information to use in the safe and / or effective administration of said cancer treatment.

[0136] Preferably, but not limited to, the cancer is of the types described below.

[0137] In certain embodiments, the method further comprises comparing the expression level of one or more markers in the exosome sample to a reference exosome expression level or a control exosome expression level of the one or more respective markers, e.g., as described below.

[0138] As with the foregoing aspects, the methods preferably include an initial step of obtaining an exosome sample from a subject, e.g., a biological sample, and / or an isolation method as described below.

[0139] In certain embodiments, the methods of the foregoing aspects further comprise the additional step of determining the subject's cancer type, which may be utilized, for example, in determining the cancer aggressiveness, prognosis, treatment regimen, and / or treatment responsiveness of the subject's cancer.

[0140] In a further aspect, the present invention provides a method for producing a composition comprising: (a) contacting a candidate agent with a cell expressing a marker selected from the group consisting of apolipoprotein E, serine protease 23, versican core protein, hyaluronan and proteoglycan link protein 3, type IV collagen alpha 1 chain, nidogen-1, connective tissue growth factor, type IV collagen alpha 2 chain, carboxypeptidase D, collagen and calcium-binding EGF domain-containing protein 1, pentraxin 3, testican-1, aminoacyl-tRNA synthase complex-interacting multifunctional protein 1, thrombospondin-1, biglycan, and any combination thereof; (b) determining whether the candidate agent modulates the expression and / or activity of the marker; The present invention provides a method for identifying or manufacturing an agent for use in treating cancer in a subject, comprising:

[0141] In certain embodiments, a candidate agent at least partially reduces, eliminates, suppresses, or inhibits the expression and / or activity of a marker.

[0142] Preferably, the agent has or exhibits few or no significant off-target and / or non-specific effects.

[0143] Preferably, the agent is an antibody or a small molecule.

[0144] Preferably, the marker is selected from the group consisting of versican core protein, nidogen-1, pentraxin 3, thrombospondin-1, and any combination thereof.

[0145] In embodiments relating to antibody inhibitors, the antibodies may be polyclonal or monoclonal, natural or recombinant. Known protocols applicable to the production, purification, and use of antibodies can be found, for example, in Chapter 2 of Coligan et al., CURRENT PROTOCOLS 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.

[0146] Generally, the antibodies of the present invention bind to or conjugate with an isolated protein, fragment, variant, or derivative of a marker. For example, the antibody can be a polyclonal antibody. Such antibodies can be prepared, for example, by injecting an isolated protein, fragment, variant, or derivative of a marker protein product into a production species, which can include mice or rabbits, to obtain polyclonal antisera. Methods for producing polyclonal antibodies are known to those skilled in the art. Exemplary protocols that can be used are described, for example, in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra, and Harlow & Lane, 1988, supra.

[0147] Monoclonal antibodies may be produced using standard methods, e.g., as described in the article by Kohler & Milstein, 1975, Nature 256, 495 (incorporated herein by reference), or by more recent modifications, e.g., as described in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra, by immortalizing spleen or other antibody-producing cells from a production species that has been inoculated with one or more of the isolated marker protein products, and / or fragments, variants, and / or derivatives thereof.

[0148] Typically, the inhibitory activity of a candidate inhibitor antibody can be assessed by in vitro and / or in vivo assays that detect or measure the expression level and / or activity of a marker protein in the presence of the antibody.

[0149] In some embodiments, modulators, such as inhibitors, may be rationally designed. These methods may involve structural analysis of the marker and the design and / or construction of molecules that bind to, interact with, or otherwise modulate the activity of the marker. These methods may particularly involve computer-assisted three-dimensional modeling of the interaction of candidate modulators with the marker.

[0150] In other embodiments of modulators such as small organic molecule inhibitors, this may involve screening large compound libraries amounting to hundreds of thousands to millions of candidate inhibitors (synthetic small organic molecules or natural products, e.g., compounds such as inhibitory peptides or proteins), which can be screened or tested for biological activity at any one of hundreds of molecular targets to discover potential new drugs or lead compounds. Screening methods may include, but are not limited to, computer-based ("in silico") screening and high-throughput screening based on in vitro assays.

[0151] Typically, the active compounds, or "hits," from this initial screening process are then successively tested in a series of other in vitro and / or in vivo tests to further characterize the active compounds, with fewer and fewer "successful" compounds at each stage being selected for further testing, and ultimately, one or more drug candidates being selected to proceed to testing in human clinical trials.

[0152] At the clinical level, screening of candidate drugs can include obtaining samples from test subjects before and after the test subjects are exposed to a test compound.Then, the level of marker protein in the sample, for example, an exosome sample, can be measured and analyzed to determine whether the level and / or activity of this marker protein changes after exposure to the candidate drug.For example, the protein product level in the sample can be determined by mass spectrometry, Western blot, ELISA, electrochemistry, and / or any other suitable means known to those skilled in the art.

[0153] In this regard, candidate agents that have been identified as being capable of reducing, eliminating, suppressing, or inhibiting the expression level and / or activity of the marker may then be administered to patients suffering from cancer. For example, if elevated activity of a biomarker is at least partially responsible for the progression and / or development of cancer, then administering a candidate agent that inhibits or reduces the activity and / or expression of that marker may treat cancer and / or reduce the risk of cancer.

[0154] In a final aspect, the invention provides an agent identified or produced according to the preceding aspects for use in accordance with the methods described herein.

[0155] With respect to the foregoing embodiments, the term "subject" includes, but is not limited to, mammals, including humans, performance animals (e.g., horses, camels, greyhounds), livestock (e.g., cattle, sheep, horses), and companion animals (e.g., cats and dogs). Preferably, the subject is a human.

[0156] All computer programs, algorithms, patent and scientific literature referred to herein is incorporated by reference.

[0157] For purposes of the present invention, the database accession numbers or unique identifiers described herein for genes or proteins such as those presented herein, and gene and / or protein sequences or sequences associated therewith, are hereby incorporated by reference.

[0158] For a more complete understanding and ability to practice preferred embodiments of the present invention, reference is made to the following non-limiting examples. [Example]

[0159] In recent years, small extracellular vesicles called exosomes have been shown to potentially serve as a 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 the cell of origin, and it is now clear that exosomes are a viable source of material for diagnostic and prognostic purposes. However, there is currently a lack of available evidence for specific markers that distinguish cancer cell-derived exosomes from normal cells. Identifying cancer-specific exosomal markers could enable the identification of cancer patients and potentially improve survival rates.

[0160] Numerous attempts have been made to identify optimal analyses of human biofluids to confirm the presence of cancer, such as analysis of tumor-secreted factors and circulating tumor DNA (ctDNA). Cancer-derived exosomes, due to their long half-life and active exosome secretion process, may serve as superior fluid biomarkers compared to cDNA, thereby demonstrating the consistent presence of cancer antigens or biomarkers. In this example, we describe a comprehensive clinical assay utilizing cancer-derived exosomal proteins for cancer diagnosis. The clinical performance of this assay is highly sensitive and specific, detecting cancer at early stages 1, thereby providing many cancer patients with the opportunity for early detection and improved survival.

[0161] cell culture Cell line authentication was performed using short tandem repeat profiling. p53 knockdown and Kras v12 overexpression (30KT p53 / KRAS Isogenic immortalized normal human bronchial epithelial cells (HBEC30KT) transformed with HBEC-1 were received from Dr. Jill Larsen. 22,27HBECs were cultured in keratinocyte serum-free medium (KSFM) supplemented with EGF (5 ng / mL) and bovine pituitary extract (50 mg / L) at 37°C under 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 under 5% CO2. Conditioned medium (CCM) was harvested from cells cultured in serum-free medium. CCM was collected by centrifugation at 100,000 g. avg Bovine exosomes were harvested from HBEC cells in KSFM depleted by overnight centrifugation at RT.

[0162] Exosome isolation and analysis Exosomes were isolated and analyzed as previously described 8,28 For exosomes for mass spectrometry analysis, the CCM was centrifuged at 300 g for 10 min at 4 °C and filtered through a 0.22 μm filter to remove floating cells and large extracellular vesicles. The clarified CCM was then concentrated to 500 μL, overlaid onto a discontinuous iodixanol density gradient, and centrifuged at 100,000 g at 4 °C. avg The exosome-containing fraction was diluted to 20 mL with PBS and centrifuged at 100,000 g for 2 hours at 4°C. avg The exosomes were then centrifuged at 100°C for 10 min. The resulting pellet was resuspended in PBS and stored at -80°C until use. Similarly, all other exosome isolates from in vitro CCM were clarified and concentrated as described above and then purified using size-exclusion chromatography. For exosome isolation from human plasma, plasma was thawed at room temperature and prepared by centrifugation at 1,500 g and 10,000 g for 10 and 20 min, respectively, to remove remaining platelets and large vesicles. The prepared plasma was overlaid onto a size-exclusion column, subsequently eluted with PBS, concentrated with an Amicon® Ultra-4 10 kDa nominal molecular weight centrifugal filter unit, and stored at -80°C until use. Exosome isolation from cell culture and human plasma was performed as previously described. 8, Western blot, tunable resistive pulse sensing (TRPS), and transmission electron microscopy.

[0163] Antibodies and reagents 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 VCAN ELISA kits were purchased from Novus Biologicals. qEV columns were purchased from Izon and stored in PBS (0.1% sodium azide) at 4°C.

[0164] Western blot analysis Western blots were performed as previously described 1,2 Briefly, proteins were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, blocked with 5% nonfat dry milk in PBS-T (0.5% Tween-20), and probed with antibodies. Protein bands were detected with enhanced chemiluminescence reagents (Amersham ECL Select).

[0165] mass spectrometry Exosome preparations were reduced by the addition of 10 mM dithiothreitol in the presence of 2% SDS, protease inhibitors (Sigma-Aldrich, P8340), and 50 mM Tris.HCl pH 8.8 (1 h at 4°C, 2 h at 22°C). Samples were then alkylated by the addition of iodoacetamide to 25 mM (1 h at 22°C) and methanol co-precipitated with trypsin (1:100 enzyme:substrate) overnight at -20°C. The pellet was resuspended in 10% acetonitrile, 40 mM ammonium bicarbonate, and digested for 8 h at 37°C, with an additional 2 h of trypsin added (1:100 enzyme:substrate).

[0166] LCMS analysis of the acidified digest (trifluoroacetic acid) was performed using a NanoAcquity UPLC (Waters) coupled to an Elite Orbitrap ETD mass spectrometer (Thermo Fisher Scientific). Two micrograms of digest were loaded onto a 20 mm × 180 μm Symmetry C trap (Waters) and separated on a 200 mm × 75 μm BEH130 1.7 μm column (Waters) over 120 min using a linear gradient (Buffer A: 0.1% formic acid in water; Buffer B: 0.1% formic acid in acetonitrile) from 2% B to 5% B over 5 min, to 30% B over 75 min, to 50% B over 10 min, to 95% B over 5 min, and a 6 min hold with re-equilibration at 2% B. The eluate from this 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 top 15 MS data were acquired in an Orbitrap with a power supply voltage of 1.8 kV, a heated capillary temperature of 275 °C, and a resolution of 120,000 s, using an AGC of 1E6. MS2 data was acquired in an ion trap with an AGC of 1E4, and a maximum injection time of 50 ms. An MS1 ​​lock mass of 445.120024 was used.

[0167] Protein identification and label-free quantification were performed using MaxQuant (version 1.4.1.23 Peak lists were extracted from Xcalibur raw files (Thermo Fisher Scientific, Germany) using MaxQuant and the embedded database search engine Andromeda 4 Peptide-to-spectrum matches (PSMs) were assigned using the Quantitative Peptide Search (PSM) algorithm. The searched database consisted of the complete Homo sapiens proteome (88,378 standard sequences downloaded from www.uniprot.org August 2013). Reverse sequences and MaxQuant contaminant databases were also searched. Label-free quantitation was performed with the instrument type set to Orbitrap, precursor mass tolerance set to 20 ppm for the initial search and 4.5 ppm for the primary search, fragment ion mass tolerance set to 0.5 Da, enzyme specificity set to trypsin / P, allowing a maximum of two missing cleavages, carbamidomethylcysteine ​​specified as a fixed modification, and protein N-terminal acetylation, asparagine / glutamine deamidation, and methionine oxidation specified as variable modifications. Secondary peptide searches and matching between runs were allowed with default settings. For identification, the PSM and protein-level FDRs were set to 0.01. Default settings were applied for all other parameters. Protein estimation and label-free quantification by spectral counting (including normalization) were performed as previously described. 5 .

[0168] Patient cohort A retrospective analysis of a patient cohort of patient plasma / serum samples collected between 2001 and 2019 was performed.

[0169] statistical analysis All calculations were performed using GraphPad Prism version 6.0, EdgeR version 2.6.10, MedCalc version 16.8.4, and IPA. Differences in protein expression values ​​from in vitro exosomes were calculated using an unpaired Student's t-test. Subcellular localization of proteins was determined using IPA (QIAGEN Inc.). Mass spectrometry-derived spectral counts were assessed using a negative-binomial exact test, where a Benjamini-Hochber adjustment was applied to control FDR. Differences with p values ​​less than 0.05 were considered significant, except for an FDR threshold of 0.001 (*p<0.05, **p<0.01, ***p<0.001).

[0170] result Generating exosomal protein signatures of cancer We hypothesized that HBECs with oncogenic mutations would secrete exosomes with different exosomal protein profiles. (p53 / KRAS) We isolated exosomes secreted by HBECs. TEM, nanoparticle tracking, and Western blotting demonstrated that the exosomes displayed a typical size distribution and contained standard exosome markers (Figure 1A-C). The proteomes of normal and transformed HBECs were then evaluated using mass spectrometry. Label-free quantification by spectral counting identified 15 extracellular proteins that were upregulated in transformed HBECs compared with normal HBECs (Figure 1D). THBS1, NID1, PTX3, and VCAN were selected for further evaluation and confirmed by ELISA in HBECs (Figure 1E).

[0171] Current clinical management of all solid malignant tumors is guided by the histopathological and / or molecular characteristics of the primary tumor. However, the expression of biomarkers for tumor classification can be highly variable even within individual tumors. During cancer progression, cancer cells can exhibit a wide variety of phenotypes, some of which are caused by epigenetic changes, oncogenic transformation, and even changes in environmental factors. Even within specific cancers (lung, brain, melanoma), tumors are highly heterogeneous diseases, reflected by various clinical and molecular classifications. Given this, we evaluated the expression of these four proteins in additional cancer cell lines to determine whether these proteins are universally upregulated in various cancer lines or specific to certain subsets of cancer cells. To address this, we isolated exosomes from a total of 22 cell lines, including non-small cell lung cancer (NSCLC), glioblastoma (GBM), colorectal (CRC), breast (BCa), prostate (PCa), melanoma (MEL), esophageal (ECa), and ovarian (OVA) cancer. Interestingly, we found that all four markers, particularly NID1, were upregulated in cancer cell-derived exosomes compared to normal HBEC exosomes, regardless of cancer type.

[0172] Evaluation of exosomal protein signatures in cancer patients We then hypothesized that oncogenically induced changes in exosomes could be used as diagnostic biomarkers 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, brain, colorectal, prostate, melanoma, stomach, and esophageal cancer. The median age of the healthy controls and patients at diagnosis was 65.5 years.

[0173] Interestingly, the combined four-protein exosome signature (THBS1, NID1, PTX3, and VCAN) was elevated in exosomes derived from cancer subjects compared with healthy controls (Figure 2A). Each protein from the exosome signature had different diagnostic capabilities in different cancer cohorts, as assessed by receiver operating characteristic (ROC) curves (Figure 3). Interestingly, using a combination of signature proteins using logistic regression, we were able to achieve excellent separation between healthy controls and cancer patients, with an area under the curve (AUC) of 0.96 (Figure 2B). Importantly, at a fixed specificity of 95%, the median sensitivity of the diagnostic exosome signature in the eight cancer types was 77.6% (95% CI: 72.0%, 82.3%). This ranged from 44% for prostate cancer to 100% for gastric cancer (Figure 2C).

[0174] Next, we sought to evaluate the ability of diagnostic exosome signatures to identify patients with early-stage cancer. For liquid biopsies to be most beneficial, patients need to be identified at the earliest possible stage to significantly improve overall cancer survival. We were able to assess the sensitivity of exosome biomarkers in stage I compared with stages II-IV for NSCLC, esophageal cancer, and gastric cancer. Importantly, sensitivity at 95% specificity was comparable to later stages for all three cancers (Figure 4), demonstrating that diagnostic exosome signatures can identify patients with early-stage cancer.

[0175] Recently, CancerSEEK (REF) demonstrated that liquid biopsies can be used to identify not only the presence of cancer but also tumor type for clinical follow-up using machine learning. Therefore, we investigated whether our exosome signature could also accurately identify the type of cancer a patient had. The accuracy of this test varied widely, with NSCLC being the most accurate predictor and gastric cancer the least accurate (Figure 5).

[0176] References 1. Lobb, R.J., et al. Optimized exosomeisolation protocol for cell culture supernatant and human plasma. J ExtracellVesicles 4, 27031 (2015). 2. Lobb, R.J., et al. Exosomes derived frommesenchymal non-small cell lung cancer cells promote chemoresistance.International journal of cancer 141, 614-620 (2017). 3. Cox, J. & Mann, M. MaxQuant enableshigh peptide identification rates, individualized p.p.b.-range mass accuraciesand proteome-wide protein quantification. Nature biotechnology 26, 1367-1372(2008). 4. Cox, J., et al. Andromeda: a peptidesearch engine integrated into the MaxQuant environment. Journal of proteomeresearch 10, 1794-1805 (2011). 5. Dave, K.A., et al. A comprehensiveproteomic view of responses of A549 type II alveolar epithelial cells to humanrespiratory syncytial virus infection. Molecular & cellular proteomics: MCP13, 3250-3269 (2014).

Claims

1. 1. A method for diagnosing cancer or diagnosing the recurrence of cancer, comprising determining the expression levels of a plurality of markers in an exosome sample obtained from a subject, wherein the markers are VCAN, NID1, PTX3, and THBS1, and the expression levels of the plurality of markers are indicative of or correlated with a diagnosis or recurrence of the cancer, and the expression levels of the plurality of markers are indicative of or correlated with the cancer type of the subject.

2. 1. A method for determining a cancer type, comprising determining expression levels of a plurality of markers in an exosome sample obtained from a subject, wherein the markers are VCAN, NID1, PTX3, and THBS1, and the expression levels of the plurality of markers are indicative of or correlated with the cancer type.

3. A method according to claim 1 or 2, wherein (i) the expression level indicates a highly aggressive cancer or a less aggressive cancer, and / or (ii) the expression level indicates a poor prognosis or a better prognosis.

4. 3. The method of claim 1 or 2, further comprising the step of comparing the expression level of the plurality of markers in the exosome sample with a reference exosome expression level of each of the plurality of markers.

5. 3. The method of claim 1 or 2, wherein the cancer and / or cancer type is selected from the group consisting of lung cancer such as NSCLC and SCLC, 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.

Images