DIAGNOSIS OF MELANOMA BY NUCLEIC ACID ANALYSIS
The present invention provides methods for diagnosing melanoma in a subject by analyzing nucleic acid molecules obtained from the subject. The present invention also provides methods for distinguishing melanoma from dysplastic nevi and/or normal pigmented skin. The methods include analyzing expression or mutations in epidermal samples, of one or more skin markers. The methods can include the use of a microarray to analyze gene or protein profiles from a sample.
1 . A method for characterizing a skin lesion in a subject comprising analyzing a nucleic acid molecule from one or more genes listed in any of Tables 1-6, or any combination thereof, in a sample of the skin lesion, thereby characterizing a skin lesion of the subject.
2 . The method of claim 1 , wherein the nucleic acid molecule is RNA.
3 . The method of claim 1 , wherein analyzing the nucleic acid molecule comprises detecting one or more mutations in the nucleic acid sequence of the nucleic acid molecule.
4 . The method of claim 3 , wherein the one or more mutations are selected from the group consisting of a substitution, a deletion, and an insertion.
5 . The method of claim 1 , further comprising amplifying the nucleic acid molecule obtained from the sample prior to analyzing.
6 . The method of claim 1 , wherein the sample is obtained by applying an adhesive tape to a target area of skin in a manner sufficient to isolate the sample adhering to the adhesive tape.
7 . The method of claim 1 , further comprising using the characterizing to determine a treatment regimen.
8 . The method of claim 2 , wherein the isolated nucleic acid molecule or an amplification product thereof, is applied to a microarray.
9 . The method of claim 8 , wherein an expression profile is detected using a microarray.
10 . The method of claim 1 , wherein the sample is obtained from a biopsy taken at the site of the skin lesion or surrounding margin.
11 . The method of claim 6 , wherein the tape comprises a rubber adhesive on a polyurethane film.
12 . The method of claim 6 , wherein about one to ten adhesive tapes or one to ten applications of a tape are applied and removed from the skin.
13 . The method of claim 6 , wherein about one to eight adhesive tapes or one to eight applications of a tape are applied and removed from the skin.
14 . The method of claim 6 , wherein about one to five adhesive tapes or one to five applications of a tape are applied and removed from the skin.
15 . The method of claim 6 , wherein the method further comprises taking a biopsy of the target area of the skin.
16 . The method of claim 2 , wherein the analyzing is performed in situ.
17 . The method of claim 1 , wherein the nucleic acid molecule is from one or more genes listed in Table 6.
18 . A method of distinguishing melanoma from dysplastic nevi or normal pigmented skin in a subject comprising analyzing a nucleic acid molecule from one or more genes listed in any of Tables 1-6, or any combination thereof, in a sample from the subject, thereby distinguishing melanoma from dysplastic nevi or normal pigmented skin in a subject.
19 . The method of claim 18 , wherein the nucleic acid molecule is RNA.
20 . The method of claim 18 , wherein analyzing the nucleic acid molecule comprises detecting one or more mutations in the nucleic acid sequence of the nucleic acid molecule.
21 . The method of claim 20 , wherein the one or more mutations are selected from the group consisting of a substitution, a deletion, and an insertion.
22 . The method of claim 18 , further comprising amplifying the nucleic acid molecule obtained from the sample prior to analyzing.
23 . The method of claim 18 , wherein the sample is obtained by applying an adhesive tape to a target area of skin in a manner sufficient to isolate the sample adhering to the adhesive tape, wherein the sample comprises nucleic acid molecules.
24 . The method of claim 19 , wherein the isolated nucleic acid molecule or an amplification product thereof, is applied to a microarray.
25 . The method of claim 22 , wherein an expression profile is detected using a microarray.
26 . The method of claim 18 , wherein the sample is obtained from a biopsy taken from a target area of skin from the subject.
27 . The method of claim 23 , wherein the tape comprises a rubber adhesive on a polyurethane film.
28 . The method of claim 23 , wherein about one to ten adhesive tapes or one to ten applications of a tape are applied and removed from the skin.
29 . The method of claim 23 , wherein about one to eight adhesive tapes or one to eight applications of a tape are applied and removed from the skin.
30 . The method of claim 23 , wherein about one to five adhesive tapes or one to five applications of a tape are applied and removed from the skin.
31 . The method of claim 23 , wherein the method further comprises taking a biopsy of the target area of the skin.
32 . The method of claim 19 , wherein the analyzing is performed in situ.
33 . A method for diagnosing melanoma in a subject comprising detecting an altered level of a target protein in a sample from the subject, as compared to the level of the target protein in a corresponding sample from a subject that does not have melanoma, wherein the protein is an expression product of a gene listed in any of Tables 1-6, or any combination thereof, thereby diagnosing melanoma in the subject.
34 . The method of claim 33 , wherein the sample is obtained by applying an adhesive tape to a target area of skin in a manner sufficient to isolate the sample adhering to the adhesive tape, wherein the sample comprises cells, and further comprising lysing the cells to extract the target protein.
35 . The method of claim 34 , wherein the tape comprises a rubber adhesive on a polyurethane film.
36 . The method of claim 34 , wherein between one and ten adhesive tapes are applied to the skin and removed from the skin.
37 . The method of claim 34 , wherein about one to eight adhesive tapes are applied and removed from the skin.
38 . The method of claim 34 , wherein about one to five adhesive tapes are applied and removed from the skin.
39 . The method of claim 34 , wherein the method further comprises taking a biopsy of the target area of the skin.
40 . The method of claim 39 , wherein protein is extracted from the biopsy sample, and the level of protein in the biopsy and the level of protein in the tape sample are analyzed.
41 . The method of claim 33 , wherein the sample is obtained by from a biopsy of a target area of skin.
42 . The method of claim 33 , further comprising obtaining a sample from uninvolved tissue of the subject.
43 . The method of claim 42 , wherein the sample from uninvolved tissue is obtained by taking a biopsy of the uninvolved skin.
44 . The method of claim 42 , wherein the sample from uninvolved tissue is obtained by:
a) applying an adhesive tape to skin of the subject in a manner sufficient to isolate a skin sample adhering to the adhesive tape, wherein the skin sample comprises cells from the stratum corneum and wherein the skin is unaffected by a disease to be tested;
b) lysing the cells to extract a protein; and
c) quantitating the extracted protein.
45 . The method of claim 42 , wherein the uninvolved skin is from the mastoid process or the upper back.
46 . The method of claim 33 , wherein the protein is an expression product of a gene listed in Table 6.
47 . A method for diagnosing melanoma in a subject, comprising:
a) providing a gene expression profile of a target area suspected of being melanoma on the skin of the subject, wherein the target area of the skin simultaneously expresses a plurality of genes at the protein level that are markers for melanoma; and
b) comparing the subject's gene expression profile to a reference gene expression profile obtained from a corresponding normal skin sample, wherein the reference gene expression profile comprises an expression value of a target gene selected from the group consisting of human mRNA for tyrosinase-related protein 1 , LIM homeobox 2 , melan-A, spermidine/spermine N1-acetyltransferase, NDRG family member 2 , carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2 , syndecan binding protein (syntenin), crystallin, alpha B, endothelin receptor type B, tetratricopeptide repeat domain 3 , WAP four-disulfide core domain 3 , cytochrome P450 (family 1 , subfamily B, polypeptide 1 ), p8 protein (candidate of metastasis 1 ), interferon gamma-inducible protein 16 , dopachrome tautomerase (dopachrome delta-isomerase, tyrosine-related protein 2 ), solute carrier family 39 (zinc transporter), member 6 , heat shock 70 kDa protein 2 , vesicle-associated membrane protein 2 (synaptobrevin 2 ), Similar to AI661453 protein, and transient receptor potential cation channel (subfamily M, member 1 ), tyrosinase (oculocutaneous albinism IA), endothelin receptor type B, tetratricopeptide repeat domain 3 , syndecan binding protein (syntenin), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog, OTU domain, ubiquitin aldehyde binding 1 , protein phosphatase 1 regulatory (inhibitor) subunit 12 A, calponin 2 , dishevelled dsh homolog 3 (Drosophila), tribbles homolog 2 (Drosophila), mucolipin 3 , actinin alpha 4 , ribosomal protein S15, CDC37 cell division cycle 37 homolog (S. cerevisiae), jumonji domain containing 3 , v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (avian), c-Maf-inducing protein, myosin heavy polypeptide 14 , Hypothetical protein MGC40222, or any combination thereof.
48 . The method of claim 47 , wherein the reference gene expression profile further comprises an expression value of any target gene listed in Tables 1-6.
49 . The method of claim 47 or 48 , wherein the reference gene expression profile is contained within a database.
50 . The method of claim 47 , wherein said comparing is carried out using a computer algorithm.
51 . A kit for characterizing a skin lesion in a subject comprising a skin sample collection device and one or more probes or primers that selectively bind to one or more nucleic acid molecules in any of Tables 1-6, or to a nucleic acid or protein expression product of a nucleic acid molecule in any of Tables 1-6.
52 . The kit of claim 51 , wherein the kit provides a probe which binds to a portion of a nucleic acid molecule in any of Tables 1-6.
53 . The kit of claim 51 , wherein the kit provides one or more primer pairs comprising a forward primer that selectively binds upstream of a gene on one strand and a reverse primer that selectively binds upstream of a gene on a complementary strand, wherein the gene is listed in any of Tables 1-6.
54 . The kit of claim 51 , wherein the skin sample collection device is a biopsy needle.
55 . The kit of claim 51 , wherein the skin sample collection device is an adhesive tape.
56 . The kit of claim 55 , wherein the adhesive tape comprises a rubber adhesive on a polyurethane film.
57 . The kit of claim 51 , further comprising a microarray containing at least a fragment of a gene or a nucleic acid or protein product of a gene identified in any of Tables 1-6 or any combination thereof.
58 . A kit for characterizing a skin lesion in a subject comprising an applicator and one or more probes or primers that selectively bind to one or more of nucleic acid molecules in any of Tables 1-6, or to a nucleic acid or protein expression product of a nucleic acid molecule in any of Tables 1-6.
59 . The kit of claim 58 , wherein the probes are detectably labeled.
60 . The method of any one of claims 1 , 18 , 33 , or 47 , wherein the subject is human.
61 . The method of any one of claims 1 , 18 , 33 , or 47 , wherein the sample is an epidermal sample.