Compositions and methods for detecting and diagnosing of melanoma

Abstract

It has been discovered that differential methylation in DNA can be used to distinguish between malignant and non-malignant skin lesions. Seven genes, 3 that are hypermethylated in melanoma, 1 that is hypermethylated in melanocytes and remains methylated in melanoma, and 3 that are hypermethylated in all tissues are provided as biomarkers. The panel demonstrates a 93% sensitivity, 97% specificity and an AUC of 95% in distinguishing between melanoma and benign lesions. Thus provided herein are melanoma biomarkers and methods of detecting them and using them to diagnose and guide treatment of subjects with the same, and monitor melanoma tumor dynamics during and after treatment.

Description

[0001] ATTORNEY DOCKET NO. PRECI 100 PCT

[0002] COMPOSITIONS AND METHODS FOR

[0003] DETECTING AND DIAGNOSING OF MELANOMA

[0004] CROSS REFERENCE TO RELATED APPLICATIONS

[0005] This application claims benefit of and priority to U.S. Provisional Application No. 63 / 715,376, filed November 1, 2024, the contents of which is incorporated by reference herein in its entirety.

[0006] REFERENCE TO THE SEQUENCE LISTING

[0007] The Sequence Listing XML submitted as a file named “PRECI_100_PCT_ST26.xml,” created on November 3, 2025, and having a size of 25,785 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

[0008] FIELD OF THE INVENTION

[0009] The disclosed invention is generally in the field of melanoma and specifically in the area of distinguishing melanoma from non-cancerous skin and skin lesions.

[0010] BACKGROUND OF THE INVENTION

[0011] Cutaneous melanoma (i.e., malignant melanocytic proliferations) is the fifth most common malignancy that accounts for around 5 % new cancer cases annually, almost 100,000 in the US alone. While early detection with treatment is generally successful, undetected melanoma can progress with metastasis and high mortality. In approximately 15% of cases, even very experienced dermatopathologists cannot confidently interpret the biopsy specimen findings to render a clear diagnosis of a benign nevus or cancerous melanoma. Diagnostic discordance of melanocytic proliferations has long been known. A large study with over 180 dermatopathologists and 240 cases establishes poor intra- and inter-observer precision and accuracy of microscopy, especially in cases spanning moderately dysplastic nevus to early melanoma. As many as 300,000 lesions such as this are not easily classified each year in the US. Because both over- and under-diagnosis of melanoma impact patient care, affordable ancillary tests on formalin fixed paraffin embedded (FFPE) tissue samples are needed to correctly classify ambiguous skin lesions.

[0012] Up to 24% of proliferative melanocytic lesions exhibit indeterminate diagnostic characteristics preventing the rendering of a definitive diagnosis. In cases with diagnostic uncertainty, guidelines support using molecular testing to differentiate benign from malignant melanocytic neoplasms, although there is little guidance on exactly what type of molecular testing should be used. Thus, clinical practice is to err on the side of malignancy, potentially resulting in patients undergoing unnecessary treatment.

[0013] Diagnosis typically proceeds according to clinical observation -> histology -> immunohistochemistry -> and molecular testing (e.g., FISH / CGH / GEP / NGS) where each escalation 1

[0014] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT increases accuracy of the diagnosis, but also incurs increasing costs. See, e.g., Hosier and Murphy, “Ancillary testing for melanoma: currenl trends and practical considerations”, Human Pathology, 140, (2023), pg. 5-21, ISSN 0046-8177, doi.org / 10.1016 / j.humpath.2023.05.002. The current test on the market, MyPath Melanoma by Castle Bioscicnccs, uses RT-qPCR to detect the RNA expression of 14 genes shown to be altered in melanoma and 9 housekeeping genes for normalization. This has a sensitivity of 90% and a specificity of 91%. Based on their studies, their test results in a 42.7% reduction in ambiguous diagnoses in hard to diagnose cases. When the results returned were benign, there was a 72% reduction in re-excision rates. These results demonstrate that a molecular diagnostic can alter clinical care if it is used clinically.

[0015] However, the current test relies on RNA which is notoriously difficult to work with, especially in FFPE samples. The test also required microdissection of area of interest for testing. Furthermore, it requires assaying 23 different genes in order to make its predictions. Both of these factors make the test more expensive than most immunohistochemistry tests. For at least these reasons, there is a need for improved means of determining if a melanocytic lesion is benign or malignant.

[0016] Thus, provided are biomarkers, and methods of detecting and using the same, to determine if a melanocytic lesion is benign or malignant.

[0017] BRIEF SUMMARY OF THE INVENTION

[0018] It has been discovered that differential methylation in DNA can be used to distinguish between malignant and non-malignant skin lesions. Seven genes, 3 that are hypermethylated in melanoma, 1 that is hypermethylated in melanocytes and remains methylated in melanoma, and 3 that are hypermethylated in all tissues. The panel demonstrates a 93% sensitivity, 97% specificity and an AUG of 95%.

[0019] Thus provided herein are melanoma biomarkers and methods of detecting them and using them to diagnose and guide treatment of subjects with the same, and monitor melanoma tumor dynamics during and after treatment.

[0020] Methods of detecting the methylation status at one or more locations in DNA from a human subject are provided. The methods can include, for example, processing DNA of a sample from the subject with a machine-based platform and detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci biomarkers provided herein. The CpG loci biomarkers can be part of, or adjacent to, the genes EVX2, PROMI and TBXT (i.e., melanoma biomarkers): TFAP2A (i.e., melanocyte biomarker); and NCOR2, LRRC8A, and TRAP1 (i.e., all-tissue biomarkers).

[0021] Thus, the methods can include detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of one, two, or all three, of genes EVX2, PROMI 2

[0022] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT and TBXT, in combination with detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of TFAP2A, in combination with detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of one, two, or all three of genes NCOR2, LRRC8A, and TRAP1.

[0023] In some forms, the methods include detecting DNA methylation at and / or close to the genomic sequences associated with any integer number of the CpG loci between 2 and 50.

[0024] Exemplary CpG loci include those within and / or including and / or being one or more of human genomic regions / positions chr2:176944088-176948446, chr4:16084196-16085735, chr6:166579974-166583423, chr6: 10419400- 10420323, chrl2:124821510-124821583, chr9: 131671004-131671084, chrl6: 3714325-3714392, chr2: 176947940-176947941, chr4: 16085180- 16085181, chr6: 166582393-166582394, chr6: 10419846- 10419846, chrl2: 124821510-124821583, chr9:131671004-131671084, and chrl6: 3714325-3714392.

[0025] In some forms, the CpG loci lies within and / or includes and / or is one or more of one or more of SEQ ID NOS: 1-14.

[0026] The processing can include, for example, one or more of PCR, digital droplet PCR, methylation specific PCR, real time methylation specific PCR, and PCR using a methylated DNA specific binding protein, quantitative PCR, quantitative real-time PCR, microarray, or DNA sequencing.

[0027] The DNA can be chemically-modified for example with sodium bisulfite or a ten-eleven translocation (TET) protein. The DNA can be purified prior to processing with the machine-based platform.

[0028] The sample can tissue optionally a melanocytic lesion, or a blood sample, optionally plasma.

[0029] Also provided are methods of determining if a biological sample includes DNA from melanoma cells. The methods can include detecting methylation in DNA of the biological sample and comparing the level of methylation to one or more controls, and determining that the biological sample includes melanoma cells when methylation is increased at one or more CpG loci of one, two, or three genes selected from the group consisting of EVX2, PROMI and TBXT.

[0030] The method can include determining that the biological sample includes melanoma cells when methylation is increased at one or more CpG loci of all three genes EVX2, PROMI and TBXT.

[0031] In some forms, methylation is normalized relative to methylation at one or more standardization CpG loci. In some forms, the standardization CpG locus is at and / or close to the genomic sequences associated with the TFAP2 gene.

[0032] 3

[0033] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT

[0034] In some forms, a determining if a biological sample includes DNA from melanoma cells includes

[0035] (i) detecting methylation in DNA of the biological sample at one or more melanoma biomarkers, one or more melanocyte biomarkers, and one or more all-tissue biomarkers;

[0036] (ii) normalizing the one or more melanoma biomarkers and the one or more melanocyte biomarkers to the one or more all tissue biomarker controls; and

[0037] (iii) preparing a ratio between the melanoma biomarker(s) and the melanocyte biomarker(s) control; wherein if the ratio is above a threshold defined by a sensitivity and / or specificity, then the sample is positive for melanoma and / or if the ratio is the same and / or below the threshold then the sample is negative for melanoma.

[0038] For example, some forms include

[0039] (i) detecting methylation in DNA of the biological sample at one or more melanoma biomarkers: EVX2, PROMI and TBXT, a melanocyte biomarker: TFAP2A, and one or more alltissue biomarkers: NCOR2, LRRC8A, and TRAP1;

[0040] (ii) normalizing the one or more melanoma biomarkers and the melanocyte biomarker to the one or more all tissue biomarkcr controls; and

[0041] (iii) preparing a ratio between the melanoma biomarker(s) and the melanocyte biomarker control; wherein if the ratio is above a threshold defined by a sensitivity and / or specificity, then the sample is positive for melanoma and / or if the ratio is the same and / or below the threshold then the sample is negative for melanoma.

[0042] In some forms, the sensitivity and / or specificity is 70% or more.

[0043] Any of the methods can further include treating the subject for melanoma when it is determined that the subject has melanoma.

[0044] Any of the methods can further include abstaining from treating the subject for melanoma when it is determined that the subject does not have melanoma.

[0045] Exemplary treatments are provided and can include, for example, one or more of surgery, radiation, immunotherapy, chemotherapy, or target drag therapy.

[0046] Any of the methods of treatment can include determining if a subject has melanoma, and treating the subject for melanoma when it is determined that the subject has melanoma, and abstaining from treating the subject for melanoma when it is determined that the subject does not have melanoma.

[0047] Reagents and kits for use in the disclosed methods are also provided.

[0048] 4

[0049] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT

[0050] In the non- limiting experiments below, disclosed compositions and methods are exemplified as a qPCR panel and associated disease classifier that assessed the methylation status of 7 different genes to distinguish between nevi with architectural disorder from melanoma in situ using DNA isolated from formalin-fixed paraffin-embedded (FFPE) samples.

[0051] The provided compositions and methods can thus be used to distinguish with high sensitivity and specificity, nevus from melanoma, and to guide clinical treatment decisions about further surgical resection of a suspicious lesion based thereon. Additionally, the DNA methylation biomarker set has the potential to be used to identify patients at risk of recurrence and to guide treatment, among other applications discussed in more detail below.

[0052] Additional advantages of the disclosed method and compositions will be set forth in part in the description that follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

[0053] BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The accompanying drawings illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0055] Figures 1A-1B show in silico validation of the melanoma marker loci using independent GEO data. Figure 1A is a boxplot showing a highly significant difference in combined DNA methylation level of the four melanoma loci between malignant melanoma samples and benign nevi. Figure IB is a plot showing Receiving Operating Characteristic (“ROC”) analysis for the same data. “AUG” - area under the curve.

[0056] Figure 2 is a violin plot showing the diagnostic panel can distinguish benign nevi / nevi with architectural disorder from melanoma in situ in clinical formalin-fixed paraffin-embedded (FFPE) samples.

[0057] Figure 3 shows molecular detection of melanoma. DNA from three FFPE benign nevi samples and three FFPE malignant melanoma in situ samples was analyzed as described in detail below. ROC analysis of panel perfomiance. Sensitivity equals 93%, specificity is 97% and the area under the curve is 95%.

[0058] Figure 4 is a flow chart of an exemplary process of detecting melanoma.

[0059] 5

[0060] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT

[0061] DETAILED DESCRIPTION OF THE INVENTION

[0062] The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0063] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0064] I. Definitions

[0065] It is to be understood that the disclosed compounds, compositions, and methods are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular forms and embodiments only and is not intended to be limiting.

[0066] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0067] Throughout this specification the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0068] As used herein, the terms “nevus” and “nevi” refer to a growth(s) on the skin that is fomred by a cluster of melanocytes (cells that make a substance called melanin, which gives color to skin and eyes). A nevus is usually dark and may be raised from the skin. Nevus can also be referred to as a mole. Nevus / nevi are typically used to refer to a benign growth. Because the disclosed methods and compositions can be used to establish the benign or malignant status, growths of indeterminant status are typically referred to herein as “melanocytic lesion,” which can be either benign (e.g., a nevus) or malignant (e.g., a melanoma).

[0069] As used herein, the term “methylation” means that a methyl group is attached to a base constituting DNA. In genomic DNA of mammalian cells, in addition to A, C, G, and T, there is the fifth base called 5 -methylcytosine (5-mC) with a methyl group attached to the fifth carbon of a cytosine ring. The methylation of 5-methylcytosine occurs only at C of CG dinucleotide (5'-mCG- 3') called CpG. Methylation of CpG inhibits the expression of alu or transposon and a repeating

[0070] 6

[0071] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT sequence of the genome. In addition, since the 5-mC of the CpG is naturally deaminated to be easily thymine (T), the CpG is a site where most epigenetic changes frequently occur in mammalian cells.

[0072] The term “genomic region”, as used herein, generally refers to identified regions of nucleic acid that arc identified by their location in the chromosome or another reference such a CpG loci.

[0073] The term “CpG islands”, as used herein, generally refers to a contiguous region of genomic DNA that satisfies the criteria of: (1) having a frequency of CpG dinucleotides corresponding to an “Observed / Expected Ratio” greater than about 0.6; and (2) having a “GC Content” greater than about 0.5. CpG islands are typically, but not always, between about 0.2 to about 3 kilobases (kb) in length having a high frequency of CpG sites. CpG islands are found at or near promoters of about 40% of mammalian genes. CpG islands are also found outside of mammalian genes. In some examples, CpG islands are found in exons, introns, promoters, enhancers, inhibitors, and transcriptional regulatory elements. CpG islands may tend to occur upstream of so-called “housekeeping genes”. CpG islands may be said to have a CpG dinucleotide content of at least about 60% of what would be statistically expected. The occurrence of CpG islands at or upstream of the 5' end of genes may reflect a role in the regulation of transcription, and methylation of CpG sites within the promoters of genes may lead to silencing. Silencing of tumor suppressors by methylation is, in turn, a hallmark of a number of human cancers.

[0074] The term “CpG shores”, as used herein, generally refers to regions extending short distances from CpG islands in which methylation may also occur. CpG shores may be found in the region about 0 to 2 kb upstream and downstream of a CpG island.

[0075] The term “CpG shelves”, as used herein, generally refers to regions extending short distances from CpG shores in which methylation may also occur. CpG shelves may generally be found in the region between about 2 kb and 4 kb upstream and downstream of a CpG island (e.g., extending a further 2 kb out from a CpG shore).

[0076] The term “hemi-methylation” or “hemimethylation”, as used herein, generally refers to the methylation state of a palindromic CpG methylation site, where only a single cytosine in one of the two CpG dinucleotide sequences of the palindromic CpG methylation site is methylated (e.g., 5'- CCMGG-3' (top strand): 3'-GGCC-5' (bottom strand)).

[0077] The term “hypermethylation”, as used herein, generally refers to the average methylation state corresponding to an increased presence of 5-mC at one or a plurality of CpG dinucleotides within a DNA sequence of a test DNA sample, relative to the amount of 5-mC found at corresponding CpG dinucleotides within another DNA sample, e.g., a normal control DNA sample or DNA sample from a subject with known disease status.

[0078] The term “hypomethylation”, as used herein, generally refers to the average methylation state corresponding to a decreased presence of 5-mC at one or a plurality of CpG dinucleotides

[0079] 7

[0080] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT within a DNA sequence of a test DNA sample, relative to the amount of 5-mC found at corresponding CpG dinucleotides within another DNA sample, e.g., a normal control DNA sample or DNA sample from a subject with known disease status.

[0081] The term “methylation state” or “methylation status”, as used herein, generally refers to the presence or absence of 5-methylcytosine (“5-mC”) at one or a plurality of CpG dinucleotides within a DNA sequence. Methylation states at one or more particular palindromic CpG methylation sites (each having two CpG dinucleotide sequences) within a DNA sequence include “unmethylated,” “fully-methylated” and “hemi-methylated.”

[0082] The term “methylated cytosine”, as used herein, generally refers to any methylated forms of the nucleic acid base cytosine that contains a methyl or hydroxymethyl functional group at the 5' position. Methylated cytosines are known to be regulators of gene transcription in genomic DNA. This term may include 5-methylcytosine and 5-hydroxymethylcytosine.

[0083] The term “methylation assay”, as used herein, generally refers to any assay for determining the methylation state of one or more CpG dinucleotide sequences within a sequence of DNA.

[0084] The term “MSP” (methylation-specific polymerase chain reaction (PCR)), as used herein, generally refers to a methylation assay, such as that described by Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996, and by U.S. Pat. No. 5,786,146, the contents of each of which arc incorporated herein by reference.

[0085] The term “methylation converted” or “converted” nucleic acid, as used herein, generally refers to nucleic acid, such as for example DNA, that has undergone a process used to convert the DNA for methylation sequencing. Examples of conversion processes include reagent-based (such as bisulfite) conversion, enzymatic conversion, or combination conversion (such as TET-assisted pyridine borane sequencing (TAPS) conversion), where unmethylated cytosines are converted into uracil prior to PCR amplification or sequencing. The conversion process may be used in methyl sequencing methods to distinguish between methylated and unmethylated cytosine bases.

[0086] The terms “subject,” “individual,” and “patient” refer to any individual who is the target of treatment using the disclosed compositions. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human. The subjects can be symptomatic or asymptomatic. The term does not denote a particular age or sex. A subject can include a control subject or a test subject.

[0087] The term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease state being treated or to otherwise provide a desired pharmacologic and / or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being administered. The effect of the effective amount

[0088] 8

[0089] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT can be relative to a control. Such controls are known in the art and discussed herein, and can be, for example the condition of the subject prior to or in the absence of administration of the drug, or drug combination.

[0090] The term “pharmaceutically acceptable” or “biocompatiblc” refers to compositions, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The phrase “pharmaceutically acceptable carrier” refers to pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, solvent or encapsulating material involved in carrying or transporting any subject composition, from one organ, or portion of the body, to another organ, or portion of the body. Each earner must be “acceptable” in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient.

[0091] The term “treating” or “preventing” a disease, disorder, or condition includes ameliorating at least one symptom of the disease or condition. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating, or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with one or more diseases or disorders are mitigated or eliminated, including, but are not limited to, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and / or prolonging survival of individuals.

[0092] II. Biomarkers for Malignant Melanocyte Proliferation

[0093] A significant portion of melanocytic lesions evaluated for suspicion of melanoma cannot be classified as definitively benign or malignant by current techniques. Current standards recommend additional molecular testing in this situation, but no consensus exists for what that testing should entail. The current commercial solution employs a 23 gene expression panel and is costly with uneven reimbursement. An alternative, out of an abundance of caution, is to surgically remove the melanocytic lesion, which can be painful and disfiguring to the subject, and maybe unnecessary as many of the indeterminant melanocytic lesions are ultimately benign.

[0094] The disclosed biomarkers of differential methylation in DNA solve this problem by providing highly accurate, sensitive, and specific means of distinguishing malignant lesions from benign ones. Its initial performance characteristics are better than the current commercially available molecular test, but the expense to perform the test is expected to be much less. Furthermore, the provided set of DNA biomarkers can also be used to monitor melanoma tumor dynamics during and after treatment.

[0095] 9

[0096] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT

[0097] The biomarkers themselves, as well as methods of detecting them not only in melanocytic lesion samples, but also non-invasive liquid biopsies (e.g., plasma), and uses thereof including, but not limited, distinguishing between benign skin lesions and malignant melanoma, and subsequent treatment guidance arc provided. Due to the less invasive nature of sample collection (e.g., blood and urine), such methods can avoid the need to prospectively removing the melanocytic lesion because a definitive diagnostic technique is unavailable or prohibitively expensive.

[0098] Different measures of diagnostic accuracy relate to the different aspects of diagnostic procedure. See, e.g., Simundic AM. Measures of Diagnostic Accuracy : Basic Definitions. EJIFCC. 2009 Jan 20;19(4):203-l 1. PMID: 27683318; PMCID: PMC4975285, which is specifically incorporated by reference herein in its entirety. Some measures are used to assess the discriminative property of the test, others are used to assess its predictive ability. While discriminative measures are mostly used by health policy decisions, predictive measures are most useful in predicting the probability of a disease in an individual.

[0099] A perfect diagnostic procedure has the potential to completely discriminate subjects with and without disease. Values of a perfect test which are above the cut-off are always indicating the disease, while the values below the cut-off are always excluding the disease. Unfortunately, a perfect test is difficult to establish in real life and therefore diagnostic procedures typically make only partial distinction between subjects with and without disease. Values above the cut-off are not always indicative of a disease since subjects without disease can also sometimes have elevated values. Such elevated values of a certain parameter of interest are called false positive values (FP). On the other hand, values below the cut-off are mainly found in subjects without disease. However, some subjects with the disease can have them too. Those values are false negative values (FN). Therefore, the cut-off divides the population of examined subjects with and without disease in four subgroups considering parameter values of interest:

[0100] • (true positive (TP) -subjects with the disease with the value of a parameter of interest above the cut-off

[0101] • (false positive (FP) -subjects without the disease with the value of a parameter of interest above the cut-off

[0102] • (true negative (TN) -subjects without the disease with the value of a parameter of interest below the cut-off

[0103] • (false negative (FN) -subjects with the disease with the value of a parameter of interest below the cut-off

[0104] Sensitivity is expressed in percentage and identifies the proportion of true positive subjects with the disease in a total group of subjects with the disease (TP / TP+FN). In other words, sensitivity is the probability of getting a positive test result in subjects with the disease (T+IB+).

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[0106] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT

[0107] Hence, it relates to the potential of a test to recognize subjects with the disease.

[0108] Specificity is a measure of a diagnostic test accuracy, complementary to sensitivity. It identifies the proportion of subjects without the disease with negative test result in total of subjects without disease (TN / TN+FP). In other words, specificity represents the probability of a negative test result in a subject without the disease (T-IB-). Therefore, specificity relates to the aspect of diagnostic accuracy that describes the test ability to recognize subjects without the disease, i.e. to exclude the condition of interest.

[0109] There is a pair of diagnostic sensitivity and specificity values for every individual cut-off. To construct a Receiving Operating Characteristic (ROC) graph, these pairs of values are plotted on the graph with the 1-specificity on the x-axis and sensitivity on the y-axis. The shape of a ROC curve and the area under the curve (AUC) can be used to estimate the discriminative power of a test. The closer the curve is located to upper-left hand comer and the larger the area under the curve, the better the test is at discriminating between diseased and non-diseased. The area under the curve can have any value between 0 and 1 and it is a good indicator of the strength of the test. A perfect diagnostic test has an AUC 1.0. whereas a nondiscriminating test has an area of 0.5.

[0110] The results in the Examples below show a sensitivity of 93%, a specificity of 97%, and an AUC of 0.95.

[0111] Thus, in some fomis, the sensitivity of the assay is at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or a 100 percent or any fractional value thereof, or range therebetween, for example, 70-100 percent; the specificity of the assay is at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or a 100 percent or any fractional value thereof, or range therebetween, for example, 70-100 percent: the area under the curve is between 0-1, for example, at least 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99, or any range therebetween; or any combination of the foregoing.

[0112] Here the assay is typically used to distinguish between malignant melanocyte proliferation and / or a melanoma and benign lesions, typically referred to herein as nevus.

[0113] A. Biomarkers for Detecting Melanoma

[0114] The top seven differentially methylated CpG loci between patients with malignant melanocyte proliferation (interchangeably referred to herein as melanoma) and those without are provided. Of the biomarkers, 3 that are hypermethylated in melanoma, 1 that is hypermethylated in melanocytes and remains methylated in melanoma, and 3 that are hypermethylated in all tissues.

[0115] As illustrated in the Examples, top performing loci were EVX2, PR0M1 and TBXT. The cumulative DNA methylation signal from these three marker loci can distinguish melanoma

[0116] 11

[0117] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT samples from benign nevi with high specificity and sensitivity (specificity 100%, sensitivity 95.1%, overall accuracy 96.0%). Thus, in some forms, the assay includes detecting methylation status at CpG loci of 1, 2, or all three loci of EVX2, PROMI, and TBXT. Referred to herein as melanoma biomarkers, the CpG loci that arc hypermethylated in melanoma arc located in or around the EVX2, PROMI, and TBXT genes.

[0118] Sequences for these genes are known in the art, see, e.g.

[0119] “Homo sapiens even-skipped homeobox 2 (EVX2), RefSeqGene on chromosome 2,” NCBI Reference Sequence: NG_012893.1, and Gene ID: 344191, the entire contents of which are incorporated by reference in their entireties, and which provides a consensus gene sequence for EVX2;

[0120] “Homo sapiens prominin 1 (PROMI), RefSeqGene on chromosome 4”, NCBI Reference Sequence: NG_011696.2 and Gene ID: 8842, the entire contents of which are incorporated by reference in their entireties, and which provides a consensus gene sequence for PROMI; and

[0121] “Homo sapiens T-box transcription factor T (TBXT), RefSeqGene on chromosome 6” and Gene ID: 6862, the entire contents of which are incorporated by reference in their entireties, and which provides a consensus gene sequence for TBXT.

[0122] The power of the disclosed assays is based in-part on differential methylation in two contexts: cancer versus normal / benign and melanocyte versus other components of skin. Thus, in some forms, the assays further include detection of methylation at additional sites, such melanocyte markers and all-tissue markers.

[0123] An example of a marker that is hypermethylated in melanocytes and stays hypermethylated in melanoma and referred to here as a melanocyte biomarker, is

[0124] “Homo sapiens transcription factor AP-2 alpha (TFAP2A), RefSeqGene on chromosome 6,” NCBI Reference Sequence: NG_016151.1 and Gene ID: 7020, the entire contents of which are incorporated by reference in their entireties, and which provides a consensus gene sequence for TFAP2A.

[0125] For example, the experiments below utilized a cycle threshold (CT) value as part of a quantitative polymerase chain reaction (qPCR) assay. A Ct value is a data point in qPCR that indicates the number of amplification cycles required to detect a target nucleic acid in a sample. A relative CT value was calculated for each target sequence by subtracting the TFAP2A CT from the target CT. Relative CTs were normalized using an on-run control from a cell line known to be hypermethylated at the relevant targets. Corrected relative CTs were used to calculate the percent methylated DNA at each locus expressed as a percentage of melanocyte derived DNA using the equation %Methylation = 100% *2 (-1 * corrected relative CT).

[0126] 12

[0127] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT

[0128] In some forms, one or more additional biomarkers can be used as a normalization standard or other control utilized to control for unmeasurable factors influencing assay performance. Examples of all-tissue biomarker sites NCOR2, LRRC8A, and TRAP1. For example, in some forms, these biomarkers arc used to 1) verify that the PCR reactions arc working within the expected parameters; 2) normalize for errors in input DNA concentration; etc.

[0129] Sequences for these genes are known in the art, see, e.g.

[0130] “Homo sapiens nuclear receptor corepressor 2 (NCOR2), RefSeqGene on chromosome 12,” NCBI Reference Sequence: NG_022928.2 and Gene ID: 9612, the entire contents of which are incorporated by reference in their entireties, and which provides a consensus gene sequence for NCOR2;

[0131] “Homo sapiens leucine rich repeat containing 8 VRAC subunit A (LRRC8A), RefSeqGene (LRG_80) on chromosome 9,” NCBI Reference Sequence: NG_009630.1 and Gene ID: 56262, the entire contents of which are incorporated by reference in their entireties, and which provides a consensus gene sequence for LRRC8A; and

[0132] “Homo sapiens TNF receptor associated protein 1 (TRAP1), RefSeqGene on chromosome 16; nuclear gene for mitochondrial product,” NCBI Reference Sequence: NG_033088.1 and Gene ID: 10131, the entire contents of which arc incorporated by reference in their entireties, and which provides a consensus gene sequence for TRAP1.

[0133] Genes can have more than one CpG loci. Thus, the methods provided herein can include detection of the methylation level at one or more CpG loci in or around one or more of the foregoing genes.

[0134] In some forms the CpG loci include or are one or more of those outlined in the following table:

[0135] According to genome build GRCh37

[0136] Thus, in some forms, the methods include detection of CpG in or at one or more of the genomic positions chr2:176944088-176948446, chr4: 16084196-16085735, chr6: 166579974-

[0137] 13

[0138] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT

[0139] 166583423, chr6: 10419400-10420323, chrl2:124821510-124821583, chr9: 131671004- E31671084, and chrl6: 3714325-3714392.

[0140] The methods typically include detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci. The detection can, but need not, encompass all of CpG at the loci, and can additionally or alternatively include detection of CpG adjacent to, but outside the loci. Such CpG can be referred to as close to the CpG loci. CpG close to the CpG loci are typically not part of another CpG loci. CpG close to the CpG loci may be within, e.g., 10, 100, 1,000, or 10,000 nucleotides, etc., from e.g., the end of the named loci. The foregoing genomic positions are provided as both a range of genomic positions, as well as all subranges therebetween, and all specific CpG dinucleotides therebetween. Thus, detection of DNA methylation for any one of the foregoing genes can include detection of methylation at any single dinucleotide, or a combination of the two, three, four, five, six, seven, eight, nine, ten, or more dinucleotide (together or separately) within the corresponding regions provided above.

[0141] For example, exemplary, non-limiting examples of CpG regions of the biomarkers disclosed herein include, but are not limited to

[0142] According to genome build GRCh37

[0143] Thus, in some forms, the methods include detection of CpG in or at one or more of the genomic position chr2: 176947940-176947941, chr4: 16085180-16085181, chr6: 166582393- 166582394, chr6:10419846-1041984, chrl2:124821510-124821583, chr9:131671004-131671084, and chrl6: 3714325-3714392. However all other specific CpG dinucleotides, and combinations thereof for each of the foregoing gene regions and there higher order combination among two, three, four, five, six, or all seven of the genes are also expressly provided.

[0144] In the exemplary assay in the experiments below, probes were used as a proxy for CpG loci, which may be larger or smaller than the probes used. It will be appreciated that probes and primers utilized in the experiments are a subset of all possible probes and primers that could be designed to

[0145] 14

[0146] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT the region, and thus although they can be used in the disclosed methods, they are also exemplary in nature and thus non-limiting. Now that the important biomarkers are provided, one of skill in the art can design suitable probes to detected them in a biological sample.

[0147] As discussed in more detail below, probes have been designed to interrogate methylation at CpG in the biomarker genes mentioned herein. Thus, in particular forms, the hypermethylation is associated with a CpG region interrogated by the following probes, such as regions including or consisting of or their reverse complement:

[0148] In particular forms, the CpG loci include one or more loci from one, two, three, four, five, six, or seven of EVX2, PROMI, TBXT, TFAP2A, NCOR2, LRRC8A, and TRAP1. In some forms, the CpG loci include one or more loci from one, two, three, or four of

[0149] EVX2, PROMI, and TBXT alone, or in combination with one or more loci from TFAP2A, or additional or alternatively in combination with one, two, or all three of NCOR2, LRRC8A, and TRAP1, preferably NCOR2.

[0150] In particular forms, the CpG loci include one or more of SEQ ID NOS:1-14.

[0151] 15

[0152] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT

[0153] Any of the disclosed methods can include analysis of methylation at one or more CpG sites of one or a combination of any 2, 3, 4, 5, 6, or 7 of the foregoing CpG biomarker sites. Some forms include analysis of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more biomarkers sites. Thus, any of the methods can include detection of methylation at any integer number of CpG biomarkcr sites between 2 and 50 inclusive, in any combination.

[0154] B. Source of the Samples

[0155] The preferred biological sample for detection of the biomarkers is a tissue and / or fluid that includes DNA or from which DNA can be isolated. In some forms, the DNA is from a skin lesion or skin sample (e.g., a melanocytic lesion) that is the target for testing. In some forms, the DNA is from blood (e.g., plasma) or urine. Thus, in some forms, the biological sample is a liquid biopsy. Such DNA can be extracted from cells or can be cell-free DNA. In some forms, the biological sample is blood or urine, and the DNA is extracted from cells therein.

[0156] A preferred biological source for detection of the biomarkers is tissue (e.g., tissue suspected of being cancerous) including biopsy material from a skin sample (e.g., a melanocytic lesion), or cells thereof.

[0157] In some forms, intact cells are subjected to biomarker detection. For example, a sample may be obtained and processed using well-known and routine clinical methods. In some aspects, the biological sample includes a plurality of cells. In certain aspects, the biological sample includes fresh or frozen tissue. In specific aspects, the biological sample includes formalin fixed, paraffin embedded (FFPE) tissue.

[0158] In some forms, the cells are permeabilized. In some forms, a cell lysate or homogenate is subjected to biomarker detection.

[0159] DNA can be purified or isolated from cells or cell-free fluids using any suitable known compositions and methods, including commercially available DNA purification kits.

[0160] C. Methods of Detecting Biomarkers

[0161] The methods can include detection of CpG methylation at part or all of the identified CpG loci and / or adjacent genomic regions e.g., 0 to 4 kilobases (kb), about 0 to 3 kb, about 0 to 2 kb, about 0 to 1 kb, about 0 to 500 base pairs (bp), about 0 to 400 bp, about 0 to 300 bp, about 0 to 200 bp, or about 0 to 100 bp upstream and / or downstream thereof. The CpG sequences that are detected can be or include CpG islands, CpG shores, and / or CpG shelves.

[0162] The size of the region subjected to detection can any suitable size so as to determine the region is methylated or unmethylated compared to a control(s). In some forms the region is at least 10, 20, 25, 50, 75, 100, 200, 250, 500, 750, 800, 1,000, 1 ,250, 1,500, 1 ,750, 2,000, 2,500, 5,000, 10,000, 12,500, 15,000, 17,500, 20,000, or 25,000 nucleotides in length, or any specific integer between about 10 and 25,000. In some forms the regions is the entire gene. In some forms, the size 16

[0163] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT of the region is one that can be easily amplified by PCR such as 50-15,000 base pairs, e.g., 200- 1,000 base pairs (e.g., for traditional PCR), 50-150 base pairs or 70-100 base pairs (e.g., for qPCR), or 4,000 to 15,000 base pairs (SeqTarget), etc.

[0164] The detection can be quantitative or qualitative. In some forms, the detection is absolute, meaning that methylation is detected or not detected. In some forms, the level of methylation is detected. Thus, some of the methods include measuring of the level of methylation level.

[0165] The DNA methylation biomarkers can be detected by PCR, digital droplet PCR, methylation specific PCR, real time methylation specific PCR, and PCR using a methylated DNA specific binding protein, quantitative PCR, quantitative real-time PCR, microarray such as DNA chips, beads, or arrays including those sold commercially, sequencing methods such as pyrosequencing, bisulfite sequencing, methylation-specific restriction digestion, mass spectroscopy, methylationspecific immunoassays, and the like, which is known in the art, but is not limited thereto.

[0166] Principles and methods of determining CpG site specific methylation and / or differential methylation at the same site(s) in various samples are known in the art. Such methods can include “conversion” of methylated DNA. Exemplary non-limited methods are discussed below, but see also, Kurdyukov and Bullock, Biology (Basel). 2016 Mar; 5(1): 3; doi: 10.3390 / biology5010003, WOOl / 26536, US2003 / 0148326A1, US2023 / 0132750, US2023 / 0109129, and US 2023 / 0101485 each of which is specifically incorporated by reference herein.

[0167] The methods can include a bisulfite treatment-dependent detection method or a bisulfite-free detection method. The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite treated DNA enzymatic deamination approaches have been developed. For example, the methylation level may be measured using a ten-eleven translocation (TET) protein as a bisulfite-free detection method. The TET protein is an enzyme that acts on DNA and is involved in chemical changes of bases.

[0168] Since methylated cytosine pairs with guanine in the same way unmethylated cytosine does, traditional sequencing methods (based on base-pairing) are not able to differentiate between methylated and unmethylated cytosines. To solve this problem, sodium bisulfite can be used to convert unmethylated cytosines to uracils, which are amplified as thymines in PCR; because methylated cytosines do not react with sodium bisulfite, they remain as cytosines in the sequence. Thus, thymines detected in bisulfite sequencing or by designing methylation specific (and / or nonmethylation specific) primers that correspond to either thymines or unmethylated cytosines in the original DNA, and alignment with the original template sequence (or the presence or absence of PCR product) easily differentiates between them.

[0169] 17

[0170] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT

[0171] More recently, an enzymatic methyl-seq (EM-seq) technique was developed, which uses TET2 in the first enzymatic step to oxidize methylated cytosines and APOBEC2 in the second enzymatic step to convert unmethylated cytosines to uracils. During the subsequent PCR amplification, oxidized mcthylcytosincs form base pairs with guanines and uracils form base pairs with adenines. Since the end products of WGBS and EM-seq (or their PCR-related alternatives) are the same (methylated cytosines stay as cytosines and unmethylated cytosines appear as thymines in the sequence), the same analysis tools can be used. Because enzymatic reactions are nondestructive, EM-seq promises better yield and higher accuracy in the measurement of methylation levels. See, e.g., Feng, et al. Epigenetics & Chromatin 13, 42 (2020). doi.org / 10.1186 / sl3072-020- 00361-9, which is specifically incorporated by reference herein in its entirety.

[0172] Alternatively, Liu et al. attempted to sequence 5mC and 5hmC directly and to retain unmodified cytosine intact (Liu, Nat Biotechnol 37, 424-429 (2019), doi.org / 10.1038 / s4L587-019- 0041-2, which is specifically incorporated by reference herein in its entirety). TET enzymes are used to oxidize 5mC and 5hmC to 5 -carboxylcytosine (5caC), and then conversion to dihydrouracil (DHU) is induced via borane reduction. A subsequent PCR reaction facilitates the conversion of DHU to thymine. The researchers termed this 5mC / 5hmC-to-T method TET-assisted pyridine borane sequencing (TAPS). An advantage is that the mild reaction conditions can preserve DNA fragments more than 10 kb long. To sequence 5mC alone, they used P-glucosyltransferase to protect 5hmC from TET oxidation and borane reduction so that only 5mC was converted to T. Alternatively, one could use potassium perruthenate to specifically oxidize 5hmC, thus enabling 5hmC-to-T conversion.

[0173] When treated with bisulfite all Cs except for methylated C are converted to T bases, whereas with the TET protein, all Cs except for methylated C or only the methylated Cs are converted to T, depending on the downstream steps.

[0174] Thus, a preparation for detecting methylation level of the CpG sites may include a compound that modifies a cytosine base or a methylation sensitive restriction enzyme, a primer or primers specific to a methylated sequence, and / or a primer or primers specific to an unmethylated allele sequence.

[0175] Pyrosequencing of bisulfite-treated DNA is based on the following principle: when the methylation occurs at a CpG dinucleotide site, 5-methylcytosine (5-mC) is formed, and in this case, the modified base is changed to uracil upon treatment with bisulfite. If the CpG dinucleotide has been methylated when the DNA extracted from the sample is treated with bisulfite, the CpG dinucleotide is preserved as cytosine and the remaining unmethylated cytosines are changed to uracils. Sequencing of the bisulfite-treated DNA may be preferably performed using a pyrosequencing method. The detailed description of pyrosequencing is known in the prior arts

[0176] 18

[0177] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT

[0178] (Ronaghi et al, Science 1998 Jul. 17, 281(5375), 363-365; Ronaghi et al, Analytical Biochemistry 1996 Nov. 1, 242(1), 84-9; Ronaghi et al. Analytical Biochemistry 2000 Nov. 15, 286 (2): 282-288; Nyr, P. Methods Mol Biology 2007, 373, 114],

[0179] Alternatively, by the bisulfitc-frcc detection method using the TET protein, only methylated C may be converted to T using the TET protein to detect the base at the methylated region (see Liu, supra). When the methylation occurs at the CpG dinucleotide site so that cytosine is formed as 5- methylcytosine (5-mC), the CpG dinucleotide has been methylated when treated with the ten-eleven translocation (TET) protein to be changed to uracil, and unmethylated cytosines are preserved.

[0180] Detection by bisulfite and / or enzyme-treated DNA is not limited only to the pyrosequencing method, but may be performed by using methods such as PCR, methylation-sensitive PCR (MSP), microarray, next generation sequencing (NGS), and the like.

[0181] PCR-based detection methods are also provided for use alone or in combination with other techniques including restriction digestion, sequencing, etc. Such methods can include the use of specifically designed primers. The primers may include a primer(s) specific CpG site(s) being methylated and / or primer(s) specific for site being unmethylated. The term “primer” refers to a nucleic acid sequence having a short free 3-terminal hydroxyl group, and a short nucleic acid sequence capable of forming a base pair with a complementary template and serving as a starting point for copying a template strand. The primer may initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffer and temperature. In addition, the primer is a sense and antisense nucleic acid with a sequence of 7 to 50 nucleotides and may incorporate an additional feature without changing the basic properties of the primer that serves as an initiation site of DNA synthesis.

[0182] Primer sequences can be designed for specific CpG sites, and further can be designed for “converted” DNA sequences. In some forms, the detection methods include at least one, and optionally two or more, selected the group consisting of a primer pair capable of specifically amplifying cytosine that is methylated to be unmodified by bisulfite or TET; a primer pair capable of specifically amplifying cytosine that is not methylated to be modified by bisulfite or TET; a primer pair capable of specifically amplifying cytosine that is methylated to be modified by a TET- based protein; and a primer pair capable of specifically amplifying cytosine that is not methylated and has not been modified by a TET-based protein.

[0183] Methylation-specific PCR method is a method of designing and using different types of primers depending on whether CpG dinucleotide is methylated in a primer to perform PCR after treating a sample DNA with bisulfite. If a primer binding site has been methylated, PCR is performed by the methylated primer, and if not methylated, PCR is performed by a normal primer. 19

[0184] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT

[0185] That is, the methylation-specific PCR method is a method of treating the sample DNA with e.g., bisulfite or a TET protein, performing PCR using two types of primers at the same time, and comparing the results.

[0186] In real time methylation specific PCR, mcthylation-spccific PCR method is converted to a real-time measurement method and to perform real time PCR by treating genomic DNA with bisulfite or a TET protein, designing PCR primers corresponding to methylation, and using these primers. Examples include, but are not limited to, detection method using a TaqMan probe complementary to an amplified base sequence and a detection method using SYBRgreen. Accordingly, the real time methylation specific PCR may selectively quantitative-analyze only methylated DNA. In this case, the real time methylation specific PCR is a method of preparing a standard curve using an in vitro methylated DNA sample, and quantitatively analyzing the methylation level by amplifying a target CpG site biomarker together with a negative control for standardization.

[0187] In an exemplary assay, chemically-modified DNA is seeded into quantitative real time PCR reactions that contain sequence-specific PCR primers for DNA amplification and sequence-specific probes to detect the target sequences, in addition to standard amplification reagents. The sequencespecific probes may contain locked nucleic acids to increase specificity and sensitivity of detection, and can be fluorescently labeled to allow for quantitation of the PCR products being amplified. This assay can be performed in any suitable format including, but not limited to, a multiplexed 96-well format. Following PCR, a post -hoc analysis of the assay can performed and a report can be prepared for the physician that indicates the results of the test. Digital droplet PCR is another technology platform that holds a certain level of appeal for analysis of DNA over that of quantitative real time PCR.

[0188] In addition or alternative, the methods can include use of a methylation-sensitive restriction enzyme. The methylation-sensitive restriction enzyme may be a restriction enzyme capable of specifically detecting the methylation of the CpG site, and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme. For example, the methylation-sensitive restriction enzyme includes Smal, SacII, EagI, Hpall, MspI, BssHII, BstUI, Notl, and the like, but is not limited thereto. Depending on methylation or unmethylation at C of the restriction enzyme recognition site, cleavage by the restriction enzyme varies and may be detected through PCR or southern blot analysis. In a method for measuring the methylation using the methylation- sensitive restriction enzyme, the methylation-sensitive restriction enzyme uses CpG dinucleotide as an action site, and does not act as the enzyme when this site is methylated. Therefore, when the sample DNA is treated with the methylation-sensitive restriction enzyme and then amplified by PCR so as to include an enzyme target site, in the case of the methylated site, the restriction enzyme does not act, 20

[0189] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT but is amplified by PCR, but the unmethylated normal site is cleaved by the restriction enzyme and not amplified by PCR, thereby measuring the methylation of a specific DNA site.

[0190] Nucleic acid chips for use in detection of the provided CpG biomarkers can include immobilized probes capable of hybridizing with fragments including CpG sites of one or more or biomarkers. In some forms, such probes are design to bind “converted” DNA.

[0191] Some methods include using a methylated DNA-specific binding protein. When a protein that specifically binds only to methylated DNA is mixed with DNA, the protein specifically binds only to the methylated DNA, so that only the methylated DNA may be selectively isolated. After mixing genomic DNA with the methylated DNA-specific binding protein, only the methylated DNA is selectively isolated. In some forms, the methods include amplifying these isolated DNAs using a PCR primer corresponding to a biomarker site, and then measuring the methylation by agarose electrophoresis. In addition, the methylation may be measured even by quantitative PCR, and the methylated DNA isolated by the methylated DNA-specific binding protein is labeled with a fluorescent dye or hybridized to a DNA chip integrated with a complementary probe to measure the methylation.

[0192] Other probe-based detection techniques can also be used. For example, in some forms, fluorescence in situ hybridization (FISH) is used. Methylation- specific fluorescence in situ hybridization (MeFISH) was developed for microscopic visualization of DNA methylation status at specific repeat sequences in individual cells. MeFISH is based on the differential reactivity of 5-methylcytosine and cytosine in target DNA for interstrand complex formation with osmium and bipyridine-containing nucleic acids (ICON). See, e.g., Li, et al., “Sequence-specific microscopic visualization of DNA methylation status at satellite repeats in individual cell nuclei and chromosomes,” Nucleic Acids Res. 2013 Oct:41(19):el86. doi: 10.1093 / nar / gkt766. Epub 2013 Aug 28. PMID: 23990328; PMCID: PMC3799461.

[0193] D. Diagnosis

[0194] 1. Single Markers

[0195] The biomarkers can be used in diagnostic and prognostic tests to assess the disease status of skin, particularly melanocytic lesions, and the effectiveness or response to treatment of disease. Based on this status, further procedures may be indicated, including additional diagnostic tests or therapeutic procedures or regimens. Representative therapies are discussed in more detail below.

[0196] The method involves, first, measuring one or more biomarkers (i.e., methylation at one or more of the disclosed CpG loci and / or the genomic sequence adjacent thereto) in a subject sample using, e.g., the methods described herein, and, second, comparing the measurement with a diagnostic amount or cut-off that distinguishes a positive or negative melanoma status. The diagnostic amount represents a measured amount of a biomarker above or below which a subject is 21

[0197] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT classified as having a particular status. For example, if the biomarker is increased (i.e., hypermethylation) in melanoma compared to a benign melanocytic lesion (e.g., nevus), then a measured amount above the diagnostic cutoff provides a diagnosis of melanoma. Likewise, if the biomarkcr is decreased (hypomcthylation) in melanoma compared to a benign melanocytic lesion (e.g., nevus), then a measured amount below the diagnostic cutoff provides a diagnosis melanoma. Similarly, if the biomarker is increased (i.e., hypermethylation) in a benign melanocytic lesion (e.g., nevus) compared to melanoma, then a measured amount above the diagnostic cutoff provides a diagnosis of a benign melanocytic lesion (e.g., nevus). Likewise, if the biomarker is decreased (hypomethylation) in a benign melanocytic lesion (e.g., nevus) compared to melanoma, then a measured amount below the diagnostic cutoff provides a diagnosis of a benign melanocytic lesion (e.g., nevus).

[0198] Of the biomarkers mentioned above, 3 that are hypermethylated in melanoma, 1 that is hypermethylated in melanocytes and remains methylated in melanoma, and 3 that are hypermethylated in all tissues. In some forms, the method includes normalizing one or more melanoma biomarkers and a melanocyte biomarker control to one or more all-tissue biomarker controls. A ratio call can be made between the melanoma biomarker(s) and a melanocyte biomarker control. If this is above the threshold defined by a particular sensitivity and specificity, then the sample is called positive. If this is the same and / or below the threshold defined by a particular sensitivity and specificity, then the sample is called positive.

[0199] In other forms, the test measurement is compared directly to measurements from subjects each with a known cancer status, which can be, for example, a single measurement or an average of pooled measures from subjects with e.g., melanoma or benign nevus. The test subject’s status can be determined by selecting that class of the known subject(s) that most closely matches test measurement.

[0200] As is well understood in the art, by adjusting the particular diagnostic cut-off used in an assay, one can increase sensitivity or specificity of the diagnostic assay depending on the preference of the diagnostician. The particular diagnostic cut-off can be determined, for example, by measuring the amount of the biomarker in a statistically significant number of samples from subjects with different cancers statuses and drawing the cut-off to suit the diagnostician's desired levels of specificity and sensitivity.

[0201] 2. Combinations of Markers

[0202] While individual biomarkers are useful diagnostic biomarkers, a combination of biomarkers may provide greater predictive value of a particular status than single biomarkers alone. Specifically, the detection of a plurality of biomarkers in a sample can increase the sensitivity and / or specificity of the test. Thus, in one form, two or more, three or more, four or more, or five or 22

[0203] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT more, or six or more, or all seven of the biomarkers can be detected and used to assess the status of malignant melanocyte proliferation in a subject. In some forms, the biomarkers include methylation at one or more of sites mentioned.

[0204] E. Determining Risk of Developing More Severe Disease

[0205] Methods for determining the risk of developing disease or more severe disease in a subject are also provided. Biomarker amounts or patterns can be characteristic of various risk states, e.g., high, medium, or low. The risk of developing a disease is determined by measuring the biomarker or biomarkers and then either submitting them to a classification algorithm or comparing them with a reference amount and / or pattern of biomarkers that is associated with the particular risk level.

[0206] F. Determining Stage of Disease

[0207] As introduced above, some forms provide methods for determining the specific stage of disease in a subject. Each stage of the disease can have a characteristic amount of a biomarker or relative amounts of a set of biomarkers (a pattern). The stage of a disease is determined by measuring the relevant biomarker or biomarkers and then either submitting them to a classification algorithm or comparing them with a reference amount and / or pattern of biomarkers that is associated with the particular stage.

[0208] G. Determining Course (Progression / Remission) of Disease

[0209] Still another form provides methods for determining the course of disease in a subject. Disease course refers to changes in disease status over time, including disease progression (worsening) and disease regression (improvement). Over time, the amounts or relative amounts (e.g., the pattern) of the biomarkers changes. This method involves measuring one or more biomarkers in a subject at least two different time points, e.g., a first time and a second time, and comparing the change in amounts, if any. The course of disease is determined based on these comparisons. Similarly, this method is useful for determining the response to treatment. If a treatment is effective, then the biomarkers will trend toward normal, while if treatment is ineffective, the biomarkers will trend toward disease indications.

[0210] H. Subject Management

[0211] In certain forms of the methods including the detection and / or analysis of one or more biomarkers further include managing subject treatment based on the status. Such management includes the actions of the physician or clinician subsequent to determining melanoma status. For example, if a physician makes a determination that sample is positive for melanoma, then a certain regime of treatment might follow. Alternatively, a determinate or an indeterminate result might be followed with further testing.

[0212] One form provides a method for selecting a subject for treatment for melanoma by detecting the presence or quantity of one or more biomarkers provided herein in a sample from a subject of an 23

[0213] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT undetermined melanoma status, a previously indeterminant melanoma status, having or suspected of having melanoma, etc., and comparing the levels of biomarker in the sample to a predetermined standard, wherein the subject is selected for treatment based on a determination of the melanoma status. For example, subjects determined not to have melanoma, may not require any further treatment. On the other hand, subjects determined to have melanoma may be given one or more treatments for melanoma. Exemplary treatment strategies are discussed in more detail below.

[0214] Additional forms relate to the communication of assay results or diagnoses or both to technicians, physicians or patients, for example. In certain forms, computers will be used to communicate assay results or diagnoses or both to interested parties, e.g.: physicians and their patients. In some forms, the assays will be performed or the assay results analyzed in a country or jurisdiction which differs from the country or jurisdiction to which the results or diagnoses are communicated.

[0215] In a preferred form a diagnosis based on the presence or absence in a test subject of any the disclosed biomarkers is communicated to the subject as soon as possible after the diagnosis is obtained. The diagnosis may be communicated to the subject by the subject's treating physician. Alternatively, the diagnosis may be sent to a test subject by email or communicated to the subject by phone. A computer may be used to communicate the diagnosis by email or phone. In certain forms, the message containing results of a diagnostic test may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications. In certain forms all or some of the method steps, including the assaying of samples, diagnosing of diseases, and communicating of assay results or diagnoses, may be carried out in diverse (e.g., foreign) jurisdictions.

[0216] I. Assessing the Effectiveness of Treatment

[0217] Methods for determining the course of melanoma in a subject are also provided. Disease course refers to changes in disease status over time, including disease progression (worsening) and disease regression (improvement). Over time, the amounts or relative amounts (e.g., the pattern) of the biomarkers changes. Accordingly, this method involves measuring one or more biomarkers in a subject at least two different time points, e.g., a first time and a second time, and comparing the change in amounts, if any. The course of disease is determined based on these comparisons. Similarly, this method is useful for determining the response to treatment. If a treatment is effective, then the biomarkers will trend toward normal or not further worsen, while if treatment is ineffective, the biomarkers will trend toward more disease indications or increasingly severe disease indications.

[0218] 24

[0219] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT

[0220] III. Methods of Treatment

[0221] Methods of treating melanoma are also provided. Such methods can be used in conjunction with any one or more of the disclosed methods otherwise provided herein, e.g., the methods of detecting one or more biomarkers indicative of melanoma, and their associated uses.

[0222] Prompt diagnosis and early institution of treatment is important to improve outcomes in melanoma.

[0223] Common treatments include, but are not limited to: surgery, immunotherapy, target drug therapy, chemotherapy, and radiation therapy.

[0224] Immunotherapy is the use of medicines to help a person’s own immune system recognize and destroy cancer cells more effectively. In some forms, the immunotherapy include administering the subject an immune checkpoint inhibitor. Melanoma cells sometimes use these checkpoints to avoid being attacked by the immune system. Checkpoint inhibitors include PD- 1 inhibitors such as Pembrolizumab (Keytruda) and nivolumab (Opdivo), PD-L1 inhibitors such as Atezolizumab (Tecentriq); CTLA-4 inhibitors such as Ipilimumab (Yervoy); LAG-3 inhibitors such as Relatlimab:

[0225] In some forms two or more checkpoint inhibitors are administered to the subject. For example, the CTLA-4 inhibitor Relatlimab is often given along with the PD- 1 inhibitor nivolumab (in a combination known as Opdualag).

[0226] In some forms, the subjects are administered a pro-inflammatory cytokine such as an interleukin. Interleukins are proteins that certain cells in the body make to boost the immune system in a general way. Lab-made versions of interleukin-2 (IL-2), such as aldesleukin, are sometimes used to treat melanoma. For advanced melanomas: IL-2 in high doses can sometimes shrink advanced melanomas when used alone. IL-2 might also be an option if checkpoint inhibitor drugs are no longer working.

[0227] In some forms, subjects are administered Tumor-infiltrating lymphocyte (TIL) therapy TILs are immune system cells called T cells that have entered (infiltrated) a tumor to attack the cancer cells. Treatments that use these cells can help shrink some melanomas. This type of treatment is also known as tumor-derived autologous T cell immunotherapy. Lifileucel (Amtagvi) is a type of TIL therapy that has been used to treat people with advanced melanomas, after other treatments have been tried. For this treatment, a melanoma tumor can be removed with surgery, white blood cells are separated out and then multiplied over a few weeks. They are then sent back to be given to the person as an infusion into a vein (IV). Once in the body, the TILs seek out and attack the melanoma cells. People getting this treatment are often first given chemotherapy for about a week to help the body accept the TILs. After getting the TILs, the person is also given IL-2 (see above), which helps these immune cells attack the cancer.

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[0230] In some forms, the subject receives oncolytic virus therapy. Oncolytic viruses can typically infect and kill cancer cells. In addition or alternative to killing the cells directly, the viruses can also alert the immune system to attack the cancer cells. Talimogene laherparepvec (Imlygic), also known as T-VEC, is an oncolytic virus that can be used to treat melanomas in the skin or lymph nodes that can’t be removed with surgery. The virus is injected directly into the tumors, typically every 2 weeks. This treatment can sometimes shrink these tumors, and it might also shrink tumors in other parts of the body.

[0231] In some forms, subjects are administered a vaccine such as Bacille Calmette-Guerin (BCG) vaccine.

[0232] In some forms, subjects are administered imiquimod cream. Imiquimod (Zyclara) is a topical drug that is put on the skin as a cream. It stimulates a local immune response against skin cancer cells. F examples, for very early (stage 0) melanomas in sensitive areas on the face, some doctors may use imiquimod if surgery isn’t able to remove all of the tumor. Imiquimod might also be an option to treat some melanomas that have spread along the skin, especially if surgery can’t be done for some reason.

[0233] In some forms, subjects (e.g., when the melanoma has a BRAF gene mutation) are treated with a targeted drug such as cobimctinib vcmurafcnib (Zclboraf), dabrafcnib (Tafinlar), and encorafenib (Braftovi).

[0234] In some forms, subjects are administered a MEK inhibitor. MEK inhibitors include trametinib (Mekinist), cobimetinib (Cotellic), and binimetinib (Mektovi).

[0235] In some forms, subjects are administered a drag that target cells with C-KIT gene changes, such as imatinib (Gleevec) and nilotinib (Tasigna).

[0236] In some forms, the subject are administered one or more chemotherapeutic drugs. Chemotherapy might be used to treat advanced melanoma after other treatments have been tried. It’s not typically used as the first treatment because newer forms of immunotherapy and targeted drugs are typically more effective. Exemplary chemotherapeutic drugs include, but are not limited to, dacarbazine (DTIC), temozolomide, nab-paclitaxel, paclitaxel, cisplatin, and carboplatin. Some of these drags are given alone, while others are more often combined (such as carboplatin and paclitaxel).

[0237] In some forms, the subject are administered radiation therapy. For example, radiation might be an option to treat some early-stage melanomas, if surgery can’t be done for some reason. Radiation might be used after surgery (as an adjuvant treatment) in some situations if there’s a high risk that the melanoma might come back. Sometimes, adjuvant radiation is given after surgery in the area where lymph nodes were removed, especially if many of the nodes contained cancer cells. This is to try to lower the chance that the cancer will come back.

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[0240] Radiation might be used to treat melanoma that has come back after surgery, either in the skin or lymph nodes, or to help treat distant spread of the disease. Radiation therapy can be used to relieve symptoms caused by the spread of melanoma to other parts of the body, especially to the brain or bones.

[0241] Surgery is the main treatment option for most melanomas, and it usually cures early-stage melanomas. Types of surgeries in excisional biopsies, wide excision surgery, Mohs surgery, and amputation. In wide excision surgery, the site of the tumor is cut out, along with a small amount of normal skin around the edges (called the margins). Mohs surgery (also known as Mohs micrographic surgery, or MMS) might be an option for some very early-stage melanomas that are in areas where a wide excision would be hard to do (such as the face or ears). In uncommon situations where the melanoma is on a finger or toe and has grown deeply, part or all of that digit might need to be amputated. In some forms, surgery includes lymph node dissection, during which the surgeon removes all of the lymph nodes in the region near the primary melanoma tumor.

[0242] If melanoma has spread (metastasized) from the skin to other organs such as the lungs or brain, the cancer is very unlikely to be curable by surgery. Even when only 1 or 2 areas of spread are found by imaging tests such as CT, MRI, or PET scans, there are likely to be others that are too small to be found by these scans. Surgery is sometimes done in these circumstances, but the goal is usually to try to control the cancer rather than to cure it.

[0243] All patients should also receive adjunctive treatment and supportive care.

[0244] IV. Kits

[0245] The materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed methods. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example disclosed are kits for bisulfite conversion and nucleic acid amplification and / or detection and sequencing, for example, HSO3-, primers, polymerase, probes, buffer, etc.

[0246] V. Data Structures and Computer Control

[0247] Disclosed are data structures used in, generated by, or generated from, the disclosed method. Data structures generally are any form of data, information, and / or objects collected, organized, stored, and / or embodied in a composition or medium. For example, computers can be utilized in one or more of the collection of detection data, determination of diagnosis, etc., and the data stored in electronic form, such as in RAM or on a storage disk.

[0248] The disclosed methods, or any part thereof or preparation thereof, can be controlled, managed, or otherwise assisted by computer control. Such computer control can be accomplished by a computer controlled process or method, can use and / or generate data structures, and can use a 27

[0249] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT computer program. Such computer control, computer controlled processes, data structures, and computer programs are contemplated and should be understood to be disclosed herein.

[0250] Uses

[0251] The disclosed methods and compositions arc applicable to numerous areas including, but not limited to, diagnosis, prognosis, patient treatment, and patient management. Other uses are disclosed, apparent from the disclosure, and / or will be understood by those in the art.

[0252] The disclosed invention can be further understood by the following numbered paragraphs:

[0253] 1. A method of detecting the methylation status at one or more locations in DNA from a human subject including processing DNA of a sample from the subject with a machine-based platform and detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of one or more genes selected from the group consisting of EVX2, PROMI and TBXT.

[0254] 2. The method of paragraph 1 further including detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of TFAP2A.

[0255] 3. The method of paragraphs 1 or 2 further including detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of one or more genes selected from the group consisting of NCOR2, LRRC8A, and TRAP1.

[0256] 4. The method of any one of paragraphs 1 -3 including detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of one, two, or all three, of genes EVX2, PROMI and TBXT, in combination with detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of TFAP2A, in combination with detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of one, two, or all three of genes NCOR2, LRRC8A, and TRAP1.

[0257] 5. The method of any one of paragraphs 1-4 including detecting DNA methylation at and / or close to the genomic sequences associated with any integer number of the CpG loci between 2 and 50.

[0258] 6. The method of any one of paragraphs 1-5, wherein the CpG loci lies within and / or includes and / or is one or more of human genomic regions / positions in the following table

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[0260] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT and / or the following table

[0261] 7. The method of any one of paragraphs 1-6, wherein the CpG loci lies within and / or includes and / or is one or more of one or more of SEQ ID NOS: 1-14.

[0262] 8. The method of any one of paragraphs 1-7, wherein the processing includes one or more of PCR, digital droplet PCR, methylation specific PCR, real time methylation specific PCR, and PCR using a methylated DNA specific binding protein, quantitative PCR, quantitative realtime PCR, microarray, or DNA sequencing.

[0263] 9. The method of any one of paragraphs 1-8, wherein the DNA is chemically- modified.

[0264] 10. The method of paragraph 9, wherein the DNA is chemically modified with sodium bisulfite or a ten-eleven translocation (TET) protein.

[0265] 11. The method of any one of paragraphs 1-10, wherein the DNA is purified prior to processing with the machine-based platform.

[0266] 12. fhe method of any one of paragraphs 1-11, wherein the sample is tissue optionally a melanocytic lesion, or a blood sample, optionally plasma.

[0267] 13. A method of determining if a biological sample includes DNA from melanoma cells including detecting methylation in DNA of the biological sample according to the method any one of paragraphs 1-12 and comparing the level of methylation to one or more controls, and determining that the biological sample includes melanoma cells when methylation is increased at one or more CpG loci of one, two, or three genes selected from the group consisting of EVX2, PROMI and TBXT.

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[0270] 14. The method of paragraph 13, including determining that the biological sample includes melanoma cells when methylation is increased at one or more CpG loci of all three genes EVX2, PROMI and TBXT.

[0271] 15. The method of paragraphs 13 or 14, wherein methylation is normalized relative to methylation at one or more standardization CpG loci.

[0272] 16. fhe method of paragraph 15, wherein the standardization CpG locus is at and / or close to the genomic sequences associated with the TFAP2 gene.

[0273] 17. A method of determining if a biological sample includes DNA from melanoma cells including

[0274] (i) detecting methylation in DNA of the biological sample at one or more melanoma biomarkers, one or more melanocyte biomarkers, and one or more all-tissue biomarkers;

[0275] (ii) normalizing the one or more melanoma biomarkers and the one or more melanocyte biomarker to the one or more all tissue biomarker controls; and

[0276] (iii) preparing a ratio between the melanoma biomarker(s) and the melanocyte biomarker(s) control; wherein if the ratio is above a threshold defined by a sensitivity and / or specificity, then the sample is positive for melanoma and / or if the ratio is the same and / or below the threshold then the sample is negative for melanoma.

[0277] 18. The method of any one of paragraphs 13-16, including

[0278] (i) detecting methylation in DNA of the biological sample at one or more melanoma biomarkers: EVX2, PROMI and TBXT, a melanocyte biomarker: TFAP2A, and one or more alltissue biomarkers: NCOR2, LRRC8A, and TRAP1;

[0279] (ii) normalizing the one or more melanoma biomarkers and the melanocyte biomarker to the one or more all tissue biomarker controls; and

[0280] (iii) preparing a ratio between the melanoma biomarker(s) and the melanocyte biomarker control; wherein if the ratio is above a threshold defined by a sensitivity and / or specificity, then the sample is positive for melanoma and / or if the ratio is the same and / or below the threshold then the sample is negative for melanoma.

[0281] 19. The method of paragraphs 17 and 18, wherein the sensitivity and / or specificity is 70% or more.

[0282] 20. The method of any one of paragraphs 13-19 further including treating the subject for melanoma when it is determined that the subject has melanoma.

[0283] 21. The method of any one of paragraphs 13-20 further including abstaining from treating the subject for melanoma when it is determined that the subject does not have melanoma.

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[0286] 22. The method of paragraphs 20 or 21, wherein treatment for melanoma includes one or more of surgery, radiation, immunotherapy, chemotherapy, or target drug therapy.

[0287] 23. A method of treating a subject including determining if the subject has melanoma according to the method of any one of paragraphs 13-22; and treating the subject for melanoma when it is determined that the subject has melanoma, and abstaining from treating the subject for melanoma when it is determined that the subject does not have melanoma.

[0288] 24. The method of paragraph 23, wherein treatment for melanoma includes one or more of surgery, radiation, immunotherapy, chemotherapy, or target drug therapy.

[0289] 25. Kit for use in the method of any one of paragraphs 1 -24.

[0290] 26. A composition or method as disclosed anywhere herein including but limited to the text including the working examples and figures.

[0291] Examples

[0292] Example 1: Identification of Biomarkers for Detection of Melanoma

[0293] Treatment decisions for melanoma are mainly based on clinical and radiological assessments. Front line detection means such as histochemistry is often inconclusive. Besides other limitations, cross-sectional imaging is only valuable if applied regularly over a relatively long period of at least 3 to 6 months. Moreover, in patients undergoing immune checkpoint blockade (ICB), interpreting imaging can be ambiguous due to pseudo progression. Therefore, complementary parameters that facilitate prognosis and monitoring response prior to and early during therapy are needed. In addition, new approaches to monitor patients after definitive treatment are also needed. Thus, alternative melanoma detection means have been developed to solve these problems.

[0294] DNA methylation is an optional covalent modification of cytosine residues in the CpG sequence context. Fundamental differences in DNA methylation patterns can exist between normal and cancer cells where a large fraction of CpGs can undergo aberrant hyper- or hypo- methylation. Since tumors have many aberrantly methylated DNA regions, a set of multiple genomic loci specifically methylated in cancer cells can be used as a marker for a test to distinguish malignant from normal cells.

[0295] Experiments were designed to determine if this relationship could be used to distinguish between benign and malignant melanoma. A new unique set of melanoma markers were identified by further filtering the original marker suite using new publicly available GEO Illumina HumanMethylation450 data from melanomas and benign nevi (GSE1208787) and melanocytes (GSE1660698). Four top performing loci (including EVX2, PROMI and TBXT) were then chosen 31

[0296] 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT for further analysis and tested in silico on independent GEO data from different Illumina platform (EPIC, GSE2020979, GSE18859310). Results presented in Figures 1A-1B show that the cumulative DNA methylation signal from the four marker loci can distinguish melanoma samples from benign nevi with high specificity and sensitivity (specificity 100%, sensitivity 95.1%, overall accuracy 96.0%).

[0297] To control for the number of melanocytes in the samples, data from GSE178416 and GSE184213 were used to identify loci that were methylated in cells expressing melanin by comparing melanocytes and melanomas to normal skin. Because these two studies were analyzed on different platforms, the probes in common between the two platforms were identified and only those probes were compared using probe-wise linear model fitting and computed empirical Bayes statistics using the limma package in R. The corresponding top 250 probes were screened for direction (methylated in melanomas and melanocytes) and the top 10 with the appropriate direction were evaluated for patterns of methylation in other tumor types using Xenabrowser and the GDC- PANCAN dataset looking at the Illumina 45 OK methylation data.

[0298] For all loci, qPCR assays were designed to the 2000 bp region surrounding the probe identified on the array. Assays were designed to detect methylated alleles after bisulfite conversion of the DNA with an optimal target length of 70 to 90 bp. Primers for amplification were screened to minimize self- and cross-annealing as well as secondary structure. A hybridization probe was also designed to further ensure specificity and all oligos (primers and probe) were screened for uniqueness in the genome.

[0299] DNA from FFPE samples was extracted using commercial kits and quantified. 20 ng of the resulting DNA was used for a 15-cycle pre-amplification step containing the primers for the genes but no probes. A 5 uL aliquot of this 200X diluted pre-amp DNA was used for the starting template in a 50 cycle PCR reaction on a 96-well plate where each column on the plate was an assay for a gene. The PCR was done on an ABI QuantStudio 5 with the following conditions: 95C for 3 min, 50 cycles of 95C 15 sec-60C 45 sec.

[0300] Table 1: PCR Primers and Probes for all loci tested

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[0305] The resulting data were analyzed in AppliedBiosystems DA2 software using default parameters to determine Ct values. The resulting Ct values were exported to a csv for further analysis. A relative CT value was calculated for each target sequence by subtracting the TFAP2A CT from the target CT. Relative CTs were normalized using an on-run control from a cell line known to be hypermethylated at the relevant targets. Corrected relative CTs were used to calculate the percent methylated DNA at each locus expressed as a percentage of melanocyte derived DNA using the equation %Methylation = 100% *2A(-1 * corrected relative CT).

[0306] A receiver operator curve (ROC) was used to determine a cutoff for each target. A sample was considered positive if any target was hypermethylated and negative only if all three biomarkers were below the cutoff for hypermethylation. The performance of this call against the ground truth of the samples was then analyzed, and yield:

[0307] Table 2: Diagnostic Results

[0308] The assay is based on differential methylation in two contexts: cancer versus normal / benign and melanocyte versus other components of skin. Both can be used together to reach the desired performance characteristics. The probes delineated are a subset of all possible probes that could be designed to the region. Of particular note is the selection of the loci and the selection of the normalization controls. The panel demonstrates a 93% sensitivity, 97% specificity and an AUG of 95%.

[0309] While qPCR offers a commercially viable way to assay samples, this assay can be adapted to work with sequencing data (Sanger, next-generation, long read, etc.) from bisulfite converted DNA. In this case, reads would be aligned to a bisulfite converted reference and the ratio of methylated to unmethylated reads calculated to give a percent methylation for each loci. This value would then be used in place of the Ct value in the pipeline outlined above.

[0310] While FFPE samples is practical source of DNA for this assay, this assay would work on fresh and flash-frozen material and plasma samples among others.

[0311] This assay has an advantage over current solutions in that it is comprised of a reduced number of genes and assesses DNA, not RNA. This makes it more robust to sample handling variations that could give false positive or false negative results. It is also cost-effective, being on par with current immunohistochemical staining techniques and well below the current market solution.

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[0314] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular forms only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0315] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific forms of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

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[0317] 45793301.1

Claims

ATTORNEY DOCKET NO. PRECI 100 PCTCLAIMSWe claim:

1. A method of detecting the methylation status at one or more locations in DNA from a human subject comprising processing DNA of a sample from the subject with a machine-based platform and detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of one or more genes selected from the group consisting of EVX2, PROM 1 and TBXT.

2. The method of claim 1 further comprising detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of TFAP2A.

3. The method of claims 1 or 2 further comprising detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of one or more genes selected from the group consisting of NC0R2, LRRC8A, and TRAP1.

4. The method of any one of claims 1-3 comprising detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of one, two, or all three, of genes EVX2, PR0M1 and TBXT, in combination with detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of TFAP2A, in combination with detecting DNA methylation at and / or close to the genomic sequences associated with one or more CpG loci of one, two, or all three of genes NC0R2, LRRC8A, and TRAP1.

5. The method of any one of claims 1-4 comprising detecting DNA methylation at and / or close to the genomic sequences associated with any integer number of the CpG loci between 2 and 50.

6. The method of any one of claims 1-5, wherein the CpG loci lies within and / or comprises and / or is one or more of human genomic regions / positions in the following table3645793301.1ATTORNEY DOCKET NO. PRECI 100 PCT and / or the following table7. The method of any one of claims 1-6, wherein the CpG loci lies within and / or comprises and / or is one or more of one or more of SEQ ID NOS: 1-14.

8. The method of any one of claims 1-7, wherein the processing comprises one or more of PCR, digital droplet PCR, methylation specific PCR, real time methylation specific PCR, and PCR using a methylated DNA specific binding protein, quantitative PCR, quantitative real-time PCR, microarray, or DNA sequencing.

9. The method of any one of claims 1-8, wherein the DNA is chemically- modified.

10. The method of claim 9, wherein the DNA is chemically modified with sodium bisulfite or a ten-eleven translocation (TET) protein.

11. The method of any one of claims 1-10, wherein the DNA is purified prior to processing with the machine-based platform.

12. The method of any one of claims 1-11, wherein the sample is tissue optionally a melanocytic lesion, or a blood sample, optionally plasma.

13. A method of determining if a biological sample includes DNA from melanoma cells comprising detecting methylation in DNA of the biological sample according to the method any one of claims 1-12 and comparing the level of methylation to one or more controls, and determining that the biological sample includes melanoma cells when methylation is increased at one or more CpG loci of one, two, or three genes selected from the group consisting of EVX2, PROMI and TBXT.

14. The method of claim 13, comprising determining that the biological sample includes melanoma cells when methylation is increased at one or more CpG loci of all three genes EVX2, PROMI and TBXT.3745793301.1ATTORNEY DOCKET NO. PRECI 100 PCT15. The method of claims 13 or 14, wherein methylation is normalized relative to methylation at one or more standardization CpG loci.

16. The method of claim 15, wherein the standardization CpG locus is at and / or close to the genomic sequences associated with the TFAP2 gene.

17. A method of determining if a biological sample includes DNA from melanoma cells comprising(i) detecting methylation in DNA of the biological sample at one or more melanoma biomarkers, one or more melanocyte biomarkers, and one or more all-tissue biomarkers;(ii) normalizing the one or more melanoma biomarkers and the one or more melanocyte biomarker to the one or more all tissue biomarker controls; and(iii) preparing a ratio between the melanoma biomarker(s) and the melanocyte biomarker(s) control; wherein if the ratio is above a threshold defined by a sensitivity and / or specificity, then the sample is positive for melanoma and / or if the ratio is the same and / or below the threshold then the sample is negative for melanoma.

18. The method of any one of claims 13-16, comprising(i) detecting methylation in DNA of the biological sample at one or more melanoma biomarkers: EVX2, PR0M1 and TBXT, a melanocyte biomarker: TFAP2A, and one or more all-tissue biomarkers: NC0R2, TRRC8A, and TRAP1;(ii) normalizing the one or more melanoma biomarkers and the melanocyte biomarker to the one or more all tissue biomarker controls; and(iii) preparing a ratio between the melanoma biomarker(s) and the melanocyte biomarker control; wherein if the ratio is above a threshold defined by a sensitivity and / or specificity, then the sample is positive for melanoma and / or if the ratio is the same and / or below the threshold then the sample is negative for melanoma.

19. The method of claims 17 and 18, wherein the sensitivity and / or specificity is 70% or more.

20. The method of any one of claims 13-19 further comprising treating the subject for melanoma when it is determined that the subject has melanoma.

21. The method of any one of claims 13-20 further comprising abstaining from treating the subject for melanoma when it is determined that the subject does not have melanoma.3845793301.1ATTORNEY DOCKET NO. PRECI 100 PCT22. The method of claims 20 or 21, wherein treatment for melanoma comprises one or more of surgery, radiation, immunotherapy, chemotherapy, or target drug therapy.

23. A method of treating a subject comprising determining if the subject has melanoma according to the method of any one of claims 13-22; and treating the subject for melanoma when it is determined that the subject has melanoma, and abstaining from treating the subject for melanoma when it is determined that the subject does not have melanoma.

24. The method of claim 23, wherein treatment for melanoma comprises one or more of surgery, radiation, immunotherapy, chemotherapy, or target drug therapy.

25. Kit for use in the method of any one of claims 1-24.3945793301.1

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