WO2010065968A1 - Marqueurs de détection du cancer - Google Patents

Marqueurs de détection du cancer Download PDF

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Publication number
WO2010065968A1
WO2010065968A1 PCT/US2009/067029 US2009067029W WO2010065968A1 WO 2010065968 A1 WO2010065968 A1 WO 2010065968A1 US 2009067029 W US2009067029 W US 2009067029W WO 2010065968 A1 WO2010065968 A1 WO 2010065968A1
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Prior art keywords
cancer
exosomes
exosome
status
patient
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PCT/US2009/067029
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English (en)
Inventor
Mark Skolnick
Jerry Lanchbury
Kirsten Timms
Chris Neff
Ryan Hoff
Hubert Wang
Susanne Wagner
Zaina Sangale
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Myriad Genetics, Inc.
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Publication of WO2010065968A1 publication Critical patent/WO2010065968A1/fr
Priority to US13/154,143 priority Critical patent/US20140113310A9/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the invention generally relates to a molecular classification of disease and particularly to molecular markers for cancer and methods of use thereof.
  • Cancer is a major health challenge. Nearly 560,000 people die from cancer annually in the United States alone, representing almost 23% of all deaths. See American Cancer Society, Cancer Facts & Figures 2008, 1 -2 (2008). Despite recent advances in molecular and imaging diagnostics, one of the most vexing aspects of cancer remains early detection. In fact, for certain types of cancer — e.g. , pancreatic adenocarcinoma — detection often occurs so late as to practically preclude any good prognosis. Thus there is an urgent need for sensitive methods of detecting cancer.
  • Small extracellular vesicles e.g. , exosomes
  • markers associated with them are often found in altered status in the samples of patients having certain diseases or disease-related pathologies as compared to samples from healthy individuals. This is especially true of epithelial cancers (e.g. , those of the lung, colon, breast, prostate, ovaries, endometrium, etc.), diabetic nephropathy, etc.
  • one aspect of the invention provides a method for detecting cancer in a patient comprising determining the status of exosomes and/or an exosome-associated marker in a sample obtained from a patient, wherein an abnormal exosome (and/or exosome-associated marker) status in the sample indicates the presence of cancer.
  • status is determined by determining the level of exosomes or an exosome-associated marker in the sample.
  • determining the level of exosomes in the sample comprises measuring the aggregate level of all exosomes in a sample.
  • status is determined by measuring the level of exosomes bearing a particular marker. This may comprise determining the status (e.g. , the level) of an exosome- associated marker.
  • exosome-associated marker may also comprise isolating exosomes from the sample and determining the status (e.g. , the level) of an exosome-associated marker.
  • the exosome-associated marker is a cancer-marker, a cancer-type marker, and/or a tissue-type marker. Examples of exosome-associated markers include those listed in Table 1.
  • Some markers are correlated with cancer of a specific type (e.g., cell type, tissue type, organ, clinical subtype, etc.).
  • a method of diagnosing a cancer in a patient comprising determining the status of an exosome-associated cancer-type marker in a sample obtained from the patient, wherein an abnormal status of the marker indicates the presence of a specific cancer type, or cancer in a specific tissue or organ, in the patient.
  • the exosome-associated marker is not necessarily correlated with cancer, but with a particular tissue type or organ. An example of such an analysis is given in FIG.2.
  • exosomes are isolated and the exosome-associated marker is then analyzed.
  • one embodiment of the invention provides a method of diagnosing a cancer in a patient comprising ( 1 ) isolating exosomes from a sample obtained from a patient and (2) determining the status of a cancer-type marker associated with the exosomes isolated in ( 1 ); wherein an abnormal status of the marker in (2) indicates the presence of indicates the presence of a specific cancer type, or cancer in a specific tissue or organ, in the patient.
  • An example of such an analysis is given in FIG.2.
  • Examples of cancer- type markers include some of those listed in Table 1 (e.g. , EGFRvIII, ErbB2).
  • Another aspect of the invention provides a method of screening for cancer in a patient comprising identifying a patient at risk of having cancer or in need of screening and determining the status of exosomes and/or an exosome- associated marker in a sample obtained from the patient, wherein an abnormal status of exosomes and/or the exosome-associated marker in the sample indicates the presence of cancer.
  • the patient is at risk for developing a specific cancer type and the abnormal status of exosomes and/or the exosome- associated marker indicates the presence of this specific cancer type.
  • Yet another aspect of the invention provides a method of detecting recurrence in a cancer patient comprising determining the status of exosomes and/or an exosome-associated marker in a sample obtained from a patient, wherein an abnormal status of exosomes and/or the exosome-associated marker in the sample indicates recurrence.
  • Still another aspect of the invention provides a diagnostic method comprising identifying a patient who is a candidate for biopsy and determining the status of exosomes and/or an exosome-associated marker in a sample obtained from the patient, wherein an abnormal status of exosomes and/or the exosome-associated marker in the sample indicates a biopsy is desirable. In some embodiments no abnormal status of exosomes and/or the exosome-associated marker indicates no biopsy is necessary.
  • One aspect of the invention provides a method of detecting a specific disease other than cancer (e.g. , rheumatoid arthritis, diabetic nephropathy) comprising determining the status of exosomes and/or an exosome-associated marker in a sample obtained from a patient, wherein an abnormal status of exosomes and/or the exosome-associated marker indicates the patient has the disease.
  • a specific disease other than cancer e.g. , rheumatoid arthritis, diabetic nephropathy
  • Exosomes can be isolated by various techniques including, but not limited to : centrifugation (e.g. , ultracentrifugation); physical filtration; affinity chromatography; contacting a sample with a solid surface (e.g. , plate, well, bead, etc.) containing an antibody against an exosome-associated marker in order to attach any exosomes to the surface; etc. or any combination of these.
  • exosome blocks comprising fixed (e.g. , formalin-fixed) exosomes embedded in some medium (e.g. , paraffin).
  • fixed exosome blocks allow for long-term storage of exosomes and also allow for analysis of the interior of exosomes.
  • FIG. 1 illustrates a method, according to the invention, for detecting cancer generally and also determining the specific cancer type
  • FIG.2 illustrates one embodiment of the invention using various biomarkers to determine which specific cancer is present in a patient.
  • FIGs. 3-7 illustrate differentiating cancer patients from cancer- free controls by determining exosome status using several different techniques.
  • blood contains various additional circulating elements. These include, but are not limited to, cellular debris (e.g., membrane fragments), free protein, protein aggregates, and small extracellular vesicles. It has been discovered that markers carried by these elements can be analyzed in order to detect cancer in patient samples. More specifically, it has been discovered that capturing these items with a capture reagent and then detecting them with a detection reagent can separate cancer patients from controls. For example, small extracellular vesicles called exosomes have been isolated from patient samples and their status analyzed by way of exosome-associated markers so as to differentiate healthy from diseased individuals. In another example cancer has been detected by subjecting plasma samples to various antibodies and integrins to capture circulating elements and analyzing these elements by way of detection antibodies.
  • cellular debris e.g., membrane fragments
  • free protein e.g., free protein aggregates, and small extracellular vesicles.
  • markers carried by these elements can be analyzed in order to detect cancer in patient samples. More specifically, it has
  • Cells form several different small vesicles within their interior that perform various functions. For example, lysosomes digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria. Some vesicles are trafficked from the cell interior to outside the cell. These include exosomes, which are found in the cell within the multi-vesicular body (MVB) and released into the extracellular matrix and into various bodily fluids such as blood, serum, urine, etc. These vesicles often bear, either inside or on their surface, various biological molecules such as protein, RNA, DNA, etc. Exosomes are secreted by a variety of cells, including those involved in immune response (T cells, B cells, dendritic cells, macrophages) and epithelial cells.
  • T cells T cells, B cells, dendritic cells, macrophages
  • epithelial cells include those involved in immune response (T cells, B cells, dendritic cells, macrophages) and epithelial cells.
  • Exosomes have been determined to be useful markers in detecting and characterizing cancer. For example, the status of exosomes in a sample has been found to correlate strongly with the presence of cancer in the patient from whom the sample was obtained. More specifically, elevated exosomes and/or exosome-associated markers in a patient sample indicate the patient has cancer. See Examples below. Further, exosome-associated markers can help in characterizing the patient's specific cancer (e.g., cell type, tissue type, organ, clinical subtype, etc.).
  • one aspect of the invention provides a method for determining whether an individual has cancer comprising determining the status of exosomes and/or an exosome-associated marker in a sample obtained from the individual, wherein an abnormal exosome (and/or exosome-associated marker) status indicates an increased likelihood of cancer.
  • the "status" of a biomolecular marker e.g. , exosomes, exosome-associated markers, etc.
  • sequence information including mutations
  • copy number e.g., sequence information, including mutations
  • post- translational modification for proteins e.g., glycosylation
  • proteins e.g., glycosylation
  • these may be assayed directly (e.g. , by assaying the expression level of a particular gene) or determined indirectly (e.g. , assaying the level of marker whose expression level is correlated to the expression level of the gene of interest).
  • determining the status of exosomes and/or an exosome-associated marker comprises determining their level in a sample.
  • the "level" of something in a sample has its conventional meaning in the art. Determining a "level” therefore includes quantitative determinations — e.g. , mg/mL, fold change, etc. Determining a "level” herein also includes more qualitative determinations — e.g. , determining the presence or absence of a marker or determining whether the level of the marker is "high,” “low” or even “present” relative to some index value.
  • Abnormal status means a marker 's status in a particular sample differs from the reference status for that marker (e.g. , in healthy samples or average diseased samples). Examples include mutated (when the sequence or structure of the marker is analyzed), elevated, decreased, present, absent, etc. For example, determining the status of an extracellular protein marker will often include determining its level in a sample (e.g., on the surface of exosomes). An abnormal status could be either lower (including undetectable) or higher levels (including anything non-zero) compared to the index value in exosome samples from healthy patients. Another example of abnormal status includes a sequence (i. e.
  • a patient has an "increased likelihood of cancer” if the status of the relevant marker in the patient's sample is correlated with cancer. Examples include mutations in particular genes correlated with cancer, a level of the marker that is closer to some cancer index value than to a normal index value, a level of the marker that exceeds some threshold value where exceeding that value is correlated with cancer, etc.
  • “increased likelihood of cancer” means a patient with an abnormal status for a marker has a higher likelihood of cancer than if the patient did not have an abnormal status.
  • An "elevated status” means that one or more of the above characteristics (e.g. , expression) of the marker of interest is higher than normal levels. Generally this means an increase in the characteristic (e.g., expression) as compared to an index value. Conversely a “low status” means that one or more of the above characteristics (e.g. , expression) is lower than normal levels. Generally this means a decrease in the characteristic (e.g. , expression) as compared to an index value. In this context, a “negative status” generally means the characteristic is absent or undetectable (which would include expression, copy number, methylation, etc.).
  • the level of a marker is determined within one or more samples as compared to some index value.
  • the index value may represent the average (e.g., mean) level in a plurality of training patients (e.g. , both diseased and healthy patients).
  • a "cancer index value” can be generated from a plurality of training patients characterized as having cancer.
  • a "cancer-free index value” can be generated from a plurality of training patients defined as not having cancer.
  • a cancer index value may represent the average level of a marker (e.g.
  • a cancer-free index value may represent the average level of the marker in patients not having cancer.
  • index values may be determined thusly:
  • a threshold value may be set for the marker.
  • the optimal threshold value is selected based on the receiver operating characteristic (ROC) curve, which plots sensitivity vs. (1 - specificity).
  • ROC receiver operating characteristic
  • the sensitivity and specificity of the test is calculated using that value as a threshold.
  • the actual threshold will be the value that optimizes these metrics according to the artisan' s requirements (e.g. , what degree of sensitivity or specificity is desired, etc.).
  • Determining the status of exosomes in a sample may also comprise assaying some marker whose status itself is correlated with exosome status.
  • This marker will often be an exosome-associated marker.
  • Exosome-associated marker means a biomolecule inside or on the surface of an exosome.
  • exosomes carry cellular biomolecules such as proteins and nucleic acids (e.g. , mRNA, microRNA, etc.) within their lipid membrane. Since exosomes are formed from membranes of the cell, they also carry molecules (e.g. , cell- surface molecules such as CD63 or EpCAM) on their surface. Additionally, since some biomolecules bind those on the surface of exosomes (e.g.
  • the invention need not be limited by the actual physical element that is measured in the claimed method as long as the status of an exosome-associated marker such as those listed in Table 1 is determined. Thus, the invention envisions detecting an exosome-associated marker even after such marker has been separated from the exosome with which it is sometimes associated.
  • the invention provides a method of detecting cancer in a patient comprising determining the status of an exosome-associated marker (e.g. , the markers listed in Table 1 ) in a sample obtained from the patient, wherein an abnormal status for such exosome-associated marker indicates an increased likelihood of cancer.
  • an exosome-associated marker e.g. , the markers listed in Table 1
  • Markers useful in this aspect include those listed in Table 1 , EpCAM, MUC l , CDH l , CK7, PSA, etc.
  • the invention provides a method of detecting cancer comprising determining the status of at least one marker chosen from the group consisting of: ADAM l O, ⁇ V ⁇ integrin, Caveolin- 1 , CD 147, CD36, CD63 , CD81 , Claudin-3 , Claudin-4, Desmocollin- 1 , EGFR, EGFRvIII, EMP-2, EpCAM, ErbB2, GP I b, HLA-DR, Hsp70, Hsp90, MFG-E8, Rab l 3 , and Tissue Factor, wherein an abnormal status indicates an increased likelihood of cancer.
  • a marker chosen from the group consisting of: ADAM l O, ⁇ V ⁇ integrin, Caveolin- 1 , CD 147, CD36, CD63 , CD81 , Claudin-3 , Claudin-4, Desmocollin- 1 , EGFR, EGFRvIII, EMP-2, EpCAM, ErbB2, GP I b, HLA
  • Patient blood samples are complex mixtures of circulating elements including, but not limited to, cellular debris (e.g. , membrane fragments), free protein, protein aggregates, and small extracellular vesicles (e.g. , exosomes).
  • the patient blood sample is enriched for exosomes and/or exosome-associated markers.
  • sample refers to any biological specimen, including any tissue or fluid, that can be obtained from, or derived from a specimen obtained from, a human subject. Such samples include, healthy or tumor tissue, bodily fluids, waste matter (e.g., urine, stool), etc.
  • the sample is blood or any substance derived therefrom — e.g. , serum or plasma.
  • the process of extracting plasma from blood, by removing the blood cells helps to enrich for exosomes and/or exosomes-associated markers. Further purifying plasma to serum, by removing fibrinogen and other clotting factors, further enriches the sample. Additional enrichment/purification may be performed by various techniques discussed in more detail below including, but not limited to, ultracentrifugation, filtration, affinity chromatography, antibody capture, cell- sorting, etc. Though the methods of the invention can differentiate cancer patients from controls in both plasma and serum samples, it has been discovered that plasma samples yield better (i.e. , clearer) differentiation. See FIG.4.
  • the invention provides a method of detecting cancer comprising enriching a sample for exosomes and determining the status of the exosomes and/or an exosome-associated marker, wherein an abnormal status for exosomes and/or the exosome-associated marker indicates an increased likelihood of cancer.
  • the exosome-associated marker is chosen from those listed in Table 1.
  • the sample is blood plasma and the sample is enriched by a technique chosen from the group consisting of ultracentrifugation, filtration, affinity chromatography, antibody capture and cell- sorting.
  • the sample is enriched using ultracentrifugation and the resulting exosomes (or other circulating elements) are coated onto a solid surface for subsequent detection (see Examples 2, 4 & 5).
  • the sample is enriched using a capture antibody specific for one of the markers listed in Table 1 and the exosomes and/or exosome-associated markers are detected using an antibody specific for another marker listed in Table 1.
  • the inventors have devised assays aimed at measuring exosomes and exosome-associated markers from patient blood, plasma and serum, some aspects of the invention need not be limited to the particular circulating element that is actually measured. Specifically, in some examples the inventors have attempted to isolate/purify exosomes from blood (or plasma or serum) samples using antibody or integrin capture, ultracentrifugation and/or filtration, followed by detection (e.g., via ELISA, ECL, flow cytometry, Bradford protein measurement, etc.). See Examples 1 , 2 & 5. In some cases, however, it is ultimately the choice of markers and/or assays that has enabled cancer detection regardless of whether exosomes themselves (or some other circulating element) have been directly measured.
  • exosomes other than using distinct capture and detection reagents e.g. , anti-CD63 antibody for capture and anti-Claudin-3 antibody for detection.
  • distinct capture and detection reagents e.g. , anti-CD63 antibody for capture and anti-Claudin-3 antibody for detection.
  • Such an assay should instead detect exosomes or small membrane fragments, protein complexes, etc. containing exosome-associated markers (e.g. , those listed in Table 1 ).
  • one aspect of the invention provides a method of detecting cancer comprising determining the status of at least two markers chosen from those listed in Table 1 in a patient sample, wherein an abnormal status for at least one of the markers indicates an increased likelihood of cancer.
  • one of the markers is used to capture circulating elements from the sample while another of the markers is used to detect (e.g. , quantify) these elements.
  • the level of the markers is determined using an antibody specific for each marker.
  • the status of the capture marker is its presence or absence, which is determined by attaching the capture marker to a solid surface via a capture reagent (e.g., antibody, nucleic acid probe, etc.), while the status of the other marker is its level as determined via a detection reagent (e.g. , antibody, nucleic acid probe, etc.).
  • a capture reagent e.g., antibody, nucleic acid probe, etc.
  • a detection reagent e.g. , antibody, nucleic acid probe, etc.
  • the exosome-associated marker is a cancer marker.
  • cancer marker means a biomarker whose status is correlated with the presence of cancer. There need not be a strong correlation between the biomarker and any specific cancer type. Examples include carcinoembryonic antigen (CEA) and epithelial membrane antigen (EMA). Another example includes EpCAM, which is hyperglycosylated in carcinoma tissue as compared to corresponding normal epithelial tissue. See, e.g., Munz et al., FRONT BiOSCI. (2008) 13 : 5195-5201 .
  • some embodiments provide a method of detecting cancer comprising providing a sample obtained from a patient and determining the level of exosomes having a general cancer marker (e.g. , hyperglycosylated EpCAM), wherein an increased level of these exosomes indicates an increased likelihood of cancer.
  • a general cancer marker e.g. , hyperglycosylated EpCAM
  • markers are correlated with cancer of a specific type (e.g., cell type, tissue type, organ, clinical subtype, etc.).
  • certain biomarkers can be used to differentiate exosomes derived from epithelial cells from other exosomes, e.g. , immune cell-derived.
  • One particularly useful marker in detecting, quantitating, collecting, and/or analyzing epithelial exosomes is EpCAM, a protein expressed on the surface of many epithelial cells.
  • Other epithelial markers useful in the invention include, but are not limited to, CDH l (cadherin 1 , type 1 , E-cadherin [epithelial]) and cytokeratin 7 (CK7 or KRT7).
  • Elevated levels of epithelial exosomes are often found in individuals having an altered physiological condition. For example, pregnant women show higher levels of epithelial exosomes in their serum, as do diabetic patients suffering from nephropathy. Further, epithelial exosomes are more abundant in samples from cancer patients than in those from cancer-free controls.
  • Another aspect of the invention provides a method of diagnosing a cancer in a patient comprising determining the status of an exosome- associated cancer-type marker in a sample obtained from the patient, wherein an abnormal status of the marker indicates the presence of a specific cancer type, or cancer in a specific tissue or organ, in the patient.
  • exosomes are isolated to some degree and the cancer-specific exosome-associated marker is then analyzed.
  • one embodiment of the invention provides a method of diagnosing a cancer in a patient comprising ( 1 ) isolating exosomes from a sample obtained from a patient and (2) determining the status of a cancer-type marker associated with the exosomes isolated in ( 1 ); wherein an abnormal status of the marker in (2) indicates the presence of indicates the presence of a specific cancer type, or cancer in a specific tissue or organ, in the patient.
  • cancer- type markers include some of those listed in Table 1 (e.g., EGFRvIII, ErbB2). An example of such an analysis is given in FIG.2.
  • cancer type means a cancer in or originating from a particular tissue or organ and/or a cancer with a particular molecular or clinical feature.
  • tissue type e.g. , squamous versus cuboidal
  • organ type e.g. , breast versus lung
  • clinical subtype e.g. , triple-negative breast cancer.
  • finding certain markers e.g. , surfactant proteins B [SFTPB] and C [SFTPC]
  • SFTPB surfactant proteins B
  • C [SFTPC] surfactant proteins
  • Breast cancer cells can show overexpression of HER2, which in turn correlates with a particular clinically-defined subtype of breast cancer. Knowing that the patient has breast cancer will generally prompt a physician to treat in a particular way (e.g. , surgery, hormone therapy, and optional adjuvant chemotherapy), while the additional knowledge that the patient has HER2-overexpressing breast cancer can prompt further personalization of that treatment (e.g. , adding trastuzumab).
  • the present invention provides various methods of determining what cancer type a patient has.
  • cancer type may be determined by more traditional diagnostic methods such as imaging (e.g., CAT scan, MRI, X-ray, etc.), physical examination (e.g. , digital rectal examination — DRE — in prostate cancer), biopsy, etc.
  • imaging e.g., CAT scan, MRI, X-ray, etc.
  • physical examination e.g. , digital rectal examination — DRE — in prostate cancer
  • biopsy e.g., a biopsy, etc.
  • one aspect of the invention provides a method of determining whether a patient has a specific cancer type, comprising determining the status of exosomes and/or an exosome-associated marker in a sample obtained from the patient, wherein an abnormal status of such exosomes and/or the exosome- associated marker indicates the presence of cancer, and performing a physical examination, imaging test, or biopsy to determine the specific cancer type.
  • molecular diagnostics may give cancer-type specificity.
  • the cancer marker in the above embodiments may simultaneously be a cancer-type marker.
  • cancer-type marker means a biomarker whose status is correlated with the presence of a specific cancer type.
  • PSA prostate-specific antigen
  • one embodiment provides the quantification of PSA-bearing exosomes as a substitute for measuring PSA in the serum, which may be a more clinically accurate measure of cancer-relevant PSA.
  • Another antigen of particular interest is CA- 125. It is frequently expressed in ovarian tumors.
  • CA- 125 is somewhat non-specific for tumor type in that it is expressed in gynecological cancers other than ovarian and in colon cancer. Thus elevated levels of CA- 125-bearing exosomes could indicate the value of more expensive and invasive tests such as an MRI of the abdominal/pelvic region, a pelvic exam, a colonoscopy and possibly biopsies.
  • EGFRvIII whose expression is correlated with a particular form of glioblastoma (i. e. , particular mutated EGFR status is correlated with a specific clinical subtype of glioblastoma).
  • glioblastoma i. e. , particular mutated EGFR status is correlated with a specific clinical subtype of glioblastoma.
  • additional examples e.g. , HER2, EGFR, KRAS, etc.
  • tissue-specific marker is a biomarker whose status is correlated with a specific tissue or organ type, though not necessarily with cancer.
  • tissue-specific markers as well as the tissues and/or organs with which they are correlated, are given in FIG.2. This additional marker is generally, though not necessarily, exosome-associated.
  • one aspect of the invention provides a method of determining whether a patient has a specific cancer type, comprising determining the status of exosomes and/or an exosome-associated marker (e.g. , those listed in Table 1 ) in a sample obtained from the patient and determining the status of at least one exosome-associated tissue-specific marker in a sample obtained from the patient, wherein an elevated level of exosomes and/or the exosome-associated marker indicates the patient has cancer and wherein a particular status of the exosome-associated tissue-specific marker indicates the patient has a specific cancer type.
  • an exosome-associated marker e.g. , those listed in Table 1
  • a particular biomolecule is exclusively or predominantly found in or produced by a particular tissue type or organ.
  • tissue type or organ examples include EpCAM (epithelial tissue), SFTPB (lung epithelium), PSA (prostate tissue), etc.
  • GPA33 glycoprotein A33 [transmembrane] is expressed in the epithelium of the colon and small intestine. See, e.g., Heath et al., PROC . NATL. ACAD . SCI. USA ( 1997) 94 :469- 474.
  • one skilled in the art could, according to the present invention, determine the level of exosomes (e.g., by determining the level of one or more of the exosome-associated markers listed in Table 1 ) and also determine whether or to what extent (e.g. , what proportion of) such exosomes also bear the GPA33 antigen. If exosome levels are elevated and some significant proportion of these exosomes also bear the GPA33 antigen, then the patient has colon cancer. In other embodiments for example, determining that exosome levels are high and PSA is present (particular PSA "status") will indicate the patient has prostate cancer. Other times, particular biomolecules are upregulated in specific tissue types or organs (i. e. , found in higher levels in some tissues or organs as compared to others).
  • one aspect of the invention provides methods comprising determining the status of a panel of markers.
  • the invention provides a method of determining whether a patient has cancer comprising determining the status of a panel of exosome-associated markers in a sample obtained from the patient, wherein a particular status of the panel of markers indicates the patient has cancer.
  • the invention provides a method of determining whether a patient has a specific cancer type, comprising determining the status of a panel of cancer-type markers in a sample obtained from the patient, wherein a particular status of the panel of markers indicates the patient has a specific cancer type.
  • the invention provides a method of determining whether a patient has a specific cancer type, comprising determining the status of exosomes and/or an exosome-associated marker in a sample obtained from the patient and determining the status of a panel of tissue-specific markers in a sample obtained from the patient, wherein an abnormal status of exosomes and/or the exosome-associated marker indicates the patient has cancer and wherein a particular status of the panel of markers indicates the patient has a specific cancer type (see, e.g., FIG.2).
  • the panel of markers comprises 2, 3 , 4, 5 ,
  • the markers may be any exosome-associated markers, including cancer-type markers, or tissue-type markers or a combination of these.
  • the panel of markers comprises two or more markers listed in Table 1.
  • the panel of markers comprises two or more markers shown in FIG.2, wherein the presence or absence (or abnormal status) of specific markers indicates, according to the flowcharts in FIG.2, the patient has cancer of a specific type.
  • the panel comprises at least two markers chosen from those listed in Table 1.
  • the status of individual markers in the panel is tested in a certain order in order to narrow down which specific cancer type is present.
  • FIG.2A-2D One example is illustrated in FIG.2A-2D.
  • CK7 cytokeratin 7
  • CK20 cytokeratin 20
  • PSA, PSAP, PSMA, Hep Par 1 , AFP, CAM 5.2, CD l O, Vimentin, RCC, and EMA may be tested [210] to determine the specific organ/tissue of origin.
  • the cancer is prostate adenocarcinoma [310] . If Hep Par 1 , AFP, and/or CAM 5.2 are present, then the cancer is hepatocellular carcinoma [311] . If CD l O, Vimentin, RCC, and/or EMA are present, then the cancer is renal cell carcinoma (clear cell type) [312] .
  • Ae l/3 , CAM 5.2, CK19, CEA (polyclonal), and EMA (or any combination thereof or any single marker) may be tested [220] to confirm that the cancer is colon adenocarcinoma. If any of these markers is found, then the cancer is colon adenocarcinoma [320] . Imaging and/or endoscopy may be performed [420] either in place of the additional marker tests [320] or as an additional confirmation. [0047] If CK7 is present and CK20 is absent as in FIG.2C [130] , then
  • the cancer is breast cancer [332] . If Chromogranin, Synaptophysin, CD56, (NCAM), and/or Leu7 are found, then the cancer is small cell/neuroendocrine carcinoma of the lung [336] . If CK5/6 is found, then the cancer may be squamous cell carcinoma of the lung [337] (diagnosis may be confirmed by imaging [430] ). CEA, Mucicarmine, B72.3 , and/or Leu M l (CD 15) are found, then the cancer may be adenocarcinoma of the lung [338] (diagnosis may be confirmed by imaging [430] ).
  • the cancer may be mesothelioma [333] (if the only marker found is CK5/6, imaging [430] may be necessary). If Vimentin is found, then the cancer is endometrial cancer [334] . If CK5/6 and/or CEA are found, then the cancer may be cervical cancer [332] (confirmation, e.g. , by pap smear, may be necessary since these markers are also expressed by other CK7+/CK20- tissue types).
  • the cancer may be tested [240] to determine the specific organ/tissue of origin. If CA- 125 and/or Mesothelin are found, then the cancer may be ovarian carcinoma [340] (confirmation, e.g. , by imaging, may be necessary since CA- 125 is also expressed in other CK7+/CK20+ tissues). If 34 ⁇ E 12, Villin, and/or CA- 125 are present, then the cancer may be cholangio carcinoma (bile duct cancer) [341] (confirmation, e.g.
  • the cancer by imaging, may be necessary since CA- 125 is also expressed in other CK7+/CK20+ tissues). If Uroplakin III is found, then the cancer is urothelial carcinoma [342] . If CD l O is found, then the cancer is papillary-type renal cell carcinoma [343] . If no marker is found, then the cancer may be chromophobe renal cell carcinoma [344] (diagnosis may be confirmed microscopically).
  • a diagnosis of the desired specificity requires more than simply determining the status of exosomes and/or an exosome-associated marker.
  • Many physiological conditions other than cancer are characterized by increased exosome secretion, including rheumatoid arthritis.
  • epithelial exosomes are elevated in pregnant women and in patients with diabetic nephropathy.
  • alternative conditions must be ruled out before a diagnosis of cancer can be made. This may require additional tests specific for the given alternative condition, e.g. , a pregnancy test.
  • one aspect of the invention provides a method for determining whether a patient has cancer comprising determining the status of exosomes and/or an exosomes-associated marker in a sample from this patient and determining whether the patient has a non-cancerous condition characterized by increased exosome secretion, wherein an abnormal status of exosomes (e.g. , EpCAM-bearing exosomes, CD63-bearing exosomes, etc.) and/or the exosome- associated marker and the absence of any non-cancerous condition characterized by increased exosome secretion indicates the patient has cancer.
  • an abnormal status of exosomes e.g. , EpCAM-bearing exosomes, CD63-bearing exosomes, etc.
  • Examples of how one might determine whether a patient has a noncancerous condition characterized by increased exosome secretion include, but are not limited to, determining whether the patient is pregnant (e.g. , by a pregnancy test), determining whether the patient is diabetic (or determining whether the patient shows signs of diabetic nephropathy), etc.
  • oncosomes are especially useful according to the present invention in detecting, classifying and monitoring cancer since they are particularly prevalent in epithelial cancers such as those of the lung, colon, breast, prostate, ovaries, endometrium, etc.
  • One way to differentiate oncosomes from noncancerous exosomes is by determining the status of some exosome-associated biomarker. Generally, the methods of the invention will involve determining whether a particular marker has an abnormal status, when such abnormal status is somehow associated with cancer cells.
  • Determining whether a marker's status is abnormal in a sample will generally comprise comparing the marker's status in the patient's sample with the marker's index or reference status (e.g. , index values discussed above) in an average, normal, or healthy patient's sample.
  • the marker's index or reference status e.g. , index values discussed above
  • Another aspect of the invention provides a method of screening for cancer in a patient comprising identifying a patient at risk of having, or in need of screening for, cancer and determining the status of exosomes and/or an exosomes-associated marker in a sample obtained from the patient, wherein an abnormal status of exosomes and/or an exosomes-associated marker in the sample indicates the presence of cancer.
  • Patients may be identified as at risk of having, or in need of screening for, cancer in a variety of ways and based on numerous clinical and/or molecular characteristics.
  • One class of patients at risk of having cancer and in need of screening is those patients known to carry a germline deleterious mutation in a tumor suppressor gene.
  • Examples include, but are not limited to, BRCA l, BRCA2, PTEN, pi 6, MLHl, MSH6, APC, MYH, etc.
  • cancer-type specificity is often less crucial since, for example, a BRCAl- mutant patient whose exosome levels indicate cancer would be expected have breast or ovarian cancer rather than some other type of cancer.
  • additional tests to determine the type of cancer may be performed as discussed above.
  • Age and environmental factors may also define at-risk patients in need of screening. For example, prostate cancer is overwhelmingly found in men over 50 years of age. Thus, men over 50 may be considered patients at risk of having, or in need of screening for, prostate cancer.
  • the relatively non-invasive nature of blood, plasma, or serum detection i. e. , simple blood draw) makes such widespread screening attractive and practical.
  • the invention provides a method of detecting cancer comprising identifying a patient having a mutation in a gene selected from the group consisting of BRCA l, BRCA2, PTEN, pi 6, MLHl, MSH6, APC, and MYH; and determining the status of exosomes and/or an exosomes- associated marker in a sample obtained from the patient; wherein an abnormal status of exosomes and/or an exosomes-associated marker in the sample indicates the presence of cancer.
  • the method further comprises additional tests to determine/confirm which type of cancer is present.
  • Yet another aspect of the invention provides a method of detecting recurrence in a cancer patient comprising providing a sample obtained from the patient and determining the status of exosomes and/or an exosomes- associated marker in the sample, wherein an abnormal status of exosomes and/or an exosomes-associated marker in the sample indicates recurrence.
  • a method of detecting recurrence in a cancer patient comprising providing a sample obtained from the patient and determining the status of exosomes and/or an exosomes- associated marker in the sample, wherein an abnormal status of exosomes and/or an exosomes-associated marker in the sample indicates recurrence.
  • exosome levels are measured soon after treatment ⁇ e.g. , to determine a post-treatment baseline) and then monitored at regular intervals there after in order to catch any significant increase ⁇ e.g. , from this baseline).
  • Still another aspect of the invention provides a method comprising identifying a patient who is a candidate for biopsy and determining the status of exosomes and/or an exosomes-associated marker in a sample obtained from the patient, wherein an abnormal status of exosomes and/or an exosomes- associated marker in the sample indicates a biopsy is desirable.
  • Biopsies are often expensive, painful, and time-consuming and, furthermore, most biopsies are negative for cancer.
  • this aspect of the invention provides an effective tool which could allow one to know whether a biopsy is required (e.g. , after PSA, mammography, etc.).
  • biopsy refers to a patient suspected of having cancer, for whom a biopsy could be expected to confirm the presence of such cancer. For example, if a patient's mammography indicates the presence of an abnormal mass, methods according to this aspect of the invention can help determine whether the mass is likely to be cancerous and thus determine whether the discomfort and expense of a biopsy is warranted.
  • exosome analysis involves exosome analysis combined with more traditional diagnostic techniques.
  • physical examination e.g. , digital rectal exam for prostate cancer
  • imaging e.g. , mammography
  • biopsy may be used to confirm a diagnosis indicated by exosome analysis according to the invention.
  • exosome analysis may be combined with exosome analysis to yield a more comprehensive diagnosis.
  • an exosome screen may indicate the presence of cancer in a patient and these exosomes may be found to be CK7+/CK20- and have the marker CK5/6 associated with them.
  • a physician may take the further step of imaging to pinpoint the location of the cancer (e.g. , in or near the lung). The physician may further perform a biopsy to determine whether the cancer is squamous cell carcinoma of the lung or cancer of the mesothelial lining of the lung.
  • Various techniques can be used to enrich for exosomes and/or exosome-associated markers and to determine the status of exosomes and/or exosome-associated markers in a sample.
  • enrichment techniques include, but are not limited to : flow cytometry (i.e., cell sorting); centrifugation (e.g. , ultracentrifugation); physical filtration; affinity chromatography; contacting a sample with a solid surface (e.g., plate, well, bead, etc.) containing an antibody against an exosome-associated marker in order to capture any exosomes on the surface; etc. or any combination of these.
  • Example 5 describes one technique using ultracentrifugation combined with filtration and ECL to enrich a sample.
  • ultracentrifugation has its conventional meaning in the art.
  • ultracentrifugation comprises spinning the sample at at least 20,00Ox g, at least 30,000x g, at least 40,00Ox g, at least 50,00Ox g, at least 75 ,00Ox g, at least 100,000x g, at least 150,00Ox g, at least 250,00Ox g, at least 500,00Ox g, at least 750,00Ox g, or at least l ,000,000x g.
  • Those skilled in the art will appreciate that several of the variables in the Examples can be adjusted while still allowing for enrichment.
  • Suitable sizes and types of filtration may be used, including filtering to elute exosome-sized particles in the sample (e.g. , > 100nm, 150nm, 200nm, 250nm), filtering to capture exosome-sized particles (e.g., ⁇ 50nm, 40nm, 30nm, 20nm, I Onm), or both combined.
  • Various techniques can be used to determine the status of exosomes and/or an exosome-associated marker in a patient sample including, but are not limited to : flow cytometry; enzyme-linked immunosorbent assay (ELISA) and its numerous variations (e.g. , electrochemiluminescence or "ECL”); immunohistochemistry (IHC); etc. or any combination of these.
  • ELISA enzyme-linked immunosorbent assay
  • ECL electrochemiluminescence
  • IHC immunohistochemistry
  • flow cytometry can both sort exosomes (i.e. , separate exosomes of interest out of the sample milieu) and quantitate them, as shown in Example 1.
  • Flow cytometry is well-suited to the methods of the invention since multiple markers may be assayed at once by analyzing a panel of antigens by cell sorting using antibodies to each antigen and counting on a multichannel sorter.
  • the invention provides a method of quantitating exosomes comprising isolating exosomes from a patient sample and counting the exosomes using flow cytometry.
  • the isolation and counting may be done simultaneously, such as by using fluorescent-activated cell sorting (FACS) adapted for use with exosomes.
  • FACS fluorescent-activated cell sorting
  • ECL ELISA can enrich a sample for exosomes and/or exosome-associated markers by capturing these on a solid surface (using, e.g. , integrins, antibodies, etc.) and can then determine their level by way of a separate detection reagent (e.g. , integrins, antibodies, etc.).
  • a separate detection reagent e.g. , integrins, antibodies, etc.
  • the invention provides a method of detecting cancer comprising fixing exosomes and/or an exosome-associated marker to a solid surface and determining the level of at least one exosome-associated marker, wherein an abnormal status of the exosome- associated marker indicates an increased likelihood of cancer.
  • the exosomes and/or exosome-associated markers are fixed to the solid surface by way of a linker.
  • the linker specifically binds exosomes and/or an exosome-associated marker (e.g. , integrins, antibodies such as anti-CD63 , anti-CD36, or any antibody against any marker listed in Table 1 , etc.).
  • the solid surface can be "sticky" - i.e., the surface may be made of a material that itself binds exosomes and/or exosome-associated markers (e.g., latex, graphite).
  • IHC is used to determine the status of exosomes and/or an exosome-associated marker. This will often be in connection with exosome blocks.
  • the invention provides a method of detecting cancer comprising determining the status of an exosome-associated marker by contacting an exosome block sample from a patient with at least one antibody that specifically binds the marker, wherein an abnormal status for the marker indicates an increased likelihood of cancer.
  • exosome-associated markers may also be found within an exosome 's interior. Examples include proteins, mRNA, microRNA, DNA, etc.
  • immunological techniques are available including, but no limited to, intraexosomal staining flow cytometry (see Example 1 below), ELISA (see Example 2 below), IHC (e.g. , using exosome blocks such as in Example 3 below), etc.
  • the exosomes may be solubilized to release their contents and nucleic acids of interest may be quantitated (e.g.
  • FISH fluorescence in situ hybridization
  • exosome blocks may be prepared in a manner similar to formalin-fixed paraffin-embedded (FFPE) tissue samples, the main difference being that isolated exosomes rather than excised tissue form the basis of exosome blocks.
  • Pathologists routinely make cell pellets from formalin-fixed body fluid samples and embed them in paraffin to produce a cell block. Likewise, one may take the fixed, pelleted exosomes and embed them in paraffin to make an exosome block.
  • This tissue block can then be sliced (e.g. , with a microtome) and sections put on slides, or a tissue microarray can be constructed.
  • exosome block comprising formalin-fixed exosomes embedded in paraffin.
  • one aspect of the invention provides a method comprising determining the status of exosomes and/or an exosome-associated marker in a sample obtained from a patient, recording and/or communicating whether the status is abnormal, and recording and/or communicating that an abnormal status indicates the presence of cancer.
  • the method comprises determining the level of exosomes in a sample obtained from a patient and, if the level of exosomes is increased, recording and/or communicating that the patient has an increased likelihood of cancer.
  • Transmittable forms for communicating the results of a test can vary and can be tangible or intangible.
  • the results can be embodied in descriptive statements, diagrams, photographs, charts, images or any other visual forms. For example, graphs showing exosome level information can be used in explaining the results. Diagrams showing such information for additional markers are also useful in indicating some testing results.
  • the statements and visual forms can be recorded on a tangible medium such as papers, computer readable media such as floppy disks, compact disks, etc., or on an intangible medium, e.g. , an electronic medium in the form of email or website on Internet or intranet.
  • results can also be recorded in a sound form and transmitted through any suitable medium, e.g. , analog or digital cable lines, fiber optic cables, etc. , via telephone, facsimile, wireless mobile phone, internet phone and the like.
  • the information and data on a test result can be produced anywhere in the world and transmitted to a different location.
  • the information and data on a test result may be recorded or generated, cast in a transmittable form as described above, and then imported into the United States.
  • the present invention also encompasses a method for producing a transmittable form of information on exosome (and/or exosome-associated marker) status for at least one patient sample.
  • the method comprises the steps of ( 1 ) determining exosome (and/or exosome-associated marker) status according to the methods of the present invention; and (2) embodying the result of the determining step in a transmittable form.
  • the transmittable form is the product of such a method.
  • One aspect of the invention provides methods of treating a patient comprising determining the status of exosomes and/or an exosomes- associated marker as discussed above.
  • the treatment methods of the invention generally further comprise some action based on the exosome (and/or exosome- associated marker) status determination. Examples of actions based on the exosome determination include recommending a biopsy; prescribing a particular treatment; administering a particular therapeutic composition; prescribing, recommending, or performing surgery; monitoring for more signs of malignancy (e.g. , recommending an additional test for cancer), etc.
  • Other actions (such as communicating, e.g. , a particular diagnosis or prognosis to a patient) are also contemplated in the treatment methods of the invention as discussed above.
  • kits for practicing the methods of the present invention.
  • the kit may include a carrier for the various components of the kit.
  • the carrier can be a container or support, in the form of, e.g. , bag, box, tube, rack, and is optionally compartmentalized.
  • the carrier may define an enclosed confinement for safety purposes during shipment and storage.
  • the kit also includes at least one component useful in determining the status of exosomes and/or an exosomes-associated marker using any of the detection techniques discussed herein.
  • the kit many include probes to exosome-associated markers (e.g. , anti-CD63 antibodies) or probes to a panel of markers useful for differentiating tissue or cancer types.
  • the detection kit of this invention Various other components useful in the detection techniques may also be included in the detection kit of this invention. Examples of such components include, but are not limited to, Taq polymerase, deoxyribonucleotides, dideoxyribonucleotides other primers suitable for the amplification of a target DNA sequence, RNase A, and the like.
  • the detection kit preferably includes instructions on using the kit for practicing the methods of the present invention using human samples.
  • Exosomes may also be measured by flow cytometry.
  • Flow cytometry is generally used to measure cell-surface antigen expression and thus may be readily adapted to measuring antigens on the surface of exosomes.
  • exosomes are isolated from a sample (e.g. , serum) and then incubated with a labeled antibody (e.g. , fluorescence-labeled) against some surface marker expected to be found on the exosomes (e.g. , EpCAM). This solution is then passed through a cell sorter, which detects and "counts" the number of labeled exosomes as they flow through an optical/electronic detection apparatus.
  • a labeled antibody e.g. , fluorescence-labeled
  • Multiple labeled antibodies may be used in the same sample as cell sorters are capable of detecting multiple wavelengths of light at once.
  • Flow cytometry may also be used to detect and quantitate cytoplasmic antigens. For this it is generally necessary to fix and permeabilize cells to enable antibodies to gain access to them, after which the process is generally the same as above.
  • Plasma samples were obtained from eight patients with no cancer and from eight OVCA patients. These plasma samples were subjected to differential centrifugation to enrich for exosomes as follows and then protein was quantified by the Bradford Assay (Bradford, A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein- Dye Binding, ANAL . BIOCHEM. ( 1976) 72 :248-254) :
  • Latex beads were coated with anti-CD63 antibodies and then contacted/incubated with the exosome samples isolated above as follows :
  • PE-conjugated anti-CD63 antibody CD63 BD Pharmingen Clone # H5C6
  • Isotype control samples comprising beads with PE-conjugated IgG l antibodies were also run through the cell sorter under identical conditions.
  • the fluorescence values (Median FL2-A) for these samples are shown in Table 3 below:
  • ELISA Enzyme-Linked immunosorbent assay
  • the purpose of an ELISA is to determine if a particular protein is present in a sample and optionally, how much. EpCAM-bearing exosomes were detected and quantitated in the supernatant of epithelial cancer cell lines in the following experiment.
  • Capture antibody is a mouse monoclonal (clone 158210, R&D systems catalog # MAB 960) raised against the extracellular domain of Human EpCAM.
  • Detection antibody is a goat polyclonal (R&D systems catalog # BAF960) raised against recombinant extracellular domain of Human EpCAM.
  • Exosomes were isolated by differential centrifugation and filtration using the same process described in Example 1 .
  • Detection antibody was conjugated to biotin.
  • Detection enzyme was Horseradish-peroxidase conjugated to streptavidin (R&D cat# DY998).
  • Enzyme substrate was H2O2 + tetramethylbenzidine (R&D cat# DY999).
  • the ELISA was read on a Packard Bioscience Fusion microplate reader set at 45OnM.
  • EpCAM was measured by ELISA ( 1 ) in the Cell-conditioned media and (2) in the Cell-conditioned media depleted by centrifugation, and (3) total protein was also measured in the centrifugation pellet. Results are summarized in Table 4 below:
  • Exosome IHC will generally be carried out on an exosome block.
  • Exosome blocks are prepared by isolating exosomes from a patient sample (e.g. , plasma sample), fixing the exosome pellet (e.g. , with formalin), and then embedding the fixed pellet in some medium (e.g. , paraffin) that allows for convenient long-term storage.
  • a patient sample e.g. , plasma sample
  • fixing the exosome pellet e.g. , with formalin
  • some medium e.g. , paraffin
  • ECL ELISA electrochemiluminescence
  • a sample is contacted with a detection antibody (e.g. , anti-CD63) labeled with an electrochemiluminescent chemical moiety, the resulting sample is exposed to electrochemical energy, and the electromagnetic radiation emitted by the electrochemiluminescent chemical moiety is detected.
  • a detection antibody e.g. , anti-CD63
  • ECL electrochemiluminescence
  • ECL ELISA was used to differentiate the plasma of cancer patients from the plasma of healthy controls. Plasma samples were obtained and diluted with 1 % MSD Blocker A, without any initial enrichment for exosomes. The plasma samples used were as follows: 47 prostate cancer, 49 breast cancer, 48 colorectal cancer, 48 lung cancer, 50 ovarian cancer, and 70 healthy controls.
  • MSD Meso Scale Discovery ®
  • MSD 96 well High Bind plates were incubated with a capture antibody (BD CD63 - 4 ⁇ g/ml) or protein ( ⁇ V ⁇ integrin - 0.2 ⁇ g/ml) overnight. The plate was then washed three times with 300 ⁇ l Wash Buffer (PBS 0.01 % Tween). The plate was then passivated with 1 % MSD Blocker A for 1 hour on an orbital shaker. The plate was then washed three times with 300 ⁇ l Wash Buffer. Diluted plasma samples were then allowed to incubate in the plate for 2 hours at room temperature on an orbital shaker.
  • the plate was then washed three times with 300 ⁇ l Wash Buffer. 25 ⁇ l of the detection antibody was then loaded into each well and this was allowed to incubate for 1 hour at room temperature on an orbital shaker. The plate was then washed three times with 300 ⁇ l wash buffer.
  • CD36 Claudin-3 and Claudin-4 :
  • ECL ELISA was combined with differential centrifugation to separate ovarian cancer patients from controls. Differential centrifugation was performed as described in Example 1 above. ECL ELISA was performed on the resulting enriched samples essentially as described in Example 5 above, except that enriched exosome samples were directly captured on the MSD plate rather than on an MSD plate coated with a capture reagent. Specifically, 25 ⁇ l of each sample was the allowed to absorb onto an MSD 96 well High Bind Plate (Cat # L 11 XB-6). The plate was then passivated using MSD Blocker A (Cat # R93AA- 1 ).

Abstract

La présente invention concerne des procédés et des compositions mettant en œuvre des marqueurs moléculaires pour la détection et la caractérisation du cancer chez un patient. Des marqueurs associés à un exosome sont préférés. La présente invention concerne en outre des kits et des blocs de tissu.
PCT/US2009/067029 2008-12-05 2009-12-07 Marqueurs de détection du cancer WO2010065968A1 (fr)

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