In vitro method for the diagnosis or prognosis of colorectal cancer or its precancerous stages

Abstract

The method of the present invention means 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 field of medicine. In particular, the present invention relates to an in vitro method for the diagnosis and / or prognosis of colorectal cancer (CRC) and / or its precancerous stage. The method of the present invention comprises determining the methylation state of gene LINC00473 (preferably gene LINC00473 in a liquid biopsy obtained from a patient), wherein a higher methylation level of gene LINC00473 relative to an established reference control level indicates the presence of colorectal cancer, its precancerous stage and / or a poor prognosis of the disease. Background Art

[0002] CRC, also known as colon cancer, rectal cancer, or bowel cancer, is cancer that develops in the colon or rectum (part of the large intestine). The vast majority of colorectal cancers are adenocarcinomas. This is because the colon has many glands within its tissue. When these glands undergo many changes at the genetic level, they progress in a predictable way, transforming from being benign to becoming an aggressive, malignant colon cancer. Colon adenomas, especially advanced colorectal adenomas (AA), while being a benign version of malignant adenocarcinomas, still have malignant potential if not removed (they are often removed due to their propensity to become malignant and lead to colon cancer).

[0003] Screening is an effective way to prevent and reduce deaths from colorectal cancer, and screening is recommended starting between the ages of 50 and 75. The best-known and most commonly used colorectal cancer screening test is called the fecal immunochemical test (FIT). The FIT detects blood in a stool sample, which can be a sign of precancer or cancer. If an abnormal result is obtained, a colonoscopy is often recommended, which allows the doctor to view the inside of the colon and rectum to make a diagnosis. During a colonoscopy, if a small polyp is found, it can be removed. If a large polyp or tumor is found, a biopsy may be performed to check if it is cancerous. Gastroenterologists use colonoscopies to find and remove these adenomas and polyps to prevent them from going on to acquire genetic changes that can lead to invasive adenocarcinomas.

[0004] As explained above, although FIT is now used to screen for colorectal cancer, it is important to note that FIT has a low sensitivity for AAs, meaning that most patients of the type described may be incorrectly classified as not having the disease. Therefore, FIT fails to identify adenomas due to its low sensitivity. Furthermore, since FIT uses stool samples, its compliance is low. On the other hand, colonoscopy is an invasive technique, where the most serious complication is usually gastrointestinal perforation. Furthermore, now, colonoscopy is a procedure that involves anesthesia, and the laxatives that are usually administered during the bowel preparation for colonoscopy have been associated with a variety of digestive problems.

[0005] It is important to note that current methods for screening the general population at risk for CRC or AA are associated with high false-positive rates. As a result, a large number of unnecessary follow-up colonoscopies are now performed.

[0006] The present invention provides a clear solution to the above-mentioned problems because it focuses on the in vitro method of identifying or screening human subjects at risk of colorectal cancer or colorectal adenoma (particularly advanced colorectal adenoma). Since the method of the present invention is preferably based on liquid biopsies (such as plasma, blood or serum) obtained from patients, it is expected to improve compliance with colorectal cancer screening. In addition, the method of the present invention provides high sensitivity and specificity, which means that it is a robust and cost-effective method for detecting colorectal cancer and colorectal adenoma. Summary of the invention

[0007] Brief description of the invention

[0008] The present invention relates to an in vitro method for the diagnosis or prognosis of colorectal cancer and / or its precancerous stage (hereinafter referred to as "the method of the present invention"). It is preferably implemented in a minimally invasive biological sample liquid biopsy obtained from a patient. The method of the present invention provides high sensitivity and specificity, which means that it is a robust and cost-effective method for detecting colorectal cancer and / or its precancerous stage.

[0009] Because 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, it is associated with a low false positive percentage. Therefore, the method described in the present invention significantly helps to reduce the number of subsequent colonoscopies, thereby improving the way in which patients are currently screened or diagnosed. Once the method of the present invention is performed, if it is determined that the patient may suffer from colorectal cancer and / or a precancerous stage, the result is confirmed by colonoscopy. However, if it cannot be determined that the patient may suffer from colorectal cancer and / or a precancerous stage, a colonoscopy is not required and routine testing is recommended using the method of the present invention.

[0010] In particular, a first embodiment of the present invention relates to an in vitro method for the diagnosis of colorectal cancer and / or a precancerous stage thereof, the method comprising determining the methylation status of at least gene LINC00473 in a liquid biopsy obtained from a patient, wherein a higher methylation level of gene LINC00473 compared to a methylation reference level of gene LINC00473 measured in a healthy subject is an indication that the subject has colorectal cancer and / or a precancerous stage thereof.

[0011] A second embodiment of the present invention relates to an in vitro method for the prognosis of colorectal cancer and / or a precancerous stage thereof, the method comprising determining the methylation status of at least gene LINC00473 in a liquid biopsy obtained from a patient, wherein a higher methylation level of gene LINC00473 compared to a methylation reference level of gene LINC00473 measured in the patient is an indication of a poor prognosis for the patient.

[0012] A third embodiment of the present invention relates to the in vitro use of at least the methylation status of the gene LINC00473 in the diagnosis and / or prognosis of colorectal cancer and / or its precancerous stage.

[0013] A fourth embodiment of the present invention relates to an in vitro use of a kit for the diagnosis and / or prognosis of colorectal cancer and / or its precancerous stage, the kit comprising reagents for determining the methylation status of at least the gene LINC00473.

[0014] In a preferred embodiment, the liquid biopsy is a plasma, blood or serum sample. Plasma is particularly preferred.

[0015] In a preferred embodiment, the precancerous stage is colorectal adenoma, preferably AA.

[0016] In a preferred embodiment, the methylation status of gene LINC00473 is determined in at least one CpG in the promoter region.

[0017] In a preferred embodiment, the methylation status of gene LINC00473 is determined in at least one CpG in the promoter region within 3000 bp around the transcription start site (TSS). This means 1500 bp upstream or 1500 bp downstream of the TSS.

[0018] In a preferred embodiment, the methylation status of gene LINC00473 is determined in at least one CpG located between chromosomal positions Chr6:166402081 and Chr6:166402638 in the promoter region.

[0019] In a preferred embodiment, the methylation status of gene LINC00473 is determined in at least one CpG in the promoter region located at a chromosomal position selected from the group consisting of: 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, in a CpG selected from the group consisting of cg06545143, and / or cg08886973, and / or cg21306006.

[0020] In a preferred embodiment, the diagnosis of colorectal cancer and / or its precancerous stages is confirmed by imaging techniques, preferably colonoscopy.

[0021] According to the method of the present invention, after measuring the methylation state of gene LINC00473, a score value is obtained and the score value is 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, which is an indication that the patient may have colorectal cancer and / or a precancerous stage thereof. The threshold value has been defined in order to optimize the sensitivity and specificity values. Therefore, in a preferred embodiment, the method of the present invention comprises: a) measuring the methylation state of gene LINC00473 in a liquid biopsy obtained from a subject, b) processing the methylation value to obtain a risk score and c) wherein if it is identified that the obtained risk score value is biased or changed compared to the reference value, this indicates that the subject has colorectal cancer and / or a precancerous stage thereof.

[0022] The last embodiment of the present invention relates to a method for diagnosing and treating colorectal cancer or a precancerous stage thereof, the method comprising: a) obtaining a liquid biopsy from a human subject; b) detecting the methylation status of gene LINC00473, and c) when the methylation level of gene LINC00473 is identified to be higher than a methylation reference level of gene LINC00473 measured in a healthy subject, diagnosing the patient with colorectal cancer or a precancerous stage thereof, and performing a colonoscopy on the patient. The colonoscopy may include removing colorectal cancer or polyps.

[0023] For the purposes of this invention, the following terms are defined:

[0024] LINC00473 (long intergenic non-protein coding RNA 473) is an RNA gene belonging to the class of non-coding RNA. It can be identified in public databases as follows: HGNC: 21160. Entrez Gene: 90632. Ensambl ID: ENSG00000223414. UniProtKB: A8K010.

[0025] • The term "colorectal cancer" is a medical condition characterized by cancer of the cells of the intestine after the small intestine, ie the large intestine (colon), including the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum.

[0026] The expression "colorectal adenoma" refers to colon adenomas, also called adenomatous polyps, which are benign and precancerous stages of colorectal cancer but still carry a high risk of progression to colorectal cancer.

[0027] The expression "advanced colorectal adenoma" refers to: an adenoma that is at least 10 mm in size or has high-grade dysplasia or a villous component greater than 20% on histology; a serrated lesion that is at least 10 mm in size or has dysplasia. For a more detailed explanation of the concept of "advanced colorectal adenoma", please refer to the following scientific articles that are common 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 2017Jun 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 onserrated polyps in the colon and rectum.Gut.2017 Jul;66(7):1181-1196.doi:10.1136 / gutjnl-2017-314005.Epub 2017Apr 27.Review.PMID:28450390].

[0028] The expression "liquid biopsy" refers to any biological fluid sample that may contain tumor derivatives. In particular, a liquid biopsy is any biological fluid (e.g., plasma, serum, blood, saliva, cerebrospinal fluid or urine) sample that may contain tumor derivatives (e.g., circulating tumor DNA).

[0029] The expression "minimally invasive biological sample" refers to any sample collected from a patient's body without using harmful instruments other than a fine needle used to collect the patient's blood, and thus without causing harm to the patient. Specifically, the minimally invasive biological sample in the present invention refers to: a blood, serum or plasma sample.

[0030] • As used herein, the term "methylation" will be understood to mean the presence of a methyl group added to a cytosine base or bases in a region of nucleic acid (eg, DNA) by the action of a DNA methyltransferase.

[0031] • Therefore, the term "methylation status" as used herein refers to the methylation level measured in the patient being analyzed. "Methylation status" may be a higher / lower methylation level compared to a reference or control level.

[0032] As used herein, "CpG dinucleotide", "CpG methylation site" or equivalents should be considered to mean a cytosine linked to a guanine via a phosphodiester bond. CpG dinucleotides are targets for methylation of cytosine residues and may be present in coding or non-coding nucleic acids. Non-coding nucleic acids are understood in the art to include introns, 5'-untranslated regions, 3'-untranslated regions, promoter regions of genomic genes, or intergenic regions.

[0033] The expression "target CpG" refers to a specific CpG that is found to be differentially methylated in the present invention.

[0034] The expression "contiguous CpGs" refers to CpGs that are potentially differentially methylated and can be found up to 1500 bp upstream or downstream of the transcription start site (TSS) (3000 bp around the TSS).

[0035] As used in the present invention, "determining the methylation state of a gene" means performing the method of the present invention by measuring the methylation state of any region of a gene, which region also contains any regulatory sequence capable of increasing or decreasing the expression of the gene. In particular, the regulatory sequence includes a promoter, which is a DNA region that initiates transcription of a gene. The promoter is located near the transcription start site of the gene, on the same strand, upstream or downstream of the DNA (towards the 5' region of the sense strand).

[0036] As used herein, "control, reference or baseline level of methylation" is understood to mean the methylation level detected in the corresponding nucleic acid from a healthy patient. Therefore, if the "methylation level is higher" of the patient is measured compared to the "control, reference or baseline level of methylation" measured in a healthy patient, then at a given sensitivity and specificity, the patient may suffer from CRC or AA. "Reference value" can be a threshold value or a cutoff value. Usually, "threshold value" or "cutoff value" can be determined by experiment, experience or theory. As will be appreciated by those of ordinary skill in the art, the threshold value can also be arbitrarily selected based on existing experiments and / or clinical conditions. The threshold value must be determined so as to obtain the best sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical consequences of false positives and false negatives). Preferably, the biomarker level (or score) obtained according to the method of the present invention can be compared with a defined threshold value by those skilled in the art. Usually, the best sensitivity and specificity (and threshold value) can be determined based on experimental data using a receiver operating characteristic (ROC) curve. For example, after determining the methylation level of the biomarker in a set of references, algorithmic analysis can be used to statistically process the methylation measurement level of the biomarker in the biological sample to be tested, thereby obtaining a significant classification standard for sample classification. The full name of the ROC curve is the receiver operating characteristic curve, also known as the receiver operating characteristic curve. It is mainly used in clinical biochemical diagnostic tests. The ROC curve is a comprehensive indicator that reflects the continuous variables of the true positive rate (sensitivity) and the false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity through an image synthesis method. A series of different cutoff values ​​(thresholds or critical values, the boundary values ​​between normal and abnormal results in diagnostic tests) are set as continuous variables to calculate a series of sensitivity and specificity values. Then use sensitivity as the ordinate and specificity as the abscissa to draw the curve. The larger the area under the curve (AUC), the higher the accuracy of the diagnosis. On the ROC curve, the point closest to the upper left corner of the coordinate graph is the critical point with high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, as AUC approaches 1, the diagnostic result is getting better and better. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is good. When AUC is higher than 0.9, the accuracy is quite high. This algorithm is preferably performed by computer. Software or systems available in the art can be used to draw ROC curves, for example: MedCalc 9.2.0.1 medical statistics software, SPSS 9.0, preferably GraphPadPrism 6.

[0037] The expression "higher level 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 invention, "higher level of methylation" is determined with reference to a baseline level represented by the methylation state of a given genomic region in a sample obtained from a subject or patient. For example, a "higher level of methylation" may be at least 2% higher than a baseline level of methylation, such as at least 5% higher than a baseline level of methylation, or at least 10% higher than a baseline level of methylation, or at least 15% higher than a baseline level of methylation, or at least 20% higher than a baseline level of methylation, or at least 25% higher than a baseline level of methylation, or at least 30% higher than a baseline level of methylation, or at least 40% higher than a baseline level of methylation, or at least 50% higher than a baseline level of methylation, or at least 60% higher than a baseline level of methylation, or at least 70% higher than a baseline level of methylation, or at least 80% higher than a baseline level of methylation, or at least 90% higher than a baseline level of methylation.

[0038] • “Including” means including but not limited to whatever follows the word “including.” Thus, the use of the term “including” indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present.

[0039] • “Consisting of means “including and limited to” whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and no other elements may be present. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 .DNA methylation level of LINC00473 promoter in colorectal tissue samples. In cohorts 1 (A) and 2 (B), colorectal cancer patients showed significantly higher LINC00473 methylation levels than non-tumor controls. (A) The methylation level of LINC00473 promoter was analyzed by InfiniumHumanMethylation450K BeadChip (450K array) and (B) by pyrosequencing. DNA methylation data are expressed as β values ​​in A and as methylation percentages in B. The horizontal line represents the median of the methylation level. P, indicates the p value analyzed by the Mann-Whitney U test. Tumor, represents colorectal cancer.

[0041] Figure 2Receiver operating characteristic (ROC) curves of LINC00473 promoter methylation in colorectal tissue samples. The area under the ROC curve (AUC) in cohort 1 (A) and cohort 2 (B) showed the high diagnostic accuracy of LINC00473 promoter methylation in distinguishing colorectal cancer patients from non-tumor controls. P, indicates the p-value of the ROC curve. CI, is the confidence interval.

[0042] Figure 3 DNA methylation levels of LINC00473 promoter in colorectal tissue samples obtained from The Cancer Genome Atlas (TCGA) according to their clinical stages. Colorectal cancer patients of all stages showed significantly higher LINC00473 methylation levels than their respective non-tumor controls (cohort 1: 0.11 ± 0.006; cohort 2: 5.84% ± 0.78%) 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%). Methylation levels of LINC00473 (A) were analyzed by Infinium HumanMethylation450K BeadChip (450K array) and expressed as β values, and (B) were analyzed by pyrosequencing and expressed as methylation percentages. Asterisks (*) indicate P < 0.0001 compared to controls. p values ​​were analyzed by Mann-Whitney U test. Tumors represent colorectal cancer.

[0043] Figure 4 .DNA methylation level of LINC00473 promoter in adenoma. Adenoma and colorectal cancer patients showed significantly higher LINC00473 methylation levels than non-tumor controls. The methylation level of LINC00473 promoter was analyzed by pyrosequencing and expressed as methylation percentage. The horizontal line represents the median of methylation level. P, indicates the p value analyzed by Mann-Whitney U test.

[0044] Figure 5 Receiver operating characteristic (ROC) curve of LINC00473 promoter methylation in adenoma. The area under the ROC curve (AUC) of cohort 3 showed the high diagnostic accuracy of LINC00473 promoter methylation in distinguishing adenoma from non-tumor controls. P, indicates the p-value of the ROC curve. CI, is the confidence interval.

[0045] Figure 6DNA 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. The methylation level of LINC00473 was analyzed in triplicate by real-time PCR. The horizontal line represents the median of the methylation level. P, indicates the p value analyzed by the Mann-Whitney U test.

[0046] Figure 7 Receiver operating characteristic (ROC) curve of LINC00473 promoter methylation in circulating plasma DNA of colorectal cancer patients relative to normal controls. The area under the ROC curve (AUC) showed the high diagnostic accuracy of LINC00473 promoter methylation in distinguishing colorectal cancer patients from healthy individuals. P, indicates the p-value of the ROC curve.

[0047] Figure 8 .Kaplan-Meier survival analysis of LINC00473 promoter methylation in patients with colorectal cancer. Colorectal cancer patients were classified according to higher or lower LINC00473 methylation levels relative to the median methylation level of the group (10% methylation). Kaplan-survival analysis showed that high methylation levels of LINC00473 were significantly associated with shorter overall survival (OS) (P=0.0256). P, indicates the p value of the log-rank test. H (high) indicates a methylation level >10%; and L (low), a methylation level <10%.

[0048] Fig. 9 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. The methylation level of LINC00473 was analyzed in triplicate by real-time PCR. The horizontal line represents the median of the methylation level. P, indicates the p value analyzed by the Mann-Whitney U test.

[0049] Fig.10 Receiver operating characteristic (ROC) curve of LINC00473 promoter methylation in circulating plasma DNA of advanced colorectal adenomas relative to normal controls. The area under the ROC curve (AUC) showed the high diagnostic accuracy of LINC00473 promoter methylation in distinguishing advanced colorectal adenomas from healthy controls. P, indicates the p-value of the ROC curve. CI, is the confidence interval.

[0050] Fig.11.DNA methylation level of LINC00473 promoter in advanced colorectal adenomas from tissues. Advanced colorectal adenomas showed significantly higher LINC00473 methylation levels than non-tumor controls. The methylation level of LINC00473 promoter was analyzed by pyrosequencing and expressed as methylation percentage. The horizontal line represents the median of methylation level. P, indicates the p value analyzed by Mann-Whitney U test.

[0051] Fig.12 .Receiver operating characteristic (ROC) curve of LINC00473 promoter methylation in advanced colorectal adenomas from tissues. The area under the ROC curve (AUC) showed the high diagnostic accuracy of LINC00473 promoter methylation in distinguishing advanced colorectal adenomas from non-tumor controls. P, indicates the p-value of the ROC curve. CI, is the confidence interval.

[0052] Fig.13 .DNA methylation levels of LINC00473 promoter in circulating plasma DNA of colorectal cancer obtained by droplet digital PCR (ddPCR). Colorectal cancer patients showed significantly higher LINC00473 methylation levels than non-tumor controls. The methylation level of LINC00473 promoter was analyzed in triplicate by ddPCR. The horizontal line represents the median of the methylation level. P, indicates the p value analyzed by Mann-Whitney U test.

[0053] Fig.14 Receiver operating characteristic (ROC) curve of LINC00473 promoter methylation in circulating plasma DNA of colorectal cancer patients relative to normal controls analyzed by droplet digital PCR (ddPCR). The area under the ROC curve (AUC) showed the high diagnostic accuracy of LINC00473 promoter methylation in distinguishing colorectal cancer patients from healthy individuals. P, indicates the p-value of the ROC curve.

[0054] Fig.15 DNA methylation levels of the LINC00473 promoter in circulating plasma DNA of advanced colorectal adenomas obtained by droplet digital PCR. Patients with advanced colorectal adenomas showed significantly higher LINC00473 methylation levels than healthy controls. The methylation level of LINC00473 was analyzed in triplicate by droplet digital PCR. The horizontal line represents the median of the methylation level, and P indicates the p-value analyzed by the Mann-Whitney U test. DETAILED DESCRIPTION

[0055] Example 1. Method.

[0056] Example 1.1. Study Participants for Colorectal Tissue Analysis.

[0057] We initially analyzed 3 independent cohorts (cohort 1, cohort 2, and cohort 3) to study DNA methylation of LINC00473 in colorectal tissues. Cohort 1 represented patients with primary colorectal cancer (n=273) and non-tumor controls (n=38). Cohort 2 included 180 patients with primary colorectal cancer and 93 non-tumor controls. And in cohort 3, there were 12 patients with primary colorectal cancer, 12 adenomas, and 12 non-tumor controls.

[0058] In addition, for the purpose of this PCT application, we also analyzed a new independent cohort (cohort 4) to investigate DNA methylation of LINC00473 in colorectal tissues, which included 50 colorectal adenomas (including 20 confirmed advanced colorectal adenomas) and 10 non-tumor controls.

[0059] Example 1.2. Analysis of DNA methylation data of colorectal tissue by 450K array.

[0060] The DNA methylation results of cohort 1 were analyzed by InfiniumHumanMethylation450K BeadChip data (450K array) obtained from the public database Cancer Genome Atlas (TCGA). The 450K array (Illumina) covers >450,000 CpG sites in the human genome, and the methylation score of each CpG is expressed as a beta (β) value ranging from 0.0 (completely unmethylated) to 1.0 (completely methylated).

[0061] Example 1.3. Bisulfite pyrosequencing analysis of colorectal tissue.

[0062] The DNA methylation status of the LINC00473 promoter in cohorts 2, 3, and 4 was analyzed by bisulfite pyrosequencing. After bisulfite conversion of ~500 ng of DNA using the EZ-96 DNA methylation kit (Zymo Research Corp.), the DNA methylation level of the LINC00473 promoter was analyzed using the PyroMark Q96 system (Qiagen) according to the manufacturer's instructions. CpG site methylation quantification was obtained using Pyro Q-CpG 1.0.9 (Qiagen). Primer sequences were designed by PyroMark Assay Design 2.0 (Qiagen) (Table 1). Table 1 shows the primers for methylation analysis of LINC00473 by pyrosequencing.

[0063] Table 1

[0064]

[0065] F: forward; R: reverse; S: sequencing; nt: nucleotide

[0066] In a preferred embodiment, the primer used in the present invention, preferably the primer SEQ ID NO: 2, is biotinylated by introducing a biotin molecule.

[0067] Example 1.4. Study Participants for Liquid Biopsy Analysis.

[0068] To analyze LINC00473 in colorectal cancer liquid biopsies, the study included 26 patients with advanced colorectal cancer (stages III and IV) prospectively recruited at diagnosis (baseline), before the start of treatment and surgery. The demographic and clinical characteristics of the patients are shown in Table 2. Twenty-eight healthy controls were also included in this study. Medical oncologists at the Department of Oncology of Complejo Hospitalario Universitario de Santiago de Compostela (CHUS) obtained disease status and stage of colorectal cancer patients. Table 2 shows the clinical characteristics of colorectal cancer patients.

[0069] Table 2

[0070]

[0071] SD: Standard deviation

[0072] To analyze LINC00473 in liquid biopsies of advanced colorectal adenomas, the study included 5 healthy controls and 12 patients with advanced adenomas. All individuals for liquid biopsy analysis were recruited at the Complejo Hospitalario Universitario de Santiago de Compostela (CHUS).

[0073] To analyze LINC00473 in colorectal cancer liquid biopsies by droplet digital PCR (ddPCR), the study included five patients with advanced colorectal cancer prospectively recruited at diagnosis (baseline), before the start of treatment and surgery. Ten patients with advanced adenomas and five healthy controls were also included in the study. Patients and controls were obtained at the Complejo Hospitalario Universitario deSantiago de Compostela (CHUS).

[0074] Example 1.5. Blood sample collection and plasma separation.

[0075] Blood samples were obtained by phlebotomy using collection tubes containing EDTA as an anticoagulant. Plasma was separated from the blood samples by first centrifugation (1500 g, 10 minutes, 4° C.) of the blood tubes within 2 hours of collection. Plasma was transferred to 1.5 mL tubes and centrifuged a second time (15000 g, 10 minutes, 4° C.). Plasma was transferred to new 1.5 mL tubes and stored at -80° C.

[0076] Example 1.6. Isolation of circulating DNA from plasma samples.

[0077] After thawing, plasma samples were collected using Circulating Nucleic Acid Kit( Circulating Nucleic Acid Kit (Qiagen) and vacuum system QIAvac 24Plus (Qiagen) were used to separate circulating DNA from 2-3 mL of plasma. Finally, circulating DNA was eluted in 75 μL elution buffer, and 1 μL of the elution buffer was passed through the kit. ONE dsDNA and Quantus TM Fluorometer (Quantus TM The concentration of circulating DNA was quantified using a 4% RT-PCR amplification kit (Fluorometer; Promega). After quantification, the circulating DNA was stored at -80°C.

[0078] Example 1.7. Bisulfite conversion of circulating DNA.

[0079] For DNA bisulfite conversion, 15-50 ng of circulating DNA was used and converted using the EZ DNA Methylation-Lightning Kit (Zymo Research) according to the manufacturer's recommendations with the following thermal cycling steps: 98° C., 8 min; 54° C., 60 min; and 4° C. After column-based purification through binding, L-desulfonation, and washing steps, bisulfite-converted DNA (bis-DNA) was eluted in 15 μL elution buffer and stored at −80° C.

[0080] Example 1.8. DNA methylation analysis of the LINC00473 promoter region in liquid biopsies by real-time PCR.

[0081] The methylation status of the promoter region of the intergenic long non-protein coding RNA 473 (LINC00473; formerly code C6orf176) in circulating plasma DNA was analyzed. The non-coding gene LINC00473 (NCBI ID: 90632; UCSC ID: uc063sul.1; Ensambl ID: ENSG00000223414; HGNC ID: HGNC: 21160) is located at chromosome 6 (chr) position 6q27 and has a CpG island containing its promoter region located at chr6:166401527-166402659. In addition, the promoter region of LINC00473 overlaps with the main body of intergenic long non-protein coding RNA 602 or LINC00602 (NCBIID: 441177; UCSC ID: uc011egm.4; Ensambl ID: ENSG00000281832; HGNC ID: 43917) in the genome.

[0082] The methylation level of the LINC00473 promoter region was determined by real-time PCR and methylation-specific quantitative PCR assay (qMSP) in the StepOne Plus system (Applied Biosystems). Each reaction contained 2uL of bisulfite-converted DNA (bis-DNA) as template, 10μl Power SYBR TM Green PCR Master Mix (ThermoFisher) and 150 nM each of the forward and reverse primers (Table 3) to detect methylation or unmethylation in a total volume of 20 μl. TM Fast optical 96-well reaction plate (MicroAmp TMFast Optical 96-Well Reaction Plate; Applied Biosystems). The thermal cycling conditions in the StepOne Plus system were: 95°C, 10 minutes; followed by 94°C for 15 seconds and 60°C for 30 seconds, 50 cycles. Water was included as a non-template control in each run to demonstrate that there was no contamination during the reaction. Colorectal cancer cell line HCT-116 and normal leukocytes (NL) were used as methylated and unmethylated positive controls in each analysis, respectively. All samples and controls were analyzed in triplicate. The threshold cycle (Ct) of each reaction was determined using the software StepOne Real-Time PCR (Applied Biosystems) using specific primers for methylation and unmethylation. Based on Cottrell et al. (2007), the DNA methylation level of each sample was expressed as a percentage (%) of methylation according to the following calculation:

[0083] Methylation (%) = 100 / [1+2^(CT CG –CT TG )]

[0084] In this formula, CT CG Indicates the threshold cycle of methylation status, CT TG The threshold cycle indicates the unmethylated state. By this calculation, the methylation level can range from 0% methylation to 100% methylation. Table 3 shows the primers used for methylation analysis of LINC00473 by real-time PCR.

[0085] Table 3

[0086]

[0087] F: forward; R: reverse; nt: nucleotide; bp: base pair.

[0088] (*) Location: chr6:166402364-166402477; (#) Location:chr6:166402363-166402478.

[0089] The forward and reverse primers used for LINC00473 methylation analysis can detect the methylation status of 6 CpGs (Table 4) located in the promoter region of LINC00473. According to Infinium Human Methylation 450K and Infinium Methylation EPIC assays (Illumina), one of the CpGs (chr6: 166402416) contained in the sequence (amplicon) amplified by real-time PCR corresponds to a CpG called cg08886973, which are microarray assays capable of detecting the methylation status of more than 450,000 and 850,000 CpGs, respectively. These microarrays were also able to detect additional CpGs from the LINC00473 promoter, which were identified as cg06545143 (chr6:166402638) and cg21306006 (chr6:166402081), located 162 nucleotides upstream and 282 nucleotides downstream, respectively, of the LINC00473 amplicon analyzed in this study. The three CpGs included in this microarray analysis (cg06545143, cg08886973, cg21306006) are located in the TSS1500 promoter region of LINC00473, which is the promoter region 200 to 1500 nucleotides upstream of its transcription start site (TSS). All chromosomal positions previously indicated are based on the UCSC Genome Browser (https: / / genome.ucsc.edu / index.html) version GRCh37 / hg19 of the University of California. Table 4 shows the CpGs of LINC00473 detected by real-time PCR in methylation analysis.

[0090] Table 4

[0091]

[0092] Example 1.9. DNA methylation analysis of the LINC00473 promoter region in liquid biopsies by droplet digital PCR (ddPCR).

[0093] The methylation level of the LINC00473 promoter region was determined by droplet digital PCR (ddPCR) in the QX200 system (Bio-Rad). First, ~2 ng of bisulfite-converted DNA (bis-DNA), 25 μl of SsoAdvanced TMPreAmp Supermix (Bio-Rad), 0.5 μl of a customized Bio-Rad assay containing forward and reverse primers and probes (Bio-Rad) for methylation (Table 5), and 0.5 μl of a customized Bio-Rad assay containing forward and reverse primers and probes (Table 5) for unmethylation, were used for multiplex preamplification reactions in a total volume of 50 μl. The final volume was made up with water. Reactions were performed on a ProFlex PCR system (Applied Biosystems) in 0.2 ml PCR tubes (Axygen): 95°C for 3 minutes; 95°C for 15 seconds and 56.2°C for 4 minutes, 10 cycles; and a final hold step of 4°C.

[0094] Next, a multiplex mixed reaction was performed, which contained 2uL of the previously obtained preamplification product, 11μl of ddPCR Supermix for Probe (ddPCR Supermix for Probe; without dUTP) (Bio-Rad), 2.2μl of a customized Bio-Rad assay containing forward and reverse primers and probes for methylation (Table 5), and 2.2μl of a customized Bio-Rad assay containing forward and reverse primers and probes for unmethylation (Bio-Rad) (Table 5), with a total volume of 22μl. The final volume was made up with water. Then, 20uL of each mixed reaction and 70μl of droplet generation oil (Droplet Generation oil; Bio-Rad) were transferred to DG8 TM The sample and oil wells of the Cartridge (BIO-RAD) were loaded on the QX200 TM Droplet generator (QX200 TM 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; 95°C for 15 s and 56.2°C for 30 s, 40 cycles; 98°C for 10 min, and a final hold step at 4°C. The temperature gradient increment for all steps was 2.5°C / s. Then, the QX200 TMThe 96-well plates were read on a Droplet Reader (Bio-Rad). Water was included as a non-template control in each run to demonstrate that there was no contamination during the reaction. In each analysis, the colorectal cancer cell line HCT-116 and normal leukocytes (NL) were used as methylated and unmethylated positive controls, respectively. All controls were included in each plate with and without a pre-amplification step. All samples and controls were analyzed in triplicate. Data analysis was performed using QuantaSoft software (Bio-Rad).

[0095] The DNA methylation level of each sample was expressed as the methylation percentage (%) according to the following calculation:

[0096] Methylation (%) = [M / (U+M)] × 100

[0097] In this formula, M (methylated) indicates the number of copies / μl of the target DNA molecule of the methylated probe (FAM probe), and U (unmethylated) indicates the number of copies / μl of the target DNA molecule of the unmethylated probe (HEX probe). By this calculation, the methylation level can range from 0% methylation to 100% methylation. Table 5 shows the sequences of primers and probes used for methylation analysis of LINC00473 by ddPCR. Based on these sequences, two customized Bio-Rad analytical reagents containing primers and probes for methylation and unmethylation, respectively, were obtained from Bio-Rad according to the manufacturer's recommendations: the ratio of primers to probes: 1.8, the primer concentration in the final reaction was 450nM, and the probe concentration in the final reaction was 250nM.

[0098] Table 5

[0099]

[0100] F: forward; R: reverse; P: probe; nt: nucleotide; bp: base pair.

[0101] FAM: fluorescein; HEX: hexachlorofluorescein

[0102] (*) Location: chr6:166402364-166402477; (#) Location:chr6:166402363-166402478.

[0103] The forward and reverse primers and probes used for methylation analysis of LINC00473 by ddPCR were able to detect the methylation status of 9 CpGs (Table 6) located in the promoter region of LINC00473. According to the Infinium HumanMethylation 450K and Infinium MethylationEPIC assays (Illumina), one of the CpGs (chr6: 166402416) corresponds to the CpG called cg08886973, is included in the amplified sequence (amplicon), and is detected with the methylated and unmethylated probes indicated in Table 5. All CpG positions of LINC00473 detected in the methylation analysis by ddPCR are shown in Table 6. All chromosomal positions indicated in Table 6 are based on the UCSC Genome Browser (https: / / genome.ucsc.edu / index.html) of the University of California, GRCh37 / hg19 version.

[0104] Table 6

[0105]

[0106] Example 1.10. Statistical analysis.

[0107] The Kolmogorov-Smirnov test was first used to assess the normality of the data distribution. Subsequently, the nonparametric Mann-Whitney U test was used for data comparison. To evaluate the diagnostic accuracy of LINC00473 methylation in detecting colorectal cancer, a receiver operating characteristic (ROC) curve was performed. The Youden index (J), which allows obtaining the cutoff point of methylation 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). To evaluate the survival analysis, the Kaplan-Meier method was performed, and the log-rank (Mantel-Cox) test was used to examine the differences between the methylated and unmethylated groups. Statistical analysis and graphical representation were performed using SPSS or GraphPad Prism 7.0 software. All p values ​​presented were calculated using a two-tailed test and were considered significant when p < 0.05.

[0108] Example 2. Results.

[0109] Example 2.1. Tissue samples.

[0110] Example 2.1.1. Methylation analysis of the LINC00473 promoter in tumor tissue can detect colorectal cancer patients.

[0111] After analysis by the InfiniumHumanMethylation450K BeadChip (450K array) from the international public database Cancer Genome Atlas (TCGA), the DNA methylation levels of the LINC00473 promoter region of colorectal tissue samples (273 patients with stage I, II, III and IV colorectal cancer, 38 non-tumor controls) were obtained (cohort 1). In this analysis, the methylation data of the 450K array were expressed as beta (β) average values, which were defined as 0.0 to 1.0. We focused our analysis on the CpG in the LINC00473 promoter region, identified as cg08886973 in the 450K array. The analysis showed that the methylation levels of colorectal cancer patients (0.51±0.011) were significantly (p<0.0001) higher than those of non-tumor controls (0.11±0.006) ( Figure 1 A). 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 1 B). In this case, the methylation values ​​obtained by pyrosequencing are expressed as a methylation percentage (%), which is defined as 0% to 100%. In summary, these data indicate that the methylation level of the analyzed LINC00473 promoter region in colorectal tissues can distinguish between non-tumor individuals and colorectal cancer patients.

[0112] Analysis of the methylation level of LINC00473 in colorectal tissue samples from cohort 1 by receiver operating characteristic curve (ROC) showed very high diagnostic accuracy in 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; CI 95%: 0.91-0.97) ( Figure 2 A). Similar results were obtained from cohort 2 (AUC = 0.90; p < 0.0001; CI 95%: 0.86-0.94) ( Figure 2 B). These results indicate that analyzing the methylation status of the LINC00473 promoter region in colorectal tissue samples has high diagnostic accuracy in identifying patients with colorectal cancer.

[0113] Importantly, when we compared the data from 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 ) were stratified by the stage of colorectal cancer patients in the cohort, the results also showed that the methylation level of colorectal cancer patients in each stage was significantly higher (p<0.0001) compared with non-tumor controls (cohort 1, n=38; cohort 2, n=93).

[0114] Example 2.1.2. Methylation analysis of the LINC00473 promoter in colorectal tissue allows identification of adenomas and advanced colorectal adenomas.

[0115] The DNA methylation levels of the LINC00473 promoter region in colorectal tissues of 12 non-tumor controls, 12 adenoma patients, and 12 colorectal cancer patients (cohort 3) were analyzed by bisulfite pyrosequencing and expressed as methylation percentage. These results showed that the methylation level in adenoma (28.21% ± %) was significantly higher (p = 0.0036) than that in healthy controls (7.28% ± 0.81%) ( Figure 4 ). As expected, colorectal cancer patients (32.83% ± 5.19%) also showed significant (P = 0.002) differences in methylation levels relative 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 analyzed LINC00473 promoter region in colorectal tissues can distinguish non-tumor individuals from adenoma patients, suggesting that the methylation status of the LINC00473 promoter can be used for early detection of colorectal cancer.

[0116] Analysis of the methylation level of LINC00473 in colorectal tissue samples of non-tumor and adenoma samples of cohort 3 showed a very high diagnostic accuracy in distinguishing adenoma from non-tumor controls by receiver operating characteristic curve (ROC), 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 a high diagnostic accuracy in identifying patients with colorectal adenomas who are at risk of developing colorectal cancer.

[0117] In addition, to evaluate the methylation status of colorectal adenomas and advanced colorectal adenomas, we analyzed the DNA methylation level of the LINC00473 promoter region in a new independent tissue sample cohort with 10 non-tumor controls and 50 adenomas, including 20 confirmed advanced colorectal adenomas, by bisulfite pyrosequencing. The analysis showed that the methylation level in adenomas (20.96% ± 15.33%) was significantly (p < 0.0001) higher than that in healthy controls (5.70% ± 0.82%). Specifically, the analysis showed that the methylation level of advanced colorectal adenomas (26.35% ± 19.66%) was significantly (p < 0.0003) higher than that of controls (5.70% ± 0.82%) ( Fig.11 ). In fact, receiver operating characteristic curve (ROC) analysis also showed a very high diagnostic accuracy in distinguishing advanced colorectal adenomas from non-neoplastic controls, with an AUC of 0.88 (p<0.0008; 95% CI: 0.75-1.00)( Fig.12 These results confirmed that the methylation level of the LINC00473 promoter region can be used to detect adenomas, especially for detecting advanced colorectal adenomas.

[0118] Example 2.2. Liquid biopsy

[0119] Example 2.2.1. Methylation analysis of the LINC00473 promoter in liquid biopsies to detect colorectal cancer patients.

[0120] The DNA methylation levels (%) of the LINC00473 promoter region in the plasma of 28 controls and 26 colorectal cancer patients were analyzed by real-time PCR, showing that the methylation level in colorectal cancer patients (25.87% ± 32.73%) was significantly (p < 0.0001) higher than that in healthy controls (0.003% ± 0.009%) ( Figure 6 ). Higher LINC00473 methylation levels were detected in 21 of 26 colorectal cancer patients (81%) compared with healthy individuals. These data suggest that the methylation level of the LINC00473 promoter region analyzed in liquid biopsies can distinguish between healthy individuals and colorectal cancer patients.

[0121] Analysis of LINC00473 methylation levels in plasma by receiver operating characteristic curve (ROC) showed very high diagnostic accuracy in 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 and using the Youden index value, a methylation cutoff value of 0.30% was obtained for LINC00473 in plasma, which allowed the detection of colorectal cancer with a sensitivity of 81% (CI 95%: 61%-93%) and a specificity of 100% (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 non-invasive analysis of the methylation status of the LINC00473 promoter region in liquid biopsies has a high diagnostic accuracy in identifying patients with colorectal cancer. Table 7 shows the characteristics of LINC00473 promoter region methylation analysis as a diagnostic test for colorectal cancer detection.

[0122] Table 7

[0123]

[0124] VPP: positive predictive value; VPN: negative predictive value

[0125] In addition, the droplet digital PCR (ddPCR) method was used to evaluate the DNA methylation level (%) of the LINC00473 promoter region in the plasma of colorectal cancer patients. For this purpose, we used 5 controls and 5 colorectal cancer patients. These analyses showed that the methylation level of colorectal cancer patients (41.19% ± 28.79%) was significantly (p = 0.005) higher than that of healthy controls (0.0% ± 0.0%) ( Fig.13 ). Higher methylation levels of LINC00473 were detected in 5 of 5 colorectal cancer patients (100%) relative to healthy individuals, indicating that the methylation level of the LINC00473 promoter region analyzed by ddPCR in liquid biopsy is also able to distinguish between healthy individuals and colorectal cancer patients. In addition, receiver operating characteristic curve (ROC) analysis showed that the methylation level of LINC00473 in plasma obtained by ddPCR had a very high diagnostic accuracy in 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)( Fig.14 These results suggest that non-invasive analysis of the methylation status of the LINC00473 promoter region in liquid biopsies by ddPCR has high diagnostic accuracy in identifying patients with colorectal cancer.

[0126] Example 2.2.2. The DNA methylation level of the LINC00473 promoter region in liquid biopsies predicts the outcome of colorectal cancer patients.

[0127] The clinical impact of LINC00473 promoter methylation levels on survival in plasma samples of 25 colorectal cancer patients from Table 2 was studied using available clinical data. Colorectal cancer patients were classified according to the median methylation level (methylation level of 10%) of the groups presented as follows: i) high LINC0047 methylation level (H: methylation level>10%) or ii) low LINC00473 methylation level (L: methylation level<10%). According to this classification of patients, Kaplan-Meir analysis showed that the presence of high LINC00473 methylation level 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 showed that the methylation status of LINC00473 in liquid biopsies could predict the clinical outcomes of patients with colorectal cancer, indicating that analysis of LINC00473 methylation in plasma could be used as a prognostic biomarker in the diagnosis of colorectal cancer.

[0128] Example 2.2.3. Methylation analysis of the LINC00473 promoter in liquid biopsies to detect patients with advanced colorectal adenoma.

[0129] The DNA methylation levels (%) of the LINC00473 promoter region in the plasma of 5 healthy controls and 12 patients with advanced colorectal adenoma were analyzed by real-time PCR, showing that the methylation level in patients with advanced colorectal adenoma (0.0676%±0.1195%) was significantly (p=0.0262) higher than that in healthy controls (0.0001%±0.0002%) ( Fig. 9 ). These data suggest that the methylation level of the LINC00473 promoter region analyzed in liquid biopsies can distinguish healthy individuals from patients with advanced colorectal adenomas.

[0130] Analysis of the methylation level of LINC00473 in plasma by receiver operating characteristic curve (ROC) showed a very high diagnostic accuracy in distinguishing patients with advanced colorectal adenoma from healthy controls, with an area under the curve (AUC) of 0.85 (p = 0.00269; CI 95%: 0.65-1.0)( Fig.10). Based on ROC curve analysis, a methylation cutoff value of 0.000115 was obtained for LINC00473 in plasma, allowing 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 detecting advanced colorectal adenomas. These results indicate that non-invasive analysis of the methylation status of the LINC00473 promoter region in liquid biopsies has high diagnostic accuracy in identifying patients with advanced colorectal adenomas. Table 8 shows the characteristics of LINC00473 promoter region methylation analysis as a diagnostic test for detection of advanced colorectal adenomas.

[0131] Table 8

[0132]

[0133] VPP: positive predictive value; VPN: negative predictive value

[0134] In addition, the DNA methylation levels (%) of the LINC00473 promoter region in the plasma of 5 healthy controls and 10 patients with advanced colorectal adenoma were evaluated by droplet digital PCR. These results showed that the methylation levels in patients with advanced colorectal adenoma (0.94% ± 0.99%) were significantly (p = 0.038) higher than those in healthy controls (0.0% ± 0.0%) ( Fig.15 ), indicating that the methylation level of the LINC00473 promoter region analyzed by ddPCR in liquid biopsies can also distinguish healthy individuals from patients with advanced colorectal adenomas. Sequence Listing <110> Gallego Health Services Santiago de Compostela Health Institute Foundation <120> In vitro method for the diagnosis or prognosis of colorectal cancer or its precancerous stages <130> 905170 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 1 ggggaggyga taggtttggt tttagtt 27 <210> 2 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 2 acacaccaaa aatcttttct acc 23 <210> 3 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 3 gataggtttg gttttagtta ta 22 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 4 tacggttttg ggtcgttagc 20 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 5 cgaaaccaac acgaacgata 20 <210> 6 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 6 gtatggtttt gggttgttag tgg 23 <210> 7 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 7 ccaaaaccaa cacaaacaat aac 23 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 8 taacgttgcg ttatttgggc 20 <210> 9 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 9 ttagttataa tgttgtgtta tttgggt 27

Claims

1. Use of a reagent for determining the methylation status of the promoter region of the gene LINC00473 located between chromosome positions Chr6:166401527 and Chr6:166402659 in a liquid biopsy in the preparation of a kit for the diagnosis of colorectal adenoma.

2. The use according to claim 1, wherein the colorectal adenoma is an advanced colorectal adenoma.

3. The use according to claim 1, wherein the reagent further comprises a reagent for determining the methylation status of the gene LINC00473 in additional CpGs in the promoter region. 4 . The use according to claim 3 , wherein the CpG is at least one CpG in the promoter region located between chromosome positions Chr6:166402081 and Chr6:166402638.

5. The use according to claim 4, wherein the chromosomal position is selected from at least one of the following positions: Chr6: 166402638, Chr6: 166402474, Chr6: 166402463, Chr6: 166402457, Chr6: 166402416, Chr6: 166402379, Chr6: 166402375, Chr6: 166402364, and Chr6: 166402081.

6. The use according to claim 5, wherein the chromosomal position is selected from at least one of the following positions: cg06545143, cg08886973, and cg21306006.

7. The use according to claim 1, wherein the reagent comprises primers for methylation analysis of LINC00473.

8. The use according to claim 7, wherein the primers include a primer having a nucleotide sequence of SEQ ID NO: 1, a primer having a nucleotide sequence of SEQ ID NO: 2, and a primer having a nucleotide sequence of SEQ ID NO:

3.

9. The use according to claim 8, wherein the primer is biotinylated by introducing a biotin molecule.

10. The use according to claim 7, wherein the primers include a primer having a nucleotide sequence of SEQ ID NO: 4, a primer having a nucleotide sequence of SEQ ID NO: 5, a primer having a nucleotide sequence of SEQ ID NO: 6, and a primer having a nucleotide sequence of SEQ ID NO:

7.

11. The use according to claim 7, wherein the primers include a primer having a nucleotide sequence of SEQ ID NO: 4, a primer having a nucleotide sequence of SEQ ID NOs: 5, a primer having a nucleotide sequence of SEQ ID NO: 8 and fused to fluorescein at the N-terminus, and a primer having a nucleotide sequence of SEQ ID NO: 9 and fused to hexachlorofluorescein at the N-terminus.

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