Method to detect prostate cancer in a sample

• US 8,192,931 B2
• Filed: 07/10/2009
• Issued: 06/05/2012
• Est. Priority Date: 02/07/2003
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Diagnocure Inc.
• Inventors: Fradet, Yves, Chypre, Camille, Piché, Lyson, Garon, Geneviéve
• Primary Examiner(s): AEDER, SEAN E
• Application Number:
  US12/500,749
• Publication Number:
  US 20110165573A1
• Time in Patent Office: 1,061 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The present invention relates to prostate cancer. More specifically, the present invention relates to a method to detect prostate cancer in a patient sample by detecting the RNA encoded by the gene PCA3. More particularly the present invention relates to a method for determining a predisposition, or presence of prostate cancer in a patient comprising: (a) contacting a biological sample of a patient with at least one oligonucleotide that hybridizes to a PCA3 polynucleotide; (b) detecting in the biological sample 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 therefrom determining the presence or absence of prostate cancer in the biological sample. The present invention further relates to diagnostic kits for the detection of prostate cancer or the risk of developing same in a patient comprising: (a) at least one container means having disposed therein at least one oligonucleotide probe or primer that hybridizes to one a PCA3 nucleic acid or complement thereof; (b) at least one oligonucleotide probe or primer that hybridizes with a second prostate specific nucleic acid or complement thereof; and (c) reagents enabling a detection of PCA3 and of the second prostate specific nucleic acid when PCA3 or second prostate-specific nucleic acid sequence is present.

Claims

1. A method for determining a predisposition for developing prostate cancer or a presence of prostate cancer in a patient, said method comprising:

(a) obtaining a crude urine sample from a patient, said crude urine sample comprising at least one prostate cell or nucleic acid extract thereof;

(b) performing an RNA hybridization and/or amplification reaction on mRNA contained in said crude urine sample, using a first oligonucleotide or first primer pair specific to a prostate cancer-specific 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 μ

g/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);

(c) performing a second RNA hybridization and/or amplification reaction on mRNA contained in said crude urine sample, using a second oligonucleotide or second primer pair specific to a prostate-specific mRNA molecule;

(d) detecting said PCA3 mRNA molecule and said prostate-specific mRNA molecule; and

(e) determining, based on the presence or level of said prostate cancer-specific PCA3 mRNA molecule in said crude urine sample, that;

(1) said patient has prostate cancer or has a higher risk of developing prostate cancer when an elevated level of said prostate cancer-specific PCA3 mRNA molecule is detected, as compared to a level thereof associated with a normal or non-malignant prostate state;

or

(2) said patient does not have prostate cancer or has a lower risk of developing prostate cancer when said prostate cancer-specific PCA3 mRNA molecule is not detected or is detected at a lower level, as compared to a level thereof associated with a normal or non-malignant prostate state, and when said prostate-specific mRNA molecule is detected, thereby validating the non detection or lower detection of said PCA3 mRNA molecule.

2. The method of claim 1, wherein said RNA amplification reaction is carried out in real-time.

3. The method of claim 1, wherein said detection is performed by fluorescence, chemiluminescence or colorimetry detection.

4. The method 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.

5. The method of claim 4, wherein said prostate-specific mRNA is PSA mRNA.

6. The method of claim 5, wherein said PSA mRNA hybridizes to human kallikrein 2.

7. The method of claim 1, wherein said RNA amplification method is:

(a) nucleic acid sequence-based amplification (NASBA);

(b) polymerase chain reaction (PCR);

(c) transcription-mediated amplification assay (TMA);

or

(d) ligase chain reaction.

8. The method of claim 2, wherein said RNA amplification method is:

(a) nucleic acid sequence-based amplification (NASBA);

(b) polymerase chain reaction (PCR);

(c) transcription-mediated amplification assay (TMA);

or

(d) ligase chain reaction.

9. The method of claim 1, wherein said amplification of PCA3 and said prostate-specific mRNA is performed simultaneously.

10. The method of claim 1, wherein said amplification of PCA3 is carried out using a primer pair comprised of the polynucleotide sequences set forth in SEQ ID NOs:

3 and 4.

11. The method of claim 1, wherein said detection of PCA3 is carried out using a molecular beacon that hybridizes to PCA3 under high stringency conditions.

12. The method of claim 11, wherein said molecular beacon comprises the sequence set forth in SEQ ID NO:

6.

13. The method of claim 5, wherein said amplification of PSA is carried out using a primer pair comprised of the polynucleotide sequences set forth in SEQ ID NOs:

1 and 2.

14. The method of claim 5, wherein said detection of PSA is carried out using a PSA molecular beacon that hybridizes to PSA under high stringency conditions.

15. The method of claim 14, wherein said PSA molecular beacon comprises the sequence set forth in SEQ ID NO:

5.

16. The method of claim 1, wherein said RNA is extracted from said crude sample using

(a) a silica based purification method;

or

(b) a target capture method.

17. The method of claim 16, wherein said RNA is extracted using a target capture method.

18. The method of claim 1, wherein said crude urine sample is from urine collected not following a digital rectal examination.

19. The method of claim 1, wherein said crude urine sample is from urine collected following a digital rectal exam.