JP6897970B2 - How to check for colorectal tumors - Google Patents

Description

The present invention relates to a method for inspecting the presence or absence of a colorectal tumor using a biological sample other than the affected area (tissue suspected to be colorectal cancer or precancerous lesion) of a test subject, and a kit for carrying out such a method.

According to the "Vital Statistics" conducted annually by the Ministry of Health, Labor and Welfare, malignant neoplasms (cancer) account for the leading cause of death in Japanese, followed by heart disease and pneumonia. According to the 2013 statistical results, the number of deaths from cancer exceeds 360,000 a year, and almost one in three people die from cancer.

According to the Ministry of Health, Labor and Welfare, the number of deaths from cancer could increase to more than about 450,000 by 2020. Looking at the cancer mortality rate (for a population of 100,000) by organ, colorectal cancer (total of colon cancer and rectal cancer) is the second worst after gastric cancer, and by gender, gastric cancer among men. Next to the worst, it is the worst, surpassing gastric cancer in women, and the worst, and the significance of colorectal cancer in the cause of death is also important.

The morbidity and mortality of colorectal cancer tend to increase, and when these rates are viewed by age, both men and women begin to increase in their 50s and tend to increase with increasing age. It is thought that the increase in colorectal cancer is related to the aging of the population and the westernization of eating habits. According to the "White Paper on Cancer and Statistics," the proportion (prevalence) of people with colorectal cancer from 2015 to 2019 was 254,900 for men and 184,800 for women, depending on the colorectal cancer. It is estimated that 25,800 men and 21,900 women will die.

Colorectal tumors that occur in the cecum, colon, and rectum are concepts that include benign tumors, colorectal adenomas, and colorectal cancers that are highly malignant and may have metastases. Among them, colorectal cancers are currently 2 in Japan. It is the second most prevalent cancer disease and is known to be caused by mutations in mucosal epithelial cells on the inner surface of the gastrointestinal tract. The main cause of canceration of mucosal epithelial cells is considered to be mutations in genomic DNA that control cell proliferation, and other environmental factors (risk factors), heredity, etc. are known as factors that affect canceration.

The progression of colorectal tumors is that colorectal adenomas grow in normal colorectal tissues, new colorectal adenomas in precancerous state become cancerous, and cancer cells that have metastasized from the cancerous part grow in non-cancerous colorectal tissue. This includes the formation of new colorectal cancer tissue.

Colorectal cancer often has no subjective symptoms even if it progresses, but it is almost completely cured at an early stage, so it is important to detect it at an early stage without subjective symptoms.

Rarely, there is an autosomal dominant inheritance disorder called familial adenomatous polyposis (FAP). The causative gene of this disease is the APC gene, and in the case of this disease, polyps containing a large number of tumors gradually develop in the large intestine, eventually resulting in colorectal cancer. Polyps begin to develop around the age of 10 and then increase in number and size over time. Colorectal cancer develops from this polyp. It has been found that cancer develops from around 15 years old, and that 50% of patients at 40 years old and almost 100% of patients at 60 years old develop colorectal cancer. The treatment method is basically to completely remove the large intestine mucosa before the age of 20, but since there are no specific symptoms, it is often not noticed, and by the time it is noticed, it will already be in a fatal state. ..

If there is a FAP patient in the family, it is necessary to carry out regular examinations from an early stage so that the diagnosis and treatment can be performed as early as possible. On the other hand, in the case of sporadic cases, since there are no cases in the family, there is no need for regular examinations, and even if a polyp occurs from around the age of 10, it may be fatal without being noticed. Therefore, early detection is very important. For that purpose, it is strongly desired to develop a simpler and more sensitive test method so that it can be found even in a general medical examination.

Thus, like many cancers, early detection of colorectal cancer is of utmost importance for its treatment, and many testing methods have been developed so far. The main test methods include fecal occult blood test, blood test, rectal finger examination, and diagnosis using colonoscopy, but the commonly used fecal occult blood test has a problem in early cancer detection sensitivity, and blood test (Tumor marker test contained in blood) also has the problem that it is positive only for advanced cancer, the problem that rectal finger examination can only be diagnosed within the reach of the finger, and the diagnosis by colonoscopy is also difficult to see. In each test method, there is a fundamental problem regarding the detection sensitivity especially for early cancer, such as the problem that the detection sensitivity is lowered in early stage cancer.

At the initial stage of this cancer, we focus on the multi-stage theory of canceration, in which mutations first occur in the genomic DNA of cells, and then various events including multiple genetic changes accumulate, and the cancer gradually becomes malignant and becomes cancerous. As a result, mutations in DNA and RNA, which is an expression product, and among them, DNA methylation is used as an index for canceration, which is a method for detecting cancer.

Patent Document 1 discloses a human DNA sequence related to canceration, and Patent Document 2 describes a method for detecting colorectal cancer using mutations in the APC, K-ras and p53 genes as indicators. Section 3 discloses a method for detecting gastrointestinal cancer using methylation of the APC and / or DCC gene as an index, but any of these methods has problems in terms of detection sensitivity, specificity, cost, and the like. The current situation is that it has not been clinically put into practical use.

The TWIST1 gene, which is one of the genes of interest in the present invention, is a gene isolated as a human homologue of the transcriptional regulator TWIST involved in morphogenesis of the fruit fly Drosophila melanogaster, and is a gene of H-TWIST and TWIST homolog of Drosophila. It is a gene that is also called by name. Its entity is a Basic helix-loop-helix (bHLH) type transcription factor, which is generally thought to be involved in cell lineage and differentiation during development. Mutations in the TWIST1 gene itself (coding region) are known to cause Seathre-Chotzen syndrome, and in recent years it has been suggested that they are involved in the acquisition of taxol resistance in nasopharyngeal cancer cells.

On the other hand, in studies in breast cancer patients, etc., high methylation was shown in various cancer-related genes such as Twist using methylation-sensitive PCR or methylation-specific PCR (MSP), Q-PCR method, etc. (non-methylation). Patent Documents 1 and 2) and the like have been reported, and methylation regulators selected from DNA methylation inhibitors, demethylating agents, and DNA methyltransferase activity antagonists are abnormal, including standard or malignant cells. A method of administering to the mammary duct of a patient having mammary epithelial cells has been reported (Patent Document 4).

However, methylation-sensitive PCR (MSP) methods, which use primers containing, for example, at least one CG dinucleotide, one of which is located at the 3'end, detect methylated sequences. Although excellent, it was not suitable for quantitative detection.

The present inventors are one of the methylation quantitative analysis techniques after treating DNA extracted from tissue samples of digestive system cancer, especially gastric cancer and colon cancer, with sodium bisulfite (sodium bisulfite) as a sample. A COBRA (Combined Bisulfite Restriction Analysis) assay was performed, and it was found for the first time that methylation of the TWIST1 gene promoter region occurred frequently in cancer tissues (colorectal cancer tissue examination method: Patent Document 5).

This method utilizes the phenomenon that unmethylated CpG is converted from cytosine to uracil in sodium bisulfite-treated DNA, but this conversion does not occur in methylated cytosine. Therefore, by treating a DNA sample with a heavy sulfite, amplifying it by PCR, and then treating it with a specific restriction enzyme, the PCR product using methylated DNA as a template is cleaved, but unmethylated DNA is used as a template. The PCR product can detect the difference that cytosine is not cleaved by conversion to uracil, and can quantify methylated DNA and unmethylated DNA. That is, the steps include (1) extracting DNA from the tissue sample of the subject, (2) treating the extracted DNA sample with heavy sulfite, (3) amplifying it by PCR, and (4) treating it with a restriction enzyme (4). 5) Electrophoresis. (6) The composition consists of quantitatively evaluating the electrophoretic image.

However, this method is a test method that requires a tissue sample for the methylation test of the TWIST1 gene, and in order to obtain the sample, an invasive surgical procedure of tissue collection must be performed. .. Such treatment is painful for the subject, is time-consuming and costly, and is not quantitative, especially when the degree of methylation is low. Therefore, the above-mentioned colorectal cancer histological examination method is at the laboratory level. Although it is an excellent test method, it cannot be said to be a practical test method, much less from the viewpoint of a test method as a simple screening.

In addition to the TWIST1 gene, research on genes related to colorectal cancer screening is progressing. For example, the BMP3 gene, NDRG4 gene, and KRAS gene are used as indicators for colorectal cancer screening by fecal occult blood test by methylation of each gene. (Non-Patent Document 3), and it has been reported that the methylation of the SEPT9 gene in plasma is an index for screening for colon cancer (Patent Document 6).

However, in the screening of colorectal cancer using the above gene in the method described in Non-Patent Document 3, it was necessary to combine the fecal occult blood test and the fecal DNA test. In addition, there is a problem that all stool samples of excreted stools are required for the stool DNA test, and in order to operate as an actual clinical test, it is necessary to bear a burden in terms of hygiene and sample transportation, and the BMP3 gene. , NDRG4 gene, and KRAS gene, which is expensive to measure methylation. Further, in the screening of colorectal cancer using the above gene in the method described in Patent Document 6, a plasma sample is used, but the plasma sample does not collect the blood cell component at the time of collecting the supernatant component as compared with the serum sample. Since it is necessary to carry out the work carefully, it takes time and effort, and there is a problem that the sample amount is as much as 3.5 mL.

By the way, as a simple colorectal cancer test, as described above, the fecal occult blood test (FOBT), which is a primary screening for colorectal cancer performed in a medical examination, is widely used. This is a test that can be easily performed without dietary restrictions and without pain, and although it is very convenient, there are reports that the test sensitivity is as low as 13-24%. It is also reported that 320,000 out of 330,000 positive fecal occult blood tests did not have colorectal cancer in subsequent tests. In this case, most of the positive fecal occult blood tests were colorectal cancer. Despite this, they are forced into a situation where they have to move on to the next step, a detailed inspection.

In addition, colonoscopy, enema X-ray examination, or blood test is usually performed as the next detailed examination by a person who has a positive fecal occult blood test. Of these, colonoscopy is characterized by being able to directly observe the lesion, and the position and size of the lesion can be determined. Furthermore, tissue is collected under the endoscope for pathological diagnosis and small adenomas. At the same time, treatment is possible, but it is painful and expensive as a test. In addition, enema X-ray examination is an examination in which barium (contrast medium) and air are injected into the large intestine from the anus and X-ray photography is performed. , A painful test.

In this way, the fecal occult blood test, which is performed as the primary screening, is less painful and simple, but there is a problem with the sensitivity and specificity of the test itself, and if this test is positive, there is a possibility of colorectal cancer. Although it is low, there is a problem that the detailed examination performed in the next step is invasive, expensive, and burdensome mentally, physically, and costly. In addition, the fecal occult blood test is a method for diagnosing the possibility of colorectal cancer, and the probability that precancerous colorectal adenoma and very early colorectal cancer cannot be detected and overlooked by the primary screening by this method. There was a problem that it was big.

Special Table 2004-527245 Japanese Unexamined Patent Publication No. 2007-274926 Japanese Unexamined Patent Publication No. 2006-166732 JP-A-2009-96808 International Publication No. 2010/11329 Pamphlet Japanese Patent Application Laid-Open No. 2014-520520

Evron E. et al. 2001. Detection of breast cancer cells in ductal lavage fluid by methylation-specific PCR. The Lancet 357: 1335-1336. Fackler M.J. et al.2003. DNA methylation of RASSF1A, HIN-1, RAR-b, CYCLIN D2 and TWIST in in situ and invasive lobular breast cancer. Int.J.Cancer 107: 970-975. Thomas F. Imperiale et al. 2014. Multitarget Stool DNA Testing for Colorectal-Cancer Screening. N Engl J Med 370: 1287-1297 April 3

In view of the above-mentioned current situation, the subject of the present invention is colorectal tumor, specifically colorectal cancer, using DNA methylation having high sensitivity and high specificity as an index by a non-invasive, inexpensive, and simple method. Alternatively, it is an object of the present invention to provide a primary screening test method for the presence or absence of colorectal adenoma and a kit for carrying out the same.

Conventionally, as described above, the degree of methylation is measured by measuring the methylation level after treating DNA with sodium bisulfite. Unmethylated cytosine is converted to uracil by treatment with sodium bisulfite in DNA, and methylated cytosine remains unchanged as cytosine. Currently, it is the mainstream to carry out various methylation assays by utilizing the property that the base sequence changes depending on the presence or absence of methylation, and a process including the following steps is performed to perform a quantitative analysis of methylation. Was going.

In a conventional example (Patent Document 5) in which DNA is treated with sodium bisulfite, a COBRA (Combined Bisulfite Restriction Analysis) assay was used as a methylation quantification method. The steps include (1) extraction of DNA from surgically resected tissue, (2) treatment with heavy sulfite of DNA (conversion of unmethylated cytosine in the CpG sequence in DNA to uracil), (3) purification of DNA, ( 4) methylation-specific PCR of heavy sulfite-treated DNA, (5) restriction enzyme BstUI treatment of PCR products, (6) electrophoresis, (7) quantitative methylation analysis.

The conventional treatment with sodium bisulfite of DNA has the following aspects in more detail, and is a complicated procedure.

Sodium bisulfite treatment was performed to modify the methylated DNA and use it for analysis by the bicarbonate PCR method. Distilled water was added to 2 μg of the DNA purified above to make a total volume of 50 μL, 5.5 μL of 2M NaOH was added, and then the mixture was incubated at 37 ° C. for 10 minutes. 30 μL of 10 mM hydroquinone (Sigma) and 520 μL of 3M sodium bisulfite (pH 5.0, Fisher Scientific) were added and incubated at 50 ° C. for 16 hours in the dark. A DNA sample treated with sodium bisulfite was purified using a DNA Cleanup Kit (Promega). The purification method followed the usage. After adding 5.5 μL of 3M NaOH to the purified DNA, incubate for 5 minutes, add 1 μL of 20 mg / mL glycogen, 33 μL of 10M ammonium acetate, and 260 μL of 100% ethanol to perform ethanol precipitation, and then vacuum dry after ethanol precipitation. DNA was pelletized, 100 μL of distilled water was added thereto, and the mixture was vortexed for 30 minutes to dissolve it, and then dissolved at 4 degrees overnight to prepare a caustic salt-treated DNA sample.

As described above, unmethylated CpG is converted from cytosine to uracil by treatment with sodium bisulfite, but this conversion does not occur in methylated cytosine. Therefore, the restriction enzyme BstUI treatment cleaves the PCR product using the methylated "CGCG" as a template, but does not cleave the PCR product using the unmethylated "CGCG" as a template.

The present inventors have performed methylation analysis of DNA extracted from a stool sample (hereinafter, also referred to as "stool DNA") using the DNA after the above-mentioned heavy sulfite treatment, but this method is a simple test. It turned out that there are the following major problems when aiming at.

That is, when DNA is treated with heavy sulfite, the amount of DNA extracted from feces, which was originally a small amount, is further reduced (approximately 90% reduction), and the DNA becomes a single-stranded state, which is structural. It becomes unstable, and in particular, when the DNA concentration is low, it is easily hydrolyzed, so that it is not suitable for methylation analysis, and a large amount of DNA is required for the analysis. For example, when performing quantitative PCR, it is not detected unless at least 10 copies are made, but considering that DNA is reduced by about 90% by treatment with sodium bisulfite, at least 100 copies are required for the test sample, so the test is performed. A large amount of sample is required.

As described above, since the amount of human DNA recovered from stool is very small (low concentration), treatment with sodium bisulfite causes a further decrease in yield and DNA degradation, which makes subsequent methylation analysis extremely difficult. There was a problem. In order to put a simple test using stool DNA into practical use, it is necessary to establish a methylation gene analysis technology (methylation gene measurement system) that can detect the degree of methylation without performing heavy sulfite treatment. There was found.

Therefore, as a result of diligent research, the present inventors used stool or serum samples to measure methylation TWIST1 (measurement of the degree (frequency, etc.) of methylation in the region upstream from the initiation codon of the TWIST1 gene) and methylation. BMP3 measurement (measurement of the degree of methylation (frequency, etc.) in the region upstream from the start codon of the BMP3 gene), methylation NDRG4 measurement (measurement of the degree of methylation (frequency, etc.) in the region upstream of the transcription start point of the NDRG4 gene) , Methylation SEPT9 measurement (measurement of the degree (frequency, etc.) of methylation of a part of the region encoding the SEPT9 gene) is performed through a predetermined methylation susceptibility limiting enzyme treatment step without performing heavy sulfite treatment. , And have found that the presence or absence of a colon tumor in a test subject can be predicted, and completed the present invention.

That is, the present invention is as shown below.
(1) A method for predicting the presence or absence of a large intestine tumor in a subject to be examined, wherein the following steps (a) to (d) are sequentially provided, and no sodium bisulfite treatment is performed.
(A) Step of extracting DNA in a biological sample collected from a test subject;
(B) A step of treating the DNA obtained in step (a) with a methylation susceptibility restriction enzyme;
(C) A region encoding the TWIST1 gene, BMP3 gene, NDRG4 gene, or SEPT9 gene in the DNA treated with the methylation susceptibility limiting enzyme in step (b), -1 to 1 from the start codon of any of the above genes. -1100 region or a part of -300 to -500 region from the transcription start point of any of the above genes is amplified, and the degree of methylation of 1 or 2 or more CpG sequences in the amplified DNA is measured. Process to do;
(D) When the degree of methylation measured in step (c) is equal to or greater than a predetermined value, it is predicted that the subject to be examined has a large intestine tumor, and when the degree of methylation is less than the predetermined value, the subject to be examined. The process of predicting the absence of colorectal tumors in
(2) The method according to (1) above, wherein the methylation susceptibility restriction enzyme is at least one selected from HhaI, HpaII, BstUI, Hpy99I, SacII, SmaI, BssHII, NaeI, RsrII, and NotI. ..
(3) Step (b)
(B-1) Step of treating with at least one methylation susceptibility restriction enzyme selected from HhaI, HpaII, BstUI, Hpy99I, SacII, SmaI, BssHII, NaeI, RsrII, NotI;
(B-2) After step (b-1), at least one selected from HhaI, HpaII, BstUI, Hpy99I, SacII, SmaI, BssHII, NaeI, RsrII, NotI, and step (b-1) Step of treating with a methylation susceptibility restriction enzyme different from the methylation susceptibility restriction enzyme used in
The method according to (2) above, which is a two-step process.
(4) Step (b-1) is a step of treating with HhaI, HpaII and exonuclease I (ExoI), and step (b-2) is a step of treating with BstUI. 3) The method described.
(5) The method according to any one of (1) to (4) above, wherein DNA amplification is performed by digital PCR in step (c).
(6) The region encoding the TWIST1 gene, BMP3 gene, NDRG4 gene or SEPT9 gene in step (c), the -1 to -1100 region from the start codon of any of the genes, or any of the genes. The above-mentioned (1) to (5), wherein a part of the -300 to -500 region from the transcription start point of the gene is any of the following (c1)-(c4). Method.
(C1) chr7: 19,157,854-19,157,945 shown in SEQ ID NO: 10;
(C2) chr4: 81,952,106-81,952,183 shown in SEQ ID NO: 20;
(C3) chr16: 58,497,096-58,497,237 shown in SEQ ID NO: 21;
(C4) chr17: 75,369,566-75,369,627 shown in SEQ ID NO: 22;
(7) The region encoding the TWIST1 gene, BMP3 gene, NDRG4 gene or SEPT9 gene in step (c), the -1 to -1100 region from the start codon of any of the genes, or any of the genes. A part of the −300 to −500 region from the transcription start site of the gene is amplified using any of the following primer pairs (c5)-(c8) and a set of probes. 6) The method described.
(C5) A set of primer pairs consisting of the oligonucleotides shown in SEQ ID NOs: 4 and 5 and probes consisting of the oligonucleotides shown in SEQ ID NO: 6;
(C6) A set of primer pairs consisting of the oligonucleotides shown in SEQ ID NOs: 11 and 12 and probes consisting of the oligonucleotides shown in SEQ ID NO: 13;
(C7) A set of primer pairs consisting of the oligonucleotides shown in SEQ ID NOs: 14 and 15 and probes consisting of the oligonucleotides shown in SEQ ID NO: 16;
(C8) A set of primer pairs consisting of the oligonucleotides shown in SEQ ID NOs: 17 and 18 and probes consisting of the oligonucleotides shown in SEQ ID NO: 19;
(8) The method according to any one of (1) to (7) above, wherein the biological sample collected from the test subject is stool collected from the test subject.
(9) Any of the above (1) to (8), which is characterized in predicting the presence or absence of a large intestine tumor in the test subject in combination with the degree of methylation measured in step (c) and the fecal occult blood test result. Or the method described.
(10) A region encoding the TWIST1 gene, BMP3 gene, NDRG4 gene or SEPT9 gene in DNA in a biological sample collected from a test subject, a region -1 to -1100 from the start codon of any of the genes. Alternatively, it is a kit for measuring the degree of methylation of one or two or more CpG sequences in a part of the -300 to -500 region from the transcription start point of any of the above genes, and the following (e) to (e) A kit for colon tumor examination, which comprises h).
(E) Reagent for extracting genomic DNA in a subject;
(F) Methylation susceptibility restriction enzyme for processing the genomic DNA;
(G) A region encoding the TWIST1 gene, BMP3 gene, NDRG4 gene or SEPT9 gene in the genomic DNA, a region -1 to -1100 from the starting codon of any of the genes, or any of the genes. Primer and probe set for amplifying a part of the -300 to -500 region from the transcription start site of the gene;
(H) Reagent for analyzing the degree of methylation;
(11) The region encoding the TWIST1 gene, BMP3 gene, NDRG4 gene or SEPT9 gene in step (g), the -1 to -1100 region from the start codon of any of the genes, or any of the genes. The kit according to (10) above, wherein a part of the −300 to −500 region from the transcription start point of the gene is any of the following (c1) to (c4).
(C1) chr7: 19,157,854-19,157,945 shown in SEQ ID NO: 10;
(C2) chr4: 81,952,106-81,952,183 shown in SEQ ID NO: 20;
(C3) chr16: 58,497,096-58,497,237 shown in SEQ ID NO: 21;
(C4) chr17: 75,369,566-75,369,627 shown in SEQ ID NO: 22;
(12) The kit according to (10) or (11) above, wherein the primer / probe set in step (g) is any of the following (c5) to (c8).
(C5) A set of primer pairs consisting of the oligonucleotides shown in SEQ ID NOs: 4 and 5 and probes consisting of the oligonucleotides shown in SEQ ID NO: 6;
(C6) A set of primer pairs consisting of the oligonucleotides shown in SEQ ID NOs: 11 and 12 and probes consisting of the oligonucleotides shown in SEQ ID NO: 13;
(C7) A set of primer pairs consisting of the oligonucleotides shown in SEQ ID NOs: 14 and 15 and probes consisting of the oligonucleotides shown in SEQ ID NO: 16;
(C8) A set of primer pairs consisting of the oligonucleotides shown in SEQ ID NOs: 17 and 18 and probes consisting of the oligonucleotides shown in SEQ ID NO: 19;
(13) The kit according to any one of (10) to (12) above, wherein the biological sample collected from the test subject is stool collected from the test subject.
(14) The kit according to (13) above, further comprising a container for a fecal occult blood test and a reagent.

According to the present invention, even a small amount of DNA obtained from a biological sample such as stool or serum can be easily and highly reliablely inspected for the presence or absence of a large intestine tumor without being treated with sodium bisulfite. By using this analysis technique, it is possible to perform a screening test for the presence or absence of a large intestine tumor using the same stool sample as the fecal occult blood test or using a stool sample that has been cryopreserved.

Furthermore, if it is a "combination test method" that predicts the presence or absence of colorectal tumor by combining the measured degree of methylation and the fecal occult blood test result, a stool sample for fecal occult blood test can be obtained without collecting a new stool sample. It should be a simple, highly reliable, and inexpensive primary colorectal cancer screening method that can be commonly used (reused) to produce test results with higher sensitivity and specificity regarding the presence or absence of colorectal tumors. Can be done.

As described above, by using the present invention, it is possible to predict the presence or absence of colorectal tumor with high sensitivity and high specificity as compared with the fecal occult blood test alone screening, or early detection is the prognosis after that. It is possible to predict the presence or absence of colorectal cancer, advanced adenoma, and intramucosal cancer that have a large effect.

Furthermore, by using a kit for carrying out this method, it is possible to easily perform a test for predicting the presence or absence of a large intestine tumor.

<Condition 1> The results of one-step enzymatic treatment using DNA of various methylation levels, that is, simultaneous treatment with HhaI, HpaII, ExoI, and BstUI are shown. <Condition 2> The results of electrophoresis when combined enzyme treatment using DNA of various methylation levels, that is, treatment with BstUI after HhaI, HpaII, and ExoI treatments are shown. The quantitative result by digital PCR is shown. FIG. 3A shows the number of copies of methylated TWIST1 and the control gene (hTERT) measured by digital PCR using DNA of various methylation levels, and FIG. 3B shows the number of copies (methylated TWIST1 copy number / hTERT copy number). Is shown. Raw digital PCR data showing quantitative reliability. One dot indicates the amount of fluorescence of one droplet. FIG. 4A relates to the methylated TWIST1 gene. FIG. 4B relates to the control gene (hTERT). The embodiment of the present invention is shown in a chart. Comparison of methylated TWIST1 copy counts in stool sample DNA from control group, Non-advanced adenoma patient group, Advanced adenoma / Tis (advanced adenoma or intramucosal cancer) patient group, CRC (colorectal cancer) patient group The results of comparing the groups by group are shown in the table. The result of FIG. 6 is shown graphically. It shows the methylated TWIST1 positive rate at a cutoff value of 1 or more. FIG. 8A is a graph showing the methylation TWIST1 positive rate in each stool sample DNA of the control group, the Non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group when the cutoff value is 1 or more. Indicated by. FIG. 8B shows the number of methylated TWIST1 positive samples and negative results in each stool sample DNA of the control group, the Non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group when the cutoff value was 1 or more. The number of samples is shown in the table. FIG. 8C shows the methylated TWIST1 sensitivity and specificity in each stool sample DNA of the control group, the non-advanced adenoma patient group, the advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group when the cutoff value is 1 or more. , Positive predictive value, and negative predictive value. It shows the methylated TWIST1 positive rate at a cutoff value of 2 or more. FIG. 9A is a graph showing the methylation TWIST1 positive rate in each stool sample DNA of the control group, the Non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group when the cutoff value is 2 or more. Indicated by. FIG. 9B shows the number of methylated TWIST1 positive samples in each stool sample DNA of the control group, the Non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group when the cutoff value was 2 or more. The number of negative samples is shown in the table. FIG. 9C shows the methylated TWIST1 sensitivity and specificity in each stool sample DNA of the control group, the non-advanced adenoma patient group, the advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group when the cutoff value is 2 or more. , Positive predictive value, and negative predictive value. It shows the methylated TWIST1 positive rate at a cutoff value of 5 or more. FIG. 10A is a graph showing the methylation TWIST1 positive rate in each stool sample DNA of the control group, the Non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group when the cutoff value is 5 or more. Indicated by. FIG. 10B shows the number of methylated TWIST1 positive samples in each stool sample DNA of the control group, the Non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group when the cutoff value was 5 or more. The number of negative samples is shown in the table. FIG. 10C shows the methylated TWIST1 sensitivity and specificity in each stool sample DNA of the control group, the non-advanced adenoma patient group, the advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group when the cutoff value is 5 or more. , Positive predictive value, and negative predictive value. Shows fecal occult blood test data. FIG. 11A graphically shows the occult blood positive (+) rate in each stool sample of the control group, the non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group. FIG. 11B shows the number of occult blood positive (+) samples and the number of occult blood negative (-) samples in each stool sample of the control group, the Non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group. Indicated by. FIG. 11C shows the sensitivity, specificity, positive predictive value, and negative predictive value of the occult blood test in each stool sample of the control group, the non-advanced adenoma patient group, the advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group. It is a table showing. The data in the combination test of the fecal occult blood test (FOBT) and the fecal DNA methylated TWIST1 test when the number of copies of FOBT (+) and / or methylated TWIST1 is 1 or more is shown. FIG. 12A shows positive FOBT (+) and / or methylated TWIST 1 copy count of 1 or more in each fecal sample of the control group, the Non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group. The positive (Yes) rate in the combination test is shown in a graph. FIG. 12B shows positive FOBT (+) and / or methylated TWIST 1 copy count of 1 or more in each fecal sample of the control group, the Non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group. The number of positive (Yes) samples and the number of negative (No) samples in the combination test are shown in the table. FIG. 12C shows positive FOBT (+) and / or methylated TWIST 1 copy count of 1 or more in each fecal sample of the control group, the non-advanced adenoma patient group, the advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group. The sensitivity, specificity, positive predictive value, and negative predictive value of the combination test are shown. Data when the fecal occult blood test (FOBT) and the fecal DNA methylated TWIST1 test are combined and the FOBT (+) and / or methylated TWIST1 copy number is 2 or more and the result is positive. FIG. 13A shows positive FOBT (+) and / or methylated TWIST1 copy count of 2 or more in each stool sample of the control group, the Non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group. The positive (Yes) rate in the combination test is shown in a graph. FIG. 13B shows positive FOBT (+) and / or methylated TWIST 1 copy count of 2 or more in each stool sample of the control group, the Non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group. The number of positive (Yes) samples and the number of negative (No) samples in the combination test are shown in the table. FIG. 13C shows a positive result with a FOBT (+) and / or methylated TWIST1 copy count of 2 or more in each stool sample of the control group, the non-advanced adenoma patient group, the advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group. The sensitivity, specificity, positive predictive value, and negative predictive value of the combination test are shown. Data when the fecal occult blood test (FOBT) and the fecal DNA methylated TWIST1 test are combined and the FOBT (+) and / or methylated TWIST1 copy number is 5 or more and the result is positive. FIG. 14A shows positive FOBT (+) and / or methylated TWIST1 copy count of 5 or more in each fecal sample of the control group, the Non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group. The positive (Yes) rate in the combination test is shown in a graph. FIG. 14B shows positive FOBT (+) and / or methylated TWIST 1 copy count of 5 or more in each fecal sample of the control group, the non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group. The number of positive (Yes) samples and the number of negative (No) samples in the combination test are shown in the table. FIG. 14C shows a positive result with a FOBT (+) and / or methylated TWIST1 copy count of 5 or more in the fecal samples of the control group, the Non-advanced adenoma patient group, the Advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group. The sensitivity, specificity, positive predictive value, and negative predictive value of the combination test are shown. Methylated TWIST1 copy count and TWIST1 copy count in stool sample DNA from control group, Non-advanced adenoma (non-advanced adenoma) patient group, Advanced adenoma / Tis (advanced adenoma or intramucosal cancer) patient group, CRC (colorectal cancer) patient group The result of comparing the number of copies ratio by group is shown in a graph. In FIG. 15A, the vertical axis represents the number of copies, and in FIG. 15B, the vertical axis represents the copy number ratio (methylated TWIST1 copy number / hTERT copy number). Further, in the graph, the numbers indicate the P values by the Mann-Whitney test (the same applies to FIGS. 20, 22, 24, 26, 28 described later). Based on the data of FIG. 15, the results of creating ROC curves for the control with each of the non-advanced adenoma patient group, the advanced adenoma / intramucosal cancer patient group, and the advanced adenoma / intramucosal cancer patient group are shown. FIG. 16A is a group of patients with non-advanced adenoma for control, FIG. 16B is a group of patients with advanced adenoma / intramucosal cancer for control, and FIG. 16C is a group of colorectal cancer for control (also FIGS. 21, 23, 25, 27, 29 described later). Similarly). The quantitative result by digital PCR is shown. FIG. 17A shows the number of copies of methylated BMP3 and the control gene (hTERT) measured by digital PCR using DNA of various methylation levels, and FIG. 17B shows the number of copies (methylated BMP3 copy number / hTERT copy number). Is shown. The quantitative result by digital PCR is shown. FIG. 18A shows the number of copies of methylated NDRG4 and the control gene (hTERT) measured by digital PCR using DNA of various methylation levels, and FIG. 18B shows the number of copies (methylated NDRG4 copy number / hTERT copy number). Is shown. The quantitative result by digital PCR is shown. FIG. 19A shows the number of copies of methylated SEPT9 and the control gene (hTERT) measured by digital PCR using DNA of various methylation levels, and FIG. 19B shows the number of copies (methylated SEPT9 copy number / hTERT copy number). Is shown. The results of comparing the methylated BMP3 copy number or copy number ratio in the stool sample DNA from the control group, the non-advanced adenoma patient group, the colorectal advanced adenoma or intramucosal cancer patient group, and the colorectal cancer patient group are shown in a graph. In FIG. 20A, the vertical axis represents the number of copies, and in FIG. 20B, the vertical axis represents the copy number ratio (methylated BMP3 copy number / hTERT copy number). Based on the data of FIG. 20, the results of creating ROC curves for the control with each of the non-advanced adenoma patient group, the advanced adenoma / intramucosal cancer patient group, and the stage 1 to 4 colorectal cancer (CRC) patient group are shown. The results of comparing the methylated NDRG4 copy number or copy number ratio in the stool sample DNA from the control group, the non-advanced adenoma patient group, the advanced adenoma or intramucosal cancer patient group, and the colorectal cancer patient group are shown in a graph. In FIG. 22A, the vertical axis represents the number of copies, and in FIG. 22B, the vertical axis represents the number of copies ratio (number of methylated NDRG4 copies / number of hTERT copies). Based on the data of FIG. 22, the results of creating ROC curves for the control with each of the non-advanced adenoma patient group, the advanced adenoma / intramucosal cancer patient group, and the stage 1 to 4 colorectal cancer patient groups are shown. The graph shows the results of comparing the number of copies or copy number ratio of methylated SEPT9 in stool sample DNA from the control group, non-advanced colorectal adenoma patient group, advanced colorectal adenoma or intramucosal cancer patient group, and colorectal cancer patient group. .. In FIG. 24A, the vertical axis represents the number of copies, and in FIG. 24B, the vertical axis represents the copy number ratio (methylated SEPT9 copy number / hTERT copy number). Based on the data of FIG. 24, the results of creating ROC curves for the control with each of the non-advanced adenoma patient group, the advanced adenoma / intramucosal cancer patient group, and the stage 1 to 4 colorectal cancer patient groups are shown. The graph shows the results of comparing the number of copies or copy number ratio of methylated TWIST1 in serum cell-free DNA from the control group, non-advanced colorectal adenoma patient group, advanced colorectal adenoma or intramucosal cancer patient group, and colorectal cancer patient group. Represent. In FIG. 26A, the vertical axis represents the number of copies, and in FIG. 26B, the vertical axis represents the copy number ratio (methylated TWIST1 copy number / hTERT copy number). Based on the data of FIG. 26, the results of creating ROC curves for the control with each of the non-advanced adenoma patient group, the advanced adenoma / intramucosal cancer patient group, and the stage 1 to 4 colorectal cancer patient groups are shown. Methylated NDRG4 copy number ratio (methylated NDRG4 copy number / hTERT copy number) in serum cell-free DNA from control group, colorectal non-advanced adenoma patient group, colorectal advanced adenoma or intramucosal cancer patient group, colorectal cancer patient group The results of separate comparisons are shown graphically. Based on the data of FIG. 28, the results of creating ROC curves for the control with each of the non-advanced adenoma patient group, the advanced adenoma / intramucosal cancer patient group, and the stage 1 to 4 colorectal cancer patient groups are shown.

As a method for predicting the presence or absence of a large intestine tumor of the present invention,
(A) Step of extracting DNA in a biological sample collected from a test subject;
(B) A step of treating the DNA obtained in step (a) with a methylation susceptibility restriction enzyme;
(C) A region encoding the TWIST1 gene, BMP3 gene, NDRG4 gene, or SEPT9 gene in the DNA treated with the methylation susceptibility limiting enzyme in step (b), -1 to 1 from the start codon of any of the above genes. -1100 region or a part of -300 to -500 region from the transcription start point of any of the above genes is amplified, and the degree of methylation of 1 or 2 or more CpG sequences in the amplified DNA is measured. Process to do;
(D) When the degree of methylation measured in step (c) is equal to or greater than a predetermined value, it is predicted that the subject to be examined has a large intestine tumor, and when the degree of methylation is less than the predetermined value, the subject to be examined. The process of predicting the absence of colorectal tumors in
The method is not particularly limited as long as it is a method in which (a) to (d) of (a) to (d) are sequentially provided and no heavy sulfite treatment is performed, and the test subjects are humans, mice, rats, hamsters, guinea pigs, and monkeys. , Cows, pigs, horses, rabbits, sheep, goats, cats, dogs and other mammals, preferably humans.

In the present invention, colorectal tumor is a concept including colorectal cancer or colorectal adenoma caused by transformation of normal cells in the colon or its immediate gastrointestinal tract. Rectal adenomas are suitable examples, including anal cancer and anal adenomas. Here, colorectal adenoma means a benign tumor without metastasis (non-advanced adenoma) or a precancerous colorectal tumor having cell morphological abnormality or structural atypia (advanced adenoma), and colorectal cancer is infinitely proliferative. Means a malignant tumor with metastasis. In addition, the progression of colorectal tumors is that colorectal adenomas grow in normal colorectal tissues, new colorectal adenomas in precancerous state become cancerous, and cancer cells that have metastasized from the cancerous part are found in non-cancerous colorectal tissue. It means that it proliferates in and a new colorectal cancer tissue is formed.

Since the present invention relates to a method for predicting the presence or absence of a large intestine tumor, the biological sample in the present invention is other than the large intestine tumor tissue, and when the convenience of detection is important, stool, blood, serum, plasma, saliva. , Urine and the like, stool or serum can be preferably mentioned, and stool can be mentioned more preferably. The biological sample also includes cells contained in feces, blood, serum, plasma, saliva, urine and the like. Since a small amount of DNA methylation can be detected by using the method of the present invention, for example, 0.05 to 0.5 g, preferably 0.1 to 0.3 g for stool is sufficient, and blood may be used. For example, 100 μL to 1 mL, preferably 300 μL to 500 μL is sufficient.

Examples of the method for extracting DNA in a biological sample include a phenol extraction method, a phenol / chloroform extraction method, an alkali dissolution method, and a method using a commercially available DNA extraction reagent.

The methylation susceptibility restriction enzyme in the present invention is not particularly limited as long as it is a restriction enzyme capable of distinguishing methylation / demethylation of CpG in the knowledge sequence, and more than 100 enzymes are currently known. HhaI (GCG / C), HpaII (C / CGG), BstUI (CG / CG) with a recognition sequence of 4 bases, Hpy99I (CGWCG /) with a recognition sequence of 5 bases, and SacII (CCGC / GG) with a recognition sequence of 6 bases. ), SmaI (CCC / GGG), BssHII (G / CCGGC), NaeI (GCC / GGC), RsrII (CG / GWCCG) with a recognition sequence of 7 bases, NotI (GC / GGCGCC) with a recognition sequence of 8 bases. be able to. BstUI is also sold under the name Bsh1236I by the manufacturer of restriction enzymes.

The above-mentioned methylation susceptibility restriction enzymes may be used alone, or may be used in combination of 2, 3, 4, or 5 or more types, preferably in combination of 3 or more types, and of HhaI, HpaII, and BstUI. The combination of the three types can be mentioned.

In addition, when treated with the above methylation susceptibility restriction enzyme, if single-stranded DNA is present in the template DNA, the single-stranded DNA escapes cleavage by the restriction enzyme and is amplified in the subsequent PCR reaction. , Causes false positives. To avoid this, it is preferable to add exonuclease I (ExoI) to remove the single-stranded DNA.

As a step of treating with the methylation susceptibility restriction enzyme, the DNA in the extracted biological sample may be treated with the above-mentioned methylation susceptibility restriction enzyme, but from the viewpoint that the DNA in the biological sample can be further cleaved (b-1). Treatment with at least one methylation susceptibility restriction enzyme selected from HhaI, HpaII, BstUI, Hpy99I, SacII, SmaI, BssHII, NaeI, RsrII, NotI; after step (b-2) (b-1) , Furthermore, at least one selected from HhaI, HpaII, BstUI, Hpy99I, SacII, SmaI, BssHII, NaeI, RsrII, NotI, and a methylation susceptibility different from the methylation susceptibility restriction enzyme used in step (b-1). The step of treating with a restriction enzyme; preferably a two-step step (hereinafter, also referred to as “complex enzyme treatment”), in which step (b-1) is treated with HhaI, HpaII and Exonuclease I (ExoI). It is more preferable that the step (b-2) is a step of processing with BstUI. In the case of simultaneous treatment of HhaI, HpaII, ExoI, and BstUI as described in Condition 1 of Example 1 described later, it was distinguished from this as "one-step enzyme treatment".

In the present invention, the region encoding the TWIST1 gene, the BMP3 gene, the NDRG4 gene, or the SEPT9 gene, the -1 to -1100 region from the start codon of any of the genes, or the gene of any of the genes. A part of the -300 to -500 region from the transcription start point is (1) the region encoding the TWIST1 gene, the -1 to -1100 region from the start codon of the TWIST1 gene, or the transcription start point of the TWIST1 gene-. Part of the 300--500 region, (2) the region encoding the BMP3 gene, the -1 to -1100 region from the start codon of the BMP3 gene, or the -300 to -500 region from the transcription start point of the BMP3 gene. Part, (3) region encoding the NDRG4 gene, -1 to -1100 region from the start codon of the NDRG4 gene, or part of the -300 to -500 region from the transcription start point of the NDRG4 gene, (4) SEPT9 gene Any of (1) to (4) of the region encoding the above, the -1 to -1100 region from the start codon of the SEPT9 gene, or a part of the -300 to -500 region from the transcription start point of the SEPT9 gene. means.

In the present invention, the region encoding the TWIST1 gene, the -1 to -1100 region from the start codon of the TWIST1 gene, or the -300 to -500 region from the transcription start point of the TWIST1 gene is preferably a part of the TWIST1 gene. A part of the -1 to -1100 region from the start codon of TWIST1, more preferably a region about 1000 bp upstream from the start codon of the TWIST1 gene, and more preferably a part of the -1 to -1100 region from the start codon of the TWIST1 gene. And 15, 17, 30, 61, 63, 66, 68 in the sequence of the −910-1001 region (position information chr7: 19,157,854-19,157,945 in the GRCh37 / hg19 database) from the start codon of the TWIST1 gene shown in SEQ ID NO: 10. , Or a sequence containing the 90th cytosine (c), more preferably part of the -1 to -1100 region from the start codon of the TWIST1 gene, and 15, 17, 30, in the sequence shown in SEQ ID NO: 10. A sequence containing the 61, 63, 66, 68, and 90th cytosine, most preferably the sequence shown in SEQ ID NO: 10, can be mentioned.

In the present invention, the region encoding the BMP3 gene, the -1 to -1100 region from the start codon of the BMP3 gene, or the -300 to -500 region from the transcription start point of the BMP3 gene is preferably a part of the BMP3. Part of the -1 to -1100 region from the start codon of the gene, more preferably part of the -1 to -1100 region from the start codon of the BMP3 gene, and from the start codon of the BMP3 gene shown in SEQ ID NO: 20. A sequence containing the 30, 44, or 46th cytosine in the sequence of the 333 to -256 region (position information chr4: 81,952,106-81,952,183 in the GRCh37 / hg19 database), more preferably from the start codon of the BMP3 gene- A sequence that is a part of the 1 to -1100 region and contains the thitocins at positions 30, 44, and 46 in the sequence shown in SEQ ID NO: 20, most preferably the sequence shown in SEQ ID NO: 20 can be mentioned.

In the present invention, the region encoding the NDRG4 gene, the -1 to -1100 region from the start codon of the NDRG4 gene, or the -300 to -500 region from the transcription start point of the NDRG4 gene is preferably a part of the NDRG4. A part of the -300 to -500 region from the transcription start point of the gene, more preferably a part of the -300 to -500 region from the transcription start point of the NDRG4 gene, and the NDRG4 gene transcript variant shown in SEQ ID NO: 21. 52, 54, 66, 68 in the sequence (position information chr16: 58,497,096-58,497,237 in GRCh37 / hg19 database) of the region -312 to -453 (region -24587 to -24446 from the start codon) from the transcription start point of 1. A sequence containing the 74th or 76th cytosine, more preferably a part of the −300 to −500 region from the transcription initiation site of the NDRG4 gene, and from the transcription initiation site of the NDRG4 gene transcript variant 1 shown in SEQ ID NO: 21. A sequence containing the 52, 54, 66, 68, 74, and 76th cytosine in the sequence of the −312 to -453 region, most preferably the sequence shown in SEQ ID NO: 21 can be mentioned.

In the present invention, the SEPT9 gene is preferably a part of the region encoding the SEPT9 gene, the -1 to -1100 region from the start codon of the SEPT9 gene, or the -300 to -500 region from the transcription start point of the SEPT9 gene. A part of the region encoding the SEPT9 gene, more preferably a part of the region encoding the SEPT9 gene, and a sequence of the region -14 to +48 from the start codon of the SEPT9 gene transcript variant 2 shown in SEQ ID NO: 22 (GRCh37 / hg19 database). The sequence containing the 26th, 28th, 35th, or 37th cytosine in the position information chr17: 75,369,566-75,369,627), more preferably a part of the region encoding the SEPT9 gene and shown in SEQ ID NO: 22. The sequence containing cytosine at positions 26, 28, 35, and 37 in the sequence in the region -14 to +48 from the start codon of the SEPT9 gene transcript variant 2 can be mentioned, most preferably the sequence shown in SEQ ID NO: 21.

In the present invention, the start codon means a codon that becomes a start point of protein synthesis when mRNA is translated into a protein, and the position of the start codon adenine (A) is set to "+1", and one of such adenines. The position upstream of the base is set to "-1". The transcription start point means the transcription start point of mRNA at the time of transcription, the position of the transcription start point is "+1", and the position one base upstream of the transcription start point is "-1". To do.

Examples of the DNA amplification method include a digital PCR method, a quantitative PCR method, a PCR method, a LAMP method, and a qAMP method, and the digital PCR method can be preferably mentioned. The length of the sequence to be amplified may be 20 to 300 bases, preferably 30 to 200 bases, and more preferably 50 to 150 bases.

Examples of the method for measuring the degree of methylation of one or more CpG sequences in the amplified region in the step (c) of the present invention include digital PCR method, quantitative PCR method, qAMP method, LAMP method, and CGH method. However, among them, the digital PCR method can be preferably mentioned. Further, the degree of DNA amplification and methylation may be measured at the same time by the above method. That is, for example, the degree of DNA amplification and methylation may be measured at the same time by quantitative PCR, LAMP method, or qAMP method.

When measuring the degree of methylation, it is sufficient to measure the degree of methylation of one or more CpG sequences in the amplified region, but the degree of methylation of all CpG sequences in the amplified region is measured. It is preferable to do so.

"Digital PCR" is a method for absolute quantification of the amount of sample DNA. In this method, the sample DNA is fractionated into about 20,000 small water droplets (droplets), and PCR is performed using a thermal cycler. The small water droplets containing the target DNA glow, and the small water droplets not containing the target DNA do not change. The principle is that the concentration of the gene to be measured in the sample can be obtained as an absolute numerical value by counting the number of shining small water droplets and non-shining small water droplets. Since the data is judged by the ants / pears of the target DNA of small water droplets (droplets), it is the same as 1/0 of the digital signal, so it is called "digital PCR". "Copy number count" is the number of copies of a gene by measuring the amount of fluorescence of the above 20,000 small water droplets (droplets) one by one with a droplet reader and counting the number of fluorescent droplets. It means the process of measuring the absolute value.

The degree of methylation can be determined by the number of methylated DNA copies per DNA extracted from a predetermined biological sample, by the number of methylated DNA copies per predetermined PCR reaction solution, or by the number of methylated DNA copies for internal control. The method of obtaining by the ratio can be mentioned. The ratio of methylated DNA to the internal control prevents false negatives when the biological sample contains almost no human cells, especially when the biological sample is stool, and the weight of the net weight of the stool sample for DNA extraction. It is useful in that the stool weight does not have to be constant because the human gene methylation level can be measured accurately regardless of the stool volume and the ratio obtained is theoretically the same even if the stool volume changes. Is. The internal control may be any human DNA that does not have a cleavage site by a methylation susceptibility restriction enzyme in the PCR amplification region, and human TERT and RNase P can be mentioned.

The presence or absence of colorectal tumor in the test subject is predicted to be present in the test subject when the degree of methylation measured above is equal to or higher than the predetermined value, and when the degree of methylation is less than the predetermined value. , It can be predicted that there is no colorectal tumor in the subject to be examined.

For example, as an example of determining the degree of methylation by the number of methylated DNA copies per DNA extracted from a predetermined biological sample, methylated DNA copies per DNA obtained from 4 mg of stool obtained from the test subject. When the number is 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more, it is predicted that the test subject has a colon tumor, and the number of methylated DNA copies is less than 1, less than 2, less than 3, less than 4, If it is less than or less than 5, a method of predicting that there is no colon tumor in the test subject can be mentioned.

Further, as an example of determining the degree of methylation by the number of methylated DNA copies per predetermined PCR reaction solution, the number of methylated DNA copies per 20 μL of PCR reaction solution is 1 or more, 2 or more, 3 or more, 4 or more. , Or, if the number of methylated DNA copies is less than 1, less than 2, less than 3, less than 4, or less than 5, it is predicted that the subject will have a colon tumor. Examples include methods for predicting the absence of colon tumors.

Furthermore, as an example when the degree of methylation is determined by the ratio of methylated DNA to internal control, it is obtained from the number of methylated DNA copies per DNA extracted from 4 mg of stool obtained from the test subject / the number of the test subject. The copy number ratio of TRT gene per DNA extracted from 4 mg of stool is 0.11 or more in the case of methylated TWIST1 / TRT ratio, 0.013 or more in the case of methylated BMP3 / TRT ratio, and methylated NDRG4 / TRT. If the ratio is 0.037 or more, or the methylated SEPT9 / TRT ratio is 0.071 or more, it is predicted that the test subject has a colon tumor, and the methylated DNA copy number ratio is methylated TWIST1. Less than 0.11 for / TERT ratio, less than 0.013 for methylated BMP3 / TERT ratio, less than 0.037 for methylated NDRG4 / TERT ratio, or 0 for methylated SEPT9 / TERT ratio If it is less than .071, a method of predicting that there is no colon tumor in the test subject can be mentioned.

Further, the test method for predicting the presence or absence of a colon tumor of the present invention is as follows: 1) Extraction of genomic DNA from stool sample (a'), 2) Treatment of stool DNA obtained in step a with methylation sensitivity restriction enzyme treatment (or Step (b') of complex enzyme treatment), 3) Amplify the CpG site contained in the -910-1001 region from the start codon of the TWIST1 gene using a digital PCR method with multiple primers, and methyl in the amplification product. Step of counting the number of copies of methylated DNA (analyzing the methylation pattern) (c'), 4) Counting the number of copies of methylated DNA per 20 μL of PCR reaction solution, and if it is above a predetermined value, colon cancer or A method including each step of the step (d') of obtaining a prediction result that a colon tumor is present may be used.

The kit for colon tumor examination of the present invention includes a region encoding the TWIST1 gene, BMP3 gene, NDRG4 gene or SEPT9 gene in DNA in a biological sample collected from a test subject, and the start codon of any of the above genes. A kit for measuring the degree of methylation of a part of the −300 to −500 region from the transcription initiation site of the -1 to -1100 region or any of the genes.
(E) Reagent for extracting genomic DNA in a subject;
(F) Methylation susceptibility restriction enzyme for processing the genomic DNA;
(G) A region encoding the TWIST1 gene, BMP3 gene, NDRG4 gene or SEPT9 gene in the genomic DNA, a region -1 to -1100 from the starting codon of any of the genes, or any of the genes. Primer and probe set for amplifying a part of the -300 to -500 region from the transcription start site of the gene;
(H) Reagent for analyzing the degree of methylation;
There is no particular limitation as long as it is provided with. Specific kit configurations include, for example, (1) enzymes and equipment related to the DNA extraction step, and in particular, reagents and equipment for extracting DNA from biological samples such as stool and serum (2). ) Methylation susceptibility restriction enzyme, buffer for its enzymatic reaction, (3) Reagents and equipment for DNA amplification, that is, target CGCG sequence, CCGG sequence, GCGC sequence, CCGCGG sequence, CCCGGG sequence, GCGCGC sequence. , GCCGGCC sequence, CGGWCCG sequence, and / or a primer probe set capable of amplifying a region containing a sequence such as GCGGCCGC, and an enzyme or buffer solution for amplification, etc. (4) Reagents for measuring DNA methylation Examples include kits containing the like and the like. Regarding the reagents required for each process, in addition to the method disclosed in papers as a conventional method, a commercially available kit, for example, QIAamp Stool DNA Isolation Kit (manufactured by Qiagen) for DNA extraction from stool is used. DNA can be extracted by using QIAamp Fast DNA Stool Mini Kit (manufactured by Qiagen) or the like.

In the present invention, the primer used for DNA amplification is a region encoding the TWIST1 gene, BMP3 gene, NDRG4 gene, or SEPT9 gene, a region -1 to -1100 from the start codon of any of the genes, or a region. It can be appropriately designed based on the sequence information of a part of the -300 to -500 region from the transcription start point of any of the above genes, and appropriately prepared using an appropriate oligonucleotide synthesizer. If it is designed so that one or more CpG sequences existing in the gene can be amplified, the position and length thereof are not particularly limited, but the length may be 10 to 50 mer, preferably 15 to 30 mer. it can.

In the present invention, the probe may be a nucleic acid probe that can hybridize to a part or all of the amplified DNA, and can be appropriately produced by using an appropriate oligonucleotide synthesizer, and its length is particularly limited. Although not, 7 to 30 mer, preferably 10 to 20 mer can be mentioned. Further, it is preferable that the probe is fluorescently labeled.

For example, as shown in Examples described later, SEQ ID NOs: 4 and 5 for targeting all methylation of the CGCG sequence, CCGG sequence, and GCGC sequence located in the nucleotide sequence shown in SEQ ID NO: 10 are shown. A combination of a primer pair consisting of the oligonucleotides and a probe consisting of the oligonucleotide shown in SEQ ID NO: 6 (methylated TWIST1 measurement primer / probe set :), a CGCG sequence located in the nucleotide sequence shown in SEQ ID NO: 20, and a CGCG sequence located in the nucleotide sequence shown in SEQ ID NO: 20. A combination of a primer pair consisting of the oligonucleotides shown by SEQ ID NOs: 11 and 12 and a probe consisting of the oligonucleotide shown by SEQ ID NO: 13 for targeting all methylation of the CCGG sequence (primer probe for measuring methylated BMP3). Set) and a primer pair consisting of the oligonucleotides shown in SEQ ID NOs: 14 and 15 and SEQ ID NO: 16 for targeting all methylation of the CGCG sequence and the GCGC sequence located in the nucleotide sequence shown in SEQ ID NO: 21. To target the combination of probes consisting of the oligonucleotides shown in (Methyled NDRG4 measurement primer / probe set) and all methylation of the CGCG sequence and GCGC sequence located in the nucleotide sequence shown in SEQ ID NO: 22. A combination of a primer pair consisting of the oligonucleotides shown in SEQ ID NOs: 17 and 18 and a probe consisting of the oligonucleotide shown in SEQ ID NO: 19 (primer / probe set for measuring methylated SEPT9) can be preferably shown.

Examples of the present invention are shown below, but the present invention is not limited to the examples.

(Examination of enzyme treatment conditions)
First, regarding the enzymatic treatment of DNA, the effect of different enzymatic treatment conditions on methylation analysis was examined under the following two conditions.
Condition 1: Enzymatic treatment of DNA: HhaI, HpaII, ExoI, BstUI simultaneous treatment (referred to as "one-step enzyme treatment")
Condition 2: DNA enzyme treatment: BstUI treatment after HhaI, HpaII, ExoI treatment (referred to as "complex enzyme treatment")
Here, HhaI, HpaII, and BstUI are methylation-sensitive restriction enzymes as described above, and ExoI is a 3'→ 5'single-stranded exonuclease (Exonuclease I) that degrades unnecessary single-stranded DNA. It is an enzyme that

Preparation of sample DNA sample (Preparation of TWIST1 methylation control DNA sample)
As a control of the TWIST1 methylation allele, HCT116-derived DNA (methylation control DNA) known to have high levels of TWIST1 methylation was used. The colorectal cancer cell line HCT116 was transferred to a 1.5 mL tube, and nucleic acid extraction was performed using the nucleic acid extractant SepaGene (Sanko Junyakusha). The extraction method followed the usage. The nucleic acid pellet obtained by the extraction was air-dried, and 100 μL of TE buffer was added and dissolved to prepare a TWIST1 methylation control DNA sample.

(Preparation of TWIST1 unmethylated control DNA sample)
As a control for TWIST1 unmethylated alleles, peripheral blood lymphocyte-derived DNA (unmethylated control DNA) known to have almost no TWIST1 methylation was used. Using a blood collection tube containing the anticoagulant EDTA-2Na, 0.2% NaCl was added to the collected peripheral blood, mixed by inversion, and then centrifuged at 2500 rpm for 5 minutes. The supernatant was removed with an ejector so as not to inhale the leukocytes precipitated on the bottom of the tube, and this operation was repeated until the pellets (leukocytes) became white. The whitened pellets were transferred to a 1.5 mL tube, and nucleic acid extraction was performed using a nucleic acid extractant SepaGene (Sanko Junyaku Co., Ltd.). The extraction method followed the usage. The nucleic acid pellet obtained by the extraction was air-dried, and 100 μL of TE buffer was added to dissolve the pellet, which was used as a TWIST1 unmethylation control DNA sample.

The methylation control DNA and the non-methylation control DNA are mixed in various ratios, and the mixing ratio of the HCT116-derived DNA to the peripheral blood lymphocyte-derived DNA (methylation control DNA content ratio) is 0% and 0.001%. , 0.01%, 0.1%, 1%, 10%, 50%, 100% DNA samples were prepared.

Table 1 shows the relationship between the methylation control DNA content in each sample and the mixing ratio of colon cancer cell line HCT116-derived DNA (methylated TWIST1 control) and peripheral blood lymphocyte-derived DNA (unmethylated TWIST1 control). Shown.

Next, an enzyme treatment containing a methylation susceptibility restriction enzyme was performed under the following two conditions. ExoI is an exonuclease.
<Condition 1> One-step enzyme treatment of DNA: Simultaneous treatment of HhaI, HpaII, ExoI, BstUI 1. Put 10 μL of DNA in a 1.5 mL tube.
2. Add 1 μL of GeneAmp PCR buffer II (attached to N808-0241, Life technologies).
3. Add 1 μL of 25 mM MgCl ₂ solution (attached to N808-0241, Life technologies).
4. Add 1 μL (10U) of HhaI (ER1851, Thermo).
5. Add 1 μL (10U) of HpaII (ER0512, Thermo).
6. Add 1 μL (20U) of ExoI (EN0581, Thermo).
7. Add 1 μL (10U) of BstUI (R0518L, New England Biolabs).
8. Mix by pipetting.
9. Warm at 37 degrees for 16 hours.
10. Warm at 60 degrees for 16 hours.
11. Warm at 98 degrees for 10 minutes.

<Condition 2> DNA complex enzyme treatment: BstUI treatment after HhaI, HpaII, ExoI treatment 1. Put 10 μL of DNA in a 1.5 mL tube.
2. Add 1 μL of GeneAmp PCR buffer II (attached to N808-0241, Life technologies).
3. Add 1 μL of 25 mM MgCl2 solution (attached to N808-0241, Life technologies).
4. Add 1 μL (10U) of HhaI (ER1851, Thermo).
5. Add 1 μL (10U) of HpaII (ER0512, Thermo).
6. Add 1 μL (20U) of ExoI (EN0581, Thermo).
7. Mix by pipetting.
8. Warm at 37 degrees for 16 hours.
Add 9.1 μL (10U) BstUI (R0518L, New England Biolabs).
10. Warm at 60 degrees for 16 hours.
11. Warm at 98 degrees for 10 minutes.

(Amplification by PCR reaction)
The primers (primer set) used for the PCR reaction are as follows.
TWIST1 Forward Primer 1 (SEQ ID NO: 1)
5'-GTCCTGGGCGTTTCTGAAG --3'
TWIST1 reverse primer 1 (SEQ ID NO: 2)
5'-CAGAAGGGCGAGAGAGC --3'
The amplification region by this primer pair is the following sequence region about 1000 bp upstream from the start codon of the TWIST1 gene (within the -944 to −1021 region from the start codon of the TWIST1 gene and chr7: 19,157,888-19,157,965 on the GRCh37 / hg19 database). (PCR product size: 78 bp).
(SEQ ID NO: 3) 5'-GTCCTGGGCG TTTCTGAAGA CGTGGCCGCG CCGCGGGGGC
TGAGGATTTG CGTCCCGGCC TGCTCTCTCG CCCTTCTG -3'

In Table 3, the underlined part shows the recognition sites of the following methylation susceptibility restriction enzymes. The restriction enzyme cleavage regions are GCGC for HhaI, CCGG for HpaII, and CGCG for BstUI.

1. 1. The following PCR reaction solution is prepared.
Enzyme-treated DNA 2 μL
・ 10x GeneAmp PCR buffer II 1 μL
・ 25 mM MgCl2 0.8 μL
・ 10 mM dNTPs 1.0 μL
・ 10 μM TWIST1 Forward primer 1 0.5 μL
・ 10 μM TWIST1 Reverse primer 1 0.5 μL
・ AmpliTaqGold 0.05 μL
・ Water 4.15 μL
2. Perform the PCR reaction under the following conditions.
・ 95 degrees 10 minutes ・ 40 cycles: 95 degrees 30 seconds, 60 degrees 30 seconds, 72 degrees 30 seconds ・ 72 degrees 4 minutes

(Confirmation of PCR product by electrophoresis)
Since the template DNA is treated with methylation susceptibility restriction enzymes, if the recognition sequence of each restriction enzyme is methylated, a band of 78 bp PCR amplification product is observed without being cleaved by the restriction enzyme, but the recognition site. Using the principle that if is not methylated, it will be cleaved with a restriction enzyme and no band will be observed, the presence or absence of methylated DNA and its proportion can be determined by the presence or absence of a band and its density.

The obtained PCR product was confirmed by 3% agarose gel electrophoresis containing ethidium bromide. From the above electrophoresis results, when the DNA of <Condition 1> was treated with enzymes, that is, when DNAs of various methylation levels were simultaneously treated with HhaI, HpaII, ExoI, and BstUI, a sample with a methylation level of 0% was obtained. Nevertheless, due to incomplete cleavage of the template DNA, amplification by PCR was observed (Fig. 1). Also, the correlation between the level of methylation and the density of the band was not sufficient. On the other hand, when the DNA of <Condition 2> is treated with BstUI after HhaI, HpaII, and ExoI treatment using DNA of various methylation levels, the template DNA is almost the same in the sample with 0% methylation level. It was cleaved and no amplification by PCR was observed (Fig. 2). There was also an overall correlation between methylation levels and band density. Therefore, it was clarified that DNA cleavage can be appropriately performed by performing complex enzyme treatment, and the methylation level can be quantified more accurately.

From the above, in the following examples using DNA not treated with sodium bisulfite, when the DNA is treated with an enzyme, the method of condition 2, that is, treatment with BstUI after treatment with HhaI, HpaII, and ExoI, is a complex enzyme treatment. We decided to use the method.

(Examination of primer for digital PCR and quantification of TWIST1 methylation level)
DNA known to have high levels of TWIST1 methylation (DNA derived from colon cancer cell line HCT116: methylation control DNA) and DNA known to have almost no TWIST1 methylation (DNA derived from peripheral blood lymphocytes: non-methylation) Methylation control DNA) was mixed in various proportions (Table 4). After multiple methylation susceptibility restriction enzyme treatments and exonuclease treatments (complex enzyme treatments), this DNA was examined for methylation level quantification by digital PCR using the designed primers and probes.

(1) Sample DNA Preparation of sample Similar to Example 1, HCT116-derived DNA known to have high levels of TWIST1 methylation and peripheral blood lymphocyte-derived DNA known to have almost no TWIST1 methylation were used. DNA samples in which the mixing ratio of HCT116 was 0%, 1%, 20%, 50%, 80%, and 100% with respect to the DNA derived from peripheral blood lymphocytes were prepared by mixing at various ratios (Table 4). ..

(2) Complex enzyme treatment of DNA: BstUI treatment after HhaI, HpaII, ExoI treatment Under the same conditions as <Condition 2> of Example 1, enzyme treatment containing a restriction enzyme was performed. ExoI is an exonuclease.
<Protocol>
1. Place 10 μL of DNA in a 1.5 mL tube.
2. Add 1 μL of GeneAmp PCR buffer II (attached to N808-0241, Life technologies).
3. Add 1 μL of 25 mM MgCl2 solution (attached to N808-0241, Life technologies).
4. Add 1 μL (10U) of HhaI (ER1851, Thermo).
5. Add 1 μL (10U) of HpaII (ER0512, Thermo).
6. Add 1 μL (20U) of ExoI (EN0581, Thermo).
7. Mix by pipetting.
8. Warm at 37 degrees for 16 hours.
Add 9.1 μL (10U) BstUI (R0518L, New England Biolabs).
10. Warm at 60 degrees for 16 hours.
11. Warm at 98 degrees for 10 minutes.
The cleavage sites by the methylation susceptibility restriction enzymes HhaI, HpaII, and BstUI are "GCGC", "CCGG", and "CGCG", respectively.

(3) Digital PCR processing As described above, "digital PCR" is a method for absolutely quantifying the amount of sample DNA. Here, a digital PCR system (QX100 Droplet Digital PCR system: BioRad) was used.

<Primer / probe set>
The primers and probes used are as follows (Table 2).
(TWIST1)
TWIST1 forward primer (SEQ ID NO: 4) 5'-TCCAAAGGCCAAACCGC-3'
TWIST1 Reverse Primer (SEQ ID NO: 5) 5'-CCGGGACGCAAATCCTC-3'
TWIST1 TaqMan probe (SEQ ID NO: 6) 5'-FAM-CTGAAGACGTGGCCGCGCC-TAMRA-3'
(Internal control)
hTERT forward primer (SEQ ID NO: 7) 5'-GGGTCCTCGCCTGTGTACAG-3'
hTERT Reverse Primer (SEQ ID NO: 8) 5'-CCTGGGAGCTCTGGGAATTT-3'
hTERT TaqMan probe (SEQ ID NO: 9) 5'-VIC-CACACCTTTGGTCACTC-MGB-3'
SEQ ID NO: 10 (Table 3) shows the sequence of the PCR amplification target region of TWIST1 (-910-1001 region from the TWIST1 gene start codon: position information chr7: 19,157,854-19,157,945 in the GRCh37 / hg19 database). In Table 3, the underline shows the sites that may be cleaved by the restriction enzymes HhaI, HpaII, and BstUI. However, since these restriction enzymes are methylation-sensitive restriction enzymes, if cytosine (C) is methylated, the site is not cleaved.
(SEQ ID NO: 10) 5'-TCCAAAGGCC AAACCGCGGC GGCCCAGCCC GGAGGTCCTG
GGCGTTTCTG AAGACGTGGC CGCGCCGCGG GGGCTGAGGA TTTGCGTCCC GG -3'

<Digital PCR protocol>
1. 1. The following PCR reaction solution is prepared.
・ Enzyme-treated DNA 2 μL
・ DdPCR Supermix for probes (# 186-3010, BioRad) 10 μL
・ 10 μM TWIST1 forward primer 0.5 μL
・ 10 μM TWIST1 Reverse Primer 0.5 μL
・ 5 μM TWIST1 TaqMan probe 1.0 μL
・ 10 μM hTERT Forward Primer 0.5 μL
・ 10 μM hTERT Reverse Primer 0.5 μL
・ 5 μM hTERT TaqMan probe 1.0 μL
・ Water 6.0 μL
For NTC (no template control), use 2 μL of water instead of the enzyme-treated DNA.
2. Droplet preparation ・ Set the PCR reaction solution in the AutoDG system (BioRad) to prepare a droplet.
3. 3. PCR reaction (thermal cycler)
・ 95 degrees 10 minutes ・ 40 cycles (94 degrees 30 seconds, 56 degrees 1 minute, ramp speed 2 degrees / second)
・ 98 degrees 10 minutes

(4) Count the number of copies
The PCR-reacted sample is set in the Droplet Reader (BioRad) of the QX100 Droplet Digital PCR system, and the number of copies of hTERT and methylated TWIST1 is counted by detecting the fluorescent dye derived from the TaqMan probe in each droplet. hTERT is an internal control and is used to detect human DNA, whether tumor or non-tumor. Since there are no restriction enzyme HhaI, HpaII, and BstUI cleavage sites in the PCR amplification region of hTERT, even if these restriction enzyme treatments are applied to the template DNA, the DNA in the region is not cleaved. In the presence of human-derived DNA, hTERT is amplified by PCR without being affected by methylation susceptibility restriction enzyme treatment.

(result)
<Quantitative by digital PCR> Fig. 3A, B, Fig. 4A, B
FIG. 3A is a diagram showing the relationship between the ratio of methylation control DNA (horizontal axis) and the number of copies of the methylated TWIST1 gene and hTERT gene (concentration: vertical axis). It was found that there was a positive linear correlation between the proportion of methylation control DNA and the methylated TWIST1 level. On the other hand, since the hTERT gene was not cleaved by the methylation susceptibility restriction enzyme, it showed a constant number of copies regardless of the mixing ratio of HCT116.

FIG. 3B shows "methylated TWIST1 copy number / hTERT copy number" (copy number ratio) when DNAs of various methylation levels are used. Here, the peripheral blood lymphocyte-derived DNA used as a sample having almost no TWIST1 methylation actually had a TWIST1 methylation level of 0.14%, so that the sample HCTs were 0%, 1%, 20%, and 50. The actual TWIST1 methylation levels of%, 80% and 100% were 0.14%, 0.82%, 13.4%, 35.8%, 62.2% and 81.1%, respectively. From the above results, it was confirmed that this method can accurately evaluate the TWIST1 methylation level.

4A and 4B are digital PCR raw data, in which one dot indicates the amount of fluorescence of one droplet. It supports the reliability of the data.

From the above results, it was found that by using the digital PCR of the present embodiment, a methylation level of 0.14% or more can be quantitatively measured. Moreover, it was shown that reliable quantification of TWIST1 gene methylation can be presented without chemical treatment of DNA (dusulfate treatment). In the digital PCR method, the lower limit of detection of mutant DNA is said to be 0.001% (1 / 100,000), so there is a possibility that the accuracy can be further improved compared to this method.

(Establishment of methylated TWIST1 measurement system using fecal sample DNA)
Based on the above results, the following method was established as a methylated TWIST1 measurement system as a simple test.

Examples of steps for the simple methylated TWIST1 measurement system in this case include (1) extraction of DNA (biological sample DNA) from a biological sample, (2) complex enzyme treatment of DNA, and (3) digital PCR treatment. It consists of a simple step of (4) counting the number of copies (Fig. 5). In this example, DNA was extracted from a fecal sample among biological samples. This is to aim for a more non-invasive, inexpensive and simple inspection. In addition, feces contain as many as 3 to 5 × 10 ¹¹ / g of bacteria, and 99.99% of the DNA in feces is said to be derived from bacteria. That is, the human DNA contained in feces is only about 0.01%.

(1) Extraction of DNA (stool DNA) from stool sample The sample is normal, non-advanced adenoma (non-advanced adenoma), Advanced adenoma (advanced adenoma) / Tis (intramucosal cancer), or CRC ( We basically used cryopreserved fecal samples from people who were judged to have (colorectal cancer).

Extract DNA from 51 healthy person stools (Control), 9 Non-advanced adenoma patient stools, 17 Advanced adenoma / Tis patient stools, 133 CRC patient stools, and 210 stool samples in total as follows. went. Of the colon adenomas, Advanced adenoma is an adenoma with a diameter of 1 cm or more, or an adenoma with villous components (tubulovillous or villous), or a high-grade or severe dysplasia. An adenoma with (severe or severe dysplasia), a precancerous lesion of the colon, and a non-advanced adenoma means a benign tumor that is not the Advanced adenoma described above. Tis is a very early stage (stage 0) colorectal cancer in which the cancer stays in the mucosa and does not extend to the submucosa. It is also called Carcinoma in situ. Here, Tis is referred to as "Tis". It is called "carcinoma in situ".

Advanced adenoma (advanced adenoma), which is a precancerous state, and Tis (intramucosal cancer), which is a very early stage cancer, are difficult to detect at an early stage, but if they can be detected, there is a high possibility that they can be completely treated. Then, the patients diagnosed with Advanced adenoma (advanced adenoma) or Tis (intramucosal cancer) were collectively described as "Advanced adenoma (advanced adenoma) / Tis (intramucosal cancer)" as one analysis group. Furthermore, CRC (colorectal cancer) means stage 1 to 4 colorectal cancer. The control group means a group in which there is no colon tumor in the large intestine tissue or the large intestine mucosa by endoscopy.

Sample and amount: 0.2 g each of the above 210 human fecal samples
Kit used: QIAamp Fast DNA Stool Mini Kit (Qiagen).
Method: DNA is extracted according to the protocol (Human DNA analysis) attached to the product. Place 0.2 g of stool sample in a 2 mL tube.
Add 2.1 mL of Inhibit EX buffer (included in the kit) to the stool specimen. Vortex agitation for 1 minute or until the stool sample is completely suspended.
3. 3. Warm the suspension at 70 ° C for 5 minutes. Vortex agitation for 15 seconds.
4. Centrifuge at 20,000 g for 1 minute.
5. Place 25 μl Proteinase K (included in the kit) in a new 1.5 mL tube.
6. Place 600 μL of the supernatant after step 4 in the 1.5 mL tube of step 5.
7. Add 600 μL of AL buffer (included in the kit) and vortex stir for 15 seconds.
8. Add 9.600 μL of ethanol (96-100%), which is heated at 70 ° C. for 10 minutes, to the solution and mix by vortex stirring.
10. Place 600 μL of the solution from step 9 on a QIAamp spin column (included in the kit). Close the lid and centrifuge at 20,000 g for 1 minute. Place the QIAamp spin column in a new 2 mL collection tube and discard the filtrate. Repeat step 10 until all lysates are column treated.
11. Carefully open the QIAamp spin column and add 500 μL of AW1 buffer (included in the kit). Centrifuge at 20,000 g for 1 minute. Place the QIAamp spin column in a new 2 mL collection tube and discard the collection tube containing the filtrate.
12. Carefully open the QIAamp spin column and add 500 μL of AW2 buffer (included in the kit). Centrifuge at 20,000 g for 3 minutes. Discard the collection tube containing the filtrate.
13. Place the QIAamp spin column in a new 2 mL collection tube and discard the old collection tube containing the filtrate. Centrifuge at 20,000 g for 3 minutes.
14. Transfer the QIAamp spin column to a new 1.5 mL tube and pipette 100 μL of ATE buffer (included in the kit) directly onto the QIAamp membrane. After allowing to stand at room temperature for 1 minute, centrifuge at 20,000 g for 1 minute to elute the DNA.

(2) DNA complex enzyme treatment: BstUI treatment after HhaI, HpaII, ExoI treatment <protocol>
It is the same as <Condition 2> of Example 2. ExoI is an exonuclease.
1. Place 10 μL of DNA in a 1.5 mL tube.
2. Add 1 μL of GeneAmp PCR buffer II (attached to N808-0241, Life technologies).
3. Add 1 μL of 25 mM MgCl ₂ solution (attached to N808-0241, Life technologies).
4. Add 1 μL (10U) of HhaI (ER1851, Thermo).
5. Add 1 μL (10U) of HpaII (ER0512, Thermo).
6. Add 1 μL (20U) of ExoI (EN0581, Thermo).
7. Mix by pipetting.
8. Warm at 37 degrees for 16 hours.
Add 9.1 μL (10U) BstUI (R0518L, New England Biolabs).
10. Warm at 60 degrees for 16 hours.
11. Warm at 98 degrees for 10 minutes.

(3) Digital PCR processing As described above, "digital PCR" is a method for absolutely quantifying the amount of sample DNA. Here, a digital PCR system (QX100 Droplet Digital PCR system: BioRad) was used.

<Primer, etc.>
The primers and the like used are the same as in Example 2 (Table 2).
TWIST1 forward primer (SEQ ID NO: 4)
TWIST1 reverse primer (SEQ ID NO: 5)
TWIST1 TaqMan probe (SEQ ID NO: 6)
(Internal control)
hTERT forward primer (SEQ ID NO: 7)
hTERT Reverse Primer (SEQ ID NO: 8)
hTERT TaqMan probe (SEQ ID NO: 9)
SEQ ID NO: 10 (Table 3) shows the sequence of the PCR amplification target region of TWIST1.
In Table 3, the underline shows the sites that may be cleaved by the restriction enzymes HhaI, HpaII, and BstUI. However, since these restriction enzymes are methylation-sensitive enzymes, if cytosine (C) is methylated, the site is not cleaved.
(SEQ ID NO: 10) 5'-TCCAAAGGCC AAACCGCGGC GGCCCAGCCC GGAGGTCCTG
GGCGTTTCTG AAGACGTGGC CGCGCCGCGG GGGCTGAGGA TTTGCGTCCC GG -3'

<Digital PCR protocol>
It is the same as in Example 2.
1. 1. The following PCR reaction solution is prepared.
Enzyme-treated DNA 2 μL
・ DdPCR Supermix for probes (# 186-3010, BioRad) 10 μL
・ 10 μM TWIST1 forward primer 0.5 μL
・ 10 μM TWIST1 Reverse Primer 0.5 μL
・ 5 μM TWIST1 TaqMan probe 1.0 μL
・ 10 μM hTERT Forward Primer 0.5 μL
・ 10 μM hTERT Reverse Primer 0.5 μL
・ 5 μM hTERT TaqMan probe 1.0 μL
・ Water 6.0 μL
2. Droplet preparation ・ Set the PCR reaction solution in the AutoDG system (BioRad) to prepare a droplet.
3. 3. PCR reaction (thermal cycler)
・ 95 degrees 10 minutes ・ 40 cycles (94 degrees 30 seconds, 56 degrees 1 minute, ramp speed 2 degrees / second)
・ 98 degrees 10 minutes

(4) Count the number of copies
The PCR-reacted sample is set in the Droplet Reader (BioRad) of the QX100 Droplet Digital PCR system, and the number of copies of hTERT and methylated TWIST1 is counted by detecting the fluorescent dye derived from the TaqMan probe in each droplet.
hTERT is an internal control used to detect human DNA in stool DNA.

(result)
<Number of copies per 20 μL of PCR reaction solution of methylated TWIST1 in each group>
6 and 7 show the control group, non-advanced adenoma patient group, advanced adenoma / Tis patient group, and stage 1-4 colorectal cancer (CRC) patients. It is a table and graph of the result of comparing the number of copies per 20 μL of the PCR reaction solution of methylated TWIST1 in each stool sample DNA (stool DNA) of the group by group. Since 20 μL of the PCR reaction solution contained DNA extracted from 4 mg of stool, the number of copies per 20 μL of the methylated TWIST1 PCR reaction solution was the number of copies per DNA extracted from 4 mg of methylated TWIST1 stool. Equivalent to.

As shown in the table of FIG. 6, in the stool DNA of the control group, the number of methylated TWIST1 copies was 0.0-maximum 2.800, median 0.0, average 0.2314 (sd: 0.6733), and non-advanced adenoma. In the stool DNA of the patient group, the minimum number of methylated TWIST1 copies is 0.0-maximum value 11.60, the average value is 4.289 (sd: 3.610), and in the stool DNA of the Advanced adenoma / Tis patient group, the number of methylated TWIST1 copies is the minimum. In the value 0.0-maximum value 240.0, average value 32.38 (sd: 64.70), and in the stool DNA of the CRC (adenoma) patient group, the number of methylated TWIST1 copies was minimum value 0.0-maximum value 4700, average value 65.08 (sd). : 473.1), the average value increased remarkably as it progressed from normal to non-advanced adenoma, advanced adenoma / intramucosal cancer, and stage 1 to 4 colon cancer.

Further, the methylation analysis technique included in the test method of the present embodiment does not require chemical treatment of DNA with heavy sulfite, which is a step of the prior art, so that the test can be carried out very easily. Moreover, since it can be detected even with a very small amount (1 to 5 copies per 20 μL) of methylated TWIST1 in stool, it can be said that the detection sensitivity is very high. Furthermore, since the test can be performed by methylation of only one gene, it can be said that the cost has been reduced.

In recent years, DNA methylation exists not only in the region upstream of the transcription initiation site but also in the gene region (Gene body), and it is considered that methylation of such a region has a function of preventing abnormal transcription initiation. .. Therefore, in the above example, the degree of methylation of the CpG sequence contained in the sequence shown in SEQ ID NO: 10 (-910-1001 region from the start codon of the TWIST1 gene) is measured, but the degree of methylation is not limited to this region, and the TWIST1 gene is not limited to this region. The methylation of the CpG sequence contained in the region encoding the above, the -1 to -1100 region from the start codon of each gene, or the -300 to -500 region from the transcription start site of any of the genes. It is considered possible to predict the presence or absence of a colon tumor in the test subject by measuring the degree of.

<Methylation positive rate in each group with a cutoff value of 1 or more in the number of methylated TWIST1 copies> FIG.
Methylated TWIST1 positive rate in each stool sample DNA in the control group, Non-advanced adenoma patient group, Advanced adenoma / Tis patient group, and CRC (colorectal cancer) patient group when the cutoff value is 1 or more: Positive rate ( The vertical axis) is shown in a graph (FIG. 8A), and the original data thereof is shown in a table (FIG. 8B). FIG. 8C shows the sensitivity of methylated TWIST1 detection in each stool sample DNA in the control group, the non-advanced adenoma patient group, the advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group when the cutoff value is 1 or more. , Specificity, positive predictive value, negative predictive value.

Here, the terms used in the present specification will be described. "Sensitivity" and "specificity" are indexes for evaluating the reliability of clinical tests. Sensitivity is the percentage of positives (outliers) (true positive rate) when tested on a population suffering from a particular disease. On the contrary, when a test is performed on a population not suffering from a specific disease, the specificity is the percentage showing negative (normal value) (true negative rate). In other words, the percentage of people who are actually sick who get positive is called sensitivity, and the percentage of people who are not sick who get negative is called specificity. If you try to increase the sensitivity of the test, the specificity will decrease (false positives will increase), and if you try to increase the specificity, the sensitivity will decrease (false negatives will increase).

Generally, a test kit with high sensitivity and specificity is considered to be highly reliable.
A highly sensitive test tends to be positive, and a negative test means that there is a high probability that a disease-free diagnosis can be made, which can be used for exclusion diagnosis. In addition, a test with high specificity tends to be negative, and if such a test is positive, it means that a disease can be diagnosed with a considerably high probability, which can be used for a definitive diagnosis. In addition, the percentage of those who actually have the disease among those who test positive is the "Positive Predictive Value (PPV)", and the percentage of those who do not actually suffer from the negative test. The ratio is called "Negative Predictive Value (NPV)".

According to the results of FIG. 8, even when the cutoff value is 1 or more, the specificity is high at 90%, the test sensitivity of the Non-advanced adenoma patient group is 89%, and the test sensitivity of the Advanced adenoma / Tis patient group is high. The test sensitivity of the colorectal cancer CRC patient group was 59%. If it is positive, it is possible to diagnose with a high probability that there is a large intestine lesion of Non-advanced adenoma or more even if the cutoff value is 1 or more. In this case, the positive rate of the control group is only 10%, and the risk of false positives is smaller than that of the fecal occult blood test described later. In other words, it is possible to avoid a situation in which a person without a lesion in the large intestine has to undergo a detailed examination.

As described above, in the present embodiment, when the cutoff value is 1 or more, the non-advanced adenoma patient group, the more advanced advanced adenoma / Tis patient group, and the colorectal cancer CRC patient group are detected with respect to the control group. It was found that it was excellent in sensitivity and specificity. In particular, it has been found that even those in the early stage of benign adenomas may be detected accurately.

<Methylation positive rate in each group with a cutoff value of 2 or more in the number of methylated TWIST1 copies> FIG. 9
Ratio of methylated TWIST1 positive individuals in each stool sample DNA in the control group, Non-advanced adenoma patient group, Advanced adenoma / Tis patient group, and CRC (colorectal cancer) patient group when the cutoff value was 2 or more: Positive rate ( The vertical axis) is shown in a graph (FIG. 9A), and the number of positives and negatives is shown in a table (FIG. 9B). FIG. 9C shows the sensitivity of methylated TWIST1 detection in each stool sample DNA in the control group, the non-advanced adenoma patient group, the advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group when the cutoff value is 2 or more. , Specificity, positive predictive value, negative predictive value.

According to the above results, when the cutoff value was 2 or more, the specificity was higher at 96%, and the specificity was higher than when the cutoff value was 1 or more. The test sensitivity was 67% for the Non-advanced adenoma patient group, 47% for the Advanced adenoma / Tis patient group, 47% for the colorectal cancer CRC patient group, and the positive predictive value was in order. The values were as high as 75%, 80% and 97%.

If the result is positive, it is possible to diagnose with a high probability that there is a large intestine lesion of Non-advanced adenoma or more with a cutoff value of 2 or more. In this case, the positive rate of the control group is only 3.9%, and the risk of false positives is much smaller than that of the fecal occult blood test described later. In other words, it is possible to avoid a situation in which a person without a lesion in the large intestine has to undergo a detailed examination as much as possible.

As described above, in the present embodiment, when the cutoff value is 2 or more, the non-advanced adenoma patient group, the more advanced advanced adenoma / Tis patient group, and the colorectal cancer CRC patient group are detected with respect to the control group. It was found that the specificity was excellent and the sensitivity was relatively high. In particular, it has been found that even those in the early stage of benign adenomas may be detected accurately.

<Methylation positive rate in each group with a cutoff value of 5 or more in the number of methylated TWIST1 copies> FIG.
Ratio of methylated TWIST1 positive individuals in each stool sample DNA in the control group, Non-advanced adenoma patient group, Advanced adenoma / Tis patient group, and CRC (colorectal cancer) patient group when the cutoff value was 5 or more: Positive rate ( The vertical axis) is shown in a graph (FIG. 10A), and the number of positives and negatives is shown in a table (FIG. 10B). FIG. 10C shows the sensitivity of methylated TWIST1 detection in each stool sample DNA in the control group, the non-advanced adenoma patient group, the advanced adenoma / Tis patient group, and the CRC (colorectal cancer) patient group when the cutoff value is 5 or more. , Specificity, positive predictive value, negative predictive value.

According to the above results, when the cutoff value is 5 or more, the specificity is 100%, which is the highest value. On the other hand, the test sensitivity was about 22% in the Non-advanced adenoma patient group, about 47% in the Advanced adenoma / Tis patient group, and about 33% in the colorectal cancer CRC patient group. The positive predictive value was 100% in all groups, which was even higher than when the cutoff value was 1 or more or 2 or more.

As described above, in the present embodiment, when the cutoff value is 5 or more, the non-advanced adenoma patient group, the more advanced advanced adenoma / Tis patient group, and the colorectal cancer CRC patient group are detected with respect to the control group. It was found that the specificity was 100% and the positive predictive value was 100%, showing excellent values. In particular, in the control, since there are no cases in which the number of copies of methylated TWIST1 is 5 or more in the stool sample DNA, the control group is not positive, and the positive person is at the stage of early benign adenoma or higher. Was found to be very accurate.
[Test Example 1]

The test results when the fecal occult blood test method, which is a general primary screening for colorectal cancer, is used are shown. The protocol followed that of the following products.

(Fecal occult blood test: FOBT)
(1) Preparation of stool samples 10 healthy stools (Control), 9 non-advanced adenoma patient stools, 17 Advanced adenoma / Tis (intramucosal cancer) patient stools, CRC ( 83 stool samples from patients with adenoma), a total of 119 stool samples were used. Grouping was performed by endoscopy and / or pathological examination. The 10 control group is a group of people who did not have colon lesions on endoscopy.

(2) Measurement (operation) method A fecal occult blood kit (OC-Hemodia Auto S'Eiken': Eiken Chemical Co., Ltd.) and a diagnostic device (OC sensor io: Eiken Chemical Co., Ltd.) were used. This kit and equipment are based on the immunoturbidimetric method of latex agglutination, and hardly react with non-human animal hemoglobin.
1) Set the latex emulsion and diluent in place on the device.
2) Enter the parameters into the device.
3) Dispense the standard and its diluent (Hb calibrator L'Eiken'Hb calibrator diluent'Eiken' sold separately) into a sample cup and set it in the sample rack of the device.
4) Start the device.
5) 35 μL of standard solution automatically diluted with standard diluent enters the reaction cell.
6) 100 μL of latex emulsion and 200 μL of diluent enter the reaction cell at the same time.
7) Measure the amount of change in absorbance for 180 seconds at a wavelength of 660 nm.
8) A calibration curve is created from the reaction of each standard solution.
9) Set the sample cup or stool collection container into which the test sample is dispensed as it is in the sample rack of the device.
10) Start the device.
11) 35 μL of the test sample enters the reaction cell, and the amount of change in absorbance is measured by the operations 6) and 7).
12) The concentration of the test substance can be determined from the calibration curve.
13) After the measurement is completed, the result will be printed out.

(3) Judgment method of measurement result The hemoglobin concentration (ng / mL) is determined by comparing the reaction of each sample with the calibration curve prepared in advance.

(4) Performance
1) Sensitivity: When measuring standard solutions with hemoglobin concentrations of 0 ng / mL and 20 ng / mL, the difference in the amount of change in absorbance between the two is 0.002 or more.
2) When measuring a controlled sample with known accuracy and concentration, the obtained value is within ± 15% of the displayed value.
-When testing a positive controlled sample, it shows positive, and when testing a negative controlled sample, it shows negative.
3) Simultaneous reproducibility ・ When the same sample is measured 10 times at the same time, the coefficient of variation (CV) of the obtained value is 10% or less.
-When the positive control sample and the negative control sample are repeatedly tested 5 times, the same result is shown for each.

(5) Measurement range
50 to 1,000 ng / mL (converted value in feces: 10 μg / g to 200 μg / g).

(Result) Fig. 11
Graph (Fig. 11A) regarding the occult blood positive (+) rate or the number of occult blood positive (+) samples in the stool samples of the control group, non-advanced adenoma patient group, advanced adenoma / Tis patient group, and CRC (colorectal cancer) patient group. ) Or the table (Fig. 11B). FIG. 11C shows the sensitivity, specificity, positive predictive value, and negative predictive value of each occult blood test in stool samples of the control group, non-advanced adenoma patient group, advanced adenoma / Tis patient group, and CRC (colorectal cancer) patient group. It is a table showing the rate.

<Positive occult blood test positive rate>
As also shown in FIGS. 11A and 11B, in this embodiment, the test sensitivity was 0% (0/9) in the Non-advanced adenoma patient group and 41% (7/17) in the Advanced adenoma / Tis patient group. It was 95% (79/83) in the CRC (colorectal cancer) patient group. This fecal occult blood test has a slightly higher false positive of 20% in the control group, but the specificity is 80%, and the test sensitivity of the colorectal cancer CRC patient group is 95%, which is high in both sensitivity and specificity. It was confirmed that it can be used as a test to detect colorectal cancer CRC patients to some extent. However, with this method, the test sensitivities of the Non-advanced adenoma patient group and the Advanced adenoma / Tis patient group are as low as 0% and 41%, and even if they are suffering from Non-advanced adenoma or Advanced adenoma / Tis, they are overlooked. The probability of getting rid of it is very high.

(Combination of fecal occult blood test (FOBT) and fecal DNA methylation TWIST1 test)
Once a stool sample is collected in the colorectal cancer screening test, not only the fecal occult blood test can be performed, but also DNA can be extracted from the stool sample and the methylated TWIST1 analysis technique of the present case can be applied. The combination of fecal occult blood test (FOBT) and fecal DNA methylated TWIST1 test means the above-mentioned "combination test", for example, the number of copies of methylated TWIST1 per 20 μL of PCR reaction solution of amplified DNA and feces. This is a method for predicting the presence or absence of a colon tumor by combining the fecal occult blood reaction results in the occult blood test.

Same as fecal occult blood test (Test Example 1), 10 healthy stools (Control), 9 non-advanced adenoma patient stools, Advanced adenoma / Tis (intramucosal cancer) patient stools 17 Specimens, 83 stools from CRC (colorectal cancer) patients, and stool samples from a total of 119 people were used. Regarding the method, the methylated TWIST1 measurement system was carried out in the same manner as in Example 3, and the fecal occult blood test was carried out in the same manner as in Test Example 1.

<Combination of fecal occult blood reaction result (+) and fecal DNA methylated TWIST1 copy number 1 or more> Fig. 12
FIG. 12 shows the data when the combination test of the fecal occult blood test (FOBT) and the fecal DNA methylated TWIST1 test is positive when the number of copies of FOBT (+) and / or methylated TWIST1 is 1 or more, and is the control group. , Non-advanced adenoma patient group, Advanced adenoma / Tis patient group, CRC (colorectal cancer) patient group, graph of positive (Yes) rate / negative (No) rate in the above combination test (Fig. 12A) , The number of positive (Yes) and negative (No) samples in the combination test (FIG. 12B), and the sensitivity, specificity, positive predictive value, and negative predictive value (FIG. 12C) of the combination test.

In addition, 1) when the fecal occult blood reaction result is (+) in the fecal occult blood test and the fecal DNA methylation TWIST1 copy number is 1 or more in the fecal DNA methylation TWIST1 test, 2) the fecal occult blood reaction result is (-) in the fecal occult blood test. ) And stool DNA methylation TWIST1 test stool DNA methylation TWIST1 copy number 1 or more, and 3) stool occult blood test result (+) and stool DNA methylation TWIST1 test stool DNA methylation Three cases where the number of TWIST1 copies was less than 1 were positive. That is, whether or not it was positive was determined by combining the number of copies of stool DNA methylated TWIST1 in the stool DNA methylated TWIST1 test and the fecal occult blood reaction result in the stool occult blood test.

According to the above results, when the fecal occult blood test and the fecal DNA methylated TWIST1 test are combined and the fecal DNA methylated TWIST1 copy number is 1 or more, the specificity is as low as 40%, but the test sensitivity is Non. -The values were very high, 89% in the advanced adenoma patient group, 88% in the Advanced adenoma / Tis patient group, and 100% in the colorectal cancer CRC patient group. If the result is positive here, it is clear that non-advanced adenoma patients, Advanced adenoma / Tis patients, and CRC (colorectal cancer) patients can be detected with a very high probability. In the case of a methylated TWIST1 independent test, it is possible to diagnose with a high probability that there is a large intestine lesion of Non-advanced adenoma or more even if the cutoff value is 1 or more.

<Combination of fecal occult blood reaction result (+) and fecal DNA methylated TWIST1 copy number 2 or more> FIG. 13
FIG. 13 shows the data when the combination test of the fecal occult blood test (FOBT) and the fecal DNA methylated TWIST1 test is positive when the number of copies of FOBT (+) and / or methylated TWIST1 is 2 or more, and is the control group. , Non-advanced adenoma patient group, Advanced adenoma / Tis patient group, CRC (colorectal cancer) patient group, graph of positive (Yes) rate / negative (No) rate in combination test (Fig. 13A), The number of positive (Yes) and negative (No) samples in the combination test (FIG. 13B), and the sensitivity, specificity, positive predictive value, and negative predictive value of the combination test (FIG. 13C).

In addition, 1) when the fecal occult blood reaction result is (+) in the fecal occult blood test and the fecal DNA methylation TWIST1 copy number is 2 or more in the fecal DNA methylation TWIST1 test, 2) the fecal occult blood reaction result is (-) in the fecal occult blood test. ) And stool DNA methylation TWIST1 test stool DNA methylation TWIST1 copy number 2 or more, and 3) stool occult blood test result (+) and stool DNA methylation TWIST1 test stool DNA methylation Three cases where the number of copies of TWIST1 was less than 2 were positive. That is, whether or not it was positive was determined by combining the number of copies of stool DNA methylated TWIST1 in the stool DNA methylated TWIST1 test and the fecal occult blood reaction result in the stool occult blood test.

According to the above results, when the fecal occult blood test and the fecal DNA methylated TWIST test are combined and the number of copies of the fecal DNA methylated TWIST is 2 or more, the specificity is higher at 70% and the test sensitivity is Non-. It was a very high value of 67% in the advanced adenoma patient group, 82% in the Advanced adenoma / Tis patient group, and 100% in the colorectal cancer CRC patient group. If the result is positive here, non-advanced adenoma patients, Advanced adenoma / Tis patients, and CRC (colorectal cancer) patients can be detected with a very high probability.

<Combination of fecal occult blood reaction result (+) and fecal DNA methylated TWIST1 copy number 5 or more> FIG. 14
FIG. 14 shows the data when the combination test of the fecal occult blood test (FOBT) and the fecal DNA methylated TWIST1 test is positive when the number of copies of FOBT (+) and / or methylated TWIST1 is 5 or more, and is the control group. , Non-advanced adenoma patient group, Advanced adenoma / Tis patient group, CRC (colorectal cancer) patient group, graph of positive (Yes) rate / negative (No) rate in combination test (Fig. 14A), The number of positive (Yes) and negative (No) samples in the combination test (FIG. 14B), and the sensitivity, specificity, positive predictive value, and negative predictive value of the combination test (FIG. 14C).

In addition, 1) when the fecal occult blood reaction result is (+) in the fecal occult blood test and the fecal DNA methylation TWIST1 copy number is 5 or more in the fecal DNA methylation TWIST1 test, 2) the fecal occult blood reaction result is (-) in the fecal occult blood test. ) And stool DNA methylation TWIST1 test stool DNA methylation TWIST1 copy number 5 or more, and 3) stool occult blood test result (+) and stool DNA methylation TWIST1 test stool DNA methylation Three cases where the number of TWIST1 copies was less than 5 were positive. That is, whether or not it was positive was determined by combining the number of copies of stool DNA methylated TWIST1 in the stool DNA methylated TWIST1 test and the fecal occult blood reaction result in the stool occult blood test.

According to the above results, in the combined test method for examining the presence or absence of colorectal adenoma by combining the fecal occult blood test and the fecal DNA methylated TWIST1 test, the specificity is 80% when the number of copies of the fecal DNA methylated TWIST1 is 5 or more. The test sensitivity is as low as 22% in the Non-advanced adenoma patient group, but 82% in the Advanced adenoma / Tis patient group and 99% in the colorectal cancer CRC patient group. It was a high value. The positive predictive value was very high at 88% and 98% in the Advanced adenoma / Tis patient group and the CRC (colorectal cancer) patient group, respectively. Regarding the false positive rate in the control, it can be said that the result is excellent at the same level (20%) as in the case of the fecal occult blood test (false positive 20%, FIG. 11A).

In summary, as a whole, compared to the conventional fecal occult blood test, this combination test method has a false positive rate of 20% in the control (healthy person), and further, in the CRC (colon cancer) patient group. Sensitivity, specificity, positive predictive value and negative predictive value were 95%, 80%, 98% and 67% in the fecal occult blood test, and 99%, 80%, 98% and 89% in this combination test method. This combination test method is generally higher, and the sensitivity, specificity, positive predictive value and negative predictive value in patients with Advanced adenoma / Tis are 41% and 80 in the fecal occult blood test. In this combination test method, the values were 82%, 80%, 88%, and 73%, which were even higher than those of%, 78%, and 44%.

In other words, this combination test method is a very excellent test method as a primary screening test method, and if the test method is positive, there is a risk of developing colorectal cancer as well as CRC (colorectal cancer) patients. It has been found that patients with Advanced adenoma / Tis that can be cured with high and early treatment can be detected accurately and with a very high probability.

Not only the discovery of CRC (colorectal cancer) patient group, but also the advanced adenoma / Tis patient group has an opportunity to perform treatment such as surgical treatment before becoming malignant, so its lifesaving effect can be very large. Understand. Further, as described above, the methylation analysis technique included in the inspection method of the present embodiment does not require chemical treatment by heavy sulfite treatment of DNA, which is a step of the conventional methylation analysis technique, and thus is a complicated operation. The inspection can be carried out very easily. In addition, since it is sufficient to have a very small amount (about 0.2 g) of a stool sample and a very small amount (1 to 5 copies) of methylated TWIST1 in the stool can be detected, not only the specificity but also the detection can be detected. It can be said that the sensitivity is also very high.

(Methylated TWIST1 measurement using stool sample DNA)
Control, non-advanced adenoma patients, advanced adenoma / Tis patients, new cases of colorectal cancer (CRC) DNA extraction in the same manner as in Example 3, Complex enzyme treatment of DNA, digital PCR treatment, and copy count were performed.

(result)
<Number of copies per 20 μL of PCR reaction solution of methylated TWIST1 in each group>
15A and 15B show a control group, a non-advanced adenoma patient group, an advanced adenoma / Tis patient group, and a stage 1-4 colorectal cancer (CRC) patient group. It is a graph of the result of comparing the number of copies or the ratio of the number of copies per 20 μL of the PCR reaction solution of methylated TWIST1 in each stool sample DNA (stool DNA) of each group. n indicates the number of samples. In addition, FIG. 16 shows a group of patients with non-advanced adenoma, a group of patients with advanced adenoma / Tis, and a group of patients with stage 1 to 4 colorectal cancer (CRC) for control. The result of the ROC curve is shown. According to the present invention, the number of copies increases as the control progresses from non-advanced adenoma, advanced adenoma / intramucosal cancer, and stage 1 to 4 colorectal cancer, and not only control and colorectal cancer but also control and progression. There was a significant difference in adenoma / intramucosal cancer, and it was confirmed that not only colorectal cancer but also advanced adenoma / intramucosal cancer can be detected.

(Examination of quantification of BMP3, NDRG4, and SEPT9 methylation levels with primers for digital PCR)
DNA known to have high levels of BMP3, NDRG4, and SEPT9 methylation (colorectal cancer cell line HCT116-derived DNA) and DNA known to have little BMP3, NDRG4, and SEPT9 methylation (peripheral blood lymphocytes) Sphere-derived DNA) was mixed in various proportions in the same manner as in Table 4. This DNA is treated with a plurality of methylation-sensitive restriction enzymes and exonuclease treatment (complex enzyme treatment) in the same manner as in Example 2, and then the methylation level quantification by digital PCR is examined using the designed primers and probes. did.

<Primer / probe set>
The primers and probes used are as follows.
(BMP3)
BMP3 forward primer (SEQ ID NO: 11) 5'-GGAAGGTACAGACAGATCTTGAAAACA-3'
BMP3 reverse primer (SEQ ID NO: 12) 5'-TCCACTCCAACGCTGAGAAA-3'
BMP3 TaqMan probe (SEQ ID NO: 13) 5'-FAM- CCGGGCCACACAC-TAMRA-3'
(NDRG4)
NDRG4 forward primer (SEQ ID NO: 14) 5'-CCGACCCTAAGGGCTTTTCT -3'
NDRG4 Reverse Primer (SEQ ID NO: 15) 5'-CGCTGCCGTAGTCTTTGTTTAGA-3'
NDRG4 TaqMan probe (SEQ ID NO: 16) 5'-FAM- TCTCTGCAGGTCTAAGG-TAMRA-3'
(SEPT9)
SEPT9 Forward Primer (SEQ ID NO: 17) 5'-GCCCACCAGCCATCATGT-3'
SEPT9 Reverse Primer (SEQ ID NO: 18) 5'-GTCCGAAATGATCCCATCCA-3'
SEPT9 TaqMan probe (SEQ ID NO: 19) 5'-FAM- CCGCGGTCAACGC-TAMRA-3'
(Internal control)
hTERT forward primer (SEQ ID NO: 7) 5'-GGGTCCTCGCCTGTGTACAG-3'
hTERT Reverse Primer (SEQ ID NO: 8) 5'-CCTGGGAGCTCTGGGAATTT-3'
hTERT TaqMan probe (SEQ ID NO: 9) 5'-VIC-CACACCTTTGGTCACTC-MGB-3'

Table 5 shows the sequences of the forward primers, reverse primers, TaqMan probe, and PCR amplification target regions of BMP3, NDRG4, and SEPT9 (regions -333 to -256 from the start codon of the BMP3 gene: position information in the GRCh37 / hg19 database chr4: 81,952,106-81,952,183: SEQ ID NO: 20, -300 to -500 region from NDRG4 gene transcription initiation site (-24587 to -24446 region from start codon): Position information in GRCh37 / hg19 database chr16: 58,497,096-58,497,237: SEQ ID NO: 21, Regions -14 to +48 from the start codon of the SEPT9 gene: Location information in the GRCh37 / hg19 database chr17: 75,369,566-75,369,627: SEQ ID NO: 22) is shown. In Table 5, the underline shows the sites that may be cleaved by the restriction enzymes HhaI, HpaII, and BstUI. However, since these restriction enzymes are methylation-sensitive restriction enzymes, if cytosine (C) is methylated, the site is not cleaved.

(result)
<Quantitative by digital PCR> FIGS. 17A, B, 18A, B, 19A, B
17A, 18A, and 19A are diagrams showing the number of copies of methylated BMP3, methylated NDRG4, and methylated SEPT9 and the number of TRT copies when DNAs of various methylation levels are used, respectively. 17B, 18B, and 19B are diagrams showing the ratio of the number of copies of methylated BMP3, methylated NDRG4, and methylated SEPT9 to the number of TRT copies when DNAs of various methylation levels are used. .. It was found that there is a positive linear correlation between the proportion of methylated control DNA and the number of copies of methylated BMP3, methylated NDRG4, and methylated SEPT9. On the other hand, since the hTERT gene was not cleaved by the methylation susceptibility restriction enzyme, it showed a constant number of copies regardless of the mixing ratio of HCT116. Therefore, by the complex enzyme treatment, not only TWIST1, but also the region encoding the BMP3, NDRG4, and SEPT9 genes, the -1 to -1100 region from the start codon of any of the genes, or any of the genes. It was clarified that the degree of methylation of the CpG sequence in a part of the -300 to -500 region can be quantitatively measured from the transcription start point of the gene.

(Measurement of methylated BMP3, methylated NDRG4, or methylated SEPT9 using fecal sample DNA)
Methylated BMP3, methylated NDRG4, or methylated SEPT9 was performed in the same experiment as in Example 3. DNA extraction, DNA complex enzyme treatment, digital PCR treatment, and copy count were performed in the same manner as in Example 3.

(result)
<Number of copies per 20 μL of methylated BMP3, methylated NDRG4, or methylated SEPT9 PCR reaction in each group>
20A and 20B show the PCR of methylated BMP3 in each stool sample DNA (stool DNA) of the control group, the non-advanced adenoma patient group, the advanced adenoma / intramucosal cancer patient group, and the stage 1 to 4 colorectal cancer patient group. It is a graph of the result of comparing the number of copies or the ratio of the number of copies per 20 μL of the reaction solution by group. 22A and 22B show the PCR of methylated NDRG4 in each stool sample DNA (stool DNA) of the control group, the non-advanced adenoma patient group, the advanced adenoma / intramucosal cancer patient group, and the stage 1 to 4 colorectal cancer patient group. It is a graph of the result of comparing the number of copies or the ratio of the number of copies per 20 μL of the reaction solution by group. 24A and 24B show the PCR of methylated SEPT9 in each stool sample DNA (stool DNA) of the control group, the non-advanced adenoma patient group, the advanced adenoma / intramucosal cancer patient group, and the stage 1 to 4 colorectal cancer patient group. It is a graph of the result of comparing the number of copies or the ratio of the number of copies per 20 μL of the reaction solution by group. n indicates the number of samples. Also shown in FIGS. 21, 23, and 25 are non-advanced adenoma patient groups and advanced adenoma / intramucosal cancer (Non-advanced adenoma) for control of methylated BMP3, methylated NDRG4, or methylated SEPT9 copy counts. The results of ROC curves for each of the Advanced adenoma / Tis) patient group and the Stage 1 to 4 colorectal cancer (CRC) patient groups are shown. According to the present invention, the number of copies increases as the control progresses from non-advanced adenoma, advanced adenoma / intramucosal cancer, and stage 1 to 4 colorectal cancer, and not only control and colorectal cancer but also control and progression. There was a significant difference in gonadoma / intramucosal cancer, and it was confirmed that not only colorectal cancer but also advanced adenoma / intramucosal cancer can be detected. Furthermore, it was confirmed that non-advanced adenomas can also be detected by using the methylated BMP3 / TRT ratio.

(Methylated TWIST1, NDRG4 measurement using serum cell-free DNA)
Using serum cell-free DNA, DNA extraction, DNA complex enzyme treatment, digital PCR treatment, and copy count were performed. After DNA extraction, DNA complex enzyme treatment, digital PCR treatment, and copy count were performed in the same manner as in Example 3.

Extraction of DNA from serum (serum cell-free DNA) From 400 μL of serum, an automatic nucleic acid extractor MagNA Pure Compact (Roche Diagnostics) and a dedicated DNA extraction kit MagNA pure Compact Nucleic Acid Isolation kit I (Roche Diagnostics) DNA was extracted by the protocol Total_NA_Plasma_100_400 set in the automatic nucleic acid extractor MagNA Pure Compact using Sticks), and finally 50 μL of DNA solution was obtained.

(result)
<Number of copies of methylated TWIST1 and methylated NDRG4 in each group per 20 μL of PCR reaction solution>
26A and 26B show 20 μL of a PCR reaction solution of methylated TWIST1 in each serum cell-free DNA of the control group, the non-advanced adenoma patient group, the advanced adenoma / intramucosal cancer patient group, and the stage 1 to 4 colorectal cancer patient group. Number of copies or ratio of copies per copy, FIG. 28 shows methylation in each serum cell-free DNA of the control group, non-advanced adenoma patient group, advanced adenoma / intramucosal cancer patient group, and stage 1-4 colorectal cancer patient group. It is a graph of the result of comparing the copy number ratio per 20 μL of the PCR reaction solution of NDRG4 by group. n indicates the number of samples. In addition, FIGS. 27 and 29 show the results of ROC curves for the control with each of the non-advanced adenoma patient group, the advanced adenoma / intramucosal cancer patient group, and the stage 1 to 4 colorectal cancer patient group, respectively. According to the present invention, it was confirmed that the presence or absence of colorectal cancer can be detected not only by stool but also by DNA extracted from serum.

From the above, it was found that the test method according to this embodiment has higher test sensitivity and is superior to the fecal occult blood test in detecting the CRC (colorectal cancer) patient group. In addition, it was shown that the detection of the advanced adenoma / Tis patient group is a test method showing high sensitivity and specificity, which is not found in the conventional fecal occult blood test. Furthermore, as a whole, the cost is low, the DNA test procedure is simple and easy, and the amount of stool sample is small, which is very useful as a screening method.

Therefore, the test method of this embodiment is for colorectal tumors, especially colorectal cancer and Advanced adenoma / Tis (intramucosal cancer), which are excellent in cost performance, simple in procedure, and highly specific and sensitive. It turned out to be very useful and effective as a primary screening method for.

Although a specific part of the content of the present invention has been described in detail above, such a specific description is merely a suitable embodiment for a person having ordinary knowledge in the art, thereby the present invention. It is clear that the range is not limited. Therefore, it can be said that the substantial scope of the present invention is defined by the claims and their equivalents.

By using the method for inspecting the presence or absence of colorectal tumors of the present invention, it is possible to develop a kit or device for detecting colorectal tumors such as colorectal cancer and colorectal adenoma at low cost, with high sensitivity and high specificity. It becomes. It provides a very useful tool in the detection of tumors for which early detection is paramount, especially cancer and advanced adenomas.

Claims (5)

A method for predicting the presence or absence of a large intestine tumor in a subject to be examined, wherein the following steps (a) to (d) are sequentially provided, and no sodium bisulfite treatment is performed.
(A) Step of extracting DNA in stool, serum or plasma collected from a test subject;
(B-1) The step of treating the DNA obtained in step (a) with HaI, HpaII, and exonuclease I (ExoI);
(B-2) After the step (b-1), a step of further adding BstUI for processing;
(C) A region encoding the TWIST1 gene, BMP3 gene, NDRG4 gene, or SEPT9 gene in the DNA treated with the methylation susceptibility limiting enzyme in step (b), -1 to 1 from the start codon of any of the above genes. -1100 region, or a part of -300 to -500 region from the transcription start point of any of the above genes, and any of the following (c1)-(c4) was amplified and amplified. The step of measuring the degree of methylation of one or more CpG sequences in DNA;
(C1) Chr7: 19,157,854-19,157,945 shown in SEQ ID NO: 10;
(C2) chr4: 81,952,106-81,952,183 shown in SEQ ID NO: 20;
(C3) chr16: 58,497,096-58,497,237 shown in SEQ ID NO: 21;
(C4) chr17: 75,369,566-75,369,627 shown in SEQ ID NO: 22;
(D) When the degree of methylation measured in step (c) is equal to or greater than a predetermined value, it is predicted that the subject to be examined has a large intestine tumor, and when the degree of methylation is less than the predetermined value, the subject to be examined. The process of predicting the absence of colorectal tumors in The method according to claim 1, wherein the DNA is amplified by digital PCR in the step (c). Claim 1 is characterized in that any amplification of (c1)-(c4) in step (c) is performed using any of the following primer pairs and probe sets of (c5)-(c8). Or the method according to 2.
(C5) A set of primer pairs consisting of the oligonucleotides shown in SEQ ID NOs: 4 and 5 and probes consisting of the oligonucleotides shown in SEQ ID NO: 6;
(C6) A set of primer pairs consisting of the oligonucleotides shown in SEQ ID NOs: 11 and 12 and probes consisting of the oligonucleotides shown in SEQ ID NO: 13;
(C7) A set of primer pairs consisting of the oligonucleotides shown in SEQ ID NOs: 14 and 15 and probes consisting of the oligonucleotides shown in SEQ ID NO: 16;
(C8) A set of primer pairs consisting of the oligonucleotides shown in SEQ ID NOs: 17 and 18 and probes consisting of the oligonucleotides shown in SEQ ID NO: 19; The method according to any one of claims 1 to 3, wherein the stool, serum or plasma collected from the test subject is the stool collected from the test subject. The method according to any one of claims 1 to 4 , wherein the degree of methylation measured in step (c) is combined with the fecal occult blood test result to predict the presence or absence of a large intestine tumor in the test subject.

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