Methods for early detection, prediction of treatment response and prognosis of colorectal cancer

Abstract

The present disclosure discloses a novel epigenetic biomarker set for early detection, prediction of treatment response and prognosis of colorectal cancer. Abnormal methylation of epigenetic biomarkers can be detected in tumor tissue and plasma samples of colorectal cancer patients, but not in normal individuals. The present disclosure also discloses primers and probes used herein.

Description

[0001] priority

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 017,309, filed on April 29, 2020. The entire contents of the above application are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on April 29, 2021, is named G4590-08300PCT_SeqListing.txt, and is 4 kilobytes in size. Technical Field

[0005] The present disclosure relates to epigenetic biomarkers for predicting the risk or susceptibility of colorectal cancer. Specifically, the present disclosure provides a method for early detection, prediction of treatment response and prognosis of colorectal cancer based on the methylation status of gene biomarkers. Background Art

[0006] Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body and is the leading cause of death worldwide.

[0007] Methylated DNA has been studied as a potential biomarker in tissues of most tumor types. In many cases, DNA methyltransferases add methyl groups to DNA at cytosine-phosphate-guanine (CpG) island sites to exert epigenetic control over gene expression.

[0008] US20210003575 is about the use of BMW Rep protein as a biomarker for colon cancer. US 20200291479 provides a method for evaluating the effectiveness of chemotherapy (such as oxaliplatin therapy) in patients with colorectal cancer and their survival prospects by determining the level of miR-133a in cancer tissue. US20200377959 discloses a method for detecting (e.g., screening) colorectal cancer, the method comprising: determining the methylation status of each of the following in the deoxyribonucleic acid (DNA) of a human individual: (a) a methylated locus within the gene ZNF132; (b) a first methylated locus within the gene ADAMTS2; and (c) a second methylated locus within the gene ADAMTS2; and diagnosing colorectal cancer in the human individual based on the determined methylation status.

[0009] However, current technologies for detecting colorectal cancer are unsatisfactory. Summary of the invention

[0010] The present disclosure discloses one or more novel epigenetic biomarkers for early detection, prediction of treatment response and prognosis of colorectal cancer. Abnormal methylation of epigenetic biomarkers is detected in tumor tissue and plasma samples of cancer patients, but not in normal individuals. The present disclosure also discloses primers and probes used herein.

[0011] In one embodiment, the present disclosure provides a method for detecting the methylation status of an individual, wherein the individual needs to detect the susceptibility to colorectal cancer or needs to predict the possibility, treatment response, prognosis or recurrence of colorectal cancer, the method comprising (a) providing a biological sample from the individual, and (b) determining the methylation status of a target DNA sequence comprising TMEM240 or a fragment thereof or MROH6 or a fragment thereof in the biological sample; wherein the presence of high or low methylation of the target DNA sequence of the individual indicates colorectal cancer and / or indicates the susceptibility, possibility, poor treatment response, poor prognosis or recurrence of colorectal cancer.

[0012] In one embodiment, the present disclosure provides a method for detecting an individual's susceptibility to colorectal cancer or predicting an individual's likelihood, treatment response, prognosis or recurrence of colorectal cancer, which comprises (a) providing a biological sample from an individual, and (b) determining the methylation status of a target DNA sequence comprising TMEM240 or a fragment thereof or MROH6 or a fragment thereof in the biological sample, wherein the presence of high or low methylation of the individual's target DNA sequence indicates the susceptibility, likelihood, poor treatment response, poor prognosis or recurrence of colorectal cancer.

[0013] In some embodiments, a target DNA sequence methylation-specific probe or a target DNA sequence methylation-specific primer is used to analyze the methylation status of a target DNA sequence and a control DNA sequence in a biological sample.

[0014] In one embodiment, the presence of hypermethylation or hypomethylation of the target DNA sequence of an individual is determined by comparing the methylation state of the target DNA sequence with the methylation state of a control DNA sequence. In some embodiments, the present disclosure provides a method for detecting the susceptibility of an individual to colorectal cancer or predicting the possibility, treatment response, prognosis or recurrence of colorectal cancer in an individual, comprising (a) providing a biological sample from an individual, the biological sample comprising a target DNA sequence containing TMEM240 or a fragment thereof or MROH6 or a fragment thereof; and (b) using a target DNA sequence methylation-specific probe or a target DNA sequence methylation-specific primer to determine the methylation state of the target DNA sequence and the control DNA sequence in the biological sample; (c) measuring the relative methylation state of the target DNA sequence compared to the control DNA sequence; (d) when the relative methylation state is hypermethylated or hypomethylated, identifying the individual as having susceptibility, possibility, poor treatment response, poor prognosis or recurrence of colorectal cancer. In some embodiments, when the methylation state of TMEM240 or a fragment thereof is about 30 times, about 32 times, about 34 times, about 35 times, about 36 times, about 37 times, about 38 times, about 39 times, or about 40 times higher than the methylation state of the control DNA sequence, it indicates hypermethylation as described herein. In another embodiment, the methylation state of TMEM240 or a fragment thereof is about 37.5 times higher or lower than the methylation state of the control DNA sequence, indicating colorectal cancer. In some embodiments, when the methylation state of MROH6 or a fragment thereof is about 35 times, about 37 times, about 39 times, about 40 times, about 41 times, about 42 times, about 43 times, about 44 times, about 45 times, about 46 times, about 47 times, or about 48 times higher than the methylation state of the control DNA sequence, it indicates hypermethylation as described herein. In another embodiment, the methylation state of MROH6 or a fragment thereof is about 44 times higher or lower than the methylation state of the control DNA sequence, indicating colorectal cancer. In another embodiment, the control DNA sequence is present in normal tissue.

[0015] In some embodiments, the biological sample described herein is tissue, cell, blood, urine, serum, plasma, feces, ascites, sputum, saliva, gastric juice, bile or oral mucosa.

[0016] In some embodiments, the methylation state is detected by polymerase chain reaction, nucleic acid sequencing (such as bisulfite sequencing or pyrophosphate sequencing), bisulfite conversion, mass spectrometry, methylation-specific nucleases, mass-based separation, target capture, or microarray. In a specific embodiment, the methylation state is detected by polymerase chain reaction.

[0017] In some embodiments, when used to determine the methylation status of TMEM240 or a fragment thereof in a human subject by polymerase chain reaction C tA value of less than 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 indicates hypermethylation. In some embodiments, when polymerase chain reaction C is used to determine the methylation status of MROH6 or a fragment thereof in a human subject, t When the value is less than 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45, it indicates high methylation. In a preferred embodiment of the present disclosure, when the polymerase chain reaction C for determining the methylation status of TMEM240 or a fragment thereof in a human subject is used, t A value of less than 45 indicates hypermethylation; or when polymerase chain reaction C is used to determine the methylation status of MROH6 or its fragments in human subjects t Values ​​less than 40 indicate hypermethylation.

[0018] In some embodiments, the methods described herein are used to detect methylation status in a human subject in need of detection of colorectal cancer.

[0019] Certain embodiments of the target DNA sequence methylation-specific primers for determining methylation in TMEM240 or a fragment thereof have about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identity to a sequence selected from the group consisting of SEQ ID NO: 1, 2 or 3. Certain embodiments of the target DNA sequence methylation-specific probes for determining methylation in TMEM240 or a fragment thereof have about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identity to a sequence selected from the group consisting of SEQ ID NO: 4. In some embodiments, the target DNA sequence methylation-specific primer for determining the methylation of MROH6 or a fragment thereof has a sequence that is at least 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identical to SEQ ID NO: 5 or 6. In some embodiments, the target DNA sequence methylation-specific probe for determining the methylation of MROH6 or a fragment thereof has a sequence that is at least 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% identical to SEQ ID NO: 7. In some embodiments, the target DNA sequence methylation-specific probe for determining the methylation of TMEM240 or a fragment thereof has a sequence that is at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3. In some embodiments, the target DNA sequence methylation-specific probe for TMEM240 or a fragment thereof has about 85% identity to the sequence of SEQ ID NO: 4. In some embodiments, the target DNA sequence methylation-specific primer for MROH6 or a fragment thereof has about 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 5 and 6. In some embodiments, the target DNA sequence methylation-specific probe for MROH6 or a fragment thereof has about 85% identity to the sequence of SEQ ID NO: 7.

[0020] In another embodiment, the target DNA sequence further comprises one or more DNA sequences selected from the group consisting of BEND5 or a fragment thereof and SMAD3 or a fragment thereof, or any combination thereof.

[0021] Certain embodiments of the target DNA sequences described herein include any of the following combinations of DNA sequences: TMEM240 or a fragment thereof and MROH6 or a fragment thereof; TMEM240 or a fragment thereof, MROH6 or a fragment thereof and BEND5 or a fragment thereof; TMEM240 or a fragment thereof, MROH6 or a fragment thereof, BEND5 or a fragment thereof and SMAD3 or a fragment thereof; TMEM240 or a fragment thereof, BEND5 or a fragment thereof and SMAD3 or a fragment thereof; TMEM240 or a fragment thereof, MROH6 or a fragment thereof and SMAD3 or a fragment thereof; MROH6 or a fragment thereof and BEND5 or a fragment thereof; and MROH6 or a fragment thereof, BEND5 or a fragment thereof and SMAD3 or a fragment thereof. In another embodiment, the target DNA sequence comprises TMEM240 or a fragment thereof and MROH6 or a fragment thereof.

[0022] In some embodiments, the methylation status is determined by polymerase chain reaction, and when the polymerase chain reaction C for determining the methylation status of BEND5 or a fragment thereof in a human subject is used t A value of less than 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 indicates hypermethylation. In some embodiments, the methylation status is determined by polymerase chain reaction, and when the polymerase chain reaction C for determining the methylation status of SMAD3 or a fragment thereof in a human subject is used, t A value greater than 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 indicates hypomethylation. In some embodiments of the present disclosure, when polymerase chain reaction C is used to determine the methylation status of BEND5 or a fragment thereof in a human subject, t A value of less than 45 indicates hypermethylation. In some embodiments, when polymerase chain reaction C is used to determine the methylation status of SMAD3 or a fragment thereof in a human subject, t Values ​​above 45 indicate hypomethylation.

[0023] In some embodiments of the present disclosure, the method further comprises the steps of defining a score of 1 if each gene or fragment thereof has hypermethylation or hypomethylation and defining a score of 0 if each gene or fragment thereof lacks hypermethylation or hypomethylation, and summing the scores.

[0024] In some embodiments of the present disclosure, the method further comprises:

[0025] If the Ct value of TMEM240 or its fragment is less than 45, the score of TMEM240 is defined as 1; if the Ct value of TMEM240 or its fragment is greater than or equal to 45, the score of TMEM240 is defined as 0;

[0026] If the Ct value of MROH6 or its fragment is less than 40, the score of MROH6 is defined as 1; if the Ct value of MROH6 or its fragment is greater than or equal to 45, the score of MROH6 is defined as 0;

[0027] If the Ct value of BEND5 or its fragment is less than 45, the score of BEND5 is defined as 1, and the scores are summed; if the Ct value of BEND5 or its fragment is greater than or equal to 45, the score of BEND5 is defined as 0; or

[0028] If the Ct value of SMAD3 or its fragment is higher than 45, the score of SMAD3 is defined as 1; if the Ct value of SMAD3 or its fragment is less than or equal to 45, the score of SMAD3 is defined as 0; and

[0029] The scores for TMEM240, MROH6, BEND5 and SMAD3 were summed.

[0030] In some embodiments of the present disclosure, if the sum of the scores of TMEM240, MROH6 and BEND5 is higher than 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25, the susceptibility, possibility, poor treatment response, poor prognosis or recurrence of colorectal cancer is indicated to the individual. In some embodiments of the present disclosure, if the sum of the scores of TMEM240, MROH6 and BEND5 is higher than 0.20, the susceptibility, possibility, poor treatment response, poor prognosis or recurrence of colorectal cancer is indicated to the individual.

[0031] In some embodiments, the target DNA sequence methylation-specific probe or target DNA sequence methylation-specific primer or any combination thereof as described herein is further used to determine the methylation status of the following one or more DNA sequences or any combination thereof: BEND5 or a fragment thereof and SMAD3 or a fragment thereof.

[0032] In some embodiments, the target DNA sequence methylation-specific primer for BEND5 or a fragment thereof described herein has a sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8, 9, 10 or 11. In some other embodiments, the target DNA sequence methylation-specific primer for BEND5 or a fragment thereof has a sequence of SEQ ID NO: 8, 9, 10 or 11. The target DNA sequence methylation-specific probe for BEND5 or a fragment thereof has a sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12 or 13. In another embodiment, the target DNA sequence methylation-specific probe for BEND5 or a fragment thereof has a sequence of SEQ ID NO: 12 or 13.

[0033] In some embodiments, the target DNA sequence methylation-specific primer for SMAD3 or a fragment thereof described herein has a sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14 or 15. In some other embodiments, the target DNA sequence methylation-specific primer for SMAD3 or a fragment thereof has a sequence of SEQ ID NO: 14 or 15. The target DNA sequence methylation-specific probe for SMAD3 or a fragment thereof has a sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 16. In another embodiment, the target DNA sequence methylation-specific probe for SMAD3 or a fragment thereof has a sequence of SEQ ID NO: 16.

[0034] In another embodiment, determining the methylation status further comprises the step of measuring specificity and sensitivity by weighted sum score analysis. In some other embodiments, determining the methylation status of a combination of targets TMEM240, MROH6, BEND5 and SMAD3 or fragments thereof has about 100% sensitivity and about 100% specificity and about 100% accuracy.

[0035] In another embodiment, the methods described herein further comprise the step of administering an anti-colorectal cancer agent to the subject.

[0036] The present disclosure provides a method for detecting an individual's susceptibility to colorectal cancer or predicting an individual's likelihood, treatment response, prognosis or recurrence of colorectal cancer, which comprises (a) providing a biological sample from an individual, and (b) determining the methylation status of a target DNA sequence comprising TMEM240 or a fragment thereof or MROH6 or a fragment thereof in the biological sample, wherein the presence of high or low methylation of the individual's target DNA sequence indicates the susceptibility, likelihood, poor treatment response, poor prognosis or recurrence of colorectal cancer.

[0037] In some embodiments of the present disclosure, the method comprises determining the methylation status of TMEM240 or a fragment thereof using a target DNA sequence methylation-specific primer whose sequence is at least 85% identical to SEQ ID NO: 1, 2 or 3 or a target DNA sequence methylation-specific probe whose sequence is at least 85% identical to SEQ ID NO: 4, and determining the methylation status of MROH6 or a fragment thereof using a MROH6 methylation-specific primer whose sequence is at least 85% homologous to SEQ ID NO: 5 or 6 or a MROH6 sequence methylation-specific probe whose sequence is at least 85% homologous to SEQ ID NO: 7.

[0038] In some embodiments of the present disclosure, the target DNA sequence further comprises one or more DNA sequences selected from the group consisting of BEND5 or a fragment thereof and SMAD3 or a fragment thereof, or any combination thereof.

[0039] The present disclosure provides an isolated nucleic acid molecule having a sequence selected from the group consisting of SEQ ID Nos: 1-16.

[0040] The present disclosure also provides a kit for detecting susceptibility to colorectal cancer in an individual or predicting the likelihood, treatment response, prognosis or recurrence of colorectal cancer in an individual, the kit comprising an isolated nucleic acid molecule for analyzing the methylation state of a target DNA sequence as described herein. The kit may further comprise sodium bisulfite and an adapter for complete target gene amplification, and a polynucleotide (e.g., a polynucleotide as a detectable marker) to quantify the presence of methylated and / or unmethylated cytosine residues in a target DNA sequence as described herein. In addition, the kit may further comprise a methylation-sensing restriction enzyme for complete target sequence or gene amplification.

[0041] The present disclosure provides a target DNA sequence methylation-specific primer pair for detecting the methylation status of TMEM240 or a fragment thereof, comprising SEQ ID NO: 1 and 2 or a sequence having at least 85% identity thereto; or SEQ ID NO: 1 and 3 or a sequence having at least 85% identity thereto. The present disclosure provides a target DNA sequence methylation-specific probe for detecting the methylation status of TMEM240 or a fragment thereof, comprising SEQ ID NO: 4 or a sequence having at least 85% identity thereto.

[0042] The present disclosure provides a target DNA sequence methylation-specific primer pair for detecting the methylation status of MROH6 or a fragment thereof, which comprises SEQ ID NO: 5 and 6 or a sequence having at least 85% identity thereto. The present disclosure provides a target DNA sequence methylation-specific probe for detecting the methylation status of MROH6 or a fragment thereof, which comprises SEQ ID NO: 7 or a sequence having at least 85% identity thereto.

[0043] The present disclosure provides a target DNA sequence methylation-specific primer pair for detecting the methylation status of BEND5 or a fragment thereof, comprising SEQ ID NO: 8 and 9 or a sequence having at least 85% identity thereto; or SEQ ID NO: 10 and 11 or a sequence having at least 85% identity thereto. The present disclosure provides a target DNA sequence methylation-specific probe for detecting the methylation status of BEND5 or a fragment thereof, comprising SEQ ID NO: 12 or 13 or a sequence having at least 85% identity thereto.

[0044] The present disclosure provides a target DNA sequence methylation-specific primer pair for detecting the methylation status of SMAD3 or a fragment thereof, comprising SEQ ID NO: 14 and 15 or a sequence having at least 85% identity thereto. The present disclosure provides a target DNA sequence methylation-specific probe for detecting the methylation status of SMAD3 or a fragment thereof, comprising SEQ ID NO: 16 or a sequence having at least 85% identity thereto.

[0045] The present disclosure also discloses a kit for detecting the susceptibility of colorectal cancer in an individual or predicting the possibility, treatment response, prognosis or recurrence of colorectal cancer in an individual, the kit comprising a target DNA sequence methylation-specific primer pair for detecting the methylation state of a target DNA sequence comprising TMEM240 or a fragment thereof and MROH6 or a fragment thereof. In one embodiment of the present disclosure, the kit further comprises a target DNA sequence methylation-specific probe for detecting the methylation state of a target DNA sequence comprising TMEM240 or a fragment thereof and MROH6 or a fragment thereof.

[0046] In some embodiments, the kit further comprises one or more target DNA sequence methylation-specific primer pairs, which are used to detect the methylation status of BEND5 or a fragment thereof or SMAD3 or a fragment thereof or any combination thereof. In some embodiments, the kit further comprises one or more target DNA sequence methylation-specific probes, which are used to detect the methylation status of BEND5 or a fragment thereof or SMAD3 or a fragment thereof or any combination thereof.

[0047] Simple diagram description

[0048] Figure 1A H to H are heat maps showing the differences in methylation status of target nucleic acid and promoter, exon and gene body regions of genes between tumor tissues and adjacent normal tissues (A: TMEM240 of Taiwan Province, China sample; B: MROH6 of Taiwan Province, China sample; C: BEND5 of Taiwan Province, China sample; D: SMAD3 of Taiwan Province, China sample; E: TMEM240 of TCGA sample; F: MROH6 of TCGA sample; G: BEND5 of TCGA sample; H: SMAD3 of TCGA sample).

[0049] Figure 2A Figures 4 to 5 show the differences in the methylation status of epigenetic biomarkers of target genes in plasma samples between healthy individuals and colorectal cancer patients at early detection (A: TMEM240; B: MROH6; C: BEND5; D: DNA methylation levels of TMEM240, MROH6, and BEND5).

[0050] Figure 3 Shown are the sum of DNA methylation levels (scores) of epigenetic biomarkers of target genes in plasma samples of healthy individuals and colorectal cancer patients.

[0051] Figure 4 Shown is a receiver operating characteristic (ROC) curve analysis demonstrating early detection of epigenetic biomarker methylation status of target genes in colorectal cancer patients and healthy individuals. DETAILED DESCRIPTION

[0052] Implementation

[0053] It should be understood that the present disclosure is not limited to the specific materials and methods described herein. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to limit the scope of the present disclosure, which will be limited only by the scope of the appended claims.

[0054] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "biomarker" includes mixtures of two or more biomarkers, and the like.

[0055] As used herein, the term "AUC" is an abbreviation for the area under the curve. Specifically, it refers to the area under the receiver operating characteristic (ROC) curve. The ROC curve is a graph of the true positive rate relative to the false positive rate of different possible cut-off points in a diagnostic test. It shows a trade-off between sensitivity and specificity, depending on the selected cut-off point (any increase in sensitivity will be accompanied by a decrease in specificity). The area under the ROC curve (AUC) is a measure of the accuracy of a diagnostic test (the larger the area, the better, the most preferred value is 1, the ROC curve of a random test is located on the diagonal line, and the area is 0.5; see: JPEgan. Signal Detection Theory and ROC Analysis, Academic Press, New York, 1975).

[0056] The term "biological sample" refers to a sample of tissue, cell, or body fluid isolated from a subject, including, but not limited to, for example, blood, buffy coat, plasma, serum, blood cells (e.g., peripheral blood mononuclear cells (PBMC), rod cells, neutrophils, metamyelocytes, monocytes, or T cells), fecal material, urine, bone marrow, bile, feces, ascites, sputum, cerebrospinal fluid, lymph fluid, skin samples, external secretions of the skin, respiratory, intestinal and urogenital tracts, tears, saliva, milk, organs, biopsies, and samples of in vitro cell culture components, including, but not limited to, conditioned medium obtained from cells and tissues (e.g., recombinant cells and cellular components) grown in culture.

[0057] The term "biomarker" refers to a nucleic acid molecule present in a sample obtained from a human cancer patient compared to a similar sample obtained from a control individual (e.g., an individual with a negative diagnosis or no cancer detected, a normal or healthy individual). A biomarker can be a detectable and / or quantifiable nucleic acid, nucleic acid fragment, polynucleotide, or oligonucleotide. Biomarkers include polynucleotides comprising a nucleotide sequence from a gene.

[0058] As used herein, the term "CpG island" refers to a GC-rich DNA segment in a gene body relative to the rest of the gene body. Typically, the GC content in these regions is 50% or greater, extending over hundreds of base pairs and sometimes thousands. These regions usually mark the 5' end of a gene.

[0059] As used herein, the term "early detection" of cancer refers to the possibility of discovering cancer before metastasis. Preferably, it refers to the possibility of discovering cancer before morphological changes in sample tissues or cells are observed.

[0060] As used herein, the terms "detect," "detecting," or "detection" can describe the general act of finding or identifying a detectably labeled composition, or the specific observation of a detectably labeled composition.

[0061] The term "gene" refers to a nucleic acid (e.g., DNA) sequence that includes a coding sequence necessary to produce a polypeptide, a precursor, or an RNA (e.g., a non-coding RNA, such as a ribosomal RNA, a transfer RNA, a spliceosomal RNA, a microRNA). A polypeptide or non-coding RNA may be encoded by a full-length coding sequence or by any portion of the coding sequence, as long as the desired activity or functional properties of the full-length polypeptide or polypeptide fragment (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) are retained. Therefore, a gene may include or exclude a promoter sequence, a terminator, a translational regulatory sequence (such as a ribosome binding site and an internal ribosome entry site), an enhancer, a silencer, an insulator, a boundary element, a replication origin, a matrix attachment site, and a locus control region. The term also encompasses the coding region of a structural gene and sequences located at the 5' and 3' ends, adjacent to the coding region, the distance of which at either end is about 1 kb or longer, so that the gene corresponds in length to the full-length mRNA. The term "gene" further includes cDNA and genome forms of the gene.

[0062] As used herein, the term "promoter" refers to a DNA region that is usually located upstream of a gene (towards the 5' region of the gene) and is required to initiate and drive gene transcription. A promoter allows the gene it controls to be properly activated or inhibited. A promoter may contain specific sequences that are recognized by transcription factors. These factors can bind to the promoter DNA sequence, causing the recruitment of RNA polymerase (an enzyme that synthesizes RNA from the coding region of a gene). A promoter generally specifies all gene regulatory components located upstream of a gene, including an upstream promoter, a 5'UTR, introns, and a leader sequence.

[0063] The term "exon" refers to any segment of a broken gene that is present in the mature RNA product. The term "intron" refers to any segment in DNA that has been transcribed but removed from the transcript by splicing with the exons on either side of it. Operationally, exon sequences are present in the mRNA sequence of a gene. Operationally, intron sequences are intervening sequences within the genome DNA of a gene that are sandwiched by exon sequences and typically have GT and AG splice consensus sequences at their 5' and 3' boundaries.

[0064] As used herein, the term "homology" refers to a first sequence that shares a certain degree of sequence identity with a second sequence, but whose sequence is not identical to that of the second sequence. For example, a polynucleotide comprising the wild-type sequence of a mutant gene is homologous to and not identical to the sequence of the mutant gene. In some embodiments, the degree of homology between the two sequences is sufficient to allow homologous recombination to occur therebetween under appropriately stringent conditions.

[0065] Techniques for determining nucleic acid and amino acid sequence identity include determining the nucleotide sequence of the mRNA of a gene and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Genome sequences can also be determined and compared in this manner. In general, identity refers to the exact correspondence of nucleotides to nucleotides or amino acids to amino acids of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotides or amino acids) can be compared by determining their percent identity. The percent identity of two sequences (whether nucleic acid or amino acid sequences) is the number of exact matches between the two compared sequences divided by the length of the shorter sequence and multiplied by 100.

[0066] In some embodiments, the degree of sequence similarity between polynucleotides can be determined by hybridizing the polynucleotides under conditions that allow stable duplex formation between homologous regions, followed by digestion with a single strand specific nuclease, and size determination of the digested fragments. Two nucleic acids or two polypeptide sequences are substantially homologous to each other when the sequences exhibit at least about 70%-75%, preferably 80%-82%, more preferably 85%-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity (as determined using the above method) over a defined length of the molecule. As used herein, substantially homologous also refers to sequences that show complete identity to a specified DNA or polypeptide sequence. Substantially homologous DNA sequences can be identified in a Southern hybridization experiment, for example, under stringent conditions (as defined for the particular system). See, for example, Sambrook et al., supra; Nucleic Acid Hybridization: A Practical Approach, ed. BD Hames and SJ Higgins, (1985) Oxford; Washington, DC; IRL Press).

[0067] As used herein, the term "prediction" refers to the likelihood that a patient will respond favorably or unfavorably to a drug or a group of drugs, and the extent of their response. Thus, treatment predictors are variables that are related to an individual patient's response to a particular therapy and are not related to prognosis.

[0068] As used herein, the term "methylation" refers to the presence of a methyl group added to one or more cytosine bases in a nucleic acid region (eg, genomic DNA) by the action of a DNA methyltransferase.

[0069] The term "methylation state" of a nucleic acid molecule refers to the presence or absence of one or more methylated nucleotide bases in a nucleic acid molecule. For example, a nucleic acid molecule containing methylated cytosine is considered methylated (i.e., the methylation state of the nucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered unmethylated.

[0070] The term "hypermethylation" refers to an average methylation state corresponding to an increased presence of methylated nucleotide bases of nucleic acid molecules at one or more CpG dinucleotides within the DNA sequence of a test DNA sample relative to the amount of methylated nucleotide bases of nucleic acid molecules found at the corresponding CpG dinucleotides in a normal control DNA sample.

[0071] The term "hypomethylation" refers to an average methylation state corresponding to a decrease in the presence of methylated nucleotide bases of nucleic acid molecules at one or more CpG dinucleotides within the DNA sequence of a test DNA sample relative to the amount of methylated nucleotide bases of nucleic acid molecules found at the corresponding CpG dinucleotides within a normal control DNA sample.

[0072] The term "C t "Threshold Cycle" is an abbreviation for threshold cycle and is defined as the calculated value of the cycle number when the PCR product exceeds the detection threshold.

[0073] The term "individual" refers to a human being.

[0074] The term "susceptibility" refers to a constitution or condition of the body that causes tissues to respond in a particular way to certain external stimuli and thus tends to make the individual more susceptible than usual to certain diseases.

[0075] The term "target" or "target sequence" refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided that sufficient conditions exist for binding to occur.

[0076] The term "risk" refers to the estimated probability of contracting a disease during a certain period of time, such as within the next 10 years, or during the lifetime of an individual.

[0077] As used herein, the term "prognosis" generally refers to the prediction of the likely course and outcome of a clinical condition or disease. Patient prognosis is usually performed by evaluating factors or symptoms of the disease that indicate a favorable or unfavorable course or outcome of the disease.

[0078] The term "weighted sum score" means that each possible alternative is scored by weighting the scores of all included objectives individually to emphasize the importance of different objectives.

[0079] As used herein, the term "nucleic acid molecule" (or "nucleic acid" or "polynucleotide") may refer to a polymeric form of nucleotides, which may include sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms, as well as mixed polymers of the above. Nucleotides may refer to ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. As used herein, "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide". Unless otherwise specified, nucleic acid molecules are generally at least 10 bases in length. The term may refer to RNA or DNA molecules of indefinite length. The term includes single-stranded and double-stranded forms of DNA. Nucleic acid molecules may include naturally occurring nucleotides and / or modified nucleotides, which are linked together by naturally occurring and / or non-naturally occurring nucleotide linkages.

[0080] Cancer is characterized by abnormal cell growth caused by one or more gene mutations or modifications, which leads to an imbalance in the balance between cell proliferation and cell death. In many disease processes (such as cancer), gene promoter CpG islands acquire abnormal high methylation, causing transcriptional silencing, which can be inherited to daughter cells after cell division. DNA methylation that causes silence in cancer typically occurs at multiple CpG sites in CpG islands, which are present in the promoters of protein-coding genes. Changes in DNA methylation have been considered an important component of cancer development. The clinical sensitivity and dynamic range provided by DNA methylation profiling are higher than other cancer detections. Therefore, the present disclosure provides a method and kit for early prediction, treatment response, and prognosis or recurrence monitoring of colorectal cancer.

[0081] In the present disclosure, the methylation state of the target DNA sequence or its fragment in the biological sample is measured to detect colorectal cancer or detect the susceptibility of colorectal cancer in human individuals or predict the treatment response, prognosis or recurrence of colorectal cancer in human individuals. In other embodiments, the methylation state of the target DNA sequence comprising TMEM240 or its fragment or MROH6 or its fragment in the biological sample is measured to detect colorectal cancer in human individuals or detect the susceptibility of colorectal cancer in human individuals or predict the treatment response, prognosis or recurrence of colorectal cancer in human individuals. In another embodiment, the methylation state of BEND5 or its fragment or SMAD3 or its fragment is further measured.

[0082] TMEM240 encodes transmembrane domain-containing protein transmembrane protein 240 found in the brain and cerebellum. Mutations in TMEM240 were found to cause spinocerebellar ataxia 21 (SCA21) with mental retardation, severe cognitive impairment, and hypokinetic and hyperkinetic movement disorders. In a preferred embodiment, the target DNA sequence comprises the promoter and exon 1 region of TMEM240.

[0083] The MROH6 gene encodes maestro heat-like repeat family member 6. Diseases associated with MROH6 include non-syndromic intellectual disability and autosomal recessive non-syndromic intellectual disability.

[0084] The BEND5 gene encodes a BEN domain-containing factor 5 that acts as a transcriptional repressor. In a preferred embodiment, the target DNA sequence comprises the promoter and exon 1 regions of BEND5.

[0085] The SMAD3 gene encodes SMAD family member 3 associated with transforming growth factor-β. In a preferred embodiment, the target DNA sequence comprises the promoter region of SMAD3.

[0086] In some embodiments, methylation comprises cytosine methylation sites. In some cases, cytosine methylation comprises 5-methylcytosine (5-mCyt) and 5-hydroxymethylcytosine. In some cases, cytosine methylation sites are present in CpG dinucleotide motifs. In other cases, cytosine methylation sites are present in CHG or CHH motifs in which adenine, cytosine or thymine are present. In some cases, one or more CpG dinucleotide motifs or CpG sites form CpG islands (short DNA sequences rich in CpG dinucleotides). In some cases, CpG islands typically, but not always, have a length between about 0.2 and about 1 kb. In some cases, methylation comprises CpG island methylation.

[0087] In some embodiments, the methylation status is analyzed by: methylation-specific enzymatic digestion; bisulfite sequencing; an analysis selected from promoter methylation, CpG island methylation, MSP, HeavyMethyl, MethyLight, and Ms-SNuPE; and other methods that rely on detecting amplified DNA. TM " refers to a fluorescence-based real-time PCR technology. MethylLight is described in Eads et al., Cancer Res. 59:2302-2306, 1999, which is incorporated herein by reference.

[0088] The term "HeavyMethyl" assay refers to an assay in which a methylation-specific blocking probe (also referred to herein as a blocker) encompassing CpG positions between or encompassed by amplification primers enables methylation-specific selective amplification of a nucleic acid sample.

[0089] The term "Ms-SNuPE" refers to Methylation-sensitive Single Nucleotide Primer Extension. MsSNuPE is described in Gonzalgo and Jones, Nucleic Acids Res. 25:2529-2531, 1997, which is incorporated herein by reference.

[0090] The term "MSP" refers to Methylation-specific PCR. MSP is described in Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996 and U.S. Pat. No. 5,786,146, each of which is incorporated herein by reference.

[0091] Bisulfite modification of DNA is a method for assessing CpG methylation status. 5-methylcytosine is the most frequent covalent base modification in eukaryotic cell DNA. However, the position of 5-methylcytosine cannot be directly identified by sequencing or hybridization methods because 5-methylcytosine has the same base pairing behavior as cytosine. In addition, the epigenetic information carried by 5-methylcytosine is completely lost during, for example, PCR amplification. Bisulfite sequencing is a method for analyzing whether 5-methylcytosine exists in DNA, which is based on a specific reaction of bisulfite with cytosine, and then after alkaline hydrolysis, cytosine is converted to uracil, and the base pairing behavior of uracil corresponds to thymine. However, 5-methylcytosine remains unmodified under the aforementioned conditions. Therefore, the original DNA is converted in such a way that methylcytosine, which is initially indistinguishable from cytosine due to hybridization behavior, can be used as the only remaining cytosine, which can be detected using molecular biology techniques (such as amplification and hybridization, or sequencing).

[0092] In one embodiment, the methylation status is detected by polymerase chain reaction, nucleic acid sequencing (such as bisulfite sequencing or pyrophosphate sequencing), bisulfite conversion, mass spectrometry, methylation-specific nucleases, mass-based separation, target capture, or microarray. In one embodiment, the methylation status is detected by amplifying the methylated CpG of the target gene using primers. In another embodiment, methylation is detected by PCR, methylation-specific PCR (MSP), real-time methylation-specific PCR, quantitative methylation-specific PCR (QMSP), PCR using a methylated DNA-specific binding protein, or quantitative PCR.

[0093] In one embodiment of the present disclosure, a target DNA sequence methylation-specific primer capable of amplifying the methylated CpG of the gene described herein can be used. The target DNA sequence methylation-specific primer comprises at least one or more CpG dinucleotides in the region hybridizing with the methylated CpG of the gene. Specifically, the target DNA sequence methylation-specific primer for amplifying the methylated CpG of the gene comprises a sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or a higher percentage of homology with a sequence selected from the group consisting of the following sequences as shown in Table 1.

[0094] In one embodiment of the present disclosure, a target DNA sequence methylation-specific probe capable of hybridizing to a methylated CpG of a gene described herein may be used. The target DNA sequence methylation-specific probe capable of hybridizing to a methylated CpG of a gene comprises at least one or more CpG dinucleotides in the region hybridizing to the methylated CpG of the gene. Specifically, the probe may comprise a sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or a higher percentage of homology with a sequence selected from the group consisting of the following sequences as shown in Table 1.

[0095] Table 1

[0096]

[0097]

[0098] In one embodiment, detecting the methylation status of the target DNA sequence comprises detecting the presence of hypermethylation in the target DNA sequence relative to a normal state of the target gene.

[0099] In some embodiments, the biological sample is tissue, cell, blood, urine, serum, plasma, feces, ascites, sputum, saliva, gastric juice, bile, or oral mucosa of a human subject suspected of having colorectal cancer or a human subject to be tested.

[0100] As used herein, the term "a person in need of cancer testing" refers to an individual who has received an initial diagnosis (e.g., a CT scan showing a mass or increased biomarker levels), but whose cancer stage or the presence or absence of methylated genes indicative of cancer is unknown. The term further includes a person who has had cancer (e.g., an individual in remission).

[0101] In some embodiments, the accuracy of a test to predict status is measured by the sensitivity of the assay, the specificity of the assay, or the area under the receiver operating characteristic (ROC) curve (AUC). For example, the larger the area under the ROC curve, the more accurate or effective the predictive value of the test.

[0102] In one embodiment, the weighted sum score is measured to determine the methylation status in the nucleic acid sequence and the gene as an indicator. For a variety of decision criteria, the weighted sum model (weighted sum model, WSM) is known to be the most preferred and simplest multi-criteria decision analysis (MCDA) / multi-criteria decision method for evaluating multiple alternatives. According to the present disclosure, the weighted sum score analysis shows that, compared with the control, the combination of TMEM240, MROH6, BEND5 and SMAD3 shows a sensitivity of about 100% and a specificity of about 96%.

[0103] In some embodiments, one or more biomarkers disclosed herein show statistical differences of at least p<0.05 in different samples. Detection tests using such biomarkers can show an AUC of at least 0.9.

[0104] In some embodiments, the hypermethylation state of the epigenetic biomarkers in the DNA sequences described herein is associated with a "poor" prognosis or with the likelihood that the individual will have an adverse reaction to a drug or a group of drugs, which leads to worsening of the cancer and / or refractoryness to one or more therapeutic agents. In some cases, a "poor" prognosis refers to the likelihood that the individual will not respond to a drug or a group of drugs, leading to worsening of the cancer. In some cases, a "poor" prognosis refers to a survival period of less than 5 years to less than 1 month for the individual. In some cases, a "poor" prognosis refers to the survival period of an individual, wherein after treatment, the survival period of the individual is less than 5 years to less than 1 month. In some cases, a "poor" prognosis further refers to the likelihood that the individual will develop a cancer that is refractory to one or more drugs.

[0105] In some embodiments, the present disclosure provides an isolated nucleic acid molecule having a sequence selected from the group consisting of SEQ ID Nos: 1-16.

[0106] In some embodiments, the present disclosure provides a kit for detecting the methylation status of a human individual who needs to be tested for susceptibility to colorectal cancer, or for predicting the likelihood, treatment response, prognosis or recurrence of colorectal cancer in a human individual, the kit comprising an isolated nucleic acid molecule having a sequence selected from a group consisting of SEQ ID No: 1 to 4 or 5 to 7, for analyzing the methylation status of a target DNA sequence comprising TMEM240 or a fragment thereof or MROH6 or a fragment thereof.

[0107] In some preferred embodiments of the present disclosure, the target DNA sequence further comprises one or more DNA sequences selected from the group consisting of BEND5 or a fragment thereof and SMAD3 or a fragment thereof or any combination thereof, and the kit further comprises an isolated nucleic acid molecule having a sequence selected from the group consisting of SEQ ID No: 8 to 16, for analyzing the methylation status of the target DNA sequence.

[0108] In some cases, the kit includes a plurality of target DNA sequence methylation-specific primers or target DNA sequence methylation-specific probes for detecting or measuring the methylation state / level of one or more target DNA sequences. In some cases, such kits include at least one polynucleotide hybridized with at least one methylation biomarker sequence described herein and at least one reagent for detecting gene methylation. Reagents for detecting methylation include, for example, sodium bisulfate, polynucleotides designed for hybridization with sequences as marker sequence products (if the marker sequence is not methylated (e.g., contains at least one CU conversion)), and / or methylation-sensitive or methylation-dependent restriction enzymes. In some cases, the kit provides a solid carrier in the form of an analytical device adapted for use in analysis. In some cases, the kit further includes a detectable label optionally connected to a polynucleotide (e.g., a probe) in the kit. In some embodiments, the kit further includes a processing unit for obtaining a weighted sum score as described herein.

[0109] Optionally, the kit also includes one or more detectably labeled polypeptides capable of hybridizing to the amplified portion. In some embodiments, the kit comprises primers sufficient to amplify the target DNA sequences described herein, and optionally comprises detectably labeled polynucleotides capable of hybridizing to each amplified DNA region or a portion thereof. The kit may further comprise a methylation-dependent or methylation-sensitive restriction enzyme and / or sodium bisulfite.

[0110] In some embodiments, the kit comprises sodium bisulfite, primers and adapters for amplifying the entire target gene, and polynucleotides (e.g., detectably labeled polynucleotides) to quantify the presence of converted methylated sequences and or converted unmethylated sequences of at least one cytosine in a DNA region of an epigenetic biomarker described herein.

[0111] In some embodiments, the kit comprises a methylation sensing restriction enzyme, primers and adapters for amplifying the entire target gene, and polynucleotides to quantify the number of copies of at least a portion of a DNA region of an epigenetic marker described herein. In some embodiments, the kit comprises a methylation binding moiety and one or more polynucleotides to quantify the number of copies of at least a portion of a DNA region of a marker described herein.

[0112] The disclosure described and claimed herein has a variety of attributes and embodiments, including (but not limited to) those attributes and embodiments illustrated or described or mentioned in this embodiment. It is not intended to be all-inclusive and the disclosure described and claimed herein is not limited to or restricted to the features or embodiments identified in this embodiment, which are included for the purpose of illustration and not limitation. It is readily recognized by those skilled in the art that various components and parameters may be changed or modified or substituted for known equivalents to some extent without departing from the scope of this disclosure. It should be understood that such modifications and equivalents are incorporated herein as if individually set forth. This disclosure also includes all steps, features, compositions and compounds mentioned or indicated in this specification, either individually or collectively, and any and all combinations of any two or more of the steps or features.

[0113] Although the present disclosure has been described by way of exemplary embodiments, various changes and modifications may occur to those skilled in the art. It is intended that the present disclosure encompass such changes and modifications as fall within the scope of the appended claims.

[0114] Examples

[0115] Materials and Methods

[0116] Sample preparation

[0117] Blood samples were collected using ETDA-K2 tubes and PAXgene blood ccfDNA (circulating free DNA) tubes (Qiagen, Hilden, Germany, 768165) designed specifically for in vitro diagnostic ccfDNA testing. Samples collected using ETDA-K2 tubes (BD, Plymouth, UK, 367525) were immediately centrifuged at 2000 × g for 10 minutes at 4 ° C. Within 2 hours, the supernatant from each sample was transferred to a new centrifuge tube and centrifuged at 6000 × g for 30 minutes at 4 ° C and then stored at -80 ° C. Samples collected using PAXgene blood ccfDNA tubes were kept at room temperature (15-25 ° C) until used within 3 days, and then centrifuged at 2000 × g for 10 minutes at 4 ° C, and then centrifuged at 6000 × g for 30 minutes at 4 ° C for separation of plasma. The plasma of each sample was divided into 1.6 mL aliquots and immediately frozen at -80 ° C until further use.

[0118] Cancer Genome Atlas Portal

[0119] The Western cohort data is based on data generated by The Cancer Genome Atlas (TCGA) research network from the Genomic Data Commons (GDC) data portal. The Cancer Genome Atlas (TCGA) is a collaboration between the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI) that has produced a comprehensive multidimensional map of key genomic changes in 33 cancer types. The TCGA dataset, which contains more than two petabytes of genomic data, is now accessible to the cancer research community to improve cancer prevention, diagnosis and treatment.

[0120] Genomic DNA extraction

[0121] Genomic DNA was extracted from matched primary tumor and adjacent colorectal tissue pairs from the same patient using the QIAamp DNA Mini Kit (Qiagen, Bonn, Germany, catalog number 51306) according to the manufacturer's instructions. After DNA quantification, purity was verified by measuring the A260 / A280 ratio (range 1.8 to 2.0) using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc, Wilmington, DE, USA).

[0122] Circulating Free DNA Extraction Manual

[0123] Circulating free DNA (cfDNA) was extracted from plasma samples using the MagMAX free DNA isolation kit (Thermo Fisher Scientific, Austin, TX, USA) or the capture cfDNA serum / plasma kit (CatchGene, New Taipei City, Taiwan Province, China) according to the manufacturer's recommended protocol. The ccfDNA sample has a clear fragment size peak between 140 and 200 bp. The DNA isolation kit provides the highest yield and low molecular weight fraction. Plasma was immediately separated from 10 mL of peripheral blood within 2 hours. After DNA quantification, purity was verified by measuring the A260 / A280 ratio (range 1.8 to 2.0) using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA).

[0124] By KingFisher TM Automated circulating free DNA extraction and bisulfite conversion using Duo Prime

[0125] At KingFisher TM ccfDNA extraction and bisulfite conversion were performed using an automated method on the Duo Prime purification system (ThermoFisher Scientific, Singapore) according to the manufacturer's instructions. This method enables simultaneous automated bead-based DNA extraction of up to six samples. TM The workflow was adapted as described in the instruction manual supplied with the free DNA isolation kit (Thermo Fisher Scientific, Austin, TX, USA, A29319). ccfDNA was extracted from 1.6 mL of plasma and eluted in 60 μL of molecular biology grade water (Corning, NY, USA, 46-000-CM). Bisulfite conversion cleanup was also performed on this machine for semi-automated analysis. TMThe instruction manual supplied with the MagPrep kit (Zymo Research, Irvine, CA, USA, D5046) was used to develop an automated protocol for bisulfite conversion cleanup. After utilizing the automated method, the extracted ccfDNA was incubated in a 60°C incubator with sodium bisulfite (6M) and hydroquinone (10mM) for 30 minutes. We used 60 μL ccfDNA for bisulfite conversion, and the bisulfite-converted ccfDNA was dissolved in 100 μL molecular biology grade water. The automated sample method was performed using a 24-deep well plate (ThermoFisher Scientific, Vantaa, Finland, 95040470). Methylation-specific real-time PCR was performed immediately using the dissolved ccfDNA converted with bisulfite.

[0126] Automated extraction of circulating free DNA using LabTurbo 24C

[0127] The automated ccfDNA extraction method was performed using the LabTurbo 24Compact system (Taigen Bioscience Co., Taipei, Taiwan) according to the manufacturer's instructions. The workflow followed the instruction manual supplied with the Labturbo Circulating DNA Mini Kit (Catalog No. AIOLCD1600, Taigen Bioscience Co., Taipei, Taiwan), where vacuum-based fully automated DNA extraction was performed simultaneously for up to 24 samples. ccfDNA was extracted from 1.6 mL of plasma and eluted in 60 μL of molecular biology grade water (46-000-CM, Corning, NY, USA). MethylationEPIC BeadChip Array for Whole Genome Methylation Analysis

[0128] The MethylationEPIC BeadChip (EPIC) array covers 850,000 CpG sites, including >90% of CpGs and 99% of Refseq genes of HM450 and an additional 413,743 CpGs. The EPIC array has been validated against the 450K platform for blood samples. Whole genome methylation analysis was performed using the MethylationEPIC BeadChip array (Illumina, San Diego, CA, USA). Bisulfite conversion was performed on 500 ng of DNA using the EpiTect Fast DNA Bisulfite Kit (QIAGEN, Bonn, Germany, catalog number 59826) according to the manufacturer's instructions. The methylation score for each CpG site is presented as a "β" value ranging from 0 (unmethylated) to 1 (fully methylated) by determining the ratio of the methylation signal intensity relative to the sum of the methylated and unmethylated signal outputs. Infinium MethylationEPIC BeadChip data were analyzed using GenomeStudio Methylation Module version 2011.1. The Infinium MethylationEPIC BeadChip was analyzed using both Infinium I and Infinium II. The Infinium I analysis design uses 2 bead types per CpG locus: 1 for each methylated and unmethylated state. The Infinium II design uses 1 bead type where methylation status is determined in a single base extension step after hybridization (right). Using heatmapper software, a heat map is used to visualize the different methylated CpG heat maps of the target gene. A gradient scale heat map is used to visualize DNA methylation levels from low to high.

[0129] Probe-based quantitative methylation-specific PCR (qMSP)

[0130] After bisulfite conversion of DNA according to the manufacturer's recommended protocol, TaqMan quantitative methylation-specific PCR (qMSP) was used in conjunction with LightCycler 96 (Roche Applied Science, Penzberg, Germany) to measure the DNA methylation levels of TMEM240, MROH6, BEND5, and SMAD3. TMqMSP was performed using the probe No-ROX set (Bioline, London, UK, catalog number BIO-86020) with specific primers and methyl-TaqMan probes for the candidate genes. Normalized DNA methylation values ​​calibrated to the control group were obtained using the LightCycler relative quantification software (version 1.5, Roche Applied Science). The β-actin (ACTB) gene was used as a methylation-independent DNA control. The primers / probes for the ACTB gene were designed to contain no CpG sites (as a control for input DNA). The primers / probes for the candidate genes were designed on their methylated promoter regions, especially on regions that were identified to be differentially expressed between normal and tumor tissues. According to the sequencing results, a successful PCR reaction can only be performed when all CpG sites are methylated. The target gene is considered to be hypermethylated when the methylation level in colorectal tumors (relative to the ACTB gene) is at least 2-fold higher than that in paired normal colorectal tissue samples. The specificity of the candidate gene methylation end product is confirmed by bisulfite sequencing. The primers and probes used for qMSP are listed in Table 1 .

[0131] Statistical analysis

[0132] Pearson's chi-squared test, Mann-Whitney U test, Wilcoxon test, and Spearman's rank correlation analyses were performed using SPSS (IBM, Armonk, NY, USA). Pearson's chi-squared test was used to compare colorectal cancer patients according to candidate gene methylation, RNA expression, and other clinical data. Paired-sample Wilcoxon test and t-test were used to compare DNA methylation differences between tumors and matched adjacent normal tissues, between different cancer types, and between surgical treatments of colorectal cancer patients. Spearman's rank correlation was used to analyze the methylation levels of tumor and plasma samples.

[0133] To evaluate the various biomarkers, Kang's nonparametric stepwise classification method was used to evaluate the accuracy of identifying patients with colorectal cancer when the proposed gene biomarkers were used. In addition to accuracy, other commonly used metrics for evaluating classification, such as the area under the receiver operating characteristic curve (AUC), sensitivity, specificity, false positive rate, and false negative rate, were also reported.

[0134] Example 1: Methylation status of target DNA sequences in colorectal cancer tissues

[0135] β values ​​based on data from the Illumina methylation 450K array were generated by the Cancer Genome Atlas (TCGA) research network. Target nucleic acids and genes were selected when the β value of normal tissue was less than 0.15; the Δβ value (the value of tumor minus the value of normal tissue) was higher than 0.5 or lower than 0.25. The methylation status Δβ values ​​(T) of the target DNA sequences are shown in Table 2.

[0136] Table 2

[0137] MROH6 TMEM240 BEND5 SMAD3 Colorectal cancer 0.50 0.73 0.53 0.09

[0138] β value (T), methylation status of tumor tissue; β value (T) ≥ 0.5 will be calculated as a hypermethylated biomarker and β value (T) ≤ 0.25 will be calculated as a hypomethylated biomarker.

[0139] Figure 1 shows the differences in methylation status (β value) of target nucleic acids and genes between tumor tissues and adjacent normal tissues (n=97). Based on data based on the Illumina Methylation 450K array, darker colors indicate tissues with higher methylation status.

[0140] Example 2: Early detection of epigenetic biomarker methylation status of target genes in plasma samples of healthy individuals and colorectal cancer patients

[0141] Extraction of circulating free DNA from plasma. Briefly, 3.5 mL of plasma was immediately separated from 10 mL of peripheral blood. After the extraction of circulating free DNA (cfDNA) from plasma obtained from colorectal cancer patients and healthy individuals, cfDNA was performed by bisulfite conversion. Probe-based methylation-specific real-time PCR (qMSP) was used for cfDNA methylation analysis.

[0142] The data obtained from the qMSP analysis were processed according to the following guidelines. t If the value is less than 45 cycles for the cyclic methylated TMEM240 gene, less than 40 cycles for the cyclic methylated MROH6 gene, less than 45 cycles for the cyclic methylated BEND5, and higher than 45 cycles for the cyclic methylated SMAD3, the score is defined as 1, respectively. Otherwise, the score is defined as 0.

[0143] Human subjects were defined as CRC patients when the average total qMSP scores of TMEM240, MROH6, and BEND5 were higher than 0.2.

[0144] FIG. 2 shows the early detection differences in the methylation status of epigenetic biomarkers of target genes in plasma samples of healthy individuals and colorectal cancer patients. Figure 3 The sum of DNA methylation levels (scores) of epigenetic biomarkers of target genes in plasma samples of healthy individuals and colorectal cancer patients is shown. Figure 4 Receiver operating characteristic (ROC) curve analysis shown in indicates early detection of epigenetic biomarker methylation status of target genes in colorectal cancer patients and healthy individuals. [Sequence Listing] <110> Alligator Biomedical Co., Ltd. <120> Methods for early detection, prediction of treatment response and prognosis of colorectal cancer <130> none <140> US 63 / 017,309 <141> 2021-04-29 <160> 16 <170> PatentIn Version 3.5 <210> 1 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Introduction <400> 1 tttagaatta tgaagattat ggtgttc 27 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Introduction <400> 2 aaaactcaac atcgaaccga 20 <210> 3 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Introduction <400> 3 cgaccccgcc cgatatccat aa 22 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 4 tttagaatta tgaagattat ggtgttc 27 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 ggtgagtttt tgatttgtaa ttgtc 25 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 atctcgtacc gctactacta cgc 23 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 7 gtcgggggtt gttgatttta gtagcgtt 28 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 gtttgggttt tggggagtc 19 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 gatcgaacaa ctcaacccg 19 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Introduction <400> 10 gtttttgtgc ggtttttgga 20 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Introduction <400> 11 aaccgcgaac gaaaactaaa 20 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 12 cgaaaataaa aatccgacga 20 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 13 ttgttacgcg ttgttcgtgt 20 <210> 14 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Introduction <400> 14 gaataaggtc gttagttatt atcgt 25 <210> 15 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 15 aatcaaatct acccgaatcg aa 22 <210> 16 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 16 gaaagaaaga aagaaagtaa attttatttt taagcg 36

Claims

1. A use of a methylation-specific primer or probe for a target DNA sequence comprising TMEM240, BEND5 and MROH6, which is used to prepare a drug for early detection of colorectal cancer in an individual, wherein the presence of high methylation of the target DNA sequence comprising TMEM240, BEND5 and MROH6 indicates colorectal cancer.

2. The method of claim 1, wherein the detection comprises providing a biological sample from the individual, and wherein the biological sample is tissue, cell, urine, serum, plasma, feces, ascites, sputum, saliva, gastric juice, bile or oral mucosa.

3. The use according to claim 2, wherein the tissue is blood.

4. The method of claim 2, wherein the detection further comprises the steps of defining a score of 1 if each gene is hypermethylated and a score of 0 if each gene is lacking hypermethylation, and summing the scores.

5. The use according to claim 1, wherein the presence of hypermethylation of the target DNA sequence in the individual is determined by comparing the methylation status of the target DNA sequence with the methylation status of a control DNA sequence.

6. The use according to claim 5, wherein the methylation status is determined by polymerase chain reaction.

7. The use according to claim 1, wherein the polymerase chain reaction C for determining the methylation status of MROH6 in the individual t Values ​​less than 45 indicate hypermethylation.

8. The use according to claim 1, wherein the polymerase chain reaction C for determining the methylation status of MROH6 in the subject t Values ​​less than 40 indicate hypermethylation. 9 . The use according to claim 1 , wherein the methylation-specific primer for the target DNA sequence comprising MROH6 comprises the sequences of SEQ ID NOs: 5 and 6. 10 . The use according to claim 1 , wherein the methylation-specific probe for the target DNA sequence comprising MROH6 comprises the sequence of SEQ ID NO:

7.

11. The use according to claim 1, wherein the methylation status is determined by polymerase chain reaction, and wherein the polymerase chain reaction C used to determine the methylation status of TMEM240 in the subject is t A value of less than 50 indicates hypermethylation; or when the polymerase chain reaction C used to determine the methylation status of BEND5 in the subject t Values ​​less than 50 indicate hypermethylation.

12. The use according to claim 1, wherein the methylation status is determined by polymerase chain reaction, and wherein the polymerase chain reaction C used to determine the methylation status of TMEM240 in the subject is t A value of less than 45 indicates hypermethylation; or when the polymerase chain reaction C used to determine the methylation status of BEND5 in the subject t Values ​​less than 45 indicate hypermethylation.

13. The use according to claim 1, in: The methylation-specific primers for the target DNA sequence of TMEM240 include a primer pair of SEQ ID NOs: 1 and 2 or SEQ ID NOs: 1 and 3; and The methylation-specific primers for the target DNA sequence of BEND5 include a primer pair of SEQ ID NOs: 8 and 9 or SEQ ID NOs: 10 and 11. 14 . The use according to claim 1 , wherein the methylation-specific probe for the target DNA sequence of TMEM240 comprises the sequence of SEQ ID NO: 4; and the methylation-specific probe for the target DNA sequence of BEND5 comprises a sequence selected from the group consisting of SEQ ID NO: 12 to 13.

15. The use according to any one of claims 1 to 14, wherein the methylation status is determined by a further step of measuring specificity and sensitivity by weighted sum score analysis.

16. An isolated nucleic acid molecule consisting of a sequence for detecting the methylation status of a target DNA sequence of TMEM240, MROH6 and BEND5.

17. The nucleic acid molecule according to claim 16, in: The sequence used to detect the methylation status of the target DNA sequence of TMEM240 is a primer pair of SEQ ID NOs: 1 and 2 or SEQ ID NOs: 1 and 3, or a specific probe of SEQ ID NO: 4; The sequence used to detect the methylation status of the target DNA sequence of MROH6 is the primer pair SEQ ID NO: 5 and 6, or the specific probe SEQ ID NO: 7; and The sequence used to detect the methylation status of the target DNA sequence of BEND5 is the primer pair of SEQ ID NOs: 8 and 9 or SEQ ID NOs: 10 and 11, or the specific probe SEQ ID NOs: 12 or 13.

18. A kit for early detection of colorectal cancer in an individual, the kit comprising a methylation-specific primer pair for detecting the methylation status of target DNA sequences of TMEM240, BEND5 and MROH6.

19. The kit of claim 18, wherein the methylation-specific primer pair for the target DNA sequence of TMEM240 is a primer pair of SEQ ID NOs: 1 and 2 or SEQ ID NOs: 1 and 3, the methylation-specific primer pair for the target DNA sequence of MROH6 is a primer pair of SEQ ID NOs: 5 and 6, and the methylation-specific primer pair for the target DNA sequence of BEND5 is a primer pair of SEQ ID NOs: 8 and 9 or SEQ ID NOs: 10 and 11.

20. The kit of claim 18, further comprising a methylation-specific probe for detecting the methylation status of the target DNA sequence for MROH6. 21 . The kit according to claim 20 , wherein the target DNA sequence methylation-specific probe for MROH6 is the specific probe SEQ ID NO:

7.

22. The kit of claim 18 or 19, wherein the kit further comprises methylation-specific probes for detecting the methylation status of the target DNA sequences for TMEM240, BEND5 and MROH6, wherein the methylation-specific probe for the target DNA sequence for TMEM240 is the specific probe SEQ ID NO:4, the methylation-specific probe for the target DNA sequence for BEND5 is the specific probe SEQ ID NO:12 or 13; and the methylation-specific probe for the target DNA sequence for MROH6 is the specific probe SEQ ID NO:7.

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