Biomarker composition for predicting therapeutic responsiveness or prognosis of prostate cancer

Abstract

The present invention relates to a biomarker capable of predicting mCRPC which is resistant to the anticancer agent docetaxel or ADT. According to the present invention, the PMSA, PSA, and EpCAM genes exhibit specific expression levels in mCRPC exhibiting poor prognosis due to resistance to docetaxel, and thus can be effectively used as a biomarker for predicting the prognosis of prostate cancer exhibiting resistance to anticancer agents or the therapeutic responsiveness of the prostate cancer to an anticancer agent. Moreover, the PMSA, PSA, and AR-V7 genes exhibit specific expression levels in mCRPC exhibiting poor prognosis due to resistance to secondary hormone agents and high CTC counts, and thus can be effectively used as a biomarker for predicting the therapeutic responsiveness or prognosis of prostate cancer exhibiting resistance to ADT. Thus, the present invention makes it possible to first analyze the expression of the genes and then determine the treatment direction of mCRPC accordingly, and thus can contribute significantly to improving the survival rate of mCRPC patients.

Description

Biomarker composition for predicting treatment response or prognosis of prostate cancer

[0001] The present invention relates to a biomarker composition capable of predicting whether prostate cancer is resistant to anticancer drugs or androgen deprivation therapy, or the prognosis after anticancer drug or androgen deprivation therapy treatment.

[0002] The prostate is a male-only organ that produces part of semen and is located below the bladder and adjacent to the rectum. Prostate cancer is most often caused by cancer of the gland cells within the prostate, and it frequently metastasizes to the lymph nodes and bones. Approximately 90% of prostate cancers are driven by androgens produced by the body.

[0003] Prostate cancer ranks sixth in incidence among Korean men and is the second most common urological cancer after bladder cancer. Notably, 10-50% of clinically localized prostate cancers are diagnosed as advanced prostate cancer. While androgen deprivation therapy (HRT) is the established standard treatment for advanced prostate cancer, most cases develop into hormone-refractory prostate cancer (HRPC) within two years. Metastatic castrate-resistant prostate cancer (mCRPC) is defined as cancer that progresses despite hormone levels reduced to the level of castration. Previously, the survival time for mCRPC was only 1.5 to 2 years, but the recent introduction of new second-line hormonal agents has increased the survival time to 4-5 years. Second-line hormonal agents selectively bind to the androgen receptor (AR) and inhibit its signaling pathways through multiple steps. It does not affect the production or blood concentration of androgens, but rather binds to androgen receptors, thereby disrupting their function. However, some mCRPC cases develop hormone-independent cancer cells. In these cases, androgen receptor inhibitors are ineffective, resulting in significantly lower survival rates. In these cases, chemotherapy or targeted therapy is used. However, if mCRPC patients' genetic characteristics are diagnosed early enough to predispose them to secondary hormone therapy, a wider range of preemptive treatment options can be implemented, potentially helping to extend patient life.

[0004] Furthermore, in cases where androgen receptor inhibitors are ineffective in mCRPC, cytotoxic chemotherapy remains the only option. Among these chemotherapy regimens, docetaxel-based chemotherapy is known to be effective. However, as with other anticancer drugs, resistance to taxanes, including docetaxel and paclitaxel, has also been reported. Resistance is a particularly serious problem in cancers such as prostate, breast, and ovarian cancers.

[0005] Therefore, drug resistance to docetaxel is a critical issue in the treatment of various cancers, including prostate cancer. In fact, when attempting chemotherapy in cancer patients, if a particular anticancer drug fails to work, resistance to other anticancer drugs frequently develops. Furthermore, combination chemotherapy, which involves administering multiple anticancer drugs with different mechanisms of action simultaneously during initial treatment, often fails to respond. This severely limits the range of available anticancer drugs, posing a significant challenge in cancer chemotherapy. Predicting anticancer drug sensitivity is becoming an essential element in determining the treatment strategy for cancer patients. However, the complex interplay of factors related to the biological response to specific drugs, the diversity of treatments and administration methods, and the difficulty of securing a large sample volume make predicting anticancer drug responsiveness challenging.

[0006] 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.

[0007] Therefore, there is an urgent need to develop a biomarker that can accurately identify mCRPC resistant to chemotherapy or secondary hormone therapy.

[0008] The purpose of the present invention is to provide a biomarker composition for predicting treatment responsiveness or prognosis of prostate cancer.

[0009] In addition, another object of the present invention is to provide a composition for predicting treatment responsiveness or prognosis of prostate cancer.

[0010] In addition, another object of the present invention is to provide a kit for predicting treatment responsiveness or prognosis of prostate cancer.

[0011] In addition, another object of the present invention is to provide a kit for screening prostate cancer treatment agents.

[0012] In addition, another object of the present invention is to provide a method for providing information necessary for predicting treatment responsiveness or prognosis of prostate cancer.

[0013] In addition, another object of the present invention is to provide a method for screening a prostate cancer treatment agent.

[0014] In order to achieve the above purpose, the present invention provides a biomarker composition for predicting treatment response or prognosis of prostate cancer, comprising at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), a protein encoded by the at least one gene, or isolated circulating tumor cells (CTC).

[0015] In addition, in order to achieve the other object mentioned above, the present invention provides a composition for predicting anticancer treatment response or prognosis of prostate cancer, which comprises an agent for measuring one or more genes selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), a protein encoded by the one or more genes, or isolated circulating tumor cells (CTCs).

[0016] In addition, to achieve the above another object, the present invention provides a kit for predicting treatment response or prognosis of prostate cancer, comprising an agent for measuring at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), a protein encoded by the at least one gene, or isolated circulating tumor cells (CTC).

[0017] In addition, in order to achieve the above another object, the present invention provides a kit for screening a prostate cancer treatment agent, which comprises at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), a protein encoded by the at least one gene, or an agent for measuring isolated circulating tumor cells (CTCs).

[0018] In addition, in order to achieve the above another object, the present invention provides a method for providing information necessary for predicting the treatment responsiveness or prognosis of prostate cancer, comprising the steps of: a) confirming the expression level of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), the expression level of a protein encoded by the gene, or the concentration of isolated circulating tumor cells (CTCs) 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.

[0019] In addition, in order to achieve the above another object, the present invention provides a method for screening a prostate cancer treatment agent, comprising the step of treating a sample with a candidate substance and selecting a candidate substance that inhibits the expression or activity of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), or inhibits at least one gene selected from the group consisting of growth, proliferation, and metastasis of CTCs.

[0020] The present invention relates to a biomarker composition for predicting treatment responsiveness or prognosis of prostate cancer, and it was confirmed that the expression levels of prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), and epithelial cell adhesion molecule (EpCAM) in circulating tumor cells (CTCs) isolated from a patient are related to prostate cancer resistant to anticancer drugs. In addition, it was confirmed that the concentration of CTCs isolated from a patient and / or the expression levels of prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), and AR-V7 (Androgen receptor variant 7) in the CTCs are related to prostate cancer resistant to androgen deprivation therapy (ADT), and thus the above genes are provided as biomarkers for predicting treatment responsiveness of prostate cancer to anticancer drugs or ADT or the prognosis of prostate cancer.

[0021] Figure 1 shows the results of classifying the experimental group of mCRPC patients with a good prognosis after treatment and the experimental group of mCRPC patients with a poor prognosis after treatment through analysis of the best PSA response in mCRPC patients treated with docetaxel, and comparing cases showing high expression of each gene (PSMA, PSA, AR, AR-V7, EpCAM, KRT19, and CTC#).

[0022] In mCRPC patients treated with docetaxel, the best PSA response analysis was used to classify the experimental group of mCRPC patients with a good prognosis after treatment and the experimental group of mCRPC patients with a poor prognosis after treatment, and the results were compared between cases showing high expression of PSMA, PSA, and EpCAM and cases showing high expression of AR, AR-V7, KRT19, and CTC#.

[0023] Figure 2 shows the results of analyzing the genes of each experimental group using a heatmap, classifying mCRPC patients treated with docetaxel into a good prognosis group and a poor prognosis group, and showing the number of CTCs isolated from each experimental group.

[0024] Figure 3 shows the results of analyzing the correlation between survival of an experimental group of mCRPC patients with a good prognosis after docetaxel treatment and an experimental group of mCRPC patients with a poor prognosis after docetaxel treatment and the expression of PSMA, PSA, or EpCAM mRNA, which are biomarker genes according to the present invention, using a Kaplan-Meier curve.

[0025] Figure 4 shows the results of analyzing the association between survival and the expression of known prostate cancer biomarkers AR, AR-V7, KRT19, or CTC# in the experimental group of mCRPC patients with a good prognosis after docetaxel treatment and the experimental group of mCRPC patients with a poor prognosis after docetaxel treatment using Kaplan-Meier curves.

[0026] Figure 5 shows the results of analyzing the correlation between the survival of an experimental group of mCRPC patients with a good prognosis after docetaxel treatment and an experimental group of mCRPC patients with a poor prognosis after docetaxel treatment and the expression of PSMA, PSA, or EpCAM, which are biomarker genes according to the present invention, using a Cox proportional-hazards model.

[0027] Figure 6 shows the results of classifying the experimental group of mCRPC patients with a good prognosis after treatment and the experimental group of mCRPC patients with a poor prognosis after treatment through analysis of the best PSA response in mCRPC patients who received secondary hormone therapy, and comparing cases showing high expression of each gene (PSMA, PSA, AR, AR-V7, EpCAM, KRT19, and CTC#).

[0028] Figure 7 shows the results of analyzing the genes of each experimental group using a heatmap, classifying mCRPC patients who received secondary hormone therapy into a good prognosis group and a poor prognosis group, and showing the number of CTCs isolated from each experimental group.

[0029] Figure 8 shows the results of analyzing the correlation between the survival of a group of mCRPC patients with a good prognosis and a group of mCRPC patients with a poor prognosis who received secondary hormone therapy and the expression of PSMA, PSA, AR-V7 mRNA or the number of CTCs, which are biomarkers according to the present invention, using a Kaplan-Meier curve.

[0030] Figure 9 shows the results of analyzing the association between survival and the expression of AR, EpCAM, or KRT19, which are known prostate cancer biomarkers, using Kaplan-Meier curves in the experimental group of mCRPC patients with a good prognosis and the experimental group of mCRPC patients with a poor prognosis who received secondary hormone therapy.

[0031] Figure 10 shows the results of analyzing the correlation between the survival of a group of mCRPC patients with a good prognosis and a group of mCRPC patients with a poor prognosis who received secondary hormone therapy and the expression of PSMA, PSA, AR-V7 mRNA or the number of CTCs, which are biomarkers according to the present invention, using a Cox proportional-hazards model.

[0032] Hereinafter, the present invention will be described in more detail.

[0033] The present invention provides a biomarker composition for predicting treatment response or prognosis of prostate cancer, comprising at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), a protein encoded by the at least one gene, or isolated circulating tumor cells (CTC).

[0034] In the biomarker composition according to the present invention, the prostate cancer may be metastatic cancer, and more preferably, metastatic castrate-resistant prostate cancer (mCRPC).

[0035] In addition, the above treatment may be anticancer drug administration or androgen deprivation therapy (ADT).

[0036] In the present invention, the term "biomarker for predicting treatment responsiveness" or "biomarker for predicting prognosis" refers to a substance that can predict / diagnose the responsiveness of a subject to anticancer drugs or ADT treatment in prostate cancer treated with anticancer drugs or ADT, 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 symptoms / diseases that do not improve after anticancer drugs or ADT treatment. For the purpose of the present invention, the biomarker for predicting anticancer drug treatment responsiveness or prognosis of prostate cancer is a gene PSMA, PSA, EpCAM, AR-V7 or an isolated CTC, wherein the mRNA or protein expression level of the gene increases or the number of the CTC increases in prostate cancer that has anticancer drug or ADT resistance. 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.

[0037] In the biomarker composition according to the present invention, the anticancer agent may be a taxane-based anticancer agent. Preferably, but not limited to, it may be at least one selected from the group consisting of docetaxel, paclitaxel, and cabazitaxel. More preferably, it may be docetaxel, but is not limited thereto.

[0038] The above docetaxel is a compound having a structure represented by the following chemical formula 1, and its anticancer activity is derived from targeting the microtubules of the mitotic spindle, thereby interfering with the alignment and separation of chromosomes in the spindle, blocking cell cycle progression, and activating the cell death pathway. Although docetaxel is a more potent anticancer agent than paclitaxel, the pathways involved in its cytotoxicity are not well defined, and many cancers often develop resistance to it, which often reduces the effectiveness of the treatment. Patients with mCRPC are also sometimes treated with docetaxel before second-line hormonal therapy. However, since it is not possible to confirm whether mCRPC patients are resistant to docetaxel, in some cases the desired therapeutic effect may not be achieved.

[0039]

[0040] According to one embodiment of the present invention, in mCRPC patients treated with docetaxel, it was confirmed that when high expression of PSMA, PSA, and EpCAM genes was shown, the prognosis was poor and the survival rate was low.

[0041] Therefore, in the present invention, at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), and epithelial cell adhesion molecule (EpCAM) or a protein encoded by the at least one gene is characterized in that it can predict the anticancer treatment responsiveness or prognosis of prostate cancer.

[0042] Therefore, prior to docetaxel treatment of mCRPC patients, genetic analysis using the biomarker composition according to the present invention can predict whether the patient has resistance to docetaxel or the prognosis according to treatment, thereby contributing to increasing the patient's survival.

[0043] Additionally, in the biomarker composition according to the present invention, the ADT may be a secondary hormone agent. The secondary hormone agent is a substance that can selectively bind to the androgen receptor and inhibit the signal transduction mechanism of the androgen receptor in multiple stages, and refers to a substance that eliminates the function of androgen without affecting the production or blood concentration of androgen. In the present invention, the secondary hormone agent may be used without limitation as long as it is a secondary hormone agent known in the art, and preferably may be at least one selected from the group consisting of enzalutamide, apalutamide, bicalutamide, darolutamide, flutamide, abiratarone, ARN-509, ODM-201, and galeterone, and more preferably may be enzalutamide or abiratarone, but is not limited thereto.

[0044] In the above mCRPC, if resistance to secondary hormone therapy is shown, the survival rate is significantly lower, but it is not possible to accurately confirm in advance whether an mCRPC patient has resistance to secondary hormone therapy, and in some cases, the targeted treatment effect may not be achieved.

[0045] According to one embodiment of the present invention, in mCRPC patients receiving secondary hormone therapy, it was confirmed that when high expression of PSMA, PSA, and AR-V7 genes was shown or the number of CTCs was high, the prognosis was poor and the survival rate was low.

[0046] Therefore, in the present invention, one or more genes selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), and AR-V7 (Androgen receptor variant 7), a protein encoded by the one or more genes, or isolated circulating tumor cells (CTC) are characterized in that they can predict the ADT treatment responsiveness or prognosis of prostate cancer.

[0047] Therefore, prior to secondary hormonal treatment of mCRPC patients, genetic analysis using the biomarker composition according to the present invention can predict whether the patient has resistance to secondary hormonal agents or the prognosis according to treatment, thereby contributing to increasing the patient's survival.

[0048] In the biomarker composition of the present invention, the isolated circulating tumor cells (CTCs) may have a cut-off concentration value of 10.2 to 10.8 CTCs / ml. The cut-off may preferably be 10.4 to 10.8 CTCs / ml, and more preferably 10.6 CTCs / ml.

[0049] If the CTC concentration of the sample is higher than the above cut-off concentration, it can be determined that the sample is resistant to secondary hormone therapy.

[0050] In addition, the present invention provides a composition for predicting anticancer treatment response or prognosis of prostate cancer, comprising an agent for measuring at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), a protein encoded by the at least one gene, or isolated circulating tumor cells (CTC).

[0051] The above formulation is a formulation that can be measured at the mRNA level, and may be at least one selected from the group consisting of antisense oligonucleotides, primers, and probes that specifically bind to any one or more genes selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and AR-V7 (Androgen receptor variant 7), but is not limited thereto.

[0052] 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.

[0053] 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.

[0054] In addition, the above formulation is a formulation that can be measured at the protein level, and may be at least one selected from the group consisting of a protein encoded by one or more genes selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), or an antibody, antibody fragment, aptamer, avidity multimer, and peptidomimetics that specifically bind to a surface marker of isolated CTC, but is not limited thereto.

[0055] 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.

[0056] 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.

[0057] The above "surface marker" refers to a protein specifically expressed on the surface of CTCs, and any CTC-specific marker known in the art can be used without limitation. Examples of the surface marker include, but are not limited to, epithelial markers, mesenchymal markers, beta-catenin, cadherin-11, GSK-3 beta, MMP2, SUMO2, Twist-1, ZEB1, CD321, CD45, or CD66b. In one embodiment of the present invention, epithelial cell adhesion molecule (EpCAM) was used as a surface marker.

[0058] 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.

[0059] 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, PSA, or AR-V7 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 thereto in the present invention.

[0060] 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.).

[0061] 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.

[0062] 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, PSA, EpCAM, AR-V7 gene, which is a biomarker of the present invention, or a CTC surface marker, 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.

[0063] In addition, the present invention provides a kit for predicting treatment response or prognosis of prostate cancer, comprising an agent for measuring at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), a protein encoded by the at least one gene, or isolated circulating tumor cells (CTC).

[0064] 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.

[0065] The term "kit for predicting treatment responsiveness or prognosis of prostate cancer" used in the present invention refers to a kit containing the composition for predicting treatment responsiveness or prognosis of prostate cancer of the present invention. Accordingly, it may be a kit for predicting anticancer drug or ADT treatment responsiveness or prognosis of prostate cancer. The expression "kit for predicting anticancer drug or ADT treatment responsiveness or prognosis of prostate cancer" may be used interchangeably or interchangeably with "composition for predicting anticancer drug or ADT treatment responsiveness or prognosis of prostate cancer."

[0066] In addition, the present invention provides a kit for screening a prostate cancer treatment agent, comprising a preparation for measuring at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), a protein encoded by the at least one gene, or isolated circulating tumor cells (CTCs).

[0067] In the screening kit of the present invention, the prostate cancer may be metastatic castrate-resistant prostate cancer (mCRPC) that is resistant to anticancer drugs or androgen deprivation therapy (ADT).

[0068] The above kit can be used to screen for, but is not limited to, a composition for enhancing the therapeutic effect of an anticancer drug, preferably docetaxel, or an inhibitor for suppressing the progression or metastasis of cancer in mCRPC with anticancer drug resistance.

[0069] In addition, the above kit can screen for, but is not limited to, a composition for enhancing the therapeutic effect of ADT, preferably a secondary hormonal agent, and an inhibitor for suppressing the progression or metastasis of cancer in mCRPC with ADT resistance.

[0070] In addition, the present invention provides a method for providing information necessary for predicting treatment responsiveness or prognosis of prostate cancer, comprising the steps of: a) confirming the expression level of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), the expression level of a protein encoded by the gene, or the concentration of isolated circulating tumor cells (CTCs) 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.

[0071] In a method for providing information necessary for predicting treatment responsiveness or prognosis of prostate cancer of the present invention, a step of determining resistance to an anticancer drug or ADT may be additionally included based on the expression level of PSMA, PSA, EpCAM and / or AR-V7 genes, which are biomarkers of the present invention, or the concentration of CTCs, and it is preferable that the prostate cancer is metastatic castrate-resistant prostate cancer (mCRPC) that is resistant to an anticancer drug or ADT. In addition, the normal control group may be a group of mCRPC patients who do not show resistance to anticancer drug or ADT treatment.

[0072] More specifically, in the method for providing information of the present invention, in mCRPC patients who have received anticancer treatment, if they show high expression of PSMA, PSA, and EpCAM genes compared to a group of mCRPC patients who do not show resistance to anticancer drugs, they can be determined to have resistance to anticancer drugs.

[0073] In addition, in the method for providing information of the present invention, in mCRPC patients who received ADT treatment, if the expression level of PSMA, PSA, EpCAM and / or AR-V7 genes is high, or the number of CTCs is high, compared to a group of mCRPC patients who did not show resistance to ADT, it can be determined that the patient is resistant to ADT.

[0074] 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 or circulating tumor cells (CTCs), but is not limited thereto.

[0075] In addition, the present invention provides a method for screening a prostate cancer treatment agent, comprising the step of treating a sample with a candidate substance and selecting a candidate substance that inhibits the expression or activity of at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), or inhibits at least one gene selected from the group consisting of growth, proliferation, and metastasis of CTCs.

[0076] In the screening method for a prostate cancer treatment agent of the present invention, it is preferable that the prostate cancer is metastatic castrate-resistant prostate cancer (mCRPC) that is resistant to anticancer drugs or ADT.

[0077] The above anticancer agent may be a taxane series anticancer agent, preferably at least one selected from the group consisting of docetaxel, paclitaxel, and cabazitaxel, and more preferably docetaxel, but is not limited thereto.

[0078] The present invention can screen for, but is not limited to, candidate substances that can enhance the therapeutic effect of anticancer drugs, preferably docetaxel, in mCRPC with anticancer drug resistance, or candidate substances that inhibit cancer progression or metastasis. 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.

[0079] In addition, the ADT may be a secondary hormone agent. In the present invention, the secondary hormone agent may be any known secondary hormone agent without limitation, and preferably may be at least one selected from the group consisting of enzalutamide, apalutamide, bicalutamide, darolutamide, flutamide, abiratarone, ARN-509, ODM-201, and galeterone, and more preferably may be enzalutamide or abiratarone, but is not limited thereto.

[0080] The present invention can screen for, but is not limited to, a candidate substance that can enhance the therapeutic effect of docetaxel or a second-line hormonal agent in mCRPC with anticancer drug or ADT resistance, or a candidate substance that inhibits the progression or metastasis of cancer. According to the screening method of the present invention, the candidate substance to be analyzed can first be brought into contact with cancer cells that include the gene, the protein encoded by the gene, or CTCs above a cutoff value. 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, the activity of the protein, or the activity of the CTC.

[0081] 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.

[0082] 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 or circulating tumor cells (CTCs) isolated therefrom.

[0083]

[0084] 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.

[0085] 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.

[0086] 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.

[0087]

[0088] Experimental Example 1. Sample Preparation and Genetic Analysis

[0089] 1-1. Patient classification and sample collection

[0090] A total of 49 patients with mCRPC who received docetaxel treatment were recruited, and peripheral blood samples were collected from them. Among the mCRPC patients, 15 were classified into a PSA-positive experimental group (a good prognosis group) and 34 were classified into a PSA-negative experimental group (a poor prognosis group). The PSA nadir value was compared with the baseline PSA level of patients treated with docetaxel. A decrease of 50% or more from the baseline PSA level was considered a good prognosis, and a decrease of 50% or less was considered a poor prognosis. 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). The characteristics and clinical information are shown in Table 1.

[0091] In addition, a total of 60 patients with mCRPC who received secondary hormone therapy were recruited, and peripheral blood samples were collected from them. These mCRPC patients were classified into two groups: 35 patients with a positive PSA response (a good prognosis group) and 25 patients with a negative PSA response (a poor prognosis group) based on Best PSA response analysis (see Figure 6 ). 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.

[0092] The administered secondary hormonal agents were Abiraterone and Enzalutamide, and among the 109 mCRPC samples, 60 patients (62 patients) receiving secondary hormonal agents were analyzed for whom clinical prognosis could be observed.

[0093] Metastatic stage (Metastatic stage)mCRPC (N=109)Age, median, IQR, years77.0 (72-80)PSA, median, IQR, ng / ml26.1 (5.7-131)Gleason Score (%)64 (3.7)713 (11.9)≥892 (84.4)Node metastasis (%)Yes101 (92.7)No8 (7.3)Bone metastasis (%)Yes107 (98.2)No2 (1.8)Visceral metastasis (%)Yes77 (70.6)No32 (29.4)Prior therapy (%)None0 (0.0)Radiation45 (41.3)Taxane29 (26.6)GnRH agonist / antagonist109 (100.0)Antiandrogen91 (83.5)Abiraterone32(29.4)Enzalutamide30 (27.5)

[0094]

[0095] 1-2. Isolation of blood cancer cells

[0096] 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.

[0097]

[0098] 1-3. Genetic Analysis

[0099] 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.

[0100] 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.

[0101]

[0102]

[0103] Example 1. Best PSA response analysis

[0104] The recruited patient group was divided into a group with a good prognosis and a group with a poor prognosis by performing the Best PSA response analysis, a known prostate cancer diagnosis method. The analysis method of the Best PSA response is to compare the patient's baseline PSA level and the lowest PSA level (PSA Nadir) value (Baseline PSA - PSA Nadir / Baseline PSA), express it as a percentage, and then x -1 It is to quantify the value. In other words, if the Baseline PSA value shows an increase rate of 50% or more compared to the PSA Nadir value, the patient is classified as a group with a poor prognosis or a group with a poor response to drugs.

[0105] As a result, as shown in Fig. 1, the group with high gene expression levels (cut-off value: PSMA=68 copies / μl, PSA=144 copies / μl, AR=357 copies / μl, AR-V7=0.16 copies / μl, EpCAM=26.3 copies / μl, KRT19=0.39 copies / μl, CTC#=13.7 CTCs / ml) was displayed for each gene. As the values ​​of the graph went to the right (above the -50% threshold), it was found that there were many groups with high expression levels of PSMA, PSA, and EpCAM genes. The patient group with a response value of -50% or higher was determined to be a patient group with a poor prognosis after taking secondary sex hormones. Through this, 35 patients were classified into the experimental group with a positive PSA reaction (the experimental group with a good prognosis) and 25 patients were classified into the experimental group with a negative PSA reaction (the experimental group with a poor prognosis).

[0106]

[0107] Example 2. Genetic analysis related to prognosis in mCRPC patients treated with docetaxel or second-line hormonal agents.

[0108] For each experimental group for docetaxel prepared above, the number of isolated blood CTCs and genetic analysis through a heatmap were counted and shown in Figure 2.

[0109] In heatmap analysis, the expression level of each gene was displayed according to the increase in the number of CTCs, and the expression level was displayed by converting it to a log scale without a reference value of the gene. The number of CTCs was determined by performing immunofluorescence staining according to a method known in the art after separation, and the cell nucleus was stained with blue (DAPI), cytokeratin (pan-cytokeratin) green, and CD45 red. When blue and green were detected, it was determined to be a CTC, and when blue and red were detected, it was determined to be a leukocyte.

[0110] A heatmap is a representation of a data matrix converted into a z-score, with the lowest value expressed in red and the highest value expressed in bright green.

[0111] As a result, a greater number of CTCs were identified in the PSA-negative group than in the PSA-positive group. Furthermore, many subjects in the PSA-negative group showed expression of PSMA, PSA, or EpCAM. Therefore, we confirmed that PSMA, PSA, or EpCAM are expressed in mCRPC with a poorer prognosis than in mCRPC with a good prognosis after docetaxel treatment.

[0112] In addition, for each experimental group for secondary hormone therapy, the number of isolated blood CTCs and genetic analysis through a heatmap were counted and shown in Fig. 7. As shown in Fig. 7, the group was divided into a group with good PSA response and a group with poor PSA response, and each group was sorted in the direction of increasing number of CTCs. The gene expression level of each sample was also displayed in a heatmap in the sorted order, and the heatmap was expressed by converting the data matrix into a z-score, and a lower expression level was indicated in red and a higher expression level in green. In the group with a poor prognosis, the number of CTCs was measured to be much higher than in the group with a good prognosis, and even among samples with similar numbers, the expression levels of the PSMA, PSA, and AR-V7 genes were measured to be higher in the group with a poor prognosis.

[0113] As a result, a greater number of CTCs were identified in the PSA-negative group than in the PSA-positive group. Furthermore, many subjects in the PSA-negative group showed expression of PSMA, PSA, or AR-V7. Therefore, we confirmed that PSMA, PSA, or AR-V7 are expressed in mCRPC with a poorer prognosis than in mCRPC with a good prognosis after secondary hormone therapy.

[0114]

[0115] Example 3. Kaplan-Meier survival analysis

[0116] 3-1. Kaplan-Meier survival analysis for PSMA, PSA, or EpCAM expression

[0117] In each experimental group for docetaxel, the correlation between the expression of PSMA, PSA or EpCAM and the 50-month survival of each experimental group was analyzed using a Kaplan-Meier curve and is shown in Figure 3.

[0118] As a result, in PSA-Progression-Free Survival (PFS), PSMA (p-value: <0.001), PSA (p-value: 0.003), and EpCAM (p-value: <0.001) were confirmed. In Radiological-PFS, PSMA (p-value: <0.001), PSA (p-value: 0.005), and EpCAM (p-value: <0.001) were confirmed. In Clinical-PFS, PSMA (p-value: <0.001), PSA (p-value: <0.001), and EpCAM (p-value: <0.001) were confirmed. In terms of overall survival (the proportion of patients diagnosed as being expected to survive after a certain time, including deaths not caused by a specific disease as well as deaths caused by a specific disease), PSMA (p-value: 0.009), PSA (p-value: 0.003), and EpCAM (p-value: 0.002) were identified.

[0119] When the expression of PSMA, PSA, or EpCAM was high, the progression-free survival period was shorter than when it was low, and the overall survival period was also shorter.

[0120] Therefore, the above three PSMA, PSA, and EpCAM genes were determined to be useful markers for diagnosing resistance to docetaxel in mCRPC.

[0121]

[0122] 3-2. Kaplan-Meier survival analysis for AR, AR-V7, KRT19, or CTC# expression

[0123] In addition, in each experimental group for docetaxel, the correlation between the expression of AR, AR-V7, KRT19 or the number of CTCs (CTC#) and the 50-month survival of each experimental group was analyzed using a Kaplan-Meier curve and is shown in Figure 4.

[0124] As a result of judging statistical significance, the P value was 0.05 or higher, so AR, AR-V7, KRT19, or CTC# were judged to be markers that are not useful for diagnosing resistance to docetaxel.

[0125]

[0126] 3-3. Kaplan-Meier survival analysis regarding PSMA, PSA, AR-V7 expression, or number of CTCs

[0127] In addition, in each experimental group for secondary hormone therapy, the correlation between the expression of PSMA, PSA or AR-V7, the number of CTCs (hereinafter referred to as CTCs# or CTC#) and the 50-month survival of each experimental group was analyzed using a Kaplan-Meier curve and is shown in Figure 8.

[0128] As a result, in PSA-Progression-Free Survival (PFS), PSMA (p-value: 0.008), PSA (p-value: 0.002), AR-V7 (p-value: 0.046), and CTCs# (p-value: <0.001) were identified. In Radiological-PFS, PSMA (p-value: <0.001), PSA (p-value: 0.009), AR-V7 (p-value: 0.039), and CTCs# (p-value: <0.001) were identified. In Clinical-PFS, PSMA (p-value: <0.001), PSA (p-value: <0.001), AR-V7 (p-value: 0.011), and CTCs# (p-value: 0.001) were identified. In Overall survival (the proportion within a diagnosis expected to survive after a certain time, including not only deaths caused by a specific disease but also deaths not caused by a specific disease), PSMA (p-value< 0.001), PSA (p-value< 0.001), AR-V7 (p-value: 0.018), and CTCs# (p-value: 0.002) were identified.

[0129] It was confirmed that patients with high PSMA, PSA, or AR-V7 expression or a high number of CTCs had a shorter progression-free survival period and overall survival period than those with low expression.

[0130] Therefore, the three PSMA, PSA, and AR-V7 genes and the number of CTCs were determined to be useful markers for diagnosing resistance to secondary hormonal agents in mCRPC.

[0131]

[0132] 3-2. Kaplan-Meier survival analysis for AR, EpCAM, or KRT19 expression

[0133] In addition, in each experimental group for secondary hormone therapy, the correlation between the expression of AR, EpCAM, or KRT19 and the 50-month survival of each experimental group was analyzed using a Kaplan-Meier curve and is shown in Figure 9.

[0134] As a result, in PSA-PFS, AR (p-value: 0.824), EpCAM (p-value: 0.708), and KRT19 (p-value: 0.089) were identified. In Radiological-PFS, AR (p-value: 0.482), EpCA (p-value: 0.094), and KRT19 (p-value: 0.089) were identified. In Clinical-PFS, AR (p-value: 0.660), EpCAM (p-value: 0.033), and KRT19 (p-value: 0.114) were identified. Unlike the biomarker according to the present invention, the AR, EpCAM, or KRT19 genes showed a high p-value (a value higher than 0.05), and it was confirmed that they did not show statistical significance.

[0135]

[0136] Example 4. Cox survival analysis

[0137] In 49 experimental groups for docetaxel, the association between PSMA, PSA, and EpCAM gene expression and survival was confirmed again using the Cox proportional-hazards model. The results are shown in Fig. 5, and a higher HR (hazard ratio) value indicates a higher risk of death, and a lower P-value indicates higher accuracy (cut-off value: PSMA=68 copies / μl, PSA=144 copies / μl, AR=357 copies / μl, AR-V7=0.16 copies / μl, EpCAM=26.3 copies / μl, KRT19=0.39 copies / μl, CTC#=13.7 CTCs / ml).

[0138] According to Figure 5, in PSA-PFS, the HR value of EpCAM-mRNA was the highest at 3.59, and the HR value of PSMA-mRNA was the second highest at 3.20. In addition, PSA-mRNA had the third highest value at 2.64. In addition, in Radiological-PFS, the HR value of EpCAM-mRNA was the highest at 3.80, and the HR value of PSMA-mRNA was the second highest at 3.67. PSA-mRNA had the third highest value at 2.67. In addition, in clinical progression, the HR value of EpCAM-mRNA was significantly significant at 8.33, and the HR value of PSMA-mRNA was the second highest at 7.01. PSA-mRNA had the third highest value at 6.71.

[0139] Additionally, for overall survival, the HR value of PSA-mRNA was 3.95, the HR value of PSMA-mRNA was 3.25, and the HR value of EpCAM was 3.90.

[0140] Therefore, it was clearly confirmed that PSMA, PSA, and EpCAM according to the present invention showed high expression in mCRPC patients with poor prognosis even after docetaxel treatment, and had an impact on the high mortality rate of mCRPC patients treated with docetaxel.

[0141] In addition, in 60 experimental groups for secondary hormone therapy, the association between PSMA, PSA, and AR-V7 gene expression and CTCs# and survival was confirmed again using the Cox proportional-hazards model. The results are shown in Fig. 10, and a higher HR (hazard ratio) value indicates a higher risk of death, and a lower P-value indicates higher accuracy (cut-off value: PSMA=6 copies / μl, PSA=10 copies / μl, AR=250 copies / μl, AR-V7=0.136 copies / μl, EpCAM=7.45 copies / μl, KRT19=0.32 copies / μl, CTC#=10.6 CTCs / ml).

[0142] According to Fig. 10, in PSA-PFS, the HR value of CTC# was the highest at 2.67, and the HR value of PSA-mRNA was the second highest at 2.52. In addition, PSMA-mRNA showed an HR value of 2.28, and AR-V7 mRNA showed an HR value of 1.82. In addition, in Radiological-PFS, the HR value of PSMA-mRNA was the highest at 4.75, and the HR value of CTC# was the second highest at 3.24. In addition, the HR value of PSA-mRNA was 2.51, and the HR value of AR-V7 mRNA was 2.07. In addition, in clinical progression, the HR value of PSMA-mRNA was 6.30, which was significantly significant, and the HR value of PSA-mRNA was 5.59, which was the second highest. The HR value of CTC# was 3.73, and the HR value of EpCAM mRNA was 2.36.

[0143] Additionally, for overall survival, the HR value of PSMA-mRNA was 7.00, the HR value of PSA-mRNA was 6.55, and the HR value of CTC# was 3.57. In addition, the HR value of AR-V7 mRNA was 2.52, and the HR value of EpCAM mRNA was 2.99.

[0144] In the above clinical progression and overall survival, EpCAM mRNA also showed a significant HR value, but no significant HR value was confirmed in PSA-PFS and Radiological-PFS, so it was determined that it was not a consistent biomarker gene.

[0145] Therefore, it was clearly confirmed that PSMA, PSA, and AR-V7 genes and CTC counts according to the present invention were highly expressed in mCRPC patients with poor prognosis even after secondary hormonal treatment, and that they had an impact on the high mortality rate of mCRPC patients who received secondary hormonal treatment.

[0146]

[0147] Thus, according to the present invention, in the experimental group resistant to the anticancer drug docetaxel, the genes of PSMA, PSA and EpCAM were expressed at a higher level than in the experimental group not resistant to docetaxel, and it was thought that this would also have an effect on the mortality rate.

[0148] Therefore, the genes of PSMA, PSA, and EpCAM according to the present invention can be usefully utilized as a technology that can accurately determine whether mCRPC has anticancer drug resistance. Therefore, in the treatment of mCRPC, the expression of the genes can be analyzed first, and then the treatment direction can be determined accordingly, which can greatly contribute to improving the survival rate of mCRPC patients.

[0149] In addition, in the experimental group resistant to secondary hormone therapy, ADT, the genes for PSMA, PSA, and AR-V7, and the number of CTCs were expressed higher than in the experimental group not resistant to secondary hormone therapy, and it was thought that this would also affect the mortality rate.

[0150] Therefore, the genes of PSMA, PSA, and AR-V7 and the number of CTCs according to the present invention can be usefully utilized as a technology that can accurately determine whether mCRPC has resistance to secondary hormone therapy. Therefore, in the treatment of mCRPC, the expression of the genes can be analyzed first and then the treatment direction can be determined accordingly, which can greatly contribute to improving the survival rate of mCRPC patients.

[0151]

[0152] 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 predicting treatment response or prognosis of prostate cancer, comprising at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), a protein encoded by the at least one gene, or isolated circulating tumor cells (CTC).

2. In paragraph 1, A biomarker composition for predicting treatment responsiveness or prognosis of prostate cancer, characterized in that the prostate cancer is metastatic castrate-resistant prostate cancer (mCRPC).

3. In paragraph 1, A biomarker composition for predicting treatment response or prognosis of prostate cancer, characterized in that the treatment is an anticancer drug or androgen deprivation therapy (ADT) treatment.

4. In paragraph 3, A biomarker composition for predicting treatment response or prognosis of prostate cancer, characterized in that the anticancer agent is a taxane series anticancer agent.

5. In paragraph 3, A biomarker composition for predicting treatment response or prognosis of prostate cancer, characterized in that the anticancer agent is at least one selected from the group consisting of docetaxel, paclitaxel, and cabazitaxel.

6. In paragraph 1, A biomarker composition for predicting treatment responsiveness or prognosis of prostate cancer, characterized in that at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), and epithelial cell adhesion molecule (EpCAM) or a protein encoded by the at least one gene can predict treatment responsiveness or prognosis of prostate cancer.

7. In paragraph 3, A biomarker composition for predicting treatment response or prognosis of prostate cancer, characterized in that the above ADT is a secondary hormonal treatment.

8. In paragraph 7, A biomarker composition for predicting treatment response or prognosis of prostate cancer, characterized in that the secondary hormone agent is at least one selected from the group consisting of enzalutamide, apalutamide, bicalutamide, darolutamide, flutamide, abiratarone, ARN-509, ODM-201, and galeterone.

9. In paragraph 1, A biomarker composition for predicting treatment response or prognosis of prostate cancer, characterized in that the above-mentioned separated circulating tumor cells (CTCs) have a cut-off concentration value of 10.2 to 10.8 CTCs / ml.

10. In paragraph 1, A biomarker composition for predicting treatment responsiveness or prognosis of prostate cancer, characterized in that one or more genes selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), and androgen receptor variant 7 (AR-V7), a protein encoded by the one or more genes, or isolated circulating tumor cells (CTCs) can predict ADT treatment responsiveness or prognosis of prostate cancer.

11. A composition for predicting the anticancer treatment response or prognosis of prostate cancer, comprising an agent for measuring at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), a protein encoded by the at least one gene, or isolated circulating tumor cells (CTC).

12. In paragraph 11, A composition for predicting treatment responsiveness or prognosis of prostate cancer, characterized in that the above formulation comprises 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), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM) and androgen receptor variant 7 (AR-V7).

13. In paragraph 11, A composition for predicting treatment responsiveness or prognosis of prostate cancer, 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.

14. In paragraph 11, A composition for predicting treatment responsiveness or prognosis of prostate cancer, characterized in that the agent is at least one selected from the group consisting of a protein encoded by at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), or an antibody, antibody fragment, aptamer, avidity multimer, and peptidomimetics that specifically binds to a surface marker of isolated CTC.

15. In paragraph 11, A composition for predicting treatment responsiveness or prognosis of prostate cancer, 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.

16. A kit for predicting treatment response or prognosis of prostate cancer, comprising an agent for measuring one or more genes selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), a protein encoded by the one or more genes, or isolated circulating tumor cells (CTCs).

17. A kit for screening a prostate cancer treatment agent, comprising a preparation for measuring at least one gene selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), a protein encoded by the at least one gene, or isolated circulating tumor cells (CTC).

18. In paragraph 17, A kit for screening for a prostate cancer treatment agent, wherein the prostate cancer is metastatic castrate-resistant prostate cancer (mCRPC) that is resistant to anticancer drugs or androgen deprivation therapy (ADT). 19.a) A step of checking the expression level of one or more genes selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and androgen receptor variant 7 (AR-V7), the expression level of a protein encoded by the gene, or the concentration of isolated circulating tumor cells (CTC) in a sample isolated from a test subject; and b) A method for providing information necessary for predicting treatment responsiveness or prognosis of prostate cancer, comprising a step of comparing the corresponding results of the corresponding gene in a normal control sample.

20. In paragraph 19, A method for providing information necessary for predicting treatment response or prognosis of prostate cancer, wherein the prostate cancer is metastatic castrate-resistant prostate cancer (mCRPC) that is resistant to anticancer drug administration or androgen deprivation therapy (ADT).

21. In paragraph 19, A method for providing information necessary for predicting treatment responsiveness or prognosis of prostate cancer, 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.

22. In paragraph 19, A method for providing information necessary for predicting treatment response or prognosis of prostate cancer, wherein the sample isolated from the above test subject is a circulating tumor cell (CTC).

23. Treating a sample with a candidate substance and inhibiting the expression or activity of one or more genes selected from the group consisting of prostate-specific membrane antigen (PSMA), prostate specific antigen (PSA), epithelial cell adhesion molecule (EpCAM), and AR-V7 (Androgen receptor variant 7), or A method for screening a prostate cancer treatment agent, comprising the step of selecting a candidate substance that inhibits at least one selected from a group consisting of growth, proliferation, and metastasis of CTCs.

24. In paragraph 23, A method for screening a prostate cancer treatment agent, wherein the prostate cancer is metastatic castrate-resistant prostate cancer (mCRPC) that is resistant to anticancer drugs or androgen deprivation therapy (ADT).

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