Abstract:
Genes for thirteen DNA damage repair or DNA damage response enzymes can be epigenetically silenced in cancers. The silencing of nucleic acids encoding a DNA repair or DNA damage response enzyme can be used prognostically and for selecting treatments that are well tailored for an individual patient. Combinations of these markers can also be used to provide prognostic information. Kits for testing epigenetic silencing can be used to determine a prognosis or a therapeutic regimen.

Description:
[0001]    This application claims the benefit of U.S. Provisional Application Ser. No. 60/702,976 filed Jul. 28, 2005, the disclosure of which is expressly incorporated herein. 
     
    
     TECHNICAL FIELD OF THE INVENTION 
       [0002]    This invention is related to the area of cancer prognosis and therapeutics. In particular, it relates to aberrant methylation patterns of particular genes in cancers. 
       BACKGROUND OF THE INVENTION 
       [0003]    DNA Methylation and its Role in Carcinogenesis 
         [0004]    The information to make the cells of all living organisms is contained in their DNA. DNA is made up of a unique sequence of four bases: adenine (A), guanine (G), thymine (T) and cytosine (C). These bases are paired A to T and G to C on the two strands that form the DNA double helix. Strands of these pairs store information to make specific molecules grouped into regions called genes. Within each cell, there are processes that control what gene is turned on, or expressed, thus defining the unique function of the cell. One of these control mechanisms is the addition of a methyl group onto a cytosine (C) base. The methyl group tagged C can be written as mC. 
         [0005]    DNA methylation plays an important role in determining whether some genes are expressed or not. By turning genes off that are not needed, DNA methylation is an essential control mechanism for the normal development and functioning of organisms. Alternatively, abnormal DNA methylation is one of the mechanisms underlying the changes observed with aging and development of many cancers. 
         [0006]    Cancers have historically been linked to genetic changes caused by chromosomal mutations within the DNA. Mutations, hereditary or acquired, can lead to the loss of expression of genes critical for maintaining a healthy state. Evidence now supports the theory that a relatively large number of cancers originate, not from mutations, but from inappropriate DNA methylation. In many cases, hyper-methylation of DNA incorrectly switches off critical genes, such as tumor suppressor genes or DNA repair genes, allowing cancers to develop and progress. This non-mutational process for controlling gene expression is described as epigenetics. 
         [0007]    DNA methylation is a chemical modification of DNA performed by enzymes called methyltransferases, in which a methyl group (m) is added to certain cytosines (C) of DNA. This non-mutational (epigenetic) process (mC) is a critical factor in gene expression regulation. See, J. G. Herman, Seminars in Cancer Biology, 9: 359-67, 1999. 
         [0008]    Although the phenomenon of gene methylation has attracted the attention of cancer researchers for some time, its true role in the progression of human cancers is just now being recognized. In normal cells, methylation occurs predominantly in regions of DNA that have few CG base repeats, while CpG islands, regions of DNA that have long repeats of CG bases, remain non-methylated. Gene promoter regions that control protein expression are often CpG island-rich. Aberrant methylation of these normally non-methylated CpG islands in the promoter region causes transcriptional inactivation or silencing of certain tumor suppressors in human cancers. 
         [0009]    Genes that are hypermethylated in tumor cells are strongly specific to the tissue of origin of the tumor. Molecular signatures of cancers of all types can be used to improve cancer detection, the assessment of cancer risk and response to therapy. Promoter hypermethylation events provide some of the most promising markers for such purposes. 
         [0010]    Promoter Gene Hypermethylation: Promising Tumor Markers 
         [0011]    Information regarding the hypermethylation of specific promoter genes can be beneficial to diagnosis, prognosis, and treatment of various cancers. Methylation of specific gene promoter regions can occur early and often in carcinogenesis making these markers ideal targets for cancer diagnostics. 
         [0012]    Methylation patterns are tumor specific. Positive signals are always found in the same location of a gene. Real time PCR-based methods are highly sensitive, quantitative, and suitable for clinical use. DNA is stable and is found intact in readily available fluids (e.g., serum, sputum, stool and urine) and paraffin embedded tissues. Panels of pertinent gene markers may cover most human cancers. 
         [0013]    Diagnosis 
         [0014]    Key to improving the clinical outcome in patients with cancer is diagnosis at its earliest stage, while it is still localized and readily treatable. The characteristics noted above provide the means for a more accurate screening and surveillance program by identifying higher-risk patients on a molecular basis. It could also provide justification for more definitive follow up of patients who have molecular but not yet all the pathological or clinical features associated with malignancy. 
         [0015]    Predicting Treatment Response 
         [0016]    Information about how a cancer develops through molecular events could allow a clinician to predict more accurately how such a cancer is likely to respond to specific therapeutic treatments. In this way, a regimen based on knowledge of the tumor&#39;s sensitivity can be rationally designed. Prior studies have shown that hypermethylation of the MGMT promoter in glioma patients is indicative of a good response to therapy, greater overall survival and a longer time to progression. 
         [0017]    There is a continuing need in the art for new prognostic markers for determining appropriate therapies for treating cancer to improve management of patient care. 
       SUMMARY OF THE INVENTION 
       [0018]    One embodiment of the invention is a method of predicting a clinical response to a DNA-damaging anti-neoplastic treatment in a cancer patient. Epigenetic silencing of a nucleic acid encoding a DNA repair or DNA damage response enzyme is determined. The nucleic acid is isolated from the cancer patient. The DNA repair or DNA damage response enzyme is selected from the group consisting of: BRCA1 (breast cancer 1, early onset, aka BRCC1, IRIS, PSCP, RNF53), ADPRTL3 (poly (ADP-ribose) polymerase family, member 3, aka PARP3, ADPRTL2, IRT1, hPARP-3, pADPRT-3), XRCC3 (X-ray repair complementing defective repair in Chinese hamster cells 3), RECQL5 (RecQ protein-like, aka FLJ90603, RECQ5), POLB (Polymerase (DNA directed), beta), FANCG (Fanconi anemia, complementation group G, aka FAG, XRCC9), MSH2 (mutS homolog 2, colon cancer, nonpolyposis type 1 ( E. coli ), aka COCA1, FCC1, HNPCC, HNPCC1), HUS1 (HUS1 checkpoint homolog ( S. pombe )), ERCC3 (excision repair cross-complementing rodent repair deficiency, complementation group 3 (xeroderma pigmentosum group B complementing) aka BTF2, GTF2H, RAD25, TFIIH, XPB), RAD9A (RAD9 homolog A ( S. pombe , aka RAD9), and LIG4 ( Homo sapiens  ligase IV, DNA, ATP-dependent (LIG4), transcript variant 1). If epigenetic silencing is determined, a more favorable clinical response to the DNA-damaging anti-neoplastic treatment is predicted. 
         [0019]    Another embodiment of the invention is a method of treating a cancer patient. Epigenetic silencing of a nucleic acid encoding a first DNA repair or DNA damage response enzyme isolated from the cancer patient is determined. The DNA repair or DNA damage response enzyme is selected from the group consisting of: BRCA1, ADPRTL3, XRCC3, RECQL5, POLB, FANCG, MSH2, HUS1, ERCC3, RAD9A, and LIG4. The cancer patient is treated with a DNA-damaging anti-neoplastic treatment if epigenetic silencing is determined. 
         [0020]    Still another embodiment of the invention is a kit for assessing methylation in a test sample. The kit comprises a reagent that (a) modifies methylated cytosine residues but not non-methylated cytosine residues, or that (b); modifies non-methylated cytosine residues but not methylated cytosine residues. The kit also comprises a pair of oligonucleotide primers that specifically hybridizes under amplification conditions to a gene selected from the group consisting of BRCA1, ADPRTL3, XRCC3, RECQL5, POLB, FANCG, MSH2, HUS1, ERCC3, RAD9A, and LIG4. 
         [0021]    These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with tools and methods for detection, prognosis, therapy, and drug selection pertaining to neoplastic cells and cancers. 
       BRIEF DESCRIPTION OF THE TABLES 
       [0022]    Table 1 lists genes encoding DNA damage repair or response enzymes, methylation of which is indicative of prognosis and DNA-damaging treatment susceptibility. 
         [0023]    Table 2 lists reference sequences for enzymes involved in DNA damage repair or DNA damage response. 
         [0024]    Table 3 lists combinations of two and three of the genes encoding DNA repair enzymes, methylation of which is indicative of prognosis and DNA-damaging treatment susceptibility. Similar combinations can be made using RAD9A and LIG4 with the other genes. 
         [0025]    Table 4 shows Ct values collected for 21 different assays representing 10 different candidate markers and different treatment conditions 
         [0026]    Table 5 shows normalized Ct values collected for 21 different assays representing 10 different candidate markers and different treatment conditions 
         [0027]    Table 6 shows difference of Ct values for resistant and untreated cell lines 
         [0028]    Table 7 shows conditions showing a Ct value difference &gt;1.5 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0029]    The inventors have identified a set of genes encoding DNA damage repair or response enzymes, transcription of which is epigenetically silenced in some cancers. Moreover, the transcriptional silencing of these genes indicates increased susceptibility to DNA-damaging anti-neoplastic treatments. The identified genes are shown in Table 1 with exemplary reference sequences. Combinations of two or three of these genes are shown in Table 2. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Genes encoding DNA damage repair or DNA damage response enzymes 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 BRCA1 
                 17q21 
                 NM_007295 (SEQ ID NO: 1) 
               
               
                   
                   
                   
                 NM_007294 (SEQ ID NO: 61), 
               
               
                   
                   
                   
                 NM_007296 (SEQ ID NO: 62), 
               
               
                   
                   
                   
                 NM_007297 (SEQ ID NO: 63), 
               
               
                   
                   
                   
                 NM_007298 (SEQ ID NO: 64), 
               
               
                   
                   
                   
                 NM_007299 (SEQ ID NO: 65), 
               
               
                   
                   
                   
                 NM_007300 (SEQ ID NO: 66), 
               
               
                   
                   
                   
                 NM_007301 (SEQ ID NO: 67), 
               
               
                   
                   
                   
                 NM_007302 (SEQ ID NO: 78), 
               
               
                   
                   
                   
                 NM_007303 (SEQ ID NO: 69), 
               
               
                   
                   
                   
                 NM_007304 (SEQ ID NO: 70, 
               
               
                   
                   
                   
                 NM_007305 (SEQ ID NO: 71), 
               
               
                   
                   
                   
                 NM_007306 (SEQ ID NO: 72) 
               
               
                 2 
                 ADPRTL3 
                 3p22.2-p21.1 
                 NM_005485 (SEQ ID NO: 2), 
               
               
                   
                   
                   
                 NM_005485 (SEQ ID NO: 73) 
               
               
                 3 
                 XRCC3 
                 14q32.3 
                 NM_005432 (SEQ ID NO: 3) 
               
               
                 4 
                 RECQL5 
                 17q25.2-q25.3 
                 NM_004259 (SEQ ID NO: 4) 
               
               
                 5 
                 POLB 
                 8p11.2 
                 NM_002690 (SEQ ID NO: 5) 
               
               
                 6 
                 FANCG 
                 9p13 
                 NM_004629 (SEQ ID NO: 6) 
               
               
                 7 
                 MSH2 
                 2p22-p21 
                 NM_000251 (SEQ ID NO: 7) 
               
               
                 8 
                 HUS1 
                 7p13-p12 
                 NM_004507 (SEQ ID NO: 8) 
               
               
                 9 
                 ERCC3 
                 2q21 
                 NM_000122 (SEQ ID NO: 9) 
               
               
                 10 
                 MGMT 
                 10q26 
                 NM_002412 (SEQ ID NO: 10) 
               
               
                 11 
                 RAD9A 
                 11q13.1-q13.2 
                 NM_004584 (SEQ ID NO: 11) 
               
               
                 12 
                 LIG4 
                 13q33-q34 
                 NM_002312/NM_206937 
               
               
                   
                   
                   
                 (SEQ ID NO: 12-13) 
               
               
                   
               
               
                 Encoded amino acids are shown in SEQ ID NO: 14-26, respectively 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Reference sequences for proteins involved in DNA damage repair or DNA damage 
               
               
                 response 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 BRCA1: NP_009225(SEQ ID NO: 44), NP_009226(SEQ ID NO: 14), NP_009227(SEQ 
               
               
                   
                 ID NO: 45), NP_009228(SEQ ID NO: 46), NP_009229(SEQ ID NO: 47), 
               
               
                   
                 NP_009230(SEQ ID NO: 48), NP_009231(SEQ ID NO: 49), NP_009232(SEQ ID NO: 
               
               
                   
                 50), NP_009233(SEQ ID NO: 51), NP_009234(SEQ ID NO: 52), NP_009237 (SEQ ID 
               
               
                   
                 NO: 53) 
               
               
                 2. 
                 ADPRTL3: NP_001003931(SEQ ID NO: 54), NP_001003935(SEQ ID NO: 
               
               
                   
                 55), NP_005476 (SEQ ID NO: 15) 
               
               
                 3. 
                 XRCC3: NP_005423 (SEQ ID NO: 16) 
               
               
                 4. 
                 RECQL5: NP_0042503(SEQ ID NO: 17), NP_001003716(SEQ ID NO: 56), 
               
               
                   
                 NP_001003715 (SEQ ID NO: 57) 
               
               
                 5. 
                 POLB: NP_002681 (SEQ ID NO: 18) 
               
               
                 6. 
                 FANCG: NP_004620 (SEQ ID NO: 19) 
               
               
                 7. 
                 MSH2: NP_000242 (SEQ ID NO: 20) 
               
               
                 8. 
                 HUS1: NP_683762 (SEQ ID NO: 58) 
               
               
                 9. 
                 ERCC3: NP_000113 (SEQ ID NO: 22) 
               
               
                 10. 
                 MGMT: NP_002403 (SEQ ID NO: 23) 
               
               
                 11. 
                 RAD9: NP_689655(SEQ ID NO: 59), NP_004575 (SEQ ID NO: 24) 
               
               
                 12. 
                 LIG4: NP_002303(SEQ ID NO: 25), NP_996820 (SEQ ID NO: 60) 
               
               
                   
               
             
          
         
       
     
         [0030]    Encoding nucleotides for SEQ ID NO: 44-60 are shown in SEQ ID NO: 27-43, respectively 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Combinations of Two and Three Genes Encoding DNA Repair Enzymes 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 NM_007295, NM_005485 
               
               
                 2. 
                 NM_007295, NM_005432 
               
               
                 3. 
                 NM_007295, NM_004259 
               
               
                 4. 
                 NM_007295, NM_002690 
               
               
                 5. 
                 NM_007295, NM_004629 
               
               
                 6. 
                 NM_007295, NM_000251 
               
               
                 7. 
                 NM_007295, NM_004507 
               
               
                 8. 
                 NM_007295, NM_000122 
               
               
                 9. 
                 NM_007295, NM_002412 
               
               
                 10. 
                 NM_007295, NM_004584 
               
               
                 11. 
                 NM_007295, NM_002312 
               
               
                 12. 
                 NM_005485, NM_005432 
               
               
                 13. 
                 NM_005485, NM_004259 
               
               
                 14. 
                 NM_005485, NM_002690 
               
               
                 15. 
                 NM_005485, NM_004629 
               
               
                 16. 
                 NM_005485, NM_000251 
               
               
                 17. 
                 NM_005485, NM_004507 
               
               
                 18. 
                 NM_005485, NM_000122 
               
               
                 19. 
                 NM_005485, NM_002412 
               
               
                 20. 
                 NM_005485, NM_004584 
               
               
                 21. 
                 NM_005485, NM_002312 
               
               
                 22. 
                 NM_005432, NM_004259 
               
               
                 23. 
                 NM_005432, NM_002690 
               
               
                 24. 
                 NM_005432, NM_004629 
               
               
                 25. 
                 NM_005432, NM_000251 
               
               
                 26. 
                 NM_005432, NM_004507 
               
               
                 27. 
                 NM_005432, NM_000122 
               
               
                 28. 
                 NM_005432, NM_002412 
               
               
                 29. 
                 NM_005432, NM_004584 
               
               
                 30. 
                 NM_005432, NM_002312 
               
               
                 31. 
                 NM_004259, NM_002690 
               
               
                 32. 
                 NM_004259, NM_004629 
               
               
                 33. 
                 NM_004259, NM_000251 
               
               
                 34. 
                 NM_004259, NM_004507 
               
               
                 35. 
                 NM_004259, NM_000122 
               
               
                 36. 
                 NM_004259, NM_002412 
               
               
                 37. 
                 NM_004259, NM_004584 
               
               
                 38. 
                 NM_004259, NM_002312 
               
               
                 39. 
                 NM_002690, NM_004629 
               
               
                 40. 
                 NM_002690, NM_000251 
               
               
                 41. 
                 NM_002690, NM_004507 
               
               
                 42. 
                 NM_002690, NM_000122 
               
               
                 43. 
                 NM_002690, NM_002412 
               
               
                 44. 
                 NM_002690, NM_004584 
               
               
                 45. 
                 NM_002690, NM_002312 
               
               
                 46. 
                 NM_004629, NM_000251 
               
               
                 47. 
                 NM_004629, NM_004507 
               
               
                 48. 
                 NM_004629, NM_000122 
               
               
                 49. 
                 NM_004629, NM_002412 
               
               
                 50. 
                 NM_004629, NM_004584 
               
               
                 51. 
                 NM_004629, NM_002312 
               
               
                 52. 
                 NM_000251, NM_004507 
               
               
                 53. 
                 NM_000251, NM_000122 
               
               
                 54. 
                 NM_000251, NM_002412 
               
               
                 55. 
                 NM_000251, NM_004584 
               
               
                 56. 
                 NM_000251, NM_002312 
               
               
                 57. 
                 NM_004507, NM_000122 
               
               
                 58. 
                 NM_004507, NM_002412 
               
               
                 59. 
                 NM_004507, NM_004584 
               
               
                 60. 
                 NM_004507, NM_002312 
               
               
                 61. 
                 NM_000122, NM_002412 
               
               
                 62. 
                 NM_000122, NM_004584 
               
               
                 63. 
                 NM_000122, NM_002312 
               
               
                 64. 
                 NM_002412, NM_004584 
               
               
                 65. 
                 NM_002412, NM_002312 
               
               
                 66. 
                 NM_004584, NM_002312 
               
               
                 67. 
                 NM_007295, NM_005485, NM_005432 
               
               
                 68. 
                 NM_007295, NM_005485, NM_004259 
               
               
                 69. 
                 NM_007295, NM_005485, NM_002690 
               
               
                 70. 
                 NM_007295, NM_005485, NM_004629 
               
               
                 71. 
                 NM_007295, NM_005485, NM_000251 
               
               
                 72. 
                 NM_007295, NM_005485, NM_004507 
               
               
                 73. 
                 NM_007295, NM_005485, NM_000122 
               
               
                 74. 
                 NM_007295, NM_005485, NM_002412 
               
               
                 75. 
                 NM_007295, NM_005485, NM_004584 
               
               
                 76. 
                 NM_007295, NM_005485, NM_002312 
               
               
                 77. 
                 NM_007295, NM_005432, NM_004259 
               
               
                 78. 
                 NM_007295, NM_005432, NM_002690 
               
               
                 79. 
                 NM_007295, NM_005432, NM_004629 
               
               
                 80. 
                 NM_007295, NM_005432, NM_000251 
               
               
                 81. 
                 NM_007295, NM_005432, NM_004507 
               
               
                 82. 
                 NM_007295, NM_005432, NM_000122 
               
               
                 83. 
                 NM_007295, NM_005432, NM_002412 
               
               
                 84. 
                 NM_007295, NM_005432, NM_004584 
               
               
                 85. 
                 NM_007295, NM_005432, NM_002312 
               
               
                 86. 
                 NM_007295, NM_004259, NM_002690 
               
               
                 87. 
                 NM_007295, NM_004259, NM_004629 
               
               
                 88. 
                 NM_007295, NM_004259, NM_000251 
               
               
                 89. 
                 NM_007295, NM_004259, NM_004507 
               
               
                 90. 
                 NM_007295, NM_004259, NM_000122 
               
               
                 91. 
                 NM_007295, NM_004259, NM_002412 
               
               
                 92. 
                 NM_007295, NM_004259, NM_004584 
               
               
                 93. 
                 NM_007295, NM_004259, NM_002312 
               
               
                 94. 
                 NM_007295, NM_002690, NM_004629 
               
               
                 95. 
                 NM_007295, NM_002690, NM_000251 
               
               
                 96. 
                 NM_007295, NM_002690, NM_004507 
               
               
                 97. 
                 NM_007295, NM_002690, NM_000122 
               
               
                 98. 
                 NM_007295, NM_002690, NM_002412 
               
               
                 99. 
                 NM_007295, NM_002690, NM_004584 
               
               
                 100. 
                 NM_007295, NM_002690, NM_002312 
               
               
                 101. 
                 NM_007295, NM_004629, NM_000251 
               
               
                 102. 
                 NM_007295, NM_004629, NM_004507 
               
               
                 103. 
                 NM_007295, NM_004629, NM_000122 
               
               
                 104. 
                 NM_007295, NM_004629, NM_002412 
               
               
                 105. 
                 NM_007295, NM_004629, NM_004584 
               
               
                 106. 
                 NM_007295, NM_004629, NM_002312 
               
               
                 107. 
                 NM_007295, NM_000251, NM_004507 
               
               
                 108. 
                 NM_007295, NM_000251, NM_000122 
               
               
                 109. 
                 NM_007295, NM_000251, NM_002412 
               
               
                 110. 
                 NM_007295, NM_000251, NM_004584 
               
               
                 111. 
                 NM_007295, NM_000251, NM_002312 
               
               
                 112. 
                 NM_007295, NM_004507, NM_000122 
               
               
                 113. 
                 NM_007295, NM_004507, NM_002412 
               
               
                 114. 
                 NM_007295, NM_004507, NM_004584 
               
               
                 115. 
                 NM_007295, NM_004507, NM_002312 
               
               
                 116. 
                 NM_007295, NM_000122, NM_002412 
               
               
                 117. 
                 NM_007295, NM_000122, NM_004584 
               
               
                 118. 
                 NM_007295, NM_000122, NM_002312 
               
               
                 119. 
                 NM_007295, NM_002412, NM_004584 
               
               
                 120. 
                 NM_007295, NM_002412, NM_002312 
               
               
                 121. 
                 NM_007295, NM_004584, NM_002312 
               
               
                 122. 
                 NM_005485, NM_005432, NM_004259 
               
               
                 123. 
                 NM_005485, NM_005432, NM_002690 
               
               
                 124. 
                 NM_005485, NM_005432, NM_004629 
               
               
                 125. 
                 NM_005485, NM_005432, NM_000251 
               
               
                 126. 
                 NM_005485, NM_005432, NM_004507 
               
               
                 127. 
                 NM_005485, NM_005432, NM_000122 
               
               
                 128. 
                 NM_005485, NM_005432, NM_002412 
               
               
                 129. 
                 NM_005485, NM_005432, NM_004584 
               
               
                 130. 
                 NM_005485, NM_005432, NM_002312 
               
               
                 131. 
                 NM_005485, NM_004259, NM_002690 
               
               
                 132. 
                 NM_005485, NM_004259, NM_004629 
               
               
                 133. 
                 NM_005485, NM_004259, NM_000251 
               
               
                 134. 
                 NM_005485, NM_004259, NM_004507 
               
               
                 135. 
                 NM_005485, NM_004259, NM_000122 
               
               
                 136. 
                 NM_005485, NM_004259, NM_002412 
               
               
                 137. 
                 NM_005485, NM_004259, NM_004584 
               
               
                 138. 
                 NM_005485, NM_004259, NM_002312 
               
               
                 139. 
                 NM_005485, NM_002690, NM_004629 
               
               
                 140. 
                 NM_005485, NM_002690, NM_000251 
               
               
                 141. 
                 NM_005485, NM_002690, NM_004507 
               
               
                 142. 
                 NM_005485, NM_002690, NM_000122 
               
               
                 143. 
                 NM_005485, NM_002690, NM_002412 
               
               
                 144. 
                 NM_005485, NM_002690, NM_004584 
               
               
                 145. 
                 NM_005485, NM_002690, NM_002312 
               
               
                 146. 
                 NM_005485, NM_004629, NM_000251 
               
               
                 147. 
                 NM_005485, NM_004629, NM_004507 
               
               
                 148. 
                 NM_005485, NM_004629, NM_000122 
               
               
                 149. 
                 NM_005485, NM_004629, NM_002412 
               
               
                 150. 
                 NM_005485, NM_004629, NM_004584 
               
               
                 151. 
                 NM_005485, NM_004629, NM_002312 
               
               
                 152. 
                 NM_005485, NM_000251, NM_004507 
               
               
                 153. 
                 NM_005485, NM_000251, NM_000122 
               
               
                 154. 
                 NM_005485, NM_000251, NM_002412 
               
               
                 155. 
                 NM_005485, NM_000251, NM_004584 
               
               
                 156. 
                 NM_005485, NM_000251, NM_002312 
               
               
                 157. 
                 NM_005485, NM_004507, NM_000122 
               
               
                 158. 
                 NM_005485, NM_004507, NM_002412 
               
               
                 159. 
                 NM_005485, NM_004507, NM_004584 
               
               
                 160. 
                 NM_005485, NM_004507, NM_002312 
               
               
                 161. 
                 NM_005485, NM_000122, NM_002412 
               
               
                 162. 
                 NM_005485, NM_000122, NM_004584 
               
               
                 163. 
                 NM_005485, NM_000122, NM_002312 
               
               
                 164. 
                 NM_005485, NM_002412, NM_004584 
               
               
                 165. 
                 NM_005485, NM_002412, NM_002312 
               
               
                 166. 
                 NM_005485, NM_004584, NM_002312 
               
               
                 167. 
                 NM_005432, NM_004259, NM_002690 
               
               
                 168. 
                 NM_005432, NM_004259, NM_004629 
               
               
                 169. 
                 NM_005432, NM_004259, NM_000251 
               
               
                 170. 
                 NM_005432, NM_004259, NM_004507 
               
               
                 171. 
                 NM_005432, NM_004259, NM_000122 
               
               
                 172. 
                 NM_005432, NM_004259, NM_002412 
               
               
                 173. 
                 NM_005432, NM_004259, NM_004584 
               
               
                 174. 
                 NM_005432, NM_004259, NM_002312 
               
               
                 175. 
                 NM_005432, NM_002690, NM_004629 
               
               
                 176. 
                 NM_005432, NM_002690, NM_000251 
               
               
                 177. 
                 NM_005432, NM_002690, NM_004507 
               
               
                 178. 
                 NM_005432, NM_002690, NM_000122 
               
               
                 179. 
                 NM_005432, NM_002690, NM_002412 
               
               
                 180. 
                 NM_005432, NM_002690, NM_004584 
               
               
                 181. 
                 NM_005432, NM_002690, NM_002312 
               
               
                 182. 
                 NM_005432, NM_004629, NM_000251 
               
               
                 183. 
                 NM_005432, NM_004629, NM_004507 
               
               
                 184. 
                 NM_005432, NM_004629, NM_000122 
               
               
                 185. 
                 NM_005432, NM_004629, NM_002412 
               
               
                 186. 
                 NM_005432, NM_004629, NM_004584 
               
               
                 187. 
                 NM_005432, NM_004629, NM_002312 
               
               
                 188. 
                 NM_005432, NM_000251, NM_004507 
               
               
                 189. 
                 NM_005432, NM_000251, NM_000122 
               
               
                 190. 
                 NM_005432, NM_000251, NM_002412 
               
               
                 191. 
                 NM_005432, NM_000251, NM_004584 
               
               
                 192. 
                 NM_005432, NM_000251, NM_002312 
               
               
                 193. 
                 NM_005432, NM_004507, NM_000122 
               
               
                 194. 
                 NM_005432, NM_004507, NM_002412 
               
               
                 195. 
                 NM_005432, NM_004507, NM_004584 
               
               
                 196. 
                 NM_005432, NM_004507, NM_002312 
               
               
                 197. 
                 NM_005432, NM_000122, NM_002412 
               
               
                 198. 
                 NM_005432, NM_000122, NM_004584 
               
               
                 199. 
                 NM_005432, NM_000122, NM_002312 
               
               
                 200. 
                 NM_005432, NM_002412, NM_004584 
               
               
                 201. 
                 NM_005432, NM_002412, NM_002312 
               
               
                 202. 
                 NM_005432, NM_004584, NM_002312 
               
               
                 203. 
                 NM_004259, NM_002690, NM_004629 
               
               
                 204. 
                 NM_004259, NM_002690, NM_000251 
               
               
                 205. 
                 NM_004259, NM_002690, NM_004507 
               
               
                 206. 
                 NM_004259, NM_002690, NM_000122 
               
               
                 207. 
                 NM_004259, NM_002690, NM_002412 
               
               
                 208. 
                 NM_004259, NM_002690, NM_004584 
               
               
                 209. 
                 NM_004259, NM_002690, NM_002312 
               
               
                 210. 
                 NM_004259, NM_004629, NM_000251 
               
               
                 211. 
                 NM_004259, NM_004629, NM_004507 
               
               
                 212. 
                 NM_004259, NM_004629, NM_000122 
               
               
                 213. 
                 NM_004259, NM_004629, NM_002412 
               
               
                 214. 
                 NM_004259, NM_004629, NM_004584 
               
               
                 215. 
                 NM_004259, NM_004629, NM_002312 
               
               
                 216. 
                 NM_004259, NM_000251, NM_004507 
               
               
                 217. 
                 NM_004259, NM_000251, NM_000122 
               
               
                 218. 
                 NM_004259, NM_000251, NM_002412 
               
               
                 219. 
                 NM_004259, NM_000251, NM_004584 
               
               
                 220. 
                 NM_004259, NM_000251, NM_002312 
               
               
                 221. 
                 NM_004259, NM_004507, NM_000122 
               
               
                 222. 
                 NM_004259, NM_004507, NM_002412 
               
               
                 223. 
                 NM_004259, NM_004507, NM_004584 
               
               
                 224. 
                 NM_004259, NM_004507, NM_002312 
               
               
                 225. 
                 NM_004259, NM_000122, NM_002412 
               
               
                 226. 
                 NM_004259, NM_000122, NM_004584 
               
               
                 227. 
                 NM_004259, NM_000122, NM_002312 
               
               
                 228. 
                 NM_004259, NM_002412, NM_004584 
               
               
                 229. 
                 NM_004259, NM_002412, NM_002312 
               
               
                 230. 
                 NM_004259, NM_004584, NM_002312 
               
               
                 231. 
                 NM_002690, NM_004629, NM_000251 
               
               
                 232. 
                 NM_002690, NM_004629, NM_004507 
               
               
                 233. 
                 NM_002690, NM_004629, NM_000122 
               
               
                 234. 
                 NM_002690, NM_004629, NM_002412 
               
               
                 235. 
                 NM_002690, NM_004629, NM_004584 
               
               
                 236. 
                 NM_002690, NM_004629, NM_002312 
               
               
                 237. 
                 NM_002690, NM_000251, NM_004507 
               
               
                 238. 
                 NM_002690, NM_000251, NM_000122 
               
               
                 239. 
                 NM_002690, NM_000251, NM_002412 
               
               
                 240. 
                 NM_002690, NM_000251, NM_004584 
               
               
                 241. 
                 NM_002690, NM_000251, NM_002312 
               
               
                 242. 
                 NM_002690, NM_004507, NM_000122 
               
               
                 243. 
                 NM_002690, NM_004507, NM_002412 
               
               
                 244. 
                 NM_002690, NM_004507, NM_004584 
               
               
                 245. 
                 NM_002690, NM_004507, NM_002312 
               
               
                 246. 
                 NM_002690, NM_000122, NM_002412 
               
               
                 247. 
                 NM_002690, NM_000122, NM_004584 
               
               
                 248. 
                 NM_002690, NM_000122, NM_002312 
               
               
                 249. 
                 NM_002690, NM_002412, NM_004584 
               
               
                 250. 
                 NM_002690, NM_002412, NM_002312 
               
               
                 251. 
                 NM_002690, NM_004584, NM_002312 
               
               
                 252. 
                 NM_004629, NM_000251, NM_004507 
               
               
                 253. 
                 NM_004629, NM_000251, NM_000122 
               
               
                 254. 
                 NM_004629, NM_000251, NM_002412 
               
               
                 255. 
                 NM_004629, NM_000251, NM_004584 
               
               
                 256. 
                 NM_004629, NM_000251, NM_002312 
               
               
                 257. 
                 NM_004629, NM_004507, NM_000122 
               
               
                 258. 
                 NM_004629, NM_004507, NM_002412 
               
               
                 259. 
                 NM_004629, NM_004507, NM_004584 
               
               
                 260. 
                 NM_004629, NM_004507, NM_002312 
               
               
                 261. 
                 NM_004629, NM_000122, NM_002412 
               
               
                 262. 
                 NM_004629, NM_000122, NM_004584 
               
               
                 263. 
                 NM_004629, NM_000122, NM_002312 
               
               
                 264. 
                 NM_004629, NM_002412, NM_004584 
               
               
                 265. 
                 NM_004629, NM_002412, NM_002312 
               
               
                 266. 
                 NM_004629, NM_004584, NM_002312 
               
               
                 267. 
                 NM_000251, NM_004507, NM_000122 
               
               
                 268. 
                 NM_000251, NM_004507, NM_002412 
               
               
                 269. 
                 NM_000251, NM_004507, NM_004584 
               
               
                 270. 
                 NM_000251, NM_004507, NM_002312 
               
               
                 271. 
                 NM_000251, NM_000122, NM_002412 
               
               
                 272. 
                 NM_000251, NM_000122, NM_004584 
               
               
                 273. 
                 NM_000251, NM_000122, NM_002312 
               
               
                 274. 
                 NM_000251, NM_002412, NM_004584 
               
               
                 275. 
                 NM_000251, NM_002412, NM_002312 
               
               
                 276. 
                 NM_000251, NM_004584, NM_002312 
               
               
                 277. 
                 NM_004507, NM_000122, NM_002412 
               
               
                 278. 
                 NM_004507, NM_000122, NM_004584 
               
               
                 279. 
                 NM_004507, NM_000122, NM_002312 
               
               
                 280. 
                 NM_004507, NM_002412, NM_004584 
               
               
                 281. 
                 NM_004507, NM_002412, NM_002312 
               
               
                 282. 
                 NM_004507, NM_004584, NM_002312 
               
               
                 283. 
                 NM_000122, NM_002412, NM_004584 
               
               
                 284. 
                 NM_000122, NM_002412, NM_002312 
               
               
                 285. 
                 NM_000122, NM_004584, NM_002312 
               
               
                 286. 
                 NM_002412, NM_004584, NM_002312 
               
               
                   
               
               
                 Although accession numbers and particular sequences are named in the combinations above, they represent the gene or protein generically, including the disclosed variant sequences. 
               
             
          
         
       
     
         [0031]    DNA-damaging anti-neoplastic treatments, according to the invention include radiation therapies as well as chemotherapies. These may cause, inter alia, single strand, or double strand breaks, modifications of particular bases, dimerization of adjacent bases, etc. Radiation therapies that damage DNA include radiation generated by an external beam, modulated radiation therapy, stereotactic radiosurgery, stereotactic radiotherapy. Chemotherapies that damage DNA include alkylating agents, platinum compounds, anthracyclines, antimetabolites, and etoposides. The alkylating agents include busulfan, N-methyl-N′-nitrosoguanidine, N-methul-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, ifosfamide, dacarbazine (DTIC), mechlorethamine (nitrogen mustard), melphalan, and temozolomide. The antimetabolites include 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine (ara-C), fludarabine, and pemetrexed. and 6-thioguanine. The platinum compounds are exemplified by carboplatin and cisplatin. The anthracyclines are exemplified by daunorubicin, doxorubicin (Adriamycin), epirubicin, idarubicin, and mitoxantrone. The etoposides are exemplified by epipodophyllotoxine etoposide, topotecan, irinotecan, etoposide (VP-16), and teniposide. 
         [0032]    Epigenetic silencing of a nucleic acid encoding a DNA repair or DNA damage response enzyme can be determined by any method known in the art. One method is to determine that a nucleic acid which is expressed in normal cells is expressed at a lower level or not expressed in tumor cells. This method does not, on its own, however, indicate that the silencing is epigenetic, as the mechanism of the silencing could be genetic, for example, by somatic mutation. One method to determine that the silencing is epigenetic is to treat with a reagent, such as DAC (5′-deazacytidine) and observe that the silencing is reversed, i.e., that the expression of the gene is reactivated or restored. Another means to determine epigenetic silencing is to determine the presence of methylated CpG dinucleotide motifs in the silenced gene. These may reside near the transcription start site, for example, within about 1 kbp, within about 750 bp, or within about 500 bp, or within about 250 bp, or within about 200 bp, or within about 100 bp. 
         [0033]    Expression of a nucleic acid encoding a DNA repair or DNA damage response enzyme can be assessed using any means known in the art. Either mRNA or protein can be measured. Methods employing hybridization to nucleic acid probes can be employed for measuring specific mRNAs. Such methods include using nucleic acid probe arrays and using Northern blots. Messenger RNA can also be assessed using amplification techniques, such as RT-PCR. Specific proteins can be assessed using any convenient method. Most such methods will employ antibodies which are specific for the particular DNA damage repair or response enzyme. The antibodies may optionally be attached to a solid support, such as an array. The sequences of the mRNA (cDNA) and proteins of the markers of the present invention are provided in the sequence listing. While nucleotide and amino acid sequences of particular allelic forms are disclosed herein, any cDNA or protein which is &gt;95, 96, 97, 98, or 99% identical may be used. Alternatively spliced forms may be used as well. 
         [0034]    Methylation-sensitive restriction endonucleases can be used to detect methylated CpG dinucleotide motifs. Such endonucleases may either preferentially cleave methylated recognition sites relative to non-methylated recognition sites or preferentially cleave non-methylated relative to methylated recognition sites. Examples of the former are Acc III, Ban I, BstN I, Msp I, and Xma I. Examples of the latter are Acc II, Ava I, BssH II, BstU I, Hpa II, and Not I. Alternatively, chemical reagents can be used which selectively modify either the methylated or non-methylated form of CpG dinucleotide motifs. 
         [0035]    Modified products can be detected directly, or after a further reaction which creates products which are easily distinguishable. Means which detect altered size and/or charge can be used to detect modified products, including but not limited to electrophoresis, chromatography, and mass spectrometry. Examples of such chemical reagents for selective modification include hydrazine and bisulfite ions. Hydrazine-modified DNA can be treated with piperidine to cleave it. Bisulfite ion-treated DNA can be treated with alkali. 
         [0036]    One way to distinguish between modified and unmodified DNA is to hybridize oligonucleotide primers which specifically bind to one form or the other of the DNA. After hybridization, an amplification reaction can be performed and amplification products assayed. The presence of an amplification product indicates that a sample hybridized to the primer. The specificity of the primer indicates whether the DNA had been modified or not, which in turn indicates whether the DNA had been methylated or not. For example, bisulfite ions modify non-methylated cytosine bases, changing them to uracil bases. Uracil bases hybridize to adenine bases under hybridization conditions. Thus an oligonucleotide primer which comprises adenine bases in place of guanine bases would hybridize to the bisulfite-modified DNA, whereas an oligonucleotide primer containing the guanine bases would hybridize to the non-modified (methylated) cytosine residues in the DNA. Amplification using a DNA polymerase and a second primer yield amplification products which can be readily observed. Such a method is termed MSP (Methylation Specific PCR). The amplification products can be optionally hybridized to specific oligonucleotide probes which may also be specific for certain products. Alternatively, oligonucleotide probes can be used which will hybridize to amplification products from both modified and nonmodified DNA. 
         [0037]    Another way to distinguish between modified and nonmodified DNA is to use oligonucleotide probes which may also be specific for certain products. Such probes can be hybridized directly to modified DNA or to amplification products of modified DNA. Oligonucleotide probes can be labeled using any detection system known in the art. These include but are not limited to fluorescent moieties, radioisotope labeled moieties, bioluminescent moieties, luminescent moieties, chemiluminescent moieties, enzymes, substrates, receptors, or ligands. 
         [0038]    Test samples for diagnostic, prognostic, or personalized medicine uses can be obtained from surgical samples, such as biopsies or fine needle aspirates, from paraffin embedded tissues, from a body fluid such as bone marrow, blood, serum, lymph, cerebrospinal fluid, saliva, sputum, stool, urine, or semen. This list of sources is not meant to be exhaustive, but rather exemplary. 
         [0039]    Although accuracy and sensitivity may be achieved by using a combination of markers, such as 5 or 6 markers, practical considerations may dictate use of smaller combinations. Any combination of markers (repair enzymes) for a specific cancer may be used which comprises 2, 3, 4, 5, 6, 7, 8, or 9 of the identified markers. These may be combined with other markers known in the art, for example MGMT. Each of the combinations for two and three markers is listed in Table 3. Other combinations of four, five, or more markers, for example, can be readily and specifically envisioned given the specific disclosures of individual markers provided herein. 
         [0040]    Kits according to the present invention are assemblages of reagents for testing methylation. They are typically in a package which contains all elements, optionally including instructions. The package may be divided so that components are not mixed until desired. Components may be in different physical states. For example, some components may be lyophilized and some in aqueous solution. Some may be frozen. Individual components may be separately packaged within the kit. The kit may contain reagents, as described above for differentially modifying methylated and non-methylated cytosine residues. Typically the kit will contain oligonucleotide primers which specifically hybridize to regions within 1 kb of the transcription start sites of the genes identified in Table 1. Typically the kit will contain both a forward and a reverse primer for a single gene. If there is a sufficient region of complementarity, e.g., 12, 15, 18, or 20 nucleotides, then the primer may also contain additional nucleotide residues or other chemical moieties that do not interfere with hybridization but may be useful for other manipulations. Exemplary of such other residues may be sites for restriction endonuclease cleavage, for ligand binding or for factor binding or linkers. Other moieties may include detectable labels or specific binding moieties, such as biotin. The oligonucleotide primers may or may not be such that they are specific for modified methylated residues. The kit may optionally contain oligonucleotide probes. The probes may be specific for sequences containing modified methylated residues or for sequences containing non-methylated residues. The kit may optionally contain reagents for modifying methylated cytosine residues. The kit may also contain components for performing amplification, such as a DNA polymerase and deoxyribonucleotides. Means of detection may also be provided in the kit, including detectable labels on primers or probes. Kits may also contain reagents for detecting gene expression for one of the markers of the present invention (Table 1). Such reagents may include probes, primers, or antibodies, for example. In the case of enzymes or ligands, substrates or binding partners may be sued to assess the presence of the marker. 
         [0041]    In one aspect of this invention, the gene is contacted with hydrazine, which modifies cytosine residues, but not methylated cytosine residues. Then the hydrazine treated gene sequence is contacted with a reagent such as piperidine, which cleaves the nucleic acid molecule at hydrazine modified cytosine residues, thereby generating a product comprising fragments. By separating the fragments according to molecular weight, using, for example, an electrophoretic, chromatographic, or mass spectrographic method, and comparing the separation pattern with that of a similarly treated corresponding non-methylated gene sequence, gaps are apparent in the fragment pattern due to positions in the test gene that contained methylated cytosine residues. The presence of gaps is indicative of methylation of a cytosine residue in the CpG dinucleotide in the target gene of the test cell. 
         [0042]    Bisulfite ions, for example, sodium bisulfite, convert non-methylated cytosine residues to bisulfite modified cytosine residues. The bisulfite ion treated gene sequence can be exposed to alkaline conditions, which convert bisulfite modified cytosine residues to uracil residues. Sodium bisulfite reacts readily with the 5,6-double bond of cytosine (but poorly with methylated cytosine) to form a sulfonated cytosine reaction intermediate that is susceptible to deamination, giving rise to a sulfonated uracil. The sulfonate group can be removed by exposure to alkaline conditions, resulting in the formation of uracil. The DNA can be amplified, for example, by PCR, and sequenced to determine whether CpG sites are methylated in the DNA of the sample. Uracil is recognized as a thymine by Taq polymerase and, upon PCR, the resultant product contains cytosine only at the position where 5-methylcytosine was present in the starting template DNA. One can compare the amount or distribution of uracil residues in the bisulfite ion treated gene sequence of the test cell with a similarly treated corresponding non-methylated gene sequence. A decrease in the amount or distribution of uracil residues in the gene from the test cell indicates methylation of cytosine residues in CpG dinucleotides in the gene of the test cell. The amount or distribution of uracil residues also can be detected by contacting the bisulfite ion treated target gene sequence, following exposure to alkaline conditions, with an oligonucleotide that selectively hybridizes to a nucleotide sequence of the target gene that either contains uracil residues or that lacks uracil residues, but not both, and detecting selective hybridization (or the absence thereof) of the oligonucleotide. 
         [0043]    The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention. 
       EXAMPLE 
       [0044]    Cell lines resistant to chemotherapeutic agents and their untreated (non-resistant) counterparts were tested for the presence of methylated alleles of the repair genes ERCC3, FanG, MSH2, PARP, polB, RAD9, RecQ, XRCC3, HUS1, and BRCA1 
         [0045]    The testing was done using a real-time methylation specific PCR [MSP] based on SybrGreen detection for all genes except the MGMT gene. The methylation status of the MGMT gene was assessed using a real time detection method based on beacon detection. 
         [0046]    Based on the difference between methylation levels in the resistant and non-resistant variant of the tests, new markers can be defined. 
         [0047]    We measured the copy numbers of methylated alleles using a real-time PCR system. Copy numbers were normalized against β-Actin. Methylated allele copy numbers were compared for resistant and sensitive cell lines. Only those markers for which methylated allele copy numbers were significantly (at least three fold) and consistently different (seen in the majority of cases) were retained. 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 CELL LINE NAME 
                   
               
               
                   
                 OF UNTREATED 
                 RESISTANT 
               
               
                   
                 CELL LINE 
                 VARIANT TO AGENT 
               
               
                   
                   
               
             
             
               
                   
                 Glc4 
                 Adriamycin (doxorubicin) 
               
               
                   
                 Glc4 
                 Cisplatin 
               
               
                   
                 Tera 
                 Cisplatin 
               
               
                   
                 A2780 
                 Cisplatin, various levels of 
               
               
                   
                   
                 resistance 
               
               
                   
                   
               
             
          
         
       
     
       Data Analysis: 
     SybrGreen Based Detection of Methylation in Cancer Cell Lines 
       [0048]    Ct values [point at which fluorescence signals collected pass a threshold common to all samples in the same run] are determined for all the cell lines (non treated and resistant) [see Table 4] 
         [0049]    Assuming identical amplification efficiencies of the assays in this study, Ct values are normalized by subtracting the Ct values determined for the gene β-Actin (never methylated) from the Ct values collected for each gene under each condition [see Table 5] 
         [0050]    The difference between the normalized Ct values collected for each gene in the non treated and resistant cell lines is calculated [see Table 6] 
         [0051]    All genes showing a Ct value difference larger 1.5 (equivalent of a 2.8 fold copy number difference after normalization) are listed and can be regarded as markers of resistance [see Table 7] 
       CONCLUSION 
       [0052]    Applying the data analysis scheme detailed above we conclude that the methylation status of the DNA repair genes ERCC3 (NM — 000122), FanG (NM — 004629), MSH2 (NM — 000251), RAD9 (NM — 004584), RecQL5 (NM — 001003715), XRCC3 (NM — 005432), HUS1 (NM — 004507), and BRCA1 (NM — 007294) correlates in a positive way with resistance to chemotherapeutic agents as exemplified using Adriamycin-resistant cell lines (derived from Glc4 cell line) and Cisplatin-resistant cell lines (derived from cell lines GCL4, Tera, A2780). 
       Data: 
     RAW Ct Values Collected 
       [0053]      
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Ct values collected for 21 different assays representing 10 different candidate 
               
               
                 markers und different treatment conditions 
               
             
          
           
               
                 Gene 
                 Glc4 
                 Glc4 ADR 
                 Glc4 CDDP 
                 Tera 
                 Tera CDDP 
                 A2780 
                 A2780/CP70 
                 A2780/C30 
                 A2780/C200 
               
               
                   
               
               
                 b-Actin 
                 23.3 
                 21.8 
                 24.6 
                 22.6 
                 22.2 
                 22.3 
                 22.8 
                 21.9 
                 21.3 
               
               
                 ERCC3_1 
                 36.4 
                 38.7 
                 38.2 
                 35.9 
                 36.9 
                 37.3 
                 36.6 
                 35.8 
                 38.0 
               
               
                 ERCC3_2 
                 35.4 
                 32.6 
                 34.0 
                 31.5 
                 32.3 
                 31.0 
                 30.6 
                 33.3 
                 31.9 
               
               
                 FanG_1 
                 37.9 
                 36.3 
                 36.0 
                 33.4 
                 34.6 
                 35.2 
                 33.9 
                 34.4 
                 35.0 
               
               
                 FanG_2 
                   
                 37.1 
                 36.8 
                 35.2 
                 35.7 
                 37.3 
                 36.5 
                 33.8 
                 35.2 
               
               
                 MSH2_1 
                 35.6 
                 32.0 
                 34.2 
                 30.7 
                 31.5 
                 31.7 
                 31.2 
                 31.9 
                 32.8 
               
               
                 MSH2_2 
                   
                   
                 39.7 
                   
                 37.6 
                 36.0 
                 36.6 
                 38.2 
                 37.7 
               
               
                 PARP_1 
                 33.8 
                 31.0 
                 34.2 
                 31.1 
                 30.7 
                 31.4 
                 30.4 
                 30.5 
                 31.5 
               
               
                 PARP_2 
                 26.3 
                 25.8 
                 27.5 
                 25.7 
                 26.1 
                 27.3 
                 26.8 
                 28.5 
                 29.1 
               
               
                 polB_1 
                 34.0 
                 32.5 
                 33.6 
                 31.2 
                 31.2 
                 32.4 
                 31.4 
                 33.1 
                 32.5 
               
               
                 polB_2 
                   
                   
                   
                 38.1 
                   
                 34.2 
                 34.4 
                 38.2 
                 34.0 
               
               
                 RAD9_1 
                   
                 34.8 
                   
                 36.4 
                 39.3 
                 36.7 
                 35.1 
                 36.9 
                 39.8 
               
               
                 RAD9_2 
                 34.7 
                 38.1 
                   
                 37.0 
                 33.3 
                 37.9 
                 34.3 
                 36.1 
                 35.2 
               
               
                 RecQ_1 
                 35.6 
                 36.2 
                 36.7 
                 37.3 
                 39.4 
                 34.6 
                 33.3 
                 32.6 
                 33.5 
               
               
                 RecQ_2 
                 36.6 
                 36.2 
                 37.4 
                 36.1 
                 36.4 
                 34.6 
                 31.9 
                 31.8 
                 32.7 
               
               
                 XRCC3_1 
                 39.6 
                 35.2 
                 37.4 
                 37.0 
                 35.7 
                 35.8 
                 35.1 
                 33.8 
                 36.2 
               
               
                 XRCC3_2 
                 38.8 
                 39.4 
                 37.9 
                 39.0 
                 38.2 
                   
                 38.8 
                 38.8 
               
               
                 HUS1_1 
                 37.9 
                 34.2 
                 37.1 
                 34.1 
                 33.8 
                 34.3 
                 33.0 
                 33.1 
                 33.8 
               
               
                 HUS1_2 
                 23.8 
                 22.5 
                 25.6 
                 23.3 
                 23.1 
                 23.1 
                 22.7 
                 22.4 
                 22.0 
               
               
                 BRCA1_1 
                 29.9 
                 26.8 
                 29.2 
                 28.1 
                 28.6 
                 26.5 
                 26.6 
                 27.0 
                 27.9 
               
               
                 BRCA1_2 
                 33.5 
                 30.9 
                 33.2 
                 31.5 
                 30.3 
                 29.4 
                 28.6 
                 30.1 
                 30.6 
               
               
                 BRCA1_3 
                 39.9 
                 37.6 
                 35.1 
                 35.8 
                 38.1 
                 35.7 
                 36.0 
                 41.3 
                 37.9 
               
               
                   
               
             
          
         
       
     
       Normalized Ct Values 
       [0054]      
         [0000]                                                                                                        TABLE 5                   Normalized Ct values collected for 21 different assays representing 10 different       candidate markers und different treatment conditions            Gene   Glc4   Glc4 ADR   Glc4 CDDP   Tera   Tera CP   A2780   A2780/CP70   A2780/C30   A2780/C200                    ERCC3_1   13.1   16.8   13.6   13.3   14.6   15.0   13.8   13.9   16.7       ERCC3_2   12.1   10.7   9.4   8.9   10.1   8.8   7.8   11.5   10.6       FanG_1   14.6   14.5   11.4   10.8   12.4   13.0   11.1   12.5   13.7       FanG_2       15.3   12.2   12.6   13.5   15.1   13.7   12.0   13.9       MSH2_1   12.3   10.2   9.6   8.1   9.3   9.4   8.5   10.1   11.5       MSH2_2           15.1       15.4   13.7   13.8   16.4   16.4       PARP_1   10.5   9.2   9.6   8.5   8.5   9.2   7.7   8.7   10.2       PARP_2   3.0   4.0   2.9   3.1   3.9   5.0   4.0   6.6   7.7       polB_1   10.7   10.6   9.0   8.6   8.9   10.1   8.6   11.3   11.2       polB_2               15.5       11.9   11.7   16.3   12.7       RAD9_1       13.0       13.8   17.1   14.4   12.4   15.1   18.5       RAD9_2   11.4   16.3       14.4   11.1   15.6   11.6   14.3   13.9       RecQ_1   12.4   14.3   12.1   14.7   17.2   12.3   10.6   10.7   12.2       RecQ_2   13.3   14.4   12.8   13.5   14.2   12.3   9.1   10.0   11.3       XRCC3_1   16.3   13.3   12.8   14.4   13.5   13.5   12.3   12.0   14.8       XRCC3_2   15.5   17.6   13.3   16.4   16.0       16.0   16.9       HUS1_1   14.6   12.4   12.5   11.5   11.6   12.0   10.2   11.2   12.5       HUS1_2   0.6   0.7   1.0   0.7   0.9   0.8   −0.1   0.5   0.7       BRCA1_1   6.6   4.9   4.6   5.5   6.3   4.2   3.9   5.1   6.6       BRCA1_2   10.3   9.1   8.6   8.9   8.0   7.2   5.8   8.2   9.3       BRCA1_3   16.6   15.8   10.5   13.2   15.9   13.5   13.2   19.4   16.6                    
Difference Untreated vs. Resistant Cell Lines
 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Difference of Ct values for resistant and untreated cell lines 
               
             
          
           
               
                 Gene 
                 Glc4 
                 Glc4 ADR 
                 Glc4 CDDP 
                 Tera 
                 Tera CP 
                 A2780 
                 A2780/CP70 
                 A2780/C30 
                 A2780/C200 
               
               
                   
               
             
          
           
               
                 ERCC3_1 
                   
                 −3.69 
                 −0.47 
                   
                 −1.32 
                   
                 1.24 
                 1.13 
                 −1.62 
               
               
                 ERCC3_2 
                   
                 1.32 
                 2.65 
                   
                 −1.15 
                   
                 0.92 
                 −2.75 
                 −1.83 
               
               
                 FanG_1 
                   
                 0.08 
                 3.17 
                   
                 −1.57 
                   
                 1.81 
                 0.44 
                 −0.73 
               
               
                 FanG_2 
                   
                 ND 
                 ND 
                   
                 −0.86 
                   
                 1.34 
                 3.10 
                 1.21 
               
               
                 MSH2_1 
                   
                 2.14 
                 2.67 
                   
                 −1.11 
                   
                 0.96 
                 −0.66 
                 −2.12 
               
               
                 MSH2_2 
                   
                 ND 
                 ND 
                   
                 ND 
                   
                 −0.09 
                 −2.66 
                 −2.65 
               
               
                 PARP_1 
                   
                 1.37 
                 0.98 
                   
                 0.02 
                   
                 1.52 
                 0.48 
                 −1.05 
               
               
                 PARP_2 
                   
                 −0.98 
                 0.09 
                   
                 −0.79 
                   
                 0.95 
                 −1.66 
                 −2.76 
               
               
                 polB_1 
                   
                 0.08 
                 1.75 
                   
                 −0.34 
                   
                 1.49 
                 −1.15 
                 −1.12 
               
               
                 polB_2 
                   
                 ND 
                 ND 
                   
                 ND 
                   
                 0.25 
                 −4.43 
                 −0.81 
               
               
                 RAD9_1 
                   
                 ND 
                 ND 
                   
                 −3.31 
                   
                 2.03 
                 −0.66 
                 −4.10 
               
               
                 RAD9_2 
                   
                 −4.87 
                 ND 
                   
                 3.26 
                   
                 4.01 
                 1.34 
                 1.66 
               
               
                 RecQ_1 
                   
                 −1.97 
                 0.23 
                   
                 −2.50 
                   
                 1.78 
                 1.64 
                 0.18 
               
               
                 RecQ_2 
                   
                 −1.06 
                 0.52 
                   
                 −0.75 
                   
                 3.17 
                 2.30 
                 0.94 
               
               
                 XRCC3_1 
                   
                 2.92 
                 3.47 
                   
                 0.96 
                   
                 1.24 
                 1.58 
                 −1.31 
               
               
                 XRCC3_2 
                   
                 −2.09 
                 2.18 
                   
                 0.39 
                   
                 ND 
                 ND 
                 ND 
               
               
                 HUS1_1 
                   
                 2.29 
                 2.15 
                   
                 −0.08 
                   
                 1.78 
                 0.80 
                 −0.52 
               
               
                 HUS1_2 
                   
                 −0.15 
                 −0.41 
                   
                 −0.23 
                   
                 0.89 
                 0.28 
                 0.17 
               
               
                 BRCA1_1 
                   
                 1.65 
                 1.98 
                   
                 −0.85 
                   
                 0.33 
                 −0.95 
                 −2.41 
               
               
                 BRCA1_2 
                   
                 1.15 
                 1.68 
                   
                 0.88 
                   
                 1.38 
                 −1.05 
                 −2.08 
               
               
                 BRCA1_3 
                   
                 0.77 
                 6.03 
                   
                 −2.69 
                   
                 0.23 
                 −5.95 
                 −3.13 
               
               
                   
               
             
          
         
       
     
         [0055]    Conditions showing a Ct value difference of the normalized Ct values for resistant and non treated cell lines larger 1.5 corresponding to a methylated allele copy number difference of 2.8-fold: 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Conditions showing a Ct value difference &gt;1.5 
               
             
          
           
               
                   
                 GLC4/ADR 
                 GLC4/CDDP 
                 TERA 
                 TERA/CP 
                 A2780 
                 A2780/CP70 
                 A2780/C30 
                 A2780/C200 
               
               
                   
                   
               
             
          
           
               
                 ERCC3_1 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 ERCC3_2 
                   
                 1 
               
               
                 FanG_1 
                   
                 1 
                   
                   
                   
                 1 
               
               
                 FanG_2 
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 MSH2_1 
                 1 
                 1 
               
               
                 MSH2_2 
               
               
                 PARP_1 
                   
                   
                   
                   
                   
                 1 
               
               
                 PARP_2 
               
               
                 polB_1 
                   
                 1 
               
               
                 polB_2 
               
               
                 RAD9_1 
                   
                   
                   
                   
                   
                 1 
               
               
                 RAD9_2 
                   
                   
                   
                 1 
                   
                 1 
                   
                 1 
               
               
                 RecQ_1 
                   
                   
                   
                   
                   
                 1 
                 1 
               
               
                 RecQ_2 
                   
                   
                   
                   
                   
                 1 
                 1 
               
               
                 XRCC3_1 
                 1 
                 1 
                   
                   
                   
                   
                 1 
               
               
                 XRCC3_2 
                   
                 1 
               
               
                 HUS1_1 
                 1 
                 1 
                   
                   
                   
                 1 
               
               
                 HUS1_2 
               
               
                 BRCA1_1 
                 1 
                 1 
               
               
                 BRCA1_2 
                   
                 1 
               
               
                 BRCA1_3 
                   
                 1 
               
               
                   
               
             
          
         
       
     
       REFERENCES 
       [0056]    The disclosure of each reference cited is expressly incorporated herein.
   Reeves et al., U.S. Pat. No. 6,596,493   Sidransky, U.S. Pat. No. 6,025,127   Sidransky, U.S. Pat. No. 5,561,041   Nelson et al., U.S. Pat. No. 5,552,277   Herman, et al., U.S. Pat. No. 6,017,704   Baylin et al, U.S. Patent Application Publication No. 2003/0224040 A1   Belinsky et al., U.S. Patent Application Publication No. 2004/0038245 A1   Sidransky, U.S. Patent Application Publication No. 2003/0124600 A1   Sidransky, U.S. Patent Application Publication No. 2004/0081976 A1   Sukumar et al., U.S. Pat. No. 6,756,200 B2   Herman et al., U.S. Patent Application Publication No. 2002/0127572 A1