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WO-2026097007-A2 - COMPOSITIONS AND METHODS FOR DETECTING AND DIAGNOSING OF MELANOMA

WO2026097007A2WO 2026097007 A2WO2026097007 A2WO 2026097007A2WO-2026097007-A2

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.

Inventors

  • BUSSEY, KIMBERLY J.
  • ROUTH, Joshua
  • VRBA, LUKAS
  • HUM, Aaron
  • BERNERT, RICHARD
  • OSHIRO, Marc Makoto
  • Nelson, Mark A.

Assignees

  • PRECISION EPIGENOMICS, INC.

Dates

Publication Date
20260507
Application Date
20251103
Priority Date
20241101

Claims (20)

  1. 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. 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. 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. 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. 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. 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 table 36 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT and/or the following table
  7. 7. 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. 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. 9. The method of any one of claims 1-8, wherein the DNA is chemically- modified.
  10. 10. The method of claim 9, wherein the DNA is chemically modified with sodium bisulfite or a ten-eleven translocation (TET) protein.
  11. 11. The method of any one of claims 1-10, wherein the DNA is purified prior to processing with the machine-based platform.
  12. 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. 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. 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. 37 45793301.1 ATTORNEY DOCKET NO. PRECI 100 PCT
  15. 15. The method of claims 13 or 14, wherein methylation is normalized relative to methylation at one or more standardization CpG loci.
  16. 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. 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. 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. 19. The method of claims 17 and 18, wherein the sensitivity and/or specificity is 70% or more.
  20. 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.

Description

ATTORNEY DOCKET NO. PRECI 100 PCT COMPOSITIONS AND METHODS FOR DETECTING AND DIAGNOSING OF MELANOMA CROSS REFERENCE TO RELATED APPLICATIONS 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. REFERENCE TO THE SEQUENCE LISTING 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). FIELD OF THE INVENTION 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. BACKGROUND OF THE INVENTION 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. 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. Diagnosis typically proceeds according to clinical observation -> histology -> immunohistochemistry -> and molecular testing (e.g., FISH/CGH/GEP/NGS) where each escalation 1 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. 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. Thus, provided are biomarkers, and methods of detecting and using the same, to determine if a melanocytic lesion is benign or malignant. BRIEF SUMMARY OF THE INVENTION 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%. 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. 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 cl