Method for the prognosis of melanoma

Abstract

The present invention provides a method for the prognosis of melanoma in isolated samples of subjects diagnosed of or suspected of suffering from melanoma. The method includes the analysis of the methylation of the promoter sequences of certain genes. The invention also provides simplified kits comprising means for detecting the methylation in the promoters of the genes of interest.

Description

[0001] Title: Method for the prognosis of melanoma

[0002] FIELD OF THE INVENTION

[0003]

[0001] This invention pertains in general to the field of medical diagnosis. It relates more specifically to the diagnosis and prognosis of melanoma.

[0004] BACKGROUND OF THE INVENTION

[0005]

[0002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0006]

[0003] Accurate estimation of the risk of recurrence in cutaneous melanoma patients is of importance as undertreatment of high-risk melanoma patients can lead to disease recurrence and death, while overtreatment of low-risk melanoma patients with adjuvant therapy can lead to toxicity and unnecessary health care costs. Currently, the predictors used to identify patients at high-risk of disease recurrence are Breslow thickness, ulceration, (micro)satellites, and number and size of lymph node metastases (assessed by sentinel lymph node biopsy, SLNB). However, these prognosticators are suboptimal in that: A) The probability of dying from melanoma for stage 1A patients is approximately 2-5%. These patients are currently not being identified; B) 6-28% of SLNB-negative patients still develop metastases; C) SLNB is an invasive surgical procedure with serious side effects such as lymphedema, numbness and pain; D) 35% of SLNB eligible patients do not undergo the procedure, SLNB is not equally available, and its prognostic value is currently under debate. Therefore, better prognosticators to predict the risk of recurrence for melanoma patients are urgently needed to optimize individual melanoma management.

[0007]

[0004] Nowadays, some of the proposed methods to determine this risk of recurrence, and thus to establish a prognostic for melanoma, include gene expression signatures. Some signatures are currently being developed by for example Castle Biosciences, SkylineDx and Exact Sciences (https: / / pubmed.ncbi.nlm.nih.gov / 32405608 / and https: / / pubmed.ncbi.nlm.nih.gov / 32844403 / ) (https: / / castletestinfo.com / products / decisiondx-melanoma / scientific-references / ). However, these gene expression signatures cannot be run in-house, but samples have to be sent to specialized laboratories to be analyzed.

[0008]

[0005] Recent evidence is also accumulating on epigenetic biomarkers that have prognostic significance (see Hoshimoto S, Kuo CT, Chong KK, et al. AIM1 and LINE- 1 epigenetic aberrations in tumor and serum relate to melanoma progression and disease outcome. J Invest Dermatol. 2012;132:1689-1697; and Cesinaro AM, Sartori G, Migaldi M, et al. Prognostic significance of MGMT gene promoter methylation in differently treated metastatic melanomas. Pathology. 2012;44:313-317).

[0009]

[0006] Epigenetic alterations have been associated with the development of various human cancers, including melanoma, and they consist of DNA methylation aberrations, microRNAs (miRNA) expression patterns, posttranslational histone modifications, and chromatin remodeling.

[0010]

[0007] The inventors previously identified Lymphocyte antigen 75 (LY75) promoter methylation as a prognostic biomarker to identify melanoma patients at high risk of disease recurrence. In the European Patent EP 3303625 (Universiteit Maastricht et al.), the inventors showed that the methylation of the promoter was a strong predictor of poor melanoma prognosis, and identified patients with aggressive disease at diagnosis independent of other prognostic markers (i.e., Breslow thickness, ulceration, (micro)satellites, and SLNB. The inventors found that LY75 promoter methylation (HR_4.442; 95%-CI; 2.307-8.553, p< 001), together with ulceration (HR=2.262; 95%- Cl 1.164-4.396, P=.O16), and metastatic disease at diagnosis (HR=5.069; 95%-CI 2.489-10.325, P<.001) were significant predictors of melanoma survival.

[0011]

[0008] The prognosis of melanoma by means of the analysis of the methylation status of at least one cytosine residue in a methylation relevant region of at least 16 genes selected from a group is disclosed in the U.S. patent application with publication number US 2013 / 9316931 A1 . Data were obtained from samples of patients diagnosed of melanoma stage 111 C.

[0012]

[0009] The qualities of a good and robust biomarker, defined as a measurable molecular signal that correlates with a certain information of the status of a cell, include a good sensitivity, a good specificity, reliability, and preferably the possibility of a fast mode to be analyzed in a cost-effective manner in the routine of the clinical laboratories. However, the finding of these biomarkers, in particular biomarkers that reliably predict the outcome once a cancer has been diagnosed, has proven to be difficult and they are still lacking.

[0013]

[0010] In light of this, new markers, products, compositions, methods and uses for the diagnosis of cancer with impact in the clinics (i.e., in the selection of an adequate treatment) would be highly desirable, but are not yet readily available. Accordingly, the technical problem underlying the present invention can been seen in the provision of such markers, products, compositions, methods and uses for complying with any of the aforementioned needs, or at least providing the public with a useful choice. The technical problem is solved by the embodiments characterized in the claims and herein below.

[0014] SUMMARY OF THE INVENTION

[0015]

[0011] As embodied and broadly described herein, the present invention is directed to the surprising finding that the methylation status of the promoter sequence of certain genes gives meaningful information regarding the outcome of a subject previously diagnosed of melanoma cancer. In particular, the epigenetic markers give reliable information about the risk of suffering recurrences and / or metastases in a subject simultaneously or previously diagnosed of melanoma.

[0016]

[0012] The current invention can help guide prognostication and treatment decisions, as these biomarkers can predict whether a melanoma patient is at high risk of suffering recurrences and / or metastases. Finally, this has an impact on the life expectancy of the subject.

[0017]

[0013] Advantageously, the method of the invention can be applied in any routine diagnostics lab, as in most of its embodiments it requires a technique that is widely used throughout the diagnostic labs, which makes it an easy addition to existing infrastructure. In addition, costs are low compared to the analysis of a gene expression signature or profile, both in handling, time and shipping costs, as well as in reagents and equipment.

[0018]

[0014] Noteworthy is that the method of the invention improves the reliability of the previously proposed biomarker defined by the methylation status of the promoter of the LY75 gene.

[0019]

[0015] One aspect of the invention relates, thus, to an in vitro method for the prognosis of a subject diagnosed or suspected of suffering melanoma, the method comprising the step of determining in an isolated sample of the subject whether one or more methylation(s) are present in one or more gene promoter sequence(s) selected from the group consisting of the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene; and wherein, if it is determined that one or more methylation(s) is / are present in the one or more of the gene promoters, the subject is classified with a prognosis of a shorter life expectancy in relation to a subject that does not comprises the one or more methylations, and / or the subject is classified with a prognosis of recurrence (i.e., melanoma recurrence) and / or metastasis.

[0020]

[0016] This is the first time any of the promoters of the listed genes, and specifically the methylation status of the same, have been significatively associated to the prognosis of melanoma. The methylation status is to be understood as the presence or absence of methylated nucleotides in nucleic acid fragments (i.e., promoter of a gene), and optionally the quantification of the said methylation (i.e., number of methylated nucleotides and / or number of methylated sequences in a sample).

[0021]

[0017] The human genes which promoters are referred to are identified in a Table that follows in this description with their corresponding ENSBL ID of the Ensembl release 112 - May 2024 © EMBL-EBI, retrievable at https: / / www.ensembl.org / index.html, and which includes a gene description.

[0022]

[0018] Moreover, all the previously listed promoters of these genes provide as single inventive concept that, in combination with the determining of the methylation status of the promoter of the LY75 gene, an accurate and reliable prognosis (i.e., recurrence free-survival) is provided as illustrated in the examples. Indeed, the combination of the methylation status of LY75 with and of any of the listed genes provide for unexpected and better hazard ratios in relation to the determining of the methylation status of the promoter of LY75 alone.

[0023]

[0019] The invention relates, thus, to in-vitro methods for the prognosis of a subject diagnosed or suspected of suffering melanoma, the methods comprising the step of determining in an isolated sample of the subject whether one or more of a gene promoter as those indicated in the paragraph above, comprises one or more methylations; and wherein if it is determined that the one or more of the promoters comprises one or more methylations, the subject is classified with a prognosis of a shorter life expectancy in relation to a subject that does not comprise the one or more methylations. The in-vitro methods for the prognosis of a subject diagnosed or suspected of suffering melanoma comprise, thus, determining the presence or absence of methylated gene promoters in the isolated sample of a subject, the gene promoters selected from one or more of the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene.

[0024]

[0020] Once a prognosis is determined from the isolated sample of a subject suffering from melanoma, the selection of an adequate therapy may be made also in a more accurate mode. It can be predicted if a subject needs therapy and if one does not, based on the prognosis. Under- and overtreatment can be prevented with the method of the invention.

[0025]

[0021] Therefore, the invention also relates to a method of deciding or recommending to initiate a therapy for a subject diagnosed or suspected of suffering melanoma, the method comprising:

[0026] (a) carrying out the in-vitro method for the prognosis as defined above in the previous aspect for the prognosis of a subject diagnosed or suspected of suffering melanoma; and

[0027] (b) if the subject is classified with a prognosis of a shorter life expectancy and / or of recurrence of melanoma and / or metastasis in relation to a subject that does not comprises the one or more methylations, then initiation of a treatment is recommended, preferably where the treatment comprises adjuvant therapy.

[0028]

[0022] As will be illustrated in the examples below, the inventors found reliable and highly specific markers for the determining of a likely outcome regarding life expectancy, risk of recurrence and of metastasis of a subject diagnosed or suspected of suffering melanoma. Therefore, it is also another aspect of the invention the use of a methylated gene promoter selected from methylated gene promoter of NR5A2 gene, methylated gene promoter of ASB18 gene, methylated gene promoter of DUOX2 gene, methylated gene promoter of LINC00682 gene, methylated gene promoter of LMX1A gene, methylated gene promoter of NKX2 gene, methylated gene promoter of OSR1 gene, methylated gene promoter of PDX1 gene, methylated gene promoter of CLDN10 gene, and combinations thereof, as marker(s) of the prognosis of a subject diagnosed or suspected of suffering melanoma.

[0029]

[0023] The term marker or biomarker are used interchangeably along this description and, as previously indicated, it relates to a molecular signal that gives information about the status of a cell or tissue.

[0030]

[0024] In addition, a diagnostic kit test based on the biomarkers provided by the present invention can ameliorate the current process of diagnosis and prognosis, conferring to the isolated samples the ability of providing valuable diagnostic and prognostic information of the disease.

[0031]

[0025] Therefore, yet another aspect of the invention is a kit that comprises one or more reagent(s) means for determining methylation in a gene promoter selected from one or more of the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene, and optionally reagent(s) means for determining methylation in one or more gene promoters selected from: the promoter of LY75 gene; the promoter of ABCC9 gene; the promoter of AKR1 B1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANXA2R gene; the promoter of AOX1 ; the promoter of ARAP2; the promoter of ASCL2; the promoter of ATAD1 or CFL1 P1 ; the promoter of ATF7IP2; the promoter of BARHL1 ; the promoter of BTBD8; the promoter of CAPN14 or EHD3; the promoter of CARD11 ; the promoter of CAV3 or OXTR; the promoter of CD109; the promoter of CD8BP; the promoter of CDCA2; the promoter of CH17; the promoter of CLIN01305; the promoter of DGLIOK; the promoter of DIP2C; the promoter of DMRTA2; the promoter of DNAJC1 ; the promoter of EFS; the promoter of EGR3 gene; the promoter of EMX2 gene; the promoter of FADS6 gene; the promoter of FAM163B; the promoter of FAM218A or TRIM61 ; the promoter of FOXP2; the promoter of GNAS; the promoter of GRHL2; the promoter of H2AFJ; the promoter of HIC1 ; the promoter of HIST1 H2AG and / or HIST1 H2BJ; the promoter of HIST1 H2AI and / orHIST1 H2BL; the promoter of HMGB2; the promoter of INPPA5A; the promoter of INTS10; the promoter of ISLR2; the promoter of ITGA3; the promoter of K CNJ3; the promoter of KCTD21 ; the promoter of KIAA1217; the promoter of KIF13B; the promoter of KLF13; the promoter of KLHL; the promoter of KNJC3; the promoter of LBX2; the promoter of LINC00261 ; the promoter of LINC01230; the promoter of LINC01305; the promoter of LRRK1 ; the promoter of LRRN4; the promoter of LYL1 ; the promoter of MAD1 L1 ; the promoter of MAP9; the promoter of MFSD8; the promoter of NFIC; the promoter of NGB; the promoter of NODAL; the promoter of PARM1 ; the promoter of PAX2; the promoter of PCDHB10; the promoter of PCDHB2; the promoter of PCDHGA1 ; the promoter of PDPN; the promoter of PNPLA6; the promoter of PRDM13; the promoter of PRDM6; the promoter of PSMB5; the promoter of RFX4; the promoter of RHOD; the promoter of RPSAP58; the promoter of S1 PR5; the promoter of SCCPDH; the promoter of SCGB3A1 ; the promoter of SHISA9; the promoter of SLC30A2; the promoter of SMIM14; the promoter of SNX10; the promoter of SVIL; the promoter of TBC1 D30; the promoter of TBX15; the promoter of TH; the promoter of THBS1 ; the promoter of TNFSF11 ; the promoter of TRIM26; the promoter of TSPAN2; and the promoter of VSTM2B; and wherein the said one or more reagent means, are the only means comprised in the kit for the determining of methylation in a gene promoter.

[0032]

[0026] The kit of the previous aspect is, thus, a purposed-device to carry out the method of the invention as previously disclosed. Therefore, another aspect of the invention relates to the use of a kit for the prognosis of a subject diagnosed or suspected of suffering melanoma, wherein the kit is as defined above and comprises one or more reagent means for determining methylation in a gene promoter selected from one or more of the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene, and optionally reagent means for determining methylation in one or more gene promoters selected from: the promoter of LY75 gene; the promoter of ABCC9 gene; the promoter of AKR1 B1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANXA2R gene; the promoter of AOX1 ; the promoter of ARAP2; the promoter of ASCL2; the promoter of ATAD1 or CFL1 P1 ; the promoter of ATF7IP2; the promoter of BARHL1 ; the promoter of BTBD8; the promoter of CAPN14 or EHD3; the promoter of CARD11 ; the promoter of CAV3 or OXTR; the promoter of CD109; the promoter of CD8BP; the promoter of CDCA2; the promoter of CH17; the promoter of CLIN01305; the promoter of DGLIOK; the promoter of DIP2C; the promoter of DMRTA2; the promoter of DNAJC1 ; the promoter of EFS; the promoter of EGR3 gene; the promoter of EMX2 gene; the promoter of FADS6 gene; the promoter of FAM163B; the promoter of FAM218A or TRIM61 ; the promoter of FOXP2; the promoter of GNAS; the promoter of GRHL2; the promoter of H2AFJ; the promoter of HIC1 ; the promoter of HIST1 H2AG and / or HIST1 H2BJ; the promoter of HIST1 H2AI and / orHIST1 H2BL; the promoter of HMGB2; the promoter of INPPA5A; the promoter of INTS10; the promoter of ISLR2; the promoter of ITGA3; the promoter of K CNJ3; the promoter of KCTD21 ; the promoter of KIAA1217; the promoter of KIF13B; the promoter of KLF13; the promoter of KLHL; the promoter of KNJC3; the promoter of LBX2; the promoter of LINC00261 ; the promoter of LINC01230; the promoter of LINC01305; the promoter of LRRK1 ; the promoter of LRRN4; the promoter of LYL1 ; the promoter of MAD1 L1 ; the promoter of MAP9; the promoter of MFSD8; the promoter of NFIC; the promoter of NGB; the promoter of NODAL; the promoter of PARM1 ; the promoter of PAX2; the promoter of PCDHB10; the promoter of PCDHB2; the promoter of PCDHGA1 ; the promoter of PDPN; the promoter of PNPLA6; the promoter of PRDM13; the promoter of PRDM6; the promoter of PSMB5; the promoter of RFX4; the promoter of RHOD; the promoter of RPSAP58; the promoter of S1 PR5; the promoter of SCCPDH; the promoter of SCGB3A1 ; the promoter of SHISA9; the promoter of SLC30A2; the promoter of SMIM14; the promoter of SNX10; the promoter of SVIL; the promoter of TBC1 D30; the promoter of TBX15; the promoter of TH; the promoter of THBS1 ; the promoter of TNFSF11 ; the promoter of TRIM26; the promoter of TSPAN2; and the promoter of VSTM2B; and wherein the said one or more reagent means are the only means comprised in the kit for the determining of methylation in a gene promoter

[0033]

[0027] Preferably, the reagents encompassed in the kits of the invention are those that allow for the analysis of methylation of nucleic acids, directly or indirectly. Moreover, these reagents may form part of assays with differing underlying principles of the physics, such as those selected from the group consisting of an immunoassay, a bioluminescence assay, a fluorescence assay, a chemiluminescence assay, electrochemistry assay, mass spectrometry, and combinations thereof.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035]

[0028] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0036]

[0029] Figures 1-9: Kaplan-Meier curves of melanoma-specific survival of patients grouped according to promoter methylation.

[0037]

[0030] In Figure 1 (A) the Kaplan-Meier of melanoma-specific survival of patients grouped according to LY75 promoter methylation; in (B) the Kaplan-Meier of melanoma-specific survival of patients grouped according to ASB18 promoter methylation; in (C) the Kaplan-Meier of melanoma-specific survival of patients grouped according to ASB18 promoter methylation in combination with LY75 promoter methylation.

[0038]

[0031] Figures 2-9 illustrate in (A) the Kaplan-Meier of melanoma-specific survival of patients grouped, respectively, according to DLIOX2, LINC00682, LMX1A, NKX2, NR5A2, OSR1 , PDX1 , and CLDN10 promoter methylation; and in (B) the Kaplan-Meier of melanoma-specific survival of patients grouped according to the promoter methylation of the gene indicated in (A) in combination with LY75 promoter methylation. Combination test was considered positive in case one or both markers were methylated. Negative was for no methylation of any of the markers.

[0039]

[0032] Figure 10 - 17 Kaplan-Meier curves of melanoma-specific survival of from a second cohort (validation, n=145) of patients grouped according to promoter methylation

[0040]

[0033] Figure 10 (A) the Kaplan-Meier of melanoma-specific survival of patients grouped according to LY75 promoter methylation; in (B) the Kaplan-Meier of melanoma-specific survival of patients grouped according to ASB18 promoter methylation; in (C) the Kaplan-Meier of melanoma-specific survival of patients grouped according to ASB18 promoter methylation in combination with LY75 promoter methylation

[0041]

[0034] Figures 11 - 17 illustrate in (A) the Kaplan-Meier of melanoma-specific survival of patients grouped in the second validation cohort (n=145), respectively, according to DUOX2, LINC00682, LMX1A, NR5A2, OSR1 , PDX1 , and CLDN10 promoter methylation; and in (B) the Kaplan-Meier of melanoma-specific survival of patients grouped according to the promoter methylation of the gene indicated in (A) in combination with LY75 promoter methylation. Combination test was considered positive in case one or both markers were methylated. Negative was for no methylation of any of the markers.

[0042]

[0035] Figure 18 is the Kaplan-Meier of melanoma-specific survival of patients grouped according to the promoter methylation of one of the genes DLIOX2, LMX1A and NR5A2. If at least one of the promoters of these three genes is found methylated, the test is positive.

[0043] DESCRIPTION

[0044] Definitions

[0045]

[0036] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0046]

[0037] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0038] For purposes of the present invention, the following terms are defined below.

[0047]

[0039] As used herein, the singular form terms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a molecule” or “a methylation”, or “a gene promoter sequence” includes a combination of two or more molecules or methylations, or gene promoter sequences, and the like.

[0040] As used herein, “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0048]

[0041] As used herein, "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... , etc..

[0049]

[0042] The invention lies in the ability to analyze the presence and amount of methylations in an isolated DNA sample (i.e., nucleic acid sample) of a subject suspected of suffering, or already diagnosed of suffering melanoma. This analysis of methylations in nucleic acids can be carried out by conventional techniques (i.e., conventional methods) the skilled person will know, some of them listed and briefly disclosed in the following sections. As herewith used, the term “one or more methylation(s) is / are present” is in particular to be understood the concluding fact that at least a cytosine was methylated in the nucleic acid fragment of the gene promoter that was analyzed. The term is also referred as the “methylation status” of a nucleic acid. For this concluding fact, the skilled person will understand that depending on the used method for the analysis of the methylation, a sort of a cut-off value or reference signal / value will be correlated with the presence of the methylation and, optionally, with the amount of methylations in a particular nucleic acid fragment or sample comprising that nucleic acid fragment. Depending on the desired specificity and sensitivity of the method, the cut-off (or cutoff) value / reference may be adjusted. Thus, the method of the invention also encompasses the comparison of the determined methylation(s) in an isolated test sample, in comparison with a “control or reference value or level”.

[0050]

[0043] For the establishment of cut-off values or reference levels or values Receiver operating characteristic (ROC) curves are usually created to assess the discriminative ability of a candidate markers (i.e., the methylation and optionally amount of methylation in a promoter of a gene). A cut-off value for a marker is usually determined based on the highest positive likelihood ratio (LR). In some examples, and in an embodiment of the method of the invention, a sample is considered methylated when its Cycle threshold value (Ct-value) from a Polymerase Chain Reaction (PCR) is below a predetermined cutoff. In other examples and depending on the technology employed to detect the methylated nucleotides in a nucleic acid fragment, a sample is considered methylated when its methylation value is equal or below the predetermined cutoff. The determining of cut-offs or reference values is under the common practice of the skilled person in the art. Moreover, as widely acknowledged specific cutoffs or reference values or levels may be applied depending on the features of a tested population.

[0051]

[0044] The term "reference control value or level" referred to in the methods of the any of the aspects of the invention is to be understood as a predefined value of a given molecular marker (i.e., methylation / methylated gene promoter) or a combination of the given molecular markers (i.e., methylation in two or more of the listed promoters of the genes). The samples are taken from a subject or group of subjects wherein the presence, absence, stage, histological subtype or grade, or course of the disease has been properly performed previously. This value is used as a threshold to discriminate subjects wherein the condition to be analyzed is present (i.e., methylation) from those wherein such condition is absent (i.e., no methylation). This reference control level is also useful for determining whether the subject has to initiate a medical regimen and how effective the regimen is. The subject or subjects from whom the "reference control value / level" is derived may include subject / s wherein the condition is absent, subjects wherein the condition is present, or both. The skilled person in the art, making use of the general knowledge, is able to choose the subject or group of subjects more adequate for obtaining the reference control level for each of the methods of the present invention.

[0052]

[0045] The term "diagnosis" is known to the person skilled in the art. As used herein "diagnosis" is understood as becoming aware or detecting of a particular medical condition complication or risk in a subject; the determination of the nature of the disease or condition; or the distinguishing of one disease or condition from another. It refers both to the process of attempting to determine or identify the possible disease or disorder, and to the opinion reached by this process. A diagnosis, in the sense of diagnostic procedure, can be regarded as an attempt at classification of an individual's condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made.

[0053]

[0046] The term “prognosis" as used herein refers to the prediction of the probable progression and outcome of a disease (i.e., outcome or progression of melanoma). It includes: tumor grading (attempt to express in replicable terms the level of cell differentiation in tumor as increasing anaplasia usually correlates with the aggressiveness of the tumor), tumor staging (attempt to express in replicable terms the extent of the tumor in the patient). As used herein prognosis means, in particular survival rate after diagnosis, wherein a poor prognosis means a low survival rate or low life expectancy; risk of recurrence and / or of metastasis.

[0054]

[0047] The “survival rate” is a part of survival analysis. It is the proportion of people in a study or treatment group still alive at a given period of time after diagnosis. It is a method of describing prognosis in certain disease conditions, and can be used for the assessment of standards of therapy. The survival period is usually reckoned from date of diagnosis or start of treatment. Survival rates are based on the population as a whole and cannot be applied directly to an individual. Survival rates may be calculated and expressed as overall survival rates, and “cumulative survival rates”, among others. The “cumulative survival” of a cohort of patients (e.g., patients with methylation in the promoter of a gene) defines the probability of surviving within a predetermined period of time. Cumulative survival is usually displayed in Kaplan-Meier curves, which displays the probability of survival (event did not occur) as a function of time. For the analysis of survival rates and associated basic concepts in survival analysis see Harrell, F. E. (2001). Regression modelling strategies: with applications to linear models, logistic regression, and survival analysis (Vol. 608). New York: Springer. The recurrence-free survival, defined as the time from cancer diagnosis until diagnosis of metastatic disease or end of follow-up, is one of the outcome measure of the survival analyses.

[0055]

[0048] The term “Cox proportional hazards regression”, or just “Cox regression”, is conceptually similar to multivariable linear or logistic regression. Cox regression examines survival as a function of several different independent variables (IVs), and the statistical significance of each of these IVs is assessed for the outcome of interest (occurrence of the event). In Cox regression, the analysis yields a “hazard ratio (HR)” that is interpreted like a relative risk. Thus, values below 1 indicate a lower risk of occurrence of the event relative to the comparison group, values above 1 indicate a higher risk, and a value of 1 indicates an identical risk.

[0056]

[0049] As used herein, the term “life expectancy”, relates to the estimate of the average number of additional time (months, years) that a person diagnosed of melanoma can expect to live in relation to this circumstance (i.e., diagnosis of melanoma).

[0050] As used herein, “conventional techniques”, or “conventional methods”, or “methods known to the skilled person” refer to a situation wherein the methods of carrying out the conventional techniques used in methods as disclosed herein will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology and related fields are well-known to those of skill in the art and are discussed, in various handbooks and literature references.

[0057]

[0051] As used herein, “comprising” or “to comprise” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. It also encompasses the more limiting “to consist of”.

[0058]

[0052] As used herein, “identity” or “sequence identity” refers to the degree of relatedness between two or more nucleic acid sequences (polynucleotide sequences), and for extension also to two or more amino acid sequences, as determined by comparing the sequences. The comparison of sequences and determination of sequence identity may be accomplished using a mathematical algorithm; those skilled in the art will be aware of computer programs available to align two sequences and determine the percent identity between them. The skilled person will appreciate that different algorithms may yield slightly different results.

[0059]

[0053] Thus, the “percent identity” between a query nucleic acid sequence and a subject nucleic acid sequence is the “identities” value, expressed as a percentage, that is calculated by, for example, the BLASTN algorithm when a subject nucleic acid sequence has 100 % query coverage with a query nucleic acid sequence after a pair- wise BLASTN alignment is performed. Such pairwise BLASTN alignments between a query nucleic acid sequence and a subject nucleic acid sequence are performed by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off. The query sequence may be 100 % identical to the subject sequence, or it may include up to a certain integer number of nucleotide alterations as compared to the subject sequence such that the % identity is less than 100%. For example, the query sequence is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subject sequence.

[0054] The “CpG island” or “CG sites” or “CG dinucleotides”, all used in this description as synonyms, are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' — > 3' direction. CpG sites occur with high frequency in genomic regions called CpG islands (or CG islands). CpG islands (or CG islands) are regions with a high frequency of CpG sites. Though objective definitions for CpG islands are limited, the usual formal definition is a region with at least 200 bp, a GC percentage greater than 50%, and an observed-to-expected CpG ratio greater than 60%.

[0060]

[0055] As used herein “bisulfite-modified DNA” as synonymous of “bisulfite DNA conversion” relates to a process used to differentiate between methylated and unmethylated cytosines in DNA for epigenetic studies. It involves treating singlestranded DNA with sodium bisulfite, or an equivalent bisulfite, disulfite or hydrogen sulfite salts, which chemically converts all unmethylated cytosines (\(C\)) into uracils (\(U\)), while methylated cytosines (\(CA{m}\)) remain unchanged. After amplification (for example by PCR), the uracils are read as thymines (\(T\)). By comparing the original and modified sequences, researchers can identify the locations of the original methylated cytosines, which are the only bases that remain as cytosines. .

[0061]

[0056] As used herein “reagents for bisulfite DNA conversion” refer to methylationspecific reagents. The reagents for bisulfite DNA conversion comprise, among others, reagents that in combination modify a nucleotide of the nucleic acid molecule as a function of the methylation state of the nucleic acid molecule, or that can change the nucleotide sequence of a nucleic acid molecule in a manner that reflects the methylation state of the nucleic acid molecule. Examples of such reagents include, but are not limited to, a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent, such a bisulfite salt, such a sodium bisulfite. Bisulfite reagents comprise bisulfite, disulfite, hydrogen sulfite, or combinations thereof. Methods of treating a nucleic acid molecule with such a reagent can include contacting the nucleic acid molecule with the reagent, coupled with additional steps, if desired, to accomplish the desired change of nucleotide sequence.

[0062] Detailed description

[0057] The invention is defined herein, and in particular in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[0063]

[0058] It is contemplated that any method, use, or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and / or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[0064]

[0059] As embodied and broadly described herein, the present invention is directed to the surprising finding that, by means of the analysis of the methylation status of promoter sequences / regions of certain genes from an isolated sample of a subject suffering from melanoma or suspected thereof, an accurate prognosis regarding the life expectancy (i.e. , statistical measure of the estimate of the average remaining years of life at a given age), and / or risk of recurrence and / or of metastasis can be predicted. From the analysis of a cohort of patients, the inventors have found with combinations of markers with high hazard ratios, many of the combinations improving the prognostic value of other prior art markers.

[0065]

[0060] The finding is noteworthy because it provides relevant information to select as soon as possible an adequate medical (i.e., therapeutical) regimen for the subject.

[0066]

[0061] As said, the invention relates to an in-vitro method for the prognosis of a subject diagnosed or suspected of suffering melanoma, the method comprising the step of determining in an isolated sample of the subject whether one or more methylation(s) are present in a gene promoter sequence selected from the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene; and wherein, if it is determined that one or more methylation(s) is / are present in the one or more gene promoters, the subject is classified with a prognosis of a shorter life expectancy in relation to a subject that does not comprises the one or more methylations, and / or the subject is classified with a prognosis of recurrence of melanoma and / or metastasis.

[0062] In an embodiment of the method for the prognosis, the presence or absence of methylation in the promoters of the genes is determined all along the promoter sequence, including one or more of the core promoter, the proximal promoter and the distal promoter. In another embodiment, the presence or absence of methylation in the promoters of the genes is determined in the CpG islands of said promoters, also in one or more of the core promoter, the proximal promoter and the distal promoter.

[0067]

[0063] When used herein, the term “promoter” refers to the genomic region upstream of the 5’-untranslated region (5’IITR) of a gene that controls the expression of the codified protein. Preferably the upstream region consists or comprises of a region of 100 - 1000 bases upstream of the 5’IITR of the gene.

[0068]

[0064] The promoter of a gene or, equivalently, gene promoter refers to a sequence of DNA to which certain proteins bind allowing initiating the transcription of a single RNA from the DNA downstream of it. There are three main portions that make up a promoter: core promoter, proximal promoter, and distal promoter. The core promoter region is located most proximal to the start codon and contains the RNA polymerase binding site, TATA box (in eukaryotes and archaea), and transcription start site (TSS). RNA polymerase will bind to this core promoter region stably and transcription of the template strand can initiate. The TATA box is a DNA sequence (5'-TAT AAA-3') within the core promoter region where general transcription factor proteins and histones can bind. Histones are proteins found in eukaryotic cells that package DNA into nucleosomes. Histone binding prevents the initiation of transcription whereas transcription factors promote the initiation of transcription. The most 3' portion (closest to the gene's start codon) of the core promoter is the TSS which is where transcription actually begins. TATA box is generally positioned in a window of up to 50 nucleotides upstream the TSS. Further, upstream from the core promoter one can find the proximal promoter which contains many primary regulatory elements. The proximal promoter is found approximately 250 base pairs upstream from the TSS and it is the site where general transcription factors bind. The final portion of the promoter region is called the distal promoter which is upstream of the proximal promoter. The distal promoter also contains transcription factor binding sites, but mostly contains regulatory elements.

[0069]

[0065] In another embodiment, the one or more methylations in the promoter of the genes herewith disclosed (i.e., NR52A, ASB18, DU0X2, UNC00682. LMX1A, NKX2, OSR1, PDX1) are determined throughout the region in the genes defined by the flank (outside) primers for pre-amplification PCR, and / or the (inside) primers for a qualitative methylation-specific PCR, and optionally the probes, as disclosed in Table 3 below for each of the genes. Other sequences of the primers also capable to amplify these regions of the respective genes may be used. As an embodiment / example, the region of the promoter of the gene NR5A2, wherein the one or more methylations are determined, is defined by the sequence spanning and amplified by primers (outside or flank) of sequence SEQ ID NO: 1 and SEQ ID NO: 2, and / or defined by the primers (inside or qMSP) of sequence SEQ ID NO: 3 and SEQ ID NO: 4. Corresponding examples / embodiments are directly deduced from Table 3 for the other genes and corresponding primers.

[0070]

[0066] In an embodiment, determining methylation in one or more gene promoter sequences in an isolated sample of a subject comprises the steps of:

[0071] (i) carrying out in DNA of the isolated sample a bisulfite DNA conversion to obtain treated promoter regions; and

[0072] (ii) amplifying those treated promoter regions, preferably amplifying CpG islands in the promoters, by means of primers that allow amplification of regions in the promoters flanked by primers that comprise or consists of the pairs of primers: SEQ ID NO: 1-2 and optionally SEQ ID NO: 3-4 for the promoter of NR5A2 gene; SEQ ID NO:6-7, and optionally SEQ ID NO: 8-9 the promoter of ASB18 gene; SEQ ID NO: 11-22 and optionally SEQ ID NO: 13-14 for the promoter of DLIOX2 gene; SEQ ID NO: 16 - 17 and optionally SEQ ID NO: 18-19 for the promoter of LINC00682 gene; SEQ ID NO: 21-22 and optionally SEQ ID NO: 23-24 for the promoter of LMX1A gene; SEQ ID NO: 26-27 and optionally SEQ ID NO: 28-29 for the promoter of NKX2 gene; SEQ ID NO: 31-32 and optionally SEQ ID NO: 33-34 for the promoter of OSR1 gene; SEQ ID NO: 36-37 and optionally SEQ ID NO: 38-39 for the promoter of PDX1 gene; and SEQ ID NO: 41-42 and optionally SEQ ID NO: 43-44 for the promoter of CLDN10 gene; and SEQ ID NO: 46-47 and optionally SEQ ID NO: 48- 49 for the promoter of LY75 gene; and

[0073] (iii) detecting methylation in the amplified treated promoter region(s) by means of specific probes.

[0074]

[0067] Thus, the primers may comprise any sequence meanwhile they hybridize totally or partially with the corresponding complementary sequences as the primers of SEQ ID NO: 1-4, 6-9, 11-14, 16-19, 21-24, 26-29, 31-34, 36-39, 41-44, and 46-49 do; and which flank the regions of interest within the promoters of the indicated genes.

[0075]

[0068] In a preferred embodiment, the primers comprise or consists of the pairs of primers: SEQ ID NO: 1-2 and optionally SEQ ID NO: 3-4 for the promoter of NR5A2 gene; SEQ ID NO:6-7, and optionally SEQ ID NO: 8-9 the promoter of ASB18 gene; SEQ ID NO: 11-22 and optionally SEQ ID NO: 13-14 for the promoter of DLIOX2 gene; SEQ ID NO: 16 - 17 and optionally SEQ ID NO: 18-19 for the promoter of LINC00682 gene; SEQ ID NO: 21-22 and optionally SEQ ID NO: 23-24 for the promoter of LMX1A gene; SEQ ID NO: 26-27 and optionally SEQ ID NO: 28-29 for the promoter of NKX2 gene; SEQ ID NO: 31-32 and optionally SEQ ID NO: 33-34 for the promoter of OSR1 gene; SEQ ID NO: 36-37 and optionally SEQ ID NO: 38-39 for the promoter of PDX1 gene; and SEQ ID NO: 41-42 and optionally SEQ ID NO: 43-44 for the promoter of

[0076] CLDN10 gene; and SEQ ID NO: 46-47 and optionally SEQ ID NO: 48-49 for the promoter of LY75 gene.

[0077]

[0069] In another preferred embodiment, the specific probes are nucleic acid sequences that comprise or consists in SEQ ID NO: 5 for the promoter of NR5A2 gene; SEQ ID NO: 10 for the promoter of ASB18 gene; SEQ ID NO: 15 for the promoter of DUOX2 gene; SEQ ID NO: 20 for the promoter of LINC00682 gene; SEQ ID NO: 25 for the promoter of LMX1A gene; SEQ ID NO: 30 for the promoter of NKX2 gene; SEQ ID NO: 35 for the promoter of OSR1 gene; SEQ ID NO: 40 for the promoter of PDX1 gene; and SEQ ID NO: 45 for the promoter of CLDN10 gene; and SEQ ID NO: 50 for the promoter of LY75 gene.

[0078]

[0070] In an embodiment of the method for the prognosis according to the aspect of the invention, the presence or absence of one or more methylation(s) (i.e., one or more methylated gene promoters, with one or more methylated nucleotide residues in each promoter) is determined by comparison with a cut-off or reference value. For example, in an embodiment, the gene promoter of the one or more genes is considered methylated when its Cycle threshold value (Ct-value) from a Polymerase Chain Reaction (PCR) is below a predetermined cutoff.

[0079]

[0071] As indicated for the setting of cutoffs or reference values, ROC curves are employed in an embodiment. These ROC curves allow, among others, to set a particular desired sensitivity and specificity correlated with a cutoff.

[0072] In an embodiment of the method, the sample is a biopsy melanoma sample selected from a biopsy of a cutaneous melanoma tumor, and / or a metastatic biopsy sample of the melanoma, preferably a sentinel lymph node biopsy (SLNB). In another embodiment is a liquid biopsy, preferably selected from blood, plasma, serum, interstitial fluid, urine, and saliva. The skilled person in the art will recognize that other samples that may comprise cells or secreted substances of the cells originated or derived from the melanoma can be used in the method of the invention. Other examples of possible biologic samples refer to any sample taken from a subject or derived from it, including, without limitation, primary cultures of autologous neoplastic melanoma cells, neoplastic cells purified from fresh autologous melanoma tissues or from cryopreserved cell suspensions by immuno-sorting (e.g. using antibodies specific for cancer cells) or other means, cryopreserved melanoma tissues, frozen melanoma samples, or other types of fixed tumor tissue, micro- / macro-dissected tumor tissues, circulating tumor melanoma cells present in body fluids. The sample refers to either biological fluids, including, without limitation, any fluid present in the subject in which acellular DNA or cancer cells may be present. The samples can be obtained from a subject using any of the methods known in the art.

[0080]

[0073] In yet another embodiment of the method for the prognosis of a subject diagnosed or suspected of suffering melanoma, an additional step is performed selected from the group consisting of determining tumor ulceration, determining metastatic disease at diagnosis, disease stage, and Breslow thickness of the melanoma (see Keung, E. Z., & Gershenwald, J. E. (2018). The eighth edition American Joint Committee on Cancer (AJCC) melanoma staging system: implications for melanoma treatment and care. Expert Review of Anticancer Therapy, 18(8), 775- 784. https: / / doi.org / 10.1080 / 14737140.2018.148924). For the analysis of all these additional features conventional protocols and techniques area available.

[0081]

[0074] The analysis or determination of these additional features from the subject aims to increase the correct classification of the result of the methylation analysis in the gene promoters, as well as to better stratify the analyzed samples. Other clinical parameters and / or features of the subject can optionally be determined to increase the accuracy of the in vitro prognosis method. Among these features the age of the subject, and the origin / race of the population interest may be considered.

[0075] In yet another embodiment of the method of the invention, it further includes one or more steps for determining one or more of the clinical and / or pathological parameters selected from sex, age at diagnosis, Melanoma subtype, Localization primary tumor, Lateralization primary tumor, Morphology, Clark level, Breslow thickness, Ulceration, Regression, Tumor-infiltrating lymphocytes, Microsatellites, Lymphangioinvasion, Perineural growth, Pre-existent nevi, Mitosis, TNM stage, among other clinical and / or pathological parameters.

[0082]

[0076] Thus, the outcome or result or conclusion of the determining of the one or more methylation(s) present (i.e., methylation status) in a gene promoter sequence selected from one or more of the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DUOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene is, in some embodiments, combined with one or more clinical and / or pathological parameters of the subject. Preferred clinical and / or pathological parameters for the combination with the methylation status are selected from sex, age at diagnosis, Melanoma subtype, Localization primary tumor, Lateralization primary tumor, Morphology, Clark level, Breslow thickness, Ulceration, Regression, Tumor-infiltrating lymphocytes , Microsatellites, Lymphangioinvasion, Perineural growth, Pre-existent nevi, Mitosis, TNM stage.

[0083]

[0077] The combination of the methylation status of the one or more of the promoters of the genes NR5A2, ASB18, DU0X2, UNC00682, LMX1A, NKX2, OSR1, PDX1 CLDN10, and optionally of other genes also listed in this description, with the one or more clinical and / or pathological parameters of the subject provide an accurate selection or recommendation of a therapy regimen for the subject. In addition, it is useful to change a previously applied therapeutic regimen if this is considered necessary with the combination of data retrieved from a sample and from the subject.

[0078] In an embodiment of the in vitro method for the prognosis of melanoma according to the invention, the subject is previously diagnosed of a primary melanoma or of a secondary melanoma.

[0084]

[0079] In yet another embodiment of the methods of the invention for the prognosis of melanoma, the melanoma is a melanoma at any stage, thus, it is a melanoma selected from any one from stage 0 to stage 4.

[0080] Melanoma staging is based on the American Joint Committee on Cancer (AJCC) staging system that uses three key pieces of information for assigning Tumor- Node-Metastasis (TNM) classifications. It uses or considers tumor size, involvement of lymph node, and metastasis. The staging of the melanoma is used to help understand how far the melanoma has advanced through the body.

[0085]

[0081] In also another embodiment of the method of the invention, the additional step comprises the determination of Breslow thickness, which determines the thickness of the melanoma, from the epidermis to the subcutaneous fat.

[0086]

[0082] Also in another embodiment, the method further comprises determining in the isolated sample whether one or more methylation(s) is / are present in one or more gene promoters selected from: the promoter of LY75 gene (ENSG00000054219, Ensembl release 112 - May 2024 © EMBL-EBI); the promoter of ABCC9 gene; the promoter of AKR1 B1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANXA2R gene; the promoter of AOX1 gene; the promoter of ARAP2; the promoter of ASCL2 gene; the promoter of ATAD1 or CFL1 P1 ; the promoter of ATF7IP2 gene; the promoter of BARHL1 gene; the promoter of BTBD8 gene; the promoter of CAPN14 or EHD3 gene; the promoter of CARD11 gene; the promoter of CAV3 or OXTR gene; the promoter of CD109 gene; the promoter of CD8BP gene; the promoter of CDCA2 gene; the promoter of CH17 gene; the promoter of CLIN01305 gene; the promoter of DGLIOK gene; the promoter of DIP2C gene; the promoter of DMRTA2 gene; the promoter of DNAJC1 gene; the promoter of EFS gene; the promoter of EGR3 gene; the promoter of EMX2 gene; the promoter of FADS6 gene; the promoter of FAM163B gene; the promoter of FAM218A or TRIM61 gene; the promoter of FOXP2 gene; the promoter of GNAS gene; the promoter of GRHL2 gene; the promoter of H2AFJ gene; the promoter of HIC1 gene; the promoter of HIST1 H2AG gene and / or HIST1 H2BJ gene; the promoter of HIST1 H2AI gene and / orHIST1 H2BL gene; the promoter of HMGB2 gene; the promoter of INPPA5A gene; the promoter of INTS10 gene; the promoter of ISLR2 gene; the promoter of ITGA3 gene; the promoter of KCNJ3 gene; the promoter of KCTD21 gene; the promoter of KIAA1217 gene; the promoter of KIF13B gene; the promoter of KLF13 gene; the promoter of KLHL gene; the promoter of KNJC3 gene; the promoter of LBX2 gene; the promoter of LINC00261 gene; the promoter of LINC01230 gene; the promoter of LINC01305 gene; the promoter of LRRK1 gene; the promoter of LRRN4 gene; the promoter of LYL1 gene; the promoter of MAD1 L1 gene; the promoter of MAP9 gene; the promoter of MFSD8 gene; the promoter of NFIC gene; the promoter of NGB gene; the promoter of NODAL gene; the promoter of PARM1 gene; the promoter of PAX2 gene; the promoter of PCDHB10 gene; the promoter of PCDHB2 gene; the promoter of PCDHGA1 gene; the promoter of PDPN gene; the promoter of PNPLA6 gene; the promoter of PRDM13 gene; the promoter of PRDM6 gene; the promoter of PSMB5 gene; the promoter of RFX4 gene; the promoter of RHOD gene; the promoter of RPSAP58 gene; the promoter of S1 PR5 gene; the promoter of SCCPDH gene; the promoter of SCGB3A1 gene; the promoter of SHISA9 gene; the promoter of SLC30A2 gene; the promoter of SMIM14 gene; the promoter of SNX10 gene; the promoter of SVIL gene; the promoter of TBC1 D30 gene; the promoter of TBX15 gene; the promoter of TH gene; the promoter of THBS1 gene; the promoter of TNFSF11 gene; the promoter of TRIM26 gene; the promoter of TSPAN2 gene; and the promoter of VSTM2B gene.

[0087]

[0083] Inventors have determined that the following genes are linked with the prognosis according to at least in silico analysis using the Skin cutaneous melanoma (SKCM) dataset within The Cancer Genome Atlas (TCGA), which contained Illumina lnfinium-450K array methylation data of 104 primary melanoma tissue samples.

[0088] Methylated samples correlate with a short life expectancy due to melanoma condition (i.e. , poor survival).

[0089]

[0084] Table 1. Genes linked with prognosis of melanoma.

[0090]

[0085] In a preferred embodiment of the method for the prognosis of a subject diagnosed or suspected of suffering melanoma, it comprises determining whether one, two, three, four, five, six, seven, eight and nine gene promoter(s) comprise(s) one or more methylation(s), preferably wherein the method comprises determining whether one or more methylation(s) is / are present in the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1 A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene. The subject is classified with a prognosis of a shorter life expectancy in relation to a subject that does not comprises the one or more methylations, and / or the subject is classified with a prognosis of recurrence of melanoma and / or metastasis, when one or more methylation(s) are detected in one of the promoters of the genes.

[0091]

[0086] In also another embodiment of the method of the invention, it comprises determining whether one or more methylation(s) is / are present in a set of gene promoters selected from the group consisting of: a) Promoter of LY75 gene and promoter of ASB18 gene; b) Promoter of LY75 gene and promoter of DU0X2 gene; c) Promoter of LY75 gene and promoter of LINC00682 gene; d) Promoter of LY75 gene and promoter of LMX1A gene; e) Promoter of LY75 gene and promoter of NKX2 gene; f) Promoter of LY75 gene and promoter of NR5A2 gene; g) Promoter of LY75 gene and promoter of OSR1 gene; h) Promoter of LY75 gene and promoter of PDX1 gene; and i) Promoter of LY75 gene and promoter of CLDN10 gene.

[0092]

[0087] In this embodiment, according to which a set of two genes including LY75 are analyzed to determine whether one or more methylation(s) is / are present in the promoters, the subject is classified with a prognosis of a shorter life expectancy in relation to a subject that does not comprise the one or more methylations, and / or the subject is classified with a prognosis of recurrence of melanoma and / or metastasis, when one or more methylation(s) are detected in one or in two of the promoters of the genes. As will be illustrated in the examples below, the analysis of the sets of two genes highly improved the hazard ratio values to correctly classify the subject previously diagnosed of melanoma.

[0093]

[0088] In also another embodiment of the method of the invention, it comprises determining whether one or more methylation(s) is / are present in the binary sets of gene promoters defined by the combination of two of the genes NR5A2 gene, ASB18 gene, DU0X2 gene, LINC00682 gene, LMX1A gene, NKX2 gene, OSR1 gene, PDX1 gene, and CLDN10 gene. In this embodiment, according to which a set of two genes are analyzed to determine whether one or more methylation(s) is / are present in the promoters, the subject is classified with a prognosis of a shorter life expectancy in relation to a subject that does not comprises the one or more methylations, and / or the subject is classified with a prognosis of recurrence of melanoma and / or metastasis, when one or more methylation(s) are detected in one or in two of the promoters of the genes. As will be illustrated in the examples below, the analysis of the sets of two genes highly improved the hazard ratio values to correctly classify the subject previously diagnosed of melanoma.

[0094]

[0089] Therefore, the method of the invention comprises determining the presence or absence of methylated gene promoters within binary sets defined by the combination of two of the genes NR5A2 gene, ASB18 gene, DU0X2 gene, LINC00682 gene, LMX1A gene, NKX2 gene, OSR1 gene, PDX1 gene, and CLDN10 gene.

[0095]

[0090] These binary sets are: NR5A2 gene and ASB18 gene; NR5A2 gene and DU0X2 gene; NR5A2 gene and LINC00682 gene; NR5A2 gene and NKX2 gene; NR5A2 gene and OSR1 gene; NR5A2 gene and PDX1 gene; NR5A2 gene and CLDN10 gene; ASB18 gene and DU0X2 gene; ASB18 gene and LINC00682 gene; ASB18 gene and LMX1A gene; ASB18 gene and NKX2 gene; ASB18 gene and OSR1 gene; ASB18 gene and PDX1 gene; ASB18 gene and CLDN10 gene; DU0X2 gene and LINC00682 gene; DU0X2 gene and LMX1A gene; DU0X2 gene and NKX2 gene; DU0X2 gene and OSR1 gene; DU0X2 gene and PDX1 gene; DU0X2 gene and CLDN10 gene; LINC00682 gene and LMX1A gene; LINC00682 gene and NKX2 gene; LINC00682 gene and OSR1 gene; LINC00682 gene and PDX1 gene; LINC00682 gene and CLDN10 gene; LMX1A gene and NKX2 gene; LMX1A gene and OSR1 gene; LMX1A gene and PDX1 gene; LMX1A gene and CLDN10 gene; NKX2 gene and OSR1 gene; NKX2 gene and PDX1 gene; NKX2 gene and CLDN10 gene; OSR1 gene and PDX1 gene; OSR1 gene and CLDN10 gene; and PDX1 gene and CLDN10 gene.

[0096]

[0091] In another embodiment, the method comprises determining the presence or absence of methylated gene promoters in each of these binary sets in combination with the determining of the presence or absence of methylated gene LY75 promoter, which provide ternary sets.

[0097]

[0092] In another embodiment, the method of the invention comprises determining the presence or absence of methylated gene promoters within ternary sets defined by the combination of three of the genes NR5A2 gene, ASB18 gene, DU0X2 gene, LINC00682 gene, LMX1A gene, NKX2 gene, OSR1 gene, PDX1 gene, and CLDN10 gene, and including (NR5A2, ASB18, DUOX2), (NR5A2, ASB18, LINC00682), (NR5A2, ASB18, LMX1A), (NR5A2, ASB18, NKX2), (NR5A2, ASB18, OSR1), (NR5A2, ASB18, PDX1), (NR5A2, ASB18, CLDN10), (NR5A2, DUOX2, LINC00682), (NR5A2, DUOX2, LMX1A), (NR5A2, DUOX2, NKX2), (NR5A2, DUOX2, OSR1), (NR5A2, DUOX2, PDX1), (NR5A2, DUOX2, CLDN10), (NR5A2, LINC00682, LMX1A), (NR5A2, LINC00682, NKX2), (NR5A2, LINC00682, OSR1), (NR5A2, LINC00682, PDX1), (NR5A2, LINC00682, CLDN10), (NR5A2, LMX1A, NKX2), (NR5A2, LMX1A, OSR1), (NR5A2, LMX1A, PDX1), (NR5A2, LMX1A, CLDN10), (NR5A2, NKX2, OSR1), (NR5A2, NKX2, PDX1), (NR5A2, NKX2, CLDN10), (NR5A2, OSR1 , PDX1), (NR5A2, OSR1 , CLDN10), (NR5A2, PDX1 , CLDN10), (ASB18, DUOX2, LINC00682), (ASB18, DUOX2, LMX1A), (ASB18, DUOX2, NKX2), (ASB18, DUOX2, OSR1), (ASB18, DUOX2, PDX1), (ASB18, DUOX2, CLDN10), (ASB18, LINC00682, LMX1A), (ASB18, LINC00682, NKX2), (ASB18, LINC00682, OSR1), (ASB18, LINC00682, PDX1), (ASB18, LINC00682, CLDN10), (ASB18, LMX1A, NKX2), (ASB18, LMX1A, OSR1), (ASB18, LMX1A, PDX1), (ASB18, LMX1A, CLDN10), (ASB18, NKX2, OSR1), (ASB18, NKX2, PDX1), (ASB18, NKX2, CLDN10), (ASB18, OSR1 , PDX1), (ASB18, OSR1 , CLDN10), (ASB18, PDX1 , CLDN10), (DUOX2, LINC00682, LMX1A), (DUOX2, LINC00682, NKX2), (DUOX2, LINC00682, OSR1), (DUOX2, LINC00682, PDX1), (DUOX2, LINC00682, CLDN10), (DUOX2, LMX1A, NKX2), (DUOX2, LMX1A, OSR1), (DUOX2, LMX1A, PDX1), (DUOX2, LMX1A, CLDN10), (DUOX2, NKX2, OSR1), (DUOX2, NKX2, PDX1), (DUOX2, NKX2, CLDN10), (DUOX2, OSR1 , PDX1), (DUOX2, OSR1 , CLDN10), (DUOX2, PDX1 , CLDN10), (LINC00682, LMX1A, NKX2), (LINC00682, LMX1A, OSR1), (LINC00682, LMX1A, PDX1), (LINC00682, LMX1A, CLDN10), (LINC00682, NKX2, OSR1), (LINC00682, NKX2, PDX1), (LINC00682, NKX2, CLDN10), (LINC00682, OSR1 , PDX1), (LINC00682, OSR1 , CLDN10), (LINC00682, PDX1 , CLDN10), (LMX1A, NKX2, OSR1), (LMX1A, NKX2, PDX1), (LMX1A, NKX2, CLDN10), (LMX1A, OSR1 , PDX1), (LMX1A, OSR1 , CLDN10), (LMX1A, PDX1 , CLDN10), (NKX2, OSR1 , PDX1), (NKX2, OSR1 , CLDN10), (NKX2, PDX1 , CLDN10), and (OSR1 , PDX1 , CLDN10). In one embodiment, the ternary combination is, preferably NR5A2, DLIOX2, LMX1A. In another embodiment, the method comprises determining the presence or absence of methylated gene promoters in each of these ternary sets in combination with the determining of the presence or absence of methylated gene LY75 promoter, which provide quaternary sets.

[0098]

[0093] Several methods for the analysis of the presence and optionally the quantification of methylation in nucleic acids, and in particular in the genes or gene promoters, are widely known by the skilled person in the art. An example of possible methods are the (multiplex) methylation-specific polymerase chain reaction analysis; the southern hybridization with methylation-sensitive endonucleases; whole genome / targeted / nanopore bisulfite sequencing; pyrosequencing; epityper; methylation-Sensitive Restriction Enzymes (MSRE); methylated DNA Immunoprecipitation (MeDIP); methyl-CpG Binding Domain (MBD) Capture; Illumina HumanMethylation450 BeadChip (450K Array); llumina Infinium Methylation EPIC Array (850K Array); Twist Bioscience's NGS (with enzymatic conversion followed by targeted sequencing); and Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA). The in vitro method of the invention for the prognosis of melanoma is not limited to any particular method for the analysis of the methylation, and is to be understood that any assay or protocol for that aim, preferably from one or more of the assays listed in this paragraph, is encompassed in the scope of the invention.

[0099]

[0094] The methods for the determining of methylation and quantification of the methylation make use of several conventional techniques including sequencing, mass spectrometry, and immunoprecipitation, among others.

[0100]

[0095] In an embodiment of the method for the prognosis of the invention, any of the previously listed methods for the analysis of the presence and the quantification of methylation in nucleic acids are used.

[0101]

[0096] In a preferred embodiment, the determining in an isolated sample from the subject whether one or more methylation(s) is / are present in one or more gene promoter, preferably the determining in an isolated melanoma sample, or in a liquid biopsy, is performed by a multiplex methylation-specific polymerase chain reaction analysis, more preferably by a nested multiplex methylation-specific polymerase chain reaction.

[0102]

[0097] For information regarding the methylation-specific polymerase chain reaction analysis see, for example Herman JG, Graff JR, Mydhanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9821-6. doi: 10.1073 / pnas.93.18.9821. PMID: 8790415; PMCID: PMC38513.

[0103]

[0098] With the aim to make the method as feasible as possible for the subject and also for the physician that has to interpret the results, the method of the invention comprises, in an embodiment, a step in which after the determination whether one or more methylation(s) are present in a gene promoter, the following computer- implemented steps are carried out: said methylation(s) are given a value and / or a score, and optionally are computed in a mathematical formula to obtain a computed value; wherein in function of the said level(s), score(s) and or computed value(s), a decision is taken about the prognosis, and / or decision of recommending initiating a treatment .

[0104]

[0099] The implementation of the method as a computer-implemented method, provides also with the option of training any algorithm under a machine-learning or artificial intelligence context. In any case, the accurate prognosis of the sample allows for a fast selection of a therapy or follow-on to be recommended.

[0105]

[0100] As indicated, the invention also relates to a method of deciding or recommending to initiate a therapy for a subject diagnosed or suspected of suffering melanoma, the method comprising:

[0106] (a) carrying out the in vitro method for the prognosis as defined above in the previous aspect for the prognosis of a subject diagnosed or suspected of suffering melanoma; and

[0107] (b) if the subject is classified with a prognosis of a shorter life expectancy and / or of recurrence of melanoma and / or of metastasis, in relation to a subject that does not comprises the one or more methylations, then initiation of a treatment is recommended, preferably where the treatment comprises adjuvant therapy.

[0108]

[0101] An adjuvant therapy includes an additional treatment given after the primary treatment for a disease. In melanoma, adjuvant therapy is sometimes used after surgery to reduce the risk of melanoma returning. As discussed, some patients who undergo surgery for their melanoma may be at high risk of their melanoma coming back. Adjuvant therapy can help delay or prevent the recurrence of melanoma.

[0109]

[0102] In an embodiment of the method of deciding or recommending to initiate a therapy for a subject diagnosed or suspected of suffering melanoma, the therapy is selected from one or more of: surgery to remove the tumor, which is applicable to all stages of melanoma; chemotherapy; radiation therapy; immunotherapy, preferably with immune checkpoint modulators (i.e., inhibitors or agonists of checkpoints); targeted therapy, preferably with one or more of dabrafenib, vemurafenib, encorafenib that block the activity of proteins made by mutant BRAF genes, and trametinib, cobimetinib, binimetinib, that block proteins called MEK1 and MEK2 which affect the growth and survival of cancer cells, oncolytic virus therapy, preferably with Talimogene laherparepvec, and angiogenesis inhibitors; and vaccine therapies.

[0110]

[0103] In another embodiment, the recommended therapy comprises the administering of one or more of the currently approved by the FDA drugs selected from the group consisting of: Aldesleukin, Amtagvi (Lifileucel), Binimetinib, Braftovi (Encorafenib), Cobimetinib Fumarate, Cotellic (Cobimetinib Fumarate), Dabrafenib Mesylate, Dacarbazine, Encorafenib, Hepzato (Melphalan Hydrochloride), IL-2 (Aldesleukin), Imlygic (Talimogene Laherparepvec), lnterleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alfa-2b), Ipilimumab, Keytruda (Pembrolizumab), Kimmtrak (Tebentafusp-tebn), Lifileucel, Mekinist (Trametinib Dimethyl Sulfoxide), Mektovi (Binimetinib), Melphalan Hydrochloride, Nivolumab, Nivolumab and Relatlimab-rmbw, Opdivo (Nivolumab), Opdualag (Nivolumab and Relatlimab-rmbw), Pembrolizumab, Proleukin (Aldesleukin), Recombinant Interferon Alfa-2b, Tafinlar (Dabrafenib Mesylate), Talimogene Laherparepvec, Tebentafusp-tebn, Trametinib Dimethyl Sulfoxide, Vemurafenib, Yervoy (Ipilimumab), and Zelboraf (Vemurafenib).

[0111]

[0104] In another embodiment of the method of deciding or recommending to initiate a therapy for a subject diagnosed or suspected of suffering melanoma, the treatment comprises neoadjuvant immunotherapy. Neoadjuvant immunotherapy includes a therapy that is carried out before surgery, while the tumor is still in place. The main goals of a neoadjuvant immunotherapy consists in: shrinking the tumor and make surgery easier or more effective; to prime the immune system by exposing it to the full antigenic landscape of the intact tumor, potentially generating a more robust, systemic antitumor immune response that could control micrometastases earlier; and assessment of pathological responses. Neoadjuvant therapies that may apply for the treatment of melanoma include the treatment with one or more of nivolumab (anti- PD1), Ipilimumab (CTLA-4 blockade), and relatlimab.

[0112]

[0105] In yet another embodiment, the treatment comprises the combination of neoadjuvant and adjuvant immunotherapies.

[0106] One of the goals of the invention is that due to a reliable and accurate prognosis of the subject suffering from melanoma, the subject can be stratified and classified for an effective treatment as soon as possible. This avoids the application of unnecessary therapeutical regimens that, ultimately, are non-feasible for the subject, that are also cost-effective, and / or result in significant comorbidity(ies). It also avoids undertreatment of patients with high-risk melanoma’s and the possible subsequent consequences such as the development of recurrences and / or metastases and the associated effects on quality of life and health care costs.

[0113]

[0107] In an embodiment of the method of deciding or recommending to initiate a therapy for a subject diagnosed or suspected of suffering melanoma, the method comprises:

[0114] (a) carrying out the in vitro method for the prognosis as defined above in the previous aspect for the prognosis of a subject diagnosed or suspected of suffering melanoma; and

[0115] (b) if the subject is classified with a prognosis of a shorter life expectancy and / or of recurrence of melanoma and / or of metastasis, in relation to a subject that does not comprises the one or more methylations, then initiation of a treatment is recommended, preferably where the treatment comprises adjuvant therapy; and

[0116] (c) wherein for the selection of a recommended treatment, one or more clinical and / or pathological parameters and / or features of the subject are taken in consideration, said clinical or pathological parameters selected from the group consisting of at least one or more of the sex, age at diagnosis, Melanoma subtype, Localization primary tumor, Lateralization primary tumor, Morphology, Clark level, Breslow thickness, Ulceration, Regression, Tumor-infiltrating lymphocytes , Microsatellites, Lymphangioinvasion, Perineural growth, Preexistent nevi, Mitosis, and TNM stage.

[0117]

[0108] The description encompasses also a method of treatment of a subject diagnosed or suspected of suffering melanoma, the method comprising:

[0118] (a) carrying out the in vitro method for the prognosis as defined above in the previous aspect for the prognosis of a subject diagnosed or suspected of suffering melanoma; and

[0119] (b) if the subject is classified with a prognosis of a shorter life expectancy in relation to a subject that does not comprises the one or more methylations, and / or if the subject is classified with a prognosis of recurrence of melanoma and / or of metastasis, then a treatment is initiated, preferably where the treatment comprises adjuvant therapy.

[0120]

[0109] With the step of the in vitro method of prognosis, as said, an outcome regarding the risk of suffering recurrences and / or metastases is determined, and this can help to decide on the most appropriate therapy regimen, which can include one or more therapeutical approaches in combination.

[0121]

[0110] In an embodiment of the method of treatment herewith disclosed, other clinical parameters and / or pathological parameters and / or features of the subject can optionally be determined to increase the accuracy of the in vitro prognosis method, as said, but also to decide and / or to initiate or select a therapy / treatment (i.e. , therapy regimen).

[0122]

[0111] In an embodiment of the method of treatment herewith disclosed, the one or more of the clinical and / or pathological parameters and / or features of the subject are selected from sex, age at diagnosis, Melanoma subtype, Localization primary tumor, Lateralization primary tumor, Morphology, Clark level, Breslow thickness, Ulceration, Regression, Tumor-infiltrating lymphocytes , Microsatellites, Lymphangioinvasion, Perineural growth, Pre-existent nevi, Mitosis, TNM stage, among other clinical and / or pathological parameters.

[0123]

[0112] With the combination of the information regarding the methylation status of the one or more of the gene promoters, and the one or more of the clinical and / or pathological parameters and / or features of the subject specific decisions regarding the treatment are taken.

[0124]

[0113] As a normal practice, for example, for those subjects suffering from a primary melanoma and that have been submitted to a radically resection (i.e., radically resected primary melanoma patients), the classification as subjects with a particular risk (e.g., high risk) of metastasis and recurrence, and ultimately reduced life expectancy, is carried out based on the outcome of the routine sentinel lymph node biopsy procedure. If the patient is classified as (high) risk of recurrence / metastasis the administering of adjuvant therapy mainly based on immune therapy (e.g., nivolumab or pembrolizumab) is initiated. If low risk of metastasis / recurrence is expected according to the outcome of the sentinel lymph node biopsy procedure, no adjuvant therapy is initiated. However, if the information regarding the methylation status of the one or more of the gene promoters, classifies the subject with a prognosis of a shorter life expectancy in relation to a subject that does not comprises the one or more methylations, and / or with a prognosis of recurrence and / or prognosis of metastasis; and in combination the outcome of the routine sentinel lymph node biopsy procedure classifies the subject with a no risk of recurrence and / or metastasis, the subject is also treated or recommended to be treated, with adjuvant therapy, such as immune therapy, including in an embodiment one or more of nivolumab and pembrolizumab.

[0125]

[0114] This is an example of practical application of the method of the invention in combination with other clinical and / or pathological parameters, which combination results in a clinical decision (treatment with adjuvant therapy) that had not been taken without the analysis of the methylation status of the one or more of the gene promoters.

[0126]

[0115] Herewith disclosed is a method of detecting methylation in one or more gene promoter sequences in an isolated sample of a subject, or a method of characterizing an isolated sample of a subject, the method comprising: determining in the isolated sample of a subject whether one or more methylation(s) are present in one or more gene promoter sequence(s), by means of one or more of a (multiplex) methylationspecific polymerase chain reaction analysis, preferably by a nested multiplex methylation-specific polymerase chain reaction; a southern hybridization with methylation-sensitive endonucleases; a whole genome / targeted / nanopore bisulfite sequencing; pyrosequencing; epityper; methylation-Sensitive Restriction Enzymes (MSRE); methylated DNA Immunoprecipitation (MeDIP); methyl-CpG Binding Domain (MBD) Capture; Illumina HumanMethylation450 BeadChip (450K Array); Illumina Infinium Methylation EPIC Array (850K Array); Twist Bioscience's NGS; and Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA); wherein the one or more gene promoter sequence(s) are selected from the group consisting of the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene, and optionally the promoter of LY75 gene.

[0127]

[0116] In an embodiment of the method of detecting methylation in one or more gene promoter sequences in an isolated sample of a subject, or of the method of characterizing an isolated sample of a subject, the determining in the isolated sample of a subject whether one or more methylation(s) are present in one or more gene promoter sequence(s) comprises the steps of:

[0128] (i) carrying out in DNA of the isolated sample a bisulfite DNA conversion to obtain treated promoter regions; and

[0129] (ii) amplifying those treated promoter regions, preferably amplifying CpG islands in the promoters, by means of primers that allow amplification of regions in the promoters flanked by primers that comprise or consists of the pairs of primers: SEQ ID NO: 1-2 and optionally SEQ ID NO: 3-4 for the promoter of NR5A2 gene; SEQ ID NO:6-7, and optionally SEQ ID NO: 8-9 the promoter of ASB18 gene; SEQ ID NO: 11-22 and optionally SEQ ID NO: 13-14 for the promoter of DLIOX2 gene; SEQ ID NO: 16 - 17 and optionally SEQ ID NO: 18-19 for the promoter of LINC00682 gene; SEQ ID NO: 21-22 and optionally SEQ ID NO: 23-24 for the promoter of LMX1A gene; SEQ ID NO: 26-27 and optionally SEQ ID NO: 28-29 for the promoter of NKX2 gene; SEQ ID NO: 31-32 and optionally SEQ ID NO: 33-34 for the promoter of OSR1 gene; SEQ ID NO: 36-37 and optionally SEQ ID NO: 38-39 for the promoter of PDX1 gene; and SEQ ID NO: 41-42 and optionally SEQ ID NO: 43-44 for the promoter of CLDN10 gene; and SEQ ID NO: 46-47 and optionally SEQ ID NO: 48- 49 for the promoter of LY75 gene; and

[0130] (iii) detecting methylation in the amplified treated promoter region(s) by means of specific probes.

[0131]

[0117] In a preferred embodiment, the primers comprise or consists of the pairs of primers: SEQ ID NO: 1-2 and optionally SEQ ID NO: 3-4 for the promoter of NR5A2 gene; SEQ ID NO:6-7, and optionally SEQ ID NO: 8-9 the promoter of ASB18 gene; SEQ ID NO: 11-22 and optionally SEQ ID NO: 13-14 for the promoter of DLIOX2 gene; SEQ ID NO: 16 - 17 and optionally SEQ ID NO: 18-19 for the promoter of LINC00682 gene; SEQ ID NO: 21-22 and optionally SEQ ID NO: 23-24 for the promoter of LMX1A gene; SEQ ID NO: 26-27 and optionally SEQ ID NO: 28-29 for the promoter of NKX2 gene; SEQ ID NO: 31-32 and optionally SEQ ID NO: 33-34 for the promoter of OSR1 gene; SEQ ID NO: 36-37 and optionally SEQ ID NO: 38-39 for the promoter of PDX1 gene; and SEQ ID NO: 41-42 and optionally SEQ ID NO: 43-44 for the promoter of

[0132] CLDN10 gene; and SEQ ID NO: 46-47 and optionally SEQ ID NO: 48-49 for the promoter of LY75 gene.

[0118] In another preferred embodiment, the specific probes are nucleic acid sequences that comprise or consists in SEQ ID NO: 5 for the promoter of NR5A2 gene; SEQ ID NO: 10 for the promoter of ASB18 gene; SEQ ID NO: 15 for the promoter of DUOX2 gene; SEQ ID NO: 20 for the promoter of LINC00682 gene; SEQ ID NO: 25 for the promoter of LMX1A gene; SEQ ID NO: 30 for the promoter of NKX2 gene; SEQ ID NO: 35 for the promoter of OSR1 gene; SEQ ID NO: 40 for the promoter of PDX1 gene; and SEQ ID NO: 45 for the promoter of CLDN10 gene; and SEQ ID NO: 50 for the promoter of LY75 gene.

[0133]

[0119] The nucleic acid sequences of the listed primers and probes in this description are to be understood as encompassing in some embodiments those functional primers and probes at least 80 % identical, or at least 85 % identical, or at least 90% identical, or at least 95 % identical, or 100% identical to the indicated SEQ ID NOs.

[0134]

[0120] As will be illustrated in the examples below, the inventors found reliable and highly specific markers for the determining of a likely outcome of a subject diagnosed or suspected of suffering melanoma.

[0135]

[0121] Therefore, and as previously indicated, another aspect of the invention is the use of a methylated gene promoter selected from methylated gene promoter of NR5A2 gene, methylated gene promoter of ASB18 gene, methylated gene promoter of DLIOX2 gene, methylated gene promoter of LINC00682 gene, methylated gene promoter of LMX1A gene, methylated gene promoter of NKX2 gene, methylated gene promoter of OSR1 gene, methylated gene promoter of PDX1 gene, methylated gene promoter of CLDN10 gene, and combinations thereof, as marker(s) of the prognosis of a subject diagnosed or suspected of suffering melanoma.

[0136]

[0122] The embodiments disclosed for any of the previous aspects of the invention, in relation to a method of prognosis of a subject diagnosed or suspected of suffering melanoma, and / or to a method for deciding or recommending to initiate a therapy for a subject diagnosed or suspected of suffering melanoma, do also apply to the other aspect of the invention and related with the use of the marker(s), or to any kit for the analysis of the methylation in the one or more of the promoters of the genes.

[0137]

[0123] In relation to the kit, it is advantageous to provide simplified kits that can be used if possible at ambulatory level or in the clinical labs in an easy and reliable mode.

[0138]

[0124] With this aim, the invention provides a kit that comprises one or more reagent means for determining methylation in a gene promoter selected from one or more of: the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene, and optionally reagent means for determining methylation in one or more gene promoters selected from: the promoter of LY75; the promoter of ABCC9 gene; the promoter of AKR1 B1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANXA2R gene; the promoter of AOX1 ; the promoter of ARAP2;-the promoter of ASCL2; the promoter of ATAD1 or CFL1 P1 ; the promoter of ATF7IP2; the promoter of BARHL1 ; the promoter of BTBD8; the promoter of CAPN14 or EHD3; the promoter of CARD11 ; the promoter of CAV3 or OXTR; the promoter of CD109; the promoter of CD8BP; the promoter of CDCA2; the promoter of CH17; the promoter of CLIN01305; the promoter of DGLIOK; the promoter of DIP2C; the promoter of DMRTA2; the promoter of DNAJC1 ; the promoter of EFS; the promoter of EGR3 gene; the promoter of EMX2 gene; the promoter of FADS6 gene; the promoter of FAM163B; the promoter of FAM218A or TRIM61 ; the promoter of FOXP2; the promoter of GNAS; the promoter of GRHL2; the promoter of H2AFJ; the promoter of HIC1 ; the promoter of HIST1 H2AG and / or HIST1 H2BJ; the promoter of HIST1 H2AI and / orHIST1 H2BL; the promoter of HMGB2; the promoter of INPPA5A; the promoter of INTS10; the promoter of ISLR2; the promoter of ITGA3; the promoter of K CNJ3; the promoter of KCTD21 ; the promoter of KIAA1217; the promoter of KIF13B; the promoter of KLF13; the promoter of KLHL; the promoter of KNJC3; the promoter of LBX2; the promoter of LINC00261 ; the promoter of LINC01230; the promoter of LINC01305; the promoter of LRRK1 ; the promoter of LRRN4; the promoter of LYL1 ; the promoter of MAD1 L1 ; the promoter of MAP9; the promoter of MFSD8; the promoter of NFIC; the promoter of NGB; the promoter of NODAL; the promoter of PARM1 ; the promoter of PAX2; the promoter of PCDHB10; the promoter of PCDHB2; the promoter of PCDHGA1 ; the promoter of PDPN; the promoter of PNPLA6; the promoter of PRDM13; the promoter of PRDM6; the promoter of PSMB5; the promoter of RFX4; the promoter of RHOD; the promoter of RPSAP58; the promoter of S1 PR5; the promoter of SCCPDH; the promoter of SCGB3A1 ; the promoter of SHISA9; the promoter of SLC30A2; the promoter of SMIM14; the promoter of SNX10; the promoter of SVIL; the promoter of TBC1 D30; the promoter of TBX15; the promoter of TH; the promoter of THBS1 ; the promoter of TNFSF11 ; the promoter of TRIM26; the promoter of TSPAN2; and the promoter of VSTM2B; and wherein the said one or more reagent means are the only means comprised in the kit for the determining of methylation in a gene promoter.

[0139]

[0125] The reagent means comprised in the kit for determining methylation in one or more gene promoter, comprise in an embodiment, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the total amount of reagents for assaying the said methylation.

[0140]

[0126] Therefore, in a preferred embodiment, the simplified kit comprises reagent means that consists only of reagent means for determining methylation in a gene promoter selected from one or more of: the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene.

[0141]

[0127] In another preferred embodiment, the kit comprises as only reagent means, those that consists of reagent means for determining methylation of the gene promoters of NR5A2 gene, of ASB18 gene, of DLIOX2 gene, of LINC00682 gene, of LMX1A gene, of NKX2 gene, of OSR1 gene, of PDX1 gene, and of CLDN10 gene.

[0142]

[0128] In also another embodiment of the kit of the invention, it comprises as only reagent means, those that consists of reagent means for determining methylation of:

[0143] (a) Promoter of LY75 gene and promoter of ASB18 gene; or

[0144] (b) Promoter of LY75 gene and promoter of DLIOX2 gene; or

[0145] (c) Promoter of LY75 gene and promoter of LINC00682 gene; or

[0146] (d) Promoter of LY75 gene and promoter of LMX1A gene; or

[0147] (e) Promoter of LY75 gene and promoter of NKX2 gene; or

[0148] (f) Promoter of LY75 gene and promoter of NR5A2 gene; or

[0149] (g) Promoter of LY75 gene and promoter of OSR1 gene; or

[0150] (h) Promoter of LY75 gene and promoter of PDX1 gene; or

[0151] (i) Promoter of LY75 gene and promoter of CLDN10 gene.

[0129] In another embodiment, the kit comprises as only reagent means, those that consist of reagent means for determining methylation of the gene promoter of LY75 gene, and for determining methylation of the gene promoters of NR5A2 gene, of ASB18 gene, of DLIOX2 gene, of LINC00682 gene, of LMX1A gene, of NKX2 gene, of OSR1 gene, of PDX1 gene, and of CLDN10 gene.

[0152]

[0130] In an embodiment of the kit of the invention, the one or more reagent means are selected from probes, nucleic acid (i.e., DNA, RNA, PNA) primers, aptamers, polymerases, buffers for the polymerization of nucleic acids, and nucleotides, preferably deoxi nucleotide triphosphates (dNTPs). Primers and aptamers in the kits are, preferably, for the specific amplification of at least the promoter regions of the genes of interest.

[0153]

[0131] The kits are adapted to comprise preferably as only reagent means those that are used for the methylation determination, and optionally for the quantification of the said methylation.

[0154]

[0132] They are in a preferred embodiment adapted to comprise the means for the carry out of a (multiplex) methylation-specific polymerase chain reaction analysis, more preferably a nested multiplex methylation-specific polymerase chain reaction.

[0155]

[0133] In another preferred embodiment, the kits are adapted for the analysis of the methylation by southern hybridization with methylation-sensitive endonucleases.

[0156]

[0134] Therefore, the kits comprise, in an embodiment, those reagents for carrying out a polymerase chain reaction and bisulfite-modified DNA (also named in this description as bisulfite DNA conversion, and known by the skilled person in the art).

[0157]

[0135] The skilled person in the art will be aware of the previously cited technologies for the determining of the DNA methylation.

[0158]

[0136] In an embodiment of the kit, the kit consists of probes, and nucleic acid primers, and optionally reagents for bisulfite DNA conversion for determining methylation, in a gene promoter selected from one or more of the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1 A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene, and optionally for determining methylation in one or more gene promoters selected from the promoter of LY75 gene; and wherein the probes, the nucleic acid primers, and optionally the reagents for bisulfite DNA conversion for determining methylation are preferably provided together in separate containers, said containers disposed in a common packaging and or common distribution means. The packaging may be any bag or bigger container, as well as any fasting means that hold together the containers with the primers, the containers with the probes and optionally the containers with the reagents for bisulfite DNA conversion for determining methylation.

[0159]

[0137] In a preferred embodiment, the kit consists of probes, and nucleic acid primers, and optionally reagents for bisulfite DNA conversion for determining methylation in two, three, four five, six, seven, eight and nine of the gene promoters.

[0160]

[0138] In a preferred embodiment of this kit that consists of probes, nucleic acid primers, and optionally reagents for bisulfite DNA conversion for determining methylation, the kit further comprises probes, and nucleic acid primers, and optionally reagents for bisulfite DNA conversion for determining methylation of LY75 gene promoter.

[0161]

[0139] In another preferred embodiment of this kit that consists of probes, nucleic acid primers, and optionally reagents for bisulfite DNA conversion for determining methylation, the kit further comprises probes, and nucleic acid primers, and optionally reagents for bisulfite DNA conversion for determining methylation in one or more gene promoters selected from the promoter of LY75 gene; the promoter of ABCC9 gene; the promoter of AKR1 B1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANXA2R gene; the promoter of AOX1 gene; the promoter of ARAP2; the promoter of ASCL2 gene; the promoter of ATAD1 or CFL1 P1 ; the promoter of ATF7IP2 gene; the promoter of BARHL1 gene; the promoter of BTBD8 gene; the promoter of CAPN14 or EHD3 gene; the promoter of CARD11 gene; the promoter of CAV3 or OXTR gene; the promoter of CD109 gene; the promoter of CD8BP gene; the promoter of CDCA2 gene; the promoter of CH17 gene; the promoter of CLIN01305 gene; the promoter of DGLIOK gene; the promoter of DIP2C gene; the promoter of DMRTA2 gene; the promoter of DNAJC1 gene; the promoter of EFS gene; the promoter of EGR3 gene; the promoter of EMX2 gene; the promoter of FADS6 gene; the promoter of FAM163B gene; the promoter of FAM218A or TRIM61 gene; the promoter of FOXP2 gene; the promoter of GNAS gene; the promoter of GRHL2 gene; the promoter of H2AFJ gene; the promoter of HIC1 gene; the promoter of HIST 1 H2AG gene and / or HIST1 H2BJ gene; the promoter of HIST1 H2AI gene and / orHIST1 H2BL gene; the promoter of HMGB2 gene; the promoter of INPPA5A gene; the promoter of INTS10 gene; the promoter of ISLR2 gene; the promoter of ITGA3 gene; the promoter of KCNJ3 gene; the promoter of KCTD21 gene; the promoter of KIAA1217 gene; the promoter of KIF13B gene; the promoter of KLF13 gene; the promoter of KLHL gene; the promoter of KNJC3 gene; the promoter of LBX2 gene; the promoter of LINC00261 gene; the promoter of LINC01230 gene; the promoter of LINC01305 gene; the promoter of LRRK1 gene; the promoter of LRRN4 gene; the promoter of LYL1 gene; the promoter of MAD1 L1 gene; the promoter of MAP9 gene; the promoter of MFSD8 gene; the promoter of NFIC gene; the promoter of NGB gene; the promoter of NODAL gene; the promoter of PARM1 gene; the promoter of PAX2 gene; the promoter of PCDHB10 gene; the promoter of PCDHB2 gene; the promoter of PCDHGA1 gene; the promoter of PDPN gene; the promoter of PNPLA6 gene; the promoter of PRDM13 gene; the promoter of PRDM6 gene; the promoter of PSMB5 gene; the promoter of RFX4 gene; the promoter of RHOD gene; the promoter of RPSAP58 gene; the promoter of S1 PR5 gene; the promoter of SCCPDH gene; the promoter of SCGB3A1 gene; the promoter of SHISA9 gene; the promoter of SLC30A2 gene; the promoter of SMIM14 gene; the promoter of SNX10 gene; the promoter of SVIL gene; the promoter of TBC1 D30 gene; the promoter of TBX15 gene; the promoter of TH gene; the promoter of THBS1 gene; the promoter of TNFSF11 gene; the promoter of TRIM26 gene; the promoter of TSPAN2 gene; and the promoter of VSTM2B gene.

[0162]

[0140] In another embodiment of the kit, the kit comprises, or consists of, one or more of:

[0163] (a) A set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 1 and 2, optionally a set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 3 and 4; and / or the nucleic acid probe of SEQ ID NO: 5;

[0164] (b) A set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 6 and 7, optionally a set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 8 and 9; and / or the nucleic acid probe of SEQ ID NO: 10

[0165] (c) A set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 11 and 12, optionally a set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 13 and 14; and / or the nucleic acid probe of SEQ ID NO: 15

[0166] (d) A set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 16 and 17, optionally a set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 18 and 19; and / or the nucleic acid probe of SEQ ID NO: 20

[0167] (e)A set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 21 and 22, optionally a set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 23 and 24; and / or the nucleic acid probe of SEQ ID NO: 25

[0168] (f) A set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 26 and 27, optionally a set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 28 and 29; and / or the nucleic acid probe of SEQ ID NO: 30

[0169] (g)A set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 31 and 32, optionally a set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 33 and 34; and / or the nucleic acid probe of SEQ ID NO: 35

[0170] (h)A set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 36 and 37, optionally a set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 38 and 39; and / or the nucleic acid probe of SEQ ID NO: 40

[0171] (i)A set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 41 and 42, optionally a set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 43 and 44; and / or the nucleic acid probe of SEQ ID NO: 45

[0172] (j)A set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 46 and 47, optionally a set of primers comprising, defined or which consist, respectively, of SEQ ID NO: 48 and 49; and / or the nucleic acid probe of SEQ ID NO: 50.

[0173]

[0141] In a preferred embodiment, the kit comprises, or consists of, two, three, four, five, six, seven, eight, nine or ten of the sets.

[0174]

[0142] In another embodiment of the kit, the kit comprises, or consists of, (a) one or more of a set(s) of primers and probes defined by or consisting of SEQ ID NOs: 1-45 in Table 3; and (b) a set(s) of primers and probes defined by or consisting of SEQ ID NOs: 46-50. These kits include the primers and probes for the determining of the methylation in one or more of the promoters of genes NR5A2, ASB18, DU0X2, LINC00682, LMX1A, NKX2, OSR1, and PDX1 and the primers and probes for the determining of the methylation in the promoter of LY75 gene.

[0175]

[0143] An example of the kit includes, thus, a set(s) of primers and probes defined by or consisting of SEQ ID NOs: 1-5, which allow the analysis of the methylation in promoter of gene NR5A2; and (b) a set(s) of primers and probes defined by or consisting of SEQ ID NOs: 46-50, which allow the analysis of the methylation in promoter of gene LY75. Other examples are directly derivable from the combination of the primers and probes for the other genes indicated in Table 3 with LY75 gene.

[0176]

[0144] In yet another embodiment of the kit that consists of probes, and nucleic acid primers, and optionally reagents for bisulfite DNA conversion for determining methylation, the kit further comprises, preferably in separate containers, one or more of polymerases, buffers for the polymerization of nucleic acids, and nucleotides, preferably deoxi nucleotide triphosphates. In a particular embodiment of the kit according to the invention, the kit further comprises instructions for its use and / or for the carrying out of any of the in vitro method for the prognosis of melanoma, or of the in vitro method of deciding or recommending initiating a therapy for a subject diagnosed or suspected of suffering melanoma, as previously disclosed.

[0177]

[0145] The kit may comprise one or more of the reagents in a solid support and each reagent in a separate container. These kits comprise all what is needed to carry out the methylation analysis in the commonly used equipment for the PCR analysis or for a southern blot analysis.

[0178]

[0146] The kit of the previous aspect is thus a tool to carry out the methods of the invention as previously disclosed.

[0179]

[0147] Therefore, another aspect of the invention relates to the use of a kit for the prognosis of a subject diagnosed or suspected of suffering melanoma, wherein the kit is as defined above and comprises one or more reagent means for determining methylation in a gene promoter selected from one or more of: the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene, and optionally reagent means for determining methylation in one or more gene promoters selected from: the promoter of LY75; the promoter of ABCC9 gene; the promoter of AKR1 B1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANXA2R gene; the promoter of AOX1 ; the promoter of ARAP2;-the promoter of ASCL2; the promoter of ATAD1 or CFL1 P1 ; the promoter of ATF7IP2; the promoter of BARHL1 ; the promoter of BTBD8; the promoter of CAPN14 or EHD3; the promoter of CARD11 ; the promoter of CAV3 or OXTR; the promoter of CD109; the promoter of CD8BP; the promoter of CDCA2; the promoter of CH17; the promoter of CLIN01305; the promoter of DGLIOK; the promoter of DIP2C; the promoter of DMRTA2; the promoter of DNAJC1 ; the promoter of EFS; the promoter of EGR3 gene; the promoter of EMX2 gene; the promoter of FADS6 gene; the promoter of FAM163B; the promoter of FAM218A or TRIM61 ; the promoter of FOXP2; the promoter of GNAS; the promoter of GRHL2; the promoter of H2AFJ; the promoter of HIC1 ; the promoter of HIST1 H2AG and / or HIST1 H2BJ; the promoter of HIST1 H2AI and / orHIST1 H2BL; the promoter of HMGB2; the promoter of INPPA5A; the promoter of INTS10; the promoter of ISLR2; the promoter of ITGA3; the promoter of K CNJ3; the promoter of KCTD21 ; the promoter of KIAA1217; the promoter of KIF13B; the promoter of KLF13; the promoter of KLHL; the promoter of KNJC3; the promoter of LBX2; the promoter of LINC00261 ; the promoter of LINC01230; the promoter of LINC01305; the promoter of LRRK1 ; the promoter of LRRN4; the promoter of LYL1 ; the promoter of MAD1 L1 ; the promoter of MAP9; the promoter of MFSD8; the promoter of NFIC; the promoter of NGB; the promoter of NODAL; the promoter of PARM1 ; the promoter of PAX2; the promoter of PCDHB10; the promoter of PCDHB2; the promoter of PCDHGA1 ; the promoter of PDPN;; the promoter of PNPLA6; the promoter of PRDM13; the promoter of PRDM6; the promoter of PSMB5; the promoter of RFX4; the promoter of RHOD; the promoter of RPSAP58; the promoter of S1 PR5; the promoter of SCCPDH; the promoter of SCGB3A1 ; the promoter of SHISA9; the promoter of SLC30A2; the promoter of SMIM14; the promoter of SNX10; the promoter of SVIL; the promoter of TBC1 D30; the promoter of TBX15; the promoter of TH; the promoter of THBS1 ; the promoter of TNFSF11 ; the promoter of TRIM26; the promoter of TSPAN2; and the promoter of VSTM2B; and wherein the said one or more reagent means are the only means comprised in the kit for the determining of methylation in a gene promoter.

[0180]

[0148] Particular reagents have been listed previously when referred to the kit of the invention.

[0181]

[0149] According to aspects and embodiments of the invention, diagnosis and prognosis of melanoma can be performed using a mathematical algorithm that assesses directly or indirectly a detectable level of methylation in one or more of the promoters of the genes previously listed, either in conjunction with or independent of other clinical and / or pathological parameters and / or features of the subject, to correctly categorize an individual sample as originating from a healthy patient, or from a particular stage of melanoma. In the sense of the invention, the term "algorithm" is also synonymous of panel or decision diagrams, predictors and combinatory of data to correctly categorize an individual sample.

[0182]

[0150] The classification algorithm may be as simple as determining whether or not methylation is present and / or which amount of methylation is present. When multiple biomarkers as the ones of the sets listed above are used, the classification algorithm may be a linear regression formula. Alternatively, the classification algorithm may be the product of any of a number of learning algorithms. In the case of complex classification algorithms, it may be necessary to perform the algorithm on the data, thereby determining the classification, using a computer, e.g., a programmable digital computer. In either case, one can then record the status on tangible medium, for example, in computer-readable format such as a memory drive or disk or simply printed on paper. The result also could be reported on a computer screen. This algorithm is used as prognostic (and if not previously diagnosed, also as diagnostic) method, and is in particular part of the kits for carrying out the methods disclosed in former aspects.

[0183]

[0151] After the determination of the presence (and optionally amount) of methylation for the prognosis of melanoma in function of the said presence / amount, score and / or computed value, a classification of the sample (from the subject) is done as a sample with a particular prognostic. The classification is optionally carried out in combination with the one or more of the clinical and / or pathological parameters of the subject, which can also be given a score and / or computed value.

[0152] The clinical and / or pathological parameters and / or features of the subject that are assessed, in an embodiment, with the presence and optionally level of methylation in the classification algorithm include, among others one or more of sex, age at diagnosis, Melanoma subtype, Localization primary tumor, Lateralization primary tumor, Morphology, Clark level, Breslow thickness, Ulceration, Regression, Tumorinfiltrating lymphocytes , Microsatellites, Lymphangioinvasion, Perineural growth, Preexistent nevi, Mitosis, and TNM stage.

[0184]

[0153] The in vitro methods of the invention provide prognostic information. In one embodiment, the methods of the invention further comprise the steps of (i) collecting the diagnostic and / or prognostic information, and (ii) saving the information in a data carrier.

[0185]

[0154] In the sense of the invention a "data carrier" is to be understood as any means that contain meaningful information data for the prognosis of melanoma, such as paper. For example, the carrier may comprise a storage medium, such as a ROM, for example a CD ROM or a semiconductor ROM, or a magnetic recording medium, for example a floppy disc or hard disk. Further, the carrier may be a transmissible carrier such as an electrical or optical signal, which may be conveyed via electrical or optical cable or by radio or other means. When the diagnosis / prognosis data are embodied in a signal that may be conveyed directly by a cable or other device or means, the carrier may be constituted by such cable or other device or means. Other carriers relate to USB devices and computer archives. Examples of suitable data carrier are paper, CDs, USB, computer archives in PCs, or sound registration with the same information.

[0186]

[0155] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and / or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[0187]

[0156] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

[0188]

[0157] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

[0189]

[0158] It will be understood that all details, embodiments, and preferences discussed with respect to one aspect of embodiment of the invention is likewise applicable to any other aspect or embodiment of the invention and that there is therefore not need to detail all such details, embodiments, and preferences for all aspect separately.

[0190]

[0159] Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention. Further aspects and embodiments will be apparent to those skilled in the art.

[0191] EXAMPLES

[0192]

[0160] Example 1. New markers for the prognosis of melanoma samples

[0193]

[0161] Marker identification

[0194]

[0162] An In silico analyis using public methylome data from The Cancer Genome Atlas (TCGA) to identify prognostic melanoma methylation markers that, in combination with LY75, could better predict melanoma prognosis was performed. This in silico analysis was performed using the Skin cutaneous melanoma (SKCM) dataset within TCGA, which contained Illumina lnfinium-450K array methylation data of 104 primary melanoma tissue samples. To investigate the prognostic value of a DNA methylation, Cox proportional-hazards regression models were created for all 396,065 available Infinium probes, using the coxph function from the R package survival. In addition, the primary tumor samples were split into two groups, one with no methylation and one with methylation, using the median beta value for each probe as a cutoff, and plotted the corresponding Kaplan-Meier survival curves of both groups, using the survfit (survival package) and ggsurvplot (survminer package) functions. For each of the analyses, p values were corrected for multiple hypothesis testing using the Benjamini-Hochberg method, which controls the false discovery rate (FDR). This approach yielded a list of candidate biomarkers in which DNA methylation was correlated to poor survival.

[0195]

[0163] Patient samples

[0196]

[0164] Promoter CpG island methylation was examined in a series of formalin-fixed, paraffin-embedded (FFPE) tissues. This series consisted of primary melanomas (n=47) of adult patients undergoing surgical resection without adjuvant treatment at the Maastricht University Medical Centre, The Netherlands or University Hospital Leuven, Belgium. All samples were collected between 1983 and 2010 and retrospectively collected from the respective pathology archives, with a median followup of 62.0 months (range 1-225 months). Collection, storage and use of all tissues and patient data were performed in agreement with the ‘Code for Proper Secondary Use of Human Tissue in the Netherlands’, and approved by the Maastricht Pathology Tissue Collection scientific committee and the Medical Ethical Committee of Maastricht University Medical Center (2021-2575 / 2022-3520).

[0197] Detailed clinicopathological information of this melanoma series is shown in Table 2.

[0198] Table 2. Clinicopathological characteristics of 47 melanoma patients with follow-up

[0199] ALM; acral lentiginous melanoma, FU; follow-up, LMM; lentigo malignant melanoma, mm; millimeter, NMM; nevoid malignant melanoma, SSMM; superficial spreading malignant melanoma, SD; standard deviation, TILs; tumor-infiltrating lymphocytes, TTM; time to metastasis

[0200]

[0165] Tissue sample collection and preparation

[0201]

[0166] FFPE samples were cut into 5-10 pm thick sections using a microtome and transferred onto microscope slides. The slides were incubated overnight at 56°C for adhesion of the tissue to the slide. Hematoxylin-eosin (H&E) stained slides were made for each sample, and the tumor tissue and percentage were indicated by a dermatopathologist. Only samples that exceeded 50% melanoma were included.

[0202]

[0167] DNA isolation and bisulfite conversion

[0203]

[0168] Prior to the DNA isolation process, 5 to 10 FFPE tissue sections were deparaffinized using a deparaffination series of 2x5 minutes Xylene, 2x3 minutes 100% EtOH, 1 minute 96% EtOH and 1 minute 70% EtOH. The tissue was subsequently lysed in a sterile 1.5 mL Eppendorf tube containing 180 pL ATL buffer and 20 pL proteinase K. DNA was isolated using the QIAamp DNA mini kit (Qiagen) according to the manufacturer’s protocol. DNA concentrations and purity were measured using Nanodrop (Thermo Scientific) and Quantus (Promega).

[0204]

[0169] Sodium bisulfite conversion of isolated DNA was performed using the Epitect Bisulfite Kit (Qiagen). A desired input of 100 ng in 20 pl per sample was maintained, and the samples were diluted in sterile water when necessary. DNA was bisulfite converted according to the manufacturer’s instructions.

[0205]

[0170] Nested qMSP

[0206]

[0171] Using nested qMSP, the DNA methylation status of the candidate biomarkers was measured. For the pre-amplification PCR, two separate flank mixes were prepared of which each mix contained primer sets for 5 candidate markers. Per sample, a reaction mix of 14.65 pL sterile water, 2.5 pL Magic buffer, 1.25 pL deoxynucleotide triphosphates (dNTPs) (GE Healthcare), 1.25 pL forward flank mix primer, 1.25 pL reverse flank mix primer and 0.1 pL Immolase Taq (GC biotech) was prepared. To perform the amplification, 4 pL of the bisulfite converted sample was added to 21 pL reaction mix. The amplification was performed with a thermal cycler (3 minutes at 95°C, 30 seconds at 95°C, 30 seconds at 56°C, 30 seconds at 72°C, previous three steps were repeated 35 times, 4 minutes at 72°C) (Bio-Rad). The preamplification PCR product was diluted 1 : 1000.

[0207]

[0172] The second step was qMSP using a TaqMan probe. For qMSP on pre-amplified DNA, reaction mix was prepared by combining 15.3 pL sterile water, 2.5 pL Magic buffer, 1 pL dNTPs, 0.75 pL pM 10 forward primer for gene of interest, 0.75 pL 10 pM reverse primer for gene of interest, 0.5 pL 5 pM probe for gene of interest, and 0.2 pL HS Taq (Bioline) per sample. Per 21 pL of reaction mix, 4 pL of the diluted sample was added. Amplification was executed using a quantitative thermal-cycler (3 minutes at 95 °C, 15 seconds at 95°C and 30 seconds at 60°C, previous two steps were repeated 40 times) (Bio-Rad). In vitro methylated DNA (IVD) was used as a positive control, and a commercially available unmethylated control (Millipore) was used as a negative control, as well as no template controls (NTC) in all steps of the process. All nested qMSP experiments were performed in triplo. The combination of primers and probes for each gene promoter allowed the method of the invention to be applied in any diagnostics lab for a fast and reliable output of the results.

[0208]

[0173] Next Table 3 includes the primers and probes used for the determining of methylation in the promoters of the genes:

[0209]

[0174] Data analysis

[0175] Receiver operating characteristic (ROC) curves with corresponding area under the curve (AUC) and 95% confidence intervals (95%-CI) were created to assess the discriminative ability of the candidate markers. A cutoff value for each marker was determined based on the highest positive likelihood ratio (LR). A sample was considered methylated when its Ct-value (Cycle threshold value) was below the predetermined cutoff. Combinations of a candidate biomarker with LY75 were tested to further improve the prognostic value of the existent LY75 methylation test. Such a combination test was considered positive in case one or both markers were methylated. Next Table 4 shows the Ct-value cutoffs for analyzed genes, which promoter methylation correlates with the prognosis of melanoma in the assayed cohort of patients.

[0210]

[0176] Table 4. Ct-value cutoffs

[0177] The outcome measure of the survival analyses was recurrence-free survival, defined as the time from cancer diagnosis until diagnosis of metastatic disease or end of follow-up. Kaplan-Meier analyses with Log Rank tests and Cox Proportional Hazard analyses were used to evaluate the association between promoter DNA methylation and recurrence-free survival, resulting in hazard ratios (HRs) and their corresponding 95%-CI.

[0211]

[0178] All statistical analyses were performed using Graphpad Prism and SPSS, and were considered significant when p<0.05.

[0212]

[0179] The inventors have measured the DNA methylation in the promoters of the abovementioned genes (i.e., biomarkers) in a first series of 47 samples. The Figures 1-9, which illustrate Kaplan-Meier curves including Log-Rank p-values are shown for each marker separate, as well as the combination with LY75. In addition, the table below (Table 5) provides the results of Cox-proportional hazard analyses; the Hazard

[0213] Ratio with corresponding 95% Confidence Interval and p-values are displayed.

[0214]

[0180] Table 5.

[0181] It can be concluded from the herewith provided data, that the methylation status in one or more of the gene promoter sequences of ASB18, DU0X2, LINC00682, LMX1A, NKX2, NR5A2, 0SR1, PDX1, CLDN10 genes has prognostic value on its own (all 9 biomarkers are prognostic by themselves and in combination with each other), but can also be used to further improve the prognostic value of the LY75 test (MLADx) by adding these markers and creating a panel of markers.

[0215]

[0182] Example 2. Validation cohort

[0216]

[0183] The results of the previous example were validated in a different cohort (n=145), using the same materials and methodologies as previously disclosed. The validation cohort was an independent study population consisting of 145 primary melanoma patients.

[0217]

[0184] Next Table 6 shows a detailed clinicopathological information of the subjects.

[0218] Table 6. Patient characteristics

[0219]

[0185] Data analysis

[0220]

[0186] As in Example 1 , receiver operating characteristic (ROC) curves with corresponding area under the curve (AUC) and 95% confidence intervals (95%-CI) were created to assess the discriminative ability of the candidate markers. A cutoff value for each marker was determined based on the highest positive likelihood ratio (LR). A sample was considered methylated when its methylation value was higher than the predetermined cutoff. Combinations of a candidate biomarker with LY75 were tested to further improve the prognostic value of the existent LY75 methylation test. Such a combination test was considered positive in case one or both markers were methylated. Next Table 7 shows the Ct-value cutoffs for analyzed genes, which promoter methylation correlates with the prognosis of melanoma in the assayed cohort of patients. Methylation in NKX2 gene promoter was also analyzed, but due to an experimental issue data could not be derived

[0221]

[0187] Table 7. Ct-value cutoffs

[0222]

[0188] As in Example 1 the outcome measure of the survival analyses was recurrence-free survival, defined as the time from cancer diagnosis until diagnosis of metastatic disease or end of follow-up. Kaplan-Meier analyses with Log Rank tests and Cox Proportional Hazard analyses were used to evaluate the association between promoter DNA methylation and recurrence-free survival, resulting in hazard ratios (HRs) and their corresponding 95%-CI.

[0223]

[0189] All statistical analyses were performed using Graphpad Prism and SPSS, and were considered significant when p<0.05.

[0224]

[0190] The inventors have measured the DNA methylation in the promoters of the abovementioned genes (i.e., biomarkers) in a second series of 145 samples. The Figures 10 - 17, which illustrate Kaplan-Meier curves including Log-Rank p-values are shown for each marker separate, as well as the combination with LY75. In addition, the table below (Table 8) provides the results of Cox-proportional hazard analyses; the Hazard Ratio with corresponding 95% Confidence Interval and p-values are displayed. This table also includes the Hazard Ratio with corresponding 95% Confidence Interval and p-value of a ternary combination, i.e., DUOX2 and / or LMX1A and / or NR5A2.

[0225]

[0191] Table 8. Cox-proportional hazard analysis

[0226]

[0192] Further, the inventors have analyzed other combinations. The ternary combination DLIOX2, LMX1A and NR5A2 resulted in the following Univariate Cox proportional hazards analysis:

[0227]

[0193]

[0228]

[0194] The data of the validation cohort allow to conclude and confirm that the methylation status in one or more of the gene promoter sequences of ASB18, DU0X2, UNC00682, LMX1A, NR5A2, 0SR1, PDX1, CLDN10 genes has prognostic value on its own (all biomarkers are prognostic by themselves and in combination with each other), but can also be used to further improve the prognostic value of the LY75 test (MLADx) by adding these markers and creating a panel of markers.

[0229]

[0195] Although most of the combinations have been exemplified with LY75, the herewith provided HR values for each of the genes makes any combination of any of them in a panel of al least two, three, four, five, six, seven, eight, nine or then of them useful sets in an invitro method for the prognosis of a subject diagnosed or suspected of suffering melanoma.

[0230]

[0196] Further, the analysis of the methylation status of ternary combinations comprising three from ASB18, DUOX2, LINC00682, LMX1A, NKX2, NR5A2, OSR1 , PDX1 , and CLDN10 are also advantageous, as illustrated with the results from the ternary combination including the analysis of the methylation of the promoters of the genes DU0X2 and / or LMX1 A and / or NR5A2, where, as indicated in Table 8 and illustrated in Figure 18, if one or more (>1) of these 3 genes is methylated, the test is positive (N=145, 17 events (=any recurrence)).

[0231]

[0197] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

[0232]

[0198] Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description, or embodiment of the present invention is disclosed, taught, or suggested in the relevant art.

Claims

CLAIMS1. An in vitro method for the prognosis of a subject diagnosed or suspected of suffering melanoma, the method comprising the step of determining in an isolated sample of the subject whether one or more methylation(s) are present in one or more gene promoter sequence(s) selected from the group consisting of the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene; and wherein, if it is determined that one or more methylation(s) is / are present in the one or more gene promoters, the subject is classified with a prognosis of a shorter life expectancy in relation to a subject that does not comprises the one or more methylations, and / or the subject is classified with a prognosis of recurrence and / or prognosis of metastasis.

2. The in vitro method according to claim 1 , wherein the sample is a biopsy melanoma sample selected from a biopsy of a cutaneous melanoma tumor, and / or a metastatic biopsy sample of the melanoma, preferably a sentinel lymph node biopsy (SLNB); or is a liquid biopsy.

3. The in vitro method according to any one of claims 1-2, wherein an additional step is performed selected / selecting data from the group consisting of determining tumor ulceration, determining metastatic disease at diagnosis, disease stage, determining Breslow thickness of the melanoma, determining melanoma subtype, determining localization of primary tumor, determining lateralization of primary tumor, determining morphology of tumor, determining Clark level, determining regression, determining tumor-infiltrating lymphocytes, determining microsatellites in tumor, determining lymphangioinvasion, determining perineural growth, determining pre-existent nevi, determining mitosis, tumor / node / metastasis (TNM) stage, determining age and / or sex of the subject, and combinations thereof.

4. The in vitro method according to any one of claims 1-3, wherein the method further comprises determining in the isolated sample whether one or more methylation(s) is / are present in one or more gene promoters selected from: the promoter of LY75 gene; the promoter of ABCC9 gene; the promoter of AKR1 B1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANXA2R gene; the promoter of AOX1 gene; the promoter of ARAP2; the promoter of ASCL2 gene; the promoter of ATAD1 or CFL1 P1 ; the promoter of ATF7IP2 gene; the promoter of BARHL1 gene; the promoter of BTBD8 gene; the promoter of CAPN14 or EHD3 gene; the promoter of CARD11 gene; the promoter of CAV3 or OXTR gene; the promoter of CD109 gene; the promoter of CD8BP gene; the promoter of CDCA2 gene; the promoter of CH17 gene; the promoter of CLIN01305 gene; the promoter of DGLIOK gene; the promoter of DIP2C gene; the promoter of DMRTA2 gene; the promoter of DNAJC1 gene; the promoter of EFS gene; the promoter of EGR3 gene; the promoter of EMX2 gene; the promoter of FADS6 gene; the promoter of FAM163B gene; the promoter of FAM218A or TRIM61 gene; the promoter of FOXP2 gene; the promoter of GNAS gene; the promoter of GRHL2 gene; the promoter of H2AFJ gene; the promoter of HIC1 gene; the promoter of HIST1 H2AG gene and / or HIST1 H2BJ gene; the promoter of HIST1 H2AI gene and / orHIST1 H2BL gene; the promoter of HMGB2 gene; the promoter of INPPA5A gene; the promoter of INTS10 gene; the promoter of ISLR2 gene; the promoter of ITGA3 gene; the promoter of KCNJ3 gene; the promoter of KCTD21 gene; the promoter of KIAA1217 gene; the promoter of KIF13B gene; the promoter of KLF13 gene; the promoter of KLHL gene; the promoter of KNJC3 gene; the promoter of LBX2 gene; the promoter of LINC00261 gene; the promoter of LINC01230 gene; the promoter of LINC01305 gene; the promoter of LRRK1 gene; the promoter of LRRN4 gene; the promoter of LYL1 gene; the promoter of MAD1 L1 gene; the promoter of MAP9 gene; the promoter of MFSD8 gene; the promoter of NFIC gene; the promoter of NGB gene; the promoter of NODAL gene; the promoter of PARM1 gene; the promoter of PAX2 gene; the promoter of PCDHB10 gene; the promoter of PCDHB2 gene; the promoter of PCDHGA1 gene; the promoter of PDPN gene; the promoter of PNPLA6 gene; the promoter of PRDM13 gene; the promoter of PRDM6 gene;the promoter of PSMB5 gene; the promoter of RFX4 gene; the promoter of RHOD gene; the promoter of RPSAP58 gene; the promoter of S1 PR5 gene; the promoter of SCCPDH gene; the promoter of SCGB3A1 gene; the promoter of SHISA9 gene; the promoter of SLC30A2 gene; the promoter of SMIM14 gene; the promoter of SNX10 gene; the promoter of SVIL gene; the promoter of TBC1 D30 gene; the promoter of TBX15 gene; the promoter of TH gene; the promoter of THBS1 gene; the promoter of TNFSF11 gene; the promoter of TRIM26 gene; the promoter of TSPAN2 gene; and the promoter of VSTM2B gene.

5. The in vitro method according to any one of claims 1-4, wherein the method comprises determining whether one, two, three, four, five, six, seven, eight and nine gene promoter(s) comprise(s) one or more methylation(s), preferably wherein the method comprises determining whether one or more methylation(s) is / are present in the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene.

6. The in vitro method according to any one of claims 1-5, wherein the method comprises determining whether one or more methylation(s) is / are present in a set of gene promoters selected from the group consisting of: a) Promoter of LY75 gene and promoter of ASB18 gene; b) Promoter of LY75 gene and promoter of DLIOX2 gene; c) Promoter of LY75 gene and promoter of LINC00682 gene; d) Promoter of LY75 gene and promoter of LMX1A gene; e) Promoter of LY75 gene and promoter of NKX2 gene; f) Promoter of LY75 gene and promoter of NR5A2 gene; g) Promoter of LY75 gene and promoter of OSR1 gene; h) Promoter of LY75 gene and promoter of PDX1 gene; and i) Promoter of LY75 gene and promoter of CLDN10 gene.

7. The in vitro method according to any one of claims 1 - 6, wherein the determining in an isolated sample from the subject whether one or more methylation(s) is / are present in one or more gene promoter, is performed by one or more of a (multiplex) methylation-specific polymerase chain reaction analysis, preferably by a nested multiplex methylation-specific polymerase chain reaction; a southern hybridization with methylation-sensitive endonucleases; a whole genome / targeted / nanopore bisulfite sequencing; pyrosequencing; epityper; methylation-Sensitive Restriction Enzymes (MSRE); methylated DNA Immunoprecipitation (MeDIP); methyl-CpG Binding Domain (MBD) Capture; Illumina HumanMethylation450 BeadChip (450K Array); Illumina Infinium Methylation EPIC Array (850K Array); Twist Bioscience's NGS; and Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA).

8. The method according to any one of claims 1-7, wherein after the determination whether one or more methylation(s) are present in a gene promoter, the following computer-implemented steps are carried out: said methylation(s) are given a value and / or a score, and optionally are computed in a mathematical formula to obtain a computed value; wherein in function of the said level(s), score(s) and or computed value(s), a decision is taken about the prognosis, and / or decision of recommending initiating a treatment.

9. A method of deciding or recommending to initiate a therapy for a subject diagnosed or suspected of suffering melanoma, the method comprising:(a) carrying out the in vitro method for the prognosis as defined in any one of claims 1-8; and(b) if the subject is classified with a prognosis of a shorter life expectancy in relation to a subject that does not comprises the one or more methylations, then initiation of a treatment is recommended, preferably where the treatment comprises adjuvant therapy.

10. Use of a methylated gene promoter selected from methylated gene promoter ofNR5A2 gene, methylated gene promoter of ASB18 gene, methylated genepromoter of DLIOX2 gene, methylated gene promoter of LINC00682 gene, methylated gene promoter of LMX1A gene, methylated gene promoter of NKX2 gene, methylated gene promoter of OSR1 gene, methylated gene promoter of PDX1 gene, methylated gene promoter of CLDN10 gene, and combinations thereof, as marker(s) of the prognosis of a subject diagnosed or suspected of suffering melanoma.

11. A kit that comprises one or more reagent means for determining methylation in a gene promoter selected from one or more of the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene, and optionally reagent means for determining methylation in one or more gene promoters selected from the promoter of LY75 gene; the promoter of ABCC9 gene; the promoter of AKR1 B1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANXA2R gene; the promoter of AOX1 gene; the promoter of ARAP2;-the promoter of ASCL2 gene; the promoter of ATAD1 or CFL1 P1 ; the promoter of ATF7IP2 gene; the promoter of BARHL1 gene; the promoter of BTBD8 gene; the promoter of CAPN14 or EHD3 gene; the promoter of CARD11 gene; the promoter of CAV3 or OXTR gene; the promoter of CD109 gene; the promoter of CD8BP gene; the promoter of CDCA2 gene; the promoter of CH17 gene; the promoter of CLINCH 305 gene; the promoter of DGLIOK gene; the promoter of DIP2C gene; the promoter of DMRTA2 gene; the promoter of DNAJC1 gene; the promoter of EFS gene; the promoter of EGR3 gene; the promoter of EMX2 gene; the promoter of FADS6 gene; the promoter of FAM163B gene; the promoter of FAM218A or TRIM61 gene; the promoter of FOXP2 gene; the promoter of GNAS gene; the promoter of GRHL2 gene; the promoter of H2AFJ gene; the promoter of HIC1 gene; the promoter of HIST1 H2AG gene and / or HIST1 H2BJ gene; the promoter of HIST1 H2AI gene and / orHIST1 H2BL gene; the promoter of HMGB2 gene; the promoter of INPPA5A gene; the promoter of INTS10 gene; the promoter of ISLR2 gene; the promoter of ITGA3 gene; the promoter of KCNJ3 gene; the promoter of KCTD21 gene; the promoter ofKIAA1217 gene; the promoter of KIF13B gene; the promoter of KLF13 gene; the promoter of KLHL gene; the promoter of KNJC3 gene; the promoter of LBX2 gene; the promoter of LINC00261 gene; the promoter of LINC01230 gene; the promoter of LINC01305 gene; the promoter of LRRK1 gene; the promoter of LRRN4 gene; the promoter of LYL1 gene; the promoter of MAD1 L1 gene; the promoter of MAP9 gene; the promoter of MFSD8 gene; the promoter of NFIC gene; the promoter of NGB gene; the promoter of NODAL gene; the promoter of PARM1 gene; the promoter of PAX2 gene; the promoter of PCDHB10 gene; the promoter of PCDHB2 gene; the promoter of PCDHGA1 gene; the promoter of PDPN gene; the promoter of PNPLA6 gene; the promoter of PRDM13 gene; the promoter of PRDM6 gene; the promoter of PSMB5 gene; the promoter of RFX4 gene; the promoter of RHOD gene; the promoter of RPSAP58 gene; the promoter of S1 PR5 gene; the promoter of SCCPDH gene; the promoter of SCGB3A1 gene; the promoter of SHISA9 gene; the promoter of SLC30A2 gene; the promoter of SMIM14 gene; the promoter of SNX10 gene; the promoter of SVIL gene; the promoter of TBC1 D30 gene; the promoter of TBX15 gene; the promoter of TH gene; the promoter of THBS1 gene; the promoter of TNFSF11 gene; the promoter of TRIM26 gene; the promoter of TSPAN2 gene; and the promoter of VSTM2B gene; and wherein the said one or more reagent means are the only means comprised in the kit for the determining of methylation in a gene promoter.

12. The kit according to claim 11 , wherein the reagent means comprised in the kit for determining methylation in one or more gene promoter, comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the total amount of reagents for assaying the said methylation.

13. The kit according to any one of claims 11-12, which comprises reagent means that consist only of reagent means for determining methylation in a gene promoter selected from one or more of: the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoterof LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene.

14. The kit according to any one of claims 11-13, which comprises as only reagent means, those that consists of reagent means for determining methylation of:(a) Promoter of LY75 gene and promoter of ASB18 gene; or(b) Promoter of LY75 gene and promoter of DLIOX2 gene; or(c) Promoter of LY75 gene and promoter of LINC00682 gene; or(d) Promoter of LY75 gene and promoter of LMX1A gene; or(e) Promoter of LY75 gene and promoter of NKX2 gene; or(f) Promoter of LY75 gene and promoter of NR5A2 gene; or(g) Promoter of LY75 gene and promoter of OSR1 gene; or(h) Promoter of LY75 gene and promoter of PDX1 gene; or(i) Promoter of LY75 gene and promoter of CLDN10 gene.

15. The kit according to any one of claims 11-14, which comprises as only reagent means, those that consist of reagent means for determining methylation of the gene promoter of LY75 gene, and for determining methylation of the gene promoters of NR5A2 gene, of ASB18 gene, of DLIOX2 gene, of LINC00682 gene, of LMX1A gene, of NKX2 gene, of OSR1 gene, of PDX1 gene, and of CLDN10 gene.

16. The kit according to any one of claims 11 - 15, wherein the one or more reagent means are selected from probes, nucleic acid primers, aptamers, polymerases, buffers for the polymerization of nucleic acids, and nucleotides, preferably deoxi nucleotide triphosphates.

17. The kit according to any one of claims 11 - 16, which consists of probes, nucleic acid primers, and optionally reagents for bisulfite DNA conversion, for determining methylation in a gene promoter selected from one or more of the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DLIOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10gene, and optionally for determining methylation in one or more gene promoters selected from the promoter of LY75 gene; the promoter of ABCC9 gene; the promoter of AKR1 B1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANAPC1 P1 gene; the promoter of ANXA2R gene; the promoter of AOX1 gene; the promoter of ARAP2;-the promoter of ASCL2 gene; the promoter of ATAD1 or CFL1 P1 ; the promoter of ATF7IP2 gene; the promoter of BARHL1 gene; the promoter of BTBD8 gene; the promoter of CAPN14 or EHD3 gene; the promoter of CARD1 1 gene; the promoter of CAV3 or OXTR gene; the promoter of CD109 gene; the promoter of CD8BP gene; the promoter of CDCA2 gene; the promoter of CH17 gene; the promoter of CLIN01305 gene; the promoter of DGLIOK gene; the promoter of DIP2C gene; the promoter of DMRTA2 gene; the promoter of DNAJC1 gene; the promoter of EFS gene; the promoter of EGR3 gene; the promoter of EMX2 gene; the promoter of FADS6 gene; the promoter of FAM163B gene; the promoter of FAM218A or TRIM61 gene; the promoter of FOXP2 gene; the promoter of GNAS gene; the promoter of GRHL2 gene; the promoter of H2AFJ gene; the promoter of HIC1 gene; the promoter of HIST1 H2AG gene and / or HIST1 H2BJ gene; the promoter of HIST1 H2AI gene and / orHIST1 H2BL gene; the promoter of HMGB2 gene; the promoter of INPPA5A gene; the promoter of INTS10 gene; the promoter of ISLR2 gene; the promoter of ITGA3 gene; the promoter of KCNJ3 gene; the promoter of KCTD21 gene; the promoter of KIAA1217 gene; the promoter of KIF13B gene; the promoter of KLF13 gene; the promoter of KLHL gene; the promoter of KNJC3 gene; the promoter of LBX2 gene; the promoter of LINC00261 gene; the promoter of LINC01230 gene; the promoter of LINC01305 gene; the promoter of LRRK1 gene; the promoter of LRRN4 gene; the promoter of LYL1 gene; the promoter of MAD1 L1 gene; the promoter of MAP9 gene; the promoter of MFSD8 gene; the promoter of NFIC gene; the promoter of NGB gene; the promoter of NODAL gene; the promoter of PARM1 gene; the promoter of PAX2 gene; the promoter of PCDHB10 gene; the promoter of PCDHB2 gene; the promoter of PCDHGA1 gene; the promoter of PDPN gene; the promoter of PNPLA6 gene; the promoter of PRDM13 gene; the promoter of PRDM6 gene; the promoter of PSMB5 gene; the promoter of RFX4 gene; the promoter of RHOD gene; the promoter of RPSAP58 gene; the promoter of S1 PR5 gene; the promoter of SCCPDH gene; the promoter of SCGB3A1 gene; the promoter of SHISA9 gene; the promoter ofSLC30A2 gene; the promoter of SMIM14 gene; the promoter of SNX10 gene; the promoter of SVIL gene; the promoter of TBC1 D30 gene; the promoter of TBX15 gene; the promoter of TH gene; the promoter of THBS1 gene; the promoter of TNFSF1 1 gene; the promoter of TRIM26 gene; the promoter of TSPAN2 gene; and the promoter of VSTM2B gene; wherein the probes, the nucleic acid primers, and optionally the reagents for bisulfite DNA conversion for determining methylation are preferably provided in separate containers, said containers disposed in a common packaging or container that comprises the separate containers.

18. The kit according to any one of claims 11 - 17, which comprises one or more of:(i) A set of primers comprising SEQ ID NO: 1 and 2, optionally a set of primers comprising SEQ ID NO: 3 and 4; and / or the nucleic acid probe of SEQ ID NO: 5;(j) A set of primers comprising SEQ ID NO: 6 and 7, optionally a set of primers comprising SEQ ID NO: 8 and 9; and / or the nucleic acid probe of SEQ ID NO: 10;(k) A set of primers comprising SEQ ID NO: 11 and 12, optionally a set of primers comprising SEQ ID NO: 13 and 14; and / or the nucleic acid probe of SEQ ID NO: 15;(l) A set of primers comprising SEQ ID NO: 16 and 17, optionally a set of primers comprising SEQ ID NO: 18 and 19; and / or the nucleic acid probe of SEQ ID NO: 20;(m) A set of primers comprising SEQ ID NO: 21 and 22, optionally a set of primers comprising SEQ ID NO: 23 and 24; and / or the nucleic acid probe of SEQ ID NO: 25;(n) A set of primers comprising SEQ ID NO: 26 and 27, optionally a set of primers comprising SEQ ID NO: 28 and 29; and / or the nucleic acid probe of SEQ ID NO: 30;(o) A set of primers comprising SEQ ID NO: 31 and 32, optionally a set of primers comprising SEQ ID NO: 33 and 34; and / or the nucleic acid probe of SEQ ID NO: 35;(p) A set of primers comprising SEQ ID NO: 36 and 37, optionally a set of primers comprising SEQ ID NO: 38 and 39; and / or the nucleic acid probe of SEQ ID NO: 40;(q) A set of primers comprising SEQ ID NO: 41 and 42, optionally a set of primers comprising SEQ ID NO: 43 and 44; and / or the nucleic acid probe of SEQ ID NO: 45;(r) A set of primers comprising SEQ ID NO: 46 and 47, optionally a set of primers comprising SEQ ID NO: 48 and 49; and / or the nucleic acid probe of SEQ ID NO: 50.7019. The kit according to any one of claims 17 - 18 which further comprises, preferably in separate containers, one or more of polymerases, buffers for the polymerization of nucleic acids, and nucleotides, preferably deoxi nucleotide triphosphates.

20. Use of a kit as defined in any one of claims 11-19, for the prognosis of a subject diagnosed or suspected of suffering melanoma.

21. A method of detecting methylation in one or more gene promoter sequences in an isolated sample of a subject, or a method of characterizing an isolated sample of a subject, the method comprising: determining in the isolated sample of a subject whether one or more methylation(s) are present in one or more gene promoter sequence(s), by means of one or more of a (multiplex) methylation-specific polymerase chain reaction analysis, preferably by a nested multiplex methylationspecific polymerase chain reaction; a southern hybridization with methylationsensitive endonucleases; a whole genome / targeted / nanopore bisulfite sequencing; pyrosequencing; epityper; methylation-Sensitive Restriction Enzymes (MSRE); methylated DNA Immunoprecipitation (MeDIP); methyl-CpG Binding Domain (MBD) Capture; Illumina HumanMethylation450 BeadChip (450K Array); Illumina Infinium Methylation EPIC Array (850K Array); Twist Bioscience's NGS; and Methylation- Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA); wherein the one or more gene promoter sequence(s) are selected from the group consisting of the promoter of NR5A2 gene, the promoter of ASB18 gene, the promoter of DUOX2 gene, the promoter of LINC00682 gene, the promoter of LMX1A gene, the promoter of NKX2 gene, the promoter of OSR1 gene, the promoter of PDX1 gene, and the promoter of CLDN10 gene, and optionally the promoter of LY75 gene.

22. The method of claim 21 , wherein the determining in the isolated sample of a subject whether one or more methylation(s) are present in one or more gene promoter sequence(s) comprises the steps of:(i) carrying out in DNA of the isolated sample a bisulfite DNA conversion to obtain treated promoter regions; and(ii) amplifying those treated promoter, preferably amplifying CpG islands in the promoters, by means of primers that allow amplification of regions in the promotersflanked by primers that comprise or consists of the pairs of primers: SEQ ID NO: 1- 2 and optionally SEQ ID NO: 3-4 for the promoter of NR5A2 gene; SEQ ID NO:6-7, and optionally SEQ ID NO: 8-9 the promoter of ASB18 gene; SEQ ID NO: 11-22 and optionally SEQ ID NO: 13-14 for the promoter of DLIOX2 gene; SEQ ID NO: 16 - 17 and optionally SEQ ID NO: 18-19 for the promoter of LINC00682 gene; SEQID NO: 21-22 and optionally SEQ ID NO: 23-24 for the promoter of LMX1A gene; SEQ ID NO: 26-27 and optionally SEQ ID NO: 28-29 for the promoter of NKX2 gene; SEQ ID NO: 31-32 and optionally SEQ ID NO: 33-34 for the promoter of OSR1 gene; SEQ ID NO: 36-37 and optionally SEQ ID NO: 38-39 for the promoter of PDX1 gene; and SEQ ID NO: 41-42 and optionally SEQ ID NO: 43-44 for the promoter of CLDN10 gene; and SEQ ID NO: 46-47 and optionally SEQ ID NO: 48-49 for the promoter of LY75 gene; and(iii) detecting methylation in the amplified treated promoter region by means of probes.

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