Biomarker composition for diagnosing metastatic castrate-resistant prostate cancer

Abstract

The present invention is a technique relating to a biomarker enabling the accurate diagnosis or prediction of mCRPC which is the malignant progression stage of mHSPC. According to the present invention, PMSA and PSA genes exhibit specific expression levels in a mCRPC patient group, and thus said genes may be usefully employed as a biomarker enabling the diagnosis or prediction of mCRPC, thereby being capable of greatly contributing to the increasing of the survival rate of patients.

Description

Biomarker composition for diagnosing metastatic castration-resistant prostate cancer

[0001] The present invention relates to a biomarker composition capable of diagnosing metastatic castrate-resistant prostate cancer, which is a malignant stage of metastatic prostate cancer.

[0002] The prostate is a walnut-sized male reproductive organ located just below the bladder and in front of the rectum. It produces and stores a portion of semen. It is anchored anteriorly to the puboprostatic ligament, adjacent to the bladder neck (where the bladder transitions to the urethra), and inferiorly by the urogenital diaphragm. Most prostate cancers are adenocarcinomas (cancers of glandular cells), which arise from prostate cells. Types can be categorized based on the degree of differentiation of the tumor tissue and the characteristics of the cells. Prostate cancer is one of the most common urological tumors worldwide, ranking third after breast and lung cancer. While uncommon before age 50, its incidence increases rapidly after that age. With the recent increase in life expectancy, the number of elderly men in Korea has been rapidly increasing, necessitating early diagnosis and ongoing management of prostate cancer to prevent its progression. Human prostate cancer, in particular, appears to have a propensity to metastasize to bone and is known to inevitably progress from an androgen-dependent state to an androgen-resistant state, increasing patient mortality. Most prostate cancers are initially androgen-dependent (AD). Early-stage prostate cancer cells require androgens for continued proliferation. Surgical (orchiectomy) or medical (GnRH agonist or estrogen) removal of testosterone through androgen deprivation therapy (ADT), or blocking androgen action with anti-androgen agents, induces rapid apoptosis in susceptible prostate cancer cells. The positive response rate is approximately 86%, based on a decrease in prostate-specific antigen (PSA) and stabilization or reduction in tumor volume. Typically, apoptosis occurs during the first few days to a week. However, after the positive response, remaining cells undergo a period of quiescence during which they tend not to die.After 18-36 months of hormone withdrawal or blockade, prostate cancer relapses in 90% of cases. Surviving cancer cells consistently become androgen-independent or non-responsive, and androgen-independent (AI) cell proliferation ensues, leading to hormone-refractory prostate cancer (HRPC) or castration-resistant prostate cancer (CRPC). Because there are currently no effective treatments for CRPC, survival rates are significantly lower. This androgen independence, which leads to CRPC, appears to be driven by various mechanisms. Furthermore, approximately 25% of men treated for prostate cancer experience recurrence, highlighting the need for early diagnosis and effective treatment.

[0003] Meanwhile, a biomarker is an indicator that can detect changes induced within a living organism due to external influences. It is actively being researched for use in diagnosing various diseases or predicting or monitoring the efficacy of specific treatments. Biomarkers include pharmacodynamic markers (PD markers) that confirm whether a drug works in the body, and predictive markers that predict drug responsiveness before administration. The use of these markers helps in establishing clinical drug strategies. For example, predictive markers, which predict efficacy through drug action, can be utilized in the patient selection process to enable more effective drug treatment. Efficacy verification markers can be used to monitor whether a drug is working well in individual patients or whether resistance has been acquired, thereby establishing more effective treatment strategies. Furthermore, even in the absence of efficacy predictive markers, the presence of efficacy verification markers allows for early monitoring of individual patient responses to drugs in drug therapy. This facilitates early identification of those who respond to the drug and those who do not, ultimately leading to more effective and successful drug treatment. Furthermore, efficacy verification markers can be used as indicators for determining appropriate drug dosage by monitoring the degree of drug response according to administered concentration.

[0004] Therefore, there is an urgent need to develop a biomarker that can accurately diagnose metastatic castrate-resistant prostate cancer (mCRPC).

[0005] The purpose of the present invention is to provide a biomarker composition for diagnosing or predicting metastatic castrate-resistant prostate cancer (mCRPC).

[0006] In addition, another object of the present invention is to provide a composition for diagnosing or predicting mCRPC.

[0007] In addition, another object of the present invention is to provide a kit for diagnosing or predicting mCRPC.

[0008] In addition, another object of the present invention is to provide a kit for screening mCRPC inhibitors.

[0009] In addition, another object of the present invention is to provide a method for providing information necessary for diagnosing or predicting mCRPC.

[0010] In addition, another object of the present invention is to provide a method for screening mCRPC inhibitors.

[0011] In order to achieve the above purpose, the present invention provides a biomarker composition for diagnosing or predicting metastatic castrate-resistant prostate cancer (mCRPC), which comprises at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) or a protein encoded by the gene.

[0012] In addition, in order to achieve the other purpose described above, the present invention provides a composition for diagnosing or predicting mCRPC, including a preparation for measuring the expression level of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) at the mRNA or protein level.

[0013] In addition, to achieve the above-described other object, the present invention provides a kit for diagnosing or predicting mCRPC, including a preparation for measuring the expression level of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) at the mRNA or protein level.

[0014] In addition, to achieve the above another object, the present invention provides a kit for screening mCRPC inhibitors, including a preparation for measuring the expression level of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) at the mRNA or protein level.

[0015] In addition, in order to achieve the above another object, the present invention provides a method for providing information necessary for diagnosing or predicting mCRPC, including the steps of: a) confirming the expression level of mRNA or protein of one or more genes selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) in a sample isolated from a test subject; and b) comparing the result with the corresponding result of the corresponding gene in a normal control sample.

[0016] In addition, in order to achieve the above another object, the present invention provides a method for screening an mCRPC inhibitor, including the step of treating a sample with a candidate substance and selecting a candidate substance that suppresses the expression or activity of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA).

[0017] The present invention relates to a biomarker composition for diagnosing or predicting metastatic castrate-resistant prostate cancer (mCRPC), and provides the genes as biomarkers for diagnosing or predicting mCRPC by confirming that the expression levels of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) in circulating tumor cells (CTCs) isolated from a patient are related to the progression from metastatic hormone-sensitive prostate cancer (mHSPC) to mCRPC.

[0018] Figure 1 shows the results of analyzing the genes of each experimental group using a heatmap, classifying the mHSPC patient group into an mCRPC progression experimental group and an mCRPC non-progression experimental group, and showing the number of CTCs isolated from each experimental group.

[0019] Figure 2 shows the results of analyzing the correlation between survival of the mCRPC progression experimental group and the mCRPC non-progression experimental group and the expression of PSMA or PSA mRNA, which are biomarker genes according to the present invention, using a Kaplan-Meier curve.

[0020] Figure 3 shows the results of Kaplan Meier curve analysis of the association between survival in the mCRPC progression group and the mCRPC progression-free group and the expression of known prostate cancer biomarkers AR, AR-V7, KRT19, or CTC#.

[0021] Figure 4 shows the results of analyzing the correlation between survival in the mCRPC progression experimental group and the mCRPC non-progression experimental group and the expression of PSMA or PSA, which are biomarker genes according to the present invention, using the Cox proportional-hazards model.

[0022] Hereinafter, the present invention will be described in more detail.

[0023] The present invention provides a biomarker composition for diagnosing or predicting metastatic castrate-resistant prostate cancer (mCRPC), comprising at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) or a protein encoded by the gene.

[0024] In the present invention, the term “biomarker for diagnosing or predicting mCRPC” refers to a substance capable of diagnosing mCRPC or predicting the possibility of developing the same, and includes organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), whose expression pattern has a significant correlation with the possibility of developing mCRPC. For the purpose of the present invention, the biomarker for diagnosing or predicting mCRPC is a gene PSMA or PSA, which is a gene whose mRNA expression or protein expression level specifically increases in mCRPC. These markers include not only genes but also DNA or mRNA complementary to any one marker, and may be a composite marker including these markers together.

[0025] In the present invention, the metastatic castrate-resistant prostate cancer (mCRPC) refers to metastatic hormone-sensitive prostate cancer (mHSPC) that has progressed to a hormone-refractory state. In addition, the mCRPC may refer to cancer that continues to progress despite rapid reduction of androgens through secondary hormone therapy.

[0026] According to one embodiment of the present invention, it was confirmed that in mCRPC patients, when high expression of PSMA and PSA genes was shown, the prognosis was poor and the survival rate was low.

[0027] Therefore, genetic analysis using the biomarker composition according to the present invention can predict the diagnosis or possibility of developing mCRPC, thereby contributing to increasing patient survival.

[0028] In addition, the present invention provides a composition for diagnosing or predicting mCRPC, including a preparation for measuring the expression level of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) at the mRNA or protein level.

[0029] The agent for measuring at the mRNA level may be, but is not limited to, at least one selected from the group consisting of antisense oligonucleotides, primers and probes that specifically bind to at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA).

[0030] The above primer or probe may be labeled with a fluorescent substance, and the fluorescent substance is selected from the group consisting of a biotin receptor peptide, a lipoic acid receptor peptide, a fluorescent protein, a green fluorescent protein (GFP), a red fluorescent protein (RFP), a yellow fluorescent protein (YFP), an enhanced green fluorescent protein (EGFP), an enhanced yellow fluorescent protein (EYFP), a cysteine-containing peptide for ligation of an arsenic dye or for conjugating metastable technetium, 4',5'-bis(1,3,2-dithioasolan-2-yl)fluorescein (FlAsH), a peptide for conjugating europium clathrate for fluorescence resonance energy transfer (FRET)-based proximity assay, and any combination thereof. Can be selected.

[0031] In addition, the above measurement may be performed by one or more methods selected from the group consisting of polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive polymerase chain reaction (Competitive RT-PCR), nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, northern blot, DNA chip, multiplex PCR, and ddPCR, but is not limited thereto.

[0032] The agent for measuring at the protein level may be, but is not limited to, one or more selected from the group consisting of antibodies, antibody fragments, aptamers, avidity multimers, and peptidomimetics that specifically bind to a protein encoded by one or more genes selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA).

[0033] In the present invention, the antibody fragment may be any one selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, single-chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody, and diabody, but is not limited thereto.

[0034] In addition, the above measurement may be performed by one or more methods selected from the group consisting of, but not limited to, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, and protein microarray.

[0035] The term "primer" as used in the present invention refers to a short nucleic acid sequence having a short free 3-terminal hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature.

[0036] The term "probe" used in the present invention refers to a nucleic acid fragment such as RNA or DNA, ranging from a few bases to several hundred bases, capable of specifically binding to mRNA, and is labeled so as to enable detection of the presence or absence of a specific mRNA. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. In the present invention, hybridization is performed using a probe complementary to the PSMA or PSA gene, and the level of gene expression can be diagnosed based on the presence or absence of hybridization. The selection of an appropriate probe and hybridization conditions can be modified based on those known in the art, and therefore are not particularly limited in the present invention.

[0037] The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologs of a natural nucleotide, and modifications between nucleotides, such as modification with uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

[0038] In the present invention, suitable conditions for hybridizing a probe with a cDNA molecule can be determined through a series of steps through an optimization procedure. Such procedures are performed as a series of steps by those skilled in the art to establish a protocol for use in a laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing time, buffer components, and their pH and ionic strength depend on various factors such as the length and GC content of the probe and the target nucleotide sequence. Regarding detailed conditions for hybridization, for example, among stringent conditions, high stringency conditions mean hybridization at 65°C in 0.5 M NaHPO4, 7% SDS (sodium dodecyl sulfate), and 1 mM EDTA, and washing at 68°C in 0.1x SSC (standard saline citrate) / 0.1% SDS. Alternatively, high stringency conditions mean washing at 48°C in 6x SSC / 0.05% sodium pyrophosphate. Those stringent conditions mean washing at 42°C in 0.2x SSC / 0.1% SDS, for example.

[0039] The term "antibody" used in the present invention is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, the antibody refers to an antibody that specifically binds to a protein expressed from the PSMA or PSA gene, which is a biomarker of the present invention, and the method for producing the antibody can be produced using a well-known method. This also includes a partial peptide that can be produced from the protein. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, or a part thereof that has antigen binding property is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies.

[0040] In addition, the present invention provides a kit for diagnosing or predicting mCRPC, including a preparation for measuring the expression level of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) at the mRNA or protein level.

[0041] In the present invention, the kit may further include tools and / or reagents for collecting a biological sample from a subject or patient, as well as tools and / or reagents for preparing genomic DNA, cDNA, RNA, or protein from the sample. For example, the kit may include PCR primers for amplifying relevant regions of genomic DNA. The kit may include probes for genetic factors useful for pharmacogenomic profiling. Furthermore, when using such a kit, labeled oligonucleotides can be used for easy identification during analysis. In addition, the kit may further contain DNA polymerase and a labeling substance such as a dNTP (dGTP, dCTP, dATP, and dTTP), a fluorescent substance, etc.

[0042] The term "mCRPC diagnostic or predictive kit" used in the present invention refers to a kit containing the mCRPC diagnostic or predictive composition of the present invention. Therefore, the expression "mCRPC diagnostic or predictive kit" may be used interchangeably or interchangeably with "mCRPC diagnostic or predictive composition."

[0043] In addition, the present invention provides a kit for screening mCRPC inhibitors, which comprises a preparation for measuring the expression level of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) at the mRNA or protein level.

[0044] The above inhibitor may be, but is not limited to, an unknown substance that inhibits the progression of mHSPC to mCRPC or enhances the response of mHSPC to hormone therapy.

[0045] In addition, the present invention provides a method for providing information necessary for diagnosing or predicting mCRPC, comprising the steps of: a) confirming the expression level of mRNA or protein of one or more genes selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) in a sample isolated from a test subject; and b) comparing the result with the corresponding result of the corresponding gene in a normal control sample.

[0046] In the method of providing information necessary for diagnosing or predicting mCRPC of the present invention, a step of diagnosing or predicting mCRPC according to the expression level of PSMA and / or PSA genes, which are biomarkers of the present invention, may be additionally included, and the normal control group may mean a normal population that has not developed cancer, or a group of mHSPC patients that have not progressed to mCRPC.

[0047] Additionally, the sample may be one or more selected from the group consisting of blood, tissue, cells, plasma, stool, urine, and semen, but is not limited thereto. Preferably, the sample may be blood, and more preferably, circulating tumor cells (CTCs), but is not limited thereto.

[0048] In addition, the present invention provides a method for screening an mCRPC inhibitor, including the step of treating a sample with a candidate substance and selecting a candidate substance that suppresses the expression or activity of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA).

[0049] The present invention can screen for, but is not limited to, candidate substances capable of inhibiting the progression of mHSPC to mCRPC, or candidate substances capable of enhancing the response of mHSPC to hormone therapy. According to the screening method of the present invention, the candidate substance to be analyzed can first be brought into contact with cancer cells containing the gene or protein. The candidate substance refers to an unknown substance used in screening to examine whether it affects the expression level of the gene, the amount of the protein, or the activity of the protein.

[0050] The above candidate substances may include, but are not limited to, chemicals, antisense oligonucleotides, antisense-RNA, siRNA, shRNA, miRNA, antibodies specific for the protein, or natural product extracts.

[0051] In the present invention, the term "sample" is used in the broadest sense. On the one hand, it encompasses specimens or cultures (e.g., microbial cultures). On the other hand, it encompasses both biological and environmental samples. Furthermore, the sample may include samples of synthetic origin. The above biological samples include whole blood, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, leukocytes, peripheral blood mononuclear cells, buffy coat, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, trachea. The sample may be at least one selected from the group consisting of organ secretions, cells, cell extracts, and cerebrospinal fluid, but is not limited thereto. Preferably, the sample may be blood isolated from an animal, and more preferably, circulating tumor cells (CTCs) isolated therefrom.The above CTCs refer to circulating cancer cells separated from primary tumor cells and circulating in the bloodstream. They can be isolated using antibodies that specifically bind to them and magnetic beads that bind to the antibodies. For example, the antibody that specifically binds to the CTCs may be anti-EpCAM (anti-Epithelial Cell Adhesion Molecule).

[0052]

[0053] The terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents of this specification as a whole. Throughout this specification, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not mean that other components are excluded, but rather that other components may be included.

[0054] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited as references herein are incorporated herein by reference.

[0055] Hereinafter, the present invention will be described in detail with reference to the attached drawings, using exemplary embodiments. However, the following exemplary embodiments are provided as illustrative examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0056]

[0057] Experimental Example 1. Sample Preparation and Genetic Analysis

[0058] 1-1. Patient classification and sample collection

[0059] A total of 44 patients with mHSPC were recruited, and peripheral blood samples were collected. The mHSPC patients were divided into two groups: 31 patients who had not progressed to mCRPC and 13 patients who had progressed to mCRPC. All samples were approved by the institutional review boards of Haeundae-Paik Hospital (HPIRB 2018-01-005-004) and Seoul National University Bundang Hospital (B-1902-522-304). Characteristics and clinical information are presented in Table 1.

[0060] mHSPC (N=44) Age, median, IQR, years 76.0 (70-79) PSA, median, IQR, ng / ml 38.0 (13.1-680) Gleason Score (%) 61 (2.2) 77 (16.0) ≥ 836 (81.8) Node metastasis (%) Yes 38 (86.4) No 6 (13.6) Bone metastasis (%) Yes 42 (95.5) No 2 (4.5) Visceral metastasis (%) Yes 28 (63.6) No 16 (36.4) Prior therapy (%) None 10 (22.7) Radiation 9 (20.5) Taxane 0 (0.0) GnRH agonist / antagonist 15 (34.1)Antiandrogen9 (20.5)Abiraterone0 (0.0)Enzalutamide0 (0.0)

[0061] Among mHSPC patients, those who developed resistance to androgen deprivation therapy (ADT) were considered to have progressed to mCRPC. In Table 1, among the prior therapy groups, 13 patients in the GnRH agonist / antagonist and antiandrogen treatment groups developed resistance and progressed to the mCRPC group.

[0062]

[0063] 1-2. Isolation of blood cancer cells

[0064] The peripheral blood of each experimental group classified above was subjected to a density gradient to remove red blood cells, treated with Ficoll solution (1.119 g / mL), and centrifuged at 700 g for 30 minutes. After centrifugation, the white blood cell and circulating tumor cell (CTC) (i.e., blood cancer cell) layers were separated, washed with 10 mL of PBS buffer, and centrifuged at 200 g for 10 minutes. After washing once more with 200 μL of PBS buffer, the white blood cell and CTC layers were obtained by centrifuging at 200 g for 5 minutes. After this, 20 μL of EpCAM cocktail solution (antibody other than anti-EpCAM antibody antigen and Dextran) capable of attaching to EpCAM, a CTC-specific antigen, was treated, and incubated for 1 hour under ice conditions to bind the anti-EpCAM of the cocktail to the EpCAM antigen of blood cancer cells, and 20 μL of nanometer-sized immunomagnetic beads (magnetic nanobeads with a diameter of several tens of nm coated with anti-dextran antibodies) capable of specifically binding to EpCAM were treated, and incubated for 1 hour and 30 minutes under ice conditions, thereby binding the dextran and anti-dextran of the cocktail, ultimately inducing binding in the form of 'cancer cell-anti-EpCAM antibody-dextran-magnetic nanobead'. After this, lateral magnetic phoretic separation was performed using a microparticle separation device using magnetic phoresis described in Korean Patent Registration No. 10-1622342. 0.2% BSA PBS buffer and the blood sample prepared above were each flowed at a rate of 2 mL / h through the above device, and the separated sample was collected in a 1.5 mL e-tube.

[0065]

[0066] 1-3. Genetic Analysis

[0067] The expression of genes PSMA, PSA, AR (Androgeen Receptor), AR-V7 (Androgen receptor variant 7), EpCAM, and KRT19 (Cytokeratin 19) in the CTC samples isolated as described above was analyzed using the ddPCR (Droplet digital PCR) method. Specifically, the CTC samples were first centrifuged at 200 g for 5 minutes, then treated with 300 μL of cell disruption solution, and total RNA was isolated using the Dynabeads mRNA Direct Kit (Invitrogen). Then, for cDNA synthesis, a final 20 μL of cDNA was synthesized using Bionner's AccuPower CycleScript RT PreMix (dT20). To increase the efficiency of the final gene detection using ddPCR, a pre-amplification process was applied. The primer information used for the above pre-amplification is shown in Table 2 below. 5 μL of diluted cDNA template was mixed with the forward and reverse primers according to the protocol in a multiplex PCR supermix (AccuPower Multiplex PCR PreMix, Bioneer) to create a final 50 μL preamplification sample. The PCR conditions used were as follows: a ramp rate of 4°C / s, an initial denaturation step at 95°C for 10 min, followed by 18 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 1 min, and extension at 72°C for 30 s. To ensure complete amplification, the final extension step was performed at 72°C for 10 min.20 μL of the PCR mixture containing the above pre-amplified sample was injected into the sample inlet of a droplet-generating cartridge (DG8 Cartridges for QX100 / QX200 droplet generator, Bio-RAD), and 70 μL of droplet generation oil (Droplet generation oil for EvaGreen, Bio-RAD) was injected into the oil inlet. Then, to form droplets, the droplet generating cartridge was mounted on the droplet generator (QX200 Droplet generator, Bio-Rad), and droplets were formed for 2 minutes. The formed droplets were transferred to a 96-well PCR plate and sealed with a sealer. PCR was performed using a PCR cycler (GeneAmp PcR System 9700, Applied Biosystems) at 95°C for 5 min (Enzyme activation), 40 cycles of 95°C for 30 s (denaturation) + 56°C for 1 min (Annealing and extension), and 4°C for 5 min + 90°C for 5 min (signal stabilization), and analyzed using a droplet reader (QX200 Droplet reader, Bio-RAD). Information on the primers used for ddPCR is also described in Table 2 below.

[0068] Gene Nucleotide sequence Primer sequence Product size (bp) Sequence number PSANM_004476.2 NM_001014986.2 NM_001193471.2 NM_001193472.2 NM_001351236.1 Pre-amplification F: 5'-AGCATTTTTGGATGAATTGAAAGC-3'6431 R: 5'-TGGGTAACCTGGTGTGAGA-3'2 ddPCR F: 5'-ATGAATTGAAAGCTGAGAACATCAAGA-3'1883 R: 5'-GGGATGAGTCTTATTTGGGTAGGAC-3'4 PSANM_001648.2 NM_001030048.1 Pre-amplification F: 5'-GGTGATGACTCCAGCCAC-3'4355R: 5'-CACGATGGTGTCCTTGATCC-3'6ddPCRF: 5'-ACCAGAGGAGTTTCTTGACCCCAAA-3'1617R: 5'-CCCCAGAATCACCCGAGCAG-3'8ARNM_000044.4NM_001011645.3Pre-amplificationF: 5'-TCTCTCAAGAGTTTGGATGGC-3'3909R: 5'-ACTGGGTGTGGAAATAGATGG-3'10ddPCRF: 5'-CAGCCTATTGCGAGAGAGCTG-3'12511R: 5'-GAAAGGATCTTGGGCACTTGC-3'12AR-V7NM_001348061.1NM_001348064.1Pre-amplificationF: 5'-GTGCGCCAGCAGAAATGA-3'44013R: 5'-TCAGGTGCTTTTTTCATCAACATAA-3'14ddPCRF: 5'-GTTCGGGCTTCTGCTTGC-3'12515R: 5'-GCTCTCATCGCAGTCAGGA-3'16EpCAMNM_002354.2Pre-amplificationF: 5'-CTCGCGTTCGGGCTTC-3'75517R: 5'-TCAGGTGCTTTTTTCATCAACATAA-3'18ddPCRF: 5'-GTTCGGGCTTCTGCTTGC-3'28719R: 5'-GCTCTCATCGCAGTCAGGA-3'20KRT19NM_002276.4Pre-amplificationF: 5'-CGACTACAGCCACTACTACAC-3'69521R: 5'-CACTATCAGCTCGCACATCG-3'22ddPCRF: 5'-TTTGAGACGGAACAGGCTCT-3'21123R: 5'-AATCCACCTCCACACTGACC-3'24.

[0069]

[0070] Example 1. Genetic analysis associated with mCRPC progression

[0071] For each experimental group prepared above, the number of isolated blood CTCs and genetic analysis through heatmap were counted and shown in Figure 1.

[0072] The cutoff value for the presence or absence of CTCs was set to 2.2 CTCs / mL, which is 0.1 more than the maximum isolated number of 2.1 CTCs / mL in a healthy donor sample. The heatmap of gene expression levels for each sample was sorted by increasing the number of CTCs. The heatmap is expressed by converting the data matrix into z-scores, with the lowest value expressed in bright red and the highest value in bright green.

[0073] As a result, a greater number of CTCs were identified in the mCRPC progression-free group than in the mCRPC progression group. Furthermore, many subjects in the mCRPC progression-free group showed PSMA or PSA expression. Therefore, we confirmed that PSMA or PSA is expressed in patients with mCRPC progression.

[0074]

[0075] Example 2. Kaplan-Meier survival analysis

[0076] 2-1. Kaplan-Meier survival analysis for PSMA or PSA expression

[0077] The correlation between the expression of PSMA or PSA and the 50-month survival of each experimental group was analyzed using a Kaplan-Meier curve and is shown in Figure 2.

[0078] As a result, the experimental group showing high expression of PSMA and PSA mRNA showed a shorter progression-free survival (PFS). In addition, the p-value of PSMA was 0.019 and the p-value of PSA was 0.026, confirming statistical significance. The criteria for the high and low gene groups were set at the median value of each gene, and PSMA was 1 copy / μL and PSMA was 0.9 copies / μL.

[0079] Therefore, the above two PSMA and PSA genes were determined to be useful markers for diagnosing mCRPC.

[0080]

[0081] 2-2. Kaplan-Meier survival analysis for AR, AR-V7, EpCAM, KRT19, or CTC# expression

[0082] In addition, the association between the expression of AR, AR-V7, EpCAM, KRT19, or CTC# and the 50-month survival of each experimental group was analyzed using Kaplan-Meier curves and is shown in Figure 3.

[0083] As a result, AR (p-value: 0.374), AR-V7 (p-value: 0.635), EpCAM (p-value: 0.268), KRT19 (p-value: 0.943), and CTC# (p-value: 0.827) were identified. Unlike the biomarker according to the present invention, the AR, AR-V7, EpCAM, KRT19, or CTC# showed a p-value of 0.05 or higher, confirming that they did not show statistical significance.

[0084]

[0085] Example 3. Cox survival analysis

[0086] In addition, in 44 experimental groups, the association between PSMA and PSA gene expression and survival rate was confirmed again using the Cox proportional-hazards model. The results are shown in Fig. 4, and a higher HR (hazard ratio) value means a higher risk of death, and a lower P-value means higher accuracy (cut-off value: PSMA=1 copies / μl, PSA=0.9 copies / μl, AR=200 copies / μl, AR-V7=0.03 copies / μl, EpCAM=5.3 copies / μl, KRT19=0.29 copies / μl, CTC#=8.8 CTCs / ml).

[0087] According to Figure 4, the HR value of PSMA-mRNA was the highest at 3.83, and the HR value of PSA-mRNA was the second highest at 3.47.

[0088] Therefore, it was clearly confirmed that PSMA and PSA according to the present invention showed high expression in mCRPC patients and had an impact on the high mortality rate of mCRPC patients.

[0089]

[0090] Thus, according to the present invention, in the mCRPC patient group, the PSMA and PSA genes were found to have higher expression than in the patient group that did not progress to mCRPC, and it was thought that this would also have an effect on the mortality rate.

[0091] Therefore, the PSMA and PSA genes according to the present invention can be usefully utilized as a technology that can accurately diagnose and predict mCRPC, and by analyzing the expression of the genes in mHSPC patients, the possibility of progression to mCRPC can be confirmed in advance, which can greatly contribute to improving the survival rate of patients.

[0092]

[0093] As described above, specific embodiments of the present invention have been described in detail. However, those skilled in the art who understand the spirit of the present invention will be able to easily suggest other inventions that are backwards or other embodiments included within the scope of the spirit of the present invention by adding, changing, or deleting other components within the scope of the same spirit. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the scope of the claims described below rather than the detailed description described above, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. A biomarker composition for diagnosing or predicting metastatic castrate-resistant prostate cancer (mCRPC), comprising at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) or a protein encoded by the gene.

2. In paragraph 1, The above mCRPC is a biomarker composition for diagnosing or predicting mCRPC, characterized in that metastatic hormone-sensitive prostate cancer (mHSPC) has progressed to hormone-refractory.

3. A composition for diagnosing or predicting mCRPC, comprising a preparation for measuring the expression level of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) at the mRNA or protein level.

4. In paragraph 3, A composition for diagnosing or predicting mCRPC, characterized in that the agent measured at the mRNA level is at least one selected from the group consisting of antisense oligonucleotides, primers, and probes that specifically bind to at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA).

5. In paragraph 3, A composition for diagnosing or predicting mCRPC, wherein the above measurement is performed by one or more methods selected from the group consisting of polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive polymerase chain reaction (Competitive RT-PCR), nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, northern blot, DNA chip, multiplex PCR, and ddPCR.

6. In paragraph 3, A composition for diagnosing or predicting mCRPC, characterized in that the agent measured at the protein level is at least one selected from the group consisting of antibodies, antibody fragments, aptamers, avidity multimers, and peptidomimetics that specifically bind to a protein encoded by at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA).

7. In paragraph 3, A composition for diagnosing or predicting mCRPC, wherein the above measurement is performed by one or more methods selected from the group consisting of Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, and protein microarray.

8. A kit for diagnosing or predicting mCRPC, comprising a preparation for measuring the expression level of one or more genes selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA), at the mRNA or protein level.

9. A kit for screening mCRPC inhibitors, comprising a preparation for measuring the expression level of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) at the mRNA or protein level.

10. In paragraph 9, A kit for screening mCRPC inhibitors, wherein the inhibitor inhibits the progression of mHSPC to mCRPC. 11.a) A step of checking the expression level of mRNA or protein of one or more genes selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA) in a sample isolated from a test subject; and b) A method for providing information necessary for diagnosing or predicting mCRPC, comprising a step of comparing the corresponding results of the corresponding gene in a normal control sample.

12. In paragraph 11, A method for providing information necessary for diagnosing or predicting mCRPC, wherein the sample isolated from the above test subject is at least one selected from the group consisting of blood, tissue, cells, plasma, stool, urine, and semen.

13. In paragraph 11, A method for providing information necessary for diagnosing or predicting mCRPC, wherein the sample isolated from the above test subject is a circulating tumor cell (CTC).

14. A method for screening an mCRPC inhibitor, comprising the step of treating a sample with a candidate substance and selecting a candidate substance that suppresses the expression or activity of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA) and prostate specific antigen (PSA).

15. In paragraph 14, A method for screening an mCRPC inhibitor, wherein the inhibitor inhibits the progression of mHSPC to mCRPC.

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