WO2008047947A1 - Method for determining effect of cisplatin administration and kit for determining effect of cisplatin administration - Google Patents

Abstract

It is determined whether or not cisplatin administration has a therapeutic effect with high accuracy and specificity. A method for determining an effect of cisplatin administration comprises a step a in which the expression of at least one gene selected from the group consisting of PDE3B (phosphodiesterase 3B) gene, PDGFC (platelet derived growth factor C) gene, PKD2 (polycystic kidney disease 2) gene, NRG1 (neuregulin 1) gene and LUM (lumican) gene in a biological sample collected from a subject to be diagnosed is measured, and a step b in which an effect of cisplatin administration is determined based on the expression level of the gene obtained as the measurement result.

Description

 TECHNICAL FIELD Field of the Invention

 The present invention relates to a method for determining the administration effect of cisplatin used as an anticancer agent and a kit for determining the effect of cisplatin administration.

 Light

Background art

 Cisplatin (abbreviated as “CDDP”) is the central agent of anticancer drugs and chemotherapy in all types of cancer. The side effects caused by cisbratin administration appear in nearly 100% of patients, but the clinical effect is not always good. The effects of cisplatin administration vary from case to case and are known to be ineffective for some patients. For this reason, many patients suffer from very serious side effects, but cancer often progresses without a therapeutic effect, resulting in a poor prognosis.

 Therefore, if the therapeutic effect of cisplatin can be determined before administration, it is a very great gospel for the patient. In addition, identify genes that are specifically upregulated in patients with resistance to cisplatin administration (drug resistance genes) and genes that are upregulated in patients with sensitivity to cisplatin administration (drug sensitivity genes). By doing so, it is possible to develop a new effective anticancer drug treatment method that overcomes drug resistance.

 However, several genes related to cisbratin resistance have been announced to date

(Bordo et al., 1994 (non-patent literature 1); Gottesman et al., 1996 (non-patent literature 2); Loe et al., 1996 (non-patent literature 3); Jin et al., 1998 (non-patent literature 4); Perez et al., 1998 ( Non-patent document 5); Hinoshita et al., 2000 (Non-patent document 6)), but there is currently no gene that can determine the effect of cisplatin administration in actual clinical practice.

 Non-Patent Literature 1 Bordow SB, Haber M, Madafiglio J, Cheung B, Marshall GM,

Norris MD. Cancer Res. Oct 1; 54 (19): 5036-5040. 1994 Non-Patent Document 2 Gottesman MM, Pastan I, Ambudkar SV. Curr Opin Genet Dev.

0ct; 6 (5): 610-617. 1996

 Non-Patent Document 3 Loe D. W., Deeley R. G., Cole S. P. Eur. J. Cancer, 32A: 945-957, 1996.

 Non-Patent Document 4 Jin S, Scotto KW. Mol Cell Biol. Jul; 18 (7): 4377-4384. 1998 Non-Patent Document 5 Perez RP. Eur J Cancer. Sep; 34 (10): 1535-1542. 1998 Non Patent Document 6 Hinoshita E, Uchiumi T, Taguchi K, Kinukawa N, Tsuneyoshi

M, Maehara Y, Sugimachi K, Ku ano M. Clin Cancer Res. Jun; 6 (6): 2401-2407.

2000 Disclosure of the Invention

 In view of the above circumstances, the present inventors have aimed to provide a method for determining the effect of cisplatin administration and a kit for determining the effect of cisplatin administration, which can determine the presence or absence of the therapeutic effect of cisplatin administration.

 In order to achieve the above-mentioned object, the present inventor has identified a gene group exhibiting resistance to or sensitivity to cisplatin by using a cell line exhibiting extremely excellent resistance to cisplatin, and has completed the present invention. .

 That is, the present invention includes the following.

 (1) PDE3B (phosphodiesterase 3B) gene, PDGFC, piatelet derived growth factor C) gene, PKD2 (Polycystic kidney disease-2 gene) gene, NRG1 (neuregulin l) gene And a step of measuring the expression of at least one gene selected from the group consisting of the LUM (Lumican) gene and a step of determining the administration effect of cisbratin based on the expression level of the gene obtained as a result of the measurement and a method for determining the effect of administration of cisbratine comprising b.

 (2) In the step a, the amount of mRNA of the gene is measured, and the method for determining the effect of cisplatin administration according to (1).

(3) In the step a, the amount of protein that is a product of the gene is measured. The method for determining the effect of cisplatin administration according to (1). (4) In the above step a, the expression level of at least one gene other than the PDE3B gene, the gene, the PKD2 gene, the NRG1 gene and the LUM gene among the genes shown in Table 1 described below is further increased. (1) The method for determining the cisplatin administration effect according to (1).

 (5) Selected from the group consisting of PDE3B (phosphodiesterase 3B) gene, PDGFC (platelet derived growth factor C) gene, PKD2 (Polycystic kidney disease-2 gene) gene, NRG1 (neuregulin 1) gene and LUM (Lumican) gene A kit for determining the efficacy of cisbratin administration, comprising a means for measuring the expression of at least one gene.

 (6) The kit for determining cisplatin administration effect according to (5), wherein the means is a polynucleotide that specifically hybridizes to mRNA of the gene or cDNA derived from the mRNA.

 (7) The cisbulatin administration effect determination kit according to (6), wherein the polynucleotide is fixed to a support.

 (8) The kit for determining the effect of cisplatin administration according to (5), wherein the means is an antibody that specifically binds to a protein that is a product of the gene.

 (9) It further comprises means for measuring the expression of at least one gene other than the PDE3B gene, the gene, the PKD2 gene, the NRG1 gene, and the LUM gene among the genes shown in Table 1 described later. (5) The cisplatin administration effect determination kit according to (5).

 This specification includes the contents described in the specification and Z or drawings of Japanese Patent Application No. 2006-286285, which is the basis of the priority of the present application. Brief Description of Drawings

 FIG. 1 is a characteristic diagram showing the results of MTT assay in Sa-3 and Sa-3R strains as representative examples of resistant strains established in the Examples.

Fig. 2 shows the expression of ABC transporters (MDR1, MRP1, and MRP2) in Sa-3 and Sa-3R strains as a representative example of resistant strains established in the Examples by RT-PCR. It is a characteristic view which shows the result.

 FIG. 3 is a Venn diagram showing the number of genes that specifically enhance expression and decrease in expression in resistant strains identified as a result of microarray analysis using the resistant strain and its parent strain established in the Examples.

 Fig. 4 is a characteristic diagram showing the four networks obtained as a result of a Pausley analysis of 199 genes identified as genes related to cisplatin resistance.

 FIG. 5 is a characteristic diagram (A) showing the results of RT-PCR performed on the lumican gene as a representative example of the five genes identified in the Examples (A) and a photograph (B) showing the results of Western blotting.

 FIG. 6 is a photograph showing the results of immunohistological staining of a clinical sample for the lumican gene as a representative example of the five genes identified in the examples.

 FIG. 7 is a characteristic diagram showing the result of obtaining the IHC score distribution for the PDE3E gene.

 Fig. 8 is a characteristic diagram showing the results of IHC score distribution for the lumican gene.

 FIG. 9 is a characteristic diagram showing the result of obtaining the distribution of IHC scores for the PDGFC gene.

 FIG. 10 is a characteristic diagram showing the results of obtaining the IHC score distribution for the PKD2 gene.

 Fig. 11 is a characteristic diagram showing the results of obtaining the IHC score distribution for the NRG1 gene. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the method for determining the effect of cisplatin administration and / or the kit for determining the effect of cisbratin administration according to the present invention will be described in detail.

This method for determining the effect of cisplatin administration is obtained as a result of the step a in which the expression of at least one or more genes selected from the following five genes is measured in a biological sample collected from a subject to be diagnosed. Based on the gene expression level And b determining the effect of administering bratin.

 PDE3B (phosphodiesterase 3B) gene (SEQ ID NO: 1)

 PDGFC (platelet derived growth factor C) gene (distributed IJ number 3)

 PKD2 (Polycystic kidney disease-2 gene) gene

 NRG1 (neuregulin l) gene (SEQ ID NO: 7)

 LUM (Lumican) gene (SEQ ID NO: 9)

 The amino acid sequence of the protein encoded by the PDE3B gene is shown in SEQ ID NO: 2. The amino acid sequence of the protein encoded by the PDGFC gene is shown in SEQ ID NO: 4. The amino acid sequence of the protein encoded by the PKD2 gene is shown in SEQ ID NO: 6. The amino acid sequence of the protein encoded by the NRG1 gene is shown in SEQ ID NO: 8. The amino acid sequence of the protein encoded by the LUM gene is shown in SEQ ID NO: 10.

 These PDE3B gene, PDGFC gene, PKD2 gene, NRG1 gene and L gene were subjected to a microarray analysis and a retrospective test using clinical specimens using cisbratin-resistant and cisbratin-sensitive strains, which will be described in detail later. As a result, the identified gene. Further, in the method for determining the effect of cisplatin administration according to the present invention, in addition to the expression levels of these genes, the expression levels of at least one or more genes obtained from the gene group listed in Table 1 are measured, In addition to the expression levels of the five genes, the expression levels of these genes may be used for the determination.

table 1

表 1において 「No.」 の欄は各遺伝子に割り振った番号を示し、 「Comraon」 の欄 は一般的な遺伝子の名称を示している。 また、 「Genbank」 の欄は Genbankの提供 するデータベースにおける当該遺伝子の登録番号であり、 「Systematic」の欄は当 該遺伝子の系統名を示し、 「Map」 の欄は当該遺伝子の染色体上の位置を示してい る。 さらに、 「Description」 の欄は遺伝子に関する説明又は当該遺伝子の産物で あるタンパク質に関する説明である。

In Table 1, the “No.” column shows the number assigned to each gene, and the “Comraon” column shows the names of common genes. The column “Genbank” is the registration number of the gene in the database provided by Genbank, the column “Systematic” indicates the strain name of the gene, and the column “Map” is the position of the gene on the chromosome. Shows The In addition, the “Description” column is a description of the gene or a description of the protein that is the product of the gene.

 The genes listed in Table 1 are composed of genes identified as a result of microarray analysis using cisplatin-resistant and cisplatin-sensitive strains, which will be described in detail later. Here, the cisplatin-resistant strain is a cell line established from an oral squamous cell carcinoma-derived cell line or an oropharyngeal squamous cell carcinoma-derived cell line using cisplatin resistance as an index, and a cell line of a patient that does not respond to cisplatin administration It has a meaning as a model. In addition, the cis-bratin sensitive strain means the oral squamous cell carcinoma-derived cell line or the oropharyngeal squamous cell carcinoma-derived cell line itself, which is the parent strain of the cis-bratin resistant strain.

 That is, the genes listed in Table 1 are composed of genes that are specifically enhanced in expression and genes that are specifically attenuated in cisplatin resistant strains. In Table 1, the genes whose expression was specifically enhanced in the cisplatin resistant strains are Nos. 1 to 164, and the genes whose expression was specifically attenuated in the cisplatin resistant strains are Nos. 165 to 199.

 In addition, for the genes listed in Table 1, the results of the above microarray analysis were used to construct a statistically meaningful deficit by performing an ingenuity pathway analysis. Is identified.

 Specifically, among the gene groups listed in Table 1, 51 gene groups form four networks, and these four networks form a large network.

Table 2

ここで、 判定対象者としては、 特に限定されず、 各種癌に罹患した患者、 各種 癌を疑われた者及び健常者のいずれであっても良い。 また、 本発明に係るシスプ ラチン投与効果判定方法は、 判定対象者の対して直接何らかの処置を施すもので はなく、 診断対象者から採取した生体由来試料を用いて実施する。

Here, the determination target person is not particularly limited, and may be any of a patient suffering from various cancers, a person suspected of various cancers, and a healthy person. In addition, the cisplatin administration effect determination method according to the present invention is not performed directly on the determination target person, but is performed using a biological sample collected from the diagnosis target person.

In the cisplatin administration effect determination method according to the present invention, the biological sample is determined Although it will not specifically limit if gene expression analysis in a subject is possible, For example, the protein extract derived from a tissue, a cell, a bodily fluid, urine, and other biological samples can be mentioned. Here, body fluid means blood, lymph fluid, tissue fluid (tissue fluid, intercellular fluid, interstitial fluid), body cavity fluid, serous cavity fluid, pleural effusion, ascites, pericardial fluid, cerebrospinal fluid (spinal fluid), joint fluid ( Synovial fluid), eye aqueous humor (aqueous humor), digestive fluid, knee fluid, intestinal fluid, semen and amniotic fluid. In addition, the biological sample may be one or a plurality of protein extracts derived from tissues, cells, body fluids, urine, and other biological samples.

 The tissue includes a part of the tissue obtained at the time of surgery performed for the treatment of cancer-affected patients, a part of the tissue collected by biopsy etc. from a diagnosis subject suspected of cancer, a patient with cancer This means that it includes a part of the tissue collected by biopsy for the purpose of distinguishing between primary and metastatic.

 In addition, as a cell in the cisplatin administration effect determination method according to the present invention, a cell isolated from each of the above tissues can be used. As the body fluid, plasma or serum separated from the blood, urine, lymph fluid, cerebrospinal fluid or ascites can be used. In the method for determining the cisplatin administration effect according to the present invention, cells or protein extracts isolated from sputum or the like can be used as other organism-derived samples.

 Specifically, in order to measure the expression of the above-described gene in a biological sample collected from a judgment subject, for example, the amount of mRNA for the gene to be measured is measured or the amount of protein that is the product of the gene to be measured is measured. Just measure.

As a method for measuring the amount of mRNA for the gene to be measured, a known method for detecting the expression of a gene can be used. For example, the Northern plotting method can be used to detect the mRNA level of the gene to be measured. In addition, a polynucleotide having a DNA sequence that hybridizes under stringent conditions to the gene to be measured can be used as a probe. In order to detect the amount of mRNA of the gene to be measured using the probe, it can be appropriately performed using a known method. For example, when preparing a probe, a label such as a fluorescent label is appropriately added to the probe, and this is hyper-hydidized with mRNA (or cDNA synthesized from mRNA) isolated from a biological sample collected from the subject. To do. afterwards, By measuring the fluorescence intensity derived from the hyper-prised probe, the mRNA level of the gene to be measured can be detected. The probe can be used by being immobilized on a support such as a glass bead or a glass substrate. That is, it can also be used in the form of a microarray or DNA chip in which probes prepared for a gene to be measured (a plurality of genes) are immobilized on a support.

 Note that “hybridizes under stringent conditions” means, for example, 1 XSSC (0.15 MN a Cl, 0.015 M sodium taenoate), 0.1 at 42 ° C. This means that the hybridization is maintained even by washing at 42 ° C with a buffer containing% SDS (Sodium dodecyl sulfate). In addition, there are various factors other than the above temperature conditions as factors affecting the stringency of hybridization, and those skilled in the art can combine various factors together with the stringency of high predication illustrated above. It is possible to achieve an equivalent stringency.

 On the other hand, as a method for measuring the amount of protein that is the product of the gene to be measured, a known protein detection method can be used. Specifically, various methods using an antibody against the protein to be measured can be applied.

 As long as the protein to be measured is an antigen and binds to the antigen, the antibody is not particularly limited, and a mouse antibody, a rat antibody, a rabbit antibody, a Hedge antibody, etc. can be used as appropriate. The antibody may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferable in that a homogeneous antibody can be stably produced. Polyclonal antibodies and monoclonal antibodies can be prepared by methods well known to those skilled in the art.

A hybridoma producing a monoclonal antibody can be basically produced using a known technique as follows. That is, a desired antigen or a cell that expresses the desired antigen is used as a sensitizing antigen, and this is immunized according to a normal immunization method. It can be prepared by fusing and screening for monoclonal antibody-producing cells (hypridoma) by conventional screening methods. Hybridomas can be produced, for example, by the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46) etc.

 Here, when producing a monoclonal antibody, the product of the gene described above can be used as an antigen, and a cell expressing a fragment of the product of the gene described above can be used as an antigen. These proteins or fragments of the protein are described in, for example, Molecuar Cloning: A Laboratory Manual 2nd edition 1st-3rd Sambrook, J. et al., Cold Spring Harber Laboratory Press publication New York 1989. According to the method, those skilled in the art can easily obtain it. Cells expressing these proteins or fragments of the proteins were also described in Molecuar Cloning: A Laboratory Manual, 2nd edition, 1st 1-3 brook Sambrook, j., Cold Spring Harber Laboratory Press, published in New York, 1989. According to the method, it can be easily obtained by those skilled in the art.

 The resulting monoclonal antibodies can be used for the quantification of proteins to be measured, such as the enzyme enzyme assay (ELISA), enzyme immunodot assay, radioimmunoassay, aggregation based assay, or other well-known methods. It can be used as a test reagent by the existing immunoassay method. In addition, the monoclonal antibody is preferably labeled. For labeling, examples of the labeling compound include enzymes, fluorescent substances, chemiluminescent substances, radioactive substances, and staining substances known in the art.

 By the way, in the method for determining cisplatin administration effect according to the present invention, the above-described PDE3B (phosphodiesterase 3B) gene, PDGFC (platelet derived growth factor C) fe, PKD2 (Polycystic kidney disease-2 gene) gene, NRG1 (neuregulin 1) gene and LUM (Lumican) gene are selected. However, the method for determining the effect of cisbratin administration according to the present invention is not limited to these five genes, and the genes listed in Table 1 Other genes selected from the group may be added as measurement targets. In particular, as a measurement target gene, in addition to the five genes described above, it is more preferable to select another gene selected from the gene group listed in Table 2.

As described above, after measuring the expression level of the gene to be measured selected from the above-described gene group in the biological sample collected from the diagnosis subject, In the method for determining the effect of splatin administration, the presence or absence of the effect of cisbra and tin administration is determined based on the expression level.

 Specifically, after measuring the expression level of the gene to be measured by any of the methods described above, the expression level of the gene is evaluated. The expression level may be evaluated by setting a reference value for each gene to be measured and comparing it with this reference value. As the reference value, a relative value with respect to the expression level of the constitutively expressed gene can be set. In addition, the expression level of the gene to be measured may be evaluated using an immunohistochemical staining (IHC) score as described above, and a value that can evaluate false positive and false negative may be evaluated as a reference value. According to the determination method and determination kit for cisplatin administration effect according to the present invention, it is possible to determine the presence or the degree of the effect of cisplatin administration for a determination target person with very high accuracy and / or excellent specificity. Can do. Here, the sensitivity means a positive rate in a patient group who succeeds in cisplatin administration. Specificity means the negative rate in the group of patients who are not successful with cisplatin.

 In particular, in the method for determining the effect of cisplatin administration according to the present invention, diagnosis is performed with higher sensitivity and / or higher specificity by increasing the types of genes to be measured (for example, 6 or more genes). be able to.

 According to the method for determining the effect of cisbulatin administration according to the present invention, the effect of cisplatin administration can be confirmed by gene expression analysis (mRNA level or protein level) using a biological sample collected from an anticancer agent / cancer patient before starting chemotherapy. Because it can be judged more objectively and specifically, it can prevent the administration of cisbratin, which is an excessive burden on patients who cannot expect the administration effect, and is useful for effective treatment policy for the patient. Knowledge can be provided.

 EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope which concerns on this invention is not limited to a following example.

<Establishment of resistant strains>

First, in this example, a cisbratin resistant strain was established. Specifically, for oral squamous cell carcinoma-derived cell lines (H-1 and Sa-3 strains) provided by the Department of Dentistry and Oral Surgery, Wakayama Medical University School of Medicine, cisplatin (hereinafter sometimes referred to as CDDP) By continuously contacting the CDDP stepwise from a low concentration of 0.1 fg / ml. Thus, a cell line having resistance to cisbratin was established. The cisplatin resistant strain established from the H-1 strain was named H-1R strain, and the cisplatin resistant strain established from the Sa-3 strain was named Sa-3R strain.

 In addition, CDP treatment was similarly applied to a cell line derived from squamous cell carcinoma of the oropharynx (KB strain) to establish a cisbratin resistant strain. The cisbratin resistant strain established from KB strain was named KBR strain.

 For these cisplatin resistant strains (H-1R strain, Sa-3 strain and KBR strain), the relative resistance to each parental strain sensitive to cisplatin was examined. Specifically, each resistant strain and each parent strain were treated with CDDP (the concentration of CDDP was 0.05 to: LO g / ml), and then MTT assay was performed. The 50% survival (death) concentration (IC50) was determined for each resistant strain and each parental strain. The relative resistance of H-1 and H-1R strains is about 10 times, the relative resistance of Sa-3 and Sa-3R strains is about 7.5 times, relative to KB and KBR strains Resistance was 8.6 times. These three cell lines did not change their resistance even when they were freeze-thawed or cultured for one month without CDDP.

 Figure 1 shows the results of MTT assembly for Sa-3 and Sa-3R strains as representative examples. The profile file similar to the profile shown in Fig. Γ was shown for other resistant strains and their parent strains.

 Although the size of the resistant strain is slightly larger than that of the parent strain, the basic shape, growth rate, and colony forming ability were not significantly changed in the resistant strain and the parent strain. The same properties were maintained.

 In order to prove that the established resistant strains are genetically resistant to C D D P, the expression state of ABC transporter gene in the drug resistance mechanism was analyzed by RT-PCR. ABC transporters (ATP-binding cassettes) are present in cell membranes, are involved in drug transport, and have been reported to be particularly associated with drug resistance. The expression of ABC transporters (MDR1, MRP1, and MRP2), which are said to be related to CDDP resistance, was examined. Specifically, the primers shown in Table 3 below were used in each RT-PCR of MDR1, MRP1, and MRP2. For comparison, GAPDH was detected by RT-PCR.

Table 3 primer

 Forward Reverse size

MDE-l 5'-AAGGTTAGTACGAAAGAGGCTGTG-3 '5'-GGGTAGAAACAATAGTGAAAACAA-3 * 2 5bp

MKP-1 5'-GGACGTGGACTTGCTTCTGA-3 'δ'-G GTGG AGATTG GTTG ATGG G-3' 222bp

MRP-2 5 '-GTAGTGGA GAATGATAATGTGAGG-3 "5'-G TAGTGG ATCAATGATAATCTGAGC-S * 204bp

GAPDH 5'-GATCTGTGCCGCCTGTGGTGA-3 "5'-GGATGAGGTTGGGCAGAGCG-3 '306bp

As a result of RT-PCR, in each resistant strain established in this example, strong expression was observed in three types of ABC transpoters. FIG. 2 shows RT-PCR results for Sa-3 and Sa-3R strains as representative examples. From this result, it became clear that each resistant strain established in this example is a cell line genetically resistant to CDP. In the previous reports related to CDDP resistance, the resistance of the resistant strain was about twice, and it was a cell line that possessed only low tolerance that could be regarded as an experimental error range. The resistant strains established in this example had higher drug resistance than the previous cell lines and were considered very useful for analysis.

Microarray analysis>

 Next, in this example, by microarray analysis using a resistant strain and its parent strain, genes that are specifically enhanced in the resistant strain and genes that are specifically attenuated in the resistant strain are identified. Identified. Specifically, for microarray analysis, Affymetrix U133 Plus 2.0 with 54675 probes immobilized was used as the microarray. For microarray analysis, GeneChip Operating Software 1.1 (Affymetrix) and GeneSpring 6.1 (Silicon Genetics) were used as analysis software.

For microarray analysis, first, the gene group in which the expression of the resistant strain Sa-3R was observed to be 1.5 times higher than that of its parent strain Sa-3, and the parent strain H- We identified a gene group in which expression of 1.5 times or more was observed compared to 1 strain and a gene group in which expression of resistant strain KBR strain was observed to be 1.5 times or more compared to its parental KB strain. A gene group common to these three groups was identified as a gene group (16 4 genes) whose expression was specifically enhanced in cis-bratin resistant strains (see Fig. 3). On the other hand, the gene group in which the expression of the resistant strain Sa-3R was observed to be 1.5 times lower than that of its parent Sa-3 strain, and the resistant strain H-1R was compared with its parent H-1. 1. The gene group in which expression of 5 times or less was observed and the gene group in which the expression of resistant strain KBR was observed 1.5 times or less compared to its parent KB strain were identified. A gene group common to these three groups was identified as a gene group (35 genes) whose expression was specifically attenuated in cisbratin-resistant strains (see Fig. 3).

 A total of 199 genes could be identified as genes related to cisbratin resistance in cancer cells.

<Pas: Analysis>

 Next, in this example, a pathologic analysis was performed on 199 genes identified as genes related to cisplatin resistance, and what kind of network was formed with 199 genes was examined. Specifically, in the pathway analysis, Ingenuity Pathway Analysis (Ingenuity systems Sento) was used as the soft air. As a result, it was found that 50 out of 1999 genes formed 4 networks, and these 4 networks formed one larger network. The genes that make up these four networks are summarized in Table 4.

Table 4

Network Genes Functons Score

ANXA1, C1QA, C1R, CD47, CNPS5,

 COPS8, CUL2, CYFIP2, DCN, EGFR, Cancer

 ERBB3, ERBB4, FMR1, FOS, FXR1, Cell-To-Cell Signaling

 GNB1, GNG10, GRB2, H2-KA, INSR, and Interaction

 26

 MAP1B, MMP2, MMP9, MMP13, NR3C1 Cellular Movement

 NGR1, PCDHGC3, PDE3B, ΡΡΠ),

 RAPGEF1, SNX2, SOCS1, SRC, VPS29,

 WASF2

 AES, ATF3, ATRX, CBX5, CDK 2A,

 CENPJI, DDR1, E2F4, E4F1, GTSE1,

 ILIA, IL7R, ITGB5, Painting PI, MMP2, Cancer

 MMP9, image P13, NFKB1, PDGFA, Tumor Morphology 22

 PDGFC, PLAGL1, PL 2, RBI, RBBP9, Cell Cycle

 RELA, RRM2B, SMC2L1, SRI, STAT5A,

 TBX3, TFPI2, TK1, TP53, USP7, WIG1A1

ABCC1, BAX, CAPN2, CASP8, CCNL2 _:

 CDC42, CNP,

 CTNNB1, DYRK1A, ERBB2, HMGA1,

 Cancer

 CNAB2, MBP, MMP2, MSN, MYC,

 Cell Eeath 11

 MYCN, MYLK, NFATC2, NPM1, OSIA:

 Hematological eisease

 PAK2, PIP5K1B, PLEC1, PRKCA,

PRKCZ, QKI, RAC1, RBMS1, RDX ₅

 SFRS2, TLE4, TP53, VBL2, V

 AKT2, BACH1, BARD1, BRCA1, CEBTPB:

 CYP1B1, ESR1, ETV6, FBLN1, FOXC2, Gene Expression

 HBA1, HBA2, HBB, HAD, HBE1, HBG1: Connective Tissue

 HBG2, HBQ1, HBZ, JUNB, L PEP, MB: Development 11

 MYOG, NCOR1, NR2F1, NR2F2, PGR, and Function

 P D2, PPARD, PPARG, PPARGC1A, Viral Function

 RFC1, RXARA, TNNI1, TP53

Also shows ^four network obtained as a result of Roh sway analysis in FIG. Table 4及Pi 4 ⁵ 0 genes constituting the network shown in concluded that the related genes strongly phenotype that put that CDDP resistant to resistant strains.

> Backward test>

Next, in this example, candidate genes capable of predicting the success rate of anticancer agents by clinical specimens were searched from these 50 genes as a retrospective test. First, by confirming mRNA expression by RT-PCR, 5 genes (PDE3B, PDGFC, PKD2, NRGl and Lumican) were selected from among the genes expressed in the resistant strains among the 50 genes. I put it out.

 Anti-human PDE3B goat polyclonal antibody (Santa Cruz) as an antibody against PDE3B, Anti-human PDGFC goat polyclonal antibody (Santa Cruz) as an antibody against PDGFC, Anti-human PKD2 Usagi polyclonal as an antibody against PD2 An antibody (manufactured by ABGENT), an anti-human PKD2 Usagi polyclonal antibody (manufactured by Santa Cruz) as an antibody against NRG1, and an anti-human Lumican goat polyclonal antibody (manufactured by Santa Cruz) as an antibody against Lumican were prepared. It was confirmed by Western blotting using the prepared antibody that the expression of these genes was enhanced in the resistant strain at the protein level.

 The results of RT-PCR and Western plotting performed on the lumican gene among the five genes described above are shown as representative examples in FIGS. 5 (A) and (B). As shown in Fig. 5 (A) and (B), it can be seen that the lumican gene is specifically expressed in the resistant strain compared to the parent strain. Similar results were obtained for other genes except the Lumican gene. ,

In addition, clinical specimens derived from cases of complete remission (CR case: Complete Response case), partial remission (PR case: Partial Response case) and unchanged case (NC case: No Change case) as a result of cisplatin administration were used. The state of expression was examined by immunohistological staining. Immunohistological staining method, a fixed paraffin-embedded tissue in formalin and sectioned at a thickness of 4 / m, after dehydrated and deparaffinized by reacting 0. 3% H ₂ 0 ₂ and 30 minutes Endogenous peroxidase was removed and the reaction with non-specific protein was inhibited with 1.5% blocking serum (Santa Cruz Biotechnology). Thereafter, 1: 100-fold diluted antibody Lin7C monoclonal antibody (Santa Cruz Biotechnology) was reacted for 30 minutes at room temperature and in a wet state. After washing three times with a phosphate buffer and reacting with Envision reagent (Dako), color was detected with 3,3-diaminobenzidine tetrahydrochloride (Dako) and detected. Finally, hematoxylin staining was performed for comparison.

The IHC score was used for the evaluation of staining in the immunohistological staining method. The IHC score is observed with an objective lens of 40 times, and the percentage of positive cells out of the total number of cells in one field and the intensity of staining (for example, 3 levels (weak, medium and strong) are evaluated. 1-3 Is the sum of the numbers multiplied by. This is done with 10 randomly selected visual fields, and the IHC score is calculated as the average value. The IHC score is a method of judging the degree of staining that is generally used at present as an excellent score indicating staining intensity and staining range.

 Figure 6 shows a representative example of the results of immunohistological staining of clinical samples for the luraican gene. Increased expression of lumi can protein was confirmed in clinical specimens resistant to cisplatin.

 In addition, IHC scores were obtained using 10 NC cases, 22 PR cases, and 16 CR cases for cisplatin administration, and the distribution of IHC scores was obtained for each of the five genes described above. Figure 7 shows the result of the IHC score distribution for the PDE3E gene, Figure 8 shows the result of the IHC score distribution for the lumican gene, and Figure 9 shows the result of the IHC score distribution for the PDGFC gene. The results of obtaining the IHC score distribution for the PKD2 gene are shown in FIG. 10, and the results of obtaining the IHC score distribution for the NRG1 gene are shown in FIG.

 As shown in FIGS. 7 to 11, it was revealed that the IHC score and the evaluation of response to cisplatin administration were correlated for all the above five genes.

 In addition, Table 5 summarizes the presence or absence of the expression and treatment effect assessment for the above five genes. In Table 5, “10” indicates that the expression at the protein level was positive for each gene, and “one” indicates that the expression at the protein level was negative for each gene.

Table 5 ―

As can be seen from Table 5, the success of cisbratine administration and the five genes mentioned above It became clear that there was a correlation with the expression of.

<Summary of results>

 As described above, first, from the results of microarray analysis>, 199 genes with high reliability as genes related to cisbratin resistance could be identified. From the results of <pathway analysis>, 51 genes with higher reliability were selected as genes related to cisplatin resistance. Furthermore, the results of <retrospective study> demonstrated that the effect of cisbratin administration can be determined from the expression level of 5 genes included in 51 genes. From these results, it can be evaluated that a useful method that can accurately determine the administration effect of cisbratin for various cancers for each patient has been developed. Industrial applicability

 According to the present invention, the presence or absence of a response due to administration of cisplatin can be determined with very good accuracy. Therefore, the method for determining the cisplatin administration effect and the kit for determining the cisplatin administration effect according to the present invention can determine whether or not a treatment including cisplatin administration is applied to the subject patient.

 All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

The scope of the claims 1. PDE3B (phosphodiesterase 3B), PDGFC ^ platelet derived growth factor C) gene, PKD2 (Polycystic kidney disease-2 gene) gene, NRG1 (neuregulin 1) gene Measuring the expression of at least one gene selected from the group consisting of the LUM (Lumican) gene, and a.  Step b for determining the effect of administration of cisbratin based on the expression level of the gene obtained as a result of the measurement,  A method for determining the effect of cisplatin administration comprising  2. The method according to claim 1, wherein in step a, the mRNA level of the gene is measured.  3. The method for determining the effect of administering cisplatin according to claim 1, wherein in step a, the amount of protein that is a product of the gene is measured.  4. In step a, further measure the expression level of at least one gene other than the PDE3B gene, the PDGFC gene, the PKD2 gene, the NRG1 gene, and the LUM gene in the gene group shown in Table 1. The method for determining the cisplatin administration effect according to claim 1. table 1 File Name

5.PDE3B (phosphodiesterase 3B) gene, PDGFC (platelet derived growth factor C) gene, PKD2 (Polycystic kidney disease-2 gene) gene, NRGl cisplatin administration efficacy, including means for measuring the expression of at least one gene selected from the group consisting of (neuregulin l) gene and LUM (Lumican) gene Fruit determination kit.  6. The kit according to claim 5, wherein the means is a polynucleotide that specifically hybridizes to mRNA of the gene or cDNA derived from the mRNA.  7. The kit for determining a cisplatin administration effect according to claim 6, wherein the polynucleotide is fixed to a support.  8. The kit according to claim 5, wherein the means is an antibody that specifically binds to a protein that is a product of the gene.  9. Further includes means for measuring the expression of at least one gene other than the PDE3B gene, the PDGFC gene, the PKD2 gene, the NRG1 gene and the LUM gene among the genes shown in Table 2. The cisplatin administration effect determination kit according to claim 5, Table 2