WO2010100899A1 - Procédé de test génétique pour le cancer par analyse de l'expression d'un gène lié au cancer à l'aide de monocytes contenus dans un échantillon de sang - Google Patents

Procédé de test génétique pour le cancer par analyse de l'expression d'un gène lié au cancer à l'aide de monocytes contenus dans un échantillon de sang Download PDF

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WO2010100899A1
WO2010100899A1 PCT/JP2010/001417 JP2010001417W WO2010100899A1 WO 2010100899 A1 WO2010100899 A1 WO 2010100899A1 JP 2010001417 W JP2010001417 W JP 2010001417W WO 2010100899 A1 WO2010100899 A1 WO 2010100899A1
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cancer
protein
mage
receptor
cytokeratin
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藤崎浩治
川崎広明
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株式会社ジーンサイエンス
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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
    • 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

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  • the present invention relates to a cancer genetic testing method, and more particularly to a cancer genetic testing method based on an expression analysis of a cancer-related gene obtained from a mononuclear cell contained in a blood sample.
  • Cancer is one of the major lifestyle-related diseases and is the leading cause of death in Japan. There are cancers that develop and progress due to environmental factors and cancers that develop and develop due to genetic factors, and the onset factors and progression processes are diverse. In cancers that develop and progress due to genetic factors, the multistage carcinogenic mechanism of colorectal cancer is well known. Colorectal cancer progresses from a polyp (adenoma) state as a precancerous lesion to malignant colorectal cancer in multiple stages.
  • This multi-stage cancer is first mutated in the RAS gene, which is one of the oncogenes, after the APC gene, a gene responsible for a dominant genetic disease called familial adenomatous adenomatosis, is inactivated and a polyp is generated.
  • the cell proliferation action is activated, and then the p53 tumor suppressor gene is inactivated and progresses to malignant colon cancer (Non-patent Document 1).
  • Such abnormal activation (overexpression) of oncogenes and inactivation (decrease in expression) of tumor suppressor genes that would normally suppress cell carcinogenesis are observed in the process of cell carcinogenesis. This is why cancer is said to be a genetic disease caused by accumulation of gene mutations, amplifications and deletions.
  • diagnostic imaging methods such as MRI (magnetic resonance imaging), PET (positron tomography), SPECT (single photon emission tomography), and X-ray CT (X-ray computed tomography) Is frequently used.
  • These diagnostic imaging methods are basically methods for detecting cancer by finding the difference between normal tissue and diseased tissue, and can be said to be highly accurate cancer testing methods because the diseased tissue can be visually recognized.
  • a method for examining a cancer antigen (tumor marker) released in a blood sample using, for example, ELISA (enzyme immunoassay) is simple and widely used.
  • the detection level capable of image diagnosis in the above-described image diagnosis method is a cancer cell population that has developed to a size of 5 mm or more, and cancer cells that have reached this level are deteriorated levels associated with invasion and angiogenesis. .
  • the concentration of the target protein as a tumor marker in the blood sample needs to be high, and it is difficult to say that the test method is generally highly sensitive.
  • an inspection method capable of detecting a cancer cell population at a detection level (5 mm or less) impossible with image diagnostic methods is desired.
  • cancer is accompanied by various gene abnormalities, it is considered that early detection and early treatment of cancer can be achieved by detecting these gene abnormalities.
  • the present invention has been made in view of the above circumstances, and an object of the present invention is to provide a genetic test method capable of predicting the existence risk of a cancer cell population and a therapeutic effect with high accuracy and early or prognosis. Is to provide.
  • the cancer genetic testing method includes a step of separating mononuclear cells from a blood sample collected from a subject, a step of extracting total RNA from the separated mononuclear cells, and The method includes a step of synthesizing complementary DNA from total RNA and a step of performing expression analysis on a cancer-related gene in the synthesized complementary DNA.
  • Mononuclear cells existing in the blood are classified into migrating mononuclear leukocytes represented by lymphocytes and monocytes and mononuclear phagocytic cell groups represented by macrophages.
  • Lymphocytes are mainly classified into B cells, T cells, and NK (natural killer) cells.
  • B cells produce antibodies that bind to various antigens and attack pathogens that have entered from the outside.
  • T cells are further classified into helper T cells that assist B cell antibody production, conversely suppressor T cells that suppress B cell antibody production, killer T cells that attach to pathogens and destroy them directly.
  • NK cells are lymphocytes having a tumor lysis function.
  • Monocytes do not contain special granules such as those found in granulocytes, but they have phagocytic ability and exhibit a variety of functions in biological defense reactions. Macrophages are thought to have been converted monocytes generated in the bone marrow and are also called phagocytic cells. Macrophages are strong in phagocytic digestion (phagocytosis) of foreign substances such as bacteria and cells that have undergone apoptosis, and are one of the main roles in immune function.
  • phagocytic digestion phagocytosis
  • macrophages fragment the foreign matter taken up by phagocytosis and present the antigen by binding this fragment to MHC-II on the cell surface. Then, antigen presentation by macrophages activates helper T cells and produces cytokines such as interleukins and lymphokines. Interleukins and cytokines produced by helper T cells activate macrophages and activate B cells that recognize the same antigen as the antigen presented. Activated B cells differentiate into antibody-producing cells and proliferate to produce antibodies against the antigen. The antibody produced by the antibody producing cell binds to the antigen to form an antibody-antigen complex. In this way, the foreign substance to which the antibody is bound is easily recognized by macrophages, and thus is efficiently phagocytosed.
  • cancer cells that have induced apoptosis or necrosis are efficiently captured by the immune function maintained by the mononuclear cells. Therefore, in the present invention, since expression analysis of cancer-related genes based on total RNA of mononuclear cells isolated from a blood sample collected from a subject is performed, it is possible to predict the existence risk and therapeutic effect of a cancer cell population. It becomes possible.
  • the mononuclear cells targeted by the cancer genetic testing method of the present invention are monocytes and / or macrophages.
  • Monocytes and macrophages have phagocytic ability as described above.
  • macrophages are phagocytosed by cancer cells that have been damaged by the above-mentioned NK cells or killer T cells and induced apoptosis.
  • NK cells or killer T cells apoptosis
  • macrophages themselves may damage cancer cells, thereby inducing apoptosis and phagocytosing the cancer cells.
  • cancer cells that have induced apoptosis and necrosis are eventually phagocytosed by macrophages. Cells can be obtained efficiently.
  • the expression analysis in the cancer genetic testing method of the present invention is characterized in that it is a real-time polymerase chain reaction (RT-PCR) analysis. Furthermore, the expression analysis in the cancer genetic testing method according to the present invention is a nucleic acid microarray analysis.
  • RT-PCR real-time polymerase chain reaction
  • a real-time-PCR (RT-PCR) analysis or a nucleic acid microarray analysis is performed for the expression analysis of cancer-related genes.
  • RT-PCR real-time-PCR
  • the partial nucleic acid sequence of a cancer-related gene used as a primer or the partial nucleic acid sequence of a cancer-related gene used as a probe in a nucleic acid microarray analysis It can be designed and synthesized accordingly. Therefore, it is possible to perform an accurate test according to the cancer to be tested, and it is possible to perform an expression analysis with high reproducibility and high sensitivity.
  • the cancer-related genes targeted by the cancer genetic testing method of the present invention include head and neck cancer, pancreatic cancer, thyroid cancer, biliary tract cancer, lung cancer, non-small cell lung cancer, kidney cancer, stomach cancer, liver cancer, colon cancer, rectum Selected from the group consisting of cancer, transitional cell carcinoma, Merkel cell carcinoma, breast cancer, uterine cancer, ovarian cancer, leukemia, esophageal cancer, skin cancer, bladder cancer, prostate cancer, brain tumor, osteosarcoma, and neuroblastoma It is a related gene.
  • the cancer-related genes targeted by the cancer genetic testing method of the present invention include bcl-2, CA125, CD44, c-kit, c-met, c-myc, COX-2, Cyclin D1, Cytokeratin-7, Cytokeratin-19, Cytokeratin-20, E2F1, E2F3, FGFR2, Gli1, GPC3, hCGbeta, Her2 / Neu, HLF-1a, HnRNP A2 / B1, hTERT, L-myc, MAGEA4, MAGEA4, MAGEMP4 -2, MMP-9, Muc-1, Muc-4, NSE, RCAS1, Survivin, Thyroglobulin, VEGF-A, VEGF-C, AFP, CEA, CGA, EGFR, MAGE A1, MAGE A3 / A6 MUC-7, ProGRP, PSA, SCC, and can be used at least one cancer-associated gene is selected from one of the group consisting of WT1.
  • cancer-related genes targeted by the lung cancer genetic testing method of the present invention are bcl-2, CA125, CD44, c-kit, c-met, c-myc, COX-2, Cyclin D1, Cytokeratin-7, Cytokeratin-19, Cytokeratin-20, E2F1, E2F3, FGFR2, Gli1, GPC3, hCGbeta, Her2 / Neu, HLF-1a, HnRNP A2 / B1, hTERT, L-myc, MAGEA4, MAGEA4, MAGEMP4 -2, MMP-9, Muc-1, Muc-4, NSE, RCAS1, Survivin, Thyroglobulin, VEGF-A, VEGF-C, AFP, CEA, CGA, EGFR, MAGE A1, MAGE A3 / A , MUC-7, ProGRP, PSA, SCC, and can be used at least one cancer-associated gene is selected from one of the group consisting of WT1.
  • cancer-related genes targeted by the lung cancer genetic testing method of the present invention are COX-2, Cyclin D1, Cytokeratin-19, E2F1, E2F3, FGFR2, Gli1, hCGbeta, Her2 / Neu, HLF-1a, HnRNP A2. / B1, MAGE A4, MAGE A12, mdm2, MMP-9, Muc-1, RCAS1, Survivin, Thyroglobulin, VEGF-A, CEA, CGA, and EGFR selected at least one cancer-related Genes can be used.
  • the cancer-related genes targeted by the breast cancer genetic testing method of the present invention include bcl-2, CA125, CD44, c-kit, c-met, c-myc, COX-2, Cyclin D1, Cytokeratin-7, Cytokeratin-19, Cytokeratin-20, E2F1, E2F3, FGFR2, Gli1, GPC3, hCGbeta, Her2 / Neu, HLF-1a, HnRNP A2 / B1, hTERT, L-myc, MAGEA4, MAGEA4, MAGEMP4 -2, MMP-9, Muc-1, Muc-4, NSE, RCAS1, Survivin, Thyroglobulin, VEGF-A, VEGF-C, AFP, CEA, CGA, EGFR, MAGE A1, MAGE A3 / A , MUC-7, ProGRP, PSA, SCC, and can be used at least one cancer-associated gene is selected from one of the group consisting of WT1.
  • cancer-related genes targeted by the genetic testing method for breast cancer of the present invention include c-kit, COX-2, Cyclin D1, Cytokeratin-19, Cytokeratin-20, E2F1, E2F3, FGFR2, hCGbeta, Her2 / Neu, At least one cancer-related gene selected from any of the group consisting of HIF-1a, HnRNP A2 / B1, MAGE A4, MAGE A12, mdm2, MMP-9, Muc-1, Thyroglobulin, VEGF-A, and EGFR Can be used.
  • the cancer-related genes targeted by the gastric cancer genetic testing method of the present invention include bcl-2, CA125, CD44, c-kit, c-met, c-myc, COX-2, Cyclin D1, Cytokeratin-7, Cytokeratin-19, Cytokeratin-20, E2F1, E2F3, FGFR2, Gli1, GPC3, hCGbeta, Her2 / Neu, HLF-1a, HnRNP A2 / B1, hTERT, L-myc, MAGEA4, MAGEA4, MAGEMP4 -2, MMP-9, Muc-1, Muc-4, NSE, RCAS1, Survivin, Thyroglobulin, VEGF-A, VEGF-C, AFP, CEA, CGA, EGFR, MAGE A1, MAGE A3 / A , MUC-7, ProGRP, PSA, SCC, and can be used at least one cancer-associated gene is selected from one of the group consisting of WT1.
  • Cancer-related genes targeted by the colorectal cancer genetic testing method of the present invention include bcl-2, CA125, CD44, c-kit, c-met, c-myc, COX-2, Cyclin D1, Cytokeratin-7. , Cytokeratin-19, Cytokeratin-20, E2F1, E2F3, FGFR2, Gli1, GPC3, hCGbeta, Her2 / Neu, HIF-1a, HnRNP A2 / B1, hTERT, L-myc, MAGEA12AMAG MMP-2, MMP-9, Muc-1, Muc-4, NSE, RCAS1, Survivin, Thyroglobulin, VEGF-A, VEGF-C, AFP, CEA, CGA, EGFR, MAGE A1, MAGE A3 / 6, MUC-7, ProGRP, PSA, SCC, and can be used at least one cancer-associated gene is selected from one of the group consisting of WT1.
  • cancer-related genes targeted by the genetic testing method for liver cancer of the present invention include bcl-2, CA125, CD44, c-kit, c-met, c-myc, COX-2, Cyclin D1, Cytokeratin-7, Cytokeratin-19, Cytokeratin-20, E2F1, E2F3, FGFR2, Gli1, GPC3, hCGbeta, Her2 / Neu, HLF-1a, HnRNP A2 / B1, hTERT, L-myc, MAGEA4, MAGEA4, MAGEMP4 -2, MMP-9, Muc-1, Muc-4, NSE, RCAS1, Survivin, Thyroglobulin, VEGF-A, VEGF-C, AFP, CEA, CGA, EGFR, MAGE A1, MAGE A3 / A , MUC-7, ProGRP, PSA, SCC, and can be used at least one cancer-associated gene is selected from one of the group consisting of WT1.
  • the cancer-related genes targeted by the cancer genetic testing method of the present invention are 14-3-3-z (ZETA), ADAM12, ⁇ -catenin, Bcl-xl, B-myb, C3G, CD44, CDC25A, CDC25B, CDCP-1, CDK2, CDK4, CDK5, CTAP III / NAP-2, Cyclin B1, Cyclin E1, CYP2A6, DNA Polk (Kappa), DPD, DSG3, DYRK2, EphA2, ERBB3, ERBB3, ERBB4, ERBB4 FOXA1 (HNF3alpha), FOXM1, GPR87, hCdc6, hCdt1, HMGA2, Hrad17, Ki-67, KRT5, KRT6A, KRT6B, KRT14, Mesothelin / ERC, MMP-7, MMP-11 Mucin5 (MUC5), Nanog, N-myc, NOTCH3, PAX9, PCNA, PDGF-B, PIK3
  • cancer-related genes targeted by the lung cancer genetic testing method of the present invention include 14-3-3-z (ZETA), ADAM12, ⁇ -catenin, Bcl-xl, B-myb, C3G, CD44, CDC25A, CDC25B, CDCP-1, CDK2, CDK4, CDK5, CTAP III / NAP-2, Cyclin B1, Cyclin E1, CYP2A6, DNA Polk (Kappa), DPD, DSG3, DYRK2, EphA2, ERBB3, ERBB3, ERBB4, ERBB4 FOXA1 (HNF3alpha), FOXM1, GPR87, hCdc6, hCdt1, HMGA2, Hrad17, Ki-67, KRT5, KRT6A, KRT6B, KRT14, Mesothelin / ERC, MMP-7, MMP-1 , Mucin5 (MUC5), Nanog, N-myc, NOTCH3, PAX9, PCNA, PDGF-B,
  • cancer-related genes targeted by the breast cancer genetic testing method of the present invention are FGFR4 (Fibroblast growth factor 4), SERENBP1 (Selenium binding protein 1), POSTN (Periostin), OsteBlastPI, LUC.
  • LRBA LPS-responsive vesicle trafficking, beach and anchor containment IL13RA1 Interleuk in 13 receptor, alpha 1, ESR1 Estrogen receptor 1, MFAP2 Microfibrillar-associated protein 2, STAT1 Signal transducer and activator of transcription 1,91kDa, PPFIA1 Protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), alpha 1, MYO1C Myosin IC, PPFIA4 Protein tyrosine phosphate, receptor type, f poly eptide (PTPRF), interacting protein (liprin), alpha 4, RAB31 RAB31, member RAS oncogene family, INPP4B Inositol polyphosphate-4-phosphatase, type II, 105kDa, ESR1 Estrogen receptor 1, and MMP11 from Matrix metalloproteinase 11 (stromelysin 3) At least one cancer-related gene selected from any
  • the cancer-related genes targeted by the gastric cancer genetic testing method of the present invention are TFF1, COX-2, ⁇ -Catenin, c-myc, c-jun, APOC1, YF13H12, CDH17, FUS, APOE, S100A11, GRO1.
  • cancer-related genes targeted by the colorectal cancer genetic testing method of the present invention are CCSA (Colon cancer-specific antigen) -2, CCSA (Colon cancer-specific antigen) -3, CCSA (Colon cancer-specific antigen). -4, CCSA (Colon cancer-specific antigen) -5, CP (Cancer-placementa) -1, Alpha-catenin, REG1A (Regenerating isletted-private 1 alpha), DPEP1 (Dipeptidase 1) , HERV-H, NLF1 uclear localized factor 1 33.1, FOXQ1 Forkhead box Q1 24.4, MSX2 Msh homeobox homologue 2 22.2, ASCL2 Achaete-scute complex-like 2 17.3, MSX1 Msh homeobox homologue 1 8.5, IRX3 Iroquois homeobox protein3 8.4, GRHL3 Grainyhead-like 3, 7.9, TRIM29 Tripartite motif-continging 29 7.4, ETV
  • DUSP4 Dual specificity phosphatase 4 7.4, REG4 Regenerating islet-delivered family
  • member 4.6.8 PHLDA1 Plextrin homology-likeness-like A, member 1 6.0, LCN2 Lipocalin 2 (oncogene 24p3) 5.7, RTEL1 Regulator of telomere elongation helicity 1, 5.6, TGFBI Transforming inc.
  • the cancer-related genes targeted by the genetic testing method for liver cancer of the present invention are Fibronincinc X02761; K00799; K02273, Tubulin alpha 1 subunit K00558, Matrix metalloproteinase 14 D26512; repair protein; RAD23A D21235, Ubiquitin-conjugating enzyme E2, M74524, Trafficking protein Neutropil gelatinase-associated lipocalin recursor lipocalin 2 X99133, TRAM protein X63679, ADP / ATP carrier protein J02683, Transcription factor High mobility group protein M23619, Growth factor Insulin-stimulated protein kinase 1 U08316, GTP binding protein Transforming protein rhoA H12 L25080, IFN response Interferon gamma antagonist A25270, Cytokines Macrophage inhibitory cyto ine 1 AF019770, Down-regulated genes in HCCs tumor tissues, Immune system IgG, Ig, I
  • cancer-related genes targeted by the genetic testing method for liver cancer related to HBV or HCV are CDC28 protein kinase 2 X54942, CDC27HS protein U00001, Extracellular matrix Ingredient beta 4X57887M6187 J05211, Metabolic pathway Procalagen C proteinase M22488 + U50330, Growth factor Platelet-derived growth factor A subunit X06374, CMP-N-acetylet alactosamide-alpha-2,6-sialytransferase Betaine-homocysteine S-methyl-transferase U50929, Dihydro-orotate dehydrogenaseb M94065, Cytidine deaminase L27943, Aldehyde oxidaseb L11005, Aminoacylase 1 L07548, Methylenetetrahydrofolate dehydrogenaseb J04031, Metallothionein-IIIb X62822, Immune system IgG receptor
  • a partial nucleic acid sequence of a cancer-related gene used as a primer in a real-time-PCR (RT-PCR) analysis or a partial nucleic acid sequence of a cancer-related gene used as a probe in a nucleic acid microarray analysis is appropriately selected.
  • RT-PCR real-time-PCR
  • the cancer genetic testing method of the present invention is based on a significant difference between the expression level of a cancer-related gene obtained from a blood sample of a healthy person and the expression level of a cancer-related gene obtained from a blood sample of a patient. It is characterized by predicting the existence risk and / or therapeutic effect of a cancer cell population.
  • absolute or relative between the expression level of a cancer-related gene in a healthy person and the expression level of a cancer-related gene in a patient obtained from real-time-PCR (RT-PCR) analysis or nucleic acid microarray analysis Since the determination is based on a significant difference in the expression level, the existence risk of the cancer cell population and / or the therapeutic effect can be predicted easily and with high accuracy.
  • RT-PCR real-time-PCR
  • the cancer genetic testing method of the present invention predicts the existence risk and / or therapeutic effect of a cancer cell population based on the analysis of the expression level of a cancer-related gene that is usually hardly observed in healthy individuals. It is characterized by.
  • the risk of cancer cell populations and / or therapeutic effects are predicted based on the expression level of cancer-related genes that are normally almost unexpressed. It is not necessary to analyze the expression level of the gene, and it is possible to predict the existence risk of the cancer cell population and / or the therapeutic effect quickly and with high accuracy.
  • the cancer genetic testing method of the present invention it is possible to predict the existence risk of a cancer cell population and the therapeutic effect with high accuracy and early or prognosis.
  • FIG. 1 is a flowchart for explaining processing steps of a cancer genetic testing method according to an embodiment of the present invention.
  • a buffy coat layer separation step 10 by centrifugation of a blood sample a mononuclear cell separation step 20 by density gradient centrifugation, Extraction step 30 of total RNA from nucleated cells, synthesis step 40 of complementary DNA (hereinafter referred to as cDNA) by reverse transcription reaction from total RNA, and real-time PCR (RT-) for cancer-related genes PCR) analysis step 50 and DNA microarray analysis step 60 as a nucleic acid microarray for cancer-related genes.
  • cDNA complementary DNA
  • RT- real-time PCR
  • the buffy coat layer separation step 10 by centrifugation of a blood sample is a step of fractionating a buffy coat layer rich in white blood cells by centrifugation from a whole blood sample collected from a subject. For example, when a whole blood sample is centrifuged (2400 ⁇ g, 10 minutes) at room temperature, the whole blood sample is separated into three layers. The middle layer fractionated at this time is a buffy coat layer, which is a fraction rich in leukocytes. Then, the buffy coat layer is carefully separated, and then mononuclear cells are separated by density gradient centrifugation.
  • the mononuclear cell separation step 20 by density gradient centrifugation is a step of separating mononuclear cells from the buffy coat layer obtained by centrifugation of the whole blood sample.
  • the density gradient centrifugation is not particularly limited. For example, sucrose, sedimentation rate method using density gradient by dextran or histopac, Ficoll method, cesium chloride, cesium sulfate, etc. It can be carried out by a method such as isodensity centrifugation using a density gradient with an aqueous solution.
  • a blood separation solution such as Lymphoprep (registered trademark) manufactured by Cosmo Bio, the separation step can be performed in one step. It can be done according to this.
  • the extraction step 30 of total RNA from mononuclear cells is not particularly limited, but total RNA can be extracted by a known or similar method. For example, when using a commercially available RNA extraction kit, it can carry out according to description of the attached instruction manual.
  • the cDNA synthesis step 40 by reverse transcription reaction from total RNA is not particularly limited.
  • CDNA can be synthesized by a method using a gene-specific primer, or by using a random primer, or a known or similar method, such as starting synthesis of cDNA from the entire region of mRNA.
  • RT-PCR real-time PCR
  • the partial nucleic acid sequence in the cancer-related gene of the primer used in the real-time-PCR (RT-PCR) analysis step 50 or the probe used in the DNA microarray analysis step 60 is known in the art or by a method analogous thereto.
  • the partial nucleic acid sequence to be used is an oligonucleotide, it can be prepared by an organic chemical method, and when it is a partial nucleic acid sequence having a relatively large size, a cell system or a cell-free system It can produce by the biological technique of.
  • the length of the partial nucleic acid sequence is not particularly limited as long as the synthesized cDNA is sufficiently long to allow hybridization, but is at least 15 bp, preferably at least 20 bp, more preferably at least 25 bp, and even more preferably. Is at least 30 bp.
  • RT-PCR real-time-PCR
  • fluorescence is emitted in proportion to the amount of cancer-related gene amplified by the PCR reaction, and the change in fluorescence intensity with time is measured.
  • the method of emitting fluorescence is not particularly limited.
  • SYBR Green which is a dye that binds to DNA
  • TaqMan probe that specifically binds to an amplified cancer-related gene is used.
  • a method known in the art or a method analogous thereto can be used.
  • the measured expression level of the cancer-related gene of the patient is compared with the expression level of the cancer-related gene of the healthy person, and determined with an absolute or relative significant difference. Based on these determination results Presence risk of cancer cell population and / or therapeutic effect is predicted.
  • the measured expression level of the cancer-related gene of the patient may be collated with the expression level data of the cancer-related gene accumulated in advance.
  • the DNA microarray analysis step 60 1) synthesis of double-stranded DNA based on the cDNA synthesized in the cDNA synthesis step 40 by reverse transcription reaction from total RNA, and 2) the obtained double-stranded DNA RNA amplification by in vitro transcription reaction based on 3) RNA labeling of healthy person-derived RNA and patient-derived RNA, 4) Cancer-related genes using both fluorescently labeled RNAs as probes 5) The expression change of the cancer-related gene of the patient relative to a healthy person is determined from the fluorescence intensity of the fluorescently labeled RNA hybridized with the probe. And the existence risk of a cancer cell population and / or a therapeutic effect are estimated based on these determination results.
  • DNA microarray used in the present embodiment a form known in the art or an equivalent form thereof can be used.
  • an array in which a partial diffusion sequence of a cancer-related gene is directly synthesized on a support. Affymetrix method
  • an array in which a partial nucleic acid sequence of a cancer-related gene is immobilized on a support Stanford method, or the like can be used.
  • nucleic acid molecules are silicon-based while repeating the process of covering and exposing the silicon substrate as a support with a light-shielding plate called a mask by photolithography technology and solid-phase nucleic acid synthesis technology.
  • An array is made by extending one base at a time. According to this method, since the synthesized partial nucleic acid sequence can be fixed perpendicularly to the support, there are advantages such as high hybridization efficiency and excellent quantification and reproducibility.
  • the Stanford method an array is prepared by spotting cDNA prepared in advance or synthetic oligonucleotide directly on a support.
  • a puffy coat layer rich in leukocytes was obtained from a blood sample collected from a subject by centrifugation. Then, the puffy coat layer was suspended, and gently layered on an equal amount of histopack solution (manufactured by Sigma), followed by density gradient centrifugation to collect mononuclear cells.
  • RNA was extracted from the obtained mononuclear cells using Easy-spin TM Total RNA Extraction Kit (manufactured by iNtron) according to the description in the attached instruction manual.
  • 4 and 5 are tables showing the results of the expression analysis of cancer-related genes in lung cancer patients and healthy individuals for each cancer-related gene extracted from the 22 cancer-related genes shown in FIG. As shown in FIG. 4 and FIG. 5, the proportion of subjects exceeding the reference range of one or more cancer-related genes among the extracted cancer-related genes is about 20% for healthy subjects and about about 20% for lung cancer patients. 90%.
  • FIG. 7 and FIG. 8 are tables showing the results of the expression analysis of cancer-related genes in breast cancer patients and healthy individuals for each cancer-related gene extracted from the 19 cancer-related genes shown in FIG. As shown in FIG. 7 and FIG. 8, the proportion of subjects who exceeded the reference range of one or more cancer-related genes among the extracted cancer-related genes was 22% for healthy subjects, and about 20% for breast cancer patients. 82%.
  • FIG. 9 is a table summarizing the evaluation of the cancer genetic testing method according to the present invention for lung cancer or breast cancer.
  • the specificity was 80% and the sensitivity was about 90% in the detection of lung cancer. Further, in the discrimination of breast cancer, the specificity was 78% and the sensitivity was about 82% as the detection rate.
  • the used genetic testing method for cancer according to the present invention is considered useful as a testing method for lung cancer or breast cancer.
  • lung cancer-related genes were extracted for lung cancer risk assessment, and 19 breast cancer-related genes were extracted for breast cancer risk assessment.
  • the gene group applied to is not limited to this.
  • lung cancer risk assessment in addition to the aforementioned lung cancer-related genes, 14-3-3-z (ZETA), ADAM12, ⁇ -catenin, Bcl-xl, B-myb, C3G, CD44, CDC25A, CDC25B , CDCP-1, CDK2, CDK4, CDK5, CTAP III / NAP-2, Cyclin B1, Cyclin E1, CYP2A6, DNA Polk (Kappa), DPD, DSG3, DYRK2, EphA2, ERBB3, ERBB4, EGF2, AGF1A (HNF3alpha), FOXM1, GPR87, hCdc6, hCdt1, HMGA2, Hrad17, Ki-67, KRT5, KRT6A, KRT6B, KRT14, Mesothelin / ERC, MMP-7, MM -11, Mucin5 (MUC5), Nanog, N-myc, NOTCH3, PAX9, PCNA, PDGF-B, PI
  • FGFR4 Fibroblast growth factor receptor 4
  • SERENBP1 Senium binding protein 1
  • POSTN PeriostinClinsePlN, FastBlastClinPlSlP
  • HSP-47 heat shock protein 47
  • CBP1 collagen binding protein 1
  • KDELR3 DEAD Asp-Glu-Ala-Adipolopopp
  • MS Parathymosin
  • HIST2H2BE Histone 2, H2be
  • TUSC3 Tuor suppressor candidate 3
  • ZNF516 Zinc finger protein 516
  • ACAD11, INPP4B, ESR1 Estrogen receptor 1
  • ACAA2 Myosin VB CYB5A (Cytochrome b-5
  • CLIP4 CAP-GLY domain containing linker family family, member
  • LRBA LPS-responsive vesicle trafficking, beach and anchor containment IL13RA1 Interle ukin 13 receptor, alpha 1, ESR1 Estrogen receptor 1, MFAP2 Microfibrillar-associated protein 2, STAT1 Signal transducer and activator of transcription 1,91kDa, PPFIA1 Protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), alpha 1, MYO1C Myosin IC, PPFIA4 Protein tyrosine phosphate, receptor type, f po ypeptide (PTPRF), interacting protein (liprin), alpha 4, RAB31 RAB31, member RAS oncogene family, INPP4B Inositol polyphosphate-4-phosphatase, type II, 105kDa, ESR1 Estrogen receptor 1, and MMP11 from Matrix metalloproteinase 11 (stromelysin 3) At least one cancer-related gene selected
  • At least one cancer-related gene selected from can be used.
  • liver cancer bcl-2, CA125, CD44, c-kit, c-met, c-myc, COX-2, Cyclin D1, Cytokeratin-7, Cytokeratin-19, Cytokeratin-20, E2F1, E2F3, FGFR2, Gli1, GPC3, hCGbeta, Her2 / Neu, HIF-1a, HnRNP A2 / B1, hTERT, L-myc, MAGE A4, MAGE A12, mdm2, MDR1, MMP-2, MMP-9, Muc-1 , Muc-4, NSE, RCAS1, Survivin, Thyroglobulin, VEGF-A, VEGF-C, AFP, CEA, CGA, EGFR, MAGE A1, MAGE A3 / A6, MUC-7, ProGRP PSA, SCC, and in addition to WT1, Fibronectinc X02761; K00799; K02273, Tubulin al
  • HBV or for liver cancer risk assessment associated with HCV, CDC28 protein kinase 2 X54942, CDC27HS protein U00001, Extracellular matrix Integrin beta 4 X53587; X52186, Desmoplakin I & II M77830; J05211, Metabolic pathway Procallagen C proteinase M22488 + U50330, Growth factor Platelet-derived growth factor A subunit X06374, CMP-N-acetylneuraminate-beta-galactosamide alpha-2,6-sialytransferase Betaine-homocysteine S-methyl-transferase U50929, Dihydro-orotate dehydrogenaseb M94065, Cytidine deaminase L27943, Aldehyde oxidaseb L11005, Aminoacylase 1 L07548, Methylenetetrahydrofolate dehydrogenaseb J04031, Metallothionein-III
  • cancer-related genes described above may be used, and cancer risk assessment may be performed based on the expression profile of a single cancer-related gene. Cancer risk assessment may be performed based on the expression profiles of cancer-related genes as a group.

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Abstract

L'invention porte sur un procédé de test génétique qui peut prédire le risque de l'apparition d'une masse de cellules cancéreuses ou l'efficacité d'une thérapie avec une précision élevée dans un stade précoce ou après pronostic. De façon spécifique, l'invention porte sur un procédé de test génétique pour le cancer, qui comprend : une étape (10) de séparation d'une couche leuco-plaquettaire à partir d'un échantillon de sang par centrifugation ; une étape (20) de séparation des monocytes par centrifugation à gradient de densité ; une étape (30) d'extraction de l'ARN total des monocytes ; une étape (40) de synthèse de l'ADN complémentaire à partir de l'ARN total par réaction de transcription inverse ; une étape (50) de réalisation d'une analyse par PCR en temps réel (RT-PCR) pour un gène lié au cancer ; et une étape (60) de réalisation d'une analyse sur micropuce d'ADN pour un gène lié au cancer par l'emploi d'une micropuce d'ADN en tant que micropuce d'acide nucléique.
PCT/JP2010/001417 2009-03-02 2010-03-02 Procédé de test génétique pour le cancer par analyse de l'expression d'un gène lié au cancer à l'aide de monocytes contenus dans un échantillon de sang WO2010100899A1 (fr)

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JP2011033495A (ja) * 2009-08-03 2011-02-17 Koto Biseibutsu Kenkyusho:Kk 予後予測のための大腸癌組織の検査方法
WO2012176175A1 (fr) * 2011-06-24 2012-12-27 Universite De Geneve Nouvelles utilisations des inhibiteurs de nanog et procédés s'y rapportant
WO2013016673A3 (fr) * 2011-07-27 2013-05-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Utilisation de l'expression de dpep1 et tpx2 pour l'évaluation du traitement ou de la durée de survie de patients atteints d'un adénocarcinome du canal pancréatique
EP2678676A2 (fr) * 2011-02-22 2014-01-01 Yale University Classificateur basé sur l'expression protéique, dans la prédiction de la récurrence d'un adénocarcinome
JP2014027898A (ja) * 2012-07-31 2014-02-13 Yamaguchi Univ 肝細胞がん発症リスクの判定方法
CN106868104A (zh) * 2015-12-10 2017-06-20 益善生物技术股份有限公司 肺癌循环肿瘤细胞分型鉴定试剂盒
KR20170092671A (ko) * 2014-12-08 2017-08-11 버그 엘엘씨 전립선암의 진단 및 치료에서 필라민을 포함하는 마커의 용도
US9797905B2 (en) 2012-06-27 2017-10-24 Berg Llc Use of markers in the diagnosis and treatment of prostate cancer
JP2018504609A (ja) * 2015-02-05 2018-02-15 クイーン メアリー ユニバーシティ オブ ロンドン 膵癌のバイオマーカー
CN107828871A (zh) * 2015-06-24 2018-03-23 湖北工业大学 基于肽核酸探针的Wnt信号通路中FoxM1基因P271Q突变的检测试剂
US10041126B2 (en) * 2012-01-27 2018-08-07 Vib Vzw Monocyte biomarkers for cancer detection
WO2020034061A1 (fr) * 2018-08-13 2020-02-20 Beijing Percans Oncology Co., Ltd. Biomarqueurs pour la thérapie anticancéreuse
WO2020097901A1 (fr) * 2018-11-16 2020-05-22 Beijing Percans Oncology Co. Ltd. Biomarqueurs pour la cancérothérapie
WO2020200323A1 (fr) * 2019-04-04 2020-10-08 清华大学 Marqueur de cellules très précoces du cancer gastrique et marqueur de cellules précoces de lésion précancéreuse gastrique et leur application dans un nécessaire de diagnostic
CN113970638A (zh) * 2021-10-24 2022-01-25 清华大学 确定胃癌极早期发生风险及评估胃癌前病变进展风险的分子标志及其在诊断试剂盒中的应用

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KR102180623B1 (ko) * 2019-01-03 2020-11-18 고려대학교 산학협력단 암의 골전이 진단용 조성물 및 이를 이용한 암의 골전이 진단방법

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JP2011033495A (ja) * 2009-08-03 2011-02-17 Koto Biseibutsu Kenkyusho:Kk 予後予測のための大腸癌組織の検査方法
EP2678676A2 (fr) * 2011-02-22 2014-01-01 Yale University Classificateur basé sur l'expression protéique, dans la prédiction de la récurrence d'un adénocarcinome
EP2678676A4 (fr) * 2011-02-22 2014-10-29 Univ Yale Classificateur basé sur l'expression protéique, dans la prédiction de la récurrence d'un adénocarcinome
WO2012176175A1 (fr) * 2011-06-24 2012-12-27 Universite De Geneve Nouvelles utilisations des inhibiteurs de nanog et procédés s'y rapportant
US9518098B2 (en) 2011-06-24 2016-12-13 Universite De Geneve Uses of NANOG inhibitors and related methods
WO2013016673A3 (fr) * 2011-07-27 2013-05-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Utilisation de l'expression de dpep1 et tpx2 pour l'évaluation du traitement ou de la durée de survie de patients atteints d'un adénocarcinome du canal pancréatique
US10041126B2 (en) * 2012-01-27 2018-08-07 Vib Vzw Monocyte biomarkers for cancer detection
US9797905B2 (en) 2012-06-27 2017-10-24 Berg Llc Use of markers in the diagnosis and treatment of prostate cancer
JP2014027898A (ja) * 2012-07-31 2014-02-13 Yamaguchi Univ 肝細胞がん発症リスクの判定方法
KR102657306B1 (ko) 2014-12-08 2024-04-12 버그 엘엘씨 전립선암의 진단 및 치료에서 필라민을 포함하는 마커의 용도
KR20170092671A (ko) * 2014-12-08 2017-08-11 버그 엘엘씨 전립선암의 진단 및 치료에서 필라민을 포함하는 마커의 용도
US10539566B2 (en) 2014-12-08 2020-01-21 Berg Llc Use of markers including filamin A in the diagnosis and treatment of prostate cancer
JP2018504609A (ja) * 2015-02-05 2018-02-15 クイーン メアリー ユニバーシティ オブ ロンドン 膵癌のバイオマーカー
CN107828871A (zh) * 2015-06-24 2018-03-23 湖北工业大学 基于肽核酸探针的Wnt信号通路中FoxM1基因P271Q突变的检测试剂
CN106868104A (zh) * 2015-12-10 2017-06-20 益善生物技术股份有限公司 肺癌循环肿瘤细胞分型鉴定试剂盒
WO2020034061A1 (fr) * 2018-08-13 2020-02-20 Beijing Percans Oncology Co., Ltd. Biomarqueurs pour la thérapie anticancéreuse
WO2020097901A1 (fr) * 2018-11-16 2020-05-22 Beijing Percans Oncology Co. Ltd. Biomarqueurs pour la cancérothérapie
WO2020200323A1 (fr) * 2019-04-04 2020-10-08 清华大学 Marqueur de cellules très précoces du cancer gastrique et marqueur de cellules précoces de lésion précancéreuse gastrique et leur application dans un nécessaire de diagnostic
CN111781356A (zh) * 2019-04-04 2020-10-16 清华大学 一种胃癌极早期细胞标志和胃癌前病变早期细胞标志及其在诊断试剂盒中的应用
CN111936858A (zh) * 2019-04-04 2020-11-13 清华大学 一种胃癌极早期细胞标志和胃癌前病变早期细胞标志及其在诊断试剂盒中的应用
CN113970638A (zh) * 2021-10-24 2022-01-25 清华大学 确定胃癌极早期发生风险及评估胃癌前病变进展风险的分子标志及其在诊断试剂盒中的应用
CN113970638B (zh) * 2021-10-24 2023-02-03 清华大学 确定胃癌极早期发生风险及评估胃癌前病变进展风险的分子标志及其在诊断试剂盒中的应用

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