Methods for testing the efficacy of PARP inhibitors against ovarian cancer

Abstract

The present invention addresses the problem of providing a method for testing the effectiveness of a PARP inhibitor against ovarian cancer. Said problem is solved by a method for testing the effectiveness of a PARP inhibitor against ovarian cancer, the method comprising (1) a step for measuring, in a biological sample collected from a subject having ovarian cancer, the copy number of at least one gene selected from the group consisting of PIK3CA, RB1, GABRA6, BRCA2, ARID1A, NOTCH3, CTNNB1, BRAF, MYC, CSMD3, NF1, PTEN, PUM3, ELP4, JAG1, BARD1, MSH2, RPP2R1A, and MDM4.

Description

[Technical Field]

[0001] The present invention relates to a method for testing the effectiveness of a PARP inhibitor against ovarian cancer, and the like. [Background technology]

[0002] In recent years, PARP inhibitors have been introduced in Japan as a therapeutic agent for ovarian cancer. Currently, the selection criteria for PARP inhibitors are hereditary ovarian cancer positivity or a positive result in the MyChoice™ diagnostic system (Non-Patent Document 1). However, approximately 70% of patients fail both selection criteria, and myChoice is an algorithm created using breast cancer patient data. It has been reported that there are patients for whom PARP inhibitors are effective even if they test negative, and patients for whom PARP inhibitors are ineffective even if they test positive. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] myChoice Diagnostic System Attachment November 24, 2020 (2nd Edition) Summary of the Invention [Problem to be solved by the invention]

[0004] An objective of the present invention is to provide a method for testing the effectiveness of a PARP inhibitor against ovarian cancer. [Means for solving the problem]

[0005] In light of the above-mentioned problems, the present inventors have conducted extensive research and have found that the above-mentioned problems can be solved by (1) a method for testing the efficacy of a PARP inhibitor against ovarian cancer, comprising the step of measuring the copy number of (A) four genes A, namely, CTNNB1, GABRA6, MYC, and RB1, and / or (B) at least one gene B selected from the group consisting of CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, PIK3CA, BRCA2, CSMD3, NF1, PTEN, PUM3, ELP4, JAG1, BARD1, MSH2, RPP2R1A, and MDM4, in a biological sample collected from a subject with ovarian cancer. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects.

[0006] Item 1. (1) In a biological sample collected from a subject with ovarian cancer, (A) four genes A, CTNNB1, GABRA6, MYC, and RB1, and / or (B) at least one gene B selected from the group consisting of CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, PIK3CA, BRCA2, CSMD3, NF1, PTEN, PUM3, ELP4, JAG1, BARD1, MSH2, RPP2R1A, and MDM4 A method for testing the effectiveness of a PARP inhibitor against ovarian cancer, comprising measuring the copy number of

[0007] Item 2. The method according to Item 1, wherein the genes whose copy numbers are measured in step (A) include gene A.

[0008] Item 3. The method according to Item 1, wherein the genes whose copy numbers are measured in step (A) include gene A and / or gene B other than BRCA2.

[0009] Item 4. The method according to any one of Items 1 to 3, wherein the biological sample is extracellular vesicles purified from a body fluid, a body fluid containing extracellular vesicles, or ovarian cancer tissue.

[0010] Item 5. Furthermore, (2) determining the effectiveness of a PARP inhibitor on ovarian cancer in the subject based on the copy number of the gene A and / or the gene B measured in the step (1); 5. The method according to any one of Items 1 to 4, comprising:

[0011] Item 6. The method according to Item 5, wherein the step (2) comprises: (2a) determining that a PARP inhibitor is effective against ovarian cancer in the subject and / or deciding to administer a PARP inhibitor to the subject, when the copy number of the gene A and / or the gene B measured in the step (1) is equal to or greater than a cutoff value.

[0012] Item 7. The method according to any one of Items 1 to 6, wherein the subject is a human.

[0013] Item 8. The method according to any one of Items 1 to 7, wherein the method for measuring the copy number of the gene A and / or the gene B is a digital PCR method.

[0014] Item 9. (A) Four genes A, namely CTNNB1, GABRA6, MYC, and RB1, and / or (B) at least one gene B selected from the group consisting of CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, PIK3CA, BRCA2, CSMD3, NF1, PTEN, PUM3, ELP4, JAG1, BARD1, MSH2, RPP2R1A, and MDM4 A test agent for the effectiveness of PARP inhibitors against ovarian cancer, including a copy number detection agent.

[0015] Item 9A. For use in testing the effectiveness of PARP inhibitors against ovarian cancer, (A) four genes A, CTNNB1, GABRA6, MYC, and RB1, and / or (B) at least one gene B selected from the group consisting of CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, PIK3CA, BRCA2, CSMD3, NF1, PTEN, PUM3, ELP4, JAG1, BARD1, MSH2, RPP2R1A, and MDM4 Copy number detection agent.

[0016] Section 9B. (A) Four genes A, CTNNB1, GABRA6, MYC, and RB1, and / or (B) at least one gene B selected from the group consisting of CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, PIK3CA, BRCA2, CSMD3, NF1, PTEN, PUM3, ELP4, JAG1, BARD1, MSH2, RPP2R1A, and MDM4 Use of a copy number detection agent for the manufacture of a test agent for the effectiveness of a PARP inhibitor against ovarian cancer.

[0017] Section 9C. (A) Four genes A, CTNNB1, GABRA6, MYC, and RB1, and / or (B) at least one gene B selected from the group consisting of CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, PIK3CA, BRCA2, CSMD3, NF1, PTEN, PUM3, ELP4, JAG1, BARD1, MSH2, RPP2R1A, and MDM4 20. Use of a copy number detection agent for testing the efficacy of PARP inhibitors against ovarian cancer.

[0018] Item 10. The test agent according to Item 9, wherein the detection agent is a probe or primer for the gene. [Effects of the Invention]

[0019] According to the present invention, a method for testing the effectiveness of a PARP inhibitor against ovarian cancer can be provided. [Brief explanation of the drawings]

[0020] [Figure 1] The results of Test Example 2 are shown. The vertical axis indicates copy number, and the horizontal axis indicates the gene to be measured. "tumor" indicates the copy number in DNA extracted from ovarian cancer tissue, and "other" indicates the copy number in DNA extracted from ascites S-EV samples (volume indicates the volume of ascites extracted). [Figure 2] This shows the ROC curve for Test Example 3. The genes used as indicators are shown at the top. The gray area in the figure indicates the 95% confidence interval. [Figure 3] This shows the ROC curve for Test Example 3. The genes used as indicators are shown at the top. The gray area in the figure indicates the 95% confidence interval. [Figure 4] This shows the ROC curve for Test Example 3. The genes used as indicators are shown at the top. The gray area in the figure indicates the 95% confidence interval. [Figure 5] This shows the ROC curve for Test Example 3. The genes used as indicators are shown at the top. The gray area in the figure indicates the 95% confidence interval. [Figure 6] This shows the ROC curve for Test Example 3. The genes used as indicators are shown at the top. The gray area in the figure indicates the 95% confidence interval. [Figure 7] This shows the ROC curve for Test Example 3. The genes used as indicators are shown at the top. The gray area in the figure indicates the 95% confidence interval. [Figure 8] This shows the ROC curve for Test Example 3. The genes used as indicators are shown at the top. The gray area in the figure indicates the 95% confidence interval. [Figure 9] This shows the ROC curve for Test Example 3. The genes used as indicators are shown at the top. The gray area in the figure indicates the 95% confidence interval. [Figure 10] This shows the ROC curve for Test Example 3. The genes used as indicators are shown at the top. The gray area in the figure indicates the 95% confidence interval. [Figure 11] This shows the ROC curve for Test Example 3. The genes used as indicators are shown at the top. The gray area in the figure indicates the 95% confidence interval. [Figure 12] 1 shows the ROC curve for Test Example 3. The genes used as indicators are shown at the top. DETAILED DESCRIPTION OF THE INVENTION

[0021] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0022] 1. Testing method In one aspect, the present invention relates to (1) a method for testing the effectiveness of a PARP inhibitor against ovarian cancer (sometimes referred to herein as the "testing method of the present invention"), which comprises the step of measuring the copy number of (A) four genes A, namely, CTNNB1, GABRA6, MYC, and RB1, and / or (B) at least one gene B selected from the group consisting of CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, PIK3CA, BRCA2, CSMD3, NF1, PTEN, PUM3, ELP4, JAG1, BARD1, MSH2, RPP2R1A, and MDM4, in a biological sample collected from a subject with ovarian cancer. This method will be described below.

[0023] 1-1. Process (1) Ovarian cancers include, but are not limited to, superficial, epithelial, and stromal malignant tumors (e.g., serous (cystic) adenocarcinoma, mucinous (cystic) adenocarcinoma, endometrioid adenocarcinoma, clear cell adenocarcinoma, adenocarcinoma-fibroma (each of the above types), adenosarcoma, mesodermal mixed tumor, [Müllerian mixed tumor] [carcinosarcoma], malignant Brenner tumor, transitional cell carcinoma, undifferentiated carcinoma, etc.), sex cord-stromal tumors (e.g., fibrosarcoma, Sertoli-stromal cell tumor (poorly differentiated), etc.), germ cell tumors (e.g., dysgerminoma, yolk sac tumor [endodermal sinus tumor], embryonal carcinoma [embryonic carcinoma], polyembryoma, choriocarcinoma, mature cystic teratoma with malignant transformation, immature teratoma (G3), etc.), carcinoma, sarcoma, malignant lymphoma (primary), secondary (metastatic) tumors, etc. Ovarian cancer of all classes, grades, and stages according to various classification criteria relating to the progression, state, etc. of ovarian cancer can be the subject of testing.

[0024] The subject is a living organism that is the target of the testing method of the present invention, and the species of the subject is not particularly limited. Examples of the subject species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, and preferably humans.

[0025] The condition of the subject is not particularly limited as long as the subject has ovarian cancer. Examples of the subject include a subject for which it is unknown whether a PARP inhibitor is effective, a subject for which a PARP inhibitor has already been determined to be effective by another method, a subject for which a PARP inhibitor has already been determined to be ineffective by another method, and a subject undergoing treatment with a PARP inhibitor.

[0026] The biological sample is not particularly limited as long as it is derived from a living organism and can contain genomic DNA derived from ovarian cancer. Examples of biological samples include body fluids such as whole blood, serum, plasma, ascites, follicular fluid, menstrual blood, saliva, cerebrospinal fluid, synovial fluid, urine, interstitial fluid, sweat, tears, and saliva, as well as samples derived from these body fluids. Biological samples can also be biological tissues, preferably ovarian cancer tissues, or samples derived from these tissues. Samples derived from body fluids / tissues are not particularly limited as long as they are samples prepared from body fluids / tissues, and examples include samples obtained from body fluids / tissues by concentrating or purifying nucleic acids or extracellular vesicles contained in the body fluids / tissues.

[0027] Body fluids can be collected from a subject by methods known to those skilled in the art. For example, whole blood can be collected by drawing blood using a syringe or the like. Serum is a portion of whole blood from which blood cells and specific blood coagulation factors have been removed, and can be obtained, for example, as the supernatant after clotting of whole blood. Plasma is a portion of whole blood from which blood cells have been removed, and can be obtained, for example, as the supernatant when whole blood is centrifuged under conditions that do not cause clotting.

[0028] Extracellular vesicles are not particularly limited as long as they are membrane vesicles that are secreted, released, or the like from cells. Extracellular vesicles are generally defined as membrane vesicles that are responsible for local or systemic intercellular signaling by transporting intracellular proteins and genetic information (mRNA, microRNA, etc.) to the outside of cells. Examples of extracellular vesicles include exosomes, microvesicles, apoptotic bodies, ectosomes, microparticles, and secreted microvesicles. From the viewpoint of test accuracy, etc., exosomes are particularly preferred as extracellular vesicles.

[0029] Extracellular vesicles can be purified, separated, concentrated, etc. from body fluids according to or in accordance with known methods. Methods for purifying, separating, concentrating, etc. extracellular vesicles include, for example, ultracentrifugation (e.g., pellet-down method, sucrose cushion method, density gradient centrifugation, etc.), methods using immunoaffinity carriers, gel filtration, field-flow fractionation, FACS, etc. Purification, separation, concentration, etc. of extracellular vesicles can also be performed using commercially available kits. These methods may be used alone or in combination of two or more.

[0030] The biological sample may preferably be extracellular vesicles purified from a body fluid, a body fluid containing extracellular vesicles, ovarian cancer tissue, or the like.

[0031] In step (1), the copy number of (A) four genes A, namely, CTNNB1, GABRA6, MYC, and RB1, and / or (B) at least one gene B selected from the group consisting of CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, PIK3CA, BRCA2, CSMD3, NF1, PTEN, PUM3, ELP4, JAG1, BARD1, MSH2, RPP2R1A, and MDM4 (herein, gene A and gene B are sometimes collectively referred to as "target genes") is measured.

[0032] In one embodiment of the present invention, it is particularly preferable that the target gene includes gene A, from the viewpoint of particularly high efficacy prediction ability. In this case, the efficacy prediction ability can be improved by further combining gene A with another gene. The other gene is preferably gene B. Furthermore, the other gene may be a gene whose CV score was 0.6 or less in Test Example 1 described below. Such genes preferably include AKT2, CCND2, KRAS, RAD51, MLH1, etc.

[0033] When the target gene includes gene A, the target gene further includes (BX) at least one gene BX selected from the group consisting of ARID1A, BRAF, NOTCH3, PIK3CA, BRCA2, CSMD3, NF1, PTEN, PUM3, ELP4, JAG1, BARD1, MSH2, RPP2R1A, and MDM4, and / or (C) at least one gene C selected from the group consisting of AKT2, CCND2, KRAS, RAD51, and MLH1; As a preferred example, the target genes may include gene A, BARD1, and RAD51.

[0034] In one aspect of the present invention, among gene B, from the viewpoint of high predictive ability of efficacy, preferred examples include CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, PIK3CA, BRCA2, etc., more preferred examples include CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, BRCA2, etc., and particularly preferred examples include CTNNB1, GABRA6, MYC, RB1, BRCA2, etc.

[0035] In one aspect of the present invention, from the viewpoint of high predictive ability of efficacy, the target genes include at least two (preferably at least three, more preferably at least four) genes selected from the group consisting of CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, PIK3CA, and BRCA2 (preferably the group consisting of CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, and BRCA2, more preferably the group consisting of CTNNB1, GABRA6, MYC, RB1, and BRCA2).

[0036] In one embodiment of the invention, the gene of interest, gene B, does not include BRCA2.

[0037] In one aspect of the present invention, among the target genes, PIK3CA, RB1, GABRA6, BRCA2, ARID1A, NOTCH3, CTNNB1, BRAF, MYC, etc. are preferred, from the viewpoint of their high contribution to predicting the effectiveness of PARP inhibitors, and PIK3CA, RB1, GABRA6, BRCA2, ARID1A, etc. are particularly preferred.

[0038] The number of target genes whose copy numbers are measured in step (1) may be one or more, but from the viewpoint of test accuracy, etc., the number may be preferably two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, or nineteen or more. From the viewpoint of efficiency, the number of target genes is preferably, for example, ten or less, eight or less, six or less, or five or less.

[0039] The PIK3CA gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 5290. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that are observed between individuals.

[0040] The RB1 gene is known, and in the case of humans, for example, the gene with NCBIGene ID: 5925 corresponds to this. The nucleotide sequence of this gene can be determined based on known genetic information. This gene also includes those containing mutations that are observed between individuals.

[0041] The GABRA6 gene is known, and in the case of humans, for example, the gene with NCBIGene ID: 2559 corresponds to this. The nucleotide sequence of this gene can be determined based on known genetic information. This gene also includes those containing mutations that are observed between individuals.

[0042] The BRCA2 gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 675. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that are found between individuals.

[0043] The ARID1A gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 8289. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations found between individuals.

[0044] The NOTCH3 gene is known, and in the case of humans, for example, the gene with NCBIGene ID: 4854 corresponds to this. The nucleotide sequence of this gene can be determined based on known genetic information. This gene also includes those containing mutations that are observed between individuals.

[0045] The CTNNB1 gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 1499. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that occur between individuals.

[0046] The BRAF gene is known, and in the case of humans, for example, the gene with NCBIGene ID: 673 corresponds to this. The nucleotide sequence of this gene can be determined based on known genetic information. This gene also includes those containing mutations that are observed between individuals.

[0047] The MYC gene is known, and in the case of humans, for example, the gene with NCBIGene ID: 4609 is an example. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that are observed between individuals.

[0048] The CSMD3 gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 114788. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations found between individuals.

[0049] The NF1 gene is known, and in the case of humans, for example, the gene with NCBIGene ID: 578 corresponds to this. The nucleotide sequence of this gene can be determined based on known genetic information. This gene also includes those containing mutations that are observed between individuals.

[0050] The PTEN gene is known, and in the case of humans, for example, the gene with NCBIGene ID: 5278 corresponds to this. The nucleotide sequence of this gene can be determined based on known genetic information. This gene also includes those containing mutations found between individuals.

[0051] The PUM3 gene is known, and in the case of humans, for example, the gene with NCBIGene ID: 9933 corresponds to this. The nucleotide sequence of this gene can be determined based on known genetic information. This gene also includes those containing mutations that are observed between individuals.

[0052] The ELP4 gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 26610. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that occur between individuals.

[0053] The JAG1 gene is known, and in the case of humans, for example, the gene with NCBIGene ID: 182 corresponds to this. The nucleotide sequence of this gene can be determined based on known genetic information. This gene also includes those containing mutations that are observed between individuals.

[0054] The BARD1 gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 580. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that are observed between individuals.

[0055] The MSH2 gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 4436. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that occur between individuals.

[0056] The RPP2R1A gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 5518. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that occur between individuals.

[0057] The MDM4 gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 4194. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that are observed between individuals.

[0058] The AKT2 gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 208. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that occur between individuals.

[0059] The CCND2 gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 894. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that are observed between individuals.

[0060] The KRAS gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 3845. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that are observed between individuals.

[0061] The RAD51 gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 5888. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that occur between individuals.

[0062] The MLH1 gene is known, and in the case of humans, for example, the gene has NCBIGene ID: 4292. The nucleotide sequence of the gene can be determined based on known genetic information. The gene also includes those containing mutations that occur between individuals.

[0063] The copy number of a target gene refers to the number of the target gene in genomic DNA. The copy number of a gene is usually 2. In the present invention, it has been found that the copy number of a target gene in ovarian cancer can be used as an indicator for predicting the effectiveness of a PARP inhibitor.

[0064] The method for measuring the copy number of a target gene is not particularly limited, and any method used as a CNV analysis technique can be used. Specific examples of the measurement method include digital PCR, sequencing, real-time PCR, array comparative genomic hybridization, comparative genomic hybridization, and fluorescent in situ hybridization. Among these, digital PCR is particularly preferred from the viewpoint of measurement accuracy, etc.

[0065] In the method for measuring the copy number of a target gene, a primer, a probe, or the like for the target gene can be used as a detection agent.

[0066] The detection agent may be modified as long as its function is not significantly impaired. Modifications include the addition of labels such as fluorescent dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, biotin, etc.

[0067] Fluorescent dyes suitable for use in the present invention include those typically used to label nucleotides for the detection and quantification of nucleic acids. Examples include, but are not limited to, HEX (4,7,2',4',5',7'-hexachloro-6-carboxylfluorescein, a green fluorescent dye), fluorescein, NED (trade name, manufactured by Applied Biosystems, a yellow fluorescent dye), 6-FAM (trade name, manufactured by Applied Biosystems, a yellow-green fluorescent dye), and rhodamine or its derivatives (e.g., tetramethylrhodamine (TMR)). Nucleotides can be labeled with fluorescent dyes using any suitable known labeling method (see Nature Biotechnology, 14, 303-308 (1996)). Alternatively, commercially available fluorescent labeling kits (e.g., Oligonucleotide ECL 3'-Oligolabeling System, manufactured by Amersham-Pharmacia) can be used.

[0068] The detection agent can also be immobilized on any solid phase for use. In this case, for example, the copy number can be measured using a substrate on which the detection agent is immobilized (e.g., a microarray chip on which a probe is immobilized).

[0069] The solid phase used for immobilization is not particularly limited as long as it can immobilize polynucleotides, etc., and examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries, and other substrates. The immobilization of the detection agent to the solid phase is not particularly limited. For example, in the case of a microarray, a commercially available spotter (e.g., manufactured by Amersham) can be used. Immobilization methods are well known in the art depending on the type of immobilized probe (e.g., photolithographic technology (Affymetrix) or in situ synthesis of oligonucleotides using inkjet technology (Rosetta Inpharmatics)).

[0070] The primers, probes, etc. are not particularly limited as long as they selectively (specifically) recognize the target gene.

[0071] Specific examples of primers and probes include the polynucleotides described in (a) below and the polynucleotides described in (b) below: (a) a polynucleotide having at least 15 consecutive bases in the base sequence of the target gene and / or a polynucleotide complementary to the polynucleotide; and (b) a polynucleotide having at least 15 bases that hybridizes under stringent conditions to the base sequence of the target gene or a base sequence complementary thereto; At least one selected from the group consisting of:

[0072] A complementary polynucleotide or complementary base sequence (complementary strand, reverse strand) refers to a polynucleotide or base sequence that is complementary to the full-length polynucleotide sequence of a target gene, or a partial sequence thereof having at least 15 consecutive bases in length (hereinafter, for convenience, these are also referred to as the "positive strand"), based on base pairing such as A:T and G:C. However, such a complementary strand is not limited to a completely complementary sequence to the base sequence of the target positive strand, but may also have a complementary relationship to the target positive strand that allows hybridization under stringent conditions. Here, stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid to which the complex or probe binds, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego, CA). For example, typical post-hybridization washing conditions include approximately 1×SSC, 0.1% SDS, and 37°C. It is preferable that the complementary strand maintains its hybridization state with the target positive strand even when washed under these conditions. While not particularly limited, more stringent hybridization conditions include approximately 0.5×SSC, 0.1% SDS, and 42°C, and even more stringent hybridization conditions include approximately 0.1×SSC, 0.1% SDS, and 65°C. Specifically, examples of such complementary strands include a strand consisting of a nucleotide sequence that is completely complementary to the nucleotide sequence of the target positive strand, and a strand consisting of a nucleotide sequence that shares at least 90%, preferably 95%, more preferably 98% or more, and even more preferably 99% or more identity with the target positive strand.

[0073] Primers, probes, etc. can be designed, for example, based on the nucleotide sequence of a target gene using various design programs. Specifically, candidate sequences for primers or probes obtained by applying the nucleotide sequence of the target gene to a design program, or sequences containing at least a portion of such sequences, can be used as primers or probes.

[0074] The base length of a primer, probe, or the like is not particularly limited as long as it has a length of at least 15 consecutive bases as described above, and can be appropriately set depending on the application. For example, when used as a primer, the base length can be, for example, 15 to 50 bases, and when used as a probe, the base length can be, for example, 15 to 150 bases.

[0075] According to the testing method of the present invention including step (1), it is possible to provide the copy number of the target gene, which is a testing indicator for the effectiveness of a PARP inhibitor against ovarian cancer, thereby assisting in the testing, etc.

[0076] 1-2. Process (2) In one embodiment, the testing method of the present invention preferably further comprises the step of (2) determining the effectiveness of a PARP inhibitor against ovarian cancer in the subject based on the copy number of the gene A and / or the gene B measured in the step (1).

[0077] More specifically, step (2) can include (2a) determining that a PARP inhibitor is effective against ovarian cancer in the subject and / or deciding to administer a PARP inhibitor to the subject when the copy number of the gene A and / or the gene B measured in step (1) is equal to or greater than a cutoff value.

[0078] The cutoff value is set in advance based on statistical analysis or ROC analysis of the copy number data of the target gene in the PARP inhibitor-effective and PARP inhibitor-ineffective groups of the evaluation population, using a database that tracks the copy number of the target gene and the efficacy of PARP inhibitors. Alternatively, the cutoff value can be set on a case-by-case basis. When the cutoff value is determined by statistical analysis, for example, the median, arithmetic mean, or other average value of the copy number data of the target gene in the evaluation population can be used. When the cutoff value is determined by ROC analysis, for example, the cutoff value based on ROC analysis can be the copy number of the target gene at the point on the ROC curve where the distance between the point on the vertical axis (sensitivity or true positive) of the ROC curve graph that is 1.0 and the point on the horizontal axis (1 - specificity) that is 0.0 is the shortest. Alternatively, the cutoff value can be derived from the Youden index of the ROC curve (Cancer 1950;3:32-35.). Once established, the database of the evaluation population may be used to set the cutoff value without any changes. Alternatively, new ovarian cancer patients, including the subjects of the present invention, may be included in the evaluation population, and the database of the evaluation population may be updated as appropriate and used to set the cutoff value.

[0079] The cutoff value for the copy number of the target gene can be, for example, as follows: PIK3CA: 2.05±0.20 (preferably ±0.10, more preferably ±0.05, and even more preferably ±0.02) RB1: 1.33 ± 0.20 (preferably ± 0.10, more preferably ± 0.05, even more preferably ± 0.02) GABRA6: 1.68 ± 0.20 (preferably ± 0.10, more preferably ± 0.05, even more preferably ± 0.02) BRCA2: 1.81±0.20 (preferably ±0.10, more preferably ±0.05, even more preferably ±0.02) ARID1A: 1.68 ± 0.20 (preferably ± 0.10, more preferably ± 0.05, even more preferably ± 0.02) NOTCH3: 1.49 ± 0.20 (preferably ± 0.10, more preferably ± 0.05, even more preferably ± 0.02) CTNNB1: 1.99±0.20 (preferably ±0.10, more preferably ±0.05, even more preferably ±0.02) BRAF: 2.47±0.20 (preferably ±0.10, more preferably ±0.05, even more preferably ±0.02) MYC: 2.82 ± 0.20 (preferably ± 0.10, more preferably ± 0.05, even more preferably ± 0.02) CSMD3: 1.22 ± 0.20 (preferably ± 0.10, more preferably ± 0.05, even more preferably ± 0.02) NF1: 1.15±0.20 (preferably ±0.10, more preferably ±0.05, even more preferably ±0.02) PTEN: 1.77±0.20 (preferably ±0.10, more preferably ±0.05, even more preferably ±0.02) PUM3: 1.46 ± 0.20 (preferably ± 0.10, more preferably ± 0.05, even more preferably ± 0.02) ELP4: 1.46±0.20 (preferably ±0.10, more preferably ±0.05, even more preferably ±0.02) JAG1: 1.45 ± 0.20 (preferably ± 0.10, more preferably ± 0.05, even more preferably ± 0.02) BARD1: 2.15 ± 0.20 (preferably ± 0.10, more preferably ± 0.05, even more preferably ± 0.02) MSH2: 1.45±0.20 (preferably ±0.10, more preferably ±0.05, even more preferably ±0.02) RPP2R1A: 1.40±0.20 (preferably ±0.10, more preferably ±0.05, even more preferably ±0.02) MDM4: 1.74±0.20 (preferably ±0.10, more preferably ±0.05, even more preferably ±0.02) AKT2: 1.01±0.20 (preferably ±0.10, more preferably ±0.05, even more preferably ±0.02) CCND2: 1.60 ± 0.20 (preferably ± 0.10, more preferably ± 0.05, even more preferably ± 0.02) KRAS: 1.67±0.20 (preferably ±0.10, more preferably ±0.05, even more preferably ±0.02) RAD51: 0.57 ± 0.20 (preferably ± 0.10, more preferably ± 0.05, even more preferably ± 0.02) MLH1: 0.57±0.20 (preferably ±0.10, more preferably ±0.05, even more preferably ±0.02).

[0080] The PARP inhibitor is not particularly limited as long as it is a drug that inhibits poly (ADP-ribose) polymerase (PARP). Examples of PARP inhibitors include olaparib, rucaparib, niraparib, talazoparib, atamparib, capivasertib, fluzoparib, pamiparib, stenoparib, veliparib, and venadaparib. In addition to these, examples include AZD9574, CBX15, IMP4297 (JS109), OX401, KT-2000, KT-3000, KT-4000, RP12146, RBN012759 (RBN3143), OPAL0001 (OPL0001), etc., or compounds having the same structure as these. Among the above-mentioned PARP inhibitors, olaparib, niraparib, etc. are preferred, with olaparib being more preferred.

[0081] The PARP inhibitor can be administered to a subject for whom a PARP inhibitor is determined to be effective in step (2) and / or a subject for whom it has been decided that a PARP inhibitor should be administered. The method of administering the PARP inhibitor is not particularly limited, and an appropriate method for each PARP inhibitor (e.g., a method described in the package insert) can be used.

[0082] 2. Testing kits In one aspect, the present invention relates to a test agent for the effectiveness of a PARP inhibitor on ovarian cancer (sometimes referred to herein as the "test agent of the present invention"), which comprises an agent for detecting the copy number of a target gene. This will be described below.

[0083] For the target gene, detection agent, etc., please follow the explanation in the previous section.

[0084] The test agent of the present invention may be in the form of a composition containing a detection agent. The composition may contain other components as needed. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0085] The test agent of the present invention may be in the form of a kit containing the detection agent. The kit may also contain instruments, reagents, etc. that can be used to carry out the test method of the present invention.

[0086] Examples of the apparatus include test tubes, microtiter plates, agarose particles, latex particles, purification columns, and glass slides.

[0087] The reagent may be, for example, a detection agent for a reference gene. [Example]

[0088] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0089] Test Example 1: Examination of the effectiveness of PARP inhibitors against ovarian cancer 1 <1-1. Patient Selection> To predict the efficacy of PARP inhibitors, we collected ovarian cancer patients (HGSC (high-grade serious carcinoma) patients) who received a PARP inhibitor (olaparib) from 2018 to 2021 (n=49). Seven patients who received the treatment continuously for more than one year were defined as "responders," and seven patients who discontinued treatment within six months due to recurrence or disease progression were defined as "non-responders" to the PARP inhibitor.

[0090] <1-2. DNA extraction> Total DNA was extracted from responder and non-responder ovarian cancer tissues (frozen or FFPE tissues) using DNeasy Blood & Tissue Kits (Qiagen) for frozen ovarian cancer tissues and QIAamp DNA FFPE Tissue Kit (Qiagen) for FFPE tissues according to the manufacturer's protocol. The concentration of extracted DNA was quantified using a Qubit 4.0 Fluorometer (Invitrogen).

[0091] <1-3. Selection of target genes for analysis> Based on the Ovarian Cancer Moon Shot database (Cell Rep 31, 107502, doi:10.1016 / j.celrep.2020.03.066 (2020)), reports on homologous recombination repair (Clin Cancer Res 24, 569-580, doi:10.1158 / 1078-0432.Ccr-17-1621 (2018)), and reports on response to PARP inhibitors (Cancers (Basel) 13, doi:10.3390 / cancers13061296 (2021)), 30 genes were selected as the original panel for analysis by ddPCR.

[0092] <1-4. Droplet digital PCR> The copy number variation (CNV) of 30 selected genes was measured using the extracted DNA using a QX200 Auto DG droplet digital PCR system (Bio-Rad) according to the manufacturer's instructions. A 22 μL reaction mixture containing 11 μL 2x ddPCR Supermix for Probes (Bio-Rad), 1.1 μL FAM-labeled target probe, 1.1 μL HEX-labeled reference probe, 1 μL restriction enzyme, 5 μL DNA template, and nuclease-free water was used for the ddPCR assay. The RPP30 probe for ddPCR copy number measurement was used for the reference gene. The restriction enzyme used for ERBB2 was MseI. HaeIII was used for the other target genes. MseI and HaeIII were diluted 5-fold with CutSmart and then 10-fold with nuclease-free water. The DNA used was at least 1 ng. PCR reactions were performed under the following cycling conditions: 95°C for 10 minutes; 40 cycles (94°C for 30 seconds, 60°C for 1 minute); 98°C for 10 minutes; 12°C hold. Plates were read using a Bio-Rad QX200 droplet reader (Bio-Rad) and analyzed using Quanta Soft software (Bio-Rad).

[0093] After measuring CNV values ​​using ddPCR, the status was analyzed using machine learning with cross validation (CV) to evaluate the contribution of these genes to PARP inhibitor response. Thirty genes were ranked based on their CV scores, and a CV score of >0.6 was considered significant and contributed to predicting PARP inhibitor response. The results are shown in Table 1.

[0094] [Table 1]

[0095] It has been found that the effectiveness of PARP inhibitors against ovarian cancer can be evaluated by measuring the copy number of at least one gene selected from the group consisting of PIK3CA, RB1, GABRA6, BRCA2, ARID1A, NOTCH3, CTNNB1, BRAF, MYC, CSMD3, NF1, PTEN, PUM3, ELP4, JAG1, BARD1, MSH2, RPP2R1A, and MDM4 in a biological sample collected from a subject with ovarian cancer.

[0096] Test Example 2: Correlation between CNV in ovarian cancer tissue and CNV in EVs in body fluids To isolate extracellular vesicles (EVs) from the ascites of ovarian cancer patients (n=49) in Experiment 1, 1 ml of ascites sample was used from each patient. Each sample was centrifuged at 500 x g for 15 minutes at 4°C to pellet floating cells. The supernatant was centrifuged at 10,000 x g for 40 minutes at 4°C. The pellet was resuspended and centrifuged again at 10,000 x g for 40 minutes at 4°C. The pellet was resuspended in PBS and stored at 4°C as large EVs (L-EVs). The supernatant was filtered through a 0.22 μm filter to remove remaining large vesicles. The filtered medium was centrifuged in an ultracentrifuge at 100,600 x g for 70 minutes at 4°C. The pellet was resuspended in PBS and centrifuged again at 100,600 x g for 70 minutes at 4°C. The final pellet was resuspended in PBS and stored at 4°C as small EVs (S-EVs) containing exosomes.

[0097] DNA was extracted from the obtained small EV samples and ovarian cancer tissues of ovarian cancer patients (n=49) in the same manner as in Test Example 1, and the copy numbers of the selected 30 genes were measured by droplet digital PCR.

[0098] The results are shown in Figure 1. We found that CNV in ovarian cancer tissues correlated with CNV in body fluid EVs.

[0099] Test Example 3. Analysis of AUC based on copy number ROC analysis was performed to distinguish between responders and non-responders in Test Example 1 using the copy number of nine genes (CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, PIK3CA, and BRCA2) selected in Test Example 1 as an indicator. The ROC curves and AUCs when the copy number of each gene is used as an index are shown in Figures 2 to 10. The ROC curves and AUCs when the copy numbers of four of the nine genes are used in combination as an index are shown in Figure 11. The ROC curves and AUCs when the copy numbers of two more genes (Test Example 1) in addition to the four genes are used in combination as an index are shown in Figure 12. It was found that each gene alone gave a relatively high AUC, and some genes alone gave significantly higher AUCs. It was also found that combining genes gave even higher AUCs.

Claims

1. (1) In a biological sample collected from a subject having ovarian cancer, (A) four genes A, CTNNB1, GABRA6, MYC, and RB1, and / or (B) at least one gene B selected from the group consisting of CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, PIK3CA, BRCA2, CSMD3, NF1, PTEN, PUM3, ELP4, JAG1, BARD1, MSH2, RPP2R1A, and MDM4 measuring the copy number of the genes whose copy numbers are to be measured in the step (1) include the gene A and / or a gene B other than BRCA2; A method for testing the efficacy of PARP inhibitors against ovarian cancer.

2. The method according to claim 1, wherein the gene whose copy number is measured in step (1) includes gene A.

3. 2. The method of claim 1, wherein the biological sample is extracellular vesicles purified from a body fluid, a body fluid containing extracellular vesicles, or ovarian cancer tissue.

4. Furthermore, (2) a step of assisting in determining the effectiveness of a PARP inhibitor for ovarian cancer in the subject based on the copy number of the gene A and / or the gene B measured in the step (1); The method according to any one of claims 1 to 3, comprising:

5. the step (2) comprising: (2a) determining that a PARP inhibitor is effective against ovarian cancer in the subject and / or assisting in the decision to administer a PARP inhibitor to the subject when the copy number of the gene A and / or the gene B measured in the step (1) is equal to or greater than a cutoff value; The method of claim 4, comprising:

6. The method according to any one of claims 1 to 3, wherein the subject is a human.

7. The method according to any one of claims 1 to 3, wherein the method for measuring the copy number of the gene A and / or the gene B is a digital PCR method.

8. (A) four genes A, CTNNB1, GABRA6, MYC, and RB1, and / or (B) at least one gene B selected from the group consisting of CTNNB1, GABRA6, MYC, RB1, ARID1A, BRAF, NOTCH3, PIK3CA, BRCA2, CSMD3, NF1, PTEN, PUM3, ELP4, JAG1, BARD1, MSH2, RPP2R1A, and MDM4 a copy number detection agent for the detection agent includes an agent for detecting the copy number of the gene A and / or the gene B other than BRCA2; A test drug for the effectiveness of PARP inhibitors against ovarian cancer.

9. The test agent according to claim 8 , wherein the detection agent is a probe or primer for the gene A and / or the gene B.

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