Methods and kits for screening for colorectal neoplasia

Abstract

The present disclosure provides methods for diagnosing colorectal neoplasia in a subject, screening for the onset or risk of onset of colorectal neoplasia, or assessing the development or prognosis of colorectal neoplasia, methods for monitoring treatment response in a subject undergoing treatment for colorectal neoplasia, and kits for use in the methods.

Description

[Technical Field]

[0001]

[0001] This disclosure relates generally to the biomedical field. In particular, this disclosure relates to methods for diagnosing colorectal neoplasia in a subject, screening for the onset or risk of onset of colorectal neoplasia, or assessing the development or prognosis of colorectal neoplasia, methods for monitoring treatment response in a subject undergoing treatment for colorectal neoplasia, and kits for use in the methods. [Background technology]

[0002]

[0002] Early detection of colorectal neoplasia at the precancerous, advanced adenoma stage or early cancer stage has been shown to significantly reduce patient mortality. Current colorectal neoplasia screening through colonoscopy or molecular testing on stool / blood samples is invasive or has very few markers, therefore, patient compliance with cancer screening and detection sensitivity are limited.

[0003]

[0003] Therefore, there is an increasing need to develop methods and / or kits that can efficiently read out epigenetic information from limited amounts of cell-free DNA derived from biological samples and that can be easily deployed and robustly implemented in clinical laboratories. Summary of the Invention [Means for solving the problem]

[0004] In one aspect, the present disclosure provides a method of diagnosing, screening for the development or risk of development of, or assessing the development or prognosis of a colorectal neoplasia in a subject, comprising: (I) treating DNA obtained from a biological sample with a reagent capable of distinguishing between unmethylated and methylated sites in DNA, thereby obtaining treated DNA; (II) quantifying the individual methylation levels of a set of target markers in the treated DNA of step (I), wherein the target markers are selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5; (III) comparing the methylation level of at least one target marker of the set of target markers each quantified in step (II) with a corresponding reference level, wherein the same or a higher methylation level of one or more of the target markers compared to its corresponding reference level indicates that the subject has a colorectal neoplasia, or has developed or is at risk for developing a colorectal neoplasia, or has or is at increased likelihood of developing a colorectal neoplasia, or has or is at risk for a poor prognosis for a colorectal neoplasia; The present invention provides a method comprising:

[0005] In another aspect, the present disclosure provides a method of diagnosing, screening for the development or risk of development of, or assessing the development or prognosis of a colorectal neoplasia in a subject, comprising: (I) treating DNA obtained from a biological sample with a reagent capable of distinguishing between unmethylated and methylated sites in DNA, thereby obtaining treated DNA; (II) quantifying individual methylation levels of a set of target markers in the treated DNA of step (I), wherein at least two target markers are selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, PKNOX2, VAV3, NDRG4 and IRF4, and at least two target markers are selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, INTERGENIC REGION 1, TMEFF2, INTERGENIC REGION 4, NKX2-6, INTERGENIC REGION 5, SLC24A2, INTERGENIC REGION 2, INTERGENIC REGION 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP; (III) comparing the methylation level of at least one target marker of the set of target markers each quantified in step (II) with a corresponding reference level, wherein the same or a higher methylation level of one or more of the target markers compared to its corresponding reference level indicates that the subject has a colorectal neoplasia, or has developed or is at risk for developing a colorectal neoplasia, or has or is at increased likelihood of developing a colorectal neoplasia, or has or is at risk for a poor prognosis for a colorectal neoplasia; The present invention provides a method comprising:

[0006]

[0006] In some embodiments, the set of target markers disclosed herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more target markers.

[0007] In some embodiments, step (II) of the present disclosure comprises: (i) pre-amplifying at least a portion of at least one target marker of a set of target markers in the treated DNA obtained from step (I) with a pre-amplification primer pool, wherein the set of target markers is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5; (ii) quantifying the individual methylation levels of the set of target markers in the DNA obtained from substep (i); Includes.

[0008]

[0008] In some embodiments, the methods of the present disclosure further comprise the step of obtaining DNA from a biological sample from the subject prior to step (I).

[0009] In another aspect, the present disclosure provides a method of diagnosing, screening for the development or risk of development of, or assessing the development or prognosis of a colorectal neoplasia in a subject, comprising: (a) obtaining a biological sample containing DNA from a subject; (b) treating the DNA in the biological sample obtained from step (a) with a reagent capable of distinguishing between unmethylated and methylated sites in DNA, thereby obtaining treated DNA; (c) pre-amplifying at least a portion of at least one target marker within the treated DNA obtained from step (b) with a pre-amplification primer pool, wherein at least a portion of at least one (e.g., each) of the target marker(s) is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5; with or without step (c); (d) if step (c) is present, individually quantifying the methylation level of one (e.g., each) target marker based on the DNA obtained from step (c); if step (c) is absent, individually quantifying the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b), wherein the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. 5; and including one or more markers selected from the group consisting of: (e) comparing the methylation level of at least one (e.g., each) target marker from step (d) with a corresponding reference level, wherein the same or a higher methylation level of one or more of the target marker(s) relative to its corresponding reference level indicates that the subject has a colorectal neoplasia, or has developed or is at risk for developing a colorectal neoplasia, or has or is at increased likelihood of developing a colorectal neoplasia, or has or is at risk for a poor prognosis for a colorectal neoplasia; The present invention provides a method comprising:

[0010]

[0010] In some embodiments, the at least one target marker in step (c) or step (d) of the above method comprises multiple target markers, and the multiple target markers comprise at least two markers selected from the group consisting of Septin9, BCAT1, and IKZF1.

[0011] In another aspect, the present disclosure provides a method of monitoring treatment response in a subject undergoing treatment for a colorectal neoplasia, comprising: (a) obtaining a biological sample containing DNA from a subject; (b) treating the DNA in the biological sample obtained from step (a) with a reagent capable of distinguishing between unmethylated and methylated sites in DNA, thereby obtaining treated DNA; (c) pre-amplifying at least a portion of at least one target marker within the treated DNA obtained from step (b) with a pre-amplification primer pool, wherein at least a portion of at least one (e.g., each) of the target marker(s) is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5; with or without step (c); (d) if step (c) is present, individually quantifying the methylation level of at least one (e.g., each) target marker based on the DNA obtained from step (c); if step (c) is not present, individually quantifying the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b), wherein the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. 5; and including one or more markers selected from the group consisting of: (e) comparing the methylation level of at least one (e.g., each) target marker from step (d) with a corresponding methylation level of one or more of the target marker(s) obtained from the same subject prior to the treatment, quantified by repeating steps (a), (b), optionally step (c), and (d) on a biological sample containing DNA obtained from the subject prior to the treatment, wherein a lower methylation level of one or more of the target marker(s) compared to its corresponding methylation level prior to the treatment indicates that the subject is responsive to the treatment; The present invention provides a method comprising:

[0012]

[0012] In some embodiments, the at least one target marker in step (c) or step (d) of the above method comprises multiple target markers, and the multiple target markers comprise at least two markers selected from the group consisting of Septin9, BCAT1, and IKZF1.

[0013] In some embodiments, the plurality of target markers further comprises one or more additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4, and IRF4. In some embodiments, the plurality of target markers further comprises one or more additional markers selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, INTERGENIC REGION 1, TMEFF2, INTERGENIC REGION 4, NKX2-6, INTERGENIC REGION 5, SLC24A2, INTERGENIC REGION 2, INTERGENIC REGION 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP.

[0014]

[0014] In some embodiments, each target marker comprises: a) a respective region defined by the Hg19 coordinates shown below;

[0015] [Table 1-1]

[0016] [Table 1-2]

[0017] and 5 kb upstream of the respective start site and 5 kb downstream of the respective end site of each of the above regions, or b) the bisulfite-converted counterpart of a), or c) the MSRE-treated counterpart of a). Contains, or is a), b), or c).

[0018] In some embodiments, the DNA in the biological sample obtained from step (a) comprises genomic DNA or cell-free DNA. In some embodiments, the cell-free DNA comprises circulating tumor DNA. In some embodiments, the target marker in the cell-free DNA is present in the biological sample in an amount of 1 ng, 0.8 ng, 0.6 ng, 0.4 ng, 0.2 ng, 0.1 ng, 0.08 ng, or 0.04 ng or less. In some embodiments, the target marker in the cell-free DNA is present in the biological sample at a concentration below the level of sensitivity of a detection assay for the target marker.

[0019] In some embodiments, the DNA obtained from substep (i) or step (c) is diluted with a diluent prior to substep (ii) or step (d).

[0020] In some embodiments, the biological sample is selected from the group consisting of tissue sections, biopsies, paraffin-embedded tissue, body fluids, colonic effluent, surgical resection samples, isolated blood cells, cells isolated from blood, and any combination thereof. In some embodiments, the body fluid is selected from the group consisting of whole blood, serum, plasma, urine, mucus, saliva, ascites, pleural fluid, pleural fluid, synovial fluid, cerebrospinal fluid, thoracentesis fluid, abdominal fluid, and any combination thereof. In some embodiments, the biological sample is obtained from the subject's plasma. In some embodiments, the colonic effluent is selected from the group consisting of a stool sample and an enema lavage sample.

[0021] In some embodiments, the reagent capable of distinguishing between unmethylated and methylated sites in DNA selectively modifies unmethylated cytosine residue(s) at CpG site(s) to produce modified residue(s), but does not significantly modify methylated cytosine residue(s). In some embodiments, the reagent capable of distinguishing between unmethylated and methylated sites in DNA comprises a bisulfite reagent. In some embodiments, the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, bisulfite salts, and any combination thereof.

[0022] In some embodiments, the reagent capable of distinguishing between unmethylated and methylated sites in DNA selectively cleaves at residues that are unmethylated but not cleave at residues that are methylated, or selectively cleaves at residues that are methylated but not cleave at residues that are unmethylated. In some embodiments, the reagent capable of distinguishing between unmethylated and methylated sites in DNA is a methylation-sensitive restriction enzyme (MSRE). In some embodiments, the MSRE is selected from the group consisting of HpaII, SalI, SalI-HF®, ScrFI, BbeI, NotI, SmaI, XmaI, MboI, BstBI, ClaI, MluI, NaeI, NarI, PvuI, SacII, HhaI, and any combination thereof.

[0023] In some embodiments, the pre-amplification primer pool comprises at least one methylation-specific primer pair, in which the at least one methylation-specific primer pair comprises a forward primer and a reverse primer, each comprising an oligonucleotide sequence that hybridizes under stringent, moderately stringent, or highly stringent conditions to at least 9 consecutive nucleotides of one of the target marker(s), wherein the at least 9 consecutive nucleotides of one of the target marker(s) comprises at least one CpG site.

[0024] In some embodiments, the pre-amplification primer pool further comprises a control primer pair for amplifying a control marker, in some embodiments, the control marker is selected from the group consisting of ACTB, GAPDH, tubulin, ALDOA, PGK1, LDHA, RPS27A, RPL19, RPL11, ARHGDIA, RPL32, C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP, and VPS29.

[0025]

[0022] In some embodiments, at least one methylation-specific primer pair comprises one or more pairs of nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54, and 170 / 171, as shown in Table 2 below.

[0026]

[0023] In some embodiments, in step (c), at least one target marker is amplified in the presence of one or more blocker oligonucleotides.

[0027] In some embodiments, quantification is performed by polymerase chain reaction (PCR) (e.g., real-time PCR, digital PCR), nucleic acid sequencing, mass-based separation (e.g., electrophoresis, mass spectrometry), or target capture (e.g., hybridization, microarray). In some embodiments, quantification is performed by real-time PCR, and optionally, the real-time PCR is multiplex real-time PCR.

[0028] In some embodiments, when step (c) is present, the quantification in step (d) comprises amplifying the DNA obtained from step (c) using a quantification primer pair(s) and a DNA polymerase, whereby at least a portion of the obtained DNA is amplified. In some embodiments, when step (c) is not present, the quantification in step (d) comprises amplifying at least one target marker in the treated DNA obtained from step (b) using a quantification primer pair(s) and a DNA polymerase.

[0029] In some embodiments, when step (c) is present, the quantification primer pair(s) used in step (d) are capable of hybridizing to at least 9 contiguous nucleotides of the DNA obtained from step (c) under stringent, moderately stringent, or highly stringent conditions. In some embodiments, when step (c) is not present, the quantification primer pair(s) used in step (d) are capable of hybridizing to at least 9 contiguous nucleotides of at least one target marker in the treated DNA obtained from step (b) under stringent, moderately stringent, or highly stringent conditions.

[0030] In some embodiments, when step (c) is present, at least one of the quantification primer pair(s) used in step (d) is identical to at least one of the methylation-specific primer pair(s) in the pre-amplification primer pool of step (c). In some embodiments, when step (c) is present, the quantification primer pair(s) used in step (d) is designed to amplify at least a portion of the DNA obtained from step (c). In some embodiments, when step (c) is not present, the quantification primer pair(s) used in step (d) is designed to amplify at least a portion of at least one target marker in the treated DNA obtained from step (b).

[0031] In some embodiments, step (d) is carried out in the presence of a detecting agent. In some embodiments, the detecting agent is selected from the group consisting of a fluorescent probe, an intercalating dye, a chromophore-labeled probe, a radioisotope-labeled probe, and a biotin-labeled probe. In some embodiments, the fluorescent probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 57-85, 172. In some embodiments, the fluorescent probe is labeled at its 5'-end with a fluorescent dye (e.g., FAM, HEX / VIC, TAMRA, Texas Red, or Cy5) and at its 3'-end with a quencher (e.g., BHQ1, BHQ2, BHQ3, DABCYL, or TAMRA).

[0032]

[0029] In some embodiments, step (e) includes comparing the Ct value(s) of the target marker(s) of step (d) with reference Ct values, wherein a same or lower Ct value of at least one target marker compared to its corresponding reference Ct value indicates that the subject has a colorectal neoplasia, has developed or is at risk for developing a colorectal neoplasia, or has or has an increased likelihood of developing a colorectal neoplasia, or has a poor prognosis or is at risk for a poor prognosis for a colorectal neoplasia; or a higher Ct value of at least one target marker compared to its corresponding Ct value prior to treatment indicates that the subject being treated for a colorectal neoplasia is responsive to the treatment.

[0033]

[0030] In some embodiments, the pre-amplification comprises 5 to 30 cycles of reaction, each cycle comprising a reaction at 85 to 99°C for 5 seconds to 5 minutes followed by a reaction at 40 to 80°C for 5 seconds to 5 minutes.

[0034] In some embodiments, when step (c) is present, the quantification in step (d) comprises determining a methylation level based on the presence or level of multiple CpG dinucleotides, TpG dinucleotides, or CpA dinucleotides in the DNA obtained from step (c). In some embodiments, when step (c) is not present, the quantification in step (d) comprises determining a methylation level based on the presence or level of multiple CpG dinucleotides, TpG dinucleotides, or CpA dinucleotides at at least one target marker in the processed DNA obtained from step (b). In some embodiments, when step (c) is present, the quantification in step (d) comprises determining a methylation level of a cytosine residue(s) based on the presence or level of one or more CpG dinucleotides in the DNA obtained from step (c). In some embodiments, when step (c) is not present, the quantification in step (d) comprises determining a methylation level of a cytosine residue(s) based on the presence or level of one or more CpG dinucleotides at at least one target marker in the processed DNA obtained from step (b). In some embodiments, when step (c) is present, the quantification in step (d) is carried out by dividing the DNA obtained from step (c) into multiple fractions. In some embodiments, when step (c) is not present, the quantification in step (d) is carried out by dividing at least one target marker in the treated DNA obtained from step (b) into multiple fractions.

[0035]

[0032] In some embodiments, the reference level in step (e) is determined based on clinical samples obtained from a group of individuals who have or are at risk of having a colorectal neoplasia and a group of individuals who are not at risk of or are free from having a colorectal neoplasia.

[0036] In some embodiments, the colorectal neoplasia is colorectal cancer, large colorectal adenoma, and / or sessile serrated polyp. In some embodiments, the colorectal neoplasia is precancerous. In some embodiments, the subject is a human.

[0037] In another aspect, the present disclosure provides a kit for diagnosing, screening for the development or risk of development of, or assessing the development or prognosis of a colorectal neoplasia, comprising: (a) a first reagent for treating DNA, the first reagent being capable of distinguishing between unmethylated and methylated sites in DNA; (b) Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5, as appropriate. a first primer pool comprising at least one primer pair for pre-amplifying at least one target sequence in at least one target marker selected from the group consisting of: 5, wherein the at least one primer pair can hybridize to at least 9 consecutive nucleotides of the at least one target sequence treated with a first reagent under stringent, moderately stringent, or highly stringent conditions; the first primer pool, wherein the target sequence comprises at least one CpG site; and (c) a second reagent, when the first primer pool is present, for quantifying the methylation level of at least one (e.g., each) target marker pre-amplified by the first primer pool; and when the first primer pool is absent, for quantifying the methylation level of at least one (e.g., each) target marker in DNA treated with the first reagent, wherein the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, and INTERGENIC REGION 4. a second reagent comprising one or more markers selected from the group consisting of INTERGENIC REGION 4, and INTERGENIC REGION 5; A kit comprising:

[0038]

[0035] In some embodiments, the at least one target marker comprises a plurality of target markers, and the plurality of target markers comprises at least two markers selected from the group consisting of Septin9, BCAT1, and IKZF1.

[0039] In some embodiments, when the first primer pool is present, the second reagent comprises a second primer pool comprising a plurality of quantification primer pairs capable of hybridizing to at least 9 contiguous nucleotides of at least one target sequence pre-amplified by the first primer pool under stringent, moderately stringent, or highly stringent conditions. In some embodiments, when the first primer pool is not present, the second reagent comprises a third primer pool comprising a plurality of quantification primer pairs capable of hybridizing to at least 9 contiguous nucleotides of at least one target sequence of said at least one target marker in the DNA treated by the first reagent under stringent, moderately stringent, or highly stringent conditions.

[0040] In some embodiments, at least one of the quantification primer pairs in the second primer pool is identical to at least one of the primer pairs in the first primer pool. In some embodiments, when the first primer pool is present, the quantification primer pair of the second primer pool is designed to amplify at least a portion of at least one target sequence pre-amplified by the first primer pool. In some embodiments, when the first primer pool is absent, the quantification primer pair of the third primer pool is designed to amplify at least a portion of at least one target sequence of at least one target marker in the DNA treated with the first reagent.

[0041]

[0038] In some embodiments, the first, second, or third primer pool comprises at least one methylation-specific primer pair.

[0042] In some embodiments, the first and second pools of primers are packaged in a single container or in separate containers. In some embodiments, the kit further comprises one or more blocker oligonucleotides.

[0043] In some embodiments, the kit further comprises a detection agent. In some embodiments, the detection agent is selected from the group consisting of a fluorescent probe, an intercalating dye, a chromophore-labeled probe, a radioisotope-labeled probe, and a biotin-labeled probe. In some embodiments, the fluorescent probe comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 57-85, 172. In some embodiments, the fluorescent probe is labeled at its 5' end with a fluorescent dye (e.g., FAM, HEX / VIC, TAMRA, Texas Red, or Cy5) and at its 3' end with a quencher (e.g., BHQ1, BHQ2, BHQ3, DABCYL, TAMRA, or Iowa Black Dark Quencher).

[0044] In some embodiments, the kit further comprises a DNA polymerase and / or a container suitable for containing a biological sample from a subject. In some embodiments, the kit further comprises instructions for use and / or interpretation of the kit results.

[0045] In some embodiments, the first reagent comprises a bisulfite reagent or a methylation-sensitive restriction enzyme (MSRE). In some embodiments, the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, bisulfite, and any combination thereof. In some embodiments, the MSRE is selected from the group consisting of HpaII, SalI, SalI-HF®, ScrFI, BbeI, NotI, SmaI, XmaI, MboI, BstBI, ClaI, MluI, NaeI, NarI, PvuI, SacII, HhaI, and any combination thereof.

[0046] In some embodiments, when the first primer pool is present, the first primer pool comprises a plurality of primer pairs for pre-amplifying at least one target sequence in the plurality of target markers, wherein the plurality of target markers comprises at least two markers selected from the group consisting of Septin9, BCAT1, and IKZF1, and further comprises one or more additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4, and IRF4. In some embodiments, when the first primer pool is not present, the third primer pool comprises a plurality of primer pairs for amplifying at least one target sequence in the plurality of target markers, wherein the plurality of target markers comprises at least two markers selected from the group consisting of Septin9, BCAT1, and IKZF1, and further comprises one or more additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4, and IRF4. In some embodiments, the plurality of target markers further comprises one or more additional markers selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, INTERGENIC REGION 1, TMEFF2, INTERGENIC REGION 4, NKX2-6, INTERGENIC REGION 5, SLC24A2, INTERGENIC REGION 2, INTERGENIC REGION 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP.

[0047] In some embodiments, each target marker comprises: a) a respective region defined by the Hg19 coordinates shown below;

[0048] [Table 2-1]

[0049] [Table 2-2]

[0050] and 5 kb upstream of the respective start site and 5 kb downstream of the respective end site of each of the above regions, or b) the bisulfite-converted counterpart of a), or c) the MSRE-treated counterpart of a). Contains, or is a), b) or c).

[0051]

[0045] In some embodiments, when a first primer pool is present, the first primer pool comprises at least one primer pair comprising or consisting of at least one pair of nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54, and 170 / 171 as shown in Table 2 below; and optionally, the second primer pool comprises at least one primer pair that is identical to at least one of the primer pairs in the first primer pool. In some embodiments, when the first primer pool is absent, the third primer pool comprises at least one primer pair comprising or consisting of at least one pair of nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54, and 170 / 171 as shown in Table 2 below.

[0052] In some embodiments, the first primer pool, the second primer pool, or optionally the third primer pool further comprises a primer pair for amplifying a control marker, in some embodiments, the control marker is selected from the group consisting of ACTB, GAPDH, tubulin, ALDOA, PGK1, LDHA, RPS27A, RPL19, RPL11, ARHGDIA, RPL32, C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP, and VPS29.

[0053]

[0047] In some embodiments, the kit further comprises a plurality of containers, each for receiving a fraction of the second primer pool.

[0054]

[0048] In another aspect, the present disclosure provides the use of a kit of the present disclosure in the manufacture of a diagnostic kit for diagnosing colorectal neoplasia in a subject, screening for the onset or risk of onset of colorectal neoplasia, or assessing the development or prognosis of colorectal neoplasia, or monitoring treatment response in a subject undergoing treatment for colorectal neoplasia.

[0055] In another aspect, the present disclosure provides a method of diagnosing, screening for the development or risk of development of, or assessing the development or prognosis of a colorectal neoplasia in a subject, comprising: (a) obtaining a biological sample containing DNA from a subject; (b) treating the DNA in the biological sample obtained from step (a) with a reagent capable of distinguishing between unmethylated and methylated CpG site(s) in DNA, thereby obtaining treated DNA; (c) pre-amplifying at least a portion of at least one target marker within the treated DNA obtained from step (b) with a pre-amplification primer pool, wherein at least a portion of at least one (e.g., each) of the target marker(s) is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5; with or without step (c); (d) if step (c) is present, individually quantifying the methylation level of at least one (e.g., each) target marker based on the DNA obtained from step (c); if step (c) is not present, individually quantifying the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b), wherein the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. 5; and including one or more markers selected from the group consisting of: (e) comparing the methylation level of at least one (e.g., each) target marker, respectively, from step (d) with a corresponding reference level, wherein the same or a higher methylation level of the at least one target marker compared to its corresponding reference level indicates that the subject has a colorectal neoplasia, or has developed or is at risk for developing a colorectal neoplasia, or has or is at increased likelihood of developing a colorectal neoplasia, or has or is at risk for a poor prognosis for a colorectal neoplasia; The present invention provides the use of a reagent for quantifying the methylation level of a target marker in the manufacture of a kit for use in a method comprising:

[0056] In another aspect, the present disclosure provides a method of monitoring treatment response in a subject undergoing treatment for a colorectal neoplasm, comprising: (a) obtaining a biological sample containing DNA from a subject; (b) treating the DNA in the biological sample obtained from step (a) with a reagent capable of distinguishing between unmethylated and methylated CpG site(s) in DNA, thereby obtaining treated DNA; (c) pre-amplifying at least a portion of at least one target marker within the treated DNA obtained from step (b) with a pre-amplification primer pool, wherein at least a portion of at least one (e.g., each) of the target marker(s) is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5; with or without step (c); (d) if step (c) is present, individually quantifying the methylation level of at least one (e.g., each) target marker based on the DNA obtained from step (c); if step (c) is not present, individually quantifying the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b), wherein the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. 5; and including one or more markers selected from the group consisting of: (e) comparing the methylation level of at least one (e.g., each) target marker from step (d) with a corresponding methylation level of one or more of the target marker(s) obtained from the same subject prior to the treatment, quantified by repeating steps (a), (b), optionally step (c), and (d) on a biological sample containing DNA obtained from the subject prior to the treatment, wherein a lower methylation level of one or more of the target marker(s) compared to its corresponding methylation level prior to the treatment indicates that the subject is responsive to the treatment; The present invention provides the use of a reagent for quantifying the methylation level of a target marker in the manufacture of a kit for use in a method comprising: [Brief explanation of the drawings]

[0057] [Figure 1]

[0051] Figure 1 shows the validation of methylation-specific primers for the target marker PKNOX2 (Figure 1A) and the control marker ACTB (Figure 1B). The Y-axis shows the ΔRn value, which was determined by subtracting the baseline fluorescence intensity from the fluorescence intensity at the indicated cycle. The X-axis shows the cycle number. As shown in Figure 1A, in the mixed DNA composition, the Ct value decreased as the percentage of converted methylated DNA increased, indicating that the primers used for preamplification of PKNOX2 were methylation-specific. As shown in Figure 1B, the curves for each DNA composition overlapped, indicating that the Ct value did not change even with an increase in the percentage of converted methylated DNA, which is consistent with the primers used for preamplification of the control marker ACTB being non-methylation-specific. [Figure 2]

[0052] Figure 2 shows the methylation abundance of the control marker ACTB and the target markers SALL1 and PKNOX2 in white blood cells (WBCs, indicated by solid circles "●"), paracancerous tissues (para tissues, indicated by solid boxes "■"), advanced adenoma tissues (AA tissues, indicated by solid equilateral triangles "▲"), and colorectal cancer tissues (CRC tissues, indicated by solid inverted triangles "▼"). The Y-axis shows the Ct value, and the X-axis shows the names of the control and target markers. A higher Ct value indicates a lower methylation abundance of the marker. Therefore, Figure 2 indicates that the methylation abundance of the target marker in WBCs was significantly lower than that in tissue samples. In particular, the methylation abundance of the target marker was lower in paracancerous tissues than in advanced adenoma tissues and colorectal cancer tissues. [Figure 3]

[0053] Figure 3 shows the distribution of the control marker ACTB and the target markers SALL1 and BCAN in biological samples obtained from a colorectal cancer patient population (CRC plasma, indicated by a solid circle "●") and a colorectal endoscopy-negative population (healthy plasma, indicated by a solid equilateral triangle "▲"). The Y-axis shows the Ct value, and the X-axis shows the names of the control marker and target marker. A lower Ct value indicates a higher methylation level of that marker. Therefore, Figure 3 shows that the methylation level of each target marker in the colorectal cancer patient population is significantly higher than that in the colorectal endoscopy-negative patient population. [Figure 4]

[0054] Figure 4 shows the AUC values ​​for all 13 target markers tested. The Y-axis represents the number of occurrences with the same AUC value, and the X-axis represents the AUC value. The AUC values ​​range from 0 to 1, with larger AUC values ​​representing higher classification power. As shown in the figure, all tested markers (i.e., NDRG4, Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, and INTERGENIC REGION 1) had AUCs in the range of 0.8 to 0.9, demonstrating the classification power for distinguishing CRC from control groups. [Figure 5]

[0055] Figure 5 shows the ROC curve for the combination of markers SALL1, BCAT1, and Septin9. The Y-axis indicates the true positive rate (i.e., sensitivity), and the X-axis indicates the false positive rate (i.e., 1 - specificity). The solid line represents the ROC curve, and the dotted line represents the 45-degree diagonal line. Points above the diagonal line represent good classification results (i.e., better than random), and points below the line represent bad results (i.e., worse than random). Therefore, the combination of target markers SALL1, BCAT1, and Septin9 has high sensitivity and high specificity in classifying colorectal neoplasms. [Figure 6-1]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-2]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-3]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-4]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-5]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-6]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-7]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-8]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-9]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-10]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-11]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-12]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-13]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-14]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-15]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-16]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-17]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-18]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-19]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-20]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-21]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-22]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-23]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-24]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-25]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-26]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-27]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-28]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-29]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-30]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-31]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-32]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-33]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-34]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-35]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-36]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-37]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-38]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-39]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-40]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-41]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-42]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-43]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-44]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-45]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-46]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-47]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-48]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-49]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-50]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-51]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-52]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-53]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-54]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-55]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-56]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-57]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-58]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-59]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-60]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-61]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-62]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-63]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-64]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-65]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-66]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-67]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-68]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-69]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-70]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-71]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-72]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-73]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-74]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-75]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-76]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-77]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-78]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-79]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-80]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-81]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-82]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-83]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-84]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-85]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-86]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-87]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-88]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-89]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-90]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-91]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-92]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-93]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-94]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-95]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-96]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-97]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-98]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-99]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-100]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-101]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-102]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-103]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-104]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-105]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-106]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-107]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-108]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. [Figure 6-109]

[0056] Figure 6 shows the base sequences of exemplary partial regions of target markers. DETAILED DESCRIPTION OF THE INVENTION

[0058]

[0057] Various aspects and embodiments of the present disclosure are disclosed below, but those skilled in the art can make various equivalent changes and modifications without departing from the spirit and scope of the subject matter of this application. The various aspects and embodiments disclosed herein are provided for illustrative purposes only and are not intended to limit the present disclosure. The actual scope of protection of this application is defined by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. All literature, patents, and patent applications cited in this disclosure are incorporated herein by reference in their entirety.

[0059] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural forms of the same unless the context clearly dictates otherwise. Thus, for example, reference to a "reagent" includes a plurality of reagents.

[0060]

[0059] Throughout this specification and the claims that follow, unless the context otherwise requires, the words "comprise," "contain," or "include," and variations such as "comprises," "consisting," "containing," "includes," "including," etc., will be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0061]

[0060] Cancer diagnosis has traditionally relied on the detection of a single marker (e.g., a gene mutation). However, cancer is a disease state in which a single marker typically cannot detect or distinguish many pathologies. Furthermore, the level of a single marker in a biological sample is usually very limited, further reducing the diagnostic specificity and / or diagnostic sensitivity of cancer. Therefore, assays that recognize only a single marker have shown limited predictive value.

[0062] One aspect of the present disclosure is to perform pre-amplification of at least a portion of at least one target marker prior to individually quantifying the methylation level of at least one (e.g., each) target marker based on DNA obtained from the pre-amplification, such that at least a portion of the at least one target marker is pre-amplified. Such a pre-amplification step is believed to increase the amount(s) / level(s) of the target marker(s) and has been found to significantly increase the diagnostic specificity and / or diagnostic sensitivity of colorectal neoplasms. Another aspect of the present disclosure is to simultaneously quantify the methylation levels of multiple target markers in a biological sample, so as to increase the diagnostic specificity and / or diagnostic sensitivity of colorectal neoplasms. In certain embodiments, multiple target markers are not pre-amplified before being quantified. In certain embodiments, multiple target markers are pre-amplified before being quantified. In particular, the inventors of the present disclosure have surprisingly found that simultaneous quantification of the methylation levels of multiple target markers in a biological sample, or a combination of a pre-amplification step and a quantification step, significantly increases the diagnostic specificity and / or diagnostic sensitivity of colorectal neoplasia, allowing for the early detection of, for example, precancerous adenoma or early cancerous stages of colorectal neoplasia. As will be understood by those skilled in the art, in the context of diagnosis, "sensitivity" defines the proportion of correctly identified positive results, i.e., the percentage of subjects correctly identified as having the disease in question. Meanwhile, "specificity" defines the proportion of correctly identified negative results, i.e., the percentage of subjects correctly identified as not having the disease in question. method In one aspect, the present disclosure provides a method of diagnosing, screening for the development or risk of development of, or assessing the development or prognosis of a colorectal neoplasia in a subject, comprising: (I) treating DNA obtained from a biological sample with a reagent capable of distinguishing between unmethylated and methylated sites in DNA, thereby obtaining treated DNA; (II) quantifying the individual methylation levels of a set of target markers in the treated DNA of step (I), wherein the target markers are selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5; (III) comparing the methylation level of at least one target marker of the set of target markers each quantified in step (II) with a corresponding reference level, wherein the same or a higher methylation level of one or more of the target markers compared to its corresponding reference level indicates that the subject has a colorectal neoplasia, or has developed or is at risk for developing a colorectal neoplasia, or has or is at increased likelihood of developing a colorectal neoplasia, or has or is at risk for a poor prognosis for a colorectal neoplasia; The present invention provides a method comprising:

[0063] In another aspect, the present disclosure provides a method of diagnosing, screening for the development or risk of development of, or assessing the development or prognosis of a colorectal neoplasia in a subject, comprising: (I) treating DNA obtained from a biological sample with a reagent capable of distinguishing between unmethylated and methylated sites in DNA, thereby obtaining treated DNA; (II) quantifying individual methylation levels of a set of target markers in the treated DNA of step (I), wherein at least two target markers are selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, PKNOX2, VAV3, NDRG4 and IRF4, and at least two target markers are selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, INTERGENIC REGION 1, TMEFF2, INTERGENIC REGION 4, NKX2-6, INTERGENIC REGION 5, SLC24A2, INTERGENIC REGION 2, INTERGENIC REGION 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP; (III) comparing the methylation level of at least one target marker of the set of target markers each quantified in step (II) with a corresponding reference level, wherein the same or a higher methylation level of one or more of the target markers compared to its corresponding reference level indicates that the subject has a colorectal neoplasia, or is at or at risk for developing a colorectal neoplasia, or will develop or has an increased likelihood of developing a colorectal neoplasia, or has or is at risk for a poor prognosis for a colorectal neoplasia; The present invention provides a method comprising:

[0064] In another aspect, the present disclosure provides a method of monitoring treatment response in a subject undergoing treatment for a colorectal neoplasia, comprising: (I) treating DNA obtained from a biological sample with a reagent capable of distinguishing between unmethylated and methylated sites in DNA, thereby obtaining treated DNA; (II) quantifying the individual methylation levels of a set of target markers in the treated DNA of step (I), wherein the target markers are selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5; (III) comparing the methylation level of at least one target marker of the set of target markers, each quantified in step (II), with a corresponding methylation level of one or more of the target marker(s) obtained from the same subject prior to the treatment, quantified by repeating steps (I) and (II) with a biological sample containing DNA obtained from the subject prior to the treatment, wherein a lower methylation level of one or more of the target marker(s) compared to its corresponding methylation level prior to the treatment indicates that the subject is responsive to the treatment; The present invention provides a method comprising:

[0065] In another aspect, the present disclosure provides a method of monitoring treatment response in a subject undergoing treatment for a colorectal neoplasia, comprising: (I) treating DNA obtained from a biological sample with a reagent capable of distinguishing between unmethylated and methylated sites in DNA, thereby obtaining treated DNA; (II) quantifying individual methylation levels of a set of target markers in the treated DNA of step (I), wherein at least two target markers are selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, PKNOX2, VAV3, NDRG4 and IRF4, and at least two target markers are selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, INTERGENIC REGION 1, TMEFF2, INTERGENIC REGION 4, NKX2-6, INTERGENIC REGION 5, SLC24A2, INTERGENIC REGION 2, INTERGENIC REGION 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP; (III) comparing the methylation level of at least one target marker of the set of target markers each quantified in step (II) with a corresponding methylation level of one or more of the target marker(s) obtained from the same subject prior to the treatment, quantified by repeating step (I) and step (II) with a biological sample containing DNA obtained from the subject prior to the treatment, wherein a lower methylation level of one or more of the target marker(s) compared to its corresponding methylation level prior to the treatment indicates that the subject is responsive to the treatment; The present invention provides a method comprising:

[0066]

[0066] In some embodiments, the set of target markers disclosed herein includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more target markers.

[0067] In some embodiments, step (II) of the present disclosure comprises: (i) pre-amplifying at least a portion of at least one target marker of a set of target markers in the treated DNA obtained from step (I) with a pre-amplification primer pool, wherein the set of target markers is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5; (ii) quantifying the individual methylation levels of the set of target markers in the DNA obtained from substep (i); Includes:

[0068] In some embodiments, substep (i) of step (II) is present. In some embodiments, substep (i) of step (II) is absent. In some embodiments, the method further comprises obtaining DNA from a biological sample from the subject prior to step (I).

[0069] In another aspect, the present disclosure provides a method of diagnosing, screening for the development or risk of development of, or assessing the development or prognosis of a colorectal neoplasia in a subject, comprising: (a) obtaining a biological sample containing DNA from a subject; (b) treating the DNA in the biological sample obtained from step (a) with a reagent capable of distinguishing between unmethylated and methylated sites in DNA, thereby obtaining treated DNA; (c) pre-amplifying at least a portion of at least one target marker within the treated DNA obtained from step (b) with a pre-amplification primer pool, wherein at least a portion of at least one (e.g., each) of the target marker(s) is pre-amplified, the at least one target marker comprising one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5; with or without step (c); (d) if step (c) is present, individually quantifying the methylation level of at least one (e.g., each) target marker based on the DNA obtained from step (c); if step (c) is not present, individually quantifying the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b), wherein the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. 5; and including one or more markers selected from the group consisting of: (e) comparing the methylation level of at least one (e.g., each) target marker, respectively, from step (d) with a corresponding reference level, wherein the same or a higher methylation level of one or more of the target marker(s) relative to its corresponding reference level indicates that the subject has a colorectal neoplasia, or is at or at risk for developing a colorectal neoplasia, or will develop or has an increased likelihood of developing a colorectal neoplasia, or has or is at risk for a poor prognosis for a colorectal neoplasia; The present invention provides a method comprising:

[0070] In another aspect, the present disclosure provides a method of monitoring treatment response in a subject undergoing treatment for a colorectal neoplasia, comprising: (a) obtaining a biological sample containing DNA from a subject; (b) treating the DNA in the biological sample obtained from step (a) with a reagent capable of distinguishing between unmethylated and methylated sites in DNA, thereby obtaining treated DNA; (c) pre-amplifying at least a portion of at least one target marker within the treated DNA obtained from step (b) with a pre-amplification primer pool, wherein at least a portion of at least one (e.g., each) of the target marker(s) is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5; with or without step (c); (d) if step (c) is present, individually quantifying the methylation level of at least one (e.g., each) target marker based on the DNA obtained from step (c); if step (c) is not present, individually quantifying the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b), wherein the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. 5; and including one or more markers selected from the group consisting of: (e) comparing the methylation level of at least one (e.g., each) target marker from step (d) with a corresponding methylation level of one or more of the target marker(s) obtained from the same subject prior to treatment, quantified by repeating steps (a), (b), optionally step (c), and (d) on a biological sample containing DNA obtained from the subject prior to treatment, wherein a lower methylation level of one or more of the target marker(s) compared to its corresponding methylation level prior to treatment indicates that the subject is responsive to the treatment; The present invention provides a method comprising:

[0071] As used herein, the terms "screen for" and variations such as "screens for" or "screening for" refer to the identification of a pathological state, disease or condition, such as the identification of a colorectal neoplasm, or the identification of a subject with a colorectal neoplasm who may benefit from a particular treatment regimen. In this disclosure, the terms "screen," "screening for," "diagnose," and "diagnosis" may be used interchangeably.

[0072] As used herein, the term "neoplasm" should be understood to refer to a lesion, tumor, or other encapsulated or unencapsulated mass or other growth form containing neoplastic cells. "Neoplastic cells" should be understood to refer to cells exhibiting abnormal growth. The term "growth" should be understood in its broadest sense and includes that relating to proliferation. In this regard, an example of abnormal cell growth is uncontrolled proliferation of a cell. Another example is failed apoptosis in a cell, thus extending its normal lifespan. Neoplastic cells can be benign or malignant. In some embodiments, the subject neoplasm is an adenoma or adenocarcinoma. Without limiting the present invention to any one theory or mode of action, adenomas are generally benign tumors of epithelial origin that are derived from epithelial tissue or exhibit clearly defined epithelial structures. These structures may exhibit a glandular appearance. Adenomas may contain malignant cell populations within the adenoma, such as those that occur when a benign adenoma or benign neoplastic lesion progresses to a malignant adenocarcinoma. In some embodiments, the neoplasm is malignant, such as a cancer. In some embodiments, the neoplasm is non-malignant, such as an adenoma.

[0073] As used herein, the term "colorectal neoplasm" refers to a neoplasm occurring in the colon, rectum, and / or appendix. In some embodiments, the colorectal neoplasm is colorectal cancer, a large colorectal adenoma, and / or a sessile serrated polyp. In some embodiments, the colorectal neoplasm is a precancerous condition.

[0074] As used herein, the term "precancerous condition" refers to a neoplasm that exhibits histological changes associated with an increased risk of developing cancer. Examples of such conditions include cell proliferative disorders with high-grade dysplasia, e.g., adenomatous polyps of the colon, in the context of colorectal cell proliferative disorders.

[0075] As used herein, the term "onset" in the context of a neoplasm, such as an adenoma or adenocarcinoma, should be understood as relating to one or more cells of the subject exhibiting dysplasia. In this regard, an adenoma or adenocarcinoma may be fully developed, in that a mass of dysplastic cells has developed. Alternatively, an adenoma or adenocarcinoma may be at a very early stage, in that relatively few abnormal cell divisions have occurred at the time of diagnosis. The present disclosure also extends to assessing a subject's risk for developing a colorectal neoplasm, such as colorectal cancer.

[0076] As used herein, the term "assess" or "assessment" refers to the ability to distinguish between samples from subjects affected and unaffected by colorectal neoplasia, or between samples from subjects at different stages of colorectal neoplasia. In some embodiments, the assessment relates to determining whether the subject's tumor has entered a developmental stage or whether there is a high likelihood that the subject's tumor has entered a developmental stage. In some embodiments, the assessment relates to the classification of the subject's tumor (e.g., stage I, stage II, stage III, stage IV, etc.). In some embodiments, the assessment relates to determining whether the subject's tumor has become less developed or more severe. In some embodiments, the assessment can help evaluate the likelihood of clinical benefit from a treatment. In some embodiments, the assessment may relate to whether and / or the likelihood that a patient will improve following treatment, e.g., treatment with a particular therapeutic agent. The assessment methods of the present disclosure can be used clinically to make treatment decisions by selecting the most appropriate treatment modality for any particular patient. The assessment methods of the present disclosure can be valuable tools in assessing whether a patient is likely to survive long-term following a given treatment regimen, such as a treatment regimen including, for example, administration of a given therapeutic agent or combination, surgical intervention, steroid treatment, etc.

[0077]

[0077] Those skilled in the art understand that discrimination or classification cannot aim to be correct in 100% of the samples analyzed. However, discrimination requires that a statistically significant amount of the samples analyzed be correctly classified. A statistically significant amount can be established by those skilled in the art using different statistical tools, such as, but not limited to, confidence intervals, p-value determination, Student's test, or Fisher's discriminating functions. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. In certain embodiments, the confidence interval is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the p-value is less than 0.1, 0.05, 0.01, 0.005, or 0.0001.

[0078]

[0078] As used herein, the term "development" refers to changes in cellular morphology and physiology along a genetically determined pathway, e.g., the natural progressive process of physical maturation from an earlier, lower or early stage to a later, more complex or advanced stage.

[0079] As used herein, the term "prognosis" refers to predicting the likelihood of an outcome of a disease condition, including, for example, recurrence, relapse, and drug resistance of the disease (e.g., cancer). The term also refers to predicting the likelihood of clinical benefit from treatment. In some embodiments, a statistical algorithm is used to provide a prognosis for a disease of interest. For example, the prognosis can be surgery, development of a clinical subtype of cancer (e.g., solid tumors such as colorectal cancer, melanoma, and renal cell carcinoma), development of one or more clinical factors, or recovery from the disease. The prognosis can be a poor prognosis (e.g., likely to recur or become drug resistant) or a good prognosis.

[0080] As used herein, the term "responsiveness" refers to a subject's beneficial response to treatment. A subject's responsiveness to treatment can be assessed using any endpoint that indicates benefit to the subject, including, but not limited to, (1) some degree of inhibition of disease progression, including slowing and complete halting; (2) a reduction in the number of disease episodes and / or symptoms; (3) a reduction in lesion size; (4) inhibition (i.e., reduction, slowing, or complete halting) of disease cell infiltration into adjacent peripheral organs and / or tissues; (5) inhibition (i.e., reduction, slowing, or complete halting) of disease spread; (6) some degree of alleviation of one or more symptoms associated with the disorder; (7) an increase in the length of disease-free presentation following treatment; (8) a reduction in the autoimmune response, which may or may not result in regression or disappearance of disease lesions, e.g., progression-free survival; (9) increased overall survival; (10) a higher response rate; and / or (11) a reduced mortality rate at a given time point following treatment. The terms "benefit" or "beneficial" are used in the broadest sense and refer to any desired effect.

[0081]

[0081] In the present disclosure, the detailed descriptions of steps (a), (b), (c), and (d) apply to both the method of diagnosing a colorectal neoplasia, screening for the onset or risk of onset of a colorectal neoplasia, or assessing the development or prognosis of a colorectal neoplasia in a subject, and the method of monitoring treatment response in a subject undergoing treatment for a colorectal neoplasia. Meanwhile, step (e) for both methods is described separately. Furthermore, in the present disclosure, step (I) of the present disclosure is identical to or at least similar to step (b) of the present disclosure. Furthermore, substep (i) of step (II) of the present disclosure is identical to or at least similar to step (c) of the present disclosure; and substep (ii) of step (II) of the present disclosure is identical to or at least similar to step (d) of the present disclosure. Furthermore, step (III) of the present disclosure is identical to or at least similar to step (e) of the present disclosure. Accordingly, step (I) and step (b) are collectively referred to below as "step (b)", substep (i) and step (c) of step (II) are collectively referred to below as "step (c)", substep (ii) and step (d) of step (II) are collectively referred to below as "step (d)", and step III and step (e) are collectively referred to below as "step (e)". Step (a)

[0082] In step (a) of the method according to the present disclosure, a biological sample containing DNA from a subject is obtained.

[0082]

[0083] As used herein, the term "biological sample" refers to a biological composition obtained from or derived from a subject of interest containing cells and / or other molecular entities (e.g., DNA) to be characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological characteristics. Biological samples include, but are not limited to, cells, tissues, organs, and / or biological fluids of a subject obtained by any method known to those of skill in the art. In some embodiments, the biological sample is selected from the group consisting of tissue sections, biopsies, paraffin-embedded tissues, body fluids, colonic effluent, surgical resection samples, isolated blood cells, cells isolated from blood, and any combination thereof. In some embodiments, the body fluid is selected from the group consisting of whole blood, serum, plasma, urine, mucus, saliva, ascites, pleural fluid, pleural fluid, synovial fluid, cerebrospinal fluid, thoracentesis fluid, abdominal fluid, and any combination thereof. In some embodiments, the colonic effluent is selected from the group consisting of a stool sample and an enema lavage sample. The selection of the type of sample that is most suitable for testing according to the methods disclosed herein depends on the nature of the situation. In some embodiments, the biological sample is obtained from the whole blood of a subject. In some embodiments, the biological sample is obtained from the plasma of a subject. Those skilled in the art will recognize various methods for preparing plasma from whole blood. For example, in some embodiments, plasma is obtained by centrifuging the whole blood from a subject one, two, three, four, five or more times.

[0083]

[0084] As used herein, the term "subject" includes both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. A "subject" may be a livestock animal, such as a cow, pig, sheep, poultry, and horse; or a rodent, such as a rat or mouse; or a non-human primate, such as an ape, monkey, or rhesus monkey; or a domestic animal, such as a dog or cat. In some embodiments, the subject is a human or a non-human primate. In some embodiments, the subject is a human. The terms "subject" and "individual" may be used interchangeably in this disclosure.

[0084]

[0085] In some embodiments, DNA is isolated from biological samples. Isolation and purification of DNA from biological samples can be performed using various methods known in the art, including the use of commercially available kits. For example, DNA is isolated from cells and tissues by dissolving the starting material under highly denaturing and reducing conditions, some using proteolytic enzymes, purifying the resulting nucleic acid fraction using a phenol / chloroform extraction step, and recovering the nucleic acids from the aqueous phase by dialysis or ethanol precipitation (see, e.g., Sambrook, J., Fritsch, EF in T. Maniatis, CSH, Molecular Cloning, 1989). In another example, several reagent systems now exist, particularly for purifying DNA fragments from agarose gels and isolating plasmid DNA from bacterial lysates, but also for isolating longer strands of nucleic acid (genomic DNA, total cellular RNA) from blood, tissues, or cell cultures. Many of these commercially available purification systems are based on the fairly well-known principle of binding nucleic acids to mineral supports in the presence of different chaotropic salts. In these systems, suspensions of finely ground glass powder, diatomaceous earth, or silica gel are used as carrier materials. Several other methods for isolating and purifying DNA from biological samples are described, for example, in U.S. Patent No. 7,888,006 and European Patent Application Publication No. 1,626,085. The choice of method is influenced by several factors, including time, cost, and the amount of DNA required.

[0085]

[0086] In some embodiments, the DNA contained in biological sample comprises genomic DNA.As used herein, the term "genomic DNA" refers to the DNA that contains the complete genome of cell or organism, and its fragment or part.Genomic DNA is a large piece of DNA that originates from a subject (for example, more than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200 or 300 kb), and can have natural modifications such as DNA methylation.

[0086]

[0087] In some embodiments, the DNA contained in a biological sample comprises cellular DNA. As used herein, the term "cellular DNA" refers to DNA present in cells in vivo or DNA obtained from in vivo cells and separated, isolated, or otherwise manipulated in vitro, unless the DNA has been removed from the cell in vivo.

[0087]

[0088] In some embodiments, the DNA contained in a biological sample includes cell-free DNA. As used herein, the term "cell-free DNA" refers to DNA fragments present in vivo outside of cells. The term can also be used to refer to DNA fragments obtained from in vivo extracellular sources and separated, isolated, or otherwise manipulated in vitro. DNA fragments in cell-free DNA typically range in length from about 100 to 200 base pairs, which likely relates to the length of the DNA stretch wrapped around a nucleosome. Cell-free DNA includes, for example, cell-free fetal DNA and circulating tumor DNA. Cell-free fetal DNA circulates in the body, such as in the blood of a pregnant mother, and represents the fetal genome, while circulating tumor DNA circulates in the body, such as in the blood of a cancer patient. In some embodiments, cell-free DNA may be substantially free of the subject's cellular DNA. For example, the cell-free DNA can contain less than about 1,000 ng per mL, less than about 100 ng per mL, less than about 10 ng per mL, or less than about 1 ng per mL of cellular DNA.

[0088]

[0089] Cell-free DNA can be prepared using conventional techniques known in the art. For example, cell-free DNA from a blood sample can be obtained by centrifuging the blood sample for about 3-30 minutes, about 3-15 minutes, about 3-10 minutes, or about 3-5 minutes at speeds of about 200-20,000 g, about 200-10,000 g, about 200-5,000 g, about 300-4,000 g, etc. For example, in some embodiments, cell-free DNA from a blood sample can be obtained by centrifuging plasma or serum from a subject one, two, three, four, five, or more times. In some embodiments, the blood sample can be obtained by microfiltration to separate cells and their fragments from a cell-free fraction containing soluble DNA. Customarily, microfiltration can be performed using a filter such as a 0.22 μm membrane filter, e.g., a 0.1 μm to 0.45 μm membrane filter.

[0089]

[0090] In some embodiments, cell-free DNA extraction from whole blood, serum, or plasma for analysis is performed using commercially available DNA extraction products. Such extraction methods claim high recovery rates (>50%) of circulating DNA, and some products (e.g., the QIAamp Circulating Nucleic Acid Kit, manufactured by Qiagen) claim to extract small DNA fragments. Typical sample volumes used range from 1 to 5 mL of serum or plasma.

[0090]

[0091] In some embodiments, the cell-free DNA comprises circulating tumor DNA. Circulating tumor DNA ("ctDNA") is tumor-derived fragmented DNA that is not associated with cells in bodily fluids (e.g., blood, urine, saliva, sputum, stool, pleural fluid, cerebrospinal fluid, etc.). Typically, ctDNA is highly fragmented, with an average length of approximately 150 base pairs. ctDNA generally comprises a very small proportion of cell-free DNA in bodily fluids (e.g., plasma); for example, ctDNA may constitute less than about 10% of plasma DNA. Typically, this percentage is less than about 1%, e.g., less than about 0.5% or about 0.0%. The total amount of plasma DNA is generally very low, e.g., approximately 10 ng / mL of plasma. The amount of ctDNA varies between individuals and depends on the tumor type, its location, and, in cancerous tumors, the stage of the cancer. However, ctDNA is typically extremely rare in body fluids and can only be detected by extremely sensitive and specific techniques. Detecting ctDNA can be useful for detecting and diagnosing tumors, guiding tumor-specific treatments, monitoring cancer treatments, and monitoring cancer remission. Step (b)

[0092] In step (b) of the method according to the present disclosure, the DNA in the biological sample obtained from step (a) is treated with a reagent capable of distinguishing between unmethylated and methylated sites in the DNA, thereby obtaining treated DNA.

[0091]

[0093] DNA methylation is a biological process in which methyl groups are added to DNA molecules (e.g., to cytosine bases or bases of DNA molecules) (e.g., by the action of DNA methyltransferase enzymes). In mammals, DNA methylation is mostly found at the 5' position of cytosine-phosphate-guanine (CpG) dinucleotides (i.e., "CpG sites"), which, when found at the 5'-CpG-3' dinucleotide within the promoter or first exon of a gene, leads to epigenetic inactivation of the gene. It is well documented that DNA methylation plays an important role in regulating gene expression, tumorigenesis, and other genetic and epigenetic diseases.

[0092]

[0094] As used herein, the term "methylated cytosine residue" refers to the derivative of cytosine residue, in which a methyl group is attached to the carbon atom (e.g., C5 atom) of the cytosine ring.In contrast to "methylated cytosine residue", the term "non-methylated cytosine residue" refers to the non-derivatized cytosine residue, in which a methyl group is not attached to the carbon atom (e.g., C5 atom) of the cytosine ring.The CpG site in which the cytosine residue is methylated is the methylated CpG site, and the CpG site in which the cytosine residue is not methylated is the non-methylated CpG site.

[0093]

[0095] In some embodiments, the reagent used in step (b) can distinguish between unmethylated and methylated CpG site(s) in DNA, thereby obtaining treated DNA. The reagent may selectively act on unmethylated cytosine residue(s) but not significantly on methylated cytosine residue(s); or the reagent may selectively act on methylated cytosine residue(s) but not significantly on unmethylated cytosine residue(s). As a result, the original DNA is converted to treated DNA in a methylation-dependent manner, and the treated DNA may be distinguishable from the original DNA by its hybridization behavior.

[0094]

[0096] For example, some reagents may selectively convert unmethylated cytosine residue(s) to uracil, thymine, or another base that does not resemble cytosine for hybridization, while leaving methylated cytosine residue(s) unconverted. In another example, some reagents may selectively cleave at residues when they are methylated or selectively cleave at residues when they are unmethylated.

[0095]

[0097] As used herein, the term "treated DNA" refers to DNA that has been treated with a reagent that can distinguish between unmethylated and methylated sites in DNA, i.e., the DNA methylation status in the DNA has been altered.

[0096]

[0098] In certain embodiments, the reagents of step (b) selectively modify unmethylated cytosine residue(s) at CpG site(s) to produce modified residue(s), but do not significantly modify methylated cytosine residue(s).

[0097]

[0099] In some embodiments, the reagent in step (b) comprises a bisulfite reagent. As used herein, the term "bisulfite reagent" refers to a reagent containing bisulfite, disulfite, bisulfite, or any combination thereof, which is useful as disclosed herein for distinguishing between methylated and unmethylated CpG dinucleotide sequences. In this disclosure, treatment of DNA with a bisulfite reagent is also referred to as a "bisulfite reaction" or "bisulfite treatment," which refers to a reaction for converting unmethylated cytosine residues, particularly unmethylated cytosine residues in nucleic acids, to uracil base(s), thymine base(s), or other base(s) that do not resemble cytosine(s) in terms of hybridization behavior, in the presence of bisulfite ions, which does not significantly convert methylated cytosine residues. In other words, bisulfite treatment is useful for distinguishing between methylated and unmethylated CpG dinucleotides.

[0098]

[0100] The bisulfite reaction for the detection of methylated cytosine residues is described in detail in Frommer, M. et al., Proc Natl Acad Sci USA 89 (1992) 1827-31 and Grigg, G. and Clark, S., Bioessays 16 (1994) 431-6. The bisulfite reaction contains a deamination step and a desulfonation step (Grigg and Clark, supra). The statement that methylated cytosine residues are not significantly converted takes into account the fact that it cannot be excluded that only a small percentage of methylated cytosine residues (e.g., less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 11%, less than 12%, less than 13%, less than 14%, less than 15%, less than 16%, less than 17%, less than 18%, less than 19%, less than 20%) are converted to uracil, thymine, or another base(s) that do not resemble cytosine(s) in terms of hybridization behavior, however, this statement is intended to refer exclusively to the conversion of only unmethylated cytosine residues.

[0099]

[0101] Those skilled in the art know how to carry out bisulfite treatment, in particular the deamination and desulfonation steps, by referring, for example, to Frommer M., et al., supra, or Grigg and Clark, supra, which disclose the main parameters of bisulfite treatment. The influence of incubation time and temperature on deamination efficiency and parameters affecting DNA degradation are disclosed.

[0100]

[0102] In some embodiments, the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, bisulfite, and any combination thereof. In some embodiments, the bisulfite reagent is sodium bisulfite. In some embodiments, the bisulfite reagent is commercially available, such as the MethylCode™ Bisulfite Conversion Kit, EpiMark™ Bisulfite Conversion Kit, EpiJET™ Bisulfite Conversion Kit, EZ DNA Methylation-Gold™ Kit, etc. In some embodiments, the bisulfite reaction is performed according to the kit's instructions.

[0101]

[0103] In some embodiments, the reagent in step (b) selectively cleaves at the residue if it is unmethylated but not if it is methylated, or selectively cleaves at the residue if it is methylated but not if it is unmethylated.

[0102]

[0104] In some embodiments, the reagent in step (b) is a methylation-sensitive restriction enzyme (MSRE).

[0103]

[0105] The term "methylation-sensitive restriction enzyme" refers to an enzyme that selectively digests nucleic acids depending on the methylation state of its recognition site. For such restriction enzymes that specifically cleave if the recognition site is neither methylated nor hemimethylated, cleavage does not occur or occurs with significantly reduced efficiency if the recognition site is methylated. For such restriction enzymes that specifically cleave if the recognition site is methylated, cleavage does not occur or occurs with significantly reduced efficiency if the recognition site is unmethylated. In some embodiments, the recognition sequence of a methylation-sensitive restriction enzyme contains a CG dinucleotide (e.g., cgcg or cccggg). In some embodiments, the recognition sequence of a methylation-sensitive restriction enzyme does not cleave if the cytosine residue of the CG dinucleotide is methylated at the C5 carbon atom.

[0104]

[0106] In some embodiments, the MSRE is selected from the group consisting of HpaII, SalI, SalI-HF®, ScrFI, BbeI, NotI, SmaI, XmaI, MboI, BstBI, ClaI, MluI, NaeI, NarI, PvuI, SacII, HhaI, and any combination thereof.

[0105]

[0107] Methods are known in the art in which a series of restriction enzyme reagents comprising methylation-sensitive restriction enzymes, or methylation-sensitive restriction enzymes that distinguish between methylated and unmethylated CpG dinucleotides within a target region, are utilized to determine methylation, such as, but not limited to, differential methylation hybridization ("DMH").

[0106]

[0108] In some embodiments, the DNA in step (a) may be cleaved prior to treatment with a methylation-sensitive restriction enzyme. Such methods are known in the art and can include both physical and enzymatic means. Particularly preferred is the use of one or more restriction enzymes that are not methylation-sensitive and whose recognition sites are AT-rich and do not contain CG dinucleotides. The use of such enzymes allows for the preservation of CpG sites and CpG-rich regions in the fragmented DNA. In some embodiments, such restriction enzymes are selected from the group consisting of MseI, BfaI, Csp6I, Tru1I, Tru9I, MaeI, XspI, and any combination thereof. Step (C)

[0109] In step (c) of the method according to the present disclosure, at least one target marker in the treated DNA obtained from step (b) is pre-amplified with a pre-amplification primer pool, thereby pre-amplifying at least a portion of at least one (e.g., each) of the target marker(s). In the present disclosure, step (c) may be referred to as a pre-amplification step. Without wishing to be bound by any theory, it is believed that step (c) is not necessary to achieve the objectives of the present invention. In some embodiments, step (c) of the method according to the present disclosure is present. In some embodiments, step (c) of the method according to the present disclosure is absent.

[0107]

[0110] One purpose of pre-amplifying target marker(s) is to increase the amount(s) of target marker(s) in the processed DNA, for example, from low amounts of target marker(s). As used herein, the term "amplification" and variations such as "amplifying," "amplified," and "amplifies" generally refer to any process that results in an increase in the copy number of a molecule or set of related molecules. When applied to polynucleotide molecules, amplification typically refers to producing multiple copies of a polynucleotide molecule, or a portion of a polynucleotide molecule, starting from a small amount of polynucleotide, where the amplified material (amplicon, PCR amplicon) is typically detectable. Polynucleotide amplification encompasses a variety of chemical and enzymatic processes. The generation of multiple DNA copies from one or a few copies of a template RNA or DNA molecule in a polymerase chain reaction (reverse transcription PCR, PCR), a strand displacement amplification (SDA) reaction, a transcription-mediated amplification (TMA) reaction, a nucleic acid sequence-based amplification (NASBA) reaction, or a ligase chain reaction (LCR) is a form of amplification.

[0108]

[0111] As used herein, the term "target marker" refers to a nucleic acid or gene region of interest whose methylation level indicates colorectal neoplasia (e.g., colorectal cancer), or indicates the onset or risk of onset of colorectal neoplasia (e.g., colorectal cancer), or indicates the development or prognosis of colorectal neoplasia (e.g., colorectal cancer). The terms "marker" and "gene" may be used interchangeably in this disclosure. The terms "marker" and "gene" are intended to include all of their transcriptional variants (e.g., the term "Septin9" includes, for example, its truncated transcript Q9HC74) and all of their promoter and regulatory elements. As those skilled in the art will recognize, some genes are known to exhibit allelic variation or single nucleotide polymorphisms ("SNPs") between subjects. SNPs include insertions and deletions of varying sizes and simple sequence repeats such as dinucleotide and trinucleotide repeats. Thus, the present disclosure should be understood to extend to any form of marker / gene resulting from any other mutation, polymorphism, or allelic variation. Furthermore, the terms "marker" and "gene" should be understood to include both the sense and antisense sequences of the marker or gene.

[0109]

[0112] The term "target marker" as used herein is broadly interpreted to encompass both 1) the original marker (with a particular methylation state) found in a biological sample or genomic DNA and 2) its processed sequence (e.g., its bisulfite-converted or MSRE-treated counterpart). Bisulfite-converted counterparts differ from target markers in genomic sequences in that one or more unmethylated cytosine residues are converted to uracil base(s), thymine base(s), or other base(s) that do not resemble cytosine(s) in terms of hybridization behavior. MSRE-treated counterparts differ from target markers in genomic sequences in that the sequence is cleaved at one or more MSRE cleavage sites.

[0110]

[0113] In some embodiments, the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one target marker comprises one or more markers (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 markers) selected from the group consisting of NDRG4, Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, and INTERGENIC REGION 1. In some embodiments, the at least one target marker comprises 13 markers selected from the group consisting of NDRG4, Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, and INTERGENIC REGION 1. In some embodiments, the at least one target marker comprises 11 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, NDRG4, SDC2, PKNOX2, TMEFF2, and INTERGENIC REGION 1. In some embodiments, the at least one target marker comprises 10 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, BCAN, NDRG4, SDC2, PKNOX2, TMEFF2, and INTERGENIC REGION 1.In some embodiments, the at least one target marker comprises 10 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, NDRG4, SDC2, PKNOX2, and TMEFF2. In some embodiments, the at least one target marker comprises 9 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, BCAN, NDRG4, SDC2, PKNOX2, and TMEFF2. In some embodiments, the at least one target marker comprises 7 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, and NDRG4. In some embodiments, the at least one target marker comprises 6 markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, BCAN, and NDRG4. In some embodiments, the at least one target marker comprises six markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, IRF4, and BCAN. In some embodiments, the at least one target marker comprises five markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, and BCAN. In some embodiments, the at least one target marker comprises five markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, and IRF4. In some embodiments, the at least one target marker comprises three markers selected from the group consisting of SALL1, BCAT1, and Septin9.

[0111]

[0114] In some embodiments, the at least one target marker can be up to one target marker (i.e., one marker, but no more than one marker). In some embodiments, the at least one target marker is Septin9. In some embodiments, the at least one target marker is BCAT1. In some embodiments, the at least one target marker is IKZF1. In some embodiments, the at least one target marker is NDRG4. In some embodiments, the at least one target marker is BCAN. In some embodiments, the at least one target marker is PKNOX2. In some embodiments, the at least one target marker is VAV3. In some embodiments, the at least one target marker is IRF4. In some embodiments, the at least one target marker is POU4F2. In some embodiments, the at least one target marker is SALL1. In some embodiments, the at least one target marker is TMEFF2. In some embodiments, the at least one target marker is ASCL4. In some embodiments, the at least one target marker is FGF12. In some embodiments, the at least one target marker is INTERGENIC REGION1.

[0112]

[0115] In some embodiments, the at least one target marker comprises multiple target markers. In some embodiments, the multiple target markers comprise at least two or three markers selected from the group consisting of Septin9, BCAT1, and IKZF1. In some embodiments, the multiple target markers of the present disclosure further comprise one, two, three, four, or five additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4, and IRF4. In some embodiments, the multiple target markers of the present disclosure further comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) additional markers selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, INTERGENIC REGION 1, TMEFF2, INTERGENIC REGION 4, NKX2-6, INTERGENIC REGION 5, SLC24A2, INTERGENIC REGION 2, INTERGENIC REGION 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP.

[0113]

[0116] In some embodiments, the multiple target markers of the present disclosure include Septin9 and BCAN, BCAT1, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: BCAN, BCAT1, IKZF1, NDRG4, PKNOX2, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one additional target marker comprises NDRG4, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4.

[0114]

[0117] In some embodiments, the multiple target markers of the present disclosure are BCAT1 as well as BCAN, Septin9, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of BCAN, Septin9, NDRG4, IKZF1, PKNOX2, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, Septin9, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0115]

[0118] In some embodiments, the multiple target markers of the present disclosure are IKZF1 as well as BCAN, Septin9, BCAT1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: BCAN, Septin9, BCAT1, PKNOX2, NDRG4, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, Septin9, and / or BCAT1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0116]

[0119] In some embodiments, the multiple target markers of the present disclosure include BCAN as well as Septin9, BCAT1, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, PKNOX2, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises NDRG4, VAV3, and / or IRF4.

[0117]

[0120] In some embodiments, the multiple target markers of the present disclosure include VAV3 and Septin9, BCAT1, IKZF1, BCAN, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, BCAN, PKNOX2, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, NDRG4, and / or IRF4.

[0118]

[0121] In some embodiments, the multiple target markers of the present disclosure include IRF4 as well as Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, BCAN, NDRG4, PKNOX2, VAV3, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or NDRG4.

[0119]

[0122] In some embodiments, the multiple target markers of the present disclosure include PKNOX2 and Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, BCAN, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0120]

[0123] In some embodiments, the multiple target markers of the present disclosure include POU4F2 and Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, PKNOX2, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4.

[0121]

[0124] In some embodiments, the multiple target markers of the present disclosure include SALL1 and Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, PKNOX2, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4.

[0122]

[0125] In some embodiments, the multiple target markers of the present disclosure include TMEFF2 as well as Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, ASCL4, SALL1, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, NDRG4, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4.

[0123]

[0126] In some embodiments, the multiple target markers of the present disclosure include ASCL4 as well as Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, TMEFF2, SALL1, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, NDRG4, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4.

[0124]

[0127] In some embodiments, the multiple target markers of the present disclosure include FGF12 and Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, TMEFF2, SALL1, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, ASCL4, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, NDRG4, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4.

[0125]

[0128] In some embodiments, the multiple target markers of the present disclosure include INTERGENIC REGION 1 and INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, NDRG4, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4.

[0126]

[0129] In some embodiments, the multiple target markers of the present disclosure include NDRG4 and Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, TMEFF2, SALL1, SLC24A2, NKX2-6, KCNA6, SOX1, HS3ST2, ASCL4, KCTD8, HMX1, MARCH11, CRHBP, FGF12, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, BCAN, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4.

[0127]

[0130] In this disclosure, the markers / genes in question are described herein by reference to both their name and their chromosomal coordinates, which are consistent with the Human Genome Database version Hg19 published in February 2009 (referred to herein as "Hg19 coordinates").

[0128]

[0131] In the present disclosure, it should be understood that target markers also include intergenic regions, which are designated "INTERGENIC REGION 1," "INTERGENIC REGION 2," "INTERGENIC REGION 3," "INTERGENIC REGION 4," and "INTERGENIC REGION 5," and are defined by their respective chromosomal coordinates. For example, in the present disclosure, INTERGENIC REGION 1 refers to the region defined by chr6:19679885-19693988; INTERGENIC REGION 2 refers to the region defined by chr10:130082033-130087148; INTERGENIC REGION 3 refers to the region defined by chr10:133107880-133113966; INTERGENIC REGION 4 refers to the region defined by chr7:152620588-152624685; and INTERGENIC REGION 5 refers to the region defined by chr8:70945014-70949177.

[0129]

[0132] In some embodiments, each target marker is: a) a respective region defined by the Hg19 coordinates shown below:

[0130] [Table 3-1]

[0131] [Table 3-2]

[0132] and 5 kb upstream of the respective start sites and 5 kb downstream of the respective end sites of each of the above regions, or b) the bisulfite-converted counterpart of a), or c) the MSRE-treated counterpart of a), or a), b), or c). The Hg19 coordinates listed above and the specific nucleotide sequences 5 kb upstream of the respective start sites and 5 kb downstream of the respective end sites of each of the above regions are available in public databases such as the UCSC Genome Browser, Ensemble, and NCBI websites.

[0133] In some embodiments, each target marker also includes all its variants, where the variants share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, i.e., nucleic acid sequences from the same region that have one or more deletions, additions, substitutions, inverted sequences, etc., compared to the marker / gene regions described herein. Thus, the present disclosure should be understood to extend to variants that achieve the same results, despite the fact that minor genetic variations between the actual nucleic acid sequences may exist between subjects.

[0134]

[0134] As used herein, the term "percent (%) sequence identity" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences to achieve the maximum number of identical amino acids (or nucleic acids) and, if necessary, introducing gaps. In other words, the percent (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence to which the sequence is being compared by the total number of amino acid residues (or bases) in the candidate sequence or in the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be accomplished using publicly available tools such as, for example, BLASTN, BLASTp (available at the US National Center for Biotechnology Information (NCBI) website; see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available at the European Bioinformatics Institute website; see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. One skilled in the art may use the default parameters provided by the tool or may customize the parameters to suit the alignment, such as by selecting an appropriate algorithm.

[0135] In step (c) provided herein, at least a portion of at least one (e.g., each) of the target marker(s) is pre-amplified. In certain embodiments, the pre-amplified portion of the target marker is within a subregion of the target marker.

[0136] Without limiting the present disclosure to any one theory or mode of action, it is believed to be particularly useful to measure the methylation level of target markers in subregions containing a high density of CpG dinucleotides, which are often hypermethylated in colorectal neoplasms, such as colorectal cancer. This finding makes subregions particularly useful targets for analysis because it simplifies the screening process due to both the shorter, more clearly defined regions of DNA that require analysis and, further, the fact that results from these regions provide significantly more conclusive results regarding the presence or absence of hypermethylation than would be obtained if analysis were performed across the entire Hg19 region of the target marker. Thus, this finding both simplifies the diagnostic, screening / monitoring process and increases the sensitivity and specificity of colorectal neoplasm diagnosis. In some embodiments, the subregion of each target marker is a) a sequence defined by the Hg19 coordinates shown below:

[0137] [Table 4]

[0138] and 5 kb upstream of the respective start site and 5 kb downstream of the respective end site of each of the above regions, or b) the bisulfite converted counterpart of a), or c) the MSRE treated counterpart of a).

[0139] In certain embodiments, the subregion of each target marker comprises or is a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 86-112, 167, or a bisulfite-converted counterpart thereof, or an MSRE-treated counterpart thereof. In certain embodiments, the bisulfite-converted counterpart of the subregion of the target marker comprises or is a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 113-166, 168, 169. The SEQ ID NOs for the subregions of each target marker are set forth in Table 1 below, and the sequences are provided in Figure 6.

[0140] [Table 5-1]

[0141] [Table 5-2]

[0142] In certain embodiments, a subregion of NDRG4 comprises a sequence selected from SEQ ID NOs: 86, 113, and 140; a subregion of BCAT1 comprises a sequence selected from SEQ ID NOs: 87, 114, and 141; a subregion of IKZF1 comprises a sequence selected from SEQ ID NOs: 88, 115, and 142; a subregion of Septin9 comprises a sequence selected from SEQ ID NOs: 89, 116, and 143; a subregion of SDC2 comprises a sequence selected from SEQ ID NOs: 90, 117, and 144; a subregion of VAV3 comprises a sequence selected from SEQ ID NOs: 89, 116, and 143; a subregion of TMEFF2 comprises a sequence selected from SEQ ID NOs: 92, 119, and 146; a subregion of SALL1 comprises a sequence selected from SEQ ID NOs: 93, 120, and 147; a subregion of BCAN comprises a sequence selected from SEQ ID NOs: 94, 121, and 148; a subregion of POU4F2 comprises a sequence selected from SEQ ID NOs: 95, 122, and 149; a subregion of PKNOX2 comprises a sequence selected from SEQ ID NOs: 96, 123, and 150; A partial region of SCL4 comprises a sequence selected from SEQ ID NOs: 97, 124, and 151; a partial region of KCNA6 comprises a sequence selected from SEQ ID NOs: 98, 125, and 152; a partial region of SOX1 comprises a sequence selected from SEQ ID NOs: 99, 126, and 153; a partial region of HS3ST2 comprises a sequence selected from SEQ ID NOs: 100, 127, and 154; a partial region of FGF12 comprises a sequence selected from SEQ ID NOs: 101, 128, and 155; a partial region of KCTD8 comprises a sequence selected from SEQ ID NOs: 102, 129, and 156. a subregion of HMX1 comprises a sequence selected from SEQ ID NOs: 103, 130, and 157; a subregion of MARCH11 comprises a sequence selected from SEQ ID NOs: 104, 131, and 158; a subregion of CRHBP comprises a sequence selected from SEQ ID NOs: 105, 132, and 159; a subregion of NKX2-6 comprises a sequence selected from SEQ ID NOs: 106, 133, and 160; a subregion of SLC24A2 comprises a sequence selected from SEQ ID NOs: 107, 134, and 161; a subregion of INTERGENIC REGION 1 comprises a sequence selected from SEQ ID NOs: 108, 135, and 162;A subregion of INTERGENIC REGION 2 comprises a sequence selected from SEQ ID NOs: 109, 136, and 163; a subregion of INTERGENIC REGION 3 comprises a sequence selected from SEQ ID NOs: 110, 137, and 164; a subregion of INTERGENIC REGION 4 comprises a sequence selected from SEQ ID NOs: 111, 138, and 165; a subregion of INTERGENIC REGION 5 comprises a sequence selected from SEQ ID NOs: 112, 139, and 166; and / or a subregion of IRF4 comprises a sequence selected from SEQ ID NOs: 167, 168, and 169.

[0143] In some embodiments, the target marker in cell-free DNA is present in a biological sample at an amount of 1 ng or less, 0.9 ng or less, 0.8 ng or less, 0.7 ng or less, 0.6 ng or less, 0.5 ng or less, 0.4 ng or less, 0.3 ng or less, 0.2 ng or less, 0.1 ng or less, 0.09 ng or less, 0.08 ng or less, 0.07 ng or less, 0.06 ng or less, 0.05 ng or less, 0.04 ng or less, 0.03 ng or less, 0.02 ng or less, or 0.01 ng or less. In some embodiments, the target marker in cell-free DNA is present in a biological sample at a percentage of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% or less. In some embodiments, the target marker in cell-free DNA is present in biological sample at a concentration that is below the sensitivity level of the detection assay for the target marker. "Detection assay sensitivity" is a measure of the ability of the detection assay to distinguish small differences in analyte concentration / amount. If the target marker in cell-free DNA present in biological sample is below the sensitivity level of the detection assay, it will prevent the quantification of the methylation level of any target marker in the sample using conventional methods. In contrast, the method disclosed herein is useful and advantageous for detecting very low amounts of target marker in a sample. In some embodiments, the target marker in cell-free DNA is present in biological sample at an amount of 0.08ng or less or 0.04ng or less.

[0144] In some embodiments, the DNA obtained from step (c) is diluted with a diluent prior to the next step (i.e., step (d)). In some embodiments, the diluent is selected from the group consisting of nuclease-free water, Tris-EDTA buffer, and any other buffer that is free of PCR inhibition. In some embodiments, the pre-amplified DNA of step (c) is added directly to the next step (i.e., step (d)) without prior dilution.

[0145]

[0141] At least one target marker in the treated DNA is preamplified with a preamplification primer pool. As used herein, the term "primer" refers to a single-stranded oligonucleotide that can act as an initiation point for template-dependent DNA synthesis under appropriate conditions, e.g., buffer and temperature, in the presence of four different nucleoside triphosphates and a polymerization agent, e.g., DNA polymerase. The length of a primer in any given case depends, for example, on the intended use of the primer and generally ranges from 15 to 30 nucleotides. Shorter primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with such a template. A primer site is the region of the template to which the primer hybridizes. A primer pair is a set of primers that includes a 5' forward primer that hybridizes to the 5' end of the sequence to be amplified and a 3' reverse primer that hybridizes to the complement of the 3' end of the sequence to be amplified. Those skilled in the art can design primers according to the marker(s) to be amplified based on common knowledge in the art (see, for example, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995). In addition, several software packages are publicly available for designing optimal probes and / or primers for various assays, for example, Primer 3 is available from the Center for Genome Research, Cambridge, Mass., USA. Obviously, the potential use of a probe or primer should be considered during its design. For example, the primer designed for the purpose of the present invention may contain at least one CpG site, or the amplification product obtained from the primer may contain at least one CpG site.Tools for designing primers for detecting DNA methylation status are also available in the art, such as MethPrimer (Li LC and Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002 November;18(11):1427-31). In the present disclosure, pre-amplification primers are used as a pool to pre-amplify any target marker(s) within the processed DNA (at least a portion of at least one (e.g., each) of the target marker(s) or a subregion of at least one target marker).

[0146] As used herein, the term "oligonucleotide" is defined as a molecule containing two or more nucleotides (e.g., deoxyribonucleotides or ribonucleotides), preferably at least five nucleotides, more preferably at least about 10-15 nucleotides, and even more preferably at least about 15-30 nucleotides, or longer (e.g., oligonucleotides are typically less than 200 residues long (e.g., 15-100 nucleotides), although as used herein, the term is intended to encompass longer polynucleotide chains). The exact size depends on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides are often referred to by their length. For example, a 24-residue oligonucleotide is called a "24-mer." Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes. Oligonucleotides can be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof.

[0147] As used herein, the terms "complementary" or "complementarity" refer to hybridization or base pairing between nucleotides or nucleic acids, such as, for example, between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are generally A and T (or A and U) or C and G. Two single-stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand, optimally aligned and compared, with appropriate nucleotide insertions or deletions, pair with at least about 80%, usually at least about 90%-95%, and more preferably about 98%-100%, of the nucleotides of the other strand. Alternatively, complementarity exists when an RNA or DNA strand hybridizes with its complement under selective hybridization conditions. Typically, selective hybridization occurs when there is at least about 65% complementarity, preferably at least about 75%, and more preferably at least about 90% complementarity over a stretch of at least 14-25 nucleotides. See M. Kanehisa, Nucleic Acids Res. 12:203 (1984), which is incorporated herein by reference.

[0148] In some embodiments, the pre-amplification primer pool comprises at least one methylation-specific primer pair. In some embodiments, the pre-amplification primer pool comprises multiple methylation-specific primer pairs. In some embodiments, the pre-amplification step is performed by methylation-specific PCR ("MSP"), which is PCR using methylation-specific primers. This technique (i.e., MSP) is described in Herman et al., "Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands." Proc Natl Acad Sci USA. 1996 Sep. 3;93(18):9821-6, and U.S. Patent No. 6,265,171.

[0149]

[0145] As used herein, the term "methylation-specific primer pair" refers to a primer pair specifically designed to recognize CpG site(s) to amplify specific target marker(s) in processed DNA using differences in methylation. The primers act only on molecules with a specific methylation state or without a specific methylation state. For example, a primer may be an oligonucleotide that can specifically hybridize to a specific CpG site with methylation in a methylation-specific manner under stringent, moderately stringent, or highly stringent conditions, but cannot hybridize to a specific CpG site without methylation; therefore, the primer is considered to specifically amplify target markers with methylation at specific CpG sites. In another example, a primer may be an oligonucleotide that can specifically hybridize to a specific unmethylated CpG site in a methylation-specific manner under stringent, moderately stringent, or highly stringent conditions, but cannot hybridize to a specific methylated CpG site. Therefore, the primer is considered to specifically amplify an unmethylated target marker at a specific CpG site. Therefore, in the present disclosure, the use of a methylation-specific primer pair(s) for pre-amplification of at least one target marker in treated DNA enables differentiation between methylated and unmethylated CpG sites. The methylation-specific primer pair of the present disclosure contains at least one primer that hybridizes to a bisulfite-treated CpG dinucleotide. Therefore, the sequence of the primer specific to methylated DNA contains at least one CpG dinucleotide, and the sequence of the primer specific to unmethylated DNA contains a "T" at a C position in the CpG and / or an "A" at a G position in the CpG.

[0150]

[0146] In some embodiments, at least one methylation-specific primer pair comprises a forward primer and a reverse primer, each comprising an oligonucleotide sequence that hybridizes to at least 9 consecutive nucleotides of one of the target marker(s) (or subregions of the target marker(s)) under stringent, moderately stringent, or highly stringent conditions, wherein the at least 9 consecutive nucleotides of one of the target marker(s) (or subregions of the target marker(s)) comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) CpG site.

[0151] As used herein, the terms "hybridize" and variations thereof, such as "hybridizing," "hybridizes," or "hybridization," may refer to the process by which two single-stranded polynucleotides non-covalently bind to form a stable double-stranded polynucleotide. In one embodiment, the resulting double-stranded polynucleotide may be a "hybrid" or "duplex." "Hybridization conditions" typically include a salt concentration of approximately less than 1 M, often less than about 500 mM, and may be less than about 200 mM. "Hybridization buffer" includes a buffered salt solution, such as 5% SSPE, or other such buffers known in the art. Hybridization temperatures can be as low as 5°C, but are typically greater than 22°C, more typically greater than about 30°C, and typically greater than 37°C. Hybridization is often performed under stringent conditions, i.e., conditions under which a sequence hybridizes to its target sequence but not to other non-complementary sequences. Stringent conditions are sequence-dependent and will vary under different circumstances. For example, a longer fragment may require a higher hybridization temperature for specific hybridization than a shorter fragment. Because other factors, including base composition and length of complementary strands, the presence of organic solvents, and the degree of base mismatch, can affect the stringency of hybridization, the combination of parameters is more important than the absolute measure of any one parameter alone. Generally, stringent conditions are selected to be approximately 5°C lower than the melting temperature (Tm) for a specific sequence at a defined ionic strength and pH.

[0152] Tm may be the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. Several formulas for calculating the Tm of nucleic acids are well known in the art. As indicated by standard standards, a simple estimate of the Tm value can be calculated by the formula, Tm = 81.5 + 0.41(% G + C), when the nucleic acid is in an aqueous solution of 1 M NaCl (see, e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other standards (e.g., Allawi and SantaLucia, Jr., Biochemistry, 36:10581-94 (1997)) include alternative calculation methods that take into account structural and environmental features, as well as sequence features, to calculate Tm.

[0153]

[0149] Generally, hybrid stability is a function of ion concentration and temperature. Typically, hybridization reactions are performed under relatively low stringency conditions, followed by washes at varying but relatively high stringency. Exemplary stringent conditions include a pH of about 7.0 to about 8.3, a salt concentration of at least 0.01 M to 1 M sodium ion (or other salt), and a temperature of at least 25°C. For example, conditions of 5xSSPE (750 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA, at pH 7.4) and a temperature of approximately 30°C are suitable for allele-specific hybridization, although the appropriate temperature will depend on the length and / or GC content of the hybridized region. In one embodiment, the "stringency of hybridization" when determining the percentage mismatch can be as follows: 1) high stringency: 0.1x SSPE, 0.1% SDS, 65°C; 2) medium stringency: 0.2x SSPE, 0.1% SDS, 50°C (also referred to as moderate stringency); and 3) low stringency: 1.0x SSPE, 0.1% SDS, 50°C. It is understood that equivalent stringency can be achieved using alternative buffers, salts, and temperatures. For example, moderately stringent hybridization can refer to conditions that allow a nucleic acid molecule, such as a probe, to bind to a complementary nucleic acid molecule. Hybridized nucleic acid molecules generally have at least 60% identity, including, for example, at least any of 70%, 75%, 80%, 85%, 90%, or 95% identity. Moderately stringent conditions can refer to conditions equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by a wash in 0.2x SSPE, 0.2% SDS at 42°C. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by a wash in 0.1x SSPE and 0.1% SDS at 65°C. Low stringency hybridization can refer to conditions equivalent to hybridization in 10% formamide, 5x Denhardt's solution, 6x SSPE, 0.2% SDS at 22°C, followed by a wash in 1x SSPE, 0.2% SDS at 37°C.Denhardt's solution contains 1% Ficoll, 1% polyvinylpyrrolidone, and 1% bovine serum albumin (BSA). 20x SSPE (sodium chloride, sodium phosphate, EDTA) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025M EDTA. Other suitable moderate-stringency and high-stringency hybridization buffers and conditions are well known to those skilled in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, NY (1989); and Ausubel et al., Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons (1999).

[0154] In some embodiments, the pre-amplification primer pool further comprises a control primer pair for amplifying a control marker. Typically, a control marker is a nucleic acid with known characteristics (e.g., a known sequence, a known copy number per cell) for use in comparison with an experimental target (e.g., a nucleic acid of unknown concentration). The control may be an endogenous, preferably invariant, gene to which the test or target nucleic acid in the assay can be normalized. Such normalization controls for sample-to-sample variations that may arise, for example, in sample processing, assay efficiency, etc., and allow for accurate sample-to-sample data comparisons to quantify amplification efficiency and bias.

[0155] In some embodiments, the control marker is selected from the group consisting of ACTB, GAPDH, tubulin, ALDOA, PGK1, LDHA, RPS27A, RPL19, RPL11, ARHGDIA, RPL32, C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP, and VPS29. In some embodiments, the sequences of the control primer pair are set forth in SEQ ID NOs: 55 and 56 in Table 2 below.

[0156] In some embodiments, at least one methylation-specific primer pair comprises one or more pairs of nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54, and 170 / 171 as shown in Table 2 below. SEQ ID NOs for primer pair(s) used in the present disclosure are represented in the form "SEQ ID NOs: n / m." For example, SEQ ID NO:1 / 2 refers to a primer pair having the nucleic acid sequences set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively, as shown in Table 2 below.

[0157] The primer pairs shown in SEQ ID NOs: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54, and 170 / 171 correspond to the markers NDRG4, BCAT1, IKZF1, Septin9, SDC2, VAV3, TMEFF2, SALL1, BCAN, POU4F2, PKNOX2, INTERGENIC REGION 1, ASCL4, INTERGENIC REGION 2, and INTERGENIC REGION 3, respectively. 2, INTERGENIC REGION 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 4, NKX2-6, SLC24A2, INTERGENIC REGION 5, IRF4.

[0158] In some embodiments, in step (c), at least one target marker is amplified in the presence of one or more blocker oligonucleotides. The use of such blocker oligonucleotides is described by Yu et al., BioTechniques 23:714-720, 1997. The blocker sequence is hybridized to the processed DNA simultaneously with the preamplification primer pair(s). Preamplification of the target marker is terminated at the 5' position of the blocker sequence, such that preamplification of the target marker is suppressed in the presence of a sequence complementary to the blocker sequence. The blocker sequence may be designed to hybridize to the processed DNA in a methylation state-specific manner. For example, in detecting methylated nucleic acids within a population of unmethylated nucleic acids, suppression of amplification of nucleic acids that are unmethylated at the position of interest may be achieved by using a blocker sequence that includes "CpA" or "TpA" at the position of interest, as opposed to "CpG" when suppression of amplification of methylated nucleic acids is desired.

[0159] In PCR methods using blocker oligonucleotides, efficient disruption of polymerase-mediated amplification requires that the blocker oligonucleotide not be extended by the polymerase. Preferably, this is achieved through the use of blockers that are 3'-deoxyoligonucleotides, or oligonucleotides derivatized with other than a "free" hydroxyl group at the 3' position. For example, 3'-O-acetyl oligonucleotides represent a preferred class of blocker molecules.

[0160] Furthermore, polymerase-mediated degradation of the blocker oligonucleotide should be prevented. Preferably, such prevention involves the use of a polymerase lacking 5'-3' exonuclease activity or, for example, the use of a modified blocker oligonucleotide having a thiolate bridge at its 5' end, which renders the blocker molecule nuclease-resistant. Certain applications may not require such 5' modifications of the blocker. For example, if the blocker- and primer-binding sites overlap, thereby preventing primer binding (e.g., with excess blocker), degradation of the blocker oligonucleotide is substantially prevented. This is because the polymerase does not extend the primer toward and into the blocker (in the 5'-3' direction), a process that normally results in degradation of the hybridized blocker oligonucleotide.

[0161] A particularly preferred blocker / PCR embodiment for purposes of this disclosure and practiced herein involves the use of peptide nucleic acid (PNA) oligomers as blocking oligonucleotides. Such PNA blocker oligomers are ideally suited because they cannot be degraded or extended by polymerases.

[0162] In certain embodiments, at least one target marker is pre-amplified with a DNA polymerase. As used herein, the term "DNA polymerase" refers to an enzyme that catalyzes the synthesis of polydeoxyribonucleotides from monodeoxyribonucleoside triphosphates (dNTPs), performing the most fundamental functions of DNA replication, repair, and, in some cases, cell differentiation.

[0163] Examples of DNA polymerases in prokaryotes include DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV, and DNA polymerase V. DNA polymerases I, II, and III are known in E. coli. DNA polymerase III appears to be most important in genome replication. DNA polymerase I is important because it has the ability to remove unpaired bases at the ends of growing strands. Retroviruses have a unique DNA polymerase, i.e., reverse transcriptase, that uses an RNA template to synthesize DNA. For eukaryotes, examples of DNA polymerases are polymerases α, β, λ, γ, σ, μ, δ, ε, η, ι, κ, ζ, θ, and Rev1. Animal cells have DNA polymerases responsible for DNA replication in the nucleus and mitochondria.

[0164] The PCR reagents used in the pre-amplification step can be any commercially available PCR mix that can be used to amplify treated DNA (e.g., KAPA2G Fast Multiplex PCR Kit, Luna® Universal Probe qPCR Master Mix, EpiTect MethyLight PCR Kit, etc.). Alternatively, one skilled in the art can prepare PCR reagents in the laboratory using Mg 2+ PCR reagents containing dNTPs, DNA polymerase, etc. may be prepared. Those skilled in the art may select appropriate PCR reaction systems and conditions according to their actual needs. In some embodiments, the pre-amplification in step (c) comprises 5 to 30 cycles of reaction, each cycle comprising a reaction at 85 to 99°C for 5 seconds to 5 minutes, followed by a reaction at 40 to 80°C for 5 seconds to 5 minutes. In some embodiments, the pre-amplification in step (c) comprises 10 to 20 cycles of reaction, each cycle comprising a reaction at 90 to 99°C for 15 seconds to 2 minutes, followed by a reaction at 45 to 60°C for 30 seconds to 3 minutes. In some embodiments, the pre-amplification in step (c) comprises 15 cycles of reaction, each cycle comprising a reaction at 95°C for 30 seconds, followed by a reaction at 56°C for 60 seconds. Step (d)

[0161] In step (d) of the method described herein, if step (c) is present, the methylation level of at least one target marker is individually quantified based on the DNA obtained from step (c); if step (c) is not present, the methylation level of at least one target marker is individually quantified in the treated DNA obtained from step (b). In the present disclosure, step (d) may also be designated as a quantification step.

[0165] As used herein, the term "methylation state" or "methylation status" refers to the presence, absence, and / or quantity of methylation at a particular nucleotide or nucleotides within a DNA region. The methylation state of a particular DNA sequence (e.g., a target marker described herein) can indicate the methylation state of all bases within the sequence, or can indicate a subset of base pairs within the sequence (e.g., the methylation state of cytosine residues or the methylation state of one or more particular restriction enzyme recognition sequences), or can indicate information regarding the methylation density of a region within the sequence without providing precise information about where methylation occurs within the sequence. The methylation state can optionally be expressed or indicated by a "methylation level." Methylation levels can be generated, for example, by quantifying the amount of intact DNA present after restriction digestion with a methylation-sensitive restriction enzyme. In this example, when a specific sequence in DNA is quantified using quantitative PCR, an amount of template DNA approximately equal to that of a mock-treated control indicates that the sequence is not highly methylated, while an amount of template substantially less than that occurring in a mock-treated sample indicates the presence of methylated DNA at that sequence. Thus, the methylation level, e.g., from the example above, represents a methylation state and can therefore be used as a quantitative indicator of methylation state. This is particularly useful when it is desirable to compare the methylation state of a sequence in a sample to a threshold level.

[0166] Methylation states at one or more specific CpG methylation sites (each having two CpG dinucleotide sequences) within a DNA sequence include "unmethylated," "fully methylated," and "hemimethylated." The term "hemi-methylation" or "hemimethylation" refers to the methylation state of double-stranded DNA in which only one strand is methylated. The term "hypermethylation" refers to the average methylation state corresponding to the increased presence of 5-methylcytosine at one or more CpG dinucleotides in the DNA sequence of a test DNA sample relative to the amount of 5-methylcytosine found at the corresponding CpG dinucleotide in a normal control DNA sample. The methylation state of a residue can be a qualitative or quantitative readout, as indicated, for example, by the methylation level. In this disclosure, the terms "methylation state" and "methylation level" may be used interchangeably. According to the present disclosure, it is possible to simultaneously determine two or more different methylation levels.

[0167] As described herein, when step (c) is present, the methylation level of at least one (e.g., each) target marker is individually quantified based on the DNA obtained from step (c); when step (c) is not present, the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b) is individually quantified, wherein the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, and BCAN. In some embodiments, the at least one target marker comprises five markers selected from the group consisting of Septin9, BCAT1, IKZF1, VAV3, and IRF4. In some embodiments, the at least one target marker comprises at least two, three, four, five, six, or seven markers selected from the group consisting of Septin9, BCAT1, IKZF1, NDRG4, BCAN, VAV3, IRF4, or any combination thereof. The detailed description of "target marker" in step (c) above (including, but not limited to, the definition of target marker, specific combinations of target markers, etc.) also applies to the "target marker" in "at least one target marker in the treated DNA obtained from step (b)" in step (d) (when step (c) is not present). The methylation level / status of one or more CpG dinucleotide sequences within a DNA sequence (eg, target markers) can be determined by a variety of assays known in the art.

[0168]

[0165] In some embodiments, the quantification in step (d) is performed by PCR (e.g., real-time PCR, digital PCR), nucleic acid sequencing, mass-based separation (e.g., electrophoresis, mass spectrometry), or target capture (e.g., hybridization, microarray).

[0169] In some embodiments, when step (c) is present, the methylation level of at least one of the target marker(s) is individually quantified based on the DNA obtained from step (c) by using MSP (see Herman, supra), e.g., by using one or more primers that specifically hybridize to the unconverted sequence under moderate and / or high stringency conditions, so that an amplification product is produced only if the template contains a methylated cytosine at a CpG site.

[0170] In some embodiments, the quantification in step (d) is performed by real-time PCR. Non-limiting examples of real-time PCR include Cottrell et al., Nucl. Acids Res. 32:e10, 2003; MethyLight™ PCR, Eads et al., Cancer Res. 59:2302-2306, 1999; Headloop PCR, HeavyMethyl™ PCR as described by Rand et al., Nucl. Acids Res. 33:e127, 2005.

[0171] As used herein, the term "HeavyMethyl™ PCR" refers to a real-time PCR technique recognized by those skilled in the art that uses one or more non-extendable nucleic acid (e.g., oligonucleotide) blockers that bind methylation-specifically to bisulfite-treated nucleic acids (i.e., the blockers specifically bind to non-mutated DNA under moderate to high stringency conditions). The amplification reaction is optionally methylation-specific, but is performed using one or more primers that flank the one or more blockers. In the presence of unmethylated nucleic acids (i.e., non-mutated DNA), the blockers bind and no PCR product is produced. For example, the TaqMan™ assay, as described in Holland et al., Proc. Natl. Acad. Sci. USA, 88:7276-7280, 1991, is essentially used to determine the methylation level of nucleic acids in a sample.

[0172] As used herein, the term "MethyLight™ PCR" refers to a skilled-intelligible, fluorescence-based, real-time PCR technology that utilizes dual-labeled fluorescent oligonucleotide probes, called TaqMan™ probes, designed to hybridize to CpG-rich sequences located between the forward and reverse amplification primers. The TaqMan™ probe contains a fluorescent "reporter moiety" and a "quencher moiety" covalently attached to linker moieties (e.g., phosphoramidites) attached to the nucleotides of the TaqMan™ oligonucleotide. During PCR amplification, the TaqMan™ probe hybridized to the CpG-rich sequence is cleaved by the 5' nuclease activity of Taq polymerase, generating a signal that can be detected in real time during the PCR reaction. In this method, Molecular Beacons can be used as detection probes, and the system does not depend on the 5'-3' exonuclease activity of the DNA polymerase used (see Mhlanga and Malmberg, Methods 25:463-471, 2001).

[0173]

[0170] As used herein, the term "Headloop PCR" refers to a real-time PCR recognized by those skilled in the art that selectively amplifies a target nucleic acid but suppresses amplification of non-amplified target variants by extension of the 3' stem-loop, forming a hairpin structure that can no longer provide a template for further amplification.

[0174]

[0171] In certain embodiments, the real-time PCR is multiplexed real-time PCR.

[0175]

[0172] As used herein, the terms "multiplex" or "multiplexing" may refer to an assay or other analytical method in which the presence and / or amount of multiple targets, e.g., multiple nucleic acid sequences, can be assayed simultaneously by using multiple markers, each having at least one different detection property, e.g., a fluorescent property (e.g., excitation wavelength, emission wavelength, emission intensity, FWHM (full width at half maximum), or fluorescence lifetime) or a unique nucleic acid or protein sequence property.

[0176] In some embodiments, the quantification in step (d) is performed by nucleic acid sequencing. Exemplary methods for nucleic acid sequencing are known in the art; see, for example, Frommer et al., Proc. Natl. Acad. Sci. USA, 89:1827-1831, 1992; Clark et al., Nucl. Acids Res. 22:2990-2997, 1994. For example, comparing the base sequence obtained using a bisulfite-treated sample with a sequence obtained using a non-bisulfite-treated sample or with a known base sequence of the region of interest can facilitate identification of methylated cytosine(s) in the DNA sequence. If a thymine residue is detected at a cytosine site in the bisulfite-treated sample compared to the untreated sample, this may be considered a mutation due to bisulfite treatment, i.e., the presence of a methylated cytosine at that site.

[0177] Methods for determining the base sequence of DNA are known in the art, and include, for example, the dideoxy chain termination or Maxam-Gilbert method (see Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed., CSHP, New York 1989)), pyrosequencing (see Uhlmann et al., Electrophoresis, 23:4072-4079, 2002), solid-phase pyrosequencing (see Landegren et al., Genome Res., 8(8):769-776, 1998), solid-phase minisequencing (see, e.g., Southern et al., Genomics, 13:1008-1017, 1992), minisequencing using FRET (see, e.g., Chen and Kwok, Nucleic Acids Res., 25:347-353, 1997), sequencing by ligation, and ultra-deep sequencing (Marguiles et al., Nature, 437(7057):376-80 (2005)).

[0178]

[0175] In certain embodiments, the quantification in step (d) is performed by mass-based separation (eg, electrophoresis, mass spectrometry).

[0179] For example, as described in Xiong and Laird, Nucl. Acids Res., 25:2532-2534, 2001, the presence of methylated cytosine residues is essentially detected by bisulfite restriction analysis (COBRA). This method exploits the difference in restriction enzyme recognition sites between methylated and unmethylated nucleic acids after treatment with a compound, e.g., bisulfite, that selectively mutates unmethylated cytosine residues. For example, the restriction endonuclease TaqI cleaves the sequence TCGA, whereas after bisulfite treatment of unmethylated nucleic acids, the sequence becomes TTGA and, as a result, is not cleaved. Digested and / or undigested nucleic acids are then detected using detection means known in the art, such as electrophoresis and / or mass spectrometry.

[0180]

[0177] As another example, different techniques are used to detect differences in nucleic acids in amplification products based on differences in nucleotide sequence and / or secondary structure after treatment with compounds that selectively mutate unmethylated cytosine residues, such as methylation-specific single-strand conformation analysis (MS-SSCA) (Bianco et al., Hum. Mutat., 14:289-293, 1999), methylation-specific denaturing gradient gel electrophoresis (MS-DGGE) (Abrams and Stanton, Methods Enzymol., 212:71-74, 1992), and methylation-specific denaturing high-performance liquid chromatography (MS-DHPLC) (Deng et al., Chin. J. Cancer Res., 12:171-191, 2000).

[0181]

[0178] In some embodiments, the quantification in step (d) is performed by target capture (eg, hybridization, microarray).

[0182] Suitable hybridization detection methods are known in the art and include, for example, Southern, dot blot, slot blot, and other nucleic acid hybridization procedures. (Kawai et al., Mol. Cell. Biol., 14:7421-7427, 1994; Gonzalgo et al., Cancer Res., 57:594-599, 1997). In some embodiments, the probes used in hybridization assays are detectably labeled. In some embodiments, the nucleic acid-based probes used in hybridization assays are unlabeled. Such unlabeled probes can be immobilized on a solid support, such as a microarray, and hybridized to detectably labeled target nucleic acid molecules.

[0183]

[0180] A methylation-specific microarray is an example of a microarray that is useful for distinguishing between sequences in which the cytosine residue(s) has been inverted and sequences in which the cytosine residue(s) has not been inverted. (See Adorjan et al., Nucl. Acids Res., 30:e21, 2002.) Hybridization-based analysis can also be used on nucleic acids after treatment with methylation-sensitive restriction enzymes.

[0184]

[0181] As yet another example, the methylation status of CpG dinucleotide sequences within a DNA sequence can be ascertained by oligonucleotide probes that hybridize to bisulfite-treated DNA simultaneously with PCR amplification primers (where the primers can be either methylation-specific or standard).

[0185] In some embodiments, step (d) is carried out in the presence of a detection agent. As used herein, the term "detection agent" is an agent used in the quantification step to detect the presence, absence, or amount of a nucleic acid.

[0186]

[0183] Various detection agents known in the art can be used in the present disclosure. In some embodiments, the detection agent is selected from the group consisting of a fluorescent probe, an intercalating dye, a chromophore-labeled probe, a radioisotope-labeled probe, and a biotin-labeled probe.

[0187]

[0184] In some embodiments, the fluorescent probe is selected from the group consisting of SEQ ID NOs: 57-85, 172, as shown in Table 2 below.

[0188]

[0185] In some embodiments, the fluorescent probe is labeled at its 5' end with a fluorescent dye (e.g., FAM, HEX / VIC, TAMRA, Texas Red, or Cy5) and at its 3' end with a quencher (e.g., BHQ1, BHQ2, BHQ3, DABCYL, or TAMRA).

[0189]

[0186] Labeling can be direct or indirect. In direct labeling, a label is directly (covalently or non-covalently) linked to the reagent. In indirect labeling, a secondary reagent is bound (covalently or non-covalently) to the primary reagent. The secondary reagent must specifically bind to the primary reagent. The secondary reagent may be linked to an appropriate label and / or may be a target (receptor) for a tertiary reagent that binds to the secondary reagent. The use of secondary, tertiary, or higher-order reagents is often to increase signal intensity. Suitable secondary and higher-order reagents include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The reagent or substrate may also be "tagged" with one or more tags, as known in the art.

[0190] In some embodiments, when step (c) is present, the quantification in step (d) comprises amplifying the DNA obtained from step (c) using a quantification primer pair(s) and a DNA polymerase, whereby at least a portion of the obtained DNA is amplified. In some embodiments, when step (c) is not present, the quantification in step (d) comprises amplifying at least one target marker in the treated DNA obtained from step (b) using a quantification primer pair(s) and a DNA polymerase.

[0191]

[0188] As used herein, the term "quantification primer pair(s)" refers to the primer pair(s) used in the quantification step.

[0192] In some embodiments, when step (c) is present, the quantification primer pair(s) used in step (d) are capable of hybridizing to at least 9 contiguous nucleotides of the DNA obtained from step (c) under stringent, moderately stringent, or highly stringent conditions. In some embodiments, when step (c) is absent, the quantification primer pair(s) used in step (d) are capable of hybridizing to at least 9 contiguous nucleotides of at least one target marker in the treated DNA obtained from step (b) under stringent, moderately stringent, or highly stringent conditions. In some embodiments, when step (c) is present, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more) of the quantification primer pair(s) used in step (d) is identical to at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more) of the methylation-specific primer pair(s) in the pre-amplification primer pool of step (c).

[0193] In some embodiments, when step (c) is not present, the quantification primer pair(s) used in step (d) are designed to amplify at least a portion of at least one target marker in the treated DNA obtained from step (b). In some embodiments, when step (c) is present, the quantification primer pair(s) used in step (d) are designed to amplify at least a portion of the DNA obtained from step (c), i.e., steps (c) and (d) are designed as nested PCR.

[0194]

[0191] Nested PCR is a modified form of PCR designed to improve sensitivity and specificity. Nested PCR involves two sequential PCR reactions, each containing two primer sets. The first amplification is performed to generate a first amplicon, and the second amplification is performed using a primer pair in which one or both of the primers anneal to a site within the region defined by the first primer pair; i.e., the second primer pair is considered to be "nested" within the first primer pair. In this way, background amplification products from the first PCR reaction that do not contain the correct internal sequence are not further amplified in the second PCR reaction.

[0195] In some embodiments, when step (c) is present, the quantification in step (d) comprises determining the methylation level of at least one (e.g., each) target marker(s) based on the presence or level of multiple CpG dinucleotides, TpG dinucleotides, or CpA dinucleotides in the DNA obtained from step (c). In some embodiments, when step (c) is not present, the quantification in step (d) comprises determining the methylation level of at least one (e.g., each) target marker based on the presence or level of multiple CpG dinucleotides, TpG dinucleotides, or CpA dinucleotides at the at least one target marker in the processed DNA obtained from step (b). In some embodiments, when step (c) is present, the quantification in step (d) comprises determining the methylation level of cytosine residue(s) based on the presence or level of one or more CpG dinucleotides in the DNA obtained from step (c). In some embodiments, if step (c) is not present, the quantification in step (d) comprises determining the methylation level of cytosine residue(s) based on the presence or level of one or more CpG dinucleotides at at least one target marker in the processed DNA obtained from step (b). In some embodiments, if step (c) is present, the quantification in step (d) comprises determining the methylation level of cytosine residue(s) based on the presence or level of one or more TpG dinucleotides in the DNA obtained from step (c). In some embodiments, if step (c) is not present, the quantification in step (d) comprises determining the methylation level of cytosine residue(s) based on the presence or level of one or more TpG dinucleotides at at least one target marker in the processed DNA obtained from step (b). In some embodiments, if step (c) is present, the quantification in step (d) comprises determining the methylation level of cytosine residue(s) based on the presence or level of one or more CpA dinucleotides in the DNA obtained from step (c).In some embodiments, when step (c) is absent, the quantification in step (d) comprises determining the methylation level of cytosine residue(s) based on the presence or level of one or more CpA dinucleotides at at least one target marker in the processed DNA obtained from step (b).

[0196] In some embodiments, when step (c) is present, the quantification step is carried out by dividing the DNA obtained from step (c) into multiple fractions. In some embodiments, when step (c) is not present, the quantification step is carried out by dividing at least one target marker in the treated DNA obtained from step (b) into multiple fractions. In some embodiments, multiple different quantification experiments are performed on multiple fractions, and different sets of DNA obtained from step (c) (or at least one target marker in the treated DNA obtained from step (b)) are quantified in one of the multiple fractions, if present in the fractions. In some embodiments, a control marker is quantified in each of the fractions. Step (e)

[0194] In step (e) of the method for diagnosing colorectal neoplasia in a subject, screening for the onset or risk of onset of colorectal neoplasia, or assessing the development or prognosis of colorectal neoplasia, the methylation level of at least one (e.g., each) target marker from step (d) is compared to a corresponding reference level, and the same or a higher methylation level of one or more of the target marker(s) compared to its corresponding reference level indicates that the subject has a colorectal neoplasia, or is developing or at risk for developing a colorectal neoplasia, or will develop or has an increased likelihood of developing a colorectal neoplasia, or has a poor prognosis or is at risk for a poor prognosis for a colorectal neoplasia.

[0197]

[0195] In step (e) of the method for monitoring treatment response in a subject undergoing treatment for colorectal neoplasia, the methylation level of at least one (e.g., each) target marker from step (d) is compared with a corresponding methylation level of one or more target markers obtained from the same subject prior to treatment, quantified by repeating steps (a), (b), and optionally step (c), and (d) on a biological sample containing DNA obtained from the subject prior to treatment, and a lower methylation level of one or more target markers compared to their corresponding methylation level prior to treatment indicates that the subject is responsive to treatment.

[0198]

[0196] Step (e) of the methods described herein may also be referred to as the comparison step.

[0199] As used herein, the terms "compare," "comparing," "compared," or "comparison" refer to comparing the methylation level of at least one (e.g., each) of the target marker(s) from the quantification step contained in the test biological sample being analyzed with the corresponding reference level. It should be understood that the terms used herein refer to the comparison of corresponding parameters or values, e.g., absolute amounts are compared with absolute reference amounts, while concentrations are compared with reference concentrations, or intensity signals obtained from a test sample are compared with the same type of intensity signal from a reference sample. The comparison may be performed manually or with the assistance of a computer. In the case of computer-assisted comparison, the determined quantity value may be compared by a computer program with values ​​corresponding to appropriate references stored in a database. The computer program may further evaluate the results of the comparison and automatically provide the desired evaluation in an appropriate output format. Based on a comparison of the methylation level of at least one (e.g., each) of the target marker(s) from the quantification step with the corresponding reference level, it is possible to identify subjects having a colorectal neoplasia, having or at risk of developing a colorectal neoplasia, or having or having an increased likelihood of developing a colorectal neoplasia, or having or at risk of having a poor prognosis for a colorectal neoplasia, and it is also possible to monitor treatment response in subjects undergoing treatment for a colorectal neoplasia.

[0200]

[0198] As used herein, the term "reference value" refers to a threshold value for including or excluding colorectal neoplasia in a subject, or the onset or risk of onset of colorectal neoplasia, or a threshold value for monitoring the treatment response of a subject undergoing treatment for colorectal neoplasia.

[0201] For example, with respect to a method for diagnosing colorectal neoplasia in a subject, screening for the onset or risk of onset of colorectal neoplasia, or assessing the development or prognosis of colorectal neoplasia, if the methylation level of one or more target marker(s) in the test sample is the same as or higher than the corresponding reference level, the subject is considered to have colorectal neoplasia, to be developing or at risk for developing colorectal neoplasia, or to develop or have an increased likelihood of developing colorectal neoplasia, or to have or be at risk for a poor prognosis for colorectal neoplasia. In some embodiments, the methylation level of one or more target marker(s) in the test sample is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more than its corresponding reference level. In the present disclosure, for diagnosing colorectal neoplasia, screening for the onset or risk of onset of colorectal neoplasia, or assessing the development or prognosis of colorectal neoplasia, it is not necessary that the methylation level of each and every target marker be the same as or higher than its corresponding reference level. Rather, it will be sufficient if the methylation level of at least one target marker quantified in the quantification step is the same as or higher than its corresponding reference level.

[0202]

[0200] As another example, with respect to a method of monitoring treatment response in a subject undergoing treatment for colorectal neoplasia, if the methylation level of one or more target marker(s) in a test sample is lower than its corresponding methylation level before treatment for colorectal neoplasia, the subject is considered to be likely to be responding to treatment. In some embodiments, the methylation level of one or more target marker(s) in a biological sample obtained after treatment for colorectal neoplasia is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% compared to the corresponding methylation level before treatment for colorectal neoplasia. In the present disclosure, it is not necessary for the methylation level of each and every target marker to be lower than the corresponding methylation level before treatment for colorectal neoplasia to indicate that a subject undergoing treatment for colorectal neoplasia is responding to treatment. Rather, it is sufficient that the methylation level of at least one target marker in a biological sample obtained after treatment for a colorectal neoplasia is lower than the corresponding methylation level before treatment for a colorectal neoplasia.

[0203]

[0201] Reference levels of methylation of target markers may be obtained from one or more reference samples, where the reference levels are obtained from experiments performed in parallel with experiments for testing the sample of interest. Alternatively, the reference levels may be obtained in a database containing a collection of data, standards, or levels from one or more reference samples or disease reference samples. In some embodiments, such a collection of data, standards, or levels is normalized so that it can be used for comparison purposes with data from one or more samples. "Normalizing" or "normalization" is the process by which measured raw data is converted into data that can be directly compared with other such normalized data. Normalization is used to overcome assay-specific errors caused by factors that vary from assay to assay, such as variations in loading, binding efficiency, detection sensitivity, and various other errors.

[0204] In some embodiments, the reference database includes methylation levels of target markers from one or more reference samples and / or other laboratory and clinical data. In some embodiments, the reference database includes methylation levels of the target markers, each normalized as a percentage of the methylation level of a control marker tested under the same conditions as the reference sample. To compare with such normalized methylation levels of the target markers, the methylation levels of the target markers in the test sample are also measured and calculated as a percentage of the methylation level of the control marker tested under the same conditions as the test sample.

[0205] In some embodiments, the reference database is established by compiling reference level data from reference samples obtained from healthy subjects and / or non-neoplastic subjects (i.e., subjects known not to have a neoplasia). In some embodiments, the reference database is established by compiling reference level data from reference samples from individuals undergoing treatment for colorectal neoplasia. In some embodiments, the reference database is established by compiling data from reference samples from individuals at different stages of colorectal neoplasia, as evidenced, for example, by different methylation levels of target markers.

[0206]

[0204] The reference level may be selected by one skilled in the art depending on the desired sensitivity and specificity. Means for determining an appropriate reference level are known to those skilled in the art; for example, the reference level can be determined from data collected from clinical studies.

[0207]

[0205] In some embodiments, the reference level in step (e) is determined based on clinical samples obtained from a group of individuals who have or are at risk of having a colorectal neoplasia and a group of individuals who are not at risk of or are free from having a colorectal neoplasia.

[0208]

[0206] One skilled in the art can determine whether an individual has or is at risk of having a colorectal neoplasia based on a variety of factors, such as age, sex, medical history, family history, symptoms, etc.

[0209] In some embodiments, the methylation level of the target marker and the reference level are expressed as cycle thresholds (i.e., Ct values). As used herein, the term "Ct value" refers to the cycle number at which the fluorescence of the PCR product can be detected above the background signal. The Ct value is inversely proportional to the amount of the target marker in the sample, i.e., a lower Ct value means a higher amount of the target marker in the sample.

[0210]

[0208] For example, in step (e) of a method for diagnosing a colorectal neoplasm in a subject, screening for the onset or risk of developing a colorectal neoplasm, or assessing the development or prognosis of a colorectal neoplasm, the Ct value(s) of the target marker(s) in step (d) are compared with reference Ct values, and a Ct value of at least one target marker that is the same or lower than its corresponding reference Ct value indicates that the subject has a colorectal neoplasm, is developing or is at risk for developing a colorectal neoplasm, or will develop or has an increased likelihood of developing a colorectal neoplasm, or has a poor prognosis or is at risk for a poor prognosis for a colorectal neoplasm. In some embodiments, if the Ct value of at least one of the plurality of target markers in step (d) is lower than its corresponding reference Ct value by cycles 2 to 10 (e.g., cycles 2, 3, 4, 5, 6, 7, 8, 9, 10), the subject is determined to have a colorectal neoplasia, to have developed or be at risk for developing a colorectal neoplasia, or to have or be at increased likelihood of developing a colorectal neoplasia, or to have or be at risk for a poor prognosis for a colorectal neoplasia.

[0211]

[0209] As used herein, the term "increased likelihood" refers to an overall increase of 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level of likelihood that a subject will develop a colorectal neoplasia or a poor prognosis for a colorectal neoplasia compared to the subject from whom the reference sample was obtained.

[0212]

[0210] As another example, in step (e) of the method of monitoring treatment response in a subject receiving treatment for colorectal neoplasia, the Ct value(s) of the target marker(s) of step (d) are compared to reference Ct values, and a higher Ct value of at least one target marker relative to its corresponding Ct value before treatment indicates that the subject receiving treatment for colorectal neoplasia is responding to the treatment. In some embodiments, the subject is determined to be responding to treatment for colorectal neoplasia if the Ct value of at least one of the multiple target markers of step (d) is higher than its corresponding reference Ct value before treatment by cycles 2 to 10 (e.g., cycles 2, 3, 4, 5, 6, 7, 8, 9, 10). kit In another aspect, the present disclosure provides a kit for diagnosing, screening for the development or risk of development of, or assessing the development or prognosis of a colorectal neoplasia, comprising: (a) a first reagent for treating DNA, the first reagent being capable of distinguishing between unmethylated and methylated sites in DNA; (b) Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5, as appropriate. a first primer pool comprising at least one primer pair for pre-amplifying at least one target sequence in at least one target marker selected from the group consisting of: 5, wherein the at least one primer pair can hybridize to at least 9 consecutive nucleotides of the at least one target sequence treated with a first reagent under stringent, moderately stringent, or highly stringent conditions; the first primer pool, wherein the target sequence comprises at least one CpG site; and (c) a second reagent, when the first primer pool is present, for quantifying the methylation level of at least one target marker (e.g., each) pre-amplified by the first primer pool; and when the first primer pool is not present, for quantifying the methylation level of at least one target marker (e.g., each) in DNA treated with the first reagent, wherein the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, and INTERGENIC REGION 4. a second reagent comprising one or more markers selected from the group consisting of INTERGENIC REGION 4, and INTERGENIC REGION 5; Also provided is a kit comprising:

[0213]

[0212] In some embodiments, the at least one target marker comprises multiple target markers, and the multiple target markers comprise at least two (e.g., 2, 3) markers selected from the group consisting of Septin9, BCAT1, and IKZF1.

[0214] In some embodiments, when the first primer pool is present, the second reagent comprises a second primer pool comprising a plurality of quantification primer pairs capable of hybridizing to at least 9 contiguous nucleotides of at least one target sequence pre-amplified by the first primer pool under stringent, moderately stringent, or highly stringent conditions. In some embodiments, when the first primer pool is not present, the second reagent comprises a third primer pool comprising a plurality of quantification primer pairs capable of hybridizing to at least 9 contiguous nucleotides of at least one target sequence of said at least one target marker in the DNA treated by the first reagent under stringent, moderately stringent, or highly stringent conditions.

[0215] In some embodiments, when the first primer pool is present, at least one of the quantification primer pairs in the second primer pool is identical to at least one of the primer pairs in the first primer pool. In some embodiments, when the first primer pool is present, the quantification primer pair of the second primer pool is designed to amplify at least a portion of at least one target sequence pre-amplified by the first primer pool. In some embodiments, when the first primer pool is not present, the quantification primer pair of the third primer pool is designed to amplify at least a portion of at least one target sequence of at least one target marker in the DNA treated with the first reagent. In some embodiments, the first, second, or third primer pool comprises at least one methylation-specific primer pair.

[0216] In some embodiments, the first and second pools of primers are packaged in a single container or in separate containers. In some embodiments, the kit further comprises one or more blocker oligonucleotides.

[0217] In some embodiments, the kit further comprises a detection agent. In some embodiments, the detection agent is selected from the group consisting of a fluorescent probe, an intercalating dye, a chromophore-labeled probe, a radioisotope-labeled probe, and a biotin-labeled probe. In some embodiments, the fluorescent probe comprises an oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 57-85, 172. In some embodiments, the fluorescent probe is labeled at its 5' end with a fluorescent dye (e.g., FAM, HEX / VIC, TAMRA, Texas Red, or Cy5) and at its 3' end with a quencher (e.g., BHQ1, BHQ2, BHQ3, DABCYL, TAMRA, or Iowa Black Dark quencher).

[0218] In some embodiments, the kit further comprises a DNA polymerase and / or a container suitable for containing a biological sample from a subject. In some embodiments, the kit further comprises instructions for use and / or interpretation of the kit results.

[0219] In some embodiments, the kit may include a reaction buffer optimized for polymerase-mediated primer extension, such as PCR, packaged in a separate container. Preferred are those for detecting the expression of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, INTERGENIC REGION 1, TMEFF2, INTERGENIC REGION 4, NKX2-6, INTERGENIC REGION 5, SLC24A2, NDRG4, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, INTERGENIC REGION 5, SLC24A2, NDRG4, INTERGENIC REGION 6, INTERGENIC REGION 7, INTERGENIC REGION 8, INTERGENIC REGION 9, INTERGENIC REGION 10, INTERGENIC REGION 11, INTERGENIC REGION 12, INTERGENIC REGION 13, INTERGENIC REGION 14, INTERGENIC REGION 15, INTERGENIC REGION 16, INTERGENIC REGION 17, INTERGENIC REGION 18, INTERGENIC REGION 19, INTERGENIC REGION 20, INTERGENIC REGION 21, INTERGENIC REGION 22, INTERGENIC REGION 23, INTERGENIC REGION 24, INTERGENIC REGION 25, INTERGENIC REGION 26, INTERGENIC REGION 27, INTERGENIC REGION 28, INTERGENIC REGION 29, INTERGENIC REGION 29, INTERGENIC REGION 30, INTERGENIC REGION 31, INTERGENIC REGION 26, INTERGENIC REGION 29, INTER 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP.

[0220] In some embodiments, the first reagent comprises a bisulfite reagent or a methylation-sensitive restriction enzyme (MSRE). In some embodiments, the bisulfite reagent is selected from the group consisting of ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, bisulfite salt, and any combination thereof. In some embodiments, the bisulfite reagent is sodium bisulfite. In some embodiments, the MSRE is selected from the group consisting of HpaII, SalI, SalI-HF®, ScrFI, BbeI, NotI, SmaI, XmaI, MboI, BstBI, ClaI, MluI, NaeI, NarI, PvuI, SacII, HhaI, and any combination thereof.

[0221]

[0220] In some embodiments, the first primer pool comprises at least one methylation-specific primer pair for pre-amplifying at least one target sequence in at least one target marker selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5.

[0222] In some embodiments, the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. 5 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 markers).

[0223] In some embodiments, the at least one target marker can be up to one target marker (i.e., one marker but no more than one marker). In some embodiments, the at least one target marker is Septin9. In some embodiments, the at least one target marker is BCAT1. In some embodiments, the at least one target marker is IKZF1. In some embodiments, the at least one target marker is NDRG4. In some embodiments, the at least one target marker is BCAN. In some embodiments, the at least one target marker is PKNOX2. In some embodiments, the at least one target marker is VAV3. In some embodiments, the at least one target marker is IRF4. In some embodiments, the at least one target marker is POU4F2. In some embodiments, the at least one target marker is SALL1. In some embodiments, the at least one target marker is TMEFF2. In some embodiments, the at least one target marker is ASCL4. In some embodiments, the at least one target marker is FGF12. In some embodiments, the at least one target marker is INTERGENIC REGION1.

[0224] In some embodiments, the at least one target marker comprises multiple target markers. In some embodiments, the multiple target markers comprise at least two or three markers selected from the group consisting of Septin9, BCAT1, and IKZF1. In some embodiments, the multiple target markers of the present disclosure further comprise one, two, three, four, or five additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4, and IRF4. In some embodiments, the multiple target markers of the present disclosure further comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) additional markers selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, INTERGENIC REGION 1, TMEFF2, INTERGENIC REGION 4, NKX2-6, INTERGENIC REGION 5, SLC24A2, INTERGENIC REGION 2, INTERGENIC REGION 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP.

[0225] In some embodiments, the multiple target markers of the present disclosure are Septin9 and BCAN, BCAT1, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: BCAN, BCAT1, IKZF1, NDRG4, PKNOX2, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one additional target marker comprises BCAN, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4.

[0226] In some embodiments, the multiple target markers of the present disclosure are BCAT1 as well as BCAN, Septin9, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of BCAN, Septin9, NDRG4, IKZF1, PKNOX2, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, Septin9, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0227] In some embodiments, the multiple target markers of the present disclosure are IKZF1 and BCAN, Septin9, BCAT1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: BCAN, Septin9, BCAT1, PKNOX2, NDRG4, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, Septin9, and / or BCAT1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0228] In some embodiments, the multiple target markers of the present disclosure include BCAN as well as Septin9, BCAT1, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, PKNOX2, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises NDRG4, VAV3, and / or IRF4.

[0229] In some embodiments, the multiple target markers of the present disclosure include VAV3 and Septin9, BCAT1, IKZF1, BCAN, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, BCAN, PKNOX2, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, NDRG4, and / or IRF4.

[0230] In some embodiments, the multiple target markers of the present disclosure include IRF4 as well as Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, BCAN, NDRG4, PKNOX2, VAV3, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or NDRG4.

[0231] In some embodiments, the multiple target markers of the present disclosure include PKNOX2 and Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, BCAN, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0232] In some embodiments, the multiple target markers of the present disclosure include NDRG4 and Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, TMEFF2, SALL1, SLC24A2, NKX2-6, KCNA6, SOX1, HS3ST2, ASCL4, KCTD8, HMX1, MARCH11, CRHBP, FGF12, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, BCAN, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4.

[0233]

[0232] In some embodiments, each target marker comprises: a) a respective region defined by the Hg19 coordinates shown below;

[0234] [Table 6-1]

[0235] [Table 6-2]

[0236] and 5 kb upstream of the respective start site and 5 kb downstream of the respective end site of each of the above regions, or b) the bisulfite-converted counterpart of a), or c) the MSRE-treated counterpart of a). Contains, or is a), b), or c).

[0237]

[0233] In some embodiments, when a first primer pool is present, the first primer pool comprises at least one primer pair comprising or consisting of at least one pair of nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54, and 170 / 171 as shown in Table 2 below; and optionally, the second primer pool comprises at least one primer pair that is identical to at least one of the primer pairs in the first primer pool. In some embodiments, when the first primer pool is absent, the third primer pool comprises at least one primer pair comprising or consisting of at least one pair of nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54, and 170 / 171 as shown in Table 2 below.

[0238] In some embodiments, the first primer pool, the second primer pool, or optionally the third primer pool further comprises a primer pair for amplifying a control marker. In some embodiments, the control marker is selected from the group consisting of ACTB, GAPDH, tubulin, ALDOA, PGK1, LDHA, RPS27A, RPL19, RPL11, ARHGDIA, RPL32, C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP, and VPS29.

[0239]

[0235] In some embodiments, the kit further comprises multiple containers, each for receiving a fraction of the second primer pool.

[0240]

[0236] In some embodiments, the kit further comprises standard reagents for performing CpG site-specific methylation analysis, said analysis comprising one or more of the following techniques: MS-SNuPE, MSP, MethyLight™, HeavyMethyl™, COBRA, and nucleic acid sequencing.

[0241]

[0237] In some embodiments, the kit may include additional reagents selected from the group consisting of buffers (e.g., restriction enzyme, PCR, storage or wash buffers); DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns) and DNA recovery components.

[0242] In one embodiment, the kit of the present disclosure comprises: (a) bisulfite reagent, (b) Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, INTERGENIC REGION 1, TMEFF2, INTERGENIC REGION 4, NKX2-6, INTERGENIC REGION 5, SLC24A2, NDRG4, INTERGENIC REGION 2, INTERGENIC REGION a first primer pool comprising a plurality of methylation-specific primer pairs for pre-amplifying at least two target sequences among a plurality of target markers, the plurality of target markers comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more) markers selected from the group consisting of 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP; a first primer pool, wherein the differential primer pairs comprise or consist of at least two pairs of nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 / 2, 3 / 4, 5 / 6, 7 / 8, 9 / 10, 11 / 12, 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 51 / 52, 53 / 54, and 170 / 171 as set forth in Table 2 below; (c) a second reagent, when the first primer pool is present, for quantifying the methylation level of at least one (e.g., each) of the plurality of target markers pre-amplified by the first primer pool, the second reagent comprising a second primer pool comprising a plurality of quantification primer pairs capable of hybridizing to at least 9 consecutive nucleotides of the plurality of target markers pre-amplified by the first primer pool under stringent conditions, moderately stringent conditions, or highly stringent conditions; In the absence of a second reagent, the second reagent is for quantifying the methylation level of at least one (e.g., each) target marker in the DNA treated with the first reagent, wherein the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC a second reagent comprising one or more markers selected from the group consisting of REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5, wherein the second reagent comprises a third primer pool comprising a plurality of quantification primer pairs capable of hybridizing to at least 9 consecutive nucleotides of at least one target sequence of at least one target marker in the DNA treated with the first reagent under stringent, moderately stringent, or highly stringent conditions. may include:

[0243]

[0239] Kits of the present disclosure may also include other components, such as buffers or solutions suitable for blocking, washing, or coating, packaged in separate containers.

[0244]

[0240] The kits of the present disclosure may further comprise one or more of the following components known in the art for DNA enrichment: a protein component, said protein selectively binding to methylated DNA; a triplex-forming nucleic acid component, one or more linkers, optionally in a suitable solution; a substance or solution for performing ligation, e.g., a ligase, a buffer; a substance or solution for performing column chromatography; a substance or solution for performing immunological-based enrichment (e.g., immunoprecipitation); a substance or solution for performing nucleic acid amplification, e.g., PCR; a dye or dyes, containing a coupling reagent, if applicable, in solution, if applicable; a substance or solution for performing hybridization; and / or a substance or solution for performing a washing step. use

[0241] In another aspect, the present disclosure provides use of a kit of the present disclosure in the manufacture of a diagnostic kit for diagnosing a colorectal neoplasm in a subject, screening for the onset or risk of onset of a colorectal neoplasm, or assessing the development or prognosis of a colorectal neoplasm, or monitoring treatment response in a subject undergoing treatment for a colorectal neoplasm.

[0245]

[0242] In another aspect, the present disclosure provides a method of diagnosing, screening for the development or risk of development of, or assessing the development or prognosis of a colorectal neoplasia in a subject, comprising: (a) obtaining a biological sample containing DNA from a subject; (b) treating the DNA in the biological sample obtained from step (a) with a reagent capable of distinguishing between unmethylated and methylated CpG site(s) in DNA, thereby obtaining treated DNA; (c) pre-amplifying at least a portion of at least one target marker within the treated DNA obtained from step (b) with a pre-amplification primer pool, wherein at least a portion of at least one (e.g., each) of the target marker(s) is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5; with or without step (c); (d) if step (c) is present, individually quantifying the methylation level of at least one (e.g., each) target marker based on the DNA obtained from step (c); if step (c) is not present, individually quantifying the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b), wherein the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. 5; and including one or more markers selected from the group consisting of: (e) comparing the methylation level of at least one (e.g., each) target marker, respectively, from step (d) with a corresponding reference level, wherein the same or a higher methylation level of one or more of the target marker(s) relative to its corresponding reference level indicates that the subject has a colorectal neoplasia, or has developed or is at risk for developing a colorectal neoplasia, or has or is at increased likelihood of developing a colorectal neoplasia, or has or is at risk for a poor prognosis for a colorectal neoplasia; The present invention provides the use of a reagent for quantifying the methylation level of a target marker in the manufacture of a kit for use in a method comprising:

[0246] In another aspect, the present disclosure provides a method of monitoring treatment response in a subject undergoing treatment for a colorectal neoplasia, comprising: (a) obtaining a biological sample containing DNA from a subject; (b) treating the DNA in the biological sample obtained from step (a) with a reagent capable of distinguishing between unmethylated and methylated CpG site(s) in DNA, thereby obtaining treated DNA; (c) pre-amplifying at least a portion of at least one target marker within the treated DNA obtained from step (b) with a pre-amplification primer pool, wherein at least a portion of at least one (e.g., each) of the target marker(s) is pre-amplified, and the at least one target marker comprises one or more markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5; with or without step (c); (d) if step (c) is present, individually quantifying the methylation level of at least one (e.g., each) target marker based on the DNA obtained from step (c); if step (c) is not present, individually quantifying the methylation level of at least one (e.g., each) target marker in the treated DNA obtained from step (b), wherein the at least one target marker is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. 5; and including one or more markers selected from the group consisting of: (e) comparing the methylation level of at least one (e.g., each) target marker from step (d) with a corresponding methylation level of one or more of the target marker(s) obtained from the same subject prior to the treatment, quantified by repeating steps (a), (b), optionally step (c), and (d) on a biological sample containing DNA obtained from the subject prior to the treatment, wherein a lower methylation level of one or more of the target marker(s) compared to its corresponding methylation level prior to the treatment indicates that the subject is responsive to the treatment; The present invention provides the use of a reagent for quantifying the methylation level of a target marker in the manufacture of a kit for use in a method comprising:

[0247] In some embodiments, the at least one target marker in step (c) above is selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. 5 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 markers).

[0248] In some embodiments, the at least one target marker in step (c) above can be up to one target marker (i.e., one marker, but not more than one). In some embodiments, the at least one target marker is Septin9. In some embodiments, the at least one target marker is BCAT1. In some embodiments, the at least one target marker is IKZF1. In some embodiments, the at least one target marker is BCAN. In some embodiments, the at least one target marker is PKNOX2. In some embodiments, the at least one target marker is VAV3. In some embodiments, the at least one target marker is IRF4. In some embodiments, the at least one target marker is NDRG4. In some embodiments, the at least one target marker is POU4F2. In some embodiments, the at least one target marker is SALL1. In some embodiments, the at least one target marker is TMEFF2. In some embodiments, the at least one target marker is ASCL4. In some embodiments, the at least one target marker is FGF12. In some embodiments, at least one target marker is INTERGENIC REGION 1.

[0249] In some embodiments, the at least one target marker in step (c) above comprises a plurality of target markers. In some embodiments, the plurality of target markers comprises at least two or three markers selected from the group consisting of Septin9, BCAT1, and IKZF1. In some embodiments, the plurality of target markers of the present disclosure further comprises one, two, three, four, or five additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4, and IRF4. In some embodiments, the multiple target markers of the present disclosure further comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) additional markers selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, INTERGENIC REGION 1, TMEFF2, INTERGENIC REGION 4, NKX2-6, INTERGENIC REGION 5, SLC24A2, INTERGENIC REGION 2, INTERGENIC REGION 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP.

[0250] In some embodiments, the multiple target markers of the present disclosure are Septin9 and BCAN, BCAT1, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: BCAN, BCAT1, IKZF1, NDRG4, PKNOX2, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one additional target marker comprises BCAN, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4.

[0251] In some embodiments, the multiple target markers of the present disclosure are BCAT1 as well as BCAN, Septin9, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of BCAN, Septin9, NDRG4, IKZF1, PKNOX2, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, Septin9, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0252] In some embodiments, the multiple target markers of the present disclosure are IKZF1 and BCAN, Septin9, BCAT1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: BCAN, Septin9, BCAT1, PKNOX2, NDRG4, VAV3, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include NDRG4, Septin9, and / or BCAT1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0253] In some embodiments, the multiple target markers of the present disclosure include BCAN as well as Septin9, BCAT1, IKZF1, VAV3, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, VAV3, NDRG4, IRF4, PKNOX2, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4.

[0254] In some embodiments, the multiple target markers of the present disclosure include VAV3 and Septin9, BCAT1, IKZF1, BCAN, IRF4, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, BCAN, PKNOX2, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0255] In some embodiments, the multiple target markers of the present disclosure include IRF4 as well as Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, BCAN, NDRG4, PKNOX2, VAV3, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4.

[0256] In some embodiments, the multiple target markers of the present disclosure include PKNOX2 and Septin9, BCAT1, IKZF1, BCAN, VAV3, IRF4, POU4F2, SALL1, SDC2, ASCL4, TMEFF2, SLC24A2, NDRG4, NKX2-6, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, BCAN, VAV3, NDRG4, IRF4, or any combination thereof. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one (e.g., at least 1, 2, or 3) additional target markers include BCAN, VAV3, and / or IRF4.

[0257] In some embodiments, the multiple target markers of the present disclosure include NDRG4 and Septin9, BCAT1, IKZF1, VAV3, IRF4, BCAN, POU4F2, PKNOX2, SDC2, TMEFF2, SALL1, SLC24A2, NKX2-6, KCNA6, SOX1, HS3ST2, ASCL4, KCTD8, HMX1, MARCH11, CRHBP, FGF12, INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. In some embodiments, the at least one additional target marker comprises at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) additional target markers selected from the group consisting of: Septin9, BCAT1, IKZF1, PKNOX2, VAV3, IRF4, BCAN, or any combination thereof. In some embodiments, the at least one additional target marker comprises Septin9, BCAT1, and / or IKZF1. In some embodiments, the at least one additional target marker comprises BCAN, VAV3, and / or IRF4. Embodiment

[0255] The biological materials, various clones and expression plasmids, media, enzymes, buffers, various culture methods, protein extraction and purification methods, and other molecular biological manipulation methods used in all examples are all well known to those skilled in the art. For details, see "Molecular Cloning: A Laboratory Manual" edited by Sambrook et al. (Cold Spring Harbor, 1989) and "Short Protocols in Molecular Biology" (Frederick M. Ausubel et al., translated by Yan Ziying et al., Science Press (Beijing), 1998).

[0258] Example 1 Validation of methylation-specific primers For the initial proof-of-concept, we selected bisulfite-converted reference DNA to evaluate primer / probe specificity. Customized primer / probe sets were designed for 28 target markers (i.e., NDRG4, BCAT1, IKZF1, Septin9, SDC2, VAV3, IRF4, TMEFF2, SALL1, BCAN, POU4F2, PKNOX2, ASCL4, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, NKX2-6, SLC24A2, and five intergenic regions, including INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5). As a proof-of-concept experiment, we created mixtures of DNA that was fully methylated at all CpG sites and fully unmethylated DNA (10%, 25%, 50%, and 100%), with a total input of 4 ng. These mixtures were evaluated in triplicate for 28 target markers using primers and probes with the sequences shown in Table 2. The experimental method is detailed below.

[0259]

[0257] Bisulfite-converted fully methylated DNA and bisulfite-converted fully unmethylated DNA were purchased from Qiagen (EpiTect Control DNA) and mixed to provide mixed DNA compositions containing 100%, 50%, 25%, and 10% fully methylated DNA in fully unmethylated DNA, respectively, where the total amount of DNA in each mixed DNA composition was 4 ng.

[0260] The mixed DNA composition was amplified by PCR in the presence of methylation-specific primer pairs (see Table 2) and detection probes (see Table 2) specific for 28 target markers (i.e., NDRG4, BCAT1, IKZF1, Septin9, SDC2, VAV3, IRF4, TMEFF2, SALL1, BCAN, POU4F2, PKNOX2, ASCL4, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, NKX2-6, SLC24A2, and five intergenic regions, including INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5). The control marker, ACTB, was also amplified by PCR using non-methylation-specific primers (see Table 2) and detection probes (see Table 2). Each of the 28 target markers and one control marker was amplified in a separate detection assay. The detection probes for the different markers were labeled with different fluorophores (FAM, HEX, VIC, TAMRA, Texas Red, or Cy5) and corresponding quenchers (BHQ1, BHQ2, BHQ3, DABCYL, or TAMRA). In the PCR reaction, each primer was at a final concentration of 500 nM, and each detection probe was at a final concentration of 200 nM.

[0261] [Table 7-1]

[0262] [Table 7-2]

[0263] [Table 7-3]

[0264]

[0259] A PCR reaction system was prepared containing 10 μL of the mixed DNA composition (4 ng DNA), 2.5 μL of a premix solution containing the above primers and probes, and 12.5 μL of a PCR reagent mix (Luna® Universal Probe qPCR Master Mix (NEB)).

[0265] The PCR reaction was carried out as follows: 95°C for 5 minutes, followed by 50 cycles of 95°C for 15 seconds and 56°C for 40 seconds (fluorescence detection was performed during this period). Different fluorescence was detected in the corresponding fluorescence channels using an ABI 7500 Real-Time PCR System.

[0266]

[0261] Results The Ct (cycle threshold) values ​​for each PCR reaction were calculated, and the Ct values ​​for the PCR reactions of each marker in the different mixed DNA compositions were analyzed. The results showed that for each tested marker, the methylation-specific primer pairs used in the PCR reactions gave Ct values ​​that decreased proportionally as the percentage of converted methylated DNA in the mixed DNA composition increased. For all tested markers, the percentage of methylated template showed a high correlation (correlation coefficient R > 0.9 for all tested markers) and linearity with the expected Ct values, indicating that the primers used for preamplification of the target markers were methylation-specific. This correlation is evidenced by the horizontal shift of the curves shown in Figure 1A (obtained with methylation-specific primers for PKNOX2) compared to the overlapping curves shown in Figure 1B (obtained with non-methylation-specific primers for the control marker, ACTB). The results for other methylation-specific primers tested for markers other than PKNOX2 were similar to those shown in Figure 1A and are not shown here.

[0267] Example 2 Comparison of methylation abundance of target markers in different tissues

[0263] To demonstrate the applicability and specificity of the selected target markers in tumor samples, 28 markers were tested in colorectal cancer tissues (CRC tissues), advanced adenoma tissues (AA tissues), and paracancerous tissues (para tissues) from colorectal cancer patients, as well as white blood cells (WBCs) from patients with negative colorectal endoscopy as controls. The details of the experimental method are as follows.

[0268] To explore the potential of these target markers in diagnosing and screening for colorectal neoplasia, we detected the methylation abundance of the target markers in DNA samples from different cells and tissues. The target markers tested in this example include NDRG4, BCAT1, IKZF1, Septin9, SDC2, VAV3, IRF4, TMEFF2, SALL1, BCAN, POU4F2, PKNOX2, ASCL4, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, NKX2-6, SLC24A2, and five intergenic regions, including INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5.

[0269]

[0265] The procedure includes the following steps:

[0270] 1. DNA samples were obtained from leukocytes, paracancerous tissues, advanced adenoma tissues, and colorectal cancer tissues, with 10 biological samples for each type (i.e., a total of 40 samples). Leukocyte DNA was extracted using the Qiagen QIAamp DNA Mini Kit, and tissue DNA was extracted using the Qiagen QIAamp DNA FFPE Tissue Kit according to the manufacturer's instructions.

[0271]

[0267] 2. The DNA sample obtained in step 1 above was treated with a bisulfite reagent (MethylCode™ Bisulfite Conversion Kit) to obtain converted DNA.

[0272] 3. Fluorescent PCR was performed on the converted DNA. Briefly, the converted DNA obtained in step 2 was amplified by PCR in the presence of methylation-specific primer pairs (see Table 2) and detection probes (see Table 2) specific for NDRG4, BCAT1, IKZF1, Septin9, SDC2, VAV3, IRF4, TMEFF2, SALL1, BCAN, POU4F2, PKNOX2, ASCL4, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, NKX2-6, SLC24A2, and five intergenic regions, including INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5. The control marker, ACTB, was also amplified by PCR using non-methylation-specific primers (see Table 2) and detection probes (see Table 2). The detection probes for different markers were labeled with different fluorophores. In the PCR reaction system, each primer was at a final concentration of 500 nM, and each detection probe was at a final concentration of 200 nM.

[0273]

[0269] A PCR reaction system was prepared containing 10 μL of the converted DNA, 2.5 μL of a premix solution containing the above primers and probe, and 12.5 μL of a PCR reagent mix (Luna® Universal Probe qPCR Master Mix (NEB)).

[0274]

[0270] The PCR reaction was carried out as follows: 95°C for 5 minutes, followed by 10 cycles of 95°C for 30 seconds and 56°C for 60 seconds (fluorescence detection was performed during this period). Different fluorescence was detected in the corresponding fluorescence channels using an ABI 7500 Real-Time PCR System.

[0275]

[0271] 4. Ct values ​​were calculated for samples obtained from leukocytes, paracancerous tissues, advanced adenoma tissues, and colorectal cancer tissues, and then combined and compared. The Ct value of undetermined wells was set at 50.

[0276]

[0272] Results

[0273] As a result, it was found that the methylation abundance of the target markers of the present disclosure (NDRG4, BCAT1, IKZF1, Septin9, SDC2, VAV3, IRF4, TMEFF2, SALL1, BCAN, POU4F2, PKNOX2, ASCL4, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, CRHBP, NKX2-6, SLC24A2, and five intergenic regions including INTERGENIC REGION 1, INTERGENIC REGION 2, INTERGENIC REGION 3, INTERGENIC REGION 4, and INTERGENIC REGION 5) in leukocytes from colorectal endoscopy-negative individuals was significantly lower (p<0.01) (see Figure 2), taking SALL1 and PKNOX2 as examples. Furthermore, significant differences were observed for the other tested target markers (p<0.01), and the results are not shown here. In particular, the abundance of methylated target markers was lower in paracancerous tissues than in advanced adenoma tissues and colorectal cancer tissues. These results suggest that each tested target marker may be applicable to the diagnosis and screening of colorectal neoplasia using leukocyte samples.

[0277] Example 3 Quantification of methylation target markers using cell-free DNA To validate the clinical performance of methylation markers on CRC plasma samples, the method disclosed herein (also referred to as the pre-amplification method) was used to test 13 markers (i.e., NDRG4, Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, and INTERGENIC REGION 1) in 88 clinically diagnosed CRC plasma samples and 107 colorectal endoscopy-negative plasma control samples. Of the 88 clinically diagnosed CRC plasma samples, 15 samples were obtained from subjects diagnosed with stage I CRC, 26 samples were obtained from subjects diagnosed with stage II CRC, 28 samples were obtained from subjects diagnosed with stage III CRC, and 19 samples were obtained from subjects diagnosed with stage IV CRC.

[0278]

[0275] Pre-amplification method

[0276] The pre-amplification method includes the following steps:

[0279]

[0277] 1. Cell-free DNA (cfDNA) samples were obtained from 1 to 4 ml of plasma samples using the QIAamp Circulating Nucleic Acid Kit (Qiagen).

[0280]

[0278] 2. 20 ng cfDNA was used as input and bisulfite converted using a bisulfite reagent (MethylCode™ Bisulfite Conversion Kit) to obtain converted cfDNA.

[0281]

[0279] 3. The converted cfDNA sample was pre-amplified. Briefly, the converted cfDNA obtained in step 2 was pre-amplified by PCR reaction in the presence of methylation-specific primer pairs (see Table 2) specific for NDRG4, Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, and INTERGENIC REGION 1. In the PCR reaction system, each primer was at a final concentration of 200 nM.

[0282]

[0280] The 25 μL PCR mix consisted of 10 μL of converted cfDNA, 2.5 μL of premix solution containing the above primers, and 12.5 μL of PCR reagent mix (Luna® Universal Probe qPCR Master Mix (NEB)).

[0283]

[0281] The PCR reaction was carried out using a ProFlex™ PCR System (Thermo Fisher) as follows: 95°C for 3 minutes, followed by 8 cycles of 95°C for 30 seconds and 56°C for 60 seconds.

[0284]

[0282] 4. The products obtained from step 3 above were diluted 10-fold and then used for several multiplex fluorescent PCR detections specific for NDRG4, Septin9, BCAT1, IKZF1, BCAN, VAV3, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2 and INTERGENIC REGION 1.

[0285] The qPCR mix consisted of 10 μL of the product diluted in step 3, 2.5 μL of primer / probe pool, and 12.5 μL of PCR reagent mix (Luna® Universal Probe qPCR Master Mix (NEB)). A non-CpG ACTB region was used as an internal control for each reaction well (see Table 2). Detection probes for different markers were labeled with different fluorophores. In the PCR reaction system, each primer had a final concentration of 500 nM, and each detection probe had a final concentration of 200 nM.

[0286] The PCR reaction was carried out as follows: 95°C for 5 minutes, followed by 50 cycles of 95°C for 15 seconds and 56°C for 40 seconds (fluorescence detection was performed during this period). Different fluorescence was detected in the corresponding fluorescence channels using an ABI 7500 Real-Time PCR System.

[0287]

[0285] Results

[0286] Samples without an amplification signal were assigned a Ct value of 50. Each tested marker was assigned a reference Ct value. If the Ct value of any test marker was equal to or lower than the corresponding reference Ct value, the sample was classified as a positive sample. Figure 3 shows the distribution of Ct values ​​for the target markers SALL1 and BCAN in the CRC-bearing and colorectal endoscopy-negative populations. As shown in Figure 3, the methylation levels of the target markers SALL1 and BCAN in the CRC-bearing population were significantly higher than those in the colorectal endoscopy-negative population (p values ​​= 2.14E-4 and 1.07E-8 for SALL1 and BCAN, respectively). The results for other target markers were similar (p < 0.01) and are not shown.

[0288]

[0287] Table 3 below shows the comparative results using five target markers (i.e., Septin9, BCAT1, IKZF1, BCAN, and VAV3) in the pre-amplification method. As shown in Table 3, the pre-amplification method exhibited ultra-high sensitivity (86.4%) for CRC and high specificity (90.7%) in the colorectal endoscopy-negative population, far surpassing existing commercial markers such as Septin9, which has a sensitivity of 48.2% for CRC in clinical trial samples (see TRChurch et al., Gut.;63:317-325 (2014)). Among the 13 target markers, other marker combinations (e.g., the combination of Septin9, BCAT1, IKZF1, VAV3, BCAN, and NDRG4; the combination of Septin9, BCAT1, IKZF1, VAV3, BCAN, NDRG4, SDC2, PKNOX2, and TMEFF2; the combination of Septin9, BCAT1, IKZF1, VAV3, BCAN, NDRG4, SDC2, PKNOX2, TMEFF2, and INTERGENIC REGION 1) were also analyzed, and the sensitivity for CRC was found to be no less than 85% and the specificity for negative colorectal endoscopy was no less than 90%.

[0289] [Table 8]

[0290]

[0288] We also compared the sensitivity of the pre-amplification method and the Septin9 alone method in classifying CRC. The Septin9 alone method performed similarly to the pre-amplification method, except that the target marker was only Septin9.

[0291] As shown in Table 4, the sensitivity of the pre-amplification method for CRC stage I, stage II, stage III, and stage IV was 73.3%, 80.8%, 89.3%, and 100%, respectively. On the other hand, the sensitivity of the Septin9 monotherapy method for CRC stage I, stage II, stage III, and stage IV was 26.7%, 65.4%, 75.0%, and 79%, respectively. Therefore, the sensitivity of the pre-amplification method is significantly improved compared to the Septin9 monotherapy method.

[0292] [Table 9]

[0293] The Ct values ​​of each tested target marker were quantified to identify the presence or absence of methylated copies in CRC samples. Alternatively, the ΔCt values ​​of each tested target marker relative to the internal control, ACTB, can be calculated to represent the relative methylation level. Importantly, all tested markers have the classification power to separate CRC from controls, with AUCs ranging from 0.8 to 0.9 (as shown in Figure 4). Different algorithms, including linear discriminant analysis, SVM, random forest, linear regression, and logistic regression, were used to construct classifiers for early cancer detection. Various marker combinations were used to achieve optimized performance. Figure 5 shows the ROC curve for one combination (SALL1, BCAT1, and Septin9). The ROC curves for other combinations are similar to Figure 5 and are not shown here.

[0294] Example 4 Comparison of LOD between pre-amplification and direct qPCR methods To compare the LODs of the preamplification and direct qPCR methods, we tested 13 target markers (i.e., VAV3, NDRG4, Septin9, BCAT1, IKZF1, BCAN, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, and INTERGENIC REGION 1) using both the preamplification and direct qPCR methods. The direct qPCR method was performed similarly to the preamplification method, except that the preamplification step was omitted. In both methods, the 13 target markers were preamplified / amplified simultaneously, but quantification was performed separately for each target marker. The LOD comparison between the preamplification and direct qPCR methods for the target marker VAV3 is shown below. Similar LOD comparisons between the preamplification and direct qPCR methods were also performed for the other 12 target markers, but are not shown here.

[0295] Briefly, CRC tissue DNA was spiked into blood cell DNA at 0.5% and 0.2% ratios, and 40 ng of DNA was treated with bisulfite (MethylCode™ Bisulfite Conversion Kit). Half of the converted DNA was pre-amplified and then used in qPCR (i.e., pre-amplification method), while the other half was used directly in qPCR (i.e., direct qPCR method). The final primer concentration in the pre-amplification step was 50 nM. The 25 μL PCR mix consisted of 10 μL of converted DNA, 2.5 μL of premix solution containing the above primers, and 12.5 μL of PCR reagent mix (Luna® Universal Probe qPCR Master Mix (NEB)). The PCR program consisted of 95°C for 3 minutes, followed by eight cycles of 95°C for 30 seconds and 56°C for 60 seconds. The product obtained after the pre-amplification step was diluted 10-fold and used for qPCR. The qPCR mix consisted of 10 μL of template DNA, 2.5 μL of primer / probe pool, and 12.5 μL of LUNA master mix. The qPCR program consisted of 95°C for 5 minutes, followed by 50 cycles of 95°C for 15 seconds and 56°C for 40 seconds (fluorescence detection interval), and was run on an ABI 7500 Real-Time PCR System. Four replicates were run in parallel. The results are shown in Table 5 below.

[0296] [Table 10]

[0297]

[0293] Compared with direct qPCR, the pre-amplification method showed improved LOD (0.50% vs. 0.20% CRC DNA percentage), stability, and detection sensitivity, as shown in Table 5. The pre-amplification method for the other 12 target markers (i.e., NDRG4, Septin9, BCAT1, IKZF1, BCAN, POU4F2, SALL1, PKNOX2, SDC2, ASCL4, TMEFF2, and INTERGENIC REGION 1) showed comparable results to direct qPCR and are not shown here.

[0298] Example 5 Quantification of methylated target markers using cell-free DNA and comparison with methods without pre-amplification.

[0299] To validate the clinical performance of methylation markers for CRC plasma samples, five markers (Septin9, BCAT1, IKZF1, BCAN, and VAV3) were tested in 32 clinically diagnosed CRC plasma samples and 29 colorectal endoscopy-negative plasma control samples using both pre-amplification and no-preamplification methods. The no-preamplification method was performed similarly to the pre-amplification method, except that it did not include the pre-amplification and dilution steps. Of the 32 clinically diagnosed CRC plasma samples, two were obtained from subjects diagnosed with stage I CRC, nine from subjects diagnosed with stage II CRC, 13 from subjects diagnosed with stage III CRC, five from subjects diagnosed with stage IV CRC, and three from subjects diagnosed with unknown stage CRC.

[0300]

[0295] The experiment included the following steps.

[0301]

[0296] 1. Cell-free DNA (cfDNA) samples were obtained from 3-5 ml plasma samples using the QIAamp Circulating Nucleic Acid Kit (Qiagen).

[0302]

[0297] 2. If the DNA was less than 40 ng, the cfDNA was divided into two aliquots and used as input for bisulfite conversion using a bisulfite reagent (MethylCode™ Bisulfite Conversion Kit). Two parallel reactions were performed, one with 10 μL elution for the preamplification method and the other with 20 μL elution, to obtain the converted cfDNA. If the DNA was 40 ng or more, 20 ng of cfDNA was used for both reactions, and the elution procedure was the same as above.

[0303]

[0298] 3. In the pre-amplification method, the converted cfDNA sample in one reaction (10 μL elution) was pre-amplified. Briefly, the converted cfDNA sample obtained from the sample in Step 2 above was pre-amplified by PCR reaction in the presence of methylation-specific primer pairs specific for Septin9, BCAT1, IKZF1, BCAN, and VAV3 (see Table 2). In the PCR reaction system, each primer was at a final concentration of 200 nM. The pre-amplification program, dilution, and qPCR assay were the same as in Example 3.

[0304] 4. For the no-preamplification method, the converted cfDNA sample from the other reaction (20 μL elution) was used to perform qPCR assays in two separate wells, each containing 10 μL of converted DNA. The qPCR mix and program were the same as for the preamplification method.

[0305]

[0300] 5. A non-CpG ACTB region was used as an internal control for each reaction well (see Table 2). Detection probes for different markers were labeled with different fluorophores. In the PCR reaction system, each primer was at a final concentration of 500 nM, and each detection probe was at a final concentration of 200 nM.

[0306]

[0301] Results

[0302] Samples without an amplification signal were assigned a Ct value of 50. Each tested marker was assigned a reference Ct value. If the Ct value of any tested marker was equal to or lower than the corresponding reference Ct value, the sample was classified as a positive sample.

[0307] Table 6 shows the comparison results using five target markers (Septin9, BCAT1, IKZF1, BCAN, and VAV3) between the pre-amplification method and the method without pre-amplification. As shown in Table 6, the pre-amplification method showed ultra-high sensitivity (96.9%) for CRC and high specificity (93.1%) for the colorectal endoscopy-negative population, while the sensitivity and specificity of the method without pre-amplification were 84.4% and 93.1%, respectively. In addition, the sensitivity of the method without pre-amplification was much higher than that of the Septin9 method alone.

[0308] [Table 11]

[0309] To validate the clinical performance of methylation markers for CRC plasma samples, more markers, including any combination of markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, NDRG4, and IRF4, will be tested in clinically diagnosed CRC plasma samples and colorectal endoscopy-negative plasma control samples, both with and without the pre-amplification method described above. For example, any one of the following combinations will be tested: (1) Septin9, (2) Septin9 and BCAT1; (3) Septin9 and IKZF1; (4) Septin9 and NDRG4; (5) Septin9 and BCAN; (6) Septin9 and VAV3; (7) Septin9 and IRF4; (8) BCAT1 and IKZF1; (9) BCAT1 and NDRG4; (10) BCAT1 and BCAN; (11) BCAT1 and VAV3; (12) BCAT1 and IRF4; (13) IKZF1 and NDRG4; (14) IKZF1 and BCAN; (15) IKZF1 and VAV3; (16) IKZF1 and IRF4; (17) NDRG4 and BCAN; (18) NDRG4 and VAV3; (19) NDRG4 and IRF4; (20) BCAN and and VAV3; (21) BCAN and IRF4; (22) VAV3 and IRF4; (23) Septin9, BCAT1, and IKZF1; (24) BCAT1, IKZF1, and NDRG4; (25) IKZF1, NDRG4, and BCAN; (26) NDRG4, BCAN, and VAV3; (27) BCAN, VAV3, and IRF4; (28) Septin9, BCAT1, and NDRG4; (29) Septin9, BCAT1, and BCAN; (30) Septin9, BCAT1, and VAV3; (31) Septin9, BCAT1, and IRF4; (32) BCAT1, IKZF1, and BCAN; (33) BCAT1, IKZF1, and VAV3; (34) BCAT1, IKZF1, and IRF4.

[0310] Example 6 CRC detection by quantification of CRC methylation target markers (Septin9, BCAT1, IKZF1, VAV3 and IRF4) using cell-free DNA.

[0311] To evaluate the clinical performance of more marker combinations, five markers (Septin9, BCAT1, IKZF1, VAV3, and IRF4) were tested in 286 clinically diagnosed CRC plasma samples and 112 colorectal endoscopy-negative plasma control samples using the method disclosed herein (also referred to as the pre-amplification method). Of the 286 clinically diagnosed CRC plasma samples, 48 ​​samples were obtained from subjects diagnosed with stage I CRC, 113 samples were obtained from subjects diagnosed with stage II CRC, 107 samples were obtained from subjects diagnosed with stage III CRC, and 18 samples were obtained from subjects diagnosed with stage IV CRC.

[0312]

[0306] The experimental method was the same as in Example 3.

[0313]

[0307] Results

[0308] Samples without an amplification signal were assigned a Ct value of 50. Each tested marker was assigned a reference Ct value. If the Ct value of any tested marker was equal to or lower than the corresponding reference Ct value, the sample was classified as a positive sample.

[0314]

[0309] As shown in Table 7, the pre-amplification method (quantification of CRC methylation markers Septin9, BCAT1, IKZF1, VAV3, and IRF4) demonstrated ultra-high sensitivity for CRC (84.3%) and high specificity (90.3%) in the colorectal endoscopy-negative population.

[0315] [Table 12]

[0316]

[0310] As shown in Table 8, the pre-amplification method (quantification of CRC methylation markers Septin9, BCAT1, IKZF1, VAV3, and IRF4) had a sensitivity of 62.5%, 85.8%, 88.8%, and 100% for CRC stage I, stage II, stage III, and stage IV, respectively.

[0317] [Table 13]

[0318] To evaluate the clinical performance of more marker combinations, more markers, including any combination of markers selected from the group consisting of Septin9, BCAT1, IKZF1, BCAN, VAV3, NDRG4, and IRF4, are tested using the methods disclosed above in plasma samples from clinically diagnosed CRC patients and plasma control samples from patients with negative colorectal endoscopy. For example, any one of the following combinations is tested: (1) Septin9, (2) Septin9 and BCAT1; (3) Septin9 and IKZF1; (4) Septin9 and NDRG4; (5) Septin9 and BCAN; (6) Septin9 and VAV3; (7) Septin9 and IRF4; (8) BCAT1 and IKZF1; (9) BCAT1 and NDRG4; (10) BCAT1 and BCAN; (11) BCAT1 and VAV3; (12) BCAT1 and IRF4; (13) IKZF1 and NDRG4; (14) IKZF1 and BCAN; (15) IKZF1 and VAV3; (16) IKZF1 and IRF4; (17) NDRG4 and BCAN; (18) NDRG4 and VAV3; (19) NDRG4 and IRF4; (20) BCAN and and VAV3; (21) BCAN and IRF4; (22) VAV3 and IRF4; (23) Septin9, BCAT1, and IKZF1; (24) BCAT1, IKZF1, and NDRG4; (25) IKZF1, NDRG4, and BCAN; (26) NDRG4, BCAN, and VAV3; (27) BCAN, VAV3, and IRF4; (28) Septin9, BCAT1, and NDRG4; (29) Septin9, BCAT1, and BCAN; (30) Septin9, BCAT1, and VAV3; (31) Septin9, BCAT1, and IRF4; (32) BCAT1, IKZF1, and BCAN; (33) BCAT1, IKZF1, and VAV3; (34) BCAT1, IKZF1, and IRF4.

Claims

1. 1. A method for diagnosing a colorectal neoplasia in a subject, screening for the onset or risk of onset of a colorectal neoplasia, or providing an index for assessing the development or prognosis of a colorectal neoplasia, comprising: (I) treating DNA obtained from a biological sample with a reagent capable of distinguishing between unmethylated and methylated sites in DNA, thereby obtaining treated DNA; (II) quantifying the individual methylation levels of subregions of the set of target markers within the processed DNA of step (I); (III) comparing the methylation level of a subregion of at least one target marker of said set of target markers quantified in step (II), respectively, with a corresponding reference level, wherein the same or a higher methylation level of one or more of said target markers compared to its corresponding reference level provides an indication that said subject has a colorectal neoplasia, or is developing or at risk for developing a colorectal neoplasia, or is developing or has an increased likelihood of developing a colorectal neoplasia, or has or is at risk for a poor prognosis for a colorectal neoplasia; Including, wherein said set of target markers comprises Septin9, BCAT1, and IKZF1; The partial regions of Septin9, BCAT1, and IKZF1 each comprise any of the following, or any of the following: a) The region defined by the Hg19 coordinates shown below: Table 1 or b) the bisulfite-converted counterpart of a), or c) the MSRE-treated counterpart of a); The method according to claim 1,

2. The method of claim 1 , wherein the set of target markers further comprises BCAN or VAV3.

3. The method of claim 1 , wherein the set of target markers further comprises BCAN and VAV3.

4. Step (II) (i) pre-amplifying at least a portion of at least one target marker of a set of target markers in the treated DNA obtained from step (I) with a pre-amplification primer pool, wherein the set of target markers includes Septin9, BCAT1, and IKZF1; (ii) quantifying the individual methylation levels of subregions of the set of target markers within the DNA obtained from substep (i); The method of claim 1 , comprising:

5. 1. A method for diagnosing a colorectal neoplasia in a subject, screening for the onset or risk of onset of a colorectal neoplasia, or providing an index for assessing the development or prognosis of a colorectal neoplasia, comprising: (a) obtaining a biological sample containing DNA from the subject; (b) treating the DNA in the biological sample obtained from step (a) with a reagent capable of distinguishing between unmethylated and methylated sites in the DNA, thereby obtaining treated DNA; (c) pre-amplifying at least a portion of at least one target marker in the treated DNA obtained from step (b) with a pre-amplification primer pool, wherein at least a portion of at least one of the target marker(s) is pre-amplified, and the at least one target marker comprises Septin9, BCAT1, and IKZF1; with or without step (c); (d) if step (c) is present, quantifying the methylation level of a subregion of the at least one target marker individually based on the DNA obtained from step (c); if step (c) is not present, quantifying the methylation level of a subregion of the at least one target marker individually in the treated DNA obtained from step (b), wherein the at least one target marker comprises Septin9, BCAT1, and IKZF1; The partial regions of Septin9, BCAT1, and IKZF1 each comprise any of the following, or any of the following: a) The region defined by the Hg19 coordinates shown below: Table 2 or b) the bisulfite-converted counterpart of a), or c) the MSRE-treated counterpart of a); and (e) comparing the methylation level of the subregion of at least one target marker from step (d), respectively, with a corresponding reference level, wherein the same or a higher methylation level of one or more of said target marker(s) compared to its corresponding reference level provides an indication that said subject has a colorectal neoplasia, or has developed or is at risk for developing a colorectal neoplasia, or has or is at increased likelihood of developing a colorectal neoplasia, or has or is at risk for a poor prognosis for a colorectal neoplasia; A method comprising:

6. 1. A method for providing an index for monitoring treatment response in a subject undergoing treatment for a colorectal neoplasia, comprising: (a) obtaining a biological sample containing DNA from the subject; (b) treating the DNA in the biological sample obtained from step (a) with a reagent capable of distinguishing between unmethylated and methylated sites in the DNA, thereby obtaining treated DNA; (c) pre-amplifying at least a portion of at least one target marker in the treated DNA obtained from step (b) with a pre-amplification primer pool, wherein at least a portion of at least one of the target marker(s) is pre-amplified, and the at least one target marker comprises Septin9, BCAT1, and IKZF1; with or without step (c); (d) if step (c) is present, quantifying the methylation level of a subregion of the at least one target marker individually based on the DNA obtained from step (c); if step (c) is not present, quantifying the methylation level of a subregion of the at least one target marker individually in the treated DNA obtained from step (b), wherein the at least one target marker comprises Septin9, BCAT1, and IKZF1; The partial regions of Septin9, BCAT1, and IKZF1 each comprise any of the following, or any of the following: a) The region defined by the Hg19 coordinates shown below: Table 3 or b) the bisulfite-converted counterpart of a), or c) the MSRE-treated counterpart of a); and (e) comparing the sub-region methylation level of at least one target marker from step (d) with a corresponding methylation level of one or more sub-regions of said target marker(s) obtained from the same subject prior to said treatment, quantified by repeating steps (a), (b), and optionally step (c), and (d) on a biological sample containing DNA obtained from said subject prior to said treatment, wherein a lower methylation level of one or more sub-regions of said target marker(s) compared to its corresponding methylation level prior to treatment provides an indication that said subject is responsive to treatment; A method comprising:

7. the at least one target marker further comprises one or more additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4, and IRF4; and / or the at least one target marker further comprises one or more additional markers selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, INTERGENIC REGION 1, TMEFF2, INTERGENIC REGION 4, NKX2-6, INTERGENIC REGION 5, SLC24A2, INTERGENIC REGION 2, INTERGENIC REGION 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP; The method of claim 5.

8. Each of said target markers comprises: a) a respective region defined by the Hg19 coordinates shown below; Table 4-1 Table 4-2 and 5 kb upstream of the respective start site and 5 kb downstream of the respective end site of each of the above regions, or b) the bisulfite converted counterpart of a), or c) the MSRE treated counterpart of a). The method of any one of claims 1 to 7, comprising, or being a), b), or c).

9. The method of any one of claims 1 to 8, wherein the DNA comprises genomic DNA or cell-free DNA.

10. 10. The method of claim 9, wherein the cell-free DNA comprises circulating tumor DNA.

11. 11. The method of claim 9 or 10, wherein the target marker in the cell-free DNA is present in the biological sample in an amount of less than or equal to 1 ng, 0.8 ng, 0.6 ng, 0.4 ng, 0.2 ng, 0.1 ng, 0.08 ng, or 0.04 ng.

12. the biological sample is selected from the group consisting of tissue sections, biopsies, paraffin-embedded tissues, body fluids, colonic effluents, surgical resection samples, isolated blood cells, cells isolated from blood, and any combination thereof; The method according to any one of claims 1 to 11.

13. the bodily fluid is selected from the group consisting of whole blood, serum, plasma, urine, mucus, saliva, ascites, pleural fluid, thoracic fluid, synovial fluid, cerebrospinal fluid, thoracentesis fluid, abdominal fluid, and any combination thereof; The method of claim 12.

14. 14. The method of any one of claims 1 to 13, wherein the reagent capable of distinguishing between unmethylated and methylated sites in the DNA selectively modifies unmethylated cytosine residue(s) at CpG site(s) to produce modified residue(s), but does not significantly modify methylated cytosine residue(s).

15. the pre-amplification primer pool comprises at least one methylation-specific primer pair; The method according to any one of claims 4 to 7.

16. the at least one methylation-specific primer pair comprises a forward primer and a reverse primer, each comprising an oligonucleotide sequence that hybridizes under stringent, moderately stringent, or highly stringent conditions to at least 9 consecutive nucleotides of one of the target marker(s), wherein the at least 9 consecutive nucleotides of one of the target marker(s) comprises at least one CpG site; 16. The method of claim 15.

17. If step (c) is present, said quantification in step (d) comprises amplifying said DNA obtained from step (c) using a quantification primer pair(s) and a DNA polymerase, whereby said at least a portion of said obtained DNA is amplified; if step (c) is not present, said quantification in step (d) comprises amplifying said at least one target marker in said treated DNA obtained from step (b) using a quantification primer pair(s) and a DNA polymerase; or if step (c) is present, the quantification primer pair(s) used in step (d) are capable of hybridizing to at least 9 contiguous nucleotides of the DNA obtained from step (c) under stringent, moderately stringent, or highly stringent conditions; if step (c) is not present, the quantification primer pair(s) used in step (d) are capable of hybridizing to at least 9 contiguous nucleotides of the at least one target marker in the treated DNA obtained from step (b) under stringent, moderately stringent, or highly stringent conditions. If step (c) is present, at least one of the quantification primer pair(s) used in step (d) is identical to at least one of the methylation-specific primer pair(s) in the pre-amplification primer pool of step (c), or When step (c) is present, the quantification primer pair(s) used in step (d) are designed to amplify at least a portion of the DNA obtained from step (c); when step (c) is not present, the quantification primer pair(s) used in step (d) are designed to amplify at least a portion of the at least one target marker in the treated DNA obtained from step (b).

7. The method according to claim 5 or 6.

18. 7. The method of claim 6, wherein step (e) comprises comparing the Ct value(s) of the target marker(s) of step (d) with reference Ct values, wherein a same or lower Ct value of at least one target marker compared to its corresponding reference Ct value indicates that the subject has, has developed or is at risk for developing a colorectal neoplasia, or has or is at increased likelihood of developing a colorectal neoplasia, or has a poor prognosis or is at risk for a poor prognosis for a colorectal neoplasia; or a higher Ct value of at least one target marker compared to its corresponding reference Ct value prior to the treatment indicates that the subject being treated for a colorectal neoplasia will be responsive to the treatment.

19. When step (c) is present, the quantifying in step (d) comprises determining a methylation level based on the presence or level of a plurality of CpG, TpG, or CpA dinucleotides in the DNA obtained from step (c); when step (c) is not present, the quantifying in step (d) comprises determining a methylation level of a subregion of at least one target marker based on the presence or level of a plurality of CpG, TpG, or CpA dinucleotides at the at least one target marker in the treated DNA obtained from step (b); If step (c) is present, said quantifying in step (d) comprises determining a methylation level of cytosine residue(s) based on the presence or level of one or more CpG dinucleotides in said DNA obtained from step (c); if step (c) is not present, said quantifying in step (d) comprises determining a methylation level of cytosine residue(s) based on the presence or level of one or more CpG dinucleotides in said at least one target marker in said treated DNA obtained from step (b), or When step (c) is present, the quantification in step (d) is carried out by dividing the DNA obtained from step (c) into a plurality of fractions; when step (c) is not present, the quantification in step (d) is carried out by dividing the at least one target marker in the treated DNA obtained from step (b) into a plurality of fractions. The method according to any one of claims 5 to 18.

20. The method of any one of claims 1 to 19, wherein the colorectal neoplasm is colorectal cancer, large colorectal adenoma, and / or sessile serrated polyp.

21. 1. A kit for diagnosing, screening for the onset or risk of onset of, or assessing the development or prognosis of a colorectal neoplasia, comprising: (a) a first reagent for treating DNA, the first reagent being capable of distinguishing between unmethylated and methylated sites in the DNA; (b) optionally, a first primer pool comprising at least one primer pair for pre-amplifying at least one target sequence in at least one target marker comprising Septin9, BCAT1, and IKZF1, wherein the at least one primer pair can hybridize to at least 9 consecutive nucleotides of the at least one target sequence treated with the first reagent under stringent, moderately stringent, or highly stringent conditions; the target sequence comprising at least one CpG site; and (c) a second reagent, when the first primer pool is present, for quantifying a methylation level of a partial region of the at least one target marker pre-amplified by the first primer pool, and when the first primer pool is not present, for quantifying a methylation level of a partial region of the at least one target marker in the DNA treated with the first reagent, wherein the at least one target marker comprises Septin9, BCAT1, and IKZF1; The partial regions of Septin9, BCAT1, and IKZF1 each comprise any of the following, or any of the following: a) The region defined by the Hg19 coordinates shown below: Table 5 or b) the bisulfite-converted counterpart of a), or c) the MSRE-treated counterpart of a); a second reagent which is Kit including:

22. 22. The kit of claim 21, wherein, when the first primer pool is present, the second reagent comprises a second primer pool comprising a plurality of quantification primer pairs capable of hybridizing to at least 9 contiguous nucleotides of the at least one target sequence pre-amplified by the first primer pool under stringent, moderately stringent, or highly stringent conditions; and when the first primer pool is not present, the second reagent comprises a third primer pool comprising a plurality of quantification primer pairs capable of hybridizing to at least 9 contiguous nucleotides of the at least one target sequence of the at least one target marker in the DNA treated with the first reagent under stringent, moderately stringent, or highly stringent conditions.

23. 23. The kit of claim 22, wherein at least one of the quantification primer pairs in the second primer pool is identical to at least one of the primer pairs in the first primer pool.

24. 24. The kit of claim 23, wherein, when the first primer pool is present, the quantification primer pair of the second primer pool is designed to amplify at least a portion of the at least one target sequence pre-amplified by the first primer pool; and when the first primer pool is not present, the quantification primer pair of the third primer pool is designed to amplify at least a portion of the at least one target sequence of the at least one target marker in the DNA treated with the first reagent.

25. The kit of any of claims 22 to 24, wherein the first, second, or third primer pool comprises at least one methylation-specific primer pair.

26. the at least one target marker further comprises one or more additional markers selected from the group consisting of BCAN, PKNOX2, VAV3, NDRG4, and IRF4; and / or the at least one target marker further comprises one or more additional markers selected from the group consisting of POU4F2, SALL1, SDC2, ASCL4, INTERGENIC REGION 1, TMEFF2, INTERGENIC REGION 4, NKX2-6, INTERGENIC REGION 5, SLC24A2, INTERGENIC REGION 2, INTERGENIC REGION 3, KCNA6, SOX1, HS3ST2, FGF12, KCTD8, HMX1, MARCH11, and CRHBP; The kit according to any one of claims 21 to 25.

27. Each of said target markers comprises: a) a respective region defined by the Hg19 coordinates shown below; Table 6-1 Table 6-2 and 5 kb upstream of the respective start site and 5 kb downstream of the respective end site of each of the above regions, or b) the bisulfite converted counterpart of a), or c) the MSRE treated counterpart of a).

27. The kit of any one of claims 21 to 26, comprising, or a), b), or c).

Images