PCA3 messenger RNA species in benign and malignant prostate tissues
View Patent ↗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.
1. A method of determining the malignancy status of prostate cells contained in a sample undergoing testing, said method comprising:
(a) amplifying a first PCA3 nucleic acid across nucleotide positions 26 and 255 of SEQ ID NO:1 and a second PCA3 nucleic acid across nucleotide positions 26 and 27 of SEQ ID NO:2 from mRNAs obtained from prostate cells contained in said sample if such first and/or second nucleic acids are present, whereby said first and second PCA3 nucleic acids are synthesized in relative abundance dependent on the malignant and non-malignant status of the prostate cells,
wherein the amplification of said first PCA3 nucleic acid 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 the amplification of said second PCA3 nucleic acid 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)
i) determining a malignant status of the prostate cells in said sample by detecting a higher level of said second PCA3 nucleic acid as compared to said first PCA3 nucleic acid; or
ii) determining a non-malignant status of the prostate cells in said sample by detecting a higher level of said first PCA3 nucleic acid as compared to said second nucleic acid; thereby determining the malignancy status of said prostate cells in said sample.
2. The method of claim 1 , wherein after step (a) and before step (b) there is an additional step of separating said first and second PCA3 nucleic acids amplified in step a) from each other.
3. The method of claim 1 , wherein after step (a) and before step (b) there is an additional step of contacting said first and second PCA3 nucleic acids with a detectably labeled hybridization probe.
4. The method of claim 1 , wherein said amplification in step a) comprises using a pair of oligonucleotides comprising a first primer complementary to a sequence contained within PCA3 exon 4a of said first and second PCA3 mRNAs.
5. The method of claim 4 , wherein said pair of oligonucleotides comprises a second primer that comprises a sequence contained within PCA3 exon 3.
6. The method of claim 5 , wherein each of said first and second primers is at least 12 nucleotides in length.
7. The method of claim 6 , wherein said amplifying in 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.
8. The method of claim 5 , wherein each of said first and second primers is at least 10 nucleotides in length.
9. The method of claim 5 , wherein each of said first and second primers is at least 15 nucleotides in length.
10. The method of claim 5 , wherein each of said first and second primers is at least 20 nucleotides in length.
11. The method of claim 5 , wherein each of said first and second primers is at least 18 to about 50 nucleotides in length.
12. The method of claim 5 , wherein each of said first and second primers is at least 20 to about 35 nucleotides in length.
13. The method of claim 1 , wherein said amplifying in 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.
14. The method of claim 1 , wherein said determining in step b) 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 μg/ml denatured salmon sperm DNA.
15. The method of claim 14 , wherein said probe hybridizes to at least 15 consecutive nucleotides of said exon 3-exon 4a junction.
16. The method of claim 14 , wherein said probe hybridizes to at least 20 consecutive nucleotides of said exon 3-exon 4a junction.