Method for assisting diagnosis of advanced adenoma in large intestine, and method for assisting diagnosis of either advanced adenoma in large intestine or colorectal cancer

Abstract

The present invention addresses the problem of providing a novel method capable of diagnosing advanced adenoma in the large intestine, or a novel method capable of diagnosing either advanced adenoma in the large intestine or colorectal cancer.　As one aspect, provided is a method for assisting the diagnosis of advanced adenoma in the large intestine in a subject, the method including (i) a step A for measuring a methylation level of CpG present in a region lying from +1 to +300 of a transcription start point in a somatostatin (SST) gene in DNA extracted from feces collected from the subject, (ii) a step B for measuring a quantitative level of a specific internal control gene in the DNA extracted from feces collected from the subject, and (iii) a step C for calculating a corrected methylation level by correcting the methylation level of CpG in the SST gene which has been measured in step A by the quantitative level of the specific internal control gene which has been measured in step B, wherein the assistance of the diagnosis is performed by comparing the corrected methylation level which has been calculated in step C with a predetermined cut-off value.

Description

Method for assisting in the diagnosis of advanced colon adenoma, and method for assisting in the diagnosis of either advanced colon adenoma or colon cancer

[0001] The present invention relates to a method for aiding in the diagnosis of advanced colon adenoma, and a method for aiding in the diagnosis of either advanced colon adenoma or colon cancer.

[0002] Colorectal cancer is a disease with high mortality and morbidity rates in Japan. In Japan, colorectal cancer was the leading cause of cancer incidence in 2019, with 155,625 cases, and the second leading cause of cancer death in 2021, with 52,418 cases. It is known that if colorectal cancer can be detected at the advanced adenoma, early stage, or precancerous lesion stage, curative surgery can be achieved in approximately 95% or more of cases, making early detection and treatment extremely important.

[0003] Fecal occult blood testing is an existing method for detecting colorectal tumors. While this test can detect colorectal cancer with high accuracy, it suffers from a low detection rate for advanced adenomas. Specifically, the sensitivity of fecal occult blood testing for colorectal cancer is 65.8%-73.8%, while the sensitivity for advanced adenomas is only 23.8%-27.1% (see Non-Patent Documents 1 and 2).

[0004] The Cologuard® kit is a test that claims to improve the diagnostic performance of advanced adenomas. This kit is a colorectal tumor testing kit that combines a fecal occult blood test with a fecal DNA extract test and was approved by the U.S. Food and Drug Administration (FDA) in 2014. The Cologuard DNA test involves KRAS gene mutations, methylation analysis of the BMP3 and NDRG4 genes, and measurement of the ACTB gene copy number. While the Cologuard test has a 92.3% sensitivity for colorectal cancer, its sensitivity for advanced adenomas is only 42.4% (see Non-Patent Document 2, cited above). Additionally, this test requires a stool sample (approximately 200 g) containing the entire volume of a single bowel movement. Japan lacks a testing system capable of handling such a large volume of stool samples. Considering the burden of hygiene and sample transportation, this test is difficult to implement as is. Furthermore, the large number of genes to be analyzed necessitates separate gene mutation and methylation analysis, resulting in high testing costs.

[0005] Meanwhile, the present inventors have previously developed a highly sensitive DNA methylation analysis technique and reported that the methylated Twist homolog 1 (TWIST1) gene is a marker for colorectal tumors (see Patent Document 1 and Non-Patent Document 3). Although a fecal DNA test targeting the methylated TWIST1 gene was combined with a fecal occult blood test, the false-positive rate in the control group (group without colorectal tumors) was somewhat high at 19.7%, leaving room for improvement (see Non-Patent Document 4). Furthermore, in Patent Document 1, there are six CpG sequences within the transcriptional regulatory region of TWIST1, and HhaI, HpaII, and BstUI were used as the methylation-sensitive restriction enzymes corresponding to each CpG to cleave each CpG. However, since cleavage tends to be incomplete when all restriction enzymes are used at once in methylation-sensitive restriction enzyme treatment, it was necessary to perform restriction enzyme treatment in two stages, adding restriction enzymes to increase the cleavage efficiency of the restriction enzymes and thereby improve the accuracy of methylation detection.

[0006] In addition, the present inventors have disclosed a method for predicting the presence or absence of colorectal tumors by detecting CpG methylation of the TWIST1, NDRG4, BMP3, or SEPT9 genes in stool or serum (see Patent Document 2), a method for diagnosing cancer by measuring the CpG methylation level in double-stranded DNA fragments derived from the RUNX3 gene (see Patent Document 3), and the usefulness of a combination of serum CpG methylated SEPT9 and AFP in diagnosing hepatocellular carcinoma (see Non-Patent Documents 5 and 6). Furthermore, the present inventors have disclosed a method for predicting the presence or absence of precancerous lesions or cancer based on the concentration or amount of double-stranded DNA in a biological sample or the copy number of the human telomerase reverse transcriptase (hTERT) gene in a biological sample (see Patent Document 4).

[0007] Other research groups have also reported elevated methylation levels of the somatostatin (SST) gene in colorectal cancer tissue (see Non-Patent Document 7). However, only colorectal cancer was the subject of verification, and advanced colorectal adenomas were not included in the verification, and methylated SST in feces was not verified at all. In addition, although the relationship between the SST gene and various cancers has been disclosed, advanced colorectal adenomas have not been investigated (see Non-Patent Document 8).

[0008] International Publication No. 2010 / 113529 Pamphlet International Publication No. 2017 / 043497 Pamphlet Japanese Patent Application Laid-Open No. 2020-014415 Japanese Patent Application Laid-Open No. 2019-106990

[0009] Morikawa, Tamiya et al. A comparison of the immunochemical fecal occult blood test and total colonoscopy in the asymptomatic population. Gastroenterology vol. 129,2 (2005): 422-8. doi:10.1016 / j.gastro.2005.05.05Imperiale, Thomas F et al. Multitarget stool DNA testing for colorectal-cancer screening. The New England journal of medicine vol. 370,14 (2014): 1287-97. doi:10.1056 / NEJMoa1311194Suehiro Y, Zhang Y, Hashimoto S, et al. Highly sensitive faecal DNA testing of TWIST1 methylation in combination with faecal immunochemical test for haemoglobin is a promising marker for detection of colorectal neoplasia. Ann Clin Biochem 2018; 55: 59-68.Suehiro Y, et al. Fecal DNA Testing of TWIST1 Methylation Identifies Patients with Advanced Colorectal Adenoma Missed by Fecal Immunochemical Test for Hemoglobin. Clinical and translational gastroenterology vol. 11,6 (2020): e00176. doi:10.14309 / ctg.0000000000000176Kotoh Y, Suehiro Y, Saeki I, et al.Novel Liquid Biopsy Test Based on a Sensitive Methylated SEPT9 Assay for Diagnosing Hepatocellular Carcinoma. Hepatol Commun 2020; 4: 461-470. Yamazaki A. et al., Hepatocellular Carcinoma Screening by Liquid Biopsy Using Highly Sensitive DNA Methylation Analysis Technology: Comparison of Diagnostic Performance Between Methylated SST and Methylated SEPT9, Yamaguchi Medical Journal, Vol. 70, No. 3, pp. 89-98, 2021. Mehdi Manoochehri, et al. SST Gene Hypermethylation Acts as a Pan-Cancer Marker for Pancreatic Ductal Adenocarcinoma and Multiple Other Tumors: Toward Its Use for Blood-Based Diagnosis. Mol Oncol 2020 Jun;14(6):1252-1267. doi: 10.1002 / 1878-0261.12684. Mori Y, et al. A Genome-Wide Search Identifies Epigenetic Silencing of Somatostatin, Tachykinin-1, and 5 Other Genes in Colon Cancer. GASTROENTEROLOGY 2006; 131:797-808.

[0010] If colon cancer can be discovered and treated early or at the precancerous stage, it can be almost completely cured. However, to date, no test method has been established that can specifically detect advanced colon adenomas, which have a high risk of cancer progression, with high accuracy. As described above, fecal occult blood testing is a common method for detecting colon cancer, but the problem with this test is that it has a low detection rate for advanced colon adenomas, which have a high risk of malignant progression. Therefore, an object of the present invention is to provide a new method for diagnosing advanced colon adenomas, or a new method for diagnosing either advanced colon adenomas or colon cancer.

[0011] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have discovered that correcting the methylation level of the SST gene in DNA extracted from stool with the quantitative level of an internal control gene is useful as a diagnostic aid for advanced colon adenoma. Furthermore, by statistically analyzing the value obtained by correcting the methylation level of the SST gene in DNA extracted from stool with the quantitative level of the internal control gene and the result of a fecal occult blood test as indicators, they have found that this is useful as a diagnostic aid for either advanced colon adenoma or colon cancer compared with the fecal occult blood test alone, and have completed the present invention.

[0012] That is, the present invention is as follows: [1] A method for assisting in the diagnosis of advanced colorectal adenoma in the subject, comprising: (i) a step A of measuring the methylation level of CpGs present in the region of +1 to +300 of the transcription start site of the somatostatin (SST) gene in DNA extracted from stool collected from the subject; (ii) a step B of measuring the quantitative level of a predetermined internal control gene in the DNA extracted from the stool collected from the subject; and (iii) a step C of calculating a corrected methylation level by correcting the methylation level of CpGs of the SST gene measured in step A with the quantitative level of the predetermined internal control gene measured in step B; wherein the corrected methylation level calculated in step C is compared with a predetermined cutoff value. [2] The method according to [1] above, wherein in step C, the corrected methylation level is calculated by dividing the methylation level of CpGs of the SST gene measured in step A by the quantitative level of the predetermined internal control gene measured in step B. [3] The method according to [1] or [2] above, wherein the quantitative level of the internal control gene is the copy number of the internal control gene. [4] The method according to any one of [1] to [3] above, wherein in step A, the methylation level of CpG present in the region +1 to +300 of the transcription start site of the SST gene is measured by treating the SST gene with at least one restriction enzyme selected from Hha I, Hpa II, Bst UI, BmgT120 I, Aat II, BssH II, Cfr10 I, Hae II, Hin1 I, Sac II, and VpaK11B I, and then amplifying the region containing the CpG recognized by the restriction enzyme. [5] The method according to any one of [1] to [4] above, wherein in step A, the cytosine of CpG present in the region of +1 to +300 of the transcription start site in the SST gene is the cytosine at position 187,670,340 or 187,670,342 of human chromosome 3 (Chr3). [6] The method according to any one of [1] to [5] above, wherein the subject is determined to be negative in a fecal occult blood test.[7] A method for assisting in the diagnosis of either advanced colorectal adenoma or colorectal cancer, comprising: (i) step A measuring the methylation level of CpG present in the region of +1 to +300 of the transcription start site of the somatostatin (SST) gene in DNA extracted from stool collected from the subject; (ii) step B measuring the quantitative level of a predetermined internal control gene in the DNA extracted from the stool collected from the subject; (iii) step C correcting the methylation level of CpG of the SST gene measured in step A by the quantitative level of the predetermined internal control gene measured in step B to calculate a corrected methylation level; and (iv) step D performing a fecal occult blood test and determining whether the result is positive or negative based on a predetermined cutoff value; wherein the method performs multivariate analysis using the methylation level measured in step A and / or the corrected methylation level calculated in step C and the result of the fecal occult blood test determined in step D as indicators. [8] The method according to [7] above, wherein in step C, a corrected methylation level is calculated by dividing the methylation level of CpG of the SST gene measured in step A by the quantitative level of a predetermined internal control gene measured in step B. [9] The method according to [7] or [8] above, wherein in step A, the methylation level of CpG present in the region of +1 to +300 of the transcription start site of the SST gene is measured by treating the region with at least one restriction enzyme selected from Hha I, Hpa II, Bst UI, BmgT120 I, Aat II, BssH II, Cfr10 I, Hae II, Hin1 I, Sac II, and VpaK11B I, and then amplifying the region containing the CpG recognized by the restriction enzyme.

[10] The method according to any one of [7] to [9] above, wherein the cytosine of CpG in the region of +1 to +300 of the transcription start site in the SST gene is the cytosine at position 187,670,340 or 187,670,342 of human chromosome 3 (Chr3).

[11] The method comprises: (i) step A of measuring the methylation level of CpG present in the region of +1 to +300 of the transcription start site of the somatostatin (SST) gene in DNA extracted from stool collected from the subject; (ii) step B of measuring the quantitative level of a predetermined internal control gene in the DNA extracted from stool collected from the subject; (iii) step C of calculating a corrected methylation level by correcting the methylation level of CpG of the SST gene measured in step A by the quantitative level of the predetermined internal control gene measured in step B; (iv) step D of performing a fecal occult blood test and determining whether the result is positive or negative based on a predetermined cutoff value; (v) step E of determining whether the methylation level measured in step A is positive or negative based on a predetermined cutoff value; and (vi) step F of determining whether the corrected methylation level calculated in step C is positive or negative based on a predetermined cutoff value; A method for assisting in the diagnosis that the subject has either advanced colorectal adenoma or colorectal cancer when at least one of the results of discrimination in step D and the results of discrimination in steps E and F is positive.

[12] The method according to

[11] above, characterized in that in step C, the methylation level of CpG of the SST gene measured in step A is divided by the quantitative level of a predetermined internal control gene measured in step B to calculate a corrected methylation level.

[0013] The present invention makes it possible to aid in the diagnosis of advanced colon adenomas, which are likely to be overlooked in colon cancer screening.

[0014] Figure 1 provides supplementary explanation of the corrected mSST values ​​used in the examples. Figure 2A shows the distribution of mSST values ​​in a control group, a non-advanced adenoma group, an advanced adenoma group, and a colorectal cancer group. Figure 2B shows the distribution of human TERT (hTERT) values ​​in a control group, a non-advanced adenoma group, an advanced adenoma group, and a colorectal cancer group. Figure 2C shows the distribution of corrected mSST values ​​in a control group, a non-advanced adenoma group, an advanced adenoma group, and a colorectal cancer group. Figure 3 shows the results of graphing the results of Table 2, divided into "control group," "non-advanced adenoma group," "advanced adenoma group," and "colorectal cancer group." Figure 4 shows the distribution of predicted values ​​based on formula (III) in a control group, a non-advanced adenoma group, and an advanced adenoma group, and the results of multiple comparisons between the predicted values ​​in each group using Dunn's test. Fig. 5 shows the distribution of predicted values ​​based on formula (IV) for a control group, a non-advanced adenoma group, an advanced adenoma group, and a colorectal cancer group, plotted, and further shows the results of a multiple comparison of the predicted values ​​between each group using Dunn's test. Fig. 6 shows the results of verifying the discriminatory ability of each prediction formula between the control group and the disease group using ROC analysis (control group (control group + non-advanced adenoma group) vs. disease group (advanced adenoma group + colorectal cancer group)).

[0015] The contents of all patent and non-patent literature cited herein are hereby incorporated by reference in their entirety.

[0016] The method of the present invention for assisting in the diagnosis of advanced colon adenoma comprises: (i) step A of measuring the methylation level of CpG present in the region of +1 to +300 of the transcription start site of the somatostatin (SST) gene in DNA extracted from stool collected from the subject; (ii) step B of measuring the quantitative level of a predetermined internal control gene in the DNA extracted from the stool collected from the subject; and (iii) step C of calculating a corrected methylation level by correcting the methylation level of CpG of the SST gene measured in step A by the quantitative level of the predetermined internal control gene measured in step B; and is not particularly limited as long as it is a method for assisting in the diagnosis of advanced colon adenoma in the subject by comparing the corrected methylation level with a predetermined cutoff value, and hereinafter this method will also be referred to as "the present method for assisting in the diagnosis of advanced colon adenoma."

[0017] Furthermore, a method A of the present invention for assisting in the diagnosis of either advanced colon adenoma or colon cancer comprises: (i) a step A of measuring the methylation level of CpG present in the region of +1 to +300 of the transcription start site of the somatostatin (SST) gene in DNA extracted from stool collected from a subject; (ii) a step B of measuring the quantitative level of a predetermined internal control gene in the DNA extracted from stool collected from the subject; (iii) a step C of calculating a corrected methylation level by correcting the methylation level of CpG of the SST gene measured in the step A by the quantitative level of the predetermined internal control gene measured in the step B; and (iv) a step D of performing a fecal occult blood test and determining whether the test is positive or negative based on a predetermined cutoff value. There are no particular limitations on the method as long as it is a method for assisting in the diagnosis of either advanced colon adenoma or colon cancer by performing multivariate analysis using as indicators the methylation level measured in step A and / or the corrected methylation level calculated in step C, and the results of the fecal occult blood test determined in step D, and it is hereinafter also referred to as "the present method A for assisting in the diagnosis of either advanced colon adenoma or colon cancer."

[0018] Furthermore, a method B of the present invention for assisting in the diagnosis of either advanced colon adenoma or colon cancer comprises: (i) a step A of measuring the methylation level of CpG present in the region of +1 to +300 of the transcription start site of the somatostatin (SST) gene in DNA extracted from stool collected from a subject; (ii) a step B of measuring the quantitative level of a predetermined internal control gene in the DNA extracted from stool collected from the subject; (iii) a step C of calculating a corrected methylation level by correcting the methylation level of CpG of the SST gene measured in step A by the quantitative level of the predetermined internal control gene measured in step B; (iv) a step D of performing a fecal occult blood test and determining whether the result is positive or negative based on a predetermined cutoff value; (v) a step E of determining whether the methylation level measured in step A is positive or negative based on a predetermined cutoff value; and (vi) a step F of determining whether the corrected methylation level calculated in step C is positive or negative based on a predetermined cutoff value. There are no particular limitations on the method as long as it assists in the diagnosis that the subject has either an advanced adenoma of the colon or colon cancer when at least one of the results determined in step D and the results determined in steps E and F is positive, and hereinafter this method is also referred to as "method B for assisting in the diagnosis of either an advanced adenoma of the colon or colon cancer in this case."

[0019] The subject herein may be a mammal such as a human, monkey, pig, dog, cat, horse, sheep, mouse, or rabbit, with a human being being preferred. Examples of such a subject include a subject whose status as to whether or not he or she has advanced colon adenoma or colon cancer is unknown, or a subject whose status as to whether or not he or she has advanced colon adenoma or colon cancer is unknown. Such subjects also include subjects who have previously suffered from advanced colon adenoma or colon cancer and have subsequently recovered from the advanced colon adenoma or colon cancer, but whose status as to whether or not they have advanced colon adenoma or colon cancer at the time of the test is unknown. Furthermore, the subject may be a subject who has tested negative in a fecal occult blood test.

[0020] As used herein, colorectal cancer refers to a malignant tumor that arises from the transformation of normal cells in the large intestine or the gastrointestinal tract immediately adjacent to it and has the potential for unlimited proliferation, invasiveness, and metastasis, including, for example, cecal cancer, colon cancer, and rectal cancer, as well as anal cancer. Advanced colon adenoma refers to a colon tumor with cellular morphological abnormalities or structural atypia, such as an adenoma with a diameter of 10 mm or more, an adenoma with histopathologically villous or tubulovillous components, or an adenoma with severe dysplasia. Benign colon tumors without the potential for unlimited proliferation, invasiveness, and metastasis, such as an adenoma with a diameter of less than 10 mm, adenoma without histopathologically villous or tubulovillous components, and adenoma without high-grade dysplasia, are considered non-advanced colon adenomas and are distinguished from advanced colon adenomas.

[0021] In the present method for assisting in the diagnosis of advanced colon adenoma, and in the present method A or B for assisting in the diagnosis of either advanced colon adenoma or colon cancer, the amount of stool is sufficient as long as it is in an amount that allows the DNA methylation level to be measured, and this amount can be 0.05 to 0.5 g, preferably 0.1 to 0.3 g.

[0022] Methods for extracting DNA from stool include phenol extraction, phenol-chloroform extraction, alkaline dissolution, and methods using commercially available DNA extraction reagents.

[0023] As used herein, the transcription start point refers to the site where transcription of mRNA starts, and corresponds to the position corresponding to the first base of mRNA. In this specification, the transcription start point is represented as "+1," and the position one base upstream of the transcription start point is represented as "-1." Therefore, +300 of the transcription start point means a position 299 bases downstream when the transcription start point is represented as +1.

[0024] In the present specification, the transcription start site of the somatostatin (SST) gene is located at position 187,670,394 on human chromosome 3 according to location information from the location information database (GRCh38 / hg38), and the region from +1 to +300 of the transcription start site of the SST gene is located at positions 187,670,095 to 187,670,394 on human chromosome 3 according to location information from GRCh38 / hg38. The region from +1 to +300 of the transcription start site of the SST gene may be a region that is highly methylated (CpG island), and may be the region from +3 to +280, +7 to +268, +10 to +150, +20 to +100, +40 to +90, or +50 to +60 of the transcription start site of the SST gene. The region from +7 to +268 of the transcription initiation site in the SST gene corresponds to positions 187,670,127 to 187,670,388 of human chromosome 3 shown in SEQ ID NO:1. Furthermore, the cytosine of CpG in the region of +1 to +300 from the transcription start site in the SST gene is preferably a cytosine of CpG recognized by at least one restriction enzyme selected from Hha I, Hpa II, Bst UI, BmgT120 I, Aat II, BssH II, Cfr10 I, Hae II, Hin1 I, Sac II, and VpaK11B I, and more preferably a cytosine at position 187,670,340 (+55 from the transcription start site) or position 187,670,342 (+53 from the transcription start site) (position 214 or 216 in SEQ ID NO: 1) on human chromosome 3. CpG islands can be identified, for example, at the following website (URL: genome.ucsc.edu / cgi-bin / hgc?hgsid=1793514212_1Ch0xAYmu91SbzU4CgcDI4uMtzZ2&db=hg38&c=chr3&l=187668840&r=187671840&o=187670126&t=187670388&g=cpgIslandExt&i=CpG%3A+25). The coding sequence for the SST gene is read from 5' to 3' on the complementary strand of genomic DNA.

[0025] As used herein, CpG methylation refers to a state in which a methyl group is attached to the carbon atom at the 5th position of the cytosine (C) residue in a CpG sequence, which is a two-base sequence (dinucleotide) in which a guanine (G) base appears next to a cytosine (C) base in DNA. Here, the "p" represents the phosphodiester bond between cytosine and guanine.

[0026] The CpG methylation level herein can be evaluated based on the number of DNA fragments in which cytosines in the CpGs are methylated (copy number) when a predetermined region of DNA encoding the SST gene is amplified to obtain DNA fragments. Specifically, when evaluation is performed using a predetermined restriction enzyme treatment, examples of the copy number include the number of copies per unit amount of restriction enzyme solution (e.g., per 1 μL), the number of copies per total amount of restriction enzyme-treated solution, and the number of copies per unit amount of DNA source sample (e.g., per 1 g of stool).

[0027] As used herein, the method for measuring the methylation level of CpG is not particularly limited, but examples include a method in which a DNA fragment is treated with a methylation-sensitive restriction enzyme, followed by amplifying a region containing CpG recognized by the restriction enzyme. Examples of methylation-sensitive restriction enzymes include at least one restriction enzyme selected from Hha I (GCG / C), Hpa II (C / CGG), Bst UI (also called BshI1236I or AccII depending on the manufacturer) (CG / CG), BmgT120 I (GG / NCC), Aat II (GACGT / C), BssH II (G / CGCGC), Cfr10 I (R / CCGGY), Hae II (RGCGC / Y), Hin1 I (GR / CGYC), Sac II (CCGC / GG), and VpaK11B I (G / GWCC(G)), and two or more types may be used in combination. Note that the above "N" means A, G, T, or C, "R" means A or G, "Y" means C or T, and "W" means A or T. The methylation-sensitive restriction enzyme is a methylation-sensitive restriction enzyme that can cleave any of the CpGs present in the region of +1 to +300 of the transcription start site in the SST gene.

[0028] Furthermore, when treating with the methylation-sensitive restriction enzyme, if single-stranded DNA is present in the template DNA, the single-stranded DNA will escape cleavage by the restriction enzyme and will be amplified in the subsequent PCR reaction, resulting in false positives. To avoid this, it is preferable to add exonuclease I (ExoI) to remove the single-stranded DNA. Furthermore, when treating with the methylation-sensitive restriction enzyme, from the viewpoint of improving the efficiency of the methylation-sensitive restriction enzyme reaction and the PCR amplification, the DNA may be fragmented by including a restriction enzyme that cannot cleave the base sequence in the amplification target region, including CpG.

[0029] When performing restriction enzyme treatment, the temperature and time can be adjusted appropriately depending on the optimal pH of the restriction enzyme used. Furthermore, as described in the application by the present inventors (Japanese Patent Application No. 2023-043439), the second stage of treatment may be performed by changing the temperature conditions without releasing the sealed state, such as by opening the lid of the reaction vessel after the first stage of enzyme treatment, or as described in Patent Document 2 above, after the first stage of treatment, the lid of the reaction vessel may be opened and a restriction enzyme may be added, and then the temperature conditions may be changed to perform the second stage of enzyme treatment.

[0030] The region containing the CpG recognized by the restriction enzyme can be amplified by known DNA amplification methods using restriction enzyme-treated DNA as a template, including PCR methods such as digital PCR (polymerase chain reaction) and real-time PCR, and loop-mediated isothermal amplification (LAMP). The length of the sequence to be amplified is 20 to 300 bases, preferably 30 to 200 bases, and more preferably 50 to 150 bases.

[0031] The primers for amplifying the region containing CpG recognized by the restriction enzyme and the probe for detecting the amplified nucleic acid can be appropriately selected depending on the methylation-sensitive restriction enzyme used. A primer set, probe, or labeled product thereof for individually measuring the methylation of each cytosine of CpG recognized by the restriction enzyme, which is contained in part or all of the region from +1 to +300 of the transcription start site in the SST gene, may be used. Alternatively, a primer set, probe, or labeled product thereof for commonly measuring the methylation of all regions containing CpG recognized by the restriction enzyme may be used.

[0032] The primers may amplify a region containing a CpG recognized by a methylation-sensitive restriction enzyme used to measure the methylation level of the CpG, and the probe may recognize a base sequence within the region containing the CpG. When multiple methylation-sensitive restriction enzyme treatments are used, all of the CpGs recognized by the methylation-sensitive restriction enzymes may be amplified with a single primer set, or a primer set recognizing each CpG may be used individually to amplify each region containing the CpG.

[0033] When Hha I and Bst UI are used as the methylation-sensitive restriction enzymes and 104 bp from −27 to +77 from the transcription start site is amplified as the PCR amplification region of methylated SST, preferred examples of the primers include a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2, or a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 2 in which one or several bases have been substituted, deleted, inserted, or added; a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3, or a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 3 in which one or several bases have been substituted, deleted, inserted, or added; a probe consisting of the nucleotide sequence shown in SEQ ID NO: 4, or a probe consisting of the nucleotide sequence shown in SEQ ID NO: 4 in which one or several bases have been substituted, deleted, inserted, or added; and labeled versions thereof. The above-mentioned "base sequence in which one or several bases have been substituted, deleted, inserted, or added" means a base sequence in which any number of bases have been substituted, deleted, inserted, or added, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2, and most preferably 1.

[0034] The above primer set can be used to amplify a sequence containing both the cytosine at position 187,670,340 (+55 from the transcription start site of the SST gene) on human chromosome 3 recognized by Hha I and the cytosine at position 187,670,342 (+53 from the transcription start site of the SST gene) recognized by Bst UI. Furthermore, the probe shown in SEQ ID NO: 4 can be used to recognize DNA in which both the cytosine at position 187,670,340 (+55 from the transcription start site of the SST gene) on human chromosome 3 recognized by Hha I and the cytosine at position 187,670,342 (+53 from the transcription start site of the SST gene) recognized by Bst UI are methylated.

[0035] Examples of the labeling substances for the forward primer label, the reverse primer label, and the probe label include enzymes such as peroxidase (e.g., horseradish peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase, and acetylcholinesterase; fluorescent substances such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride, and tetramethylrhodamine isothiocyanate; green fluorescent protein (GFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (Red Fluorescent Protein), and the like. Examples of such fluorescent reporters include fluorescent proteins such as radioisotopes (RFP), luciferase, and the like; radioisotopes such as 3H, 14C, 125I, and 131I; biotin, avidin, or chemiluminescent substances; and combinations of reporter fluorescent substances and quencher fluorescent substances or structures (for example, a combination of 5-FAM [5-carboxyfluorescein] and 5-TAMRA [5-carboxytetramethylrhodamine], a combination of VIC and MGB [minor groove binder], and a combination of 5-FAM and MGB).

[0036] The above-mentioned "measuring the CpG methylation level" may not only measure the CpG methylation in the region of +1 to +300 of the transcription start site in the amplified SST gene, but also the CpG methylation in the corresponding region of the complementary strand.

[0037] In the above-mentioned method for measuring CpG methylation levels, it is preferable not to perform sodium bisulfite treatment (bisulfite treatment). Bisulfite treatment treats single-stranded DNA, which easily degrades the DNA, resulting in the loss of approximately 90% of the DNA due to denaturation. Therefore, to achieve the detection limit of approximately 10 copies in quantitative PCR, 100 or more copies of target DNA are required in the DNA before bisulfite treatment. When quantitatively analyzing absolute copy numbers using quantitative PCR, a calibration curve using a standard substance must be created, but this poses the problem of extremely low reliability of measurements at low copy numbers. On the other hand, digital PCR does not require the creation of a calibration curve, and accurate absolute number measurements are possible even at low copy numbers.

[0038] The internal control gene used herein may be any gene present in two copies per cell, such as the human TERT gene, the human RNase P gene, etc. Examples of the quantitative level of the internal control gene include the copy number of the internal control gene, the Tt value when amplified by the LAMP method, and the cycle threshold value (Ct value, Cq value) in quantitative PCR.

[0039] The corrected methylation level herein can be determined by correcting the methylation level of the CpG of the SST gene measured in step A above with the quantitative level of a predetermined internal control gene measured in step B above. The correction may be any correction that determines the methylation level of the CpG of the SST gene measured in step A relative to the quantitative level of the predetermined internal control gene. Preferably, the corrected methylation level can be determined by dividing the methylation level of the CpG of the SST gene measured in step A above by the quantitative level of the predetermined internal control gene measured in step B above. Although the methylated SST value is affected by the amount and properties of the stool at the time of collection, correcting, preferably dividing, by the quantitative level of the internal control gene makes it possible to evaluate stool samples of any type and amount (e.g., watery stool, solid stool) (see Figure 1 below).

[0040] The method for measuring the quantitative level of the internal control gene is not particularly limited, and examples include a method of amplifying a partial region of the internal control gene. It is preferable to perform the amplification of the partial region of the internal control gene in the same reaction vessel as the measurement of the CpG methylation level, from the viewpoints of preventing a decrease in test accuracy due to differences in the amount of DNA in the vessel and improving work efficiency. Furthermore, when measuring the quantitative level of the internal control gene in the same reaction vessel as the measurement of the CpG methylation level as described above, the partial region of the internal control gene to be amplified can be a region that does not contain a base sequence recognized by a methylation-sensitive restriction enzyme contained in the reaction vessel.

[0041] When the quantitative level of the hTERT gene is measured as an internal control gene in the same reaction vessel as that for measuring the CpG methylation level, and when the reaction vessel contains Hha I and Bst UI as methylation-sensitive restriction enzymes, preferred primers used to amplify a partial region of hTERT include a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 6, or a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 6 in which one or several bases have been substituted, deleted, inserted, or added; a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 7, or a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 7 in which one or several bases have been substituted, deleted, inserted, or added; a probe consisting of the nucleotide sequence shown in SEQ ID NO: 8, or a probe consisting of the nucleotide sequence shown in SEQ ID NO: 8 in which one or several bases have been substituted, deleted, inserted, or added; and labeled versions thereof.

[0042] The predetermined cutoff value in the present method for assisting in the diagnosis of advanced colorectal adenoma can be set to increase sensitivity and specificity. For example, the predetermined cutoff value can be calculated by creating a receiver operating characteristic (ROC) curve using statistical analysis software based on the "corrected methylation level calculated in step C in DNA extracted from stool collected from patients with advanced colorectal adenoma" and the "corrected methylation level calculated in step C in DNA extracted from stool collected from control patients (no tumors detected in colonoscopy), non-advanced colorectal adenoma, and / or colorectal cancer patients, preferably from control patients, non-advanced colorectal adenoma, and colorectal cancer patients," and calculating the cutoff value using the Youden Index. Alternatively, the predetermined cutoff value can be calculated based on the mean value, the mean value ± standard deviation (SD), the mean value ± 2SD, the mean value ± 3SD, the median, the 25th percentile of the median, and the 75th percentile of the median for each methylation level.

[0043] The predetermined cutoff value for the fecal occult blood test used in step D in method A or method B for assisting in the diagnosis of either advanced colon adenoma or colon cancer can be set so as to increase sensitivity and specificity. For example, an ROC curve can be created using statistical analysis software based on "the hemoglobin concentration (ng / mL) in a stool specimen obtained by a fecal occult blood test for either advanced colon adenoma or colon cancer, or for each patient" and "the hemoglobin concentration (ng / mL) in a stool specimen obtained by a fecal occult blood test for either a control or non-advanced colon adenoma, or for each patient," and calculation can be performed using the Youden Index, or the cutoff value can be calculated based on the mean value of the hemoglobin concentration for each patient, the mean value ± standard deviation (SD), the mean value ± 2SD, the mean value ± 3SD, the median, the 25% percentile of the median, and the 75% percentile. If the value is equal to or greater than the cutoff value, it can be determined that the test is positive for "either advanced colon adenoma or colon cancer," and if the value is less than the cutoff value, it can be determined that the test is negative for "either advanced colon adenoma or colon cancer."

[0044] The cutoff value for the fecal occult blood test in Method A or B for assisting in the diagnosis of either advanced colon adenoma or colon cancer can also be set based on previously reported literature values. Specific examples include hemoglobin concentrations of 100 ng / ml and 50 ng / ml.

[0045] The predetermined cutoff value in step E of method B for assisting in the diagnosis of either advanced colon adenoma or colon cancer can be set to increase sensitivity and specificity. For example, an ROC curve can be created using statistical analysis software based on "the CpG methylation level measured in step A in DNA extracted from stool collected from either advanced colon adenoma or colon cancer, or from each patient" and "the CpG methylation level measured in step A in DNA extracted from stool collected from either a control or non-advanced colon adenoma, or from each patient," and calculation can be performed using the Youden Index. Alternatively, calculation can be performed based on the mean value, the mean value ± standard deviation (SD), the mean value ± 2SD, the mean value ± 3SD, the median, the 25% percentile value of the median, and the 75% percentile value of each methylation level. A value equal to or greater than the cutoff value can be determined as positive for "either advanced colon adenoma or colon cancer," while a value below the cutoff value can be determined as negative for "either advanced colon adenoma or colon cancer."

[0046] Furthermore, the predetermined cutoff value in step F of method B for assisting in the diagnosis of either advanced colon adenoma or colon cancer can be set so as to increase sensitivity and specificity. For example, an ROC curve can be created using statistical analysis software based on "the corrected methylation level calculated in step C in DNA extracted from stool collected from either advanced colon adenoma or colon cancer, or from stool collected from each patient" and "the corrected methylation level calculated in step C in DNA extracted from stool collected from either a control or non-advanced colon adenoma, or from each patient," and calculation can be performed using the Youden Index. Alternatively, calculation can be performed based on the mean value, the mean value ± standard deviation (SD), the mean value ± 2SD, the mean value ± 3SD, the median, the 25% percentile value of the median, and the 75% percentile value of each corrected methylation level. A value equal to or greater than the cutoff value can be determined as positive for "either advanced colon adenoma or colon cancer," while a value below the cutoff value can be determined as negative for "either advanced colon adenoma or colon cancer."

[0047] In this specification, examples of multivariate analysis include logistic regression analysis, linear discriminant analysis, and multiple regression analysis. Furthermore, for continuous variables, the Mann-Whitney U test can be used for two-group testing, the Kruskal-Wallis test and Dunn's test can be used for multiple comparisons, and the chi-square test and Fisher's exact test can be used for testing categorical variables. The indices used in the multivariate analysis are not particularly limited as long as they include the methylation level measured in step A and / or the corrected methylation level calculated in step C, and a fecal occult blood test, and may also include age and gender. Furthermore, the methylation level measured in step A and / or the corrected methylation level calculated in step C may be logarithmically or power-transformed to obtain a normally distributed value, or a binarized value based on a predetermined cutoff value or a predetermined rule. The "methylation level measured in the step A and / or the corrected methylation level calculated in the step C, and the result of the fecal occult blood test discriminated in the step D" may be the methylation level measured in the step A and the result of the fecal occult blood test discriminated in the step D, the corrected methylation level calculated in the step C and the result of the fecal occult blood test discriminated in the step D, or the methylation level measured in the step A, the corrected methylation level calculated in the step C, and the result of the fecal occult blood test discriminated in the step D. The fecal occult blood test may use a continuous variable or a ranked value such as a binary or ternary value. The methylation level (mSST value) may be determined to be mSST positive when it is equal to or higher than a predetermined cutoff value, and the corrected mSST may be determined to be corrected mSST positive when it is equal to or higher than a predetermined cutoff value. Hereinafter, the value obtained by binarizing either the mSST value or the corrected mSST value, or both, based on the above-mentioned specified cutoff value, with 1 indicating positive and 0 indicating negative, will also be referred to as the "binarized mSST value + corrected mSST value."

[0048] When performing logistic regression analysis as the multivariate analysis, for example, a prediction model formula for calculating a predicted value (P) is created using logistic regression analysis with the presence or absence of a disease as the dependent variable and at least two independent variables: the corrected mSST value, or the binarized mSST value + corrected mSST value, and a fecal occult blood test. The usefulness of this prediction model formula for assisting in the diagnosis of either advanced colon adenoma or colon cancer is then verified. Next, for the prediction model formula determined to be effective as a result of the verification, the values ​​of each variable are substituted for the independent variable terms used to create the model formula to calculate a predicted value, and the diagnosis of the presence or absence of a disease can be aided based on the predicted value. Specifically, when the predicted value is calculated based on the corrected methylation level calculated in step C, the fecal occult blood test result, age, and gender, a prediction model formula is created based on the following formula (I) by logistic regression analysis using the presence or absence of either advanced colon adenoma or colon cancer as the dependent variable and the corrected mSST value calculated in step C, the fecal occult blood test result, age, and gender as the independent variables. Then, the area under the curve (AUC) value obtained by ROC curve analysis using the predicted value derived from the model formula is used to verify whether the prediction model formula is useful for assisting in the diagnosis of either advanced colon adenoma or colon cancer. Next, the corrected mSST value, fecal occult blood test result, age, and gender obtained from the subject can be substituted into the prediction model formula determined to be effective as a result of the verification to calculate the predicted value p. When the predicted value p is calculated based on the binarized mSST value + corrected mSST value, fecal occult blood test, and age, a prediction model formula can be created based on, for example, formula (II) instead of formula (I), and the predicted value p can be calculated based on that prediction model formula.

[0049]

[0050] In the above formula (I), "β0" is the intercept (constant term), "β1 to β4" are regression coefficients, the calculated value can be substituted for "corrected mSST value", 1 can be substituted for positive and 0 can be substituted for negative for "fecal occult blood test", age can be substituted for "age", and male can be substituted for 1 and female can be substituted for gender.

[0051]

[0052] In the above formula (II), "β0" is the intercept (constant term), "β1 to β3" are regression coefficients, "(mSST value + corrected mSST value)" is the binarized mSST value + corrected mSST value, and either or both of the mSST value or the corrected mSST value are assigned 1 if positive and 0 if negative, respectively. For "fecal occult blood test," 1 is assigned if positive and 0 is assigned if negative, respectively. For "age," age can be assigned.

[0053] In diagnosing advanced colon adenoma in the present method for assisting in the diagnosis of advanced colon adenoma, the methylation level of the CpG of the SST gene measured in step A is corrected, preferably divided, by the quantitative level of a predetermined internal control gene measured in step B to calculate a corrected methylation level, which is then compared with a predetermined cutoff value. If the corrected methylation level is equal to or greater than the predetermined cutoff value, the subject can be assessed as having a high probability of having advanced colon adenoma, and if the corrected methylation level is less than the predetermined cutoff value, the subject can be assessed as having a low probability of having advanced colon adenoma.

[0054] In the present method A for assisting in the diagnosis of either an advanced colon adenoma or colon cancer, the closer the value (predicted value) obtained by the multivariate analysis is to 1, the higher the possibility that the patient is "afflicted with either an advanced colon adenoma or colon cancer," while the closer the predicted value is to 0, the lower the possibility that the patient is "afflicted with either an advanced colon adenoma or colon cancer." Therefore, it is possible to diagnose whether the patient is afflicted with either an advanced colon adenoma or colon cancer based on the value (predicted value).

[0055] Furthermore, a cutoff value for determining whether or not a subject is "afflicted with either advanced colon adenoma or colon cancer" is calculated based on the predicted value calculated from the prediction model formula created based on the above formula (I) or (II). If the predicted value is higher than the cutoff value, the subject can be evaluated as having a high possibility of being affected by either advanced colon adenoma or colon cancer, and if the predicted value is equal to or lower than the cutoff value, the subject can be evaluated as having a low possibility of being affected by either advanced colon adenoma or colon cancer. Furthermore, a cutoff value X for the predicted value for distinguishing between control and non-advanced adenoma and advanced adenoma is calculated based on the predicted values ​​calculated from the above formulas (I) and (II), and a cutoff value Y for distinguishing between advanced adenoma and colorectal cancer is calculated based on the predicted values ​​for the advanced adenoma group and the colorectal cancer group, respectively. In this way, if the predicted value calculated from the above formula (I) or (II) for a subject is less than the cutoff value X, it can be assessed that there is a high possibility that the subject has either a control or a non-advanced adenoma, or that there is a high possibility that the subject is not suffering from either an advanced adenoma or colorectal cancer; if the predicted value is equal to or greater than the cutoff value X and less than the cutoff value Y, it can be assessed that there is a high possibility that the subject is suffering from advanced adenoma; and if the predicted value is equal to or greater than the cutoff value Y, it can be assessed that there is a high possibility that the subject is suffering from colorectal cancer. The cutoff value can be calculated, for example, by creating an ROC curve using statistical analysis software based on the predicted values ​​calculated by formula (I) or (II) based on the values ​​of each independent variable of each patient in the control group, non-advanced adenoma group, advanced adenoma group, and colorectal cancer group, and then calculating it using the Youden Index, or by calculating it based on the mean value of the predicted values ​​based on each group, the mean value ± standard deviation (SD), the mean value ± 2SD, the mean value ± 3SD, the median, the 25% percentile value of the median, or the 75% percentile value. In other words, by setting the cutoff values ​​X and Y in Method A for assisting in the diagnosis of either advanced colon adenoma or colorectal cancer, it can also be made into a "method for assisting in the diagnosis of advanced colon adenoma."

[0056] In the present method B for assisting in the diagnosis of either advanced colon adenoma or colon cancer, when at least one of the "result of the fecal occult blood test determined in step D" and the "methylation level of CpG of the SST gene determined in step E and / or the corrected methylation level calculated in step F" is positive, the method can assist in the diagnosis that the subject has either advanced colon adenoma or colon cancer. On the other hand, when both the "result of the fecal occult blood test determined in step D" and the "methylation level of CpG of the SST gene determined in step E and / or the corrected methylation level calculated in step F" are negative, the method can assist in the diagnosis that the subject does not have either advanced colon adenoma or colon cancer.

[0057] In the present method B for assisting in the diagnosis of either an advanced adenoma of the colon or colon cancer, if the results of the discrimination in step D and the results of the discrimination in steps E and F are both positive, the method may assist in the diagnosis that the subject has either an advanced adenoma of the colon or colon cancer.

[0058] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0059] The control group consisted of 79 patients who had no tumors detected during colonoscopy. There were 43 patients with non-advanced colon adenomas, 117 patients with advanced colon adenomas, and 126 patients with colon cancer (stage I-IV according to the 8th edition of the TNM classification (UICC)). Stool samples for fecal occult blood testing were collected using an S stool collection container (Eiken Chemical Co., Ltd.), and stool samples for methylation analysis of DNA extracted from the stool (stool DNA testing) were collected using a stool collection cup. In the following examples, advanced colon adenomas may be simply referred to as "advanced adenomas," and non-advanced colon adenomas may be simply referred to as "non-advanced adenomas."

[0060] (1) Fecal occult blood test. Stool samples collected in an S stool collection container were used, and the hemoglobin concentration in the stool samples was measured using OC-Hemodia (registered trademark) Auto S 'Eiken' (Eiken Chemical Co., Ltd.) as the measurement reagent and OC Sensor io (registered trademark) (Eiken Chemical Co., Ltd.) as the measurement device. The cutoff value for the fecal occult blood test was set to 100 ng / mL, a commonly used hemoglobin concentration, and values ​​above the cutoff value were determined to be positive for the fecal occult blood test (reference for fecal occult blood cutoff: Ministry of Health, Labor and Welfare website www.mhlw.go.jp / shingi / 2007 / 06 / dl / s0626-13i_0002.pdf (accessed October 2, 2023)).

[0061] (2) Stool DNA Testing Approximately 200 mg of stool was collected from the stool sample in the collection cup, and DNA was extracted using a QIAamp Fast DNA stool mini kit (Qiagen) according to the protocol provided with the kit. The copy number of methylated SST of the target gene and the copy number of the hTERT gene as an internal control gene were measured using the CORD assay (combined restriction enzymes and digital PCR; see Non-Patent Document 3 and Patent Application No. 2023-043439), an improved version of a highly sensitive methylation analysis method developed by the present inventors. This CORD assay involves adding a methylation-sensitive restriction enzyme to stool-extracted DNA and incubating it. This cleaves unmethylated DNA, while methylated DNA is spared. Digital PCR amplifies only uncleaved methylated DNA, allowing methylation to be analyzed by measuring its copy number.

[0062] The following is an improved method for the highly sensitive methylation analysis described above. 1. Place 10 μL of DNA sample in a 1.5 mL tube. 2. Add 1 μL of AmpliTaq Gold buffer II (AmpliTaq Gold DNA Polymerase Kit: Thermo Fisher Scientific). 3. Add 25 mM MgCl. ２3. Add 1 μL of solution (N808-0241: AmpliTaq Gold DNA Polymerase Kit: Thermo Fisher Scientific). 4. Add 1 μL (10 U) of HhaI (Thermo Fisher Scientific). 5. Add 1 μL (10 U) of HpaII (Thermo Fisher Scientific). 6. Add 1 μL (20 U) of ExoI (Thermo Fisher Scientific). 7. Add 1 μL (10 U) of BstUI (New England BioLabs). 8. Mix by pipetting and then close the tube lid to seal. 9. Heat at 37°C for 1 hour. 10. Heat at 60°C for 16 hours. 11. Heat at 98°C for 10 minutes.

[0063] The HhaI and BstUI are methylation-sensitive restriction enzymes that recognize and cleave the base sequences 5'-GCGC-3' and 5'-CGCG-3', respectively. Because the methylated base sequences remain uncleaved, they can be amplified by PCR. This makes it possible to analyze the methylation of cytosines at positions 187,670,340 (+55 from the transcription start point) and 187,670,342 (+53 from the transcription start point) on human chromosome 3. HpaII was used to fragment DNA.

[0064] The digital PCR system used was the QX200 Droplet Digital PCR System (Bio-Rad).

[0065] After the enzyme reaction was completed, 2 μL of the enzyme-reacted DNA solution treated under the above conditions, 8 μL of water, and 12 μL of PCR reaction solution were mixed to prepare a DNA-containing PCR reaction solution. This PCR reaction solution consisted of 2 μL of a primer-probe mix containing 10 μM forward primer, 10 μM reverse primer, and 10 μM probe for amplifying a predetermined region of the SST gene and measuring a predetermined methylated cytosine level, and 5 μM forward primer, 5 μM reverse primer, and 2.5 μM probe for amplifying a predetermined region of the hTERT gene, and 10 μL of 2×ddPCR Master Mix (Bio-Rad). Multiplex PCR was performed using the above DNA-containing PCR reaction solution.

[0066] The nucleotide sequences of the primers for amplifying human chromosome 3 187,670,318-187,670,421 and the probes for detecting positions 187,670,340 (+55 from the transcription start site) and 187,670,342 (+53 from the transcription start site) on human chromosome 3 are as follows. The nucleotide sequence shown in SEQ ID NO: 5 is amplified by the above primers. The probe used was a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 4, with the fluorescent substance FAM linked to the 5' end and the quencher BHQ1 linked to the 3' end. Forward primer: 5'-GAGTGGCTGGTCAAACTCTA-3' (SEQ ID NO: 2) Reverse primer: 5'-AAAACAGAGGGAGACGGTTG-3' (SEQ ID NO: 3) Probe: 5'-FAM-TGCTGCCTGCTGATCCGCGCC-BHQ1-3' (SEQ ID NO: 4)

[0067] The base sequences of the primers for amplifying the human TERT gene as an internal control gene and the probe for detecting it are as follows. The base sequence shown in SEQ ID NO: 9 is amplified by the above primers. The probe used was a polynucleotide consisting of the base sequence shown in SEQ ID NO: 8, to which the fluorescent substance VIC was linked at the 5' end and the quencher MGB was linked at the 3' end. The amplified base sequence shown in SEQ ID NO: 9 does not contain base sequences recognized by the methylation-sensitive restriction enzymes HhaI, BstUI, and HpaII. Forward primer: 5'-GGGTCCTCGCCTGTGTACAG-3' (SEQ ID NO: 6) Reverse primer: 5'-CCTGGGAGCTCTGGGAATTT-3' (SEQ ID NO: 7) Probe: 5'-VIC-CACACCTTTGGTCACTC-MGB-3' (SEQ ID NO: 8)

[0068] Table 1 shows the base sequences of the primers, probes, and PCR amplification products. In Table 1, the bold "C" in the base listed in the column for the base sequence of the PCR amplification product in SST in the lower section indicates the cytosine at position 187,670,340 (+55 from the transcription start site) on human chromosome 3, which is a site that can be cleaved (GCG / C) by the restriction enzyme HhaI, or the cytosine at position 187,670,342 (+53 from the transcription start site), which is a site that can be cleaved (CG / CG) by BstUI. However, because these restriction enzymes are methylation-sensitive restriction enzymes, if the cytosine (C) is methylated, that site will not be cleaved.

[0069]

[0070] From this DNA-containing PCR reaction solution, an Automated Droplet Generator (Bio-Rad) was used to prepare droplets (droplets), and using a thermal cycler, the mixture was preheated at 95 ° C. for 10 minutes, followed by 40 cycles of thermal denaturation at 94 ° C. for 30 seconds and annealing at 56 ° C. for 60 seconds to amplify the DNA, and finally heated at 98 ° C. for 10 minutes. After DNA amplification, the fluorescent dye derived from the TaqMan probe in each droplet was detected using a QX200 Droplet Reader (Bio-Rad) and QuantaSoft software (Bio-Rad), and the number of DNA fragments in which cytosine is methylated in CpG in the amplified region of the SST gene (copy number) was measured, and the methylated SST copy number (mSST value) was calculated. Furthermore, the mSST value was divided by the hTERT copy number to calculate the value (methylated SST copy number / hTERT copy number: hereinafter also referred to as "corrected mSST value").

[0071] (3) Statistical Analysis Using Fecal Occult Blood Test and Fecal DNA Test as Indicators In multivariate analysis, statistical analysis was performed on the relationship between the measured or calculated indices (mSST value, corrected mSST value, binarized mSST + corrected mSST, binarized fecal occult blood test) and the subjects' clinicopathological background. GraphPad Prism ver. 9 (GraphPad Software) and StatFlex ver. 7 (Artec) were used for the analysis. For continuous variables, the Mann-Whitney U test was used to test two groups, and the Kruskal-Wallis test and Dunn's test were used for multiple comparisons. Chi-square test and Fisher's exact test were used to test categorical variables. Multiple logistic regression analysis was used for multivariate analysis. For each test marker, ROC analysis was performed to compare the diagnostic performance of the "advanced adenoma group" or "either the advanced adenoma group or the colorectal cancer group" with that of the control group. The area under curve (AUC) was calculated based on the ROC curve. Statistical significance was determined at p<0.05. Furthermore, a cutoff value was set based on the measurement value at an arbitrary specificity, and the sensitivity at that time was calculated.

[0072] (i) Univariate analysis The diagnostic performance (sensitivity, specificity) of fecal occult blood tests, mSST values, and corrected mSST values ​​for "advanced adenoma" or "either advanced adenoma or colorectal cancer" was examined for both single and combined tests. The cutoff values ​​were set as the measurement values ​​at the 95th percentile of specificity (mSST value cutoff: 67.9, corrected mSST value cutoff: 0.50). A supplementary explanation of the corrected mSST values ​​is provided in Figure 1.

[0073] Next, the distribution of mSST values ​​is shown in Figure 2A, the distribution of hTERT values ​​in Figure 2B, and the distribution of corrected mSST values ​​in Figure 2C for the control, non-advanced adenoma, advanced adenoma, and colorectal cancer (stages I-IV) groups. To determine whether there were differences in mSST and corrected mSST values ​​between groups, a Dunn's multiple comparison test was performed. The advanced adenoma group had significantly higher mSST values ​​than the control group only (Figure 2A). On the other hand, the advanced adenoma group had significantly higher corrected mSST values ​​than the control, non-advanced adenoma, and colorectal cancer groups (Figure 2C). These results demonstrate that corrected mSST is a biomarker specific to advanced adenoma. The horizontal bars in Figures 2A, 2B, and 2C represent the median values ​​for each group.

[0074] Furthermore, the sensitivity and specificity of each group were verified under the three conditions of "fecal occult blood test alone," "fecal DNA test alone," and "combined test of fecal DNA test and fecal occult blood test." The results are shown in Table 2.

[0075]

[0076] (i)-1 Fecal occult blood test alone The test sensitivity was 7.0% for the non-advanced adenoma group, 29.1% for the advanced adenoma group, 91.3% for the colorectal cancer group, and 61.3% for the advanced adenoma group + colorectal cancer group. The specificity was 87.3% when the control group alone was used as the control group, and 89.3% when the control group and the non-advanced adenoma group (control group + non-advanced adenoma group) were used as the control groups.

[0077] (i)-2 Fecal DNA Testing Alone For fecal DNA testing, the cutoff value was defined as the measurement value with 95% specificity to reduce the number of false positives. Specifically, the cutoff values ​​for the mSST and corrected mSST were set at 67.9 and 0.50, respectively. A positive result was determined for the fecal DNA test when either the mSST or corrected mSST was equal to or greater than the cutoff value, or when both were equal to or greater than the cutoff value. The test sensitivity was 14.0% for the non-advanced adenoma group, 39.3% for the advanced adenoma group, 30.2% for the colorectal cancer group, and 34.6% for the advanced adenoma and colorectal cancer groups. The specificity was 92.4% when the control group alone was used as the control group, and 90.2% when the control group and non-advanced adenoma group were used as the control groups.

[0078] (i)-3 Combination Test of Fecal DNA Test and Fecal Occult Blood Test A positive result for either the fecal occult blood test or the fecal DNA test, or a positive result for both the fecal occult blood test and the fecal DNA test, was considered a "combined test positive." The test sensitivity was 20.9% for the non-advanced adenoma group, 61.5% for the advanced adenoma group, 94.4% for the colorectal cancer group, and 78.6% for the advanced adenoma group and colorectal cancer group. The specificity was 79.7% when the control group alone was used as the control group, and 79.5% when the control group and the non-advanced adenoma group were used as the control groups. The test sensitivity for advanced adenoma was 61.5% when the fecal DNA test and the fecal occult blood test were combined, significantly improving the test sensitivity compared to 29.1% for the fecal occult blood test alone shown in (i)-1 (Table 2).

[0079] Based on the results in Table 2, the subjects were divided into a "control group," a "non-advanced adenoma group," a "advanced adenoma group," and a "colon cancer group," and the results were graphed and shown in Figure 3. Figure 3 also confirmed that the fecal DNA test can detect the advanced adenoma group with high sensitivity. It was also revealed that when at least one of the fecal occult blood test and the DNA test is positive, there is a high possibility that the subject is either an advanced adenoma of the colon or colon cancer, and when both the fecal occult blood test and the fecal DNA test are positive ("both tests positive" in Figure 3), there is an extremely high possibility that the subject is either an advanced adenoma or colon cancer.

[0080] (ii) Multivariate Analysis To identify factors that independently influence the presence or absence of disease, a disease prediction model was created by multiple logistic regression analysis using the presence or absence of disease as the dependent variable and mSST value, corrected mSST value, fecal occult blood test, age, and gender as independent variables. Next, an ROC curve analysis was performed using the predicted values ​​derived from the model. The area under the curve (AUC) value obtained by ROC curve analysis was used to verify whether this prediction model is useful for assisting in the diagnosis of advanced adenoma. Furthermore, the test sensitivity and specificity for advanced adenoma and colorectal cancer (hereinafter also referred to as "advanced adenoma + colorectal cancer") were verified. The creation and verification of the above model are specifically described below.

[0081] First, a multiple logistic regression analysis was performed using the presence or absence of disease (control + non-advanced adenoma vs. advanced adenoma) as the dependent variable and the adjusted mSST value, fecal occult blood test, age, and gender as independent variables. For statistical processing, the adjusted mSST value was calculated for qualitative data, and the fecal occult blood test and gender were dichotomized (fecal occult blood test: positive = 1, negative = 0; gender: male = 1, female = 0). The results of the multiple logistic regression analysis revealed that the adjusted mSST value, fecal occult blood test, age, and gender were independent predictors of advanced adenoma. Next, a prediction model formula combining these independent variables was derived as shown in the following formula (III):

[0082]

[0083] The corrected mSST values, fecal occult blood test results, age, and sex for each of the control group, non-advanced adenoma group, and advanced adenoma group were substituted into formula (III) to calculate predicted values. The distribution of the predicted values ​​was plotted, and the results of multiple comparisons between the predicted values ​​of each group using Dunn's test are shown in Figure 4. As shown in Figure 4, the advanced adenoma group had significantly higher predicted values ​​than the control group and the non-advanced adenoma group. In Figure 4, the horizontal bars for each group represent the median. The p-value (predicted value) calculated using formula (III) ranges from 0 to 1, with values ​​closer to 1 indicating a higher likelihood of advanced adenoma, and values ​​closer to 0 indicating a higher likelihood of the control group (control (tumor-free) or non-advanced adenoma).

[0084] The results in FIG. 4 reveal that there are significant differences in the predicted values ​​calculated by the above formula (III) between the control group vs. the advanced adenoma group and between the non-advanced adenoma group vs. the advanced adenoma group.

[0085] Next, a prediction model equation was created for the qualitative data by binarizing the mSST value + corrected mSST value. Separately, a multiple logistic regression analysis was performed with the presence or absence of disease (control + non-advanced adenoma vs. advanced adenoma + colorectal cancer) as the dependent variable, and the independent variables were the binarized mSST value + corrected mSST value (mSST value + corrected mSST value), fecal occult blood test, and age. For statistical processing, the qualitative data were binarized as (mSST value + corrected mSST value) (positive = 1, negative = 0) and fecal occult blood test (positive = 1, negative = 0). The results of the multiple logistic regression analysis revealed that (mSST value + corrected mSST value), fecal occult blood test, and age were independent predictors of advanced adenoma. A prediction model equation combining these independent variables was then derived, as shown in Equation (IV) below.

[0086]

[0087] Next, the predicted values ​​were calculated by substituting the (mSST value + corrected mSST value), fecal occult blood test, and age for each of the control group, non-advanced adenoma group, advanced adenoma group, and colorectal cancer group into the above formula (IV). The distribution of the predicted values ​​was plotted, and the results of multiple comparisons between the groups based on formula (IV) using Dunn's test are shown in Figure 5. In Figure 5, the horizontal bar for each group represents the median. Note that the p-value (predicted value) calculated using formula (IV) ranges from 0 to 1, with values ​​closer to 1 indicating a higher likelihood of advanced adenoma or colorectal cancer, and values ​​closer to 0 indicating a higher likelihood of the control group (no tumor or non-advanced adenoma).

[0088] 5, the predicted values ​​calculated by the above formula (IV) were significantly higher in the advanced adenoma group and the colon cancer group than in the control group and the non-advanced adenoma group. Specifically, it was revealed that there were significant differences in the predicted values ​​between the control group vs. the advanced adenoma group, and between the non-advanced adenoma group vs. the advanced adenoma group. It was also revealed that there were significant differences in the predicted values ​​between the control group vs. the colon cancer group, the non-advanced adenoma group vs. the colon cancer group, and the advanced adenoma group vs. the colon cancer group.

[0089] Next, ROC analysis (control group (control group + non-advanced adenoma group) vs. disease group (advanced adenoma group + colorectal cancer group)) was used to verify the ability of the prediction formulas (III) and (IV) above to distinguish between the control group and the disease group. Two combinations of independent variables were used: Formula (III): (a) corrected mSST value + fecal occult blood test + age + sex Formula (IV): (b) (mSST value + corrected mSST value) + fecal occult blood test + age. The results are shown in Figure 6.

[0090] The AUC by ROC analysis was 0.8909 for pattern (a) and 0.9019 for pattern (b), both of which were high values.

[0091] Based on the results of Figures 4 to 6, it has become clear that the p-value (predicted value) calculated by formula (III) or formula (IV) can assist in the diagnosis of either advanced colon adenoma or colon cancer. In addition, as described above, there was a significant difference in the predicted values ​​calculated by formula (IV) between the advanced adenoma group and the colon cancer group. Therefore, by calculating a cutoff value X for the predicted value for distinguishing between control and non-advanced adenoma and advanced adenoma based on the predicted values ​​of the control (tumor-free) group, the non-advanced adenoma group, and the advanced adenoma group, and by calculating a cutoff value Y for distinguishing between advanced adenoma and colon cancer based on the predicted values ​​of the advanced adenoma group and the colon cancer group, it becomes possible to distinguish between "control or non-advanced adenoma," "advanced adenoma," or "colon cancer" once the predicted value of the subject based on formula (IV) is known. That is, it has become clear that if the predicted value based on the above formula (IV) of a subject is less than the above cutoff value X, it can assist in the diagnosis of either a control or non-advanced adenoma; if it is equal to or greater than the above cutoff value X and less than the above cutoff value Y, it can assist in the diagnosis of advanced adenoma; and if it is equal to or greater than the above cutoff value Y, it can assist in the diagnosis of colorectal cancer.

[0092] Furthermore, Table 3A shows the test sensitivity for advanced adenoma when cutoff values ​​were set to achieve specificity of 95%, 90%, 85%, and 80% by ROC analysis, and Table 3B shows the test sensitivity for advanced adenoma and colorectal cancer when cutoff values ​​were set to achieve specificity of 95%, 90%, 85%, and 80%. The test sensitivity for advanced adenoma was 42.7%, 56.4%, 61.5%, and 68.4%, respectively. The test sensitivity for advanced adenoma + colorectal cancer was 61.7%, 72.0%, 75.7%, and 81.9%, respectively. Note that for the results of Table 3A, a prediction model formula was derived using the presence or absence of disease (control + non-advanced adenoma vs. advanced adenoma) as the dependent variable and the same independent variable as that used to derive formula (IV), and the values ​​of each variable were substituted into the independent variable terms of the prediction model formula to calculate the predicted value and determine the test sensitivity. In addition, for the results in Table 3B, the dependent variable was the presence or absence of disease (control + non-advanced adenoma vs. advanced adenoma + colorectal cancer), and the value of each variable was substituted into the independent variable term of the model formula (IV) to calculate a predicted value and determine the test sensitivity.

[0093]

[0094]

[0095] Furthermore, when the cutoff value is set at a specificity of 90%, the test sensitivity and AUC values ​​for advanced adenoma are shown in Table 3C, and the test sensitivity and AUC values ​​for advanced adenoma and colorectal cancer are shown in Table 3D. Here, for the results on the left side of Table 3C, the dependent variable is the presence or absence of disease (control + non-advanced adenoma vs. advanced adenoma), and the values ​​of each variable are substituted into the independent variable terms of the model formula (III) to calculate the predicted value and determine the test sensitivity. Also, for the results on the right side of Table 3C, the dependent variable is the presence or absence of disease (control + non-advanced adenoma vs. advanced adenoma), and the independent variables are the same as those used to derive formula (IV), to derive a prediction model formula, and the values ​​of each variable are substituted into the independent variable terms of the prediction model formula to calculate the predicted value and determine the test sensitivity. Furthermore, for the results on the left side of Table 3D, a prediction model formula was derived using the presence or absence of disease (control + non-advanced adenoma vs. advanced adenoma + colorectal cancer) as the dependent variable and the same independent variables as those used to derive formula (III), and the values ​​of each variable were substituted into the independent variable terms of the prediction model formula to calculate a predicted value and determine the test sensitivity.Furthermore, for the results on the right side of Table 3D, the dependent variable was used the presence or absence of disease (control + non-advanced adenoma vs. advanced adenoma + colorectal cancer) as the dependent variable, and the values ​​of each variable were substituted into the independent variable terms of the formula (IV) model formula to calculate a predicted value and determine the test sensitivity.

[0096]

[0097]

[0098] In the combined test of univariate analysis (i)-3, when the control group + non-advanced adenoma group was used as the control group, the specificity was 79.5%, and the sensitivity for advanced adenoma + colorectal cancer was 78.6% (Table 2). However, when the above prediction model formula was used for multivariate analysis, as mentioned above, the sensitivity at a specificity of 80% was 81.9%, and a 3.3% improvement in test sensitivity was observed without a decrease in specificity (Table 3B).

[0099] Furthermore, the results of a comparison of the sensitivity and specificity of the method of the present invention with that of conventional fecal occult blood tests and Cologuard are shown in Table 4. In Table 4, the sensitivity and specificity with Cologuard are listed based on the data described in Non-Patent Document 2. In addition, in Table 4, "binary determination of fecal occult blood test + binary determination of fecal DNA test (combination)" lists the combined results in Table 2. Furthermore, in Table 4, for "binary determination of fecal occult blood test + binary determination of fecal DNA test (multivariate analysis)," the sensitivity and specificity were determined by performing ROC analysis using the predicted value calculated based on the above formula (IV).

[0100]

[0101] Table 4 confirms that the fecal DNA test using fecal occult blood test + mSST has higher sensitivity for detecting advanced adenomas than conventional techniques. Furthermore, efficient detection of advanced adenomas can contribute to the selection of people who truly need detailed examinations such as colonoscopy.

Claims

1. Step A of measuring the methylation level of CpG in the region of +1 to +300 of the transcription start point in the somatostatin (SST) gene in DNA extracted from feces collected from a subject; Step B of measuring the quantitative level of a predetermined internal control gene in the DNA extracted from the feces collected from the subject; Step C of calculating a corrected methylation level by correcting the methylation level of CpG in the SST gene measured in Step A with the quantitative level of the predetermined internal control gene measured in Step B; A method for assisting in the diagnosis of advanced adenoma of the large intestine in the subject by comparing the corrected methylation level calculated in Step C with a predetermined cut-off value.

2. The method according to claim 1, wherein in Step C, the corrected methylation level is calculated by dividing the methylation level of CpG in the SST gene measured in Step A by the quantitative level of the predetermined internal control gene measured in Step B.

3. The method according to claim 1 or 2, wherein the quantitative level of the internal control gene is the copy number of the internal control gene.

4. The method according to any one of claims 1 to 3, wherein in Step A, the methylation level of CpG in the region of +1 to +300 of the transcription start point in the SST gene is measured by treating with at least one restriction enzyme selected from Hha I, Hpa II, Bst UI, BmgT120 I, Aat II, BssH II, Cfr10 I, Hae II, Hin1 I, Sac II, and VpaK11B I, and then amplifying the region containing CpG recognized by the restriction enzyme.

5. The method according to any one of claims 1 to 4, wherein in Step A, the cytosine of CpG in the region of +1 to +300 of the transcription start point in the SST gene is the cytosine at position 187,670,340 or 187,670,342 of human chromosome 3 (Chr3).

6. The method according to any one of claims 1 to 5, wherein the subject is a subject determined to be negative in the fecal occult blood test.

7. (i) Step A of measuring the methylation level of CpG in the region from +1 to +300 of the transcription start point in the somatostatin (SST) gene in DNA extracted from feces collected from a subject; (ii) Step B of measuring the quantitative level of a predetermined internal control gene in the DNA extracted from the feces collected from the subject; (iii) Step C of calculating a corrected methylation level by correcting the methylation level of CpG in the SST gene measured in Step A with the quantitative level of the predetermined internal control gene measured in Step B; (iv) Step D of performing a fecal occult blood test and discriminating whether it is positive or negative based on a predetermined cut-off value; and performing multivariate analysis using, as an index, the methylation level measured in Step A and / or the corrected methylation level calculated in Step C and the result of the fecal occult blood test discriminated in Step D to assist in the diagnosis of either advanced adenoma of the large intestine or colorectal cancer.

8. The method according to claim 7, wherein in Step C, the corrected methylation level is calculated by dividing the methylation level of CpG in the SST gene measured in Step A by the quantitative level of the predetermined internal control gene measured in Step B.

9. The method according to claim 7 or 8, wherein in Step A, the methylation level of CpG in the region from +1 to +300 of the transcription start point in the SST gene is measured by treating with at least one restriction enzyme selected from Hha I, Hpa II, Bst UI, BmgT120 I, Aat II, BssH II, Cfr10 I, Hae II, Hin1 I, Sac II, and VpaK11B I, and then amplifying the region containing CpG recognized by the restriction enzyme.

10. The method according to any one of claims 7 to 9, wherein the cytosine of CpG in the region from +1 to +300 of the transcription start point in the SST gene is the cytosine at position 187,670,340 or 187,670,342 of human chromosome 3 (Chr3).

11. Step A: measuring the methylation level of CpG in the region of +1 to +300 of the transcription start point in the somatostatin (SST) gene in DNA extracted from feces collected from a subject; Step B: measuring the quantitative level of a predetermined internal control gene in the DNA extracted from the feces collected from the subject; Step C: calculating a corrected methylation level by correcting the methylation level of CpG of the SST gene measured in Step A with the quantitative level of the predetermined internal control gene measured in Step B; Step D: performing a fecal occult blood test and determining whether it is positive or negative based on a predetermined cut-off value; Step E: determining whether the methylation level measured in Step A is positive or negative based on a predetermined cut-off value; Step F: determining whether the corrected methylation level calculated in Step C is positive or negative based on a predetermined cut-off value; and a method for assisting in the diagnosis that when at least one of the results determined in Step D and the results determined in Step E and Step F is positive, the subject has either advanced adenoma of the large intestine or colorectal cancer.

12. The method according to claim 11, wherein in Step C, the corrected methylation level is calculated by dividing the methylation level of CpG of the SST gene measured in Step A by the quantitative level of the predetermined internal control gene measured in Step B.

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