Detection of mage-a expression

Abstract

An oligonucleotide, primer or probe comprises the nucleotide sequences of any of SEQ ID NO. 5, 6, 7, 2, 3, 4, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 25. The oligonucleotides are useful for the detection of the methylation status of a gene, in particular the MAGE-A 3 gene. The oligonucleotides are useful in primer pairs, kits and methods for determining the methylation status of the MAGE-A 3 gene and for diagnosing cancer, directing therapy and selecting subjects for treatment. The primer or probe can comprise a loop or hairpin structure and can be used in real-time methylation specific PCR.

1 . An oligonucleotide comprising a primer or a probe, a primer or a probe comprising or consisting essentially of or consisting of the nucleotide sequence of any of SEQ ID NO. 5, 6, 7, 2, 3, 4, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 25 which oligonucleotide, primer or probe is useful for the detection of the methylation status of a gene. 
     
     
         2 . An oligonucleotide, primer or probe according to  claim 1  comprising, consisting essentially of or consisting of the nucleotide sequence of any of SEQ ID NO. 5, 6, 7, 2, 3, 4 or 25 which oligonucleotide, primer or probe is useful for the detection of the methylation status of a gene. 
     
     
         3 . An oligonucleotide, primer or probe according to  claim 1  comprising or consisting essentially of or consisting of the following contiguous sequences in 5′ to 3′ order.
 (a) a first nucleotide sequence of between approximately 6 and 30 nucleotides, wherein a nucleotide within said first nucleotide sequence is labelled with a first moiety selected from the donor moiety and the acceptor moiety of a molecular energy transfer pair, wherein the donor moiety emits fluorescence at one or more particular wavelengths when excited, and the acceptor moiety absorbs and/or quenches said fluorescence emitted by said donor moiety; 
 (b) a second, single-stranded nucleotide sequence comprising, consisting essentially of or consisting of between approximately 3 and 20 nucleotides; 
 (c) a third nucleotide sequence comprising, consisting essentially of or consisting of between approximately 6 and 30 nucleotides, wherein a nucleotide within said third nucleotide sequence is labelled with a second moiety selected from said donor moiety and said acceptor moiety, and said second moiety is the member of said group not labelling said first nucleotide sequence, wherein said third nucleotide sequence is complementary in reverse order to said first nucleotide sequence such that a duplex can form between said first nucleotide sequence and said third nucleotide sequence such that said first moiety and second moiety are in proximity such that, when the donor moiety is excited and emits fluorescence, the acceptor moiety absorbs and quenches said fluorescence emitted by said donor moiety; and 
 (d) at the 3′ end of the primer, a fourth, single-stranded nucleotide sequence comprising, consisting essentially of or consisting of between approximately 8 and 40 nucleotides that comprises or consists essentially of or consists of at its 3′ end a sequence of any of SEQ ID NO. 5, 7, 2, 4, 8, 11, 13 or 25 (and able to prime synthesis by a nucleic acid polymerase of a nucleotide sequence complementary to a nucleic acid strand comprising the portion of the unmethylated DNA of the MAGE A3 gene); 
 wherein, when said duplex is not formed, said first moiety and said second moiety are separated by a distance that prevents molecular energy transfer between said first and second moiety. 
 
     
     
         4 . An oligonucleotide, primer or probe according to  claim 1  comprising or consisting essentially of or consisting of the following contiguous sequences in 5′ to 3′ order.
 (a) a first nucleotide sequence of between approximately 6 and 30 nucleotides, wherein a nucleotide within said first nucleotide sequence is labelled with a first moiety selected from the donor moiety and the acceptor moiety of a molecular energy transfer pair, wherein the donor moiety emits fluorescence at one or more particular wavelengths when excited, and the acceptor moiety absorbs and/or quenches said fluorescence emitted by said donor moiety; 
 (b) a second, single-stranded nucleotide sequence comprising, consisting essentially of or consisting of between approximately 3 and 20 nucleotides; 
 (c) a third nucleotide sequence comprising, consisting essentially of or consisting of between approximately 6 and 30 nucleotides, wherein a nucleotide within said third nucleotide sequence is labelled with a second moiety selected, from said donor moiety and said acceptor moiety, and said second moiety is the member of said, group not labelling said first nucleotide sequence, wherein said third nucleotide sequence is complementary in reverse order to said first nucleotide sequence such that a duplex can form between said first nucleotide sequence and said third nucleotide sequence such that said first moiety and second moiety are in proximity such that, when the donor moiety is excited and emits fluorescence, the acceptor moiety absorbs and quenches said fluorescence emitted by said donor moiety; and 
 (d) at the 3′ end of the primer, a fourth, single-stranded nucleotide sequence comprising, consisting essentially of or consisting of between approximately 8 and 40 nucleotides that comprises or consists essentially of or consists of at its 3′ end a sequence of any of SEQ ID NO. 14, 16, 17 or 19 (and able to prime synthesis by a nucleic acid polymerase of a nucleotide sequence complementary to a nucleic acid strand comprising the portion of the methylated DNA of the MAGE A3 gene); 
 wherein when said duplex is not formed, said first moiety and said second moiety are separated by a distance that prevents molecular energy transfer between said first and second moiety. 
 
     
     
         5 . An oligonucleotide, primer or probe according to  claim 3  wherein the fourth, single-stranded nucleotide sequence comprises, consists essentially of or consists of between approximately 8 and 40 nucleotides that comprises at its 3′ end a sequence of any SEQ ID NO. 5, 7, 2, 4 or 25. 
     
     
         6 . Primer pair comprising a primer according to  claim 1 . 
     
     
         7 . Primer pair comprising a primer according to  claim 5 . 
     
     
         8 . Primer pair comprising or consisting essentially of or consisting of the nucleotide sequence of SEQ ID NO. 6 and 7; SEQ ID NO. 2 and 3; SEQ ID NO. 9 and 4; SEQ ID NO. 12 and 13; SEQ ID NO. 14 and 15; or SEQ ID NO. 17 and 18. 
     
     
         9 . Primer pair according to  claim 8  comprising or consisting essentially of or consisting of the nucleotide sequence of SEQ ID NO. 6 and 7 or SEQ ID NO. 2and 3. 
     
     
         10 . A kit for detecting the methylation status of a gene comprising at least one oligonucleotide, primer or probe as defined in  claim 1 . 
     
     
         11 . An oligonucleotide, primer or probe as defined in  claim 1 , wherein said gene is the MAGE-A3 gene. 
     
     
         12 . Method of detecting the presence and/or amount of unmethylated Mage-A3 gene in a DNA-containing sample, comprising:
 (a) contacting/treating the DNA-containing sample with a reagent which selectively modifies unmethylated cytosine residues in the DNA to produce detectable modified residues but which does not modify methylated cytosine residues   (b) amplifying at least a portion of the unmethylated gene of interest using at least one primer pair, at least one primer of which is designed to bind only to the sequence of unmethylated DNA following treatment with the reagent, wherein at least one primer in the primer pair comprises, consists essentially of, or consists of the nucleotide sequence of any of SEQ ID NO. 5, 6,7, 2, 3, 4, 8, 9, 11,12, 13 or 25.   
     
     
         13 . Method according to  claim 12  wherein at least one primer in the primer pair comprises, consists essentially of, or consists of the nucleotide sequence of any of SEQ ID NO. 5, 6, 7, 2, 3, 4 or 25. 
     
     
         14 . Method of detecting the presence and/or amount of methylated Mage-A3 gene in a DNA-containing sample, comprising:
 (a) contacting/treating the DNA-containing sample with a reagent which selectively modifies unmethylated cytosine residues in the DNA to produce detectable modified residues but which does not modify methylated cytosine residues   (b) amplifying at least a portion of the methylated gene of interest using at least one primer pair, at least one primer of which is designed to bind only to the sequence of methylated DNA following treatment with the reagent, wherein at least one primer in the primer pair comprises, consists essentially of, or consists of the nucleotide sequence of any of SEQ ID NO. 14, 15, 16, 17, 18 or 19.   
     
     
         15 . Method of diagnosing cancer or predisposition to cancer comprising detecting the methylation status of the MAGE-A3 gene in a sample by using an oligonucleotide, primer or probe as defined in  claim 1 , wherein the presence of unmethylated MAGE-A3 in the sample is indicative for cancer or predisposition to cancer. 
     
     
         16 . Method for identifying and/or selecting a patient suitable for treatment with a MAGE-A3 immunotherapeutic comprising detecting the methylation status of the MAGE-A3 gene in a sample of the patient by using an oligonucleotide, primer or probe as defined in  claim 1 , wherein if the MAGE-A3 gene is unmethylated the subject is identified and/or selected for treatment with the MAGE-A3 immunotherapeutic. 
     
     
         17 . Method for predicting the likelihood of successful treatment of cancer comprising detecting the methylation status of the MAGE-A3 gene in a sample of the patient by using an oligonucleotide, primer or probe as defined in  claim 1 , wherein if the gene is unmethylated the likelihood of successful treatment with a MAGE-A3 immunotherapeutic is higher than if the gene is methylated. 
     
     
         18 . Method of selecting a suitable treatment regimen for cancer comprising detecting the methylation status of the MAGE-A3 gene in a sample of the patient by using an oligonucleotide, primer or probe as defined in  claim 1 , wherein if the gene is unmethylated, an immunotherapeutic is selected for treatment. 
     
     
         19 . Method of treating cancer in a subject comprising administration of an immunotherapeutic, wherein the subject has been selected for treatment on the basis of measuring the methylation status of a MAGE-A3 gene by using an oligonucleotide, primer or probe as defined in  claim 1 . 
     
     
         20 . Method of treating a patient comprising: measuring the methylation status of a MAGE-A3 gene by using an oligonucleotide, primer or probe as defined in  claim 1 , and then administering to the patient a composition comprising MAGE-A3. 
     
     
         21 . Method of treating a patient susceptible to recurrence of a MAGE-A3 expressing tumour, the patient having been treated to remove tumour tissue, the method comprising: measuring the methylation status of a MAGE-A3 gene in the tumour tissue by using an oligonucleotide, primer or probe as defined in  claim 1 , and then administering to the patient a composition comprising MAGE-A3. 
     
     
         22 .- 42 . (canceled)