Diagnostic marker and therapy resistance marker for urothelial cancer

Abstract

To enable detection of urothelial cancer, to provide a diagnostic marker for urothelial cancer, to enable prediction as to whether a patient is resistant to BCG therapy, and to provide a BCG therapy resistance marker.SOLUTION: Methods of detecting urothelial cancer diagnostic marker and / or BCG therapy resistance marker are provided, the methods comprising a step of detecting PRAME of a sample collected from a subject, where the sample is of a urothelial tumor or of urinary cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a diagnostic marker for urothelial carcinoma and a marker for resistance to BCG therapy. [Background technology]

[0002] Urothelial carcinoma is divided into bladder cancer, renal pelvis cancer, and ureter cancer depending on the site of origin, with bladder cancer accounting for approximately 90% of all cases. The main initial symptoms are blood in the urine and pain during urination, and as the disease progresses, symptoms such as severe pain in the lower back, back, and flank may occur.

[0003] Bladder cancer is mainly classified into non-muscle invasive and muscle invasive types based on the depth of invasion. Currently, the treatment methods of choice for urothelial cancer are anticancer drugs, transurethral resection of the bladder tumor (TURBT), and radical cystectomy. For non-muscle invasive bladder cancer, intravesical instillation of attenuated Mycobacterium bovis (BCG) is widely used.

[0004] PRAME is expressed in malignant melanoma and was discovered as an antigen recognized by cytotoxic T lymphocytes. PRAME expression is basically restricted to non-neoplastic testes and is rarely observed in other normal somatic tissues. Due to these properties, PRAME has been shown to be useful as a diagnostic marker for several malignant tumors (Patent Document 1). It has been reported that PRAME is also expressed in urothelial carcinoma (Non-Patent Document 1), but there is little histological information, and its usefulness as a diagnostic marker for urothelial carcinoma was unknown. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2020-91175

[0006] [Non-Patent Document 1] Kaczorowski M. et al., “PRAME Expression in Cancer.A Systematic Immunohistochemical Study of >5800 Epithelial and Nonepithelial Tumors”, Am J Surg Pathol, 46(2022) Summary of the Invention [Problem to be solved by the invention]

[0007] There is a need for diagnostic markers for urothelial carcinoma. Diagnosis of urothelial carcinoma is primarily performed by urine cytology, but there are few markers useful for urine cytology, and false-negative results are frequent. Therefore, there is a particular need for markers useful for urine cytology.

[0008] Furthermore, there are groups of people who are resistant to BCG treatment, which is the cause of poor prognosis in cancer patients, but it has been difficult to predict resistance to BCG treatment.

[0009] Therefore, an objective of the present invention is to detect urothelial cancer and provide a diagnostic marker for urothelial cancer.Another objective of the present invention is to predict whether a subject is resistant to BCG therapy and to provide a BCG therapy resistance marker. [Means for solving the problem]

[0010] As a result of extensive research, the present inventors have found that PRAME can be a diagnostic marker for urothelial carcinoma and a marker for resistance to BCG therapy. The present invention includes the following aspects. Section 1. A method for detecting a diagnostic marker for urothelial cancer, comprising the step of detecting PRAME in a specimen collected from a subject, wherein the specimen is a urothelial tumor or urinary cells. Section 2. A method for assisting in the diagnosis of urothelial cancer, comprising the steps of measuring the expression level of PRAME in a sample collected from a subject, and determining that the subject has urothelial cancer if the expression level exceeds a standard value, wherein the sample is a urothelial tumor or urinary cells. Section 3. A method for detecting a BCG therapy resistance marker for urothelial cancer, comprising the step of detecting PRAME in a sample collected from a subject, wherein the sample is a urothelial tumor or urinary cells. Section 4. A method for assisting in the diagnosis of resistance to BCG treatment of urothelial cancer, comprising the steps of measuring the expression level of PRAME in a sample collected from a subject, and determining that the subject is resistant to BCG treatment of urothelial cancer if the expression level exceeds a reference value, wherein the sample is a urothelial tumor or urinary cells. Section 5. A test reagent for detecting PRAME in a sample collected from a subject as a biomarker for urothelial carcinoma, the test reagent comprising an antibody or nucleic acid for detecting PRAME. Section 6. A test reagent for detecting PRAME in a sample collected from a subject as a BCG therapy resistance marker, the test reagent comprising an antibody or nucleic acid for detecting PRAME. Section 7. A device for detecting urothelial carcinoma, comprising a processing unit, The processing unit obtaining a PRAME measurement value for a sample collected from the subject; The obtained measurement value is compared with the reference value, A detection device that outputs a display indicating that the subject has urothelial carcinoma if the acquired measurement value is higher than the reference value, and / or outputs a display indicating that the subject does not have urothelial carcinoma if the acquired measurement value is lower than the reference value. Section 8. A diagnostic device for BCG therapy resistance, comprising a processing unit, The processing unit obtaining a PRAME measurement value for a sample collected from the subject; The obtained measurement value is compared with the reference value, A diagnostic device that outputs a display indicating that the subject is resistant to BCG treatment for urothelial carcinoma when the obtained measurement value is higher than the reference value, and / or outputs a display indicating that the subject is not resistant to BCG treatment for urothelial carcinoma when the obtained measurement value is lower than the reference value. Section 9. Item 9. The method, test reagent or device according to any one of Items 1 to 8, wherein the specimen is urinary cells. Section 10. Use of PRAME present in urothelial tumors or urinary cells as a diagnostic marker for urothelial carcinoma. Section 11. Use of PRAME present in urothelial tumors or urinary cells as a marker for resistance to BCG therapy in urothelial carcinoma. Section 12. A diagnostic marker for urothelial carcinoma consisting of PRAME. Section 13. PRAME is a marker of resistance to BCG therapy in urothelial carcinoma. [Effects of the Invention]

[0011] Detecting PRAME in urothelial tumors or urinary cells can detect urothelial cancer, and detecting PRAME in urothelial tumors or urinary cells can predict whether a subject is resistant to BCG therapy. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows an example of an overview of a system 1000 for detecting urothelial carcinoma and a system 2000 for diagnosing resistance to BCG therapy. [Figure 2] 1 shows an example of a block diagram of a device 10 for detecting urothelial carcinoma and a device 20 for diagnosing resistance to BCG therapy. [Figure 3] An example of the operation of the urothelial cancer detection device 10 will be described. [Figure 4] An example of the operation of the diagnostic device 20 for BCG therapy resistance will be described. [Figure 5] FIG. 1 shows the PRAME mRNA expression levels in bladder tumors when subjects diagnosed with bladder cancer were classified into non-muscle invasive and muscle invasive. [Figure 6] FIG. 1 shows the PRAME mRNA expression levels in bladder tumors of subjects diagnosed with non-muscle invasive bladder cancer and with a poor prognosis. [Figure 7] FIG. 1 shows progression-free survival and disease-specific survival according to PRAME mRNA expression levels in bladder tumors of subjects diagnosed with non-muscle-invasive bladder cancer. [Figure 8] FIG. 1 shows the recurrence-free survival and progression-free survival of subjects with non-muscle-invasive bladder cancer who underwent BCG treatment, according to the PRAME mRNA expression level in bladder tumors. [Figure 9] FIG. 1 shows the results of Cox regression analysis of PRAME mRNA expression levels and progression-free survival in subjects with non-muscle-invasive bladder cancer who underwent BCG treatment. [Figure 10] FIG. 1 shows bladder tissue stained for PRAME protein by immunohistochemical staining. DETAILED DESCRIPTION OF THE INVENTION

[0013] <Terminology> As used herein, the term "PRAME" refers to a type of antigen known as preferentially expressed antigen in melanoma, and has been reported to be unexpressed in normal tissues other than the testis. PRAME is also known as CT130, MAPE, OIP-4, or OIP4, and is expressed from a gene registered with the National Center for Biotechnology Information under Gene ID: 23532. Diagnostic markers and BCG therapy resistance markers consisting of PRAME include proteins or mRNA expressed from the gene. Furthermore, diagnostic markers and BCG therapy resistance markers also include variants thereof, in addition to proteins or mRNA expressed from the gene.

[0014] As used herein, the term "urothelial tumor" is not limited as long as it is a tumor derived from cells present in the urothelium. Urothelial tumors include benign tumors and may also include malignant tumors such as urothelial carcinoma.

[0015] As used herein, the term "urothelial cancer" refers to cancer derived from cells present in the urothelium, and may include bladder cancer, ureter cancer, and the like.

[0016] As used herein, the term "resistance" refers to a state in which, even when a treatment that is considered to be effective is administered, the treatment effect is small or the treatment effect is weakened and symptoms recur.

[0017] 1. Diagnostic markers and BCG treatment resistance markers, and methods for detecting them 1.1 Diagnostic markers and BCG treatment resistance markers The present invention relates to a biomarker for urothelial cancer and a detection method thereof. The detection method comprises detecting PRAME contained in urothelial tumor or urinary cells collected from a subject. The PRAME protein or PRAME mRNA contained in urothelial tumor or urinary cells can be detected as a biomarker (diagnostic marker) for determining whether the subject has urothelial cancer. Furthermore, the PRAME protein or PRAME mRNA contained in urothelial tumor or urinary cells can be detected as a biomarker (BCG treatment resistance marker) for determining whether the subject is resistant to BCG treatment for urothelial cancer.

[0018] 1.2 Detection Method Detection of PRAME contained in urothelial tumors or urinary cells is not limited as long as it is performed using urothelial tumor cells, urothelial tumor tissue, or urinary cells collected from a subject. Urothelial tumor cells or urothelial tumor tissue can be collected, for example, by surgical resection or biopsy from a primary or metastatic lesion, endoscopic resection or biopsy, or isolation from pleural effusion. Determination of whether a tissue is tumorous or normal can be performed by macroscopic observation, microscopic observation, or the like. Alternatively, tumor tissue may be determined using cell proliferation activity as an indicator. When determining whether a tissue is tumorous using cell proliferation activity as an indicator, for example, if the labeling index of BrdU or Ki-67 protein in the tissue to be tested is higher than that of normal tissue, the tissue to be tested can be determined to be tumorous. Urinary cells are preferred as the specimen because they are inexpensive and non-invasive.

[0019] Urothelial tumor cells or urothelial tumor tissue or urinary cells collected from a subject are pretreated according to the method for detecting PRAME.

[0020] PRAME contained in urothelial tumors or urinary cells can be detected as protein or mRNA.

[0021] Methods for detecting PRAME as a protein include known methods such as immunostaining and Western blotting, and methods for detecting PRAME as mRNA include known methods such as in situ hybridization, RT-PCR (including quantitative RT-PCR), microarrays, and RNA-Seq.

[0022] When immunostaining or in situ hybridization is performed using urothelial tumor tissue, the urothelial tumor tissue is pretreated by fixing it with a known fixative such as formalin or paraformaldehyde, followed by preparation of a paraffin-embedded block. Alternatively, the urothelial tumor tissue is embedded in a resin for preparing frozen blocks, such as OCT Compound (registered trademark), either with or without fixation, to prepare a frozen block. The prepared paraffin-embedded block or frozen block is then thinly sliced ​​to prepare tissue sections, which are then subjected to immunostaining or in situ hybridization. The urothelial tumor tissue embedded in the block may consist of only the tumor tissue, but may also contain, for example, normal tissue.

[0023] When immunostaining or in situ hybridization is performed using urothelial tumor cells or urinary cells, the cells are smeared or collected on a slide glass and fixed with formalin, paraformaldehyde, ethanol, or the like as a pretreatment.

[0024] When detecting PRAME as a protein by Western blotting or the like, urothelial tumor tissue, urothelial tumor cells, or urinary cells are lysed in a predetermined lysis buffer as a pretreatment. The sample lysed in the lysis buffer is used as the test sample. When urinary cells are used, the lysis step may be omitted.

[0025] When detecting PRAME as mRNA using RT-PCR, microarray, RNA-Seq, etc., total RNA or mRNA is extracted from urothelial tumor tissue, urothelial tumor cells, or urinary cells as a pretreatment. If necessary, the extracted total RNA or mRNA may be used as a template for reverse transcription to synthesize complementary DNA (cDNA). The total RNA, mRNA, or cDNA is used as the test sample.

[0026] The primary antibody used to detect PRAME by immunostaining or Western blotting is not limited as long as it can detect PRAME. Examples include SIGMA-ALDRICH's anti-PRAME antibody produced in rabbit Prestige Antibodies (registered trademark) Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution (HPA045153), anti-MAPE (C-TERMINAL) antibody produced in rabbit purified immunoglobulin, buffered aqueous solution (SAB1306515), anti-PRAME antibody produced in rabbit purified immunoglobulin, buffered aqueous solution (SAB1401590), anti-PRAME antibody produced in mouse purified immunoglobulin, buffered aqueous solution (SAB1407222), and anti-PRAME antibody produced in rabbit affinity isolated antibody (SAB2108300). The primary antibody bound to PRAME can be detected by a reaction between an enzyme-labeled secondary antibody that binds to the primary antibody and the enzyme and its substrate. When immunostaining is performed, the tissue section may be deparaffinized and immersed in water, and then treated with a proteolytic enzyme such as trypsin before immunostaining.

[0027] Methods for preparing probes used in in situ hybridization are known, and commercially available probes may also be used.

[0028] Commercially available primers (which may include a probe in the case of quantitative RT-PCR) can be used for RT-PCR, and commercially available microarrays can also be used.

[0029] RNA-Seq can obtain the number of PRAME mRNA reads using a next-generation sequencer (for example, manufactured by Illumina).

[0030] When detecting PRAME by immunostaining or in situ hybridization, the presence or absence of PRAME can be detected by a human observing the tissue specimen subjected to immunostaining or in situ hybridization using a microscope, slide scanner, or the like. When an immunostaining or in situ hybridization signal is confirmed in tumor cells in the tissue specimen or in urinary cells, PRAME can be determined (confirmed) to have been detected. When even one cell containing PRAME is detected in the cells, it may be determined that "PRAME has been detected" or that "PRAME expression is positive." Alternatively, for example, when the number of cells present in a predetermined section of the microscope or slide scanner is taken as 100%, it may be determined that "PRAME has been detected" or that "PRAME expression is positive" when 10% or more, preferably 5% or more, and more preferably 1% or more of the cells contain PRAME.

[0031] When detecting PRAME by Western blotting, RT-PCR, or RNA-Seq, if PRAME is detected in a sample extracted from tumor tissue, tumor cells, or urinary cells, it may be determined that "PRAME is detected" or "PRAME expression is positive." Alternatively, by comparing the amount of PRAME protein or PRAME mRNA in a test sample derived from tumor cells or tumor tissue with that derived from normal cells or normal tissue, it may be determined that "PRAME is detected" or "PRAME expression is positive" if the amount of PRAME protein or PRAME mRNA in the test sample derived from tumor cells or tumor tissue is higher than the amount of PRAME protein or PRAME mRNA in the test sample derived from normal cells or normal tissue. Alternatively, if the amount of PRAME protein or PRAME mRNA in a test sample derived from tumor cells or tumor tissue is similar to the amount of PRAME protein or PRAME mRNA in the test sample derived from normal cells or normal tissue, it may be determined that "PRAME is not detected" or "PRAME expression is negative." Here, "high value" refers to a value that is 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more, and even more preferably 5 times or more higher. "Similar" refers to a value of approximately 0.8 to 1.1 times higher. Furthermore, before comparing the amount of PRAME protein or PRAME mRNA, the amount of protein or RNA in each test sample may be normalized with the amount of protein or mRNA derived from a housekeeping gene such as GAPDH, β2-microglobulin, or β-actin. Protein amount may be expressed in terms of mass or concentration, or may be expressed in terms of the luminescence intensity of a substrate, etc. mRNA amount may be expressed in terms of the number of mRNA copies or reads, or may be expressed in terms of fluorescence intensity, etc.

[0032] In another embodiment, a reference value for the amount of PRAME protein or RNA may be determined in advance, and if the amount of PRAME protein or RNA in a test sample derived from tumor tissue, tumor cells, or urinary cells is higher than the reference value, it may be determined that "PRAME is detected" or "PRAME expression is positive." Alternatively, if the amount of PRAME protein or RNA in a test sample derived from tumor tissue, tumor cells, or urinary cells is lower than the reference value, it may be determined that "PRAME is not detected" or "PRAME expression is negative." The reference value is not limited as long as it is a value that can determine whether the amount of PRAME protein or PRAME mRNA is detected or whether expression is positive, and can be determined by known methods. A value that can determine whether the amount of PRAME protein or PRAME mRNA is detected or whether expression is positive can also be determined using an ROC (receiver operating characteristic) curve, discriminant analysis, mode method, Kittler method, 3σ method, p-tile method, etc. Furthermore, examples of reference values ​​include sensitivity, specificity, negative predictive value, positive predictive value, and first quartile.

[0033] The method for detecting a diagnostic marker for urothelial cancer may further comprise the step of determining that the urothelial tumor cells or urinary cells are urothelial cancer cells if PRAME is detected, or may comprise the step of determining that the urothelial tumor cells or urinary cells are not urothelial cancer cells if PRAME is not detected.

[0034] The method for detecting a BCG treatment resistance marker for a urothelial tumor may further comprise the step of determining that the subject having the urothelial tumor is resistant to BCG treatment if PRAME is detected, or may comprise the step of determining that the subject having the urothelial tumor is not resistant to BCG treatment if PRAME is not detected.

[0035] Furthermore, the method for detecting a urothelial cancer diagnostic marker and a BCG treatment resistance marker may include a step of determining the necessary treatment method depending on whether or not PRAME is detected in urothelial tumor cells. For example, if PRAME is detected, a decision may be made to administer radiation therapy and / or chemotherapy in addition to surgical resection of the tumor, or a decision may be made to discontinue BCG treatment. If PRAME is not detected, a decision may be made to conduct follow-up after surgical resection of the tumor. Furthermore, if PRAME is detected, a decision may be made to conduct follow-up for 5 years after surgery. If PRAME is not detected, a decision may be made to conduct follow-up for 10 years after surgery.

[0036] The present invention relates to a method for assisting in the diagnosis of urothelial cancer and / or a method for assisting in the diagnosis of resistance to BCG therapy of urothelial cancer, which comprises measuring the expression level of PRAME in a specimen collected from a subject, and determining that the subject has urothelial cancer and / or is resistant to BCG therapy for urothelial cancer if the expression level exceeds a reference value. The specimen used is not limited as long as it is urothelial tumor cells, urothelial tumor tissue, or urinary cells, but it is preferable to use urinary cells as the specimen because it is inexpensive and non-invasive.

[0037] 2. Testing reagents The present invention relates to a test reagent for detecting PRAME, a diagnostic marker for urothelial carcinoma, present in a sample collected from a subject. The test reagent can also be used to detect BCG therapy resistance markers for urothelial carcinoma. The test reagent can include a reagent for detecting PRAME protein and / or a reagent for detecting PRAME mRNA.

[0038] The reagent for detecting PRAME protein contains one or more antibodies (e.g., primary antibodies) capable of binding to at least a portion of PRAME protein. The "antibody" may be a polyclonal antibody, a monoclonal antibody, or a fragment thereof (e.g., Fab, F(ab'), F(ab)2, etc.). The immunoglobulin class and subclass are not particularly limited. Furthermore, the antibody may be one screened from an antibody library, or may be a chimeric antibody, scFv, or the like. Furthermore, the antibody does not necessarily need to be purified, and may be an antiserum containing the antibody, ascites fluid, an immunoglobulin fraction fractionated therefrom, or the like.

[0039] The antibody contained in the test reagent may be in a dry state or dissolved in a buffer such as phosphate-buffered saline. Furthermore, the test reagent may contain at least one of a stabilizer such as β-mercaptoethanol or DTT, a protectant such as albumin, a surfactant such as polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (10) octylphenyl ether, or a preservative such as sodium azide.

[0040] The antibody that binds to PRAME may be labeled with an enzyme or a fluorescent dye, or may be immobilized on a microplate, magnetic beads, or the like.

[0041] The PRAME protein detection reagent may be provided as a test kit containing the test reagent and a package insert describing how to use the reagent or providing a URL for a web page describing how to use the reagent. Furthermore, when the antibody that binds to PRAME is an unlabeled primary antibody, the test kit may contain a secondary antibody labeled with an enzyme or fluorescent dye. Furthermore, the test kit may contain a substrate that reacts with the enzyme.

[0042] The PRAME mRNA detection reagent contains a nucleic acid that hybridizes with all or part of PRAME mRNA or PRAME cDNA. The nucleic acid is preferably a detection nucleic acid (DNA or RNA) that functions as a primer and / or a probe. The length of the detection nucleic acid is not particularly limited.

[0043] If the detection nucleic acid is a primer used in a PCR reaction, the sequence that hybridizes with PRAME mRNA or PRAME cDNA is preferably 50 mer or less, more preferably 30 mer or less, and even more preferably about 15 to 25 mer. The primer may contain a sequence that does not hybridize with PRAME mRNA or PRAME cDNA. Furthermore, the primer may be labeled with a fluorescent dye or the like.

[0044] In addition to primers, RT-PCR can also use a quantification probe that is degraded during the PCR reaction and is used for real-time quantification of PCR products. There are no limitations on the quantification probe as long as it hybridizes with PRAME mRNA or PRAME cDNA. The quantification probe is preferably a nucleic acid of approximately 5 to 20 mer that contains a sequence that hybridizes with PRAME mRNA or PRAME cDNA. Furthermore, it is preferable that one end of the quantification probe is labeled with a fluorescent dye and the other end is labeled with a quencher for the fluorescent dye.

[0045] If the detection nucleic acid is used as a capture probe in a microarray or the like, the sequence that hybridizes with PRAME mRNA or PRAME cDNA is preferably about 100 mer, more preferably about 60 mer, and even more preferably about 20 to 30 mer. The capture probe may contain a sequence that does not hybridize with PRAME mRNA or PRAME cDNA. Furthermore, the capture probe is preferably immobilized on a chip.

[0046] When the detection nucleic acid is a probe for in situ hybridization, the sequence of the detection nucleic acid that hybridizes with PRAME mRNA may be an oligonucleotide of about 15 to 100 mer, or a polynucleotide of more than 100 mer. The polynucleotide may be DNA or RNA. A labeling substance such as digoxigenin or a fluorescent dye may be bound to the probe for in situ hybridization. The probe may also contain a sequence that does not hybridize with PRAME mRNA.

[0047] The PRAME mRNA detection reagent may be provided as a test kit containing the test reagent and a package insert describing how to use the reagent or listing the URL of a webpage describing how to use the reagent. Furthermore, when detecting PRAME mRNA or PRAME cDNA by RT-PCR, the test kit may contain a nucleic acid amplification reagent (including polymerase, buffer, dNTPs, etc., even if it is heat-stable DNA), reverse transcriptase, etc. The nucleic acid amplification reagent may contain a dye such as SYBER GREEN (registered trademark), as needed. When detecting PRAME mRNA or PRAME cDNA by microarray, the test kit may contain a hybridization buffer, a washing buffer, etc. When detecting PRAME mRNA by in situ hybridization, the test kit may contain a protease such as proteinase K, a hybridization buffer, a washing buffer, etc.

[0048] 3. Urothelial cancer detection device and BCG treatment resistance diagnostic device 3.1 Configuration of urothelial cancer detection device One embodiment of the present invention relates to a urothelial cancer detection system 1000 and a urothelial cancer detection device 10.

[0049] FIG. 1 is a schematic diagram of a detection system 1000 for urothelial cancer, and in one embodiment, the detection system 1000 for urothelial cancer may include an analysis device 5a or an analysis device 5b in addition to a detection device 10 for urothelial cancer.

[0050] 2 shows a block diagram of the urothelial cancer detection device 10. The urothelial cancer detection device 10 may be connected to an input unit 111, an output unit 112, and a storage medium 113.

[0051] In the urothelial carcinoma detection device 10, a processing unit 101, a main memory unit 102, a ROM (read only memory) 103, an auxiliary memory unit 104, a communication interface (I / F) 105, an input interface (I / F) 106, an output interface (I / F) 107, and a media interface (I / F) 108 are connected to each other via a bus 109 so as to be able to communicate data with each other. The main memory unit 102 and the auxiliary memory unit 104 may collectively be simply referred to as a memory unit. The memory unit stores the measurement values ​​and reference values ​​in a volatile or non-volatile manner.

[0052] The processing unit 101 is the CPU of the urothelial carcinoma detection device 10. The processing unit 101 may be a GPU. The processing unit 101 executes a computer program stored in the auxiliary storage unit 104 or the ROM 103 and processes acquired data, thereby causing the urothelial carcinoma detection device 10 to function.

[0053] The ROM 103 is configured by a mask ROM, PROM, EPROM, EEPROM, or the like, and stores computer programs executed by the processing unit 101 and data used therefor. The processing unit 101 may be an MPU 101. The ROM 103 stores a boot program executed by the processing unit 101 when the urothelial carcinoma detection device 10 is started up, as well as programs and settings related to the operation of the hardware of the urothelial carcinoma detection device 10.

[0054] The main memory unit 102 is configured by a RAM (Random Access Memory) such as an SRAM or a DRAM. The main memory unit 102 is used to read out computer programs recorded in the ROM 103 and the auxiliary memory unit 104. The main memory unit 102 is also used as a working area when the processing unit 101 executes these computer programs.

[0055] The auxiliary storage unit 104 is configured by a hard disk, a semiconductor memory element such as a flash memory, an optical disk, etc. The auxiliary storage unit 104 stores various computer programs, such as an operating system and application programs, to be executed by the processing unit 101, and various setting data used to execute the computer programs. Specifically, the auxiliary storage unit 104 stores reference values ​​and the like in a non-volatile manner.

[0056] The communication I / F 105 is composed of serial interfaces such as USB, IEEE1394, and RS-232C, parallel interfaces such as SCSI, IDE, and IEEE1284, analog interfaces including D / A converters and A / D converters, and a network interface controller (NIC). Under the control of the processing unit 101, the communication I / F 105 receives data from the analyzers 5a and 5b or other external devices, and transmits or displays information stored or generated by the urothelial carcinoma detection device 10 to the analyzers 5a and 5b or externally as necessary. The communication I / F 105 may communicate with the analyzers 5a and 5b or other external devices via a network.

[0057] The input I / F 106 is configured from, for example, a serial interface such as USB, IEEE1394, or RS-232C, a parallel interface such as SCSI, IDE, or IEEE1284, and an analog interface including a D / A converter or an A / D converter. The input I / F 106 accepts character input, clicks, voice input, etc. from the input unit 111. The accepted input content is stored in the main memory unit 102 or the auxiliary memory unit 104.

[0058] The input unit 111 is composed of a touch panel, a keyboard, a mouse, a pen tablet, a microphone, etc., and is used to input text or voice to the urothelial cancer detection device 10. The input unit 111 may be connected to the urothelial cancer detection device 10 from outside, or may be integrated with the urothelial cancer detection device 10.

[0059] The output I / F 107 is configured, for example, from an interface similar to the input I / F 106. The output I / F 107 outputs information generated by the processing unit 101 to the output unit 112. The output I / F 107 outputs information generated by the processing unit 101 and stored in the auxiliary storage unit 104 to the output unit 112.

[0060] The output unit 112 is composed of, for example, a display, a printer, etc., and displays the measurement results sent from the analyzers 5a and 5b, various operation windows in the urothelial cancer detection device 10, analysis results, etc.

[0061] The media I / F 108 reads, for example, application software stored in the storage medium 113. The read application software is stored in the main memory unit 102 or the auxiliary memory unit 104. The media I / F 108 also writes information generated by the processing unit 101 to the storage medium 113. The media I / F 108 writes information generated by the processing unit 101 and stored in the auxiliary memory unit 104 to the storage medium 113.

[0062] The storage medium 113 is configured with a flexible disk, a CD-ROM, a DVD-ROM, etc. The storage medium 113 is connected to the media I / F 108 by a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, etc. The storage medium 113 may store application programs and the like for the computer to execute operations.

[0063] The processing unit 101 may acquire application software and various settings necessary for controlling the urothelial carcinoma detection device 10 via a network instead of reading them from the ROM 103 or the auxiliary storage unit 104. The application program may be stored in the auxiliary storage unit of a server computer on the network, and the urothelial carcinoma detection device 10 may access this server computer, download the computer program, and store it in the ROM 103 or the auxiliary storage unit 104.

[0064] An operating system that provides a graphical user interface environment, such as Windows (registered trademark) manufactured and sold by Microsoft Corporation, is installed in the ROM 103 or the auxiliary storage unit 104. The application program according to the second embodiment runs on the operating system. In other words, the urothelial carcinoma detection device 10 may be a personal computer or the like.

[0065] The urothelial cancer detection system 1000 does not need to be installed in one location, and the urothelial cancer detection device 10 and the analyzers 5a and 5b may be located in different locations and connected via a network. Furthermore, the urothelial cancer detection device 10 may be an apparatus that does not require an operator by omitting the input unit 111 and the output unit 112.

[0066] The analytical device 5a is a device for measuring the amount or concentration of protein, and includes a sample storage area 51, a reaction section 52, and a detection section 53. The cell lysate placed in the sample storage area 51 is dispensed into a microplate on which an antigen capture antibody is immobilized and placed in the reaction section 52, and incubated. After removing unreacted antigens as needed, a detection antibody is dispensed into the microplate and incubated. After removing unreacted antigens as needed, a substrate for detecting the detection antibody is dispensed into the microplate, the microplate is moved to the detection section 53, and a signal generated by the reaction of the substrate is measured.

[0067] Another embodiment of the analytical device 5a is a device for measuring the expression level of mRNA by microarray analysis, in which the reverse transcription reaction product set in the sample storage area 51 is dispensed onto a microarray chip set in the reaction section 52, hybridization is performed, and after washing, it is moved to the detection section 53 and the signal is detected.

[0068] Another embodiment of the analytical device 5a is a device for measuring the expression level of mRNA by RT-PCR, in which a reverse transcription reaction product set in a sample storage area 51 is dispensed into a microtube set in a reaction section 52, and then quantitative PCR reagents are dispensed into the microtube. While the PCR reaction is carried out in the reaction section 52, a signal in the tube is detected in a detection section 53.

[0069] The analysis device 5b is a device for measuring the expression level of mRNA by the RNA-Seq method, and includes a sequence analysis unit 54. A sample that has undergone a reaction for RNA-Seq is set in the sequence analysis unit 54, and the base sequence is analyzed within the sequence analysis unit 54.

[0070] The analytical device 5b is a fully automated Western blotting device for measuring the amount of protein by Western blotting, and is equipped with a chemiluminescent signal detection unit 54. A sample of urothelial tumor cells, urothelial tumor tissue, or urinary cells dissolved in a lysis buffer is placed in a predetermined position in the automated Western blotting device, and SDS-PAGE, blotting onto a membrane, antibody reaction, and chemiluminescence are performed, followed by analysis by the chemiluminescent signal detection unit 54, and the signal intensity is quantified.

[0071] The analyzers 5a and 5b are connected to the urothelial cancer detection device 10 by wire or wirelessly. The analyzer 5a A / D converts the protein measurement value or the mRNA measurement value and transmits it as digital data to the urothelial cancer detection device 10. Similarly, the analyzer 5b A / D converts the mRNA measurement value and transmits it as digital data to the urothelial cancer detection device 10. This allows the urothelial cancer detection device 10 to obtain the protein measurement value or the mRNA measurement value as digital data that can be processed.

[0072] 3.2 Configuration of diagnostic device for BCG treatment resistance One embodiment of the present invention relates to a BCG therapy resistance diagnostic system 2000 and a BCG therapy resistance diagnostic device 20.

[0073] FIG. 1 is an overview of a diagnostic system 2000 for BCG therapy resistance, which in one embodiment may include an analysis device 5a or an analysis device 5b in addition to a diagnostic device 20 for BCG therapy resistance.

[0074] FIG. 2 shows a block diagram of the BCG therapy resistance diagnostic device 20. The configuration of the BCG therapy resistance diagnostic device 20 is the same as that of the urothelial carcinoma detection device 10. Therefore, the description in 3.1 above can be incorporated herein. In this case, the processing unit 101, main memory unit 102, ROM 103, auxiliary memory unit 104, communication interface (I / F) 105, input interface (I / F) 106, output interface (I / F) 107, media interface (I / F) 108, bus 109, input unit 111, output unit 112, and storage medium 113 should be replaced with processing unit 201, main memory unit 202, ROM 203, auxiliary memory unit 204, communication interface (I / F) 205, input interface (I / F) 206, output interface (I / F) 207, media interface (I / F) 208, bus 209, input unit 211, output unit 212, and storage medium 213. The analyzers 5a and 5b are similar to the urothelial carcinoma detection device 10.

[0075] 3.3 Operation of the urothelial cancer detection device 3 shows an example of a flowchart of an operation example of the urothelial carcinoma detection device 10. The processing unit 101 of the urothelial carcinoma detection device 10 starts processing for detecting urothelial carcinoma when the operator inputs a command to start processing through the input unit 111. In step S11, the processing unit 101 acquires the amount of PRAME protein or PRAME mRNA in a sample collected from a subject from the analysis device 5a or analysis device 5b, or a value reflecting these, as a PRAME measurement value. Alternatively, the operator may input through the input unit 111 whether PRAME expression is positive or negative (or whether PRAME is detected) by immunostaining or in situ hybridization, and the processing unit 101 acquires this input as a PRAME measurement value.

[0076] Next, in step S12, the processing unit 101 compares the reference value of PRAME stored in the storage unit with the measurement value acquired in step S11.

[0077] If the acquired measurement value is higher than the reference value in step S13, the processing unit 101 proceeds to step S14 (YES), determines that the subject has urothelial carcinoma, and outputs a display indicating the determination result to the output unit 112 (step S16). In the present invention, "outputting a display" broadly includes displaying the determination result on a display, outputting the determination result as audio, transmitting the determination result as a signal to the outside, and printing a label or the like indicating the determination result. Also, if the acquired measurement value is lower than the reference value in step S13, the processing unit 101 proceeds to step S15 (NO), determines that the subject does not have urothelial carcinoma, and outputs a display indicating the determination result to the output unit 112 (step S16). If the subject is determined to have urothelial carcinoma, the display includes information indicating "urothelial carcinoma" or "suggesting urothelial carcinoma." Information "suggesting urothelial carcinoma" may include "suggesting that the subject is not urothelial carcinoma." If it is determined that the cancer is not urothelial cancer, the display includes information indicating "not urothelial cancer" or "suggesting that it is not urothelial cancer." Information "suggesting that it is not urothelial cancer" may include "suggesting that it is urothelioma." The information may be a mark such as an X, a circle, or an exclamation mark.

[0078] For details of the reference value, the comparison method, and the method for determining whether a measured value is higher or lower than the reference value, the explanation in 1.2 above is incorporated herein by reference.

[0079] 3.4 Operation of the diagnostic device for BCG treatment resistance 3 shows an example of a flowchart of an operation example of the BCG therapy resistance diagnostic device 20. The processing unit 201 of the BCG therapy resistance diagnostic device 20 starts processing for detecting urothelial carcinoma when the operator inputs a command to start processing from the input unit 211. In step S21, the processing unit 201 acquires the amount of PRAME protein or PRAME mRNA in a sample collected from a subject from the analysis device 5a or analysis device 5b, or a value reflecting these, as a PRAME measurement value. Alternatively, the operator may input from the input unit 211 whether PRAME expression is positive or negative (or whether PRAME is detected) by immunostaining or in situ hybridization, and the processing unit 201 acquires this input as a PRAME measurement value.

[0080] Next, in step S22, the processing unit 201 compares the reference value of PRAME stored in the storage unit with the measurement value acquired in step S21.

[0081] If the acquired measurement value is higher than the reference value in step S23, the processing unit 201 proceeds to step S24 (YES), determines that the subject is resistant to BCG therapy for urothelial carcinoma, and outputs a display indicating the determination result to the output unit 212 (step S26). If the acquired measurement value is lower than the reference value in step S23, the processing unit 201 proceeds to step S25 (NO), determines that the subject is not resistant to BCG therapy, and outputs a display indicating the determination result to the output unit 212 (step S26). If the subject is determined to be resistant to BCG therapy, the display includes information indicating "BCG therapy resistant" or "suggesting BCG therapy resistance." If the subject is determined not to be resistant to BCG therapy, the display includes information indicating "not resistant to BCG therapy" or "suggesting not resistant to BCG therapy." The information may be a mark such as an x, a circle, or an exclamation mark.

[0082] For details of the reference value, the comparison method, and the method for determining whether a measured value is higher or lower than the reference value, the explanation in 1.2 above is incorporated herein by reference. [Example]

[0083] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples in any way.

[0084] 1. Sample Urothelial tumor tissue specimens and urine cytology specimens collected at Osaka Medical and Pharmaceutical University Hospital were used in the study.

[0085] 2. Gene Expression Analysis Gene expression analysis was performed using whole-exome sequencing (NovaSeq 6000, Illumina) on genomic DNA extracted from urothelial tumor tissue samples. Gene expression levels were calculated as TPM (transcripts per million).

[0086] 3. Immunostaining The tissue samples were fixed in formalin and prepared as tissue specimens. All fixed tissue samples were embedded in paraffin, thin sections were prepared, and the tissue specimens were subjected to hematoxylin-eosin staining and immunostaining.

[0087] Immunostaining was performed using an automated staining system (Discovery, Roche Diagnostics, Basel, Switzerland) according to the protocol provided with the system. The primary antibody used to stain PRAME was an anti-PRAME antibody produced in rabbit Prestige Antibodies® Powered by Atlas Antibodies, an affinity isolated antibody, in buffered aqueous glycerol solution (HPA045153, SIGMA-ALDRICH). The secondary antibody used was a peroxidase-labeled anti-rabbit immunoglobulin antibody, and diaminobenzidine (DAD) was used for color development.

[0088] 4.Statistical analysis Statistical analysis was performed using JMP Start Statistics version 14 (Statistical Discovery Software; SAS Institute, Cary, NC, USA).

[0089] Example 1 We measured PRAME mRNA expression levels in bladder tumors collected from 459 patients diagnosed with bladder cancer and examined the correlation with various factors. The results are shown in Table 1. It can be seen that PRAME mRNA expression in bladder tumors is significantly increased when the bladder cancer is muscle-invasive or has a high pathological grade. Furthermore, as can be seen in Figure 5, only about one-quarter of patients with non-muscle-invasive bladder cancer had a TPM of 1 or higher, while just over 30% of patients with muscle-invasive bladder cancer had a TPM of 1 or higher. [Table 1]

[0090] Example 2 We examined the association between progression-free survival (PFFS) and various factors in 143 patients diagnosed with non-muscle-invasive bladder cancer. Univariate and multivariate analyses were performed using Cox regression analysis. The results are shown in Table 2. It can be seen that PFFS is strongly influenced by PRAME mRNA expression in bladder tumors. Furthermore, as shown in Figure 6, among patients diagnosed with non-muscle-invasive bladder cancer and with a poor prognosis, approximately half of the bladder tumors had high levels of PRAME mRNA. Furthermore, as shown in Figure 7, high levels of PRAME mRNA expression in bladder tumors tend to be associated with shorter PFFS and cancer-specific survival. [Table 2]

[0091] Example 3 The prognosis of 47 patients diagnosed with non-muscle invasive bladder cancer who underwent BCG treatment was observed. The results are shown in Figures 8 and 9. Among the BCG-treated group, those with high PRAME mRNA expression in bladder tumors had significantly shorter recurrence-free survival and progression-free survival, indicating that PRAME mRNA expression in bladder tumors has a strong influence on progression-free survival.

[0092] Example 4 Bladder tissue stained for PRAME protein by immunohistochemical staining is shown in Figure 10. In bladder cancer tissue, PRAME mRNA expression and protein expression were significantly correlated. These results demonstrate that PRAME is useful as a diagnostic marker for urothelial carcinoma and a marker for BCG treatment resistance.

[0093] Example 5 Immunocytochemical staining for PRAME was performed on urine cytology specimens from bladder cancer patients who had been determined to be PRAME-positive by immunohistochemical staining of urothelial tumor tissue specimens. PRAME-positive findings were observed in 60% (6 out of 10 cases) of the cancer cells present in the urine cytology specimens. These results demonstrate that PRAME, which is present in non-invasive urinary cells, is useful as a diagnostic marker for urothelial carcinoma and a marker for BCG treatment resistance.

Claims

1. A method for detecting a diagnostic marker for urothelial cancer, comprising the step of detecting PRAME in a specimen collected from a subject, wherein the specimen is a urothelial tumor or urinary cells.

2. A method for assisting in the diagnosis of urothelial cancer, comprising the steps of measuring the expression level of PRAME in a sample collected from a subject, and determining that the subject has urothelial cancer if the expression level exceeds a standard value, wherein the sample is a urothelial tumor or urinary cells.

3. A method for detecting a BCG therapy resistance marker for urothelial cancer, comprising the step of detecting PRAME in a specimen collected from a subject, wherein the specimen is a urothelial tumor or urinary cells.

4. A method for assisting in the diagnosis of resistance to BCG therapy of urothelial cancer, comprising the steps of measuring the expression level of PRAME in a sample collected from a subject, and determining that the subject is resistant to BCG therapy for urothelial cancer if the expression level exceeds a reference value, wherein the sample is a urothelial tumor or urinary cells.

5. A test reagent for detecting PRAME in a sample collected from a subject as a biomarker for urothelial carcinoma, the test reagent comprising an antibody or nucleic acid for detecting PRAME.

6. A test reagent for detecting PRAME in a sample collected from a subject as a BCG therapy resistance marker, the test reagent comprising an antibody or nucleic acid for detecting PRAME.

7. A device for detecting urothelial carcinoma, comprising a processing unit, The processing unit obtaining a measurement of PRAME in a sample taken from the subject; The obtained measurement value is compared with the reference value, A detection device that outputs a display indicating that the subject has urothelial carcinoma if the acquired measurement value is higher than the reference value, and / or outputs a display indicating that the subject does not have urothelial carcinoma if the acquired measurement value is lower than the reference value.

8. A diagnostic device for BCG therapy resistance, comprising a processing unit, The processing unit obtaining a measurement of PRAME in a sample taken from the subject; The obtained measurement value is compared with the reference value, A diagnostic device that outputs a display indicating that the subject is resistant to BCG treatment for urothelial carcinoma when the obtained measurement value is higher than the reference value, and / or outputs a display indicating that the subject is not resistant to BCG treatment for urothelial carcinoma when the obtained measurement value is lower than the reference value.

9. The method according to any one of claims 1 to 4, wherein the specimen is a urinary cell.

10. Use of PRAME present in urothelial tumors or urinary cells as a diagnostic marker for urothelial carcinoma.

11. Use of PRAME present in urothelial tumor or urinary cells as a marker for resistance to BCG therapy in urothelial carcinoma.

12. A diagnostic marker for urothelial carcinoma consisting of PRAME.

13. PRAME, a marker of resistance to BCG therapy in urothelial carcinoma.

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