Detection of hypermethylated genes for diagnosing colorectal cancer

Abstract

The present invention relates to a method for diagnosing or identifying a colorectal cancer in a subject, through the detection of the abnormal hypermethylation levels of specific genes in a biological sample of said subject. The inventors indeed identified DNA methylation biomarkers that, alone or in combination, can help diagnosing or following-up colorectal cancer patients. Further, it can be used for determining, and / or adapting a suitable therapeutic regimen for a subject diagnosed for colorectal cancer. The present invention also relates to kits comprising primers or probes to detect, diagnose, or identify hypermethylated genes.

Description

[0001] DETECTION OF HYPERMETHYLATED GENES FOR DIAGNOSING COLORECTAL CANCER

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of oncology. More particularly, the present invention relates to a method for detecting or monitoring a colorectal cancer (CRC) in a subject, through the detection of the abnormal hypermethylation levels of specific genes in a biological sample of said subject. The inventors indeed identified a panel of DNA methylation biomarkers that, alone or in combination, can help diagnosing or following-up CRC patients.

[0004] BACKGROUND OF THE INVENTION

[0005] The colorectal cancer (CRC) represents an important challenge in terms of public health. In fact, the CRC is the third cancer in terms of incidence with nearly 2 million of new cases in 2020. There were also more than 916,000 deaths, representing more than 9% of the world's mortality in 2020. Surgery is the most common treatment for all stages of colon cancer. Most of the time, surgery is followed by adjuvant chemotherapy (ACT) in patients with histological high-risk stage II and III. ACT can decrease the risk of recurrence, but the survival is contrasted.

[0006] The high mortality rate can be explained by a late diagnosis, limited monitoring tools and a low adaptability of treatments. This illustrates the real need for a new method, noninvasive, cost- effective and sensitive for the diagnosis and monitoring of CRC patients.

[0007] Thus, an improvement in diagnostic and tracking methods could allow the reduction of its mortality by allowing its detection at an early stage. The diagnostic methods now available have important limits, such as their invasiveness, cost or low sensitivity, especially at early stages. There is still a need for methods for early diagnosis and accurate monitoring of colorectal cancer.

[0008] SUMMARY OF THE INVENTION

[0009] A first aspect of the invention relates to an in vitro method for detecting or monitoring a colorectal cancer (CRC) in a subject, preferably a human subject, which method comprises detecting, or determining the level of methylation of at least one of AQP5-AS1, ZSCAN23, or COL4A1 genes, in a biological sample from the subject, wherein the biological sample contains genomic DNA.

[0010] In a particular embodiment, the method comprises detecting, or determining the level of methylation of AQP5-AS1 and ZSCAN23 genes. In another particular embodiment, the method comprises detecting, or determining the level of methylation of AQP5-AS1 and COL4A1 genes. In another particular embodiment, the method comprises detecting, or determining the level of methylation of ZSCAN23 and COL4A1 genes.

[0011] In a particular embodiment, the method comprises detecting, or determining the level of methylation of the combination of AQP5-AS1, ZSCAN23, and COL4A1 genes. In a particular embodiment, the method further comprises detecting, or determining the level of methylation of NR5A2, C9orf50, WIFI or NPY gene. In a particular embodiment, the method further comprises detecting, or determining the level of methylation of at least one of ADARB2, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, or LRRC4 (SND1) genes.

[0012] In a particular embodiment, the method comprises detecting, or determining the level of methylation of the combination of AQP5-AS1, ZSCAN23 and COL4Algenes, and at least two, preferably three, preferably four genes selected in the group consisting of : NR5A2, C9orf50, WIFI, NPY, ADARB2, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and LRRC4 (SND1) genes.

[0013] In a particular embodiment, the method comprises detecting, or determining the level of methylation of the combination of AQP5-AS1, ZSCAN23, and COL4A1 genes, and at least two, preferably three, preferably four genes selected in the group consisting of : NR5A2, C9orf50, WIFI, and NPY.

[0014] In a particular embodiment, said level or amount of methylation is determined in the nucleotide region(s) of :

[0015] - SEQ ID NO:40 in the ZSCAN23 gene,

[0016] - SEQ ID NO:41 in the C9orf50 gene,

[0017] - SEQ ID NO:42 in the AQP5-AS1 gene,

[0018] - SEQ ID NO:43 in the NR5A2 gene,

[0019] - SEQ ID NO:44 in the ADARB2 gene, - SEQ ID NO:45 in the COL4A1 gene,

[0020] - SEQ ID NO:46 in the NPY gene,

[0021] - SEQ ID NO:47 in the WIFI gene,

[0022] - SEQ ID NO:48 in the CPNE8 gene,

[0023] - SEQ ID NO:49 in the LINC00693 (RBMS3) gene,

[0024] - SEQ ID NO:50 in the LINC00900 gene,

[0025] - SEQ ID NO:51 in the DYDC2 gene, and / or

[0026] - SEQ ID NO: 52 in the LRRC4 (SND1) gene.

[0027] In a particular embodiment, said level or amount of methylation is determined in the nucleotide region(s) of :

[0028] - SEQ ID NO 53 in the ZSCAN23 gene,

[0029] - SEQ ID NO:54 in the C9orf50 gene,

[0030] - SEQ ID NO 55 in the AQP5-AS1 gene,

[0031] - SEQ ID NO:56 in the NR5A2 gene,

[0032] - SEQ ID NO:57 in the ADARB2 gene,

[0033] - SEQ ID NO:58 in the COL4A1 gene,

[0034] - SEQ ID NO:59 in the NPY gene,

[0035] - SEQ ID NO: 60 in the WIFI gene,

[0036] - SEQ ID NO:61 in the CPNE8 gene,

[0037] - SEQ ID NO:62 in the LINC00693 (RBMS3) gene,

[0038] - SEQ ID NO: 63 in the LINC00900 gene,

[0039] - SEQ ID NO:64 in the DYDC2 gene, and / or

[0040] - SEQ ID NO: 65 in the LRRC4 (SND1) gene.

[0041] In a particular embodiment, said level or amount of methylation is determined by using :

[0042] - the primers of SEQ ID NO: 1 and 2, and the probe of SEQ ID NO:27 for the ZSCAN23 gene,

[0043] - the primers of SEQ ID NO:3 and 4, and the probe of SEQ ID NO:28 for the C9orf50 gene,

[0044] - the primers of SEQ ID NO:5 and 6, and the probe of SEQ ID NO:29 for the AQP5-AS1 gene,

[0045] - the primers of SEQ ID NO: 7 and 8, and the probe of SEQ ID NO:30 for the NR5A2 gene,

[0046] - the primers of SEQ ID NO: 9 and 10, and the probe of SEQ ID NO: 31 for the ADARB2 gene,

[0047] - the primers of SEQ ID NO: 11 and 12, and the probe of SEQ ID NO:32 for the COL4A1 gene,

[0048] - the primers of SEQ ID NO: 13 and 14, and the probe of SEQ ID NO:33 for the NPY gene,

[0049] - the primers of SEQ ID NO: 15 and 16, and the probe of SEQ ID NO:34 for the WIFI gene,

[0050] - the primers of SEQ ID NO: 17 and 18, and the probe of SEQ ID NO:35 for the CPNE8 gene - the primers of SEQ ID NO: 19 and 20, and the probe of SEQ ID NO:36 for the LINC00693 (RBMS3) gene,

[0051] - the primers of SEQ ID NO:21 and 22, and the probe of SEQ ID NO:37 for the LINC00900 gene,

[0052] - the primers of SEQ ID NO:23 and 24, and the probe of SEQ ID NO:38 for the DYDC2 gene, and / or

[0053] - the primers of SEQ ID NO:25 and 26, and the probe of SEQ ID NO:39 for the LRRC4 (SND1) gene.

[0054] In a particular embodiment, the sample is a body fluid, preferably selected from the group consisting of plasma, serum, blood, urine and feces, still preferably wherein the sample is a plasma or serum sample and the DNA is circulating cell-free DNA (ccfDNA), preferably circulating tumor DNA (ctDNA).

[0055] In particular, if said gene(s) are hypermethylated as compared with reference value(s), then said subject is diagnosed or identified as suffering from a colorectal cancer.

[0056] In a particular embodiment, the method is used for an early diagnosis in a subject who is at risk of developing a colorectal cancer.

[0057] In a particular embodiment, the method is used for an early diagnosis of a cancerous lesion in the colon in a subject who has been developing metastasis from a primary tumor of unknown origin.

[0058] In a particular embodiment, the method is used for assessing the risk of relapse.

[0059] In a particular embodiment, the method is used for monitoring the progress of colorectal cancer in a subject diagnosed with colorectal cancer, which method preferably comprises a) determining the level of methylation of any of said genes, in a biological sample of said subject, at a first time point, b) determining the level of methylation of said genes selected previously in the step a), in a biological sample of said subject, at a second time point, and c) comparing the level of methylation determined in step a) to the level or amount determined in step b) or to reference values. LEGENDS TO THE DRAWINGS

[0060] Figure 1 shows DNA methylation of selected biomarkers, ZSCAN23, C9orf50, AQP5-AS1, NR5A2, ADARB2, COL4A1, NPY, WIFI, CPNE8 ; LINC00693 (RBMS3); LINC00900 ; DYDC2 ; LRRC4 (SND1) by ddPCR (n=20 to 26). Paired non-parametric Wilcoxon test was used for the analysis of the hypermethylation difference between tumor and adjacent normal tissue DNA.

[0061] Fi ure 2 shows DNA methylation of selected biomarkers, ZSCAN23, C9orf50, AQP5-AS1, NR5A2, ADARB2, COL4A1, NPY and WIFI in CRC patients by ddPCR. The difference of DNA methylation in CRC patients plasma (n=l 7) and healthy individuals plasma (n=10 to 20) was tested by ddPCR. Mann-whitney test was used for the analysis of the significance.

[0062] DETAILED DESCRIPTION OF THE INVENTION

[0063] The inventors have identified a methylation signature of colorectal cancer.

[0064] More particularly a subject of the invention is an in vitro method for detecting or monitoring a colorectal cancer in a subject, which method comprises detecting, or determining the level of, methylation of specific genes, alone or in combination, in a biological sample from the subject, wherein the biological sample contains genomic DNA. More particularly, a hypermethylation at the selected genomic sites is indicative of a colorectal cancer.

[0065] Colorectal cancer

[0066] The methods of the invention encompass diagnosing and / or monitoring colorectal cancer (CRC) in a subject. All histologic types, stages and grades are encompassed.

[0067] CRC starts in the cells of the colon or in the rectum. The most common one is adenocarcinomas of the colon and rectum but a CRC can include primary colorectal lymphomas, Gastrointestinal stromal tumors, Leiomyosarcomas and Carcinoid tumors. The most common staging system for CRC is TNM Classification, describing the tumor, nodes and metastasis (TMN). According to the severity of the cancer, a number (0 to 4) is associate with the letter. A stage 0 indicates a carcinoma in situ (NO, MO). A stage I indicates a localized cancer (T1-T2, NO, MO). A stage II refers to a locally advanced cancer, at an early stage (T2- T4, NO, MO). A stage III indicates a locally advanced cancer, at a late stage (T1-T4, N1-N3, MO). And a stage IV refers to a Metastatic cancer (T1-T4, N1-N3, Ml).

[0068] Subject

[0069] According to the invention, the terms "subject", "individual", and "patient" are used interchangeably herein and refer to a mammal that may be healthy (without any symptoms of colorectal cancer), thought to develop colorectal cancer, suspected of suffering from colorectal cancer or suffering from colorectal cancer. Said subject for example presents at least one of the following symptoms: a change in bowel habits, such as diarrhea, constipation, or narrowing of the stool, abnormally enlarging abdomen, rectal bleeding, blood in the stool, persistent cramping or abdominal (belly) pain, nausea or vomiting, weakness and fatigue, unexplained weight loss. Said subject may also have suffered from a colorectal cancer in the past, has been treated, and is monitored for potential disease recurrence. Said subject may also seem to be healthy but is at risk of developing a colorectal cancer or because a member of his family is suffering or has suffered from the same disease. Risk factors include older age, male sex, high intake of fat, sugar, alcohol, red meat, processed meats, obesity, smoking, and a lack of physical exercise.

[0070] In one embodiment, the subject is a mammal, such as a human, canine, murine, feline, bovine, ovine, swine or caprine. In a preferred embodiment, the subject is a human.

[0071] Sample

[0072] The methods of the invention require a biological sample from the subject, wherein the biological sample contains genomic DNA.

[0073] The sample may be a body fluid, a tissue sample, or a combination thereof, from the subject.

[0074] The sample may comprise cell-free DNA. As used herein, the expression “biological sample” refers to solid tissues such as, for example, a gastrointestinal biopsy or to fluids, body effluents and excretions.

[0075] In a preferred embodiment, the sample is a body fluid, preferably selected from the group consisting of plasma, serum, blood, urine, or feces. Still preferably the sample is a plasma or serum sample and the DNA is circulating cell-free DNA (ccfDNA).

[0076] In another embodiment, the sample is a tissue sample. For instance, the sample may be obtained from a colorectal tissue. Colorectal tissue or cells may be taken, for example, by biopsy, resection of tumor samples, tissue samples prepared by endoscopic means, needle-biopsies of organs.

[0077] After collection, samples are prepared prior to detection of biomarkers. Sample preparation includes isolation of nucleic acids. These isolation procedures involve separation of nucleic acids from insoluble components (e.g., cytoskeleton) and cellular membranes. Typically, colorectal tissues or cells may be treated with a lysis buffer solution prior to isolation of nucleic acids. A lysis buffer solution is designed to lyse tissues, cells, lipids and other biomolecules potentially present in the raw tissue samples. Nucleic acids can be conveniently extracted from biological samples, e.g. obtained from colorectal tissues, using standard extraction methods that are known in the art. Standard extraction methods include the use of a chemical agent such as guanidinium thiocyanate, phenol-chloroform extraction, guanidine-based extraction, and the like. Commercial nucleic acid extraction kits may be employed.

[0078] Methylation status

[0079] According to the invention, the methylation status of the target genes serves as a biomarker for colorectal cancer. CpG islands at the selected genomic sites are more particularly targeted.

[0080] DNA methylation in the human genome occurs mostly at the cytosine residues in a CpG dinucleotide at the carbon-5 position, resulting in 5-methylcytosine. CpG dinucleotides are rare in the human genome (~1%). CpG dinucleotides are often found to be in clusters of more than 200 bases with more than 50% of G+C content and a ratio of CpG frequencies of at least 0.6, which are known as CpG islands. Approximately 60% of the human gene promoters are associated with CpG islands. The CpG islands within the promoter regions are usually unmethylated in normal cells, except for some involved in tissue differentiation. In general, CpG island methylation is associated with transcriptional silencing. In cancer both global hypomethylation (decrease of overall DNA methylation) and localized hypermethylation, such as methylation of the promoter and the first exon of tumor suppressor genes, have been observed. DNA hypomethylation occurs at many genomic sequences, such as repetitive elements, retrotransposons, introns and similar elements, resulting in genomic instability, and can account for the activation of some proto-oncogenes and lead to loss of imprinting, as in the case of the IGF2 gene (encoding IGF -2) in Wilms' tumor.

[0081] Methylation of cytosine (C) nucleotides in CpG dinucleotide sequences (CpG sites; G= guanosine) of the human DNA is a known phenomenon. Moreover, abnormal methylation (hypermethylation and / or hypomethylation) of specific genomic areas is present in virtually all cancer types. Genomic regions with altered methylation states in test samples compared to controls samples are commonly referred to as "differentially methylated regions" (DMRs).

[0082] A "CpG island" as used herein refers to regions of DNA with a high G / C content and a high frequency of CpG dinucleotides relative to the whole genome. Also used interchangeably in the art is the term "CG island." The 'p' in "CpG island" refers to the phosphodiester bond between the cytosine and guanine nucleotides. Methylation may refer to methylation and / or hydroxymethylation of DNA.

[0083] A number of techniques has been proposed to detect DNA methylation. The review of 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): 15029-58) :

[0084] Methylation specific-PCR (MS-PCR) is a method in which two sets of PCR primers are specifically designed to amplify methylated and unmethylated DNA regions of interest. The detection of PCR products is originally performed by gel electrophoresis. This technique has been replaced by Quantitative MS-PCR (qMS-PCR), in which PCR amplification is monitored in real time by the incorporation of fluorescent molecules. This improvement allows for precise quantification of the DNA methylation levels of numerous specific regions and avoids the long electrophoresis step. Quantitative multiplex MS-PCR (QM-MS-PCR) and one step MS-PCR (OS-MS-PCR) are also available to co-amplify specific genes in tissues from different origins or to determine DNA methylation levels of a specific region without the DNA extraction procedure. qMS-PCR techniques are simple, rapid, inexpensive, highly sensitive and easily standardized. They are currently one of the most commonly used techniques for cancer diagnosis in clinical use. Methylation-sensitive high-resolution melting (MS-HRM) is based on the fact that the nucleotide sequence of PCR products of bisulfite-treated DNA will differ depending on the methylation status of the DNA region of interest. The methylation level is determined by comparing the melting dissociation curves to standard PCR products of the same region containing known methylated CpG sites. COBRA, for combined bisulfite restriction analysis, uses the ability of bisulfite conversion to create new restriction enzyme sites or to maintain consensus sites of MSRE. After amplification, PCR products are digested with appropriate MSRE. The proportion of digested PCR products is compared to undigested PCR products by poly-acrylamide gel electrophoresis and image quantification software. This technique is reliably applied to DNA obtained from formalin-fixed paraffin embedded (FFPE) tissue sample. Moreover, this approach allows for the assessment of the DNA methylation of a large number of biological samples. More recently, high throughput approaches have been developed. For instance, Methyl Light is a high throughput quantitative methylation assay that uses fluorescent-based real time PCR (TaqMan®, Applied Biosystems, Forster City, CA, USA) in combination to bisulfite treatment. Also combined with bisulfite treatment pyrosequencing is a quantitative DNA sequencing method in which light is emitted as a result of an enzymatic reaction representing each time a nucleotide is incorporated into the growing DNA chain. These quantitative techniques detect low amounts of methylated DNA in heterogeneous DNA preparation. Easily standardized, rapid and inexpensive, these techniques are increasingly used for clinical purpose.

[0085] More recently, next-generation sequencing (NGS) technologies significantly increased the resolution level of DNA methylation profiles. NGS can also be adapted to immuno-precipitated DNA fragment (Methyl DNA Immuno-precipitation sequencing also called MeDIP seq). Ultimately NGS permits the sequencing of the entire genome after bisulfite conversion. Beside these NGS approaches, high throughput single nucleotide polymorphism (SNP) genotyping systems are suitable for DNA methylation analysis from bisulfite-converted genomic DNA. Further, it is possible to use other methods well known to the skilled person, such as methods which directly analyze the unmodified DNA (for example nanopore sequencing), methods using specific restriction enzymes, methylated sequence enrichment methods (e.g. EpiMark methylated Enrichment kit, New England), immunoprecipitation methods. All these methods (qMS-PCR, MS-HRM, COBRA, MSRE, Methyl Light, NGS, SNP genotyping, pyrosequencing, microarray, ICE-cold PCR, nanopore etc.) can be used for determining the methylation of the markers of the invention.

[0086] In a preferred aspect of the invention, the detecting of methylation is performed by employing a digital PCR, preferably a digital droplet PCR (ddPCR).

[0087] As used herein, "digital PCR" refers to an assay that provides an end-point measurement that provides the ability to quantify nucleic acids without the use of standard curves, as is used in real-time PCR. Digital PCR includes a variety of formats, including employing droplet digital PCR, BEAMing (beads, emulsion, amplification, and magnetic), microwell plates, bulk emulsion droplets, microfluidic compartments and nanoliter- or picoliter-scale droplets produced with microfluidics.

[0088] "Droplet digital PCR" (ddPCR) refers to a digital PCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in- oil droplet partitions that support PCR amplification (Hindson, et al, 2011. Analytical Chemistry 83, 8604-8610; Pekin, et al, 2011, Lab on a Chip 11, 2156-66; Pinheiro, et al, 2012, Anal Chem 84, 1003-1011). A single ddPCR reaction may be comprised of at least 13,000- 20,000 partitioned droplets per well.

[0089] Upon evaluating a methylation state, the methylation state is often expressed as the fraction or percentage of individual strands of DNA that is methylated at a particular site (e.g., at a single nucleotide, at a particular region or locus, at a longer sequence of interest, e.g., up to a -100- bp, 200-bp, subsequence of a DNA) relative to the total population of DNA in the sample comprising that particular site. Traditionally, the amount of the unmethylated nucleic acid is determined by PCR using calibrators. In a particular embodiment, a known amount of DNA is e.g. bisulfite treated and the resulting methylation-specific sequence is determined using any technique of exponential amplification.

[0090] The method of the invention requires to detect the “level of methylation”, “methylation level” or the “amount of methylation” in CpG sites. According to the present invention, the terms “level of methylation” or “amount of methylation” refer to the determination of a quantitative measure. Thus, the terms “level of methylation” or “amount of methylation” can be used interchangeably.

[0091] In one exemplary embodiment, the present method can use a set of primers. As used herein, the term "primers" designates isolated nucleic acid molecules that can specifically hybridize or anneal to 5' or 3' regions of a target genomic region (plus and minus strands, respectively, or vice-versa). In general, they are from about 10 to 30 nucleotides in length and anneal at both extremities of a region containing about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. As they have to be used by pairs, they are often referred to as "primers pair" or "primers set".

[0092] "Specific hybridization" is observed when a define molecule does not hybridize with any other genomic region than its target genomic region. Preferably, it hybridizes with its target region in high stringency conditions, i.e., when the temperature and ionic strength conditions are chosen so as to allow the hybridization between two complementary DNA fragments.

[0093] In one exemplary embodiment, the present method can use methods for detecting gene expression (e.g., dPCR) that use fluorogenic probes to improve the specificity and / or the sensitivity of the detection of the PCR products that accumulate during PCR. Such assays are for example TaqMan® gene expression assays using probes containing minor groove binding (MGB) moiety that enhances the Tm differential between matched and mismatched probes. In addition, these MGB probes may contain a non-fluorescent quencher (NFQ) that enhances spectral resolution when using multiple dyes in a reaction. The probes that can be used in this preferred embodiment are reflected on Table 2 below and in SEQ ID NO:27-39.

[0094] As used herein, the term "probes" designates molecules that are capable of specifically hybridizing a genomic region of interest. They are useful to highlight the presence of said genomic region in biological samples. These probes may comprise at least one non-natural nucleotide, e.g., a peptide nucleic acid (PNA), a peptide nucleic acid having a phosphate group (PHONA), a bridged nucleic acid or locked nucleic acid (BNA or LNA), and a morpholino nucleic acid. Non-natural nucleotides also 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'-0-methyl-type DNA or RNA. In a preferred embodiment, the probes of the invention comprise at least 15 consecutive nucleotides. In a more preferred embodiment, the molecules which can be used as a probe according to the present invention have a total minimum size of 15 nucleotides. In an even more preferred embodiment, these molecules comprise between 15 and 30 nucleotides (in total).

[0095] For certain uses, the probes and primers of the invention may be labeled - directly or indirectly - with a detectable label. Said label may be of any kind, depending on the experiment which is to be performed. Said label may be a radioactive isotope (such as32 P,33 P,35 S,3 H orl25 1, or a nonradioactive entity which is selected from ligands (such as biotin, avidin or streptavidin), dioxygenin, haptens, colorants and luminescent agents (such as radioluminescent, chemiluminescent, bioluminescent, fluorescent or phosphorescent agents). Preferably, 6- carboxyfluorescein (FAM), VIC, HEX and tetramethylrhodamine (TAM RA) are used. Nonlabeled polynucleotide sequences may also be used, directly, as a probe or primer, for example in PCR- based processes (e.g., in quantitative PCR).

[0096] Exemplary primers and probes are summarized in Tables 1 and 2 below.

[0097] Table 1 : useful primers for determining the level of methylation of the biomarker(s) of the invention.

[0098] Table 2: useful probes for determining the level of methylation of the biomarker(s) of the invention (FAM stands for "fluorescein amidite" ; MGB stands for "minor groove binder"; NFQ stands for "non-fluorescent quencher") Tarset genes

[0099] The term "gene" refers to a nucleic acid sequence that comprises coding sequences necessary for the production of an RNA, or of a polypeptide or its precursor. The term "gene" encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends, e.g., for a distance of about 1 kb on either end, such that the gene corresponds to the length of the full- length mRNA, e.g., comprising coding, untranslated regions, structural and other sequences and regulatory sequences, e.g. promoters. The sequences that are located 5' of the coding region and that are present on the mRNA are referred to as 5' non-translated or untranslated sequences. The sequences that are located 3' or downstream of the coding region and that are present on the mRNA are referred to as 3' nontranslated or 3' untranslated sequences. The term "gene" in the present invention more particularly refers to genomic forms of a gene. In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' ends of the sequences that are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, posttranscriptional cleavage, and polyadenylation.

[0100] The inventors have shown that hypermethylation of the promoter or other regions of specific genes can be used as a sensitive and specific biomarker of colorectal cancer in patients suffering thereof, even at early stage. The below listed genes are also referred to as the "biomarkers”. They can be used individually, or in combination.

[0101] • ZSCAN23

[0102] ZSCAN23 (Zinc Finger And SCAN Domain Containing 23) is a protein coding gene. ZSCAN23 enables sequence-specific double-stranded DNA binding activity. ZSCAN23 is predicted to be involved in 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 chromosome 6, from position 28,431,930 to 28,443,502, reverse strand (Hg38 coordinates).

[0103] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of the genomic DNA that consists of chr6: 28431930-28443502 (Hg38 coordinates). In a particular embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr6: 28431930-28443502 (Hg38 coordinates).

[0104] In a preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr6: 28442840-28443839 (Hg38 coordinates). In a more preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr6: 28443240-28443439 (Hg38 coordinates).

[0105] 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:53.

[0106] In a preferred embodiment, the level or amount of methylation of the ZSCAN23 gene is determined by using the primers of SEQ ID NO: 1 and SEQ ID NO:2.

[0107] In a preferred embodiment, the level or amount of methylation of the ZSCAN23 gene is determined by using the probe of SEQ ID NO:27.

[0108] In a more preferred embodiment, the level or amount of methylation of the ZSCAN23 gene is determined by using the primers of SEQ ID NO: 1 and SEQ ID NO:2 and the probe of SEQ ID NO:27.

[0109] 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.

[0110] C0L4A1 COL4A1 (collagen type IV alpha 1 chain) is a protein coding gene. This gene encodes a type IV collagen alpha protein. Type IV collagen proteins are integral components of basement membranes. This gene is also known as “BSVD”; “BSVD1”; “RATOR”; “PADMAL”; or "COL4Als”. Its DNA sequence is located on chromosome 13, from position 110,148,963 to 110,307,157, reverse strand (Hg38 coordinates).

[0111] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of the genomic DNA that consists of chrl3: 11014963-110307157 (Hg38 coordinates).

[0112] In a particular embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl3: 11014963-110307157 (Hg38 coordinates).

[0113] In a preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl3: 110306660-110307659 (Hg38 coordinates). In a more preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl3: 110307060-110307259 (Hg38 coordinates).

[0114] In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:45. In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:58.

[0115] In a preferred embodiment, the level or amount of methylation of the COL4A1 gene is determined by using the primers of SEQ ID NO: 11 and SEQ ID NO: 12.

[0116] In a preferred embodiment, the level or amount of methylation of the COL4A1 gene is determined by using the probe of SEQ ID NO:32. In a more preferred embodiment, the level or amount of methylation of the COL4A1 gene is determined by using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 and the probe of SEQ ID NO:32.

[0117] 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.

[0118] • AQP5-AS1

[0119] AQP5-AS1 (AQP5 and AQP2 antisense RNA 2) designates a non-coding RNA. The AQP5- AS1 gene is also known as “MIAC” or “micropeptide inhibiting actin cytoskeleton”. Its DNA sequence is located on chromosome 12, from position 49,951,512 to 49,962,924 (Hg38 coordinates).

[0120] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of the genomic DNA that consists of chrl2: 49951512-49962924 (Hg38 coordinates).

[0121] In a particular embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl2: 49951512-49962924 (Hg38 coordinates).

[0122] In a preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl2: 49961035-49962034 (Hg38 coordinates). In a more preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl2: 49961440-49961629 (Hg38 coordinates).

[0123] In a 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 of a polymorphic variant sequence that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:42. In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO:42. In a 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 of a polymorphic variant sequence that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO: 55. In a preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO:55.

[0124] In a preferred embodiment, the level or amount of methylation of the AQP5-AS1 gene is determined by using the primers of SEQ ID NO:5 and SEQ ID NO:6.

[0125] In a preferred embodiment, the level or amount of methylation of the AQP5-AS1 gene is determined by using the probe of SEQ ID NO:29.

[0126] In a more preferred embodiment, the level or amount of methylation of the AQP5-AS1 gene is determined by using the primers of SEQ ID NO:5 and SEQ ID NO:6 and the probe of SEQ ID NO:29.

[0127] 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.

[0128] • NR5A2

[0129] NR5 A2 (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-Flbeta”, “LRH-1”, “LRH1”, “hBIF”, or “hBlF-2” Its DNA sequence is located on chromosome 1, from position 200,027,614 to 200,177,420, forward strand (Hg38 coordinates).

[0130] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of the genomic DNA that consists of chrl: 200027614-200177420 (Hg38 coordinates).

[0131] In a particular embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl : 200027614-200177420 (Hg38 coordinates).

[0132] In a preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl : 200038400-200039399 (Hg38 coordinates). In a more preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr9: 200038800-200038999 (Hg38 coordinates).

[0133] In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:43. In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:56.

[0134] In a preferred embodiment, the level or amount of methylation of the NR5A2 gene is determined by using the primers of SEQ ID NO: 7 and SEQ ID NO: 8.

[0135] In a preferred embodiment, the level or amount of methylation of the NR5A2 gene is determined by using the probe of SEQ ID NO:30.

[0136] In a more preferred embodiment, the level or amount of methylation of the NR5A2 gene is determined by using the primers of SEQ ID NO:7 and SEQ ID NO:8 and the probe of SEQ ID NO:30.

[0137] 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.

[0138] • C9orf50

[0139] C9orf50 (chromosome 9 open reading frame 50) is a protein coding gene. This gene is also known as “FLJ35803”. Its DNA sequence is located on chromosome 9, from position 129,612,225 to 129,621,101, reverse strand (Hg38 coordinates).

[0140] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of the genomic DNA that consists of chr9: 129612225-129621101 (Hg38 coordinates). In a particular embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr9: 129612225 to 129621101 (Hg38 coordinates).

[0141] In a preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr9: 129620034-129621033 (Hg38 coordinates). In a more preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr9: 129620434: 129620633 (Hg38 coordinates).

[0142] In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:41. In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:54.

[0143] In a preferred embodiment, the level or amount of methylation of the C9orf50 gene is determined by using the primers of SEQ ID NO:3 and SEQ ID NO:4.

[0144] In a preferred embodiment, the level or amount of methylation of the C9orf50 gene is determined by using the probe of SEQ ID NO:28.

[0145] In a more preferred embodiment, the level or amount of methylation of the C9orf50 gene is determined by using the primers of SEQ ID NO:3 and SEQ ID NO:4 and the probe of SEQ ID NO:28.

[0146] 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.

[0147] 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 play a regulatory role in RNA editing. This gene is also known as “ADAR3”, “RED2”, or “hRED2”. Its DNA sequence is located on chromosome 10, from position 1,177,313 to 1,737,525, reverse strand (Hg38 coordinates).

[0148] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of the genomic DNA that consists of chrlO: 1177313 to 1737525 (Hg38 coordinates).

[0149] In a particular embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrlO: 1177313 to 1737525 (Hg38 coordinates).

[0150] In a preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrlO: 1737180-1738179 (Hg38 coordinates). In a more preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrlO: 1737580-1737779 (Hg38 coordinates).

[0151] In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:44. In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:57.

[0152] In a preferred embodiment, the level or amount of methylation of the ADARB2 gene is determined by using the primers of SEQ ID NO:9 and SEQ ID NO: 10.

[0153] In a preferred embodiment, the level or amount of methylation of the ADARB2 gene is determined by using the probe of SEQ ID NO:31. In a more preferred embodiment, the level or amount of methylation of the ADARB2 gene is determined by using the primers of SEQ ID NO:9 and SEQ ID NO: 10 and the probe of SEQ ID N0:31.

[0154] 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.

[0155] • NPY

[0156] NPY (neuropeptide Y) is a protein coding gene. This gene encodes a neuropeptide that is 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 chromosome 7, from position 24,284,188 to 24,291,862, forward strand (Hg38 coordinates).

[0157] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of the genomic DNA that consists of chr7: 24284188-24291862 (Hg38 coordinates).

[0158] In a particular embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr7: 24284188-24291862 (Hg38 coordinates).

[0159] In a preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr7: 24283690-24284689 (Hg38 coordinates). In a more preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr7: 24284090-24284289 (Hg38 coordinates).

[0160] 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:59.

[0161] In a preferred embodiment, the level or amount of methylation of the NPY gene is determined by using the primers of SEQ ID NO: 13 and SEQ ID NO: 14.

[0162] In a preferred embodiment, the level or amount of methylation of the NPY gene is determined by using the probe of SEQ ID NO:33.

[0163] In a more preferred embodiment, the level or amount of methylation of the NPY gene is determined by using the primers of SEQ ID NO: 13 and SEQ ID NO: 14 and the probe of SEQ ID NO:33.

[0164] 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.

[0165] • WIFI

[0166] WIFI (WNT inhibitory factor 1) is a protein coding gene. The protein encoded by this gene functions to inhibit WNT proteins, which are extracellular signaling molecules that play a role in embryonic development. This gene is also known as “WIF-1”. Its DNA sequence is located on chromosome 12, from position 65,050,626 to 65,121,305, reverse strand (Hg38 coordinates). NPY (neuropeptide Y) is a protein coding gene. This gene encodes a neuropeptide that is 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 chromosome 12, from position 65,050,626 to 65,121,305, reverse strand (Hg38 coordinates).

[0167] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of the genomic DNA that consists of chrl2: 65050626-65121305 (Hg38 coordinates).

[0168] In a particular embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr!2: 65050626-65121305 (Hg38 coordinates). In a preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl2: 65120760-65121759 (Hg38 coordinates). In a more preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl2: 65121160:65121359 (Hg38 coordinates).

[0169] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO:47 in the WIFI gene, or of a polymorphic variant sequence that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:47. In a preferred embodiment, the level or amount of methylation is determined in the nucleotide region of SEQ ID NO: 60 in the WIFI gene, or of a polymorphic variant sequence that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:60.

[0170] In a preferred embodiment, the level or amount of methylation of the WIFI gene is determined by using the primers of SEQ ID NO: 15 and SEQ ID NO: 16.

[0171] In a preferred embodiment, the level or amount of methylation of the WIFI gene is determined by using the probe of SEQ ID NO:34.

[0172] In a more preferred embodiment, the level or amount of methylation of the WIFI gene is determined by using the primers of SEQ ID NO: 15 and SEQ ID NO: 16 and the probe of SEQ ID NO:34.

[0173] Preferably, the level or amount of methylation of the WIFI 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.

[0174] • CPNE8

[0175] CPNE8 (copine 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 in the C-terminus. Its DNA sequence is located on chromosome 12, from position 38,646,822 to 38,907,430, reverse strand. (Hg38 coordinates).

[0176] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of the genomic DNA that consists of chrl2: 38646822-38907430, (Hg38 coordinates).

[0177] In a particular embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl2: 38646822-38907430, (Hg38 coordinates).

[0178] In a preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl2: 38904820-38905819 (Hg38 coordinates). In a more preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl2: 38905220-38905419 (Hg38 coordinates).

[0179] 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, 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 WIFI gene, or of a polymorphic variant sequence that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:61.

[0180] In a preferred embodiment, the level or amount of methylation of the CPNE8 gene is determined by using the primers of SEQ ID NO: 17 and SEQ ID NO: 18.

[0181] In a preferred embodiment, the level or amount of methylation of the CPNE8 gene is determined by using the probe of SEQ ID NO:35.

[0182] In a more preferred embodiment, the level or amount of methylation of the CPNE8 gene is determined by using the primers of SEQ ID NO: 17 and SEQ ID NO: 18 and the probe of SEQ ID NO:35. 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.

[0183] • LINC00693 (RBMS3)

[0184] LINC00693 or RBMS3 (RNA binding motif single stranded interacting protein 3) or LINC00693 (RBMS3) is a protein coding gene. This gene encodes an RNA-binding protein that belongs to the c-myc gene single-strand binding protein family. This gene is also known as “PYY4”. Its DNA sequence is located on chromosome 3, from position 28,574,791 to 30,010,391, forward strand (Hg38 coordinates).

[0185] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of the genomic DNA that consists of chr3: 28574791-30010391 (Hg38 coordinates).

[0186] In a particular embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr3 : 28574791-30010391 (Hg38 coordinates).

[0187] In a preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr3 : 28574870-28575869 (Hg38 coordinates). In a more preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr3: 28575270-28575469 (Hg38 coordinates).

[0188] In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:49. In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:62. 1

[0189] In a preferred embodiment, the level or amount of methylation of the LINC00693 (RBMS3) gene is determined by using the primers of SEQ ID NO: 19 and SEQ ID NO:20.

[0190] In a preferred embodiment, the level or amount of methylation of the LINC00693 (RBMS3) gene is determined by using the probe of SEQ ID NO:36.

[0191] In a more preferred embodiment, the level or amount of methylation of the LINC00693 (RBMS3) gene is determined by using the primers of SEQ ID NO: 19 and SEQ ID NO:20 and the probe of SEQ ID NO:36.

[0192] 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.

[0193] • LINC00900

[0194] LINC00900 (long intergenic non-protein coding RNA 900) is a non-coding RNA gene, and is affiliated with the IncRNA class. Its DNA sequence is located on chromosome 11, from position 115,753,889 to 115,760,646, reverse strand (Hg38 coordinates).

[0195] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of the genomic DNA that consists of chrl 1 : 115753889-115760646 (Hg38 coordinates).

[0196] In a particular embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl l : 115,753,889 to 115,760,646 (Hg38 coordinates).

[0197] In a preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl l : 115759620-115760619 (Hg38 coordinates). In a more preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrl 1 : 115759620-115760619 (Hg38 coordinates). In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:50. In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO: 63.

[0198] In a preferred embodiment, the level or amount of methylation of the LINC00900 gene is determined by using the primers of SEQ ID NO:21 and SEQ ID NO:22.

[0199] In a preferred embodiment, the level or amount of methylation of the LINC00900 gene is determined by using the probe of SEQ ID NO:37.

[0200] In a more preferred embodiment, the level or amount of methylation of the LINC00900 gene is determined by using the primers of SEQ ID NO:21 and SEQ ID NO:22 and the probe of SEQ ID NO:37.

[0201] 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.

[0202] • DYDC2

[0203] DYDC2 (DPY30 domain containing 2) is a protein coding gene. This gene encodes a member of a family of proteins that contains a DPY30 domain. This gene is also known as “MGC16186”, or “bA36D19”. Its DNA sequence is located on chromosome 10, from position 80,344,745 to 80,368,073, forward strand. (Hg38 coordinates).

[0204] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of the genomic DNA that consists of chrlO: 80344745-80368073 (Hg38 coordinates).

[0205] In a particular embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrlO: 80344745-80368073 (Hg38 coordinates). In a preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrlO: 80356110-80357109 (Hg38 coordinates). In a more preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chrlO: 80356510-80356709 (Hg38 coordinates).

[0206] In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:51. In a 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO:64.

[0207] In a preferred embodiment, the level or amount of methylation of the DYDC2 gene is determined by using the primers of SEQ ID NO:23 and SEQ ID NO:24.

[0208] In a preferred embodiment, the level or amount of methylation of the DYDC2 gene is determined by using the probe of SEQ ID NO:38.

[0209] In a more preferred embodiment, the level or amount of methylation of the DYDC2 gene is determined by using the primers of SEQ ID NO:23 and SEQ ID NO:24 and the probe of SEQ ID NO:38.

[0210] 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.

[0211] LRRC4 (SND1)

[0212] LRRC4 (leucine rich repeat containing 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 chromosome 7, from position 128,027,071 to 128,032,107, reverse strand (Hg38 coordinates).

[0213] In a particular embodiment, the level or amount of methylation is determined in the nucleotide region of the genomic DNA that consists of chr7: 128027071-128032107 (Hg38 coordinates).

[0214] In a particular embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr7: 128027071-128032107 (Hg38 coordinates).

[0215] In a preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr7: 128031906-128032905 (Hg38 coordinates). In a more preferred embodiment, the method advantageously comprises determining the methylation status of one or more CpG dinucleotides in a region of said genomic DNA that consists of chr7: 128031906-128032905 (Hg38 coordinates).

[0216] 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, 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 that has at least 90% sequence identity, preferably at least 95%, still preferably at least 98%, with SEQ ID NO: 65.

[0217] In a preferred embodiment, the level or amount of methylation of the LRRC4 (SND1) gene is determined by using the primers of SEQ ID NO:25 and SEQ ID NO:26.

[0218] In a preferred embodiment, the level or amount of methylation of the LRRC4 (SND1) gene is determined by using the probe of SEQ ID NO:39.

[0219] In a more preferred embodiment, the level or amount of methylation of the LRRC4 (SND1) gene is determined by using the primers of SEQ ID NO:25 and SEQ ID NO:26 and the probe of SEQ ID NO:39. 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.

[0220] Methods

[0221] In a first aspect, the present invention therefore relates to an in vitro method for detecting or monitoring a colorectal cancer (CRC) in a subject, said method comprising determining in a biological sample of said subject the level or amount of methylation of at least one gene, from the group of genes that are described above.

[0222] In a particular embodiment, said method comprises determining the level or amount of methylation of at least one of ZSCAN23, COL4A1 or AQP5-AS1 gene in a biological sample from the subject, wherein the biological sample contains genomic DNA.

[0223] In a particular embodiment, said method comprises determining the level or amount of methylation of at least ZSCAN23 gene in a biological sample from the subject, wherein the biological sample contains genomic DNA.

[0224] In a particular embodiment, said method comprises determining the level or amount of methylation of at least COL4A1 gene in a biological sample from the subject, wherein the biological sample contains genomic DNA.

[0225] In a particular embodiment, said method comprises determining the level or amount of methylation of at least AQP5-AS1 gene in a biological sample from the subject, wherein the biological sample contains genomic DNA.

[0226] In a particular embodiment, said method comprises determining the level or amount of methylation of at least ZSCAN23 and AQP5-AS1 genes. In another particular embodiment, said method comprises determining the level or amount of methylation of at least ZSCAN23 and COL4Algenes. In another particular embodiment, said method comprises determining the level or amount of methylation of at least AQP5-AS1 and COL4Algenes. In a preferred embodiment, the method comprises determining the level of methylation of the combination of ZSCAN23, COL4A1 and AQP5-AS1 genes.

[0227] In particular, combinations of biomarkers according to the invention can be:

[0228] - ZSCAN23 gene and COL4A1 gene ;

[0229] - ZSCAN23 gene and AQP5-AS1 gene ;

[0230] COL4A1 gene and AQP5-AS1 gene ;

[0231] - ZSCAN23 gene and NR5A2 gene ;

[0232] - ZSCAN23, COL4A1 and AQP5-AS1 genes ;

[0233] - ZSCAN23, COL4A1 , AQP5-AS1 and NR5A2 genes.

[0234] In particular, combinations of biomarkers according to the invention can be:

[0235] - ZSCAN23 gene and COL4A1 gene ;

[0236] - ZSCAN23 gene and AQP5-AS1 gene ;

[0237] - ZSCAN23 gene and NR5A2 gene ;

[0238] - ZSCAN23, COL4A1 and AQP5-AS1 genes ;

[0239] - ZSCAN23, COL4A1, AQP5-AS1 and NR5A2 genes.

[0240] In a particular embodiment, the method comprises determining the level of methylation of ZSCAN23, and at least two, preferably three, preferably four, more preferably at least 5 genes selected in the group consisting of : C9orf50, AQP5-AS1, NR5A2, ADARB2, COL4A1, NPY, WIFI, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and LRRC4 (SND1) genes. In a preferred embodiment, the method comprises determining the level of methylation of ZSCAN23, and at least two, preferably three, preferably four, more preferably at least 5 genes selected in the group consisting of : AQP5-AS1, COL4A1, C9orf50, NR5A2, NPY and WIFI. In a preferred embodiment, the method comprises determining the level of methylation of ZSCAN23, and at least two, preferably three, preferably four genes selected in the group consisting of : AQP5-AS1, COL4A1, C9orf50, NR5A2 and WIFI.

[0241] In a particular embodiment, the method comprises determining the level of methylation of COL4A1, and at least two, preferably three, preferably four, more preferably at least 5 genes selected in the group consisting of : ZSCAN23, AQP5-AS1, NR5A2, ADARB2, C9orf50, NPY, WIFI, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and genes LRRC4 (SND1). In a preferred embodiment, the method comprises determining the level of methylation of COL4A1, and at least two, preferably three, preferably four, more preferably at least 5 genes selected in the group consisting of : AQP5-AS1, ZSCAN23, C9orf50, NR5A2, NPY and WIFI. In a preferred embodiment, the method comprises determining the level of methylation of COL4A1, and at least two, preferably three, preferably four genes selected in the group consisting of : AQP5-AS1, ZSCAN23, C9orf50, NR5A2 and WIFI.

[0242] In a particular embodiment, the method comprises determining the level of methylation of AQP5-AS1, and at least two, preferably three, preferably four, more preferably at least 5 genes selected in the group consisting of : C9orf50, ZSCAN23, NR5A2, ADARB2, COL4A1, NPY, WIFI, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2 and LRRC4 (SND1) genes. In a preferred embodiment, the method comprises determining the level of methylation of AQP5- AS1, and at least two, preferably three, preferably four, more preferably at least 5 genes selected in the group consisting of : COL4A1, ZSCAN23, C9orf50, NR5A2, NPY and WIFI. In a preferred embodiment, the method comprises determining the level of methylation of AQP5- AS1, and at least two, preferably three, preferably four genes selected in the group consisting of : COL4A1, ZSCAN23, C9orf50, NR5A2 and WIFI.

[0243] In a particular embodiment, the method comprises determining the level of methylation of :

[0244] - ZSCAN23 and COL4A1 genes ;

[0245] - ZSCAN23 and AQP5-AS1 genes ;

[0246] COL4Aland AQP5-AS1 genes ; or

[0247] - ZSCAN23, COL4Aland AQP5-AS1 genes, wherein the method further comprises detecting, or determining the level of methylation of at least one gene, preferably two genes, preferably three genes selected in the group consisting in: NR5A2, C9orf50, NPY or WIFI.

[0248] In a particular embodiment, the method comprises determining the level of methylation of :

[0249] - ZSCAN23 and COL4Algenes ;

[0250] - ZSCAN23 and AQP5-AS1 genes ;

[0251] COL4Aland AQP5-AS1 genes ; or

[0252] - ZSCAN23, COL4Aland AQP5-AS1 genes, wherein the method further comprises detecting, or determining the level of methylation of NR5A2 gene. In a particular embodiment, the method comprises determining the level of methylation of :

[0253] - ZSCAN23 and COL4Algenes ;

[0254] - ZSCAN23 and AQP5-AS1 genes ;

[0255] COL4A1 and AQP5-AS1 genes ; or

[0256] - ZSCAN23, COL4A1 and AQP5-AS1 genes, wherein the method further comprises detecting, or determining the level of methylation of C9orf50 gene.

[0257] In a particular embodiment, the method comprises determining the level of methylation of :

[0258] - ZSCAN23 and COL4Algenes ;

[0259] - ZSCAN23 and AQP5-AS1 genes ;

[0260] COL4A1 and AQP5-AS1 genes ; or

[0261] - ZSCAN23, COL4A1 and AQP5-AS1 genes, wherein the method further comprises detecting, or determining the level of methylation of WIFI gene.

[0262] In a particular embodiment, the method comprises determining the level of methylation of :

[0263] - ZSCAN23 and COL4Algenes ;

[0264] - ZSCAN23 and AQP5-AS1 genes ;

[0265] COL4A1 and AQP5-AS1 genes ; or

[0266] - ZSCAN23, COL4A1 and AQP5-AS1 genes, wherein the method further comprises detecting, or determining the level of methylation of NPY gene.

[0267] In a particular embodiment, the method comprises determining the level of methylation of :

[0268] - ZSCAN23 and COL4A1 genes ;

[0269] - ZSCAN23 and AQP5-AS1 genes ;

[0270] COL4A1 and AQP5-AS1 genes ; or

[0271] - ZSCAN23, COL4A1 and AQP5-AS1 genes, wherein the method further comprises detecting, or determining the level of methylation of at least two, preferably three, preferably four, more preferably at least 5 genes selected in the group consisting of : NR5A2, ADARB2, C9orf50, NPY, WIFI, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, and genes LRRC4 (SND1).

[0272] In a particular embodiment, the method comprises determining the level of methylation of : - ZSCAN23 and COL4A1 genes ;

[0273] - ZSCAN23 and AQP5-AS1 genes ;

[0274] COL4A1 and AQP5-AS1 genes ; or

[0275] - ZSCAN23, COL4A1 and AQP5-AS1 genes, wherein the method further comprises detecting, or determining the level of methylation of at least two, preferably three, preferably four genes selected in the group consisting of : NR5A2, C9orf50, NPY, and WIFI.

[0276] In particular, combinations of biomarkers according to the invention can be:

[0277] - ZSCAN23 gene and C9orf50 gene ;

[0278] - ZSCAN23 gene and AQP5-AS1 gene ;

[0279] C9orf50 gene and AQP5-AS1 gene ;

[0280] - ZSCAN23 gene and NR5A2 gene ;

[0281] - ZSCAN23, C9orf50 and AQP5-AS1 genes ;

[0282] - ZSCAN23, C9orf50, AQP5-AS1 and NR5A2 genes.

[0283] In particular, combinations of biomarkers according to the invention can be:

[0284] - ZSCAN23 gene and C9orf50 gene ;

[0285] - ZSCAN23 gene and AQP5-AS1 gene ;

[0286] - ZSCAN23 gene and NR5A2 gene ;

[0287] - ZSCAN23, C9orf50 and AQP5-AS1 genes ;

[0288] - ZSCAN23, C9orf50, AQP5-AS1 and NR5A2 genes.

[0289] In a particular embodiment, the method comprises determining the level of methylation of at least two genes, wherein said two genes are AQP5-AS1 and ZSCAN23 ; AQP5-AS1 and COL4A1 ; AQP5-AS1 and NR5A2 ; AQP5-AS1 and C9orf50 ; AQP5-AS1 and NPY ; AQP5- AS1 and WIFI ; ZSCAN23 and COL4A1 ; ZSCAN23 and NR5A2 ; ZSCAN23 and C9orf50 ; ZSCAN23 and NPY ; ZSCAN23 and WIFI ; COL4A1 and NR5A2 ; COL4A1 and C9orf50 ; COL4A1 and NPY ; or COL4A1 and WIFI genes.

[0290] In a preferred embodiment, the method comprises determining the level of methylation of at least two genes, wherein said two genes are AQP5-AS1 and ZSCAN23 ; AQP5-AS1 and COL4A1 ; AQP5-AS1 and NR5A2 ; AQP5-AS1 and C9orf50 ; AQP5-AS1 and NPY ; AQP5- AS1 and WIFI ; ZSCAN23 and COL4A1 ; ZSCAN23 and NR5A2 ; ZSCAN23 and C9orf50 ; ZSCAN23 and WIFI ; COL4A1 and NR5A2 ; COL4A1 and C9orf50 ; or COL4A1 and WIFI genes. In a preferred embodiment, the method comprises determining the level of methylation of at least two genes, wherein said two genes are AQP5-AS1 and C9orf50 ; AQP5-AS1 and NPY ; AQP5-AS1 and WIFI ; C9orf50 and ZSCAN23 ; COL4A1 and C9orf50 ; COL4A1 and NR5A2 ; COL4A1 and WIFI ; NR5A2 and AQP5-AS1 ; NR5A2 and ZSCAN23 ; or ZSCAN23 and WIFI.

[0291] In a particular embodiment, the method comprises determining the level of methylation of at least three genes, wherein said 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 WIFI ; AQP5-AS1, COL4A1 andNR5A2 ; AQP5-AS1, COL4A1 and C9orf50 ; AQP5-AS1, COL4A1 and NPY ; AQP5-AS1, COL4A1 and WIFI ; AQP5-AS1, NR5A2 and C9orf50 ; AQP5-AS1, NR5A2 and NPY ; AQP5-AS1, NR5A2 and WIFI ; AQP5-AS1, C9orf50 and NPY ; AQP5-AS1, C9orf50 and WIFI ; AQP5-AS1, NPY and WIFI ; ZSCAN23, COL4A1 and NR5A2 ; ZSCAN23, COL4A1 and C9orf50 ; ZSCAN23, COL4A1 and NPY ; ZSCAN23, COL4A1 and WIFI ; ZSCAN23, NR5A2 and C9orf50 ; ZSCAN23, NR5A2 and NPY ; ZSCAN23, NR5A2 and WIFI ; ZSCAN23, C9orf50 and NPY ; ZSCAN23, C9orf50 and WIFI ; ZSCAN23, NPY and WIFI ; COL4A1, NR5A2 and C9orf50 ; COL4A1, NR5A2 and NPY ; COL4A1, NR5A2 and WIFI ; COL4A1, C9orf50 and NPY ; COL4A1, C9orf50 and WIFI ; or COL4A1, NPY and WIFI.

[0292] In a preferred embodiment, the method comprises determining the level of methylation of at least three genes, wherein said three genes are AQP5-AS1, ZSCAN23 and COL4A1 ; AQP5- AS1, ZSCAN23 and C9orf50 ; AQP5-AS1, ZSCAN23 and NPY ; AQP5-AS1, ZSCAN23 and WIFI ; AQP5-AS1, COL4A1 and NR5A2 ; AQP5-AS1, COL4A1 and NPY ; AQP5-AS1, COL4A1 and WIFI ; AQP5-AS1, NR5A2 and C9orf50 ; AQP5-AS1, NR5A2 and NPY ; AQP5-AS1, NR5A2 and WIFI ; AQP5-AS1, C9orf50 and WIFI ; AQP5-AS1, NPY and WIFI ; ZSCAN23, COL4A1 and NR5A2 ; ZSCAN23, COL4A1 and C9orf50 ; ZSCAN23, COL4A1 and WIFI ; ZSCAN23, NR5A2 and C9orf50 ; ZSCAN23, NR5A2 and NPY ; ZSCAN23, NR5A2 and WIFI ; ZSCAN23, C9orf50 and WIFI ; COL4A1, NR5A2 and C9orf50 ; COL4A1, NR5A2 and WIFI ; or COL4A1, C9orf50 and WIFI. In a preferred embodiment, the method comprises determining the level of methylation of at least three genes, wherein said three genes are AQP5-AS1, C9orf50 and WIFI ; AQP5-AS1, C9orf50 and ZSCAN23 ; AQP5- AS1, NPY and WIFI ; AQP5-AS1, NPY and ZSCAN23 ; AQP5-AS1, ZSCAN23 and WIFI ; C9orf50, ZSCAN23 and WIFI ; COL4A1, AQP5-AS1 and NPY ; COL4A1, AQP5-AS1 and WIFI ; COL4A1, C9orf50 and WIFI ; COL4A1, C9orf50 and ZSCAN23 ; COL4A1, NR5A2 and AQP5-AS1 ; COL4A1, NR5A2 and C9orf50 ; COL4A1, NR5A2 and WIFI ; COL4A1, NR5A2 and ZSCAN23 ; NR5A2, AQP5-AS1 and C9orf50 ; NR5A2, AQP5-AS1 and NPY ; NR5A2, AQP5-AS1 and WIFI ; NR5A2, C9orf50 and ZSCAN23 ; NR5A2, NPY and ZSCAN23 ; or NR5A2, ZSCAN23 and WIFI.

[0293] In a particular embodiment, the method comprises determining the level of methylation of at least four genes, wherein said four genes are AQP5-AS1, ZSCAN23, COL4A1 and NR5A2 ; AQP5-AS1, ZSCAN23, COL4A1 and C9orf50 ; AQP5-AS1, ZSCAN23, COL4A1 and NPY ; AQP5-AS1, ZSCAN23, COL4A1 and WIFI ; AQP5-AS1, ZSCAN23, NR5A2 and C9orf50 ; AQP5-AS1, ZSCAN23, NR5A2 and NPY ; AQP5-AS1, ZSCAN23, NR5A2 and WIFI ; AQP5-AS1, ZSCAN23, C9orf50 and NPY ; AQP5-AS1, ZSCAN23, C9orf50 and WIFI ; AQP5-AS1, ZSCAN23, NPY and WIFI ; AQP5-AS1, COL4A1, NR5A2 and C9orf50 ; AQP5- AS1, COL4A1, NR5A2 and NPY ; AQP5-AS1, COL4A1, NR5A2 and WIFI ; AQP5-AS1, COL4A1, C9orf50 and NPY ; AQP5-AS1, COL4A1, C9orf50 and WIFI ; AQP5-AS1, COL4A1, NPY and WIFI ; AQP5-AS1, NR5A2, C9orf50 and NPY ; AQP5-AS1, NR5A2, C9orf50 and WIFI ; AQP5-AS1, NR5A2, NPY and WIFI ; AQP5-AS1, C9orf50, NPY and WIFI ; ZSCAN23, COL4A1, NR5A2 and C9orf50 ; ZSCAN23, COL4A1, NR5A2 and NPY ; ZSCAN23, COL4A1, NR5A2 and WIFI ; ZSCAN23, COL4A1, C9orf50 and NPY ; ZSCAN23, COL4A1, C9orf50 and WIFI ; ZSCAN23, COL4A1, NPY and WIFI ; ZSCAN23, NR5A2, C9orf50 and NPY ; ZSCAN23, NR5A2, C9orf50 and WIFI ; ZSCAN23, NR5A2, NPY and WIFI ; ZSCAN23, C9orf50, NPY and WIFI ; COL4A1, NR5A2, C9orf50 and NPY ; COL4A1, NR5A2, C9orf50 and WIFI ; COL4A1, NR5A2, NPY and WIFI ; or COL4A1, C9orf50, NPY and WIFI.

[0294] In a preferred embodiment, the method comprises determining the level of methylation of at least four genes, wherein said four genes are AQP5-AS1, ZSCAN23, COL4A1 and NR5A2 ; AQP5-AS1, ZSCAN23, COL4A1 and WIFI ; AQP5-AS1, ZSCAN23, NR5A2 and C9orf50 ; AQP5-AS1, ZSCAN23, NR5A2 and WIFI ; AQP5-AS1, ZSCAN23, C9orf50 and WIFI ; AQP5-AS1, ZSCAN23, NPY and WIFI ; AQP5-AS1, COL4A1, NR5A2 and NPY ; AQP5- AS1, COL4A1, NR5A2 and WIFI ; AQP5-AS1, COL4A1, C9orf50 and WIFI ; AQP5-AS1, COL4A1, NPY and WIFI ; AQP5-AS1, NR5A2, C9orf50 and WIFI ; AQP5-AS1, NR5A2, NPY and WIFI ; AQP5-AS1, C9orf50, NPY and WIFI ; ZSCAN23, COL4A1, NR5A2 and C9orf50 ; ZSCAN23, COL4A1, NR5A2 and WIFI ; ZSCAN23, COL4A1, C9orf50 and WIFI ; ZSCAN23, NR5A2, C9orf50 andNPY ; ZSCAN23, NR5A2, C9orf50 and WIFI ; ZSCAN23, NR5A2, NPY and WIFI ; ZSCAN23, C9orf50, NPY and WIFI ; or COL4A1, NR5A2, C9orf50 and WIFI. In a preferred embodiment, the method comprises determining the level of methylation of at least four genes, wherein said four genes are AQP5-AS1, C9orf50, NPY and WIFI ; AQP5-AS1, NPY, ZSCAN23 and WIFI ; COL4A1, AQP5-AS1, C9orf50 and WIFI ; COL4A1, AQP5-AS1, NPY and WIFI ; COL4A1, AQP5-AS1, ZSCAN23 and WIFI ; COL4A1, NR5A2, AQP5-AS1 and NPY ; COL4A1, NR5A2, AQP5-AS1 and WIFI ; COL4A1, NR5A2, AQP5-AS1 and ZSCAN23 ; COL4A1, NR5A2, C9orf50 and WIFI ; COL4A1, NR5A2, C9orf50 and ZSCAN23 ; NR5A2, AQP5-AS1, NPY and WIFI ; NR5A2, AQP5-AS1, ZSCAN23 and WIFI ; or NR5A2, C9orf50, NPY and ZSCAN23.

[0295] In a particular embodiment, the method comprises determining the level of methylation of at least five genes, wherein said five genes are AQP5-AS1, ZSCAN23, COL4A1, NR5A2 and C9orf50 ; AQP5-AS1, ZSCAN23, COL4A1, NR5A2 and NPY ; AQP5-AS1, ZSCAN23, COL4A1, NR5A2 and WIFI ; AQP5-AS1, ZSCAN23, COL4A1, C9orf50 and NPY ; AQP5- AS1, ZSCAN23, COL4A1, C9orf50 and WIFI ; AQP5-AS1, ZSCAN23, COL4A1, NPY and WIFI ; AQP5-AS1, ZSCAN23, NR5A2, C9orf50 and NPY ; AQP5-AS1, ZSCAN23, NR5A2, C9orf50 and WIFI ; AQP5-AS1, ZSCAN23, NR5A2, NPY and WIFI ; AQP5-AS1, ZSCAN23, C9orf50, NPY and WIFI ; AQP5-AS1, COL4A1, NR5A2, C9orf50 and NPY ; AQP5-AS1, COL4A1, NR5A2, C9orf50 and WIFI ; AQP5-AS1, COL4A1, NR5A2, NPY and WIFI ; AQP5-AS1, COL4A1, C9orf50, NPY and WIFI ; AQP5-AS1, NR5A2, C9orf50, NPY and WIFI ; ZSCAN23, COL4A1, NR5A2, C9orf50 and NPY ; ZSCAN23, COL4A1, NR5A2, C9orf50 and WIFI ; ZSCAN23, COL4A1, NR5A2, NPY and WIFI ; ZSCAN23, COL4A1, C9orf50, NPY and WIFI ; ZSCAN23, NR5A2, C9orf50, NPY and WIFI ; or COL4A1, NR5A2, C9orf50, NPY and WIFI.

[0296] In a preferred embodiment, the method comprises determining the level of methylation of at least five genes, wherein said five genes are AQP5-AS1, ZSCAN23, COL4A1, NR5A2 and WIFI ; AQP5-AS1, ZSCAN23, COL4A1, C9orf50 and WIFI ; AQP5-AS1, ZSCAN23, COL4A1, NPY and WIFI ; AQP5-AS1, ZSCAN23, NR5A2, C9orf50 and WIFI ; AQP5-AS1, ZSCAN23, NR5A2, NPY and WIFI ; AQP5-AS1, ZSCAN23, C9orf50, NPY and WIFI ; AQP5-AS1, COL4A1, NR5A2, C9orf50 and WIFI ; AQP5-AS1, COL4A1, NR5A2, NPY and WIFI ; AQP5-AS1, COL4A1, C9orf50, NPY and WIFI ; AQP5-AS1, NR5A2, C9orf50, NPY and WIFI ; ZSCAN23, COL4A1, NR5A2, C9orf50 and WIFI ; or ZSCAN23, NR5A2, C9orf50, NPY and WIFI. In a preferred embodiment, the method comprises determining the level of methylation of at least five genes, wherein said five genes are COL4A1, AQP5-AS1, C9orf50, NPY and WIFI ; COL4A1, AQP5-AS1, C9orf50, ZSCAN23 and WIFI ; COL4A1, AQP5-AS1, NPY, ZSCAN23 and WIFI ; COL4A1, NR5A2, AQP5-AS1, C9orf50 and WIFI ; COL4A1, NR5A2, AQP5-AS1, NPY and WIFI ; or COL4A1, NR5A2, AQP5-AS1, ZSCAN23 and WIFI.

[0297] In a particular embodiment, the method comprises determining the level of methylation of at least six genes, wherein said six genes are AQP5-AS1, ZSCAN23, COL4A1, NR5A2, C9orf50 and NPY ; AQP5-AS1, ZSCAN23, COL4A1, NR5A2, C9orf50 and WIFI ; AQP5-AS1, ZSCAN23, COL4A1, NR5A2, NPY and WIFI ; AQP5-AS1, ZSCAN23, COL4A1, C9orf50, NPY and WIFI ; AQP5-AS1, ZSCAN23, NR5A2, C9orf50, NPY and WIFI ; AQP5-AS1, COL4A1, NR5A2, C9orf50, NPY and WIFI ; or ZSCAN23, COL4A1, NR5A2, C9orf50, NPY, WIFI.

[0298] In a preferred embodiment, the method comprises determining the level of methylation of at least six genes, wherein said six genes are AQP5-AS1, ZSCAN23, COL4A1, NR5A2, C9orf50 and WIFI ; AQP5-AS1, ZSCAN23, COL4A1, NR5A2, NPY and WIFI ; AQP5-AS1, ZSCAN23, COL4A1, C9orf50, NPY and WIFI ; AQP5-AS1, ZSCAN23, NR5A2, C9orf50, NPY and WIFI ; or AQP5-AS1, COL4A1, NR5A2, C9orf50, NPY and WIFI. In a preferred embodiment, the method comprises determining the level of methylation of at least six genes, wherein said six genes are COL4A1, AQP5-AS1, C9orf50, NPY, ZSCAN23 and WIFI ; or COL4A1, NR5A2, AQP5-AS1, C9orf50, NPY and WIFI.

[0299] In a particular embodiment, the method comprises determining the level of methylation of at least seven genes, wherein said seven genes are AQP5-AS1, ZSCAN23, COL4A1, NR5A2, C9orf50, NPY and WIFI genes.

[0300] In a particular embodiment, the method comprises : a) determining the level or amount of methylation of at least one biomarker or a combination of biomarkers, in a biological sample of said subject ; and b) comparing the level or amount of methylation determined in step a) to reference value(s) ; wherein if said biomarker(s) are hypermethylated as compared with reference value(s), then said subject is diagnosed or identified as suffering from a colorectal cancer.

[0301] In the context of the invention, the reference value is obtained in a “reference sample” from a healthy subject or in a “reference sample” from a subject suffering from colorectal cancer previous to a treatment or performed in an earlier time of the treatment.

[0302] An "hypermethylation" is determined for example if the methylation value (amount) or status (level) of one of the biomarkers of the invention is significantly higher in the biological sample of the tested subject as compared with the methylation value (amount) or status (level) of the corresponding biomarker measured in a reference sample. A significantly higher amount or level of methylation of at least one of the biomarkers of the invention in the biological sample of a subject as compared with the normal amount or level of methylation in the reference sample is an indication that the tested subject has a colorectal cancer, has a high risk to have colorectal cancer or does not respond to a treatment.

[0303] A "significantly higher amount or level of methylation" refers to a methylation amount or level that is greater than the sum of the average and standard error of the assay employed to assess said amount or level.

[0304] The present invention also relates to a method for monitoring the progress of colorectal cancer in a subject that has been diagnosed for colorectal cancer, said method comprising: a) determining the level or amount of methylation of at least one biomarker or a combination of biomarkers, in a biological sample of said subject, at a first time point, b) determining the level or amount of methylation of said biomarker(s) selected previously in the step a), in a biological sample of said subject, at a second time point, and c) comparing the level or amount of methylation determined in step a) to the level or amount determined in step b). It can be concluded that the malignancy of the colorectal cancer is worsening if the level or amount of methylation determined in step b) is significantly higher than the level or amount determined in step a). In other words, the tested subject has a disease that evolves badly, even though he / she may be treated already.

[0305] In another aspect, the biomarkers of the invention can be used to predict the outcome of colorectal cancer patients. The present invention also relates to a method for predicting the clinical outcome of a subject afflicted with colorectal cancer, said method comprising: 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 said subject, and comparing same to a reference value, b) predicting the clinical outcome based on the comparison of step a).

[0306] If at least one gene or a combination of these genes is / are significantly hypermethylated in the biological sample of the tested subject as compared to the same biomarker(s) in the reference sample, then the tested subject is likely to have a bad clinical outcome (short survival, metastasis, etc.).

[0307] The present invention also relates to a method for adapting the therapeutic regimen for a subject suffering from colorectal cancer, said method comprising: a) determining the level or amount of methylation of at least one biomarker or of a combination of biomarkers as described above, in a biological sample of said subject, b) comparing same to a level or amount of methylation of the same biomarker(s) as selected in step a), in a biological sample of said subject after surgery or after a treatment has been administrated to the subject, and c) adapting / modifying the therapeutic regimen of said subject based either on a) only or on the comparison of step a) and b).

[0308] In particular, said surgery or therapeutic regimen is acknowledged to be efficient if the level or amount of said biomarker(s) is significantly inferior or equal to the level or amount of said biomarker(s) determined before said treatment. By contrast, said therapeutic regimen should be changed if the level or amount of said biomarker(s) has increased under treatment as compared with the level or amount of said biomarker(s) determined before said treatment (or determined in an earlier time of treatment). This aspect of treatment strategy is a crucial goal in a context of personalized medicine in order to improve survival while maintaining the quality of life and avoiding needless toxic effects of an ineffective treatment.

[0309] It is further disclosed a method for identifying or screening a therapeutic agent or candidate therapeutic agent, for use in treating a colorectal cancer in a subject, which method comprises administering the subject with said therapeutic agent and detecting, or determining the level of, methylation of the biomarkers of the invention, in a biological sample from the subject, wherein the biological sample contains genomic DNA, wherein a decreased of methylation after administration of the therapeutic agent is indicative of an agent that has a benefit on the colorectal cancer in the subject.

[0310] Further disclosed is a method for treating a subject that has been diagnosed with a colorectal cancer using the method of diagnosis of the invention, wherein the subject is then administered with an effective amount of a therapeutic agent, including chemotherapy and / or immunotherapy, or is subjected to surgery and / or radiation.

[0311] The chemotherapeutic drug is typically an agent selected for example from an alkylating agent, an antimetabolite, a topoisomerase inhibitor, a platin based component, a specific kinase inhibitor, a hormone, a cytokine, an antiangiogenic agent, an antibody, an immunotherapy or a vascular disrupting agent.

[0312] Further disclosed is a method for an early diagnosis in a subject who is at risk of developing a colorectal cancer.

[0313] Further disclosed is a method for an early diagnosis of a cancerous lesion in the colon in a subject who has been developing metastasis from a primary tumor of unknown origin.

[0314] Further disclosed is a method for assessing the risk of relapse.

[0315] Kit

[0316] Another aspect of the invention relates to diagnostic kits and tools for determining the hypermethylation biomarkers of the invention in order to diagnose colorectal cancer.

[0317] Kits are also provided which comprise probes and / or primers, and optionally additional reagents, useful for determining the methylation status at the genomic sites described above.

[0318] As used herein, the term "primers" designates isolated nucleic acid molecules that can specifically hybridize or anneal to 5' or 3' regions of a target genomic region (plus and minus strands, respectively, or vice-versa). In general, they are from about 10 to 30 nucleotides in length and anneal at both extremities of a region containing about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. As they have to be used by pairs, they are often referred to as "primers pair" or "primers set" (cf. the pairs SEQ ID NO: 1-2; SEQ ID NO:3-4, SEQ ID NO:5-6; SEQ ID NO:7-8; SEQ ID NO:9-10; SEQ ID NO: 11-12, SEQ ID NO: 13-14; SEQ ID NO:15-16; SEQ ID NO: 17-18; SEQ ID NO: 19-20; SEQ ID NO:21-22; SEQ ID NO:23-24; SEQ ID NO:25-26).

[0319] As used herein, the term "probes" designates molecules that are capable of specifically hybridizing a genomic region of interest (e.g., of SEQ ID NO:40 to SEQ ID NO:52). They are useful to highlight the presence of said genomic region in biological samples. These probes may comprise at least one non-natural nucleotide, e.g., a peptide nucleic acid (PNA), a peptide nucleic acid having a phosphate group (PHONA), a bridged nucleic acid or locked nucleic acid (BNA or LNA), and a morpholino nucleic acid. Non-natural nucleotides also 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'-0-methyl-type DNA or RNA.

[0320] In a preferred embodiment, the probes of the invention comprise at least 15, consecutive nucleotides which are complementary of bisulfited SEQ ID NO:27 - SEQ ID NO:39 or fragments thereof. In a more preferred embodiment, the molecules which can be used as a probe according to the present invention have a total minimum size of 15 nucleotides. In an even more preferred embodiment, these molecules comprise between 15 and 30 nucleotides (in total).

[0321] For certain uses, the probes and primers of the invention may be labeled - directly or indirectly - with a detectable label. Said label may be of any kind, depending on the experiment which is to be performed. Said label may be a radioactive isotope (such as32 P,33 P,35 S,3 H orl25 1, or a nonradioactive entity which is selected from ligands (such as biotin, avidin or streptavidin), dioxygenin, haptens, colorants and luminescent agents (such as radioluminescent, chemiluminescent, bioluminescent, fluorescent or phosphorescent agents). Preferably, 6- carboxyfluorescein (FAM), VIC, HEX and tetramethylrhodamine (TAM RA) are used. Nonlabeled polynucleotide sequences may also be used, directly, as a probe or primer, for example in PCR- based processes (e.g., in quantitative PCR).

[0322] "Specific hybridization" is observed when a define molecule does not hybridize with any other genomic region than its target genomic region. Preferably, it hybridizes with its target region in high stringency conditions, i.e., when the temperature and ionic strength conditions are chosen so as to allow the hybridization between two complementary DNA fragments. By way of illustration, high stringency conditions can be as follows. The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42°C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5*SSC (1*SSC corresponds to a 0.15 M NaCI+0.015 M sodium citrate solution), 50% of formamide, 7% of sodium dodecyl sulfate (SDS), 10*Denhardt's, 5% of dextran sulfate and 1% of salmon sperm DNA; (2) actual hybridization for 20 hours at a temperature dependent on the size of the probe (i.e. 42°C. for a probe of size>100 nucleotides), followed by two 20-minute washes at 20°C. in 2*SSC+2% SDS and one 20-minute wash at 20°C. in 0.1*SSC+0.1% SDS. The final wash is carried out in 0.1*SSC+0.1% SDS for 30 minutes at 60°C for a probe of size>100 nucleotides. The high stringency hybridization conditions described above for a polynucleotide of defined size will be adjusted by those skilled in the art for oligonucleotides of greater or smaller size.

[0323] As used herein, the term "kit" refers to any system for delivering materials. In the context of the invention, it includes systems that allow the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and / or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. The present kit can also include one or more reagents, buffers, hybridization media, nucleic acids, primers, nucleotides, probes, molecular weight markers, enzymes, solid supports, databases, computer programs for calculating dispensation orders and / or disposable lab equipment, such as multi-well plates, in order to readily facilitate implementation of the present methods. Enzymes that can be included in the present kits include nucleotide polymerases and the like. Solid supports can include beads and the like whereas molecular weight markers can include conjugatable markers, for example biotin and streptavidin or the like.

[0324] The kit of the invention more preferably contains primers and / or probes targeting specifically the nucleotide region of :

[0325] - SEQ ID NO:40 in the ZSCAN23 gene,

[0326] - SEQ ID NO:41 in the C9orf50 gene,

[0327] - SEQ ID NO:42 in the AQP5-AS1 gene,

[0328] - SEQ ID NO:43 in the NR5A2 gene,

[0329] - SEQ ID NO:44 in the ADARB2 gene,

[0330] - SEQ ID NO:45 in the COL4A1 gene, - SEQ ID NO:46 in the NPY gene,

[0331] - SEQ ID NO:47 in the WIFI gene,

[0332] - SEQ ID NO:48 in the CPNE8 gene,

[0333] - SEQ ID NO:49 in the LINC00693 (RBMS3) gene,

[0334] - SEQ ID NO:50 in the LINC00900 gene,

[0335] - SEQ ID NO:51 in the DYDC2 gene, and / or

[0336] - SEQ ID NO: 52 in the LRRC4 (SND1) gene.

[0337] In a preferred embodiment, the kit of the invention contains primers and / or probes specifically targeting at least the nucleotide region of :

[0338] - SEQ ID NO:40 in the ZSCAN23 gene,

[0339] - SEQ ID NO:45 in the COL4A1 gene, or

[0340] - SEQ ID NO:42 in the AQP5-AS1 gene.

[0341] In a preferred embodiment, the kit of the invention contains primers and probes specifically targeting the nucleotide region of :

[0342] - SEQ ID NO:40 in the ZSCAN23 gene, and

[0343] - SEQ ID NO:42 in the AQP5-AS1 gene.

[0344] In a preferred embodiment, the kit of the invention contains primers and probes specifically targeting the nucleotide region of :

[0345] - SEQ ID NO:45 in the COL4A1 gene, and

[0346] - SEQ ID NO:42 in the AQP5-AS1 gene.

[0347] In a preferred embodiment, the kit of the invention contains primers and probes specifically targeting the nucleotide region of :

[0348] - SEQ ID NO:40 in the ZSCAN23 gene, and

[0349] - SEQ ID NO:45 in the COL4A1 gene.

[0350] In a preferred embodiment, the kit of the invention contains primers and probes specifically targeting the nucleotide region of :

[0351] - SEQ ID NO:40 in the ZSCAN23 gene,

[0352] - SEQ ID NO:45 in the COL4A1 gene, and

[0353] - SEQ ID NO:42 in the AQP5-AS1 gene. In a preferred embodiment, the kit of the invention contains :

[0354] - primers of SEQ ID NO: 1 and SEQ ID NO:2 for determining the level or amount of methylation in the ZSCAN23 gene,

[0355] - primers of SEQ ID NO:3 and SEQ ID NO:4 for determining the level or amount of methylation in the C9orf50 gene,

[0356] - primers of SEQ ID NO: 5 and SEQ ID NO: 5 for determining the level or amount of methylation in the AQP5-AS1 gene,

[0357] - primers of SEQ ID NO: 7 and SEQ ID NO: 8 for determining the level or amount of methylation in the NR5 A2 gene,

[0358] - primers of SEQ ID NO:9 and SEQ ID NO: 10 for determining the level or amount of methylation in the ADARB2 gene,

[0359] - primers of SEQ ID NO: 11 and SEQ ID NO: 12 for determining the level or amount of methylation in the C0L4A1 gene,

[0360] - primers of SEQ ID NO: 13 and SEQ ID NO: 14 for determining the level or amount of methylation in the NPY gene,

[0361] - primers of SEQ ID NO: 15 and SEQ ID NO: 16 for determining the level or amount of methylation in the WIFI gene,

[0362] - primers of SEQ ID NO: 17 and SEQ ID NO: 18 for determining the level or amount of methylation in the CPNE8 gene.

[0363] - primers of SEQ ID NO: 19 and SEQ ID NO:20 for determining the level or amount of methylation in the LINC00693 (RBMS3) gene,

[0364] - primers of SEQ ID NO:21 and SEQ ID NO:22 for determining the level or amount of methylation in the LINC00900 gene,

[0365] - primers of SEQ ID NO:23 and SEQ ID NO:24 for determining the level or amount of methylation in the DYDC2 gene, and / or

[0366] - primers of SEQ ID NO:25 and SEQ ID NO:26 for determining the level or amount of methylation in the LRRC4 (SND1) gene.

[0367] In a preferred embodiment, the kit of the invention contains :

[0368] - the probe of SEQ ID NO:27 for determining the level or amount of methylation in the ZSCAN23 gene,

[0369] - the probe of SEQ ID NO:28 for determining the level or amount of methylation in the C9orf50 gene, - the probe of SEQ ID NO:29 for determining the level or amount of methylation in the AQP5-

[0370] AS1 gene,

[0371] - the probe of SEQ ID NO:30 for determining the level or amount of methylation in the NR5A2 gene,

[0372] - the probe of SEQ ID NO:31 for determining the level or amount of methylation in the AD ARB 2 gene,

[0373] - the probe of SEQ ID NO:32 for determining the level or amount of methylation in the COL4A1 gene,

[0374] - the probe of SEQ ID NO:33 for determining the level or amount of methylation in the NPY gene,

[0375] - the probe of SEQ ID NO:34 for determining the level or amount of methylation in the WIFI gene,

[0376] - the probe of SEQ ID NO:35 for determining the level or amount of methylation in the CPNE8 gene,

[0377] - the probe of SEQ ID NO:36 for determining the level or amount of methylation in the LINC00693 (RBMS3) gene,

[0378] - the probe of SEQ ID NO:37 for determining the level or amount of methylation in the LINC00900 gene,

[0379] - the probe of SEQ ID NO:38 for determining the level or amount of methylation in the DYDC2 gene, and / or

[0380] - the probe of SEQ ID NO:39 for determining the level or amount of methylation in the LRRC4 (SND1) gene.

[0381] The present invention also relates to a microarray carrying nucleotides targeting specifically the nucleotide region of :

[0382] - SEQ ID NO:40 in the ZSCAN23 gene,

[0383] - SEQ ID NO:41 in the C9orf50 gene,

[0384] - SEQ ID NO:42 in the AQP5-AS1 gene,

[0385] - SEQ ID NO:43 in the NR5A2 gene,

[0386] - SEQ ID NO:44 in the ADARB2 gene,

[0387] - SEQ ID NO:45 in the COL4A1 gene,

[0388] - SEQ ID NO:46 in the NPY gene,

[0389] - SEQ ID NO:47 in the WIFI gene,

[0390] - SEQ ID NO:48 in the CPNE8 gene. - SEQ ID NO:49 in the LINC00693 (RBMS3) gene,

[0391] - SEQ ID NO:50 in the LINC00900 gene,

[0392] - SEQ ID NO:51 in the DYDC2 gene, and / or

[0393] - SEQ ID NO: 52 in the LRRC4 (SND1) gene.

[0394] In a preferred embodiment, said microarray contains any primers of SEQ ID NO: 1 to 26 and / or probes of SEQ ID NO:27 to 39.

[0395] According to the invention, a "nucleic microarray" consists of different nucleic acid probes that are attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica- based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes can be nucleic acids such as cDNAs ("cDNA microarray") or oligonucleotides ("oligonucleotide microarray"), and the oligonucleotides may be about 10 to about 40 base pairs or less in length. Typically, the exemplary primers and probes mentioned above can be attached to said substrate. In a preferred embodiment, the nucleic acid microarray of the invention is an oligonucleotide microarray carrying oligonucleotides that can specifically hybridize with one, two, or three of the methylation regions of SEQ ID NO:40 to 52.

[0396] To determine if the biomarker of the invention is methylated or not, the tested sample is labelled, contacted with the nucleic acid microarray of the invention in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The presence of labelled hybridized complexes on the nucleic acid microarray is then determined. Many variants of the microarray hybridization technology are available to the man skilled in the art.

[0397] In particular, any available software developed for the design of microarray oligonucleotides may be used, such as, for instance, the OligoArray software (available at http: / / berry.engin.umich.edu / oligoarray / ), the GoArrays software (available at http: / / www.isima.fr / bioinfo / goarrays / ), the Array Designer software (available at http: / / www.premierbiosoft.com / dnamicroarray / index.html), the Primer3 software (available at http: / / frodo.wi.mit.edu / primer3 / primer3_code.html), or the Promide software (available at http: / / oligos.molgen.mpg.de / ), MethPrimer (http: / / www.urogene.org / cgi- bin / methprimer / methprimer.cgi) . The present invention also relates to the use of the kits described above, or of the microarrays described above, or of the primers described above, or of the probes described above, for:

[0398] • diagnosing or identifying colorectal cancer in a subject,

[0399] • predicting the clinical outcome in a subject afflicted with colorectal cancer,

[0400] • determining the therapeutic regimen of a subject with colorectal cancer, and / or

[0401] • monitoring the progress of colorectal cancer in a subject being diagnosed for colorectal cancer, in a biological sample of a subject, as proposed in the methods of the invention.

[0402] As used herein, the terms "in vitro” and "ex vivo " are equivalent and refer to studies or experiments that are conducted using biological components (e.g., cells or population of cells) that have been isolated from their usual host organisms (e.g., animals or humans).

[0403] The Examples and Figures illustrate the invention without limiting its scope.

[0404] EXAMPLES

[0405] EXAMPLE 1

[0406] MATERIALS AND METHODS

[0407] Patients and healthy control individuals

[0408] Matched tumor and adjacent non-tumor tissue biopsies from 26 CRC patients (stage I to IV) were included in this study. Plasma samples from healthy individuals (n = 10 to 20 for each assays depending on samples availability) and plasma from advanced CRC patients (stage IV, n = 17) were collected in EDTA or STRECK tubes. This study was approved by the local ethics committee and informed written consent was obtained from all the patients.

[0409] Tumor sample preparation, storage, DNA extraction, and quantification

[0410] Tumor and adjacent non-tumor tissue biopsies were flash frozen in liquid nitrogen immediately after resection until further analysis. Each tumor was reviewed by a pathologist and the tumor cell content was assessed by hematoxylin-eosin-safran staining. DNA was extracted with the QIAampDNAMini Kit (Qiagen) according to the manufacturer’s instructions. DNA concentration was measured by Qubit 2.0 fluorometer (Invitrogen, Life Technologies) with the use of the dsDNA BR Assay (Invitrogen). Extracted DNA samples were stored at -20 °C before testing.

[0411] Plasma sample preparation, storage, DNA extraction, and quantification

[0412] Plasma samples of healthy individuals were received in dry ice, aliquoted and immediately frozen at -80 °C. Before extraction, plasma samples were centrifuged at 3000g for 10 min and then extracted with the use of the QIAmp Circulating Nucleic Acid Kit (Qiagen) or the ccfDNA Plasma kit (Promega) by RSC Maxwell instrument according to the manufacturer’s instructions. Plasma form CRC patients were extracted with the use of the ccfDNA Plasma kit (Promega) by RSC Maxwell instrument or the QIAmp Circulating Nucleic Acid Kit (Qiagen). The quantity of DNA was measured by Qubit 2.0 fluorometer (Invitrogen, Life Technologies) with the use of the dsDNA HS Assay (Invitrogen). Extracted DNA samples were stored at - 20°C before testing.

[0413] Buffy coat preparation, DNA extraction, and quantification

[0414] Buffy coat was prepared from whole blood of patients that do not suffer from CRC or from healthy individuals and then extracted with the use of the QIAmp Circulating Nucleic Acid Kit (Qiagen) according to the manufacturer’s instructions. The quantity of DNA was measured by Qubit 2.0 fluorometer (Invitrogen, Life Technologies) with the use of the dsDNA BR Assay (Invitrogen). Extracted DNA samples were stored at -20 °C before testing.

[0415] Analysis and identification of DNA methylation biomarkers

[0416] Following methyl-seq and TCGA analysis, R scripts were performed to finally choose 13 candidates genes : ZSCAN23, C9orf50 (NTMT1), AQP5-AS1, NR5A2, ADARB2, COL4A1, NPY, WIFI, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2, LRRC4 (SND1).

[0417] Tissue DNA, buffy coat DNA and Plasma ccfDNA bisulfite conversion

[0418] Tissue DNA, buffy coat DNA and plasma ccfDNA were modified by bisulfite using the EZ DNA Methylation-Gold Kit (Zymo Research). In brief, bisulfite reaction was carried out at 98°C for 12 min and 64°C for 2 h35 min. The cleanup of bisulfite-converted DNA followed the recommendations of the manufacturer and converted DNA was eluted in M-Elution Buffer and stored at -20 °C.

[0419] Detection of methylation changes of selected biomarkers by droplet-based digital PCR

[0420] The hypermethylation of selected biomarkers in tumor DNA was evaluated by ddPCR. Duplex format was used to analyze hypermethylation with albumin for normalizing the DNA amount. Primers and probes were as listed in Tables 1 and 2.

[0421] DNA methylation of targeted sequences was analyzed by ddPCR using the QX-200 platform (BIO-RAD Technologies). In brief, the mixture of PCR reagents (BIO-RAD Technologies) was prepared following manufacturer’s recommendations.

[0422] A triplex panel was developed to detect the ZSCAN23 and C9orf50 methylated target sequences as well as the unmethylated Albumin sequence as a reference gene. A triplex panel was developed to detect the AQP5-AS1 and NR5A2 methylated target sequences as well as the unmethylated Albumin sequence as a reference gene. A triplex panel is performed, as described in [Tai eb J, 2021], to detect the WIFI and NPY methylated target sequences as well as the unmethylated Albumin sequence as a reference gene.

[0423] A duplex assay detecting, ADARB2 ; COL4A1 ; WIFI ; NPY ; CPNE8 ; LINC00693 (RBMS3) ; LINC00900 ; DYDC2 ; LRRC4 (SND1), methylated target sequence as well as the unmethylated Albumin sequence as a reference gene was also developed.

[0424] The PCR step for ddPCR was performed on a BIO-RAD Cl 000 or SI 000 using the following program: 10 min at 95°C (using a 2.5°C / second ramp rate), followed by 45 cycles of: 94°C, 30s and 58.4°C, 1 min (using a 2.5°C / second ramp rate), with an ultimate step of 10 min at 98°C. After completion, the emulsions were either stored at 4°C or processed immediately to measure the end-point fluorescence signal from each droplet. Data was analyzed using the Quantasoft BIO-RAD software as described by the manufacturer. Populations were clustered according to fluorescence levels, allowing to precisely count both tumor and normal amplifiable DNA molecules. For the different assays, limit of blank (LOB) were calculated as described previously [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): p. 1722-31] using commercial DNA extracted from whole Blood (Promega) for both duplex and triplex developed assays. It is defined by the frequency of positive droplets measured in normal control DNA samples with no hyper-methylated DNA present (n= 13 to 22 for each assay). The calculated LOB was subtracted from each sample for calculating their methylation level.

[0425] The sample analysis was performed following the procedure described earlier [Taly, V., et al. 2013], Samples were considered positive when the number of observed droplets was higher than LOB value. The methylation level of each sample was calculated as the ratio of the number of droplets containing methylated sequences over the number of droplets containing albumin sequences.

[0426] Two DNA controls were used for ensuring the proper realization of the modification treatment (Positive control: universal hypermethylated DNA and negative control: normal human genomic DNA).

[0427] Measurement of the methylation level of selected biomarkers in plasma circulating cell free DNA (ccfDNA) from healthy individuals and CRC patients

[0428] DNA methylation of selected biomarkers in plasma from healthy individuals or CRC patients was measured by ddPCR with the use of same reaction conditions as described before. Duplex or triplex format was used to analyze hypermethylation with albumin for normalizing the DNA amount. The same primers and probes were used as listed in the Table 1 and 2 above.

[0429] Calculation of detection sensitivity and specificity

[0430] The sum of the average and standard deviation of methylation level in non-tumor tissue DNA was used as the threshold for calculating sensitivity and specificity of each selected biomarker. Sensitivity is the percentage of the patients showing higher methylation level in tumor tissues than the threshold. Specificity is the percentage of the patients showing lower methylation level in non-tumor tissues than the threshold.

[0431] Statistical analysis Statistical analyses were performed using Prism Software (GraphPad Software Inc.) and R software 3.6.3 Studio. A P value < 0.05 was considered as significant. For the analysis of the hypermethylation difference between normal and adjacent tissues, paired non-parametric Wilcoxon test was used. For the analysis of the hypermethylation difference between healthy and CRC plasma ccfDNA, Mann-whitney test was used.

[0432] RESULTS

[0433] Hypermethylation of the selected biomarkers in DNA from CRC patients by ddPCR

[0434] As shown in Figure 1, methylation level of the selected biomarkers (ZSCAN23, C9orf50, AQP5-AS1, NR5A2, ADARB2, COL4A1, WIFI, NPY, CPNE8, LINC00693 (RBMS3), LINC00900, DYDC2 and LRRC4 (SND1)) was significantly increased in CRC tumor tissue DNA, compared with those in non-tumor tissue DNA.

[0435] These biomarkers show high specificity and sensitivity when assessed alone by ddPCR on tumor tissues compared to healthy tissues of CRC patients, as reflected in Table 3 below:

[0436] Table 3: Sensitivity and specificity of the biomarkers when assessed alone by ddPCR on tumor tissues and adjacent healthy tissues of CRC patients. Data presented are based on methylation level (%).

[0437] Figure 2 shows that the methylation level of the biomarkers ZSCAN23, C9orf50, AQP5-AS1, NR5A2, ADARB2, COL4A1, WIFI, NPY was significantly increased in plasma of CRC patients (ccfDNA), compared to the methylation level of the same biomarkers in plasma of healthy individuals. Combination of different biomarkers to reach a higher sensitivity and specificity

[0438] To reach a higher detection sensitivity and specificity, different biomarkers can be combined. As shown in Table 4 below, the detection sensitivity and specificity by ddPCR can for example be improved by combining ZSCAN23 with C9orf50, AQP5-AS1, and / or NR5A2.

[0439] Table 4: Detection sensitivity and specificity of different combination of selected biomarkers when assessed by ddPCR. Sensitivity and specificity are evaluated based in ddPCR analysis of tumor tissues and adjacent healthy tissues of CRC patients

[0440] Methylation profiles of the selected biomarkers in plasma ccfDNA and buffy coat fraction from healthy individuals and CRC patients by ddPCR

[0441] The purpose of these potential biomarkers is to detect DNA hypermethylation in CRC patients but not in healthy individuals. In order to validate this, the methylation level in plasma ccfDNA from healthy individuals was investigated by ddPCR. No significant level of methylation was observed in healthy plasma ccfDNA for these potential biomarkers (see Table 5 below). Moreover, DNA extracted from Buffy Coat (from healthy individuals) was also tested with the markers and almost no methylation of the tested markers was observed in these samples (Table 6). The number of the samples ran for each biomarker depended on the availability of DNA. As mentioned above for LOB calculation, commercial DNA extracted from whole Blood (Promega) was also used to validate the assay. Added to buffy coat DNA, these controls were performed to ensure that the markers were not positive in cells contained in the blood ensuring no false positive in case of blood cell hemolysis (for example due to pre-analytical sample handling).

[0442] However, when tested on DNA extracted from plasma of healthy subjects and CRC patients, methylation was observed only for colon patients both using duplex and triplex developed assays leading to a potential specificity of 100% for the detection of CRC in blood samples.

[0443] Table 5: Methylation level of the selected biomarkers in healthy plasma circulating cell free (ccfDNA) measured by ddPCR (n=10 to 20 depending on the availability).

[0444] Table 6: Methylation level of the selected biomarkers in healthy buffy coat DNA measured by ddPCR.

[0445] EXAMPLE 2

[0446] MATERIALS AND METHODS

[0447] Healthy control individuals and Patients Plasma samples from 25 healthy individuals were purchased from the society BIOPREDIC International. Blood was collected on EDTA tubes and centrifuged within 4 hours of collection according to the supplier's procedure. Plasma samples were received in dry ice, aliquoted and immediately frozen at -80 °C. Blood samples from 54 healthy individuals were purchased from EFS (Etablissement Frangais du Sang) on STRECK tubes. Plasma samples were separated from the cellular fraction, within 10 days of collection, by centrifugation at 1900g for 15 min followed by a second centrifugation at 6000g for 10 min and frozen at -80 °C.

[0448] Advanced CRC patients (stage IV, n = 73) were collected in EDTA tubes and centrifuged at 3000g and frozen at -80 °C. These samples were analyzed retrospectively from a prospective study approved by the local ethics committees and informed written consent was obtained from all the patients.

[0449] DNA extraction and quantification

[0450] Before extraction, plasma samples were centrifuged at 3000g for 15 min and then extracted with the use of the QIAmp Circulating Nucleic Acid Kit (Qiagen). The quantity of DNA was measured by Qubit 2.0 fluorometer (Invitrogen, Life Technologies) with the use of the dsDNA HS Assay (Invitrogen). Extracted DNA samples were stored at -20°C before testing.

[0451] Plasma ccfDNA bisulfite conversion

[0452] Plasma ccfDNA was modified by bisulfite treatment using the EZ DNA Methylation-Lightning Kit (Zymo Research). Bisulfite reaction was carried out at 98°C for 8 min and 54°C for IhOO. The cleanup of bisulfite-converted DNA followed the recommendations of the manufacturer and converted DNA was eluted in M-Elution Buffer and stored at -20 °C.

[0453] Two DNA controls were used for controlling the proper realization of the modification treatment (positive control: universal hypermethylated DNA and negative control: normal human genomic DNA).

[0454] Detection of methylation changes of selected biomarkers by droplet-based digital PCR

[0455] The hypermethylation of seven biomarkers (ZSCAN23, C9orf50 (NTMT1), AQP5-AS1, NR5A2, COL4A1, NPY and WIFI) in circulating tumor DNA was evaluated by ddPCR. A multiplex panel was used to analyze hypermethylation using a non-methylated sequence within the Albumin gene as a reference and for normalizing the DNA amount. DNA methylation of targeted sequences was analyzed by ddPCR using the 6-color naicat platform (Stilla Technologies). The mixture of PCR reagents (Stilla Technologies) was prepared following manufacturer’s recommendations.

[0456] A multiplex panel was developed to detect the AQP5-AS1, ZSCAN23, COL4A1, WIFI, NPY, NR5A2 and C9orf50 methylated target sequences as well as the unmethylated Albumin sequence as a reference gene. The PCR steps for ddPCR were performed following manufacturer’s recommendations. Data was analyzed using the Crystal Miner software as described by the manufacturer. Populations were clustered according to fluorescence levels, allowing to precisely count amplifiable DNA molecules corresponding to both methylated target sequences and unmethylated control sequence (ALB).

[0457] For each methylated marker, a limit of blank (LOB) was calculated as described previously [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): p. 1722-31] using commercial DNA extracted from whole Blood (Promega). LOB is defined by the frequency of positive droplets measured in normal control DNA samples with no hyper-methylated DNA present (n= 29). The calculated LOB was subtracted from each sample for calculating their methylation level.

[0458] The sample analysis was performed following the procedure described earlier [Taly, V., et al. 2013], Samples were considered positive when the number of observed droplets was higher than LOB value. The methylation level of each sample was calculated as the ratio of the number of droplets containing methylated sequences over the number of droplets containing albumin sequences.

[0459] Calculation of detection sensitivity and specificity

[0460] The sensitivity and specificity were calculated using the concentration (ng / mL of plasma) and / or percentage of methylated markers detected in healthy subjects and comparing them with the values obtained for colorectal cancer patients. Analysis was performed on R Studio by constructing ROC curves (R version 4.3.3, package “proc”). AUC (area under the curve) were calculated as well as the specificity and sensitivity of each marker and marker combination. Statistical analysis

[0461] Statistical analyses were performed using Prism Software (GraphPad Software Inc.) and R software 3.6.3 Studio. A P value < 0.05 was considered as significant.

[0462] In this example, performances were evaluated according to the highest concentration observed for the combination of biomarkers (i.e. performance is based on the concentration corresponding to the positive marker with the highest concentration).

[0463] RESULTS

[0464] The results confirm that AQP5-AS1, COL4A1, and ZSCAN23 biomarkers have high specificity and sensitivity when evaluated alone in the CRC patient cohort, as shown in Table 7 below:

[0465] Table 7: Evaluation of AQP 5- AS 1, COL4A1 and ZSCAN23 biomarkers when assessed alone by ddPCR in the CRC patient cohort. ROC curves were constructed as described above and corresponding AUC (area under the curve) calculated. The best combination of specificity and sensitivity was calculated by the Youden's J statistic (columns “bestSpe” and “bestSen”). The optimal cut-off is the threshold that maximizes the distance to the identity (diagonal) line. spe95Sen: Sensitivity (%) at 95% Specificity. Data are rounded to two decimals.

[0466] Combination of biomarkers

[0467] The selected biomarkers can be used in combination to achieve a higher detection sensitivity. As shown in Table 8 below, the sensitivity is improved by combining ZSCAN23, COL4A1, and AQP5-AS1 with each other. Table 8: Evaluation of AQP5-AS1, C0L4A1 and ZSCAN23 biomarkers when assessed in combination by ddPCR in the CRC patient cohort. ROC curves were constructed as described above and corresponding AUC (area under the curve) calculated. The best combination of specificity and sensitivity was calculated by the Youden's J statistic (columns “bestSpe” and “bestSen”). The optimal cut-off is the threshold that maximizes the distance to the identity (diagonal) line. spe95Sen: Sensitivity (%) at 95% Specificity. Data are rounded to two decimals.

[0468] The results in Table 9 below show that ZSCAN23, C0L4A1 and / or AQP5-AS1 biomarkers have very good sensitivity and / or specificity when combined with other biomarkers (i.e. NR5A2, C9orf50, NPY and WIFI).

[0469] Table 9: Evaluation of AQP 5- AS 1, COL4A1 and / or ZSCAN23 biomarkers when assessed in combination with other biomarkers (NR5A2, C9orf50, WIFI andNPY), by ddPCR in the CRC patient cohort. ROC curves were constructed as described above and corresponding AUC (area under the curve) calculated. The best combination of specificity and sensitivity was calculated by the Youden's J statistic (columns “bestSpe” and “bestSen”). The optimal cut-off is the threshold that maximizes the distance to the identity (diagonal) line. spe95Sen: Sensitivity (%) at 95% Specificity. Data are rounded to two decimals. Not all combinations are shown.

Claims

CLAIMS1. An in vitro method for detecting or monitoring a colorectal cancer (CRC) in a subject, preferably a human subject, which method comprises detecting, or determining the level of methylation of at least one of AQP5-AS1, ZSCAN23, or COL4A1 genes, in a biological sample from the subject, wherein the biological sample contains genomic DNA.

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

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

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

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

6. The method of any one of claims 1 to 5, which method further comprises detecting, or determining the level of methylation of at least one of NR5A2, C9orf50, WIFI and NPY genes.

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

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

9. The method of any of claims 1 to 7, comprising detecting, or determining the level of methylation of the combination of AQP5-AS1, ZSCAN23, and COL4A1 genes, and at leasttwo, preferably three, preferably four genes selected in the group consisting of : NR5A2, C9orf50, WIFI, and NPY.

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

11. The method of any of claims 1 to 10, wherein said level or amount of methylation is determined by using :- the primers of SEQ ID NO: 1 and 2, and the probe of SEQ ID NO:27 for the ZSCAN23 gene,- the primers of SEQ ID NO:3 and 4, and the probe of SEQ ID NO:28 for the C9orf50 gene,- the primers of SEQ ID NO:5 and 6, and the probe of SEQ ID NO:29 for the AQP5-AS1 gene,- the primers of SEQ ID NO: 7 and 8, and the probe of SEQ ID NO:30 for the NR5A2 gene,- the primers of SEQ ID NO: 9 and 10, and the probe of SEQ ID NO: 31 for the ADARB2 gene,- the primers of SEQ ID NO: 11 and 12, and the probe of SEQ ID NO:32 for the COL4A1 gene,- the primers of SEQ ID NO: 13 and 14, and the probe of SEQ ID NO:33 for the NPY gene,- the primers of SEQ ID NO: 15 and 16, and the probe of SEQ ID NO:34 for the WIFI gene,- the primers of SEQ ID NO: 17 and 18, and the probe of SEQ ID NO:35 for the CPNE8 gene- the primers of SEQ ID NO: 19 and 20, and the probe of SEQ ID NO:36 for the LINC00693 (RBMS3) gene,- the primers of SEQ ID NO:21 and 22, and the probe of SEQ ID NO:37 for the LINC00900 gene,- the primers of SEQ ID NO:23 and 24, and the probe of SEQ ID NO:38 for the DYDC2 gene, and / or- the primers of SEQ ID NO:25 and 26, and the probe of SEQ ID NO:39 for the LRRC4 (SND1) gene.

12. The method according to any of claims 1 to 11, wherein the sample is a body fluid, preferably selected from the group consisting of plasma, serum, blood, urine and feces, still preferably wherein 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 of claims 1 to 12, wherein if said gene(s) are hypermethylated as compared with reference value(s), then said subject is diagnosed or identified as suffering from a colorectal cancer.

14. The method according to any of claims 1 to 12, for an early diagnosis in a subject who is at risk of developing a colorectal cancer.

15. The method according to any of claims 1 to 12, for an early diagnosis of a cancerous lesion in the colon in a subject who has been developing metastasis from a primary tumor of unknown origin.

16. The method according to any of claims 1 to 12, for assessing the risk of relapse.

17. The method according to any of claims 1 to 12, for monitoring the progress of colorectal cancer in a subject diagnosed with colorectal cancer, which method preferably comprises a) determining the level of methylation of any of said genes, in a biological sample of said subject, at a first time point, b) determining the level of methylation of said genes selected previously in the step a), in a biological sample of said subject, at a second time point, and c) comparing the level of methylation determined in step a) to the level or amount determined in step b) or to reference values.

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