WO2012121324A1 - Method for selecting effective group of cancer treatments combining use of three agents of taxane-based anticancer agent, platinum complex-based anticancer agent, and pyrimidine fluoride-based anticancer agent - Google Patents

Abstract

The objective of the present invention is to provide: a method for selecting an effective group of cancer treatments combining the use of the three agents of a taxane-based anticancer agent, a platinum complex-based anticancer agent, and a pyrimidine fluoride-based anticancer agent; and a reagent for predicting the efficacy of a cancer treatment combining the use of the three agents of a taxane-based anticancer agent, a platinum complex-based anticancer agent, and a pyrimidine fluoride-based anticancer agent. The method selects an effective group of cancer treatments combining the use of the three agents of a taxane-based anticancer agent, a platinum complex-based anticancer agent, and a pyrimidine fluoride-based anticancer agent by using as an indicator the amount of expression product of at least one gene selected from the group consisting of PDGFB, PCGF3, CISH, ANXA5, ANTXR2, B4GALT5, PLK2, ATP7B, AM116A, HECA, JMJD2A, STYX, EGR1, AVPI1, and ERLIN1.

Description

A method for selecting an effective group for cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent.

The present invention relates to a method for selecting an effective group for cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent, and a taxane anticancer agent. The present invention relates to a reagent for predicting the effect of cancer treatment using a combination of cancer, platinum complex anticancer agent, and fluoropyrimidine anticancer agent.

Surgical therapy, drug therapy (chemotherapy), etc. are known as cancer treatment methods. Surgery is a treatment that removes cancerous tissue, and is effective when the degree of cancer progression is low or when no cancer metastasis is observed. However, it is difficult to completely remove the cancer tissue when the cancer is highly advanced, when cancer metastasis is observed, or when there is a possibility of cancer metastasis. Therefore, a treatment method combining surgery and drug therapy, or a treatment method using only drug therapy is required.

Pharmacological therapy is usually performed by administering an anticancer drug or the like. Anticancer agents generally act on DNA synthesis or some DNA function, and exert anticancer effects by suppressing the growth of cancer cells or killing cancer cells. Thus, since the anticancer agent acts on the function that cells in the living body have universally, it can also act on normal cells and cause side effects in the living body.

On the other hand, cancers are diverse due to differences in genetic predisposition of patients, and it is known that there are patient groups in which administration of specific anticancer drugs is effective and patient groups ineffective. Therefore, administering or continuing to administer an anticancer drug to a patient for whom the administration of the anticancer drug is not effective leads to a decrease in the patient's quality of life due to the above-mentioned side effects. This leads to an increase in the cost of treatment, which is not preferable. In view of such a situation, development of methods for selecting patient groups for which treatment with various anticancer agents is effective and development of effect prediction markers are being promoted.

For example, Patent Document 1 discloses that the expression level of PDGFB gene tends to be relatively high in prostate cancer cells having resistance to docetaxel, which is a taxane anticancer agent, and treatment with docetaxel. It describes that the expression level of the PDGFB gene can be used as an index in the method for selecting an effective group.

Non-patent document 1 discloses that the expression level of the PDGFB gene tends to be relatively high in liver cancer cells having resistance to cisplatin, which is a platinum complex anticancer agent, and cisplatin. It describes that the expression level of the PDGFB gene can be used as an index in the method for selecting an effective treatment group.

Furthermore, Non-Patent Document 2 states that the expression level of ATP7B gene tends to be relatively low in colorectal cancer cells resistant to fluorouracil, which is a fluoropyrimidine anticancer agent, and It is described that the expression level of the ATP7B gene can be used as an index in the method for selecting an effective group for treatment with fluorouracil.

WO2010 / 037859

Clinical Cancer Research. 2009; 15 (10: 3462-3471) Cancer Research. 2008; 68 (13: 4977-4982), “Single-Cell Transcription Site Activation Predicts Chemotherapy Response in Human Colorectal Tumors”

The present invention relates to a method for selecting an effective group for cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent, and a taxane anticancer agent. An object of the present invention is to provide a reagent for predicting the effect of cancer treatment using a combination of cancer, platinum complex anticancer agent, and fluoropyrimidine anticancer agent.

When each of the above three types of anticancer agents is used alone, the effective group can be selected using the expression level of the specific gene as an index, as described in Patent Document 1 and Non-Patent Documents 1 and 2 above. It is known. Specifically, the expression level of PDGFB gene is relatively low in the effective group of taxane anticancer agents, and the expression level of PDGFB gene is relatively low in the effective group of platinum complex anticancer agents. It is known that the expression level of ATP7B gene is relatively high in the effective group of fluoropyrimidine anticancer agents. However, even when the above three kinds of anticancer agents are used in combination, the expression level of the same gene can be used as an index, and further, there is a difference between the expression level in the effective group and the expression level in the ineffective group. It is not known at all whether or not it shows a similar tendency.

As a result of diligent research, the present inventors, in an effective group of cancer treatments using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent, Compared to the ineffective group, the expression level of PDGFB gene and JMJD2A gene tends to be high, and PCGF3 gene, CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, It was found that the expression levels of STYX gene, EGR1 gene, AVPI1 gene, and ERLIN1 gene tend to be low.

Surprisingly, the tendency that the expression level of PDGFB gene tends to be higher in the effective group than in the non-effective group is that when the taxane anticancer agent and the platinum complex anticancer agent are used alone ( The tendency was the opposite of Patent Document 1 and Non-Patent Document 1). In addition, the fact that the expression level of the ATP7B gene tends to be lower in the effective group than in the non-effective group was the reverse tendency when the fluorinated pyrimidine anticancer agent was used alone (Non-patent Document 2). .

Based on the above findings, the present inventors have conducted further earnest research, and as a result, PDGFB gene, PCGF3 gene, CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, JMJD2A A taxane anticancer agent, a platinum complex anticancer agent, using as an index the amount of the expression product of at least one gene selected from the group consisting of the gene, STYX gene, EGR1 gene, AVPI1 gene, and ERLIN1 gene, And it discovered that the effective group of the cancer treatment which used together 3 types of fluorinated pyrimidine type | system | group anticancer agent can be selected, and came to complete this invention.

That is, the present invention has the following configuration.

Item 1. Cancer treatment using the following steps (a), (b), and (c) in combination with a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent. To select effective groups of:
(A) PDGFB gene, PCGF3 gene, CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, JMJD2A gene in biological samples containing cancer tissues of cancer patients, Measuring the amount of an expression product of at least one gene selected from the group consisting of STYX gene, EGR1 gene, AVPI1 gene, and ERLIN1 gene;
(B) the amount of the expression product obtained in the step (a) (hereinafter collectively referred to as “gene expression level”), taxane anticancer agent, platinum complex anticancer agent, and fluorinated pyrimidine Comparing the amount of the corresponding gene expression product in the ineffective group of cancer treatment combined with three anticancer agents (hereinafter collectively referred to as “control gene expression level”), and (c ) Step of determining the cancer patient as an effective group when the following criteria (A) and (B) are satisfied:
(A) When the measured gene is PDGFB gene or JMJD2A gene, the gene expression level is higher than the control gene expression level, and (B) the measured gene is PCGF3 gene, CISH gene, ANXA5 If the gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, STYX gene, EGR1 gene, AVPI1 gene, or ERLIN1 gene, the gene expression level is lower than the control gene expression level Based on

Item 1-2. A gene for measuring the amount of expression product in step (a) is:
(I) PDGFB gene, or (ii) PDGFB gene, and PCGF3 gene, CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, JMJD2A gene, STYX gene, EGR1 gene, Item 2. The method according to Item 1, wherein the method is at least one gene selected from the group consisting of an AVPI1 gene and an ERLIN1 gene.

Item 2. Genes for measuring the amount of expression product in step (a) are PDGFB gene and PCGF3 gene, and CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, JMJD2A gene, STYX Item 3. The method according to Item 1 or Item 1-2, wherein the method is at least one selected from the group consisting of a gene, an EGR1 gene, an AVPI1 gene, and an ERLIN1 gene.

Item 3. Item 1 or 2 characterized in that the gene for measuring the amount of expression product in the step (a) is PDGFB gene, PCGF3 gene, CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, and PLK2 gene. the method of.

Item 4. Items 1 to 3, wherein the taxane anticancer agent is docetaxel, the platinum complex anticancer agent is cisplatin, or the fluoropyrimidine anticancer agent is tegafur. 4. The method according to any one of 3.

Item 5. Items 1 to 3, wherein the taxane anticancer agent is docetaxel, the platinum complex anticancer agent is cisplatin, and the fluoropyrimidine anticancer agent is tegafur. 4. The method according to any one of 3.

Item 6. The method according to any one of Items 1 to 5, wherein the cancer is stomach cancer.

Item 7. A probe or primer comprising a polynucleotide having a base sequence of 15 bases or longer that specifically hybridizes with a continuous base sequence of at least 15 bases in the base sequence shown in any of SEQ ID NOs: 1 to 15 , A reagent for predicting the effects of cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent.

Item 7-2. Probe or primer is
(I) a probe or primer comprising a polynucleotide having a base sequence of 15 bases or longer, which specifically hybridizes with a continuous base sequence of at least 15 bases in the base sequence shown in SEQ ID NO: 1, or (II ) Probes or primers comprising a polynucleotide having a base sequence of 15 bases or longer and specifically hybridizing with a continuous base sequence of at least 15 bases in the base sequence shown in SEQ ID NO: 1 and SEQ ID NOs: 2 to 15 A probe or primer consisting of a polynucleotide having a base sequence of 15 bases or more, which specifically hybridizes with a continuous base sequence of at least 15 bases in the base sequence shown in any one of
Item 8. The reagent according to Item 7.

Item 8. 3 of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent comprising an antibody that recognizes a polypeptide comprising the amino acid sequence shown in any of SEQ ID NOs: 16 to 30 A reagent for predicting the effects of cancer treatment using a combination of species.

Item 8-2. Antibody
(III) an antibody that recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16, or (IV) an antibody that recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16, and any one of SEQ ID NOs: 17 to 30 An antibody that recognizes a polypeptide consisting of the amino acid sequence shown in
Item 9. The reagent according to Item 8.

Item 9. Item 7 or wherein the taxane anticancer agent is docetaxel, the platinum complex anticancer agent is cisplatin, or the fluoropyrimidine anticancer agent is tegafur. 9. The reagent according to any one of 8.

Item 10. Item 7 or, wherein the taxane anticancer agent is docetaxel, the platinum complex anticancer agent is cisplatin, and the fluoropyrimidine anticancer agent is tegafur. 9. The reagent according to any one of 8.

Item 11. Item 11. The reagent according to any one of Items 7 to 10, wherein the cancer is gastric cancer.

Item 12. A probe or primer comprising a polynucleotide having a base sequence of 15 bases or more, which specifically hybridizes with a continuous base sequence of at least 15 bases in the base sequence shown in any of SEQ ID NOs: 1 to 15;
Use for predicting the effects of cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent.

Item 13. A probe or primer comprising a polynucleotide having a base sequence of 15 bases or more, which specifically hybridizes with a continuous base sequence of at least 15 bases in the base sequence shown in any of SEQ ID NOs: 1 to 15;
Use as a reagent for predicting the effects of cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent.

Item 14. A probe or primer comprising a polynucleotide having a base sequence of 15 bases or more, which specifically hybridizes with a continuous base sequence of at least 15 bases in the base sequence shown in any of SEQ ID NOs: 1 to 15;
Use for the manufacture of a reagent for predicting the effect of cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent.

Item 15. An antibody that recognizes a polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 16 to 30;
Used to predict the effects of cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent.

Item 16. An antibody that recognizes a polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 16 to 30;
Use as a reagent for predicting the effects of cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent.

Item 17. An antibody that recognizes a polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 16 to 30;
Use for the manufacture of a reagent for predicting the effect of cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent.

According to the present invention, an effective group for cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent is selected with high probability. Can do. Furthermore, it consists of PDGFB gene and PCGF3 gene, CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, JMJD2A gene, STYX gene, EGR1 gene, AVPI1 gene, and ERLIN1 gene By using the expression level of at least one gene selected from the group as an index, an effective group can be selected with higher probability. Furthermore, by using the expression levels of the PDGFB gene, the PCGF3 gene, the CISH gene, the ANXA5 gene, the ANTXR2 gene, the B4GALT5 gene, and the PLK2 gene as an index, the effective group can be selected with higher probability. For patients selected as an effective group, cancer treatment is actively performed using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent. Can lead to recovery of the patient's cancer.

For patients who were not selected as effective groups, that is, non-effective groups, a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent were used in combination. Therefore, it is possible to perform another treatment method that is more effective for the patient group without actively performing the cancer treatment. This is effective in terms of both reducing the side effects that may occur due to the administration of the anticancer agent and reducing the increase in the cost of treatment caused by unnecessary (ineffective) treatment.

: It is the experimental result which investigated the difference in the expression level in an effective group and an ineffective group about the PDGFB gene, the CISH gene, the ANTXR2 gene, the PLK2 gene, and the EGR1 gene by immunostaining.

: Shows the results of studying the usefulness of cancer treatment as an effective group selection marker.

(1) Screening method of the present invention The screening method of the present invention is effective for cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent. This is a method for selecting a group, and includes steps (a), (b), and (c) described later.

(1-1) Selection target according to the selection method of the present invention Selection targets according to the selection method of the present invention include taxane anticancer agents, platinum complex anticancer agents, and fluoropyrimidine anticancer agents. It is an effective group for cancer treatment using 3 types in combination.

As long as the cancer type is a cancer type capable of performing cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent. For example, but not limited to, stomach cancer, liver cancer, colorectal cancer, breast cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, lung cancer, head and neck Examples include cancer, breast cancer, biliary tract cancer, bile duct cancer, and prostate cancer. Among these, from the viewpoint of accuracy of the screening method of the present invention, stomach cancer, esophageal cancer, head and neck cancer, breast cancer, lung cancer, prostate cancer, and cervical cancer are preferable, and stomach cancer, esophageal cancer, head and neck cancer are preferable. Part cancer, breast cancer, and lung cancer are more preferable, and stomach cancer is particularly preferable. Further, as the degree of progression of cancer, cancer that is preferably classified as a degree of progression of stage III or higher, more preferably cancer that is classified as stage IV.

The treatment method is a cancer treatment method using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent.

Examples of taxane-based anticancer agents include docetaxel, paclitaxel (taxol), taxadiene, baccatin III, taxinin A, breviforiol, taxapine D, and the like. From the viewpoint of accuracy of the screening method of the present invention, docetaxel Or paclitaxel is preferred, and docetaxel is more preferred. The dose of the taxane anticancer agent is, for example, 10 to 200 mg / m ² , preferably 20 to 120 mg / m ² , more preferably 40 to 100 mg / m ² per day. The unit “mg / m ² ” indicates the dose per 1 m ^{2 of} body surface area.

Examples of the platinum complex anticancer agent include cisplatin, carboplatin, nedaplatin, and oxaliplatin, and cisplatin is preferable from the viewpoint of accuracy of the screening method of the present invention. The dose of the platinum complex anticancer agent is, for example, 10 to 200 mg / m ² , preferably 20 to 120 mg / m ² , more preferably 40 to 100 mg / m ² per day. The unit “mg / m ² ” indicates the dose per 1 m ^{2 of} body surface area.

Examples of the fluorinated pyrimidine anticancer agent include tegafur, fluorouracil, and flucytosine. From the viewpoint of accuracy of the screening method of the present invention, tegafur or fluorouracil is preferable, and tegafur is more preferable. When tegafur is used as the fluoropyrimidine anticancer agent, it is preferable to use gimeracil and oteracil potassium together, and the molar ratio of tegafur: gimeracil: oteracil potassium is, for example, 0.5-2: 0. A preparation formulated at 2 to 0.8: 0.5 to 2, particularly preferably a preparation formulated at 1: 0.4: 1. The dose of the fluorinated pyrimidine anticancer agent is, for example, 10 to 200 mg / m ² , preferably 20 to 120 mg / m ² , more preferably 40 to 100 mg / m ² per day. The unit “mg / m ² ” indicates the dose per 1 m ^{2 of} body surface area.

The treatment mode of the combined cancer treatment is particularly limited as long as it is a known method as a treatment mode of administering all of the fluoropyrimidine anticancer agent, the taxane anticancer agent, and the platinum complex anticancer agent. However, usually, a treatment schedule of about 1 to 4 weeks is set as one course, and this course is repeated a plurality of times. From the viewpoint of the accuracy of the screening method of the present invention, the treatment schedule is such that the fluoropyrimidine anticancer agent is used for a certain period, preferably 7 to 28 days, more preferably 10 to 21 days, still more preferably 12 to After daily administration for 16 days, an anticancer agent non-administration period is set for a certain period, preferably 4 to 10 days, more preferably 6 to 8 days. The schedule which administers a taxane type anticancer agent and a platinum complex type | system | group anticancer agent in any one of these is mentioned.

The target to be selected by the selection method of the present invention is a patient group in which a treatment method using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent is effective. . More specifically, when a combination treatment of the above three anticancer agents is actually performed, preferably when the above course of treatment is repeated a plurality of times, the cancer area is reduced, or cancer This is a group of patients with a downstage. The effective group is more preferably a patient group having a tumor reduction rate of 3% or more, more preferably 6% or more, particularly preferably 10% or more when the above course of treatment is actually performed once.

(1-2) Step (a)
The step (a) of the present invention comprises a PDGFB gene, a PCGF3 gene, a CISH gene, an ANXA5 gene, an ANTXR2 gene, a B4GALT5 gene, a PLK2 gene, an ATP7B gene, a FAM116A gene in a biological sample containing cancer tissue of a cancer patient, This is a step of measuring the amount of an expression product of at least one gene selected from the group consisting of HECA gene, JMJD2A gene, STYX gene, EGR1 gene, AVPI1 gene, and ERLIN1 gene.

The biological sample is not particularly limited as long as it contains cancer tissue of a cancer patient, and depending on the type of cancer, body fluid (blood, urine, etc.), tissue, extract thereof, and culture of collected tissue, etc. Is mentioned. Moreover, the collection method of a biological sample can be suitably selected by a method according to the type of biological sample and the cancer type.

The genes to be measured in the step (a) are PDGFB gene, PCGF3 gene, CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, JMJD2A gene, STYX gene, EGR1 gene, AVPI1 A gene and at least one gene selected from the group consisting of the ERLIN1 gene.

From the viewpoint of the accuracy of the selection method of the present invention, the gene to be measured in step (a) is preferably
(I) PDGFB gene, or (ii) PDGFB gene, and PCGF3 gene, CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, JMJD2A gene, STYX gene, EGR1 gene, Examples include at least one gene selected from the group consisting of the AVPI1 gene and the ERLIN1 gene.

The gene to be measured in the step (a) is more preferably from the viewpoint of accuracy of the selection method of the present invention,
(Iii) PDGFB gene and PCGF3 gene, or (iv) PDGFB gene and PCGF3 gene, and CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, JMJD2A gene, STYX gene, Examples thereof include at least one gene selected from the group consisting of EGR1 gene, AVPI1 gene, and ERLIN1 gene.

From the viewpoint of the accuracy of the selection method of the present invention, the gene to be measured in the step (a) is more preferably
(V) PDGFB gene, PCGF3 gene, and CISH gene, or (vi) PDGFB gene, PCGF3 gene, and CISH gene, and ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, JMJD2A Examples include at least one gene selected from the group consisting of a gene, a STYX gene, an EGR1 gene, an AVPI1 gene, and an ERLIN1 gene.

The gene to be measured in the step (a) is more preferably from the viewpoint of the accuracy of the selection method of the present invention,
(Vii) PDGFB gene, PCGF3 gene, CISH gene, and ANXA5 gene, or (viii) PDGFB gene, PCGF3 gene, CISH gene, and ANXA5 gene, and ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA Examples include at least one gene selected from the group consisting of a gene, JMJD2A gene, STYX gene, EGR1 gene, AVPI1 gene, and ERLIN1 gene.

The genes to be measured in the step (a) are still more preferably PDGFB gene, PCGF3 gene, CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, and PLK2 gene from the viewpoint of accuracy of the selection method of the present invention. The gene to be measured in the step (a) is particularly preferably a PDGFB gene, a PCGF3 gene, a CISH gene, an ANXA5 gene, an ANTXR2 gene, a B4GALT5 gene, a PLK2 gene, an ATP7B gene, from the viewpoint of the accuracy of the selection method of the present invention. FAM116A gene, HECA gene, JMJD2A gene, STYX gene, EGR1 gene, AVPI1 gene, and ERLIN1 gene.

The official gene names of these genes, gene symbols, SEQ ID NOs (SEQ ID NOs: 1 to 15) showing the base sequences of these genes, and SEQ ID NOs (SEQ ID NOs: 16 to 30) showing the amino acid sequences are shown in Table 1 below. Shown in

The expression product of the gene to be measured is mRNA transcribed from the gene sequence of the gene to be measured or a protein translated from the mRNA.

The measurement method of the gene expression product is not particularly limited as long as it can measure the amount of mRNA or protein, and a known measurement method can be used. Examples of such measurement methods include measuring the amount of mRNA, for example, PCR method, RT-PCR method, Northern blot method, in-situ hybridization method, microarray method, etc. Examples include Western blotting and immunostaining. A biological sample is prepared by performing an appropriate process according to these measurement methods. In the method for measuring a gene expression product, the reagent according to the present invention described later can be used as a reagent containing a primer, a probe, or an antibody.

(1-3) Step (b)
The step (b) of the present invention comprises the amount of the expression product obtained in the step (a) (hereinafter collectively referred to as “gene expression level”), a taxane anticancer agent, and a platinum complex anticancer. The amount of the corresponding gene expression product in the non-effective group of cancer treatments combined with three types of drugs and fluoropyrimidine anticancer agents (hereinafter collectively referred to as “control gene expression level”) It is a process to do.

The ineffective group of cancer treatments using a combination of taxane anticancer agent, platinum complex anticancer agent, and fluoropyrimidine anticancer agent is effective as a treatment method using the above three types in combination. This is a group of patients that are not. More specifically, it is a patient group in which reduction of the cancer site or cancer downstage was not observed as a result of repeating the above-described treatment course a plurality of times. The ineffective group is more preferably a patient group in which the tumor reduction rate is less than 3%, more preferably less than 6%, and particularly preferably less than 10% as a result of performing the above-mentioned treatment course once.

The amount of the corresponding gene expression product in the ineffective group is the amount of the expression product in the ineffective group of the same gene as that measured in the step (a). That is, when the amount of the gene expression product measured in step (a) is the amount of the PDGFB gene expression product, the amount of the corresponding gene expression product is a cancer that is known to be an ineffective group. It is the amount of the expression product of the PDGFB gene in a biological sample containing cancer tissue of a patient.

The comparison method is not particularly limited as long as the amount of the gene expression product can be compared, and can be appropriately selected according to the measurement method of the gene expression product.

(1-3) Step (c)
Step (c) of the present invention is a step of determining the cancer patient as an effective group when the following criteria (A) and (B) are satisfied.

Reference (A) means that when the measured gene is a PDGFB gene or JMJD2A gene, the gene expression level is higher than the control gene expression level.

Reference (B) means that the measured gene is PCGF3 gene, CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, STYX gene, EGR1 gene, AVPI1 gene, or In the case of the ERLIN1 gene, the gene expression level is lower than the control gene expression level.

For example, when the genes whose expression products are measured in step (a) are the PDGFB gene and the PCGF3 gene, in step (c), the PDGFB gene expression level is higher than the PDGFB gene control gene expression level. And when the gene expression level of the PCGF3 gene is lower than the gene expression level of the PCGF3 gene control, the test cancer patient is determined as an effective group.

The determination method of the level of expression is appropriately selected according to the measurement method and / or the comparison method.

(2) Reagent of the present invention The reagent of the present invention predicts the effect of cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent. As described later, it is divided into a reagent containing a primer or a probe or a reagent containing an antibody.

(2-1) Use of Reagent The use of the reagent of the present invention is for cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent. This is to determine the effect. That is, the reagent of the present invention is an effective group for cancer treatment in which the test patient uses a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent. It is used to determine whether there is a non-effective group. The effect can be determined by using the reagent of the present invention in the above-described screening method of the present invention.

As long as the cancer type is a cancer type capable of performing cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent. For example, but not limited to, stomach cancer, liver cancer, colorectal cancer, breast cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, lung cancer, head and neck Examples include cancer, breast cancer, biliary tract cancer, bile duct cancer, and prostate cancer. Among these, stomach cancer, esophageal cancer, head and neck cancer, breast cancer, lung cancer, prostate cancer, and cervical cancer are preferable, stomach cancer, esophageal cancer, head and neck cancer, breast cancer, and lung cancer are more preferable, Gastric cancer is particularly preferred. In addition, the cancer progression is preferably a cancer classified as a progression of stage II or higher, more preferably a cancer classified as a progression of stage III or higher, more preferably a classification of stage IV Cancer is mentioned.

The treatment method is a cancer treatment method using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent.

Examples of taxane-based anticancer agents include docetaxel, paclitaxel (taxol), taxadiene, baccatin III, taxinin A, breviforiol, and taxapine D. Docetaxel or paclitaxel is preferable, and docetaxel is more preferable. The dose of the taxane anticancer agent is, for example, 10 to 200 mg / m ² , preferably 20 to 120 mg / m ² , more preferably 40 to 100 mg / m ² per day. The unit “mg / m ² ” indicates the dose per 1 m ^{2 of} body surface area.

Examples of the platinum complex anticancer agent include cisplatin, carboplatin, nedaplatin, and oxaliplatin, with cisplatin being preferred. The dose of the platinum complex anticancer agent is, for example, 10 to 200 mg / m ² , preferably 20 to 120 mg / m ² , more preferably 40 to 100 mg / m ² per day. The unit “mg / m ² ” indicates the dose per 1 m ^{2 of} body surface area.

Examples of the fluoropyrimidine anticancer agent include tegafur, fluorouracil, and flucytosine. Tegafur or fluorouracil is preferable, and tegafur is more preferable. When tegafur is used as the fluoropyrimidine anticancer agent, it is preferable to use gimeracil and oteracil potassium together, and the molar ratio of tegafur: gimeracil: oteracil potassium is, for example, 0.5-2: 0. A preparation formulated at 2 to 0.8: 0.5 to 2, particularly preferably a preparation formulated at 1: 0.4: 1. The dose of the fluorinated pyrimidine anticancer agent is, for example, 10 to 200 mg / m ² , preferably 20 to 120 mg / m ² , more preferably 40 to 100 mg / m ² per day. The unit “mg / m ² ” indicates the dose per 1 m ^{2 of} body surface area.

The treatment mode of the combined cancer treatment is particularly limited as long as it is a known method as a treatment mode of administering all of the fluoropyrimidine anticancer agent, the taxane anticancer agent, and the platinum complex anticancer agent. However, usually, a treatment schedule of about 1 to 4 weeks is set as one course, and this course is repeated a plurality of times. From the viewpoint of the accuracy of the screening method of the present invention, the treatment schedule is such that the fluoropyrimidine anticancer agent is used for a certain period, preferably 7 to 28 days, more preferably 10 to 21 days, still more preferably 12 to After daily administration for 16 days, an anticancer agent non-administration period of a certain period, preferably 4 to 10 days, more preferably 6 to 8 days, is provided, and the middle of the administration period of the fluorinated pyrimidine anticancer agent (If the administration period is 14 days, preferably any one of the 5th to 11th days, preferably any one of the 7th to 9th days), the taxane anticancer agent and the platinum complex A schedule for administration of cancer drugs.

(2-2) Reagent containing primer or probe The reagent containing the primer or probe of the present invention specifically has a continuous base sequence having a length of at least 15 bases within the base sequence shown in any one of SEQ ID NOs: 1 to 15. A probe or primer comprising a polynucleotide having a base sequence of 15 bases or more that hybridizes (provided that, when the polynucleotide is RNA, the base “t” in the sequence is replaced with “u”) It is a reagent containing.

The probe or primer sequence contained in the reagent is a base of 15 bases or longer that specifically hybridizes with a continuous base sequence of at least 15 bases in the base sequence shown in any of SEQ ID NOs: 1 to 15 There is no particular limitation as long as it consists of a polynucleotide having a sequence.

Such a polynucleotide has a length of at least 15 bases to a total base length of any of the base sequences shown in any one of SEQ ID NOs: 1 to 15, preferably 20 bases in the base sequence shown in any of SEQ ID NOs: 1 to 15. A continuous base sequence of the entire base length of the base sequence represented by any one of SEQ ID NOs: 1 to 15, more preferably 30 base lengths to the entire base length of any of the base sequences represented by any of SEQ ID NOs: 1 to 15, Are designed as polynucleotides having a base length corresponding to the above.

Specific hybridization means that a specific hybrid is formed under a stringent hybridization condition and a non-specific hybrid is not formed. Stringent hybridization conditions can be determined based on the melting temperature (Tm) of a nucleic acid that forms a hybrid according to a conventional method. As washing conditions that can maintain a specific hybridized state, the conditions are usually about “1 × SSC, 0.1% SDS, 37 ° C.”, more strictly “0.5 × SSC, 0.1% SDS, 42 ° C.” Strictly, “0.1 × SSC, 0.1% SDS, 65 ° C.” is the condition.

The polynucleotide preferably has a base sequence complementary to a continuous base sequence having a length of at least 15 bases of the base sequence shown in any one of SEQ ID NOs: 1 to 15, but the specific hybridization is performed as described above. If possible, they need not be completely complementary. Such a polynucleotide is preferably 70% or more in base sequence as compared to a polynucleotide comprising a base sequence of at least 15 bases continuous in the base sequence shown in any of SEQ ID NOs: 1 to 15 or a complementary polynucleotide thereof. The polynucleotide is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more. Here, the identity of the base sequence can be calculated by identity search, sequence alignment program, BLAST, FASTA, ClustalW, or the like.

In addition, these polynucleotides can be prepared according to a conventional method using, for example, a commercially available nucleotide synthesizer based on the total base length of the base sequence represented by any of SEQ ID NOs: 1 to 15. It can also be prepared by PCR using the entire base length of the base sequence shown in any of SEQ ID NOs: 1 to 15 as a template.

As a probe or primer contained in the reagent, preferably,
(I) a probe or primer comprising a polynucleotide having a base sequence of 15 bases or more that specifically hybridizes with a continuous base sequence of at least 15 bases in the base sequence shown in SEQ ID NO: 1 (hereinafter referred to as “ It may be expressed as “a probe or primer that recognizes SEQ ID NO: 1”), or (II) specifically hybridizes with a continuous base sequence of at least 15 bases in the base sequence shown in SEQ ID NO: 1 Specifically hybridize with a probe or primer comprising a polynucleotide having a base sequence of 15 bases or more in length and a continuous base sequence of at least 15 bases in the base sequence shown in any of SEQ ID NOs: 2 to 15. A probe or primer comprising a polynucleotide having a base sequence of 15 bases or more in length; It is.

As a probe or primer contained in the reagent, more preferably,
(V) a probe or primer that recognizes SEQ ID NO: 1 and a probe or primer that recognizes SEQ ID NO: 2, or (VI) a probe or primer that recognizes SEQ ID NO: 1 and a probe or primer that recognizes SEQ ID NO: 2, and Examples include probes or primers that recognize any of SEQ ID NOs: 3 to 15.

As a probe or primer contained in the reagent, more preferably,
(VII) a probe or primer that recognizes SEQ ID NO: 1, a probe or primer that recognizes SEQ ID NO: 2, and a probe or primer that recognizes SEQ ID NO: 3, or (VIII) a probe or primer that recognizes SEQ ID NO: 1, SEQ ID NO: And a probe or primer that recognizes SEQ ID NO: 3, and a probe or primer that recognizes any of SEQ ID NOs: 4 to 15.

More preferably as a probe or primer contained in the reagent,
(IX) a probe or primer that recognizes SEQ ID NO: 1, a probe or primer that recognizes SEQ ID NO: 2, a probe or primer that recognizes SEQ ID NO: 3, and a probe or primer that recognizes SEQ ID NO: 4, or (X) SEQ ID NO: A probe or primer that recognizes 1, a probe or primer that recognizes SEQ ID NO: 2, a probe or primer that recognizes SEQ ID NO: 3, a probe or primer that recognizes SEQ ID NO: 4, and any of SEQ ID NOs: 5 to 15 Or a probe or primer.

Even more preferably, the probe or primer included in the reagent recognizes SEQ ID NO: 1, a probe or primer that recognizes SEQ ID NO: 2, a probe or primer that recognizes SEQ ID NO: 3, or recognizes SEQ ID NO: 4. Examples include a probe or primer, a probe or primer that recognizes SEQ ID NO: 5, a probe or primer that recognizes SEQ ID NO: 6, and a probe or primer that recognizes SEQ ID NO: 7.

The probe or primer included in the reagent is particularly preferably a probe or primer that recognizes SEQ ID NO: 1, a probe or primer that recognizes SEQ ID NO: 2, a probe or primer that recognizes SEQ ID NO: 3, or a probe that recognizes SEQ ID NO: 4. Or a primer, a probe or primer that recognizes SEQ ID NO: 5, a probe or primer that recognizes SEQ ID NO: 6, a probe or primer that recognizes SEQ ID NO: 7, a probe or primer that recognizes SEQ ID NO: 8, or a probe that recognizes SEQ ID NO: 9 Alternatively, a primer, a probe or primer that recognizes SEQ ID NO: 10, a probe or primer that recognizes SEQ ID NO: 11, a probe or primer that recognizes SEQ ID NO: 12, a probe or primer that recognizes SEQ ID NO: 13, or a probe that recognizes SEQ ID NO: 14 Over Bed or primer, and a probe or primer and the like that recognize SEQ ID NO: 15.

The reagent containing the primer or probe of the present invention contains at least one primer or probe composed of the above-mentioned unlabeled or labeled polynucleotide. In addition to primers or probes, other reagents necessary for the measurement of gene expression products in the above-described screening method of the present invention, such as hybridization reagents and buffers, may be included as appropriate. .

(2-3) Reagent containing antibody The reagent containing the antibody of the present invention is a reagent containing an antibody that recognizes a polypeptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 16 to 30.

The antibody used in the present invention is not limited as long as it can recognize and detect a polypeptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 16 to 30, and may be any of a monoclonal antibody and a polyclonal antibody. Good. Specifically, “detectable” means that the presence of a protein can be detected by a known protein detection method, and preferably the presence of a protein can be detected by Western blotting or immunostaining. The antibody may be an antibody prepared by using a polypeptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 16 to 30 as an immunizing antigen, or consisting of the amino acid sequence shown in any of SEQ ID NOs: 16 to 30. It may be an antibody having an antigen-binding property to a polypeptide consisting of at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence constituting the polypeptide. Such a polypeptide can be usually synthesized by a known method based on the amino acid sequence shown in any of SEQ ID NOs: 16 to 30 and the base sequence encoding it. For example, a chemical synthesis method using an amino acid synthesizer or a genetic engineering method can be used.

The antibody according to the present invention can be produced according to a conventional method (for example, Current protocol in Molecular Biology edit. Ausubel et al. (1987), Publish. John Wiley and Sons. Section 11.12-11.13). For example, in the case of a polyclonal antibody, using the above-mentioned polypeptide expressed and purified in Escherichia coli according to a conventional method, or using a polypeptide synthesized so as to have these partial amino acid sequences according to a conventional method, an experimental animal is immunized, It can be obtained from the serum of the immunized animal according to a conventional method. On the other hand, for example, in the case of a monoclonal antibody, an experimental animal is immunized with the above-mentioned polynucleotide expressed and purified in Escherichia coli according to a conventional method, or with a polypeptide synthesized with these partial amino acid sequences according to a conventional method. Then, spleen cells obtained from the experimental animals and myeloma cells are fused to synthesize hybridoma cells, which can be obtained from the cells.

The antibody contained in the reagent is preferably (III) an antibody that recognizes a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16 (hereinafter sometimes expressed as "an antibody that recognizes SEQ ID NO: 16") Or (IV) an antibody that recognizes a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 16, and
Examples thereof include an antibody that recognizes a polypeptide consisting of the amino acid sequence shown in any of SEQ ID NOs: 17 to 30.

As an antibody contained in the reagent, more preferably,
(XI) an antibody that recognizes SEQ ID NO: 16 and an antibody that recognizes SEQ ID NO: 17, or (XII) an antibody that recognizes SEQ ID NO: 16, and an antibody that recognizes SEQ ID NO: 17, and any one of SEQ ID NOs: 18 to 30 Antibody that recognizes.

More preferably, as an antibody contained in the reagent,
(XIII) an antibody that recognizes SEQ ID NO: 16, an antibody that recognizes SEQ ID NO: 17, and an antibody that recognizes SEQ ID NO: 18, or (XIV) an antibody that recognizes SEQ ID NO: 16, an antibody that recognizes SEQ ID NO: 17, and a sequence Examples thereof include an antibody recognizing number 18 and an antibody recognizing any of SEQ ID NOs: 19 to 30.

More preferably as an antibody contained in the reagent,
(XV) an antibody that recognizes SEQ ID NO: 16, an antibody that recognizes SEQ ID NO: 17, an antibody that recognizes SEQ ID NO: 18, and an antibody that recognizes SEQ ID NO: 19, or (XVI) an antibody that recognizes SEQ ID NO: 16, SEQ ID NO: An antibody recognizing SEQ ID NO: 18, an antibody recognizing SEQ ID NO: 19, and an antibody recognizing any of SEQ ID NOS: 20-30.

More preferably, as an antibody contained in the reagent, an antibody that recognizes SEQ ID NO: 16, an antibody that recognizes SEQ ID NO: 17, an antibody that recognizes SEQ ID NO: 18, an antibody that recognizes SEQ ID NO: 19, or a sequence that recognizes SEQ ID NO: 20 Examples thereof include an antibody, an antibody that recognizes SEQ ID NO: 21, and an antibody that recognizes SEQ ID NO: 22.

As the antibody contained in the reagent, particularly preferably, an antibody that recognizes SEQ ID NO: 16, an antibody that recognizes SEQ ID NO: 17, an antibody that recognizes SEQ ID NO: 18, an antibody that recognizes SEQ ID NO: 19, or an antibody that recognizes SEQ ID NO: 20 An antibody that recognizes SEQ ID NO: 21, an antibody that recognizes SEQ ID NO: 22, an antibody that recognizes SEQ ID NO: 23, an antibody that recognizes SEQ ID NO: 24, an antibody that recognizes SEQ ID NO: 25, an antibody that recognizes SEQ ID NO: 26, a sequence Examples thereof include an antibody that recognizes No. 27, an antibody that recognizes SEQ ID No. 28, an antibody that recognizes SEQ ID No. 29, and an antibody that recognizes SEQ ID No. 30.

The reagent containing the antibody of the present invention contains at least one of the above-mentioned unlabeled or labeled antibodies. Further, in addition to antibodies, other reagents necessary for the measurement of gene expression products in the above-described screening method of the present invention, such as antigen-antibody reaction reagents and buffers, may be included as appropriate.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

1. Classification of effective group and ineffective group 20 patients with gastric cancer classified in stage III or stage IV are treated with 3 types of docetaxel, cisplatin, and tegafur as anticancer agents. The effective group and the ineffective group were classified.

Table 2 below shows the age, sex, and progression of cancer of patients who were treated.

Treatment is fluorinated pyrimidine anticancer drugs tegafur, gimeracil (prevents metabolization of tegafur other than fluorouracil), and oteracil potassium (fluorouracil reduces gastrointestinal toxicity) The anticancer agent TS-1 (Takuma Pharmaceutical Co., Ltd.) (80 mg / m ² ) containing a molar ratio of 1: 0.4: 1 is orally administered for 2 weeks, and the 8th day of the oral administration period Intravenous administration of docetaxel (60 mg / m ² ), which is a taxane anticancer agent, and cisplatin (60 mg / m ² ), which is a platinum complex anticancer agent. The treatment was carried out according to a treatment schedule of a total of 3 weeks, with a weekly non-cancer drug administration period.

After completion of the above three-week treatment schedule, the metastatic lesion was photographed by computed tomography (Computed Tomography), and the major axis of the metastatic tumor was measured. The major axis of the metastasis measured before the treatment was compared with the major axis of the metastasis measured after the treatment, and the tumor reduction rate (the major axis of the metastasis after treatment / the major axis of the metastasis before treatment) was determined.

患者 Patients with tumor reduction rate of 10% or more according to the above 3 week treatment schedule were classified into effective groups, and those with less than 10% were classified into ineffective groups. The results are shown in Table 2 above. Patients with patient numbers 1-11 were classified into the effective group, and patients with patient numbers 12-19 were classified into the ineffective group.

2. Analysis of difference in gene expression between effective group and ineffective group Analyzing difference in gene expression between effective group and ineffective group classified in “1. Classification of effective group and ineffective group” above Therefore, gene expression levels in gastric cancer tissues of patients belonging to the effective group and the ineffective group were analyzed by a microarray method and an immunostaining method.

2-1. Microarray method (1) Perform upper gastrointestinal endoscopy before treatment, biopsy the gastric cancer tissue, and immediately store it frozen (2) Prepare a fresh frozen sliced section, and prepare a gastric cancer cell or cell by microdissection method Collect only normal gland ducts (3) Extract RNA from collected tissues according to conventional methods (4) Create cDNA from RNA using reverse transcriptase (5) Fragment cDNA and label with fluorescent dye Cy3 (6) Hybridization with a DNA chip loaded with 41,000 whole human genomes (7) Image images were created by scanning the DNA chip and quantified.

From the microarray results, the difference in gene expression level between the effective group and the ineffective group was examined, and 29 genes significantly different between the two groups were selected by t-test. Of these, 15 genes with high absolute expression levels in the effective group or the ineffective group, that is, 15 genes expected to be easily identified by staining were selected.

These 15 genes are PDGFB gene, PCGF3 gene, CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, JMJD2A gene, STYX gene, EGR1 gene, AVPI1 gene, and ERLIN1 gene.

Table 3 shows the microarray data for these 15 genes. In the table, the numerical value indicates the gene expression level. From this result, it became clear that the expression level in the effective group tends to be higher than the expression level in the ineffective group for the PDGFB gene and the JMJD2A gene. In addition, for the PCGF3 gene, CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, STYX gene, EGR1 gene, AVPI1 gene, and ERLIN1 gene, the expression level in the effective group is It became clear that the expression level tends to be lower than that in the ineffective group.

2-2. Immunostaining method Next, whether or not the above-mentioned expression tendency of mRNA was also observed at the protein level was evaluated by immunostaining method.

The genes whose protein expression levels were compared between the effective group and the non-effective group are PDGFB gene, CISH gene, ANTXR2 gene, PLK2 gene, EGR1 gene, ATP7B gene, and HECA gene.

Immunostaining was performed by the streptavidin-biotin-peroxidase method. That is, a thin slice of 2 μm was prepared from a formalin-fixed specimen of gastric cancer tissue, deparaffinized with alcohol, and then endogenous peroxidase was inactivated with 0.3% H 2 O 2. After immersing in 0.01 M citrate buffer for antigen activation treatment at 95 ° C. for 15 minutes, a primary antibody was added and reacted at 4 ° C. overnight. The slide was thoroughly washed with phosphate buffer (PBS), reacted with a biotinylated secondary antibody, reacted with peroxidase / streptavidin, and then colored with Diaminobenzidine tetrahydrochloride (DAB).

The results are shown in FIG. From this result, it was clarified that the PDGFB gene expression level in the effective group tends to be higher than the expression level in the ineffective group. It was also revealed that the expression level in the effective group tends to be lower than the expression level in the ineffective group for the CISH gene, ANTXR2 gene, PLK2 gene, EGR1 gene, ATP7B gene, and HECA gene.

3. Accuracy of the selection method using the expression level of each gene as an index As the expression level index of the 15 genes, the above-mentioned “1. Effective group and ineffective group” are determined by leave-one-out cross-validation method and N-fold method. Each patient in the patient group classified as “effective” or “ineffective” in the “classification” was predicted to be an effective group or an ineffective group.

«Leave-one-out cross validation first extracts one case from all 19 cases, and creates a prediction score of whether one case is valid or ineffective from the data of the remaining 18 cases. This operation is performed 19 times in total for each case, and the effect is predicted from the total prediction score.

The N-fold method randomly divides all 19 cases into N groups (N = -192-19), takes one case as test data, and predicts whether the remaining N-1 cases are valid or ineffective Create This operation is repeated N times, and the effect is predicted from the total prediction score.

The results are shown in Table 4 below.

From this result, it is clear that almost all of the 15 genes can be selected with high accuracy of about 70% or more by using the expression level of one gene as an index. It became. In addition, it was revealed that the expression level of PDGFB and PCGF3 can be selected more accurately by using as an index. Furthermore, it became clear that selection can be performed with extremely high accuracy of about 95% by using as an index the expression level of a gene group combining PDGFBGF and PCGF3 and another gene.

4). Examination of usefulness as an effective group selection marker for cancer treatment Effective group selection for cancer treatment using a combination of taxane anticancer agent, platinum complex anticancer agent and fluoropyrimidine anticancer agent The usefulness of the gene found by the above experiment as a marker was examined.

Specifically, the PDGFB gene, PCGF3 gene, CISH gene, or ANXA5 gene expression in the cell is knocked down using siRNA, and the fluoropyrimidine anticancer agent or taxane anticancer of the cell Changes in drug sensitivity to drugs were examined.

In the above experiments, it has been found that the expression level of the PDGFB gene tends to be higher in the effective group than in the ineffective group. Therefore, by knocking down the PDGFB gene in the cell, it is predicted that the cell will artificially become an ineffective group (group with low drug sensitivity).

On the other hand, it has been found that the expression level of PCGF3 gene, CISH gene, or ANXA5 gene tends to be lower in the effective group than in the ineffective group. Therefore, by knocking down the PCGF3 gene, CISH gene, or ANXA5 gene in the cell, it is predicted that the cell will artificially become an effective group (group with high drug sensitivity).

The details of the experiment are shown below.

Using human cultured gastric cancer cell line MKN45, these genes were knocked down using siRNA against PDGFB gene, PCGF3 gene, CISH gene, or ANXA5 gene. The siRNA sequences used are as shown in Table 5 below.

Specifically, MKN45 cells were cultured in 96-well plates at 1.5 x 10 / well each, and transfection was performed by adding siPORT Amine 0.45 μl and siRNA 30 to 30 μM (/ 100 μl OptiMEM medium) as transfection reagents. It was. After culturing at 37 ° C and 5% CO2 for 6 hours, the culture solution was replaced with RPMI1640 with Fetal calf serum (FCS) added to a concentration of 10%, and 5-FU (TS-1 is in vivo And finally becomes 5-FU, which is the active form), and added with docetaxel, sputum or cisplatin, and further cultured for 48 hours, followed by MTT assay. 5-FU (the final active substance of TS-1) has a final concentration of 0 μM, 7.8 μM, 15.6 μM, 31.3 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, or 1000 μM, respectively. Cisplatin is 0μM, 0.0240μM, 0.0980μM, 0.390μM, 1.560μM, 250μM, 250μM, 1000μM, 1000μM, 0.61000μM, or 0.61pM, 2.44pM, 9.77pM, 39.0pM It added so that it might become.

The cell viability obtained by MTT assay was calculated assuming that the control cells to which no drug was added were 100%. FIG. 2 shows the results of drugs changed by knockdown of each gene. As control cells, MKN45 cells supplemented with the same concentration of random siRNA were used. FIG. 2 shows the drug concentration at 50% viability as IC50 with respect to cell viability when no drug is added.

2. From FIG. 2, the sensitivity to 5-FU increased in the cells in which the ANXA5 gene was knocked down, and the sensitivity to 5-FU was similarly increased in the cells in which the CISH gene was knocked down. In addition, sensitivity to docetaxel was enhanced in cells in which the PCGF3 gene was knocked down. On the other hand, sensitivity to 5-FU decreased in cells in which the PDGFB gene was knocked down. There was no change in sensitivity to other drugs. These results were consistent with the initial prediction described above. That is, it was found that the PDGFB gene, the PCGF3 gene, the CISH gene, or the ANXA5 gene are effective as an effective group selection marker for cancer treatment.

Claims (11)

Cancer treatment using the following steps (a), (b), and (c) in combination with a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent. To select effective groups of:
(A) in a biological sample containing cancer tissue of a cancer patient,
(I) PDGFB gene, or (ii) PDGFB gene, and PCGF3 gene, CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, JMJD2A gene, STYX gene, EGR1 gene, Measuring the amount of an expression product of at least one gene selected from the group consisting of the AVPI1 gene and the ERLIN1 gene;
(B) the amount of the expression product obtained in the step (a) (hereinafter collectively referred to as “gene expression level”), taxane anticancer agent, platinum complex anticancer agent, and fluorinated pyrimidine Comparing the amount of the corresponding gene expression product in the ineffective group of cancer treatment combined with three anticancer agents (hereinafter collectively referred to as “control gene expression level”), and (c ) Step of determining the cancer patient as an effective group when the following criteria (A) and (B) are satisfied:
(A) When the measured gene is PDGFB gene or JMJD2A gene, the gene expression level is higher than the control gene expression level, and (B) the measured gene is PCGF3 gene, CISH gene, ANXA5 If the gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, STYX gene, EGR1 gene, AVPI1 gene, or ERLIN1 gene, the gene expression level is lower than the control gene expression level Based on Genes for measuring the amount of expression product in step (a) are PDGFB gene and PCGF3 gene, and CISH gene, ANXA5 gene, ANTXR2 gene, B4GALT5 gene, PLK2 gene, ATP7B gene, FAM116A gene, HECA gene, JMJD2A gene, STYX The method according to claim 1, wherein the method is at least one selected from the group consisting of a gene, an EGR1 gene, an AVPI1 gene, and an ERLIN1 gene. The gene for measuring the amount of an expression product in the step (a) is a PDGFB gene, a PCGF3 gene, a CISH gene, an ANXA5 gene, an ANTXR2 gene, a B4GALT5 gene, and a PLK2 gene according to claim 1, Method. The taxane anticancer agent is docetaxel, the platinum complex anticancer agent is cisplatin, or the fluoropyrimidine anticancer agent is tegafur. 4. The method according to any one of 3 to 3. 2. The taxane anticancer agent is docetaxel, the platinum complex anticancer agent is cisplatin, and the fluoropyrimidine anticancer agent is tegafur. 4. The method according to any one of 3 to 3. The method according to any one of claims 1 to 5, wherein the cancer is gastric cancer. A reagent for predicting the effect of cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent,
(I) a probe or primer comprising a polynucleotide having a base sequence of 15 bases or longer, which specifically hybridizes with a continuous base sequence of at least 15 bases in the base sequence shown in SEQ ID NO: 1, or (II ) Probes or primers consisting of a polynucleotide having a base sequence of 15 bases or longer and specifically hybridizing with a continuous base sequence of at least 15 bases in the base sequence shown in SEQ ID NO: 1 and SEQ ID NOs: 2 to 15 A reagent comprising a probe or primer comprising a polynucleotide having a base sequence of 15 bases or longer, which specifically hybridizes with a continuous base sequence of at least 15 bases in the base sequence shown in any of the above. A reagent for predicting the effect of cancer treatment using a combination of a taxane anticancer agent, a platinum complex anticancer agent, and a fluoropyrimidine anticancer agent,
(III) an antibody that recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16, or (IV) an antibody that recognizes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16,
A reagent comprising an antibody that recognizes a polypeptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 17 to 30. The taxane anticancer agent is docetaxel, the platinum complex anticancer agent is cisplatin, or the fluoropyrimidine anticancer agent is tegafur. Or the reagent in any one of 8. 8. The taxane anticancer agent is docetaxel, the platinum complex anticancer agent is cisplatin, and the fluoropyrimidine anticancer agent is tegafur. Or the reagent in any one of 8. The reagent according to any one of claims 7 to 10, wherein the cancer is gastric cancer.

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