JP7144037B2 - Diagnostic biomarkers for cancer - Google Patents

Description

The present invention relates to a method for determining the presence or absence of cancer development and a kit for determining the presence or absence of cancer development.

According to the "Vital Statistics" conducted annually by the Ministry of Health, Labor and Welfare, cancer is the leading cause of death in Japan, followed by heart disease in second place and pneumonia in third place. According to the 2013 statistical results, the number of deaths due to cancer exceeds 360,000 per year, which means that almost one in three people die of cancer. Looking at cancer mortality rates by organ in recent years, although there are some differences between men and women, colon cancer (colon cancer and rectal cancer), lung cancer, liver cancer, and stomach cancer tend to be the highest in that order.

In cancer treatment, early detection is extremely important, and many cancer examination methods have been developed so far. For example, the fecal occult blood test is widely used for colorectal cancer, but has the problem of low detection sensitivity and difficulty in early detection. In addition, although endoscopic examination is widely used for colorectal cancer and gastric cancer, there is a problem that subjects are highly resistant to endoscopic examination.

In recent years, cancer determination methods have been reported that focus on the relationship between cancer and double-stranded DNA fragments in serum or plasma samples. For example, a method for judging cancer using the size pattern of double-stranded DNA fragments containing repeated sequences (LINE1) in a serum sample as an indicator has been reported (Patent Document 1). Also, a method for determining gastric cancer has been reported, using the methylation level of a RUNX3 gene-derived double-stranded DNA fragment in a serum sample as an indicator (Non-Patent Document 1). However, in this literature, the number of gastric cancer patients is as small as 4 cases, and there is a possibility that the reliability is low, and it is unclear whether or not early cancer can be diagnosed. In addition, Non-Patent Document 2 reports that RUNX3 gene-derived double-stranded DNA fragments in serum samples collected from stage I early gastric cancer patients are methylated. However, the ratio of methylated patients was as low as about 19% (4/21), and there was a problem with detection sensitivity.

Japanese Patent Publication No. 2012-521772

Tan, S.H. et al, Oncol Rep. 2007, 18(5):1225-1230Oncol Rep. Lu, X.X. et al, Cancer. 2012, 118(22):5507-5517Oncol Rep.

An object of the present invention is to provide a diagnostic biomarker for cancer that can accurately determine the presence or absence of early cancer.

In order to solve the above problems, the present inventors measured the cytosine methylation level at the cytosine/guanine (CG) site of RUNX3 gene-derived double-stranded DNA fragments contained in cancer patient-derived serum samples. However, since many of the RUNX3 gene-derived double-stranded DNA fragments have DNA polymorphism, it was necessary to start by identifying the CG site common to the entire RUNX3 gene-derived double-stranded DNA fragment. In addition, even if the CG site common to the entire RUNX3 gene-derived double-stranded DNA fragment could be identified, many methylated cytosines unrelated to cancer were found, making it extremely difficult to identify cancer-specific methylated cytosines. rice field. The three types of RUNX3 gene-derived double-stranded DNA fragments defined in claim 1 etc. also had many DNA polymorphisms, and identification was thought to be difficult. We found that methylation of specific cytosines is associated with the development of stage I early gastric cancer. Furthermore, when measuring the ratio of double-stranded DNA fragments derived from the human telomerase reverse transcriptase gene (hTERT gene, hereinafter simply referred to as "TERT gene") contained in cancer patient-derived serum samples, the methylation level and It was confirmed that the presence or absence of stage I early gastric cancer can be determined more accurately by using a combination of these ratios as an index, compared to the case where each index is used alone. The present invention has been completed based on these findings.

That is, the present invention is as follows.
[1] A cancer determination method comprising the following steps (a) to (c).
(a) measuring the cytosine methylation level in one or more RUNX3 gene-derived double-stranded DNA fragments in a serum sample or plasma sample collected from a subject,
The RUNX3 gene-derived double-stranded DNA fragment comprises a double-stranded DNA fragment comprising the nucleotide sequence shown in SEQ ID NO: 1,
The above step, wherein the cytosine is the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 1;
(b) comparing the methylation level measured in step (a) to said methylation level in a cancer-free control;
(c) assessing that said subject is likely to have cancer when the methylation level measured in step (a) is higher than the methylation level in a control subject;
[2] A cancer determination method comprising the following steps (A) to (C).
(A) Measuring the cytosine methylation level and the proportion of TERT gene-derived double-stranded DNA fragments in one or more RUNX3 gene-derived double-stranded DNA fragments in a serum sample or plasma sample collected from a subject a step of
The RUNX3 gene-derived double-stranded DNA fragment comprises a double-stranded DNA fragment comprising the nucleotide sequence shown in SEQ ID NO: 1,
The above step, wherein the cytosine is the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 1;
(B) comparing the methylation level and the percentage of double-stranded DNA fragments measured in step (A) with said methylation level and said percentage of DNA fragments, respectively, in a cancer-free control;
(C) when at least one of the methylation level and the proportion of double-stranded DNA fragments measured in step (A) is higher than the methylation level and the proportion of double-stranded DNA fragments, respectively, in the control subject; Assessing that the subject is likely to have cancer;
[3] the RUNX3 gene-derived double-stranded DNA fragment further comprises a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO:2 and a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO:3;
In steps (a) and (A), the methylation level of the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 2 and the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 3 is further measured. The method according to the above [1] or [2].
[4] The method according to any one of [1] to [3] above, wherein the cancer is gastric cancer or pancreatic cancer.
[5] The method of [4] above, wherein the gastric cancer is stage I gastric cancer.
[6] The method of any one of [2] to [5] above, wherein the TERT gene-derived double-stranded DNA is a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO:4.
[7] Forward and reverse primers and/or probes for measuring cytosine methylation levels in one or more RUNX3 gene-derived double-stranded DNA fragments in serum or plasma samples, or labels thereof A cancer determination kit comprising:
The RUNX3 gene-derived double-stranded DNA fragment comprises a double-stranded DNA fragment comprising the nucleotide sequence shown in SEQ ID NO: 1,
The kit, wherein the cytosine is the 54th cytosine in the nucleotide sequence shown in SEQ ID NO:1.
[8] Forward and reverse primers and/or probes for measuring cytosine methylation levels in one or more RUNX3 gene-derived double-stranded DNA fragments in serum or plasma samples, or labels thereof things and
A cancer determination kit comprising forward primers, reverse primers and/or probes for measuring the ratio of TERT gene-derived double-stranded DNA fragments in serum or plasma samples, or labeled products thereof. hand,
The RUNX3 gene-derived double-stranded DNA fragment comprises a double-stranded DNA fragment comprising the nucleotide sequence shown in SEQ ID NO: 1,
The kit, wherein the cytosine is the 54th cytosine in the nucleotide sequence shown in SEQ ID NO:1.
[9] the RUNX3 gene-derived double-stranded DNA fragment further comprises a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO:2 and a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO:3;
Forward primer and reverse primer, and / or probes, or their labels, the methylation level of the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 2 and the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 3 The kit according to [7] or [8] above, which is for further measuring.
[10] The kit of [7] to [9] above, wherein the cancer is gastric cancer or pancreatic cancer.
[11] The kit of [10] above, wherein the gastric cancer is stage I gastric cancer.
[12] The kit of any one of [8] to [11] above, wherein the double-stranded DNA derived from the TERT gene is a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO:4. .

As another embodiment of the present invention,
A step of measuring the cytosine methylation level in one or more RUNX3 gene-derived double-stranded DNA fragments in a serum sample or plasma sample collected from a subject,
The RUNX3 gene-derived double-stranded DNA fragment comprises a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 1 (hereinafter sometimes referred to as "RUNX3 gene-derived double-stranded DNA fragment 1"), and Optionally, a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 2 (hereinafter sometimes referred to as "RUNX3 gene-derived double-stranded DNA fragment 2") and a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 3 A single-stranded DNA fragment (hereinafter sometimes referred to as "RUNX3 gene-derived double-stranded DNA fragment 3") (hereinafter collectively referred to as "RUNX3 gene-derived double-stranded DNA fragments 1 to 3") there is),
The cytosine is the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 1 (hereinafter sometimes referred to as "this cytosine 1"), the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 2 (hereinafter, "this cytosine 2”), and the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 3 (hereinafter sometimes referred to as “this cytosine 3”) (hereinafter, these cytosines are collectively referred to as “this cytosine 1 ~ 3”), a cancer determination method including the above steps;
Measure the methylation level of the subject cytosine 1 in one or more RUNX3 gene-derived double-stranded DNA fragments 1 in a serum sample or plasma sample collected from a subject and the ratio of TERT gene-derived double-stranded DNA fragments optionally, further measuring the methylation level of the subject cytosines 2-3 in the RUNX3 gene-derived double-stranded DNA fragments 2-3 in the serum or plasma sample, wherein the step (A ), including cancer determination methods; and
A method of collecting data for determining (diagnosing) cancer, including the above steps (a) to (c); and
A method of collecting data for determining (diagnosing) cancer, including the above steps (A) to (C); and
Treatment of cancer treatment ( For example, a method for diagnosing cancer, optionally including the step (p) of applying radiation therapy, anticancer drug therapy, etc.; and
Treatment for treating cancer ( For example, a method for diagnosing cancer, optionally including the step (p) of applying radiation therapy, anticancer drug therapy, etc.; and
for use in cancer determination (diagnostic) methods,
Forward and reverse primers and/or probes for measuring the methylation level of the subject cytosine 1 in one or more RUNX3 gene-derived double-stranded DNA fragments 1 in serum or plasma samples, or labels thereof optionally, said forward primer and reverse primer for further measuring the methylation level of said cytosines 2-3 in RUNX3 gene-derived double-stranded DNA fragments 2-3 in said serum or plasma sample and/or probes or labels thereof;
for use in cancer determination (diagnostic) methods,
Forward and reverse primers and/or probes for measuring the methylation level of the subject cytosine 1 in one or more RUNX3 gene-derived double-stranded DNA fragments 1 in serum or plasma samples, or labels thereof and a forward primer and reverse primer and/or probe for measuring the proportion of TERT gene-derived double-stranded DNA fragments in a serum sample or plasma sample, or a label thereof, wherein the forward Primers and reverse primers and/or probes, or labels thereof, optionally determine the methylation level of the subject cytosines 2-3 in the RUNX3 gene-derived double-stranded DNA fragments 2-3 in the serum sample or plasma sample. The combination, which is for further measurement; and
A cancer-determining biomarker comprising methylation of the subject cytosine 1 in the RUNX3 gene-derived double-stranded DNA fragment 1, optionally the subject cytosines 2-3 in the RUNX3 gene-derived double-stranded DNA fragment 2-3 wherein the methylation level of the cytosine 1-3 in a serum or plasma sample taken from a person with cancer is greater than the methylation level of the cytosine 1 in a control person without cancer said biomarker, which is high compared to a methylation level of ~3;
Methylation of the subject cytosine 1 in RUNX3 gene-derived double-stranded DNA fragment 1 for use as a biomarker in a cancer determination (diagnosis) method, optionally comprising RUNX3 gene-derived double-stranded DNA fragments 2-3 in combination with the methylation of the cytosines 2-3 of this subject, and the methylation level of the cytosines 1-3 in a serum sample or plasma sample collected from a person suffering from cancer increased methylation of the subject cytosine 1 compared to levels of methylation of the subject cytosines 1-3 in controls who have not been tested; and
A combination of the subject cytosine 1 methylation in RUNX3 gene-derived double-stranded DNA fragment 1 and a TERT gene-derived double-stranded DNA fragment for use as a biomarker in a cancer determination (diagnosis) method, and any in the RUNX3 gene-derived double-stranded DNA fragment 2-3, the methylation of the cytosine 2-3 is used in combination, and the cytosine 1 in the serum or plasma sample collected from a person suffering from cancer At least one of the methylation level of ~3 and the ratio of TERT gene-derived double-stranded DNA fragments is higher than the methylation level of cytosines 1-3 and the TERT gene-derived double-stranded DNA fragment, respectively, in cancer-free controls. The combination, which is high compared to the proportion of single-stranded DNA fragments;
can be mentioned.

According to the present invention, it is possible to accurately determine the presence or absence of the onset of cancer, especially stage I early gastric cancer. and contribute to early treatment.

FIG. 1A is a diagram showing the results of measuring the ratio of methylated cytosines 1 to 3 in RUNX3 gene-derived double-stranded DNA fragments 1 to 3 contained in serum samples from a gastric cancer group and a healthy group. The vertical axis indicates the number of methylated cytosines 1-3 per 40 μL of serum. A white circle (○) in the figure indicates each subject. “**” in the figure indicates that there is a statistically significant difference (p=0.0019). FIG. 1B is a diagram showing an ROC (Receiver Operating Characteristic) curve created based on the results obtained in FIG. 1A. FIG. 2A is a diagram showing the results of measuring the percentage of TERT gene-derived double-stranded DNA fragments contained in serum samples from a gastric cancer group and a healthy group. The vertical axis indicates the number of TERT gene-derived double-stranded DNA fragments per 40 μL of serum. A white circle (○) in the figure indicates each subject. "**" in the figure indicates that there is a statistically significant difference (p=0.0056). FIG. 2B is a diagram showing an ROC curve created based on the results obtained in FIG. 2A. FIG. 3A is a diagram showing the results of measuring the ratio of methylated cytosines 1 to 3 in RUNX3 gene-derived double-stranded DNA fragments 1 to 3 contained in serum samples from a pancreatic cancer group and a healthy group. . The vertical axis indicates the number of methylated cytosines 1-3 per 40 μL of serum. A white circle (○) in the figure indicates each subject. FIG. 3B is a diagram showing an ROC curve created based on the results obtained in FIG. 3A.

As the cancer determination method of the present invention,
The methylation level of the subject cytosine 1 in the RUNX3 gene-derived double-stranded DNA fragment 1 in a serum or plasma sample collected from a subject, and optionally, the RUNX3 gene-derived double-stranded DNA fragment in said serum or plasma sample Measuring the methylation level of the cytosines 2 and 3 of interest in 2 and 3 (hereinafter collectively referred to as "the methylation level of the RUNX3 fragment in the subject") and, if necessary, the step of quantifying (a ); compare the "RUNX3 fragment methylation level in the subject" with the methylation level in a cancer-free control (hereinafter sometimes referred to as "RUNX3 fragment methylation level in the control"); step (b); and when "the methylation level of the RUNX3 fragment in the subject" is higher than the "methylation level of the RUNX3 fragment in the control", the subject is highly likely to be suffering from cancer. A method including the steps (a) to (c) of the step (c) of evaluating (hereinafter sometimes referred to as "determination method 1"); and
"Methylation level of RUNX3 fragment in subject" and ratio of TERT gene-derived double-stranded DNA fragment in serum sample or plasma sample collected from subject (hereinafter sometimes referred to as "percentage of TERT fragment in subject") Step (A) of measuring and optionally quantifying; and the ratio of the TERT gene-derived double-stranded DNA fragments in controls not suffering from cancer (hereinafter sometimes referred to as "the ratio of TERT fragments in controls"); Either one or both of the "methylation level of RUNX3 fragment in" and "percentage of TERT fragment in subject" are higher than "methylation level of RUNX3 fragment in control" and "percentage of TERT fragment in control", respectively A method comprising steps (A) to (C) of step (C) for evaluating that the subject is highly likely to be suffering from cancer (hereinafter referred to as "determination method 2") be);
If so, there is no particular limitation (Determination method 1 and Determination method 2 may be collectively referred to as "determination method" hereinafter). This determination method is a method for assisting a doctor in diagnosing the presence or absence of cancer, and does not include the act of diagnosis by a doctor.

In addition, as the cancer determination kit of the present invention,
Determination of cancer, including forward primers and reverse primers (hereinafter sometimes referred to as "primer sets") and / or probes for measuring "methylation level of RUNX3 fragment in a subject", or labels thereof A kit for use in (diagnosis) (hereinafter sometimes referred to as "this kit 1");
Primer sets and / or probes for measuring the "methylation level of RUNX3 fragment in a subject", or labeled products thereof,
A kit for use in determining (diagnosing) cancer, including a primer set and/or a probe for measuring the "percentage of TERT fragments in a subject," or a label thereof (hereinafter, "this kit 2" sometimes called);
There is no particular limitation as long as it is (the present kit 1 and the present kit 2 may be collectively referred to as "the present kit" hereinafter). The present kit is a use invention relating to a kit for judging cancer, and these kits include components generally used in this type of judgment kit, such as carriers, pH buffers, stabilizers, instruction manuals, A package insert, such as instructions for determining cancer, is usually included.

In most of the subjects, the blood samples contained only the RUNX3 gene-derived double-stranded DNA fragment 1 among the RUNX3 gene-derived double-stranded DNA fragments 1 to 3, as shown in the present Example described later. ing. Therefore, in the present invention, the cytosine in the RUNX3 gene-derived double-stranded DNA fragment to be measured may be only the subject cytosine 1 in the RUNX3 gene-derived double-stranded DNA fragment 1, but in order to further improve the determination accuracy, RUNX3 Preferably, the subject cytosine 2 in the gene-derived double-stranded DNA fragment 2 and the subject cytosine 3 in the RUNX3 gene-derived double-stranded DNA fragment 3 are further included.

Using the methylation level of the cytosines 1 to 3 in the RUNX3 gene-derived double-stranded DNA fragments 1 to 3 and the ratio of the TERT gene-derived double-stranded DNA fragments as indicators, it is possible to determine whether the subject is suffering from cancer. Therefore, the methylation of the subject cytosines 1 to 3 in the double-stranded DNA fragments 1 to 3 derived from the RUNX3 gene and the double-stranded DNA fragment derived from the TERT gene can serve as biomarkers for cancer determination.

In the present invention, cancer includes, for example, colon cancer (colon cancer or rectal cancer); stomach cancer; liver cancer; brain tumor; small cell cancer); esophageal cancer; duodenal cancer; small bowel cancer; skin cancer; breast cancer; cancer; head and neck cancer; gallbladder cancer; cholangiocarcinoma; testicular cancer; ovarian cancer; Sarcoma including Ewing sarcoma, malignant hemangioendothelioma, malignant Schwannoma, osteosarcoma, soft tissue sarcoma; hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma blastoma such as blastoma; germ cell tumor; lymphoma; leukemia); more preferred.

In other words, the serum sample or plasma sample can be said to be a blood sample from which blood cells (eg, red blood cells, white blood cells, platelets) have been removed.

The subject is not particularly limited as long as it is a human, and examples thereof include a subject whose cancer is unknown. Such subjects include those who have had cancer in the past and who have been completely cured of cancer, but whose cancer status is unknown at the time of testing.

In the present invention, "methylation of cytosine" means a state in which the 5-position carbon of the cytosine base is methylated.

In the present invention, the RUNX3 gene-derived double-stranded DNA fragments 1 to 3 and the TERT gene-derived double-stranded DNA fragment to be measured are fragmented double-stranded DNA present outside the cell, It differs from the RUNX3 and TERT genes present on genomic DNA. The lengths of the RUNX3 gene-derived double-stranded DNA fragments 1 to 3 and the TERT gene-derived double-stranded DNA fragment are, for example, in the range of about 140 to 170 base lengths, considering that genomic DNA is fragmented on a nucleosome basis. , and/or a multiple thereof, eg, 280 to 340 bases long if doubled, and 420 to 510 bases long if tripled.

The TERT gene-derived double-stranded DNA fragment may be a fragmented TERT gene-derived double-stranded DNA contained in blood. Specifically, the TERT gene containing the nucleotide sequence shown in SEQ ID NO: 4 derived double-stranded DNA fragments. For the nucleotide sequence of the TERT gene-derived double-stranded DNA fragment contained in blood, DNA is isolated and purified from a serum sample or plasma sample according to standard methods, and subjected to a next-generation sequencer (e.g., MiniSeq manufactured by Illumina, Inc.). MiSeq, NextSeq, HiSeq and HiSeq X series; Roche 454 GS FLX sequencer manufactured by Roche).

As used herein, the "methylation level of the RUNX3 fragment" may be any value that indicates the degree of methylation of the methylated cytosines 1-3. The number (percentage) of subject cytosines 1-3 and the number (concentration) of methylated subject cytosines 1-3 per volume of serum or plasma sample can be mentioned. Further, in the present specification, the "percentage of TERT fragments" is specifically the number (concentration) of TERT fragments per volume of serum or plasma sample. These "methylation level of RUNX3 fragment" and "percentage of TERT fragment" may be absolute values or relative values.

In the above steps (a) and (A), the method for measuring the cytosine methylation level in the RUNX3 gene-derived double-stranded DNA fragments 1 to 3 can be by detecting and quantifying the methylation level of the cytosines 1 to 3. There are no particular limitations as long as the method is a hybridization method (for example, MBD2 [methyl-CpG-binding domain protein 2], chromatin immunoprecipitation [ChIP] using an anti-MBD2 antibody, etc.) to concentrate methylated DNA, DNA microarray analysis method); analysis method using a methylation-sensitive restriction enzyme; bisulfite method using hydrogen sulfite (bisulfite) treatment;

The method of analysis using the above-mentioned methylation-sensitive restriction enzyme utilizes a phenomenon in which cytosine in the recognition sequence of the restriction enzyme is methylated, making it impossible to cleave DNA. The methylation-sensitive restriction enzyme used here is such that the nucleotide sequence before and after the cytosines 1-3 in the RUNX3 gene-derived double-stranded DNA fragments 1-3 is "CCpGG" (where "C" is cytosine). , "Cp" indicates the subject cytosines 1-3 that can be methylated, and "G" indicates guanine.), HpaII normally targets such sequences. As a comparison control for HpaII, a methylation-insensitive restriction enzyme MspI targeting "CCGG" may be used. The reaction (treatment) time with HpaII can be appropriately selected, and is, for example, within the range of 1 to 48 hours, preferably 5 to 24 hours, more preferably 12 to 20 hours. Also, the reaction (treatment) temperature with HpaII can be appropriately selected, and is, for example, within the range of 30 to 39°C, preferably within the range of 35 to 38°C. After treatment with a methylation-sensitive restriction enzyme, the cleavage efficiency of the cytosines 1-3 site, that is, the methylation level of the cytosines 1-3 can be measured by LAMP (Loop-Mediated Isothermal Amplification) method; Southern hybridization; Real-time PCR (polymerase) chain reaction), PCR such as droplet digital PCR (ddPCR);

The ddPCR method is a method for absolute quantification of the amount of target DNA in a sample. In this method, target DNA in a sample is fractionated into about 20,000 small droplets, and PCR is performed using a thermal cycler. Droplets containing target DNA light up and those without do not change. The principle is that the concentration of the gene to be measured in the sample can be obtained as an absolute value by counting the number of small droplets that are illuminated and the number of small droplets that are not illuminated. Since data is determined by ant/none of the target DNA of small droplets, it is the same as 1/0 of the digital signal, so it is called "digital PCR". In this Example, which will be described later, the number of methylated cytosines 1 to 3 and the number of TERT gene-derived double-stranded DNA fragments were determined by measuring the above 20,000 small droplets (droplets) with a droplet reader. It can be quantified by measuring the amount of fluorescence one by one and counting the number of droplets emitting fluorescence. The ratio (concentration) and the ratio (concentration) of TERT gene-derived double-stranded DNA fragments contained in serum can be calculated.

Examples of the real-time PCR method include the intercalator method, the 5'-nuclease method, and the cycling probe method.

In the determination method, when measuring the methylation of the cytosines 1 to 3, a primer set, probe, or labeled product thereof that individually measures the methylation of the cytosines 1 to 3 may be used, It is preferable to measure using a primer set, probe, or label thereof that can commonly measure all methylation of cytosines 1 to 3 of the present invention, and a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 5, or SEQ ID NO: A forward primer consisting of a nucleotide sequence in which one or several nucleotides are substituted, deleted, inserted, or added to the nucleotide sequence shown in 5; a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 6, or SEQ ID NO: 6 A reverse primer consisting of a nucleotide sequence in which one or several nucleotides are substituted, deleted, inserted, or added in the nucleotide sequence shown in ; a probe consisting of the nucleotide sequence shown in SEQ ID NO: 7, or shown in SEQ ID NO: 7 A probe consisting of a nucleotide sequence in which one or several nucleotides are substituted, deleted, inserted, or added to the nucleotide sequence shown; and labels thereof can be preferably exemplified.

In the present invention, both to be compared (the methylation level of the RUNX3 fragment between the subject and the control; the ratio of the TERT fragment between the subject and the control; the methylation level of the RUNX3 fragment between the cancer patient and the control; or The percentage of TERT fragments between cancer patients and controls) correspond to each other. The methylation level of the RUNX3 fragment and the ratio of the TERT fragment in the control person may be values measured each time based on the serum or plasma sample collected from the control person when the present determination method is carried out. , may be values measured in advance. Moreover, the two to be compared are preferably serum samples or plasma samples prepared by substantially the same method, or obtained by substantially the same measurement method.

In the above steps (c) and (C), in order to evaluate (determine) whether or not the "RUNX3 fragment methylation level in the subject" is higher than the "RUNX3 fragment methylation level in the control", Any threshold value (cutoff value) can be set as appropriate, and such threshold values include, for example, the average value of "methylation level of RUNX3 fragment in controls", the average value ± standard deviation (SD), the average Value ±2SD, said mean ±3SD, median value of "methylation level of RUNX3 fragment in controls", said median ±SD, said median ±2SD, said median ±3SD and the like. In addition, the threshold is set so that sensitivity (percentage of subjects with cancer that can be correctly identified as positive) and specificity (percentage of controls without cancer that can be identified as correctly negative) is high. 2, based on the data of "methylation level of RUNX3 fragment in subjects" and "methylation level of RUNX3 fragment in controls", calculated using ROC (Receiver Operating Characteristic) curve using statistical analysis software You can also

In the above step (C), an arbitrary threshold (cutoff value) is set to evaluate (determine) whether or not the "ratio of TERT fragments in subjects" is higher than the "ratio of TERT fragments in controls". It can be set as appropriate, and such thresholds include, for example, the average value of "ratio of TERT fragments in controls", the average value ± standard deviation (SD), the average value ± 2SD, the average value ± 3SD, " The median value of the "percentage of TERT fragments in controls", said median value ±SD, said median value ±2SD, said median value ±3SD, and the like can be mentioned. In addition, the threshold is set so that sensitivity (percentage of subjects with cancer that can be correctly identified as positive) and specificity (percentage of controls without cancer that can be identified as correctly negative) is high. Alternatively, it can be calculated using a ROC curve using statistical analysis software, based on the data of "percentage of TERT fragments in subjects" and the data of "percentage of TERT fragments in controls".

In the above step (c), when the "RUNX3 fragment methylation level in the subject" is higher than the "RUNX3 fragment methylation level in the control", the subject is highly likely to be suffering from cancer. Evaluate (determine), and when "the RUNX3 fragment methylation level in the subject" is not higher (lower) than the "RUNX3 fragment methylation level in the control", the subject is likely to have cancer. is evaluated (determined) as low.

In the above step (C), the "RUNX3 fragment methylation level in the subject" is higher than the "RUNX3 fragment methylation level in the control", or the "percentage of the TERT fragment in the subject" is higher than the "control When it is higher than the ratio of TERT fragment in "or both, the subject is evaluated (determined) as having a high possibility of suffering from cancer, and the "methylation level of RUNX3 fragment in the subject" is , when "the RUNX3 fragment methylation level in controls" is not higher (lower) and the "percentage of TERT fragments in subjects" is not higher (lower) than the "percentage of TERT fragments in controls", The subject is evaluated (determined) as having a low possibility of suffering from cancer.

In the Kit, the forward primer and reverse primer for measuring the methylation level of cytosine in the RUNX3 gene-derived double-stranded DNA fragments 1-3 are the template DNAs of the RUNX3 gene-derived double-stranded DNA fragments 1-3. Among them, any one that specifically amplifies at least a part or all of the cytosines 1 to 3 of interest, specifically, the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence shown in SEQ ID NO: 2, and the nucleotide sequence shown in SEQ ID NO: 3, where the first nucleotide is upstream and the last nucleotide is downstream, the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence shown in SEQ ID NO: 2, and SEQ ID NO: 3 A forward primer that partially or fully anneals (hybridizes) on the upstream side of the nucleotide sequence shown in , the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence shown in SEQ ID NO: 2, and the nucleotide sequence shown in SEQ ID NO: 3 reverse primers that anneal in part or in whole downstream of the nucleotide sequence to be annealed. These forward primers and reverse primers specifically amplify three fragments of RUNX3 gene-derived double-stranded DNA fragment 1, RUNX3 gene-derived double-stranded DNA fragment 2, and RUNX3 gene-derived double-stranded DNA fragment 3. Preferably, specifically, a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 5, or a nucleotide in which one or several nucleotides are substituted, deleted, inserted, or added in the nucleotide sequence shown in SEQ ID NO: 5 A forward primer consisting of a sequence, a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 6, or a nucleotide in which one or several nucleotides are substituted, deleted, inserted, or added in the nucleotide sequence shown in SEQ ID NO: 6 A reverse primer consisting of a sequence can be mentioned.

In the kit, the forward primer and reverse primer for measuring the ratio of the TERT gene-derived double-stranded DNA fragment are used to specifically amplify part or all of the TERT gene-derived double-stranded DNA fragment, which is the template DNA. Specifically, in the nucleotide sequence shown in SEQ ID NO: 4, when the first nucleotide is upstream and the last nucleotide is downstream, the upstream side of the nucleotide sequence shown in SEQ ID NO: 4 , a forward primer partially or entirely annealed (hybridized), and a reverse primer partially or entirely annealed downstream of the nucleotide sequence shown in SEQ ID NO:4. More specifically, these forward primers and reverse primers are forward primers consisting of the nucleotide sequence shown in SEQ ID NO: 8, or substitutions or deletions of one or several nucleotides in the nucleotide sequence shown in SEQ ID NO: 8. , the forward primer consisting of the inserted or added nucleotide sequence, the reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 9, or the nucleotide sequence shown in SEQ ID NO: 9, one or several nucleotides are substituted or deleted , inserted or added reverse primers.

In the present kit, the length of the forward primer and the reverse primer is, for example, 10 to 40 nucleotides, preferably 12 to 35 nucleotides, more preferably 14 to 30 nucleotides, more preferably 15 to It is 25 bases long.

Considering that the polymerase used in PCR forms a complex at the 3′ end of the primer and extends the DNA strand, the nucleotide at the 3′ end of the primer is the template DNA It is preferably designed to anneal with RUNX3 gene-derived double-stranded DNA fragments 1 to 3 and TERT gene-derived double-stranded DNA fragments, and the stability of the polymerase / primer complex at the 3' end of the primer is more preferably designed to be G or C in order to increase

In the present kit, the probe for measuring the cytosine methylation level in the RUNX3 gene-derived double-stranded DNA fragments 1-3 is directly annealed (hybridized) to the RUNX3 gene-derived double-stranded DNA fragments 1-3, When measuring the methylation level of the subject cytosines 1 to 3 (for example, hybridization method), of the RUNX3 gene-derived double-stranded DNA fragments 1 to 3, which are template DNAs, part or all containing at least the subject cytosines, Any product that specifically anneals (hybridizes) and also anneals to secondary products (eg, PCR products) obtained from RUNX3 gene-derived double-stranded DNA fragments 1 to 3, which serve as indicators of methylation (hybridization), and when measuring the methylation level of the subject cytosines 1 to 3 (for example, a method of analysis using a methylation-sensitive restriction enzyme, a bisulfite method using bisulfite treatment), the secondary product It may be anything that specifically anneals (hybridizes) to part or all of it, specifically, a probe that anneals (hybridizes) to part or all of the nucleotide sequence shown in SEQ ID NO: 1, or a sequence Examples include probes that anneal (hybridize) to part or all of the nucleotide sequence shown in No. 2 and probes that anneal (hybridize) to part or all of the nucleotide sequence shown in SEQ ID NO:3. As such probes, those that specifically anneal to three fragments, namely, RUNX3 gene-derived double-stranded DNA fragment 1, RUNX3 gene-derived double-stranded DNA fragment 2, and RUNX3 gene-derived double-stranded DNA fragment 3, are preferred. Specifically, a probe consisting of the nucleotide sequence shown in SEQ ID NO: 7, or a nucleotide sequence in which one or several nucleotides are substituted, deleted, inserted, or added to the nucleotide sequence shown in SEQ ID NO: 7. can be mentioned.

In the kit, the probe for measuring the proportion of the TERT gene-derived double-stranded DNA fragment is specifically annealed (hybridized) to part or all of the TERT gene-derived double-stranded DNA fragment, which is the template DNA. Specific examples include probes that anneal (hybridize) to part or all of the nucleotide sequence shown in SEQ ID NO:4. More specifically, such probes include a probe consisting of the nucleotide sequence shown in SEQ ID NO: 10, or a nucleotide sequence shown in SEQ ID NO: 10 in which one or several nucleotides are substituted, deleted, inserted, or added. Probes consisting of a sequence of nucleotides identified by the

In the present invention, "a nucleotide sequence in which one or several nucleotides are substituted, deleted, inserted, or added" is, for example, within the range of 1 to 5, preferably within the range of 1 to 3, more preferably means a nucleotide sequence in which between 1 and 2 nucleotides have been substituted, deleted, inserted or added.

In the present kit, labeling substances in the labeled forward primer, labeled reverse primer, and labeled probe include peroxidase (for example, horseradish peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose - Enzymes such as 6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase; fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl Fluorescent substances such as chloride or tetramethylrhodamine isothiocyanate, Green Fluorescence Protein (GFP), Cyan Fluorescence Protein (CFP), Blue Fluorescence Protein (BFP), Yellow Fluorescence Protein (Yellow Fluorescence Protein (YFP), Red Fluorescence Protein (RFP), fluorescent proteins such as luciferase; radioisotopes such as ^3H , ^14C , ^125I or ^131I ; biotin, avidin, or chemiluminescence Substance; a combination of a reporter fluorescent substance and a quencher fluorescent substance or structure (for example, a combination of 5-FAM [5-Carboxyfluorescein] and 5-TAMRA [5-Carboxytetramethylrhodamine], a combination of VIC and MGB [Minor Groove Binder] ) can be mentioned.

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

1. Judgment method using serum samples derived from gastric cancer patients 1-1 Materials and methods [serum samples]
23 gastric cancer patients in the gastric cancer group (18 males, 5 females; average age 72 years [44-91 years old]) and 18 healthy subjects in the healthy group (10 males, 8 females; average age 52 years [ Serum samples were prepared from a total of 41 subjects (41 to 60 years old) according to a standard method. Serum samples were pre-collected at Yamaguchi University Hospital, Saint Hill Hospital, or Ajisu Kyoritsu Hospital between November 25, 2016 and December 20, 2017, prior to upper gastrointestinal endoscopy. Stored at -80°C until extraction. In order to avoid contamination of the blood with cancer cells, when biopsies were performed, blood sampling was performed no later than 3 weeks after the biopsies. Healthy control subjects had no history of any cancer and were determined to be free of gastric adenoma and cancer by upper gastrointestinal endoscopy. Gastric cancer patients are patients diagnosed with stage I early gastric cancer by endoscopic resection with no history of cancer other than gastric cancer. Of the 23 gastric cancer patients, 22 were well-differentiated (Well) gastric cancer patients and 1 was moderately differentiated (Moderate) gastric cancer. In addition, the average tumor size of gastric cancer patients was 13 mm (7-44 mm). In addition, the stage of gastric cancer was classified according to the International Union for Cancer (UICC). ). In addition, all of the above subjects were Japanese. The purpose of this study, the complications and risks of upper gastrointestinal endoscopy were clarified verbally and in written consent in face-to-face interviews. The above healthy subjects volunteered to participate, were limited to those who understood the risks of upper gastrointestinal endoscopy, and signed an informed consent form. The study protocol was approved by the Institutional Review Boards of Yamaguchi University Graduate School of Medicine, Saint Hill Hospital, and Ajisu Kyoritsu Hospital, and informed consent was obtained from the above subjects.

[Preparation of DNA in subject-derived serum sample]
Thaw the serum sample derived from the above subject stored at -80 ° C., from each serum sample 0.4 mL, using MagNA Pure Compact Nucleic Acid Isolation Kit I (manufactured by Roche), according to the protocol attached to the product, 50 μL of elution buffer DNA was extracted in The concentration of extracted DNA was quantified using a Qubit 2.0 fluorometer (manufactured by Thermo Fisher Scientific). Peripheral blood leukocyte-derived DNA was used as a control for unmethylated DNA, and EpiScope methylated HCT116gDNA (manufactured by Takara Bio) was used as a control for hypermethylated DNA.

[CORD (Combined Restriction Digital PCR) Assay]
To detect methylation of double-stranded DNA fragments in subject-derived serum samples, a two-step CORD assay (Suehiro Y, Zhang Y, Hashimoto S, Takami T, Higaki S, Shindo Y, et al. Highly sensitive faecal DNA testing of TWIST1 methylation in combination with faecal immunochemical test for haemoglobin is a promising marker for detection of colorectal neoplasia. Ann Clin Biochem. 2017:4563217691064.") was performed. Specifically, in the first step, 10 μL of elution buffer containing extracted DNA (equivalent to 80 μL of serum) is added with 1 μL of GeneAmp 10 x PCR Buffer II, 1 μL of 25 mmol/L magnesium chloride, and Two methylation-sensitive restriction enzymes (10 units of HhaI [recognition site: GCGC] and 10 units of HpaII [recognition site: CCGG]) and 20 units of exonuclease I (ExoI) (all from Thermo Fisher Scientific) product) was added, and DNA degradation treatment was performed at 37° C. for 16 hours. Here, ExoI was used to degrade single-stranded DNA contained in serum samples. Next, in a second step, another type of methylation-sensitive restriction enzyme (10 units of BstUI [recognition site: CGCG] [New England Biolabs]) was added and further incubated at 60° C. for 16 hours. was subjected to DNA degradation treatment. After restriction enzyme treatment, the samples were heat-treated at 98° C. for 10 minutes.

The RUNX3 gene-derived double-stranded DNA fragment 1, the RUNX3 gene-derived double-stranded DNA fragment 2, the RUNX3 gene-derived double-stranded DNA fragment 3, and the TERT gene-derived double-stranded DNA fragment (see Table 1) were prepared by Droplet Digital. Amplification and quantification were carried out by the PCR (ddPCR) method (reference “High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem. 2011;83(22):8604-10.”). Specifically, 8 μL of DNA sample after restriction enzyme treatment (equivalent to 40 μL of serum), 1×ddPCR Supermix for Probes (manufactured by BioRad), and a primer set (forward primer and reverse primer) of 0.25 μmol/L and Using 0.125 μmol / L probe (see Table 2), droplets are generated by Automated Droplet Generator (manufactured by BioRad), PCR is performed using QX100 droplet Digital PCR system (manufactured by BioRad), and data analysis is performed. , was performed using QuantaSoft software (manufactured by BioRad).

[Statistical processing]
A statistically significant difference between the gastric cancer group and the healthy group was calculated using Mann-Whitney U test, chi-square test, Fisher's test and linear regression analysis. In addition, when the P value was less than 0.05, it was evaluated that there was a statistically significant difference. Statistical analysis was performed using GraphPad InStat Ver. 3 and GraphPad Prism Ver. 6 statistical software (manufactured by GraphPad Software).

1-2 Results [Methylation level of the subject cytosines 1 to 3 in serum samples]
RUNX3 gene-derived double-stranded DNA fragment 1 and RUNX3 gene-derived double-stranded DNA fragment 2 each have one recognition site (CCGG) for the methylation-sensitive restriction enzyme HpaII. When the cytosines 1 to 3) enclosed in the boxes are methylated, cleavage by HpaII is inhibited, so both fragments are amplified and detected by the ddPCR method. On the other hand, if the subject cytosines 1-3 are not methylated, both fragments are cleaved by HpaII, and thus both fragments are not amplified or detected by the ddPCR method.

First, the 1000 Genomes Project (genome data from over 2500 individuals from 26 populations worldwide, all analyzed using whole-genome sequencing, deep exome sequencing, and high-density microarray genotyping). This information is available on the Internet), and the abundance ratios of the RUNX3 gene-derived double-stranded DNA fragment 1 and the RUNX3 gene-derived double-stranded DNA fragment 2 were investigated and found to be 99.42% to 100%, respectively. and 0-0.58% (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/?gts=rs532454563).
Next, the methylation levels of the subject cytosines 1 to 3 in both fragments and RUNX3 gene-derived double-stranded DNA fragment 3 were analyzed. As a result, as shown in FIG. 1A, the median ratio of methylated cytosines 1-3 (the number of methylated cytosines 1-3 per 40 μ serum) in the gastric cancer group was 6.0. (0-26), significantly increased compared to the median value of the healthy group (1.8 [0-9.8]). This result indicates that the methylation levels of the cytosines 1 to 3 in the RUNX3 gene-derived double-stranded DNA fragments 1 to 3 present in the serum samples of gastric cancer patients are higher than those of healthy subjects.

Moreover, when the ROC curve (AUC) was created based on the result of FIG. 1A, the AUC value showed a high value of 0.7488. In addition, as a cut-off value for distinguishing between the gastric cancer group and the healthy group, 5.9 was set, and the sensitivity (percentage of test positive people in the gastric cancer group) and specificity (in the healthy group test negatives) were 52.2% (12/23) and 94.4% (17/18), respectively.

As a result, the methylation level of the cytosines 1 to 3 in the RUNX3 gene-derived double-stranded DNA fragments 1 to 3 in a blood sample (serum sample or plasma sample) excluding blood cells is measured, and an appropriate cutoff value is set, the methylation level of the cytosines 1 to 3 can be used as an index to determine gastric cancer (especially early gastric cancer).

[Percentage of TERT gene-derived double-stranded DNA fragments in serum samples]
Since the TERT gene-derived double-stranded DNA fragment does not have recognition sites for any of the above three types of methylation-sensitive restriction enzymes (HhaI, HpaII, and BstUI), the TERT gene-derived double-stranded DNA fragment was used as a serum sample. It is amplified and detected by the ddPCR method depending on its proportion in the medium.

Analysis of the ratio of TERT gene-derived double-stranded DNA fragments in serum samples revealed that, as shown in FIG. 2A, the ratio of TERT gene-derived double-stranded DNA fragments in the gastric cancer group The median value of the number of strand DNA fragments) was 922 (348-2520), which was significantly increased compared to the median value of the healthy group (282 [122-1260]). This result indicates that the percentage of TERT gene-derived double-stranded DNA fragments present in the serum samples of gastric cancer patients is higher than that of healthy subjects.

Moreover, when the ROC curve (AUC) was created based on the result of FIG. 2A, the AUC value showed a high value of 0.8490. In addition, when 1271 was set as the cutoff value for distinguishing between the gastric cancer group and the healthy group, the sensitivity (percentage of test positive people in the gastric cancer group) and specificity (test negative in the healthy group , were 34.8% (8/23) and 100% (18/18), respectively.

This result was obtained by measuring the ratio of TERT gene-derived double-stranded DNA fragments in a blood sample (serum sample or plasma sample) excluding blood cells, and setting an appropriate cut-off value. It shows that gastric cancer (especially early gastric cancer) can be determined using the ratio of fragments as an index.

[Combination of the methylation level of the subject cytosines 1 to 3 in the serum sample and the ratio of the TERT gene-derived double-stranded DNA fragment]
Next, it was verified whether the gastric cancer group and the healthy group could be accurately determined by combining the methylation level of the cytosines 1 to 3 in the serum sample and the ratio of the TERT gene-derived double-stranded DNA fragment. did. Each cut-off value is the same as described above, and in the gastric cancer group, at least the subjects who are positive for either the methylation of the cytosines 1 to 3 or the percentage of double-stranded DNA fragments derived from the TERT gene are tested positive. In the healthy group, the subjects who were negative for both the methylation of the cytosines 1-3 and the ratio of the double-stranded DNA fragment derived from the TERT gene were evaluated as test-negative subjects. The degrees were 60.1% (14/23) and 94.4% (17/18) respectively.

This result shows the methylation level of the cytosines 1-3 in the RUNX3 gene-derived double-stranded DNA fragments 1-3 in the blood sample (serum sample or plasma sample) excluding blood cells, and the TERT gene-derived double-stranded DNA After measuring the ratio of fragments and setting an appropriate cut-off value for each, the methylation level of the cytosines 1 to 3 and the ratio of double-stranded DNA fragments derived from the TERT gene are used as indices, and each alone is used as an index. In comparison, it shows that gastric cancer (especially early gastric cancer) can be determined more accurately.

2. Judgment method using serum samples derived from pancreatic cancer patients 2-1 Materials and methods [serum samples]
26 pancreatic cancer patients in the pancreatic cancer group (11 males, 15 females; average age 70 [46-85 years old]) and 30 healthy subjects in the healthy group (15 males, 15 females; average age 49). From a total of 56 subjects aged [39-60], serum samples were prepared according to a standard method. Serum samples were collected from Yamaguchi University Hospital, Saint Hill Hospital, or Ajisu Kyoritsu Hospital between November 25, 2016 and December 20, 2017 prior to invasive examinations such as upper gastrointestinal endoscopy or surgery. and stored at -80°C until DNA extraction was performed. Cases in which a biopsy was performed were excluded to avoid contamination of the blood with cancer cells. Healthy control subjects had no history of any cancer and were determined to be free of gastric adenoma and cancer by upper gastrointestinal endoscopy. A patient with pancreatic cancer is a patient who has no medical history of cancer other than pancreatic cancer and who has been diagnosed with stage 1 to 4 pancreatic cancer based on diagnostic imaging or surgical findings. Of the 26 pancreatic cancer patients, 2 were well differentiated (Well) pancreatic cancer patients, 15 were moderately differentiated (Moderate) pancreatic cancer patients, and 1 were poorly differentiated (Poor) pancreatic cancer patients. There were 8 pancreatic cancer patients with other histological types or unknown histological types. In addition, the stage of pancreatic cancer was classified according to the International Union for Cancer (UICC) (Document "Sobin LH, Gospodarowicz MK, Wittekind C, editors. TNM Classification of Malignant Tumors, 7th ed. Oxford: Wiley-Blackwell; 2009." reference). In addition, all of the above subjects were Japanese. The purpose of this study, the complications and risks of upper gastrointestinal endoscopy were clarified verbally and in written consent in face-to-face interviews. The above healthy subjects volunteered to participate, were limited to those who understood the risks of upper gastrointestinal endoscopy, and signed an informed consent form. The study protocol was approved by the Institutional Review Boards of Yamaguchi University Graduate School of Medicine, Saint Hill Hospital, and Ajisu Kyoritsu Hospital, and informed consent was obtained from the above subjects.

The DNA in the subject-derived serum sample was prepared according to the method described in the section [Preparation of DNA in subject-derived serum sample] in Example 1 above, and the RUNX3 gene-derived double-stranded DNA fragment 1 and the RUNX3 gene-derived double-stranded DNA fragment 1 were prepared. The methylation level of this cytosine 2 in strand DNA fragment 2 was analyzed according to the method described in the item [CORD (Combined Restriction Digital PCR) assay] of Example 1 above.

2-2 Results As shown in FIG. 3A, the median ratio of methylated cytosines 1 to 3 (the number of methylated cytosines 1 to 3 per 40 μ serum) in the pancreatic cancer group was 4. .0 (0.0-26.0), an increase compared to the median value of the healthy group (2.1 [0.0-9.8]). This result indicates that the methylation levels of the cytosines 1 to 3 in the RUNX3 gene-derived double-stranded DNA fragments 1 to 3 present in the serum samples of pancreatic cancer patients are higher than those of healthy subjects.

Moreover, when the ROC curve (AUC) was created based on the result of FIG. 3A, the AUC value showed a high value of 0.6410. In addition, when 5.9 was set as the cutoff value for distinguishing between the pancreatic cancer group and the healthy group, the sensitivity (percentage of test-positive persons in the pancreatic cancer group) and specificity ( The proportion of test-negative persons in the healthy group) was 34.6% (9/26) and 93.3% (28/30), respectively.

As a result, the methylation level of the cytosines 1 to 3 in the RUNX3 gene-derived double-stranded DNA fragments 1 to 3 in a blood sample (serum sample or plasma sample) excluding blood cells is measured, and an appropriate cutoff value is set, the methylation level of the cytosines 1 to 3 can be used as an index to determine pancreatic cancer.

INDUSTRIAL APPLICABILITY The present invention contributes to early detection and early treatment of cancer.

Claims (12)

A method for assisting determination of gastric cancer or pancreatic cancer, comprising the following steps (a) to (c).
(a) measuring the cytosine methylation level in one or more RUNX3 gene-derived double-stranded DNA fragments in a serum sample or plasma sample collected from a subject,
The RUNX3 gene-derived double-stranded DNA fragment comprises a double-stranded DNA fragment comprising the nucleotide sequence shown in SEQ ID NO: 1,
The above step, wherein the cytosine is the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 1;
(b) comparing the methylation level measured in step (a) to said methylation level in a control subject not suffering from gastric or pancreatic cancer;
(c) assessing that said subject is likely to have gastric cancer or pancreatic cancer when the methylation level measured in step (a) is higher than the methylation level in a control subject; A method for assisting determination of gastric cancer, comprising the following steps (A) to (C).
(A) measuring the concentration of the TERT gene-derived double-stranded DNA fragment in a serum sample or plasma sample collected from a subject;
(B) comparing the concentration of the double-stranded DNA fragment measured in step (A) with the concentration of said DNA fragment in a control subject not suffering from gastric cancer;
(C) When the concentration of the double-stranded DNA fragment measured in step (A) is higher than the concentration of the double-stranded DNA fragment in the control subject, the subject is evaluated as having a high possibility of suffering from gastric cancer. the step of In step (A), further measuring the methylation level of cytosine in one or more RUNX3 gene-derived double-stranded DNA fragments in the serum sample or plasma sample,
in step (B), further comparing the methylation level measured in step (A) to said methylation level in a control subject not suffering from gastric cancer;
In step (C), when the methylation level measured in step (A) is higher than the methylation level in the control subject, further assessing that the subject is likely to suffer from gastric cancer.
3. The method of claim 2, wherein
wherein the RUNX3 gene-derived double-stranded DNA fragment comprises a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 1, and the cytosine is the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 1. The method as described above. The RUNX3 gene-derived double-stranded DNA fragment further comprises a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 2 and a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 3,
In steps (a) and (A), the methylation level of the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 2 and the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 3 is further measured. 4. The method according to claim 1 or 3. The method according to any one of claims 1 to 4, wherein the gastric cancer is stage I gastric cancer. 4. The method according to claim 2 or 3 , wherein the TERT gene-derived double-stranded DNA is a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO:4. forward and reverse primers and/or probes for measuring cytosine methylation levels in one or more RUNX3 gene-derived double-stranded DNA fragments in serum or plasma samples, or labeled products thereof , a kit for determining gastric cancer or pancreatic cancer,
The RUNX3 gene-derived double-stranded DNA fragment comprises a double-stranded DNA fragment comprising the nucleotide sequence shown in SEQ ID NO: 1,
The kit, wherein the cytosine is the 54th cytosine in the nucleotide sequence shown in SEQ ID NO:1. A kit for determining gastric cancer, comprising a forward primer, a reverse primer and/or a probe for measuring the concentration of a TERT gene-derived double-stranded DNA fragment in a serum sample or a plasma sample, or a label thereof. forward and reverse primers and/or probes for measuring cytosine methylation levels in one or more RUNX3 gene-derived double-stranded DNA fragments in serum or plasma samples, or labeled products thereof; 9. The kit of claim 8, wherein the RUNX3 gene-derived double-stranded DNA fragment comprises a double-stranded DNA fragment comprising the nucleotide sequence shown in SEQ ID NO: 1,
The kit, wherein the cytosine is the 54th cytosine in the nucleotide sequence shown in SEQ ID NO:1. The RUNX3 gene-derived double-stranded DNA fragment further comprises a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 2 and a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 3,
Forward primer and reverse primer, and / or probes, or their labels, the methylation level of the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 2 and the 54th cytosine in the nucleotide sequence shown in SEQ ID NO: 3 10. The kit according to claim 7 or 9, which is for further measuring the . The kit according to any one of claims 7 to 10, wherein the gastric cancer is stage I gastric cancer. 10. The kit according to claim 8 or 9 , wherein the TERT gene-derived double-stranded DNA is a double-stranded DNA fragment containing the nucleotide sequence shown in SEQ ID NO:4.

Images