WO2018008153A1 - Method for determining possibility of onset of colon cancer - Google Patents

Abstract

The present invention is a method for determining the possibility of the onset of colon cancer in human ulcerative colitis patients, wherein the method for determining the possibility of onset of colon cancer includes a measurement step for measuring the methylation rate of one or more CpG sites selected from the group consisting of CpG sites in base sequences represented by SEQ ID NOS: 1-80 in DNA recovered from a biological sample collected from a human ulcerative colitis patient, and a determination step for determining the possibility of onset of colon cancer in the human ulcerative colitis patient on the basis of the methylation rate measured in the measurement step and a preset reference value or preset multivariate discriminant.

Description

How to determine the likelihood of developing colorectal cancer

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.

Generally, examination for early detection of colorectal cancer is usually performed by endoscopy. However, it is generally difficult to visually detect early colorectal cancer because it depends largely on the skill of the operator. In particular, in patients with ulcerative colitis, detection of early colon cancer is very difficult because inflammation of the intestinal mucosa is originally severe. In addition, endoscopy has a problem that it is highly invasive and burdens the patient.

On the other hand, 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.

International Publication No. 2014/151551

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.

As a result of intensive studies to solve the above problems, 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.

That is, 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.
[1] 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 Have
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 of at least one CpG site among the CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80.
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 a high possibility of developing colorectal cancer.
[4] In the measurement step, the methylation rate of the CpG site in the base sequence represented by SEQ ID NOs: 1 to 32 is measured,
In 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.
[5] 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 When 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, [1] A method for determining the likelihood of developing colorectal cancer according to any one of [4].
[6] In the measurement step, the methylation rate of the CpG site in the base sequences represented by SEQ ID NOs: 1 to 16 is measured,
In 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. Alternatively, 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 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.
[7] Among the CpG sites in the base sequences represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 16 in the determination step, CpG sites having a methylation rate equal to or lower than a preset reference value When the sum of the number and 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: 3 to 10 and 13 is 3 or more, The method for determining the likelihood of developing colorectal cancer according to any one of [1] to [3] and [6], wherein the human ulcerative colitis patient is determined to have a high possibility of developing colorectal cancer.
[8] In the measurement step, the methylation rate of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 9 is measured,
In 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 Possibility that the human ulcerative colitis patient develops colorectal cancer when one or more of the CpG sites in the represented base sequence has a methylation rate that is equal to or higher than a preset reference value 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.
[9] In the determination step, among the CpG sites in the base sequences represented by SEQ ID NOs: 1 and 2, the number of CpG sites having a methylation rate equal to or lower than a preset reference value, and SEQ ID NOs: 3 to 9 When the sum of the CpG sites in the base sequence represented by the number of CpG sites having a methylation rate equal to or higher than a preset reference value is 3 or more, the human ulcerative colitis patient is colon cancer The method for determining the likelihood of developing colorectal cancer according to any one of [1] to [3] and [8], wherein it is determined that there is a high possibility of having developed.
[10] In the measurement step, 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.
In the determination step, 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.
[11] In the determination step, 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 SEQ ID NO: When the sum of the CpG sites in the base sequences represented by 33 to 44, 46 to 63 and 66 and the number of CpG sites having a methylation rate equal to or higher than a preset reference value is 2 or more, The method for determining the likelihood of developing colorectal cancer according to any one of [1] to [3] and [10], wherein the human ulcerative colitis patient is determined to have a high possibility of developing colorectal cancer.
[12] In the measurement step, 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. Measure and
In the determination step, 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 When 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 method for determining the likelihood of developing colorectal cancer according to any one of [1] to [3] above, wherein it is determined that a human ulcerative colitis patient is likely to develop colorectal cancer.
[13] In the determination step, 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; The sum of the CpG sites in the base sequences represented by SEQ ID NOs: 33, 35, 36, 43, 68 to 76, and 78 with a methylation rate equal to or higher than a preset reference value is 2 When the above is true, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer, and the possibility of developing colorectal cancer according to any one of [1] to [3] and [12] Judgment method.
[14] When the sum is 5 or more, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer, [5], [7], [9], [9] [11] or [13].
[15] 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. In the case described above, 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,
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. In the case described above, 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.
[17] Any of the above [1] to [16], wherein 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.
[18] The method for determining the likelihood of developing colon cancer according to any of [1] to [17], wherein the biological sample is intestinal tissue.
[19] 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.
[20] 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;
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 of one end of the first clamping piece and the one end of the second clamping piece,
The large intestine of the above-mentioned [19], which is collected by a large intestine mucosa collection kit having a larger outer diameter of the collection tool introduction part of 30 to 70 mm and a length in the rotation axis direction of 50 to 150 mm. 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 of one end of the first clamping piece and the one end of the second clamping piece,
A large intestine mucosa collection kit having a larger outer diameter of the collection tool introduction part of 30 to 70 mm and a length in the rotation axis direction of 50 to 150 mm.
[23] 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.
[26] The colonic mucosa collection kit according to any one of [22] to [25], wherein edge portions of the first clamping surface and the second clamping surface are serrated.
[27] 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.

By examining the methylation rate of a specific CpG site in genomic DNA of a biological sample collected from a patient with ulcerative colitis by the method for determining the likelihood of developing colon cancer according to the present invention, the possibility of developing colon cancer Can be determined.
In addition, 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. 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. 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. 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. 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.

<Method for determining the likelihood of developing colorectal cancer>
The cytosine base of the CpG site in genomic DNA can undergo methylation modification at the 5th carbon. In the present invention and the present specification, 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 (hereinafter sometimes referred to as the “method of determining the present invention”) is a method for determining the likelihood of developing colorectal cancer in human ulcerative colitis patients. Among the CpG sites in genomic DNA, 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. By using the methylation rate of the CpG site as a marker used to determine the likelihood of developing colorectal cancer in patients with ulcerative colitis, early colorectal cancer in patients with ulcerative colitis that is extremely difficult to visually distinguish It can be detected objectively and with high sensitivity, and early detection can be expected.

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.

Among the determination methods according to the present invention, 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. A measurement step of measuring methylation rates of a plurality of specific CpG sites as markers in the present invention in DNA collected from a biological sample collected from a human ulcerative colitis patient, And a determination step of determining a possibility of developing colon cancer in the human ulcerative colitis patient based on the measured methylation rate and a reference value previously set for each CpG site.

As 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 larger the difference between the two groups, the more reliably the presence or absence of colorectal cancer can be detected. 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.

As 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. For example, 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. By using a CpG site with a small difference in methylation rate between the non-cancerous and cancerous sites of the large intestine as a marker, 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 (hereinafter sometimes collectively referred to as “32CpG sets”) 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. Among them, 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, This is a CpG site in the base sequence represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32 (“+” in the table). 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 (hereinafter, sometimes collectively referred to as “34 CpG set”) 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. Among them, 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.

Eighteen CpG sites in parentheses in the base sequences represented by SEQ ID NOs: 33, 35, 36, 43, and 67 to 80 (hereinafter sometimes collectively referred to as “18 CpG set”) are described in Example 3 below. In the comprehensive DNA methylation analysis, the methylation rate is greatly different between the non-cancer ulcerative colitis patient group and the carcinogenic ulcerative colitis patient group. Among them, the CpG site in the base sequence represented by SEQ ID NOs: 67, 77, 79, and 80 has a methylation rate significantly lower in patients with carcinogenic ulcerative colitis than in non-cancer ulcerative colitis patients. ("-" In the table), those with carcinogenic ulcerative colitis patients whose methylation rate is considerably higher than those with non-cancer ulcerative colitis are SEQ ID NOs: 33, 35, 36, 43, 68-76, and 78. The CpG site in the base sequence represented by ("+" in the table). The CpG site used as a marker is not limited to these 18 CpG sites, and includes other CpG sites in the base sequences represented by SEQ ID NOs: 33, 35, 36, 43, and 67-80. .

For each CpG site, 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. 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. Measured in the case of CpG sites marked as “-” in Tables 1 to 3 in the 32CpG set and CpG sites marked as “−” in Tables 4 to 8 out of the 34CpG set and 18CpG set. When the methylation rate is equal to or lower than a preset reference value, it is determined that a 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. Specifically, 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.

As an example of how to obtain 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.

In addition, for multiple non-cancer ulcerative colitis patients and multiple carcinogenic ulcerative colitis patients, DNA methylation of the rectal mucosa was measured respectively, and the mean or median etc. After calculating a numerical value representative of methylation and variation of the non-cancer ulcerative colitis patient group, a threshold value that distinguishes both values in consideration of the variation can be obtained and used as a reference value.

As 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. As 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. 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.

In 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 When one or more of the CpG sites in the base sequences represented by 44, 46 to 63, 66, 68 to 76, and 78 have a methylation rate equal to or higher than a preset reference value, the human Determine that patients with ulcerative colitis are more likely to develop colorectal cancer. In the determination method according to the present invention, 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 Among the CpG sites in the represented nucleotide sequence, the number of CpG sites having a methylation rate equal to or lower than a preset reference value, and SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, 32 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. .

When 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. In the determination method according to the present invention, 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.

When the 34CpG set is used as a marker in the present invention, in the determination step, 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. In the determination method according to the present invention, 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.

When the 18CpG set is used as a marker in the present invention, in the determination step, 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 When 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. In 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. When 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.

In the present invention, 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. In the CpG site of the 32CpG set, the CpG site of the 16CpG set, and the CpG site of the 9CpG set, all of 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. On the other hand, although 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.

In the determination method according to the present invention, 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.

In the multivariate discriminant used in the present invention, a reference discriminating value for discriminating between a carcinogenic ulcerative colitis patient and a non-cancer ulcerative colitis patient is set in advance. For the standard discriminant value, 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.

When performing determination using a multivariate discriminant, specifically, in the measurement step, 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. When 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. For example, as shown in Example 2 below, among the 34 CpG sets, 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 In the case of using a multivariate discriminant created by logistic regression using the three CpG sites of the CpG site in the base sequence to be used as markers and the methylation rate of these three CpG sites as variables, For human ulcerative colitis patients, the likelihood of developing colon cancer can be determined with a sensitivity of about 96% and a specificity of about 92%. In clinical examinations and the like, if the number of CpG sites for measuring the methylation rate is large, labor and cost may be excessive. By selecting a 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. Alternatively, 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.

In addition, the biological sample may be in a state where DNA can be extracted, and may be subjected to various pretreatments. For example, it may be a formalin fixed paraffin embedded (FFPE) tissue. 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.

<Colonial mucosa collection kit>
The kit for collecting large intestine mucosa according to the present invention 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, and FIG. 1 (B) is a perspective view of the applied state. It is. FIG. 1C is a partially enlarged view of the distal end portion having the clamping surface of the sampling tool 2A. As shown in FIG. 1 (A), 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.

Both are close to each other by the force applied to the first sandwiching piece 3a and the second sandwiching piece 3b. For this reason, by applying force to the first clamping piece 3a and the second clamping piece 3b in a state where the first clamping surface 5a and the second clamping surface 5b of the sampling tool 2A are in contact with the large intestine mucosa, The large intestine mucosa can be clamped by the first clamping surface 5a and the second clamping surface 5b. More specifically, the edge 6a of the first clamping surface 5a and the edge 6b of the second clamping surface 5b are in contact with the large intestine mucosa sandwiched therebetween. In this state, by separating the collection tool 2A from the large intestine mucosa, the large intestine mucosa sandwiched between the first clamping surface 5a and the second clamping surface 5b is torn and collected.

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.

In order to collect the colonic mucosa with relatively little tissue damage, 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.

When the first sandwiching surface 5a and the second sandwiching surface 5b are cup-shaped, 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. In the case of 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. For example, by setting 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. In the case of a sawtooth shape, 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. When the edge 6a and the edge 6b are in contact with each other by applying force to the first holding piece 3a and the second holding piece 3b, the tip of the protrusion 8a fits into the tube 9a. When 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. FIG. 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. Specifically, as shown in FIG. 1D, a virtual plane in which the connecting portion 4 side from the center of the first holding piece 3a and the connecting portion 4 side from the center of the second holding piece 3b are placed. Bent to intersect P. The bending angle θ ₁ is preferably 10 to 50 °, more preferably 20 to 40 °, and even more preferably 25 to 35 °. Further, 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. When 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.

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, and FIG. 2 (B) is a plan view of the sampling tool 2C. It is. FIG. 2C is an enlarged view of the protruding portion 8b of the sampling tool 2C, and 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. FIG.

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. In this case, 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. (Fig. 2D). 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. It is preferable to adjust so that the 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. When 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)). By this buffer part, the sampling tool 2 can maintain the state which the latching claw of the projection part 8b latched to the overhang | projection part of the opening edge part of the cylinder part 9b more stably. Even when the first sandwiching piece 3a and the second sandwiching piece 3b are further applied, the elastic portion 10b at the tip is deformed by pressing, so that the first sandwiching surface 5a and the second sandwiching surface 5b are bonded. (FIG. 2E).

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, and FIG. 3B is a bottom view seen from the slit side of the collection assisting tool 11A. As shown in FIG. 3A, 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. From the viewpoint of ease of insertion into the anus, the outer diameter of the hand edge 16 is 30 to 70 mm, preferably 40 to 50 mm. Further, from the viewpoint of easy introduction of the collection tool 2 into the surface of the large intestine mucosa, the outer diameter of the distal edge portion 15 is preferably 10 to 30 mm, and more preferably 15 to 25 mm. Similarly, 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. For example, the slit 13 is preferably on the side close to the gripping portion 14 as shown in FIG. Moreover, the number of the slits 13 provided in the collection tool introduction part 12 may be one, or two or more.

Since the collection tool 2 passes through the slit 13 and reaches the surface of the large intestine mucosa, 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. 1 (B)) of the first clamping surface 5a and the second clamping surface 5b of the sampling tool 2 in a state where the edge portion 6a and the edge portion 6b are in contact with each other is 3. In the case of ˜8 mm, the width L ₂ (see FIG. 3B) of the slit 13 on the front edge portion 15 side is preferably 7 to 15 mm, and the width L _{3 of} the slit 13 on the hand edge portion 16 side (FIG. 3). (See (B)) is preferably 10 to 20 mm. In addition, two or more slits may be formed on the wall surface of the collection tool introduction part 12.

One end of the gripping part 14 is connected to the vicinity of the hand edge 16 of the collection tool introduction part 12 in a direction away from 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, and 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. As shown in the grip 14, 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. First, 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).

Next, the present invention will be described in more detail with reference to examples and the like, but the present invention is not limited thereto.

[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.

<Comprehensive analysis of methylation level of CpG site>
(1) Biopsy and DNA extraction Mucosal tissues were collected from three large intestines of the same patient, and formalin-fixed paraffin-embedded (FFPE) samples were prepared according to a conventional method. The collection sites were the cecum, rectum, and cancer for UC cancer patients, and the cecum, transverse colon, and rectum for non-cancer UC patients. A section was cut out from the FFPE sample, and DNA was extracted using a QIAmp DNA FFPE tissue kit (manufactured by Qiagen).

(2) Quality evaluation of DNA sample The concentration of the obtained DNA was determined as follows. Specifically, the fluorescence intensity of each sample was measured using Quant-iT PicoGreen dsDNA Assay Kit (manufactured by Life Technologies), and the concentration was calculated using the λ-DNA calibration curve attached to the kit.
Next, each sample was diluted to 1 ng / μL with TE (pH 8.0), and real-time PCR was performed using Illumina FFPE QC Kit (manufactured by Illumina) and Fast SYBR Green Master Mix (manufactured by Life Technologies). Ct value was determined. The difference in Ct value between the sample and the positive control (hereinafter referred to as ΔCt value) was calculated for each sample, and the quality was evaluated. Samples with a ΔCt value of less than 5 were judged to have good quality and proceeded to the subsequent steps.

(3) Bisulfite treatment Bisulfite treatment was performed on DNA samples using EZ DNA Methylation Kit (manufactured by ZYMO RESEARCH).

(4) Repair of degraded DNA and amplification of whole genome Degraded DNA was repaired by using Infinium HD FFPE Restore Kit (manufactured by Illumina) for the DNA after bisulfite treatment. The repaired DNA is alkali-denatured, neutralized, added with an enzyme and primers for whole genome amplification, and incubated at 37 ° C. Incubation Oven (manufactured by Illumina) for 20 hours or more at isothermal temperature. Was amplified.

(5) 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.

(6) 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.

(7) Labeling reaction and scanning The probe on the BeadChip after hybridization was subjected to an extension reaction to bind a fluorescent dye. Subsequently, the BeadChip was scanned with an iSCAN system (manufactured by Illumina), and methylated fluorescence intensity and unmethylated fluorescence intensity were measured. At the end of the experiment, it was confirmed that all the scan data was available and that the scan was performed normally.

(8) Quantification of DNA methylation level and comparative analysis Scan data was analyzed using DNA methylation analysis software GenomeStudio (Version: V2011.1). The DNA methylation level (β value) was calculated by the following formula.

[Β value] =
[Methylated fluorescence intensity] ÷ ([Methylated fluorescence intensity] + [Unmethylated fluorescence intensity] +100)

When the methylation level is high, the β value approaches 1 and when the methylation level is low, the β value approaches 0. DiffScore calculated by GenomeStudio was used for comparative analysis of the DNA methylation level of the UC cancer patient rectal sample group (n = 8) to the non-cancer UC patient rectal sample group (n = 8). When both DNA methylation levels are close, DiffScore approaches 0, showing a positive value when the level is high in UC cancer patients, and a negative value when low. The greater the difference in methylation level between the two groups, the greater the absolute value of DiffScore. In addition, a value (Δβ value) obtained by subtracting the average β value of the non-cancer UC patient rectal sample group (n = 8) from the average β value of the UC cancer patient rectal sample group (n = 8) was also used for the comparative analysis.

In addition, 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.

DNA methylation quantification;
Normalization: Yes (Controls)
Subtract Background: Yes Content Descriptor: HumanMethylation450_15017482_v.1.2.bpm

DNA methylation level comparative analysis;
Normalization: Yes (Controls)
Subtract Background: Yes Content Descriptor: HumanMethylation450_15017482_v.1.2.bpm
Ref Group: Comparative analysis 4. Group-3
Error Model: Illumina custom
Compute False Discovery Rate: None

(9) Multivariate analysis Using the results of quantitative and comparative analysis of DNA methylation level, statistical analysis software R (Version: 3.0.1, 64bit, Windows (registered trademark)), DiffScore calculation, cluster analysis And principal component analysis.

R script for cluster analysis:
> data.dist <-as.dist (1-cor (data.frame, use = "pairwise.complete.obs", method = "p"))
> hclust (data.dist, method = "complete")
# data.frame: Data frame consisting of CpG (rows) x samples (columns) # 1-Pearson correlation coefficient is defined as a distance and implemented by complete linkage method

R script for principal component analysis:
> prcomp (t (data.frame), scale = T)
# data.frame: data frame consisting of CpG (row) x sample (column)

<Selection of CpG biomarker>
(1) Extraction of CpG biomarker candidates As a means for selecting GpG biomarker candidates from comprehensive DNA methylation analysis data, narrowing based on DiffScore and Δβ values has been reported (BMC Med genomics vol.4, p. 50, 2011; Sex Dev vol.5, p.70, 2011). The former sets the absolute value of DiffScore to more than 30 and the absolute value of Δβ value to more than 0.2, and the latter sets the absolute value of DiffScore to more than 30 and the absolute value of Δβ value to more than 0.3. Biomarker candidates are extracted. In accordance with these methods, biomarker candidates were extracted from 485,577 CpG sites mounted on BeadChip.

Specifically, first, 72,905 CpG sites having an absolute DiffScore value of more than 30 were selected from 485,577 CpG sites. BeadChip also had 86 CpG sites located in each gene region of miR-1, miR-9, miR-124, miR-137, miR-34b / c described in Patent Document 1. However, of these, the absolute value of DiffScore was more than 30 and 27.
Next, 32 CpG sites having an absolute value of Δβ value exceeding 0.3 were extracted from the 72,905 CpG sites. Hereinafter, the 32 CpG sites are collectively referred to as a “32 CpG set”. At this point, all CpG sites located in each gene region described in Patent Document 1 have been excluded.
Furthermore, in order to discriminate cancer patients without missing, the cancer patient samples were narrowed down to those with little fluctuation in DNA methylation level. That is, the unbiased variance var of the β 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. Hereinafter, 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. Hereinafter, 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. In the table, CpG sites with # in the “16CpG” column indicate those included in the 16CpG set, and CpG sites with # in the “9CpG” column indicate those included in the 9CpG set.

(2) Multivariate analysis of clinical samples using CpG biomarker candidates Cluster analysis and principal component analysis of all 48 samples were performed using the 32CpG set, 16CpG set, and 9CpG set. As shown in 6E, in any CpG set, in the cluster analysis, all UC cancer patient samples were accumulated in the same cluster (in the frame in the figure). As shown in FIGS. 6B, 6D, and 6F, in the principal component analysis (vertical axis is the second principal component), the UC cancer patient sample (●) and the non-cancer UC patient sample (▲) are the first principal component ( Independent clusters were formed in the horizontal axis direction. That is, in any CpG set, 24 UC cancer patient samples and 24 non-cancer UC patient samples could be clearly distinguished. On the other hand, the cluster analysis and the principal component analysis were similarly performed using 27 CpG sites selected from the CpG sites located in each gene region described in Patent Document 1, and the results are shown in FIGS. 7A and 7B. Thus, a UC cancer patient sample and a non-cancer UC patient sample could not be clearly distinguished. From these results, 32CpG described in Table 9 is extremely useful as a biomarker of the onset of colorectal cancer in patients with ulcerative colitis, and by using these, the presence or absence of onset of colorectal cancer in patients with ulcerative colitis It is clear that can be determined by high sensitivity and specificity.

[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.

(1) Extraction of CpG biomarker candidates Next, CpG biomarker candidates were extracted from comprehensive DNA methylation analysis data.
Specifically, first, 324 CpG sites having an absolute value of Δβ value exceeding 0.2 were extracted from 485,577 CpG sites.

Next, the following two types of logistic regression models were created.
(1) Top 100 CpG sites were selected from 324 CpG sites by 2 groups of t-test tests, and 161,700 logistic regressions based on combinations of all 3 CpGs selected from 100 CpGs model.
(2) 52,326 logistic regression models based on all combinations of two CpGs selected from 324 CpG sites.

Regarding the discriminants of both logistic regression models, CpG sites that satisfy each of the following four criteria were selected, and the frequency of appearing CpG sites was calculated.
[Standard 1] Sensitivity is over 90%, specificity is over 90%, and coefficient p value of discriminant is less than 0.05.
[Standard 2] Sensitivity is more than 90%, specificity is more than 90%, discriminant coefficient p-value is less than 0.05, and AIC (Akaike's information criterion) is less than 30.
[Standard 3] Sensitivity is over 95%, specificity is over 85%, and coefficient p value of discriminant is less than 0.05.
[Standard 4] Sensitivity is over 95%, specificity is over 85%, discriminant coefficient p-value is less than 0.05, and AIC is less than 30.

When the top 10 CpG sites were selected for each of the four criteria, 34 CpG sites (34 CpG sets) listed in Tables 4 to 6 were selected. The results for each CpG site are shown in Table 10.

(2) Multivariate analysis of clinical samples using CpG biomarker candidates Based on the methylation level of the 34CpG set, cluster analysis and principal component analysis of all 48 samples were performed. As a result, in the cluster analysis (FIG. 8A), the majority of UC cancer patient samples accumulated in the same cluster (in the frame in the figure). In the principal component analysis (FIG. 8B, the vertical axis is the second principal component), the UC cancer patient sample (●) and the non-cancer UC patient sample (▲) are independent clusters in the first principal component (horizontal axis) direction. Formed. That is, in any CpG set, 24 UC cancer patient samples and 24 non-cancer UC patient samples could be clearly distinguished.

(3) Evaluation of the likelihood of developing colon cancer in clinical samples using CpG biomarker candidates.
Among the 34 CpG sets, a CpG site (cg10931190) in the base sequence represented by SEQ ID NO: 34, a CpG site (cg136767149) in the base sequence represented by SEQ ID NO: 37, and a base sequence represented by SEQ ID NO: 56 The accuracy of the determination of the presence or absence of colon cancer in ulcerative colitis patients was examined using the methylation rate of 3 CpG sites in the CpG site (cg14516100) as a marker.

Specifically, based on a logistic regression model using numerical values (β values) of methylation levels of the three CpG sites of specimens collected from rectums of 24 UC cancer patients and 24 non-cancer UC patients. A discriminant was created to discriminate between UC cancer patients and non-cancer UC patients. As a result, the sensitivity (the ratio of patients who were evaluated as positive among UC cancer patients) was 95.8%, and the specificity (the ratio of patients who were evaluated as negative among non-cancer UC patients) was 91.7%. The positive predictive value (the ratio of UC cancer patients out of patients evaluated as positive) was 92%, and the negative predictive value (the ratio of non-cancer UC patients out of patients evaluated as negative) was 95.6. %, Both of which were as high as 90% or more. FIG. 9 shows a ROC (Receiver-Operating-Characteristic) curve. The AUC (area under the ROC curve) was 0.98. Based on these results, it was confirmed that the possibility of developing colon cancer in ulcerative colitis patients can be evaluated with high sensitivity and specificity based on the methylation rate of several CpG sites selected from the 34CpG set. It was done.

[Example 3]
The DNA methylation level (β 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 (β value).

(1) Extraction of CpG biomarker candidates Specifically, 172 CpG sites having an absolute value of Δβ value exceeding 0.2 were first extracted from 485,577 CpG sites. Next, two types of logistic regression models were created from the 172 CpG sites in the same manner as in Example 2, and the top 10 CpG sites were selected for each of the four criteria. As a result, 18 CpG sites (18 CpG set) described in Table 7 and Table 8 were selected. The results for each CpG site are shown in Table 11.

(2) Multivariate analysis of clinical samples using CpG biomarker candidates Based on the methylation level of the 18CpG set, cluster analysis and principal component analysis of all 64 samples were performed. As a result, in the cluster analysis (FIG. 10A), the majority of UC cancer patient samples were accumulated in the same cluster (in the frame in the figure). In the principal component analysis (FIG. 10B, the vertical axis is the second principal component), the UC cancer patient sample (●) and the non-cancer UC patient sample (▲) are independent clusters in the first principal component (horizontal axis) direction. Formed. That is, in any CpG set, 32 samples of UC cancer patients and 32 samples of non-cancer UC patients could be clearly distinguished.

DESCRIPTION OF SYMBOLS 1 ... Colorectal mucosa sampling kit 2, 2A, 2B, 2C ... Collection tool, 3a ... 1st clamping piece, 3b ... 2nd clamping piece, 4 ... Connection part, 5 ... clamping surface, 5a ... 1st Clamping surface, 5b... 2nd clamping surface, 6a, 6a '.. edge portion of first clamping surface 5a, 6b, 6b' .. edge portion of second clamping surface 5b, 7a. 7b ... second bent portion, 8a, 8b ... projection portion, 9a, 9b ... cylindrical portion, 10a ... buffer portion, 10b ... elastic portion, 11, 11A, 11B ... collecting aid, 12 ... collecting device introduction portion, 13 ... slit, 14 ... gripping part, 15 ... tip edge part, 16 ... hand edge part.

Claims (27)

1. A method for determining the likelihood 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 Have
   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 of one or more CpG sites among the CpG sites in the base sequences represented by SEQ ID NOs: 1 to 80.
   A method for determining the likelihood of developing colorectal cancer.
2. The method for determining the likelihood of developing colorectal cancer according to claim 1, wherein in the measurement step, the methylation rate of 2 to 10 CpG sites is measured.
3. In the determination step, the bases represented by SEQ ID NOs: 1, 2, 11, 12, 14-18, 21-24, 26, 27, 29, 31, 45, 64, 65, 67, 77, 79, and 80 One or more of the CpG sites in the sequence have 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, 32 to 44 , 46-63, 66, 68-76, and 78, when one or more of the CpG sites in the base sequence have a methylation rate equal to or higher than a preset reference value, the human ulcer The method for determining the likelihood of developing colon cancer according to claim 1 or 2, wherein a patient with ulcerative colitis is determined to have a high possibility of developing colon cancer.
4. In the measurement step, the methylation rate of the CpG site in the base sequence represented by SEQ ID NOs: 1 to 32 is measured,
   In 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 4. The method according to claim 1, 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. The method for determining the likelihood of developing colorectal cancer according to one item.
5. In the determination step, a methylation rate is set in advance 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 that are equal to or less than the reference value and the methylation rate of CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10, 13, 19, 20, 25, 28, 30, and 32 are preliminarily determined. The human ulcerative colitis patient is determined to have a high possibility of developing colorectal cancer when the sum of the number of CpG sites equal to or greater than a set reference value is 3 or more. 5. The method for determining the likelihood of developing colorectal cancer according to any one of 4 above.
6. In the measurement step, the methylation rate of the CpG site in the base sequence represented by SEQ ID NOs: 1 to 16 is measured,
   In 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. Alternatively, 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 method for determining the likelihood of developing colorectal cancer according to any one of claims 1 to 3, wherein it is determined that there is a high possibility of developing colorectal cancer.
7. In the determination step, among the CpG sites in the base sequences represented by SEQ ID NOs: 1, 2, 11, 12, 14 to 16, the number of CpG sites having a methylation rate equal to or lower than a preset reference value; The human ulcer when the sum of the CpG sites in the base sequences represented by SEQ ID NOs: 3 to 10, 13 and the number of CpG sites having a methylation rate equal to or higher than a preset reference value is 3 or more The method for determining the likelihood of developing colorectal cancer according to any one of claims 1 to 3 and 6, wherein it is determined that a patient with ulcerative colitis has a high possibility of developing colorectal cancer.
8. In the measurement step, the methylation rate of CpG sites in the base sequences represented by SEQ ID NOs: 1 to 9 is measured,
   In 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 Possibility that the human ulcerative colitis patient develops colorectal cancer when one or more of the CpG sites in the represented base sequence has a methylation rate that is equal to or higher than a preset reference value The method for determining the likelihood of developing colorectal cancer according to any one of claims 1 to 3, wherein it is determined that the value is high.
9. In the determination step, among the CpG sites in the base sequences represented by SEQ ID NOs: 1 and 2, the number of CpG sites having a methylation rate equal to or lower than a preset reference value, and SEQ ID NOs: 3 to 9 When the sum of the number of CpG sites having a methylation rate equal to or higher than a preset reference value among CpG sites in the base sequence is 3 or more, the human ulcerative colitis patient develops colorectal cancer The method for determining the likelihood of developing colorectal cancer according to any one of claims 1 to 3 and 8, wherein it is determined that there is a high probability that the patient has a colorectal cancer.
10. In the measurement step, 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,
    In the determination step, at least one of the CpG sites in the base sequences represented by SEQ ID NOs: 45, 64, and 65 has a methylation rate equal to or lower than a preset reference value, or SEQ ID NO: 32 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 claims 1 to 3, wherein it is determined that there is a high possibility of developing colorectal cancer.
11. In the determination step, 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 SEQ ID NOs: 32-44 46 to 63 and 66, the sum of the number of CpG sites having a methylation rate equal to or higher than a preset reference value among CpG sites in the base sequences represented by 46 to 63 and 66 is 2 or more. The method for determining the possibility of developing colorectal cancer according to any one of claims 1 to 3 and 10, wherein it is determined that a patient with ulcerative colitis is highly likely to develop colorectal cancer.
12. In the measurement step, the methylation rate 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 is measured,
    In the determination step, 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 When 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 method for determining the likelihood of developing colorectal cancer according to any one of claims 1 to 3, wherein it is determined that a human ulcerative colitis patient is likely to develop colorectal cancer.
13. In the determination step, 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 SEQ ID NO: 33 , 35, 36, 43, 68 to 76, and 78, 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 sequence represented by 2 or more The method for determining the likelihood of developing colorectal cancer according to any one of claims 1 to 3 and 12, wherein the human ulcerative colitis patient is determined to have a high possibility of developing colorectal cancer.
14. The colorectal cancer according to claim 5, 7, 9, 11, or 13, wherein when the sum is 5 or more, it is determined that the human ulcerative colitis patient is likely to develop colorectal cancer. How to determine the likelihood of onset.
15. 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 determination method of the possibility of colon cancer onset of Claim 1 or 2 which determines with the possibility that the said human ulcerative colitis patient has developed colon cancer when it is above.
16. The multivariate discriminant is a variable of 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, 67-80. Including as
    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 determination method of the possibility of colon cancer onset of Claim 1 or 2 which determines with the possibility that the said human ulcerative colitis patient has developed colon cancer when it is above.
17. The multivariate discriminant is a logistic regression equation, a linear discriminant, a formula created with a naive Bayes classifier, or a formula created with a support vector machine. A method for determining the likelihood of developing colorectal cancer.
18. The method for determining the likelihood of developing colon cancer according to any one of claims 1 to 17, wherein the biological sample is intestinal tissue.
19. The method for determining the likelihood of developing colorectal cancer according to any one of claims 1 to 18, wherein the biological sample is a rectal mucosa tissue.
20. 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;
    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 of one end of the first clamping piece and the one end of the second clamping piece,
    The sample collected by the colonic mucosa collection kit having a larger outer diameter of the collection tool introduction part of 30 to 70 mm and a length in the rotation axis direction of 50 to 150 mm. A method for determining the likelihood of developing colorectal 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;
    21. The method for determining the likelihood of developing colorectal cancer according to claim 20, further comprising a second bent part on an end side where the second holding surface is formed with respect to a center part of the second holding piece.
22. A kit for collecting large intestine mucosa 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 of one end of the first clamping piece and the one end of the second clamping piece,
    A large intestine mucosa collection kit having a larger outer diameter of the collection tool introduction part of 30 to 70 mm and a length in the rotation axis direction of 50 to 150 mm.
23. 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;
    The kit for collecting large intestine mucosa according to claim 22, further comprising a second bent portion on an end 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 claim 22 or 23, wherein both the first clamping surface and the second clamping surface are cup-shaped.
25. The colonic mucosa collection kit according to any one of claims 22 to 24, wherein an inner diameter of the cup-shaped edge is 2 to 3 mm.
26. The colonic mucosa collection kit according to any one of claims 22 to 25, wherein the edge portions of the first clamping surface and the second clamping surface are serrated.
27. Onset of colorectal cancer in patients with ulcerative colitis, 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 DNA methylation rate analysis marker used to determine the possibility.

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