WO2007043531A1 - Marqueur génétique destiné à être utilisé dans le diagnostic de sensibilité à un agent antitumoral ou à une chimiothérapie concurrente et utilisation de celui-ci - Google Patents

Marqueur génétique destiné à être utilisé dans le diagnostic de sensibilité à un agent antitumoral ou à une chimiothérapie concurrente et utilisation de celui-ci Download PDF

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Publication number
WO2007043531A1
WO2007043531A1 PCT/JP2006/320225 JP2006320225W WO2007043531A1 WO 2007043531 A1 WO2007043531 A1 WO 2007043531A1 JP 2006320225 W JP2006320225 W JP 2006320225W WO 2007043531 A1 WO2007043531 A1 WO 2007043531A1
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Prior art keywords
polynucleotide
seq
combination therapy
sensitivity
radiation
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PCT/JP2006/320225
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English (en)
Japanese (ja)
Inventor
Hirofumi Doi
Kensaku Imai
Yasuo Uemura
Masakazu Fukusima
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Celestar Lexico-Sciences, Inc.
Taiho Pharmaceutical Co., Ltd.
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Application filed by Celestar Lexico-Sciences, Inc., Taiho Pharmaceutical Co., Ltd. filed Critical Celestar Lexico-Sciences, Inc.
Priority to JP2007539949A priority Critical patent/JPWO2007043531A1/ja
Publication of WO2007043531A1 publication Critical patent/WO2007043531A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a genetic marker for susceptibility diagnosis of an anti-tumor agent and radiation therapy, and more specifically, to examine the presence or absence of a specific polynucleotide in a biological sample collected from a subject such as a cancer patient. Whether or not it is sensitive to the combination therapy of tegafur, gimeracil and oteracil potassium combined antitumor agent and radiation
  • the present invention relates to a polynucleotide that functions as a genetic marker for rapidly diagnosing whether or not the combination of the above-described antitumor agent and radiation therapy is effective for treating cancer in a subject, and use thereof.
  • TS-1 tegafur 'gimeracil' oteracil potassium
  • S-1 S-1
  • Non-patent Document 1 an excellent cancer treatment method by combining the antitumor agent with radiation therapy is known.
  • Patent Document 1 Japanese Patent Publication No. 5-64123
  • Patent Document 2 Japanese Patent Publication No. 5-80451
  • Patent Document 3 Japanese Patent No. 2538422
  • Patent Document 4 Japanese Patent No. 2614164
  • the present invention has been made in view of the above-mentioned problems, and is a means for quickly and accurately diagnosing the sensitivity of an anti-tumor agent containing tegafur and gimeracil potassium terracil potassium and radiation therapy. It is intended to provide.
  • the present inventors provide a polynucleotide used as a primer or probe for the polynucleotide as the gene marker, a polypeptide encoded by the polynucleotide, and an antibody that recognizes the polypeptide.
  • the present inventors have found that it is useful for diagnosing the above-mentioned combination therapy sensitivity more quickly and accurately.
  • diagnostic agents and diagnostic kits containing the above primers, probes, and antibodies can more easily diagnose sensitivity.
  • the present invention has been completed based on such knowledge.
  • the present invention has the following [1] to [13] forces.
  • [1] A polynucleotide represented by the following (A) or (B).
  • (C) a nucleotide sequence that is hybridized under a highly stringent condition with the polynucleotide according to (A) or (B), and a tegafur / gimeracil / oteracil potassium-conjugated antitumor agent and radiation
  • one or a plurality of bases includes a base sequence in which deletion, substitution, insertion, and Z or addition are present, and tegafur 'gimeracil 'Polynucleotides that function as genetic markers for diagnosing sensitivity to combination therapy with anti-tumor agents containing oteracil potassium and radiation
  • (C) a nucleotide sequence that is hybridized under a highly stringent condition with the polynucleotide according to (A) or (B), and a tegafur / gimeracil / oteracil potassium-conjugated antitumor agent and radiation
  • one or a plurality of bases includes a base sequence in which deletion, substitution, insertion, and Z or addition are present, and tegafur 'gimeracil 'Polynucleotides that function as genetic markers for diagnosing sensitivity to combination therapy with anti-tumor agents containing oteracil potassium and radiation
  • a polynucleotide comprising at least 10 consecutive nucleotides in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 70 in the Sequence Listing
  • polynucleotide according to (1) or (2) above which comprises a nucleotide sequence in which one or more bases are missing, substituted, inserted, and Z or added, and [1] A polynucleotide that functions as a probe or primer for the described polynucleotide
  • step (II) RNA or complementary polynucleotide bound to the polynucleotide in step (I) The step of measuring the presence or absence of the above-mentioned [3] to [5] using the polynucleotide described in any one of the above as an indicator
  • step (ii) a step of measuring the presence or absence of the polypeptide bound to the antibody or its partial peptide in step (i) using the antibody as an index
  • Step (iii) Based on the measurement results of step (ii), a diagnosis of the sensitivity of combination therapy with tegafur and gimeracil * oteracil potassium combined antitumor agent and radiation
  • FIG. 1 is a diagram showing an outline of the procedure up to the computer analysis after the creation of a cDNA library in the examples.
  • FIG. 2 is a diagram showing an outline of a procedure of array annotation processing in the embodiment.
  • FIG. 3 is a diagram showing an outline of a procedure for mapping genomes to clones and thereby clustering in Examples.
  • FIG. 4 is a graph showing the ratio of the amplification amount of the radiation treatment group and the combination treatment group in the example to the amplification amount of the control group.
  • the present invention relates to an antitumor agent containing tegafur, gimeracil, and oteracil potassium, and a genetic marker for diagnosing radiation therapy sensitivity.
  • the combination therapy of the antitumor agent and radiation means a cancer treatment method using a combination of radiotherapy with the antitumor agent.
  • the antitumor agent used in the combination therapy targeted by the present invention contains tegafur, gimeracil and oteracil potassium together with a pharmaceutically acceptable carrier, and tegafur: gimeracil: It is characterized by containing oteracil potassium in a molar ratio of 1: 4: 4: 1.
  • tegafur is a known drug that is known to release the active substance 5-fluorouracil (5-FU) upon receiving an in vivo activity.
  • the production method is not particularly limited, and a known method, for example, the method described in JP-B-49-10510 can be followed.
  • Gimeracil (2,4-dihydroxy-5-cloguchil pyridine) is a known compound that exerts an action to enhance the antitumor effect of 5-FU.
  • the production method is not particularly limited, and those produced according to known methods can be used.
  • Oteracil potassium (oxonic acid potassium) suppresses side effects such as inflammation caused by the above-mentioned tegafur.
  • Oteracil potassium itself is a known compound.
  • the pharmaceutically acceptable carrier include various types of commonly used drugs such as fillers, extenders, binders, disintegrants, surfactants, lubricants and the like. Can be illustrated.
  • an antitumor agent a commercially available product can be used. Specifically, it is manufactured and sold by Taiho Pharmaceutical Co., Ltd.! The commercially available products “TS1 capsule 20” and “TS1 capsule 25”.
  • TS-1 is a registered trademark of Taiho Pharmaceutical Co., Ltd., one of the applicants of the present application).
  • the dosage unit form of the antitumor agent is not particularly limited as long as it is a non-injectable form, and can be selected according to the therapeutic purpose. Specifically, tablets, coated tablets, pills, powders, granules And oral agents such as pushells, solutions, suspensions, emulsions, and parenterals such as suppositories, ointments, patches, etc., and these administration agents are conventional formulations commonly known in this field. Formulated by the method.
  • the administration method of the antitumor agent is not particularly limited as long as it is non-injection administration such as enteral administration, oral administration, rectal administration, buccal administration, transdermal administration, etc. It is determined according to gender and other conditions, the degree of symptoms of the patient, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are administered orally. Suppositories are administered rectally. The ointment is applied to the skin, oral mucosa and the like.
  • the administration conditions of the antitumor agent can be appropriately selected depending on the usage, age, sex and other conditions, the degree of disease, and the like.
  • the amount of tegafur is about 0.1 to lOOmgZkgZ days, preferably about 1 to 30 mgZkgZ days, and the amount of gimeracil is about 0.1 to about LOOmgZkgZ days, preferably 1 to 50 mgZkgZ.
  • the amount of oteracil potassium is about 0.1 to about LOOmgZkgZ days, preferably about 1 to 40 mgZkgZ days.
  • These preparations of the present invention can be administered once a day or divided into about 2 to 4 times a day.
  • the amount of tegafur is about 1 to about LOOmgZkg, inserted once or twice a day at intervals of 6 to 12 hours and administered.
  • radiation therapy is cancer treatment by irradiating an affected area with radiation such as X-rays.
  • Radiation irradiation conditions can be appropriately selected depending on age, sex, degree of disease, and the like. For example, radiation therapy is performed 20-30 times (about 4-6 weeks) in one course. Can be set to 40-60 Gy for the entire network.
  • the irradiation site is not particularly limited as long as it contains a tumor tissue, but it is preferable to perform local irradiation on the tumor site.
  • the subject of the combination therapy of the antitumor agent and radiation is a cancer patient.
  • the types of cancer are not particularly limited, for example, head and neck cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gallbladder's bile duct cancer, spleen cancer, lung cancer, breast cancer, bladder cancer, prostate cancer, cervical cancer, etc. Is mentioned.
  • lung cancer is particularly preferred, with head and neck cancer, lung cancer and spleen cancer being preferred.
  • Conditions for performing the above-described combination therapy with an antitumor agent and radiation can be appropriately set depending on conditions such as age, sex, and degree of disease.
  • combination therapy sensitivity means individual differences in therapeutic effects in cancer patients who have undergone combination therapy. In other words, when a high therapeutic effect is obtained by combination therapy, the sensitivity is high. On the other hand, when a sufficient therapeutic effect is not obtained or no therapeutic effect is obtained, the sensitivity is low. .
  • Diagnosis of sensitivity to combination therapy means evaluation, identification, detection, judgment, and prediction of the presence / absence or level (level) of sensitivity to the above combination therapy based on some criteria.
  • the gene marker means a genetic index or predisposing factor for determining the sensitivity of a living body. That is, in the present invention, the gene marker for diagnosing susceptibility to a combination therapy of the above-described antitumor agent and radiation means that, as a result of the combination therapy, the expression level of the gene changes in response to sensitivity, etc. It indicates a genetic indicator or predisposing factor.
  • the present invention relates to a gene marker for diagnosing the sensitivity of such a combination therapy of a predetermined antitumor agent and radiation, and a polynucleotide (gene marker polynucleotide) functioning as the marker, Polynucleotides encoded by nucleotides, antibodies that recognize the polypeptides, polynucleotides that can serve as primers or probes for genetic marker polynucleotides (probes or polynucleotides for primers), and predetermined antitumors using these It provides a method for diagnosing susceptibility to combination therapy of drugs and radiation. These will be described in detail below. [0028] [1] Gene marker polynucleotide
  • the present invention first provides a novel polynucleotide.
  • the polynucleotide of the present invention is useful as a polynucleotide that functions as a genetic marker for diagnosing the sensitivity of a combination therapy of tegafur, gimeracil, and oteracil potassium, and a combination therapy with radiation (hereinafter referred to as a gene marker polynucleotide). .
  • the polynucleotide of the present invention can function as a genetic marker for diagnosing the sensitivity of a combination therapy of the antitumor agent and radiation. This is for patients whose expression level of each polynucleotide is sensitive to the above combination therapy of antitumor agents and radiation! This is due to the fact that it has a property that changes specifically before and after the combination therapy is performed. That is, the expression level of the polynucleotide of the present invention changes only in cancer patients having sensitivity to combination therapy and only when the combination therapy is applied.
  • the change in the expression level when the combination therapy is performed includes both an increase and a decrease in the expression level. That is, as a first example, if the expression level of the gene marker polynucleotide increases after the combination therapy compared to before the implementation, it is diagnosed that the combination therapy sensitivity is high when the expression level increases, and the expression level It is diagnosed that the combination therapy sensitivity is low (no change in the expression level). On the other hand, as a second example, when the expression level of the gene marker polynucleotide is decreased or not expressed at all after the combination therapy, it is diagnosed that the combination therapy sensitivity is high when it is decreased or not expressed at all. Conversely, the expression level will not decrease! / ⁇ (No change in expression level), low sensitivity to combination therapy! Diagnosed as sputum. Changes in the expression level after combination therapy can be confirmed by quantitative PCR.
  • Such changes in expression level differ depending on the base sequence constituting the marker, and even if the same marker is used, the expression level varies depending on the expression conditions, the target organism, the cancer type, etc. There are things to do.
  • RNA or DNA there is no particular limitation on whether it is RNA or DNA, its base length, whether it is single-stranded or double-stranded, circular or linear.
  • DNA it does not matter whether it is cDNA, genomic DNA, or synthetic DNA.
  • RNA it does not matter whether it is total RNA, mRNA, rRNA, or synthetic RNA.
  • a single strand it may be a sense strand or an antisense strand. It may be a double-stranded DNA, a double-stranded RNA, or a DNA: RNA hybrid.
  • the functional region is different, and may include, for example, an expression control region, a coding region, an exon, or an intron.
  • polynucleotide of the present invention include those represented by the following (A) to (D).
  • (C) a gene comprising a nucleotide sequence that can be hybridized under a highly stringent condition with the polynucleotide described in (i) or (ii) above, and a gene for diagnosing susceptibility to a combination therapy of the antitumor agent and radiation Polynucleotide that functions as a marker
  • the polynucleotide comprises one or more bases deleted, substituted, inserted, and inserted or added, and the antitumor agent and Polynucleotides that function as genetic markers for diagnosing susceptibility to combination therapy with radiation
  • the above (i) polynucleotide includes a base sequence described in any one of SEQ ID NOs: 1 to 70 in the sequence listing, and may comprise only the base sequence.
  • these polynucleotides are derived from humans by analyzing the sequence data from among those whose expression level specifically changes before and after the combination therapy with the antitumor agent and radiation. It has been confirmed that it is new.
  • SEQ ID NOs: 1 to 7, 9 to 15, 17 to 21, and 23 to 36 are preferable because they are easy to detect as markers because they are excellent in specificity in quantitative PCR.
  • polynucleotides containing the nucleotide sequences set forth in SEQ ID NOs: 1 to 33 in the Sequence Listing are selected as polynucleotides that may have the property of increasing the expression level when sensitivity to combination therapy is high (if present). It has been done.
  • a polynucleotide containing the nucleotide sequence of SEQ ID NOs: 34 to 36 may have a property that the expression level is reduced or not expressed at all when the sensitivity to combination therapy is high (existing). Selected as a nucleotide.
  • nucleotide sequences described in SEQ ID NOs: 37 to 70 are mRNA sequences predicted from the nucleotide sequences described in SEQ ID NOs: 1 to 9, 11 to 24, and 26 to 36, respectively, of the above. It is the base sequence expressed as NA.
  • the polynucleotide (B) includes a base sequence complementary to the polynucleotide described in (A). That is, it may be included in a part of the base sequence complementary to the polynucleotide described in (A) above, or only the complementary base sequence that is strong may be used.
  • the polynucleotide (C) includes a base sequence that hybridizes with the polynucleotide described in (A) or (B) under a highly stringent condition, and diagnoses the sensitivity to the combined treatment of the antitumor agent and radiation. It functions as a genetic marker for this purpose.
  • Nobbreviation can be performed according to a conventional method. Moreover, when using a commercially available library, it can be performed according to the method described in the attached instruction manual.
  • the stringent conditions are, for example, conditions in which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
  • 1 X SSC is a condition washing ⁇ of typical Southern hybrida Izeshiyon, 0. 1 0/0 SDS, preferably ⁇ or 0. 1 XS SC, salt equivalent to 0. 1% SDS concentration
  • the condition for maintaining the hybridized state even after washing with (1) is mentioned.
  • C polynucleotide examples include, for example, at least 70%, preferably 90%, more preferably 95%, particularly preferably, the polynucleotides described in (A) and (B) above.
  • the base sequence homology can be calculated by homology search, sequence alignment program, BLAST, FASTA, ClustalW, or the like.
  • the polynucleotide described in (A) or (B) above has a nucleotide sequence in which one or more bases are deleted, substituted, inserted, and Z or added. In addition, it functions as a gene marker for diagnosing the sensitivity of the above antitumor agent and radiation therapy.
  • the number of bases that may be deleted, substituted, inserted, and Z or added is one or more, for example, 1 to: about L00, preferably about 1 to 30, more preferably 1 to About 10, more preferably 1 to 5.
  • Deletions, substitutions, insertions, and Z or additions The position of the base is not particularly limited.
  • (D) polynucleotide specifically, for example, at least 70%, preferably 90%, more preferably 95%, particularly preferably 97% with the polynucleotide described in (A) or (B) above.
  • a polynucleotide comprising a base sequence having homology can be calculated in the same manner as described in (C) above.
  • Such a gene marker polynucleotide of the present invention has a function as a gene marker by being targeted for detection of a change in expression level before and after the combination therapy of the antitumor agent and radiation. Demonstrate. To determine the change in the expression level, the expression level in the subject before the combination therapy or the expression level in another patient who is insensitive or sensitive to the combination therapy is used as a standard. This can be done by calculating the statistical significance of these.
  • the present invention also provides a diagnostic method for susceptibility to the above-mentioned combination therapy, characterized by examining the presence or absence of such a gene marker polynucleotide, the details of which are as described in the following [5]. .
  • detection of the expression level of the gene marker polynucleotide of the present invention is performed by collecting a biological sample from a subject, and expressing the gene marker polynucleotide expression product in the biological sample, for example, RNA or complementary thereto. This can be done by measuring the amount of DNA. In such detection, it is convenient to use a primer or probe designed based on the gene marker polynucleotide.
  • the present invention also provides a polynucleotide useful as a primer or probe useful for such sensitivity diagnosis, and the contents thereof are as described in detail in the following [2].
  • the detection of the expression level of the gene marker polynucleotide of the present invention involves measuring the amount of the polypeptide encoded by the gene marker polynucleotide prepared from the biological sample collected from the subject. This is also feasible. In such detection, it is convenient to use an antibody that recognizes the polypeptide.
  • the present invention also provides polypeptides and antibodies useful for such sensitivity diagnosis, the contents of which are as described in detail in the following [3].
  • the gene marker polynucleotide of this invention can be obtained by a conventional method. For example, it can be amplified by PCR using the primers detailed in [2] below.
  • the present invention relates to a probe or primer of the gene marker polynucleotide of the present invention described in [1] above in diagnosing the sensitivity of a combination therapy of tegafur 'gimeracil' oteracil potassium-containing antitumor agent and radiation.
  • a polynucleotide (hereinafter, referred to as a probe or primer polynucleotide) to be used as a DNA.
  • the probe or primer polynucleotide of the present invention is not particularly limited as long as it can detect or amplify the gene marker polynucleotide of the present invention of [1] above.
  • Southern hybridization DNA Any probe that can serve as a probe for a chip or a primer for PCR such as RT-PCR may be used.
  • a polynucleotide that can specifically bind to all or part of the gene marker polynucleotide is preferable.
  • its form there is no particular limitation on whether it is RNA force DNA, its base length, single-stranded or double-stranded, circular or linear. In the case of DNA, it does not matter whether it is cDNA, genomic DNA, or synthetic DNA.
  • RNA it does not matter whether total RNA, mRNA, rRNA, or synthetic RNA. Furthermore, in the case of a single strand, it may be a sense strand or an antisense strand. good. Further, it does not ask whether the functional region is different, and may include, for example, an expression control region, a coding region, an exon, or an intron.
  • the probe or primer polynucleotide of the present invention can be designed from the gene marker polynucleotide of the present invention of [1] above.
  • a polynucleotide having at least 10 bases or more among target polynucleotides of the gene marker polynucleotide of the present invention [1] can be used.
  • the amplification product when used as a primer, for example, when PCR is used for gene amplification.
  • the amplification product may be designed as a pair of polynucleotides containing at least 10 base pairs of the gene marker polynucleotide of the present invention of [1] above.
  • the other primer is It is only necessary to select an arbitrary sequence force 5 ′ from the sequence complementary to the primer in the complementary strand of the target polynucleotide.
  • the base length of the primer is not particularly limited as long as it can specifically bind to the gene marker polynucleotide of the present invention of [1] above, but is preferably a nucleic acid fragment of 10 to 150 bases.
  • a polynucleotide comprising at least 10 consecutive nucleotides in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 70 in the Sequence Listing
  • polynucleotide according to (1) or (2) above which comprises a nucleotide sequence in which one or more bases are missing, substituted, inserted, and Z or added, and in the above [1] Polynucleotides that function as probes or primers for the described genetic marker polynucleotides
  • the polynucleotide (1) includes at least 10 consecutive nucleotides in the nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 70 in the Sequence Listing.
  • the above (1) polynucleotide when used as a primer, it preferably contains 10 to 150 bases, especially 10 to LOO bases, particularly 15 to 50 bases.
  • the above (1) polynucleotide when used as a probe, it preferably contains 10 bases or more, more preferably 20 to 300 bases.
  • the polynucleotide (2) includes a base sequence complementary to the polynucleotide described in (1). That is, it may be included in a part of the base sequence complementary to the polynucleotide described in the above (1), or only a powerful complementary base sequence may be used. There may be.
  • the polynucleotide (2) when used as a primer, it preferably contains 10 to 150 bases, especially 10 to LOO bases, particularly 15 to 50 bases.
  • when used as a probe it preferably contains 10 bases or more, more preferably 20 to 300 bases.
  • the polynucleotide (3) includes a nucleotide sequence that hybridizes with the polynucleotide described in (1) or (2) under high stringency conditions, and the gene marker polynucleotide described in [1] above It functions as a probe or primer.
  • polynucleotide specifically, for example, the polynucleotide described in (1) and (2) is at least 70%, preferably 90%, more preferably 95%, particularly preferably 97%.
  • examples thereof include polynucleotides consisting of nucleotide sequences having the same homology.
  • the base sequence homology can be calculated in the same manner as described in [1] above.
  • the polynucleotide (3) when used as a primer, it preferably contains 10 to 150 bases, especially 10 to L00 bases, particularly 15 to 50 bases.
  • it when used as a probe, it preferably contains 10 bases or more, more preferably 20 to 300 bases.
  • the polynucleotide includes the base sequence in which one or more bases are deleted, substituted, inserted, and Z or added in the polynucleotide according to (1) or (2), and It functions as a probe or primer of the gene marker polynucleotide described in [1] above.
  • the number of bases that may be deleted, substituted, inserted, and Z or added is preferably 1 or more, more preferably 1 to: L0, and further preferably 1 to 5.
  • Specific examples of such (4) polynucleotide include, for example, at least 70%, preferably 90%, more preferably 95%, and particularly preferably 97% of the polynucleotide described in (1) and (2). Examples thereof include a polynucleotide having homology and a base sequence ability. salt The base sequence homology can be calculated in the same manner as described in [1] above.
  • the polynucleotide (4) when used as a primer, it preferably contains 10 to 150 bases, especially 10 to LOO bases, particularly 15 to 50 bases.
  • the polynucleotide (4) when used as a probe, it preferably contains 10 bases or more, more preferably 20 to 300 bases.
  • primer or probe polynucleotide of the present invention include a polynucleotide comprising the base sequence set forth in any of SEQ ID NOs: 71 to 142.
  • polynucleotides having the nucleotide sequences shown in SEQ ID NOs: 71 to 142 as described below are useful as probes and primers for the above gene marker polynucleotides, particularly polynucleotides containing the nucleotide sequences described in SEQ ID NOs: 1 to 36. Yes, preferably useful as a primer.
  • Polynucleotide fOla (SEQ ID NO: 71), rOla (SEQ ID NO: 72); polynucleotide f 02a (SEQ ID NO: 73), r02a (SEQ ID NO: 74); polynucleotide f03a (SEQ ID NO: 75), r03a (SEQ ID NO: 76); Polynucleotide f04b (SEQ ID NO: 77), r04b (SEQ ID NO: 78); Polynucleotide f05a (SEQ ID NO: 79), r05a (SEQ ID NO: 80); Polynucleotide f 06a (SEQ ID NO: 81), r06a (SEQ ID NO: 82); Poly Nucleotides f 07a (SEQ ID NO: 83), r07a (SEQ ID NO: 84); Polynucleotides f08b (SEQ ID NO: 85), r08b (SEQ ID
  • polynucleotide f 13a (SEQ ID No. 95), r 13a (SEQ ID No.) 96); polynucleotide f 14a (SEQ ID NO: 97), r 14a (SEQ ID NO: 98); fl5 b (SEQ ID NO: 99), rl5b (SEQ ID NO: 100); polynucleotide f 16a (SEQ ID NO: 101), rl6 a (sequence) No.
  • polynucleotide fl7a (SEQ ID NO: 103), rl7a (SEQ ID NO: 104); Polynucleotide f 18a (SEQ ID NO: 105), rl8a (SEQ ID NO: 106); Polynucleotide f 19a (SEQ ID NO: 107), r 19a (SEQ ID NO: 108); polynucleotide f 20a (SEQ ID NO: 109), r20 a (SEQ ID NO: 110); polynucleotide f21a (SEQ ID NO: 111), r21a (SEQ ID NO: 112); polynucleotide f 22b (SEQ ID NO: 113) R22b (SEQ ID NO: 114); polynucleotide f23b ( SEQ ID NO: 115), r23a (SEQ ID NO: 116); polynucleotide f 24a (SEQ ID NO: 117),
  • Each of these polynucleotides was designed from the gene marker polynucleotides of SEQ ID NOs corresponding to the numbers in the names of the polynucleotides among the gene-powered polynucleotides containing the nucleotide sequences set forth in SEQ ID NOS: 1-36. Is.
  • Each polynucleotide name containing “f” can be used as a forward primer, and “r” can be used as a reverse primer! That is, each polynucleotide can be used as a primer for a corresponding gene marker polynucleotide as a primer pair for each semicolon segment in the above list.
  • the primer was designed in the same manner as the nucleotide sequence set forth in SEQ ID NO: 71-142 from the nucleotide sequence set forth in SEQ ID NO: 1-36. Polynucleotides can be designed.
  • Such a primer or probe polynucleotide of the present invention is useful for the synthesis and detection of the expression level of the gene marker polynucleotide described in [1] above. It contributes to quick and accurate diagnosis of sensitivity.
  • the present invention provides such a method for susceptibility diagnosis, the contents of which will be described later. As described in [5].
  • primer or probe polynucleotide of the present invention in a sensitive diagnostic kit, it is possible to more easily diagnose the sensitivity of the combination therapy of the antitumor agent and radiation.
  • the present invention also provides diagnostic agents and kits for such sensitivity diagnosis, and diagnostic methods, the contents of which are as described in [4] below.
  • the present invention provides a polypeptide encoded by the gene marker polynucleotide described in [1] above, and an antibody that recognizes the polypeptide. As long as it is encoded by the gene marker polynucleotide described in [1] above, it is indicated by a specific amino acid sequence corresponding to the polynucleotide (A) to (D) described in [1] above. In addition to polypeptides, mutants, derivatives, mature forms and amino acid modified forms thereof are included on the condition that they exhibit substantially the same function as the polypeptide. Variants include naturally occurring allelic variants, non-naturally occurring variants, and variants having amino acid sequences modified by artificial deletion, addition, or substitution.
  • mutants include those that are at least 70%, preferably 80%, more preferably 95%, and even more preferably 97% homologous to a polypeptide having no mutation.
  • the amino acid modifications include naturally occurring amino acid modifications and non-naturally occurring amino acid modifications, and specifically include phosphorylated amino acids.
  • the polypeptide of the present invention can be obtained by a conventional method based on the gene marker polynucleotide described in [1] above.
  • the polynucleotide can be cloned, ligated into a vector plasmid, transformed into a host cell such as Escherichia coli, and the resulting transformed cell is cultured and recovered from the culture. .
  • human cells such as test subjects or tissue power can also be purified and produced by a conventional method. For example, after homogenizing a subject's tissue or cells, extraction with an acid or the like is performed, and the extract is chromatographed such as reverse phase chromatography or ion exchange chromatography. It can be purified and isolated by combining fees.
  • the polypeptide of the present invention is a polypeptide encoded by the genetic marker polynucleotide described in [1] above, the genetic marker polynucleotide prepared by subject test force is collected.
  • the expression level can be detected by measuring the amount of the polypeptide encoded by. In other words, if the expression level of the polypeptide encoded by the gene marker polynucleotide increases after the combination therapy than before the combination therapy, it is diagnosed that the sensitivity of the combination therapy is higher and the amount of the polypeptide increases. Diagnosis of low sensitivity to combination therapy when there is no increase (no change).
  • the expression level of the polypeptide encoded by the gene marker polynucleotide decreases or does not express at all after the combination therapy, it is diagnosed that the sensitivity of the combination therapy is high when it decreases or does not express at all, and conversely decreases. Is not confirmed (no change in the expression level), the sensitivity to combination therapy is low!
  • the form of the antibody of the present invention is not particularly limited, and may be V of the above-described polypeptide of the present invention, monoclonal antibody having any of the antigens as antigens, or polyclonal antibody! Further, it may be an antibody having an antigen binding property to a polypeptide having a partial amino acid sequence having at least 10 consecutive amino acids, particularly 15 amino acids, particularly 20 amino acids in the amino acid sequence of the polynucleotide of the present invention.
  • the antibody can be produced according to a conventional method.
  • a polyclonal antibody an experimental animal is immunized with the above-mentioned polypeptide expressed and purified in Escherichia coli according to a conventional method, or with a polypeptide synthesized with these partial amino acid sequences according to a conventional method.
  • it can be obtained from the serum of the immunized animal according to a conventional method.
  • the polypeptide can be used to immunize experimental animals, and spleen cells obtained from the experimental animals can be fused with osteomyeloma cells to synthesize hyperpridoma cells and obtained from the cells.
  • the polypeptide and antibody of the present invention contribute to rapid and accurate diagnosis of the combination therapy sensitivity of the above-described antitumor agent and radiation, and are used as diagnostic drugs and diagnostic kits for combination therapy sensitivity. It can be used widely.
  • the present invention provides a method for susceptibility diagnosis using such an antibody, the contents of which are as described in [5] below.
  • the antibody of the present invention in a sensitivity diagnostic agent or kit, it is possible to more easily diagnose the sensitivity of the above-described combination therapy with the antitumor agent and radiation.
  • the present invention also provides diagnostic agents and kits for such sensitivity diagnosis, and diagnostic methods, the contents of which are as described in [4] below.
  • the present invention provides a diagnostic agent characterized by comprising at least the polynucleotide for primer or probe described in [2] above, or the antibody described in [3] above, and a diagnostic kit. Is.
  • the diagnostic agent and diagnostic kit of the present invention contain at least the primer or probe polynucleotide described in [2] above, or the antibody described in [3] above, and are known diagnostics.
  • An appropriate dosage form can be obtained according to a conventional method which may contain reagents used for the preparation of biological samples from subjects and detection of amplification products.
  • the above combination therapy of the subject is detected by detecting the gene marker polynucleotide contained in the biological sample of the subject or the polypeptide encoded by the polynucleotide. Can be diagnosed.
  • the present invention is characterized by examining the presence or absence of the gene marker polynucleotide described in [1] above or the polypeptide described in [3] above in a biological sample collected from a subject.
  • the present invention also provides an antitumor agent containing tegafur, gimeracil and oteracil potassium, and a method for diagnosing radiation therapy sensitivity.
  • the subject may be a cancer patient who has already undergone the above-described combination therapy of an antitumor agent and radiation, or a cancer patient who has not been performed.
  • the type of cancer is not particularly limited as long as it is a cancer type that can be treated by the above-described combination therapy with an antitumor agent and radiation.
  • head and neck cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gallbladder, bile duct cancer examples include spleen cancer, lung cancer, breast cancer, bladder cancer, prostate cancer, cervical cancer, and the like, and lung cancer, in which head and neck cancer, lung cancer, and spleen cancer are preferred.
  • the biological sample is not particularly limited as long as it is a subject's own sample and may contain a tumor.
  • Body fluid blood, urine, etc.
  • tissue extract thereof, culture of collected tissue, etc.
  • blood, particularly serum is preferable.
  • a method for collecting a biological sample can be appropriately selected according to a method according to the biological sample or the cancer type.
  • the biological sample applied to the diagnostic method of the present invention may be a cancer sample that has already been collected from cancer patients who have already undergone the above-mentioned combination therapy of antitumor agent and radiation. What was collected from the patient's body may have been subjected to the combination therapy of the above-mentioned antitumor agent and radiation.
  • the time when the biological sample is collected when speaking is preferably the time when the effect of the combination therapy is expected to be exerted. Usually, it is 30 minutes to 5 hours, preferably 1 hour to 3 hours after the combination therapy.
  • the presence or absence of the gene marker polynucleotide described in [1] above or the presence or absence of the polypeptide described in [3] above is examined.
  • the presence or absence of the gene marker polynucleotide described in [1] above is set as the detection target will be described below.
  • step (II) Step of measuring the presence or absence of RNA or complementary polynucleotide bound to the polynucleotide in step (I) using the above-described probe or primer polynucleotide as an indicator.
  • step (I) RNA prepared from a biological sample collected from a subject or a complementary polynucleotide transcribed from the RNA and the above-described probe or primer polynucleotide are combined.
  • RNA from the biological sample and preparation of a complementary polynucleotide transcribed from RNA can be performed by conventional methods.
  • RNA to the probe or primer polynucleotide can be performed by a conventional method in consideration of the conditions where all or a part of both can be easily bound.
  • step (II) in step (I), the presence of the RNA or complementary polynucleotide bound to the probe or primer polynucleotide is determined using the above-described probe or primer polynucleotide. Measure as an indicator.
  • the probe or primer polynucleotide is labeled with a labeling substance such as a radioisotope or a fluorescent substance in advance and then bound to RNA or a complementary polynucleotide. Signals derived from substances can be measured with radiation detectors or fluorescence detectors.
  • Such a diagnostic method of the present invention can be performed using Northern blotting, RT-PCR, DNA chip analysis, in situ hybridization, and the like.
  • the above-described probe or primer polynucleotide is used as a probe, and the strength of the biological sample collected by the subject is determined whether the gene marker polynucleotide is expressed in the prepared RNA, Its expression level can be detected.
  • RNA prepared by a subject is transferred to a nylon membrane or the like, and labeled with the probe in advance and hybridized with the probe (step (1)). Subsequently, the presence or absence of a double-stranded polynucleotide and RNA is measured using the signal derived from the probe label as an index (step (ii)). In addition, it is also possible to carry out according to the protocol of the kit using a commercially available kit for Northern blotting.
  • the gene marker polynucleotide in cDNA prepared from R R prepared from a biological sample collected from a subject using the above-described probe or primer polynucleotide as a primer. The presence or absence of nucleotide expression and its expression level can be detected.
  • cDNA prepared from RNA derived from a subject is used as a cage, the above primer is hybridized with this, and PCR is performed according to a conventional method (corresponding to step (I)). Amplified double-stranded DNA can be detected (corresponding to step (ii)). It is also possible to use a commercially available RT-PCR kit according to the protocol of the kit.
  • RNA prepared by a subject is transferred to a nylon membrane or the like, and labeled with the probe in advance and hybridized with the probe (step (1)). Subsequently, the presence or absence of the double-stranded polynucleotide and RNA formed is measured using the signal derived from the probe label as an index (step (II)).
  • step (III) the antitumor agent and the radiation are obtained from the measurement result of the step (II). Diagnose sensitivity of combination therapy. As a result of the measurement, diagnosis of sensitivity to combination therapy
  • the measured RNA or complementary polynucleotide measured in (II) is the same as the RNA or complementary polynucleotide measured in the same manner in other subjects who are insensitive or sensitive to the combination therapy before or after the combination therapy. Nucleotide measurement values and similar measurement values can be used as a reference by calculating numerical values in advance and comparing them to calculating statistical significance.
  • the combination therapy sensitivity is high.
  • no increase in the expression level is confirmed! (No change in the expression level)
  • a gene marker polynucleotide whose expression level decreases when combined therapy is used as an indicator
  • the decrease in the expression level is not confirmed! In the case of (no change in the expression level), the sensitivity to combination therapy is diagnosed as low.
  • EIA enzyme immunoassay
  • RIA radioisotope Immunological detection methods
  • fluorescent immunization fluorescent immunization
  • the immune reaction between the antibody and the polypeptide or partial peptide may be either a competitive method or a sandwich method.
  • the sandwich method particularly an ELISA using a solid phase antigen or a solid phase antibody, is preferred.
  • Examples of the diagnostic method include (i) to (iii).
  • step (ii) Step of measuring the presence or absence of the polypeptide bound to the antibody or its partial peptide in step (i) using the antibody as an index
  • step (iii) Step of diagnosing the sensitivity of the above-mentioned antitumor agent and radiation combination therapy from the measurement result of step (ii)
  • the prepared biological sample force is collected from the subject force, and the prepared polypeptide and the antibody are combined.
  • the preparation of the biological sample strength polypeptide can be carried out by appropriately combining known fractionation and purification methods.
  • step (ii) in step (i), the presence or absence of a polypeptide bound to the antibody or a partial peptide thereof is measured using the antibody as an index.
  • the antibody is preliminarily labeled with a labeling substance such as a radioisotope or fluorescent substance, and the force is bound to the polypeptide to detect the signal derived from the labeling substance. This can be done by measuring with a detector or a fluorescence detector.
  • step (iii) the sensitivity of the combination therapy with the antitumor agent and radiation is diagnosed from the measurement result of step (ii). Measured results
  • the diagnosis of the sensitivity to combination therapy is as follows: @ ( ⁇ ) is used to determine the measured value of the polypeptide before subjecting the combination therapy to the subject or for other insensitivity or sensitivity to the combination therapy. It can be carried out by calculating a statistically significant difference by comparing a measured value of a polypeptide measured in the same manner or a similar measured value in advance with a numerical value as a reference.
  • the combination therapy is highly sensitive.
  • the increase in the expression level is not confirmed (no change in the expression level)
  • the polypeptide of [3] above whose expression level decreases when combined therapy is used as an index
  • the polypeptide of [3] above whose expression level decreases when combined therapy is used as an index
  • the expression level decreases or is not expressed at all.
  • the decrease in the expression level is not confirmed! ⁇ ⁇
  • the combination therapy sensitivity is low! Diagnosed as sputum.
  • the diagnostic method of the present invention By carrying out the diagnostic method of the present invention, the sensitivity of the combination therapy in a subject can be diagnosed, whereby the continuity and Z or feasibility of the combination therapy can be predicted.
  • the power to use the above combination therapy further depends on the sensitivity of the combination therapy obtained by the diagnostic method of the present invention. I can judge.
  • the above combination therapy when the above combination therapy is not performed in the subject, whether or not the above combination therapy should be performed can be predicted from the combination therapy sensitivity obtained by the diagnostic method of the present invention.
  • the patient By providing the above combination therapy only to a patient who is highly likely to continue and Z or feasible, the patient can receive appropriate treatment, and thus the treatment efficiency in the overall medical care and Economic power is also useful.
  • the S-1 drug solution used as the test solution for the following tests was prepared as follows. That is, first, tegafur, gimeracil, and potassium oxalate were suspended in a 0.5% hydroxypropyl methylcellulose (HPMC) solution at 0.83 mg / mU 0.25 mgZml and 0.82 mgZml, respectively, and at about room temperature. Stir for 10 minutes. Then, it was sonicated under ice-cooling to obtain a dose of 8.3 mg ZkgZday S-1 drug solution. This dose of S-1 is the maximum non-toxic dose when orally administered to mice for 14 days.
  • HPMC hydroxypropyl methylcellulose
  • irradiation position Using an MBR-1505R2 radiation irradiation device manufactured by Hitachi Medical Corporation, set the irradiation conditions (irradiation position) so that one irradiation per mouse is 2 Gy and 5 Gy, and transplanted to the right thigh of the mouse Local irradiation was performed on the tumor lines. Avoid whole-body irradiation during irradiation. In order to avoid this, the mouse was placed in a storage box made of lead so that only the right foot was exposed to radiation.
  • Human lung cancer strains (LC-11 and Lu-99) were isolated by transplanting to the right thigh of BALBZcA-nu mice aged 5-6 weeks under the skin of the back of mice of the same strain in advance. A piece of scissors approximately 2 mm square in physiological saline was transplanted subcutaneously using a transplant needle and bred for at least 1 to 2 weeks. After that, each group (6 mice per group) was divided into 4 groups so that the mean and standard deviation (SD) of the tumor volume were as uniform as possible, and then each was divided into the control group and the radiation alone group. (X ray), drug alone group (S-1) and drug and radiation combination group (S-1 + X my), and further divided into 3 groups or 4 groups depending on the treatment period. The group began drug administration and radiation. Table 1 shows the relationship between the strains derived from each of the 30 specimen groups, treatment details, and treatment periods in “ ⁇ ”.
  • the above-mentioned S-1 drug solution was used at a rate of 8.3 ml per 1 kg body weight, once a day, using an oral sonde for the treatment period shown in Table 1. Orally administered.
  • the method described under the above irradiation conditions within about 1 hour after administration of the S-1 drug. 2Gy was applied according to the above.
  • tumor tissues were collected from each group on the second, ninth, twelfth, and fifteenth days of the start of the study, and the origin strains, treatment details, and treatment periods shown in Table 1 were collected. Thirty kinds of specimens classified separately were used for the following processes.
  • RNA was extracted from each group to prepare polyA + RNA.
  • mixed groups were prepared from the 30 sample groups according to the treatment method, and the Control group (non-radiation irradiation, S-1 non-administration), S-1 treatment group (non-irradiation, S-1 administration), and radiation treatment
  • the Control group non-radiation irradiation, S-1 non-administration
  • S-1 treatment group non-irradiation, S-1 administration
  • radiation treatment Four cDNA libraries were prepared for the group (irradiation, S-1 non-administration) and the combination treatment group (irradiation, S-1 administration).
  • RNA sample total RNA
  • RNA sample was treated with Proteinase K. That is, proteinase K (l nvitrogen) was added to 1 mg of RNA sample and reacted at 37 ° C for 1 hour to inactivate RNase and then heated to 60 ° C for 10 minutes. Proteinase K was inactivated.
  • proteinase K l nvitrogen
  • RNA sample obtained by phenol treatment and ethanol precipitation was further subjected to the following steps.
  • the sample obtained in step 2.3 was treated with a DNA-degrading enzyme (DNase I: Takara Bio). That is, 1 unit of DNase I was added to 1 mg of the purified RNA sample and reacted at 37 ° C for 30 minutes. After completion of the reaction, RNA was recovered by phenol treatment and ethanol precipitation.
  • DNase I DNA-degrading enzyme
  • RNA thus recovered was subjected to the following steps as a DNase I-treated total RNA sample.
  • polyA + RNA was extracted from the DNase I-treated total RNA sample obtained in step 2.4.
  • Rigo dT used Oligotex—dT30 (Takara Bio) to absorb and extract mRNA. The operation procedure was performed according to the protocol of the product. The mRNA was purified by ethanol precipitation after extraction.
  • PolyA + RNA which has obtained the strength of 30 sample groups (see Table 1), is treated with Control, S-1 treatment, radiation treatment, S-1 and radiation treatment as shown in Table 2 “Mixed group”.
  • Control group S-1 treatment group
  • radiation treatment group S-1 and radiation treatment as shown in Table 2 “Mixed group”.
  • Table 2 shows the relationship between the 30 sample groups and the 4 mixed groups. Note that “ ⁇ ” in Table 2 indicates the correspondence of the derived strain, treatment content, and treatment period of each sample group. [0089] [Table 2]
  • First strand synthesis and cDNA library creation were performed using oligo dT primers using the polyA + RNA of each of the four mixed groups (Table 2) prepared in Step 2.5 as a template.
  • the following sequence of operations up to the creation of the cDNA library was performed using the cDNA Construction Kit (Takara Bio Inc.) according to the operation manual of the product.
  • the four cDNAs obtained from each of the four mixing groups were obtained.
  • the library was named as follows. That is, the Control group is called TH05C, the S-1 treatment group is TH05S, the radiation treatment group is TH05X, and the combination treatment group is TH05SX (see Figure 1).
  • This cDNA fragment was treated with restriction enzymes Not I (5 units) and EcoRI (5 units) at 37 ° C for 1 hour. After that, using a gel filtration column (Invitrogen, trade name: cDNA Size Fraction Column), size fractionation was performed according to the operation manual of the same product, and cDNA around 500 bp was extracted using electrophoresis and absorbance at 260 nm as indices. Further, ethanol precipitation was performed to purify a cDNA fragment having both ends cleaved with Not I and Eco RI (hereinafter referred to as a purified cDNA sample).
  • a gel filtration column Invitrogen, trade name: cDNA Size Fraction Column
  • pBlueScript II KS (—) (Stratagene) vector was treated with restriction enzymes Not I and EcoRI. After electrophoresis using 1% agarose, the vector was excised and purified.
  • Sequencing was performed on the clones derived from the four types of libraries obtained in Step 2 above. That is, after aligning E. coli clones from each library, PCR amplification is performed using a single vector primer to amplify the inserted cDNA fragment. did. Thereafter, the amplified cDNA fragment was purified, and a sequencing reaction was performed using this as a saddle shape, followed by sequencing.
  • agar plate obtained in step 2.9 colonies formed on the agar plate were colonized on the LB medium in advance using a colony pick device Q—PIX (Genetix). Inoculate and align. Shake overnight at 37 ° C. This is to reduce the difference between each well in the following work by suppressing the variation in growth among clones by arranging and re-culturing. Subsequent series of reactions were performed using 96 multiwell plates.
  • the culture solution in each well of the multiwell plate obtained in step 3.2 was heat-treated at 98 ° C. for 3 minutes to destroy the cell membrane of E. coli.
  • the plasmid DNA contained in the heat-treated solution (lysate solution) of the fungus is used as a saddle, and the primers (KS—F, KS-R) derived from the sequence near the multi-ring region of the pBlueScript II KS (—) vector are used.
  • PCR reaction was performed to amplify the inserted cDNA fragment.
  • PCR reaction solution composition fungus lysate solution; 10 ⁇ L, 10X PCR buffer; 5 L, primer; 10 pmol each, dNTPs; 20 nmol each, EX
  • Taq (Takara Bio Inc.); Sterilized water was added to 1 unit to make 50 ⁇ L.
  • the PCR thermal cycle was 94 ° C ⁇ 30 seconds ⁇ 60 ° C ⁇ 30 seconds ⁇ 70 ° C ⁇ 1 minute 30 times.
  • Sequencing reaction was performed using the cDNA fragment amplified and purified in step 3.3 as a cage.
  • BigDye terminator vl. 1 (Applied Biosystems) was used, and the reaction was carried out according to the operation manual of the product.
  • the sequence reaction product was purified using Sephadex G-50 (Amersham) according to the operation manual of the product.
  • TH05SX combination treatment group
  • TH05S S-1 treatment group
  • TH05C Control group
  • TH05X radiation treatment group
  • sequence data of each clone obtained in the above DNA sequencing section! First, a series of sequence cleaning consisting of quality trimming, vector trimming, cross-contamination confirmation (detection 'exclusion), etc. After processing, the inserted sequence was cut out (extracted). Next, sequence annotation is performed based on the obtained insertion sequence to determine whether each clone is a known gene or a new gene, and the derived gene species and the derived organism species (human or mouse). Of the difference).
  • Figure 2 shows the outline of the sequence annotation process.
  • a sequence cleaning process is performed, the sequence data output from the sequencer is processed for each clone obtained in the above DNA sequence section, and the cDNA fragment as the target of the sequence annotation process is extracted. It was. Since it is necessary to remove the cloning vector sequence in order to identify the cDN A fragment sequence, for example, Chou HH, Holmes MH .; DNA sequence quality trimming and vector removal. Bioinformatics. 2001 Dec; 17 (12): Refer to the description of 1093- 1104. Perform vector trimming or vector removal, quality trimming for removing low quality sequences, and detection and elimination of cross contamination. was also performed.
  • the unstable base sequences present near the beginning and end of the reading sequence were removed from both sides, and a highly accurate portion was extracted as a sequence.
  • sequence data that may contain a plurality of clones was detected and excluded at the stage of this processing, and excluded from the target of subsequent sequence annotation processing.
  • sequence annotation process was performed for the sequence data that was determined to have no quality problem by the following procedure for identifying the origin of genes and species.
  • human known gene mRNA sequence database and mouse known gene mRNA sequence database were used.
  • the known gene sequence databases used in this process are GenBank, RefSeq, and LocusLink of October 8, 2004.
  • cDNA fragment sequence data obtained in step 4.2 above a homology search using transcripts (mRNA) is performed on human known gene sequence data and mouse known gene sequence data available from NCBI. It was. The obtained search results were analyzed, and the gene corresponding to the sequence data of the cDNA fragment obtained in 4.2 was identified. Single gene at this stage It was judged that the sequence uniquely identified as a single species of the species could determine not only the gene but also the derived species, and the result was annotated.
  • mRNA transcripts
  • sequence data that was determined to be a clone of a human gene and not a known gene, that is, a clone as a candidate for a new gene, from each cDNA library.
  • step 4 Compared with mRNA registered in the public database in step 4 above, the ability to exclude sequence data of clones corresponding to known genes
  • the positions of known genes and clones in the public database at the genome level The position of the cluster of the sequence data is collated, the sequence data of the clone corresponding to the known gene is excluded, and new gene candidates are selected.
  • a clone having a homology of 98% or more between the clone sequence data and the genome sequence and mapped to only one locus site in one direction of one chromosome or mitochondria was extracted.
  • human genome data the database NCBI reference Build 35.1 (representative genome sequence of each chromosome and mitochondria) was used.
  • BLAST for analysis of mapping to genomic data, see McGinnis S., Madden T. L .; "BLAST: at the core of a powerful and diverse set of sequence analysis tools.
  • the position information on the genome of the obtained cluster can be predicted as the locus site of a new gene candidate.
  • the clone with the longest portion mapped to the genomic data is used as the representative sequence of the cluster.
  • Example 1 a confirmation experiment was conducted in which 36 clones (SEQ ID NOs: 1 to 36) obtained in practice could be actually amplified by the quantitative PCR method.
  • 5 g of DNase I-treated total RNA of Day 2 (second day from the start of the test) derived from human lung cancer cell line LC 11 used in Example 1 was used.
  • Table 1 On Day 2, no RNA preparation was performed on the xenograft sample in the S-1 treatment group, so the Dnase I treatment from each of the control group, radiation treatment group, and combination treatment group was completed. Total RNA 5 g was used.
  • Cells were cultured from the glycerol stock of each sample group obtained in Example 1, and plasmid DNA was extracted.
  • the plasmid DNA extraction reagent is Wizard plus Minpreps (Promega Was used.
  • the reagent was Oligo (dT) 12-18 primer (Invitrogen) 500 ng, Superscript II RT (Invitrogen) 200 U and the reaction was performed on a 20 ul scale.
  • primers fOla SEQ ID NO: 71
  • rOla SEQ ID NO: 72
  • primers f02a SEQ ID NO: 73
  • r02a SEQ ID NO: 74
  • Primers f03a SEQ ID NO: 75
  • r03a SEQ ID NO: 76
  • Primers f04b SEQ ID NO: 77
  • r04b SEQ ID NO: 78
  • Primer f 16a (SEQ ID NO: 101), rl6a (SEQ ID NO: 102) corresponding to the clone of SEQ ID NO: 16; Primer f 17a (SEQ ID NO: 103), rl7a (SEQ ID NO: 103) corresponding to the clone of SEQ ID NO: 17 104)
  • Primers f 18a (SEQ ID NO: 105), rl8a (SEQ ID NO: 106) corresponding to the clone of SEQ ID NO: 18; primers f 19a (SEQ ID NO: 107), rl9a (SEQ ID NO: 108) corresponding to the clone of SEQ ID NO: 19;
  • Primers f 20a (SEQ ID NO: 109), r20a (SEQ ID NO: 110) corresponding to the clone of SEQ ID NO: 20; primers f 21a (SEQ ID NO: 111), r21a (SEQ ID NO: 112) corresponding to the
  • the software parameters were set according to the protocol attached to TaKaRa Ex Taq R-PCR version 2.1 (Takarabio). From the candidates listed, primer pairs were selected in consideration of both the primer design guidelines recommended by Takara Neo and Applied Systems. The selected primers were subjected to a blast search and confirmed that no other gene sequences were amplified. Primer synthesis was requested from Invitrogen. The synthesized primer sequences are listed in Table 3. No. in Table 3 indicates the sequence number of the clone corresponding to each primer. [9 ⁇ 0]
  • the vertical type is a series of dilutions of plasmid DNA in 5 stages, ie, 10 pmolZul, lpmol / ul, 100 fmol / ul, 10 fmol / ul, lfmolZul, and 50 times the first strand cDNA (from the combination treatment group) Each diluted solution was used in lul. The reaction volume was 50 ul. The PCR program was 95 ° C, lmin ⁇ (95 ° C, 15sec ⁇ 60 ° Clmin) x 45cycles, and a default dissociation curve creation program was added. The primer pass / fail is determined by the dissociation curve.
  • SEQ ID NOs: 8, 16, and 22 had strong amplification of by-products and were unable to confirm specific amplification. This is because the clone of SEQ ID NO: 8 has a short sequence length of 197 bases obtained in the EST analysis of Example 1, and the clones of SEQ ID NO: 16 and 22 have many repeated sequences in the sequence. it is conceivable that. On the other hand, specific amplification was confirmed in 33 clones excluding SEQ ID NOs: 8, 16, and 22.
  • the reaction system was the same as the reaction system in the above-described primer specificity confirmation, except that the amount of the template DNA sample for the calibration curve was different from that of the first strand cDNA sample.
  • a calibration curve is prepared using samples obtained by serially diluting plasmid DNA in 6 stages, that is, samples containing 100 pmolZul, 10 pmol / ul, lpmol / ul, and 100 fmol / uU lOfmol / uU lfmol / ul.
  • the number of copies in the sample lul diluted 10-fold from the first strand cDNA was quantified.
  • GAPD Human GAPD
  • dalyceraldehyde 3-phosphate dehydrogenase was selected as a gene for the internal standard.
  • Primer for detecting this gene is Takarabio Used off-the-shelf products sold more.
  • the present inventors have confirmed in advance that DNA amplification does not occur when mouse first strand cDNA is subjected to PCR using this primer.
  • the rautoj setting was selected using the software supplied with the ABI7500 device.
  • the calibration curve was created and the number of copies in each sample was also calculated on the same software.
  • the copy number calculation formula is as follows, and the number of copies of double-stranded plasmid DNA is calculated.
  • the size of plasmid DNA actually varies from gene to gene, but was calculated as 3,500 bp (pbluescript II KS—: 3, OOObp, average length of insert: 500 bp).
  • GAPD [Calculation! Calculated as 4,700bp (pCR2.1: 4,200bp, insert: 500bp) only.
  • the number of moles of double-stranded DNA is the number of moles of double-stranded DNA.
  • Table 4 shows 33 clones (excluding SEQ ID Nos. 8, 16, and 22 of 36 clones) and internal standard of the control group, the radiation treatment group, and the combination treatment group of LC-11 on Day 2.
  • the double-stranded DNA copy number calculated for GAPD of the gene is shown.
  • Fig. 4 shows the ratio of the expression level in the control group to the expression level in the radiation treatment group and the combination treatment group.
  • Example 1 The clones selected in (1) were found to be useful as markers for sensitivity to combination therapy because their expression levels changed after combination therapy.
  • SEQ ID NOs: 37 to 70 these are sequences predicted from SEQ ID NOs: 1 to 9, 11 to 24, and 26 to 36. Therefore, if primers are designed in the same manner as the above clones, they are useful as markers. It can be fully analogized.
  • the present invention it is possible to quickly and accurately diagnose the sensitivity of combination therapy of tegafur / gimeracyl / oteracil potassium-containing antitumor agent and radiation. Therefore, early determination as to whether or not the combination therapy should be performed on cancer patients can be made early, and the efficiency of cancer treatment can be improved.

Abstract

L'invention concerne des moyens pour le diagnostic rapide et précis de la sensibilité à un agent antitumoral contenant du Tégafur/Gimeracil/Oteracil potassium ou à une chimiothérapie concurrente. Ainsi l'invention concerne : un polynucléotide qui peut servir de marqueur génétique pour le diagnostic de la sensibilité à un agent antitumoral ou à une chimiothérapie concurrente ; un polynucléotide qui peut servir de sonde ou d'amorce pour le polynucléotide marqueur génétique ; un polypeptide codé par le polynucléotide marqueur génétique et un anticorps capable de reconnaître le polypeptide ; et un agent de diagnostic, un kit de diagnostic et un procédé pour le diagnostic de la sensibilité à une chimiothérapie concurrente qui utilisent le polynucléotide pour la sonde ou l'amorce ou l'anticorps.
PCT/JP2006/320225 2005-10-07 2006-10-10 Marqueur génétique destiné à être utilisé dans le diagnostic de sensibilité à un agent antitumoral ou à une chimiothérapie concurrente et utilisation de celui-ci WO2007043531A1 (fr)

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JP2020096578A (ja) * 2018-12-18 2020-06-25 株式会社リコー 核酸解析方法及び核酸解析プログラム、並びに、ライブラリー調製用デバイス

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* Cited by examiner, † Cited by third party
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CN102993087A (zh) * 2011-09-10 2013-03-27 山东新时代药业有限公司 一种替吉奥组方中杂质化合物及其制备方法和用途
JP2020096578A (ja) * 2018-12-18 2020-06-25 株式会社リコー 核酸解析方法及び核酸解析プログラム、並びに、ライブラリー調製用デバイス
JP7317311B2 (ja) 2018-12-18 2023-07-31 株式会社リコー ライブラリー調製用デバイス

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