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WO2018008153A1 - Procédé de détermination de la possibilité de début d'un cancer du côlon - Google Patents

Procédé de détermination de la possibilité de début d'un cancer du côlon Download PDF

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Publication number
WO2018008153A1
WO2018008153A1 PCT/JP2016/070330 JP2016070330W WO2018008153A1 WO 2018008153 A1 WO2018008153 A1 WO 2018008153A1 JP 2016070330 W JP2016070330 W JP 2016070330W WO 2018008153 A1 WO2018008153 A1 WO 2018008153A1
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WIPO (PCT)
Prior art keywords
cpg sites
methylation rate
seq
nos
colorectal cancer
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PCT/JP2016/070330
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English (en)
Japanese (ja)
Inventor
楠　正人
裕二 問山
光司 田中
荒木　俊光
彰 三井
竹鼻　健司
努 梅澤
Original Assignee
有限会社ハヌマット
Ｅａファーマ株式会社
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Application filed by 有限会社ハヌマット, Ｅａファーマ株式会社 filed Critical 有限会社ハヌマット
Priority to PCT/JP2016/070330 priority Critical patent/WO2018008153A1/fr
Priority to EP21152410.3A priority patent/EP3845669A1/fr
Priority to US16/315,961 priority patent/US20190241970A1/en
Priority to PCT/JP2017/024956 priority patent/WO2018008740A1/fr
Priority to EP17824343.2A priority patent/EP3483282A4/fr
Priority to KR1020197000171A priority patent/KR20190045146A/ko
Priority to EP21211939.0A priority patent/EP4023162A2/fr
Priority to JP2018526453A priority patent/JP7094881B2/ja
Priority to CN201780041963.8A priority patent/CN109415771A/zh
Publication of WO2018008153A1 publication Critical patent/WO2018008153A1/fr
Priority to US17/500,346 priority patent/US20220022851A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a method for determining the possibility of developing colorectal cancer in a patient with human ulcerative colitis, and a kit for collecting a rectal mucosa specimen used in the method.
  • This application claims the priority based on Japanese Patent Application No. 2015-137966 for which it applied to Japan on July 9, 2015, and uses the content here.
  • Ulcerative colitis is an inflammatory bowel disease whose cause is unknown, which mainly causes ulcers and erosions in the colonic mucosa. Complete cure is very difficult, and remission and relapse are repeated. Symptoms include local symptoms of the large intestine such as diarrhea, abdominal pain and mucous stool, and systemic symptoms such as fever, vomiting, tachycardia and anemia. Patients with ulcerative colitis are more likely to develop colorectal cancer. Therefore, early detection and treatment of colorectal cancer is important in patients with ulcerative colitis.
  • Patent Document 1 discloses methylation of five miRNA genes miR-1, miR-9, miR-124, miR-137, and miR-34b / c in neoplastic tissues in patients with ulcerative colitis.
  • the methylation rate is significantly higher than that of the non-neoplastic ulcerative colitis tissue. Therefore, the methylation rate of the five miRNA genes in the biological sample collected from the colonic mucosa, which is a non-cancerous part, is It can be used as a marker for the development of colorectal cancer in patients with ulcerative colitis.
  • the present invention relates to a method for determining the possibility of developing colorectal cancer in a patient with human ulcerative colitis by a method that is less invasive than endoscopy and less burdensome on the patient, and a rectum provided for the method
  • An object is to provide a kit for collecting a mucosal specimen.
  • the present inventors have comprehensively investigated the methylation rate of CpG sites (cytosine-phosphodiester bond-guanine) in the genomic DNA of ulcerative colitis patients.
  • the present invention was completed by finding 80 CpG sites having a marked difference in methylation rate between patients who developed colorectal cancer and patients who did not develop colorectal cancer.
  • the present invention provides the following methods [1] to [27] for determining the likelihood of developing colon cancer, a DNA methylation rate analysis marker, and a colonic mucosa collection kit.
  • a method for determining the possibility of developing colorectal cancer in a patient with human ulcerative colitis One or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80 in DNA collected from biological samples collected from human ulcerative colitis patients
  • a measurement process for measuring the methylation rate Determination step of determining the possibility of developing colon cancer in the human ulcerative colitis patient based on the methylation rate measured in the measurement step and a preset reference value or a preset multivariate discriminant
  • the reference value is a value for discriminating between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient, each set for the methylation rate of each CpG site,
  • the multivariate discriminant includes, as a variable, the methylation rate
  • a method for determining the likelihood of developing colorectal cancer [2] The method for determining the likelihood of developing colorectal cancer according to [1], wherein in the measuring step, the methylation rate of 2 to 10 CpG sites is measured. [3] In the determination step, represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, 31, 45, 64, 65, 67, 77, 79, and 80 One or more of the CpG sites in the nucleotide sequence to be methylated has a methylation rate equal to or lower than a preset reference value, or SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, When one or more of the CpG sites in the base sequences represented by 32-44, 46-63, 66, 68-76, and 78 have a methylation rate equal to or higher than a preset reference value, The method for determining the likelihood of developing colorectal cancer according to [1] or [2], wherein the human ulcerative colitis patient is determined to have
  • the methylation rate of the CpG site in the base sequence represented by SEQ ID NOs: 1 to 32 is measured
  • the determination step one or more of the CpG sites in the base sequence represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, and 31 are methyl Of the CpG site in the base sequence represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32
  • the above [1] to [3] wherein when the methylation rate is equal to or higher than a preset reference value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • a method for determining the likelihood of developing colorectal cancer is determining the likelihood of developing colorectal cancer.
  • Methylation rate among CpG sites in the base sequences represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, and 31 in the determination step Is the number of CpG sites whose pre-set reference value is less than or equal to a preset reference value, and methylation among CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32
  • the sum of the number of CpG sites with a rate equal to or higher than a preset reference value is 3 or more, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • the methylation rate of the CpG site in the base sequences represented by SEQ ID NOs: 1 to 16 is measured,
  • the determination step at least one of the CpG sites in the base sequence represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 16 has a methylation rate equal to or lower than a preset reference value.
  • the human ulcerative colitis patient when one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10 and 13 have a methylation rate equal to or higher than a preset reference value, the human ulcerative colitis patient
  • the methylation rate of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 9 is measured
  • the determination step one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 1 and 2 have a methylation rate equal to or lower than a preset reference value, or SEQ ID NOs: 3 to 9
  • the method for determining the likelihood of developing colorectal cancer according to any one of [1] to [3], wherein it is determined that the value is high.
  • the human ulcerative colitis patient is colon cancer
  • the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 33 to 66 is measured.
  • one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 45, 64, and 65 have a methylation rate equal to or lower than a preset reference value, or SEQ ID NO: 33
  • a patient with human ulcerative colitis when one or more of the CpG sites in the base sequences represented by -44, 46-63, and 66 have a methylation rate equal to or higher than a preset reference value The method for determining the likelihood of developing colorectal cancer according to any one of the above [1] to [3], wherein it is determined that there is a high possibility of developing colorectal cancer.
  • the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 33, 35, 36, 43, and 67-80 is determined.
  • at least one of the CpG sites in the base sequences represented by SEQ ID NOs: 67, 77, 79, and 80 has a methylation rate equal to or lower than a preset reference value, or a sequence
  • one or more of the CpG sites in the base sequences represented by the numbers 33, 35, 36, 43, 68 to 76, and 78 have a methylation rate that is equal to or higher than a preset reference value.
  • the multivariate discriminant includes, as a variable, the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 33 to 66, In the measurement step, the multivariate discriminant measures the methylation rate of the CpG site including the methylation rate as a variable, In the determination step, a discriminant value that is a value of the multivariate discriminant is calculated based on the methylation rate measured in the measurement step and the multivariate discriminant, and the discriminant value is set in advance as a reference discriminant value.
  • the method for determining the likelihood of developing colorectal cancer according to [1] or [2] above, wherein it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • [16] Methylation of one or more CpG sites selected from the group consisting of CpG sites in the base sequence represented by SEQ ID NOs: 33, 35, 36, 43, 67-80, wherein the multivariate discriminant is Including rate as a variable
  • the multivariate discriminant measures the methylation rate of the CpG site including the methylation rate as a variable
  • a discriminant value that is a value of the multivariate discriminant is calculated based on the methylation rate measured in the measurement step and the multivariate discriminant, and the discriminant value is set in advance as a reference discriminant value.
  • the method for determining the likelihood of developing colorectal cancer according to [1] or [2] above, wherein it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • the multivariate discriminant is a logistic regression equation, a linear discriminant, an equation created by a naive Bayes classifier, or an equation created by a support vector machine A method for determining the likelihood of developing colorectal cancer.
  • the method for determining the likelihood of developing colorectal cancer according to any one of [1] to [18], wherein the biological sample is rectal mucosal tissue.
  • the rectal mucosa tissue is A collection tool and a collection aid;
  • the sampling tool is A plate-like first clamping piece in which a first clamping surface for clamping the colonic mucosa is formed at one end;
  • a plate-like second clamping piece in which a second clamping surface for clamping the colonic mucosa is formed at one end;
  • Have At least one of the first clamping surface and the second clamping surface is cup-shaped
  • the collection aid is A frustoconical sampling tool introduction part having a through hole in the rotation axis direction and a slit on the side wall;
  • a rod-shaped gripping part Have One end
  • a method for determining the likelihood of developing cancer [21] The sampling tool is Having a first bent portion on the end side where the first clamping surface is formed with respect to the center of the first clamping piece; [20] The method for determining the likelihood of developing colorectal cancer according to [20] above, wherein the second bent portion is provided on the end side where the second holding surface is formed with respect to the center portion of the second holding piece. [22] A colonic mucosa collection kit comprising a collection tool and a collection aid.
  • the sampling tool is A plate-like first clamping piece in which a first clamping surface for clamping the colonic mucosa is formed at one end; A plate-like second clamping piece in which a second clamping surface for clamping the colonic mucosa is formed at one end; A connecting portion that connects the first clamping piece and the second clamping piece in a state of being opposed to each other at an end portion where the first clamping surface and the second clamping surface are not formed; Have At least one of the first clamping surface and the second clamping surface is cup-shaped,
  • the collection aid is A frustoconical sampling tool introduction part having a through hole in the rotation axis direction and a slit on the side wall; A rod-shaped gripping part; Have One end of the gripping part is connected to the vicinity of the edge part of the larger outer diameter of the sampling tool introduction part, The slit is provided from the edge of the smaller outer diameter of the sampling tool introduction part toward the edge of the larger outer diameter, The width of the slit is wider than the width
  • the sampling tool is Having a first bent portion on the end side where the first clamping surface is formed with respect to the center of the first clamping piece; [22] The colonic mucosa collection kit according to [22], further including a second bent portion on an end portion side where the second holding surface is formed with respect to a center portion of the second holding piece. [24] The colonic mucosa collection kit according to [22] or [23], wherein both the first clamping surface and the second clamping surface are cup-shaped. [25] The kit for collecting large intestine mucosa according to any of [22] to [24] above, wherein an inner diameter of the cup-shaped edge is 2 to 3 mm.
  • a DNA fragment comprising a DNA fragment having a partial base sequence containing one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80, and comprising a patient with ulcerative colitis
  • a marker for DNA methylation rate analysis used for determining the possibility of developing colorectal cancer.
  • the rectal mucosa collection kit according to the present invention enables the rectal mucosa to be collected relatively safely and simply from the patient's anus.
  • FIG. 1 is an explanatory diagram of a sampling tool 2A which is an embodiment of the sampling tool and a sampling tool 2B which is a modified example thereof.
  • FIG. 2 is an explanatory diagram of a sampling tool 2C which is a modification of the sampling tool 2A.
  • FIG. 3 is an explanatory diagram of a collection assisting tool 11 ⁇ / b> A that is one embodiment of the collection assisting tool 11.
  • FIG. 4 is an explanatory diagram of a collection assistance tool 11B that is a modification of the collection assistance tool 11A.
  • FIG. 5 is an explanatory diagram of a usage mode of the rectal mucosa collection kit.
  • FIG. 1 is an explanatory diagram of a sampling tool 2A which is an embodiment of the sampling tool and a sampling tool 2B which is a modified example thereof.
  • FIG. 2 is an explanatory diagram of a sampling tool 2C which is a modification of the sampling tool 2A.
  • FIG. 3 is an explanatory diagram of a collection assisting tool
  • FIG. 6A shows the result of cluster analysis based on the methylation level of CpG sites in the 32CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 6B shows the result of principal component analysis based on the methylation level of CpG sites in the 32 CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 6C shows the result of cluster analysis based on the methylation level of CpG sites in the 16 CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 6D shows the result of principal component analysis based on the methylation level of CpG sites in the 16 CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 6A shows the result of cluster analysis based on the methylation level of CpG sites in the 32CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 6C shows the result of cluster analysis based on the methylation
  • FIG. 6E shows the result of cluster analysis based on the methylation level of CpG sites in the 9CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 6F is the result of principal component analysis based on the methylation level of CpG sites in the 9CpG set selected as a result of comprehensive DNA methylation analysis in Example 1.
  • FIG. 7A shows that in Example 1, among the CpG sites in the five miRNA genes miR-1, miR-9, miR-124, miR-137, and miR-34b / c, the absolute value of DiffScore is 30. It is the result of the cluster analysis based on the methylation level of 27 CpG sites which were super.
  • FIG. 7A shows that in Example 1, among the CpG sites in the five miRNA genes miR-1, miR-9, miR-124, miR-137, and miR-34b / c, the absolute value of DiffScore is 30. It is the result of the cluster analysis
  • Example 7B shows that in Example 1, among the CpG sites in the five miRNA genes miR-1, miR-9, miR-124, miR-137, and miR-34b / c, the absolute value of DiffScore is 30. It is the result of the principal component analysis based on the methylation level of 27 CpG sites which were super.
  • FIG. 8A is the result of cluster analysis based on the methylation level of CpG sites in the 34 CpG set selected as a result of comprehensive DNA methylation analysis in Example 2.
  • FIG. 8B shows the result of principal component analysis based on the methylation level of CpG sites in the 34 CpG set selected as a result of comprehensive DNA methylation analysis in Example 2.
  • FIG. 8A is the result of cluster analysis based on the methylation level of CpG sites in the 34 CpG set selected as a result of comprehensive DNA methylation analysis in Example 2.
  • FIG. 8B shows the result of principal component analysis based on the methylation level of CpG
  • FIG. 9 shows the CpG site (cg10931190) in the base sequence represented by SEQ ID NO: 34, the CpG site (cg136767149) in the base sequence represented by SEQ ID NO: 37, and SEQ ID NO: 56 in Example 2.
  • FIG. 3 is an ROC curve of a test for the presence or absence of colon cancer in a patient with ulcerative colitis, using as a marker the methylation rate of three CpG sites in the CpG site (cg14516100) in the base sequence.
  • FIG. 10A shows the result of cluster analysis based on the methylation level of CpG sites in the 18 CpG set selected as a result of comprehensive DNA methylation analysis in Example 3.
  • FIG. 10B shows the result of principal component analysis based on the methylation level of CpG sites in the 18 CpG set selected as a result of comprehensive DNA methylation analysis in Example 3.
  • the cytosine base of the CpG site in genomic DNA can undergo methylation modification at the 5th carbon.
  • the methylation rate of a CpG site refers to the amount of methylated cytosine base (methylated cytosine) and the methylation among CpG sites in a biological sample collected from an individual organism.
  • the amount of untreated cytosine base (unmethylated cytosine) is measured, and means the ratio (%) of the amount of methylated cytosine to the sum of both.
  • the method for determining the likelihood of developing colorectal cancer according to the present invention is a method for determining the likelihood of developing colorectal cancer in human ulcerative colitis patients.
  • the methylation rate of ulcerative colitis patients who have not developed colon cancer (non-cancer ulcerative colitis patients) group and ulcerative colitis patients who developed colon cancer (carcinogenesis) It is characterized by using a marker that is significantly different from the ulcerative colitis patient group.
  • Using the methylation rate of CpG sites serving as these markers as an index it is determined whether a human ulcerative colitis patient is likely to develop colorectal cancer.
  • the determination of the likelihood of developing colon cancer in a patient with human ulcerative colitis based on the methylation rate of the CpG site as a marker may be performed based on the measured methylation rate of the CpG site itself, or the CpG site as a marker Alternatively, a multivariate discriminant including a methylation rate as a variable may be used based on a discriminant value obtained from this multivariate discriminant.
  • the determination method based on the measured methylation rate of the CpG site itself is specifically a method for determining the likelihood of developing colon cancer in a patient with human ulcerative colitis.
  • the CpG site used as a marker in the present invention those having a methylation rate greatly different between the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group are preferable.
  • the CpG site used as a marker in the present invention has a significantly higher methylation rate in patients with carcinogenic ulcerative colitis than in patients with non-cancer ulcerative colitis, that is, the methylation rate increases due to the onset of colon cancer.
  • the methylation rate of the carcinogenic ulcerative colitis patient may be significantly lower than that of the non-cancer ulcerative colitis patient, that is, the methylation rate may be lowered by the onset of colon cancer.
  • the CpG site used as a marker in the present invention those having a small difference in methylation rate between a non-cancerous site and a cancerous site of the large intestine in the same carcinogenic ulcerative colitis patient are more preferable. Even if a biological sample collected from a non-cancerous part of a patient with carcinogenic ulcerative colitis is used by using such a methylation rate of CpG site as an index, the biological sample collected from the cancerous part As in the case of using, the presence or absence of onset of colorectal cancer can be determined with high sensitivity.
  • the mucous membrane in the deep part of the large intestine must be collected using an endoscope or the like, and the burden on the patient is large, but the rectal mucosa near the anus can be collected relatively easily.
  • colon cancer develops using the rectal mucosa near the anus as a biological sample, regardless of where the cancerous site is formed. Can be detected without omission.
  • the CpG site used as a marker in the present invention is specifically one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80. Each base sequence is shown in Tables 1-8. In the base sequences in the table, CG in parentheses is a CpG site detected by the comprehensive DNA methylation analysis shown in Examples 1 to 3. A DNA fragment having a base sequence containing these CpG sites can be used as a DNA methylation rate analysis marker for determining the possibility of developing colon cancer in patients with ulcerative colitis.
  • the 32 CpG sites in parentheses in the base sequences represented by SEQ ID NOs: 1 to 32 were used in the comprehensive DNA methylation analysis in Example 1 described later.
  • the methylation rate is greatly different between the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group.
  • those with carcinogenic ulcerative colitis patients whose methylation rate is considerably lower than those of non-cancer ulcerative colitis are SEQ ID NOs: 1, 2, 11, 12, 14-18, 21-24, 26, 27, CpG sites in the base sequences represented by 29 and 31 (in the table, “ ⁇ ”), and the methylation rate of carcinogenic ulcerative colitis patients is significantly higher than that of non-cancer ulcerative colitis patients,
  • the CpG site used as a marker is not limited to these 32 CpG sites, and includes other CpG sites in the nucleotide sequences represented by SEQ ID NOs: 1-32.
  • 34 CpG sites in parentheses in the base sequences represented by SEQ ID NOs: 33 to 66 were used in the comprehensive DNA methylation analysis in Example 2 described later.
  • the methylation rate is greatly different between the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group.
  • the CpG sites in the nucleotide sequences represented by SEQ ID NOs: 45, 64, and 65 are those in which the methylation rate of carcinogenic ulcerative colitis patients is considerably lower than that of non-cancer ulcerative colitis patients (Table Among the nucleotide sequences represented by SEQ ID NOs: 33 to 44, 46 to 63, and 66, the methylation rate of carcinogenic ulcerative colitis patients is considerably higher than that of non-cancer ulcerative colitis patients. It is a CpG site in the middle ("+" in the table).
  • the CpG sites used as markers are not limited to these 34 CpG sites, and other CpG sites in the base sequences represented by SEQ ID NOs: 33 to 66 are also included.
  • a reference value for distinguishing between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient is set in advance. Measured in the case of CpG sites marked “+” in Tables 1 to 3 in the 32 CpG set and CpG sites marked “+” in Tables 4 to 8 in the 34 CpG set and 18 CpG set.
  • the methylation rate is equal to or higher than a preset reference value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • the reference value of each CpG site is experimentally determined as a threshold value that can measure the methylation rate of the CpG site in the carcinogenic ulcerative colitis patient group and the non-cancer ulcerative colitis patient group, and distinguish both groups. be able to.
  • the standard value for methylation of an arbitrary CpG site is obtained by a general statistical method. Examples thereof are shown below, but the method of determining the reference value in the present invention is not limited to these.
  • the reference value for example, for any CpG site, patients who have not been diagnosed with colon cancer by pathological examination using biopsy tissue in endoscopic examination among patients with ulcerative colitis (non- DNA methylation of rectal mucosa of patients with cancer ulcerative colitis is measured. After measuring about several patients, the numerical value which represents methylation of these patient groups by the average value or the median value, etc. can be calculated, and this can be made into a reference value.
  • DNA methylation of the rectal mucosa was measured respectively, and the mean or median etc.
  • a threshold value that distinguishes both values in consideration of the variation can be obtained and used as a reference value.
  • the CpG site used as a marker in the present invention only the CpG site in the base sequence represented by SEQ ID NOs: 1 to 16 may be used. These 16 CpG sites (hereinafter, collectively referred to as “16CpG set”) are, in the 32CpG set, the methylation rate between the non-cancerous site and the cancerous site of the colon of carcinogenic ulcerative colitis patients. The difference is small.
  • the CpG site used as a marker in the present invention it is also preferable to use only the CpG site in the nucleotide sequence represented by SEQ ID NOs: 1 to 9.
  • 9CpG set These nine CpG sites (hereinafter, collectively referred to as “9CpG set”) are, of the 16CpG set, the methylation rate between the non-cancerous site and the cancerous site of the colon of carcinogenic ulcerative colitis patients. The difference is smaller.
  • the determination step it is represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, 31, 45, 64, 65, 67, 77, 79, and 80.
  • One or more of the CpG sites in the base sequence have a methylation rate not higher than a preset reference value, or SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, 32 to
  • the sum of the number of CpG sites with a methylation rate equal to or higher than a preset reference value among CpG sites in the base sequences represented by ⁇ 44, 46-63, 66, 68-76 and 78 is 2 or more In the case of preferably 3 or more, more preferably 5 or more, it is possible to make a more accurate determination by determining that the human ulcerative colitis patient has a high possibility of developing colon cancer. .
  • the 32CpG set is used as a marker in the present invention, that is, when the methylation rate of the 32CpG set is measured in the measurement step, in the determination step, SEQ ID NOs: 1, 2, 11, 12, 14 to One or more of the CpG sites in the base sequences represented by 18, 21 to 24, 26, 27, 29, and 31 have a methylation rate equal to or lower than a preset reference value, or SEQ ID NO: When one or more CpG sites in the base sequence represented by 3 to 10, 13, 19, 20, 25, 28, 30, and 32 have a methylation rate equal to or higher than a preset reference value Furthermore, it is determined that the human ulcerative colitis patient has a high possibility of developing colorectal cancer.
  • methylation is performed among CpG sites in the base sequences represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 18, 21 to 24, 26, 27, 29, and 31.
  • the number of CpG sites whose rate is less than or equal to a preset reference value, and methyl of the CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32 When the sum of the number of CpG sites with a conversion rate equal to or higher than a preset reference value is 3 or more, preferably 5 or more, the human ulcerative colitis patient may develop colon cancer By determining that the value is high, the determination can be performed with higher accuracy.
  • one or more of the CpG sites in the base sequences represented by SEQ ID NOs: 45, 64, and 65 have a methylation rate.
  • the methylation rate is preset for at least one of the CpG sites in the base sequence represented by SEQ ID NOs: 33-44, 46-63, and 66, which are below the preset reference value. If it is equal to or higher than the reference value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • the number of CpG sites having a methylation rate equal to or lower than a preset reference value among the CpG sites in the base sequences represented by SEQ ID NOs: 45, 64, and 65, the number of CpG sites having a methylation rate equal to or lower than a preset reference value, and the sequence Among the CpG sites in the base sequences represented by the numbers 33 to 44, 46 to 63, and 66, the sum with the number of CpG sites whose methylation rate is not less than a preset reference value is preferably 2 or more, preferably 3 As described above, when it is more preferably 5 or more, it can be determined with higher accuracy by determining that the human ulcerative colitis patient has a high possibility of developing colorectal cancer.
  • one or more CpG sites in the base sequence represented by SEQ ID NOs: 67, 77, 79, and 80 are methylated.
  • the rate is below a preset reference value, or one or more of the CpG sites in the base sequence represented by SEQ ID NOs: 33, 35, 36, 43, 68 to 76, and 78 are methylated.
  • the rate is equal to or higher than a preset reference value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • the determination method according to the present invention among the CpG sites in the base sequences represented by SEQ ID NOs: 67, 77, 79, and 80, the number of CpG sites having a methylation rate equal to or lower than a preset reference value And the sum of the number of CpG sites having a methylation rate equal to or higher than a preset reference value among the CpG sites in the base sequences represented by SEQ ID NOs: 33, 35, 36, 43, 68 to 76, and 78.
  • it is 2 or more, preferably 3 or more, more preferably 5 or more, it is determined that the human ulcerative colitis patient has a high possibility of developing colorectal cancer, thereby making a more accurate determination Can do.
  • one or more CpG sites selected from the group consisting of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80 are used as markers.
  • the CpG sites used as markers in the present invention are all 80 CpG sites in parentheses in the base sequences represented by SEQ ID NOs: 1 to 80 (hereinafter sometimes collectively referred to as “80 CpG set”). May be the 32CpG set, the 16CpG set, the 9CpG set, the 34CpG set, or the 18CpG set.
  • the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group have small dispersion of the methylation rate.
  • the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group are excellent in that they have a high discriminating ability.
  • the 34CpG set and the 18CpG set are slightly less specific than the CpG site of the 32CpG set, the 16CpG set, and the CpG site of the 9CpG set, the sensitivity is very high. It is very suitable for primary screening test for ulcerative colitis.
  • the determination step is based on the methylation rate measured in the measurement step and a preset multivariate discriminant, and is capable of developing colon cancer in the human ulcerative colitis patient. Gender can be determined.
  • the multivariate discriminant includes, as a variable, the methylation rate of one or more CpG sites among the CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80.
  • the multivariate discriminant used in the present invention can be obtained by a general method used to discriminate between the two groups.
  • Examples of the multivariate discriminant include, but are not limited to, a logistic regression equation, a linear discriminant, a formula created with a naive Bayes classifier, or a formula created with a support vector machine. .
  • These multivariate discriminants include, for example, one of the CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80 for the carcinogenic ulcerative colitis patient group and the non-cancer ulcerative colitis patient group or The methylation rate of two or more CpG sites can be measured, and the resulting methylation rate can be used as a variable to prepare a standard method.
  • a reference discriminating value for discriminating between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient is set in advance.
  • the discriminant value which is the value of the multivariate discriminant used for the carcinogenic ulcerative colitis patient group and the non-cancer ulcerative colitis patient group, is obtained. It can be experimentally determined as a threshold value for distinguishing both groups by comparing the discriminant values of the cancer ulcerative colitis patient group.
  • the multivariate discriminant to be used measures the methylation rate of a CpG site containing the methylation rate as a variable, and the determination step And calculating a discriminant value which is a value of the multivariate discriminant based on the methylation rate measured in the measurement step and the multivariate discriminant, and based on the discriminant value and a preset reference discriminant value.
  • the degree of possibility that a human ulcerative colitis patient whose CpG site methylation rate was measured has developed colorectal cancer is determined.
  • the discriminant value is greater than or equal to a preset reference discriminant value, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer.
  • the multivariate discriminant used in the present invention is preferably a formula containing as a variable the methylation rate of one or more CpG sites selected from the group consisting of the 34 CpG sites, and selected from the group consisting of the 34 CpG sites. More preferably, the formula includes only the methylation rate of one or more CpG sites as a variable, and only the methylation rate of 2 to 10 CpG sites arbitrarily selected from the group consisting of the 34 CpG sites is used as a variable. More preferably, the formula includes only the methylation rate of 2 to 5 CpG sites arbitrarily selected from the group consisting of the 34 CpG sites as a variable.
  • the multivariate discriminant used in the present invention is preferably a formula containing as a variable the methylation rate of one or more CpG sites selected from the group consisting of the 18CpG sites, and selected from the group consisting of the 18CpG sites. More preferably, the formula includes only the methylation rate of one or more CpG sites as a variable, and only the methylation rate of 2 to 10 CpG sites arbitrarily selected from the group consisting of the 18 CpG sites is used as a variable. More preferably, the formula includes only the methylation rate of 2 to 5 CpG sites arbitrarily selected from the group consisting of the 18 CpG sites as a variable.
  • the CpG sites constituting the 34 CpG set and the 18 CpG set are arbitrarily selected from 2 to 10, preferably 2 to 5 CpG sites from these sets, and even when only the selected CpG sites are used. With sufficient sensitivity and specificity, it is possible to determine the possibility of developing colon cancer in human ulcerative colitis patients.
  • the CpG site in the base sequence represented by SEQ ID NO: 34 the CpG site in the base sequence represented by SEQ ID NO: 37, and the sequence represented by SEQ ID NO: 56
  • the likelihood of developing colon cancer can be determined with a sensitivity of about 96% and a specificity of about 92%.
  • labor and cost may be excessive.
  • CpG site As a marker from the CpG sites constituting the 34CpG set and the 18CpG set, it is an appropriate number of CpG sites that can be measured in clinical examinations, and is accurate to human ulcerative colon. The possibility of developing colorectal cancer in patients with inflammation can be determined.
  • the biological sample used in the determination method according to the present invention is a biological sample collected from a patient with human ulcerative colitis and is not particularly limited as long as it contains the genomic DNA of the patient.
  • blood, plasma, serum, tears, saliva, or the like may be used, or tissue pieces collected from other tissues such as the digestive tract mucosa or the liver may be used.
  • the biological sample used in the determination method according to the present invention is preferably the large intestine mucosa because it more strongly reflects the state of the large intestine, and can be collected in the rectal mucosa because it can be collected with relatively low invasiveness. More preferably.
  • the rectal mucosa of the large intestine can be easily collected using, for example, a kit for collecting large intestine mucosa described later.
  • the biological sample may be in a state where DNA can be extracted, and may be subjected to various pretreatments.
  • it may be a formalin fixed paraffin embedded (FFPE) tissue.
  • FFPE formalin fixed paraffin embedded
  • Extraction of DNA from a biological sample can be performed by a conventional method, and various commercially available DNA extraction / purification kits can also be used.
  • the method for measuring the methylation rate of a CpG site is not particularly limited as long as it is a method capable of distinguishing and quantifying a methylated cytosine base and an unmethylated cytosine base for a specific CpG site.
  • the methylation rate of the CpG site can be measured by appropriately modifying methods known in the art as they are or as necessary. Examples of methods for measuring the methylation rate of CpG sites include bisulfite sequencing, COBRA (Combined Bisulfite Restoration Analysis), and qAMP (quantitative analysis of DNA methylation using real-time PCR). In addition, you may perform using the MIAM (Microarray-based Integrated Analysis of Methylation by Isoschizomers) method.
  • the kit for collecting large intestine mucosa comprises a collection tool for collecting with the rectal mucosa sandwiched therein, and a collection aid for expanding the anus and allowing the collection tool to reach the surface of the large intestine mucosa from the anus.
  • the colonic mucosa collection kit according to the present invention will be described below with reference to FIGS.
  • FIG. 1 (A) to FIG. 1 (C) are explanatory views of a sampling tool 2A which is an embodiment of the sampling tool 2 of the colonic mucosa sampling kit 1.
  • FIG. FIG. 1 (A) is a perspective view of a state in which the first holding piece 3a and the second holding piece 3b of the sampling tool 2A are not applied
  • FIG. 1 (B) is a perspective view of the applied state.
  • FIG. 1C is a partially enlarged view of the distal end portion having the clamping surface of the sampling tool 2A.
  • the sampling tool 2A includes a first clamping piece 3a, a second clamping piece 3b, a connecting portion 4, a first clamping surface 5a, and a second clamping surface 5b. And having.
  • the first clamping piece 3a is a plate-like member in which a first clamping surface 5a for clamping the colonic mucosa is formed at one end, and the second clamping piece 3b is a large intestine at one end. It is a plate-like member on which a second clamping surface 5b that clamps the mucous membrane is formed.
  • the first sandwiching piece 3a and the second sandwiching piece 3b are connected to each other at the end where the first sandwiching surface 5a and the second sandwiching surface 5b are not formed in the connecting portion 4 in a state of facing each other. Yes.
  • the shape of the first sandwiching piece 3a and the second sandwiching piece 3b may be plate-like or rod-like, and may be formed to have a certain length for collecting the rectal mucosa. Don't stick to the shape.
  • the length of the first clamping piece 3a and the second clamping piece 3b is preferably 50 to 250 mm, more preferably 100 to 200 mm, still more preferably 70 to 200 mm, and even more preferably 70 to 150 mm. Since the first clamping piece 3a and the second clamping piece 3b have a length within the above range, the colonic mucosa is easily clamped and collected from the anus.
  • At least one of the first clamping surface 5a and the second clamping surface 5b is preferably cup-shaped. Since at least one of them is a cup shape, when the edge 6a of the first clamping surface 5a and the edge 6b of the second clamping surface 5b are in contact with each other, a space is formed inside. Of the large intestine mucosa sandwiched between the first sandwiching surface 5a and the second sandwiching surface 5b, the portion accommodated in the space does not take much load when the large intestine mucosa is torn, thus destroying the tissue. Can be suppressed. As shown in FIG. 1, since both are cup-shaped, it is easier to collect the mucosa of the large intestine, and tissue destruction can be suppressed.
  • the inner diameters of the marginal portion 6a and the marginal portion 6b may be set to such a size that a necessary amount of large intestine mucosa can be collected.
  • the large intestine mucosa used in the determination method according to the present invention it is sufficient if a small amount of mucosa can be collected.
  • the inner diameters of the edge portion 6a and the edge portion 6b to 1 to 5 mm, preferably 2 to 3 mm, a sufficient amount of the large intestine mucosa can be collected without excessively damaging the large intestine mucosa.
  • the edge portion 6a and the edge portion 6b may be flat as long as they can be brought into close contact with each other, but are preferably serrated as shown in FIG.
  • the large intestine mucosa can be cut and collected with a relatively weak force by being sandwiched between the edge 6a 'and the edge 6b'.
  • the protruding portion 8a may be formed on the inner side of one of the first holding piece 3a and the second holding piece 3b, and the cylindrical portion 9a may be formed on the other side so as to face each other.
  • the tip of the protrusion 8a fits into the tube 9a.
  • the tip of the protrusion 8a is fitted into the tube portion 9a, it is possible to stably collect the large intestine mucosa without shifting the peripheral edge 6a and the peripheral edge 6b when the collection tool 1 is separated from the large intestine mucosa. it can.
  • FIG. 1D is an explanatory diagram of a sampling tool 2B which is a modification of the sampling tool 2A. More specifically, the first clamping piece 3a and the second clamping piece 3b of the sampling tool 2B are applied with force.
  • the 1st clamping piece 3a may have the 1st bending part 7a in the edge part side in which the 1st clamping surface 5a is formed rather than the center part.
  • the 2nd clamping piece 3b may have the 2nd bending part 7b in the edge part side in which the 2nd clamping surface 5b is formed rather than the center part.
  • the first sandwiching piece 3a and the second sandwiching piece 3b are inclined while maintaining a state in which they are opposed to each other on the distal end side where the sandwiching surface is formed with respect to the center portion, thereby assisting collection. It becomes easy to penetrate the slit 13 of the tool 11 and contact the colonic mucosa.
  • the bending angle ⁇ 1 is preferably 10 to 50 °, more preferably 20 to 40 °, and even more preferably 25 to 35 °.
  • the length from the first bent portion 7a to the tip portion of the first clamping surface 5a and the length from the second bent portion 7b to the tip portion of the second holding surface 5b are 20 to 60 mm. 30 to 50 mm is more preferable.
  • the length from the bent portion to the distal end portion of the clamping surface is within the above range, it is easier to collect the mucous membrane in a state where it passes through the slit 13 of the collection assisting tool 11.
  • FIG. 2 (A) to 2 (E) are explanatory views of a sampling tool 2C, which is another modification of the sampling tool 2A.
  • FIG. 2 (A) is a front view showing a state in which the first clamping piece 3a and the second clamping piece 3b of the sampling tool 2 are not pressed
  • FIG. 2 (B) is a plan view of the sampling tool 2C.
  • FIG. 2C is an enlarged view of the protruding portion 8b of the sampling tool 2C
  • FIG. 2D is an opening edge of the cylindrical portion 9b where the locking claw of the protruding portion 8b is at the tip of the sampling tool 2C.
  • FIG. 2E is a plan view showing a state in which the first clamping surface 5a and the second clamping surface 5b are bonded to each other at the distal end portion of the sampling tool 2C.
  • the collection tool 2 When collecting mucosal tissue from the rectum of the subject, the collection tool 2 is more in a closed state than in a state in which the distance between the first holding piece 3a and the second holding piece 3b is open. It is easy to penetrate the slit 13. Therefore, as shown by the protrusion 8b in FIG. 2, the protrusion of the collection tool 2 may be a locking claw. There may be one or more locking claws of the protruding portion 8b, and any number may be used as long as it can be locked to the overhanging portion of the opening edge of the cylindrical portion 9b.
  • the cylindrical portion 9b for mating the protruding portion 8b is provided with a protruding portion on the opening edge radially inward, and the locking claw of the protruding portion 8b protrudes from the opening edge of the cylindrical portion 9b.
  • the height of the locking claw of the protrusion 8b is such that the tip of the first clamping surface 5a and the second clamping surface 5b are close to each other when locked to the overhanging portion of the tube portion 9b.
  • the first sandwiching surface 5a and the second sandwiching surface 5b are not bonded to each other, and the first sandwiching piece 3a and the second sandwiching piece 3b are further applied, whereby the tip of the protruding portion 8b.
  • first clamping surface 5a and the second clamping surface 5b can be bonded without penetrating through the bottom of the cylindrical portion 9b. This stabilizes the front end portions of the first clamping surface 5a and the second clamping surface 5b close to each other without applying force to the first clamping piece 3a and the second clamping piece 3b of the sampling tool 2. be able to.
  • the collection tool 2 penetrates the slit 13 of the collection assisting tool 11 in the state of FIG. 2D and the tip part contacts the rectal mucosa tissue, the first clamping piece 3a and the second clamping piece 3b are applied with force. Then, a part of the mucosal tissue is sandwiched and the state shown in FIG. 2E is obtained, and the mucosal tissue is collected.
  • the sampling tool 2 may be provided with a buffering portion 10a corresponding to a portion between the connecting portion and the bent portion in the first holding piece 3a and the second holding piece 3b, respectively.
  • the buffer portion 10a is provided with an elastic portion 10b at the tip thereof, and is bonded to each other by the elastic portion b in a state where the locking claw of the protruding portion 8b is locked to the protruding portion of the opening edge portion of the cylindrical portion 9b ( FIG. 2 (D)).
  • the sampling tool 2 can maintain the state which the latching claw of the projection part 8b latched to the overhang
  • FIG. 3 (A) and 3 (B) are explanatory views of a collection assisting tool 11A which is an embodiment of the collection assisting tool 11.
  • FIG. FIG. 3A is a perspective view seen from the lower side of the collection assisting tool 11A
  • FIG. 3B is a bottom view seen from the slit side of the collection assisting tool 11A.
  • the collection assisting tool 11A has a collection tool introducing part 12, a slit 13, and a gripping part 14.
  • the collection tool introduction part 12 is a truncated cone-shaped member having a through hole in the rotation axis direction, and has a slit 13 on the side wall.
  • the sampling tool introduction unit 12 is inserted into the anus from the distal edge 15 having a small outer diameter, and the sampling tool 2 is inserted from the proximal edge 16 having a large outer diameter.
  • the outer diameter of the hand edge 16 is 30 to 70 mm, preferably 40 to 50 mm.
  • the outer diameter of the distal edge portion 15 is preferably 10 to 30 mm, and more preferably 15 to 25 mm.
  • the length of the collection tool introducing portion 12 in the rotation axis direction is 50 to 150 mm, preferably 70 to 130 mm, and more preferably 80 to 120 mm.
  • the slit 13 is provided from the front edge portion 15 of the sampling tool introduction portion 12 toward the hand edge portion 16.
  • the presence of the slit 13 reaching the distal edge 15 in a part of the side wall of the collection tool introducing portion 12 increases the freedom of movement of the distal end portion of the collection tool 2 in the intestine, and in the rectum having a complicated internal structure, Large intestine mucosa can be collected more easily.
  • the slit 13 may be set at any position of the collection tool introduction unit 12.
  • the slit 13 is preferably on the side close to the gripping portion 14 as shown in FIG.
  • the number of the slits 13 provided in the collection tool introduction part 12 may be one, or two or more.
  • the width of the slit 13 is the same as that of the first clamping surface 5a of the collection tool 2 in a state where the edge 6a and the edge 6b are in contact with each other. It is designed wider than the width of the second clamping surface 5b. Further, the width of the slit 13 may be constant, but as shown in FIG. 3B, it is preferable that the slit 13 becomes wider from the front edge portion 15 toward the hand edge portion 16 side. For example, the width L 1 (see FIG.
  • the width L 2 (see FIG. 3B) of the slit 13 on the front edge portion 15 side is preferably 7 to 15 mm
  • the width L 3 of the slit 13 on the hand edge portion 16 side is preferably 10 to 20 mm.
  • two or more slits may be formed on the wall surface of the collection tool introduction part 12.
  • the length of the gripping portion 14 is preferably 50 to 150 mm, more preferably 70 to 130 mm, from the viewpoint of ease of gripping with a hand.
  • the shape of the gripping portion 14 may be any shape as long as it is easy to grip, and may be, for example, a plate shape, a rod shape, or other shapes.
  • FIG. 4 is an explanatory diagram of a collection assisting tool 11B which is a modification of the collection assisting tool 11A.
  • 4A is a perspective view seen from the upper side of the collection assisting tool 11B
  • FIG. 4B is a perspective view seen from the lower side.
  • 4 (C) to 4 (G) are a front view, a plan view, a bottom view, a left side view, and a right side view of the collection assisting tool 11B, respectively.
  • the gripping part of the collection assisting tool may be a hollow rod having an opening at the bottom and reinforced with a rib.
  • FIG. 5 is an explanatory diagram showing a mode of use of the colonic mucosa collection kit 1 according to the present invention.
  • the collection assisting tool 11 is inserted from the distal edge portion 15 into the anus of the subject from whom the colonic mucosa is collected.
  • the sampling tool 2 is introduced from the opening on the side of the hand edge 16 in a state where the grip 14 is held and stabilized with one hand.
  • the introduced collection tool 2 penetrates the slit 13 from the tip and reaches the surface of the large intestine mucosa.
  • the colonic mucosa can be collected by pulling out the sampling tool 2 from the slit 13 while the colonic mucosa is sandwiched between the clamping surface 5a and the clamping surface 5b of the sampling tool 2 (the clamping surface 5).
  • Example 1 Among the patients with ulcerative colitis, 8 patients (7 men, 1 woman) who were diagnosed with colon cancer by pathological diagnosis by biopsy tissue in endoscopy and who underwent surgery (7 men, 1 woman) DNA in colonic mucosa collected from 8 patients (7 males, 1 female) who were surgically treated other than cancer (non-cancer UC patients) who were refractory to medical treatment ulcerative colitis In contrast, the methylation rate of the CpG site was comprehensively analyzed.
  • the average age of 8 UC cancer patients was 47.1 ⁇ 12.4 years, and the average disease duration was 11.4 ⁇ 7.3 years.
  • the average age of the 8 non-cancer UC patients was 44.3 ⁇ 16.4 years, and the average disease duration was 6.5 ⁇ 5.2 years.
  • Fragmentation and purification of whole genome amplified DNA Fragmentation enzyme was added to the whole genome amplified DNA and reacted at 37 ° C. for 1 hour with a Microsample Incubator (SciGene). To the fragmented DNA, a coprecipitation agent and 2-propanol were added and centrifuged to precipitate the DNA.
  • Hybridization Hybridization buffer was added to the precipitated DNA, and the mixture was reacted with Hybridization Oven (manufactured by Illumina) at 48 ° C. for 1 hour to dissolve the DNA.
  • the lysed DNA was denatured into single strands by incubating with a Microsample Incubator (manufactured by SciGene) at 95 ° C. for 20 minutes, and then dispensed onto a BeadChip. The reaction was carried out for 16 hours or more in Hybridization Oven at 48 ° C., and the probe on the BeadChip and the single-stranded DNA were hybridized.
  • [ ⁇ value] [Methylated fluorescence intensity] ⁇ ([Methylated fluorescence intensity] + [Unmethylated fluorescence intensity] +100)
  • GenomeStudio and software Methylation Module (Version: 1.9.0) were used for DNA methylation quantification and DNA methylation level comparison analysis.
  • the setting conditions of GenomeStudio are as follows.
  • the cancer patient samples were narrowed down to those with little fluctuation in DNA methylation level. That is, the unbiased variance var of the ⁇ value of 24 samples of UC cancer patients (3 sites ⁇ 8 samples of each site) was obtained, and 16 CpG sites having an unbiased variance var value smaller than 0.05 were selected.
  • the 16 CpG sites are collectively referred to as a “16 CpG set”.
  • Nine CpG sites with an unbiased variance var value smaller than 0.03 were further narrowed down from the 16 CpG set.
  • these nine CpG sites are collectively referred to as a “9CpG set”.
  • Table 9 shows the results of each CpG site in the 32CpG set.
  • CpG sites with # in the “16CpG” column indicate those included in the 16CpG set
  • CpG sites with # in the “9CpG” column indicate those included in the 9CpG set.
  • Example 2 In addition to the patients with ulcerative colitis of Example 1, 24 patients (UC cancer patients) who were diagnosed with colorectal cancer by pathological diagnosis with biopsy tissue by endoscopy and who underwent surgery, and medical treatment Comprehensive CpG site methylation rate for DNA in colon mucosa collected from 24 patients with refractory ulcerative colitis who have undergone surgery other than cancer (non-cancer UC patients) was analyzed.
  • the DNA used for the analysis of the methylation rate of the CpG site was extracted from the FFPE sample collected from the rectal mucosal tissue of a patient with ulcerative colitis in the same manner as in Example 1, and the whole genome was amplified. Quantification and comparative analysis of DNA methylation levels were performed, and DiffScore calculation, cluster analysis, and principal component analysis were performed using the results.
  • CpG biomarker candidates were extracted from comprehensive DNA methylation analysis data. Specifically, first, 324 CpG sites having an absolute value of ⁇ value exceeding 0.2 were extracted from 485,577 CpG sites.
  • FIG. 9 shows a ROC (Receiver-Operating-Characteristic) curve.
  • the AUC area under the ROC curve
  • Example 3 The DNA methylation level ( ⁇ value) of each CpG site of the specimen collected from the rectum of the ulcerative colitis patient obtained in Example 1 and the DNA methylation of each CpG site of the ulcerative colitis patient obtained in Example 2 CpG biomarker candidates were extracted from the conversion level ( ⁇ value).

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Abstract

La présente invention concerne un procédé pour déterminer la possibilité de début d'un cancer chez des patients humains atteints de rectocolite hémorragique, le procédé pour déterminer la possibilité de début d'un cancer du côlon comprenant une étape de mesure pour mesurer le taux de méthylation d'un ou de plusieurs sites CpG choisis dans le groupe constitué par les sites CpG dans des séquences de base représentées par les séquences SEQ ID NO : 1 à 80 dans l'ADN récupéré à partir d'un échantillon biologique collecté sur un patient humain atteint de rectocolite hémorragique et une étape de détermination pour déterminer la possibilité de début d'un cancer du côlon chez le patient humain atteint de rectocolite hémorragique sur base du taux de méthylation mesuré dans l'étape de mesure et d'une valeur de référence préétablie ou d'un discriminant préétabli à plusieurs variables.
PCT/JP2016/070330 2016-07-08 2016-07-08 Procédé de détermination de la possibilité de début d'un cancer du côlon WO2018008153A1 (fr)

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PCT/JP2016/070330 WO2018008153A1 (fr) 2016-07-08 2016-07-08 Procédé de détermination de la possibilité de début d'un cancer du côlon
EP21152410.3A EP3845669A1 (fr) 2016-07-08 2017-07-07 Procédé d'évaluation de la probabilité de survenue d'un cancer du gros intestin
US16/315,961 US20190241970A1 (en) 2016-07-08 2017-07-07 Method for determining likelihood of colorectal cancer development
PCT/JP2017/024956 WO2018008740A1 (fr) 2016-07-08 2017-07-07 Procédé d'évaluation de la probabilité de survenue d'un cancer du gros intestin
EP17824343.2A EP3483282A4 (fr) 2016-07-08 2017-07-07 Procédé d'évaluation de la probabilité de survenue d'un cancer du gros intestin
KR1020197000171A KR20190045146A (ko) 2016-07-08 2017-07-07 대장암 발증 가능성의 판정 방법
EP21211939.0A EP4023162A2 (fr) 2016-07-08 2017-07-07 Procédé d'évaluation de la probabilité de survenue d'un cancer du gros intestin
JP2018526453A JP7094881B2 (ja) 2016-07-08 2017-07-07 大腸癌発症可能性の判定方法
CN201780041963.8A CN109415771A (zh) 2016-07-08 2017-07-07 大肠癌发病可能性的判定方法
US17/500,346 US20220022851A1 (en) 2016-07-08 2021-10-13 Method for determining likelihood of colorectal cancer development

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