PCA3 Messenger RNA Species in Benign and Malignant Prostate Tissues

• US 20080261228A1
• Filed: 04/07/2008
• Published: 10/23/2008
• Est. Priority Date: 09/29/1999
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Diagnocure Inc.
• Inventors: Fradet, Yves, Chypre, Camille, Busse, Ursula
• Granted Patent: US 7,655,408 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A61P 13/08 of the prostate
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/4748 Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
• CPC Class Codes: C07K 16/3069 Reproductive system, e.g. ovaria, uterus, testes, prostate
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2333/47 Assays involving proteins of known structure or function as defined in the subgroups
• CPC Class Codes: G01N 2800/56 Staging of a disease; Further complications associated with the disease
• CPC Class Codes: G01N 33/57434 of prostate

Abstract

This invention concerns the discovery of two distinct PCA3 mRNA sequences. One of these sequences corresponds to a short PCA3 mRNA molecule whereas the other PCA3 RNA molecule is longer as it comprises an additional sequence between exon 3 and exon 4a. The short RNA is associated with prostate cancer whereas the long RNA sequence is associated with a non-malignant state of the prostate. Based on the differential expression levels of these two PCA3 RNA sequences, protocols for the diagnosis of prostate disease are provided. The invention also relates to therapeutic approaches to prostate cancer.

Claims

1. A method of distinguishing differentially expressed PCA3 nucleic acids that indicate the malignancy status of prostate cells contained in a sample undergoing testing, said method comprising the steps of:

(a) performing a nucleic acid amplification reaction, using spliced RNAs obtained from prostate cells contained in said sample as templates, to amplify PCA3 nucleic acid sequences across nucleotide positions 26 and 255 of SEQ ID NO;

1 and nucleotide positions 26 and 27 of SEQ ID NO;

2, whereby there are synthesized first and second amplification products in relative abundance dependent on the malignant and non-malignant status of prostate cells,

wherein said first amplification product results from amplification of a first PCA3 mRNA that comprises an additional sequence between PCA3 exon 3 and PCA3 exon 4a, said additional sequence consisting essentially of nucleotides 27 to 254 of SEQ ID NO;

1, and

wherein said second amplification product results from amplification of a second PCA3 mRNA that comprises PCA3 exon 3 joined to PCA3 exon 4a without including said additional sequence; and

(b) detecting one of said first and second amplification products independent of the other, thereby distinguishing differentially expressed PCA3 nucleic acids that indicate the status of prostate cells contained in said sample.

2. The method of claim 1, wherein after step (a) and before step (b) there is an additional step for separating said first and second amplification products from each other.

3. The method of claim 1, wherein after step (a) and before step (b) there is an additional step for contacting said first and second amplification products with a detectably labeled hybridization probe.

4. The method of claim 1, wherein said nucleic acid amplification reaction in step (a) comprises an amplification pair of oligonucleotides.

5. The method of claim 4, wherein said amplification pair of oligonucleotides comprises a first primer complementary to a sequence contained within PCA3 exon 4a of said first and second PCA3 mRNAs.

6. The method of claim 5, wherein said amplification pair of oligonucleotides further comprises a second primer that comprises a sequence contained within PCA3 exon 3, said second primer being complementary to extension products of the first primer when using the first and second PCA3 mRNAs as templates in a primer extension reaction.

7. The method of claim 6, wherein each of said first and second primers is at least 12 nucleotides in length.

8. The method of claim 7, wherein the nucleic acid amplification reaction of step (a) comprises a reaction selected from the group consisting of a nucleic acid sequence-based amplification reaction (NASBA), a polymerase chain reaction (PCR), a transcription-based amplification reaction, a strand displacement amplification reaction (SDA), and a Q replicase reaction.

9. The method of claim 1, further comprising a step for (c) quantifying relative amounts of said first and second amplification products synthesized in said nucleic acid amplification reaction.

10. The method of claim 9, further comprising a step for (d) determining from the relative amounts of said first and second amplification products quantified in step (c) whether said sample comprises prostate cancer cells.

11. The method of claim 6, wherein each of said first and second primers is at least 10 nucleotides in length.

12. The method of claim 6, wherein each of said first and second primers is at least 15 nucleotides in length.

13. The method of claim 6, wherein each of said first and second primers is at least 20 nucleotides in length.

14. The method of claim 6 wherein each of said first and second primers is at least 18 to about 50 nucleotides in length.

15. The method of claim 6, wherein each of said first and second primers is at least 20 to about 35 nucleotides in length.

16. The method of claim 1, wherein the nucleic acid amplification reaction of step (a) comprises a reaction selected from the group consisting of a nucleic acid sequence-based amplification reaction (NASBA), a polymerase chain reaction (PCR), a transcription-based amplification reaction, a strand displacement amplification reaction (SDA), and a Q replicase reaction.

17. The method of claim 1, wherein said detecting of said second PCA3 amplification product comprises binding with a probe which hybridizes under high stringency conditions to at least 10 consecutive nucleotides of a sequence comprising nucleotides 26 and 27 of SEQ ID NO:

2, which define the exon 3-exon 4a junction, wherein said high stringency conditions comprise a hybridization at 65°

C. in 5×

SSC, 5×

Denhardt'"'"'s solution, 1% SDS, and 100 μ

g/ml denatured salmon sperm DNA.

18. The method of claim 17, wherein said probe hybridizes to at least 15 consecutive nucleotides of said exon 3-exon 4a junction.

19. The method of claim 17, wherein said probe hybridizes to at least 20 consecutive nucleotides of said exon 3-exon 4a junction.

20. A kit for distinguishing between a malignant and non malignant PCA3 mRNA, comprising:

a) a first primer which hybridizes under high stringency conditions to or upstream of nucleotide position 26 of SEQ ID NO;

1 or 2; and

b) a second primer which hybridizes under high stringency conditions to or downstream of nucleotide position 27 of SEQ ID NO;

2 or nucleotide position 255 of SEQ ID NO;

1, wherein nucleotide positions 26 and 27 of SEQ ID NO;

2 or 26 and 255 of SEQ ID NO;

1 define the PCA3 exon 3-exon 4a junction,

whereby said primers in a) and b) define a primer pair which enables an amplification of a PCA3 mRNA across said exon 3-exon 4a junction to produce a PCA3 amplicon, and wherein

a first PCA3 amplicon comprising an additional sequence between exon 3 and exon 4a, with said additional sequence being as set forth from nucleotides 27 to 254 of SEQ ID NO;

1, is associated with a non-malignant state of the prostate;

a second PCA3 amplicon lacking said additional sequence between exon 3 and exon 4a, is associated with a malignant state of the prostate;

and wherein said high stringency conditions comprise a hybridization at 65°

C. in 5×

SSC, 5×

Denhardt'"'"'s solution, 1% SDS, and 100 μ

g/ml denatured salmon sperm DNA

21. The kit of claim 20, further comprising a probe together with a suitable reagent, wherein said probe hybridizes under high stringency conditions to at least 10 consecutive nucleotides of a region comprising a PCA3 exon 3-exon 4a junction, said PCA3 exon 3-exon 4a junction being defined by nucleotide positions 26 and 27 of SEQ ID NO:

2 or 26 and 255 of SEQ ID NO;

1, and wherein said high stringency conditions comprise a hybridization at 65°

C. in 5×

SSC, 5×

Denhardt'"'"'s solution, 1% SDS, and 100 μ

g/ml denatured salmon sperm DNA.

22. The kit of claim 20, wherein said amplification of said PCA3 mRNA associated with a malignant state of the prostate and said amplification of said PCA3 mRNA associated with a non-malignant state of the prostate are performed simultaneously.