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WO2013113762A1 - Procédés et kits pour prédire le risque d'avoir un mélanome cutané chez un sujet - Google Patents

Procédés et kits pour prédire le risque d'avoir un mélanome cutané chez un sujet Download PDF

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Publication number
WO2013113762A1
WO2013113762A1 PCT/EP2013/051810 EP2013051810W WO2013113762A1 WO 2013113762 A1 WO2013113762 A1 WO 2013113762A1 EP 2013051810 W EP2013051810 W EP 2013051810W WO 2013113762 A1 WO2013113762 A1 WO 2013113762A1
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risk
subject
melanoma
inhibitors
cutaneous melanoma
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PCT/EP2013/051810
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English (en)
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Nadem Soufir
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INSERM (Institut National de la Santé et de la Recherche Médicale)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to in vitro methods and kits for predicting the risk of having cutaneous melanoma in a subject.
  • Cutaneous melanoma is a serious form of skin cancer in humans. It arises from the pigment cells (melanocytes), usually in the skin. Melanoma is currently increasing at the fastest rate of all cancers in the United States. Without even including melanoma in-situ, it is the seventh most common serious cancer in the United States 1 .
  • Melanoma is currently predicted by assessing risk factors.
  • Risk factors for melanoma are a family history of melanoma, the presence of dysplastic nevi, patient history of melanoma, weakened immune system, many ordinary nevi, exposure levels to ultraviolet radiation, exposure to severe sunburns especially as a child or teenager, and fair skin.
  • the invention aims to provide a novel method for the prognosis of melanoma using new genetic bio markers
  • the present invention relates to a method of determining whether a subject is at risk of having or developing a cutaneous melanoma, comprising testing for said subject the presence of at least one genetic alteration in the PA K2 gene, wherein the presence of said genetic alteration indicates an increased risk of having or developing cutaneous melanoma.
  • the present invention relates to a method of determining whether a subject is at risk of having or developing a cutaneous melanoma, comprising testing for said subject the presence of at least one genetic alteration in the PARK2 gene, wherein the presence of said genetic alteration indicates an increased risk of having or developing cutaneous melanoma.
  • a "subject" in the context of the present invention can be a male or female.
  • a subject can also be one who has not been previously diagnosed as having cutaneous melanoma.
  • the subject having or being at risk of having or developing a cutaneous melanoma may be a substantially healthy subject, which means that the subject has not been previously diagnosed or identified as having or suffering from cutaneous melanoma.
  • said subject may also be one that is asymptomatic for cutaneous melanoma.
  • an "asymptomatic" subject refers to a subject that does not exhibit the traditional symptoms of cutaneous melanoma.
  • said subject may be one that is at risk of having or developing a cutneous melanoma, as defined by clinical indicia such as for example fair skin, a history of sunburn, excessive ultraviolet (UV) light exposure, living closer to the equator or at a higher elevation, a family history of melanoma, a weakened immune system.
  • the subject suffers from Parkinson's disease.
  • “Risk” in the context of the present invention relates to the probability that an event will occur over a specific time period, as in the conversion to a cutaneous melanoma, and can mean a subject's "absolute” risk or “relative” risk.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion.
  • Alternative continuous measures which may be assessed in the context of the present invention include time to melanoma conversion and therapeutic geographic atrophy form of melanoma conversion risk reduction ratios.
  • Determining whether a subject is at risk of having or developing a cutaneous melanoma in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that cutaneous melanoma may occur.
  • Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of melanoma, such as fair skin, a history of sunburn, excessive ultraviolet (UV) light exposure, living closer to the equator or at a higher elevation, a family history of melanoma, a weakened immune system...
  • the methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to cutaneous melanoma, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for cutaneous .
  • the invention can be used to discriminate between normal and other subject cohorts at higher risk for cutaneous melanoma.
  • the presence of the genetic alteration is tested from a sample obtained from the subject.
  • sample in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject.
  • Bodily fluids useful in the present invention include blood, urine, saliva or any other bodily secretion or derivative thereof.
  • blood includes whole blood, plasma, serum, circulating epithelial cells, constituents, or any derivative of blood.
  • the sample comprises nucleic acids, wherein nucleic acids may be genomic DNA, heterogenous nuclear RNA (hnRNA, also referred as incompletely processed single strand of ribonucleic acid) and/or cDNA.
  • hnRNA heterogenous nuclear RNA
  • PARK2 gene has its general meaning in the art and refers to the parkinson protein 2 also known as E3 ubiquitin protein ligase (parkin) (Gene ID: 5071; NG_008289.1). Typically the mRNA sequence of this gene is deposited in the database Genbank under accession number NM 004562.2. The corresponding polypeptide sequence is deposited in databases under accession number NP 004553.2. The precise function of this gene is unknown; however, the encoded protein is a component of a multiprotein E3 ubiquitin ligase complex that mediates the targeting of substrate proteins for proteasomal degradation. Mutations in this gene are known to cause Parkinson disease and autosomal recessive juvenile Parkinson disease.
  • a “genetic alteration” has its general meaning in the art and refers to a genomic polymorphic site. Each genetic alteration has at least two sequence variations characteristic of particular alleles at the polymorphic site. Thus, a genetic alteration implies that there is association to at least one specific allele of that particular genetic alteration.
  • the alteration can comprise any allele of any variant type found in the genome, including splicing mutations, SNPs and copy number variations (insertions, deletions, duplications). Genetic alterations can be of any measurable frequency in the population.
  • DDB2 gene or protein mutations identified in DDB2 gene or protein are designated pursuant to the nomenclature of Dunnen and Antonarakis (Dunnen et Antonarakis (2000) Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutation. 15 :7-12; Erratum in: Hum Mutat 2002 ;20(5):403). As defined by Dunnen and Antonarakis at the nucleic acid level, substitutions are designated by ">”. Deletions are designated by “del” after the deleted interval (followed by the deleted nucleotides). Insertions are designated by "ins,” followed by the inserted nucleotides.
  • Intron mutations are designated by the intron number (preceded by 'TVS") or cDNA position; positive numbers starting from the G of the GT splice donor site, whereas negative numbers starting from the G of the AG splice acceptor site.
  • the mutation is best designated by the nucleotide number of the genomic references.
  • the term "Allele” has the meaning which is commonly known in the art, that is, an alternative form of a gene (one member of a pair) that is located at a specific position on a specific chromosome which, when translated result in functional or dysfunctional (including non- existent) gene products. Typically, the genetic alteration of the invention compromises the function of the gene product.
  • a genetic alteration of the invention typical confers a loss of function.
  • the genetic alteration is a mutation that truncates at least 10%, 15%, 20%, 25%, 30%, 40%), 50%), 60%), or more of the PARK2 gene product.
  • Other loss function genetic alterations include region mutations in one or more essential functional domains or essential conserved structures of the PARK2 gene product.
  • Essential or conserved structures may include those involved in ubiquitin ligase's function and include but are not limited to RING1 , IBR and RING2 domains of PARKIN which are essential for ubiquitin ligase's function.
  • polymorphism or "allelic variant” means a mutation in the normal sequence of a gene, Allelic variants can be found in the exons, introns, or the coding region of the gene, or in the sequences that control expression of the gene.
  • a "Single Nucleotide Polymorphism” or "SNP” is a DNA sequence variation occurring when a single nucleotide at a specific location in the genome differs between members of a species or between paired chromosomes in an individual. Most SNP polymorphisms have two alleles. Each individual is in this instance either homozygous for one allele of the polymorphism (i.e. both chromosomal copies of the individual have the same nucleotide at the SNP location), or the individual is heterozygous (i.e. the two sister chromosomes of the individual contain different nucleotides).
  • the SNP nomenclature as reported herein refers to the official Reference SNP (rs) ID identification tag as assigned to each unique SNP by the National Center for Biotechno logical Information (NCBI).
  • the SNPs that are associated to a high risk of having or developing a cutaneous melanoma are depicted in table 1.
  • copy number variation has its general meaning in the art.
  • the nature of the CNVs described herein is such that certain regions of PARK2 gene are present in alternate copy number in certain subjects.
  • the segment may be deleted, or it may be present in more than one copy on each particular allele.
  • the absolute breakpoint at which the variation begins and ends can be difficult to define due to experimental limitations. Experimentally, what is determined is the last polymorphism (or probe) that is outside the CNV segment upstream (5') of the segment, the first polymorphism within the CNV segment, the last polymorphism within the CNV segment and the first polymorphism outside the CNV segment downstream (3') of the segment.
  • the first two markers and the last two markers (or nucleotide probes) in the above will be adjacent markers.
  • the resulting CNV can be defined minimally as including the segment that begins with the first marker consistent with the CNV and ends with the last marker consistent with the CNV.
  • Such a definition is however not inclusive of the physical boundaries of the CNV segment.
  • An alternative way of defining the CNV is provided by the region flanked by the two polymorphisms that are inconsistent with the CNV (i.e. outside the CNV segment), but adjacent to the two polymorphisms corresponding to the first and last polymorphisms assayed within the CNV segment. The latter definition is inclusive of the actual boundaries of the CNV.
  • the CNVs that are associated to a high risk of having or developing a cutaneous melanoma are depicted in table 2.
  • subject at risk for cutaneous melanoma is one in whom a particular genetic alteration is present in the PARK2 gene.
  • carriers of the genetic alteration are at a different risk for cutaneous melanoma than non-carriers.
  • significance associated with risk of a genetic alteration is measured by a relative risk (RR).
  • significance associated with a genetic alteration is measured by an odds ratio (OR).
  • the significance is measured by a percentage.
  • a significant increased risk is measured as a risk (relative risk and/or odds ratio) of at least 1.2, including but not limited to: at least 1.5, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, 1.8, at least 1.9, at least 2.0, at least 2.5, at least 3.0, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, at least 10.0, and at least 15.0.
  • a risk (relative risk and/or odds ratio) of at least 2.0 is significant.
  • a risk of at least 3.0 is significant.
  • a risk of at least 4.0 is significant.
  • a relative risk of at least 5.0 is significant.
  • a significant increase in risk is at least 10.0 is significant.
  • other values for significant risk are also contemplated, e.g., at least 2.5, 3.5, 4.5, 5.5, or any suitable other numerical values, and such values are also within scope of the present invention.
  • a significant increase in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, and 1500%.
  • a significant increase in risk is at least 100%. In other embodiments, a significant increase in risk is at least 200%, at least 300%, at least 400%, at least 500%, at least 700%, at least 800%, at least 900% and at least 1000%. Other cutoffs or ranges as deemed suitable by the person skilled in the art to characterize the invention are however also contemplated, and those are also within scope of the present invention.
  • a significant increase in risk is characterized by a p-value, such as a p-value of less than 0.05, less than 0.01 , less than 0.001 , less than 0.0001, less than 0.00001, less than 0.000001, less than 0.0000001, less than 0.00000001, or less than 0.000000001.
  • the method of the invention comprises testing for said subject the presence of at least one genetic alteration in linkage disequilibrium with at least one genetic alteration of the invention, wherein the presence of said genetic alteration indicates an increased risk of having or developing cutaneous melanoma.
  • linkage disequilibrium refers to a population association among alleles at two or more loci. It is a measure of co-segregation of alleles in a population. Linkage disequilibrium or allelic association is the preferential association of a particular allele or any other genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population. Accordingly, two particular alleles at different loci on the same chromosome are said to be in LD if the presence of one of the alleles at one locus tends to predict the presence of the other allele at the other locus.
  • Linked variants are readily identified by determining the degree of linkage disequilibrium (LD) between the allele genotyped for one SNP and a candidate linked allele at a polymorphic site located in the chromosomal region where said SNP is locatrd or elsewhere on the chromosome.
  • the candidate linked variant may be an allele of a polymorphism that is currently known.
  • Other candidate linked variants may be readily identified by the skilled artisan using any technique well-known in the art for discovering polymorphisms.
  • One of the most frequently used measures of linkage disequilibrium is r, which is calculated using the formula described by Devlin et al. (Genomics, 29(2):311-22 (1995)).
  • r is the measure of how well an allele X at a first locus predicts the occurrence of an allele Y at a second locus on the same chromosome. The measure only reaches 1.0 when the prediction is perfect (e.g. X if and only if Y).
  • Detecting the specific genetic alterations according to the invention can be accomplished by methods known in the art for detecting sequences at polymorphic sites.
  • standard techniques for genotyping for the presence of SNPs can be used, such as fluorescence-based techniques (e.g., Chen, X. et al, Genome Res. 9(5): 492-98 (1999); Kutyavin et al, Nucleic Acid Res. 34:el28 (2006))., utilizing PCR, LCR, Nested PCR and other techniques for nucleic acid amplification.
  • SNP genotyping include, but are not limited to, TaqMan genotyping assays and SNPIex platforms (Applied Biosystems), gel electrophoresis (Applied Biosystems), mass spectrometry (e.g., MassARRAY system from Sequenom), minisequencing methods, realtime PCR, Bio-Plex system (BioRad), CEQ and SNPstream systems (Beckman), array hybridization technology (e.g., Affymetrix GeneChip; Perlegen), BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays), array tag technology (e.g., Parallele), and endonuclease-based fluorescence hybridization technology (Invader; Third Wave).
  • Applied Biosystems Applied Biosystems
  • Gel electrophoresis Applied Biosystems
  • mass spectrometry e.g., MassARRAY system from Sequenom
  • minisequencing methods minisequencing methods, realtime PCR, Bio-Plex
  • Some of the available array platforms including Affymetrix SNP Array 6.0 and Illumina CNV370- Duo and 1M BeadChips, include SNPs that tag certain CNVs. This allows detection of CNVs via surrogate SNPs included in these platforms. Thus, by use of these or other methods available to the person skilled in the art, one or more alleles at genetic alterations can be identified.
  • the determination of the said polymorphism may be determined by nucleic acid sequencing, PCR analysis or any genotyping method known in the art.
  • methods include, but are not limited to, chemical assays such as allele specific hybridization, primer extension, allele specific oligonucleotide ligation, sequencing, enzymatic cleavage, flap endonuclease discrimination; and detection methods such as fluorescence, chemiluminescence, and mass spectrometry.
  • the presence or absence of said polymorphism may be detected in a RNA or DNA sample, preferably after amplification.
  • the isolated RNA may be subjected to couple reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that are specific for the polymorphism or that enable amplification of a region containing the polymorphism.
  • reverse transcription and amplification such as reverse transcription and amplification by polymerase chain reaction (RT-PCR)
  • RT-PCR polymerase chain reaction
  • primers that are specific for the polymorphism or that enable amplification of a region containing the polymorphism.
  • conditions for primer annealing may be chosen to ensure specific reverse transcription (where appropriate) and amplification; so that the appearance of an amplification product be a diagnostic of the presence of the polymorphism according to the invention.
  • RNA may be reverse-transcribed and amplified, or DNA may be amplified, after which a mutated site may be detected in the amplified sequence by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.
  • a cDNA obtained from RNA may be cloned and sequenced to genotype the polymorphism (or identify the allele).
  • nucleic acid molecule may be tested for the presence or absence of a restriction site.
  • a base polymorphism creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR genotype the polymorphism.
  • RNA sequencing includes, but are not limited to, direct sequencing, restriction fragment length polymorphism (RFLP) analysis; hybridization with allele-specific oligonucleotides (ASO) that are short synthetic probes which hybridize only to a perfectly matched sequence under suitably stringent hybridization conditions; allele-specific PCR; PCR using mutagenic primers; ligase-PCR, HOT cleavage; denaturing gradient gel electrophoresis (DGGE), temperature denaturing gradient gel electrophoresis (TGGE), single- stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (Kuklin et al, 1997).
  • RFLP restriction fragment length polymorphism
  • ASO allele-specific oligonucleotides
  • Direct sequencing may be accomplished by any method, including without limitation chemical sequencing, using the Maxam-Gilbert method ; by enzymatic sequencing, using the Sanger method ; mass spectrometry sequencing ; sequencing using a chip-based technology; and real-time quantitative PCR.
  • DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers.
  • PCR polymerase chain reaction
  • RCA rolling circle amplification
  • InvaderTMassay or oligonucleotide ligation assay (OLA).
  • OLA may be used for revealing base polymorphisms.
  • two oligonucleotides are constructed that hybridize to adjacent sequences in the target nucleic acid, with the join sited at the position of the polymorphism.
  • DNA ligase will covalently join the two oligonucleotides only if they are perfectly hybridized to one of the allele.
  • useful nucleic acid molecules in particular oligonucleotide probes or primers, according to the present invention include those which specifically hybridize the one of the allele of the polymorphism.
  • Oligonucleotide probes or primers may contain at least 10, 15, 20 or 30 nucleotides. Their length may be shorter than 400, 300, 200 or 100 nucleotides.
  • Detection of CNVs can be done by a range of techniques suitable for such purpose.
  • techniques that can selectively determine whether a particular chromosomal segment is present or absent in an individual can be used for genotyping CNVs.
  • the technique is able to quantify the amount of segment present, i.e. determine whether a segment is deleted, duplicated, triplicated, etc. in the individual.
  • Taqman assays can be used (Bieche, I. et al. Int J Cancer 78:661-6 (1998)), as well as Fluorescent In Situ Hybridization (FISH) techniques.
  • a range of genotyping technologies can also be used, such as Molecular Inversion Probe array technology (e.g., Affymetrix SNP Array 6.0), and BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays, e.g. HumanHap chips, Human lM-Duo), as can other platforms such as Nimblegen HD2.1, High-Definition CGH arrays (Agilent Technologies), tiling array technology (Affymetrix).
  • Information about amplitude of particular probes, which is representative of particular alleles provides a quantitative dosage information for the particular allele, and by consequence dosage information about the CNV in question, since the marker is selected as a marker representative of the CNV and is typically physically located within the CNV.
  • the CNV is a deletion
  • the absence of particular marker alleles is representative of the deletion.
  • the CNV is a duplication (or a higher order copy number variation)
  • the signal intensity representative of the allele correalating with the CNV is representative of the copy number.
  • the genetic variation is detected at the protein level.
  • a variety of methods can be used for detecting genetic variations at protein level, including enzyme linked immunosorbent assays (ELISA), Western blots, immunoprecipitations and immunofluorescence.
  • ELISA enzyme linked immunosorbent assays
  • a sample from a subject is assessed for the presence of an alteration in the polypeptide encoded by the nucleic acid having the genetic alteration according to the invention.
  • Such alteration can, for example, be an alteration in the quantitative polypeptide expression (i.e., the amount of polypeptide produced).
  • An alteration in the composition of a polypeptide can be an alteration in the qualitative polypeptide expression (e.g., expression of a mutant polypeptide). Both such alterations (quantitative and qualitative) can also be present.
  • an alteration in the expression or composition of the polypeptide can be the result of a particular CNV.
  • an antibody e.g., an antibody with a detectable label
  • a particular target polypeptide e.g., a polypeptide encoded by a nucleic acid associated with a CNV as described herein
  • Antibodies can be polyclonal or monoclonal.
  • An intact antibody, or a fragment thereof e.g., Fv, Fab, Fab', F(ab')2 can be used.
  • labeled with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a primary antibody using a labeled secondary antibody (e.g., a fluorescently-labeled secondary antibody) and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.
  • the level or amount of polypeptide in the sample obtained from the subject is compared with the level or amount of the polypeptide in a control sample. Typically, when a level or amount of the polypeptide in the sample is lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant, is indicative of a genetic alteration in the in the PA K2 gene.
  • the method of the invention is performed by a laboratory that will generate a test report.
  • the test report will thus indicates whether the genetic alteration is present or absent, and preferably indicates whether the patient is heterozygous or homozygous for genetic alteration. Accordingly, if the patient is homozygous for the risk allele, then the test report further indicates that the patient is positive for a genetic alteration associated with a high risk of having or developing cutaneous melanoma. If the patient is heterozygous for the risk allele, then the test report further indicates that the patient is positive for a genetic alteration associated with a risk of having or developing cutaneous melanoma.
  • the test result will include a probability score, which is derived from running a model that include the risk factor determined for the genetic alteration of the invention that are tested.
  • the risk factor determined for a genetic alteration of the invention may be pondered by a coefficient depending on what is the contribution of said genetic alteration in the determination of the risk in comparison with another genetic alteration.
  • the method for calculating the score is based on statistical studies performed on various cohorts of patients.
  • the score may also include other various patient parameters (e.g., age, gender, weight, race, test results for other genetic risk factors or other typical risk factors such as fair skin, history of sunburn, excessive ultraviolet (UV) light exposure, living closer to the equator or at a higher elevation, family history of melanoma, or weakened immune system.
  • the weight given to each parameter is based on its contribution relative to the other parameters in explaining the inter-individual variability of having melanoma in the relevant disease population.
  • the test report may be thus generated by a computer program for establishing such a score. This probability score may be used as a guide in selecting a therapy or treatment regimen for the subject.
  • one or more melanoma treatments or prophylactic regimens may be prescribed to said subject.
  • Subjects genotyped as having one or more of the alleles described herein that are associated with increased risk of melanoma often are prescribed a prophylactic regimen designed to minimize the occurrence of melanoma.
  • An example of a prophylactic regimen often prescribed is directed towards minimizing ultraviolet (UV) light exposure.
  • UV ultraviolet
  • Such a regimen may include, for example, prescription of a lotion applied to the skin that minimizes UV penetration and/or counseling individuals of other practices for reducing UV exposure, such as by wearing protective clothing and minimizing sun exposure.
  • a treatment regimen is specifically prescribed and/or administered to individuals who will most benefit from it based upon their risk of developing melanoma assessed by the method of the invention.
  • the treatment sometimes is preventative (e.g., is prescribed or administered to reduce the probability that a melanoma arises or progresses), sometimes is therapeutic, and sometimes delays, alleviates or halts the progression of a melanoma.
  • Any known preventative or therapeutic treatment for alleviating or preventing the occurrence of a melanoma can be prescribed and/or administered.
  • the treatment sometimes is or includes a drug that reduces melanoma, including, for example, cisplatin, carmustine (BCNU), vinblastine, vincristine, and bleomycin.
  • the melanoma treatment is surgery.
  • Surgery to remove (excise) a melanoma is the standard treatment for this disease. It is necessary to remove not only the tumor but also some normal tissue around it in order to minimize the chance that any cancer will be left in the area. It is common for lymph nodes near the tumor to be removed during surgery because cancer can spread through the lymphatic system. Surgery is generally not effective in controlling melanoma that is known to have spread to other parts of the body. In such cases, doctors may use other methods of treatment, such as chemotherapy, biological therapy, radiation therapy, or a combination of these methods.
  • a further aspect of the invention relates to a method for diagnosing cutaneous melanoma comprising detecting at least one genetic alteration of the invention in a nucleic acid sample obtained from the patient.
  • Kits useful in the methods of the invention comprise components useful in any of the methods described herein, including for example, primers for nucleic acid amplification, hybridization probes the genetic alteration detection, restriction enzymes (e.g., for RFLP analysis), nucleic acid probes, optionally labelled with suitable labels (e.g., fluorescent labels), allele-specific oligonucleotides (e.g., SNP-allele specific, or CNV-allele specific probes), antibodies that bind to an altered polypeptide encoded by a nucleic acid of the invention as described herein or to a non-altered (native) polypeptide encoded by a nucleic acid of the invention as described herein, means for amplification of CNVs or fragments of CNVs as described herein, means for analyzing the nucleic acid sequence of nucleic acids comprising CNVs as described herein, means for analyzing the amino acid sequence of a polypeptide encoded by a CNV, or a nucleic acid associated with
  • kits can for example include necessary buffers, nucleic acid primers for amplifying nucleic acids, and reagents for allele-specific detection of the fragments amplified using such primers and necessary enzymes (e.g., DNA polymerase). Additionally, kits can provide reagents for assays to be used in combination with the methods of the present invention, e.g., reagents for use with other diagnostic assays for cutaneous melanoma.
  • the invention pertains to a kit for assaying a sample from a subject to detect the presence of a CNV, wherein the kit comprises reagents necessary for selectively detecting at least one particular CNV in the PARK2 gene of the subject.
  • the invention pertains to a kit for assaying a sample from a subject to detect the presence of at least particular allele of at least one polymorphism associated with a CNV in the genome of the subject.
  • the reagents comprise at least one contiguous oligonucleotide that hybridizes to a fragment of the genome of the subject comprising at least CNV, or at least one polymorphism in LD with a CNV.
  • the reagents comprise at least one pair of oligonucleotides that hybridize to opposite strands of a genomic segment obtained from a subject, wherein each oligonucleotide primer pair is designed to selectively amplify a fragment of the genome of the subject that includes at least one CNV, or a fragment of a CNV.
  • the fragment is at least 20 nucleotides in size.
  • the fragment is at least 30 nucleotides in size, at least 50 nucleotides in size, at least 100 nucleotides in size, at least 200 nucleotides in size, at least 300 nucleotides in size, at least 500 nucleotides in size, at least 1000 nucleotides in size, at least 5000 nucleotides in size, or at least 10000 nucleotides in size. It is however contemplated that the fragment can be of any other suitable size appropriate for use in kits useful to practice the present invention.
  • oligonucleotides or nucleic acids can be designed using portions of the nucleic acid sequence of a CNV, or of a genomic region of a CNV that is LD with the CNV (e.g., a flanking region of a CNV).
  • the kit comprises one or more labeled nucleic acids capable of allele-specific detection of one or more specific genetic alterations or haplotypes in LD with a CNV, and reagents for detection of the label.
  • kits for detecting SNP markers comprises a detection oligonucleotide probe, that hybridizes to a segment of template DNA containing a SNP polymorphisms to be detected, an enhancer oligonucleotide probe and an endonuclease.
  • the detection oligonucleotide probe comprises a fluorescent moiety or group at its 3' terminus and a quencher at its 5' terminus, and an enhancer oligonucleotide, is employed, as described by Kutyavin et al. (Nucleic Acid Res. 34:el28 (2006)).
  • the fluorescent moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties.
  • the detection probe is designed to hybridize to a short nucleotide sequence that includes the SNP polymorphism to be detected.
  • the SNP is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe.
  • the enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe.
  • the probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template.
  • the gap creates a synthetic abasic site that is recognized by an endonuclease, such as Endonuclease IV.
  • the enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch.
  • assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed.
  • the detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the probe is from 10-50 nucleotides in length, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art.
  • the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection, and primers for such amplification are included in the reagent kit.
  • PCR Polymerase Chain Reaction
  • the amplified DNA serves as the template for the detection probe and the enhancer probe.
  • the DNA template is amplified by means of Whole Genome Amplification (WGA) methods, prior to assessment for the presence of specific genetic alterations as described herein. Standard methods well known to the skilled person for performing WGA may be utilized, and are within scope of the invention.
  • reagents for performing WGA are included in the reagent kit.
  • modified bases including modified A and modified G.
  • modified bases can be useful for adjusting the melting temperature of the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its complementary T can be used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their complementary C base in a double stranded DNA molecule.
  • modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person.
  • a further aspect of the invention relates to a compound selected from the group consisting of cyclin-dependent kinase II (CDK2) inhibitors, cylcin E inhibitors, inhibitors of cyclin-dependent kinase II( CDK2) expression and , inhibitors of cyclin E expression for the treatment of cutaneous melanoma in subject having an genetic alteration according to the invention.
  • CDK2 cyclin-dependent kinase II
  • CDK2 has its general meaning in the art and refers to a protein serine/threonine kinase that is required for progression of cells through the Gl and S phases of the cell cycle. Inhibition of CDK2 in normal cells results in a reversible cell cycle arrest.
  • the cyclin-dependent kinase II inhibitor of the invention may comprise any suitable compound having inhibitory action on cyclin-dependent kinase II activity, i.e. a compound that is effective to suppress cyclindependent kinase II activity.
  • Cyclin-dependent kinase II inhibitors are well known in the art (Duca JS. Recent advances on structure-informed drug discovery of cyclin-dependent kinase-2 inhibitors. Future Med Chem. 2009 Nov;l(8): 1453-66. Review.).
  • said inhibitors may be selected from the group consisting of substituted oxindole derivatives described in International Patent Application No.
  • PCT/EP98/05559 filed Sep 3, 1998 for "Substituted Oxindole Derivatives,", purine derivatives described in International Publication WO97/20842 of CNRS Center Natural Research; pyridylpyrimidinamine derivatives described in International Publication WO95/09852 of Ciba-Geigy (Novartis); 2,6,9- trisubstituted compounds described in International Publication WO98/05335 of CV Therapeutics; 4H-l-benzopyran-4-one derivatives described in German Patent 3836676 of Hoechst AG; 2-thiol and 2-oxo-flavopiridol analogues described in U.S. Pat. No.
  • CDK2 inhibitors In addition to the use of specific compounds shown to be CDK2 inhibitors, the present invention also extends to the use of pharmaceutically acceptable salts, solvates, biohydrolyzable carbonates, biohydrolyzable ureides, biohydrolyzable esters, biohydrolyzable amides, biohydrolyzable carbamates, affinity reagents or prodrugs thereof in either crystalline or amorphous form.
  • CDK2 inhibitors described in International Patent Application PCT/EP98/05559 the entire disclosure of which is incorporated herein by reference
  • CDK2 inhibitors described in US7,482,342 CDK2 inhibitors described in US7122552
  • Inhibitors of expression for use in the present invention may be based on anti-sense oligonucleotide constructs.
  • Anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of CDK2 or cyclin E mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of CDK2 or cyclin E, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding CDK2 or cyclin E can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).
  • Small inhibitory RNAs can also function as inhibitors of expression for use in the present invention.
  • CDK2 or cyclin E gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that CDK2 or cyclin E gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ.
  • phosphodiester bonds of the siRNAs of the invention are advantageously protected. This protection is generally implemented via the chemical route using methods that are known by art.
  • the phosphodiester bonds can be protected, for example, by a thiol or amine functional group or by a phenyl group.
  • the 5'- and/or 3'- ends of the siRNAs of the invention are also advantageously protected, for example, using the technique described above for protecting the phosphodiester bonds.
  • the siRNAs sequences advantageously comprises at least twelve contiguous dinucleotides or their derivatives.
  • RNA derivatives with respect to the present nucleic acid sequences refers to a nucleic acid having a percentage of identity of at least 90% with erythropoietin or fragment thereof, preferably of at least 95%, as an example of at least 98%, and more preferably of at least 98%.
  • percentage of identity between two nucleic acid sequences, means the percentage of identical nucleic acid, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being randomly spread over the nucleic acid acids sequences.
  • best alignment or “optimal alignment” means the alignment for which the determined percentage of identity (see below) is the highest. Sequences comparison between two nucleic acids sequences are usually realized by comparing these sequences that have been previously align according to the best alignment; this comparison is realized on segments of comparison in order to identify and compared the local regions of similarity.
  • the identity percentage between two sequences of nucleic acids is determined by comparing these two sequences optimally aligned, the nucleic acids sequences being able to comprise additions or deletions in respect to the reference sequence in order to get the optimal alignment between these two sequences.
  • the percentage of identity is calculated by determining the number of identical position between these two sequences, and dividing this number by the total number of compared positions, and by multiplying the result obtained by 100 to get the percentage of identity between these two sequences.
  • shRNAs short hairpin RNA
  • shRNAs can also function as inhibitors of expression for use in the present invention.
  • Ribozymes can also function as inhibitors of expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of CDK2 or cyclin E mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable.
  • antisense oligonucleotides and ribozymes useful as inhibitors of expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis.
  • anti-sense R A molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and preferably cells expressing CDK2 or cyclin E.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus
  • adenovirus adeno-associated virus
  • SV40-type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • vaccinia virus
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • viruses for certain applications are the adenoviruses and adeno-associated viruses
  • AAV viruses which are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • AAV serotypes AAVl to 12
  • Recombinant AAV are derived from the dependent parvovirus AAV2 (Choi, VW J Virol 2005; 79:6801-07).
  • the adeno-associated virus type 1 to 12 can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species (Wu, Z Mol Ther 2006; 14:316- 27).
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and micro encap sulation.
  • the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • the promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes
  • a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable.
  • the promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
  • the compound is administered to said subject with a therapeutically effective amount.
  • a therapeutically effective amount is meant a sufficient amount of the active ingredient to treat macular edema at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the compound may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the compound of the invention can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the compound of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.
  • parenteral administration such as intravenous or intramuscular injection
  • other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration ; liposomal formulations ; time release capsules ; and any other form currently used.
  • EXAMPLE 1 PARKIN INACTIVATION PLAYS AN IMPORTANT ROLE IN MELANOCYTE ONCOGENESIS AND PREDISPOSES TO CUTANEOUS MELANOMA
  • Parkinson's disease is a neurodegenerative disorder characterized by a loss of melanin-positive dopaminergic neurons in the substantia nigra (SN).
  • PARK2 PARKIN
  • EOPD early onset and/or familial forms of PD
  • CM Cutaneous melanoma
  • PARK2 encodes an E3 ubiquitin ligase that is involved in cyclin E degradation.
  • PARK2 has also recently been shown to be an important tumor suppressor gene implicated in tumor development. We therefore investigated its impact on CM susceptibility and CM oncogenesis.
  • PARK2 coding region was sequenced in 370 CM patients recruited from oncogenetic survey and that matched one of following criteria: familial CM, multiple CM and CM ⁇ 25 years old, and in 16 melanoma cell lines.
  • the functional effect of PARK2 mutations was evaluated by in silico prediction tools.
  • the presence of CNV in PARK2 was investigated by MLPA and qPCR in 370 CM patients, 25 melanoma cell lines and 37 primary tumors.
  • the frequencies of point mutations and CNVs in PARK2 was obtained for 2060 healthy controls derived from ten publications in which PARK2 has been exhaustively studied (sequencing and CNV analysis) and were used as controls.
  • Statistical analysis was carried out by comparing PARK2 abnormalities between patients and controls.
  • PARK2 CNVs were mostly clustered in RING 1 , IBR and RING2 domains of PARKIN which are essential for ubiquitin ligase's function.
  • PD cases were retrospectively identified in 2/17 melanoma families harboring a PARK2 mutation, either in the index case or at least one of their first degree relatives.
  • PARK2 CNVs were present in 76% of melanoma cell lines and in 60% of primary tumors.

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Abstract

La présente invention concerne un procédé de détermination qu'un sujet est ou non à risque d'avoir ou de développer un mélanome cutané, comprenant le test pour ledit sujet de la présence d'au moins une modification génétique dans le gène PARK2, la présence de ladite modification génétique indiquant un risque augmenté d'avoir ou de développer un mélanome cutané. La présente invention concernant en outre un composé choisi dans le groupe constitué d'inhibiteurs de kinase cycline-dépendante II (CDK2), d'inhibiteurs de cycline E, d'inhibiteurs de l'expression de kinase cycline-dépendante II (CDK2) et d'inhibiteurs de l'expression de cycline E pour le traitement du mélanome cutané chez un sujet ayant une modification génétique selon l'invention.
PCT/EP2013/051810 2012-01-31 2013-01-30 Procédés et kits pour prédire le risque d'avoir un mélanome cutané chez un sujet WO2013113762A1 (fr)

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