Testing method for bladder cancer

Abstract

[Problem] The present invention addresses the problem of developing a new testing method enabling the non-invasive and highly accurate diagnosis of bladder cancer. [Solution] To provide a testing method for bladder cancer using a somatic mutation specific to bladder cancer as a biomarker.

Description

Method for Detecting Bladder Cancer

[0001] The present invention relates to a method for detecting the possibility of bladder cancer.

[0002] Bladder cancer is a general term for cancers that occur in the bladder. Most (more than 90%) of bladder cancers are urothelial carcinomas that develop in the urothelium covering the inside of the bladder. Urothelial carcinomas are classified into muscle-invasive carcinomas and non-muscle-invasive carcinomas according to the depth of cancer invasion. In non-muscle-invasive carcinomas, bladder preservation is possible, but long-term follow-up observation is required for intravesical recurrence.

[0003] Urine cytology is widely used as a test for bladder cancer. While it has a high specificity, its sensitivity ranges from 35% to 70%, and the sensitivity decreases particularly in cases of low atypia and recurrent tumors. Cystoscopy is excellent in terms of sensitivity and specificity, but it has problems in terms of invasiveness, such as pain, risk of cystitis, hematuria, and mental distress.

[0004] As molecular tumor markers that may be used for the diagnosis of bladder cancer, urinary BTA, NMP22, BFP, telomerase, CA-50, type IV collagen, TPA, and FDP are known. However, none of them are bladder cancer-specific proteins, and their levels also increase in other diseases such as pyelonephritis. Due to their limited sensitivity and specificity, their clinical usage frequency is low.

[0005] Against this background, there is a need for a method that is less invasive and can diagnose bladder cancer with high accuracy. In recent years, a new test method has been proposed for diagnosing bladder cancer by measuring the membrane protein EphA2 of extracellular vesicles (EVs) secreted into the urine of bladder cancer patients (Non-Patent Document 1).

[0006] Br J Cancer. 2022 Oct;127(7):1312-1323

[0007] An object of the present invention is to develop a new test method for diagnosing bladder cancer non-invasively and with high accuracy.

[0008] The inventors conducted their own proteomic analysis using LC / MS on three types of samples: bladder cancer tissue, extracellular viable (EV) secreted from bladder cancer tissue cultured in serum-free medium, and EV in urine. As a result, they identified several bladder cancer-specific somatic mutations. They then developed a bladder cancer test using these somatic mutations as biomarkers, thus completing the present invention.

[0009] In other words, this disclosure relates, in one embodiment, to a method for determining the possibility of bladder cancer, comprising the step of detecting one or more bladder cancer-specific protein mutations selected from the group consisting of (1) to (47) below, from a fluid-derived sample or tissue-derived sample taken from a subject. (1) E485K mutation in CUL1 (SEQ ID NO: 1) (2) D183N mutation in PRDX6 (SEQ ID NO: 2) (3) D98N mutation in CELF1 (SEQ ID NO: 3) (4) R135K mutation in RALA (SEQ ID NO: 4) (5) F73L mutation in FAM167B (SEQ ID NO: 5) (6) E186K mutation in HSPB1 (SEQ ID NO: 6) (7) S1252C mutation in SPTBN1 (SEQ ID NO: 7) (8) P120S mutation in ACTR3 (SEQ ID NO: 8) (9) E294D mutation in RBM25 (SEQ ID NO: 9) (10) D313N mutation in SRP54 (SEQ ID NO: 10) (11) E233K mutation in INTS8 (SEQ ID NO: 11) (12) Q171K mutation in CARS2 (SEQ ID NO: 12) (13) G22R mutation in PDCD5 (SEQ ID NO: 13) (14) G835R mutation in TRPM4 (SEQ ID NO: 14) (15) E648Q mutation in MUC4 (SEQ ID NO: 15) (16) R452T mutation in DHX15 (SEQ ID NO: 16) (17) D122H mutation in CSE1L (SEQ ID NO: 17) (18) E36K mutation in HIST2H2BE (SEQ ID NO: 18) (19) D212H mutation in RPN1 (SEQ ID NO: 19) (20) D901N mutation in SVIL (SEQ ID NO: 20) (21) L21V mutation in CHRAC1 (SEQ ID NO: 21) (22) R201Q mutation in SMARCB1 (SEQ ID NO: 22) (23) G200E mutation in TAF15 (SEQ ID NO: 23) (24) E104K mutation in ITGB1 (SEQ ID NO: 24) (25) G794V mutation in MCM6 (SEQ ID NO: 25) (26) D133N mutation in CTTN (SEQ ID NO: 26) (27) D321H mutation in LCP1 (SEQ ID NO: 27) (28) K102N mutation in TKT (SEQ ID NO: 28) (29) R639H mutation in PLCD1 (SEQ ID NO: 29) (30) R460-A461 deletion in VCL (SEQ ID NO: 30)(31) R39G mutation in PTBP3 (SEQ ID NO: 31) (32) E708K mutation in IARS2 (SEQ ID NO: 32) (33) D235H mutation in NLN (SEQ ID NO: 33) (34) E235Q mutation in GATAD2B (SEQ ID NO: 34) (35) R167G mutation in MYO19 (SEQ ID NO: 35) (36) M1432V mutation in MYOF (SEQ ID NO: 36) (37) R130K mutation in EBAG9 (SEQ ID NO: 37) (38) L37V mutation in BCAT2 (SEQ ID NO: 38) (39) R342Q mutation in DNAJB12 (SEQ ID NO: 39) (40) Y167H mutation in HNRNPDL (SEQ ID NO: 40) (41) G873A mutation in LNPEP (SEQ ID NO: 41) (42) D6N mutation in RPS27L (SEQ ID NO: 42) (43) K109T mutation in HIST1H2BO (SEQ ID NO: 43) (44) T83S mutation in QARS (SEQ ID NO: 44) (45) V96L mutation in STAM (SEQ ID NO: 45) (46) L980-E986 deletion in MYH14 (SEQ ID NO: 46) (47) H186Y mutation in ERGIC2 (SEQ ID NO: 47)

[0010] One embodiment of the present disclosure is characterized in that if the bladder cancer-specific mutant protein is detected in a sample taken from the subject, it is determined that there is a high probability that bladder cancer is present.

[0011] In one embodiment of the present disclosure, the bodily fluid-derived sample is characterized in that the bodily fluid-derived sample is a urine-derived sample or a blood-derived sample.

[0012] In one embodiment of the present disclosure, the urine-derived sample is characterized in that it is an extracellular vesicle (EV) in the urine.

[0013] In one embodiment of the present disclosure, the urine-derived sample is characterized in that it is urine sediment.

[0014] In one embodiment of the present disclosure, the blood-derived sample is characterized in that it is an extracellular vesicle (EV) in the blood.

[0015] In one embodiment of the present disclosure, the tissue-derived sample is characterized in that it is a sample derived from bladder tissue.

[0016] In one embodiment of this disclosure, the detection of the bladder cancer-specific protein mutation is performed by mass spectrometry.

[0017] In one embodiment of the present disclosure, the detection of the bladder cancer-specific protein mutation is performed by an immunological assay using an antibody that specifically binds to the protein having the bladder cancer-specific protein mutation.

[0018] Another embodiment of this disclosure relates to a method for monitoring bladder cancer recurrence, which includes performing the above-described examination method in subjects after treatment for bladder cancer.

[0019] One embodiment of the present disclosure is characterized in that, in a subject who has undergone treatment for bladder cancer, the above-described examination method is performed using a sample taken before the treatment and a sample taken after the treatment, and the results of each are compared.

[0020] In one embodiment of the present disclosure, the examination method of the present disclosure is characterized in that it is used in combination with other methods for detecting bladder cancer.

[0021] In one embodiment of the present disclosure, the other method for detecting bladder cancer is characterized by cystoscopy, urine cytology, ultrasound, CT scan, pelvic MRI, or bladder mucosal biopsy.

[0022] In one embodiment of the present disclosure, the other method for detecting bladder cancer is a molecular tumor marker test for bladder cancer.

[0023] In one embodiment of the present disclosure, the molecular tumor marker is characterized in that it is BTA, NMP22, BFP, cytokeratin 8 / cytokeratin 18 (CK8-18), telomerase, CA-50, type IV collagen, TPA, or FDP in urine.

[0024] Inventions that arbitrarily combine one or more of the features of this disclosure described above are also included in the scope of the present invention.

[0025] Since the somatic mutations found in this disclosure are specific to bladder cancer, their use theoretically enables a test with 100% specificity. Furthermore, by using multiple discovered somatic mutations as a "panel" in the test, the sensitivity of the test can be increased. Moreover, since the testing method of the present invention can be performed using urine-derived or blood-derived samples, it is non-invasive and enables a test that places less burden on the patient.

[0026] Figure 1 shows an overview of the experiment in Example 1. Figure 2 shows the bladder cancer-specific somatic mutations found in this disclosure. Figure 3A shows an overview of recurrence monitoring using mutant proteins in urinary EV. Figure 3B shows that mutant proteins in urinary EV were significantly reduced postoperatively.

[0027] This disclosure relates to a method for testing for bladder cancer, comprising the step of detecting one or more bladder cancer-specific protein mutations from a fluid-derived or tissue-derived sample taken from a subject.

[0028] The term "bladder cancer" as the subject of the examinations related to this disclosure may refer to cancer that develops in the bladder. While the majority of bladder cancers are urothelial carcinomas that develop in the urothelium lining the inside of the bladder, the examinations related to this disclosure are not limited to urothelial carcinomas and may also include squamous cell carcinoma, adenocarcinoma, and small cell carcinoma that develop in the bladder.

[0029] The tests described herein may be intended to assist a physician in diagnosing bladder cancer and may be performed before the physician makes a diagnosis. Furthermore, the tests described herein may be performed by healthcare professionals other than physicians (e.g., nurses, clinical laboratory technicians, or technicians at testing companies), provided that this does not violate laws and regulations.

[0030] The condition of the subjects subject to the examinations described herein is not particularly limited. Examples of subjects include those whose status as having bladder cancer is unknown, those who have already been determined to have bladder cancer by other means, those who have already been determined to not have bladder cancer by other means, those undergoing treatment for bladder cancer, and those who have completed treatment for bladder cancer.

[0031] Since the somatic mutations found in this disclosure are specific to bladder cancer, it is theoretically possible to use them to perform tests with 100% specificity. That is, if bladder cancer-specific mutant proteins are detected in a sample taken from a subject, it may be determined that there is a high probability that bladder cancer is present in the subject's body. Alternatively, a certain threshold value may be set to exclude nonspecific detections, and if bladder cancer-specific mutant proteins above this threshold value are detected in a sample taken from a subject, it may be determined that there is a high probability that bladder cancer is present in the subject's body.

[0032] The test relating to this disclosure may detect at least one of the multiple somatic mutations found, but preferably it may be a "panel test" that simultaneously detects multiple somatic mutations found. By using the multiple somatic mutations found as a "panel" in the test, the sensitivity of the test can be increased.

[0033] The results of the examinations disclosed herein can be used for determining the necessity of treatment, selecting treatment strategies, evaluating treatment effectiveness, monitoring recurrence after treatment, elucidating the pathogenesis of bladder cancer, predicting the prognosis of bladder cancer, and stratifying patients, among other purposes.

[0034] The species of organisms subject to the tests relating to this disclosure are not limited to any particular species, and include, for example, mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, with humans being preferred.

[0035] The examinations described herein can be performed using tissue samples taken from the subject (for example, tissue samples taken from the subject's bladder). For example, a sample taken from the subject's bladder mucosa during a cystoscopy (endoscopic examination) may be used for the examination. By using tissue biopsy samples in both pathological examinations and the examinations described herein, the accuracy of the examinations can be improved without increasing the burden on the subject.

[0036] The tests relating to this disclosure can be performed using bodily fluid samples collected from the subject. For example, the precipitate obtained by centrifuging urine (urinary sediment) contains bladder epithelial cells, etc., and such urinary sediment can be used in the tests relating to this disclosure.

[0037] Furthermore, as shown in the examples of this specification, mutant proteins, including bladder cancer-specific somatic mutations found in this disclosure, are encapsulated in extracellular vesicles (EVs) and secreted extracellularly (for example, in urine or blood). Therefore, EVs can be purified, separated, concentrated, etc., from urine or blood and used in the tests related to this disclosure.

[0038] In this disclosure, "extracellular vesicles (EVs)" are not particularly limited as long as they are membrane vesicles secreted or released from cells. Extracellular vesicles may be classified into exosomes, microvesicles, apoptotic bodies, etc., depending on the differences in their intracellular production mechanisms, but all of these are included in the definition of "extracellular vesicles" in this disclosure.

[0039] Methods for isolating extracellular vesicles from samples collected from the target include, but are not limited to, PEG precipitation, isolation methods using ultracentrifuges, and immunoprecipitation using extracellular vesicle capture molecules. Among these, immunoprecipitation using extracellular vesicle capture molecules is preferred because it allows for the selective separation of extracellular vesicles easily without pretreatment.

[0040] Molecules for capturing extracellular vesicles may include substances that specifically bind to extracellular vesicles. Examples of substances that specifically bind to extracellular vesicles include PS-binding substances (PS-binding proteins or their modifications) that bind to phosphatidylserine (PS), which constitutes extracellular vesicles. More specifically, examples of PS-binding substances include Tim4, which captures PS in the presence of metal ions such as calcium ions.

[0041] Alternatively, since extracellular vesicles have various antigens on their surfaces, extracellular vesicles can be captured by using a specific binding substance for the antigen. Examples of antigens expressed on the surface of extracellular vesicles include, but are not limited to, CD9, CD63, and CD81. Examples of substances that specifically bind to these antigens include antibodies, aptamers, and the like.

[0042] The method for detecting bladder cancer-specific protein mutations from a sample collected from a subject is not particularly limited as long as it can specifically detect the bladder cancer-specific protein mutations. For example, a mass spectrometry method for detecting a peptide containing a target mutation, an immunological assay (immunoassay) using an antibody that specifically recognizes a protein containing a mutation, and the like can be mentioned.

[0043] Non-limiting examples of mass spectrometry methods that may be used in the method of the present disclosure include tandem quadrupole mass spectrometry (MS / MS) method, direct injection mass spectrometry, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) method, capillary electrophoresis mass spectrometry (CE-MS) method, inductively coupled plasma mass spectrometry (ICP-MS) method, pyrolysis mass spectrometry (Py-MS) method, ion mobility mass spectrometry, and time-of-flight mass spectrometry (TOF MS) method.

[0044] When using a mass spectrometry method, a pretreatment for separating substances present in extracellular vesicles may be performed before mass spectrometry. As the separation method, various chromatographic separation techniques can be used. More specifically, for example, liquid chromatography (LC), high performance liquid chromatography (HPLC), and the like can be mentioned, but are not limited thereto. Among them, liquid chromatography or high performance liquid chromatography is preferred.

[0045] Non-limiting examples of immunological assays that may be used in the method of the present disclosure include immunohistochemical staining method, ELISA method, sandwich ELISA method, EIA method, RIA method, and Western blotting method.

[0046] The method for obtaining an antibody that specifically binds to a protein having a bladder cancer-specific protein mutation and is used in an immunological assay is not limited, and those skilled in the art can obtain an antibody according to a conventional method. For example, after immunizing an animal with a protein (full length) having a bladder cancer-specific protein mutation or a peptide fragment containing the mutation site as an immunogen, the desired antibody can be obtained by selecting, purifying, concentrating, etc. the antibody according to a conventional method in this field. The animal used for immunization is not particularly limited as long as it has the ability to produce antibodies, and may be a mammal usually used for immunization such as a mouse, a rat, or a rabbit, or a bird such as a chicken may be used.

[0047] The test according to the present disclosure may be used in combination with other test methods that are being carried out in the art to detect bladder cancer. By using a plurality of test methods in combination, bladder cancer can be detected with higher accuracy. Other methods for detecting bladder cancer include cystoscopy, urine cytology, ultrasonography, CT examination, pelvic MRI examination, and bladder mucosa biopsy. For example, urine sediment collected from the test urine collected for urine cytology may be used in the test according to the present disclosure, or the bladder mucosa collected during cystoscopy may be used in the test according to the present disclosure.

[0048] In addition, another method for detecting bladder cancer may be a molecular tumor marker test for bladder cancer. Non-limiting examples of molecular tumor marker tests that may be used in combination with the test according to the present disclosure include tests for BTA, NMP22, BFP, cytokeratin 8 - cytokeratin 18 (CK8-18), telomerase, CA-50, type IV collagen, TPA, or FDP in urine. These molecular tumor markers are not necessarily bladder cancer-equivalent proteins, but the sensitivity of the test can be increased by using them in combination with the test according to the present disclosure.

[0049] Note that the terms used in this specification are used to describe specific embodiments and are not intended to limit the invention.

[0050] Furthermore, the term "includes" as used herein means that the described items (components, steps, elements, numbers, etc.) exist, unless the context clearly requires a different interpretation, and does not exclude the existence of other items (components, steps, elements, numbers, etc.).

[0051] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as they are broadly understood by those skilled in the art to which this disclosure belongs. Unless otherwise explicitly defined, terms used herein should be construed to have a meaning consistent with that of this specification and the art relating to it, and should not be interpreted in an idealized or overly formal sense.

[0052] While terms such as "first," "second," etc., may be used to describe various elements, it is understood that these elements should not be limited by these terms. These terms are used solely to distinguish one element from another, and it is possible, without departing from the scope of this disclosure, to refer to the first element as the second element, and similarly, the second element as the first element.

[0053] The present disclosure will be described in more detail below with reference to examples; however, the present disclosure can be embodied in various forms and should not be construed as being limited to the examples described herein.

[0054] [Example 1: Detection of Bladder Cancer-Specific Somatic Mutations] Overview of the Study (See Figure 1) In this study, bladder cancer tissue, blood cells (buffy coat), and urine were provided by five bladder cancer patients, and the experiment was conducted according to the following procedure: (1) Identify somatic mutant DNA sequences from whole-exome sequencing analysis using bladder cancer tissue and blood cells. (2) Convert the DNA sequences from (1) into amino acid sequences to create a protein sequence database for each individual. (3) Perform proteomic analysis by LC / MS on three types of samples: bladder cancer tissue, EV secreted from bladder cancer tissue cultured in serum-free medium, and EV in urine. (4) Identify mutant proteins by performing a sequence search on the database incorporating somatic mutations constructed in (2).

[0055] Study Details 1. Materials and Methods 1-1. Sample Collection and Preparation Preoperative urine, cancerous tissue, and postoperative blood were collected from five patients who underwent transurethral resection of bladder tumors at the University of Tokyo Hospital. The tumors were frozen with liquid nitrogen immediately after surgical resection. All tumor tissues were diagnosed as urothelial carcinoma by pathologists. This study was conducted in accordance with the Declaration of Helsinki and was approved by the "Ethics Committee of the Cancer Institute Hospital of the Japan Cancer Research Foundation" (Approval Number 2022-GB-055) and the "Ethics Committee of the University of Tokyo Hospital" (Approval Number 2022353Ge). All patients in this study cohort gave written informed consent before participating in the study.

[0056] 1-2. Whole Genome Sequencing and Database Construction of Mutant Proteins Genomic DNA was extracted from cancer tissue and buffy coats of five patients. Following the manufacturer's protocol, DNA was isolated from buffy coats from 2–10 mg of frozen cancer and 8.5 ml of blood using the QIAamp DNA Mini Kit (QIAGEN). Blood was collected using cell-free DNA collection tubes (Roche). DNA libraries were prepared using the SureSelect Human All Exons V6 kit (Agilent Technology) and sequenced using Illumina Novaseq. Sequencing reads were aligned to the human genome hg19, and missense and frameshift mutations were identified using the Genomon 2 pipeline (https: / / genomon.readthedocs.io / ja / latest / ). Somatic mutations were filtered according to the following criteria: (i) tumor variant read count > 3, (ii) normal variant read count < 3, (iii) tumor variant allele frequency > 0.05, and (iv) repeat sequences excluded. Missense and frameshift mutations were converted to amino acid sequences using the Neoantimon R package software, and a customized database of mutant proteins was constructed. This software constructs the corresponding RNA sequences using the Reference Sequence Database and then constructs the sequences of the mutant peptides. To identify mutant peptides, a customized database is built for each patient, consisting of mutant peptides compiled with Neoantimon software and the Swiss-Prot human proteome database (20,386 entries) on the UniProt website.

[0057] 1-3. Approximately 5 mg of protein-extracted frozen cancer tissue was dissolved on ice in 100 μL of lysis buffer containing [20 mM] HEPES, [150 mM] NaCl, 1% NP-40, 0.1% SDS, and 10% glycerol. Before preparing the lysis solution, complete Protein Inhibitor Cocktail (Sigma-Aldrich) was added. The cancer tissue was lysed using BioMasher II (Funakoshi). After centrifugation at 15,000 g for 15 minutes, the supernatant was used for peptide separation. The protein concentration of the cancer tissue lysis solution was measured using Micro BCA Protein Assay Kit (Thermo Fisher Scientific), and 10 μg was used for peptide isolation.

[0058] 1-4. Separation of Tissue Exudative EV and Urine EV Tissue exudative EV (Te-EV) was isolated from the culture medium of cancer tissue. First, 2 mm square frozen cancer tissue was thawed on ice, rinsed with PBS, and then sterilized in 1.5 mL of RPMI-1640 at 37°C and 5% CO2. ２ The culture medium was incubated for 3 hours with gentle rotation under the specified conditions. The culture medium was mixed with 10% exosome-removed fetal bovine serum (FBS) (Funakoshi) and 1% penicillin-streptomycin. Urine samples were collected from each patient before surgery at the University of Tokyo Hospital. The urine samples were centrifuged at 2,000 g for 10 minutes, and the supernatant was stored at -80°C. Exosomes were isolated from the Te-EV supernatant and 5 ml of urine using Magcapture beads (Fujifilm) that capture phosphatidylserine displayed on the EV surface by the Tim4 protein. Before separating the exosomes, the urine was concentrated to 1 ml using an AmiconUltra 100K molecular weight cutoff concentrator (Merck). The concentrated urine and Tim4 beads were incubated for 1 hour with gentle rotation. EV was purified in three washing steps, and the protein was extracted with Laemmli's SDS sample buffer.

[0059] 1-5. Western Blotting Analysis Te-EV and protein (10 μg) isolated from urinary EV of bladder cancer patients were dissolved in 30 μl of Laemmli's SDS sample buffer. The protein was separated on a 10% SDS polyacrylamide gel (Thermo Fisher Scientific) and transferred to a membrane. The membrane was blocked with 4% block Ace (KAC) and incubated with anti-CD9 antibody (SHI-EXO-MO1, Cosmo Bio) and anti-CD63 antibody (SHI-EXO-MO1, Cosmo Bio). After primary antibody administration, the membrane was incubated with HRP-labeled anti-mouse IGG (NA9310V, GE Healthcare) antibody and detected using Western Lightning ECL Pro (Nacalai Tesque). Band intensity was measured using the ChemiDoc imaging system (Bio-Rad).

[0060] 1-6. Nanoparticle Tracking Analysis Te-EVs and urinary EVs were isolated from cancer tissue cultures and preoperative urine of bladder cancer patients using Magcapture beads, according to the manufacturer's protocol. Isolated samples were diluted 1:50 with PBS. EV size was analyzed using Zetaview (Particle Metrix). Settings were: maximum area: 1000, minimum area: 5, minimum brightness: 30, laser wavelength: 488 nm.

[0061] 1-7. Peptide isolation: Protein digestion is performed using S-Trap. ＴＭThe procedure was performed using a microcolumn (ProtiFi) according to the manufacturer's instructions. Cancer tissue lysate (10 μg) and isolated EV were dissolved in 30 μl of Laemmli SDS sample buffer. After adding 16 μl of SDS sample buffer, the protein was reduced with 5 mM tris(2-carboxyethyl)phosphine hydrochloride at 55°C for 15 minutes, and then alkylated with 40 mM iodoacetamide in the dark for 45 minutes. The alkylation buffer was acidified with phosphoric acid to a final concentration of 2.5% phosphoric acid. The membrane binding solution and washing buffer (100 mM triethylammonium bicarbonate [TEAB] in 90% methanol) were mixed and centrifuged at 4,000 g for 1 minute before being transferred to an S-Trap microcolumn. After washing the filtration column three times, the protein was digested in 20 μl of Trypsin / LysC Mix (Promega) in 50 mM TEAB at 47°C for 2 hours. Additional elution was performed with 40 μl of 50 mM TEAB, 40 μl of 0.2% formic acid, and 40 μl of 50% acetonitrile. The eluted peptide was dried using Speed-Vac and stored at -30°C until LC-MS analysis.

[0062] 1-8. LC-MS / MS Analysis Digested peptides were resuspended in water containing 2% acetonitrile and 0.1% formic acid and analyzed using a Thermo Orbitrap Lumos Fusion Tribrid mass spectrometer with a FAIMS-Pro interface (Thermo Scientific) and a Vanquish Neo UHPLC system (Thermo Scientific). Samples were trapped in a pre-column (Pepmap Neo C18 5 μm, 300 μm × 5 mm 1500 bar, Thermo Scientific). The captured samples were separated using an analytical column (Aurora UPHLC Column, C18, 0.075 × 250 mm, 1.6 μm FSC, IonOpticks). Mobile phase A was 0.1% formic acid, and mobile phase B was 0.1% formic acid in acetonitrile. Using a Vanquish Neo system, at a flow rate of 200 nl / min, the mixture was increased from 2% to 30% B by 115 minutes, increased to 95% B by 117 minutes, held for 3 minutes, and then returned to 2% B. Between each sample analysis, two 12-minute washing steps were performed to prevent peptide carryover. Gas-phase fractionation was performed using the FAIMS-Pro interface (ThermoScientific). MS / MS data were obtained using nine correction voltage (CV) sets. The CV settings for each sample run were as follows: Set 1 (CV = -40, -60, -80V), Set 2 (CV = -50, -70, -90V), Set 3 (CV = -45, -55, -65V). Full MS scans were performed on 350–1,500 m / z peptides in the Orbitrap at a resolution of 120K, followed by MS2 acquisition at a resolution of 15K. The maximum implantation time for the full MS scan was 50 ms, and the auto-gain control (AGC) was set to 4e5. The peptide charge states were specified from 2 to 5, and fragmentation was performed by high-energy collision dissociation (HCD) at 30% collision energy. A centroid MS2 scan was obtained using a separation window of 1.6 m / z. The maximum ion implantation time for the MS2 scan was 22 ms, and the AGC by the ion trap was 3e4.

[0063] 1-9. LC-MS / MS Data Analysis and Identification of Mutant Peptides Using Proteome Discover version 3.0 (ThermoScientific), a customized personalized database was used to perform global peptide identification, including mutant peptides. The obtained Raw files were processed with Spectrum Selector, and peptide identification was performed using Mascot and Sequest HT. Sequest search was combined with precursor detector, spectral filter, and INFERYS rescoring. One or two cleavage errors were tolerated in the search for trypsin peptides. Carbamethylation was defined for cysteine, and oxidative modification for methionine. The mass tolerance ranges for precursors and fragments were set to 10 ppm and 0.6 Da, respectively. The false detection rate (FDR) was set to 0.01 (Strict), and high-confidence peptides were identified using a percolator. PSMs annotated with mutant sequences were identified as mutant peptides.

[0064] 1-10. Validation of Identified Mutant Peptides Stable isotope-labeled peptides were artificially synthesized and used to validate the identified mutant peptides. The C-terminal lysine or arginine was labeled with 13C / 15N, making them 8 Da or 10 Da heavier, respectively, compared to the endogenous amino acid. The synthetic peptides were labeled, and MS2 spectra were obtained using 100 fmol of the synthetic peptide. MS / MS data were obtained in a similar manner to the identification of the endogenous peptide using nine CV sets, but the linear gradient was changed from 2% to 30% B to 2% to 40% B. Peak areas and intensities of the MS1 spectra were automatically calculated by Freestyle software 1.8 SP2, and optimized CVs were used after absolute quantification of the mutant peptides.

[0065] 1-11. Absolute Quantification of Mutant Peptides in Urinary EV MS / MS data was obtained by parallel reaction monitoring (PRM) analysis to calculate the abundance of mutant proteins in urinary EV. The CV, which represents the top peak area of ​​the precursor ion, was optimized. The optimized CV of the synthetic peptide was used to obtain the endogenous peak of the mutant peptide. The monoisotopic m / z of the mutant peptide was calculated using the MS-product website (University of California).

[0066] 2. Results: Based on the analysis described above, we successfully identified 39 mutant proteins from bladder cancer tissue, 28 mutant proteins from EV secreted from bladder cancer tissue cultured in serum-free medium, and 4 mutant proteins from urinary EV (Figure 2).

[0067]

[0068] These results represent the first report on bladder cancer-specific somatic mutations. The mutated protein discovered in this study could be used as a bladder cancer-specific biomarker. Furthermore, this study confirmed that mutated proteins containing bladder cancer-specific somatic mutations are encapsulated in extracellular proteins (EVs) and secreted extracellularly. In other words, bladder cancer can be non-invasively screened for by detecting bladder cancer-specific somatic mutations in EVs contained in bodily fluids (e.g., urine and blood).

[0069] [Example 2: Post-treatment recurrence monitoring using bladder cancer-specific somatic mutations as markers] To evaluate the applicability of the bladder cancer-specific somatic mutation detected in Example 1 for diagnosis, urine samples were examined pre-operatively, 3 months post-operatively, and 6 months post-operatively from one case in which the mutant protein LCP1-D321H was detected in urinary extravasation (EV) (Figure 3A). Stable isotope-labeled peptide standards were prepared and absolute quantification was performed by LC / MS, yielding values ​​of 0.286, 0.025, and 0.037 fmol / μl, respectively, and a significant decrease in values ​​was confirmed post-operatively (Figure 3B). This result was consistent with the diagnosis of recurrence-free status obtained by cystoscopy at 6 months post-operatively.

[0070] As described above, it was confirmed that the mutant protein found in this study can be used as a bladder cancer-specific biomarker. Furthermore, it was confirmed that the mutant protein found in this study can be detected from EV contained in bodily fluids (e.g., urine and blood).

Claims

1. A testing method for determining the possibility of bladder cancer, comprising the step of detecting one or more bladder cancer-specific protein mutations selected from the following group consisting of (1) to (47) in a fluid-derived sample or tissue-derived sample taken from a subject: (1) E485K mutation in CUL1 (SEQ ID NO: 1) (2) D183N mutation in PRDX6 (SEQ ID NO: 2) (3) D98N mutation in CELF1 (SEQ ID NO: 3) (4) R135K mutation in RALA (SEQ ID NO: 4) (5) F73L mutation in FAM167B (SEQ ID NO: 5) (6) E186K mutation in HSPB1 (SEQ ID NO: 6) (7) S1252C mutation in SPTBN1 (SEQ ID NO: 7) (8) P120S mutation in ACTR3 (SEQ ID NO: 8) (9) E294D mutation in RBM25 (SEQ ID NO: 9) (10) D313N mutation in SRP54 (SEQ ID NO: 10) (11) E233K mutation in INTS8 (SEQ ID NO: 11) (12) Q171K mutation in CARS2 (SEQ ID NO: 12) (13) G22R mutation in PDCD5 (SEQ ID NO: 13) (14) G835R mutation in TRPM4 (SEQ ID NO: 14) (15) E648Q mutation in MUC4 (SEQ ID NO: 15) (16) R452T mutation in DHX15 (SEQ ID NO: 16) (17) D122H mutation in CSE1L (SEQ ID NO: 17) (18) E36K mutation in HIST2H2BE (SEQ ID NO: 18) (19) D212H mutation in RPN1 (SEQ ID NO: 19) (20) D901N mutation in SVIL (SEQ ID NO: 20) (21) L21V mutation in CHRAC1 (SEQ ID NO: 21) (22) R201Q mutation in SMARCB1 (SEQ ID NO: 22) (23) G200E mutation in TAF15 (SEQ ID NO: 23) (24) E104K mutation in ITGB1 (SEQ ID NO: 24) (25) G794V mutation in MCM6 (SEQ ID NO: 25) (26) D133N mutation in CTTN (SEQ ID NO: 26) (27) D321H mutation in LCP1 (SEQ ID NO: 27) (28) K102N mutation in TKT (SEQ ID NO: 28) (29) R639H mutation in PLCD1 (SEQ ID NO: 29) (30) R460-A461 deletion in VCL (SEQ ID NO: 30) (31) R39G mutation in PTBP3 (SEQ ID NO: 31)(32) E708K mutation in IARS2 (SEQ ID NO: 32) (33) D235H mutation in NLN (SEQ ID NO: 33) (34) E235Q mutation in GATAD2B (SEQ ID NO: 34) (35) R167G mutation in MYO19 (SEQ ID NO: 35) (36) M1432V mutation in MYOF (SEQ ID NO: 36) (37) R130K mutation in EBAG9 (SEQ ID NO: 37) (38) L37V mutation in BCAT2 (SEQ ID NO: 38) (39) R342Q mutation in DNAJB12 (SEQ ID NO: 39) (40) Y167H mutation in HNRNPDL (SEQ ID NO: 40) (41) G873A mutation in LNPEP (SEQ ID NO: 41) (42) D6N mutation in RPS27L (SEQ ID NO: 42) (43) K109T mutation in HIST1H2BO (SEQ ID NO: 43) (44) T83S mutation in QARS (SEQ ID NO: 44) (45) V96L mutation in STAM (SEQ ID NO: 45) (46) L980-E986 deletion in MYH14 (SEQ ID NO: 46) (47) H186Y mutation method in ERGIC2 (SEQ ID NO: 47).

2. A method according to claim 1, wherein if the bladder cancer-specific mutant protein is detected in a sample taken from the subject, it is determined that there is a high probability that bladder cancer is present.

3. The method according to claim 1, wherein the bodily fluid-derived sample is a urine-derived sample or a blood-derived sample.

4. The method according to claim 3, wherein the urine-derived sample is an extracellular vesicle (EV) in the urine.

5. The method according to claim 3, wherein the urine-derived sample is urine sediment.

6. The method according to claim 3, wherein the blood-derived sample is an extracellular vesicle (EV) in the blood.

7. The method according to claim 1, wherein the tissue-derived sample is a sample derived from bladder tissue.

8. The method according to claim 1, wherein the detection of the bladder cancer-specific protein mutation is performed by mass spectrometry.

9. The method according to claim 1, wherein the detection of the bladder cancer-specific protein mutation is performed by an immunoassay using an antibody that specifically binds to the protein having the bladder cancer-specific protein mutation.

10. A method for monitoring the recurrence of bladder cancer, comprising performing the method according to claim 1 in a subject after treatment for bladder cancer.

11. A method according to claim 10, comprising performing the method according to claim 1 on a subject who has undergone treatment for bladder cancer, using a sample taken before the treatment and a sample taken after the treatment, and comparing the results of each.

12. The method according to claim 1, which is used in combination with other methods for detecting bladder cancer.

13. The method according to claim 12, wherein the other method for detecting bladder cancer is cystoscopy, urine cytology, ultrasound, CT scan, pelvic MRI, or bladder mucosal biopsy.

14. The method according to claim 12, wherein the other method for detecting bladder cancer is a molecular tumor marker test for bladder cancer.

15. The method according to claim 14, wherein the molecular tumor marker is BTA, NMP22, BFP, cytokeratin 8 / cytokeratin 18 (CK8-18), telomerase, CA-50, type IV collagen, TPA, or FDP in urine.

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