Dna methylation markers and methods of use

Abstract

The present invention provides methods for identifying metastases by detecting nucleic acid hypermethylation of one or more genes in one or more samples, and in particular in the lymph nodes. The invention further relates to DNA methylation as a predictor of disease recurrence and patient prognosis, specifically in the field of cancer biology.

1 . A method for identifying metastases in a subject comprising detecting nucleic acid hypermethylation of one or more genes in one or more samples, wherein detecting nucleic acid hypermethylation identifies metastases. 
     
     
         2 . The method of  claim 1 , wherein the sample comprises cells or tissues selected from the group consisting of: tumor, lymph nodes, bone marrow and blood. 
     
     
         3 - 5 . (canceled) 
     
     
         6 . A method for identifying metastases in a subject comprising detecting nucleic acid hypermethylation of one or more genes in tumor tissue or lymph node, wherein the genes are selected from the group consisting of: genes involved in tumor suppression, DNA repair, apoptosis, anti-proliferation, ras signaling, adhesion, differentiation, development, and cell cycle regulation, wherein detecting nucleic acid hypermethylation identifies metastases. 
     
     
         7 - 11 . (canceled) 
     
     
         12 . A method for identifying micrometastases in a subject comprising detecting nucleic acid hypermethylation of at least one or more genes in a sample comprising tumor and lymph nodes, wherein the sample genes are selected from the group consisting of: H-cadherin, p16, APC, RASSF1A, MGMT, DAPK, and ASC, and wherein detecting nucleic acid methylation identifies micrometastases. 
     
     
         13 . The method of  claim 12 , wherein hypermethylation of at least two of the genes is detected. 
     
     
         14 . The method of claim  11 , wherein at least two of the genes are selected from p-16 and H-cadherin, H-cadherin and APC, APC and p16, or RASSf1A and p16. 
     
     
         15 . The method of  claim 1   claim 1 , wherein the detection of metastases is used to detect or diagnose a proliferative disease. 
     
     
         16 - 19 . (canceled) 
     
     
         20 . A method for detecting or diagnosing a proliferative disease in a subject comprising detecting nucleic acid hypermethylation of one or more genes in one or more samples, wherein detecting nucleic acid hypermethylation is used to detect or diagnose a proliferative disease. 
     
     
         21 . A method for predicting the recurrence of a proliferative disease in a subject comprising: detecting nucleic acid hypermethylation of one or more genes wherein detecting nucleic acid hypermethylation of one or more genes is a predictor of the recurrence of a proliferative disease. 
     
     
         22 . The method of  claim 21 , wherein hypermethylation of one or more genes is detected in tumor or lymph nodes. 
     
     
         23 . (canceled) 
     
     
         24 . A method for staging or re-staging a proliferative disease in a subject comprising: detecting nucleic acid hypermethylation of one or more genes wherein detecting nucleic acid hypermethylation is used for staging or re-staging a proliferative disease. 
     
     
         25 - 26 . (canceled) 
     
     
         27 . A method for determining the prognosis of a subject suffering from a proliferative disease comprising: detecting nucleic acid hypermethylation of one or more genes wherein the detection of nucleic acid hypermethylation is used for determining the prognosis of a subject suffering from a proliferative disease. 
     
     
         28 - 34 . (canceled) 
     
     
         35 . A method for detecting or diagnosing a proliferative disease in a subject comprising:
 extracting nucleic acid from one or more cell or tissue samples;   detecting nucleic acid hypermethylation of one or more genes in the sample; and   identifying the nucleic acid hypermethylation state of one or more genes, wherein nucleic acid hypermethylation of genes indicates a proliferative disease.   
     
     
         36 . A method for predicting the recurrence of a proliferative disease in a subject comprising:
 extracting nucleic acid from one or more cell or tissue samples;   detecting nucleic acid hypermethylation of one or more genes in the sample; and   identifying the nucleic acid hypermethylation state of one or more genes, wherein nucleic acid hypermethylation of genes is indicative of the recurrence of a proliferative disease.   
     
     
         37 . A method for staging or re-staging a proliferative disease in a subject comprising:
 extracting nucleic acid from one or more cell or tissue samples;   detecting nucleic acid hypermethylation of one or more genes in the sample; and   identifying the nucleic acid hypermethylation state of one or more genes, wherein nucleic acid hypermethylation of genes is used for staging or re-staging of a proliferative disease.   
     
     
         38 - 42 . (canceled) 
     
     
         43 . A method of treating a subject having or at risk for having a proliferative disease comprising:
 identifying nucleic acid hypermethylation of one or more genes, where nucleic acid hypermethylation indicates having or a risk for having a proliferative disease; and   administering to the subject a therapeutically effective amount of a demethylating agent,   thereby treating a subject having or at risk for having a proliferative disease.   
     
     
         44 - 54 . (canceled) 
     
     
         55 . A method of identifying an agent that de-methylates hypermethylated nucleic acid comprising:
 identifying one or more cell or tissue samples with hypermethylated nucleic acid;   extracting the hypermethylated nucleic acid;   contacting the nucleic acid with one or more nucleic acid de-methylating candidate agents and a control agent;   identifying the nucleic acid hypermethylation state, wherein nucleic acid de-methylation of genes in the sample by the candidate agent compared to the control indicates a demethylating agent,   
       thereby identifying an agent that de-methylates hypermethylated nucleic acid. 
     
     
         56 - 70 . (canceled) 
     
     
         71 . A kit for identifying the nucleic acid hypermethylation state of one or more genes comprising gene specific primers for use in polymerase chain reaction (PCR), and instructions for use. 
     
     
         72 . A kit for detecting metastases by detecting nucleic acid hypermethylation of one or more genes, the kit comprising gene specific primers for use in polymerase chain reaction (PCR), and instructions for use. 
     
     
         73 . hThe kit of  claim 72 , wherein the metastases are micrometastases. 
     
     
         74 - 82 . (canceled)