Method to detect prostate cancer in a sample

Abstract

The present invention provides methods to detect prostate cancer by detecting the RNA encoded by PCA3. The disclosure provides a method for determining a predisposition, or presence of prostate cancer comprising: (a) contacting a sample with at least one oligonucleotide that hybridizes to a PCA3 polynucleotide; (b) detecting an amount of PCA3 and second prostate-specific polynucleotides; and (c) comparing the amount of PCA3 polynucleotide that hybridizes to the oligonucleotide to a predetermined cut off value, and determining the presence or absence of prostate cancer. Diagnostic kits are provided for detecting prostate cancer or the risk of developing same comprising: (a) at least one container means containing at least one oligonucleotide probe or primer that hybridizes to PCA3 (b) at least one oligonucleotide probe or primer that hybridizes with a second prostate specific nucleic acid; and (c) reagents for detecting PCA3 and the second prostate specific nucleic acid.

1 . A urine-based method for assessing a predisposition to or a presence of prostate cancer in a subject, said method comprising:
 (a) obtaining a urine sample from said subject, said urine sample comprising PCA3 RNA or cDNA prepared therefrom;   (b) performing a first hybridization and/or amplification reaction on said urine sample using at least a first oligonucleotide, wherein said first oligonucleotide enables the determination of whether said PCA3 RNA or cDNA comprises or lacks an intron at the PCA3 exon 3-exon 4a junction corresponding to positions:
 (i) 26 and 255 of SEQ ID NO: 7; 
 (ii) 26 and 27 of SEQ ID NO: 8; 
 (iii) 446 and 447 of SEQ ID NO: 9; or 
 (iv) 468 and 469 of SEQ ID NO: 10 or 13; 
   (c) detecting the presence or level of said PCA3 RNA or cDNA that lacks said intron; and   (d) determining that:
 (1) said subject has prostate cancer or has a higher risk of developing prostate cancer when an elevated level of PCA3 RNA that lacks said intron is detected, as compared to a level thereof associated with a normal or non-malignant prostate state; or 
 (2) said subject does not have prostate cancer or has a lower risk of developing prostate cancer when said PCA3 RNA that lacks said intron is not detected or is detected at a level that is comparable to or below a level thereof associated with a normal or non-malignant prostate state. 
   
     
     
         2 . The urine-based method of  claim 1  comprising performing a second hybridization and/or amplification reaction on said urine sample using at least a second oligonucleotide that is specific to a prostate-specific RNA molecule. 
     
     
         3 . The urine-based method of  claim 2 , wherein said prostate-specific RNA molecule is: PSA, human kallikrein 2, PSMA, transglutaminase 4, acid phosphatase, PCGEM1 mRNA or a prostate-specific PCA3 RNA that is not associated with prostate cancer. 
     
     
         4 . The urine-based method of  claim 2 , wherein said prostate-specific RNA molecule is PSA mRNA. 
     
     
         5 . The urine-based method of  claim 4 , wherein said PSA mRNA hybridizes to human kallikrein 2. 
     
     
         6 . The urine-based method of  claim 1 , wherein said first oligonucleotide hybridizes under high stringency conditions to the PCA3 nucleotide sequence of SEQ ID NO: 9 or 10, or to the full complement thereof, at a site spanning at least one PCA3 exon-exon junction, wherein said PCA3 exon-exon junction corresponds to nucleotide positions:
 (i) 98-99, 263-264, 446-447, or 985-986 of SEQ ID NO: 9; or   (ii) 120-121, 285-286, 468-469, 1007-1008, 2066-2067, or 2622-2623 of SEQ ID NO: 10.   
     
     
         7 . The urine-based method of  claim 1 , wherein said first oligonucleotide hybridizes under high stringency conditions to the PCA3 nucleotide sequence of SEQ ID NO: 9 or 10, or to the full complement thereof, at a site spanning at least the PCA3 exon-exon junction corresponding to nucleotide positions 446-447 SEQ ID NO: 9, or positions 468-469 of SEQ ID NO: 10. 
     
     
         8 . The urine-based method of  claim 6 , wherein said high stringency conditions comprise hybridization in 0.2×SSC and 0.1% SDS at 68° C. 
     
     
         9 . The urine-based method of  claim 1 , wherein said first oligonucleotide comprises, or is the full complement of, at least 10 contiguous nucleotides of SEQ ID NO: 9 or 10 and spans at least one PCA3 exon-exon junction, wherein said PCA3 exon-exon junction corresponds to nucleotide positions:
 (i) 98-99, 263-264, 446-447, or 985-986 of SEQ ID NO: 9; or   (ii) 120-121, 285-286, 468-469, 1007-1008, 2066-2067, or 2622-2623 of SEQ ID NO: 10.   
     
     
         10 . The urine-based method of  claim 9 , wherein said first oligonucleotide comprises, or is the full complement of, at least 12 contiguous nucleotides of SEQ ID NO: 9 or 10. 
     
     
         11 . The urine-based method of  claim 9 , wherein said first oligonucleotide comprises, or is the full complement of, at least 15 contiguous nucleotides of SEQ ID NO: 9 or 10. 
     
     
         12 . The urine-based method of  claim 9 , wherein said first oligonucleotide comprises, or is the full complement of, at least 18 contiguous nucleotides of SEQ ID NO: 9 or 10. 
     
     
         13 . The urine-based method of  claim 9 , wherein said first oligonucleotide spans at least the PCA3 exon-exon junction corresponding to nucleotide positions 446-447 SEQ ID NO: 9, or positions 468-469 of SEQ ID NO: 10. 
     
     
         14 . The urine-based method of  claim 1 , wherein said first hybridization and/or amplification reaction is carried out:
 (a) in real-time;   (b) by nucleic acid sequence-based amplification (NASBA);   (c) by polymerase chain reaction (PCR);   (d) by transcription-mediated amplification assay (TMA);   (e) by ligase chain reaction; or   (f) any combination of (a) to (e).   
     
     
         15 . The urine-based method of  claim 1 , wherein said first and said second hybridization and/or amplification reactions are performed simultaneously and/or in the same container. 
     
     
         16 . The urine-based method of  claim 1 , wherein said urine sample is collected following a digital rectal examination. 
     
     
         17 . The urine-based method of  claim 1 , wherein said urine sample comprises PCA3 RNA extracted using a silica-based purification method or a target capture method. 
     
     
         18 . The urine-based method of  claim 1 , wherein said urine sample is contacted with an agent for stabilizing and/or protecting prostate cells and/or RNA contained in said urine sample. 
     
     
         19 . The urine-based method of  claim 1 , wherein the detecting in (c) comprises determining the relative level of PCA3 RNA or cDNA that lacks said intron. 
     
     
         20 . The urine-based method of  claim 1 , further comprising: (e) selecting and/or adapting a prostate cancer treatment regimen for said subject based on the determination in (d).