Classifications

• • 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
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the present disclosure relates to methods for detecting cancer, methods for predicting the prognosis of cancer, biomarkers and methods for using the same, and kits for detecting cancer or predicting the prognosis of cancer.
• Extracellular vesicles are a general term for vesicles with a diameter of about 100 nm that are surrounded by a lipid bilayer membrane and are produced by cells in vivo or in culture and secreted outside the cell.
• Extracellular vesicles express tetraspanins (TSPAN) and adhesion molecules on their membranes, and contain nucleic acids such as mRNA and miRNA, skeletal proteins, proteins such as various enzymes, lipids, metabolic products, etc.
• TSPAN tetraspanins
• extracellular vesicles maintain the molecular structure of the cells that produce them, so for example, extracellular vesicles derived from cancer cells have the same molecular structure as the cancer cells. For this reason, extracellular vesicles are expected to be used as blood diagnostic materials for cancer.
• the present disclosure aims to provide a method for detecting cancer or predicting the prognosis of cancer, which can detect cancer specifically, with high sensitivity, simply and quickly.
• a method for detecting cancer or predicting the prognosis of cancer comprising: The method comprises the step of detecting tetraspanin 10 in a sample derived from a subject's body fluid.
• a biomarker for detecting cancer or predicting the prognosis of cancer This is a biomarker characterized by containing tetraspanin 10.
• a kit for detecting cancer or predicting the prognosis of cancer comprising: The kit is characterized by comprising a substance for detecting tetraspanin 10 in a sample derived from a body fluid.
• the present disclosure can achieve the above-mentioned objective and provide a method for detecting cancer or predicting the prognosis of cancer that can detect cancer specifically, with high sensitivity, simply and quickly.
• Figure 1A shows the results of analyzing tetraspanin 10 mRNA expression in various normal tissues based on FANTOM5 published in THE HUMAN PROTEIN ATLAS. The vertical axis shows the amount of TSPAN10 mRNA expression (normalized tags per million).
• Figure 1B shows the results of analyzing the expression of tetraspanin 10 mRNA in various normal blood cells based on FANTOM5 published in THE HUMAN PROTEIN ATLAS. The vertical axis shows the amount of TSPAN10 mRNA expression (normalized tags per million).
• Figure 1C shows the results of analyzing tetraspanin 10 mRNA expression in various cancer tissues based on data published in The Cancer Genome Atlas (TCGA).
• the vertical axis shows the amount of TSPAN10 mRNA expression (normalized tags per million).
• 2 is a diagram showing the results of ELISA using anti-TSPAN10 antibody for serum samples from a control group, a breast cancer stage 0/I group, and a breast cancer stage IV group in Test Example 1.
• the vertical axis indicates absorbance at a wavelength of 450 nm.
• 3 is a diagram showing a receiver operating characteristic (ROC) curve as a result of the validation of the analytical method by the ELISA method in Test Example 1.
• the vertical axis indicates sensitivity, and the horizontal axis indicates specificity.
• 4 is a diagram showing changes in ELISA detection values using anti-TSPAN10 antibody before and after treatment in Test Example 2.
• the vertical axis indicates absorbance at a wavelength of 450 nm.
• 5 is a diagram showing the results of analyzing the expression of TSPAN10 protein and p53 protein in extracellular vesicles of cancer patients based on the database published in the Te-EVs Proteome Library in Test Example 3.
• the vertical axis indicates the relative expression level of p53 protein, and the horizontal axis indicates the relative expression level of TSPAN10 protein.
• 6 is a diagram showing the results of ELISA using anti-TSPAN10 antibody and anti-p53 antibody for serum samples from a control group and an advanced breast cancer patient in Test Example 4.
• the vertical axis indicates absorbance at a wavelength of 450 nm.
• FIG. 7 is a diagram showing changes in ELISA detection values using anti-TSPAN10 antibody before and after treatment in Test Example 5.
• the vertical axis indicates absorbance at a wavelength of 450 nm, and the horizontal axis indicates the period (months) elapsed since the start of preoperative chemotherapy.
• 8 is a diagram showing the results of analyzing tetraspanin 10 mRNA expression in colorectal cancer based on the data published by TCGA in Test Example 6.
• the vertical axis shows the amount of TSPAN10 mRNA expression (normalized tags per million).
• the vertical axis indicates the survival rate
• the horizontal axis indicates the survival time (days).
• the vertical axis indicates the survival rate
• the horizontal axis indicates the survival time (days).
• 10 is a diagram showing the results of ELISA using anti-TSPAN10 antibody performed on serum samples from the control group and the colon cancer stage III/IV group in Test Example 7.
• the vertical axis indicates absorbance at a wavelength of 450 nm.
• 11 is a diagram showing a receiver operating characteristic (ROC) curve as a result of the validation of the analytical method by the ELISA method in Test Example 7.
• the vertical axis indicates sensitivity, and the horizontal axis indicates specificity.
• FIG. 12 is a diagram showing the results of analyzing the expression of tetraspanin 10 mRNA and MUC16 mRNA in various cancer tissues based on the data published by TCGA in Test Example 8.
• the vertical axis shows the expression level (FPKM) of TSPAN10 mRNA or MUC16 mRNA.
• 13 is a diagram showing the results of ELISA using anti-TSPAN10 antibody and anti-MUC16 antibody for serum samples from the control group and the pancreatic cancer stage III group in Test Example 9.
• the vertical axis indicates absorbance at a wavelength of 450 nm.
• 14 is a diagram showing a receiver operating characteristic (ROC) curve as a result of the validation of the analytical method by the ELISA method in Test Example 9.
• ROC receiver operating characteristic
• the vertical axis indicates sensitivity, and the horizontal axis indicates specificity.
• 15 is a diagram showing the results of ELISA using anti-TSPAN10 antibody and anti-MUC16 antibody for serum samples from the control group and the cancer group in Test Example 10. The vertical axis indicates absorbance at a wavelength of 450 nm.
• 16 is a diagram showing the results of calculating the average absorbance measurement values for each cancer type and graphing them from the results of the analytical method validation by ELISA in Test Example 10. The vertical axis shows the average absorbance value at a wavelength of 450 nm.
• 17 is a diagram showing a receiver operating characteristic (ROC) curve as a result of the validation of the analytical method by the ELISA method in Test Example 10.
• ROC receiver operating characteristic
• the vertical axis indicates sensitivity, and the horizontal axis indicates specificity.
• 18 is a transmission electron microscope (TEM) image of extracellular vesicles in serum of a stage III colon cancer patient in Test Example 11.
• the arrow indicates a gold colloid label.
• the scale bar is 100 nm.
• the biomarker of the present disclosure is a biomarker for detecting cancer or predicting the prognosis of cancer, and includes tetraspanin 10.
• the biomarker can be determined to be afflicted with cancer or can be predicted to have a poor prognosis for cancer.
• the method of using the biomarker disclosed herein is a method of using tetraspanin 10 in a body fluid-derived sample as a biomarker for detecting cancer or predicting the prognosis of cancer.
• cancer refers to a condition that originates from cells that compose the body, grows uncontrollably, and spreads abnormally to the surrounding area, or metastasizes or invades other parts of the body, causing a serious impact on life.
• cancer includes “carcinoma.”
• prognosis refers to the outcome of a subject.
• the outcome of a subject is an outcome due to the effects of the cancer that the subject has.
• poor prognosis refers to at least one selected from the group consisting of cancer being prone to metastasis or infiltration, being prone to recurrence, and a high probability that a subject with cancer will die from cancer.
• the subject may consider or select a treatment method such as surgical resection of the cancer, radiation therapy, or chemotherapy.
• good prognosis refers to at least one selected from the group consisting of cancer not metastasizing or infiltrating and being localized, cancer growing slowly, and a low probability that a subject with cancer will die from cancer.
• the biomarker preferably consists essentially of tetraspanin 10, and more preferably is tetraspanin 10.
• biomarker may be tetraspanin 10 itself, or may have one or more bases in the nucleic acid sequence encoding tetraspanin 10, or one or more residues in the amino acid sequence encoding tetraspanin 10 protein, that have been deleted, added, inserted, and/or substituted to the extent that does not impair the effects of the present disclosure.
• is tetraspanin 10 means that the biomarker is tetraspanin 10 itself, and the nucleic acid sequence encoding tetraspanin 10 or the amino acid sequence encoding the tetraspanin 10 protein does not have any deletions, additions, insertions, and/or substitutions.
• Tetraspanin (TSPAN) family proteins are membrane proteins that have a structure that penetrates the cell membrane four times, and are cell surface receptors present on the cell membrane. Tetraspanin family proteins form complexes with adhesion molecules such as integrins, and also form complexes with different types of tetraspanins. In humans, 33 types of tetraspanin family protein members are known, including CD9, CD63, CD81, CD82, and CD151, but tetraspanin 10 (also called oculospanin) is a different protein from these.
• the present inventors analyzed FANTOM5, which is published in THE HUMAN PROTEIN ATLAS (an open access database by Atlas Antibodies, https://www.proteinatlas.org/about/download, the contents of which are incorporated herein by reference in their entirety), and found that, among normal human tissues, tetraspanin 10 is highly expressed only in the retina (see FIG. 1A). They also found that tetraspanin 10 is not expressed in normal human blood cells (see FIG. 1B).
• tetraspanin 10 is expressed in many types of human cancer tissues, regardless of the type of cancer, based on data from The Cancer Genome Atlas (TCGA) (an open access database, https://www.cancer.gov/ccg/research/genome-sequencing/tcga, the contents of which are incorporated herein by reference in their entirety) (see Figure 1C).
• TCGA Cancer Genome Atlas
• the retina is an environment isolated from the circulating blood by the blood-retina barrier, and it is unlikely that retina-derived tetraspanin 10 is expressed in the blood. Therefore, if the subject is suffering from cancer or has previously suffered from cancer, it is believed that tetraspanin 10 in a sample derived from the subject's body fluid is derived from cancer cells.
• tetraspanin 10 is specifically expressed in extracellular vesicles derived from cancer cells and can be used as a biomarker for detecting cancer or predicting the prognosis of cancer, and further that cancer can be detected specifically, highly sensitively, simply, and quickly.
• the tetraspanin 10 biomarker may be a DNA sequence encoding tetraspanin 10, a gene sequence encoding tetraspanin 10, or a gene product thereof.
• Gene products of the gene sequence encoding tetraspanin 10 include RNA sequences, proteins, and amino acid sequences encoding the proteins.
• biomarker is a DNA sequence encoding tetraspanin 10 or a gene sequence encoding tetraspanin 10
• the biomarker may have the full length of the DNA sequence encoding tetraspanin 10 or a part of it.
• An example of DNA encoding human tetraspanin 10 protein is GeneBank Accession No. 83882.
• the biomarker having a portion of the DNA sequence encoding the tetraspanin 10 is not particularly limited as long as it does not impair the effects of the present disclosure, and examples thereof include those in which one or more deoxyribonucleic acid bases have been deleted, added, inserted, and/or substituted in GeneBank Accession No. 83882.
• biomarkers in which one or two or more deoxyribonucleic acid bases have been deleted, added, inserted, and/or substituted are not particularly limited as long as the biomarkers can be used to detect cancer or predict the prognosis of cancer, and can be appropriately selected depending on the purpose.
• deoxyribonucleic acid sequences having 95% or more sequence identity to GeneBank Accession No.: 83882 deoxyribonucleic acid sequences having 90% or more sequence identity, deoxyribonucleic acid sequences having 85% or more sequence identity, deoxyribonucleic acid sequences having 80% or more sequence identity, deoxyribonucleic acid sequences having 75% or more sequence identity, deoxyribonucleic acid sequences having 70% or more sequence identity, etc.
• deoxyribonucleic acid sequences having 85% or more sequence identity are more preferred, deoxyribonucleic acid sequences having 90% or more sequence identity are even more preferred, and deoxyribonucleic acid sequences having 95% or more sequence identity are particularly preferred.
• the biomarker having a portion of the DNA sequence encoding tetraspanin 10 is preferably a DNA sequence containing a gene sequence encoding tetraspanin 10.
• the biomarker When the biomarker is a DNA sequence containing a gene sequence encoding tetraspanin 10, it may have a portion of the gene sequence encoding tetraspanin 10, as long as the effect of the present disclosure is not impaired.
• Examples of the biomarker having a portion of the gene sequence encoding tetraspanin 10 include a gene sequence encoding tetraspanin 10 in which one or more deoxyribonucleic acid bases have been deleted, added, inserted, and/or substituted.
• RNA sequence as the gene product of the gene sequence encoding tetraspanin 10 When the biomarker is an RNA sequence as a gene product of a gene sequence encoding tetraspanin 10, the biomarker may have the entire length of the RNA sequence as a gene product of the gene sequence encoding tetraspanin 10, or may have a part of it.
• RNA sequences as gene products of gene sequences encoding human tetraspanin 10 include GeneBank Accession Nos. NM_001290212 (1,840 bp) and NM_031945.5 (1,887 bp).
• GeneBank Accession No. NM_001290212 consists of the base sequence shown in SEQ ID NO: 1.
• GeneBank Accession No. NM_031945.5 consists of the base sequence shown in SEQ ID NO: 2.
• RNA sequence as the gene product of the gene sequence encoding tetraspanin 10 is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include mRNA, ncRNA, tRNA, etc. These may be used alone or in combination of two or more types.
• the biomarker having a part of an RNA sequence as a gene product of a gene sequence encoding the tetraspanin 10 is not particularly limited as long as it does not impair the effects of the present disclosure, and examples thereof include those in which, for example, one or more ribonucleic acid bases are deleted, added, inserted, and/or substituted in GeneBank Accession No. NM_001290212 or NM_031945.5.
• the biomarker having a part of an RNA sequence as a gene product of a gene sequence encoding the tetraspanin 10 may be RNAi, siRNA, shRNA, miRNA, RISC, etc. based on GeneBank Accession No. NM_001290212 or NM_031945.5.
• biomarkers in which one or two or more ribonucleic acid bases have been deleted, added, inserted, and/or substituted are not particularly limited as long as the biomarker can be used to detect cancer or predict the prognosis of cancer, and can be appropriately selected depending on the purpose.
• ribonucleic acid sequences having 95% or more sequence identity to GeneBank Accession Number: NM_001290212 or NM_031945.5, 90% or more sequence identity, 85% or more sequence identity, 80% or more sequence identity, 75% or more sequence identity, 70% or more sequence identity, etc. may be mentioned.
• ribonucleic acid sequences having 80% or more sequence identity to GeneBank Accession Number: NM_001290212 or NM_031945.5 are preferred, ribonucleic acid sequences having 85% or more sequence identity are more preferred, ribonucleic acid sequences having 90% or more sequence identity are even more preferred, and ribonucleic acid sequences having 95% or more sequence identity are particularly preferred.
• the biomarker is a protein as a gene product of a gene sequence encoding tetraspanin 10 or an amino acid sequence encoding said protein
• the biomarker may have the entire length of the amino acid sequence encoding the protein as a gene product of a gene sequence encoding tetraspanin 10, or may have a part of the amino acid sequence.
• An example of a human tetraspanin 10 protein is GeneBank Accession No. AAH21923.1 (235 amino acids).
• Examples of human tetraspanin 10 proteins include Q9H1Z9 (Y218H) (355 amino acids) and Q9H1Z9 (R187H) (355 amino acids) in the protein database Uniport (https://www.uniprot.org/).
• GeneBank Accession No. AAH21923.1 consists of the amino acid sequence represented by SEQ ID NO:3.
• the biomarker having a portion of an amino acid sequence encoding a protein as a gene product of the gene sequence encoding the tetraspanin 10 includes, in addition to Q9H1Z9 and Q9H1Z9, there are no particular limitations as long as the effects of the present disclosure are not impaired, and examples include those in which, for example, one or more amino acid residues have been deleted, added, inserted, and/or substituted in GeneBank Accession Number: AAH21923.1, Q9H1Z9, or Q9H1Z9.
• examples of the biomarker having a portion of an amino acid sequence encoding a protein as a gene product of the gene sequence encoding the tetraspanin 10 include the amino acid sequences shown in any of SEQ ID NOs: 4 to 7 described below.
• Biomarkers in which one or more amino acid residues have been deleted, added, inserted, and/or substituted in the amino acid sequence shown by GeneBank Accession No. AAH21923.1, Q9H1Z9, or Q9H1Z9, or any of SEQ ID NOs: 4 to 7, are not particularly limited as long as the biomarker can be used to detect cancer or predict the prognosis of cancer, and can be appropriately selected depending on the purpose.
• AAH21923.1, Q9H1Z9, or Q9H1Z9, or any of SEQ ID NOs: 4 to 7 may be included.
• an amino acid sequence having 80% or more sequence identity to the amino acid sequence shown in GeneBank Accession No. AAH21923.1, Q9H1Z9, or Q9H1Z9, or any of SEQ ID NOs: 4 to 7, is preferred, an amino acid sequence having 85% or more sequence identity is more preferred, an amino acid sequence having 90% or more sequence identity is even more preferred, and an amino acid sequence having 95% or more sequence identity is particularly preferred.
• the biomarker preferably comprises an amino acid sequence shown in any one of SEQ ID NOs: 4 to 7, and more preferably consists of an amino acid sequence shown in any one of SEQ ID NOs: 4 to 7.
• SEQ ID NO: 4 SCVKYLIFLSNFPFSLLGLLALAIGLWGLAVKGSLGSDLGGPLPADPMLG
• SEQ ID NO: 5 SCVKYLIFLSNFPFSLLGLLALAIGLWGLAVKGSLGSDLGGPLPTDPMLG
• the biomarker is preferably a protein expressed in extracellular vesicles, an amino acid sequence encoding the protein, RNA or DNA encoding the amino acid sequence, or RNA or DNA contained in extracellular vesicles, and more preferably a protein expressed in extracellular vesicles derived from cancer cells, an amino acid sequence encoding the protein, RNA or DNA encoding the amino acid sequence, or RNA or DNA contained in extracellular vesicles derived from cancer cells.
• Extracellular vesicles are a general term for vesicles with a heterogeneous lipid bilayer structure secreted from almost all living cells.
• Examples of extracellular vesicles include circulating microvesicles (cMVs), microvesicles, exosomes, nanovesicles, dexosomes, blebs, vesicles, prostasomes, microparticles, intraluminal vesicles, membrane fragments, intraluminal endosomal vesicles, endosomal vesicles, exocytosis vehicles, endosomal vesicles, apoptotic bodies, multivesicular bodies, secretory vesicles, phospholipid vesicles, liposomal vesicles, argosomes, texasomes, secretosomes, trellosomes, melanosomes, oncosomes, and exocytosis vehicles.
• cMVs circulating microves
• a method for forming extracellular vesicles is described below. Inside a cell, a small internal vesicle is formed that contains proteins, mRNA, microRNA (miRNA), etc. in the cytoplasm. The internal vesicle then fuses with the cell membrane of the cell and is released to the outside as an extracellular vesicle. The released extracellular vesicles move, for example, via the blood, toward a destination determined by the attachment of a specific ligand on their surface.
• mRNA microRNA
• the extracellular vesicles that reach the target cell are either introduced via the endocytosis pathway or fused with the target cell membrane to directly release the extracellular vesicle components, i.e., components such as proteins derived from the cell that formed the extracellular vesicles, into the cytoplasm.
• the DNA molecules inside the extracellular vesicles represent the entire genome and can also reflect the state (e.g., mutation state, etc.) of the cancer cell that released the extracellular vesicles.
• tetraspanin 10 is not expressed in normal tissues other than the retina, nor is it expressed in normal blood cells. However, it is expressed in cancer tissues of many types of cancer. Therefore, tetraspanin 10 in extracellular vesicles in the blood may be useful as a biomarker for detecting cancer or predicting cancer prognosis.
• the types of cancer that can be detected or predicted prognosis by the biomarkers are not particularly limited, and examples thereof include breast cancer, colon cancer, pancreatic cancer, head and neck cancer, esophageal cancer, gastric cancer, lung cancer, thyroid cancer, uterine cancer (including uterine body cancer and cervical cancer), ovarian cancer, melanoma, kidney cancer, liver cancer, epithelial cancer, rectal cancer, colon cancer, papillary renal cell carcinoma, head and neck squamous cell carcinoma, serous cystadenocarcinoma, chromophobe renal cell carcinoma, prostate cancer, lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, renal non-clear cell carcinoma, hepatocellular carcinoma, testicular germ cell tumor, pancreatic adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, colon adenocarcinoma, prostate cancer, brain cancer, skin cancer, bone cancer,
• the type of cancer that can be detected or the prognosis can be predicted by the biomarker is preferably a solid cancer, and more preferably at least one type selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, head and neck cancer, esophageal cancer, gastric cancer, lung cancer, thyroid cancer, uterine cancer, ovarian cancer, melanoma, kidney cancer, liver cancer, epithelial cancer, rectal cancer, colon cancer, papillary renal cell carcinoma, head and neck squamous cell carcinoma, serous cystadenocarcinoma, chromophobe renal cell carcinoma, prostate cancer, lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, renal non-clear cell carcinoma, hepatocellular carcinoma, testicular germ cell tumor, pancreatic adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, and colon adenocarcinoma.
• the cancer stage that can be detected or the prognosis can be predicted by the biomarker is not particularly limited and can be appropriately selected depending on the purpose.
• the "stage" of cancer means the stage based on the TMN classification.
• stage 0 refers to cancer at stage 0 and stage I in the "TMN classification," an international standard.
• the TNM classification determines the stage by combining three elements: “T (Tumor),” i.e., the size of the lump; “N (Lymph Note),” i.e., the state of metastasis to lymph nodes; and “M (Metastasis),” i.e., metastasis to other organs.
• T Tumor
• N Lymph Note
• M Metastasis
• stage I breast cancer is T: lump is 2 cm or less
• N no metastasis to lymph nodes
• M no metastasis to other organs.
• the more advanced the stage of cancer the more difficult it is to treat and the lower the survival rate, so it is important to detect it as early as possible.
• the survival rate is high for all cancers when treated at stage 0 or stage I.
• the 5-year and 10-year survival rates are both 100% at stage 0, but at stage IV, the 5-year survival rate drops to 38.7% and the 10-year survival rate drops to 19.4% (see National Cancer Center, "2009 10-year survival rate and 2013-14 5-year survival rate compilation for all in-hospital cancer registries," December 24, 2021). Therefore, the biomarker is very useful for early diagnosis and early treatment of cancer.
• the biomarkers are particularly useful for detecting early-stage cancers, including early-stage colon adenocarcinoma and early-stage rectal adenocarcinoma, and early-stage breast cancer.
• tetraspanin 10 in extracellular vesicles derived from cancer cells can be confirmed, for example, by concentrating extracellular vesicles from a body fluid-derived sample and confirming the presence or absence of expression of tetraspanin 10 in the concentrated extracellular vesicles.
• the method for concentrating the extracellular vesicles from the body fluid-derived sample is not particularly limited and can be appropriately selected from known methods, such as size exclusion chromatography, density gradient centrifugation, fractional centrifugation, nanomembrane ultrafiltration, immunoadsorption capture, affinity purification, affinity capture, affinity selection, immunoassay, ELISA, microfluidics separation, and flow cytometry. These methods may be performed alone or in combination of two or more. These concentration methods concentrate extracellular vesicles regardless of the presence or absence of tetraspanin 10 expression.
• the method for confirming the presence or absence of tetraspanin 10 expression in concentrated extracellular vesicles is not particularly limited, but can be suitably confirmed by a method similar to the method for detecting cancer or predicting the prognosis of cancer disclosed below.
• the biomarker of the present disclosure can be suitably used as a diagnostic marker for diagnosing whether or not a patient has cancer, a cancer onset probability predicting marker for predicting whether or not a patient has the possibility of developing cancer, a prognosis predicting marker for predicting the prognosis of cancer, a pharmacodynamic marker for analyzing the effect of a drug or compound (including existing drugs and clinical trials of new drugs, etc.) on cancer (i.e., if the value of the cancer biomarker decreases after administration compared to before administration of the drug, the therapeutic effect of the drug is recognized).
• the biomarker of the present disclosure is suitably used in vitro.
• prognostic markers for predicting the prognosis of cancer include prognostic observation markers for confirming the effectiveness of cancer treatment (e.g., surgery, radiotherapy, etc.) in patients undergoing cancer treatment (i.e., the effectiveness of the treatment is recognized when the value of the biomarker decreases after treatment compared to before treatment), monitoring markers for follow-up observation of the presence or absence of recurrence (including metastasis) after cancer treatment (i.e., there is a possibility of recurrence when the value of the biomarker increases during follow-up observation compared to immediately after treatment), treatment effect prediction markers for predicting the effectiveness of a specific treatment (i.e., the effectiveness of a specific treatment is recognized when the value of the biomarker decreases after the start of a specific treatment compared to before the start of the treatment), and markers for providing an index for selecting a cancer treatment method (i.e., companion diagnostic markers used in companion diagnosis).
• prognostic observation markers for confirming the effectiveness of cancer treatment e.g., surgery
• the subject for application of the biomarker is preferably humans, but is not limited thereto, and can also be used in non-human mammals such as monkeys, mice, rats, hamsters, dogs, cats, rabbits, cows, and pigs.
• the condition of the subject to which the biomarker is applied is not particularly limited and can be appropriately selected depending on the purpose. Examples include subjects whose cancer status is unknown, subjects who have already been determined to have cancer by another method, subjects who have already been determined to not have cancer by another method, subjects undergoing cancer treatment, subjects who have already undergone cancer treatment, etc.
• the method for detecting cancer or predicting cancer prognosis of the present disclosure includes a step of detecting tetraspanin 10 in a sample derived from a subject's body fluid (hereinafter, sometimes referred to as a "first detection step"), and further includes other steps as necessary. Note that the method for detecting cancer or predicting cancer prognosis of the present disclosure is preferably performed in vitro.
• the first detection step is a step of detecting tetraspanin 10 in a sample derived from a body fluid of a subject.
• the tetraspanin 10 detected in the first detection step is preferably a biomarker of the present disclosure.
• the subject is preferably a human, but is not limited thereto, and may be a non-human mammal such as a monkey, mouse, rat, hamster, dog, cat, rabbit, cow, or pig.
• the condition of the subject is not particularly limited and can be appropriately selected depending on the purpose. Examples include subjects whose status is unknown as to whether they have cancer, subjects whose status has already been determined to have cancer by another method, subjects whose status has already been determined to not have cancer by another method, and subjects undergoing cancer treatment.
• the body fluid from which the body fluid-derived sample is collected is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include peripheral blood, serum, plasma, ascites, lymph, cerebrospinal fluid, saliva, bone marrow, urine, synovial fluid, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal secretion, amniotic fluid, breast milk, etc. These may be used alone or in combination of two or more.
• the body fluid sample may be collected at any time of day.
• the method for collecting the body fluid sample is not particularly limited, and can be appropriately selected from among known methods depending on the type of sample to be collected.
• the body fluid-derived sample may be the body fluid itself, or may be a sample obtained by subjecting the body fluid to some treatment (e.g., separation, purification, addition of drugs, etc.).
• some treatment e.g., separation, purification, addition of drugs, etc.
• blood samples such as peripheral blood, serum, and plasma are preferred as the body fluid-derived sample, with serum being more preferred.
• serum being more preferred.
• the method for processing the body fluid sample is not particularly limited, and can be appropriately selected from among known methods depending on the type of sample to be collected, the detection method, etc.
• the subject's body fluid-derived sample preferably contains extracellular vesicles, and more preferably contains cancer-derived extracellular vesicles.
• the method of detecting cancer or predicting the prognosis of cancer disclosed herein may be performed using extracellular vesicles concentrated from the subject's body fluid-derived sample instead of the subject's body fluid-derived sample.
• extracellular vesicles examples include circulating microvesicles (cMVs), microvesicles, exosomes, nanovesicles, dexosomes, blebs, vesicles, prostasomes, microparticles, intraluminal vesicles, membrane fragments, intraluminal endosomal vesicles, endosome-like vesicles, exocytosis vehicles, endosomal vesicles, endosomal vesicles, apoptotic bodies, multivesicular bodies, secretory vesicles, phospholipid vesicles, liposomal vesicles, argosomes, texasomes, secretosomes, trellosomes, melanosomes, oncosomes, exocytosis vehicles, etc.
• the body fluid-derived sample may contain one type of these alone or two or more types.
• the size of the extracellular vesicles is not particularly limited, but the diameter is preferably 10 nm to 2,000 nm, more preferably 20 nm to 1,500 nm, even more preferably 20 nm to 1,000 nm, even more preferably 20 nm to 500 nm, and particularly preferably 50 nm to 150 nm.
• the method for detecting tetraspanin 10 in a sample derived from the subject's body fluid is not particularly limited as long as it can specifically detect tetraspanin 10 and can be appropriately selected depending on the purpose, but is preferably a method capable of detecting the biomarker of the present disclosure.
• Examples of the method for detecting the biomarker include methods for detecting tetraspanin 10 protein, such as ELISA (Enzyme-Linked Immuno Sorbent Assay), immunostaining, and Western blotting; methods for detecting RNA sequences as gene products of gene sequences encoding tetraspanin 10, such as RNA-seq analysis, RT-PCR (including quantitative RT-PCR), nucleic acid chip analysis, and Northern blotting; and methods for detecting DNA sequences encoding tetraspanin 10, such as PCR, digital PCR, Southern blotting, and microarray. These methods may be used alone or in combination of two or more.
• a protein sample e.g., the blood sample, etc.
• an anti-TSPAN10 antibody e.g., the blood sample, etc.
• a sample expressing tetraspanin 10 protein in the protein sample is captured with the anti-TSPAN10 antibody, which is then detected by utilizing an enzyme reaction using an enzyme that can be used in a known ELISA method.
• a protein sample e.g., the blood sample
• a sample derived from the subject's body fluid is electrophoresed by SDS-PAGE in a conventional manner
• the protein sample is transferred to a hydrophobic membrane such as a nylon membrane in a conventional manner
• the hydrophobic membrane is reacted with an anti-TSPAN10 antibody, followed by reaction with a secondary antibody labeled with an enzyme such as HRP (horseradish peroxidase), and detection is performed by a chemiluminescence method or a color development method utilizing the enzyme activity.
• HRP horseradish peroxidase
• RNA-seq analysis method for example, cDNA of tetraspanin 10 is prepared from RNA in a sample derived from the subject's body fluid in a standard manner, and sequence analysis is performed using a next-generation sequencer or the like.
• methods include methods for obtaining expression level information by performing mapping, gene expression analysis, expression level analysis, etc. based on the data.
• cDNA is prepared from RNA in the subject's body fluid sample according to a conventional method, and a pair of nucleic acid primers (a positive strand that binds to the -strand of the cDNA, and a reverse strand that binds to the +strand) are hybridized with the cDNA so that the target region can be amplified using the cDNA as a template, and PCR is performed according to a conventional method to detect the resulting amplified double-stranded DNA.
• a pair of nucleic acid primers a positive strand that binds to the -strand of the cDNA, and a reverse strand that binds to the +strand
• the amplified double-stranded DNA can be detected by a method of detecting labeled double-stranded DNA produced by performing the PCR using a nucleic acid primer that has been labeled with RI or a fluorescent substance in advance, or by transferring the produced double-stranded DNA to a nylon membrane or the like according to a conventional method and hybridizing it with a labeled nucleic acid probe for detection.
• nucleic acid chip analysis method for example, a nucleic acid chip to which a nucleic acid probe (single-stranded or double-stranded) complementary to at least a part of a DNA sequence encoding tetraspanin 10 is attached is prepared, and the resulting double-stranded strand is detected by hybridizing the nucleic acid with RNA in a sample derived from the subject's body fluid or with a nucleic acid prepared from the RNA by a conventional method.
• a nucleic acid chip to which a nucleic acid probe (single-stranded or double-stranded) complementary to at least a part of a DNA sequence encoding tetraspanin 10 is attached is prepared, and the resulting double-stranded strand is detected by hybridizing the nucleic acid with RNA in a sample derived from the subject's body fluid or with a nucleic acid prepared from the RNA by a conventional method.
• a nucleic acid probe complementary to at least a part of a DNA sequence encoding tetraspanin 10 is labeled with a radioisotope ( 32 P, 33 P, etc.: RI) or a fluorescent substance, and then hybridized with mRNA in a sample derived from the subject's body fluid that has been transferred to a nylon membrane or the like in a standard manner, and the formed double strand between the nucleic acid probe and the mRNA in the sample derived from the subject's body fluid is detected by a radiation detector, a fluorescence detector, or the like by detecting a signal derived from the labeling substance of the nucleic acid probe (a labeling substance such as RI or a fluorescent substance).
• a radioisotope 32 P, 33 P, etc.: RI
• a fluorescent substance a fluorescent substance
• PCR method for example, a pair of nucleic acid primers (a positive strand that binds to the -strand of the cDNA, and a reverse strand that binds to the +strand) are hybridized with the DNA in the subject's body fluid sample or cDNA prepared in a conventional manner from the RNA in the subject's body fluid sample so that the target region can be amplified, and the PCR method is performed in a conventional manner to detect the amplified double-stranded DNA obtained.
• a pair of nucleic acid primers a positive strand that binds to the -strand of the cDNA, and a reverse strand that binds to the +strand
• the amplified double-stranded DNA can be detected by a method of detecting labeled double-stranded DNA produced by performing the PCR using a nucleic acid primer that has been labeled in advance with RI or a fluorescent substance, or a method of transferring the produced double-stranded DNA to a nylon membrane or the like in a conventional manner and hybridizing it with a labeled nucleic acid probe for detection.
• a nucleic acid probe complementary to at least a part of a DNA sequence encoding tetraspanin 10 is labeled with a radioisotope ( 32 P, 33 P, etc.: RI) or a fluorescent substance, and then hybridized with DNA in a sample derived from the subject's body fluid that has been transferred to a nylon membrane or the like in a standard manner, and the formed double strand between the nucleic acid probe and the DNA in the sample derived from the subject's body fluid is detected by a radiation detector, a fluorescence detector, or the like by detecting a signal derived from the labeling substance of the nucleic acid probe (a labeling substance such as RI or a fluorescent substance).
• a radioisotope 32 P, 33 P, etc.: RI
• a fluorescent substance a fluorescent substance
• the type of cancer that can be detected or the prognosis of which can be predicted in the method for detecting cancer or predicting the prognosis of cancer is not particularly limited, and examples thereof include the same types of cancer that can be detected or the prognosis of which can be predicted by the biomarker.
• the other steps in the method for detecting cancer or the method for predicting the prognosis of cancer are not particularly limited and can be appropriately selected depending on the purpose.
• Examples of the other steps include a determination step, a step of collecting the body fluid-derived sample from the subject or a healthy individual before the first detection step and the second detection step described below (hereinafter, may be referred to as a "sample collection step"), a step of subjecting the collected body fluid-derived sample to some kind of treatment (hereinafter, may be referred to as a “sample treatment step”), a step of analyzing the obtained detection value or quantification value using known analysis software after the determination step (hereinafter, may be referred to as an "analysis step”), and a step of determining a necessary treatment method depending on the detection result of tetraspanin 10 (hereinafter, may be referred to as a "treatment method determination step”).
• the determination step is broadly divided into a "determination method of the first aspect" in which the subject is determined to be suffering from cancer if the tetraspanin 10 is detected, a “determination method of the second aspect” in which the subject is determined to be suffering from cancer or to have a poor prognosis if the detection value of the tetraspanin 10 is higher than a reference value, and a “determination method of the third aspect” in which the expression level of tetraspanin 10 in the subject is compared with the expression level of tetraspanin 10 in a healthy subject.
• the method of determining whether or not the subject is suffering from cancer comprises, in the method of detecting cancer, determining that the subject is suffering from cancer when the tetraspanin 10 is detected.
• the determining step in the method of determining whether or not the subject is suffering from cancer is performed after the first detection step.
• the determining step in the determination method of the first aspect may be a step of determining that the subject is not affected by cancer if tetraspanin 10 is not detected in the method for detecting cancer.
• the method for predicting the prognosis of cancer preferably uses the determination method of the second aspect or the determination method of the third aspect described below.
• the method of the second aspect includes a step of determining that the subject is suffering from cancer or that the prognosis of the subject is poor when the detected value of tetraspanin 10 is higher than a reference value.
• the determining step in the method of the second aspect is performed after the first detection step.
• the determining step in the determination method of the second aspect may be a step of determining that the subject is not suffering from cancer or that the subject has a good prognosis when the detection value of tetraspanin 10 is lower than a reference value.
• the reference value is not particularly limited, and can be determined by a known method, so long as it is a value that can determine whether or not tetraspanin 10 is detected in a sample derived from a subject's body fluid. Specific examples of methods for determining the reference value include receiver operating characteristic curve (ROC), discriminant analysis, mode method, Kittler method, 3 ⁇ method, p-tile, etc.
• ROC receiver operating characteristic curve
• the reference value is not particularly limited, and can be, for example, sensitivity, specificity, positive predictive value, negative predictive value, etc.
• the determination method of the third aspect includes a step of quantifying tetraspanin 10 in a sample derived from the subject's body fluid (hereinafter sometimes referred to as the "first quantification step”), a step of detecting tetraspanin 10 in a sample derived from the body fluid of a healthy individual (hereinafter sometimes referred to as the "second detection step"), a step of quantifying tetraspanin 10 in the sample derived from the body fluid of the healthy individual (hereinafter sometimes referred to as the "second quantification step”), and a step of determining that the subject is suffering from cancer or has a poor prognosis if the quantitative value of tetraspanin 10 of the subject is higher than the quantitative value of tetraspanin 10 of the healthy individual (hereinafter sometimes referred to as the "determination step”).
• the tetraspanin 10 quantified in the first quantification step, the tetraspanin 10 detected in the second detection step, and the tetraspanin 10 quantified in the second quantification step are preferably biomarkers of the present disclosure.
• the sample derived from the subject's body fluid contains extracellular vesicles and the tetraspanin 10 expressed in the extracellular vesicles of the subject is detected
• the sample derived from the healthy subject's body fluid contains extracellular vesicles and the tetraspanin 10 in the sample derived from the healthy subject's body fluid is detected.
• the second detection step may be performed using extracellular vesicles concentrated from the sample derived from the healthy subject's body fluid instead of the sample derived from the healthy subject's body fluid.
• the first quantification step is a step of quantifying tetraspanin 10 in a sample derived from the subject's body fluid.
• the first quantification step is performed after the first detection step.
• Quantification of tetraspanin 10 in a sample derived from the subject's body fluid is usually performed by measuring the content of tetraspanin 10 in the sample derived from the subject's body fluid.
• the content of tetraspanin 10 may be quantified by mass or concentration, or may be quantified based on the luminescence intensity of the substrate, for example, when detecting tetraspanin 10 protein in the first detection step.
• concentration is not limited to absolute concentration, but also includes relative concentration, mass per unit volume, raw data measured to know absolute concentration, and the like.
• the content of tetraspanin 10 may be quantified based on the number of RNA copies or reads, or may be quantified based on fluorescence intensity, for example, when an RNA sequence is detected as a gene product of a gene sequence encoding tetraspanin 10 in the first detection step.
• the method for quantifying tetraspanin 10 in the subject's body fluid-derived sample is not particularly limited as long as it is a method that can quantify tetraspanin 10, and can be appropriately selected depending on the detection method for tetraspanin 10 in the first detection step, and tetraspanin 10 can be quantified by a conventional method from the detection value of tetraspanin 10 in the first detection step.
• a standard sample such as a solution containing tetraspanin 10 at a known concentration
• a calibration curve is drawn in advance to quantify the concentration of tetraspanin 10 contained in the sample derived from the subject's body fluid
• a method in which a predetermined amount of a standard tetraspanin 10 adjusted to several levels is mixed with the sample derived from the subject's body fluid and the concentration of tetraspanin 10 contained in the sample derived from the subject's body fluid is quantified by an internal standard method.
• the second detection step is a step of detecting tetraspanin 10 in a sample derived from a body fluid of a healthy subject.
• the second detection step can be performed in the same manner as the first detection step, except that the sample derived from the body fluid of the subject to be measured in the first detection step is changed to a sample derived from the body fluid of a healthy subject. Note that, in terms of accurate quantification, it is preferable that the detection method in the first detection step and the detection method in the second detection step are performed in the same manner.
• the "healthy individual” in the second detection step is preferably an individual who is clearly not suffering from the cancer and has not developed the cancer, as determined by conventionally known cancer diagnostic methods such as ultrasound, mammography, and MRI (magnetic resonance imaging), and it is even more preferable that the "healthy individual” is an individual who has no blood relatives with a history of suffering from the cancer.
• the second quantitative determination step is a step of quantifying tetraspanin 10 in the sample derived from the body fluid of the healthy subject.
• the second quantification step can be performed in the same manner as the first quantification step, except that the first quantification step is changed from quantifying tetraspanin 10 in the sample derived from the subject's body fluid detected in the first detection step to quantifying tetraspanin 10 in the sample derived from the body fluid of the healthy subject detected in the second detection step.
• the quantification method in the first quantification step and the quantification method in the second quantification step are performed by the same method.
• the determination step is a step of determining that the subject is suffering from cancer or that the prognosis of the subject is poor when the quantitative value of tetraspanin 10 of the subject is higher than the quantitative value of tetraspanin 10 of the healthy subject.
• the determination step may also be a step of determining that the subject is not suffering from cancer or that the prognosis of the subject is good when the quantitative value of tetraspanin 10 of the subject is similar to or lower than the quantitative value of tetraspanin 10 of the healthy subject.
• the quantitative value of tetraspanin 10 of the subject is “quantitative value A” and the quantitative value of tetraspanin 10 of the healthy subject is “quantitative value B"
• the quantitative value A is preferably 1.05 times or more, more preferably 1.1 times or more, even more preferably 1.5 times or more, and particularly preferably 2 times or more, of the quantitative value B.
• the protein amount or RNA amount in the subject's body fluid-derived sample and the body fluid-derived sample from the healthy subject may be normalized with the protein amount or mRNA amount derived from a housekeeping gene such as GAPDH, ⁇ 2-microglobulin, or ⁇ -actin.
• the sample collection step is a step of collecting the body fluid-derived sample from the subject or the healthy individual before the first detection step and the second detection step.
• the method for collecting the body fluid sample is not particularly limited, and can be appropriately selected from among known methods depending on the type of body fluid.
• the body fluid is a blood sample
• a method of collecting the sample with a syringe can be used.
• the sample treatment step is a step of subjecting the body fluid-derived sample collected in the sample collection step to some kind of treatment.
• the method for processing the body fluid-derived sample is not particularly limited and can be appropriately selected from known methods depending on the type of body fluid, such as separation processing, purification processing, drug addition processing, etc. These may be performed alone or in combination of two or more types.
• the body fluid is a blood sample
• serum may be separated and processed by a known method.
• the analysis step is a step of analyzing the detected or quantified values obtained after the determination step using known analysis software.
• the analysis step if the determination step is performed using the determination method of the first aspect or the determination method of the second aspect, the obtained detection value is analyzed. Also, in the analysis step, if the determination step is performed using the determination method of the third aspect, the obtained quantitative value is analyzed. Therefore, the analysis software in the analysis step can be appropriately selected depending on the detection value or quantitative value obtained by the determination method in the determination step.
• the treatment method determination step is a step of determining a necessary treatment method according to the detection result of the tetraspanin 10.
• the "detection result" in the treatment method determination step includes the following: These include a detected value, a detected value in the determination method of the first aspect and the determination method of the second aspect of the determining step, and a quantitative value in the determination method of the third aspect of the determining step.
• tetraspanin 10 is detected in the subject or if the subject is determined to be suffering from cancer, it may be decided to perform surgical removal of the cancer, to administer radiation therapy and/or chemotherapy, or to perform a combination of these treatments.
• the prognosis of the subject may be decided to perform or continue surgical resection of the cancer, to perform or continue radiation therapy and/or chemotherapy, to conduct follow-up after surgical resection of the cancer, to conduct follow-up after radiation therapy and/or chemotherapy, to conduct follow-up while continuing radiation therapy and/or chemotherapy, the duration of these follow-ups, etc.
• tetraspanin 10 is not detected in the subject, if the subject is determined not to be suffering from cancer, or if the prognosis of the subject is determined to be good, it may be decided to perform follow-up observation, and the duration of such follow-up observation, etc.
• the method for detecting cancer or the method for predicting the prognosis of cancer disclosed herein can be suitably used as, for example, a diagnostic method for diagnosing whether or not a patient is affected by cancer, a predictive method for predicting whether or not a patient has the possibility of developing cancer, a therapeutic effect observation method for confirming the therapeutic effect in a patient undergoing cancer treatment (e.g., surgery, radiotherapy, etc.) (i.e., the therapeutic effect is observed when the value of the tetraspanin 10 decreases after the treatment compared to before the treatment), a monitoring method for following up the presence or absence of recurrence (including metastasis) after cancer treatment (i.e., there is a possibility of recurrence when the value of the tetraspanin 10 increases during follow-up compared to immediately after the treatment), a pharmacodynamic method for analyzing the effect of a drug or compound (including clinical trials of existing drugs and new drugs, etc.) against cancer (i.e., the therapeutic effect
• the treatment can be appropriately selected from known cancer treatments, such as expectant therapy, surgical resection, radiation therapy (including external radiation therapy and internal radiation therapy), radioactive iodine (I-125), palladium, iridium, hormone therapy, leuprolide, goserelin, buserelin, antiandrogens, flutamide, bicalutamide, megestrol acetate, nilutamide, ketoconazole, aminoglutethimide, gonadotropin-releasing hormone (GnRH), estrogen, cryotherapy, chemotherapy, biological therapy, ultrasound irradiation, and proton beam irradiation.
• radiation therapy including external radiation therapy and internal radiation therapy
• radioactive iodine (I-125) radioactive iodine
• palladium iridium
• hormone therapy including external radiation therapy and internal radiation therapy
• leuprolide goserelin, buserelin, antiandrogens
• flutamide bicalutamide
• kit The kit of the present disclosure is a kit for detecting cancer or predicting the prognosis of cancer, which comprises a substance for detecting tetraspanin 10 in a body fluid-derived sample, and further comprises other components as necessary.
• the detection substance is not particularly limited and can be appropriately selected depending on the form of the body fluid-derived sample, and examples thereof include nucleic acids, DNA molecules, RNA molecules, antibodies, antibody fragments, aptamers, peptoids, zDNA, peptide nucleic acids (PNA), locked nucleic acids (LNA), lectins, peptides, dendrimers, membrane protein labeling substances, chemical substances, etc. These may be used alone or in combination of two or more.
• the detection substance is preferably a substance that specifically binds to tetraspanin 10 as a surface antigen of extracellular vesicles or that can detect the expression of tetraspanin 10 as a surface antigen of extracellular vesicles.
• tetraspanin 10 protein detection substance examples include antibodies capable of binding to tetraspanin 10.
• the antibody capable of binding to tetraspanin 10 is not particularly limited as long as it can detect tetraspanin 10 protein and does not impair the effects of the present disclosure, and examples thereof include polyclonal antibodies, monoclonal antibodies, human antibodies, chimeric antibodies, humanized antibodies, and the like that can bind to tetraspanin 10 protein.
• the antibody capable of binding to tetraspanin 10 is preferably one that recognizes an amino acid sequence represented by any one of SEQ ID NOs: 4 to 7 as an epitope sequence.
• the method for obtaining an antibody capable of binding to tetraspanin 10 is not particularly limited and can be appropriately selected depending on the purpose.
• the method include a method in which tetraspanin 10 protein is administered as an antigen to an animal (e.g., rabbit, chicken, pig, mouse, rat, etc.) and the antibody is purified from the serum obtained from the animal by a known method, and a method using a commercially available product.
• Examples of commercially available antibodies capable of binding to tetraspanin 10 include tetraspanin 10 polyclonal antibody (manufactured by Proteintech), anti-TSPAN10 antibody (SAB2102589, rabbit host antibody, manufactured by Sigma), etc. These may be used alone or in combination of two or more kinds.
• the antibody capable of binding to tetraspanin 10 may be in a dry state or dissolved in a buffer such as phosphate-buffered saline.
• nucleic acid detection substance used to detect a nucleic acid sequence (RNA sequence or DNA sequence) encoding tetraspanin 10 is not particularly limited as long as it does not impair the effects of the present disclosure, but is preferably at least one type selected from the group consisting of a nucleic acid primer capable of specifically hybridizing to tetraspanin 10 and a nucleic acid probe capable of specifically hybridizing to tetraspanin 10.
• “capable of specifically hybridizing” includes, for example, a nucleic acid primer or nucleic acid probe having a sequence complementary to the full length or a portion of the nucleic acid sequence encoding tetraspanin 10, a nucleic acid primer or nucleic acid probe capable of hybridizing under stringent conditions to the full length or a portion of the nucleic acid sequence encoding tetraspanin 10, etc. These may be used alone or in combination of two or more types.
• stringent conditions means that hybridization is carried out in a hybridization solution of 5xSSC or an equivalent salt concentration at a temperature of 37°C to 42°C for about 12 hours, and that after preliminary washing with 5xSSC or a solution of an equivalent salt concentration, washing is carried out with 1xSSC or an equivalent salt concentration. In order to obtain higher stringency, washing can be carried out in a solution of 0.1xSSC or an equivalent salt concentration.
• the length of the nucleic acid of the nucleic acid detection substance is not particularly limited and can be appropriately selected depending on the purpose.
• the sequence capable of specifically hybridizing with a DNA sequence encoding tetraspanin 10 is preferably 50 mer or less, more preferably 30 mer or less, and even more preferably 15 mer to 25 mer.
• the nucleic acid primer as the nucleic acid detection substance may contain a sequence that does not encode tetraspanin 10 or does not hybridize with a DNA sequence encoding tetraspanin 10.
• the sequence capable of specifically hybridizing with a DNA sequence encoding tetraspanin 10 is preferably 50 mer or less, more preferably 30 mer or less, and even more preferably 15 mer to 25 mer.
• the nucleic acid probe as the nucleic acid detection substance may contain a sequence that does not encode tetraspanin 10 or does not hybridize with a DNA sequence encoding tetraspanin 10.
• it is preferable that one end of the nucleic acid probe as the nucleic acid detection substance is labeled with a fluorescent dye, and the other end of the nucleic acid probe is labeled with a quencher for the fluorescent dye.
• the detection substance may be labeled with any labeling agent.
• labeling agent include radioisotopes, enzymes, fluorescent substances, luminescent substances, biotin, magnetic labels, enzymes, chemiluminescent probes, metal particles, non-metallic colloidal particles, polymer dye particles, dye molecules, dye particles, electrochemically active species, semiconductor nanocrystals, other nanoparticles (e.g., quantum dots, gold particles, etc.), fluorophores, etc.
• luminescent substances examples include green fluorescent protein (GFP) or variants thereof (e.g., cyan fluorescent protein, yellow fluorescent protein, etc.), luciferase, etc.
• GFP green fluorescent protein
• variants thereof e.g., cyan fluorescent protein, yellow fluorescent protein, etc.
• luciferase e.g., luciferase
• Fluorescent substances include, for example, rare earth chelates (e.g., europium chelates, etc.), rhodamine, FITC, 5-carboxyfluorescein, 6-carboxyfluorescein, TAMRA, dansyl, Lissamine, cyanine, phycoerythrin, Texas Red, Cy3, Cy5, dapoxyl, NBD, Cascade Yellow, dansyl, PyMPO, pyrene, 7-dimethylaminocoumarin-3-carboxylic acid or other coumarin derivatives, Marina Blue, Pacific Blue, Cascade Blue, 2-anthracene sulfony, PyMPO, 3, Examples include 4,9,10-perylene-tetracarboxylic acid, 2,7-difluorofluorescein, 5-carboxyfluorescein, TexasRed-X, AlexaFluor 430, 5-carboxytetramethylrhodamine (5-TAMRA), 6-carboxytetra
• the radioisotope is not particularly limited and may be appropriately selected from known isotopes, such as 3H , 11C , 14C , 18F, 32P , 35S , 64Cu , 68Ga , 86Y , 99Tc , 111In , 123I , 124I , 125I , 131I , 133Xe , 177Lu , 211At , and 213Bi .
• the detection substance is preferably contained in a container.
• the container There are no particular limitations on the container as long as it is capable of containing the detection substance, and it can be appropriately selected from known containers according to the purpose, such as tubes and glass vials.
• kits are not particularly limited as long as they do not impair the effects of the present disclosure, and can be appropriately selected depending on the purpose.
• detection substances other than the detection substance of the biomarker; equipment (e.g., tubes, pipettes, syringes, petri dishes, multi-well plates, reagents, etc.) for recovering or extracting a biological material to be detected, preferably a sample derived from a body fluid of a living organism to be detected (hereinafter, sometimes referred to as a "target sample"), equipment (e.g., separation membranes, filters, columns, reagents, etc.) for separating or purifying the target sample, equipment for extracting the target sample; internal standard reagents; ELISA method, immunostaining method, Western blotting method, RNA-seq analysis method, RT-PCR method (quantitative RT-PCR), etc.
• equipment e.g., tubes, pipettes, syringes, petri dishes, multi-well plates, reagent
• CR various reagents usually used in nucleic acid chip analysis, Northern blotting, PCR, digital PCR, Southern blotting, microarrays, etc.
• reagents usually used in nucleic acid chip analysis e.g., polymerase, buffer, dNTP, reverse transcriptase, dye, hybridization buffer, washing buffer, protease, etc.
• secondary antibodies used in these methods substrates for detecting enzyme reactions; stabilizers such as ⁇ -mercaptoethanol and DTT; protective agents such as albumin; preservatives such as polyoxyethylene (20) sorbitan monolaurate, surfactants, and sodium azide; instructions for the kit; instructions for evaluating whether or not the patient is suffering from cancer or has the potential to develop cancer.
• these may be used alone, or two or more types may be used in combination.
• the other detection substances are not particularly limited, and examples thereof include a PCNA detection substance for detecting PCNA (proliferating cell nuclear antigen), which is a marker for extracellular vesicles. These may be used alone or in combination of two or more.
• PCNA detection substance for detecting PCNA proliferating cell nuclear antigen
• the PCNA detection substance is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include an anti-PCNA antibody that recognizes PCNA, a nucleic acid primer or a nucleic acid probe having a sequence complementary to the full length or a part of the nucleic acid sequence encoding the PCNA protein, etc. These may be used alone or in combination of two or more kinds.
• the kit of the present disclosure cancer can be detected specifically, highly sensitively, simply and quickly, and therefore can be used for detecting cancer or predicting the prognosis of cancer. Therefore, it is possible to evaluate whether a subject to be detected using the kit of the present disclosure has cancer or not, and whether the prognosis is poor or not, and further, it is possible to evaluate cancer at an early stage. Therefore, the kit of the present disclosure can be suitably used as a cancer diagnosis kit or a cancer diagnosis auxiliary kit for determining whether a subject has cancer or not, and as a prognosis prediction kit for determining whether the prognosis of cancer is poor or not.
• the kit of the present disclosure can be suitably used to carry out the method of detecting cancer or predicting the prognosis of cancer of the present disclosure. Therefore, the method of using the kit of the present disclosure is the same as the method of detecting cancer or predicting the prognosis of cancer of the present disclosure.
• An anti-TSPAN10 antibody solution was prepared by diluting an anti-TSPAN10 antibody (TSPAN10 polyclonal antibody, manufactured by Proteintech) in PBS (manufactured by Nacalai Tesque, Inc.) to 300 ng/50 ⁇ L.
• the epitope sequence recognized by the anti-TSPAN10 antibody is the amino acid sequence shown in SEQ ID NO:4.
• Biotinylated anti-TSPAN10 antibody polyclonal rabbit anti-human OCSP/TSPAN10 antibody (Biotin, aa74-123, WB) LS-C453227, manufactured by LSBio) was diluted in PBS to 100 ng/100 ⁇ L to prepare biotinylated anti-TSPAN10 antibody solution A antibody solution.
• the anti-TSPAN10 antibody solution was added to an immunoplate (surface treatment: MaxiSorp TM , manufactured by Thermo Fisher Scientific) at 50 ⁇ L/well, and the plate was left to stand at 25° C. for 2 hours to immobilize the anti-TSPAN10 antibody on the immunoplate.
• a blocking buffer SuperBlock TM Blocking Buffer, manufactured by Thermo Fisher Scientific was added to the immunoplate on which the anti-TSPAN10 antibody was immobilized at 300 ⁇ L/well, and the plate was left to stand at 25° C. for 30 minutes.
• biotinylated anti-TSPAN10 antibody solution A was added to the washed wells at 100 ⁇ L/well, rotated at 300 rpm for 1 minute using a mixer (Eppendorf ThermoMixer (registered trademark) C, manufactured by Eppendorf) at 25° C., and then allowed to stand for 1 minute, and this process was repeated for 3 hours.
• the biotinylated anti-TSPAN10 antibody solution A was removed from the wells, and 400 ⁇ L of PBS was added and then removed, and this was repeated three times to wash the wells.
• a poly-HRP label (Streptavidin Poly-HRP40 Conjugate, Horseradish Peroxidase (Fitzgerald Industries International, Inc.) diluted 50,000-fold with PBS was added at 100 ⁇ L/well.
• the solution containing the poly-HRP label was removed from the wells, and 400 ⁇ L of PBS was added and then removed three times for washing.
• TMB substrate (SureBlue TM TMB 1-Component Microwell Peroxidase Substrate, sera After reacting for 5 minutes at 25° C., the reaction was stopped with 2 M hydrochloric acid. Then, the absorbance was measured at a wavelength of 450 nm using a microplate reader (Epoch 2, Agilent Technologies).
• ROC receiver operating characteristic
• the sensitivity calculated by the ELISA method using the following formula 1-1 was 84%
• the specificity calculated by the following formula 1-2 was 50%
• the positive predictive value calculated by the following formula 1-3 was 64%
• the negative predictive value calculated by the following formula 1-4 was 75%
• the prevalence calculated by the following formula 1-5 was 52%.
• the positive rate of CEA an existing representative tumor marker, for stage 0/I breast cancer is 6.8% (see Shiozaki, Shigehiro et al., "Clinical Significance of Tumor Markers for Breast Cancer," Journal of the Japanese Society of Clinical Surgery, Vol. 55, No. 5, pp. 1077-1082, 1994), and TSPAN10 has been shown to be useful as a biomarker for early cancer detection.
• Obtaining serum samples A total of seven serum samples were obtained from breast cancer patients (subjects 1 to 7). In addition, no recurrence of breast cancer was confirmed in any of the subjects at the time of blood collection after treatment through CT scans, ultrasound scans, and tumor marker tests.
• Subject 1 This subject had stage II breast cancer according to the TNM classification, and serum samples were taken before and 14 months after treatment (surgery and chemotherapy) was started (post-treatment).
• Subject 2 This subject had stage II breast cancer according to the TNM classification, and serum samples were taken before and 28 months after treatment (surgery and chemotherapy) was started (post-treatment).
• Subject 3 This subject had stage III breast cancer according to the TNM classification, and serum samples were taken before and 24 months after treatment (surgery and chemotherapy) was started (post-treatment).
• Subject 4 This subject had stage III breast cancer according to the TNM classification, and serum samples were taken before and 19 months after treatment (surgery and chemotherapy) was started (post-treatment).
• Subject 5 This subject had stage II breast cancer according to the TNM classification, and serum samples were taken before and 17 months after treatment (surgery and chemotherapy) was started (post-treatment).
• Subject 6 This subject had stage II breast cancer according to the TNM classification, and serum samples were taken before and 9 months after treatment (surgery and chemotherapy) was started (post-treatment).
• Subject 7 This subject had stage III breast cancer according to the TNM classification, and serum samples were taken before and 11 months after treatment (surgery and chemotherapy) was started (post-treatment).
• Detection by ELISA In the detection by ELISA in Test Example 1, detection by ELISA was performed in the same manner as in Test Example 1, except that the serum samples were changed to the serum samples of Subjects 1 to 7.
• TSPAN10 and p53 expression in extracellular vesicles ⁇ Analysis of TSPAN10 and p53 protein expression in extracellular vesicles> Using the Te-EVs Proteome Library (https://repository.jppostdb.org/entry/JPST000867, the contents of which are incorporated herein by reference in their entirety), the expression of TSPAN10 protein and p53 protein was analyzed in a total of 34 extracellular vesicles from 17 cancer patients (2 samples per case).
• biotinylated anti-p53 antibody solution -- Anti-p53 antibody (p53 antibody (DO-1), sc-126, manufactured by Santa Cruz Biotechnology) was labeled with biotin using an antibody/protein labeling kit (Biotin Labeling Kit-NH 2 , manufactured by Dojindo Laboratories, Inc.) to obtain a biotinylated anti-p53 antibody.
• the obtained biotinylated anti-p53 antibody was diluted in PBS to 100 ng/100 ⁇ L to prepare a biotinylated anti-p53 antibody solution.
• Serum samples from one patient with stage III breast cancer according to the TNM classification were collected before preoperative chemotherapy (0 month), 3 months, and 6 months after the start of preoperative chemotherapy with anticancer drugs (EC therapy (administration of epirubicin and cyclophosphamide) and docetaxel + trastuzumab + pertuzumab therapy). Then, 7 months after the start of preoperative chemotherapy, breast cancer was removed and serum samples were collected 1 month after surgery. Furthermore, 1 month after the start of surgery, postoperative adjuvant therapy with trastuzumab, pertuzumab, and anastrozole was started, and serum samples were collected 5 months, 9 months, and 12 months after the start of adjuvant therapy.
• Test Example 4 Detection by ELISA Detection by ELISA in Test Example 4 was performed in the same manner as in Test Example 4, except that the serum sample was changed to the above serum sample.
• the detection results by ELISA are shown in Figure 7. Compared to before preoperative chemotherapy, the absorbance measured by ELISA decreased after preoperative chemotherapy, surgery, and postoperative adjuvant therapy. This indicates that TSPAN10 is useful as a biomarker for predicting cancer prognosis.
• TSPAN10 mRNA expression in colon adenocarcinoma and rectal adenocarcinoma tissues As shown in Figure 1C, the expression level of TSPAN10 mRNA was low in colon cancer tissues including colon adenocarcinoma and rectal adenocarcinoma among various cancer tissues. Therefore, a more detailed analysis of TSPAN10 mRNA expression in colon adenocarcinoma and rectal adenocarcinoma tissues was performed.
• FIG. 8 The analysis results of the expression level of tetraspanin 10 mRNA are shown in FIG. 8. It was found that the expression level of tetraspanin 10 mRNA increases with the progression of the stage. As a result of the Jonckheere-Terpstra test, the p-value was 3.74-3 , and there was a tendency for the expression of tetraspanin 10 mRNA to increase with the progression of the stage of colorectal cancer, that is, with the progression of colorectal cancer. In FIG. 8, "FPKM” indicates the corrected expression level count data (FPKM) of RNA-seq.
• a cutoff value (0.071) was determined using Youden's index from the FPKM, and patients were classified into a "high-risk group” and a "low-risk group” based on the cutoff value.
• a cutoff value (0.07) was determined using Youden's index from the FPKM, and patients were classified into "high-risk group” and "low-risk group” based on the cutoff value.
• TSPAN10 may be useful as a biomarker for colorectal cancer.
• Detection by ELISA Detection by ELISA was performed in the same manner as in Test Example 1, except that the serum samples in Test Example 1 were changed to those of the control group and the colon cancer stage III/IV group.
• ROC receiver operating characteristic
• the sensitivity calculated by the ELISA method using the following formula 2-1 was 77%
• the specificity calculated by the following formula 2-2 was 83%
• the positive predictive value calculated by the following formula 2-3 was 83%
• the negative predictive value calculated by the following formula 2-4 was 77%
• the prevalence calculated by the following formula 2-5 was 52%. It is known that the positive rate of CEA, an existing representative tumor marker, for stage III colon cancer is 20% to 30%, and the positive rate for stage IV colon cancer is 73% (see Miyashita Tomoharu et al., Journal of the Japanese Society of Coloproctology, 2000, Vol. 53, No. 2, pp.
• TSPAN10 and MUC16 may be useful as a biomarker for various cancers.
• biotinylated anti-MUC16 antibody solution was prepared by diluting a biotinylated anti-CA125 mouse monoclonal antibody (clone M11), Elecsys® CA 125M, manufactured by Roche, in PBS to 100 ng/100 ⁇ L.
• the receiver operating characteristic (ROC) curve as a result of the validation of the analytical method using the ELISA method is shown in Figure 14.
• the area under the ROC curve (AUC) was 0.891, and the 95% confidence interval was 0.726-1.
• a cutoff value (0.650) was determined using Youden's index, and the results of the number of people divided into "positive” and "negative” for ELISA detection are shown in Table 3 below.
• the sensitivity calculated by the ELISA method using the following formula 3-1 was 75%
• the specificity calculated by the following formula 3-2 was 100%
• the positive predictive value calculated by the following formula 3-3 was 100%
• the negative predictive value calculated by the following formula 3-4 was 80%
• the prevalence calculated by the following formula 3-5 was 50%. It is known that the positive rate of serum CA125, an existing representative tumor marker, for stage III pancreatic cancer is 70% (see Takemori Yasuhiro et al., Journal of the Japanese Society of Gastroenterology, 1987, Vol. 84, No. 10, pp.
• Sensitivity [Pancreatic cancer stage III group and ELISA detection positive] / [Total of pancreatic cancer stage III group] ⁇ 100 (Equation 3-1)
• Specificity [control group and ELISA detection negative] / [total of control group] ⁇ 100 (Equation 3-2)
• Positive predictive value [Pancreatic cancer stage III group and ELISA detection positive] / [Total of ELISA detection positive] ⁇ 100 (Equation 3-3)
• Negative predictive value [control group and ELISA detection negative] / [total of ELISA detection negative] ⁇ 100 (Equation 3-4)
• Test Example 10 Validation of analytical method for various cancers using TSPAN10 and MUC16
• control group 12 were sera from healthy subjects
• 59 were sera from patients with various cancers at stage III or stage IV according to the TNM classification (hereinafter sometimes referred to as the "cancer group”).
• the cancer group included two sera from patients with head and neck cancer at stage IV according to the TNM classification, 11 sera from patients with esophageal cancer at stage IV according to the TNM classification, 13 sera from patients with gastric cancer at stage IV according to the TNM classification, 12 sera from patients with lung cancer (one at stage I according to the TNM classification, two at stage II according to the TNM classification, two at stage III according to the TNM classification, and seven at stage IV according to the TNM classification), 12 sera from patients with thyroid cancer at an unknown stage according to the TNM classification, one serum from a patient with cervical cancer at stage IV according to the TNM classification, one serum from a patient with ovarian cancer at stage IV according to the TNM classification, one serum from a patient with malignant melanoma at an unknown stage according to the TNM classification, one serum from a patient with renal cancer at stage IV according to the TNM classification, and five sera from patients with liver cancer at stage III according to the TNM classification.
• Detection by ELISA In the detection by ELISA in Test Example 9, detection by ELISA was performed in the same manner as in Test Example 9, except that the serum samples were changed to the serum samples from the control group and the cancer group.
• ROC receiver operating characteristic
• Test Example 11 Detection of extracellular vesicles in serum
• Test Examples 1 to 10 suggest that cancer can be detected or the prognosis of cancer can be predicted using the serum of cancer patients. It was also suggested that this is due to the release of extracellular vesicles derived from cancer cells into the serum. Therefore, the expression of TSPAN10 in extracellular vesicles in the serum of cancer patients was confirmed by the following method.
• the serum sample was purified using an exosome purification column (EVSecond, manufactured by GL Sciences Inc.) according to the product protocol. Specifically, a packing material was added to the column, and the storage solution was removed after thorough mixing. Next, 700 ⁇ L of fetal bovine serum (FBS) for cell culture was added to the column for blocking. After removing the FBS, PBS was added to the column and washed three times. Next, 300 ⁇ L of the serum sample was loaded, and 100 ⁇ L of PBS was added to elute, which was repeated 12 times to obtain elution fractions 1 to 12.
• EVSecond fetal bovine serum
• Detection by ELISA Detection by ELISA was performed in the same manner as in Test Example 1, except that the serum sample was changed to the elution fractions 1 to 12. As a result, high absorbance was obtained in the elution fractions 8 to 10 eluted in the 7th to 10th elutions.
• Anti-TSPAN10 antibody (SAB2102589, rabbit host antibody, manufactured by Sigma) was labeled with biotin using an antibody/protein labeling kit (Biotin Labeling Kit-NH 2 , manufactured by Dojindo Laboratories, Inc.) to obtain biotinylated anti-TSPAN10 antibody solution B.
• the epitope sequence recognized by the anti-TSPAN10 antibody (SAB2102589) is the amino acid sequence shown in SEQ ID NO:5.
• the beads were washed once with 1 mL of PBS, and the tetraspanin 10-positive extracellular vesicles captured on the beads were eluted with Exosome Elution Buffer included in the exosome purification kit (MagCaptureTM Exosome Isolation Kit PS Ver. 2, Fujifilm Wako Pure Chemical Industries, Ltd.).
• a method for detecting cancer or predicting the prognosis of cancer comprising: The method comprises the step of detecting tetraspanin 10 in a sample derived from a subject's body fluid.
• the method according to ⁇ 1> above further comprising the step of determining that the subject is suffering from cancer when the tetraspanin 10 is detected in the method for detecting cancer.
• ⁇ 3> The method according to ⁇ 1> above, further comprising a step of determining that the subject is suffering from cancer or that the prognosis of the subject is poor when the detection value of the tetraspanin 10 is higher than a reference value.
• ⁇ 4> The method according to any one of ⁇ 1> to ⁇ 3>, wherein the sample derived from the subject's body fluid contains extracellular vesicles, and the detecting step detects the tetraspanin 10 expressed in the extracellular vesicles.
• ⁇ 5> A step of quantifying tetraspanin 10 in a sample derived from the subject's body fluid; detecting tetraspanin 10 in a sample derived from a body fluid of a healthy subject; Quantifying tetraspanin 10 in a sample derived from a body fluid of the healthy subject; determining that the subject is suffering from cancer or has a poor prognosis when the quantitative value of tetraspanin 10 in the subject is higher than the quantitative value of tetraspanin 10 in the healthy subject;
• the method according to ⁇ 5> wherein the sample derived from the subject's body fluid contains extracellular vesicles, and the step of detecting tetraspanin 10 in the sample derived from the subject's body fluid detects the tetraspanin 10 expressed in the extracellular vesicles of the subject; and the sample derived from the healthy subject's body fluid contains extra
• ⁇ 7> The method according to any one of ⁇ 1> to ⁇ 6>, wherein the detection step detects the tetraspanin 10 by an ELISA method.
• the cancer is at least one selected from the group consisting of breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, esophageal cancer, gastric cancer, lung cancer, thyroid cancer, uterine cancer, ovarian cancer, malignant melanoma, kidney cancer, liver cancer, epithelial cancer, rectal cancer, colon cancer, papillary renal cell carcinoma, head and neck squamous cell carcinoma, serous cystadenocarcinoma, chromophobe renal cell carcinoma, prostate cancer, lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, renal non-clear cell carcinoma, hepatocellular carcinoma, testicular germ cell tumor, pancreatic adenocarcino
• a biomarker for detecting cancer or predicting the prognosis of cancer comprising: This is a biomarker characterized by containing tetraspanin 10.
• tetraspanin 10 is expressed in extracellular vesicles.
• the cancer is at least one selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, head and neck cancer, esophageal cancer, gastric cancer, lung cancer, thyroid cancer, uterine cancer, ovarian cancer, melanoma, renal cancer, liver cancer, epithelial cancer, rectal cancer, colon cancer, papillary renal cell carcinoma, head and neck squamous cell carcinoma, serous cystadenocarcinoma, chromophobe renal cell carcinoma, prostate cancer, lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, renal non-clear cell carcinoma, hepatocellular carcinoma, testicular germ cell tumor, pancreatic adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, and colorectal adenocarcinoma.
• ⁇ 12> The biomarker according to ⁇ 11>, which is for detecting early-stage colorectal cancer or early-stage breast cancer.
• ⁇ 13> A method for detecting tetraspanin 10 in a body fluid-derived sample, comprising using the tetraspanin 10 as a biomarker for detecting cancer or predicting the prognosis of cancer.
• ⁇ 14> The method according to ⁇ 13>, wherein the body fluid-derived sample contains extracellular vesicles.
• ⁇ 15> The method according to ⁇ 13> or ⁇ 14>, wherein the cancer is at least one selected from the group consisting of breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, esophageal cancer, gastric cancer, lung cancer, thyroid cancer, uterine cancer, ovarian cancer, melanoma, renal cancer, liver cancer, epithelial cancer, rectal cancer, colon cancer, papillary renal cell carcinoma, head and neck squamous cell carcinoma, serous cystadenocarcinoma, chromophobe renal cell carcinoma, prostate cancer, lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, renal non-clear cell carcinoma, hepatocellular carcinoma, testicular germ cell tumor, pancreatic adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, and colorectal adenocarcinoma.
• the cancer is at least one selected from
• a kit for detecting cancer or predicting the prognosis of cancer comprising: The kit is characterized by comprising a substance for detecting tetraspanin 10 in a sample derived from a body fluid.
• the detection substance is at least one selected from the group consisting of an antibody capable of binding to tetraspanin 10, a nucleic acid primer capable of specifically hybridizing to tetraspanin 10, and a nucleic acid probe capable of specifically hybridizing to tetraspanin 10.

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