Lung cancer methylation markers

Abstract

The present invention discloses a method of diagnosing lung cancer by using methylation specific markers from a set, having diagnostic power for lung cancer diagnosis and distinguishing lung cancer types in diverse samples; as well as methods to identify sets of prognostic and diagnostic value.

1 .- 15 . (canceled) 
     
     
         16 . A nucleic acid primer or hybridization probe set specific for at least one potentially methylated region of at least one marker gene suitable to diagnose or predict lung cancer or a lung cancer type. 
     
     
         17 . The set of  claim 16 , wherein the at least one the marker gene is further defined as WT1, SALL3, TERT, ACTB, or CPEB4. 
     
     
         18 . The set of  claim 16 , wherein the lung cancer is adenocarcinoma or squamous cell carcinoma. 
     
     
         19 . The set of  claim 16 , further comprising a nucleic acid primer or hybridization probe specific for at least one additional marker gene defined as ABCB1, ACTB, AIM1L, APC, AREG, BMP2K, BOLL, C5AR1, C5orf4, CADM1, CDH13, CDX1, CLIC4, COL21A1, CPEB4, CXADR, DLX2, DNAJA4, DPH1, DRD2, EFS, ERBB2, ERCC1, ESR2, F2R, FAM43A, GABRA2, GAD1, GBP2, GDNF, GNA15, GNAS, HECW2, HIC1, HIST1H2AG, HLAG, HOXA1, HOXA10, HSD17B4, HSPA2, IRAK2, ITGA4, JUB, KCNJ15, KCNQ1, KIF5B, KL, KRT14, KRT17, LAMC2, MAGEB2, MBD2, MSH4, MT1G, MT3, MTHFR, NEUROD1, NHLH2, NKX2-1, ONECUT2, PENK, PITX2, PLAGL1, PTTG1, PYCARD, RASSF1, S100A8, SALL3, SERPINB5, SERPINE1, SERPINI1, SFRP2, SLC25A31, SMAD3, SPARC, SPHK1, SRGN, TERT, THRB, TJP2, TMEFF2, TNFRSF10C, TNFRSF25, TP53, ZDHHCI1, ZNF256, ZNF711, F2R, HOXA10, KL, SALL3, SPARC, TNFRSF25, or WT1. 
     
     
         20 . The set of  claim 16 , further defined as a nucleic acid primer or hybridization probe set comprising nucleic acid primers or hybridization probes being specific for potentially methylated regions of at least 50% of the marker genes in at least one of the following combinations:
 WT1, DLX2, SALL3, TERT, PITX2, HOXA10, F2R, CPEB4, NHLH2, SMAD3, ACTB, HOXA1, BOLL, APC, MT1G, PENK, SPARC, DNAJA4, RASSF1, HLA-G, ERCC1, ONECUT2, APC, ABCB1, ZNF573, KCNJ15, ZDHHC11, SFRP2, GDNF, PTTG1, SERPINI1, and TNFRSF10C;   WT1, PITX2, SALL3, F2R, DLX2, TERT, HOXA10, MSH4, NHLH2, GNA15, PENK, RASSF1, BOLL, HOXA1, ONECUT2, ABCB1, SPARC, MT1G, HSPA2, SFRP2, PYCARD, GAD1, C5orf4, C5AR1, GNDF, ZDHHC11, SERPINE1, NKX2-1, PITX2, C5AR1, GDNF, ZDHHC11, SERPINE1, NKX2-1, PITX2, C5AR1, ZNF256, FAM43A, SFRP2, MT3, SERPINE1M, CLIC4, TNFRSF10C, GABRA2, MTHFR, ESR2, NEUROG1, PITX2, PLAGL1, TMEFF2, PTTG1, CADM1, S100A8, EFS, JUB, ITGA4, MAGEB2, ERBB2, SRGN, GNAS, TJP2, KCNJ15, SLC25A31, ZNF573, TNFRSF25, APC, KCNQ1, LAMC2, SPHK1 DNAJA4, APC, MBD2, ERCC1 HLA-G, CXADR, TP53, ACTB, KL, SMAD3, HIST1H2AG, and CPEB4;   WT1 DLX2, SALL3, TERT, TNFRSF25, ACTB, SMAD3, and CPEB4;   WT1, DLX2, SALL3, TERT, PITX2, TNFRSF25, KL, ACTB, SMAD3, and CPEB4;   WT1, PITX2, SALL3, DLX2, TERT, HOXA10, RASSF1, SPARC, IRAK2, ZNF711, DNAJA4, HLA-G, CXADR, TP53, ACTB, and CPEB4;   WT1, PITX2, SALL3, F2R, TERT, HOXA10, RASSF1, SPARC, IRAK2, ZNF711, DRD2, DNAJA4, CXADR, TP53, ACTB, and CPEB4;   WT1, ACTB, DLX2, PITX2, SALL3, HOXA10, TERT, CPEB4, HLA-G, SPARC, RASSF1, DNAJA4, CXADR, TP53, IRAK2, and ZNF711;   F2R, ZNF256, CDH13, SERPINB5, KRT14, DLX2, AREG, THRB, HSD17B4, SPARC, HECW2, and COL21A1;   KL, HIST1H2AG, TJP2, SRGN, CDX1, TNFRSF25, APC, HIC1, APC, GNA15, ACTB, WT1, KRT17, AIM1L, DPH1, PITX2, PITX2, KIF5B, BMP2K, GBP2, NHLH2, GDNF, and BOLL;   WT1, DLX2, SALL3, TERT, PITX2, HOXA10, F2R, CPEB4, NHLH2, SMAD3, ACTB, HOXA1, BOLL, APC, MT1G, PENK, SPARC, DNAJA4, RASSF1, HLA-G, ERCC1, ONECUT2, APC, ABCB1, ZNF573, KCNJ15, ZDHHC11, SFRP2, GDNF, PTTG1, SERPINI1, and TNFRSF10C;   HOXA10 and NEUROD1;   WT1, PITX2, SALL3, F2R, TERT, HOXA10, RASSF1, SPARC, IRAK2, ZNF711, DRD2, DNAJA4, CXADR, TP53, ACTB, CPEB4, DLX2, TNFRSF25, KL, and SMAD3;   TNFRSF25, SALL3, RASSF1, TERT, SPARC, F2R, HOXA10, ZNF711, and PITX2   SALL3, PITX2, SPARC, F2R, TERT, RASSF1, HOXA10, CXADR, and KL   SALL3, SPARC, PITX2, F2R, TERT, RASSF1, HOXA10, and KL;   SALL3, PITX2, SPARC, F2R, HOXA10, DRD2, ACTB, DNAJA4, CXADR, KL;   SALL3, SPARC, PITX2, F2R, TERT, RASSF1, HOXA10, TNFRSF25, DNAJA4, TP53, CXADR, and KL;   SPARC, SALL3, F2R, PITX2, RASSF1, HOXA10, TERT, KL, and TNFRSF25;   SALL3, SPARC, PITX2, F2R, TERT, RASSF1, HOXA10, KL, TNFRSF25, CXADR; and   HOXA10, RASSF1, and F2R.   
     
     
         21 . The set of  claim 16 , further defined as comprising not more than 100000 probes or primer pairs. 
     
     
         22 . The set of  claim 16 , further defined as comprising not more than 100000 probes or primer pairs. 
     
     
         23 . The set of  claim 22 , further defined as comprising immobilized probes on a solid surface. 
     
     
         24 . The set of  claim 22 , wherein the primer pairs and probes are specific for a methylated upstream region of an open reading frame of the marker genes. 
     
     
         25 . The set of  claim 22 , wherein the probes or primers are specific for methylation in the genetic regions defined by any of SEQ ID NOs 1081 to 1440 including the adjacent up to 500 base pairs corresponding to any of gene marker IDs 1 to 359. 
     
     
         26 . The set of  claim 25 , wherein the probes or primers are of SEQ ID NOs 1 to 1080. 
     
     
         27 . A method of identifying or predicting a lung cancer or a lung cancer type in a patient, comprising:
 obtaining a set of nucleic acid primers or hybridization probes of  claim 16 ;   using the set to determine the methylation status of genes for which the members of the set are specific in a sample of DNA from the patient; and   comparing the methylation status of the genes with the status of a confirmed lung cancer type positive and/or negative state, thereby identifying lung cancer or lung cancer type, if any, in the patient.   
     
     
         28 . The method of  claim 27 , wherein the methylation status is determined by methylation specific PCR analysis, methylation specific digestion analysis and either or both of hybridization analysis to non-digested or digested fragments or PCR amplification analysis of non-digested or digested fragments. 
     
     
         29 . A method of determining a subset of diagnostic markers for potentially methylated genes from the genes of gene marker IDs 1-359 of Table 1, suitable for the diagnosis or prognosis of lung cancer or lung cancer type, comprising:
 a) obtaining data of the methylation status of at least 50 random genes selected from the 359 genes of gene marker IDs 1-359 in at least 1 sample of a confirmed lung cancer or lung cancer type state and at least one sample of a lung cancer or lung cancer type negative state;   b) correlating the results of the obtained methylation status with the lung cancer or lung cancer type;   c) optionally repeating the obtaining a) and correlating b) steps for a different combination of at least 50 random genes selected from the 359 genes of gene marker IDs 1-359; and   d) selecting as many marker genes which in a classification analysis have a p-value of less than 0.1 in a random-variance t-test, or selecting as many marker genes which in a classification analysis together have a correct lung cancer or lung cancer type prediction of at least 70% in a cross-validation test;   
       wherein the selected markers form the subset of diagnostic markers. 
     
     
         30 . The method of  claim 29 , wherein a) is further defined as comprising obtaining data of the methylation status of at least 50 random genes selected from the 359 genes of gene marker IDs 1-359 in at least 5 samples of a confirmed lung cancer or lung cancer type state. 
     
     
         31 . The method of  claim 29 , wherein the correlated results for each gene b) are rated by their correct correlation to the disease or tumor type positive state, preferably by p-value test, and selected in step d) in order of the rating. 
     
     
         32 . The method of  claim 29 , wherein not more than 40 marker genes are selected in step d) for the subset. 
     
     
         33 . The method of  claim 29 , wherein the step a) of obtaining data of the methylation status comprises determining data of the methylation status by methylation specific PCR analysis, methylation specific digestion analysis, or hybridization analysis to non-digested or digested fragments, or PCR amplification analysis of non-digested or digested fragments. 
     
     
         34 . A method of identifying or predicting a lung cancer or a lung cancer type in a patient, comprising:
 providing a set of a diagnostic subset of markers identified by a method of  claim 29 ;   using the set to determine methylation status of genes for which the members of the set are specific in a sample comprising DNA from the patient; and   comparing the methylation status of the genes with the status of a confirmed lung cancer type positive and/or negative state, thereby identifying lung cancer or lung cancer type, if any, in the patient.   
     
     
         35 . The method of  claim 34 , wherein the methylation status is determined by methylation specific PCR analysis, methylation specific digestion analysis and either or both of hybridization analysis to non-digested or digested fragments or PCR amplification analysis of non-digested or digested fragments.