Method to detect prostate cancer in a sample
View Patent ↗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 composition comprising:
(a) a urine sample from a subject having or suspected of having prostate cancer, said urine sample comprising at least one prostate cell or nucleic acid extract thereof;
(b) a first oligonucleotide or first primer pair for performing an RNA hybridization and/or amplification reaction on mRNA contained in said urine sample, said first oligonucleotide or first primer pair being specific for a prostate cancer associated PCA3 mRNA molecule which is:
(i) a polynucleotide molecule comprising the sequence of SEQ ID NO: 9, 10 or 13;
(ii) a polynucleotide molecule that hybridizes under high stringency conditions to (i), wherein said high stringency conditions comprise a hybridization at 65° C. in 6×SSC or 5×SSPE, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured carrier DNA and a washing at 65° C. in 0.2×SSC/0.1% SDS; or
(iii) a polynucleotide molecule fully complementary to (i) or (ii); and
(c) a second oligonucleotide or second primer pair for performing a second RNA hybridization and/or amplification reaction on mRNA contained in said urine sample, said second oligonucleotide or second primer pair being specific for a prostate-specific mRNA molecule,
wherein at least one oligonucleotide in the composition is a labeled oligonucleotide, and the at least one labeled oligonucleotide includes at least one of (i) the first oligonucleotide, (ii) a primer of the first primer pair, (iii) the second oligonucleotide, or (iv) a primer of the second primer pair.
2. The composition of claim 1 , wherein said urine sample comprises malignant prostate cells or a nucleic acid extract thereof.
3. The composition of claim 1 , wherein said urine sample comprises crude urine.
4. The composition of claim 1 , wherein said urine sample comprises or is from:
(a) crude urine collected following a digital rectal examination; or
(b) crude urine collected not following a digital rectal examination.
5. The composition of claim 1 , further comprising reagents for amplifying and/or detecting said PCA3 mRNA molecule and said prostate-specific mRNA molecule.
6. The composition of claim 1 , wherein said first primer pair comprises oligonucleotides comprising the sequences set forth in SEQ ID NOs: 3 and 4.
7. The composition of claim 1 , wherein said prostate-specific mRNA is: PSA, human kallikrein 2, PSMA, transglutaminase 4, acid phosphatase, PCGEM1 mRNA or a prostate-specific PCA3 mRNA that is not associated with prostate cancer.
8. The composition of claim 1 , wherein said prostate-specific mRNA is PSA mRNA.
9. The composition of claim 8 , wherein said PSA mRNA hybridizes to human kallikrein 2.
10. The composition of claim 8 , wherein said second primer pair comprises oligonucleotides comprising the sequence set forth in SEQ ID NOs: 1 and 2.
11. The composition of claim 5 , wherein said reagents enable simultaneous amplification of PCA3 and said prostate-specific mRNA.
12. The composition of claim 5 , wherein said reagents are suitable for nucleic acid amplification which is at least one of:
(a) real-time amplification;
(b) nucleic acid sequence-based amplification (NASBA);
(c) polymerase chain reaction (PCR);
(d) transcription-mediated amplification assay (TMA); or
(e) ligase chain reaction.
13. The composition of claim 5 , wherein said reagents are suitable for fluorescence, chemiluminescence or colorimetry detection.
14. The composition of claim 1 , wherein the composition comprises a promoter-primer suitable for transcription-associated amplification of the prostate cancer associated PCA3 mRNA.
15. The composition of claim 1 , wherein said urine sample comprises RNA extracted from crude urine using:
(a) a silica based purification method; or
(b) a target capture method.
16. The composition of claim 4 , wherein at least one labeled oligonucleotide is labeled with a radionuclide, ligand, enzyme, enzyme substrate, reactive group, chromophore, luminescent compound, or fluorescent compound.
17. The composition of claim 4 , wherein the first oligonucleotide or first primer pair is labeled.
18. The composition of claim 4 , wherein the second oligonucleotide or first primer pair is labeled.
19. A composition comprising:
(a) a urine sample from a subject having or suspected of having prostate cancer, said urine sample comprising at least one prostate cell or nucleic acid extract thereof;
(b) a first primer pair including a first promoter-primer for performing an RNA amplification reaction on mRNA contained in said urine sample, the first promoter-primer being specific for a prostate cancer associated PCA3 mRNA molecule which is:
(i) a polynucleotide molecule comprising the sequence of SEQ ID NO: 9, 10 or 13;
(ii) a polynucleotide molecule that hybridizes under high stringency conditions to (i), wherein said high stringency conditions comprise a hybridization at 65° C. in 6×SSC or 5×SSPE, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured carrier DNA and a washing at 65° C. in 0.2 SSC/0.1% SDS; or
(iii) a polynucleotide molecule fully complementary to (i) or (ii); and
(c) a second primer pair including a second promoter-primer for performing a second RNA amplification reaction on mRNA contained in said urine sample, said second oligonucleotide or second primer pair being specific for a prostate-specific mRNA molecule.
20. The composition of claim 19 , wherein the urine sample comprises malignant prostate cells or a nucleic acid extract thereof and/or crude urine.