Patent Application
20220275453
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 370093_404USPC_SEQUENCE_LISTING.txt. The text file is 2.13 KB, was created on May 11, 2022, and is being submitted electronically via EFS-Web.
The present invention refers to the medical field. Particularly, the present invention refers to an in vitro method for the diagnosis and/or prognosis of colorectal cancer (CRC) and/or a pre-cancerous stage thereof. The method of the invention comprises the determination of the methylation status of the gene LINC00473, preferably in a liquid biopsy obtained from the patient, wherein a higher level of methylation of the gene LINC00473 with respect to an established reference control level, is indicative of the presence of colorectal cancer, a pre-cancerous stage thereof and/or of poor prognosis of the disease.
CRC, also known as colon cancer, rectal cancer, or bowel cancer, is the development of cancer in the colon or rectum (parts of the large intestine). The vast majority of colorectal cancers are adenocarcinomas. This is because the colon has numerous glands within the tissue. When these glands undergo a number of changes at the genetic level, they proceed in a predictable manner as they move from benign to an invasive, malignant colon cancer. The adenomas of the colon, particularly advanced colorectal adenoma (AA), are a benign version of the malignant adenocarcinomas but still with malignant potential if not removed (they are usually removed because of their tendency to become malignant and to lead to colon cancer).
Screening is an effective way for preventing and decreasing deaths from colorectal cancer and is recommended starting from the age of 50 to 75. The best known and most frequently used screening test for colorectal cancer is called Fecal Immunochemical Test (FIT). FIT detects blood in the stool samples which can be a sign of pre-cancer or cancer. If abnormal results are obtained, usually a colonoscopy is recommended which allows the physician to look at the inside of the colon and rectum to make a diagnosis. During colonoscopy, small polyps may be removed if found. If a large polyp or tumor is found, a biopsy may be performed to check if it is cancerous. The gastroenterologist uses a colonoscopy to find and remove these adenomas and polyps to prevent them from continuing to acquire genetic changes that will lead to an invasive adenocarcinoma.
Although, as explained above, FIT is nowadays used for screening colorectal cancer, it is important to note that FIT offers a low sensitivity for AA which means that most of said kind of patients can be wrongly classified as not having the disease. Consequently, FIT is not able to identify adenomas due to its low sensitivity. Moreover, since FIT uses stool samples, it offers a low compliance. On the other hand, colonoscopy is an invasive technique wherein the most severe complication generally is the gastrointestinal perforation. Moreover, colonoscopy is nowadays a procedure involving anesthesia, and the laxatives which are usually administered during the bowel preparation for colonoscopy are associated with several digestive problems.
It is important to note that the methods used today for screening general population at risk of suffering for CRC or AA are associated with a high rate of false positives. Consequently, a high amount of unnecessary follow-up colonoscopies are nowadays performed.
The present invention offers a clear solution to the problems cited above because it is focused on an in vitro method for identifying or screening human subjects at risk of suffering from colorectal cancer or colorectal adenomas (particularly advanced colorectal adenomas). Since the method of the invention is preferably based on a liquid biopsy obtained from the patient (for example plasma, blood or serum), it is expected to improve compliance to colorectal cancer screening. Moreover, the method of the invention offers high sensitivity and specificity, which means that it is a strong and cost-effective method for the detection of both colorectal cancer and colorectal adenomas.
The present invention refers to an in vitro method (hereinafter “method of the invention”) for the diagnosis or prognosis of colorectal cancer and/or a pre-cancerous stage thereof. It is preferably implemented in a liquid biopsy obtained from the patient, which is a minimally-invasive biological sample. The method of the invention offers high sensitivity and specificity, which means that it is a strong and cost-effective method for the detection of both colorectal cancer and/or a pre-cancerous stage thereof.
Since the method of the invention has higher sensitivity and specificity as compared to the method used today (FIT) for screening general population at risk of suffering from CRC or AA, it is associated with a lower percentage of false positives. Consequently, the method described in the present invention clearly helps in reducing the number of follow-up colonoscopies, thus improving the way that the patients are nowadays screened or diagnosed. Once the method of the invention is performed, if it is determined that the patients might be suffering from colorectal cancer and/or precancerous stage, the result is confirmed by colonoscopy. However, if it is not determined that the patient might be suffering from colorectal cancer and/or precancerous stage, there is no need to perform a colonoscopy and routine testing with the method of the invention is recommended.
Particularly, the first embodiment of the present invention refers to an in vitro method for the diagnosis of colorectal cancer and/or a pre-cancerous stage thereof, which comprises determining the methylation status of at least the gene LINC00473 in a liquid biopsy obtained from the patient, wherein a higher level of methylation of the gene LINC00473, as compared with a reference level of methylation of the gene LINC00473 measured in healthy subjects, is an indication that the subject is suffering from colorectal cancer and/or a pre-cancerous stage thereof.
The second embodiment of the invention refers to an in vitro method for the prognosis of colorectal cancer and/or a pre-cancerous stage thereof, which comprises determining the methylation status of at least the gene LINC00473 in a liquid biopsy obtained from the patient, wherein a higher level of methylation of the gene LINC00473, as compared with a reference level of methylation of the gene LINC00473 measured in the patient, is an indication that the patient has a poor prognosis.
The third embodiment of the present invention refers to the in vitro use of at least the methylation status of the gene LINC00473 for the diagnosis and/or prognosis of colorectal cancer and/or a pre-cancerous stage thereof.
The fourth embodiment of the present invention refers to the in vitro use of a kit comprising reagents for the determination of at least the methylation status of the gene LINC00473 for the diagnosis and/or prognosis of colorectal cancer and/or a pre-cancerous stage thereof.
In a preferred embodiment, the liquid biopsy is a sample of plasma, blood or serum. Plasma is particularly preferred.
In a preferred embodiment, the pre-cancerous stage is colorectal adenoma, preferably AA.
In a preferred embodiment, the methylation status of the gene LINC00473 is determined in at least a CpG of the promoter region.
In a preferred embodiment, the methylation status of the gene LINC00473 is determined in at least a 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 gene LINC00473 is determined in at least a CpG of the promoter region located between the chromosomal positions Chr6: 166402081 and Chr6: 166402638.
In a preferred embodiment, the methylation status of the gene LINC00473 is determined in at least a CpG of the promoter region located at a chromosomal position selected from the group comprising: 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 comprising: cg06545143, and/or cg08886973 and/or cg21306006.
In a preferred embodiment, the diagnosis of the colorectal cancer and/or a pre-cancerous stage thereof is confirmed by an image technique, preferably colonoscopy.
According to the method of the invention, after measuring the methylation status of the gene LINC00473, a score value is obtained and this score value is compared with a threshold value which defines the diagnostic rule. If this score value is higher than the threshold, then the corresponding sample is classified as a positive sample, which is an indication that the patient might be suffering from colorectal cancer and/or pre-cancerous stage thereof. The threshold value has been defined in order to optimize sensitivity and specificity values. Consequently, in a preferred embodiment, the method of the invention comprises: a) Measuring methylation status of the gene LINC00473, in a liquid biopsy obtained from the subject, b) processing the methylation values in order to obtain a risk score and c) wherein if a deviation or variation of the risk score value obtained is identified, as compared with a reference value, this is indicative that the subject is suffering from colorectal cancer and/or a pre-cancerous stage thereof.
The last embodiment of the present invention refers to a method for the diagnosis and treatment colorectal cancer or a pre-cancerous stage thereof, which comprises: a) obtaining a liquid biopsy from a human subject; b) detecting the methylation status of the gene LINC00473, and c) diagnosing the patient with colorectal cancer or a pre-cancerous stage thereof when a higher level of methylation of the gene LINC00473 is identified, as compared with a reference level of methylation of the gene LINC00473 measured in healthy subjects, and performing a colonoscopy to the patient. The colonoscopy may comprise the removal of the colorectal cancer or the polyps.
For the purpose of the present invention the following terms are defined:
We initially analyzed 3 independent cohorts (Cohort 1, Cohort 2, and Cohort 3) to study the DNA methylation of LINC00473 in colorectal tissues. Cohort 1 are represented by primary colorectal cancer patients (n=273) and non-tumor controls (n=38). The Cohort 2 includes 180 primary colorectal cancer patients and 93 non-tumor controls. And in the Cohort 3 there are 12 primary colorectal cancer patients, 12 adenomas and 12 non-tumor controls.
Moreover, for the purpose the present PCT application, we also analysed a new independent cohort (Cohort 4) to study the DNA methylation of LINC00473 in colorectal tissues, which includes 50 colorectal adenomas (comprising 20 confirmed advanced colorectal adenomas) and 10 non-tumor controls.
The results of DNA methylation of Cohort 1 were analyzed from the Infinium Human Methylation 450K BeadChip data (450K array) obtained in the public database The Cancer Genome Atlas (TCGA). The 450K array (Illumina) covers >450,000 CpG sites along the human genome, and methylation score of each CpG is represented as beta (β)-values that ranged between 0.0 (completely unmethylated) and 1.0 (completely methylated).
The DNA methylation status of LINC00473 promoter in the Cohorts 2, 3 and 4 were analyzed by bisulfite pyrosequencing. After bisulfite conversion of ˜500 ng of DNA with EZ-96 DNA Methylation kit (Zymo Research Corp.), the DNA methylation levels of LINC00473 promoter were analyzed with a 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 (Table 1) were designed with PyroMark Assay Design 2.0 (Qiagen). Table 1 shows the primers for methylation analyses of LINC00473 by pyrosequencing.
In a preferred embodiment, the primers used in the present invention, preferably the primer of SEQ ID NO: 2, are biotinylated by means of the incorporation of a molecule of biotin.
For the analysis of LINC00473 in liquid biopsy of colorectal cancer, the study included 26 advanced colorectal cancer patients (stage III and IV) prospectively recruited at the time of diagnosis (baseline), before the beginning of the treatment and surgery. Demographic and clinical characteristics of the patients are represented in Table 2. Twenty-eight healthy controls were also included in the study. The disease status and staging of colorectal cancer patients was obtained by the medical oncologists of the Oncology Department at Complejo Hospitalario Universitario de Santiago de Compostela (CHUS). Table 2 shows clinical characteristics of the colorectal cancer patients.
For the analysis of LINC00473 in liquid biopsy of advanced colorectal adenoma, the study included 5 healthy controls and 12 advanced adenoma patients. All the individuals for liquid biopsy analysis were recruited at the Complejo Hospitalario Universitario de Santiago de Compostela (CHUS).
For the analysis of LINC00473 in liquid biopsy of colorectal cancer by droplet digital PCR (ddPCR), the study included 5 advanced colorectal cancer patients prospectively recruited at the time of diagnosis (baseline), before the beginning of the treatment and surgery. Ten advanced adenomas and 5 healthy controls were also included in the study. The patients and controls were obtained at Complejo Hospitalario Universitario de Santiago de Compostela (CHUS).
Blood samples were obtained by phlebotomy using collection tubes containing EDTA as anticoagulant. Plasma was isolated from blood samples within 2 h of collection with a first centrifugation of blood tubes (1,500 g, 10 min, 4° C.). Plasma was transferred to a 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.
After defrosting the plasma samples, circulating DNA was isolated from 2-3 mL of plasma using the QIAamp® Circulating Nucleic Acid Kit (Qiagen) and the vacuum system QIAvac 24 Plus (Qiagen) following the manufacturer's recommendations. Circulating DNA was finally eluted in 75 μL of elution buffer and 1 μL of this elution was used to quantify the concentration of circulating DNA by the kit QuantiFluor® ONE dsDNA with the Quantus™ Fluorometer (Promega). After the quantification, circulating DNA was stored at −80° C.
For DNA bisulfite conversion, 15-50 ng of circulating DNA were used, and conversion was performed with EZ DNA Methylation-Lightning Kit (Zymo Research) according to manufacturer's recommendations following these thermocycling steps: 98° C. 8 min, 54° C. 60 min and 4° C. After column-based purification with the steps of binding, L-desulphonation and washing, bisulfite-converted DNA (bis-DNA) was eluted in 15 μL of elution buffer and stored at −80° C.
The methylation status of the promoter region of the long intergenic non-protein coding RNA 473 (LINC00473; previous symbol C6orfl76) 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 in chromosome (chr) 6 at position 6q27, and has a CpG island containing its promoter region, which is located in chr6: 166401527-166402659. In addition, the promoter region of LINC00473 overlaps in the genome with the body of the long intergenic non-protein coding RNA 602 or LINC00602 (NCBI ID: 441177; UCSC ID: uc011egm.4; Ensambl ID: ENSG00000281832; HGNC ID: HGNC:43917).
The methylation levels of the promoter region of LINC00473 were determined by real-time PCR with a quantitative methylation-specific PCR assay (qMSP) in a StepOne Plus system (Applied Biosystems). Each reaction contained 2 uL of bisulfite-converted DNA (bis-DNA) as a template, 10 μl Power SYBR™ Green PCR Master Mix (ThermoFisher) and 150 nM each forward and reverse primers (Table 3) to detect methylation or unmethylation in a total volume of 20 μl. Reactions were performed in a MicroAmp™ Fast Optical 96-Well Reaction Plate (Applied Biosystems). Thermocycling conditions in 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 non-template control in each run to verify the absence of contamination during reactions. The colorectal cancer cell line HCT-116 and normal leukocytes (NL) were used in each analysis as positive controls for methylation and unmethylation, respectively. All the samples and controls were analyzed in triplicates. The threshold cycle (Ct) was determined for each reaction with the specific primers for methylation and unmethylation using the software StepOne Real-Time PCR (Applied Biosystems). The DNA methylation level of each sample was expressed as a percentage of methylation (%) according to the following calculation based on Cottrell et al. (2007):
Methylation (%)=100/[1+2{circumflex over ( )}(CTCG−CTTG)]
In this formula, CTCG represent the threshold cycle of the methylation status and CTTG indicates the threshold cycle of the un-methylation status. With this calculation methylation levels can be between ranges of 0% to 100% of methylation. Table 3 shows the primers for methylation analyses of LINC00473 by real-time PCR.
(*) Position: chr6: 166402364-166402477;
(#) Position: chr6: 166402363-166402478.
The forward and reverse primers used for the methylation analysis of LINC00473 allows to detect the methylation status of 6 CpGs (Table 4) located at the promoter region of LINC00473. One of the CpGs (chr6: 166402416) contained in the sequence amplified (amplicon) by real-time PCR corresponds with the CpG termed cg08886973 according to the Infinium Human Methylation 450K and Infinium MethylationEPIC assays (Illumina), which are microarray assays able to detect the methylation status of more than 450,000 and 850,000 CpGs, respectively. These microarrays are also able to detect other CpGs from the LINC00473 promoter identified as cg06545143 (chr6: 166402638) and cg21306006 (chr6: 166402081) that are located, respectively, 162 nucleotides upstream and 282 nucleotides downstream the amplicon of LINC00473 analyzed in this study. The three CpGs included in this type of microarray assays (cg06545143, cg08886973, cg21306006) are located in the TSS1500 promoter region of LINC00473, which is the region of the promoter between 200 and 1500 nucleotides upstream its transcription start site (TSS). All the chromosomal positions previously indicated are according to the version GRCh37/hg19 of the UCSC Genome Browser from the University of California (https://genome.ucsc.edu/index.html). Table 4 shows the CpGs of LINC00473 that are detected in the methylation analyses by real-time PCR.
The methylation levels of the promoter region of LINC00473 were determined by droplet digital PCR (ddPCR) in a QX200 system (Bio-Rad). First, a multiplex preamplification reaction was performed using ˜2 ng of bisulfite-converted DNA (bis-DNA), 25 μl SsoAdvanced™ PreAmp Supermix (Bio-Rad), 0.5 μl of a custom Bio-Rad assay containing the forward and reverse primers and the probe for methylation (Bio-Rad) (Table 5), and 0.5 μl of a custom Bio-Rad assay containing the forward and reverse primers and the probe for unmethylation (Table 5), in a total volume of 50 μl. Final volume was completed with water. Reactions were performed in 0.2 ml PCR tubes (Axygen) on a 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 hold step of 4° C.
Next, a multiplex mix reaction was performed containing 2 uL of the pre-amplification product previously obtained, 11 μl ddPCR Supermix for Probes (No dUTP) (Bio-Rad), 2.2 μl of a custom Bio-Rad assay containing the forward and reverse primers and the probe for methylation (Table 5), and 2.2 μl of a custom Bio-Rad assay containing the forward and reverse primers and the probe for unmethylation (Bio-Rad) (Table 5), in a total volume of 22 μl. Final volume was completed with water. Then, 20 uL of each mix reaction and 70 μl of Droplet Generation oil (Bio-Rad) were transferred, respectively, to the sample and oil wells of a DG8™ Cartridge (BIO-RAD) and loaded into the QX200™ Droplet Generator (Bio-Rad). Drops were generated into the droplet well of the cartridge, transferred to a ddPCR 96-well plate (Bio-Rad) and loaded in 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 hold step of 4° C. The temperature ramp increment was 2.5° C./s for all steps. Then, the 96-well plate was read on a QX200™ Droplet Reader (Bio-Rad). Water was included as non-template control in each run to verify the absence of contamination during reactions. The colorectal cancer cell line HCT-116 and normal leukocytes (NL) were used in each analysis as positive controls for methylation and unmethylation, respectively. All controls were included in each plate with and without the preamplification step. All the samples and controls were analysed in triplicates. Analysis of the data was performed with the QuantaSoft software (Bio-Rad).
The DNA methylation level of each sample was expressed as a percentage of methylation (%) according to the following calculation:
Methylation (%)=[M/(U+M)]×100
In this formula, M (Methylation) represents the copies/l of the target DNA molecule for the methylation probe (FAM probe), and U (Unmethylation) indicates the copies/l of the target DNA molecule for the unmethylation probe (HEX probe). With this calculation methylation levels can be between ranges of 0% to 100% of methylation. Table 5 shows the sequences of primers and probes for methylation analyses of LINC00473 by ddPCR. Based on these sequences two custom Bio-Rad assays containing the primers and probe for methylation and unmethylation, respectively, were obtained from Bio-Rad according to manufacturer's recommendations: Primer to probe ratio: 1.8, primer concentration in final reaction 450 nM and probe concentration in final reaction 250 nM.
(*) Position: chr6: 166402364-166402477;
(#) Position: chr6: 166402363-166402478.
The forward and reverse primers, and the probe used for the methylation analysis of LINC00473 by ddPCR allows to detect the methylation status of 9 CpGs (Table 6) located at the promoter region of LINC00473. One of the CpGs (chr6: 166402416), which corresponds with the CpG termed cg08886973 according to the Infinium Human Methylation 450K and Infinium MethylationEPIC assays (Illumina), is contained in the sequence amplified (amplicon) and is detected with the methylated and unmethylated probe indicated in Table 5. All the CpGs positions of LINC00473 that are detected in the methylation analyses by ddPCR are represented in Table 6. All the chromosomal positions indicated in Table 6 are according to the version GRCh37/hg19 of the UCSC Genome Browser from the University of California (https://genome.ucsc.edu/index.html).
The Kolmogorov-Smirnov test was first used to evaluate the normality of the distribution of the data. Subsequently, the nonparametric Mann-Whitney U test was used for the comparison of the data. To assess the diagnostic accuracy of LINC00473 methylation to detect colorectal cancer a Receiver Operating Characteristic (ROC) curve was performed. The Youden index (J), which allows to obtain the cutoff point for methylation that provides the greatest combination of sensitivity and specificity, was calculated according to the formula: J=Sensitivity+Specificity-1 (Fluss et al., 2005). To measure the effectiveness of the diagnostic test, the positive (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 differences between the methylated and unmethylated group. The SPSS or GraphPad Prism 7.0 software was used for statistical analysis and graphic representation. All expressed β-values were calculated with two-tailed tests and were considered significant when p<0.05.
The DNA methylation levels of LINC00473 promoter region of colorectal tissue samples (273 colorectal cancer patients at stage I, II, III and IV; and 38 non-tumor controls) were obtained after the analysis by Infinium Human Methylation 450K BeadChip (450K array) from the international public database The Cancer Genome Atlas (TCGA) (Cohort 1). In this analysis the methylation data of 450K array are expressed as average beta (β)-values, which are defined from 0.0 to 1.0. We focused our analysis in the CpG of 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 non-tumor controls (0.11±0.006) (
The analysis of the methylation levels of LINC00473 in the colorectal tissue samples from the Cohort 1 by receiver operating characteristic curve (ROC) showed a very high diagnostic accuracy to differentiate 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) (
The DNA methylation levels of LINC00473 promoter region in colorectal tissues of 12 non-tumor controls, 12 adenomas and 12 colorectal cancer patients (Cohort 3) were analyzed by bisulfite pyrosequencing and expressed as % of methylation. These results showed significantly (p=0.0036) higher methylation levels in adenomas (28.21%±%) than in healthy controls (7.28%±0.81%) (
The analysis of the methylation levels of LINC00473 in the colorectal tissue samples from non-tumor and adenoma samples of Cohort 3 by receiver operating characteristic curve (ROC) showed a very high diagnostic accuracy to differentiate adenomas from non-tumor controls with an area under the curve (AUC) of 0.84 (p=0.0047; 95% CI: 0.66-1.00) (
Moreover, in order to evaluate the methylation status of colorectal adenomas and advanced colorectal adenomas, we analysed the DNA methylation levels of LINC00473 promoter region by bisulfite pyrosequencing in a new independent cohort of tissue samples with 10 non-tumor controls and 50 adenomas, which 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 controls (5.70%±0.82%) (
The DNA methylation levels (%) of LINC00473 promoter region in 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%) (
The analysis of the methylation levels of LINC00473 in plasma by receiver operating characteristic curve (ROC) showed a very high diagnostic accuracy to differentiate 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) (
Moreover, DNA methylation levels (%) of LINC00473 promoter region in plasma of colorectal cancer patients were also evaluated using the droplet digital PCR (ddPCR) methodology. For this purpose, we used 5 controls and 5 colorectal cancer patients. These analysis showed significantly (p=0.005) higher methylation levels in colorectal cancer patients (41.19%±28.79%) than in healthy controls (0.0%±0.0%) (
The clinical impact on survival of the methylation levels of LINC00473 promoter was studied in plasma samples of 25 colorectal cancer patients from Table 2 with available clinical data. Colorectal cancer patients were classified respect to the median methylation level of the group (methylation level of 10%) as presenting i) High level of methylation in LINC00473 (H: methylation level >10%) or ii) Low level of methylation in LINC00473 (L: methylation level <10%). According to this classification of the patients, Kaplan-Meir analysis showed that the presence of high levels of methylation in 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] (
The DNA methylation levels (%) of 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%) (
The analysis of the methylation levels of LINC00473 in plasma by receiver operating characteristic curve (ROC) showed a very high diagnostic accuracy to differentiate 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) (
Moreover, DNA methylation levels (%) of LINC00473 promoter region in plasma were also evaluated by droplet digital PCR in 5 healthy controls and 10 advanced colorectal adenoma patients. These 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%) (
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19382290.5 | Apr 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/060517 | 4/15/2020 | WO |
