Detecting the presence or absence of multiple types of cancer

Abstract

Provided herein are techniques for screening for multiple types of cancer from a biological sample, in particular, but not exclusively, methods, compositions and related uses for simultaneously detecting the presence of multiple types of cancer (e.g., liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer, and uterine cancer) from a biological sample (e.g., a stool sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample or a urine sample).

Description

Detailed Description of the Invention

[0001] [Technical field] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 143,611, filed January 29, 2021, and U.S. Provisional Patent Application No. 63 / 278,889, filed November 12, 2021, each of which is incorporated by reference in its entirety.

[0002] In the present specification, a technique for screening multiple types of cancer from biological samples is provided.In particular, the present invention relates to a method, composition, and related use for simultaneously detecting the presence of multiple types of cancer (e.g., liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer, and uterine cancer) from biological samples (e.g., stool samples, tissue samples, organ secretion samples, CSF samples, saliva samples, blood samples, plasma samples, or urine samples).

[0003] [Background technology] Very often, health care professionals are only able to make a cancer diagnosis after symptoms have developed, at which point it may be too late for curative treatment. Screening programs such as Pap-smears for cervical cancer and mammograms for breast cancer are intended to overcome this problem by detecting cancer at an earlier stage. However, such tests are typically only available to a subset of the population (those at highest risk), are limited to a small number of cancers, and have varying compliance rates. These methods can also be invasive or uncomfortable, potentially discouraging participation.

[0004] Therefore, there is an urgent need for improved diagnostic tools for detecting multiple types of cancer from a single adult sample.

[0005] The present invention addresses this need.

[0006] [Summary of the invention] Provided herein are techniques for screening for multiple types of cancer from a biological sample, in particular, but not exclusively, methods, compositions and related uses for simultaneously detecting the presence of multiple types of cancer (e.g., liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer, and uterine cancer) from a biological sample (e.g., a stool sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample or a urine sample).

[0007] Indeed, as described in Example I, experiments conducted in the course of identifying embodiments of the present invention identified sets of methylated DNA markers (MDMs), sets of protein markers, and combinations of MDMs and protein markers for simultaneously detecting the presence of multiple types of cancer (e.g., liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer, uterine cancer) from a biological sample (e.g., a stool sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample, or a urine sample).

[0008] In particular, such experiments have identified the following combinations of MDMs and protein markers and / or panels of MDMs and protein markers for detecting multiple types of cancer (e.g., liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer, uterine cancer) from biological samples (e.g., stool samples, tissue samples, organ secretion samples, CSF samples, saliva samples, blood samples, plasma samples, or urine samples): Methylation DNA markers: FAIM2, CDO1, SIM2, CHST2_7890, SFMBT2, PPP2R5C, ARHGEF4, TSPYL5, ZNF671, B3GALT6, FER1L4, HOXB2, BARX1, TBX1, SHOX2, EMX1, CLEC11A, HOXA1, GRIN2D, CAPN2, NDRG4, TRH, PRKCB, SHISA9, ZNF781, ST8SIA1, IFFO1, HOXA9, HOPX, OSR2, QKI, RYR2, GPRIN1, ZNF569, CD1D, NTRK3, VAV3, and FAM59B (see Figure 3, Tables 5, 13 and 14, Example I); Methylation DNA markers: FAIM2, CDO1, SIM2, CHST2_7890 , SFMBT2, PPP2R5C, ARHGEF4, TSPYL5, ZNF671, B3GALT6, FER1L4, HOXB2, BARX1, TBX1, SHOX2, EMX1, CLEC11A, HOXA1, GRIN2D, CAPN2, NDRG4, TRH, PRKCB, SHISA9, ZNF781, and ST8SIA1 (see Figure 3, Example I); Methylated DNA markers: GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, IFFO1, HOXA9, HOPX (see Table 5, Example I); Methylated DNA markers: GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, ST8SIA1, HOXA1, HOXB2, BARX1, CLEC11A, ARHGEF4, IFFO1, HOXA9, OSR2, QKI, RYR2, GPRIN1, ZNF569, SHISA9, CD1D, NTRK3, VAV3, FAM59B (see Tables 13 and 14, Example I); Methylated DNA marker: FAIM2, CHST2_7890 , ZNF671, GRIN2D, CDO1 (see FIG. 3 and Example I); Methylated DNA markers: ZNF671, GRIN2D, NDRG4 , SHOX2, B3GALT6 (see Example I); Methylation DNA markers: CDO1, GRIN2D, SHOX2, OSR2, QKI, SIM2, TRH, CAPN2, SFMBT2, CHST2_7890 , ST8SIA1, HOXA1, FER1L4, FAIM2, IFFO1, EMX1, ZNF671, PRKCB, HOXB2, BARX1, PPP2R5C, and TSPYL5 (see Example I); Protein markers: CEA, CA125, CA19.9, AFP, and CA-15-3 (see Example I). Protein markers: CEA, CA125, and CA19.9 (see FIG. 2 and Example I); and Protein markers: CEA, CA125, CA19.9, and AFP (see FIG. 4 and Example I).

[0009] As described herein, the technology provides sets of methylated DNA markers (MDMs) and subsets thereof (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, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38), sets of protein markers (e.g., sets of 2, 3, 4, 5), and combinations of MDMs and protein markers for simultaneously detecting the presence of multiple types of cancer from a biological sample (e.g., a stool sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample, or a urine sample).

[0010] In certain embodiments, a method for characterizing a biological sample, e.g., a biological sample from a human subject, comprising: a) Methylation markers in biological samples: FAIM2, CDO1, SIM2, CHST2_7890measuring the methylation level of one or more methylation markers selected from SFMBT2, PPP2R5C, ARHGEF4, TSPYL5, ZNF671, B3GALT6, FER1L4, HOXB2, BARX1, TBX1, SHOX2, EMX1, CLEC11A, HOXA1, GRIN2D, CAPN2, NDRG4, TRH, PRKCB, SHISA9, ZNF781, ST8SIA1, IFFO1, HOXA9, HOPX, OSR2, QKI, RYR2, GPRIN1, ZNF569, CD1D, NTRK3, VAV3, and FAM59B; b) measuring the expression and / or activity levels of one or more protein markers selected from CEA, CA125, CA19.9, AFP, and CA-15-3 in the biological sample.

[0011] In some embodiments, when the methylation level of one or more methylation markers is measured, the measured methylation level of the one or more methylation markers is compared to the methylation level of the corresponding one or more methylation markers in a control sample that does not have the particular type of cancer; and / or When the expression and / or activity levels of one or more protein markers are measured, the measured expression and / or activity levels of the one or more protein markers are compared to the corresponding expression and / or activity levels of the one or more protein markers in a control sample that does not contain a particular type of cancer.

[0012] In some embodiments, the method comprises: i) the methylation level measured for said one or more methylation markers is higher than the methylation level measured in each control sample; and ii) determining that the human subject has multiple types of cancer if the expression and / or activity levels of one or more protein markers are higher than the expression and / or activity levels measured in the respective control samples.

[0013] In some embodiments, the two or more types of cancer are any type of cancer, hi some embodiments, the two or more types of cancer are selected from liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer, and uterine cancer.

[0014] In some embodiments, measuring the methylation level of one or more methylation markers comprises treating DNA from the biological sample with a bisulfite-free base resolution sequencing method for directly detecting 5-methylcytosine and 5-hydroxymethylcytosine.

[0015] In some embodiments, measuring the methylation level of one or more methylation markers comprises treating DNA from the biological sample with a reagent that modifies DNA in a methylation-specific manner. In some embodiments, the reagent that modifies DNA in a methylation-specific manner is a borane reducing agent, e.g., the borane reducing agent can be 2-picoline borane. In some embodiments, the reagent comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent. In some embodiments, the reagent is a bisulfite reagent, and the treatment produces bisulfite-treated DNA.

[0016] In some embodiments, the treated DNA is amplified with a set of primers specific for one or more methylation markers. In some embodiments, the set of primers for each of the one or more selected methylation markers is selected from the group listed in Table 2. In some embodiments, the set of primers specific for each of the one or more selected methylation markers can bind to an amplicon bound by a primer sequence of a particular methylation marker gene listed in Table 2, and the amplicon bound by a primer sequence to a methylation marker gene listed in Table 2 is at least a portion of a gene region of a methylation marker listed in Table 1. In some embodiments, the set of primers specific for each of the one or more selected methylation markers is a set of primers that specifically binds to at least a portion of a gene region that includes the chromosomal coordinates of a methylation marker listed in Table 1.

[0017] In some embodiments, measuring the methylation level of one or more methylation markers comprises multiplex amplification.

[0018] In some embodiments, measuring the methylation level of at least one methylation marker comprises using one or more methods selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-specific DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, flap endonuclease assay, PCR-flap assay, and bisulfite genomic sequencing PCR.

[0019] In some embodiments, measuring the methylation level of one or more methylation markers comprises measuring the methylation of a CpG site for each of the one or more methylation markers. In some embodiments, the CpG site is in a coding region or a regulatory region.

[0020] In some embodiments, the one or more methylation markers are described by the genomic coordinates shown in Table 1.

[0021] In some embodiments, the biological sample is a fecal sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample, or a urine sample.

[0022] In some embodiments, the human subject has or is suspected of having cancer.

[0023] In some embodiments, the one or more methylation markers are selected from one of the following groups: FAIM2, CDO1, SIM2, CHST2_7890 , SFMBT2, PPP2R5C, ARHGEF4, TSPYL5, ZNF671, B3GALT6, FER1L4, HOXB2, BARX1, TBX1, SHOX2, EMX1, CLEC11A, HOXA1, GRIN2D, CAPN2, NDRG4, TRH, PRKCB, SHISA9, ZNF781, and ST8SIA1; GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, IFFO1, HOXA9, and HOPX; GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, S T8SIA1, HOXA1, HOXB2, BARX1, CLEC11A, ARHGEF4, IFFO1, HOXA9, OSR2, QKI, RYR2, GPRIN1, ZNF569, SHISA9, CD1D, NTRK3, VAV3, FAM59B; CDO1, GRIN2D, SHOX2, OSR2, QKI, SIM2, TRH, CAPN2, SFMBT2, CHST2_7890 , ST8SIA1, HOXA1, FER1L4, FAIM2, IFFO1, EMX1, ZNF671, PRKCB, HOXB2, BARX1, PPP2R5C and TSPYL5; ZNF671, GRIN2D, NDRG4, SHOX2, B3GALT6; and FAIM2, CHST2_7890 , ZNF671, GRIN2D, and CDO1.

[0024] In certain embodiments, a method for preparing a deoxyribonucleic acid (DNA) fraction from a biological sample useful for analyzing one or more genetic loci involved in one or more chromosomal abnormalities is provided, comprising: (a) Extracting genomic DNA from biological samples (b) producing a portion of genomic DNA extracted by the following method; (i) Processing of extracted genomic DNA; (ii) amplification of treated genomic DNA using individual primers specific for one or more of the following methylation markers: FAIM2, CDO1, SIM2; CHST2_7890 , SFMBT2, PPP2R5C, ARHGEF4, TSPYL5, ZNF671, B3GALT6, FER1L4, HOXB2, BARX1, TBX1, SHOX2, EMX1, CLEC11A, HOXA1, GRIN2D, CAPN2, NDRG4, TRH, PRKCB, SHISA9, ZNF781, ST8SIA1, IFFO1, HOXA9, HOPX, OSR2, QKI, RYR2, GPRIN1, ZNF569, CD1D, NTRK3, VAV3, and FAM59B; (c) analyzing one or more loci in the generated fraction of extracted genomic DNA by measuring the methylation level for each of the one or more methylation markers.

[0025] In some embodiments, treating the extracted genomic DNA comprises treating the extracted genomic DNA with a reagent that methylation-specifically modifies the DNA.

[0026] In some embodiments, the reagent that methylation-specifically modifies DNA is a borane reducing agent, for example, the borane reducing agent can be 2-picoline borane. In some embodiments, the reagent comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent. In some embodiments, the reagent is a bisulfite reagent, and the treatment produces bisulfite-treated DNA.

[0027] In some embodiments, the set of primers specific for one or more methylation markers is selected from the group set forth in Table 2. In some embodiments, the set of primers specific for each of the selected one or more methylation markers is capable of binding to an amplicon bounded by the primer sequences of the specific methylation marker genes set forth in Table 2, and the amplicon bounded by the primer sequences of the methylation marker genes set forth in Table 2 is at least a portion of a genetic region of a methylation marker set forth in Table 1. In some embodiments, the set of primers specific for each of the selected one or more methylation markers is a primer set that specifically binds to at least a portion of a genetic region that includes the chromosomal coordinates of a specific methylation marker set forth in Table 1.

[0028] In some embodiments, measuring the methylation level of one or more methylation markers comprises multiplex amplification.

[0029] In some embodiments, measuring the methylation level of at least one methylation marker comprises using one or more methods selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-specific DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, flap endonuclease assay, PCR-flap assay, and bisulfite genomic sequencing PCR.

[0030] In some embodiments, measuring the methylation level of one or more methylation markers comprises measuring the methylation of a CpG site for the one or more methylation markers. In some embodiments, the CpG site is in a coding region or a regulatory region.

[0031] In some embodiments, the one or more methylation markers are described by the genomic coordinates shown in Table 1.

[0032] In some embodiments, the biological sample is a fecal sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample, or a urine sample.

[0033] In some embodiments, the biological sample is from a human subject, hi some embodiments, the human subject has or is suspected of having cancer.

[0034] In some embodiments, the one or more methylation markers are selected from one of the following groups: FAIM2, CDO1, SIM2, CHST2_7890 , SFMBT2, PPP2R5C, ARHGEF4, TSPYL5, ZNF671, B3GALT6, FER1L4, HOXB2, BARX1, TBX1, SHOX2, EMX1, CLEC11A, HOXA1, GRIN2D, CAPN2, NDRG4, TRH, PRKCB, SHISA9, ZNF781, and ST8SIA1; GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, IFFO1, HOXA9, and HOPX; GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, S T8SIA1, HOXA1, HOXB2, BARX1, CLEC11A, ARHGEF4, IFFO1, HOXA9, OSR2, QKI, RYR2, GPRIN1, ZNF569, SHISA9, CD1D, NTRK3, VAV3, FAM59B; CDO1, GRIN2D, SHOX2, OSR2, QKI, SIM2, TRH, CAPN2, SFMBT2, CHST2_7890 , ST8SIA1, HOXA1, FER1L4, FAIM2, IFFO1, EMX1, ZNF671, PRKCB, HOXB2, BARX1, PPP2R5C and TSPYL5; ZNF671, GRIN2D, NDRG4 , SHOX2, B3GALT6; and FAIM2, CHST2_7890 , ZNF671, GRIN2D, and CDO1.

[0035] In some embodiments, each of the one or more analyzed loci is associated with any type of cancer. In some embodiments, each of the one or more analyzed loci is associated with two or more cancers. In some embodiments, each of the one or more analyzed loci is associated with one or more of liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer, and uterine cancer.

[0036] In certain embodiments, the technology relates to assessing the presence and methylation status of one or more of the methylation markers described herein in a biological sample (e.g., a stool sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample, or a urine sample). These methylation markers include one or more differentially methylated regions (DMRs) as discussed herein, e.g., as provided in Table 1 and FIG. 1. The methylation status is assessed in embodiments of the technology. Thus, the technology provided herein is not limited to a method of measuring the methylation status of a gene, and thus, the methylation status of a gene may be measured by any method known in the art.

[0037] In some embodiments, the plurality of different target regions comprises a reference target region, and in certain preferred embodiments, the reference target region comprises β-actin and / or ZDHHC1, and / or B3GALT6.

[0038] Compositions and kits for carrying out any of the methods described herein are also provided. For example, in some embodiments, reagents (e.g., primers, probes) specific for one or more methylation markers and / or proteins are provided, either alone or in sets (e.g., a set of primer pairs for amplifying multiple markers). Additional reagents for carrying out detection assays (e.g., QuARTS, PCR, enzymes for sequencing, buffers, positive and negative controls, bisulfite, TET (Ten-Eleven Translocation) enzymes (e.g., human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria TET (NgTET), Coprinopsis cinerea (CcTET), or variants thereof), borane reducing agents, or other assays) may also be provided. In some embodiments, the kits include reagents capable of methylation-specifically modifying DNA (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents) (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, TET (Ten-Eleven Translocation) enzymes (e.g., human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria TET (NgTET), Coprinopsis cinerea (CcTET)), or variants thereof), borane reducing agents), and / or agents capable of detecting expression or activity levels of protein markers described herein. In some embodiments, kits are provided that include one or more reagents necessary, sufficient, or useful for carrying out the methods. Reaction mixtures containing the reagents are also provided. Further provided are master mix reagent sets that contain multiple reagents that can be added to each other and / or to a test sample to complete a reaction mixture. In some embodiments, the kit includes a control nucleic acid that includes one or more sequences from DMR1-38 (from Table 1) and has a methylation status associated with a subject having a particular type of cancer.In some embodiments, the kit comprises a sample collector for obtaining a sample from a subject (e.g., a stool sample; a tissue sample; a plasma sample, a serum sample, a whole blood sample). In some embodiments, the kit comprises an oligonucleotide as described herein.

[0039] Methods and materials are provided herein for detecting the presence of one or more methylation markers and / or the presence of aneuploidy in a biological sample obtained from a subject. In some embodiments, the presence of one or more methylation markers and / or the presence of aneuploidy are tested simultaneously (e.g., in one test procedure, including embodiments where the test procedure itself can include multiple individual test methods of the system). In some embodiments, the presence of one or more methylation markers and / or the presence of aneuploidy of one or more classes of biomarkers are tested sequentially (e.g., in two or more different test procedures performed at two or more different time points, including embodiments where the test procedure itself can include multiple individual test methods of the system). In some embodiments of both simultaneous and sequential testing for the presence of one or more methylation markers and / or the presence of aneuploidy, the testing may be performed on a single sample or on two or more different samples (e.g., two or more different samples obtained from the same subject).

[0040] [Brief description of the drawings] [Figure 1] Information on the marker chromosomal regions and associated primers and probes used for the various methylated DNA markers listed in Table 1. The naturally occurring sequence (WT) and bisulfite modified sequence (BST) of the PCR target region are shown. [Figure 2] Three proteins (CEA, CA125, CA19-9) and five MDMs (ZNF671, GRIN2D, NDRG4 , SHOX2, B3GALT6) resulted in an overall sensitivity of 87% for all cancers with an area under the receiver operating characteristic curve (AUC) of 0.95 and a specificity of 95%. [Figure 3] Combinations of five MDMs (FAIM2, CHST2_7890, ZNF671, GRIN2D, CDO1) yielded an overall sensitivity of 74% for all cancers with a specificity of 94%. [Figure 4] The combination of four proteins (CEA, CA125, CA19.9, AFP) yielded an overall sensitivity of 62% for all cancers with a specificity of 96%.

[0041] [Mode for carrying out the invention] definition To facilitate understanding of the present technology, several terms and phrases are defined below. Further definitions are set forth throughout the detailed description.

[0042] Throughout this specification and the claims, the following terms have the meanings expressly associated therewith, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used herein may, but does not necessarily, refer to the same embodiment. Additionally, the phrase "in another embodiment" as used herein may, but does not necessarily refer to a different embodiment. Thus, as described below, various embodiments of the invention can be readily combined without departing from the scope or spirit of the invention.

[0043] Additionally, as used herein, the term "or" is an inclusive "or" operator and is equivalent to the term "and / or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for based on additional unlisted factors, unless the context clearly dictates otherwise. Additionally, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0044] The transitional phrase "consisting essentially of," when used in the claims of this application, limits the scope of the claim to certain substances or steps "and which do not materially affect the basic and novel characteristic(s)" of the claimed invention, as stated in In re Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976). For example, a composition "consisting essentially of" recited elements may contain unrecited contaminants at levels that are present but do not alter the function of the recited composition when compared to a pure composition, i.e., a composition "consisting of" the recited ingredients.

[0045] The term "one or more" as used herein refers to a number greater than 1. For example, the term "one or more" encompasses any of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, 50 or more, 100 or more, or even greater numbers.

[0046] The terms "one or more and less than a greater number," "two or more and less than a greater number," "three or more and less than a greater number," "four or more and less than a greater number," "five or more and less than a greater number," "six or more and less than a greater number," "seven or more and less than a greater number," "eight or more and less than a greater number," "nine or more and less than a greater number," "ten or more and less than a greater number," "eleven or more and less than a greater number," "twelve or more and less than a greater number," "thirteen or more and less than a greater number," "fourteen or more and less than a greater number," or "fifteen or more and less than a greater number" are not limited to the greater number. For example, the greater number may be 10,000, 1,000, 100, 50, etc. For example, the larger number can be about 50 (e.g., 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 32, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2).

[0047] The terms "one or more methylation markers" or "one or more DMRs" or "one or more genes" or "one or more markers" or "multiple methylation markers" or "multiple markers" or "multiple genes" or "multiple DMRs" are likewise not limited to specific numerical combinations. Indeed, any numerical combination of methylation markers is contemplated (e.g., 1-2 methylation markers, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 1-27, 1-28, 1-29, 1-30, 1-31, 1-32, 1-33, 1-34, 1-35, 1-36, 1-37, 1-38, 1-39, 1-40, 1-41, 1-42, 1-43, 1-44, 1-45, 1-46, 1-47, 1-48, 1-49, 1-50, 1-51, 1-52, 1-53, 1-54, 1-55, 1-56, 1-57, 1-58, 1-59, 1-60, 1-61, 1-62, 1-63, 1-64, 1-65, 1-66, 1-67, 1-68, 1-69, 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80, 1-81, 1-82, 1-83 38) (e.g., 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-21, 2-22, 2-23, 2-24, 2-25, 2-26, 2-27, 2-28, 2-29, 2-30, 2-31, 2-32, 2-33, 2-34, 2-35, 2-36, 2-37, 2-38) (e.g., 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3- 11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 3-21, 3-22, 3-23, 3-24, 3-25, 3-26, 3-27, 3-28, 3-29, 3-30, 3-31, 3-32, 3-33, 3-34, 3-35, 3-36, 3-37, 3-38) (e.g., 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 4-20 , 4-21, 4-22, 4-23, 4-24, 4-25, 4-26, 4-27, 4-28, 4-29, 4-30, 4-31, 4-32, 4-33, 4-34, 4-35, 4-36, 4-37, 4-38) (e.g., 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 5-21, 5-22, 5-23, 5-24, 5-25, 5-26, 5-27, 5-28, 5-29, 5-30,5-31, 5-32, 5-33, 5-34, 5-35, 5-36, 5-37, 5-38) (e.g., 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 6-20, 6-21, 6-22, 6- 23, 6-24, 6-25, 6-26, 6-27, 6-28, 6-29, 6-30, 6-31, 6-32, 6-33, 6-34, 6-35, 6-36, 6-37, 6-38) (e.g., 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 7-14, 7-15, 7-16 , 7-17, 7-18, 7-19, 7-20, 7-21, 7-22, 7-23, 7-24, 7-25, 7-26, 7-27, 7-28, 7-29, 7-30, 7-31, 7-32, 7-33, 7-34, 7-35, 7-36, 7-37, 7-38) (e.g., 8-9, 8-10, 8~11, 8~12, 8~13, 8~14, 8~15, 8~16, 8~17, 8~18, 8~19, 8~20, 8~21, 8~22, 8~23, 8~24, 8~25, 8~26, 8~27, 8~28, 8~29, 8~30, 8~31, 8~32, 8~33, 8~34, 8~35, 8-36, 8-37, 8-38) (e.g., 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29, 9-30 , 9-31, 9-32, 9-33, 9-34, 9-35, 9-36, 9-37, 9-38) (e.g., 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-21, 10-22, 10-23, 1 0~24, 10~25, 10~26, 10~27, 10~28, 10~29, 10~30, 10~31, 10~32, 10~33, 10~34, 10~35, 10~36, 10~37, 10~38) (e.g. 11~12, 11~13, 11~14, 11~15, 11~16, 1 1~17, 11~18, 11~19, 11~20, 11~21, 11~22, 11~23, 11~24, 11~25, 11~26, 11~27, 11~28, 11~29, 11~30, 11~31, 11~32, 11~33, 11~34, 11~35, 11~36, 11~37,11-38) (e.g., 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 12-31, 12-32, 12-33, 12-34, 12-35, 12-36, 12-37, 12-38) (e.g., 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23, 13-24, 13-25, 13-26, 13-27, 13-28, 13-29, 13-30, 13-31, 13-32, 13-33, 13-34, 13-35, 13-36, 13-37, 13-38) (e.g., 14-15, 14-16, 14-17, 14-18, 14-19, 14-20, 14-21, 14-22, 14-23, 14-24, 14-25, 14-26, 14-27, 14-28, 14-29, 14-30, 14-31, 14-32, 14-33, 14-34, 14-35, 14-36, 14-37, 14-38) (e.g., 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 15-31, 15-32, 15-33, 15-34, 15-35, 15-36, 15-37, 15-38) (e.g., 16-17, 16~18, 16~19, 16~20, 16~21, 16~22, 16~23, 16~24, 16~25, 16~26, 16~27, 16~28, 16~29, 16~30, 16~31, 16~32, 16~33, 16~34, 16~35, 16~36, 16~37, 16~38 )(For example, 17~18, 17~19, 17~20, 17~21, 17~22, 17~23, 17~24, 17~25, 17~26, 17~27, 17~28, 17~29, 17~30, 17~31, 17~32, 17~33, 17~34, 17~35, 17~36, 17~37 , 17-38) (e.g., 18-19, 18-20, 18-21, 18-22, 18-23, 18-24, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 18-31, 18-32, 18-33, 18-34, 18-35, 18-36, 18-37,18-38) (e.g., 19-20, 19-21, 19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 19-31, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38) (e.g., 20-21, 20-22, 20-23, 20-24, 20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 20-31, 20-32, 20-33, 20-34, 20-35, 20-36, 20-37, 20-38) (e.g., 21-22, 21-23, 21-24, 21-25, 21-26, 21-27, 21-28, 21-29, 21-30, 21-31, 21-32, 21-33, 21-34, 21-35, 21-36, 21-37, 21-38) (e.g., 22-23, 22-24, 22-25, 22-26 , 22-27, 22-28, 22-29, 22-30, 22-31, 22-32, 22-33, 22-34, 22-35, 22-36, 22-37, 22-38) (e.g., 23-24, 23-25, 23-26, 23-27, 23-28, 23-29, 23-30, 23-31 , 23-32, 23-33, 23-34, 23-35, 23-36, 23-37, 23-38) (e.g., 24-25, 24-26, 24-27, 24-28, 24-29, 24-30, 24-31, 24-32, 24-33, 24-34, 24-35, 24-36, 24-37, 24-38) (e.g., 25-26, 25-27, 25-28, 25-29, 25-30, 25-31, 25-32, 25-33, 25-34, 25-35, 25-36, 25-37, 25-38) (e.g., 26-27, 26-28, 26-29, 26-30, 26-31 , 26-32, 26-33, 26-34, 26-35, 26-36, 26-37, 26-38) (e.g., 27-28, 27-29, 27-30, 27-31, 27-32, 27-33, 27-34, 27-35, 27-36, 27-37, 27-38) (e.g., 28-29, 28-30, 28-31, 28-32, 28-33, 28-34, 28-35, 28-36, 28-37, 28-38) (e.g., 29-30, 29-31, 29-32, 29-33, 29-34, 29-35, 29-36, 29-37, 29-38) (e.g., 30-31,30-32, 30-33, 30-34, 30-35, 30-36, 30-37, 30-38) (e.g., 31-32, 31-33, 31-34, 31-35, 31-36, 31-37, 31-38) (e.g., 32-33, 32-34, 32-35, 32-36, 32-37, 32-38) (e.g., 33-34, 33-35, 33-36, 33-37, 33-38) (e.g., 34-35, 34-36, 34-37, 34-38) (e.g., 35-36, 35-37, 35-3 8) (e.g., 36-37, 36-38) (e.g., 37-38) (e.g., 38 or less; 37 or less; 36 or less; 35 or less; 34 or less; 33 or less; 32 or less; 31 or less; 30 or less; 29 or less; 28 or less; 27 or less; 26 or less; 25 or less; 24 or less; 23 or less; 22 or less; 21 or less; 20 or less; 19 or less; 18 or less; 17 or less; 16 or less; 15 or less; 14 or less; 13 or less; 12 or less; 11 or less; 10 or less; 9 or less; 8 or less; 7 or less; 6 or less; 5 or less; 4 or less; 3 or less; 2 or 1). ,

[0048] The term "one or more protein markers" is likewise not limited to a particular numerical combination. Indeed, any numerical combination of protein markers is contemplated (e.g., 1-2, 1-3, 1-4, 1-5 protein markers) (e.g., 2-3, 2-4, 2-5) (e.g., 3-4, 3-5) (e.g., 4-5) (e.g., 5 or less; 4 or less; 3 or less; 2 or 1).

[0049] The terms "multiple types of cancer" or "one or more types of cancer" or "multiple different types of cancer" are similarly not limited to a particular combination of numbers. Indeed, any type of numerical combination of cancer (e.g., liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, kidney cancer, and uterine cancer) is contemplated (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13 types of cancer) (e.g., 13 or less; 12 or less; 11 or less; 10 or less; 9 or less; 8 or less; 7 or less; 6 or less; 5 or less; 4 or less; 3 or less; 2 or 1).

[0050] As used herein, "nucleic acid" or "nucleic acid molecule" generally refers to any ribonucleic acid or deoxyribonucleic acid, which may be unmodified or modified DNA or RNA. "Nucleic acid" includes, but is not limited to, single-stranded and double-stranded nucleic acids. As used herein, the term "nucleic acid" also includes DNA as described above that contains one or more modified bases. Thus, DNA with a backbone modified for stability or other reasons is a "nucleic acid". As used herein, the term "nucleic acid" encompasses chemically, enzymatically, or metabolically modified forms of nucleic acid, as well as chemical forms of DNA characteristic of, for example, viruses and cells, including single cells and complex cells.

[0051] The term "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refers to a molecule that contains two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be produced by any technique, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine, and guanine.

[0052] As used herein, the term "locus" or "region" of a nucleic acid refers to a small region of a nucleic acid, e.g., a gene on a chromosome, a single nucleotide, a CpG island, and the like.

[0053] The terms "complementary" and "complementarity" refer to nucleotides (e.g., a single nucleotide) or polynucleotides (e.g., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence 5'-AGT-3' is complementary to the sequence 3'-TCA-5'. Complementarity may be "partial," in which case only a portion of the nucleic acid bases match according to the base-pairing rules. Alternatively, there may be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between strands of nucleic acids affects the efficiency and strength of hybridization between strands of nucleic acids. This is particularly important in amplification reactions and in detection methods that depend on binding between nucleic acids.

[0054] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA or of a polypeptide or its precursor. A functional polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so long as the desired activity or functional property of the polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, etc.) is retained. When used in reference to a gene, the term "portion" refers to a fragment of that gene. These fragments can range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, a "nucleotide sequence comprising at least a portion of a gene" can include a fragment of a gene or the entire gene.

[0055] The term "gene" also includes the coding region of a structural gene and includes sequences (e.g., including coding, regulatory, structural and other sequences) located adjacent to the coding region at both the 5' and 3' ends, at a distance of about 1 kb on either end, such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated or 5' untranslated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated or 3' untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. In some organisms (e.g., eukaryotes), genomic forms or clones of a gene contain coding regions interrupted by non-coding sequences, referred to as "introns" or "intervening regions" or "intervening sequences". Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA) and introns may contain regulatory elements such as enhancers. Introns are removed or "spliced ​​out" from the nuclear or primary transcript so that they are absent in the messenger RNA (mRNA) transcript, which functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0056] In addition to containing introns, genomic forms of a gene may also include sequences located both 5' and 3' to the sequences present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the untranslated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage, and polyadenylation.

[0057] When referring to a gene, the term "wild type" refers to a gene that has the characteristics of a gene isolated from a naturally occurring source. When referring to a gene product, the term "wild type" refers to a gene product that has the characteristics of a gene product isolated from a naturally occurring source. When used with respect to a protein, the term "wild type" refers to a protein that has the characteristics of a naturally occurring protein. When used with respect to an object, the term "naturally occurring" refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source and is present in an organism (including viruses) that has not been intentionally modified by the hand of man in a laboratory is naturally occurring. A wild type gene is often that gene or allele that is most frequently observed in a population, and is therefore arbitrarily referred to as the "normal" or "wild type" form of that gene. In contrast, when referring to a gene or gene product, the term "modified" or "mutated" refers to a gene or gene product, respectively, that exhibits modifications in sequence and / or functional properties (i.e., altered characteristics) when compared to a wild type gene or gene product. It is noted that naturally occurring mutants can be isolated, which are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

[0058] The term "allele" refers to genetic variations, including but not limited to variants and mutations, polymorphic loci, and single nucleotide polymorphic loci, frameshifts, and splice variants. Alleles may occur naturally in a population or may arise during the life of any particular individual in a population.

[0059] Thus, when used in reference to a nucleotide sequence, the terms "variant" and "mutant" refer to a nucleic acid sequence that differs by one or more nucleotides from another, normally related, nucleotide sequence. A "mutation" is a difference between two different nucleotide sequences, typically one sequence being a reference sequence.

[0060] The term "primer" refers to an oligonucleotide, whether naturally occurring from a restriction digest or synthetically produced, that can act as an initiation point for synthesis when placed under conditions that induce synthesis of a primer extension product that is complementary to a strand of a nucleic acid template (e.g., at an appropriate temperature and pH in the presence of nucleotides and an inducing agent such as DNA polymerase). The primer is preferably single-stranded to maximize the efficiency of amplification, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands and then used to prepare the extension product. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to prime the synthesis of the extension product in the presence of the inducing agent. The exact length of the primer will depend on a number of factors, including temperature, primer source, and method of use. In some embodiments, the primer pair is specific for a particular MDM (e.g., an MDM in Table 1) and specifically binds to at least a portion of the genetic region that contains the MDM (e.g., the chromosomal coordinates in Table 1).

[0061] The term "probe" refers to an oligonucleotide (e.g., a sequence of nucleotides) that is either naturally occurring, as in a purified restriction digest, or produced synthetically, recombinantly, or by PCR amplification, which can hybridize to another oligonucleotide of interest. Probes can be single-stranded or double-stranded. Probes are useful for the detection, identification, and isolation of specific gene sequences (e.g., "capture probes"). It is contemplated that any probe used in the present invention can, in some embodiments, be labeled with any "reporter molecule" and thus detectable in any detection system, including, but not limited to, enzymatic (e.g., ELISA, and enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

[0062] The term "target" as used herein refers to a nucleic acid that is to be sorted out from other nucleic acids, for example by probe binding, amplification, isolation, capture, etc. For example, when used in reference to the polymerase chain reaction, "target" refers to the region of nucleic acid bound by the primers used for the polymerase chain reaction, whereas when used in assays in which the target DNA is not amplified, for example, in some embodiments of an invasion cleavage assay, the target includes the site where the probe and the invading oligonucleotide (e.g., INVADER oligonucleotide) bind to form an invasion cleavage structure, which allows the presence of the target nucleic acid to be detected. A "segment" is defined as a region of nucleic acid within the target sequence.

[0063] Thus, as used herein, "non-target", when used to describe a nucleic acid such as, for example, DNA, refers to a nucleic acid that may be present in a reaction but is not the subject of detection or characterization by the reaction. In some embodiments, a non-target nucleic acid may refer to a nucleic acid present in a sample, e.g., that does not contain a target sequence, while in some embodiments, a non-target may refer to an exogenous nucleic acid, i.e., a nucleic acid that is not derived from a sample that contains or is suspected of containing a target nucleic acid, and that is added to a reaction, e.g., to normalize the activity of an enzyme (e.g., a polymerase) to reduce variation in the performance of the enzyme during the reaction.

[0064] As used herein, "methylation" refers to cytosine methylation at the C5 or N4 positions of cytosine, the N6 position of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is usually unmethylated because typical in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA in which the original template was unmethylated or methylated, respectively.

[0065] As used herein, the term "amplification reagents" refers to the reagents necessary for amplification, excluding primers, nucleic acid template, and amplification enzymes (deoxyribonucleoside triphosphates, buffers, etc.) Typically, amplification reagents are placed and contained in a reaction vessel along with other reaction components.

[0066] As used herein, the term "control" when used in reference to nucleic acid detection or analysis refers to a nucleic acid with known characteristics (e.g., known sequence, known copy number per cell) for use in comparison to an experimental target (e.g., a nucleic acid of unknown concentration). A control can be an endogenous, preferably invariant, gene that can normalize a test or target nucleic acid in an assay. Such normalization controls for possible sample-to-sample variations, e.g., in sample processing, assay efficiency, etc., allow for accurate sample-to-sample data comparisons. Genes that find use to normalize nucleic acid detection assays to human samples include, for example, β-actin, ZDHHC1, and B3GALT6 (see, e.g., U.S. Patent Application Nos. 14 / 966,617 and 62 / 364,082, each of which is incorporated herein by reference). As used herein, "ZDHHC1" refers to a gene located in human DNA on Chr16 (16q22.1) and encoding a protein characterized as zinc finger, DHHC-type containing 1, which belongs to the DHHC palmitoyltransferase family.

[0067] The control may be external. For example, in quantitative assays such as qPCR, QuARTS, etc., a "calibrator" or "calibration control" is a nucleic acid of known sequence, e.g., having the same sequence as a portion of the experimental target nucleic acid, and a known concentration or series of concentrations (e.g., serial dilution control target to generate curved calibration in quantitative PCR). Typically, the calibration control is analyzed using the same reagents and reaction conditions as used for the experimental DNA. In certain embodiments, the measurement of the calibrator is performed simultaneously with the experimental assay, e.g., in the same thermal cycler. In preferred embodiments, multiple calibrators may be included in a single plasmid, such that different calibrator sequences are easily provided in equimolar amounts. In particularly preferred embodiments, the plasmid calibrator is digested, e.g., with one or more restriction enzymes to release the calibrator portion from the plasmid vector. See, e.g., WO 2015 / 066695, which is incorporated herein by reference.

[0068] As used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, which does not exist in recognized typical nucleotide bases.For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at the 5th position of its pyrimidine ring.Thus, cytosine is not a methylated nucleotide, but 5-methylcytosine is a methylated nucleotide.In another example, thymine contains a methyl moiety at the 5th position of its pyrimidine ring, but since thymine is a typical nucleotide base of DNA, for the purposes of this specification, thymine is not considered to be a methylated nucleotide when it exists in DNA.

[0069] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

[0070] As used herein, the "methylation status," "methylation profile," and "methylation state" of a nucleic acid molecule refer to the presence or absence of one or more methylated nucleotide bases in a nucleic acid molecule. For example, a nucleic acid molecule that contains a methylated cytosine is considered to be methylated (e.g., the methylation state of the nucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered to be unmethylated.

[0071] As used herein, the term "methylation level" as applied to a methylation marker refers to the amount of methylation within a particular methylation marker. Methylation level may also refer to the amount of methylation within a particular methylation marker compared to an established standard or control. Methylation level may also refer to whether one or more cytosine residues present in a CpG context have a methylation group. Methylation level may also refer to the fraction of cells in a sample that have or do not have a methylation group on such cytosine. Alternatively, methylation level may describe whether a single CpG dinucleotide is methylated.

[0072] The methylation state of a particular nucleic acid sequence (e.g., a genetic marker, or a DNA region, as described herein) can indicate the methylation state of all bases in the sequence, or it can indicate the methylation state of a subset of these bases (e.g., one or more cytosines) within the sequence, or it can indicate information about the methylation density of a region within the sequence, with or without providing precise information of the position within the sequence where methylation occurs.

[0073] The methylation state of a nucleotide locus in a nucleic acid molecule refers to the presence or absence of a methylated nucleotide at a particular locus in a nucleic acid molecule.For example, the methylation state of a cytosine at the 7th nucleotide in a nucleic acid molecule is methylated if the nucleotide present at the 7th nucleotide in the nucleic acid molecule is 5-methylcytosine.Similarly, the methylation state of a cytosine at the 7th nucleotide in a nucleic acid molecule is unmethylated if the nucleotide present at the 7th nucleotide in the nucleic acid molecule is cytosine (and is not 5-methylcytosine).

[0074] The methylation state can be optionally expressed or indicated by a "methylation value" (e.g., methylation frequency, fraction, proportion, percent, etc.). Methylation values ​​can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing amplification profiles after a bisulfite reaction, or by comparing sequences of bisulfite-treated and untreated nucleic acids, or by comparing TET-treated and untreated nucleic acids. Thus, a value, e.g., a methylation value, represents a methylation state and can thus be used as a quantitative indicator of methylation state across multiple copies of a locus. This is of particular use when it is desirable to compare the methylation state of sequences in a sample to a threshold or reference value.

[0075] As used herein, "methylation frequency" or "percent (%) methylation" refers to the number of instances where a molecule or locus is methylated compared to the number of instances where the molecule or locus is unmethylated.

[0076] The term "methylation score" as used herein refers to a score that indicates the detected methylation events in a marker or panel of markers compared to the median methylation events for the marker or panel of markers from a random population of mammals that do not have the particular neoplasm of interest (e.g., a random population of 10, 20, 30, 40, 50, 100 or 500 mammals). The elevated methylation score in a marker or panel of markers can be any score, as long as the score is greater than the corresponding reference score. For example, the elevated methylation score in a marker or panel of markers can be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more greater than the reference methylation score.

[0077] Thus, a methylation state describes the state of methylation of a nucleic acid (e.g., a genomic sequence). Additionally, a methylation state refers to characteristics of a nucleic acid segment at a particular genomic locus that are related to methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues in this DNA sequence are methylated, the location of the methylated C residue(s), the frequency or percentage of methylated C across any particular region of the nucleic acid, and allelic differences in methylation due to, for example, differences in allelic origin. The terms "methylation status", "methylation profile", and "methylation state" also refer to the relative concentration, absolute concentration, or pattern of methylated C or unmethylated C across any particular region of a nucleic acid in a biological sample. For example, if a cytosine (C) residue(s) in a nucleic acid sequence is methylated, it may be referred to as having "hypermethylation" or "increased methylation", whereas if a cytosine (C) residue(s) in a DNA sequence is unmethylated, it may be referred to as having "hypomethylation" or "decreased methylation". Similarly, if a cytosine (C) residue(s) in a nucleic acid sequence is methylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual), the sequence is considered to have hypermethylated or increased methylation compared to the other nucleic acid sequence. Alternatively, if a cytosine (C) residue(s) in a DNA sequence is unmethylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual), the sequence is considered to have hypomethylated or decreased methylation compared to the other nucleic acid sequence. In addition, as used herein, the term "methylation pattern" refers to the collection of sites of methylated and unmethylated nucleotides across a nucleic acid region. Two nucleic acids may have the same or similar methylation frequency or percentage, but different methylation patterns, if the number of methylated and unmethylated nucleotides is the same or similar across the region, but the positions of the methylated and unmethylated nucleotides are different.Sequences are referred to as "differentially methylated" or as having "differences in methylation" or as having "differential methylation states" when they differ in the degree (e.g., one has increased or decreased methylation compared to the other), frequency, or pattern of methylation. The term "differential methylation" refers to the difference in the level or pattern of nucleic acid methylation in a cancer-positive sample compared to the level or pattern of nucleic acid methylation in a cancer-negative sample. It may also refer to the difference in the level or pattern between patients whose cancer has recurred after surgery and those whose cancer has not recurred. Differential methylation and specific levels or patterns of DNA methylation are prognostic and predictive biomarkers, for example, once precise cutoffs or predictive characteristics have been defined.

[0078] Methylation state frequencies can be used to describe a population of individuals or a sample derived from a single individual. For example, a nucleotide locus with a methylation state frequency of 50% is methylated in 50% of cases and unmethylated in 50% of cases. Such frequencies can be used, for example, to describe the extent to which a nucleotide locus or nucleic acid region is methylated in a population of individuals or a collection of nucleic acids. Thus, if the methylation in a first population or pool of nucleic acid molecules is different from the methylation in a second population or pool of nucleic acid molecules, the methylation state frequency of the first population or pool will be different from the methylation state frequency of the second population or pool. Such frequencies can also be used, for example, to describe the extent to which a nucleotide locus or nucleic acid region is methylated in a single individual. For example, such frequencies can be used to describe the extent to which a group of cells from a tissue sample is methylated or unmethylated at a nucleotide locus or nucleic acid region.

[0079] Typically, methylation of human DNA occurs on dinucleotide sequences containing adjacent guanines and cytosines, where the cytosine is located 5' to the guanine (also referred to as CpG dinucleotide sequences). Many of the cytosines within CpG dinucleotides are methylated in the human genome, but some remain unmethylated in specific CpG dinucleotide-rich genomic regions known as CpG islands (see, e.g., Antequera et al. (1990) Cell 62:503-514).

[0080] As used herein, "CpG island" or "cytosine-phosphate-guanine island" refers to a G:C-rich region of genomic DNA that contains an increased number of CpG dinucleotides compared to the total genomic DNA. A CpG island can be at least 100, 200, or more base pairs long, where the G:C content of the region is at least 50% and the ratio of observed CpG frequency to expected frequency is 0.6, and in some cases, a CpG island can be at least 500 base pairs long, where the G:C content of the region is at least 55% and the ratio of observed CpG frequency to expected frequency is 0.65. The observed CpG frequency to expected frequency can be calculated according to the method provided in Gardiner-Garden et al (1987) J.Mol.Biol.196:261-281. For example, the observed CpG frequency to the expected frequency can be calculated according to the formula R=(A×B) / (C×D), where R is the ratio of the observed CpG frequency to the expected frequency, A is the number of CpG dinucleotides in the analyzed sequence, B is the total number of nucleotides in the analyzed sequence, C is the total number of C nucleotides in the analyzed sequence, and D is the total number of G nucleotides in the analyzed sequence. Methylation status is usually determined in CpG islands, e.g., in promoter regions. However, it will be appreciated that other sequences in the human genome are prone to DNA methylation, such as CpA and CpT (see Ramsahoye (2000) Proc. Natl. Acad. Sci. USA 97:5237-5242; Salmon and Kaye (1970) Biochim. Biophys. Acta. 204:340-351; Grafstrom (1985) Nucleic Acids Res. 13:2827-2842; Nyce (1986) Nucleic Acids Res. 14:4353-4367; Woodcock (1987) Biochem. Biophys. Res. Commun. 145:888-894).

[0081] As used herein, a "methylation specific reagent" refers to a reagent that modifies the nucleotides of a nucleic acid molecule as a function of the methylation status of the nucleic acid molecule, or a compound or composition or other agent that is capable of altering the nucleotide sequence of a nucleic acid molecule in a manner that reflects the methylation state of the nucleic acid molecule. Methods of treating a nucleic acid molecule with such reagents can comprise contacting the nucleic acid molecule with the reagent, and can be optionally combined with additional steps that achieve the desired change in the nucleotide sequence. Such methods can be applied in a manner in which unmethylated nucleotides (e.g., each unmethylated cytosine) are modified into a different nucleotide. For example, in some embodiments, such reagents can deaminate unmethylated cytosine nucleotides to generate deoxyuracil residues. Examples of such reagents include, but are not limited to, methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, bisulfite reagents, TET enzymes, and borane reducing agents.

[0082] Alteration of a nucleic acid nucleotide sequence with a methylation specific reagent can also result in a nucleic acid molecule in which each methylated nucleotide is modified to a different nucleotide.

[0083] The term "methylation assay" refers to any assay for determining the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence. The term "MS AP-PCR" (Methylation-sensitive Arbitrarily Primed Polymerase Chain Reaction) refers to an art-recognized technique that allows a global scan of the genome using CG-rich primers to focus on regions most likely to contain CpG dinucleotides, and is described in Gonzalgo et al. (1997) Cancer Research 57:594-599.

[0084] The term "MethyLight™" refers to the art-recognized fluorescence-based real-time PCR technology described by Eads et al. (1999) Cancer Res. 59:2302-2306.

[0085] The term "HeavyMethyl™" refers to an assay in which methylation-specific blocking probes (also referred to herein as blockers) covering the CpG positions between or covered by the amplification primers enable methylation-specific selective amplification of a nucleic acid sample. The term "HeavyMethyl™ MethyLight™" assay refers to the HeavyMethyl™ MethyLight™ assay, which is a variation of the MethyLight™ assay in which the MethyLight™ assay is combined with a methylation-specific blocking probe that covers the CpG positions between the amplification primers.

[0086] The term "Ms-SNuPE" (Methylation-Sensitive Single Nucleotide Primer Extension) refers to the art-recognized assay described by Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-2531.

[0087] The term "MSP" (methylation specific PCR) refers to the art-recognized methylation assay described by Herman et al. (1996) Proc. Natl. Acad. Sci. USA 93:9821-9826 and by U.S. Patent No. 5,786,146.

[0088] The term "COBRA" (Combined Bisulfite Restriction Analysis) refers to an art-recognized methylation assay described in Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534.

[0089] The term "MCA" (Methylated CpG Island Amplification) refers to the methylation assay described by Toyota et al. (1999) Cancer Res. 59:2307-12 and in WO00 / 26401A1.

[0090] As used herein, a "selected nucleotide" refers to one of the four nucleotides commonly occurring in a nucleic acid molecule (C, G, T, and A for DNA; C, G, U, and A for RNA) and can include methylated derivatives of the commonly occurring nucleotide (e.g., if C is a selected nucleotide, then both methylated and unmethylated C are included in the meaning of the selected nucleotide), while a methylated selected nucleotide specifically refers to a methylated commonly occurring nucleotide and an unmethylated selected nucleotide specifically refers to an unmethylated commonly occurring nucleotide.

[0091] The term "methylation-specific restriction enzyme" refers to a restriction enzyme that selectively digests nucleic acids depending on the methylation state of its recognition site. In the case of a restriction enzyme that specifically cleaves when the recognition site is unmethylated or hemimethylated (methylation-sensitive enzyme), cleavage does not occur (or will occur with significantly reduced efficiency) if the recognition site is methylated on one or both strands. In the case of a restriction enzyme that specifically cleaves only when the recognition site is methylated (methylation-dependent enzyme), cleavage does not occur (or will occur with significantly reduced efficiency) if the recognition site is unmethylated. Preferably, it is a methylation-specific restriction enzyme, the recognition sequence of which includes a CG dinucleotide (e.g., a recognition sequence such as CGCG or CCCGGG). More preferred for some embodiments are restriction enzymes that do not cleave when the cytosine in this dinucleotide is methylated at the carbon atom C5.

[0092] As used herein, the "sensitivity" of a given marker (or set of markers used together) refers to the percentage of samples reporting DNA methylation values ​​above a threshold that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a positive is defined as a histologically confirmed neoplasm reporting a DNA methylation value above a threshold (e.g., a range associated with disease) and a false negative is defined as a histologically confirmed neoplasm reporting a DNA methylation value below a threshold (e.g., a range associated with non-disease). Thus, the sensitivity value reflects the probability that a DNA methylation measurement of a given marker obtained from a known diseased sample will be within the range of disease-associated measurements. As defined herein, the clinical relevance of a calculated sensitivity value represents an estimate of the probability that a given marker will detect the presence of a clinical condition when applied to a subject with that clinical condition.

[0093] As used herein, the "specificity" of a given marker (or set of markers used together) refers to the percentage of non-neoplastic samples that report DNA methylation values ​​below a threshold that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a negative is defined as a histologically confirmed non-neoplastic sample that reports a DNA methylation value below the threshold (e.g., in the range not associated with disease) and a false positive is defined as a histologically confirmed non-neoplastic sample that reports a DNA methylation value above the threshold (e.g., in the range associated with disease). Thus, the specificity value reflects the probability that a DNA methylation measurement of a given marker obtained from a known non-neoplastic sample will be within the range of non-disease associated measurements. As defined herein, the clinical relevance of a calculated specificity value represents an estimate of the probability that a given marker will detect the absence of a clinical condition when applied to patients who do not have that clinical condition.

[0094] The term "AUC" used herein is an abbreviation for "area under the curve". In particular, AUC refers to the area under the receiver operating characteristic (ROC) curve. The ROC curve is a plot of the true positive rate against the false positive rate for different possible cut points of a diagnostic test. The ROC curve shows the trade-off between sensitivity and specificity depending on the cut point selected (any increase in sensitivity will be accompanied by a decrease in specificity). The area under the ROC curve (AUC) is a measure of the accuracy of a diagnostic test (the larger the area, the better, with 1 being optimal, and a random test has a ROC curve that is located on the diagonal, with an area of ​​0.5. See: J.P.Egan. (1975) Signal Detection Theory and ROC Analysis, Academic Press, New York).

[0095] As used herein, the term "neoplasm" refers to any new and abnormal growth of tissue. Thus, a neoplasm can be a pre-malignant neoplasm or a malignant neoplasm.

[0096] The term "neoplasm-specific marker" as used herein refers to any biological material or element that can be used to indicate the presence of a neoplasm. Examples of biological materials include, but are not limited to, nucleic acids, polypeptides, carbohydrates, fatty acids, cellular components (e.g., cell membranes and mitochondria), and whole cells. In some cases, the marker is a specific nucleic acid region (e.g., a gene, an intragenic region, a specific locus, etc.). A region of a nucleic acid that is a marker may be referred to, for example, as a "marker gene," "marker region," "marker sequence," "marker locus," etc.

[0097] As used herein, the term "adenoma" refers to a benign tumor of glandular origin. These growths are benign, although over time they can progress to become malignant.

[0098] The terms "precancerous" or "preneoplastic" and their equivalents refer to any cell proliferative disorder that has undergone malignant transformation.

[0099] The "site" of a neoplasm, adenoma, cancer, etc., is the tissue, organ, cell type, anatomical region, body part, etc. in a subject in which the neoplasm, adenoma, cancer, etc. is located.

[0100] As used herein, the application of a "diagnostic" test includes the detection or identification of a disease state or condition in a subject, determining the likelihood that a subject will suffer from a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to a treatment, determining the prognosis (or likelihood of progression or regression) of a subject with a disease or condition, and determining the effectiveness of a treatment for a subject with a disease or condition. For example, diagnostics can be used to detect the presence or likelihood of a subject suffering from a neoplasm, or the likelihood that such a subject will respond successfully to a compound (e.g., a pharmaceutical, e.g., a drug) or other treatment.

[0101] The term "isolated", when used with respect to a nucleic acid, such as "isolated oligonucleotide", refers to a nucleic acid sequence that is identified and separated from at least one contaminating nucleic acid with which it is normally associated in its natural source. An isolated nucleic acid exists in a form or context that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids, such as DNA and RNA, are found in the state in which they naturally occur. Examples of non-isolated nucleic acids include a given DNA sequence (e.g., a gene) found on a host cell chromosome adjacent to adjacent genes, an RNA sequence, such as a particular mRNA sequence that encodes a particular protein, that is found in a cell as a mixture with many other mRNAs that encode many proteins, and the like. However, an isolated nucleic acid that encodes a particular protein includes, by way of example, such a nucleic acid in a cell that normally expresses that protein, where the nucleic acid is in a chromosomal location that is different from that of the natural cell, or is otherwise adjacent to a nucleic acid sequence that is different from that found in nature. An isolated nucleic acid or oligonucleotide may exist in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is used to express a protein, the oligonucleotide will at least comprise a sense or coding strand (i.e., the oligonucleotide may be single-stranded), but may also comprise both a sense and an antisense strand (i.e., the oligonucleotide may be double-stranded). The isolated nucleic acid can be combined with other nucleic acids or molecules after isolation from its natural or typical environment. For example, the isolated nucleic acid can be present in a host cell in which it is placed, for example, for heterologous expression.

[0102] The term "purified" refers to a molecule, either a nucleic acid or an amino acid sequence, that has been removed, isolated, or separated from its natural environment. Thus, an "isolated nucleic acid sequence" can be a purified nucleic acid sequence. "Substantially purified" molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated. As used herein, the term "purified" or "purify" also refers to the removal of contaminants from a sample. The removal of contaminating proteins increases the percentage of the polypeptide or nucleic acid of interest in the sample. In another example, a recombinant polypeptide is expressed in a plant, bacterial, yeast, or mammalian host cell, and the polypeptide is purified by the removal of host cell proteins, thereby increasing the percentage of the recombinant polypeptide in the sample.

[0103] The term "composition comprising" a given polynucleotide sequence or polypeptide refers broadly to any composition that contains the given polynucleotide sequence or polypeptide. Compositions can include aqueous solutions containing salts (e.g., NaCl), detergents (e.g., SDS), and other components (e.g., Denhardt's solution, milk powder, salmon sperm DNA, etc.).

[0104] The term "sample" is used in its broadest sense. In one sense, a sample can refer to an animal cell or tissue. In another sense, a sample refers to a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from plants or animals (including humans) and encompass fluids, solids, tissues, and gases. Environmental samples include environmental materials such as surface materials, soil, water, and industrial samples. These examples should not be construed as limiting the types of samples applicable to the present invention.

[0105] As used herein, a "remote sample," as used in some contexts, refers to a sample that is indirectly collected from a site that is not the source of the cells, tissues, or organs of the sample. For example, if sample material derived from the pancreas is evaluated in a fecal sample, the sample is a remote sample.

[0106] As used herein, the term "patient" or "subject" refers to an organism that is the subject of the various tests provided by the present technology. The term "subject" includes animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human. Furthermore, with respect to diagnostic methods, the preferred subject is a vertebrate subject. The preferred vertebrates are warm-blooded, and the preferred warm-blooded vertebrates are mammals. The preferred mammal is most preferably a human. As used herein, the term "subject" includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. As such, the present technology is provided for the diagnosis of mammals, such as humans, and those mammals that are economically important, such as animals raised on farms for human consumption, important because they are endangered, such as the Amur tiger, and / or animals that are socially important to humans, such as animals kept as pets or in zoos. Examples of such animals include, but are not limited to, carnivores such as cats and dogs; ruminants and / or ungulates, including pigs, hogs, and boars; pinnipeds, and horses. Thus, diagnostics and treatments of livestock, including, but not limited to, domesticated pigs, ruminants, ungulates, horses (including race horses), and the like, are further provided. The subject matter disclosed herein further includes a system for diagnosing lung cancer in a subject. The system may be provided as a commercially available kit that may be used, for example, to screen for lung cancer risk or to diagnose lung cancer in a subject from whom a biological sample has been collected. An exemplary system provided in accordance with the present technology includes assessing the methylation status of the markers described herein.

[0107] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of a reaction assay, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides in appropriate containers, enzymes, etc.) and / or supporting materials (e.g., buffers, written instructions for performing the assay, etc.) from one location to another. For example, a kit includes one or more enclosures (e.g., boxes) that contain the relevant reaction reagents and / or supporting equipment. As used herein, the term "fragmentation kit" refers to a delivery system that includes two or more separate containers, each of which contains a portion of the overall kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in the assay, while a second container contains an oligonucleotide. The term "fragmentation kit" is intended to encompass, but is not limited to, kits that contain analyte-specific reagents (ASRs) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act. Indeed, any delivery system that includes two or more separate containers, each of which contains a portion of the overall kit components, is included in the term "fragmentation kit." In contrast, a "mixed kit" refers to a delivery system that contains all components of a reaction assay in a single container (e.g., in a single box that houses each of the desired components). The term "kit" includes both subdivided and mixed kits.

[0108] As used herein, the term "information" refers to any collection of facts or data. With respect to information stored or processed using a computer system(s), including but not limited to the Internet, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term "information about a subject" refers to facts or data about a subject (e.g., a human, a plant, or an animal). The term "genomic information" refers to information about a genome, including but not limited to nucleic acid sequences, genes, methylation rates, allele frequencies, RNA expression levels, protein expression, phenotypes correlated with genotypes, and the like. "Allele frequency information" refers to facts or data about allele frequencies, including but not limited to the identity of the allele, statistical correlation between the presence of the allele and characteristics of the subject (e.g., a human subject), the presence or absence of the allele in an individual or population, the percentage of the likelihood of the allele being present in an individual with one or more particular characteristics, and the like.

[0109] Detailed Description Provided herein are techniques for screening for multiple types of cancer from a biological sample, in particular, but not exclusively, methods, compositions and related uses for simultaneously detecting the presence of multiple types of cancer (e.g., liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer, and uterine cancer) from a biological sample (e.g., a stool sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample or a urine sample).

[0110] Indeed, as described in Example I, experiments conducted during the process of identifying embodiments of the present invention included validation studies of the utility and performance of a combination panel of methylated DNA markers (MDM) and proteins for multi-cancer detection by testing an independent set of case / control samples with a panel of purified markers. Such experiments identified a set of methylated DNA markers (MDM), a set of protein markers, and a combination of MDM and protein markers for simultaneously detecting the presence of multiple types of cancer (e.g., liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer, uterine cancer) from a biological sample (e.g., a stool sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample, or a urine sample).

[0111] In certain aspects, the technology provides compositions and methods for identifying, determining and / or classifying multiple types of cancer from a biological sample (e.g., a stool sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample or a urine sample). The methods generally include 1) determining the methylation status of at least one methylation marker in a biological sample isolated from a subject and / or 2) the expression and / or activity level of at least one protein marker in the biological sample, where a change in the methylation status of the marker and / or the expression and / or activity level of the protein marker indicates the presence, class or site of a particular type of cancer. Generally, such methods are not limited to detecting the presence or absence of a particular type of cancer. In some embodiments, the types of cancer include, but are not limited to, liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer and uterine cancer.

[0112] In certain embodiments of the present technology, a method is provided that includes the following steps: 1) contacting nucleic acid (e.g., genomic DNA) in a biological sample obtained from a subject with at least one reagent or set of reagents that distinguish between methylated and unmethylated nucleotides (e.g., CpG dinucleotides) in at least one methylation marker; and / or contacting a biological sample obtained from a control with a set of reagents necessary to measure the expression and / or activity levels of one or more protein markers; and 2) detecting the presence or absence of multiple types of cancer (eg, with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0113] In certain embodiments of the present technology, a method is provided that includes the following steps: 1) Measure one or both of the following: a) the methylation level of one or more genes or methylation markers in a biological sample from a human individual by treating genomic DNA in the biological sample with a reagent that methylation-specifically modifies DNA; and b) the expression and / or activity levels of one or more protein markers; 2) amplifying the treated genomic DNA using a set of primers for one or more selected genes or methylation markers; and 3) determining the methylation level of said one or more genes or methylation markers.

[0114] In some embodiments of the present technology, a method is provided that includes steps 1-3 and / or step 4: 1) measuring the amount of one or more methylation marker genes in DNA from a biological sample; 2) measuring the amount of at least one reference marker in the DNA; 3) calculating a value of the amount of said at least one methylation marker gene measured in said DNA as a percentage of the amount of said reference marker gene measured in said DNA, said value being indicative of the amount of at least one methylation marker DNA measured in the biological sample; 4) Measuring the expression and / or activity levels of one or more protein markers in the biological sample.

[0115] In some embodiments of the present technology, a method is provided that includes steps 1-3 and / or step 4: 1) measuring the methylation level of CpG sites of one or more genes in a biological sample of a human individual by treating genomic DNA in the biological sample with bisulfite, a reagent capable of methylation-specifically modifying DNA; 2) amplifying the modified genomic DNA using a set of primers directed to one or more selected genes; 3) determining the methylation level of CpG sites in the selected gene or genes; 4) Measuring the expression and / or activity levels of one or more protein markers in the biological sample.

[0116] In certain embodiments, the present technology provides a method for characterizing a biological sample, the method comprising: (a) Measuring either or both of the following: i) determining the methylation level of CpG sites of one or more genes in a biological sample of a human individual by treating genomic DNA in the biological sample with bisulfite; amplifying the bisulfite-treated genomic DNA using a set of primers for the selected one or more genes; and determining the methylation level of the CpG sites; and ii) the expression and / or activity levels of one or more protein markers; (b) Measuring either or both of the following: i) the methylation level relative to the methylation level of the corresponding gene set in a control sample without a particular type of cancer; ii) the expression and / or activity level of one or more protein markers relative to the expression and / or activity level of a corresponding set of protein markers in a control sample not having a particular type of cancer; and (c) determining that the individual has a particular type of cancer if either or both of the following occur: i) the methylation level measured in said one or more genes is higher than the methylation level measured in each control sample; and ii) the expression and / or activity levels of one or more protein markers are higher than the expression and / or activity levels measured in a respective control sample.

[0117] In certain embodiments, the present technology provides methods that include one or both of the following: (i) measuring the methylation level of one or more genes in a biological sample by treating genomic DNA in the biological sample with bisulfite; amplifying the bisulfite-treated genomic DNA using a set of primers for the selected one or more genes; and determining the methylation level of the one or more genes; and ii) measuring the expression and / or activity levels of one or more protein markers.

[0118] In certain embodiments, the subject technology provides a method of screening for one or more types of cancer in a sample obtained from a subject, the method comprising: 1) One or both of the following: i) assaying the methylation status of one or more DNA methylation markers; and ii) measuring the expression and / or activity levels of one or more protein markers; and 2) Identifying a subject as having one or more types of cancer if: i) the methylation status of said marker differs from the methylation status of said marker assayed in a subject not having said one or more types of cancer; and / or ii) the expression and / or activity level of one or more protein markers differs from the expression and / or activity level of said protein markers assayed in subjects not having said one or more types of cancer.

[0119] In certain embodiments, the present technology provides a method comprising: i) measuring the methylation level of one or more genes in a biological sample of a human individual by treating genomic DNA in the biological sample with a reagent that methylation-specifically modifies DNA; amplifying the treated genomic DNA using a set of primers for the selected one or more genes; and determining the methylation level of the one or more genes; and / or ii) measuring the expression and / or activity levels of one or more protein markers.

[0120] In certain embodiments, the present technology provides a method for characterizing a biological sample, the method comprising: a) measuring the amount of at least one methylation marker gene in DNA extracted from said biological sample; treating genomic DNA in the biological sample with bisulfite; amplifying the bisulfite-treated genomic DNA with primers specific for a CpG site of each marker gene, wherein the primers specific for each marker gene are capable of binding to an amplicon bounded by the primer sequences of the marker genes listed in Table 2, and wherein the amplicon bounded by the primer sequences of the marker genes listed in Table 2 is at least a portion of a gene region of a methylation marker gene listed in Table 1; determining the methylation level of the CpG site for one or more genes; and / or b) determining the expression and / or activity levels of one or more protein markers in the biological sample.

[0121] In certain embodiments, the present technology provides a method comprising: a) extracting genomic DNA from a biological sample of a human individual having or suspected of having cancer, thereby measuring the methylation level of one or more methylation marker genes in the DNA extracted from the biological sample; treating the extracted genomic DNA with bisulfite; amplifying the bisulfite-treated genomic DNA with primers specific for one or more genes, wherein the primers specific for one or more genes are capable of binding to at least a portion of the bisulfite salt-treated genomic DNA for the chromosomal regions of the markers listed in Table 1; and measuring the methylation level of one or more methylation marker genes; and / or b) determining the expression and / or activity levels of one or more protein markers in the biological sample.

[0122] In certain embodiments, the present technology provides a method comprising: a) extracting genomic DNA from a biological sample of a human individual having or suspected of having cancer, treating the extracted genomic DNA with bisulfite, amplifying the bisulfite-treated genomic DNA with separate primers specific for CpG sites for one or more of said methylation markers, and measuring the methylation level of said CpG sites for each of said one or more methylation markers; and / or b) determining the expression and / or activity levels of one or more protein markers in the biological sample.

[0123] In certain embodiments, the present technology provides a method for preparing a DNA fraction from a biological sample of a human individual useful for analyzing one or more genetic loci involved in one or more chromosomal abnormalities, the method comprising: (a) Extraction of genomic DNA from a biological sample from a human individual; (b) generating a fraction of extracted genomic DNA by the following method; (i) treatment of extracted genomic DNA with a reagent that specifically modifies DNA by methylation; (ii) amplification of bisulfite-treated genomic DNA using separate primers specific for one or more methylation markers; (c) analyzing one or more loci in the generated fraction of extracted genomic DNA by measuring the methylation level of CpG sites for each of one or more methylation markers.

[0124] In certain embodiments, the present technology provides a method for preparing a DNA fraction from a biological sample of a human individual useful for analyzing one or more genetic loci involved in one or more DNA fragments, the method comprising: (a) Extraction of genomic DNA from a biological sample from a human individual; (b) generating a fraction of extracted genomic DNA by the following method; (i) treatment of extracted genomic DNA with a reagent that specifically modifies DNA by methylation; (ii) amplification of bisulfite-treated genomic DNA using separate primers specific for one or more methylation markers; and (c) analyzing one or more DNA fragments in the resulting fraction of extracted genomic DNA by measuring the methylation level of CpG sites for each of one or more methylation markers.

[0125] Such methods are not limited to specific methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers.

[0126] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers comprise a base in a DMR selected from the group consisting of DMRs 1-38 provided in Table 1.

[0127] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs and / or DNA methylation markers are FAIM2, CDO1, SIM2, CHST2_7890, SFMBT2, PPP2R5C, ARHGEF4, TSPYL5, ZNF671, B3GALT6, FER1L4, HOXB2, BARX1, TBX1, SHOX2, EMX1, CLEC11A, HOXA1, GRIN2D, CAPN2, NDRG4, TRH, PRKCB, SHISA9, ZNF781, ST8SIA1, IFFO1, HOXA9, HOPX, OSR2, QKI, RYR2, GPRIN1, ZNF569, CD1D, NTRK3, VAV3, and FAM59B.

[0128] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs and / or DNA methylation markers are FAIM2, CDO1, SIM2, CHST2_7890 , SFMBT2, PPP2R5C, ARHGEF4, TSPYL5, ZNF671, B3GALT6, FER1L4, HOXB2, BARX1, TBX1, SHOX2, EMX1, CLEC11A, HOXA1, GRIN2D, CAPN2, NDRG4, TRH, PRKCB, SHISA9, ZNF781, and ST8SIA1.

[0129] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers are selected from GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, IFFO1, HOXA9, and HOPX.

[0130] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers are selected from GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, ST8SIA1, HOXA1, HOXB2, BARX1, CLEC11A, ARHGEF4, IFFO1, HOXA9, OSR2, QKI, RYR2, GPRIN1, ZNF569, SHISA9, CD1D, NTRK3, VAV3, and FAM59B.

[0131] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers include FAIM2, CHST2_7890 , ZNF671, GRIN2D, and CDO1.

[0132] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers are ZNF671, GRIN2D, NDRG4 , SHOX2, and B3GALT6.

[0133] In some embodiments, the one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers are selected from the group consisting of CDO1, GRIN2D, SHOX2, OSR2, QKI, SIM2, TRH, CAPN2, SFMBT2, CHST2_7890 , ST8SIA1, HOXA1, FER1L4, FAIM2, IFFO1, EMX1, ZNF671, PRKCB, HOXB2, BARX1, PPP2R5C, and TSPYL5.

[0134] Such methods are not limited to a particular protein marker.

[0135] In some embodiments, the one or more protein markers are selected from CEA, CA125, CA19.9, AFP, and CA-15-3.

[0136] In some embodiments, the one or more protein markers are selected from CEA, CA125, and CA19.9.

[0137] In some embodiments, the one or more protein markers are selected from CEA, CA125, CA19.9, and AFP.

[0138] Such methods are not limited to screening for any particular type of cancer.

[0139] In some embodiments, the cancer is any type of cancer. A non-limiting exemplary list of cancers relevant to the described methods includes pancreatic cancer, acute myeloid leukemia (AML), breast cancer, prostate cancer, lymphoma, skin cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head and neck cancer, breast cancer, ovarian cancer, lung cancer, small cell lung cancer, Wilms' tumor, cervical cancer, testicular cancer, bladder cancer, pancreatic cancer, gastric cancer, colon cancer, prostate cancer, genitourinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, and ovarian cancer. Cancer, adrenocortical carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma.

[0140] In some embodiments, the types of cancer include, but are not limited to, liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, and gastric cancer.

[0141] In some embodiments, the cancer is selected from liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer, and uterine cancer.

[0142] In some embodiments, the cancer is selected from liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, renal cancer, and uterine cancer.

[0143] Such methods are not limited to a particular sample or biological sample type. For example, in some embodiments, the sample or biological sample is a stool sample, a tissue sample, a blood sample (e.g., a stool sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample, or a urine sample), a feces or a urine sample. In some embodiments, the sample comprises blood, serum, plasma, gastric secretions, pancreatic juice, a cerebrospinal fluid (CSF) sample, a gastrointestinal biopsy sample, and / or cells recovered from stool. The sample or biological sample may comprise cells, secretions, or tissues from lymph nodes, breast, liver, bile duct, pancreas, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gallbladder, anus, and / or peritoneum. In some embodiments, the sample or biological sample comprises cell fluid, ascites, urine, feces, gastric secretions, pancreatic juice, fluid obtained during endoscopy, blood, mucus, or saliva.

[0144] Different cancers are predicted by different combinations of markers, for example as identified by statistical techniques for the specificity and sensitivity of prediction. This technique further provides methods for identifying predictive combinations, and validated predictive combinations, for several cancers.

[0145] Such methods are not limited to the type of subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0146] Such methods are not limited to a particular manner or technique for measuring protein expression and / or activity. Techniques for measuring protein expression and / or activity levels are known in the art. Indeed, any known technique for measuring protein expression and / or activity levels is contemplated and incorporated herein.

[0147] Such methods are not limited to a particular format or technique for determining the methylation characterization, measurement or assay for one or more methylation markers, methylation marker genes, genes, DMRs and / or DNA methylation markers. In some embodiments, such techniques are based on analysis of the methylation state (e.g., CpG methylation state) of at least one marker, marker region or marker base that comprises a DMR.

[0148] In some embodiments, measuring the methylation status of a methylation marker in a sample comprises determining the methylation status of a single base. In some embodiments, measuring the methylation status of a marker in a sample comprises determining the degree of methylation at a plurality of bases. Further, in some embodiments, the methylation status of a methylation marker comprises an increase in methylation of the marker compared to a normal methylation state of the marker. In some embodiments, the methylation status of a marker comprises a decrease in methylation of the marker compared to a normal methylation status of the marker. In some embodiments, the methylation status of a marker comprises a different pattern of methylation of the marker compared to a normal methylation status of the marker.

[0149] Further, in some embodiments the marker is a region of 100 bases or less, the marker is a region of 500 bases or less, the marker is a region of 1000 bases or less, the marker is a region of 5000 bases or less, or in some embodiments the marker is 1 base. In some embodiments the marker is in a promoter with high CpG density.

[0150] In certain embodiments, methods of analyzing nucleic acids for the presence of 5-methylcytosine include treating DNA with reagents that modify DNA in a methylation-specific manner, examples of such reagents include, but are not limited to, methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, bisulfite reagents, TET enzymes, and borane reducing agents.

[0151] A frequently used method for analyzing nucleic acids for the presence of 5-methylcytosine is based on the bisulfite method described by Frommer, et al. for the detection of 5-methylcytosine in DNA (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-31, expressly incorporated herein by reference in its entirety for all purposes) or their variations. The bisulfite method for mapping 5-methylcytosine is based on the finding that cytosine, but not 5-methylcytosine, reacts with bisulfite ions (also known as bisulfite). The reaction is usually carried out according to the following steps: first, cytosine reacts with bisulfite to form sulfonated cytosine. Then, spontaneous deamination of the sulfonated reaction intermediate results in sulfonated uracil. Finally, the sulfonated uracil is desulfonated under alkaline conditions to form uracil. Detection is possible because uracil base pairs with adenine (and thus behaves like thymine), whereas 5-methylcytosine base pairs with guanine (and thus behaves like cytosine). This allows for the discrimination of methylated from unmethylated cytosines, for example, by bisulfite genomic sequencing (Grigg G, & Clark S, Bioessays (1994) 16:431-36; Grigg G, DNA Seq. (1996) 6:189-98), methylation specific PCR (MSP) as disclosed, for example, in U.S. Pat. No. 5,786,146, or by using assays involving sequence specific probe cleavage, for example, the QuARTS flap endonuclease assay (see, for example, Zou et al. (2010) "Sensitive quantification of methylated markers with a novel methylation specific technology" Clin Chem 56:A199, and U.S. Pat. Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392).

[0152] Some prior art techniques involve encapsulating the DNA to be analyzed in an agarose matrix, thereby preventing DNA diffusion and renaturation (bisulfite reacts only with single-stranded DNA), and replacing the precipitation and purification steps by rapid dialysis (Olek A, et al. (1996) "A modified and improved method for bisulfite based cytosine methylation analysis" Nucleic Acids Res. 24:5064-6). It is therefore possible to analyze individual cells for their methylation status, demonstrating the usefulness and sensitivity of the method. An overview of prior methods for detecting 5-methylcytosine is provided by Rein, T., et al. (1998) Nucleic Acids Res. 26:2255.

[0153] Bisulfite techniques involve bisulfite treatment followed by amplification of short, specific fragments of known nucleic acids followed by assaying the products for individual cytosine positions by sequencing (Olek & Walter (1997) Nat. Genet. 17:275-6) or primer extension reactions (Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-31; WO 95 / 00669; U.S. Pat. No. 6,251,594). Some methods use enzymatic digestion (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-4). Detection by hybridization has also been described in the art (Olek et al., WO 99 / 28498). Additionally, the use of bisulfite technology for methylation detection on individual genes has been described (Grigg & Clark (1994) Bioessays 16:431-6,; Zeschnigk et al. (1997) Hum Mol Genet. 6:387-95; Feil et al. (1994) Nucleic Acids Res. 22:695; Martin et al. (1995) Gene 157:261-4; WO 9746705; WO 9515373).

[0154] Various methylation assay techniques can be used in conjunction with bisulfite treatment according to the techniques of the present invention. These assays allow for the determination of the methylation status of one or more CpG dinucleotides (e.g., CpG islands) within a nucleic acid sequence. Such assays include, among other techniques, sequencing of bisulfite-treated nucleic acids, PCR (for sequence-specific amplification), Southern blot analysis, and the use of methylation-specific restriction enzymes, such as methylation-sensitive or methylation-dependent enzymes.

[0155] For example, genome sequencing has been simplified for the analysis of methylation patterns and 5-methylcytosine distribution by using bisulfite treatment (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-1831). Furthermore, restriction enzyme digestion of PCR products amplified from bisulfite converted DNA is used in the assessment of methylation status, for example as described by Sadri & Hornsby (1997) Nucl. Acids Res. 24:5058-5059 or by the method known as COBRA (combined bisulfite restriction analysis) (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534).

[0156] The COBRA™ analysis is a quantitative methylation assay useful for determining DNA methylation levels at specific loci with small amounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997). Briefly, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA. Methylation-dependent sequence differences are first introduced into genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992). PCR amplification of the bisulfite-converted DNA is then performed using primers specific for the CpG island of interest, followed by restriction endonuclease digestion, gel electrophoresis, and detection using specifically labeled hybridization probes. Methylation levels in the original DNA samples are represented by the relative amounts of digested and undigested PCR products, providing a linear method for quantification over a wide range of DNA methylation levels. In addition, the method can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.

[0157] Exemplary reagents for COBRA™ analysis (e.g., as may be found in a typical COBRA™-based kit) may include, but are not limited to: PCR primers for specific loci (e.g., specific genes, markers, DMRs, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.); restriction enzymes and appropriate buffers; gene hybridization oligonucleotides; control hybridization oligonucleotides; kinase labeling kits for oligonucleotide probes; and labeled nucleotides. Additionally, bisulfite conversion reagents may include: DNA denaturation buffers; sulfonation buffers; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffers; and DNA recovery components.

[0158] Assays such as "MethyLight™" (fluorescence-based real-time PCR technology) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPE™ (methylation-sensitive single nucleotide primer extension) reactions (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997), methylation-specific PCR ("MSP"; H Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Patent No. 5,786,146), and methylated CpG island amplification ("MCA"; Toyota et al., Cancer Res. 59:2307-12, 1999) may be used alone or in combination with one or more of these methods.

[0159] The "HeavyMethyl™" assay, a technique, is a quantitative method to assess methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific blocking probes ("blockers") that cover the CpG positions between or covered by the amplification primers allow for methylation-specific selective amplification of nucleic acid samples.

[0160] The term "HeavyMethyl™ MethyLight™" assay refers to the HeavyMethyl™ MethyLight™ assay, which is a variation of the MethyLight™ assay in which the MethyLight™ assay is combined with a methylation-specific blocking probe that covers the CpG positions between the amplification primers. The HeavyMethyl™ assay can also be used in combination with methylation-specific amplification primers.

[0161] Typical reagents for HeavyMethyl™ analysis (e.g., as may be found in a typical MethyLight™-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, or bisulfite-treated DNA sequences or CpG islands, etc.); blocking oligonucleotides; optimized PCR buffers and deoxynucleotides; and Taq polymerase.

[0162] MSP (methylation specific PCR) allows the assessment of the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation sensitive restriction enzymes (Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996, U.S. Patent No. 5,786,146). Briefly, DNA is modified with sodium bisulfite, which converts unmethylated but not methylated cytosines to uracil, and these products are then amplified with primers specific for methylated versus unmethylated DNA. MSP requires only small amounts of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents for MSP analysis (e.g., as may be found in a typical MSP-based kit) may include, but are not limited to, methylated and unmethylated PCR primers for specific loci (e.g., specific genes, markers, gene regions, marker regions, bisulfite treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides, and specificity probes.

[0163] The MethyLight™ assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (e.g., TaqMan®) and does not require further manipulation after the PCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight™ process begins with a mixed sample of genomic DNA that is converted into a mixed pool of methylation-dependent sequence differences according to standard procedures in a sodium bisulfite reaction (the bisulfite process converts unmethylated cytosine residues to uracil). Fluorescence-based PCR is then performed in a "biased" reaction, for example, with PCR primers that overlap known CpG dinucleotides. Sequence discrimination occurs both at the level of the amplification process and at the level of the fluorescence detection process.

[0164] The MethyLight™ assay is used as a quantitative test for methylation patterns in nucleic acids, e.g., genomic DNA samples, in which sequence discrimination occurs at the level of probe hybridization. In the quantitative version, the PCR reaction results in methylation-specific amplification in the presence of fluorescent probes that overlap specific putative methylation sites. An unbiased control for input DNA amount is provided by a reaction in which neither the primers nor the probe overlap any CpG dinucleotides. Alternatively, a qualitative test for genomic methylation is performed by probing biased PCR pools with either control oligonucleotides that do not cover known methylation sites (e.g., fluorescent-based versions of HeavyMethyl™ and MSP technologies) or oligonucleotides that cover potential methylation sites.

[0165] The MethyLight™ process is used with any suitable probe (e.g., “TaqMan®” probe, Lightcycler® probe, etc.). For example, in some applications, double-stranded genomic DNA is treated with sodium bisulfite and subjected to one of two sets of PCR reactions using a TaqMan® probe, e.g., an MSP primer and / or a HeavyMethyl blocker oligonucleotide and a TaqMan® probe. The TaqMan® probe is dual-labeled with fluorescent “reporter” and “quencher” molecules and is designed to be specific for relatively high GC content regions such that the probe melts at a temperature approximately 10° C. higher than the forward or reverse primer during the PCR cycle. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing / extension step. The Taq polymerase will eventually reach the annealed TaqMan® probe to enzymatically synthesize a new strand during PCR. Taq polymerase 5' to 3' endonuclease activity then displaces the TaqMan® probe by digesting it, releasing a fluorescent reporter molecule for quantitative detection of its unquenched signal using a real-time fluorescence detection system.

[0166] Typical reagents for MethyLight™ analysis (e.g., as may be found in a typical MethyLight™-based kit) may include, but are not limited to, PCR primers directed to specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffer and deoxynucleotides; and Taq polymerase.

[0167] The QM™ (Quantitative Methylation) Assay is an alternative quantitative test for methylation patterns in genomic DNA samples, in which sequence discrimination occurs at the level of probe hybridization. In this quantitative version, the PCR reaction results in unbiased amplification in the presence of fluorescent probes that overlap specific putative methylation sites. An unbiased control for the input DNA amount is provided by a reaction in which neither the primers nor the probe overlap any CpG dinucleotides. Alternatively, a quantitative test for genomic methylation is achieved by probing a biased PCR pool with either control oligonucleotides that do not cover known methylation sites (fluorescence-based versions of HeavyMethyl™ and MSP techniques) or oligonucleotides that cover potential methylation sites.

[0168] The QM™ process can be used with any suitable probe in the amplification process, e.g., "TaqMan®" probe, Lightcycler® probe. For example, double-stranded genomic DNA is treated with sodium bisulfite and receives unbiased primers and a TaqMan® probe. The TaqMan® probe is dual-labeled with fluorescent "reporter" and "quencher" molecules and is designed to be specific for relatively high GC content regions such that the probe melts at a temperature approximately 10° C. higher than the forward or reverse primers during the PCR cycle. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing / extension step. The Taq polymerase will eventually reach the annealed TaqMan® probe to enzymatically synthesize new strands during PCR. The Taq polymerase 5' to 3' endonuclease activity then displaces the TaqMan® probe by digesting it, releasing a fluorescent reporter molecule for quantitative detection of its unquenched signal using a real-time fluorescence detection system.Typical reagents for QM™ analysis (e.g., as may be found in a typical QM™-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite treated DNA sequences, CpG islands, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase.

[0169] The Ms-SNuPE™ technique is a quantitative method that involves single-base primer extension, after evaluating the difference in methylation at specific CpG sites based on bisulfite treatment of DNA (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly, genomic DNA is reacted with sodium bisulfite, converting unmethylated cytosines to uracil, while leaving 5-methylcytosines unchanged. Amplification of the desired target sequence is then performed using PCR primers specific to the bisulfite-converted DNA, and the resulting products are isolated and used as templates for methylation analysis at the CpG sites of interest. Small amounts of DNA can be analyzed (e.g., microdissected pathology sections), which avoids the use of restriction enzymes to determine the methylation status at CpG sites.

[0170] Typical reagents for Ms-SNuPETM analysis (e.g., as may be found in a typical Ms-SNuPETM-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides; gel extraction kits; positive control primers; Ms-SNuPE™ primers for specific loci; reaction buffers (for Ms-SNuPE reactions); and labeled nucleotides. Additionally, bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery reagent or kit (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffer; and DNA recovery components.

[0171] Reduced representation bisulfite sequencing (RRBS) begins with bisulfite treatment of nucleic acids to convert all unmethylated cytosines to uracils, followed by restriction enzyme digestion (e.g., with an enzyme that recognizes sites containing CG sequences, such as MspI), and completing the sequencing of the fragments after coupling to an adaptor ligand. The choice of restriction enzyme enriches for fragments in CpG-dense regions, reducing the number of redundant sequences that may map to multiple gene locations during the analysis. As such, RRBS reduces the complexity of the nucleic acid sample by selecting a subset of restriction fragments for sequencing (e.g., by size selection using preparative gel electrophoresis). In contrast to whole genome bisulfite sequencing, all fragments generated by restriction enzyme digestion contain DNA methylation information for at least one CpG dinucleotide. As such, RRBS provides an assay to assess the methylation status of one or more genomic loci, since it enriches the sample for promoters, CpG islands, and other genomic features, including frequent restriction enzyme cleavage sites in these regions.

[0172] A typical protocol for RRBS includes the steps of digesting a nucleic acid sample with a restriction enzyme such as MspI, filling in overhangs and A-tailing, ligating adapters, bisulfite conversion, and PCR. See, e.g., Meissner et al. (2005) "Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution" Nat Methods 7:133-6; Meissner et al. (2005) "Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis" Nucleic Acids Res. 33:5868-77.

[0173] In some embodiments, a quantitative allele-specific real-time target and signal amplification (QuARTS) assay is used to evaluate the methylation status. In each QuARTS assay, three reactions occur in succession, the primary reaction includes amplification (reaction 1) and target probe cleavage (reaction 2), and the secondary reaction includes FRET cleavage and fluorescent signal generation (reaction 3). When the target nucleic acid is amplified with a specific primer, a specific detection probe with a flap sequence is loosely bound to the amplicon. The presence of a specific invasive oligonucleotide at the target binding site releases the flap sequence by cleavage between the detection probe and the flap sequence by a 5' nuclease, such as FEN-1 endonuclease. The flap sequence is complementary to the non-hairpin portion of the corresponding FRET cassette. Thus, the flap sequence functions as an invasive oligonucleotide on the FRET cassette, resulting in cleavage between the FRET cassette fluorophore and the quencher, generating a fluorescent signal. The cleavage reaction can cleave multiple probes per target, thereby releasing multiple fluorophores per flap, resulting in exponential signal amplification. QuARTS can detect multiple targets in a single reaction well by using FRET cassettes with different dyes. See, e.g., Zou et al. (2010) "Sensitive quantification of methylated markers with a novel methylation specific technology" Clin Chem 56:A 199), and U.S. Patent Nos. 8,361,720; 8,715,937; 8,916,344; and 9,212,392, each of which is incorporated herein by reference for all purposes.

[0174] The term "bisulfite reagent" refers to a reagent comprising bisulfite, disulfite, hydrogen sulfite, or a combination thereof, useful for distinguishing between methylated and unmethylated CpG dinucleotide sequences, as disclosed herein. Methods of said treatment are known in the art (e.g., PCT / EP2004 / 011715 and WO2013 / 116375, each of which is incorporated by reference in its entirety). In some embodiments, the bisulfite treatment is carried out in the presence of a denaturing solvent, such as, but not limited to, n-alkylene glycol or diethylene glycol dimethyl ether (DME), or in the presence of dioxane or a dioxane derivative. In some embodiments, the denaturing solvent is used at a concentration of 1% to 35% (v / v). In some embodiments, the bisulfite reaction is carried out in the presence of a scavenger, such as, but not limited to, a chroman derivative, e.g., 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, or trihydroxybenzoic acid and its derivatives, e.g., gallic acid (see PCT / EP2004 / 011715, which is incorporated by reference in its entirety). In certain preferred embodiments, the bisulfite reaction involves treatment with ammonium bisulfite, e.g., as described in WO 2013 / 116375.

[0175] In some embodiments, the treated DNA fragments are amplified using a set of primer oligonucleotides (see, for example, Table 2) according to the invention and an amplification enzyme. Amplification of several DNA segments can be carried out simultaneously in one and the same reaction vessel. Usually, the amplification is carried out using the polymerase chain reaction (PCR). The amplicons are usually 100 to 2000 base pairs long.

[0176] In another embodiment of the method, the methylation status of CpG positions in or near markers containing DMRs (e.g., DMRs 1-38, Table 1) can be detected using methylation-specific primer oligonucleotides. This technique (MSP) is described in U.S. Pat. No. 6,265,171 to Herman. The use of methylation status-specific primers for the amplification of bisulfite-treated DNA allows differentiation between methylated and unmethylated nucleic acids. MSP primer pairs contain at least one primer that hybridizes to bisulfite-treated CpG dinucleotides. Thus, the sequence of the primer contains at least one CpG dinucleotide. MSP primers specific for unmethylated DNA contain a "T" at the C position in the CpG.

[0177] Such methods are not limited to a particular type or kind of primer or primer pair associated with one or more methylation markers, methylation marker genes, genes, DMRs and / or DNA methylation markers. In some embodiments, the primers or primer pairs are listed in Table 2 (SEQ ID NOs: 1-126). In some embodiments, the primers or primer pairs specific for each methylation marker gene can bind to an amplicon bounded by the primer sequences of the marker genes listed in Table 2, and the amplicon bounded by the primer sequences of the marker genes listed in Table 2 is at least a portion of a gene region of a methylation marker gene listed in Table 1. In some embodiments, the primers or primer pairs of the methylation markers are primer sets that specifically bind to at least a portion of a gene region that includes the chromosomal coordinates of a particular methylation marker listed in Table 1.

[0178] In another embodiment, the invention provides a method for converting oxidized 5-methylcytosine residues in cell-free DNA to dihydrouracil residues (Liu et al., 2019, Nat Biotechnol. 37, pp. 424-429; US Patent Publication No. 202000370114), which comprises reacting oxidized 5mC residues selected from 5-formylcytosine (5fC), 5-carboxymethylcytosine (5caC), and combinations thereof, with a borane reducing agent. The oxidized 5mC residue may be naturally occurring or, more typically, may be the result of prior oxidation of a 5mC or 5hmC residue, such as oxidation of 5mC or 5hmC by a TET family enzyme (e.g., TET1, TET2, or TET3), or chemical oxidation of 5mC or 5hmC by, for example, potassium perruthenate (KRuO4) or an inorganic peroxo compound or composition, such as copper(II) peroxotungstate (e.g., Okamoto et al. (2011) Chem. Commun. 47:11231-33) and combinations of copper(II) perchlorate / 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) (see, e.g., Matsushita et al. (2017) Chem. Commun. 53:5756-59).

[0179] The borane reducing agent can be characterized as a complex of borane and a nitrogen-containing compound selected from a nitrogen heterocycle and a tertiary amine. The nitrogen heterocycle can be monocyclic, bicyclic, or polycyclic, but is typically monocyclic, in the form of a 5- or 6-membered ring containing a nitrogen heteroatom and optionally one or more additional heteroatoms selected from N, O, and S. The nitrogen heterocycle can be aromatic or alicyclic. Preferred nitrogen heterocycles herein include 2-pyrroline, 2H-pyrrole, 1H-pyrrole, pyrazolidine, imidazolidine, 2-pyrazoline, 2-imidazoline, pyrazole, imidazole, 1,2,4-triazole, 1,2,4-triazole, pyridazine, pyrimidine, pyrazine, 1,2,4-triazine, and 1,3,5-triazine, any of which may be unsubstituted or substituted with one or more non-hydrogen substituents. Typical non-hydrogen substituents are alkyl groups, particularly lower alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, etc. Exemplary compounds include pyridine borane, 2-methylpyridine borane (also called 2-picoline borane), and 5-ethyl-2-pyridine.

[0180] The reaction of borane reducing agents with oxidized 5mC residues in cell-free DNA is advantageous insofar as non-toxic reagents and mild reaction conditions can be used. Neither bisulfite nor other potential DNA-degrading reagents are required. Furthermore, the conversion of oxidized 5mC residues to dihydrouracils with borane reducing agents can be carried out in a "one-pot" or "one-tube" reaction without the need to isolate intermediates. This is very important since the conversion involves multiple steps, namely (1) reduction of the alkene bond linking C-4 and C-5 in the oxidized 5mC, (2) deamination, and (3) either decarboxylation if the oxidized 5mC is 5caC or transformation if the oxidized 5mC is 5fC.

[0181] In addition to the method of converting oxidized 5-methylcytosine residues in cell-free DNA to dihydrouracil residues, the present invention also provides a reaction mixture related to the above-mentioned method. The reaction mixture includes a sample of cell-free DNA containing at least one oxidized 5-methylcytosine residue selected from 5caC, 5fC, and combinations thereof, and a borane reducing agent effective for either reducing, deaminating, and decarboxylating or transforming at least one oxidized 5-methylcytosine residue. The borane reducing agent is a complex of borane and a nitrogen-containing compound selected from nitrogen heterocycles and tertiary amines, as described above. In a preferred embodiment, the reaction mixture is substantially free of bisulfite, i.e., substantially free of bisulfite ions and bisulfite salts. Ideally, the reaction mixture is free of bisulfite.

[0182] In a related aspect of the invention, a kit for converting 5mC residues in cell-free DNA to dihydrouracil residues is provided, the kit comprising a reagent for blocking 5hmC residues, a reagent for oxidizing 5mC residues beyond hydroxymethylation to provide oxidized 5mC residues, and a borane reducing agent effective to either reduce, deaminate, and decarboxylate or transform the oxidized 5mC residues. The kit may also include instructions for using the components to practice the above method.

[0183] In another embodiment, a method is provided that utilizes the oxidation reaction described above, which allows for detecting the presence and location of 5-methylcytosine residues in cell-free DNA, and includes the following steps: (a) modifying 5hmC residues in the fragmented, adaptor-ligated cell-free DNA to provide affinity tags thereon, which affinity tags enable removal of the modified 5hmC-containing DNA from the cell-free DNA; (b) removing the modified 5hmC-containing DNA from the cell-free DNA, leaving DNA containing unmodified 5mC residues; (c) oxidizing unmodified 5mC residues to obtain DNA containing oxidized 5mC residues selected from 5caC, 5fC, and combinations thereof; (d) contacting the DNA containing the oxidized 5mC residue with a borane reducing agent effective to reduce, deaminate, decarboxylate or modify the oxidized 5mC residue, thereby providing DNA containing a dihydrouracil residue in place of the oxidized 5mC residue; (e) amplifying and sequencing DNA containing dihydrouracil residues; (f) determining the 5-methylation pattern from the sequencing results obtained in (e).

[0184] In another embodiment, a method for identifying 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) in a target nucleic acid comprises the steps of: providing a biological sample containing a target nucleic acid; Modifying a target nucleic acid, comprising: converting 5mC and 5hmC in the nucleic acid sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC) by contacting the nucleic acid sample with a TET enzyme such that one or more 5caC or 5fC residues are generated; and converting the 5caC and / or 5fC to dihydrouracil (DHU) by treating the target nucleic acid with a borane reducing agent to provide a modified nucleic acid sample comprising a modified target nucleic acid; and A method is provided which includes detecting a sequence of a modified target nucleic acid; wherein, compared to the target nucleic acid, a cytosine (C) to thymine (T) transition or a cytosine (C) to DHU transition in the sequence of the modified target nucleic acid provides either a 5mC or a 5hmC position in the target nucleic acid.

[0185] In some embodiments, the borane reducing agent is 2-picoline borane.

[0186] In some embodiments, detecting the sequence of the modified target nucleic acid comprises one or more of chain termination sequencing, microarray, high throughput sequencing, and restriction enzyme analysis.

[0187] In some embodiments, the TET enzyme is selected from the group consisting of human TET1, TET2, and TET3; mouse Tet1, Tet2, and Tet3; Naegleria TET (NgTET); and Coprinopsis cinerea (CcTET).

[0188] In some embodiments, the method further comprises blocking one or more modified cytosines, hi some embodiments, the blocking comprises adding a sugar to 5hmC.

[0189] In some embodiments, the method further comprises amplifying the copy number of one or more nucleic acid sequences.

[0190] In some embodiments, the oxidizing agent is potassium perruthenate or Cu(II) / TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl).

[0191] Cell-free DNA is extracted from a body sample from a subject, where the body sample is typically whole blood, plasma or serum, most typically plasma, but the sample can also be urine, saliva, mucosal discharge, sputum, stool or tears. In some embodiments, the cell-free DNA is derived from a tumor. In other embodiments, the cell-free DNA is derived from a patient with a disease or other pathogenic condition. The cell-free DNA may or may not be derived from a tumor.

[0192] It should be noted that in step (a), the cell-free DNA to be modified at 5hmC residues is purified, fragmented, and adapter-ligated. DNA purification in this context can be performed using any suitable method known to those of skill in the art and / or described in the relevant literature, and although the cell-free DNA itself may be highly fragmented, further fragmentation may be desirable, for example, as described in US Patent Application Publication No. 2017 / 0253924. Cell-free DNA fragments generally range in size from about 20 nucleotides to about 500 nucleotides, more typically from about 20 nucleotides to about 250 nucleotides. The purified cell-free DNA fragments modified in step (a) are end-repaired using conventional means (e.g., restriction enzymes) so that the fragments have blunt ends at each 3' and 5' end. In a preferred method, the blunt-ended fragments are also provided with 3' overhangs containing a single adenine residue using a polymerase such as Taq polymerase, as described in WO 2017 / 176630. This facilitates the subsequent ligation of a selected universal adaptor, i.e., an adaptor such as a Y adaptor or a hairpin adaptor that ligates to both ends of the cfDNA fragments and contains at least one molecular barcode. The use of adaptors also allows for selective PCR enrichment of adaptor-ligated DNA fragments.

[0193] In step (a), the "purified, fragmented cell-free DNA" then contains adaptor-ligated DNA fragments. Modification of 5hmC residues in these cell-free DNA fragments with an affinity tag as specified in step (a) is performed to allow subsequent removal of the modified 5hmC-containing DNA from the cell-free DNA. In one embodiment, the affinity tag comprises a biotin moiety, such as biotin, desthiobiotin, oxybiotin, 2-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, etc. The use of the biotin moiety as an affinity tag allows for easy removal with streptavidin, e.g., streptavidin beads, magnetic streptavidin beads, etc.

[0194] Tagging of 5hmC residues with biotin moieties or other affinity tags is achieved by covalent attachment of a chemoselective group to the 5hmC residues in the DNA fragment, which can react with the functionalized affinity tag to link the affinity tag to the 5hmC residue. In one embodiment, the chemoselective group is UDP glucose-6-azide, which undergoes spontaneous 1,3-cycloaddition reaction with alkyne-functionalized biotin moieties, as described in Robertson et al. (2011) Biochem Biophys.Res.Comm.411(1):40-3, U.S. Patent No. 8,741,567 and WO 2017 / 176630. Thus, the addition of an alkyne-functionalized biotin moiety results in the covalent attachment of a biotin moiety to each 5hmC residue.

[0195] Then, in step (b), in one embodiment, streptavidin in the form of streptavidin beads or magnetic streptavidin beads, etc., can be used to pull down the affinity-tagged DNA fragments and, if necessary, can be saved for later analysis. The supernatant remaining after removal of the affinity-tagged fragments contains DNA with unmodified 5mC residues and no 5hmC residues.

[0196] In step (c), the unmodified 5mC residue is oxidized to obtain 5caC and / or 5fC residues using any suitable means. The oxidizing agent is selected to oxidize the 5mC residue beyond hydroxymethylation, i.e. to provide 5caC and / or 5fC residues. The oxidation can be performed enzymatically using a catalytically active TET family enzyme. "TET family enzyme" or "TET enzyme" as those terms are used herein refers to a catalytically active "TET family protein" or "TET catalytically active fragment" as defined in U.S. Pat. No. 9,115,386, the disclosure of which is incorporated herein by reference. A preferred TET enzyme in this context is TET2. See Ito et al. (2011) Science 333(6047):1300-1303. The oxidation can also be performed chemically, as described in the previous section, using a chemical oxidizing agent. Examples of suitable oxidizing agents include perruthenate anions in the form of inorganic or organic perruthenates, including, but not limited to, metal perruthenates such as potassium perruthenate (KRuO4), tetraalkylammonium perruthenates such as tetrapropylammonium perruthenate (TPAP) and tetrabutylammonium perruthenate (TBAP), and polymer-supported perruthenate (PSP); and inorganic peroxo compounds and compositions such as peroxotungstate or copper(II) perchlorate / TEMPO combinations. At this point, it is not necessary to separate the 5fC-containing fragments from the 5caC-containing fragments, as long as step (e) is the next step in the method to convert both the 5fC and 5caC residues to dihydrouracil (DHU).

[0197] In some embodiments, 5-hydroxymethylcytosine residues are blocked with β-glucosyltransferase (β3GT) and 5-methylcytosine residues are oxidized with a TET enzyme effective to provide a mixture of 5-formylcytosine and 5-carboxymethylcytosine. The mixture containing both of these oxidized species can be reacted with 2-picoline borane or another borane reducing agent to obtain dihydrouracil. In a variation of this embodiment, the 5hmC-containing fragments are not removed in step (b). Rather, in "TET-assisted picoline borane sequencing (TAPS)", the 5mC-containing fragments and the 5hmC-containing fragments are enzymatically oxidized together to provide 5fC- and 5caC-containing fragments. The reaction with 2-picoline borane results in DHU residues whenever 5mC and 5hmC residues are initially present. "Chemical Assisted Picoline Borane Sequencing (CAPS)" involves selectively oxidizing 5mC-containing fragments with potassium perruthenate, leaving the 5mC residues unchanged.

[0198] The method of this embodiment has many advantages: no bisulfite is required, non-toxic reagents and reactants are used; it proceeds under mild conditions; and the entire method can be carried out in a single tube without the need to isolate intermediates.

[0199] In a related embodiment, the method includes a further step: (g), identifying hydroxymethylation patterns in the 5hmc-containing DNA removed from the cell-free DNA in step (b). This can be carried out using techniques described in detail in WO 2017 / 176630. The method can be carried out without removing or isolating intermediates in a one-tube procedure. For example, cell-free DNA fragments, preferably adaptor-ligated DNA fragments, are first functionalized with βGT-catalyzed uridine diphosphoglucose 6-azide and subsequently biotinylated via a chemoselective azide group. This procedure results in covalent attachment of biotin to each 5hmC site. In a next step, the biotinylated strand and the strand containing unmodified (native) 5mC are simultaneously pulled down for further processing. As known in the art, anti-5mC antibodies or methyl-CpG binding domain (MBD) proteins are used to pull down native 5mC-containing strands. With the 5hmC residues blocked, unmodified 5mC residues are then selectively oxidized using any suitable technique for converting 5mC to 5fC and / or 5caC, as described elsewhere herein.

[0200] The fragments obtained by amplification may carry a directly or indirectly detectable label.

[0201] In some embodiments, the label is a fluorescent label, a radionuclide, or a detachable molecular fragment that has a typical mass that can be detected by mass spectrometer.When the label is a mass label, some embodiments allow good detectability in mass spectrometer by label amplicon having a single positive or negative net charge.This detection can be performed and visualized by, for example, matrix-assisted laser desorption / ionization mass spectrometry (MALDI) or by using electron spray mass spectrometry (ESI).

[0202] Methods for isolating DNA suitable for these assay techniques are known in the art. In particular, some embodiments involve isolating nucleic acids as described in U.S. Patent No. 13 / 470,251 ("Isolation of Nucleic Acids"), which is incorporated herein by reference in its entirety.

[0203] In some embodiments, the markers described herein are used in a QUARTS assay performed on a stool sample. In some embodiments, methods are provided for generating DNA samples, particularly DNA samples that contain small volumes (e.g., less than 100 microliters, less than 60 microliters) of highly purified low abundance nucleic acids and are substantially and / or effectively free of substances that inhibit the assays (e.g., PCR, INVADER, QuARTS assays, etc.) used to test the DNA sample. Such DNA samples are used in diagnostic assays that qualitatively detect the presence or quantitatively measure the activity, expression, or amount of genes, genetic variants (e.g., alleles), or genetic modifications (e.g., methylation) present in a sample taken from a patient. For example, some cancers are correlated with the presence of certain mutant alleles or certain methylation situations, and thus detection and / or quantification of such mutant alleles or methylation situations has predictive value in cancer diagnosis and treatment.

[0204] Many useful genetic markers are present in very small amounts in samples, and many of the events that generate such markers are rare.As a result, even highly sensitive detection methods such as PCR require large amounts of DNA to provide low-abundance targets sufficient to meet or replace the detection threshold of the assay.Furthermore, the presence of even small amounts of inhibitors impairs the accuracy and precision of these assays aimed at detecting such low-abundance targets.Therefore, provided herein is a method for generating such DNA samples that provides the necessary volume and concentration control.

[0205] In some embodiments, the sample comprises stool, tissue sample, organ secretion, CSF, saliva, blood or urine. In some embodiments, the subject is a human. Such samples can be obtained by any number of means known in the art, such as those that will be apparent to those skilled in the art. Acellular or substantially acellular samples can be obtained by subjecting the sample to a variety of techniques known to those skilled in the art, including, but not limited to, centrifugation and filtration. Although it is generally preferred to obtain samples using non-invasive techniques, it is still preferred to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens. The present technology is not limited to the method used to prepare the sample and provide nucleic acid for testing. For example, in some embodiments, DNA is isolated from a sample (e.g., a stool sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample or a urine sample) using direct gene capture or related methods, as detailed in, for example, US Pat. Nos. 8,808,990 and 9,169,511, and WO 2012 / 155072.

[0206] The analysis of markers can be performed separately or simultaneously with additional markers in one test sample. For example, some markers can be combined in one test to efficiently process multiple samples and potentially provide higher accuracy of diagnosis and / or prognosis. Furthermore, those skilled in the art will recognize the value of testing multiple samples from the same subject (e.g., at successive time points). Such testing of successive samples allows the identification of changes in the methylation status of markers over time. Changes in methylation status, and the absence of changes in methylation status, can provide useful information about disease status, including, but not limited to, revealing the approximate time from the occurrence of this event, the presence and amount of salvageable tissue, suitability of drug therapy, efficacy of various therapies, and identification of the subject's outcome, including risk of future events.

[0207] It is possible to perform the analysis of biomarkers in a variety of physical formats. For example, the use of microtiter plates or automation can be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats can be developed to facilitate immediate treatment and diagnosis in a timely manner, for example, in an outpatient or emergency room setting.

[0208] Genomic DNA can be isolated by any means, including the use of commercially available kits. Briefly, biological samples in which the DNA of interest is encapsulated by cell membranes must be disrupted and dissolved by enzymatic, chemical or mechanical means. Proteins and other contaminants can then be removed from the DNA solution, for example by digestion with proteinase K. Genomic DNA is then recovered from the solution. This can be done by a variety of methods, including salting out, organic extraction, or binding of DNA to a solid support. The choice of method is influenced by several factors, including time, cost, and the amount of DNA required. All clinical sample types, including neoplastic or pre-neoplastic material, are suitable for use in the method, such as cell lines, histological slides, biopsies, paraffin-embedded tissues, body fluids, feces, tissues, colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, and combinations thereof.

[0209] The present technology is not limited to the method used to prepare samples and provide nucleic acid for testing.For example, in some embodiments, DNA is isolated from fecal samples, or from blood, or from plasma samples, for example, using the method detailed in US Patent Application No. 61 / 485386 or by related methods.

[0210] The genomic DNA sample is then treated with at least one reagent, or a series of reagents, that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker that contains a DMR (e.g., DMRs 1-38, as shown in Table 1).

[0211] In some embodiments, the reagent converts cytosine bases that are not methylated at the 5' position to uracil, thymine, or another base that differs from cytosine in terms of hybridization behavior, although in some embodiments the reagent can be a methylation-sensitive restriction enzyme.

[0212] In some embodiments, the genomic DNA sample is treated in such a manner as to convert cytosine bases that are not methylated at the 5' position to uracil, thymine, or another base that differs from cytosine in terms of hybridization behavior.

[0213] In some embodiments, this treatment is carried out with bisulfite (hydrogen sulfite, disulfite salt), followed by alkaline hydrolysis.

[0214] The treated nucleic acid is then analyzed to determine the methylation status of the target gene sequence (at least one gene, genomic sequence, or nucleotide from a marker that includes at least one DMR selected from DMRs 1-38, e.g., as shown in Table 1). Methods of analysis can be selected from those shown herein, e.g., those known in the art, including QuARTS and MSP, as described herein.

[0215] Aberrant methylation, and more specifically hypermethylation of markers that include DMRs (e.g., DMRs 1-38, e.g., as shown in Table 1), is associated with several types of cancer. Such methods are not limited to detecting the presence or absence of a particular type of cancer. In some embodiments, the types of cancer include, but are not limited to, liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer, and uterine cancer.

[0216] The present technology relates to the analysis of any sample related to multiple cancers. For example, in some embodiments, the sample includes biological fluids obtained from a patient. In some embodiments, the sample includes secretions. In some embodiments, the sample includes blood, serum, plasma, gastric secretions, pancreatic juice, gastrointestinal biopsy samples, and / or cells recovered from stool. In some embodiments, the subject is a human. Samples may include cells, secretions, or tissues from lymph nodes, breast, liver, bile duct, pancreas, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gallbladder, anus, and / or peritoneum. In some embodiments, samples include cell fluids, ascites, urine, feces, pancreatic juice, fluid obtained during endoscopy, blood, mucus, or saliva.

[0217] Such samples can be obtained by any number of means known in the art, such as those that will be apparent to those of skill in the art. For example, urine and fecal samples are readily available, while blood, ascites, serum, or pancreatic juice samples can be obtained parenterally, for example, by using a needle and syringe. Acellular or substantially acellular samples can be obtained by subjecting the sample to a variety of techniques known to those of skill in the art, including, but not limited to, centrifugation and filtration. Although it is generally preferred to obtain samples using non-invasive techniques, it remains preferred to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens.

[0218] In some embodiments, the technology relates to a method for treating a patient (e.g., a patient with any type of cancer), comprising either or both of 1) determining the methylation status of one or more methylation markers as provided herein, and 2) measuring the expression and / or activity level of one or more protein markers, and administering a treatment to the patient based on the results of determining the methylation status and / or the expression and / or activity level of the protein marker. The treatment may be administering a pharmaceutical compound, administering a vaccine, performing a surgical procedure, imaging a patient, performing another test. Preferably, the use is in methods of clinical screening, methods of prognostic evaluation, methods of monitoring the results of a therapy, methods of identifying patients most likely to respond to a particular therapeutic treatment, methods of imaging a patient or subject, and methods for drug screening and development.

[0219] In some embodiments of this technology, a method for diagnosing a particular type of cancer in a subject is provided. The terms "diagnose" and "diagnosis" as used herein refer to a method by which a person skilled in the art can estimate and even determine whether a subject suffers from a given disease or condition, or is likely to develop a given disease or condition in the future. A person skilled in the art often makes a diagnosis based on one or more diagnostic indicators, such as one or more biomarkers (e.g., one or more methylation markers, methylation marker genes, genes, DMRs, and / or DNA methylation markers disclosed herein), whose methylation status indicates the presence, severity, or absence of a condition, and / or the expression and / or activity level of one or more protein markers. Such methods are not limited to the diagnosis of a particular type of cancer. In some embodiments, the types of cancer include, but are not limited to, liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, kidney cancer, and uterine cancer.

[0220] In addition to diagnosis, clinical cancer prognosis is concerned with determining the aggressiveness of cancer and the likelihood of tumor recurrence, and planning the most effective therapy. If a more accurate prognosis can be made, or even the potential risk of developing cancer can be assessed, then appropriate therapy, and in some cases, a therapy that is less harsh for the patient, can be selected. The assessment of cancer biomarkers (e.g., determining methylation status) is useful for separating subjects with good prognosis and / or low risk of suffering from cancer, who do not require treatment or only limited treatment, from subjects who are more likely to suffer from cancer or suffer from cancer recurrence, who may benefit from more intensive treatment.

[0221] As such, "making a diagnosis" or "diagnosing" as used herein further includes determining the risk of having cancer or determining a prognosis, which can be provided to predict clinical outcome (with or without medical treatment), select an appropriate treatment (or whether the treatment is effective), or monitor a current treatment to potentially modify the treatment, based on measurements of diagnostic biomarkers (e.g., DMRs) disclosed herein. Furthermore, in some embodiments of the subject matter disclosed herein, multiple determinations of biomarkers over time can be made to facilitate diagnosis and / or prognosis. Changes in biomarkers over time can be used to predict clinical outcomes, monitor the progression of cancer or cancer subtypes, and / or monitor the effectiveness of appropriate cancer-directed treatments. In such embodiments, for example, changes in the methylation status of one or more biomarkers (e.g., DMRs) disclosed herein (and potentially one or more additional biomarkers, if monitored), and / or expression and / or activity levels of protein markers in biological samples can be expected to be seen over time during the course of an effective treatment.

[0222] The subject matter disclosed herein further provides, in some embodiments, a method for determining whether to initiate or continue prevention or treatment of cancer in a subject. In some embodiments, the method includes providing a series of biological samples from a subject over a period of time; analyzing the series of biological samples to one or both of: 1) determine the methylation status of at least one biomarker disclosed herein in each biological sample, and 2) measure the expression and / or activity level of one or more protein markers (CEA, CA125, CA19.9, AFP, CA-15-3) in the biological samples; and comparing any measurable changes in the methylation status of one or more of the biomarkers and / or protein markers in each of the biological samples. Any changes over a period of time can be used to predict the risk of developing cancer, predict clinical outcomes, determine whether to initiate or continue prevention or treatment of cancer, and whether a current treatment is effectively treating the cancer. For example, a first time point can be selected before the start of treatment, and a second time point can be selected at a time point after the start of treatment. The methylation status and expression / activity levels of the protein markers can be measured in each sample taken from different time points and qualitative and / or quantitative differences can be observed. Changes in the methylation status and / or expression / activity levels of the protein markers of the biomarker levels from different samples can be correlated with a particular cancer risk, prognosis, determination of therapeutic efficacy, and / or progression of the cancer in the subject.

[0223] In preferred embodiments, the methods and compositions of the invention are for the treatment or diagnosis of disease at an early stage, e.g., before symptoms of the disease are manifest, hi some embodiments, the methods and compositions of the invention are for the treatment or diagnosis of disease at a clinical stage.

[0224] As described, in some embodiments, multiple determinations of one or more diagnostic or prognostic biomarkers can be made, and changes in the markers over time can be used to determine a diagnosis or prognosis. For example, a diagnostic marker can be determined a first time and again a second time. In these and other embodiments, an increase in a marker from the first time to the second time can be diagnostic of a particular type or severity of cancer, or a given prognosis. Similarly, a decrease in a marker from the first time to the second time can indicate a particular type or severity of cancer, or a given prognosis. Furthermore, the degree of change in one or more markers can be related to the severity of cancer, and future adverse events. Those skilled in the art will understand that in certain embodiments, comparative measurements of the same biomarkers can be made at multiple time points, and a given biomarker can be measured at a first time point and a second biomarker at a second time point, and the comparison of these markers can provide diagnostic information.

[0225] As used herein, the phrase "determining prognosis" refers to a method that allows a person skilled in the art to predict the course or outcome of a condition in a subject. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even the ability that a given course or outcome is more or less likely to occur based on the methylation status of biomarkers (e.g., DMRs and / or protein markers). Instead, a person skilled in the art will understand that the term "prognosis" refers to the increased probability that a certain course or outcome will occur, i.e., that a course or outcome is more likely to occur in a subject that exhibits a given condition when compared to those individuals that do not exhibit the condition. For example, an individual that does not exhibit symptoms (e.g., having normal methylation status of one or more DMRs and / or expression and / or activity levels of protein markers) may have a very low probability of a given outcome (e.g., suffering from a certain type of cancer).

[0226] In some embodiments, the statistical analysis correlates the prognostic indicator with a predisposition to an adverse outcome. For example, in some embodiments, a methylation status and / or expression / activity level of a protein marker that differs from a methylation status and / or expression / activity level in a normal control sample obtained from a patient without cancer, as determined by the level of statistical significance, may signal that the subject is more likely to suffer from cancer than a subject having a level more similar to the methylation status in the control sample. Furthermore, the change in methylation status and / or protein marker expression / activity level from baseline (e.g., "normal") levels may reflect the prognosis of the subject, and the degree of change in methylation status and / or protein marker expression / activity level may be related to the severity of an adverse event. Statistical significance is often determined by comparing two or more populations to determine a confidence interval and / or p-value. See, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, which is incorporated herein by reference in its entirety. Exemplary confidence intervals of the present subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while exemplary p-values ​​are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

[0227] In other embodiments, a threshold degree of change in methylation status and / or protein marker expression / activity level of a prognostic or diagnostic biomarker disclosed herein (e.g., DMR; protein marker) can be established, and the degree of change in methylation status and / or protein marker expression / activity level of the biomarker in a biological sample is simply compared to the threshold degree of change in methylation status and / or protein marker expression / activity level. Preferred threshold changes in methylation status and / or protein marker expression / activity level of the biomarkers provided herein are about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. In yet other embodiments, a "nomogram" can be established, whereby the methylation status and / or protein marker expression / activity level of a prognostic or diagnostic indicator (biomarker or combination of biomarkers) is directly related to a trend toward a given outcome. Because reference is made to individual sample measurements rather than population averages, those skilled in the art are familiar with using such nomograms to relate the two numbers, with the understanding that the uncertainty in the measurement is the same as the uncertainty in the marker concentration.

[0228] In some embodiments, a control sample is analyzed simultaneously with the biological sample, so that results obtained from the biological sample can be compared to results obtained from the control sample. It is further contemplated that a standard curve can be provided and the assay results of the biological sample can be compared to the standard curve. Such a standard curve indicates the methylation status and / or protein marker expression / activity level status of the biomarker as a function of assay units, e.g., fluorescent signal intensity, when fluorescent labels are used. Samples taken from multiple donors can be used to provide a standard curve of control methylation status of one or more biomarkers in normal tissues, as well as "at risk" levels of one or more biomarkers in plasma taken from donors with a particular type of cancer. In certain embodiments of the method, a subject is identified as having cancer upon identification of an abnormal methylation status of one or more DMRs and / or protein marker expression / activity levels provided herein in a biological sample obtained from the subject. In other embodiments of the method, a subject is identified as having cancer upon detection of an abnormal methylation status and / or protein marker expression / activity level status of one or more such biomarkers in a biological sample obtained from the subject.

[0229] The analysis of markers may be performed separately or simultaneously with additional markers within one test sample. For example, some markers may be combined in one test to efficiently process multiple samples and potentially provide higher accuracy of diagnosis and / or prognosis. In addition, those skilled in the art will recognize the value of testing multiple samples from the same subject (e.g., at successive time points). Such testing of successive samples may allow for the identification of changes in marker methylation status and / or protein marker expression / activity level status over time. Changes in methylation status and / or protein marker expression / activity level status, as well as the absence of changes in methylation status, can provide useful information regarding disease status, including, but not limited to, identification of the approximate time from onset of an event, the presence and amount of salvageable tissue, appropriateness of drug therapy, efficacy of various therapies, and identification of subject outcomes, including risk of future events.

[0230] It is possible to perform the analysis of biomarkers in a variety of physical formats. For example, the use of microtiter plates or automation can be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats can be developed to facilitate immediate treatment and diagnosis in a timely manner, for example, in an outpatient or emergency room setting.

[0231] In some embodiments, a subject is diagnosed as having a particular type of cancer if there is a measurable difference in the methylation status and / or protein marker expression / activity level of at least one biomarker in the sample when compared to a control methylation status and / or protein marker expression / activity level status. Conversely, if no change in the methylation status and / or protein marker expression / activity level status is identified in the biological sample, the subject can be identified as not having a particular type of cancer, not at risk of cancer, or at low risk of cancer. In this regard, subjects with cancer or at risk thereof can be differentiated from subjects with cancer or at low risk thereof to subjects with substantially no cancer. Subjects at risk of developing a particular type of cancer can be placed on a more intensive and / or regular screening schedule. Meanwhile, subjects at low or substantially no risk can be avoided from being subjected to additional tests (e.g., invasive procedures) for cancer risk until future screening, such as screening performed according to the present technology, indicates that the subjects have developed a risk of cancer risk.

[0232] As mentioned above, depending on the embodiment of the method of the present technology, detecting a change in the methylation status and / or protein marker expression / activity level status of one or more biomarkers can be a qualitative or quantitative determination. Thus, diagnosing a subject as having or at risk of developing a particular cancer type indicates that a certain threshold measurement is made, e.g., that the methylation status and / or protein marker expression / activity level status of one or more biomarkers in a biological sample changes from a predetermined control methylation status and / or control protein marker expression / activity level status. In some embodiments of the present method, the control methylation status is any detectable methylation status of the biomarker. In some embodiments, the control protein marker expression / activity level status is any measurable and / or protein marker expression / activity level status of the protein marker. In other embodiments of the method, where a control sample is tested simultaneously with the biological sample, the predetermined methylation status is the methylation status in the control sample and the predetermined protein marker expression / activity level control status is the and / or protein marker expression / activity level status in the control sample. In other embodiments of the method, the predetermined methylation status and / or predetermined protein marker expression / activity level status is identified based on and / or by a standard curve. In other embodiments of the method, the predetermined methylation status and / or predetermined protein marker expression / activity level status is a specific status or range of status. Thus, the predetermined methylation status and / or predetermined protein marker expression / activity level status can be selected within acceptable ranges that would be apparent to one of skill in the art, based in part on the embodiment of the method being performed and the desired specificity, etc.

[0233] Further with respect to the diagnostic method, the preferred subject is a vertebrate subject. The preferred vertebrate is warm-blooded, and the preferred warm-blooded vertebrate is a mammal. The preferred mammal is most preferably a human. As used herein, the term "subject" includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. As such, the technology of the present invention is provided for the diagnosis of mammals such as humans, and those mammals that are economically important, such as animals raised on farms for human consumption, important because they are endangered, such as the Amur tiger, and / or animals that are socially important to humans, such as animals kept as pets or in zoos. Examples of such animals include, but are not limited to, carnivores, such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and / or ungulates, such as cows, bulls, sheep, giraffes, deer, goats, bison, and camels; and horses. Thus, further provided is diagnosis and treatment of livestock, including, but not limited to, domesticated pigs, ruminants, ungulates, horses (including racehorses), and the like.

[0234] In certain embodiments, the technology provides a method for treating a nucleic acid containing a DMR with a reagent that can modify the nucleic acid in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, a bisulfite reagent) (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, a Ten Eleven Translocation (TET) enzyme (e.g., human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria TET (NgTET), Coprinopsis cinerea (Coprinopsis cinerea (CcTET), or a variant thereof) to produce, for example, a methylation-specifically modified nucleic acid; sequencing the nucleic acid modified in a methylation-specific manner to provide a nucleotide sequence of the nucleic acid modified in a methylation-specific manner; comparing the nucleotide sequence of the nucleic acid modified in a methylation-specific manner to the nucleotide sequence of a nucleic acid comprising the DMR from a subject not having a particular type of cancer to identify differences between the two sequences; and if a difference is present, identifying the subject as having a particular type of cancer.

[0235] In some embodiments, the cancer is any type of cancer.

[0236] In some embodiments, the cancer is selected from liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer, and uterine cancer.

[0237] The present technology further provides a composition. In certain embodiments, the present technology provides a composition comprising a nucleic acid comprising a DMR and a bisulfite reagent. In certain embodiments, a composition comprising a nucleic acid comprising a DMR and one or more oligonucleotides set forth in SEQ ID NOs: 1 to 126 is provided. In certain embodiments, a composition comprising a nucleic acid comprising a DMR and a methylation-sensitive restriction enzyme is provided. In certain embodiments, a composition comprising a nucleic acid comprising a DMR and a polymerase is provided.

[0238] The technology further provides kits. The kits include embodiments of compositions, devices, apparatuses, etc. described herein, and instructions for using the kits. Such instructions describe appropriate methods for preparing an analyte from a sample, for example, methods for collecting a sample and preparing nucleic acid from the sample. The individual components of the kit are packaged in suitable containers and packaging (e.g., vials, boxes, blister packs, ampoules, jars, bottles, tubes, etc.), and the components are packaged together in a suitable container (e.g., box(es)) for convenient storage, transportation, and / or use by the user of the kit. It is understood that liquid components (e.g., buffers) may be provided in a lyophilized form that is reconstituted by the user. The kits may include controls or references to evaluate, verify, and / or ensure the performance of the kit. For example, a kit for assaying the amount of nucleic acid present in a sample may include a control that includes a known concentration of the same or another nucleic acid for comparison, and in some embodiments, may include a detection reagent (e.g., primers) specific for the control nucleic acid. The kit is suitable for use in a clinical setting, and in some embodiments, for use in a user's home. The components of the kit, in some embodiments, provide the functionality of a system for preparing a nucleic acid solution from a sample. In some embodiments, certain components of the system are provided by the user.

[0239] In certain embodiments, the technology relates to compositions (e.g., reaction mixtures). In some embodiments, compositions are provided that include a nucleic acid that includes a DMR and a reagent that can modify DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, a bisulfite reagent) (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, a TET (Ten Eleven Translocation) enzyme (e.g., human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria TET (NgTET), Coprinopsis cinerea (CcTET)), or a variant thereof). Some embodiments provide compositions that include a nucleic acid that includes a DMR and an oligonucleotide described herein. Some embodiments provide compositions that include a nucleic acid that includes a DMR and a methylation-sensitive restriction enzyme. Some embodiments provide compositions that include a nucleic acid that includes a DMR and a polymerase.

[0240] In some embodiments, the techniques described herein are associated with programmable machines designed to perform sequences of arithmetic or logical operations, such as those provided by the methods described herein. For example, some embodiments of the techniques are associated with (e.g., implemented in) computer software and / or computer hardware. In one aspect, the techniques relate to a computer that includes a form of memory, elements for performing arithmetic and logical operations, and a processing element (e.g., a microprocessor) for executing a set of instructions (e.g., the methods provided herein) for reading, manipulating, and storing data. In some embodiments, the microprocessor is part of a system for determining methylation status (e.g., one or more DMRs, e.g., DMRs 1-38 provided in Table 1); comparing methylation status; generating a standard curve; determining Ct values; calculating the proportion, frequency, or rate of methylation; identifying CpG islands; determining the specificity and / or sensitivity of an assay or marker; calculating ROC curves and associated AUCs; sequence analysis; all of which are described herein or known in the art. In some embodiments, the microprocessor is part of a system for determining the levels of protein expression and / or activity (e.g., one or more protein markers described herein); comparing the levels of protein marker expression or activity to standard non-cancerous levels; all as described herein or known in the art.In some embodiments, the microprocessor is part of a system for: 1) determining methylation status (e.g., one or more DMRs, e.g., DMRs 1-38 provided in Table 1); comparing methylation status; generating standard curves; determining Ct values; calculating methylation rates, frequencies or percentages; identifying CpG islands; determining assay or marker specificity and / or sensitivity; calculating ROC curves and associated AUCs; sequence analysis; all of which are described herein or known in the art; and 2) determining levels of protein expression and / or activity (e.g., one or more protein markers described herein); comparing levels of expression or activity of a protein marker relative to standard non-cancerous levels, all of which are described herein or known in the art.

[0241] In some embodiments, the software or hardware component receives the results of the multiple assays and determines and reports to a user a single value result indicative of cancer risk based on the results of the multiple assays (e.g., determining the methylation status of multiple DMRs, e.g., as provided in Table 1). Related embodiments calculate a risk factor (e.g., determining the expression and / or activity levels of protein markers) based on a mathematical combination (e.g., weighted combination, linear combination) of results from the multiple assays (e.g., determining the methylation status of multiple DMRs, e.g., as provided in Table 1). In some embodiments, the methylation status of the DMRs defines dimensions and can have values ​​in a multidimensional space, and the coordinates defined by the methylation status of the multiple DMRs are a result, e.g., related to cancer risk, for reporting to a user, e.g.,

[0242] In some embodiments, the techniques provided herein involve multiple programmable devices acting in concert to perform the methods as described herein.

[0243] For example, in some embodiments, multiple computers (e.g., connected by a network) can operate in parallel to collect and process data in an implementation of cluster computing or grid computing or some other distributed computer architecture that relies on complete computers (with on-board CPU, storage, power, network interfaces, etc.) being connected to a network (private, public, or the Internet), e.g., by traditional network interfaces such as Ethernet, optical fiber, etc., or by wireless networking technologies.

[0244] For example, some embodiments provide a computer including a computer-readable medium. The embodiments include a random access memory (RAM) coupled to a processor. The processor executes computer-executable program instructions stored in the memory. Processors such as these may include microprocessors, ASICs, state machines, or other processors, and may be any of a number of computer processors, such as processors from Intel Corporation of Santa Clara, California, Motorola Corporation of Schaumburg, Illinois. Processors such as these may include or be in communication with a medium, such as a computer-readable medium, that stores instructions that, for example, when executed by the processor, cause the processor to perform the steps described herein.

[0245] The computer is connected to a network in some embodiments. The computer may also include multiple external or internal devices, such as a mouse, CD-ROM, DVD, keyboard, display, or other input or output devices. Examples of computers are personal computers, digital assistants, personal digital assistants, cellular phones, mobile phones, smartphones, pagers, digital tablets, laptop computers, Internet appliances, and other processor-based devices. In general, these computers related to aspects of the technology provided herein can be any type of processor-based platform that runs on any operating system, such as Microsoft Windows, Linux, UNIX, Mac OS X, and can support one or more programs, including the technology provided herein. Some embodiments include personal computers that run other application programs (e.g., applications). Applications can be stored in memory and can include, for example, word processing applications, spreadsheet applications, email applications, instant messenger applications, presentation applications, Internet browser applications, calendar / organizer applications, and any other applications executable by a client device.

[0246] All such components, computers, and systems described herein as relating to the present technology may be logical or virtual.

[0247] In certain embodiments, the present technology provides a system for screening multiple types of cancer in a sample obtained from a subject, provided by the present technology. Exemplary embodiments of the system include, for example, a system for screening multiple types of cancer in a sample obtained from a subject (e.g., a stool sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample, or a urine sample), the system comprising: an analytical component configured to determine the methylation status of one or more methylation markers in the sample and / or determine the expression and / or activity level of one or more protein markers in the sample; a software component configured to compare the methylation status of one or more methylation markers in a sample and / or the expression and / or activity level of one or more protein markers in said sample with a control or reference sample recorded in a database; and An alert component configured to alert a user to a cancer-related condition.

[0248] In some embodiments, the alert is determined by a software component that receives results from multiple assays (e.g., determining the methylation status of one or more methylation markers) (e.g., determining expression and / or activity levels of one or more protein markers) and calculates a value or result to report based on the multiple results.

[0249] Some embodiments provide a database of weighting parameters associated with each methylation marker and / or protein marker expression and / or activity level provided herein for use in calculating a value or result and / or alert for reporting to a user (e.g., a doctor, nurse, clinician, etc.). In some embodiments, all results from the multiple assays are reported. In some embodiments, one or more results are used to provide a score, value or result indicative of the subject's cancer risk based on a combination of one or more results from the multiple assays. Such methods are not limited to a particular methylation marker.

[0250] In such methods and systems, the one or more methylation markers comprise bases in a DMR selected from the group consisting of DMRs 1-38 as provided in Table 1.

[0251] In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will appreciate that these various embodiments may be practiced with or without these specific details. In other instances, structures and mechanisms are shown in block diagram form. Moreover, those skilled in the art will readily appreciate that the particular order in which the methods are presented and performed is illustrative, and that the order can be changed and still be contemplated to be within the spirit and scope of the various embodiments disclosed herein.

[0252] experiment The following examples are illustrative of the present invention, but are not limiting. Other suitable modifications and adaptations of the various conditions and parameters normally encountered in clinical treatment will be apparent to those skilled in the art and are within the spirit and scope of the present invention. As used herein, the terms "our", "we", "I" and similar terms refer to the inventorship of the inventions described herein.

[0253] [Example] Example I This example describes experiments performed to evaluate the feasibility of a targeted assay of a panel of methylated DNA markers (MDMs) and proteins for the detection of highly lethal cancers.

[0254] Prospectively collected plasma from 180 cases of six cancer types (stages A-D liver (n=36), and TNM stages II-IV esophageal (n=18), lung (n=36), ovarian (n=30), pancreatic (n=30), and gastric (n=30)) and 257 smoking-status-, age-, and sex-matched asymptomatic controls were studied in a blinded fashion. Multiplex PCR followed by LQAS (Long Probe Quantitative Amplified Signal) assays (for general techniques see, e.g., WO 2017 / 075061 and US 15 / 841,006) were used to perform post-bisulfite quantification of 38 MDMs previously found to be common among many cancers on DNA extracted from 3 mL of plasma (see Table 1 (genomic coordinates of the regions shown in Table 1 are based on the human Feb.2009 (GRCh37 / hg19) assembly) and Figure 1; Table 2 and Figure 1 show primer and probe sequences for the markers shown in Table 1).

[0255] [Table 1]

[0256] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0257] Additionally, five protein markers (CEA, CA125, CA19-9, AFP, CA-15-3) were tested from paired serum aliquots and combined with MDM for multi-analyte analysis. Two-thirds of the cases and controls were used to develop prediction algorithms and logistic regression analyses were performed for 1) methylation markers only (Figure 3); 2) protein markers only (Figure 4); and 3) methylation and protein markers (Figure 2), as well as random forest analyses. The remaining one-third was used to validate the models.

[0258] As shown in Table 3 and Figure 2, for DMR1-26 (Table 1), three proteins (CEA, CA125, CA19-9) and five MDMs (ZNF671, GRIN2D, NDRG4 Combination of the five MDMs (FAIM2, SHOX2, B3GALT6) yielded an area under the receiver operating characteristic curve (AUC) of 0.95 and an overall sensitivity of 87% for all cancers at 95% specificity. The logistic model for the validation set of samples yielded an AUC of 0.96 and a sensitivity of 83%, with a specificity of 94% observed. Cancer-specific sensitivities ranged from 78% for lung cancer to 90% for ovarian and pancreatic cancer. Random forest and logistic analyses were highly concordant. Figure 3 shows the correlation between the five MDM combinations (FAIM2, CHST2_7890 Figure 4 shows that a combination of four proteins (CEA, CA125, CA19.9, AFP) yielded an overall sensitivity of 62% for all cancers with a specificity of 96%.

[0259] [Table 3]

[0260] Similar experiments were performed with plasma collected from 160 cases of six cancer types (esophagus (n=15), hepatocellular carcinoma (HCC) / liver (n=29), lung (n=29), ovarian (n=29), pancreas (n=30) and stomach (n=28)) and 317 age- and sex-matched asymptomatic controls tested in a blinded fashion (see Table 4 for overall stage of cancer type for each subject). Testing was performed in a blinded fashion using multiplex PCR followed by LQAS (long probe quantitative amplified signal) assay on bisulfite-converted DNA extracted from 3 mL of plasma collected in LBgard blood tubes. Protein testing was performed on paired serum aliquots and combined with MDM for multi-analyte analysis. Subjects were divided into training and validation sets with cancer type, staging, sex, and age equally represented between them. Two-thirds of the cases and controls were used to train the logistic prediction algorithm and the remaining one-third were used to validate the model.

[0261] As shown in Table 5, using stepwise logistic regression, the combination of 16 MDMs (GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, IFFO1, HOXA9, HOPX) yielded an overall sensitivity of 87% for all cancers with a specificity of 97.5%. Cancer-specific sensitivity ranged from 72% for ovarian cancer to 93% for lung cancer (see Table 5). As shown in Table 6, using stepwise logistic regression, the combination of five proteins (CEA, CA125, CA19-9, AFP, CA-15-3) yielded an overall sensitivity of 84% for all cancers with a specificity of 98%. The cancer-specific sensitivity ranged from 80% for esophageal cancer to 86% for liver cancer, ovarian cancer, pancreatic cancer, and gastric cancer (see Table 6). As shown in Table 7, the combination of 16 MDMs (GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, IFFO1, HOXA9, HOPX) with 5 proteins (CEA, CA125, CA19-9, AFP, CA-15-3) resulted in an overall sensitivity of 85% for all cancers with a specificity of 98%. The cancer-specific sensitivity ranged from 80% for esophageal cancer to 86% for liver cancer, lung cancer, ovarian cancer, and gastric cancer (see Table 7).

[0262] [Table 4]

[0263] [Table 5]

[0264] [Table 6]

[0265] [Table 7]

[0266] Similar additional experiments were performed using plasma collected from 236 cases of 13 cancer types (esophageal cancer (n=1), bladder cancer (n=20), breast cancer (n=14), cervical cancer (n=10), colorectal cancer (n=38), hepatocellular carcinoma (HCC) / liver cancer (n=13), lung cancer (n=40), ovarian cancer (n=8), pancreatic cancer (n=30), prostate cancer (n=10), renal cancer (n=20), gastric cancer (n=22) and uterine cancer (n=10)) and 146 age- and sex-matched asymptomatic controls were tested in a blinded fashion (see Table 8 for overall stage of cancer type for each subject). Table 9 shows the percent methylation cutoff and percent sensitivity for all 13 cancer types for the following MDMs: GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, ST8SIA1, HOXA1, HOXB2, BARX1, CLEC11A, ARHGEF4, IFFO1, HOXA9, OSR2, QKI, RYR2, GPRIN1, ZNF569, SHISA9, CD1D, NTRK3, VAV3, and FAM59B. Tables 10, 11, and 12 show the overall sensitivity of the 13 cancer types for the following DMRs: TRH, EMX1, and FAIM2, respectively.

[0267] As shown in Table 13, the combination of 35 MDMs (GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, ST8SIA1, HOXA1, HOXB2, BARX1, CLEC11A, ARHGEF4, IFFO1, HOXA9, OSR2, QKI, RYR2, GPRIN1, ZNF569, SHISA9, CD1D, NTRK3, VAV3, and FAM59B) in the triplex QuARTS assay resulted in an overall sensitivity of 74% for all cancers (bladder, breast, cervix, CRC, esophagus, HCC, lung, ovary, pancreas, prostate, kidney, stomach, uterus) with a specificity of 97%. Cancer-specific sensitivities ranged from 20% for the pancreas to 100% for the cervix, esophagus, ovary, and uterus.

[0268] As further shown in Table 13, 35 MDMs (GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, ST8SIA1, HOXA1, HOXB2, BARX1, CLEC11A, ARHGEF4, IFF1, ... A combination of five proteins (O1, HOXA9, OSR2, QKI, RYR2, GPRIN1, ZNF569, SHISA9, CD1D, NTRK3, VAV3, and FAM59B) and a combination of five proteins (CEA, CA125, CA19-9, AFP, CA-15-3) yielded an overall sensitivity of 78% for all cancers (bladder, breast, cervix, CRC, esophagus, HCC, lung, ovary, pancreas, prostate, kidney, stomach, uterus) with a specificity of 97%. Cancer-specific sensitivity ranged from 20% for pancreas to 100% for cervix, esophagus, ovary, and uterus.

[0269] As further shown in Table 13, the 35 MDMs (GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, ST8SIA1, HOXA1, HOXB2, The combination of the following genes (BARX1, CLEC11A, ARHGEF4, IFFO1, HOXA9, OSR2, QKI, RYR2, GPRIN1, ZNF569, SHISA9, CD1D, NTRK3, VAV3, and FAM59B) yielded an overall sensitivity of 72% for all cancers (bladder, breast, cervix, CRC, esophagus, HCC, lung, ovary, pancreas, prostate, kidney, stomach, and uterus) with a specificity of 97%. Cancer-specific sensitivity ranged from 20% for pancreas to 100% for cervix and esophagus.

[0270] As further shown in Table 13, 35 MDMs (GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, ST8SIA1, HOXA1, HOXB2, BARX1, CLEC11A, ARHGEF4, IFFO1, IFFO2, IFFO1 ... A combination of five proteins (CEA, CA125, CA19-9, AFP, CA-15-3) and a combination of five proteins (CEA, CA125, CA19-9, AFP, CA-15-3) yielded an overall sensitivity of 75% for all cancers (bladder, breast, cervix, CRC, esophagus, HCC, lung, ovary, pancreas, prostate, kidney, stomach, uterus) with a specificity of 97%. Cancer-specific sensitivity ranged from 20% for pancreas to 100% for cervix, esophagus, ovary, and uterus.

[0271] Additional studies were performed on bladder, breast, cervical, CRC, esophageal, HCC, lung, ovarian, pancreatic, renal, gastric, and uterine cancers, but excluded prostate cancer (see Table 14).

[0272] As shown in Table 14, 35 MDMs in the triplex QuARTS assay (GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, ST8SIA1, HOXA1, HOXB2, BARX1, C The combination of the following genes (LEC11A, ARHGEF4, IFFO1, HOXA9, OSR2, QKI, RYR2, GPRIN1, ZNF569, SHISA9, CD1D, NTRK3, VAV3, and FAM59B) resulted in an overall sensitivity of 77% for all cancers (bladder, breast, cervix, CRC, esophagus, HCC, lung, ovary, pancreas, kidney, stomach, uterus) (not including prostate cancer) with a specificity of 97%. Cancer-specific sensitivity ranged from 50% for kidney to 100% for cervix, esophagus, ovary, and uterus.

[0273] As further shown in Table 14, 35 MDMs in the triplex QuARTS assay (GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, ST8SIA1, HOXA1, HOXB2, BARX1, CLEC11A, ARHGEF4, IFFO1, A combination of five proteins (CEA, CA125, CA19-9, AFP, CA-15-3) and a combination of five proteins (HOXA9, OSR2, QKI, RYR2, GPRIN1, ZNF569, SHISA9, CD1D, NTRK3, VAV3, and FAM59B) resulted in an overall sensitivity of 81% for all cancers (bladder, breast, cervix, CRC, esophagus, HCC, lung, ovary, pancreas, kidney, stomach, uterus) (not including prostate cancer) with a specificity of 97%. Cancer-specific sensitivity ranged from 50% for kidney to 100% for cervix, esophagus, ovary, and uterus.

[0274] As further shown in Table 14, 35 MDMs in the MAD-LQAS assay (GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, ST8SIA1, HOXA1, HOXB2, BARX1, CL The combination of the following genes (EC11A, ARHGEF4, IFFO1, HOXA9, OSR2, QKI, RYR2, GPRIN1, ZNF569, SHISA9, CD1D, NTRK3, VAV3, and FAM59B) yielded an overall sensitivity of 74% for all cancers (bladder, breast, cervix, CRC, esophagus, HCC, lung, ovary, pancreas, kidney, stomach, and uterus) (not including prostate cancer) with a specificity of 97%. Cancer-specific sensitivity ranged from 50% for kidney to 100% for cervix and esophagus.

[0275] As further shown in Table 14, 35 MDMs (GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, ST8SIA1, HOXA1, HOXB2, BARX1, CLEC11A, ARHGEF4, IFFO1, HOXA1, and HOXB2) in the MAD-LQAS assay were identified. A combination of five proteins (CEA, CA125, CA19-9, AFP, CA-15-3) and a combination of five proteins (CEA, CA125, CA19-9, AFP, CA-15-3) yielded an overall sensitivity of 78% for all cancers (bladder, breast, cervix, CRC, esophagus, HCC, lung, ovary, pancreas, kidney, stomach, uterus) (not including prostate cancer) with a specificity of 97%. Cancer-specific sensitivity ranged from 50% for kidney to 100% for cervix, esophagus and ovary.

[0276] The experiments further led to the identification of a panel of markers for identifying multiple types of cancer from biological samples: CDO1, GRIN2D, SHOX2, OSR2, QKI, SIM2, TRH, CAPN2, SFMBT2, CHST2_7890 , ST8SIA1, HOXA1, FER1L4, FAIM2, IFFO1, EMX1, ZNF671, PRKCB, HOXB2, BARX1, PPP2R5C, and TSPYL5.

[0277] [Table 8]

[0278] [Table 9]

[0279] [Table 10]

[0280] [Table 11]

[0281] [Table 12]

[0282] [Table 13]

[0283] [Table 14]

[0284] All publications and patents mentioned in the above specification are incorporated herein by reference in their entirety for all purposes. Various modifications and variations of the compositions, methods, and applications of the described technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with certain exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are apparent to those skilled in the art of pharmacology, biochemistry, medicine, or related fields are intended to be within the scope of the appended claims. [Brief description of the drawings]

[0285] [Figure 1-1] Information on the marker chromosomal regions and associated primers and probes used for various methylated DNA markers listed in Table 1. The naturally occurring sequence (WT) and bisulfite modified sequence (BST) of the PCR target region are shown. [Figure 1-2] This is a continuation of Figure 1-1. [Figure 1-3] This is a continuation of Figure 1-2. [Figure 1-4] This is a continuation of Figure 1-3. [Figure 1-5] This is a continuation of Figure 1-4. [Figure 1-6] This is a continuation of Figure 1-5. [Figure 1-7] This is a continuation of Figure 1-6. [Figure 1-8] This is a continuation of Figure 1-7. [Figure 1-9] This is a continuation of Figure 1-8. [Figure 1-10] This is a continuation of Figure 1-9. [Figure 1-11] This is a continuation of Figure 1-10. [Figure 1-12] This is a continuation of Figure 1-11. [Figure 1-13] This is a continuation of Figure 1-12. [Figure 1-14] This is a continuation of Figure 1-13. [Figure 1-15] This is a continuation of Figure 1-14. [Figure 1-16] This is a continuation of Figure 1-15. [Figure 1-17] This is a continuation of Figure 1-16. [Figure 1-18] This is a continuation of Figure 1-17. [Figure 1-19] This is a continuation of Figure 1-18. [Diagram 2] The combination of three proteins (CEA, CA125, CA19-9) and five MDMs (ZNF671, GRIN2D, NDRG4, SHOX2, B3GALT6) yielded an overall sensitivity of 87% for all cancers with an area under the receiver operating characteristic curve (AUC) of 0.95 and a specificity of 95%. [Diagram 3] The combination of five MDMs (FAIM2, CHST2_7890, ZNF671, GRIN2D, CDO1) yielded an overall sensitivity of 74% for all cancers with a specificity of 94%. [Figure 4] The combination of four proteins (CEA, CA125, CA19.9, AFP) yielded an overall sensitivity of 62% for all cancers with a specificity of 96%.

Claims

1. A method for characterizing a sample from a subject having or suspected of having cancer, comprising: a) IFFO1, FER1L4, FAIM2, OSR2, ZNF671, EMX1, CDO1, SIM2, CHST2_7890, SFMBT2, PPP2R5C, ARHGEF4, TSPYL5, B3GALT6, HOXB2, BARX1, TBX1, SHOX2, CLEC11A, HOXA1, GRIN2D, CAPN2, NDRG4, TRH, PRKCB, SHISA9, ZNF781, ST8SI in the sample measuring the methylation level of one or more methylation markers selected from: A1, HOXA9, HOPX, QKI, RYR2, GPRIN1, ZNF569, CD1D, NTRK3, VAV3, and / or FAM59B, wherein measuring the methylation level of the one or more methylation markers comprises treating DNA from the sample with a reagent that modifies DNA in a methylation-specific manner; and b) determining in said sample the expression and / or activity level of one or more protein markers selected from CEA, CA125, CA19.9, AFP, and / or CA-15-3; The method further comprising one or both of the steps of:

2. The measuring of the methylation levels of the one or more methylation markers comprises measuring the methylation levels of one or more corresponding methylation markers in a control sample not having the particular type of cancer; and / or Measuring the expression and / or activity levels of the one or more protein markers includes measuring the expression and / or activity levels of one or more corresponding one or more protein markers in a control sample that does not contain the particular type of cancer. The method of claim 1.

3. below: i) the methylation level measured at the one or more methylation markers is higher than the methylation level measured at the corresponding marker from the control sample; and ii) the expression and / or activity level of the one or more protein markers is higher than the expression and / or activity level measured for the corresponding marker from the control sample; 3. The method of claim 2, further comprising determining that the subject has one or more types of cancer if

4. 2. The method of claim 1, wherein the one or more types of cancer are selected from liver cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, prostate cancer, renal cancer and / or uterine cancer.

5. 2. The method of claim 1, wherein the reagent that methylation-specifically modifies DNA comprises one or more of a borane reducing agent, a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and / or a bisulfite reagent.

6. 2. The method of claim 1, wherein the methylation-specific DNA-modifying reagent is a bisulfite reagent, and treatment of the DNA with the bisulfite reagent produces bisulfite-treated DNA.

7. The method of claim 6, wherein the treated DNA is amplified with a primer set specific for the one or more methylation markers.

8. The method of claim 7, wherein the set of primers specific for the one or more methylation markers is selected from the group listed in Table 2.

9. 8. The method of claim 7, wherein the primer set specific for the one or more methylation markers is capable of binding an amplicon bound by a primer sequence of a methylation marker gene listed in Table 2, and the amplicon bound by the primer sequence of the methylation marker gene listed in Table 2 is at least a portion of a gene region of a methylation marker gene listed in Table 1.

10. The method of claim 7, wherein the primer set specific to the one or more methylation markers is a primer set that specifically binds at least a portion of a gene region that includes the chromosomal coordinates of the methylation markers listed in Table 1.

11. 10. The method of claim 1, wherein measuring the methylation level of one or more methylation markers comprises multiplex amplification.

12. 2. The method of claim 1, wherein measuring the methylation level of one or more methylation markers comprises using methylation specific PCR, quantitative methylation specific PCR, methylation specific DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, flap endonuclease assay, PCR-flap assay, and / or bisulfite genomic sequencing PCR.

13. 2. The method of claim 1, wherein measuring the methylation level of one or more methylation markers comprises measuring methylation of CpG sites for the one or more methylation markers.

14. The method of claim 13 , wherein the CpG site is present in a coding or regulatory region.

15. The method of claim 1, wherein the one or more methylation markers are described by the genomic coordinates shown in Table 1.

16. 2. The method of claim 1, wherein the sample is a fecal sample, a tissue sample, an organ secretion sample, a CSF sample, a saliva sample, a blood sample, a plasma sample, or a urine sample.

17. The method of claim 1 , wherein the sample is from a human subject.

18. The one or more methylation markers are selected from the group consisting of: FAIM2, CDO1, SIM2, CHST2_7890, SFMBT2, PPP2R5C, ARHGEF4, TSPYL5, ZNF671, B3GALT6, FER1L4, HOXB2, BARX1, TBX1, SHOX2, EMX1, CLEC11A, HOXA1, GRIN2D, CAPN2, NDRG4, TRH, PRKCB, SHISA9, ZNF781, and ST8SIA1; GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, IFFO1, HOXA9, and HOPX; GRIN2D, SHOX2, ZNF671, SIM2, TRH, CAPN2, CHST2_7890, FER1L4, FAIM2, PPP2R5C, TSPYL5, NDRG4, ZNF781, CDO1, EMX1, PRKCB, SFMBT2, ST8SIA1, HOXA1, HOXB2, BARX1, CLEC11A, ARHGEF4, IFFO1, HOXA9, OSR2, QKI, RYR2, GPRIN1, ZNF569, SHISA9, CD1D, NTRK3, VAV3, and FAM59B; CDO1, GRIN2D, SHOX2, OSR2, QKI, SIM2, TRH, CAPN2, SFMBT2, CHST2_7890, ST8SIA1, HOXA1, FER1L4, FAIM2, IFFO1, EMX1, ZNF671, PRKCB, HOXB2, BARX1, PPP2R5C, and TSPYL5; ZNF671, GRIN2D, NDRG4, SHOX2, and B3GALT6; and FAIM2, CHST2_7890, ZNF671, GRIN2D, and CDO1 The method of claim 1 , wherein the compound is selected from the group consisting of

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