WO2018124735A1 - Procédé de diagnostic d'une tumeur au côlon par l'intermédiaire d'une analyse métagénomique bactérienne - Google Patents

Procédé de diagnostic d'une tumeur au côlon par l'intermédiaire d'une analyse métagénomique bactérienne Download PDF

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WO2018124735A1
WO2018124735A1 PCT/KR2017/015557 KR2017015557W WO2018124735A1 WO 2018124735 A1 WO2018124735 A1 WO 2018124735A1 KR 2017015557 W KR2017015557 W KR 2017015557W WO 2018124735 A1 WO2018124735 A1 WO 2018124735A1
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derived
colon
extracellular vesicles
bacteria
bacterial
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PCT/KR2017/015557
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Korean (ko)
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김윤근
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주식회사 엠디헬스케어
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Priority claimed from KR1020170180144A external-priority patent/KR101940426B1/ko
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Priority to CN201780081381.2A priority Critical patent/CN110382720A/zh
Priority to US16/472,986 priority patent/US20190330687A1/en
Priority to EP17887675.1A priority patent/EP3564390B1/fr
Priority to JP2019535378A priority patent/JP6914552B2/ja
Publication of WO2018124735A1 publication Critical patent/WO2018124735A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

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  • the present invention relates to a method for diagnosing colon tumors by analyzing bacterial metagenome, and more specifically, by performing bacterial metagenomic analysis using a sample derived from a subject, by analyzing the increase or decrease in the content of specific bacterial-derived extracellular vesicles. And a method for diagnosing colorectal tumors such as colorectal cancer.
  • Colon cancer or colorectal cancer is a malignant tumor of the cecum, colon and rectum that occurs on the mucous membrane of the innermost surface of the large intestine.
  • colon cancer or colorectal cancer is a malignant tumor of the cecum, colon and rectum that occurs on the mucous membrane of the innermost surface of the large intestine.
  • the frequency of eating habits has been increasing rapidly as westernized aspects of the diet recently, and the mortality rate of colorectal cancer in recent 10 years is about 80%.
  • the rate of increase is increasing.
  • the age of onset occurs most frequently in the sixties, and by site, it occurs slightly more in the rectum than in the colon.
  • Colorectal cancer can occur at any age, but over 90% of people with colorectal cancer are older than 40 years of age, and their incidence doubles every 10 years.
  • colon cancers are known to originate from colon polyp or colon adenoma. Polyps are initially an abnormal growth of the epithelium in the lining of the large intestine, protruding and appearing in 15-20% of adults. It is a common disease. In addition to colorectal polyps, family members of colorectal cancer are at increased risk for colon cancer, even if they have had ulcerative colitis for a long time. In addition, colorectal cancer is a representative cancer known to be associated with food, and is known to cause low intake of fiber, high intake of animal fat, or excessive intake of refined sugar (sugar) due to westernization of diet. .
  • cancer cells can be detected by biopsy through colonoscopy.
  • Most colon cancers are asymptomatic and therefore difficult to diagnose.
  • conventional diagnostic methods are often found when solid cancers such as colorectal cancer are advanced, it is necessary to predict colon cancer and causative factors in advance in order to prevent medical costs and death due to colorectal cancer.
  • Providing a method of prevention is an effective way.
  • Microbiota refers to a microbial community including bacteria, archaea and eukarya that exist in a given settlement.Intestinal microbiota is an important role in human physiology. It is known to have a great effect on human health and disease through interaction with human cells.
  • the symbiotic bacteria secrete nanometer-sized vesicles to exchange information about genes and proteins in other cells.
  • the mucous membrane forms a physical protective film that particles larger than 200 nanometers (nm) in size can't pass through, so that the symbiotic bacteria cannot pass through the mucosa, but bacterial-derived vesicles are usually less than 100 nanometers in size. It freely speaks to the mucous membrane and is absorbed by our body.
  • Metagenomics also called environmental genomics, can be said to be an analysis of metagenomic data obtained from samples taken from the environment (Korean Patent Publication No. 2011-0073049). Recently, it has become possible to list the bacterial composition of the human microflora by a method based on 16s ribosomal RNA (16s rRNA) sequencing. Next generation sequencing of 16s rDNA sequencing gene of 16s ribosomal RNA is performed. , NGS) platform to analyze.
  • the present inventors In order to diagnose colon tumors such as colorectal polyps and colorectal cancer, the present inventors extracted a gene from bacterial vesicles using urine and stool, which are samples derived from a subject, and performed a metagenome analysis on the colon polyps and colon. Bacterial-derived extracellular vesicles that can act as causative factors of colorectal tumors, such as cancer, have been identified, and thus the present invention has been completed.
  • an object of the present invention is to provide an information providing method for diagnosing colon tumors through metagenomic analysis of genes present in bacterial extracellular vesicles.
  • the present invention provides a method for providing information for diagnosing colon tumor, comprising the following steps:
  • the present invention provides a method for diagnosing colon tumor, comprising the following steps:
  • the present invention provides a method for predicting the risk of developing colon tumor, comprising the following steps:
  • colorectal cancer may be diagnosed by comparing the increase and decrease of the bacterial-derived extracellular vesicles in a sample derived from normal and colon cancer patients by sequencing the PCR product in step (c). have.
  • step (c) Deferribacteres, Tenericutes, Actinobacteria, Acidobacteria, Armatimonadetes, Planctomycetes, Fusobacteria, Proteobacteria, and Euryarchaeota extracellular vesicles derived from (phylum) bacteria ,
  • One or more order bacterial derived extracellular vesicles selected from the group consisting of:
  • Peptococcaceae Deferribacteraceae, Turicibacteraceae, Halomonadaceae, Clostridiaceae, Prevotellaceae, Peptostreptococcaceae, Rhodobacteraceae, Nocardioidaceae, Sphingomonadaceae, Bartonellaceae, Cellulomonadaceae, Lactobacillaceae, Rhizobiaceae, Fimbriimonadaceae, Dermacoccaceae, Leptotrichiaceae, Coriobacteriaceae, Xenococcaceae, Aeromonadaceae, Geodermatophilaceae, Bdellovibrionaceae, Moraxellaceae, Pseudomonadaceae, Streptococcaceae, One or more family or small family cells from the group consisting of Veillonellaceae, Bacteroidaceae, Aerococcaceae, Comamonadaceae, Paraprevo
  • rc4-4 Proteus, Catenibacterium, Mucispirillum, Eubacterium, Turicibacter, Alloiococcus, Halomonas, Prevotella, Dialister, Anaerostipes, SMB53, Faecalibacterium, Blautia, Capnocytophaga, Sphingomonas, Lactobacillus, Fimbriicus, Achromobacus, Achromobacsium, Achromobacsium Bdellovibrio, Alkanindiges, Roseateles, Shuttleworthia, Rhizobium, Morganella, Acinetobacter, Pseudomonas, Enterococcus, Lactococcus, Coprococcus, Bacteroides, Dorea, Streptococcus, Lachnospira, Ruminococcus, Corynebacteria, Comamocobacterium, Comamocobacterium, Lacosebacteria, Mycobacterium terpenia The increase or decrease in the content of
  • the colorectal cancer through the step of comparing the increase and decrease of the content of bacterial-derived extracellular vesicles in the colon-derived patient and colorectal cancer-derived sample by sequencing the PCR product Diagnosis can be made.
  • step (c) Spirochaetes phylum bacteria-derived extracellular vesicles
  • One or more class bacterial-derived extracellular vesicles selected from the group consisting of Spirochaetes, and Acidobacteria-6,
  • One or more order bacterial extracellular vesicles selected from the group consisting of Spirochaetales, and Myxococcales,
  • the increase or decrease in the content of one or more genus bacteria-derived extracellular vesicles selected from the group consisting of Treponema, Dialister, Oscillospira, and Eubacterium can be compared.
  • the colon polyps are diagnosed by comparing the increase or decrease of the bacterial-derived extracellular vesicles in the sample from the normal and colon polyps patient by sequencing the PCR product. Can be.
  • step (c) at least one phylum bacteria-derived extracellular vesicles selected from the group consisting of Actinobacteria, Proteobacteria, and Euryarchaeota,
  • One or more class bacterial-derived extracellular vesicles selected from the group consisting of Betaproteobacteria, Solibacteres, Gammaproteobacteria, Clostridia, Methanobacteria, and 4C0d-2,
  • One or more order bacterial-derived extracellular vesicles selected from the group consisting of Burkholderiales, Sphingomonadales, Solibacterales, Stramenopiles, Pseudomonadales, Clostridiales, Oceanospirillales, Desulfovibrionales, and Methanobacteriales,
  • Rhizobiaceae Group consisting of Rhizobiaceae, Sphingomonadaceae, Exiguobacteraceae, Moraxellaceae, Pseudomonadaceae, Streptococcaceae, Peptostreptococcaceae, Comamonadaceae, Veillonellaceae, Bacteroidaceae, Paraprevotellaceae, Ruminococcaceae, Corynebacteriaceae, Christensenellaceae, Odoribacteraceae, Deofolioaceae, Deofolioaceae Extracellular vesicles derived from abnormal families, or
  • Sphingomonas Alkanindiges, Roseateles, Rhizobium, Morganella, Proteus, Exiguobacterium, Acinetobacter, Pseudomonas, SMB53, Lactococcus, Coprococcus, Streptococcus, Bacteroides, Ruminococcus, Corynebacterium, Odoribacter, Clonastriia Triacerocitra, Clonastriia Viracerosia, Clonastriia psi
  • the increase or decrease in the content of one or more genus bacteria-derived extracellular vesicles selected from the group consisting of Dialister, Phascolarctobacterium, Sutterella, Halomonas, Roseomonas, and Methanobrevibacter can be compared.
  • the subject sample may be feces or urine.
  • Extracellular vesicles secreted by the bacteria present in the environment can be absorbed into the body and directly affect inflammation and cancer development, and colon polyps and colorectal cancer are difficult to diagnose effectively because they are difficult to diagnose early.
  • Predicting the risk of colon tumors such as colorectal polyps and colorectal cancer through metagenomic analysis of bacterial or bacterial-derived extracellular vesicles using a human-derived sample according to the present invention, early diagnosis and prediction of risk groups of colon tumors and appropriate management By delaying the onset time or preventing the onset, and after the onset can be diagnosed early can reduce the incidence of colon tumors and increase the treatment effect.
  • metagenome analysis predicts causative factors in patients diagnosed with colorectal polyps or colorectal cancer, thereby avoiding exposure to causative factors and improving colon polyps and colorectal cancer, or preventing recurrence.
  • Figure 1 is for evaluating the distribution of bacteria-derived extracellular vesicles in the body
  • Figure 1a after the administration of oral intestinal bacteria (Bacteria) and bacteria-derived vesicles (EV) in the mouth hourly (0, 5min, 3h, 6h, and 12h) is a photograph taken of their distribution
  • Figure 1b is a 12 hours after oral administration of intestinal bacteria (Bacteria) and bacteria-derived extracellular vesicles (EV) to the urine and various organs (heart, Lung, liver, kidney, spleen, adipose tissue, and muscles), and the photographs of the distribution of the bacterial and extracellular vesicles.
  • Figure 2 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the phylum level by separating bacterial-derived vesicles from colon cancer patients and normal feces.
  • EVs bacteria-derived vesicles
  • FIG. 3 shows the distribution of bacterial-derived vesicles (EVs) with significant diagnostic performance at a class level by separating bacterial-derived vesicles from colon cancer patients and normal stool.
  • EVs bacterial-derived vesicles
  • EVs bacteria-derived vesicles
  • EVs bacteria-derived vesicles
  • FIG. 6 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level by separating bacterial-derived vesicles from colon cancer patients and normal stool.
  • EVs bacteria-derived vesicles
  • FIG. 7 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the phylum level by separating the bacteria-derived vesicles from colon cancer patients and normal urine, and performing a metagenome analysis.
  • EVs bacteria-derived vesicles
  • FIG. 8 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at a class level by separating bacterial-derived vesicles from colorectal cancer patients and normal urine.
  • EVs bacteria-derived vesicles
  • FIG. 9 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the order level by separating the bacteria-derived vesicles in colon cancer patients and normal urine, and performing a metagenome analysis.
  • EVs bacteria-derived vesicles
  • FIG. 10 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the family level after separation of bacteria-derived vesicles from colon cancer patients and normal urine.
  • EVs bacteria-derived vesicles
  • 11 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level after separating the bacteria-derived vesicles in colon cancer patients and normal urine.
  • EVs bacteria-derived vesicles
  • FIG. 7 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the phylum level by separating the bacteria-derived vesicles in the urine of colon cancer patients and colon polyps patients .
  • EVs bacteria-derived vesicles
  • EVs bacteria-derived vesicles
  • FIG. 9 is a result showing the distribution of bacteria-derived vesicles (EVs) of significant diagnostic performance at the order (neck) by separating the bacteria-derived vesicles in the urine of colorectal cancer patients and colon polyps patients, performing a metagenome analysis .
  • EVs bacteria-derived vesicles
  • FIG. 10 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the family level by separating the bacteria-derived vesicles in the urine of colorectal cancer patients and colon polyps patients, and performing a metagenome analysis. .
  • EVs bacteria-derived vesicles
  • 11 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level after separating the bacteria-derived vesicles in the urine of patients with colorectal cancer and colon polyps. .
  • EVs bacteria-derived vesicles
  • EVs bacteria-derived vesicles
  • FIG. 13 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the class level by separating the bacteria-derived vesicles in the stool of colon cancer patients and colon polyps patients, and performing a metagenomic analysis .
  • EVs bacteria-derived vesicles
  • EVs bacteria-derived vesicles
  • EVs bacteria-derived vesicles
  • 16 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level after separation of bacteria-derived vesicles in the bowel cancer patients and colon polyps patients stool. .
  • EVs bacteria-derived vesicles
  • 17 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the order level by separating the bacteria-derived vesicles in the urine of colorectal cancer patients and colon polyps patients after performing a metagenome analysis .
  • EVs bacteria-derived vesicles
  • 19 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the phylum level by separating bacterial-derived vesicles from colon polyps and normal feces.
  • EVs bacteria-derived vesicles
  • EVs bacteria-derived vesicles
  • 21 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the order level by separating bacterial-derived vesicles from colon polyps and normal feces, and performing a metagenome analysis.
  • EVs bacteria-derived vesicles
  • EVs bacteria-derived vesicles
  • EVs bacteria-derived vesicles
  • Figure 24 shows the distribution of bacterial-derived vesicles (EVs) with significant diagnostic performance at the phylum level by separating bacterial-derived vesicles from colon polyps and normal urine.
  • EVs bacterial-derived vesicles
  • FIG. 25 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at a class level after separation of bacterial-derived vesicles from colon polyps and normal urine.
  • EVs bacteria-derived vesicles
  • FIG. 26 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the neck level after separation of bacteria-derived vesicles from colon polyps and normal urine.
  • EVs bacteria-derived vesicles
  • FIG. 27 shows the distribution of bacterial-derived vesicles (EVs) with significant diagnostic performance at the family level after separation of bacterial-derived vesicles from colon polyps and normal urine.
  • EVs bacterial-derived vesicles
  • FIG. 28 shows the distribution of bacterial-derived vesicles (EVs) with significant diagnostic performance at genus level after isolation of bacterial-derived vesicles from colon polyps and normal urine.
  • EVs bacterial-derived vesicles
  • the present invention relates to a method for diagnosing colorectal tumors such as colon polyps and colorectal cancer through bacterial metagenome analysis, and the present inventors extract genes from extracellular vesicles derived from bacteria using a sample derived from a subject, Bacterial-derived extracellular vesicles that could act as causative factors of colorectal tumors such as colon polyps and colorectal cancer were identified.
  • the present invention comprises the steps of (a) extracting DNA from the extracellular vesicles isolated from the subject sample;
  • It provides an information providing method for diagnosing colon tumors comprising comparing the increase and decrease of the content of bacterial-derived extracellular vesicles in a sample derived from normal people and colon polyps through sequencing of the PCR product.
  • diagnosisd colorectal cancer means to determine whether or not colon cancer is likely to develop, whether or not colon cancer is relatively high, or whether colorectal cancer has already occurred in a patient.
  • the method of the present invention can be used to prevent or delay the onset of the disease through special and appropriate management as a patient at high risk of developing colorectal cancer for any particular patient.
  • the methods of the present invention can be used clinically to determine treatment by early diagnosis of colon cancer and selecting the most appropriate treatment regimen.
  • the term "diagnosing colon polyps" means determining whether a colon polyp is likely to develop, whether the colon polyp is relatively high, or whether a polyp has already occurred.
  • the method of the present invention can be used to prevent or delay the onset of the disease through special and appropriate management as a patient at high risk of developing colon polyps for any particular patient.
  • the methods of the present invention can be used clinically to determine treatment by early diagnosis of colon polyps and selecting the most appropriate treatment regimen.
  • metagenome used in the present invention, also referred to as “metagenome”, refers to the total of the genome including all viruses, bacteria, fungi, etc. in an isolated area such as soil, animal intestine, It is mainly used as a concept of genome explaining the identification of many microorganisms at once using sequencer to analyze microorganisms which are not cultured.
  • metagenome does not refer to one species of genome or genome, but refers to a kind of mixed dielectric as the genome of all species of one environmental unit. This is a term from the point of view of defining a species in the course of the evolution of biology in terms of functional species as well as various species that interact with each other to create a complete species.
  • rapid sequencing is used to analyze all DNA and RNA, regardless of species, to identify all species in one environment, and to identify interactions and metabolism.
  • metagenome analysis was preferably performed using bacterial-derived extracellular vesicles isolated from serum.
  • meta-genomic analysis was performed on genes present in the extracellular vesicles in feces and urine of normal people, colon polyps, and colon cancer patients. Analyzes at the order, family, and genus levels, respectively, identified bacterial vesicles that could actually cause colon cancer and colon polyps development.
  • the present invention as a result of analyzing the bacteria-derived vesicles metagenome present in the stool at the gate level, Deferribacteres, Tenericutes, Actinobacteria, Acidobacteria, Armatimonadetes, Planctomycetes, and Fusobacteria door-derived vesicles derived from colon cancer patients There was a significant difference between and normal (see Example 4).
  • the bacterium-derived vesicle metagenome present in the feces at the river level Deferribacteres, Mollicutes, 4C0d-2, Bacilli, Alphaproteobacteria, Saprospirae, Fimbriimonadia, Acidobacteria-6, Solibacteres, Vesicles derived from Coriobacteriia, Oscillatoriophycideae, and Fusobacteriia bacterium showed significant differences between colon cancer patients and normal individuals (see Example 4).
  • the result of analyzing the bacterial-derived vesicle metagenome present in the stool at the neck level RF32, YS2, Deferribacterales, Turicibacterales, RF39, Oceanospirillales, Rhizobiales, Lactobacillales, Rhodobacterales, Saprospirales, Sphingomonadales, Fimbriimonadales, iii1-15, Solibacterales, Coriobacteriales, Chroococcales, Fusobacteriales, and Bdellovibrionales neck bacterial-derived vesicles showed significant differences between colon cancer patients and normal individuals (see Example 4).
  • the bacterium-derived vesicles metagenome present in the feces at an excessive level Peptococcaceae, Deferribacteraceae, Turicibacteraceae, Halomonadaceae, Clostridiaceae, Prevotellaceae, Peptostreptococcaceae, Rhodobacteraceae, Nocardioidaceae, Sphellomoaceae, Barton Cellulomonadaceae, Lactobacillaceae, Rhizobiaceae, Fimbriimonadaceae, Dermacoccaceae, Leptotrichiaceae, Coriobacteriaceae, Xenococcaceae, Aeromonadaceae, Geodermatophilaceae, and Bdellovibrionaceae were significantly different between colon cancer patients and normal colon cancer patients (see Example 4).
  • bacteria-derived vesicle metagenome present in the feces at the genus level, rc4-4, Proteus, Catenibacterium, Mucispirillum, Eubacterium, Turicibacter, Alloiococcus, Halomonas, Prevotella, Dialister, Anaerostipes, SMB53, Faecalibacterium, Blautia, Capnocytophaga, Sphingomonas, Lactobacillus, Fimbriimonas, Dermacoccus, Achromobacter, Novosphingobium, Sneathia, Agrobacterium, Blastomonas, Bdellovibrio, Alkanindiges, Roseateles, and the subcutaneous intestines of the genus There was a difference (see Example 4).
  • the bacterium-derived vesicles metagenome present in the urine was analyzed at the neck level. There was a significant difference between cancer patients and normal subjects (see Example 5).
  • the bacteria-derived vesicles metagenome present in the urine at an excessive level Moraxellaceae, Pseudomonadaceae, Streptococcaceae, Turicibacteraceae, Veillonellaceae, Bacteroidaceae, Aerococcaceae, Comamonadaceae, Clostridiaceae, Paraprevotellaceae, Christensenellaceae, Ruminococcaceae, Corynebacteriaceae, Gordoniaceae, Mycobacteriaceae, Desulfovibrionaceae, Halomonadaceae, Alcaligenaceae, Barnesiellaceae, Methanobacteriaceae, and Rikenellaceae and bacterial-derived vesicles showed significant differences between colon cancer patients and normal individuals (see Example 5).
  • the bacteria-derived vesicle metagenome present in the urine at the genus level Rhizobium, Proteus, Morganella, Acinetobacter, Pseudomonas, SMB53, Enterococcus, Lactococcus, Turicibacter, Coprococcus, Bacteroides, Dorea, Streptococcus, Lachnospira, Ruminococcus, Corynebacterium, Comamonas, Gordonia, Paraprevotella, Mycobacterium, Roseburia, Dialister, Slackia, Escherichia, Phascolarctobacterium, Sutterella, Virgibacillus, Eggerthella, Halomonasoc, Citrobacteriococcus, Miobacteriumobacero Derived vesicles were significantly different between colorectal cancer patients and normal subjects (see Example 5).
  • the vesicles derived from Spirochaetes, and Acidobacteria-6 strong bacteria were significant between colon cancer patients and colon polyps patients. There was a difference (see Example 6).
  • Eubacterium neck bacteria-derived vesicles had a significant difference between colon cancer patients and colon polyps patients (execution See Example 7).
  • bacteria-derived vesicles metagenome present in the feces at the genus level as a result of analyzing the bacteria-derived vesicles metagenome present in the feces at the genus level, Sphingomonas, Alkanindiges, and Roseateles bacteria-derived vesicles significant differences between the colon polyps and normal people (See Example 8).
  • the bacterium-derived vesicle metagenome present in the urine as a result of the analysis, Gammaproteobacteria, Clostridia, Methanobacteria, and 4C0d-2 strong bacteria-derived vesicles between the colon polyps and normal people There was a significant difference (see Example 9).
  • the bacterial-derived vesicle metagenome present in the urine as a result of neck analysis, Stramenopiles, Pseudomonadales, Clostridiales, Oceanospirillales, Desulfovibrionales, and Methanobacteriales vesicles derived from the neck bacteria and normal colon colon patients There was a significant difference between them (see Example 9).
  • the bacterium-derived vesicle metagenome present in the urine at an exaggerated level Exiguobacteraceae, Moraxellaceae, Pseudomonadaceae, Rhizobiaceae, Streptococcaceae, Peptostreptococcaceae, Comamonadaceae, Veillonellaceae, Bacteroidaceae, Paraprevotellaceae, Ruminococcaceae Corynebacteriaceae, Christensenellaceae, Odoribacteraceae, Desulfovibrionaceae, Halomonadaceae, Alcaligenaceae, Barnesiellaceae, Methanobacteriaceae, and Rikenellaceae showed significant differences between colonic and normal patients (see Example 9).
  • the bacteria-derived vesicle metagenome present in the urine at the genus level Rhizobium, Morganella, Proteus, Exiguobacterium, Acinetobacter, Pseudomonas, SMB53, Lactococcus, Coprococcus, Streptococcus, Bacteroides, Differences between the normal vesicles of the genus of the genus of the genus of the strains of the genus (See Example 9).
  • the present invention through the results of the above embodiment, by performing a metagenomic analysis on the bacterial-derived extracellular vesicles isolated from feces and urine bacteria significantly changed in colorectal cancer patients compared to normal and colon polyps patients Derived vesicles were identified, and metagenome analysis confirmed that colon cancer can be diagnosed by analyzing the increase or decrease in the content of bacterial derived vesicles at each level.
  • the present invention is a bacterial-derived vesicle with a significantly changed content in colorectal patients compared to the normal by performing a metagenome analysis on the bacterial-derived extracellular vesicles isolated from feces and urine through the results as described above
  • the metagenomic analysis confirmed that colon polyps could be diagnosed by analyzing the increase and decrease of the content of bacterial-derived vesicles at each level.
  • the fluorescently labeled 50 ⁇ g of bacteria and bacteria-derived vesicles were administered in the same manner as above 12 hours.
  • Blood, Heart, Lung, Liver, Kidney, Spleen, Adipose tissue, and Muscle were extracted from mice.
  • the intestinal bacteria (Bacteria) were not absorbed in each organ, whereas the intestinal bacteria-derived extracellular vesicles (EVs) were urine, heart, lung as shown in FIG. And distribution in liver, kidney, spleen, adipose tissue, and muscle.
  • first stool and urine were put in a 10 ml tube and centrifuged (3,500 xg, 10min, 4 ° C) to settle the suspended solids to recover only the supernatant, followed by a new 10 ml. Transferred to the tube. After removing the bacteria and foreign substances from the recovered supernatant using a 0.22 ⁇ m filter, transfer to centripreigugal filters (50 kD) and centrifuged at 1500 xg, 4 °C for 15 minutes to discard the material smaller than 50 kD and 10 ml Concentrated until.
  • centripreigugal filters 50 kD
  • PCR was performed using the 16S rDNA primer shown in Table 1 to amplify the gene and perform sequencing (Illumina MiSeq sequencer). Output the result as a Standard Flowgram Format (SFF) file, convert the SFF file into a sequence file (.fasta) and a nucleotide quality score file using GS FLX software (v2.9), check the credit rating of the lead, and window (20 bps) The part with the average base call accuracy of less than 99% (Phred score ⁇ 20) was removed.
  • SFF Standard Flowgram Format
  • the Operational Taxonomy Unit performed UCLUST and USEARCH for clustering according to sequence similarity. Specifically, the clustering is based on 94% genus, 90% family, 85% order, 80% class, and 75% sequence similarity. OTU's door, river, neck, family and genus level classifications were performed, and bacteria with greater than 97% sequence similarity were analyzed using BLASTN and GreenGenes' 16S DNA sequence database (108,453 sequences) (QIIME).
  • Example 3 By the method of Example 3, vesicles were isolated from stool of 29 colon cancer patients and 358 normal patients, and then metagenome sequencing was performed. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
  • Bacterial-derived vesicles in feces at the order level were analyzed by RF32, YS2, Deferribacterales, Turicibacterales, RF39, Oceanospirillales, Rhizobiales, Lactobacillales, Rhodobacterales, Saprospirales, Sphingomonadales, Fimbriimonadales, iii1-15, Solibactles, Corio
  • the diagnostic performance for colorectal cancer was significant (see Table 4 and Figure 4).
  • Bacterial-derived vesicles in feces at genus level were analyzed by rc4-4, Proteus, Catenibacterium, Mucispirillum, Eubacterium, Turicibacter, Alloiococcus, Halomonas, Prevotella, Dialister, Anaerostipes, SMB53, Faecalibacterium, Blautia, Capnocytopomonas, When diagnostic models were developed with one or more biomarkers of bacteria of the genus Lactobacillus, Fimbriimonas, Dermacoccus, Achromobacter, Novosphingobium, Sneathia, Agrobacterium, Blastomonas, Bdellovibrio, Alkanindiges, Roseateles, and Shuttleworthia, diagnostic performance for colorectal cancer was significant. (See Table 6 and FIG. 6).
  • Example 3 By the method of Example 3, the vesicles were isolated from the urine of 38 patients with colorectal cancer and 38 normal people and then subjected to metagenome sequencing. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
  • Example 3 By the method of Example 3, vesicles were isolated from the stool of 29 colon cancer patients and 27 colon polyp patients, and then metagenome sequencing was performed. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
  • the diagnostic performance for colon cancer was significant when the diagnostic model was developed for biomarkers of spirochaetes and Acidobacteria-6. See FIG. 13).
  • Example 7 With colon polyps Isolated from Urine Colon Cancer Patients Germ-derived parcel Metagenome Analysis-based Colorectal Cancer Diagnosis Model
  • Example 3 By the method of Example 3, the vesicles were isolated from the urine of 26 patients with colorectal cancer and 38 patients with colorectal polyps, and then metagenome sequencing was performed. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
  • Example 8 Normal people Colon polyp Separated from feces Germ-derived parcel Metagenome Analytics based Colon polyp Diagnostic Model
  • Example 3 By the method of Example 3, vesicles were isolated from stool of 27 colon cancer patients and 358 normal patients, and then metagenome sequencing was performed. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
  • Example 9 The normal person Colon polyp Isolated from urine Germ-derived parcel Metagenome Analytics based Colon polyp Diagnostic Model
  • Example 3 By the method of Example 3, the vesicles were isolated from the urine of 38 patients with colorectal cancer and 38 normal people and then subjected to metagenome sequencing. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
  • the method for diagnosing colon tumors through bacterial metagenomic analysis is performed by performing bacterial metagenomic analysis using a sample derived from a subject to analyze the increase or decrease in the content of specific bacterial-derived extracellular vesicles, such as colon polyps and colon cancer. It can be used to predict and diagnose the risk of developing a tumor. Extracellular vesicles secreted by the bacteria present in the environment can be absorbed into the body and directly affect inflammation and cancer development, and colon polyps and colorectal cancer are difficult to diagnose effectively because they are difficult to diagnose early.
  • Predicting the risk of colon tumors such as colorectal polyps and colorectal cancer through metagenomic analysis of bacterial or bacterial-derived extracellular vesicles using a human-derived sample according to the present invention By delaying the onset time or preventing the onset, and after the onset can be diagnosed early can reduce the incidence of colon tumors and increase the treatment effect.
  • the bacterial metagenomic analysis according to the present invention in patients diagnosed with colorectal polyps or colon cancer improves the progression of colon polyps and colorectal cancer or prevents recurrence by predicting the causative factors and avoiding exposure to the causative factors. It is available.

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Abstract

La présente invention concerne un procédé de prédiction de l'incidence d'une tumeur au côlon, telle qu'un polype du côlon et un cancer du côlon, et leurs facteurs causaux par analyse métagénomique d'une vésicule dérivée d'une bactérie présente dans une substance dérivée du corps humain. Plus particulièrement, la présente invention concerne un procédé de diagnostic d'un facteur causal d'une tumeur au côlon et le risque de son incidence par séquençage d'un métagénome présent dans une vésicule dérivée d'une bactérie présente dans les excréments ou l'urine. Il existe des trilliards de bactéries dans un intestin, et les bactéries sécrètent des vésicules hors de leurs cellules dans un but d'échange d'informations. Les bactéries ne sont pas absorbées dans les cellules épithéliales d'un côlon, mais les vésicules sécrétées à travers passent par la membrane muqueuse, sont absorbées dans les cellules épithéliales du côlon, sont distribuées à travers le corps par l'intermédiaire du sang, et ensuite sont excrétées à travers le foie et le rein. Les vésicules dérivées des bactéries peuvent accroître ou réduire l'apparition d'inflammation et de cancer, et la présente invention peut être utile comme procédé de diagnostic du risque d'incidence d'une tumeur du côlon ou son facteur causal par séquençage d'un métagénome génétique d'une vésicule dérivée d'une bactérie présente dans une substance dérivée du corps humain. En outre, un diagnostic précoce peut encore être posé même après l'incidence du polype du côlon ou l'apparition du cancer du côlon, et l'incidence du cancer du côlon peut être réduite, et l'effet du traitement peut être amélioré.
PCT/KR2017/015557 2016-12-28 2017-12-27 Procédé de diagnostic d'une tumeur au côlon par l'intermédiaire d'une analyse métagénomique bactérienne WO2018124735A1 (fr)

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CN201780081381.2A CN110382720A (zh) 2016-12-28 2017-12-27 通过细菌宏基因组分析来诊断结肠癌的方法
US16/472,986 US20190330687A1 (en) 2016-12-28 2017-12-27 Method for diagnosing colon tumor via bacterial metagenomic analysis
EP17887675.1A EP3564390B1 (fr) 2016-12-28 2017-12-27 Procédé de diagnostic d'une tumeur au côlon par l'intermédiaire d'une analyse métagénomique bactérienne
JP2019535378A JP6914552B2 (ja) 2016-12-28 2017-12-27 細菌メタゲノム分析を通した大腸腫瘍の判定方法

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