Intestinal bacteria marker for diagnosing kidney cancer

Abstract

The present invention addresses the problem of providing an intestinal bacteria marker for diagnosing kidney cancer.　The ratio of specific intestinal bacteria to all intestinal bacteria is calculated, and said ratio is used as an index for diagnosing kidney cancer.

Description

Gut bacterial markers for diagnosing kidney cancer

[0001] The present invention relates to a method for diagnosing kidney cancer, which includes calculating the abundance ratio of a specific intestinal bacterium to all intestinal bacteria.

[0002] Microorganisms are essentially foreign bodies to the host organism and therefore subject to elimination by the immune system. However, intestinal commensal bacteria have the characteristic of living symbiotically within the intestinal tract without being actively eliminated by the intestinal immune system. After being born in a sterile environment, mammals, including humans, become infected with various bacteria through contact with the outside world, and their intestinal flora is formed. The human intestinal tract is home to over 100 trillion of approximately 1,000 different types of intestinal commensal bacteria, which are thought to have a significant impact on the intestinal environment.

[0003] In recent years, the relationship between the intestinal microbiota and various diseases has been revealed. For example, it has been reported that the abundance of certain intestinal bacteria increases in patients with prostate cancer (Patent Document 1). A method for classifying the type of intestinal microbiota and assessing disease risk has also been reported (Patent Document 2). Furthermore, a relationship between the intestinal microbiota and clear cell renal cell carcinoma (ccRCC) has also been reported (Non-Patent Document 1). However, it has not been known until now that the intestinal bacteria described below are useful as intestinal bacterial markers for diagnosing renal cancer.

[0004] JP 2022-76117 A JP 2023-4411 A

[0005] Front Microbiol. 2022 Jul 4;13:913718.

[0006] An object of the present invention is to provide an intestinal bacteria marker for diagnosing renal cancer.

[0007] The present inventors have been conducting intensive research to solve the above-mentioned problems. Through genus-level analysis, we have identified five genera of intestinal bacteria (Ruminococcus_g4, Ruthenibacterium, Clostridium_g24, Anaerotignum, and Eggerthella) whose abundance ratios relative to the total intestinal bacteria in renal cancer patients were significantly increased compared with those in non-cancer controls. Furthermore, we have identified four species of bacteria (Clostridium bolteae, Eubacterium coprostanoligenes [PAC001050_s], Enterocloster [FCEY_s], and Anaerotignum lactafifermentas) through species-level analysis. The present inventors have found that, of these enterobacteria, one type of bacterium (the genus Anaerotignum) is useful as a diagnostic enterobacteria marker for stage I-II renal cancer, two types of bacteria (the genera Ruminococcus_g4 and Ruthenibacterium) and one type of bacterium (PAC001050_s) are useful as diagnostic enterobacteria markers for stage III-IV renal cancer, and two types of bacteria (the genus Clostridium_g24 and Eggerthella) and three types of bacteria (Clostridium bolteae, FCEY_s, and Anaerotignum lactafifermentas) are useful as diagnostic enterobacteria markers for stage I-II renal cancer and stage III-IV renal cancer, thereby completing the present invention.

[0008] That is, the present invention is as follows: [1] A method for diagnosing kidney cancer, comprising the following steps (a) and (b-1): (a) isolating genomic DNA derived from enterobacteria from a sample containing enterobacteria collected from a subject; and (b-1) calculating, based on nucleotide sequence information in the genomic DNA derived from enterobacteria, the abundance ratio of one or more bacteria selected from the group consisting of i) bacteria of the genus Clostridium having a 16S rRNA gene that has at least 90% sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, ii) bacteria of the genus Eubacterium having a 16S rRNA gene that has at least 90% sequence identity to the nucleotide sequence shown in SEQ ID NO: 2, iii) bacteria of the genus Enterocloster having a 16S rRNA gene that has at least 95% sequence identity to the nucleotide sequence shown in SEQ ID NO: 3, and iv) bacteria of the genus Anaerotignum having a 16S rRNA gene that has at least 90% sequence identity to the nucleotide sequence shown in SEQ ID NO: 4, relative to all enterobacteria, the step (b-1), wherein if the abundance ratio of the bacteria in i) is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be suffering from stage I-II or stage III-IV renal cancer; if the abundance ratio of the bacteria in ii) is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be suffering from stage III-IV renal cancer; if the abundance ratio of the bacteria in iii) is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be suffering from stage I-II or stage III-IV renal cancer; and if the abundance ratio of the bacteria in iv) is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be suffering from stage I-II renal cancer;[2] The method for diagnosing kidney cancer according to [1] above, wherein the bacterium in i) is Clostridium bolteae having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, the bacterium in ii) is Eubacterium coprostanoligenes having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO: 2, the bacterium in iii) is Enterocloster having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO: 3, and the bacterium in iv) is Anaerotignum lactafifermentas having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO: 4. [3] A method for diagnosing kidney cancer, comprising the following steps (a) and (b-2): (a) isolating genomic DNA derived from enterobacteria from a sample containing enterobacteria collected from a subject;(b-2) calculating the abundance ratio of one or more bacteria selected from the group consisting of bacteria of the genus Mediterraneibacter, bacteria of the genus Ruthenibacterium, bacteria of the genus Enterocloster, bacteria of the genus Anaerotignum, and bacteria of the genus Eggerthella relative to all intestinal bacteria based on nucleotide sequence information in genomic DNA derived from intestinal bacteria, wherein if the abundance ratio of bacteria of the genus Mediterraneibacter is increased compared to the abundance ratio in non-cancer controls or patients with stage I to II renal cancer, it indicates that the subject is likely to have stage III to IV renal cancer, if the abundance ratio of bacteria of the genus Ruthenibacterium is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is likely to have stage III to IV renal cancer, and if the abundance ratio of bacteria of the genus Enterocloster is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is likely to have stage I to II renal cancer, the step (b-2), wherein if the abundance ratio of bacteria of the genus Anaerotignum is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be suffering from stage I to II renal cancer, and if the abundance ratio of bacteria of the genus Eggerthella is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be suffering from stage I to II or stage III to IV renal cancer;[4] Bacteria of the genus Mediterraneibacter include bacteria of the genus Mediterraneibacter having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence set forth in SEQ ID NO:5; bacteria of the genus Ruthenibacterium include Ruthenibacterium lactatiformans having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence set forth in SEQ ID NO:6, and bacteria of the genus Ruthenibacterium having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence set forth in SEQ ID NO:7; bacteria of the genus Enterocloster include bacteria of the genus Enterocloster having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence set forth in SEQ ID NO:3, and Clostridium bolteae having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1; bacteria of the genus Anaerotignum include Anaerotignum having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence set forth in SEQ ID NO:8. lactatifermentans, and the Eggerthella bacterium comprises Eggerthella lenta having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO: 9. [5] The method for diagnosing kidney cancer according to any of [1] to [4] above, wherein the sample is feces. [6] The method for diagnosing kidney cancer according to any of [1] to [5] above, wherein the nucleotide sequence information in genomic DNA derived from enterobacteria is nucleotide sequence information of the 16S rRNA gene. [7] The method for diagnosing kidney cancer according to any of [1] to [6] above, wherein the subject is Japanese.

[0009] Other embodiments of the present invention include: a method for creating data for diagnosing renal cancer, comprising the steps (a) and (b-1); a method for creating data for diagnosing renal cancer, comprising the steps (a) and (b-2); a method for diagnosing renal cancer, comprising the steps (a) and (b-1); a method for diagnosing renal cancer, comprising the steps (a) and (b-2); a method for treating renal cancer or a method for preventing the progression of renal cancer, comprising the steps (a) and (b-1) and including a step of administering a treatment for treating renal cancer or a treatment for preventing the progression of renal cancer (e.g., drug therapy using an anticancer agent or the like, radiation therapy, surgical treatment, immunotherapy, etc.) to a subject who has been shown in the step (b-1) to have a high possibility of having stage I-II or stage III-IV renal cancer; a method for treating or preventing the worsening of renal cancer, which comprises the steps (a) and (b-2) above and also includes a step of administering a treatment for treating or preventing the worsening of renal cancer to a subject who has been shown in the step (b-2) to have a high probability of having stage I-II or stage III-IV renal cancer (for example, drug therapy using an anticancer agent or the like, radiation therapy, surgical treatment, immunotherapy, etc.).

[0010] According to the present invention, it is possible to accurately determine not only stage I-II renal cancer (early stage renal cancer) but also stage III-IV renal cancer (advanced renal cancer), thereby contributing to the treatment of renal cancer and the prevention of progression of renal cancer by providing appropriate treatment to more renal cancer patients or by providing appropriate measures to prevent the worsening of renal cancer.

[0011] This figure shows the results of analyzing the abundance ratio of bacteria from five genera (Ruminococcus_g4 [Fig. 1A], Ruthenibacterium [Fig. 1B], Clostridium_g24 [Fig. 1C], Anaerotignum [Fig. 1D], and Eggerthella [Fig. 1E]) relative to the total intestinal bacteria in two groups (control group and renal cancer group). "*" and "**" in the figure indicate statistically significant differences (p<0.05 and p<0.01, respectively). This figure shows the results of analyzing the abundance ratio of bacteria from five genera (Ruminococcus_g4 [Fig. 2A], Ruthenibacterium [Fig. 2B], Clostridium_g24 [Fig. 2C], Anaerotignum [Fig. 2D], and Eggerthella [Fig. 2E]) relative to the total intestinal bacteria in three groups (control group, localized group, and advanced group). The symbol "*" in the figure indicates a statistically significant difference (p<0.05). This figure shows the results of analyzing the abundance ratio of four types of bacteria (Clostridium bolteae [Fig. 3A], PAC001050_s [Fig. 3B], FCEY_s [Fig. 3C], and Anaerotignum lactafifermentas [Fig. 3D]) relative to the total intestinal bacteria in two groups (control group and renal cancer group). The symbol "*" in the figure indicates a statistically significant difference (p<0.05). This figure shows the results of analyzing the abundance ratio of four types of bacteria (Clostridium bolteae [Fig. 4A], PAC001050_s [Fig. 4B], FCEY_s [Fig. 4C], and Anaerotignum lactafifermentas [Fig. 4D]) relative to the total intestinal bacteria in three groups (control group, localized group, and advanced group). In the figure, "*" indicates that there is a statistically significant difference (p<0.05).

[0012] One aspect of the method of the present invention includes the following steps: (a) isolating genomic DNA derived from enterobacteria from a sample (sometimes referred to herein as a "subject-derived sample") containing enterobacteria collected from a subject (also referred to as a "test subject"); and (b-1) selecting, based on nucleotide sequence information in the genomic DNA derived from enterobacteria, i) bacteria of the genus Clostridium having a 16S rRNA gene that has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1, ii) bacteria of the genus Eubacterium having a 16S rRNA gene that has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 2, iii) bacteria of the genus Enterocloster having a 16S rRNA gene that has at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NO: 3, and iv) bacteria of the genus Enterocloster having a 16S rRNA gene that has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 4. a step (b-1) of calculating the abundance ratio of one or more bacteria selected from the group consisting of Anaerotignum bacteria having an rRNA gene (sometimes referred to herein as "the present intestinal bacteria 1") relative to all intestinal bacteria, wherein if the abundance ratio of the bacteria in i) is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is likely to have stage I-II or stage III-IV renal cancer; if the abundance ratio of the bacteria in ii) is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is likely to have stage III-IV renal cancer; if the abundance ratio of the bacteria in iii) is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is likely to have stage I-II or stage III-IV renal cancer; and if the abundance ratio of the bacteria in iv) is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is likely to have stage I-II renal cancer.

[0013] Another aspect of the method of the present invention comprises: (a) isolating genomic DNA derived from enterobacteria from a sample derived from a subject; and (b-2) calculating, based on nucleotide sequence information in the genomic DNA derived from enterobacteria, the abundance ratio of one or more bacteria selected from the group consisting of bacteria of the genus Mediterraneibacter, bacteria of the genus Ruthenibacterium, bacteria of the genus Enterocoloster, bacteria of the genus Anaerotignum, and bacteria of the genus Eggerthella (sometimes referred to herein as "the enterobacteria 2 of the present invention") relative to all enterobacteria, wherein if the abundance ratio of bacteria of the genus Mediterraneibacter is increased compared to the abundance ratio in non-cancer controls or patients with stage I to II renal cancer, this indicates a high possibility that the subject is suffering from stage III to IV renal cancer; and if the abundance ratio of bacteria of the genus Ruthenibacterium is increased compared to the abundance ratio in non-cancer controls, this indicates a high possibility that the subject is suffering from stage III to IV renal cancer. and step (b-2), wherein the presence ratio of Enterocloster bacteria is increased compared to that in non-cancer controls, indicating a high likelihood that the subject has stage I-II renal cancer; the presence ratio of Anaerotignum bacteria is increased compared to that in non-cancer controls, indicating a high likelihood that the subject has stage I-II renal cancer; and the presence ratio of Eggerthella bacteria is increased compared to that in non-cancer controls, indicating a high likelihood that the subject has stage I-II or stage III-IV renal cancer. The method of the present invention is a method for assisting physicians in diagnosing renal cancer and does not involve the physician's actual diagnosis. Note that the present enterobacteria 1 and the present enterobacteria 2 are sometimes collectively referred to as "the present enterobacteria" in this specification.

[0014] As used herein, "renal cancer" refers to any state in which cells in the kidney have become cancerous, and examples thereof include stage I renal cancer (i.e., a state in which the cancer is 7 cm or less in diameter and is confined to the kidney), stage II renal cancer (i.e., a state in which the cancer is more than 7 cm in diameter but is confined to the kidney), stage III renal cancer (i.e., a state in which the cancer has spread intravenously or to perirenal tissues but has not progressed to the adrenal gland, has not crossed Gerota's fascia, and has not metastasized to other organs or regional lymph nodes, or a state in which the cancer has not metastasized to other organs but has metastasized to regional lymph nodes), and stage IV renal cancer (i.e., a state in which the cancer has spread beyond Gerota's fascia or has metastasized to other organs). Suitable examples include stage I-II renal cancer (early renal cancer) and stage III-IV renal cancer (advanced renal cancer).

[0015] In this specification, a "sample containing intestinal bacteria collected from a subject" may be any biological sample derived from a subject that contains intestinal bacteria, such as feces or intestinal mucosal tissue, with feces being a preferred example because it is easy to collect and is minimally invasive.

[0016] In this specification, the "subject" may be any human, preferably a human from a cultural sphere where rice is one of the staple foods (e.g., residents of East Asia, Central Asia, South Asia, Southeast Asia, and West Asia), and more preferably a Mongoloid (e.g., Southern Mongoloid, Northern Mongoloid, Central Mongoloid, Southern Mongoloid, Eskimo, and Amerind, etc.), and specific examples include Japanese, Korean, Chinese, Taiwanese, Mongolian, Tibetan, Indochinese, and Native Americans, with Japanese being a preferred example.

[0017] As used herein, a "cancer-free control" is any human not suffering from cancer, and is preferably of the same race as the subject to be compared.

[0018] As used herein, "at least 90% sequence identity to the nucleotide sequence shown in SEQ ID NO: X" means that one or several nucleotides in the nucleotide sequence of SEQ ID NO: X have been substituted, deleted, inserted, added, or inverted (hereinafter sometimes referred to as "substitution, etc.") and that 90% or more of the entire nucleotide sequence is identical, or that there are no such substitutions, etc. and that the entire nucleotide sequence is 100% identical to the entire nucleotide sequence of SEQ ID NO: X. Furthermore, as used herein, "at least 95% sequence identity to the nucleotide sequence shown in SEQ ID NO: 3" means that one or several nucleotides in the nucleotide sequence of SEQ ID NO: 3 have been substituted, etc. and that 95% or more of the entire nucleotide sequence is identical to SEQ ID NO: 3, or that there are no such substitutions, etc. and that the entire nucleotide sequence is 100% identical to the entire nucleotide sequence of SEQ ID NO: 3. Here, "a nucleotide sequence in which one or several nucleotides have been substituted, deleted, inserted, added or inverted" means a nucleotide sequence in which, for example, 1 to 149 nucleotides, preferably 1 to 100 nucleotides, more preferably 1 to 75 nucleotides, even more preferably 1 to 50 nucleotides, more preferably 1 to 40 nucleotides, even more preferably 1 to 30 nucleotides, and more preferably 1 to 15 nucleotides have been substituted, deleted, inserted, added or inverted.

[0019] As used herein, "at least 90% sequence identity" means that the sequence identity is 90% or more, preferably 91% or more, more preferably 92% or more, even more preferably 93% or more, still more preferably 94% or more, particularly preferably 95% or more, especially more preferably 96% or more, particularly more preferably 97% or more, especially more preferably 98% or more, and most preferably 99% or more (e.g., 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, 100%, etc.). Furthermore, as used herein, "at least 95% sequence identity" means that the sequence identity is 95% or more, preferably 96% or more, more preferably 97% or more, even more preferably 98% or more, and still more preferably 99% or more (e.g., 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, 100%, etc.). Nucleotide sequence identity can be determined using a program called BLASTX (Altschul SF, et al: J Mol Biol 215: 403, 1990) based on the BLAST algorithm by Carlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc Natl Acad Sci USA 90: 5873, 1993) or a program called BLASTN (Altschul SF, et al: J Mol Biol 215: 403, 1990) based on the BLASTP program (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing nucleotide sequences using BLASTN, the parameters are, for example, score = 100 and word length = 12.

[0020] Among the Enterobacteriaceae 1 of the present invention, examples of the bacteria of category i) above include Clostridium bolteae having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:1; examples of the bacteria of category ii) above include Eubacterium coprostanoligenes having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:2; examples of the bacteria of category iii) above include Enterocloster bacteria (e.g., FCEY_s) having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:3; and examples of the bacteria of category iv) above include Anaerotignum lactafifermentas having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:4.

[0021] Examples of the Mediterraneibacter bacteria in the present Enterobacteriaceae 2 include Mediterraneibacter bacteria having a 16S rRNA gene with at least 90% sequence identity to the nucleotide sequence shown in SEQ ID NO: 5, and a population of Mediterraneibacter bacteria including Mediterraneibacter massiliensis, and more specifically, Mediterraneibacter bacteria (LT635549_s) having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 5. Furthermore, examples of the Ruthenibacterium bacteria in the present Enterobacteria 2 include Ruthenibacterium bacteria (e.g., Ruthenibacterium lactatiformans) having a 16S rRNA gene that has at least 90% sequence identity to the nucleotide sequence shown in SEQ ID NO: 6, and Ruthenibacterium bacteria populations including Ruthenibacterium bacteria having a 16S rRNA gene that has at least 90% sequence identity to the nucleotide sequence shown in SEQ ID NO: 7, and more specifically, Ruthenibacterium lactatiformans having a 16S rRNA gene that has at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 6 and Ruthenibacterium bacteria (PAC001646_s) having a 16S rRNA gene that has at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 7.Furthermore, examples of Enterocloster bacteria in the present Enterobacteriaceae 2 include Enterocloster bacteria having a 16S rRNA gene with at least 90% sequence identity to the nucleotide sequence shown in SEQ ID NO: 3, and bacteria (e.g., Clostridium bolteae) having a 16S rRNA gene with at least 90% sequence identity to the nucleotide sequence shown in SEQ ID NO: 1. More specifically, these include Enterocloster bacteria having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 3, and Clostridium bolteae having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 1 (see reference "Int J Syst EvolMicrobiol. 2020 Jan;70(1):23-34."). Furthermore, examples of the Anaerotignum bacteria in the present Enterobacteria 2 include Anaerotignum bacteria having a 16S rRNA gene with at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 8, and a population of Anaerotignum bacteria including Anaerotignum aminivorans, and more specifically, Anaerotignum lactatifermentans having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 8. Furthermore, examples of the Eggerthella bacteria in the present Enterobacteria 2 include Eggerthella bacteria having a 16S rRNA gene with at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 9, and a population of Eggerthella bacteria including Eggerthella lenta, and more specifically, Eggerthella lenta having a 16S rRNA gene with at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 9.

[0022] In the above step (a), the method for isolating enterobacteria-derived genomic DNA from a subject-derived sample is not particularly limited. For example, the subject-derived sample is first suspended in a buffer solution (e.g., Tris, HEPES, etc.) containing digestive enzymes (e.g., protease such as protease K, polysaccharide-degrading enzyme, etc.), denaturants (e.g., guanidine hydrochloride, urea), surfactants (e.g., SDS, Triton X-100, etc.), chelating agents (e.g., EDTA, EGTA, etc.), and the like, followed by a bacteriolysis treatment of the enterobacteria. To effectively perform the bacteriolysis treatment, beads for physically disrupting the cells may be added to the suspension, and a bead-based disruption treatment may be performed. The material of the beads used is not particularly limited, and examples thereof include glass, guanidium, garnet, zirconium, silica, and combinations thereof.

[0023] Next, enterobacteria-derived genomic DNA is isolated from the enterobacteria lysate after the lysis treatment. Examples of such isolation methods include those known in the art, such as methods for denaturing and removing components other than nucleic acids (e.g., phenol / chloroform method, sodium iodide method, etc.), methods for adsorbing nucleic acids to silica membranes or magnetic glass particles (e.g., spin column method, etc.), and combinations thereof. Enterobacteria-derived genomic DNA can also be isolated from a subject-derived sample using commercially available kits (e.g., Nucleospin Microbial DNA Kit [MACHEREY-NAGEL], Stool DNA Isolation Kit [Norgen Biotek], NucleoSpin DNA Stool [Takara Bio]).

[0024] In the above steps (b-1) and (b-2), examples of methods for calculating the abundance ratio of the enteric bacterium of interest relative to all enteric bacteria based on nucleotide sequence information in genomic DNA derived from enteric bacteria include performing PCR using a primer set (consisting of a forward primer and a reverse primer) designed to amplify regions in genomic DNA specific to various enteric bacteria, subjecting the resulting PCR products to sequence analysis (e.g., multiplex analysis, single-read analysis, paired-end analysis, etc.) using a sequencer such as a next-generation sequencer, and measuring the proportion of the genomic DNA of the enteric bacterium of interest relative to genomic DNA derived from all enteric bacteria based on the obtained nucleotide sequence information.

[0025] The method for identifying the enterobacteria of interest from the nucleotide sequence information after sequence analysis is not particularly limited, and any analytical method known in the art that utilizes analytical software, databases, etc. can be used. When using a database, for example, the enterobacteria of interest can be identified by performing a sequence identity search on the accumulated nucleotide sequence of the enterobacteria of interest. Specific examples of databases include GenBank, ENA, and DDBJ. Alternatively, QIIME (Quantitative Insights in Microbial Ecology), open-source software for analyzing the enterobacteria of interest, can be used.

[0026] Examples of the "region in genomic DNA specific to various enterobacteria" include a gene encoding 5S rRNA (5S rRNA gene [also referred to as 5S rDNA]), a gene encoding 16S rRNA (16S rRNA gene [also referred to as 16S rDNA]), a gene encoding 23S rRNA (23S rRNA gene [also referred to as 23S rDNA]), and the like. Since its effectiveness has been demonstrated in the present example described below, the 16S rRNA gene is a preferred example. That is, examples of the "nucleotide sequence information in genomic DNA derived from enterobacteria" include nucleotide sequence information of the 5S rRNA gene, nucleotide sequence information of the 16S rRNA gene, nucleotide sequence information of the 23S rRNA gene, and the like. Since its effectiveness has been demonstrated in the present example described below, the nucleotide sequence information of the 16S rRNA gene is a preferred example.

[0027] The "16S rRNA gene" may be the entire 16S rRNA gene or a portion thereof (for example, a region whose nucleotide sequence differs for each type of bacterium [specifically, one or more regions selected from the V1 region, V2 region, V3 region, V4 region, V5 region, V6 region, V7 region, V8 region, and V9 region]). However, since the effectiveness of this has been demonstrated in the present example described below, the V1 region and V2 region of the 16S rRNA gene are preferred examples. Here, when amplifying the V1 region and V2 region by PCR, primer sets may be arranged to sandwich each of the V1 region and V2 region. However, since both regions are linked, it is more efficient and preferable to arrange primer sets to sandwich the V1 region on the upstream side and the V2 region on the downstream side, since only one primer set is required and only one PCR is required.

[0028] As used herein, "upstream" refers to the start codon (ATG) side of the 16S rRNA gene, and "downstream" refers to the stop codon (TGA, TAG, or TAA) side of the 16S rRNA gene.

[0029] In the step (b-1), if the abundance ratio of the bacteria in iv) above is lower than the abundance ratio in patients with stage I to II renal cancer, this may be an indicator that the subject is likely to have stage III to IV renal cancer.In the step (b-2), if the abundance ratio of Enterocloster bacteria is lower than the abundance ratio in patients with stage I to II renal cancer, this may be an indicator that the subject is likely to have stage III to IV renal cancer.

[0030] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0031] 1. Methods 1-1 Renal cancer group and control group Thirty-eight patients with renal cancer who were admitted to the Department of Urology at Tohoku University Hospital between December 2020 and February 2021 for surgery and systemic treatment as their initial treatment were included in the renal cancer group (Table 1). There was no recent history of antibiotic administration. In addition, among patients receiving outpatient treatment at the Department of Nephrology and Hypertension at Tohoku University Hospital, those without diabetes as a complication and with an eGFR of 30 mL / min / 1.73 m were included. ２ These 51 patients served as the control group (Table 1). All 38 renal cancer patients were pathologically diagnosed as renal cancer, with 35 cases of ccRCC and 3 cases of papillary renal cell carcinoma (pRCC) (Table 1). Renal cancer patients diagnosed as stage III or higher according to the TNM classification were classified as the advanced group, and renal cancer patients diagnosed as stage I-II were classified as the localized group. All patients in the control and renal cancer groups were Japanese.

[0032]

[0033] 1-2 Isolation of enterobacterial genomic DNA. Feces were collected from the renal cancer group and the control group before surgery or treatment, and enterobacterial genomic DNA was isolated using a Nucleospin microbial DNA kit (Macherey-Nagel). Specifically, 100 μL of PBS containing 0.2-0.5 g of feces was mixed with 500 μL of the elution buffer provided with the kit, and the mixture was added to a Nucleospin beads tube (Macherey-Nagel) containing ceramic beads along with protease K. The bacteria were disrupted by the beads. Subsequent procedures were carried out according to the kit's instructions, and enterobacterial genomic DNA was isolated.

[0034] 1-3 Sequence analysis of 16S rRNA gene in genomic DNA derived from enterobacteria To determine the sequence of the 16S rRNA gene, genomic DNA derived from the isolated enterobacteria was analyzed using a next-generation sequencer (MiSeq, Illumina). Specifically, using genomic DNA (1 ng) derived from enterobacteria as a template, PCR was performed using the first PCR primer set (1st_PCR_27Fmod_MIX and 1st_PCR_338R_MIX [both Illumina]) and EX Taq HS (TakaRa) according to the protocol provided with the product. The V1-V2 region of the 16S rRNA gene was amplified and an adapter sequence was added. Next, using the resulting PCR product (approximately 430 bases long) as a template, PCR was performed using a second PCR primer set (2nd forward primer and 2nd reverse primer [both manufactured by Illumina]) and EX Taq HS (manufactured by TakaRa) according to the protocol provided with the product, and the amplification product of the V1-V2 region of the 16S rRNA gene, along with a sequencing adapter sequence and an index for sample identification, was obtained. Subsequently, sequencing analysis was performed using a paired-end method using a next-generation sequencer (MiSeq, manufactured by Illumina).

[0035] 1-4 Identification of the bacterial genus and species from which the 16S rRNA gene originates Based on the obtained sequence analysis data, operational taxonomic units (OTUs) were created, and the homology with representative sequences was evaluated using QIIME (Quantiative Insights in Microbial Ecology), an open-source software for gut microbiome analysis, to obtain bacterial phylogenetic data. To classify gut bacteria, expression frequencies were analyzed for each phylum, class, order, family, genus, and species. Tests were performed for differences in relative abundance of OTUs of 1% or greater within each group. Results with relative abundance less than 1% were excluded. For analyses at the genus and species levels, LDA effect sizes were calculated using LEfSe (Linear discriminant analysis effect size), and biomarkers (gut bacteria) with significant differences were searched for. Comparisons between two groups (control and renal cancer) were performed using the Wilcoxon / Kruskal-Wallis test, and comparisons between three groups (control, localized, and advanced) were performed using the Turkey-Kramer test. Enterobacteriaceae with 16S rRNA genes (V1-V2 regions) that shared 97% or more sequence identity with the nucleotide sequence of the 16S rRNA gene (V1-V2 regions) of a known bacterial species were classified as belonging to the same enterobacteriaceae. Furthermore, ALDEx2 (ANOVA-Like Differential Expression analysis version 2) analysis was performed to identify bacterial species that were increased in the renal cancer group.

[0036] 2. Results: Genera-level analysis identified five genera of intestinal bacteria (Ruminococcus_g4, Ruthenibacterium, Clostridium_g24, Anaerotignum, and Eggerthella) whose abundance ratio relative to total intestinal bacteria in the renal cancer group was significantly increased compared to the control group (Figure 1). Among these, Ruminococcus_g4 bacteria belong to the Mediterraneibacter genus, including bacteria (LT635549_s) with a 16S rRNA gene having the nucleotide sequence of SEQ ID NO: 5. Furthermore, the Ruthenibacterium genus includes Ruthenibacterium lactatiformans with a 16S rRNA gene having the nucleotide sequence of SEQ ID NO: 6 and PAC001646_s with a 16S rRNA gene having the nucleotide sequence of SEQ ID NO: 7. Furthermore, the Clostridium_g24 bacteria belong to the genus Enterocloster, which includes FCEY_s (described later) and Clostridium bolteae (described later). The Anaerotignum bacteria belong to the genus Anaerotignum, which includes Anaerotignum lactatifermentans and the like having a 16S rRNA gene having the nucleotide sequence of SEQ ID NO: 8. The Eggerthella bacteria belong to the genus Eggerthella, which includes Eggerthella lenta and the like having a 16S rRNA gene having the nucleotide sequence of SEQ ID NO: 9.

[0037] Comparison of the four genera of bacteria among the three groups (control, localized, and advanced) showed that the abundance of Ruminococcus_g4 bacteria was significantly higher in the advanced group than in the control or localized group (Figure 2A). The abundance of Ruthenibacterium bacteria was significantly higher in the advanced group than in the control group (Figure 2B). The abundance of Clostridium_g24 bacteria was significantly higher in the localized group than in the control group, but significantly lower in the advanced group than in the localized group (Figure 2C). The abundance of Anaerotignum bacteria was significantly higher in the localized group than in the control group (Figure 2D). The abundance of Eggerthella bacteria was significantly higher in the localized and advanced groups than in the control group (Figure 2E).

[0038] Furthermore, in species-level analysis, four types of bacteria (Clostridium bolteae, PAC001050_s, FCEY_s, and Anaerotignum lactafifermentas) were identified as intestinal bacteria whose abundance ratio relative to total intestinal bacteria in the renal cancer group was found to be significantly increased compared to the control group (Figure 3). Of these, Clostridium bolteae is a bacterium having a 16S rRNA gene with the nucleotide sequence of SEQ ID NO: 1. PAC001050_s is a Eubacterium coprostanoligenes having a 16S rRNA gene with the nucleotide sequence of SEQ ID NO: 2. FCEY_s is a bacterium of the genus Enterocloster having a 16S rRNA gene with the nucleotide sequence of SEQ ID NO: 3, and the nucleotide sequence of its genomic DNA has been registered (NCBI RefSeq assembly; GCF_001517625.2). The FCEY_s strain was previously classified as Clostridium bouchedurhonense, but has recently been classified as a bacterium belonging to the genus Enterocloster, the same species as Enterocloster alcoholdehydrogenati, according to the Genome Taxonomy Database (GTDB) classification (URL: https: / / gtdb.ecogenomic.org / genome?gid=GCA_001517625.2). Therefore, the FCEY_s strain may belong to the genus Enterocloster, which includes Enterocloster alcoholdehydrogenati, or it may belong to a new genus of bacteria distinct from existing Enterocloster strains. Furthermore, the Anaerotignum lactafifermentas strain is a bacterium possessing a 16S rRNA gene with the nucleotide sequence of SEQ ID NO: 4.

[0039] Comparison of the four bacteria among the three groups (control, localized, and advanced) showed that the abundance of Clostridium bolteae was significantly higher in the localized and advanced groups compared to the control group (Figure 4A). The abundance of PAC001050_s was significantly higher in the advanced group compared to the control group (Figure 4B). The abundance of FCEY_s was significantly higher in the localized and advanced groups compared to the control group (Figure 4C). The abundance of Anaerotignum lactafifermentas was significantly higher in the localized group compared to the control group, but significantly lower in the advanced group compared to the localized group (Figure 4D).

[0040] The present invention contributes to the early detection of kidney cancer, the early treatment of kidney cancer, and the prevention of kidney cancer progression.

Claims

1. A method for determining renal cancer, comprising the following steps (a) and (b-1): (a) isolating genomic DNA derived from enterobacteria from a sample containing enterobacteria collected from a subject; (b-1) calculating, based on nucleotide sequence information in the genomic DNA derived from enterobacteria, the abundance ratio of one or more bacteria selected from the group consisting of i) bacteria of the genus Clostridium having a 16S rRNA gene having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1, ii) bacteria of the genus Eubacterium having a 16S rRNA gene having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 2, iii) bacteria of the genus Enterocloster having a 16S rRNA gene having at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NO: 3, and iv) bacteria of the genus Anaerotignum having a 16S rRNA gene having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 4, relative to the total enterobacteria, the step (b-1), where if the abundance ratio of the bacteria in i) is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be suffering from stage I-II or stage III-IV renal cancer; if the abundance ratio of the bacteria in ii) is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be suffering from stage III-IV renal cancer; if the abundance ratio of the bacteria in iii) is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be suffering from stage I-II or stage III-IV renal cancer; and if the abundance ratio of the bacteria in iv) is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be suffering from stage I-II renal cancer; 2. The method for diagnosing kidney cancer according to claim 1, wherein the bacterium i) is Clostridium bolteae having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:1, the bacterium ii) is Eubacterium coprostanoligenes having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:2, the bacterium iii) is an Enterocloster bacterium having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:3, and the bacterium iv) is Anaerotignum lactafifermentas having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:

4.

3. A method for determining renal cancer, comprising the following steps (a) and (b-2). (a) isolating genomic DNA derived from enterobacteria from a sample containing enterobacteria collected from a subject; (b-2) calculating the abundance ratio of one or more bacteria selected from the group consisting of Mediterraneibacter, Ruthenibacterium, Enterocloster, Anaerotignum, and Eggerthella bacteria to all enterobacteria based on nucleotide sequence information in the genomic DNA derived from enterobacteria, wherein if the abundance ratio of Mediterraneibacter bacteria is increased compared to the abundance ratio in non-cancer controls or stage I-II renal cancer patients, it indicates that the subject is highly likely to be affected with stage III-IV renal cancer, and if the abundance ratio of Ruthenibacterium bacteria is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be affected with stage III-IV renal cancer. the step (b-2), in which, if the abundance ratio of bacteria of the genus Enterocloster is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be suffering from stage I to II renal cancer, the abundance ratio of bacteria of the genus Anaerotignum is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be suffering from stage I to II renal cancer, and the abundance ratio of bacteria of the genus Eggerthella is increased compared to the abundance ratio in non-cancer controls, it indicates that the subject is highly likely to be suffering from stage I to II or stage III to IV renal cancer; 4. The genus Mediterraneibacter includes bacteria of the genus Mediterraneibacter having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:5, the genus Ruthenibacterium includes Ruthenibacterium lactatiformans having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:6, and Ruthenibacterium having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:7, the genus Enterocloster includes bacteria of the genus Enterocloster having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:3, and Clostridium bolteae having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:1, and the genus Anaerotignum includes Anaerotignum having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:

8. The method of claim 3, wherein the bacteria of the genus Eggerthella include Eggerthella lenta having a 16S rRNA gene having at least 97% sequence identity to the nucleotide sequence shown in SEQ ID NO:

9.

5. A method for determining renal cancer described in claim 1 or 3, wherein the sample is feces.

6. A method for diagnosing renal cancer as described in claim 1 or 3, wherein the nucleotide sequence information in genomic DNA derived from enterobacteria is nucleotide sequence information of the 16S rRNA gene.

7. A method for determining renal cancer as described in claim 1 or 3, wherein the subject is Japanese.

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