Aberrantly methylated genes as markers of breast malignancy

Abstract

The invention is directed to a method of diagnosing a cell proliferative disorder of breast tissue by determining the methylation status of nucleic acids obtained from a subject. Aberrant methylation of several genes including TWIST, HOXA5, NES-1, retinoic acid receptor beta (RARβ), estrogen receptor (ER), cyclin D2, WT-1, 14.3.3 sigma, HIN-1, RASSF1A, and combinations of such genes serve as markers of breast malignancy.

1 . A method detecting primary breast cancer in a subject comprising determining the state of methylation of one or more CpG islands in the promoter of HIN-1 nucleic acids isolated from a sample comprising blood, plasma, lymph, duct cells, ductal lavage fluid, nipple aspiration fluid, breast tissue, lymph nodes, bone marrow, or a combination thereof of the subject, wherein a state of hypermethylation of one or more CpG islands in the promoter of HIN-1 nucleic acids as compared with the state of methylation of one or more CpG islands in the promoter of HIN-1 nucleic acids in comparable samples obtained from a normal subjects is indicative of primary breast cancer in the subject.  
     
     
         2 - 3 . (canceled)  
     
     
         4 . The method of  claim 1 , wherein the state of methylation of the nucleic acids is determined simultaneously.  
     
     
         5 - 8 . (canceled)  
     
     
         9 . The method of  claim 1 , wherein the sample comprises duct cells obtained by a procedure selected from ductal lavage, sentinel node biopsy, fine needle aspirate, routine operative breast endoscopy, nipple aspiration and core biopsy.  
     
     
         10 . (canceled)  
     
     
         11 . The method of  claim 1 , wherein determining the state of methylation comprises amplifying the nucleic acid by means of at least one sense primer and at least one antisense primer that distinguishes between methylated and unmethylated nucleic acids.  
     
     
         12 . The method of  claim 11 , wherein the primers hybridize with target polynucleotide sequences selected from SEQ ID NO:25-36, 41-48, and combinations thereof.  
     
     
         13 . The method of  claim 11 , wherein the primers are selected from SEQ ID NO:21-24, 37-40, and combinations thereof.  
     
     
         14 . The method of  claim 1 , further comprising contacting the nucleic acid with a methylation-sensitive restriction endonuclease.  
     
     
         15 . The method of  claim 14 , wherein the methylation-sensitive restriction endonuclease is selected from the group consisting of MspI, HpaII, BssHII, BstUI and NotI.  
     
     
         16 . A method of determining a predisposition to primary breast cancer in a subject comprising determining the state of methylation of one or more CpG islands in the promoter of HIN-1 nucleic acids isolated from a sample comprising blood, plasma, duct cells lymph, ductal ravage fluid, nipple aspiration fluid, breast tissue, lymph nodes bone marrow, or a combination thereof of the subject, 
 wherein a state of hypermethylation of the CpG islands in the promoter of HIN-1 nucleic acid(s) as compared with the state of methylation of comparable nucleic acid obtained from normal subjects is indicative of a predisposition to primary breast cancer in the subject.    
     
     
         17 - 19 . (canceled)  
     
     
         20 . The method of  claim 16 , wherein the sample comprises duct cells obtained by a procedure selected from the group consisting of ductal lavage, sentinel node biopsy, fine needle aspirate, routine operative breast endoscopy, nipple aspiration and core biopsy.  
     
     
         21 . (canceled)  
     
     
         22 . The method of  claim 16 , wherein determining the state of methylation comprises anplifying the nucleic acid(s) by means of at least one sense primer and at least one antisense primer that distinguishes between methylated and unmethylated nucleic acid.  
     
     
         23 . The method of  claim 22 , wherein the nucleic acids are amplified simultaneously.  
     
     
         24 . The method of  claim 22 , wherein the primers hybridize with target polynucleotide sequences selected from SEQ ID NO:25-36, 41-48, and combinations thereof.  
     
     
         25 . The method of  claim 24 , wherein the primers are selected from SEQ ID NO:21-24, 37-40, and combinations thereof.  
     
     
         26 . The method of  claim 16 , further comprising contacting the nucleic acid with a methylation-sensitive restriction endonuclease.  
     
     
         27 . The method of  claim 26 , wherein the methylation-sensitive restriction endonuclease is selected from the group consisting of MspI, HpaII, BssHII, BstUI and NotI.  
     
     
         28 . A method for diagnosing primary breast cancer in a subject comprising: 
 (a) contacting a nucleic acid-containing specimen selected from blood, plasma, lymph, duct cells, ductal lavage fluid, nipple aspiration fluid, breast tissue, lymph nodes bone marrow, or a combination thereof of the subject with an agent that provides a determination of the methylation state of CpG islands in the promoter of HIN-1 nucleic acids in the specimen, and    (b) identifying the methylation state of at least one CpG island in the promoter of HIN-1, wherein the CpG islands in the promoter of HIN-1 hypermethylated compared to the methylation state of the same region of the same nucleic acid in normal subjects.    
     
     
         29 - 33 . (canceled)  
     
     
         34 . The method of  claim 28 , wherein the agent is at least one sense primer and at least one antisense primer that hybridize with a target sequence in the nucleic acid.  
     
     
         35 . The method of  claim 34 , wherein the primers hybridize with target polynucleotide sequences selected from SEQ ID NO:25-36, 41-48, and combinations thereof.  
     
     
         36 . The method of  claim 34 , wherein the primers are selected from SEQ ID NO:21-24, 37-40, and combinations thereof.  
     
     
         37 - 39 . (canceled)  
     
     
         40 . The method of  claim 34 , wherein the method employs multiplex methylation-specific PCR.  
     
     
         41 . The method of  claim 40 , wherein the specimen comprises breast duct or ductal fluid.  
     
     
         42 - 44 . (canceled)