JP2012088220A - Marker for detecting gist - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a marker for detecting GIST (Gastrointestinal stromal tumor), a method for detecting or diagnosing the GIST by using the marker, and a kit for detecting or diagnosing the GIST.SOLUTION: An SOBP gene which has a low expression level in a gastric cancer and a gastric normal tissue, and on the other hand, a raised expression level in the GIST and a polypeptide encoded by the SOBP gene are used as the marker for detecting the GIST. By using a probe for detecting the GIST marker gene and an antibody against the polypeptide encoded by the GIST marker gene, the marker for detecting the GIST is detected and the detection or diagnosis of the GIST is conducted rapidly and reliably.

Description

  The present invention relates to a marker for GIST detection comprising a gene and polypeptide specifically expressed in GIST (Gastrointestinal stromal tumor), a probe for detecting the marker, and a method for detecting or diagnosing GIST using the marker.

  Gastrointestinal stromal tumors, also called GIST (Gastrointestinal stromal tumor), are mesenchymal tumors that mainly develop under the mucosa of the gastrointestinal tract. Conventionally, mesenchymal tumors that occur under the mucous membrane of the gastrointestinal tract were thought to be smooth muscle tumors, but the existence of tumors having characteristics different from leiomyomas has been revealed. Miettinen et al. Found a gastrointestinal mesenchymal tumor immunostained with anti-CD34 antibody, but named GIST, although typical smooth muscle tumors and schwannomas were not immunostained with anti-CD34 antibody (Non-patent Document 1).

  It has been reported that such GIST is a tumor of Cajal intervening cells in the gastrointestinal tract, and many GISTs can detect mutations in the c-kit gene originally expressed in Cajal intervening cells. It has been reported (Non-Patent Document 2). Furthermore, some GISTs have a mutation in the PDGFRα (platelet derived growth factor receptor alpha) gene (Non-patent Document 3).

  The mutation of the c-kit gene occurring in GIST is observed at a frequency of 70% to 90% in any of the four regions of exons 9, 11, 13, and 17. Moreover, the variation | mutation of PDGFR (alpha) gene is seen by the frequency of 5 to 10% in either of the exons 12 and 18 (nonpatent literatures 4 and 5).

  It has also been reported that c-kit gene mutations are observed even with small GISTs of 1 cm or less almost as often as large GISTs. This suggests that the mutation of the c-kit gene is a change that occurs in the early stage of tumor development and is a change acquired with malignant transformation.

  Imatinib, which is a molecular target drug, has a significant effect on GIST (Non-patent Documents 6 and 7). However, so far no solid tumors other than GIST have statistically confirmed the usefulness of imatinib. Therefore, accurate diagnosis of GIST is essential for imatinib to be used correctly.

JP 2004-233105 A

Miettinen M., Virolainen M. and Rikala M.S.Gastrointestinal stromal tumors--value of CD34 antigen in their identification and separation from true leiomyomas and schwannomas. Am. J. Surg. Pathol. 19, 207-216 (1995) Hirota, S., Isozaki, K., Moriyama, Y., Hashimoto, K., Nishida, T., Ishiguro, S., Kawano, K., Hanada, M., Kurata, A., Takeda, M., Muhammad, Tunio, G., Matsuzawa, Y., Kanakura, Y., Shinomura, Y., and Kitamura, Y. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 279, 577-580 ( 1998) Merkelbach-Bruse, S., Dietmaier, W., Fuzesi, L., Gaumann, A., Haller, F., Kitz, J., Krohn, A., Mechtersheimer, G., Penzel, R., Schildhaus, H U, Schneider-Stock, R., Simon, R., and Wardelmann, E. Pitfalls in mutational testing and reporting of common KIT and PDGFRA mutations in gastrointestinal stromal tumors. BMC Med Genet. 11, 106 (2010) Flethcer, CDM, Berman, JJ, Corless, C., Gorstein, F., Lasota, J., Longley, BJ, Miettinen, M., O'Leary, TJ, Remotti, H., Rubin, BP, Shmookler, B ., Sobin, LH, and Weiss, SW Diagnosis of Gastrointestinal Stromal Tumors: A Consensus Approach. Human Pathology, 33, 459-465 (2002) Miettinen, M., Sobin, L. H., and Sarlomo-Rikala, M. Immunohistochemical spectrum of GISTs at different sites and their differential diagnosis with reference to CD117 (KIT). Modern Pathology, 13, 1134-1142 (2000) Heinrich, MC, Corless, CL, Demetri, GD, Blanke, CD, von Mehren, M., Joensuu, H., McGreevey, LS, Chen, CJ, Van den Abbeele, AD, Druker, BJ, Kiese, B., Eisenberg, B., Roberts, PJ, Singer, S., Fletcher, CD, Silberman, S., Dimitrijevic, S., and Fletcher, JA Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J. Clin. Oncol ., 21, 4342-4349 (2003) Tarn, C., Merkel, E., Canutescu, AA, Shen, W., Skorobogatko, Y., Heslin, MJ, Eisenberg, B., Birbe, R., Patchefsky, A., Dunbrack, R., Arnoletti, JP, von Mehren, M. and Godwin, AK Analysis of KIT mutations in sporadic and familial gastrointestinal stromal tumors: therapeutic implications through protein modeling. Clinical Cancer Research, 11, 3668-3677 (2005) Schena, M., Shalon, D., Davis, R. W. and Brown, P. O.Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 270, 467-470 (1995)

  There are cases in which the diagnosis of GIST is difficult to diagnose only by pathomorphology, and it is considered that a multifaceted diagnosis by combining immunohistochemistry and gene mutation search is necessary. However, immunohistochemical methods and molecular biological methods are not versatile and accurate diagnosis may be difficult. In particular, in molecular biology, KIT protein expression is not necessarily positive in all cases of GIST, and the mutation rate of c-kit or PDGFRα gene is not 100%.

  Therefore, in order to make an accurate diagnosis of GIST, it is essential to establish a more accurate molecular biology technique, not only with the presence or absence of c-kit or PDGFRα gene mutations or KIT protein expression, but also with new New molecular biological markers have been desired.

  An object of the present invention is to provide a GIST marker, in particular, a marker for GIST detection comprising a gene and a polypeptide specifically expressed in GIST, and a method for detecting or diagnosing GIST using the marker.

  The present inventor determined the mRNA from the surgical material of multiple patients diagnosed with GIST and the surgical material of multiple patients diagnosed with gastric cancer into cancerous part (tumor part) and non-cancerous part (normal part). As a result of isolating and comprehensively obtaining and analyzing gene expression profiles by DNA microarray method, we found that SOBP (sine oculis binding protein homolog (Drosophila)) gene is specifically expressed in GIST. The inventors have found that it can be used as a GIST marker and have completed the present invention.

That is, the present invention provides the following.
1. A gene marker for GIST detection, comprising a base sequence of a SOBP gene specified by SEQ ID NO: 1 in the sequence listing.
2. 2. A DNA microarray or DNA chip for GIST marker gene expression detection, wherein at least one or more of the DNA sequence or cDNA of all or part of the base sequence of the GIST detection gene marker described in 1 above is immobilized.
3. An oligonucleotide used for detecting the expression of a SOBP gene, a GIST detection gene marker having a base sequence of a SOBP gene identified by SEQ ID NO: 1 in the sequence listing,
An oligonucleotide comprising a base sequence of the SOBP gene or a part of a complementary base sequence thereof, and forming a site-specific base pair with the SOBP mRNA or cDNA thereof.
4). A DNA microarray or DNA chip for GIST marker gene expression detection, wherein at least one oligonucleotide according to the above item 3 is immobilized.
5. A primer used for detecting a gene marker for GIST detection having a base sequence of a SOBP gene specified by SEQ ID NO: 1 in the sequence listing,
At least two kinds of primers for forming a site-specific base pair with the mRNA of the SOBP gene or its cDNA and amplifying DNA having all or part of the base sequence shown in SEQ ID NO: 1 in the sequence listing by PCR A primer set for detecting a GIST marker gene, comprising:
6). A polypeptide marker for GIST detection, comprising the whole or a part of the amino acid sequence of the SOBP protein specified by SEQ ID NO: 2 in the sequence listing.
7). The method according to claim 6, wherein the polypeptide having the amino acid sequence of the polypeptide marker for GIST detection according to 6 is induced by administering to a mammal, and the SOBP protein is detected by using an antibody that specifically binds to the polypeptide. Characteristic GIST diagnostic method.
8). 8. The GIST diagnosis method according to 7 above, wherein the antibody is a monoclonal antibody or a polyclonal antibody.
9. It is induced by administering an expression vector for mammalian cells incorporating the SOBP gene to a mammal, and specifically binds to a polypeptide comprising all or part of the amino acid sequence of the SOBP protein shown in SEQ ID NO: 2 in the Sequence Listing. A method for diagnosing GIST, comprising detecting an SOBP protein by using an antibody.
10. 10. The GIST diagnostic method according to 9 above, wherein the antibody is a monoclonal antibody or a polyclonal antibody.
11. A GIST diagnosis method characterized by using the expression level of the SOBP gene specified by SEQ ID NO: 1 in the sequence listing as an index.
12 An oligonucleotide used to detect the expression of a SOBP gene of a GIST detection gene marker having the base sequence of the SOBP gene identified by SEQ ID NO: 1 in the sequence listing, the base sequence of the SOBP gene or its complement 12. The method of GIST diagnosis according to 11, wherein the expression level index is obtained using an oligonucleotide comprising a part of the base sequence and forming a site-specific base pair with the SOBP mRNA or cDNA thereof.
13. A GIST diagnostic method for distinguishing non-GIST such as gastric cancer from GIST,
Collecting a specimen sample consisting of a patient-derived detection tissue for the purpose of GIST diagnosis;
Collecting a reference sample comprising normal tissue;
A diagnostic step of measuring and comparing the expression level or expression level of the SOBP gene or SOBP protein identified by SEQ ID NO: 1 in the sequence listing in the specimen sample and the reference sample;
GIST diagnostic method characterized by including.
14 The diagnostic step is based on the expression level or expression level of the SOBP gene or the SOBP protein in a normal tissue or a non-GIST tissue that is grasped in advance, and is determined to be GIST when higher than the reference 14. The GIST diagnosis method according to the above 13,
15. 15. The GIST diagnosis method according to any one of 11 to 14, wherein the expression level or expression level of the SOBP gene is obtained by one of RT-PCR, real-time PCR, and hybridization. .
16. 16. The GIST diagnosis method according to 15, wherein the hybridization method is a microarray method or a blot method.
17. 16. The GIST diagnostic method according to 15 above, wherein the probe used in the microarray method or blotting method is a nucleotide or a protein.
18. 18. The GIST diagnosis method according to 17 above, wherein the nucleotide is mRNA, cDNA, or a synthetic oligonucleotide.
19. A GIST diagnostic method using the expression level of the SOBP protein shown in SEQ ID NO: 2 of the Sequence Listing as an index.
20. 20. The GIST diagnosis method according to 19, wherein the SOBP protein in the specimen sample is labeled by a fluorescent antibody method and detected.
21. A pharmaceutical composition for treating GIST, comprising an antibody that specifically binds to a polypeptide comprising all or part of the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing.
22. A pharmaceutical composition for treating GIST comprising a polynucleotide comprising all or part of the base sequence shown in SEQ ID NO: 1 in the sequence listing and / or a derivative thereof.

  In the present invention, a probe for detecting a SOBP gene that is a marker gene for GIST detection, or an antibody against a polypeptide having all or part of the amino acid sequence of the SOBP protein, is used to produce a SOBP gene or a SOBP protein. GIST can be detected or diagnosed extensively and reliably using expression as an indicator.

(Expression level of SOBP gene in GIST by microarray method) Extensive gene expression analysis was performed by DNA microarray in 10 cases of GIST, 25 cases of tumor site of gastric cancer, 20 cases of non-cancerous part (normal site) obtained from gastric cancer or GIST case, The expression level ratio of the SOBP gene to the human common reference was plotted as log ₂ ratio. The black diamond represents the ratio of SOBP gene expression levels in individual cases. An asterisk (*) indicates that the P value of the difference between the two groups is statistically less than 0.0005 by t test. Furthermore, an asterisk (**) indicates that the P value of the difference between both groups is statistically less than 0.0006 by t-test.

  Unless otherwise specified, the terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs (those skilled in the art).

  Those skilled in the art will recognize many methods and materials equivalent or similar to those described herein. However, the present invention is not limited to the methods and materials described herein.

  “Measurement of gene expression level” or “detecting gene expression” is not particularly limited as long as the expression level of the gene is detected or quantified. For example, mRNA or cDNA of the gene is detected or quantified. May be. For detection or quantification of these, it is desirable to use a molecule that specifically binds to the gene or its gene product, peptide or protein. Although it does not restrict | limit especially with the molecule | numerator specifically couple | bonded with a gene or its gene product peptide or protein, It bind | bond | couples specifically with the nucleotide, DNA, cDNA, RNA, peptide, or protein which bind | bond | couples specifically with this gene. An antibody etc. can be illustrated. In addition, mRNA or protein fragments or homologues of the gene may be used for detection or quantification of the expression level of the gene.

  "DNA microarray" refers to oligonucleotides and single-stranded or double-stranded DNA arranged on a glass substrate at high density, and "DNA microarray method" refers to fluorescent labeling on the DNA microarray. A technique for qualitatively and quantitatively measuring the type and amount of nucleic acid bound to DNA by hybridization with RNA molecules.

  “Oligonucleotide” is a general term for molecules in which several nucleotides are polymerized.

  “Polynucleotide”, when used in singular or plural, refers to any polyribonucleotide or polydeoxynucleotide, which may be DNA or RNA, or may be modified DNA or RNA. DNA or RNA may be single-stranded or double-stranded, and may be a hybrid molecule containing DNA and RNA.

  “Gene marker” or “marker gene” refers to an entire gene or EST derived from that gene whose expression or level varies between certain states. If gene expression correlates with a certain state, that gene is a marker for that state.

  Specific methods for detecting or quantifying gene expression levels include Northern blotting, dot blotting, and iAFLP using labeled nucleotides, labeled cDNA or labeled RNA for probes that specifically bind to the gene. (Introduced Amplified Fragment Length Polymorphism) method, LAMP (Loop-Mediated Isothermal Amplification) method, PCR method, method of directly measuring mRNA, or the like can be used. Examples of the PCR method include RT-PCR method, real-time PCR method, and competitive PCR method.

  As the real-time PCR method, for example, cDNA is synthesized from the total RNA or mRNA in a sample using reverse transcriptase, the target region is amplified by the PCR method using the cDNA as a template, and the production process of the amplified product is performed. The method of monitoring in real time is mentioned. Examples of the reagent to be monitored in real time include SYBR (registered trademark: Moleclar Probes) Green I, TaqMan (registered trademark: Applied Biosystems) probe, and the like.

  Examples of the competitive PCR method include, for example, a method of synthesizing cDNA from total RNA or mRNA in a sample using a reverse transcriptase and reacting the cDNA and an internal standard DNA in the same reaction tube. Examples include a method of reacting by adding an RNA internal standard together with mRNA during the reaction. In addition, the sequence of the internal standard gene may be, for example, a sequence homologous to the sequence of the gene to be amplified or a non-homologous sequence.

  Examples of the method for directly measuring mRNA include nCounter Analysis system (Nanostring).

  Furthermore, specific methods for detecting or quantifying gene expression levels include DNA microarrays, DNA chips, or antibody arrays. A DNA microarray or DNA chip having at least one nucleotide or cDNA of the gene immobilized thereon is used.

  The nucleotide or cDNA may be a part corresponding to a part of the gene.

The labeling reagent used for labeling the probe is, for example, radioisotopes [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C], [ ³² P], [ ³⁵ S], enzymes, and the like. Certain β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, and fluorescent cyanine fluorescent dyes (for example, Cy2, Cy3, Cy5, Cy5.5, Cy7, Cyanine3, Cyanine5, etc.) may be used. it can.

  In addition, examples of the real-time PCR method include a method of monitoring PCR amplification products in real time using cDNA synthesized by reverse transcriptase from total RNA or mRNA in an organ (tissue) or cell as a template. . As a monitoring reagent for real-time PCR, for example, a SYBR Green I, TapMan (registered trademark: Applied Biosystems) probe or the like is used.

  Usually, a DNA microarray or DNA chip is an array or chip in which probes are fixed on a support, and the support of the DNA microarray or DNA chip may be any one that can be used for hybridization. A substrate such as glass, silicon, or plastic, a nitrocellulose film, a nylon film, or the like can be used.

  Probes used for DNA microarrays and DNA chips may be cDNA or synthetic oligo DNA.

  There are no particular restrictions on the method of using the DNA microarray or DNA chip. For example, when mRNA is purified from a biological sample and a reverse transcription reaction is performed using the mRNA as a template, a labeled cDNA can be obtained by using an appropriately labeled primer or labeled nucleotide. Hybridization between the labeled cDNA and the probe in the present invention immobilized on the surface of a DNA microarray or DNA chip, and comparing the results of hybridization with a test sample and hybridization with a control sample, GIST can be detected or diagnosed by detecting the presence or absence of the SOBP gene or measuring the expression level.

  As a labeling substance used for the preparation of the labeled cDNA, a radioisotope, a fluorescent substance, or the like can be used. For example, examples of the fluorescent substance include Cy2, Cy3, Cy5, Cy5.5, Cy7, Cyanine3, Cyanaine5, FITC (fluorescein isothiocyanate), Texas Red, and rhodamine.

  The DNA sequence information of the SOBP gene can be approached by the accession number NM_018013 or AK090879 in the gene database of NCBI (National Center for Biotechnology Information) in addition to the sequence number 1 shown in the sequence listing. In addition, the polypeptide or protein corresponding to the SOBP gene is the expression product of the above gene, and the sequence information of the amino acid sequence of the polypeptide or protein is shown in SEQ ID NO: 2 in the Sequence Listing, and in the NCBI gene database. You can also approach by session number NP_060483.

  The method for detecting or quantifying the polypeptide or protein is not particularly limited as long as it is a method for detecting or quantifying a predetermined polypeptide or protein. For example, an antibody or aptamer that specifically binds to the polypeptide or protein can be used. As the antibody, a monoclonal antibody, a polyclonal antibody, a single chain antibody, a humanized antibody, a chimeric antibody, two epitopes can be used simultaneously. Examples include bifunctional antibodies that can be recognized. These antibodies can be produced using the polypeptide or protein or a fragment thereof as an antigen using conventional protocols. Aptamers are nucleic acid molecules that specifically bind to molecules such as proteins and amino acids.

  When detecting or quantifying the polypeptide or protein present in a test sample using an antibody that specifically binds to the polypeptide or protein, immunoprecipitation, electrochemiluminescence, RIA (Radioimmunoassay), Known immunological methods such as ELISA (Enzyme-liked immunosorbent assay) and fluorescent antibody method can be used.

  As a criterion for the above determination, the expression level of the SOBP gene present in the test sample (or the expression level of the polypeptide or protein corresponding to the gene) is present in the normal control sample (or Higher than the expression level of the polypeptide or protein corresponding to the gene). For example, when a t-test is performed, P <0.05, more preferably P <0.01, still more preferably P <0.001, and still more preferably P <0.0001.

  The test method is not limited to the t test, and may be a Mann-Whitney test or a Wilcoxan signed rank test.

  In addition, the said determination criterion is not limited to a test | inspection, For example, you may use the difference of the average value of each group.

  The reference value does not need to be measured every time the expression level in the test sample is measured, for example, the expression level of the SOBP gene present in the normal control sample in various biological samples is measured in advance, The measured value can be used for comparison.

  The criteria for diagnosing GIST is, for example, setting a reference value based on the expression level of SOBP gene or protein in a previously known control sample (such as non-GIST such as gastric cancer or normal tissue of the stomach). When the expression level of the SOBP gene or protein in the breast cancer tissue as a test sample is higher than the above-mentioned standard, it is determined as triple negative breast cancer.

  The reference may be an average value or a median value of a plurality of control samples (non-GIST such as gastric cancer or normal tissue of the stomach) grasped in advance, but is not limited thereto.

  Furthermore, when performing GIST diagnosis using the GIST gene marker of the present invention, the expression level of the SOBP gene or protein is used as an index. In addition to the expression level, histological diagnosis of the test sample or other It can be comprehensively determined from gene expression levels or other clinical information. Examples of other genes include c-kit gene and PDGFRα gene.

  When the determination of GIST using the gene marker of the present invention is carried out by detecting the mRNA or transcript of the SOBP gene as described above, the specimen is not particularly limited, but for example, a tissue or pathological specimen obtained by surgery It may be a biopsy material.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

  The SOBP gene used as a GIST marker of the present invention is a cancerous part, a non-cancerous part (normal part) in a tissue removed from a stomach cancer patient by surgery, and a cancerous part, a non-cancerous part in a tissue removed from a patient diagnosed with GIST. In the (normal site), an exhaustive gene expression profile by a DNA microarray was obtained, and the expression level was specifically increased by GIST. Here, the “expression level” need not be an absolute amount, but may be a relative amount.

(Production of microarray)
A microarray is prepared using the synthetic DNA for microarray. Although there are no limitations on the microarray production method and conditions, for example, the production method described in Non-Patent Document 8 (Schena, M. et. Al., Science, 270, 467-470 (1995)) can be used.

  A human gene fragment library (manufactured by Microdiagnostic) was printed on a slide glass (manufactured by Matsunami Glass Industrial Co., Ltd., HA-coated slide glass) using a micro-volume dispensing device (manufactured by Microdiagnostic) to produce a microarray . The microarray was allowed to stand at 80 ° C. for 1 hour in a gas-phase incubator, and further irradiated with 120 mJ ultraviolet rays using a UV crosslinker (Hoefer, UVC500).

(Post-processing of microarray)
The post-treatment of the microarray was performed by the method described in the published patent publication (Japanese Patent Application Laid-Open No. 2004-233105).

(Preparation of nucleic acid sample)
Sample nucleic acid was prepared by extracting total RNA from surgical tissue using ISOGEN reagent (Nippon Gene) according to the recommended protocol. Furthermore, mRNA was purified using a Poly (A) Pure kit (Ambion) according to the protocol recommended by the company.

(Synthesis of labeled cDNA)
2.0 μg of the mRNA was labeled with a nucleic acid labeling / hybridization reagent (Microdiagnosis), reverse transcriptase
Labeled cDNA was prepared using SuperScript II (registered trademark: Life Technologies) (manufactured by Invitrogen) and Cyanine5-deoxyuridinetriphosphate (Cyanine 5-dUTP) (manufactured by Perkin Elmer). On the other hand, a human common reference (manufactured by Microdiagnostic) was used as a control. For common human reference, nucleic acid labeling / hybridization reagent (Microdiagnosis), reverse transcriptase SuperScript II (Invitrogen), Cyanine3-deoxyuridinetriphosphate (Cyanine 3-dUTP) (Perkin Elmer) are used. A labeled cDNA was prepared. The production method followed the protocol recommended by each company. Human common references are 22 types of cell lines (A431 cells, A549 cells, AKI cells, HBL-100 cells, HeLa cells, HepG2 cells, HL60 cells, IMR-32 cells, Jurkat cells, K562 cells, KP4 cells, MKN7. Cells, NK-92 cells, Raji cells, RD cells, Saos-2 cells, SK-N-MC cells, SW-13 cells, T24 cells, U251 cells, U937 cells, Y79 cells) They are mixed one by one.

(Preparation of labeled probe)
These labeled cDNAs, ie, a sample labeled with Cyanine5-dUTP and a human common reference labeled with Cyanine3-dUTP were mixed in the same tube, and then Micropure EZ (Millipore) and Microcon YM30 (registered trademark: Millipore) ( Purified by Millipore). The purification method was performed according to the protocol recommended by the company. Finally, 15 μl was prepared using a hybridization buffer and pure water attached to a nucleic acid labeling / hybridization reagent (manufactured by Microdiagnostic).

(Hybridization)
The solution was heat denatured by heating at 99 ° C. for 5 minutes and then dropped on the microarray and stored in a hybridization cassette (manufactured by Microdiagnostic). The hybridization cassette was placed in a gas-phase incubator (manufactured by Sanyo Electric Biomedica Co., Ltd.) and kept warm at 42 ° C. for 20 hours.

(Washing)
The slide glass was taken out from the hybridization cassette, and the slide glass was washed using a hybridization washing solution attached to a nucleic acid labeling / hybridization reagent (manufactured by Microdiagnostic) according to the protocol recommended by the company.

(Fluorescence intensity detection and digitization)
Fluorescence is measured on the cleaned glass slide using the scanner GenePix4000B (Axon Instrument), optically evaluated using the analysis software GenePixPro (Axon Instrument) attached to the scanner, and the fluorescence intensity is a common human reference. And expressed as a relative ratio (log ₂ ratio).

(SOBP gene expression level)
MRNA was prepared from 25 gastric cancer cases, 10 GIST cases, and 20 non-cancerous parts (normal sites) obtained from gastric cancer or GIST cases, and a comprehensive gene expression profile by a DNA microarray was obtained. As a result, the expression level of the SOBP gene was significantly increased in GIST compared with the non-cancerous part (normal site) (P <0.0006) (FIG. 1). Furthermore, the expression level was significantly increased in GIST compared with gastric cancer (tumor) (P <0.0005) (FIG. 1). The clinical information of the specimens used in this example and the expression level ratio with respect to the common reference in each specimen are shown in Table 1 below. In Table 1, the “expression level ratio” is a value obtained by dividing the expression level of the SOBP gene in the test sample (specimen) by the expression level in the human common reference and converting it to a logarithmic ratio with a base of 2. “0” represents the detection limit or less.

  Since the expression level of the SOBP gene, which is a novel GIST marker of the present invention, is low in normal tissues and gastric cancer, and the expression level is significantly increased in GIST, which is difficult to distinguish from gastric cancer and difficult to make a definitive diagnosis. By using GIST markers, it is possible to make a definitive diagnosis of GIST based on objective molecular criteria. In particular, a definitive diagnosis can be made even with a small amount of tissue such as pathological cytology before surgery. Furthermore, it can also be used as a marker for follow-up after surgery, and can be an objective judgment material when determining a postoperative treatment policy. In addition, since it is considered possible to measure the amount of SOBP protein in serum, it may be used as a postoperative tumor marker for patients diagnosed with GIST.

Claims (22)

  A gene marker for GIST detection, comprising a base sequence of a SOBP gene specified by SEQ ID NO: 1 in the sequence listing.   A DNA microarray or DNA chip for detecting GIST marker gene expression, wherein at least one DNA or cDNA having the whole or part of the base sequence of the GIST detection gene marker according to claim 1 is immobilized. An oligonucleotide used for detecting the expression of a SOBP gene, a GIST detection gene marker having a base sequence of a SOBP gene identified by SEQ ID NO: 1 in the sequence listing,
An oligonucleotide comprising a base sequence of the SOBP gene or a part of a complementary base sequence thereof, and forming a site-specific base pair with the SOBP mRNA or cDNA thereof.   A DNA microarray or DNA chip for GIST marker gene expression detection, wherein at least one oligonucleotide according to claim 3 is immobilized. A primer used for detecting a gene marker for GIST detection having a base sequence of a SOBP gene specified by SEQ ID NO: 1 in the sequence listing,
At least two kinds of primers for forming a site-specific base pair with the mRNA of the SOBP gene or its cDNA and amplifying DNA having all or part of the base sequence shown in SEQ ID NO: 1 in the sequence listing by PCR A primer set for detecting a GIST marker gene, comprising:   A polypeptide marker for GIST detection, comprising the whole or a part of the amino acid sequence of the SOBP protein specified by SEQ ID NO: 2 in the sequence listing.   A polypeptide having the amino acid sequence of the polypeptide marker for GIST detection according to claim 6 is induced by administering to a mammal, and an SOBP protein is detected by using an antibody that specifically binds to the polypeptide. GIST diagnostic method characterized by   The GIST diagnosis method according to claim 7, wherein the antibody is a monoclonal antibody or a polyclonal antibody.   It is induced by administering an expression vector for mammalian cells incorporating the SOBP gene to a mammal, and specifically binds to a polypeptide comprising all or part of the amino acid sequence of the SOBP protein shown in SEQ ID NO: 2 in the Sequence Listing. A method for diagnosing GIST, comprising detecting an SOBP protein by using an antibody.   The GIST diagnosis method according to claim 9, wherein the antibody is a monoclonal antibody or a polyclonal antibody.   A GIST diagnosis method characterized by using the expression level of the SOBP gene specified by SEQ ID NO: 1 in the sequence listing as an index.   An oligonucleotide used to detect the expression of a SOBP gene of a GIST detection gene marker having the base sequence of the SOBP gene identified by SEQ ID NO: 1 in the sequence listing, the base sequence of the SOBP gene or its complement 12. The GIST diagnosis method according to claim 11, wherein the expression level index is obtained by using an oligonucleotide comprising a part of the base sequence and forming a site-specific base pair with the SOBP mRNA or its cDNA. A GIST diagnostic method for distinguishing non-GIST such as gastric cancer from GIST,
Collecting a specimen sample consisting of a patient-derived detection tissue for the purpose of GIST diagnosis;
Collecting a reference sample comprising normal tissue;
A diagnostic step of measuring and comparing the expression level or expression level of the SOBP gene or SOBP protein identified by SEQ ID NO: 1 in the sequence listing in the specimen sample and the reference sample;
GIST diagnostic method characterized by including.   The diagnostic step is based on the expression level or expression level of the SOBP gene or the SOBP protein in a normal tissue or a non-GIST tissue that is grasped in advance, and is determined to be GIST when higher than the reference The GIST diagnosis method according to claim 13.   The GIST diagnosis according to any one of claims 11 to 14, wherein the expression level or expression level of the SOBP gene is obtained by one of RT-PCR, real-time PCR, and hybridization. Method.   The GIST diagnosis method according to claim 15, wherein the hybridization method is a microarray method or a blot method.   The GIST diagnosis method according to claim 15, wherein the probe used in the microarray method or blotting method is a nucleotide or a protein.   The GIST diagnosis method according to claim 17, wherein the nucleotide is mRNA, cDNA, or a synthetic oligonucleotide.   A GIST diagnostic method using the expression level of the SOBP protein shown in SEQ ID NO: 2 of the Sequence Listing as an index.   20. The GIST diagnostic method according to claim 19, wherein the SOBP protein in the specimen sample is labeled by a fluorescent antibody method and detected.   A pharmaceutical composition for treating GIST, comprising an antibody that specifically binds to a polypeptide comprising all or part of the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing.   A pharmaceutical composition for treating GIST comprising a polynucleotide comprising all or part of the base sequence shown in SEQ ID NO: 1 in the sequence listing and / or a derivative thereof.