WO2015064620A1 - Nouvelles fusions et méthode de détection associée - Google Patents

Nouvelles fusions et méthode de détection associée Download PDF

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WO2015064620A1
WO2015064620A1 PCT/JP2014/078728 JP2014078728W WO2015064620A1 WO 2015064620 A1 WO2015064620 A1 WO 2015064620A1 JP 2014078728 W JP2014078728 W JP 2014078728W WO 2015064620 A1 WO2015064620 A1 WO 2015064620A1
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gene
alk
zscan9
fusion
protein
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PCT/JP2014/078728
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English (en)
Japanese (ja)
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賢吾 竹内
由紀 冨樫
征士 坂田
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 JP2015545257A priority Critical patent/JP6806440B2/ja
Publication of WO2015064620A1 publication Critical patent/WO2015064620A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a novel fusion protein containing an ALK 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 ZSCAN9, 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
  • ALK anaplastic lymphoma receptor tyrosine kinase
  • ALK is a receptor tyrosine kinase, belongs to the insulin receptor superfamily, and has an extracellular domain, a single transmembrane domain, and an intracellular kinase domain (Non-patent Document 2).
  • ALK is known to constitute not only the above-mentioned EML4 but also various genes and fusion genes, and these fusion genes are also shown to be cancer causative genes (Non-Patent Documents 3 and 4). ).
  • ZSCAN9 also referred to as zinc finger and SCAN domain containing 9, ZNF193 (zinc finger protein 193) belongs to the krueppel C2H2-type zinc-finger protein family, 5 C2H2-type zinc finger domains, and 1 SCAN box domain. (Non-patent Document 5). It is not known that the gene constitutes a fusion gene.
  • 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 isolated and identified a novel fusion gene in which a part of the ZSCAN9 gene and a part of the ALK gene, which is a kinase, were fused from a sample obtained from a colon cancer patient (Examples 1 to 3).
  • the present inventors have found that the fusion gene is present in colorectal cancer patient specimens (Examples 4 to 7). Based on these findings, the present inventor provides a method for detecting an ALK fusion protein or a fusion gene encoding the protein (Examples 4 to 7), and provides a kit and a primer set therefor.
  • the present inventor provides a method for detecting a ZSCAN9 fusion protein or a fusion gene encoding the protein (Examples 4 to 7), and provides a kit and a primer set therefor.
  • a method for treating cancer comprising the step of:
  • the present invention relates to the following inventions: [1] ZSCAN9 fusion protein.
  • [4] A polynucleotide encoding the fusion protein according to any one of [1] to [3].
  • [5] A vector comprising the polynucleotide according to [4].
  • [6] A cell transformed with the vector according to [5].
  • [7] A method for detecting a ZSCAN9 fusion protein or a fusion gene encoding the fusion protein in a sample obtained from a subject.
  • the detection method according to [7], wherein the detection method includes a step of detecting cleavage of the ZSCAN9 protein or cleavage of a gene encoding the ZSCAN9 protein.
  • the detection method includes a step of detecting the presence of a fusion protein constructed from a ZSCAN9 protein and another protein or the presence of a fusion gene encoding the fusion protein.
  • the detection method according to. [10] The detection method according to any one of [7] to [9], wherein the fusion protein is a fusion protein of ZSCAN9 protein and ALK 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, (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 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 and having tumorigenicity, and (d) an amino acid sequence represented by SEQ ID NO: 2 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.
  • Detection of a ZSCAN9 fusion gene comprising a first probe capable of specifically recognizing the ZSCAN9 gene 5 ′ terminal genomic region and a second probe capable of specifically recognizing the ZSCAN9 gene 3 ′ terminal genomic region
  • a probe for detecting a ZSCAN9 fusion gene comprising a first probe capable of specifically recognizing the ZSCAN9 gene 5 ′ terminal genomic region and a second probe capable of specifically recognizing the ZSCAN9 gene 3 ′ terminal genomic region
  • a sense primer and an antisense primer designed to specifically amplify the 5′-terminal region of a polynucleotide encoding ZSCAN9 protein, and specifically amplify the 3′-terminal region of the polynucleotide A kit for detecting a ZSCAN9 fusion gene, comprising a sense primer and an antisense primer designed to be able to.
  • a kit for detecting a ZSCAN9-ALK 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 a ZSCAN9 protein and an ALK protein.
  • a ZSCAN9-ALK fusion gene 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) Detection kit: (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 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 and having tumorigenicity, and (d) an amino acid sequence represented by SEQ ID NO: 2 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.
  • a kit for detecting a ZSCAN9 fusion protein comprising an anti-ZSCAN9 antibody capable of specifically recognizing the N-terminal region of ZSCAN9 protein and an anti-ZSCAN9 antibody capable of specifically recognizing the C-terminal region of ZSCAN9 protein.
  • An antibody that specifically binds to a polypeptide in the C-terminal region of another protein that constitutes a ZSCAN9 fusion protein together with a ZSCAN9 protein, and an antibody that specifically binds to a polypeptide in the N-terminal region of the ZSCAN9 protein A kit for detecting a ZSCAN9 fusion protein.
  • a method for detecting a fusion gene of a ZSCAN9 gene and an ALK gene comprising a sense primer designed from a polynucleotide portion encoding a ZSCAN9 protein and an antisense primer designed from a polynucleotide portion encoding an ALK protein
  • the antisense primer comprises a nucleic acid molecule that anneals to the polynucleotide described in [22] under stringent conditions
  • the sense primer is stringent to the complementary strand of the polynucleotide described in [22].
  • Primer set consisting of nucleic acid molecules that anneal under conditions.
  • a primer set for detecting a fusion gene of ZSCAN9 gene and ALK gene comprising a nucleic acid molecule that anneals to a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions
  • a primer set comprising a primer and a sense primer comprising a nucleic acid molecule that anneals to a complementary strand of the polynucleotide under stringent conditions.
  • a sense primer comprising an arbitrary continuous at least 16 base oligonucleotide between base numbers 1 to 568 of SEQ ID NO: 1 and an arbitrary continuous at least 16 base oligonucleotide between base numbers 569 to 2259 of SEQ ID NO: 1
  • An antisense primer consisting of an oligonucleotide complementary to the nucleotide sequence, or a sense primer consisting of at least 16 consecutive oligonucleotides between base numbers 1 to 716 of SEQ ID NO: 12 and base number 717 of SEQ ID NO: 12
  • a primer set comprising an antisense primer consisting of an oligonucleotide that is complementary to any consecutive at least 16 base oligonucleotide between 2857 and 2857.
  • [29] The step of bringing the test substance into contact with the polypeptide according to (1) [3] 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 [29] wherein the substance that inhibits the activity and / or expression of the polypeptide is a therapeutic agent for ZSCAN9 fusion-positive cancer.
  • a pharmaceutical composition for the treatment of ZSCAN9 fusion-positive cancer comprising a substance that inhibits the activity and / or expression of a ZSCAN9 fusion protein.
  • the pharmaceutical composition according to [35], wherein the substance that inhibits the activity and / or expression of the ZSCAN9 fusion protein is a kinase inhibitor.
  • a vector comprising the polynucleotide according to [45].
  • the detection method includes a step of detecting the presence of a fusion protein constructed from an ALK protein and another protein, or the presence of a fusion gene encoding the fusion protein.
  • the detection method according to. [51] The detection method according to any one of [48] to [50], wherein the fusion protein is a fusion protein of ZSCAN9 protein and ALK 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, (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 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 and having tumorigenicity, and (d) an amino acid sequence represented by SEQ ID NO: 2 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.
  • [53] The detection method according to any one of [48] to [52], wherein the ALK fusion gene is a polynucleotide encoding the polypeptide of [44].
  • [54] The detection method according to any one of [48] to [53], wherein the fusion gene is DNA or mRNA.
  • the detection method according to [55], wherein the digestive tract is the lower digestive tract.
  • the detection method according to [55], wherein the digestive organ is a large intestine.
  • ALK fusion gene comprising a first probe capable of specifically recognizing the 5 ′ terminal genomic region of the ALK gene and a second probe capable of specifically recognizing the ALK gene 3 ′ terminal genomic region
  • a sense primer and an antisense primer designed to specifically amplify the 5 ′ end region of the polynucleotide encoding ALK protein, and specifically amplify the 3 ′ end region of the polynucleotide.
  • a kit for detecting an ALK fusion gene comprising a sense primer and an antisense primer designed to be able to.
  • a kit for detecting a ZSCAN9-ALK 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 a ZSCAN9 protein and an ALK protein.
  • a ZSCAN9-ALK fusion gene 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) Detection kit: (A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 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 and having tumorigenicity, and (d) an amino acid sequence represented by SEQ ID NO: 2 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.
  • a kit for detecting an ALK fusion protein comprising an anti-ALK antibody that can specifically recognize the N-terminal region of the ALK protein and an anti-ALK antibody that can specifically recognize the C-terminal region of the ALK protein.
  • An antibody that specifically binds to a polypeptide in the N-terminal region of another protein that constitutes an ALK fusion protein together with the ALK protein, and an antibody that specifically binds to a polypeptide in the C-terminal region of the ALK protein A kit for detecting an ALK fusion protein.
  • the kit according to [65] wherein the other protein is a ZSCAN9 protein.
  • a method for detecting a fusion gene between a ZSCAN9 gene and an ALK gene comprising a sense primer designed from a polynucleotide portion encoding a ZSCAN9 protein and an antisense primer designed from a polynucleotide portion encoding an ALK protein
  • the antisense primer comprises a nucleic acid molecule that anneals to the polynucleotide of [63] under stringent conditions
  • the sense primer is stringent to the complementary strand of the polynucleotide of [63].
  • Primer set consisting of nucleic acid molecules that anneal under conditions.
  • An antisense comprising a primer set for detecting a fusion gene of ZSCAN9 gene and ALK gene, the nucleic acid molecule annealing to a polynucleotide comprising the base sequence represented by SEQ ID NO: 1 under stringent conditions
  • a primer set comprising a primer and a sense primer comprising a nucleic acid molecule that anneals to a complementary strand of the polynucleotide under stringent conditions.
  • a sense primer composed of an arbitrary continuous at least 16 base oligonucleotide between base numbers 1 to 568 of SEQ ID NO: 1 and an arbitrary continuous at least 16 base oligonucleotide between base numbers 569 to 2259 of SEQ ID NO: 1
  • An antisense primer consisting of an oligonucleotide complementary to the nucleotide sequence, or a sense primer consisting of at least 16 consecutive oligonucleotides between base numbers 1 to 716 of SEQ ID NO: 12 and base number 717 of SEQ ID NO: 12
  • a primer set comprising an antisense primer consisting of an oligonucleotide that is complementary to any consecutive at least 16 base oligonucleotide between 2857 and 2857.
  • [70] (1) contacting the test substance with the polypeptide according to [44] 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 [70] wherein the substance that inhibits the activity and / or expression of the polypeptide is a therapeutic agent for ALK fusion-positive cancer.
  • the screening method according to [70] or [71] wherein the cancer is digestive organ cancer.
  • the screening method according to [70] or [71] wherein the cancer is gastrointestinal cancer.
  • a pharmaceutical composition for treating ALK fusion-positive cancer comprising a substance that inhibits the activity and / or expression of an ALK fusion protein.
  • the pharmaceutical composition according to [76] wherein the substance that inhibits the activity and / or expression of the ALK fusion protein is a kinase inhibitor.
  • the pharmaceutical composition according to [76] or [77], wherein the ALK fusion protein is the polypeptide according to [44].
  • the detection method of the present invention can be used as a method for detecting ALK fusion positive cancer (particularly digestive organ cancer).
  • ALK fusion positive cancer in a subject can be diagnosed, and further, it can be determined whether or not the subject is an application subject of an ALK inhibitor.
  • the detection kit and primer set of the present invention can be used in the detection method of the present invention.
  • the inhibitor screening method of the present invention it is possible to screen a drug effective for treating the fusion-positive cancer patient.
  • the substance obtained by the screening can be used as an active ingredient of a pharmaceutical composition for treating ALK fusion-positive cancer, and can also be used for the treatment of ALK fusion-positive cancer.
  • the detection method of the present invention can be used as a method for detecting a ZSCAN9 fusion-positive cancer (particularly digestive organ cancer).
  • it is possible to diagnose a ZSCAN9 fusion-positive cancer in a subject and it is possible to further determine whether or not the subject is an application subject of a ZSCAN9 inhibitor.
  • the detection kit and primer set of the present invention can be used in the detection method of the present invention.
  • the inhibitor screening method of the present invention it is possible to screen a drug effective for treating the fusion-positive cancer patient.
  • the substance obtained by the screening can be used as an active ingredient of a pharmaceutical composition for the treatment of ZSCAN9 fusion-positive cancer, and can also be used for the treatment of ZSCAN9 fusion-positive cancer.
  • FIG. 3 is a photomicrograph in place of a drawing showing the state after the fusion gene ZSCAN9ex3-ALKex20 (SEQ ID NO: 1) has been introduced into 3T3 fibroblasts and cultured for 11 days. It is the microscope picture which replaces drawing which shows the state after processing only the gene introduction
  • FIG. 5 is a photograph, instead of a drawing, showing a state 10 days after inoculating 3T3 fibroblasts introduced with the fusion gene ZSCAN9ex3-ALKex20 (SEQ ID NO: 1) subcutaneously into nude mice.
  • FIG. 5 is a graph showing changes over time in tumor size 6 to 17 days after inoculation in the nude mice shown in FIGS. 3 and 4.
  • FIG. 5 is a graph which shows the sensitivity with respect to each ALK inhibitor (crizotinib, CH5424242, TAE684, ASP3026) in Ba / F3 cell expressing a ZSCAN9-ALK fusion polypeptide.
  • 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 ZSCAN9-ALK fusion polypeptide with each ALK inhibitor.
  • the “fusion point in the ALK fusion gene” means a position where a polynucleotide derived from the ALK gene in the ALK fusion gene and a polynucleotide derived from another gene that constructs the fusion gene together with the ALK gene are combined.
  • the ZSCAN9-ALK fusion gene represented by SEQ ID NO: 1 it is a position where the 568th base and the 569th base of the base sequence are combined.
  • the “fusion point in the ALK fusion protein” means a polypeptide encoded by a polynucleotide derived from the ALK gene in the ALK fusion protein and a polynucleotide derived from the other gene that constructs the fusion gene together with the ALK gene. Means a portion where the polypeptide encoded by is bound.
  • the “fusion point in the XSCAN9 fusion gene” refers to a portion where a polynucleotide derived from the ZSCAN9 gene in the ZSCAN9 fusion gene and a polynucleotide derived from another gene that constructs the fusion gene together with the ZSCAN9 gene are combined.
  • the ZSCAN9-ALK fusion gene represented by SEQ ID NO: 1 it is a position where the 568th base and the 569th base of the base sequence are combined.
  • the “fusion point in the XSCAN9 fusion protein” refers to a polypeptide encoded by a polynucleotide derived from the ZSCAN9 gene in the ZSCAN9 fusion protein, and a polynucleotide derived from the other gene that constructs the fusion gene together with the ZSCAN9 gene. Means a portion where the polypeptide encoded by is bound.
  • ALK gene cleavage or “ALK gene is cleaved” means that ALK gene continuity is lost due to translocation or inversion of the gene, that is, ALK.
  • the gene refers to a state where the gene is divided into at least two polynucleotides including a polynucleotide containing the ALK kinase region and other polynucleotides.
  • the break point of the ALK gene is not limited as long as the protein encoded by at least one of the polynucleotides formed by cleaving the ALK gene retains ALK kinase activity.
  • a gene other than the ALK gene is cleaved or “a gene other than the ALK gene is cleaved” means that the continuity of other genes is lost due to gene translocation or inversion. A state in which another gene is divided into at least two polynucleotides.
  • cleavage of ALK protein or “ALK protein is cleaved” means that the ALK gene is cleaved as described above based on the continuity of ALK protein. Is a state in which ALK protein is divided into at least two polypeptides, ie, a polypeptide containing the ALK kinase region and another polypeptide.
  • the cleavage point of ALK protein is not limited as long as at least one of the polypeptides formed by cleaving ALK protein retains ALK kinase activity.
  • cleavage of a protein other than ALK protein or “a protein other than ALK protein is cleaved” is based on the fact that other genes are 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.
  • ZSCAN9 gene cleavage or “ZSCAN9 gene cleavage” means that the continuity of the ZSCAN9 gene is lost due to translocation or inversion of the gene, that is, ZSCAN9 It refers to the state where the gene is divided into at least two polynucleotides. It should be noted that the break point of the ZSCAN9 gene is not limited as long as the ZSCAN9 fusion protein encoded by other genes that construct the ZSCAN9 fusion gene together with the ZSCAN9 gene retains the tumorigenicity.
  • cleaved by a gene other than the XSCAN9 gene or “cleaved by a gene other than the XSCAN9 gene” means that the continuity of other genes is lost due to gene translocation or inversion. A state in which another gene is divided into at least two polynucleotides.
  • cleavage of ZSCAN9 protein or “cleavage of ZSCAN9 protein” means that the continuity of ZSCAN9 protein is based on the fact that the ZSCAN9 gene is cleaved as described above. Refers to the state in which the ZSCAN9 protein is divided into at least two polypeptides. It should be noted that the breakpoint of the ZSCAN9 protein is the function of other proteins that construct the ZSCAN9 fusion protein together with the ZSCAN9 protein in order to have the tumor-forming ability of the fusion protein (for example, when the other protein has a kinase domain, It is not limited as long as it retains (kinase activity).
  • cleaving of a protein other than the XSCAN9 protein” or “a protein other than the XSCAN9 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.
  • 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), the cleavage when the wild type protein constructs a fusion gene
  • the polynucleotide on the C-terminal side from the point is shown.
  • the 5 ′ end region comprises the 1st to 568th regions
  • the 3 ′ end region comprises the 569th to 2259th base sequences.
  • the N-terminal region is a polypeptide encoded by the 5′-terminal region
  • the C-terminal region is a polypeptide encoded by the 3′-terminal region.
  • 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 the method of Example 8 described later that a certain polypeptide “has tumorigenicity”. 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 polypeptide of the present invention is a fusion polypeptide (also referred to as “ALK fusion protein”) constructed from a polypeptide derived from ALK protein and a polypeptide derived from another protein other than ALK protein, and the ALK
  • the protein-derived polypeptide includes at least the polypeptide of the ALK kinase region in the ALK protein, and the polypeptide derived from other proteins other than the ALK protein is not particularly limited as long as it includes at least a part of the polypeptide in the other protein. .
  • the other protein as long as the ALK kinase activation is constantly maintained by fusing with a part of the ALK protein containing the ALK kinase domain, and the constructed ALK fusion protein has tumorigenic ability, There is no particular limitation.
  • the ALK fusion protein a fusion protein in which the other protein is a ZSCAN9 protein is particularly preferable.
  • a ZSCAN9 protein and an ALK constructed from an ALK protein-derived polypeptide containing at least an ALK kinase domain polypeptide and a ZSCAN9 gene-derived polypeptide containing at least a partial polypeptide of the ZSCAN9 protein It is preferably a fusion protein with a protein (hereinafter also referred to as a ZSCAN9-ALK fusion protein).
  • the polypeptide of the present invention is a fusion polypeptide (also referred to as a “ZSCAN9 fusion protein”) constructed from a polypeptide derived from a ZSCAN9 protein and a polypeptide derived from a protein other than the ZSCAN9 protein.
  • the protein-derived polypeptide includes at least a part of the polypeptide in the ZSCAN9 protein, and the polypeptide derived from other proteins other than the ZSCAN9 protein is not particularly limited as long as it includes at least a part of the polypeptide in the other protein. .
  • the constructed ZSCAN9 fusion protein has tumorigenicity.
  • the ZSCAN9 fusion protein a fusion protein in which the other protein is a protein having a kinase domain is preferable, and a fusion protein that is an ALK protein is particularly preferable.
  • a polypeptide derived from the ZSCAN9 gene comprising a polypeptide derived from the ALK protein containing at least a polypeptide of the ALK kinase region and at least a part of the polypeptide of the ZSCAN9 protein And a fusion protein of a ZSCAN9 protein and an ALK protein (hereinafter also referred to as a ZSCAN9-ALK 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, (B) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 and having tumorigenicity, (C) a polypeptide (hereinafter referred to as a homologous polypeptide) comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having tumorigenicity, and (d) a sequence In the amino acid sequence represented by No. 2, a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, and / or inserted, and having tumorigenicity (hereinafter referred to as a functional equivalent variant) Called).
  • A a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2
  • B a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 and having tumorigenicity
  • C a
  • the amino acid sequence represented by SEQ ID NO: 2 is a sequence encoded by the base sequence represented by SEQ ID NO: 1.
  • the base sequence represented by SEQ ID NO: 1 consists of the base sequence from the start codon ATG to exon 3 of the ZSCAN9 gene and from the exon 20 to the stop codon of exon 29 of the ALK gene.
  • the sequences of nucleotide numbers 1 to 568 are derived from the ZSCAN9 gene, and the sequences of nucleotide numbers 569 to 2259 are derived from the ALK gene.
  • the polynucleotide having the base sequence represented by SEQ ID NO: 1 is referred to as ZSCAN9ex3-ALKex20.
  • 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 containing an amino acid sequence having an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 2 and having tumorigenicity”.
  • a polypeptide comprising an amino acid sequence that is preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more is preferable.
  • the “identity” means a value Identity obtained by using a parameter prepared by default by a NEEDLE program (J Mol Biol 1970; 48: 443-453) search.
  • the polynucleotide of the present invention is not particularly limited as long as it is a polynucleotide (also referred to as “ALK fusion gene”) encoding the polypeptide of the present invention (ie, ALK fusion protein).
  • ALK fusion gene also referred to as “ALK fusion gene”
  • the “polynucleotide encoding the ALK fusion protein” refers to a polynucleotide consisting only of the translation region in the ALK fusion gene, the full-length genomic DNA of the ALK fusion gene, mRNA or cDNA of the ALK fusion gene. There may be.
  • the polynucleotide of the present invention is not particularly limited as long as it is a polynucleotide encoding the polypeptide of the present invention (that is, a ZSCAN9 fusion protein) (also referred to as “ZSCAN9 fusion gene”).
  • the “polynucleotide encoding the XSCAN9 fusion protein” is a polynucleotide consisting of only the translation region in the ZSCAN9 fusion gene, the full length genomic DNA of the ZSCAN9 fusion gene, or the mRNA or cDNA of the ZSCAN9 fusion gene. There may be.
  • the vector of the present invention is not particularly limited as long as it contains the polynucleotide of the present invention.
  • an appropriate vector capable of transforming a eukaryotic or prokaryotic host cell. can be prepared.
  • 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.
  • 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 is present, but the digestive organ is preferable, the digestive tract is more preferable, the gastrointestinal tract is further preferable, and the lower digestive tract is more preferable. More preferred is the large intestine. More specifically, the detection method according to the present invention is suitable for detection of gastrointestinal cancer, more preferably for detection of gastrointestinal cancer, and more preferably for detection of gastrointestinal cancer. More preferably, it can be used more suitably for detection of colorectal cancer.
  • 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 an ALK fusion gene or an ALK fusion protein comprises preparing a tissue section or a cell suspension of a sample obtained from a subject, and applying it to the cells contained in the tissue section or cell suspension. On the other hand, it can be carried out by detecting the ALK fusion gene or ALK fusion protein 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 ALK fusion gene or ALK fusion proteins may be detected.
  • the detection of the ALK fusion gene may be any of detection of genomic DNA of the ALK fusion gene, detection of mRNA that is a transcription product of the genomic DNA, or cDNA obtained using mRNA as a template.
  • a tissue section or a cell suspension of a sample obtained from a subject is prepared, and a cell contained in the tissue section or cell suspension is prepared.
  • it can be carried out by detecting a ZSCAN9 fusion gene or a ZSCAN9 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 ZSCAN9 fusion gene or A ZSCAN9 fusion protein may be detected.
  • the detection of the ZSCAN9 fusion gene may be any of detection of genomic DNA of the ZSCAN9 fusion gene, detection of mRNA that is a transcription product of the genomic DNA, or cDNA obtained using the mRNA as a template.
  • the detection method of the present invention includes a method for detecting an ALK fusion in a sample obtained from a subject, that is, a method for detecting a fusion protein (ALK fusion protein) containing an ALK kinase region, or a fusion encoding the fusion protein.
  • a method for detecting a gene is included.
  • the detection method of the present invention includes a method for detecting a ZSCAN9 fusion in a sample obtained from a subject, that is, a method for detecting a ZSCAN9 fusion protein, or a method for detecting a fusion gene encoding the fusion protein (ZSCAN9 fusion gene). Is included.
  • the detection method of the present invention includes a detection method including a step of detecting cleavage of the ALK gene or cleavage of the polypeptide encoded by the ALK gene in a digestive organ-derived sample obtained from the subject; A detection method comprising a step of detecting the presence of a fusion gene constructed from an ALK gene and another gene in a digestive organ-derived sample, or the presence of a fusion protein encoded by the fusion gene. included.
  • the detection method of the present invention includes a detection method comprising a step of detecting cleavage of the ZSCAN9 gene or cleavage of the polypeptide encoded by the ZSCAN9 gene in a sample obtained from the subject, and in the sample obtained from the subject. And a detection method comprising a step of detecting the presence of a fusion gene constructed from the ZSCAN9 gene and another gene other than that, or the presence of a fusion protein encoded by the fusion gene.
  • ALK fusion gene ⁇ Aspect for detecting ALK fusion gene>
  • 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.
  • a normal sample a sample derived from a tissue not affected by cancer
  • ALK can be detected by detecting the presence of the ALK gene or ALK protein itself. Fusions can also be detected.
  • Aspect for detecting ALK fusion gene (1) As one mode of detecting the ALK fusion gene, when the ALK fusion gene is constructed, the ALK gene is cleaved into two or more polynucleotides, and the ALK gene is cleaved, that is, ALK. By detecting the loss of continuity between the gene 5 ′ terminal region and the ALK gene 3 ′ terminal region, the ALK fusion gene can be detected.
  • an ALK fusion gene can be detected.
  • the ALK fusion gene can be detected by detecting the presence of only the ALK gene 3 ′ terminal region.
  • the ALK fusion gene is detected by specifically detecting the expression levels of the 5 ′ terminal region and the 3 ′ terminal region of the ALK 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 ALK gene is different from the expression level of the ALK gene 3 ′ terminal region, the ALK fusion gene can be detected. Alternatively, the ALK fusion gene may be detected by confirming other genes other than the ALK gene constructing the ALK fusion gene together with the ALK gene by the above method.
  • the ALK fusion gene is constructed by fusing a polynucleotide derived from an ALK gene and a polynucleotide derived from another gene other than the ALK gene. Detecting an ALK fusion gene by detecting a fusion polynucleotide comprising at least a part of a polynucleotide derived from the ALK gene and at least a part of a polynucleotide derived from another gene other than the ALK gene in succession be able to.
  • a first probe that specifically hybridizes to the 5′-terminal region of a polynucleotide derived from a gene other than the ALK gene and a hybrid that specifically hybridizes to the 3′-terminal region of the ALK gene
  • the ALK fusion gene can be detected by detecting the proximity of the two gene regions on the chromosome using the second probe.
  • the gene other than the ALK gene is ZSCAN9, that is, when the ALK fusion gene is a ZSCAN9-ALK fusion gene
  • the first probe specifically hybridizes to the 5 ′ end region of the polynucleotide derived from the ZSCAN9 gene.
  • a probe to be used may be used.
  • the ALK fusion gene is constructed based on the fact that the ALK gene-derived polynucleotide is constructed by fusing a polynucleotide derived from another gene other than the ALK gene at the fusion point.
  • a fusion polynucleotide in which at least a part of a polynucleotide derived from an ALK gene and at least a part of a polynucleotide derived from another gene other than the ALK gene in the fusion gene are continuously contained including the fusion point is detected.
  • the ALK fusion gene can be detected.
  • the ALK fusion gene can be detected by conducting a PCR reaction using the second primer that anneals automatically and confirming that a predetermined PCR product indicating the presence of the fusion point is obtained.
  • Aspect for detecting ALK fusion protein (1) when the ALK fusion gene is constructed, the ALK protein encoded by the ALK gene is also cleaved, that is, the state in which the ALK protein is cleaved, ie, the N of the ALK protein.
  • the ALK fusion protein can be detected by detecting that the terminal region and the C-terminal region are cleaved without being continuous.
  • the two regions can be detected by confirming that they are present in different proteins.
  • the ALK fusion protein may be detected by confirming, by the above method, that other proteins other than the ALK protein that constitutes the fusion protein together with the ALK protein are cleaved.
  • the ALK fusion protein can be detected by specifically detecting the expression levels of the N-terminal region and the C-terminal region of the ALK protein, respectively, and determining the ratio of the expression levels. .
  • an ALK fusion protein can be detected using as an index the difference between the expression level of the N-terminal region of the ALK protein and the expression level of the ALK protein C-terminal region.
  • the ALK fusion protein may be detected by confirming the protein other than the ALK protein constructing the ALK fusion protein together with the ALK protein by the above method.
  • the ALK fusion protein is constructed by fusing an ALK protein-derived polypeptide and a polypeptide derived from another protein other than the ALK protein.
  • the ALK fusion protein can be detected by detecting a fusion polypeptide in which at least a part of the polypeptide derived from the ALK 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 ALK protein and a second antibody that specifically binds to the C-terminal region of ALK protein are used.
  • the ALK fusion protein can be detected by confirming that the two regions are present in the same protein.
  • the ALK fusion protein is constructed by fusing an ALK protein-derived polypeptide and a polypeptide derived from another protein other than the ALK protein at the fusion point.
  • ALK fusion is detected by detecting a fusion polypeptide in which at least a part of the polypeptide derived from the ALK 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 ALK fusion protein can be detected by an immunoassay using an antibody that specifically recognizes a polypeptide containing the fusion point of the ALK fusion protein.
  • the ALK fusion protein can be detected using the activity of the ALK fusion protein as an index. Specifically, for example, after inhibiting the activity of the wild type ALK protein using a substance having an inhibitory activity against the wild type ALK protein, the kinase activity of the ALK protein is measured, and the ALK fusion protein is not included ( The ALK fusion protein can be detected using an index of high activity as compared to the case of including only wild-type ALK protein. For the measurement of the kinase activity of ALK 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 ALK may be detected.
  • the detection of the ALK fusion protein may be performed using the presence of the full-length polypeptide constituting the ALK fusion protein or the presence of the polypeptide constituting a part of the ALK fusion protein as an index. It is not limited as far as it can be confirmed.
  • the ZSCAN9 fusion gene can be detected.
  • the ZSCAN9 fusion gene can be detected.
  • the ZSCAN9 fusion gene is detected by specifically detecting the expression levels of the 5 ′ terminal region and the 3 ′ terminal region of the ZSCAN9 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 ZSCAN9 gene is different from the expression level of the ZSCAN9 gene 3′-terminal region, the ZSCAN9 fusion gene can be detected. Alternatively, the ZSCAN9 fusion gene may be detected by confirming the gene other than the ZSCAN9 gene constructing the ZSCAN9 fusion gene together with the ZSCAN9 gene by the above method.
  • ⁇ Aspect for detecting a ZSCAN9 fusion gene (1-c)> in the process of forming a ZACAN9 fusion, when duplication of at least a part of the ZSCAN9 gene or other genes other than the ZSCAN9 gene is involved, that is, a duplicated polynucleotide derived from the ZSCAN9 gene, and When a ZSCAN9 fusion gene is constructed from a duplicated polynucleotide derived from another gene other than the ZSCAN9 gene constructing the ZSCAN9 fusion gene together with the ZSCAN9 gene, the polynucleotide derived from the ZSCAN9 gene or the other gene By detecting polynucleotide duplication, the ZSCAN9 fusion gene can be detected.
  • the XSCAN9 fusion gene is constructed by fusing a polynucleotide derived from a ZSCAN9 gene and a polynucleotide derived from a gene other than the ZSCAN9 gene. Detecting a ZSCAN9 fusion gene by detecting a fusion polynucleotide comprising at least a part of a polynucleotide derived from the ZSCAN9 gene and at least a part of a polynucleotide derived from another gene other than the ZSCAN9 gene be able to.
  • a first probe that specifically hybridizes to the 3′-terminal region of a polynucleotide derived from a gene other than the ZSCAN9 gene, and a hybrid that specifically hybridizes to the 5′-terminal region of the ZSCAN9 gene The ZSCAN9 fusion gene can be detected by detecting that the two gene regions are close to each other on the chromosome using the second probe.
  • the gene other than the ZSCAN9 gene is ALK
  • the ZSCAN9 fusion gene is a ZSCAN9-ALK fusion gene
  • the first probe specifically hybridizes to the 3 ′ terminal region of the ALK gene-derived polynucleotide.
  • a probe to be used may be used.
  • the XSCAN9 fusion gene is constructed by fusing a ZSCAN9 gene-derived polynucleotide and a polynucleotide derived from another gene other than the ZSCAN9 gene at the fusion point.
  • a fusion polynucleotide in which at least a part of a polynucleotide derived from the ZSCAN9 gene and at least a part of a polynucleotide derived from a gene other than the ZSCAN9 gene in the fusion gene are continuously contained including the fusion point is detected.
  • the ZSCAN9 fusion gene can be detected.
  • a first primer that specifically anneals to a 3′-terminal region of a polynucleotide derived from a gene other than the ZSCAN9 gene, and a 5′-terminal region specific to a polynucleotide derived from the ZSCAN9 gene The ZSCAN9 fusion gene can be detected by conducting a PCR reaction using a second primer that anneals and confirming that a predetermined PCR product indicating the presence of a fusion point is obtained.
  • a ZSCAN9 fusion protein when the ZSCAN9 fusion gene is constructed, the ZSCAN9 protein encoded by the ZSCAN9 gene is also cleaved.
  • a ZSCAN9 fusion protein can be detected by detecting that the terminal region and the C-terminal region are cleaved without being continuous. Specifically, for example, using the first antibody that specifically binds to the N-terminal region of the ZSCAN9 protein and the second antibody that specifically binds to the C-terminal region of the ZSCAN9 protein, the two regions Can be detected in different proteins by detecting ZSCAN9 fusion protein.
  • the ZSCAN9 fusion protein may be detected by confirming, by the above-described method, a state in which a protein other than the ZSCAN9 protein constructing the fusion protein together with the ZSCAN9 protein is cleaved.
  • the ZSCAN9 fusion protein can be detected by specifically detecting the expression levels of the N-terminal region and the C-terminal region of the ZSCAN9 protein and determining the ratio of the expression levels. .
  • the ZSCAN9 fusion protein can be detected by using as an index the difference between the expression level of the N-terminal region of the ZSCAN9 protein and the expression level of the ZSCAN9 protein C-terminal region.
  • the ZSCAN9 fusion protein may be detected by confirming the protein other than the ZSCAN9 protein constructing the ZSCAN9 fusion protein together with the ZSCAN9 protein by the aforementioned method.
  • the ZSCAN9 fusion protein is constructed by fusing a ZSCAN9 protein-derived polypeptide and a polypeptide derived from another protein other than the ZSCAN9 protein.
  • the ZSCAN9 fusion protein can be detected by detecting a fusion polypeptide in which at least a part of the polypeptide derived from the ZSCAN9 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 C-terminal region of a protein other than the ZSCAN9 protein and a second antibody that specifically binds to the N-terminal region of the ZSCAN9 protein are used.
  • the ZSCAN9 fusion protein can be detected.
  • the ZSCAN9 fusion protein is constructed by fusing a ZSCAN9 protein-derived polypeptide and a polypeptide derived from another protein other than the ZSCAN9 protein at the fusion point. Detecting a fusion polypeptide in which at least a part of a polypeptide derived from the ZSCAN9 protein containing the fusion point and at least a part of a polypeptide derived from a protein other than the ZSCAN9 protein are continuously contained in the fusion protein.
  • the ZSCAN9 fusion protein can be detected.
  • the ZSCAN9 fusion protein can be detected by an immunoassay using an antibody that specifically recognizes a polypeptide containing the fusion point of the ZSCAN9 fusion protein.
  • the ZSCAN9 fusion protein can be detected using the activity of the ZSCAN9 fusion protein as an index.
  • a protein other than ZSCAN9 that constructs a fusion protein together with ZSCAN9 protein is a protein having an enzyme activity, it does not contain a ZSCAN9 fusion protein (including only a wild-type ZSCAN9 protein).
  • the ZSCAN9 fusion protein can be detected using the high enzyme activity as an index.
  • a method well known to those skilled in the art can be appropriately selected for measuring enzyme activity.
  • the other protein is a protein having a kinase activity (preferably an ALK protein)
  • phosphorylation is performed by a ZSCAN9 fusion protein. The phosphorylation state of the molecule undergoing the reaction may be detected.
  • the detection of the ZSCAN9 fusion protein may be performed by using the presence of the full-length polypeptide constituting the ZSCAN9 fusion protein or the presence of the polypeptide constituting a part of the ZSCAN9 fusion protein as an indicator. It is not limited as far as it can be confirmed.
  • ALK fusion gene genomic DNA, mRNA, or cDNA
  • ZSCAN9 fusion gene genomic DNA, mRNA, or cDNA
  • detection of ALK fusion protein detection of ZSCAN9 fusion protein
  • detection of ZSCAN9 fusion protein detection of ZSCAN9 fusion protein
  • ALK is used in the prepared sample.
  • a person skilled in the art can appropriately select a suitable technique for detecting a fusion gene or a ZSCAN9 fusion gene, or an ALK fusion protein or a ZSCAN9 fusion protein.
  • the detection of the ALK fusion gene or the ZSCAN9 fusion gene can be performed by either detecting the genomic DNA of the ALK fusion gene or the ZSCAN9 fusion gene, detecting mRNA that is a transcription product of the genomic DNA, or detecting cDNA obtained using the mRNA as a template. There may be.
  • hybridization technology using a hybridizing probe such as a nucleic acid probe
  • gene amplification technology using a primer that anneals to at least a part of an ALK fusion gene or ZSCAN9 fusion gene Any known technique and techniques applying 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 Example 4 or 5.
  • 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 the ALK fusion gene, as a detection probe, as will be described in detail in Example 6 below, a polynucleotide covering the 5 'terminal genomic region of the ALK gene and fluorescently labeled A combination with a polynucleotide covering the 3′-terminal genomic region of the gene and labeled with another fluorescent dye is used.
  • the two gene regions are close to each other, so that 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 fluorescence are detected. Signals derived from the dye (eg, red and green) are detected lonely. Therefore, in the FISH split assay, the presence of the ALK fusion gene is detected by detecting that the 5 ′ end genomic region of the ALK gene and the 3 ′ end genomic region of the ALK gene are not close together on the chromosome. ing.
  • the FISH split assay of the ZSCAN fusion gene as a probe for detection, as will be described in detail in Example 6 below, it is a polynucleotide that covers the 5 'terminal genomic region of the ZSCAN9 gene and is fluorescently labeled. And a combination of a polynucleotide covering the 3′-terminal genomic region of the same gene and labeled with another fluorescent dye.
  • 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 fluorescence are detected. Signals derived from the dye (eg, red and green) are detected lonely. Therefore, in the FISH split assay, the presence of the ZSCAN9 fusion gene is detected by detecting that the 5 'terminal genomic region of the ZSCAN9 gene is not close to the 3' terminal genomic region of the ZSCAN9 gene on the chromosome. ing.
  • the ALK fusion gene or the ZSCAN9 fusion gene is a ZSCAN9-ALK fusion gene, it is a polynucleotide that covers the 5 'terminal genomic region of the ZSCAN9 gene as a detection probe, as shown in Example 6 described later, and is fluorescent.
  • a ZSCAN9-ALK fusion gene can be detected by using a combination of a fluorescently labeled one and a polynucleotide covering the 3′-terminal genomic region of the same gene and labeled with another fluorescent dye can do.
  • FISH method fusion assay In the FISH fusion assay of an ALK fusion gene, for example, when the ALK fusion gene is a ZSCAN9-ALK fusion gene, the detection probe is used as a probe for detection, as described in detail in Example 5 below, and the 5 ′ terminal genome of the ZSCAN9 gene A combination of a polynucleotide that covers a region and is fluorescently labeled, and a polynucleotide that covers a 3 ′ terminal genomic region of the ALK gene and is labeled with another fluorescent dye can be used.
  • the 5 ′ end of the ZSCAN9 gene A combination of a polynucleotide covering the side genomic region and fluorescently labeled with a polynucleotide covering the 3 ′ end side genomic region of the ALK gene and labeled with another fluorescent dye it can.
  • the above FISH fusion assay and split assay can be simultaneously performed on the same pathological section.
  • another fluorescent dye for example, red
  • a 3 ′ of the ALK gene A combination of a polynucleotide covering the terminal genomic region and labeled with another fluorescent dye (for example, green) can be used. That is, each probe is labeled and used with three different color fluorescent dyes (for example, blue, red, and green).
  • the 5 'terminal genomic region of the ALK gene and the 3' terminal genomic region of the ALK gene are continuous (close), so the 5 'terminal side of the ALK gene A signal in which the fluorescence of different fluorescent labels that label the genomic region and the 3 ′ end side genomic region overlap (for example, yellow that overlaps red and green) is detected, and the 5 ′ end side genomic region of the ZSCAN9 gene is labeled
  • the signal of the fluorescent dye for example, blue
  • the ZSCAN9-ALK fusion gene when the ZSCAN9-ALK fusion gene is constructed, the 5 'terminal genomic region of the ALK gene is lost from its original position due to gene translocation, deletion or inversion, and the 5' end of the ZSCAN9 gene.
  • the side genomic region and the 3 ′ end side genomic region of the ALK gene are continuous (in some cases, including the inserted base in the middle) to form a fusion point.
  • This FISH assay in which a fusion assay and a split assay are simultaneously performed on the same pathological section, is suitable from the viewpoint of efficiency and improved determination reliability because the results of both methods can be obtained on one pathological section. is there.
  • a probe having a signal (for example, red) that shows loneliness when the fusion gene is constructed is not limited to the 5 ′ end region of the ALK gene, for example, when the fusion gene is the ZSCAN9-ALK gene, A similar effect can be obtained by designing to hybridize within the genomic region between the 'terminal genomic region and the 3' terminal genomic region of the ALK gene.
  • Probe used for detection for genome
  • it hybridizes under stringent conditions (preferably under more stringent conditions) to at least some nucleotides of the ALK fusion gene or their complementary strands. Probes that soy are preferred.
  • a nucleic acid molecule consisting of at least 32 bases consisting of 16 bases upstream and downstream of the fusion point of the ALK fusion gene (specifically, May be a probe comprising the base sequence represented by SEQ ID NO: 1 (No. 994 to No. 1025), or a complementary strand thereof.
  • a probe used for hybridization for detecting the ZSCAN9 fusion gene it hybridizes under stringent conditions (preferably under more stringent conditions) to at least some nucleotides of the ZSCAN9 fusion gene or their complementary strands. Probes that soy are preferred.
  • a nucleic acid molecule consisting of at least 32 bases consisting of 16 bases upstream and downstream of the fusion point of the ZSCAN9 fusion gene (specifically, May be a probe comprising the base sequence represented by SEQ ID NO: 1 (No. 994 to No. 1025), or a complementary strand thereof.
  • the probe that can be used in the FISH fusion assay includes a 5 'terminal genomic region of either the ZSCAN9 gene or the ALK gene.
  • a second probe capable of specifically recognizing the 3 ′ terminal genomic region of the remaining one gene preferably, the 5 ′ terminal genomic region of the ZSCAN9 gene
  • a combination of a first probe that can be specifically recognized and a second probe that can specifically recognize the 3′-terminal genomic region of the ALK gene can be used.
  • Each combination of BAC clones used in 5 can be mentioned.
  • the ALK fusion gene or the ZSCAN9 fusion gene is a ZSCAN9-ALK fusion gene
  • the first ALK gene 5′-terminal genomic region can be specifically recognized.
  • a second probe capable of specifically recognizing the 3 ′ end genomic region of the ALK gene, or a first probe capable of specifically recognizing the ZSCAN9 gene 5 ′ end genomic region, and ZSCAN9 A combination with a second probe capable of specifically recognizing the 3 ′ terminal genomic region of the gene preferably, a first probe capable of specifically recognizing the 5 ′ terminal genomic region of the ALK gene and the 3 ′ of the ALK gene Use in combination with a second probe that can specifically recognize the terminal genomic region
  • Rukoto can, more specifically, may be mentioned the combination of BAC clones used in Example 6 below.
  • 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 ALK fusion gene or ASCAN9 fusion gene to be detected.
  • a primer designed to specifically amplify at least a part of the polynucleotide of the ALK fusion gene or ASCAN9 fusion gene to be detected.
  • an ALK 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, whether or not an amplified fragment of a desired size has been obtained is determined.
  • a detecting step is included.
  • the PCR method is suitable for quantitatively detecting the ALK fusion gene or the ZSCAN9 fusion gene. Therefore, as described in ⁇ Aspect of detecting ALK fusion gene (1-b)>, the expression levels of the 5 ′ terminal region and 3 ′ terminal region of the ALK gene are specifically detected, It can use suitably for the method of detecting an ALK fusion gene by calculating
  • the expression levels of the 5 ′ terminal region and the 3 ′ terminal region of the ZSCAN9 gene are specifically detected. It can be suitably used in a method for detecting a ZSCAN9 fusion gene by determining the ratio of expression levels. Alternatively, the expression levels of the 5′-terminal region and the 3′-terminal region of other genes other than the ZSCAN9 gene constructing the ZSCAN9 fusion gene together with the ZSCAN9 gene are specifically detected, and the ratio of the expression levels is determined. By doing so, the ZSCAN9 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 ALK gene, and a sense primer designed to specifically amplify the 3 ′ terminal region of the ALK gene and Antisense primers can be used.
  • a sense primer and an antisense primer designed so as to specifically amplify the 5 ′ terminal region of the ZSCAN9 gene, and a sense primer designed so as to specifically amplify the 3 ′ terminal region of the ZSCAN9 gene and Antisense primers can be used.
  • the PCR amplification monitor (real-time PCR) method (Genome Res., 6 (10), 986, 1996) is used in the gene amplification process to detect the ALK fusion gene or the ZSCAN9 fusion gene. It is possible to perform more quantitative analysis.
  • ABI PRISM 7900 PE Biosystems
  • 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.
  • a sense primer (5′-primer) is used as an arbitrary part derived from the ZSCAN9 gene.
  • an antisense primer (3′-primer) is designed from any part derived from the ALK gene.
  • the sense primer (5′-primer) is antisenseed from any part derived from the ZSCAN9 gene.
  • Primers (3′-primers) are designed from any part derived from the ALK gene.
  • the primer set used in the detection method for detecting the ALK fusion gene of the present invention is not particularly limited as long as it can specifically amplify at least a part of the ALK fusion gene to be detected and can detect the ALK 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 ZSCAN9 fusion gene of the present invention is not particularly limited as long as it can specifically amplify at least a part of the ZSCAN9 fusion gene to be detected and can detect the ZSCAN9 fusion gene.
  • Primer design in the PCR amplification monitoring method can be performed using primer design software (eg, Primer Express; PE Biosystems).
  • 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.
  • the mRNA can be detected by a hybridization method using a probe that hybridizes to at least a part of the polynucleotide of the ALK fusion gene or the ZSCAN9 fusion gene 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.
  • ⁇ Detection of fusion protein> As a technique for detecting an ALK fusion protein or a ZSCAN9 fusion protein in a sample obtained from a subject, any technique known to those skilled in the art used for analyzing proteins, or any technique applying these techniques can be used. Also good.
  • an ALK protein or an antibody that specifically recognizes another protein other than the ALK protein that constructs the ALK fusion protein together with the ALK protein, or an ALK 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.
  • an antibody that specifically recognizes a ZSCAN9 protein, or another protein other than the ZSCAN9 protein that constructs a ZSCAN9 fusion protein together with the ZSCAN9 protein, or a ZSCAN9 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.
  • 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.
  • the polymorphism of the C-terminal region of the ALK protein is compared with the tissue section in which the fusion protein to be detected may be present.
  • immunostaining was performed using an antibody that specifically binds to the polypeptide in the N-terminal region of the ALK protein and an antibody that specifically binds to the polypeptide in the C-terminal region of the ALK protein. It is also possible to detect the presence of the fusion protein to be detected using the localization without proximity as an index. In addition, immunostaining was performed using an antibody that specifically binds to a polypeptide in the N-terminal region of the ZSCAN9 protein and an antibody that specifically binds to a polypeptide in the C-terminal region of the ZSCAN9 protein. It is also possible to detect the presence of the fusion protein to be detected using the localization without proximity 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 detection target ALK fusion protein or ZSCAN9 fusion protein is a ZSCAN9-ALK fusion protein, a cell extract in which the detection target fusion protein may be present is electrophoresed by a method well known to those skilled in the art. The protein in the cell extract is separated and blotted on the membrane. Then, the membrane blotted with the protein is immunostained with an anti-ALK antibody that binds to a polypeptide in the C-terminal region of the ALK protein and an anti-ZSCAN9 antibody that binds to the N-terminal region of the ZSCAN9 protein.
  • the presence of the fusion protein to be detected can also be detected by using as an index the binding of the anti-ALK antibody and the anti-ZSCAN9 antibody to the desired position above. 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, the presence of the fusion protein to be detected can be detected by using an anti-ALK antibody and using the antibody as an indicator that the antibody is bound to the ZSCAN9-ALK fusion protein on the membrane.
  • the presence of the fusion protein to be detected may be detected using the anti-ALK antibody binding to a position different from the position where the wild-type ALK protein is predicted on the membrane.
  • a ZSCAN9-ALK fusion protein may be detected using an anti-ZSCAN9 antibody and the same principle as when an anti-ALK antibody is used.
  • the ALK fusion protein or ZSCAN9 fusion protein to be detected is a ZSCAN9-ALK fusion protein
  • the CLK-terminal region of the ALK protein is compared with the cell extract in which the fusion protein to be detected may be present.
  • Immunoprecipitation is performed with either the anti-ALK antibody that binds to the polypeptide or the anti-ZSCAN9 antibody that binds to the polypeptide in the N-terminal region of the ZSCAN9 protein, and the remaining antibody against the precipitate is detected.
  • the presence of the fusion protein to be detected can also be detected.
  • the detection antibody 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.
  • immunoprecipitation is performed on the cell extract in which the ALK fusion protein to be detected may be present with an anti-ALK antibody that binds to a polypeptide in the C-terminal region of the ALK protein.
  • mass spectrometry the presence of a fusion protein to be detected can be detected by confirming the presence of a protein that binds to an anti-ALK antibody having a mass different from that of wild-type ALK.
  • Immunoprecipitation is performed with an anti-ZSCAN9 antibody that binds to a polypeptide in the N-terminal region of the ZSCAN9 protein on a cell extract that may contain the ZSCAN9 fusion protein to be detected, and mass analysis of the precipitate is performed.
  • mass analysis of the precipitate is performed.
  • 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 ALK fusion protein or ZSCAN9 fusion protein, and may be a monoclonal antibody or a polyclonal antibody. A combination of a monoclonal antibody and a polyclonal antibody can also be used.
  • 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, amplification products, antibodies, and the like. For example, fluorescent labels, radioactive labels, enzyme 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
  • the subject is a subject (patient) having an ALK fusion-positive cancer. And is a target for treatment with an ALK inhibitor.
  • the detection kit of the present invention includes a detection kit for detecting an ALK fusion gene or a detection kit for detecting an ALK fusion protein.
  • the detection kit of the present invention includes a detection kit of a detection target ZSCAN9 fusion gene, or a detection kit of a detection target ZSCAN9 fusion protein, or a detection target ALK fusion gene detection kit of the present invention, or
  • the ZSCAN9 fusion gene detection kit 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, or the ALK fusion gene or ZSCAN9 fusion to be detected in the detection method of the present invention.
  • Sense and antisense primers designed to specifically amplify at least a portion of the gene are included.
  • the sense and antisense primer set is a set of polynucleotides that are at least a part of the polynucleotides of the ALK fusion gene or the ZSCAN9 fusion gene and function as primers for amplification of the polynucleotide to be amplified.
  • the detection kit of the detection target ALK fusion protein or ZSCAN9 fusion protein of the present invention includes an antibody that can be used in the detection method of the present invention.
  • the kit for detecting an ALK fusion gene of the present invention has one kind of probe that can hybridize to at least a part of the polynucleotide of the ALK fusion gene or a complementary strand thereof under stringent conditions to detect the ALK fusion gene. Or it can contain in the combination of 2 or more types.
  • the kit for detecting a ZSCAN9 fusion gene of the present invention is one kind of probe capable of detecting the ZSCAN9 fusion gene by hybridizing under stringent conditions to at least a part of the polynucleotide of the ZSCAN9 fusion gene or its complementary strand. Or it can contain in the combination of 2 or more types.
  • the probe examples include any one or more of the probes described in ⁇ Technology used in detection method >>.
  • the ALK fusion gene or the ZSCAN9 fusion gene is a ZSCAN9-ALK fusion gene
  • one or more (preferably two or more) probes hybridizing to the ALK gene-derived polynucleotide, or the ZSCAN9 gene-derived polynucleotide Even if only one of one or more (preferably two or more) probes that hybridize is included, it hybridizes to one or more probes that hybridize to the ALK gene-derived polynucleotide and to the ZSCAN9 gene-derived polynucleotide.
  • the kit for detecting an ALK fusion gene of the present invention can specifically amplify at least a part of the ALK fusion gene and can contain one set of primer sets that can detect the ALK fusion gene, or a combination of two or more sets.
  • the kit for detecting a ZSCAN9 fusion gene of the present invention can specifically amplify at least a part of the ZSCAN9 fusion gene, and can contain one set of primer sets that can detect the ZSCAN9 fusion gene, or a combination of two or more sets.
  • 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 primer set for detecting a fusion gene of a ZSCAN9 gene and an ALK gene comprising a sense primer designed from a portion encoding ZSCAN9 and an antisense primer designed from a portion encoding ALK
  • the antisense primer consists of a nucleic acid molecule (preferably a nucleic acid molecule of at least 16 bases) that anneals to the “polynucleotide to be detected” under stringent conditions (preferably under more stringent conditions).
  • a primer set consisting of a nucleic acid molecule (preferably a nucleic acid molecule of at least 16 bases) that anneals to the complementary strand of the “polynucleotide to be detected” under stringent conditions (preferably under more stringent conditions) is included.
  • the primer set of the present invention includes the following primer sets (2) and / or (3).
  • a sense primer (preferably SEQ ID NO: 4) consisting of any continuous oligonucleotide consisting of at least 16 bases between base numbers 1 to 568 of SEQ ID NO: 1 (ZSCAN9ex3-ALKex20) and base numbers 569 to 2259 of SEQ ID NO: 1
  • a primer set of antisense primers (preferably SEQ ID NO: 5) consisting of oligonucleotides complementary to any consecutive at least 16 base oligonucleotides in between.
  • the base sequence of the polynucleotide represented by SEQ ID NO: 12 is a base sequence including the untranslated region (UTR) of the fusion gene represented by SEQ ID NO: 1 (ZSCAN9ex3-ALKex20). Is an array.
  • 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 an ALK fusion protein of the present invention can contain one or more combinations of antibodies that specifically bind to any site of the ALK fusion protein. Specifically, the antibodies described in ⁇ Fusion protein detection> can be exemplified.
  • the kit for detecting a ZSCAN9 fusion protein of the present invention can contain one or more combinations of antibodies that specifically bind to any site of the ZSCAN9 fusion protein. Specifically, the antibodies described in ⁇ Fusion protein detection> can be exemplified.
  • an ALK fusion protein or a ZSCAN9 fusion protein when it is a ZSCAN9-ALK fusion protein, it binds to one or more (preferably two or more) antibodies that bind to an ALK protein-derived polypeptide, or to a ZSCAN9 protein-derived polypeptide.
  • One or more antibodies that bind to an ALK protein-derived polypeptide and one or more types that bind to a ZSCAN9 protein-derived polypeptide even if only one of the above-mentioned one or more (preferably two or more) antibodies is included.
  • One or more antibodies that bind to a polypeptide containing a fusion point of an ALK fusion protein, or one or more antibodies that bind to a polynucleotide containing a fusion point of a ZSCAN9 fusion gene May be included.
  • 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 or not 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 a 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 ALK 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 ALK fusion-positive cancer or a candidate substance thereof. A step of confirming that the fusion-positive cancer has therapeutic activity can be further included.
  • the ZSCAN9 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 ZSCAN9 fusion-positive cancer or a candidate substance thereof. A step of confirming that the fusion-positive cancer has therapeutic activity can be further included.
  • 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 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). Specifically, for example, by introducing the ALK fusion gene or the ZSCAN9 fusion gene cDNA of the present invention into a recombinant vector and further introducing it into a cell, the polypeptide-expressing cell (transformed cell) Obtainable.
  • the pharmaceutical composition for treating ALK fusion-positive cancer (eg, digestive organ cancer) of the present invention contains an inhibitor for the ALK 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
  • the pharmaceutical composition for the treatment of a ZSCAN9 fusion-positive cancer of the present invention comprises an inhibitor for the ZSCAN9 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.
  • the inhibitor for the ALK fusion gene or a transcription product thereof include a kinase inhibitor, for example, an ALK inhibitor, or an inhibitor for the other gene constructing a fusion gene together with the ALK gene or a transcription material thereof.
  • the inhibitor for the ZSCAN9 fusion gene or a transcription product thereof include an inhibitor for at least one of the ZSCAN9 gene or a transcription substance thereof, or the other gene constructing a fusion gene together with the ZSCAN9 gene.
  • a kinase inhibitor can be exemplified as the inhibitor.
  • Low molecular compound include compounds described in AG879 (CAS148741-30-4), International Publications WO2008 / 045627, WO2008 / 073480, and the like.
  • inhibitors for the ZSCAN9-ALK fusion gene or transcripts thereof include, for example, crizotinib (Pfizer Inc, product name “Xalkori”), CH5424802 (Chugai Pharmaceutical Co Ltd), TAE684 (Novartis AG), ASP3026 (Astellas Pharma Inc), -3LDK-378 (Novartis AG), TSR-011 (Amgen Inc, TESARO Inc), AP-26113 (ARIAD Pharmaceuticals Inc), X-376 / 396 (Xcovery Inc), 4SC-203 (4SC AG; ProQinase GmbH), NMS-E628 (Nerviano Medical Sciences Srl), AZD-3463 (AstraZeneca plc), CEP-28122 (Cephalon Inc), CEP-37440 (Cephalon Inc), KRA-0008 (Korea Research Institute of Chemical Technology) be able to.
  • 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 ALK fusion gene or an inhibitory action on the ZSCAN9 fusion gene.
  • the double-stranded portion can be designed based on the polynucleotide base sequence containing the fusion point, for example, the base sequence containing the 568th to 569th positions of SEQ ID NO: 1.
  • 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.
  • RNAi double-stranded nucleic acid
  • 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 is not limited as long as it inhibits the transcription product of the ALK fusion gene or the transcription product of the ZSCAN9 gene, preferably the transcription product of the ZSCAN9-ALK gene. .
  • Example 1 Detection of ALK gene abnormality in clinical specimens by FISH method
  • a method in which the 5 'terminal region and 3' terminal region of the target gene are dyed with different dyes to observe gene translocation or inversion. are known.
  • This method which is a type of FISH method, is called a split assay.
  • 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.
  • ALK gene abnormality in clinical specimens was detected by FISH method split assay.
  • 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 sections were treated with the Histology FISH accessory kit (Dako), and then the BAC (bacterial-artificial-chromosome) clone (clone number) covering the green (FITC) fluorescently labeled ALK gene 5 'terminal region.
  • BAC bacterial-artificial-chromosome
  • RP11-984121 and RP11-62B19 were hybridized with a BAC clone (clone number RP11-701P18) covering the 3 ′ end region of red (TexasRed) fluorescently labeled ALK gene.
  • 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 examination of about 1500 pathological specimens, 2 specimens (derived from colon cancer patients) suggesting abnormal genomic structure in the ALK gene region were found.
  • Example 2 Identification of ALK fusion polynucleotide gene in clinical specimen 5 'of ALK gene kinase region by inverse RT-PCR method using RNA derived from tissue in which ALK genome structural abnormality was suggested by FISH analysis 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 ALKREVex22-23 (SEQ ID NO: 3) designed in the ALK 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 ALKFWDex20-21 (SEQ ID NO: 4) and reverse primer ALKREV3 (SEQ ID NO: 5)
  • second round PCR forward primer ALKFWDex21-22 (SEQ ID NO: 5) using circularized DNA as a template 6) and reverse primer ALKREV4 (SEQ ID NO: 7)
  • 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 ZSCAN9 gene was fused to the 5 ′ side of the ALK gene kinase region.
  • Example 3 Isolation of a ZSCAN9-ALK fusion polynucleotide gene in a clinical sample Using a cDNA derived from a colorectal cancer clinical sample in which an ALK genome 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, a combination of forward primer ZSCAN9-HindIII (SEQ ID NO: 8) and reverse primer ALK-EcoRI (SEQ ID NO: 9) was used. As a result of confirming the sequence of the obtained amplification product, a polynucleotide (ZSCAN9ex3-ALKex20; SEQ ID NO. 1) was obtained.
  • amino acid sequence encoded by ZSCAN9ex3-ALKex20 (SEQ ID NO: 2)
  • amino acid substitutions at three positions of I593V, K623R, and D661E were found by comparison with the registered amino acid sequence of ALK-001 (Ensemble database, Protein ID: ENSP00000373700). Admitted.
  • the first amino acid eg, “I” in I593V
  • the subsequent amino acid number eg, “593” in I593V
  • amino acid number in SEQ ID NO: 2 is the amino acid number in SEQ ID NO: 2
  • the last (For example, “V” in I593V) means an amino acid in the amino acid sequence represented by SEQ ID NO: 2, respectively.
  • Example 4 Detection of ZSCAN9-ALK 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, and the fusion gene cDNA was present in the cancer tissue. I showed that. Specifically, PCR was performed on the sample-derived RNA template using the forward primer ZSCAN9-394F (SEQ ID NO: 10) designed on the ZSCAN9 gene and the reverse primer ALK3078RR (SEQ ID NO: 11) designed on the ALK gene. Went. When the amplified product was electrophoresed, a band (492 bp) of the expected size was observed from the primer setting position, and detection of the fusion gene using clinical specimens was possible by designing primers on these genes. It was shown that.
  • Example 5 Detection of ZSCAN9-ALK fusion gene in clinical specimens by FISH fusion assay In order to confirm that the fusion gene was fused on the genome, detection was performed by 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. .
  • BAC clone (clone number CTD-2382I15) covering the 5 'terminal region of ZSCAN9 gene labeled with red (TexasRed) and the ALK gene 3' labeled with green (FITC) are labeled.
  • a fluorescence microscope BX51 (Olympus) was used.
  • Example 6 Detection of ZSCAN9 gene or ALK gene in clinical specimens by FISH method split assay According to the method described in Example 1, FISH method split assay of ZSCAN9 gene or ALK gene was performed. A pathological section was prepared in the same manner as in Example 1. The prepared unstained sections were processed with a Histology FISH accessory kit (Dako).
  • the BAC clone (clone number CTD-2382I15) covering the 5 'terminal region of the red (TexasRed) fluorescently labeled ZSCAN9 gene and the green (FITC) were fluorescently labeled.
  • Hybridization was carried out using a BAC clone (clone number RP5-874C20) covering the 3 'terminal region of the ZSCAN9 gene.
  • staining was performed with 4,6-diamino-2-phenylindole.
  • a fluorescence microscope BX51 (Olympus) was used.
  • a section in which a 5'-side signal red in this example
  • the split assay for ALK subsequently, the BAC clone (clone number RP11-701P18) covering the 5 ′ terminal region of red (TexasRed) fluorescently labeled ALK and the fluorescent labeling of green (FITC) were performed. Hybridization was performed using BAC clones (clone numbers RP11-984I21, RP11-62B19) covering the 3 ′ terminal region of the ALK gene. Subsequently, staining was performed with 4,6-diamino-2-phenylindole. For fluorescence observation, a fluorescence microscope BX51 (Olympus) was used.
  • Example 7 Detection of ZSCAN9-ALK fusion gene in clinical specimen by combination of FISH fusion assay and split assay FISH fusion assay and split assay were performed on the same pathological sections.
  • a pathological section was prepared in the same manner as in Example 1. The prepared unstained sections were processed with a Histology FISH accessory kit (Dako).
  • the BAC clone (clone number CTD-2382I15) covering the 5 'terminal region of the ZSCAN9 gene labeled with blue (Aqua) and the ALK gene 5' labeled with red (TexasRed) are labeled.
  • a BAC clone (clone number RP11-701P18) covering the terminal region and a BAC clone (clone numbers RP11-984I21, RP11-62B19) covering the 3 'terminal region of the ALK gene labeled with green (FITC).
  • FITC green
  • a fluorescence microscope BX51 (Olympus) was used.
  • a signal (blue green) adjacent to the ZSCAN9 gene 5 ′ terminal region and the ALK gene 3 ′ terminal region and loneliness away from this signal A signal (red) on the 5 ′ end side of the ALK gene was observed, indicating a genomic structural abnormality accompanied by the generation of a fusion gene. Therefore, this method can be used as a method for detecting the presence of the fusion gene.
  • Example 8 Examination of tumorigenic ability of ZSCAN9-ALK fusion polypeptide
  • ZSCAN9ex3-ALKex20 SEQ ID NO: 1
  • pLenti6 Invitrogen (registered)
  • the tumorigenicity of the ZSCAN9-ALK fusion polypeptide was examined.
  • pLenti6-ZSCAN9-ALK was introduced into the mouse fibroblast cell line NIH3T3 cells and cultured for 11 days, a transformation focus was observed as shown in FIG.
  • Example 9 Examination of susceptibility of ZSCAN9-ALK fusion polypeptide-expressing cells to each ALK 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 becomes possible to proliferate without adding IL-3 (Daley GQ and Baltimore D. Proc Natl Acad Sci USA. 1988 Dec; 85 (23): 9312-9316.).
  • each ALK inhibitor (crizotinib, CH5424802, TAE684, ASP3026) was added, and the sensitivity to each ALK inhibitor was examined by counting the number of cells after culturing for 72 hours.
  • Katayama et al. Korean T et al., Sci Transl Med, 2012 (4): 120ra17.
  • Example 10 Examination of suppression of autophosphorylation of ZSCAN9-ALK fusion polypeptide by each ALK inhibitor in ZSCAN9-ALK fusion polypeptide-expressing cells ZSCAN9-ALK fusion polypeptide by each ALK inhibitor confirmed in Example 8
  • Western blotting of extracts from each cultured cell treated with each ALK inhibitor was performed. It was. The results are shown in FIG.
  • an anti-phosphorylated ALK antibody pALK in FIG. 7; pY1604
  • an anti-ALK antibody ALK in FIG. 7 were used.
  • a fusion gene of the ZSCAN9 gene and the ALK gene exists in some digestive cancer patients and that the gene causes cancer. That is, it has been clarified that cancer patients to be treated with ALK-inhibiting drug treatment can be selected by detecting the ZSCAN9-ALK fusion gene, preferably by detecting ZSCAN9ex3-ALKex20.
  • the detection method of the present invention is useful for the determination of ALK fusion positive cancer patients.
  • the detection kit and primer set of the present invention can be used in the detection method.
  • the inhibitor screening method of the present invention can be used for screening a drug effective for treatment of the fusion-positive cancer patient.
  • the drug 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 drug 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 ZSCAN9 fusion.
  • the detection kit and primer set of the present invention can be used in the detection method.
  • the inhibitor screening method of the present invention can be used for screening a drug effective for treatment of the fusion-positive cancer patient.
  • the drug 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 drug 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: 3 to 11 in the sequence listing are synthetic primer sequences.

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Abstract

　La présente invention concerne : une méthode de détection dans laquelle des fusions (protéines de fusion et gènes de fusion qui codent les protéines de fusion), qui sont les nouveaux agents pathogènes du cancer, sont élucidées, puis des nouvelles fusions et des dérivés sont détectés sur la base des résultats ; un ensemble de trousse et amorce pour la méthode de détection susmentionnée ; une méthode de criblage des inhibiteurs de l'expression et/ou de l'activité de polypeptides qui sont les protéines de fusion ; et des compositions pharmaceutiques, qui contiennent les inhibiteurs susmentionnés, afin de traiter le cancer. Les protéines de fusion comprennent les protéines de fusion ZSCAB9 ou les protéines de fusion ALK. Dans la méthode de criblage, les protéines de fusion ou les gènes de fusion qui codent les protéines de fusion sont détectés dans un échantillon obtenu d'un sujet.
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