WO2021091277A1 - Blood-based dna methylation biomarker panel for diagnosing colorectal cancer - Google Patents

Abstract

The present application relates to a DNA methylation biomarker panel for diagnosing colorectal cancer, the panel being capable of diagnosing colorectal cancer by detecting and measuring methylation changes of genes in the blood of a patient. The blood-based DNA methylation biomarker panel for diagnosing colorectal cancer, according to the present application, diagnoses colorectal cancer through only blood collection so as to overcome side effects and the inconvenience of having to collect a patient's tissue, thereby enabling the burden on the patient to be minimized.

Description

DNA methylation biomarker panel for diagnosis of blood-based colon cancer

The present application relates to a DNA methylation biomarker panel for colorectal cancer diagnosis capable of diagnosing colorectal cancer by detecting and measuring changes in methylation of a patient's blood gene.

Cancer is a representative disease that threatens human health and is the most representative cause of death as a single disease in industrialized countries. In particular, colon cancer currently has the third highest incidence rate in Korea, the second highest in men, and the third highest in women, and the incidence and mortality rates are on the rise. In addition, considering that the lifestyle is gradually becoming westernized, the increase in colon cancer is expected to accelerate further, so active interest and research on colorectal cancer in our society are indispensable. In addition, the risk of colorectal cancer is more serious because the recurrence rate is up to 50% after cure.

Colorectal cancer is performed through fecal examination, radiographic examination, colonoscopy, etc., which are conventional examination methods for the diagnosis of colorectal cancer. However, there is a problem that this may cause pain and danger to the patient. The conventional methods as described above have a problem that not only the accuracy of diagnosis is low, but also early diagnosis of patients before the onset of colon cancer is impossible, and inconvenience to the subjects. As a method to compensate for the shortcomings of these existing diagnostic methods, development of biomarkers that can accurately diagnose colon cancer early is required, and accordingly, colon cancer is diagnosed by measuring the concentration of tumor markers in the patient's plasma. Attempts are being made to do it.

Accordingly, recently, methods for diagnosing cancer through DNA methylation measurement have been proposed. When the promoter CpG island of a specific gene is hypermethylated, the expression of that gene is blocked (gene silencing). This is the main mechanism in which the function of the gene is lost even without mutation in the protein-specific coding sequence of the gene in vivo, and the function of a number of tumor suppressor genes is lost in human cancer. It is interpreted as a cause. Therefore, searching for the methylation of the promoter CpG island of the tumor suppressor gene is of great help in cancer research, and attempts to use it for diagnosis and screening of cancer by examining it by methods such as methylation-specific PCR or automatic base analysis have been actively conducted in recent years. It is being done.

Korean Patent Laid-Open Publication No. 2007-0098034 discloses a composition for diagnosis of cancer containing a methylated promoter of a colorectal cancer-specific gene and its use. Disclosed are a cancer diagnostic composition containing a methylated promoter of a colorectal cancer-specific expression-reducing gene, a cancer diagnostic microarray in which the composition is immobilized on a substrate, and a colon cancer diagnostic kit containing the composition.

The present application is to provide a DNA methylation biomarker panel of FAM123A, PPP1R16B, TMEM90B, GLI3, and SLIT3 for diagnosis of blood-based colorectal cancer.

In one embodiment, the present application is a biomarker for methylation analysis from nucleic acids isolated from blood: FMA123A (Family with Sequence Similarity 123A), GLI3 (GLI family zinc finger 3), PPP1R16B (Protein Phosphate 1 Regulatory Subunit 16B), TMEM90B (Transmembrane Protein 90B) And it discloses a use of diagnosing colorectal cancer by measuring the methylation or degree of the CpG island region of the regulatory region of the gene of SLIT3 (Slit Guidance Ligand 3).

In one embodiment, the methylation analysis biomarker may further include THBD (Thrombomodulin) and C9orf50 (Chromosome 9 open reading frame 50).

In one embodiment, the present application provides a composition or kit for diagnosing colorectal cancer comprising a substance for detecting methylation of the methylation assay biomarker.

In one embodiment, the material is methylation specific PCR (methylation specific PCR), real time methylation specific PCR (real time methylation specific PCR), PCR using a methylated DNA specific binding protein, quantitative PCR, digital including droplet digital PCR Includes materials used for PCR, DNA chip, pyrosequencing, and bisulfite sequencing.

In one embodiment, the nucleic acid isolated from the blood for which the marker according to the present application is used is cfDNA.

In one embodiment, the nucleic acid isolated from the blood for which the marker according to the present application is used is cfDNA, and the cfDNA is used in a digital PCR method.

In one embodiment, a marker according to the present application is detected using a combination of SEQ ID NOs: 1 to 15.

In another embodiment, a marker according to the present application is detected using a combination of SEQ ID NOs: 1-21.

In another aspect, the present application provides a nucleic acid isolated from blood of a subject in need of colorectal cancer diagnosis in order to provide information necessary for colorectal cancer diagnosis: FMA123A (Family with Sequence Similarity 123A) as a methylation analysis biomarker from the nucleic acid, Measuring methylation of CpG islands in the GLI family zinc finger 3 (GLI3), Protein Phosphate 1 Regulatory Subunit 16B (PPP1R16B), Transmembrane Protein 90B (TMEM90B) and Slit guidance Ligand 3 (SLIT3) methylation marker gene regulatory regions; And compared with the control, it provides a method for detecting a specific methylation biomarker for colorectal cancer comprising the step of correlating a change in the methylation state of each gene with colorectal cancer.

In one embodiment, the analytical biomarker according to the present application may further include THBD (Thrombomodulin) and C9orf50 (Chromosome 9 open reading frame 50).

In one embodiment, in the method according to the present invention, the measurement of the methylation level comprises contacting the isolated nucleic acid with a compound that selectively modifies a non-methylated cytosine residue or selectively modifies a methylated cytosine residue, and the compound Is a hydrazine or bisulfite ion, and may include cutting the gene in contact with the hydrazine with piperidine, and treating the gene in contact with the bisulfite ion with an alkali.

In one embodiment, the measurement in the method according to the present application is methylation specific PCR (methylation specific PCR), real time methylation specific PCR (real time methylation specific PCR), PCR using methylated DNA specific binding protein, quantitative PCR, droplet digital Digital PCR including PCR, DNA chip, pyrosequencing, or bisulfite sequencing can be performed.

In one embodiment, in the method according to the present invention, the nucleic acid is cfDNA, and the measurement is performed by digital PCR.

In one embodiment, in the method according to the present application, the marker according to the present application is detected using a combination of SEQ ID NOs: 1 to 15.

In another embodiment, in a method according to the present application, a marker according to the present application is detected using a combination of SEQ ID NOs: 1-21.

In one embodiment, in the method according to the present invention, the digital PCR is ddMethyLight PCR, and sites 5'and 3'of each probe are labeled with a detectable fluorescent material.

In one embodiment, in the method according to the present application, when the ddMethyLight PCR has two or more markers in which one or more droplets are methylation-positive, or when two or more droplets are methylation-positive markers is one or more, the subject is associated with colon cancer.

In another aspect, the present application provides a method for diagnosing colorectal cancer of a subject in need of diagnosis of colorectal cancer, the method comprising: providing a nucleic acid isolated from blood of a subject in need of colorectal cancer diagnosis: FMA123A as a methylation analysis biomarker from the nucleic acid (Family with Sequence Similarity 123A), GLI3 (GLI family zinc finger 3), PPP1R16B (Protein Phosphate 1 Regulatory Subunit 16B), TMEM90B (Transmembrane Protein 90B) and SLIT3 (Slit guidance Ligand 3) methylation marker gene control region of the CpG island. Measuring methylation; And it provides a diagnostic method through the detection of a specific methylation biomarker for colorectal cancer comprising the step of correlating the change in the methylation state of each gene with colorectal cancer compared to the control.

In one embodiment, the analytical biomarker according to the present application may further include THBD (Thrombomodulin) and C9orf50 (Chromosome 9 open reading frame 50).

In one embodiment, in the method according to the present invention, the measurement of the methylation level comprises contacting the isolated nucleic acid with a compound that selectively modifies a non-methylated cytosine residue or selectively modifies a methylated cytosine residue, and the compound Is a hydrazine or bisulfite ion, and may include cutting the gene in contact with the hydrazine with piperidine, and treating the gene in contact with the bisulfite ion with an alkali.

In one embodiment, the measurement in the method according to the present application is methylation specific PCR (methylation specific PCR), real time methylation specific PCR (real time methylation specific PCR), PCR using methylated DNA specific binding protein, quantitative PCR, droplet digital Digital PCR including PCR, DNA chip, pyrosequencing, or bisulfite sequencing can be performed.

In one embodiment, in the method according to the present invention, the nucleic acid is cfDNA, and the measurement is performed by digital PCR.

In one embodiment, in the method according to the present application, the marker according to the present application is detected using a combination of SEQ ID NOs: 1 to 15.

In another embodiment, in a method according to the present application, a marker according to the present application is detected using a combination of SEQ ID NOs: 1-21.

In one embodiment, in the method according to the present invention, the digital PCR is ddMethyLight PCR, and sites 5'and 3'of each probe are labeled with a detectable fluorescent material.

In one embodiment, in the method according to the present application, when the ddMethyLight PCR has two or more markers in which one or more droplets are methylation-positive, or when two or more droplets are methylation-positive markers is one or more, the subject is associated with colon cancer.

The DNA methylation biomarker panel for diagnosis of blood-based colon cancer according to the present application diagnoses colon cancer only by blood collection, thereby overcoming the inconvenience and side effects of collecting the patient's tissue, thereby minimizing the burden on the patient.

1 is a flow chart of an experimental method for constructing a DNA methylation biomarker panel for diagnosis of blood-based colorectal cancer according to an embodiment of the present application.

2 is an image of performing Droplet Data reading according to an embodiment of the present application.

3 shows the nucleotide sequence positions of five methylation markers according to an embodiment of the present application.

4 is a bar graph showing the number of positive droplets after performing ddMethyLight for five methylation markers for the plasma of a colon cancer patient (n=91) and plasma of a normal person (n=30) according to an embodiment of the present application It is the result expressed as. It indicates that the methylation for each marker is higher in the plasma cfDNA of colon cancer patients than in the normal plasma cfDNA.

Figure 5 (a) to (c) is the sensitivity of (a) colorectal cancer plasma samples having one or more positive droplets according to an embodiment of the present application. When the number of positive droplets is more than 1, each marker shows a higher sensitivity to colon cancer patients than normal subjects. (b) This is the sensitivity of colorectal cancer plasma samples with two or more positive droplets. When the number of positive droplets is 2 or more, the sensitivity decreases. (C) The sensitivity of the control (normal) and colorectal cancer patient (CRC) samples based on the number of markers showing more than one positive droplet for each sample, 1, 2, or 3; When the number of markers showing 2 or more positive droplets for each sample is 1, 2, or 3, it is a graph showing the sensitivity of the control (normal) and colon cancer patient (CRC) samples. For each of the diagnostic criteria for colorectal cancer, the sensitivity to colorectal cancer was higher than that of the normal control group.

6 is a graph showing the number of methylated droplets of each marker according to the patient stage according to an embodiment of the present application (N, normal person). It was found that the higher the stage of colon cancer, the higher the detection rate of each marker.

7 is a graph showing the sensitivity according to the stage of colorectal cancer when the number of droplets methylated in two or more markers of five markers according to an embodiment of the present application is one or more.

8 is a graph showing the sensitivity according to the stage of colorectal cancer when the number of droplets methylated in at least one marker among five markers according to an embodiment of the present application is two or more.

9 is a graph showing the sensitivity according to the stage of colorectal cancer when the number of droplets methylated in at least two markers among seven markers according to one embodiment of the present application is at least one.

10 is a graph showing the sensitivity according to the stage of colorectal cancer when the number of droplets methylated in at least one of the seven markers according to an embodiment of the present application is two or more.

7, 8, 9, and 10 show a high sensitivity in colon cancer compared to the normal control group in each criterion, and also shows that the sensitivity increases as the stage goes up.

The present application relates to the diagnosis of colorectal cancer using a DNA methylation biomarker panel for diagnosis of blood-based colorectal cancer.

As a DNA methylation biomarker for colorectal cancer-specific blood diagnosis, FMA123A (Family with Sequence Similarity 123A), GLI3 (GLI family zinc finger 3), PPP1R16B (Protein Phosphate 1 Regulatory Subunit 16B), TMEM90B (Transmembrane Protein 90B) and SLIT3 ( Slit guidance Ligand 3) It is to diagnose colon cancer by checking whether the regulatory region of the gene is methylated.

Accordingly, in one embodiment, the present application is FMA123A (Family with Sequence Similarity 123A), GLI3 (GLI family zinc finger 3), PPP1R16B (Protein Phosphate 1 Regulatory Subunit 16B), TMEM90B (Transmembrane Protein 90B) and SLIT3 (Slit Guidance Ligand 3) It relates to a composition or kit for diagnosing colorectal cancer containing a substance for measuring the methylation level of the CpG island region of the gene regulatory region.

Colorectal cancer refers to a malignant tumor that occurs in the colon and rectum, and is called colon cancer or rectal cancer or a combination thereof, depending on the location of occurrence, as colon cancer or colorectal cancer. The stage of colorectal cancer is determined according to the degree of invasion of the tumor, the degree of metastasis to the surrounding lymph nodes, and the presence or absence of distant metastases such as liver or lung, and is classified into stage 1, stage 2, stage 3, and stage 4.

The DNA methylation biomarker for blood diagnosis specific to colon cancer of the present application can be applied to patients with colorectal cancer including rectal and colon. In particular, compared with the sensitivity of stage 1 to 3 colorectal cancer, the detection sensitivity is particularly high for stage 4 colorectal cancer. Represents.

The colon wall can be divided into a mucous layer, a submucosa layer, a muscle layer, and a serous layer from the lumen. In particular, in the case of the middle and lower rectum in the lower pelvis, there is no serous layer.

The regulatory region or promoter herein is the minimum sequence necessary to direct transcription of the gene, and may be regulated by cell type-specific, tissue-specific, or external signals or substances in a promoter-dependent gene. These promoters are located at the 5'or 3'region of the gene.

Herein, it is detected whether the methylated site of the promoter of the gene of the methylation marker is methylated, particularly the methylated CpG dinucleotide, particularly the dinucleotide in the CpG island. The CpG island refers to a DNA site in which CpG dinucleotides appear at high frequency, and in many cases is found in the promoter or 5′ exon site of the gene (Takai D, et al., Proc Natl Acad Sci USA 2002, 99:37403745). . The promoters of most human genes are located on these CpG islands and are present in a non-methylated state. The degree of methylation at these sites is very different (Hinoue T, et al., Genome Res 2011; Noushmehr H, et al., Cancer Cell 2010, 17:510522), but is related to gene inactivation or cancer. It shows a specific pattern (Soria JC et al., Cancer Res 2002, 62:351355; Eads CA et al., Cancer Res 2001, 61:34103418).

CpG islands typically have a length of about 1 to 2 kbp, and CpG islands start at the upstream of the promoter and are also present in the 3'region, including the transcriptional site downstream, e.g. enhancers, promoters and introns. It is found in several sites including. Whether or not methylation of the methylation marker herein is particularly within about 2 kbp, within about 1 kbp, particularly within 750 bp, and more particularly within 500 bp in the 5'direction from the promoter region, particularly the transcription initiation site.

In one embodiment, the methylation of the methylation marker of the present application according to the present application is detected in the CpG island at the position as described in FIG. 3.

Typically, the nucleic acid used to detect whether CpG is methylated is DNA. However, the method of the present invention may apply, for example, a sample containing DNA or RNA including DNA and mRNA, wherein the DNA or RNA may be single-stranded or double-stranded, or a DNA-RNA hybrid It may be characterized in that it is a containing sample. Mixtures of nucleic acids can also be used. The nucleic acid sequence to be detected need not be a nucleic acid present in its pure form, and the nucleic acid may be a small fraction of a large molecule, such as a portion of the whole genomic DNA.

In one embodiment, in particular, the sample used for detection of methylation of a marker according to the present application is DNA extracted from blood.

In another embodiment the sample for which the methylation of a marker according to the present invention is used for detection is in particular cfDNA extracted from blood.

In the present application, “cfDNA (cell-free DNA, cfDNA)” includes genomic fragments of various lengths present in blood, but the chromatin portion that is not protected by histone proteins is mainly cut off, showing a mode at 166 bp in length. cfDNA is mostly DNA released from hematopoietic cells in healthy people, and in cancer patients, it includes circulating tumor DNA (ctDNA) derived from cancer cells due to cancer cell death. cfDNA can be extracted from blood, and reagents/kits for extracting it are commercially available, and the method is known (Clara Perez-Barrios et al. Traansl Lung Cancer Res 5 (2016).

The DNA methylation biomarker panel for diagnosis of blood-based colorectal cancer according to the present application is based on the discovery of methylated CpG islands (island loci) specifically methylated for colorectal cancer among the changes in methylation of ctDNA. Compared to normal people, the amount of DNA (cell-free DNA, cfDNA) in the blood of colorectal cancer patients is increased, and a part of cfDNA is ctDNA originating from tumor cells. ctDNA is synchronized with genetic changes or DNA methylation changes in tumor cell genomic DNA, and a gene CpG island showing DNA methylation changes is used as a DNA methylation detection site.

In order to determine whether it is colorectal cancer using the methylation marker according to the present application, it is important to distinguish between methylation and non-methylation of the CpG region of the marker according to the present application.

In one embodiment according to the present application, a restriction enzyme sensitive to methylation is used to detect CpG dinucleotide methylation status. These restriction enzymes selectively cleave a methylated restriction enzyme recognition site or a non-methylated restriction enzyme recognition site. Examples of the former include AccIII, BanI, BstNI, MspI, or XmaI, and examples of the latter include, but are not limited to, AccII, AvaI, BssHII, BstUI, HpaII, or NotI. By analyzing the size of the product through electrophoresis after treatment with restriction enzymes, the methylation status can be determined depending on whether or not it is cleaved.

In addition, alternatively, a compound that selectively modifies the CpG dinucleotide site may be used, which may be detected directly or through an additional reaction. Electrophoresis, chromatography and mass spectrometry can be used, for example, where changes in size and/or state of charge are involved. Examples of compounds used in further reactions include hydrazine and bisulfite ions. DNA modified with hydrazine is cleaved when treated with piperidine. DNA treated with bisulfite ions can be cleaved by treatment with alkali.

In another embodiment according to the present application, various amplification methods may be used to detect CpG dinucleotide methylation status. For example, PCR (Polymerase Chain Reaction), methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, DNA microarray, pyrothermal analysis and bisulfite This can be done through sequencing. Amplification methods other than PCR include ligase chain reaction (LCR) (Barringer et al, 1990. Gene 89, 117-122), transcription amplification (WO1988/10315), and selective amplification of target sequences (US Pat.No. 6,410,276), PCR using conserved sequences (US Pat. No. 4,437,975), PCR using random primers (WO1990/06995), nucleic acid-based sequence amplification (NASBA) (US Pat. Nos. 5,409,818; 5,554,517; 6,063,603) ), and nick replacement amplification (WO2004/067726) methods.

In one embodiment, treatment with sodium bisulfite and subsequent various detection methods such as PCR-based analysis, pyronucleotide sequencing, dideoxy sequencing, high-resolution melting, single-stranded polymorphism analysis, microarray, next-generation nucleotide sequence It can be used in conjunction with analysis, MALDI-TOF mass spectrometry, to detect methylation.

Here, digital PCR (dPCR)-based analysis is used as a PCR-based analysis. Through this, it is possible to detect the presence of methylated DNA present in a small amount in the presence of an excess of unmethylated cfDNA. dPCR involves diluting a DNA sample (template) to a very low concentration and distributing the template to a large number of wells or compartments so that each well or compartment does not contain one to several or no target DNA molecules. It is a method capable of absolute quantification by performing it. ddPCR (droplet digtial PCR) is a type of digtal PCR, where the reaction is performed in droplets instead of wells, and the effective dilution factor of the template increases by more than one digit (Yu M, et al. MethyLight droplet digital PCR for detection and absolute quantification of infrequently methylated alleles.Epigenetics 2015;10:803-9). Also in this case, quantitative analysis is possible by counting the number of droplets that showed a positive reaction.

As an example of such a method, digital Methylight (dMethyLight) PCR or droplet digital Methylight, which uses a probe labeled with a fluorescent substance, in addition to a PCR primer, may be used. This method combines ddPCR and MethyLight analysis, and a methylation-specific probe and probe are used (Weisenberger DJ et al. DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight. Nucleic Acids Res 2008;36:4689-98). Through this method, a template is distributed to a plurality of partitioned reaction wells to detect whether a single allele is methylated, and the number of wells showing a positive reaction is counted to enable quantitative analysis.

In one embodiment, the methylation biomarker detection according to the present application measures the methylation level of cell-free DNA (cfDNA) in the blood of a cancer patient using a droplet digital PCR or a droplet digital PCR-based MethyLight assay (MethyLight ddPCR, or ddMethyLight). By doing so, colon cancer can be diagnosed only with simple blood collection.

In one embodiment, the combination of primers and probes for each biomarker used in the droplet digital PCR or droplet digital PCR-based MethyLight assay is as shown in SEQ ID NO: 1 to SEQ ID NO: 15.

In one embodiment, the probe according to the present application may be labeled with a labelable fluorescent material. Such a fluorescent material may be FAM, VIC, or TET as a fluorescent reporter die at 5'of the probe of the present application, and BHQ-1 or MGBNFQ as a fluorescent quencher at 3', and those skilled in the art will be able to select an appropriate one. .

In the present application, "sensitivity" refers to a case of determining that a cancer patient is cancer, and "specificity" refers to a case of determining that a patient who is not cancer is not cancerous. As a result of analysis using the droplet digital PCR technique, the positive criterion for each marker can be evaluated as a case where even one droplet is positive for methylation and two or more droplets are positive for methylation.

More specifically, the sensitivity and specificity of the methylation marker for determining colorectal cancer were evaluated using cfDNA of colon cancer patients and cfDNA of normal people.

When one or more droplets have more than one methylation-positive marker, one or more droplets have two or more methylation-positive markers, and when one or more droplets have three or more methylation-positive markers, one or more droplets are methylation-positive markers When there are more than 4 markers in which one or more droplets are methylation-positive, there are 5 markers. For each of these five cases, the sensitivity and specificity of cancer diagnosis can be evaluated.

In addition, when two or more droplets have at least one methylation-positive marker, two or more droplets have two or more methylation-positive markers, and when two or more droplets have three or more methylation-positive markers, two or more droplets are methylated. When there are 4 or more positive markers, when two or more droplets have 5 markers that are methylation positive. For each of these five cases, the sensitivity and specificity of cancer diagnosis can be evaluated.

In addition, when three or more droplets have at least one methylation-positive marker, three or more droplets have two or more methylation-positive markers, and three or more droplets have three or more methylation-positive markers, three or more droplets are methylated. When there are 4 or more positive markers, when 3 or more droplets have 5 methylation-positive markers. For each of these five cases, the sensitivity and specificity of cancer diagnosis can be evaluated.

In this way, through the evaluation of the number of 15 cases, when one or more droplets have two or more methylation-positive markers in terms of sensitivity and specificity, and when two or more droplets have one or more methylation-positive markers, it is suitable as a criterion. Specific results are shown in the following examples, FIGS. 7 and 8.

In another embodiment, the DNA methylation biomarker according to the present application includes seven markers (FAM123A, GLI3, PPP1R16B, SLIT3, TMEM90B, THBD, and C9orf50) as a panel, including two existing markers, as a panel, and sensitivity and specificity. Can be evaluated.

In this case, in one embodiment of the present application, the primer and probe combinations for each biomarker used in the droplet digital PCR or droplet digital PCR-based MethyLight assay are the same as SEQ ID NO: 1 to SEQ ID NO: 21.

The positive criterion for each marker can be evaluated as a case where even one droplet is positive for methylation and two or more droplets are positive for methylation. More specifically, in the case of evaluating the sensitivity and specificity of 91 cancer patients and 30 cfDNAs in the general population, when one or more droplets have at least one methylation-positive marker, one or more droplets have two methylation-positive markers. When more than one, one or more droplets have three or more methylation-positive markers, one or more droplets have four or more methylation-positive markers, and if one or more droplets have five or more methylation-positive markers, one or more droplets When there are 6 or more methylation-positive markers, and when one or more droplets have 7 or more methylation-positive markers. For each of these 7 cases, the sensitivity and specificity of cancer diagnosis can be evaluated.

In addition, when two or more droplets have at least one methylation-positive marker, two or more droplets have two or more methylation-positive markers, and when two or more droplets have three or more methylation-positive markers, two or more droplets are methylated. When there are 4 or more positive markers, when two or more droplets have 5 or more methylation-positive markers, when two or more droplets have 6 or more methylation-positive markers, when two or more droplets have 7 or more methylation-positive markers. For each of these 7 cases, the sensitivity and specificity of cancer diagnosis can be evaluated.

In addition, when three or more droplets have at least one methylation-positive marker, three or more droplets have two or more methylation-positive markers, and three or more droplets have three or more methylation-positive markers, three or more droplets are methylated. When there are 4 or more positive markers, when 3 or more droplets have 5 or more methylation-positive markers, when 3 or more droplets have 6 or more methylation-positive markers, when 3 or more droplets have 7 or more methylation-positive markers. For each of these 7 cases, the sensitivity and specificity of cancer diagnosis can be evaluated.

In this way, through the evaluation of the number of 21 cases, when one or more droplets have two or more methylation-positive markers in terms of sensitivity and specificity, and when two or more droplets have one or more methylation-positive markers, it is suitable as the criterion. The results are shown in Figures 9 and 10.

The composition according to the present application may thus include a substance used in the method for detecting a methylation marker according to the present application as described above.

In another aspect, the present application provides a nucleic acid isolated from blood of a subject in need of colorectal cancer diagnosis to provide information necessary for the diagnosis of colorectal cancer: FMA123A (Family with Sequence Similarity 123A), GLI3 (GLI family zinc) from the nucleic acid. measuring the methylation level of the CpG islands of the finger 3), PPP1R16B (Protein Phosphate 1 Regulatory Subunit 16B), TMEM90B (Transmembrane Protein 90B) and SLIT3 (Slit Guidance Ligand 3) methylation marker gene regulatory regions; And compared with the control, it provides a method for analyzing the methylation level of a colon cancer-specific biomarker comprising the step of correlating the change in the methylation state of each gene with colon cancer.

The method for measuring the methylation level and the criteria for determining the methylation level used in the method according to the present disclosure may be referred to as mentioned above.

Example

Example 1 Experimental method

Experiments were performed in the order shown in FIG. 1.

1) Blood collection and plasma separation

Plasma samples of normal people were posted on bulletin boards in schools and hospitals, blood was collected from 30 normal people, plasma was separated, and cell-free DNA (cfDNA) was extracted from the separated plasma. Blood samples from colon cancer patients were collected from 91 patients who visited the Colon Cancer Center at Seoul National University Hospital, and plasma was separated. All of these normal subjects and patients with colorectal cancer received research consent and received IRB (Institutional Review Board) approval.

Blood was collected from a colon cancer patient, injected into an EDTA-tube, and plasma was separated using Ficoll-Paque PLUS (GE Healthcare). After centrifugation at 1,200 rpm for 20 minutes, the plasma (upper layer) was transferred to a 1.5 ml microtube, followed by centrifugation at 13,000 rpm for 10 minutes. Plasma (upper layer) was transferred to a new 1.5ml microtube and stored in a -80°C freezer.

2) Cell-free DNA (cfDNA) extraction from plasma

QIAamp MinElute ccfDNA Mini Kit (QIAGEN) was used to extract cfDNA. Magnetic Bead Suspension 30 μl, Proteinase K 55 μl, and Bead Binding Buffer 150 μl were added to 1 ml of plasma, and then placed on a rotator and mixed at a low speed for 10 minutes. After spin-down (200 xg, 30 seconds), the tube was left in a magnetic rack for 1 minute, and the supernatant was removed. After the tube was separated from the magnetic rack, 200 μl of Bead Elution Buffer was added to the bead pellet, followed by mixing. After transferring the mixture to the Bead Elution Tube, the mixture was mixed at a speed of 300 rpm for 5 minutes on a shaker, and the tube was left in a magnetic rack for 1 minute. The supernatant was transferred to a Bead Elution Tube, and 300 μl of Buffer ACB was added and mixed, followed by spin-down. The supernatant-Buffer ACB mixture was transferred to a QIAamp UCP MinElute column, followed by centrifugation at 6000 xg for 1 minute. After adding 500 μl of Buffer ACW2 to the column in which the 2ml collection tube was inserted, centrifugation was performed at 6000 xg for 1 minute. After inserting the column into a 2ml collection tube, it was centrifuged for 3 minutes at a maximum speed (20,000 xg). After inserting the column into a 1.5ml elution tube, it was allowed to stand at 56℃ for 3 minutes. After adding 20 μl of ultra-clean water to the membrane in the middle of the column, it was left at room temperature for 10 minutes. Thereafter, centrifugation was performed for 1 minute at the maximum speed (20,000 xg). The ultra-clean water injection, left to stand at room temperature, and centrifugation at maximum speed were repeated once more under the same conditions. 40ul of the final cfDNA was extracted. (1ml Plasma/40ul cfDNA → 25ul plasma/1ul cfDNA) The extracted cfDNA was stored in a -20°C freezer.

3) Bisulfite treatment

For Bisulfite treatment EZ DNA Methylation Kit (ZYMO Research) was used. 10μl of extracted plasma cfDNA (=250ul plasma), 5μl of M-Dilution buffer, and 35μl of water were added to a 0.2ml tube, followed by mixing. After standing at 37°C for 15 minutes, 100 μl of CT Conversion Reagent (Bisulfite) was added, followed by spin-down. Put into a PCR machine and treated with Bisulfite under the following conditions: 95℃ 5 minutes → 95℃ 1 minute → 50℃ 2 hours → 95℃ 40 seconds → 50℃ 3 hours → 95℃ 40 seconds → 50℃ 5 hours → 95 ℃ 40 seconds → 50 ℃ 6 hours.

After treatment with Bisulfite, the sample was left on an ice-rack (or ice) for 10 minutes, and 400ul of M-Binding Buffer was added to the Zymo-Spin IC Column. The sample was injected into the Zymo-Spin IC column, and the column was turned upside down several times to mix. Centrifuged for 30 seconds at the highest speed (10,000 xg or more), the sample passed through the column was put back into the column and centrifuged. 100ul of M-Wash Buffer was injected into the column, and centrifuged for 30 seconds at the highest speed (10,000 xg or more). 200 μl of M-Desulphonation Buffer was injected into the column, allowed to stand at room temperature for 15 minutes, and then centrifuged for 30 seconds at the highest speed (10,000 xg or more). 200 μl of M-Wash Buffer was injected into the column and centrifuged for 30 seconds at the highest speed (10,000xg or more) was repeated twice. 10 μl of M-Elution Buffer was added to the column, allowed to stand at room temperature for 10 minutes, and then centrifuged for 30 seconds at the highest speed (10,000xg or more). 10 μl of M-Elution Buffer was added to the column, and centrifuged for 30 seconds at the highest speed (10,000 xg or more). 20 μl of M-Elution Buffer was added to the column, and centrifuged for 30 seconds at the highest speed (10,000 xg or more). The extracted cfDNA (total 40ul) was stored in a -20 ℃ freezer. (250ul Plasma/40ul cfDNA → 6.25ul plasma/1ul cfDNA)

4) Droplet digital MethyLight (ddMethyLight) analysis

The ddMethyLight analysis mixture was prepared using the composition of Table 1 and Table 2 below.

[Table 1]

[Table 2]

5'of the probe was labeled with a fluorescent reporter dye 6-FAM (6-Carboxyflourescein), and 3'was labeled with BHQ (Black Hole Quencher)-1.

Since the C-LESS-C1 probe and primer recognize a DNA strand that does not contain cytosine and are not affected by bisulfite treatment, it can react to bisulfite-converted or unconverted entire DNA, and this As a result, the relative amount of sample DNA used in the PCR reaction can be measured.

To perform droplet generation, a DG8 cartridge was inserted into the holder, and 20μl of ddMethyLight mixture and 70μl of droplet generation oil were injected into each well of the cartridge. After covering the cartridge holder with a gasket and installing the cartridge holder on the QX200 droplet generator (BIO-RAD), droplet generation started. The generated droplet 40μl was slowly dispensed into a 96-well PCR plate so that the droplet did not break. Cover the 96-well plate with the foil seal, put it in a heat sealer, and seal it at 180℃ for 5 seconds.

To perform PCR, the sealed PCR plate was put into a PCR machine, and the conditions were set as follows, and then PCR was performed: 95°C for 10 minutes (ramping rate 2°C/sec) → 94°C for 30 seconds (ramping rate 2°C/sec) sec) → 59℃ 1min (ramping rate 2℃/sec): 45 cycles → 98℃ 10min (ramping rate 2℃/sec) → 8℃.

For data reading, the PCR plate on which the PCR reaction was completed was mounted on the plate holder of the droplet reader (BIO-RAD), and QuantaSoft (BIO-RAD) was executed on a PC. After executing'flush system' and'Prime' in order, after setting the template for the sample, click and run the'Run' icon to read the data.

To confirm the analysis result, when data reading was completed, click'Analyze' to check the analysis result, and after setting the threshold in 1D Amplitude, the number of positive droplets for the target was checked. The results are shown in FIG. 2.

Example 2 Colorectal cancer specific methylation biomarker analysis according to the present application

Human Methylation EPIC Bead Chip (Illumina) with genomic DNA extracted from cancer tissues and surrounding normal intestinal tissues of colon cancer patients and genomic DNA extracted from peripheral blood mononuclear cells (PBMCs) of a normal person by the experimental method according to Example 1 Inc.) was performed to compare and analyze the NGS results obtained by comparing and analyzing 5 methylation markers (FAM123A, PPP1R16B, TMEM90B, GLI3, SLIT3) with a higher DNA methylation degree in colon cancer tissues than in normal tissues and higher DNA methylation degree than blood leukocytes Were selected.

Subsequently, primers and probes for the five selected methylation markers FAM123A, PPP1R16B, TMEM90B, GLI3, and SLIT3 were prepared as shown in FIG. 3, and the sequences are shown in Table 2. DdMethyLight was performed on the blood cfDNA of colon cancer patients (n=91) and normal people (n=30). Compared to normal subjects, many methylation markers were detected in colon cancer patients. The results are shown in FIG. 4. The number of positive droplets obtained as a result of performing ddMethyLight for 5 methylation markers for the plasma of colon cancer patients (n=91) and normal people (n=30) was displayed as a bar graph. It can be seen that the methylation of each marker is higher in plasma cfDNA of colon cancer patients than in normal plasma cfDNA.

FIG. 5 shows the detection sensitivity, and as a result of investigating the case where the detection amount (number of positive droplets) of each marker in each sample is more than one, referring to FIG. 5(a), it is detected up to 20 to 49% in colon cancer patients. However, it showed a detection sensitivity of 3 to 13% even in normal people. When the number of positive droplets is more than 1, each marker shows a higher sensitivity to colon cancer patients than normal individuals. However, when the number of positive droplets was 2 or more, the detection sensitivity of colorectal cancer patients was 10 to 22%, as shown in Fig. 5(b), and in the case of normal people, it was not detected in the remaining markers except for the PPP1R16B marker (3.3%). Did not appear. If the number of positive droplets is 2 or more, the sensitivity is lowered. Figure 5(c) is a calculation of the number of marker(s) having one or more positive droplets or two or more positive droplets among five markers for each plasma sample. When two or more markers were used as the colorectal cancer diagnosis criterion, the sensitivity was 57%, and when one or more markers with two or more positive droplets were used as the colorectal cancer diagnosis standard, the sensitivity was 47%. For each colorectal cancer diagnosis criteria, it can be seen that the sensitivity in colorectal cancer is higher than that of the normal control group.

6 shows the number of methylated droplets of each marker according to the patient's stage, and it was confirmed that the number of methylated droplets in stage 4 colon cancer was increased by a large amount as compared to the stage of the colorectal cancer patient for each marker. It indicates that the higher the stage of colon cancer, the higher the detection rate of each marker.

Figure 7 shows the sensitivity according to the colorectal cancer stage when the number of droplets methylated in two or more of the five markers is one or more, showing a sensitivity of 42 to 55% in stages 1 to 3 than the control group, and stage 4 colorectal cancer Showed a high sensitivity of 94.4%. In addition, FIG. 8 shows a sensitivity of 21 to 56% in stages 1 to 3 compared to the control group when the number of droplets methylated in at least one of the five markers is 2 or more, and a high sensitivity of 83.3% in stage 4 colon cancer. It confirmed that it shows. 7, 8, 9, 10: In each criterion, it shows a high sensitivity in colon cancer compared to the normal control group, and also shows that the sensitivity increases as the stage goes up.

Claims (17)

1. As biomarkers for methylation analysis from nucleic acids isolated from blood, FMA123A (Family with Sequence Similarity 123A), GLI3 (GLI family zinc finger 3), PPP1R16B (Protein Phosphate 1 Regulatory Subunit 16B), TMEM90B (Transmembrane Protein 90B) and SLIT3 (Slit guidance) Ligand 3) A composition for diagnosing colorectal cancer comprising a substance for measuring methylation of the CpG island region of the gene regulatory region.
2. The method of claim 1,

   The methylation analysis biomarker further comprises THBD (Thrombomodulin) and C9orf50 (Chromosome 9 open reading frame 50), a composition for diagnosing colon cancer.
3. The method according to claim 1 or 2,

   The material is methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA-specific binding protein, quantitative PCR, digital PCR including droplet digital PCR, DNA chip , Pyrosequencing, a material used for bisulfite sequencing, a composition for diagnosing colon cancer.
4. The method according to claim 1 or 2,

   The nucleic acid isolated from the blood is cfDNA, a composition for diagnosing colon cancer.
5. The method according to claim 1 or 2,

   The nucleic acid isolated from the blood is cfDNA, and the material is a material used in a digital PCR method, a composition for diagnosing colon cancer.
6. The method according to any one of claims 1 to 5,

   The material is a combination of the following primer pairs and probes for detecting CpG islands in the regulatory region of each gene, a composition for diagnosing colon cancer:

   
7. The method according to any one of claims 2 to 5,

   The material is a combination of the following primer pairs and probes for detecting CpG islands in the regulatory region of each gene, a composition for diagnosing colon cancer:

   
8. The method of claim 6 or 7,

   The digital PCR is ddMethyLight PCR, and the 5'and 3'sites of each probe are labeled with a detectable fluorescent material, a composition for diagnosing colon cancer.
9. In order to provide information necessary for the diagnosis of colorectal cancer, providing a nucleic acid isolated from the blood of a subject in need of diagnosis of colorectal cancer:

   As a methylation analysis biomarker from the nucleic acid, FMA123A (Family with Sequence Similarity 123A), GLI3 (GLI family zinc finger 3), PPP1R16B (Protein Phosphate 1 Regulatory Subunit 16B), TMEM90B (Transmembrane Protein 90B) and SLIT3 (Slit guidance Ligand 3) Measuring methylation of CpG islands in the methylation marker gene regulatory region; And

   Comprising the step of correlating the change in the methylation state of each gene compared to the control with colorectal cancer, a method of detecting a specific methylation biomarker for colorectal cancer.
10. The method of claim 9,

    The methylation analysis biomarker further comprises THBD (Thrombomodulin) and C9orf50 (Chromosome 9 open reading frame 50), a method of detecting a specific methylation biomarker for colon cancer.
11. The method of claim 9 or 10,

    The determination of the level of methylation is contacting the isolated nucleic acid with a compound that selectively modifies non-methylated cytosine residues, or selectively modifies methylated cytosine residues,

    The compound is a hydrazine or bisulfite ion, which comprises cleaving the gene in contact with the hydrazine with piperidine, and treating the gene in contact with the bisulfite ion with an alkali, colorectal cancer specific methylation Biomarker detection method.
12. The method of claim 9 or 10,

    The measurement is methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, digital PCR including droplet digital PCR, DNA chip , Pyrosequencing, or bisulfite sequencing that is performed by, a method of detecting a specific methylation biomarker for colon cancer.
13. The method of claim 12,

    The nucleic acid is cfDNA, and the measurement is performed by digital PCR.
14. The method according to any one of claims 9 to 13,

    The digital PCR is performed using a combination of the following primer pairs and probes for detecting methylation of CpG islands in the regulatory regions of each gene, a method of detecting a colorectal cancer-specific methylation biomarker:

    
15. The method according to any one of claims 10 to 13,

    The digital PCR is performed using a combination of the following primer pairs and probes for detecting methylation of CpG islands in the regulatory regions of each gene, a method of detecting a colorectal cancer-specific methylation biomarker:

    
16. The method of claim 14 or 15,

    The digital PCR is ddMethyLight PCR, and the 5′ and 3′ regions of each probe are labeled with a detectable fluorescent material.
17. The method of claim 16,

    In the ddMethyLight PCR, when one or more droplets have two or more methylation-positive markers, or when two or more droplets have one or more methylation-positive markers, the subject is associated with colorectal cancer, a colorectal cancer-specific methylation biomarker Detection method.

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