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WO2007039963A1 - Marqueur de tumeur du cerveau pour permettre le pronostic d’un patient ayant une tumeur au cerveau et son utilisation - Google Patents

Marqueur de tumeur du cerveau pour permettre le pronostic d’un patient ayant une tumeur au cerveau et son utilisation Download PDF

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Publication number
WO2007039963A1
WO2007039963A1 PCT/JP2006/312268 JP2006312268W WO2007039963A1 WO 2007039963 A1 WO2007039963 A1 WO 2007039963A1 JP 2006312268 W JP2006312268 W JP 2006312268W WO 2007039963 A1 WO2007039963 A1 WO 2007039963A1
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Prior art keywords
brain tumor
tumor marker
prognosis
protein
detecting
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PCT/JP2006/312268
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English (en)
Japanese (ja)
Inventor
Ryuya Yamanaka
Kazuto Nishio
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Niigata University
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Publication of WO2007039963A1 publication Critical patent/WO2007039963A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a brain tumor marker and a use thereof for predicting the prognosis of a brain tumor patient.
  • Tumor cells proliferate out of order due to their own genetic abnormalities and lose their order, and therapies are primarily selected based on the molecular biological characteristics of these cells. Ideal. However, histological grade (an index of malignancy determined by the number of cell divisions and tumor cell morphology) has been used as one of the factors that predict the prognosis of brain tumors. Even with the same characteristics, it was difficult to predict the prognosis of brain tumors by pathological classification, because the progress, sensitivity to treatment, and prognosis differed greatly from patient to patient. On the other hand, to understand the molecular biological characteristics of each tumor cell, it goes without saying that genetic abnormalities in tumor cells involve multiple gene abnormalities. It is essential.
  • Microarray technology is an approach that simultaneously captures a large amount of changes in the transcription level of tumor cells, and can be applied to tumor cell identity diagnosis, sensitivity to treatment, resistance to drugs, prediction of side effects, identification of new target molecules, etc. It is. By using this microarray technology and examining gene expression profiles, it has been confirmed that many genes are differentially expressed in tumor histological types and grades! RU
  • Non-Patent Documents 1 to 6 Marker genes have also been reported for brain tumors based on the expression information of certain genes (for example, Non-Patent Documents 1 to 6). Few known marker genes can be used to predict prognosis.
  • Non-Patent Document 1 Freije WA, Castro- Vargas FE, Fang Z, Horvath S, Cloughesy T, Liau LM, Mischel PS, Nelson SF. Cancer Res. 2004 Sep 15; 64 (18): 6503—10.
  • Non-Patent Document 2 Rickman DS, Bobek MP, Misek DE, Kuick R, Blaivas M, Kurnit DM, Taylor J, Hanash SM. Cancer Res. 2001 Sep 15; 61 (18): 6885—91.
  • Non-Patent Document 3 Sallinen SL, Sallinen PK, Haapasalo HK, Helin HJ, Helen PT, Schraml P, Kallioniemi OP, Kononen J. Cancer Res. 2000 Dec 1; 60 (23): 6617—22.
  • Non-Patent Document 4 Kim S, Dougherty ER, Shmulevich I, Hess KR, Hamilton SR, Trent J M, Fuller GN, Zhang W. Mol Cancer Ther. 2002 Nov; l (13): 1229— 36.
  • Non-Patent Document 5 van den Boom J, Wolter M, Kuick R, Misek DE, Youkilis AS, Wechsle r DS, Sommer C, Reifenberger G, Hanash SM. Am J Pathol. 2003 Sep; 163 (3): 1033- 43 .
  • Non-Patent Document 6 Weiner, HI., Huang H. et al, Neurosurgery. 2000 Dec. 47 (6): 1400-1409.
  • the present invention has been made in view of the circumstances as described above, and an object thereof is to provide a brain tumor marker for predicting the prognosis of a brain tumor patient and use thereof. Means for solving the problem
  • the present invention is as follows.
  • Claim 1 of the present invention is a brain tumor marker gene for predicting the prognosis of a brain tumor patient characterized by having any one of the nucleotide sequences of SEQ ID NOS: 1 to 9.
  • Claim 2 of the present invention is a probe for detecting a brain tumor marker gene, characterized in that it has a base sequence that is noblyzed under stringent conditions with any one of the brain tumor marker genes of claim 1. It is.
  • Claim 3 of the present invention is a primer for detecting a brain tumor marker gene, characterized in that it is used to specifically amplify any one of the brain tumor marker genes according to claim 1. .
  • Claim 4 of the present invention is a DNA chip characterized in that at least one probe for detecting a brain tumor marker gene according to claim 2 is immobilized on a substrate.
  • Claim 5 of the present invention is a method for predicting the prognosis of a brain tumor patient, comprising the following steps (a) and (b): (a) from a biological sample of a subject Prepared RN A or a step of measuring the expression level of all or part of the brain tumor marker gene of claim 1 in a complementary polynucleotide transcribed from the RNA, (b) the expression level is normal This is a process of predicting a large number of subjects as having a poor prognosis.
  • Claim 6 of the present invention is a kit for predicting prognosis of a brain tumor patient, comprising at least one or more selected from the following group powers (a) to (c): A probe for detecting a brain tumor marker gene according to claim 2, (b) a primer for detecting a brain tumor marker gene according to claim 3, and (c) a DNA chip according to claim 4.
  • Claim 7 of the present invention includes the following steps (a), (b), and (c): The method for screening a substance that suppresses the expression of a brain tumor marker gene according to claim 1 (A) contacting the test substance with a cell that expresses and expresses at least one of the brain tumor marker genes according to claim 1, and (b) the brain tumor marker in the cell contacted with the test substance. A step of measuring the expression level of the gene and comparing it with the expression level of the corresponding brain tumor marker gene in the control cell without contacting the test substance, (c) based on the comparison result of (b) above! Selecting a test substance that decreases the expression level of the brain tumor marker gene.
  • Claim 8 of the present invention is a brain tumor marker protein for predicting the prognosis of a brain tumor patient, comprising a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 10 to 18 It is.
  • Claim 9 of the present invention is an antibody for detecting a brain tumor marker protein, which specifically recognizes the brain tumor marker protein of claim 8.
  • Claim 10 of the present invention is characterized in that at least one brain tumor marker protein detection antibody selected from the antibody for brain tumor marker protein detection according to claim 9 is immobilized on a substrate. It is a protein chip.
  • Claim 11 of the present invention is a method for predicting the prognosis of a brain tumor patient, comprising the following steps (a) and (b): (a) a living body of a subject A step of measuring the expression level of all or part of the brain tumor marker protein according to claim 8 in the protein prepared from the sample, (b) the expression level is healthy. Predicting a large number of subjects as having a poor prognosis compared to that of.
  • Claim 12 of the present invention is a kit for predicting the prognosis of a brain tumor patient, comprising at least one selected from the group forces consisting of the following (a) to (b): (a) The protein chip according to claim 9, wherein at least one of the antibodies for detecting a brain tumor marker protein according to claim 9, and (b) the protein chip according to claim 10.
  • Claim 13 of the present invention comprises the following steps (a), (b), and (c): A screen of a substance that suppresses the expression of a brain tumor marker protein according to claim 7 -A method of: (a) contacting the test substance with a cell expressing at least one of the brain tumor marker proteins according to claim 8, (b) the brain tumor in the cell contacted with the test substance Measuring the expression level of the marker protein and comparing it with the expression level of the brain tumor marker protein corresponding to the above in the control cells not contacted with the test substance, (c) based on the comparison result in (b) above, And selecting a test substance that reduces the expression level of the brain tumor marker protein.
  • a brain tumor marker for predicting the prognosis of a brain tumor patient is provided.
  • various molecular biological materials related to this brain tumor marker gene are provided, and by using these, it is possible to predict the prognosis of a brain tumor patient.
  • diagnostics as tumor markers and drug discovery such as new anticancer agents. It is also possible to screen for substances that suppress brain tumors and advance the development of new anticancer agents.
  • FIG. 1 is a diagram showing the results of comparison of survival curves between two groups divided into two groups according to the median expression level of each gene and the expression of each gene being different.
  • FIG. 2 is a diagram showing the results of stratification at each quartile for each gene and comparison between four groups.
  • FIG. 3 shows the results of immunohistochemical analysis of the protein level expression of DDR1 gene in glioma tissue.
  • FIG. 4 is a diagram showing the correlation between DDR1 protein expression level and patient survival.
  • the “brain tumor” of the present invention refers to all tumors that develop in the cranium.
  • a typical one with a high incidence is glioma, which further includes astrocytoma, glioblastoma and the like.
  • Other brain tumors include pituitary adenomas and schwannomas.
  • the “gene” in the present invention includes DNA and RNA.
  • DNA includes, for example, cDNA genomic DNA obtained by cloning, chemical synthesis techniques, or a combination thereof.
  • Single-stranded DNA which may be double-stranded or single-stranded, may be a coding DNA that is a sense strand or an anti-coding strand that is an antisense strand.
  • the "polynucleotide” in the present specification is used to include RNA and DNA!
  • the DNA includes any of cDNA, genomic DNA, and synthetic DNA.
  • the above RNA includes total RNA, mRNA, rRNA and synthetic RNA!
  • the present inventors extract genes that are highly expressed in brain tumor tissue, and further, glioblastoma tissue that is brain tumor tissue and anaplasticity. Survival analysis was performed among glioma tissues, and brain tumor markers for predicting the prognosis of nine brain tumor patients shown in Table 1 above were identified as genes that divide survival time from genes that are highly expressed in brain tumor tissues.
  • the brain tumor marker gene for predicting the prognosis of a brain tumor patient of the present invention is a gene that is hardly expressed or not expressed in a benign brain tumor or normal brain tissue and is highly expressed only in a malignant brain tumor.
  • a gene highly expressed in a brain tumor is a gene that correlates with the survival prognosis of a brain tumor patient. Therefore, it is possible to predict the prognosis of a brain tumor patient by measuring the expression level of the brain tumor marker gene for predicting the prognosis of the brain tumor patient of the present invention.
  • a brain tumor marker for predicting the prognosis of a brain tumor patient according to the present invention is characterized in that one gene has the base sequence described in any one of SEQ ID NOs: 1 to 9.
  • Gene Accession numb in Table 1 can be approached by er.
  • the brain tumor marker gene of the present invention can be obtained by a known gene manipulation technique.
  • the probe for detecting a brain tumor marker gene of the present invention is a nucleotide sequence that is hybridized with any one of the brain tumor marker genes having the nucleotide sequence of any one of SEQ ID NOs: 1 to 9 under stringent conditions. It is characterized by having.
  • These probes for detecting a brain tumor marker gene can be appropriately prepared using a known method by selecting the whole or partial sequence of the base sequence of the brain tumor marker gene.
  • the selected partial sequence may be any portion, but is characterized in that it is a base sequence that hybridizes with a brain tumor marker gene under stringent conditions.
  • the probe for detecting a brain tumor marker gene may be labeled with a radiolabel, a fluorescent label, or the like.
  • the brain tumor marker gene detection probe can be a DNA or RNA complementary to the selected sequence based on the selected partial sequence.
  • the brain tumor marker gene of the present invention is prepared by preparing a DNA probe having an appropriate length, adding a suitable label such as a fluorescent label, and hybridizing it with a subject. Detect the number of offspring expression and predict the prognosis of patients with brain tumors.
  • a brain tumor may be detected by detecting the expression level of one gene of a brain tumor marker.
  • the conditions for "nobridizing under a stringent condition with a brain tumor marker gene" according to the nucleotide sequence of the present invention are, for example, 42 ° C. And 1 X SSC (0.15 M NaCl, 0.015 M sodium citrate), wash at 42 ° C with a buffer containing 0.1% SDS. More preferably, a washing treatment at 65 ° C with a buffer solution containing hybridization at 65 ° C and 0.1 X SSC, 0.1% SDS can be mentioned.
  • the primer for detecting a brain tumor marker gene of the present invention is used for specifically amplifying any one of the brain tumor marker genes having the base sequence described in any one of SEQ ID NOs: 1 to 9.
  • the target brain tumor marker gene can be amplified based on a known method such as PCR.
  • the primer is a nucleotide sequence of the brain tumor marker gene of the present invention (SEQ ID NOs: 1 to 9). ) Can be designed and amplified easily by conventional methods such as using commercially available primer design software.
  • the primer may be labeled with an appropriate label (for example, enzyme label, radioactive label, fluorescent label, etc.), or may be modified with piotin, phosphate, amine or the like.
  • the DNA chip of the present invention comprises a brain tumor marker having a base sequence that is hybridized under stringent conditions with any one of the brain tumor marker genes having the base sequence of any one of SEQ ID NOs: 1 to 9 It is characterized in that at least one of the gene detection probes is immobilized on a substrate such as a glass plate.
  • the DNA chip can be produced according to a production method known to those skilled in the art. By using a DNA chip, the expression level of the brain tumor marker gene of the present invention can be detected and measured.
  • the method for predicting the prognosis of a brain tumor patient includes the following steps (a) and (b): (a) RNA prepared from a biological sample of a subject Or a step of measuring the expression level of all or part of the brain tumor marker gene having the base sequence described in any one of SEQ ID NOs: 1 to 9 in a complementary polynucleotide transcribed from the RNA, (b) A step of predicting a subject as having a poor prognosis when the expression level is higher than that of a healthy subject.
  • the "subject" is a patient diagnosed as having a brain tumor, for example, by MRI examination or the like. Your excised tissue or blood.
  • the biological sample to be evaluated can be any sample containing a marker gene.
  • RNA can be prepared from a biological sample according to a conventional method, such as from a resected tissue of a patient's brain force.
  • a complementary polynucleotide transcribed from the RNA can be prepared from a tissue excised from the patient's brain or blood according to a conventional method. For example, mRNA isolated from a subject's cells is used as a saddle and cDNA is synthesized and PCR amplified. At that time, labeled dNTP can be incorporated into labeled cDNA.
  • the method for predicting the prognosis of a brain tumor patient of the present invention is carried out by detecting and measuring the expression level of any of the brain tumor marker genes of the present invention in the biological sample.
  • the salt according to any one of SEQ ID NOs: 1 to 9 in a biological sample of a healthy person When the expression level of the brain tumor marker gene having the base sequence described in any one of SEQ ID NOs: 1 to 9 in the biological sample of the subject is higher than the above reference, the prognosis is poor.
  • the detection method of the present invention includes expression of the brain tumor marker gene by a known method such as RT-PCR, Northern blot, DNA microarray analysis, in situ, and hybridization analysis. Measure the amount.
  • the probe for detecting a brain tumor marker gene or the primer for detecting a brain tumor marker gene of the present invention can be used as appropriate.
  • the DNA chip of the present invention may be used!
  • the prognosis prediction kit for a brain tumor patient of the present invention comprises at least one or more selected from the group consisting of the following (a) to (c): (a) SEQ ID NOs: 1 to 9 A probe for detecting a brain tumor marker gene having a base sequence that can be hybridized under a stringent condition with any force 1 of the brain tumor marker gene having the base sequence of any one of (1), (b) any one of SEQ ID NOS: 1 to 9 A brain tumor marker gene detection primer used to specifically amplify any one of the brain tumor marker genes having the nucleotide sequence of (c) having any one of the nucleotide sequences of SEQ ID NOS: 1 to 9 At least one probe for detecting a brain tumor marker gene having a base sequence that hybridizes with any one of the brain tumor marker genes under stringent conditions is immobilized on a substrate. DNA Chi-up.
  • a screening method for a substance that suppresses the expression of a brain tumor marker gene having the nucleotide sequence of any one of SEQ ID NOS: 1 to 9 of the present invention includes the following steps (a), (b) and (c (A) contacting the test substance with a cell expressing at least one of the brain tumor marker genes having the nucleotide sequence of any one of SEQ ID NOs: 1 to 9, (b) Measure the expression level of the brain tumor marker gene in the cell contacted with the test substance, and compare it with the expression level of the brain tumor marker gene corresponding to the above in the control cell. Step (c) A step of selecting a test substance that reduces the expression level of the brain tumor marker gene based on the comparison result of (b) above.
  • the screening method for a substance that suppresses the expression of the brain tumor marker gene of the present invention comprises: By searching for a substance that decreases the expression level of a brain tumor marker gene, a candidate substance that suppresses malignant brain tumor is provided.
  • Examples of cells used in the screening of the present invention include all cultured cells that express a brain tumor marker gene having the nucleotide sequence described in any one of SEQ ID NOs: 1 to 9. Whether or not these brain tumor marker genes are expressed in cultured cells can be easily confirmed by detecting them by a known Western plotting method or the like. Specific examples of the cultured cells include cells derived from living tissue or blood cells isolated and prepared from cancer patients, or cells into which any one of the brain tumor marker genes of the present invention has been introduced.
  • the conditions for bringing the test substance into contact with the cells are not particularly limited, but the culture conditions (temperature, pH) are such that the cells do not die and can express the brain tumor marker gene of the present invention. It is preferable to select a medium composition).
  • RNA is prepared from cells expressing at least one of the brain tumor marker genes, and the brain tumor marker gene is measured.
  • a transcribed complementary polynucleotide transcribed from the RNA cartridge may be prepared to measure the brain tumor marker gene.
  • the expression level of the brain tumor marker gene is measured by a known method such as RT-PCR method, Northern blot method, DNA microarray analysis method, in situ hybridization analysis method.
  • the probe for detecting a brain tumor marker gene or the primer for detecting a brain tumor marker gene of the present invention can be used as appropriate. Use the DNA chip of the present invention.
  • the brain tumor marker protein for predicting the prognosis of a brain tumor patient of the present invention is a protein that is hardly or not expressed in a benign brain tumor or normal brain tissue and is highly expressed only in a malignant brain tumor. Proteins that are highly expressed in brain tumors correlate with the survival prognosis of brain tumor patients.
  • the brain tumor marker protein for predicting the prognosis of a brain tumor patient of the present invention is characterized by having a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 10 to 18.
  • the gene accession numbers in Table 1 can be approached in the NCBI gene database.
  • the antibody for detecting a brain tumor marker protein of the present invention is characterized by specifically recognizing a brain tumor marker protein having a polypeptide having an amino acid sequence ability described in any one of SEQ ID NOs: 10 to 18. Since the antibody for detecting brain tumor marker protein of the present invention has a property of specifically binding to the brain tumor marker protein, the brain tumor marker protein expressed in the tissue of a subject by using the antibody for detecting brain tumor marker protein is used. Can be specifically detected.
  • the method for producing an antibody is known, and the antibody for detecting a brain tumor marker protein of the present invention can also be produced according to a conventional method.
  • the antibody for detecting a brain tumor marker protein of the present invention is a polyclonal antibody
  • the brain tumor marker protein of the present invention expressed and purified in Escherichia coli or the like according to a conventional method, or according to a conventional method.
  • the oligopeptide having the partial amino acid sequence of the brain tumor marker protein of the invention can be synthesized to immunize non-human animals such as rabbits and obtained from the sera of the immunized animals according to a conventional method.
  • a monoclonal antibody it is obtained by immunizing a non-human animal such as a mouse with the brain tumor marker protein of the present invention or an oligopeptide having a partial amino acid sequence of the protein expressed and purified in Escherichia coli according to a conventional method.
  • the obtained spleen cells and myeloma cells can be obtained from the cells prepared from cell fusion of the spleen cells and myeloma cells.
  • the antibody may be labeled with an appropriate label (for example, an enzyme label, a radioactive label, a fluorescent label, etc.), or may be appropriately modified with piotin or the like!
  • a brain tumor marker protein used as an immune antigen for the production of an antibody for detecting a brain tumor marker protein is a DNA cloning, each based on the sequence information (SEQ ID NOs: 1 to 9) of the gene provided by the present invention. It can be obtained by plasmid construction, transfection into the host, culturing of the transformant, and recovering the protein of the culture force. These operations can be performed according to methods known to those skilled in the art.
  • the protein chip of the present invention comprises at least one antibody for detecting a brain tumor marker protein that specifically recognizes a brain tumor marker protein having a polypeptide having any one of the amino acid sequences of SEQ ID NOS: 10 to 18. It is characterized by being fixed on the substrate.
  • a protein chip can be produced according to a production method known to those skilled in the art.
  • the method for predicting the prognosis of a brain tumor patient comprises the following steps (a) and (b): (a) in a protein in which a biological sample force of a subject is also prepared, A step of measuring the expression level of all or part of a brain tumor marker protein having a polypeptide having an amino acid sequence ability according to any one of SEQ ID NOs: 10 to 18, and (b) said expression level is A process of predicting a large number of subjects as having a poor prognosis compared to that of healthy individuals.
  • the "subject" is a patient diagnosed as having a brain tumor, for example, by MRI examination or the like. What is excised tissue is blood. If the patient's brain force is excised, the blood isotropic force can be prepared in accordance with conventional methods, and the biological sample force can be used to prepare the protein.
  • the method for predicting the prognosis of a brain tumor patient of the present invention is carried out by detecting and measuring any expression level of a brain tumor marker protein in the biological sample.
  • Any one of SEQ ID NOs: 10 to 18 in a biological sample of a subject based on the expression level of a brain tumor marker protein having a polypeptide consisting of the amino acid sequence of SEQ ID NOs: 10 to 18 in a biological sample of a healthy person If the expression level of the brain tumor marker protein having a polypeptide consisting of the amino acid sequence is higher than the standard, a malignant brain tumor is determined.
  • a part of a patient's tissue is collected based on a known method, and a protein is prepared therefrom according to a conventional method.
  • the expression level of the brain tumor marker protein can be detected based on, for example, a known detection method such as a Western plot method, an ELISA method, or a fluorescent antibody method.
  • a known detection method such as a Western plot method, an ELISA method, or a fluorescent antibody method.
  • the above-mentioned antibody for detecting a brain tumor marker protein can be appropriately used.
  • the expression level of brain tumor marker protein may be detected by using the above protein chip.
  • a cell force protein that expresses at least one of the brain tumor marker proteins is prepared and a brain tumor marker gene is measured.
  • the expression level of the brain tumor marker gene is measured by a known method such as a Western plot method, an ELISA method, or a protein microarray analysis method.
  • the above-mentioned antibody for detecting brain tumor marker protein can be appropriately used.
  • the expression level of brain tumor marker protein may be detected by using the protein chip.
  • the kit for predicting prognosis of a brain tumor patient of the present invention comprises at least one or more selected from the group consisting of the following (a) to (b): (a) of SEQ ID NOs: 10 to 18 At least one antibody for detecting a brain tumor marker protein that specifically recognizes a brain tumor marker protein having a polypeptide that also has an amino acid sequence ability described in any one of the above, (b) the amino acid sequence ability of any one of SEQ ID NOs: 10 to 18 A protein chip in which at least one antibody for detecting a brain tumor marker protein selected from antibodies for detecting a brain tumor marker protein that specifically recognizes a brain tumor marker protein having the polypeptide is immobilized on a substrate.
  • a screening method for a substance that suppresses the expression of a brain tumor marker protein having a polypeptide consisting of the amino acid sequence of SEQ ID NOS: 10 to 18 of the present invention includes the following steps (a), (b) and (c) comprising: (a) a test substance and a cell expressing at least one of brain tumor marker proteins having a polypeptide having an amino acid sequence ability according to any one of SEQ ID NOs: 10 to 18; (B) measuring the expression level of the brain tumor marker protein in the cell contacted with the test substance, and comparing it with the expression level of the corresponding brain tumor marker protein in the control cell not contacted with the test substance Step (c) A step of selecting a test substance that reduces the expression level of the brain tumor marker protein based on the comparison result of (b) above.
  • the screening method for a substance that suppresses the expression of a brain tumor marker protein of the present invention provides a candidate substance that suppresses a malignant brain tumor by searching for a substance that decreases the expression level of the brain tumor marker protein.
  • Examples of cells used in the screening of the present invention include all cultured cells that express a brain tumor marker protein having a polypeptide having an amino acid sequence ability described in any one of SEQ ID NOs: 10 to 18. Whether or not these brain tumor marker proteins are expressed in cultured cells can be easily confirmed by detecting them by a known Western plot method or the like.
  • Specific examples of the cultured cells include cells derived from living tissue or blood cells isolated and prepared from cancer patients, or cells into which the brain tumor marker gene of the present invention has been introduced.
  • the conditions for contacting the test substance with the cells are not particularly limited, but the culture conditions (temperature, pH) in which the cells do not die and can express the brain tumor marker protein of the present invention. It is preferable to select a medium composition).
  • the subjects were 31 cases of glioma (WHO grade: 10 cases, IV: 21 cases) that were surgically removed at the Department of Neurosurgery, Niigata University Brain Research Institute. Table 2 shows the histological diagnosis, histologic diagnosis, WHO grade, life time, survival time), ' ⁇ , Outcome).
  • radiotherapy tumor local 40 Gy, irradiation 20 Gy
  • the survival period was defined as the patient's death date or the last visit date from the date of diagnosis. Histological diagnosis was based on WHO brain tumor histology.
  • the experiments described in this specification have obtained informed consent for all target patients in accordance with the rules of the Niigata University Ethics Committee.
  • RNA quality was measured by rana ratio [28sZl8s] using Bioanalyzer 2100 (Agilent).
  • the purified total RNA was stored in 70% ethanol at 1-80 ° C until use.
  • cDN A was synthesized using an Agilent fluorescent direct label kit. Ten 1.25 1 ImM FluoroLink dCTP (Cy3—dCTP or Cy5—dCTP) using 200 g of purified total RNA and oligo dT primer with T7 promoter sequence, 42 ° C1 with 200 units of SuperScriptll reverse transcriptase After reacting for a period of time and synthesizing the first strand cDNA, the second strand cDNA was synthesized, extracted with phenol Z chloroform, and purified with Phase Loc gel. Tumor RNA was labeled with Cy5 and normal brain tissue RNA was labeled with Cy3. The obtained cDNA was recovered by ethanol precipitation and stored at -80 ° C in 70% ethanol until use. Normal brain tissue RNA purchased from Clontech was used.
  • Hybridization consists of 25 1 solution containing 2X Deposition Control Buffer 12.5 / z1, Cot-lDNA 2.5 ⁇ , Nuclease-free water 7.5; zl, cDNA 2.5 ⁇ It was conducted at 65 ° C for 17 hours. After washing the chip with 0.5% SSC / 0.1% SDS solution, the fluorescence intensity of each pixel was measured with an HP laser scanner, imaged, and tumors of normal brain tissue of each gene. Numerical values were calculated regarding the expression ratio in tissues. This experiment measured the expression of approximately 12729 genes in human brain tumor patients.
  • the standard deviation (SD) of ⁇ value (p-value), hazard ratio, and log (expression ratio) of each gene is shown. Sarasako, the median expression level of each gene was divided between two groups and examined for correlation with survival time. As a result of the discussion, the Logrank test values of the seven genes TOMM40, SLN, RNF2, LDHC, DDR1, Ksp37, and HS6ST1 were 0.05 or less, and the expression level was significantly correlated with the prognosis (Fig. 1). Furthermore, when stratified for each gene and stratified at the quartile, and the comparison between the four groups was examined, the Logrank test value for all nine genes was 0.05 or less, and the expression level significantly correlated with prognosis. (Figure 2). Therefore, it was suggested that the prognosis of brain tumor patients is predicted by examining the expression levels of the above nine genes.
  • the DDR1 gene product was stained to various degrees depending on the case. Express the expression intensity of the protein in any three fields of view as 0 to 3, and the expression area as 0, 0. 25, 0. 5, 0. 75, 1.0, and calculate the average value of the products. Level. Divided into 2 groups of low expression cases (A) with an expression level value of 1 or less and high expression cases (B) of 1 or more (a specific example is shown in Fig. 3). We examined the significant difference between the two groups. The results are shown in Fig. 4. The P value was 0.017, and when DDR1 protein was highly expressed, the prognosis was poor.

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Abstract

L’invention a pour objet un marqueur de tumeur du cerveau à utiliser dans le pronostic d’un patient ayant une tumeur au cerveau et l’utilisation dudit marqueur. Le pronostic d'un patient ayant une tumeur au cerveau peut être effectué en déterminant la quantité d'expression d'un gène marqueur de tumeur du cerveau servant au pronostic d'un patient ayant une tumeur au cerveau, dont une séquence de nucléotides est représentée dans l’une des SEQ ID NOs:1-9 dans la liste des séquences, et/ou la quantité d'expression d'une protéine marqueuse de tumeur du cerveau servant au pronostic d'un patient ayant une tumeur au cerveau, dont un polypeptide comprend un acide aminé représenté dans l’une des SEQ ID NOs:10-18 dans la liste des séquences.
PCT/JP2006/312268 2005-09-30 2006-06-20 Marqueur de tumeur du cerveau pour permettre le pronostic d’un patient ayant une tumeur au cerveau et son utilisation WO2007039963A1 (fr)

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WO2009106065A3 (fr) * 2008-02-29 2009-11-26 Meyer Helmut E Biomarqueur servant à diagnostiquer une tumeur du cerveau

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009106065A3 (fr) * 2008-02-29 2009-11-26 Meyer Helmut E Biomarqueur servant à diagnostiquer une tumeur du cerveau

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