Nucleic acid mutation detection in histologic tissue

Abstract

Methods are provided for detection of target neoplastic nucleic acids in a tissue specimen, including a tumor margin or lymph node, and reagents therefor, wherein the nucleic acids are preferably mutant tumor suppressor genes or proto oncogenes. Methods for treatment of cell proliferative diseases utilizing ribozymes or antisense oligonucleotides specific for the target mutant nucleic acids and/or replacement wild type genes are also disclosed.

1 . A method for detecting the presence of a mammalian target nucleic acid which contributes to the etiology of a neoplasm, in a tissue specimen, wherein the specimen is external to a primary neoplasm and the specimen does not exhibit morphological characteristics indicative of neoplastic pathology, and the target nucleic acid is present in the primary neoplasm and the specimen, the specimen being selected from the group consisting of a tumor margin, a regional lymph node, the method comprising extracting the nucleic acid present in the specimen and detecting the presence of the target nucleic acid.  
     
     
         2 . The method of  claim 1 , wherein the nucleic acid is amplified before detecting.  
     
     
         3 . The method of  claim 2 , wherein the amplification is by means of oligonucleotides that hybridize to the flanking regions of the target nucleic acid.  
     
     
         4 . The method of  claim 1 , wherein the target nucleic acid contains a mutation, a restriction fragment length polymorphism, a nucleic acid deletion, or a nucleic acid substitution, as compared with a corresponding wild-type nucleic acid in the specimen.  
     
     
         5 . The method of  claim 1 , wherein the target nucleic acid is selected from the group consisting of an oncogene and a tumor suppressor gene.  
     
     
         6 . The method of  claim 5 , wherein the tumor suppressor gene is selected from the group consisting of APC, DCC, NF1, NF2, Rb, RET, VHL, WT-1 and p53.  
     
     
         7 . The method of  claim 1 , wherein the neoplasm is of the head.  
     
     
         8 . The method of  claim 1 , wherein the neoplasm is of the neck.  
     
     
         9 . The method of  claim 1 , wherein the neoplasm is benign.  
     
     
         10 . The method of  claim 1 , wherein the neoplasm is malignant.  
     
     
         11 . The method of  claim 2 , wherein the amplified nucleic acid is cloned before detecting.  
     
     
         12 . A method for detecting metastases in a subject having an excised tumor comprising: 
 a) isolating tissue from a surgical margin or lymph node adjacent to said excised tumor;    b) applying to said tissue an oligonucleotide that preferentially hybridizes to a neoplastic nucleic acid, and    c) detecting the presence of said neoplastic nucleic acid, wherein the presence of said neoplastic nucleic acid indicates metastases.    
     
     
         13 . The method according to  claim 12  wherein no more than an average of about one out of every ten thousand cells of said tissue have a neoplastic nucleic acid.  
     
     
         14 . The method according to  claim 12  wherein said tissue appears normal under a microscope.  
     
     
         15 . The method according to  claim 12  wherein said neoplastic nucleic acid is a mutated tumor suppressor gene.  
     
     
         16 . The method according to  claim 15  wherein said tumor suppressor gene is the p53 gene.  
     
     
         17 . The method according to  claim 12  wherein said neoplastic acid is an oncogene.  
     
     
         18 . A method for detecting a mammalian target neoplastic nucleic acid in a tissue specimen which is external to a primary neoplasm, comprising extracting the nucleic acid present in the specimen and detecting the presence of the target nucleic acid.