Method for Providing Information for Colorectal Cancer Screening and Compositions Therefor

Abstract

The present invention relates to a method for providing information for colorectal cancer screening and a composition for colorectal cancer screening. A method for providing information for colorectal cancer screening according to an embodiment of the present invention comprises: a step of extracting DNA from a fecal sample of a subject to evaluation; a step of performing PCR using primers specific to the extracted DNA; a step of quantifying the DNA through the PCR analysis to determine the content of intestinal microorganisms; and a step of determining the degree of progression of colorectal cancer in the subject to evaluation based on the results of the determination.

Description

Technology Field

[0001] The present invention relates to a method for providing information for colorectal cancer screening and a composition for colorectal cancer screening, and more specifically, to a method and composition capable of non-invasively screening, early diagnosing, or diagnosing colorectal cancer by utilizing biomarkers. Background Technology

[0002] Cancer remains one of the leading causes of death to this day. While advancements in medical technology have brought about significant changes in cancer treatment techniques, the five-year survival rate has improved by only about 10% over the past 20 years. This can be attributed to the difficulty of timely diagnosis and treatment due to the characteristics of cancer, such as its rapid growth and metastasis.

[0003] A biomarker refers to an indicator capable of detecting changes induced within an organism due to external influences, and research is actively underway for the purpose of diagnosing various diseases such as cancer, stroke, and dementia, or predicting the efficacy of specific treatments.

[0004] Recent studies indicate that imbalances and changes in gut microbiota are associated with the development of colorectal cancer. Therefore, it is necessary to utilize the results of gut microbiota analysis to assess the condition of colorectal cancer patients and to identify the early stages of development or risk factors. Prior art literature

[0005] (Patent Document 0001) KR 10-2022-0068865 A The problem to be solved

[0006] The problem that the present invention aims to solve is as follows.

[0007] First, it is to provide biomarkers that can predict and assess the progression of colorectal cancer.

[0008] Second, the invention provides a non-invasive method and composition capable of screening for colorectal cancer.

[0009] Third, the invention provides a screening method and a composition capable of screening for colorectal cancer through a self-fecal test. The problems of the present invention are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0010] To achieve the above objective, a method for providing information for colorectal cancer screening according to various embodiments of the present invention comprises: a step of extracting DNA from a biological sample of a subject for evaluation; a step of performing PCR using primers specific to the extracted DNA; a step of quantifying the DNA through the PCR analysis to determine the content of intestinal microorganisms; and a step of determining the degree of progression of colorectal cancer in the subject for evaluation based on the results of the determination.

[0011] The intestinal microorganisms subject to identification above may be any one of the groups consisting of the genera Rhodococcus, Delftia, Stenotrophomonas, Psychrobacter, and Debosia.

[0012] The step of determining the degree of progression of colorectal cancer described above may determine the degree of progression of the subject's colorectal cancer as T in the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification method if the ratio or ranking of the content of the intestinal microorganisms found in the intestinal microbial community is above a predetermined standard, but is not limited thereto.

[0013] The step of determining the degree of progression of colorectal cancer above may determine the degree of progression of the subject to evaluation as pre-lymph node invasion if the ratio or ranking of the content of the intestinal microorganisms found in the intestinal microbial community is above a predetermined standard.

[0014] The above biological sample may be feces or tissue.

[0015] The step of determining the degree of progression of colorectal cancer above can be determined as Stage 2 if the ratio or ranking of the content of the intestinal microorganisms found in the intestinal microbial community is above a predetermined standard and the patient is female.

[0016] The step of determining the degree of progression of colorectal cancer above can be determined as Stage 1 if the ratio or ranking of the content of the intestinal microorganisms found in the intestinal microbial community is above a predetermined standard and the patient is male.

[0017] In addition, a method for providing information for colorectal cancer screening according to various embodiments of the present invention includes the step of detecting one or more functional biomarkers from a group consisting of the genus Rhodococcus, the genus Delftia, the genus Stenotrophomonas, the genus Psychrobacter, and the genus Debosia in a biological sample of a subject to evaluation.

[0018] If detection is performed in the above detection step, the degree of colorectal cancer progression of the subject being evaluated can be determined as T in the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification method, or as Stage 1 or Stage 2.

[0019] If detection is performed in the above detection step and, after a set period has elapsed, a re-detection process is conducted and one or more functional biomarkers among the groups consisting of Rhodococcus, Delftia, Stenotrophomonas, Psychrobacter, and Debosia are not detected, the degree of colorectal cancer progression of the subject being evaluated can be determined as TN or TNM, or as Stage 3 or Stage 4, among the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification methods.

[0020] In addition, a method for providing information for colorectal cancer screening according to various embodiments of the present invention comprises, in a method for classifying the colorectal cancer status of a subject to evaluation according to a TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification method, a step of extracting intestinal microorganisms from a biological sample of a subject to evaluation; and a step of confirming the content of the extracted intestinal microorganisms. The method includes the step of classifying the extracted intestinal microorganisms into a T stage if the ratio or content ranking of at least one of the genera Rhodococcus, Delftia, Stenotrophomonas, Psychrobacter, and Debosia is greater than or equal to a predetermined standard, and classifying into a TN or TNM if the ratio or content ranking of one or more selected from the groups of Rhodococcus, Delftia, Stenotrophomonas, Psychrobacter, and Debosia is less than a predetermined standard.

[0021] In addition, a method for providing information for colorectal cancer screening according to various embodiments of the present invention comprises: (a) a step of extracting intestinal microorganisms from a biological sample of a subject to evaluation; (b) a step of confirming the content of the extracted intestinal microorganisms; (c) a step of confirming whether one or more ratios or content rankings selected from the groups of Rhodococcus, Delftia, Stenotrophomonas, Psychrobacter, and Devosia among the extracted intestinal microorganisms are below a predetermined standard; (d) a step of repeating (a), (b), and (c) after a predetermined period has elapsed; and (e) a step of confirming whether the ratio or content ranking confirmed in step (d) is above a predetermined standard. and (f) if the conditions of steps (c) and (e) above are satisfied, the step of classifying the colorectal cancer status of the subject being evaluated as T among the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification methods is included.

[0022] In addition, a composition for colorectal cancer screening according to various embodiments of the present invention comprises a preparation that detects one or more functional biomarkers from the group consisting of the genus Rhodococcus, the genus Delftia, the genus Stenotrophomonas, the genus Psychrobacter, and the genus Debosia in a biological sample of a subject being evaluated.

[0023] Any limitations discussed with respect to any one embodiment of the present invention may apply to other embodiments of the present invention. Additionally, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to manufacture and use any composition of the present invention.

[0024] The above content summarizes the features and technical advantages of the present invention to better understand the detailed description below. Effects of the invention

[0025] According to the present invention, there is one or more of the following effects.

[0026] First, it can provide biomarkers that can predict and assess the extent of colorectal cancer progression.

[0027] Second, a non-invasive method and composition capable of screening for colorectal cancer can be provided.

[0028] Third, a screening method and a composition capable of screening for colorectal cancer through a self-fecal test can be provided. The effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description in the claims. Brief explanation of the drawing

[0029] Figure 1a shows the results of analyzing the beta diversity of fecal microbiota by stage (stage 1, stage 2, stage 3) of female colorectal cancer patients. Figure 1b shows the results of analyzing the beta diversity of fecal microbiota by stage (stage 1, stage 2, stage 3) of male colorectal cancer patients. Figure 2a shows the LEfSE results of analyzing the microbial communities present in feces collected from female patients according to the stage of colorectal cancer. Figure 2b shows the LEfSE results of analyzing the microbial communities present in feces collected from male patients according to the stage of colorectal cancer. Figure 3a shows the LEfSE results analyzing the microbial community present in the feces of group T. Figure 3b shows the LEfSE results analyzing the microbial community present in the feces of the TN group. Figure 4a is a table analyzing the correlation between microorganisms present in stool samples collected from female patients in Group T and clinical indicators. Figure 4b is a table analyzing the correlation between microorganisms present in stool samples collected from male patients in Group T and clinical indicators. Figure 5a is a table analyzing the correlation between microorganisms present in feces collected from female patients in the TN group and clinical indicators. Figure 5b is a table analyzing the correlation between microorganisms present in stool samples collected from male patients in the TN group and clinical indicators. Figure 6a shows the results of amplifying the genus Rhodes coccus, one of the biomarkers according to the present invention, using the qPCR method on samples from the normal group, T group, and TN group. FIG. 6b is the result of amplifying the genus Delphthia, one of the biomarkers according to the present invention, using the qPCR method on samples from the normal group, T group, and TN group. FIG. 6c is the result of amplifying Stenotrophomonas, one of the biomarkers according to the present invention, using the qPCR method on samples from the normal group, T group, and TN group. FIG. 6d is the result of amplifying the genus Deboscia, one of the biomarkers according to the present invention, using the qPCR method on samples from the normal group, T group, and TN group. FIG. 6e is the result of amplifying the genus *Psychobacter*, one of the biomarkers according to the present invention, using the qPCR method on samples from the normal group, T group, and TN group. Figure 7 is a table showing the pattern in which microbial genera present in the T group are not detected in the TN group. Figure 8 is a table showing the accuracy of the prediction results for the T group and TN group using the biomarker according to the present invention. Specific details for implementing the invention

[0030] Throughout this specification, unless otherwise required by the context, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of the described steps or elements or groups of steps or elements without excluding any other steps or elements or groups 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 described element is necessary or mandatory and that no other element may be present. “Consisting essentially of” means including any element described following the phrase and is limited to other elements that do not interfere with or affect the activity or operation described in this disclosure with respect to the described element. Thus, the phrase “consisting essentially of” indicates that the described element is necessary or mandatory, but no other element is optional and may or may not be present depending on whether or not it affects the activity or operation of the described element.

[0031] Throughout this application, the term “about” is used to indicate that a value includes an inherent deviation of error for the device, a method used to measure the value, or a deviation existing among the test subjects.

[0032] Throughout this specification, references to “one embodiment,” “an embodiment,” “a specific embodiment,” “one related embodiment,” “a specific embodiment,” “additional embodiment,” or “additional embodiment,” or any combination thereof, mean that a specific characteristic, structure, or feature described in connection with an embodiment is included in at least one embodiment of the present invention. Accordingly, the appearance of such phrases in various places throughout this specification does not necessarily refer to all identical embodiments. Additionally, a specific characteristic, structure, or feature may be combined in any suitable manner in one or more embodiments.

[0033] As used herein, the term "gut" refers to at least part of the digestive tract, including at least the stomach, small intestine, and large intestine.

[0034] As used in the present invention, the term “microorganism” refers to microorganisms, e.g., bacteria, fungi, viruses, protozoa, algae, amoebas, slime molds, or combinations thereof. For any bacteria included in the present disclosure, the present disclosure includes any strain thereof. Where a genus is specifically mentioned in the present disclosure, any species within the genus may also be included by reference, e.g., by reference in the listing. For example, where the genus Rhodococcus is mentioned, any species within the genus Rhodococcus is included in the present invention.

[0035] As used in the present invention, the term "microbiome" generally comprises a collection of microorganisms (which may also be referred to as microorganisms) within a community in a host, including a specific location and / or tissue and / or organ of the host, e.g., the intestine, e.g., bacteria, fungi, viruses, protozoa, algae, amoebas, and / or slime molds. "Probiotic" refers to one or more microorganisms introduced into the body for beneficial properties.

[0036] As used in the present invention, the term “sample” generally refers to a biological sample contained from any region of the body, for example, within the intestine. The sample may be collected from tissues or cells or from the intestinal environment. In some examples, the sample may contain or be derived from any part of body tissue, including tissue biopsies, stool, blood, lung tissue, tumors, or combinations thereof. The sample may have been isolated from the source prior to collection. In some examples, the sample is isolated during sample preparation from its primary source (cells, tissues, body fluids, e.g., blood, environmental samples, etc.). The sample may be purified, may not be purified, or may otherwise be enriched from its primary source. In some embodiments, the primary source is homogenized prior to further processing. The sample may be filtered or centrifuged to remove undesirable substances. The sample may also be purified or enriched for specific compositions within it, e.g., specific microorganisms. The sample may contain intact, fragmented, or partially degraded tissues or cells.

[0037] The terms “subject,” “test subject,” and “subject” as used in the present invention generally refer to an individual having a biological sample having undergone processing or analysis and, in some embodiments, having an associated gut microbiome.

[0038] The subject may be a patient, for example, having or suspected of having a disease (which may be referred to as a medical condition), for example, one or more cancers. The subject may be asymptomatic. The term “individual” may be used interchangeably in at least some embodiments. As used herein, “subject” or “individual” may or may not be hospitalized in a medical facility, and may or may not be treated as an outpatient at a medical facility. The individual may receive one or more medical compositions via the Internet. The individual may include a person or non-human animal of any age, and thus may include both adults and adolescents (e.g., children) and infants, and in some cases, an intrauterine individual. The subject may or may not require medical treatment; the individual may voluntarily or involuntarily participate in experiments, whether clinical or supporting basic science research. The individual may be of any gender, race, or age.

[0039] The present invention is not limited to the embodiments disclosed below but can be implemented in various different forms, and the embodiments provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0040] Specific embodiments of the present invention will be described below.

[0041] 1. Sample preparation

[0042] To investigate the association between colorectal cancer and the gut microbiome, the inventors analyzed fecal metagenomes from a group of patients who underwent therapeutic colectomy at a single institution from January 2020 to December 2022. To minimize antibiotic bias, fecal samples were collected from all patients diagnosed with colorectal cancer prior to antibiotic administration before surgery.

[0043] The criteria for participation in the study are adult patients aged 18 years or older, diagnosed with primary resectable colorectal cancer, and having undergone elective surgery.

[0044] Patients excluded from the study were those who underwent surgery due to tumor perforation or obstruction, those with microsatellite instability, those with concomitant malignancies, those with inflammatory bowel disease, and those who received preoperative chemotherapy.

[0045] Cancer types were further classified according to existing staging systems (AJCC and TNM) after determining the disease stage.

[0046] Data for the healthy participant group were obtained from the normal Korean gut microbiome dataset (accession number: PRJEB33905) produced by the Korea Food Research Institute (KFRI).

[0047] 2. Extraction of microbial genome DNA (Deoxyribonucleic Acid) and performance of 16S sequencing of colorectal cancer patients

[0048] Fecal samples were collected from each colorectal cancer patient group, flash-frozen in liquid nitrogen, and stored at -20°C. Whole microbial metagenome DNA was extracted from all samples using the QIAamp DNA microbiome kit (Qiagen, Hilden, Germany), and experiments were conducted according to the DNA extraction kit protocol. The quality of the extracted genomic DNA was verified using a bioanalyzer (Agilent 2100, Agilent Technologies, Inc., Santa Clara, CA, USA), and the samples were stored at -20°C until analysis. The Illumina platform targeted the region containing the V3-V4 high-change region of the bacterial 16S rRNA gene. PCR amplification of the target region was initiated immediately after mitochondrial DNA (mtDNA) extraction.

[0049] The 16S V3-V4 amplifier was amplified using the KAPA HiFi Hot Start Ready Mix (2×) (Roche, Penzberg, Germany). For this purpose, a pair of V3-V4 target-specific universal primers recommended by Illumina was used.

[0050] The primer sequence is as follows.

[0051] Sequence number 16S 341F primer 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGAGACAGCCTACGGGNGGCWGCAG-3' 16S 806R primer 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3'

[0052] 3. After PCR amplification, all PCR products were purified using AMPure XP beads (Beckman Coulter, California, USA). Subsequently, additional PCR amplification was performed using the Nextera XT Index Kit (Illumina, San Diego, CA, USA) to add the multiplexing index and Illumina sequencing adapter. The final PCR products were purified once again using AMPure XP beads. After constructing the amplicon library, 16S metagenome sequencing was performed using the Illumina MiSeq protocol (Illumina MiSeq, San Diego, CA, USA) in a paired-end 2 × 300 bp format.

[0053] 4. Bioinformatics Analysis

[0054] Raw sequencing data generated from a MiSeq sequencer (Illumina) was processed using plugins provided in the Quantitative Insights into Microbial Ecology version 2 (QIIME 2) pipeline. Quality-controlled amplicon sequences underwent correction and PhiX filtering, and duplication was verified using the Divisive Amplicon Denoising Algorithm 2 plugin (DADA2) of QIIME 2. This allowed for the identification of bacterial amplicon sequence variants (ASVs).

[0055] ASVs were aligned using the align-to-tree-mafft-fast tree plugin, and a phylogenetic tree was generated accordingly. Various α-diversity indices (observed features, Chao1 index, Shannon's index, Simpson's index, Pielou's equilibrium) and β-diversity indices (Bray-Curtis, unweighted UniFrac) were determined at a depth diluted to 1,980 reads per sample using the diversity plugin of QIIME 2 (normalized). Principal Coordinate Analysis (PCoA) was performed using the Bray-Curtis and unweighted UniFrac methods to investigate similarities between bacterial communities based on metadata.

[0056] Bacterial classification was performed using the SILVA 138v 99% 16S ribosomal RNA database, specifically for the V3-V4 high-variation regions of the 16S sequence. All classifications were implemented using the feature-classifier classify-sklearn plugin.

[0057] 5. Statistical Analysis

[0058] A Kruskal-Wallis (Wilcoxon rank sum) test was performed to confirm significant differences between diversity indices. Permanent multivariate analysis of variance (PERMANOVA) was applied to Bray-Curtis and unweighted UniFrac distance matrices to estimate significant differences between the β-diversity indices of bacterial communities. This analysis was performed using the 'Adonis' function (variation 999) from the vegan package of the R statistical software.

[0059] 6. Correlation Analysis

[0060] The correlation between clinical parameters and microbial communities was visualized using a correlation matrix constructed using the Spearman method. Following the normalization of the KEGG (Kyoto Encyclopedia of Genes and Genome) pathway identified through pathway analysis (see next section), the correlation between clinical parameters and the KEGG pathway was analyzed using the Pearson correlation method.

[0061] 7. Metabolic Pathway Analysis

[0062] 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.

[0063] Predicted functional genes were classified using the KEGG ontology and path analysis.

[0064] LEfSe (linear discriminant analysis effect size) analysis was performed to evaluate differential functional richness based on the predicted KEGG path.

[0065] The LEfSe method is used to identify taxonomic biomarkers contributing to groups with high rigor (LDA score ≥ 3.0, p-value < 0.1). Important biomarkers were extracted at the genus and species levels, and features marked as 'unclassified' were filtered out.

[0066] 8. Characteristics of stool microbiota in cancer patients according to cancer stage

[0067] As part of clinical factors, microbial community diversity analysis and LEfSe analysis were performed on patients without distant metastasis according to gender and cancer stage.

[0068] Among the beta diversity indices, significant differences were observed in the unweighted UniFrac analysis. In female patients, significant differences were observed between stage 1 and stage 2 (p=0.034) and between stage 2 and stage 3 (p=0.034) (Fig. 1a). In male patients, a significant difference was observed between stage 1 and stage 3 (p=0.007) (Fig. 1b). Additionally, based on LEfSe analysis, it was observed that the microbial communities identified in stage 2 of the female cancer group were also found in stage 1 of the male cancer group (Figs. 2a, 2b). These findings suggest that there is a distinct microbial community composition between various cancer stages depending on the gender of patients without metastasis. Interestingly, among these microbial communities, Delftia, Stenotrophomonas, and Sphingobacterium were reported as 'Secret Specific Core Microbiome' (CSCM) organisms. These microbiota are known to be the dominant bacteria in the proximal crypts and correspond specifically to strictly aerobic non-fermenting bacteria.

[0069] 9. Characteristics of microbial communities by gender and tumor location according to lymph node metastasis at the TNM stage in colorectal cancer patients

[0070] To investigate the characteristics of the microbiome according to the onset and stage of cancer, the patient group was divided into proximal and distal colorectal cancer groups, and LEfSe analysis was performed within each group based on sex and TNM group. In male patients with distal distal colorectal cancer originating from the distal region, CSCMs such as Delftia, Stenotrophomonas, and Acinetobacter showed an LDA score of 2.5 or higher in patients with tumor size only and no lymph node metastasis. In female patients, only Rhodococcus was consistently observed, but no general microbiome was found in the group with lymph node metastasis. In the T group originating from proximal colorectal cancer, Rhodococcus, Sphingobacterium, Delftia, and Stenotrophomonas were commonly observed in both women and men. This suggests that specific microbiomes, including Delftia, Stenotrophomonas, Sphingobacterium, and Rhodococcus, may play a role in the microbial composition associated with the origin and stage of colorectal cancer (Figs. 3a, 3b).

[0071] 10. Correlation Analysis Between Clinical Variables and Early Cancer Marker Candidate Microbiota

[0072] The inventors compared correlation patterns between clinical parameters and genus-level microbial communities specific to the T group, focusing on the microorganisms uniquely detected in the T group. In the T group of female and / or male patients, the microbial profile showed negative correlations with ALT, AST, absolute neutrophil count, creatinine, eosinophils, monocytes, platelets, and leukocytes, while showing positive correlations with albumin, BUN, hemoglobin, and proteins (Figs. 4a, 4b). In the TN group, some microorganisms were not detected in both men and women (Figs. 5a, 5b).

[0073] 11. Prediction and Validation of Microbiome Markers for Early Cancer Diagnosis

[0074] For experimental verification, qPCR was performed on five genera (Table 2). 100 samples were compared from each group: the normal group, the non-lymph node metastatic colorectal cancer group, and the lymph node metastasis group. 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, 6e).

[0075] The primer set information for detecting biomarker microorganisms at the genus level according to various embodiments of the present invention is as follows.

[0076]

[0077] When comparing cancer-specific microorganisms among all microorganisms in the sample set, there is a pattern in which specific microorganisms are rarely detected in the TN group, unlike in the T group. Among 25 types of specific bacteria associated with colorectal cancer, such as Sphingobacterium, Deftia, Stenotrophomonas, Hydrogenophaga, Carnobacterium, Rhodococcus, Pseudochrobacterium, Debosia, Acinetobacter, and Ensifer, most were rarely detected in the group with lymph node metastasis (Fig. 7).

[0078] The microbiome according to various embodiments of the present invention may be aerobic bacteria. The environment within the colon is characterized by a lack of oxygen, so it is known to be suitable for the flourishing of anaerobic fermentation microbial communities. Despite the fact that it is an environment where most aerobic microbial communities are difficult to inhabit, biomarkers according to various embodiments of the present invention were present in the early stages of colorectal cancer.

[0079] Aerobic bacteria play an important role in the intestines of early-stage cancers without lymph node metastasis. Subsequently, when lymph node metastasis occurred, the frequency of this microbial group decreased.

[0080] Predictions for the T and TN groups using target microbiota were performed using a dataset of 92 colorectal cancer patients along with 48 publicly available datasets (PRJNA507548) containing clinical information. The inventors used Catboost to predict the presence of lymph node metastasis, including publicly available data on these conditional microbes. The results showed a prediction accuracy of 80% (Fig. 8).

[0081] The following examples are included to illustrate preferred embodiments of the present invention. However, those skilled in the art will recognize that, in the context of this disclosure, numerous variations may be made to the specific embodiments disclosed and similar or similar results may still be obtained without departing from the spirit and scope of the subject matter of this disclosure.

[0082] A method for providing information for colorectal cancer screening according to various embodiments of the present invention comprises: a step of extracting DNA from a biological sample of a subject to evaluation; a step of performing PCR using primers specific to the extracted DNA; a step of quantifying the DNA through PCR analysis to determine the content of intestinal microorganisms; and a step of determining the degree of progression of colorectal cancer in the subject to evaluation based on the results of the determination. The content of specific microorganisms in the intestinal microbial community is determined through sequence analysis of the PCR product.

[0083] Tests include screening to detect or prevent colorectal cancer early, initial examinations to identify early stages or risk factors of colorectal cancer, diagnosis of cancer progression stages, diagnostic tests, and early diagnosis of colorectal cancer.

[0084] Quantifying DNA to determine the content of intestinal microorganisms is to determine 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.

[0085] Quantification 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.

[0086] Specifically, the intestinal microorganisms to be identified are any one of the groups consisting of the genera Rhodococcus, Sphingobacterium, Delftia, Stenotrophomonas, Ensifer, Psychrobacter, Acinetobacter, Brevandimonas, Pseudochrobactrum, Enterobacter, and Aeromonas.

[0087] Specifically, the step of determining the degree of colorectal cancer progression is characterized by classifying the subject's degree of progression as T in the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification system if the level of intestinal microorganisms detected in the gut microbiome exceeds a certain threshold. Specifically, the step of determining the subject's degree of colorectal cancer progression based on the verification results involves determining whether it corresponds to the T state, which is prior to lymph node invasion.

[0088] The stage of colorectal cancer progression may be classified according to the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification system.

[0089] TNM is a system that identifies tumor size (T), lymph node invasion (N), and distant organ metastasis (M).

[0090] Specifically, T (Tumor Size) indicates the size of the tumor and the degree of invasion. T0 indicates that no tumor is found, while T1, T2, T3, and T4 are classified into stages based on the size of the tumor and the extent of invasion into surrounding tissues.

[0091] N (Lymph node metastasis) - Indicates the presence of lymph node involvement. N0 indicates no lymph node infiltration, while N1 and N2 indicate varying numbers of infiltrated lymph nodes.

[0092] 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.

[0093] In various embodiments of the present invention, the classification step may be represented as a Stage.

[0094] Stage 1 refers to cases where the tumor is primarily confined to the distal layer and has not invaded surrounding tissues; it is generally classified as T1 or T2 and corresponds to cases where there is no metastasis to nearby lymph nodes. Stage 2 refers to cases where the tumor has slightly invaded nearby tissues but has not metastasized to lymph nodes; it is generally classified as T3 or T4a and corresponds to cases where there is no metastasis to lymph nodes. Stage 3 refers to cases where the tumor has metastasized to lymph nodes, and there may be several substages depending on the tumor size or degree of invasion. Stage 4 refers to cases where the tumor has metastasized to distant organs or tissues; this stage applies when the tumor has spread to other organs or to organs located far away.

[0095] Specifically, the step of determining the degree of colorectal cancer progression is characterized by determining the degree of colorectal cancer progression of the subject as pre-lymph node invasion if the level of intestinal microorganisms found in the intestinal microbiome is above a standard.

[0096] In various embodiments of the present invention, the genera Rhodococcus, Sphingobacterium, Delftia, Stenotrophomonas, Ensifer, Psychrobacter, Acinetobacter, Brevandimonas, Pseudochrobactrum, Enterobacter, and Aeromonas are biomarkers capable of determining colorectal cancer and the degree of progression of colorectal cancer, and if the level of the biomarker is above a predetermined standard, it can be determined as colorectal cancer or the degree of progression of colorectal cancer can be determined as T, Stage 1, or Stage 2.

[0097] The predetermined standard may be the microbial level of a standard control. The standard control sample may be a normal or healthy sample that has not developed colorectal cancer, or a TN, TNM, or Stage 3 or Stage 4 sample during the stage of colorectal cancer progression.

[0098] According to various embodiments of the present invention, the presence of colorectal cancer and / or the degree of progression of colorectal cancer can be determined as T, Stage 1, or Stage 2 based on whether the abundance, expression level, or concentration of a biomarker is high, very high, low, very low, corresponds to the top N%, does not correspond to the top N%, is included in the top M microorganisms, or is not included in the top M microorganisms when compared to a standard control group.

[0099] Here, N and M can be set experimentally, and in an embodiment of the present invention, M is 10.

[0100] In various embodiments, when quantifying the levels of biomarker(s) present in a sample, the levels may be determined on an absolute or relative basis. When determined on a relative basis, the levels 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 given period), level(s) found in subjects or a population of subjects free from disease or disorder, threshold values, and acceptable ranges.

[0101] Biological samples are feces or tissue. Tissue may be a tissue sample taken from inside the large intestine.

[0102] In various embodiments of the present invention, the genera Rhodococcus, Sphingobacterium, Delftia, Stenotrophomonas, Ensifer, Psychrobacter, Acinetobacter, Brevandimonas, Pseudochrobactrum, Enterobacter, and Aeromonas are biomarkers capable of determining colorectal cancer and the degree of progression of colorectal cancer, and if the level of the biomarker is above a predetermined standard, it can be determined as colorectal cancer or the degree of progression of colorectal cancer can be determined as T, Stage 1, or Stage 2.

[0103] The predetermined standard may be the microbial level of a standard control. The standard control sample may be a normal or healthy sample that has not developed colorectal cancer, or a TN, TNM, or Stage 3 or Stage 4 sample during the stage of colorectal cancer progression.

[0104] According to various embodiments of the present invention, the presence of colorectal cancer and / or the degree of progression of colorectal cancer can be determined as T, Stage 1, or Stage 2 based on whether the abundance, expression level, or concentration of a biomarker is high, very high, low, very low, corresponds to the top N%, does not correspond to the top N%, is included in the top M microorganisms, or is not included in the top M microorganisms when compared to a standard control group.

[0105] N and M can be set experimentally, and in an embodiment of the present invention, M is 10.

[0106] Specifically, the step of determining the stage of progression of colorectal cancer is characterized by determining that the level of intestinal microorganisms found in the intestinal microbiome is above a standard, and in the case of a female patient, it is determined to be Stage 2.

[0107] Specifically, the step of determining the stage of colorectal cancer progression is characterized by determining Stage 1 if the level of intestinal microorganisms detected in the gut microbiome is above a standard and the patient is male.

[0108] A method for providing information for colorectal cancer screening according to various embodiments of the present invention includes the step of detecting one or more functional biomarkers from a group 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 in a biological sample of a subject to evaluation.

[0109] Specifically, if detection occurs during the detection phase, it includes a step of determining the degree of colorectal cancer progression of the subject being evaluated as T in the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification system, or determining it as Stage 1 or Stage 2.

[0110] Specifically, if detection occurs during the detection stage, and after a set period has elapsed, a re-detection process is performed, and if one or more functional biomarkers among the groups consisting of Rhodococcus, Sphingobacterium, Delftia, Stenotrophomonas, Ensifer, Psychrobacter, Acinetobacter, Brevandimonas, Pseudochrobactrum, Enterobacter, and Aeromonas are not detected, the method includes a step of determining the degree of colorectal cancer progression of the subject being evaluated as TN or TNM, or as Stage 3 or Stage 4, among the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification methods.

[0111] A method for providing information for colorectal cancer screening according to various embodiments of the present invention comprises, in a method for classifying the colorectal cancer status of a subject to evaluation according to a TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification method, a step of extracting intestinal microorganisms from a biological sample of the subject to evaluation; and a step of confirming the content of the extracted intestinal microorganisms. and among the extracted intestinal microorganisms, if the ratio or ranking of content of at least one of the genera Rhodococcus, Sphingobacterium, Delftia, Stenotrophomonas, Ensifer, Psychrobacter, Acinetobacter, Brevandimonas, Pseudochrobactrum, Enterobacter, and Aeromonas exceeds a predetermined standard, it is classified as Stage T, and Rhodococcus, Sphingobacterium, Delftia, Stenotrophomonas, Ensifer, The method includes the step of classifying as TN or TNM if one or more selected from the genera Psychrobacter, Acinetobacter, Brevandimonas, Pseudochrobactrum, Enterobacter, and Aeromonas have a ratio or content ranking below a predetermined standard.

[0112] The defined period 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 one or more of the biomarker groups according to the various embodiments of the present invention are detected.

[0113] The ratio or content ranking may be the expression level or concentration of a biomarker in the gut microbiome.

[0114] According to various embodiments of the present invention, the presence of colorectal cancer and / or the degree of progression of colorectal cancer can be determined as TN, TNM, Stage 3, or Stage 4 based on whether the abundance, expression level, or concentration of a biomarker is high, very high, low, very low, corresponds to the top N%, does not correspond to the top N%, is included in the top M microorganisms, or is not included in the top M microorganisms when compared to a standard control group.

[0115] A method for providing information for colorectal cancer screening according to various embodiments of the present invention comprises: (a) a step of extracting intestinal microorganisms from a biological sample of a subject to evaluation; (b) a step of confirming the content of the extracted intestinal microorganisms; and (c) a step of confirming whether one or more ratios or content rankings selected from the groups of Rhodococcus, Sphingobacterium, Delftia, Stenotrophomonas, Ensifer, Psychrobacter, Acinetobacter, Brevandimonas, Pseudochrobactrum, Enterobacter, and Aeromonas among the extracted intestinal microorganisms are below a predetermined standard. (d) a step of repeating (a), (b) and (c) after a predetermined period has elapsed; (e) a step of checking whether the ratio or content ranking confirmed in step (d) is greater than or equal to a predetermined standard; and (f) a step of classifying the colorectal cancer status of the subject being evaluated as T among the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification methods when the conditions of steps (c) and (e) are satisfied.

[0116] A composition for colorectal cancer screening according to various embodiments of the present invention comprises a preparation that detects one or more functional biomarkers from the group 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 Brevendimonas, the genus Pseudochrobactrum, the genus Enterobacter, and the genus Aeromonas in a biological sample of a subject being evaluated. A diagnostic kit according to various embodiments of the present invention detects one or more of the group consisting of the biomarkers described above to provide colorectal cancer screening, early diagnosis of colorectal cancer, and information regarding the stage of colorectal cancer.

[0117] Although preferred embodiments of the present invention have been illustrated and described above, it should be understood that various variations, substitutions, and modifications may be made without departing from the spirit and scope of the design as defined by the appended claims. Furthermore, the scope of this application is not intended to be limited to specific embodiments of the processes, machines, manufactures, compositions of materials, means, methods, and steps described herein. As will be readily apparent to those skilled in the art from this disclosure, processes, machines, manufactures, compositions of materials, means, methods, and steps existing in this disclosure or to be subsequently 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 this disclosure. Accordingly, the appended claims are intended to be included within the scope of such processes, machines, manufactures, compositions of materials, means, methods, or steps.

Claims

Claim 1 A step of detecting one or more functional biomarkers from a group consisting of the genera Rhodococcus, Delftia, Stenotrophomonas, Psychrobacter, and Debosia in a biological sample of a subject for evaluation; and, if detection occurs in the above detection step, the step of determining the degree of colorectal cancer progression of the subject to evaluation as T-early colorectal cancer (T: Tumor size, N: Lymph node metastasis, M: Metastasis) with the presence of a tumor but no lymph node metastasis, or as Stage 1 according to the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification method is included; and if detection occurs in the above detection step, after a predetermined period has elapsed, a re-detection process is performed to determine one or more of the groups consisting of the genera Rhodococcus, Sphingobacterium, Delftia, Stenotrophomonas, Ensifer, Psychrobacter, Acinetobacter, Brevandimonas, Pseudochrobactrum, Enterobacter, and Aeromonas A method for providing information for a colorectal cancer screening, which further includes the step of determining the degree of progression of colorectal cancer in a subject being evaluated as Stage 3 or Stage 4, among the TNM (T: Tumor size, N: Lymph node metastasis, M: Metastasis) classification system, TN - tumor present and lymph node metastasis occurring - or TNM - tumor present and lymph node metastasis and distant metastasis occurring - if functional biomarkers are not detected. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete

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