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WO2017122815A1 - Novel fusant and method for detecting same - Google Patents

Novel fusant and method for detecting same Download PDF

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Publication number
WO2017122815A1
WO2017122815A1 PCT/JP2017/001117 JP2017001117W WO2017122815A1 WO 2017122815 A1 WO2017122815 A1 WO 2017122815A1 JP 2017001117 W JP2017001117 W JP 2017001117W WO 2017122815 A1 WO2017122815 A1 WO 2017122815A1
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WIPO (PCT)
Prior art keywords
ret
gene
rufy1
ncoa4
fusion
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PCT/JP2017/001117
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French (fr)
Japanese (ja)
Inventor
賢吾 竹内
征士 坂田
由紀 冨樫
直也 藤田
量平 片山
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公益財団法人がん研究会
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Priority to JP2017561205A priority Critical patent/JP6871869B2/en
Publication of WO2017122815A1 publication Critical patent/WO2017122815A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/99—Enzyme inactivation by chemical treatment
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • 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

Definitions

  • the present invention relates to a novel fusion protein containing a RET kinase region or a fusion gene encoding the fusion protein, and a method for detecting them.
  • the present invention relates to a novel fusion protein containing at least a part of NCOA4 or RUfy1, or a fusion gene encoding the fusion protein, and a method for detecting them.
  • Non-patent Document 1 Patent Document 1
  • Patent Document 2 Non-patent Document 2
  • RET Ret proto-oncogene
  • RET is a transmembrane receptor tyrosine kinase that binds to and activates GFR-alpha dimerized by binding to GDNF via the extracellular domain. Activation is via a cadherin-like domain of extracellular RET, and RET forms a dimer, and autophosphorylation of the intracellular tyrosine kinase domain occurs (Non-patent Document 3).
  • Non-patent literature 2 Non-patent literature 2
  • Non-Patent Document 3 Non-Patent Document 4
  • Non-Patent Document 5 The fusion product produced by the rearrangement of the RET gene and other genes has a kinase domain that is constantly phosphorylated, and continues to send signals to the MAPK pathway and the like, leading to cell canceration.
  • An object of the present invention is to provide a detection method for a fusion protein or a fusion gene encoding the fusion protein, and a detection method based on the elucidation of a fusion (fusion protein and fusion gene) that is a new causative factor of cancer.
  • Method for diagnosing cancer used method for determining subject of application of pharmaceutical composition for cancer treatment, kit and primer set for detection method, inhibitor of activity and / or expression of polypeptide as fusion protein
  • a cancer therapeutic method comprising administering the pharmaceutical composition for cancer treatment containing the inhibitor and the pharmaceutical composition for cancer treatment.
  • the present inventor has obtained, from a sample obtained from a colorectal cancer patient, a fusion gene in which a part of the NCOA4 gene and a part of the RET gene that is a kinase are fused, a part of the RUFY1 gene, and a RET that is a kinase.
  • a novel fusion gene fused with a part of the gene was isolated and identified (Examples 1 to 3), and it was found that the fusion gene was present in colorectal cancer patients (Examples 4 to 5).
  • the present inventor provides a method for detecting a RET fusion protein or a fusion gene encoding the fusion protein, and provides a kit and a primer set therefor, and the fusion protein encoding the fusion protein or the fusion protein.
  • a method for treating cancer comprising: detecting a gene, enabling determination of a cancer patient to be treated with a RET inhibitor, and administering the RET inhibitor to the cancer patient.
  • the present inventor provides an NCOA4 or RUFY1 fusion protein or a method for detecting a fusion gene encoding the fusion protein, provides a kit and a primer set therefor, and encodes the fusion protein or the fusion protein. Detecting a fusion gene that makes it possible to determine a cancer patient to be treated with an NCOA4 or RUFY1 inhibitor, and administering the NCOA4 or RUFY1 inhibitor to the cancer patient, Provide a method for treating cancer.
  • the present invention relates to the following inventions: [1] A method for detecting a RET fusion protein or a fusion gene encoding the fusion protein in a sample obtained from a subject. [2] The detection method according to [1], wherein the detection method includes a step of detecting cleavage of the RET protein or cleavage of a gene encoding the RET protein. [3] The detection method includes a step of detecting the presence of a fusion protein constructed from a RET protein and another protein or the presence of a fusion gene encoding the fusion protein. [1] The detection method according to.
  • fusion protein is a fusion protein of NCOA4 or RUFI1 protein and RET protein.
  • fusion protein is a polypeptide selected from the group consisting of the following (a) to (d): (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (NCOA4-RET) or SEQ ID NO: 4 (RUFY1-RET), (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity, (C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity; and (d) SEQ ID NO: 2 or SEQ ID NO: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted,
  • [6] The detection method according to any one of [1] to [5], wherein the RET fusion gene is a polynucleotide encoding the polypeptide according to [5].
  • [7] The detection method according to any one of [1] to [6], wherein the fusion gene is DNA or mRNA.
  • [8] The detection method according to any one of [1] to [7], wherein the sample is a digestive organ-derived sample.
  • the detection method according to [8], wherein the digestive organ-derived sample is a digestive tract-derived sample.
  • the detection method according to [8], wherein the digestive organ-derived sample is a lower digestive tract-derived sample.
  • a RET fusion gene comprising a first probe capable of specifically recognizing the RET gene 5 ′ terminal genomic region and a second probe capable of specifically recognizing the RET gene 3 ′ terminal genomic region
  • a probe for detecting a RET fusion gene comprising a first probe capable of specifically recognizing the RET gene 5 ′ terminal genomic region and a second probe capable of specifically recognizing the RET gene 3 ′ terminal genomic region
  • a sense primer and an antisense primer designed to specifically amplify the 5 ′ end region of the polynucleotide encoding the RET protein, and specifically amplify the 3 ′ end region of the polynucleotide.
  • a kit for detecting a RET fusion gene comprising a sense primer and an antisense primer designed to be able to.
  • An NCOA4-RET or RUFY1-RET fusion comprising a sense primer and an antisense primer designed to specifically amplify a polynucleotide encoding a polypeptide that is a fusion protein of NCOA4 or RUFY1 protein and RET protein Gene detection kit.
  • NCOA4-RET or RUFY1 comprising a sense primer and an antisense primer designed to specifically amplify a polynucleotide encoding a polypeptide selected from the group consisting of the following (a) to (d) -RET fusion gene detection kit: (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity, (C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity; and (d) SEQ ID NO: 2 or SEQ ID NO: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted in the amino acid sequence represented by 4, and having tumorigenic potential.
  • a kit for detecting a RET fusion protein comprising an anti-RET antibody capable of specifically recognizing the N-terminal region of the RET protein and an anti-RET antibody capable of specifically recognizing the C-terminal region of the RET protein.
  • An antibody that specifically binds to a polypeptide in the N-terminal region of another protein that constitutes the RET fusion protein together with the RET protein, and an antibody that specifically binds to a polypeptide in the C-terminal region of the RET protein A kit for detecting a RET fusion protein.
  • a fusion gene between an NCOA4 or RUFY1 gene and an RET gene comprising an antisense primer designed from a polynucleotide portion encoding a RET protein and a sense primer designed from a polynucleotide portion encoding an NCOA4 or RUFY1 protein
  • a primer set for detection wherein the antisense primer comprises a nucleic acid molecule that anneals to the polynucleotide of [16] under stringent conditions
  • the sense primer is a complementary strand of the polynucleotide of [16] Primer set consisting of nucleic acid molecules that anneal under stringent conditions.
  • a primer set for detecting a fusion gene of NCOA4 or RUFY1 gene and RET gene, which anneals to a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions A primer set comprising an antisense primer comprising a nucleic acid molecule and a sense primer comprising a nucleic acid molecule which anneals to a complementary strand of the polynucleotide under stringent conditions.
  • a primer set comprising a sense primer and an antisense primer selected from the group consisting of the following (a) to (b): (A) a sense primer composed of any consecutive at least 16 base oligonucleotides between base numbers 1 to 1984 of SEQ ID NO: 1, and any continuous at least 16 bases between base numbers 1985 to 5275 of SEQ ID NO: 1 An antisense primer consisting of an oligonucleotide that is complementary to the oligonucleotide of (B) a sense primer consisting of an oligonucleotide of at least 16 consecutive bases between base numbers 1 to 1917 of SEQ ID NO: 3 and at least 16 consecutive bases of base numbers 1918 to 5208 of SEQ ID NO: 3 An antisense primer comprising an oligonucleotide that is complementary to the oligonucleotide.
  • the screening method according to [23], wherein the substance that inhibits the activity and / or expression of the polypeptide is a therapeutic agent for RET fusion-positive cancer.
  • the screening method according to [24] wherein the cancer is digestive cancer.
  • the screening method according to [25], wherein the cancer is gastrointestinal cancer.
  • a pharmaceutical composition for treating RET fusion-positive cancer comprising a substance that inhibits the activity and / or expression of a RET fusion protein.
  • the pharmaceutical composition according to [29] wherein the substance that inhibits the activity and / or expression of the RET fusion protein is a kinase inhibitor.
  • the pharmaceutical composition according to [29] or [30], wherein the RET fusion protein is the polypeptide according to [5].
  • the pharmaceutical composition according to any of [29] to [31], wherein the cancer is gastrointestinal cancer.
  • a vector comprising the polynucleotide according to [39].
  • the detection method includes a step of detecting the presence of a fusion protein constructed from an NCOA4 or RUFY1 protein and another protein, or the presence of a fusion gene encoding the fusion protein. 42].
  • the detection method according to any of [42] to [44], wherein the fusion protein is a fusion protein of NCOA4 or RUFI1 protein and RET protein.
  • the fusion protein is a polypeptide selected from the group consisting of the following (a) to (d): (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity, (C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity; and (d) SEQ ID NO: 2 or SEQ ID NO: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted in the amino acid sequence represented by 4, and having tumorigenic potential.
  • a first probe capable of specifically recognizing the 3'-terminal genomic region of another gene that constitutes the NCOA4 or RUFY1 fusion gene together with the NCOA4 or RUFY1 gene, and the NCOA4 or RUFY1 gene 5'-terminal genomic region specific A kit for detecting an NCOA4 or RUFY1 fusion gene, comprising a second probe that can be recognized visually.
  • a sense primer and an antisense primer designed to specifically amplify the 5 ′ end region of the polynucleotide encoding NCOA4 or RUFY1 protein, and the 3 ′ end region of the polynucleotide specifically A kit for detecting an NCOA4 or RUFY1 fusion gene, comprising a sense primer and an antisense primer designed so that they can be amplified.
  • NCOA4-RET or RUFY1-RET fusion comprising a sense primer and an antisense primer designed to specifically amplify a polynucleotide encoding a polypeptide that is a fusion protein of NCOA4 or RUFY1 protein and RET protein Gene detection kit.
  • NCOA4-RET or RUFY1 comprising a sense primer and an antisense primer designed to specifically amplify a polynucleotide encoding a polypeptide selected from the group consisting of the following (a) to (d) -RET fusion gene detection kit: (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity, (C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity; and (d) SEQ ID NO: 2 or SEQ ID NO: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted in the amino acid sequence represented by 4, and having tumorigenic potential.
  • An NCOA4 comprising an anti-NCOA4 or RUFY1 antibody capable of specifically recognizing the N-terminal region of the NCOA4 or RUFY1 protein, and an anti-NCOA4 or RUFY1 antibody capable of specifically recognizing the C-terminal region of the NCOA4 or RUFY1 protein
  • a kit for detecting RUFY1 fusion protein Alternatively, a kit for detecting RUFY1 fusion protein.
  • a fusion gene of an RET gene and an NCOA4 or RUFY1 gene comprising an antisense primer designed from a polynucleotide portion encoding a RET protein and a sense primer designed from a polynucleotide portion encoding an NCOA4 or RUFY1 protein
  • a primer set for detection wherein the antisense primer comprises a nucleic acid molecule that anneals to the polynucleotide described in [57] under stringent conditions
  • the sense primer is a complementary strand of the polynucleotide described in [57] Primer set consisting of nucleic acid molecules that anneal under stringent conditions.
  • a primer set for detecting a fusion gene of NCOA4 or RUFY1 gene and RET gene, which anneals to a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions A primer set comprising an antisense primer comprising a nucleic acid molecule and a sense primer comprising a nucleic acid molecule which anneals to a complementary strand of the polynucleotide under stringent conditions.
  • a primer set comprising a sense primer and an antisense primer selected from the group consisting of the following (a) to (b): (A) a sense primer composed of any consecutive at least 16 base oligonucleotides between base numbers 1 to 1984 of SEQ ID NO: 1, and any continuous at least 16 bases between base numbers 1985 to 5275 of SEQ ID NO: 1 An antisense primer consisting of an oligonucleotide that is complementary to the oligonucleotide of (B) a sense primer consisting of an oligonucleotide of at least 16 consecutive bases between base numbers 1 to 1917 of SEQ ID NO: 3 and at least 16 consecutive bases of base numbers 1918 to 5208 of SEQ ID NO: 3 An antisense primer comprising an oligonucleotide that is complementary to the oligonucleotide.
  • [64] The step of bringing the test substance into contact with the polypeptide according to (1) [46] or a cell expressing the polypeptide, (2) analyzing whether or not the activity and / or expression of the polypeptide is inhibited; and (3) selecting the substance that inhibits the activity and / or expression of the polypeptide.
  • the screening method according to [64] wherein the substance that inhibits the activity and / or expression of the polypeptide is a therapeutic agent for cancer positive for NCOA4 or RUFY1 fusion.
  • the screening method according to [65] wherein the cancer is digestive organ cancer.
  • a pharmaceutical composition for treating NCOA4 or RUFY1 fusion-positive cancer comprising a substance that inhibits the activity and / or expression of the NCOA4 or RUFY1 fusion protein.
  • the pharmaceutical composition according to [70], wherein the substance that inhibits the activity and / or expression of the NCOA4 or RUfy1 fusion protein is a kinase inhibitor.
  • NCOA4 or RUfy1 fusion protein is the polypeptide according to [46].
  • NCOA4 or RUFI1 fusion protein A fusion protein of NCOA4 or RUFI1 and RET.
  • a vector comprising the polynucleotide according to [80].
  • a method for treating RET fusion-positive cancer wherein the substance that inhibits the activity and / or expression of a RET fusion protein is a kinase inhibitor.
  • a method for treating NCOA4 or RUFY1 fusion-positive cancer wherein the substance that inhibits the activity and / or expression of the NCOA4 or RUFY1 fusion protein is a kinase inhibitor.
  • the detection method of the present invention can be used as a method for detecting RET fusion-positive cancer (particularly digestive organ cancer).
  • the detection kit and primer set of the present invention can be used in the detection method of the present invention.
  • a substance effective for the treatment of the fusion-positive cancer patient can be screened.
  • the substance obtained by the screening method can be used as an active ingredient of a pharmaceutical composition for the treatment of RET fusion-positive cancer, and can also be used for the treatment of RET fusion-positive cancer.
  • the detection method of the present invention can be used as a method for detecting NCOA4 or RUFY1 fusion-positive cancer (particularly digestive organ cancer). Further, according to the detection method of the present invention, NCOA4 or RUFY1 fusion-positive cancer in a subject can be diagnosed, and further, it is determined whether or not the subject is an application target of an NCOA4 or RUFY1 inhibitor. it can.
  • the detection kit and primer set of the present invention can be used in the detection method of the present invention.
  • a substance effective for the treatment of the fusion-positive cancer patient can be screened.
  • the substance obtained by the screening method can be used as an active ingredient of a pharmaceutical composition for treating NCOA4 or RUFY1 fusion-positive cancer, and is also used for treating NCOA4 or RUFY1 fusion-positive cancer. be able to.
  • FIG. 5 is a photomicrograph in place of a drawing showing the state after introducing the fusion gene RUFY1-RET into NIH3T3 fibroblasts and culturing for 7 days.
  • 3 is a graph showing changes over time in tumor size 1 to 8 days after inoculation in nude mice inoculated subcutaneously with 3T3 fibroblasts introduced with the fusion gene RUFY1-RET. It is a graph which shows the sensitivity with respect to the RET inhibitor (Vandetanib) in Ba / F3 cell which expresses RUYY1-RET fusion polypeptide.
  • RET inhibitor Vandetanib
  • FIG. 5 is a photograph, instead of a drawing, showing the results of Western blotting of extracts from each cultured cell after treating Ba / F3 cells expressing a RUFY1-RET fusion polypeptide with each RET inhibitor.
  • the “fusion point in the RET fusion gene” means a portion of the RET fusion gene where a polynucleotide derived from the RET gene and a polynucleotide derived from another gene constructing the fusion gene together with the RET gene are combined.
  • the “fusion point in the NCOA4 or RUFY1 fusion gene” refers to a polynucleotide derived from the NCOA4 or RUFY1 gene in the NCOA4 or RUFY1 fusion gene and a polymorphism derived from another gene that constructs the fusion gene together with the NCOA4 or RUFY1 gene. It means the position where nucleotides are bound.
  • the fusion point is the 3 ′ of the polynucleotide derived from the NCOA4 gene. This is the position (1984/1985) where the terminal base (No. 1984) and the 5 ′ terminal base (No. 1985) of the polynucleotide derived from the RET gene are combined.
  • the fusion point is the 3 ′ of a polynucleotide derived from the RUFY1 gene. This is the position (No. 1917/1918) where the end base (No. 1917) and the 5 ′ end base (No. 1918) of the polynucleotide derived from the RET gene are combined.
  • fusion point in a RET fusion protein refers to a polypeptide encoded by a polynucleotide derived from a RET gene and a polynucleotide derived from another gene that constructs a fusion gene together with the RET gene. It means the place where the encoded polypeptide is bound.
  • the fusion point in the NCOA4 or RUFY1 fusion protein refers to constructing a fusion gene together with the polypeptide encoded by the polynucleotide derived from the NCOA4 or RUFY1 gene in the NCOA4 or RUFY1 fusion protein and the NCOA4 or RUFY1 gene. It means a portion where a polypeptide encoded by a polynucleotide derived from another gene is bound.
  • the fusion point is the C-terminal amino acid (613) of the polypeptide derived from the NCOA4 protein. No.) and the N-terminal amino acid (No. 614) of the polypeptide derived from the RET protein (No. 613/614).
  • the fusion point is the C-terminal amino acid (635th amino acid of the RUFY1 protein-derived polypeptide). No.) and the N-terminal amino acid (No. 636) of the polypeptide derived from the RET protein (No. 636/636).
  • RET gene cleavage or “RET gene cleavage” means a state in which continuity of the RET gene is lost due to translocation or inversion of the gene, ie, RET It refers to a state where the gene is divided into at least two polynucleotides, a polynucleotide containing a RET kinase region and a polynucleotide other than that.
  • the break point of the RET gene is not limited as long as the protein encoded by at least one of the polynucleotides formed by cleaving the RET gene retains the RET kinase activity.
  • cleaving of a gene other than the RET gene” or “a gene other than the RET gene is cleaved” means that the continuity of other genes is lost due to translocation or inversion of the gene. A state in which another gene is divided into at least two polynucleotides.
  • RET protein cleavage or “RET protein is cleaved” means that the RET gene is cleaved as described above based on the RET gene being cleaved as described above. Is a state in which the RET protein is divided into at least two polypeptides: a polypeptide containing the RET kinase region and another polypeptide.
  • the cleavage point of the RET protein is not limited as long as at least one of the polypeptides formed by cleaving the RET protein retains the RET kinase activity.
  • cleaving of a protein other than the RET protein” or “a protein other than the RET protein is cleaved” is based on the fact that the other gene is cleaved as described above. , Refers to a state in which the continuity of other proteins is lost, that is, a state in which other proteins are separated into at least two polypeptides.
  • NCOA4 or RUFY1 gene cleavage or “NCOA4 or RUFY1 gene is cleaved” means that the continuity of the NCOA4 or RUFY1 gene is lost due to translocation or inversion of the gene. Refers to the state of being.
  • the break point of the NCOA4 or RUFY1 gene is the function of the protein encoded by the NCOA4 or RUFY1 gene and another gene that constructs the NCOA4 or RUFY1 gene (for example, if the protein has a kinase domain, the kinase It is not limited as long as the activity is maintained.
  • nucleaved other genes other than NCOA4 or RUFY1 gene or “cleaved other genes other than NCOA4 or RUFY1 gene” means continuity of other genes due to gene translocation or inversion. Is a state in which other genes are divided into at least two polynucleotides.
  • NCOA4 or RUFY1 protein is cleaved or “NCOA4 or RUFY1 protein is cleaved” is based on the fact that the NCOA4 or RUFY1 gene is cleaved as described above. , Refers to a state in which the continuity of the NCOA4 or RUFY1 protein is lost, that is, a state in which the NCOA4 or RUFY1 protein is divided into at least two polypeptides.
  • the breakpoint of the NCOA4 or RUFY1 protein is a range that retains the functions of other proteins that construct the NCOA4 or RUFY1 fusion protein together with the NCOA4 or RUFY1 protein (for example, when the other protein has a kinase domain). It is not limited by.
  • “cleaving of a protein other than the NCOA4 or RUFY1 protein” or “a protein other than the NCOA4 or RUFY1 protein is cleaved” means that the other gene is cleaved as described above. Based on one thing, it refers to a state in which the continuity of another protein is lost, that is, a state in which the other protein is divided into at least two polypeptides.
  • the 5 ′ terminal region is a polynucleotide 5 ′ terminal from the fusion point, and in the case of a wild type gene (non-fusion gene), the wild type gene constructs the fusion gene.
  • the polynucleotide at the 5 ′ end side from the cleavage point is shown.
  • the 5 ′ terminal region may be any region of genomic DNA, mRNA, and cDNA.
  • genomic DNA it is also referred to as a 5 ′ terminal genomic region.
  • the 3 ′ terminal region is a polynucleotide 3 ′ terminal from the fusion point.
  • the wild type gene constructs the fusion gene.
  • the polynucleotide at the 3 ′ end side from the cleavage point is shown.
  • the 3 ′ terminal region may be a region in any of genomic DNA, mRNA, and cDNA.
  • genomic DNA it is also referred to as a 3 ′ terminal genomic region.
  • the N-terminal region is a polypeptide at the N-terminal side from the fusion point, and in the case of a wild type protein (a protein that is not a fusion protein), cleavage when the wild type protein constructs a fusion gene.
  • the polynucleotide on the N-terminal side from the point is shown.
  • the C-terminal region is a polypeptide at the C-terminal side from the fusion point, and in the case of a wild-type protein (a protein that is not a fusion protein), cleavage when the wild-type protein constructs a fusion gene.
  • the polynucleotide on the C-terminal side from the point is shown.
  • the 5 ′ terminal region is the first to 1984th bases, and the 3 ′ terminal region is the 1985th to 5275th bases.
  • the N-terminal region is a polycode encoded by the CDS (bases 146 to 1984 in SEQ ID NO: 1) of the 5′-terminal region of the NCOA4ex9-RETex12.
  • the 5 ′ terminal region is from the first to 1917th regions, and the 3 ′ terminal region is from the 1918-5208 base sequences.
  • the N-terminal region is a polycode encoded by the CDS (the 13th to 1917th bases of SEQ ID NO: 3) of the 5′-terminal region of the RUFY1ex16-RETex12.
  • ENS is ENST00000355710
  • NCOA4 is ENST00000578454
  • RUFY1 is ENST00000319449
  • RET is ENSP00000347942
  • NCOA4 is ENSP00000463027
  • RUFY1 is ENSP00000325594 It was.
  • stringent conditions refers to “5 ⁇ SSPE, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 200 ⁇ g / mL sperm DNA, “42 ° C. overnight” and the conditions for cleaning are “0.5 ⁇ SSC, 0.1% SDS, 42 ° C.”.
  • “More stringent conditions” means “5 ⁇ SSPE, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 200 ⁇ g / mL sperm DNA, over 42 ° C.” “Night”, the conditions for cleaning are “0.2 ⁇ SSC, 0.1% SDS, 65 ° C.”.
  • ⁇ Tumorogenicity> It can be confirmed by a known method, for example, the method of Example 4 of WO2011 / 162295 or the method of Example 6 described later, that a certain polypeptide has “tumor forming ability”. Specifically, a host (3T3 fibroblast) into which a plasmid expressing the polypeptide has been introduced is inoculated subcutaneously into nude mice, and confirmed by a method of judging the presence or absence of tumor formation. The NCOA4-RET fusion showed transformation in the introduced cells and tumor-forming ability in the introduced cell transplanted mice, suggesting that the presence of this fusion gene or its transcript is the cause of cancer at the site of expression. (See Non-Patent Document 2).
  • the detection method according to the present invention can be suitably used for detection of cancer occurring in a target organ.
  • the test site (target organ) of the subject is not limited as long as the fusion according to the present invention exists, but the digestive tract is preferable, the digestive tract is more preferable, the gastrointestinal tract is more preferable, and the lower digestive tract is more preferable. Particularly preferred is the large intestine.
  • the tissue type of the test site is not limited as long as the detection method according to the present invention can be applied, and it may be a squamous tissue or a glandular tissue, but a squamous tissue is preferable.
  • the sample obtained from the subject includes a sample collected from the subject (sample separated from the living body), specifically, any collected body fluid (preferably blood), from the subject affected area. Extracted specimens, biopsy specimens or scraped specimens, feces, urine, gastrointestinal lavage fluid and the like can be used.
  • the digestive tract washing solution may be a washing solution for the entire digestive tract, or a washing solution for the digestive tract including at least the test site, for example, a washing solution for the lower digestive tract or a washing solution for the large intestine.
  • a sample containing cells at the test site in the target organ is preferable, and an excised specimen or biopsy sample from the test site of the test subject is more preferable.
  • the method for detecting a RET fusion gene or RET fusion protein comprises preparing a tissue section or a cell suspension of a sample obtained from a subject and applying it to a cell contained in the tissue section or cell suspension.
  • it can be carried out by detecting a RET fusion gene or a RET fusion protein by techniques well known to those skilled in the art.
  • a lysate is prepared from a sample obtained from the above-mentioned subject, and a gene or protein contained therein is extracted.
  • a RET fusion gene or A RET fusion protein may be detected.
  • the detection of the RET fusion gene may be any of detection of genomic DNA of the RET fusion gene, mRNA that is a transcription product of the genomic DNA, or detection of cDNA obtained using mRNA as a template.
  • the method for detecting an NCOA4 or RUFY1 fusion gene or an NCOA4 or RUFY1 fusion protein comprises preparing a tissue section or cell suspension of a sample obtained from a subject, It can be carried out by detecting the NCOA4 or RUFY1 fusion gene or the NCOA4 or RUFY1 fusion protein on the contained cells by techniques well known to those skilled in the art. Alternatively, a lysate is prepared from a sample obtained from the aforementioned subject, and a gene or protein contained therein is extracted. In this extracted sample, an NCOA4 or RUFY1 fusion gene, Alternatively, NCOA4 or RUfy1 fusion protein may be detected.
  • the detection of the NCOA4 or RUFY1 fusion gene may be any of detection of genomic DNA of the NCOA4 or RUFY1 fusion gene, mRNA that is a transcription product of the genomic DNA, or detection of cDNA obtained using the mRNA as a template. Good.
  • the detection method of the present invention includes a method for detecting a RET fusion in a sample obtained from a subject, that is, a method for detecting a fusion protein containing a RET kinase region (also referred to as “RET fusion protein”), or the fusion protein. And a method for detecting a fusion gene encoding CHO (also referred to as “RET fusion gene”).
  • the detection method of the present invention includes a method for detecting an NCOA4 or RUFY1 fusion in a sample obtained from a subject, that is, a method for detecting an NCOA4 or RUFY1 fusion protein, or a fusion gene encoding the fusion protein (“NCOA4 or And a detection method of “RUFY1 fusion gene”.
  • the RET fusion according to the present invention comprises a RET fusion protein and a RET fusion gene.
  • the RET fusion protein according to the present invention is a fusion polypeptide constructed from a polypeptide derived from a RET protein and a polypeptide derived from another protein other than the RET protein, wherein the polypeptide derived from the RET protein is:
  • a polypeptide derived from a protein other than the RET protein including at least the polypeptide of the RET kinase region in the RET protein is not particularly limited as long as it includes at least a part of the polypeptide in the other protein.
  • the other protein is not particularly limited as long as the RET fusion protein constructed by fusing with a part of the RET protein including the RET kinase domain has a tumor forming ability.
  • the RET fusion protein it is preferable that the RET fusion protein has a tumorigenicity by constantly maintaining RET kinase activation.
  • the RET fusion protein is derived from a polypeptide derived from the RET protein and other proteins other than the RET protein as long as the RET kinase activation is constantly maintained and the constructed RET fusion protein has tumorigenicity.
  • a third polypeptide that is not any of the polypeptides may be included.
  • the third polypeptide may be located at the N-terminus or C-terminus of the RET fusion protein, or may be a polypeptide derived from a RET protein and a polypeptide derived from another protein other than the RET protein. It may be located between.
  • a fusion protein in which the other protein is NCOA4 or RUFY1 protein is particularly preferable. That is, an NCOA4 constructed from an RET protein-derived polypeptide containing at least a polypeptide in the RET kinase region and an NCOA4 or RUFY1 protein-derived polypeptide containing at least a partial polypeptide of the NCOA4 or RUFY1 protein. Or a fusion protein of RUFY1 protein and RET protein (hereinafter also referred to as NCOA4-RET fusion protein or RUFY1-RET fusion protein, NCOA4-RET or RUYY1-RET fusion protein, or NCOA4 or RUFY1-RET fusion protein) Preferably there is.
  • NCOA4 or RUFY1 fusion NCOA4 or RUFY1 fusion protein and NCOA4 or RUFY1 fusion gene>
  • the NCOA4 or RUFY1 fusion according to the present invention comprises an NCOA4 or RUFY1 fusion protein and an NCOA4 or RUFY1 fusion gene.
  • NCOA4 or RUFY1 fusion protein is a fusion polypeptide constructed from a polypeptide derived from NCOA4 or RUFY1 protein and a polypeptide derived from another protein other than NCOA4 or RUFY1 protein, wherein said NCOA4 or The polypeptide derived from RUFY1 protein includes at least a part of the polypeptide in NCOA4 or RUFY1 protein, and the polypeptide derived from other proteins other than NCOA4 or RUFY1 protein includes at least a part of the polypeptide in other proteins. There is no particular restriction.
  • the other protein is not particularly limited as long as the constructed NCOA4 or RUFY1 fusion protein has a tumor-forming ability by being fused with a part of the NCOA4 or RUFY1 protein. It is preferable that the NCOA4 or RUFY1 fusion protein has tumorigenicity by constantly maintaining the activation of the functional domain (preferably the kinase domain) of the other protein.
  • the NCOA4 or RUFY1 fusion protein is fused with a part of the NCOA4 or RUFY1 protein to constantly maintain the activation of the functional domain of other proteins other than the NCOA4 or RUFY1 protein.
  • the fusion protein may contain a third polypeptide that is neither an NCOA4 or RUFY1 protein-derived polypeptide nor a polypeptide derived from another protein other than the NCOA4 or RUFY1 protein. Good.
  • the third polypeptide may be located at the N-terminus of the NCOA4 or RUFY1 fusion protein, at the C-terminus, or a polypeptide derived from the NCOA4 or RUFY1 protein and other than the NCOA4 or RUFY1 protein. It may be located between the protein-derived polypeptide.
  • a fusion protein in which the other protein is a RET protein is particularly preferable. That is, it is constructed from a polypeptide derived from the NCOA4 or RUFY1 protein containing at least a part of the polypeptide of the NCOA4 or RUFUY1 protein, and at least a part of the polypeptide of the RET protein including a polypeptide of the RET kinase region.
  • a fusion protein of the NCOA4 or RUFY1 protein and the RET protein (hereinafter referred to as the NCOA4-RET fusion protein or the RUFY1-RET fusion protein, or the NCOA4-RET or RUFY1-RET fusion protein, or the NCOA4 or RUFY1-RET fusion protein) It is preferable that
  • the polypeptides described in the following (a) to (d) are particularly preferred: (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (NCOA4-RET) or SEQ ID NO: 4 (RUFY1-RET), (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity, (C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity (hereinafter referred to as a homologous polypeptide), and (D) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenic potential ( Hereinafter referred to as a functional equivalent variant).
  • the amino acid sequence represented by SEQ ID NO: 2 is a sequence encoded by the base sequence represented by SEQ ID NO: 1, particularly the base sequence (CDS) represented by base numbers 146 to 3193 of SEQ ID NO: 1.
  • the nucleotide sequence represented by SEQ ID NO: 1 includes the 5′-UTR sequence of the NCOA4 gene, the start codon ATG to exon 9 of the NCOA4 gene, the exon 12 of the RET gene to the stop codon of exon 20, and the 3′- of the RET gene. It consists of the base sequence of the UTR sequence.
  • nucleotide sequence represented by SEQ ID NO: 1 the nucleotide sequence of nucleotide numbers 1 to 1984 is derived from the NCOA4 gene, and the nucleotide sequence of nucleotide numbers 1985 to 5275 is derived from the RET gene.
  • a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 and a polynucleotide comprising the base sequence encoding the same This is referred to as an NCOA4ex9-RETex12 fusion (or sometimes referred to as NCOA4ex9-RETex12).
  • the amino acid sequence represented by SEQ ID NO: 4 is a sequence encoded by the base sequence represented by SEQ ID NO: 3, particularly the base sequence (CDS) represented by base numbers 13 to 3126 of SEQ ID NO: 3.
  • the nucleotide sequence represented by SEQ ID NO: 3 includes the 5′-UTR sequence of the RUFY1 gene, the start codon ATG to exon 16 of the RUFY1 gene, the exon 12 of the RET gene to the stop codon of exon 20, and the 3 ′ of the RET gene.
  • -It consists of the base sequence of the UTR sequence.
  • the sequences of base numbers 13 to 1917 are derived from the RUFY1 gene, and the sequences of base numbers 1918 to 3126 are derived from the RET gene.
  • a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 and a polynucleotide comprising the base sequence encoding the same is referred to as a RUFY1ex16-RETex12 fusion (or sometimes RUYY1ex16-RETex12).
  • the number of amino acids that can be substituted, deleted, and / or inserted is 1 to several amino acids, preferably 1 to 10, more preferably 1 to 7, and most preferably 1. ⁇ 5.
  • “Homologous polypeptide” is a “polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity”
  • a polypeptide comprising an amino acid sequence having an identity of preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more is preferable.
  • polypeptide having an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity shows the identity
  • a polypeptide having at least one substitution, deletion, and / or insertion (preferably a substitution) (a homologous polypeptide in a narrow sense) and a polypeptide having 100% identity are included.
  • the “identity” means a value Identity obtained by using a parameter prepared as a default by a NEEDLE program (J Mol Biol 1970; 48: 443-453) search.
  • the RET fusion gene according to the present invention is a polynucleotide encoding a RET fusion protein.
  • the RET fusion protein and the RET fusion gene may be collectively referred to as “RET fusion”.
  • the NCOA4 or RUFY1 fusion gene according to the present invention is a polynucleotide encoding the NCOA4 or RUFY1 fusion protein. That is, the NCOA4 fusion gene is a polynucleotide encoding the NCOA4 fusion protein, and the RUFY1 fusion gene is a polynucleotide encoding the RUFY1 fusion protein.
  • the NCOA4 or RUFY1 fusion protein and the NCOA4 or RUFY1 fusion gene may be collectively referred to as “NCOA4 or RUFY1 fusion”.
  • the NCOA4ex9-RETex12 fusion variant or the RUFY1ex16-RETex12 fusion variant is preferable.
  • the NCOA4ex9-RETex12 fusion protein variant or the RUFY1ex16-RETex12 fusion protein variant is preferable.
  • an NCOA4ex9-RETex12 fusion gene variant or a RUFY1ex16-RETex12 fusion gene variant is preferable.
  • the NCOA4ex9-RETex12 fusion variant or the RUFY1ex16-RETex12 fusion variant is preferable.
  • the NCOA4 or RUFY1 fusion protein according to the present invention the NCOA4ex9-RETex12 fusion protein variant or the RUFY1ex16-RETex12 fusion protein variant is preferable.
  • an NCOA4ex9-RETex12 fusion gene variant or a RUFY1ex16-RETex12 fusion gene variant is preferable.
  • the detection method of the present invention includes a detection method comprising a step of detecting cleavage of a RET protein or cleavage of a RET gene encoding a RET protein in a sample obtained from a subject, and in a sample obtained from the subject. And a detection method comprising a step of detecting the presence of a fusion protein constructed from the RET protein and another protein other than the RET protein, or the presence of a fusion gene encoding the fusion protein.
  • the detection method of the present invention includes a detection method including a step of detecting cleavage of NCOA4 or RUFY1 protein, or cleavage of NCOA4 or RUFY1 gene encoding NCOA4 or RUFY1 protein in a sample obtained from the subject, and the subject Detecting the presence of a fusion protein constructed from the NCOA4 or RUFY1 protein and another protein other than the NCOA4 or RUFY1 protein or the presence of a fusion gene encoding the fusion protein in the sample obtained from Detection methods are included.
  • the RET fusion gene can be detected.
  • the RET fusion gene is detected by specifically detecting the expression levels of the 5 ′ terminal region and the 3 ′ terminal region of the RET gene and determining the ratio of the expression levels. Can do. Specifically, for example, when the expression level of the 5 ′ terminal region of the RET gene is different from the expression level of the RET gene 3 ′ terminal region, the RET fusion gene can be detected. Alternatively, the RET fusion gene may be detected by confirming the gene other than the RET gene constructing the RET fusion gene together with the RET gene by the above method.
  • RET fusion gene when duplication of at least a part of the RET gene or another gene other than the RET gene is involved, that is, a duplicate polynucleotide derived from the RET gene, and RET
  • a RET fusion gene is constructed from an overlapping polynucleotide derived from another gene other than the RET gene that constructs the RET fusion gene, the polynucleotide of the RET gene or the polynucleotide derived from the other gene
  • the RET fusion gene can be detected.
  • the RET fusion gene is constructed by fusing a RET gene-derived polynucleotide with a polynucleotide derived from another gene other than the RET gene.
  • the RET fusion gene can be detected by detecting a fusion polynucleotide in which at least a part of the polynucleotide derived from the RET gene and at least a part of the polynucleotide derived from a gene other than the RET gene are continuously contained. .
  • a first probe that specifically hybridizes to a 5 ′ terminal region of a polynucleotide derived from a gene other than the RET gene, and a 3 ′ terminal region of a polynucleotide derived from the RET gene By using a second probe that specifically hybridizes and detecting that the two gene regions are close together on the chromosome, the RET fusion gene can be detected.
  • the first probe is located on the 5 ′ end side of the polynucleotide derived from the NCOA4 or RUFY1 gene.
  • a probe that specifically hybridizes to the region may be used.
  • the RET fusion gene is constructed by fusing a RET gene-derived polynucleotide and a polynucleotide derived from another gene other than the RET gene at the fusion point.
  • a fusion polynucleotide in which at least a part of the polynucleotide derived from the RET gene and at least a part of the polynucleotide derived from another gene other than the RET gene are continuously contained including the fusion point is detected.
  • the RET fusion gene can be detected.
  • a first primer that specifically anneals to a 5′-terminal region of a polynucleotide derived from a gene other than the RET gene and a specific primer that is specific to the 3′-terminal region of a polynucleotide derived from the RET gene
  • the RET fusion gene can be detected by conducting a PCR reaction using a second primer that anneals automatically and confirming that a predetermined PCR product indicating the presence of a fusion point is obtained.
  • RET fusion protein (1-a) As an embodiment for detecting the RET fusion protein, when the RET fusion gene is constructed, the RET protein encoded by the RET gene is also cleaved. By detecting that the continuity between the N-terminal region and the C-terminal region is lost, the RET fusion protein can be detected. Specifically, for example, using the first antibody that specifically binds to the N-terminal region of the RET protein and the second antibody that specifically binds to the C-terminal region of the RET protein, the two regions Can be detected by confirming that they are not present in the same protein. Alternatively, the RET fusion protein may be detected by confirming, by the above-described method, the state in which a protein other than the RET protein that constitutes the fusion protein together with the RET protein is cleaved.
  • the RET fusion protein can be detected by specifically detecting the expression levels of the N-terminal region and the C-terminal region of the RET protein and determining the ratio of the expression levels. .
  • the RET fusion protein can be detected using as an index the difference between the expression level of the N-terminal region of the RET protein and the expression level of the RET protein C-terminal region.
  • the RET fusion protein may be detected by confirming the protein other than the RET protein constructing the RET fusion protein together with the RET protein by the above method.
  • the RET fusion protein is constructed by fusing a RET protein-derived polypeptide and a polypeptide derived from another protein other than the RET protein.
  • the RET fusion protein can be detected by detecting a fusion polypeptide in which at least a part of the polypeptide derived from the RET protein and at least a part of the polypeptide derived from the other protein are continuously contained.
  • a first antibody that specifically binds to the N-terminal region of a protein other than the RET protein and a second antibody that specifically binds to the C-terminal region of the RET protein are used.
  • the RET fusion protein can be detected by confirming that the two regions are present in the same protein.
  • the RET fusion protein is constructed by fusing a RET protein-derived polypeptide and a polypeptide derived from another protein other than the RET protein at a fusion point.
  • a fusion polypeptide in which at least a part of the polypeptide derived from the RET protein containing the fusion point and at least a part of the polypeptide derived from the other protein are continuously contained in the fusion protein, Protein can be detected.
  • the RET fusion protein can be detected by an immunological assay using an antibody that specifically recognizes a polypeptide containing the fusion point of the RET fusion protein.
  • the RET fusion protein can be detected using the activity of the RET fusion protein as an index. Specifically, for example, after inhibiting the activity of the wild type RET protein using a substance having an inhibitory activity against the wild type RET protein, the kinase activity of the RET protein is measured, and the RET fusion protein is not included ( The RET fusion protein can be detected with an index of high activity as compared to the case of including only the wild-type RET protein. For the measurement of the kinase activity of the RET protein, a method well known to those skilled in the art can be appropriately selected. For example, the phosphorylation state of a molecule that is phosphorylated by RET may be detected.
  • the detection of the RET fusion protein may be performed by using the presence of the full-length polypeptide constituting the RET fusion protein or the presence of the polypeptide constituting a part of the RET fusion protein as an index. It is not limited as far as it can be confirmed.
  • a first probe that specifically hybridizes to the 5 ′ end region of the NCOA4 or RUFY1 gene and a second probe that specifically hybridizes to the 3 ′ end region of the NCOA4 or RUFY1 gene By detecting that the two gene regions are separated on the chromosome using a probe, the NCOA4 or RUFY1 fusion gene can be detected.
  • the NCOA4 or RUFY1 fusion gene may be detected by confirming the state in which the other gene constructing the fusion gene fused with the polynucleotide derived from the NCOA4 or RUFY1 gene is cleaved by the above method. .
  • the NCOA4 or RUFY1 fusion gene is detected by specifically detecting the expression levels of the 5 ′ terminal region and the 3 ′ terminal region of the NCOA4 or RUFY1 gene, respectively, and determining the ratio of the expression levels. Can be detected. Specifically, for example, when the expression level of the 5 ′ end region of the NCOA4 or RUFY1 gene is different from the expression level of the NCOA4 or RUFY1 gene 3 ′ end region, the NCOA4 or RUFY1 fusion gene can be detected. .
  • the NCOA4 or RUFY1 fusion gene may be detected by confirming the NCOA4 or RUFY1 fusion gene together with the NCOA4 or RUFY1 gene by using the above method for other genes other than the NCOA4 or RUFY1 gene.
  • the duplication of at least a part of the NCOA4 or RUFY1 gene or another gene other than the NCOA4 or RUFY1 gene is involved, ie, NCOA4 or RUFY1.
  • An NCOA4 or RUFY1 fusion gene is constructed from a duplicated polynucleotide derived from a gene and a duplicated polynucleotide derived from an NCOA4 or RUFY1 gene other than the NCOA4 or RUFY1 gene together with NCOA4 or RUFY1.
  • detecting an NCOA4 or RUFY1 fusion gene by detecting duplication of a polynucleotide derived from the NCOA4 or RUFY1 gene or the polynucleotide derived from the other gene Kill.
  • the NCOA4 or RUFY1 fusion gene is constructed by fusing a polynucleotide derived from the NCOA4 or RUFY1 gene with a polynucleotide derived from another gene other than the NCOA4 or RUFY1 gene.
  • a fusion polynucleotide comprising at least a part of a polynucleotide derived from the NCOA4 or RUFY1 gene and at least a part of a polynucleotide derived from a gene other than the NCOA4 or RUFY1 gene in the NCOA4 or RUFY1 fusion gene, By detecting, the NCOA4 or RUFY1 fusion gene can be detected.
  • a first probe that specifically hybridizes to the 5′-terminal region of a polynucleotide derived from the NCOA4 or RUFY1 gene, and 3 ′ of a polynucleotide derived from another gene other than the NCOA4 or RUFY1 gene By using a second probe that specifically hybridizes to the terminal region and detecting that the two gene regions are close together on the chromosome, the NCOA4 or RUFY1 fusion gene can be detected.
  • the second probe is located on the 3 ′ end side of the polynucleotide derived from the RET gene.
  • a probe that specifically hybridizes to the region may be used.
  • the NCOA4 or RUFY1 fusion gene is constructed by fusing the NCOA4 or RUFY1 gene-derived polynucleotide and a polynucleotide derived from another gene other than the NCOA4 or RUFY1 gene at the fusion point.
  • the NCOA4 or RUFY1 fusion gene at least a part of the polynucleotide derived from the NCOA4 or RUFY1 gene and at least a part of the polynucleotide derived from another gene other than the NCOA4 or RUFY1 gene It is possible to detect the NCOA4 or RUFY1 fusion gene by detecting a fusion polynucleotide continuously contained.
  • a first primer that specifically anneals to the 5 ′ end region of a polynucleotide derived from the NCOA4 or RUFY1 gene, and the 3 ′ end of a polynucleotide derived from another gene other than the NCOA4 or RUFY1 gene Detect an NCOA4 or RUFY1 fusion gene by performing a PCR reaction with a second primer that specifically anneals to the side region, and confirming that a predetermined PCR product indicating the presence of a fusion point is obtained. Can do.
  • a first antibody that specifically binds to the N-terminal region of NCOA4 or RUFY1 protein and a second antibody that specifically binds to the C-terminal region of NCOA4 or RUFY1 protein By confirming that the two regions are not present in the same protein, the NCOA4 or RUFY1 fusion protein can be detected.
  • the NCOA4 or RUFY1 fusion protein may be detected by confirming, by the above-described method, the state in which other proteins other than the NCOA4 or RUFY1 protein that constitutes the fusion protein together with the NCOA4 or RUFY1 protein are cleaved.
  • the NCOA4 or RUFY1 fusion protein is detected by specifically detecting the expression levels of the N-terminal region and the C-terminal region of the NCOA4 or RUFY1 protein, respectively, and determining the ratio of the expression levels. can do.
  • the detection of NCOA4 or RUFY1 fusion protein can be performed using as an indicator that the expression level of the N-terminal region of NCOA4 or RUFY1 protein is different from the expression level of the NCOA4 or RUFY1 protein C-terminal region. it can.
  • the NCOA4 or RUFY1 fusion protein may be detected by confirming the NCOA4 or RUFY1 fusion protein together with the NCOA4 or RUFY1 protein by the above-described method for other proteins other than the NCOA4 or RUFY1 protein.
  • the NCOA4 or RUFY1 fusion protein is constructed by fusing an NCOA4 or RUFY1 protein-derived polypeptide with a polypeptide derived from another protein other than the NCOA4 or RUFY1 protein.
  • detecting a fusion polypeptide in which at least a part of the polypeptide derived from the NCOA4 or RUFY1 protein and at least a part of the polypeptide derived from the other protein are continuously contained in the NCOA4 or RUFY1 fusion protein Can detect NCOA4 or RUfy1 fusion protein.
  • a first antibody that specifically binds to the N-terminal region of NCOA4 or RUFY1 protein and a second antibody that specifically binds to the C-terminal region of other proteins other than NCOA4 or RUFY1 protein By confirming that the two regions are present in the same protein, it is possible to detect the NCOA4 or RUFY1 fusion protein.
  • the NCOA4 or RUFY1 fusion protein is constructed by fusing the NCOA4 or RUFY1 protein-derived polypeptide with a polypeptide derived from another protein other than the NCOA4 or RUFY1 protein at the fusion point.
  • the NCOA4 or RUFY1 fusion protein at least a part of the polypeptide derived from the NCOA4 or RUFY1 protein containing the fusion point and at least a part of the polypeptide derived from the other protein are continuously included.
  • the NCOA4 or RUfy1 fusion protein can be detected.
  • the NCOA4 or RUFY1 fusion protein can be detected by an immunoassay using an antibody that specifically recognizes a polypeptide containing the fusion point of the NCOA4 or RUFY1 fusion protein.
  • the NCOA4 or RUFY1 fusion protein can be detected using the activity of the NCOA4 or RUFY1 fusion protein as an index.
  • the other protein other than the NCOA4 or RUFY1 protein that constructs the fusion protein together with the NCOA4 or RUFY1 protein is a protein having an enzyme activity
  • the NCOA4 or RUFY1 fusion protein is not included (wild-type NCOA4 or RUFY1 NCOA4 or RUFY1 fusion protein can be detected using the high enzyme activity as an index compared to the case of containing only protein).
  • a method well known to those skilled in the art can be appropriately selected.
  • the other protein is a protein having kinase activity (preferably a RET protein)
  • the detection of the NCOA4 or RUFY1 fusion protein may be performed using the presence of the full-length polypeptide constituting the NCOA4 or RUFY1 fusion protein or the presence of the polypeptide constituting a part of the NCOA4 or RUFY1 fusion protein as an index, It is not limited as long as the presence of the NCOA4 or RUFY1 fusion protein can be confirmed.
  • each step of detection of RET fusion gene (genomic DNA, mRNA, or cDNA), detection of NCOA4 or RUFY1 fusion gene (genomic DNA, mRNA, or cDNA), detection of RET fusion protein, detection of NCOA4 or RUFY1 fusion protein
  • RET is prepared in the prepared sample.
  • a suitable technique for detecting the fusion gene or NCOA4 or RUFY1 fusion gene, or the RET fusion protein or NCOA4 or RUFY1 fusion protein can be appropriately selected by those skilled in the art.
  • the detection of the RET fusion gene or NCOA4 or RUFY1 fusion gene is performed by detecting the genomic DNA of the RET fusion gene or NCOA4 or RUFY1 fusion gene, detecting the mRNA that is the transcription product of the genomic DNA, or using the mRNA as a template. Any of detection of cDNA obtained may be sufficient.
  • RET fusion gene genomic DNA or mRNA
  • NCOA4 or RUFY1 fusion gene genomic DNA or mRNA
  • a RET fusion gene or NCOA4 or RUFY1 fusion gene Detection of genes such as hybridization techniques using probes that hybridize to at least part (nucleic acid probes, etc.), or gene amplification techniques that use primers that anneal to at least part of RET fusion genes or NCOA4 or RUFY1 fusion genes Any technique well known to those skilled in the art and applied to these techniques can be used.
  • PCR Linear PCR
  • SDA Strand displacement amplification
  • NASBA Nucleic acid sequence-based amplification
  • ICAN Isothermal and chimeric primer-initiated amplification of nucleic acids
  • LAMP Loop-mediated isothermal amplification
  • Any technique may be used, such as a method, a TMA method (Gen-Probe's TMA system), an in situ hybridization method, a microarray method, a Northern hybridization, a Southern hybridization, a dot blot method, an RNA protection method, a DNA sequence, or an RNA sequence .
  • an in situ hybridization technique For detection of genomic DNA, an in situ hybridization technique can be suitably used. Detection using an in situ hybridization technique can be performed, for example, according to a known FISH method. Or it can implement by the fusion assay (fusion assay) which combined the chromogenic in situ hybridization (CISH) method and the silver in situ hybridization (SISH) method. Preferably, it can be detected by the FISH method split assay or FISH method fusion assay described in detail below.
  • fusion assay fusion assay
  • CISH chromogenic in situ hybridization
  • SISH silver in situ hybridization
  • DNA sequencing technology can be suitably used for detecting genomic DNA.
  • a sequencer based on the conventional Sanger method may be used, but in consideration of analysis efficiency, it is preferable to use a next-generation sequencer (for example, Metzker ML, Nat Rev Genet. 2010 Jan; 11 (See (1): 31-46).
  • Illustrative examples of the next-generation sequencer include MiSeq / HiSeq from Illumina, SOLiD system from Life Technologies, 454 sequence system (GS FLX + / GS Junior) from Roche.
  • the efficiency of analysis can be improved by enriching a region where a fusion gene may exist using a sequence capture technique or the like.
  • the sequence capture technology include Roche NimbleGen from Roche, Sure Select from Agilent Technologies, and the like.
  • representative methods for detecting genomic DNA are exemplified, but the present invention is not limited thereto.
  • ⁇ FISH method split assay> In the FISH split assay of a RET fusion gene, as a detection probe, a polynucleotide covering the 5 ′ end genomic region of the RET gene and fluorescently labeled, and the 3 ′ end genomic region of the same gene are covered. A combination with a polynucleotide labeled with another fluorescent dye is used.
  • the two gene regions are close to each other, so the two signals overlap ( For example, when red fluorescent dye and green fluorescent dye are used, it is detected as yellow), whereas when two gene regions are cleaved by translocation or inversion, two types of fluorescent dyes are detected. Signals derived from (eg red and green) are detected separately. Therefore, in the FISH split assay, the presence of the RET fusion gene is detected by detecting that the 5′-end genomic region of the RET gene and the genomic region of the RET gene 3′-end are separated on the chromosome. Yes.
  • a polynucleotide that covers the 5'-terminal genomic region of the NCOA4 or RUFY1 gene and is fluorescently labeled, and the 3 'end of the same gene A combination with a polynucleotide covering the side genomic region and labeled with another fluorescent dye is used.
  • the two gene regions (5 ′ end region and 3 ′ end region for each gene) are close to each other, so the two signals overlapped.
  • the two gene regions are cleaved by translocation or inversion while being detected as a color (for example, yellow when red and green fluorescent dyes are used)
  • Signals derived from fluorescent dyes eg, red and green
  • the FISH split assay by detecting that the 5′-end genomic region of the NCOA4 or RUFY1 gene and the 3′-end genomic region of the same gene are separated on the chromosome, the NCOA4 or RUFY1 fusion gene The presence is detected.
  • a polynucleotide that covers the 5'-terminal genomic region of the NCOA4 or RUFY1 gene and is fluorescently labeled A combination of a polynucleotide covering the 3'-end genomic region of the same gene labeled with another fluorescent dye, or a polynucleotide covering the 5'-end genomic region of the RET gene and fluorescently labeled can be detected by using a combination of the above and a polynucleotide covering the 3′-terminal genomic region of the same gene and labeled with another fluorescent dye, to detect the NCOA4 or RUFY1-RET fusion gene. it can.
  • RET fusion gene for example, when the RET fusion gene is an NCOA4 or RUFY1-RET fusion gene, it is a polynucleotide that covers the 3 'terminal genomic region of the RET gene and is fluorescently labeled as a detection probe And a combination of a polynucleotide covering the 5 'terminal genomic region of the NCOA4 or RUFY1 gene and labeled with another fluorescent dye can be used.
  • a polyprobe that covers the 3 ′ terminal genomic region of the RET gene is used as a detection probe.
  • a combination of a nucleotide that is fluorescently labeled and a polynucleotide that covers the 5'-terminal genomic region of the NCOA4 or RUFY1 gene and that is labeled with another fluorescent dye can be used.
  • the gene duplication associated with the construction of the RET fusion gene is a polynucleotide that covers at least a part of the genomic region at the 3 ′ end side of the RET gene as a detection probe.
  • fluorescently labeled ones can be used.
  • a RET fusion gene is detectable by detecting that a strong signal, for example, 2 times or more of a signal is obtained compared with the case where only a wild-type RET gene is included.
  • the 5 ′ genomic region of another gene that is fused with a polynucleotide derived from the RET gene to construct a fusion gene (for example, when the RET fusion gene is an NCOA4 or RUFY1-RET fusion gene, the NCOA4 or RUFY1 gene)
  • the detection probe may be used to detect the RET fusion gene by the above method.
  • the NCOA4 or RUFY1 fusion gene is an NCOA4 or RUFY1-RET fusion gene
  • at least one of the 5'-terminal genomic regions of the NCOA4 or RUFY1 gene is used as a detection probe. It is possible to use a fluorescently labeled polynucleotide that covers a part. Then, it is possible to detect the NCOA4 or RUFY1 fusion gene by detecting that a strong signal, for example, twice or more of the signal obtained compared to the case of containing only the wild-type NCOA4 or RUFY1 gene is obtained.
  • NCOA4 or RUFY1 fusion gene may be detected by the aforementioned method using a genomic region detection probe.
  • CGH array analysis Gene duplication associated with RET fusion gene construction or NCOA4 or RUFY1 fusion gene construction can be detected by comparative genomic hybridization (CGH) array analysis (eg, Agilent CGH / CNV Array analysis; Agilent Technologies).
  • CGH comparative genomic hybridization
  • Gene duplication associated with RET fusion gene construction or NCOA4 or RUFY1 fusion gene construction can be detected by a next-generation sequencer. Specifically, in analysis using a next-generation sequencer, it is detected that the coverage of the gene duplication portion is high (the redundancy of the relevant portion is high when the sequence of the DNA fragment to be analyzed is annotated to the reference sequence) Thus, the RET fusion gene or NCOA4 or RUFI1 fusion gene can be detected.
  • the probe used for hybridization for detecting the RET fusion gene may be a hybrid under stringent conditions (preferably under more stringent conditions) to at least some nucleotides of the RET fusion gene or their complementary strands. Probes that soy are preferred.
  • nucleic acid molecules of at least 32 bases consisting of 16 bases each upstream and downstream of the fusion point of the RET fusion gene, or their A probe containing a complementary strand may be used.
  • a probe used for hybridization for detecting the NCOA4 or RUFY1 fusion gene at least a part of the nucleotides of the NCOA4 or RUFY1 fusion gene or a complementary strand thereof is used under stringent conditions (preferably more stringent conditions). Probes that hybridize (under) are preferred.
  • a nucleic acid molecule of at least 32 bases consisting of 16 bases upstream and downstream of the fusion point of the NCOA4 or RUFY1 fusion gene.
  • a probe containing a complementary strand thereof may be used.
  • a probe that can be used in the FISH fusion assay is one of the RET gene, NCOA4 or RUFY1 gene.
  • a combination of a first probe capable of specifically recognizing the 5′-terminal genomic region and a second probe capable of specifically recognizing the 3′-terminal genomic region of the remaining one gene preferably 3 of the RET gene 'A combination of a first probe capable of specifically recognizing the terminal genomic region and a second probe capable of specifically recognizing the 5' terminal genomic region of NCOA4 or RUFI1 gene
  • a first probe capable of specifically recognizing the 5′-terminal genomic region and a second probe capable of specifically recognizing the 3′-terminal genomic region of the remaining one gene preferably 3 of the RET gene 'A combination of a first probe capable of specifically recognizing the terminal genomic region and a second probe capable of specifically recognizing the 5' terminal genomic region of NCOA4 or RUFI1 gene
  • a probe that can be used in the FISH method split assay is specifically the RET gene 5 ′ terminal genomic region.
  • a combination of a first probe that can be recognized and a second probe that can specifically recognize the 3′-end genomic region of the RET gene or a first probe that can specifically recognize the 5′-end genomic region of the NCOA4 or RUFY1 gene 1 probe and a second probe capable of specifically recognizing the 3'-terminal genomic region of NCOA4 or RUFY1 gene (preferably, a first probe capable of specifically recognizing the 5'-terminal genomic region of the RET gene Specific recognition of the probe and the 3 'end genomic region of the RET gene Can be used that combination of the second probe).
  • Detection of mRNA can be performed by analyzing mRNA itself by Northern hybridization or the like, or by analyzing complementary DNA (cDNA) synthesized using mRNA as a template by methods well known to those skilled in the art. May be.
  • a sequencing technique can be suitably used for detection of the RNA.
  • a next-generation sequencer it is preferable to use a next-generation sequencer in consideration of analysis efficiency (see, for example, Metzker ML, Nat Rev Genet. 2010 Jan; 11 (1): 31-46).
  • Illustrative examples of the next-generation sequencer include MiSeq / HiSeq from Illumina, SOLiD system from Life Technologies, 454 sequence system (GS FLX + / GS Junior) from Roche.
  • sequence capture technology examples include Roche NimbleGen from Roche, Sure Select from Agilent Technologies, and the like.
  • mRNA can be detected by a gene amplification reaction method using a primer designed to specifically amplify at least a part of the polynucleotide of the RET fusion gene or NCOA4 or RUFY1 fusion gene to be detected.
  • a primer designed to specifically amplify at least a part of the polynucleotide of the RET fusion gene or NCOA4 or RUFY1 fusion gene to be detected.
  • an amplified fragment of a desired size is further obtained. It is preferable to include a step of detecting whether or not it has been obtained.
  • the PCR method is suitable for quantitatively detecting the RET fusion gene or NCOA4 or RUFI1 fusion gene. Therefore, as described in the above ⁇ Aspect for detecting RET fusion gene (1-b)>, the expression levels of the 5 ′ terminal region and 3 ′ terminal region of the RET gene are specifically detected, A RET fusion gene can be detected by determining the ratio of expression levels. Alternatively, the expression levels of the 5′-terminal region and 3′-terminal region of other genes other than the RET gene constructing the RET fusion gene together with the RET gene are specifically detected, and the ratio of the expression levels is obtained. Thus, the RET fusion gene can be detected.
  • the expression levels of the 5 ′ terminal region and 3 ′ terminal region of NCOA4 or RUFY1 gene are specifically determined. It can be suitably used in a method for detecting an NCOA4 or RUFY1 fusion gene by detecting and determining the ratio of the expression levels. Alternatively, the expression levels of the 5 ′ terminal region and the 3 ′ terminal region of other genes other than the NCOA4 or RUFY1 gene constructing the NCOA4 or RUFY1 fusion gene together with the NCOA4 or RUFY1 gene are specifically detected. By determining the expression level ratio, the NCOA4 or RUFFY1 fusion gene can be detected.
  • PCR method and the primer design method used therefor can be performed by those skilled in the art according to a known method.
  • a sense primer and an antisense primer designed to specifically amplify the 5′-terminal region of the RET gene and a sense primer designed to specifically amplify the 3′-terminal region of the RET gene and Antisense primers can be used.
  • a sense primer and an antisense primer designed to specifically amplify the 5 ′ end region of the NCOA4 or RUFY1 gene and a design to specifically amplify the 3 ′ end region of the NCOA4 or RUFY1 gene Sense primers and antisense primers can be used.
  • a PCR amplification monitoring method for example, ABI PRISM 7900 (PE Biosystems) can be used.
  • Real-time PCR is a known method, and devices and kits for the real-time PCR are commercially available, and can be easily performed using these.
  • the sense primer (5′-primer, forward primer) is selected from NCOA4 or
  • An antisense primer (3′-primer, reverse primer) is designed from any part derived from the RET gene from any part derived from the RUFY1 gene.
  • the sense primer (5′-primer, forward primer)
  • An antisense primer (3′-primer, reverse primer) is designed from any part derived from the RET gene from any part derived from the RUFY1 gene.
  • each reaction solution is mixed with each other gene constituting the RET fusion gene by fusing with the RET gene, and the sense primer corresponding to a plurality of fusion points.
  • Multiplex PCR that detects all fusion polynucleotides can also be designed.
  • the other primers constituting the NCOA4 or RUFY1 fusion gene by fusing with the NCOA4 or RUFY1 gene and the above-mentioned sense primers corresponding to multiple fusion points are mixed.
  • the primer set used in the detection method for detecting the RET fusion gene of the present invention is not particularly limited as long as it can specifically amplify at least a part of the RET fusion gene to be detected and can detect the RET fusion gene. Is not limited, and a person skilled in the art can design based on the base sequence of the polynucleotide to be detected.
  • the primer set used in the detection method for detecting the NCOA4 or RUFY1 fusion gene of the present invention can specifically amplify at least part of the NCOA4 or RUFY1 fusion gene to be detected, and can detect the NCOA4 or RUFY1 fusion gene If it is a thing, it will not specifically limit, Those skilled in the art can design based on the base sequence of detection target polynucleotide. Primer design in the PCR amplification monitoring method can be performed using primer design software (eg, Primer Express; PE Biosystems).
  • the sense primer and the antisense primer should be set so that the size of the amplified product when amplified for mRNA or cDNA is 1 kb or less. Is appropriate.
  • mRNA can be detected by a hybridization method using a probe that hybridizes to a RET fusion gene or NCOA4 or RUfy1 fusion gene at least a part of the polynucleotide to be detected.
  • detection using a hybridization technique include Northern hybridization, dot blot method, DNA microarray method, RNA protection method and the like.
  • ⁇ Detection of fusion protein As a technique for detecting a RET fusion protein or NCOA4 or RUFY1 protein in a sample obtained from a subject, any technique known to those skilled in the art used to analyze proteins or any technique to which these techniques are applied is used. May be.
  • an antibody that specifically recognizes a RET protein or another protein other than the RET protein that constructs the RET fusion protein together with the RET protein, or a RET fusion protein is specifically used.
  • Immunoassay immunoassay
  • enzyme activity assay ELISA
  • 2-antibody sandwich ELISA fluorescence immunoassay
  • radioimmunoassay western blotting
  • immunohistochemistry examples thereof include immunoprecipitation, iAEP (intercalated antibody-enhanced polymer) method, and FRET method.
  • a mass spectrometry method or an amino acid sequence method can be used in combination with these or alone.
  • NCOA4 or RUFY1 fusion protein for example, as a method used for detection of NCOA4 or RUFY1 fusion protein, NCOA4 or RUFY1 protein, or other proteins other than NCOA4 or RUFY1 protein that construct NCOA4 or RUFY1 fusion protein together with NCOA4 or RUFY1 protein are specifically recognized.
  • Immunoassay immunoassay
  • enzyme activity assay ELISA
  • two-antibody sandwich ELISA two-antibody sandwich ELISA
  • fluorescence immunoassay radiation using antibodies or antibodies that specifically recognize NCOA4 or RUFY1 fusion protein
  • Examples include immunoassay, Western blotting, immunohistochemistry, immunoprecipitation, iAEP (intercalated antibody-enhanced polymer), and FRET.
  • a mass spectrometry method or an amino acid sequence method can be used in combination with these or alone.
  • representative methods for protein detection are exemplified, but the present invention is not limited thereto.
  • Typical methods used for detection As a detection method using an antibody, the above-mentioned known method may be used. For example, the following method can be used.
  • RET fusion protein to be detected or NCOA4 or RUFY1 fusion protein is an NCOA4 or RUFY1-RET fusion protein
  • the C-terminal of the RET protein is compared with the tissue section in which the fusion protein to be detected may be present.
  • Perform immunostaining using an anti-RET antibody that binds to a polypeptide in the side region and an anti-NCOA4 or RUFY1 antibody that binds to a polypeptide in the N-terminal region of the NCOA4 or RUFY1 protein and confirm that these antibodies are close
  • the presence of the fusion protein to be detected can also be detected as an index.
  • immunostaining was performed using an antibody that specifically binds to the polypeptide in the N-terminal region of the RET protein and an antibody that specifically binds to the polypeptide in the C-terminal region of the RET protein. It is also possible to detect the presence of the fusion protein to be detected using the presence of the distant localization as an index. In addition, immunostaining using an antibody that specifically binds to a polypeptide in the N-terminal region of NCOA4 or RUFY1 protein and an antibody that specifically binds to a polypeptide in the C-terminal region of NCOA4 or RUFY1 protein, The presence of the fusion protein to be detected can also be detected using the presence of these antibodies as an index. Alternatively, the presence of the fusion protein to be detected can be detected by performing immunostaining using an antibody that specifically binds to the polypeptide containing the fusion point.
  • ⁇ Western blotting method> when the RET fusion protein to be detected or the NCOA4 or RUFY1 fusion protein is an NCOA4 or RUFY1-RET fusion protein, a cell extract that may contain the fusion protein to be detected is obtained by a method well known to those skilled in the art. The protein in the cell extract is separated by electrophoresis using the method described above, and then blotted on a membrane.
  • the presence of the fusion protein to be detected can also be detected using as an index the binding of the anti-RET antibody and the anti-NCOA4 or RUFY1 antibody to a desired position on the membrane. It is also possible to detect the presence of a fusion protein to be detected using an antibody that specifically binds to a polypeptide containing a fusion point and using as an index that the antibody is bound to a desired position on the membrane. .
  • an anti-RET antibody can be used to detect the presence of the fusion protein to be detected using as an index that the antibody is bound to the NCOA4 or RUFY1-RET fusion protein on the membrane.
  • the presence of the fusion protein to be detected may be detected by using an anti-RET antibody binding to a position different from the position where the wild-type RET protein is predicted on the membrane.
  • An anti-NCOA4 or RUFY1 antibody may be used to detect the NCOA4 or RUFY1-RET fusion protein on the same principle as when an anti-RET antibody is used.
  • RET fusion protein to be detected or the NCOA4 or RUFY1 fusion protein is an NCOA4 or RUFY1-RET fusion protein
  • the RET protein C is added to the cell extract in which the detection target fusion protein may be present.
  • Immunoprecipitation is performed with either the anti-RET antibody that binds to the polypeptide in the terminal region or the anti-NCOA4 or RUFY1 antibody that binds to the polypeptide in the N-terminal region of the NCOA4 or RUFY1 protein, and the precipitate is By detecting with the other remaining antibody, the presence of the fusion protein to be detected can also be detected.
  • the cell extract in which the RET fusion protein to be detected is immunoprecipitated with an anti-RET antibody that binds to a polypeptide in the C-terminal region of the RET protein, and the precipitate
  • an anti-RET antibody that binds to a polypeptide in the C-terminal region of the RET protein
  • Immunoprecipitation is carried out with an anti-NCOA4 or RUFY1 antibody that binds to a polypeptide in the N-terminal region of the NCOA4 or RUFY1 protein on the cell extract in which the NCOA4 or RUFY1 fusion protein to be detected may be present.
  • the presence of the fusion protein to be detected can be detected by confirming the presence of a protein that binds to an anti-NCOA4 or RUFY1 antibody having a mass different from that of wild-type NCOA4 or RUFY1. .
  • the antibody used in the detection method according to the present invention is not particularly limited as long as it specifically binds to a desired site of the RET fusion protein or NCOA4 or RUFY1 fusion protein, and even a monoclonal antibody is a polyclonal antibody. It is also possible to use a combination of a monoclonal antibody and a polyclonal antibody.
  • the antibody may be an immunoglobulin itself or an antibody fragment that retains antigen binding ability, such as Fab, Fab ′, F (ab ′) 2 , or Fv. Any label or signal amplification method known to those skilled in the art may be used to detect antibody binding.
  • ⁇ Labeling method> In the above gene (genomic DNA, mRNA, cDNA, etc.) and protein detection methods, known techniques may be used for labeling probes, primers, amplification products, antibodies, and the like. For example, fluorescent labels, chemiluminescent labels, radioactive labels, enzyme labels, biotin labels, avidin labels and the like can be mentioned.
  • the labeling method when a probe is labeled, the labeling method may be a known method as described above. For example, when preparing a labeled nucleic acid probe from a BAC clone, nick translation, A known method such as a random prime method can be used.
  • the probe is labeled with biotin using biotin-dUTP (for example, manufactured by Roche Applied Science), and the probe is labeled by further processing phosphors, radioisotopes, enzymes, etc. bound to avidin. can do.
  • biotin-dUTP for example, manufactured by Roche Applied Science
  • the labeling method may be a known method as described above, and examples thereof include the following labeling methods.
  • Staining sensitivity can be increased by placing an intervening antibody between the first antibody that binds to the protein to be detected and the polymer reagent (Takeuchi et al., Clin Cancer Res, 2009 May 1; 15 (9) : 3143-3149).
  • FRET probe Fluorescence resonance energy transfer
  • RET fusion gene to be detected or the RET fusion protein to be detected in the detection method of the present invention is detected from a sample obtained from a subject, the subject has a RET fusion-positive cancer (patient). And is a target for treatment with a RET inhibitor.
  • ⁇ Determination of application target of treatment with NCOA4 or RUFY1 inhibitor When the detection target NCOA4 or RUFY1 fusion gene or the detection target NCOA4 or RUFY1 fusion protein is detected from a sample obtained from the subject, the subject is NCOA4 or RUFY1 fusion-positive cancer. This is a subject (patient) having a target of treatment with an NCOA4 or RUFFY1 inhibitor.
  • the detection kit of the present invention includes a detection kit for a detection target RET fusion gene or a detection kit for a detection target RET fusion protein.
  • the detection kit of the present invention includes a detection kit for a detection target NCOA4 or RUFY1 fusion gene or a detection target NCOA4 or RUFY1 fusion protein.
  • the detection kit of the RET fusion gene to be detected of the present invention or the kit for detection of the NCOA4 or RUFY1 fusion gene includes a probe that can be used in the FISH method fusion assay or FISH method split assay in the detection method of the present invention, Alternatively, sense and antisense primers designed to specifically amplify at least a part of the RET fusion gene or NCOA4 or RUFFY1 fusion gene to be detected in the detection method of the present invention are included.
  • the sense and antisense primer set is a polynucleotide set that functions as a primer for amplification of a polynucleotide to be amplified, and is a polynucleotide that is at least a part of the RET fusion gene or NCOA4 or RUFY1 fusion gene.
  • an antibody that can be used in the detection method of the present invention is included in the detection kit of the detection target RET fusion protein or NCOA4 or RUFI1 fusion protein of the present invention.
  • the kit for detecting a RET fusion gene of the present invention is one kind of probe capable of detecting the RET fusion gene by hybridizing under stringent conditions to at least a part of the polynucleotide of the RET fusion gene or its complementary strand. Or it can contain in the combination of 2 or more types.
  • the kit for detecting the NCOA4 or RUFY1 fusion gene of the present invention hybridizes under stringent conditions to at least a part of the polynucleotide of the NCOA4 or RUFY1 fusion gene, or a complementary strand thereof, and the NCOA4 or RUFY1 fusion gene Probes that can be detected can be included in one type or a combination of two or more types.
  • the probe examples include any one or more of the probes described in ⁇ Technology used in detection method >>.
  • the RET fusion gene or NCOA4 or RUFY1 fusion gene is an NCOA4 or RUFY1-RET fusion gene
  • one or more (preferably two or more) probes hybridizing to the RET gene-derived polynucleotide, or NCOA4 or RUFY1 One or more probes that hybridize to a RET gene-derived polynucleotide, and either NCOA4 or RUFY1, even if only one of one or more (preferably two or more) probes that hybridize to a gene-derived polynucleotide
  • it may comprise one or more probes that hybridize to a polynucleotide comprising a fusion
  • the kit for detecting a RET fusion gene of the present invention can specifically amplify at least a part of the RET fusion gene and can include one set of primer sets capable of detecting the RET fusion gene, or a combination of two or more sets. .
  • the kit for detecting the NCOA4 or RUFY1 fusion gene of the present invention can specifically amplify at least a part of the NCOA4 or RUFY1 fusion gene and can detect one set of primer sets that can detect the NCOA4 or RUFY1 fusion gene, or two or more sets of primer sets. Can be included in combination.
  • Examples of the primer set include any one or more of the primer sets described in ⁇ Aspect of detection method of the present invention >> or ⁇ Technique used in detection method >>.
  • a fusion gene between a RET gene and an NCOA4 or RUFY1 gene comprising an antisense primer designed from a polynucleotide part encoding a RET protein and a sense primer designed from a polynucleotide part encoding an NCOA4 or RUFY1 protein
  • a primer set for detection wherein an antisense primer is a nucleic acid molecule (preferably at least 16 bases) that anneals to a “polynucleotide to be detected” under stringent conditions (preferably under more stringent conditions).
  • a nucleic acid molecule (preferably at least 1) that anneals to the complementary strand of the “polynucleotide to be detected” under stringent conditions (preferably under more stringent conditions).
  • Primer set consisting of a nucleic acid molecule) of the bases include.
  • the primer set of the present invention includes the following primer sets (2) to (5).
  • a sense primer consisting of any consecutive oligonucleotides of at least 16 bases between base numbers 1 to 1984 of SEQ ID NO: 1 (NCOA4ex9-RETex12), and any between base numbers 1985 to 5275 of SEQ ID NO: 1
  • a primer set of antisense primers consisting of oligonucleotides that are complementary to oligonucleotides of at least 16 consecutive bases.
  • a sense primer consisting of any continuous oligonucleotide of at least 16 bases between base numbers 1 to 1917 of SEQ ID NO: 3 (RUFY1ex16-RETex12), and any of bases from base numbers 1918 to 5208 of SEQ ID NO: 3
  • a primer set of antisense primers consisting of oligonucleotides that are complementary to oligonucleotides of at least 16 consecutive bases.
  • NCOA4-1661F CTGGAGAAGACAAGTGGCTGC (SEQ ID NO: 12)
  • RET-2381R CAGGCCCCATACAATTTGAT (SEQ ID NO: 7)
  • RUFY1-1492F ATGAAACAAATGGAAGAAAGGTTG (SEQ ID NO: 13)
  • RET-2381R CAGGCCCCATACAATTTGAT (SEQ ID NO: 7)
  • a primer set for detecting the expression levels of the 5′-terminal region and the 3′-terminal region of other genes that construct a fusion gene together with the RET gene is also known.
  • the distance between the selected positions of the sense primer and the antisense sense primer is 1 kb or less, or the size of the amplification product amplified by the sense primer and the antisense sense primer Is preferably 1 kb or less.
  • the primer of the present invention usually has a chain length of 15 to 40 bases, preferably 16 to 24 bases, more preferably 18 to 24 bases, and particularly preferably 20 to 24 bases.
  • the primer set of the present invention can be used for amplifying and detecting a polynucleotide to be detected in the detection method of the present invention.
  • each primer contained in the primer set of this invention is not specifically limited, For example, it can manufacture by a chemical synthesis method.
  • the kit for detecting a RET fusion protein of the present invention can contain one or more combinations of antibodies that specifically bind to an arbitrary site of the RET fusion protein. Specifically, the antibodies described in ⁇ Fusion protein detection> can be exemplified.
  • the NCOA4 or RUFY1 fusion protein detection kit of the present invention can contain one or more combinations of antibodies that specifically bind to any site of the NCOA4 or RUFY1 fusion protein. Specifically, the antibodies described in ⁇ Fusion protein detection> can be exemplified.
  • the RET fusion protein or NCOA4 or RUFY1 fusion protein is an NCOA4 or RUFY1-RET fusion protein
  • one or more (preferably two or more) antibodies that bind to the RET protein-derived polypeptide, or NCOA4 or RUFY1 Even if only one of one or more (preferably two or more) antibodies that bind to a protein-derived polypeptide is included, one or more antibodies that bind to a RET protein-derived polypeptide, and NCOA4 or RUFY1 protein-derived
  • Polynucleotide One or more antibodies that bind may contain.
  • the method for screening an inhibitory substance of the present invention can screen a substance that inhibits the detection target polypeptide, (1) A step of bringing a test substance into contact with a polypeptide to be detected or a cell expressing the polypeptide, (2) analyzing whether the polypeptide is inhibited, and (3) selecting a substance that inhibits the polypeptide.
  • inhibitortion of polypeptide includes inhibition of activity of polypeptide and inhibition of expression of polypeptide. “Inhibition” means at least partial inhibition.
  • the screening method of the present invention includes (A) Using purified or crude polypeptide, the method using in vitro inhibition of polypeptide activity as an index, (B) a method using a cell expressing a polypeptide as an indicator of inhibition of polypeptide activity; (C) A method using the expression of the polypeptide as an index using cells expressing the polypeptide is included.
  • a test substance is added to and contacted with a polypeptide in vitro, whether or not the activity of the polypeptide is inhibited by the test substance, a control (polysaccharide not contacted with the test substance).
  • a method comprising the step of selecting a substance that inhibits the activity of the polypeptide.
  • In vitro polypeptide activity can be measured using a known kinase activity measurement method. For example, the amount of ADP produced by the kinase reaction may be used as an index, or the tyrosine phosphorylation level of the polypeptide may be used as an index. Commercially available kinase activity measurement kits can also be used.
  • the method (B) includes a step of adding a test substance to a cell expressing the polypeptide and bringing it into contact; whether the test substance inhibits the activity of the polypeptide; And a method comprising a step of selecting a substance that inhibits the activity of the polypeptide.
  • a known kinase activity measurement method can be used for the measurement of the polypeptide activity in the cells.
  • the amount of ADP produced by the kinase reaction may be used as an index, or the tyrosine phosphorylation level of the polypeptide may be used as an index.
  • a commercially available kinase activity measurement kit can also be used.
  • a test substance is added to and contacted with a cell expressing the polypeptide, whether or not the expression of the polypeptide is inhibited by the test substance, a control (contact with the test substance).
  • a method comprising a step of selecting a substance that inhibits the expression of the polypeptide.
  • Polypeptide expression in the cells can be analyzed by measuring the amount of protein or mRNA.
  • ELISA or immunoblotting can be used for measuring the amount of protein
  • RT-PCR or Northern blotting can be used for measuring the amount of mRNA.
  • the RET fusion gene is a gene having tumorigenicity. Therefore, the polypeptide inhibitor selected by the inhibitor screening method of the present invention is useful as an effective substance or a candidate substance for a pharmaceutical composition for the treatment of RET fusion-positive cancer.
  • the method may further comprise the step of confirming that the inhibitor has therapeutic activity against RET fusion positive cancer.
  • the NCOA4 or RUfy1 fusion gene is a gene having tumorigenicity. Therefore, the polypeptide inhibitor selected by the inhibitor screening method of the present invention is useful as a therapeutic agent for NCOA4 or RUFY1 fusion-positive cancer or a candidate substance thereof. Can further comprise the step of confirming that NCOA4 or RUFY1 fusion-positive cancer has therapeutic activity.
  • the confirmation step can be performed using a known evaluation system, and examples thereof include an in vitro evaluation system using cultured cells and an evaluation system using a cancer-bearing model animal transplanted with tumor cells.
  • the tumor-bearing model animal can be transplanted after a tumor tissue removed from a patient by surgery is once established as a cell line by culturing, or the tumor tissue can be directly transplanted.
  • the latter cancer-bearing model animal is known as a PDX (patient-derived xenograft) model animal, and has a gene expression profile in a subcutaneous tumor tissue that has been repeatedly passaged compared to a model animal transplanted with a cell line. Since it is more similar to the nest, it is preferable as an evaluation system.
  • the polypeptide-expressing cell can also be obtained by introducing the polynucleotide of the present invention into a desired cell according to a conventional method (for example, Molecular Cloning: A Laboratory Manual 4th Edition (2012), Cold Spring Harbor Laboratory Press). reference). Specifically, for example, the RET fusion gene of the present invention or the cDNA that is the NCOA4 or RUFY1 fusion gene is introduced into a recombinant vector, which is further introduced into a cell, whereby the polypeptide-expressing cell (transformed cell) is transformed. ) Can be obtained.
  • the pharmaceutical composition for the treatment of RET fusion-positive cancer of the present invention comprises an inhibitor for the RET fusion gene or its transcription product.
  • an inhibitor for example, a low molecular weight compound, a double-stranded nucleic acid (including siRNA), a protein (including an antibody or an antibody fragment), a peptide, or other compound
  • It is contained as an active ingredient, and if desired, a pharmaceutically acceptable carrier can be contained.
  • the pharmaceutical composition for the treatment of NCOA4 or RUFY1 fusion-positive cancer of the present invention comprises an inhibitor for the NCOA4 or RUFY1 fusion gene or a transcription product thereof.
  • an inhibitor for example, a low molecular weight compound, a double-stranded nucleic acid (including siRNA), a protein (including an antibody or an antibody fragment), a peptide, or other compound) obtained by the inhibitor screening method of the present invention is used. It is contained as an active ingredient, and if desired, a pharmaceutically acceptable carrier can be contained.
  • RET fusion gene or transcript ⁇ Inhibitor for RET fusion gene or transcript, or NCOA4 or RUFY1 fusion gene or transcript>
  • the inhibitor for the RET fusion gene or a transcription product thereof include a kinase inhibitor, for example, a RET inhibitor, or an inhibitor for the other gene that constructs a fusion gene together with the RET gene or a transcription material thereof.
  • a kinase inhibitor for example, an inhibitor of the NCOA4 or RUFY1 inhibitor, or another gene that constructs a fusion gene together with the NCOA4 or RUFY1 gene or a transcript thereof Can be mentioned.
  • Low molecular compound examples include Vandetanib (AstraZeneca Pharmaceuticals), Alectinib (F. Hoffmann-La Roche Ltd.), Cabozantinib (Exelixis Inc.), Sorafenib (Bayer Pharma AG), Pontatinib (ARIAD Pharmaceuticals), Nintendanib (Boehringer Ingelheim), Lenvatinib (Eisai Co., Ltd.), Regorafenib (Bayer Healthcare Pharmaceuticals), 1-Ter-Butyl-3-P-Tolyl-1h-Pyrazolo [3,4-D] Pyrimidin-4-Ylamine And pharmaceutically acceptable salts thereof.
  • a double-stranded nucleic acid consists of a double-stranded nucleic acid (RNA or DNA) portion and preferably an overhang at the 3 ′ end of the sense strand and the antisense strand to induce RNAi.
  • RNAi is an evolutionarily conserved phenomenon that occurs via a 21-23 base double-stranded nucleic acid generated by RNase III endonuclease (Genes Dev. 15, 485-490, 2001).
  • Each 3 ′ overhang is an arbitrary nucleic acid having 1 or 2 bases, but 2 bases are preferred.
  • the number of bases (21 to 23 bases) is the number of bases of each of the sense strand or the antisense strand containing an overhang.
  • the sense strand and the antisense strand can have the same number of bases or different numbers of bases, but preferably have the same number of bases.
  • ribonucleic acid constituting the 3 ′ overhang of the double-stranded nucleic acid for example, U (uridine), A (adenosine), G (guanosine), or C (cytidine) can be used.
  • deoxyribonucleic acid constituting the overhang for example, dT (deoxythymidine), dA (deoxyadenosine), dG (deoxyguanosine), or dC (deoxycytidine) can be used.
  • the double-stranded nucleic acid that can be used as an active ingredient of the pharmaceutical composition of the present invention is not particularly limited as long as it has an inhibitory action on the RET fusion gene or an inhibitory action on the NCOA4 or RUfy1 fusion gene.
  • the double-stranded portion includes a base sequence of a polynucleotide containing a fusion point, for example, a base sequence including 1984 to 1985 of SEQ ID NO: 1, or 1917 to 1918 of SEQ ID NO: 3. It can design based on a base sequence.
  • the duplex portion can be designed based on the base sequence of the polynucleotide encoding the kinase portion.
  • the double-stranded nucleic acid of the present invention can be produced by a conventional method (for example, J. Am. Chem. Soc., 120, 11820-11821, 1998; and Methods, 23, 206-217, 2001).
  • companies that consign and manufacture double-stranded nucleic acids for example, RNAi
  • RNAi double-stranded nucleic acid
  • a double-stranded nucleic acid can be designed by a siRNA sequence design system (commercial siDirect (registered trademark); RNAi).
  • the antibody that can be used as an active ingredient of the pharmaceutical composition of the present invention inhibits the transcription product of the RET fusion gene or the transcription product of the NCOA4 or RUFY1 fusion gene, preferably the transcription product of the NCOA4 or RUFY1-RET gene. If there is, it is not limited. For example, those that inhibit the activity of the RET fusion protein or NCOA4 or RUFY1 fusion protein, preferably the kinase activity.
  • the RUFY1-RET fusion protein that is a detection target according to the detection method of the present invention is itself a novel protein.
  • the RUFY1-RET fusion protein that is the polypeptide of the present invention is preferably a polypeptide described in the following (a) to (d): (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4, (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 and having tumorigenicity, (C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having tumorigenicity, and (d) an amino acid sequence represented by SEQ ID NO: 4 A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or inserted, and having tumorigenic potential.
  • the RUFY1-RET fusion gene to be detected according to the detection method of the present invention is itself a novel gene.
  • the polynucleotide of the present invention is not particularly limited as long as it is a polynucleotide encoding the polypeptide of the present invention (ie, RUFY1-RET fusion protein) (ie, RUFY1-RET fusion gene).
  • the “polynucleotide encoding the RUFY1-RET fusion protein” may be a polynucleotide comprising only the translation region in the RUFY1-RET fusion gene, the full-length genomic DNA of the RUYY1-RET fusion gene, or the RUFY1-RET fusion gene. It may be mRNA or cDNA of RET fusion gene.
  • the vector of the present invention is not particularly limited as long as it contains the polynucleotide of the present invention, and is prepared by incorporating the polynucleotide into an appropriate vector capable of transforming a eukaryotic or prokaryotic host cell. be able to.
  • the vector can contain a sequence necessary for expression of the polynucleotide, such as a promoter and an enhancer, and can further contain a sequence necessary for confirmation of introduction into the host, such as a drug resistance gene.
  • the transformed cell of the present invention can be prepared by transforming a suitable host cell such as a eukaryotic or prokaryotic host cell with the vector of the present invention.
  • the transformed cell of the present invention can be used for producing the polypeptide of the present invention.
  • Example 1 Detection of RET gene abnormality in clinical specimen by FISH method A method for observing gene translocation or inversion etc. by dyeing the 5 'terminal region and 3' terminal region of the target gene with different dyes are known. This method, which is a type of FISH method, is called a split assay. In the split assay, the 5 ′ terminal region and the 3 ′ terminal region of the target gene to be examined for chromosomal translocation or inversion are stained with probes labeled with different fluorescent dyes.
  • RET gene abnormality in clinical specimens was detected by FISH split assay.
  • a colon cancer tissue excised by surgery, fixed with 20% formalin and embedded in paraffin was cut to a thickness of 4 ⁇ m, and placed on a slide glass to prepare a pathological section.
  • the FISH method is based on literature (Takeuchi K, Choi YL, Soda M, Inamura K, Togashi Y, Hatano S, Enomoto M, Takada S, Yamashita Y, Satoh Y, Okumura S, Nakagawa K, Ishiano -PCR screening for EML4-ALK fusion transcripts. Clin Cancer Res. 2008; 14: 6618-6624.).
  • the prepared unstained section was treated with the Histology FISH accessory kit (Dako), followed by a BAC (bacterial-artificial-chromosome) clone (clone number) covering the 5'-terminal region of the red (TexasRed) fluorescently labeled RET gene.
  • Hybridization was performed using RP11-124O11) and a BAC clone (clone number RP11-351D16) covering the 3 'terminal region of the RET gene labeled with green (FITC).
  • FITC red fluorescently labeled
  • staining was performed with 4,6-diamino-2-phenylindole.
  • a fluorescence microscope BX51 (Olympus) was used. We found a section suggesting an abnormal genomic structure in which green and red signals were observed apart. From the examination of about 1500 pathological specimens, 2 specimens (derived from colon cancer patients) suggesting genomic structural abnormality in the RET gene region were found.
  • Example 2 Identification of RET fusion polynucleotide gene in clinical specimen 5 ' The genes present on the side were examined. First, reverse transcription and double-stranded cDNA synthesis were performed using a cDNA synthesis system (Roche) using 0.5 ⁇ g of RNA derived from a clinical specimen and a reverse transcription primer RET-2746R (SEQ ID NO: 5) designed in the RET gene kinase coding region. ). The synthesized double-stranded cDNA was purified by High Pure PCR Product Purification Kit (Roche) and then self-ligated using T4 DNA ligase (TaKaRa).
  • aKaRa T4 DNA ligase
  • Example 3 Isolation of a RUFY1-RET fusion polynucleotide gene in a clinical sample Using a cDNA derived from a colorectal cancer clinical sample in which a RET genomic structural abnormality was suggested by FISH analysis and the fused gene was identified as a template, PCR was performed using DNA polymerase (PrimeStar HS DNA polymerase), and the amplified product was cloned into pT7Blue-2.
  • a primer set for isolating the RUFY1-RET fusion polynucleotide gene a combination of the forward primer RUFY1-5′UTR (SEQ ID NO: 10) and the reverse primer RET-3′UTR (SEQ ID NO: 11) was used.
  • a polynucleotide (RUFY1ex16-RETex12) consisting of a base sequence from the start codon ATG to exon 16 of the RUFY1 gene and from the exon 12 to the stop codon of exon 20 of the RET gene. ; SEQ ID NO: 3) was obtained.
  • the 1401st C and the 1740th A in the nucleotide sequence of SEQ ID NO: 3 are reported as single nucleotide polymorphisms (rs4701135, rs72824522) without amino acid substitution, respectively.
  • NCOA4-RET fusion gene based on the results of Example 2, a polynucleotide comprising a nucleotide sequence from the start codon ATG to exon 9 of the NCOA4 gene and the stop codon of exon 12 to exon 20 of the RET gene ( NCOA4ex9-RETex12; SEQ ID NO: 1) was confirmed.
  • the sample-derived RNA template was compared with the forward primer RUYY1-1492F (SEQ ID NO: 13) designed on the RUFY1 gene and the reverse primer RET-2381R (SEQ ID NO: 7) designed on the RET gene.
  • PCR was performed using When the amplified product was electrophoresed, a band (659 bp) of the size expected from the primer setting position was observed. It has been shown that detection of fusion genes using clinical specimens is possible by designing primers on these genes.
  • Example 5 Detection of NCOA4-RET fusion gene and RUFY1-RET fusion gene in clinical specimens by FISH fusion assay To confirm that the fusion gene is fused on the genome, it is detected by FISH fusion assay Went.
  • FISH fusion assay two adjacent target gene regions are stained with probes labeled with different fluorescent dyes by chromosomal translocation or inversion. For example, by fluorescently labeling with two types of probes labeled TexasRed (red) and FITC (green), when normal (when the fusion gene has not been constructed), red and green indicate their respective signals (red and red). If the two gene regions are close to each other due to translocation or inversion, the red and green signals are detected as overlapping yellow. .
  • a red (TexasRed) fluorescent labeling BAC clone (clone number CTD-2299L6) covering the 5 'terminal region of the NCOA4 gene and green (FITC) fluorescence
  • a labeled BAC clone (clone number RP11-351D16) covering the 3 ′ terminal region of the RET gene was used.
  • a red fluorescent label (TexasRed)
  • a BAC clone (clone number RP11-410B18) covering the 5 ′ terminal region of the RUFY1 gene
  • a green (FITC) fluorescent label A combination with a BAC clone (clone number RP11-351D16) covering the 3 ′ terminal region of the gene was used.
  • a fluorescence microscope BX51 (Olympus) was used.
  • cDNA encoding RUFY1-RET fusion polypeptide was used as the cDNA encoding RUFY1-RET fusion polypeptide in SEQ ID NO: 3 obtained from a colorectal cancer clinical specimen.
  • the RUFY1-RET fusion gene was used to examine the tumorigenicity of the RUFY1-RET fusion polypeptide.
  • PLenti6-RUFY1-RET obtained by inserting the cDNA into the expression vector pLenti6 (Invitrogen (registered trademark) (Life Technologies) was introduced into the mouse fibroblast cell line NIH3T3 cells and cultured for 7 days.
  • NIH3T3 cells introduced with a vector expressing the RUFY1-RET fusion polypeptide, NIH3T3 cells treated only with a gene introduction reagent (control), and NIH3T3 cells introduced with LacZ were each subcutaneously 1 ⁇ 10 6 nude mice.
  • NIH3T3 cells introduced with a vector expressing the fusion polypeptide When inoculated one by one, tumor formation was confirmed in mice inoculated with NIH3T3 cells introduced with a vector expressing the fusion polypeptide. Tumor formation was not confirmed in mice inoculated with NIH3T3 cells (control) treated only with the gene introduction reagent.
  • the tumor size in both mice after the first day of inoculation is shown in FIG.
  • RUFY1-RET fusion polynucleotide is a causative gene of cancer because RUFY1-RET fusion polypeptide had tumorigenicity.
  • cDNA encoding a full-length RET polypeptide was introduced into NIH3T3 cells, no transformation focus was observed, confirming that the full-length RET polypeptide had no tumorigenicity.
  • Example 7 Examination of sensitivity of RUFY1-RET fusion polypeptide-expressing cells to each RET inhibitor
  • Mouse lymphoid cell line Ba / F3 cell is an IL-3-dependent cell line that is a growth factor.
  • IL-3 is required, it is known that by introducing a canceration gene (for example, a tyrosine kinase fusion gene), it can proliferate without adding IL-3 (Daley GQ and Baltimore D. Proc Natl Acad Sci USA. 1988 Dec; 85 (23): 9312-9316.).
  • each RET inhibitor (Vandetanib, Alectinib, Lenvatinib and Cabozantinib) were added and the number of cells after culturing for 72 hours was counted to examine the sensitivity to each RET inhibitor.
  • Katayama et al. Katayama R et al., Sci Transl Med, 2012 (4): 120ra17.
  • the RET inhibitor is effective for the treatment of cancer patients positive for RUFY1-RET fusion gene, and at the same time, the present evaluation system using Ba / F3 cells into which pLenti6-RUFY1-RET is introduced, It shows that it can be used for screening an effective drug for treatment of the fusion gene-positive cancer patient.
  • Example 8 Examination of suppression of autophosphorylation of RUFY1-RET fusion polypeptide by each RET inhibitor in RUFY1-RET fusion polypeptide-expressing cells RUFY1-RET fusion polypeptide by each RET inhibitor confirmed in Example 7
  • Western blotting was performed on extracts from each cultured cell treated with each RET inhibitor. It was. The results are shown in FIG.
  • an anti-phosphorylated RET antibody pRET in FIG. 7
  • an anti-RET antibody RET in FIG. 7 were used.
  • a fusion gene of the RUFY1 gene and the RET gene exists in some digestive cancer patients, and that gene causes cancer. That is, it has been clarified that cancer patients to be treated with RET-inhibiting drug treatment can be selected by detecting the RUFY1-RET fusion gene, preferably by detecting RUFY1ex16-RETex12.
  • the detection method of the present invention is useful for the determination of RET fusion-positive cancer patients.
  • the detection kit and primer set of the present invention can be used in the detection method.
  • the inhibitory substance screening method of the present invention can be used for screening a substance effective for treating the fusion-positive cancer patient.
  • the substance obtained by the screening can be used as an active ingredient of the pharmaceutical composition for treating fusion-positive cancer.
  • Cancer can be treated by administering the substance to a patient determined to be a fusion-positive cancer patient by the detection method.
  • the detection method of the present invention is useful for determination of cancer patients who are positive for NCOA4 or RUFY1 fusion.
  • the detection kit and primer set of the present invention can be used in the detection method.
  • the inhibitory substance screening method of the present invention can be used for screening a substance effective for treating the fusion-positive cancer patient.
  • the substance obtained by the screening can be used as an active ingredient of the pharmaceutical composition for treating fusion-positive cancer.
  • Cancer can be treated by administering the substance to a patient determined to be a fusion-positive cancer patient by the detection method.
  • the base sequences represented by the sequences of SEQ ID Nos: 5 to 13 in the sequence listing are synthetic primer sequences.

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Abstract

Provided are a method for elucidating a polynucleotide which is a novel gene responsible for cancer, and detecting the polynucleotide or the polypeptide coded thereby on the basis of this finding; a kit and primer set for this detection; a method for screening an inhibitor of the polypeptide; and a pharmaceutical composition for cancer treatment containing the inhibitor. This detection method detects an RET fusion protein or a fusion gene for encoding said fusion protein or an NCOA4 or RUFY1 fusion protein or a fusion gene for encoding said fusion protein in a sample from a digestive organ obtained from a subject.

Description

新規融合体及びその検出法Novel fusion and detection method thereof
　本発明は、ＲＥＴキナーゼ領域を含む新規の融合タンパク質又は該融合タンパク質をコードする融合遺伝子、及びそれらの検出方法に関する。
　本発明は、ＮＣＯＡ４又はＲＵＦＹ１の少なくとも一部を含む新規の融合タンパク質又は該融合タンパク質をコードする融合遺伝子、及びそれらの検出方法に関する。 The present invention relates to a novel fusion protein containing a RET kinase region or a fusion gene encoding the fusion protein, and a method for detecting them.
The present invention relates to a novel fusion protein containing at least a part of NCOA4 or RUfy1, or a fusion gene encoding the fusion protein, and a method for detecting them.
　染色体転座の結果、本来は別々の遺伝子が融合して融合遺伝子が作られる。これまで慢性骨髄性白血病におけるＢＣＲ－ＡＢＬ１融合体や、肺がんにおけるＥＭＬ４－ＡＬＫ融合体、肺がんを含む種々のがんにおけるＲＯＳ１融合体のように、キナーゼ遺伝子の一部を構成要素に含む融合遺伝子はしばしば発がんに本質的な役割を担い、その機能を抑制する薬剤は極めて有効な抗がん剤となることが知られている（非特許文献１、特許文献１、及び特許文献２）。
　例えば、チロシンキナーゼ阻害剤のクリゾチニブやエルロチニブの登場により、分子診断とがん治療効果の関係について、臨床で示されつつあり、分子診断による適応患者の選別により、患者を層別化した上での治療薬投与というコンセプトが広がりつつある。 As a result of the chromosomal translocation, originally fused genes are fused to create a fused gene. So far, fusion genes that include a part of the kinase gene, such as BCR-ABL1 fusion in chronic myelogenous leukemia, EML4-ALK fusion in lung cancer, and ROS1 fusion in various cancers including lung cancer, It is known that a drug that often plays an essential role in carcinogenesis and suppresses its function is a very effective anticancer agent (Non-patent Document 1, Patent Document 1, and Patent Document 2).
For example, with the advent of tyrosine kinase inhibitors crizotinib and erlotinib, the relationship between molecular diagnosis and cancer treatment effect is being shown clinically, and by selecting patients for indication by molecular diagnosis, The concept of therapeutic drug administration is spreading.
　ＲＥＴ（Ret proto-oncogene）は、膜貫通受容体型チロシンキナーゼであり、ＧＤＮＦが結合して２量体化したＧＦＲ－ａｌｐｈａと、細胞外ドメインを介して結合し、活性化される。活性化は細胞外のＲＥＴのカドヘリン様ドメインを介して、ＲＥＴが２量体を形成し、また、細胞内チロシンキナーゼドメインの自己リン酸化が起こる（非特許文献３）。
　これまでに、異なるＲＥＴ融合遺伝子が原因であるがんが複数知られている。甲状腺乳頭がんにおけるＣＣＤＣ６－ＲＥＴ、ＮＣＯＡ４－ＲＥＴ、肺がんにおけるＫＩＦ５Ｂ－ＲＥＴ、ＣＣＤＣ６－ＲＥＴ、ＮＣＯＡ４－ＲＥＴ、慢性骨髄単球白血病におけるＢＣＲ－ＲＥＴ、ＦＧＦＲ１ＯＰ－ＲＥＴ等が知られている（非特許文献２、非特許文献３、非特許文献４、及び非特許文献５）。ＲＥＴ遺伝子と他の遺伝子の再構成により生じた融合体は、恒常的にキナーゼドメインがリン酸化状態にあり、ＭＡＰＫ経路等にシグナルを送り続けることにより、細胞のがん化に繋がる。 RET (Ret proto-oncogene) is a transmembrane receptor tyrosine kinase that binds to and activates GFR-alpha dimerized by binding to GDNF via the extracellular domain. Activation is via a cadherin-like domain of extracellular RET, and RET forms a dimer, and autophosphorylation of the intracellular tyrosine kinase domain occurs (Non-patent Document 3).
To date, multiple cancers caused by different RET fusion genes are known. CCDC6-RET, NCOA4-RET in papillary thyroid cancer, KIF5B-RET, CCDC6-RET, NCOA4-RET in lung cancer, BCR-RET, FGFR1OP-RET, etc. in chronic myelomonocytic leukemia are known (Non-patent literature) 2, Non-Patent Document 3, Non-Patent Document 4, and Non-Patent Document 5). The fusion product produced by the rearrangement of the RET gene and other genes has a kinase domain that is constantly phosphorylated, and continues to send signals to the MAPK pathway and the like, leading to cell canceration.
特許第４３０３３０３号公報Japanese Patent No. 4303303 ＷＯ２０１１／１６２２９５号パンフレットWO2011 / 162295 pamphlet
　本発明の課題は、がんの新たな原因因子である融合体（融合タンパク質及び融合遺伝子）を解明したことに基づき、融合タンパク質又は当該融合タンパク質をコードする融合遺伝子の検出方法、当該検出方法を用いたがんの診断方法、がん治療用医薬組成物の適用対象者の判定方法、前記検出方法のためのキット及びプライマーセット、前記融合タンパク質であるポリペプチドの活性及び／又は発現の阻害物質のスクリーニング方法、ならびに前記阻害物質を含有するがん治療用医薬組成物及び前記がん治療用医薬組成物を投与するがん治療方法を提供することにある。 An object of the present invention is to provide a detection method for a fusion protein or a fusion gene encoding the fusion protein, and a detection method based on the elucidation of a fusion (fusion protein and fusion gene) that is a new causative factor of cancer. Method for diagnosing cancer used, method for determining subject of application of pharmaceutical composition for cancer treatment, kit and primer set for detection method, inhibitor of activity and / or expression of polypeptide as fusion protein And a cancer therapeutic method comprising administering the pharmaceutical composition for cancer treatment containing the inhibitor and the pharmaceutical composition for cancer treatment.
　本発明者は、大腸がん患者から得た検体から、ＮＣＯＡ４遺伝子の一部と、キナーゼであるＲＥＴ遺伝子の一部とが融合した融合遺伝子、及び、ＲＵＦＹ１遺伝子の一部と、キナーゼであるＲＥＴ遺伝子の一部とが融合した新規の融合遺伝子を単離同定し（実施例１～３）、当該融合遺伝子が大腸がん患者検体に存在することを見出した（実施例４～５）。
　本発明者は、これらの知見から、ＲＥＴ融合タンパク質又は該融合タンパク質をコードする融合遺伝子の検出方法を提供し、そのためのキット及びプライマーセットを提供し、この融合タンパク質又は当該融合タンパク質をコードする融合遺伝子を検出することにより、ＲＥＴ阻害物質を用いた治療の対象となるがん患者を判別することを可能とし、当該がん患者にＲＥＴ阻害物質を投与する工程を含む、がんの治療方法を提供する。
　本発明者は、これらの知見から、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質又は該融合タンパク質をコードする融合遺伝子の検出方法を提供し、そのためのキット及びプライマーセットを提供し、この融合タンパク質又は当該融合タンパク質をコードする融合遺伝子を検出することにより、ＮＣＯＡ４又はＲＵＦＹ１阻害物質を用いた治療の対象となるがん患者を判別することを可能とし、当該がん患者にＮＣＯＡ４又はＲＵＦＹ１阻害物質を投与する工程を含む、がんの治療方法を提供する。 The present inventor has obtained, from a sample obtained from a colorectal cancer patient, a fusion gene in which a part of the NCOA4 gene and a part of the RET gene that is a kinase are fused, a part of the RUFY1 gene, and a RET that is a kinase. A novel fusion gene fused with a part of the gene was isolated and identified (Examples 1 to 3), and it was found that the fusion gene was present in colorectal cancer patients (Examples 4 to 5).
Based on these findings, the present inventor provides a method for detecting a RET fusion protein or a fusion gene encoding the fusion protein, and provides a kit and a primer set therefor, and the fusion protein encoding the fusion protein or the fusion protein. A method for treating cancer, comprising: detecting a gene, enabling determination of a cancer patient to be treated with a RET inhibitor, and administering the RET inhibitor to the cancer patient. provide.
Based on these findings, the present inventor provides an NCOA4 or RUFY1 fusion protein or a method for detecting a fusion gene encoding the fusion protein, provides a kit and a primer set therefor, and encodes the fusion protein or the fusion protein. Detecting a fusion gene that makes it possible to determine a cancer patient to be treated with an NCOA4 or RUFY1 inhibitor, and administering the NCOA4 or RUFY1 inhibitor to the cancer patient, Provide a method for treating cancer.
　本発明は、以下の発明に関する：
［１］被験者から得た試料中の、ＲＥＴ融合タンパク質又は該融合タンパク質をコードする融合遺伝子の検出方法。
［２］前記検出方法が、ＲＥＴタンパク質の切断、又は、ＲＥＴタンパク質をコードする遺伝子の切断、を検出する工程を含む、［１］に記載の検出方法。
［３］前記検出方法が、ＲＥＴタンパク質とそれ以外の他のタンパク質とから構築される融合タンパク質の存在、又は、前記融合タンパク質をコードする融合遺伝子の存在、を検出する工程を含む、［１］に記載の検出方法。
［４］前記融合タンパク質が、ＮＣＯＡ４又はＲＵＦＹ１タンパク質とＲＥＴタンパク質との融合タンパク質である、［１］～［３］のいずれかに記載の検出方法。
［５］前記融合タンパク質が、以下の（ａ）～（ｄ）からなる群から選択されるポリペプチドである、［１］～［４］のいずれかに記載の検出方法：
（ａ）配列番号２（ＮＣＯＡ４－ＲＥＴ）又は配列番号４（ＲＵＦＹ１－ＲＥＴ）で表されるアミノ酸配列からなるポリペプチド、
（ｂ）配列番号２又は配列番号４で表されるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、
（ｃ）配列番号２又は配列番号４で表されるアミノ酸配列との同一性が８０％以上であるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、及び
（ｄ）配列番号２又は配列番号４で表されるアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、及び／若しくは挿入されたアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド。
［６］前記ＲＥＴ融合遺伝子が、［５］に記載のポリペプチドをコードするポリヌクレオチドである、［１］～［５］のいずれかに記載の検出方法。
［７］前記融合遺伝子が、ＤＮＡ又はｍＲＮＡである、［１］～［６］のいずれかに記載の検出方法。
［８］前記試料が、消化器由来試料である、［１］～［７］のいずれかに記載の検出方法。
［９］前記消化器由来試料が、消化管由来試料である、［８］に記載の検出方法。
［１０］前記消化器由来試料が、下部消化管由来試料である、［８］に記載の検出方法。
［１１］前記消化器由来試料が、大腸由来試料である、［８］に記載の検出方法。
［１２］ＲＥＴ遺伝子５’末端側ゲノム領域を特異的に認識できる第１のプローブと、ＲＥＴ遺伝子３’末端側ゲノム領域を特異的に認識できる第２のプローブとを含む、ＲＥＴ融合遺伝子の検出用キット。
［１３］ＲＥＴ遺伝子と共にＲＥＴ融合遺伝子を構成する他の遺伝子の５’末端側ゲノム領域を特異的に認識できる第１のプローブと、ＲＥＴ遺伝子３’末端側ゲノム領域を特異的に認識できる第２のプローブとを含む、ＲＥＴ融合遺伝子の検出用キット。
［１４］ＲＥＴタンパク質をコードするポリヌクレオチドの、５’末端側領域を特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマー、ならびに、前記ポリヌクレオチドの３’末端側領域を特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマーを含む、ＲＥＴ融合遺伝子の検出用キット。
［１５］ＮＣＯＡ４又はＲＵＦＹ１タンパク質とＲＥＴタンパク質との融合タンパク質であるポリペプチドをコードするポリヌクレオチドを特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマーを含む、ＮＣＯＡ４－ＲＥＴ又はＲＵＦＹ１－ＲＥＴ融合遺伝子の検出用キット。
［１６］以下の（ａ）～（ｄ）からなる群から選択されるポリペプチドをコードするポリヌクレオチドを特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマーを含む、ＮＣＯＡ４－ＲＥＴ又はＲＵＦＹ１－ＲＥＴ融合遺伝子の検出用キット：
（ａ）配列番号２又は配列番号４で表されるアミノ酸配列からなるポリペプチド、
（ｂ）配列番号２又は配列番号４で表されるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、
（ｃ）配列番号２又は配列番号４で表されるアミノ酸配列との同一性が８０％以上であるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、及び
（ｄ）配列番号２又は配列番号４で表されるアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、及び／若しくは挿入されたアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド。
［１７］ＲＥＴタンパク質のＮ末端側領域を特異的に認識できる抗ＲＥＴ抗体、及び、ＲＥＴタンパク質のＣ末端側領域を特異的に認識できる抗ＲＥＴ抗体を含む、ＲＥＴ融合タンパク質の検出用キット。
［１８］ＲＥＴタンパク質と共にＲＥＴ融合タンパク質を構成する他のタンパク質のＮ末端側領域のポリペプチドに特異的に結合する抗体と、ＲＥＴタンパク質のＣ末端側領域のポリペプチドに特異的に結合する抗体を含む、ＲＥＴ融合タンパク質の検出用キット。
［１９］前記他のタンパク質が、ＮＣＯＡ４又はＲＵＦＹ１タンパク質である、［１８］に記載のキット。
［２０］ＲＥＴタンパク質をコードするポリヌクレオチド部分から設計されるアンチセンスプライマー及びＮＣＯＡ４又はＲＵＦＹ１タンパク質をコードするポリヌクレオチド部分から設計されるセンスプライマーを含む、ＮＣＯＡ４又はＲＵＦＹ１遺伝子とＲＥＴ遺伝子との融合遺伝子を検出するためのプライマーセットであって、アンチセンスプライマーは［１６］に記載のポリヌクレオチドにストリンジェントな条件下でアニールする核酸分子からなり、センスプライマーは［１６］に記載のポリヌクレオチドの相補鎖にストリンジェントな条件でアニールする核酸分子からなるプライマーセット。
［２１］ＮＣＯＡ４又はＲＵＦＹ１遺伝子とＲＥＴ遺伝子との融合遺伝子を検出するためのプライマーセットであって、配列番号１又は配列番号３に示される塩基配列からなるポリヌクレオチドにストリンジェントな条件下でアニールする核酸分子からなるアンチセンスプライマー及び該ポリヌクレオチドの相補鎖にストリンジェントな条件下でアニールする核酸分子からなるセンスプライマーを含むプライマーセット。
［２２］以下の（ａ）～（ｂ）からなる群から選択される、センスプライマー及びアンチセンスプライマー、を含む、プライマーセット：
（ａ）配列番号１の塩基番号１から１９８４の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドからなるセンスプライマー、及び配列番号１の塩基番号１９８５から５２７５の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドに対して相補的であるオリゴヌクレオチドからなるアンチセンスプライマー、及び、
（ｂ）配列番号３の塩基番号１から１９１７の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドからなるセンスプライマー、及び配列番号３の塩基番号１９１８から５２０８の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドに対して相補的であるオリゴヌクレオチドからなるアンチセンスプライマー。
［２３］（１）［５］に記載のポリペプチド、又は前記ポリペプチドを発現している細胞に試験物質を接触させる工程、
（２）前記ポリペプチドの活性及び／又は発現が阻害されるか否かを分析する工程、及び
（３）前記ポリペプチドの活性及び／又は発現を阻害する物質を選択する工程
を含む、前記ポリペプチドの活性及び／又は発現を阻害する物質をスクリーニングする方法。
［２４］前記ポリペプチドの活性及び／又は発現を阻害する物質が、ＲＥＴ融合体陽性のがんの治療剤である、［２３］に記載のスクリーニング方法。
［２５］前記がんが、消化器がんである、［２４］に記載のスクリーニング方法。
［２６］前記がんが、消化管がんである、［２５］に記載のスクリーニング方法。
［２７］前記がんが、下部消化管がんである、［２５］に記載のスクリーニング方法。
［２８］前記がんが、大腸がんである、［２５］に記載のスクリーニング方法。
［２９］ＲＥＴ融合タンパク質の活性及び／又は発現を阻害する物質を含有する、ＲＥＴ融合体陽性のがんの治療用医薬組成物。
［３０］前記ＲＥＴ融合タンパク質の活性及び／又は発現を阻害する物質が、キナーゼ阻害剤である、［２９］に記載の医薬組成物。
［３１］前記ＲＥＴ融合タンパク質が、［５］に記載のポリペプチドである、［２９］又は［３０］に記載の医薬組成物。
［３２］前記がんが、消化器がんである、［２９］～［３１］のいずれかに記載の医薬組成物。
［３３］前記がんが、消化管がんである、［３２］に記載の医薬組成物。
［３４］前記がんが、下部消化管がんである、［３２］に記載の医薬組成物。
［３５］前記がんが、大腸がんである、［３２］に記載の医薬組成物。
［３６］ＲＥＴ融合タンパク質。
［３７］ＮＣＯＡ４又はＲＵＦＹ１とＲＥＴとの融合タンパク質。
［３８］以下の（ａ）～（ｄ）からなる群から選択されるポリペプチドである、［３６］に記載の融合タンパク質：
（ａ）配列番号２又は配列番号４で表されるアミノ酸配列からなるポリペプチド、
（ｂ）配列番号２又は配列番号４で表されるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、
（ｃ）配列番号２又は配列番号４で表されるアミノ酸配列との同一性が８０％以上であるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、及び
（ｄ）配列番号２又は配列番号４で表されるアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、及び／若しくは挿入されたアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド。
［３９］［３６］～［３８］のいずれかに記載の融合タンパク質をコードするポリヌクレオチド。
［４０］［３９］に記載のポリヌクレオチドを含むベクター。
［４１］［４０］に記載のベクターで形質転換された細胞。
［４２］被験者から得た試料中の、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質又は該融合タンパク質をコードする融合遺伝子の検出方法。
［４３］前記検出方法が、ＮＣＯＡ４又はＲＵＦＹ１タンパク質の切断、又は、ＮＣＯＡ４又はＲＵＦＹ１タンパク質をコードする遺伝子の切断、を検出する工程を含む、［４２］に記載の検出方法。
［４４］前記検出方法が、ＮＣＯＡ４又はＲＵＦＹ１タンパク質とそれ以外の他のタンパク質とから構築される融合タンパク質の存在、又は、前記融合タンパク質をコードする融合遺伝子の存在、を検出する工程を含む、［４２］に記載の検出方法。
［４５］前記融合タンパク質が、ＮＣＯＡ４又はＲＵＦＹ１タンパク質とＲＥＴタンパク質との融合タンパク質である、［４２］～［４４］のいずれかに記載の検出方法。
［４６］前記融合タンパク質が、以下の（ａ）～（ｄ）からなる群から選択されるポリペプチドである、［４２］～［４５］のいずれかに記載の検出方法：
（ａ）配列番号２又は配列番号４で表されるアミノ酸配列からなるポリペプチド、
（ｂ）配列番号２又は配列番号４で表されるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、
（ｃ）配列番号２又は配列番号４で表されるアミノ酸配列との同一性が８０％以上であるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、及び
（ｄ）配列番号２又は配列番号４で表されるアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、及び／若しくは挿入されたアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド。
［４７］前記ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子が、［４６］に記載のポリペプチドをコードするポリヌクレオチドである、［４２］～［４６］のいずれかに記載の検出方法。
［４８］前記融合遺伝子が、ＤＮＡ又はｍＲＮＡである、［４２］～［４７］のいずれかに記載の検出方法。
［４９］前記試料が消化器由来試料である、［４２］～［４８］のいずれかに記載の検出方法。
［５０］前記消化器由来試料が、消化管由来試料である、［４９］に記載の検出方法。
［５１］前記消化器由来試料が、下部消化管由来試料である、［４９］に記載の検出方法。
［５２］前記消化器由来試料が、大腸由来試料である、［４９］に記載の検出方法。
［５３］ＮＣＯＡ４又はＲＵＦＹ１遺伝子５’末端側ゲノム領域を特異的に認識できる第１のプローブと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子３’末端側ゲノム領域を特異的に認識できる第２のプローブとを含む、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出用キット。
［５４］ＮＣＯＡ４又はＲＵＦＹ１遺伝子と共にＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を構成する他の遺伝子の３’末端側ゲノム領域を特異的に認識できる第１のプローブと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子５’末端側ゲノム領域を特異的に認識できる第２のプローブとを含む、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出用キット。
［５５］ＮＣＯＡ４又はＲＵＦＹ１タンパク質をコードするポリヌクレオチドの、５’末端側領域を特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマー、ならびに、前記ポリヌクレオチドの３’末端側領域を特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマーを含む、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出用キット。
［５６］ＮＣＯＡ４又はＲＵＦＹ１タンパク質とＲＥＴタンパク質との融合タンパク質であるポリペプチドをコードするポリヌクレオチドを特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマーを含む、ＮＣＯＡ４－ＲＥＴ又はＲＵＦＹ１－ＲＥＴ融合遺伝子の検出用キット。
［５７］以下の（ａ）～（ｄ）からなる群から選択されるポリペプチドをコードするポリヌクレオチドを特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマーを含む、ＮＣＯＡ４－ＲＥＴ又はＲＵＦＹ１－ＲＥＴ融合遺伝子の検出用キット：
（ａ）配列番号２又は配列番号４で表されるアミノ酸配列からなるポリペプチド、
（ｂ）配列番号２又は配列番号４で表されるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、
（ｃ）配列番号２又は配列番号４で表されるアミノ酸配列との同一性が８０％以上であるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、及び
（ｄ）配列番号２又は配列番号４で表されるアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、及び／若しくは挿入されたアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド。
［５８］ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＮ末端側領域を特異的に認識できる抗ＮＣＯＡ４又はＲＵＦＹ１抗体、及び、ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＣ末端側領域を特異的に認識できる抗ＮＣＯＡ４又はＲＵＦＹ１抗体を含む、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の検出用キット。
［５９］ＮＣＯＡ４又はＲＵＦＹ１タンパク質と共にＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を構成する他のタンパク質のＣ末端側領域のポリペプチドに特異的に結合する抗体と、ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＮ末端側領域のポリペプチドに特異的に結合する抗体を含む、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の検出用キット。
［６０］前記他のタンパク質が、ＲＥＴタンパク質である、［５９］に記載のキット。
［６１］ＲＥＴタンパク質をコードするポリヌクレオチド部分から設計されるアンチセンスプライマー及びＮＣＯＡ４又はＲＵＦＹ１タンパク質をコードするポリヌクレオチド部分から設計されるセンスプライマーを含む、ＲＥＴ遺伝子とＮＣＯＡ４又はＲＵＦＹ１遺伝子との融合遺伝子を検出するためのプライマーセットであって、アンチセンスプライマーは［５７］に記載のポリヌクレオチドにストリンジェントな条件下でアニールする核酸分子からなり、センスプライマーは［５７］に記載のポリヌクレオチドの相補鎖にストリンジェントな条件でアニールする核酸分子からなるプライマーセット。
［６２］ＮＣＯＡ４又はＲＵＦＹ１遺伝子とＲＥＴ遺伝子との融合遺伝子を検出するためのプライマーセットであって、配列番号１又は配列番号３に示される塩基配列からなるポリヌクレオチドにストリンジェントな条件下でアニールする核酸分子からなるアンチセンスプライマー及び該ポリヌクレオチドの相補鎖にストリンジェントな条件下でアニールする核酸分子からなるセンスプライマーを含むプライマーセット。
［６３］以下の（ａ）～（ｂ）からなる群から選択される、センスプライマー及びアンチセンスプライマー、を含む、プライマーセット：
（ａ）配列番号１の塩基番号１から１９８４の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドからなるセンスプライマー、及び配列番号１の塩基番号１９８５から５２７５の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドに対して相補的であるオリゴヌクレオチドからなるアンチセンスプライマー、及び、
（ｂ）配列番号３の塩基番号１から１９１７の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドからなるセンスプライマー、及び配列番号３の塩基番号１９１８から５２０８の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドに対して相補的であるオリゴヌクレオチドからなるアンチセンスプライマー。
［６４］（１）［４６］に記載のポリペプチド、又は前記ポリペプチドを発現している細胞に試験物質を接触させる工程、
（２）前記ポリペプチドの活性及び／又は発現が阻害されるか否かを分析する工程、及び
（３）前記ポリペプチドの活性及び／又は発現を阻害する物質を選択する工程
を含む、前記ポリペプチドの活性及び／又は発現を阻害する物質をスクリーニングする方法。
［６５］前記ポリペプチドの活性及び／又は発現を阻害する物質が、ＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがんの治療剤である、［６４］に記載のスクリーニング方法。
［６６］前記がんが、消化器がんである、［６５］に記載のスクリーニング方法。
［６７］前記がんが、消化管がんである、［６６］に記載のスクリーニング方法。
［６８］前記がんが、下部消化管がんである、［６６］に記載のスクリーニング方法。
［６９］前記がんが、大腸がんである、［６６］に記載のスクリーニング方法。
［７０］ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の活性及び／又は発現を阻害する物質を含有する、ＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがんの治療用医薬組成物。
［７１］前記ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の活性及び／又は発現を阻害する物質が、キナーゼ阻害剤である、［７０］に記載の医薬組成物。
［７２］前記ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が、［４６］に記載のポリペプチドである、［７０］又は［７１］に記載の医薬組成物。
［７３］前記がんが、消化器がんである、［７０］～［７２］のいずれかに記載の医薬組成物。
［７４］前記がんが、消化管がんである、［７３］に記載の医薬組成物。
［７５］前記がんが、下部消化管がんである、［７３］に記載の医薬組成物。
［７６］前記がんが、大腸がんである、［７３］に記載の医薬組成物。
［７７］ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質。
［７８］ＮＣＯＡ４又はＲＵＦＹ１とＲＥＴとの融合タンパク質。
［７９］以下の（ａ）～（ｄ）からなる群から選択されるポリペプチドである、［７７］に記載の融合タンパク質：
（ａ）配列番号２又は配列番号４で表されるアミノ酸配列からなるポリペプチド、
（ｂ）配列番号２又は配列番号４で表されるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、
（ｃ）配列番号２又は配列番号４で表されるアミノ酸配列との同一性が８０％以上であるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、及び
（ｄ）配列番号２又は配列番号４で表されるアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、及び／若しくは挿入されたアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド。
［８０］［７７］～［７９］のいずれかに記載の融合タンパク質をコードするポリヌクレオチド。
［８１］［８０］に記載のポリヌクレオチドを含むベクター。
［８２］［８１］に記載のベクターで形質転換された細胞。
［８３］ＲＥＴ融合タンパク質の活性及び／又は発現を阻害する物質が、キナーゼ阻害剤である、ＲＥＴ融合体陽性のがんの治療方法。
［８４］ＲＥＴ融合タンパク質の活性及び／又は発現を阻害する物質の、ＲＥＴ融合体陽性のがんの治療用医薬組成物の製造への使用。
［８５］ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の活性及び／又は発現を阻害する物質が、キナーゼ阻害剤である、ＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがんの治療方法。
［８６］ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の活性及び／又は発現を阻害する物質の、ＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがんの治療用医薬組成物の製造への使用。 The present invention relates to the following inventions:
[1] A method for detecting a RET fusion protein or a fusion gene encoding the fusion protein in a sample obtained from a subject.
[2] The detection method according to [1], wherein the detection method includes a step of detecting cleavage of the RET protein or cleavage of a gene encoding the RET protein.
[3] The detection method includes a step of detecting the presence of a fusion protein constructed from a RET protein and another protein or the presence of a fusion gene encoding the fusion protein. [1] The detection method according to.
[4] The detection method according to any one of [1] to [3], wherein the fusion protein is a fusion protein of NCOA4 or RUFI1 protein and RET protein.
[5] The detection method according to any one of [1] to [4], wherein the fusion protein is a polypeptide selected from the group consisting of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (NCOA4-RET) or SEQ ID NO: 4 (RUFY1-RET),
(B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity,
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity; and (d) SEQ ID NO: 2 or SEQ ID NO: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted in the amino acid sequence represented by 4, and having tumorigenic potential.
[6] The detection method according to any one of [1] to [5], wherein the RET fusion gene is a polynucleotide encoding the polypeptide according to [5].
[7] The detection method according to any one of [1] to [6], wherein the fusion gene is DNA or mRNA.
[8] The detection method according to any one of [1] to [7], wherein the sample is a digestive organ-derived sample.
[9] The detection method according to [8], wherein the digestive organ-derived sample is a digestive tract-derived sample.
[10] The detection method according to [8], wherein the digestive organ-derived sample is a lower digestive tract-derived sample.
[11] The detection method according to [8], wherein the digestive organ-derived sample is a large intestine-derived sample.
[12] Detection of a RET fusion gene comprising a first probe capable of specifically recognizing the RET gene 5 ′ terminal genomic region and a second probe capable of specifically recognizing the RET gene 3 ′ terminal genomic region For kit.
[13] A first probe capable of specifically recognizing the 5′-terminal genomic region of another gene that constitutes the RET fusion gene together with the RET gene, and a second probe capable of specifically recognizing the RET gene 3′-terminal genomic region And a probe for detecting a RET fusion gene.
[14] A sense primer and an antisense primer designed to specifically amplify the 5 ′ end region of the polynucleotide encoding the RET protein, and specifically amplify the 3 ′ end region of the polynucleotide. A kit for detecting a RET fusion gene, comprising a sense primer and an antisense primer designed to be able to.
[15] An NCOA4-RET or RUFY1-RET fusion comprising a sense primer and an antisense primer designed to specifically amplify a polynucleotide encoding a polypeptide that is a fusion protein of NCOA4 or RUFY1 protein and RET protein Gene detection kit.
[16] NCOA4-RET or RUFY1 comprising a sense primer and an antisense primer designed to specifically amplify a polynucleotide encoding a polypeptide selected from the group consisting of the following (a) to (d) -RET fusion gene detection kit:
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4,
(B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity,
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity; and (d) SEQ ID NO: 2 or SEQ ID NO: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted in the amino acid sequence represented by 4, and having tumorigenic potential.
[17] A kit for detecting a RET fusion protein comprising an anti-RET antibody capable of specifically recognizing the N-terminal region of the RET protein and an anti-RET antibody capable of specifically recognizing the C-terminal region of the RET protein.
[18] An antibody that specifically binds to a polypeptide in the N-terminal region of another protein that constitutes the RET fusion protein together with the RET protein, and an antibody that specifically binds to a polypeptide in the C-terminal region of the RET protein A kit for detecting a RET fusion protein.
[19] The kit according to [18], wherein the other protein is NCOA4 or RUFI1 protein.
[20] A fusion gene between an NCOA4 or RUFY1 gene and an RET gene, comprising an antisense primer designed from a polynucleotide portion encoding a RET protein and a sense primer designed from a polynucleotide portion encoding an NCOA4 or RUFY1 protein A primer set for detection, wherein the antisense primer comprises a nucleic acid molecule that anneals to the polynucleotide of [16] under stringent conditions, and the sense primer is a complementary strand of the polynucleotide of [16] Primer set consisting of nucleic acid molecules that anneal under stringent conditions.
[21] A primer set for detecting a fusion gene of NCOA4 or RUFY1 gene and RET gene, which anneals to a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions A primer set comprising an antisense primer comprising a nucleic acid molecule and a sense primer comprising a nucleic acid molecule which anneals to a complementary strand of the polynucleotide under stringent conditions.
[22] A primer set comprising a sense primer and an antisense primer selected from the group consisting of the following (a) to (b):
(A) a sense primer composed of any consecutive at least 16 base oligonucleotides between base numbers 1 to 1984 of SEQ ID NO: 1, and any continuous at least 16 bases between base numbers 1985 to 5275 of SEQ ID NO: 1 An antisense primer consisting of an oligonucleotide that is complementary to the oligonucleotide of
(B) a sense primer consisting of an oligonucleotide of at least 16 consecutive bases between base numbers 1 to 1917 of SEQ ID NO: 3 and at least 16 consecutive bases of base numbers 1918 to 5208 of SEQ ID NO: 3 An antisense primer comprising an oligonucleotide that is complementary to the oligonucleotide.
[23] A step of bringing the test substance into contact with the polypeptide according to (1) [5] or a cell expressing the polypeptide,
(2) analyzing whether or not the activity and / or expression of the polypeptide is inhibited; and (3) selecting the substance that inhibits the activity and / or expression of the polypeptide. A method of screening for a substance that inhibits the activity and / or expression of a peptide.
[24] The screening method according to [23], wherein the substance that inhibits the activity and / or expression of the polypeptide is a therapeutic agent for RET fusion-positive cancer.
[25] The screening method according to [24], wherein the cancer is digestive cancer.
[26] The screening method according to [25], wherein the cancer is gastrointestinal cancer.
[27] The screening method according to [25], wherein the cancer is cancer of the lower gastrointestinal tract.
[28] The screening method according to [25], wherein the cancer is colon cancer.
[29] A pharmaceutical composition for treating RET fusion-positive cancer, comprising a substance that inhibits the activity and / or expression of a RET fusion protein.
[30] The pharmaceutical composition according to [29], wherein the substance that inhibits the activity and / or expression of the RET fusion protein is a kinase inhibitor.
[31] The pharmaceutical composition according to [29] or [30], wherein the RET fusion protein is the polypeptide according to [5].
[32] The pharmaceutical composition according to any of [29] to [31], wherein the cancer is gastrointestinal cancer.
[33] The pharmaceutical composition according to [32], wherein the cancer is gastrointestinal cancer.
[34] The pharmaceutical composition according to [32], wherein the cancer is cancer of the lower gastrointestinal tract.
[35] The pharmaceutical composition according to [32], wherein the cancer is colorectal cancer.
[36] RET fusion protein.
[37] A fusion protein of NCOA4 or RUFI1 and RET.
[38] The fusion protein according to [36], which is a polypeptide selected from the group consisting of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4,
(B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity,
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity; and (d) SEQ ID NO: 2 or SEQ ID NO: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted in the amino acid sequence represented by 4, and having tumorigenic potential.
[39] A polynucleotide encoding the fusion protein according to any one of [36] to [38].
[40] A vector comprising the polynucleotide according to [39].
[41] A cell transformed with the vector according to [40].
[42] A method for detecting an NCOA4 or RUFY1 fusion protein or a fusion gene encoding the fusion protein in a sample obtained from a subject.
[43] The detection method according to [42], wherein the detection method includes a step of detecting cleavage of the NCOA4 or RUFY1 protein or cleavage of a gene encoding the NCOA4 or RUFY1 protein.
[44] The detection method includes a step of detecting the presence of a fusion protein constructed from an NCOA4 or RUFY1 protein and another protein, or the presence of a fusion gene encoding the fusion protein. 42].
[45] The detection method according to any of [42] to [44], wherein the fusion protein is a fusion protein of NCOA4 or RUFI1 protein and RET protein.
[46] The detection method according to any of [42] to [45], wherein the fusion protein is a polypeptide selected from the group consisting of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4,
(B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity,
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity; and (d) SEQ ID NO: 2 or SEQ ID NO: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted in the amino acid sequence represented by 4, and having tumorigenic potential.
[47] The detection method according to any of [42] to [46], wherein the NCOA4 or RUFI1 fusion gene is a polynucleotide encoding the polypeptide according to [46].
[48] The detection method according to any one of [42] to [47], wherein the fusion gene is DNA or mRNA.
[49] The detection method according to any of [42] to [48], wherein the sample is a digestive organ-derived sample.
[50] The detection method according to [49], wherein the digestive organ-derived sample is a digestive tract-derived sample.
[51] The detection method according to [49], wherein the digestive organ-derived sample is a lower digestive tract-derived sample.
[52] The detection method according to [49], wherein the digestive organ-derived sample is a large intestine-derived sample.
[53] An NCOA4 comprising a first probe capable of specifically recognizing the NCOA4 or RUfy1 gene 5 ′ terminal genomic region and a second probe capable of specifically recognizing the NCOA4 or RUfy1 gene 3 ′ terminal genomic region. Alternatively, a kit for detecting the RUFY1 fusion gene.
[54] A first probe capable of specifically recognizing the 3'-terminal genomic region of another gene that constitutes the NCOA4 or RUFY1 fusion gene together with the NCOA4 or RUFY1 gene, and the NCOA4 or RUFY1 gene 5'-terminal genomic region specific A kit for detecting an NCOA4 or RUFY1 fusion gene, comprising a second probe that can be recognized visually.
[55] A sense primer and an antisense primer designed to specifically amplify the 5 ′ end region of the polynucleotide encoding NCOA4 or RUFY1 protein, and the 3 ′ end region of the polynucleotide specifically A kit for detecting an NCOA4 or RUFY1 fusion gene, comprising a sense primer and an antisense primer designed so that they can be amplified.
[56] NCOA4-RET or RUFY1-RET fusion comprising a sense primer and an antisense primer designed to specifically amplify a polynucleotide encoding a polypeptide that is a fusion protein of NCOA4 or RUFY1 protein and RET protein Gene detection kit.
[57] NCOA4-RET or RUFY1 comprising a sense primer and an antisense primer designed to specifically amplify a polynucleotide encoding a polypeptide selected from the group consisting of the following (a) to (d) -RET fusion gene detection kit:
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4,
(B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity,
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity; and (d) SEQ ID NO: 2 or SEQ ID NO: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted in the amino acid sequence represented by 4, and having tumorigenic potential.
[58] An NCOA4 comprising an anti-NCOA4 or RUFY1 antibody capable of specifically recognizing the N-terminal region of the NCOA4 or RUFY1 protein, and an anti-NCOA4 or RUFY1 antibody capable of specifically recognizing the C-terminal region of the NCOA4 or RUFY1 protein Alternatively, a kit for detecting RUFY1 fusion protein.
[59] An antibody that specifically binds to a polypeptide in the C-terminal region of another protein that constitutes an NCOA4 or RUFY1 fusion protein together with the NCOA4 or RUFY1 protein, and specific to a polypeptide in the N-terminal region of the NCOA4 or RUFY1 protein Kit for detection of NCOA4 or RUFY1 fusion protein comprising an antibody that binds selectively.
[60] The kit according to [59], wherein the other protein is a RET protein.
[61] A fusion gene of an RET gene and an NCOA4 or RUFY1 gene, comprising an antisense primer designed from a polynucleotide portion encoding a RET protein and a sense primer designed from a polynucleotide portion encoding an NCOA4 or RUFY1 protein A primer set for detection, wherein the antisense primer comprises a nucleic acid molecule that anneals to the polynucleotide described in [57] under stringent conditions, and the sense primer is a complementary strand of the polynucleotide described in [57] Primer set consisting of nucleic acid molecules that anneal under stringent conditions.
[62] A primer set for detecting a fusion gene of NCOA4 or RUFY1 gene and RET gene, which anneals to a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions A primer set comprising an antisense primer comprising a nucleic acid molecule and a sense primer comprising a nucleic acid molecule which anneals to a complementary strand of the polynucleotide under stringent conditions.
[63] A primer set comprising a sense primer and an antisense primer selected from the group consisting of the following (a) to (b):
(A) a sense primer composed of any consecutive at least 16 base oligonucleotides between base numbers 1 to 1984 of SEQ ID NO: 1, and any continuous at least 16 bases between base numbers 1985 to 5275 of SEQ ID NO: 1 An antisense primer consisting of an oligonucleotide that is complementary to the oligonucleotide of
(B) a sense primer consisting of an oligonucleotide of at least 16 consecutive bases between base numbers 1 to 1917 of SEQ ID NO: 3 and at least 16 consecutive bases of base numbers 1918 to 5208 of SEQ ID NO: 3 An antisense primer comprising an oligonucleotide that is complementary to the oligonucleotide.
[64] The step of bringing the test substance into contact with the polypeptide according to (1) [46] or a cell expressing the polypeptide,
(2) analyzing whether or not the activity and / or expression of the polypeptide is inhibited; and (3) selecting the substance that inhibits the activity and / or expression of the polypeptide. A method of screening for a substance that inhibits the activity and / or expression of a peptide.
[65] The screening method according to [64], wherein the substance that inhibits the activity and / or expression of the polypeptide is a therapeutic agent for cancer positive for NCOA4 or RUFY1 fusion.
[66] The screening method according to [65], wherein the cancer is digestive organ cancer.
[67] The screening method according to [66], wherein the cancer is gastrointestinal cancer.
[68] The screening method according to [66], wherein the cancer is cancer of the lower gastrointestinal tract.
[69] The screening method according to [66], wherein the cancer is colon cancer.
[70] A pharmaceutical composition for treating NCOA4 or RUFY1 fusion-positive cancer, comprising a substance that inhibits the activity and / or expression of the NCOA4 or RUFY1 fusion protein.
[71] The pharmaceutical composition according to [70], wherein the substance that inhibits the activity and / or expression of the NCOA4 or RUfy1 fusion protein is a kinase inhibitor.
[72] The pharmaceutical composition according to [70] or [71], wherein the NCOA4 or RUfy1 fusion protein is the polypeptide according to [46].
[73] The pharmaceutical composition according to any of [70] to [72], wherein the cancer is gastrointestinal cancer.
[74] The pharmaceutical composition according to [73], wherein the cancer is gastrointestinal cancer.
[75] The pharmaceutical composition according to [73], wherein the cancer is cancer of the lower gastrointestinal tract.
[76] The pharmaceutical composition according to [73], wherein the cancer is colon cancer.
[77] NCOA4 or RUFI1 fusion protein.
[78] A fusion protein of NCOA4 or RUFI1 and RET.
[79] The fusion protein according to [77], which is a polypeptide selected from the group consisting of the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4,
(B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity,
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity; and (d) SEQ ID NO: 2 or SEQ ID NO: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted in the amino acid sequence represented by 4, and having tumorigenic potential.
[80] A polynucleotide encoding the fusion protein according to any one of [77] to [79].
[81] A vector comprising the polynucleotide according to [80].
[82] A cell transformed with the vector according to [81].
[83] A method for treating RET fusion-positive cancer, wherein the substance that inhibits the activity and / or expression of a RET fusion protein is a kinase inhibitor.
[84] Use of a substance that inhibits the activity and / or expression of a RET fusion protein in the manufacture of a pharmaceutical composition for treating RET fusion-positive cancer.
[85] A method for treating NCOA4 or RUFY1 fusion-positive cancer, wherein the substance that inhibits the activity and / or expression of the NCOA4 or RUFY1 fusion protein is a kinase inhibitor.
[86] Use of a substance that inhibits the activity and / or expression of an NCOA4 or RUFY1 fusion protein in the manufacture of a pharmaceutical composition for treating an NCOA4 or RUFY1 fusion-positive cancer.
　本発明の検出方法は、ＲＥＴ融合体陽性のがん（特に消化器がん）を検出する方法として利用できる。また、本発明の検出方法によれば、被験者におけるＲＥＴ融合体陽性のがんを診断することができ、更に、ＲＥＴ阻害物質の適用対象者であるか否かを判定することができる。本発明の検出用キット及びプライマーセットは、本発明の検出方法に用いることができる。更に、本発明の阻害物質スクリーニング方法によれば、当該融合体陽性のがん患者の治療に有効な物質をスクリーニングすることができる。前記スクリーニング方法により得られた物質は、ＲＥＴ融合体陽性のがんの治療用医薬組成物の有効成分として使用することができ、また、ＲＥＴ融合体陽性のがんの治療に用いることができる。
　本発明の検出方法は、ＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがん（特に消化器がん）を検出する方法として利用できる。また、本発明の検出方法によれば、被験者におけるＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがんを診断することができ、更に、ＮＣＯＡ４又はＲＵＦＹ１阻害物質の適用対象者であるか否かを判定することができる。本発明の検出用キット及びプライマーセットは、本発明の検出方法に用いることができる。更に、本発明の阻害物質スクリーニング方法によれば、当該融合体陽性のがん患者の治療に有効な物質をスクリーニングすることができる。前記スクリーニング方法により得られた物質は、ＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがんの治療用医薬組成物の有効成分として使用することができ、また、ＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがんの治療に用いることができる。 The detection method of the present invention can be used as a method for detecting RET fusion-positive cancer (particularly digestive organ cancer). In addition, according to the detection method of the present invention, it is possible to diagnose RET fusion-positive cancer in a subject, and it is possible to determine whether or not the subject is an application subject of a RET inhibitor. The detection kit and primer set of the present invention can be used in the detection method of the present invention. Furthermore, according to the inhibitor screening method of the present invention, a substance effective for the treatment of the fusion-positive cancer patient can be screened. The substance obtained by the screening method can be used as an active ingredient of a pharmaceutical composition for the treatment of RET fusion-positive cancer, and can also be used for the treatment of RET fusion-positive cancer.
The detection method of the present invention can be used as a method for detecting NCOA4 or RUFY1 fusion-positive cancer (particularly digestive organ cancer). Further, according to the detection method of the present invention, NCOA4 or RUFY1 fusion-positive cancer in a subject can be diagnosed, and further, it is determined whether or not the subject is an application target of an NCOA4 or RUFY1 inhibitor. it can. The detection kit and primer set of the present invention can be used in the detection method of the present invention. Furthermore, according to the inhibitor screening method of the present invention, a substance effective for the treatment of the fusion-positive cancer patient can be screened. The substance obtained by the screening method can be used as an active ingredient of a pharmaceutical composition for treating NCOA4 or RUFY1 fusion-positive cancer, and is also used for treating NCOA4 or RUFY1 fusion-positive cancer. be able to.
ＮＩＨ３Ｔ３繊維芽細胞に融合遺伝子ＲＵＦＹ１－ＲＥＴを導入し、７日間培養後の状態を示す、図面に代わる顕微鏡写真である。FIG. 5 is a photomicrograph in place of a drawing showing the state after introducing the fusion gene RUFY1-RET into NIH3T3 fibroblasts and culturing for 7 days. 融合遺伝子ＲＵＦＹ１－ＲＥＴを導入した３Ｔ３繊維芽細胞を皮下に接種したヌードマウスにおける、接種後１乃至８日後の腫瘍サイズの経時的変化を示すグラフである。3 is a graph showing changes over time in tumor size 1 to 8 days after inoculation in nude mice inoculated subcutaneously with 3T3 fibroblasts introduced with the fusion gene RUFY1-RET. ＲＵＦＹ１－ＲＥＴ融合ポリペプチドを発現するＢａ／Ｆ３細胞におけるＲＥＴ阻害剤（Vandetanib）に対する感受性を示すグラフである。It is a graph which shows the sensitivity with respect to the RET inhibitor (Vandetanib) in Ba / F3 cell which expresses RUYY1-RET fusion polypeptide. ＲＵＦＹ１－ＲＥＴ融合ポリペプチドを発現するＢａ／Ｆ３細胞におけるＲＥＴ阻害剤（Alectinib）に対する感受性を示すグラフである。It is a graph which shows the sensitivity with respect to the RET inhibitor (Alectinib) in Ba / F3 cell which expresses a RUFUY1-RET fusion polypeptide. ＲＵＦＹ１－ＲＥＴ融合ポリペプチドを発現するＢａ／Ｆ３細胞におけるＲＥＴ阻害剤（Lenvatinib）に対する感受性を示すグラフである。It is a graph which shows the sensitivity with respect to the RET inhibitor (Lenvatinib) in Ba / F3 cell which expresses a RUFUY1-RET fusion polypeptide. ＲＵＦＹ１－ＲＥＴ融合ポリペプチドを発現するＢａ／Ｆ３細胞におけるＲＥＴ阻害剤（Cabozantinib）に対する感受性を示すグラフである。It is a graph which shows the sensitivity with respect to the RET inhibitor (Cabozantinib) in Ba / F3 cell which expresses RUYY1-RET fusion polypeptide. ＲＵＦＹ１－ＲＥＴ融合ポリペプチドを発現するＢａ／Ｆ３細胞を各ＲＥＴ阻害剤で処理した後、各培養細胞由来の抽出物のウェスタンブロッティングを実施した結果を示す、図面に代わる写真である。FIG. 5 is a photograph, instead of a drawing, showing the results of Western blotting of extracts from each cultured cell after treating Ba / F3 cells expressing a RUFY1-RET fusion polypeptide with each RET inhibitor.
≪定義等≫
　＜融合点＞
　本明細書における「ＲＥＴ融合遺伝子における融合点」とは、ＲＥＴ融合遺伝子における、ＲＥＴ遺伝子由来のポリヌクレオチドと、ＲＥＴ遺伝子と共に融合遺伝子を構築する他の遺伝子由来のポリヌクレオチドとが結合した箇所を意味する。
　本明細書における「ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子における融合点」とは、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子における、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来のポリヌクレオチドと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子と共に融合遺伝子を構築する他の遺伝子由来のポリヌクレオチドとが結合した箇所を意味する。 <Definitions, etc.>
<Fusion point>
In the present specification, the “fusion point in the RET fusion gene” means a portion of the RET fusion gene where a polynucleotide derived from the RET gene and a polynucleotide derived from another gene constructing the fusion gene together with the RET gene are combined. To do.
In the present specification, the “fusion point in the NCOA4 or RUFY1 fusion gene” refers to a polynucleotide derived from the NCOA4 or RUFY1 gene in the NCOA4 or RUFY1 fusion gene and a polymorphism derived from another gene that constructs the fusion gene together with the NCOA4 or RUFY1 gene. It means the position where nucleotides are bound.
　例えば、ＲＥＴ融合遺伝子、あるいは、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子が、配列番号１で表されるＮＣＯＡ４－ＲＥＴ融合遺伝子（ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２）の場合、前記融合点とは、ＮＣＯＡ４遺伝子由来のポリヌクレオチドの３’末端の塩基（第１９８４番）と、ＲＥＴ遺伝子由来のポリヌクレオチドの５’末端の塩基（第１９８５番）との結合した箇所（第１９８４／１９８５番）である。
　また、ＲＥＴ融合遺伝子、あるいは、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子が、配列番号３で表されるＲＵＦＹ１－ＲＥＴ融合遺伝子（ＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２）の場合、前記融合点とは、ＲＵＦＹ１遺伝子由来のポリヌクレオチドの３’末端の塩基（第１９１７番）と、ＲＥＴ遺伝子由来のポリヌクレオチドの５’末端の塩基（第１９１８番）との結合した箇所（第１９１７／１９１８番）である。 For example, when the RET fusion gene, or the NCOA4 or RUFY1 fusion gene is the NCOA4-RET fusion gene represented by SEQ ID NO: 1 (NCOA4ex9-RETex12), the fusion point is the 3 ′ of the polynucleotide derived from the NCOA4 gene. This is the position (1984/1985) where the terminal base (No. 1984) and the 5 ′ terminal base (No. 1985) of the polynucleotide derived from the RET gene are combined.
In the case where the RET fusion gene or the NCOA4 or RUFY1 fusion gene is a RUFY1-RET fusion gene represented by SEQ ID NO: 3 (RUFY1ex16-RETex12), the fusion point is the 3 ′ of a polynucleotide derived from the RUFY1 gene. This is the position (No. 1917/1918) where the end base (No. 1917) and the 5 ′ end base (No. 1918) of the polynucleotide derived from the RET gene are combined.
　本明細書における「ＲＥＴ融合タンパク質における融合点」とは、ＲＥＴ融合タンパク質における、ＲＥＴ遺伝子由来のポリヌクレオチドにコードされるポリペプチドと、ＲＥＴ遺伝子と共に融合遺伝子を構築する他の遺伝子由来のポリヌクレオチドにコードされるポリペプチドとが結合した箇所を意味する。
　本明細書における「ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質における融合点」とは、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質における、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来のポリヌクレオチドにコードされるポリペプチドと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子と共に融合遺伝子を構築する他の遺伝子由来のポリヌクレオチドにコードされるポリペプチドとが結合した箇所を意味する。 The term “fusion point in a RET fusion protein” as used herein refers to a polypeptide encoded by a polynucleotide derived from a RET gene and a polynucleotide derived from another gene that constructs a fusion gene together with the RET gene. It means the place where the encoded polypeptide is bound.
In the present specification, “the fusion point in the NCOA4 or RUFY1 fusion protein” refers to constructing a fusion gene together with the polypeptide encoded by the polynucleotide derived from the NCOA4 or RUFY1 gene in the NCOA4 or RUFY1 fusion protein and the NCOA4 or RUFY1 gene. It means a portion where a polypeptide encoded by a polynucleotide derived from another gene is bound.
　例えば、ＲＥＴ融合タンパク質、あるいは、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が、配列番号２で表されるＮＣＯＡ４－ＲＥＴ融合タンパク質の場合、前記融合点とは、ＮＣＯＡ４タンパク質由来のポリペプチドのＣ末端のアミノ酸（第６１３番）と、ＲＥＴタンパク質由来のポリペプチドのＮ末端のアミノ酸（第６１４番）との結合した箇所（第６１３／６１４番）である。
　また、ＲＥＴ融合タンパク質、あるいは、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が、配列番号４で表されるＲＵＦＹ１－ＲＥＴ融合タンパク質の場合、前記融合点とは、ＲＵＦＹ１タンパク質由来のポリペプチドのＣ末端のアミノ酸（第６３５番）と、ＲＥＴタンパク質由来のポリペプチドのＮ末端のアミノ酸（第６３６番）との結合した箇所（第６３５／６３６番）である。 For example, when the RET fusion protein, or the NCOA4 or RUFY1 fusion protein is the NCOA4-RET fusion protein represented by SEQ ID NO: 2, the fusion point is the C-terminal amino acid (613) of the polypeptide derived from the NCOA4 protein. No.) and the N-terminal amino acid (No. 614) of the polypeptide derived from the RET protein (No. 613/614).
In the case where the RET fusion protein or the NCOA4 or RUFY1 fusion protein is the RUFY1-RET fusion protein represented by SEQ ID NO: 4, the fusion point is the C-terminal amino acid (635th amino acid of the RUFY1 protein-derived polypeptide). No.) and the N-terminal amino acid (No. 636) of the polypeptide derived from the RET protein (No. 636/636).
　＜ＲＥＴ遺伝子又はＲＥＴタンパク質の切断＞
　更に、本明細書において、「ＲＥＴ遺伝子の切断」又は「ＲＥＴ遺伝子が切断されている」とは、遺伝子の転座又は逆位等によりＲＥＴ遺伝子の連続性が失われている状態、すなわち、ＲＥＴ遺伝子がＲＥＴキナーゼ領域を含むポリヌクレオチドと、それ以外のポリヌクレオチドと、の少なくとも２つのポリヌクレオチドとに分かれている状態を指す。なお、ＲＥＴ遺伝子の切断点（break point）は、ＲＥＴ遺伝子が切断されてできたポリヌクレオチドの少なくとも一つがコードするタンパク質がＲＥＴキナーゼ活性を保持する範囲で限定されない。
　また、「ＲＥＴ遺伝子以外の他の遺伝子の切断」又は「ＲＥＴ遺伝子以外の他の遺伝子が切断されている」とは、遺伝子の転座又は逆位等により他の遺伝子の連続性が失われている状態、すなわち、他の遺伝子が少なくとも２つのポリヌクレオチドに分かれている状態を指す。 <Cleavage of RET gene or RET protein>
Furthermore, in the present specification, “RET gene cleavage” or “RET gene cleavage” means a state in which continuity of the RET gene is lost due to translocation or inversion of the gene, ie, RET It refers to a state where the gene is divided into at least two polynucleotides, a polynucleotide containing a RET kinase region and a polynucleotide other than that. The break point of the RET gene is not limited as long as the protein encoded by at least one of the polynucleotides formed by cleaving the RET gene retains the RET kinase activity.
In addition, “cleaving of a gene other than the RET gene” or “a gene other than the RET gene is cleaved” means that the continuity of other genes is lost due to translocation or inversion of the gene. A state in which another gene is divided into at least two polynucleotides.
　また、本明細書において、「ＲＥＴタンパク質の切断」又は「ＲＥＴタンパク質が切断されている」とは、ＲＥＴ遺伝子が、前述のように切断されている状態であることに基づき、ＲＥＴタンパク質の連続性が失われている状態、すなわち、ＲＥＴタンパク質がＲＥＴキナーゼ領域を含むポリペプチドと、それ以外のポリペプチドと、の少なくとも２つのポリペプチドに分かれている状態を指す。なお、ＲＥＴタンパク質の切断点は、ＲＥＴタンパク質が切断されてできたポリペプチドの少なくとも一つがＲＥＴキナーゼ活性を保持する範囲で限定されない。
　また、「ＲＥＴタンパク質以外の他のタンパク質の切断」又は「ＲＥＴタンパク質以外の他のタンパク質が切断されている」とは、他の遺伝子が、前述のように切断されている状態であることに基づき、他のタンパク質の連続性が失われている状態、すなわち、他のタンパク質が少なくとも２つのポリペプチドに分かれている状態を指す。 In the present specification, “RET protein cleavage” or “RET protein is cleaved” means that the RET gene is cleaved as described above based on the RET gene being cleaved as described above. Is a state in which the RET protein is divided into at least two polypeptides: a polypeptide containing the RET kinase region and another polypeptide. The cleavage point of the RET protein is not limited as long as at least one of the polypeptides formed by cleaving the RET protein retains the RET kinase activity.
Further, “cleaving of a protein other than the RET protein” or “a protein other than the RET protein is cleaved” is based on the fact that the other gene is cleaved as described above. , Refers to a state in which the continuity of other proteins is lost, that is, a state in which other proteins are separated into at least two polypeptides.
　＜ＮＣＯＡ４又はＲＵＦＹ１遺伝子又はＮＣＯＡ４又はＲＵＦＹ１タンパク質の切断＞
　更に、本明細書において、「ＮＣＯＡ４又はＲＵＦＹ１遺伝子の切断」又は「ＮＣＯＡ４又はＲＵＦＹ１遺伝子が切断されている」とは、遺伝子の転座又は逆位等によりＮＣＯＡ４又はＲＵＦＹ１遺伝子の連続性が失われている状態を指す。なお、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の切断点（break point）は、ＮＣＯＡ４又はＲＵＦＹ１遺伝子と共にＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を構築する他の遺伝子がコードするタンパク質の機能（例えば、当該タンパク質がキナーゼドメインを有する場合、キナーゼ活性）を保持する範囲で限定されない。
　また、「ＮＣＯＡ４又はＲＵＦＹ１遺伝子以外の他の遺伝子の切断」又は「ＮＣＯＡ４又はＲＵＦＹ１遺伝子以外の他の遺伝子が切断されている」とは、遺伝子の転座又は逆位等により他の遺伝子の連続性が失われている状態、すなわち、他の遺伝子が少なくとも２つのポリヌクレオチドに分かれている状態を指す。 <Cleaving the NCOA4 or RUFY1 gene or the NCOA4 or RUFY1 protein>
Furthermore, in this specification, “NCOA4 or RUFY1 gene cleavage” or “NCOA4 or RUFY1 gene is cleaved” means that the continuity of the NCOA4 or RUFY1 gene is lost due to translocation or inversion of the gene. Refers to the state of being. Note that the break point of the NCOA4 or RUFY1 gene is the function of the protein encoded by the NCOA4 or RUFY1 gene and another gene that constructs the NCOA4 or RUFY1 gene (for example, if the protein has a kinase domain, the kinase It is not limited as long as the activity is maintained.
In addition, “cleaved other genes other than NCOA4 or RUFY1 gene” or “cleaved other genes other than NCOA4 or RUFY1 gene” means continuity of other genes due to gene translocation or inversion. Is a state in which other genes are divided into at least two polynucleotides.
　また、本明細書において、「ＮＣＯＡ４又はＲＵＦＹ１タンパク質の切断」又は「ＮＣＯＡ４又はＲＵＦＹ１タンパク質が切断されている」とは、ＮＣＯＡ４又はＲＵＦＹ１遺伝子が、前述のように切断されている状態であることに基づき、ＮＣＯＡ４又はＲＵＦＹ１タンパク質の連続性が失われている状態、すなわち、ＮＣＯＡ４又はＲＵＦＹ１タンパク質が少なくとも２つのポリペプチドに分かれている状態を指す。なお、ＮＣＯＡ４又はＲＵＦＹ１タンパク質の切断点は、ＮＣＯＡ４又はＲＵＦＹ１タンパク質と共にＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を構築する他のタンパク質の機能（例えば、当該他のタンパク質がキナーゼドメインを有する場合、キナーゼ活性）を保持する範囲で限定されない。
　また、「ＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質の切断」又は「ＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質が切断されている」とは、他の遺伝子が、前述のように切断されている状態であることに基づき、他のタンパク質の連続性が失われている状態、すなわち、他のタンパク質が少なくとも２つのポリペプチドに分かれている状態を指す。 Further, in the present specification, the phrase “NCOA4 or RUFY1 protein is cleaved” or “NCOA4 or RUFY1 protein is cleaved” is based on the fact that the NCOA4 or RUFY1 gene is cleaved as described above. , Refers to a state in which the continuity of the NCOA4 or RUFY1 protein is lost, that is, a state in which the NCOA4 or RUFY1 protein is divided into at least two polypeptides. The breakpoint of the NCOA4 or RUFY1 protein is a range that retains the functions of other proteins that construct the NCOA4 or RUFY1 fusion protein together with the NCOA4 or RUFY1 protein (for example, when the other protein has a kinase domain). It is not limited by.
In addition, “cleaving of a protein other than the NCOA4 or RUFY1 protein” or “a protein other than the NCOA4 or RUFY1 protein is cleaved” means that the other gene is cleaved as described above. Based on one thing, it refers to a state in which the continuity of another protein is lost, that is, a state in which the other protein is divided into at least two polypeptides.
　＜５’末端側領域／３’末端側領域、Ｎ末端側領域／Ｃ末端側領域＞
　５’末端側領域とは、融合遺伝子の場合は、融合点より５’末端側のポリヌクレオチド、野生型遺伝子（融合遺伝子でない遺伝子）の場合は、当該野生型遺伝子が融合遺伝子を構築した場合の、切断点より５’末端側のポリヌクレオチドを示す。なお、５’末端側領域は、ゲノムＤＮＡ、ｍＲＮＡ、及びｃＤＮＡのいずれにおける領域であってもよく、例えば、ゲノムＤＮＡの場合は、５’末端側ゲノム領域ともいう。
　３’末端側領域とは、融合遺伝子の場合は、融合点より３’末端側のポリヌクレオチド、野生型遺伝子（融合遺伝子でない遺伝子）の場合は、当該野生型遺伝子が融合遺伝子を構築した場合の、切断点より３’末端側のポリヌクレオチドを示す。なお、３’末端側領域は、ゲノムＤＮＡ、ｍＲＮＡ、及びｃＤＮＡのいずれにおける領域であってもよく、例えば、ゲノムＤＮＡの場合は、３’末端側ゲノム領域ともいう。 <5 ′ terminal region / 3 ′ terminal region, N terminal region / C terminal region>
In the case of a fusion gene, the 5 ′ terminal region is a polynucleotide 5 ′ terminal from the fusion point, and in the case of a wild type gene (non-fusion gene), the wild type gene constructs the fusion gene. The polynucleotide at the 5 ′ end side from the cleavage point is shown. The 5 ′ terminal region may be any region of genomic DNA, mRNA, and cDNA. For example, in the case of genomic DNA, it is also referred to as a 5 ′ terminal genomic region.
In the case of a fusion gene, the 3 ′ terminal region is a polynucleotide 3 ′ terminal from the fusion point. In the case of a wild type gene (a gene that is not a fusion gene), the wild type gene constructs the fusion gene. The polynucleotide at the 3 ′ end side from the cleavage point is shown. The 3 ′ terminal region may be a region in any of genomic DNA, mRNA, and cDNA. For example, in the case of genomic DNA, it is also referred to as a 3 ′ terminal genomic region.
　Ｎ末端側領域とは、融合タンパク質の場合は、融合点よりＮ末端側のポリペプチド、野生型タンパク質（融合タンパク質でないタンパク質）の場合は、当該野生型タンパク質が融合遺伝子を構築した場合の、切断点よりＮ末端側のポリヌクレオチドを示す。
　Ｃ末端側領域とは、融合タンパク質の場合は、融合点よりＣ末端側のポリペプチド、野生型タンパク質（融合タンパク質でないタンパク質）の場合は、当該野生型タンパク質が融合遺伝子を構築した場合の、切断点よりＣ末端側のポリヌクレオチドを示す。 In the case of a fusion protein, the N-terminal region is a polypeptide at the N-terminal side from the fusion point, and in the case of a wild type protein (a protein that is not a fusion protein), cleavage when the wild type protein constructs a fusion gene. The polynucleotide on the N-terminal side from the point is shown.
In the case of a fusion protein, the C-terminal region is a polypeptide at the C-terminal side from the fusion point, and in the case of a wild-type protein (a protein that is not a fusion protein), cleavage when the wild-type protein constructs a fusion gene. The polynucleotide on the C-terminal side from the point is shown.
　例えば、配列番号１で表されるＮＣＯＡ４－ＲＥＴ融合遺伝子（ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２）の場合、５’末端側領域は、第１～第１９８４番、３’末端側領域は、第１９８５～５２７５番の塩基配列からなるポリヌクレオチドである。配列番号２で表されるＮＣＯＡ４－ＲＥＴ融合タンパク質の場合、Ｎ末端側領域は、前記ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２の５’末端側領域のＣＤＳ（配列番号１の第１４６～１９８４番塩基）にコードされるポリペプチド（配列番号２の第１～６１３番アミノ酸）であり、Ｃ末端側領域は、前記ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２の３’末端側領域のＣＤＳ（配列番号１の第１９８５～３１９３番塩基）にコードされるポリペプチド（配列番号２の第６１４～１０１５番アミノ酸）である。 For example, in the case of the NCOA4-RET fusion gene represented by SEQ ID NO: 1 (NCOA4ex9-RETex12), the 5 ′ terminal region is the first to 1984th bases, and the 3 ′ terminal region is the 1985th to 5275th bases. A polynucleotide comprising a sequence. In the case of the NCOA4-RET fusion protein represented by SEQ ID NO: 2, the N-terminal region is a polycode encoded by the CDS (bases 146 to 1984 in SEQ ID NO: 1) of the 5′-terminal region of the NCOA4ex9-RETex12. This is a peptide (amino acids 1 to 613 of SEQ ID NO: 2), and the C-terminal region is encoded by CDS (the 1985 to 3193 bases of SEQ ID NO: 1) of the 3 'terminal region of NCOA4ex9-RETex12 Polypeptide (amino acids 614 to 1015 of SEQ ID NO: 2).
　配列番号３で表されるＲＵＦＹ１－ＲＥＴ融合遺伝子（ＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２）の場合、５’末端側領域は、第１～第１９１７番、３’末端側領域は、第１９１８～５２０８番の塩基配列からなるポリヌクレオチドである。配列番号４で表されるＲＵＦＹ１－ＲＥＴ融合タンパク質の場合、Ｎ末端側領域は、前記ＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２の５’末端側領域のＣＤＳ（配列番号３の第１３～１９１７番塩基）にコードされるポリペプチド（配列番号４の第１～６３５番アミノ酸）であり、Ｃ末端側領域は、前記ＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２の３’末端側領域のＣＤＳ（配列番号３の第１９１８～３１２６番塩基）にコードされるポリペプチド（配列番号４の第６３６～１０３７番アミノ酸）である。 In the case of the RUFY1-RET fusion gene represented by SEQ ID NO: 3 (RUFY1ex16-RETex12), the 5 ′ terminal region is from the first to 1917th regions, and the 3 ′ terminal region is from the 1918-5208 base sequences. A polynucleotide. In the case of the RUFY1-RET fusion protein represented by SEQ ID NO: 4, the N-terminal region is a polycode encoded by the CDS (the 13th to 1917th bases of SEQ ID NO: 3) of the 5′-terminal region of the RUFY1ex16-RETex12. This is a peptide (amino acids 1 to 635 of SEQ ID NO: 4), and the C-terminal region is encoded by CDS (bases 1918 to 3126 of SEQ ID NO: 3) of the 3 ′ terminal region of RUFY1ex16-RETex12 Polypeptide (amino acids 636 to 1037 of SEQ ID NO: 4).
　＜ｃＤＮＡリファレンス配列＞
　また、本明細書において、各由来遺伝子のｃＤＮＡリファレンス配列として、ＲＥＴはENST00000355710、ＮＣＯＡ４はENST00000578454、ＲＵＦＹ１はENST00000319449を、タンパク質のアミノ酸リファレンス配列としては、ＲＥＴはENSP00000347942、ＮＣＯＡ４はENSP00000463027、ＲＵＦＹ１はENSP00000325594を用いた。 <CDNA reference sequence>
In addition, in this specification, as a cDNA reference sequence of each derived gene, ENS is ENST00000355710, NCOA4 is ENST00000578454, RUFY1 is ENST00000319449, RET is ENSP00000347942, NCOA4 is ENSP00000463027, and RUFY1 is ENSP00000325594 It was.
　＜ストリンジェントな条件＞
　本明細書における「ストリンジェントな条件」とは、ハイブリダイゼーションのための条件として、「５×ＳＳＰＥ、５×Ｄｅｎｈａｒｄｔ’ｓ液、０．５％ＳＤＳ、５０％ホルムアミド、２００μｇ／ｍＬ鮭精子ＤＮＡ、４２℃オーバーナイト」、洗浄のための条件として、「０．５×ＳＳＣ、０．１％ＳＤＳ、４２℃」の条件である。「よりストリンジェントな条件」とは、ハイブリダイゼーションのための条件として、「５×ＳＳＰＥ、５×Ｄｅｎｈａｒｄｔ’ｓ液、０．５％ＳＤＳ、５０％ホルムアミド、２００μｇ／ｍＬ鮭精子ＤＮＡ、４２℃オーバーナイト」、洗浄のための条件として、「０．２×ＳＳＣ、０．１％ＳＤＳ、６５℃」の条件である。 <Stringent conditions>
As used herein, “stringent conditions” refers to “5 × SSPE, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 200 μg / mL sperm DNA, “42 ° C. overnight” and the conditions for cleaning are “0.5 × SSC, 0.1% SDS, 42 ° C.”. “More stringent conditions” means “5 × SSPE, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 200 μg / mL sperm DNA, over 42 ° C.” “Night”, the conditions for cleaning are “0.2 × SSC, 0.1% SDS, 65 ° C.”.
　＜腫瘍形成能＞
　あるポリペプチドが「腫瘍形成能を有する」ことは、公知の方法、例えば、ＷＯ２０１１／１６２２９５の実施例４の方法、あるいは、後述の実施例６の方法で確認することができる。具体的には、当該ポリペプチドを発現するプラスミドを導入した宿主（３Ｔ３繊維芽細胞）をヌードマウスの皮下に接種し、腫瘍形成の有無で判断する方法で確認する。ＮＣＯＡ４－ＲＥＴ融合体では、導入細胞における形質転換及び導入細胞移植マウスにおける腫瘍形成能が示されており、本融合遺伝子又はその転写産物の存在は、発現部位におけるがんの原因であることが示唆されている（非特許文献２参照）。 <Tumorogenicity>
It can be confirmed by a known method, for example, the method of Example 4 of WO2011 / 162295 or the method of Example 6 described later, that a certain polypeptide has “tumor forming ability”. Specifically, a host (3T3 fibroblast) into which a plasmid expressing the polypeptide has been introduced is inoculated subcutaneously into nude mice, and confirmed by a method of judging the presence or absence of tumor formation. The NCOA4-RET fusion showed transformation in the introduced cells and tumor-forming ability in the introduced cell transplanted mice, suggesting that the presence of this fusion gene or its transcript is the cause of cancer at the site of expression. (See Non-Patent Document 2).
≪本発明の検出方法に係る試料≫
　＜対象臓器＞
　本発明に係る検出方法は、対象臓器に生じるがんの検出に好適に用いることができる。被験者の被験部位（対象臓器）としては、本発明に係る融合体が存在している範囲で限定されないが、消化器が好ましく、消化管がより好ましく、胃腸が更に好ましく、下部消化管が更に好ましく、大腸が特に好ましい。
　被験部位の組織型は、本発明に係る検出方法が適用可能な範囲で制限されず、扁平上皮組織でも、腺組織でもよいが、扁平上皮組織が好ましい。 << Sample according to the detection method of the present invention >>
<Target organ>
The detection method according to the present invention can be suitably used for detection of cancer occurring in a target organ. The test site (target organ) of the subject is not limited as long as the fusion according to the present invention exists, but the digestive tract is preferable, the digestive tract is more preferable, the gastrointestinal tract is more preferable, and the lower digestive tract is more preferable. Particularly preferred is the large intestine.
The tissue type of the test site is not limited as long as the detection method according to the present invention can be applied, and it may be a squamous tissue or a glandular tissue, but a squamous tissue is preferable.
　＜被験者からの採取物＞
　本発明に係る検出方法における、被験者から得た試料としては、被験者からの採取物（生体から分離した試料）、具体的には、任意の採取された体液（好ましくは血液）、被験者患部からの摘出検体、生検試料又は擦過検体、糞便、尿、消化管洗浄液等を用いることができる。前記消化管洗浄液は、消化管全体の洗浄液であっても、あるいは、少なくとも被験部位を含む消化管の洗浄液、例えば、下部消化管の洗浄液、大腸の洗浄液であってもよい。検出感度を考慮すると、前記対象臓器における被験部位の細胞が含まれる試料が好ましく、被験者の被験部位からの摘出検体又は生検試料が更に好ましい。 <Collected from subject>
In the detection method according to the present invention, the sample obtained from the subject includes a sample collected from the subject (sample separated from the living body), specifically, any collected body fluid (preferably blood), from the subject affected area. Extracted specimens, biopsy specimens or scraped specimens, feces, urine, gastrointestinal lavage fluid and the like can be used. The digestive tract washing solution may be a washing solution for the entire digestive tract, or a washing solution for the digestive tract including at least the test site, for example, a washing solution for the lower digestive tract or a washing solution for the large intestine. In view of detection sensitivity, a sample containing cells at the test site in the target organ is preferable, and an excised specimen or biopsy sample from the test site of the test subject is more preferable.
　＜採取物の調製＞
　本発明に係るＲＥＴ融合遺伝子、又は、ＲＥＴ融合タンパク質の検出方法は、被験者から得た試料の組織切片、又は、細胞懸濁液等を作成し、組織切片又は細胞懸濁液に含まれる細胞に対し、当業者に周知の技法により、ＲＥＴ融合遺伝子、又は、ＲＥＴ融合タンパク質を検出することにより実施できる。あるいは、前述の被験者から得た試料から、可溶化液を調製し、ここに含まれる遺伝子、又は、タンパク質を抽出し、この抽出試料において、当業者に周知の技法により、ＲＥＴ融合遺伝子、又は、ＲＥＴ融合タンパク質を検出してもよい。なお、ＲＥＴ融合遺伝子の検出とは、ＲＥＴ融合遺伝子のゲノムＤＮＡの検出、当該ゲノムＤＮＡの転写産物であるｍＲＮＡ、又は、ｍＲＮＡを鋳型として得られるｃＤＮＡの検出のいずれであってもよい。 <Preparation of collected material>
The method for detecting a RET fusion gene or RET fusion protein according to the present invention comprises preparing a tissue section or a cell suspension of a sample obtained from a subject and applying it to a cell contained in the tissue section or cell suspension. On the other hand, it can be carried out by detecting a RET fusion gene or a RET fusion protein by techniques well known to those skilled in the art. Alternatively, a lysate is prepared from a sample obtained from the above-mentioned subject, and a gene or protein contained therein is extracted. In this extracted sample, a RET fusion gene or A RET fusion protein may be detected. The detection of the RET fusion gene may be any of detection of genomic DNA of the RET fusion gene, mRNA that is a transcription product of the genomic DNA, or detection of cDNA obtained using mRNA as a template.
　本発明に係るＮＣＯＡ４又はＲＵＦＹ１融合遺伝子、又は、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の検出方法は、被験者から得た試料の組織切片、又は、細胞懸濁液等を作成し、組織切片又は細胞懸濁液に含まれる細胞に対し、当業者に周知の技法により、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子、又は、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出することにより実施できる。あるいは、前述の被験者から得た試料から、可溶化液を調製し、ここに含まれる遺伝子、又は、タンパク質を抽出し、この抽出試料において、当業者に周知の技法により、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子、又は、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出してもよい。なお、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出とは、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子のゲノムＤＮＡの検出、当該ゲノムＤＮＡの転写産物であるｍＲＮＡ、又は、ｍＲＮＡを鋳型として得られるｃＤＮＡの検出のいずれであってもよい。 The method for detecting an NCOA4 or RUFY1 fusion gene or an NCOA4 or RUFY1 fusion protein according to the present invention comprises preparing a tissue section or cell suspension of a sample obtained from a subject, It can be carried out by detecting the NCOA4 or RUFY1 fusion gene or the NCOA4 or RUFY1 fusion protein on the contained cells by techniques well known to those skilled in the art. Alternatively, a lysate is prepared from a sample obtained from the aforementioned subject, and a gene or protein contained therein is extracted. In this extracted sample, an NCOA4 or RUFY1 fusion gene, Alternatively, NCOA4 or RUfy1 fusion protein may be detected. The detection of the NCOA4 or RUFY1 fusion gene may be any of detection of genomic DNA of the NCOA4 or RUFY1 fusion gene, mRNA that is a transcription product of the genomic DNA, or detection of cDNA obtained using the mRNA as a template. Good.
≪本発明の検出方法に係る検出対象≫
　本発明の検出方法には、被験者から得た試料中の、ＲＥＴ融合体の検出方法、すなわち、ＲＥＴキナーゼ領域を含む融合タンパク質（「ＲＥＴ融合タンパク質」ともいう）の検出方法、又は、前記融合タンパク質をコードする融合遺伝子（「ＲＥＴ融合遺伝子」ともいう）の検出方法が含まれる。
　本発明の検出方法には、被験者から得た試料中の、ＮＣＯＡ４又はＲＵＦＹ１融合体の検出方法、すなわち、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の検出方法、又は、前記融合タンパク質をコードする融合遺伝子（「ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子」ともいう）の検出方法が含まれる。 << Detection target according to the detection method of the present invention >>
The detection method of the present invention includes a method for detecting a RET fusion in a sample obtained from a subject, that is, a method for detecting a fusion protein containing a RET kinase region (also referred to as “RET fusion protein”), or the fusion protein. And a method for detecting a fusion gene encoding CHO (also referred to as “RET fusion gene”).
The detection method of the present invention includes a method for detecting an NCOA4 or RUFY1 fusion in a sample obtained from a subject, that is, a method for detecting an NCOA4 or RUFY1 fusion protein, or a fusion gene encoding the fusion protein (“NCOA4 or And a detection method of “RUFY1 fusion gene”.
　＜ＲＥＴ融合体：ＲＥＴ融合タンパク質とＲＥＴ融合遺伝子＞
　本発明に係るＲＥＴ融合体は、ＲＥＴ融合タンパク質とＲＥＴ融合遺伝子を含む。
　本発明に係るＲＥＴ融合タンパク質は、ＲＥＴタンパク質由来のポリペプチドと、ＲＥＴタンパク質以外の他のタンパク質由来のポリペプチドと、から構築される融合ポリペプチドであって、前記ＲＥＴタンパク質由来のポリペプチドは、ＲＥＴタンパク質における少なくともＲＥＴキナーゼ領域のポリペプチドを含み、ＲＥＴタンパク質以外の他のタンパク質由来のポリペプチドは、他のタンパク質における少なくとも一部のポリペプチドを含む範囲で特に制限されない。 <RET fusion: RET fusion protein and RET fusion gene>
The RET fusion according to the present invention comprises a RET fusion protein and a RET fusion gene.
The RET fusion protein according to the present invention is a fusion polypeptide constructed from a polypeptide derived from a RET protein and a polypeptide derived from another protein other than the RET protein, wherein the polypeptide derived from the RET protein is: A polypeptide derived from a protein other than the RET protein including at least the polypeptide of the RET kinase region in the RET protein is not particularly limited as long as it includes at least a part of the polypeptide in the other protein.
　前記他のタンパク質としては、ＲＥＴキナーゼドメインを含むＲＥＴタンパク質の一部と融合することにより、構築されたＲＥＴ融合タンパク質が腫瘍形成能を有するものであれば特に制限されない。構築されたＲＥＴ融合タンパク質において、ＲＥＴキナーゼ活性化が恒常的に維持されることにより、ＲＥＴ融合タンパク質が腫瘍形成能を有することが好ましい。 The other protein is not particularly limited as long as the RET fusion protein constructed by fusing with a part of the RET protein including the RET kinase domain has a tumor forming ability. In the constructed RET fusion protein, it is preferable that the RET fusion protein has a tumorigenicity by constantly maintaining RET kinase activation.
　また、ＲＥＴ融合タンパク質は、ＲＥＴキナーゼ活性化が恒常的に維持され、構築されたＲＥＴ融合タンパク質が腫瘍形成能を有する範囲で、ＲＥＴタンパク質由来のポリペプチド、及び、ＲＥＴタンパク質以外の他のタンパク質由来のポリペプチドのいずれでもない、第３のポリペプチドを含んでいてもよい。第３のポリペプチドは、ＲＥＴ融合タンパク質のＮ末端に位置していても、Ｃ末端に位置していても、或いは、ＲＥＴタンパク質由来のポリペプチドとＲＥＴタンパク質以外の他のタンパク質由来のポリペプチドとの間に位置していてもよい。 The RET fusion protein is derived from a polypeptide derived from the RET protein and other proteins other than the RET protein as long as the RET kinase activation is constantly maintained and the constructed RET fusion protein has tumorigenicity. A third polypeptide that is not any of the polypeptides may be included. The third polypeptide may be located at the N-terminus or C-terminus of the RET fusion protein, or may be a polypeptide derived from a RET protein and a polypeptide derived from another protein other than the RET protein. It may be located between.
　ＲＥＴ融合タンパク質としては、前記他のタンパク質が、ＮＣＯＡ４又はＲＵＦＹ１タンパク質である融合タンパク質が特に好ましい。すなわち、少なくともＲＥＴキナーゼ領域のポリペプチドを含む、ＲＥＴタンパク質由来のポリペプチドと、ＮＣＯＡ４又はＲＵＦＹ１タンパク質の少なくとも一部のポリペプチドを含む、ＮＣＯＡ４又はＲＵＦＹ１タンパク質由来のポリペプチドと、から構築された、ＮＣＯＡ４又はＲＵＦＹ１タンパク質とＲＥＴタンパク質との融合タンパク質（以下、ＮＣＯＡ４－ＲＥＴ融合タンパク質又はＲＵＦＹ１－ＲＥＴ融合タンパク質、あるいは、ＮＣＯＡ４－ＲＥＴ又はＲＵＦＹ１－ＲＥＴ融合タンパク質、あるいは、ＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合タンパク質ともいう）であることが好ましい。 As the RET fusion protein, a fusion protein in which the other protein is NCOA4 or RUFY1 protein is particularly preferable. That is, an NCOA4 constructed from an RET protein-derived polypeptide containing at least a polypeptide in the RET kinase region and an NCOA4 or RUFY1 protein-derived polypeptide containing at least a partial polypeptide of the NCOA4 or RUFY1 protein. Or a fusion protein of RUFY1 protein and RET protein (hereinafter also referred to as NCOA4-RET fusion protein or RUFY1-RET fusion protein, NCOA4-RET or RUYY1-RET fusion protein, or NCOA4 or RUFY1-RET fusion protein) Preferably there is.
　＜ＮＣＯＡ４又はＲＵＦＹ１融合体：ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質とＮＣＯＡ４又はＲＵＦＹ１融合遺伝子＞
　本発明に係るＮＣＯＡ４又はＲＵＦＹ１融合体は、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質とＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を含む。
　本発明に係るＮＣＯＡ４又はＲＵＦＹ１融合タンパク質は、ＮＣＯＡ４又はＲＵＦＹ１タンパク質由来のポリペプチドと、ＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質由来のポリペプチドと、から構築される融合ポリペプチドであって、前記ＮＣＯＡ４又はＲＵＦＹ１タンパク質由来のポリペプチドは、ＮＣＯＡ４又はＲＵＦＹ１タンパク質における少なくとも一部のポリペプチドを含み、ＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質由来のポリペプチドは、他のタンパク質における少なくとも一部のポリペプチドを含む範囲で特に制限されない。 <NCOA4 or RUFY1 fusion: NCOA4 or RUFY1 fusion protein and NCOA4 or RUFY1 fusion gene>
The NCOA4 or RUFY1 fusion according to the present invention comprises an NCOA4 or RUFY1 fusion protein and an NCOA4 or RUFY1 fusion gene.
NCOA4 or RUFY1 fusion protein according to the present invention is a fusion polypeptide constructed from a polypeptide derived from NCOA4 or RUFY1 protein and a polypeptide derived from another protein other than NCOA4 or RUFY1 protein, wherein said NCOA4 or The polypeptide derived from RUFY1 protein includes at least a part of the polypeptide in NCOA4 or RUFY1 protein, and the polypeptide derived from other proteins other than NCOA4 or RUFY1 protein includes at least a part of the polypeptide in other proteins. There is no particular restriction.
　前記他のタンパク質としては、ＮＣＯＡ４又はＲＵＦＹ１タンパク質の一部と融合することにより、構築されたＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が腫瘍形成能を有するものであれば特に制限されない。当該他のタンパク質の有する機能性ドメイン（好ましくは、キナーゼドメイン）の活性化が恒常的に維持されることにより、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が腫瘍形成能を有することが好ましい。 The other protein is not particularly limited as long as the constructed NCOA4 or RUFY1 fusion protein has a tumor-forming ability by being fused with a part of the NCOA4 or RUFY1 protein. It is preferable that the NCOA4 or RUFY1 fusion protein has tumorigenicity by constantly maintaining the activation of the functional domain (preferably the kinase domain) of the other protein.
　また、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質は、ＮＣＯＡ４又はＲＵＦＹ１タンパク質の一部と融合することによりＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質の機能性ドメインの活性化が恒常的に維持され、構築されたＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が腫瘍形成能を有する範囲で、ＮＣＯＡ４又はＲＵＦＹ１タンパク質由来のポリペプチド、及び、ＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質由来のポリペプチドのいずれでもない、第３のポリペプチドを含んでいてもよい。第３のポリペプチドは、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質のＮ末端に位置していても、Ｃ末端に位置していても、或いは、ＮＣＯＡ４又はＲＵＦＹ１タンパク質由来のポリペプチドとＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質由来のポリペプチドとの間に位置していてもよい。 Further, the NCOA4 or RUFY1 fusion protein is fused with a part of the NCOA4 or RUFY1 protein to constantly maintain the activation of the functional domain of other proteins other than the NCOA4 or RUFY1 protein. As long as the fusion protein has tumorigenicity, it may contain a third polypeptide that is neither an NCOA4 or RUFY1 protein-derived polypeptide nor a polypeptide derived from another protein other than the NCOA4 or RUFY1 protein. Good. The third polypeptide may be located at the N-terminus of the NCOA4 or RUFY1 fusion protein, at the C-terminus, or a polypeptide derived from the NCOA4 or RUFY1 protein and other than the NCOA4 or RUFY1 protein. It may be located between the protein-derived polypeptide.
　ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質としては、前記他のタンパク質が、ＲＥＴタンパク質である融合タンパク質が特に好ましい。すなわち、ＮＣＯＡ４又はＲＵＦＹ１タンパク質の少なくとも一部のポリペプチドを含む、ＮＣＯＡ４又はＲＵＦＹ１タンパク質由来のポリペプチドと、少なくともＲＥＴキナーゼ領域のポリペプチドを含む、ＲＥＴタンパク質の少なくとも一部のポリペプチドと、から構築された、ＮＣＯＡ４又はＲＵＦＹ１タンパク質とＲＥＴタンパク質との融合タンパク質（以下、ＮＣＯＡ４－ＲＥＴ融合タンパク質又はＲＵＦＹ１－ＲＥＴ融合タンパク質、あるいは、ＮＣＯＡ４－ＲＥＴ又はＲＵＦＹ１－ＲＥＴ融合タンパク質、あるいは、ＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合タンパク質ともいう）であることが好ましい。 As the NCOA4 or RUfy1 fusion protein, a fusion protein in which the other protein is a RET protein is particularly preferable. That is, it is constructed from a polypeptide derived from the NCOA4 or RUFY1 protein containing at least a part of the polypeptide of the NCOA4 or RUFUY1 protein, and at least a part of the polypeptide of the RET protein including a polypeptide of the RET kinase region. In addition, a fusion protein of the NCOA4 or RUFY1 protein and the RET protein (hereinafter referred to as the NCOA4-RET fusion protein or the RUFY1-RET fusion protein, or the NCOA4-RET or RUFY1-RET fusion protein, or the NCOA4 or RUFY1-RET fusion protein) It is preferable that
　「ＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合タンパク質」としては、下記（ａ）～（ｄ）に記載のポリペプチドが特に好ましい：
　（ａ）配列番号２（ＮＣＯＡ４－ＲＥＴ）又は配列番号４（ＲＵＦＹ１－ＲＥＴ）で表されるアミノ酸配列からなるポリペプチド、
　（ｂ）配列番号２又は配列番号４で表されるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、
　（ｃ）配列番号２又は配列番号４で表されるアミノ酸配列との同一性が８０％以上であるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド（以下、相同ポリペプチドと称する）、及び
　（ｄ）配列番号２又は配列番号４で表されるアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、及び／又は挿入されたアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド（以下、機能的等価改変体と称する）。 As the “NCOA4 or RUFY1-RET fusion protein”, the polypeptides described in the following (a) to (d) are particularly preferred:
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (NCOA4-RET) or SEQ ID NO: 4 (RUFY1-RET),
(B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity,
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity (hereinafter referred to as a homologous polypeptide), and (D) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenic potential ( Hereinafter referred to as a functional equivalent variant).
　配列番号２で表されるアミノ酸配列は、配列番号１で表される塩基配列、特には配列番号１の塩基番号１４６～３１９３で表される塩基配列（ＣＤＳ）によりコードされる配列である。配列番号１で表される塩基配列は、ＮＣＯＡ４遺伝子の５’－ＵＴＲ配列、ＮＣＯＡ４遺伝子の開始コドンＡＴＧからエクソン９まで、ＲＥＴ遺伝子のエクソン１２からエクソン２０の停止コドンまで、ＲＥＴ遺伝子の３’－ＵＴＲ配列の塩基配列からなる。配列番号１で表される塩基配列の内、塩基番号１～１９８４の配列はＮＣＯＡ４遺伝子に由来し、塩基番号１９８５～５２７５の配列はＲＥＴ遺伝子に由来する。本明細書において、配列番号２で表されるアミノ酸配列からなるポリペプチド、及び、これをコードする塩基配列からなるポリヌクレオチド（配列番号１で表される塩基配列からなるポリヌクレオチドを含む）を、ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２融合体（または、ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２という場合もある）と称する。 The amino acid sequence represented by SEQ ID NO: 2 is a sequence encoded by the base sequence represented by SEQ ID NO: 1, particularly the base sequence (CDS) represented by base numbers 146 to 3193 of SEQ ID NO: 1. The nucleotide sequence represented by SEQ ID NO: 1 includes the 5′-UTR sequence of the NCOA4 gene, the start codon ATG to exon 9 of the NCOA4 gene, the exon 12 of the RET gene to the stop codon of exon 20, and the 3′- of the RET gene. It consists of the base sequence of the UTR sequence. Of the nucleotide sequence represented by SEQ ID NO: 1, the nucleotide sequence of nucleotide numbers 1 to 1984 is derived from the NCOA4 gene, and the nucleotide sequence of nucleotide numbers 1985 to 5275 is derived from the RET gene. In the present specification, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 and a polynucleotide comprising the base sequence encoding the same (including a polynucleotide comprising the base sequence represented by SEQ ID NO: 1), This is referred to as an NCOA4ex9-RETex12 fusion (or sometimes referred to as NCOA4ex9-RETex12).
　配列番号４で表されるアミノ酸配列は、配列番号３で表される塩基配列、特には配列番号３の塩基番号１３～３１２６で表される塩基配列（ＣＤＳ）によりコードされる配列である。配列番号３で表される塩基配列は、ＲＵＦＹ１遺伝子の５’－ＵＴＲ配列、ＲＵＦＹ１遺伝子の開始コドンＡＴＧからエクソン１６まで、ＲＥＴ遺伝子のエクソン１２からエクソン２０の停止コドンまで、及びＲＥＴ遺伝子の３’－ＵＴＲ配列の塩基配列からなる。配列番号３で表される塩基配列の内、塩基番号１３～１９１７の配列はＲＵＦＹ１遺伝子に由来し、塩基番号１９１８～３１２６の配列はＲＥＴ遺伝子に由来する。本明細書において、配列番号４で表されるアミノ酸配列からなるポリペプチド、及び、これをコードする塩基配列からなるポリヌクレオチド（配列番号３で表される塩基配列からなるポリヌクレオチドを含む）を、ＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２融合体（または、ＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２という場合もある）と称する。 The amino acid sequence represented by SEQ ID NO: 4 is a sequence encoded by the base sequence represented by SEQ ID NO: 3, particularly the base sequence (CDS) represented by base numbers 13 to 3126 of SEQ ID NO: 3. The nucleotide sequence represented by SEQ ID NO: 3 includes the 5′-UTR sequence of the RUFY1 gene, the start codon ATG to exon 16 of the RUFY1 gene, the exon 12 of the RET gene to the stop codon of exon 20, and the 3 ′ of the RET gene. -It consists of the base sequence of the UTR sequence. Of the base sequence represented by SEQ ID NO: 3, the sequences of base numbers 13 to 1917 are derived from the RUFY1 gene, and the sequences of base numbers 1918 to 3126 are derived from the RET gene. In the present specification, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 and a polynucleotide comprising the base sequence encoding the same (including a polynucleotide comprising the base sequence represented by SEQ ID NO: 3), This is referred to as a RUFY1ex16-RETex12 fusion (or sometimes RUYY1ex16-RETex12).
　「機能的等価改変体」において置換、欠失、及び／又は挿入可能なアミノ酸数は、１～数個であるが、好ましくは１～１０個、更に好ましくは１～７個、最も好ましくは１～５個である。 In the “functional equivalent variant”, the number of amino acids that can be substituted, deleted, and / or inserted is 1 to several amino acids, preferably 1 to 10, more preferably 1 to 7, and most preferably 1. ~ 5.
　「相同ポリペプチド」は、「配列番号２又は配列番号４で表されるアミノ酸配列との同一性が８０％以上であるアミノ酸配列を含み、しかも、腫瘍形成能を有するポリペプチド」であるが、該同一性が、好ましくは９０％以上、より好ましくは９５％以上、更に好ましくは９８％以上であるアミノ酸配列を含むポリペプチドが好ましい。なお、「配列番号２又は配列番号４で表されるアミノ酸配列との同一性が８０％以上であるアミノ酸配列を含み、しかも、腫瘍形成能を有するポリペプチド」には、前記同一性を示し、且つ、少なくとも１つの置換、欠失、及び／又は挿入（好ましくは置換）を有するポリペプチド（狭義の相同ポリペプチド）と、同一性が１００％であるポリペプチドとが含まれる。 “Homologous polypeptide” is a “polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity” A polypeptide comprising an amino acid sequence having an identity of preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more is preferable. In addition, the “polypeptide having an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity” shows the identity, In addition, a polypeptide having at least one substitution, deletion, and / or insertion (preferably a substitution) (a homologous polypeptide in a narrow sense) and a polypeptide having 100% identity are included.
　なお、本明細書における前記「同一性」とは、ＮＥＥＤＬＥ ｐｒｏｇｒａｍ（J Mol Biol 1970; 48: 443-453）検索によりデフォルトで用意されているパラメータを用いて得られた値Ｉｄｅｎｔｉｔｙを意味する。前記のパラメータは以下のとおりである。
　　Ｇａｐ ｐｅｎａｌｔｙ ＝ １０
　　Ｅｘｔｅｎｄ ｐｅｎａｌｔｙ ＝ ０．５
　　Ｍａｔｒｉｘ ＝ ＥＢＬＯＳＵＭ６２ In the present specification, the “identity” means a value Identity obtained by using a parameter prepared as a default by a NEEDLE program (J Mol Biol 1970; 48: 443-453) search. The parameters are as follows.
Gap penalty = 10
Extended penalty = 0.5
Matrix = EBLOSUM62
　本発明に係るＲＥＴ融合遺伝子は、ＲＥＴ融合タンパク質をコードするポリヌクレオチドである。本明細書において、ＲＥＴ融合タンパク質およびＲＥＴ融合遺伝子をあわせて「ＲＥＴ融合体」と称することがある。 The RET fusion gene according to the present invention is a polynucleotide encoding a RET fusion protein. In the present specification, the RET fusion protein and the RET fusion gene may be collectively referred to as “RET fusion”.
　本発明に係るＮＣＯＡ４又はＲＵＦＹ１融合遺伝子は、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質をコードするポリヌクレオチドである。すなわち、ＮＣＯＡ４融合遺伝子は、ＮＣＯＡ４融合タンパク質をコードするポリヌクレオチドであり、ＲＵＦＹ１融合遺伝子は、ＲＵＦＹ１融合タンパク質をコードするポリヌクレオチドである。本明細書において、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質およびＮＣＯＡ４又はＲＵＦＹ１融合遺伝子をあわせて「ＮＣＯＡ４又はＲＵＦＹ１融合体」と称することがある。 The NCOA4 or RUFY1 fusion gene according to the present invention is a polynucleotide encoding the NCOA4 or RUFY1 fusion protein. That is, the NCOA4 fusion gene is a polynucleotide encoding the NCOA4 fusion protein, and the RUFY1 fusion gene is a polynucleotide encoding the RUFY1 fusion protein. In the present specification, the NCOA4 or RUFY1 fusion protein and the NCOA4 or RUFY1 fusion gene may be collectively referred to as “NCOA4 or RUFY1 fusion”.
　本発明に係るＲＥＴ融合体においては、ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２融合体バリアント、又はＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２融合体バリアントが好ましい。特に、本発明に係るＲＥＴ融合タンパク質においては、ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２融合タンパク質バリアント、又はＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２融合タンパク質バリアントが好ましい。また、本発明に係るＲＥＴ融合遺伝子においては、ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２融合遺伝子バリアント、又はＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２融合遺伝子バリアントが好ましい。 In the RET fusion according to the present invention, the NCOA4ex9-RETex12 fusion variant or the RUFY1ex16-RETex12 fusion variant is preferable. In particular, in the RET fusion protein according to the present invention, the NCOA4ex9-RETex12 fusion protein variant or the RUFY1ex16-RETex12 fusion protein variant is preferable. Further, in the RET fusion gene according to the present invention, an NCOA4ex9-RETex12 fusion gene variant or a RUFY1ex16-RETex12 fusion gene variant is preferable.
　本発明に係るＮＣＯＡ４又はＲＵＦＹ１融合体においては、ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２融合体バリアント、又はＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２融合体バリアントが好ましい。特に、本発明に係るＮＣＯＡ４又はＲＵＦＹ１融合タンパク質においては、ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２融合タンパク質バリアント、又はＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２融合タンパク質バリアントが好ましい。また、本発明に係るＮＣＯＡ４又はＲＵＦＹ１融合遺伝子においては、ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２融合遺伝子バリアント、又はＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２融合遺伝子バリアントが好ましい。 In the NCOA4 or RUFY1 fusion according to the present invention, the NCOA4ex9-RETex12 fusion variant or the RUFY1ex16-RETex12 fusion variant is preferable. In particular, in the NCOA4 or RUFY1 fusion protein according to the present invention, the NCOA4ex9-RETex12 fusion protein variant or the RUFY1ex16-RETex12 fusion protein variant is preferable. In the NCOA4 or RUFY1 fusion gene according to the present invention, an NCOA4ex9-RETex12 fusion gene variant or a RUFY1ex16-RETex12 fusion gene variant is preferable.
≪本発明の検出方法の態様（融合タンパク質及び融合遺伝子の検出方法）≫
　本発明の検出方法には、被験者から得た試料中の、ＲＥＴタンパク質の切断、又は、ＲＥＴタンパク質をコードするＲＥＴ遺伝子の切断、を検出する工程を含む検出方法と、被験者から得た試料中の、ＲＥＴタンパク質とＲＥＴタンパク質以外の他のタンパク質とから構築される融合タンパク質の存在、又は、前記融合タンパク質をコードする融合遺伝子の存在、を検出する工程を含む検出方法が含まれる。
　本発明の検出方法には、被験者から得た試料中の、ＮＣＯＡ４又はＲＵＦＹ１タンパク質の切断、又は、ＮＣＯＡ４又はＲＵＦＹ１タンパク質をコードするＮＣＯＡ４又はＲＵＦＹ１遺伝子の切断、を検出する工程を含む検出方法と、被験者から得た試料中の、ＮＣＯＡ４又はＲＵＦＹ１タンパク質とＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質とから構築される融合タンパク質の存在、又は、前記融合タンパク質をコードする融合遺伝子の存在、を検出する工程を含む検出方法が含まれる。 << Aspect of Detection Method of the Present Invention (Fusion Protein and Fusion Gene Detection Method) >>
The detection method of the present invention includes a detection method comprising a step of detecting cleavage of a RET protein or cleavage of a RET gene encoding a RET protein in a sample obtained from a subject, and in a sample obtained from the subject. And a detection method comprising a step of detecting the presence of a fusion protein constructed from the RET protein and another protein other than the RET protein, or the presence of a fusion gene encoding the fusion protein.
The detection method of the present invention includes a detection method including a step of detecting cleavage of NCOA4 or RUFY1 protein, or cleavage of NCOA4 or RUFY1 gene encoding NCOA4 or RUFY1 protein in a sample obtained from the subject, and the subject Detecting the presence of a fusion protein constructed from the NCOA4 or RUFY1 protein and another protein other than the NCOA4 or RUFY1 protein or the presence of a fusion gene encoding the fusion protein in the sample obtained from Detection methods are included.
　＜ＲＥＴ融合遺伝子を検出する態様＞
　以下、ＲＥＴ融合遺伝子を検出する態様について述べるが、これらに限定されるものではない。
　なお、以下の各態様における遺伝子の特定の領域の検出は、その例示に関わらず、あらかじめ解析した塩基配列に基づいて設計されたプローブ又はプライマーを用いて行っても、あるいは、シーケンシングによって行ってもよい。 <Aspect for detecting RET fusion gene>
Hereinafter, although the aspect which detects a RET fusion gene is described, it is not limited to these.
In addition, detection of a specific region of a gene in each of the following embodiments may be performed using a probe or primer designed based on a base sequence analyzed in advance or by sequencing, regardless of the examples. Also good.
　〔ＲＥＴ融合遺伝子を検出する態様（１）〕
　〈ＲＥＴ融合遺伝子を検出する態様（１－ａ）〉
　ＲＥＴ融合遺伝子を検出する一態様として、ＲＥＴ融合遺伝子が構築されているとき、ＲＥＴ遺伝子が２つ以上のポリヌクレオチドに切断されていることに基づき、ＲＥＴ遺伝子が切断されている状態、すなわち、ＲＥＴ遺伝子の５’末端側領域とＲＥＴ遺伝子の３’末端側領域との連続性が失われていることを検出することにより、ＲＥＴ融合遺伝子を検出することができる。
　具体的には、例えば、ＲＥＴ遺伝子の５’末端側領域に特異的にハイブリダイズする第１のプローブと、ＲＥＴ遺伝子の３’末端側領域に特異的にハイブリダイズする第２のプローブを用い、当該２つの遺伝子領域が染色体上で離れていることを検出することにより、ＲＥＴ融合遺伝子を検出することができる。
　なお、ＲＥＴ遺伝子由来のポリヌクレオチドと融合して融合遺伝子を構築している他の遺伝子が切断されている状態を前記方法で確認することにより、ＲＥＴ融合遺伝子を検出してもよい。 [Aspect for detecting RET fusion gene (1)]
<Aspect for detecting RET fusion gene (1-a)>
As one aspect of detecting the RET fusion gene, when the RET fusion gene is constructed, the RET gene is cleaved based on the fact that the RET gene is cleaved into two or more polynucleotides, ie, RET By detecting the loss of continuity between the 5 ′ end region of the gene and the 3 ′ end region of the RET gene, the RET fusion gene can be detected.
Specifically, for example, using a first probe that specifically hybridizes to the 5 ′ terminal region of the RET gene and a second probe that specifically hybridizes to the 3 ′ terminal region of the RET gene, By detecting that the two gene regions are separated on the chromosome, the RET fusion gene can be detected.
In addition, you may detect a RET fusion gene by confirming the state by which the other gene which is fuse | melted with the polynucleotide derived from a RET gene and the fusion gene is cut | disconnected by the said method.
　〈ＲＥＴ融合遺伝子を検出する態様（１－ｂ）〉
　他の一態様として、ＲＥＴ遺伝子の、５’末端側領域と、３’末端側領域の発現量をそれぞれ特異的に検出し、その発現量の比を求めることにより、ＲＥＴ融合遺伝子を検出することができる。具体的には、例えば、ＲＥＴ遺伝子の５’末端側領域の発現量と、ＲＥＴ遺伝子３’末端側領域の発現量とが異なる場合、ＲＥＴ融合遺伝子を検出することができる。
　あるいは、ＲＥＴ遺伝子と共にＲＥＴ融合遺伝子を構築しているＲＥＴ遺伝子以外の他の遺伝子について、前記方法で確認することにより、ＲＥＴ融合遺伝子を検出してもよい。 <Aspect for detecting RET fusion gene (1-b)>
In another embodiment, the RET fusion gene is detected by specifically detecting the expression levels of the 5 ′ terminal region and the 3 ′ terminal region of the RET gene and determining the ratio of the expression levels. Can do. Specifically, for example, when the expression level of the 5 ′ terminal region of the RET gene is different from the expression level of the RET gene 3 ′ terminal region, the RET fusion gene can be detected.
Alternatively, the RET fusion gene may be detected by confirming the gene other than the RET gene constructing the RET fusion gene together with the RET gene by the above method.
　〈ＲＥＴ融合遺伝子を検出する態様（１－ｃ）〉
　他の一態様として、ＲＥＴ融合遺伝子の形成過程において、ＲＥＴ遺伝子又はＲＥＴ遺伝子以外の他の遺伝子の少なくとも一部の複製（duplication）を伴う場合、すなわち、ＲＥＴ遺伝子由来の重複したポリヌクレオチドと、ＲＥＴと共にＲＥＴ融合遺伝子を構築しているＲＥＴ遺伝子以外の他の遺伝子由来の重複したポリヌクレオチドとからＲＥＴ融合遺伝子が構築されている場合、ＲＥＴ遺伝子由来のポリヌクレオチド又は前記他の遺伝子由来のポリヌクレオチドの重複を検出することにより、ＲＥＴ融合遺伝子を検出することができる。 <Mode for detecting RET fusion gene (1-c)>
In another embodiment, in the process of forming the RET fusion gene, when duplication of at least a part of the RET gene or another gene other than the RET gene is involved, that is, a duplicate polynucleotide derived from the RET gene, and RET When a RET fusion gene is constructed from an overlapping polynucleotide derived from another gene other than the RET gene that constructs the RET fusion gene, the polynucleotide of the RET gene or the polynucleotide derived from the other gene By detecting duplication, the RET fusion gene can be detected.
　〔ＲＥＴ融合遺伝子を検出する態様（２）〕
　ＲＥＴ融合遺伝子を検出する一態様として、ＲＥＴ融合遺伝子が、ＲＥＴ遺伝子由来ポリヌクレオチドと、ＲＥＴ遺伝子以外の他の遺伝子由来のポリヌクレオチドと融合して構築されていることに基づき、ＲＥＴ融合遺伝子における、ＲＥＴ遺伝子由来のポリヌクレオチドの少なくとも一部と、ＲＥＴ遺伝子以外の遺伝子由来のポリヌクレオチドの少なくとも一部とが連続して含まれる融合ポリヌクレオチドを検出することにより、ＲＥＴ融合遺伝子を検出することができる。
　具体的には、例えば、ＲＥＴ遺伝子以外の他の遺伝子由来のポリヌクレオチドの５’末端側領域に特異的にハイブリダイズする第１のプローブと、ＲＥＴ遺伝子由来のポリヌクレオチドの３’末端側領域に特異的にハイブリダイズする第２のプローブを用い、当該２つの遺伝子領域が染色体上で近接していることを検出することにより、ＲＥＴ融合遺伝子を検出することができる。ＲＥＴ遺伝子以外の他の遺伝子が、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の場合、すなわちＲＥＴ融合遺伝子がＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子である場合、第１のプローブはＮＣＯＡ４又はＲＵＦＹ１遺伝子由来のポリヌクレオチドの５’末端側領域に特異的にハイブリダイズするプローブを用いればよい。 [Aspect for detecting RET fusion gene (2)]
As one embodiment for detecting the RET fusion gene, the RET fusion gene is constructed by fusing a RET gene-derived polynucleotide with a polynucleotide derived from another gene other than the RET gene. The RET fusion gene can be detected by detecting a fusion polynucleotide in which at least a part of the polynucleotide derived from the RET gene and at least a part of the polynucleotide derived from a gene other than the RET gene are continuously contained. .
Specifically, for example, a first probe that specifically hybridizes to a 5 ′ terminal region of a polynucleotide derived from a gene other than the RET gene, and a 3 ′ terminal region of a polynucleotide derived from the RET gene By using a second probe that specifically hybridizes and detecting that the two gene regions are close together on the chromosome, the RET fusion gene can be detected. When the other gene other than the RET gene is an NCOA4 or RUFY1 gene, that is, when the RET fusion gene is an NCOA4 or RUFY1-RET fusion gene, the first probe is located on the 5 ′ end side of the polynucleotide derived from the NCOA4 or RUFY1 gene. A probe that specifically hybridizes to the region may be used.
　〔ＲＥＴ融合遺伝子を検出する態様（３）〕
　ＲＥＴ融合遺伝子を検出する一態様として、ＲＥＴ融合遺伝子が、ＲＥＴ遺伝子由来ポリヌクレオチドと、ＲＥＴ遺伝子以外の他の遺伝子由来ポリヌクレオチドとが、融合点において融合して構築されていることに基づき、ＲＥＴ融合遺伝子における、ＲＥＴ遺伝子由来のポリヌクレオチドの少なくとも一部と、ＲＥＴ遺伝子以外の他の遺伝子由来のポリヌクレオチドの少なくとも一部とが、融合点を含んで連続して含まれる融合ポリヌクレオチドを検出することにより、ＲＥＴ融合遺伝子を検出することができる。
　具体的には、例えば、ＲＥＴ遺伝子以外の他の遺伝子由来のポリヌクレオチドの５’末端側領域に特異的にアニールする第１のプライマーと、ＲＥＴ遺伝子由来のポリヌクレオチドの３’末端側領域に特異的にアニールする第２のプライマーとを用いてＰＣＲ反応を行い、融合点の存在を示す所定のＰＣＲ産物が得られることを確認することにより、ＲＥＴ融合遺伝子を検出することができる。 [Aspect for detecting RET fusion gene (3)]
As one embodiment for detecting the RET fusion gene, the RET fusion gene is constructed by fusing a RET gene-derived polynucleotide and a polynucleotide derived from another gene other than the RET gene at the fusion point. In the fusion gene, a fusion polynucleotide in which at least a part of the polynucleotide derived from the RET gene and at least a part of the polynucleotide derived from another gene other than the RET gene are continuously contained including the fusion point is detected. Thus, the RET fusion gene can be detected.
Specifically, for example, a first primer that specifically anneals to a 5′-terminal region of a polynucleotide derived from a gene other than the RET gene, and a specific primer that is specific to the 3′-terminal region of a polynucleotide derived from the RET gene The RET fusion gene can be detected by conducting a PCR reaction using a second primer that anneals automatically and confirming that a predetermined PCR product indicating the presence of a fusion point is obtained.
　＜ＲＥＴ融合タンパク質を検出する態様＞
　以下、ＲＥＴ融合タンパク質を検出する態様について述べるが、これらに限定されるものではない。 <Aspect for detecting RET fusion protein>
Hereinafter, although the aspect which detects a RET fusion protein is described, it is not limited to these.
　〔ＲＥＴ融合タンパク質を検出する態様（１）〕
　〈ＲＥＴ融合タンパク質を検出する態様（１－ａ）〉
　ＲＥＴ融合タンパク質を検出する態様として、ＲＥＴ融合遺伝子が構築されているとき、ＲＥＴ遺伝子にコードされるＲＥＴタンパク質も切断されていることに基づき、ＲＥＴタンパク質が切断されている状態、すなわち、ＲＥＴタンパク質のＮ末端側領域とＣ末端側領域との連続性が失われていることを検出することにより、ＲＥＴ融合タンパク質を検出することができる。
　具体的には、例えば、ＲＥＴタンパク質のＮ末端側領域に特異的に結合する第１の抗体と、ＲＥＴタンパク質のＣ末端側領域に特異的に結合する第２の抗体を用い、当該２つの領域が、同一のタンパク質に存在しないことを確認することにより、ＲＥＴ融合タンパク質を検出することができる。
　あるいは、ＲＥＴタンパク質と共に融合タンパク質を構築しているＲＥＴタンパク質以外の他のタンパク質が切断されている状態を前記方法により確認することで、ＲＥＴ融合タンパク質を検出してもよい。 [Aspect for detecting RET fusion protein (1)]
<Aspect for detecting RET fusion protein (1-a)>
As an embodiment for detecting the RET fusion protein, when the RET fusion gene is constructed, the RET protein encoded by the RET gene is also cleaved. By detecting that the continuity between the N-terminal region and the C-terminal region is lost, the RET fusion protein can be detected.
Specifically, for example, using the first antibody that specifically binds to the N-terminal region of the RET protein and the second antibody that specifically binds to the C-terminal region of the RET protein, the two regions Can be detected by confirming that they are not present in the same protein.
Alternatively, the RET fusion protein may be detected by confirming, by the above-described method, the state in which a protein other than the RET protein that constitutes the fusion protein together with the RET protein is cleaved.
　〈ＲＥＴ融合タンパク質を検出する態様（１－ｂ）〉
　他の一態様として、ＲＥＴタンパク質の、Ｎ末端側領域と、Ｃ末端側領域の発現量をそれぞれ特異的に検出し、その発現量の比を求めることにより、ＲＥＴ融合タンパク質を検出することができる。具体的には、例えば、ＲＥＴタンパク質のＮ末端側領域の発現量と、ＲＥＴタンパク質Ｃ末端側領域の発現量とが異なることを指標として、ＲＥＴ融合タンパク質を検出することができる。
　あるいは、ＲＥＴタンパク質と共にＲＥＴ融合タンパク質を構築しているＲＥＴタンパク質以外の他のタンパク質について、前記方法で確認することにより、ＲＥＴ融合タンパク質を検出してもよい。 <Aspect for detecting RET fusion protein (1-b)>
As another aspect, the RET fusion protein can be detected by specifically detecting the expression levels of the N-terminal region and the C-terminal region of the RET protein and determining the ratio of the expression levels. . Specifically, for example, the RET fusion protein can be detected using as an index the difference between the expression level of the N-terminal region of the RET protein and the expression level of the RET protein C-terminal region.
Alternatively, the RET fusion protein may be detected by confirming the protein other than the RET protein constructing the RET fusion protein together with the RET protein by the above method.
　〔ＲＥＴ融合タンパク質を検出する態様（２）〕
　ＲＥＴ融合タンパク質を検出する一態様では、ＲＥＴ融合タンパク質が、ＲＥＴタンパク質由来ポリペプチドと、ＲＥＴタンパク質以外の他のタンパク質由来のポリペプチドとが融合して構築されていることに基づき、ＲＥＴ融合タンパク質における、ＲＥＴタンパク質由来のポリペプチドの少なくとも一部と、前記他のタンパク質由来のポリペプチドの少なくとも一部が連続して含まれる融合ポリペプチドを検出することにより、ＲＥＴ融合タンパク質を検出することができる。
　具体的には、例えば、ＲＥＴタンパク質以外の他のタンパク質のＮ末端側領域に特異的に結合する第１の抗体と、ＲＥＴタンパク質のＣ末端側領域に特異的に結合する第２の抗体を用い、当該２つの領域が、同一のタンパク質に存在することを確認することにより、ＲＥＴ融合タンパク質を検出することができる。 [Aspect for detecting RET fusion protein (2)]
In one embodiment of detecting the RET fusion protein, the RET fusion protein is constructed by fusing a RET protein-derived polypeptide and a polypeptide derived from another protein other than the RET protein. The RET fusion protein can be detected by detecting a fusion polypeptide in which at least a part of the polypeptide derived from the RET protein and at least a part of the polypeptide derived from the other protein are continuously contained.
Specifically, for example, a first antibody that specifically binds to the N-terminal region of a protein other than the RET protein and a second antibody that specifically binds to the C-terminal region of the RET protein are used. The RET fusion protein can be detected by confirming that the two regions are present in the same protein.
　〔ＲＥＴ融合タンパク質を検出する態様（３）〕
　ＲＥＴ融合タンパク質を検出する一態様では、ＲＥＴ融合タンパク質が、ＲＥＴタンパク質由来ポリペプチドと、ＲＥＴタンパク質以外の他のタンパク質由来のポリペプチドとが融合点で融合して構築されていることに基づき、ＲＥＴ融合タンパク質における、前記融合点を含むＲＥＴタンパク質由来のポリペプチドの少なくとも一部と、前記他のタンパク質由来のポリペプチドの少なくとも一部が連続して含まれる融合ポリペプチドを検出することにより、ＲＥＴ融合タンパク質を検出することができる。
　具体的には、例えば、ＲＥＴ融合タンパク質の融合点を含むポリペプチドを特異的に認識する抗体を用いた免疫学的測定法により、ＲＥＴ融合タンパク質を検出することができる。 [Aspect (3) for detecting RET fusion protein]
In one embodiment of detecting a RET fusion protein, the RET fusion protein is constructed by fusing a RET protein-derived polypeptide and a polypeptide derived from another protein other than the RET protein at a fusion point. By detecting a fusion polypeptide in which at least a part of the polypeptide derived from the RET protein containing the fusion point and at least a part of the polypeptide derived from the other protein are continuously contained in the fusion protein, Protein can be detected.
Specifically, for example, the RET fusion protein can be detected by an immunological assay using an antibody that specifically recognizes a polypeptide containing the fusion point of the RET fusion protein.
　〔ＲＥＴ融合タンパク質を検出する態様（４）〕
　ＲＥＴ融合タンパク質を検出する一態様では、ＲＥＴ融合タンパク質の活性を指標にＲＥＴ融合タンパク質を検出することができる。
　具体的には、例えば、野生型ＲＥＴタンパク質に対して阻害活性のある物質を用いて野生型ＲＥＴタンパク質の活性を阻害した上で、ＲＥＴタンパク質のキナーゼ活性を測定し、ＲＥＴ融合タンパク質を含まない（野生型ＲＥＴタンパク質のみを含む）場合に比較して、活性が高いことを指標にＲＥＴ融合タンパク質を検出することができる。なお、ＲＥＴタンパク質のキナーゼ活性の測定には当業者に周知の方法を適宜選択することができ、例えば、ＲＥＴによりリン酸化を受ける分子のリン酸化状態を検出してもよい。 [Aspect (4) for detecting RET fusion protein]
In one embodiment of detecting the RET fusion protein, the RET fusion protein can be detected using the activity of the RET fusion protein as an index.
Specifically, for example, after inhibiting the activity of the wild type RET protein using a substance having an inhibitory activity against the wild type RET protein, the kinase activity of the RET protein is measured, and the RET fusion protein is not included ( The RET fusion protein can be detected with an index of high activity as compared to the case of including only the wild-type RET protein. For the measurement of the kinase activity of the RET protein, a method well known to those skilled in the art can be appropriately selected. For example, the phosphorylation state of a molecule that is phosphorylated by RET may be detected.
　なお、ＲＥＴ融合タンパク質の検出は、ＲＥＴ融合タンパク質を構成する全長ポリペプチドの存在、あるいは、ＲＥＴ融合タンパク質の一部を構成するポリペプチドの存在を指標として行ってもよく、ＲＥＴ融合タンパク質の存在が確認できる範囲で制限されない。 The detection of the RET fusion protein may be performed by using the presence of the full-length polypeptide constituting the RET fusion protein or the presence of the polypeptide constituting a part of the RET fusion protein as an index. It is not limited as far as it can be confirmed.
　＜ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する態様＞
　以下、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する態様について述べるが、これらに限定されるものではない。
　なお、以下の各態様における遺伝子の特定の領域の検出は、その例示に関わらず、あらかじめ解析した塩基配列に基づいて設計されたプローブ又はプライマーを用いて行っても、あるいは、シーケンシングによって行ってもよい。 <Mode for detecting NCOA4 or RUFY1 fusion gene>
Hereinafter, although the aspect which detects NCOA4 or RUFY1 fusion gene is described, it is not limited to these.
In addition, detection of a specific region of a gene in each of the following embodiments may be performed using a probe or primer designed based on a base sequence analyzed in advance or by sequencing, regardless of the examples. Also good.
　〔ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する態様（１）〕
　〈ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する態様（１－ａ）〉
　ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する一態様として、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子が構築されているとき、ＮＣＯＡ４又はＲＵＦＹ１遺伝子が２つ以上のポリヌクレオチドに切断されていることに基づき、ＮＣＯＡ４又はＲＵＦＹ１遺伝子が切断されている状態、すなわち、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の５’末端側領域とＮＣＯＡ４又はＲＵＦＹ１遺伝子の３’末端側領域との連続性が失われていることを検出することにより、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出することができる。
　具体的には、例えば、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の５’末端側領域に特異的にハイブリダイズする第１のプローブと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の３’末端側領域に特異的にハイブリダイズする第２のプローブを用い、当該２つの遺伝子領域が染色体上で離れていることを検出することにより、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出することができる。
　なお、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来のポリヌクレオチドと融合して融合遺伝子を構築している他の遺伝子が切断されている状態を前記方法で確認することにより、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出してもよい。 [Mode for detecting NCOA4 or RUFY1 fusion gene (1)]
<Mode for detecting NCOA4 or RUFY1 fusion gene (1-a)>
As one aspect of detecting the NCOA4 or RUFY1 fusion gene, when the NCOA4 or RUFY1 fusion gene is constructed, the NCOA4 or RUFY1 gene is cleaved based on the fact that the NCOA4 or RUFY1 gene is cleaved into two or more polynucleotides. By detecting that the continuity between the 5 ′ terminal region of the NCOA4 or RUFY1 gene and the 3 ′ terminal region of the NCOA4 or RUFY1 gene is lost, the NCOA4 or RUFY1 fusion gene is detected. Can be detected.
Specifically, for example, a first probe that specifically hybridizes to the 5 ′ end region of the NCOA4 or RUFY1 gene and a second probe that specifically hybridizes to the 3 ′ end region of the NCOA4 or RUFY1 gene By detecting that the two gene regions are separated on the chromosome using a probe, the NCOA4 or RUFY1 fusion gene can be detected.
In addition, the NCOA4 or RUFY1 fusion gene may be detected by confirming the state in which the other gene constructing the fusion gene fused with the polynucleotide derived from the NCOA4 or RUFY1 gene is cleaved by the above method. .
　〈ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する態様（１－ｂ）〉
　他の一態様として、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の、５’末端側領域と、３’末端側領域の発現量をそれぞれ特異的に検出し、その発現量の比を求めることにより、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出することができる。具体的には、例えば、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の５’末端側領域の発現量と、ＮＣＯＡ４又はＲＵＦＹ１遺伝子３’末端側領域の発現量とが異なる場合、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出することができる。
　あるいは、ＮＣＯＡ４又はＲＵＦＹ１遺伝子と共にＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を構築しているＮＣＯＡ４又はＲＵＦＹ１遺伝子以外の他の遺伝子について、前記方法で確認することにより、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出してもよい。 <Aspect for detecting NCOA4 or RUFY1 fusion gene (1-b)>
In another embodiment, the NCOA4 or RUFY1 fusion gene is detected by specifically detecting the expression levels of the 5 ′ terminal region and the 3 ′ terminal region of the NCOA4 or RUFY1 gene, respectively, and determining the ratio of the expression levels. Can be detected. Specifically, for example, when the expression level of the 5 ′ end region of the NCOA4 or RUFY1 gene is different from the expression level of the NCOA4 or RUFY1 gene 3 ′ end region, the NCOA4 or RUFY1 fusion gene can be detected. .
Alternatively, the NCOA4 or RUFY1 fusion gene may be detected by confirming the NCOA4 or RUFY1 fusion gene together with the NCOA4 or RUFY1 gene by using the above method for other genes other than the NCOA4 or RUFY1 gene.
　〈ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する態様（１－ｃ）〉
　他の一態様として、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の形成過程において、ＮＣＯＡ４又はＲＵＦＹ１遺伝子、あるいは、ＮＣＯＡ４又はＲＵＦＹ１遺伝子以外の他の遺伝子の少なくとも一部の複製（duplication）を伴う場合、すなわち、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来の重複したポリヌクレオチドと、ＮＣＯＡ４又はＲＵＦＹ１と共にＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を構築しているＮＣＯＡ４又はＲＵＦＹ１遺伝子以外の他の遺伝子由来の重複したポリヌクレオチドとからＮＣＯＡ４又はＲＵＦＹ１融合遺伝子が構築されている場合、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来のポリヌクレオチド又は前記他の遺伝子由来のポリヌクレオチドの重複を検出することにより、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出することができる。 <Mode for detecting NCOA4 or RUFY1 fusion gene (1-c)>
As another aspect, in the process of forming the NCOA4 or RUFY1 fusion gene, the duplication of at least a part of the NCOA4 or RUFY1 gene or another gene other than the NCOA4 or RUFY1 gene is involved, ie, NCOA4 or RUFY1. An NCOA4 or RUFY1 fusion gene is constructed from a duplicated polynucleotide derived from a gene and a duplicated polynucleotide derived from an NCOA4 or RUFY1 gene other than the NCOA4 or RUFY1 gene together with NCOA4 or RUFY1. In some cases, detecting an NCOA4 or RUFY1 fusion gene by detecting duplication of a polynucleotide derived from the NCOA4 or RUFY1 gene or the polynucleotide derived from the other gene Kill.
　〔ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する態様（２）〕
　ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する一態様として、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子が、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来ポリヌクレオチドと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子以外の他の遺伝子由来のポリヌクレオチドと融合して構築されていることに基づき、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子における、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来のポリヌクレオチドの少なくとも一部と、ＮＣＯＡ４又はＲＵＦＹ１遺伝子以外の遺伝子由来のポリヌクレオチドの少なくとも一部とが連続して含まれる融合ポリヌクレオチドを検出することにより、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出することができる。
　具体的には、例えば、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来のポリヌクレオチドの５’末端側領域に特異的にハイブリダイズする第１のプローブと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子以外の他の遺伝子由来のポリヌクレオチドの３’末端側領域に特異的にハイブリダイズする第２のプローブを用い、当該２つの遺伝子領域が染色体上で近接していることを検出することにより、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出することができる。ＮＣＯＡ４又はＲＵＦＹ１遺伝子以外の他の遺伝子が、ＲＥＴ遺伝子の場合、すなわちＮＣＯＡ４又はＲＵＦＹ１融合遺伝子がＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子である場合、第２のプローブはＲＥＴ遺伝子由来のポリヌクレオチドの３’末端側領域に特異的にハイブリダイズするプローブを用いればよい。 [Mode for detecting NCOA4 or RUFY1 fusion gene (2)]
As one mode for detecting the NCOA4 or RUFY1 fusion gene, the NCOA4 or RUFY1 fusion gene is constructed by fusing a polynucleotide derived from the NCOA4 or RUFY1 gene with a polynucleotide derived from another gene other than the NCOA4 or RUFY1 gene. A fusion polynucleotide comprising at least a part of a polynucleotide derived from the NCOA4 or RUFY1 gene and at least a part of a polynucleotide derived from a gene other than the NCOA4 or RUFY1 gene in the NCOA4 or RUFY1 fusion gene, By detecting, the NCOA4 or RUFY1 fusion gene can be detected.
Specifically, for example, a first probe that specifically hybridizes to the 5′-terminal region of a polynucleotide derived from the NCOA4 or RUFY1 gene, and 3 ′ of a polynucleotide derived from another gene other than the NCOA4 or RUFY1 gene By using a second probe that specifically hybridizes to the terminal region and detecting that the two gene regions are close together on the chromosome, the NCOA4 or RUFY1 fusion gene can be detected. When the other gene other than the NCOA4 or RUFY1 gene is a RET gene, that is, when the NCOA4 or RUFY1 fusion gene is an NCOA4 or RUFY1-RET fusion gene, the second probe is located on the 3 ′ end side of the polynucleotide derived from the RET gene. A probe that specifically hybridizes to the region may be used.
　〔ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する態様（３）〕
　ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する一態様として、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子が、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来ポリヌクレオチドと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子以外の他の遺伝子由来ポリヌクレオチドとが、融合点において融合して構築されていることに基づき、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子における、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来のポリヌクレオチドの少なくとも一部と、ＮＣＯＡ４又はＲＵＦＹ１遺伝子以外の他の遺伝子由来のポリヌクレオチドの少なくとも一部とが、融合点を含んで連続して含まれる融合ポリヌクレオチドを検出することにより、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出することができる。
　具体的には、例えば、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来のポリヌクレオチドの５’末端側領域に特異的にアニールする第１のプライマーと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子以外の他の遺伝子由来のポリヌクレオチドの３’末端側領域に特異的にアニールする第２のプライマーとを用いてＰＣＲ反応を行い、融合点の存在を示す所定のＰＣＲ産物が得られることを確認することにより、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出することができる。 [Mode for detecting NCOA4 or RUFY1 fusion gene (3)]
As one mode for detecting the NCOA4 or RUFY1 fusion gene, the NCOA4 or RUFY1 fusion gene is constructed by fusing the NCOA4 or RUFY1 gene-derived polynucleotide and a polynucleotide derived from another gene other than the NCOA4 or RUFY1 gene at the fusion point. In the NCOA4 or RUFY1 fusion gene, at least a part of the polynucleotide derived from the NCOA4 or RUFY1 gene and at least a part of the polynucleotide derived from another gene other than the NCOA4 or RUFY1 gene It is possible to detect the NCOA4 or RUFY1 fusion gene by detecting a fusion polynucleotide continuously contained.
Specifically, for example, a first primer that specifically anneals to the 5 ′ end region of a polynucleotide derived from the NCOA4 or RUFY1 gene, and the 3 ′ end of a polynucleotide derived from another gene other than the NCOA4 or RUFY1 gene Detect an NCOA4 or RUFY1 fusion gene by performing a PCR reaction with a second primer that specifically anneals to the side region, and confirming that a predetermined PCR product indicating the presence of a fusion point is obtained. Can do.
　＜ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出する態様＞
　以下、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出する態様について述べるが、これらに限定されるものではない。 <Aspect for detecting NCOA4 or RUFY1 fusion protein>
Hereinafter, although the aspect which detects NCOA4 or RUFY1 fusion protein is described, it is not limited to these.
　〔ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出する態様（１）〕
　〈ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出する態様（１－ａ）〉
　ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出する態様として、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子が構築されているとき、ＮＣＯＡ４又はＲＵＦＹ１遺伝子にコードされるＮＣＯＡ４又はＲＵＦＹ１タンパク質も切断されていることに基づき、ＮＣＯＡ４又はＲＵＦＹ１タンパク質が切断されている状態、すなわち、ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＮ末端側領域とＣ末端側領域との連続性が失われていることを検出することにより、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出することができる。
　具体的には、例えば、ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＮ末端側領域に特異的に結合する第１の抗体と、ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＣ末端側領域に特異的に結合する第２の抗体を用い、当該２つの領域が、同一のタンパク質に存在しないことを確認することにより、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出することができる。
　あるいは、ＮＣＯＡ４又はＲＵＦＹ１タンパク質と共に融合タンパク質を構築しているＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質が切断されている状態を前記方法により確認することで、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出してもよい。 [Mode for detecting NCOA4 or RUFY1 fusion protein (1)]
<Aspect for detecting NCOA4 or RUFY1 fusion protein (1-a)>
As a mode of detecting the NCOA4 or RUFY1 fusion protein, when the NCOA4 or RUFY1 fusion gene is constructed, the NCOA4 or RUFY1 protein encoded by the NCOA4 or RUFY1 gene is also cleaved based on the fact that the NCOA4 or RUFY1 protein is cleaved. By detecting that the continuity between the N-terminal region and the C-terminal region of the NCOA4 or RUFY1 protein is lost, the NCOA4 or RUFY1 fusion protein can be detected.
Specifically, for example, a first antibody that specifically binds to the N-terminal region of NCOA4 or RUFY1 protein and a second antibody that specifically binds to the C-terminal region of NCOA4 or RUFY1 protein, By confirming that the two regions are not present in the same protein, the NCOA4 or RUFY1 fusion protein can be detected.
Alternatively, the NCOA4 or RUFY1 fusion protein may be detected by confirming, by the above-described method, the state in which other proteins other than the NCOA4 or RUFY1 protein that constitutes the fusion protein together with the NCOA4 or RUFY1 protein are cleaved.
　〈ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出する態様（１－ｂ）〉
　他の一態様として、ＮＣＯＡ４又はＲＵＦＹ１タンパク質の、Ｎ末端側領域と、Ｃ末端側領域の発現量をそれぞれ特異的に検出し、その発現量の比を求めることにより、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出することができる。具体的には、例えば、ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＮ末端側領域の発現量と、ＮＣＯＡ４又はＲＵＦＹ１タンパク質Ｃ末端側領域の発現量とが異なることを指標として、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出することができる。
　あるいは、ＮＣＯＡ４又はＲＵＦＹ１タンパク質と共にＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を構築しているＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質について、前記方法で確認することにより、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出してもよい。 <Aspect for detecting NCOA4 or RUFY1 fusion protein (1-b)>
As another embodiment, the NCOA4 or RUFY1 fusion protein is detected by specifically detecting the expression levels of the N-terminal region and the C-terminal region of the NCOA4 or RUFY1 protein, respectively, and determining the ratio of the expression levels. can do. Specifically, for example, the detection of NCOA4 or RUFY1 fusion protein can be performed using as an indicator that the expression level of the N-terminal region of NCOA4 or RUFY1 protein is different from the expression level of the NCOA4 or RUFY1 protein C-terminal region. it can.
Alternatively, the NCOA4 or RUFY1 fusion protein may be detected by confirming the NCOA4 or RUFY1 fusion protein together with the NCOA4 or RUFY1 protein by the above-described method for other proteins other than the NCOA4 or RUFY1 protein.
　〔ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出する態様（２）〕
　ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出する一態様では、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が、ＮＣＯＡ４又はＲＵＦＹ１タンパク質由来ポリペプチドと、ＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質由来のポリペプチドとが融合して構築されていることに基づき、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質における、ＮＣＯＡ４又はＲＵＦＹ１タンパク質由来のポリペプチドの少なくとも一部と、前記他のタンパク質由来のポリペプチドの少なくとも一部が連続して含まれる融合ポリペプチドを検出することにより、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出することができる。
　具体的には、例えば、ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＮ末端側領域に特異的に結合する第１の抗体と、ＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質のＣ末端側領域に特異的に結合する第２の抗体を用い、当該２つの領域が、同一のタンパク質に存在することを確認することにより、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出することができる。 [Aspect (2) for detecting NCOA4 or RUFY1 fusion protein]
In one embodiment of detecting an NCOA4 or RUFY1 fusion protein, the NCOA4 or RUFY1 fusion protein is constructed by fusing an NCOA4 or RUFY1 protein-derived polypeptide with a polypeptide derived from another protein other than the NCOA4 or RUFY1 protein. In particular, detecting a fusion polypeptide in which at least a part of the polypeptide derived from the NCOA4 or RUFY1 protein and at least a part of the polypeptide derived from the other protein are continuously contained in the NCOA4 or RUFY1 fusion protein Can detect NCOA4 or RUfy1 fusion protein.
Specifically, for example, a first antibody that specifically binds to the N-terminal region of NCOA4 or RUFY1 protein and a second antibody that specifically binds to the C-terminal region of other proteins other than NCOA4 or RUFY1 protein By confirming that the two regions are present in the same protein, it is possible to detect the NCOA4 or RUFY1 fusion protein.
　〔ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出する態様（３）〕
　ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出する一態様では、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が、ＮＣＯＡ４又はＲＵＦＹ１タンパク質由来ポリペプチドと、ＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質由来のポリペプチドとが融合点で融合して構築されていることに基づき、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質における、前記融合点を含むＮＣＯＡ４又はＲＵＦＹ１タンパク質由来のポリペプチドの少なくとも一部と、前記他のタンパク質由来のポリペプチドの少なくとも一部が連続して含まれる融合ポリペプチドを検出することにより、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出することができる。
　具体的には、例えば、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の融合点を含むポリペプチドを特異的に認識する抗体を用いた免疫学的測定法により、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出することができる。 [Aspect (3) for detecting NCOA4 or RUFY1 fusion protein]
In one embodiment of detecting the NCOA4 or RUFY1 fusion protein, the NCOA4 or RUFY1 fusion protein is constructed by fusing the NCOA4 or RUFY1 protein-derived polypeptide with a polypeptide derived from another protein other than the NCOA4 or RUFY1 protein at the fusion point. In the NCOA4 or RUFY1 fusion protein, at least a part of the polypeptide derived from the NCOA4 or RUFY1 protein containing the fusion point and at least a part of the polypeptide derived from the other protein are continuously included. By detecting the fusion polypeptide, the NCOA4 or RUfy1 fusion protein can be detected.
Specifically, for example, the NCOA4 or RUFY1 fusion protein can be detected by an immunoassay using an antibody that specifically recognizes a polypeptide containing the fusion point of the NCOA4 or RUFY1 fusion protein.
　〔ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出する態様（４）〕
　ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出する一態様では、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の活性を指標にＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出することができる。
　具体的には、例えば、ＮＣＯＡ４又はＲＵＦＹ１タンパク質と共に融合タンパク質を構築するＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質が酵素活性を有するタンパク質である場合、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を含まない（野生型ＮＣＯＡ４又はＲＵＦＹ１タンパク質のみを含む）場合に比較して、当該酵素活性が高いことを指標に、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出することができる。なお、酵素活性の測定には当業者に周知の方法を適宜選択することができ、例えば、前記他のタンパク質がキナーゼ活性を有するタンパク質（好ましくはＲＥＴタンパク質）である場合、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質によりリン酸化を受ける分子のリン酸化状態を検出してもよい。 [Mode for detecting NCOA4 or RUFY1 fusion protein (4)]
In one aspect of detecting the NCOA4 or RUFY1 fusion protein, the NCOA4 or RUFY1 fusion protein can be detected using the activity of the NCOA4 or RUFY1 fusion protein as an index.
Specifically, for example, when the other protein other than the NCOA4 or RUFY1 protein that constructs the fusion protein together with the NCOA4 or RUFY1 protein is a protein having an enzyme activity, the NCOA4 or RUFY1 fusion protein is not included (wild-type NCOA4 or RUFY1 NCOA4 or RUFY1 fusion protein can be detected using the high enzyme activity as an index compared to the case of containing only protein). For the measurement of enzyme activity, a method well known to those skilled in the art can be appropriately selected. For example, when the other protein is a protein having kinase activity (preferably a RET protein), You may detect the phosphorylation state of the molecule which receives phosphorylation.
　なお、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の検出は、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を構成する全長ポリペプチドの存在、あるいは、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の一部を構成するポリペプチドの存在を指標として行ってもよく、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の存在が確認できる範囲で制限されない。 The detection of the NCOA4 or RUFY1 fusion protein may be performed using the presence of the full-length polypeptide constituting the NCOA4 or RUFY1 fusion protein or the presence of the polypeptide constituting a part of the NCOA4 or RUFY1 fusion protein as an index, It is not limited as long as the presence of the NCOA4 or RUFY1 fusion protein can be confirmed.
≪検出方法に用いる技法≫
　以下、ＲＥＴ融合遺伝子（ゲノムＤＮＡ、ｍＲＮＡ、又はｃＤＮＡ）の検出、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子（ゲノムＤＮＡ、ｍＲＮＡ、又はｃＤＮＡ）の検出、ＲＥＴ融合タンパク質の検出、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の検出の各工程及び各検出技法について、より詳細に説明するが、これらに制限されるものではない。
　なお、被験者から得た試料から、遺伝子（ゲノムＤＮＡ、又は、ｍＲＮＡ）又は、タンパク質を抽出した場合、あるいは、組織切片、又は、細胞懸濁液等を作成した場合において、調製された試料においてＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子、あるいは、ＲＥＴ融合タンパク質又はＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を検出するために好適な技法は、当業者が適宜選択することができる。 ≪Technique used for detection method≫
Hereinafter, each step of detection of RET fusion gene (genomic DNA, mRNA, or cDNA), detection of NCOA4 or RUFY1 fusion gene (genomic DNA, mRNA, or cDNA), detection of RET fusion protein, detection of NCOA4 or RUFY1 fusion protein Each detection technique is described in more detail, but is not limited thereto.
In addition, when a gene (genomic DNA or mRNA) or protein is extracted from a sample obtained from a subject, or when a tissue section or a cell suspension is prepared, RET is prepared in the prepared sample. A suitable technique for detecting the fusion gene or NCOA4 or RUFY1 fusion gene, or the RET fusion protein or NCOA4 or RUFY1 fusion protein can be appropriately selected by those skilled in the art.
　＜融合遺伝子の検出＞
　ＲＥＴ融合遺伝子、又は、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出は、ＲＥＴ融合遺伝子、又は、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子のゲノムＤＮＡの検出、当該ゲノムＤＮＡの転写産物であるｍＲＮＡの検出、又は、ｍＲＮＡを鋳型として得られるｃＤＮＡの検出のいずれであってもよい。
　被験者から得た試料中の、ＲＥＴ融合遺伝子（ゲノムＤＮＡ、又は、ｍＲＮＡ）又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子（ゲノムＤＮＡ、又は、ｍＲＮＡ）を検出する技法としては、ＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の少なくとも一部にハイブリダイズするプローブ（核酸プローブ等）を使用したハイブリダイゼーション技術、あるいは、ＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の少なくとも一部にアニールするプライマーを用いた遺伝子増幅技術等、遺伝子の検出に用いられる当業者に周知のいかなる技法、及び、これらの技法を応用した技法を用いることができる。
　すなわち、ＰＣＲ、ＬＣＲ（Ligase chain reaction）、ＳＤＡ（Strand displacement amplification）、ＮＡＳＢＡ（Nucleic acid sequence-based amplification）、ＩＣＡＮ（Isothermal and chimeric primer-initiated amplification of nucleic acids）、ＬＡＭＰ（Loop-mediated isothermal amplification）法、ＴＭＡ法（Gen-Probe’s TMA system）、インサイチュハイブリダイゼーション法、マイクロアレイ法、ノーザンハイブリダイゼーション、サザンハイブリダイゼーション、ドットブロット法、ＲＮＡプロテクション法、ＤＮＡシーケンス、ＲＮＡシーケンス等のいかなる技法を用いてもよい。 <Fusion gene detection>
The detection of the RET fusion gene or NCOA4 or RUFY1 fusion gene is performed by detecting the genomic DNA of the RET fusion gene or NCOA4 or RUFY1 fusion gene, detecting the mRNA that is the transcription product of the genomic DNA, or using the mRNA as a template. Any of detection of cDNA obtained may be sufficient.
As a technique for detecting a RET fusion gene (genomic DNA or mRNA) or NCOA4 or RUFY1 fusion gene (genomic DNA or mRNA) in a sample obtained from a subject, a RET fusion gene or NCOA4 or RUFY1 fusion gene Detection of genes such as hybridization techniques using probes that hybridize to at least part (nucleic acid probes, etc.), or gene amplification techniques that use primers that anneal to at least part of RET fusion genes or NCOA4 or RUFY1 fusion genes Any technique well known to those skilled in the art and applied to these techniques can be used.
PCR, LCR (Ligase chain reaction), SDA (Strand displacement amplification), NASBA (Nucleic acid sequence-based amplification), ICAN (Isothermal and chimeric primer-initiated amplification of nucleic acids), LAMP (Loop-mediated isothermal amplification) Any technique may be used, such as a method, a TMA method (Gen-Probe's TMA system), an in situ hybridization method, a microarray method, a Northern hybridization, a Southern hybridization, a dot blot method, an RNA protection method, a DNA sequence, or an RNA sequence .
　〔ゲノムＤＮＡの検出〕
　ゲノムＤＮＡの検出には、インサイチュハイブリダイゼーション技術を好適に用いることができる。インサイチュハイブリダイゼーション技術を利用した検出は、例えば、公知のＦＩＳＨ法に従って実施することができる。又は、クロモジェニックインサイチュハイブリダイゼーション（ＣＩＳＨ）法とシルバーインサイチュハイブリダイゼーション（ＳＩＳＨ）法を組み合わせたフュージョンアッセイ（fusion assay）で実施することができる。好適には、以下に詳述のＦＩＳＨ法スプリットアッセイ、又は、ＦＩＳＨ法フュージョンアッセイで検出することができる。 [Detection of genomic DNA]
For detection of genomic DNA, an in situ hybridization technique can be suitably used. Detection using an in situ hybridization technique can be performed, for example, according to a known FISH method. Or it can implement by the fusion assay (fusion assay) which combined the chromogenic in situ hybridization (CISH) method and the silver in situ hybridization (SISH) method. Preferably, it can be detected by the FISH method split assay or FISH method fusion assay described in detail below.
　あるいは、ゲノムＤＮＡの検出には、ＤＮＡシーケンス技術を好適に用いることができる。シーケンシングには、従来のサンガー法に基づくシーケンサーを使用してもよいが、解析の効率を考慮すると、次世代シーケンサーを使用することが好ましい（例えば、Metzker ML、Nat Rev Genet. 2010 Jan;11(1):31-46参照）。次世代シーケンサーとしては、Ｉｌｌｕｍｉｎａ社のＭｉＳｅｑ/ＨｉＳｅｑ、Ｌｉｆｅ Ｔｅｃｈｎｏｇｉｅｓ社のＳＯＬｉＤシステム、Ｒｏｃｈｅ社の４５４シーケンスシステム（ＧＳ ＦＬＸ＋／ＧＳ Ｊｕｎｉｏｒ）等が例示できる。シーケンシングにおいては、シーケンスキャプチャ技術等を用いて、融合遺伝子が存在している可能性がある領域を濃縮（enrich）することで、解析の効率を向上させることができる。シーケンスキャプチャ技術としては、Ｒｏｃｈｅ社のＲｏｃｈｅ ＮｉｍｂｌｅＧｅｎ、Ａｇｉｌｅｎｔ Ｔｅｃｈｎｏｌｏｇｉｅｓ社のＳｕｒｅ Ｓｅｌｅｃｔ等が例示できる。
　以下に、ゲノムＤＮＡの検出のための代表的な方法を例示するが、これらに限定されるものではない。 Alternatively, DNA sequencing technology can be suitably used for detecting genomic DNA. For sequencing, a sequencer based on the conventional Sanger method may be used, but in consideration of analysis efficiency, it is preferable to use a next-generation sequencer (for example, Metzker ML, Nat Rev Genet. 2010 Jan; 11 (See (1): 31-46). Illustrative examples of the next-generation sequencer include MiSeq / HiSeq from Illumina, SOLiD system from Life Technologies, 454 sequence system (GS FLX + / GS Junior) from Roche. In sequencing, the efficiency of analysis can be improved by enriching a region where a fusion gene may exist using a sequence capture technique or the like. Examples of the sequence capture technology include Roche NimbleGen from Roche, Sure Select from Agilent Technologies, and the like.
Hereinafter, representative methods for detecting genomic DNA are exemplified, but the present invention is not limited thereto.
　〈ＦＩＳＨ法スプリットアッセイ〉
　ＲＥＴ融合遺伝子のＦＩＳＨ法スプリットアッセイでは、検出用プローブとして、ＲＥＴ遺伝子の５’末端側ゲノム領域をカバーするポリヌクレオチドであって蛍光標識したものと、同遺伝子の３’末端側ゲノム領域をカバーするポリヌクレオチドであって別の蛍光色素で標識したものとの組み合わせを使用する。正常な場合（野生型ＲＥＴ遺伝子の場合）には、２つの遺伝子領域（各遺伝子ごとの５’末端側領域と３’末端側領域）が近接しているため、２つのシグナルが重なった色（例えば、赤色蛍光色素と緑色蛍光色素を使用する場合には、黄色）として検出されるのに対して、転座又は逆位により２つの遺伝子領域が切断されている場合は、２種類の蛍光色素に由来するシグナル（例えば、赤色と緑色）が別々に離れて検出される。従って、ＦＩＳＨ法スプリットアッセイでは、ＲＥＴ遺伝子の５’末端側ゲノム領域とＲＥＴ遺伝子３’末端側ゲノム領域とが染色体上で離れていることを検出することにより、ＲＥＴ融合遺伝子の存在を検出している。 <FISH method split assay>
In the FISH split assay of a RET fusion gene, as a detection probe, a polynucleotide covering the 5 ′ end genomic region of the RET gene and fluorescently labeled, and the 3 ′ end genomic region of the same gene are covered. A combination with a polynucleotide labeled with another fluorescent dye is used. In the normal case (in the case of the wild type RET gene), the two gene regions (the 5 ′ end region and the 3 ′ end region for each gene) are close to each other, so the two signals overlap ( For example, when red fluorescent dye and green fluorescent dye are used, it is detected as yellow), whereas when two gene regions are cleaved by translocation or inversion, two types of fluorescent dyes are detected. Signals derived from (eg red and green) are detected separately. Therefore, in the FISH split assay, the presence of the RET fusion gene is detected by detecting that the 5′-end genomic region of the RET gene and the genomic region of the RET gene 3′-end are separated on the chromosome. Yes.
　また、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子のＦＩＳＨ法スプリットアッセイでは、検出用プローブとして、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の５’末端側ゲノム領域をカバーするポリヌクレオチドであって蛍光標識したものと、同遺伝子の３’末端側ゲノム領域をカバーするポリヌクレオチドであって別の蛍光色素で標識したものとの組み合わせを使用する。正常な場合（野生型ＮＣＯＡ４又はＲＵＦＹ１遺伝子の場合）には、２つの遺伝子領域（各遺伝子ごとの５’末端側領域と３’末端側領域）が近接しているため、２つのシグナルが重なった色（例えば、赤色蛍光色素と緑色蛍光色素を使用する場合には、黄色）として検出されるのに対して、転座又は逆位により２つの遺伝子領域が切断されている場合は、２種類の蛍光色素に由来するシグナル（例えば、赤色と緑色）が別々に離れて検出される。従って、ＦＩＳＨ法スプリットアッセイでは、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の５’末端側ゲノム領域と同遺伝子の３’末端側ゲノム領域とが染色体上で離れていることを検出することにより、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の存在を検出している。 Moreover, in the FISH method split assay of the NCOA4 or RUFY1 fusion gene, as a detection probe, a polynucleotide that covers the 5'-terminal genomic region of the NCOA4 or RUFY1 gene and is fluorescently labeled, and the 3 'end of the same gene A combination with a polynucleotide covering the side genomic region and labeled with another fluorescent dye is used. In normal cases (in the case of wild-type NCOA4 or RUFY1 gene), the two gene regions (5 ′ end region and 3 ′ end region for each gene) are close to each other, so the two signals overlapped. If the two gene regions are cleaved by translocation or inversion while being detected as a color (for example, yellow when red and green fluorescent dyes are used) Signals derived from fluorescent dyes (eg, red and green) are detected separately. Therefore, in the FISH split assay, by detecting that the 5′-end genomic region of the NCOA4 or RUFY1 gene and the 3′-end genomic region of the same gene are separated on the chromosome, the NCOA4 or RUFY1 fusion gene The presence is detected.
　ＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子がＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子の場合、検出用プローブとして、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の５’末端側ゲノム領域をカバーするポリヌクレオチドであって蛍光標識したものと、同遺伝子の３’末端側ゲノム領域をカバーするポリヌクレオチドであって別の蛍光色素で標識したものとの組み合わせ、あるいは、ＲＥＴ遺伝子の５’末端側ゲノム領域をカバーするポリヌクレオチドであって蛍光標識したものと、同遺伝子の３’末端側ゲノム領域をカバーするポリヌクレオチドであって別の蛍光色素で標識したものとの組み合わせを使用することにより、ＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子を検出することができる。 When the RET fusion gene or NCOA4 or RUFY1 fusion gene is an NCOA4 or RUFY1-RET fusion gene, as a detection probe, a polynucleotide that covers the 5'-terminal genomic region of the NCOA4 or RUFY1 gene and is fluorescently labeled; A combination of a polynucleotide covering the 3'-end genomic region of the same gene labeled with another fluorescent dye, or a polynucleotide covering the 5'-end genomic region of the RET gene and fluorescently labeled Can be detected by using a combination of the above and a polynucleotide covering the 3′-terminal genomic region of the same gene and labeled with another fluorescent dye, to detect the NCOA4 or RUFY1-RET fusion gene. it can.
　〈ＦＩＳＨ法フュージョンアッセイ〉
　ＲＥＴ融合遺伝子のＦＩＳＨ法フュージョンアッセイでは、例えば、ＲＥＴ融合遺伝子がＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子の場合、検出用プローブとして、ＲＥＴ遺伝子の３’末端側ゲノム領域をカバーするポリヌクレオチドであって蛍光標識したものと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の５’末端側ゲノム領域をカバーするポリヌクレオチドであって別の蛍光色素で標識したものとの組み合わせを使用することができる。正常な場合（野生型ＲＥＴ遺伝子の場合）には、２種類の蛍光色素に由来するシグナル（例えば、赤色と緑色）が別々に離れて検出されるのに対して、転座又は逆位により２つの遺伝子領域が近接している場合は、２つのシグナルが重なった色（例えば、黄色）として検出される。 <FISH method fusion assay>
In the FISH fusion assay of a RET fusion gene, for example, when the RET fusion gene is an NCOA4 or RUFY1-RET fusion gene, it is a polynucleotide that covers the 3 'terminal genomic region of the RET gene and is fluorescently labeled as a detection probe And a combination of a polynucleotide covering the 5 'terminal genomic region of the NCOA4 or RUFY1 gene and labeled with another fluorescent dye can be used. In normal cases (in the case of the wild-type RET gene), signals derived from two types of fluorescent dyes (for example, red and green) are detected separately, whereas translocation or inversion 2 When two gene regions are close to each other, two signals are detected as overlapping colors (for example, yellow).
　また、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子のＦＩＳＨ法フュージョンアッセイでは、例えば、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子がＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子の場合、検出用プローブとして、ＲＥＴ遺伝子の３’末端側ゲノム領域をカバーするポリヌクレオチドであって蛍光標識したものと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の５’末端側ゲノム領域をカバーするポリヌクレオチドであって別の蛍光色素で標識したものとの組み合わせを使用することができる。正常な場合（野生型ＮＣＯＡ４又はＲＵＦＹ１遺伝子の場合）には、２種類の蛍光色素に由来するシグナル（例えば、赤色と緑色）が別々に離れて検出されるのに対して、転座又は逆位により２つの遺伝子領域が近接している場合は、２つのシグナルが重なった色（例えば、黄色）として検出される。 Further, in the FISH fusion assay of the NCOA4 or RUFUY1 fusion gene, for example, when the NCOA4 or RUFY1 fusion gene is an NCOA4 or RUFY1-RET fusion gene, a polyprobe that covers the 3 ′ terminal genomic region of the RET gene is used as a detection probe. A combination of a nucleotide that is fluorescently labeled and a polynucleotide that covers the 5'-terminal genomic region of the NCOA4 or RUFY1 gene and that is labeled with another fluorescent dye can be used. In normal cases (in the case of wild-type NCOA4 or RUFY1 gene), signals derived from two types of fluorescent dyes (eg, red and green) are detected separately, whereas translocation or inversion When two gene regions are close to each other, the two signals are detected as overlapping colors (for example, yellow).
　〈ＦＩＳＨ法を用いた遺伝子重複の検出〉
　ＲＥＴ融合遺伝子構築に伴う遺伝子重複は、例えば、ＲＥＴ融合遺伝子が、ＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子の場合、検出用プローブとして、ＲＥＴ遺伝子の３’末端側ゲノム領域の少なくとも一部をカバーするポリヌクレオチドであって蛍光標識したものを使用することができる。そして、野生型ＲＥＴ遺伝子のみを含む場合に比較して強いシグナル、例えば、２倍以上のシグナルが得られることを検出することで、ＲＥＴ融合遺伝子を検出することができる。
　なお、ＲＥＴ遺伝子由来のポリヌクレオチドと融合して融合遺伝子を構築している他の遺伝子（例えば、ＲＥＴ融合遺伝子がＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子の場合、ＮＣＯＡ４又はＲＵＦＹ１遺伝子）の５’側ゲノム領域の検出用プローブを使用し、前記方法で、ＲＥＴ融合遺伝子を検出してもよい。 <Detection of gene duplication using FISH method>
For example, when the RET fusion gene is an NCOA4 or RUFY1-RET fusion gene, the gene duplication associated with the construction of the RET fusion gene is a polynucleotide that covers at least a part of the genomic region at the 3 ′ end side of the RET gene as a detection probe. In addition, fluorescently labeled ones can be used. And a RET fusion gene is detectable by detecting that a strong signal, for example, 2 times or more of a signal is obtained compared with the case where only a wild-type RET gene is included.
The 5 ′ genomic region of another gene that is fused with a polynucleotide derived from the RET gene to construct a fusion gene (for example, when the RET fusion gene is an NCOA4 or RUFY1-RET fusion gene, the NCOA4 or RUFY1 gene) The detection probe may be used to detect the RET fusion gene by the above method.
　ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子構築に伴う遺伝子重複は、例えば、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子が、ＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子の場合、検出用プローブとして、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の５’末端側ゲノム領域の少なくとも一部をカバーするポリヌクレオチドであって蛍光標識したものを使用することができる。そして、野生型ＮＣＯＡ４又はＲＵＦＹ１遺伝子のみを含む場合に比較して強いシグナル、例えば、２倍以上のシグナルが得られることを検出することで、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出することができる。
　なお、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来のポリヌクレオチドと融合して融合遺伝子を構築している他の遺伝子（例えば、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子がＲＥＴ－ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の場合、ＲＥＴ遺伝子）の３’側ゲノム領域の検出用プローブを使用し、前記方法で、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出してもよい。 For example, when the NCOA4 or RUFY1 fusion gene is an NCOA4 or RUFY1-RET fusion gene, at least one of the 5'-terminal genomic regions of the NCOA4 or RUFY1 gene is used as a detection probe. It is possible to use a fluorescently labeled polynucleotide that covers a part. Then, it is possible to detect the NCOA4 or RUFY1 fusion gene by detecting that a strong signal, for example, twice or more of the signal obtained compared to the case of containing only the wild-type NCOA4 or RUFY1 gene is obtained.
3 'side of other genes that are fused with a polynucleotide derived from the NCOA4 or RUFY1 gene (for example, the RET gene when the NCOA4 or RUFY1 fusion gene is a RET-NCOA4 or RUFY1 fusion gene) An NCOA4 or RUFY1 fusion gene may be detected by the aforementioned method using a genomic region detection probe.
　〈ＣＧＨアレイ解析を用いた遺伝子重複の検出〉
　ＲＥＴ融合遺伝子構築又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子構築に伴う遺伝子重複は、比較ゲノムハイブリダイゼーション（ＣＧＨ）アレイ解析（例えば、Agilent CGH/CNV Array解析；アジレントテクノロジー社）により、検出することができる。 <Detection of gene duplication using CGH array analysis>
Gene duplication associated with RET fusion gene construction or NCOA4 or RUFY1 fusion gene construction can be detected by comparative genomic hybridization (CGH) array analysis (eg, Agilent CGH / CNV Array analysis; Agilent Technologies).
　〈次世代シーケンサーを用いた遺伝子重複の検出〉
　ＲＥＴ融合遺伝子構築又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子構築に伴う遺伝子重複は、次世代シーケンサーにより、検出することができる。具体的には、次世代シーケンサーを用いた解析において、遺伝子重複部分のカバレッジが高い（解析対象としたＤＮＡ断片のシーケンスをリファレンス配列にアノテーションした際の該当部分の冗長度が高い）ことを検出することで、ＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出することができる。 <Detection of gene duplication using next-generation sequencer>
Gene duplication associated with RET fusion gene construction or NCOA4 or RUFY1 fusion gene construction can be detected by a next-generation sequencer. Specifically, in analysis using a next-generation sequencer, it is detected that the coverage of the gene duplication portion is high (the redundancy of the relevant portion is high when the sequence of the DNA fragment to be analyzed is annotated to the reference sequence) Thus, the RET fusion gene or NCOA4 or RUFI1 fusion gene can be detected.
　〈検出に用いるプローブ（ゲノム用）〉
　ＲＥＴ融合遺伝子を検出するための、ハイブリダイゼーションに用いるプローブとしては、ＲＥＴ融合遺伝子の少なくとも一部のヌクレオチド又はそれらの相補鎖にストリンジェントな条件下で（好ましくはよりストリンジェントな条件下で）ハイブリダイズするプローブが好ましい。 <Probe used for detection (for genome)>
The probe used for hybridization for detecting the RET fusion gene may be a hybrid under stringent conditions (preferably under more stringent conditions) to at least some nucleotides of the RET fusion gene or their complementary strands. Probes that soy are preferred.
　例えば、融合点を含むＲＥＴ融合遺伝子のゲノムＤＮＡを検出する場合、ＲＥＴ融合遺伝子の融合点をはさんでその上流及び下流のそれぞれ１６塩基からなる、連続した少なくとも３２塩基の核酸分子、又はそれらの相補鎖を含むプローブを用いてもよい。 For example, when detecting genomic DNA of a RET fusion gene containing a fusion point, nucleic acid molecules of at least 32 bases consisting of 16 bases each upstream and downstream of the fusion point of the RET fusion gene, or their A probe containing a complementary strand may be used.
　ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出するための、ハイブリダイゼーションに用いるプローブとしては、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の少なくとも一部のヌクレオチド又はそれらの相補鎖にストリンジェントな条件下で（好ましくはよりストリンジェントな条件下で）ハイブリダイズするプローブが好ましい。 As a probe used for hybridization for detecting the NCOA4 or RUFY1 fusion gene, at least a part of the nucleotides of the NCOA4 or RUFY1 fusion gene or a complementary strand thereof is used under stringent conditions (preferably more stringent conditions). Probes that hybridize (under) are preferred.
　例えば、融合点を含むＮＣＯＡ４又はＲＵＦＹ１融合遺伝子のゲノムＤＮＡを検出する場合、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の融合点をはさんでその上流及び下流のそれぞれ１６塩基からなる、連続した少なくとも３２塩基の核酸分子、又はそれらの相補鎖を含むプローブを用いてもよい。 For example, when detecting genomic DNA of a NCOA4 or RUFY1 fusion gene containing a fusion point, a nucleic acid molecule of at least 32 bases consisting of 16 bases upstream and downstream of the fusion point of the NCOA4 or RUFY1 fusion gene. Alternatively, a probe containing a complementary strand thereof may be used.
　例えば、ＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子が、ＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子の場合、ＦＩＳＨ法フュージョンアッセイに用いることのできるプローブとしては、ＲＥＴ遺伝子又はＮＣＯＡ４又はＲＵＦＹ１遺伝子のいずれか一方の遺伝子の５’末端側ゲノム領域を特異的に認識できる第１のプローブと、残る一方の遺伝子の３’末端側ゲノム領域を特異的に認識できる第２のプローブとの組み合わせ（好ましくは、ＲＥＴ遺伝子の３’末端側ゲノム領域を特異的に認識できる第１のプローブと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の５’末端側ゲノム領域を特異的に認識できる第２のプローブとの組み合わせ）を用いることができる。
　一方、例えば、ＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子が、ＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子の場合、ＦＩＳＨ法スプリットアッセイに用いることのできるプローブとしては、ＲＥＴ遺伝子５’末端側ゲノム領域を特異的に認識できる第１のプローブと、ＲＥＴ遺伝子の３’末端側ゲノム領域を特異的に認識できる第２のプローブとの組み合わせ、あるいは、ＮＣＯＡ４又はＲＵＦＹ１遺伝子５’末端側ゲノム領域を特異的に認識できる第１のプローブと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子３’末端側ゲノム領域を特異的に認識できる第２のプローブとの組み合わせ（好ましくは、ＲＥＴ遺伝子の５’末端側ゲノム領域を特異的に認識できる第１のプローブと、ＲＥＴ遺伝子の３’末端側ゲノム領域を特異的に認識できる第２のプローブとの組み合わせ）を用いることができる。 For example, when the RET fusion gene or NCOA4 or RUFY1 fusion gene is an NCOA4 or RUFY1-RET fusion gene, a probe that can be used in the FISH fusion assay is one of the RET gene, NCOA4 or RUFY1 gene. A combination of a first probe capable of specifically recognizing the 5′-terminal genomic region and a second probe capable of specifically recognizing the 3′-terminal genomic region of the remaining one gene (preferably 3 of the RET gene 'A combination of a first probe capable of specifically recognizing the terminal genomic region and a second probe capable of specifically recognizing the 5' terminal genomic region of NCOA4 or RUFI1 gene) can be used.
On the other hand, for example, when the RET fusion gene or NCOA4 or RUFY1 fusion gene is an NCOA4 or RUFY1-RET fusion gene, a probe that can be used in the FISH method split assay is specifically the RET gene 5 ′ terminal genomic region. A combination of a first probe that can be recognized and a second probe that can specifically recognize the 3′-end genomic region of the RET gene, or a first probe that can specifically recognize the 5′-end genomic region of the NCOA4 or RUFY1 gene 1 probe and a second probe capable of specifically recognizing the 3'-terminal genomic region of NCOA4 or RUFY1 gene (preferably, a first probe capable of specifically recognizing the 5'-terminal genomic region of the RET gene Specific recognition of the probe and the 3 'end genomic region of the RET gene Can be used that combination of the second probe).
　〔ｍＲＮＡの検出〕
　ｍＲＮＡの検出は、ノーザンハイブリダイゼーション法等によりｍＲＮＡ自体を解析することにより行っても、又は、当業者に周知の方法によりｍＲＮＡを鋳型として合成した、相補的ＤＮＡ（ｃＤＮＡ）を解析することにより行ってもよい。
　上記ＲＮＡの検出には、シーケンス技術を好適に用いることができる。シーケンシングには、解析の効率を考慮すると、次世代シーケンサーを使用することが好ましい（例えば、Metzker ML、Nat Rev Genet. 2010 Jan;11(1):31-46参照）。次世代シーケンサーとしては、Ｉｌｌｕｍｉｎａ社のＭｉＳｅｑ/ＨｉＳｅｑ、Ｌｉｆｅ Ｔｅｃｈｎｏｇｉｅｓ社のＳＯＬｉＤシステム、Ｒｏｃｈｅ社の４５４シーケンスシステム（ＧＳ ＦＬＸ＋／ＧＳ Ｊｕｎｉｏｒ）等が例示できる。シーケンシングにおいては、後述の遺伝子増幅反応方法、シーケンスキャプチャ技術等を用いて、融合遺伝子が存在している可能性がある領域を濃縮（enrich）することで、解析の効率を向上させることができる。シーケンスキャプチャ技術としては、Ｒｏｃｈｅ社のＲｏｃｈｅ ＮｉｍｂｌｅＧｅｎ、Ａｇｉｌｅｎｔ Ｔｅｃｈｎｏｌｏｇｉｅｓ社のＳｕｒｅ Ｓｅｌｅｃｔ等が例示できる。 [Detection of mRNA]
Detection of mRNA can be performed by analyzing mRNA itself by Northern hybridization or the like, or by analyzing complementary DNA (cDNA) synthesized using mRNA as a template by methods well known to those skilled in the art. May be.
For detection of the RNA, a sequencing technique can be suitably used. For sequencing, it is preferable to use a next-generation sequencer in consideration of analysis efficiency (see, for example, Metzker ML, Nat Rev Genet. 2010 Jan; 11 (1): 31-46). Illustrative examples of the next-generation sequencer include MiSeq / HiSeq from Illumina, SOLiD system from Life Technologies, 454 sequence system (GS FLX + / GS Junior) from Roche. In sequencing, the efficiency of analysis can be improved by enriching the region where the fusion gene may exist using the gene amplification reaction method and sequence capture technology described later. . Examples of the sequence capture technology include Roche NimbleGen from Roche, Sure Select from Agilent Technologies, and the like.
　〈遺伝子増幅反応方法による検出〉
　ｍＲＮＡは、検出対象であるＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の少なくとも一部のポリヌクレオチドを特異的に増幅できるように設計したプライマーを用いた、遺伝子増幅反応方法にて検出することができる。以下に、ｍＲＮＡの検出のための代表的な方法を例示するが、これらに限定されるものではない。 <Detection by gene amplification reaction method>
mRNA can be detected by a gene amplification reaction method using a primer designed to specifically amplify at least a part of the polynucleotide of the RET fusion gene or NCOA4 or RUFY1 fusion gene to be detected. Hereinafter, typical methods for detecting mRNA are exemplified, but the present invention is not limited thereto.
　==ＰＣＲ法==
　例えば、ＰＣＲ法では、ＰＣＲ産物をアガロースゲル電気泳動によって分析し、エチジウムブロマイド染色等によって目的とするサイズの増幅断片が得られたか否かを確認できる。目的とするサイズの増幅断片が得られた場合は、被験者から得た試料において、ＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子が存在していたことになる。このように、ＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出することができる。
　本発明のＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出方法としては、被験者から得た試料中の、特定のポリヌクレオチドを遺伝子増幅反応により増幅する工程に加え、更に目的とするサイズの増幅断片が得られたか否かを検出する工程を含むことが好ましい。 == PCR method ==
For example, in the PCR method, a PCR product is analyzed by agarose gel electrophoresis, and it can be confirmed whether or not an amplified fragment of a target size is obtained by ethidium bromide staining or the like. When an amplified fragment of the target size is obtained, it means that the RET fusion gene or NCOA4 or RUFY1 fusion gene was present in the sample obtained from the subject. Thus, a RET fusion gene or NCOA4 or RUFI1 fusion gene can be detected.
As a method for detecting the RET fusion gene or NCOA4 or RUFY1 fusion gene of the present invention, in addition to the step of amplifying a specific polynucleotide in a sample obtained from a subject by a gene amplification reaction, an amplified fragment of a desired size is further obtained. It is preferable to include a step of detecting whether or not it has been obtained.
　ＰＣＲ法は、ＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を定量的に検出することに適している。
　従って、前述の<ＲＥＴ融合遺伝子を検出する態様（１－ｂ）>に記載のように、ＲＥＴ遺伝子の５’末端側領域と３’末端側領域の発現量をそれぞれ特異的に検出し、その発現量の比を求めることによってＲＥＴ融合遺伝子を検出することができる。或いは、ＲＥＴ遺伝子と共にＲＥＴ融合遺伝子を構築しているＲＥＴ遺伝子以外の他の遺伝子の５’末端側領域と３’末端側領域の発現量をそれぞれ特異的に検出し、その発現量の比を求めることによって、ＲＥＴ融合遺伝子を検出することができる。
　また、前述の<ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する態様（１－ｂ）>に記載のように、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の５’末端側領域と３’末端側領域の発現量をそれぞれ特異的に検出し、その発現量の比を求めることによってＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する方法に好適に用いることができる。あるいは、ＮＣＯＡ４又はＲＵＦＹ１遺伝子と共にＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を構築しているＮＣＯＡ４又はＲＵＦＹ１遺伝子以外の他の遺伝子の５’末端側領域と３’末端側領域の発現量をそれぞれ特異的に検出し、その発現量の比を求めることによって、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出することができる。 The PCR method is suitable for quantitatively detecting the RET fusion gene or NCOA4 or RUFI1 fusion gene.
Therefore, as described in the above <Aspect for detecting RET fusion gene (1-b)>, the expression levels of the 5 ′ terminal region and 3 ′ terminal region of the RET gene are specifically detected, A RET fusion gene can be detected by determining the ratio of expression levels. Alternatively, the expression levels of the 5′-terminal region and 3′-terminal region of other genes other than the RET gene constructing the RET fusion gene together with the RET gene are specifically detected, and the ratio of the expression levels is obtained. Thus, the RET fusion gene can be detected.
Further, as described in <Aspect for detecting NCOA4 or RUFY1 fusion gene (1-b)>, the expression levels of the 5 ′ terminal region and 3 ′ terminal region of NCOA4 or RUFY1 gene are specifically determined. It can be suitably used in a method for detecting an NCOA4 or RUFY1 fusion gene by detecting and determining the ratio of the expression levels. Alternatively, the expression levels of the 5 ′ terminal region and the 3 ′ terminal region of other genes other than the NCOA4 or RUFY1 gene constructing the NCOA4 or RUFY1 fusion gene together with the NCOA4 or RUFY1 gene are specifically detected. By determining the expression level ratio, the NCOA4 or RUFFY1 fusion gene can be detected.
　なお、ＰＣＲ法、及び、これに用いるプライマー設計法は、公知の方法に従って当業者が行うことができる。
　例えば、ＲＥＴ遺伝子の５’末端側領域を特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマー、ならびに、ＲＥＴ遺伝子の３’末端側領域を特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマーを用いることができる。
　例えば、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の５’末端側領域を特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマー、ならびに、ＮＣＯＡ４又はＲＵＦＹ１遺伝子の３’末端側領域を特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマーを用いることができる。 In addition, the PCR method and the primer design method used therefor can be performed by those skilled in the art according to a known method.
For example, a sense primer and an antisense primer designed to specifically amplify the 5′-terminal region of the RET gene, and a sense primer designed to specifically amplify the 3′-terminal region of the RET gene and Antisense primers can be used.
For example, a sense primer and an antisense primer designed to specifically amplify the 5 ′ end region of the NCOA4 or RUFY1 gene, and a design to specifically amplify the 3 ′ end region of the NCOA4 or RUFY1 gene Sense primers and antisense primers can be used.
　==リアルタイムＰＣＲ法==
　更には、ＰＣＲ法においては、遺伝子の増幅過程においてＰＣＲ増幅モニター（リアルタイムＰＣＲ）法（Genome Res., 6(10), 986, 1996）を用いることにより、ＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出において、より定量的な解析を行うことが可能である。ＰＣＲ増幅モニター法としては、例えば、ＡＢＩ ＰＲＩＳＭ７９００（ＰＥバイオシステムズ社）を用いることができる。リアルタイムＰＣＲは公知の方法であり、そのための装置及びキットは市販されており、これらを利用して簡便に行える。 == Real-time PCR method ==
Furthermore, in the PCR method, by using the PCR amplification monitor (real-time PCR) method (Genome Res., 6 (10), 986, 1996) in the gene amplification process, the RET fusion gene or NCOA4 or RUFY1 fusion gene In detection, it is possible to perform more quantitative analysis. As a PCR amplification monitoring method, for example, ABI PRISM 7900 (PE Biosystems) can be used. Real-time PCR is a known method, and devices and kits for the real-time PCR are commercially available, and can be easily performed using these.
　より具体的には、例えばＲＥＴ融合遺伝子がＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子であって、ｍＲＮＡを指標にしてＲＥＴ融合遺伝子を検出する場合、センスプライマー（５’－プライマー、フォワードプライマー）を、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来の任意の部分から、アンチセンスプライマー（３’－プライマー、リバースプライマー）を、ＲＥＴ遺伝子由来の任意の部分から設計する。
　また、例えばＮＣＯＡ４又はＲＵＦＹ１融合遺伝子がＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子であって、ｍＲＮＡを指標にしてＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出する場合、センスプライマー（５’－プライマー、フォワードプライマー）を、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来の任意の部分から、アンチセンスプライマー（３’－プライマー、リバースプライマー）を、ＲＥＴ遺伝子由来の任意の部分から設計する。 More specifically, for example, when the RET fusion gene is NCOA4 or RUFY1-RET fusion gene and the RET fusion gene is detected using mRNA as an index, the sense primer (5′-primer, forward primer) is selected from NCOA4 or An antisense primer (3′-primer, reverse primer) is designed from any part derived from the RET gene from any part derived from the RUFY1 gene.
Further, for example, when the NCOA4 or RUFY1 fusion gene is an NCOA4 or RUFY1-RET fusion gene and the NFOA4 or RUFY1 fusion gene is detected using mRNA as an indicator, the sense primer (5′-primer, forward primer) An antisense primer (3′-primer, reverse primer) is designed from any part derived from the RET gene from any part derived from the RUFY1 gene.
　==マルチプレックスＰＣＲ==
　ＲＥＴ融合遺伝子を検出するためのＰＣＲ法では、ＲＥＴ遺伝子と融合してＲＥＴ融合遺伝子を構成する他の各遺伝子、及び、複数の融合点に対応した上記センスプライマーを混ぜることにより、１反応液により全ての融合ポリヌクレオチドを検出するマルチプレックスＰＣＲ（Multiplex PCR）を設計することもできる。
　ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出するためのＰＣＲ法では、ＮＣＯＡ４又はＲＵＦＹ１遺伝子と融合してＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を構成する他の各遺伝子、及び、複数の融合点に対応した上記センスプライマーを混ぜることにより、１反応液により全ての融合ポリヌクレオチドを検出するマルチプレックスＰＣＲ（Multiplex PCR）を設計することもできる。 == Multiplex PCR ==
In the PCR method for detecting the RET fusion gene, each reaction solution is mixed with each other gene constituting the RET fusion gene by fusing with the RET gene, and the sense primer corresponding to a plurality of fusion points. Multiplex PCR that detects all fusion polynucleotides can also be designed.
In the PCR method for detecting the NCOA4 or RUFY1 fusion gene, the other primers constituting the NCOA4 or RUFY1 fusion gene by fusing with the NCOA4 or RUFY1 gene and the above-mentioned sense primers corresponding to multiple fusion points are mixed. Thus, it is possible to design a multiplex PCR that detects all fusion polynucleotides in one reaction solution.
　==質量分析による検出==
　上記遺伝子増幅反応方法を用いた検出方法において、増幅断片の解析のために、特開2012-100628号公報に記載の質量分析法を用いることができる。 == Detection by mass spectrometry ==
In the detection method using the gene amplification reaction method, mass spectrometry described in JP 2012-100628 A can be used for analysis of amplified fragments.
　==検出に用いるプライマーセット==
　本発明のＲＥＴ融合遺伝子を検出するための検出方法に用いられるプライマーセットは、検出対象であるＲＥＴ融合遺伝子の少なくとも一部を特異的に増幅でき、ＲＥＴ融合遺伝子を検出できるものであれば、特には限定されず、当業者が、検出対象ポリヌクレオチドの塩基配列に基づいて設計することができる。
　本発明のＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出するための検出方法に用いられるプライマーセットは、検出対象であるＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の少なくとも一部を特異的に増幅でき、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出できるものであれば、特には限定されず、当業者が、検出対象ポリヌクレオチドの塩基配列に基づいて設計することができる。
　ＰＣＲ増幅モニター法におけるプライマー設計は、プライマー設計ソフトウェア（例えば、Primer Express; PE Biosystems）などを利用してできる。また、ＰＣＲ産物のサイズが大きくなると増幅効率が悪くなるため、センスプライマーとアンチセンスプライマーは、ｍＲＮＡ又はｃＤＮＡを対象に増幅したときの増幅産物の大きさが１ｋｂ以下になるように設定するのが適切である。 == Primer set used for detection ==
The primer set used in the detection method for detecting the RET fusion gene of the present invention is not particularly limited as long as it can specifically amplify at least a part of the RET fusion gene to be detected and can detect the RET fusion gene. Is not limited, and a person skilled in the art can design based on the base sequence of the polynucleotide to be detected.
The primer set used in the detection method for detecting the NCOA4 or RUFY1 fusion gene of the present invention can specifically amplify at least part of the NCOA4 or RUFY1 fusion gene to be detected, and can detect the NCOA4 or RUFY1 fusion gene If it is a thing, it will not specifically limit, Those skilled in the art can design based on the base sequence of detection target polynucleotide.
Primer design in the PCR amplification monitoring method can be performed using primer design software (eg, Primer Express; PE Biosystems). In addition, since the amplification efficiency decreases as the PCR product size increases, the sense primer and the antisense primer should be set so that the size of the amplified product when amplified for mRNA or cDNA is 1 kb or less. Is appropriate.
　〈ハイブリダイゼーション法による検出〉
　ｍＲＮＡは、検出対象であるＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の少なくとも一部のポリヌクレオチドにハイブリダイズするプローブを用いた、ハイブリダイゼーション法にて検出することができる。
　ハイブリダイゼーション技術を利用した検出は、例えば、ノーザンハイブリダイゼーション、ドットブロット法、ＤＮＡマイクロアレイ法、ＲＮＡプロテクション法などが挙げられる。 <Detection by hybridization method>
mRNA can be detected by a hybridization method using a probe that hybridizes to a RET fusion gene or NCOA4 or RUfy1 fusion gene at least a part of the polynucleotide to be detected.
Examples of detection using a hybridization technique include Northern hybridization, dot blot method, DNA microarray method, RNA protection method and the like.
　==プローブ（ｍＲＮＡ用）==
　ハイブリダイゼーションに用いるプローブとしては、ＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の少なくとも一部又はそれらの相補鎖にストリンジェントな条件下で（好ましくはよりストリンジェントな条件下で）ハイブリダイズするプローブが好ましい。 == Probe (for mRNA) ==
The probe used for hybridization is preferably a probe that hybridizes under stringent conditions (preferably under more stringent conditions) to at least part of the RET fusion gene, NCOA4 or RUFY1 fusion gene, or their complementary strands. .
　＜融合タンパク質の検出＞
　被験者から得た試料中の、ＲＥＴ融合タンパク質又はＮＣＯＡ４又はＲＵＦＹ１タンパク質を検出する技法としては、タンパク質を解析するために用いられる当業者に周知いかなる技法、又は、これらの技法を応用したいかなる技法を用いてもよい。 <Detection of fusion protein>
As a technique for detecting a RET fusion protein or NCOA4 or RUFY1 protein in a sample obtained from a subject, any technique known to those skilled in the art used to analyze proteins or any technique to which these techniques are applied is used. May be.
　例えば、ＲＥＴ融合タンパク質の検出に用いる方法としては、ＲＥＴタンパク質、又は、ＲＥＴタンパク質と共にＲＥＴ融合タンパク質を構築するＲＥＴタンパク質以外の他のタンパク質を特異的に認識する抗体、あるいは、ＲＥＴ融合タンパクを特異的に認識する抗体を用いた免疫学的測定法（イムノアッセイ法）、酵素活性測定法（ＥＬＩＳＡ法）、２抗体サンドイッチＥＬＩＳＡ法、蛍光免疫測定法、放射免疫測定法、ウェスタンブロッティング法、免疫組織化学法、免疫沈降法、ｉＡＥＰ（intercalated antibody-enhanced polymer）法、ＦＲＥＴ法を例示することができる。あるいは、これらと組み合わせて、または単独で、質量分析法、アミノ酸シーケンス法を用いることができる。 For example, as a method used for detecting a RET fusion protein, an antibody that specifically recognizes a RET protein or another protein other than the RET protein that constructs the RET fusion protein together with the RET protein, or a RET fusion protein is specifically used. Immunoassay (immunoassay), enzyme activity assay (ELISA), 2-antibody sandwich ELISA, fluorescence immunoassay, radioimmunoassay, western blotting, immunohistochemistry Examples thereof include immunoprecipitation, iAEP (intercalated antibody-enhanced polymer) method, and FRET method. Alternatively, a mass spectrometry method or an amino acid sequence method can be used in combination with these or alone.
　例えば、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の検出に用いる方法としては、ＮＣＯＡ４又はＲＵＦＹ１タンパク質、又は、ＮＣＯＡ４又はＲＵＦＹ１タンパク質と共にＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を構築するＮＣＯＡ４又はＲＵＦＹ１タンパク質以外の他のタンパク質を特異的に認識する抗体、あるいは、ＮＣＯＡ４又はＲＵＦＹ１融合タンパクを特異的に認識する抗体を用いた免疫学的測定法（イムノアッセイ法）、酵素活性測定法（ＥＬＩＳＡ法）、２抗体サンドイッチＥＬＩＳＡ法、蛍光免疫測定法、放射免疫測定法、ウェスタンブロッティング法、免疫組織化学法、免疫沈降法、ｉＡＥＰ（intercalated antibody-enhanced polymer）法、ＦＲＥＴ法を例示することができる。あるいは、これらと組み合わせて、または単独で、質量分析法、アミノ酸シーケンス法を用いることができる。
　以下に、タンパク質の検出のための代表的な方法を例示するが、これらに限定されるものではない。 For example, as a method used for detection of NCOA4 or RUFY1 fusion protein, NCOA4 or RUFY1 protein, or other proteins other than NCOA4 or RUFY1 protein that construct NCOA4 or RUFY1 fusion protein together with NCOA4 or RUFY1 protein are specifically recognized. Immunoassay (immunoassay), enzyme activity assay (ELISA), two-antibody sandwich ELISA, fluorescence immunoassay, radiation using antibodies or antibodies that specifically recognize NCOA4 or RUFY1 fusion protein Examples include immunoassay, Western blotting, immunohistochemistry, immunoprecipitation, iAEP (intercalated antibody-enhanced polymer), and FRET. Alternatively, a mass spectrometry method or an amino acid sequence method can be used in combination with these or alone.
In the following, representative methods for protein detection are exemplified, but the present invention is not limited thereto.
　〔検出に用いる代表的な手法〕
　抗体を用いる検出方法としては、上記公知の方法によればよいが、例えば以下の方法を用いることができる。 [Typical methods used for detection]
As a detection method using an antibody, the above-mentioned known method may be used. For example, the following method can be used.
　〈免疫組織化学法〉
　例えば、検出対象のＲＥＴ融合タンパク質又はＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が、ＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合タンパク質の場合、検出対象の融合タンパク質が存在している可能性のある組織切片に対し、ＲＥＴタンパク質のＣ末端側領域のポリペプチドに結合する抗ＲＥＴ抗体及びＮＣＯＡ４又はＲＵＦＹ１タンパク質のＮ末端側領域のポリペプチドに結合する抗ＮＣＯＡ４又はＲＵＦＹ１抗体を用いた免疫染色を行い、それらの抗体が近接していることを指標に、検出対象の融合タンパク質の存在を検出することもできる。また、ＲＥＴタンパク質のＮ末端側領域のポリペプチドに特異的に結合する抗体と、ＲＥＴタンパク質のＣ末端側領域のポリペプチドに特異的に結合する抗体を用いた免疫染色を行い、それらの抗体が離れて局在していることを指標に、検出対象の融合タンパク質の存在を検出することもできる。また、ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＮ末端側領域のポリペプチドに特異的に結合する抗体と、ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＣ末端側領域のポリペプチドに特異的に結合する抗体を用いた免疫染色を行い、それらの抗体が離れて局在していることを指標に、検出対象の融合タンパク質の存在を検出することもできる。また、融合点を含むポリペプチドに特異的に結合する抗体を用いた免疫染色を行い、検出対象の融合タンパク質の存在を検出することもできる。 <Immunohistochemistry>
For example, when the RET fusion protein to be detected or NCOA4 or RUFY1 fusion protein is an NCOA4 or RUFY1-RET fusion protein, the C-terminal of the RET protein is compared with the tissue section in which the fusion protein to be detected may be present. Perform immunostaining using an anti-RET antibody that binds to a polypeptide in the side region and an anti-NCOA4 or RUFY1 antibody that binds to a polypeptide in the N-terminal region of the NCOA4 or RUFY1 protein, and confirm that these antibodies are close The presence of the fusion protein to be detected can also be detected as an index. In addition, immunostaining was performed using an antibody that specifically binds to the polypeptide in the N-terminal region of the RET protein and an antibody that specifically binds to the polypeptide in the C-terminal region of the RET protein. It is also possible to detect the presence of the fusion protein to be detected using the presence of the distant localization as an index. In addition, immunostaining using an antibody that specifically binds to a polypeptide in the N-terminal region of NCOA4 or RUFY1 protein and an antibody that specifically binds to a polypeptide in the C-terminal region of NCOA4 or RUFY1 protein, The presence of the fusion protein to be detected can also be detected using the presence of these antibodies as an index. Alternatively, the presence of the fusion protein to be detected can be detected by performing immunostaining using an antibody that specifically binds to the polypeptide containing the fusion point.
　〈ウェスタンブロッティング法〉
　例えば、検出対象のＲＥＴ融合タンパク質又はＮＣＯＡ４又はＲＵＦＹ１融合タンパク質がＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合タンパク質の場合、検出対象の融合タンパク質が存在している可能性のある細胞抽出液を、当業者に周知の方法により電気泳動して細胞抽出液中のタンパク質を分離した後、メンブレンにブロッティングする。
　そして、タンパク質がブロッティングされたメンブレンに対し、ＲＥＴタンパク質のＮ末端側領域のポリペプチドに結合する抗ＲＥＴ抗体及びＮＣＯＡ４又はＲＵＦＹ１タンパク質のＣ末端側領域に結合する抗ＮＣＯＡ４又はＲＵＦＹ１抗体を用いた免疫染色を行い、メンブレン上の所望の位置に、抗ＲＥＴ抗体と抗ＮＣＯＡ４又はＲＵＦＹ１抗体が結合していることを指標に、検出対象の融合タンパク質の存在を検出することもできる。
　また、融合点を含むポリペプチドに特異的に結合する抗体を用い、当該抗体がメンブレン上の所望の位置に結合していることを指標に、検出対象の融合タンパク質の存在を検出することもできる。
　あるいは、抗ＲＥＴ抗体を用い、当該抗体が、メンブレン上のＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合タンパク質に結合していることを指標に、検出対象の融合タンパク質の存在を検出することもできる。この際、メンブレン上で野生型ＲＥＴタンパク質が予測される位置とは異なる位置に抗ＲＥＴ抗体が結合することを指標に、検出対象の融合タンパク質の存在を検出してもよい。
　抗ＮＣＯＡ４又はＲＵＦＹ１抗体を用い、抗ＲＥＴ抗体を用いた場合と同じ原理で、ＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合タンパク質を検出してもよい。 <Western blotting method>
For example, when the RET fusion protein to be detected or the NCOA4 or RUFY1 fusion protein is an NCOA4 or RUFY1-RET fusion protein, a cell extract that may contain the fusion protein to be detected is obtained by a method well known to those skilled in the art. The protein in the cell extract is separated by electrophoresis using the method described above, and then blotted on a membrane.
Then, immunostaining using an anti-RET antibody that binds to a polypeptide in the N-terminal region of the RET protein and an anti-NCOA4 or RUFY1 antibody that binds to the C-terminal region of the NCOA4 or RUFY1 protein on the membrane on which the protein is blotted And the presence of the fusion protein to be detected can also be detected using as an index the binding of the anti-RET antibody and the anti-NCOA4 or RUFY1 antibody to a desired position on the membrane.
It is also possible to detect the presence of a fusion protein to be detected using an antibody that specifically binds to a polypeptide containing a fusion point and using as an index that the antibody is bound to a desired position on the membrane. .
Alternatively, an anti-RET antibody can be used to detect the presence of the fusion protein to be detected using as an index that the antibody is bound to the NCOA4 or RUFY1-RET fusion protein on the membrane. At this time, the presence of the fusion protein to be detected may be detected by using an anti-RET antibody binding to a position different from the position where the wild-type RET protein is predicted on the membrane.
An anti-NCOA4 or RUFY1 antibody may be used to detect the NCOA4 or RUFY1-RET fusion protein on the same principle as when an anti-RET antibody is used.
　〈免疫沈降法〉
　例えば、検出対象のＲＥＴ融合タンパク質又はＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が、ＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合タンパク質の場合、検出対象の融合タンパク質が存在している可能性のある細胞抽出液に対し、ＲＥＴタンパク質のＣ末端側領域のポリペプチドに結合する抗ＲＥＴ抗体又はＮＣＯＡ４又はＲＵＦＹ１タンパク質のＮ末端側領域のポリペプチドに結合する抗ＮＣＯＡ４又はＲＵＦＹ１抗体のいずれか一方の抗体で免疫沈降を行い、その沈降物に対して残るもう一方の抗体で検出することで、検出対象の融合タンパク質の存在を検出することもできる。上記の通り、免疫沈降と検出をした後、更には、検出抗体により、検出したポリペプチドが目的の検出対象ポリペプチドの大きさであることを確認することが好ましい。 <Immunoprecipitation method>
For example, when the RET fusion protein to be detected or the NCOA4 or RUFY1 fusion protein is an NCOA4 or RUFY1-RET fusion protein, the RET protein C is added to the cell extract in which the detection target fusion protein may be present. Immunoprecipitation is performed with either the anti-RET antibody that binds to the polypeptide in the terminal region or the anti-NCOA4 or RUFY1 antibody that binds to the polypeptide in the N-terminal region of the NCOA4 or RUFY1 protein, and the precipitate is By detecting with the other remaining antibody, the presence of the fusion protein to be detected can also be detected. As described above, after immunoprecipitation and detection, it is preferable to further confirm with the detection antibody that the detected polypeptide is the size of the target polypeptide to be detected.
　あるいは、検出対象のＲＥＴ融合タンパク質が存在している可能性のある細胞抽出液に対し、ＲＥＴタンパク質のＣ末端側領域のポリペプチドに結合する抗ＲＥＴ抗体で免疫沈降を行い、更にその沈降物の質量分析を行うことにより、野生型ＲＥＴと異なる質量の抗ＲＥＴ抗体と結合するタンパク質の存在を確認することで、検出対象の融合タンパク質の存在を検出することもできる。
　検出対象のＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が存在している可能性のある細胞抽出液に対し、ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＮ末端側領域のポリペプチドに結合する抗ＮＣＯＡ４又はＲＵＦＹ１抗体で免疫沈降を行い、さらにその沈降物の質量分析を行うことにより、野生型ＮＣＯＡ４又はＲＵＦＹ１と異なる質量の抗ＮＣＯＡ４又はＲＵＦＹ１抗体と結合するタンパク質の存在を確認することで、検出対象の融合タンパク質の存在を検出することもできる。 Alternatively, the cell extract in which the RET fusion protein to be detected may be present is immunoprecipitated with an anti-RET antibody that binds to a polypeptide in the C-terminal region of the RET protein, and the precipitate By performing mass spectrometry, it is possible to detect the presence of the fusion protein to be detected by confirming the presence of a protein that binds to an anti-RET antibody having a mass different from that of the wild-type RET.
Immunoprecipitation is carried out with an anti-NCOA4 or RUFY1 antibody that binds to a polypeptide in the N-terminal region of the NCOA4 or RUFY1 protein on the cell extract in which the NCOA4 or RUFY1 fusion protein to be detected may be present. By performing mass spectrometry of the sediment, the presence of the fusion protein to be detected can be detected by confirming the presence of a protein that binds to an anti-NCOA4 or RUFY1 antibody having a mass different from that of wild-type NCOA4 or RUFY1. .
　〔検出に用いる抗体〕
　なお、本発明に係る検出方法に用いる抗体は、ＲＥＴ融合タンパク質又はＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の所望の部位に特異的に結合する範囲で特に制限されず、モノクローナル抗体であってもポリクローナル抗体であってもよく、モノクローナル抗体及びポリクローナル抗体を組みあわせて用いることもできる。前記抗体としては、免疫グロブリンそれ自体であっても、抗原結合能を保持した抗体断片、例えば、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）2、又はＦｖでもよい。また、抗体の結合を検出するため、当業者に周知のいかなる標識やシグナル増幅法が用いられてもよい。 [Antibodies used for detection]
The antibody used in the detection method according to the present invention is not particularly limited as long as it specifically binds to a desired site of the RET fusion protein or NCOA4 or RUFY1 fusion protein, and even a monoclonal antibody is a polyclonal antibody. It is also possible to use a combination of a monoclonal antibody and a polyclonal antibody. The antibody may be an immunoglobulin itself or an antibody fragment that retains antigen binding ability, such as Fab, Fab ′, F (ab ′) 2 , or Fv. Any label or signal amplification method known to those skilled in the art may be used to detect antibody binding.
　＜標識手法＞
　上記遺伝子（ゲノムＤＮＡ、ｍＲＮＡ、ｃＤＮＡ等）及びタンパク質の検出方法において、プローブ、プライマー、増幅産物、抗体等の標識は公知の技術を用いればよい。例えば、蛍光標識、化学発光標識、放射性標識、酵素標識、ビオチン標識、アビジン標識等を挙げることができる。
　プローブを用いた検出方法において、プローブを標識する場合、その標識方法は上記のとおり、公知の方法によればよく、例えば、ＢＡＣクローンから、標識された核酸プローブを作製する場合、ニックトランスレーション、ランダムプライム法等の公知手法を用いることができる。またその際、ビオチン－ｄＵＴＰ（例えば、Roche Applied Science社製）を用いてプローブをビオチン標識し、アビジンと結合させた、蛍光体、放射性同位体、酵素等、を更に処理することでプローブを標識することができる。
　抗体を用いた検出方法において、抗体を標識する場合、その標識方法は上記のとおり、公知の方法によればよいが、例えば、下記の標識方法が挙げられる。 <Labeling method>
In the above gene (genomic DNA, mRNA, cDNA, etc.) and protein detection methods, known techniques may be used for labeling probes, primers, amplification products, antibodies, and the like. For example, fluorescent labels, chemiluminescent labels, radioactive labels, enzyme labels, biotin labels, avidin labels and the like can be mentioned.
In the detection method using a probe, when a probe is labeled, the labeling method may be a known method as described above. For example, when preparing a labeled nucleic acid probe from a BAC clone, nick translation, A known method such as a random prime method can be used. At that time, the probe is labeled with biotin using biotin-dUTP (for example, manufactured by Roche Applied Science), and the probe is labeled by further processing phosphors, radioisotopes, enzymes, etc. bound to avidin. can do.
In the detection method using an antibody, when the antibody is labeled, the labeling method may be a known method as described above, and examples thereof include the following labeling methods.
　〔ｉＡＥＰ（intercalated antibody-enhanced polymer）法〕
　検出対象となるタンパク質に結合する第一抗体と、ポリマー試薬の間に介在抗体を入れることにより、染色感度を上げることが可能である（Takeuchiら、Clin Cancer Res, 2009 May 1; 15(9):3143-3149）。 [IAEP (intercalated antibody-enhanced polymer) method]
Staining sensitivity can be increased by placing an intervening antibody between the first antibody that binds to the protein to be detected and the polymer reagent (Takeuchi et al., Clin Cancer Res, 2009 May 1; 15 (9) : 3143-3149).
　〔蛍光共鳴エネルギー移動（ＦＲＥＴ）〕
　２つの抗体の近接を検出する手法として、例えば、ＦＲＥＴ現象を利用したプローブ（ＦＲＥＴプローブ）を用いることができる。抗体の一つをドナー蛍光物質（ＣＦＰ等）で標識し、他の抗体をアクセプター蛍光物質（ＹＦＰ等）で標識した場合、両者が十分に近い距離にあると、ＦＲＥＴ現象により、ＹＦＰが励起状態となり、基底状態に戻る際に蛍光を発する。この蛍光を検出することで、２つの抗体が近接していることを検出することができる。 [Fluorescence resonance energy transfer (FRET)]
As a technique for detecting the proximity of two antibodies, for example, a probe using the FRET phenomenon (FRET probe) can be used. When one of the antibodies is labeled with a donor fluorescent substance (CFP, etc.) and the other antibody is labeled with an acceptor fluorescent substance (YFP, etc.), if the two are at a sufficiently close distance, the YFP is excited by the FRET phenomenon. And emits fluorescence when returning to the ground state. By detecting this fluorescence, it is possible to detect the proximity of the two antibodies.
≪ＲＥＴ阻害物質による治療の適用対象の判定≫
　本発明の検出方法における検出対象のＲＥＴ融合遺伝子又は検出対象のＲＥＴ融合タンパク質が、被験者から得た試料から検出された場合は、当該被験者は、ＲＥＴ融合体陽性のがんを有する対象（患者）であり、ＲＥＴ阻害物質による治療の適用対象となる。 ≪Determining the target of treatment with a RET inhibitor≫
When the RET fusion gene to be detected or the RET fusion protein to be detected in the detection method of the present invention is detected from a sample obtained from a subject, the subject has a RET fusion-positive cancer (patient). And is a target for treatment with a RET inhibitor.
≪ＮＣＯＡ４又はＲＵＦＹ１阻害物質による治療の適用対象の判定≫
　本発明の検出方法における検出対象のＮＣＯＡ４又はＲＵＦＹ１融合遺伝子又は検出対象のＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が、被験者から得た試料から検出された場合は、当該被験者は、ＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがんを有する対象（患者）であり、ＮＣＯＡ４又はＲＵＦＹ１阻害物質による治療の適用対象となる。 ≪Determination of application target of treatment with NCOA4 or RUFY1 inhibitor≫
When the detection target NCOA4 or RUFY1 fusion gene or the detection target NCOA4 or RUFY1 fusion protein is detected from a sample obtained from the subject, the subject is NCOA4 or RUFY1 fusion-positive cancer. This is a subject (patient) having a target of treatment with an NCOA4 or RUFFY1 inhibitor.
≪検出用キット≫
　本発明の検出用キットには、検出対象のＲＥＴ融合遺伝子の検出用キット、又は、検出対象のＲＥＴ融合タンパク質の検出用キットが含まれる。
　本発明の検出用キットには、検出対象のＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出用キット、又は、検出対象のＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の検出用キットが含まれる。 ≪Detection kit≫
The detection kit of the present invention includes a detection kit for a detection target RET fusion gene or a detection kit for a detection target RET fusion protein.
The detection kit of the present invention includes a detection kit for a detection target NCOA4 or RUFY1 fusion gene or a detection target NCOA4 or RUFY1 fusion protein.
　本発明の検出対象のＲＥＴ融合遺伝子の検出用キット、又は、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出用キットには、本発明の検出方法においてＦＩＳＨ法フュージョンアッセイ又はＦＩＳＨ法スプリットアッセイに用いることのできるプローブ、あるいは、本発明の検出方法における検出対象のＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の少なくとも一部を特異的に増幅できるように設計したセンス及びアンチセンスプライマーが含まれる。センス及びアンチセンスプライマーセットは、ＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の少なくとも一部のポリヌクレオチドであり、かつ、増幅対象であるポリヌクレオチドの増幅用のプライマーとして機能するポリヌクレオチドのセットである。 The detection kit of the RET fusion gene to be detected of the present invention or the kit for detection of the NCOA4 or RUFY1 fusion gene includes a probe that can be used in the FISH method fusion assay or FISH method split assay in the detection method of the present invention, Alternatively, sense and antisense primers designed to specifically amplify at least a part of the RET fusion gene or NCOA4 or RUFFY1 fusion gene to be detected in the detection method of the present invention are included. The sense and antisense primer set is a polynucleotide set that functions as a primer for amplification of a polynucleotide to be amplified, and is a polynucleotide that is at least a part of the RET fusion gene or NCOA4 or RUFY1 fusion gene.
　また、本発明の検出対象のＲＥＴ融合タンパク質又はＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の検出用キットには、本発明の検出方法に用いることができる抗体が含まれる。 In addition, an antibody that can be used in the detection method of the present invention is included in the detection kit of the detection target RET fusion protein or NCOA4 or RUFI1 fusion protein of the present invention.
　＜プローブ＞
　本発明のＲＥＴ融合遺伝子の検出用キットは、ＲＥＴ融合遺伝子の少なくとも一部のポリヌクレオチド、又は、その相補鎖にストリンジェントな条件下でハイブリダイズし、前記ＲＥＴ融合遺伝子を検出できるプローブを１種類、あるいは、２種類以上の組み合わせで含むことができる。
　本発明のＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出用キットは、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の少なくとも一部のポリヌクレオチド、又は、その相補鎖にストリンジェントな条件下でハイブリダイズし、前記ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出できるプローブを１種類、あるいは、２種類以上の組み合わせで含むことができる。 <Probe>
The kit for detecting a RET fusion gene of the present invention is one kind of probe capable of detecting the RET fusion gene by hybridizing under stringent conditions to at least a part of the polynucleotide of the RET fusion gene or its complementary strand. Or it can contain in the combination of 2 or more types.
The kit for detecting the NCOA4 or RUFY1 fusion gene of the present invention hybridizes under stringent conditions to at least a part of the polynucleotide of the NCOA4 or RUFY1 fusion gene, or a complementary strand thereof, and the NCOA4 or RUFY1 fusion gene Probes that can be detected can be included in one type or a combination of two or more types.
　プローブとして、前述の≪検出方法に用いる技法≫に記載したいずれか１種類以上のプローブが例示できる。
　例えば、ＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子がＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合遺伝子の場合、ＲＥＴ遺伝子由来ポリヌクレオチドにハイブリダイズする１種類以上の（好ましくは２種類以上の）プローブ、又は、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来ポリヌクレオチドにハイブリダイズする１種類以上の（好ましくは２種類以上の）プローブ、のいずれかのみを含んでも、ＲＥＴ遺伝子由来ポリヌクレオチドにハイブリダイズする１種類以上のプローブ、及び、ＮＣＯＡ４又はＲＵＦＹ１遺伝子由来ポリヌクレオチドにハイブリダイズする１種類以上のプローブ、の両方を含んでも、あるいは、ＲＥＴ融合遺伝子の融合点を含むポリヌクレオチドにハイブリダイズする１種類以上のプローブ、又は、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の融合点を含むポリヌクレオチドにハイブリダイズする１種類以上のプローブを含んでもよい。 Examples of the probe include any one or more of the probes described in << Technology used in detection method >>.
For example, when the RET fusion gene or NCOA4 or RUFY1 fusion gene is an NCOA4 or RUFY1-RET fusion gene, one or more (preferably two or more) probes hybridizing to the RET gene-derived polynucleotide, or NCOA4 or RUFY1 One or more probes that hybridize to a RET gene-derived polynucleotide, and either NCOA4 or RUFY1, even if only one of one or more (preferably two or more) probes that hybridize to a gene-derived polynucleotide One or more probes that hybridize to a polynucleotide containing a fusion point of a RET fusion gene, or one or more probes that hybridize to a gene-derived polynucleotide, or NCOA Or it may comprise one or more probes that hybridize to a polynucleotide comprising a fusion point of RUFY1 fusion gene.
　＜プライマーセット＞
　本発明のＲＥＴ融合遺伝子の検出用キットは、ＲＥＴ融合遺伝子の少なくとも一部を特異的に増幅でき、ＲＥＴ融合遺伝子を検出できるプライマーセットを１セット、あるいは、２セット以上の組み合わせで含むことができる。
　本発明のＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出用キットは、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の少なくとも一部を特異的に増幅でき、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を検出できるプライマーセットを１セット、あるいは、２セット以上の組み合わせで含むことができる。
　プライマーセットとして、前述の≪本発明の検出方法の態様≫又は≪検出方法に用いる技法≫に記載したいずれか１種類以上のプライマーセットが例示できる。 <Primer set>
The kit for detecting a RET fusion gene of the present invention can specifically amplify at least a part of the RET fusion gene and can include one set of primer sets capable of detecting the RET fusion gene, or a combination of two or more sets. .
The kit for detecting the NCOA4 or RUFY1 fusion gene of the present invention can specifically amplify at least a part of the NCOA4 or RUFY1 fusion gene and can detect one set of primer sets that can detect the NCOA4 or RUFY1 fusion gene, or two or more sets of primer sets. Can be included in combination.
Examples of the primer set include any one or more of the primer sets described in << Aspect of detection method of the present invention >> or << Technique used in detection method >>.
　本発明のプライマーセットには、好ましくは、
（１）ＲＥＴタンパク質をコードするポリヌクレオチド部分から設計されるアンチセンスプライマー及びＮＣＯＡ４又はＲＵＦＹ１タンパク質をコードするポリヌクレオチド部分から設計されるセンスプライマーを含む、ＲＥＴ遺伝子とＮＣＯＡ４又はＲＵＦＹ１遺伝子との融合遺伝子を検出するためのプライマーセットであって、アンチセンスプライマーは「検出対象ポリヌクレオチド」にストリンジェントな条件下（好ましくは、よりストリンジェントな条件下）でアニールする核酸分子（好ましくは、少なくとも１６塩基の核酸分子）からなり、センスプライマーは「検出対象ポリヌクレオチド」の相補鎖にストリンジェントな条件（好ましくは、よりストリンジェントな条件下）でアニールする核酸分子（好ましくは、少なくとも１６塩基の核酸分子）からなるプライマーセット、が含まれる。 In the primer set of the present invention, preferably,
(1) A fusion gene between a RET gene and an NCOA4 or RUFY1 gene, comprising an antisense primer designed from a polynucleotide part encoding a RET protein and a sense primer designed from a polynucleotide part encoding an NCOA4 or RUFY1 protein A primer set for detection, wherein an antisense primer is a nucleic acid molecule (preferably at least 16 bases) that anneals to a “polynucleotide to be detected” under stringent conditions (preferably under more stringent conditions). A nucleic acid molecule (preferably at least 1) that anneals to the complementary strand of the “polynucleotide to be detected” under stringent conditions (preferably under more stringent conditions). Primer set consisting of a nucleic acid molecule) of the bases include.
　また、前記プライマーセット（１）のより具体的な態様として、本発明のプライマーセットには、以下の（２）～（５）のプライマーセットが含まれる。 As a more specific embodiment of the primer set (1), the primer set of the present invention includes the following primer sets (2) to (5).
（２）配列番号１（ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２）の塩基番号１から１９８４の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドからなるセンスプライマー、及び配列番号１の塩基番号１９８５から５２７５の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドに対して相補的であるオリゴヌクレオチドからなるアンチセンスプライマーのプライマーセット。
（３）配列番号３（ＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２）の塩基番号１から１９１７の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドからなるセンスプライマー、及び配列番号３の塩基番号１９１８から５２０８の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドに対して相補的であるオリゴヌクレオチドからなるアンチセンスプライマーのプライマーセット。
（４）配列番号１で表されるＲＥＴ融合遺伝子を検出するための、以下のプライマーセット。
　　ＮＣＯＡ４－１６６１Ｆ：ＣＴＧＧＡＧＡＡＧＡＣＡＡＧＴＧＧＣＴＧＣ（配列番号１２）
　　ＲＥＴ－２３８１Ｒ：ＣＡＧＧＣＣＣＣＡＴＡＣＡＡＴＴＴＧＡＴ（配列番号７）
（５）配列番号３で表されるＲＥＴ融合遺伝子を検出するための、以下のプライマーセット。
　　ＲＵＦＹ１－１４９２Ｆ：ＡＴＧＡＡＡＣＡＡＡＴＧＧＡＡＧＡＡＡＧＧＴＴＧ（配列番号１３）
　　ＲＥＴ－２３８１Ｒ：ＣＡＧＧＣＣＣＣＡＴＡＣＡＡＴＴＴＧＡＴ（配列番号７） (2) a sense primer consisting of any consecutive oligonucleotides of at least 16 bases between base numbers 1 to 1984 of SEQ ID NO: 1 (NCOA4ex9-RETex12), and any between base numbers 1985 to 5275 of SEQ ID NO: 1 A primer set of antisense primers consisting of oligonucleotides that are complementary to oligonucleotides of at least 16 consecutive bases.
(3) a sense primer consisting of any continuous oligonucleotide of at least 16 bases between base numbers 1 to 1917 of SEQ ID NO: 3 (RUFY1ex16-RETex12), and any of bases from base numbers 1918 to 5208 of SEQ ID NO: 3 A primer set of antisense primers consisting of oligonucleotides that are complementary to oligonucleotides of at least 16 consecutive bases.
(4) The following primer set for detecting the RET fusion gene represented by SEQ ID NO: 1.
NCOA4-1661F: CTGGAGAAGACAAGTGGCTGC (SEQ ID NO: 12)
RET-2381R: CAGGCCCCATACAATTTGAT (SEQ ID NO: 7)
(5) The following primer set for detecting the RET fusion gene represented by SEQ ID NO: 3.
RUFY1-1492F: ATGAAACAAATGGAAGAAAGGTTG (SEQ ID NO: 13)
RET-2381R: CAGGCCCCATACAATTTGAT (SEQ ID NO: 7)
　また、本発明のプライマーセットは、前述の〈遺伝子増幅反応方法による検出〉==ＰＣＲ法==に記載のように、ＲＥＴ遺伝子の５’末端側領域と３’末端側領域の発現量を検出するためのプライマーセット、又は、ＲＥＴ遺伝子と共に融合遺伝子を構築する他の遺伝子の５’末端側領域と３’末端側領域の発現量を検出するためのプライマーセットであってもよい。 In addition, the primer set of the present invention detects the expression levels of the 5 ′ terminal region and 3 ′ terminal region of the RET gene as described in <Detection by gene amplification reaction method> == PCR method == above. Or a primer set for detecting the expression levels of the 5′-terminal region and the 3′-terminal region of other genes that construct a fusion gene together with the RET gene.
　これらのプライマーセット（１）～（５）においては、センスプライマーとアンチセンスセンスプライマーの選択位置の間隔が１ｋｂ以下であるか、あるいは、センスプライマーとアンチセンスセンスプライマーにより増幅される増幅産物の大きさが１ｋｂ以下であることが好ましい。
　また、本発明のプライマーは、通常、１５～４０塩基、好ましくは１６～２４塩基、更に好ましくは１８～２４塩基、特に好ましくは２０～２４塩基の鎖長を有する。 In these primer sets (1) to (5), the distance between the selected positions of the sense primer and the antisense sense primer is 1 kb or less, or the size of the amplification product amplified by the sense primer and the antisense sense primer Is preferably 1 kb or less.
The primer of the present invention usually has a chain length of 15 to 40 bases, preferably 16 to 24 bases, more preferably 18 to 24 bases, and particularly preferably 20 to 24 bases.
　本発明のプライマーセットは、本発明の検出方法において、検出対象ポリヌクレオチドを増幅及び検出するために用いることができる。また、本発明のプライマーセットに含まれる各プライマーは、特に限定されるものではないが、例えば、化学合成法によって製造することができる。 The primer set of the present invention can be used for amplifying and detecting a polynucleotide to be detected in the detection method of the present invention. Moreover, although each primer contained in the primer set of this invention is not specifically limited, For example, it can manufacture by a chemical synthesis method.
　＜抗体＞
　本発明のＲＥＴ融合タンパク質の検出用キットには、ＲＥＴ融合タンパク質の任意の部位に特異的に結合する抗体を１種類、あるいは、２種類以上の組み合わせで含むことができる。具体的には、前述の＜融合タンパク質の検出＞に記載した抗体が例示できる。
　本発明のＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の検出用キットには、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の任意の部位に特異的に結合する抗体を１種類、あるいは、２種類以上の組み合わせで含むことができる。具体的には、前述の＜融合タンパク質の検出＞に記載した抗体が例示できる。
　例えば、ＲＥＴ融合タンパク質又はＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が、ＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ融合タンパク質の場合、ＲＥＴタンパク質由来ポリペプチドに結合する１種類以上の（好ましくは２種類以上の）抗体、又は、ＮＣＯＡ４又はＲＵＦＹ１タンパク質由来ポリペプチドに結合する１種類以上の（好ましくは２種類以上の）抗体、のいずれかのみを含んでも、ＲＥＴタンパク質由来ポリペプチドに結合する１種類以上の抗体、及び、ＮＣＯＡ４又はＲＵＦＹ１タンパク質由来ポリペプチドに結合する１種類以上の抗体、の両方を含んでも、あるいは、ＲＥＴ融合タンパク質の融合点を含むポリペプチドに結合する１種類以上の抗体、又は、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の融合点を含むポリヌクレオチドに結合する１種類以上の抗体を含んでもよい。 <Antibody>
The kit for detecting a RET fusion protein of the present invention can contain one or more combinations of antibodies that specifically bind to an arbitrary site of the RET fusion protein. Specifically, the antibodies described in <Fusion protein detection> can be exemplified.
The NCOA4 or RUFY1 fusion protein detection kit of the present invention can contain one or more combinations of antibodies that specifically bind to any site of the NCOA4 or RUFY1 fusion protein. Specifically, the antibodies described in <Fusion protein detection> can be exemplified.
For example, when the RET fusion protein or NCOA4 or RUFY1 fusion protein is an NCOA4 or RUFY1-RET fusion protein, one or more (preferably two or more) antibodies that bind to the RET protein-derived polypeptide, or NCOA4 or RUFY1 Even if only one of one or more (preferably two or more) antibodies that bind to a protein-derived polypeptide is included, one or more antibodies that bind to a RET protein-derived polypeptide, and NCOA4 or RUFY1 protein-derived One or more antibodies that bind to the polypeptide, or one or more antibodies that bind to the polypeptide containing the fusion point of the RET fusion protein, or the fusion point of the NCOA4 or RUFY1 fusion gene Polynucleotide One or more antibodies that bind may contain.
≪阻害物質のスクリーニング方法≫
　＜ポリペプチドを阻害する物質をスクリーニングする工程＞
　本発明の、阻害物質のスクリーニング方法は、前記検出対象ポリペプチドを阻害する物質をスクリーニングすることができ、
（１）検出対象ポリペプチド、又は前記ポリペプチドを発現している細胞に試験物質を接触させる工程、
（２）前記ポリペプチドが阻害されるか否かを分析する工程、及び
（３）前記ポリペプチドを阻害する物質を選択する工程
を含む。 ≪Inhibitor screening method≫
<Step of screening for substances that inhibit polypeptides>
The method for screening an inhibitory substance of the present invention can screen a substance that inhibits the detection target polypeptide,
(1) A step of bringing a test substance into contact with a polypeptide to be detected or a cell expressing the polypeptide,
(2) analyzing whether the polypeptide is inhibited, and (3) selecting a substance that inhibits the polypeptide.
　本明細書において、「ポリペプチドの阻害」には、ポリペプチドの活性の阻害と、ポリペプチドの発現の阻害とが含まれる。また、「阻害」は、少なくとも一部の阻害を意味する。 In the present specification, “inhibition of polypeptide” includes inhibition of activity of polypeptide and inhibition of expression of polypeptide. “Inhibition” means at least partial inhibition.
　＜阻害物質スクリーニング工程とその指標＞
　本発明のスクリーニング方法には、
（Ａ）精製又は粗製のポリペプチドを用いて、インビトロでポリペプチドの活性阻害を指標とする方法、
（Ｂ）ポリペプチドを発現している細胞を用いて、ポリペプチドの活性阻害を指標とする方法、
（Ｃ）ポリペプチドを発現している細胞を用いて、ポリペプチドの発現阻害を指標とする方法
が含まれる。 <Inhibitor screening process and its index>
The screening method of the present invention includes
(A) Using purified or crude polypeptide, the method using in vitro inhibition of polypeptide activity as an index,
(B) a method using a cell expressing a polypeptide as an indicator of inhibition of polypeptide activity;
(C) A method using the expression of the polypeptide as an index using cells expressing the polypeptide is included.
　〔（Ａ）精製又は粗製ポリペプチドを用い、活性阻害を指標とする方法〕
　前記方法（Ａ）には、インビトロでポリペプチドに試験物質を添加して接触させる工程、前記試験物質により前記ポリペプチドの活性が阻害されたか否かを、対照（試験物質を接触させなかったポリペプチド）と比較して分析する工程、ポリペプチドの活性を阻害した物質を選択する工程を含む方法が含まれる。
　インビトロにおけるポリペプチド活性の測定は、公知のキナーゼ活性測定法を用いることができ、例えば、キナーゼ反応により生成するＡＤＰ量を指標としても、あるいは、ポリペプチドのチロシンリン酸化レベルを指標としてもよく、市販のキナーゼ活性測定キットを用いることもできる。 [(A) A method using purified or crude polypeptide and using activity inhibition as an index]
In the method (A), a test substance is added to and contacted with a polypeptide in vitro, whether or not the activity of the polypeptide is inhibited by the test substance, a control (polysaccharide not contacted with the test substance). And a method comprising the step of selecting a substance that inhibits the activity of the polypeptide.
In vitro polypeptide activity can be measured using a known kinase activity measurement method. For example, the amount of ADP produced by the kinase reaction may be used as an index, or the tyrosine phosphorylation level of the polypeptide may be used as an index. Commercially available kinase activity measurement kits can also be used.
　〔（Ｂ）ポリペプチド発現細胞を用い、活性阻害を指標とする方法〕
　前記方法（Ｂ）には、ポリペプチドを発現している細胞に試験物質を添加して接触させる工程、前記試験物質により前記ポリペプチドの活性が阻害されたか否かを、対照（試験物質を接触させなかった細胞）と比較して分析する工程、ポリペプチドの活性を阻害した物質を選択する工程を含む方法が含まれる。
　前記細胞におけるポリペプチド活性の測定は、公知のキナーゼ活性測定法を用いることができ、例えば、キナーゼ反応により生成するＡＤＰ量を指標としても、あるいは、ポリペプチドのチロシンリン酸化レベルを指標としてもよく、市販のキナーゼ活性測定キットを用いることもできる。 [(B) A method using polypeptide-expressing cells and using activity inhibition as an index]
The method (B) includes a step of adding a test substance to a cell expressing the polypeptide and bringing it into contact; whether the test substance inhibits the activity of the polypeptide; And a method comprising a step of selecting a substance that inhibits the activity of the polypeptide.
For the measurement of the polypeptide activity in the cells, a known kinase activity measurement method can be used. For example, the amount of ADP produced by the kinase reaction may be used as an index, or the tyrosine phosphorylation level of the polypeptide may be used as an index. A commercially available kinase activity measurement kit can also be used.
　〔（Ｃ）ポリペプチド発現細胞を用い、発現阻害を指標とする方法〕
　前記方法（Ｃ）には、ポリペプチドを発現している細胞に試験物質を添加して接触させる工程、前記試験物質により前記ポリペプチドの発現が阻害されたか否かを、対照（試験物質を接触させなかった細胞）と比較して分析する工程、ポリペプチドの発現を阻害した物質を選択する工程を含む方法が含まれる。
　前記細胞におけるポリペプチドの発現は、タンパク質又はｍＲＮＡの量を測定することにより分析することができる。タンパク質量の測定には、例えば、ＥＬＩＳＡ法、イムノブロット法を用いることができ、ｍＲＮＡ量の測定には、例えば、ＲＴ－ＰＣＲ法、ノーザンブロット法を用いることができる。 [(C) Method using polypeptide-expressing cells and expression inhibition as an index]
In the method (C), a test substance is added to and contacted with a cell expressing the polypeptide, whether or not the expression of the polypeptide is inhibited by the test substance, a control (contact with the test substance). And a method comprising a step of selecting a substance that inhibits the expression of the polypeptide.
Polypeptide expression in the cells can be analyzed by measuring the amount of protein or mRNA. For example, ELISA or immunoblotting can be used for measuring the amount of protein, and for example, RT-PCR or Northern blotting can be used for measuring the amount of mRNA.
　ここで、ＲＥＴ融合遺伝子は、腫瘍形成能を有する遺伝子である。従って、本発明の阻害物質スクリーニング方法で選択したポリペプチド阻害物質は、ＲＥＴ融合体陽性のがんの治療用医薬組成物の有効物質又はその候補物質として有用であり、本発明方法は、所望により、前記阻害物質がＲＥＴ融合体陽性のがんに対する治療活性を有することを確認する工程を更に含むことができる。
　また、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子は、腫瘍形成能を有する遺伝子である。従って、本発明の阻害物質スクリーニング方法で選択したポリペプチド阻害物質は、ＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがんの治療薬又はその候補物質として有用であり、本発明方法は、所望により、前記阻害物質がＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがんに対する治療活性を有することを確認する工程を更に含むことができる。
　前記確認工程は、公知の評価系を用いて実施することができ、例えば、培養細胞を用いるインビトロ評価系、腫瘍細胞を移植した担がんモデル動物を用いる評価系などを挙げることができる。前記担がんモデル動物としては、手術により患者から摘出した腫瘍組織を、一度培養により細胞株として樹立してから移植することもできるし、あるいは、前記腫瘍組織を直接、移植することもできる。後者の担がんモデル動物は、ＰＤＸ（patient-derived xenograft）モデル動物として知られており、細胞株を移植したモデル動物と比較して、継代を繰り返した皮下腫瘍組織における遺伝子発現プロファイルが原発巣とより類似しているため、評価系として好ましい。 Here, the RET fusion gene is a gene having tumorigenicity. Therefore, the polypeptide inhibitor selected by the inhibitor screening method of the present invention is useful as an effective substance or a candidate substance for a pharmaceutical composition for the treatment of RET fusion-positive cancer. The method may further comprise the step of confirming that the inhibitor has therapeutic activity against RET fusion positive cancer.
The NCOA4 or RUfy1 fusion gene is a gene having tumorigenicity. Therefore, the polypeptide inhibitor selected by the inhibitor screening method of the present invention is useful as a therapeutic agent for NCOA4 or RUFY1 fusion-positive cancer or a candidate substance thereof. Can further comprise the step of confirming that NCOA4 or RUFY1 fusion-positive cancer has therapeutic activity.
The confirmation step can be performed using a known evaluation system, and examples thereof include an in vitro evaluation system using cultured cells and an evaluation system using a cancer-bearing model animal transplanted with tumor cells. The tumor-bearing model animal can be transplanted after a tumor tissue removed from a patient by surgery is once established as a cell line by culturing, or the tumor tissue can be directly transplanted. The latter cancer-bearing model animal is known as a PDX (patient-derived xenograft) model animal, and has a gene expression profile in a subcutaneous tumor tissue that has been repeatedly passaged compared to a model animal transplanted with a cell line. Since it is more similar to the nest, it is preferable as an evaluation system.
　前記ポリペプチド発現細胞は、本発明のポリヌクレオチドを、常法に従って、所望の細胞に導入することで得ることもできる（例えば、Molecular Cloning: A Laboratory Manual 4th Edition（2012）、Cold Spring Harbor Laboratory Press参照）。具体的には、例えば、本発明のＲＥＴ融合遺伝子又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子であるｃＤＮＡを、組換えベクターに導入し、これを更に細胞に導入することで、前記ポリペプチド発現細胞（形質転換細胞）を得ることができる。 The polypeptide-expressing cell can also be obtained by introducing the polynucleotide of the present invention into a desired cell according to a conventional method (for example, Molecular Cloning: A Laboratory Manual 4th Edition (2012), Cold Spring Harbor Laboratory Press). reference). Specifically, for example, the RET fusion gene of the present invention or the cDNA that is the NCOA4 or RUFY1 fusion gene is introduced into a recombinant vector, which is further introduced into a cell, whereby the polypeptide-expressing cell (transformed cell) is transformed. ) Can be obtained.
≪阻害物質を含有するがん治療用医薬組成物≫
　本発明の、ＲＥＴ融合体陽性のがんの治療用医薬組成物は、ＲＥＴ融合遺伝子又はその転写産物に対する阻害物質を含む。例えば、本発明の阻害物質スクリーニング方法で得られる阻害物質（例えば、低分子化合物、二重鎖核酸（ｓｉＲＮＡを含む）、タンパク質（抗体又は抗体断片を含む）、ペプチド、又はそれ以外の化合物）を有効成分として含有し、所望により、製剤学的に許容される担体を含有することができる。
　本発明のＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがんの治療用医薬組成物は、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子又はその転写産物に対する阻害物質を含む。例えば、本発明の阻害物質スクリーニング方法で得られる阻害物質（例えば、低分子化合物、二重鎖核酸（ｓｉＲＮＡを含む）、タンパク質（抗体又は抗体断片を含む）、ペプチド、又はそれ以外の化合物）を有効成分として含有し、所望により、製剤学的に許容される担体を含有することができる。 ≪Pharmaceutical composition for cancer treatment containing inhibitory substance≫
The pharmaceutical composition for the treatment of RET fusion-positive cancer of the present invention comprises an inhibitor for the RET fusion gene or its transcription product. For example, an inhibitor (for example, a low molecular weight compound, a double-stranded nucleic acid (including siRNA), a protein (including an antibody or an antibody fragment), a peptide, or other compound) obtained by the inhibitor screening method of the present invention is used. It is contained as an active ingredient, and if desired, a pharmaceutically acceptable carrier can be contained.
The pharmaceutical composition for the treatment of NCOA4 or RUFY1 fusion-positive cancer of the present invention comprises an inhibitor for the NCOA4 or RUFY1 fusion gene or a transcription product thereof. For example, an inhibitor (for example, a low molecular weight compound, a double-stranded nucleic acid (including siRNA), a protein (including an antibody or an antibody fragment), a peptide, or other compound) obtained by the inhibitor screening method of the present invention is used. It is contained as an active ingredient, and if desired, a pharmaceutically acceptable carrier can be contained.
　＜ＲＥＴ融合遺伝子又は転写産物、あるいは、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子又は転写物に対する阻害物質＞
　ＲＥＴ融合遺伝子又はその転写産物に対する阻害物質としては、キナーゼ阻害剤、例えば、ＲＥＴ阻害物質、又は、ＲＥＴ遺伝子と共に融合遺伝子を構築するもう一方の遺伝子又はその転写物質に対する阻害物質を挙げることができる。
　ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子又はその転写産物に対する阻害物質としては、キナーゼ阻害剤、例えば、ＮＣＯＡ４又はＲＵＦＹ１阻害物質、又は、ＮＣＯＡ４又はＲＵＦＹ１遺伝子と共に融合遺伝子を構築するもう一方の遺伝子又はその転写物質に対する阻害物質を挙げることができる。 <Inhibitor for RET fusion gene or transcript, or NCOA4 or RUFY1 fusion gene or transcript>
Examples of the inhibitor for the RET fusion gene or a transcription product thereof include a kinase inhibitor, for example, a RET inhibitor, or an inhibitor for the other gene that constructs a fusion gene together with the RET gene or a transcription material thereof.
As an inhibitor for the NCOA4 or RUFY1 fusion gene or a transcription product thereof, a kinase inhibitor, for example, an inhibitor of the NCOA4 or RUFY1 inhibitor, or another gene that constructs a fusion gene together with the NCOA4 or RUFY1 gene or a transcript thereof Can be mentioned.
　〔低分子化合物〕
　前記阻害物質のうち、低分子化合物の具体例としては、Vandetanib（AstraZeneca Pharmaceuticals）、Alectinib (F. Hoffmann-La Roche Ltd.)、Cabozantinib（Exelixis Inc.）、Sorafenib（Bayer Pharma AG）、Ponatinib（ARIAD Pharmaceuticals）、Nintendanib（Boehringer Ingelheim）、Lenvatinib（エーザイ株式会社）、Regorafenib（Bayer Healthcare Pharmaceuticals）、1-Ter-Butyl-3-P-Tolyl-1h-Pyrazolo[3,4-D]Pyrimidin-4-Ylamine、および、これらの薬学的に許容される塩等を挙げることができる。 [Low molecular compound]
Among the inhibitors, specific examples of low molecular weight compounds include Vandetanib (AstraZeneca Pharmaceuticals), Alectinib (F. Hoffmann-La Roche Ltd.), Cabozantinib (Exelixis Inc.), Sorafenib (Bayer Pharma AG), Pontatinib (ARIAD Pharmaceuticals), Nintendanib (Boehringer Ingelheim), Lenvatinib (Eisai Co., Ltd.), Regorafenib (Bayer Healthcare Pharmaceuticals), 1-Ter-Butyl-3-P-Tolyl-1h-Pyrazolo [3,4-D] Pyrimidin-4-Ylamine And pharmaceutically acceptable salts thereof.
　〔二重鎖核酸〕
　二重鎖核酸は、二重鎖の核酸（ＲＮＡ又はＤＮＡ）部分と、好ましくはセンス鎖及びアンチセンス鎖の３’末端のオーバーハングとからなり、ＲＮＡｉを誘導する。ＲＮＡｉは進化的に保存された現象で、ＲＮａｓｅIIIエンドヌクレアーゼによって生じる２１～２３塩基の二重鎖核酸を介して起こる（Genes Dev. 15, 485-490, 2001）。３’側のオーバーハングはそれぞれ１又は２塩基の任意の核酸であるが、２塩基が好ましい。なお、前記塩基数（２１～２３塩基）は、オーバーハングを含むセンス鎖又はアンチセンス鎖の各々の塩基数である。また、センス鎖及びアンチセンス鎖は、同じ塩基数であることもできるし、異なる塩基数であることもできるが、同じ塩基数であることが好ましい。 [Double-stranded nucleic acid]
A double-stranded nucleic acid consists of a double-stranded nucleic acid (RNA or DNA) portion and preferably an overhang at the 3 ′ end of the sense strand and the antisense strand to induce RNAi. RNAi is an evolutionarily conserved phenomenon that occurs via a 21-23 base double-stranded nucleic acid generated by RNase III endonuclease (Genes Dev. 15, 485-490, 2001). Each 3 ′ overhang is an arbitrary nucleic acid having 1 or 2 bases, but 2 bases are preferred. The number of bases (21 to 23 bases) is the number of bases of each of the sense strand or the antisense strand containing an overhang. The sense strand and the antisense strand can have the same number of bases or different numbers of bases, but preferably have the same number of bases.
　二重鎖核酸の３’側オーバーハングを構成するリボ核酸としては、例えば、Ｕ（ウリジン）、Ａ（アデノシン）、Ｇ（グアノシン）、又はＣ（シチジン）を用いることができ、３’側のオーバーハングを構成するデオキシリボ核酸としては、例えば、ｄＴ（デオキシチミジン）、ｄＡ（デオキシアデノシン）、ｄＧ（デオキシグアノシン）、又はｄＣ（デオキシシチジン）を用いることができる。 As the ribonucleic acid constituting the 3 ′ overhang of the double-stranded nucleic acid, for example, U (uridine), A (adenosine), G (guanosine), or C (cytidine) can be used. As the deoxyribonucleic acid constituting the overhang, for example, dT (deoxythymidine), dA (deoxyadenosine), dG (deoxyguanosine), or dC (deoxycytidine) can be used.
　本発明の医薬組成物の有効成分として用いることのできる二重鎖核酸は、ＲＥＴ融合遺伝子に対する阻害作用又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子に対する阻害作用を有するものであれば、特に限定されない。例えば、二重鎖部分が融合点を含むポリヌクレオチドの塩基配列、例えば、配列番号１の第１９８４番～第１９８５番を含む塩基配列、あるいは、配列番号３の第１９１７番～第１９１８番を含む塩基配列に基づいて設計することができる。あるいは、二重鎖部分が、キナーゼ部分をコードするポリヌクレオチドの塩基配列に基づいて設計することができる。本発明の二重鎖核酸は、常法（例えば、J. Am. Chem. Soc., 120, 11820-11821, 1998; 及びMethods, 23, 206-217, 2001）により製造することができる。また、二重鎖核酸を委託製造する会社（例えば、ＲＮＡｉ社）は、当業者によく知られており、二重鎖核酸の製造に利用できる。また、ｓｉＲＮＡ配列設計システム（商用ｓｉＤｉｒｅｃｔ（登録商標）；ＲＮＡｉ社）により、二重鎖核酸を設計することができる。 The double-stranded nucleic acid that can be used as an active ingredient of the pharmaceutical composition of the present invention is not particularly limited as long as it has an inhibitory action on the RET fusion gene or an inhibitory action on the NCOA4 or RUfy1 fusion gene. For example, the double-stranded portion includes a base sequence of a polynucleotide containing a fusion point, for example, a base sequence including 1984 to 1985 of SEQ ID NO: 1, or 1917 to 1918 of SEQ ID NO: 3. It can design based on a base sequence. Alternatively, the duplex portion can be designed based on the base sequence of the polynucleotide encoding the kinase portion. The double-stranded nucleic acid of the present invention can be produced by a conventional method (for example, J. Am. Chem. Soc., 120, 11820-11821, 1998; and Methods, 23, 206-217, 2001). In addition, companies that consign and manufacture double-stranded nucleic acids (for example, RNAi) are well known to those skilled in the art and can be used for the production of double-stranded nucleic acids. In addition, a double-stranded nucleic acid can be designed by a siRNA sequence design system (commercial siDirect (registered trademark); RNAi).
　〔タンパク質・抗体〕
　本発明の医薬組成物の有効成分として用いることのできる抗体は、ＲＥＴ融合遺伝子の転写産物又はＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の転写物、好ましくは、ＮＣＯＡ４又はＲＵＦＹ１－ＲＥＴ遺伝子の転写産物を阻害するものであれば、限定されない。例えば、ＲＥＴ融合タンパク質又はＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の活性、好ましくはキナーゼ活性を阻害するものが挙げられる。 [Protein / Antibody]
The antibody that can be used as an active ingredient of the pharmaceutical composition of the present invention inhibits the transcription product of the RET fusion gene or the transcription product of the NCOA4 or RUFY1 fusion gene, preferably the transcription product of the NCOA4 or RUFY1-RET gene. If there is, it is not limited. For example, those that inhibit the activity of the RET fusion protein or NCOA4 or RUFY1 fusion protein, preferably the kinase activity.
《本発明のポリペプチド、ポリヌクレオチド、ベクター、形質転換細胞》
　＜本発明のポリペプチド＞
　本発明の検出方法に係る検出対象であるＲＵＦＹ１－ＲＥＴ融合タンパク質は、それ自体、新規タンパク質である。
　本発明のポリペプチドであるＲＵＦＹ１－ＲＥＴ融合タンパク質としては、下記（ａ）～（ｄ）に記載のポリペプチドが好ましい：
（ａ）配列番号４で表されるアミノ酸配列からなるポリペプチド、
（ｂ）配列番号４で表されるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、
（ｃ）配列番号４で表されるアミノ酸配列との同一性が８０％以上であるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、及び
（ｄ）配列番号４で表されるアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、及び／若しくは挿入されたアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド。 << Polypeptide, Polynucleotide, Vector, Transformed Cell of the Present Invention >>
<Polypeptide of the present invention>
The RUFY1-RET fusion protein that is a detection target according to the detection method of the present invention is itself a novel protein.
The RUFY1-RET fusion protein that is the polypeptide of the present invention is preferably a polypeptide described in the following (a) to (d):
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4,
(B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 and having tumorigenicity,
(C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having tumorigenicity, and (d) an amino acid sequence represented by SEQ ID NO: 4 A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or inserted, and having tumorigenic potential.
　＜本発明のポリヌクレオチド＞
　本発明の検出方法に係る検出対象であるＲＵＦＹ１－ＲＥＴ融合遺伝子は、それ自体、新規遺伝子である。
　本発明のポリヌクレオチドは、本発明のポリペプチド（すなわち、ＲＵＦＹ１－ＲＥＴ融合タンパク質）をコードするポリヌクレオチド（すなわち、ＲＵＦＹ１－ＲＥＴ融合遺伝子）である限り、特に限定されるものではない。なお、「ＲＵＦＹ１－ＲＥＴ融合タンパク質をコードするポリヌクレオチド」は、ＲＵＦＹ１－ＲＥＴ融合遺伝子において翻訳領域のみからなるポリヌクレオチドであっても、ＲＵＦＹ１－ＲＥＴ融合遺伝子のゲノムＤＮＡ全長であっても、ＲＵＦＹ１－ＲＥＴ融合遺伝子のｍＲＮＡやｃＤＮＡであってもよい。 <Polynucleotide of the present invention>
The RUFY1-RET fusion gene to be detected according to the detection method of the present invention is itself a novel gene.
The polynucleotide of the present invention is not particularly limited as long as it is a polynucleotide encoding the polypeptide of the present invention (ie, RUFY1-RET fusion protein) (ie, RUFY1-RET fusion gene). The “polynucleotide encoding the RUFY1-RET fusion protein” may be a polynucleotide comprising only the translation region in the RUFY1-RET fusion gene, the full-length genomic DNA of the RUYY1-RET fusion gene, or the RUFY1-RET fusion gene. It may be mRNA or cDNA of RET fusion gene.
　＜本発明のベクター＞
　本発明のベクターは、前記本発明のポリヌクレオチドを含む限り、特に限定されるものではなく、真核生物又は原核生物の宿主細胞を形質転換できる適当なベクターに当該ポリヌクレオチドを組み込むことにより調製することができる。前記ベクターは、当該ポリヌクレオチドの発現に必要な配列、例えば、プロモーター、エンハンサーを含むことができ、更に、宿主への導入確認に必要な配列、例えば、薬剤耐性遺伝子を含むことができる。 <Vector of the present invention>
The vector of the present invention is not particularly limited as long as it contains the polynucleotide of the present invention, and is prepared by incorporating the polynucleotide into an appropriate vector capable of transforming a eukaryotic or prokaryotic host cell. be able to. The vector can contain a sequence necessary for expression of the polynucleotide, such as a promoter and an enhancer, and can further contain a sequence necessary for confirmation of introduction into the host, such as a drug resistance gene.
　＜本発明の形質転換細胞＞
　本発明の形質転換細胞は、本発明のベクターにより、適当な宿主細胞、例えば、真核生物又は原核生物の宿主細胞を形質転換することにより調製することができる。本発明の形質転換細胞は、本発明のポリペプチドを製造するのに用いることができる。 <Transformed cells of the present invention>
The transformed cell of the present invention can be prepared by transforming a suitable host cell such as a eukaryotic or prokaryotic host cell with the vector of the present invention. The transformed cell of the present invention can be used for producing the polypeptide of the present invention.
　以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。
　なお、実施の形態及び実施例に特に説明がない場合には、J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J.G. Seidman, J. A. Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd.などの標準的なプロトコール集に記載の方法、あるいはそれを修飾したり、改変した方法を用いる。また、市販の試薬キットや測定装置を用いている場合には、特に説明が無い場合、それらに添付のプロトコールを用いる。 EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.
Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Standards such as Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.
［実施例１］臨床検体でのＲＥＴ遺伝子異常のＦＩＳＨ法による検出
　目的遺伝子の５’末端側領域及び３’末端側領域を異なる色素で染め、遺伝子の転座又は逆位等を観察する方法が知られている。ＦＩＳＨ法の一種であるこの方法はスプリットアッセイ（split assay）と呼ばれている。スプリットアッセイでは、染色体転座又は逆位等を調べたい目的遺伝子の５’末端側領域及び３’末端側領域のそれぞれを異なる蛍光色素で標識したプローブで染める。例えば、テキサスレッド（TexasRed）（赤）標識及びＦＩＴＣ（緑）標識した２種類のプローブにて蛍光標識することにより、正常な場合（融合遺伝子が構築されていない場合）は１つの黄色のシグナル（緑色と赤色のシグナルが近傍に存在している状態）として検出され、転座又は逆位等が起きている場合は、離れた緑色と赤色のシグナルとして検出される。 [Example 1] Detection of RET gene abnormality in clinical specimen by FISH method A method for observing gene translocation or inversion etc. by dyeing the 5 'terminal region and 3' terminal region of the target gene with different dyes Are known. This method, which is a type of FISH method, is called a split assay. In the split assay, the 5 ′ terminal region and the 3 ′ terminal region of the target gene to be examined for chromosomal translocation or inversion are stained with probes labeled with different fluorescent dyes. For example, by fluorescent labeling with two types of probes labeled Texas Red (red) and FITC (green), one normal yellow signal (when the fusion gene is not constructed) ( When green and red signals are present in the vicinity), and translocation or inversion occurs, they are detected as separated green and red signals.
　臨床検体でのＲＥＴ遺伝子異常をＦＩＳＨ法スプリットアッセイで検出した。手術により摘出され、２０％ホルマリンで固定化されパラフィンに包埋された大腸がん組織を４μｍの厚さで切り、スライドガラス上に乗せ病理切片を作製した。ＦＩＳＨ法は文献（Takeuchi K, Choi YL, Soda M, Inamura K, Togashi Y, Hatano S, Enomoto M, Takada S, Yamashita Y, Satoh Y, Okumura S, Nakagawa K, Ishikawa Y, Mano H. Multiplex reverse transcription-PCR screening for EML4-ALK fusion transcripts. Clin Cancer Res. 2008;14:6618-6624.）の方法に従い行った。作製した未染色の切片は、Ｈｉｓｔｏｌｏｇｙ　ＦＩＳＨアクセサリーキット（Dako社）で処理し、続いて、赤（TexasRed）の蛍光標識したＲＥＴ遺伝子５’末端側領域をカバーするＢＡＣ（bacterial artificial chromosome）クローン（クローン番号　RP11-124O11）と、緑（ＦＩＴＣ）の蛍光標識したＲＥＴ遺伝子３’末端側領域をカバーするＢＡＣクローン（クローン番号　RP11-351D16）とを用いてハイブリダイゼーションした。更に続いて４，６－ジアミノ－２－フェニルインドール（4,6-diamino-2-phenylindole）で染色を行った。蛍光観察は蛍光顕微鏡ＢＸ５１（Olympus社）を使用した。緑と赤のシグナルが離れて観察されるゲノム構造異常が示唆される切片を見出した。約１５００例の病理検体での検討から２例のＲＥＴ遺伝子領域のゲノム構造異常を示唆する検体（結腸がん患者由来）を見出した。 RET gene abnormality in clinical specimens was detected by FISH split assay. A colon cancer tissue excised by surgery, fixed with 20% formalin and embedded in paraffin was cut to a thickness of 4 μm, and placed on a slide glass to prepare a pathological section. The FISH method is based on literature (Takeuchi K, Choi YL, Soda M, Inamura K, Togashi Y, Hatano S, Enomoto M, Takada S, Yamashita Y, Satoh Y, Okumura S, Nakagawa K, Ishiano -PCR screening for EML4-ALK fusion transcripts. Clin Cancer Res. 2008; 14: 6618-6624.). The prepared unstained section was treated with the Histology FISH accessory kit (Dako), followed by a BAC (bacterial-artificial-chromosome) clone (clone number) covering the 5'-terminal region of the red (TexasRed) fluorescently labeled RET gene. Hybridization was performed using RP11-124O11) and a BAC clone (clone number RP11-351D16) covering the 3 'terminal region of the RET gene labeled with green (FITC). Subsequently, staining was performed with 4,6-diamino-2-phenylindole. For fluorescence observation, a fluorescence microscope BX51 (Olympus) was used. We found a section suggesting an abnormal genomic structure in which green and red signals were observed apart. From the examination of about 1500 pathological specimens, 2 specimens (derived from colon cancer patients) suggesting genomic structural abnormality in the RET gene region were found.
［実施例２］臨床検体でのＲＥＴ融合ポリヌクレオチド遺伝子の同定
　ＦＩＳＨ法解析によりＲＥＴゲノム構造異常が示唆された組織由来のＲＮＡをテンプレートに、インバースＲＴ－ＰＣＲ法により、ＲＥＴ遺伝子キナーゼ領域の５’側に存在する遺伝子を調べた。
　まず、逆転写及び二本鎖ｃＤＮＡ合成は、臨床検体由来のＲＮＡ０．５μｇと、ＲＥＴ遺伝子キナーゼコード領域に設計した逆転写プライマーＲＥＴ－２７４６Ｒ（配列番号５）とを用いて、cDNA Synthesis System（Roche）により行った。合成した二本鎖ｃＤＮＡを High Pure PCR Product Purification Kit（Roche）により精製した後、Ｔ４ＤＮＡリガーゼ（TaKaRa）を用いて自己環状化（self-ligation）した。環状化ＤＮＡを鋳型として、第１回ＰＣＲ（フォワードプライマーＲＥＴ－２２７１Ｆ（配列番号６）とリバースプライマーＲＥＴ－２３８１Ｒ（配列番号７）との組合せ）と、第２回ＰＣＲ（フォワードプライマーＲＥＴ－２６０５Ｆ（配列番号８）とリバースプライマーＲＥＴ－２２０８Ｒ（配列番号９）との組合せ）とを行った後、得られたＰＣＲ産物を用いてダイレクトシークエンスを行った。本実施例及び以下の実施例で使用したプライマーの塩基配列を表１に示す。
　その結果、ＮＣＯＡ４遺伝子又はＲＵＦＹ１遺伝子の一部がＲＥＴ遺伝子キナーゼ領域の５’側に融合していることが明らかとなった。 [Example 2] Identification of RET fusion polynucleotide gene in clinical specimen 5 ' The genes present on the side were examined.
First, reverse transcription and double-stranded cDNA synthesis were performed using a cDNA synthesis system (Roche) using 0.5 μg of RNA derived from a clinical specimen and a reverse transcription primer RET-2746R (SEQ ID NO: 5) designed in the RET gene kinase coding region. ). The synthesized double-stranded cDNA was purified by High Pure PCR Product Purification Kit (Roche) and then self-ligated using T4 DNA ligase (TaKaRa). First round PCR (combination of forward primer RET-2271F (SEQ ID NO: 6) and reverse primer RET-2381R (SEQ ID NO: 7)) and second round PCR (forward primer RET-2605F ( SEQ ID NO: 8) and reverse primer RET-2208R (SEQ ID NO: 9)), and then direct sequencing was performed using the obtained PCR product. Table 1 shows the base sequences of the primers used in this example and the following examples.
As a result, it was revealed that a part of the NCOA4 gene or RUFY1 gene was fused to the 5 ′ side of the RET gene kinase region.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
［実施例３］臨床検体でのＲＵＦＹ１－ＲＥＴ融合ポリヌクレオチド遺伝子の単離
　ＦＩＳＨ法解析によりＲＥＴゲノム構造異常が示唆され、融合する遺伝子が同定された大腸がん臨床検体由来のｃＤＮＡを鋳型として、ＤＮＡポリメラーゼ（PrimeStar HS DNAポリメラーゼ）を用いてＰＣＲを行い、増幅産物をｐＴ７Ｂｌｕｅ－２にクローニングした。ＲＵＦＹ１－ＲＥＴ融合ポリヌクレオチド遺伝子の単離用プライマーセットとしては、フォワードプライマーＲＵＦＹ１－５’ＵＴＲ（配列番号１０）とリバースプライマーＲＥＴ－３’ＵＴＲ（配列番号１１）との組合せを用いた。
　得られた増幅産物（３１８７ｂｐ）の配列を確認した結果、ＲＵＦＹ１遺伝子の開始コドンＡＴＧからエクソン１６までと、ＲＥＴ遺伝子のエクソン１２からエクソン２０の停止コドンまでの塩基配列からなるポリヌクレオチド（ＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２；配列番号３）が取得できた。なお、配列番号３の塩基配列における第１４０１番のＣ、第１７４０番のＡは、それぞれ、アミノ酸置換を伴わない一塩基多型（rs4701135、rs72824522）として報告されている。
　また、ＮＣＯＡ４－ＲＥＴ融合遺伝子については、前記実施例２の結果から、ＮＣＯＡ４遺伝子の開始コドンＡＴＧからエクソン９までと、ＲＥＴ遺伝子のエクソン１２からエクソン２０の停止コドンまでの塩基配列からなるポリヌクレオチド（ＮＣＯＡ４ｅｘ９－ＲＥＴｅｘ１２；配列番号１）であることを確認した。 [Example 3] Isolation of a RUFY1-RET fusion polynucleotide gene in a clinical sample Using a cDNA derived from a colorectal cancer clinical sample in which a RET genomic structural abnormality was suggested by FISH analysis and the fused gene was identified as a template, PCR was performed using DNA polymerase (PrimeStar HS DNA polymerase), and the amplified product was cloned into pT7Blue-2. As a primer set for isolating the RUFY1-RET fusion polynucleotide gene, a combination of the forward primer RUFY1-5′UTR (SEQ ID NO: 10) and the reverse primer RET-3′UTR (SEQ ID NO: 11) was used.
As a result of confirming the sequence of the obtained amplification product (3187 bp), a polynucleotide (RUFY1ex16-RETex12) consisting of a base sequence from the start codon ATG to exon 16 of the RUFY1 gene and from the exon 12 to the stop codon of exon 20 of the RET gene. ; SEQ ID NO: 3) was obtained. The 1401st C and the 1740th A in the nucleotide sequence of SEQ ID NO: 3 are reported as single nucleotide polymorphisms (rs4701135, rs72824522) without amino acid substitution, respectively.
For the NCOA4-RET fusion gene, based on the results of Example 2, a polynucleotide comprising a nucleotide sequence from the start codon ATG to exon 9 of the NCOA4 gene and the stop codon of exon 12 to exon 20 of the RET gene ( NCOA4ex9-RETex12; SEQ ID NO: 1) was confirmed.
［実施例４］ＮＣＯＡ４－ＲＥＴ融合遺伝子、ＲＵＦＹ１－ＲＥＴ融合遺伝子の検出
　表１に示すプライマーを用いて、融合部を含む領域を直接増幅するＲＴ－ＰＣＲで融合遺伝子の検出を行い、融合遺伝子ｃＤＮＡががん組織に存在していることを示した。具体的には、ＮＣＯＡ４－ＲＥＴ融合遺伝子に関しては、検体由来ＲＮＡテンプレートに対して、ＮＣＯＡ４遺伝子上に設計したフォワードプライマーＮＣＯＡ４－１６６１Ｆ（配列番号１２）とＲＥＴ遺伝子上に設計したリバースプライマーＲＥＴ－２３８１Ｒ（配列番号７）とを使用してＰＣＲを行った。増幅産物を電気泳動したところ、プライマー設定位置から予想されるサイズのバンド（４２４ｂｐ）が観察された。ＲＵＦＹ１－ＲＥＴ融合遺伝子に関しては、検体由来ＲＮＡテンプレートに対して、ＲＵＦＹ１遺伝子上に設計したフォワードプライマーＲＵＦＹ１－１４９２Ｆ（配列番号１３）とＲＥＴ遺伝子上に設計したリバースプライマーＲＥＴ－２３８１Ｒ（配列番号７）とを使用してＰＣＲを行った。増幅産物を電気泳動したところ、プライマー設定位置から予想されるサイズのバンド（６５９ｂｐ）が観察された。臨床検体を用いた融合遺伝子の検出が、これらの遺伝子上にプライマーを設計することによって可能であることが示された。 [Example 4] Detection of NCOA4-RET fusion gene and RUFY1-RET fusion gene Using the primers shown in Table 1, the fusion gene was detected by RT-PCR that directly amplifies the region containing the fusion region. Was present in cancer tissue. Specifically, for the NCOA4-RET fusion gene, the forward primer NCOA4-1661F (SEQ ID NO: 12) designed on the NCOA4 gene and the reverse primer RET-2381R designed on the RET gene (with respect to the specimen-derived RNA template) PCR was performed using SEQ ID NO: 7). When the amplified product was electrophoresed, a band (424 bp) of the size expected from the primer setting position was observed. For the RUFY1-RET fusion gene, the sample-derived RNA template was compared with the forward primer RUYY1-1492F (SEQ ID NO: 13) designed on the RUFY1 gene and the reverse primer RET-2381R (SEQ ID NO: 7) designed on the RET gene. PCR was performed using When the amplified product was electrophoresed, a band (659 bp) of the size expected from the primer setting position was observed. It has been shown that detection of fusion genes using clinical specimens is possible by designing primers on these genes.
［実施例５］臨床検体でのＮＣＯＡ４－ＲＥＴ融合遺伝子、ＲＵＦＹ１－ＲＥＴ融合遺伝子のＦＩＳＨ法フュージョンアッセイによる検出
　融合遺伝子がゲノム上で融合していることを確認するため、ＦＩＳＨ法フュージョンアッセイにて検出を行った。
　ＦＩＳＨ法フュージョンアッセイでは染色体転座又は逆位等によって近接する二つの目的遺伝子領域を異なる蛍光色素で標識したプローブで染める。例えば、ＴｅｘａｓＲｅｄ（赤）標識及びＦＩＴＣ（緑）標識した２種類のプローブにて蛍光標識することにより、正常な場合（融合遺伝子が構築されていない場合）は赤と緑はそれぞれのシグナル（赤色と緑色のシグナルが離れて存在している状態）として検出され、転座又は逆位等が起きることにより２つの遺伝子領域が近接している場合は、赤色と緑色のシグナルが重なり黄色として検出される。 [Example 5] Detection of NCOA4-RET fusion gene and RUFY1-RET fusion gene in clinical specimens by FISH fusion assay To confirm that the fusion gene is fused on the genome, it is detected by FISH fusion assay Went.
In the FISH fusion assay, two adjacent target gene regions are stained with probes labeled with different fluorescent dyes by chromosomal translocation or inversion. For example, by fluorescently labeling with two types of probes labeled TexasRed (red) and FITC (green), when normal (when the fusion gene has not been constructed), red and green indicate their respective signals (red and red). If the two gene regions are close to each other due to translocation or inversion, the red and green signals are detected as overlapping yellow. .
　具体的には、ＮＣＯＡ４－ＲＥＴ融合遺伝子に関しては、赤（TexasRed）の蛍光標識をした、ＮＣＯＡ４遺伝子５’末端側領域をカバーするＢＡＣクローン（クローン番号　CTD-2299L6）と、緑（FITC）の蛍光標識をした、ＲＥＴ遺伝子３’末端側領域をカバーするＢＡＣクローン（クローン番号　RP11-351D16）との組み合わせを使用した。
　ＲＵＦＹ１－ＲＥＴ融合遺伝子に関しては、赤（TexasRed）の蛍光標識をした、ＲＵＦＹ１遺伝子５’末端側領域をカバーするＢＡＣクローン（クローン番号　RP11-410B18）と、緑（FITC）の蛍光標識をした、ＲＥＴ遺伝子３’末端側領域をカバーするＢＡＣクローン（クローン番号　RP11-351D16）との組み合わせを使用した。
　蛍光観察は蛍光顕微鏡ＢＸ５１（Olympus社）を使用した。実施例４において融合遺伝子が陽性であった病理切片上でのフュージョンアッセイの結果、ＮＣＯＡ４遺伝子５’末端側領域とＲＥＴ遺伝子３’末端側領域との近接したシグナル（黄）、あるいは、ＲＵＦＹ１遺伝子５’末端側領域とＲＥＴ遺伝子３’末端側領域との近接したシグナル（黄）が観察され、融合遺伝子が染色体転座又は逆位等により生成されたことが確かめられた。
　この方法が当該融合遺伝子の存在を検出する方法として使えることがわかった。 Specifically, for the NCOA4-RET fusion gene, a red (TexasRed) fluorescent labeling BAC clone (clone number CTD-2299L6) covering the 5 'terminal region of the NCOA4 gene and green (FITC) fluorescence A combination with a labeled BAC clone (clone number RP11-351D16) covering the 3 ′ terminal region of the RET gene was used.
For the RUFY1-RET fusion gene, a red fluorescent label (TexasRed), a BAC clone (clone number RP11-410B18) covering the 5 ′ terminal region of the RUFY1 gene, and a green (FITC) fluorescent label, A combination with a BAC clone (clone number RP11-351D16) covering the 3 ′ terminal region of the gene was used.
For fluorescence observation, a fluorescence microscope BX51 (Olympus) was used. As a result of the fusion assay on the pathological section in which the fusion gene was positive in Example 4, a signal (yellow) adjacent to the 5 ′ terminal region of the NCOA4 gene and the 3 ′ terminal region of the RET gene, or the RUFY1 gene 5 A close signal (yellow) between the 'terminal region and the RET gene 3' terminal region was observed, confirming that the fusion gene was generated by chromosomal translocation or inversion.
It was found that this method can be used as a method for detecting the presence of the fusion gene.
［実施例６］ＲＵＦＹ１－ＲＥＴ融合ポリペプチドの腫瘍形成能の検討
　本実施例では、ＲＵＦＹ１－ＲＥＴ融合ポリペプチドをコードするｃＤＮＡとして、実施例３において大腸がん臨床検体から取得した配列番号３のＲＵＦＹ１－ＲＥＴ融合遺伝子を使用して、ＲＵＦＹ１－ＲＥＴ融合ポリペプチドの腫瘍形成能を検討した。前記ｃＤＮＡを発現ベクターｐＬｅｎｔｉ６（Invitrogen（登録商標）（Life Technologies社））に挿入して得られたｐＬｅｎｔｉ６－ＲＵＦＹ１－ＲＥＴをマウス繊維芽細胞株ＮＩＨ３Ｔ３細胞に導入し７日間培養したところ、図１に示すように形質転換フォーカスが観察された。遺伝子導入試薬のみの処理をしたＮＩＨ３Ｔ３細胞（ｃｏｎｔｒｏｌ）、ＬａｃＺを導入したＮＩＨ３Ｔ３細胞においては、形質転換フォーカスは認められなかった（図示せず）。すなわち、ＲＵＦＹ１－ＲＥＴ融合ポリペプチドを発現するベクターを導入した場合に限り、形質転換フォーカスが観察された。 [Example 6] Examination of tumor-forming ability of RUFY1-RET fusion polypeptide In this example, cDNA encoding RUFY1-RET fusion polypeptide was used as the cDNA encoding RUFY1-RET fusion polypeptide in SEQ ID NO: 3 obtained from a colorectal cancer clinical specimen. The RUFY1-RET fusion gene was used to examine the tumorigenicity of the RUFY1-RET fusion polypeptide. PLenti6-RUFY1-RET obtained by inserting the cDNA into the expression vector pLenti6 (Invitrogen (registered trademark) (Life Technologies)) was introduced into the mouse fibroblast cell line NIH3T3 cells and cultured for 7 days. A transformation focus was observed as shown. In NIH3T3 cells (control) treated only with the gene introduction reagent and NIH3T3 cells introduced with LacZ, transformation focus was not observed (not shown). That is, the transformation focus was observed only when a vector expressing the RUFY1-RET fusion polypeptide was introduced.
　ＲＵＦＹ１－ＲＥＴ融合ポリペプチドを発現するベクターを導入したＮＩＨ３Ｔ３細胞、遺伝子導入試薬処理のみをおこなったＮＩＨ３Ｔ３細胞（ｃｏｎｔｒｏｌ）、ＬａｃＺを導入したＮＩＨ３Ｔ３細胞を、それぞれ、ヌードマウスの皮下に１×１０６個ずつ接種したところ、融合ポリペプチドを発現するベクターを導入したＮＩＨ３Ｔ３細胞を接種したマウスにおいて腫瘍形成が確認された。遺伝子導入試薬処理のみをおこなったＮＩＨ３Ｔ３細胞（ｃｏｎｔｒｏｌ）を接種したマウスにおいては、腫瘍形成は確認されなかった。接種１日目以降の両マウスにおける腫瘍サイズを図２に示す。
　この結果より、ＲＵＦＹ１－ＲＥＴ融合ポリペプチドが腫瘍形成能を持っていたことから、ＲＵＦＹ１－ＲＥＴ融合ポリヌクレオチドは、がんの原因遺伝子であることが示された。
　なお、全長のＲＥＴポリペプチドをコードするｃＤＮＡをＮＩＨ３Ｔ３細胞に導入したところ、形質転換フォーカスは観察されず、全長のＲＥＴポリペプチドは腫瘍形成能を有しないことが確認された。 NIH3T3 cells introduced with a vector expressing the RUFY1-RET fusion polypeptide, NIH3T3 cells treated only with a gene introduction reagent (control), and NIH3T3 cells introduced with LacZ were each subcutaneously 1 × 10 6 nude mice. When inoculated one by one, tumor formation was confirmed in mice inoculated with NIH3T3 cells introduced with a vector expressing the fusion polypeptide. Tumor formation was not confirmed in mice inoculated with NIH3T3 cells (control) treated only with the gene introduction reagent. The tumor size in both mice after the first day of inoculation is shown in FIG.
From this result, it was shown that RUFY1-RET fusion polynucleotide is a causative gene of cancer because RUFY1-RET fusion polypeptide had tumorigenicity.
When cDNA encoding a full-length RET polypeptide was introduced into NIH3T3 cells, no transformation focus was observed, confirming that the full-length RET polypeptide had no tumorigenicity.
［実施例７］ＲＵＦＹ１－ＲＥＴ融合ポリペプチド発現細胞の各ＲＥＴ阻害剤に対する感受性の検討
　マウスリンパ系細胞株Ｂａ／Ｆ３細胞は、増殖因子であるＩＬ－３依存性細胞株であり、その増殖にＩＬ－３を必要とするが、がん化遺伝子（例えば、チロシンキナーゼ融合遺伝子）を導入することにより、ＩＬ－３を添加しなくても増殖できるようになることが知られている（Daley GQ and Baltimore D. Proc Natl Acad Sci USA. 1988 Dec;85(23):9312-9316.）。
　本実施例では、親株Ｂａ/Ｆ３細胞及び実施例６で作製したｐＬｅｎｔｉ６－ＲＵＦＹ１－ＲＥＴを導入したＢａ／Ｆ３細胞について、細胞数２０００の状態から、所定濃度の各ＲＥＴ阻害剤（Vandetanib、Alectinib、Lenvatinib、Cabozantinib）を添加し、７２時間培養した後の細胞数をカウントすることにより、各ＲＥＴ阻害剤に対する感受性を検討した。より具体的な試験方法については、例えば、Ｋａｔａｙａｍａらの文献（Katayama R et al., Sci Transl Med, 2012(4):120ra17）を参照することができる。 [Example 7] Examination of sensitivity of RUFY1-RET fusion polypeptide-expressing cells to each RET inhibitor Mouse lymphoid cell line Ba / F3 cell is an IL-3-dependent cell line that is a growth factor. Although IL-3 is required, it is known that by introducing a canceration gene (for example, a tyrosine kinase fusion gene), it can proliferate without adding IL-3 (Daley GQ and Baltimore D. Proc Natl Acad Sci USA. 1988 Dec; 85 (23): 9312-9316.).
In this example, with respect to the parent strain Ba / F3 cells and the Ba / F3 cells introduced with pLenti6-RUFY1-RET prepared in Example 6, from a state of 2000 cells, each RET inhibitor (Vandetanib, Alectinib, Lenvatinib and Cabozantinib) were added and the number of cells after culturing for 72 hours was counted to examine the sensitivity to each RET inhibitor. For more specific test methods, reference can be made to, for example, Katayama et al. (Katayama R et al., Sci Transl Med, 2012 (4): 120ra17).
　結果を図３～図６に示す。コントロールである親株Ｂａ／Ｆ３細胞（ＩＬ－３濃度＝０．５ｎｇ／ｍＬ）では、各ＲＥＴ阻害剤の濃度が細胞毒性を示す過剰量を超えない場合には、細胞増殖能に影響は認められなかった。一方、ｐＬｅｎｔｉ６－ＲＵＦＹ１－ＲＥＴを導入したＢａ／Ｆ３細胞（ＩＬ－３非添加）では、いずれのＲＥＴ阻害剤においても、濃度依存的に細胞増殖が顕著に抑制された。
　この結果は、ＲＥＴ阻害剤が、ＲＵＦＹ１－ＲＥＴ融合遺伝子陽性のがん患者の治療に有効であることを示すと同時に、ｐＬｅｎｔｉ６－ＲＵＦＹ１－ＲＥＴを導入したＢａ／Ｆ３細胞を用いる本評価系が、当該融合遺伝子陽性のがん患者の治療に有効な薬剤をスクリーニングするために用いることができることを示すものである。 The results are shown in FIGS. In the control parental Ba / F3 cells (IL-3 concentration = 0.5 ng / mL), when the concentration of each RET inhibitor does not exceed the excess amount indicating cytotoxicity, the cell proliferation ability is affected. There wasn't. On the other hand, in Ba / F3 cells into which pLenti6-RUFY1-RET was introduced (without IL-3 addition), cell growth was remarkably suppressed depending on the concentration of any RET inhibitor.
This result indicates that the RET inhibitor is effective for the treatment of cancer patients positive for RUFY1-RET fusion gene, and at the same time, the present evaluation system using Ba / F3 cells into which pLenti6-RUFY1-RET is introduced, It shows that it can be used for screening an effective drug for treatment of the fusion gene-positive cancer patient.
［実施例８］ＲＵＦＹ１－ＲＥＴ融合ポリペプチド発現細胞における各ＲＥＴ阻害剤によるＲＵＦＹ１－ＲＥＴ融合ポリペプチドの自己リン酸化抑制の検討
　実施例７で確認した、各ＲＥＴ阻害剤によるＲＵＦＹ１－ＲＥＴ融合ポリペプチド発現細胞の増殖抑制が、ＲＵＦＹ１－ＲＥＴ融合ポリペプチドのキナーゼ活性が阻害されたことによるものであることを確認するために、各ＲＥＴ阻害剤で処理した各培養細胞由来抽出物のウェスタンブロッティングを行った。
　結果を図７に示す。抗体としては、抗リン酸化ＲＥＴ抗体（図７におけるｐＲＥＴ）、抗ＲＥＴ抗体（図７におけるＲＥＴ）を使用した。ＲＥＴのポリペプチド量については、各ＲＥＴ阻害剤の処理の有無（及び処理濃度）に関してほとんど差異は認められなかった。一方、ＲＵＦＹ１－ＲＥＴ融合ポリペプチドのリン酸化は、各ＲＥＴ阻害剤による処理により、濃度依存的に顕著に減少しており、ＲＵＦＹ１－ＲＥＴ融合ポリペプチドのキナーゼ活性が阻害されることにより、ＲＵＦＹ１－ＲＥＴ融合ポリペプチドの自己リン酸化が抑制されることが確認された。 [Example 8] Examination of suppression of autophosphorylation of RUFY1-RET fusion polypeptide by each RET inhibitor in RUFY1-RET fusion polypeptide-expressing cells RUFY1-RET fusion polypeptide by each RET inhibitor confirmed in Example 7 In order to confirm that the growth suppression of the expressed cells was due to inhibition of the kinase activity of the RUFY1-RET fusion polypeptide, Western blotting was performed on extracts from each cultured cell treated with each RET inhibitor. It was.
The results are shown in FIG. As the antibody, an anti-phosphorylated RET antibody (pRET in FIG. 7) and an anti-RET antibody (RET in FIG. 7) were used. Regarding the amount of RET polypeptide, little difference was observed in the presence or absence (and treatment concentration) of each RET inhibitor. On the other hand, phosphorylation of the RUFY1-RET fusion polypeptide is markedly reduced in a concentration-dependent manner by treatment with each RET inhibitor, and the RUFY1-RET fusion polypeptide is inhibited by inhibiting the kinase activity. It was confirmed that autophosphorylation of the RET fusion polypeptide was suppressed.
　以上より、本発明において、一部の消化器がん患者においてＲＵＦＹ１遺伝子とＲＥＴ遺伝子との融合遺伝子が存在し、その遺伝子ががんの原因となっていることが明らかとなった。即ち、ＲＥＴ阻害薬物治療の対象となるがん患者を、ＲＵＦＹ１－ＲＥＴ融合遺伝子を検出することで、好ましくは、ＲＵＦＹ１ｅｘ１６－ＲＥＴｅｘ１２を検出することで選別できることが明らかとなった。 As described above, in the present invention, it has been clarified that a fusion gene of the RUFY1 gene and the RET gene exists in some digestive cancer patients, and that gene causes cancer. That is, it has been clarified that cancer patients to be treated with RET-inhibiting drug treatment can be selected by detecting the RUFY1-RET fusion gene, preferably by detecting RUFY1ex16-RETex12.
　本発明の検出方法は、ＲＥＴ融合体陽性のがん患者の判定に有用である。本発明の検出用キット及びプライマーセットは、前記検出方法に用いることができる。本発明の阻害物質スクリーニング方法は、当該融合体陽性のがん患者の治療に有効な物質をスクリーニングするのに用いることができる。前記スクリーニングにより得られる物質は、当該融合体陽性のがんの治療用医薬組成物の有効成分として使用することができる。前記検出方法により、当該融合体陽性のがん患者と判定された患者に、前記物質を投与することにより、がんを治療することができる。
　本発明の検出方法は、ＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがん患者の判定に有用である。本発明の検出用キット及びプライマーセットは、前記検出方法に用いることができる。本発明の阻害物質スクリーニング方法は、当該融合体陽性のがん患者の治療に有効な物質をスクリーニングするのに用いることができる。前記スクリーニングにより得られる物質は、当該融合体陽性のがんの治療用医薬組成物の有効成分として使用することができる。前記検出方法により、当該融合体陽性のがん患者と判定された患者に、前記物質を投与することにより、がんを治療することができる。
　以上、本発明を特定の態様に沿って説明したが、当業者に自明の変形や改良は本発明の範囲に含まれる。 The detection method of the present invention is useful for the determination of RET fusion-positive cancer patients. The detection kit and primer set of the present invention can be used in the detection method. The inhibitory substance screening method of the present invention can be used for screening a substance effective for treating the fusion-positive cancer patient. The substance obtained by the screening can be used as an active ingredient of the pharmaceutical composition for treating fusion-positive cancer. Cancer can be treated by administering the substance to a patient determined to be a fusion-positive cancer patient by the detection method.
The detection method of the present invention is useful for determination of cancer patients who are positive for NCOA4 or RUFY1 fusion. The detection kit and primer set of the present invention can be used in the detection method. The inhibitory substance screening method of the present invention can be used for screening a substance effective for treating the fusion-positive cancer patient. The substance obtained by the screening can be used as an active ingredient of the pharmaceutical composition for treating fusion-positive cancer. Cancer can be treated by administering the substance to a patient determined to be a fusion-positive cancer patient by the detection method.
As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.
　配列表の配列番号５～１３の配列で表される塩基配列は、合成プライマー配列である。 The base sequences represented by the sequences of SEQ ID NOs: 5 to 13 in the sequence listing are synthetic primer sequences.

Claims (72)

  1. 　被験者から得た消化器由来試料中の、ＲＥＴ融合タンパク質又は該融合タンパク質をコードする融合遺伝子の検出方法。 A method for detecting a RET fusion protein or a fusion gene encoding the fusion protein in a digestive organ-derived sample obtained from a subject.
  2. 　前記検出方法が、ＲＥＴタンパク質の切断、又は、ＲＥＴタンパク質をコードする遺伝子の切断、を検出する工程を含む、請求項１に記載の検出方法。 The detection method according to claim 1, wherein the detection method includes a step of detecting cleavage of the RET protein or cleavage of a gene encoding the RET protein.
  3. 　前記検出方法が、ＲＥＴタンパク質とそれ以外の他のタンパク質とから構築される融合タンパク質の存在、又は、前記融合タンパク質をコードする融合遺伝子の存在、を検出する工程を含む、請求項１に記載の検出方法。 2. The detection method according to claim 1, wherein the detection method includes a step of detecting the presence of a fusion protein constructed from a RET protein and other proteins or the presence of a fusion gene encoding the fusion protein. Detection method.
  4. 　前記融合タンパク質が、ＮＣＯＡ４又はＲＵＦＹ１タンパク質とＲＥＴタンパク質との融合タンパク質である、請求項１～３のいずれか一項に記載の検出方法。 The detection method according to any one of claims 1 to 3, wherein the fusion protein is a fusion protein of NCOA4 or RUFI1 protein and RET protein.
  5. 　前記融合タンパク質が、以下の（ａ）～（ｄ）からなる群から選択されるポリペプチドである、請求項１～４のいずれか一項に記載の検出方法：
    （ａ）配列番号２（ＮＣＯＡ４－ＲＥＴ）又は配列番号４（ＲＵＦＹ１－ＲＥＴ）で表されるアミノ酸配列からなるポリペプチド、
    （ｂ）配列番号２又は配列番号４で表されるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、
    （ｃ）配列番号２又は配列番号４で表されるアミノ酸配列との同一性が８０％以上であるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、及び
    （ｄ）配列番号２又は配列番号４で表されるアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、及び／若しくは挿入されたアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド。     The detection method according to any one of claims 1 to 4, wherein the fusion protein is a polypeptide selected from the group consisting of the following (a) to (d):
    (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 (NCOA4-RET) or SEQ ID NO: 4 (RUFY1-RET),
    (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity,
    (C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity; and (d) SEQ ID NO: 2 or SEQ ID NO: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted in the amino acid sequence represented by 4, and having tumorigenic potential.
  6. 　前記ＲＥＴ融合遺伝子が、請求項５に記載のポリペプチドをコードするポリヌクレオチドである、請求項１～５のいずれか一項に記載の検出方法。 The detection method according to any one of claims 1 to 5, wherein the RET fusion gene is a polynucleotide encoding the polypeptide according to claim 5.
  7. 　前記融合遺伝子が、ＤＮＡ又はｍＲＮＡである、請求項１～６のいずれか一項に記載の検出方法。 The detection method according to any one of claims 1 to 6, wherein the fusion gene is DNA or mRNA.
  8. 　前記消化器が、消化管である、請求項１～７のいずれか一項に記載の検出方法。 The detection method according to any one of claims 1 to 7, wherein the digestive organ is a digestive tract.
  9. 　前記消化器が、下部消化管である、請求項８に記載の検出方法。 The detection method according to claim 8, wherein the digestive organ is a lower digestive tract.
  10. 　前記消化器が、大腸である、請求項８に記載の検出方法。 The detection method according to claim 8, wherein the digestive organ is a large intestine.
  11. 　ＲＥＴ遺伝子５’末端側ゲノム領域を特異的に認識できる第１のプローブと、ＲＥＴ遺伝子３’末端側ゲノム領域を特異的に認識できる第２のプローブとを含む、ＲＥＴ融合遺伝子の検出用キット。 A kit for detecting a RET fusion gene, comprising a first probe capable of specifically recognizing the RET gene 5 'terminal genomic region and a second probe capable of specifically recognizing the RET gene 3' terminal genomic region.
  12. 　ＲＥＴ遺伝子と共にＲＥＴ融合遺伝子を構成する他の遺伝子の５’末端側ゲノム領域を特異的に認識できる第１のプローブと、ＲＥＴ遺伝子３’末端側ゲノム領域を特異的に認識できる第２のプローブとを含む、ＲＥＴ融合遺伝子の検出用キット。 A first probe capable of specifically recognizing the 5 ′ terminal genomic region of another gene that constitutes the RET fusion gene together with the RET gene, and a second probe capable of specifically recognizing the RET gene 3 ′ terminal genomic region A kit for detecting a RET fusion gene.
  13. 　ＲＥＴタンパク質をコードするポリヌクレオチドの、５’末端側領域を特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマー、ならびに、前記ポリヌクレオチドの３’末端側領域を特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマーを含む、ＲＥＴ融合遺伝子の検出用キット。 Sense primer and antisense primer designed to specifically amplify the 5 ′ end region of the polynucleotide encoding RET protein, and to specifically amplify the 3 ′ end region of the polynucleotide A kit for detecting a RET fusion gene, comprising a designed sense primer and antisense primer.
  14. 　ＮＣＯＡ４又はＲＵＦＹ１タンパク質とＲＥＴタンパク質との融合タンパク質であるポリペプチドをコードするポリヌクレオチドを特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマーを含む、ＮＣＯＡ４－ＲＥＴ又はＲＵＦＹ１－ＲＥＴ融合遺伝子の検出用キット。 Detection of an NCOA4-RET or RUFY1-RET fusion gene comprising a sense primer and an antisense primer designed to specifically amplify a polynucleotide encoding a polypeptide that is a fusion protein of NCOA4 or RUFY1 protein and RET protein For kit.
  15. 　以下の（ａ）～（ｄ）からなる群から選択されるポリペプチドをコードするポリヌクレオチドを特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマーを含む、ＮＣＯＡ４－ＲＥＴ又はＲＵＦＹ１－ＲＥＴ融合遺伝子の検出用キット：
    （ａ）配列番号２又は配列番号４で表されるアミノ酸配列からなるポリペプチド、
    （ｂ）配列番号２又は配列番号４で表されるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、
    （ｃ）配列番号２又は配列番号４で表されるアミノ酸配列との同一性が８０％以上であるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、及び
    （ｄ）配列番号２又は配列番号４で表されるアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、及び／若しくは挿入されたアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド。     NCOA4-RET or RUFY1-RET fusion comprising a sense primer and an antisense primer designed to specifically amplify a polynucleotide encoding a polypeptide selected from the group consisting of the following (a) to (d) Gene detection kit:
    (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4,
    (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity,
    (C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and having tumorigenicity; and (d) SEQ ID NO: 2 or SEQ ID NO: A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted in the amino acid sequence represented by 4, and having tumorigenic potential.
  16. 　ＲＥＴタンパク質のＮ末端側領域を特異的に認識できる抗ＲＥＴ抗体、及び、ＲＥＴタンパク質のＣ末端側領域を特異的に認識できる抗ＲＥＴ抗体を含む、ＲＥＴ融合タンパク質の検出用キット。 A kit for detecting a RET fusion protein comprising an anti-RET antibody that can specifically recognize the N-terminal region of the RET protein and an anti-RET antibody that can specifically recognize the C-terminal region of the RET protein.
  17. 　ＲＥＴタンパク質と共にＲＥＴ融合タンパク質を構成する他のタンパク質のＮ末端側領域のポリペプチドに特異的に結合する抗体と、ＲＥＴタンパク質のＣ末端側領域のポリペプチドに特異的に結合する抗体を含む、ＲＥＴ融合タンパク質の検出用キット。 An antibody that specifically binds to a polypeptide in the N-terminal region of another protein that constitutes the RET fusion protein together with the RET protein, and an antibody that specifically binds to a polypeptide in the C-terminal region of the RET protein A kit for detecting a fusion protein.
  18. 　前記他のタンパク質が、ＮＣＯＡ４又はＲＵＦＹ１タンパク質である、請求項１７に記載のキット。 The kit according to claim 17, wherein the other protein is NCOA4 or RUFY1 protein.
  19. 　ＲＥＴタンパク質をコードするポリヌクレオチド部分から設計されるアンチセンスプライマー及びＮＣＯＡ４又はＲＵＦＹ１タンパク質をコードするポリヌクレオチド部分から設計されるセンスプライマーを含む、ＲＥＴ遺伝子とＮＣＯＡ４又はＲＵＦＹ１遺伝子との融合遺伝子を検出するためのプライマーセットであって、アンチセンスプライマーは請求項１５に記載のポリヌクレオチドにストリンジェントな条件下でアニールする核酸分子からなり、センスプライマーは請求項１５に記載のポリヌクレオチドの相補鎖にストリンジェントな条件でアニールする核酸分子からなるプライマーセット。 To detect a fusion gene between a RET gene and an NCOA4 or RUFY1 gene, comprising an antisense primer designed from a polynucleotide portion encoding a RET protein and a sense primer designed from a polynucleotide portion encoding an NCOA4 or RUFY1 protein Wherein the antisense primer comprises a nucleic acid molecule that anneals to the polynucleotide of claim 15 under stringent conditions, and the sense primer is stringent to the complementary strand of the polynucleotide of claim 15. Primer set consisting of nucleic acid molecules that anneal under various conditions.
  20. 　ＮＣＯＡ４又はＲＵＦＹ１遺伝子とＲＥＴ遺伝子との融合遺伝子を検出するためのプライマーセットであって、配列番号１又は配列番号３に示される塩基配列からなるポリヌクレオチドにストリンジェントな条件下でアニールする核酸分子からなるアンチセンスプライマー及び該ポリヌクレオチドの相補鎖にストリンジェントな条件下でアニールする核酸分子からなるセンスプライマーを含むプライマーセット。 A primer set for detecting a fusion gene of an NCOA4 or RUFY1 gene and a RET gene, from a nucleic acid molecule that anneals to a polynucleotide comprising the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions A primer set comprising an antisense primer and a sense primer comprising a nucleic acid molecule that anneals to a complementary strand of the polynucleotide under stringent conditions.
  21. 　以下の（ａ）～（ｂ）からなる群から選択される、センスプライマー及びアンチセンスプライマー、を含む、プライマーセット：
    （ａ）配列番号１の塩基番号１から１９８４の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドからなるセンスプライマー、及び配列番号１の塩基番号１９８５から５２７５の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドに対して相補的であるオリゴヌクレオチドからなるアンチセンスプライマー、及び、
    （ｂ）配列番号３の塩基番号１から１９１７の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドからなるセンスプライマー、及び配列番号３の塩基番号１９１８から５２０８の間の任意の連続する少なくとも１６塩基のオリゴヌクレオチドに対して相補的であるオリゴヌクレオチドからなるアンチセンスプライマー。     Primer set comprising a sense primer and an antisense primer selected from the group consisting of the following (a) to (b):
    (A) a sense primer composed of any consecutive at least 16 base oligonucleotides between base numbers 1 to 1984 of SEQ ID NO: 1, and any continuous at least 16 bases between base numbers 1985 to 5275 of SEQ ID NO: 1 An antisense primer consisting of an oligonucleotide that is complementary to the oligonucleotide of
    (B) a sense primer composed of an arbitrary at least 16 base oligonucleotide between base numbers 1 to 1917 of SEQ ID NO: 3 and an arbitrary continuous at least 16 bases between base numbers 1918 to 5208 of SEQ ID NO: 3 An antisense primer comprising an oligonucleotide that is complementary to the oligonucleotide.
  22. （１）請求項５に記載のポリペプチド、又は前記ポリペプチドを発現している細胞に試験物質を接触させる工程、
    （２）前記ポリペプチドの活性及び／又は発現が阻害されるか否かを分析する工程、及び
    （３）前記ポリペプチドの活性及び／又は発現を阻害する物質を選択する工程
    を含む、前記ポリペプチドの活性及び／又は発現を阻害する物質をスクリーニングする方法。     (1) The step of bringing the test substance into contact with the polypeptide according to claim 5 or a cell expressing the polypeptide,
    (2) analyzing whether or not the activity and / or expression of the polypeptide is inhibited; and (3) selecting the substance that inhibits the activity and / or expression of the polypeptide. A method of screening for a substance that inhibits the activity and / or expression of a peptide.
  23. 　前記ポリペプチドの活性及び／又は発現を阻害する物質が、ＲＥＴ融合体陽性のがんの治療剤である、請求項２２に記載のスクリーニング方法。 The screening method according to claim 22, wherein the substance that inhibits the activity and / or expression of the polypeptide is a therapeutic agent for RET fusion-positive cancer.
  24. 　前記がんが、消化器がんである、請求項２３に記載のスクリーニング方法。 The screening method according to claim 23, wherein the cancer is digestive organ cancer.
  25. 　前記がんが、消化管がんである、請求項２４に記載のスクリーニング方法。 The screening method according to claim 24, wherein the cancer is gastrointestinal cancer.
  26. 　前記がんが、下部消化管がんである、請求項２４に記載のスクリーニング方法。 The screening method according to claim 24, wherein the cancer is a cancer of the lower digestive tract.
  27. 　前記がんが、大腸がんである、請求項２４に記載のスクリーニング方法。 The screening method according to claim 24, wherein the cancer is colon cancer.
  28. 　ＲＥＴ融合タンパク質の活性及び／又は発現を阻害する物質を含有する、ＲＥＴ融合体陽性のがんの治療用医薬組成物。 A pharmaceutical composition for treating RET fusion positive cancer, comprising a substance that inhibits the activity and / or expression of a RET fusion protein.
  29. 　前記ＲＥＴ融合タンパク質の活性及び／又は発現を阻害する物質が、キナーゼ阻害剤である、請求項２８に記載の医薬組成物。 The pharmaceutical composition according to claim 28, wherein the substance that inhibits the activity and / or expression of the RET fusion protein is a kinase inhibitor.
  30. 　前記ＲＥＴ融合タンパク質が、請求項５に記載のポリペプチドである、請求項２８又は２９に記載の医薬組成物。 30. The pharmaceutical composition according to claim 28 or 29, wherein the RET fusion protein is the polypeptide according to claim 5.
  31. 　前記がんが、消化器がんである、請求項２８～３０のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 28 to 30, wherein the cancer is digestive cancer.
  32. 　前記がんが、消化管がんである、請求項３１に記載の医薬組成物。 The pharmaceutical composition according to claim 31, wherein the cancer is gastrointestinal cancer.
  33. 　前記がんが、下部消化管がんである、請求項３１に記載の医薬組成物。 The pharmaceutical composition according to claim 31, wherein the cancer is a cancer of the lower gastrointestinal tract.
  34. 　前記がんが、大腸がんである、請求項３１に記載の医薬組成物。 The pharmaceutical composition according to claim 31, wherein the cancer is colon cancer.
  35. 　ＲＵＦＹ１とＲＥＴとの融合タンパク質。 Fusion protein of RUFY1 and RET.
  36. 　以下の（ａ）～（ｄ）からなる群から選択されるポリペプチドである、請求項３５に記載の融合タンパク質：
    （ａ）配列番号４で表されるアミノ酸配列からなるポリペプチド、
    （ｂ）配列番号４で表されるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、
    （ｃ）配列番号４で表されるアミノ酸配列との同一性が８０％以上であるアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド、及び
    （ｄ）配列番号４で表されるアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、及び／若しくは挿入されたアミノ酸配列を含み、しかも腫瘍形成能を有するポリペプチド。     The fusion protein according to claim 35, which is a polypeptide selected from the group consisting of the following (a) to (d):
    (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4,
    (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 and having tumorigenicity,
    (C) a polypeptide comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having tumorigenicity, and (d) an amino acid sequence represented by SEQ ID NO: 4 A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or inserted, and having tumorigenic potential.
  37. 　請求項３５又は３６のいずれか一項に記載の融合タンパク質をコードするポリヌクレオチド。 A polynucleotide encoding the fusion protein according to any one of claims 35 and 36.
  38. 　請求項３７に記載のポリヌクレオチドを含むベクター。 A vector comprising the polynucleotide according to claim 37.
  39. 　請求項３８に記載のベクターで形質転換された細胞。 A cell transformed with the vector according to claim 38.
  40. 　被験者から得た消化器由来試料中の、ＮＣＯＡ４融合タンパク質又は該融合タンパク質をコードする融合遺伝子の検出方法。 A method for detecting an NCOA4 fusion protein or a fusion gene encoding the fusion protein in a digestive organ-derived sample obtained from a subject.
  41. 　前記検出方法が、ＮＣＯＡ４タンパク質の切断、又は、ＮＣＯＡ４タンパク質をコードする遺伝子の切断、を検出する工程を含む、請求項４０に記載の検出方法。 The detection method according to claim 40, wherein the detection method includes a step of detecting cleavage of the NCOA4 protein or cleavage of a gene encoding the NCOA4 protein.
  42. 　前記検出方法が、ＮＣＯＡ４タンパク質とそれ以外の他のタンパク質とから構築される融合タンパク質の存在、又は、前記融合タンパク質をコードする融合遺伝子の存在、を検出する工程を含む、請求項４０に記載の検出方法。 41. The detection method according to claim 40, wherein the detection method comprises a step of detecting the presence of a fusion protein constructed from the NCOA4 protein and other proteins or the presence of a fusion gene encoding the fusion protein. Detection method.
  43. 　前記融合遺伝子が、ＤＮＡ又はｍＲＮＡである、請求項４０～４２のいずれか一項に記載の検出方法。 The detection method according to any one of claims 40 to 42, wherein the fusion gene is DNA or mRNA.
  44. 　前記消化器由来試料が、消化管由来試料である、請求項４１～４４のいずれか一項に記載の検出方法。 The detection method according to any one of claims 41 to 44, wherein the digestive organ-derived sample is a digestive tract-derived sample.
  45. 　前記消化器由来試料が、下部消化管由来試料である、請求項４４に記載の検出方法。 45. The detection method according to claim 44, wherein the digestive organ-derived sample is a lower digestive tract-derived sample.
  46. 　前記消化器由来試料が、大腸由来試料である、請求項４４に記載の検出方法。 45. The detection method according to claim 44, wherein the digestive organ-derived sample is a large intestine-derived sample.
  47. 　被験者から得た試料中の、ＲＵＦＹ１融合タンパク質又は該融合タンパク質をコードする融合遺伝子の検出方法。 A method for detecting a RUFY1 fusion protein or a fusion gene encoding the fusion protein in a sample obtained from a subject.
  48. 　前記検出方法が、ＲＵＦＹ１タンパク質の切断、又は、ＲＵＦＹ１タンパク質をコードする遺伝子の切断、を検出する工程を含む、請求項４７に記載の検出方法。 48. The detection method according to claim 47, wherein the detection method comprises a step of detecting cleavage of RUFY1 protein or cleavage of a gene encoding RUFY1 protein.
  49. 　前記検出方法が、ＲＵＦＹ１タンパク質とそれ以外の他のタンパク質とから構築される融合タンパク質の存在、又は、前記融合タンパク質をコードする融合遺伝子の存在、を検出する工程を含む、請求項４７に記載の検出方法。 48. The detection method according to claim 47, wherein the detection method comprises a step of detecting the presence of a fusion protein constructed from the RUFY1 protein and other proteins or the presence of a fusion gene encoding the fusion protein. Detection method.
  50. 　前記融合遺伝子が、ＤＮＡ又はｍＲＮＡである、請求項４７～４９のいずれか一項に記載の検出方法。 The detection method according to any one of claims 47 to 49, wherein the fusion gene is DNA or mRNA.
  51. 　前記試料が、消化器由来試料である、請求項４７～５０のいずれか一項に記載の検出方法。 The detection method according to any one of claims 47 to 50, wherein the sample is a digestive organ-derived sample.
  52. 　前記消化器由来試料が、消化管由来試料である、請求項５１に記載の検出方法。 The detection method according to claim 51, wherein the digestive organ-derived sample is a digestive tract-derived sample.
  53. 　前記消化器由来試料が、下部消化管由来試料である、請求項５１に記載の検出方法。 The detection method according to claim 51, wherein the digestive organ-derived sample is a lower digestive tract-derived sample.
  54. 　前記消化器由来試料が、大腸由来試料である、請求項５１に記載の検出方法。 The detection method according to claim 51, wherein the digestive organ-derived sample is a large intestine-derived sample.
  55. 　ＮＣＯＡ４又はＲＵＦＹ１遺伝子５’末端側ゲノム領域を特異的に認識できる第１のプローブと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子３’末端側ゲノム領域を特異的に認識できる第２のプローブとを含む、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出用キット。 NCOA4 or RUFY1 fusion comprising a first probe capable of specifically recognizing the NCOA4 or RUFY1 gene 5 ′ terminal genomic region and a second probe capable of specifically recognizing the NCOA4 or RUFY1 gene 3 ′ terminal genomic region Gene detection kit.
  56. 　ＮＣＯＡ４又はＲＵＦＹ１遺伝子と共にＮＣＯＡ４又はＲＵＦＹ１融合遺伝子を構成する他の遺伝子の３’末端側ゲノム領域を特異的に認識できる第１のプローブと、ＮＣＯＡ４又はＲＵＦＹ１遺伝子５’末端側ゲノム領域を特異的に認識できる第２のプローブとを含む、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出用キット。 A first probe capable of specifically recognizing the 3′-end genomic region of the other gene constituting the NCOA 4 or RUFY1 fusion gene together with the NCOA 4 or RUFY1 gene, and specifically recognizing the 5′-end genomic region of the NCOA4 or RUFY1 gene A kit for detecting an NCOA4 or RUFY1 fusion gene, comprising a second probe that can be produced.
  57. 　ＮＣＯＡ４又はＲＵＦＹ１タンパク質をコードするポリヌクレオチドの、５’末端側領域を特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマー、ならびに、前記ポリヌクレオチドの３’末端側領域を特異的に増幅できるように設計したセンスプライマー及びアンチセンスプライマーを含む、ＮＣＯＡ４又はＲＵＦＹ１融合遺伝子の検出用キット。 Sense primer and antisense primer designed to specifically amplify the 5 ′ end region of the polynucleotide encoding NCOA4 or RUFY1 protein, and specifically amplify the 3 ′ end region of the polynucleotide A kit for detecting an NCOA4 or RUFY1 fusion gene comprising a sense primer and an antisense primer designed as described above.
  58. 　ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＮ末端側領域を特異的に認識できる抗ＮＣＯＡ４又はＲＵＦＹ１抗体、及び、ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＣ末端側領域を特異的に認識できる抗ＮＣＯＡ４又はＲＵＦＹ１抗体を含む、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の検出用キット。 An NCOA4 or RUFY1 fusion comprising an anti-NCOA4 or RUFY1 antibody capable of specifically recognizing the N-terminal region of the NCOA4 or RUFY1 protein, and an anti-NCOA4 or RUFY1 antibody capable of specifically recognizing the C-terminal region of the NCOA4 or RUFY1 protein Protein detection kit.
  59. 　ＮＣＯＡ４又はＲＵＦＹ１タンパク質と共にＮＣＯＡ４又はＲＵＦＹ１融合タンパク質を構成する他のタンパク質のＣ末端側領域のポリペプチドに特異的に結合する抗体と、ＮＣＯＡ４又はＲＵＦＹ１タンパク質のＮ末端側領域のポリペプチドに特異的に結合する抗体を含む、ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の検出用キット。 An antibody that specifically binds to a polypeptide in the C-terminal region of another protein that constitutes the NCOA4 or RUFY1 fusion protein together with the NCOA4 or RUFY1 protein, and specifically binds to a polypeptide in the N-terminal region of the NCOA4 or RUFY1 protein A kit for detecting an NCOA4 or RUFY1 fusion protein, which comprises an antibody to be treated.
  60. （１）請求項５に記載のポリペプチド、又は前記ポリペプチドを発現している細胞に試験物質を接触させる工程、
    （２）前記ポリペプチドの活性及び／又は発現が阻害されるか否かを分析する工程、及び
    （３）前記ポリペプチドの活性及び／又は発現を阻害する物質を選択する工程
    を含む、前記ポリペプチドの活性及び／又は発現を阻害する物質をスクリーニングする方法。     (1) The step of bringing the test substance into contact with the polypeptide according to claim 5 or a cell expressing the polypeptide,
    (2) analyzing whether or not the activity and / or expression of the polypeptide is inhibited; and (3) selecting the substance that inhibits the activity and / or expression of the polypeptide. A method of screening for a substance that inhibits the activity and / or expression of a peptide.
  61. 　前記ポリペプチドの活性及び／又は発現を阻害する物質が、ＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがんの治療剤である、請求項６０に記載のスクリーニング方法。 61. The screening method according to claim 60, wherein the substance that inhibits the activity and / or expression of the polypeptide is a therapeutic agent for cancer positive for NCOA4 or RUfy1 fusion.
  62. 　前記がんが、消化器がんである、請求項６１に記載のスクリーニング方法。 The screening method according to claim 61, wherein the cancer is digestive organ cancer.
  63. 　前記がんが、消化管がんである、請求項６２に記載のスクリーニング方法。 The screening method according to claim 62, wherein the cancer is gastrointestinal cancer.
  64. 　前記がんが、下部消化管がんである、請求項６２に記載のスクリーニング方法。 The screening method according to claim 62, wherein the cancer is a cancer of the lower digestive tract.
  65. 　前記がんが、大腸がんである、請求項６２に記載のスクリーニング方法。 The screening method according to claim 62, wherein the cancer is colon cancer.
  66. 　ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の活性及び／又は発現を阻害する物質を含有する、ＮＣＯＡ４又はＲＵＦＹ１融合体陽性のがんの治療用医薬組成物。 A pharmaceutical composition for the treatment of cancer that is positive for NCOA4 or RUFY1 fusion, comprising a substance that inhibits the activity and / or expression of NCOA4 or RUFY1 fusion protein.
  67. 　前記ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質の活性及び／又は発現を阻害する物質が、キナーゼ阻害剤である、請求項６６に記載の医薬組成物。 68. The pharmaceutical composition according to claim 66, wherein the substance that inhibits the activity and / or expression of the NCOA4 or RUfy1 fusion protein is a kinase inhibitor.
  68. 　前記ＮＣＯＡ４又はＲＵＦＹ１融合タンパク質が、請求項５に記載のポリペプチドである、請求項６６又は６７に記載の医薬組成物。 68. The pharmaceutical composition according to claim 66 or 67, wherein the NCOA4 or RUfy1 fusion protein is the polypeptide according to claim 5.
  69. 　前記がんが、消化器がんである、請求項６６～６８のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 66 to 68, wherein the cancer is gastrointestinal cancer.
  70. 　前記がんが、消化管がんである、請求項６９に記載の医薬組成物。 70. The pharmaceutical composition according to claim 69, wherein the cancer is gastrointestinal cancer.
  71. 　前記がんが、下部消化管がんである、請求項６９に記載の医薬組成物。 70. The pharmaceutical composition according to claim 69, wherein the cancer is cancer of the lower gastrointestinal tract.
  72. 　前記がんが、大腸がんである、請求項６９に記載の医薬組成物。 70. The pharmaceutical composition according to claim 69, wherein the cancer is colon cancer.
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