WO2008047947A1 - Procédé de détermination de l'effet d'une administration de cisplatine et kit permettant de déterminer l'effet d'une administration de cisplatine - Google Patents

Procédé de détermination de l'effet d'une administration de cisplatine et kit permettant de déterminer l'effet d'une administration de cisplatine Download PDF

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WO2008047947A1
WO2008047947A1 PCT/JP2007/070864 JP2007070864W WO2008047947A1 WO 2008047947 A1 WO2008047947 A1 WO 2008047947A1 JP 2007070864 W JP2007070864 W JP 2007070864W WO 2008047947 A1 WO2008047947 A1 WO 2008047947A1
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gene
cisplatin
effect
expression
genes
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Japanese (ja)
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Hideki Tanzawa
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National University Corporation Chiba University
Koshin. Co., Ltd.
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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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/142—Toxicological screening, e.g. expression profiles which identify toxicity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4722—Proteoglycans, e.g. aggreccan
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/49—Platelet-derived growth factor [PDGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Definitions

  • the present invention relates to a method for determining the administration effect of cisplatin used as an anticancer agent and a kit for determining the effect of cisplatin administration.
  • Cisplatin (abbreviated as “CDDP”) is the central agent of anticancer drugs and chemotherapy in all types of cancer.
  • CDDP Cisplatin
  • Non-Patent Literature 1 Bordow SB, Haber M, Madafiglio J, Cheung B, Marshall GM,
  • Non-Patent Document 2 Gottesman MM, Pastan I, Ambudkar SV. Curr Opin Genet Dev.
  • Non-Patent Document 3 Loe D. W., Deeley R. G., Cole S. P. Eur. J. Cancer, 32A: 945-957, 1996.
  • Non-Patent Document 4 Jin S, Scotto KW. Mol Cell Biol. Jul; 18 (7): 4377-4384. 1998
  • Non-Patent Document 5 Perez RP. Eur J Cancer. Sep; 34 (10): 1535-1542. 1998
  • Non Patent Document 6 Hinoshita E, Uchiumi T, Taguchi K, Kinukawa N, Tsuneyoshi
  • the present inventors have aimed to provide a method for determining the effect of cisplatin administration and a kit for determining the effect of cisplatin administration, which can determine the presence or absence of the therapeutic effect of cisplatin administration.
  • the present inventor has identified a gene group exhibiting resistance to or sensitivity to cisplatin by using a cell line exhibiting extremely excellent resistance to cisplatin, and has completed the present invention. .
  • the present invention includes the following.
  • PDE3B phosphodiesterase 3B gene
  • PDGFC piatelet derived growth factor C
  • PKD2 Polycystic kidney disease-2 gene
  • NRG1 neutral receptor l gene
  • step a the amount of mRNA of the gene is measured, and the method for determining the effect of cisplatin administration according to (1).
  • step a the amount of protein that is a product of the gene is measured.
  • the method for determining the effect of cisplatin administration according to (1) is further increased.
  • the method for determining the cisplatin administration effect according to (1) is further increased.
  • kits for determining the efficacy of cisbratin administration comprising a means for measuring the expression of at least one gene.
  • kit for determining cisplatin administration effect according to (5), wherein the means is a polynucleotide that specifically hybridizes to mRNA of the gene or cDNA derived from the mRNA.
  • FIG. 1 is a characteristic diagram showing the results of MTT assay in Sa-3 and Sa-3R strains as representative examples of resistant strains established in the Examples.
  • Fig. 2 shows the expression of ABC transporters (MDR1, MRP1, and MRP2) in Sa-3 and Sa-3R strains as a representative example of resistant strains established in the Examples by RT-PCR. It is a characteristic view which shows the result.
  • FIG. 3 is a Venn diagram showing the number of genes that specifically enhance expression and decrease in expression in resistant strains identified as a result of microarray analysis using the resistant strain and its parent strain established in the Examples.
  • Fig. 4 is a characteristic diagram showing the four networks obtained as a result of a Pausley analysis of 199 genes identified as genes related to cisplatin resistance.
  • FIG. 5 is a characteristic diagram (A) showing the results of RT-PCR performed on the lumican gene as a representative example of the five genes identified in the Examples (A) and a photograph (B) showing the results of Western blotting.
  • FIG. 6 is a photograph showing the results of immunohistological staining of a clinical sample for the lumican gene as a representative example of the five genes identified in the examples.
  • FIG. 7 is a characteristic diagram showing the result of obtaining the IHC score distribution for the PDE3E gene.
  • Fig. 8 is a characteristic diagram showing the results of IHC score distribution for the lumican gene.
  • FIG. 9 is a characteristic diagram showing the result of obtaining the distribution of IHC scores for the PDGFC gene.
  • FIG. 10 is a characteristic diagram showing the results of obtaining the IHC score distribution for the PKD2 gene.
  • Fig. 11 is a characteristic diagram showing the results of obtaining the IHC score distribution for the NRG1 gene.
  • This method for determining the effect of cisplatin administration is obtained as a result of the step a in which the expression of at least one or more genes selected from the following five genes is measured in a biological sample collected from a subject to be diagnosed. Based on the gene expression level And b determining the effect of administering bratin.
  • PDE3B phosphodiesterase 3B gene (SEQ ID NO: 1)
  • PDGFC platelet derived growth factor C gene (distributed IJ number 3)
  • PKD2 Polycystic kidney disease-2 gene
  • NRG1 (neuregulin l) gene (SEQ ID NO: 7)
  • LUM (Lumican) gene (SEQ ID NO: 9)
  • the amino acid sequence of the protein encoded by the PDE3B gene is shown in SEQ ID NO: 2.
  • the amino acid sequence of the protein encoded by the PDGFC gene is shown in SEQ ID NO: 4.
  • the amino acid sequence of the protein encoded by the PKD2 gene is shown in SEQ ID NO: 6.
  • the amino acid sequence of the protein encoded by the NRG1 gene is shown in SEQ ID NO: 8.
  • the amino acid sequence of the protein encoded by the LUM gene is shown in SEQ ID NO: 10.
  • the “No.” column shows the number assigned to each gene
  • the “Comraon” column shows the names of common genes.
  • the column “Genbank” is the registration number of the gene in the database provided by Genbank
  • the column “Systematic” indicates the strain name of the gene
  • the column “Map” is the position of the gene on the chromosome. Shows
  • the “Description” column is a description of the gene or a description of the protein that is the product of the gene.
  • the genes listed in Table 1 are composed of genes identified as a result of microarray analysis using cisplatin-resistant and cisplatin-sensitive strains, which will be described in detail later.
  • the cisplatin-resistant strain is a cell line established from an oral squamous cell carcinoma-derived cell line or an oropharyngeal squamous cell carcinoma-derived cell line using cisplatin resistance as an index, and a cell line of a patient that does not respond to cisplatin administration It has a meaning as a model.
  • the cis-bratin sensitive strain means the oral squamous cell carcinoma-derived cell line or the oropharyngeal squamous cell carcinoma-derived cell line itself, which is the parent strain of the cis-bratin resistant strain.
  • the genes listed in Table 1 are composed of genes that are specifically enhanced in expression and genes that are specifically attenuated in cisplatin resistant strains.
  • the genes whose expression was specifically enhanced in the cisplatin resistant strains are Nos. 1 to 164, and the genes whose expression was specifically attenuated in the cisplatin resistant strains are Nos. 165 to 199.
  • 51 gene groups form four networks, and these four networks form a large network.
  • the determination target person is not particularly limited, and may be any of a patient suffering from various cancers, a person suspected of various cancers, and a healthy person.
  • the cisplatin administration effect determination method according to the present invention is not performed directly on the determination target person, but is performed using a biological sample collected from the diagnosis target person.
  • the biological sample is determined Although it will not specifically limit if gene expression analysis in a subject is possible, the protein extract derived from a tissue, a cell, a bodily fluid, urine, and other biological samples can be mentioned.
  • body fluid means blood, lymph fluid, tissue fluid (tissue fluid, intercellular fluid, interstitial fluid), body cavity fluid, serous cavity fluid, pleural effusion, ascites, pericardial fluid, cerebrospinal fluid (spinal fluid), joint fluid ( Synovial fluid), eye aqueous humor (aqueous humor), digestive fluid, knee fluid, intestinal fluid, semen and amniotic fluid.
  • the biological sample may be one or a plurality of protein extracts derived from tissues, cells, body fluids, urine, and other biological samples.
  • the tissue includes a part of the tissue obtained at the time of surgery performed for the treatment of cancer-affected patients, a part of the tissue collected by biopsy etc. from a diagnosis subject suspected of cancer, a patient with cancer This means that it includes a part of the tissue collected by biopsy for the purpose of distinguishing between primary and metastatic.
  • a cell in the cisplatin administration effect determination method according to the present invention a cell isolated from each of the above tissues can be used.
  • the body fluid plasma or serum separated from the blood, urine, lymph fluid, cerebrospinal fluid or ascites can be used.
  • cells or protein extracts isolated from sputum or the like can be used as other organism-derived samples.
  • the amount of mRNA for the gene to be measured is measured or the amount of protein that is the product of the gene to be measured is measured. Just measure.
  • a known method for detecting the expression of a gene can be used.
  • the Northern plotting method can be used to detect the mRNA level of the gene to be measured.
  • a polynucleotide having a DNA sequence that hybridizes under stringent conditions to the gene to be measured can be used as a probe. In order to detect the amount of mRNA of the gene to be measured using the probe, it can be appropriately performed using a known method.
  • a label such as a fluorescent label is appropriately added to the probe, and this is hyper-hydidized with mRNA (or cDNA synthesized from mRNA) isolated from a biological sample collected from the subject. To do. afterwards, By measuring the fluorescence intensity derived from the hyper-prised probe, the mRNA level of the gene to be measured can be detected.
  • the probe can be used by being immobilized on a support such as a glass bead or a glass substrate. That is, it can also be used in the form of a microarray or DNA chip in which probes prepared for a gene to be measured (a plurality of genes) are immobilized on a support.
  • hybridizes under stringent conditions means, for example, 1 XSSC (0.15 MN a Cl, 0.015 M sodium taenoate), 0.1 at 42 ° C. This means that the hybridization is maintained even by washing at 42 ° C with a buffer containing% SDS (Sodium dodecyl sulfate).
  • SDS sodium dodecyl sulfate
  • a known protein detection method can be used as a method for measuring the amount of protein that is the product of the gene to be measured. Specifically, various methods using an antibody against the protein to be measured can be applied.
  • the antibody is not particularly limited, and a mouse antibody, a rat antibody, a rabbit antibody, a Hedge antibody, etc. can be used as appropriate.
  • the antibody may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferable in that a homogeneous antibody can be stably produced.
  • Polyclonal antibodies and monoclonal antibodies can be prepared by methods well known to those skilled in the art.
  • a hybridoma producing a monoclonal antibody can be basically produced using a known technique as follows. That is, a desired antigen or a cell that expresses the desired antigen is used as a sensitizing antigen, and this is immunized according to a normal immunization method. It can be prepared by fusing and screening for monoclonal antibody-producing cells (hypridoma) by conventional screening methods. Hybridomas can be produced, for example, by the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46) etc.
  • the product of the gene described above can be used as an antigen
  • a cell expressing a fragment of the product of the gene described above can be used as an antigen.
  • proteins or fragments of the protein are described in, for example, Molecuar Cloning: A Laboratory Manual 2nd edition 1st-3rd Sambrook, J. et al., Cold Spring Harber Laboratory Press publication New York 1989. According to the method, those skilled in the art can easily obtain it. Cells expressing these proteins or fragments of the proteins were also described in Molecuar Cloning: A Laboratory Manual, 2nd edition, 1st 1-3 brook Sambrook, j., Cold Spring Harber Laboratory Press, published in New York, 1989. According to the method, it can be easily obtained by those skilled in the art.
  • the resulting monoclonal antibodies can be used for the quantification of proteins to be measured, such as the enzyme enzyme assay (ELISA), enzyme immunodot assay, radioimmunoassay, aggregation based assay, or other well-known methods. It can be used as a test reagent by the existing immunoassay method.
  • the monoclonal antibody is preferably labeled.
  • the labeling compound include enzymes, fluorescent substances, chemiluminescent substances, radioactive substances, and staining substances known in the art.
  • the above-described PDE3B (phosphodiesterase 3B) gene, PDGFC (platelet derived growth factor C) fe, PKD2 (Polycystic kidney disease-2 gene) gene, NRG1 (neuregulin 1) gene and LUM (Lumican) gene are selected.
  • the method for determining the effect of cisbratin administration according to the present invention is not limited to these five genes, and the genes listed in Table 1 Other genes selected from the group may be added as measurement targets.
  • a measurement target gene in addition to the five genes described above, it is more preferable to select another gene selected from the gene group listed in Table 2.
  • the presence or absence of the effect of cisbra and tin administration is determined based on the expression level.
  • the expression level of the gene is evaluated.
  • the expression level may be evaluated by setting a reference value for each gene to be measured and comparing it with this reference value.
  • a relative value with respect to the expression level of the constitutively expressed gene can be set.
  • the expression level of the gene to be measured may be evaluated using an immunohistochemical staining (IHC) score as described above, and a value that can evaluate false positive and false negative may be evaluated as a reference value.
  • IHC immunohistochemical staining
  • the determination method and determination kit for cisplatin administration effect it is possible to determine the presence or the degree of the effect of cisplatin administration for a determination target person with very high accuracy and / or excellent specificity. Can do.
  • the sensitivity means a positive rate in a patient group who succeeds in cisplatin administration.
  • Specificity means the negative rate in the group of patients who are not successful with cisplatin.
  • diagnosis is performed with higher sensitivity and / or higher specificity by increasing the types of genes to be measured (for example, 6 or more genes). be able to.
  • the effect of cisplatin administration can be confirmed by gene expression analysis (mRNA level or protein level) using a biological sample collected from an anticancer agent / cancer patient before starting chemotherapy. Because it can be judged more objectively and specifically, it can prevent the administration of cisbratin, which is an excessive burden on patients who cannot expect the administration effect, and is useful for effective treatment policy for the patient. Knowledge can be provided.
  • a cisbratin resistant strain was established. Specifically, for oral squamous cell carcinoma-derived cell lines (H-1 and Sa-3 strains) provided by the Department of Dentistry and Oral Surgery, Wakayama Medical University School of Medicine, cisplatin (hereinafter sometimes referred to as CDDP) By continuously contacting the CDDP stepwise from a low concentration of 0.1 fg / ml. Thus, a cell line having resistance to cisbratin was established.
  • the cisplatin resistant strain established from the H-1 strain was named H-1R strain
  • the cisplatin resistant strain established from the Sa-3 strain was named Sa-3R strain.
  • CDP treatment was similarly applied to a cell line derived from squamous cell carcinoma of the oropharynx (KB strain) to establish a cisbratin resistant strain.
  • the cisbratin resistant strain established from KB strain was named KBR strain.
  • H-1R strain, Sa-3 strain and KBR strain For these cisplatin resistant strains (H-1R strain, Sa-3 strain and KBR strain), the relative resistance to each parental strain sensitive to cisplatin was examined. Specifically, each resistant strain and each parent strain were treated with CDDP (the concentration of CDDP was 0.05 to: LO g / ml), and then MTT assay was performed. The 50% survival (death) concentration (IC50) was determined for each resistant strain and each parental strain. The relative resistance of H-1 and H-1R strains is about 10 times, the relative resistance of Sa-3 and Sa-3R strains is about 7.5 times, relative to KB and KBR strains Resistance was 8.6 times. These three cell lines did not change their resistance even when they were freeze-thawed or cultured for one month without CDDP.
  • Figure 1 shows the results of MTT assembly for Sa-3 and Sa-3R strains as representative examples.
  • the profile file similar to the profile shown in Fig. ⁇ was shown for other resistant strains and their parent strains.
  • the size of the resistant strain is slightly larger than that of the parent strain, the basic shape, growth rate, and colony forming ability were not significantly changed in the resistant strain and the parent strain. The same properties were maintained.
  • FIG. 2 shows RT-PCR results for Sa-3 and Sa-3R strains as representative examples. From this result, it became clear that each resistant strain established in this example is a cell line genetically resistant to CDP. In the previous reports related to CDDP resistance, the resistance of the resistant strain was about twice, and it was a cell line that possessed only low tolerance that could be regarded as an experimental error range. The resistant strains established in this example had higher drug resistance than the previous cell lines and were considered very useful for analysis.
  • genes that are specifically enhanced in the resistant strain and genes that are specifically attenuated in the resistant strain are identified. Identified. Specifically, for microarray analysis, Affymetrix U133 Plus 2.0 with 54675 probes immobilized was used as the microarray. For microarray analysis, GeneChip Operating Software 1.1 (Affymetrix) and GeneSpring 6.1 (Silicon Genetics) were used as analysis software.
  • the gene group in which the expression of the resistant strain Sa-3R was observed to be 1.5 times higher than that of its parent strain Sa-3, and the parent strain H- We identified a gene group in which expression of 1.5 times or more was observed compared to 1 strain and a gene group in which expression of resistant strain KBR strain was observed to be 1.5 times or more compared to its parental KB strain.
  • a gene group common to these three groups was identified as a gene group (16 4 genes) whose expression was specifically enhanced in cis-bratin resistant strains (see Fig. 3).
  • the gene group in which expression of 5 times or less was observed and the gene group in which the expression of resistant strain KBR was observed 1.5 times or less compared to its parent KB strain were identified.
  • a gene group common to these three groups was identified as a gene group (35 genes) whose expression was specifically attenuated in cisbratin-resistant strains (see Fig. 3).
  • a total of 199 genes could be identified as genes related to cisbratin resistance in cancer cells.
  • GNB1 GNG10
  • GRB2 H2-KA
  • INSR Interaction
  • MAP1B MAP1B, MMP2, MMP9, MMP13, NR3C1 Cellular Movement
  • PAK2 PAK2, PIP5K1B, PLEC1, PRKCA,
  • AKT2, BACH1, BARD1, BRCA1, CEBTPB AKT2, BACH1, BARD1, BRCA1, CEBTPB:
  • HBA1, HBA2, HBB, HAD, HBE1, HBG1 Connective Tissue
  • candidate genes capable of predicting the success rate of anticancer agents by clinical specimens were searched from these 50 genes as a retrospective test.
  • 5 genes PDE3B, PDGFC, PKD2, NRGl and Lumican
  • Anti-human PDE3B goat polyclonal antibody (Santa Cruz) as an antibody against PDE3B, Anti-human PDGFC goat polyclonal antibody (Santa Cruz) as an antibody against PDGFC, Anti-human PKD2 Usagi polyclonal as an antibody against PD2
  • An antibody manufactured by ABGENT
  • an anti-human PKD2 Usagi polyclonal antibody (manufactured by Santa Cruz) as an antibody against NRG1
  • an anti-human Lumican goat polyclonal antibody (manufactured by Santa Cruz) as an antibody against Lumican were prepared. It was confirmed by Western blotting using the prepared antibody that the expression of these genes was enhanced in the resistant strain at the protein level.
  • FIGS. 5 (A) and (B) The results of RT-PCR and Western plotting performed on the lumican gene among the five genes described above are shown as representative examples in FIGS. 5 (A) and (B). As shown in Fig. 5 (A) and (B), it can be seen that the lumican gene is specifically expressed in the resistant strain compared to the parent strain. Similar results were obtained for other genes except the Lumican gene. ,
  • the IHC score was used for the evaluation of staining in the immunohistological staining method.
  • the IHC score is observed with an objective lens of 40 times, and the percentage of positive cells out of the total number of cells in one field and the intensity of staining (for example, 3 levels (weak, medium and strong) are evaluated. 1-3 Is the sum of the numbers multiplied by. This is done with 10 randomly selected visual fields, and the IHC score is calculated as the average value.
  • the IHC score is a method of judging the degree of staining that is generally used at present as an excellent score indicating staining intensity and staining range.
  • Figure 6 shows a representative example of the results of immunohistological staining of clinical samples for the luraican gene. Increased expression of lumi can protein was confirmed in clinical specimens resistant to cisplatin.
  • IHC scores were obtained using 10 NC cases, 22 PR cases, and 16 CR cases for cisplatin administration, and the distribution of IHC scores was obtained for each of the five genes described above.
  • Figure 7 shows the result of the IHC score distribution for the PDE3E gene
  • Figure 8 shows the result of the IHC score distribution for the lumican gene
  • Figure 9 shows the result of the IHC score distribution for the PDGFC gene.
  • the results of obtaining the IHC score distribution for the PKD2 gene are shown in FIG. 10
  • the results of obtaining the IHC score distribution for the NRG1 gene are shown in FIG.
  • Table 5 summarizes the presence or absence of the expression and treatment effect assessment for the above five genes.
  • “10” indicates that the expression at the protein level was positive for each gene, and “one” indicates that the expression at the protein level was negative for each gene.
  • the presence or absence of a response due to administration of cisplatin can be determined with very good accuracy. Therefore, the method for determining the cisplatin administration effect and the kit for determining the cisplatin administration effect according to the present invention can determine whether or not a treatment including cisplatin administration is applied to the subject patient.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Wood Science & Technology (AREA)
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  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

Selon l'invention, on détermine avec une exactitude et une spécificité élevées si une administration de cisplatine présente ou non un effet thérapeutique. L'invention concerne un procédé de détermination d'un effet d'une administration de cisplatine qui comprend une étape (a) dans laquelle l'expression d'au moins un gène choisi dans le groupe comprenant le gène PDE3B (phosphodiestérase 3B), le gène PDGFC (Platelet Derived Growth Factor C ; facteur de croissance dérivé des plaquettes C), le gène PKD2 (Polycystic Kidney Disease 2 ; maladie polykystique des reins), le gène NRG1 (neuréguline 1) et le gène LUM (lumican) est mesurée dans un échantillon biologique collecté chez un sujet à diagnostiquer, et une étape (b) dans laquelle un effet d'une administration de cisplatine est déterminé en fonction du taux d'expression du gène obtenu en tant que résultat du mesurage.
PCT/JP2007/070864 2006-10-20 2007-10-19 Procédé de détermination de l'effet d'une administration de cisplatine et kit permettant de déterminer l'effet d'une administration de cisplatine WO2008047947A1 (fr)

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JP2008539900A JPWO2008047947A1 (ja) 2006-10-20 2007-10-19 シスプラチン投与効果判定方法及びシスプラチン投与効果判定キット

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JP2006-286285 2006-10-20
JP2006286285 2006-10-20

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009242378A (ja) * 2008-03-13 2009-10-22 Chiba Univ シスプラチン効果増強剤及び抗癌剤キット
JP2014526680A (ja) * 2011-09-12 2014-10-06 ユニヴェルシテイト ヘント 結腸直腸ガンのニューレグリン−１に基づく予後予測及び治療層別化

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Title
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NAKAMURA M. ET AL.: "46. cDNA Microarray o Mochiita Koganzai Cisplatin (CDDP) no Kanjusei ni Kan'yo suru Idenshigun no Kensaku", CHIBA MEDICAL JOURNAL, vol. 80, no. 2, 2004, pages 88, XP003022228 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009242378A (ja) * 2008-03-13 2009-10-22 Chiba Univ シスプラチン効果増強剤及び抗癌剤キット
JP2014526680A (ja) * 2011-09-12 2014-10-06 ユニヴェルシテイト ヘント 結腸直腸ガンのニューレグリン−１に基づく予後予測及び治療層別化

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