Method of detecting tumor-associated hypermethylated DNA in plasma or serum

Abstract

This invention relates to detection of specific extracellular nucleic acid in human or animal blood plasma or serum associated with disease. Specifically, the invention relates to detection of nucleic acid derived from mutant oncogenes or other tumor-associated DNA including non-mutated hypermethylated DNA, and to methods of detecting and monitoring extracellular mutant oncogenes or tumor-associated DNA including non-mutated hypermethylated DNA found in blood plasma or serum. In particular, the invention relates to the detection, identification, or monitoring of the existence, progression or clinical status of neoplasia in humans or other animals that contain a mutation that is associated with the neoplasm through detection of the non-mutated hypermethylated nucleic acid of the neoplasm in plasma or serum fractions.

1 . A method of detecting tumor-associated hypermethylated DNA in blood plasma or serum from a human without cancer, the method comprising the steps of extracting DNA from blood plasma or serum from a human without cancer and assaying said DNA or DNA derived therefrom for the tumor-associated hypermethylated DNA or a product or signal derived therefrom using an assay that has an amplification step. 
     
     
         2 . A method of detecting tumor-associated hypermethylated DNA in blood plasma or serum from a human without clinically diagnosed cancer, the method comprising the steps of:
 a) extracting DNA from blood plasma or serum from a human without clinically diagnosed cancer, wherein a portion of the extracted DNA comprises a tumor-associated hypermethylated DNA;   b) assaying for said tumor-associated hypermethylated DNA or a product or signal derived therefrom using an amplification method that produces an amplified product or signal indicative of the presence of the tumor-associated hypermethylated DNA; and   c) qualitatively or quantitatively detecting the amplified product or signal.   
     
     
         3 . The method of  claim 2 , wherein DNA is extracted from blood plasma or serum from a human without cancer in step (a) using an extraction method that is selected from: a gelatin-based extraction method; glass bead, silica particle or diatom-based extraction method; guanidinium thiocyanate-based extraction method; phenol-chloroform-based extraction method; selective retention on a size exclusion column; or a salting-out method. 
     
     
         4 . The method of  claim 2 , wherein the amplified product or signal is detected in step (c) using a detection method that is gel electrophoresis, ELISA and immunological-based detection, detection of a fluorescent or radioisotope or antigenic or chromogenically-labeled probe, Southern blot analysis, reverse dot blot hybridization, electrochemiluminescence, or high performance liquid chromatography. 
     
     
         5 . A method of evaluating a human without cancer for a tumor-associated hypermethylated DNA in blood plasma or serum from said human, the method comprising the steps of obtaining blood plasma or serum from a human without cancer, extracting DNA from said blood plasma or serum and assaying said DNA or DNA derived therefrom for the tumor-associated hypermethylated DNA or a product or signal derived therefrom using an assay that has an amplification step, whereby said human without cancer is evaluated for a tumor-associated hypermethylated DNA in blood plasma or serum from said human. 
     
     
         6 . A method of evaluating a human without clinically diagnosed cancer for a tumor-associated hypermethylated DNA in blood plasma or serum from said human, the method comprising the steps of:
 a) extracting DNA from blood plasma or serum from a human without clinically diagnosed cancer;   b) assaying for a tumor-associated hypermethylated DNA or a product or signal derived therefrom using an amplification method that produces an amplified product or signal indicative of the presence of the tumor-associated hypermethylated DNA; and   c) qualitatively or quantitatively determining the presence of the amplified product or signal, whereby a human without clinically diagnosed cancer is evaluated for said tumor-associated hypermethylated DNA.   
     
     
         7 . The method of  claim 6 , wherein DNA is extracted from blood plasma or serum from a human without clinically diagnosed cancer in step (a) using an extraction method that is selected from: a gelatin-based extraction method; glass bead, silica particle or diatom-based extraction method; guanidinium thiocyanate-based extraction method; phenol-chloroform-based extraction method; selective retention on a size exclusion column; or a salting-out method. 
     
     
         8 . The method of  claim 6 , wherein the amplified product or signal is detected in step (c) using a detection method that is gel electrophoresis, ELISA and immunological-based detection, detection of a fluorescent or radioisotope or antigenic or chromogenically-labeled probe, Southern blot analysis, reverse dot blot hybridization, electrochemiluminescence, or high performance liquid chromatography. 
     
     
         9 . A method of detecting tumor-associated hypermethylated DNA in blood plasma or serum from a human with a premalignant disease or condition, the method comprising the steps of extracting DNA from blood plasma or serum from a human with a premalignant disease or condition and assaying said DNA or DNA derived therefrom for the tumor-associated hypermethylated DNA or a product or signal derived therefrom using an assay that has an amplification step. 
     
     
         10 . The method of  claim 9 , wherein DNA is extracted from blood plasma or serum from a human with a premalignant disease or condition using an extraction method that is selected from: a gelatin-based extraction method; glass bead, silica particle or diatom-based extraction method; guanidinium thiocyanate-based extraction method; phenol-chloroform-based extraction method; selective retention on a size exclusion column; or a salting-out method.