Detection of hypermethylated genes for the diagnosis of colorectal cancer.

Abstract

The present invention relates to a method for diagnosing or identifying colorectal cancer in a subject through the detection of abnormal hypermethylation levels of specific genes in a biological sample. The inventors have identified DNA methylation biomarkers that can be used alone or in combination to aid in the diagnosis or follow-up of colorectal cancer patients. Furthermore, the method can be used to determine and / or adapt an appropriate treatment plan for a subject diagnosed with colorectal cancer. The present invention further relates to a kit comprising primers or probes for detecting, diagnosing, or identifying hypermethylated genes.

Description

Technical Field

[0001] The present invention relates to the field of oncology. In particular, the present invention relates to a method for detecting or monitoring colorectal cancer (CRC) in a subject through the detection of abnormal hypermethylation levels of specific genes in a biological sample thereof. The inventors have actually identified DNA methylation biomarkers that can be useful for the diagnosis or follow-up of CRC patients alone or in combination.

Background Art

[0002] Colorectal cancer (CRC) is an important issue from the perspective of public health. In fact, CRC is the third most common cancer in terms of incidence, with nearly 2 million new cases in 2020. There are also more than 916,000 deaths, representing a global mortality rate of over 9% in 2020. Surgical treatment is the most common treatment for all stages of colon cancer. In most cases, adjuvant chemotherapy (ACT) follows surgical treatment in patients with histologically high-risk stages II and III. ACT can reduce the risk of recurrence, but survival is in contrast.

[0003] The high mortality rate can be explained by the late diagnosis, limited monitoring tools, and low adaptability of treatment. This indicates a real need for new non-invasive, economic, and highly sensitive methods for the diagnosis and monitoring of CRC patients.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

[0005] Therefore, improvements in diagnostic and follow-up methods could reduce mortality by enabling early detection. Currently available diagnostic methods have significant limitations, such as their invasiveness, cost, or particularly low sensitivity in the early stages. The need for methods for early diagnosis and accurate monitoring of colorectal cancer remains. [Means for solving the problem]

[0006] A first aspect of the present invention relates to an in vitro method for detecting or monitoring colorectal cancer (CRC) in a subject, preferably a human subject, comprising the steps of detecting the methylation of at least one AQP5-AS1, ZSCAN23, or COL4A1 gene in a biological sample containing genomic DNA from the subject, or determining the level of methylation.

[0007] In one particular embodiment, the method includes a step of detecting methylation of the AQP5-AS1 and ZSCAN23 genes, or a step of determining the level of methylation. In another particular embodiment, the method includes a step of detecting methylation of the AQP5-AS1 and COL4A1 genes, or a step of determining the level of methylation. In yet another particular embodiment, the method includes a step of detecting methylation of the ZSCAN23 and COL4A1 genes, or a step of determining the level of methylation.

[0008] In a particular embodiment, the method includes the step of detecting methylation of a combination of the AQP5-AS1, ZSCAN23, and COL4A1 genes, or the step of determining the level of methylation. In a particular embodiment, the method includes the step of detecting methylation of the NR5A2, C9orf50, WIF1, or NPY gene, or the step of determining the level of methylation. In a particular embodiment, the method further includes the step of detecting methylation of at least one of the ADARB2, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, or LRRC4 (SND1) genes, or the step of determining the level of methylation.

[0009] In one particular embodiment, the method includes the steps of detecting methylation of at least two, preferably three, preferably four genes selected from the group consisting of a combination of the AQP5-AS1, ZSCAN23, and COL4A1 genes, and the NR5A2, C9orf50, WIF1, NPY, ADARB2, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and LRRC4 (SND1) genes, or determining the level of methylation.

[0010] In one particular embodiment, the method includes the steps of detecting methylation of a combination of the AQP5-AS1, ZSCAN23, and COL4A1 genes, and of at least two, preferably three, preferably four genes selected from the group consisting of NR5A2, C9orf50, WIF1, and NPY, or determining the level of methylation.

[0011] In a particular embodiment, the level or amount of methylation is - Sequence ID 40 in the ZSCAN23 gene, - Sequence ID 41 in the C9orf50 gene, - Sequence ID 42 in the AQP5-AS1 gene, - Sequence ID 43 in the NR5A2 gene, - Sequence ID 44 in the ADARB2 gene, - Sequence ID No. 45 in the COL4A1 gene, - Sequence ID 46 in the NPY gene, - Sequence ID 47 in the WIF1 gene, - Sequence ID 48 in the CPNE8 gene, - Sequence ID 49 in the LINC00693(RBMS3) gene, - Sequence ID 50 in the LINC00900 gene, - Sequence ID 51 in the DYDC2 gene, and / or - Sequence ID No. 52 in the LRRC4(SND1) gene It is determined in the nucleotide region.

[0012] In a particular embodiment, the level or amount of methylation is - Sequence ID 53 in the ZSCAN23 gene, - Sequence ID 54 in the C9orf50 gene, - Sequence ID 55 in the AQP5-AS1 gene, - Sequence ID 56 in the NR5A2 gene, - Sequence ID 57 in the ADARB2 gene, - Sequence ID 58 in the COL4A1 gene, - Sequence ID 59 in the NPY gene, - Sequence ID 60 in the WIF1 gene, - Sequence ID 61 in the CPNE8 gene, - Sequence ID 62 in the LINC00693(RBMS3) gene, - Sequence ID 63 in the LINC00900 gene, - Sequence ID 64 in the DYDC2 gene, and / or - determined in the nucleotide region of SEQ ID NO: 65 in the LRRC4 (SND1) gene is determined in the nucleotide region of.

[0013] In a particular embodiment, said level or amount of methylation is - primers of SEQ ID NOs: 1 and 2 and probe of SEQ ID NO: 27 for the ZSCAN23 gene, - primers of SEQ ID NOs: 3 and 4 and probe of SEQ ID NO: 28 for the C9orf50 gene, - primers of SEQ ID NOs: 5 and 6 and probe of SEQ ID NO: 29 for the AQP5-AS1 gene, - primers of SEQ ID NOs: 7 and 8 and probe of SEQ ID NO: 30 for the NR5A2 gene, - primers of SEQ ID NOs: 9 and 10 and probe of SEQ ID NO: 31 for the ADARB2 gene, - primers of SEQ ID NOs: 11 and 12 and probe of SEQ ID NO: 32 for the COL4A1 gene, [[ID=P20]] - primers of SEQ ID NOs: 13 and 14 and probe of SEQ ID NO: 33 for the NPY gene, - primers of SEQ ID NOs: 15 and 16 and probe of SEQ ID NO: 34 for the WIF1 gene, - primers of SEQ ID NOs: 17 and 18 and probe of SEQ ID NO: 35 for the CPNE8 gene, - primers of SEQ ID NOs: 19 and 20 and probe of SEQ ID NO: 36 for the LINC00693 (RBMS3) gene, - primers of SEQ ID NOs: 21 and 22 and probe of SEQ ID NO: 37 for the LINC00900 gene, - primers of SEQ ID NOs: 23 and 24 and probe of SEQ ID NO: 38 for the DYDC2 gene and / or - primers of SEQ ID NOs: 25 and 26 and probe of SEQ ID NO: 39 for the LRRC4 (SND1) gene are used to determine.

[0014] In one particular embodiment, the sample is a body fluid, preferably selected from the group consisting of plasma, serum, blood, urine, and feces, and more preferably the sample is a plasma or serum sample, and the DNA is circulating cell-free DNA (ccfDNA), preferably circulating tumor DNA (ctDNA).

[0015] In particular, if the aforementioned gene is hypermethylated compared to the reference value, the subject will be diagnosed with or identified as having colorectal cancer.

[0016] In one specific embodiment, this method is used for early diagnosis in individuals at risk of developing colorectal cancer.

[0017] In one specific embodiment, this method is used for the early diagnosis of cancerous lesions in the colon in subjects who have metastasized from a primary tumor of unknown origin.

[0018] In one specific embodiment, this method is used to assess the risk of recurrence.

[0019] In one particular embodiment, the method is used to monitor the progression of colorectal cancer in subjects diagnosed with colorectal cancer, and the method is preferably, a) A step of determining the level of methylation of any of the genes in the target biological sample at a first time point, b) A step of determining the level of methylation of the gene previously selected in step a) in the target biological sample at a second time point, and c) A step of comparing the methylation level determined in step a) with the level or amount determined in step b), or a reference value. Includes. [Brief explanation of the drawing]

[0020] [Figure 1]This paper shows the DNA methylation of selected biomarkers, ZSCAN23, C9orf50, AQP5-AS1, NR5A2, ADARB2, COL4A1, NPY, WIF1, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and LRRC4 (SND1), as determined by ddPCR (n=20-26). A paired nonparametric Wilcoxon test was used to analyze the differences in hypermethylation between tumor DNA and adjacent normal tissue DNA. [Figure 2] This study shows DNA methylation of selected biomarkers, ZSCAN23, C9orf50, AQP5-AS1, NR5A2, ADARB2, COL4A1, NPY, and WIF1 in CRC patients using ddPCR. Differences in DNA methylation between CRC patient plasma (n=17) and healthy control plasma (n=10-20) were examined using ddPCR. The Mann-Whitney U test was used for significance analysis. [Modes for carrying out the invention]

[0021] The inventors identified a methylation signature for colorectal cancer.

[0022] In particular, the subject of this invention is an in vitro method for detecting or monitoring colorectal cancer in a subject, the method comprising the steps of detecting methylation of a specific gene alone or in combination in a biological sample containing genomic DNA from the subject, or determining the level of methylation. In particular, hypermethylation at a selected genomic site indicates colorectal cancer.

[0023] Colon cancer The method of the present invention includes the diagnosis and / or monitoring of colorectal cancer (CRC) in a subject. This includes all histological types, stages, and grades.

[0024] CRC originates in the cells of the colon or rectum. The most common are adenocarcinomas of the colon and rectum, but CRC can also include primary colorectal lymphoma, gastrointestinal stromal tumors, leiomyosarcoma, and carcinoid tumors.

[0025] The most common staging system for CRC is the TNM classification, which describes tumor, lymph node, and metastatic (TMN) cancer. The severity of the cancer is associated with a number (0-4) and a letter. Stage 0 indicates carcinoma in situ (N0, M0). Stage I indicates localized cancer (T1-T2, N0, M0). Stage II refers to early-stage locally advanced cancer (T2-T4, N0, M0). Stage III refers to late-stage locally advanced cancer (T1-T4, N1-N3, M0). Stage IV refers to metastatic cancer (T1-T4, N1-N3, M1).

[0026] subject According to the present invention, the terms “subject,” “individual,” and “patient” are interchangeable herein and refer to mammals that may be healthy (without any symptoms of colorectal cancer), are considered likely to develop colorectal cancer, are suspected of having colorectal cancer, or have colorectal cancer. The subject exhibits, for example, at least one of the following symptoms: changes in bowel habits, e.g., diarrhea, constipation, or thin stools; abnormal abdominal distension; rectal bleeding; bloody stools; persistent cramping pain or abdominal pain; nausea or vomiting; fatigue and malaise; or unexplained weight loss. The subject may also have a history of colorectal cancer, have received treatment for it, and are being monitored for the possibility of disease recurrence. The subject may also appear healthy but be at risk of developing colorectal cancer, or have a family member with or have had the same disease. Risk factors include age, male gender, high intake of fat, sugar, alcohol, red meat, and processed meat, obesity, smoking, and lack of exercise.

[0027] In one embodiment, the subject is a mammal, such as a human, dog, mouse, cat, cow, sheep, pig, or goat. In a preferred embodiment, the subject is a human.

[0028] sample The method of the present invention requires a biological sample containing genomic DNA from a subject.

[0029] The sample may be a body fluid, tissue sample, or a combination thereof from the subject. The sample may contain cell-free DNA. As used herein, the term “biological sample” refers to solid tissue, such as a gastrointestinal biopsy, or fluids, body effluent, and excretion.

[0030] In one preferred embodiment, the sample is a body fluid, preferably selected from the group consisting of plasma, serum, blood, urine, or feces. More preferably, the sample is a plasma or serum sample, and the DNA is circulating cell-free DNA (ccfDNA).

[0031] In other embodiments, the sample is a tissue sample. For example, the sample may be obtained from colon tissue. Colon tissue or cells may be obtained, for example, by biopsy of tumor samples, tissue samples, excision, or needle biopsy of organs, prepared by endoscopic means.

[0032] After collection, the sample is prepared prior to the detection of biomarkers. Sample preparation includes the isolation of nucleic acids. The isolation procedure involves separating nucleic acids from insoluble components (e.g., the cytoskeleton) and cell membranes. Typically, colon tissue or cells may be treated with a lysis buffer solution prior to the isolation of nucleic acids. The lysis buffer solution is designed to dissolve tissues, cells, lipids, and other biomolecules that may be present in the crude tissue sample. Nucleic acids can be conveniently extracted from biological samples, for example, colon tissue, using standard extraction methods known in the art. Standard extraction methods include the use of chemical agents such as guanidine thiocyanate, phenol-chloroform extraction, and guanidine-based extraction. Commercially available nucleic acid extraction kits may also be used.

[0033] Methylation state According to the present invention, the methylation status of a target gene functions as a biomarker for colorectal cancer. CpG islands in selected genomic regions are particularly targeted.

[0034] DNA methylation in the human genome typically occurs at the 5-carbon of cytosine residues in CpG dinucleotides, resulting in 5-methylcytosine. CpG dinucleotides are rare in the human genome (approximately 1%). CpG dinucleotides are frequently found within clusters of over 200 base pairs with a G+C content of over 50% and a CpG frequency of at least 0.6, and are known as CpG islands. Approximately 60% of human gene promoters are associated with CpG islands. CpG islands within promoter regions are usually unmethylated in normal cells, except in some cells involved in tissue differentiation. Generally, CpG island methylation is associated with transcriptional silencing. In cancer, both global hypomethylation (decreased overall DNA methylation) and local hypermethylation, such as methylation of the first exon of promoters and tumor suppressor genes, have been observed. DNA hypomethylation occurs in many genomic sequences, such as repeating elements, retrotransposons, introns, and analogous elements, leading to genomic instability and potentially causing activation of several proto-oncogenes, including imprinting defects, as seen in the case of the IGF2 gene (encoding IGF-2) in Wilms' tumor.

[0035] Methylation of cytosine (C) nucleotides in CpG dinucleotide sequences (CpG sites, G = guanosine) of human DNA is a known phenomenon. Furthermore, abnormal methylation (hypermethylation and / or hypomethylation) in specific genomic regions is present in virtually all cancer types. Genomic regions in test samples that have altered methylation states compared to control samples are generally called "differentially methylated regions (DMRs)."

[0036] As used herein, "CpG island" refers to a region of DNA that has a higher G / C content and a higher frequency of CpG dinucleotides compared to the whole genome. The term "CG island" is also used interchangeably in this field. In "CpG island," "p" refers to the phosphate diester bond between a cytosine nucleotide and a guanine nucleotide. Methylation may refer to methylation and / or hydroxymethylation of DNA.

[0037] Several methods have been proposed to detect DNA methylation. A review by Delpu et al. discloses some of them (Delpu Y et al., DNA methylation and cancer diagnosis. Int J Mol Sci. 2013 Jul 18;14(7), pp. 15029-58 (Non-Patent Literature 1)).

[0038] Methylation-specific PCR (MS-PCR) is a method that specifically designs two sets of PCR primers to amplify target methylated and unmethylated DNA regions. Detection of PCR products is traditionally performed by gel electrophoresis. This method has been replaced by quantitative MS-PCR (qMS-PCR), where PCR amplification is monitored in real time by incorporating fluorescent molecules. This improvement allows for accurate quantification of DNA methylation levels in numerous specific regions, avoiding lengthy electrophoresis steps. Quantitative multiplex MS-PCR (QM-MS-PCR) and one-step MS-PCR (OS-MS-PCR) are also available for co-amplifying specific genes in tissues from different origins or for determining DNA methylation levels in specific regions without DNA extraction procedures. The qMS-PCR method is simple, rapid, inexpensive, highly sensitive, and easily standardized. Currently, the qMS-PCR method is one of the most commonly used techniques for cancer diagnosis in clinical applications. Methylation-sensitive high-resolution melting (MS-HRM) is based on the fact that the nucleotide sequence of PCR products of bisulfite-treated DNA differs depending on the methylation status of the target DNA region. The methylation level is determined by comparing the melting curve to that of a standard PCR product of the same region containing known methylated CpG sites. Bisulfite restriction enzyme analysis (COBRA) utilizes the ability of bisulfite conversion to create new restriction enzyme sites or maintain the consensus sites of MSREs. After amplification, the PCR product is digested with an appropriate MSRE. The proportion of digested PCR products is compared to undigested PCR products using polyacrylamide gel electrophoresis and image quantification software. This method is reliably applicable to DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples. Furthermore, this approach enables the evaluation of DNA methylation in a large number of biological samples. Very recently, high-throughput approaches have been developed.For example, Methyl Light is a high-throughput quantitative methylation assay that uses fluorescence-based real-time PCR (TaqMan®, Applied Biosystems, Foster City, CA, USA) in combination with bisulfite treatment. Also used in combination with bisulfite-treated pyrosequencing is a quantitative DNA sequencing method in which light is emitted as a result of each enzymatic reaction in which nucleotides are incorporated into the growing DNA strand. This quantitative method detects small amounts of methylated DNA in heterogeneous DNA preparations. These readily standardized, rapid, and inexpensive methods are increasingly being used for clinical purposes.

[0039] Very recently, next-generation sequencing (NGS) technology has significantly improved the resolution of DNA methylation profiles. NGS can also be applied to immunoprecipitated DNA fragments (methyl DNA immunoprecipitation sequencing, also known as MeDIPseq). Ultimately, NGS enables sequencing of the entire genome after bisulfite conversion. In addition to these NGS approaches, high-throughput single nucleotide polymorphism (SNP) genotyping systems are suitable for DNA methylation analysis from bisulfite-converted genomic DNA. Furthermore, other methods well known to those skilled in the art can be used, such as methods for directly analyzing unmodified DNA (e.g., nanopore sequencing), methods using specific restriction enzymes, methylation sequence enrichment methods (e.g., EpiMark methylation enrichment kits, New England), and immunoprecipitation.

[0040] All of these methods (qMS-PCR, MS-HRM, COBRA, MSRE, Methyl Light, NGS, SNP genotyping, pyrosequencing, microarrays, ICE-cold PCR, nanopore, etc.) can be used to determine the methylation of the markers of the present invention.

[0041] In a preferred embodiment of the present invention, methylation is detected using digital PCR, preferably digital droplet PCR (ddPCR).

[0042] As used herein, “digital PCR” refers to an assay that provides an endpoint measurement that gives the ability to quantify nucleic acids without the use of calibration curves used in real-time PCR. Digital PCR encompasses a variety of forms, including droplet digital PCR, beaming (beads, emulsions, amplification, and magnetism), microwell plates, bulk emulsion droplets, microfluidic compartments, and the use of nanoliter or picoliter-scale droplets produced by microfluidics.

[0043] "Droplet digital PCR" (ddPCR) refers to a digital PCR assay that measures the absolute amount of nucleic acid molecules by counting the number of nucleic acid molecules encapsulated in individual, volume-defined, oil-in-water partitions that support PCR amplification (Hindson et al., 2011. Analytical Chemistry 83, pp. 8604-8610 (Non-Patent Literature 2); Pekin et al., 2011. Lab on a Chip 11, pp. 2156-2166 (Non-Patent Literature 3); Pinheiro et al., 2012. Analytical Chem 84, pp. 1003-1011 (Non-Patent Literature 4)). A single ddPCR reaction can consist of at least 13,000 to 20,000 partitioned droplets per well.

[0044] After evaluating the methylation status, it is frequently expressed as a ratio or percentage of the total DNA population in the sample containing the specific site (e.g., in a single nucleotide, in a specific region or locus, or in a longer target sequence, e.g., up to 100 bp, 200 bp, or in a DNA subsequence) to the individual strands of DNA that are methylated at that specific site. Typically, the amount of unmethylated nucleic acid is determined by PCR using a standard substance. In a particular embodiment, a known amount of DNA is treated, for example, with bisulfite, and the resulting methylation-specific sequence is determined using one of the exponential amplification techniques.

[0045] The method of the present invention requires the detection of the "level of methylation," "amount of methylation," or "amount of methylation" at the CpG site. According to the present invention, the terms "level of methylation" or "amount of methylation" refer to a determination relating to quantitative measurement. Therefore, the terms "level of methylation" or "amount of methylation" can be used interchangeably.

[0046] In one exemplary embodiment, the method may use a set of primers. As used herein, the term “primer” refers to an isolated nucleic acid molecule that can specifically hybridize or anneal to the 5' or 3' region of a target genomic region (the positive and negative strands, respectively, and vice versa). Generally, primers are about 10–30 nucleotides long and anneal to both ends of a region containing about 50–200 nucleotides. Under appropriate conditions and with appropriate reagents, such primers enable amplification of nucleic acid molecules containing the nucleotide sequences adjacent to the primer. Since primers must be used in pairs, they are often referred to as a “primer pair” or “primer set.”

[0047] "Specific hybridization" is observed when a defined molecule does not hybridize to any other genomic region other than its target genomic region. Preferably, the defined molecule hybridizes to its target region under high stringency conditions, i.e., when temperature and ionic intensity conditions are selected to allow hybridization between two complementary DNA fragments.

[0048] In one exemplary embodiment, the method may use a method for detecting gene expression (e.g., dPCR) that uses a fluorescent probe to improve the specificity and / or sensitivity of detecting PCR products that accumulate during PCR. Such an assay is, for example, the TaqMan® gene expression assay, which uses probes containing a sub-groove junction (MGB) moiety that enhances the Tm difference between matched and mismatched probes. Furthermore, these MGB probes may contain a non-fluorescent quencher (NFQ) that enhances spectral resolution when multiple dyes are used in the reaction. Probes that may be used in this preferred embodiment are shown in Table 2 and SEQ ID NOs: 27-39 below.

[0049] As used herein, the term "probe" refers to a molecule that can specifically hybridize to a genomic region of interest. Probes are useful for highlighting the presence of such genomic region in a biological sample. These probes may include at least one non-natural nucleotide, such as peptide nucleic acids (PNA), phosphate-containing peptide nucleic acids (PHONA), cross-linked nucleic acids or locked nucleic acids (BNA or LNA), and morpholino nucleic acids. Non-natural nucleotides may further include chemically modified nucleic acids or nucleic acid analogs, such as methylphosphonate-type DNA or RNA, phosphorothioate-type DNA or RNA, phosphoramidate-type DNA or RNA, and 2'-O-methyl-type DNA or RNA.

[0050] In one preferred embodiment, the probe of the present invention comprises at least 15 consecutive nucleotides. In a more preferred embodiment, a molecule that can be used as a probe according to the present invention has a total minimum number of 15 nucleotides. In an even more preferred embodiment, those molecules comprise between 15 and 30 nucleotides (total).

[0051] For specific uses, the probes and primers of the present invention may be directly or indirectly labeled with a detectable label. The label may be of any type depending on the experiment to be performed. The label may be a radioactive isotope (e.g., 32P, 33P, 35S, 3H, or 125L), or a non-radioactive entity selected from ligands (e.g., biotin, avidin, or streptavidin), dioxygenin, haptens, colorants, and luminescent agents (e.g., radioluminescent, chemiluminescent, bioluminescent, fluorescent, or phosphorescent agents). Preferably, 6-carboxyfluorescein (FAM), VIC, HEX, and tetramethylrhodamine (TAM RA) are used. Unlabeled polynucleotide sequences may also be used directly, for example, as probes or primers in PCR-based processes (e.g., quantitative PCR).

[0052] Exemplary primers and probes are summarized in Table 1 and Table 2 below.

[0053] [Table 1]

[0054] [Table 2]

[0055] target genes The term "gene" refers to a nucleic acid sequence containing a coding sequence necessary for the production of RNA or polypeptides or their precursors. The term "gene" includes the coding region of a structural gene, and the gene includes sequences located at both the 5' and 3' ends adjacent to the coding region, for example, at approximately 1 kb intervals, such that the gene corresponds to the length of a full-length mRNA containing, for example, the coding region, untranslated region, structural sequence and other sequences, and regulatory sequences, such as promoters. A sequence located at the 5' end of the coding region and present on mRNA is called the 5' untranslated sequence. A sequence located at the 3' or downstream of the coding region and present on mRNA is called the 3' untranslated sequence. In this invention, the term "gene" specifically refers to the genomic morphology of a gene. In addition to the inclusion of introns, the genomic morphology of a gene may further include sequences located at both the 5' and 3' ends of the sequence present on the RNA transcript. These sequences are called "flanking" sequences or regions (these flanking sequences are located at the 5' or 3' end of the untranslated sequence present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence gene transcription. The 3' flanking region may contain sequences that manage transcription termination, post-transcriptional cleavage, and polyadenylation.

[0056] The inventors have shown that hypermethylation of the promoter or other regions of specific genes can be used as a highly sensitive and specific biomarker for colorectal cancer, even in its early stages, in patients with colorectal cancer. The genes listed below are also referred to as “biomarkers.” These genes can be used individually or in combination.

[0057] ZSCAN23 ZSCAN23 (containing zinc fingers and a SCAN domain) is a protein-coding gene. ZSCAN23 provides sequence-specific double-stranded DNA binding activity. ZSCAN23 is predicted to be involved in the regulation of transcription by RNA polymerase II. This gene is also known as "ZNF390," "ZNF453," "dJ29K1.3," or "dJ29K1.3.1." Its DNA sequence is located on the reverse strand at positions 28,431,930–28,443,502 of chromosome 6 (Hg38 coordinates).

[0058] In one particular embodiment, the level or amount of methylation is determined in the nucleotide region of genomic DNA consisting of chr6:28431930~28443502 (Hg38 coordinates).

[0059] In one particular embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr6:28431930~28443502 (Hg38 coordinates).

[0060] In one preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr6:28442840~28443839 (Hg38 coordinates). In a further preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr6:28443240~28443439 (Hg38 coordinates).

[0061] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 40 in the ZSCAN23 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 40. In a preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 53 in the ZSCAN23 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 53.

[0062] In one preferred embodiment, the level or amount of methylation of the ZSCAN23 gene is determined using the primers of SEQ ID NO: 1 and SEQ ID NO: 2.

[0063] In one preferred embodiment, the level or amount of methylation of the ZSCAN23 gene is determined using the probe of SEQ ID NO: 27.

[0064] In a more preferred embodiment, the level or amount of methylation of the ZSCAN23 gene is determined using the primers of SEQ ID NO: 1 and SEQ ID NO: 2 and the probe of SEQ ID NO: 27.

[0065] Preferably, the level or amount of methylation of the ZSCAN23 gene is determined by ddPCR using the primers of SEQ ID NO: 1 and SEQ ID NO: 2 and the probe of SEQ ID NO: 27.

[0066] COL4A1 COL4A1 (type IV collagen alpha 1 chain) is a protein-coding gene. This gene encodes type IV collagen alpha protein, an essential component of the basement membrane. This gene is also known as "BSVD," "BSVD1," "RATOR," "PADMAL," or "COL4A1." Its DNA sequence is located on the reverse strand at positions 110,148,963 to 110,307,157 of chromosome 13 (Hg38 coordinates).

[0067] In one particular embodiment, the level or amount of methylation is determined in a nucleotide region of genomic DNA consisting of chr13:11014963~110307157 (Hg38 coordinates).

[0068] In one particular embodiment, the method advantageously includes the step of determining the methylation status of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr13:11014963~110307157 (Hg38 coordinates).

[0069] In one preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr13:110306660 to 110307659 (Hg38 coordinates). In a further preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr13:110307060 to 110307259 (Hg38 coordinates).

[0070] In one particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 45 in the COL4A1 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 45. In one preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 58 in the COL4A1 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 58.

[0071] In one preferred embodiment, the level or amount of methylation of the COL4A1 gene is determined using the primers of SEQ ID NO: 11 and SEQ ID NO: 12.

[0072] In one preferred embodiment, the level or amount of methylation of the COL4A1 gene is determined using the probe of SEQ ID NO: 32.

[0073] In a more preferred embodiment, the level or amount of methylation of the COL4A1 gene is determined using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 and the probe of SEQ ID NO: 32.

[0074] Preferably, the level or amount of methylation of the COL4A1 gene is determined by ddPCR using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 and the probe of SEQ ID NO: 32.

[0075] AQP5-AS1 AQP5-AS1 (AQP5 and AQP2 antisense RNA 2) is a non-coding RNA. The AQP5-AS1 gene is also known as "MIAC" ​​or "actin cytoskeleton inhibitory micropeptide". Its DNA sequence is located at positions 49,951,512–49,962,924 on chromosome 12 (Hg38 coordinates).

[0076] In one particular embodiment, the level or amount of methylation is determined in a nucleotide region of genomic DNA consisting of chr12:49951512~49962924 (Hg38 coordinates).

[0077] In one particular embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr12:49951512~49962924 (Hg38 coordinates).

[0078] In one preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr12:49961035~49962034 (Hg38 coordinates). In a further preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr12:49961440~49961629 (Hg38 coordinates).

[0079] In one particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 42 in the AQP5-AS1 gene, or in a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 42. In one particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 42. In one preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 55 in the AQP5-AS1 gene, or in a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 55. In one preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 55.

[0080] In one preferred embodiment, the level or amount of methylation of the AQP5-AS1 gene is determined using the primers of SEQ ID NO: 5 and SEQ ID NO: 6.

[0081] In one preferred embodiment, the level or amount of methylation of the AQP5-AS1 gene is determined using the probe of SEQ ID NO: 29.

[0082] In a more preferred embodiment, the level or amount of methylation of the AQP5-AS1 gene is determined using the primers of SEQ ID NO: 5 and SEQ ID NO: 6 and the probe of SEQ ID NO: 29.

[0083] Preferably, the level or amount of methylation of the AQP5-AS1 gene is determined by ddPCR using the primers of SEQ ID NO: 5 and SEQ ID NO: 6 and the probe of SEQ ID NO: 29.

[0084] NR5A2 NR5A2 (nuclear receptor subfamily 5, group A member 2) is a protein-coding gene. The protein encoded by this gene is a DNA-binding zinc finger transcription factor. This gene is also known as "B1F2", "FTF", "FTZ-F1", "FTZ-F1 beta", "LRH-1", "LRH1", "hB1F", or "hB1F-2". Its DNA sequence is located on the forward strand of chromosome 1 at positions 200,027,614 to 200,177,420 (Hg38 coordinates).

[0085] In one particular embodiment, the level or amount of methylation is determined in a nucleotide region of genomic DNA consisting of chr1:200027614~200177420 (Hg38 coordinates).

[0086] In a particular embodiment, the method advantageously includes the step of determining the methylation status of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr1:200027614~200177420 (Hg38 coordinates).

[0087] In one preferred embodiment, the method advantageously includes a step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr1:200038400 to 200039399 (Hg38 coordinates). In a further preferred embodiment, the method advantageously includes a step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr9:200038800 to 200038999 (Hg38 coordinates).

[0088] In one particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 43 in the NR5A2 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 43. In one preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 56 in the NR5A2 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 56.

[0089] In one preferred embodiment, the level or amount of methylation of the NR5A2 gene is determined using the primers of SEQ ID NO: 7 and SEQ ID NO: 8.

[0090] In one preferred embodiment, the level or amount of methylation of the NR5A2 gene is determined using the probe of SEQ ID NO: 30.

[0091] In a more preferred embodiment, the level or amount of methylation of the NR5A2 gene is determined using the primers of SEQ ID NO: 7 and SEQ ID NO: 8 and the probe of SEQ ID NO: 30.

[0092] Preferably, the level or amount of methylation of the NR5A2 gene is determined by ddPCR using the primers of SEQ ID NO: 7 and SEQ ID NO: 8 and the probe of SEQ ID NO: 30.

[0093] C9orf50 C9orf50 (open reading frame 50 of chromosome 9) is a protein-coding gene. This gene is also known as "FLJ35803". Its DNA sequence is located on the reverse strand at positions 129,612,225 to 129,621,101 of chromosome 9 (Hg38 coordinates).

[0094] In one particular embodiment, the level or amount of methylation is determined in a nucleotide region of genomic DNA consisting of chr9:129612225~129621101 (Hg38 coordinates).

[0095] In one particular embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr9:129612225~129621101 (Hg38 coordinates).

[0096] In one preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr9:129620034 to 129621033 (Hg38 coordinates). In a further preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr9:129620434 to 129620633 (Hg38 coordinates).

[0097] In one particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 41 in the C9orf50 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 41. In one preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 54 in the C9orf50 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 54.

[0098] In one preferred embodiment, the level or amount of methylation of the C9orf50 gene is determined using the primers of SEQ ID NO: 3 and SEQ ID NO: 4.

[0099] In one preferred embodiment, the level or amount of methylation of the C9orf50 gene is determined using the probe of SEQ ID NO: 28.

[0100] In a more preferred embodiment, the level or amount of methylation of the C9orf50 gene is determined using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 and the probe of SEQ ID NO: 28.

[0101] Preferably, the level or amount of methylation of the C9orf50 gene is determined by ddPCR using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 and the probe of SEQ ID NO: 28.

[0102] ADARB2 ADARB2 (adenosine deaminase RNA-specific B2 (inactive)) is a protein-coding gene. This gene encodes a member of the double-stranded RNA adenosine deaminase family of RNA editing enzymes and may be involved in the regulation of RNA editing. This gene is also known as "ADAR3," "RED2," or "hRED2." Its DNA sequence is located on the reverse strand at positions 1,177,313–1,737,525 of chromosome 10 (Hg38 coordinates).

[0103] In one particular embodiment, the level or amount of methylation is determined in a nucleotide region of genomic DNA consisting of chr10:1177313~1737525 (Hg38 coordinates).

[0104] In a particular embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in a region of the genomic DNA consisting of chr10:1177313~1737525 (Hg38 coordinates).

[0105] In one preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr10:1737180~1738179 (Hg38 coordinates). In a further preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr10:1737580~1737779 (Hg38 coordinates).

[0106] In one particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 44 in the ADARB2 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 44. In one preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 57, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 57.

[0107] In one preferred embodiment, the level or amount of methylation of the ADARB2 gene is determined using the primers of SEQ ID NO: 9 and SEQ ID NO: 10.

[0108] In one preferred embodiment, the level or amount of methylation of the ADARB2 gene is determined using the probe of SEQ ID NO: 31.

[0109] In a more preferred embodiment, the level or amount of methylation of the ADARB2 gene is determined using the primers of SEQ ID NO: 9 and SEQ ID NO: 10 and the probe of SEQ ID NO: 31.

[0110] Preferably, the level or amount of methylation of the ADARB2 gene is determined by ddPCR using the primers of SEQ ID NO: 9 and SEQ ID NO: 10 and the probe of SEQ ID NO: 31.

[0111] NPY NPY (neuropeptide Y) is a protein-coding gene. This gene encodes a neuropeptide widely expressed in the central nervous system and influences many physiological processes, including cortical excitability, stress response, food intake, circadian rhythms, and cardiovascular function. This gene is also known as "PYY4". Its DNA sequence is located on the forward strand at positions 24,284,188–24,291,862 of chromosome 7 (Hg38 coordinates).

[0112] In one particular embodiment, the level or amount of methylation is determined in the nucleotide region of genomic DNA consisting of chr7:24284188~24291862 (Hg38 coordinates).

[0113] In a particular embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr7:24284188~24291862 (Hg38 coordinates).

[0114] In one preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr7:24283690~24284689 (Hg38 coordinates). In a further preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr7:24284090~24284289 (Hg38 coordinates).

[0115] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 46 in the NPY gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 46. In a preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 59 in the NPY gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 59.

[0116] In one preferred embodiment, the level or amount of methylation of the NPY gene is determined using the primers of SEQ ID NO: 13 and SEQ ID NO: 14.

[0117] In one preferred embodiment, the level or amount of methylation of the NPY gene is determined using the probe of SEQ ID NO: 33.

[0118] In a more preferred embodiment, the level or amount of methylation of the NPY gene is determined using the primers of SEQ ID NO: 13 and SEQ ID NO: 14 and the probe of SEQ ID NO: 33.

[0119] Preferably, the level or amount of methylation of the NPY gene is determined by ddPCR using the primers of SEQ ID NO: 13 and SEQ ID NO: 14 and the probe of SEQ ID NO: 33.

[0120] WIF1 WIF1 (WNT suppressor 1) is a protein-coding gene. The protein encoded by this gene functions to inhibit WNT proteins, which are important extracellular signaling molecules in embryonic development. This gene is also known as "WIF-1". Its DNA sequence is located on the reverse strand at positions 65,050,626 to 65,121,305 of chromosome 12 (Hg38 coordinates). NPY (neuropeptide Y) is a protein-coding gene. This gene encodes a neuropeptide widely expressed in the central nervous system and influences many physiological processes, including cortical excitability, stress response, food intake, circadian rhythms, and cardiovascular function. This gene is also known as "PYY4". Its DNA sequence is located on the reverse strand at positions 65,050,626 to 65,121,305 of chromosome 12 (Hg38 coordinates).

[0121] In one particular embodiment, the level or amount of methylation is determined in a nucleotide region of genomic DNA consisting of chr12:65050626~65121305 (Hg38 coordinates).

[0122] In one particular embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr12:65050626~65121305 (Hg38 coordinates).

[0123] In one preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr12:65120760~65121759 (Hg38 coordinates). In a further preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr12:65121160~65121359 (Hg38 coordinates).

[0124] In one particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 47 in the WIF1 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 47. In one preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 60 in the WIF1 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 60.

[0125] In one preferred embodiment, the level or amount of methylation of the WIF1 gene is determined using the primers of SEQ ID NO: 15 and SEQ ID NO: 16.

[0126] In one preferred embodiment, the level or amount of methylation of the WIF1 gene is determined using the probe of SEQ ID NO: 34.

[0127] In a more preferred embodiment, the level or amount of methylation of the WIF1 gene is determined using the primers of SEQ ID NO: 15 and SEQ ID NO: 16 and the probe of SEQ ID NO: 34.

[0128] Preferably, the level or amount of methylation of the WIF1 gene is determined by ddPCR using the primers of SEQ ID NO: 15 and SEQ ID NO: 16 and the probe of SEQ ID NO: 34.

[0129] CPNE8 CPNE8 (copin 8) is a protein-coding gene. This gene is one of several genes that encode a calcium-dependent protein containing two N-terminal type II C2 domains and an integrin A domain-like sequence at the C-terminus. Its DNA sequence is located on the reverse strand at positions 38,646,822–38,907,430 on chromosome 12 (Hg38 coordinates).

[0130] In one particular embodiment, the level or amount of methylation is determined in a nucleotide region of genomic DNA consisting of chr12:38646822~38907430 (Hg38 coordinates).

[0131] In one particular embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr12:38646822~38907430 (Hg38 coordinates).

[0132] In one preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr12:38904820~38905819 (Hg38 coordinates). In a further preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr12:38905220~38905419 (Hg38 coordinates).

[0133] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 48 in the CPNE8 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 48. In a preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 61 in the WIF1 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 61.

[0134] In one preferred embodiment, the level or amount of methylation of the CPNE8 gene is determined using the primers of SEQ ID NO: 17 and SEQ ID NO: 18.

[0135] In one preferred embodiment, the level or amount of methylation of the CPNE8 gene is determined using the probe of SEQ ID NO: 35.

[0136] In a more preferred embodiment, the level or amount of methylation of the CPNE8 gene is determined using the primers of SEQ ID NO: 17 and SEQ ID NO: 18 and the probe of SEQ ID NO: 35.

[0137] Preferably, the level or amount of methylation of the CPNE8 gene is determined by ddPCR using the primers of SEQ ID NO: 17 and SEQ ID NO: 18 and the probe of SEQ ID NO: 35.

[0138] LINC00693(RBMS3) LINC00693, or RBMS3 (RNA-binding motif single-strand interaction protein 3), or LINC00693(RBMS3) is a protein-coding gene. This gene encodes an RNA-binding protein belonging to the c-myc gene single-strand binding protein family. This gene is also known as "PYY4". Its DNA sequence is located on the forward strand at positions 28,574,791–30,010,391 of chromosome 3 (Hg38 coordinates).

[0139] In one particular embodiment, the level or amount of methylation is determined in a nucleotide region of genomic DNA consisting of chr3:28574791~30010391 (Hg38 coordinates).

[0140] In one particular embodiment, the method advantageously includes the step of determining the methylation status of one or more CpG dinucleotides in a region of the genomic DNA consisting of chr3:28574791~30010391 (Hg38 coordinates).

[0141] In one preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr3:28574870~28575869 (Hg38 coordinates). In a further preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr3:28575270~28575469 (Hg38 coordinates).

[0142] In one particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 49 in the LINC00693(RBMS3) gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 49. In one preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 62 in the LINC00693(RBMS3) gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 62.

[0143] In one preferred embodiment, the level or amount of methylation of the LINC00693(RBMS3) gene is determined using the primers of SEQ ID NO: 19 and SEQ ID NO: 20.

[0144] In one preferred embodiment, the level or amount of methylation of the LINC00693(RBMS3) gene is determined using the probe of SEQ ID NO: 36.

[0145] In a more preferred embodiment, the level or amount of methylation of the LINC00693(RBMS3) gene is determined using the primers of SEQ ID NO: 19 and SEQ ID NO: 20 and the probe of SEQ ID NO: 36.

[0146] Preferably, the level or amount of methylation of the LINC00693(RBMS3) gene is determined by ddPCR using the primers of SEQ ID NO: 19 and SEQ ID NO: 20 and the probe of SEQ ID NO: 36.

[0147] LINC00900 LINC00900 (long intergene non-protein-coding RNA 900) is a non-coding RNA gene belonging to the lncRNA class. Its DNA sequence is located on the reverse strand at positions 115,753,889 to 115,760,646 of chromosome 11 (Hg38 coordinates).

[0148] In one particular embodiment, the level or amount of methylation is determined in a nucleotide region of genomic DNA consisting of chr11:115753889~115760646 (Hg38 coordinates).

[0149] In a particular embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr11:115,753,889 to 115,760,646 (Hg38 coordinates).

[0150] In one preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr11:115759620~115760619 (Hg38 coordinates). In a further preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr11:115759620~115760619 (Hg38 coordinates).

[0151] In one particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 50 in the LINC00900 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 50. In one preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 63 in the LINC00900 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 63.

[0152] In one preferred embodiment, the level or amount of methylation of the LINC00900 gene is determined using the primers of SEQ ID NO: 21 and SEQ ID NO: 22.

[0153] In one preferred embodiment, the level or amount of methylation of the LINC00900 gene is determined using the probe of SEQ ID NO: 37.

[0154] In a more preferred embodiment, the level or amount of methylation of the LINC00900 gene is determined using the primers of SEQ ID NO: 21 and SEQ ID NO: 22 and the probe of SEQ ID NO: 37.

[0155] Preferably, the level or amount of methylation of the LINC00900 gene is determined by ddPCR using the primers of SEQ ID NO: 21 and SEQ ID NO: 22 and the probe of SEQ ID NO: 37.

[0156] DYDC2 DYDC2 (DPY30 domain-containing 2) is a protein-coding gene. This gene encodes a member of the family of proteins containing the DPY30 domain. This gene is also known as "MGC16186" or "bA36D19". Its DNA sequence is located on the forward strand at positions 80,344,745–80,368,073 of chromosome 10 (Hg38 coordinates).

[0157] In one particular embodiment, the level or amount of methylation is determined in a nucleotide region of genomic DNA consisting of chr10:80344745~80368073 (Hg38 coordinates).

[0158] In a particular embodiment, the method advantageously includes the step of determining the methylation status of one or more CpG dinucleotides in a region of the genomic DNA consisting of chr10:80344745~80368073 (Hg38 coordinates).

[0159] In one preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr10:80356110~80357109 (Hg38 coordinates). In a further preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr10:80356510~80356709 (Hg38 coordinates).

[0160] In one particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 51 in the DYDC2 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 51. In one preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 64 in the DYDC2 gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 64.

[0161] In one preferred embodiment, the level or amount of methylation of the DYDC2 gene is determined using the primers of SEQ ID NO: 23 and SEQ ID NO: 24.

[0162] In one preferred embodiment, the level or amount of methylation of the DYDC2 gene is determined using the probe of SEQ ID NO: 38.

[0163] In a more preferred embodiment, the level or amount of methylation of the DYDC2 gene is determined using the primers of SEQ ID NO: 23 and SEQ ID NO: 24 and the probe of SEQ ID NO: 38.

[0164] Preferably, the level or amount of methylation of the DYDC2 gene is determined by ddPCR using the primers of SEQ ID NO: 23 and SEQ ID NO: 24 and the probe of SEQ ID NO: 38.

[0165] LRRC4(SND1) LRRC4 (Leucine-Rich Repeat 4) is a protein-coding gene. The exact function of the protein encoded by this gene is unknown. This gene is also known as "NAG14" or "NGL-2". Its DNA sequence is located on the reverse strand at positions 128,027,071 to 128,032,107 of chromosome 7 (Hg38 coordinates).

[0166] In one particular embodiment, the level or amount of methylation is determined in a nucleotide region of genomic DNA consisting of chr7:128027071~128032107 (Hg38 coordinates).

[0167] In a particular embodiment, the method advantageously includes the step of determining the methylation status of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr7:128027071 to 128032107 (Hg38 coordinates).

[0168] In one preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr7:128031906~128032905 (Hg38 coordinates). In a further preferred embodiment, the method advantageously includes the step of determining the methylation state of one or more CpG dinucleotides in the region of the genomic DNA consisting of chr7:128031906~128032905 (Hg38 coordinates).

[0169] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 52 in the LRRC4(SND1) gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 52. In a preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 65 in the LRRC4(SND1) gene, or of a polymorphic variant sequence having at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity with SEQ ID NO: 65.

[0170] In one preferred embodiment, the level or amount of methylation of the LRRC4(SND1) gene is determined using the primers of SEQ ID NO: 25 and SEQ ID NO: 26.

[0171] In one preferred embodiment, the level or amount of methylation of the LRRC4(SND1) gene is determined using the probe of SEQ ID NO: 39.

[0172] In a more preferred embodiment, the level or amount of methylation of the LRRC4(SND1) gene is determined using the primers of SEQ ID NO: 25 and SEQ ID NO: 26 and the probe of SEQ ID NO: 39.

[0173] Preferably, the level or amount of methylation of the LRRC4(SND1) gene is determined by ddPCR using the primers of SEQ ID NO: 25 and SEQ ID NO: 26 and the probe of SEQ ID NO: 39.

[0174] method Accordingly, in a first aspect, the present invention relates to an in vitro method for detecting or monitoring colorectal cancer (CRC) in a subject, comprising the step of determining the level or amount of methylation of at least one gene from the group of genes in a biological sample of the subject.

[0175] In one particular embodiment, the method includes the step of determining the level or amount of methylation of at least one gene, ZSCAN23, COL4A1, or AQP5-AS1, in a biological sample containing genomic DNA from a subject.

[0176] In one particular embodiment, the method includes a step of determining the level or amount of methylation of at least ZSCAN23 in a biological sample containing genomic DNA from a subject.

[0177] In one particular embodiment, the method includes a step of determining the level or amount of methylation of at least COL4A1 in a biological sample containing genomic DNA from a subject.

[0178] In one particular embodiment, the method includes a step of determining the level or amount of methylation of at least AQP5-AS1 in a biological sample containing genomic DNA from a subject.

[0179] In one particular embodiment, the method includes a step of determining the level or amount of methylation of at least the ZSCAN23 and AQP5-AS1 genes. In another particular embodiment, the method includes a step of determining the level or amount of methylation of at least the ZSCAN23 and COL4A1 genes. In yet another particular embodiment, the method includes a step of determining the level or amount of methylation of at least the AQP5-AS1 and COL4A1 genes.

[0180] In a preferred embodiment, the method includes a step of determining the level of methylation of a combination of the ZSCAN23, COL4A1, and AQP5-AS1 genes.

[0181] In particular, the combination of biomarkers according to the present invention is - ZSCAN23 gene and COL4A1 gene, - ZSCAN23 gene and AQP5-AS1 gene, - COL4A1 gene and AQP5-AS1 gene, - ZSCAN23 gene and NR5A2 gene, - ZSCAN23, COL4A1 and AQP5-AS1 genes, - ZSCAN23, COL4A1, AQP5-AS1, and NR5A2 genes It is possible.

[0182] In particular, the combination of biomarkers according to the present invention is - ZSCAN23 gene and COL4A1 gene, - ZSCAN23 gene and AQP5-AS1 gene, - ZSCAN23 gene and NR5A2 gene, - ZSCAN23, COL4A1 and AQP5-AS1 genes, - ZSCAN23, COL4A1, AQP5-AS1, and NR5A2 genes It is possible.

[0183] In a particular embodiment, the method includes determining the methylation levels of ZSCAN23 and at least two, preferably three, preferably four, and more preferably at least five genes selected from the group consisting of the genes C9orf50, AQP5-AS1, NR5A2, ADARB2, COL4A1, NPY, WIF1, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and LRRC4 (SND1). In a preferred embodiment, the method includes determining the methylation levels of ZSCAN23 and at least two, preferably three, preferably four, and more preferably at least five genes selected from the group consisting of AQP5-AS1, COL4A1, C9orf50, NR5A2, NPY, and WIF1. In a preferred embodiment, the method includes determining the methylation levels of ZSCAN23 and at least two, preferably three, preferably four genes selected from the group consisting of AQP5-AS1, COL4A1, C9orf50, NR5A2, and WIF1.

[0184] In a particular embodiment, the method includes determining the methylation levels of COL4A1 and at least two, preferably three, preferably four, and more preferably at least five genes selected from the group consisting of ZSCAN23, AQP5-AS1, NR5A2, ADARB2, C9orf50, NPY, WIF1, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and LRRC4 (SND1). In a preferred embodiment, the method includes determining the methylation levels of COL4A1 and at least two, preferably three, preferably four, and more preferably at least five genes selected from the group consisting of AQP5-AS1, ZSCAN23, C9orf50, NR5A2, NPY, and WIF1. In a preferred embodiment, the method includes determining the methylation levels of COL4A1 and at least two, preferably three, preferably four genes selected from the group consisting of AQP5-AS1, ZSCAN23, C9orf50, NR5A2, and WIF1.

[0185] In a particular embodiment, the method includes determining the methylation levels of at least two, preferably three, preferably four, and more preferably at least five genes selected from the group consisting of AQP5-AS1 and the genes C9orf50, ZSCAN23, NR5A2, ADARB2, COL4A1, NPY, WIF1, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and LRRC4 (SND1). In a preferred embodiment, the method includes determining the methylation levels of at least two, preferably three, preferably four, and more preferably at least five genes selected from the group consisting of AQP5-AS1 and the genes COL4A1, ZSCAN23, C9orf50, NR5A2, NPY, and WIF1. In a preferred embodiment, the method includes determining the methylation levels of AQP5-AS1 and at least two, preferably three, and preferably four genes selected from the group consisting of COL4A1, ZSCAN23, C9orf50, NR5A2, and WIF1.

[0186] In a particular embodiment, this method is - ZSCAN23 and COL4A1 genes, - ZSCAN23 and AQP5-AS1 genes, - COL4A1 and AQP5-AS1 genes, or - ZSCAN23, COL4A1, and AQP5-AS1 genes This includes a step of determining the level of methylation, Herein, the method further includes the steps of detecting methylation of at least one gene, preferably two genes, preferably three genes, selected from the group consisting of NR5A2, C9orf50, NPY, or WIF1, or determining the level of methylation.

[0187] In a particular embodiment, this method is - ZSCAN23 and COL4A1 genes, - ZSCAN23 and AQP5-AS1 genes, - COL4A1 and AQP5-AS1 genes, or - ZSCAN23, COL4A1, and AQP5-AS1 genes This includes a step of determining the level of methylation, Herein, the method further includes a step of detecting methylation of the NR5A2 gene, or a step of determining the level of methylation.

[0188] In a particular embodiment, this method is - ZSCAN23 and COL4A1 genes, - ZSCAN23 and AQP5-AS1 genes, - COL4A1 and AQP5-AS1 genes, or - ZSCAN23, COL4A1, and AQP5-AS1 genes This includes a step of determining the level of methylation, Herein, the method further includes a step of detecting methylation of the C9orf50 gene, or a step of determining the level of methylation.

[0189] In a particular embodiment, this method is - ZSCAN23 and COL4A1 genes, - ZSCAN23 and AQP5-AS1 genes, - COL4A1 and AQP5-AS1 genes, or - ZSCAN23, COL4A1, and AQP5-AS1 genes This includes a step of determining the level of methylation, Herein, the method further includes a step of detecting methylation of the WIF1 gene or a step of determining the level of methylation.

[0190] In a particular embodiment, this method is - ZSCAN23 and COL4A1 genes, - ZSCAN23 and AQP5-AS1 genes, - COL4A1 and AQP5-AS1 genes, or - ZSCAN23, COL4A1, and AQP5-AS1 genes This includes a step of determining the level of methylation, Herein, the method further includes a step of detecting methylation of the NPY gene or a step of determining the level of methylation.

[0191] In a particular embodiment, this method is - ZSCAN23 and COL4A1 genes, - ZSCAN23 and AQP5-AS1 genes, - COL4A1 and AQP5-AS1 genes, or - ZSCAN23, COL4A1, and AQP5-AS1 genes This includes a step of determining the level of methylation, Herein, the method further includes the steps of detecting methylation of at least two, preferably three, preferably four, and more preferably at least five genes selected from the group consisting of NR5A2, ADARB2, C9orf50, NPY, WIF1, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and LRRC4 (SND1), or determining the level of methylation.

[0192] In a particular embodiment, this method is - ZSCAN23 and COL4A1 genes, - ZSCAN23 and AQP5-AS1 genes, - COL4A1 and AQP5-AS1 genes, or - ZSCAN23, COL4A1, and AQP5-AS1 genes This includes a step of determining the level of methylation, Herein, the method further includes the steps of detecting methylation of at least two, preferably three, preferably four genes selected from the group consisting of NR5A2, C9orf50, NPY, and WIF1, or determining the level of methylation.

[0193] In particular, the combination of biomarkers according to the present invention is - ZSCAN23 gene and C9orf50 gene, - ZSCAN23 gene and AQP5-AS1 gene, - C9orf50 gene and AQP5-AS1 gene, - ZSCAN23 gene and NR5A2 gene, - ZSCAN23, C9orf50, and AQP5-AS1 genes, - ZSCAN23, C9orf50, AQP5-AS1, and NR5A2 genes It is possible.

[0194] In particular, the combination of biomarkers according to the present invention is - ZSCAN23 gene and C9orf50 gene, - ZSCAN23 gene and AQP5-AS1 gene, - ZSCAN23 gene and NR5A2 gene, - ZSCAN23, C9orf50, and AQP5-AS1 genes, - ZSCAN23, C9orf50, AQP5-AS1, and NR5A2 genes It is possible.

[0195] In a particular embodiment, the method includes the step of determining the methylation level of at least two genes, the two genes being AQP5-AS1 and ZSCAN23, AQP5-AS1 and COL4A1, AQP5-AS1 and NR5A2, AQP5-AS1 and C9orf50, AQP5-AS1 and NPY, AQP5-AS1 and WIF1, ZSCAN23 and COL4A1, ZSCAN23 and NR5A2, ZSCAN23 and C9orf50, ZSCAN23 and NPY, ZSCAN23 and WIF1, COL4A1 and NR5A2, COL4A1 and C9orf50, COL4A1 and NPY, or COL4A1 and WIF1 genes.

[0196] In a preferred embodiment, the method includes the step of determining the methylation level of at least two genes, the two genes being AQP5-AS1 and ZSCAN23, AQP5-AS1 and COL4A1, AQP5-AS1 and NR5A2, AQP5-AS1 and C9orf50, AQP5-AS1 and NPY, AQP5-AS1 and WIF1, ZSCAN23 and COL4A1, ZSCAN23 and NR5A2, ZSCAN23 and C9orf50, ZSCAN23 and WIF1, COL4A1 and NR5A2, COL4A1 and C9orf50, or COL4A1 and WIF1 genes. In a preferred embodiment, the method includes the step of determining the methylation levels of at least two genes, the two genes being AQP5-AS1 and C9orf50, AQP5-AS1 and NPY, AQP5-AS1 and WIF1, C9orf50 and ZSCAN23, COL4A1 and C9orf50, COL4A1 and NR5A2, COL4A1 and WIF1, NR5A2 and AQP5-AS1, NR5A2 and ZSCAN23, or ZSCAN23 and WIF1.

[0197] In a particular embodiment, the method includes the step of determining the methylation levels of at least three genes, wherein the three genes are AQP5-AS1, ZSCAN23 and COL4A1; AQP5-AS1, ZSCAN23 and NR5A2; AQP5-AS1, ZSCAN23 and C9orf50; AQP5-AS1, ZSCAN23 and NPY; AQP5-AS1, ZSCAN23 and WIF1; AQP5- AS1, COL4A1 and NR5A2; AQP5-AS1, COL4A1 and C9orf50; AQP5-AS1, COL4A1 and NPY; AQP5-AS1, COL4A1 and WIF1; AQP5-AS1, NR5A2 and C9orf50; AQP5-AS1, NR5A2 and NPY; AQP5-AS1, NR5A2 and WIF1; AQP5-AS1, C9orf50 and NPY; AQP5-AS1, C9orf50 and WIF1; AQP5-AS1, NPY and WIF1; ZSCAN23, COL4A1 and NR5A2; ZSCAN23, COL4A1 and C9orf50; ZSCAN23, COL4A1 and NPY; ZSCAN23, COL4A1 and WIF1; ZSCAN23, NR5A2 and C9orf50; ZSCAN23, NR5A2 and NPY; ZSCAN23, NR5A2 and WIF1; ZSCAN23, C9orf50 and NPY; ZSCAN23, C9orf50 and WIF1; ZSCAN23, NPY and WIF1; COL4A1, NR5A2 and C9orf50; COL4A1, NR5A2 and NPY; COL4A1, NR5A2 and WIF1; COL4A1, C9orf50 and NPY; COL4A1, C9orf50 and WIF1; or COL4A1, NPY and WIF1.

[0198] In a preferred embodiment, the method includes the step of determining the methylation levels of at least three genes, wherein the three genes are AQP5-AS1, ZSCAN23 and COL4A1; AQP5-AS1, ZSCAN23 and C9orf50; AQP5-AS1, ZSCAN23 and NPY; AQP5-AS1, ZSCAN23 and WIF1; AQP5-AS1, COL4A1 and NR5A2; AQP5-AS1, COL4A1 and NPY; AQP5-AS1, COL4A1 and WIF1; AQP5-AS1, NR5A2 and C9orf50; AQP5-AS1, NR5A2 and NPY; AQP5-AS1 , NR5A2 and WIF1; AQP5-AS1, C9orf50 and WIF1; AQP5-AS1, NPY and WIF1; ZSCAN23, COL4A1 and NR5A2; ZSCAN23, COL4A1 and C9orf50; ZSCAN23, COL4A1 and WIF1; ZSCAN23, NR5A2 and C9orf50; ZSCAN23, NR5A2 and NPY; ZSCAN23, NR5A2 and WIF1; ZSCAN23, C9orf50 and WIF1; COL4A1, NR5A2 and C9orf50; COL4A1, NR5A2 and WIF1; or COL4A1, C9orf50 and WIF1.In a preferred embodiment, the method includes the step of determining the methylation levels of at least three genes, wherein the three genes are AQP5-AS1, C9orf50 and WIF1; AQP5-AS1, C9orf50 and ZSCAN23; AQP5-AS1, NPY and WIF1; AQP5-AS1, NPY and ZSCAN23; AQP5-AS1, ZSCAN23 and WIF1; C9orf50, ZSCAN23 and WIF1; COL4A1, AQP5-AS1 and NPY; COL4A1, AQP5-AS1 and WIF1; COL4A1, C9orf5 0 and WIF1; COL4A1, C9orf50 and ZSCAN23; COL4A1, NR5A2 and AQP5-AS1; COL4A1, NR5A2 and C9orf50; COL4A1, NR5A2 and WIF1; COL4A1, NR5A2 and ZSCAN23; NR5A2, AQP5-AS1 and C9orf50; NR5A2, AQP5-AS1 and NPY; NR5A2, AQP5-AS1 and WIF1; NR5A2, C9orf50 and ZSCAN23; NR5A2, NPY and ZSCAN23; or NR5A2, ZSCAN23 and WIF1.

[0199] In a particular embodiment, the method includes the step of determining the methylation level of at least four genes, the four genes being AQP5-AS1, ZSCAN23, COL4A1 and NR5A2; AQP5-AS1, ZSCAN23, COL4A1 and C9orf50; AQP5-AS1, ZSCAN23, COL4A1 and NPY; AQP5-AS1, ZSCAN23, COL4A1 and WIF1; AQP5-AS1, ZSCAN23, NR5A2 and C9orf50; AQP5-AS1, ZSCAN23, NR5A2 and NPY; AQP5- AS1, ZSCAN23, NR5A2 and WIF1; AQP5-AS1, ZSCAN23, C9orf50 and NPY; AQP5-AS1, ZSCAN23, C9orf50 and WIF1; AQP5-AS1, ZSCAN23, NPY and WIF1; AQP5-AS1, COL4A1, NR5A2 and C9orf50; AQP5-AS1, COL4A1, NR5A2 and NPY; AQP5-AS1, COL4A1, NR5A2 and WIF1; AQP5-AS1, COL4A1, C9orf50 and NPY; AQP5-AS1, COL4A1, C9o rf50 and WIF1; AQP5-AS1, COL4A1, NPY and WIF1; AQP5-AS1, NR5A2, C9 or f50 and NPY; AQP5-AS1, NR5A2, C9 or f50 and WIF1; AQP5-AS1, NR5A2, NPY and WIF1; AQP5-AS1, C9 or f50, NPY and WIF1; ZSCAN23, COL4A1, NR5A2 and C9 or f50; ZSCAN23, COL4A1, NR5A2 and NPY; ZSCAN23, COL4A1, NR5A2 and WIF1; ZSCAN23, COL4A1, C9 orf50 and NPY; ZSCAN23, COL4A1, C9orf50 and WIF1; ZSCAN23, COL4A1, NPY and WIF1; ZSCAN23, NR5A2, C9orf50 and NPY; ZSCAN23, NR5A2, C9orf50 and WIF1; ZSCAN23, NR5A2, NPY and WIF1; ZSCAN23, C9orf50, NPY and WIF1; COL4A1, NR5A2, C9orf50 and NPY; COL4A1, NR5A2, C9orf50 and WIF1; COL4A1, NR5A2, NPY and WIF1;Alternatively, these may be COL4A1, C9orf50, NPY, and WIF1.

[0200] In a preferred embodiment, the method includes the step of determining the methylation level of at least four genes, the four genes being AQP5-AS1, ZSCAN23, COL4A1 and NR5A2; AQP5-AS1, ZSCAN23, COL4A1 and WIF1; AQP5-AS1, ZSCAN23, NR5A2 and C9orf50; AQP5-AS1, ZSCAN23, NR5A2 and WIF1; AQP5-AS1, ZSCAN23, C9orf50 and WIF1; AQP5-AS1, ZSCAN23, NPY and WIF1; AQP5-AS1, COL4A1, NR5A2 and NPY; AQP5-AS1, COL4A1, NR5A2 and WIF1; AQP 5-AS1, COL4A1, NPY and WIF1; AQP5-AS1, NR5A2, C9orf50 and WIF1; AQP5-AS1, NR5A2, NPY and WIF1; AQP5-AS1, C9orf50, NPY and WIF1; ZSCAN23, COL4A1, NR5A2 and C9orf50; ZSCAN23, COL4A1, NR5A2 and WIF1; ZS CAN23, COL4A1, C9orf50 and WIF1; ZSCAN23, NR5A2, C9orf50 and NPY; ZSCAN23, NR5A2, C9orf50 and WIF1; ZSCAN23, NR5A2, NPY and WIF1; ZSCAN23, C9orf50, NPY and WIF1; or COL4A1, NR5A2, C9orf50 and WIF1.In a preferred embodiment, the method includes the step of determining the methylation level of at least four genes, the four genes being AQP5-AS1, C9orf50, NPY and WIF1; AQP5-AS1, NPY, ZSCAN23 and WIF1; COL4A1, AQP5-AS1, C9orf50 and WIF1; COL4A1, AQP5-AS1, NPY and WIF1; COL4A1, AQP5-AS1, ZSCAN23 and WIF1; COL4A1, NR5A2, AQ P5-AS1 and NPY; COL4A1, NR5A2, AQP5-AS1 and WIF1; COL4A1, NR5A2, AQP5-AS1 and ZSCAN23; COL4A1, NR5A2, C9orf50 and WIF1; COL4A1, NR5A2, C9orf50 and ZSCAN23; NR5A2, AQP5-AS1, NPY and WIF1; NR5A2, AQP5-AS1, ZSCAN23 and WIF1; or NR5A2, C9orf50, NPY and ZSCAN23.

[0201] In a particular embodiment, the method includes the step of determining the methylation level of at least five genes, wherein the five genes are AQP5-AS1, ZSCAN23, COL4A1, NR5A2 and C9orf50; AQP5-AS1, ZSCAN23, COL4A1, NR5A2 and NPY; AQP5-AS1, ZSCAN23, COL4A1, NR5A2 and WIF1; AQP5-AS1, ZSCAN23, COL4A1, C9orf5 0 and NPY; AQP5-AS1, ZSCAN23, COL4A1, C9orf50 and WIF1; AQP5-AS1, ZSCAN23, COL4A1, NPY and WIF1; AQP5-AS1, ZSCAN23, NR5A2, C9orf50 and NPY; AQP5-AS1, ZSCAN23, NR5A2, C9orf50 and WIF1; AQP5-AS1, ZSCAN23, NR5A2, NPY and WIF1; AQP5-AS1, ZSC AN23, C9orf50, NPY and WIF1; AQP5-AS1, COL4A1, NR5A2, C9orf50 and NPY; AQP5-AS1, COL4A1, NR5A2, C9orf50 and WIF1; AQP5-AS1, COL4A1, NR5A2, NPY and WIF1; AQP5-AS1, COL4A1, C9orf50, NPY and WIF1; AQP5-AS1, NR5A2, C9orf50, NPY and WIF1; ZSCA N23, COL4A1, NR5A2, C9orf50 and NPY; ZSCAN23, COL4A1, NR5A2, C9orf50 and WIF1; ZSCAN23, COL4A1, NR5A2, NPY and WIF1; ZSCAN23, COL4A1, C9orf50, NPY and WIF1; ZSCAN23, NR5A2, C9orf50, NPY and WIF1; or COL4A1, NR5A2, C9orf50, NPY and WIF1.

[0202] In a preferred embodiment, the method includes the step of determining the methylation level of at least five genes, the five genes being AQP5-AS1, ZSCAN23, COL4A1, NR5A2 and WIF1; AQP5-AS1, ZSCAN23, COL4A1, C9orf50 and WIF1; AQP5-AS1, ZSCAN23, COL4A1, NPY and WIF1; AQP5-AS1, ZSCAN23, NR5A2, C9orf50 and WIF1; AQP5-AS1, ZSCAN23, NR5A2, NPY and WIF1; AQP 5-AS1, ZSCAN23, C9orf50, NPY and WIF1; AQP5-AS1, COL4A1, NR5A2, C9orf50 and WIF1; AQP5-AS1, COL4A1, NR5A2, NPY and WIF1; AQP5-AS1, COL4A1, C9orf50, NPY and WIF1; AQP5-AS1, NR5A2, C9orf50, NPY and WIF1; ZSCAN23, COL4A1, NR5A2, C9orf50 and WIF1; or ZSCAN23, NR5A2, C9orf50, NPY and WIF1. In a preferred embodiment, the method includes the step of determining the methylation level of at least five genes, the five genes being COL4A1, AQP5-AS1, C9orf50, NPY and WIF1; COL4A1, AQP5-AS1, C9orf50, ZSCAN23 and WIF1; COL4A1, AQP5-AS1, NPY, ZSCAN23 and WIF1; COL4A1, NR5A2, AQP5-AS1, C9orf50 and WIF1; COL4A1, NR5A2, AQP5-AS1, NPY and WIF1; or COL4A1, NR5A2, AQP5-AS1, ZSCAN23 and WIF1.

[0203] In a particular embodiment, the method includes the step of determining the methylation level of at least six genes, wherein the six genes are AQP5-AS1, ZSCAN23, COL4A1, NR5A2, C9orf50 and NPY; AQP5-AS1, ZSCAN23, COL4A1, NR5A2, C9orf50 and WIF1; AQP5-AS1, ZSCAN23, COL4A1, NR5A2, N PY and WIF1; AQP5-AS1, ZSCAN23, COL4A1, C9orf50, NPY and WIF1; AQP5-AS1, ZSCAN23, NR5A2, C9orf50, NPY and WIF1; AQP5-AS1, COL4A1, NR5A2, C9orf50, NPY and WIF1; or ZSCAN23, COL4A1, NR5A2, C9orf50, NPY, WIF1.

[0204] In a preferred embodiment, the method includes the step of determining the methylation level of at least six genes, wherein the six genes are AQP5-AS1, ZSCAN23, COL4A1, NR5A2, C9orf50 and WIF1; AQP5-AS1, ZSCAN23, COL4A1, NR5A2, NPY and WIF1; AQP5-AS1, ZSCAN23, COL4A1, C9orf50, NPY and WIF1; or AQP5-AS1, COL4A1, NR5A2, C9orf50, NPY and WIF1. In a preferred embodiment, the method includes the step of determining the methylation levels of at least six genes, wherein the six genes are COL4A1, AQP5-AS1, C9orf50, NPY, ZSCAN23, and WIF1; or COL4A1, NR5A2, AQP5-AS1, C9orf50, NPY, and WIF1.

[0205] In one particular embodiment, the method includes the step of determining the methylation level of at least seven genes, the seven genes being AQP5-AS1, ZSCAN23, COL4A1, NR5A2, C9orf50, NPY, and WIF1.

[0206] In a particular embodiment, the method includes a) determining the level or amount of methylation of at least one biomarker or combination of biomarkers in the biological sample of the subject; and b) comparing the level or amount of methylation determined in step a) with a reference value, wherein if the biomarker is hypermethylated compared to the reference value, the subject is diagnosed or identified as having colorectal cancer.

[0207] In relation to the present invention, reference values ​​are obtained from "reference samples" from healthy individuals, or from "reference samples" from subjects with colorectal cancer, either prior to or earlier than the treatment.

[0208] "Hypermethylation" is determined, for example, when the methylation value (amount) or state (level) of one of the biomarkers of the present invention is significantly higher in the biological sample under test compared to the methylation value (amount) or state (level) of the corresponding biomarker measured in the reference sample. A significantly high amount or level of methylation of at least one of the biomarkers of the present invention in the biological sample under test, compared to the normal amount or level of methylation in the reference sample, is an indication that the subject under test has colorectal cancer, is at high risk of developing colorectal cancer, or is unresponsive to treatment.

[0209] "A significantly high amount or level of methylation" refers to an amount or level of methylation that is higher than the sum of the mean and standard errors of the assay used to evaluate the amount or level.

[0210] The present invention further relates to a method for monitoring the progression of colorectal cancer in a subject diagnosed with colorectal cancer, comprising: a) determining the level or amount of methylation of at least one biomarker or combination of biomarkers in a biological sample of the subject at a first time point; b) determining the level or amount of methylation of the biomarker previously selected in step a) in the biological sample of the subject at a second time point; and c) comparing the level or amount of methylation determined in step a) with the level or amount determined in step b). If the level or amount of methylation determined in step b) is significantly higher than the level or amount determined in step a), it can be concluded that the malignancy of the colorectal cancer is progressing. In other words, the subject has a disease that worsens, even if it has already been treated.

[0211] In other embodiments, the biomarkers of the present invention may be used to predict outcomes in patients with colorectal cancer. The method further relates to a method for predicting the clinical outcome of a subject with colorectal cancer, comprising the steps of: a) determining the level or amount of methylation of at least one gene selected from the group of biomarkers listed above in a biological sample of the subject and comparing it to a reference value; and b) predicting the clinical outcome based on the comparison in step a).

[0212] If at least one gene or combination of genes is significantly hypermethylated in the biological sample under test compared to the same biomarker in the reference sample, the subject is likely to have a poor clinical outcome (short survival time, metastasis, etc.).

[0213] The present invention further relates to a method for adapting a treatment plan for a patient suffering from colorectal cancer, a) A step of determining the level or amount of methylation of at least one biomarker, or a combination of biomarkers, in the target biological sample. b) A step of comparing it with the level or amount of methylation of the same biomarker selected in step a) in the biological sample of the subject after the surgical procedure or after the subject has been administered the treatment. c) A step of adapting / modifying the treatment plan for the subject based on step a) alone, or based on a comparison of steps a) and b). Regarding methods including

[0214] In particular, the surgical procedure or treatment plan is considered efficient if the level or amount of the biomarker is significantly lower than or equal to the level or amount of the biomarker determined before the procedure. Conversely, if the level or amount of the biomarker increases during the procedure compared to the level or amount of the biomarker determined before the procedure (or determined earlier in the procedure), the treatment plan should be modified. This aspect of the treatment strategy is a crucial goal in the context of personalized medicine in order to improve survival while maintaining quality of life and avoiding the unnecessary toxic effects of ineffective procedures.

[0215] A method for identifying or screening therapeutic agents or candidate therapeutic agents for use in the treatment of colorectal cancer in a subject is further disclosed, the method comprising the steps of administering the therapeutic agent to a subject, and detecting the methylation of the biomarker of the present invention in a biological sample containing genomic DNA from the subject, or determining the level of methylation, wherein a decrease in methylation after administration of the therapeutic agent indicates a drug that has a benefit against colorectal cancer in the subject.

[0216] A method for treating a subject diagnosed with colorectal cancer using the diagnostic method of the present invention is further disclosed, wherein the subject is subsequently administered an effective dose of a therapeutic agent, including chemotherapy and / or immunotherapy, or undergoes surgical procedures and / or radiation.

[0217] Chemotherapy agents are typically selected from, for example, alkylating agents, antimetabolites, topoisomerase inhibitors, platin-based components, specific kinase inhibitors, hormones, cytokines, anti-angiogenic agents, antibodies, immunotherapeutic agents, or vasolytic agents.

[0218] Further methods for early diagnosis in individuals at risk of developing colorectal cancer will be disclosed.

[0219] Further disclosures are made regarding a method for the early diagnosis of cancerous lesions in the colon in subjects with metastases from a primary tumor of unknown origin.

[0220] Further methods for assessing the risk of recurrence will be disclosed.

[0221] kit Another aspect of the present invention relates to a diagnostic kit and tool for determining hypermethylation of the biomarker of the present invention for the purpose of diagnosing colorectal cancer.

[0222] A kit is also provided that includes probes and / or primers, and optionally additional reagents, useful for determining the methylation status at the aforementioned genomic sites.

[0223] As used herein, the term "primer" refers to an isolated nucleic acid molecule that can specifically hybridize or anneal to the 5' or 3' region of a target genomic region (the positive and negative strands, respectively, and vice versa). Generally, primers are about 10–30 nucleotides long and anneal to both ends of a region containing about 50–200 nucleotides. Under appropriate conditions and with appropriate reagents, such primers enable amplification of nucleic acid molecules containing the nucleotide sequences adjacent to the primer. Since primers must be used in pairs, they are often referred to as "primer pairs" or "primer sets" (see SEQ ID NOs: 1–2, 3–4, 5–6, 7–8, 9–10, 11–12, 13–14, 15–16, 17–18, 19–20, 21–22, 23–24, 25–26 of the pairs).

[0224] As used herein, the term "probe" refers to a molecule that can specifically hybridize to a target genomic region (e.g., SEQ ID NOs. 40 to 52). Probes are useful for highlighting the presence of the genomic region in a biological sample. These probes may include at least one non-natural nucleotide, e.g., peptide nucleic acid (PNA), phosphate-containing peptide nucleic acid (PHONA), cross-linked nucleic acid or locked nucleic acid (BNA or LNA), and morpholino nucleic acid. Non-natural nucleotides may further include chemically modified nucleic acids or nucleic acid analogs, e.g., methylphosphonate-type DNA or RNA, phosphorothioate-type DNA or RNA, phosphoramidate-type DNA or RNA, and 2'-O-methyl-type DNA or RNA.

[0225] In one preferred embodiment, the probe of the present invention comprises at least 15 consecutive nucleotides complementary to bisulfite-treated SEQ ID NOs. 27 to 39 or fragments thereof. In a more preferred embodiment, the molecule that can be used as a probe according to the present invention has a total minimum number of 15 nucleotides. In an even more preferred embodiment, the molecule comprises between 15 and 30 nucleotides (total).

[0226] For specific uses, the probes and primers of the present invention may be directly or indirectly labeled with a detectable label. The label may be of any type depending on the experiment to be performed. The label may be a radioactive isotope (e.g., 32P, 33P, 35S, 3H, or 125L), or a non-radioactive entity selected from ligands (e.g., biotin, avidin, or streptavidin), dioxygenin, haptens, colorants, and luminescent agents (e.g., radioluminescent agents, chemiluminescent agents, bioluminescent agents, fluorescent agents, or phosphorescent agents). Preferably, 6-carboxyfluorescein (FAM), VIC, HEX, and tetramethylrhodamine (TAM RA) are used. Unlabeled polynucleotide sequences may also be used directly, for example, as probes or primers in PCR-based processes (e.g., quantitative PCR).

[0227] "Specific hybridization" is observed when a defined molecule does not hybridize to any other genomic region other than its target genomic region. Preferably, the defined molecule hybridizes to its target region under high stringency conditions, i.e., when temperature and ionic intensity conditions are selected to allow hybridization between two complementary DNA fragments. For example, high stringency conditions may be as follows. DNA-DNA or DNA-RNA hybridization may be carried out in two steps. (1) Pre-hybridization for 3 hours at 42°C in phosphate buffer (20mM, pH 7.5) containing 5x SSC (1x SSC is equivalent to 0.15M NaCl + 0.015M sodium citrate solution), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10x Denhardt, 5% dextran sulfate and 1% salmon sperm DNA; (2) Actual hybridization for 20 hours at a temperature appropriate to the probe size (i.e., 42°C for probes with a size of >100 nucleotides), followed by two washes of 2x SSC + 2% SDS at 20°C for 20 minutes each, and one wash of 0.1x SSC + 0.1% SDS at 20°C for 20 minutes each. The final wash is performed at 60°C for 30 minutes in 0.1x SSC + 0.1% SDS for probes with a size of >100 nucleotides. The aforementioned high-stringency hybridization conditions for polynucleotides of a specified size can be adjusted by those skilled in the art for larger or smaller oligonucleotides.

[0228] As used herein, the term “kit” refers to any system for delivering materials. In connection with the present invention, a kit includes a system that enables the storage, transport, and delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or support materials (e.g., buffers, written instructions for performing the assay, etc.) from one location to another. For example, a kit includes one or more housings (e.g., boxes) containing the relevant reaction reagents and / or support materials. The kit may also include one or more reagents, buffers, hybridization media, nucleic acids, primers, nucleotides, probes, molecular weight markers, enzymes, solid supports, a database, a computer program for calculating the distribution order, and / or disposable laboratory equipment, such as multiwell plates, to facilitate the implementation of the method. Enzymes that may be included in the kit include nucleotide polymerases, etc. Solid supports may include beads, etc., while stool sample markers may include conjugatable markers, such as biotin and streptavidin, etc.

[0229] The kit of the present invention provides comfort, - Sequence ID 40 in the ZSCAN23 gene, - Sequence ID 41 in the C9orf50 gene, - Sequence ID 42 in the AQP5-AS1 gene, - Sequence ID 43 in the NR5A2 gene, - Sequence ID 44 in the ADARB2 gene, - Sequence ID No. 45 in the COL4A1 gene, - Sequence ID 46 in the NPY gene, - Sequence ID 47 in the WIF1 gene, - Sequence ID 48 in the CPNE8 gene, - Sequence ID 49 in the LINC00693(RBMS3) gene, - Sequence ID 50 in the LINC00900 gene, - Sequence ID 51 in the DYDC2 gene, and / or - Primer and / or probe that specifically targets the nucleotide region of SEQ ID NO: 52 in the LRRC4 (SND1) gene is contained.

[0230] In a preferred embodiment, the kit of the present invention comprises at least - SEQ ID NO: 40 in the ZSCAN23 gene, - SEQ ID NO: 45 in the COL4A1 gene, or - SEQ ID NO: 42 in the AQP5-AS1 gene is contained.

[0231] In a preferred embodiment, the kit of the present invention - SEQ ID NO: 40 in the ZSCAN23 gene, and - SEQ ID NO: 42 in the AQP5-AS1 gene is contained.

[0232] In a preferred embodiment, the kit of the present invention - SEQ ID NO: 45 in the COL4A1 gene, and - SEQ ID NO: 42 in the AQP5-AS1 gene is contained.

[0233] In a preferred embodiment, the kit of the present invention - SEQ ID NO: 40 in the ZSCAN23 gene, and - SEQ ID NO: 45 in the COL4A1 gene is contained.

[0234] In a preferred embodiment, the kit of the present invention - SEQ ID NO: 40 in the ZSCAN23 gene, - SEQ ID NO: 45 in the COL4A1 gene, and - SEQ ID NO: 42 in the AQP5-AS1 gene It contains primers and probes that specifically target the nucleotide region.

[0235] In one preferred embodiment, the kit of the present invention is - Primers of SEQ ID NO: 1 and SEQ ID NO: 2 for determining the level or amount of methylation in the ZSCAN23 gene. - Primers of SEQ ID NO: 3 and SEQ ID NO: 4 for determining the level or amount of methylation in the C9orf50 gene. - Primers of SEQ ID NOs. 5 and 6 for determining the level or amount of methylation in the AQP5-AS1 gene. - Primers of SEQ ID NO: 7 and SEQ ID NO: 8 for determining the level or amount of methylation in the NR5A2 gene. - Primers of SEQ ID NO: 9 and SEQ ID NO: 10 for determining the level or amount of methylation in the ADARB2 gene. - Primers of SEQ ID NO: 11 and SEQ ID NO: 12 for determining the level or amount of methylation in the COL4A1 gene. - Primers of SEQ ID NO: 13 and SEQ ID NO: 14 for determining the level or amount of methylation in the NPY gene. - Primers of SEQ ID NO: 15 and SEQ ID NO: 16 for determining the level or amount of methylation in the WIF1 gene. - Primers of SEQ ID NO: 17 and SEQ ID NO: 18 for determining the level or amount of methylation in the CPNE8 gene. - Primers of SEQ ID NO: 19 and SEQ ID NO: 20 for determining the level or amount of methylation in the LINC00693 (RBMS3) gene. - Primers of SEQ ID NO: 21 and SEQ ID NO: 22 for determining the level or amount of methylation in the LINC00900 gene. - Primers of SEQ ID NOs. 23 and 24 for determining the level or amount of methylation in the DYDC2 gene, and / or - Primers for Sequence ID No. 25 and Sequence ID No. 26 to determine the level or amount of methylation in the LRRC4(SND1) gene. It contains.

[0236] In one preferred embodiment, the kit of the present invention is - Probe of Sequence ID No. 27 for determining the level or amount of methylation in the ZSCAN23 gene. - A probe of SEQ ID NO: 28 for determining the level or amount of methylation in the C9orf50 gene. - A probe of Sequence ID No. 29 for determining the level or amount of methylation in the AQP5-AS1 gene. - A probe of SEQ ID NO: 30 for determining the level or amount of methylation in the NR5A2 gene. - A probe of SEQ ID NO: 31 for determining the level or amount of methylation in the ADARB2 gene. - A probe of SEQ ID NO: 32 for determining the level or amount of methylation in the COL4A1 gene. - A probe of Sequence ID No. 33 for determining the level or amount of methylation in the NPY gene. - A probe of SEQ ID NO: 34 for determining the level or amount of methylation in the WIF1 gene. - A probe of SEQ ID NO: 35 for determining the level or amount of methylation in the CPNE8 gene. - A probe of Sequence ID No. 36 for determining the level or amount of methylation in the LINC00693 (RBMS3) gene. - A probe of SEQ ID NO: 37 for determining the level or amount of methylation in the LINC00900 gene. - A probe of SEQ ID NO: 38 for determining the level or amount of methylation in the DYDC2 gene, and / or - A probe for sequence number 39 to determine the level or amount of methylation in the LRRC4(SND1) gene. It contains.

[0237] The present invention further, - Sequence ID 40 in the ZSCAN23 gene, - Sequence ID 41 in the C9orf50 gene, - Sequence ID 42 in the AQP5-AS1 gene, - Sequence ID 43 in the NR5A2 gene, - Sequence ID 44 in the ADARB2 gene, - Sequence ID No. 45 in the COL4A1 gene, - Sequence ID 46 in the NPY gene, - Sequence ID 47 in the WIF1 gene, - Sequence ID No. 48 in the CPNE8 gene - Sequence ID 49 in the LINC00693(RBMS3) gene, - Sequence ID 50 in the LINC00900 gene, - Sequence ID 51 in the DYDC2 gene, and / or - Sequence ID No. 52 in the LRRC4(SND1) gene This invention relates to a microarray carrying nucleotides that specifically target the nucleotide region of a given object.

[0238] In one preferred embodiment, the microarray contains a primer of any of SEQ ID NOs: 1 to 26 and / or a probe of SEQ ID NOs: 27 to 39.

[0239] According to the present invention, a "nucleic acid microarray" consists of different nucleic acid probes attached to a substrate which may be a microchip, a glass slide, or microsphere-sized beads. The microchip may consist of a polymer, plastic, resin, polysaccharide, silica material or silica-based material, carbon, metal, inorganic glass, or nitrocellulose. The probes may be nucleic acids, for example, cDNA ("cDNA microarray") or oligonucleotides ("oligonucleotide microarray"), the oligonucleotides may be about 10 to about 40 base pairs in length or less. Typically, the exemplary primers and probes described above may be attached to the substrate. In a preferred embodiment, the nucleic acid microarray of the present invention is an oligonucleotide microarray supporting oligonucleotides that can specifically hybridize to one, two, or three methylation regions of SEQ ID NOs. 40 to 52.

[0240] To determine whether the biomarker of the present invention is methylated, a test sample is labeled and contacted with the nucleic acid microarray of the present invention under hybridization conditions to effect formation of a complex between the target nucleic acid that is complementary to the probe sequence attached to the microarray surface. Next, the presence of the labeled hybridized complex on the nucleic acid microarray is determined. Many variants of microarray hybridization techniques are available to those skilled in the art.

[0241] In particular, any available software developed for the design of microarray oligonucleotides can be used, for example, OligoArray software (available from http: / / berry.engin.umich.edu / oligoarray / ), GoArrays software (available from http: / / www.isima.fr / bioinfo / goarrays / ), Array Designer software (available from http: / / www.premierbiosoft.com / dnamicroarray / index.html), Primer3 software (available from http: / / frodo.wi.mit.edu / primer3 / primer3_code.html), or Promide software (available from http: / / oligos.molgen.mpg.de / ), software such as MethPrimer (http: / / www.urogene.org / cgi-bin / methprimer / methprimer.cgi).

[0242] The present invention further provides, as proposed in the method of the present invention, · diagnosing or identifying colorectal cancer in a subject, · predicting the clinical outcome in a subject suffering from colorectal cancer, · determining a treatment plan for a subject having colorectal cancer, and / or · monitoring the progression of colorectal cancer in a subject diagnosed with colorectal cancer in a biological sample of the subject This relates to the use of the aforementioned kit, or the use of the aforementioned microarray, or the use of the aforementioned primer, or the use of the aforementioned probe.

[0243] As used herein, the terms “in vitro” and “ex vivo” are equivalent and refer to studies or experiments conducted using biological components (e.g., cells or populations of cells) isolated from their usual host organism (e.g., animals or humans).

[0244] The embodiments and drawings illustrate the present invention without limiting its scope. [Examples]

[0245] (Example 1) Materials and methods Patients and healthy control subjects This study included paired biopsies of tumor and adjacent non-tumor tissue from 26 CRC patients (stages I-IV). Plasma samples from healthy individuals (n=10-20 per assay, depending on sample availability) and from unresectable advanced CRC patients (stage IV, n=17) were collected in EDTA or STRECK tubes. This study was approved by the local ethics committee, and written informed consent was obtained from all patients.

[0246] Tumor sample preparation, storage, DNA extraction, and quantification. Tumor and adjacent non-tumor tissue biopsies were rapidly frozen in liquid nitrogen immediately after resection until further analysis. Each tumor was re-examined by a pathologist, and tumor cell content was evaluated by hematoxylin, eosin, and saffron staining. DNA was extracted using the QIAampDNAMini kit (Qiagen) according to the manufacturer's instructions. DNA concentration was measured using a Qubit 2.0 fluorometer (Invitrogen, Life Technologies) with the dsDNA BR assay (Invitrogen). Extracted DNA samples were stored at -20°C until testing.

[0247] Plasma sample preparation, storage, DNA extraction, and quantification. Plasma samples from healthy individuals were received in dry ice, divided into fixed volumes, and immediately frozen at -80°C. Before extraction, the plasma samples were centrifuged at 3000g for 10 minutes, and then extracted according to the manufacturer's instructions using either the QIAmp circulating nucleic acid kit (Qiagen) or the ccfDNA plasma kit (Promega) with an RSC Maxwell instrument. Plasma from CRC patients was extracted using either the ccfDNA plasma kit (Promega) or the QIAmp circulating nucleic acid kit (Qiagen) with an RSC Maxwell instrument. The amount of DNA was measured using a Qubit 2.0 fluorometer (Invitrogen, Life Technologies) with a dsDNA HS assay (Invitrogen). The extracted DNA samples were stored at -20°C until testing.

[0248] Buffy coat preparation, DNA extraction, and quantification. Buffy coat was extracted from whole blood of patients without CRC or from healthy individuals, and then extracted using the QIAmp Circulating Nucleic Acid Kit (Qiagen) according to the manufacturer's instructions. The amount of DNA was measured using a Qubit 2.0 fluorometer (Invitrogen, Life Technologies) with a dsDNA BR assay (Invitrogen). The extracted DNA samples were stored at -20°C until testing.

[0249] Analysis and identification of DNA methylation biomarkers Following methyl-seq and TCGA analysis, R scripting was performed, and ultimately 13 candidate genes were selected: ZSCAN23, C9orf50 (NTMT1), AQP5-AS1, NR5A2, ADARB2, COL4A1, NPY, WIF1, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and LRRC4 (SND1).

[0250] Bisulfite conversion of tissue DNA, buffy coat DNA, and plasma ccfDNA Tissue DNA, buffy coat DNA, and plasma ccfDNA were modified with bisulfite using the EZ DNA Methylation-Gold kit (Zymo Research). In short, the bisulfite reaction was performed at 98°C for 12 minutes and at 64°C for 2 hours and 35 minutes. The bisulfite-converted DNA was purified according to the manufacturer's recommendations, and the converted DNA was eluted in M ​​elution buffer and stored at -20°C.

[0251] Detection of methylation changes of selected biomarkers by droplet-based digital PCR. Hypermethylation of selected biomarkers in tumor DNA was evaluated by ddPCR. Albumin was used to normalize DNA volume, and a two-part format was employed to analyze hypermethylation. Primers and probes are listed in Table 1 and Table 2.

[0252] DNA methylation of the target sequence was analyzed by ddPCR using the QX-200 platform (BIO-RAD Technologies). In short, a mixture of PCR reagents (BIO-RAD Technologies) was prepared according to the manufacturer's recommendations.

[0253] A triad panel was developed to detect ZSCAN23 and C9orf50 methylation target sequences, as well as unmethylated albumin sequences as reference genes. A triad panel was also developed to detect AQP5-AS1 and NR5A2 methylation target sequences, as well as unmethylated albumin sequences as reference genes. A triad panel was performed to detect WIF1 and NPY methylation target sequences, as well as unmethylated albumin sequences as reference genes, as described in Taieb J, 2021.

[0254] We also developed a pair assay to detect ADARB2, COL4A1, WIF1, NPY, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, LRRC4 (SND1) methylation target sequences, as well as unmethylated albumin sequences as reference genes.

[0255] The PCR steps for ddPCR were performed on a BIO-RAD C1000 or S1000 using the following program: 10 minutes at 95°C (using a ramp rate of 2.5°C / sec), followed by 45 cycles of 94°C, 30 seconds, and 58.4°C, 1 minute (using a ramp rate of 2.5°C / sec), with a final step of 10 minutes at 98°C. After completion, the emulsions were stored at 4°C or processed immediately, and the endpoint fluorescence signal from each droplet was measured. The data were analyzed using Quantasoft BIO-RAD software as described by the manufacturer. Populations were clustered by fluorescence level to allow for accurate counting of both tumor-aggregatable DNA molecules and normal-aggregatable DNA molecules.

[0256] For different assays, using commercially available DNA (Promega) extracted from whole blood, the upper limit of blanks (LOBs) were calculated for both the developed diploid and triploid assays, as previously described [Taly, V. et al., Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. Clin Chem, 2013. 59(12):1722-31 (Non-Patent Literature 5)]. The LOB was defined by the frequency of positive droplets measured in normal control DNA samples (n=13-22 per assay) that did not contain hypermethylated DNA. The calculated LOB was subtracted from each sample to determine its methylation level.

[0257] Sample analysis was performed according to previously described procedures [Taly, V. et al., 2013 (Non-Patent Literature 5)]. A sample was considered positive if the number of observed droplets was higher than the LOB value. The methylation level of each sample was calculated as the ratio of the number of droplets containing methylated sequences to the number of droplets containing albumin sequences.

[0258] To ensure proper modification, two DNA controls were used (positive control: universally hypermethylated DNA and negative control: normal human genomic DNA).

[0259] Measurement of methylation levels of selected biomarkers in circulating cell-free DNA (ccfDNA) from healthy individuals and CRCs. DNA methylation of selected biomarkers in plasma from healthy individuals or CRC patients was measured by ddPCR using the same reaction conditions as described above. Albumin was used to normalize DNA levels, and a two- or three-component format was used to analyze hypermethylation. The same primers and probes listed in Table 1 and Table 2 were used.

[0260] Calculation of detection sensitivity and specificity The sum of the mean and standard deviation of methylation levels in non-tumor tissue DNA was used as the threshold for calculating the sensitivity and specificity of each selected biomarker. Sensitivity is the percentage of patients who showed methylation levels above the threshold in tumor tissue. Specificity is the percentage of patients who showed methylation levels below the threshold in non-tumor tissue.

[0261] statistical analysis Statistical analysis was performed using Prism software (GraphPad Software Inc.) and R software 3.6.3 Studio. A p-value ≤ 0.05 was considered statistically significant. The paired nonparametric Wilcoxon test was used to analyze differences in hypermethylation between normal tissue and adjacent tissue. The Mann-Whitney test was used to analyze differences in hypermethylation between healthy plasma ccfDNA and CRC plasma ccfDNA.

[0262] result Hypermethylation of selected biomarkers in DNA from CRC patients by ddPCR. As shown in Figure 1, the methylation levels of selected biomarkers (ZSCAN23, C9orf50, AQP5-AS1, NR5A2, ADARB2, COL4A1, WIF1, NPY, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and LRRC4 (SND1)) were significantly elevated in CRC tumor tissue DNA compared to non-tumor tissue DNA.

[0263] As shown in Table 3 below, these biomarkers, when evaluated individually by ddPCR, exhibit higher specificity and sensitivity in tumor tissue compared to healthy tissue from CRC patients.

[0264] [Table 3]

[0265] Figure 2 shows that the methylation levels of the biomarkers ZSCAN23, C9orf50, AQP5-AS1, NR5A2, ADARB2, COL4A1, WIF1, and NPY were significantly elevated in the plasma of CRC patients (ccfDNA) compared to the methylation levels of the same biomarkers in the plasma of healthy individuals.

[0266] Combinations of different biomarkers to achieve higher sensitivity and specificity. To achieve higher sensitivity and specificity, different biomarkers can be combined. As shown in Table 4 below, the detection sensitivity and specificity by ddPCR can be improved, for example, by combining ZSCAN23 with C9orf50, AQP5-AS1 and / or NR5A2.

[0267] [Table 4]

[0268] Methylation profiles of selected biomarkers in plasma ccfDNA and buffy coat fractions from healthy individuals and CRC patients, as determined by ddPCR. The purpose of these potential biomarkers is to detect DNA hypermethylation in CRC patients but not in healthy individuals. To verify this, methylation levels in plasma ccfDNA from healthy individuals were investigated by ddPCR. For these potential biomarkers, no significant levels of methylation were observed in healthy plasma ccfDNA (see Table 5 below). Furthermore, DNA extracted from buffy coat (from healthy individuals) was also tested for the markers, and little methylation of the test markers was observed in these samples (Table 6). The number of samples performed for each biomarker depended on the availability of DNA. As mentioned above regarding LOB calculations, commercially available DNA (Promega) extracted from whole blood was also used to validate this assay. These controls were performed to ensure that the markers were not positive in cells contained in blood, in addition to buffy coat DNA, and to confirm that there were no false positives due to hemolysis of blood cells (e.g., due to sample handling before analysis).

[0269] However, when tested using DNA extracted from the plasma of healthy individuals and CRC patients, methylation was observed only in colon patients, regardless of whether the developed duplex assay or triplex assay was used, resulting in 100% potential specificity for detecting CRC in blood samples.

[0270] [Table 5]

[0271] [Table 6]

[0272] (Example 2) Materials and methods Healthy control subjects and patients Plasma samples from 25 healthy individuals were obtained from Society Bioredic International. Blood was collected in EDTA tubes and centrifuged within 4 hours of collection according to the supplier's procedure. Plasma samples were received in dry ice, portioned out, and immediately frozen at -80°C. Blood samples from 54 healthy individuals were obtained in STRECK tubes from EFS (Etablissement Francais du Sang). Within 10 days of collection, the plasma samples were separated from the cell fraction by centrifugation at 1900g for 15 minutes, followed by a second centrifugation at 6000g for 10 minutes, and then frozen at -80°C. Unresectable advanced CRC patients (stage IV, n=73) were collected in EDTA tubes, centrifuged at 3000g, and frozen at -80°C. These samples were retrospectively analyzed from prospective studies approved by the local ethics committee, and written informed consent was obtained from all patients.

[0273] DNA extraction and quantification Prior to extraction, plasma samples were centrifuged at 3000g for 15 minutes, and then extracted using the QIAmp Circulating Nucleic Acid Kit (Qiagen). The amount of DNA was measured using a Qubit 2.0 fluorometer (Invitrogen, Life Technologies) with a dsDNA HS assay (Invitrogen). The extracted DNA samples were stored at -20°C until testing.

[0274] Plasma ccfDNA bisulfite conversion Plasma ccfDNA was modified by bisulfite treatment using the EZ DNA Methylation-Lightning Kit (Zymo Research). The bisulfite reaction was carried out at 98°C for 8 minutes and at 54°C for 1 hour. The bisulfite-converted DNA was purified according to the manufacturer's recommendations, and the converted DNA was eluted in M ​​elution buffer and stored at -20°C.

[0275] To verify the proper implementation of the modification process, two DNA controls were used (positive control: universally hypermethylated DNA and negative control: normal human genomic DNA).

[0276] Detection of methylation changes of selected biomarkers by droplet-based digital PCR Hypermethylation of seven biomarkers (ZSCAN23, C9orf50(NTMT1), AQP5-AS1, NR5A2, COL4A1, NPY, and WIF1) in circulating tumor DNA was evaluated by ddPCR. A multiplex panel was used to analyze hypermethylation, using unmethylated sequences within the albumin gene as a reference and for DNA quantity normalization.

[0277] DNA methylation of the target sequence was analyzed by ddPCR using the 6-color naica® platform (Stilla Technologies). The PCR reagent mixture (Stilla Technologies) was prepared according to the manufacturer's recommendations.

[0278] A multiplex panel was developed to detect AQP5-AS1, ZSCAN23, COL4A1, WIF1, NPY, NR5A2, and C9orf50 methylated target sequences, as well as unmethylated albumin sequences as reference genes. PCR steps for ddPCR were performed according to the manufacturer's recommendations. Data were analyzed using Crystal Miner software as described by the manufacturer. Populations were clustered by fluorescence level to enable accurate counting of amplified DNA molecules corresponding to both methylated target sequences and unmethylated control sequences (ALBs).

[0279] For each methylation marker, the upper limit of blank (LOB) was calculated using commercially available DNA (Promega) extracted from whole blood, as previously described [Taly, V. et al., Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. Clin Chem, 2013. 59(12):1722-31 (Non-Patent Literature 5)]. The LOB was defined by the frequency of positive droplets measured in a normal control DNA sample (n=29) that did not contain hypermethylated DNA. The calculated LOB was subtracted from each sample to determine its methylation level.

[0280] Sample analysis was performed according to previously described procedures [Taly, V. et al., 2013 (Non-Patent Literature 5)]. A sample was considered positive if the number of observed droplets was higher than the LOB value. The methylation level of each sample was calculated as the ratio of the number of droplets containing methylated sequences to the number of droplets containing albumin sequences.

[0281] Calculation of detection sensitivity and specificity Sensitivity and specificity were calculated using the concentrations (ng / mL plasma) and / or percentages of methylation markers detected in healthy individuals, and compared with values ​​obtained for colorectal cancer patients. The analysis was performed using R Studio by constructing ROC curves (R version 4.3.3, package "proc"). The AUC (Area Under the Curve) was calculated in the same way as the specificity and sensitivity of each marker and marker combination.

[0282] statistical analysis Statistical analysis was performed using Prism software (GraphPad Software Inc.) and R software version 3.6.3 Studio. A p-value ≤ 0.05 was considered statistically significant. In this example, performance was evaluated based on the highest concentration observed for each biomarker combination (i.e., performance was based on the concentration corresponding to the positive marker with the highest concentration).

[0283] result The results, as shown in Table 7 below, confirm that the AQP5-AS1, COL4A1, and ZSCAN23 biomarkers have high specificity and sensitivity when evaluated individually in the CRC patient cohort.

[0284] [Table 7]

[0285] combination of biomarkers Selective biomarkers can be used in combination to achieve higher detection sensitivity. As shown in Table 8 below, combining ZSCAN23, COL4A1, and AQP5-AS1 improves sensitivity.

[0286] [Table 8]

[0287] The results in Table 9 below demonstrate that the ZSCAN23, COL4A1, and / or AQP5-AS1 biomarkers exhibit excellent sensitivity and / or specificity when combined with other biomarkers (i.e., NR5A2, C9orf50, NPY, and WIF1).

[0288] [Table 9A]

[0289] [Table 9B]

Claims

1. An in vitro method for detecting or monitoring colorectal cancer (CRC) in a subject, preferably a human subject, comprising the steps of detecting the methylation of at least one AQP5-AS1, ZSCAN23, or COL4A1 gene in a biological sample containing genomic DNA from the subject, or determining the level of methylation.

2. The method according to claim 1, comprising the steps of detecting methylation of the AQP5-AS1 and ZSCAN23 genes, or determining the level of methylation.

3. The method according to claim 1, comprising the steps of detecting methylation of the AQP5-AS1 and COL4A1 genes, or determining the level of methylation.

4. The method according to claim 1, comprising the steps of detecting methylation of the ZSCAN23 and COL4A1 genes, or determining the level of methylation.

5. The method according to any one of claims 1 to 4, comprising the steps of detecting methylation of a combination of AQP5-AS1, ZSCAN23, and COL4A1 genes, or determining the level of methylation.

6. The method according to any one of claims 1 to 5, further comprising the steps of detecting methylation of at least one of the NR5A2, C9orf50, WIF1, and NPY genes, or determining the level of methylation.

7. The method according to any one of claims 1 to 6, further comprising the steps of detecting methylation of at least one gene of ADARB2, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, or LRRC4 (SND1), or determining the level of methylation.

8. The method according to any one of claims 1 to 7, comprising the steps of detecting methylation of at least two, preferably three, preferably four genes selected from the group consisting of a combination of the AQP5-AS1, ZSCAN23, and COL4A1 genes, and the NR5A2, C9orf50, WIF1, NPY, ADARB2, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and LRRC4 (SND1) genes, or determining the level of methylation.

9. The method according to any one of claims 1 to 7, comprising the steps of detecting methylation of at least two, preferably three, preferably four genes selected from the group consisting of AQP5-AS1, ZSCAN23, and COL4A1 genes, and NR5A2, C9orf50, WIF1, and NPY, or determining the level of methylation.

10. The level or amount of methylation is - Sequence ID 40 in the ZSCAN23 gene, - Sequence ID 41 in the C9orf50 gene, - Sequence ID 42 in the AQP5-AS1 gene, - Sequence ID 43 in the NR5A2 gene, - Sequence ID 44 in the ADARB2 gene, - Sequence ID 45 in the COL4A1 gene, - Sequence ID 46 in the NPY gene, - Sequence ID 47 in the WIF1 gene, - Sequence ID 48 in the CPNE8 gene, - Sequence ID 49 in the LINC00693(RBMS3) gene, - Sequence ID 50 in the LINC00900 gene, - Sequence ID 51 in the DYDC2 gene, and / or - Sequence ID No. 52 in the LRRC4(SND1) gene Determined in the nucleotide region, The level or amount of methylation is preferably, - Sequence ID 53 in the ZSCAN23 gene, - Sequence ID 54 in the C9orf50 gene, - Sequence ID 55 in the AQP5-AS1 gene, - Sequence ID 56 in the NR5A2 gene, - Sequence ID 57 in the ADARB2 gene, - Sequence ID 58 in the COL4A1 gene, - Sequence ID 59 in the NPY gene, - Sequence ID 60 in the WIF1 gene, - Sequence ID 61 in the CPNE8 gene, - Sequence ID 62 in the LINC00693(RBMS3) gene, - Sequence ID 63 in the LINC00900 gene, - Sequence ID 64 in the DYDC2 gene, and / or - Sequence ID No. 65 in the LRRC4(SND1) gene Determined in the nucleotide region, The method according to any one of claims 1 to 9.

11. The level or amount of methylation is - Primers for the ZSCAN23 gene (SEQ ID NOs: 1 and 2), and probe for SEQ ID NOs:

27. - Primers for the C9orf50 gene (SEQ ID NOs: 3 and 4), and probe for SEQ ID NOs:

28. - Primers for the AQP5-AS1 gene, SEQ ID NOs. 5 and 6, and probe for SEQ ID NO.

29. - Primers for the NR5A2 gene, SEQ ID NOs. 7 and 8, and probe for SEQ ID NO.

30. - Primers for the ADARB2 gene, SEQ ID NOs. 9 and 10, and probe for SEQ ID NO.

31. - Primers for the COL4A1 gene, SEQ ID NOs. 11 and 12, and probe for SEQ ID NOs.

32. - Primers for the NPY gene, SEQ ID NOs. 13 and 14, and probe for SEQ ID NOs.

33. - Primers for the WIF1 gene (SEQ ID NOs. 15 and 16), and probe for SEQ ID NO.

34. - Primers for the CPNE8 gene, SEQ ID NOs. 17 and 18, and probe for SEQ ID NO.

35. - Primers for the LINC00693(RBMS3) gene, SEQ ID NOs. 19 and 20, and probe for SEQ ID NO.

36. - Primers for the LINC00900 gene, SEQ ID NOs. 21 and 22, and probe for SEQ ID NO.

37. - Primers for the DYDC2 gene, SEQ ID NOs. 23 and 24, and probe for SEQ ID NOs. 38, and / or - Primers for SEQ ID NOs. 25 and 26, and probe for SEQ ID NO. 39, for the LRRC4(SND1) gene. The method according to any one of claims 1 to 10, determined using

12. The method according to any one of claims 1 to 11, wherein the sample is a body fluid, preferably a body fluid selected from the group consisting of plasma, serum, blood, urine, and feces, more preferably the sample is a plasma or serum sample, and the DNA is circulating cell-free DNA (ccfDNA), preferably circulating tumor DNA (ctDNA).

13. The method according to any one of claims 1 to 12, wherein if the gene is hypermethylated compared to a reference value, the subject is diagnosed with or identified as having colorectal cancer.

14. The method according to any one of claims 1 to 12 for early diagnosis in subjects at risk of developing colorectal cancer.

15. The method according to any one of claims 1 to 12 for the early diagnosis of cancerous lesions in the colon in subjects who have metastasized from a primary tumor of unknown origin.

16. A method according to any one of claims 1 to 12 for assessing the risk of recurrence.

17. a) A step of determining the level of methylation of any of the genes in the target biological sample at a first time point, b) A step of determining the level of methylation of the gene previously selected in step a) in the target biological sample at a second time point, and c) A step of comparing the methylation level determined in step a) with the level or amount determined in step b), or a reference value. A method according to any one of claims 1 to 12 for monitoring the progression of colorectal cancer in a subject diagnosed with colorectal cancer, preferably comprising:

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