Methods for melanoma detection

Abstract

The present invention is directed to methods for detecting a melanoma, methods for determining whether a melanoma is stable or progressive, methods for evaluating the extent of surgery resection in a subject having a melanoma, and methods for determining a response by a subject having a melanoma to a therapy.

1 . A method for detecting a melanoma in a subject in need thereof, comprising:
 determining the expression level of at least 29 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific to detect the expression of the at least 29 biomarkers, wherein the at least 29 biomarkers comprise ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, YY2, and at least one housekeeping gene;   normalizing the expression level of each of ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, and YY2 to the expression level of the at least one housekeeping gene, thereby obtaining a normalized expression level of each of ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, and YY2;   inputting each normalized expression level into an algorithm to generate a score;   comparing the score with a first predetermined cutoff value; and   producing a report, wherein the report identifies the presence of a melanoma in the subject when the score is equal to or greater than the first predetermined cutoff value or identifies the absence of a melanoma in the subject when the score is below the first predetermined cutoff value, wherein the first predetermined cutoff value is 20 on a scale of 0 to 100.   
     
     
         2 . The method of  claim 1 , wherein the at least one housekeeping gene is selected from the group consisting of ALG9, SEPN, YWHAQ, VPS37A, PRRC2B, DOPEY2, NDUFB11, ND4, MRPL19, PSMC4, SF3A1, PUM1, ACTB, GAPD, GUSB, RPLPO, TFRC, MORF4L1, 18S, PPIA, PGK1, RPL13A, B2M, YWHAZ, SDHA, HPRT1, TOX4, and TPT1. 
     
     
         3 . The method of  claim 2 , wherein the at least one housekeeping gene comprises TOX4 and TPT1. 
     
     
         4 . The method of  claim 3 , wherein the normalized expression level is obtained by:
 normalizing the expression level of each of ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, and YY2 to the expression level of TOX4, thereby obtaining a first normalized expression level of each of ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, and YY2;   normalizing the expression level of each of ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, and YY2 to the expression level of TPT1, thereby obtaining a second normalized expression level of each of ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, and YY2;   averaging the first normalized expression level and the second normalized expression level to obtain the normalized expression level.   
     
     
         5 . The method of  claim 1 , having a sensitivity of greater than 90%. 
     
     
         6 . The method of  claim 1 , having a specificity of greater than 90%. 
     
     
         7 . The method of  claim 1 , wherein the melanoma is progressive. 
     
     
         8 . The method of  claim 1 , wherein at least one of the at least 29 biomarkers is RNA, cDNA, or protein. 
     
     
         9 . The method of  claim 8 , wherein when the biomarker is RNA, the RNA is reverse transcribed to produce cDNA, and the produced cDNA expression level is detected. 
     
     
         10 . The method of  claim 1 , wherein the expression level of the biomarker is detected by forming a complex between the biomarker and a labeled probe or primer. 
     
     
         11 . The method of  claim 10 , wherein when the biomarker is protein, the protein is detected by forming a complex between the protein and a labeled antibody. 
     
     
         12 . The method of  claim 11 , wherein the label is a fluorescent label. 
     
     
         13 . The method of  claim 8 , wherein when the biomarker is RNA or cDNA, the RNA or cDNA is detected by forming a complex between the RNA or cDNA and a labeled nucleic acid probe or primer. 
     
     
         14 . The method of  claim 13 , wherein the label is a fluorescent label. 
     
     
         15 . The method of  claim 13 , wherein the complex between the RNA or cDNA and the labeled nucleic acid probe or primer is a hybridization complex. 
     
     
         16 . The method of any one of  claims 1 - 15 , wherein the test sample is blood, serum, plasma, or neoplastic tissue. 
     
     
         17 . The method of  claim 1 , wherein the first predetermined cutoff value is derived from a plurality of reference samples obtained from subjects free of a neoplastic disease. 
     
     
         18 . The method of  claim 17 , wherein each reference sample is blood, serum, plasma, or non-neoplastic tissue. 
     
     
         19 . The method of  claim 1 , further comprising treating the subject identified as having a melanoma with surgery or drug therapy. 
     
     
         20 . The method of  claim 1 , wherein the subject in need thereof is a subject diagnosed with a melanoma, a subject having at least one melanoma symptom, or a subject having a predisposition or familial history for developing a melanoma. 
     
     
         21 . The method of  claim 1 , wherein a subject is human. 
     
     
         22 . The method of  claim 1 , wherein the algorithm is XGB, RF, glmnet, cforest, CART, treebag, knn, nnet, SVM-radial, SVM-linear, NB, NNET, or mlp. 
     
     
         23 . A method for determining whether a melanoma in a subject is stable or progressive, comprising:
 determining the expression level of at least 29 biomarkers from a test sample from the subject by contacting the test sample with a plurality of agents specific to detect the expression of the at least 29 biomarkers, wherein the at least 29 biomarkers comprise ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, YY2, and at least one housekeeping gene;   normalizing the expression level of each of ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, and YY2 to the expression level of the at least one housekeeping gene, thereby obtaining a normalized expression level of each of ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, and YY2;   inputting each normalized expression level into an algorithm to generate a score;   comparing the score with a second predetermined cutoff value; and   producing a report, wherein the report identifies that the melanoma is progressive when the normalized expression level is equal to or greater than the second predetermined cutoff value or identifies that the melanoma is stable when the normalized expression level is below the second predetermined cutoff value, wherein the second predetermined cutoff value is 50 on a scale of 0 to 100.   
     
     
         24 . The method of  claim 23 , wherein the at least one housekeeping gene is selected from the group consisting of ALG9, SEPN, YWHAQ, VPS37A, PRRC2B, DOPEY2, NDUFB11, ND4, MRPL19, PSMC4, SF3A1, PUM1, ACTB, GAPD, GUSB, RPLPO, TFRC, MORF4L1, 18S, PPIA, PGK1, RPL13A, B2M, YWHAZ, SDHA, HPRT1, TOX4, and TPT1. 
     
     
         25 . The method of  claim 24 , wherein the at least one housekeeping gene comprises TOX4 and TPT1. 
     
     
         26 . A method for evaluating the extent of surgical resection in a subject having a melanoma, comprising:
 determining the expression level of at least 29 biomarkers from a test sample from the subject after the surgical resection by contacting the test sample with a plurality of agents specific to detect the expression of the at least 29 biomarkers, wherein the at least 29 biomarkers comprise ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, YY2, and at least one housekeeping gene;   normalizing the expression level of each of ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, and YY2 to the expression level of the at least one housekeeping gene, thereby obtaining a normalized expression level of each of ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, and YY2;   inputting each normalized expression level into an algorithm to generate a score;   comparing the score with a third predetermined cutoff value; and   producing a report, wherein the report identifies that the surgical resection does not remove the entire melanoma when the normalized expression level is equal to or greater than the third predetermined cutoff value or identifies that the surgical resection removes the entire melanoma when the normalized expression level is below the third predetermined cutoff value, wherein the third predetermined cutoff value is 20 on a scale of 0 to 100.   
     
     
         27 . The method of  claim 26 , wherein the report further identifies that the risk of melanoma recurrence is high when the normalized expression level is equal to or greater than the third predetermined cutoff value or identifies that the risk of melanoma recurrence is low when the normalized expression level is below the third predetermined cutoff value. 
     
     
         28 . The method of  claim 26 , wherein the at least one housekeeping gene is selected from the group consisting of ALG9, SEPN, YWHAQ, VPS37A, PRRC2B, DOPEY2, NDUFB11, ND4, MRPL19, PSMC4, SF3A1, PUM1, ACTB, GAPD, GUSB, RPLPO, TFRC, MORF4L1, 18S, PPIA, PGK1, RPL13A, B2M, YWHAZ, SDHA, HPRT1, TOX4, and TPT1. 
     
     
         29 . The method of  claim 28 , wherein the at least one housekeeping gene comprises TOX4 and TPT1. 
     
     
         30 . A method for determining a response by a subject having a melanoma to a therapy, comprising:
 determining a first expression level of at least 28 biomarkers from a first test sample from the subject at a first time point by contacting the first test sample with a plurality of agents specific to detect the expression of the at least 28 biomarkers, wherein the 28 biomarkers comprise ATL1, ATP6V0D, C1ORF21, CFLAR, CFLAR-AS1, CHP1, DDX55, DMD, DNAJC9, ENOSF1, FANCL, HJURP, HLA-DOA, HLA-DRA, HNRNPA3P1, IL23A, IQGAP1, LOC494127, LOC646471, LOH12CR, PBXIP1, RNF5, SERTAD2, SLC35G5, SPATS2L, TDRD7, TXK, and YY2;   determining a second expression level of the at least 28 biomarkers from a second test sample from the subject at a second time point by contacting the second test sample with a plurality of agents specific to detect the expression of the at least 28 biomarkers, wherein the second time point is after the first time point and after the administration of the therapy to the subject;   comparing the first expression level with the second expression level; and   producing a report, wherein the report identifies that the subject is responsive to the therapy when the second expression level is significantly decreased as compared to the first expression level.   
     
     
         31 . The method of  claim 30 , wherein the first time point is prior to the administration of the therapy to the subject. 
     
     
         32 . The method of  claim 30 , wherein the first time point is after the administration of the therapy to the subject. 
     
     
         33 . The method of  claim 30 , wherein the therapy comprises an immunotherapy. 
     
     
         34 . The method of  claim 30 , wherein the therapy comprises a targeted therapy. 
     
     
         35 . The method of  claim 34 , wherein the targeted therapy comprises a BRAF inhibitor.