Detection of melanoma

Abstract

To provide a technique for screening primary skin melanoma, and a method, composition and related application for detecting the presence of primary skin melanoma.SOLUTION: A method performs: measuring a methylation level of one or more genes in a biological sample of a human individual through: treating a genome DNA in the biological sample by a reagent which modifies DNA in a methylation-specific form; amplifying the treated genome DNA by using a primer set to the one or more selected genes; and determining the methylation levels of the one or more genes by polymerase chain reaction, nucleic acid sequencing, mass analysis, methylation-specific nuclease, separation based on mass, and target capturing.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 019,753, filed May 4, 2020, which is incorporated by reference in its entirety.

[0002] [Technical field] Provided herein are techniques for screening for primary cutaneous melanoma, particularly but not exclusively, methods, compositions, and related uses for detecting the presence of primary cutaneous melanoma.

[0003] [Background technology] Melanoma is a severe form of human skin cancer. It arises from pigment cells (melanocytes), usually in the skin. The incidence of melanoma is increasing at the highest rate of any cancer in the United States, with a lifetime risk of 1 in 68. Melanoma accounts for only 4% of all skin cancers, but 80% of all deaths due to skin cancer. It has long been recognized that identifying and diagnosing melanoma is important for curing early stage disease. There are several types of melanoma, defined by the site where it first appears, including skin and eye melanoma, and rarely the gastrointestinal tract or lymph nodes.

[0004] Primary cutaneous melanoma (PCM) is a commonly fatal malignancy associated with 80% of skin cancer-related deaths (see Miller, AJ and MC Mihm Jr, New England Journal of Medicine, 2006. 355(1): p. 51-65). PCM diagnoses are predicted to be over 150,000 cases (in situ and invasive) in 2017 (American Cancer Society, 2017), with prognosis related to disease stage at diagnosis, with 5-year overall survival rates of 98.1% for patients with localized disease, 63.6% for patients with regional disease, and 16.1% for patients with distant metastases (American Cancer Society, 2017).

[0005] Current disease surveillance for patients with nonmetastatic PCM requires outpatient visits to a healthcare provider and regular PET / CT imaging. The cost of this procedure already exceeds many patient-contributed funds, and some payers are unable to or are skeptical of PET / CT reimbursement. Lower socioeconomic status (SES), as evidenced by Medicaid insurance, has been shown to significantly delay melanoma treatment (see Adamson, AS, et al., JAMA Dermatol, 2017.153(11):p.1106-1113).

[0006] Novel affordable outpatient monitoring solutions such as those described in this application are needed because sophisticated hospital-based imaging approaches will only become more expensive over time. Indeed, effective screening methods are urgently needed to reduce the number of severe melanoma patients. Technological innovations that provide accurate, affordable, and safe screening tools for early-stage melanoma and pre-symptomatic detection of advanced melanoma are essential.

[0007] The present invention meets this need. Indeed, the present invention provides novel methylated DNA markers that distinguish between cases of melanoma and its various subtypes (e.g., metastatic melanoma, primary melanoma) in various biological samples (e.g., tissue, blood).

[0008] Summary of the Invention Blood tests for melanoma screening have typically been based on marker detection in the clear portion of blood (plasma or serum) and have been insensitive or nonspecific for early stage disease. To address these historical deficiencies, the experiments performed herein explored a rational new approach based on differential methylated DNA markers.

[0009] Marker discovery and validation was achieved through the utilization of high-quality flash-frozen biobank tissue. Feasibility of marker detection in the blood compartment was established through the use of exquisitely sensitive analytical platforms (e.g., quantitative methylation-specific PCR; qMSP). Such results demonstrate that this blood testing approach establishes the analytical sensitivity necessary to accurately predict tumor site and detect treatable stages of cancer while avoiding frequent false positives.

[0010] Methylated DNA has been investigated as a potential class of biomarker in tissues of most tumor types. In many cases, DNA methyltransferases add methyl groups to DNA at cytosine-phosphate-guanine (CpG) island sites as an epigenetic control of gene expression. In a biologically intriguing mechanism, acquired methylation events in the promoter regions of tumor suppressor genes are thought to silence expression, thereby contributing to carcinogenesis. DNA methylation may be a more chemically and biologically stable diagnostic tool than RNA or protein expression (Laird (2010) Nat Rev Genet 11:191-203). Furthermore, in other cancers, such as sporadic colon cancer, methylation markers offer superior specificity, are more extensively informative, and are more sensitive than individual DNA mutations (Zou et al (2007) Cancer Epidemiol Biomarkers Prev 16:2686-96).

[0011] Analysis of CpG islands has provided important insights when applied to animal models and human cell lines. For example, Zhang and colleagues found that amplicons from different parts of the same CpG island can have different levels of methylation (Zhang et al. (2009) PLoS Genet 5:e1000438). Moreover, methylation levels were bimodally distributed between highly methylated and unmethylated sequences, further supporting a binary switch-like pattern of DNA methyltransferase activity (Zhang et al. (2009) PLoS Genet 5:e1000438). Analysis of mouse tissues in vivo and cell lines in vitro demonstrated that only about 0.3% of high CpG density promoters (HCPs, defined as having more than 7% CpG sequences within a 300 base pair region) were methylated, whereas low CpG density regions (LCPs, defined as having less than 5% CpG sequences within a 300 base pair region) tend to be highly methylated in dynamic tissue-specific patterns (Meissner et al. (2008) Nature 454:766-70). HCPs include promoters for ubiquitous housekeeping genes and highly regulated developmental genes. Several established markers, such as Wnt2, NDRG2, SFRP2, and BMP3, were present among the HCP sites that were more than 50% methylated (Meissner et al. (2008) Nature 454:766-70).

[0012] Epigenetic methylation of DNA at cytosine-phosphate-guanine (CpG) island sites by DNA methyltransferases has been investigated as a potential class of biomarker in tissues of most tumor types.

[0013] Several methods are available to search for novel methylation markers. Microarray-based interrogation of CpG methylation is a rational, high-throughput approach, but this strategy is biased towards known regions of interest, mainly established tumor suppressor promoters. Alternative methods for genome-wide analysis of DNA methylation have been developed in the last decade. There are four basic approaches. The first uses digestion of DNA with restriction enzymes that recognize specific methylation sites, followed by several possible analytical techniques that provide methylation data limited to the enzyme recognition sites or primers used to amplify the DNA in the quantification step (such as methylation-specific PCR (MSP)). The second approach uses antibodies directed against methyl-cytosine or other methylation-specific binding domains to enrich the methylated fraction of genomic DNA, followed by microarray analysis or sequencing to map the fragments to a reference genome. This approach does not provide single-nucleotide resolution of all methylation sites within a fragment. The third approach starts with bisulfite treatment of DNA to convert all unmethylated cytosines to uracil, followed by restriction enzyme digestion and full sequencing of all fragments after binding to adaptor ligands. The choice of restriction enzyme allows for enrichment of fragments in CpG-dense regions, reducing the number of redundant sequences that may map to multiple gene locations during analysis. The fourth approach involves bisulfite-free treatment of DNA, featuring TET-assisted pyridine borane sequencing (TAPS), a bisulfite-free and base-resolution sequencing method for non-destructive and direct detection of 5-methylcytosine and 5-hydroxymethylcytosine without affecting unmodified cytosines (see Liu et al., 2019, Nat Biotechnol. 37, pp. 424-429). In some embodiments, regardless of the specific enzyme conversion approach, only methylated cytosines are converted.

[0014] Reduced Representation Bisulfite Sequencing (RRBS) provides CpG methylation status data at single nucleotide resolution for 80-90% of all CpG islands and most tumor suppressor promoters at moderate to high read coverage. In cancer case-control studies, analysis of these reads identifies variably methylated regions (DMRs). Previous RRBS analysis of pancreatic cancer specimens revealed hundreds of DMRs, many of which were never associated with carcinogenesis and many of which were not annotated. Further validation studies on an independent set of tissue samples confirmed marker CpGs that were 100% sensitive and specific in terms of performance.

[0015] Provided herein are techniques for screening for melanoma and various melanoma subtypes (e.g., metastatic melanoma, primary cutaneous melanoma), particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of melanoma and various melanoma subtypes (e.g., metastatic melanoma, primary cutaneous melanoma).

[0016] Indeed, as described in Examples I and II, experiments conducted during the course of developing embodiments of the present invention identified a novel set of variably methylated regions (DMRs) for: 1) distinguishing melanoma DNA from that derived from non-neoplastic control tissue; 2) distinguishing DNA from metastatic melanoma tissue from non-neoplastic control DNA; and 3) distinguishing DNA from primary cutaneous melanoma tissue from non-neoplastic control DNA.

[0017] Such experiments have resulted in the listing and description of 331 novel DNA methylation markers that distinguish between melanoma and benign tissue (Tables 1A, 1B, 4, 5A, 5B, 5C, 7A, and 7B; see Examples I and II), that distinguish between metastatic melanoma and benign tissue (Table 5C; see Example I), that distinguish between primary cutaneous melanoma and benign tissue (Table 5C; see Example I), and that detect melanoma in blood samples (Tables 2A, 2B, 4, 5A, 6, and 8A; Examples I and II).

[0018] From these 331 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers that are capable of distinguishing between melanoma tissue and benign skin tissue: ●c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-1106273 64, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.7111 6033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (Tables 4, 5A, 5B; see Example I); ●AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2 , LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-322377 85, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.549 70444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (Tables 7A and 7B; see Example II); ●MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-7307383 0, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-225 41948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (Tables 9, 10, 11; see Example II).

[0019] From these 331 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers that are capable of distinguishing metastatic melanoma tissue from benign tissue: MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see Table 5C, Example I), and ●MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-7307383 0, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-225 41948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (Tables 9, 10, 11; see Example II).

[0020] From these 331 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers that can distinguish primary cutaneous melanoma tissue from benign tissue: MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see Table 5C, Example I), and ●MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-7307383 0, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-225 41948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (Tables 9, 10, 11; see Example II).

[0021] From these 331 novel DNA methylation markers, further experiments identified the following markers and / or panels of markers for detecting melanoma in blood samples (e.g., plasma samples, whole blood samples, white blood cell samples, serum samples): ●c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-1106273 64, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.7111 6033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (Tables 4, 5A, 5B; see Example I); one or more markers listed in Tables 8A and 8B; and ●MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-7307383 0, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-225 41948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (Tables 9, 10, 11; see Example II).

[0022] As described herein, the technology provides a large number of methylated DNA markers and subsets thereof (e.g., sets of 2, 3, 4, 5, 6, 7, or 8 markers) that are highly discriminatory for melanoma overall and various types of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma). Experiments applied selection filters to candidate markers to identify markers that provide high signal-to-noise ratios and low background levels to provide high specificity for melanoma screening or diagnosis purposes.

[0023] In some embodiments, the technology relates to assessing the presence and methylation status of one or more of the markers identified herein in a biological sample (e.g., melanoma tissue, plasma sample). These markers include one or more variably methylated regions (DMRs) as discussed herein, e.g., as shown in Tables 1A, 2A, 7A, 8A, and 9. The methylation status is assessed in embodiments of the technology. Thus, the technology provided herein is not limited in the way in which the methylation status of a gene is measured. For example, in some embodiments, the methylation status is measured by a genome scanning method. For example, one method includes restriction enzyme landmark genome scanning (Kawai et al. (1994) Mol. Cell. Biol. 14:7421-7427), and another example includes methylation-sensitive arbitrarily primed PCR (Gonzalgo et al. (1997) Cancer Res. 57:594-599). In some embodiments, changes in methylation patterns at specific CpG sites are monitored by digestion of genomic DNA with methylation-sensitive restriction enzymes followed by Southern analysis of the regions of interest (digestion-Southern). In some embodiments, analyzing changes in methylation patterns involves a PCR-based process that includes digestion of genomic DNA with methylation-sensitive or methylation-dependent restriction enzymes prior to PCR amplification (Singer-Sam et al. (1990) Nucl. Acids Res. 18:687). In addition, other techniques have been reported that utilize bisulfite treatment of DNA as a starting point for methylation analysis. These include methylation-specific PCR (MSP) (Herman et al. (1992) Proc. Natl. Acad. Sci. USA 93:9821-9826) as well as restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA (Sadri and Hornsby (1996) Nucl. Acids Res. 24:5058-5059, and Xiong and Laird (1997) Nucl. Acids Res. 25:2532-2534). PCR techniques have been developed for the detection of genetic mutations (Kuppuswamy et al. (1991) Proc. Natl. Acad. Sci. USA 88:1143-1147), as well as for the quantification of allele-specific expression (Szabo and Mann (1995) Genes Dev. 9:3097-3108, and Singer-Sam et al. (1992) PCR Methods Appl. 1:160-163). Such techniques use an internal primer that anneals to the PCR-generated template and terminates immediately 5' to the single nucleotide to be assayed. The method using the "Quantitative Ms-SNuPE Assay" described in US Pat. No. 7,037,650 is used in some embodiments.

[0024] When assessing methylation status, the methylation status is often expressed as the proportion or percentage of an individual DNA strand that is methylated at a particular site (e.g., at a single nucleotide, at a particular region or locus, at a relatively long sequence of interest, e.g., up to about 100 bp, 200 bp, 500 bp, 1000 bp subsequence of DNA or more) compared to the DNA population in the sample that contains that particular site. Traditionally, the amount of unmethylated nucleic acid is determined by PCR using calibrators. A known amount of DNA is then bisulfite treated (or non-bisulfite treated (see Liu et al., 2019, Nat Biotechnol. 37, pp. 424-429)) and the resulting methylation-specific sequences are determined using either real-time PCR or other exponential amplification, such as the QuARTS assay (e.g., those provided by U.S. Pat. Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392, which are incorporated by reference herein).

[0025] For example, in some embodiments, the method includes generating a standard curve for an unmethylated target by using an external standard. The standard curve is composed of at least two points and relates to the real-time Ct value of unmethylated DNA to a known quantitative standard. Then, a second standard curve for a methylated target is generated from at least two points and an external standard. This second standard curve relates to the Ct of methylated DNA to a known quantitative standard. Then, the Ct values ​​of the test samples are determined for the methylated and unmethylated populations, and the genome equivalent of DNA is calculated from the standard curve generated by the first two steps. The percentage of methylation at the site of interest is calculated from the amount of methylated DNA relative to the total amount of DNA in the population, for example, (number of methylated DNA) / (number of methylated DNA+number of unmethylated DNA)×100.

[0026] 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.

[0027] Also provided herein are compositions and kits for carrying out the methods. For example, in some embodiments, one or more MDM-specific reagents (e.g., primers, probes) 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, sequencing, bisulfite, Ten-Eleven translocation (TET) enzymes (e.g., human TET1, human TET2, human TET3, mouse TET1, mouse TET2, mouse TET3, Naegleria TET (NgTET), Coprinopsis cinerea (CcTET)), or variants thereof), organoborane, or other assays, buffers, positive and negative controls) can also be provided. In some embodiments, the kit contains a reagent capable of modifying DNA in a methylation-specific manner (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 (CcTET)), or a variant thereof), an organoborane), and / or an agent capable of detecting an increase in the level of a protein marker described herein. In some embodiments, a kit is provided that contains one or more reagents necessary, sufficient, or useful for carrying out the method. 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.

[0028] In some embodiments, the technology described herein relates to a programmable machine designed to perform a sequence of arithmetic or logical operations provided by the methods described herein. For example, some embodiments of the technology relate to (e.g., are implemented in) computer software and / or computer hardware. In one aspect, the technology relates 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 sequence of instructions (e.g., the methods provided herein) to read, manipulate, and store data. In some embodiments, the microprocessor is part of a system for: determining the methylation status (e.g., of one or more DMRs, e.g., DMRs 1-331 as shown in Tables 1A, 2A, 7A, 8A, and 9); comparing the methylation status (e.g., of one or more DMRs, e.g., DMRs 1-331 as shown in Tables 1A, 2A, 7A, 8A, and 9); generating a standard curve; determining a Ct value; calculating the proportion, frequency, or percentage of methylation (e.g., of one or more DMRs, e.g., DMRs 1-331 as shown in Tables 1A, 2A, 7A, 8A, and 9); identifying CpG islands; determining the specificity and / or sensitivity of an assay or marker; calculating the ROC curve and associated AUC; and sequence analysis; all as described herein or known in the art.

[0029] In some embodiments, the microprocessor or computer uses the methylation status data in an algorithm to predict the site of cancer.

[0030] 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 shown in Tables 1A, 2A, 7A, 8A, and 9)). Related embodiments calculate a risk factor based on a mathematical combination (e.g., weighted combination, linear combination) of results from multiple assays, e.g., determining the methylation status of multiple markers (such as multiple DMRs as shown in Tables 1A, 2A, 7A, 8A, and 9). In some embodiments, the methylation status of the DMRs defines a dimension 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.

[0031] Some embodiments include storage media and memory components. The memory components (e.g., volatile and / or non-volatile memory) are used to store instructions (e.g., embodiments of the processes provided herein) and / or data (e.g., work pieces such as methylation measurements, sequences, and statistical descriptions associated therewith). Some embodiments also relate to systems that include one or more of a CPU, a graphics card, and a user interface (e.g., including an output device such as a display and an input device such as a keyboard).

[0032] Programmable machines relevant to the present technology include conventional existing technologies as well as technologies under development or yet to be developed (eg, quantum computers, chemical computers, DNA computers, optical computers, spintronics-based computers, etc.).

[0033] In some embodiments, the technology involves wired (e.g., metal cable, fiber optics) or wireless transmission media for transmitting data. For example, some embodiments relate to data transmission over a network (e.g., a local area network (LAN), a wide area network (WAN), an ad-hoc network, the Internet, etc.). In some embodiments, the programmable machines reside on the network as peers, and in some embodiments, the programmable machines have a client / server relationship.

[0034] In some embodiments, the data is stored on a computer readable storage medium, such as a hard disk, flash memory, optical media, floppy disk, or the like.

[0035] In some embodiments, the technology provided herein relates to multiple programmable devices that operate together to perform the methods described herein. For example, in some embodiments, multiple computers (e.g., connected by a network) can operate in parallel to collect and process data, for example 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.) connected to a network (private, public, or Internet) by traditional network interfaces such as Ethernet, fiber optics, etc., or by wireless network technology.

[0036] For example, some embodiments provide a computer including a computer-readable medium. An embodiment includes a random access memory (RAM) coupled to a processor. The processor executes computer-executable program instructions stored in the memory. Such a processor may include a microprocessor, an ASIC, a state machine, or other processor, and may be any of a number of computer processors, such as processors from Intel Corporation of Santa Clara, California and Motorola Corporation of Schaumburg, Illinois. Such a processor may include or be in communication with a medium, e.g., a computer-readable medium, that stores instructions that, when executed by the processor, cause the processor to perform the steps described herein.

[0037] Examples of computer-readable media include, but are not limited to, electronic, optical, magnetic, or other storage or transmission devices capable of providing computer-readable instructions to a processor. Other examples of suitable media include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROMs, RAMs, ASICs, configured processors, any optical media, any magnetic tape or other magnetic media, or any other media from which a computer processor can read instructions. Various other forms of computer-readable media can also transmit or convey instructions to a computer, including routers, both wired and wireless, private or public networks, or other transmission devices or channels. Instructions can include code from any suitable computer programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, and JavaScript.

[0038] The computer is connected to a network in some embodiments. The computer may also include several external or internal devices, such as a mouse, CD-ROM, DVD, keyboard, display, or other input or output devices. Examples of computers include personal computers, digital assistants, personal digital assistants, cellular phones, mobile phones, and the like. Examples of computer-based applications include personal computers, mobile phones, smartphones, pagers, digital tablets, laptop computers, Internet appliances, and other processor-based devices. Generally, computers relevant to aspects of the technology provided herein may be any type of processor-based platform running any operating system capable of supporting one or more programs including the technology provided herein, e.g., Microsoft Windows, Linux, UNIX, Mac OS X, etc. Some embodiments include personal computers running other application programs (e.g., applications). Applications may be stored in memory and may 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.

[0039] All such components, computers, and systems described herein as relating to the present technology may be logical or virtual.

[0040] Thus, provided herein are techniques for methods of screening for melanoma and / or various forms of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma) in a sample obtained from a subject, the methods comprising assaying the methylation status of a marker in a sample (e.g., melanoma tissue) obtained from the subject (e.g., tissue sample, plasma sample), and identifying the subject as having melanoma and / or various forms of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma) if the methylation status of the marker differs from the methylation status of the marker assayed in a subject without such cancer, wherein the marker comprises a base in a variably methylated region (DMR) selected from the group consisting of DMRs 1-331 as shown in Tables 1A, 2A, 7A, 8A, and 9.

[0041] In some embodiments, where the sample obtained from the subject is skin tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without melanoma, it is indicative that the subject has melanoma: c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13 .29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-71116122, MAX .chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853 , MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (see Tables 4, 5A, 5B).

[0042] In some embodiments, where the sample obtained from the subject is skin tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without melanoma, it is indicative that the subject has melanoma: MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KC NQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see Tables 9, 10, 11).

[0043] In some embodiments, where the sample obtained from the subject is skin tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without melanoma, the subject is indicated to have melanoma: AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-32237785, MAX.chr10 .62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.54970444-54970469, MAX.chr19. 16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.1 29425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (see Tables 7A and 7B).

[0044] In some embodiments, where the sample obtained from the subject is skin tissue, the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without metastatic melanoma, indicating that the subject has metastatic melanoma: MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see Table 5C, Example I).

[0045] In some embodiments, where the sample obtained from the subject is skin tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject that does not have metastatic melanoma, the methylation state of one or more of the following markers is indicative of the subject having metastatic melanoma: MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A , KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3 , HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB , FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see Tables 9, 10 and 11).

[0046] In some embodiments, where the sample obtained from the subject is skin tissue, the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject who does not have primary cutaneous melanoma, indicating that the subject has primary cutaneous melanoma: MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see Table 5C, Example I).

[0047] In some embodiments, where the sample obtained from the subject is skin tissue, and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without primary cutaneous melanoma, the subject is indicated to have primary cutaneous melanoma: MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4 _A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME 3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2N B, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see Tables 9, 10, and 11).

[0048] In some embodiments, the sample obtained from the subject is a blood sample (e.g., a plasma sample, a whole blood sample, a white blood cell sample, a serum sample), and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without melanoma, indicating that the subject has melanoma: c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-7111612 2, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-6092185 3, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (Tables 4, 5A, 5B, 6; see Example I).

[0049] In some embodiments, the sample obtained from the subject is a blood sample (e.g., a plasma sample, a whole blood sample, a white blood cell sample, a serum sample), and the methylation state of one or more of the following markers differs from the methylation state of one or more markers assayed in a subject without melanoma, indicating that the subject has melanoma: MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921 853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541 948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FO XL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see Tables 9, 10, and 11).

[0050] In some embodiments, where the sample obtained from the subject is a blood sample (e.g., a plasma sample, a whole blood sample, a white blood cell sample, a serum sample), and the methylation status of one or more of the following markers differs from the methylation status of one or more markers assayed in a subject without melanoma, this indicates that the subject has melanoma: one or more markers listed in Table 8A.

[0051] The present technology relates to the identification and differentiation of melanoma and / or various forms of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma). Some embodiments involve the use of multiple markers, e.g., 1, 2, 3, 2-11-100 or 120 or 331 markers (e.g., 1-4, 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, 1-25, 1-50, 1-75, 1-100, 1-200, 1-300, 1-400, 1-500, 1-600, 1-750 ... , 1-331 pieces) (e.g., 2-4, 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, 2-25, 2-50, 2-75, 2-100, 2-200, 2-300, 2-331 pieces) (e.g., 3-4, 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, 3-25, 3-50, 3-75, 3-100, 3-200, 3-300, 3-331) (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, 4-25, 4-5 0, 4 to 75, 4 to 100, 4 to 200, 4 to 300, 4 to 331) (e.g., 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 11, 5 to 12, 5 to 13, 5 to 14, 5 to 15, 5 to 16, 5 to 17, 5 to 18, 5 to 19, 5 to 20, 5 to 25, 5 to 50, 5 to 75, 5 to 100, 5 to 200, 5 to 300, 5 to 331).

[0052] The technology is not limited to the methylation state that is assessed. In some embodiments, assessing the methylation state of a marker in a sample includes determining the methylation state of one base. In some embodiments, assaying the methylation state of a marker in a sample includes determining the degree of methylation at a plurality of bases. Further, in some embodiments, the methylation state of a marker includes an increase in methylation of the marker compared to the normal methylation state of the marker. In some embodiments, the methylation state of a marker includes a decrease in methylation of the marker compared to the normal methylation state of the marker. In some embodiments, the methylation state of a marker includes a different pattern of methylation of the marker compared to the normal methylation state of the marker.

[0053] 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 high CpG density promoter.

[0054] The present technology is not limited by the type of sample. For example, in some embodiments, the sample is a fecal sample, a tissue sample (e.g., a skin tissue sample), lymphatic tissue, a deep tissue biopsy, a blood sample (e.g., plasma, serum, whole blood), feces, or a urine sample. In some embodiments, the sample is a fine needle aspirate. In some embodiments, the sample is taken using a sampling device such as an adhesive swab or tape to collect cells on the skin surface. Malignant melanoma is characterized by spreading to other organs and deep tissue types, particularly lymph nodes, as well as other organs such as the lungs, liver, bone, or brain.

[0055] Furthermore, the present technology is not limited to the method used to determine the methylation status. In some embodiments, the assaying includes using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture. In some embodiments, the assaying includes using methylation-specific oligonucleotides. In some embodiments, the present technology uses massively parallel sequencing (e.g., next-generation sequencing), such as sequencing-by-synthesis, real-time (e.g., single molecule) sequencing, bead emulsion sequencing, nanopore sequencing, etc. to determine the methylation status.

[0056] The present technology provides reagents for detecting DMRs, for example, in some embodiments, a set of oligonucleotides is provided that includes sequences shown by SEQ ID NOs: 1-167 (see Tables 3, 10, and 11). In some embodiments, oligonucleotides are provided that include sequences complementary to chromosomal regions that have bases in the DMR, for example, oligonucleotides that are highly sensitive to the methylation status of the DMR.

[0057] The present technology provides a panel of various markers used to identify melanoma, for example, in some embodiments, the markers are c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-7 3073830, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (see Tables 4, 5A, 5B).

[0058] The present technology provides a panel of various markers used to identify melanoma, for example, in some embodiments, the markers are MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, The chromosomal regions with the following annotations are included: MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see Tables 9, 10, 11).

[0059] The present technology provides a panel of various markers used to identify melanoma, for example, in some embodiments, the markers are AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535- 14926715, MAX.chr16.54970444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.210806 70-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82 The present invention includes chromosomal regions with the following annotations: 543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (see Tables 7A and 7B).

[0060] The present technology provides a panel of various markers used to identify metastatic melanoma, for example, in some embodiments, the markers include chromosomal regions with the following annotations: MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see Table 5C, Example I).

[0061] The present technology provides a panel of various markers that are used to identify metastatic melanoma, for example, in some embodiments, the markers are MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B , MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see Tables 9, 10, 11).

[0062] The present technology provides a panel of various markers used to identify primary cutaneous melanoma, for example, in some embodiments, the markers include chromosomal regions with the following annotations: MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see Table 5C, Example I).

[0063] The present technology provides a panel of various markers that are used to identify primary cutaneous melanoma, for example, in some embodiments, the markers are MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_ B, including chromosomal regions with the following annotations: MAX.chr10.22541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see Tables 9, 10, 11).

[0064] The present technology provides a panel of various markers used to identify melanoma, for example, in some embodiments, the markers include chromosomal regions having annotations listed in Table 8A (see, e.g., Example II).

[0065] Embodiments of kits are provided, for example kits that include a reagent capable of modifying DNA in a methylation-specific manner (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 (CcTET)), or a variant thereof, an organoborane) and a control nucleic acid that includes one or more sequences from DMR1-331 (of Tables 1A, 2A, 7A, 8A, and 9) and has a methylation status associated with a subject without cancer. In some embodiments, the kit includes a bisulfite reagent and an oligonucleotide as described herein. In some embodiments, the kit includes reagents capable of modifying DNA in a methylation-specific manner (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 (CcTET)), or a variant thereof, an organoborane), and a control nucleic acid that includes one or more sequences from DMR1-331 (of Tables 1A, 2A, 7A, 8A, and 9) and has a methylation status associated with a subject having a particular type of cancer. Some kit embodiments include a sample collector for obtaining a sample from a subject (e.g., a fecal sample, a tissue sample, a plasma sample, a serum sample, a whole blood sample), reagents capable of modifying DNA in a methylation-specific manner (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 (CcTET)), or a variant thereof), an organoborane), and an oligonucleotide as described herein.

[0066] The present technology relates to embodiments of 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 Ten Eleven Translocation (TET) 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, an organoborane). 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.

[0067] Additional related method embodiments are provided for screening for melanoma and / or various forms of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma) in a sample obtained from a subject (e.g., a stool sample, a skin tissue sample, a fine needle aspirate, a deep tissue sample, a plasma sample, a serum sample, a whole blood sample), for example, the method includes determining the methylation status of a marker in a sample that includes a base in a DMR that is one or more of DMRs 1-331 (of Tables 1A, 2A, 7A, 8A and 9), comparing the methylation status of the marker of the subject sample to the methylation status of the marker of a normal control sample from a subject without melanoma (e.g., melanoma, and / or a form of melanoma: metastatic melanoma, primary cutaneous melanoma), and determining a confidence interval and / or p-value for the difference in methylation status of the subject sample and the normal control sample. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% or 99.99% and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001. Some embodiments of the method provide for reacting the DMR-containing nucleic acid with a reagent capable of modifying the nucleic acid in a methylation-specific manner (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 (CcTET)), or a variant thereof, an organoborane) to, for example, produce a methylation-specifically modified nucleic acid; sequencing the methylation-specifically modified nucleic acid to obtain a nucleotide sequence of the methylation-specifically modified nucleic acid; comparing the nucleotide sequence of the methylation-specifically modified nucleic acid to a nucleotide sequence of a DMR-containing nucleic acid from a subject that does not have the particular type of cancer to identify differences between the two sequences; and, if differences are present, identifying the subject as having melanoma and / or a form of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma).

[0068] A system for screening for melanoma in a sample obtained from a subject is provided by the present technology. Exemplary embodiments of the system include a system for screening for melanoma and / or a type of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma) in a sample obtained from a subject (e.g., a stool sample, a skin tissue sample, a fine needle aspirate, a deep tissue sample, a plasma sample, a serum sample, a whole blood sample), the system includes an analysis component configured to determine a methylation status of the sample, a software component configured to compare the methylation status of the sample with the methylation status of a control or reference sample recorded in a database, and an alert component configured to alert a user of a melanoma-associated methylation status. The alert is determined in some embodiments by a software component that receives results from a plurality of assays (e.g., determining the methylation status of a plurality of markers, e.g., DMRs, e.g., as shown in Tables 1A, 2A, 7A, 8A, and 9), calculates a value or result, and reports based on the plurality of results. Some embodiments provide a database of weighting parameters associated with each DMR provided herein for use in calculating a value or result and / or alert reporting to a user (e.g., a doctor, nurse, clinician, etc.). In some embodiments, all of the results from the multiple assays are reported, and in some embodiments, one or more results are used to provide a score, value, or result based on a combined one or more results from the multiple assays that is indicative of cancer risk in a subject.

[0069] In some embodiments of the system, the sample comprises a nucleic acid that comprises a DMR. In some embodiments, the system further comprises a component for isolating the nucleic acid, a component for collecting the sample, e.g., a component for collecting a fecal sample, etc. In some embodiments, the system comprises a nucleic acid sequence that comprises a DMR. In some embodiments, the database comprises nucleic acid sequences from subjects that do not have melanoma and / or a particular type of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma). Also provided are nucleic acids, e.g., sets of nucleic acids, each nucleic acid having a sequence that comprises a DMR. In some embodiments, the set of nucleic acids, each nucleic acid having a sequence from a subject that does not have melanoma and / or a particular type of melanoma. Related system embodiments include sets of nucleic acids as described and databases of nucleic acid sequences related to the set of nucleic acids. Some embodiments further include a reagent capable of modifying DNA in a methylation-specific manner (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 (CcTET)), or a variant thereof), an organoborane). Some embodiments also include a nucleic acid sequencer.

[0070] In certain embodiments, methods are provided for characterizing a sample (e.g., a stool sample, a skin tissue sample, a fine needle aspirate, a deep tissue sample, a plasma sample, a serum sample, a whole blood sample) from a human patient. For example, in some embodiments, such embodiments include obtaining DNA from the human patient sample, assaying the methylation status of DNA methylation markers that include bases in a variably methylated region (DMR) selected from the group consisting of DMRs 1-331 of Tables 1A, 2A, 7A, 8A, and 9, and comparing the assayed methylation status of the one or more DNA methylation markers to a reference methylation level of the one or more DNA methylation markers from human patients who do not have melanoma and / or a particular type of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma).

[0071] Such methods are not limited to a particular type of sample from a human patient. In some embodiments, the sample is a skin tissue sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a fecal sample, a tissue sample, a skin tissue sample, a blood sample (e.g., a fecal sample, a skin tissue sample, a fine needle aspirate, a deep tissue sample, a plasma sample, a serum sample, a whole blood sample).

[0072] In some embodiments, such methods include multiple (e.g., 1-4, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-16, 1-17, 1-18, 1-19, 1-20, 1-25, 1-50, 1-75, 1-100, 1-200, 1-300, 1-331) (e.g., 2-4, 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, 2-25, 2-50, 2-75, 2-100, 2-200, 2-300, 2-331) ~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, 2~25, 2~50, 2~75, 2~100, 2~200, 2~300, 2~331) (e.g., 3~4, 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, 3~25, 3~50, 3~75, 3~100, 3~200, 3~300, 3~331) (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, 4~25, 4~50, 4~75, 4~1 00, 4-200, 4-300, 4-331) (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, 5-25, 5-50, 5-75, 5-100, 5-200, 5-300, 5-331). In some embodiments, such methods include assaying 2-11 DNA methylation markers. In some embodiments, such methods include assaying 12-120 DNA methylation markers. In some embodiments, such methods include assaying 2-331 DNA methylation markers. In some embodiments, such methods include assaying the methylation status of one or more DNA methylation markers in a sample, including determining the methylation status of a single base. In some embodiments, such methods include assaying the methylation status of one or more DNA methylation markers in a sample, including determining the degree of methylation at a plurality of bases.In some embodiments, such methods include assaying the methylation state of the forward strand or assaying the methylation state of the reverse strand.

[0073] In some embodiments, the DNA methylation marker is a region of 100 bases or less. In some embodiments, the DNA methylation marker is a region of 500 bases or less. In some embodiments, the DNA methylation marker is a region of 1000 bases or less. In some embodiments, the DNA methylation marker is a region of 5000 bases or less. In some embodiments, the DNA methylation marker is a single base. In some embodiments, the DNA methylation marker is in a high CpG density promoter.

[0074] In some embodiments, the assaying comprises using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture.

[0075] In some embodiments, the assaying comprises the use of a methylation-specific oligonucleotide. In some embodiments, the methylation-specific oligonucleotide is selected from the group consisting of SEQ ID NOs: 1-80 (Table 3).

[0076] In some embodiments, c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-71116122, MA Chromosomal regions having annotations selected from the group consisting of X.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (see Tables 4, 5A, 5B, and 6) comprise DNA methylation markers.

[0077] In some embodiments, AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-32237785, MAX.c hr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.54970444-54970469 , MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (see Tables 7A and 7B) comprise DNA methylation markers.

[0078] In some embodiments, MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HOPX , MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see Tables 9, 10, 11).

[0079] In some embodiments, the chromosomal regions having annotations listed in Table 8A (see Example II) comprise DNA methylation markers.

[0080] In some embodiments, a chromosomal region having an annotation selected from the group consisting of MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see Table 5C, Example I) comprises a DNA methylation marker.

[0081] In some embodiments, such methods comprise determining the methylation status of two DNA methylation markers. In some embodiments, such methods comprise determining the methylation status of a pair of DNA methylation markers provided in Tables 1A, 2A, 7A8A, and / or 9.

[0082] In certain embodiments, the technology provides methods for characterizing samples obtained from human patients (e.g., fecal samples; skin tissue samples; fine needle aspirates, deep tissue samples, plasma samples, serum samples, whole blood samples). In some embodiments, such methods include determining the methylation status of DNA methylation markers in samples that contain bases in a DMR selected from the group consisting of DMRs 1-331 of Tables 1A, 2A, 7A, 8A, and 9, comparing the methylation status of the DNA methylation markers of the patient sample to the methylation status of the DNA methylation markers of a normal control sample from a human subject that does not have melanoma and / or a particular form of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma), and determining a confidence interval and / or p-value for the difference in the methylation status of the human patient and normal control samples. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% or 99.99% and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

[0083] In certain embodiments, the technology provides a method for characterizing a sample obtained from a human subject (e.g., a stool sample; a skin tissue sample; a fine needle aspirate, a deep tissue sample, a plasma sample, a serum sample, a whole blood sample), the method comprising reacting a nucleic acid comprising a DMR with a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent) to produce a methylation-specifically modified nucleic acid, sequencing the methylation-specifically modified nucleic acid to obtain a nucleotide sequence of the methylation-specifically modified nucleic acid, and comparing the nucleotide sequence of the methylation-specifically modified nucleic acid to the nucleotide sequence of a nucleic acid comprising a DMR from a subject without melanoma to identify differences between the two sequences.

[0084] In certain embodiments, the technology provides a system for characterizing a sample obtained from a human subject (e.g., a fecal sample; a skin tissue sample; a fine needle aspirate, a deep tissue sample, a plasma sample, a serum sample, a whole blood sample), the system including an analysis component configured to determine a methylation state of the sample, a software component configured to compare the methylation state of the sample with the methylation states of control or reference samples recorded in a database, and an alert component configured to determine a single value based on a combination of the methylation states and alert a user of a melanoma-associated methylation state. In some embodiments, the sample includes a nucleic acid that includes a DMR.

[0085] In some embodiments, such systems further comprise a component for isolating nucleic acid, hi some embodiments, such systems further comprise a component for collecting the sample.

[0086] In some embodiments, the sample is a stool sample, a tissue sample, a skin tissue sample, a fine needle aspirate, a deep tissue sample, a lymph node sample, a blood sample (e.g., a plasma sample, a whole blood sample, a serum sample), or a urine sample.

[0087] In some embodiments, the database comprises nucleic acid sequences that contain DMRs, hi some embodiments, the database comprises nucleic acid sequences from subjects that do not have melanoma.

[0088] In some embodiments, the sample is a fecal sample, a tissue sample (e.g., skin tissue), a blood sample (e.g., a plasma sample, a whole blood sample, a serum sample), 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 feces. In some embodiments, the subject is a human. The sample may comprise cells, secretions, or tissues from lymph glands, breast, liver, bile duct, pancreas, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gallbladder, anus, and / or peritoneum. In some embodiments, the sample comprises cell fluids, ascites, urine, feces, stomach sections, pancreatic juice, bodily fluids obtained during endoscopy, blood.

[0089] Further embodiments will be apparent to those of ordinary skill in the relevant arts based on the teachings contained herein.

[0090] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11).

[0091] [Definition] To facilitate understanding of the present technology, several terms and phrases are defined below. Further definitions are set forth throughout the detailed description.

[0092] Throughout this specification and 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 refer to the same embodiment, but not necessarily the same embodiment. Additionally, the phrase "in another embodiment" as used herein may refer to different embodiments, but not necessarily the same embodiment. Thus, as described below, various embodiments of the invention can be readily combined without departing from the scope or spirit of the invention.

[0093] 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."

[0094] The transitional phrase "consisting essentially of," when used in the claims of this application, shall be construed as follows: In re Herz, 537 F.2d 549,551-52,190 USPQ As stated in 461, 463 (CCPA 1976), patent claims are limited to certain substances or steps "and which do not materially affect the basic and novel characteristic(s)" of the claimed invention. For example, a composition "consisting essentially of" recited elements may contain unrecited contaminants at levels that, although present, do not alter the function of the recited composition when compared to a pure composition, i.e., a composition "consisting of" the recited ingredients.

[0095] 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 such chemically, enzymatically, or metabolically modified forms of nucleic acid, as well as the chemical forms of DNA characteristic of viruses and cells (including, for example, simple and complex cells).

[0096] The term "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refers to a molecule having 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.

[0097] 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.

[0098] The terms "complementary" and "complementarity" refer to nucleotides (e.g., a single nucleotide) or polynucleotides (e.g., a sequence of nucleotides) related by 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 nucleic acid strands affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and in detection methods that depend on binding between nucleic acids.

[0099] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises the 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. The term "portion," when used in reference to a gene, refers to a fragment of that gene. Fragments can range in size from a few nucleotides to the entire sequence of the gene minus one nucleotide. Thus, "nucleotides comprising at least a portion of a gene" can include a fragment of a gene or the entire gene.

[0100] The term "gene" also encompasses the coding region of a structural gene and includes sequences located adjacent to the coding region at both the 5' and 3' ends, e.g., at a distance of about 1 kb on either end, such that a gene corresponds to the length of the full-length mRNA (e.g., including coding, regulatory, structural and other sequences). 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 the coding region interrupted by non-coding sequences, termed "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, and therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0101] In addition to containing introns, genomic forms of a gene may also contain sequences located on both the 5' and 3' end of 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.

[0102] The term "wild type", when referring to a gene, refers to a gene that has the characteristics of a gene isolated from a naturally occurring source. The term "wild type", when referring to a gene product, refers to a gene product that has the characteristics of a gene product isolated from a naturally occurring source. The term "naturally occurring", when used with respect to an object, 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, the term "modified" or "mutant", when referring to a gene or gene product, refers to a gene or gene product that exhibits modifications (e.g., altered characteristics) in sequence and / or functional properties, respectively, when compared to the wild type gene or gene product. Note that naturally occurring mutants can be isolated and are identified by the fact that they have altered characteristics when compared to the wild type gene or gene product.

[0103] 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 mutations. Alleles may occur naturally in a population or may arise during the life of any particular individual in a population.

[0104] Thus, the terms "variant" and "mutant," when used in reference to a nucleotide sequence, refer to a nucleic acid sequence that differs from another, usually related, nucleotide sequence by one or more nucleotides. "Diversity" is the difference between two different nucleotide sequences, usually one of the sequences being a reference sequence.

[0105] "Amplification" is a special case of nucleic acid replication involving template specificity. It is in contrast to non-specific template replication (e.g., replication that is template-dependent but not dependent on a specific template). Template specificity is distinguished herein from fidelity of replication (e.g., synthesis of the appropriate polynucleotide sequence) and nucleotide (ribonucleotide or deoxyribonucleotide) specificity. Template specificity is often described in terms of "target" specificity. Target sequences are "targets" in the sense that they are sought to be sorted out from other nucleic acids. Amplification techniques are primarily designed for this sorting.

[0106] The term "amplifying" or "amplification" in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of a polynucleotide, usually starting from a small amount of polynucleotide (e.g., a single polynucleotide molecule), and the amplification product or amplicon is generally detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. The production of multiple DNA copies from one or a few copies of a target or template DNA molecule in a polymerase chain reaction (PCR) or ligase chain reaction (LCR, see, e.g., U.S. Patent No. 5,494,810, which is incorporated herein by reference in its entirety) is a form of amplification. Additional types of amplification include allele-specific PCR (see, e.g., U.S. Pat. No. 5,639,611, which is incorporated by reference in its entirety), assembly PCR (see, e.g., U.S. Pat. No. 5,965,408, which is incorporated by reference in its entirety), helicase-dependent amplification (see, e.g., U.S. Pat. No. 7,662,594, which is incorporated by reference in its entirety), hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and 5,338,671, each of which is incorporated by reference in its entirety), inter-sequence specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186, which is incorporated by reference in its entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Res., 16:8186, which is incorporated by reference in its entirety), and ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Res., 16:8186, which is incorporated by reference in its entirety). Research, 25:1854-1858 (1997); U.S. Pat. No. 5,508,169, each of which is incorporated herein by reference in its entirety), methylation-specific PCR (see, e.g., Herman, et al., (1996) PNAS 93(13) 9821-9826, which is incorporated herein by reference in its entirety), miniprimer PCR, multiplex ligation-dependent probe amplification (see, e.g., Schouten, et al., (2002) Nucleic Acids Research 30(12):e57, which is incorporated herein by reference in its entirety), multiplex PCR (see, e.g., Chamberlain, et al., (1988) Nucleic Acids Research 16(23)11141-11156; Ballabio, et al., (1990) Human Genetics 84(6)571-573; Hayden, et al., (2008) BMC Genetics 9:80, each of which is incorporated herein by reference in its entirety), nested PCR, overlap extension PCR (see, e.g., Higuchi, et al., (1988) Nucleic Acids Research 16(15)7351-7367, which is incorporated herein by reference in its entirety), real-time PCR (see, e.g., Higuchi, et al. al., (1992) Biotechnology 10:413-417; Higuchi, et al., (1993) Biotechnology 11:1026-1030, each of which is incorporated herein by reference in its entirety), reverse transcription PCR (see, e.g., Bustin, SA (2000) J. Molecular Endocrinology 25:169-193, which is incorporated herein by reference in its entirety), solid-phase PCR, thermal asymmetric interlaced PCR, as well as touchdown PCR (see, e.g., Don, et al., Nucleic Acids Research (1991) 19(14) 4008; Roux, K. (1994) Biotechniques 16(5) 812-814; Hecker, et al., (1996) Biotechniques 20(3)478-485, each of which is incorporated herein by reference in its entirety. Polynucleotide amplification can also be performed using digital PCR (see, e.g., Kalinina, et al., Nucleic Acids Research.25;1999-2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96;9236-41, (1999); International Patent Publication No. WO05023091A2; U.S. Patent Application Publication No. 20070202525, each of which is incorporated herein by reference in its entirety.

[0107] The term "polymerase chain reaction" ("PCR") refers to the method of K.B. Mullis, U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188, which describes a method for increasing the concentration of a segment of a target sequence in a mixture of genomic or other DNA or RNA without cloning or purification. This process for amplifying a target sequence consists of introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by thermal cycling in the presence of DNA polymerase in a precise order. The two primers are complementary to their respective strands of the double-stranded target sequence. To effect amplification, the mixture is denatured and the primers are then annealed to their complementary sequences within the target molecule. After annealing, the primers are extended with polymerase to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing, and extension constitute one "cycle" and there can be many "cycles") to obtain a highly concentrated amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore this length is a controllable parameter. Due to the repetitive aspect of the process, this method is referred to as "polymerase chain reaction" ("PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified" and they are "PCR products" or "amplicons". Those skilled in the art will understand that the term "PCR" encompasses many variations of the originally described method, such as using real-time PCR, nested PCR, reverse transcription PCR (RT-PCR), single primer and arbitrarily primed PCR, etc.

[0108] Template specificity is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under the conditions in which they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Q-beta replicase, MDV-1 RNA is a specific template for the replicase (Kacian et al., Proc. Natl. Acad. Sci. USA, 69:3038

[1972] ). No other nucleic acid is replicated by the amplification enzyme. Similarly, in the case of T7 RNA polymerase, the amplification enzyme has stringent specificity for its own promoter (Chamberlin et al, Nature, 228:227

[1970] ). In the case of T4 DNA ligase, the enzyme will not ligate two oligonucleotides or polynucleotides if there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (Wu and Wallace (1989) Genomics 4:560). Finally, thermostable template-dependent DNA polymerases (e.g., Taq and Pfu DNA polymerases) have been found to exhibit a high degree of specificity for the sequences bound by and thereby defined by the primers, due to their ability to function at high temperatures, which provide thermodynamic conditions that favor primer hybridization with the target sequence but not with non-target sequences (HA Erlich (ed.), PCR Technology, Stockton Press

[1989] ).

[0109] As used herein, the term "nucleic acid detection assay" refers to any method for determining the nucleotide composition of a nucleic acid of interest. Nucleic acid detection assays include DNA sequencing, probe hybridization, structure-specific cleavage assays (e.g., INVADER assay (Hologic, Inc.) and methods described in, for example, U.S. Patent Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev et al., Nat. Biotech., 17:292 (1999); Hall et al., PNAS, USA, 97:8272 (2000), and U.S. Pat. No. 9,096,893, each of which is incorporated by reference in its entirety for all purposes); enzymatic mismatch cleavage methods (e.g., Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, and 5,851,770, each of which is incorporated by reference in its entirety); the polymerase chain reaction (PCR) described above; branched hybridization methods (e.g., Chiron, U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, each of which is incorporated by reference in its entirety); rolling circle replication (e.g., Nos. 6,210,884, 6,183,960, and 6,235,502, which are incorporated herein by reference in their entirety; NASBA (e.g., U.S. Pat. No. 5,409,818, which is incorporated herein by reference in its entirety); molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, which is incorporated herein by reference in its entirety); E-sensor technology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, which are incorporated herein by reference in their entirety); cycling probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, which are incorporated herein by reference in their entirety); Examples of suitable hybridization techniques include, but are not limited to, the Behring signal amplification method (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, which are incorporated herein by reference in their entireties); the ligase chain reaction (e.g., Baranay Proc. Natl. Acad. Sci USA 88,189-93 (1991)); and the sandwich hybridization method (e.g., U.S. Pat. No. 5,288,609, which is incorporated herein by reference in its entirety).

[0110] The term "amplifiable nucleic acid" refers to a nucleic acid that can be amplified by any amplification method. "Amplifiable nucleic acid" is generally intended to include a "sample template."

[0111] The term "sample template" refers to nucleic acid originating from a sample that is analyzed for the presence of a "target" (defined below). In contrast, "background template" is used in reference to nucleic acid other than the sample template that may or may not be present in the sample. Background template is most often inadvertent. Background template may be the result of carryover, or it may be due to the presence of nucleic acid contaminants that are sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in the test sample.

[0112] The term "primer" refers to an oligonucleotide, either naturally occurring, for example as a nucleic acid fragment from a restriction digest, or synthetically produced, which can serve as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid template strand is induced (e.g., in the presence of nucleotides and an inducer such as DNA polymerase, and at a suitable temperature and pH). 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 sufficiently long to prime the synthesis of the extension product in the presence of the inducer. The exact length of the primer will depend on many factors, including temperature, source of primer, and use of the method.

[0113] The term "probe" refers to an oligonucleotide (e.g., a sequence of nucleotides), either naturally occurring, as in a purified restriction digest, or produced synthetically, recombinantly, or by PCR amplification, that 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" such that it is detectable by 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.

[0114] The term "target" as used herein refers to a nucleic acid that is sought 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.

[0115] 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, for example, in a sample that does not contain a target sequence, while in some embodiments, a non-target nucleic acid may refer to an exogenous nucleic acid, i.e., a nucleic acid that does not originate from a sample that contains or is suspected of containing a target nucleic acid, and a nucleic acid that is added to a reaction, for example, to normalize the activity of an enzyme (e.g., a polymerase) to reduce the variability of the enzyme's performance in the reaction. 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 not methylated because typical in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" may also refer to amplified DNA in which the original template was not methylated, or amplified DNA in which the original template was methylated, respectively.

[0116] As used herein, "amplification reagents" refers to the reagents necessary for amplification other than primers, nucleic acid template, and amplification enzymes (deoxyribonucleoside triphosphates, buffers, etc.) Typically, amplification reagents, along with other reaction components, are placed and contained in a reaction vessel.

[0117] As used herein, the term "control" used in relation to nucleic acid detection or analysis refers to a nucleic acid with known characteristics (e.g., known sequence, known copy number per cell) and used for comparison with an experimental target (e.g., a nucleic acid of unknown concentration). A control can be an endogenous, preferably invariant gene that can normalize the test or target nucleic acid in an assay. Such normalization controls allow accurate inter-sample data comparisons for sample-to-sample variations that may occur, for example, in sample processing, assay efficiency, etc. Genes that can be used to normalize nucleic acid detection assays for 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).

[0118] The control may be exogenous. For example, in quantitative assays such as qPCR, QuARTS, etc., a "calibrator" or "calibration control" is a nucleic acid of known sequence, e.g., a nucleic acid having the same sequence as a portion of an experimental target nucleic acid, and a known concentration or series of concentrations (e.g., a serially diluted control target for the generation of a calibration curve in quantitative PCR). Typically, the calibration control is analyzed using the same reagents and reaction conditions as those 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, so 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., WO2015 / 066695, which is incorporated herein by reference.

[0119] As used herein, "ZDHHC1" refers to a gene that is located in human DNA on Chr 16 (16q22.1) and encodes a protein characterized as zinc finger DHHC type-containing 1 that belongs to the DHHC palmitoyltransferase family. 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. Typical in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template, and therefore cannot be used to elucidate the methylation patterns of in vitro DNA amplification methods. In vitro amplified DNA is usually unmethylated. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA whose original template was unmethylated or methylated, respectively.

[0120] Thus, as used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, which is not present 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 present in DNA.

[0121] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

[0122] As used herein, the "methylation state," "methylation profile," and "methylation status" 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.

[0123] In some embodiments, the sample is a fecal sample, a tissue sample (e.g., skin tissue), a blood sample (e.g., a plasma sample, a whole blood sample, a serum sample), 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 feces. In some embodiments, the subject is a human. The sample may comprise cells, secretions, or tissues from lymph glands, breast, liver, bile duct, pancreas, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gallbladder, anus, and / or peritoneum. In some embodiments, the sample comprises cell fluids, ascites, urine, feces, stomach sections, pancreatic juice, bodily fluids obtained during endoscopy, blood.

[0124] 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.

[0125] Thus, a methylation state represents the state of methylation of a nucleic acid (e.g., a genomic sequence). Moreover, a methylation state refers to the 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 state", "methylation profile", and "methylation status" 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 hypermethylation 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 hypomethylation or decreased methylation compared to the other nucleic acid sequence. Furthermore, as used herein, the term "methylation pattern" refers to the collection of sites of methylated and unmethylated nucleotides across a region of a nucleic acid. Two nucleic acids may have the same or similar methylation frequency or percent methylation, 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 said to be "variably methylated" or to have "differential methylation" or to have "differential methylation states" if they differ in the degree (e.g., one has increased or decreased methylation compared to the other), frequency, or pattern of methylation. The term "variable 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. Variable 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.

[0126] 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.

[0127] As used herein, "nucleotide locus" refers to the position of a nucleotide in a nucleic acid molecule. The nucleotide locus of a methylated nucleotide refers to the position of the methylated nucleotide in a nucleic acid molecule.

[0128] Typically, methylation of human DNA occurs on dinucleotide sequences containing adjacent guanines and cytosines, where the cytosine is located 5' to the guanine (also called CpG dinucleotide sequences). Although many of the cytosines within CpG dinucleotides are methylated in the human genome, some remain unmethylated in specific CpG dinucleotide-rich genomic regions known as CpG islands (see, for example, Antequera et al. al. (1990) Cell 62:503-514).

[0129] As used herein, "CpG 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 (Ramsahoye (2000) Proc. Natl. Acad. Sci. USA 97:5237-5242; Salmon (see, for example, 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).

[0130] As used herein, a "methylation specific reagent" refers to a reagent that modifies the nucleotides of a nucleic acid molecule depending on the methylation state of the nucleic acid molecule, or a methylation specific reagent refers to a compound or composition or other agent that can change 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 include contacting the nucleic acid molecule with the reagent and, if desired, linking to additional steps to 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, and bisulfite reagents.

[0131] 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.

[0132] As used herein, the term "UDP-glucose modified with a chemoselective group" refers to a uridine diphosphoglucose molecule functionalized with a functional group, particularly at the 6-hydroxyl position, that can react with an affinity tag via click chemistry.

[0133] The term "oxidized 5-methylcytosine" refers to an oxidized 5-methylcytosine residue oxidized at the 5-position. Thus, oxidized 5-methylcytosine residues include 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxymethylcytosine. The oxidized 5-methylcytosine residues that undergo reaction with organoboranes according to one embodiment of the present invention are 5-formylcytosine and 5-carboxymethylcytosine.

[0134] 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.

[0135] The term "MS AP-PCR" (methylation-sensitive arbitrarily primed polymerase chain reaction) refers to an art-recognized technique that uses CG-rich primers to globally scan the genome and focus on regions most likely to contain CpG dinucleotides, and is described by Gonzalgo et al. (1997) Cancer Research 57:594-599.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] The term "COBRA" (Combined Bisulfite Restriction Analysis) refers to an art-recognized methylation assay described by Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534.

[0142] 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.

[0143] 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.

[0144] 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. For restriction enzymes that specifically cleave when the recognition site is unmethylated or hemimethylated (methylation-sensitive enzymes), cleavage will not occur (or will occur with significantly reduced efficiency) if the recognition site is methylated on one or both strands. For restriction enzymes that specifically cleave only when the recognition site is methylated (methylation-dependent enzymes), cleavage will not occur (or will occur with significantly reduced efficiency) if the recognition site is unmethylated. Methylation-specific restriction enzymes are preferred, whose recognition sequences contain CG dinucleotides (e.g., recognition sequences such as CGCG or CCCGGG). Further preferred for some embodiments are restriction enzymes that do not cleave when the cytosine in this dinucleotide is methylated at the carbon atom C5.

[0145] As used herein, "different nucleotide" refers to a nucleotide that is chemically different from the selected nucleotide, and typically results in that the different nucleotide has different Watson-Crick base pairing properties than the selected nucleotide, so that the commonly occurring nucleotide that is complementary to the selected nucleotide is not the same as the commonly occurring nucleotide that is complementary to the different nucleotide. For example, if C is the selected nucleotide, U or T can be the different nucleotide, as exemplified by the complementarity of C to G and the complementarity of U or T to A. As used herein, a nucleotide that is complementary to a selected nucleotide or a different nucleotide refers to a nucleotide that base pairs with the selected nucleotide or the different nucleotide under high stringency conditions with a higher affinity than the base pairing of the complementary nucleotide with three of the four commonly occurring nucleotides. One example of complementarity is the Watson-Crick base pairing of DNA (e.g., AT and CG) and RNA (e.g., AU and CG). Thus, for example, G base pairs with C under high stringency conditions with greater affinity than G base pairs with G, A, or T, and thus, when C is a selected nucleotide, G is the complementary nucleotide to the selected nucleotide.

[0146] 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 neoplasia reporting a DNA methylation value above the threshold (e.g., in the disease-associated range), and a false negative is defined as a histologically confirmed neoplasia reporting a DNA methylation value below the threshold (e.g., in the disease-unassociated range). Thus, the sensitivity value reflects the probability that a DNA methylation measurement value 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.

[0147] 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 non-disease associated range), 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 disease associated range). 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 in 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.

[0148] The term "AUC" as 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 various 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).

[0149] The term "neoplasm," as used herein, refers to any new abnormal growth of tissue. Thus, a neoplasm can be a premalignant or a malignant neoplasm.

[0150] 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.

[0151] 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.

[0152] The terms "precancerous" or "preneoplastic" and their equivalents refer to any cell proliferative disorder that has undergone malignant transformation.

[0153] A "site" of a neoplasm, adenoma, cancer, etc. is a tissue, organ, cell type, anatomical region, body part, etc. in a subject's body in which the neoplasm, adenoma, cancer, etc. is located.

[0154] As used herein, the application of a "diagnostic" test includes detecting or identifying 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 therapy, determining the prognosis (or likelihood of progression or regression) of a subject with a disease or condition, and determining the effect of a treatment on 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 favorably to a compound (e.g., a pharmaceutical, e.g., a drug) or other treatment.

[0155] 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 minimally contain a sense or coding strand (i.e., the oligonucleotide may be single-stranded), but may also contain both a sense and an antisense strand (i.e., the oligonucleotide may be double-stranded). The isolated nucleic acid is combined with other nucleic acids or molecules after isolation from its natural or normal environment. For example, the isolated nucleic acid may be present in a host cell, e.g., placed therein for heterologous expression.

[0156] 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 "to 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.

[0157] 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.).

[0158] The term "sample" is used in its broadest sense. In one sense, a sample can refer to animal cells or tissues. 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.

[0159] As used herein, a "remote sample," when used in some contexts, relates to a sample that is retrieved from a site other than the source of the sample's cells, tissues, or organs.

[0160] As used herein, the term "patient" or "subject" refers to an organism subjected to 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 the diagnostic method, 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. Thus, the present technology allows for the diagnosis of mammals, such as humans, as well as mammals of importance because they are endangered, such as the Amur tiger, mammals of economic importance, such as animals raised on farms for human consumption, and / or animals of social importance 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; pinnipeds; and horses. Thus, further provided are diagnostics and treatments for livestock, including, but not limited to, domesticated pigs, ruminants, ungulates, horses (including race horses), and the like. The subject matter disclosed herein further includes a system for diagnosing lung cancer in a subject. The system may be provided, for example, as a commercially available kit that may be used 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 according to the present technology includes assessing the methylation status of the markers described herein.

[0161] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of a reaction assay, such a delivery system includes a system that allows 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 enclosed containers (e.g., boxes) that contain the relevant reaction reagents and / or supporting materials. As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each of which contains a small portion of the total components of the kit. 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 "fragmented kit" is intended to encompass, but is not limited to, a kit that contains an analyte-specific reagent (ASR) 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 small portion of all the components of the kit, is included in the term "fragmented kit." In contrast, a "composite kit" refers to a delivery system that contains all the 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 fragmented and composite kits.

[0162] As used herein, the term "nevus" or "mole" refers to a non-cancerous skin growth that is typically composed of color (pigment) producing cells (melanocytes or nevus cells). Moles can appear singly or in groups anywhere on the skin.

[0163] As used herein, the term "lesion" refers to a mole that is under investigation (e.g., suspected to be cancerous or diagnosed as cancerous) and may or may not be cancerous. In some embodiments, "lesion" is used interchangeably with "mole" or "nevus."

[0164] As used herein, the term "melanoma" or "malignant melanoma" refers to a severe form of skin cancer that may affect only the skin or that can spread (metastasize) through the blood or lymphatic system to organs and bones. Melanoma may develop in an existing mole, or in another birthmark on the skin or on the skin without a birthmark.

[0165] As used herein, the term "metastatic melanoma" refers to melanoma that has spread to other tissues or organs.

[0166] 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 relating to 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 percentages, allele frequencies, RNA expression levels, protein expression, phenotypes associated with genotypes, and the like. "Allele frequency information" refers to facts or data about allele frequencies, including but not limited to allele identity information, statistical correlations between the presence of alleles and characteristics of a subject (e.g., a human subject), the presence or absence of alleles in an individual or population, the percentage likelihood of an allele being present in an individual with one or more particular characteristics, and the like.

[0167] Detailed Description In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will appreciate that these various embodiments may be practiced without or with 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 within the spirit and scope of the various embodiments disclosed herein.

[0168] Provided herein are technologies for melanoma screening, including, but not limited to, methods, compositions, and related applications for detecting the presence of melanoma and / or specific forms of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma). As the technology is described herein, the section headings used are for organizational purposes only and should not be construed as limiting the subject matter in any manner.

[0169] Indeed, as described in Examples I and II, experiments conducted during the course of identifying embodiments related to the present invention identified a novel set of 331 variably methylated regions (DMRs) for distinguishing cancer from non-neoplastic control DNA in melanoma-derived DNA. From these 331 novel DNA methylation markers, further experiments identified markers that can distinguish various types of melanoma from normal tissue. For example, individual sets of DMRs were identified that can distinguish 1) metastatic melanoma tissue from normal tissue, and 2) primary cutaneous melanoma tissue from normal tissue.

[0170] Although the disclosure herein refers to certain illustrated embodiments, it is to be understood that these embodiments are presented by way of example and not by way of limitation.

[0171] In certain aspects, the technology provides compositions and methods for identifying, determining, and / or classifying cancers such as melanoma and / or melanoma subtypes (e.g., metastatic melanoma, primary cutaneous melanoma). The methods include determining the methylation status of at least one methylation marker in a biological sample isolated from a subject (e.g., a stool sample; a skin tissue sample; a fine needle aspirate, a deep tissue sample, a plasma sample, a serum sample, a whole blood sample), where a change in the methylation status of the marker is indicative of the presence, class, or site of melanoma and / or its subtypes. Certain embodiments relate to markers comprising variably methylated regions (DMRs, e.g., DMRs 1-331, see Tables 1A, 2A, 7A, 8A, and 9) used in the diagnosis (e.g., screening) of melanoma and various types of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma).

[0172] In addition to the embodiments provided herein in which methylation analysis of at least one marker, region of a marker, or base of a marker that comprises a DMR (e.g., DMRs 1-331) set forth in Tables 1A, 2A, 7A, 8A, and 9 is analyzed, the present technology also provides panels of markers that comprise at least one marker, region of a marker, or base of a marker that comprises a DMR that are useful for detecting cancer, particularly melanoma.

[0173] Some embodiments of the technology are based on the analysis of the CpG methylation status of at least one marker, region of a marker, or base of a marker that comprises a DMR.

[0174] In some embodiments, the technology allows for the use of reagents that modify DNA in a methylation-specific manner (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents) in combination with one or more methylation assays to determine the methylation status of CpG dinucleotide sequences within at least one marker that comprises a DMR (e.g., DMR1-331, see Tables 1A, 2A, 7A, 8A, and 9). Genomic CpG dinucleotides can be methylated or unmethylated (alternatively known as up-methylated and down-methylated, respectively). However, the methods of the invention are suitable for the analysis of biological samples of heterogeneous nature, such as low concentrations of tumor cells, or biological material therefrom, within a background of distant samples (e.g., blood, organ waste, or feces). Thus, when analyzing the methylation status of CpG positions within such samples, quantitative assays can be used to determine the level (e.g., percentage, proportion, ratio, proportion, or degree) of methylation at a particular CpG position.

[0175] According to the present technology, determining the methylation status of CpG dinucleotide sequences in markers containing DMRs is useful for both the diagnosis and characterization of cancers such as melanoma.

[0176] Marker Combinations 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 reacts with bisulfite ions (also known as bisulfite), but 5-methylcytosine does not. The reaction is usually carried out according to the following steps: first, cytosine reacts with bisulfite salt to form sulfonated cytosine. Then, spontaneous deamination of the sulfonated reaction intermediate gives rise to 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).

[0177] 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.

[0178] Bisulfite technology usually involves amplifying short specific fragments of known nucleic acids after bisulfite treatment, then assaying the products to analyze individual cytosine positions either by sequencing (Olek & Walter (1997) Nat. Genet. 17:275-6) or primer extension reaction (Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-31, WO95 / 00669, U.S. Patent 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., WO99 / 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; WO9746705; WO9515373).

[0179] Various methylation assay procedures can be used in conjunction with bisulfite treatment according to the present technology. These assays can determine the methylation state 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 acid, PCR (for sequence-specific amplification), Southern blot analysis, and the use of methylation-specific restriction enzymes, e.g., methylation-sensitive or methylation-dependent enzymes.

[0180] 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 to assess methylation status, for example as described by Sadri & Hornsby (1997) Nucl. Acids Res. 24:5058-5059, or as embodied in the method known as COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534).

[0181] COBRA™ analysis is a quantitative methylation assay that is 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 specific labeled hybridization probes. Methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR products in a linear manner over a wide range of DNA methylation levels. Moreover, the technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.

[0182] Typical reagents for COBRA™ analysis (e.g., those that 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 denaturing buffers; sulfonation buffers; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffers; and DNA recovery components.

[0183] 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", 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.

[0184] The "HeavyMethyl™" assay technology is a quantitative method to assess methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific blocking probes ("blockers") covering the CpG positions between or covered by the amplification primers allow for methylation-specific selective amplification of nucleic acid samples.

[0185] 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.

[0186] Typical reagents for HeavyMethyl™ analysis (e.g., those that may be found in a typical MethyLight™-based kit) may include, but are not limited to, PCR primers for a specific locus (e.g., a specific gene, marker, region of a gene, region of a marker, bisulfite-treated DNA sequence, CpG island, or bisulfite-treated DNA sequence or CpG island, etc.); blocking oligonucleotides; optimized PCR buffer and deoxynucleotides; and Taq polymerase.

[0187] 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 cytosines to uracil (but not methylated cytosines), and the product is then amplified with primers specific for methylated DNA compared to 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., those that 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, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides, and specific probes.

[0188] The MethyLight™ assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (e.g., TaqMan®) without the need for 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 by standard procedures into a mixed pool of methylation-dependent sequence differences during 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 is performed both at the level of the amplification process and at the level of the fluorescence detection process.

[0189] The MethyLight™ assay is used as a quantitative test of methylation patterns in nucleic acids, e.g., genomic DNA samples, where sequence discrimination is performed at the level of probe hybridization. In the quantitative version, the PCR reaction results in methylation-specific amplification in the presence of fluorescent probes overlapping 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 of 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.

[0190] 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, for example, MSP primers and / or HeavyMethyl blocker oligonucleotides and a TaqMan® probe using 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 about 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 5' to 3' endonuclease activity of Taq polymerase then removes the TaqMan® probe by digesting it and releasing the fluorescent reporter molecule for quantitative detection of the now unquenched signal of the fluorescent reporter molecule using a real-time fluorescence detection system.

[0191] Typical reagents for MethyLight™ analysis (e.g., those that 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 buffers and deoxynucleotides; and Taq polymerase.

[0192] The QM™ (Quantitative Methylation) assay is an alternative quantitative test of methylation patterns in genomic DNA samples, where sequence discrimination is performed at the level of probe hybridization. In this quantitative version, the PCR reaction results in unbiased amplification in the presence of fluorescent probes overlapping 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 qualitative test of genomic methylation is performed by probing the biased PCR pool 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.

[0193] 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 subjected to unbiased primers and 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 about 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. Taq polymerase will eventually reach the annealed TaqMan® probe to enzymatically synthesize new strands during PCR. The 5' to 3' endonuclease activity of Taq polymerase then removes the TaqMan® probe by digesting it to release the fluorescent reporter molecule for quantitative detection of the now unquenched signal of the fluorescent reporter molecule using a real-time fluorescence detection system. Typical reagents for QM™ analysis (e.g., as might be found in a typical QM™-based kit) can 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.

[0194] Ms-SNuPE™ technology is a quantitative method for evaluating methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by single nucleotide primer extension (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosines to uracil, while leaving 5-methylcytosines unchanged. Amplification of the desired target sequence is then performed using PCR primers specific for 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 methylation status at CpG sites.

[0195] Typical reagents for Ms-SNuPE™ analysis (e.g., those that may be found in a typical Ms-SNuPE™-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 buffers; sulfonation buffers; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffers; and DNA recovery components.

[0196] 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 Mspl), and full sequencing of the fragments after binding 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. Thus, 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. Thus, RRBS enriches samples for promoters, CpG islands, and other genomic traits that have a high frequency of restriction enzyme cleavage sites in these regions, thereby providing an assay for assessing the methylation status of one or more genomic loci.

[0197] A typical protocol for RRBS includes the steps of digesting a nucleic acid sample with a restriction enzyme such as MspI, filling in the overhangs and A-tailing, ligating adapters, bisulfite conversion, and a PCR step. See, for example, 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.

[0198] 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 invading oligonucleotide at the target binding site releases the flap sequence by a 5' nuclease, e.g., FEN-1 endonuclease, cleaving between the detection probe and the flap sequence. The flap sequence is complementary to the non-hairpin portion of the corresponding FRET cassette. Thus, the flap sequence functions as an invading 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, for example, Zou et al. (2010) “Sensitive quantification of methylated "Clin Chem 56:A199," as well as U.S. Pat. Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392, each of which is incorporated by reference herein for all purposes.

[0199] The term "bisulfite reagent" refers to a reagent that includes bisulfite, disulfite, hydrogen sulfite, or a combination thereof, and is useful for distinguishing between methylated and unmethylated CpG dinucleotide sequences, as disclosed herein. Methods of such treatment are known in the art (e.g., PCT / EP2004 / 011715 and WO2013 / 116375, each of which is incorporated herein 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, such as 6-hydroxy-2,5,7,8,-tetramethylchroman 2-carboxylic acid or trihydroxybenzoic acid and its derivatives, such as gallic acid (see PCT / EP2004 / 011715, which is incorporated by reference in its entirety). In certain preferred embodiments, the bisulfite reaction includes treatment with ammonium bisulfite, for example, as described in WO2013 / 116375.

[0200] In some embodiments, the treated DNA fragments are amplified using a set of primer oligonucleotides according to the invention (see, for example, Table V) as well as 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.

[0201] In some embodiments, the technology relates to assessing the methylation status of markers including the DMRs of Tables 1A, 2A, 7A, 8A, and 9 (e.g., DMR numbers 1-331). In some embodiments, assessing the methylation status of multiple markers increases the specificity and / or sensitivity of screening or diagnosis for identifying a neoplasm (e.g., melanoma) in a subject.

[0202] 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; see US Patent Publication No. 202000370114). The method includes reacting an oxidized 5mC residue selected from 5-formylcytosine (5fC), 5-carboxymethylcytosine (5caC), and combinations thereof, with an organoborane. The oxidized 5mC residue may be naturally occurring, or, more typically, is 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, for example, by inorganic peroxo compounds or compositions such as potassium perruthenate (KRuO4) or peroxotungstic acid (see, e.g., Okamoto et al. (2011) Chem. Commun. 47:11231-33) and a combination of copper(II) perchlorate / 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) (see Matsushita et al. (2017) Chem. Commun. 53:5756-59).

[0203] Organoboranes may be characterized as complexes of boranes and nitrogen-containing compounds selected from nitrogen heterocycles and tertiary amines. The nitrogen heterocycles may be monocyclic, bicyclic, or polycyclic, but are 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 heterocycles may 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 known as 2-picoline borane), and 5-ethyl-2-pyridine.

[0204] The reaction of oxidized 5mC residues in cell-free DNA with organoboranes is advantageous insofar as non-toxic reagents and mild reaction conditions are available; no bisulfite or other potentially DNA-degrading reagents are required. Furthermore, the conversion of oxidized 5mC residues to dihydrouracils with organoboranes can be performed in a "one-pot" or "one-tube" reaction without isolation of any intermediates. This conversion is of great importance since it involves multiple steps, namely (1) reduction of the alkene bond connecting C-4 and C-5 in the oxidized 5mC, (2) deamination, and (3) either decarboxylation if the oxidized 5mC is 5caC or deformylation if the oxidized 5mC is 5fC.

[0205] 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 for the aforementioned method. The reaction mixture comprises a sample of cell-free DNA containing at least one oxidized 5-methylcytosine residue selected from 5caC, 5fC, and combinations thereof, and an organoborane effective to reduce, deaminate, and decarboxylate or deformylate at least one oxidized 5-methylcytosine residue. As previously described, an organoborane is a complex of a borane and a nitrogen-containing compound selected from a nitrogen heterocycle and a tertiary amine. In a preferred embodiment, the reaction mixture is substantially free of bisulfite, meaning substantially free of bisulfite ions and bisulfite salts. Ideally, the reaction mixture is free of bisulfite.

[0206] In a related aspect of the invention, a kit is provided for converting 5mC residues in cell-free DNA to dihydrouracil residues, wherein the kit includes a reagent for blocking 5hmC residues, a reagent for oxidizing 5mC residues beyond hydroxymethylation to provide oxidized 5mC residues, and an organoborane effective to reduce, deaminate, and decarboxylate or deformylate the oxidized 5mC residues. The kit may also include instructions for using the components to practice the methods described above.

[0207] 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 therefor, wherein the affinity tags enable removal of 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 residues with an organoborane effective to reduce, deaminate, and either decarboxylate or deformylate the oxidized 5mC residues, thereby providing DNA containing dihydrouracil residues in place of the oxidized 5mC residues; and (e) amplifying and sequencing DNA containing dihydrouracil residues; (f) The 5-methylation pattern is determined from the sequencing results of (e).

[0208] Cell-free DNA is extracted from a body sample from a subject, the body sample is typically whole blood, plasma, or serum, most typically plasma, but the sample may also be urine, saliva, mucosal excretion, sputum, feces, 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. It should be noted that in step (a), the cell-free DNA in which the 5hmC residues are modified is in purified, fragmented form, and ligated with adaptors. DNA purification in this context can be performed using any suitable method known to those skilled in the art and / or described in the relevant literature, and the cell-free DNA itself can be highly fragmented, although sometimes further fragmentation may be desirable, for example, as described in US Patent Publication No. 2017 / 0253924. The 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) such that the fragments have blunt ends at the 3' and 5' ends, respectively. In a preferred method, the blunted fragments are also provided with 3' overhangs containing a single adenine residue using a polymerase such as Taq polymerase, as described in WO2017 / 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 cell-free DNA fragments and contains at least one molecular barcode. The use of adaptors also allows for selective PCR enrichment of the adaptor-ligated DNA fragments.

[0209] 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 affinity tags is performed to allow for subsequent removal of modified 5hmC-containing DNA from the cell-free DNA, as described in step (a). In one embodiment, the affinity tag comprises a biotin moiety, such as biotin, desthiobiotin, oxybiotin, 2-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, and the like. The use of a biotin moiety as an affinity tag facilitates removal using streptavidin, such as streptavidin beads, magnetic streptavidin beads, and the like.

[0210] 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 undergo reaction with the affinity tag functionalized 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 an alkyne-functionalized biotin moiety, as described in Robertson et al. (2011) Biochem. Biophys. Res. Comm. 411(l):40-3, U.S. Patent No. 8,741,567, and WO2017 / 176630. Thus, the addition of the alkyne-functionalized biotin moiety results in the covalent attachment of the biotin moiety to each 5hmC residue.

[0211] The affinity tagged DNA fragments can then be delivered to step (b) in one embodiment using streptavidin in the form of streptavidin beads, magnetic streptavidin beads, etc., and set aside for later analysis, if desired. The supernatant remaining after removal of the affinity tagged fragments contains unmodified 5mC residues and DNA that does not have 5hmC residues.

[0212] In step (c), the unmodified 5mC residue is oxidized to provide 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 carried out enzymatically using a catalytically active TET family enzyme. The term "TET family enzyme" or "TET enzyme" as 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.

[0213] Oxidation can be carried out chemically, but can also be carried out using chemical oxidizing agents, as described in the previous section. Examples of suitable oxidizing agents include, but are not limited to: perruthenate anions in the form of inorganic or organic 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, and in the next step of the process, step (e), converts both the 5fC and 5caC residues to dihydrouracil (DHU).

[0214] In some embodiments, 5-hydroxymethylcytosine residues are blocked with β-glucosyltransferase (β3GT), while 5-methylcytosine residues are oxidized with a TET enzyme effective to provide a mixture of 5-formylcytosine and 5-carboxymethylcytosine. A mixture containing both of these oxidized species can be reacted with 2-picoline borane or another organoborane to obtain dihydrouracil. In a variation on this embodiment, 5hmC-containing fragments are not removed in step (b). Rather, in "TET-assisted picoline borane sequencing (TAPS)", 5mC-containing fragments and 5hmC-containing fragments are enzymatically oxidized together to provide 5fC- and 5caC-containing fragments. Reaction with 2-picoline borane results in DHU residues where 5mC and 5hmC residues were originally present. "Chemically assisted picoline borane sequencing (CAPS)" selectively oxidizes 5hmC-containing fragments with potassium perruthenate, leaving the 5hmC residues unchanged.

[0215] The method of the present embodiment has many advantages: no bisulfite is required, non-toxic reagents and reactants are used, and the process proceeds under mild conditions. Moreover, the entire process can be carried out in a single tube without the need for isolation of any intermediates.

[0216] In a related embodiment, the method described above comprises a further step: (g) identifying hydroxymethylation patterns in the 5hmC-containing DNA removed from the cell-free DNA in step (b). This can be done using the techniques described in detail in WO2017 / 176630. This process can be done in a one-tube method without intermediate removal or isolation. For example, first, the cell-free DNA fragments, preferably the adaptor-ligated DNA fragments, undergo functionalization with βGT-catalyzed uridine diphosphoglucose 6-azide, followed by biotinylation via chemoselective azide groups. This procedure results in covalent attachment of biotin to each 5hmC site. In the next step, the biotinylated strand and the strand containing unmodified (native) 5mC are simultaneously delivered for further processing. The native 5mC-containing strand is delivered using an anti-5mC antibody or a methyl-CpG binding domain (MBD) protein, as known in the art. Once the 5hmC residues are blocked, unmodified 5mC residues are then selectively oxidized using any suitable technique to convert 5mC to 5fC and / or 5caC, as described elsewhere herein.

[0217] The fragments obtained by amplification can carry a directly or indirectly detectable label. In some embodiments, the label is a fluorescent label, a radionuclide, or a detachable molecular fragment with 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 having a single positive or negative net charge of the labeled amplicon. Detection can be performed and visualized, for example, by matrix-assisted laser desorption ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).

[0218] Methods for isolating DNA suitable for these assay techniques are known in the art. In particular, some embodiments involve the isolation of nucleic acids as described in U.S. Patent Application Serial No. 13 / 470,251 ("Isolation of Nucleic Acids"), which is incorporated herein by reference in its entirety.

[0219] 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 that are substantially and / or effectively free of substances that inhibit 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 states, and thus detection and / or quantification of such mutant alleles or methylation states has predictive value in cancer diagnosis and treatment.

[0220] Many useful genetic markers are present in samples in very small amounts, and many of the events that generate such markers are rare.As a result, even highly sensitive detection methods such as PCR require a large amount of DNA to provide enough low-abundance targets to meet or invalidate 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.

[0221] In some embodiments, the sample comprises feces, tissue samples (e.g., skin tissue), organ secretions, 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 of skill in the art. 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 may still be preferred to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens. The techniques are not limited to the methods used to prepare the sample and provide nucleic acids for testing. For example, in some embodiments, DNA is isolated from fecal samples, or from blood, or from plasma samples, using direct gene capture, such as those detailed in U.S. Pat. Nos. 8,808,990 and 9,169,511, and WO2012 / 155072, or by related methods.

[0222] 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 a series of samples allows for the identification of changes in the methylation status of markers over time. The absence of changes in methylation status, in addition to changes in methylation status, can provide useful information regarding disease status, including but not limited to identifying the approximate time from the onset of an event, the presence and amount of recoverable tissue, the appropriateness of drug therapy, the effectiveness of various therapies, and identifying the subject's outcome, including the risk of future events.

[0223] Analysis of biomarkers can be performed 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.

[0224] It is contemplated that embodiments of the present technology are provided in the form of a kit. The kit includes embodiments of the compositions, devices, apparatus, etc. described herein, and instructions for use of the kit. Such instructions describe appropriate methods for preparing an analyte from a sample, e.g., methods for collecting a sample and preparing nucleic acid from the sample. 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 kit 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.

[0225] Different cancers are predicted by different combinations of markers, for example as identified by statistical techniques for the specificity and sensitivity of prediction. The present technology provides methods for identifying predictive combinations and validated predictive combinations for several cancers.

[0226] 〔method〕 In some embodiments of the present technology, the following steps are performed: 1) contacting nucleic acid obtained from a subject (e.g., genomic DNA isolated from a bodily fluid such as blood or plasma, fine needle aspirate, deep tissue, or skin tissue, etc.) with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker that contains a DMR (e.g., DMRs 1-331, e.g., those shown in Tables 1A, 2A, 7A, 8A, and 9); 2) detecting melanoma, metastatic melanoma, or primary cutaneous melanoma (e.g., with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0227] In some embodiments of the present technology, the following steps are performed: 1) Nucleic acid obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma, fine needle aspirate, deep tissue, or skin tissue) is subjected to PCR for the following: c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-6249281 2, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-7111612 2, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33, and 2) detecting melanoma (e.g., with a sensitivity of 80% or greater and a specificity of 80% or greater): In some embodiments of the present technology, the following steps are performed: 1) Nucleic acid obtained from a subject (e.g., genomic DNA isolated from a bodily fluid such as blood or plasma, fine needle aspirate, deep tissue, or skin tissue, etc.) is isolated from MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4 _A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HO with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker comprising a chromosomal region having an annotation selected from the group consisting of PX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763; and 2) detecting melanoma (e.g., with a sensitivity of 80% or greater and a specificity of 80% or greater). In some embodiments of the present technology, the following steps are performed: 1) Nucleic acid obtained from a subject (e.g., genomic DNA isolated from a body fluid such as blood or plasma, fine needle aspirate, deep tissue, or skin tissue) is subjected to PCR amplification using a PCR product that is capable of detecting the following: AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HC G4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693 -32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.54970444-549704 69, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MA X.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A , PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides in at least one marker comprising a chromosomal region having an annotation selected from the group consisting of: 2) detecting melanoma (e.g., with a sensitivity of 80% or greater and a specificity of 80% or greater). In some embodiments of the present technology, the following steps are performed: 1) contacting nucleic acid obtained from a subject (e.g., genomic DNA isolated from a bodily fluid such as blood or plasma, fine needle aspirate, deep tissue, or skin tissue, etc.) with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker that comprises a chromosomal region having an annotation selected from the group consisting of MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A; and 2) detecting metastatic melanoma (e.g., with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0228] In some embodiments of the present technology, the following steps are performed: 1) Nucleic acid obtained from a subject (e.g., genomic DNA isolated from a bodily fluid such as blood or plasma, fine needle aspirate, deep tissue, or skin tissue, etc.) is isolated from MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4 _A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-22541948, ME3, HO with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker comprising a chromosomal region having an annotation selected from the group consisting of PX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763; and 2) detecting metastatic melanoma (e.g., with a sensitivity of 80% or greater and a specificity of 80% or greater). In some embodiments of the present technology, a method is provided that includes the following steps: 1) measuring the methylation level of one or more genes in a biological sample of a human individual through treating genomic DNA in the biological sample with a reagent that modifies DNA in a methylation-specific manner (e.g., where the reagent is a bisulfite reagent, a methylation-sensitive restriction enzyme, or a methylation-dependent restriction enzyme), wherein the one or more genes are selected from one of the following groups: ●c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-11062736 4, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.711160 33-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (Tables 4, 5A, 5B, and 6; see Example I); ●MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-730738 30, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874- 22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see Tables 9, 10, 11); ●AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2 , LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-322377 85, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.549 70444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (Tables 7A and 7B; see Example II); one or more markers listed in Tables 8A and 8B (Example II); and MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see Table 5C, Example I); 2) amplifying the treated genomic DNA using a set of primers for one or more selected genes; and 3) determining the methylation level of one or more genes by polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, and target capture.

[0229] In some embodiments of the present technology, a method is provided that includes the following steps: 1) measuring the amount of at least one methylation marker gene in DNA from a sample, the one or more genes being selected from one of the following groups: ●c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-11062736 4, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.711160 33-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (Tables 4, 5A, 5B, and 6; see Example I); ●MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-730738 30, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874- 22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see Tables 9, 10, 11); ●AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2 , LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-322377 85, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.549 70444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (Tables 7A and 7B; see Example II); one or more markers listed in Tables 8A and 8B (Example II); and MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see Table 5C, Example I); 2) measuring the amount of at least one reference marker in the DNA; and 3) calculating a value of the amount of at least one methylation marker gene measured in the DNA as a percentage of the amount of a reference marker gene measured in the DNA, said value being indicative of the amount of at least one methylation marker DNA measured in the sample.

[0230] In some embodiments of the present technology, a method is provided that includes the following steps: 1) measuring the methylation level of CpG sites of one or more genes in a biological sample from a human individual by treating genomic DNA in the biological sample with a bisulfite reagent (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent) that can modify DNA in a methylation-specific manner; 2) amplifying the modified genomic DNA using a set of primers for one or more selected genes; and 3) determining the methylation level of the CpG sites by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; The one or more genes are selected from one of the following groups: ●c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-11062736 4, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr2.71116 033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 (Tables 4, 5A, 5B, and 6; see Example I); ●MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-730738 30, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874- 22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763 (see Tables 9, 10, 11); ●AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2 , LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-322377 85, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr16.549 70444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73 (Tables 7A and 7B; see Example II); one or more markers listed in Tables 8A and 8B (Example II); and MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A (see Table 5C, Example I).

[0231] In any of these methods, determining the methylation level for any of such markers is accomplished using the primers listed in Table 3.

[0232] Preferably, the sensitivity of such a method is about 70% to about 100%, or about 80% to about 90%, or about 80% to about 85%. Preferably, the specificity is about 70% to about 100%, or about 80% to about 90%, or about 80% to about 85%.

[0233] Genomic DNA can be isolated by any means, including the use of commercially available kits. Briefly, if the DNA of interest is encapsulated by a cell membrane, the biological sample 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. The 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, skin tissue, lymphatic tissue or aspirates, brain, lung, liver, bone or other deep organ tissues, colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, and combinations thereof.

[0234] 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 skin tissue samples or fecal samples, or from blood or from plasma samples, by direct gene capture, for example, as described in US Patent Application No. 61 / 485386, or by related methods.

[0235] The genomic DNA sample is then treated with at least one reagent, or a series of reagents, that distinguishes between methylated and unmethylated CpG dinucleotides within at least one marker that comprises a DMR (e.g., DMRs 1-331 as shown in Tables 1A, 2A, 7A, 8A, and 9).

[0236] 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, however, in some embodiments, the reagent may be a methylation-sensitive restriction enzyme.

[0237] In some embodiments, the genomic DNA sample is treated in such a way that cytosine bases that are not methylated at the 5' position are converted to uracil, thymine, or another base that differs from cytosine in terms of hybridization behavior. In some embodiments, this treatment is carried out with bisulfite (hydrogen sulfite, disulfite) followed by alkaline hydrolysis.

[0238] 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 a DMR, e.g., at least one DMR selected from DMRs 1-331, e.g., as set forth in Tables 1A, 2A, 7A, 8A, and 9). Methods of analysis can be selected from those known in the art, including those set forth herein, e.g., QuARTS and MSP, as described herein.

[0239] Aberrant methylation, and more specifically, hypermethylation of markers that include DMRs (eg, DMRs 1-331, as shown in Tables 1A, 2A, 7A, 8A, and 9), is associated with melanoma.

[0240] The technology relates to the analysis of any sample related to melanoma. For example, in some embodiments, the sample includes tissue (e.g., skin tissue) and / or bodily 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, skin tissue biopsies, deep tissue biopsies, microdissected cells from fine needle aspirates, and / or cells recovered from feces. In some embodiments, the sample includes skin tissue. In some embodiments, the subject is a human. The sample may include cells, secretions, or tissues from the ovaries, breast, liver, bile duct, pancreas, stomach, colon, skin, rectum, esophagus, small intestine, appendix, duodenum, polyps, gallbladder, anus, lymph nodes, brain, bone, and / or peritoneum. In some embodiments, the sample includes cell fluids, ascites, urine, feces, pancreatic juice, fluid obtained during endoscopy, blood, mucus, or saliva. In some embodiments, the sample is a fecal sample. In some embodiments, the skin cells are collected using adhesive tape or other sticky surface.

[0241] Such samples can be obtained by any number of means known in the art, including 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 various 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 may still be preferable to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens.

[0242] In some embodiments, the technology relates to a method of treating a patient (e.g., a patient with melanoma) (e.g., a patient with one or more metastatic melanomas, primary cutaneous melanomas), the method comprising determining the methylation status of one or more DMRs provided herein, and administering a treatment to the patient based on the results of determining the methylation status. The treatment can be administering a pharmaceutical compound, administering a vaccine, administering an immunotherapy, performing a surgery, imaging the 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.

[0243] In some embodiments of the present technology, a method for diagnosing melanoma 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 whether a subject may 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 biomarkers (e.g., DMRs disclosed herein), whose methylation status indicates the presence, severity, or absence of a condition.

[0244] 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 evaluated, appropriate therapy, and in some cases, a therapy that is less harsh for the patient, can be selected. Assessment of cancer biomarkers (e.g., determining methylation status) is useful for separating subjects who have a good prognosis and / or are at a low risk of developing cancer and do not require therapy or require limited therapy from subjects who are more likely to develop cancer or have cancer recurrence and may benefit from more aggressive treatment.

[0245] Thus, "making a diagnosis" or "diagnosing" as used herein further includes determining the risk of developing cancer or determining a prognosis, which may allow predicting a clinical outcome (with or without medical treatment), selecting an appropriate treatment (or whether a treatment is effective), or monitoring a current treatment and possibly modifying the treatment based on a measure of a diagnostic biomarker (e.g., DMR) 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. The temporal changes in the biomarkers can be used to predict clinical outcomes, monitor the progression of melanoma, and / or monitor the effectiveness of an appropriate therapy directed against the cancer. In such embodiments, for example, it may be expected to ascertain changes in the methylation status of one or more biomarkers (e.g., DMR) disclosed herein (and optionally one or more additional biomarker(s) if monitored) in a biological sample over time during the course of an effective therapy.

[0246] 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 obtaining a series of biological samples from a subject over a period of time, analyzing the series of biological samples, determining the methylation status of at least one biomarker disclosed herein in each of the biological samples, and comparing any measurable changes in the methylation status of one or more of the biomarkers in each of the biological samples. Any changes in the methylation status of the biomarkers over a period of time can be used to predict the risk of developing cancer, predict clinical outcomes, determine whether to initiate or continue cancer prevention or therapy, and whether a current therapy 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 can be measured in each of the samples taken at different time points, and qualitative and / or quantitative differences are recorded. Changes in the methylation status of the biomarker levels from the different samples can be correlated with melanoma risk, prognosis, determining treatment efficacy, and / or cancer progression in the subject.

[0247] 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.

[0248] As already mentioned, in some embodiments, multiple determinations of one or more diagnostic or prognostic biomarkers can be made, and the change in the marker over time can be used to determine the diagnosis or prognosis. For example, the diagnostic marker can be determined a first time and again a second time. In such embodiments, an increase in the marker from the first time to the second time can diagnose a particular type or severity of cancer, or a given prognosis. Similarly, a decrease in the 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, but also a given biomarker can be measured at one time point and a second biomarker at a second time point, and the comparison of these markers can provide diagnostic information.

[0249] As used herein, the phrase "determine prognosis" refers to a method by which a person skilled in the art can predict the course or outcome of a condition of 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 to predict that a given course or outcome is more or less likely to occur based on the methylation status of a biomarker (e.g., DMR). 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, compared to those individuals that do not exhibit the condition. For example, an individual that does not exhibit a condition (e.g., has normal methylation status of one or more DMRs) may have a very low probability of a given outcome (e.g., suffer from melanoma).

[0250] 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 that differs from that in a normal control sample obtained from a patient without cancer may indicate 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, as determined by the level of statistical significance. Furthermore, the change in methylation status from the baseline (e.g., "normal") level may reflect the subject's prognosis, and the degree of change in methylation status may be related to the severity of an adverse event. Statistical significance is often determined by comparing two or more populations and determining a confidence interval and / or p-value. See, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, incorporated herein by reference in its entirety. See York, 1983. 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.

[0251] In other embodiments, a threshold degree of change in the methylation status of a prognostic or diagnostic biomarker (e.g., DMR) disclosed herein can be established, and the degree of change in the methylation status of the biomarker in a biological sample is simply compared to the threshold degree of change in the methylation status. Preferred threshold changes in the methylation status 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 of a prognostic or diagnostic indicator (biomarker or combination of biomarkers) is directly related to the predisposition associated with a given outcome. Because reference is made to individual sample measurements rather than population averages, one skilled in the art is familiar with using such nomograms to relate the two numerical values, with the understanding that the uncertainty of this measurement is the same as the uncertainty of the marker concentration.

[0252] 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 presents the methylation status of the biomarker according to the assay unit, e.g., the fluorescent signal intensity if a fluorescent label is used. Samples taken from multiple donors can be used to obtain standard curves for the control methylation status of one or more biomarkers in normal tissue and for the "at risk" level of one or more biomarkers in tissue taken from a donor with dysplasia or from a donor with melanoma. In certain embodiments of the method, a subject is identified as having dysplasia upon identification of an aberrant methylation status of one or more DMRs provided herein in a biological sample taken from the subject. In other embodiments of the method, detection of an aberrant methylation status of one or more of such biomarkers in a biological sample taken from the subject identifies the subject as having cancer.

[0253] 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 a series of samples allows for the identification of changes in the methylation status of markers over time. The absence of changes in methylation status, in addition to changes in methylation status, can provide useful information regarding disease status, including but not limited to identifying the approximate time from the onset of an event, the presence and amount of recoverable tissue, the appropriateness of drug therapy, the effectiveness of various therapies, and identifying the subject's outcome, including the risk of future events.

[0254] Analysis of biomarkers can be performed 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.

[0255] In some embodiments, a subject is diagnosed as having melanoma when there is a measurable difference in the methylation status of at least one biomarker in the sample compared to the control methylation status. Conversely, if no change in the methylation status in the biological sample is identified, the subject may be identified as not having melanoma, not at risk of cancer, or at low risk of cancer. In this regard, subjects with cancer or at risk thereof may be differentiated from subjects with low to substantially no cancer or at risk thereof. Those subjects with a risk of developing melanoma may be placed on a more intensive and / or regular screening schedule, including endoscopic surveillance. On the other hand, subjects with low to substantially no risk may be avoided from being subjected to further testing (e.g., invasive procedures) for melanoma until future screening, e.g., screening performed according to the present technology, indicates that the subjects are at risk for melanoma.

[0256] As mentioned above, depending on the embodiment of the method of the present technology, detecting a change in the methylation state of one or more biomarkers can be a qualitative determination or it can be a quantitative determination. Thus, the step of diagnosing a subject as having melanoma or at risk of developing melanoma indicates that a certain threshold measurement is made, e.g., that the methylation state of one or more biomarkers in a biological sample is different from a predetermined control methylation state. In some embodiments of the present method, the control methylation state is any detectable methylation state of the biomarker. In other embodiments of the method, where a control sample is tested simultaneously with the biological sample, the predetermined methylation state is the methylation state in the control sample. In other embodiments of the present method, the predetermined methylation state is based on and / or identified by a standard curve. In other embodiments of the present method, the predetermined methylation state is a specific state or range of states. Thus, the predetermined methylation state can be selected, in part, based on the embodiment of the method to be performed and the desired specificity, etc., within acceptable limits that will be apparent to one of skill in the art.

[0257] 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. Thus, the technology allows for the diagnosis of mammals such as humans, as well as mammals of importance due to being endangered, such as the Amur tiger, mammals of economic importance, such as animals raised on farms for human consumption, and / or animals of social importance 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.

[0258] The subject matter disclosed herein further includes a system for diagnosing melanoma and / or specific forms of melanoma (e.g., metastatic melanoma, primary cutaneous melanoma) in a subject. The system may be provided as a commercially available kit that may be used, for example, to screen for melanoma risk or to diagnose melanoma in a subject from whom a biological sample has been collected. Exemplary systems provided in accordance with the present technology include assessing the methylation status of DMRs as shown in Tables 1A, 2A, 7A, 8A, and 9.

[0259] [Example] Example I Materials and Methods Tissues and blood were obtained from the Mayo Clinic Biospecimen Repository under institutional IRB oversight. Samples were selected in strict compliance with subject study approval and inclusion / exclusion criteria. Cancers consisted of 21 metastatic melanomas. Control groups included 15 non-neoplastic skin epidermal samples, 16 benign melanotic leukocytic nevi, and 36 whole blood-derived leukocytes. Tissues were macro-dissected and reviewed for histology by an expert pathologist. Samples were age-matched, randomized, and blinded. DNA was purified using QIAamp DNA Tissue Mini kits and QIAamp DNA Blood Mini kits (Qiagen, Valencia CA), respectively. DNA was repurified with AMPure XP beads (Beckman-Coulter, Brea CA) and quantified with PicoGreen (Thermo-Fisher, Waltham MA). DNA integrity was assessed using qPCR.

[0260] RRBS sequencing libraries were prepared according to a modified Meissner protocol (see Gu et al. Nature Protocols 2011 Apr;6(4):468-81). Samples were aligned in a 4-plex format and sequenced on an Illumina HiSeq by the Mayo Genomics Facility. Sequencing was performed on a 2500 instrument (Illumina, San Diego CA). Reads were processed by Illumina pipeline modules for image analysis and base calling. Secondary analysis was performed using SAAP-RRBS, a bioinformatics suite developed by Mayo. Briefly, reads were cleaned using Trim-Galore and aligned to the GRCh37 / hg19 reference genome constructed with BSMAP. For CpGs with ≥10x coverage and base quality score ≥20, methylation rates were determined by calculating C / (C+T) or, conversely, G / (G+A) for reads mapping to the opposite strand.

[0261] Individual CpGs were ranked by their hypermethylation ratio, i.e., the number of methylated cytosines relative to the total number of cytosines at that location, at a given locus. For cases, the ratio had to be ≥ 0.20 (20%); for tissue controls, ≤ 0.05 (5%) tissue-to-tissue analysis; ≥ 0.20 (20%) tissue-to-buffy coat analysis; and for buffy coat controls, ≤ 0.01 (1%). CpGs that did not meet these criteria were discarded. Candidate CpGs were subsequently binned by genomic location into DMRs (variably methylated regions) ranging from approximately 40 to 220 bp with a minimum cutoff of 5 CpGs per region. DMRs with excessively high CpG density (> 30%) were excluded to avoid GC-related amplification issues during the validation phase. For each candidate region, a 2D matrix was created comparing individual CpGs sample-by-sample for both cases and controls. These CpG matrices were then compared to reference sequences to assess whether genomically contiguous methylated sites were discarded during the initial filtering. From this subset of regions, final selection required, at the per-sample level, coordinated and contiguous (in cases) hypermethylation of individual CpGs across the DMR sequences. Conversely, control samples were required to be at least one-tenth as methylated as cases, and the CpG pattern had to be more random and less coordinated. At least 10% of cancer samples were required to have at least 50% hypermethylation at all CpG sites within the DMR.

[0262] In a separate analysis, we derived DMRs based on the mean methylation values ​​of CpGs using a proprietary DMR identification pipeline and regression package. Differences in mean methylation percentages were compared between malignant melanoma cases, tissue controls, and buffy coat controls, and tiled reading frames within 100 base pairs of each mapped CpG were used to identify DMRs with less than 5% methylation in controls. DMRs were analyzed only if the total coverage depth was an average of 10 reads per subject and the variance between subgroups was greater than 0. Assuming a biologically relevant increase in odds ratio greater than 3-fold and a coverage depth of 10 reads, ≥18 samples per group were required to achieve 80% power at a 5% significance level with a two-sided test and assuming a binomial variance inflation factor of 1.

[0263] After regression, DMRs were ranked by the difference in p-value, area under the receiver operating characteristic curve (AUC), and fold change between cases and all controls. No adjustment for false positives was performed at this stage because independent validation was planned a priori.

[0264] A subset of DMRs was selected for further expansion. Criteria were primarily logistic-derived area under the ROC curve measures that provide a performance assessment of the discriminatory potential of the region. AUC0.85 was selected as the cutoff. Additionally, methylation fold change (mean cancer hypermethylation rate / mean control hypermethylation rate) was calculated, with a lower limit of 10 for tissue-to-tissue comparisons and 20 for tissue-to-buffy coat comparisons. P values ​​had to be less than 0.01. DMRs had to be listed in both the average and individual CpG selection process. Quantitative methylation-specific PCR (qMSP) primers were designed for the candidate regions using MethPrimer (Li LC and Dahiya R. Bioinformatics 2002 Nov;18(11):1427-31) and QC-tested with 20ng (6250 equivalents) of positive and negative genomic methylation controls. Multiple annealing temperatures were tested for optimal discrimination. Validation was performed on qMSP in two stages. The first stage consisted of retesting of sequenced DNA samples. This was done to establish that the DMRs were indeed discriminatory and not the result of overfitting of a very large next-generation sequencing (NGS) dataset. The second stage utilized an even larger set of independent samples: metastatic melanomas from 35 patients, primary melanomas from 26 patients, and 73 control samples (47 benign precursor lesions or normal skin, and 26 healthy buffy coat samples).

[0265] Tissues were identified as before by expert clinical and pathological review. DNA purification was performed using the Qiagen QIAmp FFPE tissue kit. For the bisulfite conversion step, the EZ-96 DNA Methylation kit (Zymo Research, Irvine CA) was used. 10 ng of converted DNA (per marker) was amplified using SYBR Green detection on a Roche 480 LightCycler (Roche, Basel Switzerland). Serially diluted universal methylated genomic DNA (Zymo Research) was used as a quantification standard. A CpG-independent ACTB (β-actin) assay was used as input reference and normalization control. Results were expressed as copies methylated (specific marker) / copies of ACTB.

[0266] Results were analyzed logistically for the performance of individual MDMs (methylated DNA markers). For marker combinations, two techniques were used. First, the rPart technique was applied to the entire MDM set, restricted to combinations of three MDMs, and then the rPart predicted cancer probability was calculated. The second approach used random forest regression (rForest) used to estimate the cross-validation error for the entire MDM panel (1 / 3 of the data for testing) by generating 500 individual rPart models fitted to a bootstrap sample of the original data (approximately 2 / 3 of the data for training), which was repeated 500 times to avoid spurious splits that would under- or overestimate the true cross-validation measure. The results were then averaged over the 500 repetitions.

[0267] result Utilizing a proprietary methodology of sample preparation, sequencing, analysis, and filtering, we identified and qualified variably methylated regions (DMRs) that accurately identify skin cancers and are prevalent in the clinical laboratory setting. From tissue-to-tissue analysis, 124 hypermethylated malignant melanoma (MM) DMRs were identified (Tables 1A and IB). They included MM-specific regions as well as regions targeting a more universal cancer spectrum. Tissue-to-white blood cell (buffy coat) analysis yielded 38 hypermethylated MM+ epidermis tissue DMRs with less than 1% noise in WBCs (Tables 2A and 2B).

[0268] From the tissue and buffy marker panels, 40 candidate markers were selected for the initial pilot. Methylation-specific PCR assays were developed and tissue samples were tested twice; sequenced (frozen) and a larger independent cohort (FFPE). Short amplicon primers (<150 bp) were designed to target the most discriminatory CpGs within the DMR and tested on controls to ensure that fully methylated fragments were reliably and linearly amplified; i.e., they did not amplify unmethylated and / or unconverted fragments. The 80 primer sequences are listed in Table 3.

[0269] Stage 1 validation results were logistically analyzed to determine AUC and fold change. Tissue and buffy coat control analyses were performed separately. Results are highlighted in Table 4. The degree of red shading indicates the discriminatory strength of the marker assay. Many assays discriminated MM from buffy coat samples 100%, and one assay had 100% discrimination in MM vs. control tissue analysis.

[0270] These results provided a rich source of high-performing candidates for consideration in independent sample testing. Of the initial 40 assays, 32 were selected. Most had AUCs falling within the range of 0.90-1.00, while others had very large fold-change (FC) numbers (with little background) and / or demonstrated complementarity with other methylated DNA markers (MDMS). All assays demonstrated high analytical performance, including linearity, efficiency, sequence specificity (assessed using melting curve analysis), and robust amplification.

[0271] In the second validation, the entire sample and marker set was run in one batch, similar to the previous step. Approximately 10 ng of FFPE-derived sample DNA was run for each of the approximately 350 markers in total. The MM vs. normal tissue and buffy coat results for individual MDMs are listed in Table 5A. In the receiver operator characteristic analysis of individual marker candidates, the area under the curve (AUC) for MM vs. control tissue comparisons ranged from 0.43 to 0.97 (Table 5B); the median AUC was 0.835. A cross-validated panel of five MDMs (MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763 (using primers SEQ ID NOs: 47 and 48), MAX.chr7.155259597-155259763 (using primers SEQ ID NOs: 49 and 50), FOXL2NB, and HOXA9_A) yielded a sensitivity of 33 / 35 cases (94.3% (95% CI, 86.6 to 100%)) for metastatic melanoma and 22 / 26 cases (84.6% (95% CI, 70.7 to 98.5%)) for primary melanoma, with a specificity of 100% (Table 5C). For the MM vs. buffy coat comparison, the AUC ranged from 0.80 to 0.98, and the methylation fold change ratio was >20 with a median of 64 (Table 6).

[0272] Whole methylome sequencing, stringent filtering criteria, and biological validation yielded excellent candidate MDMs for malignant melanoma. When assayed in tissues, a panel of five novel MDMs (MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763 (using primers SEQ ID NOs: 47 and 48), MAX.chr7.155259597-155259763 (using primers SEQ ID NOs: 49 and 50), FOXL2NB, and HOXA9_A) that were not detectable in normal buffy coat achieved very high discrimination between melanoma and benign control tissues.

[0273] [Table 1-1]

[0274] [Table 1-2]

[0275] [Table 1-3]

[0276] [Table 1-4]

[0277] [Table 2-1]

[0278] [Table 2-2]

[0279] [Table 2-3]

[0280]

Table 2-4

[0281]

Table 3-1

[0282]

Table 4-1

[0283]

Table 5-1

[0284]

Table 5-2

[0285]

Table 5-3

[0286]

Table 6-1

[0287]

Table 6-2

[0288]

Table 7-1

[0289]

Table 7-2

[0290]

Table 7-3

[0291] [Table 7-4]

[0292] [Table 8]

[0293] [Table 9]

[0294] [Table 10-1]

[0295] [Table 10-2]

[0296] Example II. Materials and Methods Whole-genome bisulfite sequencing (WGBS) is an NGS approach that differs from the usual RRBS method in that the entire human genome is sequenced. RRBS is an enrichment method that uses restriction endonucleases specific for CpG-rich cleavage sites to generate fragments from transcriptional regulatory regions of the genome. These CpG islands have been reported to show differential or altered methylation profiles in many clinical conditions, especially cancer. The advantage of RRBS is that it reduces the size of the genome to 1-2% of the total 3.2 billion nucleotides, significantly reducing costs while capturing the majority of promoters and CpG islands. The disadvantage is that it misses a potentially significant number of regulatory regions that are CpG-rich but do not contain endonuclease binding sequences or are not part of a CpG island. Furthermore, the results are somewhat biased by the performance and efficiency of the enzymes, which vary from sample to sample. WGBS uses physical shearing to shear the genome into 200-300 bp fragments that are suitable for NGS. Its advantage is that all fragments, regardless of CpG content, are theoretically sequenced, potentially increasing the number of tumor-specific biomarkers. The disadvantage is that most of the genome is discarded because it is unregulated or not suitable for biomarker discovery. Also, WGBS is expensive to perform with a comparable depth of vertical coverage. The advent of high-performance sequencers and flow cells, as well as advances in general NGS technology, have mitigated the cost to some extent, but it is still not as affordable as RRBS and other enrichment sequencing protocols.

[0297] Samples were the same as those used in the RRBS study, allowing for direct comparison. Tissues and blood were obtained from the Mayo Clinic Biospecimen Repository under institutional IRB oversight. Samples were selected in strict compliance with subject study approval and inclusion / exclusion criteria. Cancers consisted of 21 metastatic melanomas. Control groups included 15 non-neoplastic skin epidermal samples, 16 benign melanotic leukocytic nevi, and 36 whole blood-derived leukocytes. Tissues were macro-dissected and reviewed for histology by an expert pathologist. Samples were age-matched, randomized, and blinded. DNA was purified using QIAamp DNA Tissue Mini kits and QIAamp DNA Blood Mini kits (Qiagen, Valencia CA), respectively. DNA was repurified with AMPure XP beads (Beckman-Coulter, Brea CA) and quantified with PicoGreen (Thermo-Fisher, Waltham MA). DNA integrity was assessed using qPCR.

[0298] Sequencing libraries were prepared according to the method of Ulrich et al. (see Nature Protocols. 10, 473-483 (2015)) with modifications. Briefly, 100ng of DNA was sheared with a Covaris sonicator to a target size of 200bp. Fragments >600bp were size fractionated using 0.6X Ampuri beads (Beckman). The supernatant was then amplified to 1.4X to purify the remaining fragments with a lower limit of 100bp. Ends were repaired with End-It DNA End Repair Kit (Epicentre) and purified with 1.4X Ampur beads, followed by a-tailing using Klenow (3'-5' exo- (NEB) followed by 1.4X purification. Methylated adapters (NEXTflex Bisulfite-Seq Barcodes-Bioo Scientific) were ligated with T4 DNA ligase (NEB), bisulfite treated twice using the Epitect 96 Kit (Qiagen), and bead purified. Libraries were then tested with SYBR Green qPCR to determine optimal enrichment cycles, and then enriched for either 10, 13, 16 or 18 cycles using the KAPA HiFi HotStart Uracil+ReadyMix Kit (Kapa Biosystems). Enriched libraries were bead purified in a 1:1 ratio, indexed together in groups of 24, re-purified, and run on the Bioanalyzer. Quantification was performed using the 2100 (Agilent) and qPCR (Kapa). Concentrations ranged from 5 to 80 nM.

[0299] Paired-end 150-cycle sequencing was performed on a Nova-Seq using one S4 flow cell (Illumina) per 24 samples. Reads were processed by Illumina pipeline modules for image analysis and base calling. Secondary analysis was performed using SAAP-RRBS, a bioinformatics suite developed by Mayo. Briefly, reads were cleaned using Trim-Galore and aligned to the GRCh37 / hg19 reference genome constructed with BSMAP. Methylation rates were determined for CpGs with ≥5x coverage and base quality score ≥20 by calculating C / (C+T) or, conversely, G / (G+A) for reads mapping to the opposite strand.

[0300] A proprietary DMR identification pipeline and regression package was utilized to derive DMRs based on the average methylation values ​​of CpGs. Differences in average methylation percentages were compared between malignant melanoma cases, tissue controls, and buffy coat controls, and tiled reading frames within 100 base pairs of each mapped CpG were used to identify DMRs with less than 5% methylation in controls; DMRs were analyzed only if the total coverage depth was an average of 5 reads per subject and the variance between subgroups was greater than 0.

[0301] After regression, DMRs were ranked by the difference in p-value, area under the receiver operating characteristic curve (AUC), and fold change between cases and all controls. No adjustment for false positives was performed at this stage because independent validation was planned a priori.

[0302] Individual CpGs were ranked by their hypermethylation ratio, i.e., the number of methylated cytosines relative to the total number of cytosines at that location, at a given locus. For cases, the ratio had to be ≥ 0.20 (20%); for tissue controls, ≤ 0.05 (5%) tissue-to-tissue analysis; ≥ 0.30 (30%) tissue-to-buffy coat analysis; and for buffy coat controls, ≤ 0.01 (1%). CpGs that did not meet these criteria were discarded. Candidate CpGs were subsequently binned by genomic location into DMRs (variably methylated regions) ranging from approximately 30 to 300 bp with a minimum cutoff of 5 CpGs per region. DMRs with excessively high CpG density (> 30%) were excluded to avoid GC-related amplification issues during the validation phase. For each candidate region, a 2D matrix was created comparing individual CpGs sample-by-sample for both cases and controls. These CpG matrices were then compared to the reference sequence to assess whether genomically contiguous methylated sites had been discarded during the initial filtering. From this subset of regions, final selection required, at the per-sample level, coordinated and contiguous (in cases) hypermethylation of individual CpGs across the DMR sequence. Conversely, control samples were required to be at least one-tenth as methylated as cases, and the CpG pattern had to be more random and less coordinated.

[0303] result Using the WGBS methodology with sample preparation and sequencing modifications, combined with a proprietary analytical pipeline and filters (outlined in Methods), we identified and confined variably methylated regions (DMRs) that accurately identify skin cancers and are prominent in the clinical laboratory setting. The WGBS DMRs showed substantial (~50%) overlap with the RRBS DMRs, and performance between the two studies correlated very well. However, there were many high-performing biomarkers that were not seen in the RRBS data. From tissue-to-tissue analysis, 48 ​​hypermethylated malignant melanoma (MM) DMRs were identified (Table 7A). All had AUCs >0.90 and FC ratios >10 (Table 7B). Tissue-to-white blood cell (buffy coat) analysis yielded 111 hypermethylated MM+ epidermis tissue DMRs with less than 1% noise in WBCs (Table 8A). All 111 had AUCs >0.95 and FC ratios >50 (Table 8B). The DMRs included in these tables did not appear in the RRBS study.

[0304] [Table 11-1]

[0305] [Table 11-2]

[0306] [Table 12-1]

[0307] [Table 12-2]

[0308] [Table 13-1]

[0309] [Table 13-2]

[0310] [Table 13-3]

[0311] [Table 14-1]

[0312] [Table 14-2]

[0313] [Table 14-3]

[0314] TELQAS Oligonucleotides Target Enrichment Long Probe Quantitative Amplification Signal (TELQAS) (Kisiel JB, (See, e.g., et al., Hepatology. 2018 Aug 31) oligos (forward invasive primers, reverse primers, flap probes) were designed to CpG motifs within several markers, as shown in FIG. 1 and Table 9, to detect the presence or absence of such markers in, for example, tissue and plasma samples. Table 10 refers to the primers and Table 11 refers to the probes configured for use in the TELQAS assay.

[0315] [Table 15]

[0316] [Table 16-1]

[0317] [Table 16-2]

[0318] [Table 16-3]

[0319] [Table 16-4]

[0320] [Table 17-1]

[0321] [Table 17-2]

[0322] [Incorporation by Reference] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

[0323] [Equivalent] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative rather than limiting the invention described herein. The scope of the present invention is therefore indicated by the appended claims rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. 〔others〕 [Item 1] Measuring the methylation level of one or more genes in a biological sample from a human individual, treating genomic DNA in said biological sample with a reagent that modifies DNA in a methylation-specific manner; amplifying the treated genomic DNA using a set of primers for the one or more selected genes; and determining a methylation level of the one or more genes by polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation specific nucleases, mass-based separation, and target capture; The one or more genes are selected from the group: ●c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110 627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX. chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33; or ●AGRN、BMP8B、c17orf46_B、c17orf57_B、C2CD4A、C2CD4D_B、CDYL、CMAH、DENND2D.D LEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LD LRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693 -32237785; MAX.chr10.62492374-62492793; MAX.chr11.14926535-14926715.MAX. chr16.54970444-54970469; MAX.chr19.16439332-16439390; MAX.chr20.2108067 0-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719. MAX.chr8.82543163-82543213 PARP15 PRKAG2 PROC_A PROM1_B PTGER4_A PTP4 A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73; ●MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73 073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22 541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763; or ●MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A; or one or more markers listed in Table 8A; The method is selected from the group consisting of [Item 2] 2. The method of claim 1, wherein the DNA is treated with a reagent that modifies the DNA in a methylation-specific manner. [Item 3] 3. The method of claim 2, wherein the reagent comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent. [Item 4] 4. The method of claim 3, wherein the DNA is treated with a bisulfite reagent to produce bisulfite-treated DNA. [Item 5] 2. The method of claim 1, wherein the measuring comprises multiplex amplification. [Item 6] 2. The method according to item 1, wherein measuring the amount of at least one methylation marker gene 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. [Item 7] 2. The method of claim 1, wherein the sample comprises one or more of a plasma sample, a blood sample, a fine needle aspirate sample, or a tissue sample (e.g., skin tissue). [Item 8] 2. The method of claim 1, wherein the set of primers for the selected one or more genes is listed in Table 3 or Table 10. [Item 9] 2. The method according to item 1, wherein the method is used to detect the presence or absence of melanoma in a biological sample derived from the human. [Item 10] 1. A method for characterizing a sample, comprising: a) measuring the amount of at least one methylation marker gene in DNA from said sample, said at least one methylation marker gene being selected from the group consisting of: ●c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110 627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX. chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33; or ●MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73 073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22 541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763; or ●AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LD LRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693 -32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX. chr16.54970444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73; or ●MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A; or One or more markers listed in Table 8A the measuring being one or more genes selected from: b) determining the amount of at least one reference marker in said DNA; and c) 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 said at least one methylation marker DNA measured in said sample; The method comprising: [Item 11] 11. The method of claim 10, wherein the at least one reference marker comprises one or more reference markers selected from B3GALT6 DNA, ZDHHC1 DNA, and β-actin DNA, and non-cancerous DNA. [Item 12] 11. The method of claim 10, wherein the sample comprises one or more of a plasma sample, a blood sample, a fine needle aspirate sample, or a tissue sample (e.g., skin tissue). [Item 13] 11. The method of claim 10, wherein the DNA is extracted from the sample. [Item 14] 11. The method of claim 10, wherein the DNA is treated with a reagent that modifies the DNA in a methylation-specific manner. [Item 15] 15. The method of claim 14, wherein the reagent comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent. [Item 16] 16. The method of claim 15, wherein the DNA is treated with a bisulfite reagent to produce bisulfite-treated DNA. [Item 17] 16. The method of claim 15, wherein the modified DNA is amplified using a set of primers for the selected one or more genes. [Item 18] Item 18. The method of item 17, wherein the set of primers for the selected one or more genes is listed in Table 3 or Table 10. [Item 19] 11. The method of claim 10, wherein measuring the amount of the methylation marker gene comprises using one or more of the following: polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, and target capture. [Item 20] 20. The method of claim 19, wherein the measuring comprises multiplex amplification. [Item 21] 20. The method according to claim 19, wherein measuring the amount of the at least one methylation marker gene 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. [Item 22] 11. The method according to claim 10, wherein the method is used to detect the presence or absence of melanoma in the biological sample from the human. [Item 23] 1. A method for characterizing a biological sample, comprising: (a) measuring the amount of at least one methylation marker gene in DNA from said sample, said at least one methylation marker gene being; MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A, or MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874-225419 48, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763; b) determining the amount of at least one reference marker in said DNA; and c) 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 said sample; The method comprising: [Item 24] 24. The method of claim 23, wherein the at least one reference marker comprises one or more reference markers selected from B3GALT6 DNA, ZDHHC1 DNA, and β-actin DNA, and non-cancerous DNA. [Item 25] 24. The method of claim 23, wherein the sample comprises one or more of a plasma sample, a blood sample, a fine needle aspirate sample, or a tissue sample (e.g., skin tissue). [Item 26] 24. The method of claim 23, wherein the DNA is extracted from the sample. [Item 27] 24. The method of claim 23, wherein the DNA is treated with a reagent that modifies the DNA in a methylation-specific manner. [Item 28] 28. The method of claim 27, wherein the reagent comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent. [Item 29] 29. The method of claim 28, wherein the DNA is treated with a bisulfite reagent to produce bisulfite-treated DNA. [Item 30] 28. The method of claim 27, wherein the modified DNA is amplified using a set of primers for the selected one or more genes. [Item 31] 31. The method of claim 30, wherein the set of primers for the selected one or more genes is listed in Table 3 or Table 10. [Item 32] 11. The method of claim 10, wherein measuring the amount of the methylation marker gene comprises using one or more of the following: polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, and target capture. [Item 33] 33. The method of claim 32, wherein the measuring comprises multiplex amplification. [Item 34] 33. The method according to item 32, wherein measuring the amount of at least one methylation marker gene 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. [Item 35] 24. The method of claim 23, wherein the method is used to detect the presence or absence of metastatic melanoma in the biological sample from the human. [Item 36] 1. A method for characterizing a biological sample, comprising: (a) the following groups in biological samples from human individuals: ●c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110 627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX. chr2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33; or ●MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73 073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22 541874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763; or ●AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LD LRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693 -32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX. chr16.54970444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73; or ●MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A; or One or more markers listed in Table 8A The methylation level of the CpG site of one or more genes selected from treating genomic DNA in said biological sample with bisulfite; amplifying the bisulfite-treated genomic DNA using a set of primers for the selected gene or genes; and determining the methylation level of said CpG sites by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; To measure through; (b) comparing the methylation level to the methylation level of a corresponding set of genes in a control sample that does not have melanoma; and (c) determining that the individual has melanoma if the methylation level measured in the one or more genes is higher than the methylation level measured in each of the control samples; The method comprising: [Item 37] 37. The method of claim 36, wherein the set of primers for the selected one or more genes is listed in Table 3 or Table 10. [Item 38] 37. The method of claim 36, wherein the biological sample is a plasma sample, a blood sample, a needle aspirate sample, or a tissue sample (e.g., skin tissue). [Item 39] 37. The method of claim 36, wherein the one or more genes are described by the genomic coordinates set forth in Tables 1A, 2A, 7A, 8A, and / or 9. [Item 40] 37. The method of claim 36, wherein the CpG site is present in a coding region or a regulatory region. [Item 41] 37. The method of claim 36, wherein measuring the methylation level of the CpG site of the one or more genes comprises determining a methylation score of the CpG site and determining a methylation frequency of the CpG site. [Item 42] 1. A method for characterizing a biological sample, comprising: (a) the following groups in biological samples from human individuals: The methylation levels of CpG sites of one or more genes selected from MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A are treating genomic DNA in the biological sample with bisulfite; amplifying the bisulfite-treated genomic DNA using a set of primers for the selected gene or genes; and determining the methylation level of said CpG sites by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; To measure through; (b) comparing the methylation level to the methylation level of a corresponding set of genes in a control sample not having clear cell melanoma; and (c) determining that the individual has metastatic melanoma if the methylation level measured in the one or more genes is higher than the methylation level measured in each of the control samples; The method comprising: [Item 43] Item 43. The method of item 42, wherein the set of primers for the selected one or more genes is listed in Table 3. [Item 44] 43. The method of claim 42, wherein the biological sample is a plasma sample, a blood sample, a needle aspirate sample, or a tissue sample (e.g., skin tissue). [Item 45] 43. The method of claim 42, wherein the one or more genes are described by the genomic coordinates set forth in Table 1A and / or 2A. [Item 46] 43. The method of claim 42, wherein the CpG site is present in a coding region or a regulatory region. [Item 47] 43. The method of claim 42, wherein measuring the methylation level of the CpG site of the one or more genes comprises determining a methylation score of the CpG site and determining a methylation frequency of the CpG site. [Item 48] 1. A system for characterizing a sample obtained from a human subject, comprising: an analytical component configured to determine the methylation status of the sample; a software component configured to compare the methylation status of the sample with the methylation status of a control sample or a reference sample recorded in a database; and an alert component configured to determine a single value based on a combination of methylation states and alert a user to a melanoma-associated methylation state; The system comprising: [Item 49] 49. The system of claim 48, wherein the sample comprises a nucleic acid comprising a DMR. [Item 50] 49. The system of item 48, further comprising a component for isolating nucleic acids. [Item 51] Item 49. The system of item 48, further comprising a component for collecting a sample. [Item 52] 50. The system of claim 48, wherein the sample is a stool sample, a tissue sample, a skin tissue sample, a fine needle aspirate sample, a blood sample, a serum sample, a plasma sample, or a urine sample. [Item 53] 49. The system of claim 48, wherein the database comprises nucleic acid sequences that contain DMRs. [Item 54] 50. The system of claim 48, wherein the database comprises nucleic acid sequences from subjects who do not have melanoma. [Item 55] 1) a bisulfite reagent; 2) a control nucleic acid comprising a sequence of a DMR selected from the group consisting of DMR1 to 331 of Tables 1A, 2A, 7A, 8A, and 9, and having a methylation status associated with a subject without melanoma; Including the kit. [Item 56] A kit comprising a bisulfite reagent and an oligonucleotide according to SEQ ID NOs: 1 to 80. [Item 57] A kit comprising a sample collector for obtaining a sample from a subject, a reagent for isolating nucleic acid from the sample, a bisulfite reagent, and an oligonucleotide according to SEQ ID NOs: 1-80. [Item 58] 58. The kit of item 57, wherein the sample is a stool sample, a tissue sample, a skin tissue sample, a fine needle aspirate sample, a blood sample, a serum sample, a plasma sample, or a urine sample. [Item 59] A composition comprising a nucleic acid containing a DMR and a bisulfite reagent. [Item 60] A composition comprising a nucleic acid containing a DMR and an oligonucleotide according to SEQ ID NOs: 1-167. [Item 61] A composition comprising a nucleic acid containing a DMR and a methylation-sensitive restriction enzyme. [Item 62] A composition comprising a nucleic acid comprising a DMR and a polymerase. [Item 63] 1. A method for screening for melanoma in a sample obtained from a subject, comprising: 1) assaying the methylation status of the marker in a sample obtained from the subject; and 2) identifying the subject as having melanoma if the methylation status of the marker differs from the methylation status of the marker assayed in subjects without neoplasia; The method, wherein the marker comprises a base in a variably methylated region (DMR) as provided in Tables 1A, 2A, 7A, 8A, and 9. [Item 64] 64. The method of claim 63, comprising assaying a plurality of markers. [Item 65] 64. The method of claim 63, comprising assaying 2 to 11 markers. [Item 66] 64. The method of claim 63, comprising assaying 12 to 560 markers. [Item 67] 64. The method of claim 63, wherein assaying the methylation status of the marker in the sample comprises determining the methylation status of a single base. [Item 68] 64. The method of claim 63, wherein assaying the methylation status of the marker in the sample comprises determining the degree of methylation at a plurality of bases. [Item 69] 64. The method of claim 63, wherein the methylation status of the marker comprises an increase or decrease in methylation of the marker relative to a normal methylation status of the marker. [Item 70] 64. The method of claim 63, wherein the methylation status of the marker comprises a different pattern of methylation of the marker relative to a normal methylation status of the marker. [Item 71] 64. The method of claim 63, comprising assaying the methylation state of the forward strand or assaying the methylation state of the reverse strand. [Item 72] Item 64. The method of item 63, wherein the marker is a region of 100 bases or less. [Item 73] 64. The method of claim 63, wherein the marker is a region of 500 bases or less. [Item 74] Item 64. The method of item 63, wherein the marker is a region of 1000 bases or less. [Item 75] 64. The method of claim 63, wherein the marker is a region of 5000 bases or less. [Item 76] Item 64. The method of item 63, wherein the marker is a single base. [Item 77] 64. The method of claim 63, wherein the marker is a high CpG density promoter. [Item 78] 64. The method of claim 63, wherein the sample is a stool sample, a tissue sample, a skin tissue sample, a blood sample, a fine needle aspirate sample, or a urine sample. [Item 79] 64. The method of claim 63, wherein said assaying comprises using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture. [Item 80] 64. The method of claim 63, wherein said assaying comprises the use of a methylation-specific oligonucleotide. [Item 81] 1. A method for characterizing a biological sample, comprising: treating genomic DNA in the biological sample with bisulfite; amplifying the bisulfite treated genomic DNA using primers specific for CpG sites for MAX.chr20.21080958-21081038, primers specific for CpG sites for MAX.chr7.155259597-155259763, primers specific for CpG sites for FOXL2NB, and primers specific for CpG sites for HOXA9_A; wherein a primer specific for MAX.chr7.155259597-155259763 can bind to the amplicon bounded by SEQ ID NOs: 47 and 48 or 49 and 50, wherein the amplicon bounded by SEQ ID NOs: 47 and 48 or 49 and 50 is at least a portion of a genetic region that includes chromosome 7 coordinates 155259597-155259763; and a primer specific for MAX.chr20.21080958-21081038 can bind to the amplicon bounded by SEQ ID NOs: 39 and 40, the amplicon bounded by SEQ ID NOs: 39 and 40 being at least a portion of a genetic region including chromosome 20 coordinates 21080958-21081038; Also, the primer specific for FOXL2NB can bind to the amplicon bounded by SEQ ID NOs: 5 and 6, the amplicon bounded by SEQ ID NOs: 5 and 6 being at least a portion of the gene region including chromosome 3 coordinates 138663981-138664076; and A primer specific for HOXA9_A can bind to the amplicon bounded by SEQ ID NOs: 19 and 20, the amplicon bounded by SEQ ID NOs: 19 and 20 being at least a portion of a genetic region including chromosome 7 coordinates 27209628-27209739; measuring the methylation levels of CpG sites for one or more of MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; The method comprises measuring methylation levels of CpG sites for one or more of MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A in a biological sample from a human individual via a method according to the present invention. [Item 82] 82. The method of claim 81, wherein the biological sample is a blood sample, a tissue sample, a fine needle aspirate sample, or a skin tissue sample. [Item 83] 82. The method of claim 81, wherein the CpG site is present in a coding region or a regulatory region. [Item 84] 82. The method of claim 81, wherein measuring the methylation level of the CpG sites for one or more of MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A comprises determining a methylation score for the CpG sites and determining a methylation frequency for the CpG sites. [Item 85] 1. A method for characterizing a biological sample, comprising: treating genomic DNA in the biological sample with bisulfite; amplifying the bisulfite-treated genomic DNA using primers specific for CpG sites for the one or more markers; wherein the primers specific for each of the markers are capable of binding to an amplicon bounded by each of the primer sequences listed in Table 3, and the amplicon bounded by each of the primer sequences is at least a portion of a genetic region that includes each of the chromosomal regions listed in Table 1A or Table 2A; determining the methylation level of the CpG sites for one or more of the markers by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; Through c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr1 0.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-2 9106917, MAX.chr17.73073682-73073830, MAX.chr2.71116033-71116122, MAX.ch r20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33 in a biological sample from a human individual. [Item 86] 86. The method of claim 85, wherein the biological sample is a blood sample, a tissue sample, a fine needle aspirate sample, or a skin tissue sample. [Item 87] 86. The method of item 85, wherein the CpG site is present in a coding region or a regulatory region. [Item 88] 86. The method of claim 85, wherein measuring the methylation level of a CpG site for the one or more markers comprises determining a methylation score of the CpG site and determining a methylation frequency of the CpG site. [Item 89] 1. A method for characterizing a biological sample, comprising: treating genomic DNA in the biological sample with bisulfite; amplifying the bisulfite-treated genomic DNA using primers specific for CpG sites for the one or more markers; wherein the primers specific for each of the markers are capable of binding to an amplicon bounded by each of the primer sequences listed in Table 3, and the amplicon bounded by each of the primer sequences is at least a portion of a gene region that includes each of the chromosomal regions listed in Table 8A; determining the methylation level of the CpG sites for one or more of the markers by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; The method comprises measuring the methylation level of CpG sites for one or more markers listed in Table 8A in a biological sample from a human individual via: [Item 90] 90. The method of claim 89, wherein the biological sample is a blood sample, a tissue sample, a fine needle aspirate sample, or a skin tissue sample. [Item 91] 90. The method of claim 89, wherein the CpG site is present in a coding region or a regulatory region. [Item 92] 90. The method of claim 89, wherein measuring the methylation level of a CpG site for the one or more markers comprises determining a methylation score of the CpG site and determining a methylation frequency of the CpG site. [Item 93] 1. A method for preparing a deoxyribonucleic acid (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, comprising: (a) extracting genomic DNA from a biological sample from a human individual; (b) (i) Treating the extracted genomic DNA with bisulfite; (ii) amplifying the bisulfite-treated genomic DNA using separate primers specific for CpG sites for one or more markers listed in Tables 1A, 2A, 7A, 8A, and 9; Producing a fraction of extracted genomic DNA by (c) analyzing one or more loci in the resulting fraction of extracted genomic DNA by measuring the methylation level of the CpG sites for each of the one or more markers; The method comprising: [Item 94] 94. The method of claim 93, wherein measuring the methylation level of the CpG site for each of the one or more markers is determined by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, or bisulfite genomic sequencing PCR. [Item 95] 94. The method of claim 93, wherein amplifying the bisulfite-treated genomic DNA using primers specific to CpG sites for each of the one or more markers is a set of primers that specifically bind to at least a portion of a gene region for the markers shown in Tables 1A, 2A, 7A, 8A, and / or 9. [Item 96] 94. The method of claim 93, wherein 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. [Item 97] 94. The method of claim 93, wherein each of the one or more analyzed loci is associated with melanoma and / or a subtype of melanoma. [Item 98] The one or more markers are c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.c 94. The method of claim 93, wherein the ribozyme is selected from the group consisting of hr.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33. [Item 99] The one or more markers are selected from the group consisting of AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.c 94. The method of claim 93, wherein the ribozyme is selected from the group consisting of hr16.54970444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73. [Item 100] The one or more markers are MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.2254 94. The method of claim 93, wherein the nucleotide sequence is selected from MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763. [Item 101] 94. The method of claim 93, wherein the one or more markers are selected from MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A. [Item 102] 1. A method for preparing a deoxyribonucleic acid (DNA) fraction from a biological sample of a human individual, useful for analyzing one or more DNA fragments involved in one or more chromosomal abnormalities, comprising: (a) extracting genomic DNA from a biological sample from a human individual; (b) (i) treating the extracted genomic DNA with bisulfite; (ii) amplifying the bisulfite-treated genomic DNA using separate primers specific for CpG sites for one or more markers listed in Tables 1A and 2A; To generate extracted genomic DNA fractions via: (c) analyzing one or more DNA fragments in the resulting fraction of extracted genomic DNA by measuring the methylation level of the CpG sites for each of the one or more markers; The method comprising: [Item 103] 103. The method of claim 102, wherein measuring the methylation level of the CpG site for each of the one or more markers is determined by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, or bisulfite genomic sequencing PCR. [Item 104] 103. The method of claim 102, wherein amplifying the bisulfite-treated genomic DNA using primers specific to CpG sites for each of the one or more markers is a set of primers that specifically bind to at least a portion of a gene region for the markers shown in Table 1A and / or 2A. [Item 105] 103. The method of claim 102, wherein 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. [Item 106] 103. The method of claim 102, wherein each of the analyzed DNA fragments is associated with melanoma and / or a subtype of melanoma. [Item 107] The one or more markers are selected from the group consisting of c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX.chr1.110627096-110627364, MAX.chr10.22541869-22541953, MAX.chr10.62492690-62492812, MAX.chr13.29106812-29106917, MAX.chr17.73073682-73073830, MAX.chr18.73073682-73073830, MAX.chr19.73073682-73073830, MAX.chr11.73073682-73073830, MAX.chr12.73073682-73073830, MAX.chr13.73073682-73073830, MAX.chr14.73073682-73073830, MAX.chr15.73073682-73073830, MAX.chr16.73073682-73073830, MAX.chr17.73073682-73073830, MAX.chr18.73073682-73073830, MAX.chr19.73073682-73073 103. The method of claim 102, wherein the chromosome number is selected from r2.71116033-71116122, MAX.chr20.21080958-21081038, MAX.chr5.42992655-42992768, MAX.chr5.60921627-60921853, MAX.chr6.29521537-29521696, MAX.chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33. [Item 108] The one or more markers are selected from the group consisting of AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENND2D, DLEU2, DSCR6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486-1072508, MAX.chr1.32237693-32237785, MAX.chr10.62492374-62492793, MAX.chr11.14926535-14926715, MAX.chr12.1072486-1072508, MAX.chr1.32237693-32237785, MAX.chr13.1072486-1072508, MAX.chr14.1072486-1072508, MAX.chr15.1072486-1072508, MAX.chr16.1072486-1072508, MAX.chr17.1072486-172486 r16.54970444-54970469, MAX.chr19.16439332-16439390, MAX.chr20.21080670-21081280, MAX.chr4.113432264-113432298, MAX.chr7.129425668-129425719, MAX.chr8.82543163-82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PXD2A, SLC2A2, SLC35D3, TBR1, TBX2, TCP11, TFAP2A, and TRIM73. [Item 109] The one or more markers are MAX.chr10.62492680-62492822, TMEM30B_B, MAX.chr11:14926602-14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682-73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627-60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.2254 1874-22541948, ME3, HOPX, MAX.chr20.3229151-3229791, CLIC5, MAX.chr13.29106812-29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096-110627364, MAX.chr2.71116033-71116122, MAX.chr20.21080958-21081038, and MAX.chr7.155259597-155259763. [Item 110] 103. The method of claim 102, wherein the one or more markers are selected from MAX.chr20.21080958-21081038, MAX.chr7.155259597-155259763, FOXL2NB, and HOXA9_A. [Brief description of the drawings]

[0324] [Figure 1-1] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-2] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-3] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-4]FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-5] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-6] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-7] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-8] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-9] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-10] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-11] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-12] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-13] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-14]FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-15] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-16] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-17] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11). [Figure 1-18] FIG. 1 provides information on the marker chromosomal regions used for the methylated DNA markers listed in Table 9, as well as the associated primers and probes (Tables 10 and 11).

Claims

[Claim 1] Measuring the methylation level of one or more genes in a biological sample from a human individual, treating genomic DNA in said biological sample with a reagent that modifies DNA in a methylation-specific manner; amplifying the treated genomic DNA using a set of primers for the one or more selected genes; and determining a methylation level of the one or more genes by polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation specific nucleases, mass-based separation, and target capture; The one or more genes are selected from the group: ●c10orf55, c2orf82, FOXL2NB, CLIC5, FAM174B_B, FLJ22536_A, FOXP1, GALNT3_A, HOPX, HOXA9_A, ITPKA, KCNQ4_A, LMX1B, MAX. chr1.110627096-110627364, MAX. chr10.22541869-22541953, MAX. chr10.62492690-62492812, MAX. chr13.29106812-29106917, MAX. chr17.73073682-73073830, MAX. chr2.71116033-71116122, MAX. chr20.21080958-21081038, MAX. chr5.42992655-42992768, MAX. chr5.60921627-60921853, MAX. chr6.29521537-29521696, MAX. chr7.155259597-155259763, ME3, MIR155HG, NFATC2_A, OCA2, OXT, RNF207_A, RUNX2, SIX4_A, STX16, TAL1, TMEM30B, and TSPAN33; or ● AGRN, BMP8B, c17orf46_B, c17orf57_B, C2CD4A, C2CD4D_B, CDYL, CMAH, DENN D2D, DLEU2, DSC R6, FLJ22536_B, FLJ45983, FOXF2, GALNT3_B, HCG4P6, HOXA9_B, KIFC2, LDLRAD2, LY75, LYL1, LYN, MAPK13, MAX.chr1.1072486 - 1072508, MAX.chr1.32237693 - 32237785, MAX.chr10.62492374 - 62492793, MAX.chr11.14926535 - 14926715, MAX.chr16.54970444 - 54970469, MAX.chr19.16439332 - 16439390, MAX.chr20.21080670 - 21081280, MAX.chr4.113432264 - 113432298, MAX.chr7.129425668 - 129425719, MAX.chr8.82543163 - 82543213, PARP15, PRKAG2, PROC_A, PROM1_B, PTGER4_A, PTP4A3, SDCCAG8, SH3PX D2A, SLC2A2, SLC35D3, TB R1, TBX2, TCP11, TFAP2A, and TRIM73; or ● MAX.chr10.62492680 - 62492822, TMEM30B_B, MAX.chr11:14926602 - 14926831, HOXA9_9650, C10orf55_B, MAX.chr17.73073682 - 73073830, TSPAN33, FAM174B_C, MAX.chr5.60921627 - 60921853, SIX4_A, KCNQ4_A, FOXP1, C2orf82_B, TAL1_B, BTBD19_B, MAX.chr10.22541874 - 22541948, ME3, HOPX, MAX.chr20.3229151 - 3229791, CLIC5, MAX.chr13.29106812 - 29106917, FOXL2NB, FLJ22536_A, MAX.chr1.110627096 - 110627364, MAX.chr2.71116033 - 71116122, MAX.chr20.21080958 - 21081038, and MAX.chr7.155259597 - 155259763; or MAX. chr20.21080958-21081038, MAX. chr7.155259597-155259763, FOXL2NB, and HOXA9_A; or one or more markers listed in Table 8A; The method is selected from the group consisting of

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