In vitro method for diagnosing or prognosing colorectal cancer or its precancerous stages

Abstract

The method of the present invention involves determining the methylation status of the promoter of the gene LINC00473 in a liquid biopsy obtained from a patient.

Description

[Technical Field]

[0001] The present invention relates to the medical field. In particular, the present invention relates to an in vitro method for diagnosing and / or prognosing colorectal cancer (CRC) and / or its precancerous stages. The method of the present invention preferably comprises determining the methylation status of the gene LINC00473 in a liquid biopsy obtained from a patient, wherein a higher level of methylation of the gene LINC00473 relative to an established reference control level is indicative of the presence of colorectal cancer, its precancerous stages, and / or a poor prognosis of the disease. [Background technology]

[0002] CRC (also known as colon cancer, rectal cancer, or bowel cancer) is the development of cancer in the colon or rectum (part of the large intestine). The majority of colorectal cancers are adenocarcinomas. This is due to the large number of glands within the colon's tissue. These glands undergo numerous changes at the genetic level, leading to a predictable progression from benign to invasive, malignant colon cancer. Colon adenomas, particularly advanced colorectal adenomas (AA), are benign versions of malignant adenocarcinomas but can become malignant if not removed (they are usually removed due to their tendency to become malignant and lead to colon cancer).

[0003] Screening is an effective way to prevent colorectal cancer and reduce deaths from it, and it is recommended that screening begin between the ages of 50 and 75. The best-known and most frequently used screening test for colorectal cancer is called a fecal immunochemical test (FIT). FIT detects blood in stool samples, which may be a sign of precancerous or cancerous disease. If an abnormal result is obtained, a colonoscopy is usually recommended, allowing a doctor to view the inside of the colon and rectum and make a diagnosis. During a colonoscopy, small polyps can be removed if found. Larger polyps or tumors can be biopsied to determine whether they are cancerous. Gastroenterologists use colonoscopy to find and remove these adenomas and polyps, preventing them from continuing to acquire genetic changes that lead to invasive adenocarcinoma.

[0004] As explained above, FIT is currently used to screen for colorectal cancer. However, it is important to note that FIT has low sensitivity for adenomas, meaning that most patients in this category may be erroneously classified as disease-free. As a result, FIT fails to identify adenomas due to its low sensitivity. Furthermore, FIT requires the use of fecal samples, resulting in low compliance. On the other hand, colonoscopy is an invasive technique, and the most serious complication is generally gastrointestinal perforation. Furthermore, colonoscopy today is a procedure that requires anesthesia, and the laxatives typically administered during bowel preparation for colonoscopy are associated with several digestive problems.

[0005] It is important to note that the methods used today to screen the general population at risk for CRC or AA are associated with a high rate of false positives, resulting in a large number of unnecessary follow-up colonoscopies being performed today.

[0006] The present invention provides a clear solution to the above-mentioned problems, as it focuses on an in vitro method for identifying or screening human subjects at risk of developing colorectal cancer or colorectal adenoma (particularly advanced colorectal adenoma). Because the method of the present invention is preferably based on a liquid biopsy (e.g., plasma, blood, or serum) obtained from the patient, improved compliance with colorectal cancer screening is expected. Furthermore, the method of the present invention provides high sensitivity and specificity, which means it is a powerful and cost-effective method for the detection of both colorectal cancer and colorectal adenoma. Summary of the Invention

[0007] The present invention relates to an in vitro method for diagnosing or prognosing colorectal cancer and / or its precancerous stages (hereinafter "the method of the present invention"). The method of the present invention is preferably performed on a minimally invasive biological sample, a liquid biopsy obtained from a patient. The method of the present invention provides high sensitivity and specificity, which means that the method of the present invention is a powerful and cost-effective method for detecting both colorectal cancer and / or its precancerous stages.

[0008] The method of the present invention has higher sensitivity and specificity than the method (FIT) currently used to screen the general population at risk of CRC or AA, and is therefore associated with a lower percentage of false positives.As a result, the method described in the present invention clearly helps to reduce the number of follow-up colonoscopies, thus improving the screening or diagnostic method for today's patients.After the method of the present invention is performed, if it is determined that the patient may have colorectal cancer and / or precancerous stage, the result is confirmed by colonoscopy.However, if it is not determined that the patient may have colorectal cancer and / or precancerous stage, colonoscopy does not need to be performed, and routine examination using the method of the present invention is recommended.

[0009] In particular, a first embodiment of the present invention relates to an in vitro method for diagnosing colorectal cancer and / or precancerous stages thereof, comprising determining the methylation status of at least the gene LINC00473 in a liquid biopsy obtained from a patient, wherein a higher level of methylation of the gene LINC00473 compared to a reference level of methylation of the gene LINC00473 measured in healthy control subjects indicates that the subject is suffering from colorectal cancer and / or precancerous stages thereof.

[0010] A second embodiment of the present invention relates to an in vitro method for prognosing colorectal cancer and / or its precancerous stages, comprising determining the methylation status of at least the gene LINC00473 in a liquid biopsy obtained from a patient, wherein a higher level of methylation of the gene LINC00473 compared to a reference level of methylation of the gene LINC00473 measured in the patient indicates a poor prognosis for the patient.

[0011] A third embodiment of the invention relates to the in vitro use of the methylation status of at least the gene LINC00473 for the diagnosis and / or prognosis of colorectal cancer and / or its precancerous stages.

[0012] A fourth embodiment of the present invention relates to the in vitro use of a kit comprising reagents for determining the methylation status of at least the gene LINC00473 for the diagnosis and / or prognosis of colorectal cancer and / or its precancerous stages.

[0013] In preferred embodiments, the liquid biopsy is a sample of plasma, blood or serum, with plasma being particularly preferred.

[0014] In a preferred embodiment, the precancerous stage is colorectal adenoma, preferably AA.

[0015] In a preferred embodiment, the methylation status of the gene LINC00473 is determined at least at the CpGs of the promoter region.

[0016] In a preferred embodiment, the methylation status of gene LINC00473 is determined at CpGs in the promoter region that are at least 3000 bp surrounding the transcription start site (TSS), meaning 1500 bp upstream or 1500 bp downstream of the TSS.

[0017] In a preferred embodiment, the methylation status of the gene LINC00473 is determined at least at the CpGs of the promoter region located between chromosomal positions Chr6:166402081 and Chr6:166402638.

[0018] In a preferred embodiment, the methylation status of gene LINC00473 is determined at a CpG in the promoter region located at a chromosomal location selected from the group comprising at least Chr6:166402638, and / or Chr6:166402474, and / or Chr6:166402463, and / or Chr6:166402457, and / or Chr6:166402416, and / or Chr6:166402379, and / or Chr6:166402375, and / or Chr6:166402364, and / or Chr6:166402081, preferably at a CpG selected from the group comprising cg06545143, and / or cg08886973, and / or cg21306006.

[0019] In a preferred embodiment, the diagnosis of colorectal cancer and / or its precancerous stages is confirmed by imaging techniques, preferably colonoscopy.

[0020] According to the method of the present invention, after measuring the methylation status of the gene LINC00473, a score value is obtained and compared with a threshold value defining a diagnostic rule. If the score value is higher than the threshold value, the corresponding sample is classified as a positive sample, indicating that the patient is likely to be affected by colorectal cancer and / or its precancerous stages. The threshold value is defined to optimize the sensitivity and specificity values. Thus, in a preferred embodiment, the method of the present invention comprises: a) measuring the methylation status of the gene LINC00473 in a liquid biopsy obtained from a subject; b) processing the methylation value to obtain a risk score; and c) identifying a deviation or variation in the obtained risk score value compared to a reference value, which indicates that the subject is affected by colorectal cancer and / or its precancerous stages.

[0021] A final embodiment of the present invention relates to a method for diagnosing and treating colorectal cancer or a precancerous stage thereof, comprising: a) obtaining a liquid biopsy from a human subject; b) detecting the methylation status of gene LINC00473; and c) diagnosing the patient with colorectal cancer or a precancerous stage thereof and performing a colonoscopy on the patient if a higher level of methylation of gene LINC00473 is identified compared to a reference level of methylation of gene LINC00473 measured in healthy subjects. The colonoscopy may include removal of colorectal cancer or polyps.

[0022] For purposes of the present invention, the following terms are defined: LINC00473 (Long Intergenic Non-Protein Coding RNA 473) is an RNA gene and belongs to the non-coding RNA class. LINC00473 can be identified in public databases as: HGNC: 21160. Entrez Gene: 90632. Ensambl ID: ENSG00000223414. UniProtKB: A8K010. The term "colorectal cancer" refers to a medical condition characterized by cancer of cells of the intestinal tract below the small intestine (i.e., the large intestine (colon), including the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum). The expression "colorectal adenoma" refers to adenomas of the colon, also called adenomatous polyps, which are a benign, precancerous stage of colorectal cancer but still pose an increased risk of progressing to colorectal cancer. The expression "advanced colorectal adenoma" refers to an adenoma having a size of at least 10 mm, or an adenoma with histologically high grade dysplasia or villous component of more than 20%, or a serrated lesion or dysplasia at least 10 mm in size. For a more detailed explanation of the concept of "advanced colorectal adenoma", please refer to the following scientific articles related to the general knowledge in the field of the present invention: [Rex DK, Boland CR, Dominitz JA, Giardiello FM, Johnson DA, Kaltenbach T, Levin TR, Lieberman D, Robertson DJ. Colorectal Cancer Screening: Recommendations for Physicians and Patients From the US Multi-Society Task Force on Colorectal Cancer. Gastroenterology. 2017 Jul;153(1):307-323. doi: 10.1053 / j.gastro.2017.05.013. Epub 2017 Jun 9. PMID: 28600072], [East JE, Atkin WS, Bateman AC, Clark SK, Dolwani S, Ket SN, Leedham SJ, Phull PS, Rutter MD, Shepherd NA, Tomlinson I, Rees CJ. British Society of Gastroenterology position statement on serrated polyps in the colon and rectum. Gut. 2017 Jul;66(7):1181-1196. doi: 10.1136 / gutjnl-2017-314005. Epub 2017 Apr 27. Review. PMID: 28450390]. The term "liquid biopsy" refers to any sample of bodily fluid that may contain tumor-derived material. In particular, a liquid biopsy is any sample of bodily fluid (e.g., plasma, serum, blood, saliva, cerebrospinal fluid, or urine) that may contain tumor-derived material, such as circulating tumor DNA. The expression "minimally invasive biological sample" refers to any sample taken from a patient's body that is harmless to the patient because it does not require the use of any harmful equipment other than a thin needle used to draw blood from the patient. Specifically, in the present invention, a minimally invasive biological sample refers to a blood, serum, or plasma sample. As used herein, the term "methylation" is understood to mean the presence of a methyl group added to one or more cytosine bases in a region of nucleic acid, e.g., DNA, by the action of a DNA methyltransferase enzyme. Thus, as used herein, the term "methylation status" refers to the level of methylation measured in the patient being analyzed. The "methylation status" may be a higher / lower level of methylation compared to a reference level or control level. As used herein, "CpG dinucleotide," "CpG methylation site," or equivalents shall be taken to refer to a cytosine linked to a guanine by a phosphodiester bond. A CpG dinucleotide is a target for methylation of cytosine residues and may be present within coding or non-coding nucleic acid. Non-coding nucleic acid is understood in the art to include introns, 5' untranslated regions, 3' untranslated regions, promoter regions, or intergenic regions of a genomic gene. The expression "target CpG" refers to a specific CpG that is found to be differentially methylated in the present invention. The expression "contiguous CpGs" refers to CpGs that may be differentially methylated and may be found up to 1500 bp upstream or downstream of the transcription start site (TSS) (3000 bp surrounding the TSS). As used herein, "determining the methylation status of a gene" means performing the method of the present invention by measuring the methylation status of any region of the gene, including any regulatory sequences that can increase or decrease the expression of said gene. In particular, said regulatory sequences include a promoter, which is a region of DNA that initiates transcription of a gene. A promoter is located near the transcription start site of a gene, on the same strand, upstream or downstream of the DNA (towards the 5' region of the sense strand).As used herein, a "control level, reference level, or baseline level of methylation" is understood to mean the level of methylation detected in the corresponding nucleic acid from a healthy patient. Therefore, if a "higher level of methylation" is measured in this patient compared to the "control level, reference level, or baseline level of methylation" measured in the healthy patient, the patient is likely to have CRC or AA at a given sensitivity and specificity. The "reference value" can be a threshold or cutoff value. Typically, the "threshold" or "cutoff value" can be determined experimentally, empirically, or theoretically. As will be recognized by those skilled in the art, the threshold value can also be arbitrarily selected based on existing experimental and / or clinical conditions. The threshold value must be determined to obtain optimal sensitivity and specificity depending on the function of the test and the benefit / risk balance (false-positive and false-negative clinical outcomes). Preferably, those skilled in the art may compare the biomarker level (or score) obtained according to the method of the present invention with a defined threshold value. Typically, the optimal sensitivity and specificity (and therefore the threshold value) can be determined using a receiver operating characteristic (ROC) curve based on experimental data. For example, after determining the methylation level of a biomarker in a reference group, algorithmic analysis can be used to statistically process the measured methylation level of the biomarker in the biological sample of the test subject to obtain classification criteria with significance for sample classification. The official name of the ROC curve is the receiver operating characteristic curve, also known as the receiver operating characteristic curve. ROC curves are primarily used in clinical biochemical diagnostic tests and are comprehensive indicators that reflect the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). ROC curves reveal the relationship between sensitivity and specificity through image synthesis. A series of different cutoff values ​​(thresholds or critical values, the boundary values ​​between normal and abnormal results of a diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. The curve is then plotted using sensitivity as the vertical coordinate and specificity as the horizontal coordinate.The larger the area under the curve (AUC), the higher the diagnostic accuracy. In an ROC curve, the point closest to the upper left corner of the coordinate system is the critical point with both high sensitivity and high specificity. The AUC value of an ROC curve is 1.0 to 0.5. When the AUC is greater than 0.5, the closer the AUC is to 1, the better the diagnostic result. When the AUC is 0.5 to 0.7, the accuracy is low. When the AUC is 0.7 to 0.9, the accuracy is good. When the AUC is greater than 0.9, the accuracy is very high. This algorithmic method is preferably performed using a computer. Existing software or systems in the art, such as MedCalc 9.2.0.1 medical statistics software, SPSS 9.0, or preferably GraphPad Prism 6, can be used to plot the ROC curve. The phrase "higher levels of methylation" refers to a statistically significant increase in the relative amount of methylation of a nucleic acid, e.g., DNA, compared to the amount of methylation measured in a subject / patient used as a control / reference. Thus, in the present disclosure, "higher levels of methylation" are determined relative to the baseline level represented by the methylation status of a given genomic region in a sample obtained from a subject or patient. For example, a "higher level of methylation" can be at least 2% greater than the baseline level of methylation, e.g., at least 5% greater than the baseline level of methylation, or at least 10% greater than the baseline level of methylation, or at least 15% greater than the baseline level of methylation, or at least 20% greater than the baseline level of methylation, or at least 25% greater than the baseline level of methylation, or at least 30% greater than the baseline level of methylation, or at least 40% greater than the baseline level of methylation, or at least 50% greater than the baseline level of methylation, or at least 60% greater than the baseline level of methylation, or at least 70% greater than the baseline level of methylation, or at least 80% greater than the baseline level of methylation, or at least 90% greater than the baseline level of methylation. "Comprising" means including, but not limited to, what follows the word "comprises." Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present. "Consisting only of" means "including and limiting" what precedes the phrase "consisting only of." Thus, the phrase "consisting only of" indicates that the listed elements are required or mandatory, and that no other elements may be present. [Brief explanation of the drawings]

[0023] [Figure 1] Figure 1 shows DNA methylation levels of the LINC00473 promoter in colorectal tissue samples. In cohort 1 (A) and cohort 2 (B), colorectal cancer patients showed significantly higher LINC00473 methylation levels than non-tumor controls. LINC00473 promoter methylation levels were analyzed by (A) Infinium HumanMethylation450K BeadChip (450K array) and (B) pyrosequencing. DNA methylation data are expressed as beta values ​​in A and % methylation in B. Horizontal lines represent median methylation levels. P indicates p-value analyzed by Mann-Whitney U test. Tumors represent colorectal cancer. [Figure 2] Figure 1 shows receiver operating characteristic (ROC) curves for LINC00473 promoter methylation in colorectal tissue samples. The area under the ROC curve (A) and the area under the ROC curve (AUC) for Cohort 1 (A) and Cohort 2 (B) indicate the high diagnostic accuracy of LINC00473 promoter methylation for distinguishing colorectal cancer patients from non-tumor controls. P indicates the p-value of the ROC curve. CI is the confidence interval. [Figure 3]Figure 1 shows DNA methylation levels of the LINC00473 promoter in colorectal tissue samples according to clinical stage according to The Cancer Genome Atlas (TCGA). All stages of colorectal cancer patients showed significantly higher LINC00473 methylation levels in (A) Cohort 1 (I: 0.55 ± 0.023; II: 0.53 ± 0.018; III: 0.45 ± 0.028; IV: 0.47 ± 0.031) and (B) Cohort 2 (I: 38.54% ± 7.67%; II: 34.31% ± 2.11%; III: 32.14% ± 2.84%; IV: 35.31% ± 3.95%) than their respective non-tumor controls (Cohort 1: 0.11 ± 0.006; Cohort 2: 5.84% ± 0.78%). The methylation levels of LINC00473 were analyzed by (A) Infinium HumanMethylation450K BeadChip (450K array) and expressed as β values, and (B) pyrosequencing and expressed as % methylation. An asterisk (*) indicates P<0.0001 vs. control. p-values ​​were analyzed by Mann-Whitney U test. Tumors represent colorectal cancer. [Figure 4] Figure 1 shows DNA methylation levels of the LINC00473 promoter in adenomas. Adenoma and colorectal cancer patients showed significantly higher LINC00473 methylation levels than non-tumor controls. LINC00473 promoter methylation levels were analyzed by pyrosequencing and expressed as % methylation. The horizontal line represents the median methylation level. P indicates the p-value analyzed by the Mann-Whitney U test. [Figure 5] Figure 1 shows the receiver operating characteristic (ROC) curve for LINC00473 promoter methylation in adenomas. The area under the ROC curve (AUC) in Cohort 3 indicates the high diagnostic accuracy of LINC00473 promoter methylation for distinguishing adenomas from non-tumor controls. P indicates the p-value of the ROC curve. CI is the confidence interval. [Figure 6]Figure 1 shows DNA methylation levels of the LINC00473 promoter in circulating plasma DNA of colorectal cancer patients and healthy controls. Colorectal cancer patients showed significantly higher LINC00473 methylation levels than healthy controls. LINC00473 methylation levels were analyzed in triplicate by real-time PCR. The horizontal line represents the median methylation level. P indicates the p-value analyzed by Mann-Whitney U test. [Figure 7] Figure 1 shows the receiver operating characteristic (ROC) curve for LINC00473 promoter methylation in circulating plasma DNA of colorectal cancer patients versus normal controls. The area under the ROC curve (AUC) indicates the high diagnostic accuracy of LINC00473 promoter methylation for distinguishing colorectal cancer patients from healthy individuals. P indicates the p-value of the ROC curve. [Figure 8] Figure 1 shows Kaplan-Meier survival analysis of LINC00473 promoter methylation in colorectal cancer patients. Colorectal cancer patients were classified according to whether they had higher or lower levels of LINC00473 methylation relative to the median methylation level of the group (10% methylation). Kaplan survival analysis showed that high LINC00473 methylation levels were significantly associated with shorter overall survival (OS) (P=0.0256). P refers to the p-value of the log-rank test. H (high) indicates a methylation level greater than 10%, and L (low) indicates a methylation level less than 10%. [Figure 9] Figure 1 shows DNA methylation levels of the LINC00473 promoter in circulating plasma DNA of patients with advanced colorectal adenoma and healthy controls. Patients with advanced colorectal adenoma showed significantly higher LINC00473 methylation levels than healthy controls. LINC00473 methylation levels were analyzed in triplicate by real-time PCR. The horizontal line represents the median methylation level. P indicates the p-value analyzed by the Mann-Whitney U test. [Figure 10]Figure 1 shows the receiver operating characteristic (ROC) curve for LINC00473 promoter methylation in circulating plasma DNA of advanced colorectal adenomas versus normal controls. The area under the ROC curve (AUC) indicates the high diagnostic accuracy of LINC00473 promoter methylation for distinguishing advanced colorectal adenomas from healthy controls. P indicates the p-value of the ROC curve. CI is the confidence interval. [Figure 11] Figure 1 shows DNA methylation levels of the LINC00473 promoter in advanced colorectal adenomas from tissues. Advanced colorectal adenomas showed significantly higher LINC00473 methylation levels than non-tumor controls. LINC00473 promoter methylation levels were analyzed by pyrosequencing and expressed as % methylation. The horizontal line represents the median methylation level. P indicates p-value analyzed by Mann-Whitney U test. [Figure 12] Figure 1 shows the receiver operating characteristic (ROC) curve for LINC00473 promoter methylation in advanced colorectal adenomas from tissue. The area under the ROC curve (AUC) indicates the high diagnostic accuracy of LINC00473 promoter methylation for distinguishing advanced colorectal adenomas from non-tumor controls. P indicates the p-value of the ROC curve. CI is the confidence interval. [Figure 13] Figure 1 shows DNA methylation levels of the LINC00473 promoter in circulating plasma DNA of colorectal cancer patients by droplet digital PCR (ddPCR). Colorectal cancer patients showed significantly higher LINC00473 methylation levels than non-tumor controls. LINC00473 promoter methylation levels were analyzed in triplicate by ddPCR. The horizontal line represents the median methylation level. P indicates the p-value analyzed by the Mann-Whitney U test. [Figure 14]Figure 1 shows the receiver operating characteristic (ROC) curve for LINC00473 promoter methylation in circulating plasma DNA of colorectal cancer patients versus normal controls, analyzed by droplet digital PCR (ddPCR). The area under the ROC curve (AUC) indicates the high diagnostic accuracy of LINC00473 promoter methylation for distinguishing colorectal cancer patients from healthy individuals. P indicates the p-value of the ROC curve. [Figure 15] Figure 1 shows DNA methylation levels of the LINC00473 promoter in circulating plasma DNA of advanced colorectal adenomas by droplet digital PCR. Patients with advanced colorectal adenomas showed significantly higher LINC00473 methylation levels than healthy controls. LINC00473 methylation levels were analyzed in triplicate by droplet digital PCR. The horizontal line represents the median methylation level, and P indicates the p-value analyzed by the Mann-Whitney U test. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0024] Example 1. Method Example 1.1. Study participants for colorectal tissue analysis We first analyzed three independent cohorts (Cohort 1, Cohort 2, and Cohort 3) to investigate DNA methylation of LINC00473 in colorectal tissues. Cohort 1 represents primary colorectal cancer patients (n = 273) and non-tumor controls (n = 38). Cohort 2 includes 180 primary colorectal cancer patients and 93 non-tumor controls. Cohort 3 includes 12 primary colorectal cancer patients, 12 adenoma patients, and 12 non-tumor controls.

[0025] Furthermore, for the purposes of this PCT application, we also analyzed a new independent cohort (Cohort 4) to investigate DNA methylation of LINC00473 in colorectal tissues. Cohort 4 included 50 colorectal adenoma patients (including 20 confirmed advanced colorectal adenoma patients) and 10 non-tumor controls.

[0026] Example 1.2. Analysis of DNA methylation data of colorectal tissue with 450K arrays DNA methylation results for Cohort 1 were analyzed from the Infinium HumanMethylation450K BeadChip data (450K array) obtained from the public database The Cancer Genome Atlas (TCGA). The 450K array (Illumina) covers over 450,000 CpG sites along the human genome, and the methylation score for each CpG is expressed as a beta (β) value ranging from 0.0 (fully unmethylated) to 1.0 (fully methylated).

[0027] Example 1.3. Bisulfite pyrosequencing analysis of colorectal tissue The DNA methylation status of the LINC00473 promoter in Cohorts 2, 3, and 4 was analyzed by bisulfite pyrosequencing. Approximately 500 ng of DNA was bisulfite converted using the EZ-96 DNA Methylation Kit (Zymo Research Corp.), and the DNA methylation level of the LINC00473 promoter was analyzed using the PyroMark Q96 system (Qiagen) according to the manufacturer's instructions. Quantitation of CpG site methylation was obtained using Pyro Q-CpG 1.0.9 (Qiagen). Primer sequences (Table 1) were designed using PyroMark Assay Design 2.0 (Qiagen). Table 1 shows the primers used for methylation analysis of LINC00473 by pyrosequencing.

[0028] [Table 1]

[0029] In a preferred embodiment, the primer used in the present invention, preferably the primer of SEQ ID NO: 2, is biotinylated by incorporation of a molecule of biotin.

[0030] Example 1.4. Study Participants for Liquid Biopsy Analysis For the analysis of LINC00473 in colorectal cancer liquid biopsies, this study included 26 patients with advanced colorectal cancer (stage III and stage IV) who were prospectively recruited at the time of diagnosis (baseline) before the initiation of treatment and surgery. Patient demographic and clinical characteristics are shown in Table 2. Twenty-eight healthy controls were also included in the study. The disease status and staging of the colorectal cancer patients were obtained by medical oncologists at the Oncology Department of the Complejo Hospitalario Universitario de Santiago de Compostela (CHUS). Table 2 shows the clinical characteristics of the colorectal cancer patients.

[0031] [Table 2]

[0032] For the analysis of LINC00473 in liquid biopsies of advanced colorectal adenomas, this study included five healthy controls and 12 patients with advanced adenomas. All subjects for liquid biopsy analysis were recruited at the General Hospital of the University of Santiago de Compostela (CHUS).

[0033] For the analysis of LINC00473 in colorectal cancer liquid biopsies by droplet digital PCR (ddPCR), the study included five patients with advanced colorectal cancer who were prospectively recruited at the time of diagnosis (baseline) before the start of treatment and surgery. Ten patients with advanced adenoma and five healthy controls were also included in the study. Patients and controls were obtained at the General Hospital of the University of Santiago de Compostela (CHUS).

[0034] Example 1.5. Blood Sample Collection and Plasma Separation Blood samples were obtained by phlebotomy using collection tubes containing EDTA as an anticoagulant. Plasma was separated from the blood samples within 2 hours of collection by first centrifugation of the blood tubes (1500 g, 10 min, 4°C). The plasma was transferred to a 1.5 mL tube and centrifuged a second time (15000 g, 10 min, 4°C). The plasma was transferred to a new 1.5 mL tube and stored at -80°C.

[0035] Example 1.6. Isolation of circulating DNA from plasma samples After thawing the plasma samples, circulating DNA was isolated from 2-3 mL of plasma using the QIAamp™ Circulating Nucleic Acid Kit (Qiagen) and the QIAvac 24 Plus vacuum system (Qiagen) according to the manufacturer's recommendations. Finally, circulating DNA was eluted with 75 μL of elution buffer, and 1 μL of this eluate was used to quantify the circulating DNA concentration using the QuantiFluor™ ONE dsDNA kit with a Quantus™ Fluorometer (Promega). After quantification, the circulating DNA was stored at -80°C.

[0036] Example 1.7. Bisulfite conversion of circulating DNA DNA bisulfite conversion was performed using 15 ng to 50 ng of circularized DNA using the EZ DNA Methylation-Lightning Kit (Zymo Research) according to the manufacturer's recommendations, following these thermocycling steps: 98°C for 8 min, 54°C for 60 min, and 4°C for 6 min. After column-based purification, including binding, L-desulfonation, and washing steps, the bisulfite-converted DNA (bis-DNA) was eluted with 15 μL of elution buffer and stored at -80°C.

[0037] Example 1.8. DNA methylation analysis of the LINC00473 promoter region in liquid biopsies by real-time PCR The methylation status of the promoter region of long intergenic non-protein-coding RNA 473 (LINC00473; formerly symbolized C6orf176) was analyzed in circulating plasma DNA. The non-coding gene LINC00473 (NCBI ID: 90632; UCSC ID: uc063sul.1; Ensambl ID: ENSG00000223414; HGNC ID: HGNC:21160) is located on chromosome 6 at position 6q27, with a CpG island encompassing its promoter region located at chr6:166401527-166402659. Furthermore, the promoter region of LINC00473 overlaps genomically with the body of long intergenic non-protein-coding RNA 602 or LINC00602 (NCBI ID: 441177; UCSC ID: uc011egm.4; Ensambl ID: ENSG00000281832; HGNC ID: HGNC:43917).

[0038] The methylation level of the promoter region of LINC00473 was determined by real-time PCR using a quantitative methylation-specific PCR assay (qMSP) on a StepOne Plus system (Applied Biosystems). Each reaction contained 2 μL of bisulfite-converted DNA (bis-DNA) as template, 10 μL of Power SYBR™ Green PCR Master Mix (ThermoFisher), and 150 nM each of forward and reverse primers (Table 3) to detect methylation or non-methylation in a total volume of 20 μL. Reactions were performed in a MicroAmp™ Fast Optical 96-Well Reaction Plate (Applied Biosystems). The thermocycling conditions for the StepOne Plus system were as follows: 95°C for 10 minutes, followed by 50 cycles of 94°C for 15 seconds and 60°C for 30 seconds. Water was included as a non-template control in each run to confirm the absence of contamination during the reaction. The colorectal cancer cell line HCT-116 and normal leukocytes (NL) were used in each analysis as positive controls for methylation and non-methylation, respectively. All samples and controls were analyzed in triplicate. The threshold cycle (Ct) of each reaction was determined using primers specific for methylation and non-methylation using the software StepOne Real-Time PCR (Applied Biosystems). The DNA methylation level of each sample was expressed as a percentage (%) of methylation according to the following calculation based on Cottrell et al. (2007): Methylation (%) = 100 / [1+2^(CT CG -CT TG )]

[0039] In this formula, CT CG represents the threshold cycle of the methylation status, and CT TG indicates the threshold cycle for the unmethylated state. In this calculation, the methylation level can range from 0% to 100% methylation. Table 3 shows the primers for methylation analysis of LINC00473 by real-time PCR.

[0040] [Table 3]

[0041] The forward and reverse primers used for methylation analysis of LINC00473 allow the detection of the methylation status of six CpGs in the promoter region of LINC00473 (Table 4). One of the CpGs (chr6:166402416) contained in the sequence amplified by real-time PCR (amplicon) corresponds to a CpG designated cg08886973 by the Infinium Human Methylation 450K assay and the Infinium Methylation EPIC assay (Illumina), which are microarray assays capable of detecting methylation levels of over 450,000 CpGs and 850,000 CpGs, respectively. These microarrays also detect additional CpGs from the LINC00473 promoter, identified as cg06545143 (chr6:166402638) and cg21306006 (chr6:166402081), located 162 nucleotides upstream and 282 nucleotides downstream of the LINC00473 amplicon analyzed in this study, respectively. The three CpGs included in this type of microarray assay (cg06545143, cg08886973, and cg21306006) are located in the TSS1500 promoter region of LINC00473, which is the region of the promoter 200 to 1500 nucleotides upstream of its transcription start site (TSS). All chromosomal locations shown above are based on version GRCh37 / hg19 of the UCSC Genome Browser (https: / / genome.ucsc.edu / index.html). Table 4 shows the CpGs of LINC00473 detected by real-time PCR methylation analysis.

[0042] [Table 4]

[0043] Example 1.9. DNA methylation analysis of the LINC00473 promoter region in liquid biopsies by droplet digital PCR (ddPCR) The methylation level of the promoter region of LINC00473 was determined by droplet digital PCR (ddPCR) on a QX200 system (Bio-Rad). A multiplex preamplification reaction was performed in a total volume of 50 μl using approximately 2 ng of bisulfite-converted DNA (bis-DNA), 25 μl of SsoAdvanced™ PreAmp Supermix (Bio-Rad), 0.5 μl of a custom Bio-Rad assay containing forward and reverse primers and a methylation probe (Bio-Rad) (Table 5), and 0.5 μl of a custom Bio-Rad assay containing forward and reverse primers and an unmethylation probe (Table 5). The final volume was completed with water. The reaction was performed in a 0.2 ml PCR tube (Axygen) on a ProFlex PCR System (Applied Biosystems): 95°C for 3 minutes, 10 cycles of 95°C for 15 seconds, 56.2°C for 4 minutes, and a final hold step at 4°C.

[0044] Next, a multiplex reaction was performed in a total volume of 22 μl, containing 2 μL of the preamplification product, 11 μl of ddPCR supermix (without dUTP) for probes (Bio-Rad), 2.2 μl of a custom Bio-Rad assay containing forward and reverse primers and a methylation probe (Table 5), and 2.2 μl of a custom Bio-Rad assay containing forward and reverse primers and a non-methylation probe (Bio-Rad) (Table 5). The final volume was completed with water. Next, 20 μL of each reaction mixture and 70 μl of Droplet Generation oil (Bio-Rad) were transferred to the sample well and oil well of a DG8™ cartridge (Bio-Rad), respectively, and loaded into a QX200™ Droplet Generator (Bio-Rad). Droplets were generated in the droplet wells of the cartridge, transferred to a ddPCR 96-well plate (Bio-Rad), and loaded into a C1000 Touch thermal cycler (Bio-Rad) (95°C for 10 min, 40 cycles of 95°C for 15 s and 56.2°C for 30 s, 98°C for 10 min, and a final hold step at 4°C). The temperature ramp rate was 2.5°C / s for all steps. The 96-well plate was then read using a QX200™ Droplet Reader (Bio-Rad). Water was included as a non-template control in each run to confirm the absence of contamination in the reaction. The colorectal cancer cell line HCT-116 and normal leukocytes (NL) were used in each analysis as positive controls for methylation and non-methylation, respectively. All controls, with and without the pre-amplification step, were included on each plate. All samples and controls were analyzed in triplicate. Analysis of the data was performed using QuantaSoft software (Bio-Rad).

[0045] The DNA methylation level of each sample was expressed as a percentage (%) of methylation according to the following calculation: Methylation (%) = [M / (U+M)] × 100

[0046] In this formula, M (methylated) represents the number of copies / µL of target DNA molecules of the methylated probe (FAM probe), and U (unmethylated) represents the number of copies / µL of target DNA molecules of the unmethylated probe (HEX probe). In this calculation, the methylation level can range from 0% to 100% methylation. Table 5 shows the primer and probe sequences for methylation analysis of LINC00473 by ddPCR. Based on these sequences, two custom Bio-Rad assays containing primers and probes for methylation and unmethylation, respectively, were obtained from Bio-Rad according to the manufacturer's recommendations. The primer ratio for probe was 1.8, primer concentration in the final reaction was 450 nM, and probe concentration in the final reaction was 250 nM.

[0047] [Table 5]

[0048] The forward and reverse primers and probes used in the methylation analysis of LINC00473 by ddPCR allowed the detection of the methylation status of nine CpGs in the promoter region of LINC00473 (Table 6). One of the CpGs (chr6:166402416), corresponding to a CpG designated cg08886973 by the Infinium Human Methylation 450K Assay and the Infinium Methylation EPIC Assay (Illumina), was included in the amplified sequence (amplicon) and detected with the methylated and unmethylated probes listed in Table 5. All CpG locations in LINC00473 detected by ddPCR methylation analysis are listed in Table 6. All chromosomal locations listed in Table 6 are based on version GRCh37 / hg19 of the UCSC Genome Browser (https: / / genome.ucsc.edu / index.html).

[0049] [Table 6]

[0050] Example 1.10. Statistical Analysis The Kolmogorov-Smirnov test was first used to assess the normality of data distribution. Subsequently, the nonparametric Mann-Whitney U test was used to compare data. To assess the diagnostic accuracy of LINC00473 methylation for detecting colorectal cancer, a receiver operating characteristic (ROC) curve was constructed. The Youden index (J), which allows obtaining the methylation cutoff point that provides the maximum combination of sensitivity and specificity, was calculated according to the following formula: J = sensitivity + specificity - 1 (Fluss et al., 2005). To measure the effectiveness of the diagnostic test, the positive predictive value (PPV) and negative predictive value (NPV) were calculated: PPV = true positive / (true positive + false positive); NPV = true negative / (true negative + false negative) (Hajian-Tilaki, 2013). The Kaplan-Meier method was used to assess survival, and the log-rank (Mantel-Cox) test was used to examine differences between the methylated and unmethylated groups. SPSS or GraphPad Prism 7.0 software was used for statistical analysis and visualization. All p-values ​​shown were calculated using two-sided tests, and p<0.05 was considered significant.

[0051] Example 2. Results Example 2.1. Tissue Samples Example 2.1.1. Promoter methylation analysis of LINC00473 in tumor tissue allows detection of colorectal cancer patients DNA methylation levels of the LINC00473 promoter region in colorectal tissue samples (273 colorectal cancer patients at stages I, II, III, and IV; and 38 non-tumor controls) were obtained after analysis using the Infinium HumanMethylation 450K BeadChip (450K array) from the international public database The Cancer Genome Atlas (TCGA) (Cohort 1). In this analysis, methylation data from the 450K array were expressed as mean beta (β) values, defined as 0.0 to 1.0. We focused our analysis on the CpG in the LINC00473 promoter region, identified as cg08886973 on the 450K array. This analysis showed significantly (p<0.0001) higher methylation levels in colorectal cancer patients (0.51±0.011) than in non-tumor controls (0.11±0.006) (Figure 1A). Similar results were obtained when we analyzed the methylation status of the LINC00473 promoter in cohort 2 by bisulfite pyrosequencing (180 colorectal cancer patients: 33.93% ± 1.51%; 93 non-tumor controls: 5.84% ± 0.78%; p < 0.0001) (Figure 1B). In this case, methylation values ​​obtained from pyrosequencing were expressed as a percentage (%) of methylation, defined as 0% to 100%. Overall, these data indicate that the methylation level of the promoter region of LINC00473 analyzed in colorectal tissues can distinguish between non-tumor individuals and patients with colorectal cancer.

[0052] Receiver operating characteristic (ROC) analysis of the methylation level of LINC00473 in colorectal tissue samples from cohort 1 demonstrated a very high diagnostic accuracy for distinguishing colorectal cancer patients (I, II, III, and IV) from non-tumor controls, with an area under the curve (AUC) of 0.94 (p<0.0001; CI95%: 0.91-0.97) (Figure 2A). Similar results were obtained from cohort 2 (AUC=0.90; p<0.0001; CI95%: 0.86-0.94) (Figure 2B). These results indicate that analysis of the methylation status of the LINC00473 promoter region in colorectal tissue samples has excellent diagnostic accuracy for identifying colorectal cancer patients.

[0053] Importantly, when we stratified colorectal cancer patients according to their disease stage in Cohort 1 (I: n=41; II: n=106; III: n=58; IV: n=35) and Cohort 2 (I: n=10; II: n=84; III: n=56; IV: n=28) (Figure 3), the results also showed significantly (p<0.0001) higher methylation levels in each disease stage of colorectal cancer patients compared to non-tumor controls (Cohort 1, n=38; Cohort 2, n=93).

[0054] Example 2.1.2. Promoter methylation analysis of LINC00473 in colorectal tissue allows differentiation of adenomas and advanced colorectal adenomas DNA methylation levels of the LINC00473 promoter region in colorectal tissues from 12 non-tumor controls, 12 adenoma patients, and 12 colorectal cancer patients (Cohort 3) were analyzed by bisulfite pyrosequencing and expressed as % methylation. These results showed that the methylation level in adenomas (28.21% ± %) was significantly (p = 0.0036) higher than that in healthy controls (7.28% ± 0.81%) (Figure 4). As expected, colorectal cancer patients (32.83% ± 5.19%) also showed a significant (P = 0.002) difference in methylation levels compared to non-tumor controls. Importantly, the methylation level of LINC00473 in adenomas was similar to that in colorectal cancer patients (P = 0.3186). These data indicate that the methylation level of the promoter region of LINC00473 analyzed in colorectal tissues can distinguish between non-tumor individuals and patients with adenomas, suggesting that the methylation status of the LINC00473 promoter may be useful for the early detection of colorectal cancer.

[0055] Analysis of the methylation levels of LINC00473 in colorectal tissue samples from non-tumor and adenoma samples of Cohort 3 by receiver operating characteristic curve (ROC) showed a very high diagnostic accuracy for distinguishing adenomas from non-tumor controls with an area under the curve (AUC) of 0.84 (p = 0.0047; 95% CI: 0.66-1.00) (Figure 5). These results indicate that analysis of the methylation status of the LINC00473 promoter region in colorectal tissue samples has excellent diagnostic accuracy for identifying patients with colorectal adenomas at risk of developing colorectal cancer.

[0056] To further assess the methylation status of colorectal adenomas and advanced colorectal adenomas, we analyzed DNA methylation levels of the LINC00473 promoter region by bisulfite pyrosequencing in a new, independent cohort of tissue samples, including 10 non-tumor controls and 50 adenomas (including 20 confirmed advanced colorectal adenomas). This analysis revealed significantly (p<0.0001) higher methylation levels in adenomas (20.96%±15.33%) than in healthy controls (5.70%±0.82%). Specifically, this analysis revealed significantly (p<0.0003) higher methylation levels in advanced colorectal adenomas (26.35%±19.66%) than in controls (5.70%±0.82%) (Figure 11). Indeed, receiver operating characteristic (ROC) analysis also demonstrated a very high diagnostic accuracy for distinguishing advanced colorectal adenomas from non-tumor controls, with an AUC of 0.88 (p<0.0008; 95% CI: 0.75-1.00) (Figure 12). These results confirm that the methylation level of the promoter region of LINC00473 is useful for detecting adenomas, especially advanced colorectal adenomas.

[0057] Example 2.2. Liquid biopsy Example 2.2.1. Promoter methylation analysis of LINC00473 in liquid biopsies detects colorectal cancer patients. Real-time PCR analysis of the DNA methylation levels (%) of the LINC00473 promoter region in the plasma of 28 control subjects and 26 colorectal cancer patients revealed significantly (p<0.0001) higher methylation levels in colorectal cancer patients (25.87% ± 32.73%) than in healthy controls (0.003% ± 0.009%) (Figure 6). Higher methylation levels of LINC00473 were detected in 21 of 26 colorectal cancer patients (81%) compared with healthy individuals. These data indicate that the methylation levels of the LINC00473 promoter region analyzed by liquid biopsy can distinguish between healthy individuals and patients with colorectal cancer.

[0058] Analysis of plasma LINC00473 methylation levels by receiver operating characteristic (ROC) curve showed a very high diagnostic accuracy for distinguishing colorectal cancer patients from healthy controls, with an area under the curve (AUC) of 0.87 (p<0.0001; CI 95%: 0.76-0.99) (Figure 7). Based on ROC curve analysis, a plasma LINC00473 methylation cutoff of 0.30% was obtained using Youden's index, which allowed for the detection of colorectal cancer with 81% sensitivity (CI 95%: 61%-93%) and 100% specificity (CI 95%: 88-100) (Table 7). Using this methylation level cutoff, a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 85% were obtained for the detection of colorectal cancer. These results indicate that noninvasive analysis of the methylation status of the LINC00473 promoter region in liquid biopsies has excellent diagnostic accuracy for identifying colorectal cancer patients. Table 7 shows the characteristics of methylation analysis of the LINC00473 promoter region as a diagnostic test for colorectal cancer detection.

[0059] [Table 7]

[0060] Furthermore, we also evaluated the DNA methylation level (%) of the LINC00473 promoter region in the plasma of colorectal cancer patients using droplet digital PCR (ddPCR) methodology. For this purpose, we used five control subjects and five colorectal cancer patients. These analyses showed significantly (p=0.005) higher methylation levels in colorectal cancer patients (41.19% ± 28.79%) than in healthy controls (0.0% ± 0.0%) (Figure 13). Higher methylation levels of LINC00473 were detected in 5 out of 5 (100%) colorectal cancer patients compared to healthy individuals, indicating that the methylation level of the LINC00473 promoter region analyzed by liquid biopsy with ddPCR can also distinguish healthy individuals from patients with colorectal cancer. Furthermore, receiver operating characteristic (ROC) analysis showed that the methylation level of LINC00473 in plasma obtained by ddPCR had a very high diagnostic accuracy for distinguishing colorectal cancer patients from healthy controls, with an area under the curve (AUC) of 1.0 (p<0.0062; CI 95%: 1.0–1.0) (Figure 14). These results indicate that noninvasive analysis of the methylation status of the LINC00473 promoter region in liquid biopsies by ddPCR has excellent diagnostic accuracy for identifying colorectal cancer patients.

[0061] Example 2.2.2. DNA methylation levels in the promoter region of LINC00473 in liquid biopsies predict outcome in colorectal cancer patients The clinical impact of LINC00473 promoter methylation levels on survival was investigated in plasma samples from 25 colorectal cancer patients (Table 2) using available clinical data. Colorectal cancer patients were classified as presenting either i) high levels of LINC00473 methylation (H: methylation level >10%) or ii) low levels of LINC00473 methylation (L: methylation level <10%) relative to the median methylation level of the group (10%). According to this patient stratification, Kaplan-Meier analysis showed that the presence of high levels of LINC00473 methylation was significantly associated with shorter overall survival (OS) [hazard ratio (HR) = 3.37, 95% confidence interval (CI) = 1.19–9.93, P = 0.0256] (Figure 8). These results indicate that the methylation status of LINC00473 in liquid biopsies can predict clinical outcomes in colorectal cancer patients, suggesting that analysis of LINC00473 methylation in plasma may be useful as a prognostic biomarker at the time of colorectal cancer diagnosis.

[0062] Example 2.2.3. Promoter methylation analysis of LINC00473 in liquid biopsies detects patients with advanced colorectal adenoma Real-time PCR analysis of the DNA methylation levels (%) of the LINC00473 promoter region in the plasma of five healthy controls and 12 patients with advanced colorectal adenoma revealed significantly (p=0.0262) higher methylation levels in patients with advanced colorectal adenoma (0.0676%±0.1195%) than in healthy controls (0.0001%±0.0002%) (Figure 9). These data indicate that the methylation levels of the LINC00473 promoter region analyzed by liquid biopsy can distinguish between healthy individuals and patients with advanced colorectal adenoma.

[0063] Analysis of the methylation level of LINC00473 in plasma by receiver operating characteristic curve (ROC) showed a very high diagnostic accuracy for distinguishing patients with advanced colorectal adenomas from healthy controls, with an area under the curve (AUC) of 0.85 (p = 0.00269; CI 95%: 0.65-1.0) (Figure 10). Based on ROC curve analysis, a methylation cutoff of 0.000115 for LINC00473 in plasma was obtained, which allowed for the detection of advanced adenomas with 92% sensitivity and 80% specificity (Table 8). Using this methylation level cutoff, a PPV of 92% and an NPV of 80% were obtained for the detection of advanced colorectal adenomas. These results demonstrate that noninvasive analysis of the methylation status of the LINC00473 promoter region in liquid biopsies has excellent diagnostic accuracy for identifying patients with advanced colorectal adenomas. Table 8 shows the characteristics of methylation analysis of the LINC00473 promoter region as a diagnostic test for the detection of advanced colorectal adenoma.

[0064] [Table 8]

[0065] Furthermore, the DNA methylation level (%) of the LINC00473 promoter region in plasma was also assessed by droplet digital PCR in five healthy controls and ten patients with advanced colorectal adenoma. These results showed significantly (p=0.038) higher methylation levels in patients with advanced colorectal adenoma (0.94%±0.99%) than in healthy controls (0.0%±0.0%) (Figure 15), indicating that the methylation level of the LINC00473 promoter region analyzed by liquid biopsy by ddPCR can also distinguish between healthy individuals and patients with advanced colorectal adenoma.

Claims

1. An in vitro method for aiding in the diagnosis of colorectal cancer and / or precancerous stages thereof, comprising determining the methylation status of at least the gene LINC00473 in a liquid biopsy obtained from a patient, wherein a higher level of methylation of the gene LINC00473 compared to a reference level of methylation of the gene LINC00473 measured in healthy control subjects indicates that the subject is suffering from colorectal cancer and / or precancerous stages thereof.

2. An in vitro method for aiding in the prognosis of colorectal cancer, comprising determining the methylation status of at least the gene LINC00473 in a liquid biopsy obtained from a patient, wherein a higher level of methylation of the gene LINC00473, compared to a reference level of methylation of the gene LINC00473 measured in a control patient, indicates a poor prognosis for the patient.

3. 3. The in vitro method of claim 1 or 2, wherein the liquid biopsy is a sample of plasma, blood or serum.

4. The in vitro method according to any one of claims 1 to 3, wherein said precancerous stage is colorectal adenoma.

5. The in vitro method according to any one of claims 1 to 4, wherein the methylation status of the gene LINC00473 is determined at least at the CpGs of the promoter region.

6. 6. The in vitro method according to any one of claims 1 to 5, wherein the methylation status of the gene LINC00473 is determined at least at the CpGs of the promoter region located between the chromosomal positions Chr6:166402081 and Chr6:166402638.

7. 7. The in vitro method according to any one of claims 1 to 6, wherein the methylation status of gene LINC00473 is determined at a CpG in the promoter region located at a chromosomal location selected from the group consisting of at least Chr6:166402638, and / or Chr6:166402474, and / or Chr6:166402463, and / or Chr6:166402457, and / or Chr6:166402416, and / or Chr6:166402379, and / or Chr6:166402375, and / or Chr6:166402364, and / or Chr6:166402081.

8. 8. An in vitro method according to any one of claims 1 to 7, wherein the diagnosis of colorectal cancer and / or precancerous stages thereof is confirmed by imaging techniques.

9. In vitro use of a kit comprising reagents for determining the methylation status of at least the gene LINC00473 to aid in the diagnosis and / or prognosis of colorectal cancer and / or its precancerous stages.

10. 10. The in vitro use according to claim 9, wherein the precancerous stage is colorectal adenoma.

11. 11. The in vitro use according to claim 9 or 10, wherein the methylation status of the gene LINC00473 is determined at least at the CpGs of the promoter region.

12. 12. The in vitro use according to any one of claims 9 to 11, wherein the methylation status of the gene LINC00473 is determined at least at the CpGs of the promoter region located between the chromosomal positions Chr6:166402081 and Chr6:166402638.

13. 13. The in vitro use according to any one of claims 9 to 12, wherein the methylation status of the gene LINC00473 is determined at a CpG in the promoter region located at a chromosomal location selected from the group consisting of at least Chr6:166402638, and / or Chr6:166402474, and / or Chr6:166402463, and / or Chr6:166402457, and / or Chr6:166402416, and / or Chr6:166402379, and / or Chr6:166402375, and / or Chr6:166402364, and / or Chr6:166402081.

14. The in vitro use according to any one of claims 9 to 13, wherein the diagnosis of colorectal cancer and / or precancerous stages thereof is confirmed by imaging techniques.

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