Mitochondrial DNA Deletion Between About Residues 12317-16254 for Use in the Detection of Cancer

Abstract

The present invention relates to methods for predicting, diagnosing and monitoring cancer. The methods comprise obtaining biological samples, extracting mitochondrial DNA (mtDNA) from the samples, quantifying mitochondrial DNA mutation in the sample and comparing the level of mtDNA mutation with a reference value. The methods of the invention may also be effective in screening for new therapeutic agents and treatment regimes, and may also be useful for monitoring the response of a subject to a preventative or therapeutic treatment.

1 . A method of detecting a cancer in an individual comprising:
 a) obtaining a biological sample from the individual;   b) extracting mitochondrial DNA (mtDNA) from the sample;   c) quantifying the amount of mtDNA in the sample having a deletion in the mtDNA sequence between about residue 12317 and about residue 16254 of the human mtDNA genome; and   d) comparing the amount of mtDNA in the sample having the deletion to at least one known reference value.   
     
     
         2 . The method of  claim 1  wherein the deletion has a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         3 . The method of  claim 1  wherein the at least one known reference value is the amount of the deletion in a reference sample of mtDNA from known non-cancerous tissue or body fluid. 
     
     
         4 . The method of  claim 3  wherein an elevated amount of the deletion in the biological sample compared to the reference sample is indicative of cancer. 
     
     
         5 . The method of  claim 4  further comprising the step of comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA from known cancerous tissue or body fluid. 
     
     
         6 . The method of  claim 1  wherein the at least one known reference value is the amount of the deletion in a reference sample of mtDNA from known cancerous tissue or body fluid. 
     
     
         7 . The method of  claim 6  wherein a similar level of the deletion in the biological sample compared to the reference sample is indicative of cancer. 
     
     
         8 . The method of  claim 7  further comprising the step of comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA from known non-cancerous tissue or body fluid. 
     
     
         9 . The method of  claim 1  wherein the step of quantifying is conducted using real-time PCR. 
     
     
         10 . The method of  claim 1  wherein the quantifying of the deletion includes first amplifying a target region of mtDNA that is indicative of the deletion, and quantifying the amount of the amplified target region. 
     
     
         11 . The method of  claim 10  wherein a PCR primer having a sequence corresponding to SEQ ID NO: 4 is used as part of a pair of amplification primers for amplifying the target region. 
     
     
         12 . The method of  claim 1  wherein the cancer is prostate cancer. 
     
     
         13 . The method of  claim 1  wherein the cancer is breast cancer. 
     
     
         14 . The method of  claim 1  wherein the biological sample is a body tissue or body fluid. 
     
     
         15 . The method of  claim 14  wherein the biological sample is selected from the group consisting of breast tissue, prostate tissue and urine. 
     
     
         16 . The method of  claim 10  wherein the reference value is a cycle threshold. 
     
     
         17 . A method of monitoring an individual for the development of a cancer comprising:
 a) obtaining a biological sample;   b) extracting mitochondrial DNA (mtDNA) from the sample;   c) quantifying the amount of mtDNA in the sample having a deletion in the mtDNA sequence between about residue 12317 and about residue 16254 of the human mtDNA genome; and   d) repeating steps a) to c) over a duration of time;   
       wherein an increasing level of the deletion over the duration of time is indicative of cancer. 
     
     
         18 . The method of  claim 17  wherein the deletion has a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         19 . The method of  claim 17  further comprising at least one step selected from the group consisting of: (a) comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA from known non-cancerous tissue or body fluid; and (b) comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA from known cancerous tissue or body fluid. 
     
     
         20 . The method of  claim 17  wherein the quantifying of the deletion includes amplifying a target region of mtDNA that is indicative of the deletion, and quantifying the amount of the amplified target region. 
     
     
         21 . The method of  claim 20  wherein the step of quantifying is conducted using real-time PCR. 
     
     
         22 . The method of  claim 20  wherein a PCR primer having a sequence corresponding to SEQ ID NO: 2 is used as part of a pair of amplification primers for amplifying the target region. 
     
     
         23 . The method of  claim 17  wherein the cancer is prostate cancer. 
     
     
         24 . The method of  claim 17  wherein the cancer is breast cancer. 
     
     
         25 . The method of  claim 17  wherein the biological sample is a body tissue or body fluid. 
     
     
         26 . The method of  claim 25  wherein the biological sample is selected from the group consisting of breast tissue, prostate tissue and urine. 
     
     
         27 . The method of  claim 10  wherein the amplifying of the target region is conducted using a pair of amplification primers, one of the pair of amplification primers overlapping a splice joining regions on opposite ends of the deletion. 
     
     
         28 . A method of detecting a cancer in an individual comprising:
 a) obtaining a biological sample from the individual;   b) extracting mitochondrial DNA (mtDNA) from the sample;   c) quantifying the amount of mtDNA in the sample having a deletion corresponding to the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2; and   d) comparing the amount of mtDNA in the sample corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 to at least one known reference value.   
     
     
         29 . The method of  claim 28  wherein the at least one known reference value is the amount of the sequence corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 in a reference sample of mtDNA from known non-cancerous tissue or body fluid. 
     
     
         30 . The method of  claim 28  wherein the at least one known reference value is the amount of the sequence corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 in a reference sample of mtDNA from known cancerous tissue or body fluid. 
     
     
         31 . The method of  claim 28  wherein the step of quantifying is conducted using real-time PCR. 
     
     
         32 . The method of  claim 31  wherein the quantifying of the deletion includes first amplifying a target region of mtDNA that is indicative of the deletion, and quantifying the amount of the amplified target region. 
     
     
         33 . The method of  claim 32  wherein one of a pair of PCR primers used in the amplifying of the target region overlaps a rejoining site of the sequence corresponding to SEQ ID NO: 1 or SEQ ID NO: 2, after the sequence has re-circularized. 
     
     
         34 . The method of  claim 28  wherein the cancer is prostate cancer. 
     
     
         35 . The method of  claim 28  wherein the cancer is breast cancer. 
     
     
         36 . The method of  claim 28  wherein the biological sample is a body tissue or body fluid. 
     
     
         37 . The method of  claim 36  wherein the biological sample is selected from the group consisting of breast tissue, prostate tissue and urine. 
     
     
         38 . The method of  claim 32  wherein the reference value is a cycle threshold. 
     
     
         39 . A diagnostic kit for carrying out the method of  claim 1  comprising:
 (a) material for collecting one or more biological samples; and   (b) suitable primers and reagents for detecting the mtDNA deletion.