Customized cancer treatment method for cancer patients

Abstract

The present application relates to a cancer patient customized cancer treatment method, and more particularly, according to the present application, the expression changes of 87 target genes involved in the present application and the mutations of some of the genes are quantified and analyzed using a quantitative real-time PCR method, so that the expression pattern of each cancer patient can be profiled with high accuracy and convenience. Therefore, the cancer patient target gene expression pattern analysis method according to the present application can be used for patient individualized customized treatment by providing information that can selectively administer existing cancer treatment drugs or future developed cancer treatment drugs to cancer patients.

Description

Technical Field

[0001] This invention relates to a customized cancer treatment method for cancer patients, specifically to a customized drug screening method for cancer patients and a reagent kit for using this method. Background Technology

[0002] Cancer is a disease that threatens human health and life, accounting for approximately 13% of all deaths. In 2007, 7.6 million people worldwide died from cancer. In the United States, 1.4 million new cancer cases are reported annually in recent years, making cancer the second leading cause of death. According to the SEER report, the mortality rate for all cancer types in the United States increased from 195.4 per 100,000 people in 1950 to 204.4 per 100,000 people in 1978, before declining steadily to 184.0 per 100,000 people in 2005. This downward trend appears to be attributed to earlier cancer detection due to advancements in diagnostic technology. Early detection and treatment play a crucial role in prognosis and survival across all cancer types.

[0003] There are approximately 300 cancer treatments approved and used clinically by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). These cancer treatments are approved for indications of at least one type of cancer and are indicated by specific targets.

[0004] Traditional cancer chemotherapy selects and administers appropriate treatments based on the type and severity of the cancer, rather than on the individual patient. However, clinical results generally show significant differences in the effectiveness of this chemotherapy among patients. To overcome this problem, various methods have been proposed.

[0005] In response, the inventors have strived to develop a simple and accurate method for screening medicines suitable for individual cancer patients. The results show that by using quantitative real-time PCR (qPCR) to quantify and analyze the expression changes of 87 genes that are targets of FDA-approved cancer therapeutics and the mutations of some of these genes, the expression patterns of various cancer patients can be analyzed with high accuracy and convenience. Based on this, customized medicines for cancer patients can be screened, thus completing this application.

[0006] [Preliminary Technology Documents] [Patent Documents] Korean Patent Registration No. 10-1371697 Summary of the Invention Technical issues The purpose of this invention is to provide a method for more effectively implementing chemotherapy in cancer patients, namely, to provide a customized drug screening kit for cancer patients that utilizes cancer therapeutic agents and its uses.

[0007] Problem-solving methods To achieve the objectives of this invention, a customized drug screening kit for cancer patients is provided, comprising the following components: ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, and MAPK11. Primers or probes specifically hybridizing to at least two target genes selected from the group consisting of NEK11, NR3C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB.

[0008] Furthermore, this invention provides a customized medicine screening method for cancer patients, which includes the following steps: 1) In biological samples isolated from cancer patients, the following components were measured: ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK11. The steps involve expressing at least two target genes selected from the group consisting of NR3C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB; and 2) The step of screening for drugs that act on the target genes that were determined to be highly expressed in step 1).

[0009] Furthermore, this invention provides a customized medicine screening method for cancer patients, which includes the following steps: 1) The steps of isolating and proliferating cancer cells and normal cells from cancerous tissue and normal tissue isolated from cancer patients; 2) The step of treating the cancer cells of step 1) with a cancer treatment candidate agent; 3) The step of isolating total RNA from the cancer cells and normal cells of step 1) and the cancer cells treated with the cancer treatment candidate drug of step 2), and synthesizing cDNA using the total RNA as a template; 4) Using the cDNA synthesized in step 3) as a template, and utilizing the cDNA derived from ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK11, NR3C1, ... The steps involve amplifying the target genes by using primers or probes specifically hybridizing to at least two target genes selected from the group consisting of NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB; and... 5) After determining the expression level of the target gene amplified in step 4), compare the expression levels of the target gene in normal cells, cancer cells, and cancer cells treated with cancer therapy candidate agents.

[0010] Invention Effects In this invention, quantitative real-time PCR (qPCR) is used to quantify and analyze the expression changes of 87 genes that are targets of FDA-approved cancer therapeutics, as well as the mutations in some of these genes. This allows for highly accurate and convenient profiling of expression patterns in various cancer patients, thus providing information for customized drug screening for cancer patients. Therefore, this invention, by selectively administering existing or future cancer therapeutics to cancer patients, can be used for personalized treatment. Attached Figure Description

[0011] Figure 1a and Figure 1bAccording to an embodiment of the present invention, qPCR was performed on normal tissue and cancer tissue samples collected from three patients with renal cell carcinoma to confirm the expression patterns of 87 target genes. The first N and T are normal tissue (N) and cancer tissue (T) samples from patient No. 1 with renal cell carcinoma, the second N and T are normal tissue (N) and cancer tissue (T) samples from patient No. 2 with renal cell carcinoma, and the third N and T are normal tissue (N) and cancer tissue (T) samples from patient No. 3 with renal cell carcinoma.

[0012] Figure 2a and Figure 2b This is a graph showing the expression patterns of 87 target genes confirmed by qPCR performed on the normal human kidney cell line HK-2 and the renal cancer cell lines Caki-1, Caki-2 and ACHN. The first bar represents the HK-2 cell line, the second bar represents the Caki-1 cell line, the third bar represents the Caki-2 cell line, and the fourth bar represents the ACHN cell line.

[0013] Figure 3 This is a graph showing the cell viability of renal cell carcinoma lines Caki-1, Caki-2, and ACHN after treatment with different concentrations of regorafenib.

[0014] Figure 4 This is a graph showing the size of the tumor after administration of regorafenib to a mouse model of ACHN renal cell carcinoma transplanted tumors.

[0015] Figure 5 According to an embodiment of the present invention, qPCR was performed on normal tissue and ascites samples collected from a gastric cancer patient exhibiting symptoms of malignant ascites to confirm the expression patterns of 87 target genes. The first bar chart represents the ascites sample, and the second to sixth bars represent the normal tissue samples.

[0016] Figure 6 This is a graph illustrating the cell viability of cancer cells derived from ascites samples collected from a gastric cancer patient exhibiting symptoms of malignant ascites, after treatment with different concentrations of carfilzomib, or different concentrations of ramucirumab and paclitaxel. Detailed Implementation

[0017] The present invention will now be described in more detail.

[0018] This invention provides a customized drug screening kit for cancer patients, comprising the following components: ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK11. Primers or probes specifically hybridizing to at least two target genes selected from the group consisting of NR3C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB.

[0019] In this invention, the target genes may include those derived from ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, and I. FNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK 11. NR3C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD 1. Select at least 3 or 4 from the group consisting of PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB. The number of targets may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45, or may include all of the targets mentioned, but is not limited thereto.

[0020] In this invention, the kit can be a kit for quantitative real-time PCR.

[0021] The term "primer" refers to a nucleic acid sequence with a short free 3′ hydroxyl group that can form a base pair with a complementary template and function as the starting point for template strand replication. In other words, a primer is a single-stranded oligonucleotide that, under appropriate conditions in a suitable buffer (e.g., in the presence of four different nucleosides and polymerizing agents such as DNA and DNA polymerase) and at a suitable temperature, can initiate template-directed DNA synthesis.

[0022] The primers of this invention can be chemically synthesized using the phosphoramide solid-phase carrier method or other widely known methods. Furthermore, these primers can be modified using various techniques known in the art (e.g., addition, deletion, substitution) without affecting the detection of the target gene, and do not need to be perfectly complementary to the template, but should have sufficient complementarity for hybridization. Non-limiting examples of such modifications include methylation, end-capping, substitution with homologues of one or more natural nucleotides, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonates, triphosphates, aminophosphates, carbamates, etc.) or charged linkers (e.g., thiophosphates, dithiophosphates, etc.). The nucleic acid may contain one or more additional covalently bound residues, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalating agents (e.g., acridine, psoralen, etc.), chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequences of this invention can also be modified using markers that can directly or indirectly provide a detectable signal. Examples of labels include: radioactive isotopes, fluorescent molecules, biotin, etc.

[0023] The term "probe" refers to a linear oligomer of natural or modified monomers or linkages containing deoxyribonucleotides and ribonucleotides, capable of specifically hybridizing with a target nucleotide sequence, and which is either naturally occurring or artificially synthesized.

[0024] The nucleotide sequence of the target of this invention, which should be referenced when preparing the primers or probes, can be confirmed in GenBank, and primers or probes can be designed with reference to this sequence.

[0025] For example, the primer sets shown in Table 1 below can be used as primer sets for specific hybridization with each target gene of the present invention. Specifically, they can be used for quantitative real-time PCR.

[0026]

[0027] In this invention, the cancer can be kidney cancer, stomach cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, lymphoma, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, thyroid cancer, parathyroid cancer, ureteral cancer, or blood cancer, specifically kidney cancer, but not limited to these.

[0028] Furthermore, the cancer patients may have symptoms of advanced cancer, regardless of the type of cancer, such as ascites or pleural effusion, but are not limited to these.

[0029] In this invention, the kit may contain DNA polymerase, dNTPs, buffers, etc., for performing PCR amplification reactions. The kit may further include a user guide outlining optimal reaction conditions. The guide is a printed document explaining how to use the kit, such as methods for preparing reverse transcription buffer and PCR buffer, and suggested reaction conditions. The guide may include instructions in the form of a booklet or leaflet, a label attached to the kit, and instructions on the surface of the packaging containing the kit. Furthermore, the guide may include information publicly available or provided through electronic media such as the internet.

[0030] Furthermore, this invention provides a customized medicine screening method for cancer patients, which includes the following steps: 1) In biological samples isolated from cancer patients, the following components were measured: ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK11. The steps involve expressing at least two target genes selected from the group consisting of NR3C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB; and 2) The step of screening for drugs that act on the target genes that were determined to be highly expressed in step 1).

[0031] In the method of the present invention, the biological sample in step 1) includes a variety of biological samples, specifically blood, serum, plasma, tissue, cells, lymph, bone marrow fluid, saliva, urine, feces, eye discharge, semen, brain extract, cerebrospinal fluid, synovial fluid, pleural fluid, ascites or amniotic fluid, more specifically tissue or cells, and even more specifically cancerous tissue or cancer cells.

[0032] In the method of the present invention, the target genes in step 1) may include those derived from ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, and IFNA. R1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK11, NR3C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PS At least three or four of the following groups are selected from: MD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB. The number of target genes may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45, or may include all of the stated targets, but is not limited thereto.

[0033] In the method of the present invention, the gene expression assay in step 1) can be performed according to the quantitative real-time PCR method.

[0034] In the method of this invention, the medicine acting on the target gene in step 2) can be a cancer therapeutic agent approved by the US FDA or the European EMA and used in clinical practice, but is not limited thereto. For example, a cancer therapeutic agent approved by the US FDA and used in clinical practice is described in The Author(s) BMC Systems Biology 2017, 11(Suppl 5):87.

[0035] Meanwhile, this invention provides a customized drug screening method for cancer patients, which includes the following steps: 1) The steps of isolating and proliferating cancer cells and normal cells from cancerous tissue and normal tissue isolated from cancer patients; 2) The step of treating the cancer cells of step 1) with a cancer treatment candidate agent; 3) The step of isolating total RNA from the cancer cells and normal cells of step 1) and the cancer cells treated with the cancer treatment candidate drug of step 2), and synthesizing cDNA using the total RNA as a template; 4) Using the cDNA synthesized in step 3) as a template, and utilizing the cDNA derived from ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK11, NR3C1, ... The steps involve amplifying the target genes by using primers or probes specifically hybridizing to at least two target genes selected from the group consisting of NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB; and... 5) After determining the expression level of the target gene amplified in step 4), compare the expression levels of the target gene in normal cells, cancer cells, and cancer cells treated with cancer therapy candidate agents.

[0036] In the method of the present invention, in step 1), the method of separating cancer cells and normal cells from cancer tissue and normal tissue separated from cancer patients can be any method known in the art. For example, cancer tissue or normal tissue can be decomposed using tissue-degrading enzymes or mechanical methods, and cancer cells decomposed by tissue decomposition can be separated according to cell size, density or surface characteristics using density gradient separation method, separation using cell sorting instrument, magnetic separation method, etc.

[0037] In the method of this invention, the cancer treatment candidate agent in step 2) includes any substance, molecule, element, compound, entity, or combination thereof. For example, although not limited thereto, it includes proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, etc. Furthermore, it can be a natural product, synthetic compound, or chemical compound, or a combination of two or more substances. Specific examples include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines, oligocarbamates, saccharides, fatty acids, purines, pyrimidines, or their derivatives, structural analogs, or combinations thereof, and can be synthetic substances; other candidate agents can be natural substances.

[0038] In the method of the present invention, the method for separating total RNA in step 3) can be any method known in the art, for example, phenol extraction method.

[0039] In the method of the present invention, step 4) may include processing data from ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1 , IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NE K11, NR3C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD 1. At least 3 or 4 of the following groups are selected from: PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB. The number of target genes may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45, or may include all of the target genes, but is not limited thereto.

[0040] In addition, the primers or probes in step 4) can be primers or probes for quantitative real-time PCR.

[0041] In the method of the present invention, candidate drugs that meet the following conditions can be screened as customized medicines for cancer patients: in step 5), the expression level of the amplified target gene is increased in cancer cells compared with normal cells, and decreased in cancer cells treated with cancer treatment candidate drugs.

[0042] The present invention will now be described in detail through examples.

[0043] However, the following embodiments are only for illustrating the present invention, and the content of the present invention is not limited to the following embodiments.

[0044] <Example 1> Preparation of samples from renal cell carcinoma patients Normal and cancerous tissue samples were obtained from the specimen bank of the Department of Pathology, Wonju Christian Hospital, Yonsei University, from three patients with renal cell carcinoma. Three pairs of normal and cancerous tissue samples were placed in cryo-tubes and frozen at -70°C before RNA extraction. Total RNA extraction was performed using the commercially available RNeasy Midi Kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer's instructions. The quantity and quality of the extracted total RNA were evaluated using ultraviolet spectrophotometry (DU 530, Beckmann, USA).

[0045] 2 μg of extracted total RNA was reverse transcribed in 20 μl of 2 units of DNase I (4.2 μM MgCl2). The reaction solution consisted of 2 μg total RNA, 3.68 μl of 50 mM MgCl2, and 0.96 μl of DNase I, and the volume was adjusted to 20 μl using DEPC-water.

[0046] For the samples treated with DNAse I, reverse transcription was performed using the Superscript II Reverse Transcription Kit (Invitrogen CAT# 18064-071) under the following conditions: 20 μl of 5X First Strandbuffer, 10 μl of 100 mM DTT, 20 μl of 10 mM dNTPs, 5 μl of pdN6 (1.6 μg / μl), 0.5 μl of RTase (200 U / μl), 20 μl of RNA, and 24.5 μl of DEPC-water. The total reaction volume was 100 μl.

[0047] The reverse transcription reaction was carried out at the following temperatures: 25℃ for 10 min, 42℃ for 50 min, 72℃ for 10 min, and held at 4℃.

[0048] After the reverse transcription reaction was completed, cDNA was adjusted to 5 ng / μl using DEPC-water.

[0049] <Example 2> Quantitative Real-Time PCR Analysis for Customized Drug Screening in Cancer Patients In order to conduct customized drug screening for cancer patients, using the cDNA obtained in Example 1 above as a template, the mRNA expression profiles of 87 target genes were completed by quantitative real-time PCR (qPCR) using primers for the 87 target genes listed in Table 1 below.

[0050] Specifically, from the therapeutic targets of approximately 300 cancer therapeutic agents approved by the US FDA or European EMA, 87 molecular biological targets suitable for the present invention were screened. These cancer therapeutic agents include, for example, renal cell carcinoma therapeutic agents, such as the mTOR inhibitor everolimus, and qPCR primers for these targets were designed as shown in Table 1 below.

[0051] Table 1

[0052] Using the primers described above, qPCR reaction solution was prepared according to the composition shown in Table 2 below. 10 μl of the qPCR reaction solution was aliquoted into 384-well plates, and qPCR was performed using an ABI 7900 HT instrument under the conditions shown in Table 3 below. Gene content was quantified using SDS 2.4 software.

[0053] Table 2

[0054] Table 3

[0055] <Experimental Example 1> Target gene expression profiles of renal cell carcinoma patients obtained by qPCR analysis Using normal and cancerous tissue samples collected from three renal cell carcinoma patients in Example 1 above, qPCR was performed according to the method described in Example 2 above to complete the expression profiles of 87 target genes. Figure 1a and Figure 1b ).

[0056] The result, such as Figure 1a and Figure 1bAs shown, the expression patterns of 87 target genes differed between normal and cancerous tissues in patients with renal cell carcinoma.

[0057] The above results indicate that even in the same type of renal cell carcinoma, the expression of target genes for cancer therapy differs among different patients. Based on this, it is possible to understand the individual expression patterns of each patient, thereby achieving effective treatment.

[0058] <Experimental Example 2> Expression profiles of target genes in various renal cell carcinomas obtained through qPCR analysis and customized cancer drug screening. <2-1> Expression profiles of target genes in various renal cancer cells obtained by qPCR analysis Using three renal cancer cell lines, Caki-1, Caki-2, and ACHN cells, samples were prepared according to the same method described in Example 1 above, and qPCR was performed according to the method described in Example 2 above to complete the expression profiles of 87 target genes. Figure 2a and Figure 2b As a control group, the HK-2 cell line, derived from normal human kidney tissue, was used.

[0059] The result, such as Figure 2a and Figure 2b As shown, the expression patterns of 87 target genes differed across the three renal cell carcinoma cell lines. Specifically, for DDR2, its expression was confirmed to be significantly higher in the Caki-1 cell line, while its expression was not high in the Caki-2 and ACHN cell lines.

[0060] <2-2> In vitro and in vivo customized cancer drug screening and confirmation In order to screen customized cancer drugs based on gene expression patterns, the three renal cell carcinoma cell lines of Example <2-1> were treated with anticancer agents, and the sensitivity to the anticancer agents was evaluated.

[0061] Specifically, in 96-well plates, three renal cell carcinoma lines—Caki-1, Caki-2, and ACHN—were seeded at 4,000–5,000 cells per well. After 24 hours of culture, the cells were treated with regorafenib, an FDA-approved treatment for metastatic renal cell carcinoma, at concentrations ranging from 0 to 100 μM. After 72 hours, the number of surviving cells was quantified using SRB (Sulforhodamine B) dye. Figure 3 ).

[0062] The result, such as Figure 3As shown, Caki-1 cells with high DDR2 expression were confirmed to be less sensitive to regorafenib, while Caki-2 and ACHN cell lines with low DDR2 expression were more sensitive to regorafenib.

[0063] Furthermore, the sensitivity to regorafenib was evaluated in a mouse model of tumor transplantation using ACHN cell line.

[0064] Specifically, animal experiments were conducted in accordance with the National Health Guidelines for the Management and Use of Laboratory Animals and were approved by the Institutional Animal Management and Use Committee of Yonsei University Wonju Christian Hospital (Approval No. YWC-190917-3). The tumor model used in the experiments was established as follows: 5 × 10⁶ tumor cells were placed in the tumor cells... 6 ACHN cell lines were prepared in syringes and then subcutaneously injected into the right shoulder of 5-week-old male mice (BALB / c-nude mice (Orient Bio, Inc.)) weighing 20-25g. Two weeks later, when the tumor reached a diameter of 0.5cm, the control group (Vehicle, n=5) received the vehicle, while the experimental group (n=5) received intravenous administration of 10mg / kg regorafenib three times a week for four weeks. Tumor size was then measured. Figure 4 ).

[0065] The result, such as Figure 4 As shown, the excellent cancer treatment effect of regorafenib in the experimental group was confirmed.

[0066] The above results suggest that regorafenib is suitable as an anticancer agent in renal cell carcinoma with low DDR2 expression.

[0067] <Experimental Example 3> Target gene expression profiles of gastric cancer patients obtained through qPCR analysis and customized cancer drug screening <3-1> Target gene expression profiles of gastric cancer patients obtained by qPCR analysis Ascites and normal tissue samples were obtained from the specimen bank of the Department of Pathology, Wonju Christian Hospital, Yonsei University, from a gastric cancer patient exhibiting symptoms of malignant ascites. Then, using these samples, samples were prepared according to the same method described in <Example 1> above, and qPCR was performed according to the method described in <Example 2> above. Profiling analysis was conducted on the five most expressed target genes out of 87 target genes. Figure 5 ).

[0068] The result, such as Figure 5 As shown, the five target genes are PSMB1, PSMB2, PSMB9, CD52 and FCGR1A, which are expressed at high levels, and PSMB1, PSMB2 and PSMB9 have been confirmed as targets of carfilzomib.

[0069] <3-2> In vitro and in vivo customized cancer drug screening and confirmation Gastric cancer cells derived from ascites samples of gastric cancer patients exhibiting symptoms of malignant ascites in Example <3-1> were treated with anticancer agents, and the sensitivity to the anticancer agents was confirmed.

[0070] Specifically, gastric cancer cells from ascites samples of gastric cancer patients exhibiting symptoms of malignant ascites, derived from Example <3-1> above, were seeded at 4,000-5,000 cells per well. After culturing for 24 hours, they were treated for 16 hours with carfilzomib, an overexpression gene-targeting therapeutic agent confirmed in Example <3-1> above. The control group was treated for 24 hours with anticancer agents previously used by gastric cancer patients exhibiting symptoms of malignant ascites, namely ramucirumab and paclitaxel, at a concentration of 0-100 μM. After treatment, the number of surviving cells was quantified using SRB (Sulforhodamine B) dye. Figure 6 ).

[0071] The result, such as Figure 6 As shown, the carfilzomib treatment group demonstrated a significantly better reduction in cell viability compared to the ramucirumab and paclitaxel treatment groups.

[0072] The above results indicate that even for the same type of cancer, the expression of the target genes of the 87 cancer therapeutic agents differs among different patients. Furthermore, by using qPCR to quantify and analyze the changes in their expression and the mutations of some of these genes, it is possible to screen customized cancer drugs for cancer patients with high accuracy and convenience.

[0073] Industrial applicability The method for analyzing the expression patterns of target genes in cancer patients according to the present invention can be used for personalized treatment of cancer patients by providing information that enables the selective administration of existing or future cancer therapeutic agents.

Claims

1. A customized drug screening kit for cancer patients, comprising the following components: ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK11, N... Primers or probes specifically hybridizing to at least two target genes selected from the group consisting of R3C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB.

2. The customized drug screening kit for cancer patients according to claim 1, characterized in that, The target genes include those derived from ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK11, and NR3. At least 10 target genes selected from the group consisting of C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB.

3. The customized drug screening kit for cancer patients according to claim 2, characterized in that, The target genes include those derived from ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK11, and NR3. At least 20 target genes selected from the group consisting of C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB.

4. The customized drug screening kit for cancer patients according to claim 3, characterized in that, The target genes include those derived from ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK11, and NR3. At least 25 target genes selected from the group consisting of C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB.

5. The customized drug screening kit for cancer patients according to claim 4, characterized in that, The target genes include those derived from ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK11, and NR3. At least 35 target genes selected from the group consisting of C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB.

6. The customized drug screening kit for cancer patients according to claim 5, characterized in that, The target genes include those derived from ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK11, and NR3. At least 45 target genes selected from the group consisting of C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB.

7. The customized drug screening kit for cancer patients according to claim 1, characterized in that, The primers that specifically hybridize with each of the target genes are selected from the group consisting of primers numbered 1 to 174.

8. The customized drug screening kit for cancer patients according to claim 1, characterized in that, The kit is a quantitative real-time PCR kit.

9. The customized drug screening kit for cancer patients according to claim 1, characterized in that, The cancers selected are those comprising kidney cancer, stomach cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, lymphoma, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, thyroid cancer, parathyroid cancer, ureteral cancer, and blood cancers.

10. A personalized medicine screening method for cancer patients, comprising the following steps: 1) In biological samples isolated from cancer patients, the following substances were measured: ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, and NEK11. The steps involve expressing at least two target genes selected from the group consisting of NR3C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB. as well as 2) The step of screening for drugs that act on the target genes that were determined to be highly expressed in step 1).

11. The method for customized drug screening for cancer patients according to claim 10, characterized in that, The expression of the gene was determined using a quantitative real-time PCR method.

12. A personalized medicine screening method for cancer patients, comprising the following steps: 1) The steps of isolating and proliferating cancer cells and normal cells from cancerous tissue and normal tissue isolated from cancer patients; 2) The step of treating the cancer cells of step 1) with a cancer treatment candidate agent; 3) The step of isolating total RNA from the cancer cells and normal cells of step 1) and the cancer cells treated with the cancer treatment candidate drug of step 2), and synthesizing cDNA using the total RNA as a template; 4) Using the cDNA synthesized in step 3) as a template, and utilizing the cDNA derived from ACPP, ADA, ALK, BCR-ABL-1, BTK, CDK4, CDK6, CD19, CD3D, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GNRH1, GNRHR, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCGR, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, NEK11, NR3C1 The steps involve using primers or probes to specifically hybridize at least two target genes selected from the group consisting of NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRLR, PSMB10, PSMB1, PSMB2, PSMB8, PSMB9, PSMD1, PSMD2, PTK6, RARA, RARB, RARG, RPL3, SH2B3, SIK1, SLC2A2, SMO, SSTR2, SSTR5, TEK, TLR8, TNFSF8, TNFSF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA, and VEGFB to amplify the target genes. as well as 5) After determining the expression level of the target gene amplified in step 4), compare the expression levels of the target gene in normal cells, cancer cells, and cancer cells treated with cancer therapy candidate agents.

13. The method for customized drug screening for cancer patients according to claim 12, characterized in that, The primers or probes are primers or probes used for quantitative real-time PCR.

14. The method for customized drug screening for cancer patients according to claim 12, characterized in that, In step 5), candidate agents that meet the following criteria are screened: the expression level of the amplified target gene is increased in cancer cells compared to normal cells; and the expression level is reduced in cancer cells treated with the cancer treatment candidate agent compared to normal cells.

Images