Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays

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, and to methods of detecting and monitoring extracellular mutant oncogenes or tumor-associated 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 mutated nucleic acid of the neoplasm in plasma or serum fractions.

1. A method for determining an acquired  a predictive risk factor for a non-hematologic disease in a human without cancer having a , wherein said non-hematologic disease is a premalignancy that is an adenoma, or non-hematopoietic dysplastic or hyperplastic cells or tissue, the method comprising the steps of:
 a. purifying extracellular nucleic acid from blood from a  the human without cancer to prepare extracted extracellular nucleic acid containing DNA encoding a mutated gene or fragment thereof; and concurrently or sequentially,  
 b. enriching the extracted extracellular nucleic acid for the mutated gene DNA or a fragment thereof, wherein the mutated gene DNA or a fragment thereof is concentrated or isolated from the remaining extracted extracellular nucleic acid;  
 c. amplifying a mutated gene DNA or a fragment thereof, or amplifying a signal from the enriched mutated DNA or a fragment thereof; and  
 d. detecting the product of the amplified mutated gene DNA or the product of its amplified fragment, or the amplified signal of the mutated DNA or the amplified signal of a fragment thereof, whereby said detection determines a predictive risk factor for a non-hematologic disease in the human without cancer, wherein said non- hematologic disease is a premalignancy that is an adenoma, or non - hematopoietic dysplastic or hyperplastic cells or tissue.    
 
     
     
       2. A method according to  claim 1 , wherein the product of the amplified mutated gene DNA is detected using a detection method that is gel electrophoresis, single strand conformation polymorphism, heteroduplex analysis, denaturing gradient gel electrophoresis, mismatch cleavage assay, immunological detection methods, nucleic acid hybridization, Southern blot analysis, electrochemiluminescence, reverse dot blot detection, or high-performance liquid chromatography. 
     
     
       3. The method of  claim 1  wherein extracellular nucleic acid is purified from the plasma or serum fraction of blood. 
     
     
       4. The method of  claim 1  wherein the enriched mutated gene DNA or a fragment thereof of subpart (b) is amplified in subpart (c) using an amplification method that is polymerase chain reaction, ligase chain reaction, boomerang DNA amplification, Q-beta replication, transcription-based amplification, isothermal nucleic acid sequence based amplification, self-sustained sequence replication assay, strand displacement activation, or cycling probe technology. 
     
     
       5. A method of  claim 1  wherein mutated gene DNA or a fragment thereof is enriched in subpart (b) through an endonuclease-mediated restriction digestion. 
     
     
       6. A method of  claim 1  wherein mutated gene DNA or a fragment thereof is enriched in subpart (b) by hybridization of the mutated gene DNA or a fragment thereof to an oligonucleotide to form a hybridized complex. 
     
     
       7. A method according to  claim 6  wherein the hybridized complex is further subjected to a magnetic field. 
     
     
       8. A method according to  claim 6  wherein the hybridized complex is further subjected to an electric field. 
     
     
       9. A method according to  claim 6  wherein the endonuclease is BstNI, HinP1 I, or Msp I. 
     
     
       10. A method according to  claim 1 , wherein the mutated DNA encodes a mutated oncogene or fragment thereof. 
     
     
       11. A method according to  claim 10 , wherein the mutated oncogene is mutated K-ras. 
     
     
       12. A method according to  claim 10 , wherein the mutated oncogene is mutated APC. 
     
     
       13. A method according to  claim 1 , wherein the mutated DNA is DNA having a microsatellite alteration. 
     
     
       14. A method according to  claim 1 , wherein the non-hematologic disease is colorectal adenoma, cervical dysplasia, atypical squamous metaplasia of the lung, bronchial dysplasia, atypical hyperplasia of the breast, prostatic intraepithelial neoplasia, atypical endometrial hyperplasia, dysplastic nevi of the skin, or Barrett's esophagus. 
     
     
       15. The method of  claim 1 , further comprising the steps of:
 e) demonstrating that the mutated gene DNA is absent in normal cells from the human, thereby indicating that the predictive risk factor is an acquired predictive risk factor.  
 
     
     
       16. A method for enriching purified extracellular DNA for extracellular mutated DNA or a fragment thereof in blood of a human without cancer having a premalignancy that is an adenoma, or non-hematopoietic dysplastic or hyperplastic cells or tissue, the method comprising the steps of:
 a. purifying extracellular nucleic acid from blood of a human without cancer to prepare extracellular nucleic acid containing a mutated DNA species or fragment thereof, and concurrently or sequentially    b. hybridizing an oligonucleotide that is complementary to the mutated DNA or a mutated DNA fragment to produce a hybridized product of mutated DNA or a fragment thereof; and    c. separating the hybridized product of mutated DNA or a fragment thereof from the remaining non-hybridized extracellular nucleic acid, thereby enriching the purified extracellular DNA for the mutated DNA or a fragment thereof.    
     
     
       17. The method of  claim 16  wherein the extracellular nucleic acid is purified from the plasma or serum fraction of blood. 
     
     
       18. A method according to  claim 16  wherein the oligonucleotide is further bound to a non-biologic surface. 
     
     
       19. A method according to  claim 16  wherein in subpart (c) the hybridized product is subjected to a magnetic field. 
     
     
       20. A method according to  claim 16  wherein in subpart (c) the hybridized product is subjected to an electric field. 
     
     
       21. A method according to  claim 16 , wherein the mutated DNA is mutated oncogene DNA. 
     
     
       22. A method according to  claim 21 , wherein the mutated oncogene is mutated K-ras. 
     
     
       23. A method according to  claim 21 , wherein the mutated oncogene is mutated APC. 
     
     
       24. A method according to  claim 21 , wherein the mutated oncogene is mutated P53. 
     
     
       25. A method according to  claim 16 , wherein the mutated DNA is DNA having a microsatellite alteration. 
     
     
       26. A method of identifying a human having a  disease or  premalignancy without  premalignant condition in a human wherein the human does not have clinical symptoms by detecting a mutation in DNA from blood or a blood fraction from the human, wherein the mutation is an acquired mutation that is associated with the disease or premalignancy  premalignant condition, and wherein the disease or premalignant condition is colorectal adenoma, cervical dysplasia, atypical squamous metaplasia of the lung, bronchial dysplasia, atypical hyperplasia of the breast, prostatic intraepithelial neoplasia, atypical endometrial hyperplasia, dysplastic nevi of the skin, or Barrett's esophagus; the method comprising the steps of:
 a. purifying extracellular nucleic acid from blood or a blood fraction from a  the human without cancer to prepare extracted extracellular nucleic acid containing said mutated DNA;  
 b. amplifying the mutated DNA, or alternatively amplifying a signal from the mutated DNA; and  
 c. detecting the product of the amplified mutated DNA or the amplified signal of the mutated DNA, wherein the human is identified as having the disease or premalignancy  premalignant condition.  
 
     
     
       27. A method according to  claim 26 , wherein the evaluation of blood or a blood fraction from a human for said mutation assists in the identification of said disease or premalignant condition. 
     
     
       28. A method according to  claim 26 , wherein extracellular nucleic acid is purified from the plasma or serum fraction of blood. 
     
     
       29. A method according to  claim 26 , further comprising the step of performing a diagnostic test that is colonoscopy, sigmoidoscopy, endoscopy, bronchoscopy, radiologic imaging, ultrasonography, radionucleotide imaging, PET scanning, and evaluation of organ specific bodily fluid, stool, or lavage fluid. 
     
     
       30. A method according to  claim 26 , wherein the mutated DNA is a DNA microsatellite alteration. 
     
     
       31. The method of  claim 26 , wherein the mutated DNA of subpart (a) is amplified in subpart (b) using an amplification method that is polymerase chain reaction, ligase chain reaction, boomerang DNA amplification, Q-beta replication, transcription-based amplification, isothermal nucleic acid sequence based amplification, self-sustained sequence replication assay, strand displacement activation, or cycling probe technology. 
     
     
       32. The method of  claim 26 , wherein the product of the amplified mutated DNA is detected using a method that is gel electrophoresis, single strand conformation polymorphism, heteroduplex analysis, denaturing gradient gel electrophoresis, mismatch cleavage assay, immunological detection methods, nucleic acid hybridization, Southern blot analysis, electrochemiluminescence, reverse dot blot detection, or high-performance liquid chromatography. 
     
     
       33. A method according to  claim 26 , wherein prior to amplification in subpart (b), the mutated DNA in subpart (a) is enriched, wherein the mutated DNA is concentrated or isolated from the remaining extracted nucleic acid.