Method for individualized cancer treatment for cancer patients using digital PCR

Abstract

The present invention relates to a method for individualized cancer treatment of cancer patients using digital PCR. As a result of quantitatively analyzing the expression changes of 134 target genes using digital PCR, it was confirmed that there are differences in the expression patterns of target genes among cancer patients. In addition, it was found that this method enables simpler and more accurate analysis of expression patterns compared to qPCR. Therefore, the method for analyzing the target gene expression pattern of cancer patients according to the present invention can provide information for selectively administering existing or future-developed cancer therapeutics to individual cancer patients, and can facilitate individualized treatment for each patient.

Description

Technical Field

[0001] The present invention relates to an individualized cancer treatment method for cancer patients using digital PCR, and more specifically, to an individualized therapeutic drug screening method for cancer patients using digital PCR and a kit using the same.

Background Art

[0002] Cancer is a disease that threatens human health and life and accounts for approximately 13% of the total number of deaths. In 2007, 7.6 million people died of cancer worldwide. In the United States, it has been reported that in recent years, an estimated 1.4 million people develop new cancers every year, and cancer ranks second as a cause of death. According to the statistics of the SEER (Surveillance, Epidemiology, and End Results) report, the mortality rate of 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, and then steadily decreased to 184.0 per 100,000 people in 2005. This decreasing trend is considered to be due to the fact that early detection of cancer has become possible due to the improvement of diagnostic techniques. Early detection and early treatment play important roles in the prognosis and survival of all types of cancer.

[0003] There are approximately 300 types of cancer therapeutic drugs approved for clinical use by the US FDA (Food and Drug Administration) and the European EMA (European Medicines Agency). These cancer therapeutic drugs target specific biomarkers and are approved as indications for at least one type of cancer.

[0004] In conventional cancer chemotherapy, instead of treating each cancer patient individually, an appropriate anticancer drug is selected and administered according to the type and severity of cancer. However, clinically, it has generally been shown that the therapeutic effects of such anticancer chemotherapy vary greatly among patients, and various methods have been proposed to overcome this limitation.

[0005] On the one hand, digital PCR methods that enable absolute quantification with a small amount of gene samples have attracted attention in recent years and are in the stage of technical optimization for application to molecular diagnosis. The advantage of digital PCR methods that can achieve absolute quantification with only a few picograms (pg) or nanograms (ng) of genetic material is highly suitable for the side of gene copy number. As a result, copy number analysis methods using digital PCR for various disease-related genes have been continuously developed (Patent Document 1).

[0006] Therefore, as a result of the inventors' efforts to develop a method for simply and accurately selecting a therapeutic drug suitable for each cancer patient, by quantifying and analyzing the expression changes of 134 genes targeted by cancer therapeutic drugs approved by the US FDA using digital PCR methods, it was found that the expression patterns specific to cancer patients can be profiled with high precision and simply. This enables the selection of personalized medicine for cancer patients and led to the present invention.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a method for more efficiently performing chemotherapy on cancer patients, specifically, a kit for screening personalized therapeutic drugs for cancer patients using cancer therapeutic drug targets and its use.

Means for Solving the Problems

[0009] To achieve the object of the present invention, the present invention provides a kit for screening an individualized therapeutic agent for cancer patients, comprising: primers or probes that specifically hybridize to each of the target genes ADA and CD52; and primers or probes that specifically hybridize to each of at least 5 target genes selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ.

[0010] In addition, the present invention 1) a step of measuring the expression of at least 5 target genes selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ, essentially including ADA and CD52, in a biological sample isolated from a cancer patient; and 2) a step of screening for a drug that acts on the target genes measured as highly expressed in step 1); and provides a method for screening an individualized therapeutic agent for cancer patients.

[0011] Furthermore, the present invention 1) a step of separating and growing cancer cells and normal cells from cancer tissue and normal tissue separated from a cancer patient; 2) Treating the cancer cells in step 1) with a candidate anti-cancer agent; 3) Separating total RNA from the cancer cells and normal cells in step 1) and the cancer cells treated with the candidate anti-cancer agent in step 2), and synthesizing cDNA using the total RNA as a template; 4) Using the cDNA synthesized in step 3) as a template, primers or probes that specifically hybridize to each of the target genes ADA and CD52; and primers or probes that specifically hybridize to each of at least 5 target genes selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ; generating a plurality of droplets from the reaction solution containing the primers or probes; 5) Amplifying the target nucleic acid sequences in the droplets generated in step 4); and 6) Determining the amount of the amplified target nucleic acid by counting the number of droplets containing the target nucleic acid amplified in step 5), and comparing the amounts of the amplified target nucleic acid in normal cells, cancer cells and cancer cells treated with the candidate anti-cancer agent; providing a method for screening individualized therapeutic drugs for cancer patients.

Advantages of the Invention

[0012] The present invention provides a simple and accurate analysis for screening personalized medicine for cancer patients, which quantifies the expression changes of 134 cancer treatment target genes between normal tissues and cancer tissues in cancer patients using digital PCR and confirms the differences in the expression patterns of target genes among cancer patients. Thus, the present invention suggests that it can provide information for screening personalized therapeutic agents for cancer patients. Therefore, by selectively administering existing or future-developed cancer therapeutic agents to individual patients, it can be used for personalized treatment.

Brief Description of the Drawings

[0013]

Figure 1A

Figure 1C

Figure 2

Figure 3

Best Mode for Carrying Out the Invention

[0014] Hereinafter, the present invention will be described in more detail.

[0015] The present invention provides a kit for screening individualized therapeutic agents for cancer patients, comprising primers or probes that specifically hybridize to each of the target genes ADA and CD52; and primers or probes that specifically hybridize to each of at least 5 target genes selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ.

[0016] In the present invention, the targets essentially include ADA and CD52, and further may include at least 10, 15, 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 targets selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ, or may include all of the above targets, but is not limited thereto.

[0017] In addition, the present invention provides a kit for screening individualized therapeutic agents for cancer patients, further comprising primers or probes that specifically hybridize to each of at least 10 target genes selected from the group consisting of target genes ACPP, ADA, ALK, BCR-ABL-1, BTK, CD19, CD30, 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, MET, 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.

[0018] In the present invention, the target may include at least 10, 15, 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, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or 85 targets selected from the group consisting of ACPP, ADA, ALK, BCR-ABL-1, BTK, CD19, CD30, 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, MET, 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, or may include all of the above targets, but is not limited thereto.

[0019] In the present invention, the kit may be a kit for digital PCR (digitalPCR), specifically, it may be a kit for droplet digital PCR.

[0020] The term "digital PCR (digital polymerase chain reaction)" refers to a new approach for detecting and quantifying nucleic acids, enabling more accurate quantification and highly sensitive detection of target nucleic acid molecules compared to conventional qPCR. In conventional qPCR, an analog-based approach is used for result analysis, while digital PCR employs a digital method that generates signal values of "0" or "1", allowing for the analysis of a large number of samples, simultaneous testing of multiple samples, and simultaneous execution of various test items. The digital PCR technique does not require a standard curve for DNA samples and enables absolute quantification by applying the single-molecule counting method. This technique enables more accurate absolute quantification because the PCR reaction is performed in one droplet per well.

[0021] The term "primer" refers to a short nucleic acid sequence with a free 3'-hydroxyl group that can form base pairs with a complementary template and functions as an initiation point for replication of the template strand. In other words, a primer is a single-stranded oligonucleotide that can initiate template-directed DNA synthesis in an appropriate buffer (e.g., in the presence of four different nucleoside triphosphates, DNA, and a polymerase enzyme such as DNA polymerase) at an appropriate temperature and under appropriate conditions.

[0022] The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method or other widely known methods. Also, this primer can be modified (e.g., addition, deletion, substitution) using a number of techniques well known in the art, as long as it does not affect the detection of the target gene. The primer need not be completely complementary to the template, but sufficient complementarity is required to allow hybridization with the template. Non-limiting examples of such modifications include methylation, capping, substitution with one or more congeners of natural nucleotides, and modification of the internucleotide linkage. Examples of such linkages include modification with uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.). Nucleic acids may also contain one or more additional covalently attached moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalating agents (e.g., acridine, proflavine, etc.), chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.) and alkylating agents. The nucleic acid sequences of the present invention can further be modified with labels that can directly or indirectly provide a detectable signal, examples of such labels include radioisotopes, fluorescent molecules, biotin, etc.

[0023] The term "probe" means a linear oligomer of natural or modified monomers or linkages, including deoxyribonucleotides and ribonucleotides, which can specifically hybridize to a target nucleotide sequence and may be naturally occurring or artificially synthesized.

[0024] The nucleotide sequence of the target of the present invention to be referred to in the production of the primer or probe can be confirmed in GenBank, and this sequence can be used as a reference for designing the primer or probe.

[0025] For example, a primer set that specifically hybridizes to each of the target genes of the present invention as shown in Tables 1 and 2 below can be used. Specifically, these can be used for digital PCR, more specifically droplet digital PCR.

[0026] Specifically, the primers that specifically hybridize to each of the target genes ADA and CD52 can be selected from the group consisting of the primers shown by SEQ ID NOs: 99, 100, 111, and 112 in Table 2 below; the primers that specifically hybridize to each of the target genes ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ, and RXRγ can also be selected from the group consisting of the primers shown by SEQ ID NOs: 1 to 96 in Table 1 below. More specifically, each of the primers that specifically hybridize to each of the target genes ADA and CD52 may be a primer set consisting of the nucleotide sequences of SEQ ID NOs: 99 and 100; and a primer set consisting of the nucleotide sequences of SEQ ID NOs: 111 and 112, and each of the primers that specifically hybridize to the target genes ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ, and RXRγ is a primer set consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2; a primer set consisting of the nucleotide sequences of SEQ ID NOs: 3 and 4; a primer set consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6; a primer set consisting of the nucleotide sequences of SEQ ID NOs: 7 and 8; a primer set consisting of the nucleotide sequences of SEQ ID NOs: 9 and 10; a primer set consisting of the nucleotide sequences of SEQ ID NOs: 11 and 12;A primer set consisting of the base sequences of SEQ ID NOs: 13 and 14; a primer set consisting of the base sequences of SEQ ID NOs: 15 and 16; a primer set consisting of the base sequences of SEQ ID NOs: 17 and 18; a primer set consisting of the base sequences of SEQ ID NOs: 19 and 20; a primer set consisting of the base sequences of SEQ ID NOs: 21 and 22; a primer set consisting of the base sequences of SEQ ID NOs: 23 and 24; a primer set consisting of the base sequences of SEQ ID NOs: 25 and 26; a primer set consisting of the base sequences of SEQ ID NOs: 27 and 28; a primer set consisting of the base sequences of SEQ ID NOs: 29 and 30; a primer set consisting of the base sequences of SEQ ID NOs: 31 and 32; a primer set consisting of the base sequences of SEQ ID NOs: 33 and 34; a primer set consisting of the base sequences of SEQ ID NOs: 35 and 36; a primer set consisting of the base sequences of SEQ ID NOs: 37 and 38; a primer set consisting of the base sequences of SEQ ID NOs: 39 and 40; a primer set consisting of the base sequences of SEQ ID NOs: 41 and 42; a primer set consisting of the base sequences of SEQ ID NOs: 43 and 44; a primer set consisting of the base sequences of SEQ ID NOs: 45 and 46; a primer set consisting of the base sequences of SEQ ID NOs: 47 and 48; a primer set consisting of the base sequences of SEQ ID NOs: 49 and 50; a primer set consisting of the base sequences of SEQ ID NOs: 51 and 52; a primer set consisting of the base sequences of SEQ ID NOs: 53 and 54; a primer set consisting of the base sequences of SEQ ID NOs: 55 and 56; a primer set consisting of the base sequences of SEQ ID NOs: 57 and 58; a primer set consisting of the base sequences of SEQ ID NOs: 59 and 60; a primer set consisting of the base sequences of SEQ ID NOs: 61 and 62; a primer set consisting of the base sequences of SEQ ID NOs: 63 and 64; a primer set consisting of the base sequences of SEQ ID NOs: 65 and 66; a primer set consisting of the base sequences of SEQ ID NOs: 67 and 68; a primer set consisting of the base sequences of SEQ ID NOs: 69 and 70; a primer set consisting of the base sequences of SEQ ID NOs: 71 and 72; a primer set consisting of the base sequences of SEQ ID NOs: 73 and 74; a primer set consisting of the base sequences of SEQ ID NOs: 75 and 76; a primer set consisting of the base sequences of SEQ ID NOs: 77 and 78; a primer set consisting of the base sequences of SEQ ID NOs: 79 and 80; a primer set consisting of the base sequences of SEQ ID NOs: 81 and 82;A primer set consisting of the nucleotide sequences of SEQ ID NOs: 83 and 84; a primer set consisting of the nucleotide sequences of SEQ ID NOs: 85 and 86; a primer set consisting of the nucleotide sequences of SEQ ID NOs: 87 and 88; a primer set consisting of the nucleotide sequences of SEQ ID NOs: 89 and 90; a primer set consisting of the nucleotide sequences of SEQ ID NOs: 91 and 92; a primer set consisting of the nucleotide sequences of SEQ ID NOs: 93 and 94; and a primer set consisting of the nucleotide sequences of SEQ ID NOs: 95 and 96 may also be used.;

[0027]

[0028] In the present invention, the cancer may be renal cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain tumor, prostate cancer, osteosarcoma, head and neck cancer, skin cancer, thyroid cancer, parathyroid cancer, ureteral cancer or blood cancer, and specifically may be renal cancer, but is not limited thereto.

[0029] In addition, the cancer patient may show symptoms of advanced cancer such as ascites or pleural effusion, regardless of the type of cancer, but is not limited thereto.

[0030] In the present invention, the kit contains a DNA polymerase, dNTPs, a buffer, etc. for performing a PCR amplification reaction. The kit may further contain a user guide that details the optimal reaction conditions. The guide is a printed material that explains the usage method of the kit, such as the manufacturing methods of the reverse transcription buffer and the PCR buffer, and the presented reaction conditions. The form of the guide is a pamphlet or leaflet, a label attached to the kit, or an explanation described on the surface of the package containing the kit. Furthermore, the guide can include information disclosed or provided through an electronic medium such as the Internet.

[0031] In the present invention, digital PCR was used to quantify the expression changes of 134 cancer treatment target genes between normal tissues and cancer tissues of cancer patients, and the differences in the expression patterns of cancer treatment drug targets among individual cancer patients were clarified. Also, it was confirmed that digital PCR enables more accurate analysis compared to qPCR. Therefore, the expression profiling of the 134 cancer treatment target genes by digital PCR according to the present invention can be utilized for the selection of individualized cancer treatment drugs for cancer patients.

[0032] In addition, the present invention 1) Measuring the expression of at least 5 target genes selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ in a biological sample isolated from a cancer patient; and 2) Screening for a drug that acts on the target genes measured as highly expressed in step 1); A method for screening an individualized therapeutic agent for a cancer patient is provided.

[0033] In the method of the present invention, the biological sample in step 1) includes various biological samples, specifically, blood, serum, plasma, tissue, cells, lymph, bone marrow fluid, saliva, urine, feces, eye drops, semen, brain extract, cerebrospinal fluid, synovial fluid, thymus fluid, ascites or amniotic fluid, more specifically, tissue or cells, and even more specifically, cancer tissue or cancer cells.

[0034] In the method of the present invention, the target gene essentially includes ADA and CD52 in step 1), and may include at least 10, 15, 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 target genes selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ, or may include all of the above target genes, but is not limited thereto.

[0035] Also, in the above step 1), it may further include at least 10 target genes selected from the group consisting of ACPP, ADA, ALK, BCR-ABL-1, BTK, CD19, CD30, 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, MET, 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. Specifically, it may include at least 10, 15, 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, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or 85 targets, or may include all of the above target genes, but is not limited thereto.

[0036] In the method of the present invention, the measurement of gene expression in the above step 1) can be performed according to the digital PCR method. Specifically, it may be carried out using the droplet digital PCR method.

[0037] In the method of the present invention, the medicament acting on the target gene in step 2) may be, but is not limited to, an anticancer agent approved for clinical use by the US FDA or the European EMA. For example, the anticancer agents approved for clinical use by the US FDA are described in "The Author(s) BMC Systems Biology 2017, 11(Suppl 5):87".

[0038] More specifically, the screening method for individualized therapeutic drugs for cancer patients using the droplet digital PCR method can be carried out through the following steps: a) A step of separating cancer tissue and normal tissue from a cancer patient; b) A step of separating total RNA from the separated cancer tissue and normal tissue and synthesizing cDNA using the total RNA as a template; c) Using the synthesized cDNA as a template, primers or probes that specifically hybridize to each of the target genes ADA and CD52; and at least five target genes selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ, and RXRγ A step of generating a plurality of droplets from a reaction solution containing primers or probes that specifically hybridize to each of them; d) A step of amplifying the target nucleic acid sequence in the generated droplets; e) By counting the number of droplets containing the amplified target nucleic acid, determining the amount of the amplified target nucleic acid, comparing the amount of the amplified target nucleic acid in the normal tissue and the cancer tissue, and selecting the target gene that is highly expressed in the cancer tissue compared to the normal tissue; and f) screening a medicament that acts on the selected target gene.

[0039] In the present invention, digital PCR was used to quantify the expression changes of 134 cancer treatment target genes between normal tissues and cancer tissues of cancer patients, and the differences in the expression patterns of cancer treatment drug targets among individual cancer patients were clarified. Also, it was confirmed that digital PCR enables more accurate analysis than qPCR. Therefore, the measurement of the expression patterns of the 134 cancer treatment target genes by digital PCR of the present invention can be used for the selection of individualized cancer treatment drugs for cancer patients.

[0040] Furthermore, the present invention provides a method for screening an individualized therapeutic drug for a cancer patient, comprising the following steps: 1) separating and proliferating cancer cells and normal cells from cancer tissues and normal tissues isolated from a cancer patient; 2) treating the cancer cells of step 1) with a candidate anticancer agent; 3) separating total RNA from the cancer cells and normal cells of step 1) and the cancer cells treated with the candidate anticancer agent of step 2), and synthesizing cDNA using the total RNA as a template; 4) a primer or probe that specifically hybridizes to each of the target genes ADA and CD52; and a primer or probe that specifically hybridizes to each of at least 5 target genes selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ, and RXRγ; generating a plurality of droplets from the reaction solution containing the same. 5) Amplifying the target nucleic acid sequence in the droplets generated in step 4); and 6) Determining the amount of the amplified target nucleic acid by counting the number of droplets containing the amplified target nucleic acid in step 5), and comparing the amount of the amplified target nucleic acid in normal cells, cancer cells, and cancer cells treated with a candidate anticancer agent.

[0041] In the method of the present invention, any method known in the art can be used as the method for separating cancer cells and normal cells from cancer tissue and normal tissue obtained from a cancer patient in step 1). For example, cancer tissue or normal tissue can be decomposed using a tissue-degrading enzyme or a mechanical method, and the tissue-decomposed cancer cells can be separated based on cell size, density, or surface characteristics using density gradient separation, cell sorters, magnetic separation, etc.

[0042] In the method of the present invention, the candidate anticancer agent in step 2) may include any substance, molecule, element, compound, entity, or a combination thereof. Examples include, but are not limited to, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, etc. Further, it may be a natural product, a synthetic compound, a 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 glycine, oligocarbamates, saccharides, fatty acids, purines, pyrimidines, or derivatives, structural analogs, or combinations thereof, which may be synthetic substances, or another candidate anticancer agent may be a natural product.

[0043] In the method of the present invention, any method known in the art can be used as the method for separating total RNA in step 3), for example, the phenol extraction method can be used.

[0044] In the method of the present invention, in the step 4), it essentially contains ADA and CD52, and further contains at least 10, 15, 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 target genes selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ, or may contain all of the above-listed target genes, but is not limited thereto.

[0045] Also, in the step 4), at least 10, 15, 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, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or 85 target genes selected from the group consisting of ACPP, ADA, ALK, BCR-ABL-1, BTK, CD19, CD30, 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, MET, 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 may be included, or all of the above-listed target genes may be included, but are not limited thereto.

[0046] Furthermore, in the step 4), the primer or probe may be a primer or probe for digital PCR, specifically, it may be a primer or probe for droplet digital PCR. The droplet digital PCR is a system that divides the PCR reaction mixture into 15,000 to 20,000 droplets, performs amplification therein, and then counts the target nucleic acid. Depending on whether the target nucleic acid is amplified in each droplet, the droplets are counted as positive droplets (1) and negative droplets (0) similar to digital signals. The copy number of the target nucleic acid is calculated using the Poisson distribution and finally obtained as the copy number per sample volume (μL).

[0047] Also, the reaction mixture may contain a probe such as FAM, HEX or VIC, or a fluorescent dye such as EvaGreen. These are dispensed into the cartridge together with the oil, set in the droplet generator, and 20,000 droplets can be generated.

[0048] In the method of the present invention, in the step 5), the amplification of the target nucleic acid sequence may be performed by multiplex PCR. Multiplex PCR refers to a method of performing PCR amplification in a single reaction tube using a plurality of primer sets at the same time. For example, by performing multiplex PCR in a single reaction tube using a primer set for the target gene, the gene expression level can be measured simultaneously.

[0049] In the method of the present invention, in the step 6), the PCR plate after the completion of PCR is attached to the droplet reader of digital PCR, and the final copy number of the amplified target nucleic acid can be analyzed by checking and counting the EvaGreen fluorescence value of the droplets via QuantaSoft, but it is not limited thereto.

[0050] Ideally, the label is a fluorescent label, and the fluorescent labeling substance is FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, FITC, IRD-700 / 800, CY3, CY5, CY3.5, CY5.5, HEX, TET, TAMRA, JOE, ROX, bodipy, TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, Yakima Yellow, Alexa Fluor, PET, Biosearch Blue (registered trademark), Marina Blue, Bothell Blue, 350 FAM (registered trademark), SYBR GreenI, fluorescein, EvaGreen (registered trademark), Alexa Fluor 488 JOE (registered trademark), VIC (registered trademark), HEX (registered trademark), TET (registered trademark), CAL Fluor Gold 540, Yakima Yellow, ROX (registered trademark), CAL Fluor Red 610, Cy3.5 (registered trademark), Texas Red, 568 Cy5 (registered trademark), Quasar (registered trademark) 670, LightCycler Red 640, Alexa Fluor 633, Quasar (registered trademark) 705, LightCycler Red 705, Alexa Fluor 680, Cyto9, LC Green or LC Green Plus +, and specifically, it may be FAM, HEX, SYBR Green or EvaGreen, but is not limited thereto.

[0051] The fluorescent labeling substances have different excitation and emission wavelengths depending on their types, and there are also various usage methods. Therefore, when selecting fluorescent labeling substances to be used together in a single PCR reaction, it is necessary to determine whether they can be detected individually and select them accordingly so that different colors can be used. The specific details and selection of the fluorescent labeling substances are obvious to those skilled in the technical field to which the present invention pertains.

[0052] In the method of the present invention, in the step 6), the amount of the amplified target nucleic acid is increased in cancer cells as compared with normal cells, and is decreased in cancer cells treated with a candidate anti-cancer agent as compared with untreated cancer cells, whereby it can be screened as an individualized therapeutic agent for cancer patients.

[0053] In the present invention, digital PCR was used to quantify the expression changes of 134 cancer treatment target genes between normal tissues and cancer tissues of cancer patients, and the differences in the expression patterns of cancer treatment drug targets among individual cancer patients were clarified. In addition, it was confirmed that digital PCR enables more accurate analysis than qPCR. Therefore, by measuring the expression patterns of the 134 cancer treatment target genes by digital PCR according to the present invention, therapeutic agents suitable for individual cancer patients can be screened and utilized for screening individualized therapeutic agents for individual cancer patients.

[0054] Hereinafter, the present invention will be described in detail by way of specific examples.

[0055] However, the following examples illustrate the present invention and do not limit the scope of the present invention in any way.

[0056] Example 1: Preparation of Samples

[0057] Samples of normal tissues and cancer tissues were obtained from three renal cancer patients from the pathology specimen bank of Wonju Severance Christian Hospital. The three sets of normal tissue and cancer tissue samples were stored in cryotubes at -70°C until RNA extraction. Total RNA extraction was performed using a commercially available RNeasy Midi Kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer's protocol. The quantity and quality of the extracted total RNA were evaluated using an ultraviolet spectrophotometer (DU 530, Beckman, USA).

[0058] 2 μg of the extracted total RNA was treated with 2 units of DNAse I (4.2 μM MgCl 2のUnder the following conditions, a reverse transcription reaction was carried out in a volume of 20 μL. The reaction mixture was prepared with the following composition: 2 μg of total RNA, 3.68 μL of 50 mM MgCl2, 0.96 μL of DNAse I, and DEPC water to adjust the volume to 20 μL.

[0059] For the samples treated with DNAse I, a reverse transcription reaction was carried out using the Superscript II g Reverse Transcription Kit (cat♯ 18064 - 071 from Invitrogen) under the following conditions: 20 μL of 5X First Strand buffer, 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 volume of the reaction mixture was adjusted to 100 μL.

[0060] The reverse transcription reaction was carried out under the temperature conditions of 25 °C for 10 minutes, 42 °C for 50 minutes, 72 °C for 10 minutes, and then held at 4 °C.

[0061] After the reverse transcription reaction, the mixture was adjusted to a cDNA concentration of 5 ng / μL using DEPC water.

[0062] Example 2: Digital PCR Analysis for the Selection Panel of Personalized Medicines for Cancer Patients

[0063] For the selection of personalized medicines for cancer patients, using the cDNA obtained in Example 1 as a template, the mRNA expression profiles of 48 targets were completed by droplet digital PCR (ddPCR) method using primers specific to the 48 target genes listed in Table 1 below.

[0064] Furthermore, using primers specific to the 86 target genes listed in Table 2 below, the mRNA expression profiles of 134 targets were completed by the ddPCR method.

[0065] Specifically, a total of 48 and 86 molecular biological targets compatible with the present invention were selected from the treatment targets among approximately 300 cancer therapeutic drugs approved by the US FDA or the European EMA, including kidney cancer therapeutic drugs such as everolimus, an mTOR inhibitor. The ddPCR primers for these targets were designed as shown in Tables 1 and 2 below.

[0066] [Table 1] (Forward: Forward direction, Reverse: Reverse direction) JPEG2025518984000002.jpg244160JPEG2025518984000003.jpg240160JPEG2025518984000004.jpg243160JPEG2025518984000005.jpg47160

[0067] [Table 2] (Forward: Forward direction, Reverse: Reverse direction) JPEG2025518984000006.jpg241160JPEG2025518984000007.jpg241160JPEG2025518984000008.jpg241160JPEG2025518984000009.jpg242160JPEG2025518984000010.jpg242160JPEG2025518984000011.jpg241160JPEG2025518984000012.jpg180160

[0068] Using the above primers, a ddPCR reaction solution with the composition shown in Table 3 below was prepared. 12 μL of the above ddPCR reaction mixture was dispensed into each well of a nanoplate, and ddPCR was performed under the conditions shown in Table 4 below using a QIAcuity One ddPCR system. The gene quantity was quantified using the Quanta software program.

[0069] [Table 3] JPEG2025518984000013.jpg30128

[0070] [Table 4] JPEG2025518984000014.jpg36154

[0071] Comparative Example 1: Quantitative real-time PCR analysis

[0072] Using the cDNA obtained in Example 1 as a template, quantitative real-time PCR (qPCR) analysis was performed using the primers shown in Tables 1 and 2 above.

[0073] Specifically, a qPCR reaction mixture with the composition shown in Table 5 below was prepared. 10 μL of the qPCR reaction mixture was dispensed into 384-well plates, and qPCR was performed using an ABI 7900 HT system under the conditions shown in Table 6 below. The amount of the gene was quantified using the SDS 2.4 software program.

[0074] [Table 5] JPEG2025518984000015.jpg30128

[0075] [Table 6] JPEG2025518984000016.jpg54160

[0076] Experimental Example 1: Target gene expression profile by digital PCR analysis

[0077] Using normal tissue and cancer tissue samples obtained from three renal cancer patients, ddPCR was performed according to the method described in Example 2 above, and the expression profiles of 48 target genes were completed (see Figure 2).

[0078] Also, using a set of normal tissue and cancer tissue samples obtained from the three renal cancer patients (specifically, Samples 3 in Figures 1A - 1C and Figure 2), ddPCR was performed according to the method described in Example 2 above, and the expression profiles of 134 target genes were completed (see Figure 3).

[0079] Furthermore, qPCR was performed according to the method described in Comparative Example 1 using normal tissue and cancer tissue samples obtained from three renal cancer patients, and the expression profiles of 134 target genes were completed (see FIGS. 1A to 1C).

[0080] As a result, as shown in FIGS. 1A to 1C, FIGS. 2 and 3, it was confirmed that there were differences in the expression patterns of 134 target genes between the normal tissue and cancer tissue of renal cancer patients. In particular, when ddPCR and qPCR were performed on 48 target genes, a clearer expression pattern of the target genes was obtained in ddPCR compared to qPCR, and particularly clear expression patterns were observed for genes such as PSMB5, PDFGRβ, and PolB (see FIGS. 1A and 2).

[0081] Furthermore, it was confirmed that by using ddPCR, the expression profiles of 134 target genes in the normal tissue and cancer tissue of renal cancer patients could be obtained. Twenty-three target genes, such as ADA, B-RAF, CD52, DNMT1, HPRT1, PolA, PSMB1, PSMB5, RRM1, RRM2, SRC, TOP2A, TYMS, AR, PARP1, PARP2, CRBN, DDR2, FGFR1, GNRH1, MAP2K2, VEGFB, and TUBB, showed significantly high expression levels, showing a difference of more than five times in cancer tissue compared to normal tissue. In particular, for the target genes ADA and CD52, it was difficult to confirm the expression pattern by qPCR, but a clear expression pattern was obtained by ddPCR (see FIGS. 1A to 1C and FIG. 3).

[0082] The above results indicate that even in the same type of renal cancer, the expression of cancer treatment target genes varies from patient to patient. This suggests that an effective treatment strategy can be developed by understanding the expression pattern of each patient.

[0083] Therefore, the present invention analyzed the expression patterns of 134 cancer treatment target genes between normal tissues and cancer tissues of cancer patients, and confirmed that individualized treatment tailored to individual patients can be achieved by selectively administering existing or future-developed cancer therapeutics to cancer patients based on these expression patterns.

Industrial Applicability

[0084] In the present invention, digital PCR is used to quantify the expression changes of 134 cancer treatment target genes between normal tissues and cancer tissues of cancer patients. By accurately and efficiently analyzing this expression, it was confirmed that there are differences in the expression patterns of target genes among cancer patients. Therefore, by using a kit containing primers or probes specific to the 134 cancer treatment target genes, existing or future-developed cancer therapeutics can be selectively administered to cancer patients, enabling individualized treatment tailored to individual patients.

Claims

1. Primers or probes that specifically hybridize to each of the target genes ADA and CD52; and Primers or probes that specifically hybridize to each of at least 5 target genes selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ; A kit for screening individualized therapeutic agents for cancer patients comprising the same.

2. Wherein the targets essentially include ADA and CD52, The kit for screening individualized therapeutic agents for cancer patients according to claim 1, characterized in that it includes at least 10 targets selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ.

3. Wherein the targets essentially include ADA and CD52, A kit for screening individualized therapeutic agents for cancer patients according to claim 2, characterized by comprising at least 20 targets selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ.

4. The targets essentially include ADA and CD52, A kit for screening individualized therapeutic agents for cancer patients according to claim 3, characterized by comprising at least 25 targets selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ.

5. The targets essentially include ADA and CD52, A kit for screening an individualized therapeutic agent for a cancer patient according to claim 4, characterized by comprising at least 35 targets selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ.

6. The targets essentially include ADA and CD52, A kit for screening an individualized therapeutic agent for a cancer patient according to claim 5, characterized by comprising at least 45 targets selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ.

7. The kit for screening an individualized therapeutic agent for a cancer patient according to claim 1, wherein the primers that specifically hybridize to each of the target genes are selected from the group consisting of the primers represented by SEQ ID NOs: 1 to 96, 99, 100, 111 and 112.

8. A kit for screening an individualized therapeutic agent for a cancer patient according to claim 1, further comprising primers or probes that specifically hybridize to each of at least 10 target genes selected from the group consisting of target genes ACPP, ADA, ALK, BCR-ABL-1, BTK, CD19, CD30, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGFR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GnRH1, GnRH-R, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCG-R, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, MET, 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, TNF-SF8, TNF-SF11, TOP1MT, TOP2B, TUBA1A, TUBA4A, TUBB1, TUBB3, TUBB, TUBD1, TUBE1, TUBG1, VEGFA and VEGFB.

9. A kit for screening an individualized therapeutic agent for a cancer patient according to claim 8, further comprising at least 20 targets selected from the group consisting of ACPP, ADA, ALK, BCR-ABL-1, BTK, CD19, CD30, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGFR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GnRH1, GnRH receptor, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCG receptor, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, MET, NEK11, NR3C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRL receptor, 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.

10. A kit for screening individualized therapeutic agents for cancer patients according to claim 9, further comprising at least 40 targets selected from the group consisting of ACPP, ADA, ALK, BCR-ABL-1, BTK, CD19, CD30, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGFR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GnRH1, GnRH receptor, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCG receptor, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, MET, NEK11, NR3C1, NTRK1, PARP1, PARP2, PARP3, PDCD1, PGF, PIK3CD, PRL receptor, 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.

11. An individualized therapeutic agent screening kit for cancer patients according to claim 10, further comprising at least 60 targets selected from the group consisting of ACPP, ADA, ALK, BCR-ABL-1, BTK, CD19, CD30, CD52, C-MET, CRBN, CTLA4, CYP17A1, DDR2, ERBB4, FCGFR1A, FGF1, FGFR1, FGFR2, FGFR3, FRK, GnRH1, GnRH receptor, HDAC2, HDAC3, HPRT1, IFNAR1, IFNAR2, IL2RA, IL2RB, IL2RG, ITK, JAK1, JAK2, LDLR, LHCG receptor, LIMK1, MAP1A, MAP2, MAP2K1, MAP2K2, MAPK11, MET, 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.

12. The individualized therapeutic agent screening kit for cancer patients according to claim 8, wherein the primers specifically hybridizing to each of the target genes are selected from the group consisting of the primers represented by SEQ ID NOs: 97, 98, 101 to 110, and 113 to 268.

13. The individualized therapeutic agent screening kit for cancer patients according to claim 1, wherein the kit is a digital PCR kit.

14. The individualized therapeutic agent screening kit for cancer patients according to claim 1, wherein the kit is a droplet digital PCR kit.

15. The individualized therapeutic agent screening kit for cancer patients according to claim 1, wherein the cancer is selected from the group consisting of renal cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain tumor, prostate cancer, osteosarcoma, head and neck cancer, skin cancer, thyroid cancer, parathyroid cancer, ureteral cancer and blood cancer.

16. 1) In a biological sample isolated from a cancer patient, measuring the expression of at least 5 target genes that essentially include ADA and CD52 and are selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ and RXRγ; and 2) Screening for a medicament (drug) that acts on the target genes measured as highly expressed in step 1). A method for screening an individualized therapeutic agent for cancer patients, comprising the above steps.

17. The method for screening an individualized therapeutic agent for cancer patients according to claim 16, wherein the measurement of the gene expression is performed using a digital PCR method.

18. The method for screening an individualized therapeutic agent for cancer patients according to claim 17, wherein the measurement of the gene expression is performed using a droplet digital PCR method.

19. 1) Separating and proliferating cancer cells and normal cells from cancer tissue and normal tissue isolated from a cancer patient; 2) Treating the cancer cells in step 1) with a candidate anticancer agent; 3) Separating total RNA from the cancer cells and normal cells in step 1) and the cancer cells treated with the candidate anti-cancer agent in step 2), and synthesizing cDNA using the total RNA as a template; 4) Primers or probes that specifically hybridize to each of the target genes ADA and CD52 using the cDNA synthesized in step 3) as a template; and at least five target genes selected from the group consisting of ABL1, ABL2, ALAD, EGFR, B-RAF, CD20, CD33, CHD1, C-RAF, CSF1R, DHFR, DNMT1, EPHA2, FLT1, FLT3, FLT4, FYN, GARFT, HDAC1, HDAC6, HER2, KDR, Kit, Lck, mTOR, NK1R, PDGFRα, PDGFRβ, PolA, PolB, PSMB5, Ret, RRM1, RRM2, RRM2B, SRC, TLR7, TOP1, TOP2A, TYMS, YES, CYP19A1, AR, ERα, ERβ, RXRα, RXRβ, and RXRγ Generating a plurality of droplets from a reaction solution containing primers or probes that specifically hybridize to each; 5) Amplifying the target nucleic acid sequence in the droplets generated in step 4); and 6) Determining the amount of the amplified target nucleic acid by counting the number of droplets containing the target nucleic acid amplified in step 5), and comparing the amount of the amplified target nucleic acid in normal cells, cancer cells, and cancer cells treated with the candidate anti-cancer agent; An individualized therapeutic drug screening method for cancer patients, comprising:

20. The individualized therapeutic drug screening method for cancer patients according to claim 19, wherein the primer or probe is a primer or probe for digital PCR.

21. The individualized therapeutic drug screening method for cancer patients according to claim 20, wherein the primer or probe is a primer or probe for droplet digital PCR.

22. The amount of the amplified target nucleic acid in step 6) is increased in cancer cells as compared to normal cells and decreased in cancer cells treated with a candidate therapeutic agent as compared to untreated cancer cells, whereby the candidate agent is selected. A method for screening an individualized therapeutic agent for a cancer patient according to claim 19, characterized in that.

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