WO2006092610A2 - Markers for melanoma - Google Patents

Abstract

Methods of monitoring melanoma progression in a subject comprise, in a test sample, determining expression levels of novel marker genes. These genes are selected from, inter alia, TSPY1, CYBA and MT2A. Methods of diagnosing melanoma also include determining the methylation status of markers whose methylation status has now been shown to be linked to progression of melanoma. Microarrays, screening methods, primers and methods of treatment of melanoma are also described based around the novel marker genes.

Description

Markers for Melanoma

TECHNICAL FIELD

The present invention relates to methods and products useful for diagnosing melanoma and is based around the unexpected finding of new markers associated with melanoma and its progression.

INTRODUCTION

The incidence of cutaneous melanoma is at epidemic proportions, with rates steadily rising in Western countries over the past few decades (1, 2) . However, effective treatment for patients with advanced melanoma is currently- unavailable. Moreover, the prognosis of such patients is poor, with a 10% survival rate after 5 years. Less than a decade ago, cutaneous melanoma was described as a "black tumor" and a "black box" (3) . While considerable insights have recently been made with respect to mapping out central events in melanoma development, the molecular basis of tumor progression in this disease remains ill-defined. One approach towards understanding melanoma is to compare gene expression patterns between melanocytic cells from different stages of tumor progression. In this context, DNA microarray-based gene expression profiling has had a far- reaching impact on the study of numerous tumor types (4) , including melanoma (5) . Using this approach, new subgroups of melanoma have been identified (6) , along with a wealth of marker genes that correlate with melanoma progression and drug response (7-13) . Cutaneous melanoma is a pigmented, readily accessible lesion that has been well-defined in histopathological terms (3) . Early radial growth phase (RGP) melanomas can invade into the epidermis and papillary dermis, but have no capacity for metastasis; resection at this stage is almost completely curative. A subsequent vertical growth phase (VGP) denotes a transition to a more aggressive stage, which is capable of metastasis. Changes in gene expression occurring at the RGP/VGP transition are, thus, of great interest. However, comparative transcriptomic studies have so far been hindered in this arena, as paired RGP/VGP biopsies are not normally available (since resection of RGP melanoma is often curative and no VGP develops) .

DESCRIPTION OF THE INVENTION

The present invention relates to methods and products useful for diagnosing melanoma and is based around the unexpected finding of new markers associated with melanoma and its progression.

Accordingly, there is provided, in a first aspect of the invention, a method of diagnosing melanoma in a subject comprising, in a test sample, determining expression levels of at least one gene selected from TSPYl, CYBA, MT2A, GPNMB, BST2, GIP3, ACTG2, TACl, IFITl, TNFAIP6, MXl, S100A9, COL8A1, HLA-DQAl, CIS, LGALS3, GPM6B, AZGPl, CCL2 , RGS3, UGT2B7, ClR, ISGF3G, ANKSl, CHNl, CALDl, BRD2 , RPL37A, RPLPO, PHB, PBX2, SNRK, DAP, HOXD4 , AEBPl, ENO2 , MGP, PRKl, MSX2/HOX8, SIAT7B, NPTX2 , RARB, AFlQ, IGFBP4 , PTPRN2 , AKR1C3 wherein a statistically significant decrease in the expression of any one of TSPYl, CYBA, MT2A, GPNMB, BST2 , GIP3, ACTG2, TACl, IFITl, TNFAIP6, MXl, S100A9, COL8A1 , HLA-DQAl, CIS, LGALS3 , GPM6B, AZGPl, CCL2 , RGS3 , UGT2B7 , ClR, ISGF3G, ANKSl, CHNl, CALDl, BRD2 , RPL37A, RPLPO and/or a statistically significant increase in the expression of any one of PHB, PBX2 , SNRK, DAP, H0XD4 , AEBPl, ENO2, MGP, PRKl, MSX2/HOX8, SIAT7B, NPTX2 , RARB, AFlQ, IGFBP4, PTPRN2, AKR1C3 indicates the presence of melanoma in the subject.

Note that all genes are listed utilising standard nomenclature approved by the human genome organisation to ensure that each symbol is unique. Accession numbers providing full sequence information and further details can be found at www.gene.ucl.ac.uk/nomenclature. In addition, in- figure 2, chromosomal locations and unigene accession numbers for each of the genes referred to above are also provided. It should be noted that MT2A is also referred to in the description as MTlE and these gene names are considered to be interchangeable for the purposes of the present disclosure. The accession number for this gene is given in figure 2 as M10942.

The method is most preferably an in vitro method carried out on an isolated sample. In one embodiment the method may also include the step of obtaining the sample.

The method may preferably be utilised to determine if a potential melanoma is in early radial growth phase (RGP) or a subsequent vertical growth phase (VGP) . Thus, the methods of the invention are capable of determining the aggressiveness of a melanoma, in particular whether VGP has been reached which may lead to different treatment regimes being selected.

Prior to the present invention, it was not known that the expression of these genes is linked to the incidence of melanoma and is down- or up- regulated respectively as melanoma progresses to more aggressive forms, which are eventually capable of metastasis. Generally, the method may be utilised in order to monitor progression of a melanoma to ensure it does not progress to a potentially metastatic lesion and to allow suitable treatment if melanoma is suspected.

The test sample is most preferably a tissue sample, taken from the subject, which is suspected of being tumorigenic. In- a most preferred embodiment, the sample comprises melanocytes suspected of being a melanoma. However, any other suitable test sample in which expression of the novel markers of the present invention can be measured to indicate the presence of a melanoma in particular a melanoma in VGP, are included within the scope of the invention. Test samples for diagnostic, prognostic, or personalized medicine uses can be obtained from surgical samples, such as biopsies or fine needle aspirates, from paraffin embedded tissues, or from a body fluid.

The increased or decreased level of expression must be statistically significant in order to provide a reliable test for monitoring melanoma. Any method for determining whether the expression level of the gene is significantly increased or decreased may be utilised. Such methods are well known in the art and routinely employed. For example, statistical analyses may be performed-using an analysis of variance test. A typical P value for use in such a method would be P values of < 0.05 when^' determining whether the relative expression is statistically significant. A change in expression may be deemed significant if there is at least a 10% increase or decrease for example. The test may be made more selective by making the change at least 15%, 20%, 25%, 30%, 35%, 40% or 50%, for example, in order to be considered statistically significant.

In a preferred embodiment, the increased or decreased level of expression is determined with reference to a control sample. This control sample is preferably taken from normal (i.e. non malignant) melanocytes in the subject.

Alternatively, the control sample is taken from the same tissue as that under test at an earlier time point. This is particularly relevant for monitoring progression of a melanoma, for example in order to ensure that treatment has been effective to prevent progression to an aggressive form of the melanoma. Thus, the progression, or effective prevention of progression, of a potential melanoma for example from radial to vertical growth phase can be monitored .

Suitable additional controls may also be included to ensure that the test is working properly, such as measuring expression of a suitable reference gene in both test and control samples.

In a most preferred embodiment, the subject is a human subject. Generally the subject will be a patient wherein a potential melanoma has been identified and the method may be _. used to determine if indeed there is a potentially dangerous lesion and also to guide treatment depending upon the stage of progression of the melanoma.

The method may be carried out by determining expression of at least one of the genes listed, all of which represent novel melanoma markers. In one embodiment, expression levels of all of the genes is measured. This may be done utilising microarray technology for example (as described in more detail in the experimental section below) , which provides ^■ a convenient method of analysing expression of multiple genes at the same time and from a single test sample. Microarray technology is well known in the art and commercial entities will prepare and supply suitable arrays as. required.

Of course, expression of any number of the genes representing novel melanoma markers may be assessed, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 etc up to all of the genes may be assessed in the method of the invention. Preferably, all genes are assessed in the same test sample to prevent inter-sample viability.

In a further preferred embodiment, the method according to the present invention is carried out wherein the expression of at least TSPYl is determined. As discussed in more detail in the experimental section, the expression of TSPYl is massively down-regulated in melanoma (over 300 fold) and so it is believed that this marker is particularly useful in the diagnosis, prognosis and for monitoring the progression, of melanoma. In a still further preferred embodiment, the expression of at least CYBA and/or MT2A (MTlE) is determined. Bisulphite sequencing has revealed that the promoter regions of these genes become hypermethylated in more aggressive melanoma cell lines. Thus, changes in expression associated with methylation reflect the progression of melanoma to more aggressive forms .

Expression of a gene can be assessed using any means known in the art. Either mRNA or protein can be measured. Methods employing hybridization to nucleic acid probes can be employed for measuring specific mRNAs . Such methods include using nucleic acid probe arrays (microarray technology) and using Northern blots. Messenger RNA can also^' be assessed using amplification techniques, such as RT- PCR. Advances in genomic technologies now permit the simultaneous analysis of thousands of genes, although many are based on the same concept of specific probe-target hybridization. Sequencing-based methods are an alternative; these methods started with the use of expressed sequence tags (ESTs) , and now include methods based on short tags, such as serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS) . Differential display techniques provide yet another means of analyzing gene expression; this family of techniques is based on random amplification of cDNA fragments generated by restriction digestion, and bands that differ between two tissues identify cDNAs of interest. Specific proteins can be assessed using any convenient method including immunoassays and immuno-cytochemistry but are not limited to that. Most such methods will employ antibodies which are specific for the particular protein or protein fragments.

In one embodiment, the levels of gene expression are determined using RT-PCR. Reverse transcriptase polymerase chain reaction is a well known technique in the art which relies upon the enzyme reverse transcriptase to reverse transcribe mRNA to form cDNA, which can then be amplified in a standard PCR reaction. Protocols and kits for carrying out RT-PCR are extremely well known to those of skill in the art and are commercially available.

In a preferred embodiment, the RT-PCR is carried out in real time and in a quantitative manner. Real time quantitative RT-PCR has been thoroughly described in the literature (see Gibson et al for an early example of the technique) and a variety of techniques are possible. Examples include use of Taqman, Molecular Beacons, LightCycler (Roche), Scorpion and Amplifluour systems. All of these systems are commercially available and well characterised, and may allow multiplexing (that is, the determination of expression of multiple genes in a single sample) which is particularly advantageous in the method of the present invention.

These techniques produce a fluorescent read-out that can be continuously monitored. Real-time techniques are advantageous because they keep the reaction in a "single tube" . This means there is no need for downstream analysis in order to obtain results, leading to more rapidly obtained results. Furthermore keeping the reaction in a "single tube" environment reduces the risk of cross contamination and allows a quantitative output from the methods of the invention. This may be particularly important in the clinical setting of the present invention.

As an example, real-time quantification of PCR reactions can be accomplished using the TaqMan^® system (Applied

Biosystems) , see Holland et al; Detection of specific polymerase chain reaction product by utilising the 5 '-3' exonuclease activity of Thermus aquaticus DNA polymerase; Proc. Natl. Acad. Sci . USA 88, 7276-7280 (1991) (32), Gelmini et al . Quantitative polymerase chain reaction-based homogeneous assay with flurogenic probes to measure C-Erbb-2 oncogene amplification. Clin. Chem. 43, 752-758 (1997) (33) and Livak et al . Towards fully automated genome wide polymorphism screening. Wat. Genet. 9, 341-342 (1995) (34), incorporated herein by reference. Taqman^® probes are widely commercially available, and the Taqman^® system (Applied Biosystems) is well known in the art. Taqman^® probes anneal between the upstream and downstream primer in a PCR reaction. They contain a 5 ' -fluorophore and a 3 '-quencher. During amplification the 5 '-3' exonuclease activity of the Taq polymerase cleaves the fluorophore off the probe. Since the fluorophore is no longer in close proximity to the quencher, the fluorophore will be allowed to fluoresce. The resulting fluorescence may be measured, and is in direct proportion to the amount of target sequence that is being amplified.

In the Molecular Beacon system, see Tyagi & Kramer. Molecular beacons - probes that fluoresce upon hybridization. Nat. Biotechnol . 14, 303-308 (1996) (35) and Tyagi et al . Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16, 49-53 (1998) (36) both of which are incorporated by reference herein, the beacons are hairpin-shaped probes with an internally quenched fluorophore whose fluorescence is restored when bound to its target. The loop portion acts as the probe while the stem is formed by complimentary "arm" sequences at the ends of the beacon. A fluorophore and quenching moiety are attached at opposite ends, the stem keeping each of the moieties in close proximity, causing the fluorophore to be quenched by energy transfer. When the beacon detects its target, it undergoes a conformational change forcing the stem apart, thus separating the fluorophore and quencher. This causes the energy transfer to be disrupted to restore fluorescence.

Any suitable fluorophore is included within the scope of the invention. Fluorophores that may possibly be used in the method of the invention include, by way of example, FAM, HEX™, NED™, ROX™, Texas Red™ etc. Quenchers, for example Dabcyl and TAMRA are well known quencher molecules that may be used in the method of the invention. However, the invention is not limited to these specific examples. EDANS and DABCYL form a particularly efficient fluorophore/quencher pair (62) , as do fluorescein/DABCYL (67) .

A further real-time fluorescence based system which may be incorporated in the methods of the invention is Zeneca's Scorpion system, see Detection of PCR products using self- probing amplicons and fluorescence by Whitcorαbe et al. Nature Biotechnology 17, 804 - 807 (01 Aug 1999) (37). This reference is incorporated into the application in its entirety. The method is based on a primer with a tail attached to its 5 ' end by a linker that prevents copying of - li ¬

the 5' extension. The probe element is designed so that it hybridizes to its target only when the target site has been incorporated into the same molecule by extension of the tailed primer. This method produces a rapid and reliable signal, because probe-target binding is kinetically favoured over intrastrand secondary structures .

Amplifluour primers (as described in US Patent 5,866,336 to Nazarenko and WO98/02449 both of which are incorporated herein by reference) rely upon a similar principle to molecular beacons. However, in this case, the hairpin structure is- part of the amplification primer itself. The primer binds to a nucleic acid strand and directs synthesis and thus becomes part of the amplification product. When the complementary strand is synthesised amplification occurs through the hairpin structure. This separates the fluorophore and quencher molecules, thus leading to generation of fluorescence as amplification proceeds.

In one embodiment, the method of the invention utilises at least one primer pair taken from primer pairs comprising the sequences of :

GPNMB - forward, 5'-TGCATAAAGCCAATGTAGTCCA-S' (SEQ ID NO:

1) and reverse, 5'-CAGGGACCTCATCTTTGGAA-S' (SEQ ID NO : 2)

(373 bp amplicon) ;

CYBA - forward, 5'-TTTGTGTGCCTGCTGGAGTA-S' (SEQ ID NO : 3) and reverse, δ'-CTCCTCGCTGGGCTTCTT-S' (SEQ ID NO: 4) (369 bp amplicon) ; MXl - forward, 5 ' -AGCCACTGGACTGACGACTT-3 ' (SEQ ID NO: 5) and reverse, 5'-ACGGCACTCATGCTCCTAAA-S' (SEQ ID NO : 6)

(335 bp amplicon) ; HOXD4 - forward, 5 ' -TGACTCGeCAAGATTTTATGT-3 ' (SEQ ID NO:. 7) and reverse, B'-CACCTCGCTGGGCTCTAA-a' (SEQ ID NO: 8) (190 bp amplicon) ; RARB - forward, 5'-ACAAGGTCAAAGGAGGCAGA-SMSEQ ID NO: 9) and reverse, 5'-TTCACAAGCCATTAGGGAAA-S' (SEQ ID NO: 10) (188 bp amplicon) ;

PRKl - forward, 5'-GGGCTGTTTCTTCACATCTTC-SMSEQ ID NO: 11) and reverse, δ'-GTGGACTGGTGGGGACTG-S' (SEQ ID NO: 12) (231 bp amplicon) ;

TSPYl - forward, 5 ' -CACCACAACAGCAGCCTTA-3 ' (SEQ ID NO: 13) and reverse, 5'-TGCTCCATCATATTCAACTCA-S' (SEQ ID NO: 14) (193 bp amplicon) .

The genes listed are selected examples of the genes de-scribed which belong to the present invention and which represent novel melanoma markers. Of the genes listed together with suitable primers, TSPYl, CYBA, GPNMB and MXl are all significantly down regulated in melanoma, whereas the expression of all of H0XD4, PRKl and RARB is up regulated in melanoma. The primers may, in one embodiment, be Amplifluour primers.

It should be noted that whilst PCR is a preferred amplification method, equivalents may. also be included within the scope of the invention. Examples include isothermal amplification techniques such as NASBA, 3SR, TMA and triamplification, all of which are well known in the art and commercially available (and are described in more detail below) . Other suitable amplification methods include the ligase chain reaction (LCR) (Barringer et al, 1990) , selective amplification of target polynucleotide sequences {US Patent No. 6,410,276), consensus sequence primed polymerase chain reaction (US Patent No 4,437,975), arbitrarily primed polymerase chain reaction (WO90/06995) , nick displacement amplification (WO2004/067726) .

In a further embodiment, the method of the invention incorporates use of microarrays in order to detect expression of the genes which represent novel melanoma markers. Suitable microarrays are described in further detail below.

In one embodiment, the method of the invention is used to determine whether the melanoma is likely to have metastatic capabilities. As shown in the experimental section below, the novel marker genes are significantly down- or up- regulated in more aggressive forms of melanoma which may be capable of metastasis. Therefore, significantly decreased or increased levels of expression, respectively, may be indicative of a form of melanoma capable of metastasis (which may correspond to vertical growth phase) .

As discussed in more detail below, it has been found that the reduced expression of a selection of the novel markers of the invention can be reverted to wild type levels of expression by treatment with a suitable DNA methyltransferase inhibitor. It has also been discovered that reduced expression of a selection of markers already known to be associated with melanoma can similarly be reverted to wild type levels of expression by treatment with a suitable DNA methyltransferase inhibitor. Thus, methylation is likely to be responsible, at least indirectly, for the reduced expression of these genes seen in melanoma. For a selection of these genes, methylation appears to directly regulate their expression. CpG islands in these genes have been identified using CpGPlot (see below, including Fig 10 and the Experimental section) . Therefore, detection of the methylation status of these genes, which represent novel methylation sensitive markers for melanoma proves a useful test for diagnosis, including prognosis, of melanoma.

The CpG islands for these genes, where significant methylation occurs, have been identified using suitable predictive tools, as described in more detail below with reference to figure 10.

Accordingly, in a second aspect the invention provides a method of diagnosing melanoma in a subject comprising, in a test sample, determining the methylation status of at least one gene selected from TSPYl, CYBA, MXl, MT2A, RPL37A, HSPBl wherein hypermethylation of at least one of the genes indicates the presence of melanoma. The method is one which allows melanoma progression to be monitored, in parituclar to determine whether the melanoma is likely to have metastatic capabilities.

The method is most preferably an in vitro method carried out on an isolated sample. In one embodiment, the method may also include the step of obtaining the sample.

The subject of the invention, as for the first aspect, is most preferably a human and the features described above in respect of that aspect of the invention also apply mutatis mutandis to this aspect of the invention. As discussed above, the test sample is most preferably a tissue sample taken from the subject which is suspected of being a tumor. In a most preferred embodiment the sample comprises melanocytes suspected of being a melanoma.

However, any other suitable test sample in which methylation status of the genes, which represent the novel markers of the present invention and markers with a known link to melanoma but without any knowledge of importance of methylation for melanoma progression, can be determined to indicate the presence of a melanoma are included within the scope of the invention.

Hypermethylation is a well known term in the art which denotes increased and aberrant methylation at specific CpG sites in a gene. Hypermethylation most often occurs in the promoter regions of genes and acts to depress expression of the gene. Accordingly, in a preferred embodiment, the method is carried out wherein the promoter regions of the relevant genes are assessed to determine their methylation status .

In a particularly preferred embodiment, the CpG islands identified for the appropriate genes are investigated for their methylation status. These CpG islands are represented in SEQ ID NOs: 15 and 29 (TSPYl CpG islands), 30 and 31 (CYBA CpG islands) , 32 and 33 (RPL37A CpG islands) , 38 (MXl CpG islands) , 39 (HSPBl CpG islands) and 40 (MT2a CpG islands) .

As is discussed above in respect of the first aspect of the invention, any number of the genes which represent novel methylation related melanoma markers can be assessed in order to provide a diagnosis. For example 1, 2, 3, 4, 5 or 6 of the genes may be assessed in terms of their methylation status in the method of this aspect of the invention. In a most preferred embodiment, the methylation status of at least TSPYl is determined. In a further preferred embodiment, the methylation status of at least CYBA and/or MT2A (MTlE) is determined.

In a further preferred embodiment, the methylation status of the genes is determined using methylation specific PCR (MSP) . However, any technique may be utilised to determine the methylation status of the genes, such as, for example, the well known techniques of COBRA (for more details reference is made to the experimental section below) , bisulphite sequencing and use of arrays which can distinguish between bisulphite treated nucleic acid molecules following amplification. A review of some useful techniques is provided in Nucleic acids research, 1998, Vol. 26, No. 10, 2255-2264, which reference is incorporated herein in its entirety. DNA methylation analysis has been performed successfully with a number of techniques which include the MALDI-TOFF, MassARRAY , MethyLight, Quantitative analysis of ethylated alleles (QAMA) , enzymatic regional methylation assay (ERMA), HeavyMethyl , QBSUPT, MS-SNuPE,

MethylQuant, Quantitative PCR sequencing, Oligonucleotide- based microarray systems . Still another way for the identification of methylated CpG dinucleotides utilizes the ability of the MBD domain of the McCP2 protein to selectively bind to methylated DNA sequences (Cross et al, 1994; Shiraishi et al , 1999). Restriction enconuclease digested genomic DNA is loaded onto expressed His-tagged methyl-CpG binding domain that is immobilized to a solid matrix and used for preparative column chromatography to isolate highly methylated DNA sequences.

The MSP technique will be familiar to one of skill in the art. In the MSP approach, DNA may be amplified using primer pairs designed to distinguish methylated from unmethylated DNA by taking advantage of sequence differences as a result of sodium-bisulfite treatment (Herman et al (65) and see WO 97j^'46705 , incorporated herein by reference) . After sodium- bisulfite treatment unmethylated cytosines are converted to uracil whereas methylated cytosines remain unconverted.

A specific example of the MSP technique is designated real- time quantitative MSP (QMSP) , which permits reliable quantification of methylated DNA in real time. These methods are generally based on the continuous optical monitoring of a fluorogenic PCR and represent a specific application of the well, known and commercially -available real-time PCR techniques such as Taqman^®, Molecular

Beacons^®, Lightcycler^®, Amplifluour^® and Scorpion^® etc as described in more detail above.

Other nucleic acid amplification techniques may also be modified to detect the methylation status of at least one of the panel of genes which represent novel melanoma markers . Such amplification techniques are well known in the art, and include methods such as NASBA (Compton, 1991 (45)), 3SR (Fahy et al . , 1991 (46)) and Transcription Mediated Amplification (TMA) . Other suitable amplification methods include the ligase chain reaction (LCR) (Barringer et al, 1990) , selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence primed polymerase chain reaction (US Patent No 4,437,975), arbitrarily primed polymerase chain reaction (WO90/06995) , nick displacement amplification (WO2004/067726) . This list is not intended to be exhaustive; any nucleic acid amplification technique may be used provided the appropriate nucleic acid product is specifically amplified.

Sequence variation that reflects the methylation status at CpG dinucleotides in the original genomic DNA offers two approaches to PCR primer design. First, primers that themselves do not cover any potential sites of DNA methylation. Sequence variation at sites of differential methylation are located between the two primers. Such primers are used in bisulphite genomic sequencing, COBRA, Ms--SNuPE. Second, primers that are designed to anneal specifically with either the methylated or unmethylated version of the converted sequence. If there is a sufficient region of complementarity, e.g., 12, 15, 18, or 20 nucleotides, to the target, then the primer may also contain additional nucleotide residues that do not interfere with hybridization but may be useful for other manipulations. Exemplary of such other residues may be sites for restriction endonuclease cleavage, for ligand binding or for factor binding or linkers or repeats. The oligonucleotide primers may or may not be such that they are specific for modified methylated residues.

One way to distinguish between modified and unmodified DNA is to hybridize oligonucleotide primers which specifically bind to one form or the other of the DNA. After hybridization, an amplification reaction can be performed and amplification products assayed. The presence of an amplification product indicates that a sample hybridized to the primer. The specificity of the primer indicates whether the DNA had been modified or not, which in turn indicates whether the DNA had been methylated or not .

Another way to distinguish between modified and nonmodified DNA is to use oligonucleotide probes which may also be specific for certain products. Such probes can be hybridized directly to modified DNA or to amplification products of modified DNA. Oligonucleotide probes can be labeled using any detection system known in the art . These include but are not limited to fluorescent moieties, radioisotope labeled moieties, bioluminescent moieties, luminescent moieties, chemiluminescent moieties, enzymes, substrates, receptors, or ligands.

Amplification is achieved with the use of primers specific for the sequence of the gene whose methylation status is to be assessed. In order to provide specificity for the nucleic acid molecules, primer binding sites corresponding to a suitable region of the sequence may be selected. The skilled reader will appreciate that the nucleic acid molecules may also include sequences other than primer binding sites which are required for detection of the methylation status of the gene, for example RNA Polymerase binding sites or promoter sequences may be required for isothermal amplification technologies, such as NASBA, 3SR and TMA.

TMA (Gen-probe Inc.) is an RNA transcription amplification system using two enzymes to drive the reaction, namely RNA polymerase and reverse transcriptase. The TMA reaction is isothermal and can amplify either DNA or RNA to produce RNA amplified end products. TMA may be combined with Gen-probe's Hybridization Protection Assay (HPA) detection technique to allow detection of products in a single tube. Such single tube detection is a preferred method for carrying out the invention.

Thus, in a further embodiment the method of this aspect of the invention is carried out using a technique selected from NASBA, 3SR and TMA.

The methods described above can be used in conjunction with one another to confirm a diagnosis of melanoma including an assessment of how aggressive the melanoma is, and also in prognostic applications, since they provide complementary information about the subject.

As aforementioned, it has been shown that treatment with a specific DNA methyltransferase inhibitor leads to re- expression of specific markers associated with melanoma. These markers include both genes which appear to be regulated directly by methylation and those which are indirectly regulated (see the experimental section for further details) . The prior art teaches that Histone deacetylase inhibitors (HDAC) have a similar effect on expression of repressed genes (32, 52 and 53) where methylation causes repression and as a result it is hypothesised that treatment with a HDAC inhibitor may also cause re-expression of specific markers associated with melanoma . Accordingly, in a third aspect of the invention there is provided a method of treating melanoma in a subject comprising administering a therapeutically effective amount of a DNA methyltransferase inhibitor and/or a histone deacetylase inhibitor to the subject such that expression of at least one gene selected from TSPYl, CYBA, MT2A, BST2 , GIP3, SlOOAl, IFITl, MXl, RGS3, ISGP3G, APOD, RPL37A, HSPBl is increased.

Preferably, the subject is a human. Generally the subject will be one who has been diagnosed with melanoma; for example using the methods according to the present invention.

The method is preferably one carried out to prevent the melanoma from reaching a stage in which it is capable of mestastis. Alternatively, the method may, for example, be used to treat a melanoma which is already in the vertical growth phase (VGP) .

In a preferred embodiment, the effect of the treatment is to increase the level of gene expression to the levels of gene expression found in normal melanocytes. However, it may be that any increase in expression will be beneficial for treatment of melanoma. Of course, the DNA methyltransferase inhibitor or histone deacetylase inhibitor is provided in a therapeutically relevant amount to ensure that a controlled increase in gene expression is achieved.

In a further preferred embodiment the therapeutic agent is a DNA methyltransferase inhibitor. The agent may be one which reduces expression of DNMT genes, such as suitable antisense molecules, RNAi molecules or siRNA molecules for example. In one embodiment, the antisense molecule comprises MG98. Clinical trials by MethylGene have been carried out to study this antisense reagent that specifically inhibits DNMTl (Reid et al (2002) Selective inhibition of DNA methyltransferase enzymes as a novel strategy for cancer treatment, Curr. Opin. MoI. Ther. 4, 130-137, and see www.methylgene.com for details of phase II clinical trials in metastatic renal cancer patients) .

Alternatively, the agent may be a direct inhibitor of DNMTs. Examples include modified nucleotides such as phosphorothioate modified oligonucleotides (fig 6 of ref (68)) and nucleosides and nucleotides such as cytidine analogues. Suitable examples of cytidine analogues include 5-azacytidine, 5-aza-2 ' -deoxycytidine, 5-fluouro-2 ' - deoxycytidine, pseudoisocytidine, 5, 6-dihydro-5-azacytidine, l-β-D-arabinofuranosyl-5-azacytosine (known as fazabarine) (see figure 4 of ref (68)) .

In another embodiment, the DNA methyltransferase inhibitor comprises Decitabine. Full details of this drug can be found at www.supergen.com for example.

Additional DNMT inhibitors include S-Adenosyl-Methionine

(SAM) related compounds like ethyl group donors such as L- ethionine and non-alkylating agents such as S-adenosyl- homocysteine (SAH) , sinefungin, (S) -6-methyl-6-deaminosine fungin, 6-deaminosinefungin, N4-adenosyl -N4 -methyl-2 , 4- diaminobutanoic acid, 5 ' -methylthio-5 ' -deoxyadenosine (MTA) and 5 ' -amino-5 ' -deoxyadenosine . Further agents which may alter DNA methylation and which may, therefore, be useful in the present methods and uses include organohalogenated compounds such as chloroform etc, procianamide, intercalating agents such as mitomycin C, 4- aminobiphenyl etc, inorganic salts of arsenic and selenium and antibiotics such as kanamycin, hygromycin and cefotaxim.

However, any suitable DNA methyltransferase inhibitor which is capable of increasing the expression of at least one of the genes listed above, and thus can contribute to the treatment of melanoma, is included within the scope of the invention.

In one embodiment, the therapeutic agent is additionally or alternatively a histone deacetylase (HDAC) inhibitor.

Preferred non-limiting examples include, trichostatin A (TSA), suberoyl hydroxamic acid (SBHA), 6- (3- chlorophenylureido) caproic hydroxamic acid (3-Cl-UCHA), m- carboxycinnamic acid bishydroxylamide (CBHA) , suberoylanilide hydroxamic acid (SAHA) , azelaic bishydroxamic acid (ABHA) , pyroxamide, scriptaid, aromatic sulfonamides bearing a hydroxamic acid group, oxamflatin, trapoxin, cyclic-hydroxamic-acid containing peptides, FR901228, MS-275, MGCD0103 (see www.methylgene.com), short- chain fatty acids and N-acetyldinaline .

Combination therapy using both a DNMT inhibitor and a HDAC inhibitor may give cumulative effects greater than either agent utilised in isolation. Thus, complementary dosage regimes or combination compositions are envisaged in the present invention. The therapeutic agent may, for example, be encapsulated and/or combined with suitable carriers in solid dosage forms for oral administration which would be well known to those of skill in the art or alternatively with suitable carriers for administration in an aerosol spray. Examples of oral dosage forms include tablets, capsules and liquids.

Alternatively, the therapeutic agent may be administered parenterally. Specific examples include intradermal injection, subcutaneous injection (which may advantageously give slower absorption of the therapeutic agent) , intramuscular injection (which can provide more rapid absorption) , intravenous delivery (meaning the drug does not need to be absorbed into the blood stream from elsewhere) , sublingual delivery (for example by dissolving of a tablet under the tongue or by a sublingual spray) , rectal delivery, vaginal delivery, topical delivery, transdermal delivery and inhalation.

In a pharmaceutical composition incorporating a suitable DNA methyltransferase inhibitor or a histone deacetylase, preferred compositions include pharmaceutically acceptable carriers including, for example, non-toxic salts, sterile water or the like. A suitable buffer may also be present allowing the compositions to be lyophilized and stored in sterile conditions prior to reconstitution by the addition of sterile water for subsequent administration. The carrier may also contain other pharmaceutically acceptable excipients for modifying other conditions such as pH, osmolarity, viscosity, sterility, lipophilicity, somobility or the like. Pharmaceutical compositions which permit sustained or delayed release following administration may also be used.

Furthermore, as would be appreciated by the skilled practitioner, the specific dosage regime may be calculated according to the body surface area of the patient or the volume of body space to be occupied, dependent on the particular route of administration to be used. The amount of the composition actually administered will, however, be determined by a medical practitioner based on the circumstances pertaining to the disorder to be treated, such as the severity of the symptoms, the age, weight and response of the individual .

In a fourth aspect of the invention, there is provided the us'e of a DNA methyltransferase inhibitor or a histone deacetylase inhibitor in the manufacture of a medicament for the treatment of melanoma by increasing expression of at least one gene -selected from TSPYl, CYBA, MT2A, BST2 , GIP3, SlOOAl, IFITl, MXl, RGS3 , ISGF3G, APOD, RPL37A, HSPBl.

The discussion of the third aspect of the invention applies mutatis mutandis to this aspect, in particular that the use helps to prevent the melanoma becoming metastatic and/or effectively treats an aggressive (VGP) melanoma.

In a preferred embodiment, the use described above gives rise to a medicament which provides a therapeutic effect wherein the level of gene expression is increased to the levels of gene expression found in normal melanocytes. Since the novel markers for melanoma identified in the studies described below are numerous, there is benefit in being able to screen for expression of this panel of genes in a simple and rapid manner. Accordingly, the invention provides, in a fifth aspect, a microarray for use in the methods of the invention which involve determining levels of expression of genes, comprising probes immobilised on a solid support hybridizing with transcripts or parts thereof of at least one gene selected from TSPYl, CYBA, MT2A, GPNMB, BST2, GIP3, ACTG2 , TACl, IFITl, TNFAIP6, MXl, S100A9,

COL8A1, HLA-DQAl, CIS, LGALS3, GPM6B, AZGPl, CCL2 , RGS3, UGT2B7, ClR, ISGF3G, ANKSl, CHNl, CALDl, BRD2 , RPL37A, RPLPO, PHB, PBX2, SNRK, DAP, HOXD4 , AEBPl, ENO2 , MGP, PRKl, MSX2/HOX8, SIAT7B, NPTX2 , RARB, AFlQ, IGFBP4 , PTPRN2 , AKR1C3.

Microarrays and their means of manufacture are well known and can be manufactured to order by commercial entities such as Affymetrix, for example.

The probes are the sequences which are immobilized onto the array, by known methods, and which represent selected sequences from the genes of interest, in this case the novel markers for melanoma listed above. Probe selection and array design lie at the heart of the reliability, sensitivity, specificity, and versatility of the microarrays of the invention. The methods for selecting suitable probes would be readily apparent for one of skill in the art and may involve optimization using data collected from multiple databases, bioinformatics tools, and experiment-trained computer models. The key elements of probe selection and design are common to_. the production of all arrays, regardless of their intended application and as such would be well known to one of skill in the art. Strategies to optimize probe hybridization, for example, are invariably included in the process of probe selection. Hybridization under particular pH, salt, and temperature conditions can be optimized by taking into account melting temperatures and using empirical rules that correlate with desired hybridization behaviours.

The GeneChip arrays produced by Affymetrix involve a Perfect Match/Mismatch probe strategy. For each probe designed to be perfectly complementary to a target sequence, a partner probe is generated that is identical except for a single base mismatch in its centre. These probe pairs, called the "Perfect Match probe (PM)" and the "Mismatch probe (MM)", allow the quantitation and subtraction of signals caused by non-specific cross-hybridization. The difference in hybridization signals between the partners, as well as their intensity ratios, serve as indicators of specific target abundance. Such an array design may be applicable to, and incorporated into, the arrays of the present invention.

In order to ensure specificity of the probes in terms of accurately representing the genes listed above, the microarray preferably comprises at least 10 probes representing each gene on the array. However, other numbers of probes may be utilised provided that the expression of each gene which is selected to form part of the array can be accurately and specifically measured. In a preferred embodiment, the array includes probes which represent each and every one of the genes listed. However, this may not be necessary in order to be able to accurately diagnose melanoma. Probes representing only one or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 etc of the genes may be utilised in the array. Accordingly, in one preferred embodiment, the microarray comprises probes representing transcripts of at least the TSPYl gene. In a further embodiment, the microarray comprises probes representing transcripts of at least the CYBA and/or MT2A (MTlE) genes.

Each probe is preferably at least about 20 nucleotides in length such that a probe of sufficient length to ensure sensitivity and specificity of hybridization is provided. However, any length of probe may be utilised within the scope of the invention, provided that accurate results are achieved in terms of detecting expression of the genes which represent novel melanoma markers. Possible lengths for the probes include at least 10 nucleotides and up to 250 nucleotides and preferably between about 20 and about 50 nucleotides.

In a similar fashion, the invention also provides a microarray for use in the methods which involve determining the methylation status of the genes representing novel methylation sensitive melanoma markers comprising probes immobilised on a solid support hybridizing with methylated only or both unmethylated and methylated versions of at least one gene or parts thereof selected from TSPYl, CYBA, MT2A, MXl, RPL37A, HSPBl following bisulphite treatment. Preferably, the probes allow the methylation status of at least TSPYl to be deteremined. In an additional embodiment, the probes allow the methylation status of at least CYBA and/or MT2A (MTlE) to be determined.

As discussed above, bisulphite treatment of nucleic acid will convert unmethylated cytosine residues to uracil, but methylated cytosine residues (found at specific CpG sites) will not be converted. Thus, it is possible to design probes which will discriminate between methylated (remain as cytosine following bisulphite treatment) and unmethylated (converted to uracil) sites in terms of target hybridisation.

In one embodiment, the probes correspond to appropriate portions of the CpG islands of the genes, the nucleotide sequences of which are presented in SEQ ID NOs: 15 and 29 (TSPYl CpG islands) , 30 and 31 (CYBA-CpG islands) , 32 and 33 (RPL37A CpG islands) , 38 (MXl CpG islands) , 39 (HSPBl CpG islands) and 40 (MT2a CpG islands) .

Thus, the microarray may contain two sets of probes, one which is specific for genes methylated at particular sites and one specific for genes which are unmethylated at those particular sites. Preferably, each probe will represent only one potential site of methylation to allow full specificity of binding and detection for each methylation site, although this is not essential.

Alternatively, the microarray may be a "methylation specific" array in that it contains probes designed to solely bind targets which are methylated at the specific CpG sites in the relevant genes and thus are protected from the effects of bisulphite treatment. Such an array could be utilised in conjunction with an array which comprises probes • specific only for targets which are unmethylated at specific CpG sites. The same sample may be utilised with each array in order to determine relative levels of methylation.

Following bisulphite treatment of the sample, optionally followed by amplification (see above for details of the MSP technique etc.), the target nucleic acid is hybridised to the array in order to determine whether the genes of interest are methylated at specific sites. The positive identification of methylation is indicative of melanoma or a more aggressive form of melanoma (such as VGP melanoma) .

Either the probes or the target, or both, may be labelled with a suitable label to allow detection of hybridization of the target to its complementary probe. The most preferred type of label is a fluorescent label although other labels such as radiolabels and mass-labels may also be utilised.

The tissue sample to be used is as described above and the subject from which the sample containing the test (target) nucleic acid is derived is most preferably a human subject.

In addition, the present invention also identifies and characterises for the first time the specific methylation sites in the promoter region of the genes TSPYl, CYBA, RPL37A, MXl, HSPBl and MT2A. Due to the fact that these genes appear to be hypermethylated leading to down regulation of expression and (more aggressive forms of) melanoma, there is particular diagnostic, including prognostic, value in being able to determine the methylation status of the genes in the respective CpG island regions. Accordingly, there is provided by the invention primers which can bind to the sequence of any one of SEQ ID NOs : 15 and 29 (TSPYl CpG islands), 30 and 31 (CYBA CpG islands), 32 and 33 (RPL37A CpG islands), 38 (MXl CpG islands), 39 (HSPBl CpG islands) and 40 (MT2a CpG islands) , (see figure 8, 9 and 10 also) following bisulphite treatment. The primers may be methylation specific primers, which serve to discriminate between whether there is methylation at a particular CpG site. Thus, one set of primers will be specific for the sequence following bisulphite treatment under circumstances where the cytosine residue was (or were if more than one CpG site is covered by the primers binding site) methylated and therefore unaffected by bisulphite treatment. A second set of primers may be specific for the sequence following bisulphite treatment under circumstances where the cytosine residue was (or cytosine residues were if more than one CpG site is covered by the primer binding) unmethylated and therefore would be converted to uracil following bisulphite treatment.

A third set of primers acts as a control and is specific for the sequence prior to bisulphite treatment.

The design of such primers is well within the capability of one of skill in the art and reference is made to Herman et al and WO 97/46705 both of which are incorporated herein by reference for examples of suitable primers.

Alternatively, the primers may not bind to sites which may be methylated, provided that they amplify a sequence which includes a potential methylation site. Following amplification, the products may then be bound to an appropriate array as described above in order to determine the methylation status of the amplified products (the probes are specific for methylated or unmethylated residues and provide the distinguishing function) .

Primers are well known and their design is routine. A typical primer is of length between approximately 5 and 50 nucleotides, preferably between 10 and 30 nucleotides. Primers in use must be single stranded nucleic acid molecules and are generally designed to prevent self annealing.

In one embodiment, primers comprising, or consisting essentially of or consisting of the sequence of SEQ ID NO: 16- and 17 are provided. These primers are useful for COBRA analysis of the TSPYl CpG island in conjunction with use of a suitable restriction enzyme such as BsiEI (see experimental section below, particularly figure 4) .

By identifying the novel markers linked to melanoma, new treatments for this disease may be discovered. Thus, the expression pattern linked to melanoma and in particular progression of melanoma to more aggressive (metastatic) forms may be used as a research tool to identify new pharmaceuticals which may be used to treat, prevent or control melanoma.

Accordingly, in a further aspect the invention provides an in vitro method of identifying a compound capable of treating or reducing the effects or progression of melanoma comprising the steps of; (a) administering the compound to a melanoma sample;

(b) generating an expression profile of a panel of genes comprising at least one of TSPYl, CYBA, MT2A, GPNMB, BST2,GIP3, ACTG2, TACl, IFITl, TNFAIP6, MXl, S100A9, COL8A1 , HLA-DQAl, CIS, LGALS3 , GPM6B, AZGPl, CCL2 , RGS3, UGT2B7, ClR, ISGF3G, ANKSl, CHNl, CALDl, BRD2 , RPL37A, RPLPO, PHB, PBX2, SNRK, DAP, HOXD4 , AEBPl, ENO2 , MGP, PRKl, MSX2/HOX8, SIAT7B, NPTX2, RARB, AFlQ, IGFBP4 , PTPRN2 , AKRlC3 ;

(c) comparing the expression profile obtained in (b) with the expression profile of a corresponding panel of genes expressed in a control melanocyte sample which is not a melanoma; wherein a positive correlation of the expression profiles is indicative that the compound is capable of reducing, controlling the progression of, or preventing me-lanoma.

Similarly, the invention also provides a similar compound screening and lead identification method which involves use of non-human animals; a method of identifying a compound capable of treating or reducing the effects or progression of melanoma comprising the steps of;

(a) administering the compound to an experimental non- human animal having a melanoma; (b) generating an expression profile of a panel of genes comprising at least one of TSPYl, CYBA, MT2A, GPNMB, BST2, GIP3, ACTG2, TACl, IFITl, TNFAIP6, MXl, S100A9, COL8A1, HLA-DQAl, CIS, LGALS3 , GPM6B, AZGPl, CCL2 , RGS3 , UGT2B7, ClR, ISGF3G, ANKSl, CHNl, CALDl, BRD2 , RPL37A, RPLPO, PHB, PBX2 , SNRK, DAP, HOXD4 , AEBPl, ENO2 , MGP, PRKl,

MSX2/HOX8, SIAT7B, NPTX2 , RARB, AFlQ, IGFBP4 , PTPRN2 , AKRlC3 ; (c) comparing the expression profile obtained in (b) with the expression profile of a corresponding panel of genes expressed in a control melanocyte sample which is not a melanoma; wherein a positive correlation of the expression profiles is indicative that the compound is capable of reducing or preventing melanoma.

The experimental animal may be sacrificed following the testing.

In particular, these methods allow leads to be identified which either are effective in preventing melanoma from reaching a metastatis form and/or which are capable of regressing a melanoma from an aggressive phase, such as VGP, to- a less dangerous form of melanoma.

In one embodiment, the control sample is taken from an experimental non-human animal which does not have a melanoma. Preferably, the control sample is taken from normal melanocytes in the same non-human animal .

Preferred gene panels include at least TSPYl and/or CYBA and/or MT2A (MTIE) .

The invention also provides the compounds identified by the methods described above. These compounds may be formulated into suitable pharmaceutical compositions, including suitable carriers, for administration to a patient in need thereof. Details of such compositions are provided above, and it is thought that some of the specific DNMT and HDAC inhibitors may prove to act positively in the screening methods according to this aspect of the invention.

In addition, there is also provided a method of treating melanoma comprising administering to a subject in need thereof a therapeutically effective amount of a compound which has been identified using the screening methods of the invention.

The invention will now be further described, by way of example, with reference to the following experimental section and figures in which:

FIGURE LEGENDS

Fig. 1. Phenotypic characteristics of melanoma cells in vitro .

A. Growth of parental WM793 cells and isogenic derivatives (WM793-P1, WM793-P2, and 1205-Lu). Cells (20 , 000/well) were seeded into 12-well tissue culture plates and left to grow for 7 days. Growth rates were measured using an alamarBlue assay for cell proliferation. AU = arbitrary units.

B. Invasive capacity of melanoma cells. Invaded cells were stained with Toluidene Blue, which was extracted and evaluated at Abs540nm. P value obtained using a Student t- test . In both A and B, error bars refer to the SEM of triplicate determinations.

Fig. 2. Comparative analysis of gene expression profiles.

The 66 genes identified as consistently differentially expressed between WM793 cells and the derivative cell lines, WM793-P1 and WM793-P2, are listed above. Panel A. Duplicate data with respect to gene expression obtained from WM793, WM793-P1 and WM793-P2 cell lines (separate cultures; replicate data listed beside each other) . Pairwise comparison of gene expression between different cell lines yielded the following numbers of altered transcripts: WM793 versus WM793-P1 (129 transcripts) ; WM793 versus WM793-P2 (114 transcripts) ; WM793-P1 versus WM793-P2 (14 transcripts) . Cross- comparison of the 129 and 114 transcript list yielded 68 commonly altered transcripts, of which two {TAC1 and TYRPl) are represented by an additional probe set. On average, inter-array variability between biological replicates was observed to be 2.18% (155/7,129 probe sets) .

Panel B. Independent dataset showing expression of 66 gene cohort in untreated (WM793 and 1205-Lu) and DAC treated (all four cell lines) cells. Gene expression profile information is represented using a color-coded scheme (key provided) in which dark grey refers to genes expressed at a low level (below mean absolute intensity) and white refers to genes expressed at a high level (above mean absolute intensity) . From left to right, the tabular columns refer to the Affymetrix probe set identifier, the corresponding UniGene cluster (Build_ #166) , associated chromosomal location and Human Genome Organisation- approved gene symbol for each transcript. The additional three columns detail the fold change values between WM793 and respective derivative cells. With respect to the last column: +, CpG island found around presumed transcription start site or near upstream region; -, no CpG island found in these regions; NA, upstream sequence is unknown. Crosses signify that upstream genomic sequence were obtained using either EZRetrieve or ENSEMBL.

Fig. 3. Validation of DNA microarray results by two RT-PCR analysis methods.

The vertical white and black bars refer to genes that were identified from the prior DNA microarray study as either down-regulated or up-regulated, respectively, in derivative cells as compared to parental WM793 cells.

Genes are listed according to same order shown in Fig. 2.

A. RT-PCR analysis, via a cycle limitation limitation method, of transcripts previously shown to be down- regulated [CYBA, GPNMB, DCT, TYRPl, TYR, MXl) or up- regulated {H0XD4, PRKl₁ SIAT7B, RARB) in the derivative cell lines. Level of 18 S rRNA served as a loading control .

B. Real-time RT-PCR analysis via TaqMan Low Density Arrays. Forty-five of the 66 differentially expressed genes were assessed for expression level in the WM793 series.

Expression measurements were normalized to 18 S rRNA levels. The parental WM793 cells were defined as the calibrator to which all three derivative cell lines were compared. The three data columns to the right represent the mean normalized relative quantities of target gene expression between WM793 cells and relevant derivative cells across duplicate measurements from independent stocks of exponentially growing cells. Gene expression profile information is represented to the right using a color-coded scheme (key provided below) in which white and black refer to down-regulated and up-regulated genes (as compared to WM793 cells) , respectively. Grey indicates no change in expression level .

Pig. 4. Regulation of TSPYl gene by DNA methylation. A. Northern blot analysis of TSPYl mRNA expression. Order of parental WM793 cells and derivatives indicated.

B. Effect of DAC treatment on TSPYl gene expression in melanoma cell lines. Total RNA was extracted from untreated (- DAC) and treated (+ DAC) cells and subjected to RT-PCR analysis via a cycle limitation method. The efficiency of DAC treatment was assessed by global DNA methylation analysis (see Figure 6 and 7) . Same order of samples as in A.

C. schematic representation of 5' region of TSPYl gene, including Exon 1 (black bar) . Amplicon assessed by COBRA al-so indicated (light bar) , along with relevant restriction enzyme (BsiEI) cleavage site. Vertical bars indicate CpG sites.

D. COBRA of TSPYl gene using genomic DNA extracted from melanoma cell lines, D, and metastatic melanomas,

E. Presence of 191 bp fragment signifies DNA methylation at CpG within BsiEI site. For D, same order of samples as in A. For E, genomic DNA from 20 randomly assigned metastatic melanomas (5 male, 15 female) . Samples in lanes 1, 2, 7, 8 and 13 were derived from male patients, with the remainder being from female patients. Only male- derived samples generated PCR products by COBRA, which is as expected given the chromosomal localization of the TSPYl gene.

Pig. 5. Effect of DAC treatment on melanoma cells in vitro and in vivo. A. Cells (20, 000/well) were seeded into 12-well tissue culture plates and left to grow for 7 days, while being simultaneously treated with DAC. Growth rates were measured using an alamarBlue assay for cell proliferation. Error bars refer to SEM of triplicate determinations.

B. 1205-Lu cells were injected subcutaneously into athymic female CD-I nude mice. The relative tumor volume (volume of tumor/volume of tumor at Day 1 of measurement) was recorded daily over 22 days, following an initial growth period (mean tumor volume ~ 0.1 cm³) . At day 7, animals were either treated with DAC (15 mg/kg/mouse) or PBS alone (n = 8 animals/group) . Error bars refer to the SEM of 8 determinations. There was no significant difference in body weight of treated and untreated groups over observation period (data not shown) . Significant di-fferences (P values < 0.05) in terms of tumor growth rates were observed between treated and control groups both on an overall basis (Day 22) and over time from Days 7 - 15. Statistical analysis was performed using an analysis of variance test.

C. RT-PCR analysis of TSPYl mRNA expression in tumours explanted from untreated and DAC treated mice using a cycle limitation method.

Figure 6. Ploidy of WM793 series.

A. FISH analysis on the WM793 series using chromosome X (DXZl), Y (DYZl) and Hq probes. The number of copies of each probe (labeled with different flours) was counted in individual cells and averaged. B. Flow cytometric analysis of the WM793 series. DNA content is reflected across the X-axis (propidium iodide staining). Cell counts are indicated on the Y-axis. To determine alteration in ploidy from the parental cells, the derivative cells were mixed at equal ratios with WM793 cells prior to flow sorting.

Figure 7. Global DNA methylation status in the WM793 series .

A. Measurement of 5mC content by high performance capillary electrophoresis.

B. 5mC content as a percentage of the total cytosine pool, mC peak area * 100/ (mC peak area + C peak area) .

Results are expressed as mean values with standard errors of the mean represented by bars . A Student t test was performed for each comparison of untreated and DAC treated cells; *** indicates the difference is highly significant, as indicated by a P value < 0.001.

Figure 8 TSPYl genomic sequence.

Exons 1 through 6 exemplified by M98524, specifically the sequence of unmodified DNA (bases 834 to 1102) . Bolded text indicates the 269 bp sequence within Exon 1 amplified by PCR (following bisulfite modification of DNA) , which was used for COBRA. Italicised text indicates forward and reverse primer regions (see COBRA diagram for exact primers used to amplify bisulfite-modified DNA) . Underlined sections represent the exons.

Figure 9 Region of TSPYl gene examined by COBRA

Sequence of 269 bp PCR product from TSPYl gene (Exon 1) , derived from amplification of bisulfite-modified DNA. In this schematic, all CpGs are indicating as being hypothetically methylated, and thus cytosines are not exchanged for uracil during bisulfite modification. Bolded text refers to restriction enzyme sites (Acil, BsiEI and Taql) whose consensus sequences contain CpG, and thus may be used for COBRA. BsiEI was used to conclusively demonstrate methylation of TSPYl (see Figure 4) . Italicised text refers to exact forward and reverse primers used for PCR amplification.

Figure 10. CpG islands identified in genes for which methylation has direct effect on regulation of gene expression.

CpG island prediction of TSPYl, CYBA, RPL37A, MXl, HSPBl and MT2A using EMBOSS. Genomic DNA sequences of TSPYl, CYBA, RPL37A and MXl were retrieved from EZRetrieve (http : //siriusb.umdnj . edu : 18080/EZRetrieve/multi_r. j sp) using UniGene (TSPY=Hs_2051 ; CYBA=Hs_68877;

RPL37A=Hs_433701) or GenBank (MXl=NM_002462) IDs in search. DNA from -2,000 to +1,000 relative to the transcription start site was selected and retrieved. Ensemble was used to search for HSPBl and MT2A (http : //www. ensembl . org/Homo_sapiens/) , using gene name in search criteria. The sequence used was HSPBl=ENST00000248553 and MT2A= ENST00000245185. Here, -2,000bp prior to the first exon and 1,000 downstream of the first exon was selected. Each DNA sequence was analysed using EMBOSS at (http://www.ebi.ac.uk/emboss/cpgplot/). All settings were set at default setting except for window and length, which were set at 200bp. The results as follows, and shown as the plot results in Fig 1OA and relevant nucleotide sequences in Fig 1OB with CpG islands in lighter shade and also underlined: TSPYl

Length 342 (169..510)

Length 369 (1875..2243) [Region where COBRA was performed] Length 397 (2446..2842) Note, the second and third islands are merged in the sequence data due to their proximity to one another.

CYBA

Length 311 (1867..2177) Length 231 (2212..2442)

RPL37A

Length 313 (1811..2123) Length 413 (2138..2550)

MXl

Length 1062 (1734..2795)

HSPBl Length 1154 (1740..2893)

MT2A

Length 591 (1535..2125)

Figure 11: Schematic of methylated genes

Schematic representation of CpG dinucleotides in the promoter region and first exon of analysed genes in the WM793 series. White oval indicated unmethylated residue and black oval represents methylated residue. Arrow indicates approximate start site. EXPERIMENTAL SECTION

ABSTRACT

The incidence of melanoma is increasing rapidly, with advanced lesions generally failing to respond to conventional chemotherapy. Here, we utilized DNA microarray- based gene expression profiling techniques to identify molecular determinants of melanoma progression within a unique panel of isogenic human melanoma cell lines. When a poorly tumorigenic cell line, derived from an early melanoma, was compared with two increasingly aggressive derivative cell lines, the expression of 66 genes was significantly changed. A similar pattern of differential gene expression was found with an independently derived metastatic cell line. A considerable proportion of the differentially expressed genes found have been previously associated with melanoma development and progression, including CDKN2A, IL-24 and MAGEA4. In addition, a range of novel markers were identified that correlated here with melanoma progression. Most notable was TSPYl, a Y chromosome-specific gene that displayed extensive down- regulation in expression (between 137 and 317 fold) between the parental and derivative cell lines. Examination of a putative CpG island within the TSPYl gene demonstrated that this region was hypermethylated in the derivative cell lines, as well as metastatic melanomas from male patients. Moreover, treatment of the derivative cell lines with the DNA methyltransferase inhibitor, 2' -deoxy-5-azacytidine (DAC) , restored expression of the TSPYl gene to levels comparable to that found in the parental cells. Additional

DNA microarray studies uncovered a subset of 13 genes from the above-mentioned 66 gene cohort that displayed re- activation of expression following DAC treatment, including . TSPYl, CYBA, and MT2A. DAC suppressed tumor cell growth in vitro. Moreover, systemic treatment of mice with DAC attenuated growth of melanoma xenografts, with consequent re-expression of TSPYl mRNA. Overall, our data support the hypothesis that multiple genes are targeted, either directly or indirectly, by DNA hypermethylation during melanoma progression.

INTRODUCTION

Cutaneous melanoma is a pigmented, readily accessible lesion that has been well defined in histopathological terms (3) . Early radial growth phase (RGP) melanomas can invade into the epidermis and papillary dermis, but have no capacity for metastasis; resection at this stage is almost completely curative. A subsequent vertical growth phase (VGP) denotes a transition to a more aggressive stage, which is capable of metastasis. Changes in gene expression occurring at the RGP/VGP transition are, thus, of great interest. However, comparative transcriptomic studies have so far been hindered in this arena, as paired RGP/VGP biopsies are not normally available (since resection of RGP melanoma is often curative and no VGP develops) . Here, we utilized a unique isogenic cell line model series that allows us to circumvent the lack of availability of such paired samples from the clinic.

The parental cell line in the series, WM793, was originally isolated from a superficial spreading melanoma (14) . The patient concerned has had no re-occurrence of the disease to date, suggesting that these cells had low metastatic potential. Accordingly, WM793 cells displayed poor tumorigenicity in nude mice (15) . Notably, the WM793 . cell line was used as the basis for in vivo selection of several aggressive, tumorigenic sublines (15) . In this respect, the derivative cell lines, WM793-P1 and WM793-P2, were established after inoculation in the presence of Matrigel (a reconstituted basement membrane extract) . These isogenic sublines exhibited properties of cells isolated from advanced VGP melanoma: they were highly tumorigenic in nude mice and displayed tnulti-cytokine resistance in vitro. A further cell line (1205-Lu) was derived independently at the Wistar Institute from a lung metastasis of WM793 after subcutaneous injection into the tail veins of immunodeficient mice; these cells exhibited spontaneous metastasis (16) .

In- this study, a high-density oligonucleotide array-based approach was employed to identify genes that varied in expression level between the parental and derivative cell lines. We hoped, in particular, to elucidate key molecular determinants of the RGP-VGP transition, as well as obtain additional mechanistic insights into melanoma progression.

MATERIALS AND METHODS

Cell Lines.

Conditions for culture of the melanoma cell lines used here and their original sources have been described previously (17, 18) . In brief, WM793 and 1205-Lu cells were a gift from Prof. Meenhard Herlyn (Wistar Institute, Philadelphia) , whereas WM793-P1 and WM793-P2 cells (both Nl sublines in the WM793 derivative series; 19 Kobayashi) were a gift from Prof. Robert Kerbel (University of Toronto, Canada). Cells were maintained in Dulbecco's Modified Eagle's Medium with GlutaMAX (Invitrogen) , supplemented with 10% foetal calf serum, 100 U/mL penicillin, 100 μg/ml streptomycin and 4 μg/mL insulin (Sigma-Aldrich) .

Melanoma Biopsies.

Previously extracted DNA from 20 anonymized metastatic melanomas was kindly provided by the Department of Dermatology, University of Glasgow, UK) , according to standardized ethical procedures set out by the University of Glasgow.

Nucleic Acid Extraction. Genomic DNA and total RNA were extracted from monolayer cells in culture and melanoma biopsies using the QIAamp DNA Mini (Qiagen) kit . Total RNA was extracted from monolayer cells using the Tri Reagent (Sigma) kit.

Cell Growth and Invasion Assays.

Cell growth rates were determined using an alamarBlue assay (Biosource International) , which recorded cell proliferation each day. For invasion assays, cells were seeded into Transwell ^' filters (pore size 8 μm; Costar) coated by Matrigel (1 μg/μL; 50 μg total) , with fibronectin (5 μg/mL) used as a chemoattractant . Flow Cytometric Analysis. A total of IxIO⁶ cells were used for flow cytometric analysis of DNA content. Exponentially growing cells were isolated by trypsinization, washed with 500 μL PBS, and fixed with 500 μL of ice-cold 100% ethanol . Fixed cells were centrifuged and resuspended in 125 μL of RNase solution (1 μg/μL in 1.12% w/v of sodium citrate) and incubated at 37°C for 15 minutes. Following this, cells were incubated with 125 μl of propidium iodide, solution (0.5 μg/μL in 1.12% of sodium citrate) for 30 minutes at room temperature. Samples were then analysed on a Coulter Epics flow cytometer (Beckman Coulter) .

CGH and FISH Analysis. Comparative genomic hybridization (CGH) experiments were carried out as previously described (19) . Briefly, test and reference DNA samples were labeled by nick translation with spectrum green-dUTP and red-dUTP, respectively, under conditions recommended by the supplier (Vysis) . Labeled test (melanoma cells) and reference (normal lymphocyte) DNA (500 ng) were then denatured and hybridized to normal human metaphase , chromosomes in a solution containing 50x Cotl fractionated DNA, 50% formamide, IX SSC, and 10% dextran sulfate (Vysis) . Images were acquired and analyzed using hardware and software from Applied Imaging Inc. For fluorescence in situ hybridization (FISH) experiments, a dual-colored fluorescently labeled probe to specific regions of chromosomes X (Spectrum Green; DXZl) and Y (Spectrum Red; DYZl) was used according to the manufacturer's instructions (Vysis) . A probe to Hq (bacterial artificial chromosome clone RPIl- 163A13 from the Sanger Institute, UK) was also used to determine ploidy.

DAC Treatment In Vitro.

Seeded cells were treated with 2 μg/mL 2'-deoxy-5- azacytidine (DAC) on Days 1, 3 and 5, with fresh drug- containing medium being added at each timepoint (20) . On Days 2 and 4, drug-containing medium was exchanged for drug-free medium. On Day 6, cells were harvested and total RNA extracted as above. Global DNA Methylation Analysis.

5-methylcytosine (5mC) genomic content was determined by high-performance capillary electrophoresis, as previously described (21) . Briefly, genomic DNA samples were boiled, treated with nuclease Pl (Sigma) for 16 hours at 37⁰C, and with alkaline phosphatase (Sigma) for an additional 2 hours at 37⁰C. After hydrolysis, total cytosine and 5mC content were measured by capillary electrophoresis using a P/ACE MDQ system (Beckman-Coulter) . Relative 5mC content was expressed as a percentage of total cytosine content (methylated and non-methylated) .

DNA Micrσarray Analysis. Ten micrograms of total RNA from each cell line was re-verse transcribed into single-stranded cDNA using the Superscript Choice kit (Invitrogen) .. For this purpose, an oligo-dT primer containing a T7 RNA polymerase promoter (Genset) was utilized. Following double-stranded cDNA synthesis, biotin-labelled cRNA was generated by in vitro transcription using the BioArray RNA labelling kit (Enzo) . These complex cRNA targets, which are representative of the transcriptome of a particular sample, were hybridised against HuGeneFL arrays (7,129 probe sets) . Detection was accomplished via a streptavidin-labelled fluorochrome

(phycoerythrin) and laser scanning. Normalisation of data and inter-array comparisons of gene expression profiles was carried out using Microarray Analysis Suite (MAS) software v4.0 (Affymetrix) , together with Microsoft Access. In more detail, DNA microarray experiments were analyzed using an approach based on the Mann-Whitney pairwise comparison test (22) . To identify differentially expressed genes between any two samples, pairwise comparisons were performed using MAS. Lists of altered transcripts from different pairwise comparisons were sorted via Microsoft Access. Additional bioinformatic analysis was completed using publicly available annotation databases and software tools, notably TIGR Multiple Experiment Viewer v2.0. Available upstream genomic sequences of identified differentially expressed genes were automatically retrieved using either EZRetrieve or ENSEMBL. Putative promoter-associated CpG islands at or around presumed transcription start site were identified using CpGPlot . The raw DNA microarray data has been submitted for public access to Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and can be obtained using the following accession numbers: GSE1792 and GS-E1793.

RT-PCR Analysis via Cycle Limitation.

RNA extracts were pre-digested with DNase I prior to cDNA synthesis using the DNA-Free kit (Ambion) . Single-stranded cDNA was synthesized from 1 μg total RNA using the ImProm- II Reverse Transcription kit (Promega) . Recombinant RNasin Ribonuclease Inhibitor (20 U/20 μL reaction; Promega) was added to prevent RNase-mediated degradation. Two negative controls were also utilised, namely minus reverse transcriptase enzyme control and minus template control . Following inactivation of reverse transcriptase (RT) at 70⁰C for 15 minutes, aliquots of generated single-stranded cDNA were subjected to polymerase chain reaction (PCR) amplification via a cycle limitation approach. The following primer pair combinations were used: 18 S - forward, 5'-AGGGTTCGATTCCGGAG-S' (SEQ ID NO: 19) and reverse, S'-ACCAGACTTGCCCTCC-S' (SEQ ID NO: 20) (195 bp amplicon) ;

DCT - forward, 5'-AGTGATTCGGCAGAACATCC-S' (SEQ ID N0:21) and reverse, 5'-AGTTCCAGTAGGGCAAAGCA-S' (SEQ ID NO: 22) (368 bp amplicon) ;

GPNMB - forward, 5 ' -TGCATAAAGCCAATGTAGTCCA-3 ' (SEQ ID N0:l) and reverse, S'-CAGGGACCTCATCTTTGGAA-S' (SEQ ID NO: 2) (373 bp amplicon) ;

TYR - forward, δ'-CAGCTTTCAGGCAGAGGTTC-S' (SED ID NO: 23) and reverse, S'-GCTTCATGGGCAAAATCAAT-S' (SEQ ID NO: 24) (470 bp amplicon) ;

TYRPl - forward, 5' -CCGAAACACAGTGGAAGGTT-3 ' (SEQ ID NO: 25) and reverse, 5'-ACCAGTAACAAAGCGCCAAC-S' (SEQ ID NO: 26) (406 bp amplicon) ;

CYBA - forward, 5'-TTTGTGTGCCTGCTGGAGTA-S' (SEQ ID NO: 3) and reverse, S'-CTCCTCGCTGGGCTTCTT-S' (SEQ ID NO: 4) (369 bp amplicon) ,-

MXl - forward, 5'-AGCCACTGGACTGACGACTT-S' (SEQ ID NO: 5) and reverse, S'-ACGGCACTCATGCTCCTAAA-S' (SEQ ID NO: 6) (335 bp amplicon) ;

H0XD4 - forward, 5 ' -TGACTCGCCAAGATTTTATGT-S ' (SEQ ID NO: 7) and reverse, 5'-CACCTCGCTGGGCTCTAA-S' (SEQ ID NO: 8) (190 bp amplicon) ; RARB - forward, 5'-ACAAGGTCAAAGGAGGCAGA-S' (SEQ ID NO: 9) and reverse, S'-TTCACAAGCCATTAGGGAAA-S' (SEQ ID NO: 10) (188 bp araplicon) ;

SIAT7B - forward, 5'-GGCACATCCTACCCCAGA-S' (SEQ ID NO: 27) and reverse, S'-AAGCAACTAACCCCCATCAA-S' (SEQ ID NO: 28) (272 bp amplicon) ;

PRKl - forward, 5'-GGGCTGTTTCTTCACATCTTC-SMSEQ ID NO: 11) and reverse, S'-GTGGACTGGTGGGGACTG-S' (SEQ ID NO: 12) (231 bp amplicon) ;

TSPYl - forward, 5'-CACCACAACAGCAGCCTTA-S' (SEQ ID NO: 13) and reverse, S'-TGCTCCATCATATTCAACTCA-S' (SEQ ID NO:14) (193 bp amplicon) .

PCR products were subcloned into the pCRII-TOPO vector via the Topo TA Cloning kit (Invitrogen) , with insert- containing plasmids then subjected to automated DNA sequencing via a commercial route (MWG) .

Real-Time RT-PCR analysis using TaqMan Low Density Arrays.

Pre-designed TaqMan probe and primer sets for target genes were chosen from an on-line catalogue (Applied Biosystems) . Once selected, the sets were factory-loaded into the 384 wells of TaqMan Low Density Arrays. Array format was customized on-line with two replicates per target gene. Expression levels of target genes were normalized to concentration of 18 S rRNA. Samples were analyzed using the 7900HT system with a TaqMan Low Density Array Upgrade (Applied Biosystems) , according to the manufacturer's instructions. In short, 2.5 μL of single- stranded cDNA (to final concentration of 100 ng starting RNA; see above) was combined with 47.5 μL water and 50 μL TaqMan Universal PCR Master Mix, following by loading of 100 μL sample per port. Thermalcycling conditions were as follows: 50⁰C for 2 minutes; 94°C for- 10 minutes; 97°C for 30 seconds, and 59.7°C for 1 minute. Gene expression values were calculated based on the ΔΔCt method, where one sample was designated the calibrator, to which all other samples were analyzed. Briefly, ΔCt represents the threshold cycle (Ct) of the target minus that of 18 S rRNA and ΔΔCt represents the ΔCt of each target minus that of the calibrator. Relative quantities were determined using the equation; relative quantity = 2 ^~ΛΔ Ct. For the calibrator sample (i.e. WM793 cells), the equation is relative quantity = 2 ^~°, which is 1; therefore, every other sample is- expressed relative to this.

Northern Analysis.

Eight micrograms of total RNA from each cell line was subjected to electrophoresis through an agarose- formaldehyde gel, followed by transfer to nitrocellulose membranes (17) . A TSPYl-specific cDNA probe, derived from the above-mentioned RT-PCR amplicon, was labeled by random hexanucleotide priming, with hybridization conditions as described previously (17) . Integrity and loading of RNA were determined by probing for GAPDH expression.

Southern Blot Analysis.

EcoRI-digested DNA (15 μg) was subjected to agarose gel electrophoresis, followed by transfer to nylon membrane, as described previously (23) . Use of the radiolabeled TSPYl-specific cDNA probe was as detailed above. Loading of DNA was determined by ethidium bromide staining.

COBRA.

For combined restriction bisulfite restriction analysis (COBRA) , genomic DNA was first subjected to bisulfite modification via the CpGenome DNA Modification kit (Intergen) ; this process converts unmethylated, but not methylated, cytosine residues to uracil . The genomic DNA sequence of the TSPYl gene was retrieved from GenBank (accession number: M98524) . A putative CpG island within the first exon of the TSPYl gene was identified using CpGPlot (Figure 4C) . The following primer pairs (designed against bisulfite-modified DNA) were used to amplify this region by PCR: forward, 5 ' -GGTAGTATAGGTTTTGGTGTGTG-3 ' (SEQ ID NO: 17) and reverse, 5'-CCAACACCTCCTCCAATACAAAC-S' (SEQ IDfNO: 18). Amplified PCR products (269 bp in length) were incubated with BsiEI (20 U/20 μL PCR product) for 2 hours at 60oC, with restriction digests subsequently examined by agarose gel electrophoresis.

Human Tumor Xenografts .

Animal studies were carried out under an appropriate United Kingdom Home Office Project Licence. All work conformed to the UKCCR guidelines for the welfare of animals in experimental neoplasia studies and was approved by the University of Glasgow Ethics Committee. Approximately 1 x 10^s cells in a volume of 100 μL PBS were injected subcutaneously into the left or right flank of athymic female CD-I nude mice (Charles River) . After 19 days, when the mean tumor volume was at least 0.1 cm³, the mice were weighed daily and tumor volumes were estimated by two calliper measurements assuming spherical geometry (volume = d3 x π/6) . Seven days later, when the mean tumor volume was at least 0.25 cm³, DAC (5 mg/kg/mouse) was administered by 3 intraperitoneal injections at 3 hour intervals over the course of a day (total dose, 15 mg/kg/mouse) . The control mice were injected with PBS where 10 μL of PBS was given per gram of mouse bodyweight.

RESULTS

Phenotype of Cells In Vitro.

The behavior of the WM793-based isogenic cell line model series has been well documented in vivo (15, 16) . However, there is only limited data available -with respect to characteristic features of these cell lines in vitro (15, 24) . The growth rate of the parental WM793 cells and three isogenic derivatives was examined over a period of 7 days (Figure IA) . The derivative cell lines exhibited more rapid rates of cell growth than the parental cells . Interestingly, the 1205-Lu cells showed an intermediate rate of cell growth, which may be due to the apparent increased propensity of this cell type to detach from the surface in monolayer culture. The isogenic derivatives also displayed increased invasive capacity over WM793 cells, with the most striking difference seen between 1205-Lu and parental cells (Figure IB) .

Genomic Aberrations .

A range of chromosomal abnormalities was observed in both the parental and derivative cells (Table I) . The four cell lines exhibited some identical or similar abnormalities, illustrative of the underlying genetic relatedness of the series. Moreover, the particular generic abnormalities found match those commonly present in melanoma biopsies (3, 25, 26). For example, there is a consistent gain of material on chromosomes 1, 6 and 7, where gene amplification frequently occurs in melanoma. In addition, certain progression-related abnormalities were identified, when the parental cells were compared to its various derivative cell types. Crucially, the two main derivative types, WM793-P1/WM793-P2 and 1205-Lu, exhibit some common alterations in this context (e.g. loss of 12ql0ql3 and 18p) , supporting the concept of non-random changes. Overall, there is a marked increase in the number of abnormalities observed in the derivative cells as compared to the parental cell line. FISH and flow cytometric analysis showed evidence of increased ploidy in association with progression in the model system, which is further indicative of genomic instability (see Figure 6) . As compared to WM793 cells, for example, FISH analysis showed increases in X, Y and Hq chromosomal regions in the derivative cells that were consistent with an increase in the numbers of tetraploid, triploid or hyperdiploid cells found in these cell line populations.

Table 1 CGH analysis of melanoma cells Cell line Genomic aberrations⁰

WM793 enh lqlOqter , 2ql0q35 , 6q22q27 , 7 , 8q, 2 Oq dim 10q24

WM793-P1 enh Iqlθq32 , 2ql0q35 , 5p , 6q22q27 , 7 , 8q22 ,

13q32 , 17q21q23 , 20q dim 10q24, 12ql0ql3, 18p

WM793-P2 enh Iqlθq32 , 2ql0q35 , ^"6q22q27 , 6qlθql6 , 7 ,

13q32 , 17q21q23 , 2 Oq dim 10q24, 12ql0ql3, 18, 18p 1205-Lu enh Iq₅ 2, 5p, 7, 7p+, 8q, lip, 12p, 13, 17pl3 dim 10q24, 12ql0ql3, 18, 18p ^αenh = enhanced green to red fluorescent ratio of chromosomal region (gain); dim = diminished green to red fluorescent ratio of chromosomal region (loss). Bolded text refers to melanoma progression-related differences, i.e. variation between parental and derivative cells.

Identification of Differentially Expressed Genes.

All four melanoma cell lines were subjected to gene expression profile analysis using high-density oligonucleotide arrays. In this case, total RNA from exponentially growing cultures was used. When the parental cell line in the series, WM793, was compared with the two increasingly aggressive derivative cell lines, WM793-P1 and WM793-P2, 66 genes were commonly altered with respect to- expression level (Figure 2A) . Within this cohort, 44 genes were identified as being down-regulated. A similar pattern of differential gene expression was found with the independently derived metastatic cell line, 1205-Lu (Figure 2B) . Downstream validation of a selected number of transcripts from this cohort via two RT-PCR analysis methods showed good concordance with the DNA microarray- derived data (Figure 3) .

A considerable proportion of the identified differentially expressed genes have been previously associated with melanoma development and progression. Reduced expression of the tumor suppressor genes, CDKN2A and IL-24, was observed in all three derivative cell lines as compared to the parental cells. Moreover, several genes (TYR, TYRPl, and TYRP2 ) involved in melanin biosynthesis exhibited a similar marked reduction in transcript levels in the derivative cell types. A subset of immune-related genes (including BST2 , CIS, ClR, HLA-DQAl, TNFAIPβ and LGALS3) and interferon-related genes (including GIPl, GIP3, IFITl, CASPl, ISGF3G, DAP and MXl) were also observed to be down- regulated in the derivative cells. Several genes encoding for tumor-associated antigens, such as MAGEA4 and GAGEl, displayed increased expression in the derivative cell lines. Intriguingly, the AIMl gene, which has been previously suggested as a tumor suppressor based on correlation in expression terms with experimental reversal of tumorigenicty via chromosome transfer, showed evidence of increased expression at the RNA level in the derivative cells .

In addition, a range of novel markers were identified that correlated with melanoma progression. Most notable was TSPYl, a Y chromosome-specific gene that displayed marked down-regulation in expression (between 137 and 317 fold, as determined from DNA microarray study) between the parental and derivative cell lines (Figures 2, 3 and 4A). The TSPYl gene has previously been shown to exhibit dysregulated expression in a number of cancer types, including gonadoblastoma, as well as testicular and prostate cancer (20, 27-30) . Although Y chromosome loss has been described for certain melanomas, this is not a common event. Moreover, FISH analysis showed retention of this chromosome in all four cell lines under study (see Figure 6) . Moreover, Southern blot analysis showed no evidence for deletions or gross re-arrangement of the TSPYl gene (data not shown) . TSPYl gene expression is regulated by androgens and DNA methylation (20) . This suggested that aberrant DNA methylation may have a role transcriptional silencing of TSPYl gene expression between early and advanced melanoma cell lines.

Regulation of Gene Expression by DNA Methylation. Treatment of the derivative cell lines with DAC restored expression of the TSPYl gene to levels comparable to that found in the parental cells {Figure 4B) . Moreover, examination of a putative CpG island within the TSPYl gene demonstrated that this region was hypermethylated in all three derivative cell lines (Figure 4C and D) . In addition, hypermethylation of the TSPYl gene was observed in metastatic melanomas from 5 male patients (Figure 4E) . Further DNA microarray studies uncovered a subset of 13 out of the 44 (29.5%) down-regulated. genes that displayed consistent re-activation of expression following DAC treatment across all three derivative cell lines, including TSPYl, CYBA, and MT2A (Figure 2B and Table II) . This is in contrast to only 3.24% of all transcripts represented on the DNA microarray that exhibited elevated expression in all three derivative cell lines following

DAC treatment. However, 7 out of the 13 genes do not have 5' CpG islands (Figure 2) , so are not likely to be directly regulated by DNA methylation. This phenomenon has been reported previously by Liang et al . (31) among others. In summary, multiple transcripts that are potential markers for melanoma progression can be increased following DAC treatment suggesting that the relevant genes are suppressed in terms of expression by DNA methylation, either directly or indirectly, in the more aggressive derivative cell lines. Table 2. Melanoma progression-related genes de-repressed by DAC treatment"

Pl^b P2 Lu 793 -DAC Pl-DAC P2-DAC Lu-DAC

V V V V V V V

793 793 793 793 793 793 793

TSPYl -316.9 -316.8 -137.0 1.1 -7.0 -13.9 -13.0

CYBA -101.0 -102.0 -73.0 1.9 -39.4 -14.9 1.0

MT2A -48.2 -51.4 -6.0 2.5 -2.3 1.0 1.0

BST2 -35.7 -40.9 -23.0 2.1 -2.8 -3.2 -4.0

GIP3 -26.6 -15.9 -2.6 2.5 1.0 -1.6 1.4

SlOOAl -14.2 -16.2 -4.6 2.0 -1.4 1.0 2.5

IFITl -11.7 -8.1 -13.9 9.2 -1.4 1.0 1.0

MXl -9.3 -7.4 -8.6 4.9 1.0 -1.6 4.0

RGS3 -3.7 -4.1 -4.9 -1.9 -2.6 -3.2 -4.3

ISGF3G -3.3 -3.0 -1.9 1.2 -1.4 1.0 1.0

APOD -3.3 -3.7 -2.1 2.1 1.0 1.0 7.0

RPL37A -2.3 -2.1 -1.7 -1.6 1.0 1.0 1.0

HSPBl -2.3 -2.4 -3.2 2.0 -1.5 1.3 1.0

"Fold difference in gene expression indicated relative to untreated WM793 cells. ^bPl-, WM793-P1; P2, WM793-P2; Lu, 1205-Lu; -DAC, DAC treated cells.

DAC-Mediated Inhibition of Cell Growth In Vitro.

The growth rate of all four melanoma cell lines was examined over a period of 7 days under DAC treated conditions (Figure 5A) . As compared to untreated cells (Figure IA) , the growth of both the parental and derivative cell lines was suppressed in response to DAC (Figure 5A) , albeit with the latter cell types showing a slightly delayed response in this respect. The DAC- mediated inhibitory effect on cell growth was most marked in the case of the 1205-Lu cells. Overall, this data suggests that alteration of DNA methylation has a significant effect on cellular phenotype in our model system. However, one cannot current exclude the possibility that the DAC-mediated inhibition of cell growth may also be, at least in part, due to a direct cytotoxic action, which is independent of DNA methylation.

Suppression of Tumor Growth In Vivo Following DAC Treatment . To further examine the functional role of DNA methylation in regulating tumor growth of an aggressive derivative, we examined the effect of systemic DAC treatment on 1205-Lu xenografts in vivo (Figure 5B) . Systemic DAC treatment (15 mg/kg) significantly attenuated tumor growth in comparison to untreated mice. Again, one cannot preclude the possibility that a direct cytotoxic action by DAC on tumor growth also play a part in this effect. The inhibitory effect of DAC on tumor growth was seen to cease 7 days post-treatment. This is consistent will the reversible nature of demethylation induced by DAC in xenograft models (32) . Intriguingly, while the DAC treated tumors display renewed growth capacity following withdrawal of the demethylating agent, re-expression of TSPYl mRNA was seen to be maintained in 3 out of 4 explanted tumors (Figure 5C) , suggesting a potential use of this gene as a residual biomarker for DAC activity. In summary, DAC treatment may facilitate tumorigenic reversion of advanced melanomas.

DISCUSSION The development of culture techniques permitting the establishment and propagation of cells derived from histologically defined stages of melanoma progression has permitted major advances. However, comparative studies of RGP and VGP melanoma have been limited since it has not generally proved possible to establish cell lines from each of these stages from one individual patient. Here, we have used a unique isogenic cell line-based model system that circumvents this problem.

We compared the gene expression profile of an early melanoma cell type with a variety of isogenic derivative cell lines of increasing aggressiveness. Accordingly, the expression patterns of 66 genes were identified as correlating with melanoma progression. Amongst these, we found a large number of genes previously associated with melanoma, including several tumor suppressor genes and antigenic markers, as well as genes involved in melanin biosynthesis. These commonly encountered alterations, together with the noted similarities in terms of chromosomal aberrations observed between the cell lines used here and melanoma biopsies, adds confidence to our current model of melanoma progression. However, further work at the RNA level with melanoma biopsy samples would shed additional light on this collection of putative melanoma progression-associated markers.

Of the 66 genes examined, TSPYl displayed the most striking change in gene expression terms within the WM793 series.. The TSPYl gene is found in multiple copies (20 - 40 based on current predictions) on both the long and short arm of chromosome Y (30, 33) . The TSPYl gene is normally expressed in the germ cells of the testis and distinct subsets of spermatogonia (34, 35). Apart from an assumed activity in spermatogenesis (34, 36), the functional role of TSPYl remains to be elucidated (33) . Previous work had implicated TSPYl as a putative oncogene based on its elevated expression in some gonadoblastomas, as well as testicular and prostate cancers (27-30, 37) . This contrasts with our observation of extensive down- regulation of TSPYl gene expression during melanoma progression. In addition, Dasari et al . (20) found TSPYl gene expression to be suppressed in certain prostate cancer cells. While this study highlights the TSPYl gene as a promising marker for melanoma progression and, potentially, DAC activity, further work will be required to clarify its role in cancer. Our data, however, provides an intriguing hypothesis that there may be sex-specific markers of melanoma, which may be useful in discriminating differences in terms of disease progression between males and females (2) , with the TSPYl gene being a promising candidate in this respect .

Detailed examination of the 66 gene cohort pointed towards DNA methylation as having a potentially important role in mediating gene expression alterations between parental and derivative cell lines. Demonstration of hypermethylation at a candidate CpG island within the TSPYl gene in the derivative cell lines provided initial support for this concept. This was further backed by the noted derepression of a significant proportion of down-regulated genes following DAC treatment in vitro. Overall, our data supports the hypothesis that multiple genes are targeted, either directly or indirectly, by DNA hypermethylation in this melanoma model system.

The role of DNA methylation in carcinogenesis is complex: global hypomethylation and region-specific hypermethylation co-exist (38) . DNA hypermethylation at CpG islands is known to be associated with epigenetic silencing of tumor suppressor gene expression and may increase genomic instability (39) . DNA hypermethylation can occur at all stages of tumor development and progression (40) . Previous studies have identified a number of genes that are affected by alterations in DNA methylation patterns in melanoma cells, including CDKN2A (41), PTEN (42), APAF-I (43), MAGEAl^' (44), TIMP3 (13), GAGED2 (45) , various human leukocyte class I antigens (46) , and CASP8 (47) . Our data adds a further collection of putative methylation-sensitive genes in melanoma.

DNA microarray-based gene expression profiling technology has been previously utilized in several cancer-related model systems to identify genes that are regulated by DNA methylation (13, 44, 48, 49) . Notably, van der Velden et al . (13) identified 19 genes, including TIMP3 and TYRPl, that were differentially expressed between a demethylated de-rivative clone of a primary uveal melanoma cell line and its untreated control. Our study provides further insight by linking changes in gene expression between early and advanced melanoma with DNA methylation.

It is clear that both epigenetic and genetic events contribute to determining tumor development and progression (50) . In keeping with the proposed link between DNA hypermethylation and genomic instability, CGH analysis demonstrated an increased number of chromosomal abnormalities in the derivative cell lines, as compared to the parental cells. While this analysis revealed no major striking correlations between gene expression and specific genomic aberrations in the four cell lines examined here (Figure 2 and Table I) , whole genome CGH microarray studies might provide additional clarification of this issue. It should also be noted that DAC treatment induced complex changes in gene expression, including reduced expression of certain molecular markers in the derivative cell lines (Figure 2) , as might be expected from such a broad-spectrum agent.

There is a rapidly increasing interest in the potential of epigenetic modifiers in the treatment of cancer (51) . In many cancer types, the use of DNA methyltransferase and histone deacetylase inhibitors have shown to be useful in mediating suppression of tumor growth and increased activity of other anti-cancer agents (32, 52, 53). However, the use of such agents for melanoma therapy has been inconclusive.

In an early Phase II clinical trial, Abele et al . (54) showed only one response in a set of 20 patients with melanoma treated with DAC. Following this negative result, however, a number of studies have provided more support for the use of DAC in the treatment of melanoma (53, 55, 56). Anzai et al . (55) showed a synergistic effect between DAC and the topoisomerase I inhibitor, topotecan, against melanoma cells in vitro, suggesting that combination therapies of DAC and other drugs may have more beneficial effects than DAC alone. In agreement with this concept, Coral et al . (56) noted that DAC in combination with the inhibitory cytokine, IFN-γ, enhanced the expression of human leucocyte class I antigens together with certain co- stimulatory molecules, such as ICAM-I and LFA-3, in a panel of 12 metastatic melanoma cell lines. Moreover, DAC treatment yielded a persistent (> 60 days) expression of MAGE-I in one of the melanoma cell lines. This DAC/IFN-γ combination may enhance the immunogenic potential of melanoma cells, thereby increasing the efficacy of immunotherapy. More recently, Kozar et al . (53) showed that combined treatment of DAC and IL-12 significantly attenuated growth of B16F10 melanoma cell-derived tumors in vivo, in comparison to moderate anti-tumor effects when either agent was given alone. This further supports the use of DAC as an immunomodulatory agent for complementation with inhibitory cytokines for the treatment of melanoma.

Interestingly, Wang et al . (57) proposed that responsiveness of melanomas to immunotherapy is predetermined and may be deciphered from analysis of gene expression profiling data. Within the 66 gene cohort, we noted down-regulation of a substantial cohort of immune- related and interferon-related genes in the derivative cell lines, which may be illustrative of the previously documented resistance to various inhibitory cytokines (19 Kobayashi) .

In our study, DAC suppressed tumor cell growth in vitro. Moreover, systemic treatment of mice with DAC attenuated growth of 1205-Lu-derived xenografts, with consequent re- expression of TSPYl mRNA. While this data might, in some part, be due to a direct cytotoxic action by DAC, our overall data point towards the concept that regional DNA hypermethylation at multiple loci is likely to be involved in the epigenetic regulation of melanoma progression. DAC also has an inhibitory effect on growth of WM793 cells in vitro, as well as being able to mediate complex changes in gene expression in this cell type. Given that the WM793 cell line is representative of an early melanoma, this suggests that the phenotype of these cells is also controlled, to a certain degree, by DNA methylation events. The relative enrichment of methylation-responsive markers in the identified set of down-regulated melanoma progression-related genes across the three derivative cells, however, suggests that further hypermethylation has occurred as one progresses through the WM793 series. This hypothesis is in keeping with the current model of acquisition of epigenetic marks during tumor progression. Our data provides further support for the incorporation of demethylation agents into clinical trials. Additional work is required to determine potential for synergy with other epigenetic modifiers and conventional therapies in terms of altering gene expression and therapeutic responses. In conclusion, a better understanding of melanoma progression, as exemplified by this study, may translate into new therapeutic avenues for this intractable disease.

Bisulphite sequencing of gene promoter regions. Bisulphite sequencing was carried out for the MT2A (MTlE) and CYBA genes to determine whether there was differential methylation in the more invasive derivative cells compared to the parental cells. The protocol utilised was as follows :

Bisulphite Modification

Put one heating block at 37°C and another at 50⁰C. Make reagents fresh each time.

REAGENTS

• Sodium bisulphite 4.3M pH 5 [S9000/500g/Minimum 99% SIGMA] . Mrl25.33 o Dissolve 7.6g in 20ml dH₂O. Add^' 500μl 1OM NaOH. This should give a solution of pH 5.

• Hydroquinone 2OmM [H9003/l00g/99+% SIGMA] o Hydroquinone is photosensitive so wrap tube in tinfoil prior to beginning. Dissolve 90mg in 50ml dH₂O.

• NaOH 3M o Dissolve 1.2g in 10ml dH₂O.

DNA MODIFICATION WITH SODIUM BISULPHITE • Dilute lμg DNA in 50μl H₂O in an eppendorf .

• Add 5.7μl 3M NaOH to each sample and incubate for 10-15 mins at 37°C. o Resulting solution contains ssDNA sensitive to bisulphite. • Add 33μl 2OmM hydroquinone. This is an antioxidant that prevents intermediate oxidation. Solution should turn yellow.

• Add 530μl 4.3M sodium bisulphite pH 5 to each sample. Mix well and incubate at 50⁰C for 16-17 hrs . Ensure the tubes are in darkness. Between 16 and 17 hours is optimal for this reaction and as such the incubation time should be strictly adhered to.

PURIFICATION AND PRECIPITATION OF MODIFIED DNA Promega Wizard DNA Clean-Up System Using Vacuum Manifold (catalogue no: A7280) .

• Title both syringes and columns, and attach to manifold.

• Prepare vacuum system by blocking unused lanes, closing fluxes and vacuum.

• Insert syringes .

• Add 1 ml resin to eppendorfs containing modified DNA. o Mix resin before use. (Guanidine thiosianate)

• Add mix to syringes .

• Open vacuum and then flux. Once the resin has passed, close fluxes and vacuum to prevent drying out . • Add 3ml 80% isopropanol and allow to flow through.

• Repeat with ImI 80% isopropanol.

• Discard syringes and add columns to old eppendorfs. Centrifuge at 13,000rpm for 1 min to remove trace isopropanol . • Add columns to a new eppendorf and add 50μl H₂O. Allow to stand for 3 mins at room temp .

• Centrifuge at 13,000rpm for 1 min. DNA is in eluate .

• Add 5.7μl 3M NaOH to tubes and incubate for 15-20 mins at_37°C. This is to complete the chemical conversion of C to U and eliminate salts.

• Thaw glycogen (20mg/ml) .

• Make a master mix of 0.5μl glycogen (20mg/ml) with 17μl 1OM ammonium acetate, per sample.

• Add glycogen mix to each tube and 3 vols of ice cold absolute ethanol . Incubate at -70⁰C overnight in darkness.

• . Following incubation, centrifuge at 12,000rpm for 20 mins at 4⁰C. • Discard the supernatant, wash with ImI of ice cold 70% ethanol, and centrifuge at 4⁰C for 20 mins.

• Discard supernatant and dry pellet (speedvac: 10 mins at 45°C) .

• Resuspend with 25μl H₂O.

Final solution should be at a concentration of 25ng/μl. 50ng (2μl) is used in the PCR. Following PCR, the band is excised and purified using the QIAquick Gel Extraction Kit (Qiagen) .

Cloning of PCR Products in Smaller Volume (using the Promega pGEM-T Easy vector system II; Cat. # A1380)

LIGATION

• Keep everything on ice.

• Briefly centrifuge the pGEM vector and Control Insert DNA tubes

• Add 1.5μl PCR product to each tube. • Prepare mix (per tube) o - .2X ligation buffer 2.5 o pGEM T vector 0.5 o T4 DNA ligase 0.5

• Add 3.5μl of mix to each tube. • Incubate at 15°C for 3 hours or at 4°C overnight. (4°C O/N gives max number of transformants. )

TRANSFORMATION

• Keep everything on ice . • Centrifuge ligation products briefly.

• Take the competent cells from -80⁰C and put on ice until thawed.

• Transfer 25μl of bacterial cells to ligation products tube. • Incubate on ice for 30 mins .

• Heat-shock the samples at 42⁰C for 1 min.

• Immediately return the tubes to ice for 2 mins . • Add βOOμl room temp LB medium.

• Incubate at 37⁰C for 1 hr with shaking (400 rpm) .

• Meanwhile, take LB/Amp plates from cold room. Incubate plates for about 10 mins at 37⁰C to ensure they are not cold.

• Prepare the following mixture (per plate) : o DiMethylFormamide (AppliedChem-A3676-0500) 80μl o X-GaI 50mg/ml 20μl o IPTG IM 20μl • Apply 120μl of mix to each plate.

• Following incubation, centrifuge the samples for 1 min at 4000 rpm.

• Discard 500μl of supernatant (to concentrate the bacteria) and plate samples with 60 μi of the transformation product .

• - Incubate at 37⁰C overnight.

MINI-PREP

• Put plates in the fridge for 1 hr to stop growth and finalise colour.

• Add 4ml LB/Amp (Iμl/ml) to a 15ml falcon.

• Pick the colonies with a sterile pipette tip and add to the falcons.

• Incubate overnight at 37⁰C. • Mini prep is then carried out using the QIAprep Spin Miniprep Kit (Qiagen) and the product sequenced.

The sequencing primers utilised were as follows:

MT2A (MTlE)

BS-MTlE-S 5'-AGG AAT TTA GGT GTG GAT AGG G (SEQ ID NO: 34) BS-MTlE-a 5'-AAA TTA CCT TCC ACC CTC CTC (SEQ ID NO: 35) CYBA

BS-CYBA-s 5'-GAG AGT TGA AGT TTG GGA AGT T (SEQ ID NO : 36) BS-CYBA-a 5'-TCT TAA AAC ACC CCT CCA AAC T (SEQ ID NO:37)

As can be seen from the results presented in figure 11, both CYBA and MTlE are completely unmethylated in the parental cells (WM793) but are hypermethylated in the more aggressive derivatives (WM793-P1 and WM793-P2) . Methylation of these genes thus appears to have an important impact on melanoma progression.

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Claims

1. A method of monitoring melanoma progression, in particular for determining whether the melanoma is likely to have metastatic capabilities, in a subject comprising, in a test sample, determining expression levels of at least one gene selected from TSPYl, CYBA, MT2A, GPNMB, BST2 , GIP3, ACTG2, TACl, IFITl, TNFAIP6, MXl, SlO0A9, COL8A1, HLA-DQAl, CIS, LGALS3, GPM6B, AZGPl, CCL2 , RGS3, UGT2B7, ClR, ISGF3G, ANKSl, CHNl, CALDl, BRD2 , RPL37A, RPLPO, PHB, PBX2 , SNRK, DAP, HOXD4, AEBPl, ENO2 , MGP, PRKl, MSX2/HOX8. SIAT7B, NPTX2, RARB, AFlQ, IGFBP4, PTPRN2 , AKRlC3 ; wherein a statistically significant decrease in the level of expression of any one of TSPYl, CYBA, MT2A, GPNMB, BST2 , GIP3, ACTG2, TACl, IFITl, TNFAIP6, MXl, S100A9, COL8A1, HLA-DQAl, CIS, LGALS3, GPM6B, AZGPl, CCL2 , RGS3 , UGT2B7, ClR, ISGF3G, ANKSl, CHNl, CALDl, BRD2 , RPL37A, RPLPO and/or a statistically significant increase in the expression of any one of PHB, PBX2 , SNRK, DAP, HOXD4 , AEBPl, ENO2, MGP, PRKl, MSX2/HOX8, SIAT7B, NPTX2 , RARB, AFlQ,

IGFBP4, PTPRN2, AKR1C3 indicates the presence of melanoma in the subject.

2. A method according to claim 1 wherein the level of expression is assessed with reference to a control sample.

3. A method according to claim 2 wherein the control sample is taken from normal (i.e. non malignant) melanocytes in the subject.

4. A method according to claim 2 wherein the control sample is taken from the same tissue as the test sample at an earlier time point.

5. The method according to any one of claims 1 to 4 wherein the expression of at least TSPYl is determined.

6. The method according to any one of claims 1 to 5 wherein the expression of at lest CYBA and/or MT2A is determined.

7. The method according to any one of claims 1 to 6 wherein gene expression is determined using RT-PCR, preferably quantitative RT-PCR.

8. _" The method according to claim 7 which utilises at least one primer pair taken from primer pairs comprising the sequences of :

SEQ ID NO: 1 and SEQ ID NO : 2, SEQ ID NO : 3 and SEQ ID N0:4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO : 7 and SEQ

ID NO: 8, SEQ ID NO : 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

9. The method according to any one of claims 1 to 8 which is used to determine whether the melanoma is in early radial growth phase (RGP) or a subsequent vertical growth phase (VGP) .

10. The method according to any one of claims 1 to 9 which is used to determine the appropriate treatment for the subject .

11. A method of monitoring melanoma progression, in particular for determining whether the melanoma is likely to have metastatic capabilities, in a subject comprising, in a test sample, determining the methylation status of at least one gene selected from TSPYl, CYBA, MT2A, MXl, RPL37A, HSPBl wherein hypermethylation of at least one gene indicates the presence of melanoma.

12. A method according to claim 11 wherein the test sample is a sample taken from tissue which is suspected of being a melanoma.

13. The method according to claim 11 or 12 wherein the methylation status of at least TSPYl is determined.

14. The method according to any of claims 11 to 13 wherein the methylation status of at least CYBA and/or MT2A is determined.

15. The method according to any one of claims 11 to 14 wherein the methylation status of any one or more of the CpG islands as represented in SEQ ID NOs: 15 and 29 (TSPYl CpG islands) , 30 and 31 (CYBA CpG islands) , 32 and 33 (RPL37A CpG islands) , 38 (MXl CpG islands) , 39 (HSPBl CpG islands) and 40 (MT2a CpG islands) is determined in order to determine the methylation status of the corresponding gene .

16. The method according to any one of claims 11 to 15 which utilises MSP, preferably real-time quantitative MSP.

17. A method of treating melanoma in a subject comprising administering a therapeutically effective amount of a DNA methyltransferase inhibitor or a histone deacetylase inhibitor to the subject such that expression of at least one gene selected from TSPYl, CYBA, MT2A, BST2 , GIP3, SlOOAl, IFITl, MXl, RGS3, ISGF3G, APOD, RPL37A, HSPBl is increased.

18. The method according to claim 17 wherein the level of gene expression is increased to the levels of gene expression found in normal melanocytes .

19-. Use of a DNA methyltransferase inhibitor or a histone deacetylase inhibitor in the manufacture of a medicament for the treatment of melanoma by increasing expression of at least one gene selected from TSPYl, CYBA, MT2A, BST2 , GIP3, SlOOAl, IFITl, MXl, RGS3 , ISGF3G, APOD, RPL37A, HSPBl.

20. The use according to claim 19 wherein the level of gene expression is increased to the levels of gene expression found in normal melanocytes.

21. A microarray for use in the method of any one of claims 1 to 10 comprising probes immobilised on a solid support hybridizing with transcripts or parts thereof of at least one gene selected from TSPYl, CYBA, MT2A, GPNMB, BST2 , GIP3, ACTG2, TACl, IFITl, TNFAIP6, MXl, S100A9, COL8A1, HLA-DQAl, CIS, LGALS3, GPM6B, AZGPl, CCL2 , RGS3 , UGT2B7, ClR, ISGF3G, ANKSl, CHNl, CALDl, BRD2 , RPL37A, RPLPO, PHB, PBX2 , SNRK, DAP, HOXD4, AEBPl, ENO2 , MGP, PRKl, MSX2/HOX8, SIAT7B, NPTX2, RARB, AFlQ, IGFBP4, PTPRN2 , AKR1C3.

22. The microarray of claim 21 which comprises probes representing transcripts of at least TSPYl .

23. The microarray of claim 21 or 22 which comprises probes representing transcripts of at least CYBA and/or MT2A.

24. A microarray for use in the method of any one of claims 11 to 16 comprising probes immobilised on a solid support hybridizing with either methylated only or both unmethylated and methylated versions of at least one gene or parts thereof selected from TSPYl, CYBA, MT2A, MXl, RPL37A, HSPBl following bisulphite treatment.

25. Primers which can bind to a nucleic acid molecule comprising the sequence of any one of SEQ ID NO's 15, 29, 30, 31, 32, 33, 38, 39 and 40 following bisulphite treatment.

26. Primers according to claim 25 comprising the sequence of SEQ ID NO: 17 and/or 18.

27. A method of identifying a compound capable of treating or reducing the effects or progression of melanoma comprising the steps of;

(a) administering the compound to an experimental non- human animal having a melanoma; (b) generating an expression profile of a panel of genes comprising at least one of TSPYl, CYBA, MT2A, GPNMB, BST2, GIP3, ACTG2, TACl, IFITl, TNFAIP6, MXl, SlO0A9, COL8A1, HLA-DQAl, CIS, LGALS3, GPM6B, AZGPl, CCL2 , RGS3 , UGT2B7, ClR, ISGF3G, ANKSl, CHNl, CALDl, BRD2 , RPL37A, RPLPO, PHB, PBX2, SNRK, DAP, HOXD4 , AEBPl, ENO2 , MGP, PRKl, MSX2/HOX8, SIAT7B, NPTX2 , RARB, AFlQ, IGFBP4 , PTPRN2 , AKRlC3 ;

(c) comparing the expression profile obtained in (b) with the expression profile of a corresponding panel of genes expressed in a control melanocyte sample which is not a melanoma; wherein a positive correlation of the expression profiles is indicative that the compound is capable of reducing or preventing melanoma.

28. An in vitro method of identifying a compound capable of treating or reducing the effects or progression of melanoma comprising the steps of;

(a) administering the compound to a melanoma sample;

(b) generating an expression profile of a panel of genes comprising at least one of TSPYl, CYBA, MT2A, GPNMB,

BST2, GIP3, ACTG2, TACl, IFITl, TNFAIP6, MXl, S100A9, C0L8A1, HLA-DQAl, CIS, LGALS3, GPM6B, AZGPl, CCL2 , RGS3, UGT2B7, ClR, ISGF3G, ANKSl, CHNl, CALDl, BRD2 , RPL37A, RPLPO, PHB, PBX2, SNRK, DAP, HOXD4 , AEBPl, ENO2 , MGP, PRKl, MSX2/HOX8, SIAT7B, NPTX2 , RARB, AFlQ, ^' IGFBP4 , PTPRN2 , AKR1C3 ;

(c) comparing the expression profile obtained in (b) with the expression profile of a corresponding panel of genes expressed in a control melanocyte sample which is not a melanoma; wherein a positive correlation of the expression profiles is indicative that the compound is capable of reducing, controlling the progression -of, or preventing melanoma.

29. The method of claim 27 wherein the control sample is taken from an experimental non-human animal which does not have a melanoma.

30. The method of claim 27 wherein the control sample is taken from normal melanocytes in the same non-human animal