Diagnostic gene marker panel for colorectal cancer

Abstract

The present invention relates generally to a method of screening for the onset, predisposition to the onset and/or progression of a neoplasm. More particularly, the present invention relates to a method of screening for the onset, predisposition to the onset and/or progression of a neoplasm by screening for changes to the methylation levels of a panel of gene markers. The method of the present invention is useful in a range of applications including, but not limited to, those relating to the diagnosis and/or monitoring of colorectal neoplasms, such as colorectal adenocarcinosis.

1 . (canceled) 
     
     
         2 . A method of assessing the methylation status of DNA present in a biological sample obtained from a human individual at risk of developing or of recurrence of, or suffering from, a large intestine neoplasm, comprising:
 assessing the methylation status of an IKZF1 gene by measuring the methylation status of one or more DNA subregions located within the IKZF1 gene, by performing a bisulfite conversion of the one or more DNA subregions, amplifying the bisulfite converted IKZF1 DNA subregions or the complement thereof with oligonucleotides comprising a nucleic acid sequence that is complementary to the bisulfite converted IKZF1 DNA subregions under selective hybridization conditions, and measuring the methylation status of one or more cytosines in the one or more DNA subregions; and   assessing the methylation status of a IRF4 gene by measuring the methylation status of one or more DNA subregions located within the IRF4 gene, by performing a bisulfite conversion of the one or more DNA subregions, amplifying the bisulfite converted IRF4 DNA subregions or the complement thereof with oligonucleotides comprising a nucleic acid sequence that is complementary to the bisulfite converted IRF4 DNA subregions under selective hybridization conditions, and measuring the methylation status of one or more cytosines in the one or more DNA subregions.   
     
     
         3 . The method of  claim 2 , wherein the one or more DNA subregions located within the IKZF1 gene are SEQ ID NO: 3 or SEQ ID NO: 4 or the complements thereof. 
     
     
         4 . The method of  claim 2 , wherein the one or more DNA subregions located within the IRF4 gene is SEQ ID NO: 5 or the complement thereof. 
     
     
         5 . The method of  claim 2 , wherein
 the one or more DNA subregions located within the IKZF1 gene is within a nucleic acid sequence as set forth in SEQ ID NO: 3; and   the one or more DNA subregions located within the IRF4 gene is within a nucleic acid sequence as set forth in SEQ ID NO: 5.   
     
     
         6 . The method of  claim 2 , wherein
 measuring the methylation status of the one or more DNA subregions located within the IKZF1 gene comprises measuring the methylation of one or more cytosine residues in the gene region encompassing IKZF 1; and   measuring the methylation status of the one or more DNA subregions located within the IRF4 gene comprises measuring the methylation of one or more cytosine residues in the gene region encompassing IRF4.   
     
     
         7 . The method of  claim 2 , wherein measuring the methylation status of the one or more DNA subregions located within the IKZF1 gene consists of measuring the methylation of one or more cytosine residues on the plus strand selected from: 
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                   chr7:50343869 
                   chr7:50343872 
                   chr7:50343883 
                 
                     
                   chr7:50343889 
                   chr7:50343890 
                   chr7:50343897 
                 
                     
                   chr7:50343907 
                   chr7:50343909 
                   chr7:50343914 
                 
                     
                   chr7:50343934 
                   chr7:50343939 
                   chr7:50343950 
                 
                     
                   chr7:50343959 
                   chr7:50343805 
                   chr7:50343822 
                 
                     
                   chr7:50343824 
                   chr7:50343826 
                   chr7:50343829 
                 
                     
                   chr7:50343831 
                   chr7:50343833 
                   chr7:50343838 
                 
                     
                   chr7:50343847 
                   chr7:50343850 
                   chr7:50343858 or 
                 
                     
                   chr7:50343864 
                 
                     
                     
                 
             
                
               
               
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         or a corresponding cytosine residue at position n+1 on the opposite strand. 
       
     
     
         8 . The method of  claim 2 , wherein the large intestine neoplasm is an adenoma or an adenocarcinoma. 
     
     
         9 . The method of  claim 2 , wherein the large intestine neoplasm is a colorectal neoplasia. 
     
     
         10 . The method of  claim 2 , wherein the biological sample is selected from a fecal sample, enema wash, surgical resection, tissue biopsy, or blood sample. 
     
     
         11 . The method of  claim 2 , wherein the biological sample is a plasma sample. 
     
     
         12 . The method of  claim 2 , further comprising administering a cancer treatment to the individual when methylation is measured at one or more cytosines in at least one of the DNA subregions. 
     
     
         13 . The method of  claim 2 , wherein the oligonucleotides comprising a nucleic acid sequence that is complementary to the bisulfite converted IKZF1 DNA subregions comprise a sequence as set forth in SEQ ID NO: 18 and SEQ ID NO: 23. 
     
     
         14 . The method of  claim 2 , wherein the oligonucleotides comprising a nucleic acid sequence that is complementary to the bisulfite converted IRF4 DNA subregions comprise a sequence as set forth in SEQ ID NO: 16 and SEQ ID NO: 21. 
     
     
         15 . A method of treating a human individual suffering from or at risk of developing or of recurrence of a large intestine neoplasm, comprising:
 assessing the methylation status of an IKZF1 gene by measuring the methylation status of one or more DNA subregions located within the IKZF1 gene, wherein the subregions are SEQ ID NO: 3 or SEQ ID NO: 4 or the complements thereof, by performing a bisulfite conversion of the one or more DNA subregions, amplifying the bisulfite converted IKZF1 DNA subregions or the complement thereof with oligonucleotides comprising a nucleic acid sequence that is complementary to the bisulfite converted IKZF1 DNA subregions under selective hybridization conditions, and measuring the methylation status of one or more cytosines in the DNA subregions; and   assessing the methylation status of a IRF4 gene by measuring the methylation status of one or more DNA subregions located within the IRF4 gene, wherein the subregions are SEQ ID NO: 5 or the complements thereof, by performing a bisulfite conversion of the one or more DNA subregions, amplifying the bisulfite converted IRF4 DNA subregions or the complement thereof with oligonucleotides comprising a nucleic acid sequence that is complementary to the bisulfite converted IRF4 DNA subregions under selective hybridization conditions, and measuring the methylation status of one or more cytosines in the DNA subregion; and   administering a cancer treatment to the human individual, wherein the human individual has:   a methylated IKZF1 DNA subregion that is SEQ ID NO: 3 and/or 4; and/or   a methylated IRF4 DNA subregion that is SEQ ID NO: 5.   
     
     
         16 . The method of  claim 15 , further comprising assessing a post-treatment methylation status of DNA present in a biological sample obtained from the individual at a time point subsequent to said administering, comprising:
 measuring a methylation status of an IKZF1 DNA subregion that is SEQ ID NO: 3 by performing a bisulfite conversion of the DNA subregion, amplifying the bisulfite converted IKZF1 DNA subregion with oligonucleotides that are SEQ ID NO: 18 and SEQ ID NO: 23, and measuring the methylation status of one or more cytosines in the DNA subregion; and   measuring a methylation status of a IRF4 DNA subregion that is SEQ ID NO: 5 by performing a bisulfite conversion of the DNA subregion, amplifying the bisulfite converted IRF4 DNA subregion with oligonucleotides that are SEQ ID NO: 16 and SEQ ID NO: 21, and measuring the methylation status of one or more cytosines in the DNA subregion.   
     
     
         17 . A method of monitoring the methylation status of DNA in a sample from a human individual suffering from or at risk of developing or of recurrence of a large intestine neoplasm, comprising:
 assessing a first methylation status of DNA present in a first biological sample obtained at a first time point from a human individual suffering from or at risk of developing a large intestine neoplasm, comprising:   measuring a first methylation level status of a DNA subregion located within the IKZF1 gene, wherein the subregion is SEQ ID NO: 3 or SEQ ID NO: 4 or the complement thereof, by performing a bisulfite conversion of the DNA subregion, amplifying the bisulfite converted IKZF1 DNA subregion or the complement thereof with oligonucleotides comprising a nucleic acid sequence that is complementary to the bisulfite converted IKZF1 DNA subregion under selective hybridization conditions, and measuring the methylation status of one or more cytosines in the DNA subregion; and   measuring a first methylation status of a DNA subregion located within the IRF4 gene, wherein the subregion is SEQ ID NO: 5 or the complement thereof, by performing a bisulfite conversion of the DNA subregion, amplifying the bisulfite converted IRF4 DNA subregion or the complement thereof with oligonucleotides comprising a nucleic acid sequence that is complementary to the bisulfite converted IRF4 DNA subregion under selective hybridization conditions, and measuring the methylation status of one or more cytosines in the DNA subregion, and   assessing a second methylation status of DNA present in a second biological sample obtained from the individual at a second time point subsequent to the first time point, comprising:
 measuring a second methylation level status of an IKZF1 DNA subregion that is SEQ ID NO: 3 or SEQ ID NO: 4 or the complement thereof, by performing a bisulfite conversion of the DNA subregion and measuring the methylation status of one or more cytosines in the DNA subregion; and 
 measuring a second methylation level status of a IRF4 DNA subregion that is SEQ ID NO: 5 or the complement thereof, by performing a bisulfite conversion of the DNA subregion and measuring the methylation status of one or more cytosines in the DNA subregion. 
   
     
     
         18 . A method of assessing the methylation status of DNA present in a biological sample obtained from a human individual at risk of developing or of recurrence of, or suffering from, a large intestine neoplasm, consisting of:
 assessing the methylation status of an IKZF1 gene by measuring the methylation status of one or more DNA subregions located within the IKZF1 gene, wherein the subregions are SEQ ID NO: 3 or SEQ ID NO: 4 or the complements thereof, by performing a bisulfite conversion of the one or more DNA subregions, amplifying the bisulfite converted IKZF1 DNA subregions or the complement thereof with at least one oligonucleotide comprising a nucleic acid sequence that is complementary to a sequence that originates within the bisulfite converted IKZF1 DNA subregions under selective hybridization conditions, and detecting the methylation status of one or more cytosines in the one or more DNA subregions; and   assessing the methylation status of a IRF4 gene by measuring the methylation status of one or more DNA subregions located within the IRF4 gene, wherein the subregions are SEQ ID NO: 5 or the complements thereof, by performing a bisulfite conversion of the one or more DNA subregions, amplifying the bisulfite converted IRF4 DNA subregions or the complement thereof with at least one oligonucleotide comprising a nucleic acid sequence that is complementary to a sequence that originates within the bisulfite converted IRF4 DNA subregions under selective hybridization conditions, and measuring the methylation status of one or more cytosines in the one or more DNA subregions.   
     
     
         19 . A method of sequencing DNA present in a biological sample comprising:
 (a) sequencing one or more IKZF1 DNA subregions within the IKZF1 gene in a biological sample, wherein the IKZF1 DNA subregions are SEQ ID NO: 3 or SEQ ID NO: 4 or the complements thereof, by:
 performing a bisulfite conversion of at least a portion of the IKZF1 gene comprising the one or more IKZF1 DNA subregions, 
 amplifying the bisulfite converted portion of the IKZF1 gene comprising the one or more IKZF1 DNA subregions or the complement thereof with at least one oligonucleotide comprising a nucleic acid sequence that is complementary to a region of the bisulfite converted portion of the IKZF1 gene comprising the one or more IKZF1 DNA subregions under selective hybridization conditions, and 
 sequencing the bisulfite converted portion of the IKZF1 gene comprising the one or more IKZF1 DNA subregions; and 
   (b) sequencing a IRF4 DNA subregion within the IRF4 gene in said biological sample, wherein the IRF4 DNA subregion is SEQ ID NO: 5 or the complement thereof, by:   performing a bisulfite conversion of at least a portion of the IRF4 gene comprising the IRF4 DNA subregion,   amplifying the bisulfite converted portion of the IRF4 gene comprising the IRF4 DNA subregion or the complement thereof with at least one oligonucleotide comprising a nucleic acid sequence that is complementary to a region of the bisulfite converted portion of the IRF4 gene comprising the IRF4 DNA subregion under selective hybridization conditions, and   sequencing the bisulfite converted portion of the IRF4 gene comprising the IRF4 DNA subregion.