WO2013151026A1 - Marqueur diagnostique et méthode diagnostique pour identifier les cellules cancéreuses résistant à l'inhibiteur pi3k - Google Patents

Marqueur diagnostique et méthode diagnostique pour identifier les cellules cancéreuses résistant à l'inhibiteur pi3k Download PDF

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WO2013151026A1
WO2013151026A1 PCT/JP2013/060027 JP2013060027W WO2013151026A1 WO 2013151026 A1 WO2013151026 A1 WO 2013151026A1 JP 2013060027 W JP2013060027 W JP 2013060027W WO 2013151026 A1 WO2013151026 A1 WO 2013151026A1
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igf1r
cancer
expression level
gene
pi3k inhibitor
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矢守　隆夫
慎吾 旦
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公益財団法人がん研究会
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    • 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
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • 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
    • G01N2333/575—Hormones
    • G01N2333/65—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Definitions

  • the present invention relates to a diagnostic marker and a diagnostic method for determining whether or not a cancer cell is resistant to a PI3K inhibitor.
  • Phosphatidylinositol 3-kinase are enzymes that phosphorylate the 3-position of the inositol phospholipids to localize in biological membranes, survival of carcinogenesis and cancer growth, play an important role for cancer, such as metastatic Therefore, it is considered as a powerful target for cancer treatment.
  • the PI3K pathway is activated, and inhibition of PI3K can suppress cancer growth (Non-patent Documents 1 and 2, etc.).
  • an antitumor agent comprising such a PI3K inhibitor has been developed (Patent Document 1, etc.).
  • IGF1R insulin-like growth factor 1 receptor
  • PI3K activity was correlated with the activation and positive IGF1R, activation of the PI3K pathway activity of IGF1R It is reported that activation of IGF1R indicates activation of the PI3K pathway (Patent Document 2). Furthermore, it is known that an inhibitor of IGF1R is an antitumor agent (Non-patent Document 3).
  • one anticancer drug may not work for all cancers, and may or may not work depending on the cancer patient.
  • the effect of the drug cannot be predicted at the time of prescribing the drug, that is, before the drug is administered, and the effect is not known until the drug is used.
  • molecular target anticancer drugs such as PI3K inhibitors target target molecules with cancer abnormalities. It is considered.
  • an object of the present application is to provide a diagnostic marker and a diagnostic method for determining whether a cancer cell is resistant (treatment resistance) to a PI3K inhibitor.
  • an antitumor agent comprising a PI3K inhibitor Before prescribing an antitumor agent comprising a PI3K inhibitor to a patient by such a diagnostic marker and method, it is predicted in advance whether the drug will work, or an antitumor agent comprising a PI3K inhibitor is administered. It can be determined whether the patient's cancer has acquired drug resistance.
  • the present inventors produced anti-cancer drug-resistant cells comprising PI3K inhibitor (ZSTK474), compared gene expression of the resistant strain and the parent strain with GeneChip, and found the gene overexpressed in the resistant strain. Identified as drug resistance marker candidates.
  • the gene was screened with siRNA to identify a gene (IGF1R gene) that is functionally involved in PI3K inhibitor resistance (data not shown).
  • the expression of this IGF1R gene is commonly increased among the four acquired resistant strains (Example 1 described later), and it is found that PI3K inhibitors are not effective for cancer cells with high expression of the IGF1R gene. It was.
  • the present invention relates to a diagnostic marker for determining whether a cancer patient's cancer is resistant to a PI3K inhibitor, the diagnostic marker comprising IGF1R mRNA, cDNA or protein, It is a diagnostic marker determined by the expression level of the IGF1R gene in cancer cells or cancer tissues collected from patients. Furthermore, the present invention is a diagnostic method for determining whether or not a cancer of a cancer patient is resistant to a PI3K inhibitor when treating the cancer patient with a PI3K inhibitor, This is a diagnostic method for determination based on the expression level of the IGF1R gene in cancer cells or cancer tissues collected from patients. This diagnostic method may consist of the following steps.
  • a step of creating a tissue section after fixing a piece (xenograft) with formalin and embedding in paraffin (ii) a process of preparing a control tissue section by treating a tumor sample as a control in the same manner as in (i), (iii) a step of deparaffinizing the tissue section prepared in (i) and (ii), treating with a primary antibody specific for the IGF1R protein, staining with a secondary antibody, and quantifying the staining intensity; iv) By comparing the staining intensity obtained from the tumor sample of the cancer patient with the staining intensity obtained from the control tumor sample, it is determined whether or not the cancer of the cancer patient is resistant to the PI3K inhibitor. Judgment process
  • the vertical axis represents GI50 (drug concentration (M) required to suppress the growth of cancer cells by 50%), and the horizontal axis represents the expression level of the IGF1R gene (relative value, Example 2, FIG. 2). Show.
  • the cell line marked with ⁇ indicates the reference cell line. It is a graph which shows the relationship between the tolerance with respect to PI3K inhibitor (NVP-BEZ235) of 39 types of cancer cells untreated with PI3K inhibitor and the expression level of IGF1R protein.
  • the vertical axis represents GI50 (M), and the horizontal axis represents the expression level of the IGF1R gene (relative value, Example 2, FIG. 2).
  • the cell line marked with ⁇ indicates the reference cell line.
  • the vertical axis represents GI50 (M), and the horizontal axis represents the expression level of the IGF1R gene (relative value, Example 2, FIG. 2).
  • the cell line marked with ⁇ indicates the reference cell line.
  • xenograft tumor pieces tumor pieces obtained by transplanting human-derived cancer cells subcutaneously into nude mice
  • xenograft tumor pieces tumor pieces obtained by transplanting human-derived cancer cells subcutaneously into nude mice
  • the vertical axis represents T / C (size of tumor in the drug-treated group (Treated) / size of tumor in the non-drug-treated group (Control)) (%), and the horizontal axis represents the expression of IGF1R protein in the transplanted tumor piece. Indicates the amount.
  • the cell line marked with ⁇ indicates the reference cell line.
  • PI3K inhibitors can suppress the growth of various cancers (Non-patent Document 1, etc.), but there is a significant difference in treatment responsiveness to PI3K inhibitors in both PIK3CA and PTEN abnormal and normal cancers. (Non-patent Document 2), PIK3CA and PTEN gene abnormalities do not serve as diagnostic markers for treatment responsiveness of PI3K inhibitors.
  • the PI3K inhibitor may or may not be effective, and even a cancer cell to which the PI3K inhibitor is effective may acquire drug resistance upon administration.
  • the diagnostic marker and diagnostic method of the present invention determine whether or not such cancer cells are resistant (both acquired resistance and natural resistance) to a PI3K inhibitor. Therefore, in cancer treatment with PI3K inhibitors, PI3K inhibition is achieved for patients with low IGF1R gene expression by measuring the expression level of IGF1R gene in cancer patients using the diagnostic marker and diagnosis method of the present invention. It is effective to administer the agent.
  • PI3K inhibitor examples include the following compounds. These compounds have been confirmed to inhibit PI3K, while it has also been confirmed that inhibition of PI3K suppresses the growth of cancer cells (Non-patent Document 1, etc.).
  • the cancer to which the diagnostic marker and the diagnostic method of the present invention are applied is not particularly limited.
  • lung cancer, colon cancer, stomach cancer, breast cancer, ovarian cancer, brain tumor, kidney cancer examples include prostate cancer, liver cancer, pancreatic cancer, esophageal cancer, mesothelioma, and melanoma.
  • cancer cells or tissues are collected from a subject (cancer patient).
  • Tissue cells (cancer cells) at the target site of the subject may be collected by biopsy or the like, or if necessary, only cancer cells may be accurately collected by laser microdissection.
  • the test subject's tumor tissue may be extract
  • the expression level of the IGF1R gene in cancer cells is used as an index in order to determine whether the cancer is resistant (both acquired resistance and natural resistance) to the PI3K inhibitor.
  • Human IGF1R gene the base sequence of SEQ ID NO: 1 (GenBank Accession NM_000875, NM_015883, its coding region is from 51 to 4154 th of the base sequence.), Or, a base sequence encoding the amino acid sequence of the following human IGF1R protein .
  • Human IGF1R protein consists of the amino acid sequence of SEQ ID NO: 2 (GenBank Accession AAI43722).
  • the cancer of the cancer patient is diagnosed as resistant to the PI3K inhibitor, and if the expression level is low, the cancer of the cancer patient Is not resistant to PI3K inhibitors.
  • it is effective to administer a PI3K inhibitor to a patient with a low expression level of the IGF1R gene.
  • PI3K inhibitors are considered appropriate for cancers that do not show any expression of IGF1R, but for patients with other IGF1R expression to some extent, an appropriate threshold is set, depending on the patient's circumstances, etc. It can be said that it should be judged.
  • this IGF1R gene may be measured as an absolute amount per cell or as a relative amount. This relative amount may be determined using the expression level of the reference gene or 18S ribosomal RNA.
  • a gene that is expected to be expressed to the same extent in cancer cells resistant and non-resistant to PI3K inhibitors such as ⁇ -actin (GenBank Ac No. X00351) and GAPDH (NM_002046) May be used.
  • the expression level of IGF1R gene in cancer cells or cancer tissues collected from cancer patients Compared with the expression level of the IGF1R gene in these reference cell samples, the resistance of the patient to the PI3K inhibitor may be determined.
  • the expression level of the IGF1R gene is not more than a predetermined constant value, it is determined that the patient's cancer is not resistant to the PI3K inhibitor, and in other cases or a predetermined constant value If larger, the patient's cancer may be determined to be resistant to the PI3K inhibitor.
  • Such a reference cell sample may be selected from 39 types of human cancer cell lines (Table 3) examined for the IGF1R gene in Example 2.
  • Table 3 39 types of human cancer cell lines examined for the IGF1R gene in Example 2.
  • the IGF1R expression level in Table 3 is 1.5 or higher
  • the IGF1R expression level in Table 3 is 0.5 or lower. May be.
  • NCI-H23 cells ATCC No. CRL
  • PC-3 cells ATCC No. CRL-1435 are preferably used as a reference cell sample exhibiting a low expression level of IGF1R gene.
  • the expression level of the IGF1R gene in the specimen may be evaluated as a relative value to the expression level. This diagnosis can be performed, for example, by calculating the following ratio X (%).
  • A is the expression level of the IGF1R gene in the specimen
  • B is the expression level of the IGF1R gene of the reference cell sample showing a high expression level of the IGF1R gene
  • C is the IGF1R gene of the reference cell sample showing a low expression level of the IGF1R gene. Represents the expression level of.
  • X is, for example, 20% or less, or 10% or less, it is determined that the cancer of the sample is not resistant to the PI3K inhibitor (therapeutic compatibility with the PI3K inhibitor), and If X is otherwise, or higher than 50% or higher than 80%, the specimen's cancer is resistant to a PI3K inhibitor (therapeutic resistance that is not therapeutically compatible with a PI3K inhibitor, May be determined).
  • resistance to PI3K inhibitors can be obtained only by measuring the expression level of the IGF1R gene in a specimen without measuring such a standard for each test. It is also possible to determine to a certain extent whether or not (the presence or absence of therapeutic suitability).
  • Quantification of IGF1R gene expression can be performed by measuring the expression level of IGF1R mRNA or cDNA or the expression level of IGF1R protein.
  • the expression level of IGF1R mRNA or cDNA is measured, for example, as follows: Methods for quantifying mRNA expression include, for example, oligonucleotides consisting of IGF1R mRNA or its cDNA base sequence or a part of their complementary base sequences, which bind site-specifically to IGF1R mRNA or cDNA. And a method using a primer or probe containing an oligonucleotide to be used.
  • the primer and probe may be variously modified for detecting and quantifying mRNA as long as the oligonucleotide forms a site-specific base pair with IGF1R mRNA or its cDNA.
  • Extraction of total RNA and mRNA from tissue cells (cancer cells) at the target site can be performed based on known methods.
  • Reagent kits for extracting total RNA are commercially available from various manufacturers. For example, RNeasy mini kit (Qiagen) or TRIzol (Invitrogen) can be used.
  • any gene expression quantification method such as RT-PCR method, Northern blot method, DNA chip (manufactured by Affymetrix, etc.) can be used.
  • a real-time quantitative PCR method which is one of RT-PCR methods, is preferable in that a very small amount of DNA can be detected with high sensitivity.
  • mRNA of the IGF1R gene is reverse transcribed to produce first strand cDNA, which is used as a template for PCR amplification with primers specific to the gene.
  • Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) can be used.
  • the real-time quantitative PCR method for example, TaqMan (registered trademark) Gene Expression Assays provided by Applied Biosystems can be used.
  • Amplified products can be separated and quantified by electrophoresis or the like, but it is preferable that the quantification is performed accurately and conveniently using a real-time quantitative PCR instrument such as Applied Biosystems ABI PRISM 7000 Sequence Detection System.
  • IGF1R mRNA can be quantified using various measurement methods (DNA array, Northern blot, ATAC-PCR method, etc.).
  • a primer for PCR a fragment consisting of about 10 to 30 base sequences sandwiching at least 50 bases, preferably 100 to 1,000 bases of a polynucleotide consisting of the base sequence of SEQ ID NO: 1 is usually used. .
  • a fragment consisting of at least 15 consecutive nucleotide sequences of a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 is usually used.
  • the probe base sequence is usually 15-30 bases, preferably 20-25 bases.
  • the expression level of IGF1R protein is measured, for example, as follows: Examples of the method for quantifying IGF1R protein include a method using an antibody specific for IGF1R protein, and specifically, Western blotting, dot blotting, slot blotting, ELISA, and RIA. Can be detected. Moreover, the expression level can also be quantified by performing an immunohistochemical method. As the ELISA / RIA sample, for example, the collected cell extract or serum is used as it is, or an appropriately diluted buffer is used.
  • the cell extract is used as it is or diluted appropriately with a buffer, and a sample buffer (Sigma) containing 2-mercaptoethanol for SDS-polyacrylamide gel electrophoresis. Used in combination with other products.
  • a sample buffer Sigma
  • 2-mercaptoethanol for SDS-polyacrylamide gel electrophoresis Used in combination with other products.
  • a sample buffer for example, a collected cell extract or serum itself, or a solution diluted appropriately with a buffer solution, which is directly adsorbed on a membrane using a blotting apparatus or the like is used.
  • antibodies reactive with IGF1R protein e.g., Cell Signaling Technology, Inc.
  • IGF-I Receptor ⁇ (111A9) Rabbit mAb and SantaCruz's IGF1R ⁇ (C20) may be a commercially available antibody of (sc-713) or the like, Moreover, you may use and make an appropriate antibody.
  • the antibody is directly labeled, or the antibody is used as a primary antibody for detection in cooperation with a labeled secondary antibody that specifically recognizes the primary antibody (recognizes an antibody derived from the animal from which the antibody was produced). It is done.
  • This label is preferably an enzyme (alkaline phosphatase or horseradish peroxidase, etc.), a fluorescent dye (Alexa680, IRDye800, etc.), or biotin (however, an operation for further binding an enzyme-labeled streptavidin to the biotin of the secondary antibody) Is mentioned.
  • Various pre-labeled antibodies (or streptavidin) are commercially available as labeled secondary antibodies (or labeled streptavidin).
  • RIA an antibody labeled with a radioisotope such as 125 I is used, and the measurement is performed using a liquid scintillation counter or the like.
  • the expression level of the antigen is quantified by measuring the activity of these labeled enzymes or the fluorescence intensity of the labeled fluorescent dye excited by laser light.
  • the following immunohistochemical method is effective.
  • IHC method a tumor sample of a subject is collected, and after formalin fixation and paraffin embedding, a tissue section is prepared. For those obtained cell samples, they are transplanted subcutaneously into nude mice, and tumor pieces (xenografts) are taken out and used. This tumor section is deparaffinized, then treated with an appropriate primary antibody specific for IGF1R protein, stained with an appropriate secondary antibody, and the staining intensity is quantified.
  • the antibody that reacts with the IGF1R protein the above-mentioned antibodies can be used.
  • the stained section may be converted into a digital image using an Aperio Technologies Scanscope XT slide scanner or the like, and the staining intensity may be quantified using the company's Spectrum software or the like.
  • Various tumor pieces may be prepared as a tissue microarray, and multiple samples may be measured simultaneously on one slide, or may be measured on separate slides. In that case, it is necessary to perform the staining step under the same conditions as the control sample to be compared.
  • the treatment resistance of the PI3K inhibitor can be determined from the expression of the IGF1R protein in the obtained cancer derived from the subject. In addition, the treatment resistance of the PI3K inhibitor may be determined by comparing with the expression of control cells.
  • the kit used for quantifying the expression level of IGF1R mRNA or cDNA using the diagnostic marker of the present invention comprises a primer for amplifying cDNA of IGF1R gene and a heat-resistant DNA polymerase (such as Taq polymerase), and a detection Therefore, it comprises a probe to be paired with the amplification product.
  • a primer for amplifying cDNA of IGF1R gene and a heat-resistant DNA polymerase (such as Taq polymerase), and a detection Therefore, it comprises a probe to be paired with the amplification product.
  • a heat-resistant DNA polymerase such as Taq polymerase
  • the kit used for quantifying the expression level of the IGF1R protein using the diagnostic marker of the present invention includes, for example, a primary antibody specific for the IGF1R protein, a specific antibody specific to the primary antibody, and an appropriate amount. It consists of a secondary antibody labeled with an enzyme or chemical substance. Examples of other consumable reagents that may be included in this kit include enzymes, buffers, reaction reagents, and the like necessary for quantifying proteins.
  • Example 1 prepared PI3K inhibitor (ZSTK474) resistant cancer cells was investigated the relationship between the development of tolerance and IGF1R gene for PI3K inhibitors in cancer cells that have acquired PI3K inhibitor-resistant.
  • ZSTK474 PI3K inhibitor
  • ZSTK474 resistant cancer cell lines are referred to as SF295R, SNB-75R, SNB-78R, and OVCAR3R, respectively.
  • these cells are seeded into a 96-well plate, and the next day, 5 types of ZSTK474 of 10 -8 M, 10 -7 M, 10 -6 M, 10 -5 M and 10 -4 M are added thereto. After further incubation for 48 hours, the cells were fixed with trichloroacetic acid. Cell proliferation was measured by the sulfurhodamine B (SRB) assay (J Natl Cancer Inst. 1990 Jul 4; 82 (13): 1107-12.) And GI50 (drug concentration required to inhibit cell proliferation by 50%) Calculated. The results are shown in the table below.
  • Cancer cells that acquired resistance to PI3K inhibitors were each 12-120 times more resistant than the parent strain (drug-untreated).
  • RNeasy mini kit manufactured by Qiagen
  • the expression level of 18S ribosomal RNA was simultaneously measured, and the expression level of IGF1R in each sample was normalized with the expression level of 18S ribosomal RNA. Furthermore, a value obtained by normalizing the expression level of IGF1R in each resistant strain sample with the expression level of IGF1R in the SF295 parent strain obtained here was defined as the IGF1R expression level (relative value).
  • Probe for IGF1R 5'-FAM- CCATCTTCGTGCCCAGACCTGAAAG-TAMRA-3 ' (* FAM represents 6-carboxyfluorescein, and TAMRA represents carboxytetramethylrhodamine. The base sequence of this probe corresponds to positions 2236 to 2260 of SEQ ID NO: 1.) Reaction conditions: 95 ° C 10 minutes 1 cycle 95 ° C 15 seconds 60 ° C 1 minute 40 cycles
  • Lysis buffer (10 mmol / L Tris-HCl (PH 7.4), 50 mmol / L NaCl, 0.5% w / v NP40, 0.1% w / v SDS, 50 mmol / L Sodium fluoride, 30 mmol / L sodium pyrophosphate, 50 mmol / L sodium orthavanadate, 5 mmol / L EDTA, 0.1 trypsin inhibitor unit / ml aprotinin, 1 mmol / L phenylmethylsulfonyl fluoride) and suspend the ice using Diagenode Bioruptor. Cells were disrupted by medium sonication.
  • the protein concentration of the cell extract was quantified using a protein assay kit manufactured by Pierce, and the cell extract corresponding to 10 ⁇ g protein was developed by SDS polyacrylamide gel electrophoresis (SDS-PAGE).
  • SDS-PAGE SDS polyacrylamide gel electrophoresis
  • the resistance to PI3K inhibitor (acquired resistance) is related to the expression level of IGF1R, and a significant relationship was found that the higher the expression level of the IGF1R gene, the more resistant the PI3K inhibitor is. .
  • Example 2 the relationship between ZSTK474 resistance (natural resistance) and IGF1R gene expression in cancer cells not treated with a PI3K inhibitor was examined.
  • 39 types of human cancer cell lines were prepared as non-treated PI3K inhibitor cancer cells, cell extracts were prepared from each cancer cell line in the same manner as in Example 1, and GI50 was measured.
  • the expression level of IGF1R in each cell extract a primary antibody (SantaCruz's IGF1R ⁇ (C20) (sc-713)), using a secondary antibody labeled with Alexa488 fluorescent dye, a Licor's Odyssey Measured and quantified.
  • Odyssey supplied excitation light to the fluorescently labeled sample and detected the fluorescence emitted from the fluorescent dye.
  • the fluorescence intensity of the IGF1R band in each detected sample was quantified from an image obtained by scanning the membrane labeled with Alexa488 fluorescent dye with IGF1R expression by Western blotting using Odyssey.
  • the final quantification of the expression level of IGF1R in each sample is the intermediate value of the quantification values obtained by three independent experiments. Value (relative value).
  • the results are shown in the table below and FIG. In the table, * indicates a reference cell line. This table also shows that the PI3K inhibitor may or may not work depending on the cancer cells of each patient, not the type of cancer cells.
  • X (%) (expression level of IGF1R gene in specimen ⁇ expression level of IGF1R gene in PC-3 cell) / (expression level of IGF1R gene in NCI-H23 cell ⁇ PC-3 cell) Expression level of IGF1R gene) ⁇ 100.
  • PI3K inhibitors NVP-BEZ235 and LY294002 instead of the PI3K inhibitor ZSTK474.
  • GI50 resistance to PI3K inhibitors
  • FIGS The relationship between their resistance to PI3K inhibitors (GI50) and the expression level of the IGF1R gene is shown in FIGS. From these figures, it can be seen that the higher the expression of the IGF1R gene, the less effective the PI3K inhibitor is (that is, the resistance to the PI3K inhibitor).
  • cell lines with an IGF1R expression level of X of 20% or less, particularly 10% or less have a low GI50 and a low resistance to PI3K inhibitors.
  • Example 3 the expression of IGF1R protein in an in vivo tumor sample was detected using an immunohistochemical method (IHC method), and the relationship with ZSTK474 resistance (antitumor effect) in vivo was examined.
  • IHC method immunohistochemical method
  • ZSTK474 resistance antigen-effect in vivo was examined.
  • Such an in vivo test can be said to be a condition closer to a clinical sample than an analysis result (Example 2) using human cancer cells cultured in a test tube.
  • TMA tissue microarray
  • the results are shown in the following table and FIG. In the table, * indicates a reference cell line.
  • the cell line having an IGF1R expression level of X of 20% or less, particularly 10% or less has a low T / C and a particularly low resistance to a PI3K inhibitor.

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Abstract

L'objectif de cette invention est de pourvoir à un marqueur diagnostique et à une méthode diagnostique permettant de déterminer si le cancer chez un patient atteint de cancer est résistant ou non à l'inhibiteur PI3K quand ledit patient doit être traité avec l'inhibiteur PI3K. Pour ce faire, la présente invention se base sur le fait qu'il existe une relation selon laquelle la quantité d'expression du gène IGF1R est supérieure dans une cellule cancéreuse présentant une résistance à un inhibiteur PI3K et est inférieure dans une cellule cancéreuse non résistante à l'inhibiteur PI3K (le terme "résistance" incluant à la fois la résistance acquise et la résistance naturelle dans le cadre de la présente). Le marqueur diagnostique comprend l'ARNm ou l'ADNc du gène IGF1R ou une protéine IGF1R. La quantité d'expression du gène IGF1R dans une cellule ou un tissu cancéreux prélevé sur le patient atteint de cancer est utilisée à titre de mesure. Quand la quantité de l'expression est basse, le cancer chez le patient est considéré comme n'étant pas résistant à l'inhibiteur PI3K. D'après le résultat de ce diagnostic, il s'avère que l'administration d'un inhibiteur PI3K est efficace pour un patient chez qui la quantité d'expression du gène IGF1R est basse.
PCT/JP2013/060027 2012-04-04 2013-04-02 Marqueur diagnostique et méthode diagnostique pour identifier les cellules cancéreuses résistant à l'inhibiteur pi3k WO2013151026A1 (fr)

Applications Claiming Priority (2)

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JP2012085456 2012-04-04
JP2012-085456 2012-04-04

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JP (1) JPWO2013151026A1 (fr)
WO (1) WO2013151026A1 (fr)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHAKRABARTY, A. ET AL.: "Feedback upregulation of HER3 (ErbB3) expression and activity attenuates antitumor effect of PI3K inhibitors", P.N.A.S., vol. 109, no. 8, 21 February 2012 (2012-02-21), pages 2718 - 2723 *
DAN, S. ET AL.: "Identification of IGF1R as a Predictive Biomarker for Intrinsic Resistance to PI3K Inhibitors and a Therapeutic Target for Improving the Drug Efficacy", EUROPEAN JOURNAL OF CANCER, vol. 8, no. SUPPL., November 2012 (2012-11-01), pages 113, 370 *
ISOYAMA, S. ET AL.: "Establishment of phosphatidylinositol 3-kinase inhibitor- resistant cancer cell lines and therapeutic strategies for overcoming the resistance", CANCER SCIENCE, vol. 103, no. 11, 22 October 2012 (2012-10-22), pages 1955 - 1960 *
MURANEN, T. ET AL.: "Inhibition of PI3K/mTOR Leads to Adaptive Resistance in Matrix-Attached Cancer Cells", CANCER CELL, vol. 21, no. 2, 14 February 2012 (2012-02-14), pages 227 - 239 *

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