Diagnostic method

Abstract

The present invention concerns a method for the detection or monitoring of cancer using a biological sample selected from blood, plasma, serum, saliva, urine from an individual, said method comprising: (a) obtaining DNA from the said biological sample; (b) digesting the DNA sample with one or more methylation-sensitive restriction enzymes; (c) quantifying or detecting a DNA sequence of interest after step (b), wherein the target sequence of interest contains at least two methylation-sensitive restriction enzyme recognition sites; and (d) comparing the level of the DNA sequence from the individual to a normal standard, to detect, prognosticate or monitor cancer.

1 . A method for the detection or monitoring of cancer using a biological sample selected from blood, plasma, serum, saliva, urine from an individual, said method comprising:
 (a) obtaining DNA from the said biological sample;   (b) digesting the DNA sample with one or more methylation-sensitive restriction enzymes;   (c) quantifying or detecting a DNA sequence of interest after step (b), wherein the target sequence of interest contains at least two methylation-sensitive restriction enzyme recognition sites; and   (d) comparing the level of the DNA sequence from the individual to a normal standard, to detect, prognosticate or monitor cancer.   
     
     
         2 . The method according to  claim 1 , wherein the polymerase chain reaction (PCR) is used in step (c), and the PCR amplicon contains at least two recognition sites for the methylation-sensitive restriction enzyme used in step (b). 
     
     
         3 . The method according to  claim 1 , wherein real-time quantitative polymerase chain reaction (Q-PCR) is used in step (c) and the PCR amplicon contains at least two recognition sites for the methylation-sensitive restriction enzyme used in step (b). 
     
     
         4 . The method according to  claim 1 , wherein the DNA sequence quantified in step (c) is a sequence comprising part or all of RASSF1A, or other tumor suppressor genes or other genes which demonstrate aberrant DNA methylation patterns in cancer. 
     
     
         5 . The method according to  claim 4 , wherein the DNA sequence is selected from the promoter, exon 1, or fragments thereof of RASSF1A. 
     
     
         6 . The method according to  claim 5 , wherein the DNA sequence is residues 1142 to 1269 of SEQ ID NO: 1. 
     
     
         7 . The method according to  claim 6 , wherein:
 (a) the DNA sequence is amplified using a primer comprising the sequence shown in SEQ ID NO 2 and a primer comprising the sequence shown in SEQ ID NO: 3; and/or   (b) the DNA sequence is detected using a detectably-labelled probe comprising the sequence shown in SEQ ID NO: 4.   
     
     
         8 . The method according to  claim 1 , wherein following the enzymatic treatment step (b), each target DNA molecule of the sequence of interest in the sample is cut at at least one methylation-sensitive restriction enzyme site by the methylation-sensitive restriction enzyme(s) when present in the unmethylated state. 
     
     
         9 . The method according to  claim 1 , wherein in step (b) the sample is treated with an excess of the enzyme(s), and/or wherein the incubation time is extended. 
     
     
         10 . The method according to  claim 1 , wherein the level of the DNA sequence quantified after the enzymatic treatment is used to:
 (a) diagnose cancer in the individual   (b) predict the susceptibility to cancer of the individual;   (c) assess the stage of the cancer in the individual;   (d) predict the likelihood of overall survival for the individual;   (e) predict the likelihood of recurrence for the individual; or   (f) assess the treatment effectiveness for the individual.   
     
     
         11 . The method of  claim 10 , wherein the trend of the level of the DNA sequence after the enzymatic treatment over the time course of treatment, monitoring or post-treatment is quantified. 
     
     
         12 . The method according to  claim 11 , wherein the DNA sequence comprises part or all of RASSF1A and wherein the method is used in the diagnosis, prognosis or monitoring of hepatocellular carcinoma or nasopharyngeal carcinoma. 
     
     
         13 . The method according to  claim 12 , wherein the individual carrier. 
     
     
         14 . The method according to  claim 13 , wherein the individual is a hepatitis B carrier or a hepatitis C carrier. 
     
     
         15 . The method according to  claim 1 , wherein the method further comprises quantifying or detecting a control DNA sequence in the DNA sample that has been digested with one or more methylation-sensitive restriction enzyme(s) wherein said control sequence does not demonstrate aberrant DNA methylation patterns in cancer. 
     
     
         16 . The method according to  claim 15 , wherein at least two methylation-sensitive restriction enzyme recognition sites are present in the control sequence. 
     
     
         17 . The method according to  claim 16 , wherein the same number of methylation-sensitive restriction enzyme recognition sites are present in the control sequence and the target sequence. 
     
     
         18 . The method according to  claim 15 , wherein the control sequence is β-actin. 
     
     
         19 . The method according to  claim 18 , wherein a target sequence in the β-actin is amplified using a primer comprising the sequence shown in SEQ ID NO: 5 and a primer comprising the sequence shown in SEQ ID NO: 6. 
     
     
         20 . The method according to  claim 19 , wherein the target sequence is detected using a detectably-labelled probe comprising the sequence shown in SEQ ID NO: 7. 
     
     
         21 . The method according to  claim 15 , wherein the method is used to assess the extent or to confirm the completeness of enzyme digestion.