RU2831090C2 - Method for in vitro diagnosis or prediction of colorectal cancer or its precancerous stage - Google Patents

Abstract

FIELD: medical biotechnology.

SUBSTANCE: disclosed is a method for in vitro identification of a patient with colorectal cancer or its precancerous stage, comprising determining the methylation status of the LINC00473 gene in a liquid biopsy material obtained from a patient, where the LINC00473 gene methylation level is higher than the LINC00473 gene methylation reference level measured in healthy control subjects, is an indicator that the subject suffers from colorectal cancer or a precancerous stage or has a poor prognosis. Disclosed is the in vitro use of the methylated LINC00473 gene for the diagnosis and/or prediction of colorectal cancer or its precancerous stage based on the methylation status of the LINC00473 gene.

EFFECT: method according to the invention provides high sensitivity and specificity, which means that it is a powerful and effective method of detecting both colorectal cancer and colorectal adenomas.

15 cl, 8 tbl, 15 dwg, 2 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

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

BACKGROUND OF THE INVENTION

CRC, also known as colon cancer, rectal cancer, or colorectal cancer, is the development of a malignant tumor in the colon or rectum (parts of the large intestine). The vast majority of colorectal cancers are adenocarcinomas. This occurs because the colon has numerous glands within the tissue. When these glands undergo a series of changes at the genetic level, they predictably continue their progression from a benign to an invasive, malignant tumor of the colon. Colon adenomas, particularly advanced colorectal adenoma (AA), are a benign variant of malignant adenocarcinomas, but still have malignant potential if not removed (they are usually removed due to their tendency to become malignant and lead to colon cancer).

Screening is an effective way to prevent and reduce deaths from colorectal cancer and is recommended starting at age 50 to 75. The best known and most commonly used screening test for colorectal cancer is called the fecal immunochemical test (FIT). FIT detects blood in stool samples, which can be a sign of precancerous or malignant disease. If abnormal results are obtained, a colonoscopy is usually recommended, which allows a physician to look inside the colon and rectum to make a diagnosis. During a colonoscopy, small polyps can be removed if found. If a large polyp or tumor is found, a biopsy can be performed to check if it is cancerous. A gastroenterologist uses a colonoscopy to detect and remove these adenomas and polyps to prevent them from acquiring genetic changes that can lead to invasive adenocarcinoma.

Although, as explained above, FIT is currently used for colorectal cancer screening, it is important to note that FIT has low sensitivity for AA, which means that most of these patients may be falsely classified as disease-free. Therefore, FIT is not able to identify adenomas due to its low sensitivity. In addition, since FIT uses stool samples, it has low compliance. On the other hand, colonoscopy is an invasive procedure, where the most serious complication is usually gastrointestinal perforation. In addition, colonoscopy is currently a procedure that involves anesthesia, and laxatives that are usually administered during bowel preparation for colonoscopy are associated with serious gastrointestinal problems.

It is important to note that the methods currently used to screen the general population at risk for CRC or AA are associated with a high rate of false positive results. Consequently, a large number of unnecessary surveillance colonoscopies are currently performed.

The present invention provides a clear solution to the problems listed above, since it focuses on an in vitro method for identifying or screening human subjects at risk of suffering from colorectal cancer or colorectal adenomas (in particular, colorectal adenomas at advanced stages). Since the method of the invention is preferably based on a liquid biopsy obtained from a patient (e.g. plasma, blood or serum), improved compliance with colorectal cancer screening is expected. In addition, the method of the invention provides high sensitivity and specificity, which means that it is a powerful and cost-effective method for detecting both colorectal cancer and colorectal adenomas.

DESCRIPTION OF THE INVENTION

Brief description of the invention

The present invention relates to a methodin vitro(hereinafter in the present description "the method of the invention") of diagnosing or predicting colorectal cancer and/or its precancerous stage. It is preferably carried out in a liquid biopsy obtained from patient, which is a minimally invasive biological sample. The method of the invention provides high sensitivity and specificity, which means that it is a powerful and cost-effective method for detecting colorectal cancer and/or its precancerous stage.

Since the method of the invention has a higher sensitivity and specificity compared to the method currently used (FIT) for screening the general population at risk of suffering from CRC or AA, it is associated with a lower percentage of false positive results. Therefore, the method described by the present invention clearly contributes to a decrease in the number of surveillance colonoscopies, thereby improving the way in which patients are currently screened or diagnosed. After performing the method of the invention, if it determines that patients may suffer from colorectal cancer and / or a precancerous stage, the result is confirmed by colonoscopy. However, if it does not determine that the patient may suffer from colorectal cancer and / or a precancerous stage, there is no need to perform a colonoscopy, and conventional testing by the method of the invention is recommended.

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

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

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

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

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

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

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

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

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

In a preferred embodiment, the methylation status of the LINC00473 gene is determined at least at a CpG of 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 a CpG selected from the group consisting of: cg06545143 and/or cg08886973 and/or cg21306006.

In a preferred embodiment, the diagnosis of colorectal cancer and/or its precancerous stage is confirmed by an imaging method, preferably colonoscopy.

According to the method of the invention, after measuring the methylation status of the LINC00473 gene, a score value is obtained, and this score value is compared with a threshold value defining a diagnostic rule. If this score value is higher than the threshold, then the corresponding sample is classified as a positive sample, which is an indicator that the patient may suffer from colorectal cancer and/or its precancerous stage. The threshold value is determined to optimize the sensitivity and specificity values. Therefore, in a preferred embodiment, the method of the invention comprises: a) measuring the methylation status of the LINC00473 gene, in a liquid biopsy obtained from a subject, b) processing the methylation values to obtain a risk score, and c), wherein if a deviation or variability is identified in the obtained risk score value, compared to the reference value, this is an indicator that the subject suffers from colorectal cancer and/or a precancerous stage.

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 a geneLINC00473And c) diagnosis of colorectal cancer or its precancerous stage in a patient when a higher level of gene methylation is identifiedLINC00473, compared to the reference level of gene methylationLINC00473,measured in healthy subjects, and subjecting the patient to a colonoscopy. Colonoscopy may include removal of colorectal cancer or polyps.

For the 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 class of non-coding RNAs. It can be identified in public databases as follows: HGNC: 21160. Entrez Gene: 90632. Ensambl ID: ENSG00000223414. UniProtKB: A8K010.

• The term "colorectal cancer" is a medical condition characterized by a malignant tumor of the 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 term “colorectal adenoma” refers to colonic adenomas, also called adenomatous polyps, which represent a benign and precancerous stage of colorectal cancer, but still with a high risk of progressing to colorectal cancer.

• The term “advanced colorectal adenoma” refers to adenomas that are at least 10 mm in size or histologically have a high grade of dysplasia or a villous component greater than 20%; or serrated lesions that are at least 10 mm in size or dysplasia. For a more detailed explanation of the concept of “advanced colorectal adenoma,” see the following scientific articles that belong to the general knowledge in the field to which the present invention pertains: [ 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 biological fluids that may contain tumor-derived material. In particular, a liquid biopsy is any sample of biological fluids (eg, 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 obtained from a patient without the need for the use of dangerous instruments other than fine needles used to draw blood from the patient, and therefore without risk to the patient. Specifically, a minimally invasive biological sample refers, according to the present invention, to samples of: blood, serum or plasma.

• Within the scope of the invention, the term “methylation” may be understood to mean the presence of a methyl group added by the action of the enzyme DNA methyltransferase to a cytosine base or bases in a region of a nucleic acid, such as DNA.

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

• As used herein, a "CpG dinucleotide," a "CpG methylation site," or the equivalent, shall be taken to mean a cytosine linked to a guanine via a phosphodiester bond. CpG dinucleotides are targets for methylation of a cytosine residue and may be located within 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 a genomic gene, or intergenic regions.

• The term “CpG target” refers to a specific CpG found to be differentially methylated according to the present invention.

• The term “contiguous CpGs” refers to CpGs that may be differentially methylated and they can be found up to 1500 bp upstream or downstream of the transcription start site (TSS) (3000 bp around the TSS) .

• Within the scope of the invention, "determining the methylation status of a gene" means that the method of the invention is carried out by measuring the methylation status of any region of a gene, also comprising any regulatory sequence capable of increasing or decreasing the expression of said genes. In particular, said regulatory sequence comprises a promoter, which is a region of DNA that initiates the transcription of a gene. Promoters are localized near the transcription start sites of genes, on the same strand, upstream or downstream of the DNA (towards the 5' region of the sense strand).

• Within the scope of the invention, the "control, reference or baseline methylation level" shall be understood as meaning the methylation level detected in the corresponding nucleic acid from a healthy patient. Thus, a patient is likely to suffer from CRC or AA, with a given sensitivity and specificity, if a "higher methylation level" is measured in this patient, compared to the "control, reference or baseline methylation level" measured in a healthy patient. The "reference value" may be a threshold value or a cutoff value. Typically, the "threshold value" or "cutoff value" can be determined experimentally, empirically or theoretically. The threshold value can also be chosen arbitrarily, based on existing experimental and/or clinical conditions, as understood by a person skilled in the art. The threshold value must be determined in order to obtain optimal sensitivity and specificity, in accordance with the function of the test and the benefit/risk ratio (clinical consequences of false positive and false negative results). Preferably, a person skilled in the art can compare the levels (or values) of a biomarker obtained according to the method of the invention using a certain threshold value. Typically, the optimal sensitivity and specificity (and thus 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 biomarkers in a reference group, an algorithmic analysis can be used to statistically process the measured methylation level of biomarkers in biological samples to be tested, and thus obtain a classification standard that is meaningful for classifying the samples. The full name of the ROC curve is a receiver operating characteristic curve, which is also known as an operating characteristic curve. It is mainly used for clinical biochemical diagnostic tests. The ROC curve is a comprehensive indicator reflecting continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity using an image fusion method. A series of different cutoff values (threshold or critical values, borderline values between normal and abnormal diagnostic test results) are set as continuous variables to calculate a series of sensitivity and specificity values. Then, the sensitivity is used as the vertical coordinate and the specificity is used as the horizontal coordinate to plot the curve. The larger the area under the curve (AUC), the higher the diagnostic accuracy. On the ROC curve, the point closest to the uppermost left point of the coordinate diagram is the critical point, having both high sensitivity values and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result becomes better and better as the AUC approaches 1. When the AUC is between 0.5 and 0.7, the accuracy is low. When the AUC is between 0.7 and 0.9, the accuracy is good. When the AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably carried out using a computer. Existing software or systems in the field can be used to plot the ROC curve, such as: medical statistical software MedCalc 9.2.0.1, SPSS 9.0, preferably GraphPad Prism 6.

• The expression "higher methylation level" refers to a statistically significant increase in the relative methylation level of a nucleic acid, such as DNA, compared to the methylation level measured in a subject/patient used as a control/reference. Thus, in the present description, "higher methylation level" is defined 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 methylation level" may be at least 2% more than the baseline methylation level, such as at least 5% more than the baseline methylation level, or at least 10% more than the baseline methylation level, or at least 15% more than the baseline methylation level, or at least 20% more than the baseline methylation level, or at least 25% more than the baseline methylation level, or at least 30% more than the baseline methylation level, or at least 40% more than the baseline methylation level, or at least 50% more than the baseline methylation level, or at least 60% more than the baseline methylation level, or at least 70% more than the baseline methylation level, or at least 80% more than the baseline methylation level, or at least 90% more than baseline methylation level.

• By "comprising" is meant to include, but not limit, whatever follows the word "comprising." Thus, the use of the term "comprising" indicates that the listed elements are necessary or mandatory, but that other elements are optional and may or may not be present.

• By "consisting of" is meant "including and limited by" what follows the phrase "consisting of." Thus, the phrase "consisting of" shows that the listed elements are necessary or obligatory, and that no other elements can be present.

Brief description of the drawings

Figure 1. Promoter DNA methylation levels LINC00473 in colorectal tissue samples.In cohort 1 (A) and 2 (B), for patients with colorectal cancer showed significantly higher methylation levelsLINC00473, than in tumor-free controls. Promoter methylation levelsLINC00473analyzed (A) via the Infinium HumanMethylation450K BeadChip (450K array) and (B) by pyrosequencing. DNA methylation data are expressed as β values inA, and as % methylation inB. Horizontal lines represent median methylation levels. P, shows p value analyzed by Mann-Whitney U test. Tumor, represents colorectal cancer.

Figure 2. Receiver operating characteristic (ROC) curve for LINC00473 promoter methylation in colorectal tissue samples. The area under the ROC curve (AUC) in cohort 1 ( A ) and cohort 2 ( B ) shows the high diagnostic accuracy of LINC00473 promoter methylation in discriminating between colorectal cancer patients and tumor-free controls. P, shows the p value from the ROC curve. CI, represents the confidence interval.

Figure 3. DNA methylation levels of the LINC00473 promoter in colorectal tissue samples from The Cancer Genome Atlas (TCGA) according to their clinical stage. For patients with all stages of colorectal cancer, significantly higher LINC00473 methylation levels were shown 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 in their corresponding tumor-free controls (cohort 1: 0.11%±0.006; cohort 2: 5.84%±0.78%). LINC00473 methylation levels were analyzed ( A ) by Infinium HumanMethylation450K BeadChip (450K array) and expressed as β values, and ( B ) by pyrosequencing and expressed as % methylation. Asterisks (*) indicate P<0.0001, relative to control individuals. P value was analyzed by Mann-Whitney U test. Tumor represents colorectal cancer.

Figure 4. Promoter DNA methylation levels LINC00473 in adenomas.For patients with adenomas and significantly higher methylation levels were shown in patients with colorectal cancerLINC00473, than in tumor-free controls. Promoter methylation levelsLINC00473were analyzed by pyrosequencing and expressed as % methylation. Horizontal lines represent median methylation levels. P indicates p-value analyzed by Mann-Whitney U test.

Figure 5. Receiver operating characteristic (ROC) curve for LINC00473 promoter methylation in adenomas. The area under the ROC curve (AUC) in cohort 3 shows the high diagnostic accuracy of LINC00473 promoter methylation in discriminating between adenomas and tumor-free controls. P, shows the p value from the ROC curve. CI, represents the confidence interval.

Figure 6. Promoter DNA methylation levels LINC00473 in circulating plasma DNA of colorectal cancer patients and healthy controls.For Colorectal cancer patients show significantly higher methylation levelsLINC00473, than in healthy controls. Methylation levelsLINC00473were analyzed in triplicate by real-time PCR. Horizontal lines represent median methylation levels. P, shows p-value analyzed by Mann-Whitney U-test.

Figure 7. Receiver operating characteristic (ROC) curve for LINC00473 promoter methylation in circulating plasma DNA of colorectal cancer patients relative to normal controls. The area under the ROC curve (AUC) shows the high diagnostic accuracy of LINC00473 promoter methylation in discriminating between colorectal cancer patients and healthy controls. P, shows the p value from the ROC curve.

Figure 8. Kaplan-Meier survival analysis for LINC00473 promoter methylation in colorectal cancer patients. Colorectal cancer patients were classified according to higher or lower LINC00473 methylation levels relative to the group median methylation level (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 by the log-rank test. H (high) indicates methylation level >10%; and L (low) indicates methylation level <10%.

Figure 9. Promoter DNA methylation levels LINC00473 in circulating plasma DNA of patients with advanced colorectal adenoma and healthy controls.For Patients with late-stage colorectal adenoma show significantly higher methylation levelsLINC00473 than healthy controls. Methylation levelsLINC00473were analyzed in triplicate by real-time PCR. Horizontal lines represent median methylation levels. P, shows p-value analyzed by Mann-Whitney U-test.

Figure 10. Receiver operating characteristic (ROC) curve for LINC00473 promoter methylation in circulating plasma DNA of patients with advanced colorectal adenoma relative to normal controls. The area under the ROC curve (AUC) shows the high diagnostic accuracy of LINC00473 promoter methylation in discriminating between patients with advanced colorectal adenoma and healthy controls. P, shows the p value from the ROC curve. CI, represents the confidence interval.

Figure 11. DNA methylation levels of LINC00473 promoter in advanced colorectal adenoma tissues. Advanced colorectal adenomas showed significantly higher LINC00473 methylation levels than tumor-free controls. LINC00473 promoter methylation levels were analyzed by pyrosequencing and expressed as % methylation. Horizontal lines represent median methylation levels. P, indicates p-value analyzed by Mann-Whitney U test.

Figure 12. Receiver operating characteristic (ROC) curve for LINC00473 promoter methylation in advanced colorectal adenoma tissues. The area under the ROC curve (AUC) shows the high diagnostic accuracy of LINC00473 promoter methylation in discriminating between advanced colorectal adenomas and tumor-free controls. P, shows the p value from the ROC curve. CI, represents the confidence interval.

Figure 13. DNA methylation levels of LINC00473 promoter in circulating plasma colorectal cancer DNA by droplet digital PCR (dcPCR). Colorectal cancer patients showed significantly higher LINC00473 methylation levels than tumor-free controls. LINC00473 promoter methylation levels were analyzed in triplicate by dcPCR. Horizontal lines represent median methylation levels. P, shows p-value analyzed by Mann-Whitney U test.

Figure 14. Receiver operating characteristic (ROC) curve for 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) shows the high diagnostic accuracy of LINC00473 promoter methylation in discriminating between colorectal cancer patients and healthy controls. P, shows the p value from the ROC curve.

Figure 15. Promoter DNA methylation levels LINC00473 in circulating plasma DNA of colorectal adenomas at late stages by droplet digital PCR.For patients with colorectal adenoma at late stages shows significantly higher methylation levelsLINC00473, than in healthy controls. Methylation levelsLINC00473were analyzed in triplicate by droplet digital PCR. Horizontal lines represent median methylation levels and P shows p-value analyzed by Mann-Whitney U test.

Detailed description of the invention

Example 1. Methods.

Example 1.1. Study participants for colorectal tissue analysis.

The present inventors first analyzed three independent cohorts (cohort 1, cohort 2, and cohort 3) to examine LINC00473 DNA methylation in colorectal tissues. Cohort 1 included patients with primary colorectal cancer (n=273) and tumor-free controls (n=38). Cohort 2 included 180 patients with primary colorectal cancer and 93 tumor-free controls. And cohort 3 included 12 patients with primary colorectal cancer, 12 with adenoma, and 12 tumor-free controls.

In addition, for the purposes of this PCT application, the present inventors also analyzed a new independent cohort (Cohort 4) to study LINC00473 DNA methylation in colorectal tissues, which included 50 colorectal adenomas (including 20 confirmed advanced colorectal adenomas) and 10 tumor-free controls.

Example 1.2. Analysis of DNA methylation data from colorectal tissues using the 450K array.

DNA methylation results from cohort 1 were analyzed using the Infinium HumanMethylation450K BeadChip array (450K array) from The Cancer Genome Atlas (TCGA) public database. The 450K array (Illumina) covers >450,000 CpG sites across the human genome, and the methylation status of each CpG is expressed as a beta (β) value ranging between 0.0 (completely unmethylated) and 1.0 (completely methylated).

Example 1.3. Bisulfite pyrosequencing analysis of colorectal tissues.

The DNA methylation status of the LINC00473 promoter in cohorts 2, 3, and 4 was analyzed by bisulfite pyrosequencing. Following bisulfite conversion of ~500 ng DNA using the EZ-96 DNA methylation kit (Zymo Research Corp.), DNA methylation levels of the LINC00473 promoter were analyzed using the PyroMark Q96 system (Qiagen), according to the manufacturer’s instructions. CpG site methylation quantification was performed 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 for the LINC00473 methylation assays by pyrosequencing.

Table 1

Primer sequence (5'-3') Primer size (n.) F: SEQ ID NO: 1 27 R: SEQ ID NO: 2 23 S: SEQ ID NO: 3 22

F: Forward; R: Reverse; S: Sequencing; n: Nucleotides

In a preferred embodiment, the primers used in the present invention, preferably the primer of SEQ ID NO: 2, are biotinylated by incorporating a biotin molecule.

Example 1.4. Participants in a study for liquid biopsy analysis.

To analyze LINC00473 in liquid biopsy specimens of colorectal cancer, the study included 26 patients with advanced-stage colorectal cancer (stages III and IV) prospectively enrolled at the time of diagnosis (baseline), before treatment, and before surgery. The demographic and clinical characteristics of the patients are presented in Table 2 . Twenty-eight healthy controls were also included in the study. Disease status and staging for the colorectal cancer patients were obtained from health care providers - oncologists in the Department of Oncology of the Complejo Hospitalario Universitario de Santiago de Compostela (CHUS). Table 2 shows the clinical characteristics of the colorectal cancer patients.

Table 2

Characteristics Patients (n=26) Floor Male 17 Female 9 Age (years) Mean±SD 67±10 Stage III 4 IV 22 Histology Adenocarcinoma 24 Mucinous carcinoma 2

SD: Standard Deviation

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

To analyze LINC00473 in liquid biopsies of colorectal cancer by droplet digital PCR (dcPCR), the study included 5 patients with advanced colorectal cancer prospectively enrolled at the time of diagnosis (baseline), before treatment and surgery. Ten patients with advanced adenomas and 5 healthy controls were also included in the study. Patients and controls were obtained at the Complejo Hospitalario Universitario de Santiago de Compostela (CHUS).

Example 1.5. Collection of blood samples and isolation of plasma.

Blood samples were obtained by phlebotomy using collection tubes containing EDTA as an anticoagulant. Plasma was isolated from blood samples within 2 h of collection using a first centrifugation of the blood tubes (1500 g, 10 min, 4°C). Plasma was transferred to 1.5 ml tubes, and a second centrifugation was performed (15,000 g, 10 min, 4°C). Plasma was transferred to new 1.5 ml tubes and stored at -80°C.

Example 1.6. Isolation of circulating DNA from plasma samples.

After disruption of plasma samples, circulating DNA was isolated from 2-3 ml of plasma using the QIAamp® Circulating Nucleic Acid Isolation Kit (Qiagen) and the QIAvac 24 Plus Vacuum System (Qiagen) following the manufacturer's recommendations. Circulating DNA was finally eluted in 75 μl of elution buffer, and 1 μl of this eluate was used for quantification of circulating DNA concentration by the QuantiFluor® ONE dsDNA Kit using the Quantus™ Fluorometer (Promega). After quantification, circulating DNA was stored at -80°C.

Example 1.7. Bisulfite conversion of circulating DNA.

For bisulfite conversion of DNA, 15-50 ng of circulating DNA was used and conversion was performed using the EZ DNA Methylation-Lightning kit (Zymo Research) according to the manufacturer's recommendations, following these thermal cycling steps: 98°C for 8 min, 54°C for 60 min, and 4°C. After column-based purification with coupling, L-desulfurization, and washing steps, bisulfite-modified DNA (bis-DNA) was eluted in 15 μl of elution buffer and stored at -80°C.

Example 1.8. DNA methylation analysis of the promoter region LINC00473 in liquid biopsies using real-time PCR detection.

The methylation status of the promoter region of long intergenic non-protein-coding RNA 473 ( LINC00473; preceding symbol 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 (chr) 6 at position 6q27, and has a CpG island containing its promoter region located at chr6:166401527-166402659. In addition, the LINC00473 promoter region overlaps genomically with the major portion of long intergenic non-protein-coding RNA 602 or LINC00602 (NCBI ID: 441177; UCSC ID: uc011egm.4; Ensambl ID: ENSG00000281832; HGNC ID: HGNC:43917).

LINC00473 promoter region methylation levels were determined by real-time PCR detection using quantitative methylation-specific PCR (qMSP) assay in the StepOne Plus System (Applied Biosystems). Each reaction mixture contained 2 μl of bisulfite-modified DNA (bis-DNA) as template, 10 μl of Power SYBR™ Green Master Mix (ThermoFisher) and 150 nM each of the forward and reverse primers ( Table 3 ) for detection of methylation or absence of methylation in a total volume of 20 μl. Reactions were performed in a MicroAmp™ Fast 96-well optical reaction plate (Applied Biosystems). Thermal cycling conditions in the StepOne Plus system were 10 min at 95°C, followed by 50 cycles of 94°C for 15 s and 60°C for 30 s. Water was included as a template-free control in each experiment to verify the absence of contamination during the reactions. The colorectal cancer cell line HCT-116 and normal leukocytes (NL) were used in each assay as positive controls for methylation and non-methylation, respectively. All samples and controls were assayed in triplicate. The threshold cycle (Ct) was determined for each reaction using specific primers for methylation and non-methylation using StepOne Real-Time PCR software (Applied Biosystems). The DNA methylation level of each sample was expressed as percent methylation (%), according to the following calculation based on Cottrell et al . (2007):

Methylation (%) = 100/[1+2^(CT _CG -CT _TG )]

In this formula, CT _CG represents the threshold cycle of methylation status, and CT _TG shows the threshold cycle of unmethylation status. In this calculation, methylation levels can range between 0% and 100% methylation. Table 3 shows the primers for LINC00473 methylation assays by real-time PCR detection .

Table 3

Methylation status Primer sequence (5'-3') Primer size (n.) Amplicon size (bp) Methylated F: SEQ ID NO: 4 20 114 ^(*) R: SEQ ID NO: 5 20 Unmethylated F: SEQ ID NO: 6 23 116 ^(#) R: SEQ ID NO: 7 23

F: Forward; R: Reverse; n.: nucleotides; b.p.: base pairs.

^(*) Position: chr6: 166402364-166402477; ^(#) Position: chr6:166402363-166402478.

Forward and reverse primers used for methylation analysisLINC00473, allow detection of the methylation status of 6 CpG (Table 4), localized in the promoter regionLINC00473.One of the CpGs (chr6: 166402416) contained in the sequence (amplicon) amplified by real-time PCR corresponds to a CpG named cg08886973 according to the Infinium Human Methylation 450K and Infinium MethylationEPIC assays (Illumina), which are microarray assays capable of detecting the methylation status of more than 450,000 and 850,000 CpGs, respectively. These microarrays are also capable of detecting other CpGs from the promoterLINC00473, identified as cg06545143 (chr6: 166402638) and cg21306006 (chr6: 166402081), which are located 162 nucleotides upstream and 282 nucleotides downstream of the amplicon, respectively.LINC00473,analyzed in this study. Three CpGs included in this type of microarray analysis (cg06545143, cg08886973, cg21306006) are located in the promoter region of TSS1500 ofLINC00473,which is the promoter region between 200 and 1500 nucleotides upstream of its transcription start site (TSS). All chromosomal positions listed above correspond to the GRCh37/hg19 version of the UCSC Genome Browser from the University of California (https://genome.ucsc.edu/index.html). IN Table 4CpGs from are shownLINC00473, detected in methylation assays using real-time PCR.

Table 4

Methylation status Primer Chromosomal position Methylated or unmethylated Direct chr6:166402474
chr6:166402463
chr6:166402457 Back chr6:166402379
chr6:166402375
chr6:166402364

Example 1.9. DNA methylation analysis of the promoter region LINC00473 in liquid biopsies using droplet digital PCR (dcPCR).

LINC00473 promoter region methylation levels were determined by droplet digital PCR (dcPCR) on a QX200 system (Bio-Rad). A multiplex preamplification reaction was first performed using ~2 ng of bisulfite-modified DNA (bisDNA), 25 µl of SsoAdvanced™ PreAmp Supermix (Bio-Rad), 0.5 µl of Bio-Rad custom assay mix containing forward and reverse primers and methylation probe (Bio-Rad) ( Table 5 ), and 0.5 µl of Bio-Rad custom assay mix containing forward and reverse primers and no-methylation probe ( Table 5 ), in a total volume of 50 µl. The final volume was made up with water. Reactions were performed in 0.2 ml PCR tubes (Axygen) in the ProFlex PCR system (Applied Biosystems): 3 min at 95°C, 10 cycles of 95°C for 15 s and 56.2°C for 4 min; and a final storage step at 4°C.

The reaction was then performed in a multiplex mixture containing 2 μl of the preamplification product obtained above, 11 μl of the ready-to-use dPCR Supermix for probes (dUTP-free) (Bio-Rad), 2.2 μl of the custom-designed Bio-Rad assay mix containing forward and reverse primers and a methylation probe ( Table 5 ), and 2.2 μl of the custom-designed Bio-Rad assay mix containing forward and reverse primers and a non-methylation probe (Bio-Rad) ( Table 5 ), in a total volume of 22 μl. The final volume was made up with water. Next, 20 μl of each reaction mixture and 70 μl of droplet oil (Bio-Rad) were transferred to the sample and oil wells of the 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 96-well dPCR plate (Bio-Rad), and loaded into a C1000 Touch Thermal Cycler (Bio-Rad): 10 min at 95°C, 40 cycles of 95°C for 15 s and 56.2°C for 30 s; 98°C for 10 min, and a final storage step at 4°C. The temperature ramp rate was 2.5°C/s for all steps. The 96-well plate was then read in a QX200™ droplet reader (Bio-Rad). Water was included as a template-free control in each experiment to verify the absence of contamination during the reactions. The colorectal cancer cell line HCT-116 and normal leukocytes (NL) were used in each assay as positive controls for methylation and no methylation, respectively. All controls were included in each plate with and without the preamplification step. All samples and controls were analyzed in triplicate. Data analysis was performed using QuantaSoft software (Bio-Rad).

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

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

In this formula, M (methylation) represents the number of copies/µL of target DNA molecule for the methylation probe (FAM probe), and U (no methylation) indicates the number of copies/µL of target DNA molecule for the no methylation probe (HEX probe). In this calculation, methylation levels can range between 0% and 100% methylation. Table 5 shows the primer and probe sequences for the LINC00473 methylation assays by dPCR. Based on these sequences, two custom-designed Bio-Rad assay mixes containing primers and probe for methylation and no methylation, respectively, were obtained from Bio-Rad according to the manufacturer's recommendations: primer to probe ratio: 1.8, primer concentration in the final reaction mixture of 450 nM, and probe concentration in the final reaction mixture of 250 nM.

Table 5

Methylation status Primer and probe sequence
(5'-3') Primer/probe size (n.) Amplicon size (bp) Methylated F: SEQ ID NO: 4 20 114 ^(*) R: SEQ ID NO: 5 20 P: FAM-SEQ ID NO: 8 20 Unmethylated F: SEQ ID NO: 6 23 116 ^(#) R: SEQ ID NO: 7 23 P: HEX- SEQ ID NO: 9 27

F: Forward; R: Reverse; P: Probe; n.: nucleotides; b.p.: base pairs.

FAM: fluorescein; HEX: hexachlorofluorescein

^(*) Position: chr6: 166402364-166402477; ^(#) Position: chr6:166402363-166402478.

The forward and reverse primers and probes used for methylation analysis of LINC00473 by dPCR allow detection of the methylation status of 9 CpGs ( Table 6 ) located in the promoter region of LINC00473 . One of the CpGs (chr6:166402416), corresponding to the CpG named cg08886973 by Infinium Human Methylation 450K and Infinium MethylationEPIC assays (Illumina), is contained in the amplified sequence (amplicon) and detected using the methylation and non-methylation probes listed in Table 5 . All CpG positions of LINC00473 detected in the methylation assays by dPCR are presented in Table 6 . All chromosomal positions listed in Table 6 correspond to the GRCh37/hg19 version of the UCSC Genome Browser from the University of California ( https://genome.ucsc.edu/index.html ).

Table 6

Methylation status Primer Chromosomal position Methylated or unmethylated Direct chr6:166402474
chr6:166402463
chr6:166402457 Probe chr6:166402416
chr6:166402411
chr6:166402400 Back chr6:166402379
chr6:166402375
chr6:166402364

Example 1.10. Statistical analysis.

The Kolmogorov-Smirnov test was first used to assess the normality of data distribution. Then, the nonparametric Mann-Whitney U test was used to compare the data. To evaluate the diagnostic accuracy of LINC00473 methylation for colorectal cancer detection, a receiver operating characteristic (ROC) curve was obtained. The Youden index (J), which allows obtaining the methylation cutoff point providing the best combination of sensitivity and specificity, was calculated according to the formula: J = Sensitivity + Specificity - 1 (Fluss et al., 2005). To measure the performance 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). For survival analysis, the Kaplan-Meier method was performed, and the log-rank test (Mantel-Cox) was used to test the differences between the methylated and unmethylated groups. SPSS or GraphPad Prism 7.0 software was used for statistical analysis and graphical presentation. All expressed p values were calculated using two-tailed tests and were considered significant when p<0.05.

Example 2. Results.

Example 2.1. Fabric samples.

Example 2.1.1. Promoter methylation analysis LINC00473 in tumor tissues allows detection of patients with colorectal cancer.

DNA methylation levels of the LINC00473 promoter region for colorectal tissue samples (273 patients with stage I, II, III, and IV colorectal cancer; and 38 tumor-free controls) were obtained from the Infinium Human Methylation450K BeadChip (450K array) from The Cancer Genome Atlas (TCGA) international public database (Cohort 1). In this analysis, methylation data from the 450K array are expressed as mean values (β) defined from 0.0 to 1.0. We focused our analysis on a CpG from the LINC00473 promoter region identified in the 450K array as cg08886973. This analysis showed significantly (p<0.0001) higher methylation levels in colorectal cancer patients (0.51±0.011) than in tumor-free 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 tumor-free controls: 5.84%±0.78%; p<0.0001) ( Figure 1B ). In this case, the methylation values obtained by pyrosequencing are expressed as percentage methylation (%), defined from 0% to 100%. Together, these data demonstrate that LINC00473 promoter region methylation levels analyzed in colorectal tissues are capable of discriminating between tumor-free individuals and colorectal cancer patients.

Receiver operating characteristic (ROC) curve analysis of LINC00473 methylation levels in colorectal tissue samples from cohort 1 showed very high diagnostic accuracy for discriminating between colorectal cancer patients (I, II, III, and IV) and tumor-free controls, with an area under the curve (AUC) of 0.94 (p<0.0001; CI 95%: 0.91-0.97) ( Figure 2A ). Similar results were obtained for cohort 2 (AUC=0.90; p<0.0001; CI 95%: 0.86-0.94) ( Figure 2B ). These results indicate that analysis of LINC00473 promoter region methylation status in colorectal tissue samples has high diagnostic accuracy for identifying patients with colorectal cancer.

Importantly, when the present authors stratified colorectal cancer patients according to their 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 patients with colorectal cancer at each stage compared with tumor-free controls (cohort 1, n=38; cohort 2, n=93).

Example 2.1.2. Promoter methylation analysis LINC00473 in colorectal tissues allows identification of adenomas and colorectal adenomas at late stages.

DNA methylation levels of the LINC00473 promoter region in colorectal tissues of 12 tumor-free controls, 12 adenoma patients, and 12 colorectal cancer patients (cohort 3) were analyzed by bisulfite pyrosequencing and expressed as % methylation. The results showed significantly (p=0.0036) higher methylation levels in adenomas (28.21%±%) than 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 tumor-free controls. Importantly, LINC00473 methylation levels in adenomas were similar to colorectal cancer patients (P=0.3186). These data indicate that LINC00473 promoter region methylation levels analyzed in colorectal tissues are capable of discriminating between tumor-free individuals and adenoma patients, suggesting that LINC00473 promoter methylation status may be used for early detection of colorectal cancer.

Receiver operating characteristic (ROC) curve analysis of LINC00473 methylation levels in colorectal tissue samples from cohort 3 tumor-free and adenoma samples showed very high diagnostic accuracy for distinguishing between adenomas and tumor-free 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 LINC00473 promoter region methylation status in colorectal tissue samples has high diagnostic accuracy for identifying patients with colorectal adenomas at risk for developing colorectal cancer.

Furthermore, to 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 with 10 tumor-free controls and 50 adenoma samples that included 20 confirmed advanced colorectal adenomas. This analysis showed significantly (p<0.0001) higher methylation levels in adenomas (20.96%±15.33%) than in healthy controls (5.70%±0.82%). Specifically, this analysis showed significantly (p<0.0003) higher methylation levels in advanced colorectal adenomas (26.35%±19.66%) than in control individuals (5.70%±0.82%) ( Figure 11 ). In fact, receiver operating characteristic (ROC) curve analysis also showed very high diagnostic accuracy for discriminating between advanced colorectal adenomas and tumor-free controls with an AUC of 0.88 (p<0.0008; 95% CI: 0.75-1.00) ( Figure 12 ). These results confirm that LINC00473 promoter region methylation levels can be used to detect adenomas, specifically, advanced colorectal adenomas.

Example 2.2. Liquid biopsy

Example 2.2.1. Promoter methylation analysis LINC00473 in liquid biopsies detects patients with colorectal cancer.

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 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 (81%) colorectal cancer patients, relative to healthy controls. These data indicate that LINC00473 promoter region methylation levels analyzed in liquid biopsies are capable of discriminating between healthy individuals and colorectal cancer patients.

Receiver operating characteristic (ROC) curve analysis of LINC00473 plasma methylation levels showed very high diagnostic accuracy for discriminating between colorectal cancer patients and 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 the ROC curve analysis and using the Youden index value, a cutoff threshold of 0.30% for LINC00473 plasma methylation was obtained, allowing 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 cutoff level, 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 the noninvasive analysis of LINC00473 promoter region methylation status in liquid biopsy samples has high diagnostic accuracy for identifying patients with colorectal cancer. Table 7 shows the performance of LINC00473 promoter region methylation assay as a diagnostic test for the detection of colorectal cancer.

Table 7

Parameters Percent (%) Sensitivity 81 Specificity 100 VPP 100 VPN 85

VPP: Positive Predictive Value; VPN: Negative Predictive Value

In addition, DNA methylation levels (%) of the LINC00473 promoter region in the plasma of colorectal cancer patients were also assessed using the droplet digital PCR (dcPCR) method. For this purpose, we used 5 control individuals and 5 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 LINC00473 methylation levels were detected in 5 out of 5 (100%) colorectal cancer patients compared to healthy controls, indicating that LINC00473 promoter region methylation levels analyzed in liquid biopsies by dPCR are also capable of discriminating between healthy controls and colorectal cancer patients. Furthermore, receiver operating characteristic (ROC) curve analysis showed that plasma LINC00473 methylation levels obtained by dPCR had very high diagnostic accuracy for discriminating between colorectal cancer patients and 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 LINC00473 promoter region methylation status in liquid biopsies by qPCR has high diagnostic accuracy for identifying patients with colorectal cancer.

Example 2.2.2. DNA methylation levels from the promoter region LINC00473 Liquid biopsies predict outcome for patients with colorectal cancer.

The clinical impact of LINC00473 promoter methylation levels on survival was investigated in plasma samples from 25 colorectal cancer patients from Table 2 with available clinical data. Colorectal cancer patients were classified with respect to the group median methylation level (10% methylation level) as represented by i) high LINC00473 methylation level (H: methylation level >10%) or ii) low LINC00473 methylation level (L: methylation level <10%). According to this patient classification, Kaplan-Meier analysis showed that the presence of high methylation levels of LINC00473 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 LINC00473 methylation status in liquid biopsies is capable of predicting clinical outcome in patients with colorectal cancer, suggesting that LINC00473 methylation analysis in plasma can be used as a prognostic biomarker at the time of diagnosis of colorectal cancer.

Example 2.2.3. Promoter methylation analysis LINC00473 in liquid biopsies detects patients with late-stage colorectal adenoma.

DNA methylation levels (%) of the LINC00473 promoter region in plasma of 5 healthy controls and 12 advanced colorectal adenoma patients were analyzed by real-time PCR, showing significantly (p=0.0262) higher methylation levels in advanced colorectal adenoma patients (0.0676%±0.1195%) than in healthy controls (0.0001%±0.0002%) ( Figure 9 ). These data indicate that LINC00473 promoter region methylation levels analyzed in liquid biopsies are capable of discriminating between healthy individuals and advanced colorectal adenoma patients.

Receiver operating characteristic (ROC) curve analysis of LINC00473 plasma methylation levels showed very high diagnostic accuracy for discriminating between patients with advanced colorectal adenoma and 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 the ROC curve analysis, a cutoff value of 0.000115 was obtained for LINC00473 plasma methylation, allowing detection of advanced adenoma with a sensitivity of 92% and a specificity of 80% ( Table 8 ). Using this methylation cutoff value, a PPV of 92% and an NPV of 80% were obtained for detection of advanced colorectal adenoma. These results indicate that noninvasive analysis of LINC00473 promoter region methylation status in liquid biopsy samples has high diagnostic accuracy for identifying patients with advanced colorectal adenoma. Table 8 shows the performance of LINC00473 promoter region methylation assay as a diagnostic test for detecting advanced colorectal adenoma.

Table 8

Parameters Percent (%) Sensitivity 92 Specificity 80 VPP 92 VPN 80

VPP: Positive Predictive Value; VPN: Negative Predictive Value

In addition, DNA methylation levels (%) of the LINC00473 promoter region in plasma were also assessed by droplet digital PCR in 5 healthy controls and 10 advanced colorectal adenoma patients. The results showed significantly (p=0.038) higher methylation levels in advanced colorectal adenoma patients (0.94%±0.99%) than in healthy controls (0.0%±0.0%) ( Figure 15 ), indicating that LINC00473 promoter region methylation levels analyzed in liquid biopsies by qdPCR are also capable of discriminating between healthy controls and advanced colorectal adenoma patients.

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LIST OF SEQUENCES

<110> SERVIZO GALEGO DE SAÚDE

FUNDACIÓN INSTITUTO DE INVESTIGACIÓN SANITARIA DE SANTIAGO

DE COMPOSTELA

<120> IN VITRO METHOD FOR DIAGNOSIS OR PREDICTION OF COLORECTAL

CANCER OR ITS PRE-CANCER STAGE

<130> 905170

<160> 9

<170> PatentIn version 3.5

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Claims (15)

1. A method for in vitro identification of a patient with colorectal cancer or a precancerous stage thereof, comprising determining the methylation status of the LINC00473 gene in a liquid biopsy obtained from the patient, wherein the methylation level of the LINC00473 gene is higher than a reference methylation level of the LINC00473 gene measured in healthy control subjects, which is an indicator that the subject has colorectal cancer or a precancerous stage. 2. A method for in vitro prognostication of colorectal cancer comprising determining the methylation status of the LINC00473 gene in a liquid biopsy obtained from a patient, wherein a methylation level of the LINC00473 gene that is higher than a reference methylation level of the LINC00473 gene measured in control patients is an indicator that the patient has a poor prognosis. 3. The in vitro method according to any of the preceding claims, wherein the liquid biopsy is a sample of plasma, blood or serum. 4. The in vitro method according to any one of the preceding claims, wherein the precancerous stage is a colorectal adenoma, preferably a late stage colorectal adenoma. 5. The in vitro method according to any of the preceding claims, wherein the methylation status of the LINC00473 gene is determined at least in CpGs from the promoter region. 6. An in vitro method according to any one of the preceding claims, wherein the methylation status of the LINC00473 gene is determined at least at a CpG from the promoter region located between chromosomal positions Chr6: 166402081 and Chr6: 166402638. 7. The in vitro method according to any one of the preceding claims, wherein the methylation status of the LINC00473 gene is determined at least at a CpG from 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 a CpG selected from the group consisting of: cg06545143 and/or cg08886973, and/or cg21306006. 8. The in vitro method according to any of the preceding claims, wherein the diagnosis of colorectal cancer and/or its precancerous stage is confirmed by means of an imaging method, preferably colonoscopy. 9. In vitro use of the methylated LINC0047 3 gene for the diagnosis and/or prognosis of colorectal cancer or its precancerous stage based on the methylation status of the LINC00473 gene. 10. Use of an in vitro kit containing reagents for determining at least the methylation status of the LINC00473 gene for the diagnosis and/or prognosis of colorectal cancer or its precancerous stage. 11. The in vitro use according to any one of claims 9 or 10, wherein the precancerous stage is a colorectal adenoma, preferably a late stage colorectal adenoma. 12. The in vitro use of any one of claims 9-11, wherein the methylated LINC00473 gene is used to determine the methylation status of at least a CpG from the promoter region of the LINC00473 gene. 13. The in vitro use of any one of claims 9-12, wherein the methylated LINC00473 gene is used to determine the methylation status of at least a CpG from the promoter region of the LINC00473 gene located between chromosomal positions Chr6: 166402081 and Chr6: 166402638. 14. In vitro use according to any of paragraphs. 9-13, wherein the methylated LINC00473 gene is used to determine the methylation status of at least a CpG from the promoter region of the LINC00473 gene 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 a CpG selected from the group consisting of: cg06545143, and/or cg08886973, and/or cg21306006. 15. The in vitro use according to any one of claims 9 to 14, wherein the diagnosis of colorectal cancer and/or its precancerous stage is confirmed by means of an imaging method, preferably colonoscopy.

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