KR20220020538A - Marker for diagnosis of prostate cancer - Google Patents

Abstract

The present invention relates to a marker for diagnosis and prognosis judgement of prostate cancer. According to the present invention, since an mRNA expression amount and detection amount of PSA in blood cancer cells isolated from blood, a PSA gene in the blood cancer cells which is a marker of the present invention can be usefully used for diagnosis and prognosis judgement of prostate cancer.

Description

Marker for diagnosis of prostate cancer {MARKER FOR DIAGNOSIS OF PROSTATE CANCER}

The present invention relates to a marker for the diagnosis and prognosis of prostate cancer.

Isolation of cell types or intracellular components is required for diagnostic and therapeutic purposes in the medical field, and as a preparatory tool for final purposes or other analyses in the field of research. There are currently many classification methods for cell types or intracellular components used in laboratories and clinical laboratories. The rapid isolation of different types of particles, such as viruses, bacteria, cells and multicellular entities, is a central step in a variety of applications in the areas of pharmaceutical research, clinical diagnostics and environmental analysis. Rapidly growing knowledge in drug discovery and protein research is enabling researchers to rapidly gain a better understanding of markers of protein-protein interactions, cellular signaling pathways, and metabolic processes. Such information is difficult or impossible to obtain using traditional methods such as single protein detection methods, for example, ELISA or western blotting. Therefore, the separation of cell types or intracellular components is further required.

As a method for isolating a specific target biomolecule from a biological sample such as plasma, a method using silica, glass fiber, anion exchange resin or magnetic beads is known. Among them, according to the method using magnetic beads, magnetic beads having a probe capable of binding to a target biomolecule on the surface are introduced into a sample solution to capture the target biomolecule, and the magnetic beads are separated from the sample solution again to separate the target biomolecule. extract molecules. This method of separating target biomolecules using magnetic beads (bead based separation) has already been commercialized and is widely used to separate cells, proteins, nucleic acids, or other biomolecules. For example, US Pat. No. 6,893,881 discloses a method for isolating specific target cells using antibody-coated paramagnetic beads.

Magnetophoresis separation technology using high gradient magnetic separation (HGMS) to separate these magnetic beads has a simple structure, high efficiency, easy to use, and hydrolysis compared to dielectrophoresis. It is a field that has been continuously researched for a long time because of its advantages without characteristics. Another advantage of such magnetophoresis is that the biological specificity is maintained by the biocompatible binding between the magnetic particles and the bioanalyte, and the magnetophoretic force is not affected by the media.

The conventional magnetophoretic method includes a magnetic energy source that applies a magnetic field for magnetophoretic separation of a sample having a magnetism to be separated and a magnetic microstructure region for amplifying a magnetic field gradient applied by the external magnetic energy source, A method of separating according to a magnetic density gradient by applying a magnetic field from a magnetic energy source to a sample having a magnetic field is used.

Cancer has a high mortality rate worldwide and is the second most common cause of death in Western societies after cardiovascular disease. In particular, with the aging of the population, lung cancer is increasing due to an increase in the smoking population and air pollution. The consumption of a high-fat diet has become common due to the westernization of diet, and the rapid increase in environmental pollutants and the increase in alcohol consumption has resulted in colorectal cancer, breast cancer, etc. , prostate cancer, etc. are on a continuous increase. In this situation, there is an urgent need for the creation of anticancer substances that can contribute to the promotion of human health, the improvement of the quality of healthy life, and the promotion of human health by enabling the early prevention and treatment of cancer. Innovative cancer research focusing on a deeper understanding of the underlying mechanisms of DNA damage/repair, apoptosis, tumorigenesis, and treatment in molecular terms is strongly required to conquer cancer (Varmus, 2006; Alberts, 2008, 2011) ), which could eventually be a breakthrough in cancer therapy. In most cases, malignant tumors develop in one organ (lung, liver, kidney, stomach, large intestine, rectum, etc.) and then spread to other tissues from the primary site where they first occurred. It is called metastasis. Metastasis is a phenomenon accompanying the progression of malignant tumors. As malignant tumor cells proliferate and cancer progresses, they acquire new genetic traits necessary for metastasis, then infiltrate into blood vessels and lymph glands, circulate through blood and lymph, and settle in other tissues. After that, it will proliferate.

Despite the recent increase in early diagnosis of cancer and the development of treatments such as targeted therapy, cancer still accounts for a large portion of the cause of death, and most cancers can still only be cured through surgical removal. However, even after the primary tumor is removed, it has been confirmed that local relapse and distant metastasis of cancer occur due to the involvement of many factors. Therefore, one of the most important factors in determining cancer treatment outcomes and prognosis is to determine the presence of metastasized cancer cells at the time of initial diagnosis or during treatment. Metastasis of cancer cells to lymph nodes or bone marrow is defined as “disseminated tumor cells (DTC)”, and cancer cells circulating in the peripheral blood of cancer patients are defined as “circulating tumor cells (CTC)”. These are cells that have shed from the primary or metastatic lesion. Circulating tumor cells (CTC) are not only expected to be powerful biomarkers in cancer diagnosis, treatment prognosis analysis, micrometastasis analysis, etc. Because it has advantages, it is very promising as a future cancer diagnosis method. However, since the distribution of blood cancer cells in the blood is very low at the level of 1 cancer cell per 1 billion total cells or 1 cancer cell per 10 ⁶ to 10 ⁷ white blood cells, accurate analysis is very difficult and requires a very sophisticated analysis method.

Prostate cancer is one of the most common urinary tract tumors worldwide, and is the third most common tumor after breast cancer and lung cancer. Prostate cancer is a rare disease before the age of 50, but it increases rapidly after the age of 50. Due to the recent increase in life expectancy, the number of elderly men in Korea is rapidly increasing, and prostate cancer is diagnosed at an early stage and continuous management is required to prevent deterioration. In particular, human prostate cancer appears to have a tendency to metastasize to bone, and it is known that it inevitably progresses from an androgen-dependent state to an androgen-resistant state, increasing the patient's mortality. Most prostate cancers are initially androgen dependent (AD) (Feldman BJ et al., Nat Rev Cancer 20011:34-45. and Han M et al., J Urol 2001166:416-9.). Prostate cancer cells initially need androgens in order to continue to proliferate. Upon surgical (orchiectomy) or medical (GnRH agonist or estrogen) removal of testosterone through androgen deprivation therapy (ADT), or blockade of androgen action with anti-androgens, the response of susceptible prostate cancer cells Rapid apoptosis is induced. The positive response rate is about 86% based on reduction of prostate specific antigen (PSA) and stabilization or reduction of tumor volume. Cell death, which normally occurs, occurs during the first few days to a week. However, after a positive reaction, the remaining cells undergo a period of proliferation arrest in which they do not tend to die. After 18-36 months of hormone removal and blockade, proliferation recurs in 90% of cases. Surviving cancer cells consistently become androgen-independent or unresponsive, followed by androgen-independent (AI) cell proliferation, resulting in hormone-refractory prostate cancer (HRPC) or castration resistant prostate cancer. prostate cancer, CRPC) (Scher HI, Sawyers CL. J Clin Oncol 2006 23:8253-61.). There is no currently developed treatment for CRPC, and the patient eventually dies. Androgen-independence leading to conversion to CRPC appears to be achieved by various mechanisms. Moreover, about 25% of men treated for prostate cancer relapse and require additional treatment. Currently, it is the second leading cause of cancer death among men in the United States. Early diagnosis and treatment for this disease is needed.

An object of the present invention is to develop a prostate cancer-specific marker that can be detected in blood cancer cells, in particular, a diagnostic marker that can diagnose the onset and progression of prostate cancer.

In order to achieve the above object, the present invention provides a composition for diagnosing prostate cancer.

In addition, the present invention provides a prostate cancer diagnostic kit.

In addition, the present invention is a kit for prostate cancer inhibitor screening

In addition, the present invention provides a method for providing information necessary for the diagnosis or prognosis of prostate cancer.

According to the present invention, since the mRNA expression level and detection amount of PSA in blood cancer cells isolated from blood are specific to the stage of prostate cancer, it can be usefully used for diagnosis and prognosis of prostate cancer.

1 is a schematic diagram of the entire experimental sequence for the isolation and gene analysis of blood cancer cells (CTC).
2 shows a process of manufacturing a device for separating fine particles by controlling the direction of magnetophoresis.
3 shows an apparatus for separating fine particles by controlling the direction of the prepared magnetophoresis.
4 is a diagram showing the detection rate of the PSA gene and the detected PSA expression level from prostate cancer patients.
5 is a diagram confirming the correlation between the mRNA level of the PSA gene derived from cancer cells in the blood and the PSA level in the patient's serum.

Hereinafter, a preferred embodiment of the present invention marker for diagnosing prostate cancer will be described with reference to the accompanying drawings.

In one aspect, the present invention relates to a marker for diagnosing prostate cancer comprising a PSA gene in blood cancer cells.

In one aspect, the present invention relates to a marker for predicting prognosis of prostate cancer comprising a PSA gene in blood cancer cells.

In the present invention, the term “prognostic marker, biomarker for prognosis, or prognosis diagnostic marker” refers to a substance capable of predicting/diagnosing the risk of recurrence of prostate cancer, which recurs and develops after treatment of prostate cancer. A polypeptide or nucleic acid showing an increase or decrease in expression with a significant correlation with the likelihood of occurrence of hormone-refractory prostate cancer (HRPC) or castration resistant prostate cancer (CRPC) organic biomolecules such as (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.). For the purpose of the present invention, the marker for predicting the prognosis of prostate cancer is a gene PSA in blood cancer cells, which is a gene with an increased level of mRNA expression specifically for prostate cancer. Preferably, such a marker includes not only a gene but also DNA or mRNA complementary to any one of the markers.

In one aspect, the present invention relates to a composition for diagnosing prostate cancer, comprising an agent for measuring the expression level of a prostate specific antigen (PSA) gene in circulating tumor cells (CTC).

In one embodiment, the prostate cancer may be in Localized stages or Metastatic stages, metastatic hormone-sensitive prostate cancer (mHSPC) or metastatic castrate-resistant prostate cancer (mHSPC). resistant prostate cancer (mCRPC) is more preferable.

In one embodiment, the composition may include an agent for measuring the expression level of the PSA gene at the mRNA level or the protein level of the PSA gene, and more preferably include an agent for measuring the level of PSA gene mRNA in blood cancer cells Do.

In one embodiment, the agent measured at the mRNA level is a nucleic acid sequence of the gene, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair that specifically recognizes the nucleic acid sequence and a fragment of the complementary sequence, a probe, It may be a primer pair and a probe, and the measurement is 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, or ddPCR may be performed by one or more methods selected from the group consisting of.

In one embodiment, the agent measured at the protein level is an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetics that specifically recognizes the full protein length of a gene or a fragment thereof ), and the measurement may be Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay. , complement fixation assay (Complement Fixation Assay), FACS, mass spectrometry, or one or more methods selected from the group consisting of protein microarray.

In one embodiment, the gene is a device (biochip) for separating fine particles by controlling the direction of magnetophoresis previously registered by the applicant ('10-1622342', 'a device for separating fine particles using magnetophoresis and In the isolated blood cancer cells, the expression was increased specifically for the onset and stage of prostate cancer, and in the localized stages, each patient's serum PSA and PSA mRNA in CTC Although there was no correlation, in the metastatic group, mHSPC and mCRPC, the PSA level in the serum of each patient and the PSA mRNA level in the CTC increased in the same pattern.

As used herein, the term "detection" or "measurement" means quantifying the level (concentration) of a detected or measured object.

As used herein, the term "primer" is a nucleic acid sequence having a short free 3 hydroxyl group, capable of forming a base pair with a complementary template and starting for template strand copying. It refers to a short nucleic acid sequence that functions as a point. The primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerate or reverse transcriptase) and the four different nucleoside triphosphates in an appropriate buffer and temperature.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases in length that can form specific binding to mRNA, and is labeled to indicate the presence or absence of specific mRNA can be checked. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. In the present invention, hybridization is performed using a probe complementary to the PSA gene, and the degree of gene expression in CTC can be diagnosed based on whether hybridization occurs. The selection of suitable probes and hybridization conditions may be modified based on those known in the art, and therefore, the present invention is not particularly limited thereto.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution with one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phossotriesters, phosphoro amidates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

In the present invention, suitable conditions for hybridizing a probe with a cDNA molecule can be determined in a series of processes by an optimization procedure. These procedures are carried out as a series of procedures by those skilled in the art to establish protocols for use in the laboratory. For example, conditions such as temperature, concentration of components, hybridization and washing times, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and M.L.M. Anderson, NucleicAcidHybridization, Springer-Verlag New York Inc. N.Y. (1999). For example, high stringency among the stringent conditions is 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and 0.1×SSC (standard saline citrate)/0.1% SDS at 68° C. It means washing under conditions. Alternatively, high stringency conditions mean washing at 48° C. in 6×SSC/0.05% sodium pyrophosphate. Low stringency conditions mean washing at 42° C. in, for example, 0.2×SSC/0.1% SDS.

As used herein, the term “antibody” refers to a specific protein molecule directed against an antigenic site as a term known in the art. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a protein expressed in the PSA gene, which is a marker of the present invention, and the method for preparing the antibody may be prepared using a well-known method. This also includes partial peptides that can be made from the protein. The form of the antibody of the present invention is not particularly limited, and a part thereof is also included in the antibody of the present invention as long as it has a polyclonal antibody, a monoclonal antibody, or antigen-binding property, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention includes a special antibody such as a humanized antibody.

In one aspect, the present invention relates to a pharmaceutical composition for inhibiting or treating prostate cancer, comprising an inhibitor of PSA expression in CTC as an active ingredient.

The pharmaceutical composition according to the present invention may include any material capable of inhibiting the expression of PSA in CTC.

In the present invention, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause allergic reactions or similar reactions such as gastrointestinal disorders and dizziness when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like, and carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like. and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition according to the present invention may be formulated in a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms according to known methods, and as a representative dosage form for parenteral administration, an isotonic aqueous solution or suspension is preferred as an injectable dosage form. Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving it in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules. The pharmaceutical composition formulated in the above manner may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the “administration” refers to introducing a predetermined substance into a patient by any suitable method. and the route of administration of the substance can be administered through any general route as long as it can reach the target tissue. As used herein, the term “effective amount” refers to an amount exhibiting a preventive or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may vary depending on various factors such as the type and severity of the patient's disease, age, sex, weight, sensitivity to drugs, the type of current treatment, administration method, target cell, etc. can be easily determined by experts in In addition, the pharmaceutical composition of the present invention may be administered in combination with a conventional therapeutic agent, may be administered sequentially or simultaneously with the conventional therapeutic agent, and may be administered single or multiple. Preferably, in consideration of all of the above factors, an amount capable of obtaining the maximum effect with a minimum amount without side effects can be administered, more preferably from 1 to 10000 μg/weight kg/day, even more preferably from 10 to 1000 It can be administered repeatedly several times a day at an effective dose of mg/kg body weight/day.

In one embodiment, the PSA expression inhibitor may include a chemical substance, nucleotide, antisense, siRNA oligonucleotide or natural product extract as an active ingredient, and more preferably, a sequence complementary to the nucleotide sequence of the gene The eggplant may include an antisense or small interference RNA (siRNA) oligonucleotide as an active ingredient.

As used herein, the term "repression of expression" means to cause a decrease in expression (to mRNA) or translation (to protein) of a target gene, preferably by which the target gene expression becomes undetectable or insignificant. means to exist as

As used herein, the term "small interfering RNA (siRNA)" refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (International Patent Publication Nos. 00/44895, 01/36646, 99/32619, 01 see /29058, 99/07409 and 00/44914). Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knock-down method or as a gene therapy method. The siRNA molecule of the present invention may have a structure in which the sense strand (sequence corresponding to the mRNA sequence of PSA) and the antisense strand (sequence complementary to the mRNA sequence of PSA) are positioned opposite to each other to form a double-stranded structure, The siRNA molecule of the present invention may have a single-stranded structure having self-complementary sense and antisense strands. Furthermore, siRNA is not limited to the complete pairing of double-stranded RNA parts that are paired with each other, but pairs are formed by mismatch (corresponding bases are not complementary), bulges (there is no base corresponding to one chain), etc. There may be parts that are not achieved. In addition, as long as the siRNA end structure can inhibit the expression of the PSA gene by the RNAi effect, both a blunt end or a cohesive end are possible, and the cohesive end structure includes a 3'-end protrusion structure and a 5'-end structure. Both protrusion structures are possible. In addition, the siRNA molecule of the present invention may have a form in which a short nucleotide sequence is inserted between the self-complementary sense and antisense strands, and in this case, the siRNA molecule formed by expression of the nucleotide sequence is used for intramolecular hybridization Thus, a hairpin structure is formed, and as a whole, a stem-and-loop structure is formed. This stem-and-loop structure can be processed in vitro or in vivo to generate active siRNA molecules capable of mediating RNAi. Methods for preparing siRNA include direct synthesis of siRNA in a test tube and introduction into cells through transformation, and transfection of an siRNA expression vector or PCR-derived siRNA expression cassette prepared so that siRNA is expressed in the cell. There is a method of converting or infecting.

In one embodiment, the composition of the present invention comprising a gene-specific siRNA may include an agent that promotes the influx of the siRNA into a cell. In general, an agent that promotes nucleic acid influx can be used for the agent that promotes the influx of siRNA into the cell. For example, a parent of one of a number of sterols including cholesterol, cholate and deoxycholic acid using liposomes. It may be formulated with an oily carrier. Also poly-L-lysine (poly-L-lysine), spermine (spermine), polysilazane (polysilazane), polyethylenimine (PEI: polyethylenimine), polydihydroimidazolenium (polydihydroimidazolenium), polyallylamine (polyallylamine) ), a cationic polymer such as chitosan may be used, and succinylated PLL (succinylated PLL), succinylated PEI (succinylated PEI), polyglutamic acid, polyaspartic acid (polyaspartic acid), polyacrylic acid (polyacrylic acid), polymethacylic acid (polymethacylic acid), dextran sulfate (dextran sulfate), heparin (heparin), anionic polymers such as hyaluronic acid (hyaluronic acid) Available.

In one embodiment, when an antibody specific for the protein is used as a substance for reducing the expression and activity of the PSA protein, the antibody is directly or indirectly coupled to an existing therapeutic agent or through a linker (eg, covalently) can be combined).

As used herein, the term "antisense oligonucleotide" refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to a specific mRNA sequence, and binds to a complementary sequence in mRNA to convert PSA into protein. The antisense sequence of the present invention is complementary to PSA mRNA and refers to a DNA or RNA sequence capable of binding to PSA mRNA, translation of PSA mRNA, translocation into the cytoplasm, and maturation ) or all other essential activity for overall biological function.In addition, the antisense nucleic acid can be modified at one or more bases, sugars or backbone positions to enhance efficacy (De Mesmaeker et al. ., Curr Opin Struct Biol., 5, 3, 343-55, 1995).The nucleic acid backbone is phosphorothioate, phosphotriester, methyl phosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic Can be modified by the linkage between sugars, etc. In addition, the antisense nucleic acid can include one or more substituted sugar moieties.Antisense nucleic acid can include a modified base.The modified base includes hypoxanthine. , 6-methyladenine, 5-methylpyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosyl HMC, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, and 2,6-diaminopurine. , the antisense nucleic acid of the present invention can be chemically combined with one or more moieties or conjugates that enhance the activity and cell adsorption of the antisense nucleic acid.Cholesterol moiety, cholesteryl moiety, cholic acids, thioethers, thiocholesterols, fatty chains, phospholipids, fat soluble moieties such as, but not limited to, polyamines, polyethylene glycol chains, adamantane acetic acid, palmityl moieties, octadecylamine, hexylaminocarbonyl-oxycholesterol moieties, and the like. Oligonucleotides comprising a fat-soluble moiety and methods of preparation are well known in the art (US Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid may increase the stability to nucleases and increase the binding affinity between the antisense nucleic acid and the target mRNA. Antisense oligonucleotides can be synthesized in vitro by a conventional method and administered in vivo, or antisense oligonucleotides can be synthesized in vivo. An example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to use a vector with the origin of the recognition site (MCS) in the opposite direction to allow the antisense RNA to be transcribed. Such antisense RNA preferably has a translation stop codon in the sequence so that it is not translated into the peptide sequence.

In one aspect, the present invention relates to a prostate cancer diagnostic kit comprising the composition of the present invention.

In one embodiment, the prostate cancer may be metastatic hormone-positive prostate or metastatic castration-resistant prostate cancer.

In one embodiment, 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. there is. For example, it may include PCR primers for amplifying a relevant region of genomic DNA. The kit may include probes of genetic factors useful for pharmacogenomic profiling. In addition, in the use of such a kit, the labeled oligonucleotide can be easily identified during analysis.

In one embodiment, the kit may further contain a labeling material such as a DNA polymerase and dNTPs (dGTP, dCTP, dATP and dTTP), a fluorescent material, and the like.

In one aspect, the present invention relates to a prostate cancer inhibitor screening kit comprising an agent for measuring the expression level of the PSA gene in blood cancer cells at the mRNA or protein level.

In one embodiment, the prostate cancer may be metastatic hormone-positive prostate or metastatic castration-resistant prostate cancer.

In one embodiment, the kit can screen for inhibitors that inhibit the progression or metastasis of prostate cancer.

In one aspect, the present invention comprises the steps of determining the expression level of the mRNA or protein of the PSA gene in a sample isolated from a test subject; And it relates to a method of providing information necessary for the diagnosis or prognosis of prostate cancer, comprising the step of comparing the result with the corresponding gene of the normal control sample.

In one embodiment, the method may further include preparing a sample separated from the test subject by isolating blood cancer cells from the test subject's blood sample using a biochip.

In one embodiment, the sample isolated from the test subject may be a blood cancer cell, and more preferably, a blood cancer cell isolated from the blood isolated from the test subject using a biochip, wherein the biochip is a diaphoretic and magnetophoretic sample of the present invention. A device for separating fine particles by controlling the direction, that is, 'a device for separating fine particles using magnetophoresis and a method for separating fine particles using the same', which is '10-1622342' previously registered by the present applicant, is preferred.

In one embodiment, the prostate cancer may be metastatic hormone-positive prostate cancer or metastatic castration-resistant prostate cancer.

In one embodiment, the method may further comprise distinguishing stages of prostate cancer according to the mRNA expression level of the PSA gene, wherein the stages of prostate cancer are localized stages T2, T3 and T4, mHSPC and mCRPC can be

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

manufacturing example. Manufacture of a device that separates fine particles by controlling the direction of magnetophoresis

To form a microfluidic channel, a Ti/Cu/Cr seed layer was deposited on a 0.7 mm thick bottom glass substrate (Borofloat™, Howard Glass Co, Worchester, MA), a pattern was formed through photoresist, and the plating process was performed. A 30 μm thick ferromagnetic nickel wire was formed through the

Two ferromagnetic nickel wires were included in the lower glass substrate of the microfluidic channel part for separation, of which one wire was included at an inclination angle of 57° with respect to the flow direction of the sample, and the other wire was separated after proceeding at 71° After changing the inclination angle to 113° at the distal end of the microfluidic channel, the inclination angle was again changed to 90° to form a pattern.

In order to reduce the adhesion of cells to the surface of the microfluidic channel, the ferromagnetic nickel wire was configured to be 100 μm apart from the channel surface of the microfluidic channel for separation. A lower substrate including a ferromagnetic nickel wire as a magnetic structure was prepared by removing the photoresist, applying an epoxy adhesive, and leveling the surface.

After that, SU-8 was formed on the upper glass substrate in a microfluidic channel pattern, and a sample injection part was made using Nitril rubber O-rings (size 001-1/2, McMaster-Carr, IL, USA) to prepare the upper glass substrate. The microparticle separator using magnetic flow was finally completed by bonding the prepared lower glass substrate with UV adhesive (1187-M, DYMAX Co, Torrington, CT).

Example 1. Prostate Cancer Patient Classification, Clinical Data and Sample Collection

Five samples collected from 5 control patients without cancer and 64 samples collected from 58 prostate cancer patients were treated with localized disease according to the summary stage classification of the SEER program of the National Cancer Institute. Localized stages (T2, T3 and T4) and metastatic stages (metastatic hormone-sensitive prostate cancer (mHSPC)) and metastatic castrate belonging to progressive disease due to distant metastases -resistant prostate cancer, mCRPC)), and clinical data were collected and analyzed (Table 1).

Example 2. Separation of blood cancer cells from samples

Whole blood collected from the above patients was diluted 1 to 5 with PBS solution (Invitrogen, USA) and anticoagulated (Heparin-Agarose, H-1027, Sigma Diagnostics, USA) to prepare a blood sample and , was pre-treated by injecting EpCAM-specific antigen-reactive immunomagnetic nanobeads. The sample thus prepared was injected at a rate of 8 μl/h into the sample inlet of a device for separating microparticles by controlling the directions of dielectrophoresis and magnetophoresis prepared in Preparation Example at a rate of 8 μl/h to separate blood cancer cells (CTCs).

Example 3. PSA in CTC and PCA in Serum Correlation Analysis

After aggregating and disrupting the blood cancer cells isolated in Example 2, the cell lysate and magnetic beads having OligodT ₂₅ attached to the surface were mixed to form the poly-A tail of mRNA and the beads in the lysate. T ₂₅ was bound. Thereafter, the magnetic beads to which mRNA was bound were washed twice with wash buffer A type (removal of cell suspension) and twice with wash buffer type B (removal of other cell suspensions stuck between mRNA and mRNA stabilization). The washed mRNA-magnetic beads were desorbed with Tris-HCL buffer, and the desorbed mRNA was synthesized as cDNA (complementary DNA). Multiplex PCR was performed using the synthesized cDNA, and the PSA gene was detected by the ddPCR method of the conditions shown in Table 2 below. Here, since the ddPCR method measures an absolute value rather than a relative value, a threshold is set when measuring gene expression (the reference value of PSA 0.01), and the sample (patient) corresponding to the reference value or higher is measured for each gene. was considered to be As the reference value, a value greater than or equal to the average measured value of the control sample was used. The correlation between the PSA mRNA level in the detected blood cancer cells and the PSA level in the serum among the clinical data confirmed in Table 1 was analyzed.

The CTC-derived mRNA PSA gene expression levels of the patients were derived by liner fitting from the smallest to the largest, and then liner fitting the serum PSA protein values for each patient.

As a result of confirming the detection rate and expression level of the PSA gene through the mRNA level isolated from CTC, the PSA mRNA level of CTC increased as the clinical stage increased. In particular, the PSA gene expression level and detection rate increased in mHSPC (38.47). The expression level was increased by 3428 times with localized stage (0.07462 copies/ul) and mHSPC (255.833 copies/ul). In addition, as a result of confirming the correlation between serum PSA levels and CTC PSA mRNA levels, there was no correlation between each patient's serum PSA and CTC PSA mRNA levels in the localized stages, but the metastatic group mHSPC and In mCRPC, each patient's serum PSA level and CTC PSA mRNA level showed a pattern in which the same increased (FIG. 5).

Claims (16)

A composition for diagnosing prostate cancer, comprising an agent for measuring the expression level of a PSA (prostate specific antigen) gene in circulating tumor cells (CTC). According to claim 1, Localized stage (Localized stages) or metastatic stage (Metastatic stages) of prostate cancer, prostate cancer diagnostic composition. According to claim 1, metastatic hormone-sensitive prostate cancer (metastatic hormone-sensitive prostate cancer, mHSPC) or metastatic castrate-resistant prostate cancer (metastatic castrate-resistant prostate cancer, mCRPC), prostate cancer diagnostic composition. The composition for diagnosing prostate cancer according to claim 1, comprising an agent for measuring the expression level of the PSA gene at the mRNA level or the protein level of the PSA gene. According to claim 4, wherein the agent measured at the mRNA level, a nucleic acid sequence of the gene, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair that specifically recognizes the nucleic acid sequence and a fragment of the complementary sequence, a probe, Or a primer pair and a probe, a composition for diagnosing prostate cancer. According to claim 5, wherein the measurement is polymerase chain reaction, real-time RT-PCR (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 or ddPCR is performed by one or more methods selected from the group consisting of, prostate cancer diagnostic composition. The method according to claim 6, wherein the agent measured at the protein level is an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetic ( peptidomimetics), a composition for diagnosing prostate cancer. The method of claim 7, wherein the measurement is Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay (Immunoprecipitation assay) , Complement Fixation Assay (Complement Fixation Assay), FACS, mass spectrometry, or one or more methods selected from the group consisting of protein microarray, prostate cancer diagnostic composition. A prostate cancer diagnostic kit comprising the composition of claim 1 . A kit for screening a prostate cancer inhibitor, comprising an agent for measuring the expression level of the PSA gene in blood cancer cells at the mRNA or protein level. The kit for screening a prostate cancer inhibitor according to claim 10, which is metastatic hormone-positive prostate cancer or metastatic castration-resistant prostate cancer. The kit for screening a prostate cancer inhibitor according to claim 10, which screens an inhibitor that inhibits the progression or metastasis of prostate cancer. a) determining the expression level of the mRNA or protein of the PSA gene in the sample isolated from the test subject; and
b) a method for providing information necessary for the diagnosis or prognosis of prostate cancer, comprising the step of comparing the result with the corresponding gene in the normal control sample. The method according to claim 13, wherein the sample isolated from the test subject is a blood cancer cell, providing information necessary for diagnosis or prognosis of prostate cancer. 15. The method of claim 14, which is a blood cancer cell isolated from blood isolated from a test subject using a biochip, and provides information necessary for diagnosis or prognosis of prostate cancer. The method for providing information necessary for the diagnosis or prognosis of prostate cancer according to claim 13, which is metastatic hormone-positive prostate cancer or metastatic castration-resistant prostate cancer.

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