JP2012016294A - Composition or kit for determining adjuvant chemotherapy sensitivity of stomach cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide useful polynucleotide for determining adjuvant chemotherapy sensitivity of stomach cancer.SOLUTION: In the stomach tissue derived from a patient with sensitivity to adjuvant chemotherapy of the stomach cancer and the stomach tissue derived from the patient without the sensitivity, there is provided the polynucleotide in which a change in expression level is detected. Specifically, there is provided: the polynucleotide including a specific base sequence or including a complementary sequence thereof, and mutant polynucleotide thereof; the polynucleotide hybridizing under stringent conditions to the polynucleotide thereof; and DNA encoding the polynucleotide. There is also provided: a composition for determining the adjuvant chemotherapy sensitivity of stomach cancer including a plurality of polynucleotide selected from a group comprising the modified polynucleotide thereof; a kit; nucleic acid array; or a method for determining adjuvant chemotherapy sensitivity of stomach cancer using the composition, the kit or the nucleic acid array.

Description

  The present invention relates to a determination composition useful for determination of adjuvant chemotherapy sensitivity of gastric cancer, a method of determining gastric cancer adjuvant chemotherapy sensitivity using the composition, and gastric cancer adjuvant chemotherapy sensitivity using the composition. It relates to a judgment kit.

  In cancer surgery, even if it appears that the tumor has been removed macroscopically, invisible micrometastasis may remain, so postoperative adjuvant chemotherapy that administers an anticancer drug after surgery is May be applied. This is intended to prevent cancer from growing and recurring from minute metastases by performing adjuvant chemotherapy. In postoperative adjuvant chemotherapy performed after surgery for gastric cancer, several types of anticancer agents are mainly used, but individual anticancer agents are effective for each patient. Therefore, an anticancer drug sensitivity test is sometimes performed as a means for determining the sensitivity of various drugs. This is intended to determine sensitivity to anticancer drugs by first culturing tumor cells removed by surgery in vitro, administering various anticancer drugs to the cells, and comparing the survival rate of the cells with controls. Is.

  The anticancer drug susceptibility test for gastric cancer is carried out in five protocols including the ongoing one at the Japan Cancer Clinical Trials Promotion Organization (JACCRO) (for example, Non-Patent Document 1). Patent Document 1 discloses the sensitivity of platinum complexes such as cisplatin used in the treatment of gastric cancer by measuring the level of glutathione (GSH) in a patient's blood as a surrogate marker for glutathione-S-transferase (GST) in tumor cells. Is described. Non-Patent Documents 2 to 5 include thymidylate synthase (TS), which is an important enzyme in DNA synthesis, and dihydropyrimidine dehydrogenase (DPD), which is a degrading enzyme of fluorouracil (5-FU). )) And the relationship between the expression level of vascular endothelial growth factor (VEGF) and the sensitivity of fluorouracil drugs was reported by immunostaining and quantitative RT-PCR. ing. Non-Patent Document 6 and Non-Patent Document 7 include uracil tegafur (trade name: UFT), oteracil potassium gimeracil tegafur (fluorouracil prodrugs used in adjuvant chemotherapy for gastric cancer, The effectiveness of the product name: TS-1 (TS-1) is shown.

JP-T-2005-504322

Tetsuro Kubota, History of Weekly Medicine, Vol. 217, No. 9, 869-873 (2007) Takashi Irino et al., Japanese Society of Gastroenterological Surgery, Vol. 32, No. 7, 1955-1961 (1999) Fahrejahani E. et al. Cancer Chemotherapy and Pharmacology, 60, 437-446 (2007). Hua D. Et al., World Journal of Gastroenterology, 13 (37), 5030-5034 (2007). Boku N. Et al., Japan Journal of Clinical Oncology, 37 (4), 275-281 (2007). Sakuramoto S. Et al., New England Journal of Medicine, 357, 1810-1820 (2007). Nakajima T .; Et al., British Journal of Surgery, 94 (12), 1468-1476 (2007).

  However, the above-described anticancer drug sensitivity test has a high probability of determining an ineffective anticancer agent as high as about 90%, while the probability that an effective anticancer agent can be specified is as low as about 40 to 50%. In addition, the methods of Non-Patent Documents 2 to 5 are methods that are assumed to be difficult to implement in a medical field, and are not actually performed. Accordingly, there is currently no method that can determine an effective anticancer agent with a high probability, and development of a method that can correctly determine the effect of the anticancer agent itself is required. In addition, as shown in Non-Patent Document 7, although the availability of oteracil potassium, gimeracil, tegafur (TS-1), which is currently the first-line drug for adjuvant chemotherapy, has been shown to be useful, TS It is also true that there are patients who do not respond to -1, and it is eager to select effective drugs at an early stage and to carry out effective chemotherapy for such patients.

  The present invention relates to a determination composition that is useful for determining the sensitivity to adjuvant chemotherapy for gastric cancer, a method for determining the sensitivity to adjuvant chemotherapy for gastric cancer using the composition, and the sensitivity to adjuvant chemotherapy for gastric cancer using the composition. It is an object to provide a determination kit.

  Marker search methods for determining sensitivity to adjuvant chemotherapy for gastric cancer include gene expression in gastric cancer cells derived from patients who are sensitive to adjuvant chemotherapy and gastric cancer cells derived from patients who are not sensitive in gastric tissue removed during surgery. A method to compare the amount of protein expression or cellular metabolites by some means, or measure the amount of genes, proteins, metabolites, etc. contained in the body fluid of gastric cancer patients who are sensitive to adjuvant chemotherapy and those who are not sensitive And a comparison method.

  In recent years, gene expression analysis using a nucleic acid array such as a DNA array has been widely used as a technique for searching for such a marker. Probes using base sequences corresponding to hundreds to tens of thousands of genes are immobilized on the nucleic acid array. By adding the test sample to the nucleic acid array, the gene in the sample binds to the probe, and the amount of gene in the test sample can be known by measuring the amount of binding by some means. The gene corresponding to the probe to be immobilized on the nucleic acid array can be freely selected, and cells in the tumor and non-tumor areas derived from gastric cancer patients removed during surgery or endoscopy are used in the sample. It is possible to estimate genes related to the sensitivity to adjuvant chemotherapy for gastric cancer by comparing the amount of expressed genes.

  In order to solve the above problems, the present inventors analyzed the gene expression of patients who are sensitive to adjuvant chemotherapy for gastric cancer and the gene expression of patients who are not sensitive using a nucleic acid array, and related to the sensitivity to adjuvant chemotherapy for gastric cancer. The present invention was completed by finding a gene that can be used as a marker.

1. SUMMARY OF THE INVENTION The present invention has the following features.
In the first aspect, the present invention provides a composition for determining sensitivity to adjuvant chemotherapy for gastric cancer, comprising two or more polynucleotides selected from the group consisting of the polynucleotides shown in the following (a) to (f): (Hereinafter referred to as “a composition for determining sensitivity to adjuvant chemotherapy for gastric cancer”).

(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NOs: 1 to 22, a precursor polynucleotide thereof, a mutant polynucleotide thereof, or a modified polynucleotide thereof (b) a nucleotide sequence represented by SEQ ID NOs: 1 to 22 (C) a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NOs: 1 to 22, a variant polynucleotide thereof, or a modified polynucleotide thereof (d) represented by SEQ ID NOs: 1 to 22 (E) a polynucleotide that hybridizes under stringent conditions with any one of the polynucleotides (a) to (d) (f) above (a) to (a) DNA encoding any of the polynucleotides of (e)
In another embodiment thereof, the above-mentioned composition is such that the adjuvant chemotherapy for gastric cancer is administration of fluorouracil (5-FU) or fluorouracil (5-FU) prodrug.

  As a second aspect of the present invention, there is provided a kit for determining sensitivity to adjuvant chemotherapy for gastric cancer, comprising two or more of the polynucleotides shown in the above (a) to (f).

  In the embodiment, the polynucleotide is a polynucleotide comprising the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 22, a polynucleotide comprising the complementary sequence thereof, and hybridizing with these polynucleotides under stringent conditions. The kit as described above, which is a polynucleotide that is soybean, a DNA that encodes the polynucleotide, or a modified polynucleotide thereof.

  In yet another embodiment, the above-described kit, wherein the polynucleotide is packaged in a container separately or in any combination.

  A third aspect of the present invention is a nucleic acid array for determining sensitivity to adjuvant chemotherapy for gastric cancer, comprising two or more of the polynucleotides shown in (a) to (f) above.

As a fourth aspect, the subject-derived specimen sample is measured by measuring the expression level of the target nucleic acid in the subject-derived biological sample using the composition, the kit, the nucleic acid array, or a combination thereof. In this method, the presence or absence of sensitivity to adjuvant chemotherapy for gastric cancer is determined in vitro.
In the embodiment, the determination method uses a nucleic acid array.

  In yet another embodiment, using the above composition, the above kit, the above nucleic acid array, or a combination thereof, a target in a plurality of biological samples known to be specimen samples from gastric cancer surgical patients A first step of measuring the expression level of the nucleic acid in vitro, a second step of creating a discriminant (neural network model) using the measured value of the expression level of the target nucleic acid obtained in the first step as a teacher A step, a third step of measuring the expression level of the target nucleic acid in a specimen sample derived from a subject in vitro as in the first step, and a discriminant obtained in the second step in the third step A fourth step of substituting the obtained measured value of the expression level of the target nucleic acid and determining the presence or absence of sensitivity to adjuvant chemotherapy for gastric cancer in a specimen sample derived from the subject based on the result obtained from the discriminant Including stomach cancer This is a method for determining the sensitivity to adjuvant chemotherapy.

2. Definitions Terms used herein have the following definitions.
In this specification, “adjuvant chemotherapy of gastric cancer” is chemotherapy performed for the purpose of preventing recurrence due to a small residual tumor after curative resection of gastric cancer, and by administering an anticancer agent, postoperative recurrence suppression, It refers to treatment performed for the purpose of improving survival rate. Here, recurrence refers to a case where the reappearance of a tumor has been confirmed by a tissue diagnosis such as image diagnosis or biopsy, or surgery. Here, in the present invention, the anticancer agent is fluorouracil (5-FU) or a fluorouracil prodrug. In clinical trials to date, a prescribed amount of 5-FU per body surface area has been administered for one year after surgery (for oteracil potassium, gimeracil, tegafur (trade name: TS-1)) or for 16 months (uracil tegafur) (In the case of product name: UFT) It has been shown that the survival time is extended by administration (see Non-Patent Document 6 and Non-Patent Document 7 above), and guidelines based on it. (Revised third edition of gastric cancer treatment guidelines (draft)) has been established. Currently, when using oteracil potassium, gimeracil, tegafur (trade name: TS-1), which is an anticancer drug, which is currently selected as the first-line drug, usually within 6 weeks after surgery, waiting for recovery from surgery Administration is started. The standard daily dose is 80 mg per 1 m ² of body surface area, which is administered for 4 weeks, with 2 weeks of rest as one course, and continues for 1 year after surgery.

  In this specification, “sensitivity” indicates whether or not adjuvant chemotherapy has been effective. Postoperative adjuvant chemotherapy for patients with gastric cancer treated with fluorouracil or fluorouracil prodrug has resulted in recurrence. If the patient has survived 3 years or more after surgery (no recurrence), it is “sensitive” or “sensitive”, and if a recurrence is found (recurrence) is “insensitive” or “insensitive” Show.

  In this specification, “5-FU” is an anticancer agent (trade name) that has been used for a long time in the treatment of gastrointestinal cancer including gastric cancer and other cancers, and the general name is “fluorouracil”. It is. It is classified as a pyrimidine antimetabolite, and has a structure similar to that required for cells to divide and proliferate, and is incorporated into cancer cells instead of the original metabolites. Inhibits the synthesis of the DNA. Since it is easily broken down in the body and the duration of the effect is short, it is frequently used in combination with other drugs or continuous injection into a vein by infusion.

  In the present specification, the term “fluorouracil prodrug (5-FU prodrug)” refers to a 5-FU structure that is administered in vivo with a non-active structure and metabolized in vivo, and has a pharmacological activity. It refers to the anticancer agent shown, and is usually used as an oral administration agent. As mentioned above, 5-FU is improved for the purpose of increasing the effect and reducing side effects by staying in the body for a longer period of time because the decomposition in the body is fast and the duration of the effect is short. It is. Formulations include oteracil potassium, gimeracil, tegafur combination drug (trade name: TS-1 (TS-1)), uracil tegafur combination drug (trade name: YUFT (UFT)), doxyfluridine (trade name: Flutulon), Examples include tegafur (trade names: fturafur, sunfural, lunasin, tef seal, etc.), capecitabine (trade name: Xeloda), and the like.

  In the present specification, indications with abbreviations such as nucleotide, polynucleotide, DNA, RNA, etc. are the three common guidelines, “Guidelines for creating a description including a base sequence or amino acid sequence” (Japan Patent Office) Ed) and customary in the art.

  In the present specification, the “polynucleotide” is used as a nucleic acid including any of RNA, DNA, and modified polynucleotides thereof. The DNA includes any of cDNA, genomic DNA, and synthetic DNA. The RNA includes total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, shRNA, non-coding RNA, pri-miRNA, pre-miRNA, and synthetic RNA. The modified polynucleotide includes a polynucleotide obtained by converting a natural sugar moiety, base moiety or phosphodiester bond of a nucleic acid into a non-natural structure, such as LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), phosphorothioate. Examples include a polynucleotide having a bond, an artificial polynucleotide having a 2′-O-alkyl ribose group, and the like. In the present specification, the polynucleotide is used interchangeably with the nucleic acid.

  In this specification, “gene” includes not only RNA and double-stranded DNA, but also each single-stranded DNA such as positive strand (or sense strand) or complementary strand (or antisense strand) constituting the same. It is intended to be used. The length is not particularly limited.

  Accordingly, in the present specification, unless otherwise specified, “gene” refers to double-stranded DNA containing human genomic DNA, single-stranded DNA containing cDNA (positive strand), and single-stranded DNA having a sequence complementary to the positive strand. All of DNA (complementary strand) and fragments thereof, and human genome are included. The “gene” is not limited to the “gene” represented by a specific base sequence (or SEQ ID NO), but also RNA having a biological function equivalent to RNA encoded by these, for example, homologs (ie, homologs). , Variants such as gene polymorphisms, and “genes” encoding derivatives are included. Specifically, the “gene” encoding the homologue, variant or derivative is, specifically, the complement of any of the specific base sequences represented by SEQ ID NOs: 1 to 22 under the stringent conditions described later A “gene” having a base sequence that hybridizes with the sequence can be mentioned. The “gene” 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. In addition, the “gene” may be contained in the cell, may be released outside the cell and may exist alone, or may be encapsulated in a vesicle encapsulated in a lipid bilayer called an exosome. There may be.

  Exosomes are small vesicles secreted from cells. Exosomes are derived from multivesicular endosomes and release internal vesicles to the extracellular environment by fusing with cell membranes, and at that time, vesicles contain “genes” or “nucleic acids” such as RNA and DNA. Sometimes. It is known that exosomes are contained in body fluids such as blood, serum, plasma, urine and lymph.

  In the present specification, the “transcript” refers to RNA synthesized using a DNA sequence of a gene as a template. RNA polymerase is synthesized in such a manner that RNA polymerase binds to a site called a promoter located upstream of the gene and ribonucleotide is bound to the 3 ′ end so as to be complementary to the DNA base sequence. This RNA includes not only the gene itself but also the entire sequence from the transcription start point to the end of the poly A sequence, including the expression control region, coding region, exon or intron.

  In the present specification, unless otherwise specified, “microRNA (miRNA)” is a protein complex that is transcribed as a hairpin-like RNA precursor, cleaved by a dsRNA cleaving enzyme having RNase III cleavage activity, and referred to as RISC. 15 to 25 base RNA that is incorporated in and is involved in mRNA translational suppression. The “miRNA” contains not only the “miRNA” represented by a specific base sequence (or SEQ ID NO) but also a precursor of the “miRNA” (pre-miRNA, pri-miRNA), and is encoded by these. Also included are miRNAs that have biological functions equivalent to miRNAs, such as homologues (ie, homologs), variants such as genetic polymorphisms, and “miRNAs” that encode derivatives. The “miRNA” encoding such a precursor, homologue, variant or derivative can be specifically identified by miRBase (http://www.mirbase.org/), and the stringent described later. And “miRNA” having a base sequence that hybridizes with a complementary sequence of any one of the specific base sequences represented by SEQ ID NOs: 1 to 22 under such conditions.

  As used herein, the term “probe” refers to a polynucleotide used for specifically detecting RNA produced by gene expression or a polynucleotide derived therefrom and / or a polynucleotide comprising a base sequence complementary thereto. Includes.

  In the present specification, the “primer” includes a continuous polynucleotide and / or a complementary polynucleotide that specifically recognizes and amplifies RNA generated by gene expression or a polynucleotide derived therefrom.

  Here, a polynucleotide containing a complementary base sequence (complementary strand or reverse strand) is a full-length sequence of a polynucleotide consisting of a base sequence defined by a sequence number, or a partial sequence thereof (here, for the sake of convenience, this is a normal sequence). A polynucleotide having a base complementary relationship based on a base pair relationship such as A: T (U) and G: C. However, such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target positive strand base sequence, but has a complementary relationship that allows hybridization with the target normal strand under stringent conditions. There may be.

  As used herein, “stringent conditions” refers to conditions under which a probe hybridizes to its target sequence to a detectably greater extent (eg, at least twice background) than to other sequences. Say. Stringent conditions are sequence-dependent and depend on the environment in which hybridization is performed. By controlling the stringency of the hybridization and / or wash conditions, target sequences that are 100% complementary to the probe can be identified.

  As used herein, the term “variant” refers to a nucleic acid, a natural variant resulting from polymorphism, mutation, alternative splicing during transcription, or the nucleotide sequence represented by SEQ ID NOs: 1-22. About 90% or more, about 95% or more of a mutant comprising 1 or 2 nucleotide deletions, substitutions, additions or insertions, preferably substitutions in the complementary base sequence, or the base sequence or a partial sequence thereof. It hybridizes under stringent conditions as defined above with a variant showing about 97% or more, about 98% or more, or about 99% or more, or a polynucleotide or oligonucleotide containing the nucleotide sequence or a partial sequence thereof. Means nucleic acid.

  In the present specification, “% identity” can be determined using the above-mentioned BLAST or FASTA protein or gene search system with or without introducing a gap (Karlin, S .; 1993, Proceedings of the National Academic Sciences USA, 90, p. 5873-5877; Altschul, SF, et al., 1990, Journal of Molecular Biology, 215, p. 403-410; Pearson, WR et al., 1988, Proceedings of the National Academic Sciences USA, 85, pp. 2444-2448).

  As used herein, the term “derivative” refers to a labeled derivative such as a fluorophore, a modified nucleotide (for example, a reorganization of a nucleotide and a base containing a group such as halogen, alkyl such as methyl, alkoxy such as methoxy, thio, and carboxymethyl, Derivatives containing double bonds, deamination, substitution of oxygen molecules with sulfur molecules, etc.) and modified polynucleotides such as artificial polynucleotides (eg LNA, PNA, etc.).

  In the present specification, the “(determination) composition” refers to a composition that is used directly or indirectly to determine the sensitivity of adjuvant chemotherapy performed after surgery for a stomach cancer patient. This includes nucleotides, oligonucleotides and polynucleotides capable of specifically recognizing and binding to genes whose expression varies in vivo, particularly in gastric tumor tissue, related to the susceptibility to adjuvant chemotherapy for gastric cancer. The These nucleotides, oligonucleotides and polynucleotides are used as probes for detecting the gene expressed in vivo, in tissues or cells based on the above properties, and for amplifying the gene expressed in vivo. It can be effectively used as a primer.

  In the present specification, the “specimen sample” to be detected and diagnosed refers to biological tissues in which the gene of the present invention changes in expression due to the occurrence of gastric cancer, substances derived from those biological tissues, and substances that come into contact with the biological tissues. Specifically, it refers to gastric surgical tissue or biopsy tissue.

  In the present specification, “Sensitivity” and “Specificity” mean an index represented by the calculation described later when evaluating the reliability of a clinical test. Specifically, when a test is performed on a group that is susceptible to adjuvant chemotherapy for gastric cancer, the ratio (true positive rate) indicating positive, that is, sensitivity to adjuvant chemotherapy, is Sensitivity. Conversely, when testing is performed on a population that is not sensitive to adjuvant chemotherapy for gastric cancer, the percentage (true negative rate) indicating that it is negative, that is, not sensitive to adjuvant chemotherapy, is the specificity.

  In this specification, “Accuracy” means the divisor of the total number of cases with respect to the sum of “the number of times that a recurrence-free case was correctly predicted to be chemosensitive” and “the number of times that a recurrent case was correctly predicted not to be chemosensitive” To do.

  In the present specification, “PPV” means (number of times that a recurrence-free case is correctly predicted to be susceptible to adjuvant chemotherapy) / (number of times that it is predicted to be susceptible to adjuvant chemotherapy).

  As used herein, “NPV” means (number of times that a relapsed case was correctly predicted not to be sensitive to adjuvant chemotherapy) / (number of times predicted to be not sensitive to adjuvant chemotherapy).

  In this specification, “Efficacy” means a product of PPV and NPV. Therefore, this index becomes high when a correct diagnosis is established in patients who are sensitive to adjuvant chemotherapy or patients who are not sensitive to adjuvant chemotherapy in the examination of adjuvant chemotherapy sensitivity of gastric cancer.

  As used herein, the term “miR-148a gene” or “miR-148a” includes the hsa-miR-148a gene (miRbase Accession No. MIMAT000003) described in SEQ ID NO: 4 and other species homologs. Is done.

  As used herein, the term “miR-425 gene” or “miR-425” includes the hsa-miR-425 gene (miRbase Accession No. MIMAT0003393) described in SEQ ID NO: 1 and other species homologues. Is done.

  As used herein, the term “miR-146a gene” or “miR-146a” includes the hsa-miR-146a gene (miRbase Accession No. MIMAT000049) described in SEQ ID NO: 5 and other species homologues. Is done.

  As used herein, the term “miR-193b * gene” or “miR-193b *” refers to the hsa-miR-193b * gene (miRbase Accession No. MIMAT0004767) described in SEQ ID NO: 6 or other species homologues. Etc. are included.

  As used herein, the term “miR-141 gene” or “miR-141” includes the hsa-miR-141 gene (miRbase Accession No. MIMAT000032) described in SEQ ID NO: 2 and other species homologues. Is done.

  As used herein, the term “miR-22 gene” or “miR-22” includes the hsa-miR-22 gene (miRbase Accession No. MIMAT00000077) described in SEQ ID NO: 7 and other species homologues. Is done.

  The term “miR-92a-2 * gene” or “miR-92a-2 *” as used herein refers to the hsa-miR-92a-2 * gene (miRbase Accession No. MIMAT0004508) described in SEQ ID NO: 8. ) And other species homologues.

  As used herein, the term “miR-151-3p gene” or “miR-151-3p” refers to the hsa-miR-151-3p gene (miRbase Accession No. MIMAT000057) described in SEQ ID NO: 14 and others Species homologues and the like are included.

  As used herein, the term “miR-30d gene” or “miR-30d” includes the hsa-miR-30d gene (miRbase Accession No. MIMAT00002245) described in SEQ ID NO: 15 and other species homologues. Is done.

  As used herein, the term “miR-664 gene” or “miR-664” includes the hsa-miR-664 gene (miRbase Accession No. MIMAT0005949) described in SEQ ID NO: 9, and other species homologues. Is done.

  As used herein, the term “miR-1908 gene” or “miR-1908” includes the hsa-miR-1908 gene (miRbase Accession No. MIMAT0007881) described in SEQ ID NO: 16 and other species homologues. Is done.

  As used herein, the term “miR-200b gene” or “miR-200b” includes the hsa-miR-200b gene (miRbase Accession No. MIMAT000018) described in SEQ ID NO: 17 and other species homologues. Is done.

  As used herein, the term “miR-769-3p gene” or “miR-769-3p” refers to the hsa-miR-769-3p gene (miRbase Accession No. MIMAT0003887) described in SEQ ID NO: 10 and others. Species homologues and the like are included.

  As used herein, the term “miR-1978 gene” or “miR-1978” includes the hsa-miR-1978 gene (miRbase Accession No. MIMAT0009453) described in SEQ ID NO: 3, and other species homologues. Is done.

  As used herein, the term “miR-605 gene” or “miR-605” includes the hsa-miR-605 gene (miRbase Accession No. MIMAT0003273) described in SEQ ID NO: 11, and other species homologs. Is done.

  As used herein, the term “miR-200c gene” or “miR-200c” includes the hsa-miR-200c gene (miRbase Accession No. MIMAT000017) described in SEQ ID NO: 18 and other species homologs. Is done.

  As used herein, the term “miR-1915 gene” or “miR-1915” includes the hsa-miR-1915 gene (miRbase Accession No. MIMAT0007892) described in SEQ ID NO: 19, and other species homologs. Is done.

  As used herein, the term “miR-199b-5p gene” or “miR-199b-5p” refers to the hsa-miR-199b-5p gene (miRbase Accession No. MIMAT000002) described in SEQ ID NO: 20 and others Species homologues and the like are included.

  As used herein, the term “miR-145 * gene” or “miR-145 *” refers to the hsa-miR-145 * gene (miRbase Accession No. MIMAT0004601) described in SEQ ID NO: 12 and other species homologues. Etc. are included.

  As used herein, the term “miR-500 * gene” or “miR-500 *” refers to the hsa-miR-500 * gene (miRbase Accession No. MIMAT0002871) described in SEQ ID NO: 13 or other species homologues. Etc. are included.

  As used herein, the term “miR-29b-2 * gene” or “miR-29b-2 *” refers to the hsa-miR-29b-2 * gene (miRbase Accession No. MIMAT0004515) described in SEQ ID NO: 21. ) And other species homologues.

  As used herein, the term “miR-155 gene” or “miR-155” includes the hsa-miR-155 gene (miRbase Accession No. MIMAT00000006) described in SEQ ID NO: 22 and other species homologues. Is done.

  The present invention provides a composition useful for determining the presence or absence of adjuvant chemotherapy sensitivity for gastric cancer and a method for determining adjuvant chemotherapy sensitivity for gastric cancer using the composition, thereby providing adjuvant chemotherapy for gastric cancer. It is possible to provide a determination method that is specific to sensitivity, has a high prediction rate, and is quick and simple.

A schematic diagram of a neural network model is shown. The flow of analysis for determining the genes described in Table 1 is shown. The determination accuracy of adjuvant chemotherapy sensitivity when the polynucleotides of SEQ ID NOs: 1 to 22 corresponding to the genes listed in Table 1 are used in combination is shown. The vertical axis is the probability of determining the presence or absence of gastric cancer adjuvant chemosensitivity, and the horizontal axis is necessary to determine the presence or absence of gastric cancer adjuvant chemotherapy sensitivity by increasing the combination by 1 in the order of the sequence numbers listed in Table 1. The number of polynucleotides is shown respectively.

The present invention will be described more specifically below. Target nucleic acid used for determination of sensitivity to adjuvant chemotherapy for gastric cancer As a marker for determining sensitivity to adjuvant chemotherapy for gastric cancer or gastric cancer cells using the composition and kit for determining sensitivity to adjuvant chemotherapy for gastric cancer of the present invention as defined above The target nucleic acids include, for example, human miRNA genes containing the nucleotide sequences represented by SEQ ID NOs: 1-22 (ie, miR-148a, miR-425, miR-146a, miR-193b *, miR-141, respectively). miR-22, miR-92a-2 *, miR-151-3p, miR-30d, miR-664, miR-1908, miR-200b, miR-769-3p, miR-1978, miR-605, miR-200c , MiR-1915, miR-199b-5p, miR-145 *, miR-500 * miR-29b-2 *, and miR-155), their homologues and their transcripts, or variants thereof, or derivatives thereof. Here, genes, homologues, transcripts, mutants and derivatives are as defined above. A preferred target nucleic acid is a human miRNA gene comprising the nucleotide sequence represented by SEQ ID NO: 1-22, a transcription product thereof, more preferably the transcription product.

  In the present invention, any of the above genes that are targets for gastric cancer has an increased or decreased expression level in subjects suffering from gastric cancer as compared to healthy subjects (see Table 1 in Examples below).

  The first target nucleic acid is a miR-148a gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. So far, there has been no report that an increase in the expression of the miR-148a gene or a transcription product thereof can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The second target nucleic acid is the miR-425 gene, their homologs, their transcripts, or their variants or derivatives. So far, there has been no report that an increase in the expression of the miR-425 gene or a transcript thereof can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The third target nucleic acid is the miR-146a gene, their homologs, their transcripts, or their variants or derivatives. So far, there has been no report that an increase in the expression of the miR-146a gene or a transcription product thereof can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The fourth target nucleic acid is the miR-193b * gene, their homologs, their transcripts, or their mutants or derivatives. To date, there has been no report that increased expression of the miR-193b * gene or its transcription product can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The fifth target nucleic acid is a miR-141 gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. So far, there has been no report that increased expression of the miR-141 gene or its transcription product can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The sixth target nucleic acid is the miR-22 gene, their homologs, their transcripts, or their variants or derivatives. To date, there has been no report that increased expression of the miR-22 gene or its transcription product can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The seventh target nucleic acid is a miR-92a-2 * gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. To date, there is no known report that increased expression of the miR-92a-2 * gene or its transcript can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The eighth target nucleic acid is a miR-151-3p gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. So far, there is no known report that an increase in the expression of the miR-151-3p gene or a transcription product thereof can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The ninth target nucleic acid is a miR-30d gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. So far, there has been no report that an increase in the expression of the miR-30d gene or a transcript thereof can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The tenth target nucleic acid is a miR-664 gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. To date, there has been no report that increased expression of the miR-664 gene or its transcription product can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The eleventh target nucleic acid is a miR-1908 gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. To date, there has been no report that increased expression of the miR-1908 gene or its transcription product can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The twelfth target nucleic acid is a miR-200b gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. So far, there has been no report that an increase in the expression of the miR-200b gene or a transcription product thereof can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The thirteenth target nucleic acid is a miR-769-3p gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. So far, there has been no report that increased expression of the miR-769-3p gene or its transcription product can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The fourteenth target nucleic acid is a miR-1978 gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. So far, there has been no report that an increase in the expression of the miR-1978 gene or a transcription product thereof can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The fifteenth target nucleic acid is a miR-605 gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. To date, there has been no report that increased expression of the miR-605 gene or its transcription product can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The sixteenth target nucleic acid is a miR-200c gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. So far, there is no known report that increased expression of the miR-200c gene or its transcription product can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The seventeenth target nucleic acid is a miR-1915 gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. To date, there has been no report that increased expression of the miR-1915 gene or its transcript can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The eighteenth target nucleic acid is a miR-199b-5p gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. So far, there has been no report that increased expression of the miR-199b-5p gene or its transcription product can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The nineteenth target nucleic acid is a miR-145 * gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. So far, there is no known report that increased expression of the miR-145 * gene or its transcription product can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The twentieth target nucleic acid is a miR-500 * gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. So far, there has been no report that increased expression of the miR-500 * gene or its transcription product can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The 21st target nucleic acid is a miR-29b-2 * gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. So far, there has been no report that increased expression of the miR-29b-2 * gene or its transcription product can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

  The twenty-second target nucleic acid is a miR-155 gene, a homologue thereof, a transcription product thereof, or a variant or derivative thereof. To date, there has been no report that increased expression of the miR-155 gene or its transcription product can be a marker of sensitivity to adjuvant chemotherapy for gastric cancer.

2. Composition for Determining Adjuvant Chemotherapy Sensitivity of Gastric Cancer In the present invention, a nucleic acid composition that can be used for determining the sensitivity of adjunct chemotherapy for gastric cancer or for diagnosing adjunct chemotherapy sensitivity for gastric cancer is an adjuvant chemistry for gastric cancer. MiR-148a, miR-425, miR-146a, miR-193b *, miR-141, miR-22, miR-92a-2 *, miR-151-3p, miR as therapeutic target nucleic acids -30d, miR-664, miR-1908, miR-200b, miR-769-3p, miR-1978, miR-605, miR-200c, miR-1915, miR-199b-5p, miR-145 *, miR- 500 *, miR-29b-2 *, and miR-155, their homologues, their transcripts Or the presence of their variants or derivatives, allows the expression level or abundance qualitatively and / or quantitatively measured.

  The expression level of the target nucleic acid is increased or decreased in subjects who are not sensitive to adjuvant chemotherapy compared to subjects who are sensitive to adjuvant chemotherapy for gastric cancer. Therefore, the composition of the present invention can be effectively used for measuring the expression level of target nucleic acid in gastric cancer tissues and comparing them.

  The composition that can be used in the present invention includes a polynucleotide group comprising the nucleotide sequence represented by SEQ ID NOs: 1-22 in a living tissue of a patient suffering from gastric cancer, a precursor polynucleotide group thereof, and a complementary polynucleotide thereof. Group, their variant polynucleotide group, polynucleotide group that hybridizes with the base sequence of those polynucleotide group under stringent conditions, their modified polynucleotide group, and their base sequence One or more selected from a group of polynucleotides comprising 15 or more, preferably 20 or more, 21 or more, more preferably 22 or more consecutive bases (2 or more, for example, any number from 2 to 22, or more) A combination of the following polynucleotides.

Specifically, the composition of the present invention can include one or more of the following polynucleotides.
(1) A polynucleotide comprising the nucleotide sequence represented by SEQ ID NOs: 1-22, a precursor polynucleotide thereof, a mutant polynucleotide thereof, or a modified polynucleotide thereof.
(2) A polynucleotide comprising the base sequence represented by SEQ ID NOs: 1-22.
(3) A polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NOs: 1 to 22, a mutant polynucleotide thereof, or a modified polynucleotide thereof.
(4) A polynucleotide comprising a base sequence complementary to the base sequences represented by SEQ ID NOs: 1-22.
(5) A polynucleotide that hybridizes with each of the nucleotide sequences of the polynucleotides according to (1) to (4) above under stringent conditions, or the nucleotide sequence represented by SEQ ID NOs: 1 to 22 or a complementary sequence thereof A polynucleotide that hybridizes under stringent conditions with DNA comprising each of the base sequences.
(6) A DNA encoding each of the polynucleotides of any one of (1) to (5) above.

  In the case of the precursor polynucleotide or its complementary polynucleotide, in the base sequence of each polynucleotide, for example, but not limited to, the total number of bases of 15 to 15 consecutive, 15 to 20 bases, 15 to 21 bases, A fragment having a base number in the range of 15 to 22 bases, 15 to 23 bases, 18 to 25 bases, 18 to 30 bases, 18 to 50 bases, and the like can be included. Each base sequence represented by ˜22 or a complementary base sequence thereof is included.

  Any of the polynucleotides or fragments thereof used in the present invention may be DNA, RNA, or a modified polynucleotide.

  The polynucleotide as the composition of the present invention can be prepared using a general technique such as a DNA recombination technique, a PCR method, a method using a DNA / RNA automatic synthesizer.

  DNA recombination techniques and PCR methods are described in, for example, Ausubel et al., Current Protocols in Molecular Biology, John Willy & Sons, US (1993); Sambrook et al., Molecular Cloning A Laboratories. The techniques described can be used.

  Human-derived miR-148a, miR-425, miR-146a, miR-193b *, miR-141, miR-22, miR-92a-2 *, miR-151-3p, miR-30d, miR-664, miR-1908, miR-200b, miR-769-3p, miR-1978, miR-605, miR-200c, miR-1915, miR-199b-5p, miR-145 *, miR-500 *, miR-29b- 2 * and miR-155 are publicly known, and their acquisition methods are also known as described above. Therefore, polynucleotides as the composition of the present invention can be prepared by cloning these genes.

  The polynucleotide constituting the composition of the present invention can be chemically synthesized using an automatic DNA synthesizer. In general, the phosphoramidite method is used for this synthesis, and single-stranded DNA of up to about 100 bases can be automatically synthesized by this method. Automatic DNA synthesizers are commercially available from, for example, Polygen, ABI, and Applied BioSystems.

  Alternatively, the polynucleotide of the present invention can be prepared by a cDNA cloning method. For example, microRNA Cloning Kit Wako can be used as the cDNA cloning technique.

3. Kit for determining sensitivity to adjuvant chemotherapy for gastric cancer The present invention also includes one or more (two or more, for example from two) of the same polynucleotides (and optionally fragments thereof) as those included in the composition of the present invention. A kit for determining sensitivity to adjuvant chemotherapy for gastric cancer, comprising any number of 22 or more).

  The kit of the present invention preferably contains one or more polynucleotides selected from the polynucleotides described in 1 above.

  The kit of the present invention comprises a polynucleotide comprising the nucleotide sequence represented by SEQ ID NOs: 1-22, a precursor polynucleotide thereof, a polynucleotide comprising the complementary sequence thereof, a mutant polynucleotide thereof, a polynucleotide thereof and a string. Two or more polynucleotides that hybridize under gentle conditions, DNAs encoding those polynucleotides, or modified polynucleotides thereof can be included.

  The polynucleotide fragment that can be included in the kit of the present invention is, for example, the nucleotide sequence of a precursor polynucleotide (ie, pri-miRNA, pre-miRNA) of the polynucleotide represented by SEQ ID NOs: 1 to 22, or a complementary sequence thereof. The DNA contains 15 or more or 20 or more consecutive bases.

  In a preferred embodiment, the polynucleotide is a polynucleotide comprising the base sequence represented by any of SEQ ID NOs: 1-22, a polynucleotide comprising the complementary sequence thereof, a variant polynucleotide thereof, a polynucleotide thereof, and the like. A polynucleotide that hybridizes under stringent conditions, a DNA that encodes the polynucleotide, or a modified polynucleotide thereof.

  The mutant polynucleotide is, for example, a polynucleotide in which one or two nucleotides are substituted with another base in the base sequence represented by any of SEQ ID NOs: 1 to 22 or a complementary sequence. The mutation is not limited to the above as long as it can be hybridized with the base sequence of the marker miRNA. Mutations also include, for example, nucleotide deletions or additions.

  The modified polynucleotide includes a polynucleotide obtained by converting a natural sugar part, a base part or a phosphodiester bond of a nucleic acid into a non-natural structure, and has, for example, LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), or phosphorothioate bond. Examples include polynucleotides, artificial polynucleotides having 2′-O-alkyl ribose groups, and the like. Preferred modified polynucleotides are LNA and PNA.

  LNA is a modified RNA, and is an artificial polynucleotide having a structure in which the 2′-position and 4′-position of natural RNA are cross-linked via a methylene group (AA Koshkin et al., Tetrahedron 54). : 3607, 1998; S. Obika et al., Tetrahedron Lett., 39: 5401, 1998). Like PNA, it is excellent in enzyme stability and can hybridize with DNA or RNA. The modified polynucleotide that can be used in the method of the present invention may be a hybrid form of LNA and DNA, in which case the content of LNA may vary between 0% and 100%.

  PNA is a (poly) peptide whose main chain is a 2-aminoethylglycyl skeleton, and is an artificial polynucleotide or nucleic acid having a nucleobase in its side chain (PE Nielsen et al., Science). M. Castoldi et al., RNA, 12: 913, 2006; Takashi Ohtsuki and Mitsuo Kitamatsu, Future Materials, Vol. 9, No. 5, pp. 21-21, 2009 (NTS )). Since the main chain has no charge, a stable hybrid can be formed with RNA or DNA.

  In a preferred embodiment, the polynucleotide is a polynucleotide comprising 15 or more, preferably 20 or more, 21 or more, more preferably 22 or more, 30 or less, preferably 25 or less, more preferably 24 or less consecutive bases. be able to.

  Examples of the above combinations include polynucleotides comprising the nucleotide sequences shown in SEQ ID NOs: 1 to 22 or their complementary sequences, their mutant polynucleotides, polynucleotides that hybridize with these polynucleotides under stringent conditions, and the like As shown in Table 1 to be described later, the DNA encoding the polynucleotide and the modified polynucleotide are polysequated in order from the highest polynucleotide priority (from the first to the 22nd) (sequence number order). Combining nucleotides. The probability (Accuracy;%) of correctly determining the adjuvant chemotherapy sensitivity of gastric cancer when 22 types of polynucleotides were combined in this way was compared to the number of polynucleotides required for determining the adjuvant chemotherapy sensitivity of gastric cancer. For example, the probability is 75% for a combination of miR-148a (SEQ ID NO: 1) and miR-425 (SEQ ID NO: 2), and miR-146a (SEQ ID NO: 3), miR-193b * (SEQ ID NO: The combination of 4) (the combination of the polynucleotides of SEQ ID NOs: 1 to 4) was 80%. When miRNA was further combined, the probability up to 90% was obtained by combining up to miR-769-3p (SEQ ID NO: 13) (the combination of polynucleotides of SEQ ID NO: 1 to 13) (see FIG. 3 described later). .

  The above combinations constituting the kit of the present invention are merely examples, and all other various possible combinations are included in the present invention.

  In addition to the polynucleotides of the present invention described above, the kits of the present invention can also include known or future-found polynucleotides that allow determination of gastric cancer adjuvant chemosensitivity.

  The polynucleotides contained in the kit of the present invention are packaged individually or in any combination in a container.

  The kit of the present invention may be in a form in which the polynucleotide is bound to a solid phase such as a well plate or a polymer membrane. The bond may be a covalent bond or a non-covalent bond (eg, ionic bond, physical or hydrophobic bond, etc.). The plate surface may be subjected to a surface treatment such as poly-L-lysine coating.

  The kit may contain buffers necessary for carrying out hybridization, a well plate or a membrane, instructions for use, and the like.

4). Nucleic acid array The present invention further includes the same polynucleotides as those included in the compositions and / or kits of the present invention (or the polynucleotides described in the compositions of Section 2 and / or Kits of Section 3 above). A nucleic acid array for determining sensitivity to adjuvant chemotherapy for gastric cancer, comprising a combination of a plurality of polynucleotides from 2 (eg, any number from 2 to 22, or more).

  The substrate (carrier) of the nucleic acid array on which the polynucleotide is immobilized is not particularly limited as long as the polynucleotide can be immobilized, and examples thereof include a slide glass, a silicon chip, a polymer chip, and a nylon membrane. it can. Further, these substrates may be subjected to surface treatment such as poly L lysine coating, introduction of functional groups such as amino groups and carboxyl groups.

  The immobilization method is not particularly limited as long as it is a commonly used method, and a method of spotting nucleic acid using a high-density dispenser called a spotter or an arrayer, or a fine droplet from a nozzle by a piezoelectric element or the like. Examples thereof include a method of spraying DNA onto a substrate using a spraying apparatus (inkjet method) or a method of sequentially performing nucleotide synthesis on a substrate. When using a high-density dispenser, for example, put a different gene solution in each well of a plate with many wells, and pick up this solution with a pin (needle) and spot it on the substrate in order. by. In the ink jet method, genes are ejected from a nozzle, and the genes are aligned and arranged on a substrate at a high speed. For DNA synthesis on a substrate, the base bonded to the substrate is protected with a functional group that can be removed by light or heat, and the functional group is removed by applying light or heat only to the base at a specific site by using a mask. Let Thereafter, the step of adding a base to the reaction solution and coupling with the base on the substrate is repeated.

  For example, 3D-Gene (registered trademark) Human miRNA Oligo chip from Toray Industries, Inc. Human miRNA Microarray Kit (V2) from Agilent, miRCURY LNA (registered trademark) microRNA ARRAY from EXIQON, etc. Target gene, RNA or cDNA An existing nucleic acid array platform (substrate, etc.) capable of detecting and measuring the level can be used, and the polynucleotide of the present invention can be mounted on the platform to be used for determining sensitivity to adjuvant chemotherapy of the present invention. .

  As the polynucleotide immobilized on the nucleic acid array, all or a part of the polynucleotide of the present invention described above can be used. According to a preferred embodiment, the nucleic acid array of the present invention is a polynucleotide comprising the nucleotide sequence represented by SEQ ID NOs: 1-22 or a complementary sequence thereof, DNA encoding the polynucleotide, or the polynucleotide or DNA 2 or more, or all or more of artificial polynucleotides consisting of LNA or PNA.

  In the present invention, the polynucleotide to be immobilized may be any of cDNA, RNA, synthetic DNA, synthetic RNA, and artificial polynucleotide, or may be single-stranded or double-stranded.

  The nucleic acid array can be produced by, for example, a method of immobilizing a probe prepared in advance on the substrate surface. In the method of immobilizing a polynucleotide probe prepared in advance on a substrate surface, a polynucleotide into which a functional group is introduced is synthesized, and oligonucleotides or polynucleotides are spotted on the surface of the surface-treated substrate and covalently bonded (for example, (J. B. Lamture et al., Nucleic. Acids. Research, 1994, Vol. 22, p. 2121-2125, Z. Guo et al., Nucleic. Acids. Research, 1994, Vol. 22, p. 5456-5465) . In general, the polynucleotide is covalently bonded to the surface-treated substrate via a spacer or a crosslinker. A method is also known in which polyacrylamide gel fragments are aligned on the surface of a glass substrate and a synthetic polynucleotide is covalently bound thereto (G. Yershov et al., Proceedings of the National Academic Sciences USA, 1996, vol. 94, p. 4913). In addition, a microelectrode array was prepared on a silica nucleic acid array, and an agarose permeation layer containing streptavidin was provided on the electrode as a reaction site, and this site was charged positively to immobilize the biotinylated polynucleotide. In addition, a method that enables high-speed and precise hybridization by controlling the charge of the site is known (RG Sosnowski et al., Proceedings of the National Academic Sciences USA, 1997, 94, pp. 1119-1123).

5. Method for Determining Adjuvant Chemotherapy Sensitivity of Gastric Cancer The present invention uses the composition, kit, nucleic acid array, or a combination thereof of the present invention to determine the expression behavior of miRNA genes associated with gastric cancer adjunct chemotherapy sensitivity. An in vitro determination method that uses a sample sample of a stomach cancer patient to compare the expression levels of miRNA genes in the sample and determine the expression behavior of genes related to gastric cancer adjuvant chemotherapy sensitivity in the sample sample Wherein the target nucleic acid is detectable by the polynucleotides contained in the composition, kit or nucleic acid array.

  By detecting, determining, or diagnosing miRNA or a precursor thereof (pri-miRNA or pre-miRNA) related to the sensitivity to adjuvant chemotherapy of gastric cancer from the specimen sample by the above-described method of the present invention, sensitivity and specificity can be improved. Not only is it possible to determine high sensitivity to adjuvant chemotherapy, but also the selection of the appropriate anticancer drug for the patient enables early treatment and improvement of the prognosis. And methods that allow monitoring of the effectiveness of chemotherapeutic treatments.

  As a method for extracting miRNA gene transcripts related to gastric cancer adjuvant chemotherapy sensitivity from the sample of the present invention, RNA extraction reagents containing acidic phenols such as Trizol (life technologies) and Isogen (Nippon Gene) are used. May be. When a formalin-fixed paraffin-embedded (FFPE) specimen is used, a reagent for extracting RNA from FFPE, for example, RNeasy FFPE Kit (Qiagen), RecoverAll (TM) Total Nucleic Acid Isolation Kit for FFPE (Ambion), Agencor, Forma Pure Kit (Beckman Coulter), High Pure FFPE RNA Micro Kit (Roche), and the like can be used, but the method is not limited thereto.

  The present invention also provides the use of the composition, kit or nucleic acid array of the present invention for in vitro detection of miRNA derived from gastric cancer in a specimen sample derived from a subject.

  In the above method of the present invention, a composition, kit or nucleic acid array is used which contains the polynucleotides of the present invention as described above, either singly or in any possible combination.

  In the detection, determination or diagnosis of gastric cancer of the present invention, the polynucleotides contained in the composition, kit or nucleic acid array of the present invention can be used as a primer or a probe. When used as a primer, TaqMan (registered trademark) MicroRNA Assays from Applied Biosystems can be used, but is not limited to this method.

  Polynucleotides contained in the composition or kit of the present invention are known to specifically detect a specific gene, such as Northern blotting, Southern blotting, RT-PCR, in situ hybridization, Southern hybridization In this method, it can be used as a primer or a probe according to a conventional method. As a sample to be measured, depending on the type of detection method to be used, a part or the whole of a gastric cancer lesion part and a normal tissue part of a subject is collected by biopsy or collected from a living tissue extracted by surgery. Furthermore, total RNA prepared therefrom according to a conventional method may be used, and various polynucleotides including cDNA prepared based on the RNA may also be used.

  Alternatively, the expression level of miRNA that is a target in the present invention in living tissue can be detected or quantified using a nucleic acid array (including a DNA macroarray or a DNA chip). In this case, the composition or kit of the present invention can be used as a probe for a nucleic acid array. Such a nucleic acid array is hybridized with miRNA prepared from RNA collected from a living tissue or a DNA encoding the nucleic acid and labeled nucleic acid, and a composite of the probe and labeled DNA or RNA formed by the hybridization By detecting the body using the label of the labeled DNA or RNA as an index, the presence or absence or expression level (expression level) of the miRNA gene related to the sensitivity to adjuvant chemotherapy of gastric cancer of the present invention in living tissue is evaluated. can do. In the method of the present invention, a nucleic acid array can be preferably used, which can evaluate the presence or absence of expression of a plurality of miRNA genes or the expression level simultaneously for one biological sample.

  The composition, kit or nucleic acid array of the present invention is useful for determining sensitivity to adjuvant chemotherapy for gastric cancer. Specifically, determination of gastric cancer adjuvant chemotherapy sensitivity using the composition, kit, or nucleic acid array is performed using cells that are sensitive to gastric cancer adjuvant chemotherapy or cells that are not sensitive at the time of surgery or endoscopy. And comparing the expression level of miRNA in the sample or determining the difference in the expression level of the miRNA gene detected by the diagnostic composition in gastric cancer tissues collected at the time of surgery or endoscopy Can do. In this case, the difference in the miRNA gene expression level is not only the presence or absence of expression, but also when there is expression in both the biological tissue of a patient who is sensitive to adjuvant chemotherapy for gastric cancer and the biological tissue of a patient who is not sensitive to adjuvant chemotherapy, The case where there is a difference when the expression level between the two is compared is included.

  A method for determining whether or not gastric cancer is sensitive to adjuvant chemotherapy in a sample sample by using the composition, kit or nucleic acid array of the present invention is / is not sensitive to adjuvant chemotherapy. Is collected from a biological tissue extracted by surgery or the like, and the gene contained therein is a single or plural, preferably plural, polynucleotides selected from the polynucleotide group of the present invention. Detection using and determining by measuring the gene expression level. At this time, an FFPE sample with information such as the presence or absence of recurrence may be used. The method for determining the sensitivity to adjuvant chemotherapy for gastric cancer according to the present invention may also detect, determine or diagnose the presence or absence of recurrence of the disease when a therapeutic agent is administered to prevent the recurrence of the disease, for example, in gastric cancer patients. it can.

The method of the present invention includes, for example, the following steps (a), (b) and (c):
(A) contacting a specimen sample derived from a subject with a polynucleotide of the composition, kit or nucleic acid array of the present invention;
(B) measuring the expression level of the target nucleic acid in the biological sample using the polynucleotide as a probe;
(C) based on the result of (b), the step of determining the sensitivity to adjuvant chemotherapy of gastric cancer in the sample can be included.

  Examples of the specimen sample used in the method of the present invention include a biological tissue of a subject, such as stomach tissue and surrounding tissues. Specifically, an RNA-containing sample prepared from the tissue, or a sample containing a polynucleotide further prepared therefrom, is obtained by collecting a part or all of a subject's biological tissue with a biopsy or the like, or removing it by surgery And can be prepared according to a conventional method.

  The term “subject” as used herein refers to mammals such as, but not limited to, humans, monkeys, mice, rats, and the like, preferably humans.

  The method of this invention can change a process according to the kind of biological sample used as a measuring object.

When RNA is used as the measurement object, detection of gastric cancer (cells) is performed, for example, by the following steps (a), (b) and (c):
(A) a step of binding RNA prepared from a biological sample of a subject or a complementary polynucleotide (cDNA) reverse-transcribed therefrom with a polynucleotide of the composition, kit or nucleic acid array of the present invention;
(B) a step of measuring RNA derived from a biological sample bound to the polynucleotide or a complementary polynucleotide reversely transcribed from the RNA using the polynucleotide as a probe;
(C) based on the measurement result of (b) above, determining the sensitivity to adjuvant chemotherapy for gastric cancer,
Can be included.

  For example, various hybridization methods can be used to determine the susceptibility of gastric cancer to adjuvant chemotherapy according to the present invention. As such hybridization method, for example, Northern blot method, Southern blot method, RT-PCR method, DNA chip analysis method, in situ hybridization method, Southern hybridization method and the like can be used.

When using the Northern blot method, the presence or absence of each miRNA gene expression in RNA and its expression level can be detected and measured by using the diagnostic composition of the present invention as a probe. Specifically, determination composition of the present invention (complementary strand) labeled with a radioactive isotope (such as ^{32 P, 33 P, 35 S} ) or a fluorescent substance, transfers etc. to a nylon membrane it according to a conventional method After the hybridization with RNA derived from the living tissue of the subject, the double-strand formed in the composition for determination (DNA) and RNA is labeled with the composition for determination (radioisotope or fluorescent substance) A method of detecting and measuring a signal derived from the above by using a radiation detector (BAS-1800II, which can be exemplified by Fuji Photo Film Co., Ltd.) or a fluorescence detector (which can be exemplified by STORM860, GE Healthcare, etc.) is exemplified. be able to.

  When the quantitative RT-PCR method is used, the presence or absence of miRNA gene expression in RNA and its expression level can be detected and measured by using the above-described determination composition of the present invention as a primer. Specifically, cDNA can be prepared from RNA derived from the biological tissue of a subject according to a conventional method using a commercially available reagent for reverse transcription reaction, and each target miRNA or its precursor RNA can be amplified using this as a template. As described above, a pair of primers prepared from the determination composition of the present invention (consisting of a normal strand and a reverse strand that binds to the above cDNA) are hybridized with cDNA and subjected to PCR by a conventional method. A method for detecting the double-stranded DNA can be exemplified. As the above-mentioned reverse transcription reagent, for example, mirVana qRT-PCR miRNA Detection Kit (Ambion), TaqMan (registered trademark) MicroRNA RT Kit (Applied Biosystems), miScript Reverse Transscript (Q) is used. In addition, as a method for detecting double-stranded DNA, the above PCR is performed using a primer previously labeled with a radioisotope or a fluorescent substance, the PCR product is electrophoresed on an agarose gel, and then is detected with ethidium bromide or the like. A method of detecting by staining the double-stranded DNA, a method of detecting by detecting the produced double-stranded DNA by transferring it to a nylon membrane or the like according to a conventional method and hybridizing it with a probe as a probe be able to.

  When nucleic acid array analysis is used, a nucleic acid array in which the above-described determination composition of the present invention is attached to a substrate as a nucleic acid probe (single strand or double strand) is used. A nucleic acid immobilized on a substrate generally has a name of a nucleic acid array, a DNA chip, or a DNA array, and the DNA array includes a DNA macroarray and a DNA microarray. , Including the DNA array.

  Although hybridization conditions are not limited, For example, it is set as 30 to 60 degreeC and the conditions for 1 to 24 hours in the solution containing SSC and surfactant. Here, 1 × SSC is an aqueous solution (pH 7.2) containing 150 mM sodium chloride and 15 mM sodium citrate, and the surfactant contains SDS, Triton, Tween, or the like. More preferably, the hybridization conditions include 3 to 4 × SSC and 0.1 to 0.5% SDS. Washing conditions after hybridization include, for example, a solution containing 0.5 × SSC and 0.1% SDS at 30 ° C., a solution containing 0.2 × SSC and 0.1% SDS at 30 ° C., and 30 Conditions such as continuous washing with 0.05 × SSC solution at 0 ° C. can be mentioned. It is desirable that the complementary strand maintain a hybridized state with the target positive strand even when washed under such conditions. Specifically, as such a complementary strand, a strand composed of a base sequence that is completely complementary to the base sequence of the target positive strand, and a strand composed of a base sequence having at least 80% homology with the strand Can be illustrated.

Examples of stringent hybridization conditions when PCR is performed using the polynucleotide fragment of the composition or kit of the present invention as a primer include, for example, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1-2 mM MgCl _2. And the like, using a PCR buffer having the composition as described above, and treating for about 15 seconds to 1 minute at Tm + 5 to 10 ° C. calculated from the primer sequence. As a method for calculating Tm, Tm = 2 × (number of adenine residues + number of thymine residues) + 4 × (number of guanine residues + number of cytosine residues) and the like can be mentioned.

  For other examples of “stringent conditions” in these hybridizations, see, for example, Sambrook, J. et al. & Russel, D.C. Written by Molecular Cloning, A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, published on January 15, 2001, Volumes 7.42-7.45, Volumes 8.9-8.17, etc. And can be used in the present invention.

  The present invention also measures the expression level of a target miRNA gene in a specimen sample derived from a subject using the composition, kit, nucleic acid array, or a combination thereof of the present invention, and is a patient who is sensitive to adjuvant chemotherapy for gastric cancer. The specimen sample contains miRNA related to the adjuvant chemotherapy sensitivity of gastric cancer, using as a discriminant a neural network model with the expression level of the miRNA gene of the biological sample from the patient and the biological sample from an insensitive patient as a teacher sample, and / or Provided is a method for determining that it is not included.

  That is, the present invention further uses the composition, kit, nucleic acid array, or combination thereof of the present invention to determine that a sample sample is sensitive to adjuvant chemotherapy for gastric cancer and / or not sensitive to adjuvant chemotherapy for gastric cancer. A first step of measuring the expression level of a target nucleic acid in a plurality of biological samples known to be performed in vitro, and a measured value of the expression level of the target miRNA gene obtained in the first step as a teacher sample A second step of creating a discriminant using the neural network model performed, a third step of measuring the expression level of the target miRNA gene in a specimen sample derived from a subject in vitro, as in the first step, the second step Substituting the measured value of the expression level of the target miRNA gene obtained in the third step into the discriminant obtained in the step, and based on the result obtained from the discriminant A fourth step of determining whether the specimen sample is sensitive to adjuvant chemotherapy for gastric cancer, wherein the target miRNA is detectable by the polynucleotides contained in the composition, kit or nucleic acid array Provide the above method.

Alternatively, the method of the present invention includes, for example, the following steps (a), (b) and (c):
(A) The composition for determination, kit or the expression level of the target miRNA gene in a tissue that is known to be a tissue that is sensitive to adjuvant chemotherapy for gastric cancer and / or a tissue that is not sensitive to adjuvant chemotherapy for gastric cancer Measuring with a nucleic acid array;
(B) a step of substituting the measured value of the expression level measured in (a) into the following equations 1 to 4 to create a discriminant called a neural network model;
(C) The expression level of the target miRNA gene in the specimen sample derived from the subject is measured using the determination composition, kit or nucleic acid array according to the present invention, and these are substituted into the discriminant created in (b). Determining, based on the obtained results, that the sample is sensitive to adjuvant chemotherapy for gastric cancer / or not sensitive to adjuvant chemotherapy for gastric cancer (presence / absence),
Can be included.

  The neural network model was proposed by McCulloch, W and Pitts, W (McCulloch, W AND Pitts, W, 1943, Bulletin of Mathematical Biophysics, No. 7, p. 115-133). It is a mathematical model of the connected neural circuit, and is a powerful learning machine for nonlinear regression analysis and nonlinear discriminant analysis (pattern recognition).

An example of calculating a discriminant that can be used in the method of the present invention is shown below.
In order to determine the neural network model, the expression level of the target miRNA gene in a biological sample that is known to contain a gene derived from gastric cancer adjuvant chemosensitivity-derived miRNA or / or that does not contain gastric cancer adjuvant chemosensitivity-derived miRNA Prepared as a teacher sample, the constant of the discriminant function can be determined by the following procedure.

  The subject belongs to either a group of patients who are sensitive to adjuvant chemotherapy for gastric cancer or a group of patients who are not sensitive, and belongs to a group of patients who are sensitive to adjuvant chemotherapy for gastric cancer. Is classified into -1. When these samples are used as teacher samples, when the teacher samples can be classified by the neural network model shown in FIG. 1, the discrimination function for determining whether the specimen sample is sensitive to adjuvant chemotherapy for gastric cancer and / or not sensitive is, for example, The following formula.

  When the sample amount is defined as the covariate Xn and the expression level of the miRNA gene that determines whether or not the miRNA derived from gastric cancer adjuvant chemosensitivity miRNA is included, and there are p covariates Xn, the sample sample X Gene expression and the functions that make up each neuron

と標記でき、結合する各ニューロンの識別関数は出力信号である共変量Ｘｎと結合荷重Ｗｎの積の和で標記できる。このときに各識別関数は閾値（θ）をもち、閾値を基準にして各ニューロンを構成する関数は次式で標記できる一義的な識別関数（ｙ）を得ることができる。

The discriminant function of each neuron to be coupled can be represented by the sum of products of the covariate Xn that is the output signal and the coupling weight Wn. At this time, each discriminant function has a threshold value (θ), and a function that constitutes each neuron based on the threshold value can obtain a unique discriminant function (y) that can be expressed by the following equation.

  Classify f (Sj) by substituting the covariate X for a specimen sample that is unknown to contain or not include miRNA from gastric cancer adjuvant chemosensitivity newly given to this function (ie, +1 or −1) to identify that the sample sample contains and / or does not contain miRNA derived from gastric cancer adjuvant chemotherapy sensitivity.

As described above, two groups of teacher samples are required to create a discriminant using a neural network model for classifying unknown samples. For this teacher sample example of this invention, "expression miRNA genes obtained from patient tissue is an auxiliary chemosensitivity of gastric cancer (gene _{X 1, X 2, .. X} i, ... X n) " A set corresponding to each of the above patients, and each patient of “expressed miRNA genes (genes X ₁ , X ₂ ,... X _i ,... X _n ) obtained from tissues of patients who are not susceptible to gastric cancer adjuvant chemotherapy” 2 groups of sets corresponding to. The number of expressed miRNA genes (n) measured for each of these sets varies depending on the design of the experiment, but for each gene there is a significant difference between the two groups in any experiment, A case where the difference is small or no difference is observed. In order to improve the accuracy of the discriminant expression using the neural network model, it is necessary that there is a clear difference between the two groups as the training sample. Therefore, there is a difference in the expression level between the two groups in the gene set. It is necessary to extract and use only.

  Moreover, it is preferable to extract the nucleic acid belonging to the miRNA set used in the discriminant by the neural network model as follows. First, for multiple biological samples whose specimen samples are known to be sensitive to adjuvant chemotherapy for gastric cancer and / or not sensitive to adjuvant chemotherapy for gastric cancer, the median expression level within the group, the average value within the group, the average The difference in the expression level between the two groups of each miRNA gene is obtained using a t-test that is a parametric analysis for detecting a difference in values, a Wilcoxon test that is a non-parametric analysis, and the like. Next, miRNAs are incorporated into the set one by one in the order in which the difference in the expression level between the two groups determined here is large, and each time, Accuracy, Efficiency, PPV, and NPV are calculated. At this time, “Accuracy” is the sum of “the number of times that gastric cancer did not recur correctly predicted that it was chemosensitive” and “the number of times that gastric cancer recurred and correctly died that it was not chemosensitive” The divisor of the total number of cases for. “Efficacy” is “(number of times correctly predicted non-relapsed cases to be chemosensitive) / (number of times predicted to be chemosensitive)” and “(number of times correctly predicted relapsed cases not chemosensitive)” / (Number of times predicted not to be chemosensitive) "," PPV "is" (number of times correctly predicted non-relapsed cases to be chemosensitive) / (number of times predicted to be chemosensitive) ", “NPV” can be calculated by “(number of times that recurrence cases were correctly predicted not to be chemosensitive) / (number of times that recurrence cases were predicted not to be chemosensitivity)”. The number of miRNAs incorporated into the smaller of the number of cases until the sum of the number of cases and the number of insensitive cases is met, or the upper limit of the number of measured miRNAs is reached. Repeat until. As a condition for increasing both the PPV with the probability of determining that a non-relapsed case is chemosensitive and the NPV with the probability of determining that a relapsed case is not chemosensitive, a set of genes that maximizes efficacy is adopted.

  As a method for determining the gene set that maximizes the efficiency, when one sample is removed from all the samples and the gene set is created with the remaining samples, the determination is made on the sample that has been removed in advance. By implementing the Leave one cross out validation method that employs the miRNA set that maximizes the average of the efficiency for the sample, the accuracy of miRNA set selection by repeated analysis can be increased.

  Furthermore, the miRNA selected here is a miRNA that is the number of times that is repeatedly selected when arranged in order of increasing p value in the Wilcoxon test, and if it is the same number, the miRNA that has a small p value is selected.

  The present invention is more specifically described by the following examples. However, the present invention is not limited by this example.

1. Clinicopathological findings of experimenters Twenty gastric cancer FFPE specimens with informed consent were used. The breakdown was 11 recurrence-free cases, that is, 11 cases that were sensitive to adjuvant chemotherapy, and 9 recurrent cases, that is, cases that were not sensitive to adjuvant chemotherapy. Adjuvant chemotherapy was performed with any of 5-FU prodrugs (TS-1, UFT, Flutulon).

2. Extraction of total RNA Sample 1. From the gastric cancer FFPE specimen obtained in 1 above, a 10 μm-thick section was prepared and placed in a tube. Xylene was added to dissolve the paraffin, centrifuged to remove the xylene, and the tissue was collected. Ethanol was added to the tissue to dissolve the remaining xylene, and the ethanol was removed by centrifugation. This operation was repeated twice. After the tissue was air-dried, 100 μL of Proteinase K solution (2 μg / μL) was added and incubated at 37 ° C. overnight. Purified total RNA was obtained by removing impurities such as proteins using a silica column and removing DNA by DNase I treatment.

3. Measurement of gene expression level The oligo DNA nucleic acid array used was Toray Industries, Inc. 3D-Gene (registered trademark) Human miRNA Oligo chip. The 3D-Gene (registered trademark) Human miRNA Oligo chip of Toray Industries, Inc. was operated based on the procedure specified by the company. The hybridized DNA chip was scanned using a DNA array scanner (ScanArrayLite, PerkinElmer Japan), an image was acquired, and the fluorescence intensity was digitized using GenePix Pro5.0 (Molecular Device). Statistical processing is described in Speed T. et al. “Statistical analysis of gene expression microarray data” by Chapman & Hall / CRC, and Causton H. C. "A beginner's guide Microarray gene expression data analysis" Blackwell publishing was used as a reference and analysis was performed using logarithmic values of data obtained from image analysis after hybridization. As a result, miRNA (polynucleotide) whose expression level was decreased, decreased or increased, and increased in patients who were sensitive to adjuvant chemotherapy for gastric cancer and insensitive patients could be found. The list is shown in Table 1. It is believed that these genes can be used to determine whether a sample sample is / is not sensitive to gastric cancer adjuvant chemotherapy.

4). Predictive scoring system R (http: //cran.r), using a total of 20 cases of specimen samples of 11 cases of gastric cancer that were sensitive to adjuvant chemotherapy and 9 cases of non-sensitive cases as teacher samples A discriminant was created using -project.org/). miRNA (polynucleotide) was determined according to the flow shown in FIG. That is, first, 20 cases of teacher samples were divided into 19 cases of learning set and 1 case of test set, and miRNA gene expression levels measured using specimen samples obtained from groups that were sensitive to adjuvant chemotherapy for gastric cancer in the learning set. The p-value of the Wilcoxon test was calculated for each gene expression level between the miRNA gene expression levels measured using specimen samples obtained from patient groups that were not sensitive. Next, a neural network model using a data set incorporating genes one by one in ascending order of the Wilcoxon test obtained here is constructed, and each time the test sample sample is sensitive to adjuvant chemotherapy for gastric cancer. And / or not sensitive. The determination of whether or not this specimen sample is sensitive to adjuvant chemotherapy for gastric cancer is performed on 20 combinations of learning sets and test sets that can be separated from 20 cases of teacher samples. Predicted percentages of Accuracy, Efficiency, Sensitivity, Specificity, PPV, and NPV were calculated. The results are shown in Table 2.

  Incorporation of miRNA into this miRNA set was repeated in ascending order of the p-value of the Wilcoxon test, and the set of genes (polynucleotides) at the time when the maximum efficacy was obtained was determined. The result is shown in FIG. The vertical axis of FIG. 3 indicates the probability that adjuvant chemotherapy sensitivity of gastric cancer can be determined, and the horizontal axis indicates an increase in the order of the SEQ ID NOs shown in Table 1. ) Respectively. For example, when the number on the horizontal axis is 5, the result predicted by the combination of miRNAs (polynucleotides) of SEQ ID NOs: 1 to 5 in Table 1, and when the number is 20, the combination of miRNAs (polynucleotides) of SEQ ID NOs: 1 to 20 The results predicted by are shown. In the case of a set of 13 kinds of miRNAs (polynucleotides), the maximum efficiency was 90%. As described above, a set of miRNAs (polynucleotides) was found that can determine with high probability whether or not 5-FU prodrugs are sensitive to adjuvant chemotherapy.

  According to the present invention, since it is possible to provide a composition for determining sensitivity to adjuvant chemotherapy for gastric cancer having excellent specificity and sensitivity, sensitivity and specificity can be obtained using gastric tissue extracted during gastric cancer surgery or a stomach biopsy sample. Not only is it possible to determine the sensitivity of adjuvant chemotherapy, but also the correct selection of anti-cancer drugs that are appropriate for the patient, this will lead to early treatment and improvement in prognosis, greatly reducing the burden on the patient, and It also contributes to the reduction of

Claims (9)

A composition for determining sensitivity to adjuvant chemotherapy for gastric cancer, comprising two or more polynucleotides selected from the group consisting of the polynucleotides shown in the following (a) to (f):
(A) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NOs: 1 to 22, a precursor polynucleotide thereof, a mutant polynucleotide thereof, or a modified polynucleotide thereof (b) a nucleotide sequence represented by SEQ ID NOs: 1 to 22 (C) a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NOs: 1 to 22, a variant polynucleotide thereof, or a modified polynucleotide thereof (d) represented by SEQ ID NOs: 1 to 22 (E) a polynucleotide comprising a base sequence complementary to the base sequence to be hybridized under stringent conditions with any of the polynucleotides (a) to (d) (f) DNA encoding any of the polynucleotides of (e)   The composition according to claim 1, wherein the adjuvant chemotherapy for gastric cancer is administration of fluorouracil or a fluorouracil prodrug.   A kit for determining sensitivity to adjuvant chemotherapy for gastric cancer, comprising two or more of the polynucleotides shown in (a) to (f) according to claim 1 or 2.   A polynucleotide comprising the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 22, a polynucleotide comprising the complementary sequence thereof, a polynucleotide that hybridizes with these polynucleotides under stringent conditions, and the like. The kit according to claim 3, which is a modified polynucleotide or a DNA encoding these polynucleotides.   The kit of Claim 3 or 4 with which the said polynucleotide is packaged in the container separately or in arbitrary combinations.   A nucleic acid array for determining sensitivity to adjuvant chemotherapy for gastric cancer, comprising two or more of the polynucleotides shown in (a) to (f) according to claim 1 or 2.   Using the composition according to claim 1 or 2, the kit according to any one of claims 3 to 5, the nucleic acid array according to claim 6, or a combination thereof, a target nucleic acid in a biological sample derived from a subject is obtained. A method for determining in vitro whether or not a subject has sensitivity to adjuvant chemotherapy for gastric cancer by measuring the expression level.   The method according to claim 7, wherein a nucleic acid array is used.   Using the composition according to claim 1 or 2, the kit according to any of claims 3 to 5, the nucleic acid array according to claim 6, or a combination thereof, A first step of measuring in vitro the expression level of a target nucleic acid in a plurality of biological samples known to be present, and discrimination based on the measured value of the expression level of the target nucleic acid obtained in the first step as a teacher A second step of creating an expression (neural network model), a third step of measuring the expression level of the target nucleic acid in a specimen sample derived from a subject in vitro, as in the first step, the second step Substituting the measured value of the expression level of the target nucleic acid obtained in the third step into the discriminant obtained in step 3, and determining whether the subject is sensitive to adjuvant chemotherapy for gastric cancer based on the result obtained from the discriminant Including the fourth step of determining the stomach To determine the susceptibility of cancer to adjuvant chemotherapy.

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