Information provision method for screening for colorectal cancer, and composition for screening for colorectal cancer

Abstract

The present invention relates to an information provision method for screening for colorectal cancer, and a composition for screening for colorectal cancer. The information provision method for screening for colorectal cancer, according to an embodiment of the present invention, comprises the steps of: extracting DNA from a feces sample of a subject to be evaluated; performing PCR by using primers specific to the extracted DNA; quantifying DNA through the PCR analysis so as to identify the abundance of intestinal microbiota; and determining, according to the identification result, the progression of colorectal cancer of the subject to be evaluated.

Description

Method for providing information for colon cancer screening and composition for colon cancer screening

[0001] The present invention relates to a method for providing information for colon cancer screening and a composition for colon cancer screening, and more particularly, to a method and composition for noninvasively screening, early diagnosis, or diagnosing colon cancer using a biomarker.

[0002] Recent studies have shown that imbalances and changes in gut microbiota are associated with the development of colon cancer. Therefore, analysis of gut microbiota can be used to assess the condition of colon cancer patients and identify early stages and risk factors for colon cancer.

[0003] The problems that the present invention seeks to solve are as follows.

[0004] First, it provides a biomarker that can predict and determine the progression of colon cancer.

[0005] Second, it provides a non-invasive method and composition capable of screening for colon cancer.

[0006] Third, the present invention provides a screening method and composition capable of screening for colon cancer through a self-stool examination test. The tasks of the present invention are not limited to those mentioned above, and other tasks not mentioned will be clearly understood by those skilled in the art from the description below.

[0007] In order to achieve the above task, the information providing method for colon cancer screening according to various embodiments of the present invention comprises the steps of: extracting DNA from a biological sample of a subject for evaluation; performing PCR using specific primers on the extracted DNA; quantifying the DNA through the PCR analysis to determine the content of intestinal microorganisms; and determining the degree of colon cancer progression of the subject for evaluation based on the determination result.

[0008] According to the present invention, one or more of the following effects are achieved.

[0009] First, it can provide a biomarker that can predict and determine the degree of colon cancer progression.

[0010] Second, a non-invasive method and composition capable of screening for colon cancer can be provided.

[0011] Third, a screening method and composition capable of screening for colon cancer through a self-stool examination test can be provided.

[0012] The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description of the claims.

[0013] Figure 1a shows the results of analyzing the beta diversity of the fecal microbiome by stage (stage 1, stage 2, and stage 3) of female colorectal cancer patients.

[0014] Figure 1b shows the results of analyzing the beta diversity of the fecal microbiome by stage (stage 1, stage 2, and stage 3) of male colon cancer patients.

[0015] Figure 2a shows the LEfSE results analyzing the microbial communities present in feces collected from female patients with colorectal cancer by stage.

[0016] Figure 2b shows the LEfSE results analyzing the microbial communities present in feces collected from male patients with colorectal cancer by stage.

[0017] Figure 3a shows the LEfSE results analyzing the microbial community present in the feces of the T group.

[0018] Figure 3b shows the LEfSE results analyzing the microbial community present in the feces of the TN group.

[0019] Figures 4a and 4b are tables analyzing the correlation between microorganisms present in feces collected from female patients in group T and clinical indicators.

[0020] Figures 4c and 4d are tables analyzing the correlation between microorganisms present in feces collected from male patients in group T and clinical indicators.

[0021] Figures 5a and 5b are tables analyzing the correlation between microorganisms present in feces collected from female patients in the TN group and clinical indicators.

[0022] Figures 5c and 5d are tables analyzing the correlation between microorganisms present in feces collected from male patients in the TN group and clinical indicators.

[0023] Figure 6a shows the results of amplifying the Rhodecocus genus, one of the biomarkers according to the present invention, using qPCR for samples from the normal group, T group, and TN group.

[0024] Figure 6b shows the results of amplifying Delftia genus, one of the biomarkers according to the present invention, using qPCR for samples from the normal group, T group, and TN group.

[0025] Figure 6c shows the results of amplifying the Stenotrophomonas genus, one of the biomarkers according to the present invention, using qPCR for samples from the normal group, T group, and TN group.

[0026] Figure 6d shows the results of amplifying the Devosia genus, one of the biomarkers according to the present invention, using qPCR for samples from the normal group, T group, and TN group.

[0027] Figure 6e shows the results of amplifying the genus Psychobacteria, one of the biomarkers according to the present invention, using qPCR for samples from the normal group, T group, and TN group.

[0028] Figure 7 is a table showing a pattern in which microbial genera present in the T group are not detected in the TN group.

[0029] Figure 8 is a table showing the accuracy of the prediction performance results for the T group and the TN group using the biomarker according to the present invention.

[0030] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements without excluding any other step or element or group of steps or elements. "Consisting of" means including and is limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the stated element is required or mandatory, and that no other elements may be present. "Consisting essentially of" means including any element listed after that phrase, and is limited to other elements that do not interfere with or affect the activity or action described in the present disclosure with respect to the listed element. Thus, the phrase "consisting essentially of" indicates that the listed element is required or mandatory, but that no other elements are optional, and that their presence or absence may depend on whether or not they affect the activity or action of the listed element.

[0031] The term "microorganism" as used herein refers to a microorganism, such as a bacterium, fungus, virus, protozoa, algae, amoeba, slime mold, or a combination thereof. For any bacterium included in the present disclosure, the present disclosure includes any strain thereof. In the present disclosure, when a genus is specifically mentioned, any species within the genus may also be included by reference, for example, in a list. For example, when the genus Rhodococcus is mentioned, any species within the genus Rhodococcus is included in the present invention.

[0032] The term "microbiome" as used herein generally refers to a collection of microorganisms (which may also be referred to as microbes) within a community in a host, including a particular location and / or tissue and / or organ of the host, such as the intestines, for example, bacteria, fungi, viruses, protozoa, algae, amoebas, and / or slime molds. A "probiotic" refers to one or more microorganisms introduced into the body for their beneficial properties.

[0033] The term "sample" as used herein generally refers to a biological sample comprising any region within the body, for example, the intestines.

[0034] The terms "subject," "test subject," and "subject" as used herein generally refer to an individual having a biological sample that undergoes processing or analysis and, in some embodiments, has a gut microbiome associated therewith.

[0035] The present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms, and the present embodiments are provided only to ensure that the disclosure of the present invention is complete and to fully inform a person having ordinary skill in the art to which the present invention pertains of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0036] Hereinafter, specific embodiments of the present invention will be described.

[0037] 1. Sample preparation

[0038] To study the association between colorectal cancer and the gut microbiome, the inventors analyzed fecal metagenomes from a group of patients who underwent curative colectomy at a single institution between January 2020 and December 2022. To minimize antibiotic bias, fecal samples were collected from all patients diagnosed with colorectal cancer before preoperative antibiotic administration.

[0039] Study participation criteria included adult patients aged 18 years or older, diagnosed with primary resectable colorectal cancer, and undergoing elective surgery.

[0040] Patients excluded from the study included those who underwent surgery for tumor perforation or obstruction, those with microsatellite instability, those with other concurrent malignancies, those with inflammatory bowel disease, and those who received preoperative chemotherapy.

[0041] Cancer types were further classified according to existing staging systems (AJCC and TNM) after disease stage determination.

[0042] Data from the healthy participant group were obtained from the normal Korean gut microbiota dataset (accession number: PRJEB33905) produced by the Korea Food Research Institute (KFRI).

[0043] 2. Extraction of microbial genomic DNA (deoxyribonucleic acid) from colorectal cancer patients and 16S sequence analysis

[0044] Fecal samples were collected from each group of colorectal cancer patients, flash-frozen in liquid nitrogen, and stored at -20°C. Total microbial metagenome DNA was extracted from all samples using the QIAamp DNA microbiome kit (Qiagen, Hilden, Germany), and experiments were performed according to the DNA extraction kit protocol. The quality of the extracted genomic DNA was confirmed using a bioanalyzer (Agilent 2100, Agilent Technologies, Inc., Santa Clara, CA, USA) and stored at -20°C until analysis. The Illumina platform targeted a region containing the V3-V4 high-variability region of the bacterial 16S rRNA gene. PCR amplification of the target region was initiated immediately after mitochondrial DNA (mtDNA) extraction.

[0045] The 16S V3-V4 amplicon was amplified using KAPA HiFi Hot Start Ready Mix (2×) (Roche, Penzberg, Germany). A pair of V3-V4 target-specific universal primers recommended by Illumina was used.

[0046] The primer sequences are as follows.

[0047] Sequence number 16S 341F primer 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3' 16S 806R primer 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3'

[0048] 3. After PCR amplification, all PCR products were purified using AMPure XP beads (Beckman Coulter, California, USA). Additional PCR amplification was then performed using the Nextera XT Index Kit (Illumina, San Diego, CA, USA) to add multiplexing indexes and Illumina sequencing adapters. The final PCR products were purified once again using AMPure XP beads. After construction of the amplicon library, 16S metagenomic sequencing was performed in a paired-end 2 × 300 bp format using the Illumina MiSeq protocol (Illumina MiSeq, San Diego, CA, USA).

[0049] 4. Bioinformatics analysis

[0050] Raw sequencing data generated from a MiSeq sequencer (Illumina) were processed using plugins provided in the Quantitative Insights into Microbial Ecology version 2 (QIIME 2) pipeline. Quality-controlled amplicon sequences were proofread and PhiX-filtered, and clones were confirmed using the Divisive Amplicon Denoising Algorithm 2 plugin (DADA2) in QIIME 2. This allowed the identification of bacterial amplicon sequence variants (ASVs).

[0051] ASVs were aligned using the align-to-tree-mafft-fast tree plugin, and a phylogenetic tree was generated accordingly. Various α-diversity metrics (observed features, Chao1 index, Shannon's index, Simpson's index, Pielou's evenness) and β-diversity metrics (Bray-Curtis, unweighted UniFrac) were determined using the diversity plugin in QIIME 2 at a depth diluted to 1,980 reads per sample (normalized). Principal coordinates analysis (PCoA) was performed to investigate the similarity between bacterial communities based on metadata using the Bray-Curtis and unweighted UniFrac methods.

[0052] Bacterial classification was performed using the SILVA 138v 99% 16S ribosomal RNA database, specifically for the V3-V4 high-variability region of the 16S sequence. All classifications were implemented using the feature-classifier classify-sklearn plugin.

[0053] 5. Statistical Analysis

[0054] To determine significant differences between diversity indices, the Kruskal-Wallis (Wilcoxon rank sum) test was performed. To estimate significant differences between β-diversity indices of bacterial communities, permutational multivariate analysis of variance (PERMANOVA) was applied to the Bray-Curtis and unweighted UniFrac distance matrices. This analysis was performed using the 'Adonis' function (999 variant) in the vegan package of the R statistical software.

[0055] 6. Correlation Analysis

[0056] Correlations between clinical parameters and microbiomes were visualized using a correlation matrix generated using the Spearman method. Following normalization of the Kyoto Encyclopedia of Genes and Genome (KEGG) pathways identified through pathway analysis (see the next section), the Pearson correlation method was used to analyze the correlations between clinical parameters and KEGG pathways.

[0057] 7. Metabolic pathway analysis

[0058] Metagenome functional content was inferred based on microbial community profiles obtained from representative sequences and sequence features using the PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2) pipeline.

[0059] Predicted functional genes were classified using KEGG ontology and pathway analysis.

[0060] To evaluate differential functional enrichment based on the predicted KEGG pathways, linear discriminant analysis effect size (LEfSe) analysis was performed.

[0061] The LEfSe method was used to identify taxonomic biomarkers contributing to groups with high stringency (LDA score ≥ 3.0, p-value < 0.1). Significant biomarkers were extracted at the genus and species levels, and features marked as 'unclassified' were filtered out.

[0062] 8. Characteristics of the stool microbiome in cancer patients according to cancer stage.

[0063] As part of clinical factors, we conducted a diversity analysis of the microbial community and LEfSe analysis according to gender and cancer stage in patients without distant metastasis.

[0064] Among the beta diversity indices, unweighted UniFrac analysis revealed significant differences. In female patients, significant differences were observed between stages 1 and 2 (p=0.034) and between stages 2 and 3 (p=0.034) (Figure 1a). In male patients, significant differences were observed between stages 1 and 3 (p=0.007) (Figure 1b). Furthermore, based on LEfSe analysis, we observed that the microbiota identified in stage 2 of the female cancer group was also found in stage 1 of the male cancer group (Figures 2a, 2b). These findings suggest that there are distinct microbial community compositions between different cancer stages in patients without metastasis, depending on their gender. Interestingly, among these microbial communities, Delftia, Stenotrophomonas, and Sphingobacterium were reported as "cryptic specific core microbiota" (CSCM) organisms. These microbiota are known to be dominant bacteria in the proximal crypts and are particularly characterized by strictly aerobic, non-fermentative bacteria.

[0065] 9. Characteristics of microbial communities by gender and cancer location according to lymph node metastasis in colorectal cancer patients at TNM stage.

[0066] To investigate the characteristics of the microbiota according to cancer incidence and stage, we divided the patient population into proximal and distal colorectal cancers and performed LEfSe analysis within each group based on gender and TNM group. In male patients with distal colorectal cancer, CSCMs such as Delftia, Stenotrophomonas, and Acinetobacter were observed with an LDA score of 2.5 or higher in patients with tumor size but no lymph node metastasis. In female patients, only Rhodococcus was consistently observed, but no common microbiota was detected in the lymph node metastasis group. In the T group of proximal colorectal cancer origin, Rhodococcus, Sphingobacterium, Delftia, and Stenotrophomonas were commonly observed in both women and men. This suggests that specific microbiota, including Delftia, Stenotrophomonas, Sphingobacterium, and Rhodococcus, may play a role in the microbiota composition associated with the origin and stage of colorectal cancer (Figures 3a and 3b).

[0067] 10. Correlation Analysis between Clinical Variables and Early Cancer Marker Candidate Microbiomes

[0068] The inventors compared the correlation patterns between clinical parameters and the genus-level microbiota specific to the T group, targeting microorganisms uniquely detected in the T group. In the T group of female and / or male patients, the microbial profile showed a negative correlation with ALT, AST, absolute neutrophil count, creatinine, eosinophils, monocytes, platelets, and leukocytes, whereas it showed a positive correlation with albumin, BUN, hemoglobin, and protein (Figures 4a, 4b, 4c, and 4d). In the TN group, some microorganisms were not detected in either male or female subjects (Figures 5a, 5b, 5c, and 5d).

[0069] 11. Prediction and validation of microbiome markers for early cancer diagnosis.

[0070] For experimental validation, qPCR was performed on five genera (Table 2). A total of 100 samples were compared for each group: normal, non-lymph node metastatic colorectal cancer, and lymph node metastasis. As a result, the inventors confirmed the specific detection of Rhodococcus, Delftia, Stenotrophomonas, Devosia, and Psychrobacter among aerobic bacterial genera in samples from the non-lymph node metastatic colorectal cancer group (Figs. 6a, 6b, 6c, 6d, and 6e).

[0071] Information on primer sets for detecting biomarker microbial genus levels according to various embodiments of the present invention is as follows.

[0072]

[0073] When comparing cancer-specific microorganisms among all microorganisms in the sample set, a pattern exists in which specific microorganisms are rarely detected in the TN group, unlike the T group. Among the 25 specific bacteria associated with colorectal cancer, including Sphingobacterium, Delftia, Stenotrophomonas, Hydrogenophaga, Carnobacterium, Rhodococcus, Pseudochrobacterium, Devosia, Acinetobacter, and Ensifer, most were rarely detected in the group with lymph node metastasis (Fig. 7).

[0074] The microbiome according to various embodiments of the present invention may be aerobic bacteria. The colonic environment, characterized by a lack of oxygen, is known to be ideal for the proliferation of anaerobic fermentation microorganisms. Despite this environment being inhospitable to most aerobic microorganisms, biomarkers according to various embodiments of the present invention were found to be present in the early stages of colon cancer.

[0075] Aerobic bacteria play a significant role in the intestines of early-stage cancers without lymph node metastases. Later, when lymph node metastases developed, the frequency of these microbial groups decreased.

[0076] We used a dataset of 92 colorectal cancer patients, along with 48 publicly available datasets containing clinical information (PRJNA507548), to predict T and TN groups using target microbiota. The inventors used Catboost to predict the presence of lymph node metastases, including publicly available data on these conditional microbiota. The results showed a prediction accuracy of 80% (Figure 8).

[0077] The following examples are included to illustrate preferred embodiments of the present invention. However, those skilled in the art should recognize that, in light of the present disclosure, numerous changes can be made in the specific embodiments disclosed and still obtain similar or comparable results without departing from the spirit and scope of the subject matter of the present disclosure.

[0078] A method for providing information for colon cancer screening according to various embodiments of the present invention comprises the steps of: extracting DNA from a biological sample of a subject; performing PCR using specific primers on the extracted DNA; quantifying the DNA through PCR analysis to determine the content of intestinal microorganisms; and determining the degree of colon cancer progression of the subject based on the determination results. The content of specific microorganisms in the intestinal microbiota is determined through sequence analysis of the PCR product.

[0079] Tests include screening to detect or prevent colon cancer early, early tests to identify the early stages or risk factors of colon cancer, diagnosis of colon cancer stages, diagnostic tests, and early diagnosis of colon cancer.

[0080] Quantifying DNA to determine the content of intestinal microorganisms involves determining the amount of DNA present in a biological sample. The DNA is isolated from extracellular vesicles derived from bacteria in feces. According to various embodiments of the present invention, the biological sample is feces.

[0081] Quantitative techniques may utilize PCR (Polymerase Chain Reaction), qPCR (Quantitative Polymerase Chain Reaction), PCR using nucleic acids, latency flow methods using antigen-antibody reactions, and / or other DNA quantification methods.

[0082] Specifically, the intestinal microorganism to be identified is any one of the groups consisting of the genus Rhodococcus, the genus Sphingobacterium, the genus Delftia, the genus Stenotrophomonas, the genus Ensifer, the genus Psychrobacter, the genus Acinetobacter, the genus Brevundimonas, the genus Pseudochrobactrum, the genus Enterobacter, and the genus Aeromonas.

[0083] Specifically, the step of determining the degree of colon cancer progression is characterized by determining the degree of colon cancer progression of the subject of evaluation as T in the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification system if the level of intestinal microorganisms found in the intestinal microflora is above the standard. Specifically, the step of determining the degree of colon cancer progression of the subject of evaluation based on the confirmation result is to determine whether it corresponds to the T state before lymph node invasion.

[0084] The stage of colon cancer progression can be classified according to the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification system.

[0085] TNM is a system that specifies the size of the cancer (T), whether it has invaded lymph nodes (N), and whether it has metastasized to distant organs (M).

[0086] In detail, T (Tumor Size) - indicates the size and extent of tumor invasion. T0 indicates that no tumor is found, and T1, T2, T3, and T4 are divided into stages based on the size of the tumor and the extent of invasion into surrounding tissues.

[0087] N (Lymph node metastasis) - Indicates whether lymph nodes are invaded. N0 indicates no lymph node infiltration, while N1 and N2 indicate varying numbers of invaded lymph nodes.

[0088] M (Distant organ metastasis) - Indicates whether there is distant organ metastasis. M0 indicates no distant organ metastasis, and M1 indicates the presence of distant organ metastasis.

[0089] In various embodiments of the present invention, the classification step may be expressed as a Stage.

[0090] Stage 1 is when the tumor is mainly confined to the distal layer and has not invaded the surrounding tissues, usually T1 or T2, and has not metastasized to the surrounding lymph nodes. Stage 2 is when the tumor has slightly invaded the nearby tissues but has not metastasized to the lymph nodes, usually T3 or T4a, and has not metastasized to the lymph nodes. Stage 3 is when the tumor has metastasized to the lymph nodes, and there may be several substages depending on the tumor size and degree of invasion. Stage 4 is when the tumor has metastasized to distant organs or tissues, and this stage corresponds to when the tumor has metastasized to other organs or distant organs.

[0091] Specifically, the step of determining the degree of colon cancer progression is characterized by determining that the degree of colon cancer progression of the subject is before lymph node infiltration if the level of intestinal microorganisms found in the intestinal microflora is above the standard.

[0092] In various embodiments of the present invention, the genera Rhodococcus, Sphingobacterium, Delftia, Stenotrophomonas, Ensifer, Psychrobacter, Acinetobacter, Brevundimonas, Pseudochrobactrum, Enterobacter, and Aeromonas are biomarkers that can determine colon cancer and the progression of colon cancer, and when the level of the biomarker is above a predetermined standard, colon cancer can be determined, or the progression of colon cancer can be determined as T or Stage 1 or Stage 2.

[0093] A predetermined standard may be the microbial level of a standard control sample. The standard control sample may be a normal or healthy sample without colon cancer, or a sample with a colon cancer stage of TN, TNM, or Stage 3 or 4.

[0094] According to various embodiments of the present invention, the presence or absence of colon cancer and / or the progression of colon cancer can be determined as T or Stage 1 or Stage 2 based on whether the abundance, expression level or concentration of the biomarker is high, very high, low, very low, in the top N%, not in the top N%, included in the top M microorganisms or not included in the top M microorganisms compared to a standard control group.

[0095] Here, N and M can be set experimentally, and in the embodiment of the present invention, M is 10.

[0096] In various embodiments, when quantifying the level of a biomarker(s) present in a sample, the level may be determined on an absolute or relative basis. When determined on a relative basis, the level may be compared to a control group, which may include, but is not limited to, historical samples from the same patient (e.g., a series of samples over a period of time), level(s) found in a subject or population of subjects without the disease or disorder, threshold values, and acceptable ranges.

[0097] Biological samples include feces or tissue. Tissue samples may be taken from the inside of the colon.

[0098] In various embodiments of the present invention, the genera Rhodococcus, Sphingobacterium, Delftia, Stenotrophomonas, Ensifer, Psychrobacter, Acinetobacter, Brevundimonas, Pseudochrobactrum, Enterobacter, and Aeromonas are biomarkers that can determine colon cancer and the progression of colon cancer, and when the level of the biomarker is above a predetermined standard, colon cancer can be determined, or the progression of colon cancer can be determined as T or Stage 1 or Stage 2.

[0099] A predetermined standard may be the microbial level of a standard control sample. The standard control sample may be a normal or healthy sample without colon cancer, or a sample with a colon cancer stage of TN, TNM, or Stage 3 or 4.

[0100] According to various embodiments of the present invention, the presence or absence of colon cancer and / or the progression of colon cancer can be determined as T or Stage 1 or Stage 2 based on whether the abundance, expression level or concentration of the biomarker is high, very high, low, very low, in the top N%, not in the top N%, included in the top M microorganisms or not included in the top M microorganisms compared to a standard control group.

[0101] N and M can be set experimentally, and in the embodiment of the present invention, M is 10.

[0102] Specifically, the stage for determining the stage of colon cancer progression is characterized by the level of intestinal microorganisms found in the intestinal microflora being above the standard, and in the case of a female patient, it is determined to be Stage 2.

[0103] Specifically, the stage for determining the stage of colon cancer progression is characterized by the level of intestinal microorganisms found in the intestinal microflora being above the standard, and in the case of a male patient, it is determined to be Stage 1.

[0104] A method for providing information for colon cancer screening according to various embodiments of the present invention includes a step of detecting at least one functional biomarker from a group consisting of Rhodococcus genus, Sphingobacterium genus, Delftia genus, Stenotrophomonas genus, Ensifer genus, Psychrobacter genus, Acinetobacter genus, Brevundimonas genus, Pseudochrobactrum genus, Enterobacter genus, and Aeromonas genus in a biological sample of a subject for evaluation.

[0105] Specifically, when detection is made in the detection stage, the stage of determining the progression of colon cancer in the subject of evaluation as T in the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification system or as Stage 1 or Stage 2 is included.

[0106] Specifically, if detection is made in the detection step, after a set period of time has elapsed, a re-detection process is performed, and if at least one functional biomarker from the group consisting of Rhodococcus, Sphingobacterium, Delftia, Stenotrophomonas, Ensifer, Psychrobacter, Acinetobacter, Brevundimonas, Pseudochrobactrum, Enterobacter, and Aeromonas is not detected, the stage of determining the progression of colon cancer of the subject of evaluation as TN or TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification system, or as Stage 3 or Stage 4 is included.

[0107] A method for providing information for colon cancer screening according to various embodiments of the present invention comprises a method for classifying the colon cancer status of a subject of evaluation according to the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification system, comprising: a step of extracting intestinal microorganisms from a biological sample of the subject of evaluation; a step of confirming the content of the extracted intestinal microorganisms; And among the extracted intestinal microorganisms, if at least one of the genera Rhodococcus, Sphingobacterium, Delftia, Stenotrophomonas, Ensifer, Psychrobacter, Acinetobacter, Brevundimonas, Pseudochrobactrum, Enterobacter, and Aeromonas has a ratio or content ranking higher than a predetermined standard, it is classified as stage T, and Rhodococcus, Sphingobacterium, Delftia, Stenotrophomonas, Ensifer, A step of classifying as TN or TNM is included when the ratio or content rank of one or more selected from the group of Psychrobacter, Acinetobacter, Brevundimonas, Pseudochrobactrum, Enterobacter, and Aeromonas is below a predetermined standard.

[0108] The specified period of time may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, 13, 14, 15, 16, 17, 18 or more months after detection of any one or more of the biomarker groups by the various embodiments of the first invention.

[0109] The ratio or content ranking may be the expression level or concentration of the biomarker in the gut microbiota.

[0110] According to various embodiments of the present invention, the presence or absence of colon cancer and / or the progression of colon cancer can be determined as TN or TNM or Stage 3 or Stage 4 based on whether the abundance, expression level or concentration of the biomarker is high, very high, low, very low, in the top N%, not in the top N%, included in the top M microorganisms or not included in the top M microorganisms compared to a standard control group.

[0111] A method for providing information for colon cancer screening according to various embodiments of the present invention comprises: (a) extracting intestinal microorganisms from a biological sample of a subject for evaluation; (b) checking the content of the extracted intestinal microorganisms; (c) checking whether the ratio or content rank of one or more selected from the group consisting of Rhodococcus genus, Sphingobacterium genus, Delftia genus, Stenotrophomonas genus, Ensifer genus, Psychrobacter genus, Acinetobacter genus, Brevundimonas genus, Pseudochrobactrum genus, Enterobacter genus, and Aeromonas genus among the extracted intestinal microorganisms is below a predetermined standard; (d) a step of re-performing (a), (b), and (c) after a predetermined period of time has elapsed; (e) a step of confirming whether the ratio or content ranking confirmed in step (d) is above a predetermined standard; and (f) a step of classifying the colon cancer status of the subject of evaluation as T among the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification systems, if the conditions of steps (c) and (e) are met.

[0112] A composition for screening for colon cancer according to various embodiments of the present invention comprises an agent for detecting at least one functional biomarker from the group consisting of Rhodococcus genus, Sphingobacterium genus, Delftia genus, Stenotrophomonas genus, Ensifer genus, Psychrobacter genus, Acinetobacter genus, Brevundimonas genus, Pseudochrobactrum genus, Enterobacter genus, and Aeromonas genus in a biological sample of a subject for evaluation. A diagnostic kit according to various embodiments of the present invention detects at least one of the group consisting of the above-described biomarkers, thereby providing information on colon cancer screening, early diagnosis of colon cancer, and stage of colon cancer.

[0113] While the present invention has been illustrated and described with respect to preferred embodiments thereof, it should be understood that various changes, substitutions, and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Furthermore, the scope of the present application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions of matter, means, methods, and steps described herein. As those skilled in the art will readily recognize from this disclosure, processes, machines, manufactures, compositions of matter, means, methods, and steps existing herein or later developed that perform substantially the same function or achieve substantially the same results as the corresponding embodiments described herein may be utilized in accordance with the present disclosure. Accordingly, the appended claims are intended to include within their scope any such processes, machines, manufactures, compositions of matter, means, methods, or steps.

Claims

1. A step of extracting DNA from a biological sample of the subject of evaluation; A step of performing PCR using specific primers for the extracted DNA; A step of quantifying DNA through the above PCR analysis to confirm the content of intestinal microorganisms; and An information providing method for colon cancer screening, comprising a step of determining the degree of colon cancer progression of a subject of evaluation based on the above confirmation results.

2. In paragraph 1, A method for providing information for colon cancer screening, wherein the intestinal microorganisms to be confirmed above are any one of the groups consisting of the genus Rhodococcus, the genus Delftia, the genus Stenotrophomonas, the genus Psychrobacter, and the genus Devosia.

3. In paragraph 1, The steps for determining the progression of colon cancer are: An information providing method for colon cancer screening, characterized in that when the level of the intestinal microorganisms found in the intestinal microflora is above the standard, the progression of colon cancer of the subject of evaluation is judged as T among the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification systems.

4. In paragraph 1, The steps for determining the progression of colon cancer are: A method for providing information for colon cancer testing, characterized in that the degree of colon cancer progression in the subject of evaluation is determined to be before lymph node infiltration when the level of the intestinal microorganisms found in the intestinal microflora is above the standard.

5. An information-providing method for colon cancer screening, comprising a step of detecting at least one functional biomarker from the group consisting of Rhodococcus, Delftia, Stenotrophomonas, Psychrobacter, and Devosia in a biological sample of a subject of evaluation.

6. In paragraph 5, An information providing method for colon cancer screening, which further includes a step of judging the progression of colon cancer of the subject of evaluation as T in the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification system or as Stage 1 or Stage 2, if detection is made in the above detection step.

7. In paragraph 5, If detection is made in the above detection step, after a set period of time, if any one or more functional biomarkers from the group consisting of Rhodococcus genus, Sphingobacterium genus, Delftia genus, Stenotrophomonas genus, Ensifer genus, Psychrobacter genus, Acinetobacter genus, Brevundimonas genus, Pseudochrobactrum genus, Enterobacter genus, and Aeromonas genus are not detected through a re-detection process, a colorectal cancer test further including a step of judging the colorectal cancer progression of the subject of evaluation as TN or TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification system, or as Stage 3 or Stage 4 Method of providing information.

8. In the method of classifying the colon cancer status of the subject of evaluation according to the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification system, A step of extracting intestinal microorganisms from a biological sample of a subject of evaluation; A step for confirming the content of the extracted intestinal microorganisms; and Among the above extracted intestinal microorganisms, if at least one of the genera Rhodococcus, Delftia, Stenotrophomonas, Psychrobacter, and Devosia has a ratio or content ranking higher than a predetermined standard, it is classified as stage T. A method for providing information for colon cancer screening, comprising a step of classifying into TN or TNM when one or more ratios or content ranks selected from the group consisting of Rhodococcus, Delftia, Stenotrophomonas, Psychrobacter, and Devosia are below a predetermined standard. 9.(a) A step of extracting intestinal microorganisms from a biological sample of the subject of evaluation; (b) A step of confirming the content of the extracted intestinal microorganisms; (c) a step of confirming whether the ratio or content rank of one or more selected from the group consisting of Rhodococcus, Delftia, Stenotrophomonas, Psychrobacter, and Devosia among the extracted intestinal microorganisms is below a predetermined standard; (d) a step of re-performing (a), (b) and (c) above after a predetermined period of time has elapsed; (e) a step of confirming whether the ratio or content ranking confirmed in the above step (d) is above a predetermined standard; and (f) A method for providing information for a colon cancer examination, including a step of classifying the colon cancer status of the subject of evaluation as T among the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification systems, if the conditions of the above steps (c) and (e) are met.

10. A composition for screening for colon cancer, comprising an agent for detecting at least one functional biomarker from the group consisting of Rhodococcus, Delftia, Stenotrophomonas, Psychrobacter, and Devosia in a biological sample of a subject of evaluation.

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