Method for testing prostate cancer

Abstract

To provide a novel biomarker of prostate cancer and a method for use thereof.SOLUTION: A method for testing prostate cancer includes the step (1) of detecting at least one biomarker selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella in an enteric bacteria-containing sample taken from a subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for testing prostate cancer. [Background technology]

[0002] In Japan, prostate cancer is the cancer with the highest incidence rate among men, with approximately 100,000 new cases reported annually. In addition, with the Westernization of dietary habits and the aging of the population, the incidence rate and number of new cases of prostate cancer are expected to continue to increase.

[0003] Prostate cancer is usually diagnosed based on the concentration of prostate specific antigen (PSA) in the blood (PSA value). Although it varies depending on the age of the examinee, a PSA value of 4.0ng / mL or higher is generally considered abnormal. However, PSA values ​​can also be increased by factors such as prostate enlargement and inflammation. Therefore, all subjects with abnormal PSA values ​​have prostate cancer. Therefore, if the PSA level is abnormal, ultrasound-guided needle biopsy of the prostate is recommended. A definitive diagnosis is made using the method described above.

[0004] However, even if prostate needle biopsy is performed on subjects with abnormal PSA values, prostate cancer is only found in about 20-40% of subjects with PSA values ​​between 4.0 and 10 ng / mL, and in about 50% of subjects with PSA values ​​above 10 ng / mL. In addition, prostate needle biopsy is usually performed with a one-night, two-day hospital stay, which places a heavy burden on the subject. Furthermore, prostate needle biopsy is a highly invasive test and can cause complications such as pain, infection, bloody stools, blood in the urine, and urinary retention.

[0005] In addition, the PSA test may diagnose low-grade prostate cancer, which is unlikely to have a prognostic value. Overdiagnosis and overtreatment are problems.

[0006] Non-Patent Document 1 reports the analysis of feces from 105 patients who underwent prostate biopsy. However, this is related to the diagnosis of prostate cancer including low malignancy, and its diagnostic accuracy was also low.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a novel biomarker for prostate cancer and a method for using the same. Preferably, an object of the present invention is to provide a biomarker for high-grade prostate cancer and a method for using the same.

Means for Solving the Problems

[0009] As a result of intensive research, the present inventor has found that (1) in an intestinal bacteria-containing sample collected from a subject, At least one biomarker selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella in the A method for examining prostate cancer, which includes the step of detecting a biomarker, can solve the above problems. As a result of further research based on this finding, the present invention was completed. That is, the present invention includes the following aspects.

[0010] Item 1. (1) A method for examining prostate cancer, which includes the step of detecting at least one biomarker selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella in an intestinal bacteria-containing sample collected from a subject.

[0011] Item 2. (2) A method according to Item 1, which includes the step of determining whether the subject has prostate cancer and / or the likelihood that the subject will develop prostate cancer in the future based on the amount or concentration of the biomarker detected in the step (1).

[0012] Item 3. In the step (2), created based on the amount or concentration of the biomarker The method according to claim 2, wherein the determination is made using the obtained discriminant.

[0013] Item 4. The method according to any one of Items 1 to 3, wherein the prostate cancer is high-grade prostate cancer.

[0014] Item 5. The method according to any one of Items 1 to 4, wherein there are 5 or more of the biomarkers.

[0015] Item 6. The method according to any one of Items 1 to 5, wherein there are 10 or more of the biomarkers.

[0016] Item 7. The biomarkers are Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus , Propionispora, and Sebaldella, and the method according to any one of Items 1 to 6.

[0017] Item 8. The method according to any one of Items 1 to 7, wherein the subject is Japanese.

[0018] Item 9. An inspection drug for prostate cancer, comprising a detection agent for at least one biomarker selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella marker.

[0019] Item 10. At least one biomarker selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus in an intestinal bacteria-containing sample collected from an animal treated with a test substance A method for screening an active ingredient of a prophylactic or therapeutic agent for prostate cancer, using the amount or concentration of at least one biomarker selected from the group consisting of Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella as an index.

[0020] Item 11. At least one biomarker selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus in an intestinal bacteria-containing sample collected from an animal treated with a test substance A method for evaluating the carcinogenicity or malignancy of prostate cancer, using the amount or concentration of at least one biomarker selected from the group consisting of Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella as an index.

Advantages of the Invention

[0021] According to the present invention, a biomarker for prostate cancer can be provided. By using the biomarker, it may be possible to perform tests for prostate cancer, screen for active ingredients of prophylactic or therapeutic agents for prostate cancer, evaluate the carcinogenicity or malignancy of prostate cancer, and the like.

Brief Description of the Drawings

[0022]

Figure 1

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Mode for Carrying Out the Invention

[0023] In this specification, with respect to the expressions "containing" and "comprising", the concepts of "containing", "comprising", "consisting essentially of", and "consisting only of" are included.

[0024] 1. Method for detecting prostate cancer In one aspect of the present invention, there is provided a method for examining prostate cancer (which may also be referred to as "the examination method of the present invention" in this specification), comprising the step of detecting at least one biomarker selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella, in an intestinal bacteria-containing sample collected from a subject. This will be described below. Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus Propionispora, and Sebaldella.

[0025] 1-1. Step (1) The type of prostate cancer to be examined is not particularly limited. All classes, grades, stages, etc. in various classification criteria of prostate cancer are not particularly limited. According to the biomarker of the present invention, in particular, the presence or absence of high-grade prostate cancer can be determined. High-grade prostate cancer refers to prostate cancer with a Gleason score of 7 or more.

[0026] The subject is the target organism of the inspection method of the present invention, and its species is not particularly limited. Examples of the species of the subject include various mammalian animals such as humans, chimpanzees, monkeys, mice, rats, dogs, cats, rabbits, etc., and preferably humans. Among humans, , particularly preferably Japanese people.

[0027] The state of the subject is not particularly limited. Examples of the subject include a sample whose prostate cancer status is unknown, a sample that has already been determined by another method to have prostate cancer, a sample that has already been determined by another method not to have prostate cancer, a sample during the treatment of prostate cancer, a sample suspected of future prostate cancer, etc.

[0028] The intestinal bacteria-containing sample is not particularly limited as long as it contains intestinal bacteria. Examples of the intestinal bacteria-containing sample include rectal samples (e.g., rectal bacteria samples collected with gloves inserted during digital rectal examination or attachments to the hand, collection kits, etc.), gastrointestinal tract contents such as feces. The intestinal bacteria-containing sample may be employed alone or in combination of two or more. The intestinal bacteria-containing sample can be collected from the subject by methods known to those skilled in the art.

[0029] The detection target in step (1) is at least one biomarker selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella (in this specification, these may be collectively referred to as "target biomarkers".). ).

[0030] The target biomarker is a biomarker whose amount changes in prostate cancer, and prostate cancer can be differentiated by using this as an indicator.

[0031] Alistipes means all bacteria belonging to Alistipes. Similarly, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella each mean all bacteria belonging to the indicated genus or family.

[0032] Among the target biomarkers, at least one biomarker (BM1) selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, and Syntrophococcus is a target biomarker that increases in prostate cancer (especially high-grade prostate cancer).

[0033] Among the target biomarkers, at least one biomarker (BM2) selected from the group consisting of Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella is a target biomarker that decreases in prostate cancer (especially high-grade prostate cancer).

[0034] The number of target biomarkers in the project (1) may be only 1 type, but may also be 2 or more types, 3 or more types, 4 or more types, 5 or more types, 6 or more types, 7 or more types, 8 or more types, 9 or more types, 10 or more types, 11 or more types, 12 or more types, 13 or more types, 14 or more types, 15 or more types, 16 or more types, 17 or more types, 18 or more types, 19 or more types, or all combinations of 20 types. By combining more target biomarkers, it becomes possible to perform tests for prostate cancer (especially high-grade prostate cancer) more accurately.

[0035] In a particularly preferred embodiment of the present invention, the target biomarkers are Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuri a, Moryella, Peptostreptococcus, Propionispora, and Sebaldella, including a total of 18 species.

[0036] Detection is usually performed by measuring the amount or concentration of the target biomarker. The "concentration" is not limited to the absolute concentration, and may also be the relative concentration, the weight per unit volume, the amount per total bacterial mass or total nucleic acid amount in the sample, or the raw data measured to know the absolute concentration.

[0037] The method for detecting the target biomarker is not particularly limited as long as it can specifically detect part or all of the target biomarker. Specific detection methods include, for example, culturing intestinal bacteria in a pre-predicted selective medium and confirming the presence or absence of colonies of the target intestinal bacteria, the PCR method for detecting genes (such as the 16S rRNA gene) or nucleic acids derived from the gene that can discriminate bacterial species, the Southern hybridization method, the Northern hybridization Examples include the isosation method, DNA microarray method, etc. In addition, shotgun sequencing of DNA in a sample and methods using amplicon sequencing can also be used. Furthermore, methods based on antibodies for metabolic detection of bacterial metabolites and proteomic detection of bacterial proteins can also be used. These methods can be carried out according to or in accordance with conventional methods.

[0038] According to the inspection method of the present invention including step (1), the amount and / or concentration of a target biomarker, which is a detection index for prostate cancer (especially high-grade prostate cancer) can be provided, and thereby detection of prostate cancer (especially high-grade prostate cancer) etc. can be assisted.

[0039] The inspection result by the inspection method of the present invention including step (1) can be used for determination of treatment effect, elucidation of the pathological condition of prostate cancer (especially high-grade prostate cancer), prognosis prediction of prostate cancer (especially high-grade prostate cancer), patient stratification, selection of treatment methods (personalized medicine, treatment responsiveness), etc.

[0040] 1-2. Step (2) In one aspect, the inspection method of the present invention further includes, as (2), a step of determining whether or not the subject has prostate cancer (especially high-grade prostate cancer) and / or the possibility that the subject will develop prostate cancer (especially high-grade prostate cancer) in the future, based on the amount or concentration of the target biomarker detected in the step (1). According to the inspection method of the present invention including the step 2, it becomes possible to determine prostate cancer (especially high-grade prostate cancer).

[0041] Examples of the determination method include a method using a discriminant formula created based on the amount or concentration of a target biomarker, a method using a cut-off value of the amount or concentration of BM1 and / or BM2, etc. In the present invention, it is preferable to use the amounts or concentrations of a plurality of target biomarkers as an index, and in such a case, the method using a discriminant formula is particularly preferable.

[0042] The discriminant can be created using any discriminant analysis method that can create a discriminant for differentially discriminating prostate cancer (especially high-grade prostate cancer) from others (healthy, healthy and low-grade prostate cancer), such as Fisher's linear discriminant analysis, non-linear discriminant analysis using Mahalanobis distance, neural network, Support Vector Machine (SVM), etc., but these are not limited to specific examples. In step (2), for example, methods such as neural network, k-nearest neighbor method , decision tree, logistic regression analysis, etc. can also be used for determination. The discriminant can also use one created in advance based on the amount or concentration of the target biomarker measured from another subject, or one created based on the amount or concentration of the target biomarker measured from the subject in step (1).

[0043] The cut-off value can be appropriately set by those skilled in the art from viewpoints such as sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, etc. For example, the amount and / or concentration of the target biomarker in an intestinal bacteria-containing sample collected from a subject not suffering from prostate cancer (especially high-grade prostate cancer) can be used as a value determined each time or a predetermined value based thereon.

[0044] 2. Diagnosis of prostate cancer with higher accuracy When it is determined by the inspection method of the present invention including step (2) that the subject has prostate cancer (especially high-grade prostate cancer), by combining with the step of applying a diagnosis by a doctor of prostate cancer (especially high-grade prostate cancer) to the inspection method of the present invention, prostate cancer (especially high-grade prostate cancer) can be diagnosed with higher accuracy. Further, since the inspection method of the present invention can detect prostate cancer (especially high-grade prostate cancer) more accurately, by combining the above steps with the inspection method of the present invention, it is possible to diagnose more efficiently and accurately that the subject "has prostate cancer (especially high-grade prostate cancer)".

[0045] 3. Prevention, countermeasures, and treatment of prostate cancer When it is determined by the inspection method of the present invention including step (2) that the subject has prostate cancer (especially high-grade prostate cancer), and / or is likely to develop prostate cancer (especially high-grade prostate cancer) in the future, then further with respect to the inspection method of the present invention, or when it is diagnosed as having prostate cancer (especially high-grade prostate cancer) as described in "2. More accurate diagnosis of prostate cancer" above, then further with respect to the combination of the inspection method of the present invention and the step of applying the diagnosis by a doctor, (3) For a subject determined or diagnosed as having (or likely to develop in the future) prostate cancer (especially high-grade prostate cancer), by performing the step of preventing and / or treating prostate cancer, it becomes possible to prevent and / or treat the prostate cancer of the subject. Also, since the inspection method of the present invention can detect prostate cancer (especially high-grade prostate cancer) more accurately, by combining step (3) with respect to the inspection method of the present invention, or with respect to the combination of the inspection method of the present invention and the step of applying the diagnosis by a doctor, a subject with prostate cancer (especially high-grade prostate cancer) can be treated more efficiently and reliably, and for a subject who may develop prostate cancer (especially high-grade prostate cancer) in the future, preventive measures or countermeasures can be taken more efficiently and reliably. The treatment method of prostate cancer is not particularly limited, and various known treatment methods can be adopted. Examples of treatment methods include chemotherapy, surgical treatment, radiotherapy, immunotherapy, and the like. These can be carried out according to known methods. (or is likely to develop in the future) and diagnosed as having prostate cancer, it becomes possible to prevent and / or treat the prostate cancer of the subject. Also, since the inspection method of the present invention can detect prostate cancer (especially high-grade prostate cancer) more accurately, by combining step (3) with respect to the inspection method of the present invention, or with respect to the combination of the inspection method of the present invention and the step of applying the diagnosis by a doctor, a subject with prostate cancer (especially high-grade prostate cancer) can be treated more efficiently and reliably, and for a subject who may develop prostate cancer (especially high-grade prostate cancer) in the future, preventive measures or countermeasures can be taken more efficiently and reliably.

[0046] The treatment method of prostate cancer is not particularly limited, and various known treatment methods can be adopted. Examples of treatment methods include chemotherapy, surgical treatment, radiotherapy, immunotherapy, and the like. These can be carried out according to known methods.

[0047] The therapeutic agents used in chemotherapy are not particularly limited, and various anticancer agents can be used. Examples of anticancer agents include alkylating agents, antimetabolites, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum preparations, molecular target drugs, hormonal agents, biological agents, and the like. Examples of alkylating agents include cyclophosphamide, ifosfamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, ranimustine, and the like. Examples of antimetabolites include enocitabine, carmofur, capecitabine, tegafur, tegafur-uracil, tegafur-gimeracil-oteracil potassium, gemcitabine, cytarabine, cytarabine ocfosfate, nelarabine, fluorouracil, fludarabine, pemetrexed, pentostatin, methotrexate, cladribine, doxifluridine, hydroxycarbamide, mercaptopurine, and the like. Examples of microtubule inhibitors include alkaloid anticancer agents such as vincristine, and taxane anticancer agents such as docetaxel and paclitaxel. Examples of antibiotic anticancer agents include mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, aclarubicin , idarubicin, pirarubicin, peplomycin, mitoxantrone, amrubicin, dinostatin stimalamer, and the like. Examples of topoisomerase inhibitors include CPT-11, irinotecan, nogitecan, which have a topoisomerase I inhibitory effect, and etoposide and sobuzoxane, which have a topoisomerase II inhibitory effect. Examples of platinum preparations include, for example, cis Examples include platinum drugs such as latin, nedaplatin, oxaliplatin, and carboplatin. Examples of hormonal agents include dexamethasone, finasteride, tamoxifen, astrozole, exemestane, ethinyl estradiol, chlormadinone, goserelin, bicalutamide, flutamide, prednisolone, leuprorelin, letrozole, estramustine, toremifene, phosphoestrol, mitotane, methyltestosterone, medroxyprogesterone, and mepitiostane. Examples of biological agents include interferon α, β, and γ, interleukin 2, ubenimex, and dried BCG. Examples of molecular targeted drugs include nivolumab, pembrolizumab, rituximab, alemtuzumab, trastuzumab, cetuximab, panitumumab, imatinib, dasatinib, nilotinib, gefitinib, erlotinib, temsirolimus, bevacizumab, VEGF trap, sunitinib, sorafenib, tositumomab, bortezomib, gemtuzumab ozogamicin Examples include ising, ibritumomab tiuxetan, ibritumomab ozogamicin, tamibarotene, tretinoin, etc. In addition to the molecular targeted drugs specified here, human epidermal growth factor receptor 2 inhibitors, epidermal growth factor receptor inhibitors, Bcr-Abl tyrosine kinase inhibitors, epidermal growth factor tyrosine kinase inhibitors, mTOR inhibitors, vascular endothelial growth factor receptor 2 inhibitors ( α-VEGFR-2 antibodies), inhibitors targeting angiogenesis such as these, and various tyrosine kinase inhibitors such as MAP kinase inhibitors, inhibitors targeting cytokines, proteasome inhibitors, molecular targeted drugs such as antibody-anticancer drug conjugates, etc. can also be included. These inhibitors also include antibodies. The therapeutic agents can be used alone, in combinations of two, or in combinations of three or more.

[0048] 4. Test drug for prostate cancer ​In one aspect, the present invention relates to a prostate cancer test agent (which may also be referred to as "the test agent of the present invention" herein) containing a detection agent for at least one biomarker selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella (which may also be referred to as "the detection agent of the present invention" herein). The following is an explanation thereof. ).

[0049] Regarding Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella, prostate cancer, etc., it is the same as the definition in the above "1. Prostate cancer test method".

[0050] The detection agent of the present invention is not particularly limited as long as it can (preferably specifically) detect the target biomarker. Examples of the detection agent include primers, probes, antibodies, etc. against the gene that is the target biomarker, the gene of the bacterium that is the target biomarker, or their expression products.

[0051] The detection agent of the present invention may be modified as long as its function is not significantly impaired. Examples of the modification include the addition of a labeling substance such as a fluorescent dye, an enzyme, a protein, a radioisotope, a chemiluminescent substance, biotin, and the like.

[0052] As the fluorescent dye used in the present invention, those generally used for labeling nucleotides for the detection and quantification of nucleic acids can be preferably used. For example, HEX (4,7,2’,4’,5’,7’-hexachloro-6-carboxylfluorescein, green fluorescent dye), fluorescein, NED (trade name, manufactured by Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, manufactured by Applied Biosystems, yellow-green fluorescent dye), rhodamin or its derivative [for example, tetramethylrhodamin (TMR)] can be mentioned, but it is not limited thereto. As a method for labeling nucleotides with a fluorescent dye, an appropriate one of known labeling methods can be used (see Nature Biotechnology, 14, 303-308 (1996)). Also, a commercially available fluorescent labeling kit can be used (for example, the oligonucleotide ECL 3’-oligolabeling system manufactured by Amersham Pharmacia).

[0053] The detection agent of the present invention can also be used after being immobilized on an arbitrary solid phase. Therefore, the test agent of the present invention can be provided in the form of a substrate on which the detection agent is immobilized (for example, a microarray chip on which a probe is immobilized, etc.).

[0054] The solid phase used for immobilization is not particularly limited as long as it can immobilize polynucleotides, etc., and examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries, and other substrates. The immobilization of the detection agent to the solid phase is not particularly limited. The immobilization method is well known in the art depending on the type of immobilized probe, such as using a commercially available spotter (such as Amersham) for microarrays (e.g., photolithographic technology (Affymetrix), in situ synthesis of oligonucleotides using inkjet technology (Rosetta Inpharmatics), etc.).

[0055] The primers, probes, etc. are not particularly limited as long as they selectively (specifically) recognize the target biomarker or a nucleic acid derived therefrom. Here, "selectively (specifically) recognize" means, for example, in the Northern blot method, that the target biomarker can be specifically detected, and in the RT-PCR method, that the target biomarker or a nucleic acid derived therefrom (cDNA, etc.) is specifically amplified, but is not limited thereto, and may be any primer or probe that allows a person skilled in the art to determine that the detected or amplified product is derived from the target biomarker.

[0056] Specific examples of primers and probes include the polynucleotides listed in (a) below. a polynucleotide as described in (b) below: (a) having at least 15 consecutive bases in the base sequence of the target biomarker; and / or a polynucleotide complementary to said polynucleotide, (b) A nucleic acid sequence that is a sequence of a target biomarker or a sequence complementary thereto is stripped. The present invention includes at least one selected from the group consisting of polynucleotides having at least 15 bases that hybridize under neutral conditions.

[0057] A complementary polynucleotide or complementary base sequence (complementary strand, reverse strand) refers to the full-length sequence of a polynucleotide consisting of the base sequence of a target biomarker, or a partial sequence thereof having a base sequence of at least 15 consecutive bases in length in the base sequence (herein, for convenience, these are also referred to as "sense strands"). It means a polynucleotide or base sequence that is in a base-complementary relationship based on base pair relationships such as A:T and G:C with respect to the sense strand. However, such a complementary strand is not limited to forming a completely complementary sequence with the base sequence of the target sense strand, and may have a complementary relationship to such an extent that it can hybridize with the target sense strand under stringent conditions. Here, the stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid that binds to the complex or probe, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). For example, as the washing conditions after hybridization, conditions such as "1×SSC, 0.1% SDS, 37°C" can usually be mentioned. The complementary strand is preferably one that maintains a hybridized state with the target sense strand even when washed under such conditions. Although not particularly limited, more stringent hybridization conditions such as "0.5×SSC, 0.1% SDS, 42°C" and even more stringent hybridization conditions such as "0.1×SSC, 0.1% SDS, 65°C" can be mentioned as washing conditions. Specifically, as such a complementary strand, a strand consisting of a base sequence that is in a completely complementary relationship with the base sequence of the target sense strand, and a strand consisting of a base sequence having at least 90%, preferably 95%, more preferably 98% or more, and even more preferably 99% or more identity with the strand can be exemplified.

[0058] Primers, probes, etc. can be designed, for example, based on the base sequence of a target biomarker using various design programs. Specifically, a candidate sequence of a primer or a probe, or a sequence containing at least a part of the sequence, obtained by applying the base sequence of the target biomarker to a design program can be used as a primer or a probe.

[0059] The base length of primers, probes, etc. is not particularly limited as long as it has a length of at least 15 consecutive bases as described above, and can be appropriately set according to the application. As the base length, for example, when used as a primer, 15 bases to 35 bases can be exemplified, and when used as a probe, 15 bases to 35 bases can be exemplified.

[0060] The test agent of the present invention may contain other detection agents (for example, probes for detecting other nucleic acids, antibodies, etc.) other than the detection agent of the present invention. In this case, the test agent of the present invention may be a test agent capable of detecting other diseases and conditions in addition to prostate cancer. In this case, the detection agent of the present invention is included as a detection agent for prostate cancer testing. From this perspective, the test agent of the present invention is, in one aspect, a test agent for prostate cancer including a detection agent for prostate cancer consisting of the detection agent of the present invention.

[0061] The test agent of the present invention may be in the form of a composition. The composition may contain other components as necessary. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, etc.

[0062] The test agent of the present invention may be in the form of a kit. In addition to the above detection agent or the composition containing the same, the kit may include those that can be used for detecting a target biomarker in a sample containing intestinal bacteria of a subject. Specific examples of such things include various reagents (for example, nucleic acid extraction reagents, buffers, etc.), instruments (for example, instruments for purifying and separating samples containing intestinal bacteria), and the like.

[0063] 5. Screening method for active ingredients of preventive or therapeutic agents for prostate cancer In one aspect of the present invention, at least one selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella in a sample containing intestinal bacteria collected from an animal treated with a test substance The present invention relates to a method for screening an active ingredient of a prophylactic or therapeutic agent for prostate cancer using the amount or concentration of a biomarker as an index (in this specification, it may also be referred to as "the method for screening an active ingredient of the present invention"). Hereinafter, this will be described.

[0064] Regarding the sample containing intestinal bacteria, the target biomarker, prostate cancer, the measurement of the amount or concentration of the target biomarker, etc., they are the same as the definitions in the above "1. Method for examining prostate cancer".

[0065] The species of the animal is not particularly limited. Examples of the species of the animal include various mammalian animals such as humans, chimpanzees, monkeys, mice, rats, dogs, cats, rabbits, and the like. Preferably, a prostate cancer model animal can be used.

[0066] As the test substance, it can be widely used regardless of whether it is a naturally occurring compound or an artificially created compound. Also, not limited to purified compounds, compositions in which various compounds are mixed or extracts of animals and plants can also be used. Compounds include not only low-molecular compounds but also high-molecular compounds such as proteins, nucleic acids, and polysaccharides.

[0067] More specifically, the method for screening the active ingredient of the present invention can use, for example, a discriminant formula or a cut-off value obtained according to or analogous to the method described in step (2) of the test method of the present invention. By applying the amount or concentration of the target biomarker to the discriminant formula or the cut-off value, when it is determined that the subject does not have prostate cancer (or has a preventive or therapeutic effect on prostate cancer), the step of selecting the test substance as an active ingredient of a preventive or therapeutic agent for prostate cancer (or a candidate substance for an active ingredient of a preventive or therapeutic agent for prostate cancer) is included.

[0068] More specifically, the method for screening the active ingredient of the present invention, for example, when the biomarker used as an index is at least one biomarker (BM1) selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, and Syntrophococcus, and when the value of the above index is lower than the amount or concentration of the corresponding biomarker (control value) in the intestinal bacteria-containing sample collected from an animal not treated with the test substance, the step of selecting the test substance as an active ingredient of a preventive or therapeutic agent for prostate cancer (or a candidate substance for an active ingredient of a preventive or therapeutic agent for prostate cancer) is included.

[0069] As another specific example, the method for screening the active ingredient of the present invention includes a step of selecting the test substance as an active ingredient (or a candidate substance for the active ingredient of the prophylactic or therapeutic agent for prostate cancer) for the prophylactic or therapeutic agent for prostate cancer when the value of the above index is higher than the amount or concentration (control value) of the corresponding biomarker in the intestinal bacteria-containing sample collected from an animal not treated with the test substance. The corresponding biomarker means the same bacteria or gene as the target biomarker used as the index. "High" means, for example, that the value of the index is 2 times, 5 times, 10 times, 20 times, 50 times, 100 times the control value. "Low" means, for example, that the value of the index is 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100 of the control value. In one aspect of the present invention, at least one selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella in the intestinal bacteria-containing sample collected from an animal treated with the test substance when the amount or concentration of the corresponding biomarker in the intestinal bacteria-containing sample collected from an animal not treated with the test substance (control value).

[0070] The corresponding biomarker means the same bacteria or gene as the target biomarker used as the index.

[0071] "High" means, for example, that the value of the index is 2 times, 5 times, 10 times, 20 times, 50 times, 100 times the control value. means.

[0072] "Low" means, for example, that the value of the index is 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100 of the control value. means.

[0073] 6. Method for evaluating disease inducibility or exacerbation of the above bacteria in the intestinal bacteria-containing sample collected from an animal treated with the test substance A method for evaluating the carcinogenicity or malignancy progression of prostate cancer using the amount or concentration of a biomarker (which may also be referred to as "the method for evaluating toxicity of the present invention" in this specification). The following is an explanation thereof.

[0074] Regarding the intestinal bacteria-containing sample, the target biomarker, prostate cancer, the measurement of the amount or concentration of the target biomarker, the species of the animal, the test substance, etc., they are the same as the definitions in the above "1. Method for examining prostate cancer" and "5. Method for screening the active ingredient of a prophylactic or therapeutic agent for prostate cancer". The same applies.

[0075] More specifically, for the method for evaluating toxicity of the present invention, for example, a discriminant formula or a cut-off value obtained according to or similar to the method described in step (2) of the inspection method of the present invention can be used. By applying the amount or concentration of the target biomarker to the discriminant formula or the cut-off value, when it is determined that the subject does not have prostate cancer (or has the carcinogenicity or malignancy progression of prostate cancer), the step of selecting the test substance as a prostate cancer-inducing or malignancy-progression substance is included. More specifically, for the method for evaluating toxicity of the present invention, for example, when the biomarker used as an index is at least one biomarker (BM1) selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, and Syntrophococcus, when the value of the above index is higher than the amount or concentration (control value) of the corresponding biomarker in the intestinal bacteria-containing sample collected from an animal not treated with the test substance, the step of selecting the test substance as a prostate cancer-inducing or malignancy-progression substance is included.

[0076] More specifically, for the method for evaluating toxicity of the present invention, for example, when the biomarker used as an index is at least one biomarker (BM1) selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, and Syntrophococcus, when the value of the above index is higher than the amount or concentration (control value) of the corresponding biomarker in the intestinal bacteria-containing sample collected from an animal not treated with the test substance, the step of selecting the test substance as a prostate cancer-inducing or malignancy-progression substance is included.

[0077] As another specific example, in the toxicity evaluation method of the present invention, when the biomarker used as an index is at least one biomarker (BM2) selected from the group consisting of Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella, if the value of the above index is lower than the amount or concentration (control value) of the corresponding biomarker in the intestinal bacteria-containing sample collected from an animal not treated with the test substance, the step of selecting the test substance as an inducer or exacerbator of prostate cancer is included.

[0078] Regarding the corresponding biomarker and the degree of high / low, it is the same as the definition in "5. Screening method for active ingredients of preventive or therapeutic agents for prostate cancer".

Example

[0079] Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited by these examples.

[0080] Test Example 1. Search for prostate cancer biomarkers and determination of prostate cancer As subjects, 114 Japanese people who underwent prostate biopsy (72 Japanese subjects diagnosed with prostate cancer (low malignancy (Gleason score ≦ 6): 14 subjects, high malignancy (Gleason score = 7: 32 subjects, Gleason score ≧ 8: 26 subjects): 58 subjects, and 42 non-cancer Japanese subjects) were adopted. The backgrounds of the subjects are as follows.

[0081]

Table 1

[0082] ​​Bacterial DNA was extracted from a cryopreserved sample of a swab that had wiped the fingertip of a glove inserted into the rectum during a digital rectal examination performed before biopsy, using the QIAGEN DNeasy Power Soil Kit. Using the obtained DNA as a template, two variable regions (V1, V2) of the 16S rRNA gene were amplified by PCR. Amplicon sequencing was performed under 251bp-paired-end conditions using the Illumina MiSeq. The gut microbiota was identified at the genus or family level using the metagenomic analysis software QIIME (version 1.9.1). The sequences of the bacterial DNA were referenced against a database (Greengenes).

[0083] As part of the results, the prevalence of bacteria is shown in Figures 1 to 3. As shown in these figures, it was found that three types of bacteria, Alistipes genus bacteria, Rikenellaceae family bacteria, and Lachnospira genus bacteria, were increased in prostate cancer, especially high-grade prostate cancer.

[0084] Subsequently, 221 species of unknown names were excluded from the total 724 species identified in this analysis, and the remaining 503 species were subjected to Lasso (least absolute shrinkage and selection operator) regression analysis to discriminate high-grade prostate cancer. Lasso regression analysis identified 18 species as prostate biomarkers (bacteria that increase in high-grade prostate cancer: Lachnospira, Aeromonas, Eggerthella, Kytococcus , Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, and Syntrophococcus; Bacteria that decrease in high-grade prostate cancer: Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella) were narrowed down. Using this, a discriminant formula (FMPI) for determining high-grade prostate cancer positive by logistic regression was created. The formula is shown below.

[0085]

Number

[0086] When the presence or absence of high-grade prostate cancer was determined using the above subject and FMPI, the ROC curve is shown in FIG. 4. In FIG. 4, as a comparison target, the ROC curve when the presence or absence of high-grade prostate cancer was determined using the PSA value is also shown. As shown in FIG. 4, the diagnostic accuracy of high-grade prostate cancer using FMPI was found to be high with an AUC of 0.85 in the ROC analysis. As shown in FIG. 4, the diagnostic accuracy of high-grade prostate cancer using FMPI was found to be high with an AUC of 0.85 in the ROC analysis. The diagnostic accuracy of high-grade prostate cancer using FMPI was found to be high with an AUC of 0.85 in the ROC analysis.

[0087] Test Example 2. Determination of prostate cancer 1 As subjects, 38 Japanese men who underwent prostate biopsy (24 Japanese subjects diagnosed with prostate cancer (low-grade (Gleason score ≦ 6): 5 cases, high-grade (Gleason score = 7: 11 cases , Gleason score ≧ 8: 8 cases): 19 cases, and 14 non-cancer Japanese subjects) were adopted. The background of the subjects is as follows. The background of the subjects is as follows.

[0088]

Table 2

[0089] Bacterial DNA was extracted from a cryopreserved sample of a swab that had wiped the fingertip of a glove inserted into the rectum during a digital rectal examination performed before biopsy, using the QIAGEN DNeasy Power Soil Kit. Using the obtained DNA as a template, two variable regions (V1, V2) of the 16S rRNA gene were amplified by PCR. Amplicon sequencing was performed using an Illumina MiSeq under 251bp - paired - end conditions. The gut microbiota was identified at the genus or family level using the metagenomic analysis software QIIME (version 1.9.1). The sequences of the bacterial DNA were referenced against a database (Greengenes).

[0090] The presence or absence of high - grade prostate cancer was determined using the above - mentioned subject and the FMPI created in Test Example 1. The ROC curve for this case is shown in Figure 5. Figure 5 also shows the ROC curve for the case where the presence or absence of high - grade prostate cancer was determined using PSA values as a comparison. As shown in Figure 5, the diagnostic accuracy of high - grade prostate cancer using FMPI was found to be high even when using a subject group different from the subject group used to create the FMPI. This was the case when using a different subject group from the subject group used to create the FMPI. It was found to be high.

[0091] Test Example 3. Determination of prostate cancer 2 The presence or absence of prostate cancer was determined using the subjects of Test Example 1 (Discovery cohort) or the subjects of Test Example 2 (Test cohort) and the FMPI created in Test Example 1. The ROC curve is shown in Figure 6. As shown in Figure 6, it was found that the presence or absence of prostate cancer could also be determined with high accuracy using FMPI. It was found that it was possible to determine the presence or absence of prostate cancer with high accuracy using FMPI.

Claims

1. (1) In the samples containing enterobacteria collected from the subjects, Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moryella, Peptostreptococcus, Propionispora, and Sebaldella were detected. The method includes detecting at least one biomarker selected from the group consisting of: How to test for cancer.

2. The method of claim 1, further comprising: (2) determining whether or not the subject is suffering from prostate cancer and / or the possibility that the subject will be suffering from prostate cancer in the future based on the amount or concentration of the biomarker detected in step (1).

3. In the step (2), a discriminant prepared based on the amount or concentration of the biomarker The method of claim 2 , wherein the determination is made using a

4. The method according to any one of claims 1 to 3, wherein the prostate cancer is high-grade prostate cancer.

5. The method according to any one of claims 1 to 4, wherein the biomarkers are five or more.

6. The method according to any one of claims 1 to 5, wherein the number of biomarkers is 10 or more.

7. 7. The method of any of claims 1 to 6, wherein the biomarkers include Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moriella, Peptostreptococcus, Propionispora, and Sebaldella.

8. The method according to any one of claims 1 to 7, wherein the subject is Japanese.

9. At least one biomarker selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moriella, Peptostreptococcus, Propionispora, and Sebaldella. A prostate cancer testing agent including a detection agent.

10. The amount or concentration of at least one biomarker selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moriella, Peptostreptococcus, Propionispora, and Sebaldella in an intestinal bacteria-containing sample collected from an animal treated with a test substance is indicated. The present invention relates to a method for screening for an active ingredient of a prostate cancer preventive or therapeutic agent.

11. The amount or concentration of at least one biomarker selected from the group consisting of Alistipes, Rikenellaceae, Lachnospira, Aeromonas, Eggerthella, Kytococcus, Phascolarctobacterium, p-75-a5, Raoultella, Roseomonas, Syntrophococcus, Acidaminococcus, Anaerofilum, Atopobium, Blautia, Kocuria, Moriella, Peptostreptococcus, Propionispora, and Sebaldella in an intestinal bacteria-containing sample collected from an animal treated with a test substance is indicated. A method for evaluating the induction or aggravation of prostate cancer.

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