Method to detect prostate cancer in a sample

• US 8,546,551 B2
• Filed: 05/14/2012
• Issued: 10/01/2013
• 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, Pich, Lyson, Garon, Genevive
• Primary Examiner(s): AEDER, SEAN E
• Application Number:
  US13/470,451
• Publication Number:
  US 20120309001A1
• Time in Patent Office: 505 Days
• Field of Search: None
• US Class Current: 536/23.1
• 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 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.

Claims

1. A prostate cancer diagnostic 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 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); 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.

2. The prostate cancer diagnostic composition of claim 1, wherein said urine sample comprises malignant prostate cells or a nucleic acid extract thereof.

3. The prostate cancer diagnostic composition of claim 1, wherein said urine sample comprises crude urine.

4. The prostate cancer diagnostic 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 prostate cancer diagnostic composition of claim 1, further comprising reagents for amplifying and/or detecting said PCA3 mRNA molecule and said prostate-specific mRNA molecule.

6. The prostate cancer diagnostic 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 prostate cancer diagnostic 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 prostate cancer diagnostic composition of claim 1, wherein said prostate-specific mRNA is PSA mRNA.

9. The prostate cancer diagnostic composition of claim 8, wherein said PSA mRNA hybridizes to human kallikrein 2.

10. The prostate cancer diagnostic 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 prostate cancer diagnostic composition of claim 5, wherein said reagents enable simultaneous amplification of PCA3 and said prostate-specific mRNA.

12. The prostate cancer diagnostic composition of claim 5, wherein said reagents enable nucleic acid amplification:

(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).

13. The prostate cancer diagnostic composition of claim 5, wherein said reagents enable detection by fluorescence, chemiluminescence or colorimetry detection.

14. The prostate cancer diagnostic composition of claim 5, wherein said reagents comprise a first molecular beacon that hybridizes to PCA3 under high stringency conditions, and/or a second molecular beacon that hybridizes to said prostate-specific mRNA under high stringency conditions.

15. The prostate cancer diagnostic composition of claim 14, wherein said first molecular beacon comprises the sequence set forth in SEQ ID NO:

6.

16. The prostate cancer diagnostic composition of claim 14, wherein said second molecular beacon hybridizes to PSA mRNA under high stringency conditions and comprises the sequence set forth in SEQ ID NO:

5.

17. The prostate cancer diagnostic 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.