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WO2007077977A1 - Composition et procede permettant de predire le pronostic postoperatoire ou le risque metastasique d’un patient cancereux - Google Patents

Composition et procede permettant de predire le pronostic postoperatoire ou le risque metastasique d’un patient cancereux Download PDF

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Publication number
WO2007077977A1
WO2007077977A1 PCT/JP2006/326410 JP2006326410W WO2007077977A1 WO 2007077977 A1 WO2007077977 A1 WO 2007077977A1 JP 2006326410 W JP2006326410 W JP 2006326410W WO 2007077977 A1 WO2007077977 A1 WO 2007077977A1
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Prior art keywords
cancer
group
metastasis
polynucleotide
prognosis
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Japanese (ja)
Inventor
Takashi Takahashi
Shuta Tomida
Kiyoshi Yanagisawa
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Nagoya University NUC
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Nagoya University NUC
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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
    • 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
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a composition or method for predicting postoperative prognosis or metastatic potential in cancer patients in vitro.
  • the present invention also provides a method for screening a cancer metastasis inhibitor using cultured cancer cells.
  • Cancer is a multifactorial disease in which genes and environmental factors are complicatedly involved.
  • Various therapies such as chemotherapy, radiation therapy, surgery, and immunotherapy have been implemented, but due to the complexity of cancer onset mechanisms, it is imperative to treat mid-stage and end-stage cancer and to suppress cancer metastasis. However, it cannot be said that sufficient effects have been achieved.
  • cancer diagnosis methods such as cytology, histology, endoscope, image diagnosis, and biochemical diagnosis using cancer markers are used. It is active.
  • cancer metastasis One of the major causes that hinders cancer treatment and recurrence is cancer metastasis. Because of the motility and invasive ability of cancer, cancer spreads remotely from the primary focus to another tissue and grows. The overall picture of this transfer mechanism is largely unknown. As cancers become more malignant, and when cancers are removed, cancers are more likely to metastasize, resulting in recurrence of the cancer, so preventing cancer migration is an early diagnosis and effective treatment method for cancer. Along with development, it has become an important issue in the medical field.
  • a cancer metastasis inhibitor for example, a substance that inhibits signal transduction via the human VEGF receptor F 1 t _ l (Patent Document 1), a peptide having cell adhesion inhibitory activity (Patent Document 2), a function of purine receptor There are known purine compounds (Patent Document 3) and the like.
  • purine compounds Patent Document 3 and the like.
  • studies on genes involved in cancer metastasis suggest that matrix-degrading enzymes, vascular endothelial growth factors and their receptors are involved in metastasis.
  • a cancer metastasis-related gene discovered from a mouse cell line has been reported as a cancer metastasis-related gene (Patent Document 4).
  • RNA suppression techniques include, for example, antisense method, ribozyme, RNA interference (RNA i) and the like (Non-patent Document 1).
  • RNA i is highly specific, so basic research for its medical application is rapidly progressing, and its target is viral disease, cancer, genetic disease, etc.
  • Patent Document 2 is a description of RNA interference technique.
  • Patent Document 1 Japanese Patent Laid-Open No. 2 003- 26 1 46 0
  • Patent Document 2 Japanese Patent Application Laid-Open No. 09-143076
  • Patent Document 3 Japanese Unexamined Patent Publication No. 7-08 2 1 56
  • Patent Document 4 Japanese Table 9 8Z04 543 1
  • Non-Patent Literature 1 Daisuke Seki et al., Experimental Medicine 2 2-14 14 (extra number), 89-98 pages, 2 004, Yodosha (Tokyo, Japan)
  • Non-Patent Literature 2 Experimental Medicine 2 2-4 “RN A i Science”, 2004, Yodosha (Tokyo, Japan) Disclosure of Invention
  • metastasis is not caused by a single gene but by a coordinated interaction of a plurality of gene groups, identifying cancer metastasis-related gene markers, and The relationship between the gene marker and postoperative prognosis or metastatic potential of cancer patients was examined.
  • An object of the present invention is to provide a method and a composition for predicting the postoperative prognosis or metastatic potential of cancer patients in vitro using a cancer metastasis-related gene group as a marker.
  • Another object of the present invention is to provide a method for screening a cancer metastasis inhibitor based on inhibition or suppression of a cancer metastasis-related gene or a transcription product thereof.
  • the present invention has the following characteristics.
  • a method for predicting postoperative prognosis or metastasis potential in cancer patients in vitro the base sequence shown in SEQ ID NOs: 1 to 45 or a mutant sequence thereof in a biological sample derived from the patient
  • At least one expression level or transcription or translation product level of a cancer metastasis-related gene marker containing a marker is measured using a probe corresponding to the marker, so that the prognosis after surgery is better or the possibility of metastasis is increased.
  • To determine the postoperative prognosis or metastatic potential using the relative difference in level between the lower patient group and the patient group with worse postoperative prognosis or higher metastatic potential. Including the above method.
  • the cancer metastasis-related gene marker is composed of the nucleotide sequence shown in SEQ ID NOs: 28 to 45 or a mutant sequence thereof, the patient has a better postoperative prognosis or is less likely to transfer.
  • the method according to (1) above which is identified as a patient.
  • the cancer metastasis-related gene marker comprises the nucleotide sequence shown in SEQ ID NOs: 1 to 27 or a mutant sequence thereof, the patient is a patient with a worse prognosis after surgery or a higher possibility of metastasis.
  • a polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 45, a polynucleotide complementary thereto, a variant thereof, and a polynucleotide that hybridizes to them under stringent conditions Or the method according to any one of (1) to (3) above, which is selected from the group consisting of those fragments containing 15 or more consecutive bases.
  • the probe is an antibody against a polypeptide selected from the group consisting of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 1-45 or a polypeptide encoded by a variant thereof, or a fragment thereof, or the The method according to any one of (1) to (3) above, which is a fragment.
  • a polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 4, a complementary polynucleotide, a variant thereof, a polynucleotide that hybridizes under stringent conditions thereto, or 15 or more
  • a composition for predicting postoperative prognosis or metastasis potential in cancer patients in vitro comprising at least one probe selected from the group consisting of those fragments comprising a sequence of nucleotides.
  • Polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 28 to 45, polynucleotide complementary thereto, variant thereof, polynucleotide hybridizing under stringent conditions, or 15 or more
  • Polynucleotide comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 27, polynucleotide complementary thereto, variant thereof, polynucleotide hybridizing under stringent conditions, or 1 5 or more consecutive bases
  • a composition for predicting the postoperative prognosis or metastatic potential of cancer patients in vitro comprising at least one probe selected from:
  • composition for predicting in vitro the postoperative prognosis or metastatic potential of a cancer patient comprising at least one probe selected from the group.
  • markers as indicators, it is possible to predict the postoperative prognosis of cancer patients and the possibility of cancer metastasis, so it is possible to formulate treatment plans for patients, improve treatment outcomes for cancer, and recurrence due to metastatic cancer. It can be expected to be of great use for prevention and prognosis management. Furthermore, in cultured cancer cells, it is possible to screen for a drug that regulates its expression using the above marker as an index, which leads to the development of a drug that suppresses or prevents cancer metastasis. it can.
  • Fig. 1 shows the procedure for establishing the human highly metastatic lung cancer cell line LNM35.
  • FIG. 2 shows a comparison of the motility and invasion ability (A) in cultured cells and the ratio of lymph node metastasis and lung metastasis (B) after mouse transplantation between the LNM3 5 strain and its parent strain.
  • Figure 3 shows the correlation between the expression pattern of 45 metastasis-related genes and the proportion of survivors after surgery when applied to 50 cases of lung cancer data from the Aichi Cancer Center (Nagoya, Japan). Shown to be roughly divided into two groups.
  • Figure 3A shows the results of hierarchical clustering analysis.
  • Figure 3B shows the results of force planmeyer survival analysis (vertical axis: survival rate, horizontal axis: number of months).
  • Figure 4 shows that when applied to Harvard University (USA) 62 cases of lung cancer data (Bhattacharjee A et al., Proc Natl Acad Sci USA 98: 1 3 790-9 5, 200 1) The correlation between the expression pattern of 45 metastasis-related genes and the proportion of survivors after surgery is broadly divided into two groups.
  • Figure 4A shows the results of hierarchical clustering analysis.
  • Figure 4B shows the results of the force plan meyer survival analysis (vertical axis: survival rate, horizontal axis: number of months).
  • Figure 5 is applied to 7 9 cases of breast cancer (van't Veer LJ et al., Nature 4 1 5: 5 30-6, 2002) from the Netherlands National Cancer Institute (Netherlands).
  • Figure 5A shows the results of hierarchical clustering analysis.
  • Fig. 5B shows the results of a force plan myria survival analysis (vertical axis: survival rate, horizontal axis: number of months).
  • Figure 6 shows the recurrence and death predictors independent of stage determined by multivariate analysis with a Co x proportional hazards model for the risk of 5-year survival in lung cancer patients.
  • postoperative prognosis means determining the prognosis of a patient after surgery for cancer, typically by the survival rate at 5 years postoperatively. Prognosis is closely related to cancer metastasis, and ⁇ poor prognosis '' means that the cancer is highly invasive, metastatic, or highly advanced, while ⁇ good prognosis '' means It means such invasiveness, metastasis or low progression.
  • metastasis refers to a series of processes by which cancer cells move from the primary site to distant tissue by their motility and invasive potential, where they proliferate and form neoplasms.
  • Metastasis is a major obstacle to cancer treatment because it causes cancer recurrence. Cancer cells that have infiltrated blood vessels or lymphatic vessels are known to stay in various tissues and cause multiple cancer diseases.
  • any metastasis is targeted as long as the cancer metastasis-related gene marker of the present invention is involved.
  • lymph node metastasis can also be targeted.
  • patient refers to mammals including humans, dogs, and cats, with the preferred mammal being humans.
  • a “biological specimen” refers to a tissue, cell or body fluid taken from a mammal (preferably human), preferably a cancer tissue or cell.
  • a mammal preferably human
  • cancer tissue or cells derived from a group of patients with a better postoperative prognosis or a lower likelihood of metastasis and a worse prognosis after surgery or a higher possibility of metastasis. Any cancer can be used as long as it shows a relative difference in the expression level of the gene gene related to the present invention from those derived from different patient groups.
  • lung cancer breast cancer, colon cancer, prostate cancer, stomach cancer, esophageal cancer, liver cancer, spleen cancer, kidney cancer, uterine / cervical cancer, ovarian cancer, bladder cancer, brain tumor, thyroid gland
  • examples include, but are not limited to, cancer, lymphoma, testicular cancer, osteosarcoma, skin cancer, melanoma, and blood cancer (especially leukemia).
  • cancer metastasis-related gene marker refers to a gene that has a difference in expression level between a high metastatic cancer cell line and a low metastatic cancer cell line. It is a marker that has been released and can be used to determine the postoperative prognosis or metastatic potential of cancer patients as in the method of the present invention.
  • a “variant” is a variant that arises from a biological event such as mutation, polymorphism, alternative splicing, the degeneracy of the genetic code, or a homologue between species. Etc.
  • the mutant includes one or more, preferably one or several, on the sequence of the gene containing the nucleotide sequence represented by SEQ ID NOs: 1 to 45 related to the present invention or the polypeptide encoded by the gene. Mutants having mutations such as substitutions, deletions, additions, insertions, etc., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more identical to the sequence or a partial sequence thereof Mutants consisting of sequences having sex, and the like.
  • “several” usually means an integer of 10 or less. Identity (%) can also be determined using known B LAST and FAS TA programs with or without gaps (SF A ltschul et al., J. Mo 1. B i o. 2 1 5: 403-4 1 0, 1 990). In general, identity (%) can be calculated as a percentage of the number of matched bases over the total number of bases.
  • stringent conditions include, but are not limited to, at least 80%, preferably at least 90%, more preferably at least 95% identity. This means hybridization and washing conditions that allow nucleotide sequences to hybridize to each other. For example, hybridization and washing conditions in microarray analysis are 1M sodium chloride Z0.5%
  • the human cancer metastasis-related gene marker of the present invention was found as follows. Metastatic lung cancer cell line known as NCI — H 4 60 (American Type C 1 ture Collection; US Viryua)) (parent strain) A highly metastatic cancer cell line, LNM35, was isolated by subculture of cells from and by in vivo selection using mice based on metastatic potential (see Figure 1). On the other hand, cancer cells with even lower metastases can be obtained from NC I —H 4 60 cells by limiting dilution or treatment with a demethylating agent.
  • the LNM3 5 strain showed significantly higher levels of motility, invasion ability, lymph node metastasis, and lung metastasis compared to the parent strain or low-metastasis cancer cell line ( (See Figure 2).
  • the cancer metastasis-related marker identified as described above can be a marker not only in lung cancer but also in solid cancer such as sputum cancer.
  • One marker reported so far has been specific to a particular cancer, but it was surprising that the marker of the present invention is a common marker for various cancers.
  • Solid cancers such as head cancer, ovarian cancer, bladder cancer, brain tumor, thyroid cancer, lymphoma, testicular cancer, osteosarcoma, skin cancer, melanoma are included.
  • Further possible cancers include blood cancer (particularly leukemia).
  • the cancer metastasis-related gene marker includes not only the gene comprising the nucleotide sequence shown in SEQ ID NOs: 1 to 45 but also mutant genes thereof.
  • such variants include those produced in vivo as a result of biological events such as mutations, polymorphisms, alternative splicing and the like.
  • the genetic marker is detected at the nucleic acid level or protein level as a transcript or translation product. In the case of a transcript, it is detected as mRNA, cRNA or cDNA.
  • RNA from cells or tissues using extraction methods such as phenolic mouth form Z-soamyl alcohol, guanidinium / cesium chloride, etc., and use oligo d T cell mouth column method to extract poly A (+) RNA or mRNA If necessary, further synthesize cDNA and cRNA from mRNA (for example, F. M. A usubel et al., 'Short Protocolsin Molecular Biology (3rd edition) AC ompendiumof Methods from Curren Protocolsinolecular Biology) , 1 995, John Wiley & Sons, Inc. (USA)).
  • a translation product it is detected as a protein encoded by the gene or a variant thereof or a fragment thereof.
  • the cancer-related gene markers related to the present invention are roughly classified into two groups according to their expression patterns corresponding to the prognosis or metastatic potential of cancer patients after external surgery (see FIGS. 3 to 5). .
  • the cancer metastasis-related gene marker consists of the base sequence shown in SEQ ID NOs: 28 to 45 or a mutant sequence thereof
  • the patient has a better postoperative prognosis or is less likely to metastasize. Identified as a patient.
  • the cancer metastasis-related gene marker consists of the nucleotide sequence shown in SEQ ID NOs: 1 to 27 or a mutant sequence thereof
  • the patient Identified as a patient with a worse postoperative prognosis or with a higher likelihood of metastasis.
  • the present invention provides a method for predicting the postoperative prognosis or metastasis potential of cancer patients in vitro, which method comprises the steps of: SEQ ID NO :! Measure the expression level of at least one of the cancer metastasis-related gene markers including the nucleotide sequence shown in -45 or a mutant sequence thereof or the transcription or translation product level thereof using a probe corresponding to the marker, and This includes predicting the prognosis or metastasis after surgery using a significant level difference from a non-cancerous control sample as an index.
  • the probe used in the present invention is not particularly limited as long as it can detect the above marker, but is usually a polynucleotide or an antibody. Therefore, a composition comprising such a polynucleotide or antibody can be used to predict the postoperative prognosis or metastatic potential of cancer patients.
  • the polynucleotide probe according to the present invention includes (i) a polynucleotide having the nucleotide sequence shown in SEQ ID NOs: 1 to 45, (ii) a polynucleotide complementary to the polynucleotide of (i), (Iii) A polynucleotide variant of (i) and (ii), a polynucleotide of (iV) (i) or a polynucleotide of (ii) or a polynucleotide of (iii) hybridized under stringent conditions And (V) at least one, preferably at least 2, more preferably 3-4 selected from the group consisting of a fragment comprising 15 or more contiguous bases of said polynucleotide or variant. Includes 5 probes, for example 4-4 5 or 5-4 5 probes.
  • the cancer metastasis-related gene markers according to the present invention can be divided into the following two groups according to the prognosis status (survival
  • group 1 is a case where the postoperative prognosis of cancer patients is worse, and the markers involved in this group are the gene groups of SEQ ID NOs: 1 to 27, while group 2 is postoperatively of cancer patients The prognosis is better, and the marker involved in this group is the gene group of SEQ ID NOs: 28-45.
  • good prognosis means that cancer is highly invasive, metastatic, or advanced, and the proportion of 5-year survivors of cancer patients determined in this way is about 7 0%
  • poor prognosis means that the cancer is less invasive, metastatic, or less advanced, and the proportion of 5-year survivors of cancer patients is about 50% or less. is there.
  • At least one of the genetic markers belonging to each group preferably at least 2, more preferably 3 to all, for example, 4 to all, 5 to all markers, Inspect with the corresponding probe.
  • Probes for detecting markers belonging to Group 1 include polynucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 27, complementary polynucleotides, mutants thereof, and stringent genes. Probes selected from the group consisting of polynucleotides that hybridize under conditions, or fragments thereof containing 15 or more contiguous bases are included.
  • a probe for detecting a marker belonging to Group 2 includes a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 28 to 45, a complementary polynucleotide thereto, a variant thereof, and a string thereto. Probes selected from the group consisting of polynucleotides that hybridize under harsh conditions, or fragments thereof containing 15 or more contiguous bases are included.
  • the present invention also includes a composition containing each of the groups related to Group 1 and Group 2.
  • a variant as a probe is one or more, preferably one or several, in the base sequence represented by SEQ ID NOs: 1 to 45, as defined above, such as substitution, deletion, addition, insertion, etc.
  • Mutants having mutations, mutants comprising a sequence having identity of usually 80% or more, 85% or more, preferably 90% or more, more preferably 95% or more, 98% or more, etc. including.
  • a polynucleotide that hybridizes under stringent conditions hybridizes to the polynucleotide or variant of (i) to (iii) above under the hybridization conditions as defined above.
  • the base sequence represented by SEQ ID NOs: 1-45 may have a gene that has been altered by biological events such as sudden mutation, polymorphism, or alternative splicing.
  • the polynucleotide allows detection of the gene.
  • the fragment of the polynucleotide has a size of 15 bases to less than the total number of bases. Fragments can have any number of bases within this range, for example 20 bases or more, 30 bases or more, 50 bases or more, 70 bases or more, 100 bases or more, 150 bases or more, 20 bases or more The number of bases, such as 2500 bases or more.
  • the polynucleotide probe used in the present invention can be synthesized by a conventional chemical DNA synthesis technique or a gene recombination technique.
  • the polynucleotide is a DNA molecule having about 100 bases or less, it can be synthesized using a DNA automatic synthesizer (for example, Applied biosyms, USA) using the phosphoramidite method.
  • a DNA automatic synthesizer for example, Applied biosyms, USA
  • the polynucleotide can be produced by cDNA cloning. Obtain total RNA from the target cancer tissue, obtain poly A (+) RNA by oligo d T cellulose column treatment, and then c DN A live by reverse transcriptase-polymerase chain reaction (RT-PCR) method Create a rally from this library and register it in a data bank such as Gen Bank or ni Gene
  • an antibody may be used as a probe.
  • antibodies include the following.
  • an antibody or a fragment thereof against a polypeptide selected from the group consisting of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 1-45 or a polypeptide encoded by a variant thereof, or a fragment thereof,
  • an antibody or fragment thereof against a polypeptide selected from the group consisting of the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOS: 1 to 27 or a polypeptide encoded by a variant thereof, or a fragment thereof;
  • the present invention provides an antibody or fragment thereof described in (i), (ii) and (iii) above, preferably at least one selected from each of (ii) and (iii), preferably Is at least 2, more preferably 3 to all, eg 4
  • the antibodies used in the present invention include polyclonal antibodies, monoclonal antibodies, anti-peptide antibodies and the like.
  • Antibody fragments include F ab, (F ab ′) 2 , F c,
  • F c ', F d, F v, etc. are included. These antibody fragments can be obtained, for example, by limited degradation of the antibody with a protease such as papain or pepsin.
  • Polypeptides or fragments thereof corresponding to each gene are synthesized using protein synthesis or gene recombination techniques, and the resulting polypeptides or fragments thereof are used as antigens for rabbits, mice, rats, horses, horses, etc. Immunize animals such as sheep, goats, and hidges, and produce and purify antibodies against these antigens.
  • a polyclonal antibody immunizes the animal subcutaneously with an antigen of about 10 to 300 g, further boosts about 2 weeks later, collects blood about 3 weeks to 1 month after the first immunization, Ig G component containing the desired polyclonal antibody from serum, ammonium sulfate fractionation, ion exchange It can be made by a method that includes separation using chromatography. In order to increase specificity, the obtained IgG was bound to a column made by binding the target protein to a carrier such as cellulose or agarose, and then eluted with a high salt concentration buffer.
  • a specific polyclonal antibody can be obtained by desalting by a method such as ultrafiltration.
  • Antibody titers can be determined by conventional immunoassays, such as enzyme immunoassays (EIA, ELISA), radioimmunoassays (RIA), fluorescent antibody methods, luminescence immunoassays, etc. It can be measured by a conventional method such as a sandwich method, a latex aggregation method, a latex turbidimetric method, a red blood cell agglutination method, a latex agglutination method, etc.
  • EIA enzyme immunoassays
  • RIA radioimmunoassays
  • fluorescent antibody methods luminescence immunoassays, etc. It can be measured by a conventional method such as a sandwich method, a latex aggregation method, a latex turbidimetric method, a red blood cell agglutination method, a latex agglutination method, etc.
  • a monoclonal antibody can be prepared, for example, by the following general method.
  • the target polypeptide or a fragment thereof is administered subcutaneously to mice or rats (for example, BalbZc mice) in the same manner as polyclonal antibodies, and boosted approximately 1 to 4 times at intervals of 1 to 4 weeks. Do. When the antibody titer reaches its peak, the antigen is injected intravenously or intraperitoneally for final immunization. After 2 to 5 days, antibody-producing cells (eg, spleen cells or lymph node cells) are collected.
  • antibody-producing cells eg, spleen cells or lymph node cells
  • the antibody-producing cells are then fused to a myeloma cell line (preferably a hypoxanthine 'guanine' phosphoribosyl 'transferase (HG PRT) deficient cell line) to produce hybridoma cells and HAT (hypoxanthine, aminopterin). , Chimin) Make a selection.
  • a myeloma cell line preferably a hypoxanthine 'guanine' phosphoribosyl 'transferase (HG PRT) deficient cell line
  • HAT hypoxanthine, aminopterin
  • Chimin make a selection.
  • antibody-producing cells and myeloma cell lines are mixed at a ratio of about 1: 1 to 20: 1 in animal cell culture media such as serum-free DMEM and RPM I-1640 media.
  • a cell fusion promoter such as polyethylene glycol. Confirmation of the target antibody can be performed by the immunoassay described above.
  • Antibody purification can be performed by appropriately combining methods such as ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and gel filtration chromatography.
  • An anti-peptide antibody is an antibody against a linear peptide on the surface of a protein, and can increase immunological specificity.
  • Such peptides are for example K yte—Doolittle et al.
  • the estimated peptide can then be synthesized using a peptide synthesizer.
  • the synthesis of the target polypeptide was carried out by incorporating the c DN clone into the expression vector and transformed or transfected with the vector. It can be obtained from the prokaryotic or eukaryotic host cell by culturing the cell or culture supernatant. Commercially available expression vectors can be used. Host cells include prokaryotic cells such as bacteria (eg, Enterococcus, Bacillus subtilis, Pseudomonas bacteria, etc.), yeast (eg, Saccharomyces, Pichia, etc.), insect cells (eg, S f cells), mammalian cells (eg, CHO).
  • prokaryotic cells such as bacteria (eg, Enterococcus, Bacillus subtilis, Pseudomonas bacteria, etc.), yeast (eg, Saccharomyces, Pichia, etc.), insect cells (eg, S f cells), mammalian cells (eg, CHO).
  • the vector consists of plasmids, cosmids, phages, etc., DNA encoding the target polypeptide, promoter, enhancer if necessary, polyaduration signal, ribosome binding site, replication origin, terminator, selection marker Can be included.
  • a DNA sequence encoding a labeled peptide such as a 6 to 10 residue histidine tag, a FLAG, a GFP polypeptide, etc., can also be included.
  • the gene recombination techniques are described in Sambrook et al. (Above) and Ausbe1 et al. (Above), and the techniques described therein can be used for the present invention.
  • the target polypeptide obtained as described above is gel filtration, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, isoelectric focusing, electrophoresis, ultrafiltration, salting out, It can be purified by appropriately combining dialysis.
  • the composition may be in the form of a kit or a microarray, that is, the probe is included in the form of a kit or a microarray.
  • polynucleotides or antibodies capable of detecting the total number of markers from one or more of each of the two groups of genetic markers (Tables 1 and 2), individually or in combination of two or more It can be packaged in a suitable container.
  • the antibody is a polyclonal antibody, a monoclonal antibody, an anti-peptide antibody or the like produced by the above method, but is not limited thereto.
  • the type of antibody may be any type, class, or subclass, for example, IgG, IgM, IgE, IgD, IgA, IgGl, IgG2, IgG3, Including I g G 4, I g A l, I g A 2 and the like.
  • Antibody fragments include F ab, (F a b ′) 2 , F c, F d, F v and the like.
  • the kit may further contain reagents for performing hybridization, such as a buffer, a reverse transcriptase, and a labeled secondary antibody.
  • reagents for performing hybridization such as a buffer, a reverse transcriptase, and a labeled secondary antibody.
  • the array is a DNA microarray (also known as a DNA chip), a tissue array, or a protein microarray.
  • Each of these microarrays is bound with the above-mentioned polynucleotide as a probe or the above-described antibody or fragment thereof. That is, on the surface of the array, one of the above gene markers or a transcription or translation product can be detected, a polynucleotide that can be hybridized with a gene marker or a variant thereof, or a polynucleotide encoded by these genes. An antibody or fragment thereof that can specifically bind to a peptide or a variant or derivative thereof is bound as a probe.
  • glass or resin polymer
  • polymer polymer
  • poly L-lysine, silane or densified amino groups are introduced on the surface.
  • the polynucleotide or antibody is bound to the substrate by a spot method or an ink jet method.
  • Variants are generally 80% or more, 85% or more, preferably 90% or more at the nucleotide or amino acid level with the complete or partial sequence of the above gene or polypeptide. More preferably, it has 95% or more identity and 98% or more identity.
  • Derivatives of polypeptides include, for example, chemically modified derivatives such as glycosylation, phosphorylation, sulfation, alkylation, and acylation.
  • the method of the present invention is a method for predicting the postoperative prognosis or metastasis potential of a cancer patient after surgery, wherein the base sequence shown in SEQ ID NOs: 1 to 45 in a biological specimen derived from the patient Alternatively, at least one expression level of a cancer metastasis-related gene marker containing the mutant sequence or its transcriptional or translational product level is measured using a probe corresponding to the marker, so that the prognosis after surgery is improved.
  • Postoperative prognosis or metastasis is possible using the relative difference in level between patients with good or lower metastatic potential and those with worse postoperative prognosis or higher metastatic potential. Including determining gender.
  • the expression level is the transcription or translation product level for the expression of the above-mentioned gene in a biological specimen, and a specimen from a patient group with a better postoperative prognosis or a lower possibility of metastasis. It means the difference in the expression of the gene when comparing the expression of the gene with a specimen from a patient group with a later prognosis or a higher possibility of metastasis.
  • the difference in gene expression level can be made by measuring the presence or amount of the gene or its corresponding polypeptide as a marker. Therefore, simply measuring the presence of each marker classified into two groups can predict the postoperative prognosis or metastatic potential of cancer patients in vitro.
  • Biological specimens include cancer tissue or cells that have been removed by surgery, tissue or cells obtained by biopsy, and the like.
  • the above-mentioned polynucleotide hybridized with each marker is used.
  • the number of genes to be detected is 1 or 2 or more per group, preferably 2 or more, more preferably 3 to the total number, for example, 4 to the total number, 5 or more to the total number.
  • the number of genes to be detected The more it is, the better the prediction accuracy.
  • Hybridization should be performed by microarray method, blotting method such as Northern or Southern blot, Northern or Southern hybridization method, insitue hybridization method, quantitative RT_PCR method, etc. Can do.
  • Preferred hybridization methods are microarray, quantitative RT-PCR or blotting. Examples of preferred microarrays are DNA microarrays and protein microarrays.
  • the solid phase includes, for example, glass, polymer, and the like, and a spacer including a reactive group for covalently binding a nucleic acid can be introduced with a crosslinker. Since such chips are commercially available, it is desirable to use them.
  • the solid phase immobilization of the nucleic acid probe is not particularly limited, but a general method, for example, a method of spotting DNA using a high-density dispenser called a spotter or layerer, a droplet is ejected from a nozzle It can be carried out using methods such as an inkjet method.
  • DNA microarrays are prepared by labeling nucleic acids such as DNA or RNA in biological specimens and cDNA and cRNA derived from them with fluorescent substances such as Cy dye (Cr3 or Cy5). Hybridize with the above probe. The fluorescence intensity is read using a laser scanning reader and the data is analyzed by a computer.
  • the nucleic acid probe of the present invention is combined with a radioisotope (for example: ⁇ 2 ⁇ and
  • primers are annealed with cDNA and PCR is performed so that the target gene region can be amplified using cDNA prepared from RNA in biological samples as a template.
  • the detected double-stranded DNA is detected.
  • the ability to pre-label primers with radioisotopes or fluorescent materials, or by electrophoresis of PCR products on agarose gel and staining of double-stranded DNA with ethimubu-mide etc. Can be detected and quantified.
  • PCR conditions include, for example, denaturation: 92-94 ° C. for 30 seconds to 5 minutes; annealing: 50-55 ° C. for 30-60 seconds; extension: 68-72 ° C. It includes 30 to 40 cycles of reaction, with 30 seconds to 10 minutes as one cycle.
  • Reverse transcriptase is a commercially available enzyme such as Super Script® III (Invitrogen, USA) ⁇ AM VR everse ⁇ , ranscriptase, Promega, USA), M-ML V (RN ase H_) (Takara Shuzo) , Kyoto, Japan) can be used.
  • the hybridization may be any of DNA-DNA hybridization, DNA-RNA hybridization, and RNA-RNA hybridization.
  • Hybridization is usually performed under stringent conditions.
  • stringent conditions include, for example, 1 M sodium chloride Z0.5% (WZV) sarkosyl
  • hybridization condition may be, for example, about 45 to 50 at 2 to 6 XSSC followed by 0.2 to 2 at about 50 to 65 ° C.
  • An alternative method for measuring the expression level of the gene is an immunological method.
  • the antibodies produced as described above can be used for the detection of the target polypeptide or fragment thereof in a biological specimen.
  • Polypeptide By creating protein microarrays with many antibodies bound on a microarray substrate, or by spotting many antibodies on a filter such as a PVDF membrane, Polypeptide can be detected or quantified. Or, conventional immunological methods such as enzyme immunoassay (ELISA, EIA), fluorescent antibody method, radioimmunoassay (RIA), luminescence immunoassay, immunoturbidimetric method, latex agglutination, latex ratio A target polypeptide or a fragment thereof in a biological sample can be detected or quantified by a turbidity method, a hemagglutination reaction, a particle agglutination reaction or a Western plot method.
  • a polymer film such as polystyrene, polycarbonate, or polyethylene
  • a plate such as polystyrene, polycarbonate, or polyethylene
  • a plate such as polystyrene, polycarbonate, or polyethylene
  • a plate such as polystyrene, polycarbonate, or polyethylene
  • a plate such as polystyrene, polycarbonate, or polyethylene
  • a plate such as polystyrene, polycarbonate, or polyethylene
  • a plate such as polystyrene, polycarbonate, or polyethylene
  • a plate such as polystyrene, polycarbonate, or polyethylene
  • a plate such as polystyrene, polycarbonate, or polyethylene
  • a plate such as polystyrene, polycarbonate, or polyethylene
  • a plate such as polystyrene, polycarbonate, or polyethylene
  • a plate such as polystyrene, polycarbonate, or polyethylene
  • the antibody may be labeled, or a labeled secondary antibody may be used.
  • Labels include enzymes such as horseradish rust peroxidase and alkaline phosphatase, fluorescent substances such as fluorescein, rhodamine and derivatives thereof, luminescent substances such as luciferase and luminol, 32 P, 1 25 I, 35 Radioactive isotopes such as S are included. Labeling includes, for example, the dartal aldehyde method, the maleimide method, the pyridyl disulfide method, the chloramine T method, and the Bolton Hunter method.
  • the present invention further relates to inhibition or suppression of a cancer metastasis-related gene comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 27 or a transcription product thereof using cultured cancer cells, or the motility of cultured cancer cells and A screening method for a cancer metastasis inhibitor comprising screening a candidate drug for inhibition or suppression of Z or invasive ability is provided.
  • this method comprises preparing cultured cancer cells, culturing the cells in the presence of a candidate drug, and inhibiting or suppressing the expression of the cancer metastasis-related gene or a transcription product thereof, or Screening candidate drugs for inhibition or suppression of motility and / or invasive capacity.
  • the cell line of human cultured cancer cells is not particularly limited by the type of cancer, for example, a known metastatic human cancer cell line, MeWo; Tumor cell line, MDA-MB-435; breast cancer cell line, LNCaP or PC-3; highly metastatic human lung cancer cell line, LNM35; prostate cancer cell line, etc., which can be used in the present invention .
  • the degree of inhibition or suppression of expression of the human cancer metastasis-related gene or its transcription product can be determined by a comparative experiment with a control to which no candidate drug is added.
  • Expression levels were obtained from cancer cell lines by well-known methods (for example, extraction with phenol, blackform Z, soamyl alcohol, guanidinium Z, cesium chloride, etc., ethanol precipitation, oligo d T cellulose column chromatography, etc.).
  • a hybridization method using fluorescent or radiolabeled probes for RNA or mRNA or poly A (+) RNA, or for cDNA synthesized from RNA by the reverse transcriptase — PCR (RT-PCR) method (For example, Northern hybridization, Southern hybridization, DNA microarray, yarn and fabric microarray, etc.).
  • RT-PCR reverse transcriptase — PCR
  • the expression level is measured by measuring the intracellular level of the polypeptide encoded by the human cancer metastasis-related gene by an immunoassay using an antibody against the polypeptide or a fragment thereof, Western high-pridition, etc. Can be determined indirectly.
  • the probe is a nucleotide sequence of SEQ ID NOs: 1-45 or a sequence complementary thereto, or a sequence thereof, for example, about 20 or more, about 30 or more, 50 or more, 70 or more, 100 or less. Above, DNA having a sequence consisting of 1550 or more, 2200 or more, and 2500 or more nucleotides.
  • the probe is preferably a labeled probe bound with a fluorescent or radioactive label.
  • Fluorescent labels include, for example, fluoresamine, P-damine, their derivatives, Cy3, Cy5, etc.
  • Radiolabels include, for example, radioactive phosphorus or iow atoms.
  • An immunoassay is an analysis method that uses an antigen-antibody reaction, and is performed by a method that appropriately combines, for example, an enzyme-linked antibody method (for example, ELISA), a fluorescent antibody method, a solid phase method, a homogeneous method, and a sandwich method. Can do. These methods are well known in the art and their conventional techniques can be used in the present invention.
  • Human culture In cancer cells, the expression of the human cancer metastasis-related gene or the corresponding mRNA is significantly inhibited or suppressed by the presence of the candidate drug compared to the control without the candidate drug.
  • the candidate drug can be identified as a cancer metastasis inhibitor.
  • Candidate drug screening can also be found by examining inhibition or suppression of motility and Z or invasive potential of human cultured cancer cells, preferably metastatic cancer cells.
  • In vitro motility or invasion of cancer cells can be performed using, for example, a transwell chamber culture system.
  • a transwell chamber culture system for example, an insert (made by Becton Dickinson) with a polyethylene terephthalate film having a small pore of 8 microns in diameter is inserted into a 24-well cell culture plate, and serum-free cells inside the insert are inserted.
  • Inoculated with metastatic cancer cell lines in the medium cultured for 24 hours, and then counted by counting the number of cells that have passed through the small pores and moved to the lower membrane (Kozaki K et al., Cancer Research) 6 0: 2 5 3 5-4 0, 2 0 0 0).
  • the invasion assay uses a similar system, but can be measured by covering the top of the membrane with Matrigel before seeding the cells (Kozaki K et al., Cancer Research 60: 2). 5 3 5-4 0, 2 0 0 0).
  • Candidate agents include, but are not limited to, small molecules, peptides, polypeptides, proteins, nucleosides, oligonucleotides, polynucleotides, nucleic acids (DNA or RNA), and the like.
  • the polypeptide or protein as a candidate drug includes, for example, an antibody or a fragment thereof against the polypeptide or protein encoded by the base sequence shown in SEQ ID NOs: 1 to 27 or a mutant sequence thereof.
  • the nucleic acid includes a ribozyme capable of cleaving mRNA corresponding to the nucleotide sequence shown in SEQ ID NOs: 1 to 27 or a mutant sequence thereof, s iRNA (sma lin terf er RN RN), and the like.
  • the GC content is about 30 to about 70%, preferably about 50%
  • Criteria can be used such that all nucleotides are equal and G is not contiguous
  • the 5 ′ end nucleotide of the antisense strand is A, U, etc.
  • the ribozyme as a candidate drug is RNA having catalytic activity and has an activity of cleaving mRNA corresponding to the target human cancer metastasis-related gene according to the present invention. This cleavage inhibits or suppresses the expression of the gene.
  • the candidate triplet is Ribozymes containing a sequence complementary to the portion of the mRNA sequence to be cleaved can be used for cleaving the target mRNA.
  • Ribozymes include hammerhead type ribozymes.
  • Hammer one heads type Ribozaimu includes areas nucleotide sequence, which may only form a cavity for capturing a stable Mg 2 + ions when RN A to sensor-site bound constituting the sensor portion Nukure And a nucleotide sequence comprising a region that is complementary to the sequence surrounding the cleavage site of the target RNA.
  • a low-metastasis strain (N 15) was obtained from the parent strain N C I — H 4 60 cells by the limiting dilution method. Furthermore, by treating L NM 3 5 with a demethylating agent (5 —aza—2 '—deoxycytidine) (1 // 1 ⁇ for 2 4 hours) in the same culture medium as above, low Transfer strains (L 2 D 2 and L 2 D 3 A) could be obtained.
  • a demethylating agent (5 —aza—2 '—deoxycytidine) (1 // 1 ⁇ for 2 4 hours
  • the motility assay is a transwelch filter with a filter of 8 m. Using a bar, the number of cells migrated to the back of the filter after counting for 24 hours was counted.
  • the invasion ability is measured using a transwell chamber in which the gel is coated on a filter with 8 ⁇ m micropores and counts the number of cells that have migrated to the back of the filter after 24 hours of incubation. I went by the procedure.
  • Lymph node and lung metastases were determined by weight and number of masses, respectively.
  • the LNM3 5 strain showed the highest levels of both motility and invasion, and also showed high levels of lymph node and lung metastasis. I confirmed that there was.
  • the parent strains showed very low motility, invasive ability, lymph node metastasis, and lung metastasis, confirming that they were low metastasis cancer cell lines.
  • RNA was caged, and an oligo-d T primer and Super Scriptl I reverse transcriptase were used to produce 100 ⁇ Ci of [ 33 P] d CT. Reverse transcription in the presence of P, and c DN A microa'ray (Gen F i 1 ter Human M icroarrays manufactured by Invitrogen)
  • LNM3 5 and N 15 are each 2 times, and in the total 4 times of expression profiles, spots whose expression level (scanned spot value) is 0.1 or less are excluded from analysis due to detection limit. did.
  • the expression profiles of LNM35 and N15 were compared, and spots with an expression difference of 2 SD (twice the standard deviation) or more were extracted.
  • the expression level of the two expression profiles of LNM35 was both higher than 1.0 in the spot whose expression was enhanced by LNM35.
  • the spot whose expression was enhanced by N 15 when searching for spots that met the condition that the expression values of the two expression profiles of N 15 were both greater than 1.0, 45 Spots corresponding to these genes were extracted.
  • the expression value was determined from the average of the two times.
  • the dots shown in red or green in the figure indicate the expression status of each gene in individual cases.
  • the red color indicates that the gene expression is relatively high compared to other cases, and the green color indicates that the gene expression is relatively low.
  • the dendrograms in the figure show similarity. If the degree of similarity is high, the branches indicating the connection of the dendrogram are short, and if the degree of similarity is low, the branch indicating the connection of the dendrograms is long.
  • the cases with the highest similarity are linked in order, and are finally compiled into a single tree diagram.
  • Fig. 5A shows the results of a cluster analysis using data from 79 breast cancer patients acquired at the National Cancer Institute.
  • a clustering analysis of 79 breast cancer specimens revealed that they were classified into two large groups: left and right.
  • One group on the left is the power of many highly expressed genes (red dots)
  • the prognosis of the group in which the expression of the gene group whose expression is enhanced in LNM 35 is relatively high (Fata 1 group) is poor, and the expression of the gene group in which the expression is suppressed in LNM35 is relatively
  • the prognosis of the high group (Fa V orab 1 e group) was good, and it could be confirmed using data from human breast cancer cases obtained at the National Cancer Institute of the Netherlands.
  • the signal noise function (signal — to-noisemetrics, Liolub et al., Science, Vol. 28 6, pp 5 3 1 to 5 3 7, 1 9 99) Using.
  • the cancer case group is defined as c 1 ass 1 for cancer with good postoperative prognosis and c 1 ass 2 for cancer with poor postoperative prognosis
  • the weight S which is the signal-noise statistic is given by Is calculated.
  • ci ass i represents the average value of all expression intensity data of c 1 ass 1
  • ch ss 2 represents the average value of all expression intensity data of c 1 ass 2
  • classl represents c 1
  • the standard deviation of all expression intensity data of ass 1 is shown
  • class 2 shows the standard deviation of all expression intensity data of class 2 .
  • a weighted vote for gene x is calculated using the following W eighted-Voting formula.
  • V X S (G x -b J
  • V x is a weighted vote for gene x
  • S is a weight calculated by the above equation
  • G x is the expression intensity (or expression level) of gene X
  • b x is
  • genes (Gene) A, B, C (and above) Measure the expression intensity (or expression level) of genes (G ene) X, ⁇ , ⁇ (genes characterized by poor postoperative prognosis) The average value () and standard deviation ( ⁇ ) of the expression intensity of each gene of the five samples of the group are calculated, and the weight (S) and the center (b x ) are calculated from the above formula.
  • sample A and sample B to be typed For sample A and sample B to be typed, measure the expression intensity (or expression level) of each gene, calculate the weight (S), G x — b x from the above equation, and obtain each V x. Find the sum of V x of 6 genes.
  • the present invention can predict the possibility of cancer metastasis and prognosis in cancer patients, and thus can greatly contribute to cancer treatment planning and prognosis management. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

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Abstract

La présente invention concerne un procédé permettant de prédire in vitro le pronostic postopératoire ou le risque métastasique d’un patient cancéreux. Ledit procédé comprend la mesure du degré d’expression d’au moins un marqueur génétique lié aux métastases cancéreuses dans un échantillon biologique prélevé auprès du patient, ou la mesure du taux de produit de transcription ou de traduction du marqueur, en utilisant une sonde correspondant au marqueur. Le procédé selon l’invention comprend ensuite la détermination du pronostic postopératoire ou du risque métastasique en se basant sur la différence du taux relatif mentionné plus haut entre un groupe de patients présentant un meilleur pronostic postopératoire ou un risque métastasique plus faible et un autre groupe de patients présentant un moins bon pronostic postopératoire ou un risque métastasique plus élevé en tant qu’indication; la présente invention a également trait à une composition permettant de prédire le pronostic postopératoire ou le risque métastasique, laquelle contient une sonde telle que décrite ci-dessus.
PCT/JP2006/326410 2005-12-28 2006-12-28 Composition et procede permettant de predire le pronostic postoperatoire ou le risque metastasique d’un patient cancereux Ceased WO2007077977A1 (fr)

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